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Coenzyme Q levels decrease during aging in most tissues and in the target organs of a number of diseases. The uptake of this lipid into the blood and other tissues was investigated in 6-wk-old male Sprague-Dawley rats after 3 wk of dietary supplementation. In addition to the natural form of coenzyme Q(10), acetylated and succinylated forms were also administered. Coenzyme Q(10) was taken up into the blood, but uptake was significantly greater in rats given the succinylated ( approximately 40%), and particularly, the acetylated forms ( approximately 70%). All three forms increased significantly the total coenzyme Q concentration in both the liver ( approximately 100%) and spleen ( approximately 130%). Coenzyme Q(10) and its esterified forms were not taken up into kidney, heart, muscle or brain. Intraportal and intraperitoneal administration of succinylated coenzyme Q(10) gave results similar to those obtained in the dietary experiments. Uptake of the dietary coenzyme Q(10) into the liver and spleen did not down-regulate the endogenous synthesis, i.e., the amounts of isolated coenzyme Q(9) did not change in these tissues. Thus, esterification of coenzyme Q increases the uptake of dietary lipid into the blood; however, the derivatization does not contribute to the elevation of coenzyme Q levels in various organs. [ABSTRACT FROM AUTHOR]

The dietary uptake of alpha-tocopherol and coenzyme Q was investigated in rats. Rats were fed diets supplemented with alpha-tocopherol or coenzyme Q10 (1 g/kg diet) or an unsupplemented control diet. In control rat tissues, the content of coenzyme Q was 4-11 times higher than that of alpha-tocopherol, but in plasma, the ratio was reversed. Among the subcellular fractions of rat liver homogenate, Golgi vesicles and lysosomes had the highest alpha-tocopherol concentration, and high concentrations of coenzyme Q were observed in the outer and inner mitochondrial membranes as well as in lysosomes, Golgi vesicles and plasma membranes. The uptake of alpha-tocopherol into the liver and plasma reached a maximal level after only 2 d of supplementation, whereas in the kidney, heart, muscle and brain, the levels continued to increase throughout the 6-wk treatment period. In contrast, dietary coenzyme Q was taken up into the liver and plasma only, and not into the other organs. This lipid appeared mainly in the Golgi system, whereas alpha-tocopherol exhibited a more general cellular distribution. The decay of the supplied alpha-tocopherol was slow in the various organs, but the disappearance of coenzyme Q was rapid from both liver and plasma. Pretreatment of rats with alpha-tocopherol increased the levels of both endogenous and exogenous coenzyme Q in the liver and plasma. These results demonstrate that the uptake of alpha-tocopherol from the diet is an extensive and general phenomenon at both the tissue and cellular levels, in contrast to the selective and restricted uptake of coenzyme Q. [ABSTRACT FROM AUTHOR]

Coenzyme Q is an important mitochondrial redox component and the only endogenously produced lipid-soluble antioxidant. Its tissue concentration decreases with aging and in a number of diseases; dietary supplementation of this lipid would fulfill important functions by counteracting coenzyme Q depletion. To investigate possible uptake, rats were administered 12 mumol coenzyme Q10/100 g body wt once daily by gastric intubation. The appearance of coenzyme Q10 in various tissues and blood after 6 h, 4 d or 8 d was studied. The control group of rats received rapeseed-soybean oil (the vehicle in the experimental group). Lipids were extracted with petroleum ethermethanol, and the reduced and oxidized forms of coenzyme Q9 and Q10 were separated and quantified by reversed-phase HPLC. In the plasma, the total coenzyme Q concentration was doubled after 4 d of treatment. Coenzyme Q10 was also recovered in liver homogenates, where, as in the plasma, it was largely in the reduced form. Uptake into the spleen could be to a large extent accounted for by the blood content of this organ. No dietary coenzyme Q10 was recovered in the heart or kidney. The uptake in the whole body was 2-3% of the total dose. Coenzyme Q10 found in the liver was located mainly in the lysosomes. Dietary coenzyme Q10 did not influence the endogenous biosynthesis of coenzyme Q9. This is in contrast to dietary cholesterol, which down-regulates cholesterol biosynthesis. The dietary coenzyme Q10 level in the plasma decreased to approximately 50% after 4 d. These results suggest that dietary coenzyme Q10 may play a role primarily in the blood and that no appreciable uptake occurs into tissues. [ABSTRACT FROM AUTHOR]

