Evidence of changes in renal charge selectivity in patients with type 1 (insulin-dependent) diabetes mellitus.

Altered filtration of macromolecules due to decreased electrical charge of the glomerular basement membrane might be the initial step in the development of albuminuria in patients with Type 1 (insulin-dependent) diabetes mellitus. We therefore investigated the selectivity index, i.e. renal clearance of non-glycated plasma albumin/clearance of glycated plasma albumin in 38 patients with Type 1 diabetes mellitus. The two albumin molecules differed slightly in charge, non-enzymatic glycated albumin being more anionic at physiological pH compared with unmodified plasma albumin. Glycated albumin in plasma and urine was determined by a specific, sensitive and highly reproducible chromatographic procedure. In diabetic patients with normal urinary albumin excretion, the selectivity index was increased three-fold compared with that of non-diabetic subjects (2 p less than 0.01). A significant correlation (r = 0.53, 2 p less than 0.01) between haemoglobin A1c and selectivity index was demonstrated in these patients, indicating a change in charge-dependent renal filtration could possibly be attributed to non-enzymatic glycation of components in the glomerular basement membrane and tubuli. Diabetic patients with increased albumin excretion rate had a significantly lower selectivity index compared with patients with normal albumin excretion (2 p less than 0.01). A significant negative correlation (r = 0.85, 2 p less than 0.001, exponential curve fit) was seen between urinary albumin excretion and selectivity index in the diabetic patients, indicating that the capability of differentiating between macromolecules of different charges is again lost with increasing urinary albumin excretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Smoking and glycaemic control in male insulin dependent (type 1) diabetics.

To test if cigarette smoking influences the quality of glycaemic control 98 consecutive male patients suffering from insulin dependent diabetes with onset of the disease before 30 yr of age and more than 5 yr duration of the disease were interviewed about their actual tobacco consumption, and their concentrations of haemoglobulin A1c were determined. Forty-seven patients smoked cigarettes, the rest (51) were regarded as non-smokers. Age, duration of diabetes, relative body weight, and insulin-requirement were similar in the 2 groups. No significant difference in glycaemic control as judged from the level of stable haemoglobin A1c was found (smokers 8.8%-range 6.5-11.4, and non-smokers 8.4%-range 6.4-11.6). Thus the measurement of haemoglobin A1c levels revealed no difference in glycaemic control according to smoking habits in insulin dependent diabetics.

Comparison of six assays for glycosylated haemoglobin determination.

To examine which glycosylated haemoglobin A components and which assays are the most useful in assessing long-term control in diabetic patients we have compared glycosylated haemoglobin concentrations in normal subjects and diabetic patients measured by six different methods, three chromatographic, a colorimetric, an isoelectric focusing and an agar gel electrochromatographic method. Despite the fact that the correlation between methods was high and the precision calculated from intra-assay variations acceptable, several differences in results were found. Thus Isolab columns determined lower values than other chromatographic methods and the unstable aldimine fraction interfered in agar gel electrochromatography. The increase in HbA1 in diabetics compared with normals was less than the corresponding increase in both HbA1c and total ketoamine bound glucose. This finding was consistent with the observation that the contribution of HbA1a + b fraction to HbA1 was constant at glucose concentrations above 10 mmol/l while a linear increase in these minor haemoglobins and consequently in HbA1 occurred at glucose concentrations below this level. We conclude that HbA1c' determined either by isoelectric focusing or ion exchange chromatography are the assays of choice for the determination of glycosylated haemoglobin in clinical routine.

Glycosylated haemoglobin and steady-state mean blood glucose concentration in Type 1 (insulin-dependent) diabetes.

Since glucose control and glycosylated haemoglobin varies asyncroneously, we have studied the steady-state relationship between these two factors. In Type 1 (insulin-dependent) diabetic patients with a constant haemoglobin A1c during the preceding 2 years, 15 ambulatory blood glucose profiles during a 5-week period showed a constant glucose level and provided a precise estimate of the mean blood glucose concentration. In addition, we studied 15 non-diabetic subjects who provided three glucose profiles and had one haemoglobin A1c determination performed. A good correlation was found for a curvilinear relationship (haemoglobin A1c = 2.07 X mean blood glucose0.596, r = 0.98). This close relationship indicates that glycosylated haemoglobin is a valuable, but not very sensitive, index of glucose control.

Haemoglobin F and the assessment of glucose control by chromatographically determined HbA1c levels.

HbF and HbA1c-levels were determined in diabetic patients and non-diabetic controls to evaluate the influence of HbF on chromatographically determined HbA1c-levels. No difference in mean HbF was observed between controls and diabetic patients. However, in the combined material, women had a higher mean value (0.31%) than men (0.20%). Estimates of time average blood glucose concentrations (Cglc, mmol/l) were calculated from HbA1c-values in diabetic patients corrected for codetermined HbF. This correction resulted in a difference greater than 1 mmol/l in only 4% of diabetic men and 28% of diabetic women compared to estimates of Cglc from uncorrected HbA1c-results. We conclude, however, that the influence of HbF on chromatographically determined HbA1c-levels in most cases is of minor importance in assessing glucose control in diabetic patients.

