William E.M. Lands

A new, sensitive determination of phosphate

Abstract A new method for the determination of phosphorus using Triton X-100 instead of reducing agents is more sensitive than procedures described earlier. Organic phosphate esters do not appear to interfere with the determination of free phosphate under the conditions of the assay. All reagents are stable at room temperature and the absorbance is measured at convenient wavelengths.

Prostaglandin biosynthesis can be triggered by lipid peroxides

Studies of ferriheme cyclooxygenase, using two different assay systems, show that a variety of peroxides can trigger a rapid acceleration of cyclooxygenase activity to produce prostaglandins. Lipid hydroperoxides formed by lipoxygenase were the most potent activators tested, followed by prostaglandin Gz, which was slightly less potent. Peroxides nonspecifically generated during arachidonate autoxidation were as potent as the enzymatically formed lipid peroxides. These findings have important implications for cell function since any process which generates peroxides may activate the cyclooxygenase. Thus the balance between formation and removal of cellular lipid peroxides sets a peroxide tone that can regulate the rate of prostaglandin formation in cells.

INHIBITION OF PROSTAGLANDIN BIOSYNTHESIS BY EICOSAPENTAENOIC ACID

Eicosapentaenoic acid [20 : 5 (n-3)] is not oxidized by the purified cyclooxygenase from sheep vesicular glands in the conditions of low peroxide tone in which arachidonate [20 : 4 (n-6)] is rapidly oxygenated. When the level of peroxide in incubation mixtures is allowed to rise, there is a dramatic change in reactivity of the cyclooxygenase to react with 20 : 5 (n-3) at one-halt the rate and one-third the extent observed with 20 : 4 (n-6). Overall, the low peroxide levels expected in vivo would most probably cause the (n-3) type of fatty acid to be a general inhibitor of prostaglandin formation, through both reversible and irreversible actions at the enzyme site.

Biochemistry and physiology of n-3 fatty acids.

Considering the n‐3 fatty acids to be partial agonists relative to n‐6 fatty acids helps consolidate into a unified interpretation the many diverse reports and controversies on the actions of these two types of essential fatty acids. Some research reports illustrate the similarities between these two types and some emphasize the differences, leaving readers to evaluate the status of n‐3 fatty acids from a viewpoint that is conceptually similar to regarding a glass of water as half empty or half full. Both n‐3 and n‐6 types of fatty acids must be obtained through the diet because they are not synthesized de novo by vertebrates. Both types can support important physiological and developmental processes, can form eicosanoids (prostaglandins, leukotrienes, lipoxins, etc.), can be esterified to and hydrolyzed from tissue glycerolipids, and can be metabolically elongated and desaturated to a variety of highly unsaturated fatty acids. However, some nonesterified n‐6 acids are vigorously converted to potent n‐6 eicosanoids that exert intense agonist actions at eicosanoid receptors, whereas the n‐3 acids less vigorously form n‐3 eicosanoids that often produce less intense (partial) actions. Because both types owe their presence in vertebrate tissues to dietary intake, important physiological consequences follow the inadvertent selection of different average daily dietary supplies of these two types of polyunsaturated fatty acids.—Lands, W. E. M. Biochemistry and physiology of n‐3 fatty acids. FASEB J. 6: 2530‐2536; 1992.

Maintenance of lower proportions of (n − 6) eicosanoid precursors in phospholipids of human plasma in response to added dietary (n − 3) fatty acids

Competition between the (n - 3) and (n - 6) types of highly unsaturated fatty acids can diminish the abundance of (n - 6) eicosanoid precursors in a tissue, which in turn can diminish the intensity of tissue responses that are mediated by (n - 6) eicosanoids. The mixture of 20- and 22-carbon highly unsaturated fatty acids maintained in the phospholipids of human plasma is related to the dietary intake of 18:2 (n - 6) and 18:3 (n - 3) by empirical hyperbolic equations in a manner very similar to the relationship reported for laboratory rats (Lands, W.E.M., Morris, A. and Libelt, B. (1990) Lipids 25, 505-516). Analytical results from volunteers ingesting self-selected diets showed an inter-individual variance for the proportion of (n - 6) eicosanoid precursors in the fatty acids of plasma phospholipids of about 5%, but the variance among multiple samples taken from the same individual throughout the day was less (about 3%), closer to the experimental variance of the analytical procedure (about 1%). The reproducibility of the results makes it likely that analysis of fatty-acid composition of plasma lipids from individuals will prove useful in estimating the diet-related tendency for severe thrombotic, arthritic or other disorders that are mediated by (n - 6) eicosanoids. Additional constants and terms were included in the equations to account for the effects of 20- and 22-carbon highly unsaturated (n - 3) fatty acids in the diet. A lower constant for the 20- and 22-carbon (n - 3) fatty acids compared to that for the 18-carbon (n - 3) fatty acid in decreasing the ability of dietary 18:2 (n - 6) to maintain 20:4 (n - 6) in tissue lipids confirmed the greater competitive effectiveness of the more highly unsaturated n - 3 fatty acids in the elongation/desaturation process. Also, a lower constant for direct incorporation of 20-carbon fatty acids of the n - 6 vs. the n - 3 type indicated a greater competitive effectiveness of 20:4 (n - 6) relative to 20:5 (n - 3) in reesterification after release from tissue lipids. The equations may be used in reverse to estimate the dietary intakes of the (n - 3) and (n - 6) fatty acids by using the composition of the fatty acids that had been maintained in plasma lipids.

