Relationship between vitamin D status and vascular complications in patients with type 2 diabetes mellitus Chan-Hee Jung, Kyu-Jin Kim, Bo-Yeon Kim, Chul-Hee Kim, Sung Koo Kang, Ji-Oh Mok PII: DOI: Reference:
S0271-5317(15)00288-2 doi: 10.1016/j.nutres.2015.11.008 NTR 7562
To appear in:
Nutrition Research
Received date: Revised date: Accepted date:
14 August 2015 24 October 2015 11 November 2015
Please cite this article as: Jung Chan-Hee, Kim Kyu-Jin, Kim Bo-Yeon, Kim ChulHee, Kang Sung Koo, Mok Ji-Oh, Relationship between vitamin D status and vascular complications in patients with type 2 diabetes mellitus, Nutrition Research (2015), doi: 10.1016/j.nutres.2015.11.008
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Relationship between vitamin D status and vascular complications in patients with type 2 diabetes mellitus
Division
of
Endocrinology
and
Metabolism,
RI P
T
Chan-Hee Jung, Kyu-Jin Kim, Bo-Yeon Kim, Chul-Hee Kim, Sung Koo Kang, Ji-Oh Mok *
Department
of
Internal
Medicine,
SC
Soonchunhyang University College of Medicine, Bucheon Hospital, 1174, Jung-dong,
MA NU
Wonmi-gu, Bucheon-si, Gyeonggi-do, 420-767, Republic of Korea
Chan-Hee Jung and Kyu-Jin Kim made equal contributions as first authors to this manuscript.
ED
*Corresponding Author Ji-Oh Mok, MD, PhD
of
Endocrinology
and
Metabolism,
Department
of
Internal
Medicine,
CE
Division
PT
Address:
Soonchunhyang University College of Medicine, Bucheon Hospital
AC
1174, Jung-dong, Wonmi-gu, Bucheon-si, Gyeonggi-do, 420-767, Republic of Korea Phone : +82-32-621-5212, Fax : +82-32-621-5018 E-mail : [email protected]
1
ACCEPTED MANUSCRIPT Abbreviations 25(OH)D; 25-hydroxyvitamin D
T
T2DM; type 2 diabetes mellitus
RI P
DPN; diabetic peripheral neuropathy DN; diabetic nephropathy
SC
CV; cardiovascular
MA NU
HbA1c; glycated hemoglobin TC; Total cholesterol LDL-C; LDL-cholesterol TG; triglyceride
ED
HOMA-IR; homeostasis model assessment of insulin resistance BMI; body mass index
PT
MNSI; Michigan Neuropathy Screening Instrument
CE
CPTs; current perception thresholds eGFR; estimated glomerular filtration rate
AC
DR; diabetic retinopathy
ARB; angiotensin receptor blocker ACEI; angiotensin converting enzyme inhibitor OHA; oral hypoglycemic agent
2
ACCEPTED MANUSCRIPT Abstract We aimed to investigate the association between serum 25-hydroxyvitamin D (25(OH)D) and
T
microvascular complications in type 2 diabetes mellitus (T2DM) patients. It was
RI P
hypothesized that lower 25(OH)D would be associated with increased microvascular complications in T2DM. A total of 257 T2DM patients (111 males, 146 females) who
SC
underwent diabetic microvascular complications (peripheral neuropathy, nephropathy,
MA NU
retinopathy) studies were recruited. Patients were categorized into three groups according to vitamin D status: vitamin D sufficient (n=41, 25(OH)D ≥ 20 ng/mL), vitamin D insufficient (n=132, 10 ≤ 25(OH)D < 20 ng/mL), and vitamin D deficient (n=84, 25(OH)D < 10 ng/mL). In males, the prevalence of diabetic peripheral neuropathy (DPN) was significantly higher in
ED
patients with vitamin D deficiency than in those with insufficiency or sufficiency (38%, 11.7%, and 10%, respectively, p=0.005). In addition, the prevalence of diabetic nephropathy
PT
(DN) was significantly higher in female with vitamin D deficiency than in the other two
CE
groups (40%, 20.6%, and 0%, p=0.007). Compared to the male in the vitamin D sufficient group (reference), male in the vitamin D deficient group had an increased risk of DPN after
AC
adjusting for confounding factors (OR=7.79, 95% CI=1.52-40.05). For females, when the vitamin D sufficient group was used as a reference, those in the vitamin D deficient group had an increased risk of DN after adjusting for confounding factors (OR=4.27, 95% CI=1.5811.56). This present study found that a serum 25(OH)D level less than 10 ng/mL is independently associated with increased DPN in male patients and increased DN in female patients with T2DM.
