SANGUINARINE IN THE BLOOD AND URINE CASES OF EPIDEMIC DROPSY

8/9/2019 SANGUINARINE IN THE BLOOD AND URINE CASES OF EPIDEMIC DROPSY

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Fd Cosmet. Toxicol.Vol. 12 pp. 69%702. Pergamon Press 1974. Printed n Great Britain

S H O R T P A P E R

SANGUINARINE IN THE BLOOD AND URINECASES OF EPIDEMIC DROPSY

I. S. SHENOLIKAR, C. RUKMINI, K. A. V. R. KRISHNAMACHARIand K. SATYANARAYANA

National Institute of Nutrition. Indian Council of Medical Research,H yderabad-500007, India(Received 7 January 1974)

O F

Summary --Duri ng a recent outbreak of epidemic dropsy at Sirpur-Kagaznagar, Andhra Pradesh,samples of blood and urine were obtained from patients severely affected with symptoms of th edisease and oil samples were collected from their households. Screening of these samples for thepresence of sanguinarine indicated that all the household samples of oil, all but one of the 22 ran-dom urine samples and two of the 22 blood samples taken were positive. The two blood samplescontained 4.7 and 28.3/~g sanguinarine/100 ml serum. The levels of sanguinarine found in the edibleoils and the finding that pure argemone oil contained 5.4 mg sanguinarine/ml together indicatedthat the degree of adulteration of the edible oil samples with argemone oil ranged from 0.16 to2 " 2 7 o .

IntroductionThere have been seve ra l ou tb reak s o f ep idem ic d ropsy in Ind i a s ince 1877 and va r ious

repor ts have deal t in de ta i l wi th the epidemiologica l and c l in ica l fea tures of the d isease .T h a t e p i d e m i c d r o p s y i s c a u s e d b y t h e c o n s u m p t i o n o f a r g e m o n e o il p r e s e n t a s a n a d u l te r -an t i n mus t a rd o i l has been c lea r ly docum en ted (La l & Das G up ta , 1941), and sangu ina r -ine , the a lka lo id present in argemone o i l , has been incr iminated as the causat ive agent forthe d isease (Sarkar , 1948) . Ho w ever , no in form at ion i s so far avai lable regarding the pres-ence o f s angu ina r ine i n t he c ir cu l a t ion o r u r ine o f pa t ien t s su f fe r ing f rom ep idemic d ropsy .Th i s pape r p re sen t s t he r e su l ts o f ana lyses o f s amples o f s e rum a nd u r ine ob ta ined f rompa t i en t s s eve re ly a f f ect ed wi th ep idemic d ro psy in t he r ecen t ep idemic a t S i rpu r -K agaz na -ga r , Andhra P radesh . O the r de t a i l s r ega rd ing the ep idemic have been r epor t ed e l s ewhere(Kr i shnamacha r i & Sa tyana rayana , 1972) .Experimental

Materials. Pa i r ed se rum and u r ine samples were ob ta ine d f rom 22 pa t i en t s su f fe r ingf rom ep idemic d ropsy . B lood samples were t aken unde r f a s t i ng cond i t i ons and the se rumwas sepa ra t ed . Ran do m samples o f u r ine were ob ta ined , s ince t he co l l ec ti on o f 24 -h r s am-ples wa s impract ica l in the s i tua t ion exis t ing a t the s i te of the epidemic . In addi t ion , n inesamples o f ed ib l e o i ls were ob ta ined f rom h ouse ho lds i n wh ich the re were pa t i en t s suf fe r-ing f rom ep idemic d ropsy , and th ree fu r the r o i l s amples were ob ta ined f rom sea l ed t i n sob ta ined f rom d i f fe r en t shops a t Ka gaznag a r t ow ards t he end o f t he ep idemic .

699


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700 I.S. SHENOLIKAR,C. RUKMINI,K. A. V. R. KRISHNAMACHARIand K. SATYANARAYANAAnalytical procedures. Sanguinarine was determined in all samples of oil, serum and

urine, but the quantitative estimation of sanguinarine in the random urine samples wasconsidered to have little significance.

