CYP3A5 genotype had no impact on intrapatient variability of tacrolimus clearance in renal transplant recipients.

BACKGROUND: The risk of long-term chronic allograft nephropathy and graft loss after kidney transplantation is increased in patients with a high intrapatient variability in the clearance of tacrolimus. CYP3A5 genotype has a significant influence on the oral bioavailability of tacrolimus and is a potential influence on variability of exposure. METHODS: The study population consisted of 118 renal transplant recipients with stable renal function 12 months after transplant. The intrapatient variability of tacrolimus concentration was calculated. The patients were divided into low- and high-intraindividual variability groups using the median variability of tacrolimus clearance as the cutoff value. RESULTS: No differences in baseline characteristics were observed between the expressers (n = 37) and nonexpressers (n = 81) except for ethnicity, which is in line with previous observations. Tacrolimus dose requirement was significantly higher in patients expressing CYP3A5, confirming earlier observations (P < 0.0001). However, intraindividual variability of tacrolimus clearance was not related to CYP3A5 genotype (P = 0.3331). CONCLUSIONS: The intrapatient variability of tacrolimus clearance was not associated with CYP3A5 genotype in stable renal transplant recipients.

Osteoarthritis-like changes in the heterozygous sedc mouse associated with the HtrA1-Ddr2-Mmp-13 degradative pathway: a new model of osteoarthritis.

OBJECTIVE: To test the hypothesis that the spondyloepiphyseal dysplasia congenita (sedc) heterozygous (sedc/+) mouse, a COL2A1 mutant, is a model for the study of osteoarthritis (OA) in the absence of dwarfism and to investigate the presence of HtrA1, Ddr2, and Mmp-13 and their possible involvement in a universal mechanism leading to OA. DESIGN: Whole mount skeletons of adult animals were analyzed to determine whether sedc/+ mice exhibit dwarfism. To characterize progression of osteoarthritic degeneration over time, knee and temporomandibular joints from sedc/+ and wild-type mice were analyzed histologically, and severity of articular cartilage degradation was graded using the Osteoarthritis Research Society International (OARSI) scoring system. Immunohistochemistry was used to detect changes in expression of HtrA1, Ddr2, and Mmp-13 in articular cartilage of knees. RESULTS: As previously reported, the sedc/+ skeleton morphology was indistinguishable from wild type, and skeletal measurements revealed no significant differences. The sedc/+ mouse did, however, show significantly higher OARSI scores in knee (9, 12 and 18 months) and temporomandibular joints at all ages examined. Histological staining showed regions of proteoglycan degradation as early as 2 months in both temporomandibular and knee joints of the mutant. Cartilage fissuring and erosion were observed to begin between 2 and 6 months in temporomandibular joints and 9 months in knee joints from sedc/+ mice. Immunohistochemistry of mutant knee articular cartilage showed increased expression of HtrA1, Ddr2, and Mmp-13 compared to wild type, which upregulation preceded fibrillation and fissuring of the articular surfaces. CONCLUSIONS: With regard to skeletal morphology, the sedc/+ mouse appears phenotypically normal but develops premature OA as hypothesized. We conclude that the sedc/+ mouse is a useful model for the study of OA in individuals with overtly normal skeletal structure and a predisposition for articular cartilage degeneration.

A systematic evaluation of the core communication skills expected of a perfusionist.

