Studies on microcapsules to replace erythrocytes in the passive agglutination reaction.

Two kinds of microcapsules were made from polyurethane and polyurea. Each capsule had a mean diameter of 6 microns, a specific gravity of 1.10, and was red in color. The capsules were coated with ovalbumin condensed with glutaraldehyde. The coated capsules were agglutinated with anti-ovalbumin antibody. The polyurethane capsule (positive charge) bound more ovalbumin and showed a higher sensitivity than did the polyurea capsule (negative charge) or erythrocytes.

Photoaffinity labeling of the beta-adrenergic receptors and receptor reserve for isoprenaline.

The beta-adrenergic receptors in the taenia isolated from the guinea pig caecum were irreversibly inactivated by photoaffinity labeling with 1-isoprenaline and 2-(2-hydroxy-3-isopropylaminopropoxy)iodobenzene. The photoinactivation was eliminated in the presence of protectors of the receptors. Photoinactivation caused a considerable parallel shift in the concentration-action curve of 1-isoprenaline. The results possibly point to a certain receptor reserve for 1-isoprenaline.

Saltatory conduction of peripheral nerve impulse in clioquinol-treated rats.

By using a new method, unidimensional latency-topography, which shows the saltatory conduction pattern of an impulse along peripheral nerve fibers, the internodal length, internodal conduction time and conduction velocity were determined from the L5 ventral and/or dorsal root filaments of clioquinol-treated rats (CTR). The saltatory conduction pattern was preserved in most of the CTR fibers tested, but was not seen in some fibers. A positive correlation was seen between the conduction velocity and the internodal length in the nerve fibers of both the normal rats and CTR. Although there was no difference in the internodal length between normal rats and CTR, conduction velocities determined in CTR fibers were lower than those in normal rat fibers. Myelin length was calculated from the saltatory conduction pattern in the topography to represent the functional length of the saltatory conduction. The functional myelin length of the CTR fiber was shorter than that of normal rats. Shortening of the functional myelin length in CTR is due to the widening of the Ranvier node, which corresponds to the exposure of the Ranvier node, i.e. demyelination. It was concluded that the decrease in conduction velocity in CTR fibers was due to exposure which caused delayed excitation at the Ranvier nodes.

Liquid chromatographic determination of urinary dopamine and norepinephrine as fluorescamine derivatives.

Dopamine (DM) and norepinephrine (NE) with 3,4-dihydroxybenzylamine as internal standard in normal human urine were extracted with alumina, labeled with fluorescamine and determined fluorometrically by liquid chromatography. The method is simple and in the case of excretion of DM in urines of subjects receiving 1-dopa, even the alumina treatment was unnecessary. The C.V. of method was 4.3% and 4.5% for DM and NE, respectively. The mean contents of DM and NE in 7 normal urines were 182 and 35.5 ng/mg creatinine, respectively.

Urinary levels of vanilmandelic acid and homovanillic acid determined by high-speed liquid chromatography.

High-speed liquid chromatographic determinations of vanilmandelic acid and homovanillic acid in human urine have been made. The effects of taking refreshments on the assay were examined. HVA conjugates in urine also were hydrolyzed and determined. Urinary excretion of vanilmandelic acid, free and conjugated homovanillic acid in normal subjects and patients with neuroblastoma, phaeochromosytoma and Sipple's syndrome were determined and discussed. Urinary excretion values (mean +/- 1 S.D.) of vanilmandelic acid and homovanillic acid in 14 normal subjects were 2.0 +/- 0.7 mg/day and 4.0 +/- 1.0 mg/day, respectively.

Gas-liquid chromatographic and mass fragmentographic determination of catecholamines in human plasma.

Quantitative analytical methods for plasma catecholamines and their conjugates by the use of gas-liquid chromatography have been developed. Epinephrine and dopamine have also been determined by mass fragmentography. The contents of catecholamines in the plasma of normal adults and patients with hypertension, neuroblastoma and pheochromocytoma have been demonstrated.

Gas-liquid chromatographic and mass fragmentographic determination of 3-O-methylated catecholamines in human plasma.

Plasma 3-O-methylated catecholamines, i.e. 3-methoxytyramine, normetanephrine and metanephrine, were separated from catecholamines by passing through alumina and further purified by adsorbing on weakly acidic resin and Amberlite XAD-4. The amines were trifluoroacetylated and determined by gas chromatography or mass fragmentography. Tracer quantities of tritiated 3-MT, NMN or MN were used as internal standards for total recovery estimations. The contents of 3-O-methylated catecholamines in the plasma of normal persons and patients with hyperthyroidism, hypertension, neuroblastoma and pheochromocytoma were measured.

O-methylation of dopamine-o-sulfates with catechol-0-methyltransferase.

