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l-arginine and nitric oxide-related compounds in plasma: comparison of normal and arginine-free diets in a 24-h crossover study Oranee 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 vasodilator with antiplatelet properties. The availability of l-arginine has been suggested to be a rate-limiting factor in the production of NO in conditions such as hypercholesterolemia. It was speculated that fluctuations in plasma concentrations of l-arginine during the day may be dependent upon dietary intake of the amino acid, or other variables, and might modify the elaboration of endogenous NO. Over a 24-h period, the plasma concentrations of l-arginine and NO-related compounds (NOx) were measured during an l-arginine and nitrate/nitrite-free diet (diet A) or a nitrate/nitrite-free diet with a fixed amount of l-arginine intake (3.8 g/d) (diet B) in eight healthy volunteers during a 2-day crossover study. Subjects were randomly selected to begin with diet A or diet B and consumed the other diet on the second day. During diet A, plasma l-arginine decreased significantly from 09.00 to 16.00 (21.4 ± 2.0 to 11.9 ± 1.1 ␮g/ml ), rose slightly in the evening (to 16.6 ± 1.7␮g/ml) and gradually increased during the night. During diet B, plasma larginine showed a peak after each meal (approximately 23 ␮g/ml). Plasma NOx concentrations measured by chemiluminescence did not show any circadian variation on either diet. Plasma l-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 the substrate for the endothelial nitric oxide (NO) synthase (eNOS), which metabolizes this amino acid to l-citrulline and NO, a powerful vasodilator with antiplatelet properties.2 While the impaired availability of NO in endothelium and platelets has been associated with cardiovascular risk factors and with aging,3 experimental4–10 and clinical studies11–16 have shown that the attenuation of vascular and platelet NO activity can be reversed in some of these conditions by administration of l-arginine. In humans, maintenance of plasma l-arginine is mainly dependent on the dietary intake of l-arginine and its synthesis by the kidney.17 Some studies11,18 suggest that l-arginine therapy is associated with an increase in surrogate markers of NO production, such as plasma nitrates and exhaled NO. However, the precise relationship between plasma concentration of l-arginine and NO production is not fully understood. Since circadian patterns have been described for several phenomena occurring in the cardiovascular system, including regulation of vascular tone and a

Division of Clinical Pharmacology, bGeneral Clinical Research Center and cDivision of Cardiovascular Medicine, Stanford University Medical Center, Stanford, CA, and dGeriatric Research, Education and Clinical Center, Palo Alto VA Health Care System, Palo Alto, CA, USA Address for correspondence: Terrence F Blaschke, Division of Clinical Pharmacology, Stanford University School of Medicine, S-009, Stanford, CA 94305-5130, USA.

 Arnold 1999

platelet aggregation,19–21 physiological variations of plasma l-arginine concentrations could influence endothelial NO production and thus modify vascular tone and platelet function. Therefore, concomitant determination of changes in plasma l-arginine and plasma markers of NO production over 24 h would provide interesting information to explore this possibility. This study was designed to determine the physiologic variations of plasma concentrations of l-arginine and of NO-related compounds (NOx) over the course of 24 h and to evaluate the impact of the dietary intake of l-arginine on these parameters. Plasma NOx and plasma concentrations of l-arginine were simultaneously measured at regular intervals to assess a possible relationship between an in vivo index of NO production and the plasma concentration of its precursor.

Subjects and methods Subjects Eight healthy volunteers (four males) were enrolled in the study. Ages ranged from 23 to 59 years (with an average of 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 significant disease state, or chronic use of alcohol or any medication. All subjects were non-smokers. A complete physical examination and routine laboratory tests (SMA-20, CBC and 1358-863X(99)VM260OA

