Effects of deuterium oxide on folate metabolism in Lactobacillus casei

Transcription

1 Indian Journal of Biochemistry & Biophysics Vol. 40, June 2003, pp Effects of deuterium oxide on folate metabolism in Lactobacillus casei M S Pote, G Viswanathan and V Kesavan* Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai Received 27 August 2002; revised 16 December 2002; accepted 7 February 2003 Various folate coenzymes and their polyglutamyl derivatives involved in 1-carbon metabolism are modulated as a result of altered physiological states and also vary with respect to growth conditions. We studied the metabolic changes in folic acid and their conjugated polyglutamyl derivatives in Lactobacillus casei cells grown in the presence of D 2 O. A 40% decrease in methyltetrahydrofolyl polyglutamate derivatives was observed in the cells grown in media prepared with D 2 O- depleted water (D 2 O content, 8-10 ppm). Chromatographic analysis of folates showed significant alterations in the formyland methyltetrahydrofolate derivatives and their polyglutamylation profiles. Higher amounts of oxidized folates were also present in the cells grown in D 2 O-depleted conditions. No significant changes were observed in folates and their polyglutamate derivatives when the cells were grown in the presence of 300, 450 and 600 ppm D 2 O. The altered folate homoestasis is attributed to changes in the metabolic adaptation of cells to D 2 O-depleted environment. Coenzyme derivatives of folic acid play a key role in metabolism of one carbon transfers. They are required for the synthesis of nucleic acid precursors, purines and pyrimidines, in the metabolism of certain amino acids and in the initiation of protein synthesis 1. In nature folates exist mainly as polyglutamate forms containing 2-7 glutamic acid residues attached through γ-carboxyl linkages 2. These polyglutamyl derivatives of folates are more active in various enzymatic reactions compared to their corresponding monoglutamyl forms 1. The polyglutamylfolate spectrum and the pteridine substitution vary with respect to rate of growth and nutrition, and are affected by cyclic hormonal changes, drug reaction, diseased states and genetic disorders Growth conditions as well as exposure to amino acids such as methionine also affect the polyglutamyl chain length of folates 11. Elongation and distribution of folylpolyglutamates of longer glutamate chain lengths *Author for correspondence Tel : (022) : Fax : vkesav@apsara.barc.ernet.in Abbreviations used: N 10 -CHOH 4 PteGlu, N 10 -formyltetrahydropteroyl-glutamic acid; N 10 -CHOPteGlu, N 10 -formylpteroylglutamic acid; N 10 -CHOPteGlu 4, N 10 -formylpteroyltetraglutamic acid; N 5 -CHOH 4 PteGlu 4, N 5 -formyltetrahydropteroyltetraglutamic acid; N 5 -CHOH 4 PteGlu 2, N 5 -formyltetrahydropteroyldiglutamic acid; N 10 -CHOH 4 PteGlu 4, N 10 -formyltetrahydropteroyltetraglutamic acid; N 5 -CH 3 H 4 PteGlu; N 5 -methyltetrahydropteroylglutamic acid; N 5 -CH 3 H 4 PteGlu 3, N 5 -methyltetrahydropteroyltriglutamic acid; N 5 -CH 3 H 4 PteGlu 4, N 5 -methyltetrahydropteroyltetraglutamic acid; N 5 -CH 3 H 4 PteGlu 5, N 5 -methyltetrahydropteroylpentaglutamic acid; PteGlu, pteroylglutamic acid, PteGlu 3, pteroyltriglutamic acid; PteGlu 4, pteroyltetraglutamic acid. have also been observed in cultures of human fibroblasts in the presence of media deficient in folate 12. Heavy water (deuterium oxide, D 2 O) induces changes leading to altered physiological effects in a variety of biological processes compared to normal water, which contains 150 ppm D 2 O. Variations in concentrations of D 2 O influence cellular activity and growth. In living organisms, the deuterium/hydrogen (D/H) ratio changes during cell cycle 13. Isotope deuterium appears to have a vital role in signal transduction in cell cycle regulation 14. Heavy water has been shown to cause a sharp reduction in the rates of primary hydrogen ion transport (at the plasma membrane ATPase) and causes significant changes in physiology, cellular metabolism, and activity of enzymes 15,16. At higher concentrations of 25% and above, D 2 O has been reported to have wide range of toxic effects on nervous system, liver, and formation of different blood cells and at cellular level may affect mitosis and membrane function 17,18. The deuteriumdepleted water (containing 8-10 ppm D 2 O) significantly decrease the growth rate of fibroblast cell line and also inhibits the tumour growth 14. We report here studies on folate and polyglutamylfolate status and the metabolic changes occurring in the Lactobacillus casei cells treated either with D 2 O or with deuterium-depleted water. As L. casei utilises a wide range of substituted and unsubstituted tetrahydrofolates, and can be regularly maintained in the laboratory, a study with these cells in altered physiological conditions may provide a clear picture of the changes in folate metabolism.

