The primary aim of this randomized placebo-controlled, partially-blinded study on 269 healthy adults with mean 25(OH)D of 39.8 (11.9) nmol/L was to evaluate the relative efficacy of equi-unit D2 and D3 oral supplements given daily, 2-weekly, or 4-weekly in raising 25(OH)D level over 20 weeks. Predetermined secondary aims included comparing D2, D3, 25(OH)D2, and 25(OH)D3 levels. The primary outcome measure was adjusted area-under-the-curve between days 0 and 140 (AUC 140 ) . The main results were: 1) in the long term (20 weeks), the D3 2-weekly followed by D3 4-weekly and D2 daily regimens were superior in raising 25(OH)D levels. In the first few weeks of treatment, however, the 4-weekly followed by 2-weekly regimens were superior to all daily regimens. 2) D3 2-weekly and 4-weekly regimens were consistently superior to the corresponding D2 regimens; however, D2 daily regimen was consistently superior to D3 daily regimen. 3) 25(OH)D2 level was significantly higher in the daily compared to the 2-weekly and 4-weekly D2-treated groups, whereas, 25(OH)D3 level was lower in the daily compared to the 2-weekly and 4-weekly D3-treated groups 4) The increase in 25(OH)D level was inversely related to baseline level, however, its inverse relation to BMI appeared to be D-type and time dependent (mainly short time after D2 treatment). 5) Daily, 2-weekly, and 4-weekly D2 regimens were associated with a similar and significant decrease in 25(OH)D3 level that correlated with the increase in 25(OH)D2 level and baseline 25(OH)D level, in one participant with measurable baseline 25(OH)D2 level, D3 caused a similar decrease in 25(OH)D2 level, while in the D2/D3-treated group, 25(OH)D3 level didn’t increase. 6) The increases in D3 level in the 2-weekly and 4-weekly D3 treated groups were higher than the increases in D2 level in the corresponding D2 treated groups, the opposite was true for 25(OH)D2 and 25(OH)D3 levels. 7) Females had higher increases in D3, D2, 25(OH)D2, and 25(OH)D3 levels than males. 8) D treatment was associated with significant increases in urinary calcium and creatinine levels but not calcium/creatinine ratio.

All of the seven D supplement regimens in our study significantly increased 25(OH)D. At day 140, mean increase was 3.3 nmol/L in the placebo group and 28.6 nmol/L in the active-treatment groups. Although comparison is difficult because the dose–response curve is curvilinear, with an average of 1786 IU/day, this translates into an increase of about 16 nmol/L per 1000 IU, which is consistent with previous observations [8, 21, 26, 27, 36, 40]. In men with baseline 25(OH)D of 70 nmol/L, it was estimated that the increase in 25(OH)D level is about 17.5 nmol/L per 1000 IU daily D3 dose [40]. A review of recordings of 17,614 healthy adults participating in a preventive health program found an average increase of 12 nmol/L per 1000 IU for daily dosing interval of 0 to 1000 IU [21]. In a multicenter, retrospective data extraction study, an average daily dose of 2700 IU D3 increased 25(OH)D by 11.8 nmol/L per 1000 IU [27]. It is to be noted that the recovered content of the capsules in our study was about 90% of the label claim and that compliance with study medication was 98.4 to 100%. In our study, the increase in 25(OH)D level plateaued around days 70 and 112 in the daily and 2-weekly groups, respectively. Time to plateau ranged from 5 weeks to five months in previous studies [40, 41]. Due to our relatively frequent sampling, we were able to observe clear fluctuations in 25(OH)D levels when measured 2 weeks and 4 weeks after dosing, which may have clinical implication in term of monitoring response to therapy. Interestingly, the fluctuations were more pronounced with D2 dosing, consistent with shorter half life of 25(OH)D2 [4, 7, 42].

