Assessment of Nitric Oxide Production by Measurement of [15N]Citrulline Enrichment in Human Plasma Using High-Performance Liquid Chromatography–Mass Spectrometry

doi:10.1006/abio.1997.2515

Analytical Biochemistry

Volume 257, Issue 1, 1 March 1998, Pages 45-52

https://doi.org/10.1006/abio.1997.2515 Get rights and content

Abstract

Nitric oxide (NO) is formed by a class of NO synthases (NOS), which convert arginine into citrulline. A decreasedin vivoNO availability can be the result of an increased NO inactivation or a decreased NO production. The latter can be assessed by measurement of isotopic enrichment of plasma citrulline during infusion of isotopically labeled arginine. The potential of high-performance liquid chromatography (HPLC) coupled to mass spectrometry (MS) to determine enrichments of [¹⁵N₂]arginine and [¹⁵N]citrulline in plasma during infusion of [¹⁵N₂]arginine in humans was investigated. Two types of MS instruments were evaluated: a sector-type mass spectrometer equipped with a frit fast-atom bombardment (FAB) interface and a quadrupole instrument with electrospray ionization (ESI). FAB–MS appeared to be unsuitable for determination of isotope ratios, because background ions influenced the observed isotope ratio in an unpredictable way. In combination with either off- or on-line reversed-phase HPLC, ESI–MS proved to be a more reliable technique. However, the amount of material that is introduced in the mass spectrometer is critical and should be carefully controlled. During infusion of [¹⁵N₂]arginine in 14 healthy subjects, a mean arginine-to-citrulline conversion rate of 0.22 ± 0.07 (SD) μmol.kg⁻¹.h⁻¹was found. In 4 subjects who received an intravenous infusion with the NOS antagonistl-NMMA, the conversion rate decreased from 0.30 ± 0.14 to 0.10 ± 0.06 μmol.kg⁻¹.h⁻¹. It is concluded that ESI–MS in combination with HPLC can be successfully applied for determination of arginine and citrulline enrichments in plasma, thus providing a useful tool for assessment ofin vivoNO production.

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Cited by (23)

