Search

Introduction: Hereditary fibrinogen Aα-chain (AFib) amyloidosis is a relatively uncommon renal disease associated with a small number of pathogenic fibrinogen Aα (FibA) variants; wild-type FibA normally does not result in amyloid deposition. Proteomics is now routinely used to identify the amyloid type in clinical samples, and we report here our algorithm for identification of FibA in amyloid., Methods: Proteomics data from 1001 Congo red-positive patient samples were examined using the Mascot search engine to interrogate the Swiss-Prot database and generate protein identity scores. An algorithm was applied to identify FibA as the amyloid protein based on Mascot scores. FibA variants were identified by appending the known amyloidogenic variant sequences to the Swiss-Prot database., Results: AFib amyloid was identified by proteomics in 64 renal samples based on the Mascot scores relative to other amyloid proteins, the presence of a pathogenic variant, and coverage of the p.449-621 sequence. Contamination by blood could be excluded from a comparison of the FibA score with that of the fibrinogen β and γ chains. The proteomics results were consistent with the clinical diagnosis. Four additional renal samples did not fulfill all the criteria using the algorithm but were adjudged as AFib amyloid based on a full assessment of the clinical and biochemical results., Conclusion: AFib amyloid can be identified reliably in glomerular amyloid by proteomics using a score-based algorithm. Proteomics data should be used as a guide to AFib diagnosis, with the results considered together with all available clinical and laboratory information.

Many algorithms have been proposed for automated segmentation of white matter hyperintensities (WMH) in brain MRI. Yet, broad uptake of any particular algorithm has not been observed. In this work, we argue that this may be due to variable and suboptimal validation data and frameworks, precluding direct comparison of methods on heterogeneous data. As a solution, we present Leave-One-Source-Out Cross Validation (LOSO-CV), which leverages all available data for performance estimation, and show that this gives more realistic (lower) estimates of segmentation algorithm performance on data from different scanners. We also develop a FLAIR-only WMH segmentation algorithm: Voxel-Wise Logistic Regression (VLR), inspired by the open-source Lesion Prediction Algorithm (LPA). Our variant facilitates more accurate parameter estimation, and permits intuitive interpretation of model parameters. We illustrate the performance of the VLR algorithm using the LOSO-CV framework with a dataset comprising freely available data from several recent competitions (96 images from 7 scanners). The performance of the VLR algorithm (median Similarity Index of 0.69) is compared to its LPA predecessor (0.58), and the results of the VLR algorithm in the 2017 WMH Segmentation Competition are also presented., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Diagnosis and treatment of systemic amyloidosis depend on accurate identification of the specific amyloid fibril protein forming the tissue deposits. Confirmation of monoclonal immunoglobulin light chain amyloidosis (AL), requiring cytotoxic chemotherapy, and avoidance of such treatment in non-AL amyloidosis, are particularly important. Proteomic analysis characterises amyloid proteins directly. It complements immunohistochemical staining of amyloid to identify fibril proteins and gene sequencing to identify mutations in the fibril precursors. However, proteomics sometimes detects more than one potentially amyloidogenic protein, especially immunoglobulins and transthyretin which are abundant plasma proteins. Ambiguous results are most challenging in the elderly as both AL and transthyretin (ATTR) amyloidosis are usually present in this group. We have lately described a procedure for tissue decellularisation which retains the structure, integrity and composition of amyloid but removes proteins that are not integrated within the deposits. Here we show that use of this procedure before proteomic analysis eliminates ambiguity and improves diagnostic accuracy., Significance: Unequivocal identification of the protein causing amyloidosis disease is crucial for correct diagnosis and treatment. As a proof of principle, we selected a number of cardiac and fat tissue biopsies from patients with various types of amyloidosis and show that a classical procedure of decellularisation enhances the specificity of the identification of the culprit protein reducing ambiguity and the risk of misdiagnosis., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Abnormalities in coherent cortical circuit functioning, reflected in gamma band activity (to approximately 40 Hz), may be a core deficit in schizophrenia. The early auditory gamma band response (EAGBR) is a neurophysiologically simple probe of circuit functioning in primary auditory cortex. We examined the EAGBR in first hospitalized schizophrenia to assess whether it was reduced at first hospitalization., Method: Wavelet evoked power and intertrial phase locking of the EAGBR at Fz to standard tones during an oddball target detection task were examined in 28 first hospitalized schizophrenia patients (10 female) and 44 control subjects (17 female)., Results: At first hospitalization EAGBR trial-to-trial phase locking and evoked power were significantly reduced in patients. Although reduced overall in patients, greater total symptoms were significantly associated with greater gamma phase locking and power. Additionally, greater EAGBR power was marginally associated with greater positive factor scores, hallucinations, and thinking disturbance., Conclusions: Abnormalities of gamma band functioning in local auditory sensory circuits are present in schizophrenia at first hospitalization further evidence that basic sensory processes are impaired in schizophrenia. It remains to be determined whether the EAGBR becomes permanently impaired with disease progression, and if its reduction is specific to schizophrenia.

