Association of hsCRP and vitamin D levels with mild cognitive impairment in elderly type 2 diabetic patients

AIMS
The aim of the study was to determine the serum levels of 25-hydroxyvitamin D and high-sensitivity C-reactive protein (hsCRP) in elderly diabetic patients with and without mild cognitive impairment (MCI) and to examine factors (including 25-hydroxyvitamin D and hsCRP) associated with MCI in elderly patients with type 2 diabetes (T2DM).


METHODS
A total of 194 T2DM elders were evaluated: 62 subjects with MCI and 132 controls. Data was collected concerning biochemical parameters and biomarkers.


RESULTS
HsCRP concentration was elevated and 25-hydroxyvitamin D level was decreased in MCI patients to controls. HsCRP level was negatively correlated with 25-hydroxyvitamin D level and with MoCA score, and highly correlated with HbA1c level. The multivariable analysis indicated that less years of formal education, previous CVD and hypertension, increased number of co-morbidities, higher level of hsCRP and lower level of 25-hydroxyvitamin D, are the predisposing factors for MCI.


CONCLUSIONS
Higher hsCRP level and lower 25-hydroxyvitamin D may be regarded as a state of cognitive impairment in elderly patients with T2DM. Further prospective larger studies should be conducted to check the association between decreased vitamin D and risk of cognitive decline and to clarify whether this association may be mediated by systemic inflammation.


Introduction
Both type 2 diabetes (T2DM) and dementia are the most common disorders in late life, and patients with type 2 diabetes show an increased risk to develop the cognitive impairment. 1,2 Many studies reported these subjects have problems with verbal memory, immediate recall, delayed recall, psychomotor speed and verbal fluency. 2 Cognitive impairment may lead to difficulties in self-management and an increase risk of diabetes-related complications. 3 Mild cognitive impairment (MCI) represents the transitional stage from the cognitive changes of normal ageing to very early dementia and 10-15% of patients with MCI have progress to dementia per year compared to 1-2% of healthy controls. 4 Due to the growth of the elderly population both conditions: MCI and T2DM have recently become a focus of research.
Several mechanisms contributing to the etiology of cognitive impairment in diabetes has been proposed. One of the theories underlies the causative role for vascular pathology in diabetic brain. Some studies showed that small vessel diseases in the brain (white matter lesions and lacunae) affect cognitive function in older diabetics without overt dementia or symptomatic stroke. 5 Other hypotheses suggest that hyperglycemia lead to higher formation of advanced glycation end products (AGEs), increased glucose shunting in the hexosamine pathway, diacylglycerol activation of protein kinase C, and polyol pathway activation. 6 Vitamin D (25-hydroxvitamin D; 25[OH]D) deficiency has been associated with various neuropsychiatric symptoms, and it has also been reported to have an important role in the development of dementia. Some studies showed that serum 25-hydroxvitamin D levels are lower in subjects with impaired cognitive function and dementia than in healthy controls. 7,8 The precise mechanism associating dementia risk and vitamin D deficiency is not fully explained. Some studies have shown that low level of vitamin D has been related to increase white matter hyperintensities and larger ventricular volumes. 9,10 Another study has shown that reduced serum vitamin D concentration is associated with low level of β-amyloid in cerebrospinal fluid. 11 Recent evidence suggests that low vitamin D concentrations are associated with increased levels of inflammatory markers. In one large study performed in older adults have reported that those subjects who have low serum levels of 25(OH)D have increased inflammatory biomarker profiles, including increased CRP, plasma fibrinogen and J o u r n a l P r e -p r o o f 4 influence on the development of cognitive decline in patients with diabetes either by a direct effect on the brain or through the enhancement of atherosclerosis, vascular disease and endothelial dysfunction. 15 Although there are some studies that described separately relationship of inflammation or vitamin D levels with MCI in diabetes, data about common pathogenesis included low-grade inflammation measured by hs-CRP concentrations and serum vitamin D levels in elderly subjects with coexisting mild cognitive impairment and type 2 diabetes are lacking. Therefore, the aims of the study were twofold: Firstly, evaluate levels of serum 25-hydroxvitamin D and high-sensitivity C-reactive protein (hs-CRP) in elderly patients with T2DM with and without MCI and secondly, identify the factors (including vitamin D levels and hs-CRP levels) associated with MCI in elderly patients with T2DM.

