Ferric acetate-uranium acetate reagent in cholesterol estimations in plasma and high density lipoprotein fractions: comparison with existing methods

The use of ferric acetate-uranium acetate colour reaction for the estimation of cholesterol in the supernatants of plasma samples after precipitation of low density lipoprotein (LDL) and very low density lipoprotein (VLDL) cholesterol by heparin-MnCl2 was assessed and compared with the conventional method using the FeCl3 colour reaction and also with the method using o-phthalaldehyde as the colouring reagent. All three methods gave comparable values when total cholesterol in plasma samples was determined and also when high density lipoprotein (HDL) fractions were separated by ultracentrifugation and the cholesterol contents determined. But when heparin-MnCl2 precipitation was used for HDL separation, and the cholesterol content determined, the FeCl3 method gave significantly lower values. This could be due to interference of the cholesterol colour reaction with FeCl3, due to Mn2+ ions present in the supernatant. Addition of Mn2+ to cholesterol standards and subsequent colour development with ferric acetate-uranium acetate and FeCl3 reagents showed that Mn2+ decreased the absorbancy of the coloured complex at 560 nm only when FeCl3 was used. Percentage recovery of added cholesterol was also lower when the heparin-MnCl2 supernatant was treated with FeCl3 reagent for colour development. Use of ferric acetate-uranium acetate reagent provides a simpler and quicker method. It does not suffer from interference due to the presence of Mn2+ ions and gives results comparable to the o-phthalaldehyde method and those using ultracentrifugation as the separation procedure.     

Assignment of heme methyl 1H-NMR resonances of high-spin and low-spin ferric complexes of cytochrome p450cam using one-dimensional and two-dimensional paramagnetic signals enhancement (PASE) magnetization transfer experiments.

An 1H-NMR study of ferric cytochrome P450cam in different paramagnetic states was performed. Assignment of three heme methyl resonances of the isocyanide adduct of cytochrome P450 in the ferric low-spin state was recently performed using electron exchange in the presence of putidaredoxin [Mouro, C., Bondon, A., Jung, C., Hui Bon Hoa, G., De Certaines, J.D., Spencer, R.G.S. & Simonneaux, G. (1999) FEBS Lett. 455, 302-306]. In this study, heme methyl protons of cytochrome P450 in the native high-spin and low-spin states were assigned through one-dimensional and two-dimensional magnetization transfer spectroscopy using the paramagnetic signals enhancement (PASE) method. The order of the methyl proton chemical shifts is inverted between high-spin and low-spin states. The methyl order observed in the ferric low-spin isocyanide complexes is related to the orientation of the cysteinate ligand.     

Sources of error in the isotopic cholesterol balance method in African green monkeys consuming a cholesterol-free diet

Six African green monkeys were labeled intravenously with [1,2-(3)H]cholesterol while consuming a cholesterol-free liquid formula diet. The plasma cholesterol specific activity was compared with the specific activity of the biliary cholesterol and bile acids and with the fecal neutral steroids in order to determine whether the traditional isotopic balance method was valid for the calculation of endogenous cholesterol excretion. The specific activity of biliary cholesterol and bile acids averaged 10-15% lower than plasma cholesterol specific activity. Fecal cholesterol and coprostanone specific activities were similar to that of the biliary cholesterol, but the specific activity of fecal coprostanol was approximately 25% lower. This suggests that biliary cholesterol and bile acids were derived from a pool of hepatic cholesterol that did not completely equilibrate with the whole body exchangeable cholesterol pool. In addition, there was further reduction in the specific activity of coprostanol, the major fecal neutral steroid, presumably by cholesterol synthesized in the lower intestine and preferentially converted to coprostanol. As a result, the traditional isotopic balance procedure underestimated endogenous neutral steroid excretion by 46% and bile acid excretion by 31% in African green monkeys fed the cholesterol-free diet. Within 7 days after the addition of 1 mg cholesterol/kcal to the diet, the specific activities of plasma and biliary cholesterol and biliary bile acids were identical and there was no difference in the specific activities of the individual fecal neutral steroids. Thus, the traditional isotopic balance procedure (DPM fecal neutral steroids + bile acids/specific activity [DPM/mg] plasma cholesterol) can be used for calculation of endogenous cholesterol excretion in cholesterol-fed animals during the nonsteady state when plasma cholesterol concentrations are rapidly increasing, as well as after a new steady state has been achieved.-Henderson, G. R., and R. W. St. Clair. Sources of error in the isotopic cholesterol balance method in African green monkeys consuming a cholesterol-free diet.     

