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1 Available online at Annals of Clinical & Laboratory Science, vol. 44, no. 4, 2014 Association of the Urine Homocysteine/Creatinine Ratio to Proinflammatory Cytokine, Natural Anticoagulant, and Nitric Oxide Levels in Cerebrovascular Disease Jong Weon Choi 1, Moon Hee Lee 2, Tatsuyoshi Fujii 3, Noriyoshi Fujii 4, and Yeonsook Moon 1 Departments of 1 Laboratory Medicine and 2 Internal Medicine, College of Medicine, Inha University, Incheon, Korea, 3 School of Medicine, University of Tsukuba, Ibaraki, Japan, and 4 Department of Electrical Engineering and Bioscience, Waseda University, Tokyo, Japan Abstract. This study investigated the relationship between the urine homocysteine/creatinine (uhcy/cr) ratio and the levels of natural anticoagulants, proinflammatory cytokines, and nitric oxide (NOx) metabolites in cerebrovascular diseases. No significant differences were observed in protein C, protein S, and antithrombin III levels among subjects with serum Hcy (shcy) >15.0 µmol/l and 15.0 µmol/l. However, subjects with a uhcy/cr ratio >14.8 µmol/g Cr showed significant differences in the levels of the corresponding parameters than those with uhcy/cr ratio 14.8 µmol/g Cr. The sensitivity and specificity of the shcy level at a cutoff of 15.0 µmol/l were 32.6% and 85.7%, respectively, with a positive predictive value of 69.5%. In contrast, those values for the uhcy/cr ratio at a cutoff of 14.8 µmol/g Cr were 55.1% and 91.4% with a positive predictive value of 86.5%. The uhcy/cr ratio correlated more closely with protein C, antithrombin III, TNF-α, and NOx levels than did shcy concentrations. In short, the uhcy/cr ratio has a significant relationship with anticoagulation- and inflammation-related parameters. A measurement of the uhcy/cr ratio may provide helpful information for assessing patients with cerebrovascular diseases. Keywords: homocysteine, anticoagulation, nitric oxide, urine, cerebrovascular diseases. 461 Introduction Homocysteine (Hcy) is a highly reactive, sulfhydryl-containing amino acid. Hcy plays crucial roles as a proinflammatory factor, as a prothrombotic agent, and as a vasodilation-impairing element in patients with hyperhomocysteinemia (HHcy) [1]. Vascular endothelial cells are extremely sensitive to serum Hcy concentration. HHcy disrupts the vascular endothelium and changes the characteristics of endothelial function from anticoagulant to procoagulant [2]. Modification of the hemostatic protein induced by Hcy is one of the biotoxicities of Hcy in vascular diseases [3]. HHcy has been considered a risk factor for atherothrombotic diseases, but a causal relationship between HHcy and thrombotic vascular diseases is still debated [4]. HHcy is also found in subjects with impaired renal function [5]. Address correspondence to Jong Weon Choi, MD, PhD; Department of Laboratory Medicine, Inha University Hospital, 7-206, 3-ga, Shinheung-dong, Jung-gu, Incheon , Korea; phone: ; fax: ; e mail: jwchoi@inha.ac.kr Hcy is an endogenous compound excreted in urine and is stable up to 3 months when urine specimens are stored at -20 o C. Because of fluctuations in the concentrations of solutes, urine Hcy (uhcy) levels are expressed as the ratio to urine creatinine (Cr) concentrations [6]. Serum Hcy concentrations and their association with coronary heart diseases have been extensively studied. However, data for Hcy levels in urine specimens, especially in relation to the excretion of creatinine, are limited in cerebrovascular diseases. The present study investigated whether the corrected value of uhcy adjusted by the urine Cr level (uhcy/ Cr ratio) is significantly associated with the blood levels of natural anticoagulants, tumor necrosis factor-α (TNF-α), interleukin-5 (IL-5), and nitric oxide (NOx) metabolites in cerebral ischemia. Materials and Methods A total of 98 patients with cerebrovascular diseases [51 males and 47 females; median age =67 (range=45-79 years)] were studied. Age-matched healthy subjects (n=35) without preexisting cerebrovascular /14/ by the Association of Clinical Scientists, Inc.