2 176 INDIAN J. BIOCHEM. BIOPHYS., VOL. 40, JUNE 2003 Materials and Methods Chemicals Deuterium oxide and D 2 O-depleted water (containing 8-10 ppm D 2 O) were obtained from Heavy Water Division, Bhabha Atomic Research Centre, Mumbai 85. Growth of L. casei cells L. casei cells were grown in the media prepared with D 2 O-depleted or normal water (D 2 O content; 150 ppm, D/H ratio, 1:6600) or media containing various concentrations such as 300, 450 and 600 ppm D 2 O. After seven generations, the cells were grown in bulk in respective media and were isolated and used for the studies. Folate extraction L. casei cells were homogenized in 0.1 M phosphate buffer (ph 6.0) containing 1% ascorbate and heated in boiling water for 5 min to inactivate endogenous folyl conjugase. The samples were further extracted in the same buffer containing 1% ascorbate for 10 min at 80 C, clarified by centrifugation and volume was made to 10% (w/v). Folic acid assay Folate activity was determined by a microbiological assay using L. casei ATCC 7469 and Pediococcus cerevisiae ATCC 8081 with 5-formyl tetrahydrofolate (Sigma Chemical Co., USA) as standard (after correction for the inactive isomer), as described earlier 19. In brief, the assay mixture contained 4.7% folic acid casei medium (HiMedia Laboratories Ltd., Mumbai, India), 1 mg potassium ascorbate and samples after suitable dilutions, in a total volume of 5 ml at ph 6.0. The tubes, after sterilization, were inoculated with L. casei and P. cerevisiae cultures and incubated at 37 C for 18 hr and turbidity was measured at 560 nm in Klett- Summerson colorimeter. The conjugated folates were assayed after prior digestion of samples with purified chicken liver folyl conjugase 19. L. casei is active for a wide range of reduced and oxidized, as well as formyl and methyl forms of folates and to some extent to di- and tri-polyglutamyl forms. P. cerevisiae responds only to non-methyl and unconjugated tetrahydrofolates. A differential assay before and after external purified chicken liver folyl conjugase treatment of the samples was, therefore, used to derive the methyl- and other one-carbon tetrahydro forms of simple and conjugated folates. Characterization of folates of cells grown in various concentrations of D 2 O Aliquot of the cell extract from normal, D 2 O- depleted and 600 ppm D 2 O grown, corresponding to μg L. casei active folates was analyzed by DEAE-cellulose chromatography ( cm), using 0.1 M-0.5 M phosphate buffer (ph 6.0) containing 1% ascorbate for gradient elution, as described earlier 20. The peak fractions were assayed by differential microbiological assays with L. casei and P. cerevisiae and identified with authentic standards, as well as from elution pattern standardized earlier in our laboratory 21. This method not only characterizes the number of γ-glutamyl residues, but also the state of reduction of the pteridine ring and the nature of the 1- carbon substitutes attached. The values shown in Table 3 represent relative amounts in percentage of total folate forms. Protein estimation Protein was determined by the method of Lowry et al 22. Results and Discussion Folate profiles (L. casei activity) of the cells grown in the media prepared with D 2 O-depleted, normal water (150 ppm D 2 O) and water containing 300 and 600 ppm D 2 O are shown in Table 1. Although studies were carried out in the cells grown in the media prepared with 300, 450 and 600 ppm D 2 O, the results, to a great extent, are similar and hence the values for the cells grown with 300 and 600 ppm D 2 O are only represented. Unconjugated folate levels were decreased in the cells grown in media prepared with D 2 O-depleted water. The monoglutamylfolates showed 40% less activity when compared with the values of normal cells i.e., the cells grown