Urine was concentrated to a convenient volume on a water bath, and all samples ofurine and serum were deproteinized by the addition of trichloroacetic acid. An equalvolume of conc. HC1 was added to the supernatant solution, and the mixture was evapor-ated to dryness. The residue was extracted repeatedly with 1~o acetic acid in chloroformand the solution was made up to a known volume. All or part of this chloroform extractwas used for the detection and/or estimation of sanguinarine.Sanguinarine was detected by the procedure of Chakravarti, Choudhuri, Das Gupta &Gita Datta (1959), suitably modified for thin-layer instead of paper chromatography andusing the solvent system used by Hakim, Mijovic & Walker (1961). Quantitative deter-mination of sanguinarine in the collected samples, and in samples of pure argemone oilexpelled from the seeds in the laboratory, was carried out by the method of S. Babu, G.S. Thapar, I. S. Shenolikar & B. V. Rama Sastri (1972, to be published). For the oil analy-ses, 1 ml conc. HC1 (AR), 0'5 ml ethanol and 2 ml of the oil sample in a stoppered testtube were shaken well for 2 min and then heated on a water-bath for 5-10 rain. Afterfiltration through a wet filter-paper, the filtrate was dried by evaporation on a water-bathand the residue was taken up in chloroform containing 1~o glacial acetic acid. This chloro-form solution and the chloroform extracts prepared from urine and sera as describedabove were spotted, along with pure standard sanguinarine, on thin-layer chroma-tographic plates and developed in butanol-acetic acid-water (63: 10:27, by vol.). Plateswere viewed under an ultraviolet lamp and the golden-yellow fluorescent band at R v 0"45,which was in line with the standard sanguinarine spot, was cut out. A similar orange fluor-escent spot (dihydrosanguinarine) at the solvent front was also cut out. The pooled silicapowder was eluted with hot ethanol and made up to a known volume. The fluorescencewas read in a spectrofluorimeter at excitation maximum 335 nm and emission maximum490 nm. A standard curve was calibrated with standard sanguinarine and the levels in theunknown samples were read off from the standard curve.Resul ts and D iscuss ion

The results of screening oil, urine and serum samples for the presence of sanguinarineare summarized in Table 1. All the oil samples obtained from households in which therewere severe cases of dropsy contained detectable amounts of sanguinarine, although thesamples obtained in sealed tins from various shops gave negative results. All the urine sam-ples except one, but only two of the 22 serum samples contained detectable sanguinarine.

The extent of argemone oil adul teration in these edible oil samples was assessed fromtheir sanguinarine content. For this purpose, a reference standard of a pure sample of arge-mone oil expressed in the laboratory from argemone seeds was used. Genuine argemoneoil was found to contain 5.4 mg sanguinarine/ml. Based on this figure, the argemone oilcontent of the edible oil samples analysed in this investigation ranged between 0.16 and2"20~o. Since the exact amount of oil consumed by the patients suffering from epidemicdropsy could not be ascertained, no correlation between the consumption of the contami-nated oil and the severity of the disease could be obtained. From the oil consumption pat-tern in the epidemic (Krishnamachari & Satyanarayana, 1972), it could be surmised thata daily intake of argemone oil as low as 0.02 ml or an intake of 108/~g sanguinarine couldlead to the development of clinical manifestations. However, pure sanguinarine dissolved


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Sanguinarine in epidemic dropsy 701Table 1. Sanguinarine analyses o f samples of edible oils and o f serum and urine fro m patients with epidemic dropsy

Total no. of samples Sanguinarine levelsMaterial Containing foundtested Screened sanguinarine (#g/100 ml)Edible oil 9 9 864-11880Serum 22 2* 4-7 & 28.3Urine 22 21" + to + + + +?*Limit of detection > 0.5/~g/100 ml.+,Values in random urine samples ranging from approximate ly 0.5/~g/100 ml (+ ) to >2.0/~g/100 ml (+ + + +).

either in peanut oil or in ethylene glycol produced no signs of epidemic dropsy when in-jected into rats in a dose of 25 mg/kg body weight (equivalent to 5 ml argemone oil/kg)or given orally to monkeys in a dose of 1-6 mg/kg body weight (equivalent to 0"3 ml arge-mone oil/kg), although argemone oil containing similar amounts of sanguinarine did. Thisobservation is in accordance with that reported by Lal, Das Gupta, Agarwala & Adak(1941) and suggests that in addition to the toxic alkaloid, sanguinarine, the presence ofsome other factor(s) in argemone oil is necessary to produce the disease (Rukmini, 1971).Identification of such factor(s) and the mode of action are now under investigation.

