Measurement of rates of cholesterol synthesis using tritiated water

Rates of sterol synthesis in various tissues commonly are assessed by assaying levels of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase on isolated microsomes or by measuring the rates of incorporation of various 14C-labeled substrates or [3H]water into cholesterol by whole cell preparations in vitro or by the tissues of the whole animal in vivo. While measurement of activities of HMG-CoA reductase or rates of incorporation of 14C-labeled substrates into cholesterol give useful relative rates of sterol production, neither method yields absolute rates of cholesterol synthesis. The use of [3H]water circumvents the problem of variable and unknown dilution of the specific activity of the precursor pool encountered when 14C-labeled substrates are used and does yield absolute rates of cholesterol synthesis provided that the 3H/C incorporation ratio is known for a particular tissue. In 12 different experimental situations it has been found that from 21 to 27 micrograms atoms of 3H are incorporated into cholesterol from [3H]water in different tissues of several animal species, so that the 3H/C incorporation ratio is similar under nearly all experimental conditions and varies from 0.78 to 1.00. When administered in vivo, [3H]water rapidly equilibrates with intracellular water and is incorporated into sterols within the various organs at rates that are linear with respect to time. From such data it is possible to obtain absolute rates of cholesterol synthesis in the whole animal and in the various organs of the animal. Current data suggest, therefore, that use of [3H]water yields the most accurate rates of cholesterol synthesis both in vitro and in vivo.     

Cholesterol synthesis and esterification in cultured intestinal mucosa. Evidence for compartmentation

The current studies were undertaken to define the optimal conditions for measuring the absolute rates of cholesterol synthesis in cultured rabbit intestine and to assess whether the rate of sterol synthesis affects the esterification of locally formed or absorbed cholesterol. Using both [3H]water or [14C]octanoate (3 mM) as a precursor, sterol formation was linear during the 24 h culture, resulting in comparable estimates of the rate of synthesis equivalent to 129.5 and 118.7 nmol acetyl CoA incorporated per g per h, respectively. The presence of liposomal cholesterol or the hydroxymethylglutaryl-CoA reductase inhibitor mevinolin suppressed the rates of cholesterol synthesis by 24 and 92% of controls, respectively. Only 12% of total newly synthesized cholesterol was recovered in the medium and more than 97% was in the unesterified form, in both medium and biopsy. Even when the rate of sterol synthesis was stimulated over 90-fold by increasing concentrations of [14C]mevalonolactone, less than 8% of the label in total cholesterol was found in the sterol nucleus of the esterified cholesterol. Rather, the majority of the cholesterol ester-bound radioactivity was incorporated into the fatty acid moiety. On the other hand, there was only a limited decrease in the esterification of absorbed [3H]cholesterol both when the rate of sterol synthesis was increased with 10 mM mevalonolactone and when it was inhibited with mevinolin. The data suggest that locally synthesized and absorbed cholesterol is organized in distinct functional pools with different degrees of esterification in the mucosal epithelial cell.     

Critical analysis of the use of 14C-acetate for measuring in vivo rat cholesterol synthesis

The bulk of cholesterol produced by the liver and the gut enters the mobile pool of body cholesterol. This process is called internal secretion in contrast with the fraction of biosynthesized cholesterol directly eliminated in the feces (fecal external secretion). In rats, under various conditions, a linear relationship was found between the rates of internal secretion measured by the isotope equilibrium method (range: 10-60 mg/day) and the sum of sterol radioactivities measured in liver and intestine 70 min after a [14C]-acetate pulse. In fact, a better correlation was found between the radioactivities of liver sterols and the values for internal secretion. In this new relationship, the ordinate at the origin corresponds to a minimal internal secretion of about 10 mg/day, which implies an important extrahepatic cholesterol production, probably from the gut. Indeed, in adult male rats, fed a semi-purified sucrose-rich diet, the relative contribution of this organ to the internal secretion was higher than in adult rats fed a commercial diet and higher than in young animals, whatever the circadian period. It can be concluded that some of the discrepancies observed in the literature about the relative participation of the intestine and the liver in the internal secretion of cholesterol are probably due to differences in experimental and nutritional conditions (age and sex of the animals, diet composition, time of the circadian cycle) rather than to the cholesterol precursor used (3H2O or [14C] acetate) to assess the activity of cholesterol synthesis. Indeed, a comparative study of 3H2O and [14C]acetate incorporation into sterols of enterocytes indicated the same crypt-villus radioactive gradient, regardless of the intestinal site studied (duodenum, jejunum or ileum) and thus validated the use of [14C]acetate. Other experiments however, showed evidence of some local differences in the cytosolic dilution of labeled acetyl CoA by the endogenous cholesterol precursor in rats under various conditions (control or cholestyramine-enriched diet, parenteral nutrition). After intravenous infusion of 1,2[13C]acetate, mass fragmentography of free cholesterol isolated from liver and intestine indicated different 13C-labeling patterns of newly synthesized molecules. They indicate that cholesterol is generally synthesized from acetyl CoA with a lower 13C-content in the liver than in the intestine. The local endogenous flow of acetyl CoA used for cholesterol synthesis was about 2-fold higher in the hepatocytes than in the enterocytes. This conclusion was confirmed by the results obtained with several experimental groups exhibiting a large range of both internal secretion of cholesterol and sterol radioactivities in liver and intestine after [14C]acetate injection.(ABSTRACT TRUNCATED AT 400 WORDS)     

