The association between serum ferritin and 25-hydroxyvitamin D and metabolic syndrome in Korean women: the Korea National Health and Nutrition Examination Survey 2010–2012

The present study was conducted to assess the association between serum ferritin and 25-hydroxyvitamin D [25(OH)D] and metabolic syndrome (MetS) in Korean women. The data of a total of 9,256 adults (6,960 women without MetS and 2,296 women with MetS) aged ≥20 years from the Fifth Korean National Health and Nutrition Examination Survey (KNHANES V) (2010–2012) were analyzed. A covariance test adjusted for covariates was performed for serum ferritin levels in relation to vitamin D (vitamin D deficiency, 25(OH)D <10.0 ng/ml; vitamin D insufficiency, 25(OH)D ≥10.0, <20.0 ng/ml; vitamin D sufficiency, 25(OH)D ≥20.0 ng/ml). The key study results were as follows: First, in women without MetS, after adjusting for related variables (smoking, alcohol drinking, regular exercise, current menstruation, hormonal contraceptives, hormone-replacement therapy, SBP, DBP, BMI, WM, TC, TGs, HDL-C, FPG, AST, ALT, and age), vitamin D was positively associated with serum ferritin levels (p<0.001). Second, in women with MetS, after adjusting for related variables (except age), vitamin D was positively associated with serum ferritin levels (p = 0.041). However, when further adjusted for age, vitamin D was not associated with serum ferritin levels (p = 0.293). In conclusion, vitamin D was positively associated with serum ferritin levels in women without MetS but not in women with MetS.

Introduction I ron is a vitally important metal to the normal physiological processes of many organisms, (1) and it is essential for many metabolic processes, such as oxygen transport and DNA synthesis. (2) The serum ferritin level reflects iron stores in the body, since ferrous iron combined with apoferritin is stored by ferritin in many organisms. (3) Low serum ferritin levels are associated with diseases such as telogen effluvium, iron deficiency anemia (IDA), and bone mineral density, (4)(5)(6) while high serum ferritin levels are associated with cardiovascular disease, insulin resistance, and metabolic syndrome (MetS). (7)(8)(9) In the past, the main role of vitamin D was understood to be controlling the calcium levels and bone metabolism by its involvement in calcium and phosphate absorption in the intestines. (10) However, recently, vitamin D has also received attention regarding additional functions concerning its effects on the prevention of diseases, such as telogen effluvium, cardiovascular disease, MetS, and anemia. (11)(12)(13)(14) In terms of anemia, some studies have reported that vitamin D was positively associated with ferritin levels. (15,16) However, these results may vary depending on whether the subjects have diseases such as MetS and diabetes mellitus, because ferritin is a marker of iron stores but also an important biomarker of insulin resistance, inflammation, and oxidative stress. (17) Currently, it is unclear whether ferritin can be considered as a marker of iron stores or inflammation in subjects with insulin resistance. Serum ferritin levels are regulated by hepcidin, which plays a role in reducing iron absorption from the intestine. (18) However, some studies reported that in spite the increase of hepcidin, serum ferritin levels were increased in subjects with MetS. (19,20) Therefore, our objective in this work was to assess the association between vitamin D and ferritin levels in Korean women with and without MetS using data from the Fifth Korean National Health and Nutrition Examination Survey (KNHANES V; 2010-2012), which is representative of the population of Korea (https://knhanes.cdc.go.kr/ knhanes/index.do).

