Post-transcriptional modification of serum creatine kinase in infected intensive care patients.

Low creatine kinase (CK) activities in serum are associated with high fatality rates in intensive care patients. The underlying mechanisms for this phenomenon were investigated. No correlation was found with other biochemical markers of inflammation (CRP, alpha-1 acid glycoprotein, alpha-2 macroglobulin). In the patients' serum a factor is described which is capable of increasing the activation energy of normal CK-MM, indicating molecular changes in CK-structure. This factor is likely to be an enzyme which is present in liver tissue and in fibroblasts. Similar results were obtained after in vitro treatment of normal serum samples with arylsulfatase. Furthermore, bacterial strains isolated in the serum of intensive care patients were found to alter human CK structure. In the investigated patient group, changes in CK activation energy are influenced by serum factors other than carboxypeptidase N activity.

Mass concentration and activity concentration of creatine kinase isoenzyme MB compared in serum after acute myocardial infarction.

We compared three current methods (immunoinhibition, "Isomune-CK" immunoprecipitation, and the Tandem-E CKMB II immunoenzymometric assay) for determination of creatine kinase (CK; EC 2.7.3.2) isoenzyme MB in serum. Although results inter-correlated well, the immunoinhibition assay gave higher activity values. Atypical CK forms did not interfere with the immunoprecipitation and immunoenzymometric methods. In acute myocardial infarction the catalytic properties of CK decreased with the enzyme's age, as reflected by a steady increase in activation energy of the catalyzed reaction. In septicemia patients with very low CK and CK-MB catalytic activity, mean CK-MB mass concentration exceeded the upper reference limit, suggesting an increased rate of loss of activity concentration in these patients' sera. Because of the assay's lesser susceptibility to conformational changes at the active site of the enzyme, we suggest that measurement of CK-MB mass concentration is better suited for infarct sizing than measurement of catalytic activity.

Activation energy and lectin affinity chromatography of gamma-glutamyltransferase as a marker for enzyme heterogeneity.

Lectin affinity chromatography of gamma-glutamyl transferase (GGT,EC 2.3.2.2) is able to detect differences in the carbohydrate moiety of the enzyme. Binding of tissue GGT towards lectins is significantly different from serum GGT, showing increased galactosylation in tissue forms. Kidney GGT is less glycosylated than GGT from other tissues (liver, pancreas, prostate, vesiculae seminales). Increases in sialic acid content of GGT are associated with an increase in the activation energy of the catalyzed reaction. Differences in galactose, fucose and N-acetylhexosamine content induce much smaller effects on activation energy. In liver diseases, serum GGT is characterized by an altered affinity against lectins recognizing galactose, fucose and N-acetyglucosamine and by increased activation energy. In patients with liver disease, use of fixed temperature conversion factors can lead to erroneous calculations of serum GGT enzyme activity (errors up to 13.3%).

Measurement of autoimmune response against collagen types I, III, and IV by enzyme-linked immunosorbent assay, and its application in infective endocarditis.

In these enzyme-linked immunosorbent assays for determination of autoantibodies (IgG, IgM) against collagen type I (ACA I), III (ACA III), and IV (ACA IV), we use commercially available antigen preparations. Inhibition curves showed limited cross-reactivity between these different collagen preparations, the major interference being observed after addition of collagen type III in the ACA I procedures. Imprecision (CVs) for high- and low-titer samples ranged between 1.5% and 7.9% (within-run) and 5.5% and 12.7% (between-run) for ACA I, between 2.2-8.7% and 4.5-10.7% for ACA III, and between 1.3-5.9% and 7.4-11.6% for ACA IV. Significantly increased humoral immune response against collagen types I and III (P less than 0.001) could be demonstrated during the first month of infective endocarditis. In contrast, only borderline increases, however constant, of autoimmune response against basement-membrane collagen (ACA IV) were noticed during 90 days of follow-up.

Anti-myosin humoral immune response following cardiac injury.

A sensitive and highly specific ELISA assay was developed to determine the anti-myosin humoral immune response (AMA) in various heart diseases: acute viral myocarditis, infective endocarditis, acute myocardial infarction, and valve and coronary bypass surgery. The mean study entry AMA titer of each patient group was already significantly increased compared with age matched controls. During further follow-up (90 d) all the groups except for endocarditis showed a significant increase of AMA titer compared with their entry titer. Anti-myosin antibody titer were higher after cardiac surgery than after myocardial infarction or inflammatory heart disease. These results suggest that anti-myosin immune response is not limited to infectious processes in which the pathogen induces antibodies which cross-react with heart constituents but is merely caused by direct cardiac injury. Myosin as a major compound of heart cellular proteins turned out to be a good candidate to trigger immune response after cardiac injury.

