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10.1136/bmjopen-2023-083227
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Original Research
Nutrition and Metabolism
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Associations of serum 25-hydroxyvitamin D with hsCRP and other novel inflammatory biomarkers in children: a cross-sectional study
http://orcid.org/0000-0001-5996-2376
Dong Hongli 1dhljiyi@163.com

Wang Feiying 2946923407@qq.com

Gao Liang 21053430164@qq.com

Xu Xiaojing 218068186581@163.com

http://orcid.org/0009-0003-1202-8166
Ni Yufei 2niyffy@163.com

1 Scientific Education Section, Affiliated Maternity & Child Health Care Hospital of Nantong University, Nantong, Jiangsu, China
2 Department of Child Healthcare, Affiliated Maternity and Child Health Care Hospital of Nantong University, Nantong, Jiangsu, China
Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.

None declared.

Professor; niyffy@163.com
2024
05 9 2024
14 9 e08322715 12 2023
26 8 2024
Copyright © Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.
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Abstract

Objective

Previous studies have shown the anti-inflammatory effect of 25-hydroxyvitamin D (25(OH)D) and the crucial roles of high-sensitive C reactive protein (hsCRP) and novel inflammatory markers (red blood cell distribution width–platelet count ratio (RDWPCR), mean platelet volume–platelet count ratio (MPVPCR), neutrophil–lymphocyte ratio (NLR) and white blood cell–neutrophil ratios (WBCNR)) in several diseases, but scarce data explored the associations of 25(OH)D with hsCRP and novel inflammatory markers. This study aimed to investigate these associations in children.

Design

Cross-sectional study.

Setting

Children in China.

Participants

10141 children (mean age 14.6 months) were included.

Primary and secondary outcome measures

HsCRP, red blood cell distribution width, platelet count, mean platelet volume, neutrophil, lymphocyte and white blood cell were measured.

Results

Overall, serum 25(OH)D was inversely associated with hsCRP and novel inflammatory biomarkers. In multivariable analysis, serum 25(OH)D was inversely associated with hsCRP and novel inflammatory biomarkers (Q quartile (Q) 4 vs Q1: 1129.75 vs 2090.99 for hsCRP; 4246.94 vs 6829.89 for RDWPCR; 4863.57 vs 5545.66 for MPVPCR; 4345.76 vs 6507.46 for NLR; 2418.84 vs 2868.39 for WBCNR). Similar results also were observed in stratified analyses by sex (boys and girls). Moreover, serum 25(OH)D was inversely associated with elevated inflammation levels. After adjustment for other potential covariates, inverse associations between serum 25(OH)D and elevated inflammation levels were still observed. The corresponding ORs (95% CI) were 0.05 (0.04, 0.06) for hsCRP, 0.13 (0.11, 0.15) for RDWPCR, 0.74 (0.64, 0.85) for MPVPCR, 0.11 (0.09, 0.13) for NLR and 0.57 (0.49, 0.66) for WBCNR in the fourth quartile compared with the first quartile, respectively.

Conclusions

Generally, the graded and inverse associations of serum 25(OH)D with hsCRP and four novel inflammatory markers (RDWPCR, MPVPCR, NLR and WBCNR) were observed. The present study provided further support for the anti-inflammatory effects of 25(OH)D.

cross-sectional studies
paediatric endocrinology
nutritional support
Nantong Health Committee MS2022075 Nantong University Clinical Medicine Specialized Research Fund Project 2023LY004
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pmcSTRENGTHS AND LIMITATIONS OF THIS STUDY

This study first used a generalised linear model to compare the differences in the concentrations of inflammatory markers (eg, high-sensitive C reactive protein (hsCRP), red blood cell distribution width–platelet count ratio, mean platelet volume–platelet count ratio, neutrophil–lymphocyte ratio and white blood cell–neutrophil ratios) among sex-stratified quartiles classified by serum 25-hydroxyvitamin D (25(OH)D).

This study also further determined the associations between serum 25(OH)D and elevated inflammatory biomarkers levels using logistic regression model.

A large sample size allowed us to determine the associations of serum 25(OH)D with hsCRP and four novel inflammatory markers in children and ensured the stability and reliability of the results.

The seasonal variation of vitamin D level might be considered due to subjects were recruited over all seasons.

The limitation of this study were that the causality could not be inferred from the cross-sectional study. And the make-up of the study sample in this study had a relatively narrow population range, thus caution is needed when extrapolating the findings of this study.

