Quick Hit Summary

Nitrix oxide (NO) is one of the many factors that regulate blood flow in the human body. Key amino acids involved with the synthesis of nitric oxide include arginine, citrulline and to a lesser extent, ornithine. Based off the notion that increased blood flow enhances physical performance, the supplement industry has made millions of dollars off of Nitric Oxide/Muscle Pump supplements that contain these amino acids. In Part 1 of this series examining the "Muscle Pump" supplements, we're going to take a close look at the relationship of these amino acids and nitric oxide production. As we'll see, despite the fact that nitric oxide is synthesized directly out of arginine, dietary citrulline may be our best bet if we hope to have any chance of seeing a spike in NO production.

Nitric Oxide (NO) Article Series

In this series of articles we're going to discuss the in's and out's of the NO Supplements; covering everything from basic NO physiology to the effects of the NO amino acids in isolation, or as part of propriety blended "NO Boosting" supplements. In case you missed any of the articles, I recommend checking them out.

• Nitric Oxide Supp's Part I – Understanding the Key Players
• Nitric Oxide Supp's Part II – Let's "Pump You Up"… or Not?
• NO Supp's Part III – The "Everything But The Kitchen Sink" NO Supplements
• NO Supp's Part IV – Training Age & Supraphysiological NO Spikes
• Still to Come: NO Supp's Part V – True Potential Benefits of Citrulline Supplementation
• Still to Come: NO Supp's Part VI – True Potential Benefits of Arginine Supplementation

Those "Must Have" Supplements

Figure 1. Illustration of Nitric Oxide, the key molecule that many supplements claim to spike. Image Source²⁰

Every now and then, a supplement explodes onto the market, causing both competitive and recreational athletes to rush to the nearest neutraceutical store in order to purchase the new “must have” ergogenic aid. In due time, we often find that these products fall into 1 of 3 categories:

• 1) First are the legitimate supplements that enter the market, have research to back them up and lead to great gains in performance. An example of this would be creatine.¹

• 2) Next up are the highly hyped supplements that enter the industry, generate a lot of buzz and then fade away as people realize that the results were not quite equal to that claimed by advertisements. Think ZMA which at one time was a “hot” supplement, only to find itself next to the other “has been” supplements.

• 3) Finally there are the supplements that enter the market, research proves it to be ineffective, yet the frenzy over them continues.

With regards to this 3rd category, there is one supplement that many individuals would quickly add to the list. Yet, on the flip side of the equation, others claim that it enhances their workout to the point where they do not know how they could survive without it. What supplement could possibly cause such a great divide in opinions you ask???? Well it’s none other than the stimulant laden Muscle Pump/Nitric Oxide (NO) supplements that adorn the pages of your favorite muscle magazine and line the shelves of neutraceutical stores across the nation.

So what’s the truth behind the NO supplements? Are they truly as great as the advertisements claim or as awful as the "haters" on the various bulletin boars would have it? Over the upcoming weeks, we’re going to take an in-depth look at these NO Supplements, carefully examining the scientific literature, to see if we can settle this debate!

What is Nitric Oxide?

Figure 2. The production of Nitric Oxide. NOS = Nitric Oxide Synthase. Image created by Sean Casey

Nitric Oxide is a vasodialator². In other words, it causes blood vessels to relax, increasing blood flow to a given area. Using the logic that increased blood flow to exercising muscles is beneficial, neutraceutical manufacturers went to work, creating supplements that "pumped up" your muscles via increased NO production. The “key players”, i.e. ingredients, commonly found in these supplements are arginine, citrulline and, to a lesser extent, ornithine. Why these amino acids (AA’s) you ask?

As seen in Figure 2, NO is synthesized out of the amino acid arginine³. Using basic logic, if we increase the main substrate, we’ll get more end product. Citrulline, one of the byproducts of this reaction, can be converted back into arginine by the kidneys⁴ as well as by the cells that line the inside of blood vessels.¹⁶¹⁷ This relationship between arginine, citrulline and NO is referred to as the Citrulline – NO Cycle and is depicted in Figure 3.¹⁸ Likewise, the body can convert ornithine into citrulline; which, as just stated, can be converted back into arginine, thus, leading to increased NO production. (More on this later in this article).

