Redox changes in coenzyme Q in the millisecond time range: an approach using rapid quenching and high-performance liquid chromatography

We have combined a rapid-quenching protocol with HPLC analysis to measure the kinetics of reduction of coenzyme Q in a mitochondrial enzyme complex. The method has a time resolution of several milliseconds and will readily measure 1-20 nmol of the Q derivatives under investigation. By changing the HPLC solvent, either Q6 or Q10 can be studied.     

Studies on reduced and oxidized coenzyme Q (ubiquinones). II. The determination of oxidation-reduction levels of coenzyme Q in mitochondria,microsomes and plasma by high-performance liquid chromatography

Reduced and oxidized coenzyme Q₁₀ (Q₁₀H₂ and Q₁₀) in guinea-pig liver mitochondria were rapidly extracted and determined by high-performance liquid chromatography (HPLC). The percentages of Q₁₀H₂ as compared to the total (sum of Q₁₀ and Q₁₀H₂) were increased by the addition of respiratory substrates such as succinate, malate and β-hydroxybutyrate (State 4). The levels of Q₁₀H₂ in State 4 were increased more extensively with electron-transport inhibitors such as KCN, NaN₃ and antimycin A. These results indicate that the method for determining Q₁₀H₂ and Q₁₀ by HPLC is quite useful for investigation of the physiological function of coenzyme Q in mitochondria and other organelles. The reduced and oxidized coenzyme Q levels of rat liver mitochondria, which contain both coenzyme Q₉ and coenzyme Q₁₀, were measured simultaneously. The results suggest that coenzymes Q₉ and Q₁₀ play a similar role as an electron carriers. The liver microsomes of guinea-pig contained approx. 133 nmol total coenzyme Q₁₀ per g protein. The Q₁₀H₂ levels of microsomes were increased from 46.5 to 67.5 and 64.8% with NADH and NADPH, respectively. The plasma levels of total coenzyme Q were 0.92 μg/ml for man, 0.35 μg/ml for guinea-pig and 0.27 μg/ml for rat. The reduced coenzyme Q were also present in those plasma samples. The levels of reduced coenzyme Q were 51.1, 48.9 and 65.3%, respectively.     

High-throughput solid-phase extraction for the determination of cimetidine in human plasma

For the implementation and validation of an automated `high-throughput' solid-phase extraction (SPE) system, using microtiter solid-phase technology and a pipetting robot, a SPE method previously validated manually for cimetidine in human plasma was adapted. Sample cleanup was performed by means of SPE using Microlute extraction plates in the 96-well format, each well filled with 50 mg of Varian C₁₈ sorbent. Separation was performed by reversed-phase high-performance liquid chromatography (HPLC) with UV detection at 234 nm. The validated calibration range was from 0.100 to 5.00 mg/l, with an inaccuracy and imprecision below 20% at all concentration levels. Validation results on linearity, specificity, precision, accuracy and stability are shown and are found to be adequate. Cross-check analysis of samples from a clinical trial showed that there is a good correlation between results obtained by the automated method and results obtained by the manual method. The average sample preparation time for a technician decreased from approximately 4 min per sample to 0.6 min. A sample throughput of at least 160 samples per day can be achieved, the HPLC analysis time being the rate-limiting step.     

Evidence for coenzyme Q function in transplasma membrane electron transport

Transplasma membrane electron transport activity has been associated with stimulation of cell growth. Coenzyme Q is present in plasma membranes and because of its lipid solubility would be a logical carrier to transport electrons across the plasma membrane. Extraction of coenzyme Q from isolated rat liver plasma membranes decreases the NADH ferricyanide reductase and added coenzyme Q10 restores the activity. Piericidin and other analogs of coenzyme Q inhibit transplasma membrane electron transport as measured by ferricyanide reduction by intact cells and NADH ferricyanide reduction by isolated plasma membranes. The inhibition by the analogs is reversed by added coenzyme Q10. Thus, coenzyme Q in plasma membrane may act as a transmembrane electron carrier for the redox system which has been shown to control cell growth.     