The human pentraxin proteins, serum amyloid P component (SAP) and C-reactive protein (CRP) are important in routine clinical diagnosis, SAP for systemic amyloidosis and CRP for monitoring the non-specific acute phase response. They are also targets for novel therapies currently in development but their roles in health and disease are controversial. Thus, both for clinical use and to rigorously elucidate their functions, structurally and functionally intact, pharmaceutical grade preparations of the natural, authentic proteins are required. We report here the production from normal human donor plasma and the characterization of the first such preparations. Importantly, we demonstrate that, contrary to reports using recombinant proteins and less well characterized preparations, neither CRP nor SAP stimulate the release by human peripheral blood mononuclear cells in vitro of any TNFα, IL-6 or IL-8, nor does SAP cause release of IL-1β or IL-10. Furthermore neither of our preparations was pro-inflammatory in mice in vivo., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Objective: Diabetes has been linked with more severe periodontal disease and with coronary heart disease (CHD). The purpose of this study was to determine if periodontal infection was a significant modifier in the risk that diabetes poses for increased carotid artery intimal-medial wall thickness (IMT) and more advanced atheroma lesions as reflected in atherosclerotic plaque calcification measured by acoustic shadowing., Methods and Results: Comparisons for analyses of cardiovascular outcomes were performed based upon periodontitis and diabetes status. Periodontitis was measured using pocket depth and attachment loss at six sites per tooth. Cross-sectional data on 6048 persons aged 52-74 years were obtained from the Dental Atherosclerosis Risk in Communities Study. Participants without diabetes (n=5257) were compared to those with diabetes (n=791). Dependent variables were thick IMT (>1 mm), presence of acoustic shadowing, and prevalent CHD. All models were adjusted for the following covariates: gender, age, race/center, LDL and HDL cholesterol, BMI, triglycerides, hypertension, smoking, income and education. For multivariate model building, all non-normally distributed variables were transformed and multivariable logistic regression analyses were performed to evaluate the relationship between periodontal infection, diabetes, and cardiovascular outcomes. Individuals with diabetes and with severe periodontitis were found to be significantly more likely to have IMT>1 mm [OR=2.2, (1.4-3.5)], acoustic shadowing [OR=2.5, (1.3-4.6)], and CHD [OR=2.6, (1.6-4.2)] compared to those without diabetes or periodontal disease., Conclusion: Results from this study suggest that among people with diabetes, periodontal disease may increase the likelihood of subclinical atherosclerotic heart disease and CHD., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Bio-inspired microstructured surfaces have been developed that attach effectively to a wet surface. However, little is known about the associated tribological and adhesion properties involved. In this paper, the adhesion of a microstructured surface to peritoneal tissue is investigated as a function of preload, speeds of approach and angle of tilt to the horizontal. Results show that within a critical range of preload and speed of approach of the microstructured surface to the tissue result in good adhesion characteristics at the micro-structured surface/tissue interface. The microstructured surface adhesion to the tissue and the payload supported against gravity depend significantly on the tilt angle to the horizontal. Furthermore, a wet adhesion model was used to elucidate the adhesion mechanism of the microstructured surface to a wetted glass. These tests provide significant initial data for the design of a miniature mobile intra-abdominal device which drives the need for this adhesion study., (Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Periodontal disease is associated with diabetes, heart disease, and chronic kidney disease (CKD), relationships postulated to be due in part to vascular inflammation. A bidirectional relationship between CKD and periodontal disease is plausible, though this relationship has not been previously reported. In this study, we assessed the potential for connections between CKD and periodontal disease, and mediators of these relationships using structural equation models of data from 11,211 adults ≥ 18 years of age who participated in the Third National Health and Nutrition Examination Survey. Multivariable logistic regression models were used to test the hypothesis that periodontal disease was independently associated with CKD. Given the potential that the periodontal disease and CKD relationship may be bidirectional, a two-step analytic approach was used that involved tests for mediation and structural equation models to examine more complex direct and indirect effects of periodontal disease on CKD, and vice versa. In two separate models, periodontal disease (adjusted odds ratio of 1.62), edentulism (adjusted odds ratio of 1.83), and the periodontal disease score were associated with CKD when simultaneously adjusting for 14 other factors. Altogether, three of four structural equation models support the hypothesized relationship. Thus, our analyses support a bidirectional relationship between CKD and periodontal disease, mediated by hypertension and the duration of diabetes.

Background: The term "diabetes mellitus" describes a group of disorders characterized by elevated levels of glucose in the blood and abnormalities of carbohydrate, fat and protein metabolism. A number of oral diseases and disorders have been associated with diabetes mellitus, and periodontitis has been identified as a possible risk factor for poor metabolic control in subjects with diabetes., Methods: The authors reviewed the literature to identify oral conditions that are affected by diabetes mellitus. They also examined the literature concerning periodontitis as a modifier of glycemic control., Results: Although a number of oral disorders have been associated with diabetes mellitus, the data support the fact that periodontitis is a complication of diabetes. Patients with long-standing, poorly controlled diabetes are at risk of developing oral candidiasis, and the evidence indicates that periodontitis is a risk factor for poor glycemic control and the development of other clinical complications of diabetes. Evidence suggests that periodontal changes are the first clinical manifestation of diabetes., Conclusions: Diabetes is an important health care problem. The evidence suggests that oral health care providers can have a significant, positive effect on the oral and general health of patients with diabetes mellitus.

