Teruaki Yuzuriha

Simultaneous determination of reduced and oxidized ubiquinones

Publisher Summary Quantitative analyses of individual ubiquinones (Q) homologs in biological samples have been performed by high-performance liquid chromatography (HPLC) combined with an ultraviolet spectrometric detector (UVD) or by mass spectrometry (MS). An electrochemical detector (ECD) for HPLC was confirmed to be simple and sensitive for the determination of Q. However, only Q was determined by these methods. For the determination of QH 2 (ubiquinol) and Q in mitochondria, submitochondrial particle and cell-free bacterial homogenates, the dual-wavelength spectrometric method has been generally used. The method, however, cannot simultaneously measure the amounts of QH 2 and Q in whole tissues owing to the presence of vitamin A and other interfering compounds, which have an absorbance in the same spectral region as Q and undergo an absorption change by chemical reduction. The dual-wavelength spectrometric method cannot separately determine individual Q homologs. The analytical procedure described was developed to provide a rapid, sensitive, and direct assay method for QH 2 and Q in biological samples. This method is based on extraction from tissues, mitochondria, microsomal fractions, or plasma with organic solvents, followed by quantitation by means of reversed-phase chromatography with UVD and ECD.

Increased lung uptake of liposomes coated with polysaccharides.

Liposomes labeled with [14C]coenzyme Q10 in the lipid bilayer were coated with various polysaccharide derivatives, i.e., palmitoyl conjugates of pullulan, pullulan phosphate, amylopectin, amylopectin phosphate and amylopectin sulfate. The kinetics of disposition and the tissue distribution of [14C]coenzyme Q10 after intravenous injection of the liposomes into guinea pigs were investigated. Lung uptake of radioactivity after injection of the O-palmitoyl amylopectin- and O-palmitoyl amylopectin phosphate-coated liposomes was 5- and 3-times higher, respectively, at 30 min after injection than that of the conventional liposomes. For doubly labeled liposomes with [3H]inulin and [14C]coenzyme Q10, the 3H/14C ratios in the lung, spleen and heart were similar to one another. Urinary excretion of [3H]inulin encapsulated in O-palmitoyl amylopectin-coated liposomes was much lower than that of unencapsulated [3H]inulin. These observations suggest that the O-palmitoyl amylopectin-coated liposomes are rather stable in vivo and are taken up into tissues in the intact form.

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 beta-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 microgram/ml for man, 0.35 microgram/ml for guinea-pig and 0.27 microgram/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.

Simultaneous determination of ubiquinone-10 and ubiquinol-10 in tissues and mitochondria by high performance liquid chromatography

Abstract A method of high performance liquid chromatography with both of a UV detector and an electrochemical detector for the simultaneous determination of ubiquinone and ubiquinol was established. This method could sensitively and specifically measure the redox state of ubiquinone in mitochondria and tissues.

Transport of [14C]coenzyme Q10 from the liver to other tissues after intravenous administration to guinea pigs

After intravenous injection of [14C]coenzyme Q10 into guinea pigs, the blood level of radioactivity declined rapidly within 30 min. However, radioactivity in the blood subsequently increased, reached a peak at 8 h after the injection and thereafter decreased slowly. Since the liver uptake of radioactivity at 30 min after the injection was 87.8% of the dose and the increase of blood level was also seen in guinea pigs with bile duct fistula, it was concluded that the radioactivity taken up by the liver was redistributed to the blood and then transferred to other tissues. Tissue radioactivity levels were highest in the liver and spleen at 30 min after the injection and decreased thereafter. The levels in the blood and kidney peaked at 8 h, while those in the heart and brain peaked at 24 h and subsequently decreased. In contrast, the level in the adrenal gland was still increasing at 168 h after the injection; it rose sharply from 30 min, and at 24 h was higher than the level in any other tissue. Electrophoretic analysis showed that the radioactivity in serum at 8 h after the injection was associated with lipoproteins. It was confirmed by thin layer radiochromatography that the radioactivities in serum and liver at various times were due to coenzyme Q10 and coenzyme Q10 hydroquinone.

