한국영양학회지 (Korean J Nutr) 9; 42(5): 423~ 433 DOI.4163/kjn.9.42.5.423 엽산과비타민 B 12 결핍에의한호모시스테인혈증흰쥐의조직내비타민지표간의상관관계분석 * 민혜선 김미숙 한남대학교생명나노과학대학식품영양학과 A Critical Evaluation of the Correlation Between Biomarkers of Folate and Vitamin B 12 in Nutritional Homocysteinemia * Min, Hyesun Kim, Misook Department of Food and Nutrition, College of Bio-Nano Science, Hannam University, Daejeon 6-791, Korea ABSTRACT Folate and vitamin B12 are essential cofactors for homocysteine (Hcy) metabolism. Homocysteinemia has been related with cardiovascular and neurodegenerative disease. We examined the effect of folate and/or vitamin B12 deficiency on biomarkers of one carbon metabolism in blood, liver and brain, and analyzed the correlation between vitamin biomarkers in mild and moderate homocysteinemia. In this study, Sprague-Dawley male rats (5 groups, n = ) were fed folatesufficient diet (FS), folate-deficient diet (FD) with or 3 g homocystine (FSH and FDH), and folate-/vitamin B12-deficient diet with 3 g homocystine (FDHCD) for 8 weeks. The FDH diet induced mild homocysteinemia (plasma Hcy 17.41 ± 1.94 nmol/ml) and the FDHCD diet induced moderate homocysteinemia (plasma Hcy 44.13 ± 2.65 nmol/ ml), respectively. Although liver and brain folate levels were significantly lower compared with those values of rats fed FS or FSH (p <.1, p <.1 respectively), there were no significant differences in folate levels in liver and brain among the rats fed FD, FDH and FDHCD diet. However, rats fed FDHCD showed higher plasma folate levels (126.5 ± 9.6 nmol/l) compared with rats fed FD and FDH (21.1 ± 1.4 nmol/l, 22. ± 2.2 nmol/l)(p <.1), which is the feature of ethyl-folate trap by vitamin B12 deficiency. Plasma Hcy was correlated with hepatic folate (r = -.641, p <.1) but not with plasma folate or brain folate in this experimental condition. However, as we eliminated FDHCD group during correlation test, plasma Hcy was correlated with plasma folate (r = -.581, p <.1), hepatic folate (r = -.684, p <.1) and brain folate (r = -.321, p <.5). Hepatic S-adenosylmethionine (SAM) level was lower in rats fed FD, FDH and FDHCD than in rats fed FS and FSH (p <.1, p <.1 respectively) and hepatic S-adenosylhomocysteine (SAH) level was significantly higher in those groups. The SAH level in brain was also significantly increased in rats fed FDHCD (p <.5). However, brain SAM level was not affected by folate and/or vitamin B12 deficiency. This result suggests that dietary folate- and vitamin B12-deficiency may inhibit methylation in brain by increasing SAH rather than decreasing SAM level, which may be closely associated with impaired cognitive function in nutritional homocysteinemia. (Korean J Nutr 9; 42(5): 423 ~ 433) KEY WORDS: homocyteine, S-adenosylmethionine, S-adenosylhomocysteine, DNA methylation, cognitive function. 서 엽산과비타민 B 12 는호모시스테인이메티오닌으로메틸 접수일 :9년 6월 일 / 수정일 :9년 7월 7일채택일 :9년 7월 일 * This work was supported by the Korea Research Foundation Grant funded by the Korean Government (KRF-4-4- C85) and also supported by Hannam University Research Fund (9). To whom correspondence should be addressed. E-mail: hsmin@hnu.kr 론 화되는과정에필요한영양소로서뇌의기능에중요한역할을한다. 1-3) 호모시스테인은비필수아미노산으로서신경퇴화를촉진하여정신질환및치매를비롯한노인기뇌신경질환의독립적인위험인자로작용하는것으로보고되고있다. 4-7) 호모시스테인이신경계작용을저해한다는사실은 cystathionine β-synthase 결핍환자의지적능력손상, 뇌조직위축및경련증상등으로부터이미오래전부터알려져있었다. 8,9) 노인대상연구에서혈장내엽산및비타민 B 12 농도가낮을때혈장호모시스테인농도가높은 9 The Korean Nutrition Society
