Amyloid peptides and proteins in review.

Amyloids are filamentous protein deposits ranging in size from nanometres to microns and composed of aggregated peptide beta-sheets formed from parallel or anti-parallel alignments of peptide beta-strands. Amyloid-forming proteins have attracted a great deal of recent attention because of their association with over 30 diseases, notably neurodegenerative conditions like Alzheimer's, Huntington's, Parkinson's, Creutzfeldt-Jacob and prion disorders, but also systemic diseases such as amyotrophic lateral sclerosis (Lou Gehrig's disease) and type II diabetes. These diseases are all thought to involve important conformational changes in proteins, sometimes termed misfolding, that usually produce beta-sheet structures with a strong tendency to aggregate into water-insoluble fibrous polymers. Reasons for such conformational changes in vivo are still unclear. Intermediate aggregated state(s), rather than precipitated insoluble polymeric aggregates, have recently been implicated in cellular toxicity and may be the source of aberrant pathology in amyloid diseases. Numerous in vitro studies of short and medium length peptides that form amyloids have provided some clues to amyloid formation, with an alpha-helix to beta-sheet folding transition sometimes implicated as an intermediary step leading to amyloid formation. More recently, quite a few non-pathological amyloidogenic proteins have also been identified and physiological properties have been ascribed, challenging previous implications that amyloids were always disease causing. This article summarises a great deal of current knowledge on the occurrence, structure, folding pathways, chemistry and biology associated with amyloidogenic peptides and proteins and highlights some key factors that have been found to influence amyloidogenesis.

Parkinson's disease: prospects for improved drug therapy.

L-Dopa has long been the mainstay of therapy for Parkinson's disease but its long-term shortcomings, principally uncoordinated, spasmodic or irregular movements (dyskinesias) and fluctuating control of motor symptoms (on/off fluctuations), are well documented. The postulated neuroprotective properties of L-deprenyl, often used as an adjunct to L-dopa, are under scrutiny and doubts have also been raised regarding its safety. Alternative therapeutic approaches are clearly needed. In this review, Jim Hagan, Derek Middlemiss, Paul Sharpe and George Poste outline some new approaches to treatment, with an emphasis on novel, selective dopamine receptor agonists. In addition, Parkinson's disease is commonly thought to be caused by the neurotoxic effects of an unidentified agent but recent data indicate a greater genetic component than previously recognized. Developments in the genetics of Parkinson's disease may provide the key to the next generation of therapeutics.

Glucose-induced oxidative stress in vascular contractile cells: comparison of aortic smooth muscle cells and retinal pericytes.

Free radical-mediated damage to vascular cells may be involved in the pathogenesis of diabetic vasculopathy. The aim of this study was to compare the extent of glucose-induced oxidative stress in both vascular smooth muscle cells (VSMCs) and pericytes and the effect on antioxidant enzyme gene expression and activities. Porcine aortic VSMC and retinal pericytes were cultured in either 5 or 25 mmol/l glucose for 10 days. Intracellular malondialdehyde (MDA) was measured as a marker of peroxidative damage, and mRNA expression of CuZn-SOD, MnSOD, catalase, and glutathione peroxidase (GPX) were measured by Northern analysis. Glutathione (GSH) was also measured. There was a significant increase in MDA in VSMCs in 25 mmol/l glucose (1.34 +/- 0.11 vs. 1.88 +/- 0.24 nmol/mg protein, 5 vs. 25 mmol/l D-glucose, mean +/- SE, n = 15, P < 0.01), but not in pericytes (0.38 +/- 0.05 vs. 0.37 +/- 0.05 nmol/mg protein, n = 11). There was a significant decrease in GSH in both cell types (VSMC, 1.40 +/- 0.13 vs. 0.69 +/- 0.12 nmol/mg protein, n = 15, P < 0.001; pericytes, 1.97 +/- 0.17 vs. 0.94 +/- 0.16 nmol/mg protein, n = 11, P < 0.001). mRNA expression of CuZnSOD and MnSOD was increased only in VSMCs (by 58.5 +/- 8.1 and 41.0 +/- 6.9%, respectively, n = 8, P < 0.01). CuZnSOD protein was increased by approximately 120% (P < 0.00001). None of the antioxidant enzyme activities was altered between 5 and 25 mmol/l glucose in either cell type. Both MnSOD activities and GSH concentrations were higher in pericytes compared with VSMC under basal (5 mmol/l) conditions (P < 0.05 and P < 0.02, respectively). These results demonstrate glucose-induced reduction of GSH in both cells, but only in VSMC is there evidence of oxidant damage in the form of lipid peroxidation, implying significant differences in intracellular responses to glucose between contractile cells in the macro- and microvasculature.

