- Email: [email protected]
[43] High-performance liquid chromatographic separation of the naturally occurring folic acid derivatives
HPLC
[431
OF TISSUE
269
FOLATES
recovered by centrifugation, dissolved in I ml of 10 mM Na phosphate2% SDS-SO mM mercaptoethanol (pH 7.0), and dialyzed overnight against 1 liter 10 mit4 Na phosphate-0.1% SDS-200 mA4 NaCl (pH 7.0). Fractionation of the transport protein (at 23”) on a column (1.8 x 65 cm) of Sephacryl S-300 that has been equilibrated with the dialysis buffer gives a single, labeled component which is purified 50-fold relative to intact cells. Comments
Covalent labeling of the transport protein via the NHS ester of [3H]methotrexate shows a relatively high specificity; only 20-40% of the label is distributed among other membrane proteins. Noncovalently bound methotrexate cannot be used to label the protein during purification, since it dissociates during solubilization of the protein from the membrane. The molecular weight of the protein is 36,000, as judged by its mobility in SDS-PAGE. When various detergents are compared for their ability to extract the labeled protein,‘* Nonidet P-40 was found to be equivalent to Triton X-100, while 5- to IO-fold higher concentrations of CHAPS, cholate, and octylglucoside are required to achieve the same degree of solubilization. Acknowledgments Publication BCR-3753 from the Research Institute of Scripps Clinic. The work was supported by grants from the National Cancer Institute (CAO6522, CA32261, CA23970) and the American Cancer Society (CH-31 and CH-229). The authors are indebted to Karin Vitols for assistance in preparation of the manuscript.
[431 High-Performance Liquid Chromatographic Separation of the Naturally Occurring Folic Acid Derivatives’ By SUSAN D. WILSON and DONALD
W. HORNE
The high resolution inherent in high-performance liquid chromatography (HPLC) along with the speed and reproducibility of this technique has led to its application to the task of separation and quantitation of the r Supported by the Veterans Administration and by NIH Grants AM15289 and AM32189.
METHODS
IN ENZYMOLOGY,
VOL.
122
Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
270
PTERIDINES,
ANALOGS,
AND
PTERIN
COENZYMES
[431
naturally occurring folate derivatives .2-6An equally important aspect of this analytical methodology is a tissue extraction procedure which protects easily oxidized folates. Recently, we discovered that using ascorbate solutions at elevated temperature to extract tissue folates results in chemical interconversion of several of these derivatives.’ Procedures have now been developed which allow tissue extraction without these interconversions, baseline separation of naturally occurring folate monoglutamates by HPLC, and quantitation via Lactobacillus casei microbiological assay. These procedures have been described elsewhere.* Standard Folic Acid Deriuutives. Pteroylglutamic acid (PteGlu, folic acid), H,PteGlu, 5-CH3-HdPteGlu, 5-HCO-H4PteGlu, and H,PteGlu are available from Sigma. IO-HCO-H,PteGlu and 5,10-CHz-HdPteGlumay be synthesized by published procedures as outlined by Wilson and Home.’ These derivatives may be purified as described below using HPLC. The purified standards should be stored in 10% (w/v) sodium ascorbate at -20” (or below) under a Nz atmosphere in Thunberg tubes (Kontes) at reduced pressure. Microbiological Assay of Folutes. Assays were performed as described by Wilson and Hornes*9 with the following modifications. The glycerol-cryoprotected L. cusei inoculum cultures were prepared by directly suspending the lyophilized culture from the American Type Culture Collection (L. gasei subspeciesrhumnosus, ATCC 7469) in 1 ml of singlestrength Folic Acid Casei Medium (Difco) supplemented with 0.25 mg sodium ascorbate and 0.30 ng PteGlu per milliliter. This