Simple and sensitive high-performance liquid chromatographic method for the determination of diltiazem and six of its metabolites in human plasma

A sensitive, specific and reproducible high-performance liquid chromatographic technique is described for the simultaneous determination in human plasma of diltiazem (DZ) and six of its primary and secondary metabolites which are products of N- and O-demethylation, deacetylation and N-oxidation. The method involves addition of excess KHCO₃ to 1 ml of plasma, followed by extraction with 4 ml of ethyl acetate. The organic layer was extracted with 0.01 M HCl and the aqueous layer was dried under nitrogen and then reconstituted with 0.002 M HCl. DZ and its metabolites were free from interference and were baseline-separated. Calibration curves were linear in the concentration range studied (5–500 ng/ml for all the species). The lower limit of quantification of the assay was 5 ng/ml for DZ and the metabolites. Inter-day and intra-day coefficients of variation were less than 10%. The applicability of this procedure is shown by evaluating the kinetics of DZ and its metabolites in three patients receiving chronic DZ therapy. N-Demethyldiltiazem, deacetyldiltiazem and N-demethyldeacetyldiltiazem were found to be the major metabolites, as previously described. Deacetyldiltiazem N-oxide was found in two of the patients. The other two known but unreported metabolites in human, O-demethyldeacetyldiltiazem and N,O-didemethyldeacetyldiltiazem, were found in the plasma of all three patients.     

In vivo andin vitro methylation of lysine residues ofEuglena gracilis histone H1

We have earlier identified and purified two protein-lysine N-methyltransferases (Protein methylase III) fromEuglena gracilis [J. Biol. Chem.,260, 7114 (1985)]. The enzymes were highly specific toward histone H1 (lysine-rich), and the enzymatic products were identified as ε-N-mono-, di- and trimethyllysines. These earlier studies, however, were carried out with rat liver histone H1 as thein vitro substrate. Presently, histone H1 has been purified fromEuglena gracilis through Bio-Rex 70 and Bio-Gel P-100 column chromatography. TheEuglena histone H1 showed a single band on SDS-polyacrylamide gel electrophoresis and behaved like other histone H1 of higher animals, whereas it had a much higherR _f value than the other histones H1 in acid/urea gel electrophoresis. When theEuglena histone H1 was [methyl-³H]-labeledin vitro by a homologous enzyme (one of the twoEuglena protein methylase III) and analyzed on two-dimensional gel electrophoresis, three distinctive subtypes of histone H1 were shown to be radiolabeled, whereas five subtypes of rat liver histone H1 were found to be labeled. Finally, by the combined use of a strong cation exchange and reversed-phase Resolve C₁₈ columns on HPLC, we demonstrated thatEuglena histone H1 contains approximately 9 mol% of ε-N-methyllysines (1.40, 1.66, and 5.62 mol% for ε-N-mono-, di- and trimethyllysines, respectively). This is the first demonstration of the natural occurrence of ε-N-methyllysines in histone H1.     

Effects of diaphragmatic ischemia on the inspiratory motor drive.

To assess the effect of diaphragmatic ischemia on the inspiratory motor drive, we studied the in situ isolated and innervated left diaphragm in anesthetized, vagotomized, and mechanically ventilated dogs. The arterial and venous vessels of the left diaphragm were catheterized and isolated from the systemic circulation. Inspiratory muscle activation was assessed by recording the integrated electromyographic (EMG) activity of the left and right costal diaphragms and parasternal intercostal and alae nasi muscles. Tension generated by the left diaphragm during spontaneous breathing attempts was also measured. In eight animals, left diaphragmatic ischemia was induced by occluding the phrenic artery for 20 min, followed by 10 min of reperfusion. This elicited a progressive increase in EMG activity of the left and right diaphragms and parasternal and alae nasi muscles to 170, 157, 152, and 128% of baseline values, respectively, an increase in the frequency of breathing efforts, and no change in left diaphragmatic spontaneous tension. Thus the ratio of left diaphragmatic EMG to tension rose progressively during ischemia. During reperfusion, only the frequency of breathing efforts and alae nasi EMG recovered completely. In four additional animals, left diaphragmatic ischemia was induced after the left phrenic nerve was sectioned. Neither EMG activity of inspiratory muscles nor respiratory timing changed significantly during ischemia. In conclusion, diaphragmatic ischemia increases inspiratory motor drive through activation of phrenic afferents. The changes in alae nasi activity and respiratory timing indicate that this influence is achieved through supraspinal pathways.     

Effect of phrenic afferent stimulation on pattern of respiratory muscle activation.

