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Alkaline Phosphotase

EC 3. 1. 3. 1. Alkaline Phosphotase. 알카리성 영역에서 인산 에스터 (phpshporic ester) 을 가수분해하는 효소. Phospho-Monoesterase, Phospho-diesterase, Phospho-amydase, hydro-phosphatase. Monoester : metaphosphoric acid, pyrophosphoric acid,

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Alkaline Phosphotase

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  1. EC 3. 1. 3. 1 Alkaline Phosphotase 알카리성 영역에서 인산 에스터(phpshporic ester) 을 가수분해하는 효소 Phospho-Monoesterase, Phospho-diesterase, Phospho-amydase, hydro-phosphatase • Monoester : metaphosphoric acid, pyrophosphoric acid, • glycerol phosphate, G-6-P, 5-N, PNPP • - Diester : Cyclic, AMP, DNA, RNA • - Triester : 유기인산 농약

  2. ALP 생리적 기능 • 인산화합물을 가수분해( orthophosphoric monoester • phosphohydrolase하는 효소.(pH 9-10.5) • 인산 기의 전의 반응에 촉매 작용. • Phosphomonoester과 Alcohol의 전이 작용에 촉매. • 당, 지질의 흡수, 인산과 Ca 의 흡수 및 수송을 담당 • 핵산 합성 조절, 골 파괴 ,와 화골에 관여

  3. 골 연화(Osteomalacial), Paget병, 구루병(Rickets) 골 전이암, 골 형성 질환(Osteoblastic lesion), 간염 폐쇄성 황달, 영양실조(Malnutrition), 저 인산염 혈증(hypophosphatemia) Normal Value : 102 - 249 U /L (M), 82 – 211 U/L (F). 혈액형 O.B형에서는 지질부하의 식사에 의하여 정상인의 1,5배 가량 소장형 증가하며 혈액형과 관계가 없이 간 경변증에는 소 장형 ALP가 증가한다.

  4. Alkaline phosphatase/Acid phosphatase 1㎎ P/㎗/60min 1㎎ phenol/㎗/30min(15) 1m㏖ PNP/ L /60min 1 u㏖ phenophthalein/L/1min Bowers-McComb 1umol/37℃/1min

  5. Na-β-glycerol phosphate + H2O → glycerol + phophate    phosphate + Amm,molybdate + ANSA → blue color

  6. Folin-Ciocalteu phenyl phosphate + H2O → phenol + phosphate phenol + 4- aminoantiphyrine → pot ferricyanide → red color

  7. p-nitrophenylphosphate(4-NPP, PNPP)  +  H2O     → phosphate +  p-nitrophenol(4-NP, PNP) 4-nitrophenylphosphate (4NPP, PNPP; 과거에는 para nitrophenyl phosphate) p-nitrophenyl phosphate + H2O→ p-nitrophenol +  phosphate p-nitrophenol + NaOH → yellow color

  8. Carbonate Buffer, 0.05M pH10.1   Sodium carbonate.anhydrous  3.18gm  NaHCO3                    1.68g  Substrate phenylphosphate, Na2   0.215g 4-aminoantipyrine 0.09g boric acid            2.6g,   chloroform            1ml    Chromogenic  carbonate buffer      200ml  Standard Phenol           1.0gm,    0.1N HCl         1.0L    Kind-King

  9. Procedure

  10. Standard Curve Prepare standard volumetrically as follows (phenol 100 ㎎/㎗ = 100 K-A unit)

  11. 2. Diethanolamine(DEA) 3. EthylAminoEthanol(EAE) 4. MethylaminoEthanol(MAE) • 2-Amino-2-Methyl-1-Propanol(AMP) 5. tris(hydroxymethyl)amino methan(Tris) ALP 완충 용액

