Lack of evidence for contribution of Glu298Asp (G894T) polymorphism of endothelial nitric oxide synthase gene to plasma nitric oxide levels

Thrombosis Research 107 (2002) 129 – 134

Regular Article

Lack of evidence for contribution of Glu298Asp (G894T) polymorphism of endothelial nitric oxide synthase gene to plasma nitric oxide levels Jesung Moon a, Suin Yoon a, Eunkyung Kim a, Chol Shin b, Sangmee Ahn Jo c, Inho Jo a,* a

Division of Cardiovascular Research, Department of Biomedical Sciences, National Institute of Health, 5 Nokbun-dong, Eunpyung-gu, Seoul 122-701, South Korea b Ansan Health Center, Korea University Hospital, 516 Gojan-dong, Ansan, Kyonggi-do 425-707, South Korea c Division of Brain Research, Department of Biomedical Sciences, National Institute of Health, 5 Nokbun-dong, Eunpyung-gu, Seoul 122-701, South Korea Received 7 May 2002; received in revised form 16 August 2002; accepted 30 August 2002

Abstract Introduction: Both positive and negative associations between a rare allele of 27-bp repeat polymorphism in intron 4 of endothelial nitric oxide synthase and plasma nitric oxide (NO) levels were previously reported, and further, these conflicting results were suggested to be partly accounted for smoking status of subjects. However, the genetic contribution of Glu298Asp (G894T) polymorphism to plasma NO levels with respect to smoking status has not been published. Methods: In a group of 411 healthy Korean subjects aged 19 – 81 years, the end product of NO (NOx: nitrite plus nitrate) as an index of plasma NO levels was measured by the Griess method. The genotypes of G894T polymorphism were determined by the banding patterns on gel electrophoresis after restriction enzyme digestion. Results: Comparison of plasma NOx levels revealed no significant differences across the genotypes and alleles of G894T polymorphism, which is independently of smoking status. However, significant differences in plasma NOx levels between nonsmokers and smokers were observed ( P = 0.0040). Furthermore, only the common G allele was found to be responsible for these differences. Multiple regression analysis showed that the most independent contributing factor for plasma NOx levels was smoking ( P = 0.0119) and followed by triglycerides ( P = 0.0384). Conclusions: Our results indicate no substantial effect of G894T polymorphism on the variance of plasma NOx levels in healthy Korean population. D 2002 Elsevier Science Ltd. All rights reserved. Keywords: Endothelial nitric oxide synthase gene; Polymorphism; Plasma nitric oxide level; Korea

Nitric oxide (NO) is one of the most versatile molecules known, and plays an important role in almost every biological system. It can interact with a number of molecular targets, and these determine the profile of NO as a major biological mediator, modulator, and effector [1]. Three distinct isoforms of NO synthase (NOS), which are apparently tissue-specific, are responsible for NO biosynthesis in various tissues [2]. In endothelial cells, NO is catalyzed from the amino acid L-arginine and molecular oxygen by the endothelial NOS (eNOS). Circulating NO is primarily produced

Abbreviations: NO, nitric oxide; NOx, NO metabolite (nitrite plus nitrate); eNOS, endothelial NO synthase; eNOS, eNOS gene; iNOS, inducible NOS; eNOS4b/a polymorphism, 27-bp repeat polymorphism located in intron 4 of eNOS; G894T polymorphism, G894T missense mutation located in exon 7 (Glu298Asp) of eNOS. * Corresponding author. Tel.: +82-2-380-1521; fax: +82-2-388-0924. E-mail address: [email protected] (I. Jo).

by eNOS and it contributes to basal vascular tone and regulates blood flow and blood pressure [3 –5]. Reduction in basal NO release is known to predispose humans to hypertension, thrombosis, vasospasm, and atherosclerosis [3– 7]. In contrast, high circulating NO levels, which occur with excessive inducible NOS (iNOS) expression under pathological conditions, are generally toxic [1]. Markedly elevated NO levels are reported to be associated with endotoxic shock and exaggerated inflammatory reactions [8], and may lead to acute hepatic dysfunction [9] and predispose humans to asthma [10] and cardiomyopathy [11]. Since plasma NO released from cells rapidly autooxidizes to yield the stable metabolites of NO (NOx: nitrite plus nitrate), the level of NOx in blood may be an indicator of endogenous NO production [12]. One of the factors influencing an individual’s continuous basal plasma NOx production is reported to be eNOS gene (eNOS) polymorphism although many other environmental factors including cigarette smoking are also highly specu-

