Preparation and Purification of Lipid Hydroperoxides from Arachidonic and 3'-Linolenic Acids MAX O. FUNK, RAMDAS ISAAC, and NED A. PORTER, 1 Paul M. Gross Chemical Laboratories, Duke University, Durham, North Carolina 27706

ABSTRACT

C o m m e r c i a l soybean lipoxygenase may be used under carefully controlled reaction conditions to give high yields of l ipid hydroperoxides. Lipid hydroper- oxides so derived from ~/-linolenic or arachidonic acid may be purified by high pressure liquid chromatography. Thus, c o m m e r c i a l l ipoxygenase serves as a viable source for 100 mg quantities of lipid hydroperoxides.

INTRODUCTION

We have recently developed a method for the study of peroxy radical cyclization reac- tions based on the radical induced decomposi- t ion of unsaturated hydroperoxides. Our objec- tive has been to utilize this method in an investigation of a model reaction for the bio- synthesis of prostaglandins (1-2). Toward this end, it was necessary to obtain isomerically pure lipid hydroperoxides on a preparat ively useful scale.

Autoxidat ion of fat ty acids yields hydro- peroxides which are not only mixtures of posi- tional isomers but are racemic as well (3). Lipoxygenase catalyzed oxidation, on the other hand, has a demonstrated positional specificity and gives hydroperoxides which are enantio- merically pure (3). The enzymatic reaction was therefore the method of choice for our pur- poses.

The lipoxygenase reaction has been the sub- ject of numerous investigations (4-14). A vari- ety of factors have been shown to be important in determining the product specificity in the oxygenation of fat ty acids by lipoxygenase.

1Author to whom correspondence should be addressed.

These factors include pH, temperature, pres- ence or absence of Ca2+, method of substrate preparation, and source and homogenei ty of the enzyme. The factors which affect the isolated yield of the hydroperoxides have been studied to a lesser extent. In general, the ab- sorbance increase at 234 nm is the only estima- t ion of hydroperoxide yield. In fact, using com- mercial l ipoxygenase and conditions reported in the literature, we could prepare lipid hydro- peroxides from 7-1inolenic acid in no more than 5% isolated yield.

We report here a s tudy of the soybean l i p o x y g e n a s e oxidat ion of 3,-linolenic and arachidonic acids. The purpose of this work was to maximize the isolated yields of various lipid hydroperoxides and, in so doing, make the lipoxygenase system conveniently useful in a preparative sense. The cornerstone of this study was high performance liquid chromatography (HPLC). By the use of this technique, both the hydroperoxy fat ty acids and the corresponding methyl esters, I-III, could be obtained in pure form (Fig. 1). I, an ~-10 hydroperoxide from 3,-linolenic acid, is particularly interesting be- cause it serves as a starting material for model studies on prostaglandin biosynthesis. The cor- responding 6o-10 hydroperoxide from arachi- donic acid could not be prepared in useful yields with our reaction conditions.

MATERIALS AND METHODS

7-L ino l en i c acid, arachidonic acid, and linoleic acid (99% +) were obtained from Nu- chek Prep (Elysion, MN) and used without further purification.

Soybean lipoxygenase was obtained from Sigma Biochemical Co. (St. Louis, MO) and was assayed with linoleic acid: pH 9, 90,000 u/mg; pH 7, 45,500 u]mg. The method used was that

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FIG. 1. Hydroperoxides formed from soybean lipoxygenase and 3,-linolenic and aracbidonic acid.

113

114 MAX O. FUNK, RAMDAS ISAAC, AND NED A. PORTER

13 - ISOM ER

I I 0 2 0 M I N

FIG. 2. Analytical high performance liquid chro- matography trace for separation of ",/-linolenic hydro- peroxide isomers. Methyl ester, ultraviolet detection.

prescribed by Sigma Biochemical Co.

Substrate Solutions

Ethanol dispersion: A stock solution of the s u b s t r a t e (0.088M) was prepared in 95% e t h a n o l . A l i q u o t s we re di luted to the a p p r o p r i a t e c o n c e n t r a t i o n w i t h borate (0.047M, pH 9.0) or phosphate (0.047M, pH 7.0) buffer.

Ammonium salt: The substrate was treated with 2M NH4OH (I ml /50 /a l substrate) and was diluted with water (ca. 50 ml/50 #1 sub- strate). The pH of the solution was adjusted to 7.0 or 9.0 using 0.1N hydrochloric acid. The final concentrat ion was obtained by dilution with borate or phosphate buffer.

