4NALYTICAL

BIOCHEMISTRY

71, 265-272 (1976)

Microsomal Laurie Acid ll- and 12-Hydroxylation: New Assay Method Utilizing High Pressure Liquid Chromatography1

A

LUCY L. FAN,BETTIE SUE S. MASTERS,AND RUSSELLA. PROUGH Department

of Biochemistry, The University 5323 Harry Hines Boulevard,

of Texas Health Science Dallas, Texas 75235

Center,

Received June 23, 1975; accepted October 23, 1975 A new assay method using high pressure liquid chromatography has been developed which permits the simultaneous isolation, determination, and quantitation of lauric acid and its hydroxylated products after methylation of extracts from kidney or liver microsomal incubation mixtures. The small differences in polarity between the lauric acid, 11-hydroxy- and 12-hydroxy-lauric acid after methylation permit their separation on reverse phase columns packed with octadecyltrichlorosilane bonded to silicone polymers. The total time required for the chromatography is less than 1 hr. Using this method, the formation of hydroxylated products was shown to have a linear dependence on protein concentration and time. The K, for lauric acid and NADPH were determined to be 8 pM and 54 pM in kidney microsomes. respectively.

Laurie acid has been shown to be hydroxylated by liver and kidney microsomes to yield two hydroxylated products, 12-hydroxylauric acid (12-OH) and 1 1-hydroxylauric acid (1 l-OH) (1,2). The separation of hydroxylated products from lauric acid has been carried out by silicic acid column chromatography (1,3,4) and the separation of the two hydroxylated laurate methyl ester derivatives has been obtained by thin-layer chromatography or radio-gas chromatography. Silicic acid column chromatography cannot separate the two hydroxylated products, and thinlayer techniques require large incubation mixtures and repeated chromatography to obtain resolution of the two products. High pressure liquid chromatography (HPLC) is an efficient technique for separation of small molecules which cannot be easily separated by other methods. To date, low molecular weight fatty acids have not routinely been separated by HPLC. The HPLC technique described in this paper has the advantage of complete recovery of products and substrates and simplicity of assay as compared to the radio-gas chromatographic method (1,5). 1 Supported in part by USPHS Grant No. HL 13619 (BSSM), grant No. I-453 from the Robert A. Welch Foundation (BSSM), USPHS Grant No. HL 17134 (RAP), and a National Cancer Institute Contract No. CP 33362( RAP). Lucy L. Fan was a Predoctoral Fellow of the Robert A. Welch Foundation. 265 Copyright 0 1976 by Academic Press Inc. All rights of reproduction m any form reserved

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FAN,

MASTERS

MATERIALS

AND

PROUGH

AND METHODS

Chemicals. NADP+ and NADPH were obtained from P-L Biochemicals, Inc. Isocitrate and isocitrate dehydrogenase were purchased from the Sigma Chemical Co. [lJ4C]Lauric acid was obtained from the Amersham/Searle Corp. and purified by thin-layer chromatography. The specific activity of the lauric acid routinely used as substrate was about 0.5 mCi/mmol. Pure samples of 1I-hydroxylauric acid and 12-hydroxylaurate methyl ester were generous gifts from Dr. Sten Orrenius, Karolinska Institute, Stockholm, Sweden. 11-Hydroxylauric acid was methylated with BF,-MeOH. Both methyl laurate standards were shown by mass spectral analysis to be more than 90% pure. All other chemicals used were purchased from commercial sources and were of reagent grade. Instrumentation. The instrument used was a Waters Associates Model ALC-202/401 liquid chromatograph equipped with a refractometer. The type of column used for all separations was a 1/4 in. x 1 ft ,ubondapak/C,, column purchased from Waters Associates which is an ether-linked octadecyl side chain bonded to silica beads. The column efficiency was evaluated using biphenyl as a standard and 100% methanol at 2 ml/min flow rate as the mobile phase. Columns with a theoretical plate count of less than 2500 will not adequately separate the methyl esters of the two hydroxylaurate derivatives. Assay methods. Sprague-Dawley male rats weighing approximately 150-200 g were used unless otherwise noted; the rats were killed by decapitation. The kidneys were removed, the cortex was dissected from the medulla, and the cortex was homogenized in 8 ml of 0.25 M sucrose/g tissue. The microsomes were isolated as described previously for liver microsomal preparations (6). The final microsomal protein concentration was adjusted to 15 mg/ml in 0.05 M potassium phosphate buffer (pH 7.7) with 10m4 M EDTA. Protein was determined by the method of Lowry et al. (7). The incubation mixtures used in this study were similar to those reported by Ellin et al. (2) and each reaction mixture contained 1.5 mg kidney cortex microsomes, 50 mM Tris buffer, pH 7.5, 5 mM MgC12, 0.005 mM MnCI,, 1 mM NADP+, 5 mM DL-isocitrate, 0.36 IU pig heart isocitrate dehydrogenase, and 50 PM [lJ4C]sodium laurate in a final volume of 2 ml. The reaction mixture was incubated for 8 min at 37°C. When NADPH was used, the reaction medium lacked the NADPH regenerating system consisting of isocitrate and isocitrate dehydrogenase. The reactions were stopped by the addition of 0.8 ml of 10% H,SO, and were extracted three times with 5 ml of ether. The combined extracts were washed with water until neutral. After the ether was evaporated, the samples were dissolved in 0.5 ml benzene and methylated with 2 ml BF,-MeOH at 80-85”Cfor 6 min (8). The methylation reaction was terminated by chilling and by addition of 5 ml 10% aq KHCOB. Ether extracts of these mixtures