Airway epithelial cells are the first targets of environmental stimuli and local cytokines. Pyocyanin-induced synergism with interleukin (IL)-1 or tumour necrosis factor (TNF) in triggering IL-8 release has been documented previously. In this study, IL-8 mRNA and protein expression were examined in cultured human bronchial epithelial cells (BEAS-2B) stimulated with pyocyanin alone, and in combination with IL-1beta or phorbol 12,13-dibutyrate (PDBu) in the absence and presence of a group of glucocorticoids. IL-8 mRNA was measured by RT-PCR, and IL-8 protein by ELISA (cell supernatants). Pyocyanin alone produced no increase in IL-8 mRNA and release. However, pyocyanin upregulated the stimulatory effect of IL-1beta or PDBu on the release of IL-8 in a dose-dependent manner. The stimulatory effect of pyocyanin on the IL-1beta- or PDBu-stimulated IL-8 release was reduced in the presence of dexamethasone, budesonide, and fluticasone. Budesonide and fluticasone were 10-fold more potent than dexamethasone. The protein kinase C (PKC) inhibitor, Go6976, also significantly reduced the stimulatory effect of pyocyanin on IL-1beta, and PDBu increased IL-8 release. In conclusion, this study shows that PKC signal pathway seems to be involved in the pyocyanin-mediated upregulation of the IL-1beta and PDBu-induced IL-8 release in BEAS-2B cells. These findings suggest that a vicious cycle perpetuating inflammation may exist in the biologic milieu of bronchiectatic patients infected with Pseudomonas aeruginosa due to the production of pyocyanin. The priming action of pyocyanin appears to be blocked by glucocorticoids, thus providing in vitro data in support of the clinical efficacy of inhaled glucocorticoids as anti-inflammatory drugs.

Background: Maintenance of luminal area is essential for the optimal performance of venous bypass grafts. However, injury and response to the arterial circulation evoke vascular remodelling that favors intimal hyperplasia, with luminal encroachment and inward remodelling. Potassium channel-opening drugs reduce tissue workload and peripheral vascular resistance and through these mechanisms could favor outward or expansive remodelling of vein grafts. We tested the hypothesis that levcromakalim, a potassium channel opener, would enhance expansive remodelling in vein grafts., Methods: A randomized, double-blind, placebo-controlled trial was conducted in 33 rats with vena cava-to-aorta bypass grafts. Drugs were administered via osmotic pump for 7 days after surgery. Half the cohort had bromodeoxyuridine (BrdU) infused at day 6. Morphometric analysis was conducted of pressure perfusion-fixed grafts harvested at 1 week and 4 weeks., Results: At 1 week, lumen area was similar in both groups (1.82 +/- 0.39 mm(2) placebo vs 1.85 +/- 0.36 mm(2) levcromakalim), although medial cell density and BrdU staining were significantly increased in the placebo group. At 4 weeks, lumen area was unchanged in the placebo group (1.88 +/- 0.51 mm(2)) but had increased to 2.32 +/- 0.46 mm(2) in the levcromakalim group (P = .039 vs 1 week), with a very significant reduction in the intimal area (levcromakalim, 0.06 +/- 0.02 mm(2) vs placebo, 0.33 +/- 0.17 mm(2); P = .001)., Conclusions: Early, short-term treatment with levcromakalim favors expansive remodelling of experimental vein grafts to mimic the effect of external stenting. This expansive remodelling was associated with a reduction in medial cell proliferation at 1 week., Clinical Relevance: Critical limb ischemia can be treated by bypass surgery or angioplasty, but inward remodelling with restenosis is a common problem. There has been little previous experimental work to identify treatments associated with expansive remodelling, which would increase the chances of vessel patency. Here, in a randomized trial, we show that short-term treatment with a potassium channel opener (a class of drug that can be used to treat hypertension) results in strong, expansive remodelling, with increases the lumen area and graft size of experimental vein grafts by >25%.

Background: Diabetes mellitus and periodontal diseases are common chronic diseases in the United States. Periodontal infection may adversely affect glycemic control in people with diabetes. This article reviews the evidence regarding how treatment of periodontal diseases affects glycemic control., Types of Studies Reviewed: The review consisted of a MEDLINE literature search to identify primary research reports on the relationship between periodontal therapy and changes in glycemic control. The review identified three randomized clinical trials and nine nonrandomized clinical follow-up studies., Results: The strength, quantity and breadth of evidence are varied, precluding clear-cut guidance for determining whether treating periodontal infection has a beneficial effect on glycemic control. Despite the variation and limitations in the literature, evidence supports the concept that periodontal diseases can contribute to poorer glycemic control in people with diabetes. Although the evidence is not unequivocal, it provides sufficient support for additional investigations of the effect of preventing and treating periodontal infections on managing glycemic control., Clinical Implications: Sufficient evidence exists to incorporate oral examinations and periodontal care in management regimens for people with diabetes. It is prudent to assess patients' glycemic control status and communicate the importance of referring patients with diabetes for thorough oral health evaluations and necessary care.

Objective: Many diabetics complain of xerostomia, a condition that can affect oral health, nutritional status, and diet selection. This study's purposes were (1) to investigate the effect on salivary flow of type 2 diabetes and change in glycemic control in a group of older adults over time and (2) to compare flow rates with subjective complaints of xerostomia., Study Design: A total of 39 older adults, 24 with type 2 diabetes and 15 who were nondiabetic (controls), aged 54-90 years, participated in a 1-year follow-up study. Diabetic status was determined by means of glycosylated hemoglobin (HbA1c) levels and 2-hour glucose tolerance tests. Poor glycemic control was defined as HbA1c > 9%. Unstimulated whole, unstimulated parotid, and stimulated parotid saliva flow rates were measured for all subjects by a single examiner at baseline and 1 year later. Each subject completed a standardized xerostomia questionnaire at every visit., Results: Age, sex, and duration of diabetes did not adversely affect salivary flow rates. Subjects with poorly controlled diabetes had significantly lower stimulated parotid saliva flow rates at both visits. There were no significant changes in flow rates over time on the basis of diabetic status or glycemic control. Subjects with diabetes reported significantly more complaints of thirst but not of xerostomia at 1 year., Conclusions: These results suggest that older adults with poorly controlled diabetes may have impaired salivary flow in comparison with subjects with better controlled diabetes and nondiabetic subjects, yet they may not have concomitant xerostomic complaints. There were no significant changes in salivary flow rates or glycemic control over the 1-year period.

