eNOS and BH4; endothelial function or dysfunction. Importance of tetrahydrobiopterin (BH4)

Received: 14-Oct-2018 Accepted Date: Nov 03, 2018; Published: 12-Nov-2018

Citation: Gantzer J. eNOS and BH4; endothelial function or dysfunction. Importance of tetrahydrobiopterin (BH4). J Neurol Clin Neurosci. 2018;2(4): 01-04.

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Abstract

Tetrahydrobiopterin (BH4) is a multifunctional cofactor required for vital enzyme activity in the synthesis reactions of the neurotransmitters Dopamine and Serotonin as well as in the synthesis of the gaseous signaling agent Nitric Oxide (NO) involved in vascular health. BH4 must be maintained at continuously high levels including intracellular synthesis and recycling to prevent endothelial dysfunction with ongoing generation of free radicals and concomitant oxidative damage which leads to deleterious effects on the vascular wall including loss of vasodilation and protection against atherosclerotic pathogenesis.

Imbalances entail a drop in BH4 levels and occur in states of high blood sugar, high blood pressure, high blood lipids; their subsequent vascular disease states including diabetes, atherosclerosis, hypertension, hyperlipidemia, and hyperhomocysteinemia induce endothelial dysfunction with negative impacts on the NOS/BH4 enzyme system with resultant increased free radical formation, decreased NO production, and concomitant reduced NO vasodilatory and signaling bioactivity. In addition to vascular effects, impaired NO production also has a role in neurodegenerative diseases including impaired cerebral blood flow and decreased BDNF secretion (important for cognition, learning, and memory).

Endothelial dysfunction is oxidative stress driven by low levels of BH4 at the Nitric Oxide Synthase enzyme (NOS), called NOS-uncoupling; where NOS-Uncoupling is a perpetuating cycle of Superoxide (OO-) and Peroxynitrite (ONOO-) free radical formation. Oxidative stress in endothelial cells depletes BH4, switches NOS generation from NO to OO-, promotes formation of ONOO- from NO and OO-; dropping the intracellular ratio of BH4:BH2 inducing a feed forward cycle of more and more BH4 depletion and NOS-Uncoupling. Low levels of ONOO- exposure cause BH4 levels to drop by 60% in 500 seconds.

Vascular and cognitive health entails maintaining balanced redox ratios of BH4:BH2, sufficient arginine and citrulline levels for enzyme efficiency, as well as folate and antioxidants for cofactor rescue and anticipated free radical formation. Dietary essential vitamins and antioxidants acts as free radical scavengers and have successfully been shown to restore BH4/NO levels, endothelial NOS function, and protect against vascular and cognitive decline.

Keywords

BH4 tetrahydrobiopterin; OO- superoxide anion; ONOO- peroxynitrite; Endothelial dysfunction; Oxidative stress; Free radicals; Antioxidants.

Abbreviations

NOS: Nitric Oxide Synthase; BDNF: Brain derived Neurotrophic Factor.

Introduction

In the discussion of BH4 there are a few assumptions that need to be made. 1st assumption is the understanding of oxidative stress. Oxidative stress is a system which generates free radicals. The 2nd assumption is the understanding of what a free radical is. A free radical is a molecule which has lost its electron and wants it back so therefore steals an electron from any susceptible molecule near it. The 3rd assumption is required to talk about any system with oxidative stress and the generation of free radicals, and that is the basic understanding of redox reactions. A redox reaction occurs when a molecule gains or loses electrons. When a molecule has all of its electrons it’s said to be reduced, and when it loses its electrons it’s said to be oxidized [1-5].

The body does not want a system of free radicals except for short bursts for physiologic functions [6,7], then under homeostatic conditions the body utilizes built-in protective mechanisms of antioxidants to neutralize the free radicals. Under conditions of chronic oxidative stress with persistent generation of free radicals the intrinsic antioxidant enzyme systems get overwhelmed, thus creating a perpetual cycle of more and more free radical formation, insufficient antioxidant levels to neutralize and clear them, and more damage to local tissues from the chronic exposure of oxidative stress. Oxidative stress in blood vessels is called endothelial dysfunction. Endothelial dysfunction is oxidative stress [8-10].