The two blood samples giving a positive result were found to contain 4.7 and 28.3 #gsanguinarine/100 ml serum and were obtained from two subjects who were severelyaffected. The other blood samples contained no detectable sanguinarine. However, theurine of all except one of the patients contained this alkaloid. Earlier it was observed thatthe minimum level of sanguinarine visually detectable by the procedure employed here was10.8 ng/2 ml urine. Sanguinarine may therefore have been present at levels below the de-tectable 11 ng/2 ml in the serum samples from patients showing detectable levels in theurine. A lack of correlation between the presence of detectable levels of sanguinarine inthe serum and high levels in the urine may have been due to the fact that urine analyseswere limited to random samples. It is possible that some correlation might have beenfound if 24-hr urine samples could have been collected.

These data indicate that although no direct correlation between the serum levels of san-guinarine and the severity of epidemic dropsy could be shown, detection of sanguinarinein random samples of urine from affected persons could be used as a biochemical indexto assess the adulteration of edible oils with argemone oil in communities where the dis-ease occurs. It has been emphasized (Krishnamachari & Satyanarayana, 1972) that in thisepidemic the adulteration of the edible oils with argemone oil was deliberate. This wasclearly the case, since the oils consumed were peanut oil and sesame oil, the seeds of whichdo not resemble argemone seeds.A c k n o w l e d g e m e n t - - W e are grateful to Dr. C. Gopalan, Director, National Institute of Nutrition, for his keen inter-est in this investigation.

R E F E R E N C E SCbakravarti, R. N., Choudhuri, K. N., Das Gupta, B. & Gita Datta, A. (1959). Detection of traces of argemoneoil in mustard oil, amino test. J. Proc. Inst. Chem. G t Br. 31, 118.Hakim, S. A. E., Mijovi6, Valerie & Walker, J. (1961). Distribution of certain poppy-Fumaria alkaloids and apossible link with the incidence of glauc0ma. Nature, Lond. 189, 198.Krishnamachari, K. A. V. R. & Satyanarayana, K. (1972). Epidemic dropsy in Andhr a Pradesh. Indian J. reed.Res. 60, 741.


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702 I. S . SHENOLIKAR, C. RUKM INI, K. A. V. R. KRISHNAMACHARIand K. SATYANARAYANAL al , R . B. & Das Gu p ta , A . C . (1941) . Inves t iga t ion s in to the ep idem io logy o f ep idemic d rops y . P a r t X . A no teo n a n o u t b r e a k o f e p i d e m i c d r o p s y a s s o c i a t ed w i t h t h e u s e o f m u s t a r d o i l p r e s s e d f r o m t h e s e e d s a d u l te r a t e d .

lnd ian J . reed . Res . 29, 157.Lal , R. B., Da s Gu pta , A. C. , Agarwala~ S. P. & A dak, B. (1941). Inv es t ig a t ion s in to the epid em iolog y of epidem icdrops y . P a r t XI I I . App l i ca t ion o f b io log ica l t e s t to . l n d i a n J . r e e d . R e s . 29, 813.Rukm in i , C . (1971) . Sang u ina r ine po ten t i a t ing fac to r in a rgem one o i l . Ind ian J . reed . Res . 59, 1676.Sa rka r , S . N . (1948) . I s o la t ion f rom a rgemone o i l o f d ihydros angu ina r ine and s angu ina r ine : T ox ic i ty o f s an -gu ina r ine . N a t u r e , L o n d . 162, 265.

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SANGUINARINE IN THE BLOOD AND URINE CASES OF EPIDEMIC DROPSY