HPLC ANALYSIS

OF LAURATE

METABOLISM

267

were filtered and were evaporated under N,. The samples were redissolved in a small volume of methanol prior to chromatography. All chromatographic separations were performed at room temperature at a flow rate of 2 ml/min. The radioactive reaction products and unreacted substrate were collected using a fraction collector and measured by liquid scintillation spectrometry using a Beckman Model 230 liquid scintillation counter. The scintillation cocktail consisted of 666 ml toluene, 334 ml Triton X-100, and 5.5 g PPO/liter. A 0.2 ml aliquot of sample in 60% methanol added to 7 ml of scintillation cocktail caused a 15- 10% decrease of the counting efficiency and automatic external standardization techniques were used routinely to correct the data. Dry samples of lauric acid and its hydroxy derivatives were dissolved in benzene, the methyl esters were formed by reaction with BF,-MeOH (8), and the reaction mixtures were extracted with ether. Laurie acid and methyl laurate could be eluted from the pbondapak/C,, column with 90% acetonitrile in water, and their retention times were 2.7 and 4.3 min, respectively. Using these separation conditions to test the methylation procedure, it was noted that more than 98% of the lauric acid was methylated to yield methyl laurate. ll-Hydroxyand 12hydroxylauric acid could not be separated under a variety of solvent conditions due to peak overlapping; however, their retention times were 8 and 9 min, respectively, when eluted separately by 60% methanol in water. There was no detectable radioactivity in fractions from methylated reaction mixtures corresponding to 1 I-hydroxy- and 12-hydroxylauric acid, but two easily resolved peaks were noted which had retention times identical to those of the 1 l-hydroxy- and 12-hydroxylaurate methyl ester standards. The methyl esters of the hydroxy metabolites were separated using 60% methanol in water at 2000 psi, and methyl laurate was eluted by 100% methanol at 500 psi. Methyl laurate was retained when the 60% methanol elution solvent was employed and could be eluted only with higher methanol concentrations. The retention times of laurate, ll-hydroxylaurate, and 12-hydroxylaurate methyl ester standards were shown to be independent of each other at the concentrations which were used normally. Acetonitrile (45%) could separate the hydroxylaurate derivatives as completely as methanol (60%), but some biological extracts after methylation with BF, and methanol had limited solubility in acetonitrile solutions compared to methanol solutions. Methyl laurate could be eluted with 100% acetonitrile. Since the routine application of particulate or immiscible material can cause deterioration of small particle liquid chromatography columns, the use of 60% and 100% methanol solutions is recommended to elute the hydroxylaurate and laurate methyl esters, respectively. The results presented in this report using acetonitrile as the mobile phase are still valid. since the recovery of isotope in all cases was greater than 9570.

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AND PROUGH

RESULTS

The elution pattern of the methylated reaction mixture showed two peaks with retention times identical to the two standard methyl esters of 1I-hydroxy- and 12-hydroxylaurate measured by radioactivity and refractive index, respectively (Fig. I), when 60% methanol was used as the mobile phase. Upon elution with 100% methanol, methyl laurate was eluted from the column. The two hydroxylated products were rechromatographed separately and were found to have retention times identical with those of the two hydroxylaurate methyl ester standards. The use of refractive index for monitoring the column effluent involved the addition of 100 times higher concentrations of the hydroxylaurate methyl ester standards than the metabolites in order to detect the change in refractive index due to the hydroxylaurate derivatives. This procedure was performed to confirm, with the appropriatestandards, that the hydroxylaurate methyl esters had identical retention times with the radioactive metabolites eluted from the column. Radioisotopic analysis was used to quantitate the metabolites, and no differences were seen in the separation or analysis of the radioactive hydroxylaurate methyl esters when the respective standards were added. No hydroxylated derivatives were found when 0.8 ml of 10% H&SO, was added to the complete reaction mixture at zero time (Fig. 2A). No hydroxylated products were obtained in the absence of microsomes (Fig. 2B) or NADPH. Mass spectral analysis of the three radioactive peaks