  12.    2-Amino2,1-Propanol : 0.36mol/l pH10.4  (0.35mol/l)   Magnessium acetate : 2.84mmol/l       (2.0mmol/l)      Zinc sulfate : 1.43mmol/l              (1.0mmol/l)       N-(2-hydroxyethyl)-ethylenediamintriacetatic acid 2.84 mmol/l  (2.0mmol/l)     PNPP : 57.1mmol/l   (17mmol/l)        AMP : 0.36mol/l Reagent 2(Start reagent/Substrate)700ul Reagent 1(Buffer/activator)    2200ul IFCC/AMP Alkaline Phosphotase Bowers-McComb

  13. IFCC/AMP Alkaline Phosphotase Bowers-McComb Reagent 1(Buffer/activator)    2200ul Prewarmin . . . . . . 2 ~ 3min/37℃ 검 체(Serum) . . . . 100ul 부드럽게 혼합 Reagent 2(Start reagent/Substrate)700ul 부드럽게 혼합 . . . . . 20초 후 340nm 흡광도 측정 ……A1 A1 흡광도 측정 후 Stopwatch 작동하여 . . . . . 1min 에 도달하면 340nm 흡광도 측정 ……A2 1min간 흡광도 변화 량을 산출한다 . . . . A2 - A2 = /min

  14. 1.0 = mmol 4-NP/ml 3.0 = Total volume in Cuvet 0.1 = Volume of Serum in Cuvet 1000 = Convert mmol to umol 효소 활성 구하는 방법 ε= 1.0m㏖ 4-NP / L : 18.75

  15. 06/03/08 D1 07:47 001 156 447 ALP ---S1--- ---S2--- 22 6773 882 8309 S1ABS K R1 R2 25 6847 882 8281 24 3089 (0) (265) 0.0858/265U

  16. 측정시 주의 • EDTA, Citrate, Oxalate, Heparin. • 용혈(6x), 황달, lipemic, 검체 blank 4-NPP. • 고분자형ALP, 과립결합성의ALP 가 유리. • SH제, Phenylalanine, Cystein, Tryptophan • Mg, Co, Mn, Zn 은 ALP을 활성화.

  17. 효소 활성의 특이성 동일, 다 분자 형, 장기 별로 분포, 생리학적 • IUB 효소상임위원회 에서 인정 • Amino acid chain에 의하여 dimer, trimer, tetramer. 로 형성 • 물리적, 화학적 성질이 다르다 • 음 이온으로 charge되는 것 부터(+쪽으로 빠른것) 1번이 된다 • Isoenzyme

  18. ISOENZYME

  19. Liver   Bone      Placent      Intest Phenylalanine        -        -           +             ++            Homoarginine       +          +       -              - Urea                 +         +++      +             +          56'C/15min            +        ++        -              + 60'C/10min             +          +   -              +  Phenylalanine = placenta, intestinal ⇓ L-formoarginine = Liver, Bone⇓ Urea = Bone⇓ (대부분영향) 56℃ 65℃ = Bone ⇓ (대부분영향 ), Placenta불변

  20. ACID PHOSPHATASE • ACP는 전립선 조직과 그 분비액에 많이 함유되어있으며 • 기타 적혈구, liver, Spleen, Kidney,      BM에도 분포 • 세포 속의 라이소자임과 골지체에 다량함유 • L-tartaric acid 은 전립선 ACP 활성을 저해한다 • Fishman-Lerner method • RT(refri) 2hr, Defreezer 1wk • sodium citrate 18mg/1ml. 20% Sod, acetaet 10ul/1ml • Massage, Cathether  ACP 상승

  21. Acid Buffer solution, pH4.1   Citrate acid 19.0gm , 1N NaOH 185ml 0.1N HCl             100 ml , Distill water 1L Stock Substrate P-nitorphenylphosphate, Na2 0.4g Distill water 100ml Acid buffer 1 vol 에 stock substrate 1 vol Working acid substrate solution 0.02N NaOH Standard P-NPP 139mg/DW 100ml 0.5ml+0.05N NaOH 100ml  

  22. Acid Phosphatase • Procedure

  23. Standard Curve Prepare standard volumetrically as follows

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