Quantitation of whole molecule human chorionic gonadotropin and free beta-human chorionic gonadotropin subunit in the Abbott IMx analyser.

An assay for the quantitation of the serum levels of free and whole molecule-associated beta-subunit of human chorionic gonadotropin on the Abbott IMx analyzer is described. The assay detects human beta-chorionic gonadotropin with a sensitivity of approximately 0.5 IU/l while showing no cross-reactivity with follitropin (1000 IU/l) or thyrotropin (2.0 IU/l), and 0.02% cross-reactivity with lutropin (1000 IU/l). Haemoglobin (7.50 milligrams), bilirubin (0.50 milligrams), and triacylglycerols (10.6 milligrams) did not interfere with the assay. Pooled within-run, between-run, and total assay CVs were less than or equal to 5.2%, and less than or equal to 2.7%, and less than or equal to 6.3%, respectively. Values obtained with this assay correlated well (r = 0.98, n = 228) with those values obtained using the Hybritech TandemR-R HCG (Total Beta-HCG) IRMA. The normal range of the assay was found to be less than or equal to 5 IU/l (n = 311). The assay protocol provides results for up to 23 serum samples in approximately 47.5 minutes with the ability to report the human beta-chorionic gonadotropin concentrations of 5 specimens in approximately 15.5 minutes. We conclude that this is an acceptable assay for monitoring human beta-chorionic gonadotropin levels associated with normal pregnancy, reproductive pathology, and reproductive technology such as in vitro fertilization or embryo transfer.

Quantifying the MB isoenzyme of creatine kinase with the Abbott "IMx" immunoassay analyzer.

In this two-step automated assay of the MB isoenzyme of creatine kinase (CK-MB), developed for the Abbott "IMx" immunoassay analyzer, monoclonal anti-CK-MB antibody immobilized onto latex microparticles and polyclonal anti-CK-MM antibody coupled to alkaline phosphatase are used. Within-run CVs ranged from 3.9% to 9.0%, between-run CVs from 0.0% to 5.6%, and the sensitivity was 0.2 microgram/L. Twenty-four results can be obtained in about 37 min. Analytical recovery of CK-MB added to human serum or plasma ranged from 89% to 109%. Icteric, lipemic, or hemolyzed samples did not interfere with CK-MB recovery. Cross-reactivity with CK-MM and CK-BB was 0.012% and 0.001%, respectively. The normal reference interval was 0-5 micrograms/L. IMx CK-MB results correlated well with CK-MB enzyme activity as determined by electrophoresis (n = 203; r = 0.97; slope = 0.90; y-intercept = -4.29) and with commercial immunoassays. We think that this assay will be useful for confirmation of acute myocardial infarction, both in critical-care units and in the clinical laboratory.

Automated enzyme immunoassay for lutropin with the Abbott IMx analyzer.

An automated enzyme immunoassay for human lutropin for use with the Abbott IMx analyzer is described. The assay provides results in approximately 40 min with a sensitivity of 0.25 int. units of LH per liter for up to 23 serum or plasma samples. Cross-reactivity with follitropin (2000 int. units/L) and thyrotropin (2 int. units/L) was negligible; it was 0.016% with human choriogonadotropin (1 X 10(6) int. units/L). There was no interference by high concentrations of bilirubin (0.5 g/L), hemoglobin (7.50 g/L), or triglycerides (13.5 g/L). Intra-, inter-, and total assay CVs were less than or equal to 3.75%, less than or equal to 7.1%, and less than or equal to 7.94%, respectively. Values obtained with the IMx correlated well (r = 0.98, n = 194) with values obtained with Diagnostic Products' LH Double Antibody RIA, and Serono's LH MAIAclone assay. This assay should be useful for small to medium-size laboratories involved in the clinical diagnosis of reproductive pathology.

External fixation of injured nail bed with the INRO surgical nail splint.

The use of the nail as a splint is desirable in the management of acute nail bed injuries. However, when the nail plate is destroyed and during secondary reconstruction of the nail bed, the nail is not available for use as a splint. Several substitutes, notably silicone sheet have been used, but without much success. The INRO surgical nail, with qualities similar to the nail splint was developed for use as a substitute. During the period of study from 1983 to 1985, 89% of patients had good results.

Epitope mapping of human luteinizing hormone using monoclonal antibodies.