in media prepared in distilled water (150 ppm D 2 O), the levels being 143±28 ng folates/g cells. Polyglutamylfolate levels, obtained by the difference in activities before and after conjugase treatment of the samples prior to assay were decreased by 37% in the cells grown with D 2 O-depleted conditions, as compared to normal cells (Table 1). Total folates determined after the folyl conjugase treatment were only 61% in the cells grown in the media with D 2 O-depleted water, as compared to normal cells. The cells grown in the media prepared either with 300 or 600 ppm D 2 O, however, showed little changes in folate profiles as compared to the normal (Table 1), probably due to the low D 2 O concentration used. It has been shown that, in

3 POTE et al.: DEUTERIUM OXIDE EFFECTS ON FOLATE METABOLISM 177 Table 1 Folate levels in the L. casei cells grown in the presense of various concentrations of D 2 O [Values are mean±sem for three experiments. Values in parenthesis show the percentage over the control] Experimental Before conjugase After conjugase Conjugated conditions treatment treatment Folate (ng of folate/g of cells#) Normal water 143±28 493± (150 ppm D 2 O) 300 ppm D 2 O 162±33* 460±68* 298(85) 600 ppm D 2 O 195±8* 522±43* 327(93) D 2 O-depleted water 85±11** 305±39** 220(62) (8-10 ppm D 2 O) #Levels after and before conjugase treatment are indicative of total folate and unconjugated folate content respectively. The difference between these levels is taken as the conjugated folate content. *P>0.5; **P<0.1 Table 2 Methylfolate profiles in L. casei cells grown in various concentrations of D 2 O [Values are mean ±SEM for three experiments] Experimental L. casei P. cerevisiae Methyl folates Methyl folates conditions activity activity % of control (ng folate activity /g cells#) Normal water 493±87 26± (150 ppm) 300 ppm D 2 O 460±68 27±3* ppm D 2 O 522±43 29±1* D 2 O-depleted water 305±39 52±3** #Total folates were determined after overnight digestion of samples with chicken liver folyl conjugase (see Text). *P>0.5; **P<0.01 mammals as high as 15% replacement of body water by D 2 O is required for a noticeable alteration in physiological responses and above 20% replacement induces toxic effects 23,24. P. cerevisiae activity, which represents reduced tetrahydrofolates and their formyl derivatives, showed only 5.2% of total folates (L. casei activity) in normal cells (Table 2). These levels were elevated to 15.5% of total folates in the cells with D 2 O-depleted water. No significant changes were observed in folate levels in the cells grown with media containing either with 300 ppm or 600 ppm D 2 O. Differential microbiological assays (see Methods) which give the amount of methyl tetrahydrofolate derivatives showed (Table 2) that of the total folates present, 94.7% (467 ng folate/g cells) are methyl forms in the cells grown in media prepared with normal water. These levels were considerably decreased to 46% (253 ng folate/g cells) in the cells grown in media prepared with D 2 O- depleted water. This would probably have an adverse effect on various methylation reactions involving methyltetrahydrofolate coenzymes, such as formation of methionine from homocysteine. Methyl folate levels in the cells grown in either 300 or 600 ppm D 2 O did not show significant changes as the values were comparable to that of the control cells grown in presence of 150 ppm D 2 O (Table 2). The present studies thus indicated overall changes in monoglutamyl, polyglutamyl and total folate forms in the cells grown in media prepared with D 2 O-depleted water. However, higher concentrations of D 2 O did not change metabolic profiles of folates in L. casei. Analysis of folate profiles in the cells grown in various concentrations of D 2 O by analytical DEAEcellulose chromatography