Developmental changes in ketogenic enzyme gene expression during sheep rumen development.

Ketogenesis is the conversion of acetyl-CoA to the ketone bodies acetoacetate and beta-hydroxybutyrate (BHBA). In hepatic ketogenesis, which occurs during fasting in both nonruminant and ruminant animals, the source of acetyl-CoA is the mitochondrial oxidation of predominantly long-chain fatty acids. In the mature, fed ruminant animal, the ruminal epithelium is also capable of producing ketone bodies. In this case, the source of acetyl-CoA is the mitochondrial oxidation of butyrate produced by the microbial fermentation of feed. The purposes of this study were to determine ontogenic and dietary effects on ketogenic enzyme gene expression in developing lamb ruminal epithelium. Twenty-seven conventionally reared lambs and twenty-seven milk-fed lambs were slaughtered between 1 and 84 d of age. Six additional milk-fed lambs were weaned (the fed group) or maintained on milk replacer with a volatile fatty acid gavage (the VFA group) until 84 d of age. At slaughter, total RNA was extracted from samples of ruminal epithelium. The expression of the genes encoding acetoacetyl-CoA thiolase, the first enzyme in the ketogenic pathway, and 3-hydroxy-3-methylglutaryl-CoA synthase, the rate-limiting enzyme in the ketogenic pathway in nonruminant liver, were examined. Acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl-CoA synthase mRNA concentrations increased with age independent of diet. 3-Hydroxy-3-methylglutaryl-CoA synthase mRNA levels in ruminal epithelium obtained from milk-fed lambs were low before 42 d of age, but a marked increase occurred by 42 d of age. At 84 d of age, there were no differences in acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl-CoA synthase expression due to diet. The pattern of the expression of these genes, in particular, 3-hydroxy-3-methylglutaryl-CoA synthase, parallels the rate of production of BHBA by rumen epithelial cells isolated from the same lambs, which increased to conventionally reared adult levels at 42 d of age and did not differ with diet. In conclusion, development of the ketogenic capacity of the ruminal epithelium occurs as the animal ages, regardless of dietary treatment. Thus, the expression of the genes encoding the ketogenic enzymes are not affected by the presence of VFA in the ruminal lumen.

Sheep rumen metabolic development in response to age and dietary treatments.

This study examined the time course of rumen metabolic development in the absence of solid feed consumption and the effect of delayed solid feed consumption on sheep rumen development. Twenty-seven lambs consumed milk replacer until slaughter at nine ages from 1 to 84 d (milk group). Three additional lambs consumed milk replacer from 1 to 48 d. From 49 d until slaughter at 84 d, these lambs were weaned onto solid feed (fed group). At slaughter, rumen contents were removed for VFA analysis and rumen epithelium was preserved for morphological examination. Rumen epithelial cells were isolated and incubated in media containing 2.5 mM U-[14C]-glucose or 10 mM 1-[14C]-butyrate. Rumen VFA concentrations did not change with age in lambs given milk replacer. At 84 d of age, intraruminal VFA concentrations were elevated in lambs consuming solid feed compared to 84-d-old lambs given milk replacer (P < .05). The number of ruminal papillae per square centimeter decreased (P < .05) while papillae length and width did not change significantly with age in rumen epithelium from lambs given milk replacer. At 84 d of age, rumen epithelium from lambs in the fed group had fewer and larger papillae/per square centimeter than rumen epithelium from lambs given milk replacer (P < .05). Rates of glucose and butyrate oxidation and acetoacetate and lactate production by rumen cells isolated from lambs given milk replacer did not change with age. Beta-hydroxybutyrate (BHBA) production was undetectable before 42 d of age in lambs given milk replacer and increased to levels found in conventionally raised adults by 84 d. At 84 d there were no differences in rates of glucose and butyrate oxidation or acetoacetate and lactate production by rumen cells between the two treatment groups. Thus, the change in substrate oxidation from glucose to butyrate, indicative of rumen metabolic maturation, does not occur in the absence of solid feed consumption. However, the development of rumen ketogenesis, as evidenced by increased BHBA production, does occur in the absence of solid feed consumption. Delaying the initiation of solid feed consumption results in rumen morphological development but does not stimulate rumen metabolic development. Increased intraruminal VFA concentrations, earlier exposure to VFA, or a longer period of exposure to VFA may be required to induce the genes responsible for rumen metabolic development.

