Rat Muscle Homogenates Research Articles

Digital PCR Quantitation of Muscle Mitochondrial DNA: Age, Fiber Type, and Mutation-Induced Changes.

Definitive quantitation of mitochondrial DNA (mtDNA) and mtDNA deletion mutation abundances would help clarify the role of mtDNA instability in aging. To more accurately quantify mtDNA, we applied the emerging technique of digital polymerase chain reaction to individual muscle fibers and muscle homogenates from aged rodents. Individual fiber mtDNA content correlated with fiber type and decreased with age. We adapted a digital polymerase chain reaction deletion assay that was accurate in mixing experiments to a mutation frequency of 0.03% and quantitated an age-induced increase in deletion frequency from rat muscle homogenates. Importantly, the deletion frequency measured in muscle homogenates strongly correlated with electron transport chain-deficient fiber abundance determined by histochemical analyses. These data clarify the temporal accumulation of mtDNA deletions that lead to electron chain-deficient fibers, a process culminating in muscle fiber loss.

Quantitation of Deltamethrin in Rat Liver and Muscle Homogenates Using Dispersive Solid-Phase Extraction with GC-NCI-MS.

An improved bioanalytical method to determine pyrethroids from small-volume (300 μL) rat liver and muscle homogenates was developed, validated, and applied to small-animal studies. The method used dispersive SPE (d-SPE) to clean the samples, and GC negative chemical ion MS (GC-NCI-MS) to analyze the samples. High-quality trace analysis of pyrethroids in biological samples was necessary to assess the health risk of environmentally encountered levels. Currently used highly sensitive methods to measure pyrethroids have focused on large-volume samples related to environmental exposure (water, soil, food products) or urine; however, there are no validated methods for quantifying this class of compounds in small-volume rat liver and muscle tissue homogenates. Individual rat tissue homogenate samples (300 μL) were prepared by protein precipitation using hexane-saturated acetonitrile. The samples were mixed on a vortex mixer and decanted into a d-SPE tube containing octadecylsilyl (C18) and primary secondary amine (PSA) sorbents and magnesium sulfate. The samples were centrifuged before evaporation to dryness. Pyrethroids were extracted and reconstituted from the residue using toluene in advance of injection into an Agilent Model 6890N gas chromatograph equipped with a Model 5973 quadrupole mass analyzer. Samples were ionized via electron capture in the negative ion mode using methane as a chemical ionization gas. Six qualifying ions were monitored using selected-ion monitoring for quantitation and verification of the analyte. Cis-permethrin was used as the internal standard. Method linearity was from 1 to 500 ng/mL for muscle and liver homogenates. The inter- and intraday precision and accuracy of the method were better than 20% at the lower LOQ and better than 15% over the remainder of the linear range. The method was used to elucidate tissue time-courses of deltamethrin (DLM) disposition following oral dosing of rats with 1 to 5 mg/kg in corn oil. DLM was monitored in rat tissue samples ≤24 h following dosing. Sample cleanup with d-SPE provided more selective chromatography than previous analytical methods.

Junctophilins and μ‐calpain: partners in excitation–contraction uncoupling

Junctophilins and μ‐calpain: partners in excitation–contraction uncoupling

μ-Calpain and calpain-3 are not autolyzed with exhaustive exercise in humans

mu-Calpain and calpain-3 are Ca2+-dependent proteases found in skeletal muscle. Autolysis of calpains is observed using Western blot analysis as the cleaving of the full-length proteins to shorter products. Biochemical assays suggest that mu-calpain becomes proteolytically active in the presence of 2-200 microM Ca2+. Although calpain-3 is poorly understood, autolysis is thought to result in its activation, which is widely thought to occur at lower intracellular Ca2+ concentration levels ([Ca2+]i; approximately 1 microM) than the levels at which mu-calpain activation occurs. We have demonstrated the Ca2+-dependent autolysis of the calpains in human muscle samples and rat extensor digitorum longus (EDL) muscles homogenized in solutions mimicking the intracellular environment at various [Ca2+] levels (0, 2.5, 10, and 25 microM). Autolysis of calpain-3 was found to occur across a [Ca2+] range similar to that for mu-calpain, and both calpains displayed a seemingly higher Ca2+ sensitivity in human than in rat muscle homogenates, with approximately 15% autolysis observed after 1-min exposure to 2.5 microM Ca2+ in human muscle and almost none after 1- to 2-min exposure to the same [Ca2+]i level in rat muscle. During muscle activity, [Ca2+]i may transiently peak in the range found to autolyze mu-calpain and calpain-3, so we examined the effect of two types of exhaustive cycling exercise (30-s "all-out" cycling, n = 8; and 70% VO2 peak until fatigue, n = 3) on the amount of autolyzed mu-calpain or calpain-3 in human muscle. No significant autolysis of mu-calpain or calpain-3 occurred as a result of the exercise. These findings have shown that the time- and concentration-dependent changes in [Ca2+]i that occurred during concentric exercise fall near but below the level necessary to cause autolysis of calpains in vivo.

