Pharmacokinetics of 2-oxothiazolidine-4-carboxylate, a cysteine prodrug, and cysteine.

The pharmacokinetics of both 2-oxothiazolidine-4-carboxylate (OTZ), a prodrug of cysteine, and total blood cysteine (cysteine plus cystine) were investigated in 18 healthy volunteers. OTZ was given either as a single, 2-hour intravenous infusion (56-66 mg/kg) or similarly infused (70-100 mg/kg) every 8 hours for four doses. Blood was assayed for OTZ, total blood cysteine, and glutathione. The pharmacokinetics of OTZ were analyzed alone and simultaneously with total cysteine using the NONMEM software package (University of California at San Francisco. The pharmacokinetics of OTZ were best described by Michaelis-Menten kinetics with parallel first-order elimination. OTZ was efficiently removed from the plasma. The Michaelis-Menten route of elimination was attributed to conversion of OTZ to total cysteine. At plasma OTZ concentrations equal to the Michaelis constant Km, 84% of OTZ was converted to total cysteine. These findings suggest that OTZ administered intravenously is an efficient means of increasing total blood cysteine.

L-2-Oxothiazolidine-4-carboxylic acid prevents endotoxin-induced cardiac dysfunction.

We tested the hypothesis that treatment with the glutathione repleting agent, L-2-oxothiazolidine-4-carboxylic acid (OTZ), could prevent endotoxin-induced ventricular dysfunction. Rabbits were treated with OTZ 2.4 g/kg (10% solution subcutaneously), or an equal volume and osmolality of saline, 24 h prior to, and again (intravenously) just prior to, infusion of 1 mg/kg E. coli endotoxin (or vehicle control). Ventricular contractility was measured in isolated hearts perfused by support rabbits. Contractility did not change in control groups (Saline/Control [n = 7] or OTZ/Control [n = 7]) over 6 h. However, Emax decreased in the Saline/Endotoxin group (-16.1 +/- 4.5% from baseline, n = 7, p < 0.05) and this was prevented by pretreatment with OTZ in the OTZ/ Endotoxin group (+6.3 +/- 4.1%, n = 7, p < 0.05 by analysis of variance). To better understand the mechanism of this effect we measured myocardial glutathione concentration and found it to be greater in OTZ/Endotoxin animals (104 +/- 4 ng/g) than in the Saline/Endotoxin animals (80 +/- 3 ng/g, p < 0.05). OTZ did not appreciably alter the endotoxin-induced increase in serum concentration of tumor necrosis factor (TNF) or the endotoxin-induced increase in myocardial leukocyte content. We conclude that oxygen radicals contribute to the early decrease in left ventricular contractility after endotoxin infusion and this decrease may be prevented by OTZ.

The effects of (L)-2-oxothiazolidine-4-carboxylate on urinary oxalate excretion.

PURPOSE: A phase I study was done to evaluate the safety and pharmacokinetics of (L)-2-oxothiazolidine-4-carboxylate (OTZ). An ancillary objective was to compare the effects of treatment with 2 levels of OTZ to placebo on urinary oxalate excretion in healthy male subjects. MATERIALS AND METHODS: Individuals underwent intravenous infusion of 70 (6) or 100 (6) mg/kg, body weight OTZ, or placebo for 2 hours at 4, 8-hour intervals. Urine was collected during the 12 hours before treatment, and at 0 to 4, 4 to 8, 8 to 24, 24 to 28, 28 to 32 and 32 to 48 hours after the initial infusion. Urine samples were assayed for creatinine, oxalate, citrate, sulfate, urate, phosphate and pH. RESULTS: Urinary oxalate excretion relative to creatinine decreased significantly in the 100 mg./kg. dose group by 4.1 mg./gm. during the first 24 hours and by 4.6 mg./gm. in 24 to 48 hours compared to baseline values (p < 0.05). Slight decreases of 0.9 and 1.1 mg./gm., respectively, in the 70 mg./kg. dose group, and 1.6 and 2.3 mg./gm., respectively, in the placebo group were observed. Oxalate excretion on day 2 in the 100 mg./kg. dose group was significantly less than that in the placebo group (p = 0.04). Urinary pH decreased and sulfate excretion increased with OTZ therapy. CONCLUSIONS: Treatment with 100 mg./kg. OTZ every 8 hours decreases urinary oxalate excretion in healthy men.

Urinary excretion of 3-methyladenine after consumption of fish containing high levels of dimethylamine.

