1 Enzyme activity p H eg LD lipase

효소측정 1) Enzyme activity 측정 : 정해진 조건(p. H, 온도, 이온강도, 조인자 농도, , ) 하에서 효소반응속도를 평가하여 측정 : 충분한 기질과 조인자를 제공하여 효소가 최대한 작용할 수 있도록 한다 : 반응 조건이나 측정법이 다른 경우 서로 환산되지 않음 (->참고치 차이) eg) LD, lipase. . : 단위 IU - enzyme activity that catalyze the formation of 1 micromole of product (or the disappearance of substrate) per minute per liter of sample under optimal condition (IU/L) SI - katal (catalytic unit) : enzyme activity that catalyze the conversion of 1 mole of substrate to product per second per liter of sample (kat/L) : spectrophotometric assay eg) lactate (substrate) + NAD+ (cofactor) LD pyruvate + NADH (product) 340 nm : absorbance, 증가 : Coupled enzymatic reactions neither the product nor the substrate can be measured another coupled reaction (indicator reaction) to produce an indicator substance

AST (aspartate aminotransferase), ALT (alanine aminotransferase) - Aspartate (or alanine)의 아미노기를 α-ketoglutarate로 전달 - Measurement (coupled enzymatic assay) AST Vt B 6 ① aspartate + α-ketoglutarate oxaloacetate + glutamate ALT Vt B 6 alanine + α-ketoglutarate pyruvate + glutamate ② oxaloacetate + NADH MD malate + NAD+ pyruvate + NADH LD lactate + NAD+ 340 nm : 흡광도, 감소 - vitamin B 6 (pyridoxal-5 -phosphate, P 5 P) cofactor of AST & ALT, supplied by patient’s serum vitamin B 6 def (alcoholics) or 투석 : B 6 부족으로 AST & ALT false decrease

AST (aspartate aminotransferase), ALT (alanine aminotransferase) * De Ritis ratio (AST/ALT) - normal ratio 1. 15 - most forms of acute hepatocellular injury : AST > ALT (higher activity in hepatocyte) ongoing damage (1 -2일 후) ALT > AST (longer half-life of ALT) - Generally, chronic (extra) : ALT, - alcoholic liver dis. (AST>ALT) : Mtc AST – predominant form longer half life 기타 AST : MI, skeletal m injury, surgery, shock, CI, PI acute (intra) : AST

γ-glutamyl transferase (GGT) - Major use : discrimination of source of increased ALP (if ALP & GGT increase source : biliary tract) > x 10 in chronic cholestasis d/t PBC or sclerosing cholangitis - Chronic alcohol abuse : rough correlation between amount of alcohol intake & GGT activity : 금주를 해도 천천히 감소 (금주 1개월까지 elevation 상태) - Drugs (acetaminophen, phenytoin, carbamazepine) : microsomal enz. (GGT) activation 시키는 약물에 의해 증가

Alkaline phosphatase (ALP) - cell membrane enz. , catalyzes alkaline hydrolysis of a large variety of substrates 거의 모든 조직에 분포(liver, bone (osteoblasts), intestinal epi. , placenta, renal tubule) - lipid transport in intestine, calcification process in bone - reference rage : highly depend on age & sex 소아기부터 지속적 증가, 10대 초반에 최대치 (성인의 X 3 -4)후 감소, 남아>여아 20대 초반에 성인 농도 도달, 50세까지 남녀 비슷 50세(폐경) 이후 남<여(bone ALP 증가)

Alkaline phosphatase (ALP) - Measurement (functions only at alkaline p. H) ρ-nitrophenyl phosphate (PNPP) + H 2 O (colorless) ALP Mg 2+ , Zn 2+ p. H 10. 3 PO 43 - + ρ-nitrophenol (yellow) 405 nm : 흡광도 증가 - ALP activity in serum : mainly from liver in healthy adult (liver >> bone) (composition : age dependent) - Blood group B or O secretors : minimal amounts of intestinal ALP in serum - Preanalytical variation : pregnancy (placenta & bone ALP 증가) : X 2 -3 : 증가 - high body mass index(10%), 항간질약제(liver isoenz. ) smoking (10%, pulmonary production of placental-like ALP) : 감소 - 경구피임제(20%) 수혈 or cardiopulmonary bypass(chelation of cation by citrate)