suspension (250 ~1) was inoculated into 250 ml of the same, sterile medium and incubated at 37”for about 18 hr. An equal volume of sterile 80% (v/v) glycerol was added and the suspension stored at -70” in 2-ml aliquots. Prior to assay, these aliquots were made up to 10 ml with sterile, 0.9% (w/v) NaCl (saline), washed twice by centrifugation in saline, and resuspended in 2 ml of saline. In our hands, a 1 : 13 dilution of this suspension gave an optimal standard curve using HPLC purified (6R,S)-5-HCO-H,PteGlu from 0 to 0.5 ng per tube (calculated as the active S isomer) with an 18-hr incubation 2 K. E. McMartin, V. Virayotha, and T. R. Tephly, Arch. Biochem. Biophys. 209, 127 (1981). 3 D. S. Duch, S. W. Bowers, and C. A. Nichols, Anal. Biochem. 130, 385 (1983). 4 I. Eto and C. L. Kmmdieck, Anal. Biochem. l2fJ, 323 (1982). 5 B. Shane, Am. J. Clin. Nutr. 35, 599 (1982). 6 J. F. Gregory, D. B. Sartian, and B. P. F. Day, J. Nutr. 114, 341 (1984). ’ S. D. Wilson and D. W. Home, Proc. Nutl. Acad. Sci. U.S.A. 80, 6500-6504 (1983). * S. D. Wilson and D. W. Home, Anal. B&hem. 142, 529-535 (1984). 9 S. D. Wilson and D. W. Horne, C/in. Chem. (Winston-Salem, N.C.) 28, 1198 (1982).
I431
HPLC OF TISSUE FOLATES
271
at 37”. This procedure represents a modification of our previous method.*p9 Preparation of Liver Extracts. Male Sprague-Dawley rats were anesthetized with sodium pentobarbital, the liver removed, weighed, and minced with scissors, and 10 volumes of 100”extraction buffer (2%, w/v, sodium ascorbate, 0.2 M 2-mercaptoethanol, 50 mM HEPES, 50 mM 2-(N-cyclohexyl-amino)-ethane-sulfonic acid (Ches), pH 7.85) was added The test tube was heated at 100”for 10 min in the dark, cooled in an ice bath, and the liver was homogenized in a Teflon-glass homogenizer. The homogenate was centrifuged at 40,000 g for 20 min and the resulting supematant again centrifuged at 40,000 g for 10 min in a Sorvall RCZB centrifuge. The floating lipid layer was removed by aspiration and the supematant stored at -20” in Thunberg tubes under a N2 atmosphere at reduced pressure. Conjugase Treatment of Extracts. The source of y-glutamyl hydrolase (conjugase)used to hydrolyze folate polyglutamates was rat plasma. Heparinized blood was centrifuged at 2,000 g for 10 min at 4” and dialyzed overnight against buffer containing charcoal to remove endogenousfolates. HPLC-L. casei analysis of this preparation indicated that it contained 10 ng 5-CH3-I&PteGlu per milliliter. Aliquots of extracts were filtered through Amicon YMT membranesto remove high molecular weight compounds (synthetic PteGlud freely passed through the membrane). The filtrate was warmed to 37” and plasma conjugase (0.25 volumes) was added in three equal portions at 15min intervals. The tubes were incubated an additional 1.5 hr, heated at 100”for 5 min, centrifuged to remove precipitated protein, and stored at -20” at reduced pressure under N2 until assayed. High-Performance Liquid Chromatography. HPLC separation of folate monoglutamates was performed as described previously.9,i0Briefly, this entailed eluting folates from a Beckman-Altex Ultrasphere I.P. column with a concave ethanol gradient (8-15% ethanol, setting #8, Waters Model 660 Solvent Programmer) using tetrabutylammonium phosphate (TBAP) as an ion-pair reagent. An alternative procedure employed a Spectra-Physics SP8700Solvent Delivery System equipped with a 2-ml injection loop. Solvents employed were (A) water, (B) 25% ethanol (v/v), and (C) 80% methanol (v/v). Solvents A and B each contained 1 mM sodium ascorbate and 7 mM TBAP reagent. Solvent C was employed to remove tightly adhering substances and as a column storage solvent. Folates were eluted from the Ultrasphere I.P. column isocratically (70% lo D. W. Home, W. T. Brig@, and C. Wagner, Anal. Biochem. 116, 393 (1981).