Introduction

Inflammation, an essential risk factor of neurodevelopmental impairment during early to middle childhood,1 played a key role in various chronic diseases, such as cardiovascular diseases and type 2 diabetes mellitus.2 3 Previous study pointed out that nutrition had the potential value of nutritional therapy by exerting crucial anti-inflammatory effects.4 Therefore, the exploration of novel modified nutritional factors for controlling systematic inflammation would be of high priority.

25-hydroxyvitamin D (25(OH)D), the main form of circulating vitamin D, is characteristics of long half-life and relatively stable.5 It has been considered as the optimal indicator to reflect the nutritional status of circulating vitamin D in the body. Sîrbe et al indicated that 25(OH)D exerted pleiotropic effects on inflammation and related diseases.6 Hypersensitivity C reactive protein (hsCRP) was considered as a representative biomarker of systemic inflammation, which might be modulated by 25(OH)D.7 Prior studies aimed at exploring the associations of 25(OH)D or vitamin D supplementation with hsCRP have produced inconsistent results, with inverse810 and null associations11 in different adults. Vitamin D supplementation is the only source of circulating vitamin D for children, and the body metabolism level of children is significantly different from that of adults. Nevertheless, only few data assessed the association between 25(OH)D and hsCRP in children (positive12 and inverse1315 associations). Additionally, red blood cell distribution width–platelet count ratio (RDWPCR), mean platelet volume–platelet count ratio (MPVPCR), neutrophil–lymphocyte ratio (NLR) and white blood cell–neutrophil ratios (WBCNR) were widely known as the haematological biomarkers of systemic inflammation. The stability, sensitivity and specificity of these comprehensive indices were better than those of a single indicator.1621 The emerging evidences demonstrated the key roles of these novel inflammatory markers in some chronic diseases (eg, cardiovascular disease,22 hypertension,23 decompensation24 and type 2 diabetes nephropathy25). Nonetheless, no study has yet evaluated the associations of 25(OH)D with RDWPCR, MPVPCR, NLR and WBCNR.

To address the above questions, this hospital-based cross-sectional study examined the associations of 25(OH)D with hsCRP and four novel inflammatory markers (RDWPCR, MPVPCR, NLR and WBCNR) in children.

Materials and methods

Study participants

We conducted a hospital-based cross-sectional study. The medical records of 10 141 children with inflammatory marker measurements (The age range was 1–53 months, mean age 14.6 months) were analysed between January and December 2021 from the department of Child Healthcare, Nantong Maternal and Child Healthcare Hospital. Children aged 1 month to <5 years were included in this study, regardless of gender. Subjects were excluded according to the following predefined criteria: (1) suffering from mental disorders, cognitive impairment, blindness, deafness and other diseases; (2) receiving or having previously received treatment for major illnesses, such as radiotherapy and chemotherapy; (3) having a history of severe congenital diseases, such as congenital heart disease, congenital biliary atresia and congenital pulmonary dysplasia.

Epidemiological data and laboratory measurements

Sex, age, weight, height, vitamin D intake (eg, 400, 600 or 800 IU/day), feeding patterns (breastfeeding, artificial feeding or mixed feeding) and complementary feeding (yes or no) were collected. Height and weight were measured using an electronic column scale or electronic baby scale (Saikang Medical Measurement System (Pinghu), China) by medical workers in accordance with the standard protocol, and then all readings were accurate to 1 cm and 0.01 kg, respectively. Height and weight were measured twice, and the interval between two measurements were less than 2 cm and 0.20 kg, respectively.

Serum 25(OH)D concentration was measured using ELISA. Briefly, 10 µL serum sample were mixed with 200 µL biotin. The mixture was vortexed for 30 s (200–400 rpm), incubated at room temperature for 60 min, washed the plate with washing solution (five times) and then added 200 µL enzyme conjugates. Keep for 30 min at room temperature. The board was washed, added 100 µL substrate and sealed well. After stand at room temperature for 15 min, 100 µL termination liquid was added into the mixture, and then slightly oscillate for 20–30 s. The Rayto RT-6100 ELISA analyzer was applied to assess the serum concentration of 25(OH)D. Red blood cell distribution width, platelet count, mean platelet volume, neutrophil, lymphocyte and white blood cell were measured using automatic haematology analyser BC-7500CRP (Mindray, Shenzhen, China). The RDWPCR, MPVPCR, NLR and WBCNR were calculated.