Alkaline phosphatase (ALP) isoenzyme - 동종효소 : liver, bone, intestine, placenta - ALP isoenzyme EP : ID of tissue source of ALP Liver 1 (L 1), Bone/Placental, Liver 2 (L 2, macrohepatic), Intestinal - Liver isoenzyme : cholestatic process L 1 : some non-malignant dis. (cholestasis, cirrhosis, viral hepatitis. . ) hepatic metastasis in lung, GI & lymphoma L 2 (macrohepatic) : may be present healthy subject at lower conc. Than L 1 may be increased in above state - Bone isoenzyme : interpreted with respect to age osteoblastic bone tumors hyperactivity of osteoblasts involved in bone remodeling (Paget’s dis) - Intestinal isoenzyme : 종종 blood group B or O secretors, (cirrhosis, CRF. . ) - Placental isoenzyme : pregnancy, (smoking) Regan isoenzyme : placental isoenzyme in some patient with malignancy

F/47 liver bone other 성인 60 30 <10 소아 30 60 <10

Lactate dehydrogenase (LD) - 인체 내 모든 조직의 세포질에 존재, 해당경로의 일부 구성 - Measurement lactate + NAD+ LP method 80 -200 IU/L LD vs pyruvate + NADH PL method 200 -400 IU/L - preanalytical variation : 혈장 < 혈청 (평균 30 IU/L, d/t 혈소판으로부터 유리) 용혈 LD 증가, LD 1/LD 2 LD 5 : labile at cold (실온보관 24 hr) - Tetramer of 2 active subunits (H & M) : 5 isoenzymes LD 1 (HHHH), LD 2 (HHHM), LD 3 (HHMM), LD 4 (HMMM), LD 5 (MMMM) Myocardial tissue, RBC : high LD 1 & LD 2 (much lower LD 4 & LD 5) Liver , skeletal muscle : high LD 4 & LD 5 (much lower LD 1 & LD 2)

Lactate dehydrogenase (LD) - Causes of abnormal results 1) nonspecific : abnormal value is not specific for damage to any particular organ 2) LD, AST, ALT, CK 를 함께 고려 LD only marked increase : 혈구, kidney, lung, LN, tumors all increase (AST>ALT) : cardiac or skeletal 3) LD in liver dis. : hepatitis 증가 가능, but not-specific LD & ALP : space occupying lesion (liver meta, HCC) 4) shock & metastatic carcinoma : damage to multiple organ (mixed pattern) 4) > X 5 -10 : megaloblastic anemia, HA, sepsis, shock, cardiopulmonary arrest, advanced malignancy (esp. leukemia, lymphoma). . 5) isoenzyme EP 이용 (2 -1 -3 -4 -5) : determination of source of injury

Lactate dehydrogenase (LD) - Causes of abnormal results 4) isoenzyme EP 이용 (2 -1 -3 -4 -5) - Germ cell tumor (seminoma, dysgerminoma): LD 1 - MI : LD 1/LD 2 >1 (flipped pattern), 36시간이 지나야. . (acute Dx 사용 X) - HA, MA, renal infarct, RCC : LD 1 (often flipped) - Leukemia, lymphoma, MM, pulmonary dis. : LD 3, LD 4 증가 (LD 1, LD 2 감소) - Skeletal muscle injury, ischemic or toxic hepatic injury : LD 5, LD 4 증가 - tombstone pattern : diffuse tissue necrosis (shock, hypoxemia) <

LD isoenzyme EP 223 -497 IU/L

LD isoenzyme EP 223 -497 IU/L

Amylase - 다당류를 가수분해하는 칼슘 함유 효소 - preanalytical variation : 칼슘길항제(citrate, oxalate) 항응고제 -> false decrease saliva contamination (saliva >> serum, X 700) - isoenzymes : S type (salivary gland) & P type (pancreas) 거의 동량 serum에 존재 - Acute pancreatitis : > X 3 in serum (20 -30 hrs : peak, upto X 10 -20 within 48 -72 hrs : return to normal, if not -> continuing necrosis, pseudocyst) : 20% of patients - not increased (poor sensitivity for pancreatitis) - serum amylase << urine amylase ( X 2 -6) small MW 50 k. Da -> 사구체 여과 (혈청 증가 수시간 내에 증가 시작) - Not correlated with severity - Nonspecific increase in intraabdominal inflammation - Decreased specificity in renal failure - Normally elevated in macroamylasemia macroamylase - complex of immunoglobulin with amylase (주로 S type) high MW로 사구체 통과 못하여 serum amylase level 증가 시킴

Lipase - 중성지방을 지방산과 글리세롤로 분해하는 효소 - more specific than amylase in acute pancreatitis 4 -8시간 이후 증가하여 24시간에 peak (earlier increase) decrease within 7 -14 days (longer) - Not correlated with severity - low MW, 사구체 여과, but 근위세뇨관에서 대부분 재흡수