Two cases of inflammatory muscle disease presenting with raised serum concentrations of troponin T.

Troponins T and I are highly sensitive markers of myocardial injury. However, non-cardiac disorders, such as pulmonary embolism, renal failure, subarachnoid haemorrhage, sepsis, eclampsia, chemotherapy, and inflammatory muscle conditions (dermatomyositis and polymyositis), can also result in raised serum troponin concentrations. This article describes two cases that occurred within a month of each other in Craigavon Area Hospital, whereby conditions unrelated to myocardial ischaemia resulted in raised concentrations of cardiac markers. The first patient, in retrospect, underwent unnecessary investigation as an inpatient in the cardiac ward. Experience gained from this case led to more appropriate consultation and management of the second patient.

Increase in mean platelet volume in patients with chronic renal failure treated with erythropoietin.

AIMS: To assess whether r-HuEPO (recombinant human erythropoietin) has any effect on thrombopoiesis in patients with chronic renal failure. METHODS: This was a retrospective study of 78 patients with chronic renal failure undergoing either haemodialysis (n = 57) or intraperitoneal dialysis (n = 21). All patients had a full blood count (in EDTA) measured before starting r-HuEPO and at monthly intervals thereafter up to six months. Variables studied were haematocrit, platelet count, mean platelet volume (MPV) and platelet distribution width (PDW). Other groups of control patients were also studied--patients with chronic renal failure receiving dialysis but not r-HuEPO (n = 40) and a group of patients with normal renal function who were receiving aspirin (n = 30). RESULTS: There was a significant increase in mean haematocrit (p < 0.01) and in mean platelet volume (p < 0.001) over the six month period, but no change in either total platelet count or platelet distribution width in the patients with chronic renal failure receiving r-HuEPO. In contrast, both the control groups showed no significant change in MPV. CONCLUSIONS: The results suggest that r-HuEPO affects thrombopoiesis and may be part of a group of humoral factors contributing to megakaryocyte development and maturation. Larger platelets are more reactive and may contribute to the increased risk of thrombosis associated with r-HuEPO.

Biochemical markers of alcohol abuse.

Excessive alcohol consumption is a major health problem in the UK leading to both serious morbidity and mortality. This study compared newer potential biochemical markers of excessive alcohol consumption [carbohydrate-deficient transferrin (CDT), mitochondrial AST (mAST) and alpha glutathione-s-transferase (alpha-GST)] with conventional markers (AST, ALT, GGT, MCV). Patients (n = 85) were enrolled in the study and subdivided into several groups on the basis of alcohol consumption. Patients with non-alcoholic liver disease (NALD) (n = 40) were also enrolled. All the markers, with the exception of the ratio mAST/total AST were significantly higher in heavy drinkers/alcoholics compared to teetotallers/social drinkers (p < 0.05). mAST and AST/ALT ratio were significantly higher in alcoholics compared to NALD (p < 0.01), whereas ALT was higher in the NALD group (p < 0.05). Multivariate discriminant function analysis (Wilks method) demonstrated that the logarithmic functions of AST/ALT ratio and mAST could correctly classify 87.9% of cases into either the alcoholic or NALD groups. ROC plot analysis showed that AST, mAST and GGT were the best markers at distinguishing heavy consumption of alcohol from lesser levels and that AST/ALT ratio and mAST were the best in distinguishing alcoholics from NALD. In conclusion, none of the newer biochemical markers, with the exception of mAST, offers any major advantage over the conventional markers.

Effect of red wine consumption on lipoprotein (a) and other risk factors for atherosclerosis.

Epidemiological studies have pointed to the role of alcohol, and red wine in particular, in reducing the incidence of coronary heart disease. This study attempted to distinguish, in vivo, the effects of components specific to red wine and those of alcohol on lipoproteins, antioxidant status and membrane fluidity. Volunteers (n = 20) were given 200 ml of red wine per day for 10 days. Following a 6-week washout, this was repeated with white wine. Changes within treatment groups were analysed by paired t tests and repeated measures analysis of variance was used to distinguish effects of red wine components and alcohol. LDL was prepared by ultracentrifugation and all other assays were by conventional laboratory techniques. No effect with either treatment was detected on total cholesterol, triglycerides, HDL or measures of antioxidant status, including the susceptibility of LDL to oxidation. Red wine reduced LDL cholesterol (p < 0.01), and both treatments reduced LDL apo B (p < 0.01) and increased LDL chol:apo B ratio (p < 0.01), implying an increase in LDL size. Potential anti-atherogenic changes specific to red wine were reduction in lipoprotein (a) (p < 0.001) and increased membrane fluidity (p < 0.01). These results are not in keeping with the proposed role of red wine components in free-radical protection, but the reduction in lipoprotein (a) merits further investigation.

