Citrulline

L-Citrulline is a non-protein amino acid compound (does not get used to form structural proteins such as enzymes), and unlike L-Arginine it is not widely present in all proteins. It is highest in watermelon (where it derives its name, as watermelon's are known as Citrullus vulgaris^[2]) where it averages 2.1mg/g wet weight (although absolute numbers vary)^[3] and consuming watermelon has been noted to acutely increase both plasma arginine and citrulline (3.3kg watermelon being equivalent to 10g supplemental L-arginine)^[4][5] and to increase fasting arginine and ornithine in the range of 12-22% following consumption of 780-1560g daily.^[6]

Other food sources of L-citrulline include muskmelons, bitter melons, squashes, gourds, cucumbers, and pumpkins.^[2]

Citrulline is an amino acid that is not one of the essential amino acids nor a common dietary amino acid (named after its only good natural source, watermelons), but is interconverted in the body and serves roles similar to L-arginine and L-ornithine supplementation

Citrulline is created in the body by one of two pathways, either recycled from arginine (the conversion of arginine into nitric oxide leaves citrulline as a byproduct)^[7][8] or produced from the nitrogen (and some carbon) contained in L-glutamine,^[9] where the enzyme Ornithine transcarbamylase uses both ornithine and carbamoyl phosphate (which requires glutamine) to produce citrulline in enterocytes.^[10][11]

It appears that the arginine pathway accounts for about 10% of circulating citrulline while the glutamine pathway accounts for 90%;^[7] reducing plasma glutamine levels can reduce plasma citrulline.^[12]

Two pathways mediate production of L-Citrulline, with the citrulline that is a byproduct of nitric oxide production being a minor contributor but plasma glutamine being a major contributor

As for the urea cycle (one of the mechanisms behind the 10%), L-Arginine is converted into L-Ornithine via the arginase enzyme (giving off urea as a cofactor)^[13][14] and from there ornithine (using carbamoyl phosphate as a cofactor) is subject to the Ornithine carbamoyltransferase enzyme to produce L-Citrulline. In this sense, the metabolic pathway from arginine towards citrulline (via ornithine) causes an increase in urea and a concomitant decrease in ammonia, which was used by the Carbamoyl phosphate synthase enzyme to create carbamoyl phosphate.^[15] If need be, arginine can directly be converted into L-citrulline via a Arginine deiminase enzyme to produce, rather than require, ammonia.^[16]

The cycle is formed as citrulline then binds with L-aspartate (related to D-Aspartic acid as its isomer) to form arginosuccinate via the arginosuccinate synthase enzyme, and then the arginosuccinate lysase enzyme degrades arginosuccinate into free arginine and fumarate; arginine then reenters the urea cycle anew.^[15] Fumarate can simply enter the TCA (Krebs) cycle as an energy intermediate,^[16] and citrulline negatively regulates the arginase enzyme.^[17]

Citrulline's convertion into L-arginosuccinate and subsequent conversion into L-arginine also is involved in the nitric oxide cycle rather than the urea cycle, with the only difference being that Arginine directly converts into citrulling (by giving off a nitric oxide molecule) rather than being indirectly converted via ornithine.^[18][19]

Citrulline can be produced by arginine by one of two mechanism, either directly via arginine giving off a nitric oxide molecule (which is the path involved in the nitric oxide cycle) or indirectly via arginine's conversion into ornithine (which is involved in the urea cycle, sequestering ammonia)

Citrulline per se is more of a byproduct of arginine metabolism (nitric oxide cycle) and of ornithine metabolism (urea cycle), and merely gets converted back to arginine via arginosuccinate. That being said, supplemental citrulline also beneficially affects arginine and ornithine concentrations so their bioactivity is also relevant

The main determinent of citrulline being converted into arginine appears to merely be serum concentrations of citrulline^[20] which in a rested state are in the range of 22.4-27μmol/L.^[21][22] Due to this, increasing plasma citrulline appears to cause linear increases in arginine up until around a 5-10g dose (A C_max of 1314-2756μmol/L) while higher oral doses appear to cause less increases in arginine than expected.^[23]

L-Citrulline is taken up to a much greater degree in the gut than its counterpart L-Arginine, and results in a higher plasma level of L-Arginine via the Arginine/Ornithine/Citrulline cycle.^[24] It is absorbed through numerous sodium-dependent transporters.^[25] 

Relative to arginine, citrulline is better absorbed in the intestines and may be a better source of bodily arginine than arginine itself