So then, what is endothelial function? Endothelial function is focal vasodilation during homeostasis [8,11].

NOS-enzyme and its cofactor BH4 during homeostasis:

BH4 is a cofactor for an enzyme called Nitric Oxide Synthase, NOS. Nitric Oxide Synthase generates the gaseous signaling molecule Nitric Oxide, NO. Nitric Oxide in blood vessel maintains and controls local vasodilation alongside deterring pathogenesis of atherosclerosis, and is an intracellular signaler [6,8].

BH4 is also a redox molecule. Redox reactions occur when molecules gain or loss electrons. In the case of BH4 it gains/losses (accepts/donates) electrons as Hydrogen atoms. In redox reaction nomenclature, a molecule with all its electrons is fully reduced, and BH4 is so called TetraHydroBiopterin, a fully reduced biopterin molecule; it possesses 4 electrons seen as having 4 Hydrogen atoms. This is the physiological and functional state of BH4 [1- 3,12].

Only the fully reduced BH4 molecule can be a cofactor for NOS-enzyme to form the end-product NO for vasodilation and blood vessel homeostasis [8].

NOS-enzyme simplified and NOS-Coupling:

NOS simplified have 4 constituents: Arginine, BH4, Molecular Oxygen O2, NADPH. Arginine is the substrate. BH4 is the cofactor.

Arginine is the precursor molecule to the formation of NO, meaning Arginine will enzymatically become Nitric Oxide. The Nitrogen adjacent to the amino end group of Arginine is cleaved off and replaced with an Oxygen as Arginine becomes the amino acid Citrulline and gaseous Nitric Oxide is formed.

This is called NOS-Coupling. Arginine and Molecular Oxygen enzymatically react to make Nitric Oxide.

BH4 stabilizes the NOS-enzyme in its fully reduced form and BH4 is NOT consumed.

Arginine is coupled to Molecular Oxygen, BH4 stabilizes the enzymatic reaction; then Citrulline, NO, and BH4 remain. This is an important feature of homeostasis and defines NOS-Coupling [8,13,14].

Once again; only BH4 in its fully reduced state can act in its physiological functional role as a cofactor for NOS-enzyme production of the vasodilator NO; this occurs during homeostasis in an oxidative stress-free environment.

BH4 under conditions of oxidative stress is highly susceptible to being oxidized by free radicals. Recall free radicals have lost their electrons. Molecules with Hydrogen atoms (as electrons) are easy targets and susceptible to oxidation (free radicals steal them). This renders BH4 a free radical scavenger instead of a cofactor, because to scavenge and neutralize free radicals it must give up its electrons which are the equivalent of losing its Hydrogen atoms to the free radical. This is oxidation of BH4. The resultant molecule is either BH2 as DiHydroBiopterin (partially reduced) with 2 Hydrogen atoms or Biopterin (fully oxidized) with no Hydrogen atoms. BH2 has the same binding affinity for the NOS-enzyme but cannot act as a cofactor to stabilize the coupling of Molecular Oxygen to Arginine [1,12,15-17].

In a system of homeostasis there are virtually undetectable levels of BH2 and all measurable biopterin exists as the fully reduced BH4. In a system of oxidative stress the ratio of BH4 is significantly decreased and BH2 is not only measurable but becomes the prominent biopterin [11,12].

Therefore, it becomes important to look at the BH4:BH2 ratio. Lower BH4:BH2 ratios are correlated with more enzyme derangement and more oxidative stress [9].