Effect of volatile fatty acid infusion on development of the rumen epithelium in neonatal sheep.

The purpose of this study was to determine whether the continuous intraruminal infusion of calculated physiological concentrations of volatile fatty acids (VFA) stimulated the metabolic development of the neonatal rumen. Eight 1-wk-old lambs were assigned to one of three treatments: saline infusion (three lambs), VFA infusion (three lambs), or no infusion (two lambs). Rumen catheters were surgically implanted into lambs in the infusion groups. The amount of VFA infused, beginning at 2 wk of age, increased weekly in equal increments of 12.5% of the estimated net energy requirement until, at 6 wk of age, lambs received 50% of their estimated net energy requirement from the infused VFA. All lambs consumed milk replacer for ad libitum intake and had free access to water. The lambs that were infused with VFA tended to have longer rumen papillae. There were no differences in width or number of papillae per square centimeter across treatments. Rumen epithelial cells isolated from lambs that were infused with VFA tended to oxidize less glucose and produce more acetoacetate than did cells from lambs that were infused with saline or from uninfused lambs. beta-Hydroxybutyrate production by isolated rumen epithelial cells and concentrations of blood glucose, acetoacetate, and beta-hydroxybutyrate were not different among the three treatments. Thus, infusion of physiological concentrations of VFA appears to stimulate some aspects of rumen metabolic development.

Propionate modulation of ruminal ketogenesis.

The ruminal epithelium is the primary source, through the metabolism of butyrate, of circulating ketone bodies in fed ruminants. Volatile fatty acid metabolism was investigated in short-term (2-h) incubations of isolated sheep ruminal epithelial cells. Ruminal epithelial cells were isolated from ruminal papillae via serial tryptic digestion. Cells were incubated in the presence of various combinations of butyrate (0, .5, 1, 5, 10, or 25 mM), propionate (0, .1, .5, 1, 5, 10, 25, 50, or 100 mM), acetate (0, .5, 1, 5, 25, 50, or 100 mM), and succinate (0, 5, 15, 50 mM) to evaluate the effects of these substrates on butyrate metabolism. Variables measured included beta-hydroxybutyrate (beta-HBA) and acetoacetate (AcAc) production from butyrate, [14C]butyrate oxidation to 14CO2, and lactate and pyruvate formation from propionate and succinate. Butyrate oxidation to CO2, beta-HBA and total ketones produced (nanomoles of butyrate metabolized per 10(6) cells per minute) were linear over the 2-h incubation period (y = .805X + 2.24, r2 = .78; y = 1.02X - 9.1, r2 = .72; and y = 2.238X - 11.76, r2 = .65, respectively). Acetate inhibited beta-HBA formation from butyrate (P < .05) when present at concentrations greater than 5 mM, although a dose-dependence was not consistently exhibited. However, butyrate oxidation to beta-HBA was stimulated (P < .05) by propionate while acetoacetate production declined (P < .05), resulting in no net change in ketone body production (P > .05). A concomitant increase in lactate and pyruvate production was noted with increasing concentrations of propionate. Succinate addition also increased lactate production by the ruminal epithelial cells. In contrast to propionate, succinate addition resulted in a decrease in beta-HBA formation from butyrate (P < .05). Butyrate metabolism by isolated ruminal epithelial cells is influenced by other VFA produced in the rumen, and propionate-induced stimulation of beta-HBA from butyrate does not seem to be mediated via succinate but rather is a result of a shift in the mitochondrial NADH/NAD status.

Identification of two cDNA clones encoding small proline-rich proteins expressed in sheep ruminal epithelium.

Small proline-rich (SPRR) proteins are markers frequently associated with squamous cell differentiation. They have been proposed to be a novel group of precursor polypeptides for the cornified envelope in epidermal keratinocytes. A plus/minus screening procedure was used to identify cDNA clones expressed in mature but not in neonatal sheep ruminal epithelium. Two clones encoding SPRR proteins were identified and are reported here. Clone 27 encodes an ovine SPRR protein corresponding to the human type-II SPRR protein. Clone 26 encodes an ovine SPRR protein similar to human type-II SPRR protein, but which also contains an N-terminal His-Pro repeat similar to the paired repeats found in the Drosophila paired proteins. The unique combination of a paired domain and an SPRR protein has not been reported prior to this study. The tissue distribution indicates that specific expression of the genes corresponding to these two clones occurs in the epithelium of the ruminant forestomach, and to a lesser extent in skin epithelium. In situ hybridization demonstrated that the SPRR mRNA for both clones were localized in the stratum granulosum, in support of their putative physiological function, i.e. formation of the cornified envelope. Based on Northern blot analysis, mRNA complementary to the two clones appears in the ruminal epithelium by 1 week of age, corresponding to the formation of the stratum granulosum during ruminal epithelial development. The different patterns of changes in amount of mRNA corresponding to these clones during rumen epithelial development indicate that they play different roles in rumen epithelial development.

