Fluorometric determination of 1,2,3,4-tetrahydro-6,7-dihydroxyisoquinoline in biological materials by HPLC.

In the belief that endogenous 1,2,3,4-tetrahydro-6,7-dihydroxyisoquinoline (DA-Fp) could be a potential marker involved in the etiology of various diseases such as Parkinsonism, we attempted to develop a fluorescence method for DA-Fp. It was synthesized by condensation of dopamine with formaldehyde according to an established method. Periodate was identified by screening from various oxidation reagents as a fluorescence reagent to DA-Fp. Optimal reaction conditions were obtained with 0.25 mM NaIO4 in 0.1 M phosphate buffer (pH 8.0) at 37 degrees C for 15 min. The fluorescence spectrum of the derivative showed that we had found a new reaction specific for DA-Fp. This reaction was coupled on-line to high performance liquid chromatography (HPLC), which enabled us to achieve a highly sensitive method for determining DA-Fp. A working curve was linear from 2 to 800 pmol of DA-Fp per injection. To determine DA-Fp in biological materials, the pretreatment before HPLC was optimized by hydrolysis of its conjugate and suppression of the artifact with l-phenylephrine. Urinary excretion of DA-Fp in men was measured by this new present method. The urinary excretion of endogenous DA-Fp increased in a rabbit given L-DOPA. The DA-Fp concentration was determined in rat brain. The significance of DA-Fp in these biological materials is discussed and evaluated. We conclude that the present method will be useful for studying tetrahydroisoquinolines involved in many diseases.

[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.

Infarction of breast fibroadenoma in a postmenopausal woman.

Infarction of a breast fibroadenoma usually occurs in young females during pregnancy or lactation. This report describes a rare case of the total infarction of a fibroadenoma in a 58 year old postmenopausal woman. The chief complaint was a rapidly enlarged, slightly painful lump. Medullary carcinoma was suspected on palpation and mammography. Microscopically, the tumor was composed of a coarsely lobulated necrotic mass invaginated into an epithelial lined cyst. This was interpreted as an infarcted intracanalicular fibroadenoma, although the possibility of an infarcted phyllodes tumor cannot be entirely excluded. The significance of this rare event is the possible confusion with carcinoma. Extensive search for viable tissue is essential for the confirmation of the diagnosis.

Primary malignant lymphoma of the urinary bladder. A case report.

A patient with primary malignant lymphoma of the urinary bladder is presented. Grossly, the bladder showed multiple submucosal masses. Histologically and immunohistochemically, diffuse B-cell lymphoma of the medium-sized cell type was revealed. On the basis of clinicopathological features, the present case resembled previously recorded cases of bladder lymphoma. The pathogenesis of this primary bladder lymphoma was presumably associated with follicular or chronic cystitis. Primary lymphoma of the bladder is a condition that is very rarely included in series of extranodal lymphomas, and there is a curious sex difference in its occurrence rates between Japan and Western countries. Primary lymphoma of the bladder may be considered a lymphoma that originates fro mucosa-associated lymphoid tissue.

Sex difference of the metabolic disposition of clioquinol in rats.

The order of plasma levels of clioquinol and its conjugates in male rats after intraduodenal administration of 32.7 mumol/kg dose of clioquinol was clioquinol sulfate (C-Sul) greater than glucuronide (C-Glu) much greater than clioquinol, whereas that in female rats was C-Glu greater than C-Sul much greater than clioquinol. Total (urine + bile) recovery was almost the same among male and female rats. The percentage of excretion amounts of C-Sul (urine + bile) to the total excretion amounts for 24 h in male rats after intravenous administration of clioquinol was about twice that in female rats, while the percentage of excretion amounts of C-Glu to the total excretion amounts in male rats was smaller than that in female rats. In intravenous administration of 16.4 mumol/kg dose of C-Glu, C-Sul and clioquinol other than C-Glu were found in bile and urine of male and female rats. The percentage of excretion amounts of C-Sul and C-Glu (urine + bile) to the total excretion amounts was similar among male and female rats, respectively. In intravenous administration of 16.4 mumol/kg dose of C-Sul, C-Glu and clioquinol other than C-Sul were found in bile and urine of male and female rats, and the percentage of excretion amounts of C-Sul (urine + bile) to the total excretion amounts in male rats was 1.3 fold that in female rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in lipid peroxide concentrations in plasma and tissues by repeated administration of clioquinol to neonatal rats.

