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Biomedical subjects

M Kai

Publications and source records attributed to M Kai.

At least 163 records · Page 9Linked to original sources
PubMed1989

Determination of renin activity in human plasma by column-switching high-performance liquid chromatography with fluorescence detection.

A column-switching high-performance liquid chromatographic (HPLC) method with fluorescence detection is described for the determination of the renin activity in human plasma. The method is based on the quantification of the enzymatically produced angiotensin I. Angiotensin I liberated from a synthetic substrate (tridecapeptide of human angiotensinogen) and [Val5]-angiotensin I as an internal standard are converted into fluorescent derivatives by reaction with benzoin. The derivatives are separated from various interfering substances by column-switching HPLC using three reversed-phase columns. The limit of detection (signal-to-noise ratio = 3) of the renin activity is 2.7 pmol of angiotensin I formed per h per ml of plasma, which corresponds to approximately 820 fmol of angiotensin I injected. The column-switching method in combination with pre-column derivatization for the fluorimetric detection permits the sensitive and selective determination of the enzymatically formed angiotensin I. Hence low activities of renin in normal human plasma are readily measured.

Chromatography, High Pressure Liquid↗
PubMed1989

Cytochrome oxidase of an acidophilic iron-oxidizing bacterium, Thiobacillus ferrooxidans, functions at pH 3.5.

Cytochrome oxidase of Thiobacillus ferrooxidans was partially purified. The oxidase preparation had haems a and c, and oxidized ferrocytochrome c-552 of the bacterium. The optimal pH of the reaction was 3.5. The enzyme also oxidized the reduced form of rusticyanin, a copper protein of the bacterium. Our results indicate that the reduction of molecular oxygen by this enzyme may occur in the periplasm.

Azurin↗
PubMed1989

Combined effects of radiation and caffeine on embryonic development in mice.

The combined effect of radiation and caffeine has been studied in mouse embryos. Radiation and/or caffeine were administered to ICR mice on Day 11 of gestation. Intrauterine death, gross malformation, and fetal body weight were selected as indicators of effects. Doses of whole-body gamma irradiation were 0.5 to 2.5 Gy and those of caffeine were 100 and 250 mg/kg maternal body wt. Intrauterine mortality increased with increasing radiation dose; this trend was more remarkable in combination with caffeine. Gross malformations such as cleft palate and defects of forelegs and hindlegs appeared frequently in the fetuses treated with both radiation and caffeine. Decreased fetal weight was observed even in mice treated with 0.5 Gy of radiation or 100 mg/kg caffeine. There was a linear relationship between dose and reduction of fetal weight. The fetal weight was a sensitive, precise, and easy-to-handle indicator for the effects of growth retardation. Intrauterine mortality and frequencies of cleft palate and defects of forelegs and hindlegs were higher than the sum of those induced by radiation and by caffeine separately. The results indicated that the combined action of radiation and caffeine on intrauterine death and malformations was synergistic.

Abnormalities, Drug-Induced↗
PubMed1988

Determination of oestrogens in pregnancy urine by high-performance liquid chromatography with fluorescence detection.

A simple and sensitive high-performance liquid chromatographic method is described for the determination of three oestrogens (oestriol, oestrone and oestradiol) in pregnancy urine. Free oestrogens are extracted with chloroform from the urine sample. The phenolic group of each oestrogen in chloroform is formylated in the presence of an alkaline aqueous solution, and the resulting aldehyde is converted into a fluorescent derivative by reaction with 1,2-diamino-4,5-dimethoxybenzene. In order to determine free and conjugated oestrogens, conjugated oestrogens are hydrolysed by heating in hydrochloric acid before the extraction and then treated in the same way as free oestrogens. The fluorescent derivatives are separated on a reversed-phase column, TSKgel ODS-120T, with stepwise gradient elution using an aqueous methanol-containing phosphate buffer (pH 2.2). The lower limit of detection for each oestrogen is ca. 200 fmol per 100-microliters injection volume.

