Modulation of Mitochondrial Respiration During Early Reperfusion Reduces Cardiac Injury in Donation After Circulatory Death Hearts.

ABSTRACT: Donation after circulatory death (DCD) donors are a potential source for heart transplantation. The DCD process has unavoidable ischemia and reperfusion (I/R) injury, primarily mediated through mitochondria, which limits routine utilization of hearts for transplantation. Amobarbital (AMO), a transient inhibitor of the electron transport chain, is known to decrease cardiac injury following ex vivo I/R. We studied whether AMO treatment during reperfusion can decrease injury in DCD hearts. Sprague Dawley rat hearts subjected to 25 minutes of in vivo ischemia (DCD hearts), or control beating donor hearts, were treated with AMO or vehicle for the first 5 minutes of reperfusion, followed by Krebs-Henseleit buffer reperfusion for 55 minutes (for mitochondrial isolation) or 85 minutes (for infarct size determination). Compared with vehicle, AMO treatment led to decreased infarct size (25.2% ± 1.5% vs. 31.5% ± 1.5%; P ≤ 0.05) and troponin I release (4.5 ± 0.05 ng/mL vs. 9.3 ± 0.24 ng/mL, P ≤ 0.05). AMO treatment decreased H 2 O 2 generation with glutamate as complex I substrate in both subsarcolemmal mitochondria (SSM) (37 ± 3.7 pmol·mg -1 ·min -1 vs. 56.9 ± 4.1 pmol·mg -1 ·min -1 ; P ≤ 0.05), and interfibrillar mitochondria (IFM) (31.8 ± 2.8 pmol·mg -1 ·min -1 vs. 46 ± 4.8 pmol·mg -1 ·min -1 ; P ≤ 0.05) and improved calcium retention capacity in SSM (360 ±17.2 nmol/mg vs. 277 ± 13 nmol/mg; P ≤ 0.05), and IFM (483 ± 20 nmol/mg vs. 377± 19 nmol/mg; P ≤ 0.05) compared with vehicle treatment. SSM and IFM retained more cytochrome c with AMO treatment compared with vehicle. In conclusion, brief inhibition of mitochondrial respiration during reperfusion using amobarbital is a promising approach to decrease injury in DCD hearts.

Targeting oxidative stress with amobarbital to prevent intervertebral disc degeneration: Part I. in vitro and ex vivo studies.

BACKGROUND: Mounting evidence that oxidative stress contributes to the pathogenesis of intervertebral disc (IVD) degeneration (IDD) suggests that therapies targeting oxidative stress may slow or prevent disease progression. PURPOSE: The objective of this study was to investigate the inhibitory effects of amobarbital (Amo) on the mitochondria of nucleus pulposus (NP) cells under tert-butyl hydrogen peroxide (tBHP)-induced oxidative stress or in NP tissues under oxidative stress from tissue injury as a means of identifying therapeutic targets for IDD. STUDY DESIGN/SETTING: We tested the effects inhibiting mitochondria, a major source of oxidants, with Amo in NP cells subjected to two different forms of insult: exposure to tBHP, and physical injury induced by disc transection. N-acetylcysteine (NAC), an antioxidant known to protect NP cells, was compared to the complex I inhibitor, Amo. METHODS: NP cells were pre-treated for 2 hours with Amo, NAC, or both, and then exposed to tBHP for 1 hour. Apoptosis, necrosis, and reactive oxygen species (ROS) production were assessed using confocal microscopy and fluorescent probes (Annexin V, propidium iodide, and MitoSox Red, respectively). The activation of mitogen-activated protein kinases (MAPKs) involved in oxidative stress responses were interrogated by confocal imaging of immunofluorescence stains using phospho-specific antibodies to extracellular signal-regulated kinase (ERK), c-JUN N-terminal kinase (JNK), and p38. Mitochondrial function was assessed by imaging JC-1 staining, a probe for membrane potential. RESULTS: Amo was modestly more protective than NAC by some measures, while both agents improved mitochondrial function and lowered tBHP-induced apoptosis, necrosis, and ROS production. Activation of MAPK by tBHP was significantly suppressed by both drugs. Physically injured IVDs were treated immediately after transection with Amo or NAC for 24 hours, and then stained with dihydroethidium (DHE), a fluorescent probe for ROS production. Immunofluorescence was used to track the expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a transcription factor that induces the expression of antioxidant genes. Amo and NAC significantly reduced ROS production and increased Nrf2 expression. CONCLUSION: These findings suggest that the progression of IDD may be forestalled by Amo via protection of NP cells from oxidative stress following IVD injury. CLINICAL SIGNIFICANCE: This study will define the extent to which a novel, minimally invasive procedure targeting oxidative stress in NP cells can augment surgical interventions intended to retard IVD degeneration.

