Creatine kinase-MB mass concentration versus creatine kinase-B activity for the detection of acute myocardial infarction in patients with slightly elevated total creatine kinase activity in serum.

OBJECTIVE: The diagnostic value of creatine kinase-MB mass concentration (CK-MB mass) was compared with that of creatine kinase-B (CK-B) activity in patients with suspected acute myocardial infarction (AMI) but with total serum CK activity only slightly above the reference range. DESIGN: One hundred consecutive blood samples with total CK activity between 120 and 360 U l-1 and CK-B activity > or = 9 U l-1 were analysed. Electrophoresis of CK isoenzymes was also performed. SETTING: Patients from all departments of the hospital were included. About half of the patients originated from the coronary care unit. SUBJECTS: The blood samples derived from 49 patients. Thirteen patients had at least one serum sample with total CK activity above 360 U l-1. These and another three patients were omitted from the study. RESULTS: Acute myocardial infarction had been diagnosed clinically (with CK and CK-B methods) in 12 of 33 patients. However, using the CK-MB mass concentration of the reference method, five of these 12 patients did not have myocardial infarction whereas nine patients with small infarctions were undetected. A good correlation was seen between the results from CK-MB mass concentration and CK isoenzyme electrophoresis, but there was a poor correlation between these methods and CK-B activity including the CK-B/CK ratio. A relatively high proportion (24%) of the selected patients had increased levels of macro CK. CONCLUSION: CK-B activity was inaccurate for the detection of probably myocardial infarction in patients with slightly elevated total CK activity. Increased levels of macro CK interfering with the CK-B assay was one explanation for this observation.

Recurrent transient hyperphosphatasemia of infancy in an adult.

We report a case of recurrent transient hyperphosphatasemia in a 29-year-old man with immune deficiency. He had serum alkaline phosphatase (ALP; EC 3.1.3.1) activity 16.9- and 4.8-fold greater than the upper reference limit on two occasions; the activity returned to normal within 2 months on the first and within 1 month on the second. On both occasions we observed the typical electrophoretic pattern for ALP isoenzymes seen in transient hyperphosphatasemia of infancy. We noted no evidence of liver or bone disease. Recognition of the occurrence of transient hyperphosphatasemia of infancy in adults, although rare (it is the fifth case reported), seems as important as in children so that unnecessary extensive investigations are avoided.

Local immunoglobulin production in synovial tissue in patients with Lyme borreliosis.

A study was made to find out whether immunoglobulins are produced locally in synovial tissue in patients with Lyme borreliosis. Synovial fluid specimens from six patients with Lyme borreliosis were compared with those from 25 patients with rheumatoid arthritis, psoriatic arthritis, unspecified oligoarthritis or arthrosis (control group). Agarose electrophoresis revealed local oligoclonal IgG and IgM bands in the synovial fluid of two patients with Lyme borreliosis, but no local bands were observed in the control group. An index for local synthesis of immunoglobulins in synovial fluid was calculated in analogy with the IgG index for cerebrospinal fluid. The two patients with Lyme borreliosis in whom oligoclonal bands were seen in the synovial fluid showed the highest synovial fluid IgG indices and the highest concentrations of specific IgG antibodies against Borrelia spirochetes in synovial fluid. The presence of local oligoclonal immunoglobulin bands and a high synovial fluid IgG index suggest that immunoglobulins are produced locally within the synovial tissue in some patients with Lyme borreliosis. The increase in immunoglobulins may be a response to a local invasion of Borrelia spirochetes or may represent an immune reaction which continues after the spirochetes no longer are viable.

Further characterization of a heat-stable alkaline phosphatase with low sensitivity to L-phenylalanine.

