Strong increase in hydroxy fatty acids derived from linoleic acid in human low density lipoproteins of atherosclerotic patients

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Abstract

Linoleic acid is the most abundant fatty acid in human low density lipoproteins (LDL). Oxidation of LDL transforms linoleic acid to hydroperoxyderivatives. These are converted to 9-hydroxy-10,12-octadecadienoic acid (9-HODE) and 13-hydroxy-9,11-octadecadienoic acid (13-HODE). 9-HODE is much more abundant in oxidized LDL than other lipid peroxidation products and therefore an indicator of lipid peroxidation (LPO). In this study the 9-HODE content in the LDL of 19 obviously healthy volunteers and 17 atherosclerotic patients was investigated. The level of 9-HODE obtained from LDL of young atherosclerotic patients (aged 36–47 years) was increased by a factor of 20 when compared with samples from healthy volunteers of the same age group. The content of 9-HODE in the LDL of atherosclerotic patients aged between 69 and 94 years increased 30–100 fold when compared with young healthy individuals, but when compared with `healthy' individuals of the same age group it was only 2–3 fold increased. Obviously, as individuals grow older LDL becomes more and more oxidized. Consequently, assuming that LDL oxidation is a precondition for atherosclerosis—older individuals will suffer from atherosclerosis, even if no easy detectable visible signs of this disease are recognizable. According to 9-HODE determination, the onset of the disease starts slowly in most individuals at around 50 years of age.

Introduction

In the recent years, increasing evidence accumulated links atherosclerosis with the oxidation of unsaturated fatty acids (Esterbauer et al., 1992Jackson et al., 1993Berliner and Heinecke, 1996). Glavind et al. (1952)observed a positive relationship between aortic lipid peroxide content and atherosclerosis, by determining the hydroperoxides derived from fatty acids using a colour-reaction with 3,5-dichloro-4,4′-dihydroxyphenylenediamine (Hartmann and Glavind, 1949).

This finding was later confirmed by Brooks et al. (1970)who recognized that aortic plaques contain large amounts of cholesterol esters of 9-hydroxy-10,12-octadecadienoic acid (9-HODE) and 13-hydroxy-9,11-octadecadienoic acid (13-HODE), derived from the reduction of the corresponding linoleic acid hydroperoxides. In addition the corresponding ketones, 9-oxo-10,12-octadecadienoic acid and 13-oxo-9,11-octadecadienoic acid—were enriched. Kaduce et al. (1989)found that linoleic acid is converted to 9- and 13-HODE by the catalysis of human umbilical vein endothelial cells. Recently, Wang and Powell (1991)reported that 9- and 13-HODE-cholesterol esters were found in increased amounts in aortas and LDL of rabbits fed on a diet enriched in cholesterol, together with hydroxy metabolites of arachidonic acid. Belkner et al. (1991)also detected 9- and 13-HODE cholesterol esters in atherosclerotic plaques in humans who had died from atherosclerosis. If the amount of these esters in plaques was compared with tissues obtained from normal regions, an increase was observed that coincided with the severity of the atherosclerotic lesions.

Oxidation of unsaturated fatty acids in the course of lipid peroxidation (LPO) is not restricted to atherosclerosis. Halliwell and Grootveld (1987)listed a significant number of diseased states (including aging) in which LPO might be involved. Since direct measurement of hydroperoxides is difficult LPO is usually measured by determination of malondialdehyde (MDA), a final oxidation product of arachidonic acid. MDA reacts with thiobarbituric acid (TBA) to form a coloured product. The fluorescent intensity of the addition product is measured (Janero, 1990). Several investigators studied the amount of thiobarbituric acid reactive substances (TBARS) in the plasma of atherosclerotic patients in comparison with healthy volunteers and found increased levels of these LPO-products in atherosclerotic patients (Satoh, 1978Szczeklik and Gryglewski, 1980Goto, 1982Aznar et al., 1983Hagihara et al., 1984Jeny et al., 1986Ledwozyw et al., 1986Schimke et al., 1986Stringer et al., 1989Yalcin et al., 1989).