The urinary excretion of the DNA alkylation product, 3-methyladenine (3-MeAde), was measured in human volunteers who were on controlled diets and consumed fresh fish, or frozen-stored fish that contained 50-fold higher levels of dimethylamine (DMA), with or without ingested nitrate. DMA potentially could react with nitrosating agents in the diet or within the body, and produce the potent carcinogen N-nitrosodimethylamine (NDMA), which can then react with DNA to form several adducts including 3-MeAde. Our findings show that there was no increase in urinary levels of 3-MeAde after consumption of fish preserved by frozen storage relative to levels after consumption of fresh fish. Furthermore, consumption of 225 mg sodium nitrate (equal to the nitrate content in a large glass of beet juice) at 1 h prior to consumption of the frozen-stored fish did not increase urinary 3-MeAde levels as would be expected if nitrate enhanced endogenous nitrosation of DMA. In contrast, urinary excretion of 3-MeAde from a volunteer who was a moderate cigarette smoker (11 cigarettes per day) was approximately 3- to 8-fold higher than dietary 3-MeAde intake. These findings indicate that consumption of high levels of DMA in fish does not result in detectable levels of NDMA formation and genetic damage as measured by the urinary biomarker 3-MeAde.

The role of oxidative stress in HIV disease.

Evidence has accumulated suggesting that HIV-infected patients are under chronic oxidative stress. Perturbations to the antioxidant defense system, including changes in levels of ascorbic acid, tocopherols, carotenoids, selenium, superoxide dismutase, and glutathione, have been observed in various tissues of these patients. Elevated serum levels of hydroperoxides and malondialdehyde also have been noted and are indicative of oxidative stress during HIV infection. Indications of oxidative stress are observed in asymptomatic HIV-infected patients early in the course of the disease. Oxidative stress may contribute to several aspects of HIV disease pathogenesis, including viral replication, inflammatory response, decreased immune cell proliferation, loss of immune function, apoptosis, chronic weight loss, and increased sensitivity to drug toxicities. Glutathione may play a role in these processes, and thus, agents that replete glutathione may offer a promising treatment for HIV-infected patients. Clinical studies are underway to evaluate the efficacy of the glutathione-repleting agents, L-2-oxothiazolidine-4-carboxylic acid (OTC) and N-acetylcysteine (NAC), in HIV-infected patients.

Urinary markers for exposures to alkylating or nitrosating agents.

Investigation of urinary markers as indices of endogenous nitrosation and of gastric cancer etiology has been a major focus of our work. As part of this effort, studies have been carried out on a Colombian population at high risk for gastric cancer. In this group, nitrosoproline excretion was highly correlated with nitrate excretion in the subpopulation with advanced gastric pathology, but not in control subpopulations with more normal stomachs. Neither urinary 7-methylguanine nor 3-methyladenine was strongly related to gastric pathology or to urinary nitrate or nitrosoproline levels. More recently, as evidence has accumulated concerning the importance of nitric oxide as a cellular messenger, we have begun research toward developing markers for the presence of nitric oxide and for endogenous nitrosation via this compound. Nitric oxide is formed from arginine by activated endothelial cells as a messenger for vasodilation. We have shown that prolonged exercise leads to increased urinary nitrate and that when 15N-arginine is ingested by humans, 15N-nitrate levels increase in 24-hr urine collections. Nitrosohydroxyethylglycine and 3-nitrotyrosine were evaluated as indices for the formation of N-nitrosomorpholine and for the nitration of protein, respectively, under experimental conditions (e.g., immunostimulation) expected to enhance nitric oxide formation. Nitrotyrosine has not proved useful as a biomarker for nitration/nitrosation reactions in immunostimulated rats. Immunostimulation of rats following administration of morpholine led to increases in urinary nitrate and nitrosohydroxyethylglycine. This procedure, however, would not be appropriate for humans due to the toxicity of morpholine and the carcinogenicity of N-nitrosomorpholine.

Endogenous incorporation of nitric oxide from L-arginine into N-nitrosomorpholine stimulated by Escherichia coli lipopolysaccharide in the rat.