Keywords : Vitamin D; peripheral neuropathy; nephropathy; microvascular complication; type 2 diabetes mellitus 3
ACCEPTED MANUSCRIPT 1. Introduction In addition to its classical role in the skeleton and bone health, vitamin D has been
T
recognized to exert non-classical pleiotropic effects such as anti-inflammatory, anti-
RI P
angiogenic, anti-proliferative and immunomodulatory properties [1]. Vitamin D appears to have important effects on glucose homeostasis via actions on insulin synthesis, secretion, and
SC
inflammation [2,3]. In fact, there is considerable evidence to suggest a link between vitamin
MA NU
D deficiency and increased incidence of type 2 diabetes mellitus (T2DM) [4]. T2DM may give rise to many microvascular complications, leading to high morbidity and mortality. Diabetic nephropathy is the leading cause of chronic kidney disease and is associated with increased cardiovascular mortality [5]. Diabetic peripheral neuropathy is one
ED
of the most common of all the long-term complications of diabetes and plays a major role in the development of foot ulcers, which cause an enormous effect on quality of life for the
PT
patients [6].
CE
Therefore, it is important to define and control modifiable risk factors that contribute to diabetic complications. T2DM and vitamin D deficiency have been recognized as pandemic
AC
diseases with numerous health consequences. Moreover, vitamin D deficiency is known to be more common in those diagnosed with diabetes [7]. Previous studies have demonstrated an association between vitamin D deficiency and cardiovascular (CV) mortality, CV risk factors or subclinical atherosclerosis in T2DM patients as well as in the general population [8-11]. Recently, a link between vitamin D deficiency and microvascular complications in T2DM has been suggested [12-15]. Nevertheless, the evidence for relationship of vitamin D deficiency and diabetic microvascular complications is very limited, and the information that is available is inconsistent. We hypothesized that lower 25(OH)D would be associated with increased of microvascular 4
ACCEPTED MANUSCRIPT complications in T2DM. To test this hypothesis, the study objective was to investigate the association between 25(OH)D, a commonly used marker for vitamin D status, with
AC
CE
PT
ED
MA NU
SC
RI P
T
microvascular complications including DPN and DN in Korean patients with T2DM.
5
ACCEPTED MANUSCRIPT 2. Methods and Materials 2.1.Subjects
T
We conducted a cross-sectional study. We recruited 300 T2DM patients who underwent
RI P
evaluation for diabetic microvascular complications at the endocrinology clinic of Soonchunhyang University Bucheon Hospital, Korea from January 2009 to May 2014.
SC
Among the patients, those who took calcium or vitamin D medications or supplement, those
MA NU
with osteoporosis, and those whose serum 25(OH)D was not measured were excluded. Finally, 257 patients (111 males and 146 females) remained for current analyses. After receiving the information concerning the study, all participants provided written informed consent. The ethics committee of the hospital approved the study protocol.
ED
The participants were categorized into three groups based on vitamin D status, deficient [25(OH)D < 10 ng/mL], insufficient [10 ng/mL ≤ 25(OH)D < 20 ng/mL] and sufficient [20
PT
ng/mL ≤ 25(OH)D]. These cutoff levels were recommended by the World Health
CE
Organization (WHO) and, more recently, the Institute of Medicine [16-18].