14C-labeled substrate catabolism by human diploid fibroblasts derived from infants and adults

Untransformed diploid skin fibroblasts from eight normal adults, aged 24 to 74 years, catabolized several 14C-labeled substrates less effectively than cells from ten normal male infants. 14C-labeled substrate metabolism was quantitated either by measuring the evolution of 14CO2 from the 14C-labeled compounds or the incorporation of 14C into cellular protein via transamination of tricarboxylic acid cycle intermediates derived from the 14C-labeled substrates. With these methods, adult cells catabolized [1-14C]butyrate, [1-14C]octanoate, and 1-[2-14C]leucine at rates 44 to 64% of those found in infant cells. The oxidation of [1,4-14C]succinate and [U-14C]malate was identical in both infant and adult cells, while [2,3-14C]succinate catabolism was mildly decreased in adult cells (65-80% of control). These observations parallel those made in rat tissues and confirm that the same phenomenon occurs in cultured human fibroblasts.     

Acetoacetate is a cholesterogenic precursor for myelinating rat brain and spinal cord. Incorporation of label from [3-14C]acetoacetate, [14C]glucose and 3H2O

Rat pups, 3 weeks old, were injected i.p. with combinations of 3H2O and either [3-14C]acetoacetate or [14C]glucose. 3H/14C incorporation ratios were measured in lipid fractions of homogenates and myelin prepared from whole brain and spinal cord. Spinal cord synthesized at least twice as much fatty acids and 3-fold more sterols than whole brain. Both tissues used acetoacetate preferentially for sterol synthesis, whereas label from [14C]glucose was distributed between fatty acids and sterols in the same way as 3H from 3H2O. The relative contributions of acetoacetate to sterol synthesis in whole tissue and in the purified myelin fraction were about the same, both for the cerebrum and for the spinal cord.     

Cholesterol biosynthesis by the cornea. Comparison of rates of sterol synthesis with accumulation during early development

The origin of the cholesterol needed by the cornea for growth and cell turnover was addressed by comparing absolute rates of sterol synthesis with rates of sterol accumulation during early development of the rabbit. Linearity of incorporation of 3H2O and [14C]mevalonate into digitonin-precipitable sterols with time of incubation in vitro and a lack of accumulation of 14C in intermediates of sterol biosynthesis indicated that tritiated water can validly be used to measure rates of sterol synthesis by the cornea. The rate of sterol synthesis per unit weight of rabbit cornea was constant between 14 and 60 days of age at an average 1.03 nmol of 3H of 3H2O incorporated/mg dry cornea per 8 h. Essentially all of the synthesized cholesterol and most of the cholesterol mass was present in corneal epithelium. The cumulative sterol synthesized over the 46-day period studied exceeded the observed rate of cholesterol accumulation by sixfold. Cholesterol synthesized in excess of the growth requirement was likely used to support turnover of the epithelium which was estimated at 9 days. Removal of cholesterol from the cornea by excretion into tear fluid and clearance by high density lipoproteins are also considered.     

