Acetyl Metabolite Research Articles

Dynamics of Acetyl Homeostasis in Bacillus subtilis

Bacillus subtilis biofilms are effective models for studying prokaryotic multicellular systems. These biofilms, multicellular bacterial communities encased in a protective polysaccharide and protein matrix, are associated with several bacterial and chronic infections but can also serve beneficial purposes in agriculture and wastewater treatment. Recent investigation into post‐translational protein modification in bacteria has elucidated important regulatory mechanisms for growth and biofilm formation. Protein acetylation has been observed to regulate bacterial multicellularity, and it is closely linked to cellular metabolism by using acetyl metabolites as donors of acetyl groups. The goal of this study is to examine the role of protein lysine acetylation as a regulatory mechanism for metabolism B. subtilis. We hypothesize that bacteria use acetylation as a buffering mechanism for acetyl metabolites to balance levels and prevent metabolite toxicity by increasing acetylation during times of high acetyl metabolite levels and deacetylating proteins to restore metabolites during times of low levels. Wild type bacteria as well as knockout mutants affecting protein acetylation and the carbon overflow pathway are grown in biofilm‐inducing minimal media, and cell and supernatant samples are collected at various ODs. Acetyl‐CoA, acetyl‐phosphate, and acetate levels over the course of growth are measured using fluorometric and colorimetric metabolite test level kits. Dynamics of global lysine acetylation levels are also measured by an anti‐acetyl lysine Western blot. Work so far has focused primarily on optimizing and troubleshooting metabolite assay kits by modifying growth and sample preparation conditions. Acetate dynamics have been measured for the wild type bacteria, showing a continuous secretion of acetate into the supernatant throughout exponential growth. This result is expected, as rapid growth should coincide with continuous utilization of the carbon overflow pathway. Initial Western blots show increased acetylation in the ΔackAΔacuCΔsrtN triple knockout mutant, which is expected due to a buildup of acetyl phosphate and inhibition of deacetylase activity. Future research will also focus on analyzing mutated genes in Δpta suppressor mutants to uncover genes related to acetyl‐CoA‐mediated cell death. Findings from this study will promote the understanding of the regulatory mechanisms behind biofilm formation and potentially uncover novel information about bacterial survival strategies and programmed cell death.Support or Funding InformationThis project was funded by grants from the National Science Foundation, the Northeastern University Honors Program, and the Northeastern University Office of Undergraduate Research and Fellowships.

Preliminary observations on the excretion of acebutolol and its acetyl metabolite in the urine and faeces of man.

Preliminary observations on the excretion of acebutolol and its acetyl metabolite in the urine and faeces of man.

Solid-phase extraction and HPTLC determination of isoniazid and acetylisoniazid in serum. Comparison with HPLC.

A new solid-phase extraction (SPE) and quantitative determination of isoniazid (INH) and its acetyl metabolite (AcINH) in serum by high-performance thin-layer chromatography (HPTLC) is presented. Alkalized serum samples with nicotinamide as an internal standard are applied to an SPE cartridge containing a new SPE sorbent, [poly (divinylbenzene-co-N-vinylpyrrolidone )]. A simple procedure of conditioning, washing, and eluting steps is described. After evaporation of the eluates to dryness and reconstitution, one-dimensional HPTLC is performed on silica gel plates with ethyl acetate-methanol (70:30) as a mobile phase. Quantitation is done by densitometry. Convenient validation parameters are obtained (linearity, limits of detection and quantitation, precision, and accuracy) for INH and AcINH. The method is compared with a high-performance liquid chromatographic (HPLC) technique developed in the laboratory, and satisfactory correlation is found between data from the two techniques. The HPTLC method is sensitive and specific and is used to quantitate INH and AcINH in patient blood serum, and the results are compared with those obtained by HPLC.