Distinctive patterns of amobarbital metabolites.

This paper establishes that the relative proportion of amobarbital metabolites in urine is highly variable from person to person and that observations of plasma half-life give no indication of this variability, but it shows that a valid estimate of a given person's metabolite pattern can be obtained by studying a single urine specimen in the postdistributive phase. The two metabolites which were measured in urine accounted on the average of 9 subjects for 80% +/- 3% of the dose with a range from 66% to 94%. The two metabolites were the well known 3'-hydroxyamobarbital (COH) as a product of side chain hydroxylation and N-beta-D-glucopyranosyl amobarbital (N-glu), a glucose conjugate which at some earlier time had been mistaken for an N-hydroxylation product. Among 129 volunteer subjects, the metabolite ratio N-glu/COH showed a median value of about 0.5 with a range from 0 to 2.8. A virtual absence of N-glu was observed in one of the 129 subjects and confirmed by a second administration of amobarbital 3 mo later. Of the 14 subjects with predominant N-glu excretion 4 were of Chinese origin, while there were 6 Chinese among the 115 other subjects (p less than 0.02).

Variation in amobarbital metabolism: evaluation of a simplified population study.

Kinetic constants of amobarbital metabolism were established for 52 subjects on the basis of urinary analysis extending over several days, usually 96 hr. There was no evidence of effect of age or sex on any of the constants. C-Hydroxylation was induced by cigarette smoking as much as 100%, but glucosidation was not affected. A factor influencing the constants was ethnicity of subjects (Caucasian or Oriental). This study confirms ethnic differences in amobarbital metabolism that were reported after measuring the concentration of metabolites in single samples of urine, that is, urine specimens voided during the postdistributive phase after oral drug intake. It appears that extreme simplification of sampling methods may be contemplated in the design of metabolic investigations of populations.

Effect of human growth hormone on amobarbital metabolism in children.

Drug metabolism changes during the course of growth and development. Hormones mediate somatic growth and may mediate other developmental changes. To determine the effect of human growth hormone (hGH) on human drug metabolism, 6 hGH-deficient children were given single oral doses of amobarbital before and 6 wk after beginning hGH replacement therapy. Amobarbital was selected as a marker of hepatic microsomal oxidation. Half-lives rose from 13.89 +/- 2.78 hr to 22.75 +/- 3.97 hr, volume of distribution was unchanged, and clearance fell from 62.2 +/- 15.2 ml/kg/hr to 31.2 +/- 11.4 ml/kg/hr. Results indicate that hGH slows the metabolism of amobarbital, probably through an effect on the hepatic microsomal drug-oxidizing system.

Salivary excretion of amobarbital in man.

The concentrations of amobarbital in saliva and serum were determined in 5 normal adults following ingestion of 120 mg of sodium amobarbital. There was excellent linear relationship between amobarbital concentrations in saliva and serum (r = 0.993); salivary levels were 36.1% of serum levels. Since the pH of saliva was generally lower than that of blood in man, the degree of ionization of amobarbital in serum and saliva had to be taken into consideration. Estimation of the protein binding of amobarbital in serum from concentrations of amobarbital in saliva and serum was in good agreement with the in vitro data of equilibrium dialysis.

Deficient metabolism of debrisoquine and sparteine.

Genetic deficiencies of alicyclic hydroxylation of debrisoquine and of sparteine oxidation are independently discovered entities, each of clinical significance in its sphere. This paper reports evidence to indicate that these 2 deficiencies have the same cause. Previous investigation of one of the affected subjects had revealed normal oxidative metabolism of amobarbital and antipyrine in terms of both metabolic rates and urinary metabolite patterns. Thus the genetic defect in the metabolism of sparteine and debrisoquine is not a generalized deficiency of drug oxidation or of the cytochrome P450 system.

A case of deficiency of N-hydroxylation of amobarbital.