Mechanisms of oxidative modification of low density lipoproteins under conditions of oxidative and carbonyl stress

Low-molecular-weight aldehydes (glyoxal, methylglyoxal, 3-deoxyglucosone) generated on autooxidation of glucose under conditions of carbonyl stress react much more actively with amino groups of L-lysine and epsilon-amino groups of lysine residues of apoprotein B-100 in human blood plasma low density lipoproteins (LDL) than their structural analogs (malonic dialdehyde (MDA), 4-hydroxynonenal) resulting on free radical oxidation of lipids under conditions of oxidative stress. Glyoxal-modified LDL aggregate in the incubation medium with a significantly higher rate than LDL modified by MDA, and MDA-modified LDL are markedly more poorly absorbed by cultured human macrophages and significantly more slowly eliminated from the rat bloodstream upon intravenous injection. Studies on kinetics of free radical oxidation of rat liver membrane phospholipids have shown that ubiquinol Q(10) is the most active lipid-soluble natural antioxidant, and suppression of ubiquinol Q(10) biosynthesis by beta-hydroxy-beta-methylglutaryl coenzyme A reductase inhibitors (statins) is accompanied by intensification of lipid peroxidation in rat liver biomembranes and in LDL of human blood plasma. Injection of ubiquinone Q(10) protects the human blood plasma LDL against oxidation and prevents oxidative stress-induced damages to rat myocardium. A unified molecular mechanism of atherogenic action of carbonyl-modified LDL in disorders of lipid and carbohydrate metabolism is discussed.     

Attenuation of blood parameters in smokers and non-smokers after intake of a complex food additive

This report describes an intervention study with healthy volunteers (20 smokers, 28 non-smokers) taking a food additive mainly containing vitamin C (ascorbic acid), vitamin E (alpha-tocopherol), ubiquinone (Q10), vitamin A and zinkoxide for four weeks in a double blind, randomized and placebo controlled manner. Before and after the intervention blood was withdrawn and general blood parameters were analyzed. In addition, lipid soluble antioxidants were analyzed in blood plasma by HPLC and the water soluble antioxidative properties were tested with the enzymic xanthin/xanthinoxidase-reaction. In summary the results show that the smoker-verum group exhibit a significant down regulation of the leukocyte counts. The test for antioxidants show the following significant differences after intervention: Smokers exhibit an increase of both vitamin E and coenzyme Q10 and an attenuation of their (before intervention) clearly increased water soluble-antioxidative potential, non-smokers showed only an increase of vitamin E and trends of an increase of Q10 and water soluble-antioxidative potential. These results may contribute to the discussion of the intrinsic deficiency brought about by smoking and the possible attenuation of part of these deficiency by increasing the intake of certain vitamins or food additives.     

Enantioselective quantitation of the ecstasy compound (R)- and (S)-N-ethyl-3,4-methylenedioxyamphetamine and its major metabolites in human plasma and urine

An enantioselective HPLC method has been developed and validated for the stereospecific analysis of N-ethyl-3,4-methylenedioxyamphetamine (MDE) and its major metabolites N-ethyl-4-hydroxy-3-methoxyamphetamine (HME) and 3,4-methylenedioxyamphetamine (MDA). These compounds have been analyzed both from human plasma and urine after administration of 70 mg pure MDE-hydrochloride enantiomers to four subjects. The samples were prepared by hydrolysis of the o-glucuronate and sulfate conjugates using beta-glucuronidase/arylsulfatase and solid-phase extraction with a cation-exchange phase. A chiral stationary protein phase (chiral-CBH) was used for the stereoselective determination of MDE, HME and MDA in a single HPLC run using sodium dihydrogenphosphate, ethylendiaminetetraacetic acid disodium salt and isopropanol as the mobile phase (pH 6.44) and fluorimetric detection (lambda(ex) 286 nm, lambda(em) 322 nm). Moreover, a suitable internal standard (N-ethyl-3,4-methylenedioxybenzylamine) was synthesized and qualified for quantitation purposes. The method showed high recovery rates (>95%) and limits of quantitation for MDE and MDA of 5 ng/ml and for HME of 10 ng/ml. The RSDs for all working ranges of MDE, MDA and HME in plasma and urine, respectively, were less than 1.5%. After validation of the analytical methods in plasma and urine samples pharmacokinetic parameters were calculated. The plasma concentrations of (R)-MDE exceeded those of the S-enantiomer (ratio R:S of the area under the curve, 3.1) and the plasma half time of (R)-MDE was longer than that of (S)-MDE (7.9 vs. 4.0 h). In contrast, the stereochemical disposition of the MDE metabolites HME and MDA was reversed. Concentrations of the (S)-metabolites in plasma of volunteers were much higher than those of the (R)-enantiomers.     

