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Biomedical subjects

N Hashizume

Publications and source records attributed to N Hashizume.

At least 19 recordsLinked to original sources
PubMed2001

Is skeletal muscle damaged by the oxidative stress following anaerobic exercise?

We investigated whether the injury of skeletal muscle owing to the action of free radicals and the subsequent oxidative damage to tissues occurred during anaerobic exercise. To estimate injury to skeletal muscle, we determined certain indices of oxidative damage to skeletal muscle; i.e., leukocyte counts, concentrations of hypoxanthine, xanthine, urate, tissue- and serum-type CK-M isoforms, myoglobin, and total antioxidant capacity (TAC) of serum. Blood for these tests was collected at 3 min post-exercise. Post-anaerobic exercise concentrations of lactate were significantly increased from pre-exercise. The neutrophil and lymphocyte counts and alanine concentration were significantly increased by anaerobic exercise, even when the results were corrected for plasma volume changes; the plasma concentrations of hypoxanthine, urate, and TAC of serum were also significantly increased. The plasma concentration of xanthine was negatively correlated with TAC of serum. The activities of tissue- and serum-type CK-M were significantly increased post-exercise. When the hypoxanthine, urate, TAC of serum, myoglobin, and tissue- and serum-type CK-M were corrected for plasma volume changes, the post-exercise increases were no longer significantly different from the pre-exercise results. We suggest that these latter test results following anaerobic exercise exclude the presence of oxidative damage to skeletal muscle.

Adult↗
PubMed2001

A new quantitative analytical method of serum biotinidase activity using biocytin as a substrate and its clinical significance in Japan.

We have developed a new quantitative analytical method of serum biotinidase activity, which uses the native substrate biocytin, and to which can be applied the improved agar plate method of biotin bioassay. Assay characteristics were within acceptable ranges (intra-assay CVs, 4.44% and 1.95% at 1.82+/-0.08 and 3.08+/-0.06 pmol/min/ml; day-to-day CV, 5.92% at 2.68+/-0.16 pmol/min/ml). The enzyme activity with biocytin was stable at 4 degrees C for 90 days. The mean value of the serum biotinidase levels in 129 healthy adults was 2.71+/-0.93 pmol/min/ml. The method was clinically comparable with a colorimetric method for detection of biotinidase deficiency. Biotin supplementation treatment normalized our partial biotinidase deficiency patient's serum biotinidase activity. This normalized phenomenon has not yet been observed in a Caucasian patient. We also found that the distribution of the enzyme activities with biotinyl-p-aminobenzoate in 8 of 11 patients with suspected biotin metabolic disorders shifted to a higher level than that of the controls. Although, we have few opportunities to analyze the sera of biotin metabolic disorders in Japan, the new method are suitable for clinical research applications in combination with the colorimetric method.

Adult↗
PubMed2001

Effects of antiinflammatory drugs on migration of the rabbit corneal epithelium.

PURPOSE: To evaluate and compare the effects of diclofenac sodium 0.1% and betamethasone phosphate 0.1% on corneal wound healing. SETTING: Department of Ophthalmology, Wakayama Medical College, Wakayama, Japan. METHODS: Using the method described by Nishida et al., corneal epithelial spreading was measured in vitro in a rabbit corneal block in the presence or absence of 2 antiinflammatory agents at various doses. RESULTS: At clinical doses (1 microg/mL and 10 microg/mL), the drugs did not suppress migration of the corneal epithelium. At high doses (20 microg/mL, 50 microg/mL, and 100 microg/mL), they did inhibit the migration. There was no between-group difference in corneal epithelial migration at clinical doses. At high doses, corneal epithelial migration was inhibited in the diclofenac sodium group compared with the betamethasone group. CONCLUSIONS: At clinical doses, diclofenac sodium and betamethasone did not inhibit corneal epithelial migration. However, these drugs should be prescribed cautiously in patient with high-risk diseases such as diabetes mellitus and glaucoma.

Animals↗
PubMed2001

Postnatal development of corneal curvature and thickness in the cat.

