Residual (5-Cell Function in Children With IDDM ... - Diabetes Care

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esidual (5-Cell Function in Children With IDDM: Reproducibility of Testing and Factors Influencing Insulin Secretory Reserve

CHERIL CLARSON, BM, BS, FRCP(C), DENIS DANEMAN, MD, BCh, FRCP(C), ALLAN L. DRASH, MD, DOROTHY J. BECKER, MB, BCh, FCP(SA), AND ROBERT M. EHRLICH, MD, FRCP(C)

Reproducibility of C-peptide secretion was assessed in 20 children (group 1) by their responses to two Sustacal- (a mixed liquid meal) stimulation tests performed 7-14 days apart. For the 12 C-peptidepositive children (basal C-peptide ^0.03 pmol/ml) there were no differences in the basal or stimulated values between tests 1 and 2. The effect of exogenous insulin on C-peptide secretion was assessed in 20 other children (group 2) by their responses to two Sustacal tests, one test without and one with soluble insulin (0.25 U/kg) injected subcutaneously before testing. Eleven children were C-peptide positive and had no differences in C-peptide response between tests 1 and 2. The results from test 1 in groups 1 and 2 were combined with those from 44 others undergoing a single Sustacal test (group 3, N = 84). There was a close correlation between basal and peak C-peptide concentrations in the 44 C-peptide-positive children (r = .88, P < .001). Peak C-peptide concentrations correlated inversely with HbAi (r = —.29, P < .01), insulin dose in units per kilogram (r = —.40, P < .001), and duration of diabetes (r = .33, P < .001) and positively with age at onset of diabetes (r = .34, P < .001). The C-peptide-positive children had reduced glucose response to Sustacal, lower HbA, concentration, lower insulin requirement, later age of onset, and shorter duration of diabetes than children who were C-peptide negative. Diabetes Care 10:33-38, 1987

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he measurement of C-peptide is valuable in studying the natural course of residual (3-cell function in children with insulin-dependent diabetes mellitus (IDDM). An increase in C-peptide concentrations has been found from several days to weeks after the diagnosis of diabetes (1,2). Studies indicate that during the first 2 yr of disease, many patients have some residual (3-cell function, whereas by 6-8 yr, only traces of C-peptide are detectable (3-5). Accurate measurement of C-peptide may be valuable in assessing children with IDDM because even minimal residual P-cell function appears to be important in achieving more physiologic glucose homeostasis (3,6). It is also essential in providing baseline data for intervention studies aimed at preserving (3-cell function after the "honeymoon" (remission) period (7,8). Several important aspects of C-peptide measurements have not been adequately addressed. The reproducibility of Cpeptide concentrations obtained in response to the same stimulus has not been evaluated, only the responses to different

stimuli (5,9). The effect of exogenous insulin on C-peptide levels has also not been assessed. The relationship between fasting/basal and stimulated C-peptide levels has been investigated with conflicting results. There are reports of a significant correlation between basal and stimulated C-peptide levels in response to glucagon and glucose (10,11). However, Mirel et al. (5) found no correlation of basal concentrations and the peak attained in response to glucose, tolbutamide, or glucagon. The primary aim of this study was to evaluate two aspects of C-peptide measurement not previously reported: I) reproducibility of C-peptide response to the same physiologic stimulus and 2) the effect of exogenous insulin on the Cpeptide response. The second aim was to contribute to reported data on C-peptide by assessing i) the relationship of basal and stimulated C-peptide concentrations and 2) factors influencing continuing residual P-cell function (e.g., sex, age of onset, and duration of diabetes) and its impact on metabolic control.

