Measurement of myocardial infarct size from plasma fatty acid-binding protein or myoglobin, using individually estimated clearance rates.

OBJECTIVE: In patients with acute myocardial infarction (AMI), estimation of infarct size from the early markers, fatty acid-binding protein (FABP) and myoglobin (MYO), usually assumes average (fixed) rate constants (FCR) for protein clearance from plasma. However, individual variation in FCR is large. Renal dysfunction causes slower clearance of FABP and MYO from plasma and, hence, overestimation of infarct size in 20-25% of patients. We investigated whether or not more accurate values of infarct size could be obtained with individually estimated clearance rates. METHODS: Concentrations of FABP and MYO and, for comparison, activities of the established cardiac markers, creatine kinase (CK) and alpha-hydroxybutyrate dehydrogenase (HBDH), were assayed in serial plasma samples from 138 patients with AMI. Individual FCR values of FABP and MYO were estimated from plasma creatinine concentrations, sex and age. RESULTS: Individual FCR values varied from 0.4 to 2.4 h-1. Use of these individual FCR values significantly improved the correlation between infarct size, as estimated from FABP or MYO on the one hand, and from CK and HBDH on the other. Approximately equal estimates of infarct size were obtained for all four marker proteins. CONCLUSIONS: Using individually estimated clearance rates, renal insufficiency no longer hampers calculation of infarct size from FABP and MYO, and reliable estimates of total myocardial damage can be obtained within 24 h after first symptoms.

Thrombolytic therapy does not change the release ratios of enzymatic and non-enzymatic myocardial marker proteins.

Measurements of cardiac marker proteins in plasma from patients with acute myocardial infarction (AMI) have become important in the evaluation of recanalization therapy. The validity of this approach has however been questioned, because it was claimed that coronary reperfusion may increase the recovery in plasma of cardiac enzymes, such as creatine kinase (CK). In the present study, possible effects of thrombolytic therapy on the release of enzymatic and nonenzymatic marker proteins were investigated. Activities of CK and lactate dehydrogenase (LDH), and concentrations of myoglobin (Mb) and fatty acid-binding protein (FABP) were determined in serial plasma samples obtained from 50 patients with confirmed AMI, of whom 36 received thrombolytic therapy, and 14 did not. Treatment delay was 2.8+/-1.6 (mean+/-SD) h, and hospital delay in untreated patients was 2.7+/-1.8 h. Average infarct size, expressed in gram-equivalents of heart muscle per litre of plasma (g-eq/l), varied between 5.5 and 7.2 g-eq/l for the four marker proteins in patients treated with thrombolytic therapy, and between 4.6 and 6.4 g-eq/l in untreated patients, with a tendency to larger infarct sizes for Mb and FABP than for CK and LDH. Thrombolytic therapy, although significantly accelerating protein release rates, did not influence the release ratios. These results indicate that thrombolytic therapy has no significant effects on the recovery of cardiac marker proteins in plasma.

Malignant neoplasms in cyclosporin A-induced autoimmunity (CyA-AI).

Lethally X-irradiated LEW rats reconstituted with syngeneic bone marrow and given low-dose Cyclosporine A (CyA) for 5 weeks develop, after withdrawal of CyA, symptoms of disease resembling graft-vs.-host disease (GVHD) as seen after allogeneic bone-marrow transplantation. Symptoms of disease may include acute dermatitis and chronic disease resembling scleroderma. Since anti-class II MHC cytotoxic lymphocytes are generated in this model, it has been proposed as an anti-tumor regimen in humans. We now report that LEW rats treated according to this protocol may, after cessation of CyA administration, paradoxically develop malignant neoplasms. Of 48 experimental animals, 31 developed rapidly progressive subcutaneous and/or intracutaneous tumors commencing at 6 weeks, 13 weeks and 6 months after cessation of CyA. Tumors were of mesenchymal origin, usually high-grade sarcoma, adenocarcinoma or both mesenchymal and epithelial tumors. Such tumor incidence exceeded the incidence of tumor growth in X-irradiated controls, and in rats subjected to thymectomy prior to X-irradiation and CyA administration. CyA by itself induced no tumors. Our results show that total body X-irradiation is required for tumor development but that the presence of CyA-induced autoimmune disease increases the incidence significantly.

One-step enzyme-linked immunosorbent assay (ELISA) for plasma fatty acid-binding protein.