2 462 Table 1. Median values of proinflammatory markers and natural anticoagulants in relation to shcy levels and the uhcy/cr ratio, and the correlation coefficients between Hcy-related indices and various parameters. Patients vs. controls Patient group patient group healthy controls shcy level (µmol/l ) uhcy/cr ratio (µmol/g Cr) Serum Cr levels (mg/dl) a Correlation coefficients 15.0 > > shcy uhcy/cr (µg/l) ratio ( n = 98) (n =35) (n = 66) (n =32) (n = 44) (n =54) (n = 25) (n =25) shcy and uhcy/cr ratio shcy levels (µmol/l) 17.2( ) b 10.8( ) 10.5( ) 18.6( ) c 13.2( ) 16.5( ) c 11.4 ( ) 18.1 ( ) c r NA NA p uhcy/cr ratio (µmol/g Cr) 20.9 ( ) b 8.2 ( ) 14.2( ) 25.7( ) c 11.4( ) 28.6( ) c 18.7 ( ) 21.4( ) r NA NA p Proinflammatory markers TNF-α(pg/mL) 38.1( ) b 24.9( ) 34.5( ) 42.7( ) c 34.8( ) 43.2( ) c 39.6( ) 37.1( ) r p IL-5 (pg/ml) 16.5( ) b 10.9( ) 11.6( ) 21.5( ) c 12.0( ) 20.8( ) c 17.4( ) 15.2 ( ) r p NOx (µmol/l) 23.9( ) b 36.2( ) 27.3( ) 18.4( ) c 28.1( ) 17.5( ) c 21.5( ) 24.6( ) r p hscrp (mg/dl) 0.21( ) b 0.09( ) 0.17( ) 0.23( ) c 0.16( ) 0.24( ) c 0.20( ) 0.22( ) r p Natural anticoagulants Protein C (%) 84(48-114) b 95(84-129) 92(63-114) 90(48-107) 94(57-114) 85(48-107) c 86(51-114) 82(48-109) r p <0.001 Protein S (%) 90(54-121) 93(81-125) 90(54-121) 89(67-109) 91(63-116) 83(54-121) c 87(56-121) 92(54-117) r p AT III (%) 83(69-105) b 96(76-114) 89(69-105) 87(79-101) 93(69-105) 84(82-98) c 84(73-103) 82(69-105) r p <0.001 <0.001 Hcy-related cofactors Vitamin B12 (pg/ml) r ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) p Folate (ng/ml) 7.1( ) b 10.4( ) 8.2( ) 5.0( ) c 8.0( ) 5.1( ) c 6.9( ) 7.3( ) r p <0.001 <0.001 Renal function Serum Cr (mg/dl) 1.0( ) 0.8( ) 0.8( ) 1.1 ( ) c 0.9( ) 1.0( ) 0.7( ) 1.2( )c r p Data are expressed as medians (ranges). shcy = serum homocyteine, uhcy/cr ratio = ratio of urine Hcy to urine creatinine levels, TNF-α = tumor necrosis factor-α, IL-5 = interleukin-5, NOx = nitric oxide, hscrp = high-sensitivity C-reactive protein, AT III = antithrombin III, NA= not applicable. a The values of the 75 th percentile of serum Cr levels are compared with those of the 25 th percentile for the 98 patients included in the study. b,c Statistically significant (p<0.05), versus healthy controls and the corresponding groups, respectively, were computed by a Mann-Whitney U test.