Requirements for hydroperoxide by the cyclooxygenase and peroxidase activities of prostaglandin H synthase.

Purified prostaglandin H synthase contains cyclooxygenase activity that forms the hydroperoxide, prostaglandin G, and peroxidase activity which removes hydroperoxides. Since hydroperoxides are necessary activators of cyclooxygenase activity, the paradoxical presence of two apparently opposing activities requires careful interpretation. Kinetic studies indicate that the concentration of hydroperoxide needed for full cyclooxygenase activity is much less than that which gives 50 percent effectiveness with the peroxidase. Thus, the peroxidase activity of the synthase is very ineffective in decreasing the hydroperoxide concentration below levels that still permit rapid cyclooxygenase action.

The effect of dietary supplementation of fish oil on experimental myocardial infarction

The effect of altering the abundance of precursors and inhibitors of prostaglandin formation by dietary supplements of fish oil was investigated in dogs with experimentally induced myocardial infarction. Prior to induction, 10 male mongrel dogs were fed standard dog chow supplemented with 25% of the total calories as menhaden oil for 36 to 45 days. The fatty acid composition of th lipids in plasma and platelets changed to reflect the increased intake of polyunsaturated fatty acids of the n-3 type. Thrombosis and subsequent infarction was induced by electrical stimulation of the left circumflex coronary artery of ambulatory dogs that were monitored by telemetry. Upon stimulation of control animals, the frequency of ectopic beats rose from less than 10% at the beginning to about 80% after 19 hours. In contrast, the oil-fed dogs maintained a more normal ECG pattern, showing less than 30% ectopic beats after 19 hours. In these animals, the size of infarction (measured by formazan formation) was 3% of the left ventricle compared to 25% in the control animals. The results suggest that dietary supplementation with fish oil may be beneficial in reducing myocardial damage associated with coronary artery thrombosis.

Quantitative effects of dietary polyunsaturated fats on the composition of fatty acids in rat tissues

A method combining data on fatty acid composition into subsets is used to illustrate general relative competitive selectivities in the metabolic and transport events that maintain fatty acid compositions in tissue lipids and to minimize differences among tissues or species in the amount of individual fatty acids. Fatty acid compositions of triglycerides and phospholipids in several tissues of the rat were maintained with simple relationships between the exogenous n−3 and n−6 dietary polyunsaturated fatty acids and the endogenous n−7 and n−9 types of fatty acid. The general pattern of fatty acids in triglycerides was similar for liver, plasma and adipose tissue, averaging about 30% as saturated acids, 67% as 16- and 18-carbon unsaturated acids and only about 2% as 20- and 22-carbon highly unsaturated acids. The tissues maintained a linear relationship between the amount of 18-carbon polyunsaturated fatty acids in the diet and in the tissue triglycerides, with the proportionality constant for 18∶3n−3 being 60% of that for 18∶2n−6. The total phospholipids of liver, plasma and red blood cells maintained about 45% of the fatty acids in the form of saturated fatty acids and 20–30% as 20- and 22-carbon highly unsaturated fatty acids irrespective of very different proportions of n−3, n−6 and n−9 types of fatty acids. In all three tissues, the 20-carbon highly unsaturated fatty acids of the n−3, n−6 and n−9 type were maintained in a competitive hyperbolic relationship with apparent EC50 values for dietary 18∶2n−6 and 18∶3n−3 near 0.1% of dietary calories. The consistent quantitative relationships described in this study illustrate an underlying principle of competition among fatty acids for a limited number of esterification sites. This approach may be useful in predicting the influence of diet upon tissue levels of the substrates and antagonists of eicosanoid biosynthesis.

FACTORS REGULATING THE BIOSYNTHESIS OF VARIOUS PROSTAGLANDINS

the Karolinska Institute working in collaboration with Professor B. Samuelsson. At that time we were able to develop thin-layer techniques which allowed quantitative measurement of the many different products appearing during prostaglandin biosynthesis. This led to the identification of the unusual pros t ag land in , l l -ke to -PGFl , , f rom reaction mixtures(Granstr8m et a1,1968),and a recognition that the oxidative cyclization most probably occurs after the liberation of the essential acid(Lands & Samuelsson,l968),by action of an acylhydrolase. Last year I began again to use those methods to examine in more detail the regulation of prostaglandin biosynthesis. A useful review of the factors in the biosynthesis of prostaglandins recognized up to that point was provided by Samuelsson in that years Progress of Biochemical Pharmacology(Samuelsson,l969).

Inhibition of Prostaglandin Biosynthesis

Little evidence exists for stored prostaglandins in tissues (Jouvenaz, Nugteren, Beerthuis and Van Dorp, 1970) and a capacity of a tissue to release prostaglandins seems to reflect principally the capacity for biosynthesis of these compounds. Since prostaglandins represent a diverse family of oxygenated derivatives formed from certain polyunsaturated fatty acids, the Open image in new window Fig. 3.1 Formation and degradation of prostaglandins. biosynthesis of prostaglandins consists of many steps that involve a variety of different enzymes and cofactors. Inhibition of prostaglandin synthesis may thus occur by blocking any of the several processes that are involved. A brief summary of some of the metabolic events leading to the different prostaglandins is given in Figure 3.1. Because of the diversity of these processes and the stimuli and mediators that appear to modify these reactions it is expected that a wide variety of chemical agents will diminish the formation of prostaglandins in vivo. We will indicate some of the events of prostaglandin biosynthesis known to be influenced by chemical agents, and will suggest a logical framework for comparing the relative inhibitory activity of these different agents.