KONZO AND CASSAVA TOXICITY - stuba.skszolcsanyi/education/files/Organicka chemia II... · I thank Dr Yu-Haey Kuo (Dianna) for the initiation to the use of HPLC instruments and for

KONZO AND CASSAVA TOXICITY

A STUDY OF ASSOCIATED NUTRITIONAL FACTORS IN THE POPOKABAKA

DISTRICT, DEMOCRATIC REPUBLIC OF CONGO

Delphin DIASOLUA NGUDI

Academic Year 2004-2005

KONZO AND CASSAVA TOXICITY: A STUDY OF ASSOCIATED

NUTRITIONAL FACTORS IN THE POPOKABAKA DISTRICT,

DEMOCRATIC REPUBLIC OF CONGO

KONZO-ZIEKTE EN DE TOXICITEIT VAN MANIOK: EEN STUDIE VAN DE VOEDINGSFACTOREN IN POPOKABAKA DISTRICT,

DEMOCRATISCHE REPUBLIEK KONGO

Door

Delphin DIASOLUA NGUDI, M.Sc.

Thesis submitted in fulfilment of the requirements for the degree

of Doctor (Ph.D.) in Applied Biological Sciences

Proefschrift voorgedragen tot het bekomen van de graad van

Doctor in the Toegepaste Biologische Wetenschappen

Op gezag van: Rector: Prof. Dr. Apr. A. DE LEENHEER

Decaan: Prof. Dr. Ir. Herman VAN LANGENHOVE

Promotoren:Prof. Dr. Patrick KOLSTEREN

Prof. Dr. Ir. Fernand LAMBEIN

CITATION

“A person who has food has many problems.

A person who has no food has only one problem”

Chinese saying

DEDICATION

To Thérèse Luntala and our sons Berdit, Gaël and Beni

In memory of my Grand Oncle André Banketa and my father Bernard Ngudi-a-nkama

Promotoren: Prof. Dr. Patrick KOLSTEREN

Vakgroep Voedselveiligheid en Voedselkwaliteit, Universiteit Gent Voedings unit, Prins Leopold Instituut voor Tropische Geneeskunde, Antwerpen Prof. Dr. Ir. Fernand LAMBEIN Instituut Planten Biotechnologie voor Ontwikkelingenlanden, Universiteit Gent

Decaan: Prof. Dr. Ir. Herman VAN LANGENHOVE

Academiejaar 2004 - 2005

Delphin DIASOLUA NGUDI

KONZO AND CASSAVA TOXICITY

A STUDY OF ASSOCIATED NUTRITIONAL FACTORS IN THE POPOKABAKA

DISTRICT, DEMOCRATIC REPUBLIC OF CONGO

Proefschrift

voorgedragen tot het bekomen van de graad van

Doctor in de Toegepaste Biologische Wetenschappen

Op gezag van de rector,

Prof. Dr. Apr. A. DE LEENHEER

Nederlandse vertaling titel:

KONZO-ZIEKTE EN DE TOXICITEIT VAN MANIOK: EEN STUDIE VAN DE

VOEDINGSFACTOREN IN POPOKABAKA DISTRICT, DEMOCRATISCHE

REPUBLIEK KONGO

Diasolua Ngudi, D. (2005). Konzo and cassava toxicity: a study of associated nutritional factors in the Popokabaka District, Democratic Republic of Congo. Ph D. thesis Universiteit Gent, Belgium, 160 p ISBN 90-5989-073-6 The author and the promoter give the authorisation to consult and to copy parts of this work for personal use only. Every other use is subject to the copyright laws. Permission to reproduce any material contained in this work should be obtained from the author.

Acknowledgments

I would like to express my gratitude to all the many people who and institutions that have

contributed to this research. I am particularly grateful to my promoter Prof Dr Patrick

Kolsteren for his encouragement, his guidance and constructive criticisms. My special thanks

to Prof Dr Ir Fernand Lambein, the co-promoter of this thesis for accepting me to work under

his supervision. I am indebted to him for improving my skills, including my laboratory

techniques and for sharing his knowledge and experience on nutritional and neuro-

toxicological disease. I also thank Prof. Em. Dr. Ir André Huyghebaert, my promoter until his

retirement for handling the administrative issues of my enrolment to the university.

I thank Dr J. Howard Bradbury from the Australian University and chairperson of the

Cyanide Cassava Disease Network for his advices and for graciously providing me kits for

analyses of cyanide and thiocyanate. I thank Dr Yu-Haey Kuo (Dianna) for the initiation to

the use of HPLC instruments and for her meticulous attention in scrutiny of manuscripts. I

thank Prof Dr Thorkild Tylleskär for inspiring and encouraging me to initiate this research.

Dr Theophile Ntambwe, former Director of PRONANUT has to be thanked for his support

and follow up of my scholarship file. I am thankful to Prof Dr JP Banea Mayambu, Director

of PRONANUT for the encouragement, guidance and support. I am indebted to both of them

for providing me field facilities. The staff and colleagues of PRONANUT are greatly

acknowledged for their enthusiasm and encouragement.

Prof Dr Simon Malele ma Ludani from the Australia’s University of Southern Queensland/

Dubai and Dr Fabienne Ladrière from Médecins du Monde are thanked for their assistance

and for reading and improving this thesis. I am deeply grateful to the members of the

examination committee: Prof Dr Ir Georges Hofman, Prof Dr Ir Colin Janssen, Prof Dr Geert

Callewaert, Prof Dr Armand Christophe and Dr Ir Bruno De Meulenaer. Their criticism of

this work was very constructive.

The Belgian Cooperation and Development Ministry has exceptionally granted me a mixed

scholarship through the Belgian Technical Cooperation (BTC) in April 2002. I thank Mr

Marino Orban, Mr G. Kasende and Mrs Sarah Stijnen of BTC for their efficient management

of my dossier. Congolese Ministry of Health through PRONANUT, OXFAM – Destelbergen

(Katrien Goddemaer), GTZ (Nour Salua) and anonymous relatives and friends provided also

financial support to conduct part of my program.

Prof Em Marc Van Montagu, Prof Ann Depicker, Prof Dr Lieve Gheysen and staff of IPBO

are thanked for the hospitality and their interest in my research.

Konzo and cassava toxicity

II

Exceptional thanks to Ir. Anne-Marie De Winter, Regine Haspeslagh, Veerle Van Ongeval, Ir.

Ann Peters, Christine Graveel, Jean-Pierre Dubois, Ing. Alfons Lenaert, Myriam De Vos and

Ing. Yves De Jonge for their assistance, continuous interest and encouragement.

I am indebted to the Visie newspaper and to the VRT-film crew Jo Frere and Jasmine De

Bruycker for the interest in the topic of my research and for their objective reporting on the

disease on the disease konzo and on our research.

Piet Meyvaert, Paul Meirsman, Lieve Van Wijmeersch, Fernand Verhoeven, Erik Verhaegen,

Willy Mpoyi, Louis Kitenge, Raf Nunga, Elie and Blaise Ndosimau, Sera and Emmanuel

Kisuesue, Ir Bernard Lelou, Prof Dr Emmanuel Biey, Dr José Biey, Dr Thomas Mpiana, Dr

Clément Mulenda Tshamala, Michel Fazili and other friends, not mentioned by name, are

acknowledeged for their help and friendship.

I would like to express my cheerfully thanks to my mother, to my sisters Lili, Micheline and

Euphrasie,to my sister in law Makiese and to my brothers in law Malueki, Nsinga, Ntondo

and Yende for taking care of our sons while we were out of the country and far from them.

Many thanks to my uncle Prof Dr Mamingi Nlandu, to my brothers Domi, Sivis and Nsimba,

to my cousin Dimbu, to my sisters in law Annie, Julie and Elisée and to all my nephews and

nieces.

To my sons Berdit, Gäel and Beni, I have to apologise for not giving them all the attention

they deserved during the many busy years that research entailed. I thank Thérèse Luntala for

often coping with my absence concerning things of life and for her advice, help and love.

Last but not the least; I thank the authorities and the population of Popokabaka for their

enthousiasm and collaboration during my field trips.

Gent, June 2005

Delphin Diasolua Ngudi


III

CONTENTS

Acknowledgments......................................................................................................................I

CONTENTS........................................................................................................................... III

List of figures ........................................................................................................................... V

List of tables............................................................................................................................VI

Abbreviations and acronyms..............................................................................................VIII

Samenvatting ........................................................................................................................... X

Résumé .................................................................................................................................XIII

Summary............................................................................................................................ XVII

I Literature review.............................................................................................................. 2

I.1 Introduction.................................................................................................................... 2

I.1.1 Paraparesis and neurodegenerative diseases, what is the meaning? ...................... 2

I.1.2 The “hidden endemias” .......................................................................................... 3

I.2 Konzo.............................................................................................................................. 7

I.2.1 Background information on konzo......................................................................... 7

I.2.2 Clinical features and differential diagnosis.......................................................... 10

I.2.3 Epidemiology ....................................................................................................... 14

I.2.4 Infection or toxico-nutritional etiology? .............................................................. 15

I.3 Dietary exposure to cyanide from cassava .................................................................. 17

I.3.1 Cassava................................................................................................................. 17

I.3.2 Cyanide toxicity ................................................................................................... 27

I.4 Conclusion.................................................................................................................... 39

I.4.1 Rationale of the research ...................................................................................... 40

I.4.2 Objectives............................................................................................................. 40

II Occurrence of konzo and dietary pattern .................................................................... 43

II.1.1 Introduction .......................................................................................................... 43


IV

II.1.2 Materials and Methods ......................................................................................... 43

II.1.3 Results .................................................................................................................. 47

II.1.4 Discussion and conclusion ................................................................................... 54

III Cassava food quality and safety.................................................................................... 59

III.1 Food Safety and Amino Acid Balance in Processed Cassava "cossettes" ............... 59

III.1.1 Introduction ...................................................................................................... 59

III.1.2 Materials and methods ..................................................................................... 62

III.1.3 Results and discussion...................................................................................... 66

III.2 Residual cyanogens, free and total amino acid profiles of cooked cassava leaves

"saka- saka”......................................................................................................................... 79

III.2.1 Introduction ...................................................................................................... 79

III.2.2 Materials and methods ..................................................................................... 80

III.2.3 Results and discussion...................................................................................... 82

III.2.4 Conclusions ...................................................................................................... 92

IV Dietary cyanogen and sulphur metabolites excretion................................................. 95

IV.1.1 Introduction ...................................................................................................... 95

IV.1.2 Material and methods ....................................................................................... 96

IV.1.3 Results .............................................................................................................. 98

IV.1.4 Discussion ...................................................................................................... 100

V General discussion and conclusions............................................................................ 105

V.1 Occurrence of konzo............................................................................................... 106

V.2 Cassava foods and sulphur metabolites ................................................................. 108

V.3 Conclusions and recommendations........................................................................ 111

References ............................................................................................................................. 114

Curriculum Vitae ..................................................................................................................... ii


V

List of figures

Figure I-1: Health zones of Kwango District in Bandundu Province, DRC............................. 8

Figure I-2: From left to right: mild form, moderate form and severe form of konzo in young

subjects ............................................................................................................................. 12

Figure I-3 : Cassava roots harvested and cassava plant in the field........................................ 18

Figure I-4: Location of cassava production, 1996 (Scott et al, 2000)..................................... 23

Figure I-5: Summary of traditional cassava processing in Africa (from Banea-Mayambu,

1997c)............................................................................................................................... 27

Figure I-6: Cyanogenesis from linamarin (McMahon et al, 1995) ......................................... 29

Figure I-7: Basic processes involved in the metabolism of cyanide (ATSDR, 1997) ............ 35

Figure I-8 : Cysteine catabolism ............................................................................................. 37

Figure II-1: Distribution of onset of konzo from 1980 to 2002.............................................. 49

Figure III-1: Flow diagram of cassava cossettes processing .................................................. 60

Figure III-2: Free amino acids in cassava cossette samples ................................................... 74

Figure III-3: Protein amino acids profile of the raw and cooked cassava leaves ................... 87


VI

List of tables

Table I-1: Characteristic features of four tropical myeloneuropathies (Tylleskär et al, 1994c)5

Table I-2: WHO criteria for konzo versus newly suggested criteria (with permission and from

Tshala-Katumbay, 2001a) ................................................................................................ 11

Table II-1: Socio-demographic variables and 24hr recall food consumption of participants

among the high prevalence of konzo health area (n = 224) and the low prevalence of

konzo health area (n =263)............................................................................................... 46

Table II-2: Distribution of konzo cases per health area .......................................................... 49

Table II-3 : Degree of disability on walking and age distribution of konzo patients by gender

.......................................................................................................................................... 50

Table II-4: 24-hour recall of household food intake frequencies (%) .................................... 51

Table II-5: Seasonal food consumption availability (%) listed by the respondents ............... 53

Table III-1: Cyanogens content in cassava cossettes (mg HCN equivalent kg - 1 dry weight)67

Table III-2: Estimated daily cossettes and total cyanogens intake ......................................... 69

Table III-3: Total protein amino acids content in cassava cossettes (mg g - 1dry weight)...... 72

Table III-4: Amino acid scoring pattern of different cossette samples .................................. 73

Table III-5. Free protein amino acids content in cassava cossettes (mg g - 1 dry weight)....... 75

Table III-6. Essential Amino Acid (EAA) requirements and estimated daily intake ............. 77

Table III-7: Cyanogen content in raw and cooked cassava leaves (mg HCN equivalent kg-1

dry weight) ....................................................................................................................... 84

Table III-8: Protein content and amino acid composition of raw and cooked pounded cassava

leaves (g kg-1 dry weight) ................................................................................................. 86

Table III-9: Comparison of the essential amino acid contents of different raw and cooked

pounded cassava leaves samples and their amino acid score with the recommended FAO

reference ........................................................................................................................... 89


VII

Table III-10: Free amino acid and trigonelline content in raw and cooked cassava leaves (g

kg-1 dry weight) ................................................................................................................ 91

Table IV-1: Distribution of konzo- affected households in each health area with the number

of konzo patients given in brackets .................................................................................. 98

Table IV-2: Total cyanogens in cassava flour, thiocyanate and taurine in urine samples

collected in three konzo prevalence areas of Popokabaka (DRC). .................................. 99


VIII

Abbreviations and acronyms

α-ABA α-Amino butyric acid

AMPA α-Amino-3hydroxy-5-methyl-isoxazole-4-propionic acid

ATC 2-Aminothiazoline-4-carboxylic acid

ATSDR Agency for Toxic Substances and Disease Registry

BOAA ß-Oxalylaminoalanine

ß-ODAP ß-N-oxalyl-α,ß-diaminopropionic acid

BTC/CTB Belgian Technical Cooperation/ Coopération Technique Belge

CEPLANUT Centre National de Planification de Nutrition Humaine

CI confidence interval

CN- Cyanide

CNS Central nervous system

D. R. C. Democratic Republic of Congo

EAA Essential amino acids

FAO Food and Agriculture Organisation

GABA γ-Amino butyric acid

HCN Hydrogen cyanide

HNL α-Hydroxynitrile lyase

HPLC High Performance Liquid Chromatography

HTLV-1 Human T cell lymphocyte virus Type I

IITA International Institute for Tropical Agriculture

KCN Potassium cyanide

NaCN Sodium cyanide

OCN- Cyanate

OR Odds Ratio

PITC Phenylisothiocyanate

Ppm Part per million

Prhz Popokabaka rural health zone

PRONANUT Programme National de Nutrition

R. D. C. République Démocratique du Congo

SAA Sulphur containing amino acids

SCN- Thiocyanate

SD Standard deviation


IX

SPSS Statistical Package for Social Science

TAN Tropical ataxic neuropathy

TSP/HAM Tropical spastic paraparesis/ Human T cell lymphocyte virus

Type I-associated myelopathy

UDPG Uridine diphosphoglucose

UK United Kingdom

UNICEF United Nations for Infants and Children Emergency Funds

UNU United Nations University

USA United States of America

WHO World Health Organisation


X

Samenvatting

Konzo is een neurologische aandoening die gekenmerkt wordt door een plots beginnende en

blijvende verlamming van de benen. Dit komt vooral voor bij kinderen en vrouwen op

vruchtbare leeftijd. Wortels van bittere maniok (Manihot esculenta Crantz) en daarvan

afgeleide producten die een hoog gehalte aan cyanogenen bevatten, kunnen acute cyanide

vergiftiging veroorzaken met symptomen als braken, duizeligheid, maagpijn, flauwte,

hoofdpijn en diarree. Konzo werd toegeschreven aan een langdurige chronische inname van

cyanide in bittere maniokwortels die onvoldoende geroot werden. Maniok is een belangrijke

basis voor het dagelijkse voedsel van meer dan een half miljard mensen verspreid over de

ganse wereld, nochtans bevat het cyanogeen glycosiden, voornamelijk linamarine dat na

enzymatische omzetting tot cyanohydrine aanleiding kan geven tot het giftige blauwzuur

(HCN) na verdere enzymatische of spontane omzetting. Alhoewel er voldoende aanwijzingen

zijn voor een verband tussen de ziekte konzo en de consumptie van bittere cassava, blijft het

pathologisch mechanisme van de ziekte onduidelijk. Men vermoedt dat zowel HCN als zijn

metabolieten (2-aminothiazolin-4-carbonzuur, cyanaat en isothiocyanaat) een rol spelen in de

pathologie maar er is geen proefdier model om dit te bevestigen. Wel staat vast dat de

enzymatische omzetting van cyanide naar thiocyanaat (80% van het cyanide wordt langs die

weg gedetoxifieerd) gebruik maakt van zwavel afkomstig van de zwavelhoudende

aminozuren methionine en cysteine.

In dit werk wordt het voorkomen van de konzo ziekte in Popokabaka, in de Bandundu

provincie van de Democratische Republiek Kongo bestudeerd. Uit diezelfde regio kwam het

eerste gepubliceerde verslag over konzo in 1938. Mogelijke associaties tussen de ziekte met

gezinsgebonden factoren en met het eetpatroon van de bevolking die hoofdzakelijk maniok

eet, werden onderzocht. Konzo komt nog steeds voor in die regio met een incidentie van 1,3

‰ in 2002. Meer vrouwen dan mannen waren aangetast en er werd geen informatie over de


XI

levensverwachting van de patiënten gevonden. Zowel geslacht als burgerlijke stand

vertoonden een statistische associatie met de kans op konzo. Maniok was het dominante

bestanddeel van de voeding in praktisch elk gezin en werd minstens eenmaal per dag gegeten

als luku, een dikke pasta van maniokmeel en water. Als bijgerechten werd voornamelijk

maniok bladeren en lokaal geteelde bonen gegeten. Alhoewel die bijgerechten rijker zijn aan

eiwit is de kwaliteit van dit eiwit laag door een gebrek aan zwavelhoudende aminozuren.

De ‘cossettes’, de gerote en gedroogde maniok wortels die het voornaamste

voedingsbestanddeel vormen en de maniokbladeren die het voornaamste bijgerecht vormen in

de regio werden onderzocht. De dagelijkse hoeveelheid werd bepaald, het gehalte aan en de

dagelijkse inname van cyanogeen en de hoeveelheid zwavelhoudende aminozuren vereist

voor de detoxificatie ervan werd berekend. De vrije en totale aminozuren in de maniok

produkten werden bepaald om eventuele inherente toxinen op te sporen, om de kwaliteit van

de proteïne te bepalen en om te kunnen vergelijken met de dagelijkse vereisten voor kinderen

en volwassenen. Er werd berekend dat kinderen van 1 tot 9 jaar dagelijks 0,4 tot 1,1 mg HCN

equivalenten innemen in 241 tot 389 g maniok produkten, hoofdzakelijk van ‘cossettes’.

Matig actieve volwassen mannen en vrouwen namen 0,6 tot 1,5 mg HCN equivalenten in per

dag van 390 tot 532 g maniok produkten bereid uit ‘cossettes ‘, in de veronderstelling dat

60% van de dagelijkse energievereisten voldaan worden door maniokwortels die 1,6 tot 2,8

mg HCN equivalenten bevatten per kg droog gewicht. We vonden geen potentieel giftige niet-

proteïne aminozuren in maniokwortels. Lysine en leucine zijn de limiterende aminozuren

terwijl methionine slechts voor 13% bijdraagt in het lage gehalte van zwavelhoudende

aminozuren van de ‘cossettes’. Er werd berekend dat, indien alleen maniok gegeten wordt, de

dagelijkse behoefte aan methionine voor kinderen van 1 tot 9 jaar slechts voor 60% voldaan

is, terwijl aan de dagelijkse behoefte van de volwassenen wel voldaan wordt indien 60% van

de dagelijkse energiebehoeften komt uit maniokprodukten (het nationale gemiddelde). De


XII

maniokbladeren die wel rijker zijn aan proteïne maar eveneens arm zijn aan methionine,

kunnen dit gebrek bij kinderen in gebieden met konzo niet compenseren. De maniokbladeren

als bijgerecht kunnen ook een bijkomende bron van cyanide zijn indien dit onvoldoende lang

gekookt wordt wegens een gebrek aan brandhout, de enige beschikbare brandstof voor de

bereiding. Zwavelhoudende aminozuren zijn noodzakelijk voor de detoxifiëring van cyaniden

afkomstig van maniokwortels die onvoldoende geroot werden, maar ook van de bladeren die

onvoldoende lang gekookt werden.

In een epidemiologische studie werden urinestalen en stalen van de maniokbloem van

verschillende gezinnen onderzocht met de bedoeling de voedselveiligheid na te gaan, de

inname van cyanogenen te berekenen en een mogelijk verband na te gaan met de

zwavelmetabolieten taurine en thiocyanaat in de urine. Drie generaties na het eerste

gedocumenteerd voorkomen van konzo in deze regio blijft er nog steeds een hoog risico voor

de blootstelling aan cyanide van maniokbloem. In de helft van de gezinnen is het

cyanidegehalte in maniokbloem hoger dan de door de Wereld Gezondheid Organisatie en de

FAO aanbevolen grens van 10µg HCN equivalent/g bloem. De helft van de urinestalen

bevatten meer dan 300 µmol/l thiocyanaat. Dit wijst op een grote overmaat aan cyanide

inname Alhoewel men geen significante correlatie vond, wijst de lage concentratie taurine, de

eindmetaboliet van de zwavelhoudende aminozuren, op een hoog verbruik van zwavel voor

de detoxifiëring van cyanide met vorming van thiocyanaat.

De bevolking van Popokabaka is nog steeds blootgesteld aan een te hoog cyanogeen gehalte

uit de maniokvoeding en misschien ook aan cyanogenen uit de omgeving. Dit verhoogt het

risico voor konzo in die regio waar die verlammende ziekte nog steeds voorkomt. Een beter

gevarieerd en gebalanceerd dieet, dat rijker is aan methionine, is noodzakelijk voor een meer

efficiënte detoxifiëring van cyanide in het menselijke lichaam.


XIII

Résumé

Konzo est un désordre neurologique caractérisé par un début soudain d’une paralysie

permanente des membres inférieurs. Les enfants d’au moins trois ans et les femmes en âge de

procréer sont les plus affectés. La consommation des tubercules de manioc et de leurs

produits dérivés contenant une grande quantité de cyanogènes peut causer une intoxication

pouvant se manifester par des vomissements, des nausées, des vertiges, des douleurs

abdominales, la faiblesse, des maux de tête et la diarrhée. Konzo a été attribué à une haute

consommation de cyanure des tubercules de manioc amer insuffisamment traité. Le manioc,

aliment de base très important pour plus d’un demi milliard d’habitants de la planète, contient

des glucosides cyanogéniques, principalement la linamarine qui après conversion

enzymatique en cyanohydrines, peut libèrer spontanément ou enzymatiquement un prodruit

toxique, l’acide cyanhydrique. Bien que le lien évident entre la maladie konzo et la

consommation du manioc amer insuffisammnet traité soit établi, les mécanismes

pathogéniques du konzo restent encore à élucider. L’acide cyanhydrique et ses métabolites

(acide 2-aminothiazoline-4-carboxylique, cyanate, thiocyanate) ont été suspectés de jouer un

rôle dans la pathogénicité du konzo chez l’être humain mais il n’y a pas encore de modèle

animal pour s’en assurer ou le confirmer. En effet, la conversion enzymatique du thiocyanate

à partir du cyanure (environ 80 % de cyanure est transformé par cette voie) nécessite le soufre

provenant des acides aminés soufrés, la méthionine et la cystéine.

Notre travail rapporte l’apparition des cas de konzo à Popokabaka, province de Bandundu, R.

D. Congo, une des régions incluse dans la première publication sur le konzo en 1938. Nous

décrivons l’association entre le konzo et les facteurs socio-économiques liés au ménage et les

habitudes alimentaires des populations consommant le manioc. Le konzo continue à sévir

dans cette région de Popokabaka avec une incidence de 1,3 ‰ en 2002. La paralysie affecte

plus de femmes que d’hommes et nous n’avons trouvé aucune publication sur l’espérance de


XIV

vie (ou sur la mortalité) des personnes atteintes de konzo. Le genre et l’état civil des chefs de

ménage sont associés au degré de prévalence de konzo dans les différentes localités de la

région. L’alimentation dans la contrée est largement dominée par le manioc. Presque tous les

ménages consomment au moins une fois par jour du luku, pâte obtenue après cuisson et

malaxage de la farine de manioc dans de l’eau bouillante. Les principaux aliments

d’accompagnement du luku, les feuilles de manioc et le niébé, sont limités en acides aminés

soufrés.

Nous avons analysé les cossettes de manioc (produit dérivé des tubercules de manioc roui et

séché) et les feuilles de manioc, respectivement aliment de base et principal aliment

d’accompagnement dans la région, pour estimer la quantité de cyanogènes résiduels

consommée journellement et la quantité d’acides aminés soufrés disponibles pour la

détoxification de ces cyanogènes. Nous avons déterminé les acides aminés libres et totaux

dans les cossettes et les feuilles de manioc pour détecter la présence inhérente des acides

aminés non protéiniques potentiellement toxiques, pour évaluer la qualité de leur protéine

alimentaire et la comparer aux besoins recommandés en acides aminés des enfants et des

adultes. Nous avons trouvé une consommation journalière estimée de 0,4 à 1,1 mg HCN

équivalent dans 241 à 389 g de luku et de 0,6 à 1,5 mg HCN équivalent dans 390 à 532 g de

luku respectivement chez les enfants âgés de 1 à 9 ans et chez les adultes (masculin et

féminin) avec une activité modérée lorsque 60 % de l’énergie alimentaire journalière requise

provient du manioc de 1,6 et de 2,8 mg HCN équivalent par kg de produit sec. Aucun acide

aminé non protéinique potentiellement toxique n’a été détecté dans les produits dérivés du

manioc. La lysine et la leucine sont des acides aminés limitants et le contenu en méthionine

est très bas contribuant pour environ 13 % du total des acides aminés soufrés dans les

cossettes de manioc. Les enfants âgés de 1 à 9 ans ne peuvent pas s’attendre à satisfaire leurs

besoins recommandés en méthionine tandis que les adultes peuvent les satisfaire à partir de la


XV

quantité requise calculée pour satisfaire 60 % de l’énergie alimentaire journalière mais pas

assez pour la détoxication de cyanure et le métabolisme normal. Les feuilles de manioc,

quantitativement riches en protéines mais limitées en acides aminés soufrés, ne peuvent pas

compenser la déficience alimentaire en acides aminés soufrés occasionnée par l’aliment de

base dans les régions affectées par le konzo. Nous concluons aussi que les feuilles de manioc

peuvent être une autre source non négligeable de cyanogène alimentaire dans cette région. En

effet les feuilles de manioc requièrent pour leu détoxication une longue cuisson et avec, le

manque d’électricité et de gaz, la rareté de bois de chauffage pour la préparation des aliments,

il y a risque d’écourter le temps de cuisson et par conséquent de consommer des aliments

insuffisamments cuits.