Figure 3 The Citrulline-NO cycle. NOS = Nitric Oxide Synthase; ASS = Arginosuccinate synthetase; ASL = Arginosuccinate lyase. Image created by Sean Casey

For the history buffs out there, the presence of a substance in the wall of arteries, capable of inducing vasodialation, was first discovered in 1980 by researchers at State University of New York⁵. However, researchers did not know what the molecule was and referred to it as "endothelial derived relaxation factor"⁶. It wasn’t until later in the 80’s that researchers discovered that this molecule was actually Nitric Oxide and was synthesized out of Arginine⁷. The key pioneers involved with the discovery and analysis of this molecule were Dr. Robert F. Furchgott, Dr. Louis J Ignarro and Dr. Ferid Murad. In 1998, their efforts were awarded as they were presented with the Nobel Prize in Physiology or Medicine for their discoveries regarding "… nitric oxide as a signaling molecule in the cardiovascular system."¹⁹

Protein Digestion & The Urea Cycle

Figure 4. The dietary fate of protein. Image created by Sean Casey

Now that we have a rough understanding of how/why these amino acids are included in NO supplements, we are going to take a step back and tackle this issue from the outside-in by briefly reviewing protein digestion as well as the urea cycle…

Upon eating your favorite source of protein, food travels to your stomach before entering your small intestines (See Figure 4)⁸. In each of these locations proteins are broken down by various enzymes into amino acids which are then absorbed by the cells of the intestinal wall known as enterocytes. Within the enterocytes, some, but not all, of the amino acids are metabolized before heading to the liver via the hepatic portal vein (Hepatic = Liver).

Once reaching the liver, amino acids can be used for energy, synthesized into proteins/carbs/fat, held for other purposes or released into systemic circulation.⁸ If broken down for synthesis of carbohydrates or fat, nitrogen is stripped off the amino acid and eventually finds itself entering into the urea cycle; This prevents the toxic buildup of the metabolic waste product known as ammonia⁹. The end product of this cycle, urea, is then excreted from the body with one’s urine. As seen in Figure 5, the NO amino acids play a key role in this process as they’re inter-converted from one another via the following enzymes: Arginase (ARG), Ornithine carbamotransferase (OCT); Arginosuccinate synthetase (ASS); Arginosuccinate lyase (ASL).

Figure 5.The Urea Cycle is responsible getting rid of excess nitrogen in the body. NH2 = Amino groups; ARG = Arginase; OCT = Ornithine carbamotransferase; ASS = Arginosuccinate synthetase; ASL = Arginosuccinate lyase. Image created by Sean Casey

FYI – Ammonia, generated from the breakdown of the body’s “own” proteins (ie- endogenous) is also removed via this mechanism. Ditto for nitrogen containing metabolic waste products. This will be an important point to remember for Part IV of this series.

Arginine, Citrulline, Ornithine: From Mouth to NO Production

Figure 6. Dietary fates of the nitric oxide amino acids. ARG = Arginase; OCT = Ornithine carbamotransferase; ASS = Arginosuccinate synthetase; ASL = Arginosuccinate lyase. Image created by Sean Casey

Side note… Although not depicted in the above graph, I want to briefly mention that both dietary glutamine and proline feed into the NO amino acid cycle via conversion to ornithine. In his doctoral thesis research, Robert Christopher Kennedy Tomlinson studied the effects of dietary proline on arginine synthesis in 5 healthy men.²¹ For 48 hours prior to the trial, individuals followed a standardized diet. Additionally, these men were in a "fed" vs. fasted state throughout the 8 hr experimental trial (They consumed 150 kcal/h and a protein intake of 3.25 g/h). 5 hours into the experiment, they also started consuming proline. Final results of this trial indicated that dietary proline contributed to 4% of the total arginine flux. Using the same sample group and protocol Kennedy Tomlinson repeated the experiment but had the men ingest glutamine. Kennedy Tomlinson found that glutamine contributed to 50% of the total arginine synthesis.