Our finding that D3 is superior to D2 in raising 25(OH)D level when given 2-weekly or 4-weekly is consistent with the published literature. The superiority of D3 was seen in studies that used 50,000 IU daily [5] or weekly, [4] a bolus of 300,000 [42] or 50,000 IU, [7] and a bolus of 10,000,000 IU in cows, [43] but not in studies that used 400 IU daily, [8, 9] 1000 IU daily, [29, 36] or 2000 IU daily [10]. Nevertheless, it was also reported with daily doses of 4000 IU for 14 days [11]. A 2012 meta-analysis found that D3 is more potent than D2, interestingly the difference was significant in the 4 RCTs (48 patients) that used bolus oral or intramuscular doses but not in the 6 RCTs (146 patients) that used daily supplements [24]. The interaction between D-type and dosing schedule was clearly shown in this study; while daily D3 was less efficient than 2-weekly and 4-weekly D3 in raising 25(OHD3, daily D2 was superior to 2-weekly and 4-weekly D2 in raising 25(OH)D2 levels. It is to be noted that the formulation of the capsules in our study was based on the common unitage that 1 IU equals 25 ng crystalline D2 or D3. Since the molecular weights of D2 and D3 are 384 and 396, respectively, 25 ng D3 would be equivalent to 25.78 ng, [28] thus the potency of D2 may have been underestimated by about 3% if one considers molar equivalence rather than weight equivalence in determining potency in IUs. Our results suggest that for long term results, D2 is best given daily while D3 is best given 2-weekly. However, the 4-weekly followed by the 2-weekly (D2 or D3) regimens are clearly superior in in rapidly raising 25(OH)D levels.

We observed consistent decrease in 25(OH)D3 levels in D2 treated groups. This was observed in most [8, 33, 44] but not all [29] previous studies that fractionated 25(OH)D levels. In a meta-analysis of RCTs on the effect of UV-exposed mushrooms consumption, the increase in 25(OH)D2 level was associate with a decrease in 25(OH)D3 level [35]. Further, 1000 IU D3 daily for 11 week did not change 1,25(OH) 2 D3 level, while 1000 IU D2 daily increased 1,25(OH) 2 D2 level by 7.4 pg/ml and decreased 1,25(OH) 2 D3 level by 9.9 pg/ml [36]. A similar decrease in 1,25(OH) 2 D3 was seen in response to 4000 IU D2 daily for 8 weeks [44]. Several observations from our study may shed light on the mechanism(s) underlying these observations. We found that the D2-induced decrease in 25(OH)D3 level was similar in the daily, 2-weekly, and 4-weekly groups, that it was correlated with the increase in 25(OH)D2 level, baseline 25(OH)D level, and day 140 25(OH)D level, that there was no change in 25(OH)D3 level in the group treated with a combination of D2 and D3, and that when 25(OH)D2 level is measurable (one case), D3 treatment resulted in a similar decrease in 25(OH)D2 level. These observations suggest that the D2-induced decrease in 25(OH)D3 level may more related to the resulting 25(OH)D level rather than being specific to D2 treatment. In fact, in one study, 400 and 1000 IU D3 daily for one year resulted in an increase in 25(OH)D3 level with a concomitant decrease in 25(OH)D2 level [34]. Interestingly, in a crossover study on high-yielding dairy cows, pre-administration of 10,000,000 IU of D3 significantly reduced 25(OH)D2 response to 10,000,000 IU of D2 [43]. It may be that there is a regulatory mechanism that increases the disposal of 25(OH)D in response to increases in its level [20] and that it has been observed with D2 treatment mainly because study participants commonly don’t have measurable 25(OH)D2 levels. Since there was essentially no change in 25(OH)D3 level in the group that received combination of 1000 IU D2 and 1000 IU D3, it appears that, in a setting similar to our study (baseline 25(OH)D level around 40 nmol/L and average dose of 1800 IU daily), an amount of 25(OH)D that can be produced by 1000 IU intake is disposed daily. If such a mechanism really exists it can be exploited in defining normal 25(OH)D levels.