Lectin-affinity chromatography of serum gamma-glutamyltransferase in liver disease.

The variation of the carbohydrate chain of gamma-glutamyltransferase was studied in 45 liver patients by means of lectin affinity chromatography. Five lectins were used: concanavalin A, Ricinus communis I and II, Maclura pomifera and Ulex europaeus agglutinin. The binding towards Con A was shown to be independent from the binding towards the other lectins. Parallel variations of binding results against the galactose- and fucose-recognizing lectins were obtained. In liver steatosis, the binding results were comparable to those obtained in normal patients. Cirrhosis and metastasis patients showed a decreased binding towards Con A, while the binding against the various galactose- and fucose-recognizing lectins was increased. After neuraminidase treatment, an increased affinity towards all lectins was observed. However, differences in RCA I and RCA II binding between patients and controls still persisted. Besides sialic acid, also galactose and fucose residues contribute to serum gamma-glutamyltransferase heterogeneity.

Liver plasma membrane: the source of high molecular weight alkaline phosphatase in human serum.

This study presents biochemical, histochemical, morphological and immunological evidence that part of the high molecular weight alkaline phosphatase observed in the serum of patients with liver disease and particularly in cases of intrahepatic cholestasis or focal-, extrahepatic obstruction originates from the liver plasma membrane. The high molecular weight protein alkaline phosphatase complex contains several plasma membrane enzymes and behaves like a plasma membrane fragment after isopycnic density gradient ultracentrifugation in sucrose, cesium chloride and metrizamide. Electron microscopic examination revealed a triple-layered vesicle which retained alkaline phosphatase activity. Incubation of human liver cells with anti-serum against purified high molecular weight multienzyme complex resulted in fixation of antibodies on the plasma membrane as shown by positive plasma membrane fluorescence. These plasma membrane fragments in the serum are not of biliary origin.

A novel alkaline phosphatase isozyme in 4 patients with acute non-lymphocytic leukemia: its nature, origin and clinical significance.

A novel alkaline phosphatase (AP) isozyme has been observed in the serum of 4 patients suffering from acute non-lymphocytic leukemia (ANLL). It resembled the AP of liver/bone origin in most physico-chemical characteristics, but particular electrophoretic characteristics in agar and starch gel and a distinct molecular weight gave this novel AP isozyme its unique character. Its leukemic origin has been demonstrated by isolation from peripheral blood and bone and bone marrow blasts. The appearance of the novel AP isozyme in the patients' sera appeared to be an ominous sign as, in all four, it shortly preceded death. In patients it may have contributed to the observed resistance towards thioguanine therapy.

Occurrence of enzyme-immunoglobulin complexes in chronic inflammatory bowel disease.

The presence of circulating enzyme-immunoglobulin complexes was investigated in serum samples of patients with inflammatory bowel disease. In a series of 20 patients with ulcerative colitis AP-Ig complexes were demonstrated in three and LDH-Ig complexes were observed in two others. No AP-Ig or LDH-Ig complexes were found in the sera of 25 patients with Crohn's disease. These findings suggest a relationship between enzyme-immunoglobulin complex formation and ulcerative colitis.

Occurrence of immunoglobulin G-alkaline phosphatase complexes in human serum.

We studied three patients in whom all or part of their serum alkaline phosphatase circulated as a complex with immunoglobulin G (IgG). Serum alkaline phosphatase isozymes were visualized by their electrophoretic (in agar, agarose, and starch gel and on cellulose acetate), gel filtration, and electroimmunodiffusion behaviour. The alkaline phosphatase-IgG complex was of the liver type (two cases) and bone type (one case). The reaction pattern of alkaline phosphatase with different human tissues and with sera of heterologous origin suggests that the complex is of the antigen-antibody type. A direct genetic mechanism seems unlikely since in one patient the IgG-alkaline phosphatase complex was not present in previous serum samples. The presence of this complex has no apparent correlation with the observed abnormalities. Alkaline phosphatase linked to immunoglobulin G must be considered in the interpretation of increased serum alkaline phosphatase.

Spontaneous shedding of plasma membrane fragments by human cells in vivo and in vitro.

Human duodenal fluid, secretion fluid of a villous adenoma of the rectum, urine and culture medium of HeLa cells contain plasma membrane fragments which can be revealed by electrophoresis in different media, gel filtration on Sepharose 4-B, electron microscopy and cytochemistry. They carry plasma membrane enzymes (alkaline phosphatase EC 3.1.3.1, leucine aminopeptidase EC 3.4.1.1, 5'-nucleotidase EC 3.1.3.5, maltase EC 3.2.1.20) in the same ratio as the membranes of the cells of origin. Equilibrium density centrifugation results in recovery of these plasma membrane fragments at density 1.190 (g/ml) in CsCl, 1.165 (g/ml) in sucrose, and 1.135 (g/ml) in metrizamide. Similar plasma membrane fragments were decribed previously in the serum of certain liver patients. These observations give evidence that shedding of whole plasma membrane fragments (koinozymic shedding) is a widespread feature of viable cells.

