Cholest-3,5-dien-7-one formation in peroxidized human plasma as an indicator of lipoprotein cholesterol peroxidation potential

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ELSEVIER

Biochimica et Biophysica Acta 1255 (1995) 341-343

Biochi~ic~a et BiophysicaA~ta

Rapid Report

Cholest-3,5-dien-7-one formation in peroxidized human plasma as an indicator of lipoprotein cholesterol peroxidation potential Mikyoung Hahn, Maoyun Tang, M.T. Ravi Subbiah * Lipid Laboratory, Department of Internal Medicine, University of Cincinnati Medical Center, University Hospital, P.O. Box 670540, Cincinnati, OH 45267-0540, USA Received 22 November 1994; accepted 20 December 1994

Abstract

Lipoprotein peroxidation susceptibility is routinely evaluated using products of unsaturated fatty acids as markers (e.g., malonaldehyde). The significance and factors influencing peroxidation of cholesterol moiety of lipoproteins are relatively unknown due to lack of a reliable marker product which can be measured easily. Under the influence of Cu 2÷ ions, the major product of lipoprotein cholesterol peroxidation (isolated after saponification) was cholest-3-5-dien-7-one (CSD). Apart from gas-liquid chromatography, this compound lends itself for measurement by alternative methods. Due to lack of the 3/3-hydroxyl group, CSD was separated from the rest of the oxysterols and cholesterol by passing through digitonin-coated silica-gel G and its concentration was determined by absorption at 283 nm. The recovery of CSD by this method exceeded by 87%. The formation of CSD was also sensitive to vitamin E and therefore could be used as an index of lipoprotein cholesterol susceptibility to peroxidation. Keywords: Cholesterol peroxidation; Cholest-3,5 dien-7-one; Lipoprotein; Digitonin precipitation

Oxidation of low density lipoproteins is believed to play a key role in atherogenesis [1]. Oxidation of lipoprotein lipids basically involves two processes: (a) oxidation of unsaturated fatty acids to fatty acid hydroperoxides and a variety of aldehydes (malonaldehyde, hexanal, nonenal, etc.), and (b) oxidation of cholesterol moiety into oxysterols [2,3]. While there are a number of methods to evaluate fatty acid peroxidation using either fatty acid diene derivatives [4], or malonaldehyde [5], or 4-hydroxy nonenal [6] formation, cholesterol peroxidation was measured by the total estimation of a number of oxysterols formed by gas-liquid chromatography [3]. Recent studies from our laboratory noted [7] that, under metal catalyzed peroxidation conditions, cholest-3,5-dien-7-one (CSD) represents the major oxysterol formed with smaller amounts of cholesterol 5,6-epoxides, 26-hydroxycholesterol, 7a-hydroxycholesterol and 7-ketocholesterol. The measurement of CSD and other oxysterols made using GLC is time-consuming and may not be accessible to routine assay. Therefore, the GLC technique was compared with a simple method that was developed to measure CSD as an index of

* Corresponding author. 0005-2760/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 5 - 2 7 6 0 ( 9 5 ) 0 0 0 0 4 - 6

cholesterol peroxidation potential. To achieve this, CSD is separated from cholesterol and other oxysterols (all containing the 3/3-OH group) by digitonin precipitation and its concentration is determined by absorbance at 283 nm. Human plasma samples were obtained either from the Hoxworth Blood Center or from subjects according to protocol approved by the Institutional Review Board. We caried out deliberate plasma lipoprotein peroxidation potential determination as per the details described previously [8]. Briefly, 1 ml of plasma was incubated with cupric acetate (10/xmol/1) for varying periods (2-24 h) at 37 ° C. Aliquot of the samples were taken for the measurement of oxysterols. To extract oxysterols, we subjected aliquots of the plasma to saponification with 2 N ethanolic NaOH for 1 h [7,9] and oxysterols were extracted with chloroform/methanol (2:1) and used for GLC analysis. Alternatively, the chloroform/methanol extracts were evaporated to dryness and picked up in hexane. The hexane extract was passed through a digitonin-silica-gel G column (prepared by mixing 10 g of silica-gel G with digitonin solution (300 m g / 5 ml water)) as per modified procedure [10]. About 250 mg of the digitonin coated silica-gel G was packed into a pasteur pipette containing a glass wool plug. The hexane solution was allowed to pass

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through the column and washed with the addition of 2 ml of hexane. The hexane extract (containing only cholest3,5-dien-7-one) is evaporated to dryness and picked up in 2 ml of ethanol for determination of its absorption at 283 nm. The concentration of CSD was calculated using standard curve prepared with authentic standard (Sigma) (using a molar extinction coefficient of 17.6. 103). Absorption spectra of authentic CSD and hexane extracts of samples

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Fig. 2. Ultraviolet absorption spectra of authentic CSD (A) and hexane extract (B) of plasma (after Cu 2+ oxidation, saponification and purification through digitonin columns). Spectra measurement conditions as described in the text. 4