DEVELOPMENT OF LIPOGENESIS IN RAT BRAIN CORTEX: THE DIFFERENTIAL INCORPORATION OF GLUCOSE AND ACETATE INTO BRAIN LIPIDS IN VITRO

—The oxidation to CO₂ and the incorporation of [U-¹⁴C]glucose and [U-¹⁴C]acetate into lipids by cortex slices from rat brain during the postnatal period were investigated. The oxidation of [U-¹⁴C]glucose was low in 2-day-old rat brain, and increased by about two-fold during the 2nd and 3rd postnatal weeks. The oxidation of [U-¹⁴C]acetate was increased markedly in the second postnatal week, but decreased to rates observed in 2-day-old rat brain at the time of weaning. Both labeled substrates were readily incorporated into non-saponifiable lipids and fatty acids by brain slices from 2-day-old rat. Their rates of incorporation and the days on which maximum rates occurred were different, however, maximum incorporation of [U-¹⁴C]glucose and [U-¹⁴]acetate into lipid fractions being observed on about the 7th and 12th postanatal days, respectively. The metabolic compartmentation in the utilization of these substrates for lipogenesis is suggested. The activities of glucose-6-phosphate dehydrogenase, cytosolic NADP-malate dehydrogenase, cytosolic NADP-isocitrate dehydrogenase, ATP-citrate lyase and acetyl CoA carboxylase were measured in rat brain during the postnatal period. All enzymes followed somewhat different courses of development; the activity of acetyl CoA carboxylase was, however, the lowest among other key enzymes in the biosynthetic pathway, and its developmental pattern paralleled closely the fatty acid synthesis from [U-¹⁴C]glucose. It is suggested that acetyl CoA carboxylase is a rate-limiting step in the synthesis de novo of fatty acids in developing rat brain.     

Enzymatic synthesis of 1,3,6,8-tetrahydroxynaphthalene solely from malonyl coenzyme A by a fungal iterative type I polyketide synthase PKS1

The Colletotrichum lagenarium PKS1 gene encoding iterative type I polyketide synthase of 1,3,6,8-tetrahydroxynaphthalene (T4HN) was overexpressed in Aspergillus oryzae. SDS-PAGE analysis of the cell-free extract prepared from the transformant showed an intense band of 230000 which corresponded to the molecular weight of the deduced PKS1 protein. By using this cell-free extract, in vitro synthesis of T4HN was successfully confirmed as the first example of the fungal multi-aromatic ring polyketide synthase activity ever detected. To identify the starter unit for T4HN synthesis, (14)C-labeled acetyl CoA and/or (14)C-labeled malonyl CoA were used as substrates for T4HN synthase reaction. Observed was the incorporation of (14)C label into T4HN solely from malonyl CoA even in the absence of acetyl CoA and not from acetyl CoA. This in vitro result unambiguously identified that malonyl CoA serves as the starter as well as extender units in the formation of T4HN by fungal polyketide synthase PKS1.     

Sterol biosynthesis in the echinoderm Asterias rubens.

1. [2(-14)C]Mevalonic acid injected into the echinoderm Asterias rubens (Class Asteroidea) was effectively incorporated into the non-saponifiable lipid. 2. The most extensively labelled compounds were squalene and the 4,4-dimethyl sterols with much lower incorporations into the 4alpha-monomethyl and 4-demethyl sterol fractions. 3. Labelled compounds identified were squalene, lanosterol, 4,4-dimethyl-5alpha-cholesta-8,24-dien-3beta-ol and 4alpha-methyl-5alpha-cholest-7-en-3beta-ol; these are all intermediates in sterol biosynthesis. 4. The major sterol in A. rubens, 5alpha-cholest-7-en-3beta-ol, was also labelled showing that this echinoderm is capable of sterol biosynthesis de novo. 5. No evidence was obtained for the incorporation of [2(-14)C]mevalonic acid into the C28 and C29 components of the 4-demethyl sterols or 9beta,19-cyclopropane sterols found in A. rubens and it is assumed that these sterols are of dietary origin. 6. Another starfish Henricia sanguinolenta also incorporated [2(-14)C]mevalonic acid into squalene and lanosterol. 7. Various isolated tissues of A. rubens were all capable of incorporation of [2(-14)C]mevalonic acid into the nonsaponifiable lipid. With the body-wall and stomach tissues radioactivity accumulated in squalene and the 4,4-dimethyl sterols, but with the gonads and pyloric caecae there was a more efficient incorporation of radioactivity into the 4-demethyl sterols, principally 5alpha-cholest-7-en-3beta-ol.     

