alt/sgpt activity
DESCRIPTION
ALT/sGPT activity. xiaoli. ALT. Alanine transaminase (ALT). ALT or sGPT (serum glutamate pyruvate transaminase). GPT (ALT) catalyses the transfer of amino-groups from alanine to 2-oxoglutarate and thus the formation of glutamate and pyruvate. GPT. GOT. GPT. GOT. organ. organ. 2000. - PowerPoint PPT PresentationTRANSCRIPT
ALT/sGPT activity
xiaoli
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.
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
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
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.
Aspartate aminotransferase (AST)
AST or sGOT (serum glutamate oxaloacetate transami
nase)
AST
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)
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).
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 :
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.
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, 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.
ALT/sGPT activity
Karment’s method
Reitman-Frankel method
King’s method
Mohun’s method
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.
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]
( 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
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
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.
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,
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
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
NEXT :
URIC ACID
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.