Infection with the bacterium Helicobacter pylori is associated with altered gastric acid secretion and gastrointestinal disease. Recent work has suggested that N alpha-methylhistamine, produced by the bacterium and acting on histamine receptors in gastric tissue, might be involved. Gastric juice and tissue biopsies from infected patients have been analysed for the presence of N alpha-methylhistamine using a specific and sensitive assay based on gas chromatography mass spectrometry. N alpha-Methylhistamine was detected in five of seven samples of gastric juice from infected patients (5-180 pmol/ml) but was absent in nine uninfected subjects. The compound was not found in fundic and antral biopsies from both subject groups. Helicobacter pylori, cultured on agar and in broth with and without added histamine, was found not to produce detectable levels of N alpha-methylhistamine. Instillation of this compound at 10(-5) mol/l into the gastric lumen produced a significant increase in acid secretion in vivo while plasma gastrin concentration remained unchanged. N alpha-Methylhistamine in gastric juice appears therefore to be associated with infection, although this product is not generated directly by the bacterium. The concentrations found are below those required to affect acid secretion or gastrin production in vivo, although higher local concentrations may exist around a site of infection.

A gas chromatography-electron capture mass spectrometry assay has been developed for the histamine H3 receptor agonist, N(alpha)-methylhistamine (N(alpha)-MH). The assay is linear from 50 pg-10 ng, with a limit of detection of 50 pg/ml for gastric juice and plasma, and 50 pg/sample for bacteria (10(7)-10(8) CFU) and gastric tissue (5-10 mg wet weight). The limits of quantification are 100 pg/ml for gastric juice (%RSD=1.4) and plasma (%RSD=9.4), and 100 pg/sample for bacteria (%RSD=3.9) and tissue (%RSD=5.8). N(alpha)-MH was not present in human plasma, but low levels (1.4 ng/ml and 0.4 ng/ml) were detected in two samples of human gastric juice obtained from patients infected with Helicobacter pylori.

Objective: There is no consensus on the possible association between diabetes and salivary dysfunction in older persons with diabetes. This study's purpose was to investigate the effect of diabetes and glycemic control on salivary function in an older population., Study Design: Twenty nine persons with type 2 diabetes and 23 nondiabetic control subjects participated (age range, 54-90 years). Diabetic status was determined by a glycosylated hemoglobin (HbA(1c)) test and a 2-hour glucose tolerance test. Poor glycemic control was defined as HbA(1c) >9%. Unstimulated whole saliva, unstimulated parotid, and stimulated parotid flow rates were measured, and subjects completed a standardized xerostomia questionnaire., Results: Persons with poorly controlled diabetes had lower (P =.01) stimulated parotid flow rates than persons with well-controlled diabetes and nondiabetic control subjects. There were no significant differences in xerostomic complaints based on diabetic or glycemic control status or salivary flow rates., Conclusions: These results provide some evidence that poorly controlled diabetes may be associated with salivary dysfunction in older adults who have no concomitant complaints of xerostomia.

Background: The authors assessed the dental checkup frequency of adults living in the Detroit tricounty area and identified demographic, access and subjective factors associated with visits to a dentist made not because of a dental problem., Methods: Data are from a 1994 probability-based sample of adults who were interviewed and received an in-home oral examination. The authors used the variables of age, sex, education level, income level, dental insurance status, usual place for care, objective measures of oral health, and subjective assessments of health, pain and dental anxiety to predict the frequency of dental checkups., Results: The authors found that differences in dental checkup behavior were related to demographics, access to dental care, subjective ratings of oral and general health and other self-assessments, and clinical parameters of oral health. In multiple logistic regression analysis considering all variables simultaneously, sex, income, having a usual place for care and level of dental care anxiety were found to be associated with having dental checkups. The authors determined the statistical significance level at P < .05., Conclusions: A total of 69.7 percent of the study population reported having had a dental checkup at least once a year in the past five years. The authors found that four factors associated with infrequent dental checkups: being male, having lower income levels, not having a usual place for care and being anxious about receiving dental care., Practice Implications: Dental health professionals should consider the correlates of dental checkup frequency identified in this study and the usefulness of proposed strategies to increase and sustain regular preventive visitation patterns in their own patient populations.

Persisting poor glycemic control has been shown to be associated with the incidence and progression of diabetes-related complications. The bulk of oral health-related research has focused on the impact of diabetes on periodontal health, yet there are several lines of evidence to support the plausibility of the notion that periodontal infections contribute to problems with glycemic control. This article reviews the body of English-language literature containing reports of clinical research that has considered the relationship between treatment of periodontal diseases and improvement in glycemic control in humans. Although there is supportive clinical and epidemiologic evidence, equivocal and contrary evidence also exists. It is concluded from this review that the quantity, breadth, and strength of evidence-based knowledge are currently insufficient to establish periodontal therapy as influential in improving glycemic control in either type 1 or type 2 diabetes. Further rigorous, systematic study of the effects of treating periodontal infection on glycemic control is warranted.

The influence of low density lipoproteins (LDL) in the plasma on the regulation of cholesterol biosynthesis is not clear. We studied the changes in plasma mevalonic acid (MVA) concentration and the lathosterol/cholesterol (L/C) ratio, which are well established indices of whole body cholesterol synthesis, in four normocholesterolaemic subjects after each had undergone LDL apheresis on two occasions. LDL apheresis of 75% of the calculated plasma volume reduced LDL-cholesterol by 44% to 1.5 +/- 0.2 mmol/l without changing plasma MVA levels or L/C ratios. Apheresis of 125% of the calculated plasma volume decreased plasma LDL-cholesterol by 69% to 0.9 +/- 0.2 mmol/l, with significant increases in plasma MVA and L/C ratio on the day after the procedure. These results imply that LDL-cholesterol is an integral part of the sterol regulatory pool and suggest that plasma levels cannot be lowered below 1-1.4 mmol/l in normal subjects without upregulating cholesterol biosynthesis.