The changes in lipid peroxide concentrations in plasma and tissues after subcutaneous administration of clioquinol to clioquinol-sensitive (S-rats) and -resistant neonatal rats (R-rats) were investigated. When a fixed dose of 150 mg/kg/d of clioquinol was given to R-rats for 14 d after birth, no significant difference in lipid peroxide concentrations in plasma, liver, kidney, brain and spinal cord at 5, 10 and 15 d was observed between clioquinol-treated and untreated rats. However, with increasing doses of clioquinol to R-rats every 5 d (150----300----600 mg/kg/d), the lipid peroxide concentrations at 15 d were higher in plasma, brain and spinal cord of clioquinol-treated rats than in those of untreated rats. These results suggested that repeated administrations of large doses of clioquinol to rats increased the lipid peroxides in nerve tissues. With S-rats at 5 d after birth, the lipid peroxide concentrations in liver were approximately twice those in R-rats regardless of the clioquinol administration.

Analysis of chinoform binding to human serum albumin by an improved partition equilibrium method.

Previous methods such as equilibrium dialysis to measure chinoform binding to human serum albumin are flawed with problems due to the drug's poor solubility in neutral aqueous solutions and its strong adsorption onto the surfaces of dialysis membrane and other apparatus. Accordingly, we adopted a previously reported improved partition equilibrium method to keep off such difficulties, and developed additionally a rapid method using oil-wells. Both of these methods were used in this study. The equilibration of both partition and binding was attained after 6 h by the standard method and after 2 h by the rapid method. Two binding sites were found on the albumin molecule. One had an extremely large affinity constant of 10(8) M-1 and the other had a moderate one of 10(6) M-1. These values obtained with both methods agreed well and were sufficiently reproducible. The bound percentage of chinoform in serum was estimated at 99.999% under the condition where chinoform reveals its nervous toxicity. The binding constant had a maximum at pH 7.6, the physiological pH of serum. The pH profile suggested the participation of a histidine residue in the binding site. The inhibition experiment with palmitic acid revealed that the first binding site of chinoform was not identical with that of palmitic acid. The present method is applicable to analyses of protein binding by other drugs showing similar physicochemical properties as chinoform.

Absorption, distribution and metabolism of clioquinol in clioquinol-sensitive and -resistant neonatal rats.

Three types of rats with respect to sensitivity to clioquinol have been identified, the highly sensitive (S-rat), the intermediately sensitive, and the resistant (R-rat). In a toxicity test that lasted for 20 d after birth, the difference in sensitivity to clioquinol between S- and R-rats was confirmed by repeated subcutaneous administration of a clioquinol suspension prepared with polysorbate 80 (clioquinol dose of 150 or 300 mg/kg/d). Plasma and tissue concentrations of clioquinol and the rate of metabolism of the drug in neonatal S- and R-rats were measured. Plasma concentration of clioquinol in 1-d-old S-rats after a single subcutaneous administration was higher than that in the same age R-rats and the area under the mean plasma concentration-time curve for the S-rats was approximately twice that for the R-rats. In addition, clioquinol concentrations in liver, kidney and brain of S-rats at 9 h after the administration were more than twice those of the R-rats. From the experiments on the formation of clioquinol glucuronide and sulfate with 9000 g supernatant fraction of liver, it was suggested that the difference in the plasma concentration after the administration may be responsible for the difference in the metabolizing rate of clioquinol.

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.

Biopharmaceutical evaluation of methylcellulose as an excipient for nitroglycerin tablets.