The endogenous formation of nitrate in the rat, mouse and human occurs through cellular processes involving the oxidation of the guanido group of arginine. These processes proceed from arginine to nitric oxide with subsequent conversion to electrophilic nitrosating agents capable of forming carcinogenic nitrosamines. We have now demonstrated that endogenous nitrosamine formation can occur via cells stimulated in vivo by bacterial lipopolysaccharide (LPS). The nitrosation of morpholine given to rats by i.p. injection yields N-nitrosomorpholine (NMOR) which is subsequently oxidized in the liver. A major metabolite of NMOR, N-nitroso-(2-hydroxyethyl)glycine, was previously shown by other investigators to be excreted into urine. Treatment of rats with LPS, arginine and morpholine creates a large increase in NMOR urinary metabolites over a 24-h period. This process is not influenced by simultaneous dosage with large amounts of NaNO3. Therefore the endogenous LPS-induced formation of NMOR proceeds directly from nitric oxide prior to incorporation into the nitrate body pool. The proportion of endogenously synthesized nitric oxide incorporated into NMOR is approximately 3 x 10(-6).

Inhibition of nitrosamine formation by ascorbic acid.

Nitrosation occurs under a wide variety of conditions by reaction of most types of amines with any of a large number of nitrosating species. Nitrite can be formed in vivo via bacterial reduction of nitrate and by activated macrophages and endothelial cells. The mechanism of nitrite formation by mammalian cells is via enzymatic oxidation of arginine to NO followed by oxidation to N2O3 and N2O4. Nitrosatable amines are found in many foods and some, eg, dimethylamine, are synthesized in the body. Precursors of N-nitroso compounds are thus almost constantly present together under favorable reaction conditions in vivo and there is, consequently, considerable interest concerning possible human health risks arising from endogenous formation of this class of compounds. Among many nitrosation inhibitors, most attention has focused on ascorbic acid, which reacts with many nitrosating agents and which is virtually nontoxic. This presentation discusses the chemistry of ascorbic acid inhibition of nitrosation reactions.

Nitrate biosynthesis in rats, ferrets and humans. Precursor studies with L-arginine.

L-Arginine, the primary nitrogen source for nitric oxide synthesized by many cell types in culture and for biosynthesized nitrate in humans, is also a nitrogen source for biosynthesized nitrate in rats and ferrets. After administration of [15N2]L-arginine to rats and ferrets, [15N]NO3- was detected in urine. Escherichia coli lipopolysaccharide induced more than a 10-fold increase in urinary nitrate in rats and a parallel increase in incorporation of 15N from [15N2]L-arginine into NO3-. Bradykinin, a vasodilator which induces nitric oxide production by endothelial cells in vitro, lacked detectable effect on urinary nitrate or on incorporation of L-arginine nitrogen into nitrate in rats. A prolonged period of vasodilation brought on by an extended period of exercise increased urinary nitrate 2-fold in human subjects. In the rat, recoveries in 24 h post-dose urine collections of [15N]NO3- given i.v. and i.p. were 75 and 64% respectively, while in the ferret, recoveries of i.v. and per os [15N]NO3- doses were 49 and 34% respectively. Thus, nitrate synthesized by mammalian cells in vivo would undergo losses similar to those for exogenous nitrate.

L-arginine is a precursor for nitrate biosynthesis in humans.

Nitrogen from L-arginine was incorporated into urinary nitrate in human subjects. Two subjects given an oral dose of [15N2]L-arginine excreted 24 and 17 umol [15N]nitrate/24 hr, respectively, in their urine in the 24 hr period following the dose. This work demonstrates that L-arginine, a nitrogen source for biosynthesized nitrate in cultured cells and research animals, is a precursor for endogenously synthesized nitrate in humans.

Nitrosation by stimulated macrophages. Inhibitors, enhancers and substrates.

Macrophages and their immortalized cell lines can be activated to form nitrite and nitrate via oxidation of arginine and this is accompanied by the formation of N-nitroso compounds. The mechanism of nitrosamine formation has been investigated through the use of compounds which are known either to inhibit or enhance acid-catalyzed nitrosation. The range of nitrogen acceptors has been expanded to include ureas as well as amines of varying pKa and structure. The results are consistent with a mechanism in which NO is oxidized to N2O3 and N2O4, which are capable of nitrosating amines, but not ureas or amides, at neutral pH. This is in agreement with a recent observation that macrophage cell-free extracts can oxidize arginine to NO. The effect of ascorbic acid on intact activated macrophages is complex since nitrite formation is enhanced over a very wide range of ascorbate concentrations (5-500 microM) while nitrosation is inhibited at ascorbate concentrations greater than 50 microM.

Mechanisms of endogenous nitrosation.