Background: The respiratory-chain deficiencies are a broad group of largely untreatable diseases. Among them, coenzyme Q10 (ubiquinone) deficiency constitutes a subclass that deserves early and accurate diagnosis., Methods: We assessed respiratory-chain function in two siblings with severe encephalomyopathy and renal failure. We used high-performance liquid chromatography analyses, combined with radiolabelling experiments, to quantify cellular coenzyme Q10 content. Clinical follow-up and detailed biochemical investigations of respiratory chain activity were carried out over the 3 years of oral quinone administration., Findings: Deficiency of coenzyme Q10-dependent respiratory-chain activities was identified in muscle biopsy, circulating lymphocytes, and cultured skin fibroblasts. Undetectable coenzyme Q10 and results of radiolabelling experiments in cultured fibroblasts supported the diagnosis of widespread coenzyme Q10 deficiency. Stimulation of respiration and fibroblast enzyme activities by exogenous quinones in vitro prompted us to treat the patients with oral ubidecarenone (5 mg/kg daily), which resulted in a substantial improvement of their condition over 3 years of therapy., Interpretation: Particular attention should be paid to multiple quinone-responsive respiratory-chain enzyme deficiency because this rare disorder can be successfully treated by oral ubidecarenone.

Fibroblasts were cultured to determine the involvement of peroxisomes in cholesterol and dolichol synthesis. For this purpose, the behavior of cells from patients with Zellweger syndrome, with X-linked adrenoleukodystrophy, and from nondiseased control subjects was studied. Cells both after pretreatment with mevinolin and without pretreatment were incubated in a medium containing [3H]-mevalonate. In fibroblasts from patients with peroxisomal defects, the cholesterol content and mevalonate incorporation into cholesterol were decreased by 10-20% in comparison with control cells. Mevinolin pretreatment decreased the incorporation rate of [3H]-mevalonate into cholesterol but increased the labeling of ubiquinone and dolichol both in diseased and control cells. Squalene synthase activity was unchanged, whereas the activity of farnesyl-pyrophosphate synthase was increased in the diseased states. The results show that in patients with peroxisomal deficiency neither the amount nor the rate of synthesis of cholesterol and dolichol is reduced to any greater extent.

To investigate possible persistent nephrotoxic effects of trichloroethylene (TRI), a retrospective study was carried out on 39 workers exposed to high levels of TRI from 1956 to 1975. Total protein levels in urine, as well as serum and urine creatinine and serum urea were unchanged in comparison with the control. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was applied to differentiate between tubular and/or glomerular dysfunction. Urinary excretion of alpha-1-microglobulin and glutathione transferase (GST) alpha, as markers of proximal tubular damage, were correlated with the SDS-PAGE patterns of urinary proteins both in the TRI exposed and the control group. GST alpha was found in elevated concentrations in the urine of the TRI-exposed workers. No increase of urinary GST alpha was observed in the control group, even when alpha-1-microglobulin was elevated as a result of non-toxic damage. Both in the control and exposed groups, GST pi, a marker of distal tubular damage, was in the normal range. The results show that chronic exposure to high doses of TRI causes persistent changes to the proximal tubular system of the kidney and that GST alpha excretion into the urine is a marker well suited for quantitation of the extent of renal damage.

