1. Introduction
Vitamin D belongs to dietary micronutrients and it is known for pleiotropic
actions, going far beyond its classical function of maintenance of calcium and
phosphorous homeostasis [1]. Main source of vitamin D in humans is skin synthesis
in two stage process in which the B ring of 7-dehydrocholesterol is broken by
ultraviolet (UV) radiation from the sun and it further isomerizes to vitamin
D [2]. Vitamin D can be also obtained from the diet—vitamin D
(cholecalciferol) can be found inter alia (i.a.) in fish, while vitamin D
(ergocalciferol) is produced in variety of plants and yeast [3].
Vitamin D ingested form the diet is incorporated into chylomicrons and from
lymphatic system it enters the circulation [4]. In the bloodstream, vitamin D is
bound to vitamin D binding protein (DBP) and lipoproteins, from DBP it is
released to the liver, which is main but not the sole source of 25(OH)D, after
25-hydroxylation of vitamin D on C-25 [4]. Further metabolism of 25(OH)D occurs
in kidneys where 1-alpha-hydroxylase (CYP27B1), one of cytochrome P450
mixed-function oxidase (CYP), can be found [3]. The mutations in CYP27B1 gene
underlies pseudovitamin D deficiency caused by inadequate 1,25(OH)D
production [5]. Apart from the kidneys, CYP27B1 is also expressed in several
external sites [6].
1,25(OH)D binds to vitamin D receptor (VDR) in nearly all cells and
activates the expression of 24-hydroxylase (CYP24A1)—The main enzyme of
vitamin D catabolism. The direct products of CYP24A1 reaction are
24,25(OH)D and 1,24,25(OH)D. Thus, the determination of
24,25(OH)D in serum may be useful in diagnostics of patients with
partial or complete loss of CYP24A1 function. In those patients, the
administration of vitamin D may result in symptomatic hypercalcemia [7]. Despite
being known as “inactive” metabolite, it was proven 24,25(OH)D
exhibits biological activity—It suppresses parathyroid hormone (PTH)
secretion, regulates embryogenesis, stimulates bone remodelling and cartilage
growth/maturation [8, 9, 10, 11].
Recently it was proven that 25(OH)D is present in two stereoisomeric forms
that differ in the arrangement of groups on a single asymmetric carbon atom—C-3,
as a result of 3-epimerase reaction, while epi-25(OH)D has fewer
calcemic effects than non-epimeric 25(OH)D [12]. This alternative path was
discovered once liquid chromatography with tandem mass spectrometry (LC-MS/MS)
measurement was introduced: when the other diagnostic methods are used
(immunoassays: chemiluminescence immunoassay (CLIA), electrochemiluminescence
immunoassay (ECLIA), DBP-based assays, in-house high-performance liquid
chromatography (HPLC)), epimer may remain undetected, which may lead to
overestimation of vitamin D status [13]. The results of in vitro and
in vivo studies indicate that all main metabolites of vitamin D
constitute substrates for epimerase. Moreover, they can be further metabolised by
1-alpha-hydroxylase and 24-hydroxylase—Following the classic pathway [13].
3-epi-25(OH)D is detectable in low concentrations in serum, reaching the
highest concentration in infancy [14]. The exact function of vitamin D epimers
has not been fully understood. 3-epi-1,25-(OH)D has lower affinity to
VDR as compared to nonepimeric form, nevertheless it induces osteocalcin
expression, CYP24, suppresses the secretion of PTH and stimulates the production
of surfactant in type II pneumocytes [15, 16, 17].
Apart from essential role in blood calcium and phosphorus homeostasis, vitamin D
causes also the extra-skeletal effects: in cellular proliferation,
differentiation, and immune modulation [1]. Therefore, vitamin D has become the
subject of numerous studies in relation to its potential protective effect in
pathophysiology of diabetes, cardiovascular diseases, autoimmune diseases,
infections and cancer [1]. Thus, it is important to maintain the adequate vitamin
D level. Given the fact that skin production and regular diet is frequently
insufficient to ensure proper vitamin D level, it needs to be supplemented. Due
to limited solar exposure and outdoor activity, monotonous diet, atrophic skin
changes leading to decreased dermal production and reduced renal production, the
elderly are more prone to develop vitamin D insufficiency [18]. However, adequate
supplementation of vitamin D in elderly is a challenge. To improve patient
compliance, an attractive option is to administer higher doses of vitamin D taken
less frequently. However, questions about the effectiveness and safety of such
interventions arose. There are also significant individual differences in the
response to the same supplementation dose, which may origin from differences in
body weight, initial vitamin D concentration, polymorphism of genes involved in
vitamin D metabolism [18, 19].
