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Glutathione(γ-glutamine amino acid)

GGT. Gamma Glutamyl Transferase , GGT. γ - Glutamyl Transpeptidase, γ - GTP. EC 2. 3. 2. 2. Glutathione(γ-glutamine amino acid) + Amino acid (peptide). Glutamyl amino acid(peptide)

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Glutathione(γ-glutamine amino acid)

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  1. GGT Gamma GlutamylTransferase, GGT γ - Glutamyl Transpeptidase, γ - GTP EC 2. 3. 2. 2 Glutathione(γ-glutamine amino acid) + Amino acid (peptide) Glutamyl amino acid(peptide) + Cysteinylglycine(free amino acid)

  2. 1) GGT 생리적 기능 • γ-glutamylpeptide의 γ-glutamyl group ➡ • amino acid, peptide에 전이 작용 촉매 • 아미노산의 운송과 Glutathion 의 대사에 관여 • 세포의 glutathione의 분해와 재 합성 • Amino acid 전송

  3. GAMMA GLUTAMYL TRANSFERASE • Membrane binding enzyme • microsome, mitochondria / liver cell • 1952 : Haines • 1960 : Orlowski & Goldbarg • 1970 : glutathion oxidation • kidney, pancrease, liver, Gall Bladder, spleen, intestine, brain, heart.

  4. 2) GGT 임상적 의의 • 간 담도 질환에서 대부분 상승 • 약물에 대하여 대단히 예민 - 알콜 • 담즙과 관련한 정보, 종양성 간질환 – ALP, LAP • 원발성 전이간암, 폐색성황달, 담즙성 울혈간장애, • 담즙성 간경화, 알콜성 간염, 임신 경과에 따라 감소, • 분만직전1/2

  5. GGT Substrate • GGT 측정법은 γ-glutamyl기가 glycylglycin 공여체로 여러 가지의 합성기질 이용. 1. γ-Glutamyl-p-nitroanilide-410nm 2. γ-Glutamyl-3-carboxyl-4-nitroanilide – 415nm 3. γ-Glutamyl-3-carboxyl-4-hydroxyanilide - 635nm 4. γ-Glutamyl-p-dimethylaminoanilide– 660nm 합성기질 + Glycylglycine → γ-Glutamyl-Glycylglycine + 생성물

  6. GGT 1969년Szasz, GGPNA) Orlowski의 보고법 SSCC= Tris-HCl, MgCl2, pH7.6 1. γ-Glutamyl-p-nitroanilide r-Glu-pNA + Glycylglycine  γ-Glutamyl-Glycylglycine + P-nitroaniline (yellow) 410nm P-dimethylaminocinenamaldehyde (red) 565nm

  7. GGT 1981년 IFCC r-Glu-3C-4NA r-Glu -CANA 2. γ-Glutamyl-3-carboxyl-4-nitroanilide + Glycylglycine γ-Glutamyl-Glycylglycide  + 5-Amino-2-Nitrobenzoate(yellow)                                                            415nm r-Glu-3C-4NA mole absorbity 410nm = 7.9 x 103 mol

  8. r-GGT + Glycylglycine 기질의 안정성 우수 3. γ-Glutamyl-3-carboxyl-4-hydroxyanilide 5-aminosalicylic acid  + p-Xylenol   (Blue)                                                 635nm   Sod, Metaperiodate 

  9. r-GGT + Glycylglycine 4. γ-Glutamyl-p-dimethylaminoanilide p-dimethylaminoanilide  +  Iron  (청록색)                                         660nm Fe(CN)6NH3

  10. GGT 초속도 측정법의 반응조건 r-Glu-3C-4NA mole absorbity 410nm = 7.9 x 103 mol

  11. NORMAL VALUE Male : 10 - 55 IU/L Femal : 5 – 40 IU/L 신생아 : 40 - 250 IU/L 신생아는 성인의 10배, 유아기 성인의 2배, 음주 후 증가, 성장하면서 저하, 취학 연령에서 성인 수준, 연령증가,

