Which method for quantifying "microalbuminuria" in diabetics? Comparison of several immunological methods (immunoturbidimetric assay, immunonephelometric assay, radioimmunoassay and two semiquantitative tests) for measurement of albumin in urine.

We have compared the chemical and clinical characteristics of an immunonephelometric assay (INA), two immunoturbidimetric assays (ITA) and two semi-quantitative methods with those of a solid-phase radioimmunoassay (RIA) for measurement of urinary albumin (UA) concentration in 136 diabetic patients. INA and RIA had similar accuracy, and provided comparable results. However, RIA has slightly greater sensitivity than INA, which is easier and faster. Good agreement was also found between RIA and the two ITA methods, although one of these overestimated RIA values in the low-medium range (5-30 mg/l) of urinary albumin. ITA seems suitable for initial screening of albuminuria in diabetic patients but more sensitive procedures (such as RIA and INA) seem preferable for measurement of UA concentrations in the normal range. The two semi-quantitative methods showed high sensitivity but poor specificity, because of the large number of false positive results. About 50% of diabetic patients "positive" by these methods did not have microalbuminuria. The utility of these methods is questionable, because many samples from diabetic patients need to be reassayed by a more specific and sensitive assay such as the RIA, INA or ITA methods.

How and how long to store urine samples before albumin radioimmunoassay: a practical response.

We used three study protocols to check the dependence of albumin stability, measured by an RIA, on different temperatures, durations, and materials (i.e., assay tubes) of urine storage. Albumin values obtained for samples stored in three types of assay tubes (glass, polystyrene, and polypropylene) throughout the 2 months of the first (prospective) protocol were superimposable. The 24-h storage of six urine samples at room temperature or at 4 degrees C, as well as 72-h storage at 4 degrees C, did not affect the albumin measurement by RIA. After 2 months of storage of these same six urine samples at -20 degrees C, there was still no albumin decrease. A significant albumin decrease occurred (a mean of approximately 5% per year, throughout the range of albumin concentrations tested) when samples stored at -20 degrees C were reassayed by RIA after > or = 2 years (second protocol, retrospective). Finally, 3 of 21 (14.3%) urine pools stored at -20 degrees C for various periods (4-21 months) showed a significant albumin loss after storage; the time of storage as well the decrement rates of these 3 pools differed from each other (third protocol, retrospective). Short- and medium-term (2-6 months) freezing of urine samples at -20 degrees C does not significantly affect the stability of immunoreactive albumin. For longer preservation periods, storage of urine samples at -70 degrees C may be preferable.

Albuminuria estimated from proteinuria in diabetics. Is it a real alternative in clinical practice?

While proteinuria detectable by dip-sticks is the hallmark of overt diabetic nephropathy, urinary albumin excretion (UAE) above normal but Albustix-negative (so-called microalbuminuria) is the main characteristic of the preproteinuric phase of the diabetic renal disease. Reliable measurement of UAE requires very sensitive and accurate methods, such as radioimmunoassay (RIA), which, however, are not suitable for routine UAE analysis. The Coomassie Brilliant Blue dye binding method has been suggested to be a simple and economical way of indirectly measuring albuminuria in diabetic patients. In the present paper, we compared the chemico-clinical characteristics of a RIA method with those of the Coomassie dye binding method, in order to verify if it is really possible to calculate albuminuria from proteinuria (and vice versa) by a simple linear regression equation, as previously suggested. The RIA has shown a better sensitivity and accuracy in comparison to the dye binding method. Our study suggests that there is not a linear relationship between proteinuria and albuminuria in diabetic patients. Indeed, the ratio between the total proteinuria, as measured with the dye method, and the albuminuria, as measured by a specific and sensitive RIA, varies greatly in diabetic patients with or without glomerular nephropathy. While the dye binding method appears the best procedure for the assay of total microproteinuria, since it is precise, cheap and feasible, the RIA, due to its high sensitivity and specificity, is more suitable for early and accurate detection of microalbuminuria, as well for the close follow-up of subjects at risk of developing overt diabetic nephropathy.