3 Urine homocysteine/creatinine ratio in cerebrovascular disease 463 and cardiovascular diseases were evaluated as a control group. This study was approved by the Institutional Review Board of Inha University Hospital. We included patients with ischemic stroke (n=78) and transient ischemic attack (n=20) who showed no cognitive impairment and normal renal function (serum Cr levels 1.5 mg/dl). Stroke was defined as a neurological deficit of vascular origin that lasted longer than 24 hours and was confirmed by radiographic studies [7]. A transient ischemic attack was defined as a sudden onset of focal neurological dysfunction of vascular origin that was resolved within 24 hours [8]. Subjects excluded from study were patients with hepatitis (n=4), abnormal thyroid function (n=2), and a history of medications (n=2), especially the drugs phenytoin, methotrexate, and thiazide diuretics because these drugs may influence Hcy concentrations [9]. Serum Hcy (shcy) levels were measured by an enzymatic reaction using a commercially available kit (Catch Inc, Seattle, WA, USA) [10]. Concentrations of uhcy were analyzed by high-performance liquid chromatography using 3-mercaptopropionic acid as an internal standard, as described previously [6]. The uhcy/cr ratio was calculated with the following formula: uhcy/cr ratio (µmol/g Cr)=uHcy level (µmol/l)/urine Cr concentration (g/l) [11]. The following parameters were measured: proinflammatory markers [TNF-α, IL-5, NOx, and high-sensitivity C-reactive protein (hscrp)], natural anticoagulants [protein C, protein S, and antithrombin III (AT III)], and Hcy-related cofactors (vitamin B12 and folate). TNF-α and IL-5 were measured using commercially available enzyme-linked immunosorbent assay kits (manufactured by R&D Systems, Minneapolis, MN, USA, and BD Inc., San Diego, CA, USA, respectively). Serum hscrp levels were assayed by a turbid immunoassay method using a chemical analyzer (Hitachi 7180, Hitachi, Tokyo, Japan). NOx concentrations were determined by a reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent nitrate reductase assay. After serum nitrate was converted to nitrite by NADPHdependent nitrate reductase, the total concentration of nitrite was measured by spectrophotometry at 540 nm [12]. Patients were classified into 2 groups according to shcy levels and their uhcy/cr ratio: patients with shcy 15.0 µmol/l (n=66) and >15.0 µmol/l (n=32); and patients with uhcy/cr ratio 14.8 µmol/g Cr (n=44) and >14.8 µmol/g Cr (n=54). The cutoff points of the shcy levels and the uhcy/cr ratio were based on the 95% confidence limits of shcy concentration and uhcy/cr ratio of healthy controls. To assess the effect of renal function on shcy and uhcy/ Cr ratio, patients were further divided into 2 groups based on the 75 th and 25 th percentiles of serum Cr levels in patient populations: patients with serum Cr 0.8 mg/ dl (n=25) and 1.1 mg/dl (n=25). Data analysis was conducted using a non-parametric test (Mann-Whitney U test). Correlation coefficients were calculated by Spearman s method. All p values <0.05 were considered statistically significant. Results As shown in Table 1, shcy levels were significantly higher in the patient group than in healthy controls. In particular, median levels of the uhcy/cr ratio in patients were 2.5 times higher than those in the controls (20.9 vs. 8.2, p<0.05). Patients showed significant differences in the levels of TNF-α, IL-5, NOx, hscrp, protein C, AT III, and folate compared with controls, but no significant differences were observed in protein S and vitamin B12 levels between the 2 groups. Patients with shcy >15.0 µmol/l revealed a propensity toward decreased values in protein C, protein S, and AT III levels, compared to those with shcy 15.0 µmol/l, but no statistically significant differences were noted between the 2 groups. In contrast, medians of protein C, protein S, and AT III levels in patients with uhcy/cr ratio >14.8 µmol/g Cr were 85%, 83%, and 84%, respectively, which were significantly lower than those in patients with uhcy/cr ratio 14.8 µmol/g Cr (94%, 91%, and 93%, p <0.05, respectively). TNF-α, IL-5, and hscrp levels were increased to a significantly greater extent in patients with shcy levels >15.0 µmol/l and uhcy/cr ratio >14.8 µmol/g Cr than in patients with shcy levels 15.0 µmol/l and uhcy/cr ratio 14.8 µmol/g Cr. Serum NOx concentrations were significantly lower in the groups with elevated shcy and uhcy/cr ratio than in non-elevated groups of shcy and uhcy/cr ratio (Table 1). Sensitivity, specificity, and positive predictive value of the uhcy/cr ratio at a cutoff of 14.8 µmol/g Cr were 55.1%, 91.4%, and 86.5%, respectively, exceeding those with an shcy level at a cutoff of 15.0 µmol/l (32.6%, 85.7%, and 69.5%, respectively). Sensitivity was increased up to 66.3% when a