Here, we performed randomised controlled trial to assess the concentrations of
vitamin D metabolites in serum after oral administration of 120,000 international
units (IU) of vitamin D in the elderly patients admitted to the hospital—population
especially prone to develop vitamin D deficiency. In addition, we
assessed the percentage of adipose tissue in all patients using the dual-energy
X-ray absorptiometry method, assuming this parameter may affect the increase in
25(OH)D concentration and the pharmacokinetics of metabolites. To our
knowledge, this is the first study with early metabolite assessment after
high–dose vitamin D administration in the elderly population, which may provide
a better understanding of the vitamin D metabolism pathway in this group of
patients.
2. Materials and Methods
2.1 Study Design
The randomized, two-arms, open study which was conducted in the Department of
Internal Medicine Department of Bielański Hospital in Warsaw (Poland) between
April 2021 and August 2021. All participants were admitted to the hospital due to
emergency reasons. The exclusion criteria were hypercalcemia, nephrolithiasis,
kidney insufficiency, documented vitamin D metabolism disorders such as
sarcoidosis, parathyroid disease or genetic defects, vitamin Dsupplementation within 6 months prior to the hospitalisation. The study was
approved by Bioethics Committee of Centre of Postgraduate Medical Education
(Warsaw, Poland) on 14.04.2021 (number 19/2021).
Patients were randomized in two groups: study group receiving 120,000 IU of
vitamin D (four tablets containing 30,000 IU of vitamin D each,
Solderol, series number 2011020F, Pharma Patent Kft, Budapest, Hungary) and
control group. The randomization list was created using a computer-generated code
(https://www.randomizer.org). All participants provided written informed consent before
participation.
The primary aim of the trial was to assess the effects of a single high dose of
vitamin D (120,000 IU) on serum 25(OH)D, 25(OH)D,
24,25(OH)D, 3-epi-25(OH)D, 1,25(OH)D,
24,25(OH)D/25(OH)D ratio, and 25(OH)D/3-epi-25(OH)D
ratio concentration at baseline, 3 days and 7 days after administration, compared
to control group. The secondary aim was assessment of the influence of percentage
of fat tissue on serum concentration of metabolites and their changes after bolus
dose.
2.2 Sample Collection and Measurements of Vitamin D Metabolite Levels
Patients’ specimens were collected at three time points: at baseline, 3 days and
7 days after oral administration of 120,000 IU vitamin D. Blood samples were
collected from antecubital vein into Vacuette® blood collection
tubes (6 mL) with serum cloth activator. Some samples were missing, mainly due to
transfer of some patients to other centres and pre-analytical errors. Blood was
centrifuged using standard laboratory procedures (3500 revolutions per minute
(rpm), 10 minutes) and the serum was aliquoted and frozen at –86 °C until further
analysis. In each patient’s serum sample the levels of creatinine (CREA), PTH,
albumin and calcium were determined. CREA and calcium were measured using the
spectrophotometric method on Cobas 6000 e501 (Roche Diagnostics, Risch-Rotkreuz,
Switzerland). Albumin was measured using the spectrophotometric method on Cobas
Integra 400 plus (Roche Diagnostics, Risch-Rotkreuz, Switzerland). PTH was
determined using ECLIA assay on Cobas 8000 e801 (Roche Diagnostics,
Risch-Rotkreuz, Switzerland).