The type and amount of lipophilic antioxidants in plasma of hyperlipidemic patients are of great importance since they play a central role in preventing deleterious oxidation of blood lipids and proteins. Isolation and quantitation of lipophilic antioxidants from hyperlipidemic plasma samples meet great obstacles because of increased levels of various intermediary lipid products. This study was designed to develop a rapid and efficient extraction and separation procedure for simultaneous analysis of ubiquinone-9 and -10 as well as alpha-, delta-, and gamma-tocopherol isomers. The levels of ubiquinone-10, alpha- and gamma-tocopherol were analyzed in human plasma samples using high-performance liquid chromatography. Lipid extraction was performed by petroleum ether/methanol/water. After phase separation, ubiquinone was reduced to ubiquinol by sodium borohydride and the lipids were separated on a C18 column. A binary gradient with solvents containing lithium perchlorate was used, and an electrochemical detector was employed for quantitation. This procedure was also efficient for the analysis of antioxidant lipids in samples containing a large number of accumulated and interfering lipid intermediates. Thus, the procedure described here is useful for efficient and rapid quantitation of ubiquinones and tocopherols in human plasma samples, especially those originating from hyperlipidemic patients.

Background: Low blood levels of antioxidants are associated with an increased risk of developing coronary artery disease. Lipophilic antioxidants are transported in lipoproteins, and hypolipidaemic therapy may therefore alter their blood concentrations., Methods: The present randomized, placebo-controlled cross-over study of 21 men with combined hyperlipidaemia examines whether 10-12 weeks of gemfibrozil treatment affects the serum concentrations of the antioxidants ubiquinone-10 or alpha- or gamma-tocopherol., Results: Gemfibrozil treatment lowered plasma triglycerides and both total and very low-density lipoprotein (VLDL)-cholesterol (P < 0.001 for all by ANOVA), whereas high-density lipoprotein (HDL)-cholesterol increased (P < 0.001). The median serum levels of ubiquinone-10 decreased from 1.30 mumol L-1 (interquartile range 0.87-1.71 mumol L-1) with placebo to 0.76 mumol L-1 (0.66-0.95) with gemfibrozil treatment (P < 0.001). Corresponding levels for alpha- and gamma-tocopherol were: 68.5 mumol L-1 (51.1-84.7) vs. 40.8 mumol L-1 (30.3-55.0) and 8.6 mumol L-1 (5.2-16.7) vs. 4.3 mumol L-1 (3.5-7.0) respectively (P < 0.001 for both). The decrease in serum antioxidants was also evident when standardized for total cholesterol (P < 0.05) or LDL-cholesterol (P < 0.001). Normolipaemic control subjects had significantly lower antioxidant levels than placebo-treated patients: ubiquinone 0.63 mumol L-1 (0.41-1.05), alpha-tocopherol 34.3 mumol L-1 (27.3-45.6) and gamma-tocopherol 3.2 mumol L-1 (2.5-4.2) (P < 0.001 for all). The association of antioxidants with lipoprotein lipids was further established by positive correlations between the levels of antioxidants and those of total cholesterol (r = 0.64, P < 0.001) or total triglycerides (r = 0.71, P < 0.001)., Conclusion: Gemfibrozil treatment of men with combined hyperlipidaemia reduces serum antioxidant levels to the levels seen in healthy normolipidaemic men. The mechanisms and the relevance of this finding remain unclear and need to be addressed in further studies.

Rats were treated with various peroxisome proliferators and concomitant changes in ubiquinone levels were monitored. In addition to clofibrate and di(2-ethylhexyl)phthalate, acetylsalicylic acid, 2-ethylhexanoic acid, thyroxine and dehydroepiandrosterone were used as proliferators. Administration of these compounds increased the contents of ubiquinone in liver and, to some extent, in kidney and muscle. No change in corresponding valued for heart or brain were observed. The treatments did not influence cholesterol levels, but increased the amounts of dolichol in the liver to various extents. Treatment of rats with the catalase inhibitor aminotriazole increased the ubiquinone levels in kidney, heart and muscle but not in liver. Comparison of peroxisomal fatty acid beta-oxidation with ubiquinone amounts in liver homogenates after treatment with a number of peroxisome proliferators demonstrated a direct correlation between these two parameters. Subcellular fractionation of liver after peroxisome proliferation revealed that the ubiquinone level was increased in mitochondria and lysosomes which are the main compartments for this lipid, but an increase was also observed in both peroxisomes and microsomes. The increase in hepatic ubiquinone after treatment with various types of proliferators was related to the decrease in blood cholesterol level. These results show that the volume of the peroxisomal compartment and the ubiquinone content in animal tissues are interrelated.

