10-formyltetrahydrofolate exists in pig plasma

Nutrition Research, Vol. 20, No. 9. pp. 13551359, 2OC0 Copyright 0 2ooO Elsevier Science Inc. Printed in the USA. All rights reserved 027 I-53 I7/W$-see front matter ELSEVIER

PII: SO271-5317(00)00208-6

lo-FORMYLTETRAHYDROFOLATE

EXISTS IN PIG PLASMA

Yasuhaiu Mizuno, D.V.M., Ph. D., Takako Nasuno, D.V.M., Shihoko Okusa, D.V.M., Minoru Shimoda, D.V.M., Ph. D., and Eiichi Kokue, D.V.M., Ph. D. Department of Veterinary Medicine, Tokyo University of Agriculture & Technology, Saiwaicho 3-5-8, Fuchu, Tokyo 183-0054, Japan

We determined plasma folate concentrations by microbiological assay and high-performance liquid chromatography with electrochemical detection (HPLCECD). We observed a marked difference between total folate (53.4 * 21.0 nM, n=12) obtained by microbiological assay and the sum (26.7 + 9.9 nM, n=12) of tetrahydrofolate (H,PteGlu) and 5methyltetrahydrofolate (5-CH,-H.,PteGlu) detected by HPLC-ECD. In order to elucidate this difference, microbiological Heat or assays were performed with fractions of the HPLC eluate of pig plasma. Pig plasma after heating showed acid treatment was used for deproteinization. Those retention times were three peaks of folate activity on the chromatogram. identical to those of lo-formyltetrahydrofolate (lo-HCO-H,PteGlu), H,PteGlu and 5-CH,-H,PteGlu. It was suggested that the existence of lo-HCO-H,PteGlu produced the difference (26.7 f 16.0 nM, n=12) between total folate by microbiological assay and the sum of H,PteGlu and 5-CH,-H.,PteGlu by HPLCECD. After acid treatment, the peak with retention time similar to lo-HCOH,PteGlu became smaller than that in heat treatment, and at the same time a peak in addition to lo-HCO-H,PteGlu, H,PteGlu and 5-CH,-H.,PteGlu appeared on the chromatogram. This peak exhibited the same retention time as that of 5,10H,PteGlu and 5-CH,-H,PteGlu in methenyltetrahydrofolate (5,10_CH=H,PteGlu). pig plasma were completely recovered after heating under ascorbate concentration at 100 mM (pH 7.4). These results also supported the existence of lo-HCOH,PteGlu in pig plasma. Our data indicates that IO-HCO-H,PteGlu exists in pig plasma as a major folate derivative in addition to H,PteGlu and 5-CH,-H,PteGlu. Science Inc. 0 ?ooO Elsevaer Key words: Folate, Pig plasma, Microbiological assay, High-performance liquid chromatography

CTION Major folate derivatives in pig plasma were reported to be tetrahydrofolate (H,PteGlu) and 5-methyltetrahydrofolate (5-CH,-H,PteGlu) (1). We determined folate concentration in pig plasma using two distinctively different methods including the microbiological assay using Lactobacillus casei (L. casei, ATCC 7469) and an high-performance liquid chromatography (HPLC) with electrochemical detection (ECD) technique. L. casei responds to the widest spectrum of folate derivatives (2), whereas the HPLC-ECD technique had a capability of detecting only H,PteGlu and 5-CH,-H,PteGlu in pig plasma (3). Total folate concentrations in pig plasma were determined by microbiological assay, and H,PteGlu and 5-CH,-H,PteGlu concentrations in Corresponding author; MESON Shimoda, Department of Veterinary Medicine, Tokyo University of Agriculture & Technology, Tel & Fax; +81-42-367-5770, E-mail; [email protected] 1355

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Y. MIZUNO et al.

We found a remarkable difference between total pig plasma were determined by HPLC-ECD. folate concentrations and the sum of H,PteGlu and 5-CH,-HJ’teGlu. We, therefore, postulated the existence of an extra folate derivative(s) in addition to the previously reported H,PteGlu and 5CH,-H,PteGlu in plasma of pigs. The present study was undertaken to identify unknown folate(s) in pig plasma.

