저작자표시 - 비영리 - 변경금지 2.0 대한민국 이용자는아래의조건을따르는경우에한하여자유롭게 이저작물을복제, 배포, 전송, 전시, 공연및방송할수있습니다. 다음과같은조건을따라야합니다 : 저작자표시. 귀하는원저작자를표시하여야합니다. 비영리. 귀하는이저작물을영리목적으로이용할

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1 저작자표시 - 비영리 - 변경금지 2.0 대한민국 이용자는아래의조건을따르는경우에한하여자유롭게 이저작물을복제, 배포, 전송, 전시, 공연및방송할수있습니다. 다음과같은조건을따라야합니다 : 저작자표시. 귀하는원저작자를표시하여야합니다. 비영리. 귀하는이저작물을영리목적으로이용할수없습니다. 변경금지. 귀하는이저작물을개작, 변형또는가공할수없습니다. 귀하는, 이저작물의재이용이나배포의경우, 이저작물에적용된이용허락조건을명확하게나타내어야합니다. 저작권자로부터별도의허가를받으면이러한조건들은적용되지않습니다. 저작권법에따른이용자의권리는위의내용에의하여영향을받지않습니다. 이것은이용허락규약 (Legal Code) 을이해하기쉽게요약한것입니다. Disclaimer

2 의학박사학위논문 질량분석기를이용한 유전용혈빈혈관련적혈구막단백및 효소의새로운정량분석법개발 2016 년 8 월 서울대학교대학원의학과검사의학전공박철민

3 국문초록 유전용혈빈혈은적혈구막단백의이상이나적혈구효소의이상등이원인이다. 적혈구막단백에대한검사는고전적인전기영동법을이용해왔으나재현성이떨어지며시간과노력이많이든다. 적혈구효소이상은분광광도법을이용하여측정하였으나, 다중검사가어렵고질환원인효소가다양하여효율이떨어진다. 이에최신의질량분석기법을이용하여유전용혈빈혈의원인중적혈구막단백의이상과적혈구대사효소의이상진단에서기존검사법보다개선된검사법을개발하고, 그성능을검증하고자한다. 적혈구막단백분석은질량분석기를이용하여적혈구막단백유래펩티드에서임상적으로유의한적혈구막단백을찾았다. 기존에알려진 band3, spectrin alpha, spectrin beta, protein 4.1, protein 4.2, ankyrin 에대해식별하였다. 정밀도를평가하고기존방법과비교하였고참고범위를설정하였다. 적혈구대사효소분석은 glucose-6-phosphate dehydrogenase (G6PD), pyruvate kinase (PK), pyrimidine 5 -nucleotidase (P5 N), hexokinase (HK), triosephosphate isomerase (TPI), adenosine deaminase (AD) 등주요효소 6 가지에대해질량분석기를이용한검사법개발을위해 ion suppression 을평가하고검체희석배수와반응시간을최적화하였다. 새검사법의직선성, 정밀도를확인하였고참고범위를설정및환자예비검사를하였다. 질량분석기를이용한적혈구막단백분석에서는임상적으로유의한 i

4 6가지막단백을식별하였으며, 막단백측정의변이계수 (coefficient of variation, CV) 는 % 범위였다. 새검사법을기존검사법인 SDS- PAGE 법과비교한결과 Spectrin alpha와 beta의합 (r=0.8154, P=0.014) 과 ankyrin(r=0.8743, P=0.005) 은강한상관관계를, protein 4.1과 protein 4.2는유의한상관성이없었다. 18명의환자중 protein 4.2 9명, band 3 7명, ankyrin 6명, spectrin alpha 2명, specrn beta 4명에서결핍이있었다. 질량분석기를이용한적혈구효소검사법의직선성은모든효소가우수하였다. 정밀도평가결과각효소측정값의 CV 범위는 % 였다. 각효소의참고범위는 G6PD ( mg/min/ghb), PK ( mg/min/ghb), P5 N ( μg/min/ghb), HK ( μg/min/ghb), TPI ( μg/min/ghb), AD ( μg/min/ghb) 이었다. 16명의환자검사결과 6명에서 G6PD, 4명에서 PK, 각 1명에서 P5 N과 HK의활성도감소가관찰되었다. 본연구를통해새롭게개발된질량분석을이용한적혈구막단백과적혈구효소에대한검사법이향후유전용혈빈혈의진단에도움이될수있을것으로생각한다 주요어 : 용혈빈혈, 적혈구막단백, 적혈구효소, 질량분석기학번 : ii

5 CONTENTS Abstract... i Contents... iii List of Tables... iv List of Figures... v List of Abbreviations and Symbols... vi 서론... 1 연구재료및방법 적혈구막단백분석 적혈구효소분석 연구결과 적혈구막단백분석 적혈구효소분석 고찰 참고문헌 영문초록 iii

6 LIST OF TABLES Table 1. Patients group characteristics for erythrocyte membrane protein analysis Table 2. List of standard solutions for each enzyme reaction Table 3. Reaction conditions for each erythrocyte enzyme Table 4. Conditions for tandem mass spectrometry and multiple reaction monitoring (MRM) transitions of products and internal standards Table 5. Patients group characteristics for erythrocyte enzyme Table 6. Major identified erythrocyte membrane proteins Table 7. Precision of six erythrocyte membrane protein amounts Table 8. Reference intervals of six erythrocyte membrane proteins Table 9. Proportions of six membrane proteins in suspicious patients with erythrocyte membrane disorders Table 10. Protein deficiencies in suspicious patients with erythrocyte membrane disorders Table 11. Optimized reaction conditions of dilution rate and incubation time Table 12. Within-run and between-run precisions at low and high concentrations Table 13. Pilot study results of suspicious patients with erythrocyte enzymopathy iv

7 LIST OF FIGURES Figure 1. Reaction scheme for six erythrocyte enzymes Figure 2. Representative UPLC-MS/MS MRM chromatograms of G6PD, PK, and P5 N (Set A) Figure 3. Representative UPLC-MS/MS MRM chromatograms of HK, TPI, and AD (Set B) Figure 4. Effect of sample amount on enzyme activity for each enzyme reaction. 44 Figure 5. Effect of reaction time on enzyme activity for each enzyme reaction Figure 6. Linearity for each enzyme reactions Figure 7. Comparison of the G6PD activities measured by newly developed assay and old spectrophotometric assay v

8 LIST OF ABBREVIATIONS AND SYMBOLS ACN AD ADP AK ATP CMP DHAP DTT EDTA EMA G6P G6PD Gal-1-P GAP GPI HK NADP P5'N PEP PFK PFOA acetonitrile adenosine deaminase adenosine diphosphate adenylate kinase adenosine triphosphate cytidine monophosphate dihydroxyacetone phosphate dithiothreitol ethylenediaminetetraacetic acid eosin-5-maleimide binding test glucose-6-phosphate glucose-6-phosphate dehydrogenase galactose-1-phosphate glyceraldehyde-3-phosphate glucose phosphate isomerase hexokinase nicotinamide adenine dinucleotide phosphate pyrimidine 5' nucleotidase Phosphoenolpyruvate phosphofructokinase pentadecafluorooctanoic acid vi

9 PGK PK PMSF SDS-PAGE phosphoglycerate kinase pyruvate kinase phenylmethane sulfonyl fluoride sodium dodecyl sulfate-polyacrylamide gel electrophoresis TFA TPI UPLC-MS/MS trifluoroacetic acid triose phosphate isomerase ultra-performance liquid chromatography- tandem mass spectrometry vii

