2 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.
3 organGOTGPTheart1560007100liver14200044000skeletal990004800kidney9100019000pancreasespleenlungserum280002000140001200100007002016ALT is an enzyme produced in hepatocytes and is highly concentrated in the liver.
4 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 co-substrates are lacking there.
5 Elevated levels of ALT may indicate : alcoholic liver disease ！！ Therefore, when the liver is injured, ALT is released into the bloodstream.Elevated levels of ALT may indicate :alcoholic liver diseasecancer of the livercholestasis or congestion of the bile ductscirrhosis or scarring of the liver with loss of functiondeath of liver tissueHepatitis or inflammation of the livernoncancerous tumor of the liveruse of medicines or drugs toxic to the liver
7 Aspartate aminotransferase (AST) organGOTGPTheart1560007100liver14200044000skeletal990004800kidney9100019000pancreasespleenlungserum280002000140001200100007002016Both 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.
8 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).
9 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.Hepatitisheart attackprimary muscle diseaserecent surgerysevere burnsmuscle injury
10 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.
11 AST and ALT is in the different distribution of the hepatocytes 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.
12 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.
14 Karment’s method alanine+ - ketoglutaric acid NAD+ +lactic acid GPT glutamic acid + pyruvic acidalanine+ - ketoglutaric acidLDHNAD+ +lactic acidNADH+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.
15 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.alkaline medium
16 SPECIMENS & MATERIALS （1）Sample specimen (Serum or plasma) （2）Phosphate buffer :Dissolve Na2HPO g and KH2PO g,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
17 Mix, incubate at 37 oc for 5 mins Standard curve12345Standard solution(ml)0.000.050.100.150.200.25ALT substrate(ml)0.500.450.400.350.30PBS (ml)Mix, incubate at 37 oc for 5 mins2,4-DNPH0.5allow them at 37 oc for 20minNaOH 0.4mol/LKarman’s unit285797150200mix and stand for 10min ,measure the Abs against with distilled water at 520nm. A0、A1、A2、A3、A4、A5
18 Make standard curve: . Attention: When the activity of ALT is less than 97 karment’s unit , the direct relationship is best.Make standard curve:An-A0A5-A0.A4-A0A3-A0A2-A0A1-A0KarmanO285797150200the activity of ALT
19 2. the activity of ALT Serum(ml) 1) Incubate ALT substrate at 37 oc for 5min,TCSerum(ml)0.1-ALT substrate(ml)0.5Mix , allow them at 37 oc for 30min2,4-DNPH（ml）0.5Mix , incubate at 37 oc for 30minSerum(ml)-0.10.4mol/L NaOH(ml)5.0Mix 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.
20 Make standard curve: Normal range: 2-59 U/L . . An-A0 A5-A0 A4-A0 At-AcA2-A0A1-A0KarmanO285797150200the activity of ALT
21 Patient preparation1. 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.
23 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 50- fold 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.