NOS-enzyme derangement and nos-uncoupling

The detrimental effects of NOS-enzyme derangement are vast and functionally insufficient. Recall the simplified version of NOS has 4 constituents: Arginine BH4 O₂ NADPH. Also recall its mentioned under homeostatic conditions. This is important because the NOS-enzyme structure has a reductase end to bind NADPH and an oxidase end to bind Molecular Oxygen. These 2 events occur with or without Arginine and/or BH4 and when the reaction occurs without them it’s called NOS-Uncoupling where free radicals are formed and BH4 is oxidized [8,9,12,15].

NOS-UnCoupling generates free radicals as its end-products in the form of Reactive Species: Reactive Oxygen Species (ROS) Superoxide Anion and Reactive Nitrogen Species (RNS) Peroxynitrite.

Superoxide Anion OO- is an uncoupled Molecular Oxygen free radical. In a system where BH4 and OO- exist together, the OO- steals (oxidizes) the Hydrogen atoms (electrons) from BH4 readily oxidizing it to BH2. This creates a perpetuating cycle because BH2 cannot stabilize the reaction and more OO- gets created, oxidizing more BH4, further dropping the BH4:BH2 ratio [8,18].

Occurring simultaneously is the formation of an even more potent free radical, Peroxynitrite [24].

Peroxynitrite ONOO- is formed when NO and OO- exist in a system together. Even during decreased NOS-enzyme efficiency NO is still generated, but its bioavailability is decreased because instead of its physiological role in vasodilation NO is scavenged by OO- forming the free radical ONOOfurther dropping the BH4:BH2 ratio via oxidation of BH4 [8,18].

The fate of BH4 under oxidative stress and endothelial dysfunction is oxidization by OO- and exceedingly faster oxidation by ONOO-; even under low levels of ONOO- exposure BH4 levels dropped by 60% in 500 seconds [18]. NOS-UnCoupling negatively effects bioavailability of both BH4 and NO from exposure to free radicals whose near proximity scavenges them; BH4 rendered BH2 and NO rendered ONOO- [3,9,18].

NOS-UnCoupling is the aberrant function of the enzyme due to low substrate (Arginine NO precursor) and/or low cofactor (BH4) with consequential production of free radical reactive species in a perpetuating cycle. Endothelial dysfunction results from NOS-UnCoupling; which IS oxidative stress [8].

Taken altogether- Endothelial dysfunction from intracellular/extracellular vascular oxidative stress is correlated with oxidation of BH4, low ratio of BH4:BH2, simultaneous NOS-Uncoupling, persistent generation of OOand ONOO- free radicals, decreased NO bioavailability for vasodilation, and resultant vasoconstriction [8-18].

Endothelial dysfunction and inflammatory conditions

Chronic exposure of endothelium to free radical reactive species is a driver of disease and underlying cause of cardiovascular and neurodegenerative disease states from endothelial dysfunction and its feed forward oxidative stress. The connection is simply mechanism of action [8,20-52].

A system of low substrate and/or low cofactor induces UnCoupling with resultant generation of free radicals which then continues to deplete the cofactor BH4 further inducing more UnCoupling and generating more free radicals; worsening and contributing to the UnCoupling and oxidative stress state.

An oxidative stress state causes UnCoupling by oxidizing BH4 which renders the molecule unable to fulfill its cofactor role to stabilize the enzymatic reaction therefore lowering the BH4:BH2 ratio ultimately looping the BH4-eNOS system into the perpetuating cycle of free radical formation, low levels of BH4, and persistent oxidative stress [8-10,12].

Chronic inflammatory systemic conditions such as hyperglycemia, diabetes, hypertension, hyperhomocysteine, atherosclerosis/oxidized-LDL, viral/toxin load, and autoimmunity are sources of vascular free radicals and each individually induce NOS-UnCoupling [7,8,11,13,20].

