untitled

Clinical Pediatric Hematology-ncology Volume 20 ㆍ Number 1 ㆍ April 2013 REVIEW ARTICLE 적혈구효소검사 송정한 서울대학교의과대학분당서울대학교병원진단검사의학과 Junghan Song, M.D. Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul National University Bundang Hospital, Seongnam, Korea Among ~20 RBC enzyme deficiencies causing hereditary hemolytic anemia (HRA), deficiencies involving three RBC enzymes such as glucose-6-phosphatase, pyruvate kinase and pyrimidine 5 -nucleodiase were known to be relatively common. The methods that have been used for RBC enzyme analysis are based on the kinetic spectrophotometry. This method, however, usually requires multiple step reactions and manual manipulations which are labor-intensive and time-consuming, and carry a greater risk of error due to their complexity. To solve this problem, we had successfully developed the multiplex enzyme analysis for galactose using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). We are now trying to adopt this method to other RBC enzymes associated with HRA. The devised method will allow simple, rapid, sensitive and reproducible quantification of RBC enzymes and should be helpful for the confirmatory diagnosis of HRA caused by RBC enzyme deficiencies. Key Words: Hereditary hemolytic anemia, RBC enzyme deficiency, Mass spectrometry pissn 33-52 / eissn 33-4580 Clin Pediatr Hematol ncol 2013;20:8 12 Received on April 2, 2013 Revised on April 7, 2013 Accepted on April, 2013 Corresponding author: Junghan Song Department of Laboratory Medicine, Seoul National University Bundang Hospital, Seoul National University College of Medicine, 82, Gumi-ro 173 beon-gil, Bundang-gu, Seongnam 463-707, Korea Tel: +82-31-787-7691 Fax: +82-31-787-4015 E-mail: songjhcp@snu.ac.kr 서론적혈구포도당이대사되는과정에관여하는효소결핍은용혈빈혈을초래할수있고, 대개열성유전된다. 적혈구는미토콘드리아가없어에너지의대부분을포도당대사과정을통해얻는다. 포도당대사의 % 는 Embden-Meyerhof (EM) 경로로일어나는데포도당이유산으로대사되는과정에서 2M ATP가생성된다. 나머지 5-15% 는 hexose-monophosphate (HMP) shunt를통하여대사됨으로써적혈구가산화상태로되는것을방지한다. 즉 NH를생산하여산화혈색소를환 원시키는데, 산화제에노출되면 HMP shunt 활성이증가된다 [1]. 적혈구효소결핍을진단하는데는두가지단계가있는데, 첫단계는선별과정이고두번째단계는특정효소검사이다. 그다음유전자가확인된효소의경우유전자변이를검사할수있다. 삼투압취약성검사, 자가용혈검사등단순비특이적선별검사로대사이상유무를선별진단할수있다. 흔히결핍되는효소는 glucose-6-phosphate dehydrogenase (G6PD), pyruvate kinase (PK), pyrimidine 5 nucleotide (P5 N) 등이지만 [1,2], 당대사와핵산대사에관여하는어떤효소라도결핍되면용혈빈혈을일으킬수있기때문에의심이 8