Effects of prolonged exercise and recovery on sarcoplasmic reticulum Ca2+ cycling properties in rat muscle homogenates.

To examine the effects of exercise and exercise plus active and passive recovery on sarcoplasmic reticulum (SR) Ca2+-handling properties. Crude muscle homogenates were prepared from adult rat gastrocnemius muscle from two experiments. In one experiment, the muscle was extracted immediately after prolonged treadmill running (RUN), after a 45 min period of reduced exercise intensity (RUN+) following RUN and compared with controls (CON). In the second experiment, muscle was extracted during passive recovery following the same run protocol at 10 min (REC10), 25 min (REC25) and 45 min (REC45) and compared with CON. Sarcoplasmic reticulum Ca2+-uptake was 31% higher (P < 0.05) in RUN+ compared with CON and RUN. Higher values (P < 0.05) were also found in REC25 (48%) and REC45 (50%) compared with CON. Maximal Ca2+-ATPase was increased by 23% (P < 0.05) in RUN+ compared with CON and RUN and by 65-68% (P < 0.05) in REC25 and REC45 compared with CON. A higher (P < 0.05) Hill coefficient for Ca2+-ATPase activity was observed in RUN+ (2.3 +/- 0.2) compared with CON (1.7 +/- 0.2) or RUN (1.6 +/- 0.2), but not for any REC conditions. In addition, the coupling ratio (Ca2+-uptake/Ca2+-ATPase activity) was higher (P < 0.05) in RUN+ (2.2 +/- 0.10) compared with CON (1.9 +/- 0.05) and RUN (1.9 +/- 0.08). It is concluded that in crude homogenates, SR Ca2+-uptake and Ca2+-ATPase activity are elevated in recovery following prolonged running and that the elevation in these properties is more pronounced during passive compared with active recovery.

Neuronal nitric oxide synthase isoforms alpha and mu are closely related calpain-sensitive proteins.

The neuronal nitric oxide synthase isoform nNOSmu, which is expressed in striated muscle, differs from nNOSalpha, the major brain isoform, by the insertion of 34 amino acid residues between the calmodulin- and flavin-binding domains [J Biol Chem 271:11204-11208 (1996)]. We show here that recombinant, purified nNOSmu, despite the peptide insertion, has the same spectroscopic properties, L-arginine kcat and Km values, optimal pH, and calmodulin binding affinity constant as nNOSalpha. However, nNOSmu consumes NADPH and reduces cytochrome c at approximately half the rate of nNOSalpha. The rates of degradation of the two proteins by rat brain and muscle homogenates show that nNOSmu is degraded more slowly than nNOSalpha. The in vitro half-lives of nNOSalpha and nNOSmu are 12 and 50 min, respectively, and calpain is important for this degradation. These short in vitro half-lives suggest that the nNOS isoforms are susceptible to rapid degradation in vivo. The elevated (20-fold) levels of calpain in diseased muscle tissue in Duchenne muscular dystrophy, and the hydrolytic sensitivity of both nNOS mu and nNOSalpha to this enzyme, may contribute to the deficiency of nNOS activity in the diseased tissue.

Measurement of Sarcoplasmic Reticulum Function in Mammalian Skeletal Muscle: Technical Aspects