Medium-Chain Fatty Acids Enhanced the Excretion of Fecal Cholesterol and Cholic Acid in C57BL/6J Mice Fed a Cholesterol-Rich Diet

The objective of the present study was to investigate the cholesterol-reducing effect of medium-chain fatty acids (MCFAs) completed by elevated excretion of fecal neutral steroids and/or bile acids. Blood and liver lipid profiles, fecal neutral steroids, bile acids, and mRNA and protein expression of the genes relevant to cholesterol homeostasis were measured and analyzed in C57BL/6J mice fed a cholesterol-rich diet with 2% caprylic acid or capric acid for 12 weeks. Blood total cholesterol and low-density lipoprotein cholesterol (LDL-c) levels were reduced significantly as compared to diet with palmitic acid or stearic acid. Caprylic acid promoted the excretion of fecal neutral steroids, especially cholesterol. The excretion of fecal bile acids, mainly in the form of cholic acid was enhanced and accompanied by elevated expression of mRNA and the protein of hepatic cholesterol 7α-hydroxylase (CYP7A1). These results indicate that MCFAs can reduce blood cholesterol by promoting the excretion of fecal cholesterol and cholic acid.     

Contraceptive steroids alter the steady-state kinetics of bile acids

Contraceptive steroids increase the ratio of cholic acid to chenodeoxycholic acid in bile. This alteration may contribute to the development of cholesterol gallstones. The objective of this study was to measure the effects of contraceptive steroids on bile acid kinetics and to relate them to changes in cholesterol metabolism. Steady-state kinetics of bile acids were measured in 15 healthy women, on and off contraceptive steroids. Cholic acid synthesis increased 30.3% (P less than 0.025) and its pool increased by 37.4% (P less than 0.025). Chenodeoxycholic acid synthesis decreased 6.4% (P = 0.08) and its pool decreased by 11.8% (P less than 0.05) during use of contraceptive steroids. The fractional turnover rates of both primary bile acids did not change. The changes in kinetics of the primary bile acids were related to alterations in biliary lipid and cholesterol metabolism, separately reported. (J. Lipid Res. 1987. 28: 828-839). During use of contraceptive steroids, total bile acid pool and total bile acid synthesis correlated directly with cholesterol synthesis, assayed in mononuclear leukocytes (r = 0.50 and r = 0.54, respectively) but not with the plasma clearance of chylomicron remnants, measured with retinyl palmitate. The data indicate that contraceptive steroids directly alter the hepatic synthesis of bile acids and suggest that newly synthesized cholesterol may be a preferred substrate for bile acid synthesis during use of contraceptive steroids.     

Contraceptive steroids increase cholesterol in bile: mechanisms of action

Contraceptive steroids increase the risk of acquiring cholesterol gallstones. The factors responsible include an increase in cholesterol saturation of bile and an increase in rate of secretion of cholesterol into bile. The goal of this study was to investigate the mechanism(s) of these increases in biliary cholesterol. During the use of contraceptive steroids, cholesterol saturation of gallbladder bile and the amount of cholesterol secreted per mole of bile acid increased (P less than 0.05 and P less than 0.02, respectively). Cholesterol absorption, cholesterol synthesis, chylomicron remnant clearance, and the concentration of plasma and lipoprotein lipids were not altered by contraceptive steroids. Despite this apparent lack of effect, important correlations were present during steroid use. LDL (low density lipoprotein) cholesterol increased as dietary cholesterol increased (r = 0.58, P less than 0.025). Cholesterol synthesis correlated directly with VLDL cholesterol concentration (r = 0.64, P less than 0.01), biliary cholesterol secretion (r = 0.68, P less than 0.01) and with molar percent cholesterol in bile (r = 0.49, P = 0.06). Chylomicron remnant clearance also correlated with cholesterol secretion (r = 0.85, P less than 0.001). As either remnant uptake or synthesis increased, the effect of the other source of hepatic cholesterol on biliary cholesterol secretion diminished. These relationships were not observed in the same subjects when they were not taking the hormones. The findings suggest that both newly synthesized and dietary cholesterol contribute to the cholesterol secreted in bile. This is consistent with the hypothesis that cholesterol for secretion into bile and VLDL is derived from a common metabolic pool of free cholesterol. It is proposed that contraceptive steroids exert their effect on biliary cholesterol by increasing cholesterol entering the pool and/or by inhibiting hepatic ACAT (acylcoenzyme A:cholesterol acyltransferase) activity, a known effect of progesterone, so that an increase in free cholesterol entering the pool leads to an increase in output.     