0049-3848/02/$ - see front matter D 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 9 - 3 8 4 8 ( 0 2 ) 0 0 2 0 8 - 6

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lated [13,14]. In general, three classes of genetic variations in eNOS have been identified; those in intron regions, those in 5V-flanking DNA (promoter) and those within the open reading frame [15,16]. Considerable data demonstrate that these variations have been evaluated for possible links to cardiovascular disease in human [15,16]. Among them, the variable number of tandem repeat polymorphism located in intron 4 of eNOS (eNOS4b/a polymorphism) was reported to be significantly associated with plasma NOx levels [13,14]. In this regard, another G894T polymorphism located in exon 7 was also speculated to be associated with plasma NOx levels. This variant resulted in the amino acid change, which substitutes aspartic acid for glutamic acid at amino acid residue 298 (Glu298Asp), suggesting an alteration in its enzymatic activity and subsequently in plasma NOx production. In fact, only one epidemiologic study, with a relatively small sample size, has shown that the plasma NOx levels in the T allele carriers were significantly higher compared with those in the G allele carriers [17], but further studies, using a larger population size, need to be conducted for reaching clear conclusion. In this study, we used G894T polymorphic marker to examine if genotypic variation at the eNOS locus contributes to quantitative phenotypic variation in circulating plasma NOx levels in Korean population, and further this variation, if any, is smoking status-dependent. Our results clearly indicate that the G894T polymorphism is not linked to the physiological variation of plasma NOx levels.

1. Methods 1.1. Subjects and blood sampling We selected 480 consecutive healthy subjects among persons aged 18 –84 years who participated in a comprehensive health screening in Ansan Health Center. Subjects were then tested for genotyping of G894T polymorphism and their plasma NOx levels were measured. We excluded subjects with either diabetes mellitus, myocardial infarction, stroke, or hypertension. We also excluded persons who gave insufficient data. Therefore, complete data were available on 411 subjects (153 men and 258 women). Blood pressure was measured, hypertension defined, body mass index calculated and smoking classified as described previously [18]. All subjects refrained from cigarette smoking and food for at least 12 h and drinking for at least 8 h before the study. Informed written consent for participation was obtained from each individual, and the study protocol was approved by an Ansan Health Center review committee. The demographic and clinical features of the subjects are shown in Table 1. A 10-ml peripheral venous blood sample was drawn into the tubes containing EDTA and centrifuged within 4 h. The plasma and cellular components were stored separately at 70 jC in aliquots until analysis.

Table 1 Demographic and clinical characteristics of the subjects (N = 411) Characteristic Age (years) Male/female Non-/ex-/currentsmoker Plasma NOx levels (Amol/l) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Total cholesterol (mg/dl) Triglycerides (mg/dl) High density lipoprotein (mg/ml) Low density lipoprotein (mg/ml) Body mass index (kg/m2)

Values

Range a

44 F 13 153/258 279:28:104 53.11 F 22.80 (3.88 F 0.42)b 113 F 11 71 F 9

19 – 81

12.9 – 145.8 (2.6 – 5.0) 70 – 139 40 – 88

170.44 F 36.33

78.4 – 353.4

116.52 F 70.45

25.0 – 459.0

48.1 F 14.5

25.0 – 169.1

99.0 F 35.9

6.5 – 285.8

23 F 3

13 – 44

a

Values are means F S.D. Values in parenthesis indicate the natural log-transformed plasma NOx levels. b

1.2. Genotyping of G894T polymorphism Genomic DNA was extracted from the frozen cellular components by the phenol/chloroform extraction method. The extracted genomic DNA was stored at 4 jC until analysis. The G894T polymorphism was screened using polymerase chain reaction-restriction fragment length polymorphism analysis as described [19] with minor modifications. Primer pairs to amplify a part of the eNOS containing exon 7 by polymerase chain reaction were as follows: sense 5V-TCC CTG AGG AGG GCA TGA G-3Vand antisense 5VTGA GGG TCA CAC AGG TTC CT-3V. The resulting 457bp amplification product was digested with the restriction enzyme BanII (New England Biolabs) according to the manufacturer’s instructions. The wild type (GG) of amplification product was cleaved into two smaller fragments (320-, and 137-bp) by BanII but the homozygous variant (TT) was unaffected. The heterozygous variant (GT) yielded three fragments (457-, 320-, and 137-bp) by BanII digestion. The restricted fragments were separated on 2% agarose gels with ethidium bromide staining. Homozygous TT variant undigested by BanII was reconfirmed by digestion with another restriction enzyme MboI (New England Biolabs). 1.3. Determination of plasma NOx levels Since NO is unstable and quickly autooxidized to nitrite and nitrate, the plasma NOx levels were measured as total nitrite concentration as described in many previous studies [13,14,17] with minor modifications. Briefly, a 50 Al of