Les acides aminés soufrés sont essentiels pour la détoxication des cyanogènes résiduels

contenus dans le manioc (tubercules ou feuilles) insuffisamment traité ou cuit.

Les échantillons de manioc prélevés auprès des ménages ainsi que ceux des urines obtenus

des participants sélectionnés au hasard pour une étude épidémiologique que nous avions

effectuée ont été examinés pour évaluer l’innocuité de la farine de manioc prête à la cuisson

puis à la consommation, pour déterminer la charge en cyanogène et apprécier la relation

potentielle entre les métabolites soufrés urinaires, la taurine et le thiocyanate. Il y a un risque

élevé d’exposition alimentaire aux cyanogènes dû à la consommation du manioc

insuffisamment traité dans cette région où le konzo est rapporté depuis trois générations. La

farine de manioc prélevée dans plus de la moitié des ménages contenait un taux de cyanogène

au dessus du seuil de 10 ppm fixé par la FAO et l’OMS. Les urines de plus de la moitié des

participants contenaient plus de 300 umol de thiocyanate par litre. Ce qui suggère qu’il y a

une charge importante en cyanure. Par ailleurs, ces urines accusaient aussi une concentration

très basse en taurine (produit du métabolisme des acides aminés soufrés), suggérant que le

soufre est préférentiellement orienté dans la détoxication du cyanure par la formation du


XVI

thiocyanate, bien que nous n‘avons presque pas trouvé de corrélation entre la taurine urinaire

et le thiocyanate.

La population de Popokabaka reste toujours grandement exposé aux cyanogènes alimentaires

du manioc et peut être aussi aux cyanogènes environnementaux. La prévention de risque élevé

d’appariton de cas de konzo dans la région requiert une alimentation suffisante, variée et

équilibrée particulièrement riche en méthionine pour permettre une détoxication effective du

cyanure par l’organisme en laissant assez d’acides aminés soufrés pour le reste des besoins

métaboliques de l’organisme.


XVII

Summary

Konzo is a neurological disorder characterised by sudden onset of paralysis of the legs, which

occurs particularly in children and women of childbearing age. Consumption of cassava

(Manihot esculenta Crantz) and its products that contain large amounts of cyanogens may

cause acute cyanide poisoning with symptoms of vomiting, nausea, dizziness, stomach pains,

weakness, headache and diarrhoea. Konzo has been attributed to the high dietary cyanide

exposure from insufficiently processed roots of bitter cassava. Cassava which is an important

staple food for more than half a billion inhabitants worldwide contains cyanogenic glycosides,

mainly linamarin that after enzymatic conversion to cyanohydrins, may release spontaneously

or enzymatically the toxic hydrogen cyanide (HCN). Although evidence linking the disease

with consumption of bitter cassava has been established, the pathogenic mechanism of konzo

remains unclear. HCN and its metabolites (2-aminothiazoline-4-carboxylic acid, cyanate and

thiocyanate) have been suspected to play a role in the pathogenicity in humans but there is no

animal model to ascertain or to confirm this. The enzymatic conversion of cyanide into

thiocyanate (about 80 % of cyanide is transformed by this route) requires sulphur arising from

the sulfur containing amino acids (SAA) methionine and cysteine.

In this work, we reporte the occurrence of konzo disease in Popokabaka, Province of

Bandundu, D. R. Congo, one of the areas included in the first published report on konzo in

1938. We described associated household factors involved in the disease and the dietary

pattern of the cassava consuming populations. Konzo is still occurring in this area with an

incidence rate of 1.3‰ in 2002. The disease affected a larger proportion of females than

males but we found no reports on the life expectancy of konzo patients. Gender and marital

status of the heads of household were associated with the degree of prevalence of konzo. The

diet was largely dominated by cassava and almost all households consumed at least once daily

the luku, a stiff porridge from the cassava flour. Major foods such as cassava leaves and


XVIII

cowpeas consumed as side–dishes to the staple food luku are of poor quality in protein

especially in SAA.

We analysed processed cassava roots ‘cossettes’, as the major staple food and cassava leaves,

as the major side-dish to the staple food in the region to estimate the quantity of daily intake

of cyanogen and for calculate the amount of SAA required for its detoxification. We

determined free and total amino acids in the cassava products to investigate the presence of

potentially toxic inherent nonprotein amino acids, to evaluate the dietary protein quality and

to compare with the amino acid requirements of children and adults. We estimated that

children (1-9yr) consumed daily about 0.4 to 1.1 mg of HCN equivalent in 241 to 389 g of

cassava product from the ‘cossettes’ and moderately active female or male consumed 0.6 to

1.6 mg of HCN equivalent in 390 to 532 g cassava product from the ‘cossettes’ when 60% of

the daily energy requirement is provided by cassava roots containing between 1.6 and 2.8 mg

HCN equivalent per kg dry weight. No potentially toxic nonprotein amino acids were detected

in cassava products. Lysine and Leucine were the limiting amino acids and the methionine

content was very low and contributed only about 13 % of the total SAA in the ‘cossettes’. We

found that children of 1 to 9 years old cannot expect to meet methionine requirement whereas

adults can meet SAA requirement from the calculated quantity required to satisfy 60 % of the

daily energy from the staple food. Cassava leaves that were found to be quantitatively rich in

protein but this protein is of poor quality with SAA as the most limiting amino acids, cannot

compensate for the dietary deficiency in SAA in the staple food in konzo affected areas. We

concluded also that cassava leaves could be an additional source of dietary cyanogen in the

region. The leaves require prolonged cooking and with the unavailability of electricity or gas

and scarcity of firewood they are consumed after a short cooking time.

SAA are essential for detoxification of the residual cyanogens in the insufficiently processed

cassava roots and also in the improperly cooked cassava leaves.


XIX

In an epidemiological study, samples of cassava flour from households and samples of urine

obtained from selected participants were examined to monitor the safety of the flour intended

to be consumed, to check cyanogen overload and to assess a potential relation between

urinary sulphur metabolites taurine and thiocyanate. There is a high risk of dietary cyanogen

exposure from cassava flour in this region where konzo was first reported three generations

ago. Cassava flour from more than half of the households had total cyanogen content above

the WHO/FAO recommended safe limit (10 μg HCN equivalent/kg cassava flour). The urine

samples from half of the participants contained more than 300 μmol/l of thiocyanate. This

suggested a high cyanide overload. The low concentration in urinary taurine found suggested

that more sulphur is directed to the detoxification of cyanide by formation of thiocyanate,

although urinary taurine and thiocyanate were slightly or not correlated.

The populations of Popokabaka are still highly exposed to cyanogen dietary from cassava and

perhaps to environmental cyanogens. The increased risk of konzo in this region where the

paralytic disease is still occurring requires a more efficient post harvest processing and a

better balanced diet, particularly richer in methionine, to allow efficient detoxification of

cyanide in the body.


1

CHAPTER I:

LITERATURE REVIEW


2

I Literature review

I.1 Introduction

I.1.1 Paraparesis and neurodegenerative diseases, what is the

meaning?

Paraparesis is a common form of neurological disability in developing countries (Howlett,

1994). It is a slight paralysis or weakness of both legs resulting in mild to moderate loss of

bilateral lower extremity motor function, which may be a manifestation of spinal cord

diseases; peripheral nervous system diseases; muscular diseases; intracranial hypertension;

parasagittal brain lesions; and other conditions. Paraparesis often progresses to paraplegia,

paralysis of the legs and lower part of the trunk. Symptoms are mild and may include spastic

paraparesis of the lower limbs, ataxia hypertonia (excessive muscle tone), mild peripheral

neuropathy, and problems of urinary incontinence (Parker, 2004). Leprosy, poliomyelitis,

tuberculosis and trauma are the main causes, but a heterogeneous group of diseases with

paraparesis also exists whose occurrence is limited to the tropics and whose etiology is still

unknown (Howlett, 1994).

In the 19th century, neurologists recognised that the muscle weakness could be due to primary

disorders of muscle or secondary to loss of neuromuscular integrity, as it happens when

peripheral nerves are cut or when motor neurones degenerate. Furthermore, it was observed

that there are forms of motor neurone degeneration which selectively affect upper motor

neurone (e.g.: primary lateral sclerosis, hereditary spastic paraplegia, tropical spastic

paraparesis (TSP), neurolathyrism, konzo) or lower motor neurone (e.g.: spinal muscular

atrophies, hereditary bulbar palsy), or combination of upper and lower motor neurone (e.g.:

amyotrophic lateral sclerosis) (Donaghy, 1999; Swash et al, 1999; Talbot, 2002).


3

Neurodegeneration corresponds to any pathological condition primarily affecting neurons. In

practice, neurodegenerative disorders represent a large group of neurological disorders with

heterogeneous clinical and pathological expressions affecting specific subsets of neurons in

specific functional anatomic systems; they arise for unknown reasons and progress in a

relentless manner (Przedborski et al, 2003). A number of mechanisms appear to contribute to

the neurodegenerative process, including alterations in calcium homeostasis in the

endoplasmic reticulum which contribute to neuronal excitotoxicity and apoptosis, and

unregulated calpain (cysteine endopeptidase; EC 3.4.22.17) proteolysis, initiated by the

dysregulation of calcium ion homeostasis. Mitochondrial disfunction may also be linked to

neurodegenerative disease through free radical generation, impaired calcium buffering and the

mitochondrial permeability transition. Apoptotic and necrotic cell death are both observed in

neurodegenerative diseases. Another mechanism may be the disorganization of the

cytoskeleton leading to neuronal degeneration (Sigma-RBI®, 2001).

Motor neurone disease is a term introduced by Brain in 1962, intending to unify under one

umbrella various idiopathic degenerative motor system diseases (Swash et al, 1999). A large

number of diseases of diverse aetiology may selectively affect the motor neurons in the

central nervous system (CNS) and, among the hundreds of different neurodegenerative

disorders. So far most attention has been given to only a handful of diseases including

Alzheimer disease, Parkinson disease, Huntington disease, and amyotrophic lateral sclerosis

(Tshala-Katumbay, 2001a; Talbot, 2002; Przedborski et al, 2003). Many of the less common

or less publicized neurodegenerative disorders, though not less devastating, have remained

essentially ignored or neglected (Przedborski et al, 2003).

I.1.2 The “hidden endemias”

Tropical myeloneuropathies (Table I-1) is a term proposed by Román et al (1985) for the

“hidden endemias” of the neurodegenerative diseases that predominantly affect the spinal


4

cord and peripheral nerves with poorly known aetiology such as Tropical Spastic

Paraparesis/Human T cell Lymphocyte Virus Type I-Associated Myelopathy (TSP/HAM),

tropical ataxic neuropathy (TAN), neurolathyrism and konzo, diseases in which a virus or a

natural toxin causes selective upper motor neurone impairment (Tylleskär, 1994c; Verma and

Bradley, 2001). Upper motor neurone signs are the result of an interruption in the neural

pathway above the anterior horn cell. Characteristic of an upper motor neurone disease are:

• Weakness – the extensors are weaker than the flexors in the arms, but the reverse is

true in the legs with weakness more pronounced in flexor muscles.

• Muscle wasting is absent or slight – muscle wasting is prominent in a lower motor

neurone lesion

• Hyper-reflexia and clonus in upper motor neurone disorder – reflexes are absent or

reduced in a lower motor neurone lesion

• Spasticity

• No fasciculations in upper motor neurone disorder – fasciculations occur in a lower

motor neurone lesion.

Tropical Spastic Paraparesis/Human T cell Lymphocyte Virus Type I-Associated Myelopathy

(TSP/HAM) is a neurological disease characterised by slowly progressive spastic paraparesis

with insidious onset in adulthood. It has been found all around the world (except at the

Artics), mainly in tropical and subtropical regions. The diagnostic criterion of TSP/HAM is

seropositivity to HTLV-I (Cassab and Penalva-de-Oliveira, 2000; Maloney et al 2000;

Zaninovic’, 2001).

TAN is a form of tropical myeloneuropathy which was first described in Nigeria but also

occurs in other parts of the tropics. Its clinical presentation is characterized by gradual onset

of ataxia due to posterior column loss. The clinical diagnosis requires at least two of the

following features: bilateral optic atrophy, deafness, predominantly posterior column


5

myelopathy and polyneuropathy. The occurrence of TAN is associated with chronic moderate

dietary cyanide exposure arising from cassava (Howlett, 1994).

Table I-1: Characteristic features of four tropical myeloneuropathies (Tylleskär et al, 1994c)

Konzo Tropical ataxic

neuropathy

Neurolathyrism HTLV-I

associated

myelopathy

Geographical area Africa Africa Asia/ Africa Worldwide

Occurrence epidemic

and endemic

endemic epidemic and

endemic

endemic

Highest prevalence 3 % 3 % 3 % 0.1 %

Familial clustering yes yes yes yes

Type of onset acute slow acute slow

Course permanent progressive permanent progressive

High incidence age

group

< 40 > 40 < 40 > 40

Main neurological

findings:

Gait abnormality

Spastic

paraparesis

Ataxia

Spastic

paraparesis

Spastic

paraparesis

Peripheral

neuropathy

no yes no common

Sphincter

involvement

no no rare yes

Optic atrophy rare yes no no

Deafness no common no no

Etiology Attributed

to weeks of

high

cyanide

exposure

from

cassava

Attributed to

prolonged,

varying cyanide

exposure from

cassava

Caused by

months of high

grass pea

(Lathyrus sativus)

consumption

Caused by

chronic HTLV-I

infection


6

Neurolathyrism is an upper motor neurone disease caused by excessive and prolonged

consumption of grass pea (chickpea), Lathyrus sativus, which contains the glutamate

analogue neurotoxin ß–N-oxalyl-α,ß-diaminopropionic acid (ß-ODAP) also known as ß-

Oxalylaminoalanine(BOAA). ß-ODAP is an excitotoxic amino acid that presumably acts on

the neuronal glutamate receptors. Neurolathyrism is epidemic and endemic in geographic

areas subject to famine and drought such as Afghanistan, Bangladesh, China, Ethiopia, Nepal

and India. Neurolathyrism is characterized by spastic paraparesis of the legs with or without

sphincter disturbances (Spencer, 1999; Getahun et al, 1999).

Konzo is a distinct disease entity with selective upper motor neuron damage which is

characterised by a sudden onset of an irreversible, a non-progressive and symmetrical spastic

paraparesis or, in severely affected subjects, tetraparesis (Howlett et al, 1990; Tylleskär,

1994b; Banea-Mayambu, 1997c; Tshala-Katumbay, 2001a; Mwanza et al, 2005). Konzo has

been attributed to the high dietary cyanide exposure from insufficiently processed roots of

bitter cassava (Manihot esculenta Crantz) and reported from remote rural areas of

Mozambique, Tanzania, Cameroon, Angola, the Central African Republic and the Democratic

Republic of Congo (DRC) (Trolli, 1938; Cliff et al, 1985; Howlett et al, 1990; Banea et al,

1992a; Tylleskär et al, 1992; Tshala-Katumbay, 2001b; Bonmarin et al, 2002; Ernesto et al,

2002a). Konzo has only been reported from cassava growing and consuming areas but

affected populations constitute only a fraction of the total of over 500 million cassava-

consuming populations of the tropics. Konzo has some similarities to neurolathyrism but there

is no geographical overlap of the two diseases (Howlett et al, 1990; Tylleskär et al, 1994c;

Lambein et al, 2004). Prevalence rates for konzo vary between studies; rates between 1 and 30

per 1000 have been reported (Tylleskär et al, 1992). The total number of confirmed konzo

cases in reported studies exceeds 4000 (Tylleskär, 1994a; Bradbury, 2004). The D. R. C.

covers the largest reported number of konzo cases. The Health Ministry of D. R. C. estimated


7

the number of konzo cases in D. R. C. to be around 100,000 (R. D. C., 2000). The age and the

sex distribution of konzo show a distinct pattern. No child under the age of 2.5 years, of

which most are breast-fed, has ever been found to contract konzo. Women of child bearing

age and children 3-13 years of age run the highest risk of contracting konzo. No case of konzo

has been reported from nearby urban populations (Banea-Mayambu et al, 1997a).

I.2 Konzo

I.2.1 Background information on konzo

Formerly called epidemic spastic paraparesis (Carton et al, 1986; Rosling, 1988), konzo is a

neurological disorder that gives rise to crippling spastic paralysis of both legs (paraparesis) or

of both legs and arms (tetraparesis) in severely affected subjects (Tshala-Katumbay, 2001a;

Mwanza et al, 2005). It is an upper motor neuron disease which was first described in the

former Belgian Congo (present D. R. C.) by Dr Trolli in a published report that summarised

regrouped observations in Kwango district about two affections of unknown origin; epidemic

spastic paraparesis, “konzo” of the people in Kwango and a syndrome with oedema and

dyschromic cutaneous lesions (Trolli, 1938).

In 1936, Dr Tessitore, a district medical officer in Kahemba area reported an outbreak of an

affection that he called “amyotrophic lateral sclerosis” of which he described several cases. In

1937, Dr Mercken noted around Feshi, area neighbouring Kahemba some cases of affection

which evoke symptoms of Heine-Medin disease. Some times later in 1937, Drs Doucet and

Orlovitch reported other cases in Moyen-Wamba (the present Popokabaka and Mwela

Lemba), another Feshi neighbouring area (Figure I-1). Some subjects reported having been

affected earlier during outbreaks of the year 1928 or 1929 and 1931- 1932. The affection

appeared periodically in those areas and was well known by the local population who named

it “konzo”.


8

Figure I-1: Health zones of Kwango District in Bandundu Province, DRC


9

The word “konzo” originally khoondzo has its origin from kiyaka, the language spoken in

Kwango district and had three meanings (Trolli, 1938; Van der Beken, 1993). It means a

fetish used with traps to catch wild animals by weakening their legs, the trap itself and, tied

legs. This latter meaning is illustrated by a famous proverb in Kiyaka:

“mene, yakele khosi mutu, khoondzo watholula bidiimbu”,

This means “I was strong, but khoondzo (fetish) has weakened my legs”. As a consequence of

this proverb, konzo has come to mean “trapped” or “weakened “or “tied” legs. It is in this

sense that konzo is used to denote the paralytic disease and thus, a local belief that the disease

is related to a bad destiny or sorcery (Van der Beken, 1993).

Lucasse (1952) who did not read the report of Trolli, described and suggested the first clinical

description of konzo observed in some affected subjects 14 years ago in Kwango district, as

follows:

Bilateral paresis of the lower limbs which is accompanied by spasms in the adductor and

flexor muscles of the lower part of the body giving rise to vicious attitudes of lower limbs and

sometimes of spine (lordosis).

After a period with no further reports on konzo, the National Planning Centre of Human

Nutrition of Zaire (present D. R. C.) CEPLANUT (currently PRONANUT) in 1982 reported

hundreds of cases of spastic paraparesis from an outbreak that started in 1978 in the Central

part of the Bandundu province, neighbouring Kwango district (CEPLANUT, 1982). Another

outbreak occurred in 1983, in the northern part of the above area and up to now, outbreaks of

konzo are still occurring in the Bandundu province and other parts of D. R. C. (Tshala-

Katumbay, 2001b; Bonmarin et al, 2002).

Other outbreaks of konzo have been described in several other parts of Africa, especially in

Mozambique, Tanzania, Central African Republic, and Cameroon (Cliff et al, 1985; Howlett

et al, 1990; Tylleskär et al, 1994c; Ernesto et al, 2002a).


10

Konzo is now accepted as the scientific name for a distinct human disease entity which is

characterised by a sudden onset of a non-progressive and irreversible spastic paraparesis in a

person formerly without other symptoms (Howlett et al, 1990; Tylleskär, 1994b).

I.2.2 Clinical features and differential diagnosis

I.2.2.1 Clinical features and classification

Konzo is a distinct type of upper motor neuron disease with a typical clinical picture of

crippling spastic paraparesis (WHO, 1996). The clinical picture of konzo is identical in all

studies (Trolli, 1938; Lucasse, 1952; Carton et al, 1986; Howlett et al, 1990; Tylleskär et al,

1995; Cliff and Nicala, 1997; Tshala-Katumbay, 2001b; Bonmarin et al 2002; Ernesto et al,

2002a). The disease typically occurs in an apparently healthy person and there is no

prodromal phase or triggering illnesses. The onset is characterized by an abrupt paraparesis

occurring the first days of the illness. A common history is that of a healthy person who goes

to bed feeling well and wakes up during the night or early morning unable to stand or walk.

The paraparesis may also occur abruptly during or after manual work or a long walk. Initial

symptoms are often described as heaviness, trembling or weakness of the legs associated with

difficulty or inability to stand. Other complaints that may appear over time include weakness

in the arms or hands, difficulty in articulating speech, and blurring of vision. Sensory

symptoms of radicular low back pain, and paresthesia in the legs, can also be present but these

usually clear in the first weeks or months. Incontinence is typically absent. The disease affects

mainly children and women of childbearing age (Howlett, 1994; Tylleskär, 1994b; WHO,

1996).

Although the main clinical picture of konzo consists of the sudden onset of a non progressive

and symmetrical spastic paraparesis of the legs in affected subjects, the diagnosis of konzo is

based on the WHO criteria (WHO, 1996) given in the Table I-2.


11

These criteria are easy to use in the field to screen the population. However Tshala-Katumbay

(2001a) suggested a new version with more operational criteria in comparison with the WHO

criteria (Table I-2).

The degree of physical disability caused by konzo was classified by Lucasse (1952) and later

amended by WHO (1996) as follows:

• Mild form: when the patient does not need to regularly use any walking aid

• Moderate form: when the patient regularly uses one or two stick(s) or crutches

• Severe form: when the patient is bedridden or unable to walk without living support.

Table I-2: WHO criteria for konzo versus newly suggested criteria (with permission and from

Tshala-Katumbay, 2001a)

Criteria WHO New version

1 Visible symmetric spastic

abnormality of gait while walking

or running

Sudden onset of a non –progressive

bilateral and symmetric spastic

abnormality of gait while walking or

running

2 History of onset less than one week

followed by a non-progressive

course in a formerly healthy person

Bilaterally exaggerated knee or ankle

jerks

3 Bilaterally exaggerated knee or

ankle jerks without signs of disease

of the spine

Absence of objective sensory and genito-

urinary symptoms

4 Absence of grass pea (Lathyrus

sativus) consumption

Living under conditions of sub-acute or

chronic exposure to cyanogens and

undernutrition at the onset


12

Figure I-2: From left to right: mild form, moderate form and severe form of konzo in young

subjects

This classification (Figure I-2) is easy to use even by paramedical agents unfamiliar with the

symptoms but it is sometimes difficult to distinguish between slightly konzo affected persons

(mild form) with non-affected persons.

I.2.2.2 Differential diagnosis

Konzo with its upper motor neuron manifestations can be confused with other diseases. Using

the WHO criteria, konzo by its spastic paraparesis can easily be distinguished from causes of

flaccid paraplegia such as poliomyelitis, leprosy or trauma (Howlett, 1994). The commonest

neurological diseases to be considered in its differential diagnoses include neurolathyrism,

TAN and TSP/HAM (Table I-1).


13

Konzo is clinically very similar to neurolathyrism but differs from TAN and TSP/HAM

(Tylleskär et al, 1994c; Cliff and Nicala, 1997; Zannovic’, 2001; Lambein et al, 2004). The

socio-economic conditions of konzo and neurolathyrism patients are very similar as well.

Both diseases can be considered a sign of poverty, monotonous diet and illiteracy (Getahun et

al, 2002b). Neurolathyrism only differs from konzo with the somewhat higher age of onset,

predominance of males among the affected, sphincter involvement in some cases and the

absence of cranial nerve involvement (Tylleskär et al, 1994c). There is no geographical

overlap between the consumption of the grass pea and cassava and therefore there is no

geographical overlap of the two diseases. It would be difficult to make a differential diagnosis

between neurolathyrism and konzo if both disorders occurred in the same population

(Tylleskär et al, 1994c; Lambein et al, 2004).

Konzo and TAN have been attributed to dietary cyanide exposure from consumption of

insufficiently processed cassava roots, but rates of exposure differ in both diseases (Howlett et

al, 1990). In contrast to konzo, TAN is a progressive disorder with slow onset that mainly

affects older adults. Furthermore, konzo involves damage to upper motor neurone, whereas

TAN is mainly caused by damage of sensory neurons in the spinal cord resulting in ataxia.

TAN rarely progresses to inability to walk, whereas a high proportion of the konzo-affected

subjects are unable to walk. About half of the TAN cases have optic atrophy which is rare

among konzo cases. About one in five TAN cases has exaggerated reflexes, a sign that occurs

in all konzo cases (Howlett, 1994; Tylleskär et al, 1994c).

TSP/HAM is clinically possible to distinguish from both konzo and neurolathyrism, although

the clinical features of TSP/HAM include typical signs that are similar to both diseases such

as muscle weakness in the legs, hyperflexia, clonus and extensor plantar responses.

TSP/HAM is characterised by a chronic progressive spastic paraparesis with sphincter

disturbances, not to mild sensory loss, absence of spinal cord compression, urinary


14

incontinence, impotence and seroposivity for HTLV-I antibodies (Tylleskär, 1994b; Cliff and

Nicala, 1997; Cassab and Penalva-de-Oliveira, 2000; Maloney et al 2000; Zaninovic’, 2001).

I.2.3 Epidemiology

The occurrence of konzo is limited to geographical pockets in rural Africa and the majority of

cases occur in epidemic outbreaks during the dry season (Rosling, 1997; Bonmarin et al,

2002). Sporadic cases also occur, but they are also restricted to dry or war periods with

monotonous cassava diet. Not a single case has been identified in an urban population

(Tylleskär et al, 1995). More than 4000 cases have been confirmed from reported studies. Of

these, more than half of the cases are from D. R. C. but the reported number of cases is

undoubtedly underestimated, as case detection is incomplete in the remote rural areas

affected. The Health Ministry of D. R. C. has estimated the number of konzo cases in D. R. C.

to be around 100,000 (R. D. C., 2000). Two extensive epidemic outbreaks, each numbering

more than 1000 cases have been brought to the attention of the scientific community; the first

reported outbreak occurred in Kwango district in the southern part of Bandundu province of

D. R. C. in 1936-37, and the second in Nampula Province of Mozambique in 1981. Other

smaller outbreaks have been reported from very poor remote rural population of Central

African Republic, Mozambique, Tanzania, Cameroon, Angola, and Democratic Republic of

Congo (D. R. C.) (Howlett et al, 1990; Banea-Mayambu et al, 1992a; Tylleskär et al, 1994c;

Tshala-Katumbay, 2001b; Bonmarin et al, 2002; Ernesto et al, 2002a).