Statistical analysis

Means (±SD) and median (IQR) were used to present normally distributed and skewed continuous variables, respectively. All continuous variables were examined for normality distribution by Kolmogorov-Smirnov test. Continuous variables were rank transformed. The original data and the rank transformation of data were included in analysis, respectively. Categorical variables were presented as the frequencies (per cent). The t-test and χ2 test were used to analyse continuous and categorical variables, respectively. Partial correlation was performed to examine the correlations between serum 25(OH)D and vitamin D intake and inflammatory markers (hsCRP, RDWPCR, MPVPCR, NLR and WBCNR). Analysis of covariance was performed to compare the mean differences in the concentrations of inflammatory markers (eg, hsCRP, RDWPCR, MPVPCR, NLR and WBCNR) among sex-stratified quartiles classified by serum 25(OH)D. Linear trends in continuous variables across quartiles of serum25(OH)D were assessed with a generalised linear model. Moreover, logistic regression model was performed to determine the associations between serum 25(OH)D and elevated inflammatory biomarkers levels. Elevated inflammatory biomarkers levels were determined by hsCRP≥1.0mg/L26 and inflammatory biomarkers (arbitrarily defined as top quartile by sex). Model 1 was univariate analysis without adjustment; and model 2 adjusted for potential covariates, including sex, height, weight, ages, vitamin D intake, feeding patterns and complementary feeding. Stratified analyses were conducted in boys and girls, respectively. All analyses were conducted using SPSS V.21.0 software package (IBM SPSS Statistics, Armonk, New York, USA). Two-sided p values<0.05 were considered statistically significant.

Patient and public involvement

Patients were not involved in the design of this study.

Results

Characteristics of subjects

The subjects were divided into quartiles according to serum 25(OH)D (n=10 141) level (table 1). The means according to the quartiles of 25(OH)D were as follows: 46.46 ng/mL (quartile (Q) 1 group), 71.75 ng/mL (Q2 group), 91.55 ng/mL (Q3 group) and 117.47 ng/mL (Q4 group). The subjects with higher 25(OH)D concentration had an older age, higher height and weight.

Table 1 Baseline characteristics of children by quartiles of serum 25(OH)D

Variables	N	Q1 (n=2534)	Q2 (n=2536)	Q3 (n=2536)	Q4 (n=2535)	P trend	
25(OH)D, ng/mL	10 141	46.46±12.68	71.75±4.99	91.55±6.43	117.47±11.58	<0.001	
Sex, n (%)						0.989	
Boys	5254	1313 (51.82)	1314 (51.81)	1314 (51.81)	1313 (51.80)		
Girls	4887	1221 (48.18)	1222 (48.19)	1222 (48.19)	1222 (48.20)		
Age, months	10 141	9.45±0.25	15.72±0.25	17.12±0.25	16.25±0.25	<0.001	
Height, cm	10 141	67.63±0.32	76.96±0.32	80.48±0.32	80.20±0.32	<0.001	
Weight, kg	10 141	7.82±0.07	10.03±0.07	10.91±0.07	10.73±0.07	<0.001	
Vitamin D intake						<0.001	
 400 IU/day, n (%)	9847	2396 (94.55)	2475 (97.59)	2497 (98.46)	2479 (97.79)		
 600 IU/day, n (%)	11	2 (0.10)	0 (0.00)	3 (0.10)	6 (0.24)		
 800 IU/day, n (%)	283	136 (5.35)	61 (2.41)	36 (1.44)	50 (1.97)		
Feeding patterns						<0.001	
 Breastfeeding, n (%)	2165	863 (34.06)	613 (24.17)	399 (15.73)	290 (11.44)		
 Artificial feeding, n (%)	6154	928 (36.62)	1443 (56.90)	1798 (70.90)	1985 (78.30)		
 Mixed feeding, n (%)	1822	743 (29.32)	480 (18.93)	339 (13.37)	260 (10.26)		
Complementary feeding, n (%)						0.015	
 Yes	6518	773 (30.51)	1548 (61.04)	2010 (79.26)	2187 (86.27)		
 No	3623	1761 (69.49)	988 (38.96)	526 (20.74)	348 (13.73)		
25(OH)D25-hydroxyvitamin DQquartile

Correlation of serum 25(OH)D and vitamin D intake with hsCRP and novel inflammatory biomarkers

Table 2 showed an inverse correlation of serum 25(OH)D and vitamin D intake with hsCRP and novel inflammatory biomarkers in partial correlation analyses adjusted for sex and age. Mean partial correlation coefficients of 25(OH)D and vitamin D intake were −0.564 and −0.041 for hsCRP, −0.541 and −0.043 for RDWPCR, −0.539 and −0.043 for NLR and −0.223 and −0.041 for WBCNR, respectively. Besides, only 25(OH)D was inversely correlated with MPVPCR (r: −0.080), whereas null correlation between vitamin D intake and MPVPCR was observed.

