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http://vmj.sagepub.com/content/4/1/27The online version of this article can be found at:

 DOI: 10.1177/1358836X9900400105

1999 4: 27Vasc Medand Terrence F Blaschke

Oranee Tangphao, Stephan Chalon, Ann M Coulston, Heitor Moreno, Jr, Jason R Chan, John P Cooke, Brian B Hoffmanin a 24-h crossover study

L-arginine and nitric oxide-related compounds in plasma: comparison of normal and arginine-free diets  

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Vascular Medicine1999;4: 27–32

l-arginine and nitric oxide-related compounds in plasma:comparison of normal and arginine-free diets in a 24-hcrossover studyOranee Tangphaoa, Stephan Chalona, Ann M Coulstonb, Heitor Moreno Jra, Jason R Chanc, John P Cookec, Brian B Hoffmand and

Terrence F Blaschkea

Abstract: The amino acid l-arginine is the precursor of nitric oxide (NO), a powerful vasodilatorwith antiplatelet properties. The availability of l-arginine has been suggested to be a rate-limit-ing factor in the production of NO in conditions such as hypercholesterolemia. It was speculatedthat fluctuations in plasma concentrations of l-arginine during the day may be dependent upondietary intake of the amino acid, or other variables, and might modify the elaboration ofendogenous NO. Over a 24-h period, the plasma concentrations of l-arginine and NO-relatedcompounds (NOx) were measured during an l-arginine and nitrate/nitrite-free diet (diet A) ora nitrate/nitrite-free diet with a fixed amount of l-arginine intake (3.8 g/d) (diet B) in eight healthyvolunteers during a 2-day crossover study. Subjects were randomly selected to begin with dietA or diet B and consumed the other diet on the second day. During diet A, plasma l-argininedecreased significantly from 09.00 to 16.00 (21.4 ± 2.0 to 11.9 ± 1.1 mg/ml ), rose slightly in theevening (to 16.6 ± 1.7mg/ml) and gradually increased during the night. During diet B, plasma l-arginine showed a peak after each meal (approximately 23 mg/ml). Plasma NOx concentrationsmeasured by chemiluminescence did not show any circadian variation on either diet. Plasmal-arginine concentrations change during the day and are influenced by dietary intake.Importantly, plasma NOx do not seem to vary with this pattern in healthy individuals.

Key words: chemiluminescence; l-arginine; nitrate/blood; nitric oxide/blood

Introduction

l-arginine is considered to be a nutritionally dispensable(or non-essential) amino acid in humans.1 l-arginine is thesubstrate for the endothelial nitric oxide (NO) synthase(eNOS), which metabolizes this amino acid tol-citrullineand NO, a powerful vasodilator with antiplatelet proper-ties.2 While the impaired availability of NO in endotheliumand platelets has been associated with cardiovascular riskfactors and with aging,3 experimental4–10 and clinical stud-ies11–16have shown that the attenuation of vascular and pla-telet NO activity can be reversed in some of these con-ditions by administration ofl-arginine.

In humans, maintenance of plasmal-arginine is mainlydependent on the dietary intake ofl-arginine and its syn-thesis by the kidney.17 Some studies11,18suggest thatl-argi-nine therapy is associated with an increase in surrogatemarkers of NO production, such as plasma nitrates andexhaled NO. However, the precise relationship betweenplasma concentration ofl-arginine and NO production isnot fully understood. Since circadian patterns have beendescribed for several phenomena occurring in the cardio-vascular system, including regulation of vascular tone and

aDivision of Clinical Pharmacology,bGeneral Clinical Research Centerand cDivision of Cardiovascular Medicine, Stanford University MedicalCenter, Stanford, CA, anddGeriatric Research, Education and ClinicalCenter, Palo Alto VA Health Care System, Palo Alto, CA, USA

Address for correspondence: Terrence F Blaschke, Division of ClinicalPharmacology, Stanford University School of Medicine, S-009, Stanford,CA 94305-5130, USA.

Arnold 1999 1358-863X(99)VM260OA

platelet aggregation,19–21physiological variations of plasmal-arginine concentrations could influence endothelial NOproduction and thus modify vascular tone and platelet func-tion. Therefore, concomitant determination of changes inplasmal-arginine and plasma markers of NO productionover 24 h would provide interesting information to explorethis possibility.

This study was designed to determine the physiologicvariations of plasma concentrations ofl-arginine and ofNO-related compounds (NOx) over the course of 24 h andto evaluate the impact of the dietary intake ofl-arginineon these parameters. Plasma NOx and plasma concen-trations ofl-arginine were simultaneously measured at reg-ular intervals to assess a possible relationship between anin vivo index of NO production and the plasma concen-tration of its precursor.