Consistent with the above interpretation and with previous studies, [9, 11–16, 26, 35] we found significant negative correlation between baseline 25(OH)D level and response to treatment. A recent review found that 17 out of 20 studies documented such correlation (3 studies had inadequate sample size and variation in baseline level), which may explain up to 20% of response variation [13]. A recent systematic review of studies that used modest daily doses of D3 (200 to 800 IU), also found negative correlation, albeit not significant [26]. The negative correlation together with the non-linear response in 25(OH)D level to increasing doses of D [6, 16, 20, 21] again suggest a regulatory step mechanism [20]. In our study, the significant negative correlation between baseline and increment 25(OH)D level was first seen at day 28 (when 25(OH)D level was 59.1 (14.7) nmol/L), suggesting a threshold effect. Interestingly, pooling data of 3 RCTs, subjects with single nucleotide polymorphisms (linked to D binding proteins and 25-hydroxylase) that are associated with the lowest baseline 25(OH)D level had the smallest increase in 25(OH)D level (32). Thus in some subjects, low baseline 25(OH)D level may reflect a genetic potential rather than lifestyle influence and may be associated with lower rather than higher increment in 25(OH)D level.

Higher BMI/body fat percentage was associated with smaller response to D supplement in several studies [12–14, 18–21, 45]. BMI may be a better predictor than absolute weight [21] and was suggested as the most powerful response predictor to D supplement [19]. Up to 34.5% of response variation may be related to BMI, more apparently with higher D doses [13]. In a large retrospective study, mean increases in 25(OH)D level were 28.7, 23.6, and 20.1 nmol/L with BMI <25, 25–29, or ≥ 30 kg/m2, respectively [18]. Nevertheless, not all studies showed such negative association [13]. This may be due to the fact that higher BMI is also associated with baseline lower 25(OH)D levels, [17, 45] which is itself positively associated with response to D supplement. In our study, BMI was a significant response predictor to D2 but not D3 and only during the first 4 weeks of treatment, suggesting two additional potential modifiers of the relationship between BMI and 25(OH)D response to D supplement, D-type and time of assessment. The mechanisms underlying the association between BMI and response to D supplement are not clear. It was suggested that D and 25(OH)D may be trapped in access adipose tissue, as 25(OH)D is released with weight loss 1–6 months following bariatric surgery [46, 47]. However, total body fat storage may account for only 17% of the administered dose (extrapolated from subcutaneous fat), [4] and D and 25(OH)D may be also deposited in liver, muscle, and skin as shown in animal studies, suggesting that volume dilution may play a role [20]. The relatively lower affinity of D binding protein to D2 and 25(OH)D2 [24, 28] makes them more accessible to extra-vascular tissues, which may explain our finding.

We found that the increase in D3 level was 2–3 fold higher than the increase in D2 level (in the 2-weekly and 4-weekly treated groups). Few studies examined D2 and D3 after D2 and D3 supplementation [4, 43]. After similar doses of D2 and D3 (50,000 IU weekly for 12 weeks), subcutaneous fat D3 storage was 2 times higher than D2 storage [4]. Equivalent pharmaceutical doses of D3 and D2 in cows increased D3 level more than D2 level, respectively [43]. The difference may be related to the different structure of D2 and D3 side chains, theoretically causing differential absorption, binding to D binding proteins, inactivation by 24-hydroxylation, or activation by 25-hydroxylation. Absorption is not likely to be involved as studies of tritium-labeled D2 and D3 in healthy subjects found similar recoveries after oral dosing, however, D binding protein has double association constant to D3 compared to D2, and in vitro, mitochondrial 25-hydroxylase is 5 times faster for D3 compared to D2 [11, 24, 28]. It is to be noted that most of the ingested D is not converted to 25(OH)D; an RCT found that oral 25(OH)D3 is 4–5 more potent than D3 in raising 25(OH)D3 levels, [41] Interestingly, in our study, 25(OH)D2 levels were higher than 25(OH)D3 levels, suggesting that the lower D2 levels were due, at least in part, to higher D2 accessibility to the 25-hydroxylase enzyme because of lower affinity to D binding proteins. It is of note that the combination of D3 AUC 7 and 25(OH)D3 AUC 7 was about 50% higher than the combination of D2 AUC 7 and 25(OH)D2 AUC 7 ; indicating that a mechanism other than faster 25-hydroxylation (such as higher accessibility of D2 and 25(OH)D2 to extra-vascular tissues and faster degradation) is also involved. These mechanisms may apply only for schedules using high doses as 25(OH)D AUC 140 was higher in D2 daily than D3 daily treatment.

