Estimating antemortem drug concentrations from postmortem blood samples: the influence of postmortem redistribution.

AIMS: To compare blood drug concentrations during life with postmortem drug concentrations measured from a peripheral site and a central site. METHODS: Coroner's cases from October 1990 to July 1997 were reviewed. Six cases had data on both antemortem and postmortem blood drug concentrations. The postmortem to antemortem ratio was compared with the postmortem central to peripheral ratio, using cardiac blood as a central site and femoral blood as a peripheral site. RESULTS: Drugs that have a high postmortem central to peripheral ratio; that is, drugs that exhibit considerable postmortem redistribution, also have high postmortem to antemortem ratios. CONCLUSIONS: A large degree of error can arise from attempting to estimate antemortem drug concentrations and the ingested dose from postmortem measurements. The chosen site and technique for postmortem blood sampling can greatly influence the concentration of drug measured.

High-performance liquid chromatographic assay of plasma thalidomide: stabilization of specimens and determination of a tentative therapeutic range for chronic graft-versus-host disease.

Thalidomide is now widely used to treat chronic graft-versus-host disease, but its use is associated with non-teratogenic side effects such as peripheral neuropathy. To examine the value of monitoring plasma concentrations of the drug in such patients, we have developed a high-performance liquid chromatographic (HPLC) assay. The method uses 0.5 mL plasma, is linear to 10 mg/L and had a detection limit of 0.2 mg/L. Thalidomide in plasma specimens was unstable at physiological pH but could be stabilized for several weeks by simple acidification. We describe a protocol for monitoring patients treated with thalidomide which permits convenient transportation and storage of specimens and report, provisionally, that plasma concentrations in the range 1-7 mg/L are therapeutically effective in chronic graft-versus-host disease without adverse side effects.

Analysis of etorphine in postmortem samples by HPLC with UV diode-array detection.

Etorphine is a synthetic narcotic analgesic usually used in veterinary medicine. It possesses an analgesic potency up to 1000 times greater than morphine and is therefore used in low doses, primarily for tranquilising large animals. For veterinary use, etorphine is usually available in its commercial formulation as Immobilon, when in combination with acepromazine or methotrimeprazine. Due to the potency of etorphine, only very low doses are required to produce adverse or fatal effects. This paper describes a method for detecting and quantifying etorphine using HPLC with UV diode array detection (HPLC-DAD) and demonstrates the advantage of the technique for the detection of Immobilon at low doses. In a forensic case involving Immobilon, the etorphine concentrations measured in postmortem femoral vein and heart blood specimens were 14.5 and 23.5 micrograms/l, respectively. No etorphine was detected in the urine. To our knowledge this is the first time postmortem etorphine concentrations have been reported.

A previously unidentified acepromazine metabolite in humans: implications for the measurement of acepromazine in blood.

High-performance liquid chromatography-diode-array detection results obtained during the investigation of two cases involving acepromazine prompted us to study the stability of the drug in blood. It was found that acepromazine can undergo in vitro conversion by human red blood cells to 2-(1-hydroxyethyl)promazine, a product that has been reported as a minor urinary metabolite in horse urine but not previously identified in humans. Further, our analytical findings in the two cases examined suggest that 2-(1-hydroxyethyl)promazine may be the major unconjugated metabolite of acepromazine in humans. These findings have important implications for the analytical toxicology of acepromazine.

Applications of an HPLC-DAD drug-screening system based on retention indices and UV spectra.

An analytical database, using high-performance liquid chromatography (HPLC) with diode array ultraviolet (UV) detection, of over 250 toxicologically relevant drugs has been extensively applied to both clinical and forensic toxicology. This general drug screening system, based on a mixed-phase (octadecyl/cyanopropyl) column and gradient reversed-phase HPLC, can identify a wide range of basic, acidic, and neutral drugs and metabolites, some of which are not amenable to gas chromatography and thin-layer chromatography. Compounds are identified using both retention index (RI) value (calculated by interpolation between a series of reference drug markers) and UV spectral data. It has been previously shown that this chromatographic system provides long-term reproducibility and has potentially useful selectivity differences compared with those based on octadecylsilane columns. Development has been undertaken to improve the speed and practicality of the system for emergency toxicology screening by increasing the rate of the elution gradient, while maintaining the applicability of the RI database. The database has also been found to be an important tool in determining optimum strategies for the adaptation of the system for quantitative analyses of drugs and metabolites under isocratic conditions.

Screening clients for an augmentative and alternative communication clinic: a multitrait-multimethod approach.

