Kazuhiro Inagaki

Affinity purification and glucose specificity of aldose reductase from bovine lens

Abstract Aldose reductase, a possible key enzyme of sugar-cataract formation in diabetes, has been purified from bovine lens by a five-step procedure including affinity chromatography with Mātrex gel red A. The enzyme was purified 12,600-fold and was apparently homogeneous by polyacrylamide gel electrophoresis. The glucose specificity of the purified enzyme was studied with d -glucose anomers and d -glucitol as substrates. The ratios of the reduction rate of α- d -glucose to that of β- d -glucose at 10, 13, and 20 m m were 1.90, 1.76, and 1.72, respectively. These values were in good agreement with the ratios (1.92, 1.81, and 1.66) calculated on the basis of the rate constants reported for d -glucose mutarotation equilibrium ( J. M. Los, L. B. Simpson, and K. Wiesner, 1956, J. Amer. Chem. Soc.78, 1564–1568 ) and the assumption that aldose reductase acts on the aldehyde form of d -glucose. In addition, the composition of d -glucose produced from d -glucitol in the reverse reaction was 63% α anomer and 37% β anomer, which also agreed well with the values, 65 and 35%, respectively, calculated from the rate constants in reactions from the aldehyde form to both the α anomer and the β anomer. It was suggested from these kinetic analyses that aldose reductase acts on the aldehyde form of d -glucose (Km = 0.66 μm) but not on either the α or the β anomer of d -glucose.

Determination of fluconazole in human serum by solid-phase extraction and reversed-phase high-performance liquid chromatography.

A simple, rapid, and accurate reversed-phase octadecylsilyl high-performance liquid chromatographic method using solid-phase column extraction is described for measuring fluconazole in human serum. The column eluent was monitored by ultraviolet absorption at 210 nm. Fluconazole was extracted from diluted serum by adsorption on a small Bond-Elut C18 cartridge after the addition of UK48,134 as the internal standard and recovered by elution with methanol. The methanol was then evaporated to dryness and the residue reconstituted in 200 microliters of mobile phase and filtered prior to injecting an aliquot (50 microliters) onto an Adsorbosphere C18 column (4.6 x 250 mm, 5 microns particle size), using a mobile phase of 25 mM tris(hydroxymethyl)aminomethane-phosphate buffer (pH 7.0):acetonitrile (75:25, vol/vol). The retention times were 6.6 min for fluconazole and 9.0 min for the internal standard. The assay was precise, with inter- and intraassay coefficients of variation of less than or equal to 2.9% and less than or equal to 2.1%, respectively, and with good linearity (r = 1.000) in the range of 0.1 to 25 micrograms/ml. The duration of each analysis was 15 min and the minimum detectable serum concentration was 0.1 microgram/ml.

A wide interindividual variability of urinary 6β-hydroxycortisol to free cortisol in 487 healthy Japanese subjects in near basal condition

The frequency distribution of CYP3A activity was investigated by measuring ratios of urinary 6&bgr;-hydroxycortisol to free cortisol in 487 healthy subjects to determine whether a genetic polymorphism for this cytochrome enzyme exists in “native-born” Japanese persons. Spot urine samples (from 9:00 am to 12:00 pm) were collected for measurement of 6&bgr;-hydroxycortisol and free cortisol by high-performance liquid chromatography with a CN column after extracting with a solid-phase column (Bond-Elut C18). The frequency distribution of the urinary 6&bgr;-hydroxycortisol to free cortisol was widely distributed among subjects but with no clear bimodality by a probit plot. Furthermore, the frequency distribution assessed on a new normal test variable plot indicated the possible existence of a CYP3A sexual dimorphism. Mean 6&bgr;-hydroxycortisol levels were higher in women (n = 249) than in men (n = 238) by 1.7-fold, and this difference was statistically significant (P < 0.01). These results show that a CYP3A genetic polymorphism in Japanese persons, based on 6&bgr;-hydroxycortisol excretions, likely does not exist, but there appears to be a broad unimodal distribution of enzyme activity in the population.

Effect of Dietary Fiber on Morphine-induced Constipation in Rats

Morphine is used to alleviate chronic cancer pain. However, constipation is a major adverse effect that often detracts from the patients quality of life. In this study, we investigated the effectiveness of dietary fiber on morphine-induced constipation. Rats were fed on a normal diet or one containing either 10% or 20% apple fiber for two weeks before morphine was administered. In the control diet group, the fecal number and dry weight were decreased by treating with morphine in a dose-dependent manner. Moreover, the motility of the small and large intestines was reduced. The fecal number and weight were increased and the colon motility was promoted by dietary fiber, regardless of whether morphine was being administered. The dietary fiber increased the concentration of short-chain fatty acids (SCFAs) in the cecum. These results suggest that dietary fiber has a preventative effect on morphine-induced constipation by increasing SCFAs in the cecum, and thereby promoting colon motility in rats.