Glutathione (GSH) was measured using HPLC-electrochemical detection in bronchoalveolar lavage fluid from 28 neonates for up to 21 days after birth. GSH levels varied from 0.1-11.2 mumol l-1 (with a geometric mean concentration of 1.3 mumol l-1). GSH in epithelial lining fluid was estimated using the urea dilution method at 15.0 mumol l-1 (range 0.5-196 mumol l-1), which is significantly lower than observed in adult subjects. There was an L shaped relationship between GSH and the two markers of oxygen therapy, oxygen index and FiO2. The lowest GSH levels were associated with the group of infants with the most severe airways problems who required high oxygen.

HMG-CoA reductase inhibitors or statins are effective in both the primary and secondary prevention of coronary heart disease, the extent of benefit being proportional to the reduction in low density lipoprotein (LDL) cholesterol achieved. Atorvastatin, a newly licensed compound, reportedly lowers LDL with greater efficacy than other statins. The mechanism of this action was, therefore, explored in twenty patients with refractory familial hypercholesterolemia who received in a single-blind sequence simvastatin 40 mg/day, placebo and atorvastatin 10 mg/day each for 4 weeks. At the end of the placebo period the effects of single 40-mg doses of simvastatin and atorvastatin on plasma levels and urinary excretion of mevalonic acid, indices of HMG-CoA reductase activity, were compared. Administration of atorvastatin 10 mg daily for 1 month lowered LDL cholesterol by 32.5%, compared with placebo (P = 0.0001), which was 4.5% less than the decrease after simvastatin 40 mg daily (P = 0.33). The area under the plasma curve and urinary mevalonic acid/ creatinine ratio were both significantly less during the 24 h after a single dose of atorvastatin 40 mg than after a single dose of simvastatin 40 mg (P < 0.01). These findings suggest that the greater efficacy of atorvastatin compared with simvastatin is due to more prolonged inhibition of HMG-CoA reductase, presumably reflecting longer residence of atorvastatin or its active metabolites in the liver.

An assay based on combined microbore high-performance liquid chromatography-positive ion electrospray ionisation mass spectrometry with selected ion recording has been developed for the measurement of the antihistamine drug terfenadine in human plasma. A deuterated analogue of terfenadine was synthesised for use as an internal standard and extraction of terfenadine was carried out on C18 solid phase extraction columns. The limit of detection of terfenadine in plasma is 0.1 ng/ml and the intra-assay coefficient of variation at 1 ng/ml is 10.1%. Plasma concentrations of terfenadine measured in six normal subjects following a 120 mg oral dose are reported.

Fasting plasma mevalonic acid (MVA), an indicator of in vivo cholesterol synthesis, was measured in 35 patients with familial hypercholesterolaemia (FH) of whom 7 were treated with pravastatin 10-40 mg/day, 7 with simvastatin 10-40 mg/day and 21 with atorvastatin 80 mg/day. Reductions in low density lipoprotein (LDL) cholesterol and MVA on maximal dose therapy differed significantly between the three drugs: 34.7%, 42.9% and 54.0% (P = 0.0001), and 31.6%, 48.9% and 58.8% (P = 0.004), respectively. Patients on atorvastatin were subdivided according to whether their reduction in LDL cholesterol on treatment was above or below the mean percentage change for the whole group. Basal values of LDL cholesterol did not differ significantly, but above average responders had a significantly higher mean pre-treatment level of MVA (6.2 +/- 0.60 vs. 4.3 +/- 0.61 ng/ml, P < 0.05) than below average responders. When all three drug groups were pooled above average responders showed a significantly greater absolute decrease in MVA on treatment than below average responders (3.85 +/- 0.48 vs. 2.33 +/- 0.40 ng/ml, P < 0.05). However, there was no significant correlation between the magnitude of the decreases in LDL cholesterol and MVA. These findings suggest that FH patients who responded well to statins had a higher basal level of plasma MVA, i.e. a higher rate of cholesterol synthesis, which was more susceptible to pharmacological inhibition. The more marked cholesterol lowering effect of atorvastatin 80 mg/day presumably reflects, at least in part, its ability to inhibit HMG-CoA reductase to a greater extent than maximal recommended doses of pravastatin and simvastatin of 40 mg/day.

The 4-hydroxy-2-alkylquinolines and their N-oxides are secondary metabolites produced by Pseudomonas aeruginosa which inhibit the growth of a number of Gram-positive organisms including Staphylococcus aureus. To facilitate the identification of these compounds in biological fluids, we have developed a rapid profiling system based on gas chromatography-electron-capture mass spectrometry of the O-bistrifluoromethylbenzoyl derivatives. Using the technique, over twenty hydroxyalkylquinolines have been identified from a culture obtained from a strain of P. aeruginosa obtained from a patient with severe bronchiectasis.

In this study, 29 individuals who sustained a neck strain as a result of a car crash were drawn from a group of physiotherapy and general practices and were followed up after 6 months. The aim was to examine relationships between the state of the neck injury at the time of follow up and crash-related factors, notably crash severity and occupant awareness. Crash severity was assessed by measurement of damage to the involved vehicles, while 6-month injury status was established through physical examinations and interviews. No statistically significant associations between crash severity and 6-month injury status were found, but subjects who were unaware of the impending collision had a greatly increased likelihood of experiencing persisting symptoms of and/or signs of neck strain, compared with those who were aware (odds ratio = 15.0; 95 per cent confidence limits: 1.8, 178). While the role of crash severity in the production and duration of neck strains remains unclear, awareness appears to have a strong protective influence and may prove to be a useful prognostic indicator in clinical settings.