Table 2 Relationships of serum novel inflammatory biomarkers with 25(OH)D and vitamin D intake*

	25(OH)D	Vitamin D intake	
r′	P value	r′	P value	
hsCRP	−0.564	<0.001	−0.041	0.005	
RDWPCR	−0.541	<0.001	−0.043	0.004	
MPVPCR	−0.080	<0.001	0.019	0.222	
NLR	−0.539	<0.001	−0.043	0.005	
WBCNR	−0.223	<0.001	−0.041	0.007	
The meaning of the bold values indicated that P-values were considered statistically significant.

* Partial correlation analysis, adjusted for age and sex.

hsCRPhypersensitivity C reaction proteinMPVPCRmean platelet volume-to-platelet count ratioNLRneutrophil–lymphocyte ratios25(OH)D25-hydroxyvitamin DRDWPCRred blood cell distribution width–platelet count ratioWBCNRwhite blood cell–neutrophil ratios

Associations of serum 25(OH)D with hsCRP and novel inflammatory biomarkers

As shown in table 3 and online supplemental table 1, serum 25(OH)D was inversely associated with hsCRP and novel inflammatory biomarkers in both univariable and multivariable analysis according to the rank transformation of the data. After adjustment for the potential covariates, serum 25(OH)D was inversely associated with hsCRP and novel inflammatory biomarkers (Q4 vs Q1: 1129.75 vs 2090.99 for hsCRP; 4246.94 vs 6829.89 for RDWPCR; 4863.57 vs 5545.66 for MPVPCR; 4345.76 vs 6507.46 for NLR; 2418.84 vs 2868.39 for WBCNR). In the stratified analyses by sex (boys and girls), respectively. In boys, serum 25(OH)D was inversely associated with hsCRP and novel inflammatory biomarkers (Q4 vs Q1: 1146.23 vs 2098.25 for hsCRP; 4495.58 vs 7105.08 for RDWPCR; 5085.07 vs 5680.90 for MPVPCR; 4277.01 vs 6537.81 for NLR; 2498.13 vs 2983.22 for WBCNR). Similar inverse associations were also observed in girls (Q4 vs Q1: 1088.60 vs 2079.05 for hsCRP; 3938.40 vs 6587.51 for RDWPCR; 4564.79 vs 5407.63 for MPVPCR; 4355.82 vs 6554.33 for NLR; 2344.05 vs 2731.31 for WBCNR).

Table 3 Multivariable analysis of hsCRP and novel inflammatory biomarkers level according to quartiles of 25(OH)D.

Variables	Quartiles by 25(OH)D	P trend	
Q1	Q2	Q3	Q4	
Total populations						
 hsCRP	2090.99±25.21	1591.03±25.61*	1284.77±25.08*†	1129.75±25.70*†‡	<0.001	
 RDWPCR	6829.89±59.34	4768.05±54.92*	4440.18±55.71*†	4246.94±56.96*†	<0.001	
 MPVPCR	5545.66±62.45	4968.76±57.80*	4933.30±58.63*	4863.57±59.9*	<0.001	
 NLR	6507.46±53.12	4876.55±49.17*	4555.07±49.87*†	4345.76±50.99*†§	<0.001	
 WBCNR	2868.39±27.84	2489.16±25.77*	2380.11±26.14*¶	2418.84±26.73*	<0.001	
Boys						
 hsCRP	2098.25±35.22	1665.50±35.89*	1407.19±35.07*†	1146.23±35.97*†‡	<0.001	
 RDWPCR	7105.08±81.87	5026.86±74.78*	4726.19±75.92*§	4495.58±78.23*†	<0.001	
 MPVPCR	5680.90±88.12	5176.12±80.48*	5195.73±81.72**	5085.07±84.20*	<0.001	
 NLR	6537.81±74.89	4919.52±68.41*	4481.29±69.45*†	4277.01±71.56*†	<0.001	
 WBCNR	2983.22±40.69	2552.13±37.16*	2476.70±37.74*	2498.13±38.88*	<0.001	
Girls						
 hsCRP	2079.05±35.38	1537.88±35.89*	1184.50±35.22*†	1088.60±36.10*†	<0.001	
 RDWPCR	6587.51±86.59	4509.49±80.76*	4100.80±81.93*¶	3938.40±83.31*†	<0.001	
 MPVPCR	5407.63±89.27	4747.82±83.27*	4646.45±84.46*	4564.79±85.89*	<0.001	
 NLR	6554.33±75.34	4868.50±70.27*	4580.64±71.28*¶	4355.82±72.48*†	<0.001	
 WBCNR	2731.31±38.17	2414.73±35.60*	2286.14±36.11*	2344.05±36.72*	<0.001	
Abbreviations were shown in plus , standard error and Q, quartile. Sex, height, weight, ages, vitamin D intake, feeding patterns, and complementary feeding.. P compared with Q1; < 0.001 compared with Q1; P compared with Q2; P compared with Q2; P compared with Q3; P compared with Q3.