Chromatography, High Pressure Liquid↗
PubMed1988

High-performance liquid chromatographic determination of leucine-enkephalin-like peptide in rat brain by pre-column fluorescence derivatization involving formylation followed by reaction with 1,2-diamino-4,5-dimethoxybenzene.

Fluorimetrically reactive leucine-enkephalin, one of the opioid peptides in rat brain tissues such as the striatum, cortex and hypothalamus, was assayed by reversed-phase high-performance liquid chromatography with fluorescence detection based on pre-column derivatization of the tyrosyl residue. The tyrosine-containing peptides extracted from the tissue were first formylated with chloroform in an alkaline medium, and the resulting aldehydes were then converted into highly fluorescent derivatives by reaction with 1,2-diamino-4,5-dimethoxybenzene. The derivative of leucine-enkephalin-like peptide in tissue was separated from various other fluorescent compounds on a reversed-phase column (TSK gel ODS-120T) by isocratic elution and detected by fluorimetry. The concentrations of the leucine-enkephalin-like peptide in the tissues were 20-245 pmol/g. The method is sensitive enough to permit the quantitative determination of the endogenous peptide at concentrations as low as 5.6 pmol/g in brain tissues.

Animals↗
PubMed1988

Assay for enkephalin-degrading peptidases in rat brain tissues by high-performance liquid chromatography with on-line post-column fluorescence detection.

The activities of enkephalin-degrading peptidases such as enkephalinases A and B in rat brain tissues were simultaneously assayed by a high-performance liquid chromatographic method with fluorimetric detection with an automatic reaction system. Tyrosine and tyrosine-containing peptides produced enzymatically from the substrate, methionine-enkephaline, were separated by gradient elution on a reversed-phase column (TSK gel ODS-120T), and then converted into fluorescent derivatives for detection by reaction with hydroxylamine, cobalt(II) and borate reagents. The method permits the simple and sensitive detection of N-terminal tyrosine-containing fragments of the enkephalin peptide. The limits of detection are 5-20 pmol per assay tube for the N-terminal tyrosine-containing fragments. The enzyme activities in the regionally separated tissues were 54-191 pmol/min.mg protein for enkephalinase A and 79-153 pmol/min.mg protein for enkephalinase B, which were calculated from the formation of Tyr-Gly-Gly and Tyr-Gly, respectively, during the enzyme reaction.

Animals↗
PubMed1988

High-performance liquid chromatographic determination of forphenicine in mouse serum and muscle by pre-column fluorescence derivatization using 1,2-diamino-4,5-ethylenedioxybenzene as fluorogenic reagent.

A simple and sensitive high-performance liquid chromatographic method has been developed for the determination of forphenicine in biological samples. Forphenicine in the deproteinized sample is converted by reaction with 1,2-diamino-4,5-ethylenedioxybenzene into a fluorescent derivative. The derivative is separated on a reversed-phase column (TSK gel ODS-120T) by isocratic elution with acetonitrile-30 mM phosphate buffer (pH 6.5) (5:1, v/v) and monitored fluorimetrically. The method allows the quantification of forphenicine in serum (100 microliters) and muscle (0.1 g) of mice dosed with forphenicine or forphenicinol. The limits of detection (signal-to-noise ratio of 3) are 7.35 pmol/ml in serum and 5.36 pmol/g in muscle. The distribution of forphenicine and forphenicinol in the mouse serum and muscle after oral administration of these compounds is also described.

Animals↗
PubMed1988

A new enzymatic approach for estimating total and direct bilirubin.

We designed an enzymatic assay for total (TBil) and direct bilirubin (DBil), the principle of which involves measuring the decrease in absorbance at 450 nm produced by bilirubin oxidase from Myrothecium verrucaria. Since TBil and DBil are oxidized at pH 7.2 and 3.7, respectively, the degree of bilirubin oxidation is measurable in each case. An analysis of bilirubin by high-performance liquid chromatography, before and after the enzymatic reaction with bilirubin oxidase, verified the specificity of the enzyme. The results obtained using this method varied linearly with TBil and DBil concentrations up to at least 250 mg/L and 150 mg/L, respectively. Reducing substances, commonly used anticoagulants and hemoglobin showed no apparent interference. The degree of day-to-day precision (CV) for TBil and DBil ranged from 1.2% (206.2 mg/L) to 10.6% (3.5 mg/L) and from 1.8% (84.3 mg/L) to 12.4% (2.1 mg/L), respectively. Values measured using this new method correlated well with those obtained by Malloy-Evelyn's method and the slide method employing the Kodak Ektachem analyzer.