FIG. 1. The separation of laurate and its hydroxy metabolites by high pressure liquid chromatography. The reaction mixtures were prepared as described in the Methods section and laurate, 11-hydroxylaurate (1 l-OH), and 12-hydroxylaurate (12-OH) were in the form of methyl esters. (- 0 -), dpm of aliquots (approx 10 nmol of hydroxyated products) from the chromatography fractions of an 8-min incubation mixture containing 0.75 mg/ml of rat kidney cortex microsomes; (---), refractive index of a chromatographic run of equimolar amounts (1 pmol) of the methyl esters of 1 I- and 1Zhydroxylaurate standards added to the final sample prior to HPLC analysis. The mobile phase was 60% methanol and methyllaurate was eluted with 10% methanol.

HPLC ANALYSIS .c

30

d h

25

e 2

20

5 $

15

a 3 ? F

OF LAURATE

)

METABOLISM

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6. PROTEfN

10 5

s I

3

6 Tmw

9 (min)

12

15

FIG. 2. The effect of time and protein concentration on lsurate hydroxylation. (A) Time using 0.75 mg/ml rat kidney cortex microsomes from 350-g rats. (B) Microsomal protein (from 150-g, 12-hr starved rats) concentration using S-min incubation times (the reaction mixture contained the indicated amounts of protein in a 2-ml vol). The mobile phase was 45% acetonitrile to elute the methylated hydroxylaurates and 100% acetonitrile to elute methyllaurate.

from a metabolic reaction mixture showed mass peaks consistent with those obtained with the respective standards (1). Other endogenous compounds were apparently eluted with the 12-hydroxylaurate and thin layer chromatography subsequent to HPLC was required prior to mass spectral analysis. No attempt was made to identify these compounds since they were nonradioactive and were present in extracts of incubation mixtures which were terminated immediately after addition of NADPH. In agreement with Bjorkhem and Danielsson (l), the II- and 12-hydroxylaurates had parent peaks of 230 m/e and fragment peaks of 186 and 200 m/e, respectively. In Table 1, the recovery of 14C at each step of the method is shown, and the overall recovery of 14C was noted to be greater than 95%. The amount of hydroxylated products formed increased linearly with time up to 15 min and was proportional to the concentration of microsomal protein (0.25- 1.5 mg/ml) present in the incubation mixture (Fig. 2). The K, for lauric acid was determined to be 8 PM (Fig. 3) which is similar to TABLE RECOVERY

OF LAURIC

ACID

CONTAINING

AND RAT

1

ITS HYDROXY KIDNEY

CORTEX

METABOLITES

FROM

INCUBATES

MICROSOMES

Step”

Recovery (%)

Ether extraction from reaction mixture Water wash until neutral Ether extraction of methylated mixture HPLC separation Total of all steps

99 99 98 99 >95

u A complete description of each step is described in the Methods section.

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FAN, MASTERS

AND PROUGH

FIG. 3. The dependence of laurate hydroxylation on laurate concentration. The reaction mixtures containing 0.75 mg/ml rat kidney cortex microsomes were incubated for 8 min in the presence of varying concentrations of sodium laurate. The elution conditions are those shown in Fig. 2, and the data were analyzed using the method of Hanes (11). The rats used weighed 150 g.

a previously published value (2) obtained with kidney cortex microsomes. The K, for NADPH was found to be 54 PM (Fig. 4) which is a lower value than that obtained by Ichihara et al. utilizing rat liver microsomes (9) or a reconstituted system from kidney cortex microsomes (10). DISCUSSION

Cytochrome P-UO-mediated mixed-function oxidations of a variety of substrates, including steroids, drugs, carcinogens, and fatty acids, have been demonstrated in liver microsomes, but kidney microsomes have been shown to catalyze mainly fatty acid hydroxylation (12). The hydroxylation of physiological fatty acids such as lauric acid has been chosen to be a model system to study the enzymatic nature and physiological role of microsomal fatty acid hydroxylation.

