The effect of aspirin on C-reactive protein as a marker of risk in unstable angina.

OBJECTIVES: This study was designed to assess the interaction between aspirin and C-reactive protein (CRP) release in unstable angina. BACKGROUND: C-reactive protein release in acute coronary syndromes may be a response to myocardial necrosis or may reflect the inflammatory process that drives atherogenesis. Aspirin has the potential to influence CRP release, either by its anti-inflammatory activity or by reducing myocardial necrosis. The clinical significance of this potential interaction has not previously been tested. METHODS: We conducted a prospective cohort study of 304 consecutive patients admitted with non-ST-elevation acute coronary syndromes. Serial blood samples were obtained for CRP and troponin I assay. End points were cardiac death and nonfatal myocardial infarction during follow-up for 12 months. RESULTS: A total of 174 patients (57%) were taking aspirin before admission. Patients taking aspirin had lower troponin I concentrations throughout the sampling period, only 45 (26.0%) having concentrations >0.1 mg/l compared with 48 (37.8%) patients not taking aspirin (p = 0.03). Maximum CRP concentrations were also lower in patients taking aspirin (8.16 mg/l [3.24 to 24.5]) than in patients not taking aspirin (11.3 mg/l [4.15 to 26.1]), although the difference was not significant. However, there was significant interaction (p = 0.04) between prior aspirin therapy and the predictive value of CRP concentrations for death and myocardial infarction at 12 months. Thus, odds ratios (95% confidence intervals) for events associated with an increase of 1 standard deviation in maximum CRP concentration were 2.64 (1.22-5.72) in patients not pretreated with aspirin compared with 0.98 (0.60-1.62) in patients pretreated with aspirin. CONCLUSIONS: The association between CRP and cardiac events in patients with unstable angina is influenced by pretreatment with aspirin. Modification of the acute-phase inflammatory responses to myocardial injury is the major mechanism of this interaction.

Interaction between smoking and the glycoprotein IIIa P1(A2) polymorphism in non-ST-elevation acute coronary syndromes.

OBJECTIVES: The goal of this study was to determine the interaction between smoking and the glycoprotein IIIa P1(A2) polymorphism in patients admitted with non-ST-elevation acute coronary syndromes (ACS). BACKGROUND: An increased incidence of the P1(A2) polymorphism in smokers presenting with ST-elevation acute myocardial infarction (AMI) has recently been reported. We, therefore, postulated that, as a consequence of this interaction, fewer smokers with the P1(A2) polymorphism would present with non-ST-elevation ACS. METHODS: We performed a prospective cohort analysis of 220 white Caucasoid patients admitted with non-ST-elevation ACS fulfilling Braunwald class IIIb criteria for unstable angina who were stratified by smoking status. RESULTS: There were twice as many nonsmokers as smokers. Nonsmokers compared with smokers were older (mean [SD]; 63.9 [11.2] vs. 57.6 [10.3]; p < 0.0001), more likely to have had a previous admission with unstable angina (24.3% vs. 13.2%; p = 0.051) and AMI (45.8% vs. 30.3%; p < 0.026), more likely to have undergone revascularization (24.3% vs. 1.8%; p = 0.028) and were more likely to be on aspirin on admission (60.4% vs. 44.7%; p = 0.026). The proportion of nonsmokers positive for the P1(A2) polymorphism was equivalent to that expected for this population but was significantly reduced in smokers (28.7% vs. 10%; Pearson chi-square = 9.09, p = 0.0026). In a logistic regression model, the odds ratio (OR) for being positive for the P1(A2) polymorphism was significantly reduced by smoking (OR [interquartile range]: 0.26 [0.11 to 0.62]; p = 0.0026). CONCLUSIONS: There is a significant reduction in the P1(A2) polymorphism in smokers admitted with non-ST-elevation ACS compared with nonsmokers, which suggests an interaction between smoking and this polymorphism.

Angiotensin-converting enzyme inhibition is associated with reduced troponin release in non-ST-elevation acute coronary syndromes.