To examine the bioavailability of nitroglycerin in sublingual tablet, 5 healthy adult volunteers underwent simultaneous sublingual administration of a tablet prepared with methylcellulose and a tablet prepared with lactose containing 0.15 mg of nitroglycerin and 0.15 mg of nitroglycerin-15N3, respectively. In 4 of 5 volunteers, no difference in plasma concentration determined by gas chromatography-mass spectrometry (GC-MS) was observed between 2 tablets described above, so it was well accepted that nitroglycerin in tablets prepared with methylcellulose had a similar bioavailability to that in tablets prepared with lactose. The simultaneous administration of the 2 nitroglycerin tablets to a healthy volunteer, one of which containing the drug labeled with a stable isotope followed by GC-MS assay, seems to be the most suitable method for a precise comparison of bioavailability of the formulations.

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.

Sensitive fluorimetry of adenine-containing compounds with high-performance liquid chromatography.

A definitive method to determine adenine compounds simultaneously was established by introducing a new fluorescent reagent into high-performance liquid chromatography. Bromoacetaldehyde was the best reagent among the haloacetaldehydes examined. A quantitative reaction was obtained even for unstable ADP and ATP. A high resolution of adenine nucleotides was obtained using a column of Hitachi gel No. 3012-N. The method was applied to the measurement of cyclic AMP in urine, and ADP and ATP in brain and blood. Further, the sensitivity of the method was increased by a new fluorescence spectrophotometer constructed for micro-HPLC. Femtomole amounts of the adenine nucleotides were clearly separated.

Enterohepatic circulation of clioquinol in the rat.

The existence of the enterohepatic circulation (EHC) of clioquinol was confirmed by using paired rats, donor and recipient, which were connected to each other with a bile duct-to-duodenum cannula. The concentrations of clioquinol and its metabolites appearing in the plasma of the recipient following intraduodenal 10 mg/kg dose of clioquinol to the donor were fairly low. However, within 24 h after the administration ca. 12% of the dose was reexcreted in the bile of the recipient as clioquinol glucuronide and ca. 2% in the urine as clioquinol sulfate. From these results and the data of biliary excretion in our previous paper, the glucuronide was found to play a role on the EHC. Further, both in vitro and in situ results suggested that clioquinol glucuronide excreted in the bile may be absorbed partially after return to the parent drug in the intestinal tract and partially as such without deconjugation.

Quantitative determination of furosemide in plasma, plasma water, urine and ascites fluid by high-performance liquid chromatography.

A high-performance liquid chromatographic method using spectrofluorometric detection is described for the determination of furosemide in plasma, plasma water, urine and ascites fluid. The extraction procedure decreases interference from endogenous substances. The detection limit of furosemide is 10 ng in 0.5 ml of biological sample. The method is sufficiently sensitive for pharmacokinetic study of furosemide with normal subjects and patients with liver cirrhosis and/or renal disease after oral administration of furosemide in a retard capsule, and for study of protein binding of furosemide in patients with various diseases.

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.

Intestinal absorption and metabolism of clioquinol in the rat.

Plasma concentrations of clioquinol and its metabolites after single or repeated oral administration of clioquinol, absorption region of clioquinol in gastrointestinal tract, and intestinal metabolism were studied in rats. Plasma concentrations of clioquinol after oral administration of four different doses (20, 100, 200 and 400 mg/kg) were lower than those of the two metabolites, clioquinol glucuronide and sulfate. Mean maximal plasma concentration of unchanged drug was in the range of 1-8 nmol/ml. Clioquinol was absorbed poorly from the stomach and fairly from the small intestine. Bile was an important route for excretion of clioquinol in rats. The mesenteric venous plasma from the closed intestinal loops of both jejunal and ileal regions was analyzed for clioquinol and the metabolites and it was found that clioquinol glucuronide was formed predominantly in both regions. From the results of the present studies, intestinal metabolism of clioquinol can be pointed out as a major factor for difficulty to cause clioquinol intoxication.