Nonenzymatic glycation of human platelet membrane proteins in vitro and in vivo.

Human platelet membrane proteins (PMP), incubated in vitro in the presence of various concentrations of glucose, undergo nonenzymatic glycation, as evidenced by incorporation of [3-3H]glucose radioactivity into the acid-precipitable fraction. The time course of the reaction is linear for the first hours, and the rate of glycation depends on the glucose concentration in the medium: at a glucose concentration of 80 mmol/L, up to 60 nmol of glucose is bound per milligram of PMP. The ketoaminic nature of the glucose/protein linkages was demonstrated by the finding of 5-hydroxymethylfurfuraldehyde by liquid-chromatographic analysis of acid hydrolysates of PMP. We analyzed PMP from 13 subjects with type I poorly controlled diabetes and from 10 nondiabetics. Nonenzymatic glycation, evaluated as nanomoles of the aldehyde per milligram of protein, was much greater in diabetic patients than in nondiabetics: 1.58 +/- 0.70 vs 0.37 +/- 0.18 (mean +/- SD).

Colorimetric versus radioimmunological measurement of glycated and non-glycated serum albumin after affinity chromatography.

Affinity chromatography by m-aminophenylboronic acid has been proposed for routine measurement of glycated albumin. We assayed glycated and non-glycated fractions of serum albumin (HSA) eluted by affinity chromatography columns by both a specific RIA method for the human serum albumin (HSA) and by a colorimetric method. Sixteen diabetic patients presented a significantly higher percentage of glycated-HSA than 7 control subjects with both methods, and a strong correlation was found between the values obtained with the two methods. RIA was able to detect a significant concentration of glycated-HSA in all normal subjects, while the colorimetric method was not. The accuracy of separation between the glycated and non-glycated fractions of albumin was tested using [14C]glucose as tracer. When [14C]glycated-HSA purified by Sephadex G25 filtration was chromatographed using the m-aminophenylboronic acid, only 5.3% of the total 14C-radioactivity present in the solution was recovered in the bound fraction, while 44.0% was eluted in non-protein-bound fraction and 54.7% was retained in the column. Our findings confirm that affinity chromatography by m-aminophenylboronic acid can be a useful tool in the monitoring of short glycemic control of diabetic patients. Our data also indicate that the affinity chromatography with m-aminophenylboronic acid does not accurately discriminate between glycated and non-glycated fraction of HSA.

Exercise-induced proteinuria in well-trained athletes.

We studied the rate of urinary excretion of albumin, alpha 1-microglobulin (as an indicator of the renal tubular involvement), sodium, potassium, and creatinine in the basal state (overnight urine collection) and after physical exercise (training session) in 10 professional cyclists, to verify whether protein excretion is increased even in well-trained athletes after physical effort. In addition, we wanted to understand whether the origin of exercise-induced proteinuria was glomerular, tubular, or both. Compared with the basal state (overnight collection), exercise significantly (P less than 0.01) increased the excretion rate of albumin (4.2 +/- 2.6 micrograms/min vs 18.1 +/- 10.6 micrograms/min, mean +/- SD), Na, and K, and also the urinary volume. Creatinine output was not affected by exercise. The mean (+/- SD) overnight excretion rate of albumin by athletes was quite similar to that found for 91 healthy nonathletes at rest (4.6 +/- 2.7 micrograms/min). The mean exercise-related excretion of alpha 1-microglobulin by the athletes significantly exceeded the overnight value (6.6 vs 0.3 mg/L, P = 0.037). Our study indicates that (a) albuminuria furnishes the greater contribution to the increase in exercise-induced proteinuria; (b) the exercise proteinuria is both glomerular and tubular in origin, and is reversible; (c) the enhanced protein requirement of athletes may in part be due to the recurrent excretion of proteins in the urine after physical effort.

'Microalbuminuria' in type I (insulin-dependent) diabetic patients with and without retinopathy.

