Therapeutic response to single intravenous bolus administration of formate dehydrogenase in methanol-intoxicated rats

Toxicology Letters 161 (2006) 89–95

Therapeutic response to single intravenous bolus administration of formate dehydrogenase in methanol-intoxicated rats Arumugham Muthuvel, Rathinam Rajamani, Rathinasamy Sheeladevi ∗ Department of Physiology, Dr. A.L.M.Postgraduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai 600113, Tamil Nadu, India Received 14 April 2005; received in revised form 6 July 2005; accepted 7 July 2005 Available online 26 September 2005

Abstract Methanol remains to be a major public and environmental health hazard. Formic acid is the toxic metabolite responsible for the metabolic acidosis observed in methanol poisoning in humans, in non-human primates and in folate-depleted rodents. Cytochrome oxidase inhibition by formate leads to lactic acid accumulation, which contributes significantly to metabolic acidosis. Toxic effects in human beings are characterized by formic acidemia, metabolic acidosis, ocular toxicity, nervous system depression, blindness, coma and death. Elimination of formate is one of the principles of management in methanol poisoning. Hemodialysis facility is not readily available in all the places, in developing countries like India. Formate dehydrogenase (EC 1.2.1.2) acts directly over formate and converts formate into CO2 in the presence of NAD. Effect of single intravenous bolus infusion of formate dehydrogenase, obtained from Candida boidinii; in methanol-intoxicated folate deficient rat model was evaluated. Folate depletion induced by methotrexate (MTX) treatment. Carbicarb (Carb) (equimolar solution of sodium carbonate and sodium bicarbonate) was used to treat metabolic acidosis. Experimental design consists of seven groups, namely Saline control, methanol control, MTX control, Enzyme control, MTX-methanol control, MTX-methanol-Carb and MTX-methanol-Carb-Enz group. Male wistar rats treated with MTX (0.3 mg/kg) for a week, were injected (i.p.) with methanol (4 gm/kg), 12 h latter, Carbicarb solution was infused, following this enzyme was infused (i.v.) in bolus. Blood samples were collected every 15 min for an hour from the cannulated left jugular vein and blood methanol, formate were estimated, respectively, with HPLC and fluorimetric assay. Blood pH, blood gases pO2 , pCO2 and bicarbonate were monitored with blood gas analyzer in order to evaluate acid base status of the animal. Results obtained show that there is significant elimination of formate within 15 min. It may be concluded that single bolus infusion of formate dehydrogenase facilitates fast removal of formate, a highly toxic metabolite in methanol poisoning. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Methanol; Formate dehydrogenase; Folate; Formate; Methotrexate

1. Introduction Methanol is one of the major adulterants of illicit liquor in India causing blindness and death (Kumar

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Corresponding author. Tel.: +91 44 24480789; fax: +91 44 24926709. E-mail address: [email protected] (R. Sheeladevi).

et al., 2003; Mittal et al., 1991; Ravichandran et al., 1984). Cluster of infant deaths was reported due to topical application of methanol after a vaccination programme in Egypt (Darwish et al., 2002). It is one of the widely used industrial solvents and used in the production of many synthetic organic compounds. It is used as fuel in gasoline blends. Methanol remains to be a major public and environmental health hazard.

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A. Muthuvel et al. / Toxicology Letters 161 (2006) 89–95