Vitamin D metabolites analysis: 25(OH)D, 24,25(OH)D,
epi-25(OH)D and 25(OH)D was performed by isotope dilution mass
spectrometry. Serum samples (50 L) were subjected to protein
precipitation and specific derevatization
4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD). The analytes
were then separated using a high performance liquid chromatograph (ExionLC,
Sciex, Framingham, MA, USA) and analyzed on a triple quadrupole tandem mass
spectrometer (QTRAP®5500, Sciex, Framingham, MA, USA) using an
ESI ion source in positive ion mode. Details of the described method are included
in the publication [20].
A modified procedure [21] was used to analyze the serum calcitriol
concentration. The serum sample (200 L) was enriched with the
isotope labelled internal standard and then subjected to protein precipitation
with acetonitrile (500 L). After precipitation and centrifugation
supernatant was transferred to an eppendorf tube, and acetonitrile was removed by
under nitrogen stream. Calcitriol was extracted using twice liquid-liquid
extraction (vortex, 30 seconds) with ethyl acete (2 200
L). Pulled extract was evaporated to dryines under nitrogen stream.
Then samples were derivatized with DAPTAD reagent the same as described in the
publication [20]. After reconstitution in 50 L of MeOH:HO (1:1),
20 L of sample was injected on LC-MS/MS system. The chromatographic
separation was carried out in 45 °C in a gradient of 0.1% formic acid (mobile
phase A) and 0.1% formic acid in methanol (mobile phase B) on the column,
Cosmocore®PBr, 100 2.1 mm; 2.6 M
(Nacalai Tesque, Japan). The analysis was performed using an ESI type ion source
(electrospray) in positive ion mode. Detection was performed in MRM mode on a
QTRAP5500 with tandem mass spectrometer (Sciex, Framingham, MA, USA).
2.3 Dual-Energy X-Ray Absorptiometry (DXA) Assessment and Anthropometric
Measurements
The percentage of fat tissue was determined using DXA on Lunar Prodigy Advance
(GE Healthcare, Madison, WI, USA) and analyzed using programme Encore v18 (Adobe, San Jose, CA, USA). All
scans were performed and analyzed by one operator. BMI (body mass index) was
calculated as weight (kg) divided by the square of height (m).
2.4 Statistical Analysis
The data were statistically analyzed using 13.1 software (STATSOFT, Kraków, Poland).
Minimum and maximum values as well as median and interquartile range (IQR)
(lower quartile–upper quartile) were estimated for numerical variables, and
absolute numbers (n) and percentages (%) of the occurrence of items for
categorical variables.
Mann-Whitney U test was used to compare numerical variables between the study
group and the control group or Pearson’s chi-square test to compare gender
between those two groups.
Wilcoxon signed ranks test was used to compare numerical variables between two
time points: baseline and 3 days after vitamin D oral administration, between
baseline and 7 days after vitamin D oral administration, between 3 and 7 days
after vitamin D oral administration, separately in the study group and in the
control group.
Pearson’s correlation coefficient was used to correlate two numerical variables
between each other.
p-values 0.05 were considered statistically significant.
3. Results
Study population included 58 patients aged from 61 to 96 years old (mean age
73.7 years old, 48.26% women and 51.72% men) randomized in two groups: study
group (30 subjects) and control group (28 subjects). Baseline characteristics of
study participants and metabolites of vitamin D concentrations are summarized in
Table 1.
Table 1.Characteristics and metabolites of vitamin D at baseline.
