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In vivo arginine production and intravascular nitric oxide synthesis in hypotensive sepsis^1,2,3,4

¹ From the US Department of Agriculture, Agricultural Research Service, Children's Nutrition Research Center, Department of Pediatrics (SV and FJ); the Translational Metabolism Unit, Division of Diabetes, Endocrinology and Metabolism (JG and AB); and the Section of Pulmonary and Critical Care Medicine, Baylor College of Medicine, Houston, TX (VPB and KG), and Medical Services, Ben Taub General Hospital, Houston, TX (JG, AB, VPB, and KG)

² SV and JG contributed equally to this work.

³ Supported by funds from Burroughs Wellcome, USA; by federal funds from the US Department of Agriculture, Agricultural Research Service, under Cooperative Agreement no. 58-6250-6001; and by NIH GCRC grant no. MO1 RR00188. SV's fellowship was supported by Patronato del Hospital Infantil de México "Federico Gómez" (México) and the Fogarty International Center for AIDS International Training and Research Program grant no. D43 TW01036.

⁴ Address reprint requests to F Jahoor, Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, 1100 Bates Street, Houston, TX 77030-2600. E-mail: fjahoor{at}bcm.tmc.edu.

	ABSTRACT

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Background: Arginine is important in the response to infections and is a precursor for the synthesis of the vasodilator nitric oxide (NO). Low plasma arginine is correlated with a worse prognosis in patients with sepsis, and increased NO has been implicated in the hypotension of sepsis. Data on in vivo arginine and NO kinetics are lacking in hypotensive septic adults.

Objective: We aimed to measure in vivo arginine production and the intravascular NO synthesis rate in hypotensive septic patients.

Design: Arginine flux and the fractional and absolute synthesis rates of plasma NO were measured in fasted healthy (n = 10) and hypotensive septic (n = 6) adults by using a 6-h constant infusion of [¹⁵N₂-guanidino]arginine. Urinary excretion of the NO metabolites nitrite and nitrate (NOx) and plasma concentrations of NOx, arginine, and creatinine were also measured.

Results: All patients had hyperdynamic septic shock and impaired renal function. Compared with the control subjects, the patients had slower arginine flux (99 ± 8 compared with 50 ± 7 µmol · kg^–1 · h^–1; P < 0.01), lower plasma arginine concentrations (75 ± 8 compared with 40 ± 11 µmol/L; P < 0.01), higher plasma NOx concentrations (30 ± 4 compared with 65 ± 1.8 µmol/L), and a slower fractional synthesis rate of NOx. There was no significant difference in the absolute synthesis rate of NOx between groups. In patients with sepsis, the plasma NOx concentration correlated with the glomerular filtration rate and plasma creatinine but not with mean arterial pressure.

Conclusions: Patients with septic shock have a shortage in the availability of arginine associated with a slower production. Impaired renal excretion of NOx is a contributor to the high plasma NOx in these patients.

Key Words: Hypotensive sepsis • arginine • nitric oxide • stable isotope • kinetics • renal function

	INTRODUCTION

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In addition to its role as a substrate for protein synthesis, arginine serves several important functions during the response to infections and trauma (1). Dietary supplementation with arginine has been shown to improve immune function, reduce the incidence of sepsis, decrease the rate of post-injury weight loss, and improve wound healing in injured and infected humans (2, 3) and animals (4). Furthermore, low plasma arginine has been shown to be correlated with a worse prognosis in patients with sepsis (5). These findings suggest that there may be an overall increase in the requirement for arginine that is not met by endogenous production in stressed states. Direct experimental evidence for this is mixed. Arginine production was shown to be increased in septic pigs (6). A study in humans, however, reported that the rate of arginine production was not significantly different in pediatric septic patients than in healthy adults (6). Data on arginine production in hypotensive septic adults are lacking.