10 서론 유전용혈빈혈 (hereditary hemolytic anemia) 는유전적으로발생하는용혈빈혈의통칭으로원인에따라적혈구막의이상, 적혈구대사 ( 효소 ) 이상, 혈색소 (hemoglobin) 변이에의한것등세가지로분류한다. 유전용혈빈혈은임상적으로빈혈, 황달, 비장비대등의임상소견과망상적혈구수 (reticulocyte count), 말초혈액도말, 혈청합토글로빈, LDH, 빌리루빈등의검사소견을토대로의심할수있다 [1]. 먼저, 적혈구막의이상에의한유전용혈빈혈은대표적으로유전성구상적혈구증 (hereditary spherocytosis), 유전성타원적혈구증 (hereditary elliptocytosis), 유전성난형적혈구증 (hereditary ovalocytosis) 등이있다. 적혈구막단백과골격단백은서로유기적으로결합하여적혈구의형태를유지하고변형성을갖게한다. 이런역할을하는대표적인단백으로는 band 3, spectrin alpha, spectrin beta, ankyrin, protein 4.1, protein 4.2 등이알려져있으며, 이들막단백또는골격단백의이상에의해적혈구의상대적인표면적의감소, 세포내점도의증가, 세포막변형성의감소등이일어나게되면용혈빈혈을초래하는것으로알려졌다 [2,3]. 적혈구막단백질환의유병률은유전성구상적혈구증의경우서구에서는 1:2,000-5,000, 유전성타원적혈구증은서아프리카에서 1:50, 유전성난형적혈구증은동남아시아에서 1:4-20으로비교적흔히발생한다 [4,5]. 지금까지임상적으로적혈구막단백질환에의한 1

11 유전용혈빈혈이의심되면선별검사로전통적으로삼투압취약성검사 (osmotic fragility test) 를시행해왔으나민감도, 특이도가낮고재현성이떨어져검사의신뢰성이낮은단점이있다. 이에따라최근에는민감도, 특이도가향상된 EMA (eosin-5-maleimide)-binding test) 를권장하고있다 [6]. 적혈구막단백이상의확진검사로는전기영동법인 SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) 방법을이용해왔으나 [7], 이검사법은 1차원 PAGE를이용하여각단백질밴드의농도를비교하는것으로검사의재현성이떨어지며, 적혈구막단백질환이강력히의심되는환자에게서도검사결과가음성이나오는예가많아서그임상적유용성에한계가있다. 또한, 모든검사과정이수작업으로이루어지고검사소요시간이최대 1주일정도되어임상검사실에서진단검사로서쉽게사용하기어려운점이있다. 따라서재현성이있으면서시간과비용을줄일수있는새로운검사방법이필요하다. 적혈구대사이상이원인인유전용혈빈혈은적혈구내에너지대사과정에관여하는효소의이상이원인이다. 흔히결핍되는효소로 glucose-6-phosphate dehydrogenase (G6PD) 나 pyruvate kinase (PK), pyrimidine 5 -nucleotidase (P5 N) 등이지만 [8], hexokinase (HK), triosephosphate isomerase (TPI), adenosine deaminase (AD) 등적혈구내당대사와핵산대사에관여하는다른효소의이상이있어도용혈빈혈을일으킬수있으므로진단을위해서많은수의효소에대해검사가필요할수있다. 효소이상중 G6PD 결핍증의경우, 아프리카, 중동, 동남아지역에서최대 30% 의 2

12 유병률을보이며 [9], 우리나라는 0.9%-3.5% 정도로보고되고있다 [10]. 현재우리나라는외국에비해유병률이비교적낮으나국제결혼이나이주노동자의급속한증가등으로인해환자가점점증가할것으로예상된다. 유전용혈빈혈관련대사효소이상의진단은효소반응을이용하여효소의활성도를측정하거나분자유전학적방법으로직접효소유전자의돌연변이를검사하는방법이있다 [11]. 이중효소의활성도를측정하는검사는대부분 NAD나 NADH ( 또는 NADP나 NADPH) 의산화환원반응을 340 nm 에서 10분간의 kinetics로측정하는분광광도법이이용되었다 [12]. 그러나이방법은효소반응과정이여러단계를거쳐복잡하고모든과정을수작업으로해야하며, 효소산물이아닌조효소를측정하기때문에정확도와특이도가떨어질수있는단점이있다. 이런단점을극복하기위한방법으로최근에질량분석기를응용한검사법이개발되어다양한대사이상질환의검사에적용되고있다 [13 15]. 질량분석기를이용한검사법은단일효소반응으로생성된산물을직접특이적으로측정할수있고, 동시에여러효소검사를한꺼번에할수있는다중검사가용이한장점이있으므로, 적혈구효소검사에대해서도질량분석기를이용하면기존검사의단점을보완할수있으리라예상된다. 본연구를통해질량분석기를이용하여유전용혈빈혈의원인중적혈구막단백 (band 3, spectrin alpha, spectrin beta, protein 4.1, protein 4.2, ankyrin) 의 3

13 이상과적혈구대사효소 (G6PD, PK, P5 N, HK, TPI, AD) 의이상을보다쉽고정확하게검출할수있는검사법을개발하고, 그검사법의성능을검증하여임상에서실제로유전용혈빈혈의진단검사로활용할수있도록하고자한다. 4

14 연구재료및방법 1. 적혈구막단백분석 1.1. 시약 본연구에사용한 acetonitrile (ACN; Fisher Scientific, Pittsburgh, PA, USA) 과증류수는 HPLC grade를사용하였다. Ethylene-diaminetetraacetic acid (EDTA), phenylmethane sulfonyl fluoride (PMSF), ammonium bicarbonate, ammonium formate, dithiothreitol (DTT), iodoacetamide, trifluoroacetic acid (TFA), HCl는 Sigma Aldrich (St. Louis, MO, USA) 에서제조한제품을사용하였다. Sequencing grade의 modified trypsin 은 Promega (Medison, WI, USA) 제품을사용하였다. 저장성용해액 (hypotonic lysis solution) 은 5 mm의 Tris- HCl, 0.1 mm의 EDTA, 0.2 mm의 PMSF로제조하였고, ph 조절은 0.5 M ammonium bicarbonate을사용하였다. 5

15 1.2. 검체전처리 EDTA 용기에채취한전혈검체를생리식염수로 3회세척후원심분리하고상청액을제거하였다. 혈색소오염제거를위해총 3회에걸쳐저장성용해액 40 ml를혼합하여 15,000 rpm에서 20 분간원심분리하였다. 이후 Membrane Protein Extraction Kit (BioVision, Milpitas, CA, USA) 를이용하여단백을추출하였다. 추출한단백질은 50 mm ammonium bicarbonate와 RapiGest SF (Waters, Milford, MA, USA) 100 μl와혼합하여변성시켰다. 단백농도측정은 Quick Start Bradford Protein Assay Kit (BioRad, Hercules, CA, USA) 를이용하였다. 80 에서 20분동안가열한다음, dithiothreitol을첨가하여농도를 5mM로맞추고 60 에서 30분간가열하였다. 5분간상온에냉각시킨후, 150 mm의 iodoacetamide를혼합했고암실에 30분간두었다. Modified trypsin을 1:50(w/w) 비율로섞은다음 37 에서하룻밤반응시켰다. 분해된단백액에 1M HCl를혼합하고, 30분동안 37 에서반응시킨다음 Oasis HLB Cartridge (Waters) 으로탈염하고 SpeedVac Concentrator (Thermo Fischer Scientific, Bremen, Germany) 로용매를건조시켰다. 100 mm ammonium formate 용액을넣어최종단백의양을 500 fg으로조절하였다. 6