BACKGROUND: The objective of this systematic evaluation was to identify the sentinel standards necessary to obtain a core level of communication required of a clinical perfusionist during cardiopulmonary bypass (CPB). Once these sentinel standards were identified and a core level of communication was established (via four simulated case scenarios), a team of cardiac healthcare professionals was assembled to interpret both the accuracy of response and the speed of response encountered in each case scenario. METHODS: Four simulated case scenarios were utilized in order to replicate the typical patterns of verbal exchange that occur during surgeries using extracorporeal technology. The simulated case scenarios included CPB interactions associated with preparation, initiation, maintenance, termination and post CPB. For all CPB interactions, two variables were measured: accuracy of the perfusionist's response and speed of the perfusionist's response. The cases took place in a controlled setting within an empty operating room at The Children's Hospital of Philadelphia. Four clinical perfusionists each represented the role of the "perfusionist" in all simulated case scenarios. RESULTS: When analyzing the accuracy and speed of the responses, each clinical perfusionist recorded an average score of 96.3% or higher with all case scenarios. Since the clinical perfusionists who participated in the scenarios were primarily pediatric perfusionists, the scores were best during the pediatric case scenario, 99.3% (Case Scenario #4). The lowest scores were captured during Case Scenario #3 (96.3%) which involved a more intense adult patient scenario. CONCLUSION: The systematic evaluation of both response accuracy and response time (presented in various adult and pediatric patient case scenarios) can be beneficial within the realm of perfusion education. Students will be introduced to core communication concepts within the clinical realm. This study supports the idea that simulation and evaluation may ease the transition for students from the didactic to clinical realm in terms of communication. Further studies need to be developed in order to define "standard" CPB communication guidelines for perfusion students.

Tribal ethnicity and CYP2B6 genetics in Ugandan and Zimbabwean populations in the UK: implications for efavirenz dosing in HIV infection.

OBJECTIVES: To determine differences in CYP2B6 loss of function (LoF) single nucleotide polymorphisms (SNPs) and haplotypes between Zimbabweans and Ugandans, and within Ugandan populations (Bantu and Nilotic). METHODS: Genetic epidemiological study enrolling adult black African Ugandan and Zimbabwean patients attending a UK HIV-1 clinic, irrespective of antiretroviral therapy status. Genomic DNA was extracted from whole blood and the presence of CYP2B6 alleles was determined by direct sequencing of all nine exons of the CYP2B6 gene. Blood was also collected, where appropriate, for determination of efavirenz concentrations. Frequency of SNPs in all patients and LoF haplotype frequencies were calculated. The relationship between the number of LoF haplotype alleles possessed and efavirenz trough concentration (ETC) was determined. RESULTS: Thirty-six Zimbabweans and 74 Ugandans (58 Bantu and 16 Nilotic) were recruited. The definite haplotypes determined were *6, *18, *20 and *27 as LoF and *4 as gain of function. Among those with definite genotypes, the frequency of LoF alleles was 65% [95% confidence interval (95% CI): 51-80] of Zimbabweans versus 22% (95% CI: 12-31) of Ugandan Bantus (P = 10(-6)) and versus 39% (95% CI: 14-64) of Ugandan Nilotics (P = 0.09). Among the 19 patients with definite genotype and with available ETCs, log ETCs were associated with a greater number of LoF haplotype alleles [848 ng/mL (n = 12), 1069 ng/mL (n = 4) and 1813 ng/mL (n = 3) for 0, 1 or 2 LoF haplotypes, respectively (P = 0.016)]. CONCLUSIONS: Among Zimbabweans, LoF haplotypes constitute the majority of CYP2B6 alleles and are significantly higher in prevalence compared with Ugandans. Frequencies of LoF haplotypes and SNPs in Ugandan Nilotics appear to lie between those of Zimbabweans and Ugandan Bantus. These findings may have relevance to pharmacokinetics and dosing of efavirenz in African populations.

The acute effects of synthetic intravenous Delta9-tetrahydrocannabinol on psychosis, mood and cognitive functioning.