Konzo primarily affects children above the age of three and women in the fertile age group.

Adult males are less frequently affected. No breast-fed child (in affected populations, the

breast feeding period extends beyond two years of age) has been found to have contracted

konzo. A pronounced familial clustering of cases of konzo has been noted in all affected

populations. Prevalence varies between studies and between most affected villages.

Prevalence rates of between 1 and 30 per 1000 have been recorded (Howlett, 1994; Cliff et al,


15

1997b). The peculiar geographical, seasonal and age variation in occurrence as well as the

abrupt onset have facilitated epidemiological studies for possible etiological factors in konzo

(Howlett, 1994; Tylleskär et al, 1995; WHO, 1996; Banea-Mayambu et al, 1997a; Rosling,

1997).

I.2.4 Infection or toxico-nutritional etiology?

“The etiology of konzo is unclear. Information given by local people is useless. Often, they

say the disease is sent either by God or by the ndoki, the bad spirit of enemies” stated

Tessitore in 1930s (Trolli, 1938). Three generations later after the first report, the etiology of

konzo has not been established with certainty. The infectious etiology was proposed (Trolli,

1938; Lucasse, 1952; Carton et al, 1986) but konzo patients do not show any signs of

infections and are sero-negative to HTLV-I and other retroviruses (Tylleskär et al, 1996;

Tshala-Katumbay et al, 2001b). The facts that outbreaks are restricted to rural areas without

any secondary cases along connecting roads or in neighbouring urban areas argues against

infectious etiology. So far, all studies on konzo have failed to demonstrate an infectious

etiology (WHO, 1996; Rosling, 1997).

A toxico-nutritional hypothesis was suggested in 1930s by Georgiades who observed

similarities between konzo and lathyrism and recommended further studies on cassava

concerning the processing and detoxification methods used before its consumption. Konzo

might be caused by cyanide exposure resulting from consumption of insufficiently processed

cassava roots and simultaneous low dietary intake of sulphur containing amino acids

providing substrate to thiocyanate conversion (Trolli, 1938; Cliff et al, 1985). This hypothesis

is supported by a consistent association between temporal and geographical occurrence of

konzo and the chain of events that leads to high cyanide and low sulphur intake. This chain is:

• Intensive cultivation of bitter cassava varieties in poor rural areas,


16

• A cassava dominated diet, which is brought about by having a farming system

dominated by bitter cassava

• Shortcuts in processing as indicated by high residual levels of cyanogens in cassava

products consumed,

• High cyanide intake indicated by high urinary and serum thiocyanate levels,

• Low intake of foods rich in sulphur containing amino acids indicated by low urinary

inorganic sulphate levels (Tylleskär, 1994d; Tylleskär et al, 1995; Cliff et al, 1997a;

Rosling, 1997; Banea-Mayambu et al, 1997c)

High consumption of cassava is by itself not sufficient to cause konzo and within the affected

populations, cassava is consumed daily by everybody but only certain percentage of the

population acquire konzo (Tylleskär, 1994b). The underlying cause inducing the high

exposure to cyanide and unbalanced diet include drought, intensive trade of cassava by poor

farmers and collapse of the socio-economic fabric due to political conflicts and civil war

(Banea-Mayambu et al, 1997a; Banea-Mayambu et al, 1997b; Cliff et al, 1997b; Tshala-

Katumbay et al, 2001b). So far, the resulting diet and toxic exposure associated to konzo is

similar in all areas and konzo has not been reported from any area lacking this unbalanced

diet (Rosling, 1997).

Although the etiology and the exact cellular malfunctions induced by overconsumption of

cassava products remain unclear, cyanide (CN-), 2-aminothiazoline-4-carboxylic acid (ATC),

cyanate (OCN-) and thiocyanate (SCN-) have been suspected playing a role in the pathogenic

mechanism of konzo (Spencer, 1999; Tor-Agbidye et al, 1999):

• CN- has been suggested as a causal factor in some series of neurological disorders

because of its potential inhibitor effect on mitochondrial energy transformation

secondarily inducing neuronal dysfunction. CN- is unlikely to be responsible since the


17

outcome of sub-lethal cyanide intoxication is Parkinsonism, with changes in basal

ganglia, cerebellum and cerebral cortex;

• ATC, a minor cysteine-dependent metabolite of cyanide, has not yet been investigated

for its systemic toxicity. However, its intracerebroventricular injection in rats induces

seizures and hippocampal damage, neither of which are known to occur in konzo;

• OCN-, a normal human metabolite that is produced by the spontaneous degradation of

urea (carbomoylation), is known to cause neurodegenerative disease in humans and

animal (WHO, 2004); but these neurological conditions appear to be more closely

related to TAN rather than to konzo (Tor-Agbidye et al, 1999);

• SCN- is generally considered a major, innocuous detoxification product of cyanide.

SCN- is constantly elevated in subjects with konzo (Tylleskär, 1994, Banea-Mayambu,

1997c; Tshala-Katumbay, 2001b). Since experimental evidence shows that SCN-

increases glutamate binding to the α-amino-3-hydroxy-5-methyl-isoxazole-4-

propionic acid (AMPA) receptor and potentiates AMPA-mediated responses. This

might secondarily induce excitotoxic effects and hence neuronal disfunction or cell

death. The potential role of thiocyanate in konzo merits attention (Tor-Agbidye et al,

1999).

I.3 Dietary exposure to cyanide from cassava

I.3.1 Cassava

I.3.1.1 Classification and botany

Cassava (Manihot esculenta Crantz) is the English name given to the manioc plant, a hardy

perennial shrub belonging to the family Euphorbiaceae and ranging in height between 1 – 3

m depending on growing conditions (Brough, 1991). There are many cultivars or varieties


18

under cultivation. Cassava varieties are usually classified into sweet and bitter cultivars but no

morphological or other taxonomic characteristics seem to be associated with this

classification (Nweke and Bokanga, 1994). The genus Manihot incorporates over 200 species

of which Manihot esculenta is the most important, from the nutritional and economic points

of view (Nartey, 1978).

The shrub may have multibranched or unbranched stems, grey, green or brown in colour, with

large palmate leaves (Figure I-3). The primary leaves are unlobed, whereas the secondary

leaves are 3-lobed, and subsequent leaves develop, the lobes increase in number, reaching the

number of lobes characteristic of the cultivar. Mature stem cuttings which are universally

used as propagules and planted erect or at a particular angle for vegetative regeneration of

plants, give rise to roots at the cut end via callus tissue formation, and adventitious roots form

at nodes in the soil.

Figure I-3 : Cassava roots harvested and cassava plant in the field


19

The roots are initially fibrous, but gradually undergo enlargement. At maturity, they become

fusiform, long and slender, occasionally globose appendices of the stem in the upper

rhizosphere, seldom penetrating deeply into the soil. The mature root tuber (Figure I-3)

possesses three distinct regions, namely, the phelloderm or peel, the cortex or flesh, and the

central vascular core. The peel is generally 1-4mm thick and composed of outer epidermis, a

sub-epidermis and an inner layer readily separable from the bulk of the tuber. The cortex

consists of a mass of parenchyma cells and constitutes the region of carbohydrate storage.

Generally, the cortex lacks xylem vessels, and is therefore without fibre, but older tubers

develop hardened xylem vessels, giving rise to stringy tubers, undesirable for food. Root

pigmentation may vary with respect to variety, from light yellow, brown to pink and

intermediate shades, whereas cortex pigmentation varies from white, yellow to pink. The

tubers are the most valuable part of the plant, although in some countries the leaves are used

as green vegetable (Nartey, 1978; Brough, 1991).

I.3.1.2 Introduction and distribution of cassava in Africa

Cassava is believed to have been introduced originally in the Gulf of Benin in 1562 and along

the Congo River in 1611 from where it spread to the west coast of Africa (Nartey, 1978;

Carter et al, 1997). Later introductions in the islands of Reunion, Madagascar and Zanzibar

led its spread in East Africa. Finally, it spread inland in all directions to encompass the region

of Lake Tanganyika (Nartey, 1978; Brough, 1991).

The Portuguese first brought cassava to Africa in the form of flour or “farinha”. The

Tupinamba Indians of eastern Brazil had taught them techniques of cassava preparation and

production and, they had developed a liking for the various processed forms. Cassava flour

was used as a provision for ships plying between Africa, Europe and Brazil. The first mention

of cassava cultivation in Africa dates back to 1568. At first, it was cultivated with the sole

purpose of supplying slave ships, until 1600. In the late 19th century cassava had been


20

successfully incorporated into many farming systems of Central Africa instead of millet, yam

and plantain, the former staple food in most areas along the Congo River (Carter et al, 1997).

The ultimate wide distribution of cassava in the whole of tropical Africa was motivated by the

ability of the crop to withstand locust attacks, and to tolerate drought, poor soils and weeds.

These characteristics, together with the fact that the crop can be left without harvesting over

several years, made it a useful security against periods of famine (Nartey, 1978).

In the late 19th and 20th centuries, colonial administrators encouraged diffusion and increased

cultivation of cassava. The encouragement by the colonial governments may often have taken

place in a manner insensitive to the applicability of cassava to local farming systems and food

habits. Moreover colonial governments displayed an ambivalent attitude towards cassava.

Whilst it was introduced as an anti-famine and anti-locust crop, cassava was also thought to

promote laziness, soil depletion and malnutrition. Post-independence diffusion of the crop in

Africa has primarily been the result of local processes of migration and agricultural change.

There is ample evidence of the willingness of African farmers to experiment with the search

for new crops and varieties. Cassava’s special characteristics make it well adapted to farmers’

risk aversion strategies and allow it to be grown under a great diversity of circumstances and

changing economic conditions. The consumption of cassava leaves, in frequent rather than

sporadic form, was probably an African invention (Carter et al, 1997).

Currently, cassava is grown on wide scale between latitudes 30° north and south, the so called

“cassava belt”, an ecological zone which coincides with many of the less developed countries

where cassava is adapted to the prevailing conditions (Nartey, 1978).

I.3.1.3 Importance and advantages

Cassava (Manihot esculenta Crantz) is a shrub widely grown for its tuberous roots in tropical

regions of Africa, Asia and Latin America. Sweet and bitter cultivars are produced as food,

feed and for industrial uses.


21

Cassava roots form a staple food for an estimated 500 million people in the tropics and the

leaves are commonly consumed as a vegetable in several areas. Cassava ranks fourth on the

list of major food crops in developing countries after rice, wheat and maize (FAO, 1990).

Cassava is of great importance for food security in Africa in general, and D. R. C in

particular. Cassava possesses a number of useful agricultural traits. The crop is a relatively

efficient producer even under adverse environmental conditions such as erratic low rainfall

and low soil fertility. Cassava productivity in terms of calories per unit land area is

significantly higher than that of other staple food crops. The edible portion in percent of dry

weight of the root crop is high. Cassava is resistant to locust damage and most pests. Growth

of cassava requires a low input in the timing of labour. Except being sensitive to drought

shortly after planting, cassava requires no special planting or harvesting dates. The roots can

be stored in the ground without harvesting for a lengthy period of time, up to three years or

more after the formation of the edible roots is complete. Hence, cassava cultivation serves as

something like a household food bank that can be drawn upon when adverse agro-climatic

conditions or civil unrest limit the availability of and access to other food (Koch et al, 1994;

Scott et al, 2000).

In sub-Saharan Africa, cassava provides daily food products for nearly half of the continent’s

population. DRC is the country with the highest per capita consumption in the world, about

60 % of total daily energy intake is provided by cassava (FAO, 1990). In addition, cassava

leaves contribute 20 % of the protein in Congolese diets. The Congolese staple food

production takes mainly place on traditional farms. The Congolese smallholder farming is

characterized by reliance on family labour, on a small stock of physical capital and on a large

area of land. Women play a predominant role in farming, processing, and marketing. In rural

areas, most traditional subsistence food crops have become important as cash crops and urban

food demand is the driving force behind this evolution. Farms without an adequate access to


22

markets are generally characterized by low levels of cash income and surplus production

(Goossens, 1996). Cassava production is a commercial activity, and not merely a subsistence

agricultural activity. Cassava is an important source of cash income for poor farmers as well

as for prosperous ones. Both rich and poor farmers often sell a higher proportion of cassava

than from any other crop or income earning activity. The proportion of cassava production

marketed is a good indicator of the level of diversification in the crop production activity of

an area (Nweke and Bokanga, 1994).

I.3.1.4 Production and consumption levels

“Cassava is apparently emerging from its obscurity in the tropics and is marching northward

and southward to fill new roles in temperate climates”, is the assessment of Franklin D.

Martin quoted by Nartey (1978).

In the last two decades, cassava production grew at a more modest pace, 1.8 percent annually.

Production of cassava grew by 27 percent between 1983 and 1996 to 164 million metrics ton.

On a per capita basis, production of cassava in developing countries remained virtually

constant at 37 kg per capita, supported mainly by the per hectare production growth in Sub-

Saharan Africa. Production tends to be highly skewed toward particular regions. Slightly

more than half the global production of cassava takes place in sub- Saharan Africa, followed

by Southeast Asia with 23 percent and Latin America with 20 percent (Figure I-4).

Most statistics do not usually distinguish between sweet and bitter varieties; in some, sweet

varieties are not included as they are commonly grown as a secondary crop for home

consumption. Brazil is the largest producer of cassava in the world, but most of the crop is

consumed locally and exports are only a small portion of the total output. The same pattern

applies to other important producers, such as Nigeria, Indonesia, D. R. C., India and

Colombia. Cassava does not form an important part of the staple diet in Thailand, and that


23

country is the world’s largest exporter of cassava products. In contrast, Africa does not export

much cassava because production is almost entirely consumed as food.

Figure I-4: Location of cassava production, 1996 (Scott et al, 2000)

Sub-Saharan Africa, 51.6%

Latin America, 19.5%

Other South Asia, 0.2%

Southeast Asia, 23.0%

China, 2.2%

India, 3.5%

In the last few years most of the important producers have greatly increased their production.

Surplus production of cassava products enters international trade in different forms, such as

chips, broken dried roots, meal, flour and tapioca starch. Dried cassava roots and meal are

used as raw material for compound animal feed, while cassava starch is used for industrial

purposes, particularly the paper and textile industry; grocery tapioca is used solely for human

consumption. The principal markets for cassava products are the European Community, USA,

UK and Japan (FAO, 2004).


24

Between 1983 and 1996, increase of consumption of cassava as food has been particularly

rapid in Sub- Saharan Africa at 3.1 percent per year. The region has experienced low and

negative economic growth and booming populations (Scott et al, 2000).

Cassava is the staple food in most of D. R. C., and especially in Bandundu province where a

great number of konzo cases have been reported (Trolli, 1938; Banea-Mayambu et al, 1992a;

Tylleskär et al, 1994c; Tshala-Katumbay, 2001b; Bonmarin et al, 2002).

Between 1987 and 1990, the annual production of fresh cassava roots in Bandundu was 4

million metric tons while the national production was estimated at 17 million tons. The annual

production per household was 7.9 tons and the quantities of cassava produced are ten to

twenty times larger than for other crops in Bandundu. The cassava production per farm is

relatively stable during the year: between 560 kg and 680 kg per month in Bandundu. Farm

purchases of cassava are highest in October and November, when fields are prepared and

available labour to harvest roots is limited. Sales are highest from September to February,

when receipts from other crops are low. Nearly all households buy cassava from time to time

but the quantities are generally small and are used for immediate consumption or to be resold.

Only seven percent of the rural households are not self supporting for cassava (Goossens,

1996)

I.3.1.5 Problems associated with cassava

I.3.1.5.1 Nutritional value

Cassava roots contain around 30 to 40 % of dry matter of which starch and sugars account for

approximately 90 %. This renders cassava root, an excellent source of carbohydrate but has

extremely low levels of protein and fat (Bradbury and Holloway, 1988)

Unlike the roots, which are essentially a source of carbohydrate, fresh cassava leaves are a

good source of proteins and vitamins which can provide a valuable supplement to


25

predominantly starchy diets (Hahn, 1989). The nutrient content of cassava leaves is

comparable with other green leaves and other vegetables generally regarded as good protein

sources (Mbemba and Remacle, 1992). Vitamin A, thiamine, riboflavin, niacin and vitamin C

are of high concentration in the fresh leaves (Ravindran and Ravindran, 1988; Almazan and

Theberge, 1989)

I.3.1.5.2 Constraints in production

Besides the advantages on the production of cassava, there are many production constraints

which can include diseases, pest, weeds, soil and agronomic factors, and socio-economic

factors. The major diseases of cassava are leaf diseases, stem diseases and tuber rot.

Vertebrate pest, nematodes, mites and insects may attack or infect the roots and render them

susceptible to rot-causing organisms. Cassava can be seriously affected by early weed

infestation. Weed competition in cassava crops reduces canopy development, tuberization and

tuber yield. The important soil and agronomic factors that affect cassava production are soil

temperature and moisture (if above 30° C and if drought is frequent), soil erosion and low soil

fertility (continuous cultivation of cassava without adequate erosion control measures, can

result in severe and irreversible soil degradation), and poor cultural practices. The main socio-

economic factors affecting cassava production relate to inadequate resource allocation and

infrastructure (IITA and UNICEF, 1990).

I.3.1.5.3 Post-harvest deterioration

Cassava roots are extremely perishable. They can be kept in the ground prior to harvesting for

up to about 2 years, but once they have been harvested (removed from the stem) they begin to

deteriorate within 40 – 48 hours (IITA and UNICEF, 1990). The fresh tubers in general have

high moisture content, usually between 50 and 70 %, and hence have a relatively low

mechanical strength. They also have a very high respiratory rate, and the resultant heat


26

production softens the texture, which leads to damage. Unlike the other tuber crops, cassava

roots do not exhibit exogenous dormancy, have no function in propagation, and possess no

bud primordial from which regrowth can occur. For these reasons, cassava roots are more

perishable than other tuber crops. Mechanical damage during the harvesting and handling

stages also renders cassava root unsuitable to long-term storage. Deterioration of cassava has

an adverse effect on the processed product, and thus the crop must be stored properly (IITA

and UNICEF, 1990; Ravi et al, 1996, Ravi and Aked, 1996).

I.3.1.5.4 Cassava in human nutrition

Cassava root is an important starchy crop grown in the tropics, which constitutes the staple

food of about half of billion people and the leaves are consumed as vegetable. Roots and

leaves of cassava contain high level of cyanogenic glycosides mainly linamarin and to a lesser

extent lotaustralin which can be hazardous to the consumer and of which the potential toxicity

is a public health concern (Essers, 1995; Padmaja, 1995). Roots from the sweet varieties may

be eaten raw or cooked by boiling, steaming or roasting. Roots from the bitter varieties

required processing to remove the cyanogenic compound before consumption. The processing

methods generally adopted comprise combinations of activities such as peeling, boiling,

steaming, slicing, grating, soaking or steeping, fermenting, pounding, roasting, drying and

milling (Hahn, 1989; Padmaja, 1995). They can be transformed in the form of flour or gari

(granules) and then consumed as fufu (stiff porridge), chikwangue (wrapped steamed boiled

paste much stiffer than fufu), tapioca, dried gari or stiff paste of gari (Figure I-5).

Consumption of cassava leaves is of great significance in the nutrition of a population

subsisting primarily on cassava. The most widely practiced processing for use of leaves as

food involves crushing, parboiling in water, washing and cooking. Sometimes additional

ingredients such as pepper, palm oil, fish, peanut or other aromatic ingredients are added. The

leaves, in some parts, are sun dried then cut or pounded and finally cooked. Cooked cassava


27

leaves are served and consumed as the main side-dishes to the processed cassava roots like

fufu, chikwangue or boiled cassava root (Hahn, 1989).

Figure I-5: Summary of traditional cassava processing in Africa (from Banea-Mayambu,

1997c)

I.3.2 Cyanide toxicity

I.3.2.1 Introduction

Cyanide (CN-) most commonly occurs as hydrogen cyanide (HCN) and its salts sodium

cyanide (NaCN) and potassium cyanide (KCN). Cyanides comprise a wide range of

compounds of varying degrees of chemical complexity, all of which contain a CN moiety, to

which humans are exposed in gas, liquid, and solid form from a broad range of natural and

anthropogenic sources. Cyanogenic glycosides, producing hydrogen cyanide upon hydrolysis,

are found in a number of plant species. Cyanides are also produced by certain bacteria, fungi,

Fresh cassava roots

Soaking Heaping Cutting

Solid state fermentation

Grating

Fermentation in water

Moist fermentation

Pounding Drying Drying

-

Pressing

Boiling Pounding Pounding

Drying

Roasting

Paste Flour Flour

Pounding

Granules Flour


28

and algae. Minute amounts of cyanide in the form of vitamin B12 (cyanocobalamine) are a

necessary requirement in the human diet (ATSDR, 1989).

HCN, a colourless or pale liquid or gas with a faint bitter almond-like odour, has a molecular

weight of 27.03 and a boiling point of 25.7° C. It is miscible with water and alcohol and

slightly soluble in ether.

Cyanide is released to the environment from numerous sources. Metal finishing and organic

chemical as well as iron and steel production, and automobile exhaust are major sources of

cyanide releases in the atmosphere. Workers in a wide variety of occupations may be exposed

to cyanides. The general population may be exposed to cyanides by inhalation of

contaminated air, ingestion of contaminated drinking water, and/or consumption of a variety

of foods (ATSDR, 1989).

Among the general population, subgroups with the highest potential for exposure to cyanide

include active and passive smokers, individuals involved in large scale processing of food

high in cyanogenic glycosides and individuals consuming foods high in cyanogenic

glycosides (WHO, 2004).

I.3.2.2 Occurrence of cyanogenic glycosides

The cyanogenic glycosides are a group of nitrile-containing secondary plant compounds that

yield cyanide (cyanogenesis) following enzymatic breakdown. They are amino acid-derived

plant constituents and their functions remain to be determined in many plants; however, in

some plants they have been implicated as herbivore deterrents and as transportable forms of

reduced nitrogen (Kakes, 1994). Whereas most plants produce a small amount of cyanide

associated with ethylene production, between 3000 – 12000 plant species produce sufficient

quantities of cyanogenic compounds to be considered toxic (Poulton, 1990). The

concentrations of cyanogenic glycosides can vary widely as a result of genetic and

environmental factors, location, season, and soil types (Ermans et al, 1980). Several


29

economically important plants are highly cyanogenic, including white clover, flax (containing

linamarin), almonds, sorghum, wild lima bean, rubber tree, and cassava. The agronomically

most important cyanogenic food crop however, is cassava (McMahon et al, 1995; White et al,

1998; Vetter, 2000).

I.3.2.3 Cyanogenesis in cassava

All cassava tissues, with the exception of seeds, contain cyanogenic glycosides mainly

linamarin and lesser amounts of lotaustralin in about 10 to 1 ratio. An acyanogenic cassava

cultivar has never been found (Bokanga, 1994). Cyanogenic glycosides are

compartmentalised within the cell vacuole while the complementary hydrolytic enzymes are

located within the cytosol of the same cells (White et al, 1994). The amino acids valine and

isoleucine are the precursors used in the synthesis of linamarin and lotaustralin, respectively.

The initial step in the biosynthesis of linamarin is the N-hydroxylation of valine followed by

the formation of 2-methyl-propanal oxime and its dehydration to yield 2-methylpropionitrile.

Figure I-6: Cyanogenesis from linamarin (McMahon et al, 1995)


30

The addition of oxygen forms acetone cyanhydrin which is then glycosylated (by a soluble

UDPG-glucosyltransferase) to form linamarin (Conn, 1994). No HCN is released from intact

cyanogenic plants, the substrates (cyanogenic glycosides) and the enzymes must be located in

different compartments of the cell. Cyanogenesis is initiated in cassava when the plant tissue

is damaged. The generation of cyanide from linamarin is a two-step process involving the

initial deglycosylation of linamarin and the cleavage of acetone cyanhydrin to form acetone

and cyanide (Figure I-6). These reactions are catalysed by linamarase (a ß-glucosidase) and

by α-hydroxynitrile lyase (HNL). Since acetone cyanhydrin may enzymatically be broken

down by HNL as well as a spontaneously decompose at pH slightly >4.0 or temperature >

30°C, it has been generally assumed that the linamarase is the rate-limiting step (McMahon et

al, 1995; White et al, 1998; Vetter, 2000). In spite of the relative instability of acetone

cyanohydrin, it can coexist with intact glycosides and HCN in differently processed cassava

products. Therefore, cyanogens in cassava products can exist in three forms:

• Cyanogenic glycosides (linamarin and lotaustralin),

• Acetone cyanohydrin,

• Free HCN (Tewe, 1994).

Cassava tubers vary widely in their cyanogenic glycosides content, although most varieties

contain 15 to 400 mg HCN equivalent/ kg fresh weight. Occasionally, varieties with high

cyanide content (1300 to 2000 mg/ kg) are also encountered (Hahn, 1989).

Cassava leaves also contain high concentrations of cyanogenic glucosides and the values fall

mostly in the range of 1000 to 2000 mg HCN equivalent/ kg on a dry matter basis (Bokanga,

1994). Very high values up to 4500 mg/ kg have been occasionally reported. The high content

of cyanogenic glycosides in cassava is however a factor restricting its utilization as a food

(Padmaja, 1995).


31

I.3.2.4 Effect of processing on cyanogenic glycosides in cassava

Cassava roots are processed by a number of methods that vary widely from region to region.

Generally, all those techniques are intended to reduce toxicity and improve palatability and

storability (Tewe, 1994). Adequate processing of cassava is of prime importance in

eliminating the toxic glycosides and converting cassava into a safe food. Cyanide–yielding

substances of cassava are normally reduced to negligible levels by effective processing

(Rosling, 1988). Cassava roots provide an important source of dietary energy but they have

some limitations. Firstly roots are readily perishable if they are not processed and they cannot

be stored like cereals or other tubers (potatoes, yam). Secondly roots from the bitter varieties

cannot be consumed raw and they are unsuitable for roasting and boiling as fresh roots

because of the high levels of potentially toxic cyanogenic glycosides (linamarin and

lotaustralin). Therefore, cassava roots from such varieties must be processed before

consumption to reduce the content of toxic cyanogenic glycosides and their degradation

products (acetone cyanhydrin and free cyanide) in the final food product. Because of their

high water content, harvested roots rot if they are not processed shortly after harvesting. The

processing considerably reduces the water content (about 50 - 70 % in the freshly harvested

tuber) and thus facilitates transportation. The processing serves to make the starch of the

cassava root suitable for consumption as a major food component in the form of boiled paste,

flour or granules in the many different dishes prepared according to cultural preferences.

Cultural preferences vary a lot and the choice of processing method is thus aimed at obtaining

a cassava food product which is safe to eat and has a desired taste, flavour and texture.