Determination of polymyxin E1 in rat plasma by high-performance liquid chromatography

A precise and accurate HPLC assay for polymyxin E(1) in rat and dog plasma has been validated. Samples and standards are extracted from plasma with a 96-well C(8) extraction disk plate. Sample extracts are derivatized with dansyl chloride, and polymyxin E(1) derivative is quantitated on a C(8) column by HPLC with fluorescence detection. The assay is linear in the range of 0.050-5.00 micro g/ml for polymyxin E(1). The precision and accuracy of polymyxin E(1) plasma assay was well within the recommended limits set in the FDA Guidance for Bioanalytical Method Validation. Polymyxin E(1) stability in rat and dog plasma for 24 h at room temperature and through three freeze-thaw cycles was demonstrated.     

Redox state of glutathione and thioredoxin in differentiation and apoptosis

This study was organized by Professor Karl Folkers with the objective of finding derivatives of coenzyme Q which could be more effectively absorbed and would give better biomedical effects. In this series all the compounds are 2,3 dimethoxy, 5 methyl p benzoquinone with modified side chains in the 6 position. The modifications are primarily changes in chain length, unsaturation, methyl groups and addition of terminal phenyl groups. The test system evaluates the growth of serum deficient HL60, 3T3 and HeLa cells in the presence of coenzyme Q10 or coenzyme Q analogs. Short chain coenzyme Q homologues such as coenzyme Q2 give poor growth but compounds with saturated short aliphatic side chains from C10 to C18 produce good growth. Introduction of a single double bond at the 2' or 8' position in the aliphatic chain retains growth stimulation at low concentration but introduces inhibition at higher concentration. Introduction of a 3' methyl group in addition to the 2' enyl site in the side chain decreases the growth response and maintains inhibition. Addition of a terminal phenyl group to the side chain from C5 to C10 can produce analogs which give strong stimulation or strong inhibition of growth. The action of the analogs is in addition to the natural coenzyme Q in the cell and is not based on restoration of activity after depletion of normal coenzyme Q. The effects may be based on any of the sites in the cell where coenzyme Q functions. For example, coenzyme Q2 is known to decrease mitochondrial membrane potential whereas the analog with a 10C aliphatic side chain increases potential. Both of these compounds stimulate plasma membrane electron transport. Inhibition of apoptosis by coenzyme Q may also increase net cell proliferation and the 10C analog inhibits the permeability transition pore.     

Simultaneous determination of retinol, β-carotene and α-tocopherol in adipose tissue by high-performance liquid chromatography

A method is described for evaluation of fat-soluble vitamin in human adipose tissue with the aim to obtain, accurately and within the shortest analysis time, a time-integrated measure of exposure to vitamins from the diet. Fat tissue was deproteinized with ethanol and extracted with n-hexane. Normal-phase HPLC was performed in a Lichrosorb Si60 column with a gradient of n-hexane–2-propanol at 1 ml/min. Detection was accomplished using a diode-array system (for retinol and β-carotene) in series with a fluorescence detector (α-tocopherol). The method was validated and applied to human adipose tissue in a total of 140 subjects. The mean contents found were 0.43, 0.84, 240.3 μg/g for retinol, β-carotene and α-tocopherol, respectively. The method is sensitive enough for detecting the compounds in 1.6 mg of adipose tissue considering the lowest concentration found.     

Inhibition by coenzyme Q of ethanol- and carbon tetrachloride-stimulated lipid peroxidation in vivo and catalyzed by microsomal and mitochondrial systems

The ability of coenzyme Q to inhibit lipid peroxidation in intact animals as well as in mitochondrial, submitochondrial, and microsomal systems has been tested. Rats fed coenzyme Q prior to being treated with carbon tetrachloride or while being treated with ethanol excrete less thiobarbituric acid-reacting material in the urine than such rats not fed coenzyme Q. Liver homogenates, mitochondria, and microsomes isolated from rats treated with carbon tetrachloride and ethanol catalyze lipid peroxidation at rates which exceed those from animals also fed coenzyme Q. The rate of lipid peroxidation catalyzed by submitochondrial particles isolated from hearts of young, old, and endurance trained elderly rats was inversely proportional to the coenzyme Q content of the submitochondrial preparation in assays in which succinate was employed to reduce the endogenous coenzyme Q. Reduced, but not oxidized, coenzyme Q inhibited lipid peroxidation catalyzed by rat liver microsomal preparations. These results provide additional evidence in support of an antioxidant role for coenzyme Q.     

