Associations of C-reactive Protein with 25-hydroxyvitamin D in 24 Specific Diseases: A Cross-sectional Study from NHANES

Most diseases might be associated with acute or chronic inflammation, and the role of vitamin D in diseases has been extensively explored in recent years. Thus, we examined the associations of one of the best markers for inflammation ― C-reactive protein (CRP) with 25-hydroxyvitamin D [25(OH)D] in 24 specific diseases. We performed cross-sectional analyses among 9,809 subjects aged ≥18 years who participated in the U.S. National Health and Nutrition Examination Survey (NHANES) in 2007~2010. The generalized additive model (GAM) was used to explore the associations of CRP with 25(OH)D in different diseases, adjusted for the age, gender, examination period and race. Distributions of CRP were significantly different (P < 0.05) in gender, examination period and race, and distributions of 25(OH)D were different (P < 0.05) in the examination period and race. Generally, CRP was negatively associated with 25(OH)D for majority diseases. 25(OH)D was negatively associated with CRP generally, and the associations were disease-specific and disease category-specific. In respiratory, gastrointestinal and mental diseases, the associations tended to be approximately linear. While in metabolic diseases, the associations were nonlinear, and the slope of the nonlinear curve decreased with 25(OH)D, especially when 25(OH)D < 30 μg/L.

D in GAM. Associations between CRP and 25(OH)D were statistically significant in majority diseases. Those minority diseases with nonsignificant associations may due to the small sample sizes of patients.