Hyperglycemia as well as ONOO- decrease de novo synthesis of BH4 by inducing ubiquitination-induced GTPCH enzyme degradation; lowering levels of cofactor [16,21]. Diabetes patients present with one of the lowest levels of BH4, even oxidation to the level of undetectable BH2, where only biopterin exists, and their intrinsic enzyme defense mechanisms of Glutathione Reductase, Catalase, and Superoxide Dismutase become completely inhibited from high levels of free radicals [9,16,19]. AGEs from hyperglycemia [22], hypertension [11], hyperhomocysteine [20], and oxidized-LDL [23] each individually activate the endothelial NADPHoxidase (NOX4) with significant production of OO- and concomitant ONOO [11,20-23]. NOX4 activation in endothelium is one of the largest contributors to vascular free radicals [24]. Viral/toxin load, autoimmunity, and atherosclerosis via oxidized-LDL activate the inflammation-induced NFkB cytokine pathway generating more free radicals which stimulates LOX/COX which in turn stimulates NOX4 [7,23,25,26]. In addition, viral/ toxin load and autoimmunity activate the inducible iNOS of immune cells which depletes Arginine levels via uptake for macrophage-NO production; lowering levels of substrate [7,27].

Each one of these inflammatory states and their correlated mechanisms of free radical formation individually impact the BH4 levels indirectly as non-eNOS vascular free radicals (oxidation via exposure) and directly by NOS-UnCoupling BH4 oxidation. A person with several or all of these inflammatory states experience a compounding effect and degenerative barrage of NOS-UnCoupling, endothelial dysfunction, free radical formation and chronic oxidative stress [8-27].

Additionally, cognitive decline is largely correlated with NOSUnCoupling. The cognition neurotrophin BDNF has been found to be synthesized from cerebral endothelium and dependent on eNOS function [28,29]. STZ-induced diabetes mellitus mice showed endothelial dysfunction (UnCoupling) as well as decreased expression of the enzyme, decreased BDNF, and vascular dementia [30]. Endothelial dysfunction is correlated with traumatic brain injury as well as secondary brain injury following trauma from NOS-UnCoupling and simultaneous activation of iNOS [31- 33].

Endothelial dysfunction does not occur when there are sufficient levels of substrate/cofactor for enzyme coupling and sufficient levels of antioxidants to neutralize basal rate generation of and exposure to free radicals. This is homeostasis. To prevent and reverse endothelial dysfunction, homeostasis must be restored at these 2 levels [11,34]. Furthermore, chronic inflammatory disease states must be supported with dietary and supplemented antioxidants alongside revised lifestyles which decrease free radicals and restore antioxidant defense enzyme systems [35,36] to include improved sleep [37], exercise [38,39], and stress management [40].

Substrate levels of Arginine can be increased dietary by whey protein powder, nondairy pea protein, purified Arginine powder, purified Citrulline powder, and/or combination of Arginine/Citrulline powder; Arginine, Citrulline, and combo Arginine/Citrulline are also available as capsules [34,41]. Supplemented maternal Arginine in recipient cloned embryology improved levels of Arginine, its metabolites, NO, pregnancy rate, as well as embryo development [42]. Arginine in healthy population is considered semi-essential whereas in children and chronic disease states it is considered essential, hence the body cannot intrinsically produce it, and it must be consumed in diet or a deficiency presents [43]. BH4 supplementation is not available for purchase to the public, however in severe cases can be administered at hospitals as Sepiaterin [44]; however if the body’s oxidative stress state is not addressed (antioxidants/lifestyle) the administered BH4 is rapidly oxidized [13].

For antioxidant support all of the following are beneficial and recommended to be included in dietary choices as well as vitamin supplemented: essential B-vitamins Pyridoxine B6 [45]/Folate B9 [46,47]/ Cobalamin B12 [48], essential fat soluble vitamins Retinoic Vitamin A [49]/ Tocopherol Vitamin E [23/]Cholecalciferol Vitamin D [50], essential water soluble Ascorbate Vitamin C [51], antioxidants Resveratrol [10]/Curcumin [52], and amino acid Cysteine as NAC. All of these are proven agents to combat free radicals, restore intrinsic antioxidant enzyme systems GPx/ SOD/Catalase, replenish fully reduced BH4 levels, protect BH4 from future oxidation, and most important work synergistically to support ReCoupling of the NOS-enzyme [8,10,13,45-52].