272
PTERIDINES,
ANALOGS,
AND PTERIN COENZYMES
r431
6
0.8-
0.7 0.6-
0. I
0
lo
20
30
40
FRACTION
50
60
70
80
NUMBER
FIG. 1. High-performance liquid chromatographic separation and L. casei determination of standard folic acid derivatives. Elution conditions and L. casei assay protocol are described in the text. (I) IO-HCO-H,PteGlu, (2) H,PteGlu, (3) 5-HCO-H4PteGlu, (4) H,PteGlu, (5) S,lO-CHa-HJ’teGlu, (6) 5-CH,-H,PteGlu, and (7) PteGlu; 5-10 ng of each derivative was applied to the column. From Wilson and Home.’
DISTRIBUTION OF RAT LIVER FOLATE+~
Distribution Cofactor IO-HCO-H,PteGlu HJYeGlu 5-HCO-H$teGlu 5-CHrH4PteGlu
hzk) 1.42 2.06 0.49 2.35
-’ 0.13 2 0.16 2 0.04 f 0.22
cm’ 22.6 32.7 7.7 37.3
2 2 ‘2
1.1 1.4 0.5 2.6
o From Wilson and Home.* b The distribution of folate derivatives in rat liver was determined by HPLC-L. casei analysis of conjugase-treated extracts prepared as described in the text. The values reported represent the mean -+ SE of 15 experiments. c Percentage of total folate eluted from the column.
rp-HPLC OF FTERIDINESFROM BLOOD
[441
273
A, 30% B) for the first 25 min, followed by a linear gradient ending at 75 min (40% A, 60% B). Fractions of 1 ml were collected into tubes containing 0.1 ml of 10% (w/v) sodium ascorbate. Appropriate aliquots were chromatographed via HPLC and folates were quantitated using the L. casei microbiological assay. Figure 1 shows the elution profile when standard folates were analyzed via the HPLC-L. casei procedure described above. All standard folates were baseline resolved, a fact which makes quantitation of each derivative unambiguous. The elution position of each standard was confirmed by UV spectroscopy. The data in the table were obtained by applying our procedures (extraction, conjugase treatment, and HPLC-L. casei assay) to determining the distribution of folates in rat liver.
1441 Separation of Pteridines from Blood Cells and Plasma by Reverse-Phase High-Performance Liquid Chromatography
BYHANS-JGRGZEITLERand
BERTAANDONDONSKAJA-RENZ
Reduced pterins (e.g., biopterin, folates) are intermediates in anabolic and catabolic reactions in pteridine metabolism and serve as coenzymes for different enzymatic reactions. In the biosynthesis of melanins, neurotransmitters, and prostaglandins mixed-function oxygenases need reduced biopterins, whereas the conversion of some amino acids and the biosynthesis of purines and pyrimidines (transfer of one-carbon units) are mediated by several folate derivatives. These reactions are important in various tissues’ and in the development, differentiation, and maturation of blood cells.* The folate coenzymes in nonanimal cells are synthesized from guanosine triphosphate and are one of the systems of self-regulation that may get out of control in malignant conditions.3 The importance of various pteridines in cell metabolism of higher mammals was recognized by Kaufman in 1963.4The identification of 6-hydroxymethylpterir$ as a I C. Kutzbach and E. L. R. Stokstad, Biochim. Biophys. Acta 139, 217 (1967). 2 D. Watkins, T. E. Shapiro, and B. A. Cooper, in “Biochemical and Clinical Aspects of Pteridines” (H. C. Curtius, W. Pfleiderer, and W. Wachter, eds.), Vol. 2, p. 351. de Gruyter, Berlin, 1983. 3 A. Albert, in “Chemistry and Biology of Pteridines” (W. Pfleiderer, ed.) p. 1. de Gruyter. Berlin, 1975. 4 S. Kaufman, Proc. Natl. Acad. Sci. U.S.A. 50, 1085 (1963). 5 B. Stea, P. S. Backlund, P. B. Berkey, A. K. Cho, B. C. Halpern, R. M. Halpern, and R. A. Smith, Cancer Res. 38, 2378 (1978).