The levels and rates of biosynthesis of mevalonate pathway lipids in rat brain were investigated during development and aging. Between birth and 18 months of age there are only moderate decreases in the phospholipid and cholesterol contents but an increase in the levels of dolichyl-P and, particularly of dolichol. The amount of ubiquinone is unchanged. The rate of incorporation of [3H]leucine into protein decreases by 10% during the first year, while the incorporation of [3H]glycerol into phospholipids decreases by 20%. The high rates of [3H]mevalonate incorporation into cholesterol and dolichol after birth decreases rapidly. In contrast, the rate of incorporation into ubiquinone is constant. Squalene synthase activity decreases rapidly in the early postnatal period and at 18 months of age this activity is 10-fold lower than immediately after birth. cis-Prenyltransferase activity is also high during the first postnatal month and reaches a constant level at 4 months of age. Significantly, nonaprenyl 4-hydroxybenzoate transferase activity is high during the entire period investigated. The rate of lipid peroxidation does not change during aging. These results demonstrate that brain cholesterol and dolichol exhibit a low rate of turnover during aging, whereas ubiquinone is synthesized at a high rate and exhibits rapid turnover throughout the entire lifespan.

The proximal portion of the human kidney tubular system contains the alpha form, while the distal portion contains the pi form of glutathione transferase. These cytoplasmic proteins are released into the urine under pathological conditions, and an ELISA procedure has been developed for their quantitation. Optimal conditions with respect to concentrations of antibody and antigen and incubation times were determined. The procedure developed can detect as little as 0.5 ng enzyme per ml urine, even in the presence of high concentrations of other proteins. No cross reaction between these two isoenzymes or with a number of other proteins in the urine was observed. Antibodies interacted with these antigens in urine samples in the same manner as they interacted with the purified proteins. Storage of samples without loss of antigen required the presence of low concentrations of detergent, such as Tween 20, which both stabilized the enzymes and prevented their adsorption to the walls of the plastic tubes. The results indicate that increased urinary levels of these two enzyme proteins, as determined by the ELISA procedure, are useful markers for tubular damage.

The peroxisome proliferators perfluorooctanoic acid (PFOA; 0.02% w/w), perfluorodecanoic acid (PFDA; 0.02%, w/w), nafenopin (0.125%, w/w), clofibrate (0.5%, w/w), and acetylsalicylic acid (ASA; 1%, w/w) were administered to male C57 BL/6 mice in their diet for two weeks. Parameters for Fe3+ ADP, NADPH or ascorbic acid-initiated lipid peroxidation in vitro were measured. Approximately a twofold increase in susceptibility to lipid peroxidation was obtained for all the peroxisome proliferators tested. Cotreatment of mice with the peroxisome proliferator ASA (1%, w/w) and a catalase inhibitor, 3-amino-1,2,4-triazole (AT; 0.4%, w/w) for 7 days resulted in little inhibition of peroxisome proliferation, an elevated level of H2O2 in vivo, and total inhibition of the increased susceptibility to lipid peroxidation in vitro. No increase in lipid peroxidation in vivo was observed. Certain antioxidant enzymes (DT-diaphorase, superoxide dismutase, glutathione transferase, glutathione peroxidase, and glutathione reductase) and components (ubiquinone and alpha-tocopherol) were also measured. The results showed that there was some induction of these antioxidant enzymes and components by ASA or aminotriazole, except for glutathione peroxidase and superoxide dismutase, which were inhibited. The possible involvement of oxidative stress in the carcinogenicity of peroxisome proliferators is discussed.