Table 1. RBC enzymes associated with red cell metabolism and their substrate and products Enzyme Substrate Product Pathway Mayo/ SNUBH a) Phosphofructokinase Triose Phosphate isomerase dehydrogenase Phosphoglycerate kinase kinase Adenylate kinase peroxidase Aldolase dehydrogenase Glucose phosphate isomerase dehydrogenease Hexokinase Acetylcholinesterase Monophosphoglyceromutase Enolase Pyrimidine 5' nucleotidase reductase Lactate dehydrogenase Adenosine deaminase Fructose-6-phosphate 1,3-Disphosphoglycerate Phosphoenol pyruvate ATP 2 Fructose-1,6-diphosphate Fructose-6-phosphate Glucose Acetylthiocholine iodide 3-Phosphoglycerate 2-Phosphoglycerate CMP, AMP disulfide Adenosine Fructose-1,6-diphosphate Dihydroxyacetone phosphate 1,3-Disphosphoglycerate 3-Phosphoglycerate disulfide Pentose-5-phosphate Thiocholine 2-Phosphoglycerate Phosphoenol pyruvate Cytidine, adenine Lactate Inosine HMP HMP a) Enzyme tests performed in Mayo Clinic and Seoul National University Bundang Hospital (SNUBH)., Embden-Meyerhof passway; HMP, hexose monophosphate shunt. 될경우모든효소에대하여검사를실시하는것이적절하다. 그래서대개 G6PD, PK, PN 같이비교적빈도가높은효소에대해검사를먼저시행하고이들이이상이없을경우다른효소활성도검사를시행한다. 효소결핍이의심되면보통 20 여가지의효소를측정하는데 phosphofructokinase (PFK), triose phosphate isomerase (TPI), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), pyruvate kinase (PK), adenylate kinase (AK), glutathione reductase (GR), glutachioneperoxidase (GPx), lactate dehydrogenase (LD), aldolase (AD), glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenease (6PGDH), glucose phosphate isomerase (GPI), hexokinase (HK), acetylcholinesterase (AchE), adenosine deaminase (ADA), Monophosphoglyceromutase (MPGM), enolase (EN), pyrimidine 5 nucleotidase (P5 N) 등 20가지효소를정상대조결과와비교하여검사한다 (Table 1). 메이요클리닉에서는 G6PD, PK, GPI, HK, ADA, AK, PFK, PGK, TPI, P5 N 등의효소와 glutathione을검사하고있다 (Table 1) [3]. 분당서울대학교병원에서는일차적으로메이요클리닉에서검사하고있는 10종의효소검사에대해새로운검사법을확립하고있다. 이들효소들의기질, 대사산물, 관여하 는대사과정은 Table 1과같다. 검사방법적혈구효소검사법으로대부분 NAD나 NADH ( 혹은 N나 NH) 의산화ㆍ환원을 340 nm에서 10분간의 kinetics로측정하는분광광도법 (spectrophotometry) 이이용되었다 [4]. 그러나이방법은효소반응과정이여러단계를거쳐복잡하고모든과정을수작업을해야하며, 정확도와특이도가떨어지는단점이있다. 최근에 UPLC-MS/MS 방법이다양한효소검사에이용되고있다 [5,6]. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) 를이용한방법은단일효소반응으로생성된산물을직접특이적으로측정할수있고, 동시에여러효소검사를한꺼번에측정할수있는장점이있다. UPLC-MS/MS 방법을이용한구체적인검사법은다음과같다. 1) 검체항응고제로는 acid-citrate dextrose (ACD), heparin, EDTA가모두가가능하나 ACD 항응고제가가장좋은것으로되어있고, 효소와항응고제에따라차이가있지만, 냉장보관하면 6-20일간 Clin Pediatr Hematol ncol 9

Junghan Song 안정하다 [4]. 채혈당일에검사실에냉장상태로도착하는것을권장한다. 재검등을고려할때 10 ml 정도의전혈이필요하다. 2) 검사원리각각의효소반응에서생긴산물을 UPLC-MS/MS를이용하여정량함으로써적혈구효소활성도를측정한다. 3) 검체전처리신선전혈을 1.5 ml 튜브에가득담은후 15,000 g에서 3분원심분리한다. 상층혈장과백혈구층을제거하여버린후남아있는적혈구에동량만큼생리식염수를넣어혼합한후다시원심분리한다. 같은방법으로 1회더세척한다. 상층생리식염수를제거하고남은적혈구를적절한양으로분주하여 70 o C에서검사전까지보관한다. 4) 검사재료각검사반응에필요한시약은 Beutler가기술한책을참고하였다 [4]. 본효소반응은 1차반응만을하기때문에상기방법의일부를변형하였고, 효소반응에필요한시약은 Table 2 에요약하였다. 5) 효소반응구체적인효소반응방법은다음과같다. 표준용액, 대조검체및그외필요한시약을조제하여준비한다. 환자검체는희석액 (0.001% EDTA, 0.00005% mercaptoethanol) 을이용하여해당희석배수만큼희석하여준비한다. 한검체당 1.5 ml 튜브 3개씩준비한다. 두개는검체를반복측정하기위해, 한개는공시료로사용한다. 검체를검사항목별해당용량만큼분주 ( 이때기벽에묻지않게주의한다 ) 후각각의공시료는먼저 95 o C 5분동안가열하여효소활성도를없앤다. 공시료의가열처리가끝나면다음의단계를조작한다. 각각의효소반응에필요한시약을해당양만큼넣어잘혼합한다. 37 o C에서 30분간항온한후 95 o C에 5분가열하여효소반응을중지시킨다. 95 o C 가열이끝나면 15,000 g에서 5분간원심분리한다. 각해당항목의검사가완료되면 1번, 2번, 3번패널검사에해당하는각항목별검체의상층 20 μl를 3개의패널튜브에넣어잘혼합한다. 잘혼합된검체를 microplate에옮겨 LC-MS/MS로분석한다. 검사가끝난희석된검체는혈액자동분석기를이용하여혈색소치를측정한다. 6) LC-MS/MS 측정방법효소반응이끝난반응액에서효소반응산물은 UPLC-MS/MS 를이용하여다음과같은조건으로정량한다. 장비로는 Xevo TQ-MS (Waters, Watford, UK) 를사용하고, UPLC 컬럼은 ACQUITY UPLC HSS C18 T3 컬럼 (Waters) 을사용한다. 이동상 A로 DW에 0.1% NH 4 H를, 이동상 B로 acetonitrile에 0.1% NH4H를각각사용한다. 이동상은 1.8분까지는 A 이동상을 100% 에서 % 로유지하다가 2.0분부터 A 이동상을 10% 로낮춘후 3분까지유지하고 3.2분에다시 100% 로돌아온다. 속도는 0.3 ml/min로유지한다. 탠덤질량분석에서는 multiple reaction monitoring (MRM) 방법을이용하여효소반응산물을정량하는데구체적인 MRM transition 및질량분석기의조건은 Table 3와같다. Table 2. Substrate, RBC dilution & volume, buffer and other reagents for each enzyme reaction Panel Enzyme Substrate (μl) RBC dilution & volume (μl) Buffer (μl) Mg ++ buffer (μl) 20 mm ATP (μl) 30 mm (μl) 2 mm N (μl) D.W (μl) 1 2 3 PFK PK P5'N ADA G6PD AK GPI TPI PGK HK 20 mm F-6-P mm PEP 4.6 mm CMP 4 mm adenosine 6 mm G-6-P 20 mm AMP 20 mm F-6-P 30 mm GA3P 100 mm 3PGA 20 mm glucose 100 10 80 200 200 5 100 180 120 120 PFK, phosphofructokinase; PK, pyruvate kinase; P5 N, pyrimidine 5' nucleotidase; ADA, adenosine deaminase; G6PD, glucose- 6-phosphate dehydrogenase; AK, adenylate kinase; GPI, glucose phosphate isomerase; TPI, triose phosphate isomerase; PGK, phosphoglycerate kinase; HK, hexokinase. 10 Vol. 20, No. 1, April 2013