424 / 엽산과비타민 B 12 영양상태관련지표사이의상관관계분석 경향을나타냈으며, 연령이높을수록혈장호모시스테인농도가높았다. ) 신경정신계질환자들을대상으로조사된연구에서이환자들가운데엽산과비타민 B 12 결핍이빈번하게나타났으며, 11-13) 이두비타민의결핍이신경계질환을가져오는원인은직접적으로신경계에손상을주거나또는호모시스테인농도를증가시킴으로써간접적으로신경계손상을가져오는있는것으로보고되었다. 3,14,15) 일부연구에서호모시스테인증가되었을때 DNA의메틸화가저하되고, DNA 손상이증가되며세포사멸이증가되어신경이손상되는것으로제안되었다. 14,16,17) 즉, 엽산또는비타민 B 12 가결핍되었을때뇌조직의 S-adenosylmethionine (SAM) 농도가저하되고 S-adenosylhomocysteine (SAH) 농도가증가되어저메틸화를가져오기때문에신경이손상되는것으로제안하였다. 18,19) SAM은 DNA 메틸화뿐아니라 epinephrine 과 phosphatidylethanolamine 과같은신경전달물질의합성에필수적인역할을하므로뇌조직에서가장중요한메틸공여체로작용한다. ) 따라서엽산또는비타민 B 12 가결핍되면 SAM 합성의저하와 SAH 합성의증가로인해 SAM/SAH 비율이저하됨으로써 DNA 손상을일으켜세포사멸을가져오므로, 호모시스테인이신경계에간접적으로독성을나타내는것으로보고하였다. 14) 비타민 B 12 가결핍되면 methionine synthase (MS) 활성이저하되며, 이로인해호모시스테인으로부터메티오닌의합성이저해되기때문에호모시스테인이축적됨과동시에메티오닌대사회로에이상을초래한다. 이현상은 methyl-folate trap 가설에의해다음과같이설명되고있다. 21-23) 비타민 B 12 가결핍되면 methionine synthase 활성이저하되므로 MS의조효소로이용되는 5-methylthetrahydrofolate (5MTHF) 가다른형태로전환되지못하고축적되는데, 이는 MS에의한효소반응이 5MTHF 를다른형태로전환시키는유일한반응이기때문이다. 따라서비타민 B 12 가결핍되면세포내엽산은 5MTHF 형태로 trap되고, 그결과 DNA합성과세포분열과단백질의메틸화가저해된다. 이와동시에메티오닌의합성이저하되어 SAM 농도가저하된다. SAM은 methylenetetrahydrofolate reductase (MTHFR) 의강력한저해제이므로 SAM 농도가저하되면 MTHFR 활성이 up-regulate되므로, 5, -methylene-thf 가 5MTHF 로환원되는반응이더욱촉진되어세포내에엽산이충분량있어도메티오닌대사회로가저해되고, 엽산은 DNA 합성에이용될수없는 5MTHF 형태로 trap 된다. 21-23) 또한 5MTHF는 folylpolyglutamate synthase에대한친화력이매우낮으므로 polyglutamate 형태로잘전환되지 않아서, 엽산이세포내에효율적으로보유되지않고혈액으로방출된후소변으로배설되므로조직내총엽산함량이낮아진다. 23,24) 이런이유에서엽산결핍과비타민 B 12 의결핍시엽산대사와메티오닌대사회로의 biomarker 에나타나는변화양상이다르므로, 이들비타민결핍에의한호모시스테인혈증에있어혈장호모시스테인, 혈장및조직세포의엽산농도및간과뇌조직에서메티오닌대사회로의 biomarker 들간의상관관계가달라지나이에대한연구가미흡하다. 따라서본연구에서는흰쥐를대상으로엽산결핍식이를이용하여경미한호모시스테인혈증 (mild homocysteinemia) 을유발하고, 엽산 / 비타민 B 12 결핍식이를이용하여중위의호모시스테인혈증 (moderate homocysteinemia) 을유발한상태에서두비타민의 biomarker 인혈장호모시스테인과간과뇌조직 SAH, SAM 및혈장, 간및뇌조직엽산농도사이의상관관계를분석함으로써엽산결핍과비타민 B 12 결핍에의한호모시스테인혈증의특성을 methyl-folate trap 가설에근거하여밝히고자하였다. 재료및방법실험동물사육과실험식이 실험동물의사육및관리는 NIH Guide (NIH Publications NO. 8-23, 1996) 에따라수행되었다. Sprague Dawley 종 6 주령숫컷쥐 (187.2 ±.5 g) 마리를일본 Slc 사 (Hamamatsu, Japan) 에서분양받아 3일간고형사료를공급한후, 체중에따라난괴법으로 5군으로나누어스텐레스사육장에한마리씩분리하여실험식이로 8주간사육하였다. 사육실의온도와습도는각각 22 ± 2, ± 5% 로유지하였으며조명은 12시간주기 (9 : ~21 : ) 로조절하였고물은 2차증류수로급여하였다. 영양결핍 ( 엽산또는비타민 B 12) 에의한영양성호모시스테인혈증 (nutritional homocysteinemia) 을유도할목적으로엽산결핍식이, 비타민 B 12 결핍식이및.3% 호모시스틴첨가식이 25) 를이용하였다. 실험식이는 AIN-93M식이 (Dyets, Bethlehem, PA, USA) 를기준으로대조군은엽산충분식이 (FS) 를주었으며, 엽산충분식이에호모시스틴을첨가한식이 (FSH), 엽산결핍식이 (FD), 엽산결핍식이에호모시스틴을첨가한식이 (FDH) 및엽산 / 비타민 B 12 결핍식이에호모시스틴을첨가한식이 (FDHCD) 를각각공급하였다 (Table 1). 식이섭취량은 2일에한번씩측정하였으며, 체중은 1주일에한번씩일정한시간에측정하였다. 식이효율 (Food efficiency ratio: FER) 은일정기간의체중