REFERENCES

1. Kohen R, Nyska A. Oxidation of biological systems: Oxidative stress phenomena, antioxidants, redox reactions, and methods for their quantification. Toxicol Pathol. 2002;30(6):620-50.
2. Novo E, Parola M. Redox mechanisms in hepatic chronic wound healing and fibrogenesis. Fibrogenesis Tissue Repair. 2008;1(1):5.
3. Pryor WA, Squadrito GL. The chemistry of peroxynitrite: A product from the reaction of nitric oxide with superoxide. Am J Physiol. 1995;268:L699-722.
4. Basu P, Burgmayer SJN. Pterin chemistry and its relationship to the molybdenum cofactor. Coord Chem Rev. 2011;255(9-10):1016-1038.
5. Thöny B, Auerbach G, Blau N. Tetrahydrobiopterin biosynthesis, regeneration and functions. Biochem J. 2000; 1:1-16.
6. Godo S, Shimokawa H. Divergent roles of endothelial nitric oxide synthases system in maintaining cardiovascular homeostasis. Free Radic Biol Med. 2017;109:4-10.
7. Rath M, Müller I, Kropf P, et al. Metabolism via Arginase or nitric oxide synthase: Two competing arginine pathways in macrophages. Front Immunol. 2014;5:532.
8. Gantzer J. Tetrahydrobiopterin - clinical implications and natural solutions. Nutritional Perspectives. 2016;39(2):35-42.
9. Crabtree MJ, Smith CL, Lam G, et al. Ratio of 5,6,7,8-tetrahydrobiopterin to 7,8-dihydrobiopterin in endothelial cells determines glucose-elicited changes in NO vs. superoxide production by eNOS. Am J Physiol Heart Circ Physiol. 2008;294(4):H1530-40.
10. Xia N, Daiber A, Habermeier A, et al. Resveratrol reverses endothelial nitric-oxide synthase uncoupling in apolipoprotein E knockout mice. J Pharmacol Exp Ther. 2010;335(1):149-54.
11. Landmesser U, Dikalov S, Price SR, et al. Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension. J Clin Invest. 2003;111(8):1201.
12. Kim HL, Park YS. Maintenance of cellular tetrahydrobiopterin homeostasis. BMB Rep. 2010;43(9):584-92.
13. Alkaitis MS, Crabtree MJ. Recoupling the cardiac nitric oxide synthases: tetrahydrobiopterin synthesis and recycling. Curr Heart Fail Rep. 2012;9(3):200-10.
14. Böger, R. The emerging role of asymmetric dimethylarginine as a novel cardiovascular risk factor. Cardiovasc Res. 2003;59(4):824-33.
15. Crabtree MJ, Tatham AL, Hale AB, et al. Critical role for tetrahydrobiopterin recycling by dihydrofolate reductase in regulation of endothelial nitric-oxide synthase coupling: relative importance of the de novo biopterin synthesis versus salvage pathways. J Biol Chem. 2009;284(41):28128-36.
16. Alp NJ, Shafi Mussa, Jeffrey Khoo, et al. Tetrahydrobiopterin-dependent preservation of nitric oxide-mediated endothelial function in diabetes by targeted transgenic GTP-cyclohydrolase I over-expression. J Clin Invest. 2003;119:725-735.
17. Alp NJ, McAteer MA, Khoo J, et al. Increased endothelial tetrahydrobiopterin synthesis by targeted transgenic GTP-cyclohydrolase I overexpression reduces endothelial dysfunction and atherosclerosis in ApoE-knockout mice. Arterioscler Thromb Vasc Biol. 2004;24(3):445-50.
18. Laursen JB, Somers M, Kurz S, et al. Endothelial regulation of vasomotion in apoE-deficient mice: implications for interactions between peroxynitrite and tetrahydrobiopterin. Circulation. 2001;103(9):1282-8.
