Glycemic Variability in T1DM: Insulin Infusion vs Injections
All children suffering from type 1 diabetes and being treated at our institution were eligible for this cross-sectional observational cohort study. Inclusion criteria: age 6–18 years, duration of disease > 1 year, C-peptide below 0·3 nmol/l, intensive insulin treatment with either MDI or CSII, constant mode of insulin therapy for at least 6 months. Exclusion criteria: any febrile illness during the last three months, chronic inflammatory-/rheumatic disease (e.g. Crohn's disease, rheumatoid arthritis), hepatitis, HIV, glucocorticoid treatment, liver-, renal- or cardiac failure, hereditary dyslipidaemia, skin conditions prohibiting sensor needle insertion, inborn or acquired errors of metabolic disease except type 1 diabetes. Forty-eight patients (22 boys) consented to participate and were enrolled between January and December 2010. Patient characteristics are given in Table 1. CSII treatment was introduced in our centre in 2007 and rarely used as first line treatment before 2010. Thus, in this study, the majority of patients on CSII treatment were switched from MDI mainly to prevent recurrent hypoglycaemia and patients (or parents) wish. In patients with CSII therapy (n = 22) solely short-acting insulins were used, while patients on MDI therapy (n = 26) received a combination of either short- and long-acting insulins (n = 8), short- and intermediate-acting insulins (n = 9), normal and long-acting insulins (n = 5), or short-, normal and long-acting insulins (n = 4). Patients applied 4 to 5 insulin boli per day. The study received appropriate Ethics committee approval from the institutional review board and was performed in accordance with the Declaration of Helsinki. Subjects and/or their parents gave assent and written informed consent prior to participation, i.e. to undergo continuous glucose monitoring (CGM) for a 72-h period.
All children and adolescents were seen in our outpatient clinic in the afternoon. Demographic and clinical data including yearly assessments of retinopathy by a trained ophthalmologist as well as results from routine laboratory analysis were gathered by interview and chart review, respectively. Furthermore, blood was taken for determination of actual HbA1c, lipids and creatinine to estimate the glomerular filtration rate (eGFR) according to the Schwartz formula. Pubertal stage was assessed according to Tanner and blood pressure (BP) was measured according to the updated Task Force Report on high blood pressure by using an oscillometric device (Dinamap 1846SX; Critikon, Tampa, USA). Individual age- (height) and gender-related SD scores (SDS) for height, weight, BMI and BP were calculated as described. Patients were classified as hypertensive in case of BP values above the height and gender-related 95 percentile.
The patients and/or parents were instructed to the use of the CGM (Guardian REAL-Time, Medtronic, Northridge, USA) and to enter a minimum of two blood glucose values per day for calibration. It should be noted that none of the patients enrolled in this study had used CGM before. The sensor was placed subcutaneously and in CSII patients contralaterally to the catheter by the same investigator (CS) in all subjects. Patients on MDI therapy were asked to inject insulin contralaterally to the site of the sensor. Patients/parents kept a study diary and recorded meals, symptomatic hypo- and hyperglycaemic events and sport activities. The patients were asked to collect urine for determination of albumin, F2-isoprostanes and PGF2 during two consecutive 24-h periods. At the end of the 72-h observation period, the CGM sensor was removed and data transfer was achieved using the carelink personal therapy management software (Medtronic, Northridge, USA).