To be considered as safe for consumption, cyanogens should be removed by processing to a

level below 10 mg equivalent HCN per kilogram (ppm) dry weight of cassava product, the

recommended safe limit set by FAO/WHO (1991)


32

Although the processing steps are different for each product, they permit the glucosidase to

interact with the cyanogens and the release of cyanide. The processing leads to two end

products mostly depending on the locally available processing resources (Hahn, 1989;

O’Brien et al 1995; Ravi and Abed, 1996). Dry chips or cossettes and flour are the main

products where sunlight is abundant and wet paste is the main product where water supply is

abundant. These products need additional home preparation. Cooked paste, steamed and

toasted granules are relatively more advanced that enter the marketing system in ready-to-

serve forms, although toasted granules may need minimal preparation by soaking in hot or

cold water. These products are usually more convenient and attractive to urban consumers,

and competitive with food grains in the market place. Cassava is more often processed into

first group products for home use and into second group products for sale. However cassava is

widely marketed in form of cossettes in D. R. C. and most of the cassava products pass

through fermentation or soaking stage. The period of the fermentation usually lasts a number

of days, and varies depending on the product, processing technique and on the market for

which the product is intended for sale (Nweke and Bokanga, 1994). But where market access

has been improved, the fermentation period tends to decline; e.g. in D. R. C., the completion

of a new tarmac road to the capital city of Kinshasa resulted in an increased demand due to

improved market access. This caused the farmers producing cassava cossettes with a reduced

soaking period from three or four days to one or two days (Tylleskär et al, 1991).

Retting of cassava roots by steeping them in water causes higher losses in total cyanogens

and, makes them soft and causes the cells to rupture, releasing linamarase. Cyanogenic

glycoside removal can be enhanced by direct leaching into the soaking water (Muzanila et al,

2000). Wet fermentation has been reported to facilitate the breakdown of cyanogenic

glycosides to low total cyanogen levels, up to 13,5 % reduction during the first of day

soaking, and 65% reduction the second day of soaking (O’Brien et al, 1992) or even in one


33

experiment up to 90 % reduction after 4 days of fermentation (Padmaja, 1995). The efficiency

of fermentation or soaking of cassava root has been well documented as one of the best

methods for cyanogen elimination (O’Brien et al, 1992; Padmaja, 1995; Ravi and Padmaja,

1997). Sun-drying of cassava is generally considered to be the least efficient of the various

categories commonly practiced in Africa (Mlingi et al, 1995; Essers et al, 1996). The

cyanogenic potential of heap fermented cassava roots was significantly lower than those from

sun dried (Zvauya et al, 2002). The short-cut method of alternate pounding and drying of

cassava roots resulted in a sharp decline in glycoside levels but high cyanohydrin levels may

remain if the products are not sufficiently dried (Mlingi et al, 1995). Processing steps such as

crushing and pounding may be incorporated prior to sun-drying to increase the efficiency of

cyanogen removal. However, sun-drying alone as a processing method of highly cyanogenic

cassava varieties remains inadequate if levels are to be reduced to the recommended FAO/

WHO safe limit set at 10 mg HCN equivalent /kg dry (Essers et al, 1996; Bainbridge et al,

1998).

Fresh cassava leaves contain very high levels of cyanogenic glycosides, usually 5 to 20 times

more than the amount present in the edible parts of the roots. Effective detoxification

processing is required prior to consumption. Bokanga (1994) found after pounding cassava

leaves a reduction of the cyanogenic potential by 63 to 73 %. The rapid removal of cyanogens

from cassava leaves can be attributed to the presence in the leaves of a high level of

linamarase activity, to the extensive mechanical damage imparted to the leaves during

pounding thereby facilitating the contact between linamarase and linamarin and promoting

cyanogenesis. The removal of cyanide by altering the cooking time and initial water

temperature in the preparation of pounded cassava leaves showed that starting with water at a

temperature of 27°C, brought down the total cyanide level 3.8 times more effectively than

starting with already boiling water and, increasing cooking time from half hour to one and


34

half hour resulted in a 2.5-fold more effective reduction (Essers, 1989). Gradual heat applied

during cooking of pounded cassava leaves accelerates the evaporation of hydrogen cyanide

(boiling point at 25.7°C) and starting cooking at 27°C will increase progressively the

temperature to reach the maximum linamarase activity reported to be 55°C. Putting the leaves

into boiling water will immediately reduce drastically the activity of linamarase and may

therefore prevent liberation of cyanide from its glycosydic bond (Essers, 1989; Bokanga,

1994).

I.3.2.5 Metabolism of cyanogens in Humans

During cassava processing, cyanogenic glycosides (linamarin and lotaustralin) break down

into glucose and acetone cyanohydrin through the activity of the endogenous enzyme

linamarase. Acetone cyanohydrin gradually breaks down into HCN spontaneously

(temperature and pH dependant) or enzymatically (Figure I-6). These cyanogens (linamarin,

acetone cyanohydrin and HCN) can be reduced to negligible levels by effective processing,

but insufficiently processed cassava products contain varying amounts of cyanogens.

Following consumption, any of the three types of cyanogens may result in cyanide exposure

(Essers et al, 1992; Rosling, 1994). However the fate of the cyanogens will differ during

digestion in the gut and metabolism in the body (Carlsson et al, 1995).

The ingested linamarin is thought to be hydrolysed to glucose and acetone cyanhydrin in the

intestinal tract; hydrogen cyanide is then produced by a catalytic reaction in the intestine and

rapidly absorbed from the intestine to the blood (Sreeja et al, 2003). A part of ingested

linamarin has been found to pass through the human body unchanged, absorbed directly in the

intestine and excreted intact in urine (Carlsson et al, 1995; Carlsson et al, 1999; Sreeja et al,

2003). Ingested residual cyanhydrins are assumed to break down to cyanide in the alkaline

environment of the gut (Tylleskär et al, 1992). Cyanide is rapidly absorbed by the

gastrointestinal tract and distributed throughout the body by the blood. The major portion of


35

cyanide in blood is sequestered in the erythrocytes, and a relatively small proportion is

transported via plasma to the target organs (liver, lungs, kidney, brain, central nervous

system). Although cyanide can interact with substances such as methemoglobin in the

bloodstream, the majority of cyanide metabolism occurs within the tissues. Cyanide is

metabolized in mammalian systems by one major route and several minor routes (Figure I-7).

Figure I-7: Basic processes involved in the metabolism of cyanide (ATSDR, 1997)


36

The major route of metabolism for cyanide is detoxification in the liver by the mitochondrial

enzyme rhodanese (thiosulphate-sulphurtransferase, EC 2.8.1.1), which catalyses the transfer

of the sulfane sulphur of thiosulphate to the cyanide ion to form the less toxic thiocyanate

(Figure I-7). About 80 % of cyanide is detoxified by this route. The rate-limiting step is the

amount of thiosulphate which is produced by ß-mercaptopyruvate resulting from

transamination of cysteine. While rhodanese is present in the mitochondria of all tissues, the

species and tissue distributions of rhodanese are highly variable. In general the highest

concentrations of rhodanese are found in the liver, kidney, brain and muscle, but the supply of

thiosulphate is limited.

Cyanide is principally excreted as thiocyanate in the urine but the limiting factor in cyanide

metabolism is the concentration of the sulfur containing substrates primarily thiosulphate, but

also cystine and cysteine as product of methionine (essential amino acid) and cysteine

catabolism (Figure I-8). There are several pathways for cysteine catabolism. The more

important catabolic pathway is that via a cytochrome –P450-coupled enzyme, cysteine

dioxygenase that oxidises the cysteine sulphydyl to sulfinate, producing the intermediate

cysteinesulfinate. Cysteinesulfinate can serve as a biosynthetic intermediate undergoing

decarboxylation and oxidation to produce taurine, the bile salt precursor. The enzyme

cystathionase can also transfer the sulphur from one cysteine to another generating

thiocysteine and pyruvate. Transamination of cysteine yields ß-mercaptopyruvate which then

reacts with sulphite (SO32-) to produce thiosulphate and puryvate. Both thiocysteine and

thiosulphate can be used by the enzyme rhodanese to incorporate sulphur into cyanide,

thereby detoxifying the cyanide to thiocyanate (Hoffer, 2002; Komarnisky et al, 2003;

Stipanuk, 2004).


37

Figure I-8 : Cysteine catabolism

Methionine

Cysteine

Pyruvate

+

Thiocysteine

ß-Mercaptopyruvate

Thiosulphate

+

cyanide

Cysteine

+ or Thiocyanate

Thiocyanate

Cysteinesulfinate

Hypotaurine

Taurinerhodanese

Cysteine

NH4+ H20

Pyruvate

SO32-

3-mercaptopyruvate sulphurtransferase

aminotransferase

α-ketoglutarateglutamate


38

The level of thiocyanate normally present in body fluids is low but increases with chronic

exposure to cyanide and with smoking habits (Vesey et al. 1999, Kussendrager and Van

Hooijdonk, 2000). Thiocyanate remains the most useful chemical biomarker for dietary

cyanogen intake because it is a very stable metabolite that can be determined with relatively

cheap, specific and sensitive methods (Rosling, 1994, Ressler and Tatake, 2001). Urinary

thiocyanate is commonly used to check cyanogen overload in a population using cassava

roots and cassava products as staple food (Haque & Bradbury, 1999, Ernesto et al. 2002a).

Cyanide can also be metabolized by several minor routes, including the combination of

cyanide and hydroxycobalamin (vitamin B12) to yield cyanocobalamin (vitamin B12) and the

non-enzymatic combination of cyanide with cystine, forming 2-aminothiazoline-4-carboxylic

acid (ATC) which is excreted via the urine. However in protein-deficient subjects, in whom

sulfur amino acids are low, cyanide may conceivably be converted to cyanate (Tor-Agbidye et

al, 1999).

I.3.2.6 Effects of cassava toxicity in humans

The toxicity of cassava arises from the release of cyanide during hydrolysis of cyanogenic

glycosides by the glucosidases of intestinal microflora. Intact linamarin has also been reported

to be absorbed through the intestinal mucosa. Cyanide can also be released in vivo by

glucosidases of the liver and other tissues, causing in situ cytotoxicity (Padmaja, 1995).

Cyanide is a potent toxin that acts by inhibiting cellular respiration.

Cyanide toxicity occurs when the capacity for conversion of cyanide to thiocyanate is acutely

exceeded. This leads to inhibition of cytochrome oxidase and prevents cell respiration and

oxidative respiration. Acute toxicity results from the ingestion of lethal amounts of cyanide.

Doses of 50 to 100 mg are reported to be lethal to adults. Acute cassava poisoning, sometimes

leading to the death of whole families, has been occasionally reported in humans after

consumption of bitter cassava roots or inadequately processed cassava, usually at times when


39

the normal eating habits are affected by famine. More common are incidences of chronic

cyanide toxicity due to prolonged consumption of insufficiently processed cassava. Chronic

toxicity of cassava has been implicated in several diseases such as tropical ataxic neuropathy,

endemic goiter and konzo. Many of these conditions result from the consumption of poorly

processed cassava.

I.4 Conclusion

Konzo is a paralytic disease rarely reported and little known even in the affected area. This

symmetric paralysis of both legs affects mainly women at childbearing age and children

above three years old, among the poor rural population of remote areas of Sub-Saharan Africa

where cassava is the staple food. Affected persons live far from the big city where decisions

are made and they are of no particular interest for political authorities. Literature on konzo is

limited to some epidemiological consideration. Evidence linking the disease with high

consumption of improperly processed cassava roots has been established. In addition, a low

intake of sulphur amino acids (methionine and cysteine) needed for the metabolic

detoxification of cyanide in the human body has also been thought to be an important co-

factor in the development of konzo (Cliff et al, 1985).

Up to date, there is no medicine to cure this crippling non-progressive and irreversible

disease. Nevertheless, some investigators (Ernesto et al, 2002b) have proposed the following

strategies to prevent and to eliminate konzo:

• Introduction of other staples, vegetables, pulses and fruits to decrease the daily

cyanide intake and broaden the diet of the people,

• Improved processing of cassava roots to produce products that have less residual

cyanide,

• Introduction of low cyanide, high yielding, well-adapted, disease-resistant varieties of

cassava,


40

• and improved early warning systems of a possible konzo epidemic.

I.4.1 Rationale of the research

Konzo is a neglected and an emerging neurological crippling disease that affects the poor

segments of remote rural communities of sub-Saharan Africa. There is convincing evidence

linking konzo with high cyanide exposure (Tylleskär, 1994a; Ludolph and Spencer, 1996):

First, heavy dietary reliance on bitter cassava is strongly associated with the development of

konzo. Secondly, there is a consistent association between shortcut soaking cassava

processing and outbreaks of konzo. Thirdly, the bitter cassava contains cyanogenic

glycosides, mainly linamarin and to a lesser extent lotaustralin which upon hydrolysis release

the mitochondrial toxin cyanide, a potent inhibitor of cytochrome C oxidase (complex IV of

the mitochondrial respiratory chain). Also, in affected populations, the excretion of urinary

thiocyanate and the ratio thiocyanate/ inorganic sulphur in the blood of affected populations

are increased. (Tylleskär et al, 1991; Banea et al, 1992b; Tylleskär et al, 1992).

Definite confirmation of an etiologic role of cyanide in konzo by identification of the

mechanism in an experimental animal model, or a quasi experimental preventive intervention

is lacking. There is therefore a need to know if a wild plant (food), a vitamin deficiency,

another toxin in cassava or some other factor may be contributing to or be an essential factor

in the aetiology of konzo (Tylleskär, 1994a, Bonmarin et al, 2002).

I.4.2 Objectives

I.4.2.1 General objective

• Identify associated nutritional factors involved in konzo

I.4.2.2 Specific objectives

• To review the literature on konzo and its relation to cassava dietary exposure.


41

• To determine the prevalence and associated dietary factors with Konzo.

• To assess dietary intake with special emphasis on intake of sulfur amino acids.

• To quantify the daily intake of cyanogen, and to estimate the amount of sulphur amino

acids required for their detoxification in konzo affected areas.

• To determine free amino acids in order to evaluate the presence of inherent potentially

toxic nonprotein amino acids in the cassava products.

• To develop total protein amino acids profiles of cassava products in order to evaluate the

dietary protein quality and to compare them with the amino acid requirements of children

and adults.

• To monitor the level of dietary exposure to cyanogens from cassava in the selected konzo

affected community.

• To assess a potential relationship between urinary thiocyanate as biomarker of daily

cyanogen exposure and taurine as modulator of neuroexcitation.


42

CHAPTER II:

OCCURRENCE OF KONZO AND DIETARY PATTERN*

* This chapter will be submitted for publication in Tropical Medicine and International Health as: Delphin Diasolua Ngudi, Jean-Pierre Banea-Mayambu., Fernand Lambein and Patrick Kolsteren. Konzo and dietary pattern in cassava-consuming populations of Popokabaka, Democratic Republic of Congo


43

II Occurrence of konzo and dietary pattern

II.1.1 Introduction

In Popokabaka district (D. R. C.) where cases of konzo were first reported three generations

ago (Trolli, 1938), new cases were found and the diet has changed little. Cassava flour, the so

called “luku” or “fufu” is consumed together with a sauce prepared from cassava leaves. Meat

and fish are not eaten daily in the villages (Tshala-Katumbay, 2001a).

Protein-energy malnutrition which is a major health problem in the region has been attributed

to the combined effect of infections and inadequate diet. An unbalanced diet is suggested to

be the main risk factor for several diseases such as obesity, stroke, cancer (Thiele et al, 2004)

and a factor aggravating growth retardation in children in Bandundu Province, D. R. C.

(Banea-Mayambu et al, 2000).

The present paper reports the prevalence of konzo, the household risk factors associated and

the dietary pattern in cassava consuming populations. Dietary intake patterns and socio-

economic variables are well known indicators for assessing nutritional status of a community

(Agrahar-Murugkar and Pal, 2004).

II.1.2 Materials and Methods

II.1.2.1 Study area

The study was conducted in Popokabaka rural health zone (Prhz) (5°38’35” – 5°43’0” latitude

South, 16°34’60” – 16°37’8” longitude East), district of Kwango, province of Bandundu (1 -

8° S, 16 – 20° E), D. R. C. in February 2003 during the rainy season. Prhz covers an area of

7,949 km2 with a population of 149,227 inhabitants in 2002 (density of 19 inhabitants/ km2).

Prhz is divided into 38 health areas. The vegetation is bushy savannah with few forest

galleries where the climate is tropical with annual rainfall varying around 1200 mm and a


44

long dry season of about 5 to 6 months (Kama, 1970). The poor soil makes the rural

population focus on the cultivation of cassava, the most important cash crop and the main

staple food for this region. Prhz faces various constraints for its development. Accessibility to

Prhz is difficult especially during the rainy season and limits the communication with

Kinshasa, the capital of D. R. C. where manufactured goods and other food products can be

purchased or excess of crop production can be sold. Recrudescence of endemic pathologies

(malaria, trypanosomiasis, tuberculosis, leprosy, etc), poverty of the population and limited

number of unsafe drinking water sources are main problems encountered by Prhz (Mwela,

2002).

II.1.2.2 Subject

Four health areas in Prhz with a total population of 12,416 inhabitants in 2,069 households

(national mean size of household members is 6, see R. D. C. 2001) were selected based on the

accessibility and the reported prevalence of konzo in the areas. The Epitable calculator of

Epiinfo version 6 was used to calculate the sample size for a single proportion of a limited

community study based on the size of the population, the desired precision (0.99), the

expected prevalence of konzo (4 %), the designed effect (2) and on the confidence level (95

%). After introduction of those required data, a sample size of 2,685 inhabitants in 448

households was obtained. This sampled population was attained through heads or delegates of

household* who were enrolled in this study in a random sampling after selection of the first

participant (household) as starting point, near the main entrance road to the village or near the

health centre (clinic). The sample size was adjusted upward to 487 participating households to

avoid other factors that could decrease the yield of usable responses. No refusal was observed

during the survey. Written authorisations were obtained from the administrative and health

* A household was defined as group of persons sharing the same meals since at least 3 months before the survey.


45

authorities, oral consent and assistance were also obtained from the village leaders and from

the subjects.

The heads or delegates of household were interviewed by trained enumerators using a closed

and open-ended questionnaire designed to collect the following information: identification of

the household (name of the respondent, village, name, sex, marital status, education and

origin of head of household), socio- demographic characteristics: family size, family member

affected by konzo (name, birthday, onset, sex, degree of walking), owning land farm or

husbandry, list of all foods consumed during the previous day (morning, lunch and evening),

the origin of the cassava consumed the previous day as staple food and the duration of retting

(soaking), the composition of the “luku” and list of foods consumed often during the rainy

season and the dry season. The WHO criteria for konzo were applied to detect cases and to

confirm the diagnosis (WHO, 1996).

II.1.2.3 Statistics

Descriptive statistics were used for socio-demographic and other household related variables.

On the basis of the prevalence found, the areas were pooled into two groups: the low

prevalence area and the high prevalence area. Thus, the degree of konzo prevalence in the

health area was used to measure the risk of konzo and as a dependent variable in multivariate

analysis. Data were entered using EpiInfo (version 6.04) and analyses were performed with

SPSS (version 11.5 statistical packages for Windows). Excel 2003 for Windows was used to

plot graphs.


46

Table II-1: Socio-demographic variables and 24hr recall food consumption of participants

among the high prevalence of konzo health area (n = 224) and the low prevalence of

konzo health area (n =263)

Health area Variables

High Low OR (CI 95 %) P

Sex Female Male

81 142

70 193

1.36 (1.05 – 1.78)

0.024

Age Under 35 35 +

49 145

63 155

0.87 (0.63 – 1.20) 0.438

Marital status Unmarried Married

37 183

26 236

1.69 (1.06 – 2.71)

0.030

Education Illiterate Literate

109 114

99 161

1.28 (1.05 – 1.58)

0.021

Occupation Farmer Other

196 26

178 76

1.26 (1.15 – 1.38)

< 0.005

Native No Yes

52 172

50 213

1.22 (0.86 – 1.72)

0.266

Origin of cassava consumed in the household

Own farm Other origin

215 7

240 19

1.04 (1.00 – 1.09)

0.045

Soaking time of the cassava consumed

Less than 3 nights 3 nights +

62 153

202 38

0.34 (0.28 – 0.43)

< 0.005


47

Composition of luku consumed

Only cassava Cassava + cereal

Meat consumption Yes No

Sesame consumption Yes No

Cereals consumption Yes No

Cowpea consumption Yes No

Vegetables consumption Yes No

Cassava tuber consumption Yes No

Cassava leaves “Saka-Saka” consumption Yes No

Luku consumption Yes No

192 28

84 140

5

219

105 119

76 148

161 63

39 185

91 133

223 1

244 15

79 184

108 155

34 229

70 193

186 77

23 240

104 159

260 3

0.93 (0.87 – 0.98)

1.25 (0.97 – 1.60)

1.66 (1.50 – 1.84)

3.63 (2.57 – 5.11)

1.27 (0.97 – 1.67)

1.02 (0.91 – 1.14)

1.99 (1.23 – 3.23)

1.03 (0.83 -1.28)

1.01 (0.99 – 1.02)

0.010

0.084

< 0.005

< 0.005

0.092

0.84

0.006

0.853

0.628

II.1.3 Results

The majority (69 %) of the interviewed heads or delegates of household were male. The mean

age of respondents was 43.3 years (SD 12.0) with a range of 61 years (maximum 78 and

minimum 17). 79 % of the members of the households were native from the area. The

illiteracy rate among the heads or delegates of household was 43 % and among the literate,


48

more than 50 % did not finish the primary school (sixth grade), only 1.6 % and 0.4 % have

reached respectively secondary school (at least ninth grade school) and high school or

university, respectively. Most of the heads of household were married (86%) and small-scale

farmers (78 %) as main occupation. Other main occupation of the participants encompassed

mainly sawyer (5.5 %), teacher (4.5 %), worker (2.3 %), retailer (2.1 %), roadman (1.8 %)

and hunter (0.4%). The mean family size was 6.2 ± 2.7 (range 14; max 15 and min 1). About

75 % of female respondents were illiterate and almost all of them were farmer except three

with one retailer and two house wives with no other occupation. Table II-1 shows the

distribution of some socio-demographic variables of participants among the high prevalence

of konzo health area (n = 224) and the low prevalence health area (n =263). Degree of konzo

prevalence was statistically significant and associated with female gender [OR (95 % CI) =

1.4 (1.1 -1.8), P = 0.024], unmarried status OR (95 % CI) = 1.7 (1.1 -2.7), P = 0.030],

illiteracy [OR (95 % CI) = 1.2 (1.1 -1.6), P = 0.021], farmer as main occupation [OR (95 %

CI) = 1.3 (1.2 -1.4), P< 0.05] and slightly with consumption of cassava originated from own

farm as opposed to cassava obtained elsewhere [OR (95 % CI) = 1.0 (1.0 – 1.1), P = 0.045].

The origin of 24 hour cassava flour consumed in 93.4 % of households was from their own

farmstead and the retting time of the processed cassava was less than three nights in 57,4 % of

those families. In the households from where cassava originated from their own farmstead,

90.6 % of them consumed luku that was composed only of cassava flour and 9.4 % mixed

their cassava flour with maize flour.

Among the 3,015 individuals in the 487 households selected, 43 konzo patients were detected

in 33 (6.8 %) of the households; thus, a prevalence of 1.4% and an incidence in 2002 of 1.3 ‰

(Figure II-1). The mean number of affected family members per household was 1.30 (SD

0.6) with a range of two (minimum 1 and maximum 3). The distribution of konzo cases per


49

selected health areas is presented in Table II-2. 77 % of patients were female (male-to-female

ratio 1:3.3) and 64 % of the patients were under 15 year of age.

Figure II-1: Distribution of onset of konzo from 1980 to 2002

0

2

4

6

8

10

12

1975 1980 1985 1990 1995 2000 2005

year of onset

Case

s

Table II-2: Distribution of konzo cases per health area

Number of household

per number of konzo

cases

Health area

1 2 3

Number of konzo patients

High prevalence area

Masina

Mutsanga

22

14

8

4

2

2

1

1

0

33

21

12

Low prevalence area

Popo-Secteur

Imwela

3

3

0

2

1

1

1

1

0

10

8

2

Total 25 6 2 43


50

The majority [25/ 33 (75.8 %)] of the affected households had a single affected family

member. The degree of disability on walking and the age distribution of konzo patients by

gender are shown in Table II-3. The mild form of the disease was the most common and

found in 74 % of patients followed by the crawler or severe form (16%) and the moderate

form (9 %). No male was found to be attained by the moderate form. The earliest year of

onset of paralysis reported is 1980 and the latest 2002. Figure II-1 shows the distribution of

konzo cases by year of onset. Around half of the cases occurred between 1998 and 2002.

Table II-3 : Degree of disability on walking and age distribution of konzo patients by gender

Variables Sex Total

Female Male

Degree of disability

a. Walk without stick

b. Walk with stick

c. Can’t walk (crawl)

Total

24

4

5

33

8

-

2

10

32

4

7

43

Age (Years)

a. Under 5

b. 5 – 9

c. 10 – 14

d. 15 – 19

e. 20

Total

5

10

4

2

8

29

2

3

1

2

2

10

7

13

5

4

10

39

From the food consumption patterns of the respondents, we observed that less than 60 % ate

three main meals a day as breakfast, lunch and dinner, and their dietary pattern was based


51

basically on cassava stiff porridge, the so called fufu or luku. Table II-4 presents the

frequencies of 24 hour foods consumed; 97.9 % had their evening meal whereas 58.7 % of

households had lunch and 91.6 % had breakfast in the morning. Luku, the cassava flour stiff

porridge was the main staple food consumed at least once during the day in 99.2 % of

households. Rice was consumed as staple food in 0.6 %. Maize and other cassava roots

products (boiled or raw cassava roots) were the other staple foods consumed mainly during

lunch time as snacks.

Table II-4: 24-hour recall of household food intake frequencies (%)

Food Morning Lunch Evening

Roots and tubers

Luku (cassava flour stiff porridge)

Other cassava roots products

Sweet potato

Yam

85.1

1.6

-

-

31.2

5.6

0.2

4.3

95.4

1.2

--

Vegetables and fruits

Vegetables

Saka Saka (pounded cassava leaves)

Tomato

Mushrooms

Mbondi (Salacia pynaertii)

Mfumbwa (Gnetum africanum)

Spinach

Ngayi-Ngayi (Hibiscus sabandja)

Matembele (Sweet potato leaves)

Kikalakasa (Psophocarpus scandens leaves)

Amaranth

Other vegetables (unspecified)

Fruits

Safou

Banana

Pineapple

25.5

16.6

10.6

8.8

4.4

2.0

1.0

0.6

0.4

0.2

1.6

1.0

0.6

-

5.9

6.5

2.8

4.1

1.8

0.4

-

0.4

-

-

0.8

1.0

2

0.4

20.7

14.5

11.9

11.1

7.0

2.2

3.2

1.4

0.4

0.8

2.4

0.6

-

-


52

Legumes

Cowpeas (Vigna ungiculata)

Beans (Phaseolus vulgaris)

Voandzou (Vigna subterranea)

16.6

1.0

0.2

8.0

0.2

2.0

17.1

0.8

0.6

Cereals

Maize

Rice

4.4

0.6

13.3

1

3.8

0.2

Oleaginous grains (seeds and nuts)

Sesame

Peanut

Soybean

Pumpkins seeds (Curcubitaceae sp.)