Animals A total of 12 Goettingen minipigs (40-70 kg SW, 5-7 years old) were used in this experiment. The animals were fed a restricted amount of a feed (0.8 kg/animal) once a day and given water ad libitum. Although synthetic PteGlu was not added to the feed, folate content was 1.19 mg/kg of feed by microbiological assay after folate conjugase treatment. Venous blood was collected from the venae jugularis extema under light isoflurane anesthesia after a 24-hour fasting. After centrifugation, plasma containing sodium ascorbate at the final concentration of 20 mM was placed at -80 “C until analysis. The animal experiments were approved by the Animal Ethics Committee of the Agricultural Faculty, Tokyo University of Agriculture and Technology. Plasma folate concentrations by microbiological assay and those by HPLC-ECD Concentrations of H,PteGlu and 5-CH,-H,PteGlu in pig plasma were determined by HPLC-ECD (4). Concentrations of total and non-methylfolate in pig plasma were obtained by microbiological assay using L. casei and Enterococcus hirae (E. hirae, ATCC 8043) as test organisms, respectively (2). Methylfolate concentrations were obtained by subtracting E. hirae values from L. casei values. Pig plasma was deproteinized by heating at 121 “C for 5 min with ascorbate at the final concentration of 100 mM, and was stored at -80 “C until assay. We observed that the recoveries of H,PteGlu and 5-CH,-H,PteGlu after heating under 100 mM ascorbate concentration (pH 7.4) were 95.7 f 9.3 and 96.8 + 8.8 %, respectively. However, under 50 mM ascorbate concentration (pH 7.4), the recoveries of those folates were 61.4 f 7.7 and 77.4 f 5.7 %, respectively. Identification offolate in pig plasma Both heat and acid treatments were used as methods of deproteinization. The former treatment was that pig plasma was mixed with an equal volume of 0.2 M ascorbate solution (pH 7.4) and the mixture was heated at 121 “C for 5 min. The latter was that pig plasma was treated with an equal volume of 0.5 M perchloric acid. After centrimgation, the supernatant was subjected to HPLC separation. The column was a phenylsilyl column (Radial-Pak type 5NvpH4J.1, Waters, Milford, MA). The mobile phase was 0.1 M potassium phosphate containing 50 mM 2-mercaptoethanol and 0.4 % acetonitrile (pH 6.0) and the flow rate was 0.8 mL/min. The tractions (0.2 ml/tube) of HPLC eluate were collected and aliquots of the fractions were applied to microbiological assay using L. casei as an assay organism. Chromatograms of pig plasma were compared with that of folate standards. The folate standards contained H,PteGlu, 5CH,-H,PteGlu, IO-formyltetrahydrofolate (IO-HCO-H,PteGlu), 5-formyltetrahydrofolate (5-HCOH,PteGlu), dihydrofolate (H,PteGlu), folic acid (PteGlu), 5, IO-methenyltetrahydrofolate (5,l OCH=H,PteGlu), 5,10-methylenetetrahydrofolate (5,10-CH,-H,PteGlu). IO-HCO-H,PteGlu was prepared from 5,10-CH=H,PteGlu immediately before use, as described by Shin et al (5). PteGlu was purchased from Wako Pure Chemical (Osaka, Japan) and the other folates were from Dr. Schircks Laboratories (Jona, Switzerland). Statistical analysis Two sample t-test with Welch’s correction was used to evaluate the difference of folate

1357

FOLATE IN PIG PLASMA

concentrations between microbiological assay and HPLC-ECD when the variance was significantly different by F-test (PO.O5).

