Separation and identification of triglycerides, cholesteryl esters, cholesterol, 7-dehydrocholesterol, dolichol, ubiquinone, alpha-tocopherol, and retinol by high performance liquid chromatography with a diode array detector

A simple isocratic high performance liquid chromatography (HPLC) system is described that allows separation and identification of cholesteryl esters, triglycerides, ubiquinone, alpha-tocopherol, dolichol, cholesterol, 7-dehydrocholesterol, and retinol. This consisted of a normal phase cyanopropyl column with 0.1% isopropanol in heptane as the solvent. The effluent was monitored with an LKB model 2140 diode array detector which enabled the lipids to be identified by their characteristic absorption spectra. This system was applied to a sample of dog liver in which cholesteryl esters, retinyl esters, triglycerides, ubiquinone, dolichol, cholesterol, and retinol were identified. Retinyl esters and vitamin D esters were identified by their similarity in absorption spectra to retinol and vitamin D. A system to transfer and store the chromatograms on the VAX PDP-11 or an optical disc is also described.     

Effects of pravastatin and cholestyramine on products of the mevalonate pathway in familial hypercholesterolemia

Patients with heterozygous familial hypercholesterolemia (n = 12) were treated either with pravastatin, a specific inhibitor of HMG-CoA reductase, or cholestyramine, followed by a period of combined treatment with both drugs. Initially, these patients had increased serum levels of low density lipoprotein (LDL) cholesterol (8.77 +/- 0.48 mmol/l; SEM), lathosterol (5.32 +/- 0.60 mg/l), and ubiquinone (0.76 +/- 0.09 mg/l), while the serum dolichol concentration was in the normal range. Cholestyramine treatment (n = 6) decreased the levels of LDL cholesterol (-32%) and increased lathosterol (+125%), but did not change dolichol or ubiquinone levels in a significant manner. Pravastatin treatment (n = 6) decreased LDL cholesterol (-27%), lathosterol (-46%), and ubiquinone (-29%). In this case, the amount of dolichol in serum also showed a small but statistically insignificant decrease (-16%) after 12 weeks of treatment. Combined treatment with cholestyramine and pravastatin (n = 6) resulted in changes that were similar to, but less pronounced than, those observed during pravastatin treatment alone. In no case was the ratio between ubiquinone and LDL cholesterol reduced. Possible effects on hepatic cholesterol, ubiquinone, and dolichol concentrations were studied in untreated (n = 2), cholestyramine-treated (n = 2), and pravastatin-treated (n = 4) gallstone patients and no consistent changes could be observed. The results indicate that treatment with pravastatin in familial hypercholesterolemia decreases serum ubiquinone levels in proportion to the reduction in LDL cholesterol.     

In vitro and in vivo synthesis of dolichol and other main mevalonate products in various organs of the rat

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.     

Effects of mevinolin treatment on tissue dolichol and ubiquinone levels in the rat.

Rats were treated with mevinolin by intraperitoneal injection (15 days) or dietary administration (30 days). The cholesterol, dolichol, dolichyl phosphate and ubiquinone contents of the liver, brain, heart, muscle and blood were then investigated. The cholesterol contents of these organs did not change significantly, with the exception of muscle. Intraperitoneal administration of the drug increases the amount of dolichol in liver, muscle and blood and decreases the dolichyl-P amount in muscle. The same treatment increases the level of ubiquinone in muscle and blood and decreases this value in liver and heart. Oral administration decreases dolichol, dolichyl-P and ubiquinone levels in heart and muscle, while in liver the dolichol level is elevated and ubiquinone level lowered. In brain the amount of dolichyl-P is increased. Intraperitoneal injection of mevinolin also modifies the liver dolichol and dolichyl-P isoprenoid pattern, with an increase in shorter chain polyisoprenes. The levels of dolichol and ubiquinone in the blood do not follow the changes observed in other tissues. Incorporation of [3H]acetate into cholesterol by liver slices prepared from mevinolin-treated rats exhibited an increase, whereas in brain no change was seen. Labeling of dolichol and ubiquinone was increased in both liver and brain, but incorporation into dolichyl phosphate remained relatively stable. The results indicate that mevinolin affects not only HMG-CoA reductase but, to some extent, also affects certain of the peripheral enzymes, resulting in considerable effects on the various mevalonate pathway lipids.     