Total radical trapping antioxidant potential (TRAP) and exercise.

The relationship between physical activity, physical fitness and total radical trapping antioxidant potential (TRAP) was examined in the Northern Ireland Health and Activity Survey. This was a cross-sectional population study (n = 1600) using a two-stage probability sample of the population. TRAP was calculated using the sum of the individual serum antioxidant concentrations (urate, protein thiols, ascorbate, alpha tocopherol and bilirubin) multiplied by their respective stoichiometric values. Physical fitness was determined by estimation of VO2max by extrapolation from submaximal oxygen uptake, and physical activity was recorded by computer-assisted interview. Mean serum TRAP concentrations were significantly higher in males (653 +/- 8.2 mumol/l, mean +/- SEM) compared to females (564 +/- 8.0 mumol/l) (p < 0.0001). Both male and female smokers had significantly lower TRAP values than non-smokers (males p < 0.0001, females p = 0.02). In females, there was a positive relationship of TRAP with age (p < 0.001) and body mass index (p < 0.001) but a negative relationship with physical fitness (p < 0.05). The known beneficial effects of exercise and activity do not appear to be directly mediated through increased antioxidant status.

Biochemical detection and monitoring of alcohol abuse and abstinence.

The merits and limitations of traditional and new markers for alcohol abuse (and abstinence) are critically examined for detection and monitoring of alcoholics, hazardous drinkers and binge drinkers. The traditional markers discussed include gamma-glutamyltransferase (GGT), aspartate and alanine aminotransaminases (AST, ALT) and mean corpuscular volume (MCV); new markers include mitochondrial AST, carbohydrate-deficient transferrin (CDT), serum/urine 5-hydroxytryptophol, beta-hexosaminidase and acetaldehyde adducts. The strengths and weaknesses of several of the self-reporting screening questionnaires are also explored. No laboratory test is reliable enough on its own to support a diagnosis of alcoholism. Sensitivities and specificities vary considerably and depend on the population concerned. GGT continues to remain the test that combines greatest convenience and sensitivity: its diagnostic accuracy can be enhanced by combination with other traditional markers (AST, ALT, MCV). None of the newer markers offers significant advantage, although CDT seems to be better at monitoring patients for increased alcohol consumption or progress towards abstinence.

Ascorbate and exercise in the Northern Ireland population.

The relationship between physical activity, physical fitness and serum ascorbate was examined in the Northern Ireland Health and Activity Survey. This was a cross sectional population study (n = 1600), using a two stage probability sample, of the population of N. Ireland. Physical activity profile was recorded by computer assisted interview and physical fitness was determined by estimation of VO2 max by extrapolation from submaximal oxygen uptake. Serum ascorbate was measured using a colorimetric reaction of 2,4 dinitrophenylhydrazine with dehydroascorbate. Mean serum ascorbate was greater in females than males (p < 0.001), and was lower in smokers than non-smokers in both males (p = 0.01) and females (p < 0.001). There was no statistically significant relationship between serum ascorbate and age, social class, body mass index, physical activity or physical fitness in males but there was a relationship with age (p < 0.01) and physical fitness (p < 0.05) in females.

Ascorbate and malondialdehyde in stroke patients.

It is believed that free radical formation and subsequent oxidative damage in the form of lipid peroxidation may be a factor in the cerebral damage secondary to the ischaemia of a cerebrovascular accident (CVA). Total serum ascorbate and malondialdehyde (MDA) were measured in 45 patients with CVA on the day of admission to hospital (Time 0) and 48 hours later (Time 48 hours) and also in 45 age and sex matched controls. There was no statistical difference in total serum ascorbate between the control group (34.2 mumol/l +/- 3.1, mean +/- SEM) and the CVA patients at Time 0 (37.3 +/- 2.9) but there was a statistically significant decrease at Time 48 hours (22.7 +/- 2.0) (p < 0.001) in the CVA patients. With MDA there was no statistical difference between the patients at Time 0 (0.79 mumol/l +/- 0.06) and the control group (0.83 +/- 0.06) but there was a significant increase at Time 48 hours (1.65 +/- 0.08) (p < 0.001). These findings are in keeping with possible evidence of free radical damage in CVA.