Bilirubin↗
PubMed1987

High-performance liquid chromatography of N-terminal tyrosine-containing oligopeptides by pre-column fluorescence derivatization with hydroxylamine, cobalt (II) and borate reagents.

A pre-column fluorescence derivatization method is described for the high-performance liquid chromatographic determination of N-terminal tyrosine-containing oligopeptides involving methionine-enkephalin and leucine-enkephalin. The peptides are converted into fluorescent derivatives by heating in a weakly alkaline medium (pH 8.5) containing hydroxylamine, cobalt(II) ion and borate. The derivatives are separated on a reversed-phase column (TSKgel ODS-120T) by gradient elution of acetonitrile in a mobile phase containing borate buffer (pH 8.5) and tetra-n-butylammonium chloride, and then determined by fluorimetry. The derivatization provides a single fluorescent product for each N-terminal tyrosine-containing oligopeptide, but does not allow the production of fluorescent derivatives for peptides having no tyrosyl residue at the N-terminal. The method is selective and sensitive; the lower limits of detection for the N-terminal tyrosine-containing oligopeptides tested were 140-310 fmol per 100 microliters injected.

Boric Acids↗
PubMed1987

On-line post-column fluorescence detection for N-terminal tyrosine-containing peptides in high-performance liquid chromatography.

A detection system based on on-line post-column fluorescence derivatization is described for the determination of N-terminal tyrosine-containing peptides by reversed-phase high-performance liquid chromatography. The peptides are automatically converted into fluorescent derivatives by reaction with hydroxylamine, cobalt (II) and borate after peptide separation on a reversed-phase column (TSKgel ODS-120T) followed by passage through an ultraviolet absorbance detector. The reaction system permits the fluorescence detection at 435 nm (emission) with excitation at 335 nm for N-terminal tyrosine-containing synthetic peptides in as little as picomole amounts. The facile fluorescence detection of N-terminal tyrosine-containing fragments produced from methionine-enkephalin by enzymatic degradation using a rat brain homogenate was achieved by comparison with the ultraviolet absorption detection at 215 nm.

Animals↗
PubMed1987

On-line post-column fluorescence derivatization of arginine-containing peptides in high-performance liquid chromatography.

A selective detection system based on on-line post-column fluorescence derivatization is described for the analysis of arginine-containing peptides by high-performance liquid chromatography. The peptides are automatically converted into fluorescent derivatives with benzoin, a fluorogenic reagent for guanidino compounds, after separation on a reversed-phase column (TSKgel ODS-120T) and detection in an ultraviolet absorption detector. The system permits fluorescence detection at 435 nm emission with irradiation at 325 nm for arginine-containing peptides in as little as picomole amounts. The chromatogram obtained with fluorescence detection only shows peaks corresponding to arginine-containing peptides. The facile detection of arginine-containing fragments in the tryptic digest of beta-melanocyte stimulating hormone as a model compound could be achieved by comparison with a chromatogram obtained with ultraviolet absorption detection at 215 nm.

Arginine↗
PubMed1987

Immunocytochemical localisation of carbonic anhydrase isozyme III in equine skeletal muscle.

The location of carbonic anhydrase III (CA-III) in frozen sections of biopsies of Thoroughbred horse skeletal muscle was studied. Fibre types were determined by ATP-ase and succinate dehydrogenase staining. CA-III isozyme was detected using a peroxidase conjugated anti-CA-III antibody. CA-III was found to be localised in slow twitch oxidative fibres (ST), but was also present in fast twitch oxidative (FTH) fibres in small amounts. Fast twitch glycolytic (FT) fibres were stained lightly compared with control sections. The concentrations of CA-III in muscle and liver were 70 micrograms/mg protein and 4 micrograms/mg protein, respectively. CA-I and CA-II were not found in muscle extracts by the double immunodiffusion method.