한국영양학회지 (Korean J Nutr) 9; 42(5): 423~433 / 425 Table 1. Composition of experimental diets Ingredients (g/kg diet) Group FS FSH FD FDH FDHCD Cornstarch 465.62 462.62 465.62 462.62 462.62 Casein (>85%) 1 1 1 1 1 Dextrinized cornstarch 155 155 155 155 155 Sucrose Soybean oil Fiber (Pectin) 1) Mineral mix (AIN-93M-MX) 35 35 35 35 35 L-Cystine 1.8 1.8 1.8 1.8 1.8 Choline bitartrate (41.1% choline) 2.5 2.5 2.5 2.5 2.5 Tert-Butylhydroquinone.8.8.8.8.8 Vitamin mix (AIN-93-VX) Folate-free vitamin mix 2) Folate & B12-free vitamin mix 3) Homocystine 3 3 3 1) Five percent of pectin was added to each diet for the purpose of accelerating depletion of vitamin B12 2), 3) Folate-free vitamin mix and folate & B12-free vitamin mix were made from AIN-93-vitamin mix by omitting folate or folate & vitamin B12, respectively FS: folate-sufficient diet, FSH: folate-sufficient/.3% homocystine diet, FD: folate-deficient diet, FDH: folate-deficient/.3% homocystine diet, FDHCD: folate-deficient/vitamin B12-deficient/.3% homocystine diet 증가량을동일기간섭취한식이량으로나누어산출하였다. 시료수집및전처리실험기간이종료되기전날부터동물을 12시간절식시킨상태에서 CO2 가스로마취시킨뒤개복하여심장천자법으로헤파린처리된주사기를사용하여혈액을채취하였다. 채취한혈액은즉시 1, g에서 15분간원심분리하여혈장을분리및채취한후즉시 - 에냉동보관하였다. 간과뇌조직을적출한후무게를측정하고즉시액체질소로급속냉동시켜 - 에냉동보관하였다가생화학적분석에사용하였다. 조직및혈장의엽산분석시료의엽산함량은 Lactobacillus casei (ATCC 7469) 를이용한미생물학적분석방법을 96-well 에적용시킨방법으로분석하였다. 26) 간과뇌조직의엽산은 aultolysis 방 법에 27) 의해 polyglutamate chain 을가수분해한후분석 하였으며혈장엽산은전처리없이직접미생물학적분석법으로분석하였다. 혈장호모시스테인분석혈장호모시스테인농도는 Araki & Sako 의 HPLC 법 28) 을일부수정한방법 29) 을이용하여분석하였다. 혈장호모시스테인의티올기와 ammonium 7-fluorobenzo-2-oxa-1, 3-diazole-4-sulphonate (Wako chemicals, Osaka, Japan) 를반응시켜높은형광성을나타내면서안정한성질의형광물질을형성시켰다. ODS Hypersil analytical column (2 4.6 mm, I.D. 5 μm, Thermo-Keystone, Runcorn, Great Britain) 을이용하여분리한후 RF-AXL fluorescence detector (Shimadzu, Kyoto, Japan) 로정량하였다. 간과뇌조직의 SAM과 SAH의분석간과뇌조직내 SAM과 SAH의농도는 Wagner 등 ) 의방법의해측정하였다. 조직을.4 M perchloric acid 용액을이용하여분쇄한후원심분리하여얻은상청액을당일분석하였다. 상청액은 centrifugal filter (Millipore 사 ) 로여과한후 ODS Betasil analytical column (2 4.6 mm, I.D. 5 μm, Thermo-Keystone, Runcorn, Great Britain) 에주입하였으며, gradient mobile phase를이용하여분리하였다. SPD-A UV detector (Shimadzu, Kyoto, Japan) 를이용하여 254 nm에서의얻은흡광도의면적을이용하여 SAM과 SAH의농도를정량하였다. 통계처리실험결과의통계처리는 SPSS PC + 14. 통계 package program 을이용하여수행하였다. 실험식이처리에의한각분석항목의분석치는평균값과표준오차 (Mean ± S.E.) 로나타냈으며, 실험군간의비교는일원배치분산분석으로분석하였다. 식이에따른측정치의평균값의차이에대한유의성은 Duncan의다중비교법을이용하여사후검정하였
426 / 엽산과비타민 B 12 영양상태관련지표사이의상관관계분석 다. 변인들사이의상관관계는 Pearson test 로조사하였다. 모든통계처리는양측검정하였으며, p값이.5 미만일때통계적으로유의한것으로평가하였다. 결과식이섭취량, 체중증가량및식이효율 실험식이를 6주령흰쥐에게 8주간급여한후성장률을비교하였다 (Table 2). FD군과 FDH군의성장은 FS군과비슷한성장률을나타냈다. 그러나 FDHCD 군은다른식이군과비교할때성장이유의적으로저하되었다. 식이섭취량은 FS, FD, FDH의식이섭취량은비슷한섭취량을보였으나 FDHCD 군은유의적으로적은식이섭취량을나타냈다 (p <.5). 또한 FDHCD 군의식이효율은.218 ±.5 로서다른실험군보다유의적으로낮았다 (p <.1). 이결과로부터엽산과 B 12 가동시에결핍된식이군은낮은식이섭취량과식이효율로인해체중증가가유의적으로감소된것으로보인다. 그러나식이에.3% 호모시스틴첨가하거나엽산결핍식이공급은흰쥐의성장에유의적인영향을주지않았다. 혈장, 간및뇌조직의엽산농도혈장과간및뇌조직의엽산농도를분석한결과는 Table 3과같았다. 혈장엽산농도는엽산결핍식이군 (FD, FDH) 이각각 21.1 ± 1.4 nmol/l, 22. ± 2.2 nmol/l 으로유사했으며, 대조군 (162.3 ± 13.5 nmol/l) 의약 13% 수준으로유의적으로낮았다 (p <.1). 이와대조적으로 FD- HCD 군의혈장엽산농도는 126.5 ± 9.6 nmol/l 로서대조군보다는낮았지만, FD군과 FDH군의약 6배높은수준이었다. 간엽산농도는 FD군, FDH군에서각각 5.1 ±.4 nmol/g, 4.8 ±.4 nmol/g로대조군 (17.3 ±.78 nmol/g) 과비교할때각각대조군의 28% 또는 27% 수준으로저하되었다 (p <.1). FDHCD 군의간엽산농도는 4.4 ±.3 nmol/g 으로 FD, FDH군과유의적인차이가없었다. 