Complications associated with central venous catheters in a haematology unit.

The use of central venous catheters in patients suffering from haematological disorders has brought enormous benefits, but has been associated with an increase in septicaemia. We have reviewed septic and other complications in 43 patients who received one of three different forms of central venous catheters (type A-Hickman, type B-Portacath, type C-Pasport) during 1991. All complications were reviewed up to 18 months following insertion. The total complication rate was 31% (0.97 per 100 catheter days), and the total sepsis complication rate was 18.8% (0.49 per 100 catheter days). Type A catheters had the greatest sepsis complication rate of 29.5% (0.84 per 100 catheter days), with type B 15% (0.39 per 100 catheter days) and type C 9.9% (0.32 per 100 catheter days). Prophylactic antibiotics on the day of catheter insertion did not reduce the sepsis rate or prolong catheter survival.

An audit of therapeutic drug monitoring of anticonvulsants.

An audit of therapeutic drug monitoring (TDM) of anticonvulsants was performed to assess both its use and misuse in the management of patients with epilepsy. Over a four week period all samples received for phenytoin, carbamazepine, sodium valproate and phenobarbitone assays were included in the audit. The aims were to establish the source of the specimens, the reasons for the requests and to ascertain what action, if any, would be taken when the result of the assay was provided. A total of 163 separate assays were performed over the four week period (43 phenytoin, 74 carbamazepine, 41 valproate, 5 phenobarbitone). Only 18.7% of all requests originated from the adult neurology department. The vast majority of tests had been ordered by junior medical staff (only 10% by consultants) and approximately 50% were 'routine' with no satisfactory clinical reason for the request offered. There was a tendency to manipulate prescribed doses on the basis of drug levels alone without taking the clinical picture into consideration. These results demonstrate a general ignorance, especially amongst junior medical staff, of the value of TDM of anticonvulsants, and reinforce the need for both an educative and interpretive service to be provided by the Chemical Pathology Department.

Paradoxical decreases in high-density lipoprotein cholesterol with fenofibrate: a quite common phenomenon.

BACKGROUND: Reduced high-density lipoprotein cholesterol (HDL-C) concentration is considered to be an independent risk factor for cardiovascular morbidity and mortality. Fibrates are useful in managing dyslipidaemia; reports highlight an expected increase in HDL-C of 10-15% in conjunction with falls in plasma triglycerides of approximately 30%. Despite this, there are several reported cases of paradoxical decreases in HDL-C caused by fibrate treatment. AIM: To report the second largest observational study to date. METHODS: Fenofibrate use at a regional lipid clinic was associated with reductions in HDL-C in a considerable proportion of patients, necessitating cessation of the medication. In view of this, characteristics of the first 94 patients to be given fenofibrate were retrospectively analysed, and comparisons were made between those whose profiles responded as expected and those experiencing paradoxical decreases in HDL-C. RESULTS: 94 patients (57 male; mean (SD) age 52.5 (12.5) years; mean (SD) body mass index 28.9 (4.5) kg/m2) were assessed. After 8-12 weeks on daily fenofibrate (200 mg micronised or equivalent), 43 of the patients (46%) showed a paradoxical decrease in HDL-C (in nine the decrease was >50% from baseline). When responses to fenofibrate were compared against baseline variables, there were no significant differences between groups other than a higher baseline HDL-C (p = 0.045) in patients responding appropriately. CONCLUSIONS: Fenofibrate was associated with a reduction in HDL-C in almost half the patients studied. This is substantially more than in most studies reported to date. Other HDL-C-raising strategies need to be considered in these patients, and the mechanisms need to be explored.

Investigation of a multimarker approach to the initial assessment of patients with acute chest pain.