Endogenous formation of N-nitroso compounds has been demonstrated in both humans and experimental animals. The extent of this process has been estimated by measurement of urinary N-nitrosoproline and has been shown to be modulated by dietary precursors and inhibitors. It is now also recognized that other (non-gastric) pathways of endogenous nitrosation, including those catalysed by bacteria and mammalian cells, may exist. The mammalian cell catalysed pathway utilizes arginine as a precursor for the nitrosating agent and may occur in macrophages or endothelial cells. The estimated contribution of this pathway to normal basal endogenous nitrosation is approximately 20 nmol of N-nitrosoproline/day.

Macrophage oxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate.

Previous studies have shown that murine macrophages immunostimulated with interferon gamma and Escherichia coli lipopolysaccharide synthesize NO2-, NO3-, and citrulline from L-arginine by oxidation of one of the two chemically equivalent guanido nitrogens. The enzymatic activity for this very unusual reaction was found in the 100,000g supernatant isolated from activated RAW 264.7 cells and was totally absent in unstimulated cells. This activity requires NADPH and L-arginine and is enhanced by Mg2+. When the subcellular fraction containing the enzyme activity was incubated with L-arginine, NADPH, and Mg2+, the formation of nitric oxide was observed. Nitric oxide formation was dependent on the presence of L-arginine and NADPH and was inhibited by the NO2-/NO3- synthesis inhibitor NG-monomethyl-L-arginine. Furthermore, when incubated with L-[guanido-15N2]arginine, the nitric oxide was 15N-labeled. The results show that nitric oxide is an intermediate in the L-arginine to NO2-, NO3-, and citrulline pathway. L-Arginine is required for the activation of macrophages to the bactericidal/tumoricidal state and suggests that nitric oxide is serving as an intracellular signal for this activation process in a manner similar to that very recently observed in endothelial cells, where nitric oxide leads to vascular smooth muscle relaxation [Palmer, R. M. J., Ashton, D. S., & Moncada, S. (1988) Nature (London) 333, 664-666].

Influence of ascorbic acid dose on N-nitrosoproline formation in humans.

A relationship between ascorbic acid intake and N-nitrosoproline (NPRO) excretion in humans on a controlled diet was established. Seven healthy males were placed on a low nitrate, low ascorbic acid diet for 12 consecutive days. On days 3-12, a 5.24 mmol oral dose of sodium nitrate was administered in mid-afternoon, at least 2 h after the subject's last meal. On days 4-12, a 4.35 mmol oral dose of L-proline was administered 30 min after the nitrate dose. Ascorbic acid was given in amounts which increased daily from day 5 to day 10 (0.01-5.68 mmol; 1.76-1000 mg) with the proline. Total 24 h urines were assayed for nitrate, NPRO and total ascorbic acid. Nitrate balance was monitored using [15N]nitrate. Average endogenous nitrate synthesis was 1.28 +/- 0.43 mmol/day/person. NPRO excretion was reduced by 6 nmol/day when 0.05 mmol of ascorbic acid was administered. However, as much as 5.68 mmol ascorbic acid did not return NPRO excretion to levels observed before the nitrate and proline were administered. More than 10 times the ascorbic acid required to completely inhibit NPRO formation in vitro did not return NPRO excretion to baseline levels. These data indicate that endogenous nitrosation may be more facile than predicted by the in vitro chemistry.

Relationship between ascorbic acid dose and N-nitrosoproline excretion in humans on controlled diets.

A logarithmic dose-response relationship between ascorbic acid dose and N-nitrosoproline (NPRO) excretion in humans on a controlled diet was established. Seven healthy males were placed on a low-nitrate, low-ascorbic acid diet for 12 consecutive days and given nitrate on days 3-12 and L-proline on days 4-12, after the nitrate dose. Ascorbic acid was given in increasing amounts with the proline on days 5-10. Urine was analysed quantitatively for nitrate, NPRO and ascorbic acid. Ascorbic acid doses as low as 0.05 mmol reduced NPRO excretion by an average of 6 nmol/day; however, as much as 5.68 mmol ascorbic acid did not return NPRO excretion to levels observed before nitrate and proline were administered. Complete inhibition of endogenous NPRO formation from exogenous precursors requires more than the 2:1 molar ratio of ascorbic acid to nitrite that has been demonstrated in vitro. These data may be useful in interpreting epidemiological studies of nitrate exposure and in making dietary recommendations.