The metabolism and synthesis of cysteinyl leukotrienes by the isolated perfused pig kidney has been investigated. Kidneys were maintained for up to six hours in a recirculating perfusion system by using an oxygenated Krebs-Henseleit buffer containing albumin and the perfluorinated oxygen carrier, FC-43. Perfusion pressure was maintained at 12-13.5 kPa, with perfusion flow rates of 150-250 ml/min resulting in a urine output of between 20-180 ml/hr. Infusion of 3H-LTC4 into the renal artery resulted in rapid and complete metabolism, with the major urinary metabolites comprising LTE4, omega-hydroxy-LTE4, omega-carboxy-LTE4 and N-acetyl-omega-hydroxy-LTE4. The capacity of the isolated kidney to synthesize cysteinyl leukotrienes was monitored by measuring urinary LTE4 excretion; there was a basal urinary excretion of LTE4 (median 43 pg/min, range 8-470 pg/min). Neither lipopolysaccharide or human recombinant tumor necrosis factor alpha had any effect on basal excretion. Treatment with the calcium ionophore A23187, however, resulted in a 38.1 +/- 9.6-fold increase in urinary LTE4 excretion. We conclude that the isolated pig kidney, in the absence of circulating cells, can synthesize cysteinyl leukotrienes in the absence of circulating cells, which can then undergo extensive oxidative metabolism.

Positron emission tomography (PET) requires the use of compounds labelled with short-lived, positron-emitting isotopes (e.g., t1/2 of 11C approximately 120 min). As the concentration of unbound, non-metabolised drug is required as the input function for modeling, this presents particular problems for the study of the kinetics and metabolism of such compounds. We have now developed a rapid extraction procedure, followed by high-performance liquid chromatography using a short analytical column coupled to an on-line gamma-detector to determine the metabolism and kinetics of a non-selective beta-adrenergic antagonist of high affinity, S-4-(tert.-butylamino-2-hydroxypropoxy)benzimidazol-2-one. This antagonist is potentially well suited to the non-invasive localisation of beta-receptors in vivo. The ligand was rapidly taken up into the beta-receptor pool or excreted in urine, with less than 5% of the drug converted to metabolites. Plasma protein binding was only 16%. No significant metabolism of the ligand was observed in the anaesthetised dog, and, therefore, no correction for blood metabolite concentration is required for kinetic analysis of the 11C-labelled ligand during PET studies in this species. The analytical method reported here should be widely applicable: quantification of metabolites enables accurate estimation of the input function and is critical to the interpretation of PET data.

An assay has been developed for N tau-methylhistamine, a major metabolite of the autocoid histamine, based on gas chromatography-electron-capture negative-ion chemical ionisation mass spectrometry. N tau-Methylhistamine was extracted from urine by cation-exchange chromatography and converted to its di-(3,5-bistrifluoromethylbenzoyl) derivative. The latter has good chromatographic properties and gives a negative-ion mass spectrum with the molecular ion (M., m/z 605) as base peak. A commercially available trideuterated analogue of N tau-methylhistamine was used as internal standard. Basal urinary excretion of N tau-methylhistamine in five normal subjects was found to be 0.21 +/- 0.05 mumol/h (289 +/- 74 mumol/mol of creatinine). This value was not significantly altered in these subjects following the infusion of a subpharmacological dose of histamine. In eight atopic volunteers, basal urinary excretion of N tau-methylhistamine was also not significantly changed following challenge with inhaled allergen.

Circulating concentrations of mevalonic acid (MVA) change in parallel with, and may be used as a marker of cholesterol biosynthesis. Plasma MVA levels have been quantified using a sensitive and specific capillary gas chromatography-electron capture mass spectrometric assay. The detection limit for MVA in plasma is 100 pg/ml; the intra-assay variation is 5.11%; the inter-assay variation is 7.7%. Using this assay, the mean plasma MVA in 15 normolipidemic subjects was 2.37 +/- 1.2 ng/ml (range 0.41-5.31 ng/ml). Administration of 40 mg of simvastatin (an HMG-CoA reductase inhibitor) significantly accenutated the diurnal decrease in plasma MVA levels. This assay may be useful in investigating cholesterol synthesis rates in different dyslipidemias and individual responses of HMG-CoA reductase-inhibiting drugs.

The cysteinyl leukotrienes C4 and D4 are potent renal vasoconstrictors which may modulate glomerular function in vivo, and may therefore be important in the pathogenesis of hepatorenal syndrome. Urinary leukotriene E4, the major metabolite of leukotrienes C4 and D4, was elevated in patients with hepatorenal syndrome (17.8 ng/h) when compared with normal controls (5.1 ng/h) or subjects with renal failure alone (1.9 ng/h). Urinary leukotriene E4 was also elevated in subjects with decompensated liver disease (cirrhosis with ascites 28.6 ng/h, severe hepatocellular dysfunction 57.5 ng/h), but normal in compensated liver disease (6.7 ng/h). In the early stages of hepatorenal syndrome, leukotriene E4 excretion rate was up to 100-fold higher (560 ng/h) than in normals, and fell in parallel with creatinine clearance, indicative of the glomerular filtration rate-dependent renal excretion. Following correction for creatinine clearance, leukotriene E4, excretion was considerably higher in hepatorenal syndrome (54.1 pg/ml creatinine clearance) compared with normals (1.0 pg/ml creatinine clearance), chronic renal failure (3.2 pg/ml creatinine clearance), decompensated liver disease (ascites 7.7 pg/ml creatinine clearance, and severe hepatocellular dysfunction 11.0 pg/ml creatinine clearance), and compensated liver disease (1.9 pg/ml creatinine clearance). To interpret the significance of these findings, we determined renal clearance and endogenous metabolism of the cysteinyl leukotrienes by infusion of [3H]leukotriene C4 into a single subject with hepato-renal syndrome and two control subjects. Renal clearance of leukotriene E4, was reduced in hepatorenal syndrome (2.4 ml/min) compared with controls (greater than 17 ml/min) which together with the increased excretion rate of leukotriene E4 demonstrates that there is increased cysteinyl leukotriene production in hepatorenal syndrome. This may be one of the factors involved in its pathogenesis.