The meaning of the bold values indicated that P-values were considered statistically significant.

* p<0.001 compared with Q1.

† p<0.001 compared with Q2.

‡ p<0.001 compared with Q3.

§ p<0.05 compared with Q3.

¶ p<0.05 compared with Q2.

** p<0.05 compared with Q1.

hsCRPhigh-sensitive C reactive proteinMPVPCRmean platelet volume–platelet count ratioNLRneutrophil–lymphocyte ratioQquartileRDWPCRred blood cell distribution width–platelet count ratioWBCNRwhite blood cell–neutrophil ratios

Additionally, similar results were also found in both univariable and multivariable analysis based on the original data (online supplemental tables 2 and 3).

Associations between serum 25(OH)D and elevated inflammation levels

Additionally, we further explored the associations between serum 25(OH)D and elevated inflammation levels (table 4). Generally, serum 25(OH)D was inversely associated with elevated inflammation levels. In model 1 without adjustment, the ORs (95% CI) in the fourth quartile were 0.10 (0.08, 0.13) for hsCRP, 0.18 (0.16, 0.21) for RDWPCR, 0.71 (0.63, 0.81) for MPVPCR, 0.33 (0.29, 0.38) for NLR and 0.39 (0.34, 0.44) for WBCNR compared with the first quartile, respectively. In model 2 with adjustment for other potential covariates, inverse associations between serum 25(OH)D and elevated inflammation levels were still observed. The corresponding ORs (95% CI) were 0.05 (0.04, 0.06) for hsCRP, 0.13 (0.11, 0.15) for RDWPCR, 0.74 (0.64, 0.85) for MPVPCR, 0.11 (0.09, 0.13) for NLR and 0.57 (0.49, 0.66) for WBCNR in the fourth quartile compared with the first quartile, respectively. Table 5 and online supplemental table 4 showed the stratified analyses by sex. Inverse associations between serum 25(OH)D and elevated inflammation levels were observed in both boys and girls. In multivariable analysis, the corresponding ORs (95% CI) were 0.05 (0.03, 0.07) and 0.05 (0.03, 0.07) for hsCRP, 0.12 (0.10, 0.15) and 0.13 (0.10, 0.16) for RDWPCR, 0.78 (0.64, 0.95) and 0.71 (0.57, 0.87) for MPVPCR, 0.09 (0.07, 0.12) and 0.12 (0.09, 0.15) for NLR and 0.49 (0.40, 0.61) and 0.65 (0.52, 0.80) for WBCNR in the fourth quartile compared with the first quartile, respectively. Consistent to these findings from the rank transformation of the data, there were the inverse associations between serum 25(OH)D and elevated inflammation levels according to the original data (online supplemental tables 5–7).

Table 4 The associations of 25(OH)D with the presence of high inflammation determined by inflammatory biomarkers