The aim of Augmentative and Alternative Communication (AAC) services is to support, enhance, or provide alternative methods of communication for individuals who are not independent verbal communicators. However, relatively little evaluative research has been conducted with adult AAC users. The establishment of Freeport Hospital's AAC Clinic for adults included the development of questionnaires to gather information for the selection and provision of services for its clients. The present study addresses the issue reliability and validity of three novel measures contained within the AAC Clinic's Background Information Questionnaire using a multitrait-multimethod (MTMM) design. Results from this evaluation are discussed in terms of clinical application and policy development.

Chronic airflow obstruction. Two-pronged approach to outpatient management.

The goal of outpatient management of chronic airflow obstruction is to relieve symptoms, which can be accomplished by smoking cessation and by use of bronchodilators. The inhaled beta agonists are the first choice for bronchodilator therapy, followed by oral theophylline if beta agonists alone are ineffective. If symptoms persist, corticosteroids should be tried. Because of the serious side effects of steroids, however, objective evidence of improvement must be documented before long-term use can be justified.

Concurrent avian malaria and avipox virus infection in translocated South Island saddlebacks (Philesturnus carunculatus carunculatus).

CASE HISTORY: Outbreaks of mortality in South Island saddlebacks (Philesturnus carunculatus carunculatus) that had been translocated to two offshore islands in the Marlborough Sounds of New Zealand were investigated during the summers of 2002 and 2007. Both outbreaks were associated with a severe decrease in numbers of saddlebacks of up to 60% of approximately 200 birds. CLINICAL AND PATHOLOGICAL FINDINGS: Many of the surviving birds were in poor condition, and had skin lesions on the legs and head. Necropsy showed pale liver and lungs, and a swollen spleen. Histopathology revealed schizonts resembling Plasmodium spp. within the cytoplasm of many hepatocytes and splenic histiocytes. The skin lesions consisted of epithelial proliferations containing numerous Bollinger bodies typical of avipox virus (APV) infection. Two different APV were isolated, using PCR, from two different birds exhibiting skin lesions. Each isolate had 100% sequence homology with APV members from either Clade A or Clade B. In addition, PCR analysis revealed that the Plasmodium elongatum present in infected birds belonged to a strain that was endemic in the population of North Island saddlebacks (Philesturnus carunculatus rufusater). DIAGNOSIS: Concurrent infections with Plasmodium spp. haemoparasites and APV were identified as the likely cause of death in the birds examined. CONCLUSIONS AND CLINICAL RELEVANCE: Although the Plasmodium spp. identified is thought to be endemic to saddlebacks in New Zealand, the affected birds were likely to be immunocompromised by concurrent APV infection or through lack of genetic diversity. Both the introduced mosquito Culex quinquefasicatus and the native mosquito Culex pervigilans are likely vectors for both these diseases, and the provision of water supplies less favourable to mosquito-breeding is recommended.

Development of a high-performance liquid chromatography retention index scale for toxicological drug screening.

An efficient and practical analytical method for correcting HPLC retention data has been produced using an HPLC diode-array UV detector system. The system is based on retention indices (RI) and is to be used primarily for the identification of toxicologically relevant drugs involved in clinical and forensic toxicology. The RI correction method was chosen as it provided a slightly greater degree of reproducibility than using relative retention time (RRT), particularly for acidic and neutral drugs. Development of the system involved the establishment of the optimal chromatographic conditions and extensive studies of the stability of the system. An acetonitrile gradient elution was used with RI values determined by interpolation from a series of specifically chosen basic and acidic/neutral marker drugs which eluted at regular intervals to produce a linear RI scale. It was found that two separate RI scales were required for basic and acidic/neutral drugs. The use of multiple drug markers as primary retention index standards had not been previously applied to HPLC general drug screening and comparison with a recently published database suggests that the system may also provide improved selectivity.

Morphologic and morphometric effects of prolonged cigarette smoking on the small airways.

We studied lungs from 25 smokers and 14 lifelong nonsmokers, all over 40 yr of age, to examine the relationship of long-term cigarette smoking to histopathologic changes in the small airways. Despite considerable overlap between the 2 groups, smokers had a significantly higher score (p < 0.01) for small airway disease. The specific morphologic features separating smokers from nonsmokers were increases in goblet cell metaplasia (p < 0.001), smooth muscle hypertrophy (p < 0.05), inflammation in the walls of bronchioles (p < 0.01), and respiratory bronchiolitis (p < 0.001). The average bronchiolar diameter was not significantly different in smokers compared with nonsmokers; however, smokers had an excess of airways less than 400 microns in diameter (p < 0.03). Among smokers, the severity of small airway disease correlated with the percentage of airways that are less than 400 microns in diameter (rs = 0.63) and with the extent of centrilobular emphysema (r = 0.53). Smokers also had an increase in the proportion of bronchial gland mass (p < 0.05), but this pathologic feature was not related to the severity of either small airway disease or centrilobular emphysema. We concluded that prolonged cigarette smoking is associated with progressive pathologic changes in the small airways that may be an important cause of airflow obstruction and that may predispose to the development of centrilobular emphysema.