Materials and Methods
Study subjects. This study was performed using data from KNHANES V. KNHANES V were each conducted for 3 years (2010-2012), using a rolling sampling survey that involved a complex, stratified, multistage, probability cluster survey of a representative sample of the non-institutionalized civilian population in South Korea. The survey was composed of three parts: a health interview survey, a health examination survey, and a nutrition survey. Each survey was conducted by specially trained interviewers. The interviewers were not provided with any prior information regarding specific participants before conducting the interviews. Participants provided written informed consent to participate in this survey, and we received the data in anonymized form. In the KNHANES V (2010-2012), 25,534 individuals over age 1 were sampled for the survey. Among them, of the 19,392 subjects who participated in the KNHANES V, we limited the analyses to adults aged ≥20 years. We excluded participants 2,138 subjects whose data were missing for important analytic variables, such as serum ferritin level, 25(OH)D level, various blood chemistry tests, and information about lifestyle. We excluded I participants who had cancer (650 subjects) or hepatitis virus B (470 subjects) or hepatitis virus C (38 subjects). In addition, we excluded participants were excluded 6,840 men. Finally, 9,256 subjects were included in the statistical analysis. The KNHANES V study has been conducted according to the principles expressed in the Declaration of Helsinki. (Institutional Review Board No.  2010-02CON-21-C; 2011-02CON-06-C; 2012-01EXP-01-2C). All participants in the survey signed an informed written consent form. Further information can be found in "The KNHANES V Sample", which is available on the KNHANES website. The data from KNHANES is available on request by email if the applicant logs onto the "Korea National Health and Nutrition Examination Survey" website.
General characteristics and blood chemistry. Research subjects were classified by sex (men or women), smoking (nonsmoker or ex-smoker or current smoker), alcohol drinking (yes or no), and regular exercise (yes or no). In the smoking category, participants who smoked more than one cigarette a day, those who had previously smoked but do not presently smoke, and those who never smoked were classified into the current smoker, ex-smoker, and non-smoker groups, respectively. Alcohol drinking was indicated as "yes" for participants who had consumed at least one glass of alcohol every month over the last year. Regular exercise was indicated as "yes" for participants who had exercised on a regular basis regardless of indoor or outdoor exercise. (Regular exercises was defined as 30 min at a time and 5 times/w in the case of moderate exercise, such as swimming slowly, doubles tennis, volleyball, badminton, table tennis, and carrying light objects; and for 20 min at a time and 3 times/w in the case of vigorous exercise, such as running, climbing, cycling fast, swimming fast, football, basketball, jump rope, squash, singles tennis, and carrying heavy objects). Women health index were included current menstruation, hormonal contraceptives, and hormone-replacement therapy.
Metabolic syndrome. Metabolic syndrome was defined using the diagnostic criteria of the National Cholesterol Education Program (NCEP) based on common clinical measures including TGs, HDL-C, blood pressure, FBG, and WM. TGs over 150 mg/dl was set as the criteria for elevated TGs. The criteria for reduced HDL-C were HDL-C of less than 50 mg/dl. FBG over 100 mg/dl was set as the criteria for elevated FBG. SBP over 130 mmHg or DBP over 85 mmHg or medication were set as the criteria for elevated blood pressure. The criteria for abdominal obesity were abdominal measurements of over 80 cm, according to the Asia-Pacific criteria. (21) The presence of defined abnormalities in any three of these five measures constitutes a diagnosis of metabolic syndrome. Concentrations of serum ferritin were measured using an immunoturbidimetric Assay (IRMA-mat Ferritin; DiaSorin, Still Water, MN) using a 1470 Wizard Gamma Counter (Perkin Elmer, Turku, Finland).
Data analysis. The collected data were statistically analyzed using SPSS WIN (ver. 18.0). The distributions of the participant characteristics were converted into percentages, and the successive data were presented as averages with standard deviations. The distribution and average difference in clinical characteristics according to vitamin D status were calculated using chi-squared and an analysis of variance (ANOVA). In the case of analysis of covariance test (ANCOVA), the 4 models constructed were: 1) adjusted for smoking, alcohol drinking, regular exercise, current menstruation, hormonal contraceptives, and hormone-replacement therapy; 2) further adjusted for SBP, DBP, BMI, and WM; 3) further adjusted for TC, TG, HDL-C, FBG, AST, and ALT; 4) further adjusted for age. The significance level for all of the statistical data was set as p<0.05.

Comparisons of serum ferritin levels and anemia indices according to vitamin D in women with and without MetS.