To establish structure-function relationships for human (h) LH, 14 murine monoclonal antibodies (MABs) to hLH were characterized in terms of their affinity of binding (Ka), their specificity for intact glycoproteins and their subunits, their paratopic relationships, and their ability to interfere with the biological activity of hLH. The Ka values obtained ranged between 2.4 x 10(6) and 1.0 x 10(10) for intact hLH and between 2.3 x 10(6) and 7.5 x 10(8) liters/M for the free alpha- and beta-subunits, indicating that, in general, the antibodies showed higher avidity for the intact hormone. Six MAB recognized both the intact and free alpha-subunit of hLH, cross-reacted with intact hCG, hFSH, and hTSH, and thus appeared to be alpha-directed. Four MAB were beta-directed, recognizing both intact hLH and its free beta-subunit. One of these beta-directed MABs also cross-reacted with intact hCG, hFSH, and hTSH, while two others recognized both intact and free beta-subunits of hLH and hCG. The fourth beta-directed MAB was quite specific for intact hLH and its beta-subunit. The remaining four MABs recognized epitopes only on the intact hormone; three recognized intact hLH and hCG, and the fourth was specific for intact hLH. Their paratopic relationships tested in competitive binding studies resulted in either mutual competition or complementarity, sometimes with cooperativity. Biointerference, defined as the ability to inhibit hLH-induced testosterone biosynthesis in dispersed rat Leydig cells, indicated that three of the alpha- and one of the beta-directed antibodies neutralized the biological response of hLH in this bioassay in a dose-responsive manner. Their ability to inhibit hLH bioactivity largely paralleled their affinity constants. Our data have allowed us to establish a tentative topographic relationship of epitopes to the biological region of the molecule of hLH, foreshadowing (in additive binding studies) some of the possible combinations of antibodies that might allow us to design two- or multiple-site immunometric assays in which measurement of immunoactive LH reflects biological activity. In addition, these studies suggest that both the alpha- and beta-subunits participate in LH receptor binding and/or biological activity.

Susceptibilities of cardiac myofibrillar proteins to cathepsin D-catalyzed degradation.

The binding of various myofibrillar proteins to cathepsin D and consequent susceptibility to degradation was assessed by determination of apparent Michaelis constant (Km) values for the purified enzyme using myosin, alpha-actinin, actin, and tropomyosin as substrates. Cathepsin D, purified 1,000-fold to homogeneity from canine cardiac tissue, was incubated (0-100 min at 37 degrees C) with myofibrillar proteins isolated from homologous tissue and labeled radioactively with 14C by reductive alkylation. The reaction was terminated by addition of trichloroacetic acid (10% wt/vol), and radioactivity in the supernatant fraction was determined after centrifugation (5 min) at 100,000 g. Double reciprocal graphs (1/reaction velocity vs. 1/substrate concn) were constructed for each substrate from the linear portion of graphs of disintegrations. min-1 X ml-1 100,000-g supernatant vs. time (min). Apparent Km values (+/- SD) calculated for myosin, alpha-actinin, actin, and tropomyosin were found to be 2.7 +/- 0.3 X 10(-6) (n = 6), 10.0 +/- 2.3 X 10(-6) (n = 13), 13.0 +/- 1.3 X 10(-6) (n = 7), and 45.5 +/- 9.0 X 10(-6) (n = 6) mol/l, respectively. The results show that myofibrillar proteins differ in their binding and thus susceptibility to hydrolysis by cathepsin D in order of molecular weight (i.e., myosin greater than alpha-actinin greater than actin greater than tropomyosin). Because the relative turnover rates of myofibrillar proteins are known to be independent of molecular weight, our results suggest that the degradation of these proteins by cathepsin D is not rate limiting and that other factors including lysosomal or nonlysosomal enzymes must determine the rate-limiting steps of myofibrillar protein degradation in cardiac tissue.

Regulation of cathepsin D metabolism in rabbit heart: evidence for a role for precursor processing in the control of enzyme activity.