Oral supplementation of citrulline in humans at 0.18g/kg has been noted to double plasma arginine^[12] which has been replicated elsewhere^[22] alongside an equivalent increase in ornithine concentrations^[22] but these doublings in arginine and ornithine are alongside an 6-11 fold increase in plasma citrulline.^[12][22]

A single dose of 6g citrulline malate (0.08g/kg) in athletes prior to exercise has noted increases in plasma citrulline (173% increase), ornithine (152% increase), and arginine (123% increase) when measured after exercise, and these values were normalized with 3 hours rest.^[21] This same dose has been noted elsewhere to raise plasma citrulline and arginine to similar degrees.^[26]

Interestingly, the aforementioned studies using 0.18g/kg citrulline noted a 6-11 fold increase in citrulline alongside a mere doubling of arginine and ornithine^[12][22] while the later study using 6g (calculated to be 0.08g/kg) had a much smaller increase in citrulline but still more than doubled both arginine and ornithine.^[21] This was also noted in a dose-response study using 2g up to 15g citrulline where citrulline in plasma followed linear dose-dependence and both arginine and ornithine having less dose-dependence.^[23] The authors hypothesized that, due to the increase in arginine being less than predicted and serum citrulline being the main predictor of arginine synthesis^[20] that this indicates a rate-limiting step being reached in the kidneys.

Oral Citrulline supplementation can increase plasma urea cycle amino acids which include citrulline itself (most drastic increase), ornithine, and arginine. Ornithine and Arginine can reach up to a doubling or tripling with 5-10g citrulline supplementation and then seems to be restricted from increasing further, while citrulline simply follows linear dose-response patterns (more oral supplementation causing more serum citrulline)

Citrulline has been noted to not influence serum levels of any of the branched chain amino acids at rest,^[22] but can accelerate depletion of BCAAs induced by prolonged exercise (via increasing their utilization as fuel).^[21]

A reduction of glutamine (13% following 0.18g/kg citrulline for 7 days) has been noted with citrulline supplementation^[22] although another study noted acute usage of 6g Citrulline (0.08g/kg) failed to alter glutamine concentrations.^[21]

Other amino acids tested (glutamic acid, aspartic acid, asparagine, alanine, lysine, tryptophan, phenylalanine, L-tyrosine, histidine) are mostly unaffected.^[21]

Citrulline may deplete BCAA serum concentrations during exercise, but does not appear to do this inherently at rest. There are also some interactions with glutamine, but beyond that other amino acids do not appear to be affected

Approximately 83% of orally ingested citrulline appears to be taken up by the kidneys^[27][28][29] where it is converted into L-Arginine in the proximal tubules (via the enzymes arginosuccinate synthase and arginosuccinate lyase^[30]); this conversion of citrulline into arginine (either from supplemental citrulline or that produced as a byproduct of arginine creating nitric oxide) accounts for 5-15% of circulating arginine but 60% of total arginine (the difference being due to the ability of arginine to be incorporated into proteins).^[12][31]

The majority of L-citrulline either floats in the blood passively or gets transported to the kidneys for conversion into arginine. Due to this, most bioactivities of citrulline are secondary to arginine ingestion

Arginine has a few interactions with memory and learning, and as it is the molecule that citrulline acts vicariously through then reading that page will give a more complete view of citrulline's actions

Citrulline (and beta-alanine) have been found to be normally expressed at higher concentrations in the hippocampus of trained rats relative to untrained rats in a cognitive test.^[32]

In heart failure patients with preserved ejection fraction given 3g citrulline, pulmonary artery pressure appeared to be reduced (56.67+/-7.96mmHg to 47.67+/-8.59mmHg; 16%) alongside an improvement in right ventricular ejection fraction. Arginine was equally effective, but required a higher dose (8g),^[33] and this study is duplicated in Medline.^[34]

A study in healthy individuals given citrulline (3,200mg 60-90 minutes before testing) was able to decrease the ECG QT interval, indicative of a shortening of the time required to de/repolarize the myocardium.^[35]

5,600mg L-citrulline daily for a week in otherwise healthy middle aged men was able to significantly reduce brachial–ankle pulse wave velocity associated with increaes in serum nitric oxide metabolites but without changes in blood pressure overall,^[36] while in prehypertensive persons given 'watermelon extract' (1,350mg citrulline with 650mg arginine taken twice a day) for 6 weeks there was a reduction in aortic and brachial pulse pressure (6+/-2mmHg and 8+/-3mmHg, respectively) and a reduction in aortic systolic blood pressure by 7+/-2mmHg (brachial blood pressure and aortic diastolic unaffected).^[1]