Incubation

The substrate solution was equilibrated at 0 or 25 C and was combined with the enzyme solution (1 mg/ml). The reaction was main- tained at the correct temperature with a steady stream of 02 gassing.

Isolation

The reaction was terminated by acidifying to pH 4 and extracting with ether (3x, 100 ml). The extracts were washed with water (2x, 50 ml), dried (MgSO4), and evaporated. If the methyl ester was to be prepared, the residue was treated with diazomethane (excess, 5 ml ether, 0 C, 30 rain) and the solvent then re- moved.

Chromatography

Analytical liquid chromatography was per- formed on a Waters ALC 202 (Waters Associ- ates, Framingham, MA). 6 f t x 1/8 in. of Corasil II was used. 8 ft x 3/8 in. of Porasil A was used for preparative separations.

Silica Gel H: HF-254 (4:1), 0.25mm, was

used for thin layer chromatography (TLC). The spots were made visable under ultraviolet light, and peroxides were detected by Fe (SCN)2 , and charring with cerium sulphate.

Solvent systems: Analytical HPLC: acids, acetic acid: 2-propanol: hexane (1:5:994); m e t h y l esters, 2-propanol:hexane (1:249). T L C : a c i d s , acetic acid: 2-propanol:hexane (1:20: 229); methyl esters, e ther :hexane (1: 1). Preparative HPLC:acids, acetic acid: 2-propanol: hexane (1 : 10: 989); methyl esters, 1-propanol: hexane (1:249).

Product Identification

Hydroperoxide products were identif ied by NaBH4 reduction and hydrogenat ion to the h y d r o x y s t e a r a t e s , followed by oxidation (CrO3) to the ketostearate and mass spectral analysis (15).

RESULTS

A general method for the estimation of the conversion of fat ty acids to hydroperoxides in a l i p o x y g e n a s e catalyzed reaction has been developed. Two factors influenced the yield of a particular hydroperoxide isomer: the overall conversion of substrate to hydroperoxides and the ratio of the isomers obtained.

The conversion factor for a given set of reac- t ion conditions was estimated by TLC of the e t h e r e a l extracts. The materials generally present in the extracts were grouped into three rough categories on the basis of Rf. The hydro- peroxides were clearly identifiable on the plates both by their quenching of flourescence at 254 nm and by a strong positive response to the ferrous thiocyanate reagent. Many of the reac- tions contained residual amounts of the starting materials, which had a distinctly higher Rf than the product hydroperoxides. Other by-products of reaction were all more polar than the hydro- peroxides by TLC. Three types of compounds were therefore generally present in a given reaction mixture: substrate fat ty acid, hydro- peroxides, and polar by-products.

The relative amounts of the hydroperoxide isomers formed were determined by analytical HPLC. The product mixtures were injected without prior purification. A typical analytical separation for the 9 and t 3 hydroperoxide-7- linolenate methyl esters is presented in Figure 2. R s values were calculated according to the relationship Rs = 2(tr2 - t r l ) / (Wl+W2)where tr----retention time and w=peak width. R s values in the range 2.3-2.5 for the separation of the 9 and 13 hydroperoxide isomers of 7-1inolenic acid methyl ester were typical. Free fat ty acid hydroperoxide separations were as good as

LIPIDS, VOL. 11, NO. 2

LIPID HYDROPEROXIDES

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LIPIDS, VOL. 11, NO. 2

116 MAX O. FUNK, RAMDAS ISAAC, AND NED A. PORTER

those of the methyl esters. Linoleic fatty acid hydroperoxides could also be separated by these techniques.

TLC and HPLC data from a series of reac- tions run on T-linolenic and arachidonic acid are presented in Table I. As noted, very little difficulty was encountered in the preparation of II. The enzyme demonstrated a high degree of specificity for the 13 position of ~,-linolenic acid at pH 9 and 0 C regardless of the substrate form employed (Table I, Expt. 1-2). The crude esterification reaction mixture was virtually all hydroperoxide by TLC, and only the 13 isomer could be detected by analytical HPLC. II was also the predominant product at pH 7 and 0C when the reaction was terminated after 5 rain (Expt. 3). Preparative HPLC of this mixture re- sulted in a 34% isolated yield based on the fatty acid. Longer reaction times at pH 7 and 0 C led to the formation of polar by-products (Expt. 4) at the expense of the 13 isomer.