DIABETES CARE, VOL. 10 NO. 1, JANUARY-FEBRUARY 1987

RESIDUAL 3-CELL FUNCTION IN CHILDREN WITH IDDM/C. CLARSON AND ASSOCIATES

PATIENTS AND METHODS

We studied 84 children with IDDM, diagnosed by ketonuria at onset and by continuing insulin dependence. The mean age ± SD at testing was 13.7 ± 2.6 yr (range 7.2-18.8 yr), and the mean duration of diabetes was 3.9 ± 2.5 yr (range 0.5-11.5 yr). Subjects were chosen at random from children aged 7—18 yr attending the Diabetes Clinic at the Hospital for Sick Children, Toronto (N = 44), and the Children's Hospital of Pittsburgh, Pennsylvania (N = 40). The clinical characteristics of the subjects from the two clinics were not significantly different in terms of sex distribution, age at testing, or duration of disease. The children were studied in three groups. Group 1 consisted of 20 subjects in whom reproducibility of C-peptide secretion was assessed. Two Sustacal- (Mead Johnson, Belleville, Ontario, Canada) stimulation tests were performed 7— 14 days apart. On both occasions, the morning insulin dose was withheld until after test completion. Group 2 consisted of 20 subjects in whom the effect of exogenous insulin on reproducibility of C-peptide secretion was assessed. The first Sustacal-stimulation test was performed as in group 1. On the second occasion, 7-14 days later, soluble (crystalline) insulin (0.25 U/kg) was administered 15 min before Sustacal ingestion. Group 3 consisted of 84 subjects. The results from test 1 in groups 1 and 2 were combined with those from 44 others undergoing a single Sustacal test. Sustacal, a mixed liquid meal, was used as the stimulus for residual P-cell function because a precise, and therefore reproducible, caloric intake could be administered, and the effects of the gastrointestinal hormones on insulin secretion (enteroinsular axis) would be an integral part of the test. Sustacal contains 6.1 g/dl protein, 24 g/dl fat, and 14 g/dl carbohydrate and provides 1 kcal/ml. After an overnight fast, an indwelling catheter was placed in an antecubital vein. Blood samples were obtained at 0 (basal), 15, 30, 45, 60, 90, and 120 min after ingestion of 7 ml/kg of Sustacal in groups 1 and 2 and at 0, 60, and 120 min in group 3. Blood for C-peptide determinations was collected in tubes containing Kallikrein inactivating units of aprotinin per milliliter of blood. Serum was stored at — 20°C. C-peptide concentrations were measured by radioimmunoassay with the antiserum Ml230 (12). Intra- and interassay coefficients of variation were 5.6 and 9.1%, respectively. The lower limit of detection for this assay is 0.025 pmol/ml. All samples from one subject were analyzed in the same assay. C-peptide concentrations are reported as those obtained in unextracted serum. We (data not shown) and others (14) have found no significant differences in C-peptide concentration with and without polyethylene glycol (PEG) extraction. The PEG method in our experience has a greater variability, as shown by higher coefficients of variation (>10%). The specimens were handled in a similar manner and run by the same individual in both institutions (D.D.). In addition, all characteristics of the C-peptide assay were identical. Serum glucose was measured by a glucose oxidase method (Ektachem). HbA lc concentrations were determined

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by minicolumn chromatography after removal of the labile fraction (13). The data were analyzed on the VAX-750 computer operating system with the Minitab statistical package. Student's t test for paired means and linear regression were used for the remainder of the statistical analyses. Results are expressed as means ± SD unless otherwise stated. Patients whose basal C-peptide concentrations were ^0.03 pmol/ml were considered to have residual p-cell function (C-peptide positive), whereas those whose basal measurements were below this level were designated C-peptide negative. We chose this cutoff concentration in view of the fact that all subjects with basal C-peptide 0.03 pmol/ml showed a significant increase after Sustacal. RESULTS

