Coppell et at.: Identification of folates by iodine oxidation 155 Pteridines Vo!' 1, 1989, pp. 155 - 157 Identification of Folates by Iodine Oxidation at Acid, Neutral and Alkaline pH By A . D . Coppell, R . J. Leeming!) Haematology Department, The General Hospital, Steelhouse Lane, Birmingham B4 6NH, Great Britain J. A. Blair Biology Division, Aston University, Birmingham B4 7ET, Great Britain (Received March 1989) Summary Oxidation by iodine at pH 1.5, pH 7.0 with and without catalase and at pH 12.5, differentiated folic acid, 5- methyltetrahydrofolic acid, 10-formylfolic acid, 10-formyltetrahydrofolic acid and 5-formyltetrahydrofolic acid, when the products were assayed with Lactobacillus casei. This method is proposed as an alternative to differential microbiological assay for identifying folates. Introduction Blakley (7). 5-Methyltetrahydrofolic acid (5- The methods commonly used for measuring folates CH3THF) was obtained from Eprova, Switzerland. in biological material a re microbiological (1) or radio­ 5-Formyltetrahydrofolic acid (5-CHOTHF) was a gift isotope dilution assays (2). The identification of in­ from Lederle. 10-Formyltetrahydrofolic acid (10- dividual folates is carried out using differential mi­ CHOTHF) was prepared by acidifying 5-CHOTHF, crobiological assays with L. casei, P. cerevisiae and leaving for one hour at 25 "C in the dark then return­ S.faecalis (3). This process is time consuming, re­ ing to neutral pH. Tetrahydrofolic acid (THF) was quiring the maintenance of three stock cultures in obtained from Eprova. The iodine solution was pre­ appropriate culture media. High performance liquid pared by saturating a 2 gi l potassium iodide solution chromatography (HPLC) using electrochemical de­ with crystalline iodine. tection has been proposed as an alternative, however this is not yet a routine procedure (4). Bioautography, The Lactobacillus casei folate assay was carried out a method which combines chromatography and mi­ by a semi-automated method (8). crobiological growth response, is sensitive, specific but For iodine oxidation, 2.5 ml of 10 Jlg /ml solutions of qualitative (5). folates were placed in 5 ml volumetric flasks. For acid Folates can be differentially oxidised (6), giving prod­ and alkaline oxidation, 0.25 ml of 2M HCI a nd 2M ucts some of which may be microbiologically active NaOH respectively were added followed by 0.1 ml of and others which are inactive. This may be utilised iodine solution which was sufficient to provide an to differentiate between individual folates. excess leaving the solution yellow. After one hour at 25 DC 0.05 g of ascorbic acid was added which made the solution colourless. The solutions were returned Materials and Methods to pH 7.0 with M NaOH and M HCl and the volumes Folic acid was obtained from Sigma. 10-Formylfolic were made to 5.0 ml with 0.2M phosphate buffer pH acid (10-CHOFA) was prepared by the method of 6.1. At pH 7.0 the oxidation was repeated with the addition of 0.1 ml of 5 gi l catalase immediately before 1) Author to whom correspondence should be addressed. the iodine. Pteridines Vo!' 1 / 1989 / No.3 Copyright'(' 1989 Walter de Gruyter . Berlin ' New York Coppell et ul.: Identification of folates by iodine oxidation Table 1. Remaining L. cusei activity of folate expressed as a percentage and standard deviation after iodine oxidation at pH 1.5, pH 7.0 (before and after trea tment with catalase) and pH 12.5 for 1 hour at 25 ' C in the dark. Folate Analogue (N) pH 1.5 pH 7.0 pH 7.0 pH 12.5 without catalase with catalase Folic Acid (5) 99.2 ±1.3 0.47 ± 0.7 10.0 ± 1.7 50.8 ± 16.8 5-CH3THF (5) 0.45 ± 0.34 60.8 ± 7.2 99. 1 ± 1.04 52.0 ± 14.8 10-CHOFA (5) 99.8 ± 0.42 99.2 ± 0.61 99.4 ± 1.3 100 ± 0 10-CHOTHF (5) 99.5 ± 0.59 94.8 ± 3.9 98.6 ± 1.5 7.0 ± \.8 5-CHOTHF (5) 99.6 ± 0.59 14.4 ± 4.3 30.3 ± 1.5 7.8 ± 2.1 The experiments were repeated using manganese diox­ Assay using L. casei Folate measured ide to replace the iodine solution, 0.1 g of manganese dioxide was added in the powdered form. After ad­ (a) Total folate THF; 5-CH3THF; dition of 0.05 g ascorbic acid the material was centri­ (No oxidising 10-CHOTHF fuged at 2000 g for three minutes. THF was oxidised conditions) by iodine at acid and neutral pH and the pterin formed was quantitatively measured by HPLC (9). (b) Following acid 10-CHOTHF oxidation with iodine Results (c) Following neutral pH 5-CH3THF; The findings of the microbiological assays are given oxidation with 10-CHOTHF in Table 1. With iodine at neutral pH the activities of iodine and catalase folic acid and 5-CHOTHF were consistently reduced to 5% and 30% respectively. 5-CH3THF had an By simple rearrangement of (a), (b) and (c) it is intermediate activity at 50-70% whilst 10-CHOTHF possible to identify individual folates: and 10-CHOFA showed almost 100% activity. Under acid conditions 5-CH3THF retained less than (a) - (c) = THF 10% of its original activity whereas the other folates (c) - (b) = 5-CH3THF were unaffected. At alkaline pH, 10-CHOFA again (b) = 10-CHOTHF proved to be completely stable. Folic acid and 5- CH3THF had 50% residual activity. 10-CHOTHF The ability to identify folate analogues following io­ and 5-CHOTHF fell to 10% and THF gave 100% dine oxidation at acid, alkaline and neutral pH using pterin at acid and neutral pH. Lactobacillus casei is confirmed and is potentially of Treatment with catalase afforded some protection clinical use. By varying pH, selective inactivation oc­ against oxidation at pH 7.0. 5-CH3THF increased curs leaving other analogues unaffected and therefore from 70% to 100% activity. available for Lactobacillus casei in the assay. Subse­ quent treatment with conjugase would further enable Iodine and manganese dioxide gave similar results at the identification of polyglutamates. all three pH levels, therefore only the figures for iodine are quoted. All five folates used in this study gave responses which could be used to identify them. Folic acid and 5- CH3THF show no activity after neutral and acid oxidation respectively. 10-CHOFA can be identified Discussion because its activity remains unchanged under all the Tetrahydrofolic acid (THF), 5-CH3THF and 10- conditions tried. To separate 5-CHOTHF and 10- CHOTHF and their polyglutamates are the major CHOTHF is more difficult as they show similar re­ components of the normal folate metabolic pool in sponses. However, with neutral oxidation and the man. Folic acid, 5-CHOTHF and 10-CHOFA are not addition of catalase, 5-CHOTHF has 30% activity natural constituents of normal folate metabolism. An whilst 10-CHOTHF does not lose any activity. THF interpretation of the results if used in the clinical is rapidly and totally oxidised to pterin at pH 7.0 by situation would be as follows: iodine in potassium iodide. Pteridines / Vol. 1 / 1989 / N o.3 Coppell et at. : Identification of folates by iodine oxida tion 157 References 1. 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