Morphologic changes in the muscular pulmonary arteries: relationship to cigarette smoking, airway disease, and emphysema.

The muscular pulmonary arteries were studied by morphometric methods in 25 long-term cigarette smokers and were compared with those of 14 lifelong nonsmokers. Muscular pulmonary arteries < 500 microns in external diameter were identified and counted. The external diameter, medial thickness, and intimal thickness were measured in each of these arteries. Smokers had an increased number of transected muscular arteries < 200 microns in diameter (p < 0.03), increased medial smooth muscle (p < 0.02), and more intimal thickening (p < 0.04). Among smokers, these vascular changes correlated significantly with the severity of small airway disease and with the degree of emphysema, but not with bronchial mucous gland enlargement. We conclude that regular cigarette smoking is associated with morphologic changes in the muscular pulmonary arteries that evolve in parallel with small airway disease and emphysema. Although the functional significance of these findings is unknown, they may be important in the eventual development of pulmonary hypertension and cor pulmonale.

Lung disease in long-term cigarette smokers with and without chronic air-flow obstruction.

Despite the established role of cigarette smoking in the causation of chronic air-flow obstruction (CAO), only a small proportion of regular cigarette smokers develop significant clinical disease. We compared emphysema severity as well as pathologic and morphometric features of the peripheral conducting airways and the muscular pulmonary arteries among 3 groups of older subjects. These groups included lifelong nonsmokers (NSM), regular smokers without severe disease (SM), and smokers with an established diagnosis of CAO (SM-CAO). For most pathologic features examined there was an orderly progression in severity when comparing SM to NSM and SM-CAO to SM. Emphysema severity and scores for peripheral airways disease, except for goblet cell metaplasia, better distinguished SM-CAO from SM than did measures of bronchiole lumenal caliber. Within the SM-CAO group, the premortem percent predicted forced expiratory volume in one second (FEV1) correlated significantly with emphysema severity (r = 0.74), with average bronchiole diameter (r = 0.54), with the proportion of bronchioles with diameters less than 400 mu (r = 0.51), but not with any of the scores for bronchiolar disease. However, within this group no morphologic or pathologic feature of the small airways was an independent predictor of ventilatory function beyond that of emphysema alone. When compared with those from NSM, histological sections from SM-CAO lungs contained approximately twice as many fully muscularized artery profiles 0 to 300 mu in diameter, the arterial medial muscle layer was doubled in thickness, and the amount of arterial intimal fibrosis was tripled.(ABSTRACT TRUNCATED AT 250 WORDS)

A sensitive, real-time, RNA-specific PCR method for the detection of recombinant AAV-CFTR vector expression.

Following adeno-associated virus (AAV)-mediated transduction, cellular RNA preparations can be contaminated with AAV single-stranded DNA. The single-stranded DNA genome of recombinant AAV vectors can serve as an efficient, but undesirable, template for traditional reverse transcriptase-polymerase chain reaction (RT-PCR) methods. Consequently, recombinant AAV gene therapy presents a unique challenge to the design of sensitive and reliable methods to detect vector-derived mRNA. Several methods have been proposed to reduce the presence of single- and double-stranded vector DNA without compromising RNA specificity. For example, DNase I, although widely used, can be ineffective at completely removing the AAV single-stranded DNA genome. We have developed a sensitive real-time RNA-Specific reverse transcriptase PCR (RS-PCR) method that is independent of DNase I treatment. The RS-PCR method relies on the generation of a first-strand cDNA template using a primer with a linker sequence, X, at the 5'- end such that synthesis of second-strand cDNA incorporates the X-linker sequence into the cDNA template. The RS-PCR then utilizes forward and reverse primers targeting AAV vector sequence and the X-primer site, respectively, while a vector-specific Taqman probe makes sensitive real-time detection possible. We present data to validate the sensitivity and RNA specificity of the RS-PCR method and propose two unique endogenous control strategies by monitoring expression of both beta-glucuronidase and endogenous cystic fibrosis transmembrane conductance regulator (CFTR). Finally, we demonstrate the utility of this new RS-PCR method in detecting recombinant AAV-CFTR expression, including, an in vitro transduction assay and methods to support both preclinical and clinical trials.

The suitability of the urinary metabolite 1-hydroxypyrene as an index of poly nuclear aromatic hydrocarbon bioavailability from workers exposed to carbon black.