  12. INTERFERING SUBSTANCE 1. Serum free from hemolysis is preferred. 2. GGT activity is inhibited by citrate, oxalate, flouride 3. EDTA plasma up to 1 mg/mL blood may be used 4. Heparin produces turbidity in the reaction mixture. 5. Anti-epileptic drugs such as phenytoin (Dilantin) and barbituates may show falsely elevated 6. Specimens with values greater than 600 IU/L should be diluted 1:1 with 0.9% saline

  13. 장기 중 GGT 활성은 ? • 성숙기에는 신장, 췌장 • 태아기에는 신장 ↓생후 ↑ • 간에서는 태아기↑ 생후 ↓ • 태생기에서는 siallic acid ↑ • 성숙기에서는 siallic acid ↓

  14. 자동분석기 Glycylglycine 25.59mg/ml 220ml r-Glu-3C-4NA 10.73mg/ml 100ml Increased r-Glu-3C-4NA mole absorbity 410nm = 7.9 x 103 mol

  15. CALCULATION 3.0 = Total volume in cuvet in ml - (TV) 0.1 = Volume of serum specimen in ml – (SV) r-Glu-3C-4NA mole absorbity 410nm = 7.9 x 103 mol

  16. 5-Aminosalicylic method(용수법) ① γ-Glutamyl-3-carboxy-4-hydroxyanilide glycylglycine GGT 5-Aminosalicylic acid p-xylenol Sod, metaperiodate Quinon color 635nm

  17. 5-Aminosalicylic method(용수법) ② Reagent: 1. Substrate : γ-Glutamyl-3-carboxy-4-hydroxyanilide 2. Color reagent : P-xylenol, Sod, metaperiodate(NaIO2) 3. Standard :5-aminosalicylic(200mU/ml)

  18. LeucineAminopeptidase Amino Acids Metabolism Amino Acids Metabolism Amino Acids Metabolism Amino Acids Metabolism Amino Acids Metabolism Amino EC 3.4.11.1-4 Amino Acids Metabolism Amino Acids Metabolism Acids Amino Acids Metabolism Amino Acids Metabolism Amino Acids Metabolism Amino Acids Metabolism Amino Acids Metabolism Amino Acids Metabolism Tai-jin,kim, tjkim@hmc.hanyang.ac.krDepartment of Clinical chemistry, HanYang University College of Medicine

  19. Leucine Aminopeptidase, AP Peptide 결합을 가수분해 하는 효소 Cytosol(leucyl) Aminopeptidase, LAP EC 3. 4. 11. 1 Microsome(alanyl) Aminopeptidase, AAP EC 3. 4. 11. 2 CystinylAminopeptidase, CAP EC 3. 4. 11. 3

  20. Endopeptidase : 단백질 내부의 peptide 결합을 분해 small peptide - (trypsin, chymotrypsin) Exopeptidase : 단백분자의 N-말단, C-말단으로 부터 한 개씩 amino acid를 제거하는 효소 Aminopeptidase : Amino 말단으로부터 amino acid 한 개씩 제거하는 효소 Carboxylpeptidase : Carboxyl 말단 부터 한 개씩 amino acid을 제거하는 효소- Dipeptidase : dipeptide의 peptide 을 분해하여 AA 생성 LeucineAminopeptidase : peptide로 부터 leucine을 제거하는 효소

  21. Aminopeptidase, AP

  22. Phnathioazine, Steroid, INH, antithyroid LAP is an enzyme normally found in liver cells (hepatocytes). It is is also released into the blood after damage to liver cells, such as from drugs or infection (for example, hepatitis). Measurement of LAP in blood may serve as an indicator of liver damage 1955년 – Green 1958년 – Goldbarg/Rutenberg

  23. LAP is a proteolytic enzyme that breaks chemical bonds in proteins at specific sites next to leucine amino acids. Serum (blood) LAP is measured to diagnose liver dysfunction. However, LAP is made by virtually all tissues. Elevations are also seen in several cancers and systemic lupus erythematosus (SLE).