Early identification of acute coronary syndrome (ACS) is important to guide therapy at a time when it is most likely to be of value. In addition, predicting future risk helps identify those most likely to benefit from ongoing therapy. Cardiac troponin T (cTnT) is useful for both purposes although cannot reliably rule out ACS until 12 hours after pain onset and does not fully define future risk. In this review article we summarize our previously published research, which assessed the value of myocyte injury, vascular inflammation, hemostatic, and neurohormonal markers in the early diagnosis of ACS and risk stratification of patients with ACS. In addition to cTnT, we measured heart fatty acid binding protein (H-FABP), glycogen phosphorylase-BB, high-sensitivity C-reactive protein, myeloperoxidase, matrix metalloproteinase 9, pregnancy-associated plasma protein-A, D-dimer, soluble CD40 ligand, and N-terminal pro-brain natriuretic peptide (NT-proBNP). Of the 664 patients enrolled, 415 met inclusion criteria for the early diagnosis of acute myocardial infarction (MI) analysis; 555 were included in the risk stratification analysis and were followed for 1 year from admission. In patients presenting <4 hours from pain onset, initial H-FABP had higher sensitivity for acute MI than cTnT (73% vs. 55%; P=0.043) but was of no benefit beyond 4 hours when compared to cTnT. On multivariate analysis, H-FABP, NT-proBNP, and peak cTnT were independent predictors of 1-year death/MI. Our research demonstrated that, in patients presenting within 4 hours from pain onset, H-FABP may improve detection of ACS. Measuring H-FABP and proBNP may help improve long-term risk stratification.

Alcohol increases homocysteine and reduces B vitamin concentration in healthy male volunteers--a randomized, crossover intervention study.

BACKGROUND: Few studies have examined the effect of alcohol consumption on total homocysteine (tHcy) concentrations. AIM: To assess the effect of an 8-week intervention with vodka or red wine on plasma tHcy and B vitamin concentrations in healthy male volunteers. To assess the effect on tHcy according to methylenetetrahydrofolate reductase (MTHFR) 677C>T genotype. DESIGN AND METHODS: A randomized controlled crossover intervention study measuring tHcy and serum folate and vitamin B(12) concentrations was conducted in 78 male subjects (21-70 years). Following a 2-week washout period during which no alcohol was consumed, all subjects consumed 24 g alcohol (either 240 ml red wine or 80 ml vodka)/day for a 2-week period. Following a further 2-week washout, participants consumed the alternate intervention for 2 weeks. RESULTS: A significant increase in plasma tHcy was observed after the 2-week red wine intervention (5%, P = 0.03), and a non-significant increase in tHcy with vodka intervention (3%, P = 0.09). When the two interventions were compared, the change in tHcy did not differ between the vodka and red wine interventions (P = 0.57). There were significant decreases in serum vitamin B(12) and folate concentrations, and this decrease did not differ between interventions. The increase in tHcy observed in both interventions did not vary by MTHFR 677C>T genotype. CONCLUSION: A 2-week alcohol intervention resulted in a decrease in folate and vitamin B(12) status and an increase in plasma tHcy. The effect of alcohol intervention on tHcy, folate and vitamin B(12) concentrations did not differ between the red wine and vodka intervention groups.

Mean platelet volume in diabetes mellitus.

We measured mean platelet volume (MPV) in patients with diabetes mellitus, compared with MPV in non-diabetic control subjects. Mean MPV was significantly increased in the diabetic subjects (8.9 +/- 0.07 fL, mean +/- SEM) compared with non-diabetic subjects (8.0 +/- 0.05) (p < 0.001). Since platelet size is a determinant of platelet function, with larger platelets being more reactive per unit volume, we believe platelets may play a part in the micro- and macro-vascular complications of diabetes mellitus.

C-substituted macrocycles as candidates for radioimmunotherapy.

The reaction between aryl aldehydes, the macrocyclic ligand 6-methyl-1,4,8,11-tetraazacyclotetradecane-6-amine (L1), and NaBH3CN produces the corresponding benzyl-substituted ligands in good yield. Copper(II) complexes of the ligands derived from salicylaldehyde (L2), p-hydroxybenzaldehyde (L4), and p-carboxybenzaldehyde (L5) were structurally characterized: [CuL2](ClO4)2.3H2O (monoclinic, P2(1)/c, a = 11.915(6) A, b = 13.861(2) A, c = 17.065(8) A, beta = 102.14(2) degrees, Z = 4); [CuL4](ClO4)2 (monoclinic, P2(1)/n, a = 9.550(3) A, b = 17.977(2) A, c = 14.612(4) A, beta 96.76(1) degrees, Z = 4), and [CuL4](ClO4)2 (monoclinic, P2(1)/n, a = 9.286(2) A, b = 11.294(1) A, c = 23.609(8) A, beta 93.68(1) degrees, Z = 4). Conjugation of several CuII complexes to a protein (bovine serum albumin) has been pursued with a view to the application of these macrocycles as bifunctional chelating agents in radioimmunotherapy.