19. Xu J, Wu Y, Song P, et al. Proteasome-dependent degradation of guanosine 5'-triphosphate cyclohydrolase I causes tetrahydrobiopterin deficiency in diabetes mellitus. Circulation. 2007;116(8):944-53.
20. Vathsala ER, Connie WH Woo, Yaw L Siow, et al. Homocysteine stimulates NADPH oxidase-mediated superoxide production leading to endothelial dysfunction in rats. Canadian Journal of Physiology and Pharmacology. 2007;85(12): 1236-1247.
21. Zhao Y1, Wu J, Zhu H, et al. Peroxynitrite-dependent zinc release and inactivation of guanosine 5'-triphosphate cyclohydrolase 1 instigate its ubiquitination in diabetes. Diabetes. 2013;62(12):4247-56.
22. El-Bassossy HM, Neamatallah T, Balamash KS, et al. Arginase overexpression and NADPH oxidase stimulation underlie impaired vasodilation induced by advanced glycation end products. Biochem Biophys Res Commun. 2018;499(4):992-997.
23. Singh U, Devaraj S, Jialal I. Vitamin E, Oxidative Stress, and Inflammation. 2005;25:151-174.
24. Dusting GJ1, Selemidis S, Jiang F. Mechanisms for suppressing NADPH oxidase in the vascular wall. Mem Inst Oswaldo Cruz. 2005;100:97-103.
25. Gupta M1, Kaur G2. Aqueous extract from the Withania somnifera leaves as a potential anti-neuroinflammatory agent: a mechanistic study. J Neuroinflammation. 2016;13(1):193.
26. Zuniga FA, Ormazabal V, Gutierrez N, et al. Role of lectin-like oxidized low density lipoprotein-1 in fetoplacental vascular dysfunction in preeclampsia. Biomed Res Int. 2014;2014:353616.
27. Wink DA, Hines HB, Cheng RY, et al. Nitric oxide and redox mechanisms in the immune response. J Leukoc Biol. 2011;89(6):873-91.
28. Monnier Ak, Prigent-Tessier A, Quirié A, et al. Brain-derived neurotrophic factor of the cerebral microvasculature: a forgotten and nitric oxide-dependent contributor of brain-derived neurotrophic factor in the brain. Acta Physiol (Oxf). 2017;219(4):790-802.
29. Banoujaafar H, Monnier A, Pernet N, et al. Brain BDNF levels are dependent on cerebrovascular endothelium-derived nitric oxide. Eur J Neurosci. 2016;44(5):2226-35.
30. Utkan T, Yazir Y, Karson A, et al. Etanercept improves cognitive performance and increases eNOS and BDNF expression during experimental vascular dementia in streptozotocin-induced diabetes. Curr Neurovasc Res. 2015;12(2):135-46.
31. Kröncke D, Fehsel K, Kolb-Bachofen V. Inducible nitric oxide synthase in human diseases. Clin Exp Immunol. 1998;113(2):147-156.
32. Kozlov AV, Bahrami S, Redl H, et al. Alterations in nitric oxide homeostasis during traumatic brain injury. Biochim Biophys Acta Mol Basis Dis. 2017;1863(10 Pt B):2627-2632.
33. Garry PS, Ezra M, Rowland MJ, et al. The role of the nitric oxide pathway in brain injury and its treatment--from bench to bedside. Exp Neurol. 2015;263:235-43.
34. Morita M, Hayashi T, Ochiai M, et al. Oral supplementation with a combination of L-citrulline and L-arginine rapidly increases plasma L-arginine concentration and enhances NO bioavailability. Biochem Biophys Res Commun. 2014;454(1):53-7.
35. Steiber A, Carrero JJ. Vitamin Deficiencies in Chronic Kidney Disease, Forgotten Realms. J Ren Nutr. 2016;26(6):349-351.
36. Gropper S, Smith J, Carr T. Advanced Nutrition and Human Metabolism; 5th edition, International edition. pg 196, 348, 366.37