Variable |
Study group (n = 30) |
Control group (n = 28) |
p |
Age (years) |
70.50 (66–77) |
72 (68–82) |
0.185 |
Gender, F/M |
11 (36.67)/19 (63.33) |
17 (60.71)/11 (39.29) |
0.067 |
BMI (kg/m) |
27.79 (22.68–29.70) |
27.50 (23.98–29.75) |
0.876 |
Percentage of a fat tissue (%) |
32,70 (28–41,20) |
37.90 (29.80–48.10) |
0.492 |
CREA (mg/dL) |
0.82 (0.74–0.91) |
0.82 (0.65–0.91) |
0.575 |
PTH (pg/mL) |
37.15 (25.80–49.90) |
40.25 (30.10–56.50) |
0.635 |
Albumin (g/dL) |
3.43 (3.18–3.87) |
3.25 (2.96–3.85) |
0.450 |
Calcium (mmol/L) |
2.21 (2.14–2.34) |
2.16 (2.09–2.29) |
0.216 |
Corrected calcium (mmol/L) |
2.33 (2.27–2.39) |
2.30 (2.26–2.33) |
0.219 |
25(OH)D (ng/mL) |
12 (7.58–16.77) |
17.93 (9.42–25.41) |
0.096 |
25(OH)D (ng/mL) |
0.39 (0.29–0.52) |
0.22 (0.16–0.35) |
0.005 |
25(OH)D total (ng/mL) |
12.42 (8.01–17.09) |
18.45 (9.57–25.61) |
0.121 |
3-epi-25(OH)D (ng/mL) |
0.48 (0.26–0.69) |
0.64 (0.36–1.10) |
0.074 |
24,25(OH)D (ng/mL) |
0.44 (0.25–0.93) |
1.03 (0.46–2.20) |
0.009 |
1.25(OH)D (pg/mL) |
24.59 (13.92–37.62) |
21.58 (14.92–29.77) |
0.863 |
24,25(OH)D/25(OH)D ratio |
0.04 (0.03–0.06) |
0.06 (0.05–0.10) |
0.016 |
25(OH)D/3-epi-25(OH)D ratio |
22.92 (18.51–31.93) |
24 (18.87–29.45) |
0.863 |
Data are given in medians and interquartile ranges and for gender in number and
percentage.
p for Mann-Whitney’s U test, except from gender for which chi-square test was
used.
Abbreviations: CREA, creatinine; PTH, parathyroid hormone.
Corrected calcium = Calcium [mmol/L] + (40 – Albumin [g/L]) 0.02. |
No statistically significant differences between study and control group were
observed, apart from 25(OH)D, 24,25(OH)D concentration and
24,25(OH)D/25(OH) D ratio. At baseline vitamin D deficiency
(25(OH)D 30 ng/mL) was found in 92.8% of study participants and among those
in 30.3% was below 10 ng/mL. There was significant correlation at baseline
between serum concentrations of 25(OH)D and PTH concentration (r = –0.277,
p = 0.039) in the entire study population. In our study,
3-epi-25-(OH)D was detectable in all participants.
In the whole study population (n = 58), there were positive correlations at
baseline between serum concentrations of 25(OH)D and
24,25(OH)D, 3-epi-25(OH)D (r = 0.885, and r = 0.877,
respectively, p 0.001 for all) as shown in Fig. 1. There
was no significant correlation between 25(OH)D and 1,25(OH)D at
baseline (r = 0.241, p = 0.07) (Fig. 1). Serum concentrations of
24,25(OH)D at baseline correlated positively with 3-epi-25(OH)D
(r = 0.767, p 0.001) (Fig. 1).
Fig. 1.
Correlations between baseline concentrations. (a)
25(OH)D and 3-epi-25(OH)D. (b) 25(OH)D and
24,25(OH)D. (c) 25(OH)D and 1,25(OH)D. (d)
3-epi-25(OH)D and 24,25(OH)D in all participants. r, Pearson’s
correlation coefficient.
All subjects completed the study. Changes in the serum concentrations of vitamin
D metabolites on 3rd day and 7th day after intervention are presented in Fig. 2.
Single, oral administration of 120,000 IU of vitamin D showed to rapidly
normalize 25(OH)D (30 ng/mL) levels in most patients (56.6% study
group). All subjects, except for one patient, in the intervention group achieved
a serum 25(OH)D concentration 20 ng/mL. The highest achieved
25(OH)D level in the study group was 46.47 ng/mL. However, the individual
changes in 25(OH)D and other metabolites were variable
(Supplementary Fig. 1).
Fig. 2.
Changes between baseline, 3 days and 7 days after oral
administration of 120,000 IU of vitamin D in the study group. (a) 25(OH)D.
(b) 25(OH)D. (c) 25(OH)D total. (d) 3-epi-25(OH)D. (e)
24,25(OH)D. (f) 1,25(OH)D. (g)
24,25(OH)D/25(OH)D ratio. (h) 25(OH)D/3-epi-25(OH)D
ratio. Midpoint = median, box = IQR (Lower quartile–Upper quartile), whiskers =
Min–Max. p for Wilcoxon signed ranks test.