showed significant changes in the various forms of folates (Fig. 1A, B and C). Gross changes in the folate profile were seen in the cells grown in D 2 O-depleted media, as compared to normal cells or cells treated with 600 ppm D 2 O. The folate derivatives identified from the chromatographic peak fractions from these cell extracts represented in Fig. 1A, B and C are computed in Table 3. Thus, the peak 1 in Fig. 1A (fraction 7, Table 3) represents N 10 - CHOH 4 PteGlu (9.5%); peak 3 (fraction 18) oxidized conjugated formyl folates N 10 -CHOPteGlu 4 (17.5 %); and peak 5 (fraction 28), N 10 -CHOH 4 PteGlu 4 (3.6%). Other folates present were mainly methyl forms constituting 32.8% N 5 -CH 3 H 4 PteGlu (peak 2, fraction 13); 16.1% N 5 -CH 3 H 4 PteGlu 3 (peak 4, fraction 24); and 8.4% N 5 -CH 3 H 4 PteGlu 4 (peak 8, fraction 40). The

4 178 INDIAN J. BIOCHEM. BIOPHYS., VOL. 40, JUNE 2003 unreduced folates of 4.1% PteGlu (peak 6, fraction 30); 5.4% PteGlu 3 (peak 7, fraction 38); and 2.6% PteGlu 4 (peak 9, fraction 44) were also observed. Fig. 1 Analytical DEAE-cellulose chromatogram of D 2 O treated cells [A, Normal water; B, D 2 O-depleted water; C, 600 ppm D 2 O. ( ), Before conjugase treatment;. ( ), after conjugase treatment. See Text for details] The chromatographic profiles of folates in the cells grown in media prepared with 600 ppm D 2 O (Fig. 1B) were almost similar to that of control, except in the absence of N 10 -CHOPteGlu 2 (fraction 23, Table 3), and differences in the levels of N 10 -CHOPteGlu (fraction 18), N 5 -CH 3 H 4 PteGlu 4 (fraction 40), PteGlu 3 (fraction 38) and PteGlu 4 (fraction 44). The various forms of folates analysed in cells grown in D 2 O-depleted medium (Fig. 1C and Table 3) were: 12.6% N 10 -CHOH 4 PteGlu (peak 1, fraction 7), 15.2% N 10 -CHOPteGlu (peak 2, fraction 11), 4.9% N 5 -CHOH 4 PteGlu 2 (fraction 23), 9.5% N 10 - CHOH 4 PteGlu 4 (peak 4, fraction 18) and 7.3% N 10 - CHOH 4 PteGlu 4 (peak 7, fraction 28). The methylfolates were 13.4% N 5 -CH 3 H 4 PteGlu (peak 3, fraction 13), 7% N 5 -CH 3 H 4 PteGlu 3 (peak 6, fraction 24), 2.1% N 5 -CH 3 H 4 PteGlu 4 (peak 10, fraction 40) and 6.4% N 5 -CH 3 H 4 PteGlu 5 (peak 12, fraction 54). Significant alterations in various one-carbon substitution and polyglutamyl chain lengths in formyl and methyl tetrahydrofolate derivatives were observed in the cells grown in media with D 2 O-depleted water. Methyltetrahydrofolyl polyglutamates, the major folate form in the cells grown in the media prepared with normal water (Table 2) were lowered (almost to 23%) in D 2 O-depleted cells (Table 3, peak fractions 13, 24 and 40), while formyltetrahydrofolyl polyglutamate in the form of N 10 -CHOH 4 PteGlu 4 (fraction 28, Table 3) was increased almost 2-fold (7.2%). Unreduced folates such as PteGlu, PteGlu 3 and PteGlu 4 which were 12.1% in normal cells (Fig. 1A) were significantly enhanced to 21.6% (fractions Table 3 Folate derivatives identified from fractions after DEAE-cellulose analytical chromatography (Fig. 1). [The fractions obtained from DEAE-cellulose chromatography (Fig. 1A, B and C) were analyzed by differential microbiological assays with L. casei and P. cerevisiae, before and after chicken liver folyl conjugase digestion to identify the formyl, methyl, reduced and oxidized folate derivatives, comparing with authentic standards, as described in the Text] Peak fraction no. Folates identified Folates as % of total Normal 600 ppm D 2 O D 2 O-depleted 7 N 10 -CHOH 4 PteGlu N 10 -CHOPteGlu N 10 -CHOPteGlu N 5 -CHOH 4 PteGlu N 10 -CHOH 4 PteGlu N 5 -CH 3 H 4 PteGlu N 5 -CH 3 H 4 PteGlu N 5 -CH 3 H 4 PteGlu N 5 -CH 3 H 4 PteGlu PteGlu PteGlu PteGlu

