Quantitative determination of apogossypol, a pro-apoptotic analog of gossypol, in mouse plasma using LC/MS/MS.

A simple and selective liquid chromatography/tandem mass spectrometry (LC/MS/MS) method based on internal standard quantitation using apigenin as the internal standard has been developed and validated for the analysis of the gossypol analog apogossypol, a pro-apoptotic compound, in mouse plasma. The methodology involves protein precipitation of plasma samples followed by LC/MS/MS analysis. Ascorbic acid was added to the spiking solutions and plasma samples to stabilize the easily oxidized compound. Separation of apogossypol and the internal standard from the plasma matrix was achieved using a C18 column with a gradient elution profile consisting of 5mM ammonium acetate and methanol. The validated range of the method extended from 10 to 2000 ng/mL with accuracies of 85-115% and precision of <15%. The average recovery of apogossypol at three concentrations (50, 200 and 1000 ng/mL) assayed in triplicate using this methodology was determined to be 90.8+/-12.9%. Recovery for the internal standard (apigenin) at a concentration of 500 ng/mL was found to be 99.9+/-6.41%. Apogossypol concentrations of 50 ng/mL and above were found to be stable in extracted plasma for 24h when stored at 25 degrees C. This method has been applied to the determination of apogossypol concentrations in plasma collected from mice given an IV dose of apogossypol.

Secondary screening system for preclinical testing of human lung cancer therapies.

The National Cancer Institute has instituted a primary screening system for testing new agents against cultured cancer cell lines. The purpose of this study was to determine the feasibility of using a nude rat orthotopic (organ-specific) human lung cancer model system as an in vivo secondary screen for general evaluation of new anticancer agents and therapies active against lung cancer. To make this determination, we tested whether this system allows measurement of uptake and tumoricidal activity of anticancer therapies. Tumor-bearing lungs from 53 Rowett nude rats with orthotopically implanted human large-cell undifferentiated lung carcinoma (NCI-H460) were perfused ex vivo for 1 hour with or without each of two anticancer modalities. Lungs were perfused with blood-free perfusate alone (untreated control), perfusate with 100 micrograms/mL doxorubicin (treated positive control), or perfusate with lymphokine-activated killer cells plus human recombinant interleukin-2 (LAK/rIL-2). Weight gain during perfusion was the criterion used to quantitate lung injury. Treatment efficacy was measured by clonogenic assay after enzymatic disaggregation of the perfused tumors. Doxorubicin levels in the tumor and in the uninvolved lung were measured by high-performance liquid chromatography. Both treatment groups showed only slight increases in lung weight compared with that in the untreated control group, suggesting good lung tolerance of the procedure. Lung and tumor levels of doxorubicin were 320 +/- 21 ng/mg of tissue and 32 +/- 5 ng/mg of tissue (means +/- SE), respectively. Clonogenic assay demonstrated a fivefold to 10-fold reduction in the surviving fraction of tumor cells with doxorubicin but no change with LAK/rIL-2.(ABSTRACT TRUNCATED AT 250 WORDS)

Diversity of penetration of anti-cancer agents into solid tumours.

Failure of anti-cancer agents to reach all clonogenic cells at cytotoxic concentrations is recognized as an important form of resistance in solid tumours. Subcutaneously implanted mammary adenocarcinoma 16/C was used to evaluate the intratumour distribution of five alkylating, bioreductive alkylating and intercalating agents and two radiation sensitizers. The agents were classified according to their in vivo distribution in well- and poorly-perfused tumour regions, as delineated by lissamine green. The classifications were: (1) distribution in direct proportion to the vascular supply; (2) uniform distribution to well- and poorly-perfused tumour regions; and (3) preferential retention in the poorly-perfused tumour regions. Our current state of knowledge did not allow reliable prediction of the classification based on chemical structure or mechanism of action.

Distribution of adriamycin in mice bearing mammary adenocarcinoma 16/C.