Population
194 participants were obtained from the study previously described elsewhere. 16 We conducted a cross-sectional study among T2DM subjects patients recruited from outpatient diabetology clinic affiliated with the university hospital no 1 in Lodz, Poland from November 2013 to February 2014. As vitamin D status depends in part on sunlight exposure all participants were requited in the same season (winter). All subjects were Caucasian race.  The following tests were administrated to evaluate the participants' the cognitive functions: the Montreal Cognitive Assessment (MoCA), 17 Katz Basic Activities of Daily living (BADL) and Lawton Instrumental Activities of Daily Living (IADL) questionnaires to collect information on daily activities. 18,19 The MoCA assesses multiple cognitive domains, including attention and concentration, executive functions, immediate and delayed memory, language ability, visuoconstructional skills, conceptual thinking, calculation performance, and orientation. The normal MoCA score is ≥26, with one point added if the subject has fewer than 12 years of formal education. The MoCA is recommended to detect MCI in the elderly patients with type 2 diabetes. 20 Diagnosis of MCI was performed according criteria proposed by the MCI Working Group of the European Alzheimer"s Disease Consortium which are currently available standard test. 21,22 These criteria included absence of dementia. Therefore we excluded from the study subjects with MOCA score 19 and below and sent them to J o u r n a l P r e -p r o o f 6 We evaluated 2 groups of subjects: group 1 -patients with MCI and group 2 -patients without MCI (controls).

Covariates
The outcome variable was mild cognitive impairment. The explanatory variables are sociodemographic, clinical, and biochemical factors that were collected by patient interview,

Ethics
The study was operated in accordance with the World Medical Association"s Declaration of Helsinki. Each participant was assigned a number by which he/she was identified to keep his or her privacy. The approval was obtained from the independent local ethics committee of Medical University of Lodz. The purpose, nature, and potential risks of the experiments were fully explained to the subjects, and all subjects gave written, informed consent at the beginning of the study. We included only patients who had been fully able to understand and cooperate with study procedures.

Statistical Analysis
All data are presented as means  SD. This study was designed to detect significant changes between the diabetic patients with MCI and control (diabetic patients without MCI).
The mean prevalence of MCI in diabetic patients is 14-31 % thus with prediction of MCI in this study about 25%, using a two tails test with power of 90% and α = 0.05 a calculated minimal sample size of 60 for diabetic MCI positive patients was required to yield a statistically significant result [http://www.gpower.hhu.de/]. 2,6 Normality of distributions was assessed using the Shapiro-Wilk tests. The descriptive statistics for the categorical variables were tested using the χ 2 , and the continuous variables using the Student"s t or the Mann Whitney-U tests whenever applicable. Pearson correlation analysis for normally distributed J o u r n a l P r e -p r o o f variables and Spearman rank correlation for nonnormally distributed variables were used to assess relationships. Simple logistic regression model was done in order to select so-called independent factors which increase the selection risk of MCI in elderly patients with T2DM. Then multivariable regression model in order to select the "strongest" factor from independent risk factors. To "optimize" the multivariable model, a stepwise approach was used (backward elimination with Wald criteria). Odds ratios (OR) were computed and presented with the 95% interval of confidence (CI). A P value of less than 0.05 was considered statistically significant. Statistica 13.1 (StatSoft, Poland) was used for analysis.

General description of patients with MCI and controls
The demographic and clinical characteristics of the study group are presented in Table   1. High-sensitivity C-reactive protein level was significantly increased in patients with MCI compared to controls (p<0.001) ( Table 1). In the group of patients with MCI hsCRP concentration was highly correlated with HbA1c level (r=0.55, p<0.001). The results indicated that hsCRP level was inversely correlated with MoCA score (r=-0.59, p<0.001). A positive but weak correlation was found between this parameter and total cholesterol, or BMI.
The results are presented in Table 2.  (Table 3).