To allow a more rapid determination of heart-type fatty acid-binding protein (FABP) concentration in plasma a direct non-competitive (sandwich-type) ELISA was developed which uses high-affinity monoclonal antibodies to FABP. Total performance time of the one-step immunoassay is 45 min. The standard curve was linear between 0.2-6 micrograms/L, and the within-run and between-run coefficients of variations were below 6 and 11%, respectively. The serum FABP concentration measured in 79 healthy individuals was 1.6 (0.8) [mean (SD), range 0.3-5.0] micrograms/L. The assay can be used for rapid plasma or serum FABP measurement in the early diagnosis of acute myocardial infarction.

Estimation of myocardial infarct size from plasma myoglobin or fatty acid-binding protein. Influence of renal function.

Myoglobin (M(r) 18,000) and fatty acid-binding protein (M(r) 15,000), are low molecular mass cytoplasmic proteins that are considered useful biochemical markers for early detection or exclusion of acute myocardial infarction, and also for early estimation of infarct size. As each of these proteins shows renal clearance, we studied the influence of renal function on the estimation of infarct size from their plasma concentration curves. For this, infarct size estimated from plasma myoglobin or fatty acid-binding protein release curves was compared with that estimated with the established infarct size markers hydroxybutyrate dehydrogenase and creatine kinase, which are not influenced by changes in renal function. The discordance between infarct size estimates was related to renal function. Creatine kinase (EC 2.7.3.2), hydroxybutyrate dehydrogenase (EC 1.1.1.27), myoglobin, fatty acid-binding protein and creatinine were assayed serially in plasma samples obtained frequently and for at least 72 hours after the start of thrombolytic therapy in 20 patients with acute myocardial infarction. Cumulative release of the different cardiac markers was calculated by using a two-compartment model for circulating proteins. Mean tissue contents of 156 U/g for hydroxybutyrate dehydrogenase, 2163 U/g for creatine kinase, 2.79 mg/g for myoglobin and 0.57 mg/g wet weight for fatty acid-binding protein, were used to express infarct size in gram-equivalents of healthy myocardium per litre of plasma (g-eq/l). Mean plasma creatinine was obtained by averaging the creatinine concentrations measured in all plasma samples taken during the first 24 hours after acute myocardial infarction. A relation was found between the mean plasma creatinine concentration during the first 24 hours after acute myocardial infarction and the discordance between infarct size estimated from cumulative hydroxybutyrate dehydrogenase release, compared to infarct size estimated from cumulative myoglobin or fatty acid-binding protein release. For patients with mean plasma creatinine concentrations within the reference interval for creatinine (group 1, n = 15) a good agreement was found between infarct size estimated from myoglobin or fatty acid-binding protein plasma curves and that estimated with either hydroxybutyrate dehydrogenase or creatine kinase. However, for patients with a mean creatinine concentration above the upper reference limit (group 2, n = 5), infarct size calculated from plasma myoglobin or fatty acid-binding protein release curves was markedly overestimated, especially for larger infarcts. Estimation of infarct size from serial plasma myoglobin or fatty acid-binding protein concentrations is possible in the first 24 hours after the onset of symptoms, but only in patients with normal renal function, as estimated from plasma creatinine concentrations.

Monoclonal antibodies to human heart fatty acid-binding protein.

Heart-type fatty acid-binding protein (H-FABP), a 15 kDa non-enzymatic protein, which is abundantly present in heart and some skeletal muscles, was recently found to be a useful plasma marker for acute myocardial infarction. The BIAcore biosensor technology was used to raise and characterize a panel of 13 monoclonal antibodies against human H-FABP which did not crossreact with other FABP types of human origin. The kinetics of association and dissociation between FABP and the monoclonal antibodies was studied. By pairwise mapping several distinct epitopes could be identified.

X-irradiation of the thymus is not required for the induction of cyclosporine-induced autoimmunity.

The thymus-dependent model of cyclosporine-induced autoimmunity (CsA-AI) in the Lewis rat requires a lethal total body X-irradiation and rescue with syngeneic or autologous bone marrow and cyclosporine (CsA) administration for at least 4 weeks; two to three weeks after cessation of CsA, the animals develop a graft-versus-host-like disease. The obligatory role of the thymus in the etiology of CsA-AI has been established unequivocally, but the way in which disease is thymus dependent is a topic of debate. In the present study we demonstrate that the model of CsA-AI requires the presence of a thymus for at least 2 weeks after total body irradiation and CsA administration, but that X-irradiation of the thymus itself is not necessary to bring about disease. Transplantation of neonatal thymus shows in addition that in the absence of X-irradiation of the thymus, CsA therapy is required to generate autoreactive cells, but that disease occurs only if peripheral autoregulatory cells are eliminated by X-irradiation.