Three branch-point enzymes of the mevalonate pathway, farnesyl pyrophosphate synthase, cis-prenyltransferase and squalene synthase were characterized in rat hepatic cytosol, microsomes and peroxisomes isolated from rats after treatment with peroxisome proliferators, inducers of the endoplasmic reticulum or modulators of lipid metabolism. Cholestyramine and phenobarbital induced primarily the cytosolic farnesyl pyrophosphate synthase, whereas clofibrate and phthalates elevated the corresponding peroxisomal activity. cis-Prenyltransferase activities in microsomes were induced 4-5-fold after clofibrate, phthalate and phenobarbital administration, but these same treatments affected the peroxisomal activity to only a limited extent. Squalene synthase activity in microsomes was completely abolished, but the peroxisomal activity was unaffected after administration of cholesterol. On the other hand, clofibrate and phthalate induced only the microsomal activities. Mevinolin treatment greatly increased peroxisomal and cytosolic farnesyl pyrophosphate synthase activities, but not the mitochondrial activity, and the cis-prenyltransferase activities were elevated in peroxisomes, but not in microsomes. These results demonstrate that the branch-point enzymes in cholesterol and dolichol biosynthesis at various cellular locations are regulated differentially and that the capacities of peroxisomes and the endoplasmic reticulum to participate in the synthesis of polyisoprenoid lipids is affected profoundly by treatment with different xenobiotics.

A radioimmunoassay procedure for the quantitation of glutathione transferase-pi was developed in order to determine the levels of this protein in human urine. The enzyme was isolated from human placenta with a purification factor of 366 (compared to the original high-speed supernatant fraction), and upon gel electrophoresis, only a single band was seen. Polyclonal antisera were subsequently raised in rabbits and found to be suitable for a radioimmunoassay. Glutathione transferase-pi was localized immunohistochemically to the cells of the distal tubules, the thin loop of Henle and the collecting ducts in the kidney. In contrast, the alpha-isoenzyme was localized exclusively in the proximal tubular epithelium. Samples of urine from healthy individuals contained about 6 ng of the pi-transferase/ml. The method proved to be specific for glutathione transferase-pi, and no cross-reaction with the alpha- or mu-transferase or with other proteins occasionally appearing in urine occurred. The protein was quite stable upon storage and insensitive to variations in the urine pH. Thus, it appears that glutathione transferase-pi can be conveniently quantitated by radioimmunoassay and changes in the concentration of this protein in human urine thus monitored.

Glutathione transferase-pi released from kidney tubular epithelial cells was analyzed in the urine of recipients of renal allografts. Urinary content of alpha-class glutathione transferase was also determined for comparison. Control urine from healthy individuals contained detectable levels of the pi-isoenzyme (6.6 +/- 0.46 ng/ml, mean +/- SEM) and this concentration was not increased in the urine of patients demonstrating cyclosporine A-induced nephrotoxicity (6.3 +/- 0.29 ng/ml), in contrast to the alpha-form. Acute rejection increased excretion of the pi-isoenzyme (19.0 +/- 2.0 ng/ml), but not of the alpha-glutathione transferase. Thus, while the serum creatinine level increases in connection with both cyclosporine A-induced nephrotoxicity and acute rejection, analyses of urinary glutathione transferases distinguish well between these conditions. Acute tubular necrosis and renal transplant infarction resulted in a rapid elevation in urinary levels of both alpha- and pi-transferase. The advantages of this approach are that release of the protein into the urine occurs rapidly after tubular damage, the assay is sensitive and specific and can also distinguish between certain pathological conditions. These studies thus indicate that the urinary level of glutathione transferase-pi can be used for monitoring certain pathological processes in the kidney. Quantitation of this enzyme complements the information obtained by measurement of glutathione transferase-alpha.