Comparisons of serum ferritin levels and anemia indices according to vitamin D status are shown in Table 5  In women with MetS, after adjusting for related variables (except age), vitamin D was positively associated with the serum ferritin levels (p = 0.041). However, when further adjusted for age, vitamin D was not associated with the serum ferritin levels (p = 0.293). In terms of anemia indices by vitamin D after adjusting for related variables, vitamin D was significantly associated with Fe (p = 0.004), TIBC (p = 0.013), Hb (p<0.001), and Hct (p<0.001) in women without MetS. However, vitamin D was not associated with Fe (p = 0.817), TIBC (p = 0.494), Hb (p = 0.378), and Hct (p = 0.430) in women with MetS.

Discussion
The present study investigated the association between serum ferritin and 25(OH)D levels in Korean women with and without MetS using data from KNHANES V conducted in 2010-2012. There were several key findings of this study after adjusting for variables. Vitamin D was positively associated with serum ferritin levels in women without MetS but not in women with MetS ( Table 5).
The prevalence of vitamin D deficiency varies across ethnic groups and countries. (23) The prevalence of vitamin D deficiency or insufficiency (<20.0 ng/dl) in our results (77%) is higher than that in Germany (58%) (24) and similar to that in Scotland (78%). (25) Vitamin D is known to prevent osteoporosis, cardiovascular disease, and insulin resistance. (10,26,27) In addition, vitamin D regu-lates the hepcidin-ferroportin axis in macrophages, and the increase of vitamin D is known to reduce systemic hepcidin levels and ameliorate anemia. (28) Ferritin, which reflects the iron stores in the blood, is regulated by hepcidin. (29) Research on the association between vitamin D and ferritin is being conducted all over the world. Jeong and colleagues (15) and Andıran and colleagues (30) reported that serum 25(OH)D was positively correlated with serum ferritin levels in Korea and the US, respectively. However, Monlezun and colleagues (31) and Castro and colleagues (32) reported that serum 25(OH)D was not associated with serum ferritin levels in adults from the US and Portugal, respectively. These inconsistent results may be due to the different populations, ethnic groups/countries and the different subjects of the studies (e.g., gender, with or without diseases).
Ferritin is a biomarker of iron stores and is reduced in subjects with anemia. (3,5,8) Some studies have suggested that vitamin D is inversely associated with the incidence of IDA in women. Lee et al. (5) reported that the odds ratios for iron deficiency [serum ferritin level <12 μg/L and transferrin saturation <16%] and IDA (Hb <13 g/dl and iron deficiency) in subjects with vitamin D deficiency [25(OH)D <15 ng/ml] were 1.86 (95% CI, 1.07-3.22) and 2.59 (95% CI, 1.11-6.07) after controlling for other risk factors in healthy Korean women. In addition, Suh et al. (33) reported that 25(OH)D levels (Means ± SE) were lower in Korean women with IDA (Hb <12 g/dl and serum ferritin level <15 μg/L, 14.43 ± 0.24 ng/ml) than in those with non-IDA (Hb ≥12 g/dl and serum ferritin level ≥15 μg/L, 16.40 ± 0.13 ng/ml) (p<0.001). In the present study, in women without MetS, the vitamin D was positively associated with ferritin levels. In addition, the vitamin D was positively associated with Fe, Hb, and Hct levels. These results considered that vitamin D has a positive effect on anemia. Currently, as far as we know, there is no the research on vitamin D and ferritin in subjects with MetS. In health populations, vitamin D increases the ferritin levels by is suppressed hepcidin. However, in subjects with MetS, despite the increased of hepcidin, serum ferritin levels were increased, and both hepcidin and ferritin levels were increased as increasing of MetS components. (19,20) In the present study, ferritin and 25(OH)D level increased in women with MetS than in women without MetS, but Fe and TFS level decreased in women with MetS than in women without MetS. In women with MetS, when adjusting for related variables (except age), the vitamin D was positively associated with ferritin levels. However, when further adjusting for age, it was not statistically significant. In addition, the vitamin D was not associated with all of the anemia-related variables (e.g., Fe, TIBC, Hb, and Hct). Age is associated with vitamin D and ferritin level and incidence of anemia and MetS. (34)(35)(36)(37) In the present study, the incidence of MetS was increased as the increased of age in both women with and without MetS. We found that ferritin, 25(OH)D, Hb, and Hct were increased with increase of age in women without MetS. However, in women with MetS, ferritin and 25(OH)D were increased with increase of age but Hb and Hct were decrease.
