Participants

Forty-six children and adolescents with T1DM were recruited from the regular attendees of the Pediatric and Adolescents Diabetes Unit (PADU), Ain Shams University, and 46 healthy children were included as a control group from the healthy siblings of attendees of the outpatient clinic. They were matched for age and gender. Participants were selected by simple random sampling.

Diagnosis of T1DM was made according to the criteria of the International Society of Pediatric and Adolescent Diabetes (ISPAD) 2022 [13].

Exclusion criteria included children and adolescents with proven hyperlipidemia, hypertension, obesity, other autoimmune diseases, acute coronary syndrome, stroke, history of deep venous thrombosis or pulmonary embolism, and history of malignancy or coagulation disorders. Patients with hypocalcemia and those receiving treatment with aspirin, clopidogrel, warfarin, nonsteroidal anti-inflammatory drugs, or any drug that affects the coagulation system were also excluded.

Sample size calculation

Using the PASS 11.0 program & based on a study carried out by Sobczak et al. [2] a sample size of 92 participants in total, 46 patients & 46 controls is sufficient to detect a difference of 29.5% between groups in individual fibrin clot parameters given that power is 80% & confidence level 95% taking in consideration the 10% drop out rate.

The patient group was further subdivided into 2 groups according to the presence or absence of microangiopathy (17 patients with complications and 29 patients without complications).

Ethical considerations

The study protocol was approved by the Ethical Committee of Ain Shams University with an approval number MS 185/2022 and written informed consent was obtained from all cases and their legal guardians before participation.

ProceduresClinical assessment

All enrolled participants were subjected to detailed medical history collection with special emphasis on demographic data, age at onset of diabetes, diabetes duration, and insulin therapy. Anthropometric measures assessment was done including weight in kilograms (Kg), height in centimeters (cm), and body mass index (BMI) calculation in kg/m2 plotting them on age and gender standard percentiles [14].

The modified magnesium deficiency questionnaire (MDQ)-10 was used by a single physician to assess the symptoms of magnesium deficiency. MDQ-10 is a validated 10 questions questionnaire that showed a significant correlation with hypomagnesemia; its total score range is 0–31, with a cut-off value of >5 [15].

Fundus examination was done for children and adolescents with T1DM by direct ophthalmoscope through dilated pupils for assessment of diabetic retinopathy [16]. A complete neurological examination was performed for children and adolescents with T1DM by a single-blinded neurologist. The Toronto Clinical Scoring System (TCSS) was adopted as a screening tool for diabetic peripheral neuropathy. It consists of three parts: symptom scores, reflex scores, and sensory test scores. The maximum score is 19 points [17].

Biochemical assessment Blood sampling

About 5 ml of blood volume was withdrawn from each participant for the following investigations

a) Magnesium level:

Serum magnesium was assessed by the direct method in which a colored complex is measured at 520/800 nm at Beckman Coulter Au 480.

b) Glycosylated hemoglobin (HbA1c):

HbA1c measurement was carried out using the Tina-QuantR HbA1c kit supplied by Roche Diagnostics on Roche/Hitachi CobasR* c501 System (Roche Diagnostics International Ltd. CH-6343 Rotkreuz, Switzerland) based on turbidimetric inhibition immunoassay (TINIA).

c) PT / aPTT clotting:

A blood sample was placed in a clean tube containing sodium citrate. The citrated blood tube was centrifuged at 2500 rpm for 20 minutes and plasma was separated into a clean Eppendorf tube and used to immediately measure prothrombin time (PT= 11–13.5 seconds), partial thromboplastin time (PTT= 23–35 seconds) and calculate the INR (INR= patient PT/normal reference range of PT= 0.8–1.1). The coagulation profile included PT, PTT, and INR was performed by viscosity-based electromechanical detection systems using Stago STA4 Compact (Diagnostica Stago, France).

d) Serum levels of PAI-1:

Determination of serum levels of PAI-1 using ELISA based on double antibody sandwich tech (Bioassay Technology Laboratory; E1159HU, Shanghai Korain Biotech Co., Shanghai, China).

e) Fasting serum triglycerides (TG) and total cholesterol (TC) were assessed by quantitative enzymatic colorimetric technique (Bio Merieux-Diagnostic Chemicals Ltd., Charlottetown, CA, USA). Serum high-density lipoprotein (HDL) was measured by the phosphotungstate precipitation method (Bio Merieux kit, Marcyl’Etoile, Craponne, France). LDL cholesterol was calculated by Friedewald’s formula [18].

Urine sampling

Urinary albumin excretion in 3 consecutive early morning fasting spot urine samples was assayed for children and adolescents with T1DM by an immuno-turbidimetric method using the Beckman Colter AU 480 system (Beckman coulter, Inc. 250 s. Kraemer Blvd. Brea, CA92821, USA. Albuminuria was defined by an albumin excretion rate of > 30 mg/g creatinine, microalbuminuria as urinary albumin excretion 30–299 mg/g creatinine and macroalbuminuria as urinary albumin excretion ≥ 300 mg/g creatinine. Nephropathy was defined as having micro or macro-albuminuria [19].

Statistical analyses

Data were collected, revised, coded, and entered into the Statistical Package for Social Science (IBM SPSS) version 27. The quantitative data were presented as mean, standard deviations, and ranges when their distribution was found parametric. Qualitative variables were presented as numbers and percentages.

The comparison between the two groups regarding qualitative data was done by using the Chi-square test. The comparison between two independent groups with quantitative data and parametric distribution was done by using the independent t-test. Spearman correlation coefficients were used to assess the correlation between two quantitative parameters in the same group. Stepwise multiple linear regression analysis was performed to detect the most significant independent variables associated with hypomagnesemia among the studied children and adolescents with T1DM. The confidence interval was set to 95% and the margin of error accepted was set to 5%. So, the pvalue was considered significant at the level of <0.05.