Urine for determination of F2-isoprostane and PGF2 was available from 45 patients (25 on MDI therapy) and aliquots were initially stored at −80 °C until quantitative determination of FISO and PGF2 by gas chromatography/triple quadrupole mass spectrometry (GC-MS/MS) essentially as described previously. Per urine an aliquot was spiked with deuterated internal PGF2 standard (IS), acidified with formic acid to pH 2.6, extracted with ethyl acetate/hexane and subsequently derivatized to the correspondent pentafluorobenzyl esters. Samples were purified by means of thin-layer chromatography with ethyl acetate/hexane as developing solvent. A broad zone with Rf 0.03–0.34 was scraped off, eluted with pure ethyl acetate and subjected to preparation of trimethysilyl ethers. A Finnigan MAT TSQ700 mass spectrometer (Thermo Electron, Dreieich, Germany) equipped with a Varian 3400 gas chromatograph (Palo Alto, CA) and a CTC A200S autosampler (Zwingen, Switzerland) was used. The first quadrupole was set to monitor the [M-PFB] ions (m/z 569 for PGF2a and m/z 573 for tetradeuterated IS). Collision cell pressure was 0.2 Pa and collision energy was 12 eV). For F2-isoprostane determination, product ions [M-3TMSOH] (m/z 299 and m/z 303 for the internal standard) were monitored. In a second run for quantification of PGF2 product ions [C12H17] (m/z 161) and [C12H4H13] (m/z 165 for the tetradeuterated internal standard) and a collision energy of 16 eV were used.
Data from the 3-day CGM were used to calculate i) the mean of the daily differences (MODD), ii) the continuous overlapping net glycaemic action for one, two and four hours (CONGA-1, -2, -4), and iii) the mean of glycaemic excursions (MAGE) as described previously. In addition, the frequency of mild and severe hypoglycaemias, i.e. glucose concentrations below 3.9 mmol/l and 2·8 mmol/l, was counted. The mean glucose concentration, the standard deviation of the mean glucose concentration, the mean length of time with glucose concentration > 8·88 mmol/l, the intensity of hyperglycaemia (area under the curve (AUC) for glucose concentration > 8·88 mmol/l) as well as the HbA1c were used to generate the glucose pentagon and to calculate the Glycaemic Risk Parameter (GRP) as described before.
Data were analysed using the SPSS statistical package 15.0 (SPSS Inc. Chicago, Illinois, USA). Normal distribution was evaluated by the Kolmogorow-Smirnow test and comparison between groups were done using Student's t-Test or Mann–Whitney-U test, if appropriate. All P-values are two sided and a P-value below 0·05 was considered significant. Data are given as mean ± SD or median and range, if appropriate.
Patients and Methods
All children suffering from type 1 diabetes and being treated at our institution were eligible for this cross-sectional observational cohort study. Inclusion criteria: age 6–18 years, duration of disease > 1 year, C-peptide below 0·3 nmol/l, intensive insulin treatment with either MDI or CSII, constant mode of insulin therapy for at least 6 months. Exclusion criteria: any febrile illness during the last three months, chronic inflammatory-/rheumatic disease (e.g. Crohn's disease, rheumatoid arthritis), hepatitis, HIV, glucocorticoid treatment, liver-, renal- or cardiac failure, hereditary dyslipidaemia, skin conditions prohibiting sensor needle insertion, inborn or acquired errors of metabolic disease except type 1 diabetes. Forty-eight patients (22 boys) consented to participate and were enrolled between January and December 2010. Patient characteristics are given in Table 1. CSII treatment was introduced in our centre in 2007 and rarely used as first line treatment before 2010. Thus, in this study, the majority of patients on CSII treatment were switched from MDI mainly to prevent recurrent hypoglycaemia and patients (or parents) wish. In patients with CSII therapy (n = 22) solely short-acting insulins were used, while patients on MDI therapy (n = 26) received a combination of either short- and long-acting insulins (n = 8), short- and intermediate-acting insulins (n = 9), normal and long-acting insulins (n = 5), or short-, normal and long-acting insulins (n = 4). Patients applied 4 to 5 insulin boli per day. The study received appropriate Ethics committee approval from the institutional review board and was performed in accordance with the Declaration of Helsinki. Subjects and/or their parents gave assent and written informed consent prior to participation, i.e. to undergo continuous glucose monitoring (CGM) for a 72-h period.