Mechanism and kinetic characteristics of the uncoupling by plant steroids of biliary cholesterol from bile salt output

In male Wistar rats fed diets containing different plant steroids, including sitosterols, diosgenin, digitonin and saponin from gypsophila, biliary cholesterol secretion significantly increased 50% to 300%, whereas biliary bile salt and phospholipid showed minor changes. Both cholesterol and phospholipid outputs were coupled to biliary bile salt output in a curvi-linear relationship which could be fitted by rectangular hyperbolae, in the animals fed with different plant steroids. The theoretical maximal biliary cholesterol output significantly increased by 200% in sitosterol-fed rats and 500% in diosgenin-fed animals. No changes were found in the kinetic characteristics of biliary phospholipid outputs. Adding 2% cholesterol to the diosgenin diet abolished the increment of biliary cholesterol output induced by the plant steroid. The intraperitoneal injection of 45 mumol/kg body wt per day (3 days) diosgenin, a C27-sapogenin, and 65 mumol/kg body wt. per day (3 days) tomatidin, a C27-alkaloid, incorporated in phosphatidylcholine-taurocholate liposomes significantly increased biliary cholesterol output by 70%. These experiments indicated that the plant steroid-induced biliary cholesterol output was independent of the inputs of cholesterol from the diet and from hepatic cholesterogenesis modified by the plant steroid. It was apparent that the profound changes of biliary cholesterol secretion were the consequence of direct effects of the steroids on the intrahepatocytic regulatory mechanisms of biliary cholesterol secretion. This novel effect appears to be a universal characteristic of plant steroids, since it can be elicited by sitosterols, C27-sapogenins, C27-alkaloids, and saponins of the cholanic and beta-amirinic group.     

The Histochemical Localization of Cholesterol in Formalin-Fixed and Fresh Frozen Sections

The premixed cholesterol reagent (acetic acid, sulphuric acid, ferric chloride and phosphoric acid) employed in a spectrophotometric method for serum cholesterol determinations was applied to frozen sections of various rat and rabbit tissues. The histochemical reaction was compared with the results obtained by the classical Schultz reaction. The same bluish-green color was observed after treatment of formalin-fixed and fresh cryostat sections incubated in 2.5% iron alum at 37 C for 3 days. The premixed cholesterol reagent is stable for years at room temperature if stored in a brown bottle and is thus readily available for routine use.     

Quantitative analysis of cholesterol in 5 to 20 microliter of plasma

A gas-liquid chromatographic micromethod for quantitation of cholesterol in 20 micro l of plasma was developed using 5alpha-cholestane as an internal standard, saponification with tetramethylammonium hydroxide-isopropanol, and extraction with tetrachloroethylene-methyl butyrate. Cholesterol levels in plasma samples were calculated by comparing cholesterol-cholestane peak height ratios with those of preassayed reference plasma. Over a plasma cholesterol range of 44 to 468 mg/100 ml, the gas-liquid chromatographic micromethod and the automated ferric chloride colorimetric method gave nearly identical results (r = 0.99) in duplicate aliquots of 131 plasma samples.     