J. Moon et al. / Thrombosis Research 107 (2002) 129–134

plasma sample was reacted with 150 Al of enzyme reagents containing NADPH (50 Amol/l, in final concentration), FAD (5 Amol/l) and nitrate reductase (200 U/l) from Aspergillus niger (Sigma) at 37 jC for 30 min. After reaction, the sample was incubated with lactate dehydrogenase (10 mg/l) from rabbit muscle (Sigma) and sodium pyruvate (10 mmol/ l) at 37 jC for 10 min to oxidize NADPH. At this time, sample was also subjected to deproteinization by the addition of zinc sulfate (15 g/l), and the resulting sample was centrifuged at 1000  g at 25 jC for 15 min. One hundred microliters of the supernatant was applied to a microtiter plate well and followed by the addition of 100 Al of Griess reagent (1 g/l sulfanilamide, 0.1 g/l N-[1-naphthyl]ethylenediamine and 25 g/l phosphoric acid). After color development at 25 jC for 10 min, the absorbance was measured on a microplate reader at a wavelength of 540 nm. Each sample was assayed in duplicate wells. The absorbance at 620 nm was used as background values and the calibration curve was plotted using known amounts of potassium nitrate in distilled water. The linearity was observed in the range from 0 to 100 Amol/l. 1.4. Statistical analysis Deviation from the Hardy – Weinberg equilibrium was assessed using v2 test with 1 degree of freedom. Values are expressed as mean F standard deviation (S.D.). The plasma NOx levels were normalized for analyses by natural logarithmic transformation because of their skewed statistical distribution. The coefficient of variation was 10.8% in this study. Differences between two groups were compared by either Student’s t-test for continuous variables. For comparisons among three groups, ANOVA was accomplished. The analyses were based on an additive (abnormal T allele vs. normal G allele), a dominant (abnormal homozygote and heterozygote combined vs. normal homozygote, TT + GT vs. GG) and a recessive (abnormal homozygote vs. heterozygote and normal homozygote combined, TT vs. GT + GG) model of inheritance. The subject numbers or frequencies for three models were calculated from those of GG, GT, and TT genotypes as described [20]. The association between plasma NOx levels and other contributing factors was evaluated by the multiple regression analysis. Statistical analyses were performed using SAS 8.1 and P < 0.05 was considered to be significant.

2. Results The demographic and clinical features of the total study population (411 subjects) aged 44.11 F 13.23 (mean F S.D.) years (ranged from 19 to 81) are given in Table 1. The women were recruited more than the men (153 men and 258 women). Approximately two thirds (67.89%, 279 subjects) of studied population were nonsmokers, a quarter (25.30%, 104 subjects) current smokers and the remaining 28 subjects

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(6.81%) ex-smokers. No significant differences in demographic and clinical parameters were detected across the genotypes of G894T polymorphism (data not shown). The basal plasma NOx levels (in Amol/l) in all subjects were 53.11 F 22.80, which were not different with respect to either age ( P = 0.1140) or sex ( P = 0.0797). Genotype distributions of GG, GT, and TT in this study were 0.796 (N = 327), 0.195 (N = 80), and 0.009 (N = 4), respectively, and satisfied the Hardy –Weinberg equilibrium law (Table 2). The plasma NOx levels in the subjects with TT appeared to be higher relative to those in either GT or GG, but their differences were not statistically significant (58.93 F 25.57, 51.84 F 19.65, and 53.35 F 23.52, respectively, P = 0.8248). The frequencies of the G allele and the T allele were 0.893 and 0.107, respectively. The mean plasma NOx levels were essentially same between subjects with G allele and those with T allele. Neither dominant ( P = 0.9882) nor recessive (data not shown) effect of T allele was observed. Since it was suggested that smoking status may be an important factor to determine plasma NOx levels with respect to eNOS4b/a genotypes [15], we also examined if there was smoking status-dependent relationship between G894T polymorphism and plasma NOx levels. In this study, 28 exsmokers were included among smokers as previous study [21]. First of all, no significant association was observed in comparison of allele frequencies of G894T polymorphism between nonsmokers and smokers (additive model, P = 0.8586; dominant model, P = 0.7889). No analysis for the recessive effect of T allele was accomplished in this study since there were only few subjects carrying TT genotype among both nonsmokers and smokers. Next, when the data of either nonsmokers or smokers were analyzed separately, there were still no significant differences in plasma NOx levels across alleles of G894T polymorphism (Table 3). On the

