Drugs that target lipoxygenases and leukotrienes as emerging therapies for asthma and cancer.

Considerable amount of work has been done in the area of enzymatic and non-enzymatic oxidation of arachidonic acid. This effort resulted in understanding of the functions of lipid mediators--eicosanoids in various aspects of health and disease. A mechanism by which aspirin exerts therapeutic effects puzzled pharmacologists for a long time until John Vane, in 1971, discovered that aspirin and its congeners block formation of prostaglandins, a class of lipids that originate from oxidation of arachidonic acid by cyclooxygenase. Since that discovery the pharmacology of eicosanoids has substantially progressed, which resulted in new drugs available in clinics. In addition to many new inhibitors of cyclooxygenase, two isoforms of which are known, much effort has been given to find inhibitors of synthesis and function of leukotrienes, a class of lipids that are derived from 5-lipoxygenase. These lipids are generated in asthma and their uncontrolled biosynthesis aggravates the symptoms of asthma. A new class of drugs called lukasts, inhibitors of 5-LOX products, has been developed and entered clinics as the first new therapy to treat asthma in nearly 20 years. New discoveries in the field of lipoxygenase show great opportunities for drug development for cancer prevention and treatment as it has been established that lipoxygenases and their products are required for cancer growth. Intense research in this field is likely to produce new drugs in the near future.

Biological nitration of arachidonic acid.

When a spontaneous autoxidation of arachidonic acid to prostaglandin-like products was first described almost 40 years ago, it was thought to be an artifact that interfered with the detection of enzymatically generated prostaglandins. It has now been generally accepted that the autoxidation of arachidonic acid occurs in vivo and leads to formation of isoprostanes and other products. Sensitive methods can detect the isoprostanes as useful biological markers, which help to estimate, non-invasively, the burden of free radicals formed in pathologies resulting from oxidative stress. After the discovery of NO, it has been hypothesized that NO and its active congeners (reactive nitrogen species, RNS), such as nitrogen dioxide radical (NO2), nitrous acid, peroxynitrite, can also participate in lipid peroxidation, either as initiators or modulators of processes initiated by the hydroxyl radical. In biological systems these RNS not only originate from the biosynthesis of NO but also from exogenous sources such as polluted air and dietary nitrite. While the ability of NO2 to induce lipid peroxidation has been long known, more recent studies have discovered novel processes that have been termed lipid nitration. Polyunsaturated fatty acids appear to be readily targeted by RNS. Among the products of arachidonic acid nitration by NO2, interesting lipids have been detected, such as nitroeicosatetraenoic acids, alpha,beta-nitrohydroxyeicosatrienoic acids, and trans-arachidonic acids. The products of fatty acid nitration have the potential to function as biomarkers and/or lipid mediators of mechanisms distinct from fatty acid peroxidation but offering insight into the contribution of specific RNS such as NO2 to the damage of biological membrane resulting from nitrooxidative stress.

Determination of trans-arachidonic acid isomers in human blood plasma.

Endogenous trans fatty acids originate from diet, but recent studies also suggest that cis-trans isomerization of fatty acids is possible by nitrogen dioxide radical, a product of NO and nitrite oxidation. We developed a method for quantitative analysis of four trans-arachidonic acids (TAA) in human plasma using isotopic dilution gas chromatography/mass spectrometry (GC/MS) with deuterium-labeled internal standard. Esterification of the plasma fatty acid extract with pentafluorobenzyl (PFB) bromide followed by high-performance liquid chromatography purification yielded a fairly pure fraction containing TAA-PFB esters that was analyzed by GC/MS. Partial separation of the TAA isomers was obtained on various GC columns. Comparison of the retention time with the synthetic standards revealed that all four TAA isomers are present in human plasma. The mean concentration of TAA in human plasma was 20.2ng/ml. The levels of isomers were 12.48+/-1.28, 2.75+/-0.39, and 4.99+/-0.74ng/ml for 5E-AA + 11E-AA, 8E-AA, and 14E-AA, respectively. The identification of TAA in plasma suggests that isomerization of arachidonic acid occurs in vivo. Our method allows distinguishing between the dietary and the NO(2)-dependent mechanisms of trans fatty acid formation and will be useful in defining the role of TAA as an in vivo marker of nitrooxidative stress in clinical and experimental settings.