Discussion
Vitamin D was considered to have potential beneficial effects on reducing inflammation and alleviating pain 7,8 . Thus, our study aimed to explore the associations of CRP with 25(OH)D in 24 specific diseases among adults of NHANES using GAM. Firstly, we found that 25(OH)D was negatively associated with CRP in general. And the associations were disease-specific and disease category-specific: in respiratory, gastrointestinal and mental diseases, the associations tended to be approximately linear. While in metabolic diseases, the associations were nonlinear, and the slope of the non-linear curve decreased with 25(OH)D, especially when 25(OH)D was less than 30 μg/ L. Secondly, the cut-off value of 25(OH)D in cancer was higher than that in other diseases. 25(OH)D was negatively associated with CRP in almost all diseases, which was similar to previous studies 15,18 . One of the possible reasons is vitamin D could inhibit the progression of inflammation by reducing the secretion of inflammatory cytokines such as IL-6, which could indirectly decrease the CRP level 19 . Another reason may be that CD8 + T cells could directly destroy the damaged or infected host cells, thereby reducing the amount of pro-inflammatory cells. Meanwhile, CD8 + T cells carried higher levels of vitamin D receptor (VDR) expression, whose function was regulated by vitamin D [20][21][22] , which may also indirectly down-regulate the CRP level.
In this study, associations of CRP with 25(OH)D differed in different disease categories. Similar negative associations, which presented approximate linear, were found in respiratory, gastrointestinal diseases, mental diseases and arthritis. Previous studies based on large populations showed that there was a strong dose-response association between lower vitamin D levels and increased risk of upper respiratory infection 23,24 . The potential mechanism for this may be that vitamin D, by activating VDR, had immunomodulatory effects on structural cells in airways 25 , while these cells could trigger the pathogenesis of asthma through complex interactions with inflammatory lymphocytes 26 . Besides, the immune regulation effect of vitamin D in asthma could both reduce airway hyper-responsiveness and inflammation, and contribute to the airway remodeling 27 .
Previous studied reported that vitamin D status was associated with the composition and function of the intestinal microbiome. In addition, vitamin D and VDR regulate the innate immune response to the microbiome 28,29 . Moreover, previous evidence suggested that vitamin D may be beneficial for autoimmune and allergic pathologies 30,31 .
Vitamin D may also have influence on emotion and depression 32,33 . This could attribute to inflammation, a well-known hypothesis mechanism for depression 34  www.nature.com/scientificreports www.nature.com/scientificreports/ diseases. It is reported that low levels of vitamin D and VDR were significantly associated with higher levels of inflammatory markers, which could be attenuated by vitamin D supplementation 35 .
Further, we found nonlinearly negative associations of 25(OH)D with CRP in metabolic diseases. Previous studies also indicated that vitamin D might associate with the pathogenesis of type 2 diabetes mellitus (T2DM) 36 and dyslipidemia 37 . It was reported that the supplementation of vitamin D may reduce systemic inflammation, that is, vitamin D supplementation had beneficial effect on the CRP levels 38,39 . The main potential mechanism of progression of T2DM was β cell dysfunction due to inflammatory stress and insulin resistance, while VDR was the key regulator of inflammation and beta cell survival 40 . There was evidence that the associations between 25(OH)D level and hypertriglyceridemia may be mediated through inflammation, as these associations disappeared after CRP as a covariate 41 . It was also suggested that obesity-related inflammation could lead to abnormal function of fat cells, elevate circulating levels of free fatty acid and ectopic lipid accumulation 42 .
In addition, in metabolic diseases, we found that the nonlinear associations of CRP with 25(OH)D had a weakening tendency as 25(OH)D increasing, which is similar with several previous studies. They found that the negative association between 25(OH)D and CRP would gradually decrease when 25(OH)D level was close to or higher than 21 μg /L 43,44 . This result indicated that, when the 25(OH)D level was higher than 30 μg/L, no additional clinical benefit was obtained, which was consistent with our study. It was also noteworthy that the cut-off value of 25(OH)D above-mentioned in cancer was higher than common diseases. Moreover, it was reported that higher 25(OH)D concentrations had benefits effect on the breast cancer, lung cancer and colorectal cancer [45][46][47] . However, the optimal doses of 25(OH)D concentration has not been determined yet 48,49 .
Thus, vitamin D supplementation might be always beneficial in respiratory, gastrointestinal and mental diseases, which may not so significant in metabolic diseases. Although metabolic diseases were not considered as inflammatory diseases traditionally, inflammatory components apparent promoting the disease process were increasingly taken into investigation 12 . The negative associations of CRP with vitamin D might provide greater potential therapeutic prospects for common diseases. Moreover, the supplementation of vitamin D in cancer should be more than that in common diseases for greater benefit.
Some limitations should be noted in present study. Firstly, the study was cross-sectional, we cannot draw a causal conclusion on the associations of CRP with 25(OH)D. Secondly, several unexplained variables might influence the associations of CRP with 25(OH)D cannot be taken into account. Thirdly, the complex sampling analysis was not involved in this study.

Conclusion
In general, 25(OH)D was negatively associated with CRP, and the associations were disease-specific and disease category-specific. In respiratory, gastrointestinal and mental diseases, the associations tended to be approximately linear. While in metabolic diseases, the associations were nonlinear, and the slope of the nonlinear curve decreased with 25(OH)D, especially when 25(OH)D less than 30 μg/ L.