Methanol is well absorbed from the gastrointestinal tract mucosa as well as through the skin and lungs. Apart from oral ingestion, both inhalation and transdermal exposure can result in toxicity. It should be noted that the highest morbidity and mortality have been associated with deliberate or accidental oral ingestion of methanolcontaining mixtures (WHO, 1997). In mammalian species, methanol is metabolized to formaldehyde in the liver and by subsequent oxidative steps, formic acid and carbon dioxide are formed (Makar and Tephly, 1977; Liesivuori and Savolainen, 1991). The metabolism of formate is mediated through a tetrahydrofolate dependent path way (Eells et al., 1982). Humans (and non-human primates) are uniquely sensitive to methanol poisoning because of their low liver folate content (Johlin et al., 1987). The toxic effects in these species are characterized by formic acidemia, metabolic acidosis, ocular toxicity, nervous system depression, blindness, coma and death (Viccellio, 1998). Specifically, formic acid is the toxic metabolite responsible for the metabolic acidosis observed in methanol poisoning in humans, in non-human primates and in folate-depleted rodents (Liesivuori and Savolainen, 1991; Lee et al., 1994b). Formic acid is believed to be the toxic metabolite responsible for the ocular toxicity in methanol-poisoned humans (Sharpe et al., 1982), and is also responsible for the ocular toxicity produced in non-human primates and folatedepleted rodents (Eells, 1991; Murray et al., 1991; Eells et al., 1995). Cytochrome oxidase inhibition by formate (Nicholls, 1975) leads to lactic acid accumulation, which contributes significantly to metabolic acidosis (Smith et al., 1981; Liesivuori and Savolainen, 1991). Hence, formate takes part in metabolic acidosis directly as well as indirectly. Rodents do not develop metabolic acidosis in methanol poisoning similar to human beings owing to their high liver folate content, formate is metabolized quickly. Only, folate deficient rodents they do develop formate accumulation and acidosis (Lee et al., 1994a; Eells et al., 2000). Methotrexate (MTX), a dihydrofolate analog inhibits dihydrofolate reductase, an enzyme which is important for convertion of dihydrofolate to tetrahydrofolate. MTX treatment causes depletion of folate stores (Barford et al., 1980; Schalinske and Steele, 1996). Hence, folate dependent formate metabolism can be impaired by MTX treatment. MTX treated folate deficient rat model was utilized to study the effect of chronic methanol exposure on amino acids and on monoamines of nervous tissue (Gonzalez-Quevedo et al., 2002). The treatment protocol for methanol poisoning as reported by Barceloux et al., 2002, includes correction of

metabolic acidosis by bicarbonate infusion, administration of folinic acid (active form of folic acid) to facilitate formate metabolism, administration of alcohol dehydrogenase inhibitor, Fomepizole (alcohol dehydrogenase inhibitor) to prevent further breakdown of methanol and selective hemodialysis to eliminate methanol and formate. Hemodialysis facility is not readily available in all the places in developing countries like India. Folinic acid therapy facilitates formate metabolism indirectly by increasing the liver folate content. Formate dehydrogenase (EC 1.2.1.2) acts directly over the formate and converts formate into CO2 in the presence of NAD. This enzyme may be utilized to breakdown formate, which accumulates as a toxic metabolite in methanol poisoning. Since, enzymatic elimination is very fast, quick removal of formate may potentially be beneficial in correcting metabolic acidosis and in preventing tissue toxic effect of formate. Many enzymes are clinically used, to eliminate specific substances from the body, for example, uricase in hyperuricemia (Bomalaski and Clark, 2004), asparaginase in acute lymphoblastic leukemia (Nesbit et al., 1979) and glucocerebrosidase in Gaucher’s disease (Barton et al., 1990). Carbicarb (Carb) was used to treat the metabolic acidosis. Carbicarb is an alkalinizing agent, combination of 0.33 M sodium carbonate and 0.33 M sodium bicarbonate (Filley and Kindig, 1984; Shapiro et al., 1989; Kucera et al., 1989; Forsythe and Schmidt, 2000). Moreover, buffering by Carbicarb consumes CO2 (Adrogue and Madias, 1998), which may facilitate elimination of CO2 , a byproduct of this enzyme-based approach to eliminate formate. In this present study, efficacy of single bolus intravenous injection of formate dehydrogenase in eliminating the formate, a highly toxic metabolite in methanol poisoning, was evaluated. 2. Materials and methods 2.1. Reagents Methanol (HPLC grade) obtained from Sisco Research Laboratories, Bombay, India. All other reagents were of analytical grade. Formate dehydrogenase from Candida boidinii (10.5 units/mg protein) was purchased from Sigma–Aldrich Co., St. Louis, MO. Formate dehydrogenase enzyme from C. boidinii has molecular weight of 74,000 (Schuette et al., 1976). Minimum optimal pH of the enzyme is 7.5. Its activity was assessed according to the published method (Schuette et al., 1976). One unit of enzyme was defined as the amount of enzyme that catalyzes the conversion of one micromole of NAD to NADH per minute at pH 7.5 at 37 ◦ C.