Simple and sensitive high-performance liquid chromatography method for the determination of docetaxel in human plasma or urine

Several methods for quantification of docetaxel have been described mainly using HPLC. We have developed a new isocratic HPLC method that is as sensitive and simpler than previous methods, and applicable to use in clinical pharmacokinetic analysis. Plasma samples are spiked with paclitaxel as internal standard and extracted manually on activated cyanopropyl end-capped solid-phase extraction columns followed by isocratic reversed-phase HPLC and UV detection at 227 nm. Using this system, the retention times for docetaxel and paclitaxel are 8.5 min and 10.5 min, respectively, with good resolution and without any interference from endogenous plasma constituents or docetaxel metabolites at these retention times. The total run time needed is only 13 min. The lower limit of quantification is 5 ng/ml using 1 ml of plasma. The validated quantitation range of the method is 5–1000 ng/ml with RSDs≤10%, but plasma concentrations up to 5000 ng/ml can be accurately measured using smaller aliquots. This method is also suitable for the determination of docetaxel in urine samples under the same conditions. The method has been used to assess the pharmacokinetics of docetaxel during a phase I/II study of docetaxel in combination with epirubicin and cyclophosphamide in patients with advanced cancer.     

Fasting and lactation effect fat-soluble vitamin A and E levels in blood and their distribution in tissue of grey seals (Halichoerus grypus)

Grey seals among other phacoids represent a good model to study the mobilisation, transfer and deposition of fat-soluble components such as vitamins in lactating females and suckling pups because during the lactation period mothers may fast completely while secreting large quantities of high fat milks, and pups deposit large amounts of fat as blubber. The level of vitamins A and E in different tissues (liver, adipose tissue, kidney, heart, skeletal muscle, testis) and blood plasma of adult grey seal females and males changed as a result of fasting and lactation; changes were also observed in pups. The most obvious effects were a significant increase of retinol and a decrease of vitamin E levels in plasma of females with the onset of lactation as well as a substantial decrease in liver vitamin E. In suckling pups both retinol and vitamin E levels in plasma increased with the onset of suckling; after weaning no changes in retinol but a significant decrease in plasma vitamin E was observed. While liver vitamin A levels tended to be unaffected by suckling or post-weaning fast, liver vitamin E levels increased with the uptake of milk substantially (P<0.01) and returned at weaning to low levels similar to that in fetuses. Adipose tissue levels of vitamin A and E in both females and pups were only marginally affected by lactation, suckling or post-weaning fast. Results indicate that both plasma and liver levels of vitamin A and E are affected by the mobilisation, absorption and deposition of these components during lactation in seals to a much greater extent than adipose tissue, from which fat-soluble vitamins are mobilized at rates similar to that of lipids.     

Determination of 8:2 fluorotelomer alcohol in animal plasma and tissues by gas chromatography-mass spectrometry

Fluorotelomer alcohols (FTOHs) constitute an important group of compounds among the perfluoroalkyl substances (PFAS). The PFAS have recently been a focus of many environmental and biological studies. This generated a strong need for analytical methods for analysis of PFAS at trace levels in various environmental and biological matrices. A quantitative analytical method for analysis of 8:2 FTOH in rat plasma and rat liver, kidney, and adipose tissue using GC-MS with electron impact (EI) ionization was developed and validated. Extraction of water-diluted plasma with methyl tert-butyl ether (MTBE) was used for rat plasma. The analysis of rat liver or kidney tissues required homogenization of tissue on ice, extraction with hexane, and clean up of the extract by silica (Si) normal-phase solid phase extraction (SPE). Similarly, the adipose tissue was dissolved in n-heptane and cleaned up by Si SPE. The methods were validated by performing spike recovery experiments for each type of matrix investigated and tested on authentic samples originating from 8:2 FTOH toxicological studies.     

