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Simultaneous Measurement of One-Carbon and Polyglutamate Derivatives of Folic Acid in Rat Liver Using Enzymatic Interconversions of Folates Followed by Ternary Complex Formation with Thymidylate Synthetase and 5-Fluorodeoxyuridylic Acid: Standardization of the Method^1,2,

G. Franklin Carl^*,,3 and Mary L. Smith^*

^* Medical Research Service, Veterans Affairs Medical Center, Augusta, GA 30904 Department of Neurology, Medical College of Georgia, Augusta, GA 30912

A sensitive assay is needed for the measurement of individual folate derivatives in samples that contain low concentrations of folate. The ternary complex method for the determination of folylpolyglutamates has been combined with procedures for interconverting folate derivatives to provide a method capable of measuring 28 different folate derivatives in biological samples. The method takes advantage of the properties of the ternary complex formed with thymidylate synthetase, fluorodeoxyuridylic acid and 5,10-methylenetetrahydrofolic acid. In the presence of excess purified thymidylate synthetase and excess [³H]fluorodeoxyuridylic acid, limiting concentrations of folates were converted to 5,10-methylenetetrahydrofolate using purified folate interconverting enzymes. This process separated folate derivatives into four groups: Tetrahydrofolate + 5,10-methylenetetrahydrofolate; dihydrofolate; 5,10-methenyl-, 5-formyl-, and 10-formyltetrahydrofolates and 5-methyltetrahydrofolate. Within groups specificity was good, showing little overlap between folates. Sensitivities to 100 fmol of folate were achievable and to 1 pmol were standard. Recoveries were linear for each of the groups in this system to 50 pmol of folate. Ternary complexes containing different polyglutamates were separated by isoelectric focusing, visualized by fluorography and measured by densitometry. The densitometry was linear with folate concentration in the range 20–200 fmol for each of the polyglutamates. Primary and secondary coefficients of variation were determined. This method provides the sensitivity to measure individual folates in the femtomole range and the flexibility to determine the concentrations of 28 separate pools of folate derivatives, differentiating between derivatives of the pteridine moiety and glutamate chain length simultaneously.

KEY WORDS: • thymidylate synthetase • densitometry • folate • isoelectric focusing • fluorography

¹ Supported by the Medical Research Service of the Department of Veterans Affairs.

² The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

³ To whom correspondence should be addressed.

Manuscript received 17 May 1994. Revision accepted 30 August 1994.