BACKGROUND: Recent work suggests that heavy use of cannabis is associated with an increased risk of schizophrenia-like psychosis. However, there is a dearth of experimental studies of the effects of the constituents of cannabis, such as Delta9-tetrahydrocannabinol (THC). In a study of intravenous (i.v.) synthetic THC in healthy humans, we aimed to study the relationship of the psychotic symptoms induced by THC to the consequent anxiety and neuropsychological impairment. METHOD: Twenty-two healthy adult males aged 28+/-6 years (mean+/-s.d.) participated in experimental sessions in which i.v. THC (2.5 mg) was administered under double-blind, placebo-controlled conditions. Self-rated and investigator-rated measurements of mood and psychosis [the University of Wales Institute of Science and Technology Mood Adjective Checklist (UMACL), the Positive and Negative Syndrome Scale (PANSS) and the Community Assessment of Psychic Experiences (CAPE)] were made at baseline and at 30, 80 and 120 min post-injection. Participants also completed a series of neuropsychological tests [the Rey Auditory Verbal Learning Task (RAVLT), Digit Span, Verbal Fluency and the Baddeley Reasoning Task] within 45 min of injection. RESULTS: THC-induced positive psychotic symptoms, and participant- and investigator-rated measurements of these were highly correlated. Participants showed an increase in anxiety ratings but there was no relationship between either self- or investigator-rated positive psychotic symptoms and anxiety. THC also impaired neuropsychological performance but once again there was no relationship between THC-induced positive psychotic symptoms and deficits in working memory/executive function. CONCLUSIONS: These findings confirm that THC can induce a transient, acute psychotic reaction in psychiatrically well individuals. The extent of the psychotic reaction was not related to the degree of anxiety or cognitive impairment.

Medical and legal confusion surrounding gamma-hydroxybutyrate (GHB) and its precursors gamma-butyrolactone (GBL) and 1,4-butanediol (1,4BD).

BACKGROUND: Gamma-hydroxybutyrate (GHB) is used as a recreational drug, with significant associated morbidity and mortality; it is therefore a class C drug under the Misuse of Drugs Act (1971). However, its precursors gamma-butyrolactone (GBL) and 1,4-butanediol (1,4BD) remain legally available despite having similar clinical effects. AIM: The aim of this study was to determine whether the relative proportions of self-reported ingestions of GHB or its precursors GBL and 1,4BD were similar to those seen in analysis of seized drugs. DESIGN AND METHODS: Retrospective review of our clinical toxicology database to identify all cases of self-reported recreational GHB, GBL and 1,4BD use associated with ED presentation in 2006. Additionally all seized substances on people attending local club venues were analysed by a Home Office approved laboratory to identify any illicit substances present. RESULTS: In 2006, there were a total of 158 ED presentations, of which 150 (94.9%) and 8 (5.1%) were GHB and GBL self-reported ingestions respectively; 96.8% (153) were recreational use. Of the 418 samples seized, 225 (53.8%) were in liquid form; 85 (37.8%) contained GHB and 140 (62.2%) contained GBL. None of the seized samples contained 1,4BD and there were no self-reported 1,4BD ingestions. CONCLUSION: Self-reported GHB ingestion was much more common than GBL ingestion, whereas GBL was more commonly found in the seized samples. These differences suggest that GBL use may be more common than previously thought and we suggest that there should be further debate about the legal status of the precursors of GHB.

Can vacuum assisted venous drainage be achieved using a roller pump in an emergency? A pilot study using neonatal circuitry.

There has been much advancement in perfusion technology over its 50 years of progression. One of these techniques is vacuum-assisted venous drainage (VAVD). Many perfusionists augment venous drainage using VAVD, typically from a wall vacuum source. This study explores alternates to providing VAVD if the wall vacuum fails. In two porcine laboratories, approximately 36 in. of 3/16-in. tubing was connected to a sucker return port and placed into the roller head next to the arterial pump. The vacuum was monitored with a DLP pressure monitoring system (Medtronic). This system was connected to small-bore tubing and attached to a stopcock on top of the reservoir. The vacuum was regulated using another stopcock connected to a non-filtered luer lock port on top of the reservoir or by a segment of 3 x 0.25-in.-diameter tubing attached to the vent port with a c-clamp. Vacuum drainage was achieved, ranging from -18 mmHg to -71 mmHg by manipulating the stopcock or c-clamp. Changes in venous drainage were seen by volume fluctuations in the venous reservoir. The vacuum was adjusted to account for dramatic changes. Augmented venous drainage using a roller pump can be achieved successfully during cardiopulmonary bypass (CPB). This method of active drainage can be used in lieu of wall suction or during times of emergency if wall suction fails.