[2H2]-dopamine-3-0-sulfate (DM-3-0-S) and [2H2]-dopamine-4-0-sulfate (DM-4-0-S) were synthesized to investigate the possibility of their being substrates for catechol-0-methyltransferase (COMT). [2H5]-3-0-methyldopamine (3-0-Me-DM) and [2H5]-4-0-methyldopamine (4-0-Me-DM) were also synthesized as internal standards for the determination of enzymatic products by gas chromatography-mass spectrometry (GC-MS). [2H2]-DM-3-0-S or [2H2]-DM-4-0-S was incubated at 37 degrees for 60 min in the presence of S-adenosyl-L-methionine with a crude enzyme preparation obtained from rat liver homogenate. The incubation mixture was treated with 0.5N HCl at 100 degrees C for 1h to hydrolyze the remaining sulfate moiety. The reaction products were extracted with an Amberlite XAD-4, derivatized with pentafluoropropionic anhydride and determined by GC-MS. When [2H2]-DM-3-0-S was used as a substrate, [2H2]-3-0-Me-DM was found to be a major product accompanied by [2H2]-4-0-Me-DM as a minor product. The ratio of [2H2]-3-0-Me-DM to [2H2]-4-0-Me-DM was found to be 26:1, while the ratio was 5.4:1 when [2H2]-dopamine was used as a substrate. When [2H2]-DM-4-0-S served as a substrate, [2H2]-3-0-Me-DM was preferentially produced without detectable formation of [2H2]-4-0-Me-DM.

Determination of the partition coefficient and acid dissociation constants of iodochlorhydroxyquin by an improved partition method.

A simple and precise method for determining partition coefficients was developed utilizing a disposable injector. The method was used to determine the partition ratio of iodochlorhydroxyquin between n-decane and phosphate-buffered saline, pH 7.4. The partition ratio was found to be 1750 with a coefficient of variation of 3%. Moreover, it was found that iodochlorhydroxyquin did not associate in n-decane at a concentration less than 1 X 10(-3) M. The acid dissociation constants and the partition coefficient of the undissociated species were determined. These values corresponded well with the values obtained from spectrophotometric methods.

Qualitative analysis of ultraviolet-absorbing substances by use of fluorescent materials.

Developments of detection techniques of ultraviolet absorbing substances with fluorescent materials are reviewed.

Deterioration of spinal reflex in beagles orally ingesting clioquinol.

Spinal reflex and neuromuscular functions were investigated in the beagles ingesting clioquinol over a long period. The twitch tension, the refractory period, the summation of contraction and the discharge of the muscle spindles of the gastrocnemius muscle were not significantly damaged in the treated beagles, whereas the response with long latency of the spinal reflex disappeared completely during the administration not in the control but in the treated dogs. The meanings of disappearance of the spinal reflex in the treated dogs are discussed in relation to the degenerative change in the fasciculus gracilis at the cervical level of the spinal cord.

Interactions between Bifidobacterium bifidum N4 and Escherichia coli K-12 in their mixed cultures.

The interactions between Bifidobacterium bifidum N4 (B. bifidum) and Escherichia coli K-12 (E. coli) were investigated in their mixed cultures. Under conditions in which both bacteria grew well in their pure culture, B. bifidum inhibited the growth of E. coli even when the latter was inoculated at 10(4)-fold and preincubated for 41 hr. The inhibition in the mixed cultures appeared when the pH values were reduced below 4.6. When the lowering of pH was prevented by the addition of NaOH, no inhibition was observed. At the same initial pH of 6.5, lactic acid and acetic acid, metabolites of B. bifidum, had more inhibitory effect on the growth of E. coli than other aliphatic fatty acids. On the other hand, in the mixed cultures with E. coli, B. bifidum grew in the absence of its essential vitamins, riboflavin and pantetheine and, furthermore, aerobically.

[Differences between phthaleins and sulfonphthaleins].

Differences in color and molecular structure between phthalein-pigments and sulfonphthalein-pigments were investigated using X-ray crystallography and absorption spectrophotometry of their aqueous solutions. The molecular structure of sulfonefluorescein (H2+SF-) was determined as a zwitter ion, 2-(3-hydroxonio-6-hydroxy-3H-xanthen-9-yl)benzenesulfonate. The absorption spectra of H2+SF- demonstrated the dissociation profile of a dibasic acid, while those of fluorescein (H2FL) indicated a tribasic acid and further, at pH > 10, SF2- and FL2-, while at pH < 0, H2+SF- and H3+FL to be dominant. The spectra of H2+SF- were analyzed to obtain the values of pK1 and pK2 together with the spectrum of HSF-. Similarly, from the spectra of H3+FL the values of pK1, pK2 and pK3 together with the spectra of HFL- and H2FL were obtained. Further by adding 1/5 of the H3+FL spectrum to 2/15 of HFL- spectrum, an indicated spectrum as that of H2FL was obtained. From these results, the features of dissociation of H2SF- and H3+FL were estimated. The molecular structure of phenolsulfonphtalein (H2PS-) was determined as a zwitter-ion, alpha-(4-hydroxonio-2,5-cyclohexadiene-1-ylidene)-alpha- (4-hydroxyphenyl)-2-toluenesulfonate. The absorption spectra of H2PS- demonstrated that H2PS-, HPS- and PS2- became dominant at pH << 0, pH = 4 and pH > 11, respectively. On the contrary, phenolphthalein (H2PP) displayed only one type of absorption spectrum in the visual region, the shape of which was similar to that of PS2- while the molar extinction coefficient was smaller. The spectra were analyzed to obtain the values of pK1 = 9.05 and pK2 = 9.50. The spectra also demonstrated a slow addition reaction of OH- to PP2- and pK3 = 12 was obtained by measuring the absorbance after equilibration. From these results, the features of dissociation and coloration of H2+PS- and H2PP were estimated.