A heat-stable alkaline phosphatase, hitherto found in two families with inherited hyperphosphatasemia, was further characterized. The enzyme was similar to serum placental alkaline phosphatase from pregnant women concerning its apparent affinity constant (Km) for 4-nitrophenyl phosphate and its reactivity with H7 monoclonal anti-placental alkaline phosphatase (PLAP) antibodies, but different in the following respects: it exhibited greater heat stability, a higher pH optimum, lower sensitivity to inhibition by L-phenylalanine, and no reactivity with C2 monoclonal anti-PLAP antibodies. The low sensitivity to L-phenylalanine suggests that the enzyme might correspond to a rare phenotype of placental alkaline phosphatase found in human term placenta.

Inherited occurrence of a heat stable alkaline phosphatase in the absence of malignant disease.

We describe a family with an inherited persistent elevation of serum alkaline phosphatase activity in the absence of malignant disease, observed for at least 15 yr. Isoenzyme studies revealed that this increased activity was due to an enzyme which showed similarities to serum placental alkaline phosphatase from pregnant women having the following properties: high heat stability; reactivity to anti-placental alkaline phosphatase antiserum; lack of inhibition by L-homoarginine; moderate inhibition by EDTA; and lack of interaction with wheat germ lectin. The enzyme was less sensitive than placental alkaline phosphatase to inhibition by L-phenylalanine, L-tryptophan, L-leucine, L-leucyl-glycyl-glycine and L-phenylalanyl-glycyl-glycine. The enzyme also differed from the placental alkaline phosphatase in its electrophoretic mobility, isoelectric heterogeneity and apparent molecular mass. We conclude that the enzyme is an inherited heat stable alkaline phosphatase variant which might correspond to a rare phenotype of placental alkaline phosphatase.

Possible errors in the calculation of cell-binding parameters exemplified by the analysis of the IgG-Fc receptor system.

The binding parameters of monomeric and polymeric (immune complex with a molecular weight of 500,000 daltons) rabbit IgG to homologous Fc receptor-bearing alveolar macrophages were estimated, using corrected values for IgG and cell concentrations. Considering the maximum percentage of monomeric IgG binding to cells (2.7%) and the maximum percentage of cells binding monomeric IgG (32%) instead of the IgG and cell concentrations in the initial reaction mixture, a 36-fold increase of the equilibrium constant (K) (from 0.6 x 10(6) L/M to 21.3 x 10(6) L/M) and a 3-fold increase of the maximum number of IgG molecules able to bind to a single cell (n) (from 7.8 x 10(5) to 23.7 x 10(5] were registered. Since more than 60% of the polymeric IgG is bound to 46% of the macrophage population, the values of K (from 10.8 x 10(6) L/M to 15.6 x 10(6) L/M) and n (from 4.3 x 10(5) to 9.4 x 10(5] are only doubled by using the corrected values for IgG and cell concentrations. It results that the cytophilic fraction of the monomeric IgG, representing only 2.7% of total IgG, has a slightly higher affinity for the Fc receptors than the polymeric IgG. By considering the actual number of macrophages which bind IgG it is evident that the number of Fc receptors per cell is higher than that determined by the usual procedure which does not take into account cellular heterogeneity.

Microheterogeneity of serum alkaline phosphatase isoenzymes as revealed by isoelectric focusing.

The microheterogeneity of human serum alkaline phosphatase (ALP) was investigated by means of isoelectric focusing. Liver and bone isoenzymes focused in a similar pattern, with about 10 bands located between pH 3.7 and 4.9, but differed in the relative intensity of the various bands. Intestinal ALP exhibited 7 to 8 bands at pH 4.9-5.1, and the placental enzyme showed 2 to 3 bands at pH 4.9. Mild digestion with neuraminidase revealed that the banding of liver and bone isoenzymes was at least partly due to differences in the sialic acid content of the various fractions. Extensively desialylated liver and bone isoenzymes showed apparently identical patterns with 6 to 7 bands focused at pH 6.2-6.7. Isoelectric focusing is a useful method for characterizing the microheterogeneity of alkaline phosphatase isoenzymes. The clinical value of this method seems to be limited, however, since it did not distinguish between liver and bone isoenzymes and failed to detect 'specific' isoelectric fractions correlated to various diseases.