In youth given a cold-pressor test (known to increase blood pressure without affecting heart rate^[37][38]), and supplemental arginine has been previously shown to attenuate the rise seen in persons with high cholesterol.^[39] In otherwise healthy young men subject to the cold-pressor test after 4 weeks of 6g citrulline supplementation (two doses of 3g) there was a lesser spike in systolic blood pressure seen with the test relative to baseline in the citrulline group only.^[40]

In youth with congenital heart disease having supplemental citrulline around an operation (5 doses of 1.9g/m², which is 3.2g and 3.6g for average adult females and males), subjects with elevated citrulline concentrations in the blood appeared to be protected from developing pulmonary hypertension and there was less overall development in the citrulline treated group (due to higher serum citrulline concentrations).^[41]

Citrulline supplementation appears to reduce blood pressure and improve blood flow in situations where blood flow is otherwise hindered or blood pressure higher than normal, but citrulline does not have unidirectional reducing effects; it may be ineffective in resting normotensive persons

Citrulline at 0.1mM (physiologically relevant concentration) has been noted to increase insulin secretion from pancreatic β-cells and to augment glucose-stimulated insulin release, and these events were inhibited with a NOS inhibitor and thus secondary to nitric oxide.^[42] 10-fold the concentration (1mM) does not appear to be significantly different than 0.1mM,^[43] and it appears that both arginine as well as the urea cycle intermediate L-arginosuccinate both also have activating effects on pancreatic beta-cells.^[43]

The exercise-induced increase in insulin has been noted to be decreased with supplemental citrulline (3g taken three hours before exercise or 9g over the course of 24 hours).^[44]

Supplementation of citrulline has been noted to modulate enzymes in the glycogenolysis (glycogen breakdown) and glycolysis (converting glucose into ATP) pathways in rat skeletal muscle, which was said to shift muscle from aerobic energy metabolism (fatty acid based) to anaerobic.^[45] Elsewhere, ATP production from aerobic pathways has been noted in both rats^[46] and humans given 6,000mg citrulline for 3 days^[47] and phosphocreatine (bioactive form of creatine) has been noted to have its depletion attenuated by 28%^[46] and its replenishment rate during rest accelerated by 20%^[47] (although this resynthesis does not appear to be reliable^[46]).

Elsewhere, citrulline (as malate) has been found to increase muscular ATP efficiency^[46] which is a similar phenomena as is seen with nitrate supplementation^[48] and arginine supplementation^[49] due to nitric oxide.^[50]

At least one study in rats has noted increased force output by 23% following intake of 3g/kg daily (human equivalent of 480mg/kg).^[46]

Citrulline does appear to interact with ATP production (in a beneficial manner) via increasing the efficiency of energy production, but it is unclear whether citrulline increases aerobic or anaerobic energy metabolism (or both, depending on context)

Citrulline (as malate) is sometimes used as an acid and ammonia buffer in bacterial models^[51] and in mammals including humans.^[52] Citrulline administration does appear to stimulate hepatic ureagenesis (via Ornithine sequestering ammonia)^[52] and elsewhere has been noted to simply increase ornithine concentrations itself (by approximately double, a similar increase in ornithine and arginine).^[12][22] 6g of citrulline appears to increase plasma ornithine (200% or so) to a lesser degree than supplemental ornithine itself at a similar dosage (300-500%).^[53][54]

Ammonia normally signals for muscle fatigue when it accumulates due to inhibiting the conversion of Pyruvate to Acetyl-CoA (via activating phosphofructokinase)^[55][56] which hinders contractility of muscles.^[57] Secondary to preventing ammonia accumulation (which has been confirmed in mice^[58]), an increase in physical performance may occur.

Citrulline may also possess the properties of ornithine supplementation and buffer ammonia, but on a gram per gram basis appears to be less efficient at raising plasma ornithine concentrations than ornithine itself (which is opposite that seen with arginine, where citrulline is actually more effective)

Although citrulline does not appear to be an activator of mTOR like leucine is (mTOR activation, via S6K, induces muscle protein synthesis), citrulline does appear to restore muscle protein synthesis rates^[59][60] and muscular function following food restriction^[61] and this positive effect on muscle protein synthesis is abolished by rapamycin and thus mediated by the mTORc1 pathway.^[62][63]