Two recently developed methods for measuring the maximal rate of Ca2+ uptake and Ca2+ ATPase activity (EC 3.1.6.38) in vitro use muscle homogenate rather than isolated sarcoplasmic reticulum (SR). In this study we investigated technical aspects of these assays, including specificity and variability of the assays, the effect of different freezing treatments on maximal Ca2+ uptake and Ca2+ ATPase activity of human and rat muscle homogenate, stability of the homogenate, and the modification of the Ca2+ uptake assay to measure Ag+-induced Ca2+ release. Addition of cyclopiazonic acid (20 μm) blocked Ca2+ uptake, demonstrating specificity of the assay. Using frozen muscle homogenate, the inter- and intraassay variation for both assays was less than 9%. Whereas homogenates were stable to freezing for both Ca2+ uptake and Ca2+ ATPase activity, there was a significant (P < 0.05) decrease in activity when muscle was freeze-dried or quickly frozen in small pieces. After 1 h on ice, rat muscle homogenate Ca2+ uptake and Ca2+ ATPase activity had decreased by 6.0% (ns) and 3.6% (P < 0.05), respectively; after 3 h, activity had decreased by 15.3 and 14.7%, respectively (P < 0.01). The Ca2+ uptake assay was modified to allow measurement of Ag+-induced Ca2+ release. Following homogenate addition, after the SR vesicles were loaded with Ca2+ and [Ca2+] had declined to a plateau, AgNO3 (141 μm) was added, initiating release of Ca2+ into the assay solution. Addition of dithiothreitol (4 mM) blocked the Ag+-induced Ca2+ release, demonstrating specificity of the assay.

The role of the lymphatic system in the presystemic absorption of fluphenazine after intramuscular administration of fluphenazine decanoate in rats

Sprague Dawley rats ( n = 16) were injected intramuscularly with fluphenazine decanoate (78 mg/kg) in sesame oil. Groups of four rats were surgically prepared on days 1, 7, 15 and 32 post-injection and samples of lymph obtained from the thoracic duct. Fluphenazine was found in similar concentrations in lymph and plasma, although the decanoate was not detected in either body fluid. These data suggest that the lymphatic system plays a role in the presystemic absorption of fluphenazine after i.m. administration of fluphenazine decanoate in the rat. In vitro experiments showed that fluphenazine decanoate was very rapidly hydrolyzed when incubated with rat plasma ( t 1 2 0.6 h ) or muscle homogenates ( t 1 2 1.0 h ). Rates of hydrolysis of fluphenazine decanoate were lower in dog muscle homogenates ( t 1 2 4.3 h ), and very much lower when incubated with plasma from dog 42.9 h) or humans ( t 1 2 50.9 h).

Sequential kinetic thermometric determination of the activity of peroxidase and catalase using catechol as substrate and inhibitor for their reaction with hydrogen peroxide

A kinetic thermometric method was developed for the determination of peroxidase by its catalytic effect on the oxidation of catechol by hydrogen peroxide. The proposed method allows the determination of peroxidase activity over the range 0.05-0.3 I.U. with an R.S.D. 2.3%. The method was applied to the determination of peroxidase activity in prespiked matrices of animal origin (rat muscle and liver homogenates). The determination of the activity of peroxidase added to rat liver was found to be subject to a matrix effect that was corrected for by using an appropriate matrix-to-substrate concentration ratio. As catalase is thermometrically active at the same pH as peroxidase in its reaction with hydrogen peroxide and behaves similarly towards catechol when bound to a rat liver matrix, a procedure was developed for the sequential thermometric determination of the activity of both enzymes using catechol as substrate for peroxidase and inhibitor for catalase.

Oscillations in glycolysis: multifactorial quantitative analysis in muscle extract.

A multifactorial quantitative analysis of oscillations in glycolysis was conducted in the postmicrosomal supernatant of rat muscle homogenates incubated in the presence of yeast hexokinase. Oscillations in adenine nucleotides, D-fructose 1,6-bisphosphate, triose phosphates, L-glycerol 3-phosphate, 3HOH generation from D-[5-3H]glucose, NADH and L-lactate production were documented. The occurrence of such oscillations were found to depend mainly on the balance between the consumption of ATP associated with the phosphorylation of D-glucose, as catalyzed by both yeast and muscle hexokinase, and the net production of ATP resulting from the further catabolism of D-fructose 6-phosphate, as initiated by activation of phosphofructokinase. The oscillatory pattern was suppressed in the presence of D-fructose 2,6-bisphosphate. It is proposed that the quantitative information gathered in this study may set the scene for further studies in extracts of cells other than myocytes, e.g. hepatocytes and pancreatic islet cells, in which no oscillation of glycolysis was so far observed.

