Study on the Relationship of 25-hydroxyvitamin D and Intact Parathyroid Hormone in Chinese Young Adults

Background : Vitamin D plays an important role in bone health and other diseases, however, there is no consensus over the optimal serum 25-hydroxyvitamin D (25(OH)D) concentration. Many studies dened a level of serum 25(OH)D at which serum intact parathyroid hormone (iPTH) levels decreased and reached a plateau might be the optimal status for vitamin D suciency. This study was performed to study the relationship between 25(OH)D concentration and intact parathyroid hormone (iPTH) concentration in the Chinese non-clinical young population aged 18-44y. Methods : The serum samples of young adults aged 18-44 years old were selected from the established serum bank of Chinese Chronic Diseases and Nutrition Survey (CCDNS, 2015-2018). The serum 25(OH)D concentration was determined by high performance liquid chromatography tandem mass spectrometer, and the iPTH was determined by electronic chemiluminescence method. The relationship of 25(OH)D and iPTH were analyzed by partial correlation analysis, and the threshold of 25(OH)D was analyzed by locally weighted scatter plot smoothing method after adjusting the iPTH by age, season of blood draw, BMI, waist, sex and latitude. Results : 623 serum samples (293 male, 330 female) of 25(OH)D concentration and iPTH concentration were analyzed. Signicant higher 25(OH)D concentration was found in male than female. And the samples from the southern China had higher 25(OH)D concentration than those from the northern China. Samples from autumn had higher 25(OH)D concentration than those from spring and winter. The inversely relationship between 25(OH)D concentration and iPTH concentration was observed when the 25(OH)D was below 17.6 ng/mL for both sexes. The threshold for male was 16.2 ng/mL and it was 25.6 ng/mL for female. Conclusions : The threshold and the relationship between 25(OH)D and iPTH differs by sex among Chinese young adults aged 18-44y. Further study is needed to evaluate the sex-specic ranges of optimal vitamin D. Trial registration :


Background
Vitamin D is one of the essential micronutrients in human body. Vitamin D can stimulate the synthesis of osteopontin and alkaline phosphatase in osteoblasts and inhibit the apoptosis of osteoblasts, which is bene cial to bone formation [1]. Recent studies have found that vitamin D nutritional status is not only related to bone health, but also to cell metabolism, immune system, respiratory system and other human functions [2,3]. On the other hand, vitamin D excess can lead to hypocalcaemia [4], therefore it is very important to accurately evaluate the nutritional status of human vitamin D for early prevention and treatment of related diseases. 25-hydroxyvitamin D [25(OH)D] is the main circulation form of vitamin D in blood with a half-life of 2-3 weeks [5]. It is recognized as a reliable indicator for evaluating vitamin D status in human body [6].
However, there are still controversies about the threshold of 25(OH)D in serum for judging the nutritional status of vitamin D. There are two main popular perspectives of thresholds for vitamin D de ciency recommended by the American Institute of Medicine (IOM) and the Endocrine Society (TES), respectively [5,7]. The Institute of Medicine (IOM) of America considered that a serum 25(OH)D level of at least 20 ng/mL (50 nmol/L), meet the requirements of at least 97.5% of the population. And it de ned vitamin D de ciency as <12 ng/ml (30 nmol/L) 25(OH)D and considered levels between 12-20 ng/ml (30-50 nmol/L) as insu cient vitamin D [6,8]. TES issued serum 25(OH)D level of 30 ng/mL (75 nmol/L) was considered su cient. Serum 25(OH)D level that was lower than 20 ng/mL (50 nmol/L) as vitamin D de ciency and serum 25(OH)D level between 20-30 ng/mL (50-75nmol/L) as vitamin D insu ciency [5].
Intact Parathyroid hormone (iPTH) is a calcium-regulated hormone and it mainly acts on kidney and bone tissues. iPTH concentration has been reported inversely associated with serum 25(OH)D concentrations until a threshold, and then the iPTH concentration reaches its plateau at a minimum level above the threshold [9]. This threshold and above is often suggested as Vitamin D su ciency [10][11]. There are a number of cross-sectional and randomized controlled trials report on the inversely relationship between 25(OH)D concentration and iPTH concentration in different countries and ethnicities [10][11]. There was no consistency in the threshold values of serum 25(OH)D that varies from 25 nmol/L (10 ng/mL) to 50 nmol/L (125 ng/mL) [10]. There are still some studies in which such threshold or the negative correlation was not found.
This study is to analyze the relationship between serum 25(OH)D concentration and iPTH concentration in Chinese young adults aged 18-44y based on the Chinese Chronic Diseases and Nutrition Survey (CCDNS) 2015-2018. We try to understand if there is such a threshold for this speci c group of Chinese population.