Ventilation and electromyogram (EMG) activities of the right hemidiaphragm, parasternal intercostal, triangularis sterni, transversus abdominis, genioglossus, and alae nasi muscles were measured before and during central stimulation of the left thoracic phrenic nerve in 10 alpha-chloralose anesthetized vagotomized dogs. Pressure in the carotid sinuses was fixed to maintain baroreflex activity constant. The nerve was stimulated for 1 min with a frequency of 40 Hz and stimulus duration of 1 ms at voltages of 5, 10, 20, and 30 times twitch threshold (TT). At five times TT, no change in ventilation or EMG activity occurred. At 10 times TT, neither tidal volume nor breathing frequency increased sufficiently to reach statistical significance, although the change in their product (minute ventilation) was significant (P less than 0.05). At 20 and 30 times TT, increases in both breathing frequency and tidal volume were significant. At these stimulus intensities, the increases in ventilation were accompanied by approximately equal increases in the activity of the diaphragm, parasternal, and alae nasi muscles. The increase in genioglossus activity was much greater than that of the other inspiratory muscles. Phrenic nerve stimulation also elicited inhomogeneous activation of the expiratory muscles. The transversus abdominis activity increased significantly at intensities from 10 to 30 times TT, whereas the activity of the triangularis sterni remained unchanged. The high stimulation intensities required suggest that the activation of afferent fiber groups III and IV is involved in the response. We conclude that thin-fiber phrenic afferent activation exerts a nonuniform effect on the upper airway, rib cage, and abdominal muscles and may play a role in the control of respiratory muscle recruitment.     

A rapid method for the detection of tryptophanase in anaerobic bacteria

A total of 633 anaerobic bacteria were examined for tryptophanase production using a rapid method which distinguishes within 5 to 180 minutes between anaerobes that contain tryptophanase and those that do not. Of the 196 tryptophanase-positive isolates tested, 99% showed tryptophanase activity within 2 hours as compared with 94.4% in 24 hours by a conventional method. A total of 299 tryptophanase-negative organisms were tested. Ninety three percent of these remained negative after 24 hours as compared with 95.3% when tested with a 24-h conventional method. Additional information was obtained on the sensitivity of this test and the time-dependent production of indole by tryptophanase.     

Copper metabolism in the plaice, Pleuronectes platessa (L.)

Copper metabolism in a teleost, the plaice, Pleuronectes platessa (L.) from a natural environment has been studied. Distribution in the various tissues of the metal and of five key copper-dependent enzymes: ceruloplasmin EC 1.16.3.1; Superoxide dismutase EC 1.15.11; tryptophan oxygenase EC 1.13.1.12; cytochrome oxidase EC 1.9.3.1 and monoamine oxidase EC 1.4.3.4 have been determined. The copper distribution was found to be similar to that in mammals with the greatest concentrations in the brain and heart. Distribution of the copper enzymes is also similar to that found in mammals. A preliminary characterization of the copper enzymes showed that plaice cytochrome oxidase has a pH maximum 2 pH units more alkaline than the mammalian enzyme and that plaice tryptophan oxygenase is more sensitive to heat denaturation than the mammalian enzyme. The present data form a base-line against which studies on factors affecting the copper metabolism in a teleost can be assessed.     

Viral Pollution of Surface Waters Due to Chlorinated Primary Effluents

The role of chlorinated primary effluents in viral pollution of the Ottawa River (Ontario) was assessed by examining 282 field samples of wastewaters from two different sewage treatment plants over a 2-year period. The talc-Celite technique was used for sample concentration, and BS-C-1 cells were employed for virus detection. Viruses were detected in 80% (75/94) of raw sewage, 72% (68/94) of primary effluent, and 56% (53/94) of chlorinated effluent samples. Both raw sewage and primary effluent samples contained about 100 viral infective units (VIU) per 100 ml. Chlorination produced a 10- to 50-fold reduction in VIU and gave nearly 2.7 VIU/100 ml of chlorinated primary effluent. With a combined daily chlorinated primary effluent output of approximately 3.7 × 10⁸ liters, these two plants were discharging 1.0 × 10¹⁰ VIU per day. Because the river has a mean annual flow of 8.0 × 10¹⁰ liters per day, these two sources alone produced a virus loading of 1.0 VIU/8 liters of the river water. This river also receives at least 9.0 × 10⁷ liters of raw sewage per day and undetermined but substantial amounts of storm waters and agricultural wastes. It is used for recreation and acts as a source of potable water for some 6.0 × 10⁵ people. In view of the potential of water for disease transmission, discharge of such wastes into the water environment needs to be minimized.     

The involvement of the anticodon adjacent modified nucleoside N-(9-(BETA-D-ribofuranosyl) purine-6-ylcarbamoyl)-threonine in the biological function of E. coli tRNAile.

tRNAile was isolated from E. coli Cp 79 (leu-, arg-, thr-, his-, thiamin-, RCrel) which had been grown on a sub-optimal concentration of thr and was found to contain an average of 50% less N-[9-(beta-D-ribofuranosyl)- purin-6-ylcarbamoyl]threonine, t6Ado, than tRNAile from cells grown on an optimum concentration of thr and containing a normal complement of t6Ado. The two tRNA's were identical in their ability to be aminoacylated, to accept the 3'-terminal dinucleotide, and to form an ile-tRNAile-Tu-GTP complex. In contrast, the t6Ado-deficient-tRNA was significantly less efficient in binding to ribosomes compared to the normal tRNA. This difference was seen in the binding of deacylated tRNA and in the nonenzymatic and enzymatic binding of ile-tRNA, all in response to poly AUC. The t6Ado-deficient ile-tRNA demonstrated no binding at Mg2+ concentrations less than or equal to 10 mM, while the normal ile-tRNA bound at low Mg2+ concentrations. Tetracycline had the same effect on the normal as on the t6Ado-deficient ile-tRNA binding. As a control, the binding of phe-tRNA (which does not contain t6Ado) from normal and thr-starved cells in response to poly U was identical. It was concluded that t6Ado is required for proper codon-anticodon interaction.