Subjects and methods

SubjectsEight healthy volunteers (four males) were enrolled in thestudy. Ages ranged from 23 to 59 years (with an averageof 43 ± 12 , mean± SD). Mean weight was 74± 21 kg(mean± SD) and average height was 171± 9 cm(mean± SD). Body mass index (BMI) was 25± 7 kg/m2

(mean± SD) and total cholesterol was 190± 22 mg/dl(mean± SD). Exclusion criteria included any significantdisease state, or chronic use of alcohol or any medication.All subjects were non-smokers. A complete physical exam-ination and routine laboratory tests (SMA-20, CBC and

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28 O Tangphao et al

urinalysis) were performed. Subjects signed a written con-sent approved by the Stanford Administrative Panel onHuman Subjects in Medical Research (Stanford, CA, USA).

Study designSubjects were admitted to the General Clinical ResearchCenter at Stanford University Medical Center (GCRC) inthe morning after overnight fasting. Studies began at 09.00and were 48 h in length. An intravenous catheter wasinserted in one arm for blood collection throughout thestudy. The study was designed as a crossover study. Theorder of diet (A or B) during the 2 days was randomlyassigned. On both days, total caloric intake was 2000Kcal/day for females and 2500 Kcal/day for males. Allcooking and drinking water was double distilled in orderto minimize the NOx content from water (nitrate concen-tration can be as high as 10 mg/l in ordinary water). Drink-ing water was limited to 1000 ml/day. The amount ofnitrate in double distilled water in the laboratory was lessthan 0.1 mg/l or 100 ppb. Caffeine and any medicationswere prohibited during the study. None of the participantsexercised regularly. Physical exercise was prohibited 48 hbefore and during the study. Subjects were allowed to walkin the GCRC but were asked to stay in a supine positionfor 30 min before blood was drawn. Blood samples weredrawn via the intravenous catheter at 15 to 60 min intervalsfrom 08.00 to 24.00 and at 2- to 4-h intervals from 24.00 to08.00. Diet A was made from a combination of Microlipid

(Sherwood Medical, St Louis, MO, USA) emulsion andPolycose (Ross Products Division, Abbott Labs, Col-umbus, OH, USA). Diet A contained nol-arginine and wasvery low in nitrate and nitrite content. Subjects receivedthe liquid diet in seven equal portions throughout the dayfrom 09.00 until 22.00. Diet B was a normal food dietdesigned to provide 3.8 g ofl-arginine distributed betweenthree meals (according to USDA-ARS 1996). The amountsof l-arginine in the breakfast, lunch and dinner wereapproximately 1.0 g, 1.7 g and 1.1 g, respectively. Diet Balso contained minimal amounts of nitrate and nitrite. Mealtimes for diet B were 09.00 (after the first blood draw),13.00 and 18.00. All volunteers tolerated both diets well.

Analytic methodsPlasma samples were analyzed forl-arginine concen-trations using high performance liquid chromatography(HPLC) as developed in the authors’ laboratory.22 The coef-ficient of variation of this assay was less than 2%. The sameplasma samples were also analyzed for NOx by chemilumi-nescence as previously described.23,24 Briefly, NO gener-ated in vivo is converted into nitrite/nitrate, nitrosyl hemo-proteins, nitrosyl metal complexes andS-nitrosocompounds in the circulation. The assay method consistsof the thermolysis of all these NO-related compounds andthe detection of resulting nitrate after enzymatic reduction.

Data analysisData are expressed as mean± SEM except where statedotherwise. Average plasma concentrations ofl-arginineand NOx in each group were expressed as area under thecurve (AUC) between plasma concentrations ofl-arginineor NOx over 24 h. In order to assess diurnal variation, AUCvalues for plasma concentration versus time were dividedinto eight 3-h-intervals beginning from midnight. AUC 24-

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h and 3-h values were compared between the two dietsusing the pairedt-test. The differences between diet A anddiet B on plasmal-arginine and plasma NOx concen-trations at each time point were also tested using the pairedt-test. Statistical significance was defined asp , 0.05.

Results

Mean plasmal-arginine concentrations are shown in Figure1. At the beginning of the day, the average plasmal-argi-nine concentrations were 21.4± 2.0 and 20.6± 2.5 mg/mlfor diet A (no arginine) and diet B (normal arginine),respectively (NS). With diet A administration, plasmal-arginine concentrations decreased significantly from 09.00to 16.00 (21.4± 2.0 to 11.9± 1.1 mg/ml) and slightlyincreased in the evening and overnight. Plasmal-arginineconcentrations at 24 h were not significantly different fromplasmal-arginine concentrations at the beginning of theprevious day for diet A (21.4± 2.0 versus18.5± 1.9mg/ml).