We investigated the frequency of microalbuminuria (albumin excretion rate, AER greater than 15 micrograms/min) ('overnight' urine collection and radioimmunological evaluation) and its relation to retinopathy (assessed by fluorangiography) in 113 type I (insulin-dependent) diabetic subjects (aged 31 +/- 13 years; diabetes duration 11 +/- 7 years), all Albustix-negative. Sixty eight patients (60.2%) were free of retinal lesions, 31 (27.4%) had background retinopathy and 14 (12.4%) had proliferative retinopathy. Microalbuminuria was found in 25 patients (22%). Fifteen patients (13%) showed both retinopathy and microalbuminuria. Fifteen % (10/68) of the patients with no retinopathy and sixteen % (5/31) of those with background retinal lesions had microalbuminuria, while 29% (4/14) of the patients with proliferative retinopathy were normoalbuminuric. Among the 29 patients with diabetes for less than five years, 1 had retinopathy and 4 had microalbuminuria. Out of 15 patients with both retinopathy and microalbuminuria, 13 (87%) had had diabetes for more than 10 years. Diabetic retinopathy is more frequent than microalbuminuria (40 vs 22%). Although the linkage between retinopathy and microalbuminuria is weak, after ten years of diabetes the two complications may frequently coincide.

Increased urinary excretion of digoxin-like immunoreactive substance by insulin-dependent diabetic patients: a linkage with hypertension?

Excretion of digoxin-like immunoreactivity (DLIS) was measured by RIA in timed overnight urine collections from 91 normotensive nondiabetic subjects and 104 normotensive insulin-dependent diabetic (IDDM) patients. The mean +/- SD DLIS excretion rate for the diabetic patients significantly exceeded that for the controls (73 +/- 41 vs 63 +/- 36 pg/min, P = 0.024). In both groups, the mean DLIS excretion rates for men were significantly higher (P = 0.0014, P = 0.006) than for women. In the controls, the DLIS excretion rate significantly correlated with the urinary excretion rate of creatinine (P less than 0.01), Na+ (P less than 0.05), and K+(P less than 0.05), and with the subjects' body weight (P less than 0.01), body mass index (P less than 0.05), and systolic blood pressure (P less than 0.05). In the diabetics, the DLIS excretion rate was significantly correlated with body weight (P less than 0.05) and with urinary excretion rates for albumin (P less than 0.01), creatinine (P less than 0.01), Na+ (P less than 0.05), and K+(P less than 0.05). Our data indicate that: (a) increased amounts of a cardiac glycoside-like substance (or a group of substances) are excreted in the urine of IDDM patients; (b) the urinary excretion of DLIS seems to depend on glomerular filtration rate and physiocochemical properties of glomerular membrane, as well as on subjects' body mass; and (c) because cardiac glycoside-like substances may increase peripheral vascular resistance, increased urinary excretion of DLIS by IDDM patients may indicate a tendency to develop hypertension.

Measurement of urinary albumin excretion (UAE) in diabetic patients: immunonephelometry versus radioimmunoassay.

We have compared the chemico-clinical characteristics of an immuno-nephelometric technique (INA) with those of a solid-phase radioimmunoassay (RIA) method for the measurement of urinary albumin excretion (UAE) in diabetic patients. The UAE was evaluated in 227 diabetics; all, except 9, were Albustix-negative. The calibration of the nephelometer apparatus every time before the assay did significantly improve the accuracy and precision of the INA method. Similar values were obtained with the two methods (INA = 2.1 + 0.95 RIA, n = 227, r = 0.969) through all the ranges of albumin concentration explored. INA seems as suitable as RIA for the assay of UAE in diabetics. The two methods are comparably accurate and precise. However, RIA appears more sensitive than INA, while immunonephelometric system is easier and faster to perform (up to 200 samples can be assayed in about 1 hour, while 2-4 hours are necessary with RIA), with fewer handling steps. The INA instrumentation is automated and the reagents are more stable and less hazardous than those used in RIA. However, due to the cost of the nephelometer apparatus, the INA technique may be employed by those laboratories which have already this instrumentation or by those which will use it in the next future for the assay of other analytes in addition to albumin.