이와대조적으로뇌조직의엽산농도는엽산결핍식이군 (FD, FDH) 에서각각 599.3 ± 72.5 nmol/g, 599.1 ± 7. nmol/g 로서대조군 (4.31 ±.9 nmol/g) 의약 87~ 92% 수준으로저하되었으나 (p <.1), FDHCD 군의뇌엽산농도는 772.1 ± 145.4 nmol/g 으로대조군과동일한수준을나타냈다. 혈장호모시스테인 엽산과비타민 B 12 결핍및호모시스틴식이를 8주간급여하였을때혈장호모시스테인농도는 Fig. 1과같았다. FSH 군의혈장호모시스테인농도 (7. ±.67 nmol/ml) 는대조군 (4.95 ±.43 nmol/ml) 보다다소증가되었으나차이가유의적이아니었으므로정상식이에.3% 호모시스틴을첨가하였을때호모시스테인혈증이유발되지않았으며, FD군과 FDH군의혈장호모시스테인농도는각각 13.75 ± 3.3 nmol/ml, 17.41 ± 1.94 nmol/ml로증가되어경미한호모시스테인혈증이유발되었다 (p <.1). 그러나 FDHCD 군의혈장호모시스테인농도는 44.13 ± 2.65 nmol/ml 로서중위의호모시스테인혈증이나타났으므로, 엽산및비타민 B 12 결핍과호모시스틴첨가에의한 FDH- CD식이로심한호모시스테인혈증 (nutritional homocyst- Table 3. The effects of folate- and vitamin B12-deficient diet combined with homocystine feeding on plasma and liver folate concentration of rats Group Plasma folate (nmol/l) Liver folate (μmol/g) Brain folate (nmol/g) FS 162.3 ± 13.5 1)a2) 17.3 ±.7 a 772.3 ± 139.7 a FSH 172.9 ± 14.4 a 17.6 ± 1.4 a 81.1 ± 144.3 a FD 21.1 ± 1.4 c 5.1 ±.4 b 599.3 ± 72.5 b FDH 22. ± 2.2 c 4.8 ±.4 b 599.1 ± 7. b FDHCD 126.5 ± 9.6 b 4.4 ±.3 b 772.1 ± 145.4 a 1) Values are mean ± S.E. (n = ) 2) Values with different superscript letter within a column are significantly different at p <.5 by Duncan s multiple-range test Table 2. The effects of folate- and vitamin B12-deficient diet combined with homocystine feeding on food intake, body weight gain, food efficiency ratio, and body weight of rats Group Weight gain (g/day) Food intake (g/day) F.E.R 1) Body weight (g) FS 5.3 ±.1 2)a3) 19.78 ±.51 ab.2 ±.3 a 387.2 ± 8.4 a FSH 5.6 ±.2 a.82 ±.67 a.268 ±.4 a 1. ± 12. a FD 5.4 ±.3 a. ±.75 a.259 ±.4 a 389.5 ± 15.5 a FDH 5.6 ±.2 a 21.7 ±.51 a.267 ±.6 a 4.8 ± 13.2 a FDHCD 4. ±.1 b 18.47 ±.28 b.218 ±.5 b 314.7 ± 6.9 b F-value p <.1 p <.5 p <.1 p <.1 1) Food efficiency ratio (g body weight gain/ g feed) 2) Values are mean ± S.E. (n = ) 3) Means in the same row not sharing a common letter are significantly different among groups at p <.5 by Duncan s multiplerange test
한국영양학회지 (Korean J Nutr) 9; 42(5): 423~433 / 427 einemia) 을흰쥐에게유발시킬수있었다. 간과뇌조직의 S-adenosylmethionine 과 S-adenosylhomocysteine 엽산또는비타민 B 12 결핍및호모시스틴식이가간과뇌조직의 SAH와 SAM에미치는영향을조사한결과는 Table 4와같았다. FD군과 FDH 군의간조직의 SAH 농도는각각대조군보다 44% 및 % 씩증가되었고 (p <.1), 간 SAM 농도는대조군보다각각 72%, 71% 저하되었으며 (p <.1), 그결과두군모두 SAM/SAH 비율이대조군보다약 8% 저하되었다 (p <.1). 한편 FDHCD 군의간 SAH 농도를대조군과비교할때대조군보다 7% 증가되었고 (p <.1), SAM 농도는대조군보다 81% 저하되어 (p <.1) SAM/SAH 비율이대조군보다약 9% 저하되어매우낮은 SAM/SAH 비율을나타냈다 (p Plasma Hcy (nmol/ml) c c b FS FSH FD FDH FDHCD Fig. 1. The effects of folate- and vitamin B12-deficient diet combined with homocystine feeding on plasma homocysteine. Mean ± S.E (n = ). Bars with different superscript letter are significantly different among groups (p <.5). Folate-deficient diets (FD, FDH) elevated plasma homocysteine significantly (p <.1), whereas homocystine feeding alone did not affect plasma Hcy in rats. Mild homocysteinemia was induced by FD or FDH and moderate homocysteinemia was induced by FDHCD. FS: folate-sufficient diet, FSH: folate-sufficient/.3% homocystine diet, FD: folate-deficient diet, FDH: folate-deficient/.3% homocystine diet, FDHCD: folate-deficient/vitamin B12-deficient/.3% homocystine diet. b a <.1). 