[CANCER RESEARCH 37, 1125-1132, April 1977) Production of Formaldehyde from N5-Methyltetrahydrofolate by Normal and Leukemic Leukocytes1 Janet Thorndike and William S. Beck Department of Medicine, Harvard Medical School, and Hematology Research Laboratory, Massachusetts General Hospital, Boston, Massachusetts 02114 SUMMARY malian tissues (3, 14, 22, 25, 29, 46). It was first considered that the methyl group was transferred intact in analogy with Extracts of human normal and leukemic leukocytes con the methyl transfer known to take place from AdoMet to tam an enzyme that catalyzes a transfer of labeled methyl various amines (2). This early conclusion rested on the carbon from Ns@[i4C]methyltetnahydrofoIate to tryptamine. assumption that radioactive reaction products were N- Evidence is presented that this reaction is not attributable to methyl derivatives of the amine substrates (14, 22). How a methyltransferase but to the following reaction sequence: even, later studies identified the products as the come (a) an oxidation of N5-[―C]methyltetrahydrofolate to N5,N'°- sponding carbolines formed by spontaneous condensation [i4Clmethylenetetrahydrofolate that is catalyzed by N5,N10- of amines with HCHO (4, 17, 28, 29, 36, 42, 46). These data methylenetetrahydnofolate reductase (EC 1.1.1.68); (b) suggested that transfer of the methyl carbon from CH3- spontaneous release of [“C]fonmaldehydefrom N5,N10- H4folate to an aromatic amine occurs in 2 steps: an enzy [“C]methylenetetrahydrofolate; and (C) nonenzymatic con matic conversion pf CH3-H4folate to HCHO, followed by a densation of [‘4C]formaldehydewith tryptamine to form a nonenzymatic reaction of HCHO with amine. radioactive carboline derivative. The occurrence of this se It was then shown that, in assays for Ns,Nio@methyIene@ quence inleukocytesissuggestedby datathatshow that H4folate reductase (EC 1.1.1.68) by the method of Kutzbach the enzyme reaction is strongly stimulated by addition of and Stokstad (20), tissue extracts catalyze a conversion of flavin adenine dinucleotide and that the final product is CH3-H4folate to HCHO; i.e., CH3-H4folate is oxidized in the chromatognaphically identical to the adduct formed in the presence of an electron acceptor to N5,N'°-methylene reaction of [“C]fonmaldehydewith tryptamine. In the ab H4folate, which then dissociates to HCHO and H4folate (9, sence of tnyptamine, a product accumulates that can react 10, 20). This observation is of interest because transfer of with other HCHO acceptors, i.e., f3-phenylethylamine and the methyl carbon from CH3-H4folateto amines is stimulated dimedone; another reaction product is tetnahydnofolate. by electron acceptors such as FAD on menadione (15, 31, Production of formaldehyde is relatively more active in 44). normal lymphocytes than in normal granulocytes, but it is We have been prompted to study this reaction of CH3- even higher in lymphocytes of chronic lymphocytic leuke H4folate in human hematic tissues because of the particular mia. Activity in granulocytes from a subject with chronic importance of tissue folate metabolism and the controven myelocytic leukemia is also elevated but to a lessen extent sies that have surrounded the distinctive cobalamin-de than activity in lymphocytes of chronic lymphocytic leuke pendent reaction, in which the methyl group of CH3-H4folate mia. Formaldehyde production in leukocytes is only slightly is transferred to homocysteine (28, 39). The purposes of this stimulated by addition of various cobalamins, and activity is study are: (a) to determine whether a transfer of the methyl normal in leukocytes from a vitamin B12-deficient patient. carbon of CH3-H4folateto tryptamine occurs in human Ieu We conclude that the system is cobalamin independent. kocytes; (b) to characterize the nature of this reaction ; (c) to Thus, there exists an active pathway from N5-methyltetrahy compare levels of the reaction in leukocytes from normal drofolate to tetrahydrofolate other than the one catalyzed by individuals and subjects with leukemia and other disorders; cobalamin-dependent N5-methyltetnahydrofolate-homocys and (d) to determine to what extent a cobalamin-independ teinemethyltransfenase. ent conversion of CH3-H4folate to H4folate can occur in these cells. INTRODUCTION Transfer of the methyl carbon from CH3-H4folate2to ano MATERIALS AND METHODS matic amines has been demonstrated in a variety of mam Reagents. [‘4C]CH3-H4folate(54@Ci/@tmoIe),[2-'4C]folic I This work was supported by Research Grant CA-03728 from the National acid (58 @Ci/@moIe),and[‘4C]formaldehyde(2 @Ci/@moIe) Cancer Institute, NIH, and by the John Phyffe Richardson Fund. 2 The abbreviations used are: CH3-H4folate, N'.methyltetrahydrofolate; were obtained from Amersham/Searle Corp. , Arlington AdoMet, S-adenosylmethionine; N,N'0-methylene-H4folate, N5,N'0@methyl@ Heights, Ill. [i4C]CHHfolatewasstored undenN2at —20°as enetetrahydrofolate; H4folate, tetrahydrofolate; FAD, flavin adenine dinucleo a 4.5 mM solution in 1 M mercaptoethanol at pH 7. 