Although data are available regarding lumbar spinal somatosensory evoked potentials (SSEPs) after posterior tibial nerve stimulation in the mature rat, age-related changes spanning the period of early development have not been defined. We obtained lumbar spinal SEPs after posterior tibial nerve stimulation in 6 cohorts of animals (N = 36) ranging in age from weanling (15 days) to early adulthood (110 days) by recording from needle electrodes placed in the L1-2 and L5-6 interspinous ligaments. Absolute latencies of the major wave form components at the two recording sites declined rapidly until the mid-juvenile period (36 days) and more slowly thereafter. Mean peripheral conduction velocities (+/- S.D.) increased from 11.06 (+/- 0.02) to 33.22 (+/- 3.55) m/sec and mean central conduction velocities (+/- S.D.) increased from 6.27 (+/- 1.07) to 23.64 (+/- 3.84) m/sec from 15 to 110 days respectively. The linear relationships of central and peripheral conduction velocities to both age and weight as defined by standard regression were highly significant. No sex differences were noted for peripheral velocities at all ages studied. Central velocities revealed significant sex differences at 110 days but not earlier. This study demonstrates that lumbar spinal SEPs after posterior tibial nerve stimulation undergo a predictable evolution which can be represented by a simple cable model of a lengthening myelinated pathway.

3-Hydroxychrysene, a metabolite of the polycyclic aromatic hydrocarbon (PAH) chrysene, was metabolised by rat liver microsomal preparations obtained from Arochlor 1254-pretreated rats. Eight major metabolites were isolated by high performance liquid chromatography and characterised by u.v. spectroscopy and a variety of mass spectrometric techniques. The metabolites were unambiguously identified as 9-hydroxy-trans-1,2-dihydroxy-1,2-dihydrochrysene and 9-hydroxy-r-1,t-2,t-3,c-4-tetrahydroxy-1,2,3,4-tetrahydrochrysene and tentatively identified as 3-hydroxy-trans-5,6-dihydroxy-5,6-dihydrochrysene (since chrysene is a symmetrical molecule the 3- and 9-positions are equivalent), 9-hydroxy-trans-3,4-dihydroxy-3,4-dihydrochrysene, 1,2,3-trihydroxy-1,2,3,4-tetrahydrochrysene, an oxidised phenol and two diphenols. These results indicate that 3-hydroxychrysene can be further metabolised via a number of different pathways including those involving the formation of phenol- and triol-epoxides.

A gas chromatographic-mass spectrometric assay has been developed for the measurement of N tau-methylimidazoleacetic acid in urine and plasma. The method uses the isopropyul ester 3,5-bistrifluoromethylbenzoyl derivative of N tau-methylimidazoleacetic acid and electron capture negative ion chemical ionisation mass spectrometry. The derivative has very good chromatographic properties and a negative ion mass spectrum which contains only a molecular ion at m/z 422. When this ion is specifically monitored, an amount of derivative equivalent to 1 pg of parent compound can be detected. A deuterated analogue of N tau-methylimidazoleacetic acid was synthesised for use as an internal standard and this allowed the development of an assay for N tau-methylimidazoleacetic acid, in urine with a precision of 2.9% and in plasma with a precision of 1.5%.

Thermospray liquid chromatography-mass spectrometry has been applied in the solution of a number of problems of biological and biomedical interest. These include the analysis of phenazines from the Gram negative bacterium Pseudomonas aeruginosa, steroids released by rat adrenals and eicosanoids generated by human inflammatory cells. The application of the technique to leukotrienes in blood is discussed. Isotopic labelling prior to analysis, to facilitate identification and structure elucidation is outlined with reference to the steroids.

The high perinatal mortality among infants of women transferred from general practitioner to consultant care is well known, the implication being that if these women had been booked for consultant care several of the deaths might not have occurred. The findings of this study indicate that booking into a consultant unit did not reduce perinatal mortality and suggest that, where liaison between hospital and general practitioners is good and transfer easy, booking into a general practitioner maternity unit can be as safe for low-risk women as booking into a consultant unit.

2-Amino-3,8-dimethylimidazo(4,5-f)quinoxaline (MeIQx) is a potent mutagen found in cooked food. MeIQx and its isotopically labelled (13C, 15N2 and 14C) analogues were synthesised and used for metabolic studies in vivo. An equimolar mixture of MeIQx and its 13C, 15N2 stable isotope labelled analogue (containing tracer amounts of 14C-MeIQx) was given intraperitoneally to mice. Some 67% of the radioactivity was eliminated in urine and faeces within 24h. Four radiolabelled species were observed when urine was analysed by HPLC, corresponding to unchanged MeIQx and three more polar metabolites. Urine was analysed directly by HPLC-thermospray mass spectrometry. Four signals were observed containing the characteristic 1:1 isotopic doublet, corresponding to unchanged MeIQx, an MeIQx glucuronide, and two uncharacterized metabolites.

