Correlation of serum vitamin D level with ketoacidosis in Chinese children with type 1 diabetes mellitus

Objective: Diabetic ketoacidosis is a common complication in children with type 1 diabetes mellitus. The purposes of the present study were to explore clinical correlates of serum vitamin D level in Chinese children with type 1 diabetes. Methods: A total of 143 inpatients (boys/girls = 60/83) were recruited from Tianjin Children’s Hospital. Their demographic and clinical characteristics were collected. These patients were divided into the non-DKA group(n=43) and DKA group(n=100). Results: The positive ZnT8-ab was signicantly higher in DKA patients compared with non-DKA patients (p=0.038). There was a negative correlation between plasma glucose and the concentration of vitamin D(r =−0.188, p=0.024), although there was no signicant difference in vitamin D between two groups of T1DM patients with or without DKA (p=0.317). The multiple logistic regression revealed that sex(male) and BMI were independent risk factors to predict the deciency or insuciency of Vitamin D in T1DM children. When BMI is lower than 16 kg/m 2 according to the cut-off value of the ROC curve, it provides some implications of Vitamin D deciency or insuciency in TIDM children ( 95%CI:0.534~0.721, P=0.014). Conclusions: Our results suggested that positive ZnT8-ab was associated with a greater risk of DKA at T1DM onset. Additionally, neither vitamin D levels nor the proportion of patients with different levels of vitamin D differed between the two groups inT1DM children with or without DKA. Furthermore, Vitamin D level was negatively correlated with plasma glucose, lower BMI and male children with T1DM were prone to be decient or insucient of Vitamin D.


Background And Introduction
Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease, especially in children with a higher occurrence rate. Diabetic ketoacidosis (DKA) is a potentially life-threatening complication of T1DM and is characterized by metabolic acidosis, hyperglycemia, and ketonaemia. 1 Insulin de ciency and increased levels of the counter-regulatory hormones (glucagon, catecholamines, cortisol, and growth hormone)can lead to DKA. 2 The clinical symptoms of DKA develop over several hours and include dehydration, nausea and/or vomiting, deep respiration, breath smells of acetone, blurry vision, drowsiness, eventually loss of consciousness (coma). 1 Evidence suggested that structured diabetes self-management education had led to DKA rates decrease. 3 However, recent data have shown that DKA is still a major cause of death in patients with T1DM. 4 The development of T1DM involves many factors. The risk of DKA in established T1DM is 1-10% per patient per year. Newly diagnosed T1DM or patients with known diabetes have different risk factors for DKA. 2 A reviewed literature described potentially non-modi able and modi able risk factors for recurrent DKA, such as high HbA1c, low socioeconomic status, younger age. 1 All of these risk factors urgently demand more e cient ways for early prediction, and eventually prevention or reversal of the disease as soon as possible. Biomarkers play an important role in clinical diagnosis, therapeutic effect, and prognosis, as indicators of normal and abnormal physiological or pathological processes. 5 Now, autoantibodies (AAbs) may serve as relatively effective prognostic markers of the risk of diabetes progression. 6 However, the rate of development depends on many factors, including sex, age, genotype, type, and number of AAbs. 5 Hence, it is essential to nd additional biomarkers that can predict the progression of T1DM and identify the best timing for DKA intervention and therapy. Autoimmune thyroid disease (AITD) and T1DM are two common autoimmune diseases and can occur concomitantly. 7 Many studies have demonstrated that changes in serum thyroid hormone parameters are associated with diabetes. A study concluded that thyroid hormone parameters seemed to re ect the effects of a prolonged period of catabolism, DKA, or both. 8 Besides, vitamin D is associated with both innate and adaptive immune systems. De ciency or insu ciency of vitamin D seems to be a high-risk factor for autoimmune diseases such as T1DM and AITD. 7 In this study, we aim to explore the relationship between vitamin D level and the risk of DKA and its components in patients with T1DM.