Isolation and characterization of a cDNA clone encoding ovine type I carbonic anhydrase.

To identify genes involved in the postnatal development of sheep ruminal epithelium, a lambda gt22a cDNA library was constructed from poly(A)+ RNA isolated from mature sheep ruminal epithelium. A plus/minus screening procedure was used to identify genes expressed in mature but not in neonatal ruminal epithelium. One of the cDNA clones isolated encodes an ovine carbonic anhydrase, based on nucleotide and deduced peptide sequence analysis. The deduced peptide is most closely related to eukaryotic type I carbonic anhydrase, based on comparison with all available carbonic anhydrase sequences. Northern blot hybridization confirmed that the amount of mRNA complementary to the carbonic anhydrase cDNA clone is more than five times higher in ruminal epithelium for mature sheep (12 wk old) than in ruminal epithelium from neonatal lambs (< 12h old). Messenger RNA complementary to this cDNA clone was found only in the epithelium of the ruminant forestomach compartments (i.e., rumen, reticulum, and omasum), but small amounts of hybridizable mRNA were also found in sheep skin. This carbonic anhydrase cDNA clone will allow the study of transcriptional regulation of the carbonic anhydrase gene during ruminal epithelial development.

Interactions between gluconeogenesis and fatty acid oxidation in isolated sheep hepatocytes.

The interaction of gluconeogenesis and fatty acid oxidation in isolated sheep hepatocytes was studied. Addition of tetradecylglycidic acid, an inhibitor of carnitine palmitoyltransferase I (EC 2.3.1.21), to isolated hepatocytes inhibited gluconeogenesis from a mixture of pyruvate plus lactate and from propionate alone. Inhibition constants for tetradecylglycidic acid on gluconeogenesis were 4.77 +/- 1.00 microM and 7.25 +/- 1.52 microM, respectively, for pyruvate plus lactate and for propionate as gluconeogenic substrates. The inhibition constants were not different. At the highest substrate concentrations examined, gluconeogenesis from pyruvate plus lactate and from propionate in the presence of 10 microM tetradecylglycidic acid was 47.3 and 41.4% of their respective controls. Similar to previous observations with butyrate, caproate addition inhibited gluconeogenesis from propionate by isolated hepatocytes and was unable to prevent inhibition of gluconeogenesis induced by tetradecylglycidic acid. Carnitine palmitoyltransferase I activity was lower in mitochondria isolated from hepatocytes preincubated with insulin than in control hepatocytes. The data suggest 1) that maximum rates of gluconeogenesis in isolated sheep hepatocytes from either pyruvate plus lactate or from propionate as gluconeogenic substrates require beta-oxidation, 2) that intermediates common to the metabolism of butyrate and caproate may be involved in the inhibition of propionate conversion to glucose by isolated sheep hepatocytes, and 3) that carnitine palmitoyltransferase I activity in isolated sheep hepatocytes can be modulated by insulin treatment.

Palmitate metabolism by isolated sheep rumen epithelial cells.