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urinalysis) were performed. Subjects signed a written consent approved by the Stanford Administrative Panel on Human Subjects in Medical Research (Stanford, CA, USA). Study design Subjects were admitted to the General Clinical Research Center at Stanford University Medical Center (GCRC) in the morning after overnight fasting. Studies began at 09.00 and were 48 h in length. An intravenous catheter was inserted in one arm for blood collection throughout the study. The study was designed as a crossover study. The order of diet (A or B) during the 2 days was randomly assigned. On both days, total caloric intake was 2000 Kcal/day for females and 2500 Kcal/day for males. All cooking and drinking water was double distilled in order to minimize the NOx content from water (nitrate concentration can be as high as 10 mg/l in ordinary water). Drinking water was limited to 1000 ml/day. The amount of nitrate in double distilled water in the laboratory was less than 0.1 mg/l or 100 ppb. Caffeine and any medications were prohibited during the study. None of the participants exercised regularly. Physical exercise was prohibited 48 h before and during the study. Subjects were allowed to walk in the GCRC but were asked to stay in a supine position for 30 min before blood was drawn. Blood samples were drawn via the intravenous catheter at 15 to 60 min intervals from 08.00 to 24.00 and at 2- to 4-h intervals from 24.00 to 08.00. Diet A was made from a combination of Microlipid (Sherwood Medical, St Louis, MO, USA) emulsion and Polycose (Ross Products Division, Abbott Labs, Columbus, OH, USA). Diet A contained no l-arginine and was very low in nitrate and nitrite content. Subjects received the liquid diet in seven equal portions throughout the day from 09.00 until 22.00. Diet B was a normal food diet designed to provide 3.8 g of l-arginine distributed between three meals (according to USDA-ARS 1996). The amounts of l-arginine in the breakfast, lunch and dinner were approximately 1.0 g, 1.7 g and 1.1 g, respectively. Diet B also contained minimal amounts of nitrate and nitrite. Meal times for diet B were 09.00 (after the first blood draw), 13.00 and 18.00. All volunteers tolerated both diets well. Analytic methods Plasma samples were analyzed for l-arginine concentrations using high performance liquid chromatography (HPLC) as developed in the authors’ laboratory.22 The coefficient of variation of this assay was less than 2%. The same plasma samples were also analyzed for NOx by chemiluminescence as previously described.23,24 Briefly, NO generated in vivo is converted into nitrite/nitrate, nitrosyl hemoproteins, nitrosyl metal complexes and S-nitroso compounds in the circulation. The assay method consists of the thermolysis of all these NO-related compounds and the detection of resulting nitrate after enzymatic reduction. Data analysis Data are expressed as mean ± SEM except where stated otherwise. Average plasma concentrations of l-arginine and NOx in each group were expressed as area under the curve (AUC) between plasma concentrations of l-arginine or NOx over 24 h. In order to assess diurnal variation, AUC values for plasma concentration versus time were divided into eight 3-h-intervals beginning from midnight. AUC 24Vascular Medicine 1999; 4: 27–32

h and 3-h values were compared between the two diets using the paired t-test. The differences between diet A and diet B on plasma l-arginine and plasma NOx concentrations at each time point were also tested using the paired t-test. Statistical significance was defined as p ⬍ 0.05.

Results Mean plasma l-arginine concentrations are shown in Figure 1. At the beginning of the day, the average plasma l-arginine concentrations were 21.4 ± 2.0 and 20.6 ± 2.5 ␮g/ml for diet A (no arginine) and diet B (normal arginine), respectively (NS). With diet A administration, plasma larginine concentrations decreased significantly from 09.00 to 16.00 (21.4 ± 2.0 to 11.9 ± 1.1 ␮g/ml) and slightly increased in the evening and overnight. Plasma l-arginine concentrations at 24 h were not significantly different from plasma l-arginine concentrations at the beginning of the previous day for diet A (21.4 ± 2.0 versus 18.5 ± 1.9 ␮g/ml). With diet B, plasma l-arginine concentrations were sustained during the day and showed three peaks, one after each meal. Each peak occurred approximately 3 h after meal times as shown in Figure 1. Mean peak l-arginine concentrations were 24.4 ± 3.1, 22.2 ± 3.1 and 22.3 ± 2.4 ␮g/ml for breakfast, lunch and dinner, respectively. After the peak following the evening meal, plasma l-arginine concentrations gradually decreased below morning baseline values until a plateau was reached at 04.00. Plasma l-arginine concentrations after 24 h appeared slightly lower compared to the previous day but no statistical difference was detected (20.6 ± 2.5 versus 15.4 ± 1.2 ␮g/ml). Plasma larginine concentrations during diet A and diet B were significantly different at several time points between 10.30 and 22.00 as indicated in Figure 1. The differences in plasma l-arginine concentrations expressed as AUC values between diet A and diet B were significant for the following intervals: 09.00 to 12.00 noon, 12.00 noon to 15.00 and 15.00 to 18.00, as indicated in Figure 2. No significant fluctuations in plasma NOx concentrations over 24 h (expressed as AUC values) were detected for either diet A or diet B (Figure 3). The 24 h profiles for the plasma concentrations of l-arginine and NOx were not influenced by the sequence in which the diets were administered.