OBJECTIVES: This study was done to determine the effects of angiotensin-converting enzyme (ACE) inhibition and other clinical factors on troponin release in non-ST-elevation acute coronary syndrome (ACS). BACKGROUND: Troponin is now widely used as a marker of risk in ACS, but determinants of its release have not been defined. METHODS: This was a prospective cohort study of 301 consecutive patients admitted with non-ST-elevation ACS. Baseline clinical data were recorded, ACE gene polymorphism was determined and serial blood samples were obtained for troponin-I assay. RESULTS: Significant troponin-I release (>0.1 microg/l) was detected in 93 (31%) patients. Pretreatment with ACE inhibitors, recorded in 53 patients (17.6%), independently reduced the odds of troponin-I release (odds ratio 0.25; 95% confidence intervals 0.10 to 0.64) and was associated with lower maximum troponin-I concentrations (median [interquartile range]) compared with patients not pretreated with ACE inhibitors (0.44 microg/l [0.19 to 2.65 microg/l] vs. 4.18 microg/l [0.91 to 12.41 microg/l], p = 0.01). Pretreatment with aspirin, recorded in 173 patients (57.5%), did not significantly reduce the odds of troponin-I release after adjustment but was associated with lower maximum troponin-I concentrations compared with patients not pretreated with aspirin (2.31 microg/l [0.72 to 8.02 microg/l] vs. 5.85 microg/l [1.19 to 12.79 microg/l], p = 0.05). The ACE genotyping (n = 268) showed 81 patients (30%) DD homozygous and 77 (29%) II homozygous. There was no association between ACE genotype and troponin release. CONCLUSIONS: We conclude that ACE inhibition reduces troponin release in non-ST-elevation ACS. This is likely to be mediated by the beneficial effects of treatment on vascular reactivity and the coagulation system.

Recognition of pertussis toxin by antibodies to synthetic peptides.

Eight synthetic peptides, selected from the amino acid sequence of pertussis toxin (PT) subunits S1, S2, S3 and S4, were assessed for their ability to induce protein-recognizing and neutralizing antibodies. Seven of these peptides, prepared as conjugates of either keyhole limpet haemocyanin or tetanus toxoid, induced significant levels of antibody, all of which reacted with SDS-denatured PT on Western blots. Six of the antibodies bound to PT-coated ELISA plates; this binding was inhibited by homologous peptide antigen. However, none of the antibodies, including those directed against the N-terminus of subunit S1, were able to attenuate in vivo or in vitro toxin-dependent activity. Further investigation revealed that only one antibody, specific for the C-terminus of S1 (peptide Slc, 237-255), could recognize the conformation of native PT in solution. The other five antipeptide antibodies which reacted with PT-coated ELISA plates did not recognize PT when captured onto ELISA plates via either a monoclonal antibody or fetuin, unless the conformation of the toxin had been relaxed by reduction with dithiothreitol. Conversely, the native PT-recognizing response of peptide Slc did not bind the conformationally relaxed PT molecule. From this study, it appears likely that a peptide capable of inducing PT-neutralizing antibody must closely resemble the conformation of the cognate sequence in the native protein.

A longitudinal study of markers of bone turnover in Graves' disease and their value in predicting bone mineral density.

Whether biochemical markers can predict improvement in reduced bone mineral density (BMD) associated with thyrotoxicosis in unclear. We investigated the relationship between serum osteocalcin (OC), bone-specific alkaline phosphatase (b-ALP), serum deoxypyridinoline (Sdpd) and pyridinoline (Spyr), 24-hour urinary deoxypyridinoline (Udpd), and BMD in 17 thyrotoxic patients during 1 yr of treatment. Coinciding with euthyroidism at 4-8 weeks, there was a peak in b-ALP and OC and a prompt fall into the normal range in Udpd and Sdpd, but not Spyr, levels. Mean b-ALP continued to be raised at week 52 when it was inversely correlated with BMD. Mean BMD rose approximately 6%, P < 0.01, over 1 yr. Coupling indices were calculated as a measure of bone balance and, at diagnosis, was [minus4.26 in favor of bone resorption and rose with treatment in favor of bone formation: weeks 2: -0.23; 4: +4.01; 8: +4.37; 12: +4.44; 24: +2.32; and 52: +1.56. Bone turnover is balanced within 2 weeks of starting treatment for thyrotoxicosis. Udpd accurately indicates thyrotoxic bone resorption. Serum b-ALP indicates continuing bone formation and, at 1 yr, may provide a marker for low BMD. OC, Sdpd, and Spyr are less sensitive in documenting bone remodeling during treatment of thyrotoxicosis.