AC
2.2.Anthropometric and biochemical measurements We collected demographic data, biochemical data, clinical data, and treatment history using medical records. Height and weight were measured to the nearest 0.1 cm and 0.1 kg, respectively. Body mass index (BMI) was calculated as body weight (kg) divided by height squared (square meters). Waist circumference was taken midway between the inferior margin of the last rib and the crest of the ilium in the horizontal plane whilst in an upright position. WC was measured in duplicate with an anthropometric tape while the subjects were wearing light clothing. Data related to alcohol consumption was obtained from self-completed surveys. Alcohol 6
ACCEPTED MANUSCRIPT consumption was categorized into two groups, drinkers and non drinkers, the latter defined as those who drink alcohol more than once per month.
T
Fasting glucose, insulin, C-peptide levels, glycated hemoglobin (HbA1c), lipid profiles,
RI P
creatinine, and 25(OH)D were measured. The level of 25(OH)D was measured using a radioimmunological determination kit (CIS Bio, France). HbA1c was measured by ion-
SC
exchange high-performance liquid chromatography (Bio-Rad, Hercules, CA, USA). Serum
MA NU
creatinine level was measured by the alkaline picrate (Jaffe) method. Total cholesterol (TC), Low density lipoprotein-cholesterol (LDL-C) and triglyceride (TG) were determined using the liquid enzymatic method with an automatic biochemical analyzer (7600-110;Hitachi Inc., Tokyo, Japan) and HDL-C was measured by the selective inhibition method. Serum insulin
ED
and C-peptide were measured using an immunoradiometric assay kit (DIAsource Immunoassay, Belgium). Insulin resistance was calculated using the homeostasis model
PT
assessment of insulin resistance (HOMA-IR) according to the following formula: [fasting
CE
insulin (μIU/mL) x fasting blood glucose (mmol/L)]/22.5.
AC
2.3.Evaluation of microvascular complications Diabetic peripheral neuropathy (DPN) was diagnosed in the presence of typical symptoms using the Michigan Neuropathy Screening Instrument (MNSI) and compatible findings on neurological screening examinations such as a 10-g monofilament test, a vibration test with 128 Hz tuning fork, and an ankle reflex examination or electrophysiological studies. Although electrophysiological studies are not essential, current perception thresholds (CPTs) tests were carried out in all patients using a Neurometer (Neurotron Inc., Baltimore, MD, USA).
7
ACCEPTED MANUSCRIPT Diabetic nephropathy (DN) was defined according to albuminuria, which was measured by radioimmunoassay (Immunotech, Prague, Czech Republic). Albumin excretion was assessed
T
using a first-morning spot urine sample. However, if a first-morning specimen is not
RI P
available, second-morning specimen is considered acceptable. Urine albumin < 30 mg/g creatinine was categorized as normoalbuminuria, 30-300 mg/g creatinine as
SC
microalbuminuria, and ≥ 300 mg/g creatinine as overt proteinuria. Patients were considered
MA NU
to have nephropathy if they showed microalbuminuria or overt proteinuria [19]. Estimated glomerular filtration rate (eGFR) was calculated using the Modification of Diet in Renal Disease Study equation [20].
Diabetic retinopathy (DR) was defined based on findings of dilated pupils on fundoscopy
PT
proliferative retinopathy.
ED
carried out by an ophthalmologist. DR was defined as having nonproliferative retinopathy or
CE
2.4.Statistical analyses
Statistical analyses were performed using the SPSS Version 18 (2010) (SPSS Inc., Chicago,
AC
Illinois, USA). Values were represented as means ± standard deviation (SD) for variables that were normally distributed, median (interquartile range : IQR) for variables that were not normally distributed or as number of participants (percentages). P-values < 0.05 were considered statistically significant. To evaluate the clinical and demographic characteristics of the study subjects, analysis of variance and Kruskal-Wallis tests were used to compare the quantitative data of the three groups according to vitamin D status (deficient, insufficient, and sufficient). Chi-square test was used for categorical data. Before the test, Shapiro-Wilk test for normality and Levene’s homogeneity of variance test was conducted. Non-normally distributed variables, that is, 8
ACCEPTED MANUSCRIPT triglycerides, serum fasting insulin, fasting c-peptide, HOMA-IR, and microalbumin were natural-logarithmic transformed before analysis.