Discussion This study demonstrates that removal of l-arginine from the diet for one day in healthy individuals causes a significant decrease in plasma l-arginine concentrations during the awake period followed by a spontaneous return to normal morning basal concentrations overnight. In the same subjects, a normal amount of l-arginine in the diet (3.8 g/d) was associated with a rise in plasma l-arginine concentration after each meal. Importantly, none of these two controlled diets influenced plasma NOx concentrations, an in vivo index of NO biosynthesis. Plasma l-arginine changes reflect the balance between

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Figure 1 Average plasma l-arginine concentrations over each 24-h study period. Subjects received a nitrate/nitrite and l-argininefree diet (diet A) (closed circles) or a nitrate/nitrite-free diet with usual dietary amounts of l-arginine (diet B) (open circles). At each time point, the differences in plasma l-arginine concentrations from the same subjects were tested between diet A and diet B and significant differences were detected as indicated. * indicates statistical significance of p ⬍ 0.05; # indicates statistical significance 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 the 24-h study with diet A (white bars) or diet B (hatched bars). The differences in AUC values from the same subjects during diet A and 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 by paired t-test). Error bars represent the SEM at each time point.

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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 day observed for either diet A or diet B.

complex inter-organ processes leading to movement of the amino acid into and out of the circulation. Endogenous synthesis of arginine occurs primarily in the kidney and to a lesser extent in the liver via conversion of citrulline to arginine.17 However, the liver does not contribute significantly to the maintenance of the plasma concentrations of l-arginine, since the amino acid synthesized in this organ is routed towards its local utilization.17 The mean dietary intake of l-arginine in industrialized countries is 3– 6 g/day;1 60% of this exogenous source appears in the general circulation.17 Isotopic studies have shown that the net rate of de novo arginine synthesis in healthy humans is not affected by a 6–7 day arginine-free diet.25,26 Consequently, it has been proposed that whole-body arginine homeostasis in healthy adults may be achieved principally via a modulation in the level of dietary arginine intake and/or with regulation in the rate of its catabolism to ornithine and glutamate.25 In this 24-h study, the l-arginine-free diet was associated with a gradual decrease in plasma concentration – reaching 47% of the baseline value after 7 h. Comparison with the normal diet also demonstrated a significant decrease in the 3-h AUC intervals from 09.00 to 18.00. This phenomenon was followed by a return towards normal plasma l-arginine concentrations during the night. Our 24h observation period complements previous findings from arginine kinetic studies carried out after 6 days of a controlled diet.25,26 Information regarding plasma l-arginine concentrations provided by these tracer studies is limited but suggests that fasting morning plasma concentrations of the amino acid (measured by an HPLC method) after a 6day arginine-free diet do not differ with an arginineenriched diet and that the post-meal value spontaneously decreases during the day when l-arginine is removed from Vascular Medicine 1999; 4: 27–32