In fact, the association of anemia and ferritin is differ among the type of anemia. Ferritin is decreased in subjects with IDA but may be increased in subjects with anemia of chronic disease and inflammation. (38) Vitamin D plays a role in both the prevention of anemia and the improvement of insulin resistance and inflammation. (39,40) In subjects with IDA, vitamin D increase ferritin by down-regulation of hepcidin. However, in subjects with anemia of chronic diseases and inflammation, vitamin D may be decreased ferritin for the improvement of inflammation status. (17,41) The MetS is characterized by insulin resistance, (42) and the subjects with MetS are already status with increased inflammation and reduced bet cell function. (43,44) As described above, ferritin is an indicator of inflammation as well as a marker of iron store. Currently, in the subjects with MetS, whether ferritin is a marker of iron store or a marker of inflammation is still unclear. We considered the possibility that vitamin D may detect the degree of ferritin in the blood and regulate the level of ferritin in women. If less than constant level of ferritin in the blood such as women with IDA or without MetS, vitamin D recognizes the ferritin as a marker of iron store and increases ferritin levels by downregulation of hepcidin. (45) However, if more than constant level of ferritin in the blood such as women with MetS, vitamin D may recognize the ferritin as a marker of inflammation rather than iron store and may reduce ferritin levels by up-regulation of hepcidin, or it is also a possibility that there will be no change in ferritin level as the up-regulation and down-regulation of hepcidin may occur simultaneously. Martinelli and colleagues suggested that although hepcidin was increased in subjects with MetS, ferritin or Hb also were increased. (20) We found that 25(OH)D was not associated with ferritin as well as Fe, TIBC, Hb, and Hct in women with MetS. These results are similar to study of Luo and colleague. (46) Although the subject is not the MetS, in a study of they conducted on subjects with type 2 diabetes mellitus which is characterized by insulin resistance, vitamin D was not associated with ferritin as well as serum iron, iron saturation, and high sensitivity CRP. In the present study, we could not demonstrate the mechanisms that whether vitamin D plays a role the prevention of IDA or plays a role the improvement of inflammation in women with MetS. However, we were able to determine that vitamin D was not associated with ferritin in Korean women with MetS. The present study has some limitations. First, season is the most important determinant of serum 25(OH)D levels, but the data of the KNHANES V study (2010-2012) did not specify serum 25(OH)D levels according to season. Therefore, season could not be used as adjustment variable. Second, hepcidin is an important determinant of serum ferritin levels. However, hepcidin were not employed in the KNHANES V study. Third, serum calcium concentrations and the daily intake volume of vitamin D are important determinants of serum 25(OH)D levels, but the KNHANES V study did not measure serum calcium concentrations or daily intake volumes of vitamin D. Therefore, serum calcium concentrations and daily intake volumes of vitamin D could not be used as adjustment variables. Forth, we have not been able to investigate the medication for anemia, diabetes, dyslipidemia, and hypertension. Therefore, the medication for anemia, diabetes, dyslipidemia, and hypertension could not be used as adjustment variables. The serum 25(OH)D levels for each season, along with calcium, and hepcidin, should be included as variables for vitamin D status in future studies. Although the present study has these limitations, this is the first reported study to determine the relationship between ferritin and vitamin D in Korean adults without and with MetS. Therefore, more accurate results might be obtained by performing a cohort study by adding these variables.
In conclusion, the present study investigated the association between serum ferritin and 25(OH)D levels in Korean women with and without MetS using data from the KNHANES V conducted in 2010-2012. Vitamin D was found to increase with serum ferritin levels in women without MetS but not in women with MetS.