Methods
Sample. The NHANES, which were conducted by Centers for Disease Control and Prevention (CDC), aimed to assess the health status of the U.S. non-institutionalized civilian population. This survey utilized a complex probability sampling design, and collected information by standardized interviews, physical examinations and tests of biological samples 50,51 . A total of 20,686 subjects were enrolled in NHANES in 2007~2010. Our study focused on the relationship between CRP and 25(OH)D among adults in 24 specific diseases. For this purpose, the reluctant subjects who <18 years old (7,931 participants), unwilling to accept the measurement of 25(OH) D (1,925 participants) or CRP (4 participants) were excluded, and 1,017 participants who did not have any of 24 specific diseases were also excluded. As shown in Fig. 2, finally, a total of 9,809 participants who at least had one of diseases were involved in the study.   www.nature.com/scientificreports www.nature.com/scientificreports/ Data measurement. NHANES database sociodemographic information included age, gender, examination period and race. Examination period indicated the time that the participant measured the 25(OH)D, which was classified into either November 1st through April 30th or May 1st through October 31st. Because it was rather cold in the northern states in winter, the data were collected in the northern states in summer and southern states in winter 52 . Race was coded in 5 categories: Non-Hispanic Whites, Non-Hispanic Blacks, Mexican Americans, other Hispanics, and other non-Hispanic race including non-Hispanic multiracial.
During the NHANES physical examination, weight and height were measured in a standardized fashion. Height and weight values were automatically transmitted from stadiometers and scales to the Integrated Survey Information System database. This database was designed to reduce data errors and contained age-and sex-specific edit ranges for each body-size measure on the basis of previous NHANES data. If an entry was outside this range, the recorder was alerted that the value was unusual and required to verify the measurement 53 . Body mass index (BMI) was calculated by the weight divided by height squared (kg/m 2 ). Blood specimens were     3 55 . Patients with the following diseases were defined as having been told by a doctor or other health professional: emphysema, diabetes, high cholesterol, thyroid problem, gout, kidney stone, failing kidney, anemia, heart attack, coronary heart disease (CHD), stroke, sleeping trouble, cancer, arthritis, prostate disease. Patients with asthma, chronic bronchitis and liver condition were defined as still having the disease at the time of the investigation. Patients with following diseases were defined as having these diseases started during those 30 days: cold/ chest cold, flu/ pneumonia/ ear infection, stomach or intestinal illness, anxious 56 . Patients with depression were defined as those whose Patient Health Questionnaire (PHQ-9) score ≥10, based on the participants' symptoms over the past two weeks 57,58 . Obesity was defined those whose BMI ≥ 30 kg/m 2 59 . In addition, patients with cancer were defined that who had cancer or a malignancy of any kind.
In this study, 24 specific diseases were divided into 5 categories: a. respiratory and gastrointestinal diseases, including asthma, chronic bronchitis, cold/ chest cold, flu/ pneumonia/ ear infection, emphysema and stomach/ intestinal illness; b. metabolic diseases, including diabetes, high cholesterol, thyroid problem, gout and obesity; c. cardiovascular and cerebrovascular diseases, including heart attack, CHD and stroke; d. mental diseases, including anxious, sleeping trouble and depression; e. other diseases, including cancer, arthritis, prostate disease, failing kidney, anemia, liver condition and kidney stone. Statistical analysis. The x ± S and the M [P 25 , P 75 ] were used to describe the distribution of continuous variables, and proportion was used to describe the distribution of categorical variables. Wilcoxon rank sum test was used to compare the continuous variables, and Rao-Scott-χ 2 test was used to compare the categorical variables. CRP was log-transformed to obtain normal distributions for analysis, and back-transformed for representation in the tables. Age, gender, examination period and race were adjusted to examine the associations between log-CRP and 25(OH)D. GAM was also used to explore the associations of CRP with 25(OH)D. It was a generalization of the generalized additive model (GLM), which had great potential and flexibility in addressing non-linear relationships 60 . Two variables were linear associated when the estimated degrees of freedom (EDF) in GAM was equal to one. All statistical analyses were performed by R version 3.4.3, and the package "gam" 61 , "mgcv" 62 and "glmnet" 63 were used. Statistical significance was set at P < 0.05.
Ethics approval and consent to participate. All NHANES protocols were approved by the National Center for Health Statistics' Research Ethics Review Board, all participants signed a consent form before their participations and all research was performed in accordance with relevant guidelines/regulations. The statement of informed consent is openly available in https://www.cdc.gov/nchs/nhanes/biospecimens/participants.htm.
Informed consent. Health information collected in the NHANES is kept in strictest confidence. During the informed consent process, survey participants are assured that data collected will be used only for stated purposes and will not be disclosed or released to others without the consent of the individual or the establishment in accordance with section 308(d) of the Public Health Service Act (42 U.S.C. 242 m). Only samples from participants who have consented for future research are stored in the NHANES Biospecimen Repository and are available to researchers.