16 1.3. 액체크로마토그래피 - 질량분석기를이용한분석 전처리과정이끝난검체는액체크로마토그래피장비인 nanoacquity ultra-performance liquid chromatography (UPLC) 와질량분석기 Synapt MS (Waters) 를이용하여분석하였다. 약 600 ng의전처리된검체를주입하여첫번째 trapping column (XBridgeTM BEH C18, 5 μm, 300 μm 50mm; Waters) 를용리액 (eluent) 97:3(v:v) 20 mm ammonium formate + 용매 (A: 증류수, B:ACN) 과함께 1 μl/min 속도로통과시켰다. 첫번째 trapping column에서는 ACN(55%) 의단일농도구배를이용하였다. 1차 trapping column에서분리된펩티드는 dilution tee로옮겨지고 99.9:0.1(v:v) 0.1 formic acid + A: 증류수, B: ACN 이동상 (mobile phase) 을이용하여 20 μl/min 속도로용출하였다. 2차 trapping column (Symmetry C18, 5μm, 180 μm 20 mm; Waters) 을이용하여확보한펩티드를 analytical column (BEH C18, 1.7 μm, 75 μm 150 mm; Waters) 를이용하여분리하였다. 2-50% 의이동상의선형농도구배를통해 110분이상 300 nl/min의속도로 110분이상분리용출하였다. 용출시온도는 40 로유지하였다. 정량을위한내부표준물질로 Saccharomyces cerevisiae (P00924) 기원의 enolase를첨가하였다. m/z 값보정을위해 30% ACN + 0.1% formic acid 용액에 400 fmol/μl의 [Glu1]-Fibrinopeptide B 를혼합하여 2 μl/min으로분무하였다. 질량분석기 (MS) 는 10,000 FWHM의해상도로 positive ion mode에서실행하였고, m/z 50-1,990 범위에서연속모드로시행하였다. 탠덤 7

17 질량분석 (MS/MS) 데이터는 ev 충돌에너지범위를동시에 모니터링하여얻었다. 8

18 1.4. 데이터처리및분석 질량분석을통해얻은원시데이터파일은 PLGS 2.4 소프트웨어 (Waters) 로처리하였다. 단백식별은 Uniprot reviewed human protein database[16] 를이용하였다. 데이터베이스를통해확인된단백은가장양이많은 3 개의펩티드의 peak intensity 를이용하여정량하였다 [17]. 내부표준물질로첨가한 enolase 양과비교하여 column 에주입된각단백의양을정하였고, 최종단백의양은 column 에분주한총단백중비율에서 100 배를곱하여계산하였다. 9

19 1.5. 검사법성능평가 새검사법의성능을평가하기위해 6가지단백에대해정밀도를평가하고기존검사법인 Fairbanks SDS-PAGE 법과새검사법의검사결과를서로비교하였다. 새검사법의정밀도평가는환자검체를 5일간중복측정 (duplicate) 하여평가하였고, 기존검사법과의비교평가는유전용혈빈혈의심환자를포함한 8명의검체를분석하여 band 3에대한각각의단백의비율을구한다음기존검사법에의한결과와서로비교하였다. 10

20 1.6. 정상인잔여검체를이용한참고범위설정및환자검 체예비검사 참고범위는혈액질환이없고혈구감별계산결과가정상인 47명의임상검체를이용하여설정하였고, 그중남자는 29명이었다. 평균나이는 22.4세 ( 범위 0-61세 ) 였고, 평균혈색소농도는 13.9 g/dl ( 범위 ) 였다. 참고범위는전체단백에서차지하는각단백의비율로표기하였고, 전체결과의 2.5와 97.5 백분위로구하였다. 새검사법에대한예비검사는 18명의환자군을대상으로하였고, 개별환자의임상적특성은 Table 1에정리하였다. 11

21 Table 1. Patients group characteristics for erythrocyte membrane protein assay evaluation Case No. Sex Age (yrs) Hb (g/dl) Osmotic fragility Spherocyte Transfusion Hx* 1 M F NA +/- - 3 F M M F *Transfusion Hx means whether a patient had received erythrocyte transfusions within three months before a testing or not. Abbreviations: Hx, history; NA, not available + - +/- - + NA F 12d 9.4 NA + NA 8 M Normal M M NA M F F 4mo F M F 41d F F /

22 1.7. 통계분석 분석소프트웨어는 IBM SPSS Statistics 21 (IBM Corporation, Armonk, NY, USA) 및 Excel 2010 (Microsoft Corporation, Redmond, WA, USA) 을사용하였다. 정밀도평가결과는는측정값에대한변이계수 (CVs; coefficients of variations) 로나타내었다. 기존검사법과의비교는선형회귀분석을수행하였고, 상관계수 (correlation coefficient, r) 와유의확률을구하였다. 참고범위는정상인검체측정결과의 2.5와 97.5 백분위수로설정하였다. 13

23 2. 적혈구효소분석 2.1. 시약 본연구에사용한 ACN (Fisher Scientific, Pittsburgh, PA, USA) 과증류수는 HPLC grade를사용하였다. 반응에서필요한각효소마다필요한기질, 산물과반응에필요한 NADP, ATP, ADP, Tris buffer, HCl, EDTA, MgCl 2, KCl과이동상에서사용한 formic acid와 pentadecafluorooctanoic acid (PFOA) 는모두 Sigma Aldrich (St. Louis, MO, USA) 에서제조한제품을사용하였다. 그리고 triosephosphate isomerase의활성도를측정하기위하여사용된 α-glycerophosphate dehydrogenase 효소도역시 Sigma Aldrich에서구매하였다. 14

24 2.2. 검사원리 검사의원리는 Figure 1과같다. G6PD, PK, P5 N, HK, AD 효소의경우각각의효소반응산물인 6-phosphogluconate, pyruvate, cytidine, [ 13 C 5 ]glucose-6-phophate, inosine을액체크로마토그래피-탠덤질량분석기로직접측정하는원리이다. TPI 효소의경우에는효소반응산물인 dihydroxyactone phosphate의분자량이기질인 D-glyceraldehyde-3- phosphate와같고구조도비슷하여액체크로마토그래피- 탠덤질량분석기로는구분이불가능하기때문에부득이하게 2차효소반응을시킨후생성된 glycerol-3-phosphate를측정하였다. 15

25 Figure 1. Reaction scheme for six erythrocyte enzymes Abbreviations: G6PD, glucose-6-phosphate dehydrogenase; PK, pyruvate kinase; P5 N, pyrimidine 5 nucleotidase; HK, hexokinase; TPI, triosephosphate isomerase; AD, adenosine deaminase 16

26 2.3. 검체전처리 전혈검체를 1.5 ml 튜브에가득담고 15,000g에서 3분원심분리하였다. 상층혈장과백혈구층을제거하여버린후남아있는적혈구에동량만큼생리식염수를넣어혼합한후다시원심분리하였다. 같은방법으로 2회더세척한후, 상층액을제거하고남은적혈구를적절한양으로분주하여 -70 에서검사전까지보관하였다. 17