T
For confirming the correlations between 25(OH)D level and clinical and biochemical
RI P
variables, we calculated the correlation coefficient and p-value using Pearson’s or Spearman’s bivariate correlation analysis. A Chi-square test and Student’s t-test were used for comparing
SC
the prevalence of the vitamin D deficiency and the difference in mean level of 25(OH)D
MA NU
according to presence of microvascular complications. The independence of the association of DPN and DN with vitamin D deficiency was assessed by multivariate logistic regression, after adjusting for confounding factors which were chosen by univariate analysis and, traditionally known risk factors such as age, duration of diabetes, HbA1C, BMI, smoking,
ED
lipid, inflammatory marker, and medications.
We performed post hoc power analyses with PASS (version 12: NCSS, Kaysville, Utah, USA)
AC
CE
PT
in order to estimate the probability of detecting present effects with the current sample.
9
ACCEPTED MANUSCRIPT 3. Results 3.1. The clinical characteristics of the participants
T
The mean age of the participants was 58.8 ± 12.1 years, and the mean duration of diabetes
RI P
was 7.7 ± 7.9 years. The mean body mass index (BMI) was 25.0 ± 4.0 kg/m2, and mean 25(OH)D level was 14.4 ± 8.6 ng/mL (Supplemental Table 1). The females had a
SC
significantly lower BP, FPG, HbA1C, and TG, and longer duration and higher HDL-C than
MA NU
males (Supplemental Table 1). The participants were divided into three groups (84 vitamin D deficient patients, 132 vitamin D insufficient patients, and 41 vitamin D sufficient patients). The prevalence of deficiency and sufficiency of vitamin D in all patients was 32.7% and 15.9%, respectively. The proportion of vitamin D status according to gender was significantly
ED
different (deficient, insufficient, and sufficient, 18.9%, 54.1%, and 27% in males and 43.2%, 49.3%, and 7.5% in females, p
S0271-5317(15)00288-2 doi: 10.1016/j.nutres.2015.11.008 NTR 7562
To appear in:
Nutrition Research
Received date: Revised date: Accepted date:
14 August 2015 24 October 2015 11 November 2015
Please cite this article as: Jung Chan-Hee, Kim Kyu-Jin, Kim Bo-Yeon, Kim ChulHee, Kang Sung Koo, Mok Ji-Oh, Relationship between vitamin D status and vascular complications in patients with type 2 diabetes mellitus, Nutrition Research (2015), doi: 10.1016/j.nutres.2015.11.008
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Relationship between vitamin D status and vascular complications in patients with type 2 diabetes mellitus
Division
of
Endocrinology
and
Metabolism,
RI P
T
Chan-Hee Jung, Kyu-Jin Kim, Bo-Yeon Kim, Chul-Hee Kim, Sung Koo Kang, Ji-Oh Mok *
Department
of
Internal
Medicine,
SC
Soonchunhyang University College of Medicine, Bucheon Hospital, 1174, Jung-dong,
MA NU
Wonmi-gu, Bucheon-si, Gyeonggi-do, 420-767, Republic of Korea
Chan-Hee Jung and Kyu-Jin Kim made equal contributions as first authors to this manuscript.
