M. T. R. Subbiah

Mechanisms of Cardioprotection by Estrogens

Abstract Strong scientific evidence suggests that estrogen therapy offers considerable protection from coronary artery disease. While earlier studies focused mainly on favorable changes in plasma lipid profiles as the mechanism of estrogen action, recent studies have documented other mechanisms of action including direct effects on vascular wall. The other mechanisms include estrogens role as an antioxidant, its ability to protect from DNA damage, inhibition of cell proliferation, and altering vascular response to vasoactive agents. Considerable emphasis must be placed on research on individual components of the commonly used conjugated estrogens and estrogen structure/function relationship to target specific tissues and minimize undesirable effects while maintaining estrogens cardioprotective potency.

Oxidation of cholesterol moiety of low density lipoprotein in the presence of human endothelial cells or Cu+2 ions: Identification of major products and their effects

Oxidation of lipoproteins is believed to play a key role in atherogenesis. In this study, low density lipoproteins (LDL) was subjected to oxidation in the presence of either human umbilical vein endothelial cells or with Cu+2 ions and the major oxides formed were identified. While cholesterol-alpha-epoxide (C-alpha EP) was the major product of cholesterol peroxidation in the presence of endothelial cells, cholest-3,5-dien-7-one (CD) predominated in the presence of Cu+2 ion. Both steroids were identified by gas chromatography/mass spectrometry. HDL cholesterol was resistant to oxidation. When tested on human skin fibroblasts in culture C-alpha EP (10 micrograms/ml) caused marked stimulation of 14C-oleate incorporation into cholesterol esters, while CD stimulated cholesterol esterification only mildly. These studies show that a) C-alpha EP is the major peroxidation product of LDL cholesterol moiety in the presence of endothelial cells and b) it causes marked stimulation of cholesterol esterification in cells. C-alpha EP may play a key role in increasing cholesterol esterification noted in atherogenesis.

Specific Changes of Bile Acid Metabolism in Spontaneously Diabetic Wistar Rats

Abstract Bile acid metabolism has been investigated in a newly described animal model depicting juvenile human diabetes (spontaneously diabetic Wistar (BB) rat) and compared to normoglycemic control from the Wistar strain. Diabetic animals used were on insulin treatment except for the last 24 hr. The plasma glucose levels (mg%) of diabetic rat (D) was significantly higher than control rats (C) (150 ± 35 in C vs 340 ± 32 in D). The total bile acid pool (mg/100 g) in D was significantly (P < 0.05) higher when compared to C (9.0 ± 0.8 in C vs 14.9 ± 1.7 in D). The pool of cholic acid was significantly (P < 0.05) increased while that of chenodeoxycholic acid was significantly (P < 0.05) decreased (cholic acid: 5.9 ± 0.45 in C vs 10.06 ± 1.2 in D; chenodeoxycholic 0.90 ± 0.1 in C vs 0.57 ± 0.06 in D). This increased the cholic/chenodeoxycholic acid ratios from 6.6 ± 0.4 in controls to 19.3 ± 2.4 in diabetic rats. These studies have shown diverse alteration in the concentration of the two primary bile acids in the diabetic state.

6-Keto-PGF1α Synthesis in Diabetic Rat Aorta: Effect of Substrate Concentration and Cholesterol Feeding

Abstract The effect of short-term feeding of a 1% cholesterol diet to normal and streptozotocin-induced diabetic rats on aortic 6-keto-PGF1α synthesis from exogenous and endogenous arachidonic acid (AA) was investigated. Diabetes and cholesterol feeding (by themselves) resulted in a reduction in aortic synthesis of 6-keto-PGF1α from both exogenous and endogenous arachidonic acid. An additive effect of diabetes and cholesterol feeding together was found for synthesis of 6-keto-PGF1α from exogenous but not endogenous AA. Experiments in which the AA concentration was varied suggested that the inhibition of aortic 6-keto-PGF1α synthesis by diabetes was competitive in nature. The diabetic rat was also found to be severely compromised in its ability to handle dietary cholesterol, as evidenced by a dramatic increase in plasma total cholesterol.

High Acyl-CoA cholesterol acyl transferase activity in fetal rabbit aorta: Evidence for the presence of stimulating factor(s) in amniotic fluid

The activity of Acyl CoA-cholesterol acyl transferase was markedly high in fetal aortas when compared to maternal and adult male rabbits. This activity dropped by 50% at 1 week of age. This high activity in fetal aorta a) did not appear to be due to changes in plasma cholesterol levels or to the later development of endogenous inhibitor in the aorta, but rather b) due to stimulatory factor(s) present in amniotic fluid.

Regional aortic differences in atherosclerosis-susceptibility: Changes in prostaglandin biosynthesis and cholesterol accumulation in response to desoxycorticosterone (DOCA)-salt induced hypertension

SummaryIn spontaneously atherosclerosis-susceptible White Carneau pigeons, intimai cushions that appear at birth near the coeliac branch of aorta do not progress into atherosclerotic lesions. However, the area across from the intimai cushion (so called ‘lesion area’) a) accumulates cholesteryl esters b) synthesizes more PGE2 and c) eventually develops into complicated atherosclerotic plaques. When DOCA-salt hypertension is induced in the pigeons, the ‘intimai cushion’ area displays a) accumulation of increasing amounts of cholesteryl esters and b) increase in the synthesis of all prostaglandins (particularly PGE2) from C14-arachidonic acid and c) approaches similarity to the ‘lesion area’ in the magnitude of these changes. These results suggest that under the influence of a risk factor, the ‘intimai cushion’ can acquire biochemical properties of the atherogenic areas of the aorta.

Effect of diabetes during pregnancy on maternal and neonatal bile acid metabolism in the rat.

Abstract The effect of streptozotocin-induced diabetes in pregnant rats, on subsequent maternal and neonatal bile acid metabolism was investigated. Plasma glucose levels (mg%) of diabetic pregnant (DP) rats was significantly greater (p < 0.05) in comparison to control pregnant (CP) rats. Total bile acid pool (mg/100g body wt) in DP was significantly higher (p < 0.05) when compared to CP (DP, 29.42 ± 4.44 vs 8.14 ± 1.13 in CP) with a marked increase in cholic acid pool size in DP (DP, 22.65 ± 3.33 vs 3.50 ± 0.48 CP, p < 0.05). The fecal excretion of bile acid was significantly lower (p < 0.05) in DP rats when compared to CP rats. Thin-layer chromatographic analysis of bile acid conjugates at postpartum revealed a significant increase in glycine conjugates in bile from DP rats. Examination of the cholic acid pool (μg/100 g body wt) in neonates (2 days old) derived from the two groups of rats indicated a strikingly reduced pool of cholic acid in neonates derived from diabetic mothers (neonates from DP 853.45 μ 213.98 vs 3690.86 μ 797.26 in CP,p < 0.05). Plasma cholesterol levels (mg%) in neonates of diabetic mothers was also significantly (p < 0.05) reduced. This study demonstrates that the diabetic state during pregnancy not only alters maternal bile acid metabolism, but also affects fetal bile acid metabolism in utero such that the effects may persist into neonatal life.

Isolation and characterization of a novel lipoprotein particle from human placental extracts

While it is believed that placental tissue is very active in lipid metabolism, the nature of lipid containing particles secreted (if any) by this tissue is not known. Lipoprotein profile of human placental tissue was analysed by gel filtration, gel electrophoresis and electron microscopy. Our studies demonstrated the presence of lipoproteins with unusual compositions. A novel major lipoprotein (which eluted in the same position on plasma VLDL) was characterized. While this lipoprotein floated at density greater than 1.006 gr/ml and contained apo B (same as plasma VLDL) it differed from plasma VLDL in a) size, b) contining a significant amount of apo Al, and c) carried bulk of the cholesterol (80% in free form) and phospholipids. This study suggests that placental tissue might contain unique lipoproteins perhaps serving specific metabolic needs.

Evidence for the presence of low density and very low density lipoproteins in human amniotic fluid

Previous analysis of amniotic fluid (AF) noted only the presence of high density lipoprotein (HDL). In this study AF lipoprotein profile was examined using gel filtration column chromatography and Ouchterlony gel diffusion. Unlike previous studies which showed only the presence of HDL, we found significant amounts of low density lipoprotein (LDL) and very low density lipoprotein (VLDL). AF-LDL and AF-VLDL were identified by reactions with anti-h-apolipoprotein AI and AII antiserum and anti-h-apolipoprotein B-antiserum, respectively. Furthermore, bulk of the cholesterol mass was carried in VLDL (53.6 +/- 7.7%) and LDL (32.5 +/- 4.3%) with minor amounts (13.9 +/- 1.3%) in HDL fraction. It is concluded that human AF contains all three lipoproteins with most of the cholesterol being carried in very low density lipoprotein fraction.

Human breast milk stimulates prostaglandin synthesis in cultured human skin fibroblasts

Effect of human breast milk or its fractions on prostaglandin synthesis was investigated in cultured human skin fibroblasts. Prostaglandins released into the media were measured by radioimmunoassay. Incorporation of breast milk (2% level) into 10% fetal calf serum media (for 48 hours) stimulated the synthesis of 6-keto-PGF1 alpha (stable product of prostacyclin) by 800%. This stimulating effect of milk persisted after cold acetone extraction to remove phospholipids and potentiated further after dialysis. Stimulation by one of the commercial formulas (Similac) was less than 50% of the milk effect. Milk also stimulated PGE2 synthesis, although to a much lesser degree. These studies show for the first time that a) human breast milk contains potent factor(s) capable of influencing prostaglandin synthesis and suggest that b) these factors might have a role in the development of lipid synthetic pathways during early life.