Different routes of insulin administration do not influence serum free thiols in type 1 diabetes mellitus

Abstract Aims Intraperitoneal (IP) insulin administration is a last‐resort treatment option for selected patients with type 1 diabetes mellitus (T1DM). As the IP route of insulin administration mimics the physiology more closely than the subcutaneous (SC) route, we hypothesized that IP insulin would result in less oxidative stress (expressed as systemic level of free sulphydryl (R‐SH) content) compared to SC insulin in subjects with T1DM. Materials and methods Prospective, observational case‐control study. Serum thiol measurements were performed at baseline and at 26 weeks in age‐ and gender‐matched patients with T1DM. Serum‐free thiols, compounds with a R‐SH group that are readily oxidized by reactive oxygen species, are considered to be a marker of systemic redox status. Results A total of 176 patients, 39 of which used IP and 141 SC insulin therapy were analysed. Mean baseline R‐SH concentration was 248 (31) μmol/L. In multivariable analysis, the route of insulin therapy had no impact on baseline R‐SH levels. The estimated geometric mean concentrations of R‐SH did not differ significantly between both groups: 264 (95% CI 257, 270) for the IP group and 258 (95% CI 254, 261) for the SC group with a difference of 6 (95% CI −2, 14) μmol/L. Conclusions Based on R‐SH as a marker of systemic oxidative stress, these findings demonstrate that the route of insulin administration, IP or SC, does not influence systemic redox status in patients with T1DM.


Currently, Continuous intraperitoneal insulin infusion (CIPII) is
used as a last-resort treatment option for selected patients with type 1 diabetes mellitus (T1DM) who fail to reach glycaemic control despite intensive subcutaneous (SC) insulin therapy. With CIPII, insulin is infused directly in the intraperitoneal (IP) space resulting in higher concentrations of insulin in the portal vein catchment area, higher hepatic insulin extraction and lower peripheral plasma insulin concentrations compared with SC insulin administration. [1][2][3] The aberrant production of reactive oxygen species (ROS), due to hyperglycaemia, is considered to be the central element of oxidative stress and, ultimately, plays an important role in the pathogenesis of T1DM, its progression and ultimately micro-and macrovascular complications. [4][5][6][7] Insulin has a pivotal role in the ROS production in T1DM through its effects on glucose-, IGF1-, lipid metabolism and its direct impact on the endothelium. 8 In previous studies, IP administration of insulin resulted in better HbA1c levels, lower glycaemic variability with a lower frequency of hypoglycaemia [9][10][11][12][13][14][15] and (near-) restoration of insulin-like growth factor (IGF)-1 metabolism [16][17][18][19] as compared to SC therapy. Given these effects, it was suggested that not only the insulin level, but also the route of administration might be of importance in the regulation of the redox status in T1DM. 20,21 Indeed, in the animal model, delivering the same dose of insulin IP resulted in lower hepatic oxidative stress and inflammation as compared to continuous SC insulin delivery. 20 To date, however, there are no data on the effect of the route of insulin administration on whole-body redox status in humans. We hypothesized that the route of insulin administration affects the systemic redox status and that the IP route may have a beneficial effect compared with SC insulin therapy. We therefore investigated the effects of CIPII compared with SC insulin administration on redox status in a prospective, observational, matched case-control study in patients with T1DM.

| Study design, aims and outcomes
This multicentre study was investigator-initiated and had a prospective, observational matched case-control design. Inclusion took place at Isala hospital (Zwolle, the Netherlands) and Diaconessenhuis hospital (Meppel, the Netherlands). Primary aim of this study was to compare the effects of long-term IP insulin delivery to SC insulin delivery, with respect to glycaemic control. Aim of the present analysis was to test the hypothesis that IP insulin therapy would result in a more favourable redox status compared with SC insulin therapy.
Serum-free thiols, compounds with a free sulphydryl (R-SH) group that are readily oxidized by reactive oxygen species, were used as marker systemic redox status. From the measures available to measure ROS, we considered R-SH as an appropriate measure for oxidative stress in the current study: R-SH are a robust and powerful read-out of the systemic in vivo reduction-oxidation (redox) status. 22 In previous studies, in a variety of diseases, R-SH have been linked with oxidative stress and clinical outcome. 23 Secondary outcomes include subanalyses for MDI-and CSII-treated patients and a multivariable regression analysis with baseline R-SH concentrations as outcome variable.

| Patient selection
Cases were subjects on IP insulin therapy using an implantable insulin pump (MIP 2007D, Medtronic/MiniMed) for the past 4 years without interruptions of >30 days, in order to avoid effects related to initiating therapy. Inclusion criteria for cases were have been described in detail previously. 9 In brief, patients with T1DM, aged 18 to 70 years who fulfilled abovementioned criteria for CIPII and had an HbA1c ≥ 58 mmol/mol and/or ≥5 incidents of hypoglycaemia (defined as glucose < 4.0 mmol/L) per week, were eligible. The SC control group was age-and gender-matched to the cases and consisted of patients with T1DM, using either multiple daily subcutaneous injections (MDI) or continuous subcutaneous insulin infusion (CSII)), for the past 4 years without interruptions of >30 days and a HbA1c at time of matching ≥ 53 mmol/ mol. Exclusion criteria for the present study for both cases and controls included the following: impaired renal function (plasma creatinine ≥ 150 µmol/L or Cockcroft-Gault ≤ 50 mL/min), cardiac problems (unstable angina or myocardial infarction within the previous 12 months or NYHA class III or IV congestive heart failure), cognitive impairment, current or past psychiatric treatment for schizophrenia, cognitive or bipolar disorder, current use of oral corticosteroids or suffering from a condition which necessitated corticosteroids use more than once in the previous 12 months, alcohol or drug abuse, current gravidity or plans to become pregnant during the study. 24 The ratio of participants on the different therapies (CIPII:MDI:CSII) was 1:2:2.