27 2.4. 효소반응 표준용액의제조 각효소의산물인 6-phosphogluconate, pyruvate, cytidine, [ 13 C 5 ]glucose-6- phosphate, glycerol-3-phosphate, inosine을각각증류수에녹여서필요한가장높은표준용액을제조하여그용액을다시희석하여필요한표준용액을각각제조하였다. 가장낮은농도의표준용액은증류수를사용하였다. 각각의효소의활성을고려하여각각제조한표준용액의농도는 Table 2과같았다. 18

28 Table 2. List of standard solutions for each enzyme reaction (unit: μg/ml) Product STD 0 STD 1 STD 2 STD 3 STD 4 6-phosphate gluconate ,000 2,000 4,000 Pyruvate ,000 2,000 4,000 Cytidine [ 13 C 5 ]glucose-6-phosphate ,000 2,000 Glycerol-3-phosphate ,000 Inosine Abbreviations: STD, standard solution 19

29 적혈구효소반응 이미제조한표준용액과대조검체및그외필요한시약을조제하여준비하고, 환자검체는희석액 (0.001% EDTA, % mercaptoethanol) 을이용하여희석하여준비하였다. 한검체당 1.5 ml 튜브 3개씩준비하여 2개는중복측정 1개는공시료 (sample blank) 로사용하였다. 공시료는적혈구효소의활성도를없애기위하여 95 에서 5분동안가열한후 다음의단계를조작하였다. 각각의효소반응에필요한완충용액과 기질, 그리고그외반응에필요한시약을각각의정해진양만큼넣고 37 에서반응시켰다 (Table 3). 정해진반응시간이끝나고효소반응을중지시키기위해 95 에 5분가열하였고, 13,000g에서 5분간원심분리하였다. 원심분리가끝난후세트검사에해당하는각효소별검체의상층을 2개의세트튜브에넣어잘혼합하였다. 세트의구성은사용하는시약과기질및산물에따라서세트 A는 G6PD (10 μl), PK (40 μl), P5 N (50 μl), 세트 B는 HK (40 μl), TPI (40 μl), AD (30 μl) 으로구성하였다. 검사가끝나고남은희석검체는혈액자동분석기를이용하여혈색소치를측정하였다. 20

30 Table 3. Reaction conditions for each erythrocyte enzyme Set Enzyme Substrate erythrocyte Buffer Additional reagents Internal standard A G6PD 200 μl 20mM G6P 20μL 50 μl buffer1* 200 μl 10mM NADP 20 μl 0.2 mm [ 13 C]Gal-1-P A PK 100 μl 25mM PEP 20μL 100 μl buffer1* 100 μl 1M KCl 50 μl 30mM ADP 20 μl 0.2 mm [ 13 C]-Gal-1-P A P5 N 100 μl 4.6mM CMP 20μL 50 μl buffer1* 20 μl 1 mg/ml [ 13 C 5 ]cytidine B HK 200 μl 10mM [ 13 C 5 ]glucose 20μL 100 μl buffer1* 400 μl 20mM ATP 20 μl 0.2 mm [ 13 C]Gal-1-P B TPI 20 μl 5mM D-GAP 20μL 100 μl buffer2 50 μl α-glycerophosphate 100 μl NADH dehydrogenase 20 μl 0.2 mm [ 13 C]Gal-1-P B AD 100 μl 2mM adenosine 20μL 100 μl buffer2 20 μl 1 mg/ml [ 13 C 5 ]inosine *buffer1: 1M Tris-HCl with 5mM EDTA and 0.1M MgCl 2, ph 8.0 buffer2: 1M Tris-HCl with 5mM EDTA, ph 8.0 Abbreviations: G6PD, glucose-6-phosphate dehydrogenase; PK, pyruvate kinase; P5 N, pyrimidine 5 nucleotidase; HK, hexokinase; TPI, triosephosphate isomerase; AD, adenosine deaminase; G6P, glucose-6-phosphate; PEP, phosphoenolpyruvate; CMP, cytidine monophosphate; D-GAP, D-glyceraldehyde-3- phosphate; NADP, nicotinamide adenine dinucleotide phosphate; ADP, adenosine diphosphate; ATP, adenosine triphosphate; Gal-1-P, galactose-1-phosphate 21

31 2.5. 액체크로마토그래피 - 질량분석기를이용한분석 질량분석기는 Quattro Premier MS/MS (Waters) 를사용하였고, UPLC의 column은 ACQUITY UPLC BEH Amide ( mm, 1.7 μm, Waters) 를사용하였다. 이동상 A로증류수에 0.1% formic acid와 0.1% pentadecafluorooctanoic acid (PFOA) 를, 이동상 B로 ACN에 0.1% formic acid와 0.1% PFOA를각각사용하였다. 이동상은 2분까지는이동상 A를 100% 으로유지하다가 2분부터이동상 A를 70% 로 2.5분까지유지하고 2.5분에다시 100% 로돌아오게하였다. 유속은 0.2 ml/min을유지하게하였다. 탠덤질량분석기에서 multiple reaction monitoring (MRM) 방법을이용하여정량하였다. 구체적인 MRM transition 및질량분석조건은 Table 4과같다. 22

32 Table 4. Conditions for tandem mass spectrometry and multiple reaction monitoring (MRM) transitions of products and internal standards Collision MRM transition Enzyme Compound Cone (V) (ev) (m/z) G6PD 6-phosphogluconate PK Pyruvate P5 N Cytidine HK [ 13 C 5 ]glucose-6-phosphate TPI Glycerol-3-phosphate AD Inosine IS1* [ 13 C]glactose-1-phosphate IS2 [ 13 C 5 ]cytidine IS3 [ 13 C 5 ]inosine *IS1: internal standard for G6PD, PK, HK, and TPI. IS2: internal standard for P5 N IS3: internal standard for AD Abbreviations: G6PD, glucose-6-phosphate dehydrogenase; PK, pyruvate kinase; P5 N, pyrimidine 5 nucleotidase; HK, hexokinase; TPI, triosephosphate isomerase; AD, adenosine deaminase 23

33 2.6. Ion suppression 평가 Post-column infusion 방법을이용하여 ion suppression을평가하였다 [18]. 실제검사와같은조건으로공시료를검사하면서각효소반응의산물또는 IS을하나씩 infusion pump를이용하여일정하게질량분석기로동시에주입한다. 이때주입하고있는산물또는 IS가나오는 retention time에서의결과의변화를관찰하여 matrix의영향을평가하였다. 24

34 2.7. 적혈구검체양과반응시간에따른각효소반응에 대한효과 적혈구검체량과각효소반응에대한효과를보기위하여반응시간은 30분으로고정하고전처리과정을거친적혈구검체와희색액을이용하여희석배수를 G6PD는 5배, 10배, 20배, 40배, 그외의효소는 2배, 5배, 10배, 20배인검체를제조하여각희석배수마다세개씩검사하였다. 반대로반응시간에따른각효소반응의결과를보기위하여희석배수를고정하고각효소별 4가지반응시간조건 (G6PD, HK, AD는 10, 20, 30, 40분, PK는 10, 20, 30, 45, 60분, P5 N은 60, 120, 180, 240분, TPI는 30, 60, 90, 120분 ) 에서각반응시간마다세개씩검사하였다. 25

35 2.8. 각반응에대한정밀도평가 Within-run에대한정밀도평가를하기위해서높은농도의검체와낮은농도의검체를 5번씩측정하여 CVs 값을계산하였으며, Betweenrun에대한정밀도평가를하기위해서높은농도와낮은농도의검체를 5일연속하여측정하여 CVs값을각각구하였다. 26