Evaluation of a commercial immunoenzymometric assay for human prolactin.

A commercially available immunoenzymometric assay for human prolactin based on two monoclonal antibodies, one bound to a solid-phase bead and the other conjugated to alkaline phosphatase, was evaluated. The assay had a lower limit of detection of 2 ng/ml and between-batch coefficients of variation of less than 10% for prolactin concentrations in the range 7-152 ng/ml. Assay accuracy as judged by comparison of patient samples analysed by a RIA, assay of quality control samples and recovery experiments, was good. No significant cross-reactivities or interferences were found. The assay is precise, accurate, fast and robust with the advantage of using a non-isotopic label.

Evaluation of fluorescence excitation transfer immunoassay for measurement of specific proteins.

Fluorescence excitation transfer immunoassay is a suitable technique for the measurement of serum IgG and CRP. The reagents, once reconstituted, are stable for at least 3 months. The method shows no interference due to bilirubin, lipaemia or haemolysis up to high levels. The assay is simple to perform, reliable and offers considerable advantages over manual techniques such as radial immunodiffusion or electroimmunoassay.

Development and validation of a particle-enhanced turbidimetric immunoassay for C-reactive protein.

The development of a particle-enhanced turbidimetric immunoassay for C-reactive protein is described. The method demonstrates excellent precision, with the calibration curve remaining stable for at least 16 weeks. The method compares well with established techniques and there is no interference from a variety of autoantibodies, endogenous serum constituents or commonly used drugs.

The matrix effects on kinetic rate constants of antibody-antigen interactions reflect solvent viscosity.

This study describes the influence of different matrices on two model antibody-antigen interactions; that between beta2microglobulin and anti beta2microglobulin, and that of rabbit anti mouse Fc fragment (RAMFc) with mouse IgG. The matrices investigated were; phosphate-buffered saline pH 7.4 containing 0.05% Tween 20 detergent, horse serum, a 50:50 mixture of phosphate-buffered saline/Tween 20 and horse serum, and four glycerol solutions of differing concentrations. A recently developed optical biosensor, the IAsys, was used to monitor the interactions in real-time and provide precise determinations of k(ass), k(diss) and KA values. The results show that the rates of association and dissociation for the two different antibody:antigen models are significantly affected by the surrounding matrix. Glycerol of known viscosity was used as a matrix in both models to show that this effect is attributable to the viscosity as opposed to proteins present in the matrix. The viscosity of the matrix has also been shown to have an apparent influence upon the overall equilibrium/affinity constant for the interaction, with measurements of KA tending to increase with viscosity. The significant effects of matrix on kinetic rate constants for antibody-antigen interactions shown here have important implications in the use of immunoassays where non-equilibrium measurements are made in serum matrices.

A rapid and robust particle-enhanced turbidimetric immunoassay for serum beta 2 microglobulin.

A rapid particle-enhanced turbidimetric immunoassay (PETIA), for the measurement of serum beta 2-microglobulin is described. The method has a working range of 0.2-40 mg/l, with good precision and a correlation coefficient of 0.97 when compared with an established radioimmunoassay method. One of the major advantages of this assay is the stability of the calibration curve (up to at least 20 months). This, and the fact that no pretreatment of serum samples is necessary, makes the assay ideally suited for all types of routine determination.

Soluble CD8 stabilizes the HLA class I molecule by promoting beta2M exchange: analysis in real-time.

Human soluble CD8 (sCD8) is secreted by activated CD8+/- cytotoxic T lymphocytes (CTLs). The immunological role of sCD8 is poorly defined, however. We have studied the influence of sCD8 on HLA class I interactions by real-time analysis. Using an optical biosensor we demonstrated that the binding of sCD8 to HLA-A2 promotes exchange of beta2-microglobulin (beta2m) in order to stabilize the complex. Kinetic analysis showed that sCD8 significantly increased the affinity (K(A)) of HLA-A2 for immobilized human beta2m; from 1.14 +/- 0.04 x 10(9) M(-1) in its absence, to 2.18 +/- 0.21 x 10(9) M(-1) following preincubation with sCD8. This suggests that the sCD8:HLA class I complex is unlikely to be degraded at the cell surface. Even in the presence of exogenous peptide (HLA-A2 specific or nonspecific), sCD8 has a stabilizing influence on the HLA class I molecule. These findings point to an immunosuppressive role for sCD8, because the binding of sCD8 to HLA class I would block the binding site for CTL-bound CD8 and, therefore, interfere with T cell activation and proliferation. This may have particular significance in pathological situations where elevated levels of sCD8 are found in extracellular fluids, and sCD8 may provide an alternative approach for immunosuppressive therapy.