Simultaneous analysis of anthranilic acid derivatives in pharmaceuticals and human urine by high-performance liquid chromatography with isocratic elution

A high-performance liquid chromatographic (HPLC) method for simultaneous determination of mefenamic acid (MFA), flufenamic acid (FFA) and tolfenamic acid (TFA) is presented for application to pharmaceuticals and human urine. Isocratic reversed-phase HPLC was employed for quantitative analysis using tetra-pentylammonium bromide (TPAB) as an ion-pair reagent. Urine samples were purified by solid-phase extraction using a silica-based strong anion-exchanger, Bond-Elut SAX cartridge. The HPLC assay was carried out using a Wakosil ODS 5C18 column (5 microm, 150x4.6 mm I.D.). The mobile phase consisted of 1.9 g of TPAB dissolved in 1:1 of a mixture of acetic acid-sodium acetate buffer solution, pH 5.0, and acetonitrile (11:9, v/v). The calibration curves of MFA, FFA and TFA showed good linearity in the concentration range of 33-167 microg/ml with a wavelength of 280 nm for pharmaceuticals, and in the low concentration range (1.7-30.1 microg/ml) with a wavelength of 230 nm for biological fluids. The correlation coefficients were better than 0.9999 in all cases. The lower limits of detection (defined as a signal-to-noise ratio of about 3) were approximately 2 ng for MFA, 3.5 ng for FFA and 2.5 ng for TFA. The procedure described here is rapid, simple, selective and is suitable for routine analysis of pharmaceuticals and pharmacokinetic studies in human urine samples.

Acid load during total parenteral nutrition: comparison of hydrochloric acid and acetic acid on plasma acid-base balance.

The effects of hydrochloric acid and acetic acid on the plasma acid-base balance were investigated in four rabbits receiving parenteral nutrition. Hyperchloremic metabolic acidosis was observed only in the animals receiving total parenteral nutrition (TPN) whose pH was adjusted with hydrochloric acid. The observed acidosis was due to an excess of hydrogen ions with chloride ions as judged by the plasma-base excess and urinary net-acid excess and not by the infusion of solution having a high titratable acidity. The hydrogen ion released from the acetic acid added to TPN is consumed by the metabolism of the acetate ion and thus does not contribute to the net hydrogen-ion load. A reduction in the chloride load by using acetic acid to adjust the pH of the TPN solution when it is formulated can be safely achieved and prevents acidosis.

Development of potent aldose reductase inhibitors having a hydantoin structure

Seventeen hydantoin derivatives were tested as inhibitors of aldose reductase, an enzyme believed to participate in the initiation of diabetic complications. Nine compounds with high inhibitory activities (IC50 values against purified rat lens aldose reductase less than or equal to 1.06 X 10(-6)M) were tested further for their abilities to prevent sorbitol accumulation induced by exposure of excised rat lens and sciatic nerve to a high glucose concentration (50 mM). Seven active compounds among them inhibited sorbitol accumulation by about 50% or more at a concentration of 10(-5)M. These seven compounds were given orally to streptozotocin-induced diabetic rats at a dose of 50 mg/kg/day and were assessed for their abilities to prevent both sorbitol accumulation in two tissues (lens and sciatic nerve) and myo-inositol depletion in the sciatic nerve. 1-[(2,4,5-Trichlorophenyl)sulfonyl]hydantoin, 1-[(2,5-dichlorophenyl)sulfonyl]hydantoin, and 1-[(beta-naphthyl)sulfonyl]hydantoin were found to be the most effective: they inhibited sorbitol accumulation in the sciatic nerve completely and that in the lens by more than 92%. It is conceivable from this study that the three compounds are promising for further investigation targeted to the treatment of diabetic complications.

The Presence of A manganese-rich particle in lysosome of rat pancreas due to excess manganese treatment

Manganese (Mn) accumulates at a higher level in the pancreas than in any other organs when excess Mn is administered to the rat. The present study is carded out to analyze the intracellular localization of Mn existed in the pancreatic cell of Mn‐treated rats. Transmission electron microscope and X‐ray micro‐analysis connected with a rapid freezing fixation technique showed that a large amount of Mn was localized in tysosomal particles of the pancreatic cell of Mn‐treated rats. The Mn‐rich particles disappeared when the element‐administration was discontinued, showing that the accumulation of Mn is reversible. To confirm that Mn is in the lysosomes, a centrifugal subcellular‐fractionation and a neutron activation analysis were carried out. The result indicated that much Mn existed in the lysosomal fraction.

Quantification of pentazocine in human plasma by HPLC with electrochemical detection

Abstract A rapid and sensitive high performance liquid chromatographic method with electrochemical detector was described to measure pentazocine concentrations in plasma. The separation of pentazocine and an internal standard, levallorphan, from interfering compound in plasma was achieved by reversed phase phenyl column chromatography in combination with a solid extraction cartridge as clean-up. Analytical recoveries for pentazocine and levallorphan were determined as 88.6 % and 92.8 %, respectively. The detection limit was determined as 500 pg/ml plasma. This method has been successfully applied to the pharmacokinetic study of pentazocine in healthy male volunteers dosed rectum at 50 mg and plasma levels monitored up to 24 h after dosing.

Development of a Facts Database for Evidence- Based Adverse Reaction Diagnosis

Purpose: We have been building the CARPIS (Case Reports of Adverse Drug Reactions and Poisoning Information System) database since 1987 and have been using CARPIS primarily as a literature database. The purposes of this study were to revamp CARPIS as a fact database for adverse drug reactions (ADRs) and to develop methodology for identifying symptoms as part of evidence-based adverse reaction diagnosis. Revamp: Fact data in case reports, such as causative drugs, ADRs, and patient backgrounds, were analyzed to develop functions for the fact database and to establish different data fields. We developed our own dictionaries and data-tabulating function. Outcome: It was possible to calculate the incidence of subjective symptoms for ADRs, list causative drugs, and identify risk factors using the revamped CARPIS. The incidence of each subjective ADR symptom was compared with the incidence of objective symptoms associated with particular ADRs to identify subjective symptoms that could estimate ADRs. We succeeded in creating new information regarding the symptoms for evidence-based adverse reaction diagnosis. Conclusion: We were successful in revamping CARPIS as a novel fact database for ADRs and in developing methodology for identifying symptoms for evidence-based adverse reaction diagnosis.