The response of solid mammary adenocarcinoma 16/C to treatment with Adriamycin is highly variable and ranges from growth under treatment to complete regression. Tumour and host factors were evaluated to determine the influence of each on the response. We determined that the concentration of Adriamycin in plasma and tumour was a function of tumour size and treatment history in mice bearing mammary adenocarcinoma 16/C. The plasma concentrations following a single dose of Adriamycin (10 mg/kg) increased in proportion to tumour mass without a concurrent increase in tumour concentration. When mice bearing large tumours (greater than 1.0 g) were treated with a multidose protocol, the plasma concentrations were higher and the tumour concentrations lower following the initial dose than following subsequent doses; in tumour-free mice, prior treatment with Adriamycin did not affect the plasma level achieved after a second dose. The magnitude of the decrease in plasma and increase in tumour concentrations was a function of the initial tumour size and the treatment schedule. The increase in tumour levels represented the sum of residual Adriamycin and drug bound as a result of the dose immediately prior to analysis. At the time of the initial treatment, the Adriamycin was distributed within each tumour in proportion to vascular perfusion. The percent of the tumour mass that was well-perfused appeared to increase with repeated treatments. The results indicate that the plasma concentration of Adriamycin did not necessarily reflect the tumour exposure in the mammary adenocarcinoma 16/C model. In hosts bearing mammary adenocarcinoma 16/C--or, possibly, other tumours that produce similar effects on the host--a low initial dose of Adriamycin might modify the distribution, possibly reduce the toxicity and allow escalation of subsequent doses with increased exposure of the tumour.

Antimitotic agents: structure-activity studies with some pyridine derivatives.

Antitumor activity in mice was observed for the oxime of the previously reported ethyl [6-amino-4-[(1-methyl-2-phenyl-2-oxoethyl)amino]-5-nitropyridin -2-yl] carbamate (8) and several related compounds. These compounds are precursors of the active ethyl pyrido[3,4-b]pyrazin-7-ylcarbamates (e.g., 4), which are potent antimitotic agents. In the 5-nitropyridine series overall biological activity was reduced by replacement of the oxime moiety with a keto or alcohol group and by replacement of the 1-methyl group of the side chain with hydrogen. Reduction of the nitro group of the 5-nitropyridines containing an alcohol in the side chain to the corresponding 5-aminopyridines increased biological activity. Preliminary studies showed that the 5-nitropyridine oximes were considerably less potent than the pyridopyrazines as antimitotic agents and that the former are apparently not converted to the latter in vivo. The inhibition of the incorporation of pyrimidine nucleosides into DNA and RNA was identified as another possible mode of action of the 5-nitropyridine oximes.

Pharmacokinetics of perfluorooctanoate in cynomolgus monkeys.

The pharmacokinetics of perfluorooctanoate (PFOA) in cynomolgus monkeys were studied in a six-month oral capsule dosing study of ammonium perfluorooctanoate (APFO) and in a single-dose iv study. In the oral study, samples of serum, urine, and feces were collected every two weeks from monkeys given daily doses of either 0, 3, 10, or 20 mg APFO/kg. Steady-state was reached within four weeks in serum, urine, and feces. Serum PFOA followed first-order elimination kinetics after the last dose, with a half-life of approximately 20 days. Urine was the primary elimination route. Mean serum PFOA concentrations at steady state in the 3, 10, and 20 mg/kg-day dose groups, respectively, were 81, 99, and 156 microg/ml in serum; 53, 166, and 181 microg/ml in urine; and, 7, 28, and 50 microg/g in feces. Mean liver concentrations reached 16, 14, and 50 microg/g in the 3, 10, and 20 mg/kg groups, respectively. In the iv study, three monkeys per sex were given a single dose of 10 mg/kg potassium PFOA. Samples were collected through 123 days. The terminal half-life of PFOA in serum was 13.6, 13.7, and 35.3 days in the three male monkeys and 26.8, 29.3, and 41.7 days in the three females. Volume of distribution at steady state was 181 +/- 12 and 198 +/- 69 ml/kg for males and females, respectively. Based on the result of both the oral and iv studies, the elimination half-life is approximately 14-42 days, and urine is the primary route of excretion.

Variability of tumor response to chemotherapy. I. Contribution of host heterogeneity.