13.9

12.8

1.2

0.8

4.3

17.8

0.2

1.0

12.6

11.9

0.2

1.2

Flesh

Red meat (cow, pork, goat, lamb, wild animals)

Fish

Chicken

Insect (Caterpillar and Larva)

Milk

Egg

Wild bird (unspecified)

Grasshopper

8.7

4.7

1.2

1

0.6

0.4

-

-

4

3.2

0.8

-

-

-

-

-

11.1

9.4

1.6

1.6

-

0.4

0.2

0.2

No Food 8.4 41.3 2.1

Saka - saka (pounded cassava leaves) was the main condiment consumed as side-dish with

luku in 40 % of households followed by cowpeas (30 %) and sesame (23.2 %). Peanut and

tomato were used as ingredients to prepare their sauce. Peanuts also were consumed during

lunch time as snacks to accompany grains of maize or other cassava roots products.

Mushrooms and unconventional green leafy vegetables such as mbondi (Salacia pynaertii)

and mfumbwa (Gnetum africanum), which villagers gather from the nearby bush or forests

were popular. They were consumed as supplementary foods to the staple luku in 17.7 %, 18.1


53

% and 11.3 % of households respectively. Only 15 % of households consumed garden

vegetables.

Table II-5: Seasonal food consumption availability (%) listed by the respondents

Foods Rainy season Dry season

Sweet potato

Yam

Saka Saka (pounded cassava leaves)

Tomato

Mushrooms

Mbondi (Salacia pynaertii)

Mfumbwa (Gnetum africanum)

Spinach

Ngayi-Ngayi (Hibiscus sabandja)

Matembele (Sweet potato leaves)

Amaranth

Maize

Rice

Banana

Pineapple

Cowpeas (Vigna ungiculata)

Beans (Phaseolus vulgaris)

Sesame

Peanut

Pumpkin seeds (Curcubitaceae sp.)

Red meat (cow, pork, goat, lamb)

Rats & wild animals

Fish

Chicken

Insect (Caterpillar and Larva)

Egg

Wild bird (unspecified)

Grasshopper

3.1

12.7

76

49.5

67

36.1

21.1

30.6

15.7

9.1

29.6

54.5

0.4

24.4

23.2

50.9

18.6

36.4

54.5

5

3.3

1

18.4

0.4

18

0.6

0.4

1.2

4.8

36.9

7

4.6

4.1

13.3

21.1

1.9

1.2

1.2

3.1

5.2

0

5.2

3.1

9.1

8.7

8.9

11

52.1

68.7

78

49.5

0.6

17.4

0.2

12.7

36.7


54

Meat and fish were at lesser degree consumed to supplement the staple food. Usually they are

added in the preparation of vegetables. Consumption of fruits is less; safou, pineapple, banana

and orange were the only fruits listed and consumed by less than 1% of households the day

before the survey.

The comparison between degree of prevalence of konzo in health area and 24 hour recall food

(groups) consumption is summarised in Table II-1. There is a statistically significant

association between the prevalence of konzo with consumption of cereals ([OR (95 % CI) =

3.6 (2.6 -5.1)], [OR (95 % CI) = 1.2 (1.1 -1.4)], with consumption of tubers of cassava [OR

(95 % CI) = 2.0 (1.2 -3.2)] and with consumption of sesame [OR (95 % CI) = 1.7 (1.5 -1.8)].

No statistically significant association was found between the prevalence of konzo in health

area with consumption of meat, with consumption of cowpea, with consumption of

vegetables, with consumption of saka-saka and, with consumption of luku. The frequencies of

foods consumed by season (Table II-5) show a decrease of consumption of almost all the

foods listed by the participants from the rainy season to the dry season, except the

consumption of meat and pumpkin seeds, which increases consumption.

II.1.4 Discussion and conclusion

Konzo is still occurring in this area three generations after the first report (Trolli, 1938). The

prevalence of konzo (1.4 %) found in our study, is lower than the expected prevalence of 4 %

but in the range of that reported in the literature (Tylleskär et al, 1991; Tshala-Katumbay et al,

2001b). The overall sex and age distribution of patients in our study was similar to most of the

previous studies in the region and elsewhere (Howlett, 1994; Banea- Mayambu, 1997c,

Tshala-Katumbay et al, 2001a). Preponderance of female patients (female to male ratio 3.3: 1)

in our study is similar to almost all other studies except the ones carried out in Tanzania and

Mozambique where male cases were preponderant (Howlett, 1994; Tshala-Katumbay et al,

2001b).


55

Households whose head was illiterate carried an increased risk of konzo. More than half of

the heads of household or representatives were illiterate or did not go beyond primary school

level. Illiteracy was also found to be a factor that carries an increased risk of paralysis in the

case of neurolathyrism, a spastic paraparesis with many similarities to konzo (Lambein et al,

2004). Education drives both individual and community development, and illiterates are likely

to have low socio-economic status even in such remote rural areas. Literate people probably

have better access to information than the illiterate on food processing especially home

detoxification methods (Getahun et al, 2002b). Women, who belong to the most susceptible

group to develop konzo and who also play the primary role in the household food security,

has been found in this study to be less educated and at higher risk of konzo. Three quarters of

them were illiterate and among those who were literate, half did not finish the primary school

education. In D. R. C., more than half of the women of rural areas are illiterate (R. D. C.,

2001). Unmarried status of the head of household as a risk factor can be explained by the

excessive workloads. The main occupation as a farmer for a head of household is associated

with increased risk of konzo. This can be explained by the fact that people are relying on their

own sole culture and consumption of cassava. Increased risk of konzo is found to be

associated with consumption of cassava originating from the household’s own farm. This may

be related to poverty, as the less poor have more access to marketed commodities, which

results in a more varied diet.

The food consumption pattern of the selected households in both high and low konzo

incidence areas is dominated by cassava diet. Fufu, the processed product of cassava roots is

the staple food of almost all the households and saka-saka is the main side-dish consumed

with fufu. Cassava roots are an excellent source of carbohydrate but contain extremely low

levels of protein and fat (Bradbury and Holloway, 1988). The protein is of poor quality,

leucine and lysine are limiting amino acids. The proportion of methionine is low and the


56

chemical score of the protein is around 40 (chapter III-2). Cassava leaves have a high protein

content but also this protein is of poor quality limited in lysine, histidine and sulphur

containing amino acids (methionine and cysteine) (chapter III-1). High dietary exposure to

cyanogen was found in this area (chapter IV).

Consumption of cowpea with fufu is also popular and provided a high quantity of protein in

the diet but also in cowpeas the sulphur containing amino acids are low (Finetin, 2001).

Consumption of cereals and sesame is found in this study to be protective factors against

konzo. Similar protection by methionine rich cereals was also found for neurolathyrism

(Getahun et al, 2003). Mixing cassava with cereals and mixing legumes such as cowpeas with

cereals may thus increase the quality of the meal by optimising the balance of essential amino

acids. Consumption of the unconventional green leafy vegetable mbondi (Salacia pynaertii)

and pumpkin seeds that are rich in protein with a high content in methionine and cysteine

(Mbemba and Remacle, 1992; Finetin, 2001), should also be promoted. Consumption of fresh

or boiled cassava tubers is a risk factor for konzo. Cassava roots contain high level of

cyanogenic glucosides that should be removed by processing. Peeling and boiling cassava are

not enough to lower the toxin compounds to a safe level. The peeled roots need to be soaked

in slow running water (retting or “rouissage”) for at least three nights. Consumption of luku

made from roots soaked less than three nights was associated with increased incidence of

konzo.

Nutritional resources listed became scarce during times of dry season. Almost all crops are

rain-fed and cannot survive during the dry season unless watering or irrigation which is not

done in this area because of absence of inputs or of major river systems for irrigation,

electricity for irrigation pumps and other factors that limit irrigation. Cassava roots that

constitute the staple food resist the drought. Under drought conditions the linamarin content

of cassava roots is known to increase due to increased water stress on the cassava plant


57

(Bokanga et al, 1994). Moreover, the weather of the dry season is mostly cloudy and the mean

temperature is 20 0C or less; which may be favourable for high exposure to cyanogen. The dry

season is the period of intensive cassava trade resulting in frequently shortcuts in cassava

processing with high residual cyanogens level in the product. As the duration of dry season in

this area of particular dry tropical climate is estimated to 5 or 6 months, serious drought may

increase the cyanide intake of individuals, if non- efficient processing techniques are used, to

such a degree as to precipitate the occurrence of konzo. High prevalence of konzo has been

reported in the dry season (Banea-Mayambu et al, 1997a; Cardoso et al, 2004).

In conclusion, konzo is still occurring in this area with an incidence in 2002 of 1.3 ‰, where

women who play the principal role in the household food security are in majority illiterate.

Although konzo was reported in this area in 1938, in this study we found no cases with onset

before 1980. We found no reports on the life expectancy of konzo patients. The diet is largely

dominated by cassava and major foods consumed are of poor quality in protein especially in

sulphur containing amino acids. Methionine and cysteine are required for the detoxification of

cyanide in the body. The results obtained in this study, confirm that low intake of sulphur

containing amino acids (methionine + cysteine) is associated with incidence of konzo, as well

as with dietary cyanide exposure. Therefore the emphasis should be placed on increasing

production and access to cereals, sesame and pumpkin seeds to increase the availability of

sulphur amino acids in the diet. Vegetable gardens should be promoted to encourage the

consumption of leafy vegetables in all seasons. Appropriate information, communication and

training in cassava processing and promotion of a better balanced diet may prevent this

irreversible crippling disease, konzo.


58

CHAPTER III.

CASSAVA FOOD QUALITY AND SAFETY


59

III Cassava food quality and safety

III.1 Food Safety and Amino Acid Balance in Processed Cassava

"cossettes"*

III.1.1 Introduction

Cassava (Manihot esculenta Crantz, Euphorbiaceae) is the major staple food consumed by the

population of D. R. C. (Goossens, 1996). Processed cassava roots provide more than 60 % of the

daily energy intake (FAO, 1990).

Sweet varieties of cassava roots may be consumed directly while bitter varieties with high

content of cyanogenic glycosides are traditionally processed to reduce toxicity and to improve

palatability and storability. Many varieties of processed cassava roots with different local names

are known: cossettes, chikwangue, fufu, malemba, luku, ntuka, etc.

Cossettes, which is the most popular cassava product in D. R. C., is obtained by soaking or

immersing fresh bitter cassava roots (whole or peeled) in a stream or stationary water (near a

stream) for at least 3 days to allow them to ferment until they become soft. The fermented roots

are then taken out, peeled and sundried on mats, racks, roofs of houses, etc. Depending on the

weather, sundrying takes 2-5 days (Hahn, 1989). The dried fermented cassava root is the so-

called "cossettes" (Figure III-1). This form of cassava product is preferred because it can be

stored for a long period and can be traded over much longer distances (Goossens, 1996; Minten

and Kyle, 1999).

* This sub- chapter has been published as: Delphin Diasolua Ngudi, Yu – Haey Kuo and Fernand Lambein (2002). Food safety and amino acid balance in processed cassava roots “cossettes”. Journal of Agricultural and Food Chemistry 50, 3042 – 3049.


60

Figure III-1: Flow diagram of cassava cossettes processing

Steps of cossettes production Location

Harvesting of whole fresh bitter cassava

roots

Fields

Peeling and chopping

or not

River, stream or village

Soaking and natural fermentation in water

for 3-5 days (and peeling)

River, stream or village

Sun-drying or fire-drying

for 3-5 days

Village

Cossettes Village

Pounding or

milling

Storage and packaging in

jute or propyl-ethylene

sacks

Cassava flour

Village

Trade Local markets or city markets


61

When the roots are soaked and dried for a shorter period because of insufficient food supply or

poor agro-ecological conditions, the remaining cyanogen content can be much higher than that

after normal process. High intakes of dietary cyanogens from poorly processed cassava roots in a

diet deficient in sulphur amino acids have been implicated in the causation of konzo (Tylleskär,

1994b).

D. R. C. is the most affected country where konzo has been reported from remote rural areas of

Bulungu, Kahemba, Masi-Manimba and Popokabaka in Bandundu province (Howlett, 1994;

Tylleskär et al, 1995).

Besides the high content of cyanogens, cassava roots are also known to be poor in protein content

(Hahn, 1989). Proteins are a necessary part of the daily diet because the human body does not

store protein as it does with carbohydrate and fats. Furthermore, 9 of the 20 protein amino acids

are either not synthesized at all by our body or can only be synthesized in insufficient amounts.

Humans must obtain them from dietary sources. These are known as the dietary essential amino

acids that include histidine, isoleucine, leucine, tryptophan, lysine, methionine, phenylalanine,

threonine and valine. Failure to receive an adequate dietary supply of essential amino acids leads

to retarded growth and development in children and to disease and body deterioration in adults

(McMury and Castellion, 1996).

The objective of this study is to determine residual cyanogen in different samples of cossettes to

check the safety, to quantify the daily intake of cyanogen and to estimate the amount of sulphur

amino acids required for their detoxification. Free and total protein amino acids profiles of

cossettes are determined to evaluate the dietary protein quality and to compare with the amino

acid requirements of children and adults. Nonprotein amino acids have been reported to be

present in many commonly eaten foods and these compounds have the ability to interfere with a

wide range of fundamental biochemical processes and cause disease (Rozan et al, 2000;


62

Rubenstein, 2000). Neurolathyrism, which shares clinical similarities with konzo, has been

associated with the overconsumption of grass pea (Lathyrus sativus L., Fabaceae) which contains

a neurotoxic nonprotein amino acid, BOAA or its synonym ODAP (Howlett, 1994; Tylleskär et

al, 1994c; Getahun et al, 1999). Therefore the presence of any inherent potentially toxic

nonprotein amino acid in cossette samples is also examined.

III.1.2 Materials and methods

III.1.2.1 Plant Materials

Cossettes were purchased in five different markets (Ngaba, Lemba, Livulu, Rond Point and

Matete) of Kinshasa, capital of D. R. C. The cossettes in those markets are supplied by Bandundu

province where konzo has been reported and depending on the size, they are sold in bulk of about

10 pieces of roots. About 500g (2 or 3 pieces) of the cossettes from each market were finely

ground with an electric small laboratory grinder "Culatti" with 200 µm sieve prior to sampling

and analyses.

Cassava flour from Cameroon was purchased from an exotic food shop in Antwerp, Belgium for

comparison. Cameroon is a part of central Africa where cassava is processed like in D. R. C.

III.1.2.2 Determination of Cyanogens

A simple picrate paper kit developed by Egan et al (1997) and improved by Bradbury et al (1999)

was used for the determination of all forms of cyanogens in cassava products. Protocol B1 was

followed for the determination of total cyanogens and acetone cyanohydrin + HCN/ CN-.


63

III.1.2.2.1 Total Cyanogens

100 mg of sample was placed on top of 21 mm diameter Whatman 3 MM filter paper disc

containing 1 M phosphate buffer at pH 8 and linamarase in a flat bottomed plastic bottle

(supplied in the kit). Millipore filtered deionised water (0.5 mL) was added and a yellow picrate

paper attached to a plastic strip was immediately inserted into the vial that was closed

immediately with a screw lid and allowed to stand at room temperature for 24 h. The plastic

backing sheet was removed carefully from the picrate paper. This latter paper was immersed in

5.0 ml of deionised water for about 30 min. The absorbance of the solution was measured at 510

nm, using cuvettes of 1 cm light path against a blank, which contained a yellow solution

produced by a picrate paper not exposed to HCN/ CN-.

The total cyanogens content (expressed in ppm) was calculated by the simple equation:

Total cyanogens content = 396 x Absorbance

Other samples were prepared as above but without cassava flour, using square linamarin papers

equivalent to 50 and 400 ppm, to serve as controls.

III.1.2.2.2 Acetone Cyanohydrin + HCN/ CN-

This analysis was done following the above procedure. However 200 mg of guanidine

hydrochloride was added after the addition of the phosphate buffer pH 8 filter paper disc. The

incubation time was 3 h.

The amount of linamarin was calculated through the following equation:

Linamarin content = Total cyanogens - (acetone cyanohydrin + HCN/ CN-)


64

III.1.2.3 Determination of Amino acids

An HPLC gradient system with precolumn phenylisothiocyanate (PITC) derivatisation (Khan et

al, 1994) was used to analyse free amino acids. Total protein amino acids were determined after

sample hydrolysis.

III.1.2.3.1 Extraction of free amino acids

50 μL of DL- Allylglycine (100 nmol/ ml) was added to finely ground sample (5 g) as internal

standard. The samples were then extracted in 3 volumes of 70 % ethanol and stored overnight at

4o C. The extracts were centrifuged (34800g, 20 min) and the pellets were washed twice with 70

% ethanol. The supernatants were pooled and concentrated under vacuum and stored in a deep

freezer at - 20 o C.

III.1.2.3.2 Sample hydrolysis

The flour sample was hydrolysed under vacuum in 6 M HCl following the AOAC 982.30 E

procedure (18).

III.1.2.3.3 Amino acid analysis

Aliquots of extract or hydrolysate were concentrated and dried under vacuum (37 o C, 20 mm Hg)

then a coupling reagent (methanol: water: triethylamine; 2:2:1; v/v) was added, mixed and dried

immediately under vacuum during 10 min. After this, PITC reagent (methanol: triethylamine:

water: PITC; 7:1:1:1; v/v) was added and allowed to stand at room temperature for 20 min before

drying under vacuum. PITC derivatives were dissolved in buffer A (0.1 M ammonium acetate,

pH 6.5) and filtered through a 0.22 μm Millipore membrane.


65

20 μl of sample was injected into an HPLC (Waters model 991 equipped with photodiode array

detector) using a gradient system of buffer A (100 - 0 %) and buffer B (0.1 M ammonium acetate

containing acetonitrile and methanol, 44:46:10; v/v, pH 6.5) (0 - 100 % after 50 min). The

operating temperature was 43 o C. A reverse phase column from Alltech (Alltima C 18, 5 μm,

250 x 4.6 mm) was used. The absorbance at 254 nm was recorded and used for calculations. A

standard protein amino acid mixture (food hydrolysate A 9656, Sigma) was derivatised as above

and used as standard for calculations. The results were analysed by Millennium software (Waters,

version 1.10)

III.1.2.3.4 Tryptophan Determination

A rapid and simple acid ninhydrin method described by Gaitonde and Dovey (1970) and adapted

for colorimetric determination of tryptophan by Sodek et al (1975) was used. Cossettes samples

were partially defatted by suspension in 20 volumes of acetone and stirring occasionally for 30’.

After filtration, the powder was left to air-dry. Portions (500 mg) of defatted cassava cossettes

were extracted in a centrifuge tube with 2.0 ml of 70 % ethanol for 30’ at room temperature. The

mixture was occasionally stirred and homogenized with a glass rod. 5 mL of NaOH (0.5 %) were

then added and extraction continued for another 1 h. After centrifugation, a clear supernatant was

collected and 0.2 ml of it was taken for tryptophan assay. The acid ninhydrin method using

reagent b (250 mg of ninhydrin dissolved in 10 ml of formic acid- hydrochloric acid; 3:2; v/v)

was followed for the determination of tryptophan in the samples. Readings were made against a

reagent blank in a spectrophotometer (Shidmazu, UV-1601) at 390 nm using cuvettes of 1 cm

light path. Sample blanks contained a similar aliquot of extract together with reagent b without

ninhydrin.


66

After subtracting the absorbance value of the sample blank, the tryptophan content was read off a

standard curve. Lysozyme (Grade I from egg white; Sigma Chemical Co.) was used to construct

the standard curve. Tryptophan values obtained from this graph were then corrected for tyrosine

interference according to Zahnley and Davis (1973).

III.1.2.4 Statistics

The results were computed and compared by analysis of variance using the software package

SPSS 9.0 for Windows. Significant differences amongst means were confirmed using the Tukey

Honestly Significant Differences set at 95 % confidence interval (P < 0.05). Data are expressed

as means ± standard deviation.

III.1.3 Results and discussion

III.1.3.1 Cyanogens

The six samples of cassava cossettes had residual cyanogens below 10 mg HCN equivalent kg-1

as shown in Table III-1. This is the recommended safe limit by the Codex alimentarius (FAO/

WHO, 1991). The highest level was found in samples from Cameroon (9.37 mg HCN equivalent

kg-1) and the lowest level in samples from Rond Point (1.45 mg HCN equivalent kg-1), showing a

6.5 fold variation with a significant difference between samples from Cameroon and all other

samples (P < 0.05). No significant differences was found between samples from Matete and

Ngaba, and among samples from Rond Point, Livulu and Lemba but those last samples were

significantly different from those from Matete and Ngaba (P < 0.05).

Enzymatic determination of the cyanogenic glycoside linamarin, the major source of cyanide in

cassava, showed a variation of almost ten fold between 0.924 and 8.58 mg HCN equivalent kg-1.


67

Table III-1: Cyanogens content in cassava cossettes (mg HCN equivalent kg - 1 dry weight)*

Cossettes Total Cyanogens Acetone Cyanohydrin + HCN/

CN-

Linamarin

Matete

(N = 3)

2.772 b ± 0.396 0.264 a ±0.280 2.508 c ±0.457

Cameroon

(N = 3)

9.372 c ± 0.229 0.792 a ± 0.280 8.580 d ± 0.229

Lemba

(N = 3)

1.716 a ± 0.229 0.396 a ± 0.457 1.320 a, b ± 0.229

RondPoint

(N = 3)

1.452 a ± 0.229 0.528 a ± 0.229 0.924 a ± 0.229

Livulu

(N = 3)

1.584 a ± 0.396 0.132 a ± 0.229 1.452 a, b, c± 0.229

Ngaba

(N = 3)

2.904 b ± 0.457 0.792 a ± 0.280 2.112 b, c ± 0.243

Again the samples from Cameroon were significantly higher than all other samples (P < 0.05)

while the linamarin content of Rond Point was significantly different from Ngaba and Matete,

and also Lemba was different from Matete (P < 0.05). No significant difference was found for the

content of acetone cyanohydrin + HCN/ CN - between samples (P > 0.05), this varied 6 fold

between 0.13 and 0.79 mg HCN kg-1 in the cassava cossettes examined.

The fresh bitter cassava roots typically used in the region have total cyanogen levels of 100 to

500 mg HCN equivalent kg-1 root, even up to 1500 mg HCN kg-1 (Bradbury and Holloway, 1988;

O’Brien et al, 1992; Padmaja, 1996). Although the original content of the fresh roots from which

the cossettes were prepared is not known, it is obvious that the processing and handling of the

material resulted in a reduction of total cyanogen of at least 10-30 fold, up to 150-500 fold,

* Values are means ± standard deviation a, b, c same superscript within a column means no significant difference (P> 0.05)


68

giving a final result within the recommended safe limit set at 10 mg HCN equivalent per kg of

dry weight. The processing and handling included soaking, sundrying, storage and transportation

to the open markets in Kinshasa where the cossettes are sold in jute sack or in bulk. The samples

from Cameroon bought in Europe were packed in a plastic foil of low permeability.

The low levels of glycosides in the flour from cossettes can be explained by continued cell

desintegration and enzymatic activity of the linamarase from the cytoplasm hydrolysing the

cyanogens from the disrupted vacuoles during soaking and throughout the four days of drying

before moisture fell to low levels in these big root pieces (Banea-Mayambu, 1997). When

considering time and temperature factor, it can be assumed that even in short soaked cossettes

cyanohydrins might be lost during storage and transportation over much longer distances from

Bandundu to the markets in Kinshasa than when consumed locally in Bandundu area. The low

cyanogen exposure from cassava might explain the absence of cases of konzo in urban

consumers, while the crippling disease konzo is prevalent in remote rural areas of Bandundu

Province (Minten and Kyle, 1999; Formunyam et al, 1985; Banea-Mayambu et al, 1998). Even

the shortcut processed cassava products from Bandundu area sold in Kinshasa do not cause

clinical symptoms of cyanide exposure (Banea-Mayambu et al, 1998).

Oke (1968) reported HCN contents of 1.0 mg/ 100 g in cossettes from D. R. C. and O'Brien et al

(1992) found a variation in cyanogens content of fermented cassava roots ranging between 0 to

11.3 mg kg-1 in villages of Cameroon. In populations with cassava roots as their main staple food,

a basic daily energy need of 6276 kJ (1500 kcal) can be satisfied with 500 g dry weight cassava

root products. Adult consumers would then be exposed to approximately less than 5 mg HCN

equivalent per day comparing to the Codex alimentarius safe level of 10 mg HCN equivalent per

kg dry weight (Rosling, 1988). If cossettes as staple food provide 60 % of dietary daily energy


69

intake in D. R. C., calculated from the FAO/ WHO energy requirements (FAO/WHO/UNU,

1985), it means that for the samples from Matete about 0.7 mg HCN equivalent is present in 241

g of cassava cossettes to be consumed daily by children (1 to 3 years old) and about 1.5 mg HCN

equivalent is present in 532 g of cassava cossettes to be consumed daily by a moderately active

adult man to meet energy requirements of 3414 (816) and 7531 (1800) kJ (kcal) respectively

(Table III-2).

Table III-2: Estimated daily cossettes and total cyanogens intake

Daily cyanogens from

cossettes‡ (mg)

Subjects Daily

energy

required*

in kJ (kcal)

60 % daily

energy

required in

kJ (kcal)

Daily

cossettes

intake† (g) Matete

samples

Livulu

samples

Children 1 – 3 yr

Children 7 – 9 yr

Adult female

(moderately

active)

Adult male

(moderately

active)

5690

(1360)

9162

(2190)

9204

(2200)

12552

(3000)

3414

(816)

5497

(1314)

5523

(1320)

7531

(1800)

241

389

390

532

0.7

1.1

1.1

1.5

0.4

0.6

0.6

0.8

* from FAO/ WHO/ UNU (1985) † 100 g cassava provides 338 kcal (FAO/WHO/UNU, 1985) ‡ Total cyanogens (Table III-1) x daily cossettes intake (Table III-2)


70

III.1.3.2 Total protein amino acids

Table III-3 represents the total protein amino acids profiles in cassava cossette samples. The

overall average of the total protein amino acids is 23.7 mg/ g dry weight cassava cossettes in

which essential amino acids represent 54.6 % and the sulphur containing amino acids 9.7 %.

Alanine was the major protein amino acid in all samples except in the samples from Rond Point

where glutamic acid was the most important. This finding is in agreement with some studies done

with fresh cassava roots (Bradbury and Holloway, 1988; Firmin and Kamenan, 1996; Glew et al,

1997). This suggests that during the post-harvest processing practised, the loss of protein is

negligeable while the loss of cyanogens is considerable.