Arginine administration has also been shown to reduce blood pressure and renal vascular resistance in essential hypertensive patients with normal or insufficient renal function (7). This effect is due to vasodilation mediated by nitric oxide (NO), a broadly distributed endogenous molecule synthesized from arginine in a reaction catalyzed by nitric oxide synthase (NOS). The NO-NOS system is thought to play a role in initiating or maintaining the hypotension and resistance to vasopressor drugs commonly observed in septic shock (8). In early studies, plasma concentrations of NO metabolites were found to be markedly increased in patients with septic shock (9, 10). In addition, both the expression and activity of NOS have been found to be elevated in both in vitro studies of human tissues (11) and animal models of sepsis (12). These observations have led to the hypothesis that inhibition of NOS activity may ameliorate the hypotension associated with sepsis and hence improve clinical outcome. Despite the plausibility and attractiveness of this hypothesis, however, the experimental data do not support it. In several studies of experimental sepsis in animals, treatment with nonselective NOS inhibitors worsened organ function and survival (13, 14). A clinical trial to evaluate the NOS inhibitor N^G-methyl-L-arginine in hypotensive septic patients (13) had to be suspended because of worse survival among patients receiving the drug (15). These results suggest that the underlying hypothesis may not be true because it was based solely on static, in vitro measurements of NOS activity and NO concentrations (10) rather than on in vivo kinetic data regarding the processes that regulate NO concentrations, ie, the rates of production of arginine and the synthesis and clearance of NO.

In the only published study of in vivo NO kinetics in human sepsis, NO synthesis was faster in septic children than in healthy adults (6). There are no data, however, on the in vivo rate of production of arginine and its conversion to NO in hypotensive septic adults. The purpose of the present study was thus to measure arginine production and its rate of conversion to NO in septic patients with hypotension by using a stable-isotope-tracer technique (16). We tested the hypothesis that the rate of production of arginine and its conversion to NO are faster in hypotensive septic patients than in healthy volunteers.

	SUBJECTS AND METHODS

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Subjects
The study was reviewed and approved by the Institutional Review Board for Human Subject Research of Baylor College of Medicine & Affiliated Hospitals. Six adult patients with septic shock admitted to the medical intensive care unit (MICU) of Ben Taub General Hospital in Houston, TX, were recruited. Septic shock was defined by the criteria of the American College of Chest Physicians–Society of Critical Care Medicine Consensus Conference Committee (17). All patients had direct evidence of infection and met the criteria for severe sepsis and also satisfied the criteria for septic shock (sepsis-induced hypotension not responding to adequate fluid resuscitation and requiring dopamine at a rate >5 µg · kg^–1 · min^–1 or norepinephrine at a dose of >0.1 µg/kg). The MICU patients had been diagnosed with severe sepsis in the 24-h period before the study and had developed septic shock in the previous 12 h. Ten healthy adult volunteers participated in the study as control subjects. All control volunteers were in good health as established by medical history, physical examination, and blood chemistry measurements. They were selected to encompass a wide range of adult ages (25–75 y). Written consent was obtained from each control subject and from the spouse or nearest relative of each patient with septic shock.

Experimental protocol
In this study, the rate of synthesis of NO from arginine in the plasma compartment was estimated by determining the fractional rate of synthesis of plasma nitrate plus nitrite (NOx) from arginine and the concentration of NOx in plasma. This approach is feasible because ¹⁵N-labeled NO is an intermediate produced in the pathway in which [¹⁵N₂]guanidino arginine is converted to [¹⁵N]ureido citrulline (16). Hence, [¹⁵N₂]guanidino arginine is the only possible precursor of ¹⁵NO. Also, because the NO produced is reduced to nitrite and then to nitrate, ¹⁵NOx can only be made from ¹⁵NO. Therefore, ¹⁵NOx is an excellent surrogate measurement of ¹⁵NO. By administering [¹⁵N₂]guanidino arginine and measuring the isotopic enrichment of plasma arginine at steady state (precursor pool) and the plasma isotopic enrichment of NOx (product), one can calculate the fractional rate of synthesis of nitrite and nitrate (and hence of NO) by using the standard precursor-product equation.

Isotope tracer infusion
Tracer infusions were performed in healthy subjects in the adult General Clinical Research Center (GCRC) of Baylor College of Medicine, and the patients with sepsis were studied in the MICU of Ben Taub General Hospital. The healthy subjects were instructed by a dietitian to consume a diet free of nitrite and nitrate for 2 d before their admission to the GCRC. After fasting overnight, the subjects were admitted to the GCRC, and intravenous catheters were placed in an antecubital vein for isotope infusion and in a hand vein of the contralateral arm for blood sampling. After baseline blood and urine samples were collected, a priming dose (6 µmol/kg) of [¹⁵N₂]guanidino arginine (99.9%; Cambridge Isotope Laboratories, Woburn, MA) was administered as a bolus injection followed by a constant infusion of the isotope at 6 µmol · kg^–1 · h^–1 for 6 h.