Paradoxical response to tacrolimus assessed by interleukin-2 gene expression.

BACKGROUND: The calcineurin inhibitors cyclosporine and tacrolimus are potent immunosuppressive drugs, used mainly after organ transplantation. Methods to monitor their pharmacodynamic effects are not well established. METHODS: Whole blood samples from healthy volunteers (n = 16) were incubated for 24 hours in culture medium; each sample was preincubated for 2 hours with or without tacrolimus. An identical procedure was performed for 7 samples using blood from renal transplant patients before transplantation. Following the culture period, total RNA was isolated and quantitative reverse transcriptase polymerase chain reaction, using TaqMan probes, was employed to quantify interleukin-2 (IL-2) mRNA expression, and IL-2 mRNA copy number was reported by reference to a standard curve. RESULTS: IL-2 mRNA synthesis was suppressed by the presence of tacrolimus in most cases, compared with a control sample. However, some samples demonstrated up-regulation of mRNA expression. In the patient samples, there was up-regulation of IL-2 mRNA in two samples and, after transplantation, these patients developed acute rejection. CONCLUSION: Quantitative measurement of cytokine IL-2 regulated gene expression may represent a method to assess the efficacy of calcineurin inhibitor drugs.

Validation of high-performance liquid chromatographic-mass spectrometric method for the analysis of lidocaine in human plasma.

A sensitive and simple liquid chromatography-tandem mass spectrometry method is developed and validated for the determination of lidocaine in human plasma. Bupivacaine is used as an internal standard, and the plasma extraction is performed by a simple liquid-liquid extraction. The limit of quantitation (LOQ) is 0.5 ng/mL with a signal-to-noise ratio greater than 5. The calibration curve is linear from 0.5 to 250 ng/mL with an r2 greater than 0.99. The coefficients of variation for within- and between-assay imprecision, including LOQ, are < or = 13% and < or = 8%, respectively. The percentage of inaccuracy for within- and between-assay, including LOQ, are < or = 9% and < or = 5%, respectively. The absolute recovery of lidocaine and bupivacaine are greater than 84% and 82%, respectively. The higher sensitivity and accuracy of this method allow the measurement of low concentrations of lidocaine in plasma from a clinical study of topically applied lidocaine in healthy subjects.

Myocardial magnesium depletion associated with prolonged hypomagnesemia: a longitudinal study in heart transplant recipients.

OBJECTIVES: This study was carried out to establish prospectively the incidence and relation of hypomagnesemia and myocardial magnesium depletion after heart transplantation. BACKGROUND: No serial in vivo study of the relation of serum with tissue magnesium has been described. Myocardial magnesium depletion is associated with intracellular calcium overload, an increased incidence of cardiac arrhythmia and changes in coronary vasculature similar to those seen in the accelerated atherosclerosis that compromises graft survival after transplantation. METHODS: In a prospective study in 19 consecutive patients, serum and myocardial magnesium content were measured serially for 9 months after heart transplantation. Blood cyclosporine was assayed simultaneously. RESULTS: The incidence of hypomagnesemia was 100% during the 9-month study period, with lowest levels at 3 months (mean 0.80 vs. 0.64 mmol/liter, p less than 0.002). Myocardial magnesium depletion developed in 94% and was persistent in 55%; the lowest levels occurred at 6 months (mean 33.6 vs. 30.1, mumol/g, p less than 0.04). Hypomagnesemia predated decreases in myocardial magnesium by 2 to 6 weeks. Peak cyclosporine levels correlated positively with the decrease in serum magnesium. Clinical events were rare. CONCLUSIONS: This is the first report of serial measurement of tissue magnesium. Persistent hypomagnesemia is invariably accompanied by myocardial magnesium depletion in the transplanted heart. Reciprocal calcium overload and adverse changes in coronary vasculature would be expected from previous studies and merit further investigation. Should the implications of this study extend to the native heart, myocardial magnesium depletion may contribute to the high incidence of fatal arrhythmic events observed in patients with heart failure, who commonly have persistent hypomagnesemia.

