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ALT/sGPT activity xiaoli. Alanine transaminase (ALT) ALT or sGPT (serum glutamate pyruvate...

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ALT/sGPT activi ty xiaoli
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Page 1: ALT/sGPT activity xiaoli. Alanine transaminase (ALT) ALT or sGPT (serum glutamate pyruvate transaminase) ALT GPT (ALT) catalyses the transfer of amino-groups.

ALT/sGPT activity

xiaoli

Page 2: ALT/sGPT activity xiaoli. Alanine transaminase (ALT) ALT or sGPT (serum glutamate pyruvate transaminase) ALT GPT (ALT) catalyses the transfer of amino-groups.

Alanine transaminase (ALT)

ALT or sGPT (serum glutamate pyruvate transaminase)

ALTALT

GPT (ALT) catalyses the transfer of amino-groups from alanine to 2-oxoglutarate and thus the formation of glutamate and pyruvate.

Page 3: ALT/sGPT activity xiaoli. Alanine transaminase (ALT) ALT or sGPT (serum glutamate pyruvate transaminase) ALT GPT (ALT) catalyses the transfer of amino-groups.

ALT is an enzyme produced in hepatocytes and is highly c

oncentrated in the liver.

organ GOT GPT

heart 156000 7100

liver 142000 44000

skeletal 99000 4800

kidney 91000 19000

organ GOT GPT

pancrease

spleen

lung

serum

28000 2000

14000 1200

10000 700

20 16

Page 4: ALT/sGPT activity xiaoli. Alanine transaminase (ALT) ALT or sGPT (serum glutamate pyruvate transaminase) ALT GPT (ALT) catalyses the transfer of amino-groups.

GPT (ALT) is widely distributed in cells throughout the body. GPT (ALT) is found predominantly in the cytoplasm of hepatic parenchymal cells and is widely considered to be specifically for the liver. In addition, it is also active in the heart, skeletal muscle, pancreas, and the kidney.

GPT (ALT) activity in the liver is about 3,000 times higher than its activity in the serum. Only if cells are damaged GPT (ALT) will be excreted into the blood. In the plasma, GPT (ALT) has no biological function because the necessary substrates and co-substrates are lacking there.

Source & biological action

Page 5: ALT/sGPT activity xiaoli. Alanine transaminase (ALT) ALT or sGPT (serum glutamate pyruvate transaminase) ALT GPT (ALT) catalyses the transfer of amino-groups.

Elevated levels of ALT may indicate : alcoholic liver disease cancer of the liver cholestasis or congestion of the bile ducts cirrhosis or scarring of the liver with loss of function death of liver tissue Hepatitis or inflammation of the liver noncancerous tumor of the liver use of medicines or drugs toxic to the liver

!! Therefore, when the liver is injured, ALT is r

eleased into the bloodstream.

Page 6: ALT/sGPT activity xiaoli. Alanine transaminase (ALT) ALT or sGPT (serum glutamate pyruvate transaminase) ALT GPT (ALT) catalyses the transfer of amino-groups.

Aspartate aminotransferase (AST)

AST or sGOT (serum glutamate oxaloacetate transami

nase)

AST

Page 7: ALT/sGPT activity xiaoli. Alanine transaminase (ALT) ALT or sGPT (serum glutamate pyruvate transaminase) ALT GPT (ALT) catalyses the transfer of amino-groups.

organ

GOT GPT

heart 156000 7100

liver 142000 44000

skeletal 99000 4800

kidney 91000 19000

organ GOT GPT

pancrease

spleen

lung

serum

28000 2000

14000 1200

10000 700

20 16

Both these enzyme are found in most tissues , but the relative

amounts vary . heart muscles are richer in AST, whereas liver

contains both but more of ALT.

Aspartate aminotransferase (AST)

Page 8: ALT/sGPT activity xiaoli. Alanine transaminase (ALT) ALT or sGPT (serum glutamate pyruvate transaminase) ALT GPT (ALT) catalyses the transfer of amino-groups.

AST also reflects damage to the hepatic ce

lls and is less specific for liver disease. It can al

so be released with heart, muscle and brain dis

orders.

Therefore, this test may be ordered to help

diagnose various heart, muscle or brain disorder

s, such as a myocardial infarct (heart attack).