간조직과대조적으로 FSH, FD 및 FDH 식이군의뇌조직 SAM 농도는엽산결핍, 비타민 B 12 결핍및호모시스틴급여에의해영향을받지않고대조군과유사한수준을보였다 (Table 4). 그러나 FDHCD 군의뇌조직 SAH 농도는대조군보다 % 증가되었으며 (p <.5), 그결과 SAM/ SAH 비율은대조군보다약 28% 저하되었다 (p <.5). 비타민 biomarker 들사이의상관관계영양성호모시스테인혈증가운데엽산결핍에의한호모시스테인혈증과비타민 B 12 결핍에의한호모시스테인혈증은메티오닌대사회로의중간대사물질인호모시스테인, SAM 및 SAH 농도에각각다른영향을줄수있으며, 17) 이로인해두영양소가부족할때나타나는 vitamin biomarker 들의변화를비교하였다. 따라서전체실험군 (5개실험군 ) 과엽산과비타민 B 12 가동시에부족한 FDHCD 군을제외한 4개실험군의자료를따로분리하여, 호모시스테인혈증과관련된 biomarker 들사이의상관관계를분석하여 Pearson 의상관계수를 Fig. 2와 Table 5에각각 A, B로제시하였다. Fig. 2A에서볼수있듯이혈장호모시스테인은간엽산과는음의상관관계 (r = -.641, p <.1) 을보였으나뇌엽산또는혈장엽산과는유의적인상관관계를보이지않았다. 그러나이와대조적으로 FDHCD 군을제외시킨나머지네개의실험군자료만으로상관관계를분석하였을때혈장호모시스테인과뇌엽산 (r = -.321, p <.5), 혈장엽산 (r = -.581, p <.1) 및간엽산 (r = -.684, p <.1) 사이에모두유의적인상관관계를나타냈다. 혈장호모시스테인과간조직의 SAH 및 SAM 농도사이의상관관계는 Table 5의 A와 B와같았으며, FDHCD 군을제외시킨나머지네개의실험군자료만으로상관관계를분석하였을때뇌조직 SAH 농도들제외한모든상관계수가전체실험군자료로분석한경우보다높았다. 즉, Table 5A에서혈장호모시스테인과간조직의 SAH 및 SAM Table 4. The effects of folate- and vitamin B12-deficient diet combined with homocystine feeding on liver and brain SAM and SAH concentration of rats Liver Brain Group SAH (nmole/g) SAM (nmole/g) SAM : SAH ratio SAH (nmole/g) SAM (nmole/g) SAM : SAH ratio FS 15.88 ±.85 1)c2) 41.73 ± 3.54 a 2.64 ±.21 a 1.55 ±.9 b 13.57 ±.31 NS3) 8.85 ±.46 a FSH 15.11 ±.79 c 33.66 ± 2. b 2.26 ±.19 a 1.45 ±.9 b 14.25 ±.65 9.92 ±.47 a FD 22.91 ± 1.64 b 11.83 ± 1.76 c.53 ±.8 b 1.44 ±.14 b 13.91 ±.66 9.93 ±. a FDH 23.78 ±.91 b 12. ± 1.28 c.52 ±.6 b 1.43 ±.9 b 13.45 ±.31 9.47 ±.39 a FDHCD 32.87 ± 1.43 a 7.93 ±.73 c.25 ±.3 b 2.49 ±.42 a 14.87 ±.42 6.37 ±. b 1) Values are mean ± S.E. (n = ) 2) Values with different superscript letter within a column are significantly different at p <.5 by Duncan s multiple-range test 3) NS: not significant
428 / 엽산과비타민 B 12 영양상태관련지표사이의상관관계분석 a r = -.156, NS 1) R 2 =.3533 a r = -.581, p <.1 1 1 Plasma folate (nmol/l) Plasma folate (nmol/l) b R 2 =.418 r = -.641, p <.1 b r = -.684, p <.1 R 2 =.4846 5 15 5 15 Liver folate (μmol/g) Liver folate (μmol/g) A c r = -.9, NS Brain folate (nmol/g) B r = -.321, p <.1 R 2 =.32 c Brain folate (nmol/g) Fig. 2. The correlation between concentrations of plasma homocysteine and tissue folate. A: Data of biomarkers of FS, FSH, FD, FDH and FDCDH (5 groups) were analyzed for correlation test. Correlation coefficients between plasma homocysteine and plasma folate, a: liver folate, b: and brain folate, c. B: Data of biomarkers of FS, FSH, FD and FDH (4 groups) were analyzed for correlation test. NS: not significant. 농도사이의상관계수는 ( 각각 r =.739, p <.1; r = -.629, p <.1) 였으며, Table 5B에서의이들상관계수 ( 각각 r =.878, p <.1; r = -.657, p <.1) 보다높았다. 그러나이와대조적으로혈장호모시스테인과뇌조직 SAH 사이의상관관계는 FDHCD 군을포함하였을때에만유의적이었다 (r =.667, p <.1). 고찰 역학조사및임상연구에서호모시스테인혈증이동맥경화성심혈관계질환, 뇌졸중, 말초동맥경색질환및정맥전색의독립적인위험인자로밝혀졌으며, 3,5) 인지능력손상, 치매, 알츠하이머및우울증과같은신경계질환의위험인자로도보고되었다. 3,31-33) 호모시스테인혈증은엽산또는비타민 B 12 가결핍되거나메티오닌대사회로의일부효소활성이결여되어메티오닌대사에장애가있을때나타난다. 