On 3rd day after administration of vitamin D, participants receiving vitamin
D showed a significant increase in serum 25(OH)D and 25(OH)D total
concentrations (all p 0.001 vs. baseline), whereas no significant
changes were seen between 3rd and 7th day after intervention (Fig. 2a,c).
Interestingly, there was a significant decrease in the concentration of
25(OH)D between baseline and 7th day after intervention in the study group
(p = 0.010) (Fig. 2b). Moreover, after vitamin D administration,
3-epi-25(OH)D rose rapidly and reached a peak on 3rd day (p 0.001) (Fig. 2d). By contrast, 24,25(OH)D level rose slower and
achieved the highest concentration on 7th day during this study, with significant
growth between 3rd and 7th day (p = 0.009) (Fig. 2e).
1,25(OH)D level was determined at baseline and on the 3rd day, a
significant increase in study group was noted (p = 0.005) (Fig. 2f).
There was no statistically significant correlation between serum PTH and increase
in 1.25(OH)D between 3rd day and baseline (r = 0.038, p =
0.856).
The analysis of the ratio 24,25(OH)D to 25(OH)D showed
significant increase between 3rd and 7th day p = 0.002 (Fig. 2g).
25(OH)D to 3-epi-25(OH)D ratio reached significant decrease in the
study group, with the highest drop on 3rd day (p 0.001)
(Fig. 2h).
A percentage increase in serum 25(OH)D after supplementation was dependent
on baseline 25(OH)D: the lower concentration at baseline, the higher
increase in 25(OH)D (Fig. 3). Furthermore, there were significant positive
correlations between percentage increase in 25(OH)D and a percentage
increase serum concentration of 24,25(OH)D (r = 0.954, p
0.001), 3-epi-25(OH)D (r = 0.803, p 0.001) and
1,25(OH)D (r = 0.789, p 0.001) (Fig. 4). In the control
group, no significant changes in concentration of analyzed metabolites of vitamin
D were observed (Supplementary Table 1).
Fig. 3.
Correlation between 25(OH)D at baseline and change of
25(OH)D 7 days after oral administration of 120,000 IU of vitamin D
compared to baseline, in the study group. r, Pearson’s correlation coefficient.
Fig. 4.
Correlations of changes. (a) Percentage of 3-epi-25(OH)D
and 25(OH)D after 7 days and baseline. (b) Percentage of
24,25(OH)D and 25(OH)D after 7 days and baseline. (c)
Percentage of 1,25(OH)D after 3 days and baseline and 25(OH)D
after 7 days and baseline after oral administration of 120,000 IU of vitamin D in
the study group. r, Pearson’s correlation coefficient.
In our study, one patient demonstrated excessive increase in 25(OH)D after
administration of vitamin D, from 1.81 to 46.47 ng/mL on 3rd day, achieving the
highest concentration of 25(OH)D among all participants. In addition, this
patient also reached the highest level of 1.25(OH)D, from 5.79 to
78.45 pg/mL. Meanwhile, 24,25(OH)D and 3-epi-25(OH)D
increased significantly and reached a peak on 3rd day. Finally, on 7th day after
intervention, the concentration of 25(OH)D dropped to 27.51 ng/mL.
On 7th day after administration of 120,000 IU Vitamin D, increase of serum
calcium was statistically significant (p = 0.032) (Fig. 5). None of the
study participants developed hypercalcemia.
Fig. 5.
Changes of calcium concentration between baseline and 7 days
after oral administration of 120,000 IU of vitamin D in the study group. Midpoint = median, box = IQR (Lower quartile–Upper quartile), whiskers =
Min–Max. p for Wilcoxon signed ranks test.
The baseline concentration of analyzed metabolites of vitamin D were
neither dependent on BMI nor percentage of fat tissue. Similarly, BMI and
percentage of fat tissue were not correlated with percentage increase in
concentration of these metabolites (Supplementary Table 2).