ED
*Corresponding Author Ji-Oh Mok, MD, PhD
of
Endocrinology
and
Metabolism,
Department
of
Internal
Medicine,
CE
Division
PT
Address:
Soonchunhyang University College of Medicine, Bucheon Hospital
AC
1174, Jung-dong, Wonmi-gu, Bucheon-si, Gyeonggi-do, 420-767, Republic of Korea Phone : +82-32-621-5212, Fax : +82-32-621-5018 E-mail : [email protected]
1
ACCEPTED MANUSCRIPT Abbreviations 25(OH)D; 25-hydroxyvitamin D
T
T2DM; type 2 diabetes mellitus
RI P
DPN; diabetic peripheral neuropathy DN; diabetic nephropathy
SC
CV; cardiovascular
MA NU
HbA1c; glycated hemoglobin TC; Total cholesterol LDL-C; LDL-cholesterol TG; triglyceride
ED
HOMA-IR; homeostasis model assessment of insulin resistance BMI; body mass index
PT
MNSI; Michigan Neuropathy Screening Instrument
CE
CPTs; current perception thresholds eGFR; estimated glomerular filtration rate
AC
DR; diabetic retinopathy
ARB; angiotensin receptor blocker ACEI; angiotensin converting enzyme inhibitor OHA; oral hypoglycemic agent
2
ACCEPTED MANUSCRIPT Abstract We aimed to investigate the association between serum 25-hydroxyvitamin D (25(OH)D) and
T
microvascular complications in type 2 diabetes mellitus (T2DM) patients. It was
RI P
hypothesized that lower 25(OH)D would be associated with increased microvascular complications in T2DM. A total of 257 T2DM patients (111 males, 146 females) who
SC
underwent diabetic microvascular complications (peripheral neuropathy, nephropathy,
MA NU
retinopathy) studies were recruited. Patients were categorized into three groups according to vitamin D status: vitamin D sufficient (n=41, 25(OH)D ≥ 20 ng/mL), vitamin D insufficient (n=132, 10 ≤ 25(OH)D < 20 ng/mL), and vitamin D deficient (n=84, 25(OH)D < 10 ng/mL). In males, the prevalence of diabetic peripheral neuropathy (DPN) was significantly higher in
ED
patients with vitamin D deficiency than in those with insufficiency or sufficiency (38%, 11.7%, and 10%, respectively, p=0.005). In addition, the prevalence of diabetic nephropathy
PT
(DN) was significantly higher in female with vitamin D deficiency than in the other two
CE
groups (40%, 20.6%, and 0%, p=0.007). Compared to the male in the vitamin D sufficient group (reference), male in the vitamin D deficient group had an increased risk of DPN after
AC
adjusting for confounding factors (OR=7.79, 95% CI=1.52-40.05). For females, when the vitamin D sufficient group was used as a reference, those in the vitamin D deficient group had an increased risk of DN after adjusting for confounding factors (OR=4.27, 95% CI=1.5811.56). This present study found that a serum 25(OH)D level less than 10 ng/mL is independently associated with increased DPN in male patients and increased DN in female patients with T2DM.
Keywords : Vitamin D; peripheral neuropathy; nephropathy; microvascular complication; type 2 diabetes mellitus 3
ACCEPTED MANUSCRIPT 1. Introduction In addition to its classical role in the skeleton and bone health, vitamin D has been
T
recognized to exert non-classical pleiotropic effects such as anti-inflammatory, anti-
RI P
angiogenic, anti-proliferative and immunomodulatory properties [1]. Vitamin D appears to have important effects on glucose homeostasis via actions on insulin synthesis, secretion, and
SC
inflammation [2,3]. In fact, there is considerable evidence to suggest a link between vitamin
MA NU
D deficiency and increased incidence of type 2 diabetes mellitus (T2DM) [4]. T2DM may give rise to many microvascular complications, leading to high morbidity and mortality. Diabetic nephropathy is the leading cause of chronic kidney disease and is associated with increased cardiovascular mortality [5]. Diabetic peripheral neuropathy is one
ED
of the most common of all the long-term complications of diabetes and plays a major role in the development of foot ulcers, which cause an enormous effect on quality of life for the
PT
patients [6].
