Citrulline functions as an intermediate in the urea cycle and the nitric oxide cycle. It is produced from L-ornithine and carbamoyl phosphate in the urea cycle. Citrulline can be converted to L-arginine, which serves as the substrate for nitric oxide synthase (NOS) to produce nitric oxide. Citrulline also functions as a nitric oxide synthase inhibitor and a protective agent.

L-citrulline is an amino acid that is produced from l-arginine through the arginine-citrulline pathway, ornithine in the degradation of proline, glutamine, and glutamate. As dimethylarginine (ADMA) is broken down, L-citrulline is also produced. This process is aided by dimethylarginine dimethylaminohydrolase (DDAH), which also produces DMA as a byproduct[1]. L-
Citrulline is involved in the nitric oxide cycle and promotes vasodilation. It has antioxidant and anti-inflammatory properties. In vitro, L-citrulline is used in biochemical research to study nitric oxide production pathways and amino acid metabolism. It serves as a precursor to L-arginine, which is crucial for the urea cycle and nitric oxide synthesis.

In vivo, citrulline supplementation can increase energy levels, stimulate the immune system, and help clear ammonia, a cytotoxin. It has been shown to promote vasodilation and improve blood flow. Citrulline is used as a nutraceutical and micronutrient. It has been investigated for its potential benefits in cardiovascular health, erectile dysfunction, and athletic performance.

In vitro assays for citrulline typically measure its conversion to arginine and the production of nitric oxide. Cells (e.g., macrophages or endothelial cells) are incubated with citrulline, and arginine levels are measured by HPLC or LC-MS/MS. Nitric oxide production is measured by the Griess reaction, which detects nitrite, a stable breakdown product of nitric oxide.

Cellular assays for citrulline are performed using cell lines that express nitric oxide synthase (e.g., macrophages, endothelial cells). Cells are treated with citrulline at concentrations ranging from 100 to 1000 μM. Nitric oxide production is measured by the Griess reaction. Arginine uptake and metabolism are assessed using radiolabeled arginine or by measuring amino acid levels in the culture medium.

In vivo animal studies with citrulline are conducted in rodent models of cardiovascular disease, hypertension, and metabolic disorders. Citrulline is administered orally or intraperitoneally at doses ranging from 100 to 1000 mg/kg. Blood pressure, vascular function, and markers of oxidative stress and inflammation are measured. The compound's effects on athletic performance may be assessed in exercise models.

Citrulline is orally available and crosses the blood-brain barrier through the LAT1 transporter. It is metabolized to arginine in the kidney and other tissues. The compound has a short half-life and is primarily excreted as urea and carbon dioxide. For research use, it is typically dissolved in water or saline.

Toxicity Summary
L-citrulline is converted to L-arginine by arginine succinate synthase. L-arginine is key to the therapeutic effects of citrulline. Many activities of L-arginine, including its potential anti-atherosclerotic effects, may be related to its role as a precursor to nitric oxide (NO). NO is produced by all tissues in the body and plays a vital role in the cardiovascular, immune, and nervous systems. NO is generated from L-arginine by NO synthase (NOS), primarily mediated by 3',5'-cyclic guanosine monophosphate (cGMP). NO activates guanylate cyclase, which catalyzes the synthesis of cGMP from guanosine triphosphate (GTP). cGMP is then converted to guanosine monophosphate by cGMP phosphodiesterase. Nitric oxide synthase (NOS) is a heme-containing enzyme with a partial sequence similar to cytochrome P-450 reductase. NOS exists in multiple isoenzymes, two of which are constitutively expressed, and one is induced by immune stimulation. Constitutive NOS present in vascular endothelial cells is called eNOS, while constitutive NOS present in the brain, spinal cord, and peripheral nervous system is called nNOS. NOS induced by immune or inflammatory stimuli is called iNOS. iNOS can be constitutively expressed in certain tissues, such as lung epithelial cells. All nitric oxide synthases use NADPH (reduced nicotinamide adenine dinucleotide phosphate) and oxygen (O₂) as cosubstrates, with cofactors including FAD (flavin adenine dinucleotide), FMN (flavin mononucleotide), tetrahydrobiopterin, and heme. Interestingly, ascorbic acid appears to enhance NOS activity by increasing intracellular tetrahydrobiopterin levels. eNOS and nNOS synthesize NO in response to elevated calcium ion concentrations, or under certain conditions under non-calcium-dependent stimuli (e.g., shear stress). In vitro studies have shown that the Km value of NOS for L-arginine is in the micromolar range. The concentrations of L-arginine in endothelial cells and other cells, as well as in plasma, are in the millimolecular range. This means that under physiological conditions, NOS and its substrate L-arginine are saturated. In other words, L-arginine is not expected to be the rate-limiting step of this enzyme, and oral supplementation of this amino acid may lead to excessively high L-arginine concentrations, but this does not appear to have any effect on NO production. The reaction appears to have reached its maximum activity. However, in vivo studies have shown that under certain conditions, such as hypercholesterolemia, L-arginine can enhance endothelium-dependent vasodilation and NO production.
Citrulline is generally well tolerated with a favorable safety profile. It is a naturally occurring amino acid and is not associated with significant toxicity at therapeutic doses. Side effects are mild and include gastrointestinal discomfort. No significant organ toxicity has been reported.

[1]. Quantitative Analysis of l-Arginine, Dimethylated Arginine Derivatives, l-Citrulline, and Dimethylamine in Human Serum Using Liquid Chromatography-Mass Spectrometric Method. Chromatographia. 2018;81(6):911-921.

Pharmacodynamics
Citrulline is a non-essential amino acid and a precursor to arginine. It is claimed that citrulline supplementation can increase energy levels, stimulate the immune system, and help clear ammonia (a cytotoxin). L-Citrine is produced from L-ornithine and carbamoyl phosphate in one of the core reactions of the urea cycle. It is also a byproduct of the reaction produced from L-arginine in a NO synthase-catalyzed reaction. Although L-citrulline is an amino acid, it is not involved in protein synthesis and is not one of the amino acids encoded by DNA. While citrulline cannot be integrated into proteins during protein synthesis, some proteins are known to contain citrulline as an amino acid. These citrulline residues are produced by a class of enzymes called peptidyl arginine deiminases (PADs), which convert the amino acid arginine to citrulline. Proteins containing citrulline residues include myelin basic protein (MBP), filaggrin, and several histones.
Citrulline is a non-essential amino acid used as a dietary supplement for cardiovascular health, erectile dysfunction, and athletic performance. It is also used in biochemical research to study nitric oxide production and amino acid metabolism. The compound is available over-the-counter as a nutritional supplement.

C6H13N3O3

175.188

175.095

372-75-8

9750

White to off-white solid powder

1.3±0.1 g/cm3

386.7±42.0 °C at 760 mmHg

214 °C

187.7±27.9 °C

0.0±1.9 mmHg at 25°C

1.531

-1.53

4

4

5

12

171

1

C(C[C@@H](C(=O)O)N)CNC(=O)N

RHGKLRLOHDJJDR-BYPYZUCNSA-N

InChI=1S/C6H13N3O3/c7-4(5(10)11)2-1-3-9-6(8)12/h4H,1-3,7H2,(H,10,11)(H3,8,9,12)/t4-/m0/s1

(2S)-2-amino-5-(carbamoylamino)pentanoic acid

L-Cytrulline; L-Citrulline; Citrulline

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ≥ 50 mg/mL (~285.40 mM)

Solubility in Formulation 1: 100 mg/mL (570.81 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)