the diet. Further studies are needed to determine the precise mechanism(s) responsible for this 24-h profile of plasma larginine concentrations during an l-arginine-free diet. The authors also observed three peaks following each meal during the normal diet; this phenomenon confirms that the intestinal absorption of l-arginine has a relevant effect on its plasma concentration. This observation suggests that the effect of food intake should be considered in pharmacokinetic studies designed to evaluate plasma concentrations of l-arginine after oral supplementation. l-arginine is the substrate for endothelial NO synthesis, a reaction that is catalyzed by the constitutive endothelial enzyme eNOS.2 NO plays a key role in the regulation of vascular tone and platelet aggregation and adhesion.2,3 Changes due to hypercholesterolemia and aging, two conditions associated with impairment of the l-arginine/NO pathway, have shown benefit from l-arginine supplementation in humans.11–16 l-arginine supplementation (8– 21 g/day by the oral route) improved endothelial dysfunction and was associated with a modest increase in surrogate markers of NO in patients with hypercholesterolemia.11 Recent studies have shown that NO can be detected in plasma in several active or inactive forms appearing in pools of different sizes.27–29 Among these plasma indices of NO production, plasma NOx measured by chemiluminescence appears to include the major metabolites of NO in the human body.23 This surrogate marker has been successfully used by other investigators to demonstrate significant changes in NO production in humans.24 The authors hypothesized that physiological variations of plasma l-arginine concentrations either induced by a normal or by an arginine-free diet could influence endothelial NO production and consequently plasma NOx concen-

Plasma l-arginine and NO-related compounds in humans

trations. Since the nitrate present in the blood may derive from dietary sources such as nitrate- or nitrite-containing food or drinking water,30 a nitrate/nitrite-restricted diet was maintained during this 48 h study. The plasma concentrations of NOx measured in the study by a chemiluminescence assay23,24 did not change in response either to a 24-h arginine-free diet or to an increase in plasma concentrations of l-arginine following the meals in the normal diet. Also, a study in healthy subjects has shown that supplemental (56 mg/kg per day) l-arginine does not influence plasma nitrate concentrations or the rate of conversion of plasma arginine to urinary nitrate.31 This lack of influence of a normal or an arginine-free diet on whole-body NO production in healthy humans suggests that exogenous larginine plays a very limited role as a precursor of NO in healthy humans on a short-term basis. In support of this conclusion it has been reported that only 4% of endogenous arginine is used for NO synthesis.26 Although NO production may not be influenced by physiological variations of l-arginine in healthy subjects, this does not rule out a possible effect of supplementary l-arginine on NO metabolism in pathophysiological situations. In a previous study by the present group, evaluating the effects of oral l-arginine supplementation (14–21 g/day) over 12 weeks on the plasma concentrations and the pharmacokinetics of l-arginine in hypercholesterolemic patients, average plasma concentrations rose from 16 to 22 ␮g/ml.32 Although plasma NOx were not measured in this particular study, supplemental l-arginine in hypercholesterolemic patients has been found to be associated with a modest rise in nitrosoprotein levels, another index of NO production.11 Further studies are needed to assess whether oral supplementation of this magnitude is also associated with increased plasma nitrate concentrations in these patients or in normals. Because of the short duration of the l-argininefree diet in this study (24 h), it is conceivable that plasma NOx levels might have fallen below that seen in the normal diet if the arginine-free diet had been maintained longer. However, the rapid turnover of l-arginine and nitric oxide makes this less likely. The possibility that physiological fluctuations of plasma l-arginine concentrations over 24 h may influence endothelial NO generation and thus vascular tone in hypercholesterolemic but not in normal subjects also remains to be explored. In conclusion, plasma concentrations of l-arginine are quickly influenced by dietary l-arginine. Fluctuations of plasma l-arginine during normal and l-arginine-free diets appear to be insufficient to influence NOx concentrations over 24 h . These results suggest that in healthy individuals at rest, plasma l-arginine concentrations are not rate-limiting in the formation of NO.

Acknowledgements Funding for this project was provided by NIH grant AG05627, an American Heart Association Grant-in-Aid (JRC), NIH grants HL 08506 and HL 58683, and GCRC grant RR00070. Dr Tangphao was supported by Ananthamahidol Foundation, Bangkok, Thailand. Drs Moreno and Chalon were supported by Merck Sharp and Dohme International Fellowships in Clinical Pharmacology. Dr Cooke is the founder of CookePharma (Mountain View, CA, USA), but Vascular Medicine 1999; 4: 27–32

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

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