High performance liquid chromatography analysis of nifedipine and some of its metabolites in hypertensive patients

Techniques are described for the analysis of nifedipine and three of its metabolites; dehydronifedipine (I), dehydronifedipinic acid (II), and dehydronifedipinolactone (III) in human serum and urine. The analytes are extracted at pH 9 or 3 with ethyl acetate and chromatographed on a C8 reverse-phase column. Recoveries from spiked control serum and urine ranged from 70 to 95% depending on the polarity of the analyte. The coefficient of variation ranged from 10 to 15% (nifedipine, I, and III at 50-200 ng/mL) and 15-20% (II at 200-2000 ng/mL). Accuracy was always within the limits of precision. The metabolism of nifedipine in a group of hypertensive patients receiving long-term nifedipine therapy was compared with that reported for acute administration. Serum nifedipine levels were comparable in both cases, but contrary to previous reports, dehydronifedipine was consistently found in significant amounts. Serum levels of II were considerably elevated but III was not detected either in serum or in urine. Thus, prolonged nifedipine therapy in conjunction with other antihypertensive medications appears to be associated with significant alterations in the metabolism of nifedipine compared with acute administration.     

Calcium/Calmodulin Activation of Soybean Glutamate Decarboxylase

Recently, we provided preliminary evidence for calcium (Ca2+)/calmodulin (CaM) stimulation of plant glutamate decarboxylase (GAD; EC 4.1.1.15). In the present study, a detailed characterization of the phenomenon is described. GAD was partially purified from various soybean (Glycine max L. Merr.) tissues (developing seed coat and cotyledons, leaf, and root) in the presence of EDTA by a combination of ammonium sulfate precipitation and anion-exchange fast protein liquid chromatography. GAD activity showed a sharp optimum at pH 5.8, with about 12% of maximal activity at pH 7. It was stimulated 2- to 8-fold (depending on the tissue source) in the presence of Ca2+/CaM at pH 7 but not at pH 5.8. Furthermore, when the protease inhibitor phenylmethylsulfonyl fluoride was omitted from the purification procedure, GAD activity was insensitive to Ca2+/CaM but was similar in magnitude to CaM-stimulated activity. The stimulation by Ca2+/CaM was fully inhibited by the CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfon-amide and trifluoperazine. With saturating CaM or Ca2+, the concentrations of Ca2+ and CaM required for half-maximal stimulation were about 7 to 11 [mu]M and 25 nM, respectively. The effect of Ca2+ and CaM appeared to be through a 2.4-fold stimulation of Vmax and a 55% reduction in Km. The results suggested that GAD is activated via Ca2+ signal transduction.     

Precedence effects and the evolution of chorusing

The structured choruses produced by rhythmically signalling males in many species of acoustic animals have long-captured the imagination of evolutionary biologists. Though various hypotheses have been forwarded to explain the adaptive significance of such chorusing, none have withstood empirical scrutiny. We suggest instead that alternating and synchronous choruses represent collective epiphenomena resulting from individual males competing to jam each other''s signals. These competitions originate in psychoacoustic precedence effects wherein females only orient toward the first call of a sequence, thus selectively favouring males who produce leading calls. Given this perceptual bias, our modelling confirms that a resetting of signal rhythm by neighbours'' signals, which generates either alternation or synchrony, is evolutionarily stable provided that resetting includes a relativity adjustment for the velocity of signal transmission and selective attention toward only a subset of signalling neighbours. Signalling strategies in chorusing insects and anurans are consistent with these predicted features.     

A gas chromatography-mass spectrometry method for the quantitative analysis of melatonin in plasma and cerebrospinal fluid

A method has been developed for the quantitative analysis of melatonin in human plasma and cerebrospinal fluid. The technique involves a simple one-step extraction with chloroform and conversion of the melatonin in the extract to its N-trimethylsilyl derivative, which is then measured by means of high-sensitivity gas chromatography—mass spectrometry. Intra-assay precision for triplicate samples at the 20-pg/ml level was better than 20%, while the interassay precision for separate determinations made over the course of a week was better than 18%. In a series of human plasma samples taken over several times during a 24-hr period, a significant increase in melatonin level was noted during the hours of darkness.     

Increased metabolic heat production following chronic alpha-methyldopa therapy in hypertensives

The objective of this study was to measure metabolic heat production in mild-moderate hypertensives when given chronic alpha-methyldopa for control of hypertension and to compare this with hydrochlorothiazide. Four men and one woman, age range 29-55 years, euthyroid, and not previously on alpha-methyldopa, had their drugs replaced by placebo for 4 weeks, and then alpha-methyldopa or hydrochlorothiazide was randomized single blindly to patients. The dose of each drug was increased until the ambulatory diastolic blood pressure was less than 95 mmHg (1 mmHg = 133.322 Pa), and then the dose was maintained for 20-24 weeks. The two active drugs were then crossed over, and each medication was again titrated to attain diastolic normotension (less than 95 mmHg). Maintenance was for a comparable 20-24 weeks. At week 4 (end of placebo period), weeks 20-24 (for hydrochlorothiazide or alpha-methyldopa monotherapy), and again at week 40-48 (end of cross-over period), metabolic heat production (watts), heart rate (beats per minute), and blood pressure (millimetres of Hg) were measured in a resting sitting position for 1 h in a whole body calorimeter (air temperature 28 degrees C and a dew point temperature less than 5 degrees C) for 15 experimental sessions. In four out of five patients, metabolic heat production was significantly (P less than 0.05) higher when patients were treated with alpha-methyldopa. We speculate from our data that alpha-methylnorepinephrine, the major metabolite of alpha-methyldopa, may be thermogenic and that this should be considered when the drug is prescribed.