4. Discussion
In presented study, we assessed the effect of a single, oral, high dose
administration of cholecalciferol (120,000 IU) on serum concentration of
25(OH)D and other metabolites of vitamin D, namely: 24,25(OH)D,
3-epi-25(OH)D 1,25(OH)D, 25(OH)D, in elderly subjects,
who were admitted to the hospital due to emergency reasons. To our knowledge, it
is the first trial which assessed the early influence of high dose of
cholecalciferol on vitamin D metabolites in the elderly.
Most subjects had vitamin D deficiency, which confirms prevalence of the
deficiency and the need for supplementation in elderly. Observation time points
of changes in vitamin D metabolites serum concentrations in first week after
administration were chosen since in the prior studies large single dose of
vitamin D (50,000 IU or more) caused a rapid increase in 25(OH)D
concentration and reached a peak mostly after 3 days [22, 23, 24]. High dose of
cholecalciferol (120,000 IU) was chosen, because in our previous study on 35
patients admitted to the Department of Internal Medicine, after oral
administration of 60,000 IU of cholecalciferol, only 4 out of 35 (11.43%)
patients with baseline deficiency reached recommended serum 25(OH)D (30 ng/mL)
on the seventh day [25].
Given all the skeletal and extraskeletal benefits of adequate vitamin D status,
vitamin D testing has increased rapidly in the recent years [26]. Currently, the
serum 25(OH)D can be routinely measured using CLIA, ECLIA, HPLC, or
LC-MS/MS-based methods [27]. However, only mass spectrometry provides the optimal
detector for vitamin D metabolites separated by liquid chromatography techniques
[28]. To reliably investigate the early changes in vitamin D metabolites after
administration of 120,000 IU of cholecalciferol, we used LC-MS/MS technique,
since it allows to identify interfering substances with may otherwise complicate
accurate measurement of vitamin D status, i.a. 3-epi-(OH)D in 25(OH)D assay
or 4,25(OH)D in 1,25(OH)D assay [29, 30].
In our study, the oral administration of 120,000 IU of cholecalciferol resulted
in correction of vitamin D deficiency or insufficiency in most participants,
(56.6%) of patients reached 25(OH)D concentrations 30 ng/mL recommended
by the Endocrine Society [31]. All subjects, except for one patient, in the study
group achieved a serum 25(OH)D concentration 20 ng/mL, recommended by
the Institute of Medicine [32]. No one exceed reference value of vitamin D
(30–50 ng/mL) [33]. A meta-analysis of 30 studies using bolus dosing showed that
a single vitamin D dose of 100,000 IU offers a significant increase in
vitamin D concentrations but in most studies was insufficient to raise 25(OH)D
concentration 30 ng/mL in populations with baseline 25(OH)D concentrations
20 ng/mL. The dose higher than 200,000 IU were more effective, but the risk of
adverse events was higher [34]. Ilahi et al. [35] reported that the
elderly after administration high dose vitamin D have the lower initial peak, but
the slower pace of decline in vitamin D concentration when compared to younger
people. According to Ilahi et al. [35], 100,000 IU of vitamin D can be
safely recommended every 2 months for patients with moderate baseline 25(OH)D
concentrations.
We have consistently confirmed previous studies, that baseline 25(OH)D
concentration determines 25(OH)D response to vitamin D supplementation
[36, 37, 38, 39, 40]. Since hepatic hydroxylation of vitamin D may be a saturable
process, response to vitamin D supplementation could be affected by baseline
25(OH)D concentrations [41]. In our study, change in 25(OH)D had a
significant inverse correlation with baseline 25(OH)D concentration (r = –0.688,
p = 0.001). The highest increase in serum 25(OH)D was observed in
patients with severe deficiency (10 ng/mL), the lowest in subjects with
suboptimal concentration (20–30 ng/mL). Moreover, applied high dose did not
increase vitamin D concentration to those associated with an overdose of vitamin
D, even in patient who had baseline concentration of 25(OH)D close to 30
ng/mL. In our study the administration of high dose of cholecalciferol was
associated with statistically significant decline in 25(OH)D level
(p = 0.010). This phenomenon was already described in the study of
Hammami et al. [42], in which administration of 50,000 IU vitamin D3
resulted in decrease of 25(OH)D at 28 day in the comparison to the placebo
arm (adjusted mean difference 9.8 mmol/L, 95% confidence interval (CI) 5.2–14.4
nmol/L, p 0.001).