OBJECTIVE: To evaluate the postnatal development of central corneal curvature and thickness in the domestic cat. Animals studied Six Domestic Short-haired (DSH) kittens starting at 9 weeks of age and 6 adult cats. PROCEDURES: Kittens were evaluated biweekly to monthly for a 12-month period, starting at age 9 weeks. Corneal development was monitored by hand-held keratometry and ultrasound biomicroscopy. Standard regression analysis using a nonlinear least squares method was used to generate a formula that would predict corneal curvature as a function of age. RESULTS: Mean keratometry (K) values for the 9-week-old cats were 54.51 (+/-1.02) diopters (D) and these values steeply declined over the next 3 months to 44.95 (+/-0.90) D. Thereafter, K-values gradually decreased to reach a plateau by 12-15 months of age of 39.90 (+/-0.42) D. Because K-values still appeared to be slightly diminishing at this point, six other > 2-year-old cats were evaluated by keratometry and were found to have K-values of 38.99 (+/-0.81). Two to four diopters of astigmatism was common in young kittens whereas adult cats had a low mean degree of astigmatism (< 1 D). A formula that predicted keratometry values in diopters (K) as a function of age in weeks (w) was established as follows: K = 39.83 + 26.87 exp(-0.074 w). The central cornea increased in thickness primarily during the first 4 months of life with 9 week-old kittens having values of 0.379 (+/-0.012) mm; 16-week-old kittens, 0.548 (+/-0.021) mm and 67 week-old cats, 0.567 (+/-0.012) mm. CONCLUSIONS: The maturation process of the feline cornea proceeds over the first 1-2 years of life to attain an adult status that is characterized by a roughly spherical state of approximately 39 D corneal curvature, substantially flatter than the human cornea, and a central thickness similar to the human cornea. Research studies of the refractive or optical properties of the cornea in which cats are used as experimental animals should be conducted on animals greater than 18 months of age.

Animals↗
PubMed2000

An automated assay for measuring serum ascorbic acid with use of 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy, free radical and o-phenylenediamine.

We developed a novel, cost-effective, and automated assay for ascorbic acid (AsA) in serum using a COBAS MIRA S analyzer (Roche Diagnostic System). Our method has a wide dynamic range and covers AsA concentrations from well below the lower reference interval to well above it. AsA is oxidized by 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy, free radical (TEMPO) to dehydroascorbic acid (DAsA). The latter condenses with o-phenylenediamine (OPDA) to form a quinoxaline derivative that absorbs light at 340 nm. The change in absorbance at 340 nm is proportional to the concentration of AsA in the specimen. The automated system permitted the assay of 65 specimens per hour at a cost of approximately US$ 0.01 per specimen for reagents. The assay can be applied directly to serum specimens (direct method) and also to sera with a prior deproteinization step with metaphosphoric acid. The detection limit for the direct serum assays is 0.8 vs. 0.4 mg/l with the deproteinization method. The recovery of AsA from a supplemented serum pool was of >95% for both procedures. We used four distinct methods on 66 patients sera. The direct method for AsA correlated well with an HPLC method (r=0.964, P<0.001); the direct method also correlated well with a method that uses AsA oxidase (r=0.975, P<0. 001). The deproteinization method correlated well with HPLC (r=0.981, P<0.001), and with the AsA oxidase procedure (r=0.994, P<0.001). Ten within-day determinations on a serum pool gave a C.V. <4.3% for both the direct and deproteinization procedures. The between-day assays of the same serum pool over 10 days gave a C.V. of <6.7% by both methods.

Artifacts↗
PubMed2000

A simple and rapid method for the routine assay of total ascorbic acid in serum and plasma using ascorbate oxidase and o-phenylenediamine.

A simple and rapid analysis of total ascorbic acid (AsA) in serum and plasma and its automated analysis are described. AsA is oxidized by ascorbate oxidase (AsA oxidase) to dehydroascorbic acid that then reacts with o-phenylenediamine (OPDA) to form a quinoxaline derivative that absorbs at 340 nm. The change in absorbance is directly proportional to the total AsA concentration. The assay was validated with a linear concentration range of 0.8-80 mg/L, and the within-day and between-day assays precision did not exceed 8.6% and 12.5%, respectively. On 47 sera, the manual enzymatic procedure gave 0.2 mg/L on average lower values than those of an automated enzymatic procedure with a correlation coefficient of 0.847. On another 66 sera, results by automated enzymatic method correlated well with the HPLC method and the regression equation is Y (enzymatic, automated)=0.97 X (HPLC)+0.1, r=0.980, Sy.x=0.6 mg/L. An experienced analyst can perform about 24 manual assays per hour whereas the automated procedure gave a rate of 100 assays per hour.