METHODS
IN ENZYMOLOGY,
VOL.
122
Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
[431
OF TISSUE
269
FOLATES
recovered by centrifugation, dissolved in I ml of 10 mM Na phosphate2% SDS-SO mM mercaptoethanol (pH 7.0), and dialyzed overnight against 1 liter 10 mit4 Na phosphate-0.1% SDS-200 mA4 NaCl (pH 7.0). Fractionation of the transport protein (at 23”) on a column (1.8 x 65 cm) of Sephacryl S-300 that has been equilibrated with the dialysis buffer gives a single, labeled component which is purified 50-fold relative to intact cells. Comments
Covalent labeling of the transport protein via the NHS ester of [3H]methotrexate shows a relatively high specificity; only 20-40% of the label is distributed among other membrane proteins. Noncovalently bound methotrexate cannot be used to label the protein during purification, since it dissociates during solubilization of the protein from the membrane. The molecular weight of the protein is 36,000, as judged by its mobility in SDS-PAGE. When various detergents are compared for their ability to extract the labeled protein,‘* Nonidet P-40 was found to be equivalent to Triton X-100, while 5- to IO-fold higher concentrations of CHAPS, cholate, and octylglucoside are required to achieve the same degree of solubilization. Acknowledgments Publication BCR-3753 from the Research Institute of Scripps Clinic. The work was supported by grants from the National Cancer Institute (CAO6522, CA32261, CA23970) and the American Cancer Society (CH-31 and CH-229). The authors are indebted to Karin Vitols for assistance in preparation of the manuscript.
[431 High-Performance Liquid Chromatographic Separation of the Naturally Occurring Folic Acid Derivatives’ By SUSAN D. WILSON and DONALD
W. HORNE
The high resolution inherent in high-performance liquid chromatography (HPLC) along with the speed and reproducibility of this technique has led to its application to the task of separation and quantitation of the r Supported by the Veterans Administration and by NIH Grants AM15289 and AM32189.
METHODS
IN ENZYMOLOGY,
VOL.
122
Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
270
PTERIDINES,
ANALOGS,
AND
PTERIN
COENZYMES
[431
naturally occurring folate derivatives .2-6An equally important aspect of this analytical methodology is a tissue extraction procedure which protects easily oxidized folates. Recently, we discovered that using ascorbate solutions at elevated temperature to extract tissue folates results in chemical interconversion of several of these derivatives.’ Procedures have now been developed which allow tissue extraction without these interconversions, baseline separation of naturally occurring folate monoglutamates by HPLC, and quantitation via Lactobacillus casei microbiological assay. These procedures have been described elsewhere.* Standard Folic Acid Deriuutives. Pteroylglutamic acid (PteGlu, folic acid), H,PteGlu, 5-CH3-HdPteGlu, 5-HCO-H4PteGlu, and H,PteGlu are available from Sigma. IO-HCO-H,PteGlu and 5,10-CHz-HdPteGlumay be synthesized by published procedures as outlined by Wilson and Home.’ These derivatives may be purified as described below using HPLC. The purified standards should be stored in 10% (w/v) sodium ascorbate at -20” (or below) under a Nz atmosphere in Thunberg tubes (Kontes) at reduced pressure. Microbiological Assay of Folutes. Assays were performed as described by Wilson and Hornes*9 with the following modifications. The glycerol-cryoprotected L. cusei inoculum cultures were prepared by directly suspending the lyophilized culture from the American Type Culture Collection (L. gasei subspeciesrhumnosus, ATCC 7469) in 1 ml of singlestrength Folic Acid Casei Medium (Difco) supplemented with 0.25 mg sodium ascorbate and 0.30 ng PteGlu per milliliter. This suspension (250 ~1) was inoculated into 250 ml of the same, sterile medium and incubated at 37”for about 18 hr. An equal volume of sterile 80% (v/v) glycerol was added and the suspension stored at -70” in 2-ml aliquots. Prior to assay, these aliquots were made up to 10 ml with sterile, 0.9% (w/v) NaCl (saline), washed twice by centrifugation in saline, and resuspended in 2 ml of saline. In our hands, a 1 : 13 dilution of this suspension gave an optimal standard curve using HPLC purified (6R,S)-5-HCO-H,PteGlu from 0 to 0.5 ng per tube (calculated as the active S isomer) with an 18-hr incubation 2 K. E. McMartin, V. Virayotha, and T. R. Tephly, Arch. Biochem. Biophys. 209, 127 (1981). 3 D. S. Duch, S. W. Bowers, and C. A. Nichols, Anal. Biochem. 130, 385 (1983). 4 I. Eto and C. L. Kmmdieck, Anal. Biochem. l2fJ, 323 (1982). 5 B. Shane, Am. J. Clin. Nutr. 35, 599 (1982). 6 J. F. Gregory, D. B. Sartian, and B. P. F. Day, J. Nutr. 114, 341 (1984). ’ S. D. Wilson and D. W. Home, Proc. Nutl. Acad. Sci. U.S.A. 80, 6500-6504 (1983). * S. D. Wilson and D. W. Home, Anal. B&hem. 142, 529-535 (1984). 9 S. D. Wilson and D. W. Horne, C/in. Chem. (Winston-Salem, N.C.) 28, 1198 (1982).