Production of active lysosomal enzymes may involve limited proteolysis of inactive high molecular weight precursors. Precursor processing potentially regulates lysosomal enzyme activity. To test whether rabbit cardiac cathepsin D is first synthesized as a precursor and whether prolonged fasting (a condition affecting both cathepsin D and total cardiac protein turnover) influences precursor processing, rates of cathepsin D synthesis and processing were compared in left ventricular slices of control and 3-d-fasted rabbits incubated in vitro with [(35)S]methionine. (35)S-labeled cathepsin D was isolated by butanol-Triton X-100 extraction, immunoprecipitation, and dodecyl sulfate-polyacrylamide gel electrophoresis. Total cardiac protein synthesis was measured by tracer incorporation and normalized for differences in precursor pool size by direct measurement of [(35)S]aminoacyl-tRNA-specific radioactivity. Relative cathepsin D synthetic rates were obtained by comparing (35)S incorporation into cathepsin D with (35)S incorporation into all cardiac proteins. Enzyme processing was assessed in pulse-chase experiments and assayed by autoradiography. The results indicate that (a) rabbit cardiac cathepsin D is synthesized as a precursor (53,000 mol wt) that is processed to a 48,000-mol wt form, (b) rates of both cathepsin D and total cardiac protein synthesis are similar in control and fasted rabbits, suggesting that decreased enzyme degradation rather than increased synthesis is responsible for the elevated levels of cardiac cathepsin D in starvation, and (c) cathepsin D processing in hearts of fasted animals is incomplete, with accumulation of the precursor during pulse-chase experiments of 6 h duration. Based upon these results, a three-stage model for the regulation of cathepsin D activity in rabbit heart is proposed.

Rabbit cardiac immunoreactive cathepsin D content during starvation-induced atrophy.

To determine whether the increased activity of cathepsin D observed during starvation-induced cardiac atrophy results from activation of preexisting enzyme or synthesis of new enzyme, a solid phase double-antibody radioimmunoassay was developed for measurement of immunoreactive cathepsin D in extracts of rabbit myocardium. Cathepsin D activity was significantly increased in the hearts of animals starved for 3, 7, and 14 days (82.6 +/- 0.8, 87.2 +/- 3.8, and 95.3 +/- 3.5 U/g wet wt, respectively) compared to controls (65.5 +/- 1.4 U/g wet wt; P less than 0.001). Immunoreactive cathepsin D was increased to a greater extent (168 +/- 7, 179 +/- 16, 200 +/- 17, and 104 +/- 5 micrograms/g wet wt for 3-, 7-, and 14-day starvation and controls, respectively; P less than 0.001) than that expected on the basis of the observed increase in enzyme activity. Sephadex G100 gel filtration of cardiac lysosomal extracts from starved and control animals revealed no evidence of high or low molecular weight forms of cathepsin D. The results suggest the observed increase in cathepsin D activity during starvation-induced cardiac atrophy is accompanied by an increased synthesis and/or decreased degradation of cathepsin D protein, rather than activation of preexisting enzyme. The lower activity levels observed during starvation possibly result from alterations in the concentrations of endogenous inhibitors or activators of cathepsin D.

A double antibody radioimmunoassay for canine cardiac cathepsin D.

We have developed a sensitive double antibody radioimmunoassay for measuring canine cardiac cathepsin D. Radioiodinated cathepsin D was prepared by chloramine T oxidation using a highly purified source of enzyme. High avidity antiserum to the canine cardiac enzyme was raised in rabbits. Antibody-bound cathepsin D was separated from free enzyme using goat anti-rabbit IgG second antibody. The least amount of immunoreactive enzyme measurable in the radioimmunoassay was 2.4 ng.cm-3 as determined by antibody titration. The assay was linear for concentrations of enzyme in the range of 10 to 120 ng.cm-3. Within-assay and between-assay variations were 12%. The radioimmunoassay described was used to measure the immunoreactive cathepsin D content of the 100 000 x g supernatant fraction of canine myocardial homogenates.

Sub-mitochondrial and sub-microsomal distribution of creatine kinase in guinea pig myocardium.