5 POTE et al.: DEUTERIUM OXIDE EFFECTS ON FOLATE METABOLISM , 38 and 44, Table 3) in the cells grown in D 2 O- depleted water. An increase of 7% in the active formyl co-factor forms (peaks 7 and 28, Table 3) as well as the total formyl folates from 30.6% to 49.5% (peaks 7, 11, 18, 23 and 28, Table 3) was also observed in the cells grown in the media prepared with D 2 O-depleted water. However, the total methyl folates, the major form of folates in the cells grown in normal water were drastically reduced to 29% in the cells grown in D 2 O-depleted media (Table 3). No significant changes were noticed in total methyl folate profile (61.7%) in the cells grown in the media with 600 ppm D 2 O. Another important finding is that higher levels (31.4%) of oxidized folates were observed in the D 2 O-depleted cells (Table 3, fractions 11, 38 and 44) indicative of oxidative changes, resulting possibly at the cost of mainly methyl folate form. Oxidized folates of formyl- and methyltetrahydrofolates have been shown to be poor substrates for dihydrofolate reductase, resulting in an impaired regeneration of reduced folate co-factors 25. Further, oxidized folate co-enzymes might act as an anti-folate. Oxidized folates have also been shown to have less affinity than reduced folate co-factors for folate binding proteins which are intracellular folate carriers 26. The nonavailability of reduced folates co-factors would thus lead to altered folate homeostasis and result in an overall impairment in folate-dependent reactions. Recently, Gyongyi and Somlyai 27 have shown that depletion of the naturally occurring deuterium can result in tumour regression in mice, dogs and humans. The effect of deuterium depletion on gene expression plays a key role in tumour development. The cell cycle regulation and tumour development are sensitive to deuterium-depletion 27. In the present study, D 2 O-depletion resulted in alterations in certain 1-carbon substitution, as well as polyglutamylation of folates in L. casei cells. Acknowledgement The authors would like to thank Dr N C Verma, former Head, Radiation Biology Division, Bhaba Atomic Research Centre, Mumbai, for encouragement and advice. References 1 Stockstad E L R & Koch J (1967) Physiol Rev 47, Kesavan V & Noronha J M (1992) Experientia 48, Krumdieck C L & Eto I (1986) in Chemistry and Biology of Pteridines (Cooper B A & Whitehead V M, ed.), pp , Walter de & Gruyter Co., Berlin and New York 4 Varela Moreiras G & Selhub J (1992) J Nutr 122, Tamura T & Stockstad E L R (1993) Br J Haematol 25, Priest D G, Doig M T & Mangum M (1983) Biochim Biophys Acta 756, Nair C P P, Viswanathan G & Noronha J M (1992) J Clin Biochem Nutr 13, Carl F G, Smith M L, Fuman M, Eto I, Schatz R A & Krumdieck C L (1991) J Nutr 121, Mikol Y B, Hoover K L, Creasia D & Poirier L A (1983) Carcinogenesis 4, Krumdieck C L & Howard-Peebls P N (1983) Am J Med Genet 16, Chan P Y & Cossins E A (1980) Arch Biochem Biophys 200, Hilton J G, Cooper B A & Rosenblatt D (1979) J Biol Chem 254, Somolyai G, Jancso G, Jakli G, Vass K, Barna B, Lakics V & Gall T (1993) FEBS Lett 317, Kotyk A, Dvorakova M & Koryta J (1990) FEBS Lett 264, Doring O & Bottger M (1992) Biochem Biophys Res Commun 182, Stefanescu I, Steflea D & Titescu Gh (1999) ICSI Conference, Abstr pp Rosenbaum K, Janke K, Schnackerz K D & Cook P (1998) Biochemistry 37, Kushner D J, Baker A & Dunstall T G (1999) Can J Physiol Pharmacol 77, Kesavan V & Noronha J M (1983) Am J Clin Nutr 37, Noronha J M & Silverman M (1962) J Biol Chem 237, Narasimha Rao K.& Noronha J M (1978) Anal Biochem 88, Lowry O H, Rosenbrough N J, Farr A L & Randall R J (1951) J Biol Chem 3, Hodel A, Hebbers J O, Cottier H & Laissue J A (1982) Life Sciences 30, Bachner P, Mckay G & Rittenberg D (1964) Proc Natl Acad Sci 51, Keresztesy J C & Donaldson K O (1961) Biochem Biophys Res Commun 5, Wagner C (1982) Ann Rev Nutr 2, Gyongyi Z & Somlyai G (2000) In Vivo 14,