Increased nonsterol isoprenoids, dolichol and ubiquinone, in the Smith-Lemli-Opitz syndrome: effects of dietary cholesterol

Smith-Lemli-Opitz syndrome (SLOS) is an inherited autosomal recessive cholesterol deficiency disorder. Our studies have shown that in SLOS children, urinary mevalonate excretion is normal and reflects hepatic HMG-CoA reductase activity but not ultimate sterol synthesis. Hence, we hypothesized that in SLOS there may be increased diversion of mevalonate to nonsterol isoprenoid synthesis. To test our hypothesis, we measured urinary dolichol and ubiquinone, two nonsterol isoprenoids, in 16 children with SLOS and 15 controls, all fed a low-cholesterol diet. The urinary excretion of both dolichol (P < 0.002) and ubiquinone (P < 0.02) in SLOS children was 7-fold higher than in control children, whereas mevalonate excretion was comparable. In a subset of 12 SLOS children, a high-cholesterol diet decreased urinary mevalonate excretion by 61% (P < 0.001), dolichol by 70% (P < 0.001), and ubiquinone by 67% (P < 0.03). Our hypothesis that in SLOS children, normal urinary mevalonate excretion results from increased diversion of mevalonate into the production of nonsterol isoprenoids is supported. Dietary cholesterol supplementation reduced urinary mevalonate and nonsterol isoprenoid excretion but did not change the relative ratios of their excretion. Therefore, in SLOS, a secondary peripheral regulation of isoprenoid synthesis may be stimulated.     

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.     

Rates of cholesterol, ubiquinone, dolichol and dolichyl-P biosynthesis in rat brain slices

Slices from the brain and liver of rats were prepared and upon incubation exhibited a continuous and high capacity for incorporation of radioactive precursors into proteins and lipids. Using [3H]mevalonate as precursor, the rates of biosynthesis of cholesterol, ubiquinone, dolichol and dolichyl-P in brain slices were determined and found to be 5.5, 0.25, 0.0093 and 0.0091 nmol/h/g, respectively. Dolichol and dolichyl-P accumulate to a limited extent, but almost all of these lipids in the brain originate from de novo synthesis. The calculated half-lives for cholesterol, ubiquinone, dolichol and dolichyl-P were 4076, 90, 1006 and 171 h, respectively. The results indicate that lipids formed via the mevalonate pathway in the brain have an active and independently regulated biosynthesis.     

Depression of hepatic dolichol levels by cholesterol feeding

A study was conducted to determine whether repression of 3-hydroxy-3-methylglutaryl CoA reductase by a chronic high-cholesterol diet would deplete hepatic dolichol levels. Four-week-old male C57BL/6J mice were maintained on a control diet or a diet supplemented with 5% cholesterol. Animals from both groups were killed at various times and reductase activity and levels of free dolichol, dolichyl acyl ester, dolichyl phosphate, and ubiquinone were measured. The reductase activity was reduced by 90% within 1 week and remained depressed through 56 days. Initially, the levels of the free dolichol, acyl ester, phosphoryl ester, and ubiquinone were 7, 16, 5, and 80 micrograms/g liver, respectively. Early increases in the concentration of dolichyl phosphate and free dolichol were similar in both the cholesterol-fed and control groups. However, in the cholesterol-fed group the concentration of dolichyl acyl esters was only 50% of that in the control group by 7 days and it remained lower throughout the experiment. Total dolichol levels were lower by about 30%. Ubiquinone levels were transiently depressed at 7 days by 33% but returned to control levels by 4 weeks. After 56 days, the control values of dolichol and dolichyl phosphate remained constant whereas the dolichyl acyl ester levels continuously increased to a value of 133 micrograms/g of liver by 156 days. Subcellular fractionation of livers from 4-week-old mice indicated a lysosomal distribution of dolichol and dolichyl acyl ester and a lysosomal and microsomal distribution of dolichyl phosphate.     