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2.2. Methanol intoxication experiment Male wistar rats (230–250 g) were used. Animals were maintained in a clean rodent room. They were housed two to three per cage in cages that were fitted with stainlesssteel wire-mesh bottoms. They were maintained at a temperature of 28 ± 1 ◦ C, and under a daily photoperiod of 12-h light/dark cycle. The animals were fed with pellet diet (Hindustan Lever Ltd., Mumbai, India) and tap water ad libitum. The commercial rat feed contained 5% fat, 21% protein, 55% nitrogen free extract, 4% fibre (w/w) with adequate mineral and vitamin contents. The animals were handled according to the principles of laboratory care framed by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Govt. of India. The experimentation protocol was reviewed and approved by Institutional Ethical committee. For treatment, methanol (MeOH) was diluted in sterile saline (20% (w/v) solution) and administered intraperitoneally. Methotrexate was also diluted in sterile saline and administered subcutaneously. Carbicarb solution was prepared in sterile saline (0.33 M sodium carbonate and 0.33 M sodium bicarbonate) (Shapiro et al., 1989). The rats were divided into seven groups. Each group consists of six animals. Four groups of animals, namely MTX control, MTX-MeOH control, MTX-MeOH-Carb, MTXMeOH-Carb-Enz groups were administered with methotrexate 0.3 mg/kg (s.c.) for seven days (Gonzalez-Quevedo et al., 2002). Another three groups of animals, namely Saline control, Enzyme control, MeOH control received saline (s.c.) for one week. On the seventh day, to permit repeated sampling, the rats were anaesthetized with ketamine/xylazine (70 mg/kg (i.m.)/3 mg/kg (i.p.)). The left jugular vein was cannulated with silastic tubing (0.025 in. i.d. × 0.047 in. o.d., 20 cm long; Dow Corning Corporation). After overnight recovery from the anaesthetic drug, the animals were taken for study. (Horton et al., 1992). Animals were cannulated, a day before experiment in order to avoid possible influence of anaesthetic drugs over blood gases. On the eighth day of experiment, MTX treated animal models received 200 ␮l of methotrexate 0.3 mg/kg (s.c.). All other control animals (Saline control, MeOH control, Enzyme control) received 200 ␮l saline (s.c.). Two hours later, MTXMeOH control, MTX-MeOH-Carb, MTX-MeOH-Carb-Enz, and MeOH control were administered with methanol diluted in saline (4 gm/kg) (i.p.). Saline control, Enzyme control and MTX control groups received equivalent volume of saline (i.p.). After 12 h, MTX-MeOH-Carb and MTX-MeOH-Carb-Enz groups were infused (i.v.) with Carbicarb solution (2 ml/kg) (Shapiro et al., 1989) through rat tail vein over a period of 2 min. After 3 min, MTX-MeOH-Carb-Enz group and Enzyme Control group were administered with 200 ␮l enzyme solution (300 units of enzyme in saline) (i.v.). All other groups received equal amount of saline (i.v.). Carbicarb and enzyme infusions were done through rat tail vein and blood withdrawals were done from the cannulated left jugular vein. After

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intravenous infusion of enzyme, blood samples were collected for an hour, at every 15 min time points from the cannulated jugular vein. Prior to intravenous infusion of Carbicarb and enzyme or saline, one blood sample was taken which was considered as zero time value. In the end of experiment, the venous cannula was surgically removed under anesthesia and the animals were left in the cages after proper wound treatment. 2.3. Blood methanol measurement Hundred microliters plasma was deproteinized with equal volume of acetonitrile and centrifuged at 5000 × g for 7 min at 4 ◦ C. Supernatant was analyzed by HPLC Refractive index detector system (Shimadzu RID 10A) equipped with a Rezex ROA-organic acid column (300 mm × 7.5 mm i.d.; Phenomenex) (Dorman et al., 1994) and security guard cartridge (AJO 4490, Phenomenex). Column oven was used to maintain the temperature at 60 ◦ C. The mobile phase was 0.026 N H2 SO4 (Pecina et al., 1984; Sharma et al., 1991; Calull et al., 1992). Methanol was used as an External standard. Recovery studies of methanol from blood were done and it was found to be 92–96%. Linearity for methanol was found to be 5–500 mg/100 ml. The detector sensitivity for methanol was found to be 5 mg/100 ml. Reproducibility was more than 93%. 2.4. Blood formate measurement Protein free supernatants were prepared from the whole blood by the sequential addition of 7.5% ZnSO4 and 0.4 N NaOH. Formate analysis was performed by the coupled formate dehydrogenase–diaphorase enzymatic method (Makar and Tephly, 1982). The fluorescence of each sample was determined by Hitachi 650-10 M Fluorescence spectrophotometer. 2.5. Blood gas analysis An automated blood gas analyzer (RadiometerCopenhagen, ABL5) was used to immediately measure blood pH, pCO2 , pO2 and bicarbonate in heparinized whole blood samples. Bicarbonate values were calculated from pH and pCO2 using the Henderson–Hasselbach equation (Eells et al., 1996). 2.6. Statistical analysis All the data were statistically analyzed using one-way analysis variance (ANOVA) followed by Tukey’s Multiple comparison test. Students-t test was used when one comparison was made between two groups. In all the cases, the minimum level of significance was taken as P < 0.05. The results shown are mean ± S.D. Software SPSS (version 10) was used for statistical analysis.

