Leaders in Pharmaceutical Business Intelligence (LPBI) Group

Author and Reporter: Meg Baker, Ph.D., Registered Patent Agent

The 1998 Noble Prize for medicine was for the discovery that nitric oxide (NO) was the chemical messenger responsible for relaxing vascular tissue and thereby increasing blood flow and reducing blood pressure. Alfred Noble himself had been prescribed nitro-glycerin for heart problems over 100 years before, a compound which is metabolized to NO.

NO, a gas at room temperature, has an exceedingly short half-life in the body. Normally, NO is produced from an amino acid, L-arginine (L-Arg), a normal component of the dietary protein, and molecular oxygen (O2) by the one of the several Nitric Oxide Synthases (EC 1.14.13.39): endothelial (eNOS, NOS III), inducible (iNOS, NOS II), and neural (nNOS, NOS I). In human studies, supplementation with l-arginine improved endothelium-dependent vasodilation.

The reaction of iNOS with L-Arg to produce NO leaves another amino acid, citrulline. Excess L-Arg can also be degraded by arginase (enzyme having two isoforms, I and II) which may be coinduced with iNOS in some cell types.

Citrulline formed as a by-product of the NOS reaction can be recycled to arginine by argininosuccinate synthetase (AS) and argininosuccinate lyase (AL).

Mori (2007)  http:// www.ncbi.nlm.nih.gov/ pubmed/ 17513437 found that AS and sometimes AL are coinduced with inducible NOS (iNOS) in various cells. In these cells, NO was synthesized from citrulline (via arginine) as well as from arginine, indicating operation of the citrulline-NO cycle.

Whereas, low concentrations of NO protect cells from apoptosis, excessive NO causes apoptosis. NO causes endoplasmic reticulum (ER) stress, induces a transcription factor, CAAT/enhancer binding protein (C/EBP) homologous protein (CHOP), and leads to apoptosis.

The active site of NOS is formed by a heme-containing substrate-binding cavity, where L-arginine (Arg) and O₂ are converted to L-citrulline and NO. The electrons required for reductive O₂ activation are transferred from NADPH via the NOS-bound flavins (riboflavin, Vitamin B2) FMN and FAD. All NOS isoforms are only active as homodimers.

Generation of NO occurs in two discrete O2-requiring steps, with intermediate formation of N-hydroxy-L-arginine (NHA or NOHLA). NHA formation consumes one molecule of O₂ and two electrons. Conversion of NHA to L-citrulline and NO requires another molecule of O₂ and one more electron (http://en.wikipedia.org/wiki/Nitric_oxide_synthase).  The overall stoichiometry, reflecting the three electrons derived from NADPH, that pass through the flavin co-factors and are transferred one by one via the heme iron,  is then:

L-arginine + 3/2 NADPH + H⁺ + 2 O₂ = citrulline + nitric oxide + 3/2 NADP⁺

Another factor affecting NOS activity is the availability of essential co-factors such as tetrahydrobiopterin (BH4) (Boeger et al. Cardiovasc Res (2003) 59 (4): 824-833 http://cardiovascres.oxfordjournals.org/content/59/4/824.full, Vasquez-Vivar J., et al . Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors. Proc. Natl. Acad. Sci. USA 1998;95:9220-9225 http://www.pnas.org/content/95/16/9220.full). H₄-biopterin binds in the immediate vicinity of the heme at the dimer interface, interacting with residues from both subunits. When BH4 availability is limiting, electron transfer from NOS flavins becomes “uncoupled” from l-arginine oxidation and the ferrous-dioxygen complex formed as an intermediate in the reaction sequence, dissociates and superoxide(O2−·) is produced.

See Figure 1 in Werner et al.  2003 Exp Biol  Med 228: 1291-1302.

