Eeva-Liisa Appelkvist

Quinone-responsive multiple respiratory-chain dysfunction due to widespread coenzyme Q10 deficiency

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.

Age-related Changes in the Lipid Compositions of Rat and Human Tissues

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.

Distribution and redox state of ubiquinones in rat and human tissues

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.

Glutathione transferase alpha as a marker for tubular damage after trichloroethylene exposure

Abstract 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.

Effects of clofibrate, phthalates and probucol on ubiquinone levels

Rats were exposed through their diet to clofibrate, di(2-ethylhexyl)phthalate or probucol for 6 weeks and the levels of ubiquinone (UQ), cholesterol and dolichol were monitored in liver, muscle, heart, brain and blood. The levels of UQ-9 and -10 were increased by clofibrate and, in particular by phthalate administration. With the latter agent this increase in liver was fourfold, in muscle was twofold and levels in the heart and blood increased by 20%, whereas there was no change in the brain. Probucol led to a moderate decrease in the level of UQ in liver, muscle and blood, but not in heart or brain. The extent of reduction of UQ was not modified by any of the treatments employed. Probucol did not have any effect on tissue or blood cholesterol levels, whereas clofibrate or phthalate elicited a variable response, including both increases and decreases depending on the tissue analyzed. Phthalate treatment increased the dolichol content to some extent in all tissues and in blood, but the level of this lipid was not modified upon clofibrate or probucol treatment. These results demonstrate that tissue and blood levels of UQ can be increased by exposure to appropriate chemical agents without elevating the concentration of cholesterol.

Quantitation of glutathione transferase-pi in the urine by radioimmunoassay.

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.

Effects of inhibitors of hydroxymethylglutaryl coenzyme A reductase on coenzyme Q and dolichol biosynthesis

SummaryInhibitors of hydroxymethylglutaryl coenzyme A reductase are used clinically to decrease blood levels of low-density lipoprotein cholesterol in hypercholesterolemic patients. However, little is known about the possible effects of these inhibitors on dolichol and cholesterol synthesis. Oral administration of mevinolin to rats was found here to decrease dolichol, dolichyl-P and coenzyme Q levels in the heart and skeletal muscle and to increase the hepatic dolichol level while decreasing the coenzyme Q content in this same organ. The amounts of dolichyl-P decreased in heart and muscle and increased in brain. Intraperitoneal administration also affected the levels of these lipids. The concentrations of blood lipids were not modified in the same manner as tissue lipids. Analysis of individual enzyme activities and of incorporation of [3H]acetate into various lipids of liver and brain slices demonstrated that both up- and down-regulation of different proteins occur in various tissues, resulting in modifications in lipid synthesis. Hypercholesterolemic patients were found to have high blood coenzyme Q levels, which are decreased upon pravastatin treatment, although they are still above control values. It appears that these HMG-coenzyme A reductase inhibitors do not selectively lower cholesterol levels, but that they also modify the dolichol and coenzyme Q content and synthesis both in the liver and various other tissues.

The effects of inducers of the endoplasmic reticulum, peroxisomes and mitochondria on the amounts and synthesis of ubiquinone in rat liver subcellular membranes

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.

In vitro labeling of peroxisomal cholesterol with radioactive precursors

Peroxisomes isolated from rat liver were incubated with [3H]squalene and [3H]mevalonate and the subsequent incorporation of radioactivity into cholesterol studied. The isolated lipids became labeled after incubation with both precursors. In contrast to findings with microsomes, trypsin and detergent treatment of peroxisomes did not influence the rate of cholesterol synthesis. In addition, the luminal content of peroxisomes could alone mediate this synthetic process. Upon treatment of rats with various inducers of peroxisomes and of the endoplasmic reticulum, as well as upon feeding with cholesterol and cholestyramine, large differences in the pattern ofin vitro incorporation of [3H]mevalonate into the cholesterol of peroxisomes and microsomes were observed. Injection of this precursor also resulted in high initial labeling of peroxisomal cholesterolin vivo. These experiments indicate that cholesterol synthesis may also occur in peroxisomes.

Nonmembrane associated dolichol in rat liver

The distribution of dolichol in rat liver was studied. Upon high-speed centrifugation, 9% of the total tissue dolichol was recovered in the supernatant. Dolichol was enclosed in vesicles and in lipidic particles which were isolated by gel filtration and density gradient centrifugation. The particles had a diameter of 20 nm and contained dolichol, ubiquinone, cholesterol, phospholipid and some protein. Similar particles were recovered upon incubation of isolated hepatocytes with liposomes containing dolichol. From the lysosomal lumen, lipid particles containing dolichol, ubiquinone, cholesterol and phospholipid, but no protein, were isolated. The diameter of the particles was 20–40 nm with a molecular weight of 130 kDa. Puromycin treatment inhibited protein synthesis, but did not affect dolichol transfer from the endoplasmic reticulum to lysosomes, suggesting that the transfer is not mediated by newly synthesized apoprotein. The results indicate that a sizeable portion of the total cellular dolichol is present in cytoplasm and in lysosomal lumen. Furthermore, dolichol probably participates in the translocation process.