It has been shown recently that the overall metabolism of amobarbital in man is essentially under genetic control. The drug normally undergoes two hydroxylation reactions, leading to 3'-hydroxyamobarbital (C-OH) and N-hydroxyamobarbital (N-OH). This paper describes a sibship in which two mothers who are identical twins show a gross deficiency on N-OH elimination in urine. The whole set of sibship data suggests that this deficiency represents a recessive trait controlled by a single pair of allelic autosomal genes which regulate N-OH formation. Several methodical approaches to assess an individual's capacity for N-OH formation are illustrated. There was no evidence of compensatory or concordant regulation of the two hydroxylation reactions. The case of this family illustrates that the functional lack of a biotransformation reaction is almost certain to be overlooked if one measures only the disappearance of a multimetabolized drug and not the appearance of metabolites.

Maternal and neonatal elimination of amobarbital after treatment of the mother with barbiturates during late pregnancy.

Plasma half-lives of amobarbital were determined in newborn children of 10 mothers who had been treated with barbiturates for hypertension in pregnancy for 6 to 42 days prior to delivery. Five mothers had received amobarbital, 200 mg daily, and 5, phenobarbital, 60 to 180 mg daily. Half-lives in 7 of the babies ranged from 16.6 to 49.4 hr, comparable to those previously reported in babies of mothers who had received only a single dose of amobarbital. Thus there was no evidence of induction of amobarbital hydroxylation in these children. Two babies who had a greater than normal rise in serum bilirubin had longer half-lives (86.1 and 117.7 hr). In 1 baby whose mother had membranous glomerulonephritis, plasma amobarbital concentration did not significantly change over the period of the study.

Effect of acute and chronic exercise on hepatic drug metabolism.

Recent research indicates that physical exercise and fitness are new host factors with impact on hepatic drug metabolism, contributing to the intra- and interindividual variation in drug response. Moderate to heavy physical exercise for a few hours reduces liver blood flow as assessed by indocyanine green clearance, leading to a decreased elimination of drugs exhibiting flow-limited metabolism (high clearance drugs) such as lignocaine (lidocaine). However, hepatic elimination of drugs exhibiting capacity-limited metabolism (low clearance drugs) such as antipyrine (phenazone), diazepam and amylobarbitone (amobarbital) is not affected by acute physical exercise. Improved physical fitness as expressed by the maximum oxygen uptake seems to increase the elimination rate of the low clearance drug antipyrine and possibly also aminopyrine, while investigations of the biotransformation of high clearance drugs are contradictory. The sum of research in this recent field is rather limited and the mechanism whereby changes in physical fitness influence hepatic drug metabolism needs to be established. It is not known if other liver functions are changed. If the findings also apply for drugs with a low therapeutic index, there may be a risk of exercise-induced changes in drug efficacy and toxicity. It is suggested that future studies on host factors influencing drug metabolism should include information on physical activity.

Ethnic differences in drug metabolism.

Interethnic differences in drug-metabolising capacity may be substantial, and they are sufficiently frequent to warrant attention. Such differences may consist of different mean values of quantitative traits in separate populations, or of different frequency distributions as produced by the occurrence of genetic enzyme variants. The collection of population data requires the investigation of substantial numbers of subjects. This may be no problem if drug-metabolising enzymes occur in blood or are sufficiently stable in their tissues to allow investigation in vitro. However, if investigations require the use of probe drugs, new efforts are needed to adapt pharmacokinetic methods to make them suitable for population studies. This development of methods is further called for because genetic variants seem to be more easily detected through the assessment of particular metabolites than through the determination of pharmacokinetic parameters of the parent drug. Many studies with probe drugs comparing different populations have given results that are equivocal in terms of the nature-nurture interplay. However, a set of data with antipyrine has pointed to environmental factors as the principal determinant of differences in metabolising capacity, while data with debrisoquine have indicated monogenically controlled variation of one facet of the cytochrome P-450 system. In several instances, statistically significant differences between population means have been established by testing small numbers of subjects, numbers insufficient to establish distribution patterns that would allow the recognition of genetic polymorphism. The populations studied range from Greenlanders to South African Blacks, but most comparisons pertain to Caucasians and Orientals.

The kinetics of amylobarbitone metabolism in healthy men and women.