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Fig. 1. Gas liquid chromatographic analysis of oxysterols formed upon incubation of plasma with Cu 2+. (A) Total oxysterols formed. (B) After purification of oxysterols formed through digitonin columns which eliminate all 3/3-OH sterols. (C) Standard cholesta-3,5-dien-7-one. Column conditions as described in the text. Peak identification. 1. 5ot-cholestane: 2. Cholesterol; 3. Cholest-3,5-dien-7-one; 4. Cholesterol or- and /3epoxides; 5. 25-hydroxycholesterol; 6. 7-ketocholesterol.

was determined in ethanol (after evapaoration of hexane) in a Hitachi (Tokyo, Japan) Model 100-60 spectrophotometer. The feasibility of the procedure in terms of separation of CSD from cholesterol and other oxysterols was verified by gas-liquid chromatography using retention time relative to 5o~-cholestane [10]. A Sigma 1 (Perkin-Elmer, Palo Alto, CA) gas chromatograph with a 15 m × 0.5 mm (i.d.) DB-17 column (1 /xm thick coating) were used. The chromatographic conditions use injector 275 ° C, detector 280 ° C, column 248 ° C, carrier gas, helium, flow rate 10 ml/min. The identity of the steroids was confirmed as described previously [7]. The validity of the method for separation of CSD for other sterols was initially documented by gas chromatography. Fig. 1A shows a gas-liquid chromatographic profile of the oxysterols isolated upon incubation of human plasma with Cu 2+ for 24 h modification. As can be seen from the figure, CSD represented nearly 50% of the oxysterols formed. Although there was considerable variation in the composition of oxysterols between samples, CSD represented the major compound in all the cases. The rest of the peaks are represented by cholesterol c~-epoxide, cholesterol /3-epoxide, 25-hydroxycholesterol and 7-ketocholesterol. Purification of this extract through digitoninsilica-gel G columns removed virtually all the components (including cholesterol) except CSD (Fig. 1B), which was identified by comparison od the retention time with that of an authentic standard (Fig. 1C). The concentration of CSD was calculated by absorption at 283 nm. This wave length was chosen based on the absorption maxima of standard CSD (Fig. 2). The hexane extract showed several peaks of absorption (210, 234, and 283 nm) indicating several components (Fig. 2B). The formation of CSD from cholesterol under the influence of

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M. Hahn et al. /Biochimica et Biophysica Acta 1255 (1995) 341-343

Table 1 Cholest-3,5-dien-7-one (CSD) formation in plasma after peroxidation: comparison of two methods (mean ± S.E.) Incubating conditions

CSD formation ( / z g / m l plasma) GC

spectrophotometric

Plasma, control 1 ml Plasma + Cu 2+ (10/.zmol/I) 2h 4h 24 h

Not detectable 25.0 + 4.2 36.2 +_ 6.5 76.5 + 3.6

32.8 + 0.3 46.7 + 0.4 68.4 + .1.0

Plasma, Control + CST added (100/xg) % Recovery (GC) % Recovery (absorption at 283 nm)

99.9 _+ 1.8 87.8 _+ 1.3

1 ml of plasma was incubated with Cu z+ for varying periods and the formation of CSD was evaluated either by GLC or by absorption at 283 nm.

Table 2 Effect of vitamin E on CSD formation in plasma: comparison of two methods (mean _+ S.E.)

Incubating conditions a

Plasma + C u 2+ (10/xmol/l) Plasma + Cu 2+ (10/xmol/l) + vitamin E (100/zg)

Concentration ( / ~ g / m l plasma) GC

spectrophotometry

76.5 _+ 3.6 9.3 _+ 0.43 b

68.4 + 1.0 12.3 +_ 3.2 b

1 ml of plasma was incubated with Cu 2+ for 18 h in the presence or absence of vitamin E. The formation of CSD and total oxysterols was measured by gas liquid chromatography. b p < 0.05 for difference from incubations without vitamin E.

Cu 2÷ at different time periods was compared by both methods and the results were within reasonable limits. It was noted that the formation of cholest-3,5-dien-7-one in plasma increased at 2 and 4 h, reaching values up to 76.5 /xg/ml noted after 24 h (Table 1). Using digitonin column separation method, the recovery of CSD exceeded 87% and the results obtained were comparable to GC methods (Table 1). The formation of CSD was sensitive to the antioxidants (like vitamin E) which caused a greater than 80-90% reduction in the formation of CSD as evaluated by both the methods (Table 2). These results show that the formation of CSD can be used as a marker for cholesterol peroxidation. While its concentration can be conveniently measured by GC chromatography, a simple comparative method is described. Because of the lack of /3-OH group at position 3, this compound can be conveniently separated from cholesterol and the rest of its oxidation products. Because of the strong absorption at 283 nm (due to conjugated double bonds present in the molecule), a simple spectrophotometric determination of its concentration can be made. Recovery of added CSD by the method exceeded 87% and was comparable to the GC method. Further studies of the factors influencing CSD formation are currently underway in the laboratory.

This work was supported in part by grant HL-50881 from the National Institutes of Health. Word processing assistance of Wilma Hollon is gratefully acknowledged.

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