Km=WpM

0.5 t

I

, FIG. 4. The dependence of laurate tion mixtures containing 0.75 mg/ml incubated for 8 min in the presence conditions are those shown in Fig. Hanes (11).

hydroxylation on NADPH concentration. The reacrat kidney cortex microsomes from 300-g rats were of varying concentrations of NADPH. The elution 2, and the data were analyzed using the method of

HPLC

ANALYSIS

OF LAURATE

METABOLISM

271

The ll- and 12-hydroxylaurate metabolites have been separated and characterized by thin-layer and gas chromatographic techniques. Analysis of reaction mixtures using thin-layer chromatography required pooling materials from several incubation mixtures and repeated chromatography to achieve complete separation (1). Gas chromatographic analysis of the hydroxylaurate metabolites could not be performed using normal flame ionization detection due to the large contamination of the samples with endogenous microsomal lipids, but requires the use of a radio-gas chromatograph equipped with a gas flow radioactivity detector (1,5). Although these gas flow radioactivity detectors are unique in the analysis of radioactive vaporized samples, they have a lower sensitivity than liquid scintillation counting and are subject to extensive CO,-quenching depending upon the amount of endogenous carbon compounds present in the sample. This report presents an alternate technique of separating the 1land 12-hydroxylaurate metabolites of microsomal laurate hydroxylation reactions using an isocratic (stepwise elution) HPLC method. Although all of the separations described above involve multiple-step sample preparation, the final analysis can be performed on a single 2-ml incubation mixture using a simplified isocratic HPLC instrument consisting of a solvent delivery system and a microparticle octadecylsilane liquid chromatography column. This method further allows complete recovery of the separated metabolites of the incubation mixture. The ratio of 12- to 1 I-hydroxylation of 3: 1, determined using rat kidney cortex microsomes, differed from the previously reported values of 2:l (5). However, variation in ratio of 12- to 11-hydroxylation between 1.4 to 2.8 have been shown (12,13). The variation in this ratio may depend on the condition of the animal and will be the subject of further investigation. In liver microsomes, this ratio was found to be 1: 1 (1). In conclusion, we have developed an improved assay method utilizing HPLC techniques which permits the complete separation and quantitation by liquid scintillation spectrometry of the two hydroxylated products of microsomal lauric acid hydroxylation. This method allows complete recovery of the derivatized products using an isocratic high pressure liquid chromatograph, and the method can be less complex and expensive than the radio-gas chromatographic procedure (5). ACKNOWLEDGMENT The authors wish to express their thanks to Ms. Virginia W. Patrizi for her aid in per. forming mass spectral analyses.

REFERENCES 1. Bjorkhem, I., and Danielsson, H. (1970) Eur. J. Biochem. 17, 450-459. 2. Ellin, A., Jakobsson, S. V., Schenkman, J. B., and Orrenius, S. (1972)Arch. Biophys. 150,64-71. 3. Preiss. B., and Bloch, K. (1964) J. Bid. Chem. 239, 85-88.

Biochem.

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AND PROUGH

4. Kusunose, M., Kusunose, E., and Coon, M. J. (1%4)J. Biol. Chem. 239, 1374-1380. 5. Ellin, A., Orrenius, S., Pilotti, A., and Swahn, C. G. (1973) Arch. Biochem. Biophys. 158, 597-604. 6. Remmer, H., Griem, H., Schenkman, J. B., and Estabrook, R. W. (1967) Methods in Enzymol. 10, 703-708. 7. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (19Sl)J. Biol. Chem. 193, 265-275. 8. Burton, R. M., and Guerra, F. C. (1974) in Fundamentals of Lipid Chemistry, p. 175, BI-Science Publication Division, Webster Groves, MO. 9. Ichihara, K., Kusunose, E., and Kusunose, M. (1969) Biochim. Biophys. Acta. 176, 704-712. 10. Ichihara, K., Kusunose, E., and Kusunose, M. (1971) Biochim. Biophys. Acta. 239, 178- 189. 11. Dixon, M., and Webb, E. C. (1%4) Enzymes, 2nd ed., pp. 68-69, Academic Press, New York. 12. Orrenius, S., Ellin, A., Jakobsson, S. V., Thor, H., Cinti, D. L., Schenkman, J. B., and Estabrook, R. W. (1973) Drug Metab. and Disp. 1, 350-357. 13. Ellin, A., and Orrenius, S. (1974) FEBS Letters 50, 378-381.