36 2.9. 각반응에대한직선성평가 각효소반응마다표준용액을희석하여농도를알고있는 5 개의검체를 제조하고, 실제측정결과와예상농도를선형회귀법으로비교하여 직선성을평가하였다. 27

37 2.10. 검사법비교 기존방법과새로운검사법비교를위해 G6PD의 CAP 외부정도관리검체 5개와 G6PD 검사용정도관리물질 (Biochemical Enterprise, Milan, Italy) 3개를이용하였다. 총 8개검체를새검사법으로검사한다음 CAP 외부정도관리검체의경우 Trinity G6PD kit (Trinity Biotech, Wicklow, Ireland) 의 group 평균값을, G6PD 정도관리물질은제조회사에서지정한값과비교하였다. 28

38 2.11. 정상인잔여검체를이용한참고범위설정및환자검 체예비검사 정상인의잔여검체를이용하여검사하여얻은결과값의 2.5와 97.5 백분위수로설정하여참고범위를구하였다. 성인참고치의총대상은 21명이었다. 남자는 10명, 여자는 11명이었으며, 나이는평균 53세 ( 범위 33-81세 ) 였다. 소아의참고범위는 3개월부터 9세까지소아총 26명을대상으로구하였다. 남자 16명여자 10명이었으며, 평균나이는 4.8세였다. 또한임상적으로적혈구대사이상으로인한용혈빈혈이의심되는 16명의환자를의뢰받아검사를진행하였다. 그중 2명에서부모형제등가족의검체를분석하였다. 환자의임상정보는 Table 5에정리하였다. 29

39 Table 5. Patients group characteristics for erythrocyte enzyme analysis Case No. Sex Age (yr) Hb (g/dl) Splenomegaly Transfusion Hx* 1 M M NA NA 3 M NA 4 M M F F NA + 8 M Splenectomy - 9 F NA - 10 F NA - 11 F NA - 12 M Splenectomy - 13 F NA + 14 F NA - 15 F NA + 16 M NA - *Transfusion Hx means whether a patient had received erythrocyte transfusions within three months before a testing or not. Abbreviations: Hx, history; NA, not available 30

40 연구결과 1. 적혈구막단백분석 1.1. 적혈구막단백확인 전체 122개단백이확인되었다. 6가지막단백은그중 95% 이상을차지하였고가장흔했다. 가장흔한세포내단백인헤모글로빈은전체단백질의 1.5% 이내였다. 본실험에서확인된단백을 Table 6에나열하였다. 확인된주요단백의정보는이전연구 [19,20] 에서알려진단백과동일하였다. 31

41 Table 6. Major identified erythrocyte membrane proteins Accession Name Sequence Coverage (%) P02730 Band 3 anion transport protein 50.8 P02549 Spectrin alpha chain erythrocyte 63.0 P16157 Ankyrin P11277 Spectrin beta chain erythrocyte 61.4 P11166 Solute carrier family 2 facilitated glucose transporter membrane P11171 Protein P16452 Erythrocyte membrane protein protein P04406 Glyceraldehyde 3 phosphate dehydrogenase 60.9 P60709 Actin cytoplasmic P27105 Erythrocyte band 7 integral membrane protein 51.1 Q08495 Dematin 36.2 Q kDa erythrocyte membrane protein 48.9 P35611 Alpha adducin 17.8 P35612 Beta adducin 19.5 P68871 Hemoglobin subunit beta 67.7 P28289 Tropomodulin 39.4 Q75955 Flotillin Q14254 Flotillin

42 1.2. 질량분석검사법의분석성능 주요 6 개단백측정시변이계수 (coefficient of variation, CV) 는 % 였다. Table 7 에정밀도평가결과를정리하였다. 다른단백에비해 protein 4.1 의정밀도가상대적으로낮았다. 33

43 Table 7. Precision of six erythrocyte membrane protein amounts Proteins Mean amount (%) Within-run CVs (%) Total CVs (%) Band Spectrin alpha Spectrin beta Spectrin (alpha+beta) Ankyrin Protein Protein Abbreviations: CV, coefficient of variation 34

44 1.3. SDS-PAGE 법과비교및참고범위 질량분석기를이용한새검사법을기존방법인 Fairbanks SDS- PAGE 법과비교한결과, spectrin alpha 와 beta 의합 (r=0.8154, P=0.014) 과 ankyrin (r=0.8743, P=0.005) 은비교방법과질량분석기를이용한새검사법결과간에서로강한상관관계를보였다. 그러나, protein 4.1 (r=0.5632, P=0.147) 과 protein 4.2 (r=0.131, P=0.816) 는유의한상관성을보이지않았다. 정상인검체를이용하여산정한 6 가지단백의참고범위는 Table 8 에나타내었다. 35

45 Table 8. Reference intervals of six erythrocyte membrane proteins Protein Reference interval (%)* Band Spectrin alpha Spectrin beta Spectrin (alpha+beta) Ankyrin Protein Protein *Reference interval was calculated as 2.5 th and 97.5 th percentiles from normal controls by dividing the amount of each protein by sum of total identified proteins 36

46 1.4. 환자에대한검사적용 계산된참고범위를 18명의유전구상적혈구증의심환자에적용하여결핍유무를판단하였다. 가장흔하게결핍된단백은 protein 4.2 (50.0%, 9/18) 였다. 다음으로 band 3 (38.9%, 7/18) 와 ankyrin (33.3%, 6/18) 이결핍되었고, spectrin alpha (11.1%, 2/18) 와 spectrin beta (22.2%, 4/18) 도일부환자에서결핍이관찰되었다. Protein 4.1 은어떤환자에서도감소되지 않았다. 9 명에서는두종류이상의단백결핍이있었고, 1 명은결핍이 전혀관찰되지않았다. Table 9 에환자별막단백정량결과를나타내었고, Table 10 에환자별결핍된단백을정리하였다. 37

47 Table 9. Proportions of six membrane proteins in suspicious patients with erythrocyte membrane disorders Case No. Band 3 Spectrin alpha Spectrin beta Ankyrin Protein 4.1 Protein % 17.19% 14.44% 12.06% 4.90% 3.32% % 12.87% 10.78% 12.81% 4.27% 2.98% % 16.32% 14.02% 16.45% 4.72% 0.00% % 15.11% 11.37% 13.86% 4.00% 3.16% % 18.91% 14.43% 13.80% 4.76% 3.50% % 15.81% 13.28% 13.98% 4.32% 3.39% % 17.77% 11.34% 11.03% 5.32% 2.73% % 19.14% 13.72% 11.02% 4.43% 2.31% % 16.52% 15.84% 12.35% 4.70% 3.54% % 16.14% 14.94% 13.55% 4.81% 3.03% % 15.62% 12.36% 11.89% 4.62% 2.51% % 15.82% 12.74% 14.55% 4.32% 2.78% % 15.29% 11.13% 12.51% 4.11% 2.25% % 15.44% 15.57% 10.99% 4.40% 2.37% % 15.67% 14.72% 13.60% 3.95% 3.20% % 13.61% 12.30% 14.58% 4.38% 2.68% % 17.30% 15.24% 17.31% 4.65% 2.83% % 15.56% 13.66% 12.60% 3.80% 2.35% 38

