Elevation of Blood Vitamin Dz Levels Does Not Impede the Release of

Vitamin D3 From the Skin

Lois Y. Matsuoka, Jacob0 Wortsman, John G. Haddad, and Bruce W. Hollis

The mechanism for the transfer of fat-soluble vitamin D3 from the avascular basal cellular layers of the epidermis to dermal
capillaries and peripheral circulation is unknown, although vitamin D-binding protein (DBP) is thought to mediate this process.
To evaluate the effect of increased occupancy of vitamin D carrier(s) on vitamin D3 removal from the skin, serial serum vitamin
D2 and D3 concentrations were determined in three groups of six healthy volunteers given combinations of an oral dose of
vitamin Dz (50,000 IU) and a fixed dose of UVB radiation (27 mJ/cmZ). Serum vitamin D3 levels increased significantly following
UVB (time effect, P c .Ol by ANOVA), but the response remained unchanged after pretreatment with vitamin D2, increasing
from 3 f 1 to 14 f 5 ng/mL (mean f SEM), versus UVB alone, 5 + 1 to 16 f 5 ng/mL. Elevation of serum vitamin Dz levels was
also similar in the groups given vitamin DZ alone (< 1 to 64 f 8 ng/mL) and vitamin D2 + UVB (< 1 to 45 t 8 ng/ mL). There was
no time or treatment effect for changes in serum levels of 25.hydroxyvitamin D, 1,25dihydroxyvitamin D, or (DBP) levels
(P > .l). We conclude that vitamin D3 egress from the skin is not affected by elevated circulating vitamin D concentrations; thus,
the cutaneous release of vitamin D is probably mediated by a protein such as DBP with a high carrying capacity for the vitamin.
Copyright c: 1992 by W.B. Saunders Company

T HE MAIN SOURCE OF vitamin D detected normally ary). All subjects gave their informed consent as approved by the
in the circulation is solar light-stimulated cutaneous Jefferson Medical College (Philadelphia, PA) Institutional Review
production. In this photosynthetic process, newly formed Board.
Immediately after collection of a basal blood sample, the
previtamin D2 appears in basal avascular layers of the
experimental subjects were randomly assigned to one of three
epidermis as a result of the interaction between UVB
groups composed of six individuals each. Group 1, or D2 group,
photons (wavelengths, 290 to 320 nm) and epidermal
received vitamin Dz in a single dose of 50,000 IU (1.25 mg or 3,250
7dehydrocholesterol molecules. While still in the epider- nmol) administered in capsule form (Calciferol, Schwarz Pharma,
mis, previtamin D.7undergoes spontaneous thermal isomer- Milwaukee, WI) at approximately 9:00 AM. According to studies
ization with molecular rearrangement into the fat-soluble performed by Lo et al, the peak vitamin Dz serum concentration is
vitamin D? (cholecalciferol).L Vitamin D3 reaches the reached 12 hours after oral intake.” Group II, or Dz plus UVB
circulation through dermal capillary vessels, although the group received vitamin Dz in the same manner as group I, and 12
mechanism for its translocation from the epidermis is hours later the subjects were submitted to whole-body irradiation
unknown. It is nevertheless assumed that a plasma factor, with UVB at the fixed-dose level of 27 mJ/cm:. This dose of UVB is
group-specific component ([GC] also known as vitamin suberythematous (one minimal erythema dose for white skin type
III subjects is 33 to 36 mJ/cm’), but well above the threshold for
D--binding protein [DBP]), binds and removes vitamin Dj
detection of a statistically significant vitamin D, photosynthetic
from the epidermis.’ although direct information on this
response (15 to 18 mJ/cm’).9 The characteristics of the irradiation
phenomenon is not available. chamber and calibration of the UVB radiation delivered by the
To further investigate the mechanism of vitamin D3 photounit have already been described in detail’; with this proto-
egress from the skin, we evaluated whether elevating col, the peak vitamin D3 serum response to UVB occurs at 24
vitamin D? levels in the circulation would interfere with the hours.‘“.” Group III, or UVB group, received only irradiation with
process. To this effect, normal subjects were given a large UVB at the same dose level and time as group 11.
dose of vitamin Dz orally, followed by whole-body irradia- Blood samples were obtained again at 12, 34, and 36 hours after
tion with UVB to stimulate vitamin D3 formation. Vitamin collection of the basal samples. Serum was separated promptly and
D! (ergocalciferol) originates from dietary intake, can be stored at -20°C until analysis. Serum vitamin Dz and D3 levels
were measured by high-performance liquid chromatography using
separated from vitamin D3 by high-performance liquid
a method previously described.J The intraassay variability for
chromatography, and is metabolized along the same path-
vitamin D is less than 10%. interassay variability is leas than 16%,
way of activation as vitamin D3, into 25-hydroxyvitamin D limit of detectability is 1 ng/mL. and the sensitivity for both
and 1,25dihydroxyvitamin D.3-5 Newly formed vitamin D3 vitamins, Dz and Dj, is 0.3 ng/mL.
that is not transferred to the circulation can be photode- The concentrations of 25-hydroxyvitamin D and 1,75-dihydroxyvi-
graded locally into the biologically inactive products, 5,6- tamin D were determined as follows: serum samples underwent
transvitamin Dj, suprasterol I. and suprasterol II, if expo- initial extraction in methyl alcohol-methylene chloride, and the
suTe to light is continued.h