Phosphorylation of the cardiac isoform of calsequestrin in cultured rat myotubes and rat skeletal muscle

Calsequestrin is a high-capacity Ca2+-binding protein and a major constituent of the sarcoplasmic reticulum (SR) of both skeletal and cardiac muscle. Two isoforms of calsequestrin, cardiac and skeletal muscle forms, have been described which are products of separate genes. Purified forms of the two prototypical calsequestrin isoforms, dog cardiac and rabbit fast-twitch skeletal muscle calsequestrins, serve as excellent substrates for casein kinase II and are phosphorylated on distinct sites (Cala, S.E. and Jones, L.R. (1991) J. Biol. Chem 266, 391–398). Dog cardiac calsequestrin is phosphorylated at a 50 to 100-fold greater rate than is rabbit skeletal muscle calsequestrin, and only the dog cardiac isoform contains endogenous Pi on casein kinase II phosphorylation sites. In this study, we identified and examined both calsequestrin isoforms in rat muscle cultures and homogenates to demonstrate that the cardiac isoform of calsequestrin in rat skeletal muscle was phosphorylated in vivo on sites which are phosphorylated by casein kinase II in vitro. Phosphorylation of rat skeletal muscle calsequestrin was not detected. In tissue homogenates, cardiac and skeletal muscle calsequestrin isoforms were both found to be prominent substrates for endogeneous casein kinase II activity with cardiac calsequestrin the preferred substrate. In addition, these studies revealed that the cardiac isoform of calsequestrin was the predominant form expressed in skeletal muscle of fetal rats and cultured myotubes.

In Vitro Binding of Chloroquine to Rat Muscle Preparations

Chloroquine (7-chloro-4-[[4-(diethylamino)-1-methylbutyl]a-mino]quinoline; CQ) accumulates in the insoluble fraction of rat muscle homogenates with an equilibrium distribution ratio of 7.8 L/kg. This is of a similar magnitude to muscle:plasma ratios observed in vivo. Uptake is approximately linear, even up to concentrations of free CQ greatly exceeding toxic plasma levels. Cell structural integrity is not essential for uptake, indicating that CQ is bound to tissue constituents and not trapped inside cells by an active process. At pH 7.4, binding of ionized CQ at anionic sites on tissue predominates over lipid partitioning of the un-ionized species. Binding to phospholipids is a major component of total muscle binding.

Electron Beam-Induced Mass Loss in Freeze-Dried Tissue and Protein Samples

Quantification in biological x-ray microanalysis depends on accurate evaluation of mass loss. Although several studies have addressed the problem of electron beam induced mass loss from organic samples (eg., 1,2). uncertainty persists as to the dose dependence, the extent of loss, the elemental constituents affected, and the variation in loss for different materials and tissues. in the work described here, we used x-ray counting rate changes to measure mass loss in albumin (used as a quantification standard), salivary gland, and muscle.In order to measure mass loss at low doses (10-4 coul/cm2 ) large samples were needed. While freeze-dried salivary gland sections of the required dimensions were available, muscle sections of this size were difficult to obtain. To simulate large muscle sections, frog or rat muscle homogenate was injected between formvar films which were then stretched over slot grids and freeze-dried. Albumin samples were prepared by a similar procedure. using a solution of bovine serum albumin in water. Samples were irradiated in the STEM mode of a JEOL 100C.

The metabolic fate of branched-chain amino acids and 2-oxo acids in rat muscle homogenates and diaphragms

1. 1. After incubation of muscle preparations with [U- 14C]branched-chain amino acids or 2-oxo acids, radioactive metabolites were separated, identified and quantified. 2. 2. Homogenates of rat heart and skeletal muscle incubated with 4-methyl-2-oxopentanoate accumulated isovalerate, 3-hydroxyisovalerate and the corresponding carnitine esters. 3. 3. Incubation with 3-methyl-2-oxobutanoate resulted in the production of isobutyrate, 3-hydroxyisobutyrate and their carnitine esters. Addition of L-carnitine increased the production of the esters. 4. 4. The enzymes 3-methylcrotonyl-CoA carboxylase and 3-hydroxyisobutyric acid dehydrogenase apparently are inactive during incubation of muscle homogenates. 5. 5. With liver homogenates the degradation of both 2-oxo acids was more complete. 6. 6. Rat hemidiaphragms incubated with leucine, valine and isoleucine accumulated the corresponding branched-chain 2-oxo acids, fatty acids and hydroxylated fatty acids. 7. 7. The degradation of valine was markedly limited by the release of these metabolites. 8. 8.Considerable amounts (relatively smaller for valine) of radioactivity were also recovered in CO 2 and glutamine and glutamate. 9. 9. Incubations with branched-chain 2-oxo acids gave the same radioactive products, except for glutamine and glutamate. 10. 10. Radioactivity was never found in lactate, pyruvate or alanine. 11. 11. These data indicate that the carbon-chains of amino acids entering the citric acid cycle in muscle, are not used for oxidation or for alanine synthesis, but are converted exclusively to glutamine.