Table 2 Plasma NOx levels with respect to genotypes and alleles of the G894T polymorphism in total population (N = 411) Genotypes and alleles

N (%)

Plasma NOx level (Amol/l)

GG

327 (79.6)

GT

80 (19.5)

TT

4 (0.9)

53.35 F 23.52a ( 3.89 F 0.43)b 51.84 F 19.65 ( 3.88 F 0.38) 58.93 F 25.57 ( 4.01 F 0.41) 53.18 F 23.11 ( 3.88 F 0.43) 52.49 F 20.00 ( 3.89 F 0.38) 53.35 F 23.52 ( 3.89 F 0.43) 52.18 F 19.83 ( 3.88 F 0.38)

G allele

734 (89.3)

T allele

88 (10.7)

Without T allele (GG) With T allele (GT and TT) a

327 (79.6) 84 (20.4)

P

0.8248

0.9024

0.9882

Values are means F S.D. Values in parenthesis indicate the natural log-transformed plasma NOx levels. b

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Table 3 Plasma NOx levels with respect to genotypes and alleles of the G894T polymorphism in nonsmokers and in smokers Genotypes and alleles

Plasma NOx level (Amol/l) N

Nonsmokers

N

Smokers

Total

279

132c

GG

223

GT

53

TT

3

50.89 F 21.49a ( 3.84 F 0.42)b 50.87 F 22.01 ( 3.84 F 0.43) 50.44 F 18.92 ( 3.85 F 0.38) 60.18 F 31.16 ( 4.01 F 0.50) 0.7716 50.83 F 21.66 ( 3.84 F 0.42) 51.43 F 19.92 ( 3.87 F 0.39) 0.6475 50.87 F 22.01 ( 3.84 F 0.43) 50.96 F 19.46 ( 3.86 F 0.39) 0.7525

57.79 F 24.78 ( 3.97 F 0.41) 58.65 F 25.79 ( 3.98 F 0.42) 54.60 F 21.10 ( 3.93 F 0.38) 55.18 (4.01 F 0.50) 0.8459 58.18 F 25.23 ( 3.98 F 0.42) 54.64 F 20.33 ( 3.94 F 0.37) 0.6238 58.65 F 25.79 ( 3.98 F 0.42) 54.62 F 20.70 ( 3.93 F 0.37) 0.5837

P G allele

499

T allele

59

P Without T allele (GG) With T allele (GT and TT) P

223 56

104 27 1

235 29

104 28

P

0.0040 0.0052 0.3786 NAd

0.0001 0.4271

0.0052 0.4037

a

Values are means F S.D. Values in parenthesis indicate the natural log-transformed plasma NOx levels. c A total of 28 ex-smokers were included among smokers. d Not analyzed. b

other hand, significant differences in plasma NOx levels between nonsmokers and smokers were found ( P = 0.0040). This significance with respect to smoking status was valid only in subjects carrying the common G allele (additive effect, P = 0.0001; dominant effect, P = 0.0052). The relative capacities of the risk factors and T variant to predict plasma NOx levels were analyzed using multiple regression analysis. As a result, the T allele of G894T polymorphism were not found to be an independent contributing factor for the plasma NOx levels, while smoking ( P = 0.0019) and triglycerides ( P = 0.0384) were independent ones among all variables tested.

3. Discussion There is only limited information if G894T missense mutation (Glu298Asp) gives rise to functional alteration of eNOS enzyme activity. Recently, Tesauro et al. [22] have proven that this mutation can affect the susceptibility of eNOS to cleavage. However, it is not clear if this increased susceptibility is an in vivo event or an in vitro phenomenon. Nonetheless, the functional potential of this G894T variant was supported by an observation of a trend for a reduced eNOS activity in the T allele carriers [23]. Furthermore, Philip et al. [24] also showed that the T allele carriers were more responsive to administration of phenylephrine for vasoconstriction, resulting in an increased blood pressure.