Ruminal palmitate metabolism was examined using an isolated cell system. Palmitate oxidation to 14CO2 by rumen epithelial cells isolated from the rumens of mature sheep was linear during the course of a 2-h incubation (11.1 nmoles.million cells-1.2 h-1) and 3.6 times the rate of palmitate oxidation by cells isolated from neonatal rumen (3.1 nmoles.million cells-1.min-1). Subsequent experiments were conducted with mature rumen epithelial cells. Neither acetate (50 mM), propionate (10 mM), dibutyryl cAMP (.2 mM), nor insulin (10 mU/mL) altered palmitate oxidation to CO2. However, butyrate (10 mM) addition reduced (P less than .05), and ammonia (15 mM) tended to reduce (P less than .10), palmitate oxidation (51.6 and 82.0% of control, respectively), whereas addition of glucose (2.5 mM) increased (P less than .05) palmitate oxidation (151% of control). Of the compounds tested, only propionate, butyrate, and ammonia reduced palmitate oxidation to total acid-soluble metabolites. Propionate (10 mM) addition completely abolished palmitate oxidation to acid-soluble metabolites. Succinate addition (5 to 50 mM) increased palmitate oxidation to CO2 but exhibited no consistent effect on palmitate oxidation to either acid-soluble metabolites or beta-hydroxybutyrate. Propionate completely abolished palmitate oxidation to beta-hydroxybutyrate, suggesting that propionate-induced inhibition of palmitate oxidation is not mediated via succinate. The data indicate 1) that rumen epithelium is capable of oxidizing palmitate, 2) that ruminal palmitate oxidation may be subject to regulation by developmental factors, and 3) that palmitate metabolism seems to be influenced more by ruminally derived metabolites than by factors derived exclusively from the general circulation.

Developmental changes in glucose and butyrate metabolism by isolated sheep ruminal cells.

The ontogeny of glucose and butyrate metabolism in developing sheep ruminal epithelium was determined using an isolated ruminal cell system. Ruminal cells were isolated from 21 lambs at seven ages before weaning. Rumen weight increased in proportion to increases in body weight, except between 28 and 42 d, when rumen weight increased threefold, whereas body weight increased only 33%. Glucose oxidation rates [expressed as nmol/(1 x 10(6) ruminal cells.120 min)] by ruminal cells were low at birth (14.2 +/- 5.08), increased sharply by 14 d (71.38 +/- 16.71), and remained elevated until 42 d. Following 42 d, glucose oxidation declined to rates lower than those observed at birth (6.11 +/- 0.83). Butyrate oxidation to CO2 increased from birth (20.03 +/- 3.41) to a peak at 4 d (134.0 +/- 31.71) and decreased throughout the remainder of the preweaning period (56 d; 36.32 +/- 7.48). Butyrate inhibited glucose oxidation by ruminal cells isolated at 14, 28 and 42 d. Similarly, glucose inhibited butyrate oxidation by ruminal cells isolated from 4 d to 28 d following birth. beta-Hydroxybutyrate production [nmol/(1 x 10(6) ruminal cells.120 min)] from butyrate by ruminal cells was undetectable at birth, but measurable by 4 d (3.28 +/- 2.15). Rates of beta-hydroxybutyrate production remained unchanged through 42 d; however, by 56 d, production had increased 10-fold (36.71 +/- 0.67). The metabolic adaptations of the ruminal epithelium are intimately associated with the physical development of the tissue, and major shifts in the fate of glucose and butyrate carbon occur prior to weaning.

Technical note: isolation and characterization of sheep ruminal epithelial cells.

A system for the isolation and characterization of sheep ruminal epithelial cells has been developed. Ruminal papillae from the ventral cranial sac of 12- to 24-wk-old Dorset ram lambs were subjected to serial tryptic digestions. Initial digestions (two 15-min periods) in the series were used to remove keratinized cells of the stratum corneum, which were able to produce only small amounts of beta-hydroxybutyrate from butyrate (5.37 +/- .72 nmol/120 min/per milligram of dry cell weight). Later digest fractions, containing primarily cells from the stratum basale, exhibited high viabilities (75 to 95%) and proved capable of converting butyrate to beta-hydroxybutyrate at relatively high rates (33.6 +/- 6.7 nmol/120 min per milligram of dry cell weight). Neither acetate nor propionate underwent significant conversion to beta-hydroxybutyrate. However, addition of acetate inhibited (77.4% of control) and addition of propionate stimulated (200% of control) beta-hydroxybutyrate production. Acetate addition reduced the propionate-induced stimulation of beta-hydroxybutyrate production from butyrate (136% of control). These results are similar to those obtained from in vitro incubations of ruminal papillae and suggest that an isolated cell system may prove useful in the further study of ruminal epithelial metabolism.

Gluconeogenic dependence on ketogenesis in isolated sheep hepatocytes.