엽산과비 타민 B 12 는메티오닌대사회로에영향을줌으로써 DNA 메틸화에필요한 SAM의합성및조직내 SAH의수준을유지시킴으로써신경세포및혈관계의기능을정상으로유지하는것으로제안되었으므로, -32) 본연구에서는흰쥐를대상으로식이에엽산과비타민 B 12 를결핍시켜경미한호모시스테인혈증또는중위의호모시스테인혈증을유발하였으며 (Fig. 1), 메티오닌대사회로상의이들비타민 biomarker를분석함으로써영양성호모시스테인혈증에있어엽산결핍과비타민 B 12 결핍이이 biomarker 들간의상관관계에미치는영향을조사하였다. 엽산결핍식이군 (FD, FDH) 의혈장엽산농도는대조군의약 13% 수준으로유의적으로낮았으나 (p <.1), FD- HCD군의혈장엽산농도는 FD군과 FDH군의약 6배높은수준으로조사되어, 엽산결핍 / 비타민 B 12 결핍 / 호모시스틴군의혈장엽산농도는엽산만결핍되었을때보다크게높았다. 이결과는비타민 B 12 결핍에의한 methyl-folate
한국영양학회지 (Korean J Nutr) 9; 42(5): 423~433 / 429 Table 5. Correlation between the concentration of vitamin biomarkers in plasma, liver and brain (A) Correlation among the five groups 1) Plasma Hcy Brain folate Plasma folate Liver folate Liver SAH Liver SAM Brain SAH Brain SAM Plasma Hcy 1 -.9 -.156 -.641** -.878** -.657** -.667** -.114 Brain folate 1 -.551** -.467** -.181 -.3 -.4 -.138 Plasma folate 1 -.712** -.9** -.588** -.69 -.18 Liver folate 1 -.718** -.812** -.333 -.35 Liver SAH 1 -.673** -.525** -.8 Liver SAM 1 -.284 -.135 (B) Correlation among the four groups 2) Plasma Hcy Brain folate Plasma folate Liver folate Liver SAH Liver SAM Brain SAH Brain SAM Plasma Hcy 1 -.321* -.581** -.684** -.739** -.629** -.7 -.5 Brain folate 1 -.575** -.657** -.336* -.6** -.27 -.252 Plasma folate 1 -.8** -.774** -.741** -.44 -.66 Liver folate 1 -.714** -.767** -.55 -.148 Liver SAH 1 -.573** -.739** -.55 Liver SAM 1 -.49 -.6 1) Data of the five experimental groups including FS, FSH, FD, FDH, and FDHCD were analysed according to Pearson test and correlation coefficients were presented. *: p <.5, **: p <.1 2) Data of the four experimental groups including FS, FSH, FD, and FDH were analysed according to Pearson test and correlation coefficients were presented. *: p <.5, **: p <.1 trap 으로인해엽산이세포내에효율적으로보유되지못하고혈류로운반된후배설되기때문인것으로보인다. 21-24) 이와대조적으로뇌조직의엽산농도는엽산결핍식이군 (FD, FDH) 에서는유의적으로저하되었으나, 흥미롭게도 FDH- CD군의뇌엽산농도는대조군과동일한수준을나타냈다. 따라서뇌조직은다른조직보다엽산결핍식이에의해경미하게영향받으며엽산농도의항상성이잘유지되는것으로나타났다. 이와같이 FDHCD 군의뇌엽산농도가변화되지않은이유는확실치않으나, 뇌조직은간조직과일부다른단일탄소기대사체계를가지고있기때문인것으로사료된다. 식이에호모시스틴을.3% 수준으로첨가하였을때 (FSH) 혈장호모시스테인농도가대조군 (4.95 ±.43 nmol/ml) 보다증가하여 7. ±.67 nmol/ml 으로상승되었으나, 통계적으로유의적인차이는아니었으며또한호모시스테인혈증의수준은아닌것으로나타났다. 따라서식이에.3% 의호모시스틴만을공급하는것으로는호모시스테인혈증유발에미흡하므로호모시스틴첨가량을증량하는것이필요한것으로보인다 (Table 4). 또한엽산결핍시에도호모시스틴첨가에의한혈장호모시스테인에유의적인영향을나타내지않아서 Kim 등 25) 의연구와차이가있었다. 호모시스테인은 S-adenosylhomocysteine hydrolase 의강력한저해제로작용하므로호모시스테인이증가되면조직의 SAH 농도도증가되는것으로알려져있다. 34) 본연 구의엽산결핍군 (FD, FDH) 에서간조직내 SAM 농도는약 % 저하되고 SAH 농도는약 % 증가되었으며, 그결과 SAM/SAH 비율이대조군보다약 8% 저하되었다. 또한엽산결핍 / 비타민 B 12 결핍 / 호모시스틴군 (FDHCD) 의경우간조직내 SAM 농도는약 81% 저하되고 SAH 농도는약 7% 증가되었으며, 그결과 SAM/SAH 비율이대조군보다약 9% 저하되었다. 이와같이비타민 B 12 의결핍시 SAH 농도가특히현저하게증가되었다. 또한 SAM은 MTHFR의알로스테릭저해제로작용하므로, 엽산과비타민 B 12 의결핍으로인한 SAM/SAH 비율의저하는 MT- HFR의활성을증가시켜엽산의기능적결핍을더욱심화시키는것으로알려져있다. 35,36) 이와같이 SAM 합성이저하되면엽산조효소가 5MTHF 형태로되어메티오닌대사회로에주로이용되므로 DNA합성에필요한퓨린이나피리미딘합성에는이용되지못하게되어, 세포분열과단백질합성을제한하는결과가나타난다 (methyl-folate trap). 22) 따라서신경이나뇌조직과같은조직에서더중요한메틸화반응에메티오닌이이용될수있도록뇌조직에메티오닌이더효율적으로보유됨으로써뇌의 SAM 농도는간조직 SAM 농도와달리항상성이잘유지되는것으로보고되었다. 22) 엽산과비타민 B 12 는호모시스테인으로부터메티오닌이합성되는반응의조효소로작용하며, 메티오닌은 methionine-adenosyltransferase의작용에의해 SAM 합성에이용되므로호모시스테인이메티오닌으로재메틸화되는반응