Now that we’ve covered the digestion of proteins in general, let’s shift our attention to how dietary arginine, citrulline & ornithine are metabolized within the body. Please refer to Figure 6 which is a simplified illustration of this process.

The first major site of metabolism, faced by the NO amino acids, occurs in the enterocytes of the small intestines. Here, a portion of the consumed arginine and ornithine are converted to citrulline due to the presence of the ARG and ORN enzymes¹⁰. The arginine and ornithine which escape breakdown within the enterocytes travel to the liver. Here, further breakdown occurs as the liver takes what it needs to properly function.¹³ As a result, upon reaching systemic circulation, little dietary arginine actually remains as both the gut and liver have consumed/utilized that which is needed for their own purposes.

In contrast, citrulline fares much better as very little of it is broken down within the enterocytes due to low ASL and ASS activity¹¹ which were the key enzymes involved with citrulline breakdown (See Figure 5). As such, citrulline travels relatively unscathed to the liver. Supplement companies as well as scientist often state that citrulline is not taken up by the liver¹². However, this idea was recently challenged in a 2007 study conducted by van de Poll et al. in which researchers found that 55% of the citrulline produced in the enterocytes was filtered out of the bloodstream by the liver.¹³ The remaining citrulline eventually makes its way to the kidneys (or other tissue). Upon reaching it's destination, citrulline is converted to arginine via the ASL and ASS enzymes and released into systemic circulation¹⁴. As previously mentioned, this process is not limited to the kidneys and occurs throughout the body.¹⁶¹⁷ Thus, citrulline from the gut can be converted to arginine, leading to NO production.

As you can see, from a theoretical standpoint, if we’re trying to get arginine to our blood vessels, taking citrulline may give us the best chance of making this happen simply because it is less “filtered” than dietary arginine. Yet, is their substantial evidence to indicate that citrulline is a superior with respect to raising plasma arginine levels?

In a cross-over designed study, Schwedhelm et al. attempted to answer this question by having 20 healthy volunteers supplement their normal diets according to the following protocols¹⁵:

(Please note, each of these supplement protocols was 1 week in duration. On the 7th day, participants took only 1/2 of the daily dose.)

• Placebo
• Citrulline 750mg; 2x/day
• Citrulline 1500 mg; 2x/day
• Citrulline 3000 mg; 2x/day
• Arginine SR (slow release) 1600mg; 2x/day
• Arginine IR (immediate release) 1000 mg; 3x/day

On the 7th day of each protocol, study participants had their blood drawn at 0, 0.5, 1, 2, 4, 6, 8, 12, 16 and 24 h. As seen in Figure 7, taking 3000 mg of Citrulline on day 7, following 6 days of 6000 mg/day, led to ~3x greater serum arginine levels vs. following either of the supplemental arginine protocols. However, please note that to get this 3x increase in serum arginine AUC, they took ~2x the amount of citrulline (vs. arginine trials). If the citrulline and arginine doses were equal, citrulline increased plasma arginine levels 1.45x vs ARG SR (statistically significant) and a 1.48x vs ARG IR (non-statistically significant). To the best of my knowledge, there have not been any other studies that have examined the effects of both citrulline and arginine on blood-arginine levels in the same test subjects.

Figure 7. 24 Hour Arginine AUC. (#) indicates significantly different from ARG SR 1600. (*) indicates significantly different from ARG IR 1000. Image created by Sean Casey. Data adapted from Schwedhelm et al.¹⁵

Bottom Line

The main amino acids commonly found in NO products are arginine, citrulline and to a lesser extent, ornithine. As described above, these amino acids are highly intertwined with one another and can influence plasma arginine levels. That said, being the inquisitive mind you are, I’m sure you’re probably asking yourself…

"Do these amino acid supplements actually amplify nitric oxide production?"

To find out the answer to this question, check out Part II of this series!

References

¹ Buford TW, Kreider RB, Stout JR, Greenwood M, Campbell B, Spano M, Ziegenfuss T, Lopez H, Landis J, Antonio J. International Society of Sports Nutrition position stand: creatine supplementation and exercise. J Int Soc Sports Nutr. 2007 Aug 30;4:6.