Citrulline has failed to activate both the p44/42 MAPK pathway and the ERK1/2 pathway in skeletal muscle,^[59] while leucine itself can activate ERK1/2 and induce 4E-BP1 (downstream of mTOR) to a much larger degree than citrulline.^[59]

Citrulline positively influences the mTOR pathway, but this does not appear to be through direct activation like leucine does. If anything, citrulline normalizes suppressed mTOR signalling

For human studies, supplementation of 0.18g/kg citrulline for a week has failed to significantly modify leucine oxidation rates or whole body protein synthesis^[22] but elsewhere at the same dose has been noted to improve nitrogen balance in humans in the fed state.^[12] Urinary nitrogen in the fasted state with oral doses of citrulline between 2-15g does not appear to be acutely affected.^[23]

Mixed evidence as to whether Citrulline supplementation can help with muscle protein synthesis

Citrulline, when supplemented at dosages around 250mg/kg bodyweight, has been implicated in increasing anaerobic performance in rats^[46] and mice.^[58]

Rat studies have noted benefits with 250mg/kg citrulling (human equivalent of 40mg/kg)

In weightlifters given a training protocol (do as many reps as possible until fatigue, for 8 sets), ingestion of 8g citrulline malate alongside 10g sucrose (sucrose also supplied to placebo condition) failed to improve performance on sets 1-2 but progressively increased the amount of reps conducted relative to placebo from 10.48% to 52.92% more reps.^[64]

This study also noted that while 73.17% of the subjects reported nonresponse on set 1, all subjects reported response on set 8.^[64]

Muscle soreness has been noted to be reduced following weight training with 8g citrulline malate by 39.74% (24 hours after) and 41.79% (48 hours after) relative to placebo.^[64]

Citrulline appears to benefit weightlifting secondary to reducing fatigue from a prolonged workout; it does not appear to be too effective on the first few reps conducted

A trial in incline treadmill walking to fatigue with either 3g citrulline acutely or 9g over 24 hours (three doses of 3g) actually noted a reduction in time to exhaustion (895.4s to 888.2s; 0.8%) and increase in the rate of percieved exertion.^[44] The authors also noted that the exercise-induced increase in insulin that occured in placebo failed to occur with citrulline.^[44]

6g of citrulline malate has been found to augment the increases in neutrophil oxidative burst following prolonged cycling exercise, although there did not appear to be oxidative burst damage (MDA or DNA damage) associated with citrulline.^[26]

In trained athletes given 6g citrulline malate prior to a prolonged (137km) cycling test, the exercise-induced increase in growth hormone appeared to be increased; when measured immedaitely after exercise, the citrulline group had 66.8% higher GH concentrations which (after 3 hours of rest) was attenuated to 28%.^[21] Elsewhere, doses of 2-15g citrulline have failed to influence growth hormone when at rest when measured over 8 hours.^[23]

IGF-1 concentrations after 0.18g/kg citrulline for 7 days are not significantly affected.^[22]

Has been noted to increase growth hormone concentrations, but this may require physical exercise as a prerequisite. As such, it may be more correct to say citrulline augments growth hormone release rather than directly induces it

Insulin has been found to be unaltered by 0.18g/kg citrulline during 7 days of supplementation^[22] and during prolonged exercise preloaded with either placebo or citrulline.^[21]

Citrulline is thought to be a pro-erectile agent as it is a precursor for arginine, and arginine is the substrate from which nitric oxide is produced and then can induce cGMP (via the NO/cGMP/VEGF pathway);^[65] an increase in cGMP is also the ultimate effect of PDE5 inhibitors such as viagra or icariin from horny goat weed.^[66]

In men with erectile dysfunction as assessed by weak erections (assessed b yerectile hardness score^[67]) given 1,500mg citrulline daily (two doses of 750mg) for one month was able to benefit half of the 24 patients assessed (as assessed by 'very satisfied' with treatment) while there was merely a 8.3% improvement in placebo.^[68]

Citrulline appears to have pro-erectile properties vicariously through arginine. It is likely more potent than arginine at doing this due to having a greater bodily exposure to arginine, but the preliminary evidence right now does not suggest that it is more potent than the reference drug Viagra

Branched Chain Amino Acids (BCAAs) are a group of three amino acids (leucine, isoleucine, and valine) which are involved with skeletal muscle synthesis and degradation. Of these three, leucine is commonly said to be the most 'important' since it is the prime mediator of muscle protein synthesis (and that appears to be the most common reason people supplement BCAAs).