Plasma folate concentrations by microbiological assay and those by HPLC-ECD Relationships of plasma folate concentrations of 12 individual pigs determined by microbiological assay and HPLC-ECD are represented in Figure 1. Total folate concentrations obtained by microbiological assay (L. casei) were much higher than the sum of H,PteGlu and 5CH,-H,PteGlu obtained by HPLC-ECD in all pigs except for 2 pigs. As to methylfolate As shown in Table 1, the mean of total concentration, a marked difference was not observed. folate concentration by microbiological assay was significantly higher than the mean of the sum of The mean of methylfolate concentration by H,PteGlu and 5-CH,-H,PteGlu by HPLC-ECD. microbiological assay (L. casei values minus E. hirae values) was not significantly different from The mean difference between total 5-CH,-H,PteGlu concentration obtained by HPLC-ECD. folate and the sum of H,PteGlu and 5-CH,-H,PteGlu was 26.7 2 16.0 nM. -

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FIG. 1. Plasma Folate Concentrations of 12 Individual Pigs Mehtylfolate (right) and total folate (left) concentrations in HPLC-ECD represent 5-CH,-H,PteGlu and the sum of H,PteGlu and 5-CH,-H.,PteGlu, respectively.

TABLE 1 in Pig Plasma Determined by Microbiological Assay and HPLC-ECD Plasma concentrations (nM) Total folate Non-methylfolate Methylfolate Microbiological assay 53.4 +21.0* 43.1 f 18.9* 10.3 f 6.0 HPLC-ECD 26.7 f 9.9 16.8 + 7.4 9.9 f 3.5

Folate Concentrations

Values are mean f SD (n=12). Non-methylfolate, methylfolate and total folate in HPLC-ECD are the values of H,PteGlu, 5-CH,-H,PteGlu and the sum of H,PteGlu and 5-CH,-H,PteGlu, respectively. * indicates significant difference (~~0.01) from the value by HPLC-ECD.

Y. MIZUNO et al.

1358

Identification offolate in pig plasma Chromatographic separation of folate standards is shown in Figure 2 (top). Based on this chromatogram, eight folate derivatives were separated and identified. When pig plasma was deproteinized by heating, three peaks of folate activity were observed on the chromatogram (Figure 2, bottom). Those peaks had the same retention times as those of IO-H,PteGlu, H,PteGlu By acid and 5-CH,-H,PteGlu, respectively. (d treatment, there were four peaks of folate lb) activity (Figure 2, middle). Three of them exhibited the same retention times as those I 04 observed on the chromatogram alter heat treatment. The retention time of an extra peak was the same as that of 5,10-CH=H,PteGlu. The peak with the same retention time as that of IO-HCO-H,PteGlu was smaller than that in the case of heat treatment.

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DISCUSSIQN Acid treatment

In order to identify unknown folate(s), pig plasma was analyzed by the combination of the HPLC method and microbiological assay which allowed isolation and identification of a total of eight folate derivatives (Figure 2, top). We identified lo-HCO-H,PteGlu in pig plasma based on the following observations: 1) pig plasma after heat treatment showed only three peaks of folate activity, including IO-HCOH,PteGlu, H,PteGlu and 5-CH,-H,PteGlu on the chromatogram (Figure 2, bottom), and 2) after acid treatment, lo-HCO-H,PteGlu peak was smaller than that after heat treatment and a fourth peak in addition to IO-H,PteGlu, H,PteGlu and 5-CH,-H,PteGlu appeared on the chromatogram (Figure 2, middle). The retention time of this fourth peak was identical to that of authentic 5,10-CH=H,PteGlu, and it is known that lo-HCO-H,PteGlu is readily converted to 5,10_CH=H,PteGlu under the acidic pH.