Analysis of alpha-lipoprotein cholesterol in 50 microl of plasma

A micromethod for quantitation of alpha-lipoprotein cholesterol was devised for gas-liquid chromatography to minimize plasma sample size and facilitate lipid studies using capillary blood from children or small animals. alpha-Lipoprotein cholesterol was measured by gas-liquid chromatography in 20 micro l of the centrifuged supernate obtained after addition of 5 micro l of mixed heparin-manganese chloride solution 1:1 (v/v) to 50 micro l of plasma. Comparison of venous alpha-lipoprotein cholesterol measured by gas-liquid chromatography with venous alpha-lipoprotein cholesterol measured by conventional heparin-manganese precipitation and ferric chloride (colorimetric) cholesterol determination gave a correlation coefficient of 0.98 for 80 plasma samples. Capillary alpha-lipoprotein cholesterol and venous alpha-lipoprotein cholesterol were closely correlated in 31 patients (r = 0.97).     

Sensitive detection of specific repair endonucleases: radial diffusion assay utilizing differential alkaline denaturation of supercoiled and nicked PM2 DNA in agarose gels

An improved method for separation and quantitation of sulfated neutral and acidic steroids in human feces was developed. The procedure consists of separation of sulfated steroids on Sephadex LH-20 and hydrolysis by cholylglycine hydrolase followed by quantitation and identification of the trimethylsilylether derivatives by gas-liquid chromatography and gas-liquid chromatography-mass spectroscopy. Using this procedure, we detected no sulfated bile acids in human feces. However, sulfated cholesterol was detected in the sulfated bile acid fraction obtained from human fecal extracts. Analysis showed that cholesterol sulfate comprised 12.3, 11.2, and 31.0% of the total neutral sterol fraction in the three fecal samples. Using our procedures, cholesterol sulfate and bile acid sulfates in a biological mixture can be quantitated and identified when they are present.     

Increased ABA sensitivity results in higher seed dormancy in soft white spring wheat cultivar ‘Zak’

As a strategy to increase the seed dormancy of soft white wheat, mutants with increased sensitivity to the plant hormone abscisic acid (ABA) were identified in mutagenized grain of soft white spring wheat “Zak”. Lack of seed dormancy is correlated with increased susceptibility to preharvest sprouting in wheat, especially those cultivars with white kernels. ABA induces seed dormancy during embryo maturation and inhibits the germination of mature grain. Three mutant lines called Zak ERA8, Zak ERA19A, and Zak ERA19B (Zak ENHANCED RESPONSE to ABA) were recovered based on failure to germinate on 5 μM ABA. All three mutants resulted in increased ABA sensitivity over a wide range of concentrations such that a phenotype can be detected at very low ABA concentrations. Wheat loses sensitivity to ABA inhibition of germination with extended periods of dry after-ripening. All three mutants recovered required more time to after-ripen sufficiently to germinate in the absence of ABA and to lose sensitivity to 5 μM ABA. However, an increase in ABA sensitivity could be detected after as long as 3 years of after-ripening using high ABA concentrations. The Zak ERA8 line showed the strongest phenotype and segregated as a single semi-dominant mutation. This mutation resulted in no obvious decrease in yield and is a good candidate gene for breeding preharvest sprouting tolerance.     

Dietary sterols/steroids and the generalist caterpillar Helicoverpa zea: Physiology,biochemistry and midgut gene expression

Sterols are essential nutrients for insects because, in contrast to mammals, no insect (or arthropod for that matter) can synthesize sterols de novo. Plant-feeding insects typically generate their sterols, commonly cholesterol, by metabolizing phytosterols. However, not all phytosterols are readily converted to cholesterol. In this study we examined, using artificial diets containing single sterols/steroids, how typical (cholesterol and stigmasterol) and atypical (cholestanol and cholestanone) sterols/steroids affect the performance of a generalist caterpillar (Helicoverpa zea). We also performed sterols/steroids analyses, using GC/MS techniques, to explore the metabolic fate of these different dietary sterols/steroids. Finally, we used a microarray approach to measure, and compare, midgut gene expression patterns that arise as a function of dietary sterols/steroids. In general, H. zea performed best on the cholesterol and stigmasterol diets, with cholesterol as the dominant tissue sterol on these two treatments. Compared to the cholesterol and stigmasterol diets, performance was reduced on the cholestanol and cholestanone diets; on these latter treatments stanols were the dominant tissue sterol. Finally, midgut gene expression patterns differed as a function of dietary sterol/steroid; using the cholesterol treatment as a reference, gene expression differences were smallest on stigmasterol, intermediate on cholestanol, and greatest on cholestanone. Inspection of our data revealed two broad insights. First, they identify a number of genes potentially involved in sterol/steroid metabolism and absorption. Second, they provide unique mechanistic insights into how variation in dietary sterol/steroid structure can affect insect herbivores.     