Host factors that might be associated with the variable response of tumors to effective chemotherapy were studied in B6C3F1 mice bearing transplanted mammary adenocarcinoma 16/C tumors and treated with melphalan. Tumor response ranged from regression to an unpalpable size to growth under treatment. That biochemical resistance of the cell population was not primarily responsible for the variability was demonstrated by passage of responsive and nonresponsive tumors into new hosts followed by treatment with melphalan. When the implanted subcutaneous tumor weighed 1.0 g or less (usually 12 to 13 days postimplant), both the plasma levels of melphalan and the variability in plasma levels were similar to those observed in tumor-free mice. With tumor progression beyond 1.0 g, an increase in mean plasma levels and in variability, but not in plasma half-life, was observed. A correlation between the dose of melphalan administered, the schedule, and the percentage of tumor responses was found. There was no correlation between the plasma levels in individual mice following a given dose of melphalan and subsequent tumor response. Also, there was no correlation between the plasma levels of melphalan in individual mice following the second, third or fourth treatment in a multiple-dose treatment schedule and the response of the tumor in that mouse to previous treatments. Prior therapy (1, 2 or 3 doses administered 4 days apart) either prevented the increase in plasma levels that occurred in mice bearing untreated advanced tumors or reduced the plasma level (and the variability) to approximately that found in tumor-free mice. Whether this was a direct result of the effects of melphalan on the host or an indirect result of tumor inhibition is not known. A similar study in tumor-free mice indicated that prior treatment had only minimal effects on subsequent plasma levels. These studies indicate that heterogeneity of the host was not a major factor in variable tumor response if therapy was initiated when the tumors weighed 1.0 g or less.

Variability of tumor response to chemotherapy. II. Contribution of tumor heterogeneity.

The role of tumor-to-tumor variability in response to chemotherapy was investigated in mice bearing mammary adenocarcinoma 16/C treated with melphalan. Lissamine green, a triphenylmethane dye, was given systemically to delineate areas of perfusion in the tumors. The regions of low perfusion ranged from less than 10% to greater than 90% of the mass of individual tumors. The variation in perfusion was as large between bilateral tumors in a mouse as between tumors in different hosts. The presence of viable cells capable of continued growth in the regions of low perfusion was demonstrated by bioassay. Concentrations of melphalan following i.p. administration varied by as much as tenfold or more between regions of low and high perfusion. Concentrations of melphalan in the well-perfused regions were similar to plasma concentrations at 30 min after administration, but elimination from the plasma was more rapid. The levels of melphalan in the tumor were higher following the initial dose than following succeeding doses in a multiple dose schedule. The results indicate that tumor-to-tumor variations in perfusion and drug distribution are major factors in variable tumor response.

Distribution of nitroimidazoles and L-phenylalanine mustard in mammary adenocarcinoma 16/C tumors.

Using the triphenylmethane dye, lissamine green, as an indicator of blood perfusion, we have demonstrated that L-phenylalanine mustard (L-PAM) is differentially distributed in mice bearing mammary adenocarcinoma 16/C tumors. Following i.p. administration, concentrations of L-PAM in various regions of the tumors vary by as much as 10-fold or more between regions of low and high perfusion. Since the nitroimidazoles, metronidazole and misonidazole, increase the cytotoxicity of certain antitumor agents, these compounds were investigated for their ability to increase the distribution of L-PAM into tumor regions of low perfusion. Administration of metronidazole (400 mg/kg) or misonidazole (800 mg/kg) 1 h prior to L-PAM and lissamine green resulted in elevated plasma levels of L-PAM and increased concentrations of L-PAM in tumor regions of high perfusion. A slight increase in the normally low levels of L-PAM in tumor regions of low perfusion was observed but the increase was not statistically significant. In contrast to the uneven distribution of L-PAM, metronidazole and misonidazole were evenly distributed throughout plasma and tumor regions of both high and low perfusion. Bioassay of tumors following in vivo exposure to metronidazole and L-PAM indicated decreased viability in fragments from tumor regions of high perfusion, but not from tumor regions of low perfusion. These studies demonstrate that the nitroimidazoles increased L-PAM levels in plasma and in tumor regions of both high and low perfusion but did not induce a uniform distribution of L-PAM throughout the tumors. The nitroimidazoles may enhance the effectiveness of L-PAM as an antitumor agent by increasing the concentration of drug that reaches a tumor.

Biodistribution of radiolabeled lipid-DNA complexes and DNA in mice.

