Possible Impact of Tetrahydrobiopterin and Sepiapterin on Endothelial Dysfunction
To the Editor:
Vasquez-Vivar et al1 reported on diminished tetrahydrobiopterin (BH4) concentrations in vessels from hypercholesterolemic rabbits. This is an interesting finding because both in serum and urine from patients with coronary artery diseases and hypercholesterolemia, total plasma biopterin concentrations were found to be unchanged2 (unpublished data, 2002). However, total biopterin represents the sum of BH4, 7,8-dihydrobipterin (BH2), and fully oxidized biopterin, and one cannot exclude possible the effect of either reduced BH4 or increased BH2 concentrations on the endothelial dysfunction in these patients. In normal plasma, almost all biopterin (>95%) is present as BH4, and measurement of biologically active tetrahydro-derivative seems to be essential. Differential oxidation with iodine and subsequent high-pressure liquid chromatography (HPLC), according to Fukushima and Nixon,3 is a simple method to measure different oxidation forms of biopterin. In this method, under acidic conditions, BH4 and BH2 are oxidized to biopterin, while under basic conditions, only BH2 is oxidized to biopterin, and BH4 undergoes a side-chain cleavage to form the blue fluorescing pterin. The difference in biopterin content between the two oxidations represents the actual BH4 levels. HPLC separation of biopterin from pterin and isoxanthopterin is essential for the correct interpretation.
Another important finding of Vasquez-Vivar et al1 is the observation that supplementation with sepiapterin, an intermediate in the salvage pathway of BH4, worsens responses to endothelium-dependent agonists Ach and A23187 and that sepiapterin in high concentrations uncouples purified endothelial NO synthase (eNOS) and leads to generation of superoxide O2−. Recently, it has been shown that BH2 and sepiapterin inhibit NOS in vitro by displacing the prebound BH4 with >80% efficiency.4 BH2 and sepiapterin are metabolites that accumulate in patients with variants of BH4 deficiency, and it has been suggested that they may potentate the superoxide formation during the uncoupled reaction of NOS.5,6⇓ NO production was found to be significantly reduced in the brain in these patients.7 Particularly, patients with sepiapterin reductase deficiency, in which both BH2 and sepiapterin accumulate in the brain, have extremely low levels of nitrate and nitrite (end products of NO) in cerebrospinal fluid (Figure) and low-dose treatment with BH4 (2.5 to 5.0 mg/kg) was not efficient to restore the brain’s NO production. Thus, both the oxidation of BH4 to BH2 and sepiapterin formation promote uncoupling of the NOS reaction and stimulate superoxide and peroxynitrite production. Although patients with BH4 deficiency show no cardiovascular problems, one can speculate that, in patients with hypercholesterolemia and other diseases presenting with endothelial dysfunction, a similar situation occurs. Sepiapterin is first reduced in endothelial vessels to BH2 by sepiapterin reductase, but the capacity of dihydrofolate reductase to reduce BH2 to BH4 may be to low, resulting in formation of toxic metabolites. Incubating the vessels with methotrexate, an inhibitor of dihydrofolate reductase, may bring additional information on how sepiapterin and BH2 promote the superoxide formation.
A number of studies documented that pro-inflammatory cytokines increase BH4 formation in cultured vascular endothelial cells.8 Another factor which may influence vascular BH4 levels is vitamin C. It has been shown recently that long-term treatment with vitamin C decreases BH2 levels and increase BH4 levels in aortas of apolipoprotein-deficient mice.9 Simultaneous application of vitamin C + E, as suggested in patients with sepiapterin reductase deficiency in order to improve the efficiency of the BH4 therapy, needs further investigation.
- ↵Vasquez-Vivar J, Duquaine D, Whitsett J, Kalyanaraman B, Rajagopalan S. Altered tetrahydrobiopterin metabolism in atherosclerosis: implications for use of oxidized tetrahydrobiopterin analogues and thiol antioxidants. Arterioscler Thromb Vasc Biol. 2002; 22: 1655–1661.
- ↵Jones CL, Vasquez-Vivar J, Kalyanaraman B, Griscavage-Ennis M, Gross SS. Tetrahydropterins but not dihydropterins attenuate the reduction of superoxide from eNOS: a novel role for tetrahydrobiopterin. Pteridines. 2001; 12: 52–53.