Susceptibility and resistance to cyclosporin A-induced autoimmunity in rats.

Lethally irradiated Lewis (LEW) rats, reconstituted with syngeneic bone marrow and next given Cyclosporin A (CyA) for several weeks, develop disease (Cyclosporin A-induced autoimmunity; CyA-AI) after withdrawal of CyA. This disease resembles in terms of dermal changes the acute dermatitis and chronic scleroderma also seen in graft-versus-host disease (GVHD). In this study we report the relative resistance of the Brown Norway (BN) rat strain to the induction of CyA-AI. In contrast to LEW rats, in which CyA-AI was originally described, BN rats showed no acute dermatitis or scleroderma-like skin pathology in spite of comparable changes in the thymus and a maturation arrest of CD4+ T cells. The difference was also demonstrated functionally for whereas in LEW rats delayed-type hypersensitivity (DTH) reactions could not be elicited during CyA-AI, these were within normal limits in BN rats subjected to the same protocol; NK activity on the other hand was unaffected in both strains. The observation that BN rats developed very mild late disease as evidenced by a slight though significant weight loss suggests that the BN strain is susceptible to the disease but that lesser effector cell generation or, alternatively, stronger suppressor cell responses may prevent dermal disease. These observations may contribute to the elucidation of the mechanisms involved in this experimental autoimmune disease.

On the localization of effector cells in cyclosporin-induced autoimmunity.

Lethally irradiated rats, reconstituted with syngeneic bone marrow and given Cyclosporin A (CyA) for 6 weeks, developed disease resembling allogeneic graft-versus-host disease 2 weeks after withdrawal of CyA. Other studies have demonstrated the pivotal role of the thymus in the etiology of this CyA-induced autoimmune disease (CyA-AI). In this study the question was addressed whether inducer/effector cells of CyA-AI are generated in the thymus during or after CyA administration; whether these cells stay in the thymus, or, if they don't, whether they home to the secondary lymphoid organs. Adoptive transfer of thymocytes from donors treated for induction of CyA-AI obtained one and 14 days after cessation of CyA administration did not elicit CyA-AI in irradiated secondary recipients. Furthermore, adult thymectomy of rats immediately after the course of CyA did not influence the kinetics of development of skin pathology, although weight loss commenced later in thymectomized than in sham-thymectomized rats. Lymph node and spleen cells obtained from donors treated for induction of CyA-AI one and 14 days after withdrawal of CyA-AI caused CyA-AI upon adoptive transfer to secondary recipients, but the symptoms of acute disease (dermatitis, alopecia and weight loss) were strikingly less severe upon transfer of lymphoid cells obtained one day after stopping CyA than 14 days thereafter. Therefore, this study demonstrates that CyA-AI inducer/effector cells are generated in the thymus during the administration of CyA. These cells exit from the thymus during CyA administration; either they home predominantly peripherally (i.e. in the skin) rather than in the secondary lymphoid organs, or they leave the thymus as inducer cells which home in the lymphoid organs where they subsequently may trigger potentially autoreactive lymphocytes as probably also present in normal individuals, or both pathways may be operative.

Anti-56K: a novel, frequently occurring autoantibody specificity in connective tissue disease.

A novel frequently occurring autoantibody specificity in serum from connective tissue disease patients is described. The autoantibodies as detected by immunoblotting are directed against a 56,000 Dalton (56K) antigen, that after biochemical fractionation predominantly is found in the cytoplasmic fraction of various cell types and tissues. Attempts to localize the antigen more precisely were unsuccessful primarily because these antibodies do not produce a positive immunofluorescence pattern. The antigen in its native form is not associated with DNA or one of the common cytoplasmic or nuclear RNAs in the cell. Immunoprecipitation studies also showed that the protein is not closely associated with other proteins in a multi-component complex. Anti-56K antibodies are found in about 8% of patients with connective tissue disease, most commonly in patients with Sjögren's syndrome (14%). It is not found in patients with mixed connective tissue disease, polymyositis/dermatomyositis or scleroderma and rarely (less than 1%) in healthy control subjects. The fact that 22% of the anti-56K sera also contain La/SS-B antibodies support the idea that this antibody specificity might be characteristic for a subclass of Sjögren's syndrome patients.