Plasma L-citrulline concentrations in L-arginine–supplemented healthy dogs
2017, Journal of Veterinary Cardiology
To determine whether oral l-arginine increases plasma [l-citrulline] in dogs.
Eleven healthy staff-owned dogs were used in this study.
Dogs (n = 3) were given l-arginine (50mg/kg PO q8h) for 7 days, and plasma [l-arginine] and [l-citrulline] were analyzed by high performance liquid chromatography at baseline (BL), steady state trough, and 0.5, 1, 1.5, 2, 4, 6, and 8 h after final dosing on day 7. Eleven dogs were then treated with 100mg/kg l-arginine PO q8h for 7 days, and [l-arginine] and [l-citrulline] were measured at BL, steady state trough, and at peak 4 hrs after dosing (T4 hrs).
– Plasma [l-arginine] and [l-citrulline] peaked at T4 hrs on the 50mg/kg dosage. Target outcome, modeled after human study results, of a doubling of [l-arginine] and a 25–30% increase in [l-citrulline] from BL were not reached. After the 100mg/kg dosage, plasma [l-arginine] increased from a BL median of 160.1 μM (range, 100.2–231.4 μM) to a peak of 417.4 μM (206.5–807.3 μM) at T4 hrs, and plasma [l-citrulline] increased from a BL median of 87.8 μM (59.1–117.1 μM) to peak of 102.2 μM (47.4–192.6 μM) at T4 hrs. Ten of eleven dogs showed a doubling of plasma [l-arginine] and 4/11 dogs achieved 25–30% or greater increases in plasma [l-citrulline]. No adverse effects on heart rate or blood pressure were noted.
– Oral l-arginine dosage of 100mg/kg q8h doubles plasma [l-arginine] in healthy dogs, but conversion to l-citrulline is quite variable. Further evaluation of this dosage regimen in dogs with pulmonary hypertension is warranted.
Measurement of in vivo nitric oxide synthesis in humans using stable isotopic methods: A systematic review
2011, Free Radical Biology and Medicine
Citation Excerpt :
In the following years, new methods were developed using different tracers (arginine, oxygen) [40–42] and different routes of administration (intravenous, intragastric, oral, inhalation) [40–43] and measuring different labeled species (citrulline, nitrate, nitrite) [43], as well as sampling from different accessible pools (plasma, urine) [40,41,44]. Seven studies used an arginine–citrulline protocol [38,40,45–49], 27 studies utilized an arginine–nitrate protocol [37,39–41,43,44,50–70], and only one study utilized the oxygen–nitrate protocol [42]. The intravenous primed-constant infusion was the most utilized administration route for the tracers [36,40–50,56,59,60,63,65–68], whereas a bolus intravenous administration of labeled l-arginine was employed in a few protocols [41,55,56,58,59,65,66].
Stable isotopic methods are considered the “gold standard” for the measurement of rates of in vivo NO production. However, values reported for healthy human individuals differ by more than 1 order of magnitude. The reason for the apparent variability in NO production is unclear. The primary aim of this review was to evaluate and compare the rates of in vivo NO production in health and disease using stable isotope methods. Articles were retrieved using the PubMed electronic database. Information on concentrations, isotopic enrichments of fluxes, and conversion rates of molecules involved in the NO metabolic pathway was extracted from selected articles; 35 articles were included in the final analysis. Three protocols were identified, including the arginine–citrulline, the arginine–nitrate, and the oxygen–nitrate protocols. The arginine–citrulline protocol showed a wider variability compared to the arginine–nitrate and oxygen–nitrate protocols. The direction of the association between disease state and rate of NO production was essentially determined by the etiopathogenesis of the disorder (inflammatory, metabolic, vascular). Considerable variation in methodologies used to assess whole-body NO synthesis in humans exists. The precision of several aspects of the techniques and the validity of some assumptions made remain unknown, and there is a paucity of information about physiological rates of NO production from childhood over adolescence to old age.
Simultaneous determination of l-arginine and 12 molecules participating in its metabolic cycle by gradient RP-HPLC method. Application to human urine samples
2007, Analytica Chimica Acta
We have developed and described a highly sensitive, accurate and precise reversed-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous determination of l-arginine and 12 molecules participating in its metabolic cycle in human urine samples. After pre-column derivatization with ortho-phthaldialdehyde (OPA) reagent containing 3-mercaptopropionic acid (3MPA), the fluorescent derivatives were separated by a gradient elution and detected by fluorescence measurement at 338 nm (excitation) and 455 nm (emission). l-Arginine (ARG) and its metabolites: l-glutamine (GLN), N^G-hydroxy-l-arginine (NOHA), l-citrulline (CIT), N^G-monomethyl-l-arginine (NMMA), l-homoarginine (HARG), asymmetric N^G,N^G-dimethyl-l-arginine (ADMA), symmetric N^G,N^G′-dimethyl-l-arginine (SDMA), l-ornithine (ORN), putrescine (PUT), agmatine (AGM), spermidine (SPERMD) and spermine (SPERM) were extracted in a cation-exchange solid-phase extraction (SPE) column and after derivatization separated in a Purospher^® STAR RP-18e analytical column. The calibration curves of analysed compounds are linear within the range of concentration: 45–825, 0.2–15, 16–225, 12–285, 0.1–32, 15–235, 0.1–12, 0.1–12, 10–205, 0.02–12, 0.1–24, 0.01–10 and 0.01–8 nmol mL⁻¹ for GLN, NOHA, CIT, ARG, NMMA, HARG, ADMA, SDMA, ORN, PUT, AGM, SPERMD and SPERM, respectively. The correlation coefficients are greater than 0.9980. Coefficients of variation are not higher than 6.0% for inter-day precision. The method has been determined or tested for limits of detection and quantification, linearity, precision, accuracy and recovery. All detection parameters of the method demonstrate that it is a reliable and efficient means of the comprehensive determination of ARG and its 12 main metabolites, making this approach suitable for routine clinical applications. The levels of analysed compounds in human urine can be successfully determined using this developed method with no matrix effect.
Inaccuracies in selected ion monitoring determination of isotope ratios obviated by profile acquisition: nucleotide <sup>18</sup>O/<sup>16</sup>O measurements
2007, Analytical Biochemistry
Citation Excerpt :
The ability to detect such low enrichment may be extremely valuable for measuring the incorporation of isotopically enriched amino acids into proteins [31–33]. Overall, the results presented in Table 4 compare favorably with similar work for derivatized amino acids [34–36]. These studies used ESI with either ion trap or QMS and fast atom bombardment ionization-QMS.
Precise and accurate measurements of isotopologue distributions (IDs) in biological molecules are needed for determination of isotope effects, quantitation by isotope dilution, and quantification of isotope tracers employed in both metabolic and biophysical studies.While single ion monitoring (SIM) yields significantly greater sensitivity and signal/noise than profile-mode acquisitions, we show that small changes in the SIM window width and/or center can alter experimentally determined isotope ratios by up to 5%, resulting in significant inaccuracies. This inaccuracy is attributed to mass granularity, the differential distribution of digital data points across the m/z ranges sampled by SIM. Acquiring data in the profile mode and fitting the data to an equation describing a series of equally spaced and identically shaped peaks eliminates the inaccuracies associated with mass granularity with minimal loss of precision. Additionally a method of using the complete ID profile data that inherently corrects for “spillover” and for the natural-abundance ID has been used to determine ¹⁸O/¹⁶O ratios for 5′,3′-guanosine bis-[¹⁸O₁]phosphate and TM[¹⁸O₁]P with precisions of ∼0.005. The analysis protocol is also applied to quadrupole time-of-flight tandem mass spectrometry using [2-¹⁸O] arabinouridine and 3′-UM[¹⁸O₁]P which enhances signal/noise and minimizes concerns for background contamination.
Methods using stable isotopes to measure nitric oxide (NO) synthesis in the l-arginine/NO pathway in health and disease
2007, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Nitric oxide (NO) is an important gaseous radical involved in many physiological processes. It is produced from the amino acid l-arginine by the action of nitric oxide synthases (NOS) in what is called the l-arginine/NO pathway. Tracking its metabolic fate in biological fluids is of particular interest as it may indicate how the human body responds in health and disease. However, due to its short life span (a few seconds) it is very difficult to accurately monitor any up- or down-regulation in body fluids in vivo. As a consequence, methods have been developed based on the measurement of the NO-derived products nitrite and nitrate or on the substrate of NO, l-arginine and its simultaneously generated product, l-citrulline. Considering only a fraction of the endogenous l-arginine pool is used for the synthesis of NO, NO-production cannot be estimated by measuring changes in the concentrations of l-arginine and/or l-citrulline alone. Instead, to estimate NO-related changes in the l-arginine and/or l-citrulline pools a form of tagging these metabolites for the NOS-mediated reaction is required. The application of stable isotopes is an elegant way to track NOS-mediated changes. The present paper is focussed on the application of various combinations of chromatography and mass spectrometry to measure isotopic enrichments resulting from the conversion of l-arginine to NO and l-citrulline in a one-to-one stoichiometry. In addition, the various aspects and principles involved in the application of stable isotopes in metabolic studies in general and the study of the activity of NOS in particular are discussed.
L-arginine-nitric oxide pathway in endstage reneal disease: Authors' reply
2002, American Journal of Kidney Diseases