The higher levels of D3 compared to D2 may have important implication regardless of their impact on serum 25(OH)D level. Because of lower affinity to D binding protein, D2 and D3 have more cellular accessibility than 25(OH)D2 and 25(OH)D3 (except for the kidney, parathyroid gland, and placenta, where the megalin-cubulin system is expressed) and may have important physiological roles in breast milk and as substrates for many tissues [2].

We found that females had significantly larger (60, 55, 28%) adjusted AUC 7 than males for D2, D3, and 25(OH)D2 levels (and larger 13%,but not significant increase in 25(OH)D3 level). Females also had significantly 22% larger adjusted 25(OH)D2 AUC 140.

The results suggest about 49% better bioavailability of both D2 and D3 in females. Sex effect on response to D supplement has not been directly studied before. However, it is of note that D binding proteins are higher in females than males, in premenopausal women compared to postmenopausal women, in pregnant women, in women on oral contraceptives, [22] and in postmenopausal women hormone replacement therapy [23]. Further, estrogens increase hepatic 25-hydroxylation of D and the impact of D binding protein on response to D treatment may be partly D-type dependent [13]. The observed sex differences may be related to higher D binding protein and faster 25-hydroxyaltion in females; although a sex difference in D absorption rate cannot be excluded. It is also possible that higher body fat and lower baseline 25(OH)D levels in females may play a role.

In agreement with previous studies using even higher doses of D, [6, 48, 49] none of our participants developed hypercalcemia or hypercalciuria. In fact, the increase in urinary calcium/creatinine ratio was not significant. Nevertheless, the calcium/creatinine ratio may be misleading as there was significant increase in both calcium and creatinine urinary excretion. An increase in creatinine generation and urinary excretion has been described in patients with chronic kidney disease treated with vitamin D receptor activator, paricacitol [50]. The increase in creatinine excretion associated with D treatment casts doubt on the usefulness of ratios that include urinary creatinine (such as albumin/creatinine and calcium/creatinine) in evaluating the effect of D treatment on kidney function [51, 52] or D intoxication [53].

The strengths of this study include using repeated measurements, having a placebo arm, and ability to study several active-treatment regimens simultaneously, which enabled observing small changes and uncovering mechanistic insights. They also include effective randomization and concealment, partial blinding, frequent follow up to strengthen and verify compliance and verification of D capsule content across the study period.

Limitations

The interpretation of the results of this study may be limited by its sample size, 15% follow up loss, lower compliance with daily compared to 2-weekly and 4-weekly regimens, capsule content that is lower than label claim, and capsule formulation based on weight equipotency of D2 and D3. The rate of follow up loss was similar across the groups, the characteristics of participants who completed the study were similar to those of the entire cohort, incompliance rate was 1 to 1.6% in the daily groups, and the discrepancy between capsule content and label claim was similar across the capsules; thus these factors would not be expected to affect the main findings of the study. Although the incompliance rate was low, it was measured by capsule count, which may not be reliable. Thus the lower dose–response observed with daily D3 treatment compared to 2-weekly and 4-weekly D3 treatments could be explained at least in part by incompliance. However, such explanation is not likely given our observation that the dose–response was higher with daily D2 treatment compared to 2-weekly and 4-weekly D2 treatments and the fact that assignment to daily D2 or D3 treatment was random and blinded. The lower capsule content implies that the observed increments in D and 25(OH)D levels may have been up to 10% higher. Further, the fact that the molecular weight of D2 is about 3% higher compared to D3 indicates that our study may have underestimated response to D2 treatment. Nevertheless, such difference would not be expected to change the conclusions of the study. Further, the strength (in terms of IU) of most currently available D supplements is based on the assumption of weight rather than molar equipotency of D2 and D3. Another limitation of the study is that our findings may not be generalizable to lower or higher doses of vitamin D, or to subjects with different baseline 25 (OH)D levels, with different demographics, or with co-morbidities. Also, since the study was exploratory in nature, we have conducted multiple comparisons (including ad hoc comparisons), which would increase the rate of type 1 error. Further, the study examined surrogate endpoints (vitamin D and hydroxyvitamin D levels) rather than clinical endpoints. Finally, due to our assay sensitivity, we were not able to measure D2 and D3 levels in the daily treated groups.