48 Table 10. Protein deficiencies in suspicious patients with erythrocyte membrane disorders Case No. Observed protein deficiencies 1 band 3, ankyrin 2 spectrin alpha, spectrin beta 3 protein spectrin beta 5 band 3 6 (none) 7 spectrin beta, ankyrin, protein ankyrin, protein band 3, ankyrin 10 band 3 11 ankyrin, protein protein spectrin beta, protein ankyrin, protein band 3 16 band 3, spectrin alpha, protein band 3 18 protein

49 2. 적혈구효소분석 2.1. 각효소에대한산물정량분석 액체크로마토그래피에서각효소에대한산물과 IS 는 4 분안에잘 분리되었다 (Figure 2, 3). 그리고산물이나 IS 가나오는 retention time 에서는 ion suppression 이관찰되지않았다. 40

50 Figure 2. Representative UPLC-MS/MS MRM chromatograms of G6PD, PK, and P5 N (Set A). Abbreviations: G6PD, glucose-6-phosphate dehydrogenase; PK, pyruvate kinase; P5 N, pyrimidine 5 nucleotidase; IS, internal standard 41

51 Figure 3. Representative UPLC-MS/MS MRM chromatograms of HK, TPI, and AD (Set B). Abbreviations: HK, hexokinase; TPI, triosephosphate isomerase; AD, adenosine deaminase; IS, internal standard 42

52 2.2. 적혈구검체양과반응시간에따른각효소반응에대 한효과 전처리과정을거친적혈구검체를희석하여각효소마다의반응결과를확인하였을때 G6PD의경우희석배수가 5배에서 40배가될때까지직선성이유지되었고, P5 N의경우 2배에서 20배까지직선성이유지되는것을관찰할수있었다. 그외나머지효소인 PK, HK, TPI, AD의경우 2배에서 10배까지직선성이유지되었다 (Figure 4). 직선성이유지되는희석배수로고정하고반응시간에따른효소마다의반응결과를확인하였을때에는 P5 N의경우 240분까지직선성이유지되는것을관찰할수있었으며, PK와 TPI의경우 60분, HK와 AD의경우 40분, G6PD의경우 30분까지직선성이유지되는것을확인할수있었다 (Figure 5). 이결과를바탕으로직선성이유지되는희석배수와반응시간을고려하여각각의효소반응마다의최적의반응조건을결정하였다 (Table 11). 결정한반응조건을적용하여이후의평가를진행하였다. 43

53 Figure 4. Effect of sample amount on enzyme activity for each enzyme reaction. Each incubation time was 30 min. Abbreviations: G6PD, glucose-6-phosphate dehydrogenase; PK, pyruvate kinase; P5 N, pyrimidine 5 nucleotidase; HK, hexokinase; TPI, triosephosphate isomerase; AD, adenosine deaminase 44

54 Figure 5. Effect of reaction time on enzyme activity for each enzyme reaction. Abbreviations: G6PD, glucose-6-phosphate dehydrogenase; PK, pyruvate kinase; P5 N, pyrimidine 5 nucleotidase; HK, hexokinase; TPI, triosephosphate isomerase; AD, adenosine deaminase 45

55 Table 11. Optimized reaction conditions of dilution rate and incubation time Enzyme Dilution rate Incubation time (min) G6PD PK 5 30 P5 N HK 5 30 TPI AD 5 30 Abbreviations: G6PD, glucose-6-phosphate dehydrogenase; PK, pyruvate kinase; P5 N, pyrimidine 5 nucleotidase; HK, hexokinase; TPI, triosephosphate isomerase; AD, adenosine deaminase 46

56 2.3. 각반응에대한정밀도평가 정밀도평가를하기위해서각각의반응마다높은농도와낮은검체를이용하여 within-run과 between-run에대한 CV값을구하여 Table 12에정리하였다. Within-run CV값에서는낮은농도에서는 %, 높은농도에서는 % 를보였으며, Between-run CV값에서는낮은농도에서는 %, 높은농도에서는 % 이었다. 47

57 Table 12. Within-run and between-run precisions at low and high concentrations Enzyme Within-run CV (%) Between-run CV (%) Low High Low High G6PD PK P5 N HK TPI AD Abbreviations: G6PD, glucose-6-phosphate dehydrogenase; PK, pyruvate kinase; P5 N, pyrimidine 5 nucleotidase; HK, hexokinase; TPI, triosephosphate isomerase; AD, adenosine deaminase; CV, coefficient of variation 48

58 2.4. 각표준용액에대한직선성평가 각효소반응산물의정량을위해사용한 5가지농도의표준용액을측정하여그결과값을선형회귀방법으로직선성을평가한결과, G6PD와 PK의경우 μg/ml, HK의경우 μg/ml, TPI의경우 μg/ml, P5 N과 AD의경우 μg/ml의범위에서모두 R 2 값이 0.99 이상으로직선성이유지되었다 (Figure 6). 49

59 Figure 6. Linearity for each enzyme reactions Abbreviations: G6PD, glucose-6-phosphate dehydrogenase; PK, pyruvate kinase; P5 N, pyrimidine 5 nucleotidase; HK, hexokinase; TPI, triosephosphate isomerase; AD, adenosine deaminase 50

60 2.5. 검사법비교 G6PD 검사법비교결과, 기존방법에따른결과값과새검사법의결과 값간의상관성은결정계수 R 2 = (slope, ; intercept, ) 으로 우수하였다. (Figure 7) 51

61 New Method (mg/min/ghb) y = x R² = Old method (U/gHb) Sample* Old New Control Control Control CAP CAP CAP CAP CAP Figure 7. Comparison of the G6PD activities measured by newly developed assay and old spectrophotometric assay *Three control samples from BEN G6PD kit (Biochemical Enterprise) and five CAP proficiency testing samples in recent two years ( ) Units for old and new method results are U/gHb and mg/min/ghb, respectively. Abbreviations: CAP, College of American Pathologists; G6PD, glucose-6- phosphate dehydrogenase 52

62 2.6. 정상인잔여검체를이용한참고범위설정 성인 21명에서대해효소활성을측정하여계산한각효소의참고범위는 G6PD ( mg/min/ghb), PK ( mg/min/ghb), P5 N ( μg/min/ghb), HK ( μg/min/ghb), TPI ( μg/min/ghb), AD ( μg/min/ghb) 이었다. 소아에서의참고범위는 G6PD ( mg/min/ghb), PK ( mg/min/ghb), P5 N ( μg/min/ghb), HK ( μg/min/ghb), TPI ( μg/min/ghb), AD ( μg/min/ghb) 이었다. 53

63 2.7. 환자검체예비검사 적혈구효소이상에의한빈혈의심환자검체를검사한결과를 Table 13에정리하였다. 환자 1, 3, 5, 7, 9, 15에서 G6PD의활성이감소하였다. 환자 8, 10, 12, 15는 PK의활성도가감소하였다. 환자 3은 P5 N이감소하였고, 환자 1은 HK 활성도가감소하였다. 환자 1, 4는 AD의활성도가감소하였다. 그외나머지환자에서는모든효소의활성도가정상참고범위였다. 가족검사에서 G6PD와 P5 N이감소한환자 3의경우부, 모, 여자형제 ( 누나 ) 에대한검사결과어떤효소활성도의감소도관찰할수없었다. G6PD와 PK의활성이감소한환자 15의부, 모, 남자형제 ( 오빠 ) 의검사결과, 어머니의 G6PD 활성도가참고범위의하한이었고, 그외가족효소활성도의감소는관찰되지않았다. 54