Similar to previous studies, we observed the rapid increase in serum
1,25(OH)D, it can be explained by fast 1 alfa-hydroxylation
25(OH)D in condition of deficiency of vitamin D and secondary
hyperparathyroidism [23, 43].
No significant correlations between percentage increase in 1,25(OH)D
and PTH concentration at baseline may be the result of limited number of study
participants. However, in the study of Amrein et al. [23] it was
suggested that responsiveness of the parathyroid glands in severely ill patients
may be diminished. In our study, it seems that increase of 1,25(OH)D
was mainly attributed to the sudden rise of the available substrate for
1-alpha-hydroxylase in a population with pronounced vitamin D deficiency.
Simultaneously to dynamic increase of 25(OH)D and 1,25(OH)D,
we noted early triggering of catabolic pathway. We found that serum
24,25(OH)D concentration was positively correlated with serum
25(OH)D, which is in accordance with the study published by Kim et
al. [44], in which strong correlation between 25(OH)D and
24,25(OH)D was observed (r = 0.868, p 0.001). This
relationship may be explained by the fact that enzymatic synthesis of
24,25(OH)D is regulated by 25(OH)D concentration and vitamin D receptor
activity in CYP24A1 [44, 45]. CYP24A1 is also inducible by its substrate
1,25(OH)D, which is a protective mechanism from excess VDR pathway
activation. Catabolic role of CYP24A1 was also confirmed in the studies with
CYP24A1-null mouse—when CYP24A1 is absent, half-life of 1,25(OH)D
increases significantly, from 6 to 60 hours [46, 47]. While CYP24A1 has been
established as a crucial enzyme in vitamin D catabolism, it was also proven in
works in balance with CYP27B1, which converts 25(OH)D to 1,25(OH)D,
both in the kidney and extra-renal target-cells where it prevents excessive
exposure to 1,25(OH)D hormone [45]. In our study, we observed the
gradual increase in 24,25(OH)D concentration, with the highest
concentration of 7th day, reflecting the activation of CYP24A1. In
contrary to our study, Wagner et al. [48] indicated there is a lag in
CYP24A1 activation compared to CYP27A1, suggesting that catabolism induced by
vitamin D supplementation may occur over weeks not days. However, the
administered dose of cholecalciferol was significantly lower (28,000 IU/weekly)
and the study was conducted on healthy, young adults, which may have determined
postponed activation of catabolic CYP24A1 [48].
Since the usage of LC-MS/MS method allows to determine both 24,25(OH)D and
25(OH)D simultaneously, there is possibility to calculate
24,25(OH)D/25(OH)D ratio, widely known as the vitamin D metabolite ratio
(VMR) [49]. VMR reflects not only vitamin D degradation but also is supposed to
indicate vitamin D status and VDR activity, while CYP24A1 expression is
upregulated by 1,25(OH)D [28, 49, 50]. Nevertheless, in the study of Francic
et al. [51] on 106 hypertensive subjects receiving 2800 IU daily of
vitamin D, VDR did not predict the change in 25(OH)D after vitamin D
supplementation. Concerning the 24,25(OH)D/25(OH)D ratio the results of
this study in accordance with another published report—Saleh et al.
[52] has proven that the administration of 100,000 IU of vitamin D results in
increase in 24,25(OH)D/25(OH)D ratio 4 weeks after, which may be explained
by the fact, that in case of the excess of substrate (25(OH)D) 24,25(OH)D
is favored over the active metabolite 1,25(OH)D, which allows to
avoid 1,25(OH)D toxicity.