From the Department of Dermatoloffv, Jejjerson Medical College,

MATERIALS AND METHODS
Philadelphia, PA: the Department of Medrcine, Southern Illinois
The study population comprised 18 individuals, most of whom Ut1iversit.vSchool of Medicine, Springfield, IL: the Department of
were medical students in the third decade of life: there were three Medicine, University of Pennsylvania School of Medicine, Philadel-
women and 15 men. None had a history of hepatic or renal phia, PA; and the Department of Pediatrics, Medical Universig of
disorders, none were taking vitamin D, anticonvulsant medica- South Carolina. Charleston, SC.
tions, or corticosteroids, and none were obese or undernourished. Address reprint requests to Lois Y Matsuoka. MD. Jeflerson
All subjects were white with skin type III (sometimes burn, always Medical College. 111 S 11th St, Philadelphiu, PA 19107.
tan) according to the classification of Pathak et al7 The study was Copyright 0 1992 by W.B. Saunders Company
performed during the winter months (November through Febru- 0026-0495/921-/111-0018$03.00l0

Metabolism, Vol 41, No 11 (November), 1992: pp 1257-1260 1257

1258
lipid extracts were subjected to preparative chromatography on
silica cartridges for separation and purification of 25-hydroxyvita-
min D and 1,25_dihydroxyvitamin D. Serum 25hydroxyvitamin D
and 1,25_dihydroxyvitamin D concentrations were measured with
competitive-binding protein assays.
DBP levels were measured in duplicate by rocket immunoelectro-
phoresis using purified DBP as a standard and a monospecific
antiserum developed in rabbits.r2
Statistical Analysis
Samples with undetectable serumvitamin D concentrations ( < 1
ng/mL) were treated as having the level of 1 ng/mL. Data were
analyzed by a 3 (treatment groups) x 4 (time points) split-plot
ANOVA. Post-hoc testing for location of significant differences
was performed by paired t test.
RESULTS
Serum vitamin Ds levels (Fig 1A) increased significantly
after UVB treatment (P < .Ol by ANOVA for group x time
interaction). Post-hoc testing demonstrated that serum
vitamin D3 concentrations were significantly higher at 36
hours (24 hours after UVB) in the UVB-treated groups.
Serum vitamin D3 profiles were nevertheless similar in
groups that did and did not receive vitamin Dz (P > .Ol by
ANOVA). Serum vitamin D3 levels remained unchanged in
the group that did not receive UVB irradiation (time effect,
P > .l; treatment effect non-UVB- v UVB-treated groups,
P < .Ol).
Serum vitamin D2 levels increased markedly and signifi-
cantly after the intake of 50,000 IU vitamin Dz orally at all
measured time points (Fig 1B). Addition of UVB irradia-
tion did not affect the elevation of serum vitamin D2 levels
(treatment effect, P > .1 v vitamin DZ alone by ANOVA).
Actual vitamin D2 concentrations reached peak values at 12
hours, decreasing slightly thereafter (time effect, P < .Ol
by ANOVA). In the UVB alone group, the levels of serum
vitamin D2 remained undetectable throughout the study
period (treatment effect, P < .Ol v vitamin D-treated
groups).
Measurements of 25hydroxyvitamin D, 1,25dihydroxyvi-
tamin D, and DBP did not show significant differences
throughout time or between treatment groups for any of the
analytes (P > .l; Fig lC, D, and E).
DISCUSSION
The present report documents that formation and re-
lease of vitamin D3 from the skin proceed unaltered in the
presence of a marked elevation of the serum vitamin Dz