Creatine kinase (CK) - 에너지 대사와 관계, creatine phosphate + ADP CK creatine +ATP - Dimer of 2 subunits (M & B) - 3 isoenzymes CK-MM (CK 3, 골격근), CK-MB (CK 2, 심근), CK-BB (CK 1, 뇌와 장) serum isoenzyme EP : MM >>> MB > BB 96 -100% <5% 0% (never cross BBB) cf) Isoforms (high resolution EP) : MM - 3 isoforms (MM 1, MM 2, MM 3) , MB - 2 isoforms (MB 1, MB 2) : 조직에서 방출된 후 혈청의 carboxypeptidase에 의해 C 말단의 aa가 짤리면서 immunoreactivity 나 charge가 tissue form (unmodified form)과는 다른 동형 단백 : 방출 수 시간 내에 enzymatic cleavage -> high fraction of tissue form -> recent damage 의미

Creatine kinase (CK) - 반감기 24 hr - Causes of abnormal results : 주로 cardiac (MI) or skeletal muscle damage시 증가 - Skeletal muscle injury trauma, surgery, strenuous exercise, IM injection : mild elevation (X 5 -6), rapid increase & falls (반감기 고려) neuroleptic malignant syn. : phenothiazine or other psychotrophic agents : acute muscle injury - muscle rigidity, fever, WBC↑, elevated CK(diagnostic) chronic muscle damage : persistent elevation (mild or more extensive) : medication (HMG coenzyme A reductase inhibitors, glucocorticoids) congenital myopathies, inflammatory disorders(PM, DM) hypothyroidism, alcohol abuse rhabdomyolysis : > x 20, rapid rise & fall, myoglobinuria : serum myoglobin - insensitive & short half-life : not reliable urine myoglobin – important, toxic to kidney (-> ATN) : 원인 – drug (alcohol, cocaine), viral infection, extreme exertion, hyperthermia trauma, 하지의 ischemia, inflammatory myopathy

1. 대사와 분비 기능 1) Bilirubin - diazo reaction (van den Bergh reaction) bilirubin + diazotized sulfanilic acid -> azobilirubin (540 nm) - unconjugated bilirubin : slow reaction 반응촉진제(caffeine or methanol) 사용 X -> direct-reacting bilirubin 측정 (direct bilirubin) 사용 O -> total bilirubin 측정 - total bilirubin ≠ conjugated + unconjugated bilirubin (delta bilirubin) - delta bilirubin (biliprotein) : 알부민과 공유결합(irreversible)되어 있는 conjugated bilirubin : 반감기- 17일 (conjugated bilirubin : 24 hr 미만) : not filtered at glomerulus (conjugated bilirubin : 수용성, 사구체 여과) conjugated hyperbilirubinemia without bilirubinuria (간세포 손상 혹은 biliary obstruction 회복기에 지속성 황달 유발)

1. 대사와 분비 기능 1) Bilirubin - direct bilirubin : not equal to conjugated bilirubin (estimates of conj. bilirubin) = 70 -80% of conj. bilirubin & delta bilirubin + small % of unconj. bilirubin - direct bilirubin 검사법의 정확도 : 검체 취급과 시약 구성 - normal direct bilirubin 0 -0. 1 mg/d. L (liver or biliary tract diseases) - total bilirubin : age & sex (F<M) - bilirubinometer (bilirubin 전용 분광광도계) : bilirubin 455 nm 노란색, 신생아 친화성 소변 혹은 쓸개즙 배설 diazo reaction 알부민과 가역적 결합 알부민과 비가역적 공유결합 unconjugated 지용성 수용성 - + indirect +++ - - +

1. 대사와 분비 기능 2) Ammonia - enzymatic assay using glutamate dehydrogenase GDH ammonia + α ketoglutarate -------> glutamate + H 2 O NADPH NADH (340 nm : decrease in absorbance) - specimen handling (검체 채취와 운송의 중요성) : 검체 재취 및 운송 과정 중 대사가 진행되면 안된다…(false increase) : EDTA or heparin WB (arterial blood better than venous blood) : kept in ice water before centrifugation : 채혈 후 15분 이내 검사

2. 합성기능 1) 단백합성 - 알부민, AAT, ceruloplasmin. . 2) 응고인자 3. 간손상 1) 효소 - Cellular locations of enzymes in hepatocyte - Liver enzymes are found in specific location Cytoplasmic enz. : ALT, AST, LD Mitochondrial enz. : AST Canalicular enz. : ALP, GGT, 5’-nucleotidase - Type of liver injury determination of pattern of enzyme change 2) ICG