Studies on digoxin--14C-acetate incorporation in to digoxin and degenerative changes in the brain in rats administered digoxin

The human hypothalamus produces an endogenous membrane Na+-K+ ATPase inhibitor digoxin. Digoxin is a steroidal glycoside and could be synthesised by the isoprenoid pathway. The other metabolites of the isoprenoid pathway are cholesterol, dolichol and ubiquinone. We have tried to find out the extent of incorporation of 14C acetate into digoxin in rat brain. The effects of digoxin administration on the rat brain was also studied. The results show that the percentage incorporation of 14C acetate into digoxin is low but detectable. The maximum incorporation was observed for cholesterol, followed by dolichol and finally ubiquinone. The histopathological changes observed after digoxin administration were focal degeneration of the ganglion cells in the cerebrum and cerebellum. The carbohydrate components of the glycoproteins were reduced and the concentration of serotonin, dopamine, and epinephrine showed a significant increase. The role of digoxin in mediating neuronal cell death is discussed.     

Relief of temperature sensitivity in a concanavalin A resistant Chinese hamster ovary cell line auxotrophic for cholesterol

The concanavalin A resistant, glycosylation-deficient, Chinese hamster ovary cell variant CR-7 is auxotrophic for cholesterol owing to an inability to adequately convert lanosterol to cholesterol. It is also temperature sensitive for growth, being unable to proliferate at 39 degrees C. Temperature sensitivity was relieved by addition of mevalonolactone, dolichol, or dolichyl-P to the growth medium, provided that cholesterol was also present in amounts sufficient to overcome cholesterol auxotrophy at 34 degrees C. Other metabolites of mevalonolactone (squalene, ubiquinone, lanosterol, and isopentenyladenine) were inactive in this regard. Measurement of dolichol levels in CR-7 and wild-type cells at 34 degrees C and after exposure to 39 degrees C showed that dolichol increased at 39 degrees C to an approximately equal extent in both cell types. Dolichol, dolichyl-P, ubiquinone, and isopentenyladenine had no effect on the sensitivity of either wild-type or CR-7 cells to the cytotoxic effects of concanavalin A. Mevalonolactone or lanosterol markedly increased the resistance of CR-7 to the lectin, but had no effect on wild-type cells. This raises the possibility that the presence of unusually large amounts of lanosterol, coupled with low amounts of cholesterol, in the membranes of CR-7 may be related to its concanavalin A resistance and other characteristic phenotypic abnormalities.     

A simple method for the isolation of vitamin D metabolites from plasma extracts

A simple method has been developed using 'SEP-PAK' disposable silica cartridges to separate the major endogenous vitamin D metabolites, namely vitamin D3, 25-hydroxy vitamin D3 (25OHD3), 1,25 dihydroxy vitamin D3 (1.25 (OH)2D3) and 24,25 dihydroxyvitamin D3 (24,25 (OH) 2D3). After extraction of plasma in isopropanol-toluene (25:75) the dried extract is reconstituted in hexane; this is applied to a SEP-PAK column, and stepwise elution carried out under gravity with 0.1 divided by isopropanol in hexane (neutral lipids), 1% isopropanol in hexane (D3), 3 divided by isopropanol in hexane (25OHD3), 3.125 divided by ethanol in dichloromethane (24,25 (OH) 2D3) and 50 divided ethanol in toluene (1, 25(OH) 2D3). Complete separation of these D3 metabolites is achieved by this process and up to 40 samples can be handled at one time.If combined with a suitable ligand binding assay, the system appears to be suitable for preparation of samples prior to the routine assay of vitamin D metabolites.     

Ubiquinone, dolichol, and cholesterol metabolism in aging and Alzheimer's disease.

The lipid compositions of various regions of the human brain were investigated during aging and in Alzheimer's disease. The phospholipid amounts and compositions remained unchanged during aging. There were, however, considerable differences both in phospholipid composition and amount when the various regions were compared. The level of dolichol increased severalfold in all regions up to the age of 70, but there was no further elevation thereafter. The ubiquinone level decreased significantly in all parts of the brain upon aging. In Alzheimer's disease, the dolichol level was decreased in all regions, and particularly, in those affected by the disease. In contrast, the dolichyl-P concentration increased in those regions that exhibited morphological changes. There was no modification in cholesterol distribution, but a significant elevation in ubiquinone content was observed in most regions. The only phospholipid whose level was elevated was phosphatidylinositol, and only in those parts of the brain that were affected. The content of polyunsaturated fatty acids in phosphatidylethanolamine was greatly decreased in connection with the disease, with a parallel increase in the saturated portion. The results indicate that Alzheimer's disease results in specific and significant changes in the levels of lipid products of the mevalonate pathway in the brain.     

