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Technical Briefs |
1 Division of Medical Biochemistry, Stavanger University Hospital, Stavanger, Norway;2 Agder University College, Department of Natural Sciences, Kristiansand, Norway;3 Blood Bank and 7 Center for Clinical Research, Haukeland University Hospital, Bergen, Norway;4 Cecor AS, Haugesund, Norway; 5 Elf Petroleum Stavanger AS, Stavanger, Norway;6 Norsk Hydro Aluminum, Karmøy, Norway;
aaddress correspondence to this author at: Agder University College, Department of Natural Sciences, N-4604 Kristiansand, Norway; e-mail mohammad.a.mansoor{at}hia.no
Endothelial-cell–derived nitric oxide (NO) relaxes vascular smooth muscle cells, causes vasodilation, and inhibits platelet aggregation. It has been demonstrated that NO3 is the predominant and stable form of NO present in plasma and represents the bioavailability of NO (1)(2). NO generation is depressed in blood vessels affected by atherosclerosis, and a loss of NO activity is associated with impaired vasoreactivity, enhanced platelet aggregation, and increased endothelial cell–leukocyte interactions (3).
Recently, evidence has accumulated that suggests that increased concentrations of the amino acid homocysteine (Hcy) also increase the risk for cardiovascular disease (CVD) (4). Hcy concentrations can be lowered by supplementation with folate, vitamin B6, vitamin B2, and vitamin B12 (5)(6). In particular, folate may also contribute in the prevention of CVD because folate seems to restore impaired NO metabolism (7).
In the present investigation, we included apparently healthy persons. Individuals on all types of medications were excluded from this cross-sectional study. Blood samples were collected from participants who had been fasting for 12 h. The blood samples were centrifuged at 4 °C within 1 h and stored at –80 °C until analysis. Informed written consent was obtained from all participants. The study was approved by the Regional Ethics committee, University of Bergen, Norway.
Total homocysteine (tHcy) concentrations in plasma were measured by an HPLC method (8). Plasma NO3 concentrations were measured by capillary electrophoresis (9), and total serum cholesterol, HDL-cholesterol, and serum triglyceride concentrations were measured by a routine enzymatic colorimetric method (Roche/Hitachi). The concentrations of erythrocyte folate, serum folate, and vitamin B12 were measured on an Access Immuno Assay System (Sanofi Pasteur Diagnostics).
The program "Power and Precision" was used for the power calculations. Power computations were performed for the comparison of smokers vs nonsmokers with serum folate as the primary endpoint, based on a 2-sample t-test for ln-transformed data. All P values given are 2-tailed. The statistical program StatView for the Macintosh (Abacus Concepts) was used for all calculations.
The characteristics of the study participants are given in Table 1
. In a simple regression analysis, we found a significant positive relationship between the concentrations of serum folate and plasma NO3 (R = 0.17; P = 0.017) and a significant inverse relationship between the concentrations of serum folate and tHcy (R = 0.244; P = 0.0005). In a multiple regression analysis that included age, sex, body mass index, NO3, tHcy, and vitamin B12 as independent variables and serum folate as dependent variable, we found a significant positive relationship between the concentrations of serum folate and plasma NO3 (P = 0.0037), and a significant negative relationship between the concentrations of serum folate and tHcy (P = 0.0005; Table 1B of the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol51/issue7/). The present findings may indicate that folate is involved in the intracellular formation and release of NO into plasma. There are at least 2 mechanisms by which NO could be converted to NO3. The first possibility is the direct and rapid oxidative conversion of NO to NO3 in the presence of oxy-hemoproteins in the extracellular fluids. The second possibility is that the oxidation of NO to NO3 takes place through the formation of an NO2 intermediate, also in the presence of oxy-hemoproteins (10).
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5-Methyltetrahydrofolate (5-MTHF) is the principal form (perhaps as much as 
95%) of folate in serum; therefore, most studies have used serum folate as an appropriate marker for folate status in their aim to explore the relationship between serum folate and tHcy concentrations (11). As suggested in Fig. 1
, 5-MTHF is related to the formation of both NO and Hcy in the cell. In the presence of 5-MTHF, the enzyme dihydrobiopterin reductase can reduce quininoid dihydrobiopterin to tetrahydrobiopterin (BH4) (12). 5-MTHF also stabilizes BH4, enhances its binding to NO synthase (NOS), increases the activity of NOS, and thus regulates the ratio of NO to superoxide (O2·–) (13). Therefore, our findings may indicate that the intracellular concentrations of 5-MTHF may regulate the bioavailability of NO.
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A decrease in the folate concentration leads to an accumulation of tHcy. Oxidation of Hcy in the presence of trace elements generates O2·– and hydrogen peroxide (H2O2) (14); it has therefore been suggested that folate deficiency may contribute to an increase in the concentration of O2·–. Furthermore, increased concentrations of Hcy also reduce the availability of BH4 in vitro and thus increase the formation of O2·– (15).
In a recent study it was suggested that folic acid supplementation improves endothelial function in coronary artery disease (CAD) by a mechanism largely independent of Hcy (16). It is reasonable to assume that the improvement in endothelial function was attributable to increased formation of NO during the folic acid intervention. A different study showed that folic acid restored endothelial dysfunction in patients with hypercholesterolemia who had tHcy concentrations within the reference interval (17).
Our observation that smokers have higher plasma concentrations of NO3 than do nonsmokers is in accordance with previously published data (Fig. 1B
of the online Data Supplement) (18), but are contradictory to the findings of Node et al. (19). Oxidants, free radicals, and trace elements in cigarette smoke may activate macrophages and neutrophils in the circulation, which may generate increased amounts of NO and nitrite (20). It is also possible that cigarette smoke may prevent reaction of NO with thiols to form nitrosothiols, important reservoirs for NO in blood (21).
From our findings in the present investigation, we postulate the following possible clinical implications: Increased dietary consumption of fruits and green vegetables may increase the concentrations of folate in serum/cells and thus may increase the ratios NO/Hcy and NO/O2·–. Other possible benefits from increased concentrations of serum folate may be decreases in the concentrations of soluble circulating adhesion molecules. Increased concentrations of circulating adhesion molecules seem to be associated with the process of inflammation.
Acknowledgments
We thank Drs. Øyvind Hetland and Peter H. Evans for reading the manuscript and for useful comments.
References
The following articles in journals at HighWire Press have cited this article:
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  D. J. Veselik, S. Divekar, S. Dakshanamurthy, G. B. Storchan, J. M.A. Turner, K. L. Graham, L. Huang, A. Stoica, and M. B. Martin Activation of Estrogen Receptor-{alpha} by the Anion Nitrite Cancer Res., May 15, 2008; 68(10): 3950 - 3958. [Abstract] [Full Text] [PDF]
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