A microparticle enzyme immunoassay to measure sirolimus.

Measurement of sirolimus as a guide to therapy is widely accepted. Since the commercial introduction of the drug, the only method available to measure blood concentrations has been high-performance liquid chromatography (HPLC). Only a limited number of centers have the facilities to perform this technique and, as a result, the measurement of the drug has been performed in central laboratories, often some distance from the clinical centers. This article describes a single-center assessment of a new immunoassay to measure sirolimus, including a comparison between immunoassay results and a chromatographic technique. Calibration accuracy was good, reproducibility at 11 ng/mL was better than 6%, and sensitivity was better than 2 ng/mL; all these parameters are appropriate for routine clinical use. There was a mean positive bias of almost 20% for the measurement of sirolimus in clinical samples from kidney transplant patients receiving the drug, compared with HPLC. This bias was most likely due to cross-reactivity with metabolites of the drug and was of the order noted when an earlier configuration of this immunoassay was used in clinical practice. We conclude that, despite the analytical bias, this immunoassay offers a viable alternative to the use of HPLC and would be an assay suitable for implementing at local centers.

Cardiac consequences and treatment of disopyramide intoxication: experimental evaluation in dogs.

A slow (1.18 mumol.kg-1.mm-1) intravenous infusion of disopyramide (mol.wt 339) was given to 8 adult Beagle dogs. An initial phase of slow decline in cardiac output and broadening of the QRS complex on the ECG, with systolic blood pressure maintained above 13.5 kPa (100 mmHg), was followed by a phase of rapid circulatory failure without a correspondingly dramatic change in ECG appearances. Underventilation and cardiac arrhythmias were observed only in the agonal phase after several minutes of circulatory arrest. They were not therefore the primary cause of death, which was due to failure of myocardial contractility. Three positively inotropic drugs (isoprenaline, dopamine, and glucagon) are shown to be capable of restoring the failing circulation, provided they are given before the phase of complete circulatory standstill. In this respect isoprenaline appears superior to dopamine and glucagon.

Sirolimus for rescue and primary immunosuppression in transplanted children receiving tacrolimus.

AIMS: The role of sirolimus (SRL) as a rescue agent (n=42) and as a component of primary immunosuppression (n=8) was evaluated in a mixed population of 50 transplanted children receiving tacrolimus (liver: 26, heart: 5, intestinal: 5, liver-intestine: 9, lung: 1, bone marrow: 1, liver-kidney: 1, multivisceral: 1). Rescue indications for tacrolimus (TAC) failure were recurrent acute rejection and acute rejection complicating withdrawal of immunosuppression in posttransplant lymphoproliferative disorder (PTLD). Rescue indications for TAC toxicity were nephrotoxicity, pancreatitis, seizures, hypertrophic cardiomyopathy, and graft-versus-host disease. RESULTS: Mean age at rescue was 11.5 years and mean follow-up was 204 (range 18-800) days. As primary immunosuppression, SRL+TAC prevented early acute rejection in 7/8 children. The indication for rescue resolved in 33/42 children. In children with TAC toxicity, this was associated with decrease in TAC doses by 50%, significant improvements in renal function, and continuing decline in Epstein-Barr virus (EBV) viral load in PTLD patients. Serious adverse events led to discontinuation of SRL in 9/42 rescue patients, 3 of them also experienced acute rejection. Three additional children also experienced acute rejection on SRL therapy (overall incidence 6/50, 12%). Pharmacokinetic analysis in the first week of SRL administration suggested a short half-life (11.8+/-5.5 hr, n=21). CONCLUSIONS: SRL and reduced-dose TAC may achieve adequate immunosuppression without compromising renal function or enhancing EBV viremia significantly.