Enantioselective high-performance liquid chromatographic assay for determination of the enantiomers of a new anti-ulcer agent, E3810, in Beagle dog plasma and rat plasma

An enantioselective high-performance liquid chromatographic method for the determination of E3810, a new anti-ulcer agent, in Beagle dog plasma and rat plasma has been developed. After extraction from plasma with ethyl acetate, E3810 enantiomers were measured by reversed-phase high-performance liquid chromatography on a Chiralcel OD-R column. The enantiomers were detected by ultraviolet absorbance detection at 290 nm. The recoveries of E3810 enantiomers and internal standard were greater than 91%. The calibration curves were linear from 0.03 to 20 micrograms/ml for Beagle dog plasma and from 0.1 to 100 micrograms/ml for rat plasma. The limits of quantification of both enantiomers were 0.03 micrograms/ml for Beagle dog plasma and 0.1 micrograms/ml for rat plasma. The intra- and inter-day accuracy and precision data showed good reproducibility of the method. The assay was applied for the analysis of E3810 enantiomers in plasma after intravenous administration of racemic E3810 to Beagle dogs and rats. This method should be very useful for enantioselective pharmacokinetic studies of E3810.

Improved Drug Delivery to Target Specific Organs Using Liposomes as Coated with Polysaccharides

An assembly of a cell wall-like structure on the outmost surface of liposomes has been constructed, which makes liposomes tough against chemical and physicochemical lyses of liposomal membranes caused by external stimuli. In accordance with this idea, we have prepared partly modified polysaccharides, (0-palmitoylpullulan (OPP) and (0-palmitoylamylopectin (OPA), and coated the outmost surface of egg phosphatidylcholine liposomes by them. The efficiency in coating liposomes with the artificial cell wall has been ascertained by four different methods: (1) isolation of polysaccharide-coated liposomes by gel-filtration, (2) reduced permeability for a water soluble material, carboxyfluorescein, encapsulated in the interior of liposomes, (3) increased resistance against the enzymatic lysis with phospholipase D for the coated liposomes, and (4) decreased probability in the enzymatic digestion with pullulanase of the polysaccharide strongly bound to the surface of liposomes. These results suggest a wider usage of the polysaccharide-coated liposomes as an improved drug carrier.

Calcitonin Gene‐Related Peptide‐Binding Sites of Porcine Cardiac Muscles and Coronary Arteries: Solubilization and Characterization

Abstract: Calcitonin gene‐related peptide (CGRP)‐binding sites were solubilized, using digitonin, from the porcine spinal cord, atria, and coronary arteries. The specific binding of 125I‐human α‐CGRP to the solubilized binding sites was inhibited by human α‐ and β‐CGRP and by rat α‐CGRP, but not by angiotensin II or human calcitonin. Scatchard plot analysis of saturation gave the same KD value for CGRP in the crude membrane fractions of the tissues examined. The affinity of CGRP to the binding sites was decreased by solubilization in the atria and coronary arteries, but not in the spinal cord. Affinity labeling followed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis revealed distinct molecular sizes of the specific binding sites among the tissues; 70K for the spinal cord, 70K and 90K for the coronary arteries, and 70K and 120K for the atria. These results indicate that the molecular characteristics of the specific binding sites of CGRP in the cardiovascular system are distinct from those in the central nervous system.

Positive inotropic effects and receptors of calcitonin gene-related peptide (CGRP) in porcine ventricular muscles.

Calcitonin gene-related peptide (CGRP) in porcine ventricular muscles. positive inotropic effects in the isolated, electrically driven false tendon of the porcine heart. Specific CGRP-binding sites were present in solubilized membrane fractions; the dissociation constant (Kd) and the maximum binding (Bmax) were 50.4 pM and 180 fmol/mg protein, respectively. SDS-PAGE analysis of CGRP-binding sites revealed the molecular mass of 70 K and 120 K. Few CGRP-like immunoreactive nerves were present in the ventricular muscle layer. These results indicate that CGRP activates specific receptor sites on the ventricular muscles and causes positive inotropic responses. CGRP receptors in ventricles are likely to be activated by circulating CGRP.

Polymer Coated Liposomes for Drug Delivery to Target Specific Organs

Liposomes have gained wide acceptance in chemotherapy as potential carriers in introducing drugs and macromolecules into cells. However, several problems to be overcome still remain before the liposomal drug-delivery system achieves its usefulness in biological and medicinal usages (1, 2). First of all is to depress the permeability of water soluble materials entrapped in liposomes, and the second is to access an ability for liposomes to specifically land onto the target cells or tissues.