The effect of hydrocortisone on cholesterol metabolism of cultured human skin fibroblasts

Hydrocortisone in physiologic concentrations resulted in a reduction in sterol synthesis by cultured normal human skin fibroblasts. These changes were observed when [14C]acetate, [14C]octanoic acid and 3H2O were used as precursors. However, the incorporation of [3H]mevalonic acid lactone into digitonin-precipitable sterols was not affected by hydrocortisone, suggesting that hydrocortisone inhibits sterol synthesis at a site prior to the formation of mevalonic acid. In contrast, the activity of hydroxymethylglutaryl-CoA reductase was stimulated several-fold by the hormone. Thus, the inhibitory effect of hydrocortisone on the cholesterol synthetic pathway may be on hydroxymethylglutaryl-CoA synthase.     

Sterol and sesquiterpenoid biosynthesis during a growth cycle of tobacco cell suspension cultures

The accumulation and biosynthesis of sterols and fungal elicitor-inducible sesquiterpenoids by tobacco (Nicotiana tabacum) cell suspension cultures were examined as a function of a 10 day culture cycle. Sterols accumulated concomitantly with fresh weight gain. The rate of sterol biosynthesis, measured as the incorporation rate of [¹⁴C]acetate and [³H]mevalonate, was maximal when the cultures entered into their rapid phase of growth. Changes in squalene synthetase enzyme activity correlated more closely with thein vivo synthesis rate and accumulation of sterols than 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) enzyme activity. Cell cultures entering into the rapid phase of growth also responded maximally to fungal elicitor as measured by the production of capsidiol, an extracellular sesquiterpenoid. However, the rate of sesquiterpenoid biosynthesis, measured as the incorporation rate of [¹⁴C]acetate and [³H]mevalonate, could not be correlated with elicitor-inducible HMGR or sesquiterpene cyclase enzyme activities, nor elicitor-suppressible squalene synthetase enzyme activity.Abbreviations FPP farnesyl diphosphate - HMGR 3-hydroxy-3-methylglutaryl coenzyme A reductase     

Changes in sterol metabolism in the skin of developing chick embryo and alterations in the presence of an anticholesterolemic agent and a chemical carcinogen

Changes in sterol metabolism in the skin of chick embryo during its development were studied with embryonal chick skin and with the cultured skin tissues. Changes in sterol metabolism of the skin of chick embryo began to appear at day 17, as observed by the accumulation of dihydrolanosterol, and the ratio of dihydrolanostrol:cholesterol increased thereafter until hatching. A similar change in sterol metabolism was also observed with the cultured skin tissue of chick embryo, although the stages of development seem to have been delayed by 3 days. The active sterol metabolism of the cultured skin tissue was also confirmed by studies of incorporation of [2-14C]acetate into sterols. 20,25-Diazacholesterol almost completely inhibited the incorporation of [2-14C]acetate into C27 sterols, whereas a chemical carcinogen, 4-hydroxyaminoquinoline 1-oxide, inhibited the incorporation of [2-14C]acetate into lathosterol but not that into cholesterol.     

Effects of osmotic pressure on the oxidative metabolism of Leishmania major promastigotes.

Leishmania major promastigotes were washed and resuspended in an iso-osmotic buffer. The rate of oxidation of 14C-labeled substrates was then measured as a function of osmolality. An acute decrease in osmolality (achieved by adding H2O to the cell suspension) caused an increase in the rates of 14CO2 production from [6-14C]glucose and, to a lesser extent, from [1,(3)-14C]glycerol. An acute increase in osmolality (achieved by adding NaCl, KCl, or mannitol) strongly inhibited the rates of 14CO2 production from [1-14C]alanine,[1-14C]glutamate, and [1,(3)-14C]glycerol. The rates of 14CO2 formation from [1-14C]laurate,[1-14C]acetate, and [2-14C]glucose (all of which form [1-14C]acetyl CoA prior to oxidation) were also inhibited, but less strongly, by increasing osmolality. These data suggest that with increasing osmolality there is an inhibition of mitochondrial oxidative capacity, which could facilitate the increase in alanine pool size that occurs in response to hyper-osmotic stress. Similarly, an increase in oxidative capacity would help prevent a rebuild up of the alanine pool after its rapid loss to the medium in response to hypo-osmotic stress.     