37. Wilking M, Ndiaye M, Mukhtar H, et al. Circadian rhythm connections to oxidative stress: implications for human health. Antioxid Redox Signal. 2013;19(2):192-208.
38. Kluge M, Fetterman J, Vita J. Mitochondria and Endothelial Function. Circ Res . 2013;112(8):1171-1188.
39. Ferris LT, Williams JS, Shen CL. The effect of acute exercise on serum brain-derived neurotrophic factor levels and cognitive function. Med Sci Sports Exerc. 2007;39(4):728-34.
40. Onen Sertoz O, Tolga Binbay I, Koylu E, et al. The role of BDNF and HPA axis in the neurobiology of burnout syndrome. Prog Neuropsychopharmacol Biol Psychiatry. 2008;32(6):1459-65.
41. Morita M, Sakurada M, Watanabe F, et al. Effects of Oral L-Citrulline Supplementation on Lipoprotein Oxidation and Endothelial Dysfunction in Humans with Vasospastic Angina. Immunol Endocr Metab Agents Med Chem. 2013;13(3):214-220.
42. Li Z, Zhimin Yue, Chengfa Zhao, et al. Maternal dietary supplementation of arginine increases the ratio of total cloned piglets born to total transferred cloned embryos by improving the pregnancy rate of recipient sows. Anim Reprod Sci. 2018;196:211-218.
43. Ihlemann N, Rask-Madsen C, Perner A, et al. Tetrahydrobiopterin restores endothelial dysfunction induced by an oral glucose challenge in healthy subjects. Am J Physiol Heart Circ Physiol. 2003;285(2):H875-82.
44. Xie L, Liu Z, Lu H, et al. Pyridoxine inhibits endothelial NOS uncoupling induced by oxidized low-density lipoprotein via the PKCα signalling pathway in human umbilical vein endothelial cells. Br J Pharmacol. 2012;165(3):754-64.
45. Roe ND, He EY, Wu Z, et al. Folic acid reverses nitric oxide synthase uncoupling and cardiac dysfunction in insulin resistance: Role of Ca2+/Calmodulin-activated Protein Kinase II. Free Radic Biol Med. 2013;65:234-243.
46. Chalupsky K, Kračun D, Kanchev I, et al. Folic acid promotes recycling of tetrahydrobiopterin and protects against hypoxia-induced pulmonary vascular hypertension by recoupling endothelial nitric oxide synthase. Antioxid Redox Signal. 2015;23(14):1076-91.
47. Misra UK, Kalita J2Singh SK, Rahi SK. Oxidative Stress Markers in Vitamin B12 Deficiency. Mol Neurobiol. 2017;54(2):1278-1284.
48. Achan V, Tran CT, Arrigoni F, et al. All-trans-Retinoic acid increases nitric oxide synthesis by endothelial cells: a role for the induction of dimethylarginine dimethylaminohydrolase. Circ Res. 2002;90(7):764-9.
49. Moretti R, Caruso P, Dal Ben M, et al. Vitamin D, Homocysteine, and Folate in Subcortical Vascular Dementia and Alzheimer Dementia. Front Aging Neurosci. 2017;9:169.
50. Huang A, Vita JA, Venema RC, et al. Ascorbic acid enhances endothelial nitric-oxide synthase activity by increasing intracellular tetrahydrobiopterin. J Biol Chem. 2000;275(23):17399-406.
51. Jiménez-Osorio AS, García-Niño WR1, González-Reyes S, et al. The Effect of Dietary Supplementation With Curcumin on Redox Status and Nrf2 Activation in Patients With Nondiabetic or Diabetic Proteinuric Chronic Kidney Disease: A Pilot Study. J Ren Nutr. 2016;26(4):237-44.
52. Crow JP. Peroxynitrite scavenging by metalloporphyrins and thiolates. Free Radic Biol Med. 2000;28(10):1487-94.