Characterization of serum alkaline phosphatase isoenzymes by affinity electrophoresis in agarose gel containing lectin combined with agar gel electrophoresis.

The combined use of affinity electrophoresis in agarose gel containing lectin and of agar gel electrophoresis for the quantitation of liver, bone, biliary and intestinal alkaline phosphatase isoenzymes is described. Sera from patients with various diseases and from normal subjects (blood donors) have been analyzed. Data from normal subjects show that the bone isoenzyme is the predominant fraction (about 62%) in adults. The relative proportions of the alkaline phosphatase isoenzymes are similar in both sexes in adulthood (21-50 years). The higher alkaline phosphatase activity found in men than in women (ages 21-50 years) is due to higher values for both liver and bone isoenzymes. The difference between men and women tends to decrease after the age of 50 mainly due to an increase of the bone isoenzyme in women.

Affinity electrophoresis of human serum alkaline phosphatase isoenzymes in agarose gel containing lectin.

Separation of alkaline phosphatase isoenzymes using affinity electrophoresis in agarose gel containing lectin is described. The bone and biliary isoenzymes precipitate during electrophoresis and are clearly separated from the liver isoenzyme. The liver, intestinal and placental alkaline phosphatases are essentially not affected by the lectin. The migration distances of the precipitating bone and biliary fractions vary with their alkaline phosphatase activity. The bone isoenzyme is more heterogeneous than the biliary isoenzyme with respect to interaction with lectin forming both insoluble and soluble complexes. Affinity electrophoresis in agarose gel containing lectin can be used for quantitation by densitometry of liver and bone isoenzymes in sera containing only these two fractions but must be combined with conventional electrophoresis, preferably in agar gel, if biliary, intestinal, or placental isoenzymes are also present.

Antibodies from patients with liver diseases and from normal human or animal sera against glutaraldehyde-polymerized albumins: lack of species specificity.

By the use of glutaraldehyde-polymerized albumins of different species (human, rabbit, bovine, goat and mouse) it was demonstrated that anti-albumin antibodies in sera of patients with liver diseases and in normal human and animal (rabbit, mouse) sera are not species specific.

Immunogenicity and effector functions of glutaraldehyde-treated rabbit and mouse immunoglobulin G.

Rabbit and mouse IgG treated with glutaraldehyde (GA) were immunogenic in homologous species. Glutaraldehyde treatment induced in the IgG molecule two types of antigenic determinants. One of them was found on the monomeric fraction of GA-treated rabbit IgG (haptenic determinant) and the other on the polymeric fraction (structural determinant). The haptenic determinants were found also on monoaldehyde-treated rabbit IgG and GA-treated Fab and Fc fragments. It was demonstrated that rabbit and mouse antibodies are specific for GA-treated IgG and have species specificity. While GA treatment did not alter the antigen binding capacity of rabbit IgG antibody, its effector functions (except protein A binding) were much affected. Thus it was found that GA treatment enhances IgG ability to react with rheumatoid factor, reduces drastically its capacity to activate the complement system, abolishes the cytophilic properties of IgG and accelerates its catabolic rate. The possible blocking effect of GA on the amino acid residues (mainly Lys) situated in or very close to the effector sites of the IgG molecule is suggested.

C1q has albumin binding activity but does not interfere in the hemagglutination test for anti-albumin antibodies.

Two factors in the sera of patients with liver disease interact with polymerized human serum albumin (HSAP). These are anti-albumin antibodies (AAA) and receptors for polymerized albumin on HBsAg. Recently it was demonstrated by radioimmunoassay that purified human C1q also binds HSAP. Our data confirm the reaction between purified human C1q and HSAP by passive hemagglutination and by indirect immunofluorescence of HSAP-coated erythrocytes (E-HSAP). It is shown that although purified C1q binds HSAP, in the serum, the AAA and not C1q are responsible for the albumin binding activity in HBsAg-negative sera of patients with liver disease and of normal individuals, as detected by passive hemagglutination of E-HSAP, AAA were found to inhibit the binding of serum C1q to polymerized albumin, and hence the E-HSAP hemagglutination test for AAA titration in human sera proved valid.