The tissue biodistribution and expression of [33P]DNA-1-[2-[9-(Z)-octadecenoyloxy]ethyl]]-2-[8](Z)-heptadece nyl]-3 -[hydroxyethyl]imidazolinium chloride (DOTIM):cholesterol complexes and 33P-radiolabeled DNA expressing chloramphenicol acetyl transferase (CAT; 4.7 kB) were studied after intravenous (iv) injection in ICR mice. Mice were injected with 200 microL of complex containing DNA at 3 mg/kg or DNA alone. One group received 8 microCi of radioactivity and were sacrificed at 5 and 20 min, and 1, 2, 4 and 24 h post-dose (n = 4/time point). A second group received the equivalent of 3.9 microCi of radioactivity and were sacrificed at 20 min, and 2 and 24 h for subsequent whole body autoradiographic analysis (WBA; n = 2/time point). The tissue distribution of intact DNA was assessed by Southern blot at 24 h post-dose, whereas the integrity of complexes and DNA incubated in heparinized whole blood was studied separately. In further studies, the time course of expression in lung tissue over a 48-h period was examined, and the relative lung-expression of purified open circular (OC) versus supercoiled (SC) DNA at 24 h was evaluated. Approximately 42% of the radioactivity was found in the lungs 5 min after injection and about half this percentage was found in the liver. By 2 h, only 5% remained in the lungs, but 48% was present in the liver. No other tissue accumulated >5% of the dose throughout the duration of the study. WBA radiograms confirmed the tissue distribution results and highlighted significant accumulation of radioactivity in bone over time. Southern Blot analysis demonstrated intact DNA in many tissues 24 h after dosing. In contrast, the majority of DNA incubated in blood was degraded within 2 h, although the complexes afforded some protection relative to DNA alone. The OC DNA expressed equivalently to SC DNA in lung tissue (OC = 1035 +/- 183 pg; SC = 856 +/- 257 pg/mg soluble protein, n = 6, mean +/- SEM) at 24 h, and detectable levels of CAT were present within 2 h of dosing (21.3 +/- 7.2 pg, n >/= 8, mean +/- SD). The results confirm that DNA-DOTIM:cholesterol complexes are initially deposited in the lungs after iv administration.

Metabolism of 14C-labelled sucrose esters of stearic acid in rats.

Rats were dosed by oral gavage (250 mg/kg) with compounds containing sucrose esterified in four, six or eight positions with stearic acid. For each compound, rats excreted greater than 95% of the dose in the faeces. The extent of absorption and metabolism of radioactivity was inversely related to the degree of esterification. For rats dosed with sucrose esters labelled in the fatty acid moieties, the degree of absorption of radioactivity was highest for the tetraesterified compound (5.9% of the dose). At 120 hr after dosing with this derivative, the highest concentrations of radioactivity, aside from tissues of the gastrointestinal tract, were found in fat (183 micrograms-equivalents/g tissue), lymph nodes (117 micrograms-equivalents/g tissue) and the liver (88 micrograms-equivalents/g tissue); appreciable radioactivity appeared in the blood (3.9 micrograms-equivalents/g tissue) and collected lymph (5.0-7.6% of the dose). For rats dosed with esters labelled in the sucrose moiety, the amounts of radioactivity absorbed were lower than after dosing with the corresponding sucrose derivatives labelled in the fatty acid moieties; the absorbed radioactivity was greatest following administration of the tetraesterified compound (3.0%). Relatively little radioactivity was found in tissue samples collected from these rats. These results are consistent with limited hydrolysis of the sucrose esters, presumably to sucrose and fatty acids, prior to intestinal absorption.

Toxicity of methylsulfonylmethane in rats.

Methylsulfonylmethane (MSM) is a popular dietary supplement used in a variety of conditions including pain, inflammation, allergies, arthritis, parasitic infections and the maintenance of normal keratin levels in hair, skin and nails. Despite its popularity, there is little published toxicology data on MSM. The objective of this study was to evaluate the acute and subchronic toxicity of MSM in rats at a dose five to seven times the maximum recommended dose in humans. MSM administered in a single gavage dose of 2 g/kg resulted in no adverse events or mortality. MSM administered as a daily dose of 1.5 g/kg for 90 days by gavage resulted in no adverse events or mortality. Necropsy did not reveal any gross pathological lesions or changes in organ weights. Renal histology of treated animals was normal. It is concluded that MSM is well tolerated in rats at an acute dose of 2 g/kg and at a subacute chronic dose of 1.5 g/kg.

Disposition of sucrose octa-isobutyrate in rats, dogs and monkeys.