An interpretation of the serum alkaline phosphatase isoenzyme patterns in patients with obstructive liver disease.

Earlier studies have identified two main isoenzymes of alkaline phosphatase in the sera of patients with obstructive liver disease. This paper reports on a study of these isoenzymes in specific types of liver disease where the pathology in relation to bile duct obstruction is known. The results have been used to support the theory that in biliary obstruction the increase in serum alkaline phosphatase is in part due to regurgitation of the biliary isoenzymes.

The nature of the serum alkaline phosphatases in liver diseases.

The two main alkaline phosphatase isoenzymes in serum from patients with extrahepatic biliary obstruction have been purified. Properties of these isoenzymes, such as electrophoretic mobility, separation on gel filtration, ultracentrifugation characteristics, Michaelis constants, and sensitivity to neuraminidase have been studied and compared with the isoenzymes of liver and bile. The results show that there is an abnormal isoenzyme in the serum from these patients and that this isoenzyme is similar but not identical with the main bile isoenzyme. It is suggested that it may be associated with a lipoprotein carrier.

Quality assessment of blood glucose monitors in use outside the hospital laboratory.

The quality of analytical results required in clinical practice is generally dictated by analytical rather than clinical criteria. The increasing availability of analytical technology that can be used outside the hospital laboratory challenges this situation. In an attempt to highlight this dilemma one method of glucose analysis outside the laboratory has been assessed using a pilot external quality assessment scheme (EQAS) and the results have been compared with the performance of the same machines in the laboratory. In the laboratory reliable results were obtained when the Glucometer was used by experienced laboratory personnel giving a good correlation of results (r = 0.96) when compared with an automated method. The results of the EQAS revealed that 44% of hospital ward glucose estimations and 63% of general practice estimations would be considered unsatisfactory by conventional laboratory criteria--that is, greater than +/- 2 SD of the mean laboratory result. This level of performance must, however, be considered in relation to the number of times that a clinically misleading result is obtained. No analytical system should be used in clinical practice without a continuous, objective assessment of its performance.

Serum gamma-glutamyltransferase isoenzymes in extrahepatic biliary obstruction.

The gamma-glutamyltransferase isoenzymes in the sera of patients with extrahepatic biliary obstruction have been studied, using electrophoretic, gel filtration, and ultracentrifugation techniques, and compared with those present in normal sera. Five isoenzymes were shown to exist in patients' sera, three of which were not demonstrated in normal sera. The observations are discussed in relation to the influence of biliary regurgitation and the possible solubilisation of membrane-bound enzymes. The results are compared with those of previous studies on alkaline phosphatase.

gamma-Glutamyl transferase isoenzymes in human bile.

The gamma-glutamyl transferase isoenzymes of bile were studied using electrophoretic, gel filtration, and ultracentrifugation techniques. In view of the known association of other biliary enzymes with lipids the effects of butanol extraction were investigated. The results show the presence of four isoenzymes of gamma-glutamyl transferase in bile, differing in electrophoretic mobilities, molecular size, and density. The correlation between the properties of biliary gamma-glutamyl transferase and of alkaline phosphatase is discussed.

The nature of the alkaline phosphatases of bile.

The main alkaline phosphatase isoenzymes of human bile have been purified by DEAE-cellulose chromatography. Characteristics of the isoenzymes, such as electrophoretic mobility before and after butanol extraction, Michaelis constant, and change in electrophoretic mobility following exposure to neuraminidase have been studied and compared with isoenzymes from other sources. The results show that the main alkaline phosphatase of bile is derived from the liver. It is present as a protein phosphatidylcholine complex.

Evidence based practice: clinicians' use and attitudes to near patient testing in hospitals.