Methods
Study population 650 samples of adults aged 18-44y were collected from the Chinese population serum bank established from the CCDNS 2015-2018 by the method of simple random sampling. CCDNS is a cross-sectional survey of the civilian non-institutionalized population of China, conducted by the National Institute of Nutrition and Health and the Center for the Prevention and Control of Chronic Non-communicable Diseases, Chinese Center for Disease Control and Prevention (NINH & CPC, China CDC). All the participants of this survey were selected by using a strati ed proportional random cluster sampling. All the participants and their guardians gave their informed consent in writing to participate in the CCDNS survey.

25(OH)D and iPTH detection
Both serum 25(OH)D and serum iPTH were detected in this study. 2 mL of fasting venous blood was collected and centrifuged at 1500× g for 15 min, 20-30 min after the blood was taken. The serum was aliquot and stored in a brown vessel at −20℃ in the laboratory where the investigation area was located.
All the serum specimens from all the investigation areas were transported to the serum bank located in NINH by cold chain. All the blood samples were preserved at -70℃ in a freezer before detection. The High Performance Liquid Chromatography Tandem Mass Spectrometer (AB Sciex Pte. Ltd. USA) was used to analyze serum 25(OH)D concentration. The calibration of the assay was veri ed by using the NIST standard reference material SRM 972. The average bias was 2.3% for 25(OH)D 2 and 1.7% for 25(OH)D 3 comparing with Nist SRM 972. The iPTH concentration was measured by electronic chemiluminescence immunoassay (Roche e601, F Hoffmann-La Roche Ltd. Switzerland). The CV was 4.9% in the 51.5-53.9 pg/mL range, and 4.2% in the 186-191 pg/ml range.

Variables
A national project workgroup was established in China CDC to develop a uni ed survey and questionnaires to carry out the investigation by using uni ed equipments and methods. The basic information of the subjects (including age, sex, region type, etc.) was collected by questionnaire. China's Qinling Mountains and Huaihe River are recognized as the boundary to divide the north and the south. The latitude of each investigation area was seached from Google map. The region type are classi ed according to the level and type of economic development and the population size [12]. Body Mass Index (BMI) was calculated according to bodyweight and height. Waist was measured by the uniformed method. Body weight was measured by uniform electronic scale, and standing height was measured by a metal column type height meter.
Statistical Analysis. Serum 25(OH)D and iPTH concentrations were recorded by using P50 (P25-P75) because they were not compliant with the normal distribution according to the normality test, and then they were compared by the Kruskal-Wallis test in different subgroups. The correlation was analyzed by spearman correlation method among 25(OH)D, iPTH, season, age, sex, latitude, bmi, waist. Adjusted by age, BMI, latitude, waist and season, the correlation between the 25(OH)D concentration and the iPTH concentration was analyzed by the partial correlation analysis. The generalized additive model was adopted and applied by mgcv package in R software to generate an adjusted iPTH to adjust the potential factor that may have in uence on iPTH, 25(OH)D or both of them, such as BMI, age, latitude, waist, season of blood draw, sex. The adjusted iPTH for each sex were also made. And the adjusted iPTH concentrations were used for the following analysis. The locally weighted scatterplot smoothing (LOESS) procedure is a nonparametric method for tting a smooth curve that best characterizes the relationship between 2 continuous variables [13]. Given the reported non-linear association between iPTH and 25(OH)D, the LOESS curve was then utilized to characterize the association between them, and then the threshold was predicted. The SAS version 9.4 statistical software (SAS Institute Inc., Cary, NC, USA) and R version 3.6.1 statistical software were used. Two tailed P< 0.05 was considered as statistically signi cant.