Obtaining the reported ratio of the 9 to 13 isomers at a high level of conversion was less readily accomplished. With reaction conditions qualitatively similar to those described by Roza and Francke (10)(Expt. 5-6) and Christopher et al. (11) (Expt. 7), but with commercial lipoxy- genase rather than purified enzyme, poor yields of I were obtained. Polar by-products were identified by TLC as the major components of the oxidation mixtures. The formation of these b y - p r o d u c t s was, however, suppressed by increased dilutions and reduced reaction times (Expt. 8-12) for the substrate solubilized as the ammonium salt and oxidized at room tempera- ture and pH 7. Preparative HPLC resulted in a 25% isolated yield of I. A similar change in reaction conditions for the substrate in an e t h a n o l d i spers ion (Expt. 13) was only moderately successful in improving the yield of I.

The 6o6 hydroperoxide from arachidonic acid, III, was prepared in high yield with the pH 9 ethanol dispersion method (Expt. 14). Analy- tical HPLC showed only minute amounts of other hydroperoxides present, and TLC showed the hydroperoxide to be the major product. With conditions that maximize the r hydro- peroxide in 74inolenic acid (Expt. 15), no sig- nificant change in hydroperoxide ratio for arachidonic acid was noted, Several more polar by-products were formed with these reaction conditions, however.

DISCUSSION

The conversion of unsaturated lipids to h y d r o p e r o x i d e s by lipoxygenase enzymes appears to be an important chemical event in

plants, e.g., potato (7), wheat (8), and soybean (5), and a lipoxygenase system in mamallian blood has recently been investigated (9).

The chemical reactivity of the lipid hydro- peroxides formed in the lipoxygenase systems has not been studied in any detail. A primary difficulty in studies of lipid hydroperoxide has been the preparation and isolation of pure compounds from readily available enzymes. Methods described here overcome this diffi- culty, making quantities of pure hydroperoxide on the 100 mg scale available from commercial lipoxygenase.

The recent report that HPLC is useful in the analytical separation of the methyl ester hydro- peroxides of linoleic acid (16) has prompted us to report our studies based on this technique. We find that HPLC is a useful tool in separating hydroperoxides derived from T-linolenic and arachidonic acids. Both the methyl ester and the free fatty acid hydroperoxides may be iso- lated on a preparative scale. Separations of (50-100 mg) the 9 and 13 hydroperoxy methyl esters I and II have been achieved routinely, and separations of larger samples would appear to be straightforward.

We find that commercial lipoxygenase is a useful synthetic reagent only under carefully controlled conditions. Reaction time is of extreme importance. Long reaction times result in the formation of polar nonperoxide by- products. Contaminants in the commercial material are probably responsible for those side reactions, inasmuch as experiments reported with purified enzyme preparations apparently do not result in significantly reduced peroxide product yield in long time-scale reactions (10,11). Dilution, pH, and reaction media also affect product yield.

ACKNOWLEDGMENTS

This research was supported by the National Insti- tutes of Health and the Army Research Office, Durham, NC.

REFERENCES

1. Porter, N.A., and M.O. Funk, J. Org. Chem. 40:3614 (1975).

2. Funk, M.O., R. Isaac, and N.A. Porter, J. Amer. Chem. Soc. 97:1281 (1975).

3. Hamberg, M., Anal. Biocherm 43:515 (1971). 4. Hamberg, M., and B. Samuelsson, Biochem.

Biophys. Res. Commun. 21:531 (1965). 5. Hamherg, M., and B. Samuelsson, J. Biol. Chem.

242:5329 (1967). 6. Gardner, H.W., Lipids 10:248 (1975). 7. Galliard, T. and D.R. Phillips, Biochem. J.

124:431 (1971). 8. Graveland, A., Lipids 8:606 (1973).

LIPIDS, VOL. 11, NO. 2

LIPID HYDROPEROXIDES 117

9. Nugteren, P.H., Biochim. Biophys. Acta 380:299 (1975).

10. Roza, M., and A. Franka, Ibid. 316:76 (1973). 11. Christopher, J.P., E.K. Pistorius, F.E. Regnier,

and B. Axelrod, Ibid. 289:82 (1972). 12. Verhue, W.M., and A. Franke, Ibid. 285:43

(1972). 13. Christopher, J.P., EoK. Pistorius, and B. Axelrod,

Ibid. 284:54 (1972).

14. Smith, W.L., and W.E.M. Lands, Biochem. Bio- phys. Res. Commun. 41:846 (1970).

15. Hamberg, M., and B. Samuelsson, J. Biol. Chem. 242:5329 (1967).

16. Chan, H.W.S., and F.A.A. Prescott, Biochim. Bio- phys. Acta 380:141 (1975).

[Received S e p t e m b e r 5, 1975]

A Guide for Authors is Located in Lipids 11 (January):85(1976)

LIPIDS, VOL. 11, NO. 2