I431
HPLC OF TISSUE FOLATES
271
at 37”. This procedure represents a modification of our previous method.*p9 Preparation of Liver Extracts. Male Sprague-Dawley rats were anesthetized with sodium pentobarbital, the liver removed, weighed, and minced with scissors, and 10 volumes of 100”extraction buffer (2%, w/v, sodium ascorbate, 0.2 M 2-mercaptoethanol, 50 mM HEPES, 50 mM 2-(N-cyclohexyl-amino)-ethane-sulfonic acid (Ches), pH 7.85) was added The test tube was heated at 100”for 10 min in the dark, cooled in an ice bath, and the liver was homogenized in a Teflon-glass homogenizer. The homogenate was centrifuged at 40,000 g for 20 min and the resulting supematant again centrifuged at 40,000 g for 10 min in a Sorvall RCZB centrifuge. The floating lipid layer was removed by aspiration and the supematant stored at -20” in Thunberg tubes under a N2 atmosphere at reduced pressure. Conjugase Treatment of Extracts. The source of y-glutamyl hydrolase (conjugase)used to hydrolyze folate polyglutamates was rat plasma. Heparinized blood was centrifuged at 2,000 g for 10 min at 4” and dialyzed overnight against buffer containing charcoal to remove endogenousfolates. HPLC-L. casei analysis of this preparation indicated that it contained 10 ng 5-CH3-I&PteGlu per milliliter. Aliquots of extracts were filtered through Amicon YMT membranesto remove high molecular weight compounds (synthetic PteGlud freely passed through the membrane). The filtrate was warmed to 37” and plasma conjugase (0.25 volumes) was added in three equal portions at 15min intervals. The tubes were incubated an additional 1.5 hr, heated at 100”for 5 min, centrifuged to remove precipitated protein, and stored at -20” at reduced pressure under N2 until assayed. High-Performance Liquid Chromatography. HPLC separation of folate monoglutamates was performed as described previously.9,i0Briefly, this entailed eluting folates from a Beckman-Altex Ultrasphere I.P. column with a concave ethanol gradient (8-15% ethanol, setting #8, Waters Model 660 Solvent Programmer) using tetrabutylammonium phosphate (TBAP) as an ion-pair reagent. An alternative procedure employed a Spectra-Physics SP8700Solvent Delivery System equipped with a 2-ml injection loop. Solvents employed were (A) water, (B) 25% ethanol (v/v), and (C) 80% methanol (v/v). Solvents A and B each contained 1 mM sodium ascorbate and 7 mM TBAP reagent. Solvent C was employed to remove tightly adhering substances and as a column storage solvent. Folates were eluted from the Ultrasphere I.P. column isocratically (70% lo D. W. Home, W. T. Brig@, and C. Wagner, Anal. Biochem. 116, 393 (1981).