Short term variation of human immunoglobulin levels with an estimation of the day to day physiological variability.

In this work serum Ig levels were determined daily for 14 days in 12 subjects, every two days for 14 days in 19 subjects, every week for 1 month in 11 subjects and fortnightly in 11 subjects for 6 months. The serum IgG, IgM and IgA levels were quantitated by the linear plate method. The day-to-day reproducibility of the technique was checked for a period of one months on seven different samples. The mean coefficient of variation in the reproducibility experiments was found to be 4.8% for IgG, 8.2% for IgM and 5.2% for IgA. Since the observed variation in all groups of subjects was found to be higher than the methodological variation, it appears that physiological variability occurs in the serum Ig levels of an individual. No difference was found between the physiological variances of IgG, IgM and IgA observed in the group with daily analysis and those with determinations every two days. The physiological variations increase when longer periods are considered between consecutive determinations, but the variability of IgG and IgA in the group with fortnightly determinations is less important than that observed in the group with weekly analyses. Some evidence is presented indicating a periodic fluctuation of serum IgG and IgA levels with an amplitude of nearly 20% and with a recurrence rate of two weeks.

L-p-Bromotetramisole, a new reagent for use in measuring placental or intestinal isoenzymes of alkaline phosphatase in human serum.

L-p-Bromotetramisole is proposed as a new reagent for use in determining intestinal and (or) placental isoenzymes of alkaline phosphatase in human serum. Its main advantage over L-phenylalanine is its high discriminating potency at very low concentrations. For highest accuracy, the samples may even be "titrated" with the inhibitor. Analytical recoveries of intestinal and placental isoenzymes in serum are complete, and results correlate well with those by other methods. At the appropriate concentrations, L-p-bromotetramisole gives nearly identical results on a variety of sera, irrespective of the buffer used. Within-day CV's were 6.1 and 2.1 for 20% and 60% of the resistant isoenzyme activity, respectively. The day-to-day CV was 3.45 for a sample containing 53.3% of the resistant isoenzyme (n=15). The well-known increase in placental isoenzyme activity with time of gestation could be confirmed on 612 sera from pregnant women. The activities of the intestinal isoenzyme in sera from healty blood donors (n = 126) and hospitalized patients (n = 135) agree with previous findings obtained by the other techniques.

The separation and characterization of liver plasma membrane fragments circulating in the blood of patients with cholestasis.

Investigations on the high molecular weight isozyme of alkaline phosphatase (R type of AP), which is typically found in the serum of patients with cholestasis, have revealed that AP of the R type corresponds to the conventional liver AP which is attached to vesicular material. The isolation of these vesicles by Sepharose gel filtration is described. Several features were found to be characteristic for these vesicles: 1. The presence of the following enzymes known to be membrane bound: alkaline phosphatase (AP), 5FEET-NUCLEOTIDASE, L-leucyl-beta-naphthylamidase (LAP), and gamma-glutamyl transpeptidase (gamma-GT). 2. The absence of the following enzymes known not to be present on cell membranes: glutamic pyruvic transaminase (SGPT), glutamic oxalacetic transaminase (SGOT), lactate dehydrogenase (LDH), and acid phosphatase. 3. The typical ultrastructural appearance and the cytochemical visualization of alkaline phosphatase and 5feet-nucleotidase. It is concluded that the vesicles correspond to fragments of the liver cell membranes that appear and continue to circulate in the blood of patients with cholestasis.

The Position Of National Surveys In Proficiency Testing Of Clinical Chemistry Laboratories.

Results of the Belgian proficiency surveys (1971-1973) for clinical chemistry labs are reported. The specific position of this external type of quality control is analyzed. Special attention is given to the detection of systematic factors that could interfere with the introduction of a uniform medical record. A new procedure is described for displaying results grouped according to methods. The difference between the main methods in actual use is of the systematic type rather than a difference in precision. The Youden approach to collaborative tests has been elaborated and generalized by the introduction of correla-tion as an expression of the incidence of systematic factors. The shape of the contour encompassing 95% of the Youden couples, defines a target area often very different from the circular surface expected with uncorrelated results. The participating laboratories, as a group, appeared to work from the beginning according to the current state of the art. Improvement is only possible if basic changes in methods or in calibration are introduced. This suggests a broadening of the activities of the Belgian Society for Quality Control.