Monitoring cyclosporin: is it still important?

This paper reviews the current data which provide a rationale for the measurement of cyclosporin as a guide to therapy. Methodological problems related to sample matrix and analytical technique are considered, and the most commonly used methods considered. Factors which could influence the clinical interpretation of cyclosporin measurements are examined, including other drug therapy, compliance with therapy, cyclosporin metabolites, pharmacokinetic variables and sample timing. It is concluded that, whilst isolated measurements do not offer a definitive diagnostic tool, taken in context they can be of considerable value in optimising therapy.

Elevated serum levels of soluble interleukin-2 receptor, neopterin and beta-2-microglobulin in idiopathic dilated cardiomyopathy: relation to disease severity and autoimmune pathogenesis.

BACKGROUND: It has not been assessed whether high levels of soluble interleukin 2 receptor (sIL-2R), neopterin and beta-2 microglobulin in idiopathic dilated cardiomyopathy reflect heart failure severity and/or an active autoimmune process. The aim of this study was to relate serum levels of these markers to clinical and autoimmune features. METHODS: We studied 60 patients with idiopathic dilated cardiomyopathy, 67 controls with ischemic heart failure and 34 normals. RESULTS: Abnormal levels of sIL-2R, but not of neopterin and beta-2 microglobulin, were more frequent in idiopathic dilated cardiomyopathy than in ischemic patients (35% vs. 16%; P=0.02) or in normals (35% vs. 12%, P=0.01); mean sIL-2R levels were, however, similar in idiopathic dilated cardiomyopathy and ischemic heart failure (842+/-75 vs. 762+/-93 U/ml, P=NS). In idiopathic dilated cardiomyopathy abnormal levels of sIL-2R were associated with lower peak oxygen consumption (P=0.008), higher neopterin and HLA class II expression in the myocardium (P=0.02), but were unrelated to cardiac autoantibody status or titer. In addition, abnormal levels of neopterin were associated with adverse prognosis and higher beta-2 microglobulin; abnormal levels of beta-2 microglobulin with lower echocardiographic percent fractional shortening, higher sIL-2R and higher neopterin. CONCLUSIONS: There is no convincing evidence that abnormal sIL-2R, neopterin and/or beta-2 microglobulin are disease-specific markers of idiopathic dilated cardiomyopathy. The lack of association with cardiac autoantibodies suggests that these abnormalities are mainly related to heart failure severity rather than autoimmune pathogenesis. In keeping with this view, high levels of sIL-2R, neopterin and/or beta-2 microglobulin identified a subset of idiopathic dilated cardiomyopathy patients with advanced disease and poor prognosis.

Mitochondrial calcium deposition in association with cyclosporine therapy and myocardial magnesium depletion: a serial histologic study in heart transplant recipients.

Intramitochondrial calcification has been reported in heart transplant recipients treated with high-dose cyclosporine. Myocardial magnesium depletion is common in this group and, on the basis of extensive data from animal studies, would be expected to produce similar mitochondrial deposition of calcium. This prospective study investigated the occurrence of such calcification in biopsy specimens obtained serially in nine heart transplant recipients with simultaneous analysis of myocardial magnesium. During a mean follow-up of 32 weeks, 24 biopsy specimens were analyzed from nine patients. Mitochondrial calcium deposition was more marked in biopsy specimens from recipients with magnesium depletion (p < 0.025). Early toxic cyclosporine levels occurred in three recipients associated with a significant but reversible increase in mitochondrial calcification (p < 0.0001). Histologic rejection and use of calcium antagonists did not modify these findings. It is concluded that although cyclosporine toxicity does induce mitochondrial calcium deposition, such deposition can occur in the absence of toxicity should myocardial magnesium depletion be concurrent. Long-term follow-up will establish the clinical sequelae of such observations. However, when taken together with the results of this study, recent reports of attenuation of accelerated graft atherosclerosis by calcium antagonists may suggest that cyclosporine-induced myocardial magnesium depletion may have an etiologic role in this multifactorial process.