A relationship between LDL oxidation and atherosclerosis was detected by Steinbrecher et al. (1984)and Morel et al. (1984)who found that LDL is the main carrier of cholesterol in blood (Goldstein and Brown, 1977, Morel et al., 1984, Steinbrecher et al., 1984, Brown and Goldstein, 1986, Gey, 1986, Gotto, 1987) when incubated with cultured endothelial cells from rabbit aorta or human umbilical vein it became `oxidized'. A similar oxidized LDL was obtained from artificial oxidation induced by metal ions, e.g. Cu2+ (Heinecke et al., 1984, Morel et al., 1984, Steinbrecher et al., 1984). This oxidized LDL (oxLDL) was found to be atherogenic and cytotoxic and was assumed to be responsible for the damaging effects observed in atherosclerosis. Weisser et al. (1992)found a slight but significant raised level of TBARS in the LDL of atherosclerotic patients compared with LDL of healthy individuals. Increased amounts of hydroperoxides in the phospholipid fraction in human plasma (Miyazawa et al., 1988, Miyazawa, 1989) and LDL (Miyazawa et al., 1990) of atherosclerotic patients compared with healthy subjects was obtained using a RP-HPLC-chemiluminescence method by Miyazawa.

Considering the fact that

  • 9- and 13-HODE are enriched in atherosclerotic plaques (Brooks et al., 1970)

  • endothelial cells catalyze the generation of 9- and 13-HODE (Kaduce et al., 1989)

  • linoleic acid occurs in a ratio of about 7:1 compared with arachidonic acid in the LDL particle (Esterbauer et al., 1992)

  • linoleic acid only produces very low amounts of MDA in the TBA test (Esterbauer et al., 1989)

we expected enrichment of 9- and 13-HODE in the LDL fraction of atherosclerotic patients. This assumption was strengthened by the finding that the C-18 hydroxy fatty acids content in LDL in healthy individuals is remarkably increased in older individuals compared with younger ones (Jira et al., 1996).

To the best of our knowledge an attempt has never been made to analyze 9- or 13-HODE in LDL from atherosclerotic patients. In this paper we describe the finding that the 9-HODE content is strongly increased in the LDL of young atherosclerotic patients in comparison with healthy volunteers of the same age group.

Section snippets

Materials

N-Methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) was obtained from Machery and Nagel (Düren, Germany). All other chemicals were purchased from Fluka (Neu Ulm, Germany). Solvents were distilled before use.

Methods

Blood samples from young healthy individuals were obtained from the red cross blood service in Bayreuth. Samples of obviously healthy individuals, older than 60 years was donated by volunteers of a senior club. The volunteers were selected because of their exceptional mental and physical

Results

The primary products of LPO are hydroperoxides of polyunsaturated fatty acids. Most of these LOOHs are reduced enzymically to more stable hydroxy derivatives (LOH) under catalysis by glutathione-dependent peroxidases (Lehmann et al., 1992) or suffer in presence of Fe2+ ions, cleavage to LO radicals which react mainly with other unsaturated fatty acids (LH) by hydrogen abstraction, also producing LOH (Halliwell and Gutteridge, 1992).

The content of hydroxy acids in the LDL of healthy and

Discussion:

In previous investigations the degree of LPO in serum and LDL was investigated by measuring the MDA-content (Satoh, 1978, Szczeklik and Gryglewski, 1980, Goto, 1982, Aznar et al., 1983, Hagihara et al., 1984, Jeny et al., 1986, Ledwozyw et al., 1986, Schimke et al., 1986, Stringer et al., 1989, Yalcin et al., 1989, Weisser et al., 1992) or by determination of the peroxides (Miyazawa et al., 1988, Miyazawa, 1989, Miyazawa et al., 1990). In all these investigations a moderate increase of LPO

Acknowledgements

We thank Deutsche Forschungsgemeinschaft and Fond der Chemischen Industrie for financial support. We are obliged to M. Glaeßner for running the mass spectra and W. Kern for purification of solvents. We are also very grateful to Dr. U. Pachmann and Dr. R. Offner from the BRK Bayreuth for providing blood samples.

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