Depletion of essential isoprenoids and ER stress induction following acute liver-specific deletion of HMG-CoA reductase

HMG-CoA reductase (Hmgcr) is the rate-limiting enzyme in the mevalonate pathway and is inhibited by statins. In addition to cholesterol, Hmgcr activity is also required for synthesizing nonsterol isoprenoids, such as dolichol, ubiquinone, and farnesylated and geranylgeranylated proteins. Here, we investigated the effects of Hmgcr inhibition on nonsterol isoprenoids in the liver. We have generated new genetic models to acutely delete genes in the mevalonate pathway in the liver using AAV-mediated delivery of Cre-recombinase (AAV-Cre) or CRISPR/Cas9 (AAV-CRISPR). The genetic deletion of Hmgcr by AAV-Cre resulted in extensive hepatocyte apoptosis and compensatory liver regeneration. At the biochemical level, we observed decreased levels of sterols and depletion of the nonsterol isoprenoids, dolichol and ubiquinone. At the cellular level, Hmgcr-null hepatocytes showed ER stress and impaired N-glycosylation. We further hypothesized that the depletion of dolichol, essential for N-glycosylation, could be responsible for ER stress. Using AAV-CRISPR, we somatically disrupted dehydrodolichyl diphosphate synthase subunit (Dhdds), encoding a branch point enzyme required for dolichol biosynthesis. Dhdds-null livers showed ER stress and impaired N-glycosylation, along with apoptosis and regeneration. Finally, the combined deletion of Hmgcr and Dhdds synergistically exacerbated hepatocyte ER stress. Our data show a critical role for mevalonate-derived dolichol in the liver and suggest that dolichol depletion is at least partially responsible for ER stress and apoptosis upon potent Hmgcr inhibition.     

Influence of peroxisome proliferator-activated receptor alpha on ubiquinone biosynthesis

The control of ubiquinone biosynthesis by peroxisome proliferators was investigated using peroxisome proliferator activated receptor alpha (PPARalpha)-null mice. Administration of 2-(diethylhexyl)phthalate to control mice resulted in elevated ubiquinone levels in the liver, while dolichol, dolichyl-P and cholesterol concentrations remained unchanged. In PPARalpha-null mice, the level of these lipids were similar to control levels and administration of the peroxisome proliferator did not increase the levels of ubiquinone. The increase in ubiquinone levels was the result of increased synthesis. Induction was most pronounced in liver, kidney and heart, which have relatively high levels of PPARalpha. When the tissue concentration of hydrogen peroxide was elevated by inhibition of catalase activity with aminotriazole, the amount of ubiquinone was not increased, suggesting that the induction of ubiquinone synthesis occured through a direct mechanism. The activities of branch-point enzymes FPP-synthase, squalene synthase, cis-prenyltransferase, trans-prenyltransferase and NPHB-transferase were substantially increased in control but not in PPARalpha-null mice after treatment with peroxisome proliferators. These data suggest that the induction of ubiquinone biosynthesis after administration of peroxisome proliferators is dependent on the PPARalpha through regulation of some of the mevalonate pathway enzymes.     

A Chinese hamster ovary cell mutant F2A8 utilizes polyprenol rather than dolichol for its lipid-dependent asparagine-linked glycosylation reactions

Previous results suggested that F2A8, a glycosylation mutant of Chinese hamster ovary cells, had a lower amount of dolichyl phosphate available for asparagine-linked glycosylation reactions relative to parental cells. The steady-state amounts and identities of polyisoprenoid lipids were determined by incubating F2A8, its parental cell line B4-2-1, and wild-type Chinese hamster ovary cells for 24 h with [2-3H]mevalonate. The neutral lipids, ubiquinone, cholesterol, and cholesteryl esters, which were the most highly labeled from [3H]mevalonate, were labeled equally in all three cell types. In wild-type and B4-2-1 cells, mevalonate incorporation into the anionic glycosylated and phosphorylated derivatives of dolichol was 10-fold higher than into the neutral free dolichol and dolichyl esters. In contrast, in F2A8 cells, label accumulated in neutral polyisoprenol lipids, so that the ratio of neutral to anionic lipids was 1:1 rather than 1:10. In wild-type and B4-2-1 cells, the polyisoprenoid found as free alcohol and in phosphorylated and glycosylated forms was shown by high pressure liquid chromatography using a silica column to be primarily dolichol, a polyisoprenol that has a saturated terminal isoprene unit. In contrast, in F2A8 cells the polyisoprenoid found primarily as the free alcohol and in phosphorylated and glycosylated forms appeared to be completely unsaturated polyprenol. The distribution of chain lengths of the labeled polyisoprenols of F2A8, B4-2-1, and wild-type cells was the same as determined by high pressure liquid chromatography using a reverse-phase column, with the predominant chain length being 19 isoprene units. These results combined with our previous studies on the phenotype of the F2A8 mutant indicate that the unsaturated polyprenyl phosphate derivatives do not function as well as dolichyl phosphate derivatives in cellular glycosylation reactions.     