- ↵Rosenkranz-Weiss P, Sessa WC, Milstien S, Kaufman S, Watson CA, Pober JS. Regulation of nitric oxide synthesis by proinflammatory cytokines in human umbilical vein endothelial cells: elevations in tetrahydrobiopterin levels enhance endothelial nitric oxide synthase specific activity. 1994; 93: 1875–1876.
- ↵D’Uscio LV, Milstien S, Richardson D, Smith L, Katusic ZS. Long-term vitamin C treatment increases vascular tetrahydrobiopterin levels and nitric oxide synthase activity. Circ Res. 2003; 92: 88–95.
Inadequate tetrahydrobiopterin (BH4) concentration has been linked to impaired endothelial NO synthase (eNOS) activity and to loss of endothelial function in vascular conditions such as hypercholesterolemia. Blau and Thöny correctly contend that despite the critical importance of BH4 in NO formation from eNOS, patients with BH4 deficiency typically show no evidence of vascular disease. Studies that have evaluated the effects of BH4 supplementation have further observed divergent effects in animal models as well as in humans, with some studies reporting beneficial or no effects and a few reporting actual worsening of vascular function.1 We have recently presented an alternative hypothesis that may reconcile these apparently contradictory findings. This is that the relative concentrations of fully reduced (BH4) and oxidized forms of tetrahydrobiopterin (BH2), rather than the absolute concentration of BH4, are what governs the nature of products generated from eNOS.2 At low BH4 or high BH2 (7,8-dihydrobiopterin or sepiapterin) levels, eNOS generates superoxide that is not inhibited by saturating l-arginine or ascorbate.2 In addition, experimental BH4 deficiency (such as that induced by treatment with 2,4-diamino-6-hydroxy-pyrimidine and N-acetylserotonin, a GTP cyclohydrolase-I and sepiapterin reductase inhibitor, respectively) shows that supplementation with sepiapterin in the presence of N-acetylserotonin causes further increases in superoxide production by endothelial cells. This indicates that the BH4/BH2 ratio controls superoxide formation in the endothelium and may represent a physiologically relevant mechanism to regulate eNOS products.
Based on our study, it appears that BH4 homeostasis in atherosclerotic vascular tissue is complex. While the tissue retains the ability to generate BH4 from sepiapterin, this activity does not translate to improvements in vascular relaxation. Cotreatment with N-acetylcysteine augments BH4 concentrations beyond normal levels and yet did not ameliorate vascular function. This result indicates that BH4 metabolism is altered in experimental atherosclerosis, and that mere supplementation with sepiapterin, an oxidized precursor of BH4, alone or with antioxidants does not translate to therapeutic benefits. The mechanisms involved in the regulation of BH4 levels in the vascular wall are not known. Likewise, it is unclear how ascorbate may influence the BH4/BH2 ratio in the endothelium. It has been shown that chemical reduction of 7,8-BH2 to generate BH4 is not a mechanism by which ascorbate alters BH4/BH2 ratio.3 Also, ascorbate does not improve de novo BH4 synthesis, nor is it a first-line defense against superoxide and peroxynitrite.4 Thus a thorough understanding of the mechanisms involved in regulating the balance between the different redox forms of BH4, and the role of anti-oxidants in maintaining this balance, appears to be critical in designing rational therapeutic strategies to ameliorate endothelial dysfunction. This objective, however, will not be accomplished with the available analytical methods for BH4 analysis. Indirect methodology such as that based on BH4 oxidation to secondary products is complicated by fact that BH4 is oxidized to generate several products. Clearly, this methodology prevents full identification and quantification of the analogs involved in the process. The development of more direct methods for quantifying BH4 and its metabolites in cells and tissues is warranted.
- ↵Huraux C, Makita T, Kurz S, Yamaguchi K, Szlam F, Tarpey MM, Wilcox JN, Harrison DG, Levy JH. Superoxide production, risk factors, and endothelium-dependent relaxations in human internal mammary arteries. Circulation. 1999; 99: 53–59.
- ↵Vásquez-Vivar JM, Martásek P, Kalyanaraman B. BH4/BH2 ratio but not ascorbate controls superoxide and nitric oxide generation by eNOS. Circulation. 2000; 102 (Suppl II): 63.Abstract
- ↵Vásquez-Vivar J, Santos AM, Augusto O. Peroxynitrite-mediated formation of free radicals in human plasma: EPR-detection of ascorbyl, albumin-thiyl and uric-acid-derived free radicals. Biochem J. 1995; 314: 869–876.