Dependence of gluconeogenesis on beta-oxidation and ketogenesis from long-chain fatty acids was examined in isolated sheep hepatocytes. Hepatocytes were incubated with a combination of gluconeogenic precursors (2 mM pyruvate, 20 mM lactate, and 5 mM propionate) plus other fatty acids, in the presence and absence of tetradecylglycidic acid, an inhibitor of the carnitine palmitoyltransferase reaction. Palmitate oxidation to total acid-soluble metabolites or beta-hydroxybutyrate was markedly inhibited by the addition of tetradecylglycidic acid. In general, oxidation of palmitate to carbon dioxide was not altered by tetradecylglycidic acid. Glucose production was inhibited 28 to 50% in the presence of tetradecylglycidic acid. Addition of acetate and butyrate inhibited gluconeogenesis, but octanoate addition had a slight stimulatory effect. In the presence of tetradecylglycidic acid, butyrate, but not acetate, addition further reduced gluconeogenesis. In contrast, addition of octanoate in the presence of tetradecylglycidic acid restored gluconeogenic rates to control values. The results are consistent with observations in several nonruminant species and suggest that, as in those species, ruminant gluconeogenesis requires at least a basal rate of beta-oxidation and ketogenesis from long-chain fatty acids to support maximum gluconeogenic rates.

Heterogeneous expression of protein and mRNA in pyruvate dehydrogenase deficiency.

Deficiency of pyruvate dehydrogenase [pyruvate:lipoamide 2-oxidoreductase (decarboxylating and acceptor-acetylating), EC 1.2.4.1], the first component of the pyruvate dehydrogenase complex, is associated with lactic acidosis and central nervous system dysfunction. Using both specific antibodies to pyruvate dehydrogenase and cDNAs coding for its two alpha and beta subunits, we characterized pyruvate dehydrogenase deficiency in 11 patients. Three different patterns were found on immunologic and RNA blot analyses. (i) Seven patients had immunologically detectable crossreactive material for the alpha and beta proteins of pyruvate dehydrogenase. (ii) Two patients had no detectable crossreactive protein for either the alpha or beta subunit but had normal amounts of mRNA for both alpha and beta subunits. (iii) The remaining two patients also had no detectable crossreactive protein but had diminished amounts of mRNA for the alpha subunit of pyruvate dehydrogenase only. These results indicate that loss of pyruvate dehydrogenase activity may be associated with either absent or catalytically inactive proteins, and in those cases in which this enzyme is absent, mRNA for one of the subunits may also be missing. When mRNA for one of the subunits is lacking, both protein subunits are absent, suggesting that a mutation affecting the expression of one of the subunit proteins causes the remaining uncomplexed subunit to be unstable. The results show that several different mutations account for the molecular heterogeneity of pyruvate dehydrogenase deficiency.

Cloning and cDNA sequence of the dihydrolipoamide dehydrogenase component human alpha-ketoacid dehydrogenase complexes.

cDNA clones comprising the entire coding region for human dihydrolipoamide dehydrogenase (dihydrolipoamide:NAD+ oxidoreductase, EC 1.8.1.4) have been isolated from a human liver cDNA library. The cDNA sequence of the largest clone consisted of 2082 base pairs and contained a 1527-base open reading frame that encodes a precursor dihydrolipoamide dehydrogenase of 509 amino acid residues. The first 35-amino acid residues of the open reading frame probably correspond to a typical mitochondrial import leader sequence. The predicted amino acid sequence of the mature protein, starting at the residue number 36 of the open reading frame, is almost identical (greater than 98% homology) with the known partial amino acid sequence of the pig heart dihydrolipoamide dehydrogenase. The cDNA clone also contains a 3' untranslated region of 505 bases with an unusual polyadenylylation signal (TATAAA) and a short poly(A) track. By blot-hybridization analysis with the cDNA as probe, two mRNAs, 2.2 and 2.4 kilobases in size, have been detected in human tissues and fibroblasts, whereas only one mRNA (2.4 kilobases) was detected in rat tissues.

Isolation of a cDNA clone for the dihydrolipoamide acetyltransferase component of the human liver pyruvate dehydrogenase complex.

Dihydrolipoamide acetyltransferase (E2) forms the structural core of pyruvate dehydrogenase complex. A cDNA clone (lambda E2-1) for mammalian E2 was identified from a human liver lambda gt11 library using anti-E2 serum. Affinity-selected antibodies using the fusion protein from lambda E2-1 immuno-reacted specifically with E2 of purified pyruvate dehydrogenase complex on immuno-blot analysis. The cDNA insert was approximately 2.3 kb in length with an internal EcoR1 site generating 1.4 and 0.9 kb fragments. A synthetic 17-mer oligodeoxynucleotide mixture based on the amino acid sequence surrounding the lipoic acid-containing lysine residue in bovine kidney E2 hybridized with the 2.3 kb cDNA insert and the 1.4 kb fragment.