Role of melatonin in the induction and maintenance of sleep

Pharmacokinetic studies of melatonin in young and elderly human volunteers, and the measurement of hypnotic effects in chicks under alternate light-dark or permanent light conditions, show that melatonin is a bioprecursor of hypnotic acetyl metabolites produced by the enzymatic acetylation of both melatonin and 2-oxomelatonin under the control of serotonin N-acetyltransferases (NATs), which are present in the pineal gland. The acetyl metabolite of melatonin, which we call carbo2, is an N-acetyl-β-carboline. The electroencephalographs (EEG) architecture of the sleep produced by this compound is similar to thai of physiological sleep, and is characterized by the significant proportion of slow-wave deep sleep and rapid eye movement sleep. This is in sharp contrast to the EEG sleep architecture observed with GABAergic (GABA, γ-aminobutyric acid) compounds. Since insomnia and sleep disorders are believed to be due to a lack of NAT enzymes in the pineal gland, a new therapeutic approach of sleep disorders by administration of such hypnotic acetyl metabolites of melatonin, or synthetic analogs thereof, can be en visaged.

Thin-layer chromatographic characterization of metabolites formed from an AMPA antagonist drug candidate

A pilot study has been conducted to develop sample preparation procedure and TLC separation of radiolabeled metabolites of an AMPA (2-amino-3-(3-hydroxymethylisoxazol-4-yl)propionic acid) antagonist drug (6-(4-aminophenyl)-8-chloro-2-methyl-11 H -imidazo[1,2 c ] [2,3] benzodiazepine). The N -acetyl metabolite was identified by chromatography and mass spectrometry. Both in vivo experiments with experimental animals and in vitro study with rat hepatocytes indicated that the N-acetylation reaction is very important in the metabolism of the compound investigated. N-Acetylation behavior in the rat, dog, mouse, and rabbit were compared to enable selection of suitable species for further study.

Pharmacokinetics of Sulphamethazine and its Acetyl Metabolite in Chickens

Rajaian, H., Symonds, H.W. and Bowmer, C.J. 2001. Pharmacokinetics of sulphamethazine and its acetyl metabolite in chickens. J. Appl. Anim. Res., 19: 85–96. The aim of this study was: first, to establish the basic pharmacokinetic characteristics of sulphamethazine following single intravenous, intramuscular, intracrop and oral dose to chickens; and second, to determine the kinetic behaviour of its main metabolite, N4-acetyl sulphamethazine. Each group of five Hi-Sex chickens was given a bolus dose of sulphamethazine (100 mg kg−1) by the above routes. Blood samples were collected at various time intervals for up to 48 h after dosing and were assayed spectrophotometrically for the presence of sulphamethazine and its metabolite. The results suggest first, that the blood clearance (≈0.06 L h−1 kg−1) and elimination half-life (≈9 h) of sulphamethazine in chickens are independent of the route of administration and second, the extent of acetylation of sulphamethazine in chickens may be influenced by the route by which the drug is administered and is low for the intramuscular route.

Determination of retigabine and its acetyl metabolite in biological matrices by on-line solid-phase extraction (column switching) liquid chromatography with tandem mass spectrometry

A HPLC assay with tandem mass spectrometric detection in the positive-ion atmospheric pressure chemical ionisation (APCI) mode for the sensitive determination of retigabine [( I), D-23129] and its acetyl metabolite [( II), ADW 21-360] in plasma was developed, utilising the structural analogue (D-10328), ( III), as internal standard. Automated on-line solid-phase extraction of diluted plasma samples, based on 200-μl plasma aliquots, at pH 6.5, allowed a reliable quantification of retigabine and the acetyl metabolite down to 1 ng/ml. Injection of 500 μl of diluted plasma onto a C 2 stationary phase-based column switching system in combination with a 75 mm×4 mm reversed-phase analytical column at a flow-rate of 0.5 ml/min provided cycle times of 4 min per sample. The standard curves were linear from 1 to 1000 ng/ml using weighted linear regression analysis (1/ x 2). The method is accurate (mean accuracy ≤±10%), precise (RSD <±15%) and sensitive, providing lower limits of quantification in plasma of 1 ng/ml for retigabine ( I), and 2.5 ng/ml for the metabolite ( II) with limits of detection of 0.5 ng/ml for both analytes. Up to 200 unknowns may be analysed each 24 h per analyst.