Creatine kinase MB isoforms: a potential predictor of acute cardiac allograft rejection.

BACKGROUND: Noninvasive studies to detect or predict acute allograft rejection after heart transplantation have failed to be sufficiently reliable to substitute for endomyocardial biopsy. Isoforms of creatine kinase MB isoenzyme (MB2 and MB1) are extremely sensitive markers of ischemic myocardial damage and, in theory, may be elevated in cardiac allograft rejection when myocardial necrosis is visible on microscopy (International Society for Heart and Lung Transplantation grade 2 or greater). METHODS: We examined, prospectively, the endomyocardial biopsy specimens (n = 256) of 50 consecutive patients undergoing orthotopic heart transplantation. Blood samples for creatine kinase MB isoforms (n = 527) were taken immediately before endomyocardial biopsy and at intervals between biopsies. RESULTS: The median ratio of MB2/MB1 in plasma samples taken at the time of biopsy for grades 2 and 3 was not significantly different from the ratio from biopsy specimens graded 0 and 1 (1.65 versus 1.33; p = Not significant). The sensitivity for diagnosing a moderately severe rejection was 47% with a specificity of 58%. However, in patients with significant acute rejection (grades 2 and 3) in whom consecutive samples were collected, the MB2/MB1 ratio was significantly increased before histologic changes seen on biopsy in 13 of 16 rejection episodes by a mean of 14 days. The sensitivity for predicting rejection (grade 2 or 3) before endomyocardial biopsy was 60% with a specificity of 71% (positive predictive value 43%, negative predictive value 86%). CONCLUSIONS: Creatine kinase MB isoforms may predict the occurrence of acute rejection before histologic evidence seen on endomyocardial biopsy.

Postmortem assay of digoxin by radioimmunoassay.

Analysis of postmortem blood samples from patients previously on maintenance digoxin therapy suggests that the results are of value in assessing the degree of digitalization at the time of death. Control cases gave results within the normal therapeutic range wheras of six cases in which digoxin was suspected of being implicated in the death five had 'serum' digoxin levels above the therapeutic range. Differences in digoxin concentration were noted in blood collected from three sites in the body, and it is suggested that postmortem blood should be collected from the leg veins if assessment of antermortem digitalization is to be made.

Gas-liquid chromatographic measurement of lignocaine in small samples of canine myocardium after enzymatic digestion.

A method is described whereby lignocaine can be measured in small samples of canine myocardium. A proteolytic enzyme is used to digest the tissue, and the drug, together with an internal standard, is subsequently extracted into an organic solvent and measured by gas-liquid chromatography. The method is reproducible using tissue samples as small as 50 mg and appears to be suitable for the measurement of a number of drugs in small samples of tissue.

High-performance liquid chromatographic measurement of amiodarone and desethylamiodarone in small tissue samples after enzymatic digestion.

A method is described for the measurement of amiodarone and desethylamiodarone in small tissue samples. With the exception of fat, for which lipase is used, the tissues are digested with a proteolytic enzyme. After the addition of an internal standard the analytes are extracted from the homogeneous digest into an organic solvent and measured by high-performance liquid chromatography (HPLC) with UV detection at 240 nm. The method shows good reproducibility using tissue samples as small as 20 mg and suggests extensive accumulation of both compounds in some tissues, with particularly high concentrations in tissues associated with adverse effects of the drug.