The disposition of 200 mg/kg of 14C-labelled sucrose octa-isobutyrate (14C-SOIB), a component of sucrose acetate isobutyrate (SAIB), a beverage emulsion stabilizer, was studied in rats, dogs and monkeys. After oral administration of 14C-SOIB to three rats, 3-15% of the dose was excreted as volatile products, 1-2% appeared in urine and 78-93% was recovered in faeces. In dogs, recoveries of radiolabel in CO2, urine and faeces were approximately 1%, less than 2% and 77-94%, respectively. Monkeys excreted the majority of the dose in faeces; less than 2% of the administered radioactivity was eliminated in either CO2 or urine. The biliary excretion of radiolabel from 14C-SOIB was negligible in rats and monkeys; however, in dogs, 3-10% of the dose was excreted into bile. It was demonstrated by chromatographic analyses of faeces that 14C-SOIB was more extensively hydrolysed in the gastro-intestinal tract of rats and dogs than in monkeys. The results indicate that after oral administration, rats and dogs absorb SOIB following hydrolysis of the sugar ester in the gut. The proportion of the dose that is absorbed by the rat is oxidized to CO2. In the dog, little of the absorbed product is oxidized; rather, it is circulated through an enterohepatic pathway. In contrast, in the monkey, SOIB is not detectably hydrolysed in the gut or absorbed. These findings show that there is a species difference in the disposition of SOIB; the most salient findings relate to a difference in the disposition of SOIB in the dog compared with the rat.

The role of folates in methanol toxicity.

In the monkey and human, methanol toxicity is characterized by a metabolic acidosis and an ocular toxicity which occur coincident with an accumulation of formate in blood. In contrast, methanol insensitive species such as the rat do not accumulate formate after methanol administration. Folate-dependent reactions are involved in the oxidation of formate to CO2 in both the rat and the monkey. Monkey liver contains a significantly lower hepatic folate level than does rat liver and, thus, formate accumulation in the monkey may be related to a functional folate deficiency in this species. Formate metabolism in the monkey can be stimulated with folate administration. After methanol administration, treatment of monkeys with repetitive doses of either 5-formyltetrahydrofolic acid or folic acid results in a marked decrease in blood formate accumulation, an absence of metabolic acidosis and no blood bicarbonate depletion. Also, methanol toxicity, once established in the monkey, can be reversed with 5-formyltetrahydrofolic acid administration. The results indicate that 5-formyltetrahydrofolic acid decreases formate accumulation after methanol by stimulating the rate of formate oxidation or utilization and provide additional evidence for the involvement of folate-dependent reactions in the metabolism and toxicity of methanol in the monkey.

Distribution of clomesone in mice.

The distribution of the novel alkylating agent clomesone [2-chloroethyl (methylsulfonyl)-methane sulfonate] has been studied in mice after iv administration of 35 mg/kg. In plasma, [14C]clomesone was eliminated in an apparent single phase with a half-life of 11 minutes. The elimination of total radioactivity occurred with an initial half-life of 51 minutes, and then in prolonged phase of undetermined length. At least two organic soluble metabolites were detectable in plasma. Highest tissue concentrations of radioactivity were in liver, kidney, and small intestines where, at most times of analysis, the levels were two to three times higher than those in plasma. The rate of elimination of radiolabel from spleen, lungs, brain, and muscle, which contained about equal concentrations of radioactivity at most times after dosing, paralleled the rate of loss from plasma. A major proportion of the radioactivity in tissues was bound to trichloracetic acid-precipitable macromolecules. Within 24 hours, 77.6% of the dose was recovered in urine. High-pressure liquid chromatographic analyses of 8-hour urine collections demonstrated that less than 2% of the dose was excreted unchanged. These results demonstrate that clomesone is rapidly eliminated from plasma and that it and/or its products become extensively bound to tissue components.

Immunological properties of glycolipids from membranes of Acholeplasma laidlawii.

Glycolipids, the predominant class of lipids in the membranes of Acholeplasma laidlawii, are the haptenic determinants that react with anti-A. Laidlawii serum to fix complement. The predominant complement-fixing activity of the membrane glycolipids was associated with the monoglucoysyl diglyceride, diglucosyl diglyceride, glycerlphosphoryl diglucosyl diglyceride (GPDD), and an unknown lipid B, which did not react with ninhydrin but release glucose and glycerol and traces of phosphorus upon hydrolysis. The glycolipids monoglucosyl diglyceride and diglucosyl diglyceride or GPDD and unknown lipid B were paired as a result of their cross-reactions with selective antisera prepared with the aid of reconstituted membrane complexes containing membrane lipids. Reconstituted membrane complexes assembled from [14C]monoglucosyl diglyceride and delipidated membrane proteins gave optimal complement fixation titers before saturation of the complexes with the ]14C]monoglucosyl diglyceride. The phosphoglycolipid of the membrane, GPDD, was anticomplementary as a pure lipid, a cholesterol liposome, and a reconstituted membrane complex. This anticomplementary activity, which was caused by 3 mug of pure GPDD, affected both human and guinea pig complement. Although human C1, C4, C3, and C5 were not inhibited by GPDD, C2 was inhibited 10-fold by reconstituted membrane complexes containing 150 mug of GPDD. A role for this phosphoglycolipid is discussed in the hypothetical mechanism of inhibition of C2 attachment to SAC1, 4 sites.