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FIG. 2. Chromatograms of Folate Standards and Pig Plasma Obtained by HPLC Separation Followed by Microbiological Assay The standard solution of folate contained each folate at 10 ng/mL; (a) lo-HCO-HJ’teGlu, (b) H,PteGlu, (c) 5-HCO-H,PteGlu, (d) H,PteGlu (e) PteGlu, (f) 5,10-CH=H,PteGlu, (g) 5-CH,H,PteGlu, (h) 5,10-CH,-H,PteGlu

In the study presented here, we were able to demonstrate the presence of IO-HCOH,PteGlu in pig plasma using heat treatment and 100 mM ascorbate (pH 7.4). It is possible that IO-HCO-H,PteGlu exists in serum/plasma of other species, because it has been reported that monoglutamate and polyglutamate of IO-HCO-H,PteGlu occur naturally in various biological samples. For example, IO-HCO-H,PteGlu was detected in rat bile (5) and in human plasma alter the administration of 5-HCO-H,PteGlu or PteGlu (6, 7). In addition, polyglutamate of IO-HCOH,PteGlu (lo-HCO-H,PteGly) was reported to exist in the liver and kidney of the rat and mouse as well as in lima beans (8, 9, 10). A few groups of researchers suggested the presence of loHCO-H,PteGlu,, in ery-throcyte or serum (11, 12), although they did not identify this folate. Further investigation is necessary for the method of folate analysis for the detection of IO-HCO-

FOLATE IN PIG PLASMA

HPteGlu

1359

in the circulation of various animal species as well as in humans.

Pigs are usually given feeds supplemented with PteGlu in order to improve the folate nutriture. PteGlu is utilized in the body after reduction, methylation and formylation. Reduced folate, therefore, should be important on estimation of effects of PteGlu supplementation to feeds. Considering the existence of IO-HCO-H,PteGlu in pig plasma, the more accurate determination of serum/plasma folate concentration in pigs will be practical. In conclusion, IO-HCO-H,PteGlu occurs naturally in pig plasma as a major folate derivative in addition to H,PteGlu and 5-CH,H,PteGlu.

1. Natsuhori M, Shimoda M, Kokue E, Hayama T, Takahashi Y. Tetrahydrofolic principal congener of plasma folates in pigs. Am J Physiol 1991; 261 :R82-R86.

acid as the

2. Tamura T. Microbiological assay of folates. In: Picciano MF, Stokstad ELR, Gregory JF ed. Folic acid metabolism in health and disease, New York: Wiley-Liss, 1990: 121-37. 3. Shimoda M. Simultaneous determination of tetrahydrofolate and Ns-methyltetrajydrofolate in pig plasma by high-performance liquid chromatography with electrochemical detection. J Vet Med Sci 1992; 54(2):249-53. 4. Mizuno Y, Kokue E, Ohnishi N, Toride Y. Effect of oral administration

plasma folate levels in pigs: comparison Anim Sci 1997; 77:497-502.

of folate sources on between reduced and oxidized forms of folate. Can J

5. Shin Ho-C, Shimoda M, Kokue E. Identification of lo-formyltetrahydrofolate, and tetrahydrofolate and 5-methyltetrahydro-folate as major reduced folate derivatives in rat bile. J Chromatogr 1993; 620:39-46. 6. Priest DG, Schmitz JC, Bunni MA, Stuart RK. Pharmacokinetics of leucovorin metabolites in human plasma as a function of dose administered orally and intravenously. J Nat1 Cancer Inst 1991; 83:1806-12. 7. Schmitz JC, Stuart RK, Priest DG. Disposition of folic acid and its metabolites: with leucovorin. Clin Pharmacol Ther 1994; 55:501-8.

a comparison

8. Wilson SD, Horne DW. High-performance liquid chromatographic determination of the distribution of naturally occurring folic acid derivatives in rat liver. Anal Biochem 1984; 1421529-35. 9. Duch DS, Bowers SW, Nichol CA. Analysis of folate cofactor levels in tissues using highperformance liquid chromatography. Anal Biochem 1983; 130:385-92. 10. Seyoum E, Selhub J. Combined affinity and ion pair column chromatographies of food folate. J Nutr Biochem 1993; 4:488-94.

for the analysis

11. Iwai K, Luttner PM, Toennies G. Blood folic acid studies. J Biol Chem 1964; 239:2365-g. 12. Ratanastien K, Blair JA, Leming RJ, Cooke WT, Melikian V. Serum folates in man. J Clin Path 1977; 30:438-48. Accepted

for

publication

March 8,

‘2000.