64 Table 13. Pilot study results of suspicious patients with erythrocytes enzymopathy Case No. Sex/Age (yrs) G6PD PK P5 N HK TPI AD 1 M/ M/ M/ M/ M/ F/ F/ M/ F/ F/ F/ M/ F/ F/ F/ M/ Reference interval (Pediatric)

65 Reference interval (Adult) *Unit for all results except G6PD and PK was μg/min/ghb. G6PD and PK unit was mg/min/ghb Abbreviations: G6PD, glucose-6-phosphate dehydrogenase; PK, pyruvate kinase; P5 N, pyrimidine 5 nucleotidase; HK, hexokinase; TPI, triosephosphate isomerase; AD, adenosine deaminase 56

66 고찰 본연구로유전용혈빈혈진단에서적혈구막단백에대해질량분석법을이용한 SDS-PAGE 법을대체할새로운방법과적혈구주요효소의이상에대한보다간편하고강력한검사법을개발하고평가하였다. 적혈구막단백은세포막단백의비밀을밝히는열쇠로생각되어많은연구가이루어지고있다 [21]. 최근의연구경향은단백분리기법의발전 [19,22 24] 과고성능질량분석기의개발 [25 27] 에따라기존에알려지지않은새로운적혈구막단백을찾아내는것에중점을두고있다. 이러한연구는단백검출의민감도를향상하고적혈구의생리와발병과정에대한이해를돕는데일조하고있다 [28]. 그러나, 이렇게찾아낸새로운단백은실제환자에서의의미가불분명하므로임상진단목적으로는아직은쓸수없으며, 환자진료에오히려혼란을줄수있다. 따라서, 본연구에서는기존 SDS-PAGE 법에서검출하는실제진단에사용되는주요 6가지적혈구단백에초점을맞추어검사법개발을진행하였다. 본연구에서사용한것과같은고해상도질량분석기는생명과학연구에서단백의변화나바이오마커발견을위한단백프로파일링에널리사용되며, 다양한단백분리기술과같이결합하여이전에는불가능하였던수천가지의단백을식별할수있다 [29]. 그러나, 이러한연구의주요목적은바이오마커후보를검색하고가능한많은단백질을찾아냄으로써생리학적과정을밝히기위함이므로본연구에서와같이 57

67 진단검사법으로서특정단백을검출하는목적에서는방법특유의낮은민감도와높은부정확성으로인해비교적유용성이낮다. 다만, 유전성적혈구막단백질환에서중요한단백들은양이비교적많아민감도와재현성측면에서크게문제가되지않았다. 이는임상검사에서혈장알부민과면역글로불린과같은풍부한단백검출이용이한것과비슷하다. 적혈구막단백에관한이전연구는백혈구의오염을방지하는데노력을기울였다 [20,24,30]. 백혈구는전체혈액세포의 0.1% 에불과하므로본연구의대상인 6가지단백같은적혈구에주로존재 [31] 하며상대적으로양이풍부한단백의검사에는영향을미치지않을가능성이높다. 따라서본연구에사용된간단한백혈구오염방지방법은적혈구막의주요단백의검사목적으로는충분하다고판단된다. 새로운혈구막단백검사법의단점은총단백에서의비율을측정하는검사, 즉상대적정량이라는점이다. 현재임상에서사용되는검사대부분이절대정량법임을고려한다면이는단점이될수있다. 향후본연구에서얻은적혈구단백의질량분석결과를토대로 MRM 기반의절대정량법을개발할수있을것으로예상한다. 적혈구막단백에대한새검사법의성능평가결과정밀도는최대 8% 이하로우수하였다. Protein 4.1의정밀도가상대적으로가장낮았지만농도가낮은것을감안하면허용가능한수준으로생각된다. 참고범위설정과정에서나이나성별에따라뚜렷한차이는관찰되지않았으므로, 58

68 참고범위를구분없이하나로설정하여결과를판정하였다. 다만, 유전질환의특성상나이별성별간차이가있을수있으므로, 차후가능하다면나이별성별참고범위설정하려는노력이필요할것으로생각한다. 의심환자를대상으로적혈구단백의이상을검사한결과는 1명을제외한 17명환자에서하나이상의단백감소를검출할수있었다. Protein 4.2 감소가가장흔하게관찰되었는데, 이는유전구상적혈구증에서백인에서는드문편이나 [32,33], 일본 [34] 이나국내에서는비교적흔하게검출되는것으로알려져있다. Ankyrin은백인에서 50-60% 정도로가장흔하게관찰되는막단백이상으로본연구에서는 33.3%(6/18) 에서감소하였는데이는이전국내연구 [35] 와유사하였다. Band 3 감소는 7명 (38.9%) 으로상대적으로흔하게관찰되었는데, 기존 SDS-PAGE 법의경우 band 3에대한비율로감소여부를판단하므로소량의감소가있으면검출이어려운점 [32] 과실제로 band 3 이상으로인한유전용혈빈혈이많이보고 [36 38] 되고있는것을고려하면기존연구에서 band 3 결핍이실제보다과소평가되었을가능성을생각할수있다. 특정적혈구막단백결핍과임상증상의중증도관계에대해서많이알려진것은없으나, band 3 결핍보다 spectrin의결핍에서평균혈색소치가낮으며임상증상이심한것으로알려져있다 [5]. 본연구에서도 spectrin이감소한환자군에서 band 3 감소군에비해평균 59

69 혈색소치가낮았다 (12.9 vs. 9.4 g/dl). 또한, 2개이상의단백감소가동시에있는환자군이 1개이하의막단백감소가있는군에비해혈색소치가낮았다 (9.8 vs g/dl). 다만, 환자수가적어통계적유의성은없었다. 특정막단백결핍환자에서결핍의정도와임상중증도에대한이전연구는없었으며, 본연구에서도 band 3, ankyrin, spectrin 등모든단백에서연관관계는관찰되지않았다. 이러한막단백결핍의종류, 정도와임상증상의관계에대해서는차후에많은환자검사결과와임상정보가축적되면추가적인분석이필요할것으로생각된다. 새로운적혈구막단백검사법의검사소요시간은검체전처리과정에서최종결과보고까지 2-3일정도소요되며이는기존의 SDS-PAGE 법이수작업위주로일주일정도소요되던것에서크게단축된것으로검사실에서실제환자검사수행시큰이점으로작용할것으로생각된다. 적혈구효소이상에대한새로운검사법은우선기존분광광도법에비해효소반응단계를줄였다. 분광광도법으로검출할수있도록하기위해추가적인효소반응단계가필요한기존검사법에비해 TPI을제외한나머지효소에서한단계반응만으로검사가가능하게하였고조효소가아닌효소산물을직접측정하였다. 이는검사의정확도를높이는효과가있을것으로생각된다. 다만, 효소반응단계에기본적으로소요되는시간이있어기존방법에비해검사소요시간의이점은크지않으나, 6가지효소검사를 3가지씩 2세트로나누어한번에검사를할수 60