Subjects
This cross-sectional study was carried out in the Endocrine inpatient wards of Tianjin Children's Hospital, from June 1, 2017, to May 31, 2019. One hundred and forty-three children(boys/girls = 60/83) met the diagnosis of T1DM according to the WHO criteria, including fasting plasma glucose ≥ 7.0 mmol/l, 2-h postprandial plasma glucose ≥ 11.1 mmol/l, HbAlc ≥ 6.5%, as well as random plasma glucose concentration of ≥ 11.1 mmol/l. They were aged 10-180 months (mean age: 90.03 ± 43.07), with a mean age of onset of 74.52 ± 39.41 months (range 10-160 months). 143 patients were divided into two groups: T1DM without DKA group and T1DM with DKA group.
All the patients were obtaining medical treatment with insulin being subcutaneous injections such as Novolin and Novorapid. DKA was de ned as blood glucose concentration > 13.9 mmol/l, Venous pH < 7.3 or serum bicarbonate (HCO 3 ) < 15 mmol/L, ketonemia or ketonuria, anion gap > 12. Major medical abnormalities, including central nervous system diseases, angiocardiopathy, or life-threatening medical illnesses (infections or cancer) were excluded. All subjects were Han Chinese.
After the study procedure was explained in detail to the parents of patients included in the study, they signed the informed consent document.
Before this study began, the research protocol was approved by the Institutional Review Board of Tianjin Children's Hospital.

Clinical and laboratory data
Bodyweight and height were measured in a standardized way and body mass index (BMI, weight in kg /square of height in meters) was calculated. Blood samples were collected before an initial insulin therapy. The plasma was separated, aliquoted, and stored at -70°C before use. Plasma glucose, HbA1c, Cpeptide, triglyceride (TG), Total cholesterol (TC), HDL cholesterol (HDL-C), LDL cholesterol (LDL-C), T3, T4, and TSH levels were measured at the diagnostic laboratory of Tianjin Children's hospital.

Vitamin D assays
The level of vitamin D was measured by equipment [API 3200MD™ LC/MS/MS System, AB Sciex]. It was de ned as de cient, insu cient, and su cient if vitamin D level was ≤ 15,≤20 > 15, and > 20 ng/ml, respectively.

Clinical measures
General information, sociodemographic characteristics, and family history of all subjects were collected by a researcher. Complete medical history, physical examination, and laboratory data were also obtained for each patient. Additional information was collected from available medical records and collateral resources.

Statistical analysis
Since all the demographic and clinical variables were normally distributed in subjects (Shapiro-Wilk onesample test, all p > 0.05), comparisons of demographic and clinical data between two groups were analyzed by using independent samples t-tests for continuous variables and the chi-square test or Fisher's exact test for categorical variables. Analysis of covariance (ANCOVA) was further conducted to control for the effects of sex, age, and BMI on metabolic disturbances. Correlations between variables were assessed with Pearson's correlation coe cients. Bonferroni corrections were applied to each test to adjust for multiple testing. A binary logistic regression analysis was performed to predict risky variables.
Finally, a multiple logistic regression analysis was used to identify signi cant predictive variables associated with Vitamin D. Statistical analyses were carried out using SPSS version 21, and MedCalc version 19.0.7 was used to draw ROC curves. P < 0.05 were considered indicative of statistical signi cance.    Correlation analysis of serum 25(OH)D level and clinical measures were shown in Table 3.   Table 4. Male patients with T1DM were prone to be de cient in Vitamin D (p = 0.006). A higher level of plasma glucose and TG, and a lower level of C-peptide and T3 were more likely to lead to the de ciency or insu ciency of vitamin D (all p < 0.05). Binary logistic regression analyses found that males were 2.320 times more likely to be insu cient or de cient in Vitamin D compared with females((OR = 2.320, p = 0.017)). Moreover, the plasma glucose level in patients with insu cient or de cient Vitamin D was 1.

Multivariate logistic regression to predict the de ciency or insu ciency of Vitamin D in T1DM patients
Finally, a multiple logistic regression was performed to predict the de ciency or insu ciency of Vitamin D in T1DM patients. Two variables statistically predicted the de ciency or insu ciency of Vitamin D in T1DM children, including sex and BMI. The coe cients of these variables were shown in Table 5.