The distribution of alpha and pi class glutathione transferases in autopsy and biopsy samples of normal human tissues was investigated by immunohistochemistry. The class alpha glutathione transferases exhibited restricted distribution. Intensive staining was visible in all hepatocytes, in kidney proximal tubular cells, in the zona reticularis of adrenal cortex and in Leydig cells of testis. Staining of lesser intensity could also be observed in the gastrointestinal epithelium, exocrine pancreas and some bile and pancreas ducts. In colon and gall bladder only nuclei were stained, but in the other tissues both nuclei and cytoplasm contained alpha class glutathione transferases. Glutathione transferase pi exhibited a more general distribution and could be observed in epithelia of the respiratory, gastrointestinal and urinary tracts, in all endocrine cells investigated, and also in the exocrine glands of prostate, in smooth muscle, adipocytes, blood vessel endothelium and placenta. It was also visible in the Schwann cells of peripheral nerves and in the choroid plexus. In gall bladder and colon only nuclei were stained, while in the intrahepatic bile ducts only cytoplasm was stained. All other positive cells exhibited glutathione transferase pi in both nuclei and cytoplasm.

Isolated peroxisomes were able to utilize [3H]isopentenyl diphosphate to synthesize farnesyl diphosphate, which then was utilized as substrate by both the peroxisomal squalene synthetase and cis-prenyltransferase. The specific activity of squalene synthetase in peroxisomes was as high as in microsomes, i.e. 160 pmol/mg of protein/min. If NADPH was omitted from the assay medium, presqualene diphosphate accumulated, which indicates that the reaction occurs in two steps, as in microsomes. In the presence of NADPH, incorporation from [3H]farnesyl diphosphate was stimulated 3-fold, and the major products were squalene and cholesterol. The specific activity of cis-prenyl-transferase in peroxisomes was 4-fold higher than in microsomes, i.e. 456 pmol of isopentenyl diphosphate incorporated/mg of protein/h. There were two major products formed from farnesyl diphosphate and [3H] isopentenyl diphosphate, i.e. trans,trans,cis-geranylgeranyl diphosphate and long chain polyprenyl diphosphates. The polyprenyl diphosphates had the same chain length distribution as that of dolichol derivatives in rat liver, with the dominating polyisoprenes being C90 and C95. In contrast to the microsomal enzyme, peroxisomal cis-prenyltransferase did not require detergents for optimal activity. The enzyme was associated primarily with the peroxisomal membrane after sonication of the peroxisomes.

The distribution and redox state of ubiquinone in rat and human tissues have been investigated. A rapid extraction procedure and direct injection onto HPLC were employed. It was found in model experiments that in postmortem tissue neither oxidation nor reduction of ubiquinone occurs. In rat the highest concentrations of ubiquinone-9 were found in the heart, kidney, and liver (130-200 micrograms/g). In brain, spleen, and intestine one-third and in other tissues 10-20% of the total ubiquinone contained 10 isoprene units. In human tissues ubiquinone-10 was also present at highest concentrations in heart, kidney, and liver (60-110 micrograms/g), and in all tissues 2-5% of the total ubiquinone contained 9 isoprene units. High levels of reduction, 70-100%, could be observed in human tissues, with the exception of brain and lung. The extent of reduction displayed a similar pattern in rat, but was generally lower.

Myelin bodies were isolated from the renal cortex of gentamicin-treated rats (100 mg/kg body weight, twice daily for 3 days, i.p.) employing an initial pelleting by differential centrifugation and subsequent flotation on a discontinuous sucrose gradient. These structures were found to contain almost twice as much protein as phospholipid and SDS-polyacrylamide gel electrophoresis revealed the presence of many different polypeptides. All the major phospholipids are present, although myelin bodies contain a considerably higher proportion of phosphatidylinositol, somewhat more phosphatidylcholine and considerably lower percentages of phosphatidylserine and sphingomyelin than do normal renal phospholipids. The fatty acids of myelin body phospholipids are highly saturated (67.3-87.9%) and a striking feature is the occurrence of relatively large amounts of 22:1, presumably erucic acid, especially in sphingomyelin. Myelin bodies contain small amounts of unesterified cholesterol, unesterified dolichol and coenzymes Q9 and Q10.

