“INCREASED

PROTHROMBIN, APOLIPOPROT

EIN A-IV, AND HAPTOGLOBIN IN

THE CEREBROESPIAL FLUID OF

PATIENTS WITH HUNTINGTON´S

DISEASE.”Yen-Chu Huang.

Maria Camila Montufar Pantoja

Heyly Milena Ortega

III SEMESTER

2011

UNIVERSIDAD PONTIFICIA BOLIVARIANA

GENERAL OBJETIVE

Demonstrate how Prothrombin,

Apolipoprotein A-IV and Haptoglobin

concentrations in CSF has an important

role in the HD evolution and

compare these concentrations in HD and

control patients .

INTRODUCTION

HD is a hereditary, degenerative brain disorder. It

is caused by an unstable CAG trinucleotide.

Today there are others proteins involved in HD

development and symptoms (Apolopoprotein A-

IV, Haptoglobin , Prothrombin) and its important to

clarify through proteomics studies in CSF wich

are the concentrations (up or down) and if this

levels of biomarkers are correlated in HD.

PROTHROMBIN

Plasma protein synthesized

in the liver.

It plays an important function in

coagulation process by

reaction with the thromboplastin

enzyme , with Ca ++ cation and

V activate factor as coenzymes.

In HD prothrombin

proinflammatory agent : DISEASE

STATUS BIOMARKER

Tromboplastina

Ca++

Xa

Va

APOLIPOPROTEIN AIV (APO

AIV)

glycoprotein synthesized by the human intestine and

liver

The formation of chylomicrons acts as a signal for the

induction of Apo AIV synthesis

alters the activity of the enzymes (LPL and LCAT) of

lipoprotein metabolism and cholesterol efflux from

extrahepatic tissues

Is involved in the long-term regulation of both food

intake and body weight

HAPTOGLOBIN

protein produced by the liver

haptoglobin binds free hemoglobin (Hb) released from

erythrocytes with high affinity and thereby inhibits its

oxidative activity

The haptoglobin-hemoglobin complex will then be

removed by the reticuloendothelial system

It prevents loss of iron through the kidneys and

protecting the kidneys from damage by hemoglobin

ENFERMEDAD DE HUNITNGTON

- HD

Progressive neurodegener

ative

disorder with autosomal

dominant inheritance:

- Movement Disorders.

- Cognitive impairment.

- Psychiatric disorders.

MATERIALES Y

METODOS

LCR y suero extraido de 9 HD ( 5 hombres- 4

mujeres).

LCR y suero de 18 pacientes tipo control (7

hombres- 11 mujeres).

Cuatro grupos:

- 1 Electroforesis de dos dimensiones.

- 3 Cuantificacion de protrombina, Apo-A IV y

haptoglobina.

PREPARACION DE LAS

MUESTRAS

Muestras de LCR• Centrifugados.

• Almacenados a -80°

Muestras de Suero

• Almacenados a 4°c durante 1 hora.

• Centrifugados.

• Almacenados a -80°c.

ELECTROFORESIS DE DOS

DIMENSIONES (2-DE )

Técnica de alta resolución cuyo

objetivo es la separación de mezclas

de proteínas altamente complejas.

las proteínas son separadas por dos

criterios físicos.

- Punto isoeléctrico

(isoelectroenfoque).

- Masa molecular

por electroforesis discontinua en gel

de poliacrilamida en presencia de

SDS (SDS-PAGE).

DIGESTION EL GEL

Forma parte de la preparación de las muestras para

la espectrometría de masas, consta de cuatro

pasos:

- Decoloración.

- Reducción y alquilación.

- Corte de la proteína serina proteasa tripsina

- Extracción del péptido generado.

ESPECTROMETRIA DE MASA

técnica experimental que permite la medición de iones derivados de

moléculas

se basa en el fenómeno conocido como desbastado (sputtering,

en inglés) de partículas centradas en un blanco, que son

bombardeadas por iones, átomos o moléculas. Dependiendo del

intervalo de energía de la partícula primaria, ocurren colisiones.

el patrón de fragmentación resultante así como los

iones residuales constituyen el espectro de masas

WESTERN BLOT

Busco proteínas

especificas

Uso ac

Uso técnica SDS-

PAGE

Separación de

proteínas por

electroforesis en gel

ELISA

Ensayo por inmunoabsorción ligado a enzimas

Fosfatasa alcalina—peroxidasa de rabano

Detección ag-ac

3 tipos:

Ensayo tipo sandwich

Ensayo competitivo

Ensayo indirecto

RESULTADOS

Representative Western blots of prothrombin (72 kD)and albumin (64 kD) in CSF and serum of HD patientsand their corresponding controls.

Representative Western blots of Apo A-IV (46 kD) and

albumin (64 kD) in CSF and serum, respectively, of HD

patients and their corresponding controls (C).

RESULTADOS

Qalb, CSF haptoglobin, and serum haptoglobin

concentration in HD patients.

RESULTADOS

DISCUSSION

AUTOR OPINION YES NO

Bjorkqvist M.

The mutant huntingtin

may enchance the ability

of microglia to produce

proinflamatory mediators

(IL-6, IL-8 and TNF-a)

and contribute to

neurodegeneration in

HD.

Djoussé L.

In HD, weigth loss is a

characteristic feature in

advanced as well early

stage.

Fang Q, Strand In proteome

analyses several the

neuronal proteins

were found to be

reduced

Underwood BR A serum metabolic

profile indicative of a

pro-catabolic

phenotype in early

HD

CONCLUTIONS

The proteomic studies in CFS in patients HD andcontrol patients are very important to determinateif the increase levels of Haptoglobin,Apolipoprotein A-IV and Prothrombin arecorrelated with HD development.

It is important to recognize this proteins likespecifics biomarkers in HD; thats why theconcentrations of each one were quantified inCSF and serum; thus indirectly evaluate the BBBintegrity.

CONCLUTIONS

To be able to correlate the levels of these proteins with other mental

diseases such as Alzheimer's, multiple sclerosis, among others; allow

us to determine whether the activity of proteins mentioned in the study

are a common phenomenon in a variety of neurodegenerative-tive

disease or are specifically for HD.

Becouse of the intact values of BBB and the increase levels of proteins,

it suggest that they are produced in the CNS itself and that the relation

within de concentrations of proteins in CSF has nothing to do with the

concentration of these in serum.

MAPA CONCEPTUAL

MAPA CONCEPTUAL

BIBLIOGRAFIA

MARTINEZ, L. Biología Molecular. ED UPB.

Segunda edición. Medellín. 2011.

HUANG Y-C, TSENG M-Y, et al. Increased

prothrombin, Apolipoprotein A-IV, and

Haptoglobin in the cerebrospinal fluid of

patients with Huntington`s disease. Enero 31,

2011