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^☆: Waller, G. R.Dermer, O. C.

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Analytical Biochemistry

Regular ArticleAssessment of Nitric Oxide Production by Measurement of [15N]Citrulline Enrichment in Human Plasma Using High-Performance Liquid Chromatography–Mass Spectrometry☆

Abstract

Br. J. Anaesth.

Kidney Int.

Lancet

J. Chromatogr.

J. Chromatogr. (Biomed. Appl.)

Anal. Biochem.

Metabolism

Metabolism

J. Chromatogr. A

J. Am. Soc. Mass Spectrom.

Trends Anal. Chem.

Anal. Biochem.

Nature

J. Clin. Invest.

Proc. Natl. Acad. Sci. USA

Plasma L-citrulline concentrations in L-arginine–supplemented healthy dogs

Measurement of in vivo nitric oxide synthesis in humans using stable isotopic methods: A systematic review

Simultaneous determination of l-arginine and 12 molecules participating in its metabolic cycle by gradient RP-HPLC method. Application to human urine samples

Inaccuracies in selected ion monitoring determination of isotope ratios obviated by profile acquisition: nucleotide <sup>18</sup>O/<sup>16</sup>O measurements

Methods using stable isotopes to measure nitric oxide (NO) synthesis in the l-arginine/NO pathway in health and disease

L-arginine-nitric oxide pathway in endstage reneal disease: Authors' reply

Regular Article
Assessment of Nitric Oxide Production by Measurement of [¹⁵N]Citrulline Enrichment in Human Plasma Using High-Performance Liquid Chromatography–Mass Spectrometry☆