Ascorbate Oxidase↗
PubMed1999

Purification and characterization of protease produced by Staphylococcus aureus isolated from a diseased chicken.

A protease produced by Staphylococcus aureus, isolated from a chicken suffering from dermatitis, was purified by successive precipitation with ammonium sulfate, ion-exchange chromatography on Q-Sepharose FF, Sp-Sepharose FF and Mono-Q columns. By Mono-Q column chromatography, two proteases (protease 1 and 2) were obtained. The molecular weights of protease 1 and 2 were estimated at 23.1 and 22.7 kDa, respectively, by SDS-polyacrylamide gel electrophoresis. Their isoelectric points were 5.85 and 5.55, respectively, and they possessed antigenic similarity when examined by the immunoblotting. The N-terminal amino acid sequences of both the proteases were identical (RAQYVNQLKNFKIRETQ). The activities of both the proteases were strongly increased by reducing agents such as L-cysteine and sodium thioglycolate. Their activity was inhibited by thiol protease inhibitors, but was not inhibited by metalloprotease or serine protease inhibitors. From the results, it seems likely that these proteases, produced by S. aureus from diseased chickens, might belong to the thiol protease group.

Amino Acid Sequence↗
PubMed1999

Cytosolic NADPH-UQ reductase, the enzyme responsible for cellular ubiquinone redox cycle as an endogenous antioxidant in the rat liver.

Cellular ubiquinone (UQ) is expected to act as an endogenous antioxidant against oxidative stress. To confirm this, UQ-reductases which are necessary to regenerate ubiquinol (UQH2) were investigated in rat tissue, and a novel NADPH-dependent UQ (NADPH-UQ) reductase was found in cytosol. The cytosolic NADPH-UQ reductase activity accounted for more than 80% of UQ-10 reduction by the rat liver homogenate in the presence of NADPH. Furthermore, the NADPH-UQ reductase activities in various tissues were correlated to the redox states of UQ in the corresponding tissues. Rat liver cytosol with NADPH protected lecithin liposomes containing UQ-10, as well as UQH2-10 from AMVN (2,2'-azobis(2,4-dimethylvaleronitrile))-induced lipid peroxidation. The enzyme purified from rat liver cytosol, reduced UQ-10 in lecithin liposomes at approximately the same rate as did cytosol. These results supported that cytosolic NADPH-UQ reductase is the enzyme responsible for nonmitochondrial UQ reduction acting as an endogenous antioxidant against oxidative stress. The antioxidant role of the UQ redox cycle and NADPH-UQ reductase was discussed in relation to other cellular NADPH-dependent antioxidant enzymes.

Animals↗
PubMed1999

Proposed standard for human blood vitamin B1 value using HPLC. The Committee for Vitamin Laboratory Standards, Japan.

Standard reference ranges for all laboratory test values are mandatory. This study was designed to establish a reference range for blood vitamin B1 levels, since the normal range has not been determined in the Japanese population. We founded the Japan Committee for Vitamin Laboratory Standards, which was incorporated with the Vitamin Society of Japan and the Japanese Society of Nutrition and Food Science. We standardized whole blood vitamin B1 levels using three HPLC techniques (post-column reverse-phase HPLC, pre-column reverse-phase HPLC, and precolumn GP-HPLC). The reference range was obtained in 54 volunteers administered a 1,800 kcal diet with 2 mg of vitamin B1 (1.74 mg measured) daily to avoid marginal vitamin B1 deficiency in the population. The range for each assay was 26-47, 28-51, and 28-56 ng/ml, respectively. Our data suggest that 26-28 ng/ml is the lower limit of normal for whole blood vitamin B1, but further studies in a larger population are needed in order to obtain more definitive results.

Chromatography, High Pressure Liquid↗
PubMed1999

Threshold concentration of unbound bilirubin to induce neurological deficits in a patient with type I Crigler-Najjar syndrome.