Multiple forms of ACTH biological activity in the pars intermedia of the rat adenohypophysis.

Acid extracts of a) acutely dispersed rat pars intermedia (PI) cells, b) media after incubation of PI cells, c) whole nervosa-intermedia, and d) whole pars distalis, were chromatographed on Sephadex G-50 Fine in 1% acetic acid. Three peaks of ACTH biological activity were resolved in all four extracts. Peak I eluted in the void volume of the column, peak III co-eluted with synthetic ACTH^1–39, and peak II eluted in an intermediate position. The predominant ACTH activity derived from the PI tissue was peak I, amounting to over 70% of the total ACTH activity present in that lobe. The positions of PI peaks I and II remained unaltered after rechromatography as well as after treatment with and chromatography in 8 M urea. However, peak I of PI ACTH was further resolved into two separate peaks by chromatography on Sephadex G-100 SF. Thus pars intermedia ACTH activity appears to be composed of four separate entities, with the predominant forms being larger than ACTH^1–39.     

The separation of ribonucleic acids from Escherichia coli on lysin-agarose.

Lysine-agarose provides a simple means of separating RNA species of different moleecular weight. When RNA from Escherichia coli is added to a small column of lysine-agarose and elution is carried out at neutral pH with a shallow linear gradient of NaCl the RNA species are eluted according to size; 4S and 5S RNA species are completely separated and after a delay the 16S and 23S species are eluted as separate peaks. The process is very reproducible and the different species are eluted at a fixed salt concentration even when changes are made in the gradient, provided other conditions, under which the column is run remain constant.     

Rat liver metabollothionein interactions of silver,zinc, and cadmium

Weanling rats were fed diets either alone or with combinations of silver, elevated zinc, or elevated cadmium for 7 weeks. The rats were then killed, and the silver, zinc, and cadmium proteins isolated from the livers by gel filtration and ion exchange chromatography. When silver was fed alone in diet, low levels of this metal were eluted as two peaks from the ion exchange column. In contrast, when silver was fed with cadmium or elevated zinc, three metal-containing peaks were eluted from this ion exchange column. Amino acid analysis revealed that the major proteins binding these metals are metallothioneins, as judged by high cysteine content.     

Enhanced production of dolichol,but not dolichyl phosphate,in the earliest stages of rat liver regeneration

The regenerating liver presents a changed ability to use mevalonate 16 hr after partial hepatectomy. The dolichol content and its synthesis from mevalonate is increased, while no variation of dolichyl-P and ubiquinone parameters are detectable.The greater amount ofmevalonate utilized to form dolichol, but not dolichyl-P, in this proliferating system, raises some questions about the physiological significance of these isoprenoid compounds and about their biosynthetic sequence.     

Analysis method for lipoproteins by high-performance liquid chromatography with sulfopropyl-ligand column and magnesium ion-containing eluents

We have developed a new analysis method for lipoproteins in serum by high-performance liquid chromatography using a sulfopropyl-ligand column with eluents containing magnesium nitrate. The magnesium ion anchors lipoproteins to the ligands on the column gel. Lipoproteins are eluted from the column with a magnesium nitrate concentration gradient and detected by postcolumn reaction using a reagent containing cholesterol esterase and cholesterol oxidase. High-density lipoprotein, low-density lipoprotein, and very-low-density lipoprotein were eluted in order from the column. The within-assay and between-assay coefficients of variation for cholesterol concentration in lipoproteins were 1.1-3.7 and 1.3-5.8%, respectively. The correlation coefficients between the values of total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol obtained by the new method and those obtained by an enzymatic method using an automated chemical analyzer were 0.940, 0.979, and 0.909, respectively. The new method was successfully applied to the analysis of plasma lipoproteins of patients with hyperlipidemia.