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Table 1 Blood parameters at zero minute time point before Carbicarb and enzyme/saline infusion Parameter Blood formate (mM) Blood pH Bicarbonate (mM/L) pCO2 (mmHg)

Saline control 0.45 7.46 25.67 45.07

± ± ± ±

0.165 0.033 2.61 4.54

MeOH control 2.9 7.44 22.2 48.56

± ± 0.041 ± 1.47 ± 3.76

0.634*

MeOH-MTX control 8.07 7.27 13.4 47.34

± ± 0.017*,# ± 1.630*,# ± 3.35 0.730*,#

MTX-MeOH-Carb 7.45 7.30 13.1 46.18

± ± ± ±

0.876 0.023 2.92 3.1

MeOH-MTX-Carb-Enz 8.29 7.32 11.9 45.78

± ± ± ±

0.705 0.029 3.78 4.22

Blood parameter changes in Enzyme control, MTX control were insignificant with Saline control (data not shown). Results shown are mean ± S.D. * Significance at P < 0.05 with Saline control. # Significance at P < 0.05 with MeOH control.

3. Results Blood methanol level, 12 h after intraperitonial administration, was 249.6 ± 3.48 mM, in the MeOH control animals. In Saline control, at zero minute time point, formate level due to endogenous source was found to be 0.45 ± 0.17 mM and pH, pCO2 and bicarbonate values, respectively, were, 7.46 ± 0.03, 45.07 ± 4.5 mmHg and 25.67 ± 2.61 mM/L (Table 1). Blood formate level in MeOH control and in MTXMeOH control, at zero minute time point (Table 1), respectively, were 2.9 ± 0.63 and 8.07 ± 0.73 mM. There was significant accumulation of formate (P < 0.01) in MTX-MeOH control group at all time points with respect to Saline control (Fig. 1). In MTX-MeOH control group, the pH, bicarbonate and pCO2 values at zero minute time point, respectively, were 7.27 ± 0.023, 13.4 ± 1.63 mM/L and 47.34 ± 3.35 mmHg. There was significant decrease (P < 0.05) in the pH and bicarbonate values in the MTX-MeOH control group at all time points with respect to Saline control (Figs. 2 and 3). The changes in pCO2 level of MeOH control and MTX-

Fig. 1. Blood formate level at 15, 30, 45, 60 min time points after Carbicarb and enzyme/saline infusion. Blood formate changes in Enzyme control, MTX control were insignificant with Saline control (data not shown). Zero minute value was noted before Carbicarb and enzyme or saline infusion. Results shown are mean ± S.D. ‘a’ significance at P < 0.05 with Saline control. ‘b’ significance at P < 0.05 with MeOH control. ‘c’ significance at P < 0.01 with MTX-MeOH control.

MeOH control were insignificant, at all time points with respect to Saline control. Changes in blood parameters’ values of Enzyme control and MTX control were insignificant with respect to Saline control.