Page 9: ALT/sGPT activity xiaoli. Alanine transaminase (ALT) ALT or sGPT (serum glutamate pyruvate transaminase) ALT GPT (ALT) catalyses the transfer of amino-groups.

acute hemolytic anemia, acute pancreatitis or inflammation of the pancreas. acute renal failure or loss of kidney function. cirrhosis of the liver. Hepatitis heart attack primary muscle disease recent surgery severe burns muscle injury

Elevated levels of AST may indicate :

Page 10: ALT/sGPT activity xiaoli. Alanine transaminase (ALT) ALT or sGPT (serum glutamate pyruvate transaminase) ALT GPT (ALT) catalyses the transfer of amino-groups.

In infectious hepatitis and other inflammatory conditions affecting the liver, ALT is characteristically as high as or higher than AST, and the ALT/AST (De Ritis) ratio becomes greater than unity, >1.5.

sALT/sAST (De Ritis) ratio, normally is less than 1, about 0.7~0.9.

Ratios between ALT and AST are useful to physicians

in assessing the etiology of liver enzyme abnormalities.

Page 11: ALT/sGPT activity xiaoli. Alanine transaminase (ALT) ALT or sGPT (serum glutamate pyruvate transaminase) ALT GPT (ALT) catalyses the transfer of amino-groups.

AST and ALT is in the different distribution of the hepatocytes. ALT exists primarily in the cytoplasm of liver cell. if there is a slight liver cell damage, ALT firstly leak into the bloodstream, so that the serum ALT increased. The AST mainly in the "mitochondria“of liver cells, the mitochondria are "bubble" in the liver cell cytoplasm. if there is a slight liver cell damage, AST don`t leak into the bloodstream.

Page 12: ALT/sGPT activity xiaoli. Alanine transaminase (ALT) ALT or sGPT (serum glutamate pyruvate transaminase) ALT GPT (ALT) catalyses the transfer of amino-groups.

When the AST was significantly higher, mitochondr

ia of liver cells are injuries, indicating severe liver dama

ge, such as cirrhosis, chronic severe hepatitis, Alcohol-i

nduced hepatitis, drug-induced hepatitis. AST may also

be greater than ALT.

Doctor checking patient's transaminase, specially att

ention to the patient's AST and ALT ratio, if the ALT /

AST < 1, note a serious liver cell injury, need timely tre

atment.

Page 13: ALT/sGPT activity xiaoli. Alanine transaminase (ALT) ALT or sGPT (serum glutamate pyruvate transaminase) ALT GPT (ALT) catalyses the transfer of amino-groups.

ALT/sGPT activity

Karment’s method

Reitman-Frankel method

King’s method

Mohun’s method

Page 14: ALT/sGPT activity xiaoli. Alanine transaminase (ALT) ALT or sGPT (serum glutamate pyruvate transaminase) ALT GPT (ALT) catalyses the transfer of amino-groups.

Karment’s method

alanine+ - ketoglutaric acid

glutamic acid + pyruvic acid

GPT

LDH

NAD+ +lactic acid

NADH+H+The NADH + H+ has two absorption peaks at 260 nm and 340 nm. Upon oxidation (dehydrogenation), as NADH becomes NAD+, the absorption at 340 nm disappear. Tracing the disappearance of NADH, we can measure the enzyme reaction which is consuming the NADH.

Page 15: ALT/sGPT activity xiaoli. Alanine transaminase (ALT) ALT or sGPT (serum glutamate pyruvate transaminase) ALT GPT (ALT) catalyses the transfer of amino-groups.

ALT catalyzes the transamination from L-alanine to

- ketoglutaric acid, forming L- glutamic acid and pyruvic ac

id. ♣ The pyruvic acid which combines with 2,4 – dinitrophe

nylhydrazine(2,4-DNPH) to give a brown colored complex(p

yruvate-2,4–dinitrophenylhydrazone)in alkaline medium. ♣T

he change of absorbance of the colored complex due to the tra

nslation of pyruvic acid that is proportional to the activity of

ALT.

a- ketoglutaric acid + L-alanineALT

L- glutamic acid + pyruvic acid

pyruvic acid + 2 , 4-DNPHH2O

pyruvate-2,4- dinitrophenylhydrazonealkaline medium

Reitman-Frankel method [principle]

Page 16: ALT/sGPT activity xiaoli. Alanine transaminase (ALT) ALT or sGPT (serum glutamate pyruvate transaminase) ALT GPT (ALT) catalyses the transfer of amino-groups.

( 1) Sample specimen (Serum or plasma)

( 2) Phosphate buffer :

Dissolve Na2HPO4 13.97g and KH2PO4 2.69g,make up to

1L,pH7.4

(3) ALT substrate:

- ketoglutaric acid 30mg, L-alanine 1.78g, make up to 100

ml with PBS, pH7.4.