When looking at citrulline ingestion at rest, 0.18g/kg has been found to improve nitrogen balance in one study when ingested in the fed state^[12] while another noted a failure with the same dose, with both whole body protein synthesis and leucine oxidation rates being unaffected.^[22]

During exercise, 6g citrulline in trained cyclists prior to a 137km cycling test noted that plasma BCAAs were reduced relative to control and attributed to increase utilization of BCAAs for fuel.^[21]

Citrulline does appear to interact with BCAA metabolism in the body, although human studies seem to have differing results based on the context of the study

Similar to how citrulline is able to restore muscle protein synthesis rates^[59][60] and muscular function^[61] during aging and malnourishment in rats via mTORc1 dependent means,^[62][63] citrulline itself is a weak agonist (enough that it could be false positive from sensitizing mTORc1^[59]). Conversely, leucine itself is a potent activator of mTORc1 activity.

Citrulline may positively mediate leucine's signalling through mTOR, which theoretically suggests that they are synergistic. The application of the combination towards weight lifters has not yet been investigated, so the synergism is currently just a hypothesis rather than a demonstrated fact

L-citrulline increases levels of nitric oxide (NO), a vasodilator. Although NO effects are potent, the extent of vasodilation is limited by the short half life of NO, which is broken down by oxidation soon after it is formed.^[69] One study found that taking reduced glutathione (GSH), a reducing agent, alongside L-citrulline may help to protect against the rapid oxidative oxidative destruction of NO. In vitro studies in human umbilical vein endothelial cells demonstrated that L-citrulline (0.3mM) in combination with GSH (1mM) resulted in significantly greater nitrite levels than vehicle-treated controls or L-citrulline alone. Likewise, 3-day treatment of rats with L-citrulline (500mg/kg/day) and GSH (50mg/kg/day) by oral gavage increased plasma nitric oxide levels more than a control or L-citrulline alone. The investigators also conducted a small human trial, where resistance-trained makes were randomly assigned to orally ingest either a placebo, L-citrulline (2g/day), GSH (1g/day), or L-citrulline (2g/day) in combination with GSH (200mg/day) for 7 days, followed by performance of a resistance exercise session. Consistent with in vitro and rat studies, subjects taking GSH along with L-citrulline had significantly higher nitrite and nitric oxide levels 30 minutes after exercise relative to placebo or L-citrulline alone.^[69]

Supplementing with reduced glutathione (GSH) alongside L-citrulline may extend the half life of nitric oxide, potentially resulting in greater vasodilation compared to L-citrulline alone.

Nitrate is a small nitric oxide donor that is the main bioactive of beetroot juice.

Serum nitrite (reduced form of nitrate) appears to be increased during exercise following consumption of 6g citrulline malate, which is thought to be an indicator of increase nitric oxide production.^[21]

Sodium bicarbonate (baking soda) is an ergogenic supplement that is reported to increase physical performance, and although overall it is somewhat unreliable in doing so it appears to be effective for short duration activities characterized by metabolic acidosis ('the burn').

Citrulline administration appears to promote ureogenesis (production of urea) and secondary to this promoted the renal resorption of bicarbonate;^[52] it is thought that citrulline may promote a slight buffering effect via preserving bicarbonate.^[52]

A study assessing synergism between citrulline and bicarbonate supplementation is currently not available.

Theoretically synergistic, but has not been directly investigated at this moment in time

One study in endothelial cells (in vitro) noted that the statin tested, simvastatin, was able to increase mRNA concentrations of eNOS (the enzyme in endothelial cells that produces nitric oxide from arginine) and thus was said to act synergistically with arginine and/or citrulline for improving blood pressure in persons with high cholesterol.^[70]

As simvastatin,^[70] atorvastatin,^[71][72] and lovastatin^[73] (bioactive in red yeast rice) have all been noted to increase eNOS,^[71] iNOS,^[71][73] and nNOS^[72] it is thought that this is a common mechanisms to statin drugs in general.

Statin drugs may be able to increase the expression of the enzyme that mediates conversion of arginine into nitric oxide, and due to this there is possible synergism for anything pertaining to nitric oxide. This has not yet been tested in a living system

Citrulline appears to be well-tolerated by rats in doses up to 3g/kg bodyweight.^[58][46] 

In humans, 15g citrulline taken acutely does not appear to cause diarrhea or intestinal upset^[23] which is notably different than both ornithine and arginine that may cause diarrhea at 10g dosages when taken in boluses^[74][75] due to limited absorption of these amino acids which then proceed to the colon to cause osmotic diarrhea.^[74]