4 / 엽산과비타민 B 12 영양상태관련지표사이의상관관계분석 은 SAM의합성에필수적이다. 2,37,38) Table 4와같이간조직내메티오닌회로의메틸화관련 biomarker 들이크게변화되었으며, 또한 SAH는 SAM이메틸기를전이하는반응의생성물이며 DNA 메틸화를비롯한여러가지메틸화반응을저해하는물질이므로, 34) DNA를비롯한여러물질들의메틸화가저해될수있다. 또한 SAM은 DNA methyltransferase 의메틸기공여체이므로조직내 SAM의부족은 DNA 메틸화를저하시킴으로써신경조직의발달및유지에필요한특정유전자의발현에영향을줄수있는것으로제안되고있다. 39,) SAH는 SAH hydrolase 에의해호모시스테인과 adenosine 으로분해되지만, 호모시스테인이효율적으로제거되지않는경우 SAH hydrolase 의반응이역반응으로일어나므로호모시스테인으로부터 SAH가합성되는반응이우세하게일어난다. 39,41) 그이유는 methyltransferase 에대한친화력이 SAM 보다 SAH가훨씬높기때문이며, 이런성질때문에거의모든종류의 methyltransferase 의효소작용은 SAH 에의해크게저해된다.,41) 이와같이 SAH의 methyltransferase 에대한친화력이 SAM 보다큰성질때문에, SAH 농도가 SAM : SAH ratio 보다 methyltransferase 의활성저하와더밀접한관계가있었으며,,41) 혈장과림프구에서도 SAH 농도가높을때 DNA 메틸화수준이유의적으로낮았다. 42) 심혈관계질환자의혈장호모시스테인농도 (moderate homocysteinemia) 와혈장 SAH의농도는대조군보다높았으며, 심혈관계질환자의경우이들 biomarker 들사이의상관관계가높았다. 42,43) 간조직과달리엽산결핍식이군 (FD, FDH) 의뇌조직 SAM 농도는엽산결핍, 비타민 B 12 결핍및호모시스틴급여에의해영향을받지않고대조군과유사한수준을보였다 (Table 4). 이와같이뇌조직의 SAM 농도는엽산결핍과비타민 B 12 결핍식이에의해변화되지않은반면 SAH 농도는엽산과비타민 B 12 결핍식이에의해유의적으로영향을받는것을알수있었다. 뇌에는간조직과달리 betaine-homocysteine methyltransferase 가결여되어있으므로뇌의호모시스테인대사는간조직과동일하지않을것으로생각된다. 메티오닌대사회로의 biomarker 들간의상관관계를전체실험군 (5개실험군 ) 을대상으로분석한결과와비타민 B 12 부족군인 FDHCD 군을제외한 4개실험군의자료를따로분리하여분석한결과를비교하였다 (Table 5). 전체실험군자료로분석하였을때에는혈장호모시스테인농도는혈장엽산및뇌엽산과상관관계를나타내지않았으며, 비타민 B 12 결핍군 (FDCDH) 을제외시킨나머지 4군자료를분 석하였을때에만유의적인상관관계가나타났다 (Fig. 2, Table 5A). 이와같은결과가나타난이유는비타민 B 12 가결 핍된경우간조직의엽산농도는저하되나혈장과뇌조직의 엽산농도는상대적으로영향을받지않기때문에비타민 B 12 결핍시혈장호모시스테인, 혈장엽산및뇌엽산농도사 이의관계가변화되기때문인것으로보인다. 따라서비타 민 B 12 결핍이빈번히나타나는노인대상의역학조사또 는인체연구에서혈장내비타민 biomarker 들사이의상관 성을분석할때이를고려할필요가있을것으로사료된다. 혈장호모시스테인과간조직의 SAH 및 SAM 농도사이 의상관관계는 FDHCD 군을제외시킨나머지네개의실험 군자료만으로상관관계를분석하였을때뇌조직 SAH 농 도들제외한모든상관계수가전체실험군자료로분석한 경우보다대체로높은경향이었다 (Table 5). 이와대조적 으로혈장호모시스테인과뇌조직 SAH 사이의상관관계는 FDHCD 군을포함하였을때에만유의적이었다. 이상의결 과로부터간조직의 SAH 와 SAM 농도는혈장호모시스테 인과유사하게변화되나, 뇌조직의 SAH 와 SAM 의농도는 간조직과달리혈장호모시스테인과관련되어있지않으며 뇌조직에는 SAM 과 SAH 의독립적인조절기전이있기때 문인것으로추정된다. 또한 Table 5B 에서만혈장호모시 스테인과뇌조직 SAH 농도사이에상관관계가관찰된이 유는엽산만결핍된경우 (FD 또는 FDH) 뇌조직의 SAH 농도가증가되지않았으나 (Table 4), 엽산과비타민 B 12 가동시에결핍된 FDHCD 군에서 SAH 가현저하게증가 되었기때문인것으로보인다. 일반노인을대상으로한연구에서치매환자들의혈장 내엽산, 비타민 B 12 및 B 6 농도와인지능력점수사이에 음의상관관계가관찰되었으며, 혈장호모시스테인농도는 조직내엽산또는비타민 B 12 및 B 6 농도보다인지능력저 하와더상관성이높은것으로조사되었다. 2,17) 또한신경및 정신질환자들의뇌, 척수액내호모시스테인농도는정상 인보다높았으며, 이때뇌척수액의호모시스테인농도는 혈청호모시스테인농도와비례관계를나타냈다. 44,45) 그러 나뇌척수액호모시스테인농도는혈장호모시스테인의 1/ ~1/ 정도로낮은수준이었다. 45) 우울증환자들의적혈구엽산및혈장엽산농도는대조 군보다낮았으며, 46) 우울증이심한정도와혈청엽산, 적혈 구엽산및엽산결핍기간사이에도상관관계가보고되었 다. 47) 또한, 우울증환자의약 31% 정도가낮은혈청비타 민 B 12 수준을나타냈으며일반여성을대상으로한연구 에서도유사한결과가보고되었다. 46,48) 심한고호모시스테인혈증환자들은정신지체, 뇌위축, 발