AIM: To survey the use made of laboratory services for urgent tests and clinicians' attitudes to near patient testing. METHODS: A questionnaire was sent to clinicians working in acute hospitals within Trent and North West Thames Regions. RESULTS: 197 replies were received. Most demand came from intensive care units. Overall, clinicians requested a median of six urgent tests a day. Blood glucose and dip stick urine testing were the most commonly performed bedside tests, but 41% of clinicians did not use ward testing. The most frequently cited indication for bedside testing was the need for speed. 85% of clinicians trusted results obtained in their central hospital laboratory, but there was an almost equal division between those who did (34%) and those who did not (38%) trust the results from near patient testing. A slightly larger proportion indicated they would accept responsibility (44%) for results obtained on the ward than would not (35%). Most staff indicated that better transport to the laboratory would remove the need for near patient testing. CONCLUSIONS: Clinicians have demonstrated an apparent need for rapid response testing but there is a strong preference for rapid transport systems and central laboratory analysis rather than bedside testing as a solution to this problem. There is a need to investigate the clinical and cost-effectiveness of near patient testing as a solution to rapid response testing.

Lessons for the laboratory from a general practitioner survey.

AIMS: To assess the current performance of the clinical biochemistry service provided to general practitioners, with particular attention to result turnround times, and to identify and improvements required. METHODS: Postal questionnaire survey of general practitioners in the London Borough of Tower Hamlets who used the clinical biochemistry laboratory of the Royal London Hospital. A flow analysis study of turnround times for general practitioner samples was also performed. RESULTS: Responses to the questionnaire showed that although 82% of general practitioners thought the current quality of service provided was better than fair, the actual turnround times achieved were longer than the acceptable times required. There was also a strong demand (> 66% of responders) for additional information-such as highlighting of abnormal results-to be provided with results. There was wide variability between practitioners in their use of the laboratory (from none to > 800 requests per year), with no apparent correlation to practice size. Of the repertoire of tests requested, a surprisingly high percentage (14.3%) were for thyroid function. Flow analysis of turnround times for thyroid function tests showed that problems lay not with the time taken for analysis (only 7.8% of the total turnround time) but with the pre- and postanalytical phases, that is, the sample collection and results delivery service. CONCLUSIONS: Increasing the proportion of health care delivered in the primary care sector will inevitably increase the requirement for pathology services. Improvements in the specimen collection and results delivery service to general practitioners are needed to meet their expectations. It remains to be determined whether increased investment in these aspects of laboratory service would result in improved patient care in the primary sector.

Circulating MMP9, vitamin D and variation in the TIMP-1 response with VDR genotype: mechanisms for inflammatory damage in chronic disorders?

BACKGROUND: Vitamin-D deficiency and vitamin-D receptor genotype (VDR) are risk factors for several disorders with inflammatory components, including coronary heart disease (CHD) and diabetes, though the mechanisms involved are unclear. AIM: To examine the hypothesis that vitamin D status modulates the matrix metalloproteinase (MMP) system in a population with a high prevalence of vitamin D deficiency, a situation affecting susceptibility to CHD and diabetes. DESIGN: Prospective cross-sectional, interventional and embedded studies. METHODS: Circulating MMP2,9, the inhibitor TIMP-1 and C-reactive protein (CRP) were measured during studies of vitamin-D deficiency as a risk factor for type 2 diabetes and CHD in 171 healthy British Bangladeshi adults, free of known diabetes or major illness. Vitamin D status, VDR genotype, body-build, blood pressure, lipid and insulin profiles, glucose tolerance, fibrinogen, PAI-1, folate and homocysteine were measured. Vitamin-D-deficient subjects were re-assessed after 1 years' supplementation. MMP, TIMP-1 and CRP levels were measured in 41 subjects halfway through 5-year follow-up. Independent determinants of circulating concentrations of MMP9, TIMP-1 and CRP were assessed by multiple regression analysis. RESULTS: Vitamin D status was the sole determinant of circulating MMP9 (inversely) and an independent determinant of CRP (inversely). Determinants of TIMP-1 were MMP9, systolic blood-pressure (directly) and VDR genotype (TaqI). Significant reductions in MMP9 (-68%), TIMP-1 (-38%) and CRP (-23%) concentrations followed vitamin-D supplementation. DISCUSSION: Vitamin-D insufficiency is associated with increased circulating MMP2,9 and CRP, correctable by supplementation. This finding provides a possible mechanism for tissue damage in chronic inflammatory conditions, including CHD and diabetes.