The subjects of this study were 50 hemodialysis (HD) patients undergoing maintenance HD treatment three times weekly at an artificial kidney center at Hanyang University Seoul Hospital between August and September 2012. The inclusion criteria were as follows: (1) patients had been on HD for more than 3 months, (2) patients were over 19-years-old, (3) patients did not have cancer and showed no active inflammation or infection; (4) patients did not have renal osteodystrophy; (5) patients did not have active hepatitis or any other active liver disease. One patient dropped out due to hospitalization at another hospital while two refused to answer the questionnaire. A total 47 HD patients (19 males and 28 females) completed the questionnaire. All participants gave written informed consent, which was approved by the Institutional Review Board of Hanyang University Hospital (HYUH IRB 2012-06-023).

Blood pressure was evaluated prior to mid-week HD sessions for 1 month before blood biochemical sampling, and four results were averaged. Pre-dialysis blood pressure was measured by automated monitors (Fresenius Medical Care AG & Co, Bad Homburg, Germany). Blood samples were obtained at predialysis for this study. Serum 25(OH)D levels were determined using a chemiluminescent immunoassay (Liaison 25 OH vitamin D Total Assay, Liaison, USA); serum 1,25(OH)₂D levels were determined using a radioimmunoassay (1,25 dihydroxyvitamin D RIA-CT, Liaison, USA); intact parathyroid hormone (iPTH) was determined using an electro-chemiluminesent immunoassay (PTH, Roche, Germany); high-sensitivity C-reactive protein (hs-CRP) was determined using immunoturbidmetry (CRPH, Beckman coulter, USA). In order to compare serum 25(OH)D levels by gender, we defined vitamin D insufficiency and deficiency according to the Kidney Disease Outcome Quality Initiative (K/DOQI) guidelines from the National Kidney Foundation [16]. Vitamin D status was considered adequate (≥ 30 ng/ml), insufficient (15-< 30 ng/ml), or deficient (≤ 15 ng/ml). Vitamin D deficiency was further subdivided into moderate (10-≤ 15 ng/ml) or severe (< 10 ng/ml). Biological variables were assessed according to serum 25(OH)D level (15 ng/ml), which is a criterion of deficiency and insufficiency in the K/DOQI guidelines. Serum creatinine, albumin, blood urea nitrogen (BUN), alkaline phosphatase (ALP), calcium, and phosphorus were measured by standard laboratory methods.

Body mass index (BMI, kg/m²) was calculated using dry weight and height. Age, duration of hemodialysis, main causes of kidney disease, and types and dosages of medication were investigated from medical records at the beginning of the study. Subjects answered questionnaires about sun exposure (sunscreen usage, time of outdoor activity, and frequency of outdoor activity per week), physical activity, supplement usage, smoking habits, and alcohol consumption. Active outdoor exercisers were categorized into two groups, "yes" or "no", according to the following questions: "Do you exercise outdoors?", "Do you exercise outdoors more than 3 times per week?", "Do you exercise more than 30 minutes to 1 hour outdoors?", "Do you use sun protection during outdoor exercise or when leaving the house?", and "Do you usually wear a hat, long sleeves, or long pants when you participate in outdoor activities?"

Dietary vitamin D and calcium intakes were collected by using 24-hour recall and 2-day food diaries. Dietary data for the first day were assessed by a dietitian using 24-hour recall with plastic food models, whereas data for the second and third days were completed using 2-day food diaries. Subjects were given a scale for weighing foods. Average daily vitamin D and calcium intakes were calculated using Can-pro 4.0 (Computer Aided Nutritional Analysis Program 4.0, The Korean Nutrition Society, Korea). Subjects answered a 37-item modified food frequency questionnaire (FFQ) that was primarily derived from a 63-item FFQ developed from the Korean National Health and Nutrition Examination Surveys (KNHANES IV). The FFQ for this study was modified to estimate vitamin D intake and consumption frequencies across nine categories (almost never, 1 time/mo, 2-3 times/mo, 1 time/wk, 2-4 times/wk, 5-6 times/wk, 1 time/d, 2 times/d, and 3 times/d). The FFQ also included three categories for intake amount (1/2 serving size, 1 serving size, or 1 and 1/2 serving size). To analyze the association between serum 25(OH)D level and major vitamin D food sources, three food groups were selected based on previous studies [22] as follows: total fish (50 g/serving), including anchovy, tuna, croaker, codfish, hairtail, mackerel, and salmon; eggs (50 g/serving); and milk (200 ml/serving). All consumption frequencies were standardized into servings per week.

The study subjects were asked to take self-selected dietary supplements containing calcium and vitamin D as well as other dietary supplements such as vitamin C, omega-3, and red ginseng. Daily intake and frequency per week were recorded. Prescribed amounts of vitamin D and calcium derived from patients' medical records were collected. An active vitamin D analogue, paricalcitol (Zemplar), and a calcium supplement as a phosphate-binder (Phoslo, calcium acetate 710 mg) were used.