Based on the clinical course of a 16-year-old boy with type I Crigler-Najjar syndrome, we estimated the threshold concentration of unbound bilirubin, as assayed by the horseradish peroxidase method, that apparently induces toxicity to the brain. Before the age of 15, the patient did not manifest any neurological or behavioural dysfunction despite increased bilirubin in serum. The binding affinity and the binding capacity of the patient's serum albumin for bilirubin determined when he was about 14 years old were 10(8)(mol/L)-1 and 1.01 to 1.04 mol/L, respectively. These values were nearly the same as those of normal controls reported in the literature. The total bilirubin binding capacity was greater than the patient's total bilirubin concentration, showing that his serum albumin was not saturated with bilirubin. The reserve bilirubin binding capacity (RBBC) was estimated to be 158 mumol/L and the unbound bilirubin concentration to be 15.1 nmol/L. Concentration of unbound bilirubin peaked at 21.7 nmol/L at the age of 15 years and 11 months, i.e. 2 months before the onset of difficulties in walking and speaking. At this time, the RBBC was estimated as -64 mumol/L. A peak concentration of total bilirubin, 811 mumol/L, was observed during the period of rapid loss of the ability to walk or speak. At the age of 16 years and 1 month the RBBC decreased to -98 mumol/L and the unbound bilirubin concentration to 18.8 nmol/L. Following phototherapy, the patient's neurological state returned to normal; he could speak and walk normally. At the age of 16 years and 2 months the RBBC returned to 105 mumol/L and unbound bilirubin decreased to 16.6 nmol/L. These results suggest that maintaining the concentration of unbound bilirubin at < 20 nmol/L and the total bilirubin concentration at lower than the binding capacity of serum albumin is important for prevention of neurological deficits in Crigler-Najjar syndrome. The upper limit of unbound bilirubin in such an older patient was nearly the same as that reported for newborns.

Adolescent↗
PubMed1999

Esterification makes retinol more labile to photolysis.

Because retinyl palmitate was reported to be more stable to oxidation than retinol, we wondered if retinyl palmitate was also more resistant to photolysis as compared to free alcohol. We investigated the resistance of ethanolic solutions of retinol, retinyl palmitate, or both to air oxidation and (or) photolysis using fluorescent light. The initial concentrations were all-trans-retinol, 14 mumol/L, and all-trans-retinyl palmitate, 14 mumol/L. The concentrations of retinol and retinyl palmitate were determined by HPLC and are expressed as a percentage of their original concentrations. After 4 h of exposure to an 18 W fluorescent lamp at 15 cm from the solution, the means (SD) of the surviving analytes were 64% (3%) for retinol and 5% (2%) for retinyl palmitate in a solution containing both retinol and retinyl palmitate. Taking account of the cis isomer arising from retinyl palmitate, 29% (3%) of the retinyl palmitate survived after 4 h of photolysis. Degradation of retinyl palmitate might occur after the conversion of trans isomer to cis isomer during photolysis, however, trans isomer could be degraded with a lesser extent of isomerization. After 4 h of bubbling air through the solution in the dark, 49% (6%) of retinol and 69% (4%) of retinyl palmitate survived. Exposing retinol or retinyl palmitate separately to air oxidation, bubbling air through the solution, or photolysis, exposing them to light, we found that retinyl palmitate could retard the air oxidation of retinol (p < 0.001), but it had no effect on the light-induced degradation of retinol. We also studied the effect of the addition of approximately 1,560 mumol/L alpha-tocopherol, approximately 190 mumol/L beta-carotene and approximately 2,000 mumol/L ascorbic acid as antioxidants. In the presence of 156 mumol/L alpha-tocopherol, 87% (1%) of the retinol and 91% (4%) of the retinyl palmitate remained after air oxidation. Although the photolysis of retinol and retinyl palmitate was also inhibited by 190 mumol/L beta-carotene, alpha-tocopherol and ascorbic acid did not exert inhibiting effects. We conclude that retinyl palmitate is physico-chemically more labile to photolysis but is more resistant to air oxidation than retinol.

Air↗
PubMed1999

[Primary preventive medicine and laboratory medicine].

Nutrition plays an important role in the primary preventive medicine. In 1999, we have introduced the Dietary Reference Intakes (DRI) established by the National Academy of Sciences of U.S. into Recommended Dietary Allowances (RDAs) for the Japanese population. Where, reference intervals for the concentration of nutrient in blood are required in setting an Adequate Intakes (AI). For vitamins, also the AI is experimentally determined based on the reference intervals of healthy people, and the assessment of the nutritional status by laboratory investigations is an important part of preventive medicine. In 1997, we founded the Japan Committee for Vitamin Laboratory Standards and standardized whole blood vitamin B1 levels using HPLC method. Reference intervals for vitamin A, vitamin C and vitamin E might be presented in future report. We annually examined 15,000 participants in national nutrition survey. We proposed a use of results of this research for setting AI of vitamins. Genetic investigation should be introduced in future primary preventive medicine, and stress markers such as "biopyrrin" for oxidative stress, uropepsin, and urinary 17KS-S seems to be very important for medical examination.

