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The effects of pyrroloquinoline quinone (PQQ) and PQQ-oxazole (PQQ-glycine adduct) on DNA synthesis were examined using cultured human fibroblasts. Confluent fibroblasts were cultured in serum-free Dulbecco's modified Eagle's media, and various concentrations of PQQ and PQQ-oxazole were added to the media. After incubation for 24 h, [3H]thymidine was added to the media as an indicator for DNA synthesis of the cells. The thymidine incorporation into the cells was significantly enhanced even in the presence of very low concentrations of PQQ (0.003-0.03 microM); it remained significantly enhanced, up to 30 microM PQQ. However, the incorporation remarkably decreased at 750-1500 microM of PQQ. In contrast to the results for PQQ, DNA synthesis was not stimulated by addition of 0.003-3 microM PQQ-oxazole, but it was slightly enhanced at concentrations 15-750 microM. In morphological examination of the cultured human fibroblasts, cell density was increased by addition of 0.003-30 microM PQQ when compared with that of the control, supporting the above biochemical data. However, there were no distinct differences in morphological effect between PQQ and PQQ-oxazole.

The best conditions for extractions of free pyrroloquinoline quinone (PQQ) from crude biological samples were investigated with various organic solvents and Sep-Pak C18 cartridges. PQQ was measured with use of its native fluorescence in aqueous solution. PQQ was well extracted into n-butanol under acid conditions, and addition of NaCl did not improve the solvent extraction. PQQ, which had been extracted into n-butanol, could be re-extracted into an aqueous phase by addition of either n-heptane or pyridine, or combination of them. PQQ, which had been adsorbed to Sep-Pak C18 cartridges, could be eluted with a mixture of pyridine and water with very excellent recovery. The recovery of 1 micrograms PQQ, which had been added to 1 g human liver, brain and 1 ml plasma and had undergone the n-butanol and the Sep-Pak extractions, was 50, 75 and 105%, respectively. From the blank fluorescence, endogenous levels of free PQQ in human liver, brain and plasma were found not greater than 0.41, 0.08 and 0.13 micrograms/g or ml, respectively, if present.

A method was developed for the simultaneous determination of dopamine (DA), epinephrine (E), norepinephrine (NE), 3,4-dihydroxyphenylacetic acid (DOPAC) and 3-methoxy-4-hydroxyphenylglycol (MHPG), as well as L-3,4-dihydroxyphenylalanine (L-DOPA) with liquid chromatography (LC) using electrochemical (EC) detection. With a ODS column and a mobile phase consisting of a sodium acetate-citrate with heptasulfonic acid, this method was applied on simultaneous determination of chatechols released from thalamic slices of ddY mouse. The pretreatment of the bathing medium required only centrifugation, and the supernatant was injected directly into the LCEC system. The high potassium stimulation of catecholaminergically innervated thalamic slices led to increase in the levels of DA, NE, DOPAC and MHPG, especially of NE, but not that of L-DOPA itself. In the present study, we designed to make simultaneous determination of catechols released from thalamic slices for estimation of the physiological status of catecholaminergic neuronal activity.

Nineteen phenothiazines were tested for in vitro inhibition of human platelet type B monoamine oxidase (MAO). The inhibition potency was highly dependent on structures of their side chains. The inhibition was most potent for drugs with (hydroxyethyl-piperazinyl)propyl chains followed in decreasing order by those with (N-methylpiperazinyl)propyl, (2-dimethylamino-2-methyl)ethyl and 3-dimethylaminopropyl chains. Kinetic analyses were carried out for promazine, promethazine, perazine and perphenazine as representatives of each group; the four drugs showed competitive inhibition, and K i values of 124, 31.4, 19.2 and 22.6 μM, respectively.