Discussion
The results of this study showed that serum vitamin D level was significantly diabetic patients with MCI and 70 subjects with no MCI and they found that cognitive impairment is associated with lower level of vitamin D. 25 These data are consistent with our results, however their patients were younger with mean age around 55 years and they excluded subjects older than 70 years where cognitive impairment has greater impact on heath in general. Similar to our results the authors also showed that vitamin D levels were independently associated with MoCA score and they concluded that vitamin D might have a potential protective effect against MCI in patients with diabetes. Several biological mechanisms had been proposed that might explain the association between low serum vitamin D levels and cognitive impairment. In the recent study the authors showed that vitamin D deficiency is related to reduced hippocampal volume and disrupted structural connectivity. 26 The researchers had analyzed an existing structural and diffusion MRI dataset of elderly patients with MCI and found that low vitamin D is associated with reduced volumes of hippocampal subfields and connection deficits in elderly people with MCI, which may exacerbate neurocognitive outcomes. Other studies showed that low vitamin D concentration was associated with reduced β-amyloid levels in cerebrospinal fluid, which is a well-known risk factor for dementia. 27 Vitamin D receptors are widely expressed in the human brain areas including hippocampus, hypothalamus, prefrontal cortex and may be potentially involved in many neuropsychiatric processes. 28 In our study we have also found that in the group of patients with MCI serum 25hydroxyvitamin D levels was negatively correlated with BMI and with hsCRP level. This observation is consistent with previous investigations. In systematic review and meta-analysis of fifty-five observational studies, the authors showed the inverse relationship between serum vitamin D status and body mass index (BMI) in studies of both diabetic and non-diabetic subjects. 28 The researchers suggest that many pathologic conditions like obesity, metabolic In our study we found that hsCRP level was significantly increased in patients with MCI compared to controls. Hs-CR -an acute-phase protein is a sensitive and dynamic marker for low grade inflammation in diabetes. Other researchers had also confirmed higher concentration of hs-CRP in diabetic elderly patients with MCI compared to those without cognitive impairment. 31,32 We also notice that hsCRP concentration was highly correlated with HbA1c level. Patients with MCI were more likely be diagnosed with retinopathy and nephropathy compared to controls. Persistent hyperglycemia and development of diabetes micro complications could enhance cognitive dysfunction by aggravating inflammation.
Other authors showed that chronic hyperglycemia and increased HbA1c levels are risk factor for the cognitive impairment. 36 CRP is also associated with increased risk of development and prognosis of cardiovascular diseases and for cardiovascular events in T2DM. We showed that system. 33 We known that that stepwise analysis may greatly bias the final results for lots of reasons therefore we compared the results of our regression analysis with evidence found from literature finding. In consistent to our data some authors found that poor education and hypertension are the risk factors in MCI of T2DM patients. 34 Another researchers in multivariate logistic regression analysis showed that vascular diseases were significantly related to increase the odds of MCI and its specific subtype. 35 Chen et al revealed that low level of vitamin D, and history of hypertension are independent factors predicted MoCA score in multivariate regression analysis. 25 They concluded that vitamin D may be a potential protective factor for cognitive impairment in patients with type 2 diabetes.
This study provides important insights into common pathogenesis included low-grade inflammation measured by hs-CRP concentrations and serum vitamin D levels underlying cognitive impairment in elderly diabetic subjects however, it is not without limitations. First, it was a single-center study, most subjects lived in urban areas in central Poland, therefore the study should be extended to the inhabitants of other regions and the results may be obtained from entire Polish population. Our participants were recruited from one race/ethnicity, which may limit the generalizability of findings to other populations. Second, the study wasn"t designed as longitudinal prospective investigation. It could be interesting to check the contribution of low-grade inflammation and hypovitaminosis D to cognitive decline and latelife dementia risk, although the exact mechanism is uncertain. Thirdly because the stepwise analysis may greatly bias the final results for lots of reason some sensitivity analyses in modifying the pre-determined list of variables may be performed.

Conclusions
In summary, the current study demonstrated that T2DM elderly individuals with mild