Mitochondrial and microsomal fractions were isolated from guinea pig myocardium by differential pelleting. The mitochondrial fraction was subjected to analytical subfractionation by sucrose density gradient centrifugation and the gradient fractions assayed for marker enzymes for the various mitochondrial compartments, viz outer membrane (monoamine oxidase), intermembranous space (adenylate kinase), inner membrane (Mg2+-dependent ATPase and cytochrome c oxidase) and mitochondrial matrix (malate dehydrogenase), and for creatine kinase. Both creatine kinase and adenylate kinase were released by suspending the mitochondria in 50 mmol . litre-1 sodium phosphate buffer. Sonication or disruption with the detergent, digitonin released the adenylate kinase but the creatine kinase remained associated with the inner membranes. Subsequent salt treatment desorbed the creatine kinase from these membranes. It is concluded that creatine kinase is located to the outer aspect of the inner mitochondrial membrane. Analytical subfractionation of the microsomal fraction clearly resolved markers for the sarcolemma (5'-nucleotidase), outer mitochondrial membrane (monoamine oxidase) and endoplasmic reticulum (neutral alpha-glucosidase and RNA). Creatine kinase was localised in the endoplasmic reticulum particularly the smooth membranes.

The loss of electrically active myocardium in cardiogenic shock.

A method of precordial mapping of RS ratio changes and the appearance of Q waves that occur in acute myocardial infarction has been developed. The serial changes of R and Q waves in 40 patients suffering uncomplicated anterior infarction shows that the loss of electrically active myocardium occurs within 6 h from the onset of chest pain. The total precordial ECG changes of R and Q waves in 5 patients suffering anterior infarction and cardiogenic shock showed that the loss of electrically active myocardium continues after 6 h from the onset of chest pain, and is significantly different from the uncomplicated group. This was supported by the serial plasma MBCK activity determinations which suggest more than one episode of necrosis.

Loss of electrically active myocardium during anterior infarction in man.

A method has been developed of praecordial mapping of changes in R/S ratio and the appearance of Q waves in acute myocardial infarction. Observation of the serial changes in R and Q waves in 40 patients with uncomplicated anterior infarction shows that the loss of electrically active myocardium occurs within 6 hours of the onset of chest pain. Complications, such as recurrent chest pain, associated with extension of myocardial necrosis can be identified and assessed. The total praecordial changes in R/S ratio and Q wave amplitude correlate with the total MB fraction of creatine kinase activity released into the plasma in 20 patients after uncomplicated anterior infarction. This technique for identifying those factors that may modify the progressive loss of active myocardium in the early phase of acute infarction of the heart is noninvasive and repeatable.

Natural history and evaluation of ST segment changes and MB CK release in acute myocardial infarction.

The experimental evidence relating ST segment elevation in the electrocardiogram to the progress and extent of ischaemic myocardial damage is discussed. There are difficulties in applying this to patients: the reproducibility of praecordial mapping was tested using a multiple analysis of variance. This showed that factors such as time after the onset of myocardial infarction and posture can affect measurements of sigmaST elevation significantly. There was a pattern of changes in segmaST elevation and of changes in plasma MB CK activity in a group of patients with uncomplicated anterior infarction. A significant byt weak correlation was found between sigmaST elevation in the first hour and the total MB CK activity released into the plasma, but not at any other time. The use of sigmaST elevation as a measure of the extent of ischaemic damage is unreliable. In 5 patients with a variety of complications of acute anterior infarction, changes in sigmaST elevation werr significantly different from the uncomplicated group, and MB CK release profiles suggested further necrosis. The pattern and time course of ST segment changes may be of use in assessing the progress of ischaemic myocardial damage.

Subcellular compartmentation of creatine kinase isoenzymes in guinea pig heart.

In an attempt to determine whether a subcellular compartmentation of creatine kinase exists and if so, whether there is a different distribution of the 3 isoenzymes of CK in the cell, studies were carried out with the guinea pig heart which had been subfractionated by either isopycnic density gradient centrifugation or differential pelleting. Isoenzyme analysis of CK in the isolated subcellular fractions by electrophoresis on agarose gels, revealed that the MM isoenzyme occurred in the cytosol, myofibrils, and the microsomes while the MB isoenzyme (which is cardio-specific) was only found in the cytosol. Trace amounts of the BB isoenzyme were detected in the cytosol. Considerable CK activity was associated with the mitochondria, this did not represent the MM, the MB, or the BB isoenzymes but was a distinct and additional mitochondrial-specific form of CK. PH optima and kinetic studies were carried out to characterise and distinguish the mitochondrial isoenzyme from other CK isoenzyme activity. The evidence for a differential compartmentation of MM, MB, BB, and mitochondrial CK is discussed in relation to their possible cellular roles.