Effect of various agents and conditions on palmitate oxidation by homogenates of rat liver and rat and human skeletal muscle

1. 1. The effect of various inhibitors of fatty acid transport and of respiratory chain on palmitate oxidation was investigated in homogenates and mitochondria of rat muscle and homogenates of rat liver and human muscle. 2. 2. Inhibition of fatty acid transport by carnitine omission, malonyl-CoA. tetradecylglycidic acid and mersalyl decreased oxidation more with muscle than with rat liver. 3. 3. Antimycin and KCN decreased markedly palmitate oxidation and caused a larger accumulation of peroxisomal oxidation products. 4. 4. Inhibition of mitochondrial long-chain fatty acid transport decreased accumulation of peroxisomal products in comparison to the control. 5. 5. The effect of malonyl-CoA was dependent on the nutritional state, the pH and the palmitatc-alhumin ratio with liver homogenates. and only on the latter parameter with muscle homogenates. 6. 6. Effects observed were comparable for rat and human muscle homogenates.

The interaction between hexamethonium and tubocurarine on the rat neuromuscular junction.

The ability of hexamethonium (C6) to reverse the neuromuscular blocking action of tubocurarine (Tc) has been reinvestigated at the voltage clamped endplate of the omohyoid muscle of rat. The possibility that a weak anticholinesterase action of C6 could contribute to the paradoxical potentiation of the peak amplitude of the endplate response has been examined. C6 (50-200 microM) caused an increase in the amplitude of nerve-evoked endplate currents (e.p.cs) recorded in the presence of 0.6 microM Tc. The effect decreased with hyperpolarization of the muscle fibre. Irreversible inhibition of acetylcholinesterase resulted in a loss of the anti-curare effect of C6. C6 did not cause an increase in e.p.c. amplitude when acetylcholine (ACh) receptors were blocked irreversibly by alpha-bungaratoxin. When transmission was blocked by increased Mg2+ concentration, C6 (50-400 microM) reduced the amplitude of e.p.cs without appreciably affecting their time course. C6 caused a decrease in the amplitude of miniature endplate currents (m.e.p.cs) the effect being slightly increased when the fibre was hyperpolarized. An e-fold increase in the effectiveness of C6 occurred with approximately 58 mV hyperpolarization. High concentrations (greater than 400 microM) affected the time course of m.e.p.cs in a manner suggestive of open channel block, but this was not evident at 200 microM, the concentration that was most effective in reversing Tc block. When tested against responses to short ionophoretic pulses of agonists, C6 was less effective against ACh (EC50ca. 300 microM) than against carbachol (CCh) (EC50 100 microM). When cholinesterase was irreversibly inhibited, C6 blocked responses to both agonists equally (EC50ca. 100 microM). The effectiveness of C6 in blocking the action of CCh was reduced 10 fold in the presence of 0.6 microM Tc, implying that the two antagonists compete for the same binding site. C6 (50-200 microM) in the presence of Tc (0.6 microM) increased the response to ionophoretically applied ACh but not that to CCh. C6 was equipotent in blocking m.e.p.cs and responses to ionophoretically applied ACh whereas Tc was more potent against the exogenously applied agonist. C6 was a weak inhibitor of acetylcholinesterase activity in rat muscle homogenates (EC50 1.5 mM). The results are discussed in terms of the kinetic hypothesis advanced by Ginsborg & Stephenson (1974) to account for the Tc reversal phenomenon. It is concluded that this theory can explain most of the effect on e.p.cs, but that the weak anticholinesterase action of C6 is also a factor, particularly in the reversal of Tc block of ionophoretic responses.

Extracellular lipase and proteinase of Basidiobolus haptosporus: possible role in subcutaneous mycosis.

Properties of proteinase(s) and phospholipase A in dialyzed culture filtrate of Basidiobolus haptosporus were studied. Lysolecithin, one of the hydrolytic products of phosphatidyl choline by phospholipase A was prepared and found able to hemolyse human red blood cells and 'clear' rat skin and muscle homogenates. The proteinase was able to digest human serum proteins. The pathogenic mechanism employed by this fungus may involve hydrolysis of lecithin to yield lysolecithin, which destroys cell membranes thereby liberating intracellular contents. The protein components of these contents are then digested by the proteinase(s) yielding amino acids which along with other metabolites serve as nutrients for the growth of the pathogen.

