Effects of dark-rearing and light exposure on incorporation of [3H]lysine into a tubulin-enriched fraction in rat visual cortex.

were identical irrespective of whether ["C]or [3H]-labetalol had been administered to the animals; therefore labetalol is not metabolized by N-dealkylation. The urine samples were concentrated on Amberlite XAD-2 resin, and the metabolites were purified by t.1.c. and paper chromatography. Enzymic hydrolysis, chemical analysis, t.l.c., paper chromatography and high-voltage profile electrophoresis (Conway et al., 1973) showed that the rat and rabbit metabolite (11) was the 0-phenyl glucuronide of labetalol, and rat and rabbit metabolite (I) the glucuronide of metabolite (V). Paper chromatography with solvent butanol/acetic acid/water ( 4 : 1 : 1.6, by vol.) showed that the major dog metaboliteconsisted of two metabolites, with Rp0.54 (16Xof the dose) and RF 0.67 (25 %of the dose). After purification by t.1.c. and paper chromatography the metabolite with RF 0.54 was shown to be the 0-phenyl glucuronide. The other metabolite was a glucuronide that was slowly hydrolysed to labetalol by /?-glucuronidase but not attacked by arylsulphatase. It has been tentatively identified as a glucuronide formed by conjugation of the glucuronyl group with the secondary alcohol group of labetalol. Mobility-pH profile high-voltage electrophoresis indicated that metabolite (V) contained two phenolic groups. When the metabolite was examined by mass spectrometry the fragmentation pattern obtained confirmed the presence of an extra phenolic oxygen and suggested that the compound was 5-{l-hydroxy-2-[(3-hydroxyphenyl-l-methylpropyl)amino]ethyl}salicylamide. This compound was synthesized and shown to be identical with metabolite (V). Two male subjects who took 200mg of [3H]labetalol orally (about 3mg/kg) excreted 59 and 61 % of the dose of radioactivity in the urine during 48 h; only in one subject was unchanged drug found (4 % of the dose). Because of the high rate of first-pass metabolism of labetalol by man (Table 1) the clinical dose used for the treatment of hypertension is 600mg or more per day. The major human radioactivemetabolite had RF0.20 on t.1.c. (Fig. la), but it was only partially hydrolysed to labetalol by glucuronidase. Paper chromatography with butanol/ acetic acid/water (4: 1 : I .6, by vol.) showed that it consisted of two metabolites with RF 0.58 (1 5 % of the dose) and RF 0.7 (45 % of the dose), These were isolated, and the former was shown to be the 0-phenyl glucuronide oflabetalol(I1). The second metabolite was resistant to hydrolysis by glucuronidase and sulphatase, but was hydrolysed by HCI to give labetalol and 2-hydroxy-5-~l-hydroxy-2-[(l-methyl-3-phenylpropyl)amino]ethyl}benzoic acid. The latter is formed through the hydrolysis of the amide group of labetalol to the carboxylic acid. The major metabolite in man is therefore a conjugate of labetalol, and further work is required to characterize the conjugating group. Hydroxylabetalol was less active as an aand 8-receptor antagonist than labetalol, and the conjugates of labetalol were pharmacologically inactive.