70 있으므로각효소별로따로따로측정해야했던번거로운과정을줄일수있었다. 새로운검사법은 6가지효소를 2세트로나누어검사하도록하였는데, 이는예비실험을통해다중검사가능여부, 유병률등진단검사적특성을고려하여설정하였다. 검사결과판단을위해 6가지효소모두에참고범위를설정하였다. 나이에따라효소의활성도가다르므로성인과 10세이하소아의참고치를각각설정하였다. 소아환자가많은유전질환의특성을고려한다면향후소아유전용혈빈혈환자에좀더적합한세분화된참고범위를추가설정하는것이필요하다. 적혈구효소검사가의뢰된 16명의환자에대하여예비검사를시행하였을때 G6PD의활성도감소를보인환자는 6명 (37.5%), PK의활성도감소를보인환자는 4명 (25.0%), AD는 2명 (12.5%), P5 N과 HK는각각 1명 (6.2%) 씩감소되었다. 환자수가그리많지않음에도적혈구효소이상중가장흔한것으로알려져있는세가지효소, 즉 G6PD, P5 N, PK의결핍이모두관찰되었다. 특이한점은유전자검사를통해 PK 효소이상이확진된환자 1명에서 PK의활성도가저하되지않은결과를보였는데, 이러한결과의원인으로백혈구오염이나 mutant PK의가능성을생각할수있다. 백혈구에도 PK가다량존재하므로백혈구제거가충분치않으면 PK의검사결과에큰영향을미칠수있다 [39]. Mutant PK의경우생체내에서는활성이떨어지지만, 기질인 phosphoenolpyruvate의농도가높은 61

71 조건에서검사를시행하면정상활성도를보이는경우가있다고알려져있다. PK의활성도가정상으로나타나기때문에 PK 결핍을배제하기가어려우며, 낮은농도의기질을사용해야진단할수있다 [12]. 따라서, 검사전에백혈구제거를확인하고 mutant PK가의심될경우기질농도를낮춘조건에서추가검사를시행하는것이필요하다. 환자가족에대한검사에서 G6PD 감소를보인환자의어머니에서 G6PD 활성도가정상범위의하한을보인것이외에는모두정상범위를보였다. G6PD의경우 X염색체열성 (X-linked recessive) 이며, 그외나머지효소결핍은대부분상염색체열성으로유전 [11] 되므로효소활성도검사상환자가족은정상으로나올가능성이있으며, 보인자여부등의정확한확인을위해서는원인유전자에대한분자진단검사가필요할것으로생각한다. 현재국내에서검사법이확립된효소는 10여종류로새로운검사법에서검사가능한 6가지외에 glucose phosphate isomerase (GPI), adenylate kinase (AK), phosphofructokinase (PFK), phosphoglycerate kinase (PGK) 등이있으며, 이외에도유전용혈빈혈을일으킬수있는효소는다양하다. 이들효소에대해서는추가적인검사법개발이필요하다. 유전용혈빈혈은수혈을하는환자가많으므로이에대한검사법도수혈의영향을반드시고려해야한다. 최근수혈력이있는환자는수혈된적혈구에의해막단백이나효소이상이있더라도정상으로나올수있기때문에검사결과위음성을보일수있다. 이를해결하기위해서 62

72 수혈된적혈구의양을계산하여최종검사결과를보정하는방법을생각할수있다. 그러나, 이를위해서는수혈된적혈구의막단백의양이나효소활성도를알고있어야하며, 수혈된적혈구를정상적혈구로간주하여정상참고범위를적용하는것도정상참고범위가넓어정확도가떨어진다. 또한실제임상에서는일반적으로수혈직후가아니라수혈후시간이어느정도지나서검사를의뢰하므로수혈이후의수혈된적혈구의변화를추정하기어렵다. 이와같은문제로수혈력이있는환자에서수혈영향을반영하여검사결과를보정하는것은현실적으로어렵다고판단된다. 그러므로가능한한수혈이후많은시간이지난이후에검체채취및검사를진행하는것이수혈이검사결과에미치는영향을최소화할수있는현실적인방법으로생각된다. 덧붙여서검사시행시반드시최근수혈력에대한조사를실시하고, 먄약수혈력이있다면검사결과보고서에최근의수혈이검사결과에영향을미칠수있음을명기해야한다. 또한, 검사결과가정상이거나, 혹은비정상이라도추정진단과맞지않는경우등에서는수혈의영향이없어진이후에재검사할필요가있다. 결론적으로본연구를통해새롭게개발된질량분석을이용한검사법이앞으로유전용혈빈혈의선별과진단에실제적으로사용될수있을것으로평가되며, 향후본연구에서얻은지식과기술을토대로질량분석기등최신의검사기기를이용한향상된검사법개발이지속적으로이루어질것으로기대한다. 63

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77 Abstract Development of a new analysis method using mass spectrometry on erythrocyte membrane proteins and enzymes related to hereditary hemolytic anemia Chul Min Park Department of Medicine The Graduate School Seoul National University The causes of hereditary hemolytic anemia include erythrocyte membrane protein disorders, or erythrocyte enzymopathies. Classic conventional electrophoresis has been used to examine the erythrocyte membrane protein, but it showed lower reproducibility in addition to large consumption of time and efforts. Erythrocyte enzymopathies has been measured by using the spectrophotometry. But this method is difficult to perform multiple tests at once and its effectiveness is deteriorated due to the variety of causal enzymes. To this end, this study aims to develop and to verify the performance of a test method more improved than the existing method in diagnosis of erythrocyte membrane disorders and erythrocyte enzymopathies among the causes of hereditary hemolytic anemia by using the latest techniques of mass spectrometry. 68

78 For the analysis of erythrocyte membrane protein, clinically significant erythrocyte membrane protein from the erythrocyte membrane protein-derived peptides by using mass spectrometry. And they were distinguished from previous known band 3, spectrin alpha, spectrin beta, protein 4.1, protein 4.2, and ankyrin. The precision of new method was evaluated, compared to the previous methods and its reference range was established. For the analysis of erythrocyte metabolism enzymes, the ion suppression was evaluated for development of method by using a mass spectrometry and the test sample diluting multiple and reaction time were optimized for 6 types of main enzymes including the glucose-6-phosphate dehydrogenase (G6PD), pyruvate kinase (PK), pyrimidine 5 -nucleotidase (P5 N), hexokinase (HK), triosephosphate isomerase (TPI) and adenosine deaminase (AD). Linearity and precision of new method were confirmed while the reference range was established and the preliminary evaluation for the patients was performed. The analysis of erythrocyte membrane protein by using a mass spectrometry had identified 6 types of clinically significant membrane proteins, and the coefficients of variation (CVs) of membrane protein measurement were in the range of %. When compared the new test method to the previous test method:sds- PAGE method, the sum of spectrin alpha and beta (r=0.8154, P=0.014) and ankyrin(r=0.8743, P=0.005) had shown strong correlation while protein 4.1 and protein 4.2 had no significant correlation. Of 18 patients, there were deficiencies of protein 4.2 in 9 patients, band 3 deficiencies in 7 patients, ankyrin deficiencies in 6 patients, spectrin alpha deficiencies in 2 patients, and specrn beta deficiencies in 4 patients. All enzymes had shown excellent linearity from the rythrocyte enzyme assay performed by using a mass spectrometry. When conducted the precision 69

79 assessment, the results had shown the CVs ranges of each enzyme s measured value was %. The reference ranges of each enzyme were G6PD ( mg/min/ghb), PK ( mg/min/ghb), P5 N ( μg/min/ghb), HK ( μg/min/ghb), TPI ( μg/min/ghb) and AD ( μg/min/ghb). After the essays for 16 patients, the reduction of activities were observed for G6PD in 6 patients, for PK in 4 patients, and for P5 N and for HK was observed from each one subject. The erythrocyte membrane protein and erythrocyte enzyme assays by using newly developed mass spectrometry through this study, are considered to be helpful for the diagnosis of hereditary hemolytic anemia in the future. Keywords: hemolytic anemia, erythrocyte membrane, erythrocyte enzyme, mass spectrometry Student Number: