Content uploaded by Rakesh Sharma
Author content
All content in this area was uploaded by Rakesh Sharma
Content may be subject to copyright.
A preview of the PDF is not available
Comparison of Cholesterol Lowering Diets: Apple, Casein Cytochrom P450 protein and Cholesterol 7α Hydroxylase Activities in Hamsters
Figures
Figures - uploaded by Rakesh Sharma
Author content
All figure content in this area was uploaded by Rakesh Sharma
Content may be subject to copyright.
ResearchGate has not been able to resolve any citations for this publication.
Microsomal hydroxymethylglutaryl-CoA (HMG-CoA) reductase of rat liver is inactivated in vitro by Mg2+ and ATP or ADP in a time-dependent process that is mediated by an inactivator, present both in liver cytosol and microsomes. Inactivation of microsomal reductase is completely reversed by an activator present in liver cytosol. Restoration of microsomal reductase activity to a maximal value depends upon time and on activator concentration. Activator activity is totally inhibited by 50 mM sodium fluoride. The activity of solubilized, 1400-fold purified reductase is modified by the inactivator and activator in a manner similar to that of microsomal reductase. The inactivator and activator are proteins. The inactivator appears to have a molecular weight greater than 150,000; the partially purified activator has an apparent molecular weight of 20,000 to 30,000. Our data suggest that regulation of HMG-CoA reductase in vitro may occur via protein-mediated interconversion of forms of differing catalytic activity. Active and inactive reductase have the same K(m) for HMG-CoA and their interconversion is not accompanied by a gross change in molecular weight. The catalytic activity of HMG-CoA reductase increases 5- to 7-fold during isolation of microsomes. This increase, which appears to be due to the action of the activator, is blocked when liver tissue is immediately homogenized in the presence of sodium fluoride. Assay of reductase activity in microsomes prepared by techniques used in most laboratories thus may not accurately measure physiological activity. In rats fed a normal diet, about 85% of liver HMG-CoA reductase appears to exist in an inactive form in vivo.
The relationships between cholesterol 7 alpha-hydroxylase activity, pool of free microsomal cholesterol, and degree of substrate saturation of the enzyme were studied in untreated (n = 5), cholesterol-fed (n = 4), and cholestyramine-treated (n = 6) gallstone patients undergoing cholecystectomy. Highly accurate methods based on isotope dilution-mass spectrometry were used for assay of the cholesterol 7 alpha-hydroxylase activity and for determination of the concentration of free cholesterol in the microsomes. The cholesterol-enriched diet increased the cholesterol 7 alpha-hydroxylase activity about twofold. Cholestyramine treatment was associated with a five- to sixfold increase of the cholesterol 7 alpha-hydroxylase activity. The concentration of free microsomal cholesterol remained essentially unchanged. The apparent degree of saturation of the enzyme was calculated to be 85% in the untreated patients, 86% in the cholesterol-fed patients, and 67% in those treated with cholestyramine. A significant negative correlation was obtained between enzyme activity and apparent substrate saturation. It is concluded that the apparent substrate saturation of the cholesterol 7 alpha-hydroxylase in human liver microsomes is high but that availability of cholesterol may limit the enzyme activity to some extent a high bile acid synthesis rates.
Dietary fibers (DF) of the “cellan” type (consisting mainly or exclusively of undestroyed cells) were prepared as ethanol-dried materials from apple, cabbage, sugar-beet, soybean hulls, wheat bran, and suspension cultures of Chenopodium album L. and investigated with respect to their interactions with water, water−oil dispersions, bile acids, and oil. Water binding and retention capacities were found to be especially high in cellans obtained from thin-walled raw material. Water damp sorption by dry cellans, when analyzed according to the GAB and BET equations, shows a considerable fraction of monolayer water. At a water activity of 0.98, the cell and capillary spaces outside the walls remained in the air-filled state but the cell wall pores are filled with water. When the water content of a concentrated aqueous cellan suspension was equal to or below the water binding capacity, its rheological behavior was found to be of pseudoplastic nature. At a given dry weight concentration, yield stress and viscosity of such concentrated suspensions were highest for cellans with the highest water binding capacity. Dry cellan particles absorbed fatty oils without swelling but swell in a detergent-stabilized oil/water emulsion with a similar liquid absorption capacity as in water. In contrast to the dry or alkane-saturated cell wall, the hydrated wall is not permeable to oils in the absence of a detergent. Oil droplets may be entrapped within the cells, yielding a stable dispersion of oil in water. As DF of the cellan type absorb bile acids, preferentially glycoconjugates, from diluted solutions, they may have a potential to decrease the serum cholesterol level.
Patients with cholesterol gallstones have a reduced pool of bile acids. This study was undertaken to clarify the mechanism
by which bile acid biosynthesis does not increase to supranormal levels to cause a reexpansion of the pool. We investigated
the first two steps of the bile acid biosynthesis pathway by assaying the activities of cholesterol 7α-hydroxylase, the rate-limiting
enzyme in this pathway, and 3β-hydroxy-Δ5-C27-steroid dehydrogenase/isomerase, and by measuring the concentrations of 7α-hydroxycholesterol and 7α-hydroxy-4-cholesten-3-one
in liver specimens from ten patients with cholesterol gallstones and ten gallstone-free controls. In the patients with gallstones,
cholesterol 7α-hydroxylase activity, 3β-hydroxy-Δ5-C27 dehydrogenase/isomerase activity, and hepatic 7α-hydroxy-4-cholesten-3-one concentration did not significantly different
from levels in controls, but hepatic 7α-hydroxycholesterol concentration was more than twofold that of controls (12.9 ± 2.6
vs 5.3 ±1.2 nmol/g liver,P<0.01). The concentration of 7α-hydroxycholesterol positively correlated with the ratio of cholesterol 7α-hydroxylase activity
to 3β-hydroxy-Δ5-C27 dehydrogenase/isomerase activity (r=0.93;P<0.005) in the gallstone-free controls. In contrast, this correlation disappeared in the patients with gallstones. These results
suggest a derangement of the normal 7α-hydroxycholesterol metabolism in the patients with gallstones. The reason for the accumulation
of 7α-hydroxycholesterol remains unclear; however, it is possible that, in patients with cholesterol gallstones, the accumulated
7α-hydroxycholesterol causes inappropriate suppression of cholesterol 7α-hydroxylase activity by product inhibition.
Cholesterol 7 alpha-hydroxylase, the key enzyme in bile acid synthesis, has been implicated in atherosclerosis and gallstone disease. The aim of this study was to check if the use of hydroxypropyl-beta-cyclodextrin (HPBCD), a vehicle for solubilizing cholesterol, augmented the rate of 7 alpha-hydroxycholesterol formation in hamster liver microsomes compared to classical assays in which labeled cholesterol was delivered in Tween 80. We observed that [14C]cholesterol carried by HPBCD enhanced the sensitivity of the assay tenfold. However, linearity of 7 alpha-hydroxycholesterol formation with time was short because of the rapid transformation of 7 alpha-hydroxycholesterol into 7 alpha-hydroxy-cholesten-3-one and 7 alpha,12 alpha-dihydroxy-cholesten-3-one when NADPH alone was present in the incubation medium. In order to avoid the transformation of 7 alpha-hydroxycholesterol into 7 alpha-hydroxy-cholesten-3-one, which is essentially NAD(+)-dependent, but is also NADP(+)-dependent, NADPH (1 mmol/l) plus an NADPH-regenerating system must be present in the medium. In this improved assay, the optimal pH was 7.4 and the apparent Km for control and cholestyramine-fed hamsters had a similar value of 315 mumol/l; linearity in the formation of 7 alpha-hydroxycholesterol was also apparent after a relatively short time period (10 min), but with a markedly greater slope of the curve. With a short incubation time (6 min), microsomes from livers of hamsters (five and nine weeks old) that were fed with a commercial ground diet yielded rates of 7 alpha-hydroxycholesterol formation of 115 +/- 10 and 150 +/- 16 pmol/min.mg protein, respectively, whereas microsomes from hamsters fed with a lithogenic sucrose-rich diet (five weeks old) yielded rates of 7 alpha-hydroxycholesterol formation of 77 +/- 7 pmol/min.mg protein, which were significantly lower (-33%) than those of corresponding control hamsters. This improved cholesterol 7 alpha-hydroxylase assay is very sensitive, simple and rapid, and does not necessitate sophisticated equipment. It can be particularly useful for determining cholesterol 7 alpha-hydroxylase activity in liver biopsies from dyslipidemic or lithiasic patients.
1. Adsorption of bile salts by dietary fibre is believed to promote their excretion and hence to reduce the serum cholesterol level in man and experimental animals.
2. We have tested a number of plant fibre fractions and other related materials for their ability to adsorb bile salts from aqueous solution. The ‘insoluble’ plant fractions were from ‘dry grain’ (a residue from brewing), apple, wheat bran, lucerne ( Medicago sativa ), soya beans, mung beans ( Phaseolus mungo ), chick peas ( Cicer arietinum ), rolled oats, spinach ( Spinacia oleracea ), sunflower seeds, sawdust and sheep faeces. The other materials were cholestyramine, pectin and lignins prepared from wheat bran and from sawdust.
3. Only cholestyramine and the fibre from lucerne, soya beans, mung beans, chick peas, spinach, and sunflower seeds adsorbed enough of either sodium cholate or sodium deoxycholate for adsorption to be detectable.
4. This result conflicts with a report that the lignin component of dietary fibre is responsible for adsorption of bile salts.
5. Adsorption of bile salts, when it occurs, may depend on the presence of saponins bound to the fibre.
In agreement with previous work, treatment of rats with cholesterol, 2% in diet, stimulated the cholesterol 7 alpha-hydroxylase activity more than 2-fold. With less than 1% in diet, no significant effect was obtained. Intravenous infusion of cholesterol-enriched Intralipid had no stimulatory effect. In accordance with some recent work by other groups, it was shown that the stimulation of the cholesterol 7 alpha-hydroxylase by dietary cholesterol was associated with elevated levels of mRNA corresponding to the enzyme. Most of the stimulation of the activity induced by dietary cholesterol could not be prevented by lymphatic drainage. Feeding lymph fistulated rats with 2% cholesterol in diet stimulated the cholesterol 7 alpha-hydroxylase almost 2-fold, indicating that under the conditions employed, a major part of the cholesterol-induced stimulation of the activity was due to factor(s) unrelated to the flux of cholesterol from the intestine to the liver. There was a good correlation between the amount of cholesterol excreted in faeces and the activity of the cholesterol 7 alpha-hydroxylase. The half-life of intraperitoneally administered labelled cholic acid was significantly shorter in rats treated with 2% cholesterol in diet (t1/2 = 1.2 +/- 0.1 days) than in control rats (t1/2 = 1.9 +/- 0.18 days). A notable finding was that the weight of faeces was considerably higher in rats fed cholesterol than in the controls. It is hypothesized that a high dietary load of cholesterol causes increased binding of bile acids in the intestine and increased loss of bile acids in faeces. This leads to a reduced suppression of the cholesterol 7 alpha-hydroxylase by the bile acids. The results support the contention that the flux of bile acids rather than the flux of cholesterol from the intestine is the major direct regulator of bile acid biosynthesis.
Effects of viscous indigestible polysaccharides on the pancreas exocrine function were investigated in growing rats. Rats were fed a nonfiber diet or a diet containing approximately 5% of one of the following fibers: apple pectin, lambda-carrageenan, locust bean gum, gum xanthan, guar gum or sodium (Na) alginate. Pancreatic-bile secretion was found to be elevated in rats fed for 2 wk the highly viscous polysaccharides, sodium alginate, locust bean gum, gum xanthan and guar gum. The polysaccharides may have interfered with the digestion and absorption of nutrients, resulting in a decreased digestibility and an enlargement of digestive organs. When alginic acid and calcium alginate, insoluble polysaccharides that did not contribute to viscosity, were given to rats, they had no effect on pancreatic and biliary secretion compared with sodium alginate. The results demonstrate that consumption of viscous indigestible polysaccharides leads to changes in the exocrine pancreatic-biliary function and may depress the process of digestion and absorption. Rats may compensate for the inefficiency of digestion and absorption with a hyperplasia/hypertrophy of digestive organs and an increased secretion of digestive juice.
Rats fed a semipurified diet containing casein developed higher levels of circulating triglycerides and cholesterol than animals fed a soy protein-containing diet. The increased serum lipid levels in non-fasted rats were associated largely with the d less than 1.006 g/ml lipoprotein particles (e.g. chylomicrons or very low density-like lipoproteins). In addition, casein-fed rats exhibited higher levels of circulating insulin and depressed hepatic 7 alpha-hydroxylase levels compared to soy-fed rats. Supplementation of the casein diet with arginine, to give an arginine/lysine ratio comparable to that in the soy diet, resulted in a reduction of d less than 1.006 g/ml lipids, a reduction in serum insulin levels and an elevation in hepatic 7 alpha-hydroxylase activity. Supplementation of the soy diet with lysine also resulted in modification of these parameters toward those observed with casein diets, albeit the effects were less dramatic. The results suggest that the hyperlipidemia associated with feeding casein-based diet is associated with decreased rates of clearance of chylomicron-like lipoproteins and their component triglycerides and cholesterol. Furthermore, this is largely prevented by addition of arginine to diets containing casein as the sole protein source.