Variables	Quartiles by 25(OH)D	P trend	
Q1	Q2	Q3	Q4	
hsCRP						
 Case, n	530/761	246/762	158/763	145/761		
 Model 1	1	0.21 (0.17, 0.26)*	0.11 (0.09, 0.14)*	0.10 (0.08, 0.13)*	<0.001	
 Model 2	1	0.26 (0.21, 0.34)*	0.08 (0.06, 0.11)*	0.05 (0.04, 0.06)*	<0.001	
RDWPCR						
 Case, n	1217/2534	539/2536	414/2536	365/2535		
 Model 1	1	0.29 (0.26, 0.33)*	0.21 (0.19, 0.24)*	0.18 (0.16, 0.21)*	<0.001	
 Model 2	1	0.23 (0.20, 0.27)*	0.15 (0.13, 0.17)*	0.13 (0.11, 0.15)*	<0.001	
MPVPCR						
 Case, n	726/2534	640/2536	601/2536	565/2535		
 Model 1	1	0.84 (0.74, 0.95)†	0.77 (0.68, 0.88)**	0.71 (0.63, 0.81)*	<0.001	
 Model 2	1	0.85 (0.74, 0.96)†	0.79 (0.69, 0.91)†	0.74 (0.64, 0.85)*	<0.001	
NLR						
 Case, n	961/2534	585/2536	564/2536	426/2535		
 Model 1	1	0.49 (0.43, 0.56)*	0.47 (0.41, 0.53)*	0.33 (0.29, 0.38)*	<0.001	
 Model 2	1	0.14 (0.12, 0.17)*	0.13 (0.11, 0.15)*	0.11 (0.09, 0.13)*	<0.001	
WBCNR						
 Case, n	950/2534	612/2536	492/2536	481/2535		
 Model 1	1	0.53 (0.47, 0.60)*	0.40 (0.35, 0.46)*	0.39 (0.34, 0.44)*	<0.001	
 Model 2	1	0.68 (0.60, 0.77)*	0.57 (0.50, 0.66)*	0.57 (0.49, 0.66)*	<0.001	
Abbreviations were shown in plus Q, quartile; OR, odd ratio; , confidence interval. Model 1 was univariate analysis without adjustment. Model 2 adjusted for sex, height, ages, weight, vitamin D intake, feeding patterns, and complementary feeding. OR, 95% CI. P compared with Q1; < 0.001 compared with Q1.

The meaning of the bold values indicated that P-values were considered statistically significant.

* p<0.001 compared with Q1.

† p<0.05 compared with Q1.

hsCRPhigh-sensitive C reactive proteinMPVPCRmean platelet volume–platelet count ratioNLRneutrophil–lymphocyte ratio25(OH)D25-hydroxyvitamin DQquartileRDWPCRred blood cell distribution width–platelet count ratioWBCNRwhite blood cell–neutrophil ratios

Table 5 Multivariable analysis of the associations of 25(OH)D with the presence of high inflammation determined by inflammatory biomarkers by sex (OR, 95% CI)

Variables	Quartiles by 25(OH)D	P trend	
Q1	Q2	Q3	Q4	
hsCRP						
 Boys	1	0.27 (0.19, 0.39)*	0.12 (0.09, 0.18)*	0.05 (0.03, 0.07)*	<0.001	
 Girls	1	0.26 (0.19, 0.37)*	0.06 (0.04, 0.08)*	0.05 (0.03, 0.07)*	<0.001	
RDWPCR						
 Boys	1	0.22 (0.19, 0.27)*	0.13 (0.11, 0.17)*	0.12 (0.10, 0.15)*	<0.001	
 Girls	1	0.23 (0.19, 0.28)*	0.16 (0.13, 0.20)*	0.13 (0.10, 0.16)*	<0.001	
MPVPCR						
 Boys	1	0.88 (0.73, 1.04)	0.81 (0.67, 0.97)†	0.78 (0.64, 0.95)†	0.016	
 Girls	1	0.82 (0.68, 0.99)†	0.79 (0.65, 0.97)†	0.71 (0.57, 0.87)*	<0.001	
NLR						
 Boys	1	0.12 (0.09, 0.15)*	0.11 (0.09, 0.14)*	0.09 (0.07, 0.12)*	<0.001	
 Girls	1	0.16 (0.13, 0.21)*	0.13 (0.10, 0.17)*	0.12 (0.09, 0.15)*	<0.001	
WBCNR						
 Boys	1	0.60 (0.50, 0.72)*	0.54 (0.44, 0.66)*	0.49 (0.40, 0.61)*	<0.001	
 Girls	1	0.76 (0.63, 0.92)†	0.61 (0.50, 0.75)*	0.65 (0.52, 0.80)*	<0.001	
Abbreviations were shown in plus Q, quartile; OR, odd ratio; , confidence interval. Height, weight, ages, vitamin D intake, feeding patterns, and complementary feeding were adjusted. P compared with Q1; < 0.001 compared with Q1.

The meaning of the bold values indicated that P-values were considered statistically significant.

* p<0.001 compared with Q1.