RADICALS

The conversion of Arg to NHA and of NHA to L-citrulline and NO both depend on the presence of H₄-biopterin. In the absence of substrate or pterin, NADPH oxidation by NOS is accompanied by formation of O₂⁻ and peroxide (H₂O₂). Uncoupled eNOS is assumed to produce superoxide (O2−·) in addition to or instead of NO (·NO) which will react with itself, with NO, or with -hydroxyl, -sulfhydryl, or or side groups of proteins, lipids, or glycans. Reaction of ·NO produced by eNOS, with O2−· produced by eNOS or by other enzymes, such as NADPH and xanthine oxidases, decreases the amount of ·NO available to stimulate vascular relaxation. At the very low BH4 concentration of 100 nmol/L, recombinant human eNOS activity is fully developed. However, biopterin is formed from the pterin heterocycle also present in folic acid (Vitamin B9,
pteroyl-L-glutamate)
and which is synthesized from GTP. Human GTP cyclohydrolase I (GTPCH), is the rate-limiting enzyme in BH4 synthesis (Crabtree et al. JBC 2008, http://www.jbc.org/content/284/2/1136.full).

In addition to the NOS reaction, which generates a H₃-biopterin radical cation, a neutral H₃-biopterin radical is formed when H₄-biopterin reacts with various radicals and which can be reduced back to H₄-biopterin by ascorbate (Vitamin C). Folate species are also required to synthesize pyrimidines and purines (for DNA synthesis and repair and NADH and NADPH).

Enhancing NO Synthesis

The normal way to increase vascular nitric oxide is through vascular stress, such as exercise. As oxygen demand increases, cardiac output increases and the endothelial lining of the arteries releases nitric oxide into the blood, which, in turn, relaxes and widens the vessel wall, allowing for enhanced blood flow.

Enhancing the presence of L-Arg or the one or more of the NOS enzymes are obviously essential for NO production. However, NOS enzymes are co-valently bound to heme (heme, iron), and flavin co-factors (Vit B2), and require soluble co-factors NADPH (a dinucleotide phosphate, containing niacin, Vitamin B3), and BH4 (from Vit B9).

Foods high in Arginine and Citrulline include melons and cucumber, peanuts, salmon, and soy. Arginine is found in varying degrees (3-15% by weight) in all animal proteins. Blue-fin tuna has 1.8 g of arginine per 100 g so 2 oz. of tuna will provide about 1 g of arginine. Other sources of 1 g of L-Arg: 2.7 oz. of chicken thighs, about 4 oz. of chicken breast, 2 oz. of 75 percent lean hamburger or about 2.5 oz. of pork.

Foods rich in antioxidants and polyphenols will provide protection against free radical assault on proteins and, in particular, act to protect the NOS enzyme and cofactors. Almost all fruit and vegetables such as blueberries, cranberries, carrots, grapefruit, soybeans, apples, and spinach contain high levels of antioxidants. In addition, nuts, tea, seeds, dark chocolate, red wine, and seafood generally contain antioxidants such as resveratrol, ascorbate, and other phytochemicals. Other free radical scavengers, tocopherols (alpha-tocopherol, Vit E) work predominantly in the lipid environment such as in cell membranes, while the sulfur-containing soluble molecule, glutathione (GSH) protects the cytosolic milieu.

Supplements

Both L-Arg or L-citrulline can be purchased over the counter. Dietary L-arginine will be taken up by the intestine and transported directly to the liver by the hepatic artery as are most of the products of digestion. Much of this L-Arg will be used in metabolic steps related to the urea cycle which is co-ordinated with the kidney to rid the body of excess nitrogen and prevent ammonia concentration from building. A small amount will enter the blood stream and be used for NO synthesis.

Proargi-9 Plus® is one product being sold containing mutltigram doses of L-Arg plus L-Citrulline in combination with anti-oxidants and folate. Proargi-9 Plus® is a registered trademark and copyright of Nature’s Sunshine Products, Inc. L-arginine Plus™ is formulation with similar ingredients and stated amounts of L-Arg and L-Citrulline and is not affiliated with the makers of Proargi-9-Plus. Niteworks® is a registered trademark and copyright of Herbalife International, Inc. and is not affiliated with or a sponsor of L-arginine Plus™.