CE
Therefore, it is important to define and control modifiable risk factors that contribute to diabetic complications. T2DM and vitamin D deficiency have been recognized as pandemic
AC
diseases with numerous health consequences. Moreover, vitamin D deficiency is known to be more common in those diagnosed with diabetes [7]. Previous studies have demonstrated an association between vitamin D deficiency and cardiovascular (CV) mortality, CV risk factors or subclinical atherosclerosis in T2DM patients as well as in the general population [8-11]. Recently, a link between vitamin D deficiency and microvascular complications in T2DM has been suggested [12-15]. Nevertheless, the evidence for relationship of vitamin D deficiency and diabetic microvascular complications is very limited, and the information that is available is inconsistent. We hypothesized that lower 25(OH)D would be associated with increased of microvascular 4
ACCEPTED MANUSCRIPT complications in T2DM. To test this hypothesis, the study objective was to investigate the association between 25(OH)D, a commonly used marker for vitamin D status, with
AC
CE
PT
ED
MA NU
SC
RI P
T
microvascular complications including DPN and DN in Korean patients with T2DM.
5
ACCEPTED MANUSCRIPT 2. Methods and Materials 2.1.Subjects
T
We conducted a cross-sectional study. We recruited 300 T2DM patients who underwent
RI P
evaluation for diabetic microvascular complications at the endocrinology clinic of Soonchunhyang University Bucheon Hospital, Korea from January 2009 to May 2014.
SC
Among the patients, those who took calcium or vitamin D medications or supplement, those
MA NU
with osteoporosis, and those whose serum 25(OH)D was not measured were excluded. Finally, 257 patients (111 males and 146 females) remained for current analyses. After receiving the information concerning the study, all participants provided written informed consent. The ethics committee of the hospital approved the study protocol.
ED
The participants were categorized into three groups based on vitamin D status, deficient [25(OH)D < 10 ng/mL], insufficient [10 ng/mL ≤ 25(OH)D < 20 ng/mL] and sufficient [20
PT
ng/mL ≤ 25(OH)D]. These cutoff levels were recommended by the World Health
CE
Organization (WHO) and, more recently, the Institute of Medicine [16-18].
AC
2.2.Anthropometric and biochemical measurements We collected demographic data, biochemical data, clinical data, and treatment history using medical records. Height and weight were measured to the nearest 0.1 cm and 0.1 kg, respectively. Body mass index (BMI) was calculated as body weight (kg) divided by height squared (square meters). Waist circumference was taken midway between the inferior margin of the last rib and the crest of the ilium in the horizontal plane whilst in an upright position. WC was measured in duplicate with an anthropometric tape while the subjects were wearing light clothing. Data related to alcohol consumption was obtained from self-completed surveys. Alcohol 6
ACCEPTED MANUSCRIPT consumption was categorized into two groups, drinkers and non drinkers, the latter defined as those who drink alcohol more than once per month.
T
Fasting glucose, insulin, C-peptide levels, glycated hemoglobin (HbA1c), lipid profiles,
RI P
creatinine, and 25(OH)D were measured. The level of 25(OH)D was measured using a radioimmunological determination kit (CIS Bio, France). HbA1c was measured by ion-
SC
exchange high-performance liquid chromatography (Bio-Rad, Hercules, CA, USA). Serum
MA NU
creatinine level was measured by the alkaline picrate (Jaffe) method. Total cholesterol (TC), Low density lipoprotein-cholesterol (LDL-C) and triglyceride (TG) were determined using the liquid enzymatic method with an automatic biochemical analyzer (7600-110;Hitachi Inc., Tokyo, Japan) and HDL-C was measured by the selective inhibition method. Serum insulin
ED
and C-peptide were measured using an immunoradiometric assay kit (DIAsource Immunoassay, Belgium). Insulin resistance was calculated using the homeostasis model
PT
assessment of insulin resistance (HOMA-IR) according to the following formula: [fasting
CE
insulin (μIU/mL) x fasting blood glucose (mmol/L)]/22.5.
AC
2.3.Evaluation of microvascular complications Diabetic peripheral neuropathy (DPN) was diagnosed in the presence of typical symptoms using the Michigan Neuropathy Screening Instrument (MNSI) and compatible findings on neurological screening examinations such as a 10-g monofilament test, a vibration test with 128 Hz tuning fork, and an ankle reflex examination or electrophysiological studies. Although electrophysiological studies are not essential, current perception thresholds (CPTs) tests were carried out in all patients using a Neurometer (Neurotron Inc., Baltimore, MD, USA).