4 464 Annals of Clinical & Laboratory Science, vol. 44, no. 4, 2014 Table 2. Prevalence of elevated shcy levels and the uhcy/cr ratio in patients with cerebrovascular diseases and healthy controls. Parameters Patient group (n=98) Healthy controls (n=35) shcy levels (µmol/l) > (32.6%) a 5(14.3%) (67.4%) a 30(85.7%) uhcy/cr ratio (µmol/g Cr) > (55.1%) a 3(8.6%) (44.9%) a 32(91.4%) Combination index shcy levels >15.0 µmol/l or uhcy/cr 65(66.3%) a 4(11.4%) ratio >14.8 µmol/g Cr a p<0.05 versus healthy controls, computed by a Mann-Whitney U test. combination index of shcy levels >15.0 µmol/l and uhcy/cr ratio >14.8 µmol/g Cr was applied (Table 2). The median shcy level in patients with serum Cr levels 1.1 mg/dl (75 th percentile) was 18.1 µmol/l, which was significantly above the value in those with serum Cr levels 0.8 mg/dl (25 th percentile; 11.4 µmol/l, p<0.05); however, no significant differences were found in the uhcy/cr ratio between the 2 groups. The shcy level and uhcy/cr ratio had significant correlations with TNF-α, IL-5, NOx, and hscrp levels. The uhcy/cr ratio was more strongly correlated with the levels of protein C, AT III, TNF-α, and NOx metabolites than with shcy levels. Serum Cr levels were significantly correlated with shcy levels (r=0.23, p=0.029) but did not correlate with the uhcy/cr ratio. Discussion In this study, a new index for the uhcy/cr ratio, which was determined using uhcy levels amended by urine Cr content, was investigated. There were no significant differences in protein C, protein S, and AT III levels between elevated and non-elevated shcy groups. However, median values of the corresponding parameters were significantly lower in patients with an increased uhcy/cr ratio than in those with a decreased uhcy/cr ratio. The uhcy/ Cr ratio more strongly correlated with protein C, AT III, TNF-α, and hscrp levels than with shcy concentrations. These results suggest that the uhcy/ Cr ratio, in comparison with shcy levels, may accurately reflect the potential of thrombosis and subclinical inflammation in cerebrovascular diseases. Briani et al [13] reported that increased shcy levels were observed in 54.2% of patients who had suffered a stroke, and Stein and McBride [14] demonstrated that HHcy was detected in 42% of patients with cerebrovascular diseases. In our study, elevated shcy levels were observed in 32.6% of the patients. These discrepancies may reflect the differences in patient populations and in the severity of disease among the studies. In our study, patients with stroke, showing no cognitive impairment and normal renal function, were enrolled. In the current study, sensitivity, specificity, and positive predictive values of the uhcy/cr ratio were superior to those of shcy levels. Sensitivity was elevated when a combination index of the shcy level and the uhcy/cr ratio was used. These observations imply that measurement of the uhcy/cr ratio in conjunction with shcy levels may be useful to evaluate patients with cerebrovascular diseases. Potter et al [15] reported that HHcy is a marker for renal impairment rather than a cardiovascular risk factor. A group of investigators demonstrated that a high shcy level was closely associated with chronic

5 Urine homocysteine/creatinine ratio in cerebrovascular disease 465 renal disease [16]. In the present study, patients with increased serum Cr levels exhibited significantly higher shcy levels than those with decreased serum Cr levels; however, no significant differences were observed in the uhcy/cr ratio between the 2 groups. Further, serum Cr levels were closely correlated with shcy levels but not with the uhcy/cr ratio. These findings indicate that the uhcy/cr ratio compensated by urine Cr levels may lessen the impact of kidney function on Hcy concentrations. This is a likely explanation as to why there were no significant differences in the uhcy/cr ratio