Coenzyme Q1-catalyzed luminol chemiluminescent assay for rapid antimicrobial susceptibility testing of Mycobacterium bovis

Coenzyme Q1 is herein proposed as the best catalyst among coenzymes Q and vitamins K for quinone-catalyzed luminol chemiluminescent assays applied to rapid determination of viability or rapid antimicrobial susceptibility tests of Mycobacterium bovis. Luminol chemiluminescence intensity (LCI) was determined 10 min after the incubation of M. bovis with coenzyme Q1, and was proportional to CFU (colony-forming unit)/ml in the range of 9,000 to 2,250,000. LCI depended on the the production of the superoxide anion (O2-) rather than H2O2 during a 10-min incubation of M. bovis with coenzyme Q1, as superoxide dismutase reduced LCI more effectively than catalase. The minimal inhibitory concentrations (MICs) of 10 kinds of antituberculous agents estimated on the basis of decrease in LCI after one or two days' cultivation were in good agreement with MICs determined by turbidity analysis, which requires upwards of 1 week to complete.     

Assay of coenzyme Q(10) in plasma by a single dilution step

A new method is described for determining coenzyme Q(10) (CoQ(10)) in plasma. The method is based on oxidation of CoQ(10) in the sample by treating it with para-benzoquinone followed by extraction with 1-propanol and direct injection into the HPLC apparatus. This method achieves a linear detector response for peak area measurements over the concentration range of 0.05-3.47 microM. Diode array analysis of the peak was consistent with CoQ(10) spectrum. Supplementation of the samples with known amounts of CoQ(10) yielded a quantitative recovery of 96-98.5%; the method showed a level of quantitation of 1.23 nmol per HPLC injection (200 microl of propanol extract containing 33.3 microl of plasma). A correlation of r = 0.99 (P < 0.0001) was found with a reference electrochemical detection method. Within run precision showed a CV% of 1.6 for samples approaching normal values (1.02 microM). Day-to-day precision was also close to 2%.     

The importance of plasma membrane coenzyme Q in aging and stress responses

The plasma membrane of eukaryotic cells is the limit to interact with the environment. This position implies receiving stress signals that affects its components such as phospholipids. Inserted inside these components is coenzyme Q that is a redox compound acting as antioxidant. Coenzyme Q is reduced by diverse dehydrogenase enzymes mainly NADH-cytochrome b₅ reductase and NAD(P)H:quinone reductase 1. Reduced coenzyme Q can prevent lipid peroxidation chain reaction by itself or by reducing other antioxidants such as α-tocopherol and ascorbate. The group formed by antioxidants and the enzymes able to reduce coenzyme Q constitutes a plasma membrane redox system that is regulated by conditions that induce oxidative stress. Growth factor removal, ethidium bromide-induced ρ° cells, and vitamin E deficiency are some of the conditions where both coenzyme Q and its reductases are increased in the plasma membrane. This antioxidant system in the plasma membrane has been observed to participate in the healthy aging induced by calorie restriction. Furthermore, coenzyme Q regulates the release of ceramide from sphingomyelin, which is concentrated in the plasma membrane. This results from the non-competitive inhibition of the neutral sphingomyelinase by coenzyme Q particularly by its reduced form. Coenzyme Q in the plasma membrane is then the center of a complex antioxidant system preventing the accumulation of oxidative damage and regulating the externally initiated ceramide signaling pathway.     

Direct assay for phosphotransacetylase and acetyl-coenzyme A carboxylase by high-performance liquid chromatography

A simple and specific assay to measure the activity of two coenzyme A derivative-processing enzymes, i.e., phosphotransacetylase (EC 2.3.1.8) and acetyl-coenzyme A carboxylase (EC 6.4.1.2), is described. The assay is based on the HPLC analysis of the short-chain coenzyme A derivatives formed by the enzymatic reaction, viz., acetyl-CoA and malonyl-CoA. For this purpose, ion-pair reversed-phase HPLC conditions are optimized. Furthermore, the influence of several variables on the enzyme reaction is studied in order to get maximum activity. Due to its short analysis time, good selectivity, and chromatogram information, HPLC proves to be an excellent method for the assay of these enzymes.     

Effect of low doses of radiation on vitamin A and E levels in rat liver

It has been established that under intake of small doses of 137Cs by rat for 9 months the radioactivity of whole rat body was increased in irregular manner. Under this condition the level of fat-soluble vitamins A and E is decreased and the decrease is well correlated with a level of radionuclide accumulation by rat's body. The possible causes of decrease of the vitamins A and E level under intake of small doses of radionuclide are discussed.