Sample characteristics
Samples of poor blood quality or those with incomplete information were excluded, and the serum 25(OH)D concentration and iPTH concentration of 623 blood samples (293 male and 330 female) aged 18-44y were included in this study ( Table 1) summer was not included. In this study, the median 25(OH)D level was highest in autumn.
No significant difference was found in spring, autumn and winter in terms of the median iPTH concentration.

Serum 25(OH)D and its correlation with iPTH
The correlation results showed that was with 25(OH)D was correlated with iPTH, latitude, sex, season and age, and iPTH was correlated with BMI and waist (P<0.05) (Supplemental Table S1). The 25(OH)D concentration was correlated with iPTH, age, latitude and waist in males, while it was correlated with iPTH, season and latitude in females. The iPTH was correlated with BMI and waist in males, and it was correlated with waist in female (Supplemental Table S2 Figure   S1). As for male, the threshold was 16.2 ng/mL and it was 25.6 ng/mL for female.

Discussion
As early as 1987, researchers began to study the relationship between vitamin D and iPTH by crosssectional studies or randomized controlled trials to search the plateau period of iPTH and the corresponding 25(OH)D concentration among different groups, including the elderly, female, healthy adults, vitamin D-de cient people, etc [14][15]. Survey data from NHANES 2003NHANES -2004NHANES and 2004NHANES -2005 shows that the relationships among 25(OH)D, BMD and PTH in American adults vary according to race/ethnicity. Signi cant inverse relationship between 25(OH)D and iPTH values was only observed when 25(OH)D concentrations were below 26 ng/ml among blacks, while inverse relationship was observed above and below a 25(OH)D level of 20 ng/ml in whites and Mexican-Americansin [14]. Aloia et al. [16] found that iPTH reached plateau stage when the serum 25(OH)D concentration was between 40-50 nmol/L (16-20 ng/mL) of African American women. In addition to the above studies, there are also studies that failed to nd the threshold [10,17]. Different races and genetic backgrounds might explain the phenomenon, and it is necessary to study whether the same cutoffs are suitable for different ethnicity.
This is the rst national cross-sectional study on the threshold of 25(OH)D among Chinese population. In our study, we analyzed the threshold when iPTH reaching plateau stage by sex, because of the signi cant difference in the distribution of 25(OH)D levels between male and female. We found that the concentration of 25(OH)D was 19.2 ng/mL (48 nmol/L) while not adjusted by covariates when iPTH entered into its plateau in Chinese women aged 18-44y. It is close to the threshold of 50.8 nmol/L (20.3 ng/mL) in adult of both sex aged 20-45y in Shanghai city, China reported by Yao et al [18]. The threshold for 25(OH)D was 17.6 ng/mL after adjusted by sex, age, latitude, BMI, waist and season of blood draw. It was close to IOM' recommendation and similar with most study reported [10]. The threshold of male (16.2 ng/mL) was obviously lower than that of female (25.6 ng/mL). Both of the thresholds were within the most frequent range of literature reports. There are few studies about the threshold of male reported, scattered in the range of 15-20ng/mL, followed by 30-35ng/mL [10]. The threshold for the de ciency of sex could be better explored and analyzed with more relevant indicators, such as calcium and vitamin D intake, renal function, bone health indicators, endocrine indicators, etc. Arabi et al. [19] think that age but not gender modulate the relationship between 25(OHD) and PTH among adolescents (10-17 years) and elderly (65-85 years) of the same ethnic group living in Lebanon. No age speci c difference was found in terms of relationship between 25(OH)D and iPTH in this study, probably because of the concentrated age distribution. However, the sex differences of threshold was found in the relationship of 25(OH)D and PTH in our study. Similar sex differences were reported by a cross-sectional study conducted among adolescents aged 12-15y in North Ireland, where the researchers found a threshold at 60 nmol/L (24 ng/mL) in girls, however, no cutoff was found in boy [20]. Although 25(OH)D level of male was signi cantly higher than that of