| Study protocol
There were four study visits. During the first visit, baseline characteristics were collected using a standardized case record form.
During the second visit (5-7 days later), laboratory measurements were performed. During the third visit, 26 weeks after visit 1, clinical parameters were collected. During the fourth visit, 5-7 days after the third visit, laboratory measurements were performed.
Throughout the study period, insulin (human insulin of E. Coli origin, 400 IU/mL, trade name: Insuman Implantable ® , Sanofi-Aventis) was administered with an implantable pump for IP insulin users and patients using CSII or MDI continued their own insulin regime consisting of fast-acting insulin analogues and for MDI patients also long-acting insulin analogues or NPH insulin. All patients received standard care. The implantable insulin pump used during this study and related procedures has been described in more detail previously. 25,26

| Measurements
Demographic and clinical parameters included the following: age, gender, weight, length, blood pressure, smoking and alcohol habits, The 24-hours interstitial glucose profiles were recorded using a blinded CGM device (iPro2, Medtronic). The CGM device was inserted in the periumbilical area, and in pump users contralateral to the (implanted) insulin pump. Patients were instructed to perform a minimum of 4 blood glucose self-measurements daily during the CGM period, using a blood glucose metre (Contour XT; Bayer) to calibrate the sensor. All procedures related to the CGM were performed by one, trained physician (PRvD).

| Statistical analysis
Results were expressed as mean (with standard deviation (SD)) or median (with interquartile range [IQR]) for normally distributed and non-normally distributed data, respectively. A significance level of 5% (two-sided) was used. Normality was examined with Q-Q plots. Differences between the IP and SC groups averaged over the study period and in time were estimated using the general linear model.
A regression model based on covariate analysis (ANCOVA) was applied in order to adjust for possible baseline imbalances. In the model, the fixed factors CIPII and SC insulin therapy were used as determinants. The difference in scores was determined based on the b-coefficient of the particular (CIPII or SC) group. Significance of the b-coefficient was investigated with the Wald test based on a P < .05.
The quantity of the b-coefficient, with a 95% CI, gives the difference between both treatment modalities over the study period adjusted for baseline differences. Subsequently, all variables that associated with R-SH with a P-value of <.1 were included in the multivariable linear regression using backward selection. The quality of the model was described using the accuracy of the prediction by the adjusted R 2 value. In order to avoid collinearity, only the coefficient of variation (CV) of the CGM measurements was used. The CV measures intraday variation in glucose patterns, is defined as the SD divided by the mean of blood glucose values and is advocated to be the most optimal measure of glycaemic variability. [30][31][32] Statistical analyses were performed using SPSS (IBM SPSS Statistics for Windows, Version 20.0. , IBM Corp). The study protocol was registered prior to the start of the study (NCT01621308 and NL41037.075.12) and approved by the local medical ethics committee. All patients gave informed consent.  Table 1).   Baseline R-SH concentrations were normally distributed with a mean concentration of 248 (31) μmol/L (see Appendix S1). According to the multivariate model, factors that had an independent, inverse relation with baseline R-SH concentrations were age and BMI (see Table 2), whereas fasting glucose and albumin concentrations had a positive relation.  Table 3). This increase was present in both the IP Route of insulin administration (SC = 1) 0.112 .143

| D ISCUSS I ON
The findings in this 26-week study suggest that the route of insulin administration, IP or SC, does not influence the systemic redox status in patients with T1DM. Although R-SH concentrations at baseline and at the end of the study were higher among T1DM patients treated with IP insulin, this was not significantly different as compared to the group of patients treated with SC insulin.
In the only previous study (by Dal   findings. And although we prespecified redox status as a secondary outcome in the study protocol, no separate power calculation was performed to detect potential relevant differences in R-SH. By using the directions of the 95% confidence intervals, one could hypothesize that there are undetected differences in R-SH between the IP and SC group. Finally, the lack of information with regard to other plasma antioxidant species such as ascorbate, uric acid and smallmolecular-weight thiols and markers of inflammation (due to cost constraints) should be mentioned.
In conclusion, the findings in this study demonstrate that the route of insulin administration, IP or SC, does not influence systemic redox status in subjects with T1DM, at least, measured as per R-SH group detection.

ACK N OWLED G EM ENTS
The authors would like to thank Marian Bulthuis and the department of Clinical Chemistry of Isala for their help in the laboratory analysis.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no financial or other relationships that might lead to a conflict of interest. PRv.D. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

E TH I C A L A PPROVA L
The study protocol was in compliance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. The study protocol was registered prior to the start of the study (NCT01621308 and NL41037.075.12) and approved by the local medical ethics committee. All patients gave informed consent.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data will be made available by the authors upon (reasonable) request.