Regulation of glucosamine synthetase activity by cholesterol and hydrocortisone

The effects of cholesterol and hydrocortisone (cortisol) on the activity of purified glucosamine synthetase from rat liver was studied in vitro. It was found that the enzyme activity is increased by cholesterol and inhibited by hydrocortisone. These steroids block the allosteric effect of vitamin K1 on the enzyme. There is evidence testifying to the allosteric type of cholesterol and hydrocortisone effects on glucosamine synthetase.     

Measurements of cholesterol turnover, synthesis, and absorption in man, carried out by isotope kinetic and sterol balance methods

We have estimated the daily synthesis of cholesterol in man by measuring the excretion of cholesterol and its conversion products during periods of controlled sterol intake (sterol balance method), using isotopic or chromatographic procedures (or a combination of the two). Estimates of daily synthesis by this method are based on the premise that the subject is in the metabolic steady state; i.e., the synthesis of cholesterol is equal to the balance (or difference) between the intake of cholesterol and the excretion of cholesterol and its products. To test this premise, we carried out sterol balances in 11 patients; simultaneously, after administration of isotopic cholesterol, turnover was calculated according to previously described models (one-pool, two-pool, or isotopic steady state models for the distribution of radioactive cholesterol within various pools of the body). With calculations based on the one-pool model, turnover rates were considerably higher than estimates based on all other models, and reasons are given for considering these to be overestimates. Good agreement was obtained between results calculated from the two-pool model and those based on sterol balance data; neither method is theoretically preferable to the other. However, with the sterol balance method supplemented by isotopic techniques, valid measurements of cholesterol absorption can be obtained; this in turn permits the essential distinction to be made between daily synthesis and daily turnover of cholesterol when the diet contains cholesterol. In addition, the use of chromatographic isolation procedures provides an accurate measurement of the balance of -sitosterol. This in turn permits valid corrections to be made for losses (which may be large) of neutral steroids during intestinal transit; this is a unique advantage of the chromatographic method.     

Role of albumin for the cholesterol transport and on the steroidogenic pathways in serum-free medium newborn rat adrenocortical cell cultures

[3,4-13C]cholesterol-albumin complex incubated in serum-free medium allowed to evaluate quantitatively the transfer of cholesterol in newborn rat adrenocortical cultured cells, its accumulation as free cholesterol or cholesterol esters and its transformation into steroids which were also originated (47%) from intracellular unlabelled cholesterol. Increasing concentrations of albumin up to 5 g/l enhanced the production of total steroids but in the meantime decreased the 21-hydroxylated steroid fraction. Internalization of albumin shown by using [methyl-14C]methylated-albumin as a tracer accounted only for a minor part in the cholesterol uptake but strikingly affected the steroidogenic pathways by favoring the reductive metabolism of progesterone over the corticosteroid biosynthesis.     

Effects of membrane steroid modification on human erythrocyte glucose transport

The partial removal of cholesterol from the human erythrocyte membrane, by contact with lecithin sols, had mixed effects on the transport of d-glucose. When about 8% of the cholesterol was removed, the rate of d-glucose transfer was increased, but as cholesterol was progressively further removed, the transport was inhibited. Replacement of the depleted cholesterol by 3-ketosteroids did not restore the transport activity; but with substitution of steroids containing only a 3β-hydroxy substituent, the rate of glucose transport returned to normal. In some instances, as little as a 2% replacement of the removed cholesterol by 3β-hydroxy steroids was sufficient for full restoration of d-glucose transport. Cholesterol substitution by steroids with a more planar nucleus and a more bulky side chain than cholesterol also aided in the restoration of glucose transfer. The partial removal of cholesterol had no effect on the apparent K_m for d-glucose, but excessive membrane cholesterol led to a 4-fold decrease in d-glucose affinity. The extent of transport inhibition by a fixed phloretin treatment was independent of membrane steroid content.     