3',5'- tide; N5-formyl-H4folate, N5-formyltetrahydrofolate; FMN, riboflavin 5'-phos phate; N, N'°-methenyl-H4folate, N5,N' °-methenyltetrahydrofolate ; CLL, [3HJfoIic acid (22 @Ci/@mole)waspurchased from Schwarz/ chronic lymphocytic leukemia; CML, chronic myelocytic leukemia. Ab Mann, Onangeburg, N. Y.; N5-formyl-H4folate, calcium Ieu breviatlons of cofactors are in accordance with standard usage in The covonin, was obtained from Lederle Laboratories, Pearl Journal of Biological Chemistry. Received August 2, 1976; accepted December 29, 1976. Riven, N. V.; tryptamine hydrochloride, N-methyltnyptamine, APRIL 1977 1125 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1977 American Association for Cancer Research. J. Thorndike and W. S. Beck f3-phenylethylamine, AdoMet, FAD, and folic acid were pro disrupted and centrifuged as described for blood leuko duced by Sigma Chemical Co., St. Louis, Mo. FMN, NADH, cytes. and NADPH were obtained from Calbiochem, La Jolla, - Assay of Formaldehyde ProductIon from CH3-H4folate. Calif.; NAD and NADP were from Boehringer/Mannheim, Cell extracts were incubated with [i4C]CH3@H4foIatein the Mannheim, West Germany; cyanocobalamin was from presence of a saturating concentration of tryptammne. Dun Schwarz/Mann. Silica gel and cellulose thin-layer Chroma ing this incubation, enzymatically produced HCHO was gram sheets were purchased from Eastman Kodak Co., converted to the corresponding carboline, which was ex Rochester, N. V. tracted in toluene:isoamyl alcohol (pH 10) (14). Standard Methylcobalamin and propylcobalamin were synthesized incubation mixtures contained (in a total volume of 0.25 ml): by reduction of cyaflocobalamin with sodium borohydride, [14C]CH3-H4folate,9 nmoles; tryptamine, 5 @moles;FAD,0.5 followed by alkylation with methyl iodide and propyl bro @mole;potassium phosphate (pH 6.5), 25 @moles;andcell mide, respectively (32). Dihydrofolate and H4folate were extract,0to0.5mg protein.Afterincubationfor30mm at37° prepared by reduction of folic acid with sodium hydrosulfite in the dark, 0.5 ml of 0.5 M sodium borate (pH 10) was (11) or sodium borohydride (40), respectively. N5,N10-meth added; tryptoline was extracted with 3.5 ml of tolu enyl-H4folate was prepared by boiling H4folate in 80% formic ene:isoamyl alcohol (97:3) (‘‘tolueneextract'‘and‘‘toluene acid:0.1 M mercaptoethanol for 5 mm (37) on by treat extractible―are used in subsequent discussion to refer to ment of N5-formyl-H4folate with HCI (33). N5,N'°-methenyl this extraction). After centnifugation for 5 mm at 400 x g, [3H]H4folate, prepared by reduction of [3H]folic acid with aliquots of the toluene extract were evaporated to dryness sodium borohydride (40) and boiling with formic acid (37), and taken up in 1 ml isoamyl alcohol. Radioactivity was was purified by chromatography on cellulose thin-layer assayed in Aquasol liquid scintillation fluid in a Beckman sheets in 1 N formic acid:0.2 M mercaptoethanol, followed LS-150 scintillation counter. Incubations lacking enzyme by elution with 0.1 M mercaptoethanol. Unlabeled N5,N'°- were used as controls. Enzyme activity was expressed as methenyl-H4folate (which gives a fluorescent spot at AF nmoles HCHO formed pen hr pen mg protein. 0.24) was used as reference standard. N5,N10-['4Cjmethyl Chromatographlc Characterization of Reaction Prod ene-H4folate was prepared from [14C]HCHO and H4folate ucts. Chromatognaphic methods differed depending on (18). CH3-[―C]H4folate was synthesized by reduction of whether substrates were [“C]CH3-H4folateon CH3-[2- [2-―C]folicacid with sodium borohydnide and methylation “C)H4folate.Whenthe substrate was [‘4C]CH3-H4folatein with unlabeled HCHO in the presence of borohydride standard incubation mixtures, aliquots of toluene extracts (8); samples were purified by chromatography on cellulose were applied to a silica gel thin-layer chromatography sheet thin-layer sheets in 0.1 M potassium phosphate:0.2 M men and developed in acetone:1 N ammonium hydroxide (10:3) captoethanol, eluted with 0.2 M mercaptoethanol, and (16). In a separate reference mixture, 0.05 ml of a 40 @M evaporated to a smaller volume under N2. [@C]CH3-H4folate solution of [14C]formaldehyde was mixed with 0.2 ml of 125 was used as reference standard. mM tryptamine and 0.5 ml of 0.5 M sodium borate (pH 10) PreparatIon of Lymphocytes and Granulocytes. Leuko and extracted with 3.5 ml of toluene:isoamyl alcohol (97:3). cytes were obtained from venous blood (10 to 60 ml) of Aliquots of this toluene extract were chromatographed on ostensibly normal subjects and patients with the following the same sheet. A reference sample of nonnadioactive N- various disorders: CLL, CML, unexplained monocytosis, methyltryptamine was also developed on the same sheet. and severe cobalamin deficiency (mean corpuscular vol Sheets were dried, examined under UV, cut into portions, ume, 117 cu @m;serumcobalamin, 20 pg/mI). Some of the and assayed for radioactivity in Aquasol liquid scintillation leukemic subjects were receiving prednisone and cyclo fluid. phosphamide (CLL) or busulfan (CML); none was receiving However, when the substrate was CH3-[2-'4C]H4folate,be folic acid.