Biomarkers↗
PubMed1998

Immunolocalization of proto-oncogene products in keratocytes after epithelial ablation, alkali burn and penetrating injury of the cornea in rats.

BACKGROUND: We examined the immunolocalization of proto-oncogene products, including c-Fos and c-Jun, in the rat cornea during epithelial and stromal wound healing after simple epithelial ablation, penetrating injury or alkali burn. METHODS: Eighty-four male Wistar rats were divided into three groups and subjected to treatments as follows: (a) ablation of central corneal epithelium leaving basement membrane intact, (b) alkali burn in the central cornea with 1 N NaOH and (c) penetrating injury at the central cornea. The affected eyes were then enucleated after various intervals of healing. The frozen sections were immunohistochemically stained with the antibodies against proto-oncogene products. RESULTS: c-Fos- and c-Jun-immunoreactive cells were detected in the epithelium around the epithelial defect from 60 to 120 min after these treatments. The distribution of these cells were varied in these three types of injury. The immunoreactivities for these proteins were also detected in keratocytes after epithelial ablation. In the corneas with alkali burn, the immunoreactivities were detected in the keratocytes in the whole corneal stroma, and these immunoreactions were stronger than those observed in simple epithelial ablation. In the corneas with penetrating injury, such immunoreaction was seen only in keratocytes around the wound. CONCLUSION: These findings indicate that activator protein 1-mediated transcriptional activation for epithelial migration is initiated in the early phase after each injury, and that stromal keratocytes are also transcriptionally activated not only by alkali burn or penetrating injury but also by simple epithelial ablation in which basement membrane was not affected.

Animals↗
PubMed1998

Decline in plasma retinol in unconjugated hyperbilirubinemia treated with bilirubin adsorption using an anion-exchange resin.

When an anion-exchange resin column (Plasorba BR-350) was used for the treatment of unconjugated hyperbilirubinemia, we found an unexpected decrease in plasma retinol (vitamin A) concentrations in a patient with type I Crigler-Najjar syndrome. The purpose of our study was to investigate the mechanism of this decrease in plasma retinol. When the patient's serum bilirubin exceeded the bilirubin binding capacity of 14.7 mumol bilirubin/g serum albumin (i.e., 720 mumol/L of bilirubin), abrupt deterioration of the patient's neurologic status (suppression in his gait and speech) occurred, so the need to apply plasmapheresis to reduce the unconjugated bilirubin was indicated. Blood was drawn from the radial artery at a flow rate of 160 mL/min and pumped into a membrane plasma separator at a rate of 40 mL/min. The plasma was passed through the bilirubin adsorbent column and returned to the venous blood line of the plasma separator. Plasma samples were taken at the inlet and outlet of the bilirubin adsorbent column before and after treatment. The concentration of unconjugated bilirubin in plasma was effectively reduced by the perfusion, but plasma retinol was coincidentally decreased by the perfusion to vitamin A deficiency levels. The patient's plasma retinol was 2,127 nmol/L at the beginning of therapy and decreased to 1,492 nmol/L after repeated adsorption treatments. As the amounts of decrease in retinol (912 +/- 123 nmol/L) after the perfusion were almost equal to those in retinol-binding protein (1,010 +/- 192 nmol/L), retinol may have been removed as a form of holo retinol-binding protein. Decreases in retinol and retinol-binding protein levels were also observed in low-density lipoprotein (LDL) apheresis with a dextran sulfate column (i.e., a cation-exchange resin column). In the patient with Crigler-Najjar syndrome, retinol taken dietarily was removed by plasmapheresis. However, the patient manifested no clinical symptoms associated with vitamin A deficiency, since his liver storage of retinol could supply the loss caused by plasmapheresis treatment. We should measure plasma retinol concentrations to evaluate the loss of retinol during plasmapheresis treatment coupled with an anion-exchange resin column.

Adolescent↗