HepG2 cells were cultured in the presence of different concentrations of cyclosporin A (CsA) or Nva2-cyclosporin (Nva2-Cs) for up to 20 days. At a low concentration (2 micrograms/ml) of CsA or Nva2-Cs, the [3H]thymidine incorporation into DNA and the rate of incorporation of [3H]leucine into total protein decreased by 20-25%. Concentrations of 10 micrograms/ml resulted in a 70% reduction of the [3H]thymidine incorporation in comparison with controls. Low concentrations of CsA resulted in mitochondria in the condensed state together with autophagosomes, large vacuoles, and elevated numbers of coated vesicles, as shown by electron microscopy. Low concentrations of Nva2-Cs resulted in swollen mitochondria, increased autophagocytosis, and increased numbers of intermediate filaments and microtubules. Higher doses of these substances (5 micrograms/ml) caused disarrangement of mitochondrial cristae, vesiculation of the endoplasmic reticulum, an elevated number of free polysomes, and accelerated autophagocytosis. Labeling of phospholipids and triglycerides with [3H]glycerol and of cholesterol and dolichol with [3H]acetate was decreased after exposure of HepG2 cells to CsA, or, in particular, Nva2-Cs. Phospholipids secreted from the cells into the medium exhibited an increased level of labeling, but the specific radioactivity of the neutral lipids in the medium was significantly decreased. Treatment of HepG2 cells with either CsA or Nva2-Cs doubled the mitochondrial cytochrome oxidase and carnitine acetyl-transferase, as well as microsomal NADPH-cytochrome c reductase activities. Such treatment also increased the cyanide-insensitive beta-oxidation of fatty acids in peroxisomes, as well as cytoplasmic DT-diaphorase and glutathione transferase activities. Prolonged treatment of the cells with CsA did not result in any cumulative effect. HepG2 cells appear to be suitable for studying the effects of cyclosporins on cellular structure and metabolism and in this system the two drugs studied here exhibited similar effects.

Cholesterol biosynthesis by isolated rat liver peroxisomes was examined. Labeling of cholesterol from [3H]-mevalonate in the presence of peroxisomes required the addition of cytosol, since peroxisomes, like microsomes, apparently possess only those enzymes of cholesterol biosynthesis subsequent to the steps involving farnesyl-PP. Under the conditions employed the amounts of 4,4-dimethyl and desmethyl sterols generated by peroxisomes were equal to or exceeded those produced by the microsomes. In addition, marker enzyme analysis demonstrated minimal microsomal contamination in the peroxisomal fraction. The metabolite patterns observed by HPLC after incubation of these two fractions with [3H]mevalonate were different. Dihydrolanosterol oxidase, steroid-14-reductase, steroid-8-isomerase, and steroid-3-ketoreductase activities were present in peroxisomes. Separation of peroxisomes into membranes and contents revealed that all the synthesizing activities are associated with the membrane fraction. 7 alpha-Hydroxylase, which catalyzes the first step in the biosynthesis of bile acids, was also present in peroxisomes, but it remains to be clarified to what extent peroxisomal cholesterol is a substrate for bile acid synthesis.

Rats were treated with inducers of peroxisomes, mitochondria and the endoplasmic reticulum, as well as receiving diets and drug known to influence the mevalonate pathway. Treatment with clofibrate and 2-diethylhexylphthalate (DEHP) increased microsomal and mitochondrial ubiquinone contents, but a decrease was observed in lysosomes. In vivo labeling of this lipid with [3H]mevalonate was also elevated. The amount of cholesterol did not change upon exposure to these inducers of peroxisomes and mitochondria, but its rate of labeling was decreased. The concentration of dolichol increased only after treatment with DEHP and only in lysosomes. The inducers of the endoplasmic reticulum phenobarbital, 3-methylcholanthrene and N-nitrosodiethylamine enhanced the rate of ubiquinone synthesis and exposure to the latter two substances also elevated the amount of this lipid in microsomes. A cholesterol-rich diet increased the labeling of ubiquinone and decreased cholesterol labeling, while cholestyramine treatment had opposite effects on lipid labeling in both microsomes and mitochondria. The results demonstrate that the ubiquinone contents of the various membranes of hepatocytes change in a characteristic manner under the influence of inducers and dietary factors. Clearly, the level of ubiquinone and its biosynthesis are regulated separately from those of the other products of the mevalonate pathway, cholesterol and dolichol.