  24. LAP can also be released into blood by tumors that arise in the liver, so it may also serve as a tumor marker or indicator.Other enzymes, such as ALT, AST, ALP, LDH, and GGT, are more commonly measured for the same purpose.

  25. 생리적 변동 출생시 높은 활성치을 보이다가 급격히 저하한다. 9세이후 서서히 증가(14/11) – 성인 활성치. 성별에 의한 차이는 없다.(10%낮다). 운동에 의한 일시적 증가. 활성제 : Mg2+, Mn2+. 저해제: EDTA, oxalate (Heparine재외).

  26. LAP Substrate ① ι-Leucyl amide 기질 ①1 ι-Leucyl amide 기질 ②ι-Leucyl - β-naphthylamid 기질 ③ ι-Leucyl- p-nitroanilide 기질 ④ ι-Leucyl –3-carboxy-4-hydroxyamide 기질

  27. LAP ι-Leucyl amideι-Leucinel ＋Ammonia(NH3) GLDH Ammonia(NH3)＋Glutamyl acid              Glutamin NADH+           NAD+ NADP NADPH ① ι-Leucyl amide 기질 IndoIphenol blue

  28. LeusinDH ι-Leucinel           4-methyl-2-oxobentenacid＋ NH3 NAD+               NADH + H+ ①1 ι-Leucyl amide 기질법 Aminopeptidase ι-Leucyl amide   ι-Leucine ＋ Ammonia(NH3)

  29. ②ι-Leucyl - β-naphthylamid Goldbarg-Rutenburg M Aminopeptidase Serum 0.02㎖ / 37℃/2hr간/ β-naphthylamine 1/12ug = 1 unit 국제단위 mU/ml(U/L) = G-R × 0.2425 ι-Leucyl - β-naphthylamidι-Leucine ＋ β-naphthylamine pH 7.0 Diazo 535nm ① Bratton-Marshall reagent - diazonization ② Diazonium salt - diazonization ③ p-dimethylamin benzoldehyde ④p-dimethyaminocinnamaldehyde, 465nm (N-naphthylethylenediamine dihydrochloride).

  30. ι-Leucyl- p-nitroanilide ι-Leucine ＋ p-nitroaniline  410nm Aminopeptidase 5-aminosalicylic acid  + p-Xylenol   (Blue)                                                 635nm   Sod, Metaperiodate  ③ ι-Leucyl- p-nitroanilide Tuppy 법 p-dimethylaminocinnamaldehyde, 565nm NaNO2, N-1-naphthylethylenediamine,545nm NaNO2, m-tolydiethanol amin 530nm ④ ι-Leucyl –3-carboxy-4-hydroxyamide

  31. ι-Leucyl - β-naphthylamid 기질법 Reagent 1) Substrate : 1.37mmol/L ι-Leucyl - β-naphthylamid 2) Color reagent A : 0.5M Acetate buffer, pH 4.1 3) Color reagent B : 300mg/dl fast blue-B/0.1N HCl

  32. NORMAL VALUE Goldbarg-Rutenburg Male : 84 - 200 G-R unit Femal : 76 – 184 G-R unit ι-Leucyl amide Male : 13.0 – 28.0 IU/L Femal : 15.5 – 31.8 IU/L 간 외 담즙 울체 (폐쇄성 황달) 간 내 담즙 울체 (세담관성 간염 , 원발성 담즙성 간경변) 임신

  33. Isoenzyme • LDH, ALP-isoenzyme 방법과 동일 하게 실행한다 2. three band ( LAP-C, LAP-M, CAP ) 3. 기질 Leucyl - p-nitroanilide 4. N-naphthyl-ethylenediamine, sodium nitrite, Amm sulfomate • 기질 beta-naphthylamide 2. Fast garnet GBC, fast blue B, Fast black K Acryamide, Cellulose acetate, Agar gel

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