Yen-Chu Huang: Universidad Pontificia Bolivariana

“INCREASED PROTHROMBIN,
APOLIPOPROTEIN 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.
Xa
Ca++
 It plays an important function in Va
coagulation process by Tromboplastina
reaction with the thromboplastin

enzyme , with Ca ++ cation and
V activate factor as coenzymes.
 In HD prothrombin 
proinflammatory agent : DISEASE
STATUS BIOMARKER
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
PREPARACION DE LAS
MUESTRAS
 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.
Mu
estr
as Muestras de
de Suero
LC
R
• Ce • Almacenados
ntrif a 4°c durante
uga 1 hora.
dos • Centrifugados
. .
• Alm • Almacenados
ace a -80°c.
nad
os
a-
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 patients
and their corresponding controls.
RESULTADOS
 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.
DISCUSSION
AUTOR OPINION YES NO
The mutant huntingtin
may enchance the ability
Bjorkqvist M. of microglia to produce
proinflamatory mediators
(IL-6, IL-8 and TNF-a)
and contribute to
neurodegeneration in
HD.
In HD, weigth loss is a
characteristic feature in
Djoussé L. 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
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
MAPA CONCEPTUAL
BIBLIOGRAFIA
 MARTINEZ, L. Biología Molecular. ED UPB.
Segunda edición. Medellín. 2011.

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“INCREASED PROTHROMBIN,

APOLIPOPROTEIN A-IV, AND

Maria Camila Montufar Pantoja

UNIVERSIDAD PONTIFICIA BOLIVARIANA

 Demonstrate how Prothrombin,

 HD is a hereditary, degenerative brain disorder. It

 Plasma protein synthesized

reaction with the thromboplastin

 The formation of chylomicrons acts as a signal for the

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

 Is involved in the long-term regulation of both food

 Técnica de alta resolución cuyo

 las proteínas son separadas por dos

 Forma parte de la preparación de las muestras para

- Extracción del péptido generado.

 se basa en el fenómeno conocido como desbastado (sputtering,

 el patrón de fragmentación resultante así como los

 Representative Western blots of prothrombin (72 kD)

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

 Qalb, CSF haptoglobin, and serum haptoglobin

Fang Q, Strand In proteome

Underwood BR A serum metabolic

 The proteomic studies in CFS in patients HD and

 It is important to recognize this proteins like

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