Fig. 2. Blood pH level at 15, 30, 45, 60 min time points after Carbicarb and enzyme/saline infusion. Blood pH changes in Enzyme control, MTX control were insignificant with Saline control (data not shown). Zero minute value was noted before Carbicarb and enzyme or saline infusion. Results shown are mean ± S.D. ‘a’ significance at P < 0.05 with Saline control. ‘b’ significance at P < 0.05 with MeOH control. ‘c’ significance at P < 0.01 with MTX-MeOH control.

Fig. 3. Blood bicarbonate level at 15, 30, 45, 60 min time points after Carbicarb and enzyme/saline infusion. Blood bicarbonate changes in Enzyme control, MTX control were insignificant with Saline control (data not shown). Zero minute value was noted before Carbicarb and enzyme or saline infusion. Results shown are mean ± S.D. ‘a’ significance at P < 0.05 with Saline control. ‘b’ significance at P < 0.05 with MeOH control; ‘c’ significance at P < 0.01 with MTX-MeOH control.

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Table 2 Blood parameters at 15 min time point after Carbicarb and enzyme/saline infusion Parameter Blood formate (mM) Blood pH Bicarbonate (mM/L) pCO2 (mmHg)

Saline control 0.42 7.45 25.12 47.33

± ± ± ±

0.124 0.027 2.23 6.1

MeOH control 3.01 7.42 21.9 49.01

± ± 0.023 ± 1.03 ± 4.32

0.498*

MeOH-MTX control 8.35 7.26 13.1 45

± ± 0.02*,# ± 1.05*,# ± 5.3 0.86*,#

MTX-MeOH-Carb 7.96 7.46 28.7 51.45

± ± ± ±

0.903 0.025$ 3.26$ 5.5

MeOH-MTX-Carb-Enz 5.23 7.44 26.9 50.12

± ± ± ±

0.694$ 0.019$ 2.54$ 4.71

Blood parameter changes in Enzyme control, MTX control were insignificant with Saline control (data not shown). Results shown are mean ± S.D. * Significance at P < 0.05 with Saline control. # Significance at P < 0.05 with MeOH control. $ Significance at P < 0.01 with MTX-MeOH control.

When compared to MeOH control, there was significant accumulation of formate (P < 0.05) and significant reduction (P < 0.05) in the pH and bicarbonate values in the MTX-MeOH control group at all time points. After Carbicarb infusion followed by enzyme/saline infusion, significant elevation of pH and bicarbonate values (P < 0.01), were observed in MTX-MeOH-Carb and in MTX-MeOH-Carb-Enz group at 15, 30, 45 and 60 min time points with respect to MTX-MeOH control (Figs. 2 and 3). Formate was significantly (P < 0.01) decreased in MTX-MeOH-Carb-Enz group in relation to MTXMeOH control group at 15, 30, 45 and 60 min time points (Fig. 1). Insignificant changes were observed in the pCO2 level of MTX-MeOH-Carb-Enz group and MTX-MeOH-Carb at 15, 30, 45 and 60 min time point with respect to MTX-MeOH control (Table 2). 4. Discussion Methanol is rapidly absorbed from the gastrointestinal tract with peak absorption occurring in 30–60 min depending on the presence or absence of food in the stomach (Becker, 1983). In order to avoid fluctuation due to oral ingestion, intraperitoneal route was selected. Blood methanol was estimated to correlate with blood formate level. Gonzalez-Quevedo et al. (2002), employed the MTX treated folate deficient rat model to induce formate accumulation in order to study the effect of chronic methanol administration on amino acids and on monoamines in retina, optic nerve, and brain of rat. Methanolintoxicated rats that accumulated formate concentrations of 8–15 mM developed metabolic acidosis, retinal dysfunction, and retinal histopathological changes as reported by Eells et al. (1996). In the same study, they showed, N2 O gas exposed methanol-intoxicated rats, when exposed to methanol (4 gm/kg, i.p.) accumulated formate concentrations of 7 mM within 12 h after methanol exposure, which correlates well with our