The influence of the immunosuppressive agent cyclosporine A on protein synthesis was investigated in rat liver in vivo and in vitro. Incorporation of [14C]leucine into total protein by microsomes was inhibited 36% in the presence of cyclosporine A. Treatment of rats with cyclosporine A also resulted in decreased protein synthesis by microsomes isolated from the same animals. This inhibition was dependent on the dose administered and the duration of treatment. The inhibitory factor in this latter case appeared to be associated with the supernatant fraction. Decreased in vivo incorporation of [35S]methionine into proteins of microsomal membranes and peroxisomes, but not into microsomal luminal proteins was also observed after treatment of rats with cyclosporine A. On SDS-polyacrylamide gel electrophoresis certain of the microsomal membrane and peroxisomal proteins displayed decreased labeling. Furthermore, cyclosporine A treatment decreased the inductive effects of phenobarbital, clofibrate, and phthalate ester on microsomal and peroxisomal enzymes. It is suggested that certain of the toxic effects of cyclosporine A are exerted through inhibition of cellular protein synthesis.

The levels of cholesterol, ubiquinone, dolichol, dolichyl-P, and total phospholipids in human lung, heart, spleen, liver, kidney, pancreas, and adrenal from individuals from one-day-old to 81 years of age were investigated and compared with the corresponding organs from 2 to 300 day-old rats. The amount of cholesterol in human tissues did not change significantly during aging, but the level of this lipid in the rat was moderately elevated in the organs of the oldest animals. In human pancreas and adrenal the ubiquinone content was highest at one year of age, whereas in other organs the corresponding peak value was at 20 years of age, and was followed by a continuous decrease upon further aging. A similar pattern was observed in the rats, with the highest concentration of ubiquinone being observed at 30 days of age. Dolichol levels in human tissues increase with aging, but they increase to very different extents. In the lungs this increase is seven-fold, and in the pancreas it is 150-fold. The elevation in the dolichol contents of rat tissues ranges from 20 to 30-fold in our material. In contrast, the levels of the phosphorylated derivative of dolichol increased to a more limited extent, i.e., 2 to 6-fold in human tissues and even less in the rat. These results demonstrate that the levels of a number of lipids in human and rat organs are modified in a characteristic manner during the life-span. This is in contrast to phospholipids, which constitute the bulk of the cellular lipid mass.

The relative rate of biosynthesis of dolichol from [3H]mevalonate in nine rat organs was studied in slices and in the whole animal. This biosynthesis was also compared to that of cholesterol and ubiquinone. All tissues examined are able to synthesize dolichol, as well as ubiquinone and cholesterol. Comparison of the data from slices in vitro with the in vivo studies demonstrated relatively good agreement for dolichol and ubiquinone synthesis. Although dolichol of high specific radioactivity was recovered in the blood, redistribution between organs, such as occurs with cholesterol, appears to be insignificant. The highest rates of dolichol biosynthesis were found in kidney, spleen and liver. On the other hand, muscle makes the largest contribution to total body dolichol synthesis. Newly synthesized dolichol also appears in the bile, but excretion by this route is far from sufficient to account for dolichol turnover. Incorporation of mevalonate into the final products is mainly dependent on biosynthetic activity. For comparison of the biosynthetic rates in different organs, possible sources of errors (such as variations in the size of the precursor pool, limitation by the rate of precursor uptake or non-linear incorporation) were investigated the size of the mevalonate pool in various organs. Equilibration of this pool with exogenous mevalonate is a rapid and passive process. The size of the mevalonate pool does not determine the rates of cholesterol and dolichol biosynthesis, indicating the presence of regulatory steps in the terminal portion of these biosynthetic pathways.