An accurate and sensitive assay of [14C]octanoate oxidation and its application on tissue homogenates and fibroblasts

A procedure was developed to assay [14C]octanoate oxidation from the production of both 14CO2 and 14C-labeled acid-soluble products. Octanoic acid and its CoA and carnitine esters were eliminated from the acid-soluble products by alkaline hydrolysis of the esters and acidification and binding of the acid to Lipidex 1000. The method was evaluated with homogenates of various rat tissues and human muscles and with human fibroblasts. 14CO2 production was variable and comprised less than 3% of the total oxidation products with homogenates and 26 +/- 19% with fibroblasts. As compared to palmitate, oxidation rates of octanoate were higher in rat liver and heart homogenates, of the same magnitude in muscle homogenates, but lower in fibroblasts. The proportion of antimycin-insensitive oxidation was much lower with octanoate than with palmitate. Using the assay a case of medium-chain acyl-CoA dehydrogenase deficiency could be indicated.     

Effect of alterations of the specific activity of the intracellular acetyl CoA pool on apparent rates of hepatic cholesterogenesis

We have previously shown significant dilution of the specific activity of the intracellular acetyl CoA pool when radiolabeled acetate is used as the precursor in liver slice experiments. In the present study, using liver from animals subjected to various manipulations known to alter the rate of cholesterogenesis, the specific activity of the intramitochondrial acetyl CoA pool was 27-49% of the theoretical specific activity expected if no endogenous dilution occurred. Because the cytosolic acetyl CoA pool that gives rise to cholesterol is not in equilibrium with the intramitochondrial pool, these values cannot be used to correct the flux of labeled carbon from [(14)C]acetate into cholesterol. However, because [(14)C]octanoate is rapidly oxidized intramitochondrially to acetyl CoA, which feeds both the intra- and extramitochondrial metabolic pathways, [(14)C]octanoate can be utilized to determine true flux rates of C(2) units into cholesterol and other products. Using this substrate in liver slices from animals subjected to a variety of experimental manipulations, the specific activity of the intracellular acetyl CoA pool was 54-71% of the expected specific activity. After correction for endogenous dilution, the C(2) flux into cholesterol varied from 335 to 459 nmoles.g(-1).hr(-1) in control animals, was suppressed 10-40-fold in animals subjected to fasting and cholesterol feeding, and increased into the range of 1500 nmoles.g(-1).hr(-1) after derepression with cholestyramine feeding or biliary diversion. Data also are presented that show very good agreement between the corrected C(2) flux rate from octanoate into cholesterol and microsomal HMG CoA reductase activity in the same liver under conditions in which the synthetic rates were varied over a 100-fold range.     

Absolute rates of sterol synthesis estimated from [3H]water for bovine lens epithelial cells in culture

All cells of the avascular ocular lens derive from a monolayer of epithelial cells located on only the anterior surface of this organ. The source of the cholesterol required for the growth and division of these cells was studied by using cultures of bovine lens epithelial cells. Cells were in active growth during the third to fourth day of subculture following seeding. Absolute rates of cholesterol synthesis were estimated for the cultured cells from incorporation of [3H]water. Rates were estimated on the assumption that 0.81 atoms of 3H of [3H]water were incorporated into cholesterol per carbon atom of cholesterol, a situation where all of the NADPH would be generated by oxidative enzymatic processes. We tested this assumption by measuring the changes in sterol mass per dish of cells grown in lipoprotein-deficient media over day 3 to 4 of subculture and by simultaneously measuring the rates of incorporation of [3H]water into sterols during this period. In this situation, the increases in sterol mass should be attributable solely to de novo sterol synthesis. We calculated that an average of 0.79 atoms of 3H of [3H]water were incorporated by these cells into cholesterol per carbon atom of cholesterol. Sterol synthesis was only modestly decreased (about 30%) when the cells were cultured in media prepared with whole calf serum. Growth rates of the cells were also little affected by the absence of lipoproteins. In spite of the capacity to furnish its sterol requirements by de novo synthesis, the lens epithelial cells readily degraded 125I-labeled bovine LDL, and LDL greatly decreased sterol synthesis when added to the media at low levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of sterol but not fatty acid synthesis by valproate in developing rat brain in vivo.

The effect of administration of valproate on lipogenesis in the developing rat brain in vivo was studied. Valproate inhibited by 21-38% the rate of 3H2O incorporation into brain sterols, without significantly affecting fatty acid synthesis. Similarly, R-[2-14C]mevalonate incorporation into sterols was inhibited by 33-54%; the low rate of fatty acid synthesis under these conditions was not affected by valproate. Plasma ketone bodies decreased after treatment with valproate. Valproate inhibited (about 50%) both sterol and fatty acid synthesis in livers of weanling rats. It is concluded that valproate can specifically inhibit sterol synthesis in the brain during development, in part at a stage after mevalonate formation, and also by decreased exogenous precursor supply.     