7
ACCEPTED MANUSCRIPT Diabetic nephropathy (DN) was defined according to albuminuria, which was measured by radioimmunoassay (Immunotech, Prague, Czech Republic). Albumin excretion was assessed
T
using a first-morning spot urine sample. However, if a first-morning specimen is not
RI P
available, second-morning specimen is considered acceptable. Urine albumin < 30 mg/g creatinine was categorized as normoalbuminuria, 30-300 mg/g creatinine as
SC
microalbuminuria, and ≥ 300 mg/g creatinine as overt proteinuria. Patients were considered
MA NU
to have nephropathy if they showed microalbuminuria or overt proteinuria [19]. Estimated glomerular filtration rate (eGFR) was calculated using the Modification of Diet in Renal Disease Study equation [20].
Diabetic retinopathy (DR) was defined based on findings of dilated pupils on fundoscopy
PT
proliferative retinopathy.
ED
carried out by an ophthalmologist. DR was defined as having nonproliferative retinopathy or
CE
2.4.Statistical analyses
Statistical analyses were performed using the SPSS Version 18 (2010) (SPSS Inc., Chicago,
AC
Illinois, USA). Values were represented as means ± standard deviation (SD) for variables that were normally distributed, median (interquartile range : IQR) for variables that were not normally distributed or as number of participants (percentages). P-values < 0.05 were considered statistically significant. To evaluate the clinical and demographic characteristics of the study subjects, analysis of variance and Kruskal-Wallis tests were used to compare the quantitative data of the three groups according to vitamin D status (deficient, insufficient, and sufficient). Chi-square test was used for categorical data. Before the test, Shapiro-Wilk test for normality and Levene’s homogeneity of variance test was conducted. Non-normally distributed variables, that is, 8
ACCEPTED MANUSCRIPT triglycerides, serum fasting insulin, fasting c-peptide, HOMA-IR, and microalbumin were natural-logarithmic transformed before analysis.
T
For confirming the correlations between 25(OH)D level and clinical and biochemical
RI P
variables, we calculated the correlation coefficient and p-value using Pearson’s or Spearman’s bivariate correlation analysis. A Chi-square test and Student’s t-test were used for comparing
SC
the prevalence of the vitamin D deficiency and the difference in mean level of 25(OH)D
MA NU
according to presence of microvascular complications. The independence of the association of DPN and DN with vitamin D deficiency was assessed by multivariate logistic regression, after adjusting for confounding factors which were chosen by univariate analysis and, traditionally known risk factors such as age, duration of diabetes, HbA1C, BMI, smoking,
ED
lipid, inflammatory marker, and medications.
We performed post hoc power analyses with PASS (version 12: NCSS, Kaysville, Utah, USA)
AC
CE
PT
in order to estimate the probability of detecting present effects with the current sample.
9
ACCEPTED MANUSCRIPT 3. Results 3.1. The clinical characteristics of the participants
T
The mean age of the participants was 58.8 ± 12.1 years, and the mean duration of diabetes
RI P
was 7.7 ± 7.9 years. The mean body mass index (BMI) was 25.0 ± 4.0 kg/m2, and mean 25(OH)D level was 14.4 ± 8.6 ng/mL (Supplemental Table 1). The females had a
SC
significantly lower BP, FPG, HbA1C, and TG, and longer duration and higher HDL-C than
MA NU
males (Supplemental Table 1). The participants were divided into three groups (84 vitamin D deficient patients, 132 vitamin D insufficient patients, and 41 vitamin D sufficient patients). The prevalence of deficiency and sufficiency of vitamin D in all patients was 32.7% and 15.9%, respectively. The proportion of vitamin D status according to gender was significantly
ED
different (deficient, insufficient, and sufficient, 18.9%, 54.1%, and 27% in males and 43.2%, 49.3%, and 7.5% in females, p