Simultaneous determination of nerisopam, a novel anxiolytic agent showing polymorphic metabolism, and its N-acetyl metabolite from human plasma by a validated high-performance liquid chromatographic method

A sensitive reversed-phase high-performance liquid chromatographic method with ultraviolet absorbance detection has been developed to simultaneously determine the concentrations of nerisopam (EGIS-6775) and its N-acetyl metabolite (EGIS-7649) from human plasma. The separation of the investigated compounds and internal standard was achieved on a Nucleosil 7 C 18 column with 2 m M heptanesulphonic acid containing 0.04 M phosphoric acid-acetonitrile-methanol (70:25:5 v/v), pH 2.7 mobile phase. The detection was performed at 385 nm. The compounds were isolated from plasma by Bakerbond C 18 solid-phase extraction. The limit of quantitation was 10 ng/ml plasma for each compound investigated. The assay has been validated with respect to accuracy, precision and system suitability. All validated parameters were found to be within the necessary limits. On the basis of the sensitivity, linearity and validation parameters, the developed analytical method was found to be suitable for the determination of nerisopam and its N-acetyl metabolite from human plasma and for application in pharmacokinetic studies and human drug monitoring. The pharmacokinetic parameters obtained from twelve human volunteers are reported. It was found that nerisopam acetylation is polymorphic: the volunteers with fast or slow acetylator phenotypes produced significantly different plasma concentrations. In slow acetylator phenotypes the concentration of nerisopam was considerably higher in plasma, while the level of its acetyl metabolite was higher in plasma of fast acetylators.

Liquid chromatographic separation and measurement of optical isomers of aminoglutethimide and its acetyl metabolite in plasma, saliva, and urine.

An accurate and specific liquid chromatographic method for the separation and analysis of the R(+) and S(-) enantiomers of both aminoglutethimide (AG) and its acetylated metabolite (AcAG) in plasma, saliva, and urine is described. The separation was achieved by use of two serial Chiralcel OD columns [cellulose tris(3,5-dimethylphenyl carbamate)] with a mixture of hexane/isopropanol/methanol (65:17.5:17.5, per volume) as a mobile phase. The flow rate was 0.7 ml/min, and the compounds were detected in the effluent spectrophotometrically at 245 nm. The plasma, saliva, or urine sample (300 microliters) was extracted with dichloromethane after the addition of an equal volume of acetate buffer (pH 5.6) to the sample. The extraction recovery of the R(+) and S(-) enantiomers of AG and AcAG from plasma, saliva, and urine at different concentrations under these conditions was > 80.9%. No interference from any endogenous substance or concomitantly used drug was observed. The ratio of the peak area of R(+) and S(-) enantiomers of both AG and AcAG/internal standard was linearly (r > or = 0.995) related to concentration in the range 0.83-40.0 micrograms/ml, and the coefficient of variation (CV) at different concentrations was consistently < or = 13%. We are presently employing this method to study the pharmacokinetics of each of these enantiomers in breast cancer patients.

Pharmacokinetics of sulfadimidine and its N4‐acetyl metabolite in healthy and diseased rabbits infected with Pasteurella multocida

The pharmacokinetics of sulfadimidine (SDM) and its N4-acetyl metabolite (N4SDM) were investigated after intravenous bolus injection of a single dose (200 mg/kg) of SDM in normal and diseased New Zealand white rabbits. The apparent distribution volume at steady state, total body clearance and elimination half-life of SDM in normal animals were 0.7 +/- 0.3 l/kg, 0.57 +/- 0.24 l/kg/h and 1.6 +/- 1.3 h, respectively. Of the administered dose, 62.1% was metabolized by N4-acetylation, and 12.7 +/- 1.1 and 2.8 +/- 1.8% of the dose was excreted as free drug by the kidney and gastrointestinal tract, respectively. The 'apparent' formation and elimination half-lives of N4SDM were 0.6 +/- 0.4 and 2.2 +/- 1.1 h, respectively. The metabolite was eliminated mainly by excretion through the kidney. There was no significant effect of acute pasteurellosis on the pharmacokinetics of either SDM or N4SDM in rabbits.

Is Class III antiarrhythmic activity important?

The beginning of thought about a discrete Class III antiarrhythmic action stemmed from the observation that sotalol produced a very marked increase in the time course of repolarization with little or no change in conduction velocity in cardiac muscle. It is known that in patients with hypothyroidism or hypocalcemia, both of which are associated with a marked lengthening of the cardiac action potential, arrhythmias are exceedingly rare. The acetyl metabolite of procainamide also produces, as its sole action, a marked increase in the action-potential duration, and it has antiarrhythmic activity. It is clear that lengthening the action-potential duration is antiarrhythmic. Sotalol is a unique beta blocker in not only antagonizing sympathetic stimulation competitively, but also in exerting a potent antiarrhythmic effect by prolonging repolarization. Because of the augmented tension that is associated with the increase in action-potential duration, sotalol produces a less negative inotropic effect than that seen with other beta blockers. Sotalol is a potent broad-spectrum antiarrhythmic agent because of its Class III properties, resulting from an increase in the length of cardiac repolarization, a property that is modulated by its beta-adrenergic blocking actions.

