Transcript ALT

ALT/sGPT activity
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Alanine transaminase (ALT)
ALT
ALT or sGPT (serum
glutamate pyruvate transaminase)
GPT (ALT) catalyses the transfer of amino-groups from alanine to 2oxoglutarate and thus the formation of glutamate and pyruvate.
organ
GOT
GPT
organ
GOT
GPT
heart
156000
7100
pancrease
28000
2000
liver
142000
44000
spleen
14000
1200
skeletal
99000
4800
lung
10000
700
kidney
91000
19000
20
16
ALT is
serum
an enzyme produced in hepatocytes and is highly
concentrated in the liver.
Source & biological action
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 cosubstrates are lacking there.
Therefore, when the liver is injured, ALT is
released into the bloodstream.
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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
Aspartate aminotransferase (AST)
AST
AST or sGOT (serum
transaminase)
glutamate oxaloacetate
Aspartate aminotransferase (AST)
organ
GOT
GPT
organ
GOT
GPT
heart
156000
7100
pancrease
28000
2000
liver
142000
44000
spleen
14000
1200
skeletal
99000
4800
lung
10000
700
kidney
91000
19000
20
16
Both
serum
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.

AST also reflects damage to the hepatic
cells and is less specific for liver disease. It can
also be released with heart, muscle and brain
disorders.

Therefore, this test may be ordered to help
diagnose various heart, muscle or brain disorders,
such as a myocardial infarct (heart attack).
Elevated levels of AST may indicate :





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

Ratios between ALT and AST are useful to physicians
in assessing the etiology of liver enzyme abnormalities.
sALT/sAST (De Ritis) ratio, normally is less than 1,
about 0.7~0.9.
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.
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.
When the AST was significantly higher,
mitochondria of liver cells are injuries, indicating severe
liver damage, such as cirrhosis, chronic severe hepatitis,
Alcohol-induced hepatitis, drug-induced hepatitis. AST
may also be greater than ALT.
Doctor checking patient's transaminase, specially
attention to the patient's AST and ALT ratio, if the ALT /
AST < 1, note a serious liver cell injury, need timely
treatment.
ALT/sGPT activity
Karment’s method
Reitman-Frankel method
King’s method
Mohun’s method
Karment’s method
GPT
alanine+ - ketoglutaric acid
glutamic acid + pyruvic acid
LDH
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.
NADH+H+
NAD+ +lactic acid
Reitman-Frankel method [principle]
ALT catalyzes the transamination from L-alanine to
- ketoglutaric acid, forming L- glutamic acid and pyruvic
acid. ♣ The pyruvic acid which combines with 2,4 –
dinitrophenylhydrazine(2,4-DNPH) to give a brown colored
complex(pyruvate-2,4–dinitrophenylhydrazone)in alkaline
medium. ♣The change of absorbance of the colored complex
due to the translation of pyruvic acid that is proportional to
the activity of ALT.
a- ketoglutaric acid + L-alanine
pyruvic acid + 2 , 4-DNPH
ALT
H2O
L- glutamic acid + pyruvic acid
pyruvate-2,4- dinitrophenylhydrazone
alkaline medium
SPECIMENS & MATERIALS
（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
1. Standard curve
0
1
2
3
4
5
Standard solution(ml)
0.00
0.05
0.10
0.15
0.20
0.25
ALT substrate(ml)
PBS (ml)
0.50
0.45
0.40
0.35
0.30
0.25
0.10
0.10
0.10
0.10
0.10
0.10
0.5
0.5
Mix, incubate at 37 oc for 5 mins
2,4-DNPH
0.5
0.5
0.5
0.5
allow them at 37 oc for 20min
NaOH 0.4mol/L
Karman’s unit
5
5
5
5
5
5
0
28
57
97
150
200
mix and stand for 10min ,measure the Abs against with
distilled water at 520nm. A0、A1、A2、A3、A4、A5
Attention:
Make standard curve:
When the activity of
ALT is less than 97
karment’s unit , the direct
relationship is best.
An-A0
A5-A0
A4-A0
A3-A0
.
.
.
A2-A0
A1-A0
O
.
.
Karman
28
57
97
150
the activity of ALT
200
2. the activity of ALT
1) Incubate ALT substrate at 37 oc for 5min,
T
0.1
Serum(ml)
0.5
ALT substrate(ml)
Mix , allow them at 37 oc for 30min
2,4-DNPH（ml）
0.5
C
0.5
0.5
Mix , incubate at 37 oc for 30min
Serum(ml)
0.4mol/L NaOH(ml)
-
0.1
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.
Make standard curve:
An-A0
Normal range: 2-59 U/L
A5-A0
A4-A0
At-Ac
.
.
A3-A0
.
A2-A0
A1-A0
O
.
.
Karman
28
57
97
the activity of ALT
150
200
Patient preparation
1. It is better not drink ,not high-lipid diet ,not too tired.
2. Strenuous exercise and intramuscular injections
before testing should be avoided, since they may
increase GOT levels.
3. If hemolysis ,GPT in red cells are released ,that
increase the level of GPT of serum or plasma.
NEXT :
URIC ACID
In viral hepatitis and other forms of liver disease
associated with hepatic necrosis, serum AST and ALT levels
are elevated even before the clinical signs and symptoms of
disease (such as jaundice) appear.
Levels for both enzymes may reach values as high as
100 times the upper limit of the reference, although 20- to 50fold elevations are most frequently encountered.
Peak values of transaminase activity occur between the
seventh and 12th days; activities then gradually decrease,
reaching normal levels by the third to fifth week if recovery is
uneventful.