한국영양학회지 (Korean J Nutr) 9; 42(5): 423~433 / 431 작등광범위한임상적병변을나타낸다. 49) 이환자들의뇌신경손상의원인이호모시스테인의직접적인독성효과때문인지혈관병변을통한이차적인결과인지는확실하지않으나, 최근에호모시스테인은 N-methyl-D-aspartate (NM- DA) 수용체에 agonist 로작용하여신경장애를가져오거나, 호모시스테인의산화산물인 homocysteic acid가뇌세포에서생성된후신경자극에의해분비되어 NMDA 수용체를활성화시켜일종의흥분성신경전달물질로작용하는것으로보고되고있다. ) 비타민 B 12 의영양상태를정확히판정하는것은매우중요함에도불구하고, 총혈청비타민 B 12 의 5~% 만이 transcobalamin 에결합되어세포내로운반되므로총혈청비타민 B 12 농도는비타민 B 12 활성을나타내는적합한지표가아닌것으로지적되고있다. 따라서최근연구에서는혈장호모시스테인과혈장 methylmalonic acid 농도가비타민 B 12 결핍을측정하는지표로많이이용되고있다. 7,) 이상의결과를요약하면, 엽산결핍시와엽산결핍 / 비타민 B 12 결핍시에혈장엽산, 뇌엽산및뇌 SAH와호모시스테인농도사이의상관관계가현저한차이를보였다. 또한, 엽산 / 비타민 B 12 결핍으로인한뇌 SAH 농도의증가는뇌조직에서메틸화저해요인으로작용할수있으므로영양성호모시스테인혈증에서나타나는인지능력저하의원인으로작용할수있을것으로사료된다. 요약 본연구는흰쥐를대상으로엽산결핍또는엽산결핍 / 비타민 B 12 결핍 /.3% 호모시스틴식이의공급을통해각각경미한호모시스테인혈증과중위의호모시스테인혈증을유도한후, 혈장, 간및뇌조직내메티오닌대사회로 biomarker와엽산농도사이의상관관계를분석함으로써영양성호모시스테인혈증의특성을규명할목적으로실시하였다. 6 주령 Sprague-Dawley 숫컷쥐에게엽산이충분한식이 (FS), 엽산결핍식이 (FD), 또는동일식이에호모시스틴을첨가한식이 (FSH and FDH), 엽산결핍 / 비타민 B 12 결핍 / 호모시스틴첨가식이 (FDHCD) 를 8주간공급하였다. 1) FD와 FDH 식이군은경미한호모시스테인혈증을 (17.41 ± 1.94 nmol/ml) 나타냈으며, FDHCD 식이군은중위의호모시스테인혈증을 (44.13 ± 2.65 nmol/ml) 나타내어엽산과비타민 B 12 결핍에의한영양성호모시스테인혈증의모델로이용할수있었다. 2) FD, FDH, FDHCD 식이군의간 (p <.1) 과뇌조직 (p <.1) 내엽산농도는 FS, FSH군보다유의적으 로낮았으나, FD, FDH, FDHCD 식이군의간및뇌조직의엽산농도사이에는유의적인차이가없었다. 이와대조적으로혈장엽산농도는 FDHCD 식이군 (126.5 ± 9.6 nmol/ L) 이 FD, FDH 식이군 (21.1 ± 1.4 nmol/l, 22. ± 2.2 nmol/l)(p <.1) 보다약 6배높았으며, 이는비타민 B 12 결핍에의한 methyl-folate trap 으로인해엽산이효율적으로조직내보유되지못하고혈류로나와소변을통해배설되기때문인것으로보인다. 3) FD와 FDH 식이군의간조직의 SAH 농도는각각대조군보다 44% 및 % 씩증가되었고 (p <.1), 간 SAM 농도는각각대조군보다 72%, 71% 저하되었으며 (p <.1), 그결과두군모두 SAM/SAH 비율이대조군보다약 8% 저하되었다 (p <.1). 한편 FDHCD 식이군의간 SAH 농도를대조군과비교할때대조군보다 7% 증가되었고 (p <.1), SAM 농도는대조군보다 81% 저하되었으며 (p <.1), 그결과 SAM/SAH 비율이대조군보다약 9% 저하되어매우낮은 SAM/SAH 비율을나타냈다 (p <.1). 뇌조직 SAM 농도는엽산결핍, 비타민 B 12 결핍및호모시스틴급여에의해영향을받지않고대조군과유사한수준을보여뇌조직내 SAM의항상성을나타냈으나, FDHCD 식이군의뇌조직 SAH 농도는대조군보다 % 증가되었으며 (p <.5), 그결과 SAM/SAH 비율은대조군보다약 28% 저하되었다 (p <.5). 따라서중위의호모시스테인혈증을나타낸실험군에서만뇌조직의 SAH 농도가증가되었다. 4) 영양결핍 ( 엽산또는비타민 B 12) 에의한호모시스테인혈증의특성을조사할목적으로혈장, 간및뇌조직내메티오닌대사회로 biomarker 와엽산농도사이의상관관계를조사하였다. 혈장호모시스테인은간엽산과음의상관관계 (r = -.641, p <.1) 을보였으나, 뇌엽산또는혈장엽산과는유의적인상관관계를보이지않았다. 그러나이와대조적으로 FDHCD 식이군을제외시킨나머지네개의실험군자료만으로상관관계를분석하였을때혈장호모시스테인과뇌엽산 (r = -.321, p <.5), 혈장엽산 (r = -.581, p <.1), 간엽산 (r = -.684, p <.1) 사이에모두유의적인상관관계를나타냈다. 혈장호모시스테인과간조직의 SAH 및 SAM 농도사이의상관관계는 FDHCD 군을제외시킨나머지네개의실험군자료만으로상관관계를분석하였을때뇌조직 SAH 농도들제외한모든상관계수가전체실험군자료로분석한경우보다높았다. 따라서엽산결핍 / 비타민 B 12 결핍으로인한호모시스테인혈증 (FDHCD) 에서는혈장엽산이엽산결핍군 (FD, FDH) 보다높으면서동시에혈장호모시스테인농도도높은특성
432 / 엽산과비타민 B 12 영양상태관련지표사이의상관관계분석 을보였다. 결론적으로, 식이중엽산만결핍된경우와엽산결핍과비타민 B 12 결핍을동반할경우 methyl-folate trap 으로인해혈장엽산과호모시스테인농도패턴에차이가있었으며, 메티오닌대사회로의 biomarker 사이의상관관계와혈장엽산, 뇌엽산및뇌 SAH와호모시스테인농도사이의상관관계가차이가있었다. 또한엽산결핍과비타민 B 12 결핍으로인해나타나는뇌 SAH 농도의증가는메틸화를저해시킴으로써인지능력에영향을줄수있을것으로사료된다. Literature cited 1) Goodwin JS, Goodwin JM, Garry PJ. Association between nutritional status and cognitive functioning in a healthy elderly population. JAMA 1983; 249(21): 2917-2921 2) Riggs KM, Spiro A III, Tucker K, Rush D. Relations of vitamin B-12, vitamin B-6, folate, and homocysteine to cognitive performance in the Normative Aging Study. Am J Clin Nutr 1996; 63 (3): 6-314 3) Selhub J, Bagley LC, Miller J, Rosenberg IH. B vitamins, homocysteine, and neurocognitive function in the elderly. Am J Clin Nutr ; 71(2): 614s-6s 4) Refsum H, Ueland PM, Nygård O, Vollset SE. Homocysteine and cardiovascular disease. Annu Rev Med 1998; 49: 31-62 5) Selhub J. Public health significance of elevated homocysteine. 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