Dr. Joe Prendergast is an endocrinologist using L-Arg therapy who, over 19 years, never had to admit any of his 7200 diabetes patients to the hospital for peripheral artery disease, recommends supplemental L-Arg formulations to his patients. The combination of L-Arg with L-citrulline a longer acting NO forming product. http://www.livingwithoutdisease.com/?route=references/prendergast

Supplements of L-Arg and, in particular, in combination with L-citrulline other B-vitamins and antioxidents may be an effective way to boost vascular NO synthesis for anyone not exercising or eating a balanced diet, having a deficiency in any of the L-Arg recycling enzymes, NOS enzymes, co-factor recycling or synthetic enzymes, or other risk factor. Specific risk factors, such as inherently elevated levels of the natural NOS inhibitor ADMA (asymmetric-dimethyl-L-arginine) are beginning to be uncovered and will be the subject of another post.

 Additional References

Nitric Oxide: Biology and Pathobiology,  LJ Ignarro Editor, Sep 13, 2000 http://books.google.com/books?id=h5FugARr4bgC&dq=pterin+ring&source=gbs_navlinks_s

Mori, M. Regulation of nitric oxide synthesis and apoptosis by arginase and arginine recycling.  J Nutr. 2007 Jun;137(6 Suppl 2):1616S-1620S.   http://www.ncbi.nlm.nih.gov/pubmed/17513437

Werner, et al.  Tetrahydrobiopterin and Nitric Oxide: Mechanistic and Pharmacological Aspects Exp Biol Med December 2003 vol. 228 no. 11 1291-1302  Werner et al. Exp Biol Med 2003

Davel AP, Wenceslau CF, Akamine EH, Xavier FE, Couto GK, Oliveira HT, Rossoni LV. Endothelial dysfunction in cardiovascular and endocrine-metabolic diseases: an update.  Braz J Med Biol Res. 2011 Sep;44(9):920-32. Epub 2011 Aug 19. Davel et al. Braz J Med Biol Res 2011

Rainer H Boeger. Pharmacokinetic and pharmacodynamic properties of oral L-citrulline and L-arginine: impact on nitric oxide metabolism   Schwedhelm E, et al. Br J Clin Pharmacol. 2008_65_51-9

Louise Ignarro, UCLA, Nobel Prize Recipient, Author “NO More Heart Disease”

John Cook, Peripheral artery disease study, Author “Cardiovascular Cure”

Other aspects of Nitric Oxide involvement in biological systems in humans are covered in the following posts on this site:

Nitric Oxide in bone metabolism July 16, 2012

Author: Aviral Vatsa PhD, MBBS

http://pharmaceuticalintelligence.com/2012/07/16/nitric-oxide-in-bone-metabolism/?goback=%2Egde_4346921_member_134751669

 

Nitric Oxide production in Systemic sclerosis July 25, 2012

Curator: Aviral Vatsa, PhD, MBBS

http://pharmaceuticalintelligence.com/2012/07/25/nitric-oxide-production-in-systemic-sclerosis/?goback=%2Egde_4346921_member_138370383

 

Nitric Oxide Signalling Pathways August 22, 2012 by

Curator/ Author: Aviral Vatsa, PhD, MBBS

http://pharmaceuticalintelligence.com/2012/08/22/nitric-oxide-signalling-pathways/?goback=%2Egde_4346921_member_151245569

 

Nitric Oxide: a short historic perspective August 5, 2012

Author/Curator: Aviral Vatsa PhD, MBBS

http://pharmaceuticalintelligence.com/2012/08/05/nitric-oxide-a-short-historic-perspective-7/

 

Nitric Oxide: Chemistry and function August 10, 2012

Curator/Author: Aviral Vatsa PhD, MBBS

http://pharmaceuticalintelligence.com/2012/08/10/nitric-oxide-chemistry-and-function/?goback=%2Egde_4346921_member_145137865

 

Nitric Oxide and Platelet Aggregation August 16, 2012 by

Author: Dr. Venkat S. Karra, Ph.D.

http://pharmaceuticalintelligence.com/2012/08/16/no-and-platelet-aggregation/?goback=%2Egde_4346921_member_147475405

 

The rationale and use of inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure August 20, 2012

Author: Larry Bernstein, MD

http://pharmaceuticalintelligence.com/2012/08/20/the-rationale-and-use-of-inhaled-no-in-pulmonary-artery-hypertension-and-right-sided-heart-failure/