† p<0.05 compared with Q1.

hsCRPhigh-sensitive C reactive proteinMPVPCRmean platelet volume–platelet count ratioNLRneutrophil–lymphocyte ratio25(OH)D25-hydroxyvitamin DQquartileRDWPCRred blood cell distribution width–platelet count ratioWBCNRwhite blood cell–neutrophil ratios

Discussion

In this hospital-based cross-sectional study of children, we observed graded and inverse associations of serum 25(OH)D with hsCRP and four novel inflammatory markers (RDWPCR, MPVPCR, NLR and WBCNR). Consistently, serum 25(OH)D was inversely associated with elevated inflammation levels.

Elevated serum hsCRP level, a biomarker of asthma control in children,27 was also linked to some chronic diseases, such as gestational diabetes mellitus,28 hyperlipidemia29 and cardiovascular diseases.30 These studies indicated that lowering serum hsCRP level was a rationale for therapies and prevention of asthma and inflammatory related chronic diseases. Cojic et al pointed out that vitamin D exerted anti-inﬂammatory and antioxidative eﬀects via inﬂuencing myeloperoxidase, xanthine oxidase and catalase activity in type 2 diabetic patients with metformin therapy.31 However, the association between circulating 25(OH)D and hsCRP has not been extensively characterised in children. Brustad et al found a negative association between plasma 25(OH)D and hsCRP in Childhood aged 6 months (β: −0.004).14 Lower hsCRP value was observed in ≥25 ng/mL 25(OH)D group than that in <25 ng/mL 25(OH)D group among obese children (0.053 vs 0.356 mg/dL).13 Similarly, our study also found that serum 25(OH)D was inversely associated with hsCRP level. Nevertheless, the negative association between plasma 25(OH)D and hsCRP was not replicated in previous study (β: 0.003).14 The inverse correlation between 25(OH)D and CRP might be attenuated to null in infants with 25(OH)D above 50 nmol/L,32 and even reversed in asymptomatic adults.33 Besides, a positive correlation between cord blood 25(OH)D and hsCRP (B=1.003) in the newborn was observed, with a 10-unit increase in 25(OH)D level resulting in a 10-unit increase in hsCRP level.12 Previous study also observed a U-shaped association between 25(OH)D and hsCRP in the general population,34 indicating the anti-inflammatory and proinflammatory effects of 25(OH)D. Different subjects (a larger population-based cohort (n=633) vs a smaller high-risk cohort (n=264)) and different measurement methods of 25(OH)D might provide explanation for inconsistent results in the two cohorts.14 Multivariate linear regression models were calculated in healthy newborns without considering total vitamin D supplement of pregnant mothers,12 which might partly overestimate the underlying associations. Additionally, the differences in sample size (nthis study=10141 vs nCOPSAC2000=264,14) the subjects (newborn12 vs obese children13 vs 6 months infants14 vs 14.6 months children (this study)), statistic methods (linear regression analysis12 vs analysis of covariance (this study)) and potential covariates adjusted might also partly contribute to the heterogeneity in above studies. Although no data exist concerning mechanism of 25(OH)D and hsCRP, some cell culture models had indicated the biological background of 25(OH)D.3539 As one major metabolites of 25(OH)D, 1,25(OH)2D could modulate inflammatory processes by decreasing proinflammatory cytokines (eg, interleukin 6 (IL-6), IL-23 or tumour necrosis factor)36 37 and impairing the proliferation of dendritic cells38 and B-cells.39

Additionally, previous studies indicated that novel inflammatory biomarkers (RDWPCR, MPVPCR, NLR or WBCNR) were proposed as a representation of inflammation in some chronic diseases.4042 However, scarce data examined the associations of 25(OH)D and novel inflammatory biomarkers. Based on data from 208 patients with end-stage renal disease receiving maintenance dialysis, a significant inverse association of serum 25(OH)D and NLR was observed (25(OH)D>10 vs <10 ng/dL: 2.96 vs 3.49).43 Subgroup analysis found that NLR was still significantly lower in 25(OH)D>10 ng/dL group than that in 25(OH)D<10 ng/dL group among haemodialysis patients (8.97 vs 14.88); but the difference was not significant in peritoneal dialysis patients.43 An observed study including 100 newborns pointed out that serum 25(OH)D3 was negatively associated with NLR.44 The corresponding NLR values were 1.8 and 1.3 in the insufficient and sufficient groups, respectively.44 In line with these findings, our study also observed an inverse association between serum 25(OH)D and NLR in children. In vivo and vitro studies demonstrated that vitamin D might reduce NLR by increasing T-regulatory cell populations,45 inhibiting the production of proinflammatory cytokines and increasing the production of anti-inflammatory cytokines.4651 Moreover, our data revealed inverse associations of 25(OH)D with RDWPCR, MPVPCR and WBCNR. Although no study had yet assessed these associations until now, some biological mechanisms supported our findings. Agh et al found that vitamin D supplementation significantly reduced white blood cell counts and mean platelet volume in male rats,51 which was in agreement with the results of the present study. Toxqui et al pointed out that vitamin D-fortified flavoured skim milk could reduce red blood cell distribution width in iron-deficient menstruating women.52 This study indicated vitamin D might modulate RDWPCR via reducing red blood cell distribution width. Nonetheless, a larger sample prospective study is needed to confirm our findings.