( 4) Standard solution:

dissolve 22mg sodium pyruvate with PBS, make up to 100ml.

( 5) Spectrophotometer

SPECIMENS & MATERIALS

Page 17: ALT/sGPT activity xiaoli. Alanine transaminase (ALT) ALT or sGPT (serum glutamate pyruvate transaminase) ALT GPT (ALT) catalyses the transfer of amino-groups.

0 1 2 3 4 5

Standard solution(ml) 0.00 0.05 0.10 0.15 0.20 0.25

ALT substrate(ml) 0.50 0.45 0.40 0.35 0.30 0.25

PBS (ml) 0.10 0.10 0.10 0.10 0.10 0.10

Mix, incubate at 37 oc for 5 mins

2,4-DNPH 0.5 0.5 0.5 0.5 0.5 0.5allow them at 37 oc for 20min

NaOH 0.4mol/L 5 5 5 5 5 5

Karman’s unit 0 28 57 97 150 200

1. Standard curve

mix and stand for 10min ,measure the Abs against with distilled water at 520nm. A0、 A1、 A2、 A3、 A4、 A5

Page 18: ALT/sGPT activity xiaoli. Alanine transaminase (ALT) ALT or sGPT (serum glutamate pyruvate transaminase) ALT GPT (ALT) catalyses the transfer of amino-groups.

Make standard curve:

the activity of ALT

An-A0

Karman28 57 97 150 200

A1-A0

A2-A0

A3-A0A4-A0A5-A0

. .

.

.

.

O

Attention:

When the activity of ALT is less than 97 karment’s unit , the direct relationship is best.

Page 19: ALT/sGPT activity xiaoli. Alanine transaminase (ALT) ALT or sGPT (serum glutamate pyruvate transaminase) ALT GPT (ALT) catalyses the transfer of amino-groups.

2. the activity of ALT

T C

Serum(ml) 0.1 -

ALT substrate(ml) 0.5 0.5

Mix , allow them at 37 oc for 30min

2,4-DNPH ( ml )

0.5 0.5

Mix , incubate at 37 oc for 30min

Serum(ml) - 0.1

0.4mol/L NaOH(ml) 5.0 5.0

Mix and stand for 10min, set zero with distilled water at 520nm. measure the At、 Ac, Determine the activity of ALT in the sample from the standard curve by AT-AC.

1) Incubate ALT substrate at 37 oc for 5min,

Page 20: ALT/sGPT activity xiaoli. Alanine transaminase (ALT) ALT or sGPT (serum glutamate pyruvate transaminase) ALT GPT (ALT) catalyses the transfer of amino-groups.

Make standard curve:

the activity of ALT

An-A0

Karman28 57 97 150 200

A1-A0

A2-A0

A3-A0

A4-A0

A5-A0

. .

.

.

.

O

At-Ac

Normal range: 2-59 U/L

Page 21: ALT/sGPT activity xiaoli. Alanine transaminase (ALT) ALT or sGPT (serum glutamate pyruvate transaminase) ALT GPT (ALT) catalyses the transfer of amino-groups.

1. It is better not drink ,not high-lipid diet ,not too tired.

2. Strenuous exercise and intramuscular injections befor

e testing should be avoided, since they may increase G

OT levels.

3. If hemolysis ,GPT in red cells are released ,that increa

se the level of GPT of serum or plasma.

Patient preparation

Page 22: ALT/sGPT activity xiaoli. Alanine transaminase (ALT) ALT or sGPT (serum glutamate pyruvate transaminase) ALT GPT (ALT) catalyses the transfer of amino-groups.

NEXT :

URIC ACID

Page 23: ALT/sGPT activity xiaoli. Alanine transaminase (ALT) ALT or sGPT (serum glutamate pyruvate transaminase) ALT GPT (ALT) catalyses the transfer of amino-groups.

In viral hepatitis and other forms of liver disease associate

d with hepatic necrosis, serum AST and ALT levels are elevat

ed even before the clinical signs and symptoms of disease (s

uch as jaundice) appear.

Levels for both enzymes may reach values as high as 10

0 times the upper limit of the reference, although 20- to 50- fol

d elevations are most frequently encountered.

Peak values of transaminase activity occur between the s

eventh and 12th days; activities then gradually decrease, reac

hing normal levels by the third to fifth week if recovery is unev

entful.


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