““INCREASED PROTHROMBIN, INCREASED PROTHROMBIN, APOLIPOPROTEIN A-IV, AND APOLIPOPROTEIN A-IV, AND

HAPTOGLOBIN IN THE HAPTOGLOBIN IN THE CEREBROESPINAL FLUID OF PATIENTS CEREBROESPINAL FLUID OF PATIENTS

WITH HUNTINGTON´S DISEASE.”WITH HUNTINGTON´S DISEASE.”Yen-Chu HuangYen-Chu Huang.

Maria Camila Montufar PantojaHeyly Milena OrtegaIII SEMESTER2011

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 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.

PROTHROMBINPROTHROMBIN

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

HUNITNGTON DISEASE - HD

Progressive neurodegenerative disorder with autosomaldominant 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

ELECTROFORESIS DE DOS DIMENSIONES (2-DE )

 Técnica de alta resolución cuyo objetivo es la separación de mezclas de proteínas

- Punto isoeléctrico (isoelectroenfoque).

ánodo es ácida y la del cátodo es alcalina

- Masa molecular por electroforesis gel de poliacrilamida

DIGESTION EL GEL

Forma parte de la preparación de las muestras para la espectrometría de masas

- 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 patients and 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 and control patients are very important to determinate if the increase levels of Haptoglobin, Apolipoprotein A-IV and Prothrombin are correlated with HD development.

It is important to recognize this proteins like specifics biomarkers in HD; thats why the concentrations of each one were quantified in CSF and serum; thus indirectly evaluate the BBB integrity.

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

BIBLIOGRAFIA

MARTINEZ, L. Biología Molecular. ED UPB. Segunda edición. Medellín. 2011.

Yen-Chu Huang. Increased Prothrombin, Apolipoprotein A-IV, and Haptoglobin in the Cerebrospinal Fluid of Patients with Huntington’s Disease . Plos One. Vol 6, January 2011.

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