Comparison between males and females was carried out by independent t-test for continuous variables, and chi-square test was performed for categorical variables. For comparison of serum 25(OH)D levels, subjects were divided into two groups by serum 25(OH)D concentration, deficiency (< 15 ng/ml) and non-deficiency (≥ 15 ng/ml). Biological variables according to serum 25(OH)D level were assessed through ANCOVA adjusted for age, gender, and HD duration. To assess determinants of serum 25(OH)D concentration, simple and multiple linear regression analyses were conducted to describe the relationship between serum 25(OH)D level and possible explanatory variables. Continuous variables were dietary vitamin D intake (µg/day) and sun exposure time (h/week). Categorical variables were total fish (< 2 or ≥ 2 servings/week), milk (< 200 ml or ≥ 200 ml/week), eggs (< 150 g or ≥ 150 g/week), sun exposure (< 6 h or ≥ 6 h/week), active outdoor exercise (yes or no), sunscreen use (yes or no), gender (male or female), and BMI (< 23 or ≥ 23 kg/m²). To analyze the relationships between sun exposure or vitamin D intake and serum 25(OH)D levels, sun exposure time was set to 6 h/wk (mean value of the subjects) as a criterion for categorical variables, whereas vitamin D intake was set to 2 µg/day (median value of the subjects). Statistical analyses were conducted using PASW Statistics Version 18.0 (IBM Inc).

Serum 25(OH)D and 1,25(OH)₂D concentrations of males and females are shown in Table 2. Mean 25(OH)D and 1,25(OH)₂D levels were 13.5 ± 5.8 ng/ml and 20.6 ± 11.8 pg/ml, respectively. Only two patients (4.3%) showed sufficient vitamin D levels [25(OH)D ≥ 30 ng/ml], whereas 13 patients (27.7%) showed severe vitamin D deficiency [25(OH)D < 10 ng/ml]. Prevalence of vitamin D deficiency [25(OH)D < 15 ng/ml] in female patients was 78.6% (n = 22), whereas prevalence in males was 63.2% (n = 12). Distribution of serum 25(OH)D status was significantly different according to gender (P = 0.046). Serum albumin levels were significantly lower in females (P = 0.013) compared to males, whereas alkaline phosphatase levels were significantly higher in females (P = 0.041). Average dietary intake of vitamin D was 3.25 µg/day for males and 2.47 µg/day for females. Sun exposure time per week was 8.91 h for males and 5.40 h for females. Dietary vitamin D intake and sun exposure time were higher in males compared to females, although there were no significant differences.

Biological variables according to serum 25(OH)D levels are shown in Table 3. To compare biochemical variables according to serum 25(OH)D levels, we divided them into two vitamin D subgroups: deficient (< 15 ng/ml) and non-deficient (≥ 15 ng/ml). Concentrations of 1,25(OH)₂D in the non-deficient group were significantly higher compared to the deficient group (P < 0.001). However, iPTH and other biological variables were not significantly different between the two groups after adjusting for age, gender, and HD duration. Dietary vitamin D intake and sun exposure were also not significantly different between the non-deficient and deficient groups. Paricalcitol (Zemplar), a vitamin D analogue, was prescribed to all eight patients, and seven patients were in the deficient 25(OH)D group over the previous 3 months. Paricalcitol did not increase serum 25(OH)D levels. In terms of physical activity, regular exercise was classified as those who exercise for ≥ 30 min at least three times per week. Active outdoor exercise was classified as one who exercises outside regularly. The proportion of regular exercise was not significantly different between the two groups, whereas the proportion of active outdoor exercise in the non-deficient group (≥ 15 ng/ml) was significantly higher than that in the deficient group (< 15 ng/ml) (P = 0.022).

To evaluate the main determinants of serum 25(OH)D concentration, we analyzed dietary and sun exposure factors affecting serum 25(OH)D concentrations in HD patients by simple and multiple linear regression analyses (Tables 4 and 5). Dietary factors in the analysis were daily vitamin D intake as a continuous variable and major dietary sources of vitamin D (total fish, ≥ 2 servings/wk; milk, ≥ 200 ml/wk; eggs, ≥ 150 g/wk) as categorical variables. The results of the multiple linear regression model were adjusted for BMI and gender. Dietary intake of vitamin D and food sources did not increase serum 25(OH)D levels in HD patients. We also evaluated other determinants, including sun exposure time as a continuous variable and categorical variable (1: ≥ 6 h/wk, 0: < 6 h/wk), active outdoor exercise (1: yes, 0: no), and sunscreen use (1: yes, 0: no). From the simple linear regression analysis, the most important relative effects on serum 25(OH)D status were time of sun exposure and active outdoor exercise. Serum 25(OH)D level increased by 0.27 ng/ml for every 1 h of sun exposure per day and increased by 4.12 ng/ml for every 6 h per week. Active outdoor exercise longer than 30 min at least three times per week was another important relative determinant of serum 25(OH)D status. Specifically, serum 25(OH)D levels increased by 5.21 ng/ml for outdoor exercisers. To assess the association between sun exposure or dietary vitamin D intake and serum 25(OH)D status, dietary factors such as daily vitamin D intake and fish intake were applied to a multiple linear regression model. The results for sun exposure and dietary factors with serum 25(OH)D concentration were adjusted for BMI and gender. Sun exposure time and active outdoor exercise did not increase serum 25(OH)D levels in the multiple linear regression model. Sunscreen use was not significantly associated with serum 25(OH)D status in the simple linear model.