The metabolism of cholesterol in the presence of liver mitochondria from normal and thyroxine-treated rats

1. [26-(14)C]- and [4-(14)C]-Cholesterol were incubated with liver mitochondria from normal and thyroxine-treated rats, and the radioactivity was measured in the carbon dioxide evolved during the incubation, in a butanol extract of the incubation mixture and in a volatile fraction containing substances of low molecular weight derived from the side chain of cholesterol. The butanol extract was separated by paper chromatography into three radioactive fractions, one of which contained the steroids more polar than cholesterol. 2. The butanol extract from incubations with [4-(14)C]cholesterol contained a radioactive substance moving with the same R(F) as cholic acid on thin-layer chromatography. 3. After incubation with [26-(14)C]-cholesterol, 60-80% of the radioactivity extracted by steam-distillation of the incubation mixture at acid pH was recovered as [(14)C]propionic acid. 4. In the presence of [26-(14)C]cholesterol, mitochondria from thyroxine-treated rats produced more radioactivity in carbon dioxide and in the volatile fraction, and less radioactivity in the fraction containing the polar steroids, than did mitochondria from normal rats. In the presence of [4-(14)C]cholesterol, mitochondria from thyroxine-treated rats produced the same amount of radioactivity in the polar steroids as did normal mitochondria. 5. Thyroxine treatment had no effect on the capacity of the mitochondria to oxidize propionate to carbon dioxide. 6. These results are best explained by supposing that thyroxine stimulates a rate-limiting reaction leading to the cleavage of the side chain of cholesterol, but has little or no influence on the hydroxylations of the ring system or on the oxidation of the C(3) fragment removed from the side chain.     

Isolation of Trilobatin,a Sweet Dihydrochalcone-Glucoside from Leaves of Vitis piasezkii Maxim. and V. saccharifera Makino

The effect of melanoidin, prepared with a D-glucose and glycine system, on cholesterol metabolism in rats was examined. Male rats of the Wistar strain weighing ca. 140 g were fed a high-cholesterol diet supplemented with nondialyzable melanoidin for three weeks. The dietary melanoidin suppressed elevation of the cholesterol level in both plasma and liver, while the cholesterol levels in feces and the contents of the caecum decreased unexpectedly. Neutral steroids other than cholesterol also decreased. However, chromatograhy of fecal steroids indicated that melanoidin markedly affected the intestinal metabolism of neutral steroids, including cholesterol. On the other hand, the fecal recovery of radioactivity from orally ingested 26 [27]-¹⁴C cholesterol was promoted by the added melanoidin, although the radioactive species were not identified. This suggested that the cholesterol level decreased due to the interference of melanoidin in the intestinal absorption of cholesterol.     

Inhibition of cholesterol biosynthesis by fluorinated mevalonate analogues

The conversion of mevalonate to cholesterol in rat liver homogenates (IC50 = 0.01-1.0 mM) is inhibited by 6- (I), 6,6-di- (II), and 6,6,6-trifluoromevalonate (III), as well as 4,4-difluoromevalonate (IV). Addition of compound I, III, or IV to rat liver homogenates results in the accumulation of 5-phospho- and 5-pyrophosphomevalonate. The conversion of isopentenyl pyrophosphate to cholesterol is not inhibited by the fluorinated analogues. It thus appears likely that the decarboxylation of mevalonate 5-pyrophosphate is inhibited. Rat liver homogenates catalyze the phosphorylation of I and III. The inhibition of the decarboxylation of mevalonate 5-pyrophosphate by I and III was demonstrated directly with partially purified decarboxylase. Compound I is a remarkably effective inhibitor of the decarboxylation (Ki = 10 nM). Similar results were reported by Nave et al. [Nave, J. F., d'Orchymont, H., Ducep, J. B., Piriou F., & Jung, M. J. (1985) Biochem. J. 227, 247]. It is likely that the phosphorylated or pyrophosphorylated forms of all inhibitors tested are responsible for inhibition. We also describe a chemical method for the synthesis of mevalonate 5-pyrophosphate.