1. Sodium amylobarbitone (3.54 mg/kg) was given by intravenous injection to seven healthy men and nine healthy women who were not receiving other drugs. Serum amylobarbitone and urine hydroxyamylobarbitone concentrations were measured by gas-liquid chromatography. There was no significant difference between the groups either in the serum amylobarbitone concentration/time curves or in the urinary excretion of hydroxyamylobarbitone.2. The serum amylobarbitone concentration decayed over 48 h as a double exponential function of time; the first exponential component had a mean half-time of 0.6 h (males 0.56 +/- 0.06 h, females 0.62 +/- 0.08 h, +/- S.E.) and the second exponential component had a mean half time of 21 h (males 22.7 +/- 1.6 h, females 20.0 +/- 1.0 h, +/- S.E.).3. The urinary excretion of hydroxyamylobarbitone over 48 h accounted for 34% of the dose (males 33.8 +/- 3.2%, females 35.2 +/- 3.0%, +/- S.E.). One male and two female subjects excreted hydroxyamylobarbitone partly as a conjugate which was readily hydrolysed in acid.4. An elimination constant (k(el)) derived from the serum concentration/time curve by the application of a two compartment model was approximately proportional to beta (h(-1)), the rate constant of the second exponential component. There was a positive correlation (r=0.78, P<0.001) between beta and the mean rate of urinary excretion of hydroxyamylobarbitone during the 24 to 48 h period.

Relative potencies for barbiturate binding to the Torpedo acetylcholine receptor.

1. The structural requirements of an allosteric barbiturate binding site on acetylcholine receptor-rich membranes isolated from Torpedo electroplaques have been characterized by the ability of fourteen barbiturates to displace [14C]-amobarbitone binding. 2. The barbiturates could be grouped into two classes with ten barbiturates producing a strong inhibition of [14C]-amobarbitone binding (class one) and with four exerting minimal effects (class two). 3. Eight of the ten class one barbiturates displaced essentially all of the [14C]-amobarbitone from its binding site, while, at their respective aqueous solubility limits, two of these barbiturates (thiopentone and dimethylbutylbarbitone (DMBB) inhibited [14C]-amobarbitone binding by nearly 80%. The apparent inhibition constants (KI) for the class one barbiturates ranged from 13 microM for amobarbitone to 2.8 mM for barbitone with the other eight agents lying in the range 100-600 microM, and having the rank order pentobarbitone approximately secobarbitone greater than thiopentone greater than DMBB greater than butabarbitone approximately phenobarbitone greater than aprobarbitone greater than allylbarbitone. 4. By contrast, the class two barbiturates had minimal effects even at close to saturating concentrations. [14C]-amobarbitone binding was reduced slightly (less than 30%) by hexobarbitone, mephobarbitone and methohexitone and was enhanced slightly (less than 20%) by metharbitone. 5. All of the class two, but none of the class one barbiturates, were N-methylated.

Metabolism of amylobarbitone in patients with chronic liver disease.

1. A single dose of amylobarbitone (3.23 mg/kg) was given by intravenous injection to each of ten healthy controls and two groups of five patients with chronic liver disease. A curve of serum amylobarbitone concentration against time was prepared for each subject and the proportion of the serum amylobarbitone bound to protein determined. The urinary excretion of the metabolite hydroxyamylobarbitone, ethyl (3 hydroxyisoamyl) barbituric acid was measured.2. The degree of protein binding of serum amylobarbitone was reduced in the five patients (group I) with abnormally low concentrations of albumin in serum (<3.5 g/100 ml) but was normal in the five patients (group II) with normal serum albumin concentrations (>3.5 g/100 ml).3. The equation for a double exponential decay was fitted to the concentration/time curves for amylobarbitone free in the serum water. The mean intercepts and rate constants were used to calculate the dimensions of mathematical models based on a two compartment open system.4. The five patients (group I) who had abnormally low concentrations of albumin in serum showed impairment of amylobarbitone metabolism; the rate constant beta(h(-1)) for the second exponential decay of serum amylobarbitone concentration was reduced (P<0.01), the urinary excretion of hydroxyamylobarbitone was reduced (P<0.001) and the mean serum water clearance (C, ml/min) representing amylobarbitone elimination by metabolism was reduced.5. The five patients (group II) who had normal concentrations of albumin in serum showed no impairment of amylobarbitone metabolism. Within the total patient group there were strong and significant positive correlations between the serum albumin concentration and each of the indices of the rate of amylobarbitone metabolism.6. Both patient groups showed an increase in the first dispositional rate constant alpha(h(-1)) and in the clearance (C(t) ml/min) representing transfer between central and peripheral compartments. The physiological basis for this observation is uncertain.7. The clinical response to the single intravenous dose of amylobarbitone was not significantly greater (P=0.11) in the patient group (I) with slow amylobarbitone metabolism than in the patient group (II) with a normal rate of amylobarbitone metabolism.