Immunochemical characterization of anti-albumin antibodies in liver diseases.

Anti-albumin antibodies (AAA) were isolated from sera of hepatic patients and normal individuals by affinity chromatography on insolubilized glutaraldehyde-treated human albumin. Anti-albumin antibodies were found to belong to IgG and IgM classes in both normal and hepatic patients. The normal level of AAA increased in pathologic conditions, the increase recorded for IgM AAA being higher than that for IgG AAA. The dissociation rate of AAA from the radiolabeled antigen in normal and hepatic sera showed that the affinity of AAA was higher in normal sera than in sera of patients with chronic liver disease and acute viral hepatitis. Anti-albumin antibodies were fractionated into two populations (AAA1 and AAA2) by a two-step chromatographic procedure. AAA1 and AAA2 were found different as regards their affinity for the antigen; specifically, AAA1 affinity was higher than that of AAA2. The other difference between AAA1 and AAA2 might stand in their specificity for the haptenic and structural determinants present in the glutaraldehyde-treated albumin.

Anti-albumin antibodies in normal rabbit sera.

Antibodies reacting with homologous glutaraldehyde (GA)-modified albumin were demonstrated in normal rabbit sera (NRS) by passive hemagglutination and direct binding assay. The ability of rabbit anti-albumin antibodies (AAA) to react with homologous GA-modified albumin was found to increase with the degree of albumin polymerization. AAA did not react with GA-treated monomeric albumin. It was proved that rabbit AAA are not species-specific since they react also with heterologous GA-polymerized albumin. The possible role of AAA in the catabolism of in vivo aged albumin molecules, antigenically similar with GA-induced albumin polymers, is discussed.

Antibodies against glutaraldehyde-modified albumin in normal mouse sera.

An antigen-coated plate radioimmunoassay was used to detect antibodies against homologous glutaraldehyde-modified albumin (in monomeric or polymeric form) in normal mouse sera. Mouse antibodies reacted also with heterologous glutaraldehyde-treated albumin; for instance human albumin. Immunoelectrophoresis of purified anti-albumin antibodies and adsorption experiments on protein A-Sepharose 4B gel indicated that mouse antibodies belong mainly to the IgG class. The ability of mouse anti-albumin antibodies to react also with pyridinium compounds suggested that such structures are probably part of the new antigenic determinants induced in mouse albumin by glutaraldehyde treatment. It was assumed that anti-albumin antibodies have a physiologic role in the recognition and removal from the circulation of in vivo 'aged' mouse albumin.

An immunological hypothesis for plasma protein catabolism.

A selective mechanism of plasma protein catabolism is suggested, based on three main assumptions: (1) plasma proteins are submitted to molecular ageing thus achieving "modified" forms after a given time period; (2) the system recognizing the "modified" molecules consists of preformed physiological autoantibodies; (3) the elimination of the immune complexes formed between "modified" proteins and the corresponding autoantibodies takes place via binding to Fc receptor bearing cells.

Effect of protein A of Staphylococcus aureus on the binding of monomeric and polymeric IgG to Fc receptor-bearing cells.

The effect of protein A of Staphylococcus aureus (SpA) on the binding of rabbit IgG to the Fc receptors of mouse lymphocytes and macrophages was found to correlate with the aggregation of the IgG ligands. After binding anti-erythrocyte IgG complexed with SpA, the cells were able to attach to and kill erythrocyte indicator cells with a higher efficiency than lymphoid cells treated with anti-erythrocyte IgG alone. The amount of anti-peroxidase IgG which can be bound to effector cells was not changed by reaction with SpA. In contrast, the binding to cells of IgG-coated erythrocytes and of anti-peroxidase IgG complexed with peroxidase was substantially reduced by reaction with SpA. The results are compatible with the presence of two distinct Fc receptors, one for cytophilic monomeric IgG and another for polymeric (antigen-complexed) IgG.