Synthesis of glycosylated haemoglobin in vivo.

The synthesis of glycosylated haemoglobins in vivo was measured during 24 h of controlled hyperglycaemia in seven insulin dependent diabetics. The mean blood glucose concentration was 22 mmol/l, while electrolytes and other metabolites were kept normal by infusion of 4-23 IU of insulin during hyperglycaemia. The study confirmed the velocity and magnitude of unstable HbAlc formation previously found in vitro. The stable HbAlc formed in 24 h was an average 0.006% of total haemoglobin/mmol glucose. This compares well with the rate of HbAlc synthesis reported in normal subjects using 59Fe-kinetic measurements, and is in accordance with the concept of slow changes in stable HbAlc with time and glucose concentration. To investigate the possibility that the rate of HbAlc synthesis varies with erythrocyte age, glycosylated haemoglobins were measured in erythrocyte fractions after density separation on Percoll-Albumin gradients. We found both in normal subjects and in insulin treated diabetics that the 5% least dense cells contained 70%-80% of whole blood HbAlc. Assuming the least dense cells to be the youngest erythrocytes, this observation is inconsistent with a slow linear increase in HbAlc. Similar results were obtained in six newly diagnosed insulin dependent diabetic patients both before and after the first 30 days of insulin treatment, even though a marked decrease in young cell HbAlc would be expected with the improved glucose control observed. We therefore conclude that density separation of erythrocytes is an inadequate technique to study age related HbAlc synthesis.

An artificial betacell: assessment of the glucose analyser, infusion system and optimization of constants for the algorithms.

The glucose analyser and insulin infusion modules of the Biostator were tested. The infusion system was reliable since more than 99% of the computed volume of insulin solution was delivered by the infusion pump at infusion rates above 1/100 of maximum, and no insulin was adsorbed onto infusion bags or tubing. Blood glucose results from the Biostator were compared with routine laboratory methods during long-term feedback control. Both slope (0.73) and scatter (r=0.87) around the regression line were unsatisfactory when the recommended calibration procedure was used. Tests in fasting non-diabetic subjects showed a significant correlation between the variation in Biostator glucose read-out and the plasma protein concentration in the detector outflow. In diabetics the ratio between Biostator glucose read-out and laboratory glucose determinations declined significantly with time. These observations led to the introduction of a standardization procedure based on externally determined blood glucose concentrations. During long-term feedback experiments in diabetics this procedure resulted in a significant increase in slope (0.84) but no improvement in scatter around the regression line. Repeated OGTTs revealed a set of constants for the algorithms, which enabled normal glucose tolerance to be achieved with smaller amounts of insulin.

Rapid changes in chromatographically determined haemoglobin A1c induced by short-term changes in glucose concentration.

Chromatographically determined haemoglobin A1c concentration was measured during short-term (1-24h) changes in glucose concentration in vitro and in vivo. In vitro at 37 degrees C the HbA1c concentration increased with glucose concentration and time both in normal and diabetic erythrocytes. In normal erythrocytes incubated in 20--100 mmol/l glucose, the increases in the HbA1c concentration were maximal after 4--6 h and then stable for the next 18--20 h. During the first hour, increases in the HbA1c concentration were linear with time and on average 0.034% HbA1c x h-1 x mmol/l glucose-1. In erythrocytes, after a rapidly produced increase (2 h), HbA1c decreased to preincubation concentrations during a further incubation of the erythrocytes in a glucose-free medium at 37 degrees C for 4--6 h. The mean rate of linear decrease was 0.017% x h-1 x mmol/l glucose-1. After incubation of erythrocytes in 100 mmol/l glucose for 24 h, 1.3% HbA1c remained stable for 6 h in saline. The rapid increase in HbA1c concentration, as determined by chromatography, was not due to stable HbA1c (ketoamine linked glucose) as no increase was found in the HbA1c concentrations determined by the thiobarbiturate method. In juvenile diabetics controlled by an artificial beta-cell, rapid changes of blood glucose concentration (up to 20 mmol/l) resulted in increases in HbA1c concentration of as much as 1.9% within 12 h (mean 1.1%). Rapid in vivo increases in HbA1c concentration were reversible by normalization of the blood glucose concentration. That rapid changes in HbA1c may occur in daily diabetic life was evidenced by differences in HbA1c concentration between blood samples from out-patient diabetics incubated in saline for 16 hours at 4 degrees C and 37 degrees C (range of differences 0.2--1.4% HbA1c). The differences correlated to the blood glucose concentration at the time of sampling blood for HbA1c determination. Thus, incubation of blood at a low glucose concentration prior to determination of the glycosylated haemoglobin concentration may overcome interference from rapidly produced HbA1c.