Group I. Twelve of the 20 children studied were C-peptide positive and 7 were C-peptide negative on both occasions. Only one child was negative on one test and positive on the other (peak C-peptide on 2nd test, 0.05 pmol/ml). Table 1 illustrates basal, peak, and maximum change for C-peptide and glucose concentrations in the C-peptide-positive subjects in this group. There were no differences in these values between tests 1 and 2 for the whole group or for subjects who were C-peptide positive. Figure 1 shows the correlation between peak C-peptide concentrations in tests 1 and 2 in group 1 subjects (N = 20, r = .965, P < .001). A similar correlation was found for the basal C-peptide concentrations (r = .936). Group 2. Eleven of the 20 children in this group were C-peptide positive on both occasions. Nine children were C-peptide negative on both tests. The basal and stimulated C-peptide and glucose measurements are shown in Table 2. Although the glucose response was suppressed in test 2, there were no differences in stimulated C-peptide measurements between tests 1 and 2. Figure 2 shows the correlation between the peak C-peptide concentrations in tests 1 and 2 in group TABLE 1 Reproducibility of residual 3-cell secretory responses in C-peptide—positive children (N = 12)

C-peptide (pmol/ml) Basal Peak Amax Glucose (mM) Basal Peak Amax

Test 1

Test 2

0.09 ± 0.06 0.18 ± 0.11 0.09 ± 0.007

0.10 ± 0.06 0.17 ± 0.10 0.08 ± 0.05

10.2 ± 4.6 18.9 ± 4.9 8.7 ±3.4

9.9 ± 3.4 18.5 ± 4.3 8.4 ± 3.0

Amax, maximum change. Differences between tests 1 and 2 are not significant.

DIABETES CARE, VOL. 10 NO. 1, JANUARY-FEBRUARY 1987

RESIDUAL (3-CELL FUNCTION IN CHILDREN WITH 1DDM/C. CLARSON AND ASSOCIATES

ently of disease duration. Girls (N = 35) had lower basal C-peptide concentrations than boys (P < .05) and higher insulin requirements (P < .02). The group of 40 children who were C-peptide negative had an increased glucose response, a higher HbA, concentration, a higher insulin requirement, an earlier age of onset, and longer duration of diabetes than children who were Cpeptide positive (Table 3). Frequency of insulin injections (1 vs. 2 times/day) did not differ in the C-peptide-positive versus —negative subjects.

Peak . 3 C-peptide (pmol/ml): Test 1

.2 .3 .4 Peak C-peptide (pmol/ml):Test 2 FIG. 1. Reproducibility of Sustacal test. Correlation of peak Cpeptide concentrations in tests 1 and 2 in group 1 children.

DISCUSSION

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espite extensive investigation of residual (3-cell function in IDDM, the reproducibility of the Cpeptide response to the same stimulus has not been reported previously; only the response to different stimuli has been reported (5,9,14). There are reports of patients responding differently to various stimuli. Mirel et al. (5) found that C-peptide responses were greater after glucagon than after glucose or tolbutamide, and Heinze et al. (15) reported a significant C-peptide response to an arginine infusion but a minimal response to glucose in the same patients. Alternatively, Faber and Binder (9) found a close correlation between the C-peptide response to a standard meal and to glucagon. In addition, the effect of conventional doses of exogenous insulin on the C-peptide response has not been reported. Our results reveal highly reproducible C-peptide responses to Sustacal stimulation. The dose of insulin employed in group 2 in this study did not appear to suppress the C-peptide response, despite significant suppression of the glucose response. Therefore the data confirm that the results of a single Sustacal-stimulation test indicate residual P-cell function in a particular time frame, even in the presence of exogenous insulin in dosages such as those employed in the subjects in group 2. Note that all patients, including group 2 subjects receiving subcutaneous insulin, were significantly hyperglycemic throughout. C-peptide re-

2 subjects (N = 20, r = .974, P < .001). The correlation for basal concentrations was r = .967. Thus, the dose of soluble insulin used in test 2 was not sufficient to suppress the (maximal) C-peptide response, despite a significantly reduced glucose response. In both groups 1 and 2, frequency of insulin injections did not interfere with test reproducibility. Group 3. Forty of the 84 children in this group had basal C-peptide concentrations