between the 75 th and 25 th percentiles for serum Cr levels in subject populations. HHcy is a strong inducer of endothelial dysfunction in small vessels such as cerebral arterioles. Endothelial dysfunction is related to the suppression of endothelial NOx synthase by endogenous inhibitors [17]. In our study, serum NOx concentrations were significantly lower in patients with increased shcy levels than in those with decreased shcy levels, as well as in patients with an elevated uhcy/cr ratio rather in those with a non-elevated uhcy/cr ratio. These findings partly support the previous results of Karolczak and Olas [18], which demonstrated that links of Hcy to NOx metabolism are the main reason of biotoxicity of Hcy in cardiovascular diseases. Because NOx is a potent relaxant of vascular smooth muscle, the diminished NOx production in our study may account for the adverse effect on hemodynamic function in patients with a stroke. In summary, the uhcy/cr ratio was closely associated with natural anticoagulants, proinflammatory cytokiness, and NOx metabolites, irrespective of renal function. Measurement of the uhcy/cr ratio in conjunction with shcy levels may offer additional benefits for monitoring patients with cerebrovascular diseases. Acknowledgements References 1. Di Minno MN, Tremoli E, Coppola A, Lupoli R, Di Minno G. Homocysteine and arterial thrombosis: challenge and opportunity. Thromb Haemost 2010;103: Weiss N. Mechanisms of increased vascular oxidant stress in hyperhomocysteinemia and its impact on endothelial function. Curr Drug Metab 2005;6: Harpel PC, Zhang X, Borth W. Homocysteine and hemostasis: pathogenic mechanisms predisposing to thrombosis. J Nutr 1996;126:1285S-1289S. 4. Terwecoren A, Steen E, Benoit D, Boon P, Hemelsoet D. Ischemic stroke and hyperhomocysteinemia: truth or myth? Acta Neurol Belg 2009;109: Ferechide D, Radulescu D. Hyperhomocysteinemia in renal diseases. J Med Life 2009;2: Proksch B, Jelesnianski S, Oberrauch W, Fux R, Gleiter CH. Adaptation of a high-performance liquid chromatographic method for quantitative determination of homocysteine in urine. J Chromatogr B 2005;828: Sun Y, Chien KL, Hsu HC, Su TC, Chen MF, Lee YT. Use of serum homocysteine to predict stroke, coronary heart disease and death in ethnic Chinese. 12-year prospective cohort study. Circ J 2009;73: Tyrrell P, Swain S, Rudd A. Diagnosis and initial management of transient ischaemic attack. Clin Med 2010;10: Ueland PM, Refsum H. Plasma homocysteine, a risk factor for vascular disease: plasma levels in health, disease, and drug therapy. J Lab Clin Med 1989;114: Kellogg MD, Parker R, Ricupero A, Rifai N. Evaluation of an enzymatic homocysteine assay for the Hitachi series chemistry analyzer. Clin Chim Acta 2005;354: Kusmierek K, Glowacki R, Bald E. Analysis of urine for cysteine, cysteinylglycine, and homocysteine by high-performance liquid chromatography. Anal Bioanal Chem 2006;385: Choi JW. Nitric oxide production is increased in patients with rheumatoid arthritis but does not correlate with laboratory parameters of disease activity. Clin Chim Acta 2003;336: Briani C, Cagnin A, Gallo L, Toffanin E, Varagnolo M, Zaninotto M. Anti-heparan sulphate antibodies and homocysteine in dementia: markers of vascular pathology? J Neurol Sci 2005;229: Stein JH, McBride PE. Hyperhomocysteinemia and atherosclerotic vascular disease: pathophysiology, screening, and treatment. Arch Intern Med 1998;158: Potter K, Hankey GJ, Green DJ, Eikelboom JW, Arnolda LF. Homocysteine or renal impairment: which is the real cardiovascular risk factor? Arterioscler Thromb Vasc Biol 2008;28: Shankar A, Wang JJ, Chua B, Rochtchina E, Flood V, Mitchell P. Positive association between plasma homocysteine level and chronic kidney disease. Kidney Blood Press Res 2008;31: Wilson KM, Lentz SR. Mechanisms of the atherogenic effects of elevated homocysteine in experimental models. Semin Vasc Med 2005;5: Karolczak K, Olas B. Mechanism of action of homocysteine and its thiolactone in hemostasis system. Physiol Res 2009;58: This study was supported by a research grant from Inha University.

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324 Annals of Clinical & Laboratory Science, vol. 34, no. 3, 2004 Brief Communication: Association of Serum Insulin-Like Growth Factor-I and Erythropoiesis in Relation to Body Iron Status Jong Weon Choi