Metabolism and urinary excretion of 5-amino salicylic acid in healthy volunteers when given intravenously or released for absorption at different sites in the gastrointestinal tract.

In six healthy subjects serum concentrations of 5 amino salicylic acid (5ASA) and acetyl 5ASA were measured for up to 24 hours, and urinary excretion over 48 hours. After an intravenous injection of 3.26 mmol 5ASA serum concentrations fell rapidly with a distribution half-life of 17 +/- 2 min and an elimination half-life of 42 +/- 5 min. After 45 minutes acetyl 5ASA became the dominant compound and after seven hours serum concentrations of both components were almost unrecordable. Orally ingested 5ASA in three preparations to ensure its release in the stomach, small intestine and ileocaecal region respectively gave lower serum concentrations and urinary excretion than those obtained after an intravenous infusion. Bioavailabilities which ranged from 19% for ileocaecal release to 75% for release in the upper gastrointestinal tract, were calculated from areas under the serum concentration curves. Urinary excretion of 5ASA and its acetyl metabolite over 48 hours was 78%, 52%, 55%, and 21% respectively of the dose given intravenously and orally for gastric, small intestinal and ileocaecal release.

Implications of Metronidazole Pharmacodynamics for Therapy of Trichomoniasis

A liquid chromatographic technique for the detection of metronidazole (MDZ) and its major metabolites, combined with a vaginal sampling technique employing extraction of drug from vaginal swabs, was used to evaluate the concentration of MDZ in the vaginal fluid during therapy. Wide variation in the absolute concentration of unchanged drug was noted, although the average vaginal levels were about half the serum concentration 2 h after treatment had been initiated and comparable to the serum levels 6 and 24 h after treatment. Oral administration of MDZ invariably resulted in the presence of the drug in the serum and urine but the drug was not always detected in the vaginal secretions. The presence of the relatively inactive acetyl metabolite in the vaginal fluid of patients who failed to respond to therapy for trichomoniasis suggested the possible role of drug modification as a contributing factor in the lack of therapeutic success. The presence of MDZ in women in whom the cervix had previously been surgically removed attests to the transport or transudation of the drug across the vaginal epithelium without involvement of the secretory activity of the cervix.

Gas—liquid chromatographic assay of aminoglutethimide and a high-performance liquid chromatographic assay for its acetyl metabolite in biological fluids

A rapid, sensitive and selective gas—liquid chromatographic assay for aminoglutethimide is described. The same extraction procedure may be employed prior to a high-performance liquid chromatographic assay for acetamidoglutethimide which is also detailed. Both assays are suitable for the study of the pharmacokinetics of aminoglutethimide and acetamidoglutethimide in biological fluids in man.

Acebutolol pharmacokinetics in renal failure.

Acebutolol was administered orally in a single dose of 200 mg to 17 individuals whose renal function varied markedly. The plasma half-life and elimination rate constant for acebutolol showed a four-fold variation but these did not correlate with the degree of renal impairment. However, there was a good correlation between the renal clearance of creatinine and that of acebutolol (P less than 0.001). The half-life and elimination rate of the acetyl metabolite, diacetolol, were subject to 10-fold inter-individual variability which correlated significantly with the creatinine clearance and serum creatinine concentration. The AUC for the acetyl metabolite showed a 40-fold individual variation which also correlated with renal function. It is concluded that renal elimination is the principal route of excretion for diacetolol but not the parent compound, acebutolol.

Disposition of sulfadimethoxine in swine: inclusion of protein binding factors in a pharmacokinetic model.

