Blood specimen preparation Glucose analysis Liver function test Neutral sugar and sialic acid determination ( 由吳教授講解 )

Blood sample collection

Heparin Heparin: Direct thrombin inhibitors

Blood glucose determination

Hormonal regulation Insulin: Release from  -cells islet of Langerhans of pancreas in response to high blood glucose –Increase uptake of glucose –Increase synthesis of glycolytic enzymes, glucokinase, PFK, & PK –Inhibits gluconeogenesis Glucagon:  -cells islets of Langerhans of pancreas in response to low blood glucose –Stimulate liver (not muscle) to release glucose –Involved in long term glucose maintenance Epinephrine (adrenaline): released by adrenal glands –Active on liver and muscle –Stimulates glycogen breakdown and glycolysis

The typical human adult uses 160 g of glucose per day, with 120 g being used by the brain. The available reserve of glucose is adequate for about one day (about 20 g of circulating glucose and 190 g of glycogen). After the reserve becomes depleted, how would a starved body obtain more glucose? In animals, many precursors lead to the synthesis of glucose (gluconeogenesis). In humans, the principal precursors are glycerol from triacylglycerols and glucogenic amino acids from proteins.

Blood glucose determination Glucose oxidase 2H 2 O + Glucose + O 2 → gluconic acid + H 2 O 2 2H 2 O aminoantipyrine (AAP) → 4H 2 O + Quinoneimine Dye (OD505, pink) peroxidase

血糖的正常值 正常人空腹時的血糖值為 mg/dl 將空腹血糖異常值從 110 mg/dL 降到了 100 mg/dL ， 可能使前驅前糖尿病的診断增加约 20% 預料國內將多出近 20 萬名代謝症候群病患 2003 年下修

Liver function test GOT and GPT determination 肝功能測試 : 決定你人生是黑白或彩色

Transaminase: GOT(AST), GPT(ALT) Having the function of transferring amino group of amino acids from alpha-amino acids to alpha-keto acids, therefore, named transaminase.

GOT (Glutamyl oxaloacetic transaminase, Aspartate aminotransferase, AST) L-Aspartic Acid + Ketoglutaric Acid → Oxaloacetate + L- Glutamate GPT (Glutamyl pyruvic transaminase, Alanine aminotransferase, ALT) L-Alanine + Ketoglutaric → Pyruvate + Glutamate

One of the thousands kinds of liver enzymes, and a kind of transferase. Storage mostly in liver etc. Low serum concentration. Large amount of transaminase is released into blood mostly on liver cell damages. -Thus, detection of serum level tells the existence of liver cell damage. Transaminase: GOT(AST), GPT(ALT)

OrgansGOTGPT Myocardium Liver Kidney Pancreas Spleen Lung Red blood cell Serum 156, ,000 99,000 28,000 14,000 10, ,100 44,000 4,800 2,000 1, Concentration in organs and tissues (Karmen unit/1 gm wet weight)

Chapter 16 TCA cycle

Malate aspartate shuttle

METHOD PRINCIPLE In this method ALT catalyzes the reaction of L-alanine and  - ketoglutaric acid to form pyruvate and glutamate under controlled conditions (37  C) and pH The addition of acidic 2,4-dinitrophenylhydrazine stops the reaction and forms the 2,4- dinitrophenylhydrazone. So that it may be measured at nm.

測定血液中的 GOT 及 GPT 活性 Reitman & Frankel 比色法 ( 酮酸 -DNP 呈色法 ) –  -Keto acid –DNP (2,4-dinitrophenylhydrazine) ( 金黃色 ) –2,4-dinitrophenylhydrazone ( 紅棕色 ) RCCOOH O NH 2 NH NO 2 ＋ RC COOH N NO 2 N H+H+ OH - (Pyruvate or OXA) Aspartic acid or alanine Transaminase (GOT or GPT)

GOT 及 GPT 的正常值 Serum GOT (SF Units/ml) Serum GPT (SF Units/ml) 正常值 0~280~21 臨界值 28~4021~35 S. F. Unit (Sigma-Frankel Unit): 在 25°C ， pH 7.5 的狀態下，每分鐘能產生 4.82×10 -4  mol glutamate 的酵素活性。 I. Unit (International Unit): 在特殊狀態下，每分鐘能轉變 1  mol 受質的酵素量。 1 SF U/ml = 0.48 IU/L

GOT 及 GPT 的比較 CharacteristicGOTGPT Present in tissues other than liver More in heart than in liver; also in skeletal muscle, kidney, brain Relative low conc. in other tissue Location in hepatocyteMitochondria and cytoplasm Cytoplasm only Reference range in adult blood IU/liter5-35 IU/liter Half-life in blood12-22 hr37-57 hr Change with acute inflammatory damage Moderately sensitiveExtremely sensitive Change with primary or secondary neoplasm Substantial riseModerate or no rise Change with cirrhosisModerate riseMild or moderate rise Change with myocardial infarct Substantial riseMild or moderate rise

(GOT) (GPT)