The same tracer infusions were performed in fasted septic patients. The tracer was infused through a preexisting central venous catheter placed for monitoring purposes, and blood and urine samples were obtained from preexisting arterial and Foley catheters, respectively.

Additional blood samples were drawn at hourly intervals during the infusion. Urine samples were collected hourly by micturition in the healthy subjects and through a Foley catheter in the patients with sepsis. The urine samples were kept on ice and stored at –70 °C for further analysis.

Hemodynamic measurements
In the patients with sepsis, heart rate and mean arterial blood pressure were monitored throughout the 6-h isotope infusion period, and mean values were calculated. Cardiac index was calculated from cardiac output measured 2–4 times during the same 6-h period.

Sample analysis
The blood samples were drawn into prechilled tubes containing Na₂EDTA, the tubes were centrifuged immediately at 4 °C, and the plasma was removed and stored immediately at –70 °C for later analysis. The tracer-tracee ratio of plasma arginine was measured by negative chemical ionization gas chromatography–mass spectrometry (NCI GC-MS) by using a Hewlett-Packard HP 5989B quadrupole mass spectrometer (Palo Alto, CA). Plasma arginine was extracted by cation-exchange chromatography, and the trifluoroacetyl ester derivative was prepared by reacting the dried residue with 0.4 mL of a 4:1 mixture of dichloroethane:trifluoroacetic anhydride at 100 °C for 2 h. Tracer-tracee ratios were measured by selectively monitoring ions at mass-to-charge (m/z) ratios of 444 to 446.

The isotopic enrichment of plasma NOx was also determined by NCI GC-MS according to the method described by Tsikas (18). Briefly, the nitrate in 0.5 mL of plasma was reduced to nitrite by adding 25 mg cadmium. The mixture was acidified with 0.1 mL of 20% acetic acid and shaken at room temperature for 15 min to reduce nitrate to nitrite. After centrifugation at 1000 x g for 5 min, the supernatant fluid was removed and further extracted with acetone, the nitrite was converted to its pentafluorobenzyl derivative by reacting with 25 µL of 2,3,4,5,6-pentafluorobenzyl bromide at 50 °C for 1 h, and isotope ratios were measured by selectively monitoring ions at m/z ratios of 46 to 47.

Plasma arginine concentrations were measured by standard ion-exchange chromatography. Plasma and urinary NOx concentrations were measured by in vitro isotope dilution as described by Tsikas (19). Briefly, 0.5 mL of the baseline plasma or urine sample was spiked with a known quantity of Na¹⁵NO₃, the internal standard; the nitrate was then reduced to nitrite; and the isotopic enrichment of the nitrite was measured as described above.

Calculations
Arginine flux (rate of production) was calculated from the steady state equation as follows:

(1)

where IE_Inf and IE_pl are the isotope enrichments of the tracer in the infusate and in plasma at isotopic steady state, respectively; i is the rate of infusion of ¹⁵N₂-arginine in µmol · kg body wt^–1 · h^–1; and the units of flux are µmol · kg^–1 · h^–1.

The fractional synthesis rate (FSR) of NO was calculated according to the precursor-product equation as previously described by us (20). Essentially, when a labeled amino acid is given by constant infusion, the precursor pool enrichment reaches a constant value with time; thus, by measuring the rate of incorporation of the label into a product after a plateau is reached in the precursor pool, FSR can be obtained from the following:

(2)

where IE NOx_t2 –IE NOx_t1 is the increase in isotopic enrichment of NOx over the period (t₂ –t₁) of the infusion, and IE arg_pl is the plateau isotopic enrichment of plasma arginine. In this calculation, the plateau enrichment of arginine in plasma is assumed to represent the enrichment of all the arginine pools from which NO and hence nitrite and nitrate are synthesized. The absolute synthesis rate (ASR) of plasma NOx in the intravascular compartment was calculated as the product of the plasma NOx concentration and the FSR. The units of intravascular ASR are expressed as µmol · L plasma^–1 · h^–1.

Statistical analysis
Data are expressed as means ± SEMs. Differences between groups were detected by use of the unpaired t test. A probability of 5% (P < 0.05) was assumed to represent statistical significance. Pearson correlations between outcome variables were performed with the STATA statistical package (version 7 for WINDOWS; Stata Corporation, College Station, TX).