Animals↗
PubMed1986

Sensitive assay for serum angiotensin-converting enzyme and separation of angiotensin analogues by high-performance liquid chromatography with fluorescence detection.

A high-performance liquid chromatographic method is described for the assay of angiotensin-converting enzyme in human serum and for the separation of angiotensins and their analogues after pre-column fluorescence derivatization with benzoin. Angiotensin II, formed enzymatically from angiotensin I, is converted into a fluorescent derivative which is then separated isocratically from the substrate and biological substances in the enzyme reaction mixture on a reversed-phase column (TSK gel ODS-120T). The lower limit of detection for angiotensin II is 0.66 pmol per enzyme assay tube. The method is simple and sensitive, and requires as little as 5 microliter of human serum. Angiotensin analogues can also be separated and quantified by the chromatographic technique, and thus this method permits the use of the analogues of angiotensin I as substrates.

Angiotensin II↗
PubMed1986

High-performance liquid chromatography of tyrosine-containing peptides by pre-column derivatization involving formylation followed by fluorescence reaction with 1,2-diamino-4,5-dimethoxybenzene.

A pre-column fluorescence derivatization method is described for the high-performance liquid chromatographic determination of tyrosine-containing peptides. A tyrosyl residue in the peptide is first formylated in an alkaline medium in the presence of chloroform, and the resulting aldehyde is then converted into a fluorescent derivative by reaction with 1,2-diamino-4,5-dimethoxybenzene. The derivative is separated on a reversed-phase column (LiChrosorb RP-18) by isocratic elution with an aqueous acetonitrile-containing potassium chloride-hydrochloric acid buffer (pH 2.2) and sodium 1-hexanesulphonate. The method is selective and fairly sensitive; the lower limits of detection for the tyrosine-containing peptides tested are in the range 3.4-26.2 pmol in a 100-microliter injection volume.

Amino Acid Sequence↗
PubMed1986

Simple determination of forphenicinol in human plasma and erythrocytes by HPLC with native fluorescence detection.

A high performance liquid chromatographic method is described for monitoring forphenicinol, a possible therapeutic drug for cancer and muscular dystrophy, in human plasma and erythrocytes. Forphenicinol in the deproteinized samples was separated from interfering biogenic substances on an aminopropyl-bonded silica (Unicil NH2) column within 10 minutes with isocratic elution, and determined with fluorescence detection. The detection limits for forphenicinol in plasma and erythrocytes are 65 pmol (12.8 ng)/ml and 160 pmol (31.5 ng)/ml, respectively, corresponding to 2 pmol each in a 100 microliters injection volume. The method is very simple, and sensitive enough to permit the quantification of forphenicinol in the blood samples from man dosed with forphenicinol.

Adjuvants, Immunologic↗
PubMed1986

Dietary sardine oil increases erythrocyte membrane fluidity in diabetic patients.

The effects of dietary sardine oil rich in eicosapentaenoic acid, C20:5 (EPA), on erythrocyte membrane fluidity and membrane and plasma lipids were investigated in diabetic and control subjects. Before consumption of this oil, the levels of erythrocyte membrane fluidity were lower in the diabetic subjects, as noted in our previous work (Diabetes 1983; 32:585-91). Decreased membrane polyunsaturated fatty acid contents were evident. Daily consumption of 2700 mg of sardine oil for 8 wk increased erythrocyte membrane fluidity, as determined by electron spin resonance using the 12- or 16-stearic acid label. This increase was seen after 4 wk, and the level remained elevated for 8 wk. Membrane EPA of phospholipid acyl-chains significantly increased after 4 wk and was even more apparent after 8 wk. Membrane-free cholesterol to phospholipid molar ratios significantly decreased after 8 wk. Both the diabetic and normal subjects responded to the sardine oil in the same way. After feeding with sardine oil, there no longer were differences in erythrocyte membrane fluidity between the normal and diabetic subjects. We propose that improvement in membrane fluidity may contribute to the amelioration of altered cell membrane functions in diabetic patients.

Adult↗