present study. In our study, the blood formate level of MTX-MeOH control group, 12 h after methanol administration, was 8.07 ± 0.74 mM. There was significant accumulation of formate and fall in the pH and bicarbonate values in the MTX-MeOH control group. Decrease in the pH and the bicarbonate level clearly showed, the development of the metabolic acidosis in the MTX-MeOH control group in relation to MeOH control. Since, the MeOH control group was not folate deficient, formate metabolism was not impaired. Hence, MeOH control group did not develop metabolic acidosis though there was slight increase in the formate when compared to Saline control. In this present study, restoration of normal pH helps in two ways, first it prevents further deterioration in the acid base status of the animal and secondly, it facilitates enzymatic elimination of formate by providing a pH environment closer to the optimal pH of the enzyme. Usually, bicarbonate infusion was done as a first line of treatment to increase the pH, when there is severe metabolic acidosis due to methanol poisoning (Barceloux et al., 2002). But, bicarbonate buffering may raise the pCO2 and paradoxically worsen the acidosis (Adrogue and Madias, 1998). When compared to sodium bicarbonate, Carbicarb raises the pH far more and boosts pCO2 far less when given intravenously to animals with metabolic acidosis (Forsythe and Schmidt, 2000). Hence, in this present study, Carbicarb was used to increase the pH. In experimental lactic acidosis, Carbicarb increased blood and intracellular pH with little or no rise in the arterial or venous pCO2 (Bersin and Arieff, 1988; Kucera et al., 1989). Because, carbonate is a stronger base, it is used in preference to bicarbonate for buffering hydrogen ions, generating bicarbonate rather than CO2 (CO3 2− + H+ → HCO3 − ). Besides, more importantly the carbonate ion can react with carbonic acid, thereby consuming CO2 (CO3 2− + H2 CO3 → 2HCO3 − ) (Adrogue and Madias, 1998). Significant increase in the pH in MTX-MeOH-Carb and in MTX-MeOH-Carb-Enz

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groups with respect to MTX-MeOH control group at 15, 30, 45 and 60 min time points was due to buffering action of Carbicarb. Based on our preliminary studies with different dosages (100, 200, 300 units), the enzyme dosage (300 units) was determined and taken for study. The formate level in MTX-MeOH-Carb-enzyme group with respect to MTX-MeOH control at 15, 30, 45 and 60 min time points was significantly lower. The sudden decrease in formate level in the MTX-MeOH-Carb-Enz group was due to enzymatic breakdown of formate. Enzymatic elimination of formate by formate dehydrogenase produces CO2 as a byproduct. Though, CO2 was produced, the changes in pCO2 at 15, 30, 45 and 60 min time points in MTX-MeOH-Carb-Enz group were insignificant in relation to MTX-MeOH control. This might possibly be due to CO2 consuming action of carbonate ion of Carbicarb, as explained above. It may be concluded that fast removal of formate is possible with single bolus intravenous infusion of formate dehydrogenase. In the case of human beings, this treatment may be combined with alcohol dehydrogenase inhibitor (Fomepizole) in order to prevent further production of formate. Acknowledgements The authors thank Late Prof. A. Namasivayam for his valuable guidance and support. The financial support provided by the Indian Council of Medical Research, (IRIS.No: 9700750) New Delhi, is graciously acknowledged. References Adrogue, H.J., Madias, N.E., 1998. Management of life threatening acid–base disorders. N. Engl. J. Med. 338 (1), 26–34. Barceloux, D.G., Bond, G.R., Krenzelok, E.P., Cooper, H., Vale, J.A., 2002. The American Academy of Clinical Toxicology Adhoc Committee on the treatment guidelines for methanol poisoning. J. Toxicol. Clin. Toxicol. 40 (4), 415–446. Barford, P.A., Blair, J.A., Malghani, M.A., 1980. The effect of methotrexate on folate metabolism in the rat. Br. J. Cancer 41, 816–820. Barton, N.W., Furbish, F.S., Murray, G.J., Garfield, M., Brady, R.O., 1990. Therapeutic response to intravenous infusions of glucocerebrosidase in a patient with Gaucher’s disease. Proc. Natl. Acad. Sci. U.S.A. 87, 1913–1916. Becker, C.E., 1983. Methanol poisoning. J. Emerg. Med. 1, 51–58. Bersin, R.M., Arieff, A.I., 1988. Improved hemodynamic function during hypoxia with Carbicarb, a new agent for the management of acidosis. Circulation 77, 227–233. Bomalaski, J.S., Clark, M.A., 2004. Serum Uric acid lowering therapies: where are we heading in management of hyperuricemia and the potential role of uricase. Curr. Rheumatol. Rep. 6, 240–247.

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