The samples from Livulu had the highest total protein amino acid content (27.3 mg/ g of dry

weight cassava cossettes) with 54.2 % of essential amino acids. The samples from Lemba

contained the highest essential amino acids proportion (63.5 %) and those from Rond Point were

the lowest (50.4 %). Leucine and lysine, the purely ketogenic amino acids, were the limiting

amino acids in our samples (Table III-4). Leucine was the first limiting amino acid in the samples

from Livulu and Ngaba with an amino acid score of 0.36 and 0.45 respectively. Lysine was the

first limiting amino acid in the other samples with an amino acid score varying from 0.35 to 0.47.

Results of Firmin and Kamenan (1996) showed sulphur amino acids (methionine + cysteine) in

fresh cassava roots and leucine in fermented pulp of cassava roots as first limiting amino acid,

respectively. Yeoh and Truong (1996) found sulphur amino acids, leucine and lysine to be

limiting amino acid in different cultivars of cassava roots studied. Bradbury and Holloway (1988)

reported large differences in amino acid composition between different cultivars of cassava roots

examined and there was no essential amino acid, which was clearly the first limiting amino acid.

Nevertheless, on the average histidine was the first and leucine the second limiting amino acid.


71

Although sulphur amino acids were not the limiting amino acids in our samples, we should notice

that the proportion of methionine represented in average only 13 % (4.7 - 16.2 %) and cysteine 87

% (83.8 - 95.3 %) of total sulphur amino acids. Methionine normally supplies part of the body's

needs for cysteine. With cysteine-free diets, methionine can supply all of the body's needs for

cysteine. Cysteine can spare methionine and a certain proportion of dietary methionine is

converted to cysteine (Brody, 1994).

III.1.3.3 Free amino acids

The free amino acid pattern of cossettes samples is summarised in Table III-5. The

concentrations of free amino acids were in general very low. Arginine and sulphur amino acids

(methionine and cysteine) were not found. Histidine was not found in in the samples from

Matete, Rond Point and Ngaba. No asparagine was detected in the samples fromMatete and

Livulu.

The samples from Livulu showed the highest amount in total free amino acids (6.2 mg/ g of dry

weight cossettes) and those from Rond Point, the lowest (0.27 mg/ g of dry weight cossettes).

This represents about 23 fold variation among the few samples examined; duration and flow rate

of water during soaking leading to leaching out can probably explain this finding. Threonine was

quantitatively the most important free amino acid in five of the samples examined, while in the

samples from Rond Point sample asparagine was the most abundant (Figure III-2). Alanine

ranked the second place except in the samples from Livulu, Rond Point and Cameroon. No

known potentially toxic nonprotein amino acids were detected in our samples.


72

Table III-3: Total protein amino acids content in cassava cossettes (mg g - 1dry weight)*

Cossettes Total

protein

amino acids

Matete

(N = 4)

Cameroon

(N = 4)

Lemba

(N = 4)

RondPoint

(N = 4)

Livulu

(N = 3)

Ngaba

(N = 4)

Asp 00.200a ± 0.022 00.234 a ± 0.044 00.351 a b ± 0.074 00.480 b c ± 0.120 00.403 b ± 0.049 00.564 c ± 0.041

Glu 1.074a ± 0.257 1.255 a ± 0.079 1.343 a ± 0.209 22.016 b ± 0.186 1.702 a b ± 0.589 22.270 b ± 0.291

Ser 00.700a ± 0.157 00.688 a ± 0.058 00.749 a ± 0.112 1.053 b ±0.023 00.970 a b ± 0.272 00.691 a ± 0.066

Gly 1.342 a ± 0.175 1.254 a ± 0.231` 1.423 a b ± 0.164 1.921 b ± 0.173 1.388 a ± 0.413 1.217 a ± 0.111

His 1.218 a b ± 0.177 1.744 b ± 0.140 1.082 a ± 0.038 1.203 a ± 0.113 1.111 a ± 0.580 1.060 a ± 0.121

Arg 00.796 a ± 0.073 00.715 a ± 0.024 00.744 a ± 0.085 1.029 b ± 0.071 1.034 b ± 0.089 00.796 a ± 0.092

Thr 1.787a b ± 0.638 22.239 a b c ± 0.71 3.177 b c ± 0.807 00.945 a ± 0.335 3.508 c ± 0.907 3.493 c ± 0.594

Ala 5.124 c ± 0.869 44.309 b c ± 0.675 3.349 b ± 0.334 00.962 a ± 0.059 5.329 c ± 0.968 5.628 c ± 0.316

Pro 1.494 a b ± 0.072 1.345 a ± 0.154 1.664a b ± 0.269 1.930 b ± 0.264 1.679 a b ± 0.269 1.498 a b ± 0.110

Tyr 1.410 a ± 0.064 1.272 a ± 0.190 1.035 a ± 0.506 1.343 a ± 0.186 1.035 a ± 0.506 1.191 a ± 0.346

Val 00.961 a ± 0.287 1.153 a ± 0.485 3.306 b ± 0.363 1.104 a ± 0.449 1.733 a ± 0.783 1.374 a ± 0.343

Met 00.162 a ± 0.028 00.108 a ± 0.029 00.399 a ± 0.079 00.372 a ± 0.110 00.302 a ± 0.368 00.398 a ± 0.243

Cys 1.828 a ± 0.070 22.183 a ± 0.231 22.157 a ± 0.168 1.975 a ± 0.440 1.853 a ± 0.250 22.061 a ± 0.999

Ile 00.740 a b ± 0.148 00.554 a ± 0.237 1.604 b± 0.556 00.609 a ± 0.303 00.612 a ±0.241 00.914 a b ± 0.539

Leu 00.660 a ± 0.108 00.648 a ± 0.261 1.717 b ± 0.692 00.699 a ± 0.527 00.652 a ± 0.112 00.760 a ± 0.321

Phe 00.900a ± 0.104 00.783 a ±0.234 00.842 a ± 0.063 00.708 a ± 0.202 22.314 b ± 0.568 00.523 a ± 0.363

Lys 00.497 a ± 0.123 00.527 a ± 0.092 00.531 a ± 0.256 00.530 a ± 0.108 00.751 a ± 0.124 00.691 a ± 0.164

Try 00.741 a ± 0.004 00.754 a ± 0.022 00.877 a ± 0.188 00.780 a ± 0.026 00.907 a ± 0.018 00.740 a ± 0.012

* Values are means ± standard deviation a, b, c same superscript within a row means no significant difference (P> 0.05)


73

Table III-4: Amino acid scoring pattern of different cossette samples

FAO/ WHO1* Amino acid Scores2† Essential Amino Acid

(EAA) Children

(2-5 years)

Matete Lemba Livulu Ngaba RondPoint Cameroon

Threonine 34 2.43 3.54 3.78 3.97 1.42 3.05

Cysteine + Methionine 25 3.68 3.88 3.16 3.80 4.80 4.24

Valine 35 1.27 3.58 1.81 1.52 1.61 1.66

Isoleucine 28 1.22 2.17 0.80 1.26 1.11 0.91

Leucine 66 0.46 0.99 0.36 0.45 0.54 0.45

Tyrosine + Phenylalanine 63 1.69 1.14 1.95 1.05 1.66 1.37

Histidine 19 2.97 2.16 2.14 2.16 3.24 4.25

Lysine 58 0.40 0.35 0.47 0.47 0.47 0.37

Tryptophan 11 3.11 3.02 3.02 2.60 3.62 3.17

First limiting Amino Acid Lysine Lysine Leucine Leucine Lysine Lysine

Second limiting Amino Acid Leucine Leucine Lysine Lysine Leucine Leucine

* Recommended amino acid scoring pattern from FAO/ WHO/ UNU (1985) † Amino acid score = mg of amino acid in 1g of test protein per mg of amino acid in 1 g of reference Protein (FAO/WHO, 1991)


74

Figure III-2: Free amino acids in cassava cossette samples

0,0

10,0

20,0

30,0

40,0

50,0

60,0

70,0

Asp Glu Ser Gly His Arg Thr Ala Pro Tyr Val Met Cys Ile Leu Phe Lys Asn Gln Trp

Free amino acids

% o

f tot

al fr

ee a

min

o ac

ids

in d

iffer

ent c

osse

tte s

ampl

es

MateteCameroonLemba RondPoint Livulu Ngaba


75

Table III-5. Free protein amino acids content in cassava cossettes (mg g - 1 dry weight)*

Cossettes Free protein

amino acids Matete

(N = 4)

Cameroon

(N = 3)

Lemba

(N = 3)

RondPoint

(N = 3)

Livulu

(N = 3)

Ngaba

(N = 3)

Aspartic acid 0.005a ± 0.003 0.012 b ± 0.000 0.030 c ± 0.001 ND 0.051 d ± 0.005 0.044 d ± 0.007

Glutamic acid 0.013a ± 0.000 0.079 d ± 0.003 0.048 c ± 0.001 0.012 a ± 0.001 0.082 d ± 0.006 0.025 b ± 0.001

Serine 0.002a ± 0.000 0.008 a b ± 0.005 0.015 b c ± 0.003 0.003a ±0.002 0.029 d ± 0.001 0.023 c d ± 0.007

Glycine 0.007 a b ± 0.000 0.016 b c ± 0.003` 0.025 c ± 0.003 0.006 a ± 0.003 0.051 d ± 0.002 0.005 a b ± 0.009

Histidine ND∗ 0.017 a ± 0.000 0.048 a ± 0.050 ND 0.245 b ± 0.015 ND

Arginine ND ND ND ND ND ND

Threonine 0.370 a ± 0.119 1.106 b ± 0.056 1.135 b ± 0.212 0.072 a ± 0.005 2.171 c ± 0.344 1.523 b ± 0.264

Alanine 0.046 a ± 0.029 0.151 a b ± 0.016 0.223 c ± 0.047 0.025 a ± 0.000 0.284 b ± 0.116 0.270 b ± 0.065

Proline 0.039 b ± 0.002 0.064 c ± 0.007 0.098 e ± 0.004 0.017 a ± 0.000 0.298 f ± 0.008 0.081 d ± 0.005

Tyrosine 0.014 b ± 0.002 0.031 c ± 0.002 0.077 d ± 0.001 0.004 a ± 0.000 0.145 e ± 0.005 0.087 d ± 0.007

Valine 0.025a b± 0.000 0.043a b ± 0.001 0.076 b c ± 0.043 0.010 a ± 0.001 0.124 c± 0.010 0.056 a b ± 0.000

Methionine ND ND ND ND ND ND

Cysteine ND ND ND ND ND ND

Isoleucine 0.012 b ± 0.000 0.016 b ± 0.001 0.070 d ± 0.005 0.003 a ± 0.002 0.107 e ±0.006 0.027 c ± 0.003

Leucine 0.023 a ± 0.001 0.044 a ± 0.001 0.139 a b ± 0.002 0.007 a ± 0.000 0.541 b ± 0.000 0.081 a b ± 0.003

Phenylalanine 0.015 a b ± 0.000 0.043 a b ±0.000 0.089 c ± 0.004 0.006 a ± 0.004 1.925 d ± 0.060 0.075 b c ± 0.003

Lysine 0.010 b ± 0.000 0.024 c ± 0.002 0.036 d ±0.003 0.005 a ± 0.003 0.112 e ± 0.003 0.018 c ± 0.002

Asparagine ND 0.003 a ± 0.000 0.002 a ± 0.003 0.096 a ± 0.083 ND 0.010 b ± 0.001

Glutamine 0.006 a b ± 0.000 0.024 c d ± 0.000 0.015 b c ± 0.003 0.002 a ± 0.001 0.032 d ± 0.002 0.160 e ± 0.010

Tryptophan 0.027 a b ± 0.000 0.221 c ± 0.011 0.038 a b ± 0.026 0.006 a ± 0.003 0.005 a ± 0.004 0.063 b ± 0.008

* Values are means ± standard deviation a, b, c , d, e same superscript within a row means no significant difference (P> 0.05) ∗ ND: not detected


76

III.1.3.4 Essential Amino Acid (EAA) requirements and estimated daily

intake

Rose and Wixon (1955) demonstrated the influence of cysteine on the methionine requirement

for an adult man by determining the conditions that supported a zero or slightly positive nitrogen

(N) balance. They observed that cysteine alone without methionine resulted in a negative N

balance. A near zero N balance was observed with a diet containing 0.8 g of methionine, while N

balance was negative with 0.7 g methionine diet. Higher levels of methionine resulted in a

positive N balance. They concluded that oversupply of cysteine could give a positive N balance

with lower intake of methionine, but even then the intake of methionine remains essential. This

statement illustrates the limiting of the ability of cysteine to spare methionine. Although cysteine

can fulfill a large fraction of our requirement for sulphur amino acids, according to Altman and

Dittmer (1974) in the combination cysteine + methionine, 30 - 50 % of total requirement for

adults may be furnished by cysteine and 50 - 70 % furnished by methionine.

The expected daily methionine and sulphur amino acids intake provided by cassava cossettes

consumption, which in the case of D. R. C. represents 60 % of daily energy intake, are compared

with the suggested amino acid patterns requirement (Table III-6). It can be concluded that

children of 1 to 9 years old cannot expect to meet methionine requirement whereas adults can

meet sulphur amino acid requirement. Sulphur amino acids are required for cyanide

detoxification in the human body (Bradbury and Holloway, 1988; Rosling, 1994). A daily supply

of about 1.2 mg of dietary sulfur from S-containing amino acids is needed by the human body to

detoxify 1.0 mg of HCN (Padmaja, 1996). When the body is regularly exposed to cassava

cyanogens the increased synthesis of rhodanese, enzyme responsible for cyanide detoxification in


77

the human body by forming thiocyanate, makes extra demands on the body's reserves of sulphur

amino acids. If this demand is prolonged as in the regular consumption of cassava root

insufficiently processed, and the diet is inadequate, the synthesis of many proteins vital for bodily

Table III-6. Essential Amino Acid (EAA) requirements and estimated daily intake

EAA suggested patterns of

requirement*

Estimated daily EAA (from cossettes) intake† (mg)

(mg AA/ day) Matete samples Livulu samples

Child Child Child Child Child

> 1yr

Adult

female

Adult

male

1-3 yr 7-9 yr

Adult

female

Adult

male

1-3 yr 7-9yr

Adult

female

Adult

male

Thr 1000 305 500 431 695 697 951 845 1365 1368 1867

Cys + Met - 550 1100 - - 776 1059 - - 840 1146

Met 800 - - 39 63 - - 73 117 - -

Val 900 650 800 232 374 375 511 418 674 676 922

Ile 1000 450 700 178 288 289 394 147 238 239 326

Leu 1500 620 1100 159 257 257 351 157 254 254 347

Tyr+ Phe - 1120 1100 - - 901 1229 - - 1306 1782

Phe 800 - - 217 350 - - 558 900 - -

Lys 1600 500 800 120 193 194 264 181 292 293 400

Trp 250 157 250 179 288 289 394 219 353 354 483

functions may be impaired and lead to the development of protein deficiencies and other diseases

(Padmaja, 1996; Onwuka et al, 1992; Tor-Agbidye et al, 1998). Other food components of the

diet should contribute to a better balanced amino acid composition of the diet, especially the level

of sulphur amino acids. In the case of lathyrism, a neurodegenerative disease with similar clinical

* From Altman and Dittmer (1974) † Daily cossettes intake (Table III-2) x Amino acid (Table III-3)


78

symptoms as konzo, Lambein et al (2001) have suggested that the ratio of cereals (rich in

methionine) to Lathyrus seeds (rich in lysine and low in sulphur amino acid) may be a

determining factor in the etiology. In the regions neighbouring the konzo-affected areas in

Bandundu where traditionally corn or millet flour is mixed with cassava as staple food, no cases

of konzo have been reported. This may corroborate our views as to the importance of methionine

for a healthy balanced diet.

Hence, the recommended daily methionine allowance should be reconsidered and given

separately from total S-amino acid requirement.

III.1.3.5 Conclusion

The processed cassava roots available on the markets in Kinshasa have cyanogens content within

the safe limit recommended by FAO/ WHO. Proper processing, time and storage conditions and

traditional transport in jute sacks appear to contribute to reduce residual cyanogens in the

cossettes whereas insufficient processing and transport in airtight wrapping which prevents the

release of cyanide can probably explain the level of cyanogen found in the cossettes from

Cameroon samples.

No potentially toxic nonprotein amino acids were detected in this study.

The dietary requirements for sulphur amino acids need to be adjusted for the loss caused by

cyanide detoxification. The total sulphur amino acids availability does not give a correct value for

the requirement of the essential amino acid methionine. In the case when cassava is taken as

staple food, the low methionine content may aggravate the risk for cyanide toxicity and konzo

disease, even when the cysteine present covers the dietary requirement for sulphur amino acids.


79

III.2 Residual cyanogens, free and total amino acid profiles of cooked

cassava leaves "saka- saka” *†

III.2.1 Introduction

In the konzo-affected areas of D. R. C., processed cassava roots are prepared as described before

(chapter III-1), while cassava leaves or "saka-saka" are prepared as follows: the hard petioles are

removed, the tender leaves and the shoots are selected and may be blanched in warm/ boiled water

for a few minutes or partially dried on a pan or a pot over fire and then squeezed to remove liquid

before pounding. The spinach-like mass obtained after pounding with a traditional wooden mortar

and pestle is then cooked with some water added. Usually palm oil and salt are added and

sometimes also traditional spices and onion. (CEPLANUT, 1988; Almazan and Theberge, 1989;

Hahn, 1989).

Reports on the nutritional quality of cassava leaf protein as food are scanty and conflicting

(Bokanga, 1994). The majority of studies considered cassava leaf as animal feed and focused

mainly on cyanogen removal. Residual cyanogens and the presence of inherent potentially toxic

nonprotein amino acids were examined in this study before and after cooking pounded cassava

leaves to check their safety. The aim of this paper is also to assess the amino acid profiles and the

protein quality of cooked pounded cassava leaves as food, which is the most common daily side

dish as sauce and as main source of protein in a diet consisting of processed cassava roots as the

exclusive staple food in konzo affected areas of DRC, especially in Bandundu province.

* This sub-chapter has been published as:

Delphin Diasolua Ngudi, Yu – Haey Kuo and Fernand Lambein (2003). Cassava cyanogens and free amino acids in raw and cooked leaves. Food and Chemical Toxicology 41, 1193 – 1197.

† Delphin Diasolua Ngudi, Hu – Haey Kuo and Fernand Lambein (2003). Amino acid profiles and protein quality of cooked cassava leaves or “saka saka”. Journal of the Science of Food and Agriculture 83, 529 – 534.


80

III.2.2 Materials and methods

III.2.2.1 Sample acquisition

Deep-frozen pounded raw cassava leaves from D. R. C., about 500 to 600 g packed in plastic foil,

were purchased in five different exotic food shops in Ghent, Belgium (Dampoort, Foreign and

Ghana) and in Paris, France (Congo and Chateau).

III.2.2.2 Sample handling and culinary processing

Each packet of raw sample was divided into two parts. One part was kept as such for analysis and

the other part was subjected to the following culinary treatment on a hot plate:

About 250 ml of water was added to 100 g of raw pounded cassava leaves and allowed to boil. 10

ml of palm oil and about 1 g of salt were added when boiling started and the dish was stirred with

a wooden spoon for mixing of the ingredients. The cooked pounded cassava leaves or "saka-

saka" were ready to eat after 30 minutes of boiling (CEPLANUT, 1988). The samples were

analysed after cooling down to room temperature.

III.2.2.3 Determination of cyanogens

See section III.1.2.2

III.2.2.4 Determination of protein

The samples were extracted in 3 vol of physiological solution (NaCl 0.15 M; pH 5.96 at room

temperature) and stored overnight at 4 oC. The extracts were centrifuged (34800g, 20 min) and

the pellets were washed twice with physiological solution. The supernatants were pooled and

used for protein analyses.


81

The Bio-Rad® Protein assay kit, consisting of dye reagent concentrate and lyophilised bovine

albumin as protein standard was used to determine protein content in our samples. A standard

curve was made using several dilutions of protein standard containing 0.2 to 1.4 mg ml –1.

Analyses of protein were done as follows: 0.1 ml of sample was placed in a test tube and then 5

ml of diluted dye reagent was added and mixed several times. The absorbance was measured at

595 nm versus reagent blank within a period of 5 minutes to one hour after mixing. The

absorbance was converted to protein content using the standard curve.

III.2.2.5 Determination of amino acids

See section III.1.2.3

III.2.2.6 Tryptophan determination

See sub-section III.1.2.3.4

III.2.2.7 Protein quality evaluation

The amino acid scoring pattern recommended by FAO/WHO/UNU (1985) was used for the

evaluation of dietary protein quality as follows:

Amino acid score = mg of amino acid in 1 g of test protein 18 mg of amino acid in 1g of reference protein

The essential amino acid showing a score less than 1 was a limiting amino acid. The lowest

amino acid score (the most limiting amino acid) indicates the quality of the protein.


82

III.2.2.8 Statistics

The software package SPSS 10.0 for windows was used for the analysis of variance of the data.

The statistically significant differences among means were confirmed using the Tukey Honestly

significant differences at 95 % confidence interval (P<0.05).

III.2.3 Results and discussion

III.2.3.1 Total cyanogens

The total cyanogens content of the raw and the cooked cassava leaves samples are summarised in

Table III-9. The initial levels of total cyanogens in the raw (pounded) cassava leaves samples

ranged from 35.9 ± 0.4 to 107.5 ± 0.8 mg HCN equivalent kg–1, acetone cyanohydrin + HCN/

CN- from 5.7 ± 1.9 to 24.1 ± 4.5 mg HCN equivalent kg–1 and the linamarin from 30.2 ± 2.4 to

83.4 ±5.3 mg HCN equivalent kg–1 dry weight. Those values are up to 10- fold higher than what

we detected in the processed cassava roots (Chapter III-1). A Significant reduction (P< 0.05) in

total cyanogens was observed when the raw samples were cooked; 96 - 99 % of the total

cyanogens were removed after cooking the cassava leaves. Bokanga (1994) observed that

pounding alone reduced the cyanogenic potential by about 60 - 70 %.

After cooking, the total cyanogens varied from 0.30 ± 0.04 to 1.9 ± 0.2 mg HCN equivalent kg-1

dry weight and the acetone cyanohydrin + HCN/CN- were not detected. The residual cyanogens

were below the recommended safe limit set at 10 mg HCN equivalent kg-1 by the Codex

alimentarius (FAO/ WHO, 1991).

Although the original content and the varieties of the fresh leaves from which the samples were

pounded are not known, this decrease can be explained by the following considerations. Besides

the genetic differences of the plant varieties, the variation in cyanogen content between samples


83

can also be explained by the maturity of the leaves. Padmaja (1989) reported lower contents of

cyanide in the older leaves compared with young leaves. Cyanogen content may also depend on

the heat treatment during preparation of the leaves before pounding. The leaves may be washed

with tap water or blanched in warm water for a few minutes or partially dried over fire or

grinding before pounding (Almazan and Theberge, 1989). The heat treatment can be favorable

for cyanide reduction or can destroy the endogenous hydrolysing enzyme linamarase. Finally, the

consistency of pounding can also play a role in the reduction of cyanogens during pounding of

cassava leaves. Destruction of the cells leads to contact between the cyanogenic glucosides and

the endogenous enzyme with subsequent release of HCN. Our finding is in agreement with the

fact that the rapid removal of cyanogens from cassava can be attributed to the heat applied during

boiling which accelerates the evaporation of HCN and cyanohydrin produced by the linamarin

hydrolysis (Almazan and Theberge, 1989; Essers, 1989; Bokanga, 1994).


84

Table III-7: Cyanogen content in raw and cooked cassava leaves (mg HCN equivalent kg-1 dry weight)

Dampoort Foreign Congo Chateau Ghana

Raw

(n=4)

Cooked

(n=4)

Raw

(n=4)

Cooked

(n=4)

Raw

(n=4)

Cooked

(n=4)

Raw

(n=4)

Cooked

(n=4)

Raw

(n=4)

Cooked

(n=4)

Total

cyanogens

Acetone

cyanhydrin

+HCN/CN-

Linamarin

35.9b±0.4

5.7ab±1.9

30.2b±2.4

1.3a±0.3

NDb

1.3a±0.3

107.5d±0.8

24.2d±4.5

83.4d±5.3

0.8a±1.1

ND

0.8a±1.1

87.9c±4.5

15.3c±0.3

72.7c±4.2

0.30a±0.04

ND

0.30a±0.04

83.7c±2.4

10.5bc±2.5

73.2c±4.9

0.7a±0.1

ND

00.7a±0.1

86.1c±3.1

15.0c±3.9

71.1c±0.8

1.9a±0.2

ND

1.9a±0.2

Same letter within a row means no significant difference (P>0.05)

a Values are means ± standard deviation

b Not detected.


85

III.2.3.2 Total protein and amino acid profiles

The total protein content and the amino acids composition (g kg-1 dry weight) of five different

samples of cassava leaves before and after cooking are listed in Table III-7. The total protein

content of the raw pounded cassava leaves samples ranged from 235.8 g in the Foreign sample to

351.8 g in the Dampoort sample. Those values are within the ranges reported in the literature

(Almazan and Theberge, 1989; Hahn, 1989; Bokanga, 1994; Yeoh and Chew, 1976; Ravindran

and Ravindran, 1988). Ravindran and Ravindran (1988) observed a decrease of protein content in

cassava leaves with ageing: from 381 g kg-1 in very young cassava leaves to 286 g kg-1 in young

leaves and 197 g kg-1 in mature leaves. The total protein content in the cooked samples ranged

from 111.8 g kg-1 dry weight in the Chateau sample to 144.6 g kg-1 dry weight in the Dampoort

sample. The results showed a significant (P < 0.05) decrease by an average of 58% in protein

content after cooking pounded cassava leaves. The large volume of water added and the

prolonged cooking time (at least 30 min of boiling) necessary for this culinary treatment to

remove the bitter taste, which might lead to losses of amino acids by diffusion and by thermal

degradation, can explain this decrease (Clemente and al, 1998; De la Cruz, 1999). Cooking of

green beans in a covered pot or pressure cooker was observed to cause important losses of amino

acids compared to the raw sample (De la Cruz et al, 1999). Other authors also observed a

significant reduction of amino acids in chickpea seeds after cooking with distilled water (Attia et

al, 1994; Clemente et al, 1998). During preparation of food, the side chains of some protein-

bound amino acids can react chemically with each other or with other molecules present in the

food and those reactions can result in a reduction of nutritive value (Sherr et al, 1989).