Procedures
All children and adolescents were seen in our outpatient clinic in the afternoon. Demographic and clinical data including yearly assessments of retinopathy by a trained ophthalmologist as well as results from routine laboratory analysis were gathered by interview and chart review, respectively. Furthermore, blood was taken for determination of actual HbA1c, lipids and creatinine to estimate the glomerular filtration rate (eGFR) according to the Schwartz formula. Pubertal stage was assessed according to Tanner and blood pressure (BP) was measured according to the updated Task Force Report on high blood pressure by using an oscillometric device (Dinamap 1846SX; Critikon, Tampa, USA). Individual age- (height) and gender-related SD scores (SDS) for height, weight, BMI and BP were calculated as described. Patients were classified as hypertensive in case of BP values above the height and gender-related 95 percentile.
The patients and/or parents were instructed to the use of the CGM (Guardian REAL-Time, Medtronic, Northridge, USA) and to enter a minimum of two blood glucose values per day for calibration. It should be noted that none of the patients enrolled in this study had used CGM before. The sensor was placed subcutaneously and in CSII patients contralaterally to the catheter by the same investigator (CS) in all subjects. Patients on MDI therapy were asked to inject insulin contralaterally to the site of the sensor. Patients/parents kept a study diary and recorded meals, symptomatic hypo- and hyperglycaemic events and sport activities. The patients were asked to collect urine for determination of albumin, F2-isoprostanes and PGF2 during two consecutive 24-h periods. At the end of the 72-h observation period, the CGM sensor was removed and data transfer was achieved using the carelink personal therapy management software (Medtronic, Northridge, USA).
Consecutive Quantification of Urinary F2-isoprostanes and PGF2 by GC-MS/MS
Urine for determination of F2-isoprostane and PGF2 was available from 45 patients (25 on MDI therapy) and aliquots were initially stored at −80 °C until quantitative determination of FISO and PGF2 by gas chromatography/triple quadrupole mass spectrometry (GC-MS/MS) essentially as described previously. Per urine an aliquot was spiked with deuterated internal PGF2 standard (IS), acidified with formic acid to pH 2.6, extracted with ethyl acetate/hexane and subsequently derivatized to the correspondent pentafluorobenzyl esters. Samples were purified by means of thin-layer chromatography with ethyl acetate/hexane as developing solvent. A broad zone with Rf 0.03–0.34 was scraped off, eluted with pure ethyl acetate and subjected to preparation of trimethysilyl ethers. A Finnigan MAT TSQ700 mass spectrometer (Thermo Electron, Dreieich, Germany) equipped with a Varian 3400 gas chromatograph (Palo Alto, CA) and a CTC A200S autosampler (Zwingen, Switzerland) was used. The first quadrupole was set to monitor the [M-PFB] ions (m/z 569 for PGF2a and m/z 573 for tetradeuterated IS). Collision cell pressure was 0.2 Pa and collision energy was 12 eV). For F2-isoprostane determination, product ions [M-3TMSOH] (m/z 299 and m/z 303 for the internal standard) were monitored. In a second run for quantification of PGF2 product ions [C12H17] (m/z 161) and [C12H4H13] (m/z 165 for the tetradeuterated internal standard) and a collision energy of 16 eV were used.
Assessment of Glycaemic Control
Data from the 3-day CGM were used to calculate i) the mean of the daily differences (MODD), ii) the continuous overlapping net glycaemic action for one, two and four hours (CONGA-1, -2, -4), and iii) the mean of glycaemic excursions (MAGE) as described previously. In addition, the frequency of mild and severe hypoglycaemias, i.e. glucose concentrations below 3.9 mmol/l and 2·8 mmol/l, was counted. The mean glucose concentration, the standard deviation of the mean glucose concentration, the mean length of time with glucose concentration > 8·88 mmol/l, the intensity of hyperglycaemia (area under the curve (AUC) for glucose concentration > 8·88 mmol/l) as well as the HbA1c were used to generate the glucose pentagon and to calculate the Glycaemic Risk Parameter (GRP) as described before.
Statistics
Data were analysed using the SPSS statistical package 15.0 (SPSS Inc. Chicago, Illinois, USA). Normal distribution was evaluated by the Kolmogorow-Smirnow test and comparison between groups were done using Student's t-Test or Mann–Whitney-U test, if appropriate. All P-values are two sided and a P-value below 0·05 was considered significant. Data are given as mean ± SD or median and range, if appropriate.
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