female, iPTH did not differ signi cantly. This might also indicate that the corresponding iPTH was no longer decreased when the level of 25(OH)D increases to a certain range. The secretion of sex hormones and growth hormone might also be associated with the sex difference. The results from different studies is controversial regarding the relationships of 25(OH)D concentration with levels of sex hormones and gonadotropins [21]. Frank et al consider that the impact of sex hormone on skeletal metabolism was different between sexes [22,23]. Besides the intake of vitamin D and calcium, the nutritional status, such as vitamin K nutritional status which has synergetic effect with vitamin D, extent of outdoor activities might be others reason for the disparity between sexes in terms of 25(OH)D.
Further study should also be made to reveal the difference between sexes. We also found the disparity of 25(OH)D concentration in different seasons. Though no samples for summer in our study, the seasonal distribution of 25(OH)D concentration is consistent with our former study [24] and the report from Kroll et al. in the United States [25]. In their study, they found the lowest concentration of iPTH occurs 3.5 weeks after the 25(OH)D 3 reaches its highest concentration and vice versa. We can hardly tell the impact of relationship between iPTH concentration and 25(OH)D concentration, because our samples are limited and not distributed to every month of a year. Further study should also be made to explore the impact of seasonal factor on the relationship between iPTH and 25(OH)D.
The vitamin D insu ciency and de ciency of Chinese adults aged 18-44y was 34.5% and 19.4%, respectively, according to IOM's recommendation. The vitamin D insu ciency and de ciency reached to 30.7% and 53.9%, respectively, and only 15.4% was su cient when adopting TES's recommendation. The median 25(OH)D concentration was 19.1(13.2-26.1) ng/mL, and it is apparently lower than 10 years ago [26]. Less outdoor time, the use of sunscreen might partly explain the reduction of 25(OH)D concentration. Higher 25(OH)D concentration in southern area attributes to stronger ultraviolet radiation in southern area than in the northern area. We suggest exploring sex speci c thresholds by taking into consideration potential impact of physiological differences between male and female. Although there are signi cant differences in 25(OH)D levels between the northern and southern populations in Chinese adults, it is di cult to distinguish individuals strictly according to regions due to population mobility, topographic differences, and etc. For these reasons, we do not recommend setting different boundaries for different regions.
We acknowledge several limitations. First, the limited samples size. Although samples in this study covered all the provincial administrative regions in China, the sample size was only up to 623 cases. In particular, there were only 10 samples in April, which may also lead to the deviation of results. Since this study is an exploratory study in this eld of China, the sample size was small indeed, and we will increase the sample size in subsequent studies. Secondly, the dietary sources of vitamin D and calcium intake [26], bone health indicators, in ammatory factors, and other factors that may in uence the relationship between iPTH and 25(OH)D, were not included in this study. Due to the above factors, it may lead to bias in the relationship or threshold between iPTH and 25(OH)D in this study.

Conclusions
In summary, although the present study was hindered by some limitations, we still nd a threshold for Chinese adult aged 18-44y at 17.6 ng/mL for both sexes (16.2 ng/mL for male and 25.6 ng/mL for female, respectively), above which we consider the vitamin D status is su cient. The relationship between 25(OH)D and iPTH differs by sex, and further study is needed to evaluate the sex-speci c ranges of optimal vitamin D by increasing sample size and adding more related indicators.

Consent for publication
The data of this study did not include personal information (i.e., name or address). All participants were adults.
Not applicable.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Competing interests Figure 1 Distribution of intact parathyroid hormone (iPTH) by sex Distribution of 25 hydrovitamin D by sex