With diet B, plasmal-arginine concentrations were sus-tained during the day and showed three peaks, one aftereach meal. Each peak occurred approximately 3 h aftermeal times as shown in Figure 1. Mean peakl-arginineconcentrations were 24.4± 3.1, 22.2± 3.1 and 22.3± 2.4mg/ml for breakfast, lunch and dinner, respectively. Afterthe peak following the evening meal, plasmal-arginineconcentrations gradually decreased below morning baselinevalues until a plateau was reached at 04.00. Plasmal-argi-nine concentrations after 24 h appeared slightly lower com-pared to the previous day but no statistical difference wasdetected (20.6± 2.5 versus 15.4± 1.2mg/ml). Plasmal-arginine concentrations during diet A and diet B were sig-nificantly different at several time points between 10.30 and22.00 as indicated in Figure 1.

The differences in plasmal-arginine concentrationsexpressed as AUC values between diet A and diet B weresignificant for the following intervals: 09.00 to 12.00 noon,12.00 noon to 15.00 and 15.00 to 18.00, as indicated inFigure 2.

No significant fluctuations in plasma NOx concentrationsover 24 h (expressed as AUC values) were detected foreither diet A or diet B (Figure 3).

The 24 h profiles for the plasma concentrations ofl-argi-nine and NOx were not influenced by the sequence in whichthe diets were administered.

Discussion

This study demonstrates that removal ofl-arginine fromthe diet for one day in healthy individuals causes a signifi-cant decrease in plasmal-arginine concentrations duringthe awake period followed by a spontaneous return to nor-mal morning basal concentrations overnight. In the samesubjects, a normal amount ofl-arginine in the diet (3.8 g/d)was associated with a rise in plasmal-arginine concen-tration after each meal. Importantly, none of these two con-trolled diets influenced plasma NOx concentrations, an invivo index of NO biosynthesis.

Plasmal-arginine changes reflect the balance between

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Plasmal-arginine and NO-related compounds in humans29

Figure 1 Average plasma l-arginine concentrations over each 24-h study period. Subjects received a nitrate/nitrite and l-arginine-free diet (diet A) (closed circles) or a nitrate/nitrite-free diet with usual dietary amounts of l-arginine (diet B) (open circles). Ateach time point, the differences in plasma l-arginine concentrations from the same subjects were tested between diet A and dietB and significant differences were detected as indicated. * indicates statistical significance of p , 0.05; # indicates statisticalsignificance of p , 0.01. Error bars represent the SEM at each time point. Arrows represent meal times for diet B.

Figure 2 Average area under the curve between plasma concentrations of l-arginine versus time in eight intervals during the24-h study with diet A (white bars) or diet B (hatched bars). The differences in AUC values from the same subjects during diet Aand diet B were significant during 09.00 to 12.00 noon, 12.00 noon to 15.00 and 15.00 to 18.00, as indicated by * (p , 0.05 bypaired t-test). Error bars represent the SEM at each time point.

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30 O Tangphao et al

Figure 3 Average plasma NOx measured by chemiluminescence over 24 h with diet A (closed circles) or diet B (open circles).Error bars represent the SEM at each time point. Arrows represent meal times for diet B. There was no variation during the dayobserved for either diet A or diet B.

complex inter-organ processes leading to movement of theamino acid into and out of the circulation. Endogenous syn-thesis of arginine occurs primarily in the kidney and to alesser extent in the liver via conversion of citrulline to argi-nine.17 However, the liver does not contribute significantlyto the maintenance of the plasma concentrations ofl-argi-nine, since the amino acid synthesized in this organ isrouted towards its local utilization.17 The mean dietaryintake of l-arginine in industrialized countries is 3–6 g/day;1 60% of this exogenous source appears in the gen-eral circulation.17 Isotopic studies have shown that the netrate of de novo arginine synthesis in healthy humans is notaffected by a 6–7 day arginine-free diet.25,26 Consequently,it has been proposed that whole-body arginine homeostasisin healthy adults may be achieved principally via a modu-lation in the level of dietary arginine intake and/or withregulation in the rate of its catabolism to ornithine and glut-amate.25 In this 24-h study, thel-arginine-free diet wasassociated with a gradual decrease in plasma concen-tration – reaching 47% of the baseline value after 7 h. Com-parison with the normal diet also demonstrated a significantdecrease in the 3-h AUC intervals from 09.00 to 18.00.This phenomenon was followed by a return towards normalplasmal-arginine concentrations during the night. Our 24-h observation period complements previous findings fromarginine kinetic studies carried out after 6 days of a con-trolled diet.25,26 Information regarding plasmal-arginineconcentrations provided by these tracer studies is limitedbut suggests that fasting morning plasma concentrations ofthe amino acid (measured by an HPLC method) after a 6-day arginine-free diet do not differ with an arginine-enriched diet and that the post-meal value spontaneouslydecreases during the day whenl-arginine is removed from

