L-Arginine Test to Evaluate Insulin Secretory Reserve

ONLINE APPENDIX

Thirty-seven patients who received islet transplant subsequent to kidney transplant were retrospectively studied. The patients received an islet transplant according to ABO compatibility and a triple immunosuppressive regimen (steroids, cyclosporine, and mofetil mycophenolate) plus induction with ATG (SANGSTAT) at the time of islet transplant.

Patients were retrospectively split into long-term functioning (Long fx=17 patients with C-peptide> 1.0 ng/ml for more than 12 months) or short-term functioning (Short fx=20 patients who lost C-peptide secretion> 1 ng/ml within 12 months) groups. All the data were expressed as mean  standard error. The day of metabolic tests, subcutaneous administration of long-acting insulin was suspended the night before and replaced with shots of short-acting insulin.

ELISA

Human proinsulin levels were assayed with an ELISA kit with no cross-reactivity with human insulin (DAKO Diagnostics Ltd., UK). The minimum detectable dose was 6 pmol/l. Intra-assay CV was 3.5% and inter-assay CV was 5.5%. Serum C-peptide levels (intra-assay CV 3.0%; interassay CV 3.0%) were assayed by RIA using commercial kits (Medical System, Genoa, Italy). Human APO-1/FAS levels were assayed with an ELISA kit (BioSource Europe S.A., Belgium). No cross-reactivity with SIL-2R, sICAM-1, sVCAM-1, TNF- was demonstrated, and the minimum detectable level was <2 ng/ml. Intra-assay CV was 4.9% and inter-assay CV was 8.9%. Human sFAS Ligand levels were assayed with an ELISA kit (Bender MedSystemsDiagnostics GmbH, Austria). The minimum detectable dose was 0.1 ng/ml. Intra-assay CV was 6.1% and inter-assay CV was 7.0%. Human sIL-2R levels were assayed with an ELISA kit (BioSource Europe). The minimum detectable dose was <12 pg/ml. Intra-assay CV 5.5%, inter-assay CV 6.1%. Human Activin A levels were assayed with an ELISA kit (RD System Europe LtD, UK). The minimum detectable dose was <1.5 pg/ml. Intra-assay CV 5.0%, inter-assay CV 9.0%. Human Amylin levels were assayed with an ELISA kit (Peninsula Laboratories, [name city, state USA]). No cross-reactivity with insulin, proinsulin, glucagon, or somatostatin was demonstrated, and the minimum detectable dose was 0.05 ng/ml. An intra-assay CV of 5.0% and an inter-assay CV of 14% were demonstrated.

L-Arginine test to evaluate insulin secretory reserve

The patients were given the following tests: oral glucose tolerance test (OGTT: 75 g glucose with samples for blood glucose and plasma free insulin at 0, 30, 60, 90, 120, 180 min); L-arginine infusion (30 g, iv, as a 10% solution given over 30 min with samples for plasma free insulin at 0, 5, 10, 20, 30, 45, 60, 90, 120 min). These tests were performed with the patient in a recumbent position at 09.00 h after an overnight fast. The tests were performed at 3-6 months after transplant, 1, 2, 3, and 4 years after transplantation. Not all the patients were given all the tests at every interval: tests were performed according to the clinical conditions of the patient or the duration of follow-up. When islet-transplanted patients were not insulin-free, insulin was withdrawn at least 12 h before testing. Informed consent was obtained from all patients. Calculations: the peak of insulin secretion after i.v. glucose was calculated as the difference between the first minute serum free insulin values and basal values for each patient. The area under the curve (AUC) of serum insulin levels was calculated according to the trapezoidal method for the arginine test (delta AUC: 0–60 min).

Insulin Resistance

HOMA-IS (Homeostasis model assessment of insulin sensitivity) and HOMA-IR (Homeostasis model assessment of insulin resistance) have been used as markers of insulin resistance and are calculated as follows:

HOMA-IS= 22.5/[Fasting insulin (U/ml) x Fasting glycemia (mmol/l)]. The normal value is 1. Then we calculated the HOMA-IR= 1/HOMA-IS.

Autoantibody measurements

GADAs (antibodies to glutamic acid decarboxylase) and IA-2As (autoantibodies to protein tyrosine phosphatase isoforms IA-2) were measured using a radiobinding assay with in vitro–translated [35S]methionine-labeled GAD65 and IA-2IC (amino acids 605–979) as previously reported1. GADAs are expressed as arbitrary units. Results for IA-2As were expressed as an index defined as follows: (sample counts per minute – negative standard control counts per minute)/(positive standard control counts per minute – negative standard control counts per minute). The thresholds for positivity were determined from the 99th percentile of control subjects and corresponded to 3 U for GADAs and index 0.010 for IA-2As, GADA sensitivity 84, 86, and 88%; GADA specificity 97, 97, and 92%; IA-2A sensitivity 60, 62, and 70%; and IA-2A specificity 100, 99, and 99%, respectively. The intra- and interassay coefficients of variation of GADA for control samples designated at 10 GADA U were 9% and 17%, respectively, and for IA-2IC were 4.8% and 9.9%, respectively.

Statistical analysis

All the data were expressed as mean  standard error. Data were tested for normal distribution with the Kolmogorov-Smirnov test and for homogeneity of variances with Levene’s test. Differences between parameters were evaluated using Student’s T test when parameters were normally distributed, Mann Whitney U test when parameters were not normally distributed, and a chi-square test for categorical variables. Two-sided paired Student t-test (for parametric data) and Kruskal-Wallis test (for non-parametric data) were used to compare baseline parameters with follow-up values. Correlations were assessed with a Spearman rank correlation coefficient. A p value of less than 0.05 (by two-tailed testing) was considered an indicator of statistical significance. Analysis of data was done using the SPSS statistical package for Windows (SPSS Inc., Chicago,IL).

Table 1. The two groups of patients were similar in terms of pre-transplant characteristics and number of transplanted islets.

References

1.Bonifacio E, Genovese S, Braghi S, et al. Islet autoantibody markers in IDDM: risk assessment strategies yielding high sensitivity. Diabetologia. 1995;38:816-822.