Table 3. MS/MS conditions and MRM transitions for each compound Panel Compound Parents (m/z) Daughter (m/z) Cone (V) Collision (ev) 1 2 3 Cytidine Inosine Fructose 1,6-diphosphate Dihydroxyacetone phosphate 87.0 242.0 267.0 274.9 338.9 8.9 4.8 169.0 8.9 4.8 43.0 109.0 135.0 6.9 97.0 198.8 134.0 96.8 198.8 134.0 24 35 19 35 8 13 20 12 18 21 21 Fig. 1. Request (A) and report (B) forms for RBC enzyme analysis. 7) 검사의뢰양식및보고양식검사의뢰양식과보고양식은 Fig. 1과같다. 검사의뢰서에는환자의인적사항이외에, 임상증상, ( 추정 ) 진단명, 의뢰처관련정보를상세히기록하여야한다. 검사보고는재검여부및검사항목에따라다르지만 2-4주정도소요된다. 결론적혈구효소검사는효소결핍에의한유전성용혈빈혈을확진하는데중요한검사이다. 지금까지의검사방법으로는복 잡한과정의효소반응거쳐생성된효소반응산물을분광광도법으로정량하는방법이이용되었다. 그러나이방법은복잡하고정확도및특이도가떨어지는단점때문에정확한진단적가치를제공하지못하였다. 이번에새롭게개발되고있는 UPLC-MS/MS 방법은단일효소반응으로생성된산물을직접특이적으로측정할수있는장점이있어효소결핍에의한유전용혈빈혈을진단하는데도움이될것으로기대된다. 감사의글 This work supported by the Hereditary Hemolytic Anemia Clin Pediatr Hematol ncol

Junghan Song Working Party of the Korean Society of Hematology. 참고문헌 1. Kim HK, Lee YJ, Lee EY, Lee JN, Cho HI, Han JY. Hemolytic anemia. In: Korean Society of Laboratory Medicine. Textbook of laboratory medicine. 4th ed. Seoul, Korea: E-Public, 2009: 133-4. 2. Hah J. Red blood cell enzymopathies causing hereditary hemolytic anemia. Clin Pediatr Hematol ncol 2012;19:1-6. 3. Metabolic Hematology Laboratory. Mayo Medical Laboratories. Rochester, MN: Mayo Clinic, 20. (Accessed April, 6, 2013 at http://www.mayomedicallaboratories.com/mediax/test-info/hematology/metabolic.pdf) 4. Beutler E, ed. Red cell metabolism. A manual of biochemical methods. 2nd ed. New York, NY: Grune & Stratton, 1975. 5. Ko DH, Jun SH, Park KU, Song SH, Kim JQ, Song J. Newborn screening for galactosemia by a second-tier multiplex enzyme assay using UPLC-MS/MS in dried blood spots. J Inherit Metab Dis 20;34:409-14. 6. Ko DH, Jun SH, Park HD, et al. Multiplex enzyme assay for galactosemia using ultraperformance liquid chromatographytandem mass spectrometry. Clin Chem 2010;56:764-71. 12 Vol. 20, No. 1, April 2013