Sulfadimethoxine was administered intravenously and orally to five swine. More than 75% of the dose was excreted into urine as the acetyl metabolite with 4-6% excreted unchanged. Plasma and urine data were not consistent when a linear pharmacokinetic model was used to describe the data. Sulfadimethoxine has a high affinity for plasma protein, and the data were subsequently fitted to a nonlinear model, which included saturable protein binding. The choice of a nonlinear model was further supported by a minimum value for the Akaike information criteria. The protein binding constant obtained was 2.8 X 10(4) M-1 and the total protein binding site concentration in plasma was 4.6 X 10(-4) M. Both values are comparable with in vitro data. This result suggests that the nonlinear model involving protein binding can be successfully applied to pharmacokinetic data. The apparent biological half-life of sulfadimethoxine (free and bound) in plasma was 14 hr; however, the half-life of elimination of free drug was 1.25 hr. Following oral administration, all of the dose was absorbed with an apparent absorption half-life of 2.9 hr.

Antiarrhythmic significance of dosing intervals in beta receptor blocking therapy of hypertension with acebutolol

Six hypertensive patients with daily ventricular arrhythmias underwent a double-blind crossover study to examine whether a once daily regimen of beta receptor blockade was equipotent in antihypertensive and antiarrhythmic activity to a twice daily regimen. Acebutolol, a relatively cardioselective beta blocking compound with intrinsic sympathomimetic properties, was given in two regimens: 200 mg twice daily or 400 mg once daily. Ventricular ectopic beats were analyzed both during physical exercise and with multiple 24 hour ambulatory electrocardiographic (Holter) recordings. Serum concentrations of acebutolol and its acetyl metabolite were determined using high pressure liquid chromatography. The two regimens of acebutolol were equally potent in reducing the blood pressure and heart rate at rest and during physical exertion. The hourly heart rates during 24 hours were reduced to the same extent by both regimens. The single daily 400 mg dose did not significantly reduce the incidence of arrhythmias, whereas 200 mg twice daily evoked a significant reduction during 24 hours. Serum concentrations of acebutolol were twice as great with the twice daily regimen as with the single dose. Both treatments significantly shortened the Q-T c interval. The data suggest that, despite apparent beta receptor blockade and good blood pressure control, beta blooking agents with a relatively short plasma half-life lose their antiarrhythmic potency when administered on a once daily basis. This property seems to be more closely related to the plasma concentration of the compound than to the degree of clinically assessed beta receptor blockade.

Assay of trimethoprim, sulfamethoxazole and its N4‐acetyl metabolite in biological fluids by high‐pressure liquid chromatography

AbstractA reversed‐phase HPLC assay method was developed to determine levels of the drugs trimethoprim, sulfamethoxazole and the N4‐acetyl metabolite of the latter in human blood plasma and urine. The organic substances are extracted with ethyl acetate by a single extraction at pH 6.8. The method is rapid, accurate, sensitive and precise and well suited to routine determination of these drugs on patients during clinical pharmacokinetic investigations.

Kinetic discrimination of three sulfamethazine acetylation phenotypes.

The relationship between sulfamethazine disposition kinetics and acetylator phenotype was studied in 19 healthy subjects. Various kinetic parameters for sulfamethazine and its N4-acetylated metabolite were determined after a dose of a rapidly absorbed oral solution. When plotted on a frequency distribution histogram, the results exhibited a well-defined trimodal pattern for acetylation clearance values and overall elimination or metabolic rate constants. These data were consistent with the well-recognized acetylation polymorphism for sulfamethazine, except that they clearly subdivided the previously acknowledged "fast" acetylator mode into intermediate and rapid acetylator groups. The apparent distribution volume and renal clearance for sulfamethazine and acetylsulfamethazine did not differ significantly among the 3 phenotypes. Of special interest was the observation that rapid acetylators initially produce much greater amounts of acetyl metabolite than intermediate acetylators. The potential clinical implications of identifying rapid and intermediate acetylators are discussed in view of evidence showing that acetyl metabolites may be pharmacologically active or function as intermediates in toxic metabolic pathways.

Dose-dependent acebutolol disposition after oral administration.

The relationship between dose and area under the blood concentration-time curve has been studied in 6 healthy subjects following both oral and intravenous doses of acebutolol. There is a more than proportional increase in area with increasing oral doses, and the area over a dosing interval following multiple oral doses is greater than the total area after a single dose of the same size. The role of an acetyl metabolite in producing these effects is discussed, as is the relevance of these observations to the clinical use of acebutolol.