Levels of airborne dust have been measured in the breathing zone of five warehouse packers in a carbon black manufacturing plant on consecutive days over 1 week. Post-shift urine samples were collected on individuals over the week and the concentration of the pyrene metabolite 1-hydroxypyrene have been measured. A questionnaire was used to assess the likelihood of potential confounding factors, such as direct exposure to feedstock oil, as well as smoking and diet. The arithmetic mean both of airborne dust and of excreted 1-hydroxypyrene for each individual have been correlated (a) with the constraint that the regression line pass through the origin, and (b) without this constraint. The slope of the regression through the origin was significant and that of the second regression (not through the origin) was not. The statistical test to determine whether excretion on days after Monday was higher than that on Monday itself proved highly significant. The results suggest that 1-hydroxypyrene may be a useful indicator of exposure to the PNAs adsorbed onto carbon black.

Caffeine or theophylline for neonatal apnoea?

Caffeine, in the dose usually recommended (12.5 mg/kg loading dose and 3 mg/kg daily maintenance), and a higher dose regimen (25 mg/kg loading and 6 mg/kg daily maintenance), was compared with theophylline (7.5 mg/kg loading and 3 mg/kg thrice daily maintenance). The study was a randomised controlled trial in the treatment of a group of 44 infants of less than 31 weeks' gestation (mean gestational age 28.3 weeks) who were suffering from frequent apnoeic attacks. All three regimens produced a significant reduction in apnoeic attacks within 24 hours, but only the higher dose caffeine and theophylline groups showed a significant improvement in apnoea within eight hours. The use of caffeine for the treatment of neonatal apnoea is recommended, because a once daily dose is more easily administered, and because it was found that plasma concentrations were more predictable than those of theophylline. If used in very preterm infants, however, its is suggested that a higher dose regimen than that previously recommended be used to achieve a faster response.

Lack of effect of H2-receptor antagonists on the pharmacokinetics of alcohol consumed after food at lunchtime.

The possibility of a pharmacokinetic interaction between H2-receptor antagonists and alcohol consumed at lunchtime, was investigated in 24 healthy non-alcoholic male subjects, each receiving ranitidine 150 mg four times daily, cimetidine 400 mg four times daily, famotidine 20 mg four times daily and placebo in an open, four-way cross-over study. The subjects consumed 50 g alcohol after a standard lunch on the eighth day of dosing with study medication. Blood samples taken during the 6 h after alcohol consumption were analysed for alcohol concentrations by gas liquid chromatography using head space analysis. None of the H2-receptor antagonists had any statistically significant effects on any of the pharmacokinetic parameters for alcohol. Mean Cmax (95% CI) results for ranitidine were 547 (516, 580), cimetidine 531 (501, 563), famotidine 563 (530, 598) and placebo 529 (499, 561) mg l-1.

Longitudinal follow-up of a cohort of newborn infants treated with inhaled nitric oxide for persistent pulmonary hypertension.

OBJECTIVE: To describe the outcome of a group of term newborn infants treated with inhaled nitric oxide for severe persistent pulmonary hypertension. STUDY DESIGN: We performed a prospective longitudinal medical and neurodevelopmental follow-up of 51 infants treated as neonates for persistent pulmonary hypertension of the newborn with inhaled nitric oxide. The original number of treated infants was 87, of whom 25 died in the neonatal period; of 62 infants who survived, 51 were seen at 1 year of age and 33 completed a 2-year evaluation. Statistical analysis used population medians, means, and standard deviations for parameters assessed. Paired t tests and chi-square analysis were used to compare outcomes measured at 1 year with assessment at 2 years for the 32 infants seen at both 1- and 2-year visits. RESULTS: At 1-year follow-up median growth percentiles were 20%, 72.5%, and 50% for weight, length, and occipitofrontal circumference, respectively. Thirteen of 51 infants (25.5%) were < 5th percentile in weight. Nine of 51 infants (17.6%) had feeding problems (need for gastrostomy feeding or gastroesophageal reflux), and 14 (27.5%) had a clinical diagnosis of reactive airways disease. Infant development as measured by the Bayley Scales of Infant Development was 104 +/- 16 for the mental development index and 97 +/- 20 for the psychomotor index. Six of 51 infants (11.8%) were found to have severe neurologic handicaps, defined as a Bayley score on either the mental development or psychomotor index of < 68, abnormal findings on neurologic examination, or both. Fewer children (6.1% vs 15.7%) required supplemental oxygen at 2 years compared with 1 year, and performance on the psychomotor index of the Bayley Scales improved significantly. CONCLUSIONS: One- and 2-year follow-up of a cohort of infants with persistent pulmonary hypertension of the newborn who were treated with inhaled nitric oxide had an 11.8% (1 year) and 12.1% (2-year) rate of severe neurodevelopmental disability. There are ongoing medical problems in these infants including reactive airways disease and slow growth that merit continued close longitudinal follow-up.