Cardiovascular status in young patients with insulin-dependent diabetes mellitus.

BACKGROUND: Although the existence of diabetic cardiomyopathy in adults is firmly established, the presence of cardiac abnormalities in young diabetic patients is not universally accepted. We sought to determine the early stages of cardiac derangement and whether they are associated with renal dysfunction. METHODS AND RESULTS: Thirty-nine patients (29 boys; mean age, 17.6 +/- 3.4 years) with insulin-dependent diabetes mellitus underwent echocardiography and timed overnight urine collection. Echocardiographic evaluation consisted of left ventricular mass, performance (shortening fraction, velocity of circumferential fiber shortening, stroke volume, and cardiac index), preload (end-diastolic dimension and volume), after-load (end-systolic wall stress and systemic vascular resistance), and contractility (velocity of fiber shortening relative to wall stress). Creatinine clearance and albumin excretion were measured from the urine sample. Glycosylated hemoglobin levels were measured; height and weight were measured; and Quetelet index (weight/height2) was calculated. These data were compared with control data. Left ventricular mass (26 +/- 6 versus 22 +/- 6 g/ht2.7, P < .01), the indexes of performance, blood pressure, and contractility (0.14 +/- 0.14 versus 0.003 +/- 0.03 circumference/s, P < .0003) were significantly higher in the diabetic patients than in control subjects. To evaluate the correlates of left ventricular mass and contractility in the diabetic patients, univariate and multiple regression analyses were performed. Significant univariate correlations of mass included albumin excretion (r = .36, P < .02), glycosylated hemoglobin (r = .35, P < .04), and stroke volume (r = .34, P < .03). A multivariate model included Quetelet index, albumin excretion, and duration of diabetes. Significant univariate correlations of contractility included insulin dosage (r = -.36, P < .02), creatinine clearance (r = .40, P < .02), and Quetelet index (r = .34, P < .03). A multivariate model included insulin dosage and creatinine clearance. CONCLUSIONS: Early onset of diabetes mellitus is associated with increased left ventricular mass, performance, contractility, and blood pressure. These cardiovascular findings are correlated with increased creatinine clearance and microalbuminuria. These relations suggest that alterations in cardiovascular and renal function may occur in parallel in adolescents with insulin-dependent diabetes mellitus.

Profile of renal permselectivity by simultaneous enzyme-linked immunosorbent assay of albumin, transferrin, IgG, and alpha 1-microglobulin with a new microplate reader.

A competitive enzyme-linked immunosorbent assay (ELISA) is described for determining a renal permselectivity profile involving the urinary proteins albumin, transferrin, IgG, and alpha 1-microglobulin (alpha 1-m). The ELISA reader uses a computer-controlled array of multiplexed light-emitting diode (LED)-photodiode pairs for rapid measurements of absorbance on microplates. A 3,3'-dimethylnaphthidine reagent adapts the 3,5,3',5'-tetramethylbenzidine chromophore to monochromatic LED emission at 550 nm. We applied this ELISA to the determination of renal permselectivity in healthy children and young adults and in children with insulin-dependent diabetes mellitus. The geometric means (and SD) of protein excretion rates in a group of 85 normal subjects were as follows: albumin, 3.5 micrograms/min (1.83); transferrin, 173 ng/min (2.76); IgG, 1.11 micrograms/min (2.22), and alpha 1-m, 0.98 microgram/min (2.36).

Microplate measurement of urinary albumin and creatinine.

We describe microplate methods for measurement of human urinary albumin (HUA) by competitive enzyme-linked immunosorbant assay (ELISA) and creatinine with a modified commercial enzymatic kit. Incorporation of substrate mixing into the competitive ELISA changes the dynamic absorbance-concentration response, greatly simplifying calculations and improving sensitivity and accuracy. Measurement of creatinine in urine and plasma samples with a commercially available enzymatic kit modified for analysis by use of an inexpensive microplate reader produced values comparable in precision and accuracy to those obtained by an automated kinetic Jaffé method.

Field measurement of plasma and erythrocyte cholinesterases.

We have optimized Ellman-type plasma and erythrocyte cholinesterase assays for the field determination of pesticide exposure. The assays employ stable, premixed, inexpensive colorimetric reagents and a simplified kinetic measurement procedure using a novel battery-operable light-emitting-diode-source colorimeter (LEDSC). These methods were compared to a field tintometric method for cholinesterase currently in widespread use, using samples from organophosphate-intoxicated hospitalized patients. In comparison to the colorimetric method, the tintometric method was less precise and consistently underestimated the extent of pesticide exposure. Methods for the effective field determination of pesticide exposure using cholinesterase assays are critically evaluated with regard to their practical, biochemical, and medical relevance.

Indirect active-site titration of plasminogen activators.

A method for the determination of the molar concentration of active tissue plasminogen activator (TPA) and urokinase (UK) has been developed. The method employs the principle of back-titration of a calibrated trypsin standard with a calibrated standard solution of a chloromethyl ketone inhibitor reactive with both trypsin and activator. Both trypsin and chloromethyl ketone standards are calibrated using a guanidinobenzoyl ester active-site titrant. Less than 20 ng of activator can be accurately determined by this method. The method is used to assay commercial samples of TPA and UK, to calculate the specific activity of such samples, and to determine kinetic constants of plasma activator inhibitor.

Measurement of acetylcholinesterase in erythrocytes in the field.

We describe here a field method we developed for colorimetry of erythrocytic acetylcholinesterase (EC 3.1.1.7) in capillary blood samples. Three stable, premixed assay reagents and de-ionized water (but no centrifuge or balance) are required. This method, adapted for a microplate format, is essentially that of Ellman et al. (Biochem Pharmacol 1961;7:88-95) as modified by George and Abernethy (Clin Chem 1983;29:365-8). Assays were quantified and corrected for hematocrit by using a battery-powered colorimeter with a silicon carbide (blue) light-emitting-diode source. Advantages over existing field methods include better portability, ruggedness, greater precision, and lower cost per sample.

Methyltrypsin: a novel probe of proteinase-inhibitor interactions.

Incubation of trypsin with m-guanidinobenzenesulfonic acid methyl ester (mGBSOM) under mild conditions resulted in its quantitative and specific conversion to N-3-methylhistidinyl-57-trypsin (methyltrypsin). The interactions of alpha-2-plasmin inhibitor (alpha 2PI) and alpha-1-proteinase inhibitor (alpha 1PI) with the active-site modified enzymes methyltrypsin and dehydroalanyl-195-trypsin (anhydrotrypsin) were studied by thionine difference spectroscopy. For methyltrypsin the KA with alpha 1PI and alpha 2PI was 2.7 X 10(5) M-1 and 1.3 X 10(5) M-1, respectively, and with anhydrotrypsin, 7.0 X 10(3) M-1 and 3.2 X 10(5) M-1, respectively.

Alpha-2-plasmin inhibitor comprises a single domain.

The reaction between plasmin and alpha 2PI (alpha-2-plasmin inhibitor), which constitutes a major regulatory step in fibrinolysis, involves both interactions between the kringle structures of plasmin and a complementary site on the inhibitor, and also between the inhibitor reactive center and the enzyme active site. An attempt was made to distinguish calorimetrically the two functional domains of alpha 2PI. Only one transition was seen, both in the DSC and UV experiments, contrary to the expected two. This transition was unaffected by K4 of plasminogen but was abolished in the presence of anhydrotrypsin. This suggests the major domain structure in alpha 2PI is associated with the reactive center.