	RESULTS

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Although the patients with septic shock tended to be older than the healthy control subjects, age was not significantly different between the groups (P = 0.09; Table 1). This was expected because the control group intentionally comprised a broad age range to determine any effect of age on arginine production and NO synthesis. We did not, however, find any association between age and any of the kinetic measurements. The patients with sepsis also had significantly higher serum concentrations of creatinine, aspartate aminotransferase, and alanine aminotransferase. There was no significant difference between the groups in body mass index.

Table 2

Table 3

Figure 1

View larger version (17K): [in this window] [in a new window]   FIGURE 1. Arginine kinetics in healthy subjects (n = 10) and hypotensive patients with sepsis (n = 6). *Significantly different from healthy subjects, P < 0.01 (unpaired t test).

Figure 2

Figure 3

View larger version (27K): [in this window] [in a new window]   FIGURE 2. Plasma nitrate plus nitrite (NOx) kinetics in healthy subjects (n = 10) and hypotensive patients with sepsis (n = 6). FSR, fractional synthesis rate; ASR, absolute synthesis rate. *Significantly different from healthy subjects, P < 0.01 (unpaired t test).

View larger version (21K): [in this window] [in a new window]   FIGURE 3. Urinary nitrate plus nitrite (NOx) kinetics in healthy subjects (n = 10) and hypotensive patients with sepsis (n = 6). *Significantly different from healthy subjects, P < 0.01 (unpaired t test).

Table 4

	DISCUSSION

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The arginine flux of 50 ± 7 µmol · kg^–1 · h^–1 obtained for our adult septic patients is similar to the value of 67 ± 21 µmol · kg^–1 · h^–1 reported for septic children by Argaman et al (6). Our conclusion that arginine production is diminished in sepsis is, however, in contrast with the conclusion reached by Argaman et al (6) when they compared their values for the septic children with historical values for healthy adults. They reached this conclusion because their historical value for arginine flux in healthy adults, 68 ± 10 µmol · kg^–1 · h^–1, is much lower than that of our group of healthy adults of 99 ± 8 µmol · kg^–1 · h^–1.

The supply and hence the de novo synthesis of the dispensable amino acids is important for survival because these amino acids are precursors for the synthesis of numerous metabolites, peptides, and proteins that are necessary for physiologic homeostasis. Arginine is a good example of such an amino acid (4, 21). Although in normal health it is regarded as a dispensable amino acid, some evidence suggests that in severely stressed states such as sepsis, a higher demand for arginine exceeds its availability, thus making it a conditionally essential amino acid (6, 22). Our present finding of a smaller plasma arginine pool and a slower flux in hypotensive septic patients suggests that in this particular group of patients there is an overall increase in the requirement for arginine that is not met by its production.

In the fasted state, arginine flux represents arginine derived from protein breakdown plus de novo synthesis. Hence, the slower arginine production in these patients with sepsis can be explained by either a decrease in protein breakdown or diminished de novo synthesis. Because we and others have shown that protein breakdown is stimulated in sepsis (23, 24), it can be concluded that impaired de novo synthesis is one factor contributing to the slower arginine production in patients with septic shock. It is also possible that the stress-induced increase in arginine utilization may be contributing toward depletion of the arginine pool. For example, a study of septic children reported that arginine oxidation was twice the rate of de novo synthesis, which indicates a negative balance (6). On the other hand, because all the patients had evidence of liver damage, one cannot rule out the possibility that arginase released into the circulation plus the increased arginase activity in the macrophages of patients with sepsis may be responsible for the lower circulating arginine concentrations (25). These findings and evidence that supplemental dietary arginine improves immune function, reduces the incidence of sepsis, and improves wound healing in injured and infected humans (2, 3) and animals (4) strongly support the argument that arginine becomes an indispensable amino acid in severely stressed states such as sepsis (6, 22).

There is evidence that arginine's vasodilatory and immunomodulatory actions are wholly and partially indirect, being mediated through NO. In early studies, the plasma concentration of NOx was found to be markedly elevated in septic shock (9, 10). This led to the proposal that increased NO production may be responsible for the hypotension (9). Our present finding of higher plasma NOx concentrations in the hypotensive septic group corroborates these earlier findings. However, our finding of a slower FSR and no significant difference in the ASR of plasma NOx between the control and septic groups suggests that the larger plasma NOx pool may not be due to increased NO synthesis as suggested by others (9). On the other hand, our findings of a slower glomerular filtration rate that was inversely correlated with plasma NOx and the slower renal excretion of NOx in the septic group suggest that the elevated plasma NOx in these patients is partly due to impaired renal excretion. In another study of hypotensive septic patients, Evans et al (26) reached the opposite conclusion that elevated plasma NOx was not due to impaired excretion. This conclusion was based on their finding that the correlation coefficient between plasma creatinine and NOx failed to achieve statistical significance. Interestingly, the correlation coefficient of 0.45 reported by Evans et al (26) is exactly the same as in the present study.