86

Table III-8: Protein content and amino acid composition of raw and cooked pounded cassava leaves (g kg-1 dry weight)*

Amino acids Foreign (n= 5) Chateau (n= 5) Congo (n= 4) Dampoort (n= 5) Ghana (n= 4)

Raw Cooked Raw cooked Raw Cooked Raw cooked Raw cooked

Aspartic acid 40.8c±0.1 12.1a±1.0 33.9b±0.4 17.0a±2.5 33.9b±0.4 16.3a±3.9 37.1bc±3.2 16.0a±2.6 38.6bc±2.8 13.6a±0.4

Glutamic Acid 36.8c±0.3 12.8a±2.4 36.2c±0.7 16.7ab±3.2 37.9c±4.1 16.9ab±4.0 44.4d±4.1 19.6b±3.0 41.1cd±0.2 15.5ab±0.6

Serine 11.8b±2.1 3.9a±1.0 12.9bc±2.3 5.3a±1.6 15.1bc±2.5 4.6a±0.7 16.8c±1.7 6.0a±0.3 15.0bc±0.3 4.7a ±1.1

Glycine 11.0c±0.2 5.2a±1.2 11.5cd±0.5 5.9a±0.8 13.7de±1.3 5.5a±1.0 18.0f±1.7 8.2b±1.1 13.9e±0.5 6.3ab±0.3

Histidine 5.4b±0.9 1.2a±0.2 6.7cd±0.5 1.3a±0.7 6.6bc±0.6 1.6a±0.2 7.9de±0.7 2.0a±0.2 8.1e±0.8 1.6a±0.3

Arginine 16.8b±0.6 8.5a±1.2 16.1b±0.5 9.3a±1.8 18.4bc±2.2 7.9a±1.0 24.0d±2.4 10.6a±1.5 21.4cd±0.8 8.8a±0.6

Threonine 8.8c±0.6 4.2a±0.6 9.6c±0.4 4.7ab±1.1 11.7d±1.1 4.4ab±0.1 17.5e±0.7 6.3b±0.6 12.8d±0.6 4.5ab±0.3

Alanine 21.7bc±1.9 13.6a±3.4 20.7bc±1.9 13.9a±1.9 25.1cd±3.0 14.4a±1.1 30.9d±4.2 15.9ab±3.7 25.4cd±1.7 15.8ab±3.2

Proline 11.3b±0.4 6.4a±1.3 11.6b±0.5 6.7a±1.0 14.4c ±1.8 6.3a±0.4 17.9d±2.1 8.1a±1.1 14.9c±0.3 6.3a±0.4

Tyrosine 9.4c±0.3 4.3a±0.5 10.1c±0.4 4.7a±0.8 12.1d±1.4 4.9a±0.1 15.4e±1.5 6.6b±0.5 12.2d±0.4 4.3a±0.3

Valine 10.6c±0.2 4.9ab±1.1 11.5cd±0.4 4.8ab±0.9 12.9de±1.5 4.6a±0.5 16.8f±1.4 6.6b±0.4 14.8e±1.0 4.7ab±0.2

Methionine 3.2c ±0.4 0.3a±0.1 3.1c±0.5 ND† 3.4c±0.2 0.8ab±0.3 4.7d±0.6 1.5b±0.4 2.8c±0.4 0.2a±0.2

Cysteine ND ND ND ND ND ND ND ND ND ND

Isoleucine 7.5b±0.4 2.3a±0.9 7.8bc±0.3 2.6a±1.0 9.0bc±1.1 2.5a±0.8 11.8d±1.2 4.1a±0.8 9.8c±0.8 2.5a±1.0

Leucine 14.3c±0.3 6.3a±1.4 15.9cd±0.4 7.1ab±1.1 17.6de±1.8 5.9a±0.7 24.0f±2.3 9.7b±0.3 18.6e±0.9 7.0a±0.2

Phenylalanine 12.4b±1.8 5.4a±1.2 13.4b±0.3 5.9a±2.7 15.0b±2.0 4.3a±0.8 18.7c±1.1 6.9a±0.6 14.9b±0.8 6.0a±1.1

Tryptophan 3.5 c±0.0 1.4a±0.0 3.5 c±0.0 1.4 a±0.1 3.8 d±0.0 1.3 a±0.0 4.1 e±0.2 1.7 b±0.0 3.6 cd±0.1 1.6 b±0.0

Lysine 9.6c±0.9 4.1a±1.1 15.3d±0.7 3.7a±1.3 15.5d±1.1 5.6ab±0.3 20.5de±2.0 7.1bc±0.6 18.3e±2.2 5.1ab±0.7

Total Protein 235.8c±5.6 114.1 a±2.4 256.7 d±7.3 111.8 a±2.4 293.3e±2.7 113.1a±3.2 351.8f±7.3 144.6b±3.5 291.7e±5.3 112.2a±1.1

* Values are means ± standard deviation a,b,c,d,e,f same superscript within a row means no significant difference (P>0.05) † ND= not detected


87

Aspartic acid, glutamic acid and alanine were the major amino acids found in all the samples

studied. They represented together an overall average of 101 g and 46 g kg-1 dry weight in the

raw samples and in the cooked samples respectively. Histidine, tryptophan and methionine were

the amino acid found in lowest concentration with together an overall average of 14 g and 3.6 g

kg-1 dry weight in the raw and in the cooked samples respectively. Other authors obtained similar

profiles in all the varieties of raw cassava leaf studied (Yeoh and Chew, 1976; Ravindran and

Ravindran, 1998). Cysteine was not detected in any sample.

Figure III-3: Protein amino acids profile of the raw and cooked cassava leaves

Figure III-3 shows the profile of the individual amino acid per total protein, which is almost

similar for all the samples. No marked differences can be observed between the raw and the

cooked samples when considering individual amino acid, except alanine which increased

significantly after cooking and methionine showing the highest decrease after cooking.

0

20

40

60

80

100

120

140

160

Asp Glu Ser G ly His Arg Thr Ala Pro Tyr Val Met Ile Leu Phe Try Lys

am ino acids

g am

ino

acid

per

kg

tota

l pro

tein

Raw

cooked


88

Methionine is highly required in the konzo-affected areas for dietary cyanide detoxification. The

reactive thioether group in methionine that is involved in oxido-reduction reactions and the

thermal breakdown of methionine can explain this finding (Clemente et al, 1998; De la Cruz et al,

1999). Excessive heat treatment causes considerable nutritional damage to methionine (Geervani

and Theophilus, 1980; Shemer and Perkins, 1975).

A comparison of the total essential amino acid profiles with the FAO/WHO reference pattern

(Table III-8) showed that the raw cassava leaves samples contained 357 g to 401 g of total

essential amino acids per kg of cassava leaves protein. This is higher than the 339 g of total

essential amino acids in the recommended FAO/WHO-reference protein. The cooked samples

contained less total essential amino acids than the FAO/WHO reference ranging from 299.3 g kg-

1 total protein content in the Foreign samples to 330.2 g kg-1 total protein content in the Ghana

samples


89

Table III-9: Comparison of the essential amino acid contents of different raw and cooked pounded cassava leaves samples and their

amino acid score with the recommended FAO reference

Essential AA FAO

Ref.*

Foreign Chateau Congo Dampoort Ghana

Raw Cooked Raw cooked Raw Cooked Raw cooked Raw cooked

Histidine 19 (1.0) 23 † (1.2) 11 (0.6) 26 (1.4) 12 (0.6) 23 (1.3) 14 (0.8) 22 (1.2) 14 (0.7) 28 (1.5) 14 (0.7)

Threonine 34 (1.0) 37 (1.1) 37 (1.1) 37 (1.1) 42 (1.2) 40 (1.2) 40 (1.2) 50 (1.5) 44 (1.3) 44 (1.3) 40 (1.2)

AAA‡ 63 (1.0) 92 (1.5) 85 (1.3) 91 (1.4) 95 (1.5) 92 (1.5) 81 (1.3) 97 (1.5) 94 (1.5) 93 (1.5) 91 (1.4)

Valine 35 (1.0) 44 (1.3) 43 (1.2) 45 (1.3) 43 (1.2) 44(1.3) 41 (1.2) 48 (1.4) 46 (1.3) 51 (1.5) 42 (1.2)

SAA 25 (1.0) 13(0.5) 0.3 (0.01) 12 (0.5) ND§ 12 (0.5) 7 (0.3) 13 (0.5) 10 (0.4) 10 (0.4) 0.2 (0.01)

Isoleucine 28 (1.0) 31 (1.1) 20 (0.7) 30 (1.1) 23 (0.8) 31 (1.1) 22 (0.8) 33 (1.2) 28 (1.0) 34 (1.2) 22 (0.8)

Leucine 66 (1.0) 61 (0.9) 55 (0.8) 62 (0.9) 63 (0.9) 60 (0.9) 52 (0.8) 68 (1.0) 67 (1.0) 64 (1.0) 62 (0.9)

Tryptophan 11(1.0) 15(1.4) 12(1.1) 14 (1.3) 12(1.1) 13(1.2) 12(1.1) 12(1.1) 12(1.1) 12(1.1) 14(1.3)

Lysine 58 (1.0) 41 (0.7) 36 (0.6) 60 (1.0) 33 (0.6) 53 (0.9) 50 (0.9) 58 (1.0) 49 (0.8) 63 (1.1) 45 (0.8)

Total 339 357 299.3 377 323 368 319 401 364 399 330.2

1st limiting amino acid SAA SAA SAA SAA SAA SAA SAA SAA SAA SAA

2nd limiting amino acid

Other limiting amino

acids

Lysine

Leucine

Histidine

Lysine

Isoleucine

Leucine

Leucine Lysine

Histidine

Isoleucine

Leucine

Leucine

Lysine

Histidine

Leucine

Isoleucine

Lysine

Histidine

Lysine

Histidine

Isoleucine

Lysine

Leucine

* FAO-protein reference from FAO/ WHO/ UNU (1985) † Calculated from Table III-3: Essential amino acid per total protein, the results are expressed in g kg -1 of the protein, the amino acid scores are indicated

between brackets ‡ AAA= aromatic amino acids (Phenylalanine +Tyrosine) § ND= not detected


90

The scoring pattern in Table III-8 showed that sulphur-containing amino acids were the most

limiting amino acid in all the samples (raw and cooked) with an amino acid score varying from

0.5 to less. Other investigators obtained the same results from their raw cassava leaves samples

studied (Yeoh and Chew, 1976; Lancaster, 1983). Besides sulphur amino acids, lysine and

leucine were limiting amino acids in the raw Foreign and Congo samples and leucine in the raw

Chateau samples. The maturity of the cassava leaves (young or mature) used and preliminary

processing (blanching or partial grinding or not) before pounding were unknown and might have

an effect on lysine and leucine. In all the cooked samples except the Chateau sample, histidine

was the second limiting amino acid. Lysine, leucine and isoleucine were also second or third

limiting amino acids in some of the cooked samples.

III.2.3.3 Free amino acids

The free amino acids and trigonelline (N-methyl-nicotinic acid) pattern of the raw and cooked

cassava leaves samples are summarised in Table III-10. The total free amino acids detected and

trigonelline varied from 10.8 g kg-1 to 38.2 g kg-1 in the raw samples and from 7.4 g kg-1 to 25.6 g

kg-1 in the cooked samples. Thus, pounded cassava leaves showed a decrease in the total free

amino acids content after cooking. The highest decrease was observed in the Ghana samples

(45.4 %) followed by the Foreign samples (38.3%), the Dampoort samples (31.3 %), the Congo

samples (23.5%) and the Chateau samples (15 %). The concentration of total free amino acids

including free protein amino acids and free nonprotein amino acids was at least 6-fold higher in

the leaves than what we found in the cassava roots (Chapter III-1). Aspartic acid, glutamic acid

and alanine are the major free protein amino acids found in the samples. Methionine and cysteine

were not detected as free amino acid in any samples. γ-Amino butyric acid (GABA) and α-amino

butyric acid (α- ABA) are the free nonprotein amino acids detected in all the samples.


91

Table III-10: Free amino acid and trigonelline content in raw and cooked cassava leaves (g kg-1 dry weight)*† Amino acids Dampoort (n= 4) Foreign (n= 4) Congo (n= 4) Chateau (n= 4) Ghana (n= 4)

Raw Cooked Raw cooked Raw Cooked Raw cooked Raw cooked Aspartic acid 1.16b±0.44 0.48a±0.07 4.57e±0.24 2.23 c±0.01 1.34b±0.23 0.94a,b±0.16 3.53d±0.38 3.16d±0.23 1.98c±0.15 1.09 b±0.08 Glutamic Acid 0.21a±0.02 0.38a±0.18 4.44e±0.22 2.1 0 b,c±0.02 2.51c,d±0.13 1.92 b±0.27 5.53f,g±0.21 5.10 f±0.30 5.72f,g±0.39 2.73d±0.24 Serine 0.18a±0.01 0.13a±0.01 1.77e±0.11 1.02c±0.01 0.50 b±0.01 0.43b±0.00 1.75e±.0.08 1.87e±0.19 3.02f±0.16 1.39d±0.02 Glycine 0.28d±0.02 0.13b,c±0.02 0.18c±0.02 0.10a,b±0.00 0.07a,b±0.02 0.05a,b±0.03 0.10a,b±0.00 0.09a,b±0.06 0.52e±0.01 0.26d±0.00 Histidine ND‡ ND 0.87e±0.11 0.51c±0.03 0.19 b±0.01 0.21 b±0.03 0.72 d±0.11 0.87e±0.07 1.07f±0.06 0.53c±0.01 Arginine 0.40a±0.03 0.17a±0.02 2.55 d±0.28 1.52c±0.06 0.25a±0.01 0.26a±0.01 1.14b±0.19 1.32b,c±0.16 2.75d±0.16 1.25b,c±0.00 Threonine ND ND 0.15a±0.09 0.02a±0.00 0.24a±0.00 0.25a±0.02 0.89b±0.19 1.08b±0.19 1.01b±0.03 0.26a±0.26 Alanine 1.64a,b±0.15 1.05a±0.12 1.65a,b±0.10 0.70a±0.01 2.09b±1.47 0.79a±0.09 1.73a,b±0.12 2.59 b,c±1.22 3.67c±0.14 1.54a,b±0.01 Proline 0.51d±0.06 0.41b,c±0.02 0.48c,d±0.04 0.24a±0.00 0.37 b,c±0.06 0.31a,b±0.01 0.72e±0.05 0.64e±0.04 2.46g±0.09 1.01f±0.00 Tyrosine 0.42 b±0.03 0.27a±0.01 0.85c,d±0.09 0.45b±0.00 0.42b±0.06 0.39a,b±0.00 0.97d±0.06 0.89d±0.02 1.46e±0.10 0.72c±0.03 Valine 0.86b±0.10 0.44a±0.01 1.48c±0.08 0.85b±0.04 0.77b±0.05 0.73b±0.01 1.88d±0.08 1.74d±0.10 2.81e±0.06 1.40c±0.03 Methionine ND ND ND ND ND ND ND ND ND ND Cysteine ND ND ND ND ND ND ND ND ND ND Isoleucine 0.44±0.04 0.26±0.00 0.69±0.03 0.35±0.00 0.39±0.38 0.21±0.00 0.53±0.02 0.45±0.02 1.42±0.04 0.68±0.00 Leucine 0.65±0.04 0.37±0.01 0.75±0.04 0.43±0.00 0.24±0.02 0.26±0.00 0.57±0.03 0.51±0.02 2.21±0.08 1.12±0.00 Phenylalanine 0.51±0.05 0.39±0.01 2.09±0.21 1.10±0.01 0.71±0.14 0.67±0.10 1.44±0.33 1.38±0.15 2.39±0.07 1.16±0.00 Tryptophan 0.19±0.022 0.21±0.02 1.46±0.08 0.84±0.02 0.59±0.03 0.57±0.07 1.37±0.25 1.35±0.13 1.67±0.07 1.07±0.01 Lysine 0.28±0.02 0.07±0.00 0.55±0.07 0.45±0.00 0.12±0.01 0.18±0.02 0.33±0.04 0.41±0.07 0.98±0.06 0.67±0.02 Trigonelline 0.81±0.10 0.94±0.13 0.29±0.02 0.41±0.03 0.37±0.03 0.35±0.00 1.33±0.13 0.44±0.05 0.32±0.02 0.49±0.02 Asparagine 0.18±0.02 0.16±0.00 1.49±0.10 2.25±0.02 0.32±0.01 0.27±0.03 3.51±0.20 1.14±0.11 1.39±0.08 1.81±0.00 Glutamine 0.23±0.02 0.39±0.02 0.78±0.05 0.71±0.00 0.22±0.01 0.11±0.01 1.75±0.11 0.44±0.03 0.67±0.03 0.54±0.00 GABA 1.69±0.17 1.07±0.08 0.18±0.05 0.40±0.09 0.05±0.00 0.05±0.00 0.03±0.07 0.01±0.00 0.52±0.03 0.91±0.18 α-ABA 0.19±0.02 0.09±0.01 0.10±0.01 0.20±0.00 0.06±0.00 0.07±0.00 0.28±0.02 0.09±0.00 0.14±0.00 0.20±0.00

* Values are means ± standard deviation, a,b,c,d,e Same superscript within a row means no significant difference (P>0.05) ND= Not detected; GABA = γ-Amino butyric acid; α- ABA= α-Amino butyric acid


92

GABA is a major constituent in higher plants and its physiological function in the plant is

suggested to be involved in pH regulation, nitrogen storage, plant development and defense, as

well as a compatible osmolyte and an alternative pathway for glutamate utilisation (Shelp et al.,

1999). GABA is a major inhibitory neurotransmitter in mammalian brain and alterations in

GABAergic function have been postulated to underlie seizure pathogenesis (Goldsmith et al.,

1990). Trigonelline, which is not an amino acid but a multifunctional natural plant hormone, was

also found in all the samples. The toxicological effects of trigonelline have not been studied but

considering its multiple effects in the plant, there is a need to study its potential effect on human

health (Rozan et al., 2000).

III.2.4 Conclusions

In konzo affected areas, cassava leaves can contribute to the total uptake of cyanide in the diet

besides the cassava roots. There is no electricity or gas available and in general cooking is done

in the evening when the mothers are tired after hard work and long walking from the field. The

availability of firewood, time to cook and duration of cooking can contribute as factors to higher

dietary exposure to cyanogens in those regions.

All the raw samples had high protein content and high essential amino acids compared to the

recommended FAO/ WHO pattern but limiting in sulphur amino acids, in lysine and leucine.

Cooking lowered the protein content of the raw pounded cassava leaves studied from 285.9 g kg-1

dry weight to 119.2 g kg-1 dry weight on average, but it is still relatively high comparing to other

vegetables. Quantitatively, the cooked cassava leaves can almost fulfil the recommended daily

protein intake (FAO/WHO/UNU, 1985). This can be illustrated by the comparison between the

average daily consumption of cassava leaves in DRC estimated at 500 g, thus an average of about

60 g of protein (from our samples), and the recommended safe level of daily protein intake in


93

terms of protein qualities which is 48 g or 62 g for food protein quality of score 0.6 for adult

woman and adult man, respectively (Lancaster, 1983; FAO/WHO/UNU, 1985). Unfortunately,

our results showed that the cooked-pounded cassava leaves were deficient in at least 3 essential

amino acids (sulphur amino acids, histidine and lysine) and thus of poor quality (Friedman,

1996). Therefore the consumption of cassava leaves as the main source of dietary protein cannot

compensate the methionine deficiency in konzo-affected areas where the dietary requirement for

methionine needs to be adjusted for the loss caused by cyanide detoxification (Diasolua Ngudi,

2002). This dietary methionine requirement may be further increased if the leaves are also not

properly cooked because of high level of cyanide in the fresh leaves (Lancaster, 1983). Cereals

and legumes should be promoted as sources of sulphur amino acids and lysine respectively to

improve protein quality of the diet of the poor population at risk for konzo and thus to prevent

konzo and malnutrition.

It has been suggested that the deficiency in sulphur amino acids in unbalanced diets could be a

contributing factor in the etiology of neuro-toxico-nutritional diseases such as konzo and

neurolathyrism (Lambein et al, 2001). Neither ODAP nor other known potentially toxic

nonprotein amino acid was detected in our samples. One major peak found in our samples with

elution time of 37.6 min in HPLC analysis and absorption maximum at 265.4 nm after PITC

derivatisation was not identified.

Better information and education especially of those preparing the food could be a relatively

cheap and sustainable intervention. Considering the level of socio-economic impact of such

diseases, such intervention would save resources to those communities.


94

CHAPTER IV

DIETARY CYANOGEN AND SULPHUR METABOLITES

EXCRETION*

* This chapter has been submitted for publication in Food and Chemical Toxicology as: Delphin Diasolua Ngudi, Yu – Haey Kuo, Fernand Lambein and Patrick Kolsteren. High risk of dietary cyanogen exposure in a population living in a konzo – affected area of Democratic Republic of Congo.


95

IV Dietary cyanogen and sulphur metabolites excretion

IV.1.1 Introduction

Urinary amino acid excretion is an important tool for the diagnosis and clinical management

of disturbances of amino acid metabolism. Common indications for urine amino acid testing

include clinical presentations such as neurological deterioration, hyperammonemia, kidney

stones, metabolic acidosis, failure to thrive, inborn errors of amino acid metabolism, etc

(Bezkorovainy & Rafelson, 1996, Venta, 2001). The alteration in urinary excretion is

principally a reflection of changes that occur in plasma amino acid composition since the

concentrations of the free amino acid in urine seem to be mainly related to protein intake

(Pavy et al, 1988, Brand et al. 1997).

Urinary thiocyanate is commonly used to check cyanogen overload in a population using

cassava roots and cassava products as staple food (Haque & Bradbury, 1999, Ernesto et al.

2002a). The level of thiocyanate normally present in body fluids is low but increases with

chronic exposure to cyanide and with smoking habits (Vesey et al. 1999, Kussendrager and

Van Hooijdonk, 2000). Thiocyanate remains the most useful chemical biomarker for dietary

cyanogen intake because it is a very stable metabolite that can be determined with relatively

cheap, specific and sensitive methods (Rosling, 1994, Ressler and Tatake, 2001).

Taurine (2-amino-ethyl sulphonic acid) is an ubiquitous free amino acid highly abundant in

excitable tissues, including the heart and brain. In addition to functioning as a

neuroprotectant, antioxidant, osmoregulator and Ca2+ modulator, taurine may function as an

inhibitory neuromodulator and neurotransmitter in the central nervous system. It is an end

product from the catabolism of sulphur amino acids methionine and cysteine, and it is

excreted almost entirely in urine (Laube et al. 2002, Olive, 2002, Hou et al. 2003). The


96

urinary levels of taurine have been proposed as a potential biochemical marker of total body

protein status or of sulphur amino acids catabolism (Waterfield et al. 1995, Hou et al. 2003).

In this study, we compared the level of total cyanogen in the sampled cassava flour to the

recommended FAO/ WHO safe limit. We measured urinary thiocyanate to check cyanogen

overload in the selected community. The potential relationship between urinary taurine and

urinary thiocyanate, biomarker of daily cyanogen, was assessed and cases of konzo were

detected.

IV.1.2 Material and methods

IV.1.2.1 Subjects

Samples of cassava flour and urine were obtained and examined from about one tenth of the

participants selected randomly in an epidemiological study (Chapter I) we carried out in

February 2003 in Popokabaka rural health zone (Prhz), province of Bandundu (1° - 8° South;

16° – 20° East), Democratic Republic of Congo (D. R. C.). Three health areas were chosen in

cooperation with the chief medical doctor and the nurse supervisor of Prhz based on the

number of reported konzo cases: Popo-secteur (low prevalence area), Mutsanga (medium or

moderate prevalence area) and Masina (high prevalence area). After informed oral consent,

forty two heads of household or their delegates (11 females and 31 males; age 46 ± 12 yr,

range 20 – 76 yrs) among the participants of the above mentioned study were randomly

selected to provide samples. After the interview, each selected participant received two empty

plastic vials; one to fill up with cassava flour of the evening meal and the other with the first

morning urine of the next day. Konzo affected-households were registered and the patients

were checked for confirmation. Filled vials were collected without addition of any

preservative early in the morning of next day. Twelve participants did not return the vial with

cassava flour because they either had no evening meal or did not prepare cassava flour for the


97

evening. The collected samples were transported from the field to the laboratory at 4° C.

Samples were stored at –20 °C until assayed.

IV.1.2.2 Analytical methods

IV.1.2.2.1 Total cyanogen in cassava flour

See chapter III 1.2.2

IV.1.2.2.2 Urine sample

Urinary thiocyanate

Protocol D1 of the picrate kit method developed by Haque & Bradbury (1999) was used to

determine thiocyanate in the urine. The thiocyanate content in ppm was calculated by the

equation:

thiocyanate content (ppm) = 78 x absorbance

The thiocyanate content in µmol/l was obtained by multiplying the thiocyanate content in

ppm by 17.2. Blank and controls were prepared as described using water instead of urine and

standard paper disc loaded with thiocyanate of 68.8 or 688 µmol/l solution (4 ppm or 40

ppm), respectively. The absorbance was measured at 510 nm, using a spectrophotometer

(Shidmazu, UV 1601).

Urinary taurine

Taurine was analysed by high performance liquid chromatography as described for the other

amino acids in chapter I.

IV.1.2.3 Statistics

The data showed skewed distributions, therefore median and intervals are presented as the

observed ranges of total cyanogens, urinary thiocyanate and taurine. The Spearman’s rho

statistics were used for bivariate correlations to measure the association between two


98

variables; correlation was significant at the 0.01 level or at the 0.05 level (two-tailed). Results

were computed using Microsoft Windows Excel 2003 and statistical analyses of the data were

carried out using the software package SPSS 11.5 for Windows.

IV.1.3 Results

Out of 42 participants, 21 % were living in a household affected by at least one konzo case

(Table IV-1). No konzo cases were reported in the low prevalence area while in other areas,

13 cases of konzo were reported, from which 9 patients were in the high prevalence area and

4 other patients in the moderate prevalence area. Confirmation of the diagnosis was done by

applying the WHO criteria for konzo (WHO, 1996): a visible symmetric spastic abnormality

when walking and/or running, a history of abrupt onset (< 1 week), a non- progressive course

in a formerly healthy person, showing bilaterally exaggerated knee and/or ankle jerks without

signs of spinal disease.

Table IV-1: Distribution of konzo- affected households in each health area with the number

of konzo patients given in brackets

Number of affected household

(Number of konzo patient)

Health area

1 2 3 Total

Low prevalence

Moderate prevalence

High prevalence

0

2 (2)

4 (4)

0

1 (2)

1 (2)

0

0

1 (3)

0

3 (4)

6 (9)

Total 6 (6) 2 (4) 1(3) 9 (13)

Cyanogen content in cassava flour, thiocyanate and taurine content in the urine samples are

summarised in Table IV-2. Cyanogens were not detected in 26.7 % of samples and 46.7 % of

cassava flour samples had total cyanogens below 10 ppm (µg HCN equivalent/g cassava


99

flour), the WHO/FAO recommended safe limit (FAO/ WHO, 1991). There is a large variation

in cyanogen content, values ranged from 2.90 to 169.75 ppm with a median of 16.50 ppm.

One sample had cyanogens content above 100 ppm. This highest concentration was found in

the low prevalence area where 60 % of cassava flour samples contained total cyanogens

within the WHO/FAO recommended safe limit. In the moderate area, only 22.2 % of cassava

samples had total cyanogens within the WHO/FAO recommended safe limit compare to 54.5

% found in the high prevalence area.

Table IV-2: Total cyanogens in cassava flour, thiocyanate and taurine in urine samples

collected in three konzo prevalence areas of Popokabaka (DRC).