Methanol toxicity: treatment with folic acid and 5-formyl tetrahydrofolic acid.

After methanol administration to monkeys, an accumulation of formate in blood occurs coincident with the development of metabolic acidosis and a depletion of blood bicarbonate. Formate metabolism in monkeys depends upon and is regulated by a folate-dependent system; therefore, the effect of folic acid pretreatment and 5-formyl tetrahydrofolic acid administration on methanol toxicity was investigated. Treatment of monkeys with repetitive doses of either sodium folate (48, 24, 12, and 4 hr prior to methanol) or 5-formyl tetrahydrofolic acid (2 mg/kg at 0, 4, 8, 12, and 18 hr after methanol) resulted in a marked decrease in the levels of blood formate and an absence of both metabolic acidosis and depletion of blood bicarbonate following methanol administration. Also, 5-formyl tetrahydrofolic acid reversed methanol toxicity once it was established in the monkey. The results indicate that folate compounds decrease formate accumulation after methanol by stimulating formate oxidation or utilization and suggest a possible use for folates in the treatment of certain cases of human methanol poisoning.

Disposition of thymidine administered as large doses to rats and mice.

Blood and urine levels of thymidine and its catabolic product, thymine, have been determined for mice and rats given a single large dose of thymidine and for rats during and after infusion of large amounts of this drug. For mice given a bolus dose (400 mg/kg, 1.2 g/m2), two phases of elimination of thymidine from blood were evident, an initial phase (half-life = 4 mins) and a longer second phase (half-life = 17 mins). The initial (2-min) level in blood was 2.8 mM. For rats given an equivalent dose (150 mg/kg, 1.2 g/m2), three phases with half-lives of 2, 29, and 365 mins were observed. The initial (5-min) concentration in the blood was 0.6 mM. More of a dose of [2-14C]thymidine was converted to CO2 by rats than by mice. For both species, small amounts of radioactivity from this labeled compound became associated with macromolecules of the small intestine. Following infusion of rats with thymidine at rates of 300 and 600 mg/kg/hr for 24 hrs (60 and 120 g/m2, respectively), steady-state blood levels were approximately 0.7 and 1.5 mM, respectively. When the infusions were stopped, a phase with a half-life of 35 mins and a longer phase of indeterminate length were noted for each dose. Elimination of metabolically formed thymine from the blood of rats and excretion into the urine was mediated by a process that was apparently saturated.

Methanol poisoning and formate oxidation in nitrous oxide-treated rats.

Formic acid does not accumulate in the rat after the administration of methanol as it does in methanol-poisoned humans and monkeys. In addition, rats do not manifest the metabolic acidosis and ocular toxicity characteristic of methanol intoxication in primates. Nitrous oxide treatment was used to inhibit 5-methyltetrahydrofolate homocysteine methyltransferase (methionine synthetase, EC 4.2.99.10) in order to delineate the role of this enzyme in regulating the metabolism of formate in rats and in determining the sensitivity of this species to methanol intoxication. Nitrous oxide treatment resulted in a decrease in hepatic levels of nonmethylated tetrahydrofolate forms and an increase in 5-methyltetrahydrofolate. Rats treated with nitrous oxide exhibited a marked decrease in the rate of oxidation of formate to carbon dioxide. The rate of disappearance of formate from the blood in these animals was decreased to half the control rate. Rats treated with nitrous oxide and administered methanol accumulated formate in blood and developed metabolic acidosis. These studies support the concept of a key role of methionine synthetase in supplying the tetrahydrofolate required for the folate-dependent oxidation of formate to carbon dioxide as well as the importance of this pathway in determining the sensitivity of a species to methanol poisoning.