There were several strengths of our study. We first identified the associations of serum 25(OH)D with hsCRP and four novel inflammatory markers (RDWPCR, MPVPCR, NLR and WBCNR) in children. Emerging evidences reported the important role of hsCRP and four novel inflammatory markers in chronic diseases.4042 The present study revealed that 25(OH)D might be the modulating indicator for hsCRP and four novel inflammatory markers levels, and elucidate the health benefits of 25(OH)D on growth and development of children through improving hsCRP and four novel inflammatory markers levels. Besides, serum 25(OH)D could accurately and objectively reﬂect the metabolites of vitamin D without being inﬂuenced by random errors from dietary or supplemental assessment. Moreover, a large sample size allowed us to determine the associations of serum 25(OH)D with hsCRP and four novel inflammatory markers in children and ensured the stability and reliability of the results. Finally, the seasonal variation of vitamin D level might be considered due to subjects were recruited over all seasons. Nevertheless, some limitations were still considered. First, the causality could not be inferred from the cross-sectional study. Although the age range of included participants was relatively large, serum 25(OH)D could accurately and objectively reﬂect the metabolites of vitamin D without being inﬂuenced by random errors in dietary or supplemental assessment. Besides, 25(OH)D, a major metabolite of vitamin D, is produced by hydroxylation in liver,53 which is mainly influenced by diets or vitamin D supplements rather than inflammatory cytokines. Moreover, circulating 25(OH)D level was relatively steady with a half-life of 2–3 w.5 Therefore, cause-and-effect correlations were less likely to be overturned. Second, 25(OH)D level was measured only once in our study, but it was stable in serum and hence was generally used to estimate vitamin D level.54 Third, the underlying associations in the present study might be overestimated due to the lack of information on other drugs using (eg, docosahexaenoic acid, calcium and vitamins). Finally, the make-up of the study sample in this study had a relatively narrow population range, thus caution is needed when extrapolating the findings of this study.

Generally, the graded and inverse associations of serum 25(OH)D with hsCRP and four novel inflammatory markers (RDWPCR, MPVPCR, NLR and WBCNR) were observed. The present study provided further support for the anti-inflammatory effects of 25(OH)D. Further prospective studies are needed to replicate these findings in different populations.

supplementary material

10.1136/bmjopen-2023-083227 online supplemental file 1

Acknowledgements

We thank all research members who contributed to this study.

Data availability statement

All data relevant to the study are included in the article or uploaded as online supplemental information.

Review Process File
05 09 2024

Funding: This work was partly supported by the Nantong University Clinical Medicine Specialized Research Fund Project (No. 2023LY004) and Nantong Health Committee Foundation (No. MS2022075). The funders had no role in the design, analysis or writing of this article.

Prepublication history and additional supplemental material for this paper are available online. To view these files, please visit the journal online (https://doi.org/10.1136/bmjopen-2023-083227).

Provenance and peer review: Not commissioned; externally peer reviewed.

Patient consent for publication: Not applicable.

Patient and public involvement: Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.

Ethics approval: Ethical approval and the requirement for documentation of informed consent were waived by the ethics committee of Nantong Maternal and Child Healthcare Hospital due to the retrospective nature of the present study (Y2021055). All the procedures being performed were part of the child health care. The acquisition and use of data included in this study were permitted by the Nantong Maternal and Child Healthcare Hospital. Data were extracted from medical records. The consents were not obtained due to the difficulty in reconnection and retrospective design of the study. Consent waiver was got from the ethics committee of the ethics committee of Nantong Maternal and Child Healthcare Hospital. The private information was well protected. All the experiment protocols in this study for involving human were conducted in accordance with the Declaration of Helsinki.
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