Hepatic and nonhepatic sterol synthesis and tissue distribution following administration of a liver selective HMG-CoA reductase inhibitor, CI-981: comparison with selected HMG-CoA reductase inhibitors.

Since cholesterol biosynthesis is an integral part of cellular metabolism, several HMG-CoA reductase inhibitors were systematically analyzed in in vitro, ex vivo and in vivo sterol synthesis assays using [14C]acetate incorporation into digitonin precipitable sterols as a marker of cholesterol synthesis. Tissue distribution of radiolabeled CI-981 and lovastatin was also performed. In vitro, CI-981 and PD134967-15 were equipotent in liver, spleen, testis and adrenal, lovastatin was more potent in extrahepatic tissues than liver and BMY21950, pravastatin and PD135023-15 were more potent in liver than peripheral tissues. In ex vivo assays, all inhibitors except lovastatin preferentially inhibited liver sterol synthesis; however, pravastatin and BMY22089 were strikingly less potent in the liver. CI-981 inhibited sterol synthesis in vivo in the liver, spleen and adrenal while not affecting the testis, kidney, muscle and brain. Lovastatin inhibited sterol synthesis to a greater extent than CI-981 in the spleen, adrenal and kidney while pravastatin and BMY22089 primarily affected liver and kidney. The tissue distribution of radiolabeled CI-981 and lovastatin support the changes observed in tissue sterol synthesis. Thus, we conclude that a spectrum of liver selective HMG-CoA reductase inhibitors exist and that categorizing agents as liver selective is highly dependent upon method of analysis.     

Regulation of sterol biosynthesis in etiolated mung bean hypocotyl sections

The incorporation of radioactivity into sterols by transmethylation of methionine-[¹⁴C-methyl] was studied in mung bean hypocotyl sections. Young hypocotyl sections (1 cm) synthesized 4 times more radioactive sterols than older sections (5 cm). The transmethylation reactions may be rate limiting in older tissues. Wounding has only a quantitative effect on sterol biosynthesis, as seen by incorporation experiments with MVA-[2-¹⁴C]. Naphthalene acetic acid (NAA) stimulates sterol biosynthesis in both wounded surfaces and intact tissues of mung bean hypocotyl sections.     

Whole body and hepatic cholesterol synthesis rates in the guinea-pig: effect of dietary fat quality

The effects of polyunsaturated, monounsaturated and saturated dietary fat on total and hepatic cholesterol synthesis were studied in the guinea-pig. Male Hartley guinea-pigs were fed semi-synthetic diets containing 7.5% (w/w) of either corn oil (CO), olive oil (OL) or lard for a period of 5 weeks and rates of endogenous cholesterol synthesis were determined from the incorporation of [3H]water into digitonin-precipitable sterols (DPS) and by measurement of sterol balance. In addition, total and expressed 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activities were determined in hepatic microsomes. Rates of whole body cholesterol synthesis determined by incorporation of [3H]water into DPS were significantly lower for guinea-pigs on the CO diet with values of 18.7 +/- 1.8 mumol/h (n = 4) vs. 26.7 +/- 4.8 and 24.6 +/- 1.8 mumol/h for animals on the OL (n = 4) and lard (n = 3) diets (P less than 0.001), respectively. Hepatic cholesterol synthesis rates were significantly decreased in animals on the OL diet, whether determined from incorporation of [3H]water into DPS or by analysis of HMG-CoA reductase activity. Hepatic total and free cholesterol levels were not different for animals on the three dietary fats; however, cholesteryl ester levels were 35% lower in guinea-pigs fed the lard diet (P less than 0.02). Sterol balance measurements indicated that whole body cholesterol synthesis rates were not affected by dietary fat quality (51.9 +/- 12.2, 42.8 +/- 7.6 and 51.2 +/- 20.2 mg/kg per day for animals on the CO, OL and lard diets, respectively). This is in striking contrast to the observed reduction in cholesterol synthesis rates for animals on the polyunsaturated CO diet as determined by incorporation of [3H]water into DPS. One possible explanation for the discrepancy between the sterol balance and [3H]water incorporation data is a polyunsaturated fat-mediated effect on energy utilization, which affects the equilibration of NADPH with the body water pool such that the [3H]NADPH has a lower specific activity than body [3H]water.