The structure of beta-cell tropin, an insulin secretagogue released by the neuro-intermediate lobe of the obese (ob/ob) mouse, has recently been determined as the 22-39 moiety of ACTH. A method for the preparation of this octadecapeptide using mild solid-phase procedures followed by preparative high pressure liquid chromatography is described. The molecular weight of the synthetic peptide has been confirmed by Fast Atom Bombardment mass spectrometry. Synthetic beta-cell tropin is indistinguishable in its chromatographic, antigenic and biological properties from natural beta-cell tropin.

The purpose of this study was to establish whether women with peripheral arterial disease can be distinguished from controls on the basis of plasma lipid and apolipoprotein profiles. One group of patients with peripheral arterial disease (n = 20) was characterized by a localized aortic stenosis referred to as the 'small aorta syndrome' (SAS). The other group of patients with peripheral arterial disease (n = 23) had a diffuse segmental pattern of stenoses referred to as the 'stenosing peripheral arterial disease' (SPAD). After correcting for the effects of age and body mass index, the SAS group had elevated plasma total cholesterol (TC) levels when compared to normal controls (P less than or equal to 0.008), while the SPAD group had triacylglycerol (TG) levels different from controls (P = 0.02). Both groups of patients were characterized by reduced levels of apolipoprotein A-I (P less than or equal to 0.04) and increased levels of apolipoprotein C-III (P less than or equal to 0.002). Apolipoproteins B and E were also elevated in both groups of patients but not significantly. Mutivariate analyses indicated that the A-I/C-III ratio correctly discriminated 97.8% of the SAS and the A-I/C-III ratio plus A-I discriminated 89.8% of the SPAD patient from the controls. In addition, multivariate analyses showed that the variables age, TC/Apo B, Apo B/C-III and TG/C-III discriminated SPAD from SAS patients with a correct classification of 93.2%. Results of this study showed that the measurement of apolipoproteins A-I, B and C-III in conjunction with TC and TG is of potential use for differentiating patients with peripheral arterial disease from normal controls as well as for distinguishing patients with SAS from those with SPAD. It seems that particular patterns of peripheral arterial disease in women may be associated with slightly different alterations in the plasma lipoprotein system.

A new method is described for screening and establishing the primary structure of proteins. The procedure is both rapid and sensitive and allows study of the C-terminus of the protein with equal facility to the N-terminus. This new strategy which is illustrated here for the polypeptide hormone Insulin, has obvious applications in recombinant DNA biotechnology research, in post-translational modification and site-directed mutagenesis studies, or any other aspect of modern protein chemistry requiring accurate definition of primary structure.

A rapid and simple method for the on-line concentration and high-performance liquid chromatographic (HPLC) purification of the leukotrienes in good yield from biological fluids is described. Readily available antisera are used in conjunction with this system to give a specific and sensitive assay for leukotrienes B4, C4 and D4 with sub-nanogram limits of detection. Tritium-labelled leukotrienes are used as internal standards, both to locate the leukotrienes post-HPLC and to accurately determine recoveries.

The leukotrienes LTC4, D4, and E4 are potent bronchoconstrictor agents and are thought to have an important role in asthma. Urinary LTE4, a stable urinary end-product of LTC4 and LTD4, was measured, by means of high-performance liquid chromatography and radioimmunoassay. LTE4 excretion followed a log-normal distribution in twenty-nine healthy controls, with a geometric mean of 23.8 (95% confidence interval 19.9-28.2) ng/mmol creatinine. Urine was collected from eight atopic subjects for 3 h after antigen inhalation and a control urine collection was made a week later at the same time of day. Urinary LTE4 was significantly higher after antigen challenge than in the control sample (153.7 [87.1-271.3] vs 23.5 [13.7-69.5] ng/mmol creatinine; p less than 0.01). Urinary LTE4 was also measured in twenty patients with severe acute asthma and nine patients with seasonal allergic rhinitis. Mean urinary LTE4 was higher in the asthmatic patients (78.3 [46.5-131.8] ng/mmol creatinine) than in normal subjects (p less than 0.01), although there was substantial overlap into the normal range. The urinary LTE4 values of the rhinitis patients were within the normal range whether or not they had symptoms. LTC4 and LTD4 were also found in bronchoalveolar lavage fluid from one of the three atopic subjects challenged with antigen before lavage, and in a single patient who underwent lavage after admission with severe acute asthma. These studies provide evidence that leukotrienes are released in vivo in man after antigen challenge and in acute asthma.

A fast atom bombardment mass spectrometric protocol has been developed to determine the type of oligosaccharide chain present in glycoproteins. The procedure is based on acetolysis of the intact glycoconjugate, extraction of the peracetylated carbohydrate fragments and analysis by fast atom bombardment mass spectrometry. The molecular ions present in the FAB spectra uniquely define the composition of the oligosaccharides with respect to hexose, aminohexose and sialic acid content. High mannose oligosaccharides yield a series of peracetylated hexose oligomers whereas complex-type oligosaccharides afford a series of N-acetyl-lactosamine containing species. Fucosylation is usually not detected but sialylated oligosaccharides are readily identified and the type of sialic acid is also defined. The method has been tested on three glycoproteins of known structure - fetuin, ribonuclease B and erythrocyte Band 3 - and on a glycoprotein of unknown structure - alpha-galactosidase I, an enzyme lectin from Vicia faba. The latter is shown to contain high mannose carbohydrate chains.