Two possible explanations for the slower FSR of NO observed in these patients with sepsis are limited precursor supply and impaired NOS activity. Our data suggest that a limited arginine supply secondary to decreased production is probably a primary contributor. Another possibility is impaired cellular arginine uptake plus impaired NOS activity because of increased production of asymmetric and symmetric dimethylarginine (ADMA and SDMA, respectively). In critically ill septic patients, concentrations of both ADMA and SDMA are elevated in plasma (27) because of increased production (28) plus decreased renal excretion (29). SDMA will compete with arginine for transport by the y⁺ transporter, and ADMA is known to inhibit all isoforms of NOS.

Although it has been postulated that increased NO production may be responsible for the hypotension of septic shock (30), we are not aware of any study showing that NO production is correlated with mean arterial pressure in patients with septic shock. In the present study, no correlation existed between mean arterial pressure and plasma NOx, which suggests that increased NO synthesis may not be responsible for the hypotension of these patients with sepsis. Evans et al (26) reached a similar conclusion based on their observation that plasma NOx was not elevated in all hypotensive septic patients. They concluded that NO may not be the only factor determining blood pressure during sepsis. On the basis of this finding and evidence that NO may protect against endotoxin-induced tissue damage in the liver and kidney (14), they (26) and others (31, 32) have warned against the potential dangers of blocking NOS activity as a therapy for septic shock. These findings plus our present finding that in vivo plasma NO synthesis is probably not upregulated in septic shock may explain why treatment with nonselective NOS inhibitors worsened organ function and survival in studies of experimental sepsis (14, 33). It also offers a possible explanation for why clinical trials evaluating the NOS antagonist N^G-methyl-L-arginine in hypotensive septic patients resulted in worse survival among patients receiving the drug and had to be suspended (15).

It should be pointed out that the present study was conducted in patients 3 d after sepsis was diagnosed when there was already evidence that renal and hepatic functions were compromised to some degree. Hence, the patients' arginine and NO metabolic response may not be representative of the acute response to sepsis. At present, we are not aware of any other comparable study of arginine and NO metabolism in adults with septic shock. There are, however, data from 2 studies in septic children (6) and endotoxemic pigs (34). For example, using the conversion of ¹⁵N₂-arginine to ¹⁵N-citrulline to estimate NO production in children in whom sepsis was diagnosed 24 h earlier, Argaman et al (6) reported an NO synthesis rate that was higher than historical values from healthy adults. These authors, however, did not report plasma NOx concentrations.

Finally, one can argue that our method of calculating the ASR of plasma NO has drawbacks. First, besides the 2 major end products NO₂ and NO₃, NO also occurs in plasma as nitrosylated compounds (21). This could result in an underestimate of total plasma NO in septic patients and hence the calculated ASR. Any such underestimation would be negligible, however, because total nitrosylated compounds in plasma are in nanomolar quantities compared with micromolar quantities of NOx (35). Second, plasma volumes were not measured and it is likely that the patients with sepsis may have had larger plasma volumes because of their compromised renal function. If this was the case, then the total plasma NO synthesis rate would have been faster in the septic group. Hence, we cannot rule out the possibility that the ASR of plasma NO was faster in the patients with sepsis. This does not affect the calculation of the FSR, however, because ¹⁵N₂-arginine is the only possible precursor of ¹⁵NO, and ¹⁵NOx can only be made from ¹⁵NO.

	ACKNOWLEDGMENTS

 
We are grateful to the nurses and dietitians of the adult GCRC and the medical intensive care unit of Ben Taub General Hospital, to the staff of the clinical pharmacy, and to Margaret Frazer and Melanie Del Rosario for their excellent work and support in the conduct of the studies and analysis of the samples.

All authors contributed to all aspects of the production of this manuscript, from the design of the study, data collection, analysis and interpretation, and writing of the manuscript. None of the authors had any conflicts of interest with the funding agencies.

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