Nitric Oxide: The Nobel Prize in Physiology or Medicine 1998 Robert F. Furchgott, Louis J. Ignarro, Ferid Murad August 16, 2012

Reporter: Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2012/08/16/nitric-oxide-the-nobel-prize-in-physiology-or-medicine-1998-robert-f-furchgott-louis-j-ignarro-ferid-murad/

 

Coronary Artery Disease – Medical Devices Solutions: From First-In-Man Stent Implantation, via Medical Ethical Dilemmas to Drug Eluting Stents August 13, 2012

Author: Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2012/08/13/coronary-artery-disease-medical-devices-solutions-from-first-in-man-stent-implantation-via-medical-ethical-dilemmas-to-drug-eluting-stents/

 

Nano-particles as Synthetic Platelets to Stop Internal Bleeding Resulting from Trauma

August 22, 2012

Reported by: Dr. V. S. Karra, Ph.D.

http://pharmaceuticalintelligence.com/2012/08/22/nano-particles-as-synthetic-platelets-to-stop-internal-bleeding-resulting-from-trauma/

Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production July 19, 2012

Curator and Research Study Originator: Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2012/07/19/cardiovascular-disease-cvd-and-the-role-of-agent-alternatives-in-endothelial-nitric-oxide-synthase-enos-activation-and-nitric-oxide-production/

Macrovascular Disease – Therapeutic Potential of cEPCs: Reduction Methods for CV Risk

July 2, 2012

An Investigation of the Potential of circulating Endothelial Progenitor Cells (cEPCs) as a Therapeutic Target for Pharmacological Therapy Design for Cardiovascular Risk Reduction: A New Multimarker Biomarker Discovery

Curator: Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2012/07/02/macrovascular-disease-therapeutic-potential-of-cepcs-reduction-methods-for-cv-risk/

 

Bone remodelling in a nutshell June 22, 2012

Author: Aviral Vatsa, Ph.D., MBBS

http://pharmaceuticalintelligence.com/2012/06/22/bone-remodelling-in-a-nutshell/

Targeted delivery of therapeutics to bone and connective tissues: current status and challenges- Part, September  

AuthorL Aviral Vatsa, PhD, September 23, 2012

http://pharmaceuticalintelligence.com/2012/09/23/targeted-delivery-of-therapeutics-to-bone-and-connective-tissues-current-status-and-challenges-part-i/

Calcium dependent NOS induction by sex hormones: Estrogen

Curator: S. Saha, PhD, October 3, 2012

http://pharmaceuticalintelligence.com/2012/10/03/calcium-dependent-nos-induction-by-sex-hormones/

 

Nitric Oxide and Platelet Aggregation,

Author V. Karra, PhD, August 16, 2012

http://pharmaceuticalintelligence.com/2012/08/16/no-and-platelet-aggregation/

Bystolic’s generic Nebivolol – positive effect on circulating Endothelial Progenitor Cells endogenous augmentation

Curator: Aviva Lev-Ari, PhD, July 16, 2012

http://pharmaceuticalintelligence.com/?s=Nebivolol

 

Endothelin Receptors in Cardiovascular Diseases: The Role of eNOS Stimulation

Author: Aviva Lev-Ari, PhD, 10/4/2012

http://pharmaceuticalintelligence.com/2012/10/04/endothelin-receptors-in-cardiovascular-diseases-the-role-of-enos-stimulation/

 

Inhibition of ET-1, ETA and ETA-ETB, Induction of NO production, stimulation of eNOS and Treatment Regime with PPAR-gamma agonists (TZD): cEPCs Endogenous Augmentation for Cardiovascular Risk Reduction – A Bibliography

Curator: Aviva Lev-Ari, 10/4/2012.

http://pharmaceuticalintelligence.com/2012/10/04/inhibition-of-et-1-eta-and-eta-etb-induction-of-no-production-and-stimulation-of-enos-and-treatment-regime-with-ppar-gamma-agonists-tzd-cepcs-endogenous-augmentation-for-cardiovascular-risk-reduc/