Modification of allergenicity and immunogenicity of formate dehydrogenase by conjugation with linear mono methoxy poly ethylene glycol: improvement in detoxification of formate in methanol poisoning.

BACKGROUND: Single bolus intravenous infusion of native formate dehydrogenase (FD), isolated from Candida boidinii was found to eliminate formate, a highly toxic metabolite in methanol poisoning. In order to prevent immunological reactions which might be produced by multiple dosing of formate dehydrogenase and to prolong the serum half life of the enzyme, the N-hydroxysuccinimidyl ester of methoxy polyethylene glycol propionic acid (mPEG-SPA 5000) was conjugated to native formate dehydrogenase. METHOD: PEGylation reactions were run at 20 degrees C for 30 min in a reaction buffer (0.2 mol/l sodium phosphate buffer, pH 8.3). The PEGylated molecules were purified from unreacted PEG with Amicon Ultra-4 (10 K) and by Sephacryl S-300 HR gel-filtration chromatography. Unreacted formate dehydrogenase molecules were removed by DEAE Sepharose FF anion-exchange chromatography. PEG-FD enzyme molecules obtained from reacting ratio of FD/PEG of 1/40 had an enzyme activity of 68% of unmodified enzyme. Immunogenicity of PEGylated and native enzyme was evaluated by ELISA. Allergenicity was evaluated by active systemic anaphylaxis and passive cutaneous anaphylaxis tests. In vivo efficacy of PEG-FD or native FD was comparatively evaluated by single intravenous administration of PEG-FD or native FD in folate deficient methanol intoxicated albino rats along with Carbicarb buffer infusion. Methanol and formate were estimated at specific time points respectively with HPLC and fluorescence spectrophotometer. RESULT: PEG-FD had comparatively longer half life and lower immunogenicity than native FD. PEG-FD had better in vivo efficacy than native FD in eliminating the formate. CONCLUSION: Conjugation of mPEG-SPA 5000 with native FD reduces its immunogenicity and increases its efficacy in detoxification of formate in methanol poisoning.

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

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 CO(2) 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 (0.3mg/kg) for a week, were injected (i.p.) with methanol (4 gm/kg), 12h 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 pO(2), pCO(2) 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.