Muscle proteolytic enzyme activities in diabetic rats.

Proteolytic enzyme activities were measured in skeletal muscle of Sprague-Dawley rats with streptozotocin-induced diabetes [tail vein injection of streptozotocin (100 mg/kg), under ether anesthesia]. Assay of rat muscle homogenates from diabetic rats revealed a significant increase in alkaline serine protease activity as compared to untreated control rats and diabetic rats given insulin. There were no significant changes in lysosomal cathepsin activities in diabetic muscle as compared to controls. Gel studies of myofibrils isolated from the three groups of rats, subjected to autolysis, revealed that the serine protease had copurified with the myofibrils. Treatment of rats with compound 48/80, which degranulates mast cells, abolished the alkaline protease activity. There was no serine protease activity associated with the myofibrils isolated from compound 48/80-treated rats. Results from this study indicate that serine proteases are not involved in muscle protein breakdown in diabetes and are of mast cell origin.

Characteristics of saxitoxin binding to the sodium channel of sarcolemma isolated from rat skeletal muscle.

1. The characteristics of saxitoxin (STX) binding to the mammalian Na channel have been studied in purified sarcolemma isolated from rat skeletal muscle. 2. STX binds specifically to isolated sarcolemma with a Kd of 1.43 x 10(-9) M and Bmax of 7-8 p-mole STX bound/mg membrane protein at 0 degrees C in the presence of 140 mM-NaCl. In rat muscle homogenate under the same conditions the corresponding values are Kd = 1.53 x 10(-9) M and Bmax = 0.15-0.20 p-mole/mg protein (18-20 p-mole/g wet wt.). Membrane purification produced a fortyfold increase in STX binding site concentration per milligram protein. Calculated binding site density in isolated sarcolemma was about 30 sites/micron 2 of membrane surface. 3. Denervation (10-14 days) results in a 43% reduction in the density of high-affinity STX binding sites in purified sarcolemma, but the Kd for this class of sites is not changed. 4. In sarcolemma, the apparent Kd for STX binding is dependent on temperature pH and ionic strength. The Q10 for Kd between 0 and 40 degrees C is 1.3. Protonation of a group having a pK of 6.0 markedly raises Kd without affecting Bmax. Apparent Kd increases eightfold when ionic strength is raised from 20 to 600 mM. 5. Dissociation and association rate constants for STX binding are temperature dependent with Q10 of 2.6 and 1.9 respectively between 0 and 20 degrees C. 6. STX binding is competitively inhibited by monovalent and divalent cations under conditions of constant total ionic strength. An affinity sequence of Tl+ greater than Li+ greater than Na+ greater than K+ greater than Rb+ greater than Cs+ is seen for the monovalent cation-binding site. 7. The STX binding site is relatively stable to heat and to enzymic degradation. A specific modifier of carboxyl residues inactivates subsequent STX binding. This process can be prevented by the presence of STX during the reaction. 8. Characteristics of the STX binding site in isolated sarcolemma are compared to those reported for other isolated excitable membranes and for studies of whole muscle and muscle homogenate. Sarcolemma provides a potential source of enriched Na channels for further purification efforts in a mammalian system.

Rat intestinal 25-hydroxyvitamin D3- and 1alpha,25-dihydroxyvitamin D3-24-hydroxylase.

The 24-hydroxylation of 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3 occurs in vitro in rat intestinal homogenates. 1alpha,24,25-Trihydroxyvitamin D3 obtained by incubating 1alpha,25-dihydroxyvitamin D3 with rat intestinal homogenates was isolated and then identified by co-chromatography of the biologically generated material with synthetic 1alpha,24R,25-trihydroxyvitamin D3 on a high pressure liquid chromatography system and by ultraviolet spectroscopy, periodate cleavage, and mass spectroscopy. 24,25-Dihydroxyvitamin D3 obtained by incubating 25-hydroxyvitamin D3 with intestinal homogenates was identified by co-chromatography of the biologically generated material with authentic 24R,25-dihydroxyvitamin D3 on a high pressure liquid chromatography system and by periodate cleavage. Tissue distribution studies indicate that the process occurs in rat intestinal homogenates but not in rat muscle, liver, or fetal calvaria homogenates. Nephrectomy does not diminish the production of 1alpha,24,25-trihydroxyvitamin D3 in vivo while it reduces markedly, but does not eliminate, production of 24,25-dihydroxyvitamin D3.