² Bruckdorfer. The basics about nitric oxide. Molecular aspects of medicine.2005 vol:26 iss:1-2 pg:3 -31

³ Stuehr DJ. Enzymes of the L-arginine to nitric oxide pathway. J Nutr. 2004 Oct;134(10 Suppl):2748S-2751S; discussion 2765S-2767S.

⁴ Moinard C, Cynober L. Citrulline: a new player in the control of nitrogen homeostasis.J Nutr. 2007 Jun;137(6 Suppl 2):1621S-1625S.

⁵ Furchgott, R.F., and Zawadzki, J.V. 1980. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature, 288: 373–376.

⁶ Cherry, P.D., Furchgott, R.F., Zawadzki, J.V., and Jothianandan, D. Role of endothelial cells in relaxation of isolated arteries by bradykinin. Proc. Natl. Acad. Sci. U.S.A. 1982. 79: 2106–2110.

⁷ Palmer RM, Rees DD, Ashton DS, Moncada S. L-arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation. Biochem Biophys Res Commun. 1988 Jun 30;153(3):1251-6.

⁸ Wardlaw GM, Hampl JS, DiSilvestro RA. Perspectives in Nutrition. 6 ed. New York, New York. 2004. Pgs 235-236

⁹ Nelson DL, Cox MM. Principles of Biochemistry. 3rd ed. New York, New York. 2000

¹⁰ Moinard C, Cynober L. Citrulline: a new player in the control of nitrogen homeostasis. J Nutr. 2007 Jun;137(6 Suppl 2):1621S-1625S.

¹¹ Davis PK, Wu G. Compartmentation and kinetics of urea cycle enzymes in porcine enterocytes. Comp Biochem Physiol B Biochem Mol Biol. 1998 Mar;119(3):527-37.

¹² Windmueller HG, Spaeth AE. Am J Physiol. 1981 Dec;241(6):E473-80. Source and fate of circulating citrulline.

¹³ van de Poll MC, Siroen MP, van Leeuwen PA, Soeters PB, Melis GC, Boelens PG, Deutz NE, Dejong CH. Interorgan amino acid exchange in humans: consequences for arginine and citrulline metabolism. Am J Clin Nutr. 2007 Jan;85(1):167-72.

¹⁴ Moinard C, Cynober L. Citrulline: a new player in the control of nitrogen homeostasis. J Nutr. 2007 Jun;137(6 Suppl 2):1621S-1625S.

¹⁵ Schwedhelm E, Maas R, Freese R, Jung D, Lukacs Z, Jambrecina A, Spickler W, Schulze F, Böger RH. Pharmacokinetic and pharmacodynamic properties of oral L-citrulline and L-arginine: impact on nitric oxide metabolism. Br J Clin Pharmacol. 2008 Jan;65(1):51-9. Epub 2007 Jul 27.

¹⁶ Goodwin BL, Solomonson LP and Eichler DC. Argininosuccinate synthase expression is required to maintain nitric oxide production and cell viability in aortic endothelial cells. J Biol Chem 2004; 279: 18353-18360.

¹⁷ Karbach S, Simon A, Slenzka A, Jaenecke I, Habermeier A, Martiné U, Förstermann U, Closs EI. Relative contribution of different l-arginine sources to the substrate supply of endothelial nitric oxide synthase. J Mol Cell Cardiol. 2011 Nov;51(5):855-61. Epub 2011 Aug 2.

¹⁸ Husson A, Brasse-Lagnel C, Fairand A, Renouf Sand Lavoinne A. Argininosuccinate synthetase from the urea cycle to the citrulline-NO cycle. Eur J Biochem 2003; 270: 1887-1899.

¹⁹ Nobelprize.org. The Nobel Prize in Physiology of Medicine 1998. 2012. Accessed February 12, 2012 from:http://www.nobelprize.org/nobel_prizes/medicine/laureates/1998/illpres/

²⁰ Yikrazuul. Nitric Oxide. December 10, 2008. This image has been released into the public domain. Image accessed February 13, 2012 from: http://commons.wikimedia.org/wiki/File:Nitric_oxide.svg

²¹ Tomlinson, RCK. Arginine Synthesis in Humans. Doctoral Theses. 2011