Age-dependent disposition of amobarbital: analog computer evaluation.

Previously published observations on 4-hour and 24-hour amobarbital blood levels in two groups of subjects (ages 20--40 and over-65) were analyzed with use of an analog computer and literature data for the rate constants of absorption, distribution and metabolism. It was found that the volume of distribution did not change with age, and the increase in biologic half-life from 22.8 hours in the young subjects to 86.62 in the elderly subjects was due to a decreased rate of metabolism. When the one-point method is used, the size of the nightly dose of amobarbital should be reduced in elderly subjects from 200 mg to 50 mg in order to maintain the same steady-state blood levels found in younger subjects.

Secretion of drugs by the parotid glands of rats and human beings.

The following drugs have been demonstrated to be secreted by the parotid glands of rats and human beings: amobarbital, chlorpromazine, codeine, glutethimide, meprobamate, pentobarbital, phenobarbital, and secobarbital. Methadone could not be detected in the parotid saliva of either rats or human beings, and morphine has been demonstrated only in parotid saliva of rats.

Effect of paracetamol on amylobarbitone hydroxylation in man: a gas chromatographic method for simultaneous estimation of underivatized paracetamol and barbiturates.

A rapid and specific technique for the simultaneous gas chromatographic estimation of underivatized paracetamol and barbiturates using an alkali flame ionization detector is described which is suitable for both forensic and pharmacokinetic investigations. An improved method for estimation of 3-hydroxyamylobarbitone is also detailed. These techniques were used in an investigation of the effects of oral administration of 1 g paracetamol 8 hourly on the formation of 3-hydroxyamylobarbitone from a single oral dose of 200 mg sodium amylobarbitone. No significant changes were found in the plasma concentrations and total body clearance of amylobarbitone nor was there any alteration in the urinary elimination of 3-hydroxyamylobarbitone.

The distribution of amylobarbitone, butobarbitone, pentobarbitone and quinalbarbitone and the hydroxylated metabolites in man.

Fluid and tissue specimens collected from 30 subjects at autopsy have been assayed for their content of common sedative barbiturates and the corresponding hydroxylated metabolites by g.l.c. Where one barbiturate had been ingested an inverse relationship between lipid solubility of the drug and the distribution in fluids and tissues was observed. In most cases the liver, and in the remainder the spleen, contained the highest concentrations of barbiturate. Bile concentrations were often in excess of those in the corresponding liver. The metabolites of the four sedative barbiturates were usually present in lower amounts than the parent drugs in the fluids and tissues of most subjects but urine often contained much higher concentrations of metabolites--sometimes exceeding that of the parent drug in the liver. Administration of two or more barbiturates together did not appear to affect the distribution and metabolism of the individual drugs.

Toxicological analysis of amobarbital and glutethimide from bone tissue.

Author examined cadaver organs and bone samples (sternum, rib) of drug poisoning cases. Following suitable procedures, active drug components (amobarbital, glutethimide, and so forth) were identified by gas chromatography/mass spectrometry (GC/MS). Based on results of quantitative GC analysis, relationships were sought between the active agent concentrations measured in the organs and the bone samples.

[Determination of barbiturates from biological material]. / Stanovení barbiturátu z biologického materiálu

A trace quantitative analysis of barbiturates has been carried out in blood, urine, organs and in gastric and intestinal concents. The amount of sample required for analysis is very small [approximately 400 mul blood]. Extraction is carried out four times with the mixture of acetone and either 1:1]. The preparation of columns, packing and standards has been described. The device used for the analyses [Chrom 3] is furnished with an adjusted feeding block preventing the decomposition of samples in the doser. The column temperature is 190 degrees C, the column packing is Chromaton N-AW-DMCS coated with 3% NPGS and 0.75% trimer acid, detector FID.