272
PTERIDINES,
ANALOGS,
AND PTERIN COENZYMES
r431
6
0.8-
0.7 0.6-
0. I
0
lo
20
30
40
FRACTION
50
60
70
80
NUMBER
FIG. 1. High-performance liquid chromatographic separation and L. casei determination of standard folic acid derivatives. Elution conditions and L. casei assay protocol are described in the text. (I) IO-HCO-H,PteGlu, (2) H,PteGlu, (3) 5-HCO-H4PteGlu, (4) H,PteGlu, (5) S,lO-CHa-HJ’teGlu, (6) 5-CH,-H,PteGlu, and (7) PteGlu; 5-10 ng of each derivative was applied to the column. From Wilson and Home.’
DISTRIBUTION OF RAT LIVER FOLATE+~
Distribution Cofactor IO-HCO-H,PteGlu HJYeGlu 5-HCO-H$teGlu 5-CHrH4PteGlu
hzk) 1.42 2.06 0.49 2.35
-’ 0.13 2 0.16 2 0.04 f 0.22
cm’ 22.6 32.7 7.7 37.3
2 2 ‘2
1.1 1.4 0.5 2.6
o From Wilson and Home.* b The distribution of folate derivatives in rat liver was determined by HPLC-L. casei analysis of conjugase-treated extracts prepared as described in the text. The values reported represent the mean -+ SE of 15 experiments. c Percentage of total folate eluted from the column.
rp-HPLC OF FTERIDINESFROM BLOOD
[441
273
A, 30% B) for the first 25 min, followed by a linear gradient ending at 75 min (40% A, 60% B). Fractions of 1 ml were collected into tubes containing 0.1 ml of 10% (w/v) sodium ascorbate. Appropriate aliquots were chromatographed via HPLC and folates were quantitated using the L. casei microbiological assay. Figure 1 shows the elution profile when standard folates were analyzed via the HPLC-L. casei procedure described above. All standard folates were baseline resolved, a fact which makes quantitation of each derivative unambiguous. The elution position of each standard was confirmed by UV spectroscopy. The data in the table were obtained by applying our procedures (extraction, conjugase treatment, and HPLC-L. casei assay) to determining the distribution of folates in rat liver.
1441 Separation of Pteridines from Blood Cells and Plasma by Reverse-Phase High-Performance Liquid Chromatography
BYHANS-JGRGZEITLERand
BERTAANDONDONSKAJA-RENZ
Reduced pterins (e.g., biopterin, folates) are intermediates in anabolic and catabolic reactions in pteridine metabolism and serve as coenzymes for different enzymatic reactions. In the biosynthesis of melanins, neurotransmitters, and prostaglandins mixed-function oxygenases need reduced biopterins, whereas the conversion of some amino acids and the biosynthesis of purines and pyrimidines (transfer of one-carbon units) are mediated by several folate derivatives. These reactions are important in various tissues’ and in the development, differentiation, and maturation of blood cells.* The folate coenzymes in nonanimal cells are synthesized from guanosine triphosphate and are one of the systems of self-regulation that may get out of control in malignant conditions.3 The importance of various pteridines in cell metabolism of higher mammals was recognized by Kaufman in 1963.4The identification of 6-hydroxymethylpterir$ as a I C. Kutzbach and E. L. R. Stokstad, Biochim. Biophys. Acta 139, 217 (1967). 2 D. Watkins, T. E. Shapiro, and B. A. Cooper, in “Biochemical and Clinical Aspects of Pteridines” (H. C. Curtius, W. Pfleiderer, and W. Wachter, eds.), Vol. 2, p. 351. de Gruyter, Berlin, 1983. 3 A. Albert, in “Chemistry and Biology of Pteridines” (W. Pfleiderer, ed.) p. 1. de Gruyter. Berlin, 1975. 4 S. Kaufman, Proc. Natl. Acad. Sci. U.S.A. 50, 1085 (1963). 5 B. Stea, P. S. Backlund, P. B. Berkey, A. K. Cho, B. C. Halpern, R. M. Halpern, and R. A. Smith, Cancer Res. 38, 2378 (1978).
METHODS
IN ENZYMOLOGY,
VOL.
122
Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.