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Free radicals, lipid peroxidation, antioxidants and atherosclerosis
Oxidation is a part of the normal metabolism of the human body. In
these metabolic processes highly reactive molecules, free radicals,
are processed. Free radicals are atoms or molecules that contain one
or more unpaired electrons. These compounds have important physiologic
functions e.g. for the function of phagocytes, but they are also capable
to induce oxidative damage to essential biomolecules such as nucleic
acids, lipids and proteins. Free radicals may play a role in the development
of atherosclerosis and other degenerative diseases primarily by oxidizing
polyunsaturated fatty acids in lipoproteins, especially low-density
lipoprotein (LDL). These oxidation reactions can be prevented by a complex
antioxidant defense system in the human body, which includes enzymes
such as paraoxonase (PON), superoxide dismutase (SOD), catalase, glutathione
peroxidase, and antioxidants such as vitamin E and C, ß-carotene,
urate and thiols. Normally in the body exists a balance between the
formation of radicals and defense, but "oxidative stress"
may result when these systems fail to cope with the production of radicals.
Disturbance in the balance may, according to current knowledge, contribute
to the development of atherosclerosis and other vascular dysfunction.
Measurement of lipid peroxidation in our nutritional studies
A variety of methods are available to measure lipid peroxidation, but
no single assay is an accurate measure of the whole oxidation process.
Direct evaluation of LDL oxidation is difficult, because lipoprotein
oxidation is likely to occur in the mileum of the arteria wall. The
extent of lipid oxidation can be measured by measuring the primary and
secondary peroxidation products. In our epidemiological studies and
human supplementation trials we have used a wide variety of assays to
evaluate lipid peroxidation. In most of the trials we have measured
oxidation using plasma total peroxyl radical trapping antioxidant parameter
(TRAP), the oxidation susceptibility of VLDL+LDL or LDL to oxidation
(after induction of Cu2+), F2-isoprostanes, baseline diene conjugates
in LDL (LDL-BCD) and hydroxyl fatty acids.
Out of these different methods, isoprostanes are currently thought to
be the most valuable biomarkers of lipid peroxidation. F2-isoprostanes
are produced in vivo from the peroxidation of unsaturated fatty acids.
F2-isoprostanes have been shown to be present in increased amounts in
human atherosclerotic lesions. Also plasma, serum or urinary levels
of F2-isoprostanes have been shown to be increased in subjects with
hypercholesterolemia, liver cirrhosis and asthma.
The F2-isoprostane concentrations can be determined from serum, plasma
or urine samples by GC/MS with negative-ion chemical ionization using
a deuterium-labeled F2-isoprostane as an internal standard. F2-isoprostane
levels are presented against creatinine concentration.
Another method to measure lipid peroxidation in vivo is plasma C18 hydroxy
fatty acid concentration. In this method lipids are extracted from plasma
by the method of Folch. Extracted samples are swiftly hydrogenated by
using platinum as a catalyst, following saponification and conversion
to methyl esters. Silica SPE columns are used to purify the formed monohydroxy
fatty acid methyl esters. A tetramethylammonium hydroxide derivatization
is carried out for all the hydroxy groups. Finally, the concentrations
of monohydroxy fatty acids (OH groups at positions 8, 9, 10, 11, 12,
13, 15 and 16 of the C18 chain) are determined by GC/ MS with electron
impact mass spectroscopy. Total concentration and concentrations of
single acids are determined. C19 hydroxy fatty acid is used as an internal
standard.
In addition to F2-isoprostanes and C18 hydroxy fatty acids, the lipid
peroxidation in vivo can also be evaluated by measuring baseline (uninduced)
diene concentration. Baseline diene concentrations are considered to
be an early stage marker of polyunsaturated fatty acid oxidation. In
this method LDL is precipitated with heparin/citrate and in the following
extraction, diene concentration is measured photometrically against
hexane. Final results are presented against cholesterol concentration
of precipitated LDL. This analysis method has been used, for example,
in different supplementation studies and studies studying the effects
of physical exercise on lipid peroxidation in humans.
One of the most commonly used ex vivo methods in our clinical trials
is copper-induced serum oxidation. In this method serum is diluted with
phosphate buffered saline (PBS) and oxidation is initiated by the addition
of copper. The formation of conjugated dienes is followed by monitoring
the change in absorbance at 234 nm and lag-time to the maximum oxidation
rate (lag-time) is determined.
Another commonly used ex vivo method in our trials is plasma/LDL total
peroxyl radical trapping antioxidant parameter (TRAP). Plasma total
peroxyl radical trapping potential (TRAP) is determined with a modification
of the method by Metsä-Ketelä et al (1991).
Our results and research interests
We have studied the effects of different phenolic compounds on lipid
peroxidation. In these studies we have used phloem, chocolate, coffee,
herbs and mixture of green tea, onion and apple as a source of phenolic
compounds and we have evaluated the short-term (0-2 hours) as well as
long-term (3-4 weeks) effects. The results of these studies will be
published in the near future and therefore we cannot give detailed information
about the results. However, these results suggest that catechins and
other polyphenols may inhibit lipid peroxidation in short-term as well
as long-term in the water-soluble phase of serum. In this series of
studies ingestion of phenolic-rich compounds did not have any adverse
effects on health when evaluated by measuring the function of liver
and kidneys.
In the Antioxidant Supplementation on Atherosclerosis Prevention (ASAP)
Study we have observed that elevated plasma total homocysteine (tHcy)
levels were associated with enhanced in vivo lipid peroxidation in men,
as measured by F2-isoprostanes. In this subsample of 100 consecutive
men out of 256 male participants, serum enterolactone concentration
also correlated negatively with F2-isoprostanes. For ASAP study, plasma
F2-isoprostane concentrations were determined at the Vanderbilt University
Medical Center, Nashville, USA, in 1997.
More information about our studies:
jaakko.mursu@uku.fi
Vanharanta M, Voutilainen S, Nurmi T, Kaikkonen J, Roberts JL, Morrow
JD, Adlercreutz H, Salonen JT. Association between low serum enterolactone
and increaced plasma F2-isoprostanes, a measure of lipid peroxidation.
Atherosclerosis 2002;160:465-469. PDF
Voutilainen S, Morrow J, Roberts J, Alfthan G, Nyyssönen K,
Salonen J. Correlation between Plasma Total Homocysteine Concentration
and Plasma F2-Isoprostane in 100 men in Eastern Finland. Arteriosclerosis,
Thrombosis and Vascular Biology 1999;19:1263-1266. PDF
Salonen JT. Markers of oxidative damage and antioxidant protection:
assessment of LDL oxidation. Free Radical Research 2000;33:S41-6.
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