In our study we also determined levels of 3-epi-25(OH)D, which was present
is all subjects. 3-epimerase catalyzes the reaction of C-3 hydroxy group of the A
ring from the alpha to beta orientation, can be identified in a number of cells,
but not in the kidney [13]. Despite 3-epi-25(OH)D can be substrate for
CYP27B1 and CYP24A1, it has reduced affinity to DBP in comparison to non-epimeric
form, whereas epi-1,25-(OH)D has lower affinity to VDR, which results in
reduced transcriptional activity and fewer biological effects [15]. Since we
observed the peak of 3-epi-25(OH)D already on 3rd day, it may indicate that
production of 3-epi-25(OH)D may be early catabolic mechanism protecting
from toxicity of active forms of vitamin D, followed by the gradual increase of
activity of CYP24A1, resulting in slower increase of 24,25(OH)D
concentration.
In large cross-sectional studies, it was proven that obesity is linked to
vitamin D deficiency—25(OH)D correlates inversely with BMI, total fat mass,
visceral adiposity, and waist circumference, also in the elderly [53, 54, 55, 56]. Many
hypotheses were developed to explain this relationship—main proposed
mechanism include increased serum concentration of immunoreactive PTH, higher
vitamin D sequestration, dilution, and clearance in obesity [57, 58]. However, in
our study there was a correlation neither between the BMI nor the percentage of
fat tissue and baseline vitamin D metabolites concentrations and changes in
vitamin D metabolites concentrations after the administration of 120,000 IU of
cholecalciferol (Supplemeantary Table 1). It may be attributed to the
relatively low number of subjects in the study and differences in dietary habits
and exposure to solar UV radiation.
Low medication adherence among the elderly is a common problem and it is a
consequence of i.a. the greater burden of co-morbidity and large numbers of
prescribed drugs [59]. Concerning failure to medication adherence and prevalence
of vitamin D deficiency in geriatric population, regimens with high vitamin D
doses administered less frequently may be an advantageous option. However, the
recent studies indicated that daily vitamin D supplementation may be more
effective It is known that a single high dose of vitamin D led to greater
induction of catabolic mechanisms (through the activation of 24-hydroxylase) than
a daily vitamin D supplementation, the effect was dependent on the dose [60]. As
a result, it may induce a downregulation of 1,25(OH)D. Ketha H
et al. [61] demonstrated that 24.25(OH)D/25(OH)D
ratio after single bolus (150,000 IU) attained a significantly greater value for
at least 28 days after administration, compared with daily dose group (5000
IU/per day), in which the 24,25(OH)D/25(OH)D remained
relatively stable and lower than the baseline value. However, is worth noting
that 1,25(OH)D concentrations were not significantly different in the
two dosing groups in this study [61]. The second possible mechanism explaining
the advantage of daily supplementation is the fact that cholecalciferol with 20 h
half-life is continuously available for internalization into cells in comparison
to bolus dosing. Intact vitamin D binds to DBP much less strongly than 25(OH)D,
thus cholecalciferol enters the cell easily for activation [62]. These mechanisms
could explain divergences in outcomes depending on dosing regimen that have been
observed in intervention studies, for example a recent meta-analysis concerning
acute respiratory infections [63]. These observations require confirmation in
large-scale intervention studies, comparing the different vitamin D dosage
regimens.
5. Conclusions
In our study, oral administration of high dose of vitamin D may be safe and
effective option for vitamin D insufficiency correction in elderly patients. We
also confirmed the efficacy of catabolic mechanisms of vitamin D, namely
3-epi-25(OH)D and 24,25(OH)D production, which prevents
excessive increase of active form of vitamin D.
Author Contributions
DL, WM, WZ and PG designed the research study. DL and AS performed the research.
DL, AS, KK, DR and PG analyzed the data. DL, AS, KK, MO and PG wrote the
manuscript. All authors contributed to editorial changes in the manuscript. All
authors read and approved the final manuscript.
Ethics Approval and Consent to Participate
The study was approved by Bioethics Committee of Centre of Postgraduate Medical
Education (Warsaw, Poland) on 14.04.2021 (number 19/2021). Effect of Single High Dose of Cholecalciferol on Serum Metabolites of Vitamin D, is registered and will be posted on the ClinicalTrials.gov public website (Identifier: NCT05591170).
Acknowledgment
Not applicable.
Funding
This research received no external funding.
Conflict of Interest
The authors declare no conflict of interest.