In the same study, they postulated that such increased response could be due to the fact that T allele produces less NO despite no direct evidence. Taken together, we expected initially that G894T variant may be associated with the plasma NOx levels. However, in this study, we failed to show a significant association between them, providing no functional relevance of G894T variant in determining plasma NOx levels. Our finding is inconsistent with a recent one showing that healthy T allele carriers have increased plasma NOx levels [17]. Although these incompatible findings are not clarified at the present time, the sampling variability, mainly due to the limited sample size, may be implicated. We analyzed G894T polymorphism in a group of 411 healthy Korean subjects in this study, whereas Yoon et al. [17] analyzed a lesser sample size (128 subjects). Other important differences in environmental exposures as well as in genotype frequencies of G894T polymorphism between two studies may also be a plausible explanation. In this regard, smoking status was previously hypothesized to be related with eNOS4a allele-dependent plasma NOx levels [15]. In this study, we found that smoking significantly increased the plasma NOx levels, but we could not clarify the nature of increased plasma NOx levels. Smoking itself was reported to contain NO, which may contribute to increased plasma NOx levels. In this study, however, it is unlikely that NO inhaled from smoke would affect plasma NOx levels as described previously [25,26]. Alternatively, smoking and/or its constituent(s) were also reported to increase NO by induction of either eNOS [27 – 29] or iNOS expression. The sequential pathway activating iNOS, however, occurs transiently as far as it is in a morbid situation; otherwise, eNOS activation influences plasma NOx levels in basal state [14]. At the present time, we do not know if smoking confers such a morbid situation and, even so, we still do not know how the increased plasma NOx levels are maintained continuously without degradation. As described in Methods, we collected blood samples from subjects who had refrained from cigarette smoking and food for at least 12 h and drinking for at least 8 h before the study; therefore, smoking is likely to be excluded as a possible source of the increased plasma NOx levels observed in this study, as described in detail [12]. Further studies, however, need to be conducted for reaching clear conclusion, but these are beyond the scope of current study. Furthermore, our sampling protocol, along with the fact that diet was not controlled in this study, indicates that diet alone would probably not contribute to group differences in plasma NOx levels. One of the most interesting findings in the present study is that the differences in plasma NOx levels with respect to smoking status were only statistically significant in subjects carrying the common genotype or allele of G894T polymorphism, but not in those with the rare ones (Table 3). Based on current finding, we hypothesize that the wild-type (common) G allele carriers can be highly susceptible to smoking-induced phenotypic alterations such as an increase

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in plasma NOx levels compared with mutant-type (rare) allele carriers. Our data may be inconsistent with the previous data showing that eNOS4a mutant allele, but not common allele, of eNOS4b/a polymorphism was significantly associated with smoking-dependent risk of coronary artery disease [21]. With respect to the genotype or allele of G894T polymorphism, however, to date there are no published data available on smoking status-dependent alterations in cardiovascular events. Although the above conflicting findings are not clarified at the present time, it is suggested that the mechanism of smoking action on the increase in plasma NOx levels may be different from that on the increase in risk of coronary artery disease, especially with respect to the contribution of different eNOS polymorphisms. Our study has one important limitation, which is a finding that T allele of G894T polymorphism has an estimated frequency of only 0.107 and the lack of association between G894T polymorphism and plasma NOx levels in total population is based on only four individuals who were homozygous for this polymorphism. Furthermore, the finding of no significant differences in plasma NOx levels in subjects without G allele between nonsmokers and smokers is only valid based on few subjects who were homozygous for the rare T allele in studied population (Table 3). Therefore, a larger sample should be examined to confirm the relation between this polymorphism and plasma NOx levels. In conclusion, in a Korean population, we were unable to find any significant association of G894T polymorphism with basal plasma NOx levels, which is incompatible with the previous study [17]. From this finding, it is highly implicated that factors, such as diet and smoking, other than genetic effect of eNOS may be more importantly responsible for controlling plasma NOx levels in at least Korean population. Indeed, smoking and triglycerides were found to be major independent factors contributing for plasma NOx levels in this study. Nonetheless, comprehensive genetic approaches with other or as-yet unidentified polymorphic sites of eNOS, together with larger sample sizes, should be performed before making conclusive claims about genetic contribution of eNOS polymorphism to basal plasma NOx levels. Acknowledgements This work was supported in part by the Korean National Institute of Health intramural research grant (334-6113-211207-00) and Science Research Center grant from the Korean Science and Engineering Foundation (KOSEF) to the Nitric Oxide Radical Toxicology Research Center (NORTReC) to Dr. Inho Jo, by Korea University Institutes of Medical Science grant (2000-n6) to Dr. Chol Shin, and by the Biomedical Brain Research Center grant from the Ministry of Health and Welfare to Dr. Sangmee Ahn Jo. We thank Dr. Younjhin Ahn and Mr. Jungbok Lee for their valuable discussions regarding statistical analysis, and Ms. Jooyoung Lee for secretarial assistance.

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