Control of bovine hepatic fatty acid oxidation.

Fatty acid oxidation by bovine liver slices and mitochondria was examined to determine potential regulatory sites of fatty acid oxidation. Conversion of 1-[14C]palmitate to 14CO2 and total [14C]acid-soluble metabolites was used to measure fatty acid oxidation. Oxidation of palmitate (1 mM) was linear in both liver slice weight and incubation time. Carnitine stimulated palmitate oxidation; 2 mM dl-carnitine produced maximal stimulation of palmitate oxidation to both CO2 and acid-soluble metabolites. Propionate (10 mM) inhibited palmitate oxidation by bovine liver slices. Clofenapate, an inhibitor of fatty acid esterification, alone increased palmitate oxidation and was able to prevent the propionate-induced inhibition of palmitate oxidation by liver slices. Propionate (.5 to 10 mM) had no effect on palmitate oxidation by mitochondria, but malonyl Coenzyme A, the first committed intermediate of fatty acid synthesis, inhibited mitochondrial palmitate oxidation (inhibition constant = .3 microM). Liver mitochondrial carnitine palmitoyltransferase (EC 2.3.1.21) exhibited Michaelis constants for palmitoyl Coenzyme A and l-carnitine of 11.5 microM and .59 mM, respectively. Long-chain fatty acid oxidation in bovine liver is regulated by mechanisms similar to those in rats but adapted to the unique digestive physiology of the bovine.

Aspects of the regulation of long-chain fatty acid oxidation in bovine liver.

Factors involved in regulation of bovine hepatic fatty acid oxidation were examined using liver slices. Fatty acid oxidation was measured as the conversion of 1-[14C] palmitate to 14CO2 and total [14C] acid-soluble metabolites. Extended (5 to 7 d) fasting of Holstein cows had relatively little effect on palmitate oxidation to acid-soluble metabolites by liver slices, although oxidation to CO2 was decreased. Feeding a restricted roughage, high concentrate ration to lactating cows resulted in inhibition of palmitate oxidation. Insulin, glucose, and acetate inhibited palmitate oxidation by bovine liver slices. We suggest the regulation of bovine hepatic fatty acid oxidation may be less dependent on hormonally induced alterations in enzyme activity as observed in rat liver and more dependent upon action of rumen fermentation products or their metabolites on enzyme systems involved in fatty acid oxidation.

A technique for isolation of bovine hepatocytes.

A technique for preparing viable and functional isolated hepatocytes from cattle liver is described. The basic procedure, which was adapted from published methods established for laboratory species, employed a two-step in vitro vascular perfusion of the caudate lobe: (1) perfusion with a calcium-free buffer containing ethylene bis(oxyethylenenitrilo)tetraacetic acid (EGTA) for removal of blood cells and extracellular calcium and (2) perfusion with calcium-fortified buffer containing collagenase for cell dissociation. Hepatocyte suspensions prepared from the caudate lobes of 20 cattle possessed a mean viability of 81.3% as determined by trypan blue exclusion. Mean yield was 2.2 X 10(7) viable hepatocytes/g of liver (wet wt). Viable hepatocytes utilized O2 at a rate 2.82 times greater than nonviable hepatocytes. Biochemical function of the hepatocyte suspensions was assessed by rates of gluconeogenesis and fatty acid oxidation. Glucose production from added lactate ranged from .88 to 1.47 mumol X min-1 X g-1 of liver tissue (dry wt). Both gluconeogenic and fatty acid oxidation rates were substantially greater in isolated hepatocytes when compared with liver slices. Isolated hepatocyte contained .398 +/- .033 (SE) nmol cytochromes P-450/mg microsomal protein and .285 +/- .025 nmol cytochrome bs/mg microsomal protein, which was comparable with amounts in liver tissue from the same animals (.568 +/- .056 and .298 +/- .033 nmol/mg protein, respectively). No significant decline of either cytochrome was detectable for isolated hepatocytes for up to 5.5 h after euthanasia. The potential usefulness of isolated bovine hepatocytes in xenobiotic metabolism studies is illustrated by the epoxidation of aldrin.