Discussion
Our study con rmed that ZnT8-ab positivity was associated with a greater risk of DKA in T1DM children.
Moreover, neither vitamin D levels nor the proportion of patients with different levels of vitamin D differed between the two groups of children with T1DM. Besides, Vitamin D level was negatively correlated with plasma glucose, lower BMI and male children with T1DM were prone to be de cient or insu cient of Vitamin D.
We found that patients with DKA were younger than those without DKA, not only age of onset but also current age, which can be de ned by a consensus statement from ISPAD. 2 A review considered that AAbs were regarded as the gold standard for the prediction of T1DM progression currently. 5 In our study, we observed the prevalence of positive ZnT8-ab was at greater risk of DKA, which was in concordance with that found in the study performed by Niechcial et al. Interestingly, they found that the titers of GAD-ab and IA2-ab were lower in DKA patients but titer of ZnT8-ab was higher in DKA patients. 6 However, based on our results, the titers of the three AAbs (GAD-ab, IA2-ab, and ZnT8-ab) weren't a signi cant difference between two groups. The research demonstrated that ZnT8-ab positive patients had a higher incidence of multiple diabetes-related AAbs, which means more severe β-cell dysfunction and higher prevalence of DKA. 9 ZnT8 is a 369-amino acid pancreas-speci c zinc transporter, which was encoded by the SLC30A8 gene at the chromosome 8q14.11, and SLC30A8 is a major target of humoral autoimmunity in T1DM. 10 ZnT8 plays a key role in glucose homeostasis. A study suggested that ZnT8 was a crucial protein for both zinc accumulation and regulation of insulin secretion in pancreatic β cells. 11 Compared with other autoimmune markers, ZnT8-ab is highly βcell-speci c and ZnT8-ab expression may not occur until there is enough β-cell damage. 9 In hence, positive ZnT8-ab can be used as an indicator to predict the progression of T1DM.
In our study, we found a lower level of T3 in DKA patients. As we all know that patients with diabetes have a high risk of AITD. 12 49 of 143 T1DM patients in our study suffered from thyroid disease, such as multiple cystic nodules of the thyroid, hypothyroidism, Autoimmune Polyendocrine Syndrome. The pathogenesis of AITD involves cellular and humoral autoimmune mechanisms against the thyroid gland.
T lymphocytes in ltrate the glands, then subsequent development of various degrees of thyroid dysfunction. 7 Thyroid hormones T4 and T3 were produced by a glycosylated transmembrane protein named thyroid peroxidase, which is distributed in the apical part of follicular thyroid cells. 13 Metabolomics techniques have shown that people who develop diabetes have different levels of certain lipids when compared with people who remain non-diabetic. 14 In addition, thyroid dysfunction can lead to metabolic disorders. This in turn results in stimulation of glycogenolysis and gluconeogenesis, increased glucose absorption, and lipolysis, causing deterioration of metabolic control. 15  Currently, many studies are exploring the relationship between vitamin D and immune-related diseases, including T1DM. 16 Because of the discovery of speci c vitamin D receptors (VDR) on pancreatic β-cell, the role of active vitamin D on β-cell' function was con rmed. 17 There was evidence suggested that calcitriol maintained β-cell mass and improves islets function via several pathways. The insulin secretion process was a calcium-dependent mechanism. 18 A review concluded that vitamin D improves glucose homeostasis, and the molecular mechanism is that promoting insulin sensitivity via at least two different pathways: promoting β-cell function by ameliorating deleterious molecular mechanisms and increasing peripheral insulin sensitivity. 19 VDR expression and activity were important for all stages of T cells, ranging from development to differentiation and elicitation. Administration of 1,25(OH) 2 D 3 can enhance Treg and inhibit autoreactive T H 1, leading to a reduction in the incidence of T1DM in the diabetic mouse model. 17  Several limitations of our study should be noted.
Firstly, this study was a cross-sectional design and all subjects were recruited from a local hospital.
Hence, observations and conclusions should be treated with caution. Secondly, the concentration of Vitamin D was measured at disease onset. There is no uniform time point of Vitamin D measurements, in winter or summer time. Therefore, further accurate studies need to be conducted to validate our ndings. Thirdly, we had a comparatively small sample size of patients, which had become smaller when dividing into subgroups. Therefore, the results and conclusions in our study should be regarded as preliminary and should be con rmed after further prospective, multicenter, and large-scale trials.

Conclusion
In summary, the current study has shown that ZnT8-ab can emerge as a reliable marker for DKA in T1DM patients. Additionally, neither vitamin D levels nor the proportion of patients with different levels of vitamin D differed between the two groups of children with T1DM. Furthermore, Vitamin D level was negative correlated with plasma glucose. Lower BMI, and male children with T1DM were prone to be de cient or insu cient of Vitamin D. Further prospective and multicenter studies including large population size, more parameters, and more ethnic groups are needed to demonstrate these ndings.      Figure 1 ROC curves of BMI predicting the de ciency or insu ciency of Vitamin D in T1DM patients

Supplementary Files
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