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the diet. Further studies are needed to determine the precisemechanism(s) responsible for this 24-h profile of plasmal-arginine concentrations during anl-arginine-free diet. Theauthors also observed three peaks following each meal dur-ing the normal diet; this phenomenon confirms that theintestinal absorption ofl-arginine has a relevant effect onits plasma concentration. This observation suggests that theeffect of food intake should be considered in pharmacoki-netic studies designed to evaluate plasma concentrations ofl-arginine after oral supplementation.

l-arginine is the substrate for endothelial NO synthesis,a reaction that is catalyzed by the constitutive endothelialenzyme eNOS.2 NO plays a key role in the regulation ofvascular tone and platelet aggregation and adhesion.2,3

Changes due to hypercholesterolemia and aging, two con-ditions associated with impairment of thel-arginine/NOpathway, have shown benefit froml-arginine supplementa-tion in humans.11–16 l-arginine supplementation (8–21 g/day by the oral route) improved endothelial dysfunc-tion and was associated with a modest increase in surrogatemarkers of NO in patients with hypercholesterolemia.11

Recent studies have shown that NO can be detected inplasma in several active or inactive forms appearing inpools of different sizes.27–29 Among these plasma indicesof NO production, plasma NOx measured by chemilumi-nescence appears to include the major metabolites of NOin the human body.23 This surrogate marker has been suc-cessfully used by other investigators to demonstrate sig-nificant changes in NO production in humans.24

The authors hypothesized that physiological variations ofplasmal-arginine concentrations either induced by a nor-mal or by an arginine-free diet could influence endothelialNO production and consequently plasma NOx concen-

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Plasmal-arginine and NO-related compounds in humans31

trations. Since the nitrate present in the blood may derivefrom dietary sources such as nitrate- or nitrite-containingfood or drinking water,30 a nitrate/nitrite-restricted diet wasmaintained during this 48 h study. The plasma concen-trations of NOx measured in the study by a chemilumi-nescence assay23,24 did not change in response either to a24-h arginine-free diet or to an increase in plasma concen-trations of l-arginine following the meals in the normaldiet. Also, a study in healthy subjects has shown that sup-plemental (56 mg/kg per day)l-arginine does not influenceplasma nitrate concentrations or the rate of conversion ofplasma arginine to urinary nitrate.31 This lack of influenceof a normal or an arginine-free diet on whole-body NOproduction in healthy humans suggests that exogenousl-arginine plays a very limited role as a precursor of NO inhealthy humans on a short-term basis. In support of thisconclusion it has been reported that only 4% of endogenousarginine is used for NO synthesis.26 Although NO pro-duction may not be influenced by physiological variationsof l-arginine in healthy subjects, this does not rule out apossible effect of supplementaryl-arginine on NO metab-olism in pathophysiological situations. In a previous studyby the present group, evaluating the effects of orall-argi-nine supplementation (14–21 g/day) over 12 weeks on theplasma concentrations and the pharmacokinetics ofl-argi-nine in hypercholesterolemic patients, average plasma con-centrations rose from 16 to 22mg/ml.32 Although plasmaNOx were not measured in this particular study, sup-plementall-arginine in hypercholesterolemic patients hasbeen found to be associated with a modest rise innitrosoprotein levels, another index of NO production.11

Further studies are needed to assess whether oral supple-mentation of this magnitude is also associated withincreased plasma nitrate concentrations in these patients orin normals. Because of the short duration of thel-arginine-free diet in this study (24 h), it is conceivable that plasmaNOx levels might have fallen below that seen in the normaldiet if the arginine-free diet had been maintained longer.However, the rapid turnover ofl-arginine and nitric oxidemakes this less likely. The possibility that physiologicalfluctuations of plasmal-arginine concentrations over 24 hmay influence endothelial NO generation and thus vasculartone in hypercholesterolemic but not in normal subjectsalso remains to be explored.

In conclusion, plasma concentrations ofl-arginine arequickly influenced by dietaryl-arginine. Fluctuations ofplasmal-arginine during normal andl-arginine-free dietsappear to be insufficient to influence NOx concentrationsover 24 h . These results suggest that in healthy individualsat rest, plasmal-arginine concentrations are not rate-limit-ing in the formation of NO.

Acknowledgements

Funding for this project was provided by NIH grant AG-05627, an American Heart Association Grant-in-Aid (JRC),NIH grants HL 08506 and HL 58683, and GCRC grant RR-00070. Dr Tangphao was supported by AnanthamahidolFoundation, Bangkok, Thailand. Drs Moreno and Chalonwere supported by Merck Sharp and Dohme InternationalFellowships in Clinical Pharmacology. Dr Cooke is thefounder of CookePharma (Mountain View, CA, USA), but

Vascular Medicine1999;4: 27–32

no support for this study was provided by CookePharma.The authors gratefully acknowledge the technical assistanceof GCRC nursing and dietary staffs. The authors also thankDr Bradley Efron for assistance with the statistical analysis.

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