Konzo prevalence area

Low Moderate High Total

Total Cyanogens

(µg HCN equivalent/ g cassava flour)

n

Median

Min

Max

10

19.00

9.11

169.75

9

22.44

2.90

62.83

11

10.69

4.75

54.12

30

16.50

2.90

169.75

Urinary thiocyanate (µmol/ l) n

Median

Min

Max

12

279.28

6.26

675.27

14

400.91

41.59

1037.06

16

287.10

21.02

1101.00

42

300.74

6.26

1101.00

Taurine (mmol/ mol creatinine) n

Median

Min

Max

12

6.47

0.00

23.90

14

4.29

0.00

97.59

16

13.57

0.00

41.55

42

8.84

0.00

97.59 The urinary thiocyanate content ranged from 6.26 to 1101 µmol/l urine. The lowest

concentration (6.26 µmol thiocyanate/l urine) was found in the low prevalence area while the

highest concentration (1101 µmol thiocyanate/l urine) was found in the high prevalence area

and was excreted by a participant from a konzo-affected household. 69 % of the urine samples

had thiocyanate content above 172 µmol/l urine (10 ppm) with 13.8 % of them above values


100

of 900 µmol. All the konzo-affected household participants of the moderate area excreted

high urine concentration of thiocyanate (more than 500 µmol thiocyanate/l urine), while in the

high prevalence area only one participant had excessively high values.

The urine concentrations of taurine were low. Except the one sample with the highest

concentration (97.59 mmol/mol creatinine), the taurine concentrations of all other samples

ranged between 0 (or not detected) to 41.55 mmol/mol creatinine. More than half (61.9 %) of

the urine samples were below the reference limits (13 to 534 mmol taurine/mol creatinine)

calculated by Venta (2001). The highest concentration of taurine was found in a sample from

the moderate prevalence area. Taurine was detected in only 83.3 % of urine samples among

which 71.4 % excreted thiocyanate above 10 ppm or 172 μmol/ l urine. Urinary taurine was

slightly or not correlated to urinary thiocyanate (R2= 0.017, P = 0.415).

IV.1.4 Discussion

The identification of cases of konzo in the moderate and high prevalence areas in the present

study shows that this crippling neurodegenerative disease is still occurring in this part of

Bandundu province from where the first cases were reported three generations ago by Trolli

(1938). The study found that a high proportion of cassava flour samples contain total

cyanogens above the recommended safe limit set at 10 µg HCN equivalent/g by the Codex

alimentarius (FAO/ WHO, 1991). Even when compared to the higher acceptable limit (40

ppm) used in Indonesia, that is one of the highest cassava producer and consumer countries

worldwide (Djazuli & Bradbury, 1999), 16.6 % of our cassava flour samples are still above

this limit. The values in this study were higher than the ones we previously reported (Diasolua

Ngudi et al. 2002) on the processed cassava roots available on the markets of Kinshasa, the

capital of D. R. C., where important quantities of cassava cossettes coming from the study

areas are sold. Therefore, there is a risk of dietary exposure to cyanogen from consumption of


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cassava flour in the areas studied. Essers et al. (1998) stated that the cyanogen content in

cassava flour in rural areas of Africa usually grossly exceeds the safety limit set by the Codex

alimentarius but toxic effects are rare under normal conditions. Partly because the cyanogens

are mainly bound in glucosides which are relatively stable in the human body, and the form in

which the product is consumed (stiff paste) causes a slow release of the toxicant which can

then be detoxified gradually and more effectively by the body’s defence mechanism. Konzo

has been reported to occur most frequently when the mean cyanide content of cassava flour

exceeds 100 ppm (Lawrence, 1999). Shortcuts in the processing of cassava roots have been

reported to result in high residual levels of cyanogen substances and the consumption of such

roots leads to dietary cyanogen exposure (Banea et al. 1992).

Cyanogen exposure from cassava roots is the essential risk factor for konzo and thiocyanate

levels remains the best indicator of daily cyanide intake (Rosling, 1994, Banea-Mayambu et

al. 2000). An additional source of dietary cyanogen exposure can come from consumption of

cassava leaves which is the main source of protein in a diet consisting of processed cassava

roots in those konzo-affected areas (Chapter III). The preparation of this vegetable requires

prolonged boiling (at least 30 minutes) with additional water and firewood in order to reduce

the cyanogen content. Consumption of cassava leaves inadequately prepared and long-term

smoke inhalation from the firewood, the only fuel available, may be additional factors in the

overall cyanogen exposure. Low availability of water and firewood might be considered as

additional risk factor for cyanide exposure (Chapter III).

Half of the urine samples analysed contained levels of thiocyanate above 300 µmol/l while

Tshala-Katumbay et al. (2001b) reported that 75 % of the urine samples contained

thiocyanate levels above 300 µmol/l in the same “high prevalence area” of this study.

Tylleskär et al. (1992) also reported a high thiocyanate excretion in this region. When

comparing the study areas, we found higher cyanogen content in cassava flour from the low


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prevalence area than in cassava flour from the other two areas. However, this cyanide content

in the flour reflects only the safety of the food and the potential human exposure to cyanide,

as the excretion of thiocyanate is higher in the urine from moderate and high prevalence areas

than in the urine from the low prevalence area. The most affected population might be better

aware of a further konzo attack and take more precaution on improving the processing of

cassava, the sole staple food consumed (Cardoso et al. 2004).

The proportion of thiocyanate formed from a cyanide load will decrease if the subject is

malnourished. The conversion of cyanide to thiocyanate implies a reaction with sulphur

originating from dietary sulphur amino acids in the presence of rhodanese (thiosulphate-

sulphurtransferase, EC 2.8.1.1) as catalyst. The rate of detoxification is therefore limited by

the supply of a sulphur donor. Addition of different condiments containing sulphur amino

acids in the cassava – based diet might also reduce the risk for konzo.

The concentrations of taurine in our samples were lower than those reported as reference

limits from urine samples of participants on a normal diet (Venta, 2001) and from female

college students of Japan (Nakamura et al. 2002). More than half (61.9 %) of the urine

samples were below the reference limits of 13 to 534 mmol/ mol creatinine set by Venta

(2001). The average excretion of taurine from our samples (0.11 ± 0.15 mmol/g creatinine)

was 7 fold lower than the average excretion of taurine (0.78 ± 0.53 mmol/g creatinine) from

urine of 58 female college students of Japan (Nakamura, 2002). This may reflect a low intake

of dietary sulphur containing amino acids in the study areas. Taurine as well as thiocyanate is

an end product of the catabolism of sulphur containing amino acids metabolism involving

methionine and cysteine. Low sulphur amino acid intake can lead to low excretion of taurine

and thiocyanate. However, in this study the two metabolites were found to be slightly or not

correlated. Production of thiocyanate may affect the quantity of taurine excreted. In vivo

production of taurine in rats has been observed to be reduced preferentially over sulphate


103

production when the supply of sulphur containing amino acids is limited (Tomozawa et al.

1998). Taurine is abundant in the brain where it has multiple functions as an anti-oxidant and

neuroprotectant. The physiological effects of taurine depletion from the brain are not well

documented.

It has been mentioned before that konzo occurs when the diet contains predominantly

insufficiently processed cassava (Tylleskär et al. 1992), which is also deficient in sulphur

containing amino acids (chapter III). Intake of sulphur amino acids from other components of

the diet needs to be known to allow correct evaluation.

In conclusion, this study reveals that konzo is still occurring in this area and that there is a risk

of dietary cyanogen exposure from cassava flour. Cassava flour samples from more than half

of the selected households contained total cyanogen above the WHO/ FAO recommended

safe limit. The urine analyses suggest an overload of cyanogen. The urine samples from more

than half of the participants excreted high amounts of thiocyanate and low amounts of taurine.

The low concentrations of taurine in the urine samples may suggest that more sulphur

metabolites be directed to detoxification of cyanide by formation of thiocyanate and can also

reflect the suboptimal intake of sulphur containing amino acids in the diet. No correlation was

found between taurine and thiocyanate; this might be due to the small number of urine

samples and the large variability in the data (taurine content of 61.9 % of the samples were

below the limit reference). More samples are needed to better evaluate the relationship

between urinary taurine and thiocyanate.

Food diversification and proper cassava processing combined with better and organised public

information can contribute to decrease the high dietary cyanogen exposure and the risk for

konzo. There is a need to adjust upwards the dietary requirements for sulphur amino acids to

compensate for the demand for cyanide detoxification in cassava consuming areas.


104

CHAPTER V

GENERAL DISCUSSION AND CONCLUSIONS


105

V General discussion and conclusions

This thesis summarises research on toxicological and dietary factors involved in konzo. The

investigations were observational and focussed on possible associations between the

occurrence of the disease and the exposure to cyanogen and the composition of the diet. Field

observations and measurements and laboratory analyses were done. Cross sectional and

ecological design were used to construct an updated epidemiological picture of differential

distribution of konzo among people with different risk profiles, to measure and to explore

both exposure and outcome. The design focussed on the incidence and distribution of the

disease in the community, and on the characteristics of the population groups rather than on

the individual members. Aggregate data based on surveys of groups of people were used to

assess diet-disease relationships. The unit in which the data were collected is the household.

Urine was collected on an individual basis. The household food consumption focussed on

nutritional aspects of the diet. Although individual based studies would allow a more direct

estimation of the risk of disease in relation to exposure, population based methods might

show that populations that have a higher exposure to cyanogen also have a higher rate of

konzo but it would not necessarily follow that konzo only occurred in areas with high dietary

exposure of the population to cyanogen.

Therefore, a limited community design was used to describe konzo and to identify possible

dietary and household associated factors. We subsequently sampled cassava foods and urine

to allow the evaluation of potential etiologic exposure as well as the interrelationships among

them.

Cases of konzo were unambiguously distinguished by their physical disability.

Misclassification was not an issue in our studies. High expert medical doctor and nurse, and

the author of this thesis confirmed, after examination, all konzo cases included and further

supervised the interviews conducted by trained enumerators. However since the studies rely


106

on information obtained from the heads of household, recall bias cannot entirely be excluded.

Furthermore, in a typical rural setting like that of the study area where most of the heads of

household have a low educational level and food intake is based mainly on cassava as staple

food, the use of a food frequency questionnaire was not applicable. A qualitative 24 hour

recall was used to assess food consumption. The lack of quantitative measurements is a

limitation of the work. Estimation of food portion sizes and measurement of food intake per

kg of individual body weight could allow a better toxicological evaluation. However, the

study subjects share the same geographical environment, ethnicity and culture and we have

validated the data through repeated observation, using a set of open questions format to

investigate the 24 hour and seasonal food recall.

The studies in this thesis addressed new areas in konzo research ranging from the

identification of household factors to dietary risk and protective factors. The identification of

this wide array of associated factors led us to suggest possible measures to prevent the disease

and to recommend new directions for further research. We can not claim geographical

representation since we did not cover all konzo prone areas of the D. R. C. Underestimation

of the magnitude of the konzo problem is possible. The numbers of konzo cases in the study

area represent about 4.3 ‰ of the total number of cases in D. R. C., roughly estimated at

100,000 (R. D. C., 2000)

V.1 Occurrence of konzo

Although most of the cases reported in chapter 3 occurred in the 1990s. At present, konzo, a

preventable disease, is still occurring in Popokabaka Rural Health Zone areas, one of the

regions where cases were described in the first report on konzo in 1938 (Trolli, 1938). The

clinical picture of the affected subjects in our studies was similar to those previously

described in the same region (Trolli, 1938; Tylleskär et al 1992; Banea-Mayambu et al 2000;

Tshala-Katumbay et al, 2001; Bonmarin et al., 2002). The main symptom was non-


107

progressive symmetrical spastic paraparesis (paralysis of both legs) with sudden onset. Konzo

was already described in other parts of D. R. C. and in other sub-Saharan African countries

such as Mozambique, Tanzania, Angola, the Republic of Central Africa and Cameroon.

However, konzo does not occur in all regions with cassava-based diets even in the same

country. No konzo case has been reported in Latin America from where cassava originated

and is consumed as staple food neither in Nigeria, the big producer and per capita consumer

of cassava nor in Indonesia and Thailand where cassava is a popular food. Therefore, high

consumption of cassava root by itself is not the only or exclusive cause for konzo (Tylleskär,

1994c). The occurrence of konzo in a community reflects a deterioration of socio-economic

conditions and those who are affected by konzo are trapped in a spiral of poverty, and

educational and political neglect. Poverty and lack of education affect people’s capacity to

prevent the disease as well as their ability to live in areas having less exposure to this risk.

There is a relationship between poverty (socio-economic as well as educational) and

vulnerability, and they are mutually reinforcing.

Age and sex distributions of konzo affected subjects in our study were similar to most of the

previous studies in the same region and elsewhere (Howlett, 1994; Banea-Mayambu, 1997,

Tshala-Katumbay et al, 2001b). Children less than 14 years and women at childbearing age

are particularly susceptible to konzo. In Chapter 4, our studies compared the expected daily

intake of the sulphur amino acids methionine and cysteine provided by cassava cossettes

consumption with the suggested essential amino acid requirement, and we concluded that

children of 1 to 9 years old cannot expect to meet methionine requirement whereas adults can

meet the minimal requirement for these sulphur amino acids. No child under two years was

affected by konzo. In Bandundu province, breastfeeding is general at birth and this practice

decreases slowly until two years old: 98.6 % of children aged from 12 to 15 month and 62.7

% of those aged from 20 to 23 month old are still breastfed (R. D. C., 2001). Mother milk is


108

rich in amino acids containing sulphur. The dominance of female patients (female to male

ratio 3.3: 1) in our studies is similar to almost all other studies except the ones carried out in

Tanzania and Mozambique where male cases were preponderant (Howlett, 1994; Tshala-

Katumbay et al, 2001b, Bonmarin et al, 2002). The absence of a suitable animal model for

konzo makes it difficult to explain or to study the reasons for the high susceptibility of

females at reproductive age for konzo. The female hormones, especially 17 β-oestrogen have

been proposed as protective factor for neurolathyrism to explain the high susceptibility for

young men, but can these hormones be an aggravating factor for konzo (Lambein et al,

2004)? Rural depopulation resulting in migration of active males to bigger cities looking for

“welfare” and the primary socio-economic role played by women in the household food

security may expose women to higher workload and higher oxidative stress.

The socio-economic crisis that affected D. R. C. for several years has probably had a major

impact in this particular region and led to the persistent occurrence of konzo during several

generations. The socio-economic burden of this crippling disease on this impoverished region

is heavy (Bonmarin et al, 2002). Additional stresses such as military activity, political

conflicts and drought-provoked food shortage have been identified as factors leading to

inadequate diets which triggered konzo epidemics (Essers, 1995).

V.2 Cassava foods and sulphur metabolites

Cassava flour mixed or stirred in boiling water to obtain a stiff porridge, the so called “fufu”

or “luku” is the main daily staple food for almost all the households studied (Chapter 2).

Cassava flour is derived from the roots which in normal conditions are soaked (retted) for at

least three nights, then sun dried for 3 to 5 days, then pounded and finally sieved (Chapter 3-

1). Cassava flour is an excellent source of carbohydrate (Bradbury and Holloway, 1988).

However, we find that the protein of cassava roots is of poor quality, leucine and lysine are

limiting amino acids and also the proportion of methionine is low, giving a chemical score of


109

the protein of around 40 (Chapter 3 - 1). Our studies (Chapter 4) also reveal that more than

half of the households were soaking cassava roots for less than three nights. As a

consequence, a high proportion of households had cassava flour containing total cyanogens

above the recommended safe limit set at 10 µg HCN equivalent/g by the Codex alimentarius

(FAO/ WHO, 1991). High cyanogen exposure from frequent and exclusive consumption of

insufficiently processed cassava roots is thought to be a major etiological factor in konzo.

In this region, saka-saka (pounded cassava leaves) constitutes the main condiment consumed

as side-dish with luku (Chapter 2). Although quantitatively the main source of protein in the

diet, this protein is also of poor quality with sulphur amino acids as the most limiting amino

acids (Chapter 3-2). Cassava leaves were also found to be a potential additional source of

dietary exposure to cyanogens, apart from the cassava roots. The cooking of saka-saka

requires prolonged boiling (at least 30 minutes) with additional water and firewood in order to

reduce the cyanogen content but the supply of both water and firewood is limited (Chapter 3-

2). There is no water source in the villages, neither is there electricity nor gas. Sources of

water are located at least at 15 minutes walking (RDC, 2001) and cooking is done exclusively

with firewood which nowadays is becoming scarce in this savannah area.

No potentially toxic nonprotein amino acid was detected in cassava roots as well as in cassava

leaves (Chapter 3). Neurolathyrism, which shares clinical similarities with konzo, has been

associated with β-ODAP, a neuro-excitatory nonprotein amino acid present in the grass pea

(Lathyrus sativus) (Getahun et al, 1999).

High thiocyanate content was found in more than half of the urine samples analysed. This

suggested a high exposure to cyanide (Chapter 4). Luku and saka-saka are the known and the

main sources of dietary cyanide, but smoke inhalation from wood fires inside the primitive

housing and probably also inhalation of HCN escaped during soaking and sun drying of

cassava roots might be considered as additional sources of cyanide exposure that need further


110

study. The conversion of cyanide to thiocyanate in the human body requires sulphur

originating from dietary sulphur amino acids. The rate of detoxification is therefore limited by

the supply of a sulphur donor. When the body is regularly exposed to cyanogens, the

increased synthesis of rhodanese, enzyme responsible for cyanide detoxification in the human

body by forming thiocyanate, makes extra demands on the body's reserves of sulphur amino

acids. If this demand is prolonged as in the regular consumption of cassava insufficiently

processed, and the diet is inadequate, the synthesis of taurine may be impaired (Chapter 4).

Taurine and thiocyanate are excreted in the urine as end products of the catabolism of sulphur

containing amino acids methionine and cysteine. Production of thiocyanate may affect the

quantity of taurine excreted. In the case when insufficiently processed cassava is consumed as

staple food, the low methionine content may aggravate the risk for cyanide toxicity and konzo

disease. Moreover, the consumption of cassava leaves as the main source of dietary protein

can not compensate for the methionine deficiency in konzo-affected areas where the dietary

requirement for methionine needs to be adjusted for the loss caused by cyanide detoxification

(Chapter 4).

This dietary imbalance can be corrected if other components of the diet contribute to a better

balanced amino acid composition, especially the level of sulphur amino acids. Consumption

of cereals and sesame is found in Chapter 2 to be protective factors against konzo. Similar

protection by methionine rich cereals was also found for neurolathyrism (Getahun et al,

2003). In the regions neighbouring the konzo-affected areas in Bandundu, where traditionally

corn or millet flour is mixed with cassava as staple food, no cases of konzo have been

reported. This may corroborate our views as to the importance of methionine for a healthier

balanced diet.


111

V.3 Conclusions and recommendations

The high urinary thiocyanate levels found in our studies show that the population of

Popokabaka is still highly exposed to dietary and perhaps environmental cyanogens and to the

risk of konzo. A better balanced diet, especially richer in methionine is required to allow

efficient detoxification of cyanide in the body. Therefore, we recommend:

a) Supplementation of methionine to contribute to the detoxification of cyanide should

be done in parallel with the promotion of consumption of foods rich in methionine

locally available (cereals, sesame, soybean, pumpkin seeds, eggs, meat, etc). A study

of the effect of methionine administered on urinary levels of taurine and on

thiocyanate can help to better understand the role of taurine, as an antioxidant, a

neuroprotectant, and an inhibitory neuromodulator in the central nervous system, and

the relationship between taurine and thiocyanate in konzo.

b) Promotion of safe cassava processing to reduce significantly dietary cyanogen

exposure. Sufficient soaking (retting) combined with sun drying has been proven to be

effective in reducing cyanogen. Organised public information should be promoted.

Attention should be drawn on the duration of cassava processing especially during the

dry season which is the period of low ambient daily temperature and low water

supply. Days are cloudy and the temperatures are below 20°C; HCN is evaporated

more slowly at lower temperature as the boiling point of HCN is 25.7°C, thus removal

of HCN is not optimal during the cold dry season. The dry season is reported to be the

period of high incidence of konzo. We suggest the increase of the period of soaking

and drying during this season (at least 5 days each).

c) Promotion of consumption of luku obtained from the mixing of cassava with maize

flour or other cereals. Maize is locally available but pounding by hand to transform

grain to flour seems to be hard for the population. Therefore, an adapted milling


112

technology should be implemented. Better information on the benefit of mixing

cassava and maize or other cereals should be promoted.

d) Food diversification should be promoted especially during the dry season. Nutritional

resources are scarce during the dry season. Almost all crops are rain-fed and cannot

survive during the dry season. Watering or an irrigation system is not practicable in

this area because of absence of inputs and of major river systems for irrigation. Roads

should be better maintained to open the region to the market.

Konzo is a very much neglected disease in D. R. C., where political instability and military

activity are factors attracting the international attention. The patients suffering from this

incurable disease become a socio-economic liability for their family. Prevention of this

disease and its dramatic socio-economic consequences can be attained by simple education of

the basics of nutrition and the supply of the simple means to produce a more varied diet. A

national campaign should be organised to identify isolated pockets of konzo affected

communities and to distribute preventive information.


113

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CURRICULUM VITAE

Curriculum Vitae

Identity

Full name: Delphin DIASOLUA NGUDI

Place and date of birth: Kinshasa, May 2, 1961 Sex: Male

Nationality: Congolese (Democratic Republic of Congo)

Marital status: Married and 3 sons

Permanent address: Inga 57 Quartier 10, N’djilj/ Kinshasa, D R Congo

Present address: Filips Van Cleeflaan 386, 9000 Gent, Belgium

Email address: [email protected]

Education

Master of Science in Food Science and Technology, Universiteit Gent/ Katholieke

Universiteit Leuven, Belgium, 1999

Masters Thesis: Effect of sprouting and lactic acid fermentation on protein in finger millet

(Eleusine Coracan) and kidney beans (Phaseolus vulgaris

Promoter: Prof Dr Ir André Huyghebaert and Dr John Van Camp

Degree in Complementary Studies in Nutrition and Food Science, Universiteit Gent,

Belgium, 1997

Thematic study: Mise en place des activités de surveillance nutritionnelle à Kinshasa

Graduate in Medical Techniques: Nutrition and dietetics, Institut Supérieur de Techniques

Médicales, Kinshasa, R. D. Congo, 1983

Thematic study: Evaluation du critère de prise de poids ou du périmètre brachial pour la

récupération des enfants malnourris.

iii

Additional Training

Summer course on “biosafety assessment and regulation of agricultural biotechnology”,

Plant Biotechnology Institute for Developing Countries (IPBO), Ghent university,

Belgium, 2004.

International Training Course in Dairy Technology: “Dairy technology from rural to

industrial level”, B.A.D.C./ Universiteit Gent, Belgium, 1998

Socrates Intensive Course: Food Packaging, European Union/ Universiteit Gent, Belgium,

1998

International Training in Nutrition and Food Science, Target Program on food security:”

Micronutrients deficiencies, Université de Benin, Cotonou, Bénin,1994

Training in writing of health education school manuals, UNICEF/ Ministry of Primary

and Secondary Education, Kisantu, R. D. Congo, 1991

Training of Health Community Workers Trainers, (U.S.A.I.D./ SANRU Project),

Kimpese, R .D. Congo, 1985

Employement record

• 2000 to date: Research on cassava and konzo at Universiteit Gent, Belgium

• 1984 to date: Nutritionist and Food quality control Officer at the National Program of

Nutrition (PRONANUT/ D R. Congo) (former CEPLANUT)

• 1992 to 1996: Collaborating Assistant to the Director of CEPLANUT and Member of

the Technical Committee

• 1993 to 1995: Technical Assistant to the project TCP/ ZAI/ 2355(A) – FAO/

CEPLANUT) “Food vended street”

• 1989 to 1992: Assistant to the Activity coordinator of CEPLANUT Regional Office

of Bandundu Region in Kikwit

iv

• 1984 to 1989: Head of the Nutrition Education section and Contact for the regional

council for food and nutrition of Bandundu Province (CRANB), project

660-079 ( United States Agency for International .Development

(USAID) / CEPLANUT)

International meeting Attendance

The first International Conference on Food Systems, college of Food systems, United

Emirates University, Al- Ain (United Arab Emirates), October 19 – 21, 2003

World Health Organization (WHO) AFRO Regional awareness raising workshop on food

safety evaluation, Bamako, (Mali), December 4 – 6, 2002.

International Food Policy Research Institute (IFPRI) 2020 vision: Sustainable Food

Security for All by 2020, Bonn (Germany), September 4-6, 2001 (Participation sponsored

by GTZ- Echborn)

Publications

1. Diasolua Ngudi D., Kuo Y.H., Lambein F.: Cassava cyanogens and free amino acids

in raw and cooked leaves. Food and Chemical Toxicology 2003, 41, 1193-1197.

2. Diasolua Ngudi D., Kuo Y.H., Lambein F.: Amino acid profiles and protein quality of

cooked cassava leaves 'saka saka'. Journal of the Science of Food and Agriculture.

2003, 83, 529-534.

3. Diasolua Ngudi D., Kuo Y.-H., Lambein F. Cassava leaves, a non-negligible source of

dietary exposure to cyanogens. Cassava Cyanide Diseases Network (CCDN) News

2003, 2, 1-2.

4. Diasolua Ngudi D., Kuo Y.-H., Lambein F. : Food Safety and Amino Acid Balance in

Processed Cassava "cossettes" Journal of Agricultural and Food Chemistry 2002, 50,

3042-3049.

v

5. Lambein F., Diasolua Ngudi D., Kuo Y.-H.: Vapniarca revisited: Lessons from an

inhuman human experience. Lathyrus lathyrism newsletter 2001, 2, 5-7.(website URL:

http://go.to/lathyrus).

6. Mbithi-Mwikya S., Ooghe W., Van Camp, J., Ngudi, D., Huyghebaert A.: Amino acid

profiles after sprouting, autoclaving, and lactic acid fermentation of finger millet

(Eleusine coracan) and kidney beans (Phaseolus vulgaris L). J. Agr.Food Chem. 2000,

48, 3081- 3085.

7. Diasolua Ngudi D., Kuo Y.-H., Lambein F., Patrick Kolsteren. High risk of dietary

cyanogen exposure in a population living in a konzo – affected area of Democratic

Republic of Congo. Food and Chemical Toxicology (submitted for publication)

8. Diasolua Ngudi D., Banea-Mayambu J.-.P., Lambein F., Kolsteren P.:.Crippling konzo

in DRC, three generations later. The Lancet (submitted for publication)

Memberships

Member and country contact of cassava cyanide diseases network (CCDN)

Member of the Congolese Nutritionists and Dieticians Association and former Secretary

of the Council (1995-1996)

Member of Science Press, vzw

Our web page: http://www.sciencepress.be

Visit web from Delphin

ISBN 90-5989-073-6

Documents

KONZO AND CASSAVA TOXICITY - stuba.skszolcsanyi/education/files/Organicka chemia II... · I thank Dr Yu-Haey Kuo (Dianna) for the initiation to the use of HPLC instruments and for