level. Thus, within a concentration range that far exceeds its
physiologic levels, increased ligand occupancy of carrier
vitamin D protein is not a limiting factor for the transfer of
vitamin D3 from the skin into the blood.
In contrast to previous studies that tested only the effect
of oral doses of vitamin D2 and/or vitamin D3 on its own
metabolism,5J3 the present study used a more physiologic
approach by stimulating cutaneous vitamin D3 synthesis
with UVB radiation and providing vitamin Dz through the
oral route. The administered dose of vitamin D2 was high
enough to produce blood levels that have been previously
shown to correctly identity intestinal malabsorption syn-
dromes in five of seven patients8 Regarding the intensity of
MATSUOKA ET AL
UV irradiation used to stimulate synthesis of vitamin Ds,
our previous work with this UVB dose has demonstrated
that untanned whites exhibit a consistent serum response.9
In contrast, the vitamin D3 response to UVB is abolished in
sunscreen usersll and is attenuated in heavily pigmented
human racial groupsr4 and suntanned individuals.‘5 We
must note that the present study did not document the
increases in levels of active vitamin D metabolites that
would be anticipated from the elevations of serum vitamin
D levels, but this is most likely a consequence of the short
period evaluated.
As a fat-soluble substance, vitamin D present in plasma
circulates almost totally bound to proteins; the free vitamin
D fraction is practically negligible, and methodology for its
direct measurement is currently not available. Notwithstand-
ing, the free forms of active metabolites of vitamin D may
be functional in some in-vitro and in-vivo assay systems.r6-r9
DBP is a plasma protein with a high affinity for vitamin Dzo
that could be involved in its removal from the skin.
However, hepatic uptake of vitamin D, presumably a
receptor-mediated process, is greatest when vitamin D3 is
presented on low-density lipoprotein or chylomicron rem-
nants, and lowest when it is presented on DBP.21 DBP has
structural homology with albumin, binds free fatty acids and
actin,22l23 and provides a very large carrying capacity for
vitamin D and its metabolites, suggesting a cutaneous
function for this protein as the explanation for the present
results. Of note, while the levels of vitamin D2 observed in
these experiments were far below the total sterol-binding
capacity of DBP in serum,24 vitamin D intoxication occurs
at sterol levels that do not saturate the sterol-binding
capacity of serum?
Determination of DBP levels was performed to exclude
spurious results due to abnormal levels of the protein.26,27 A
quantitative abnormality was excluded by the finding of
similar and unchanged DBP levels between and within
groups. Qualitative defects in DBP molecules are highly
improbable, since the main allele products, GC1 fast,
GC’ F’ow,GC 2, or the GC 1 anodal and GC 1 cathodal
isotypes, exhibit similar binding affinity for [3H]25-hydroxyvi-
tamin D3.28
The syndrome associated with vitamin D intoxication has
been ascribed to high circulating concentrations of 25
hydroxyvitamin D.16 Since neither 1,25_dihydroxyvitamin
Dl” nor vitamin D itself (present report) are capable of
regulating the cutaneous photosynthetic process, it is appar-
ent that vitamin D3 release from the skin will not be
suppressed in patients with coexisting vitamin D toxicity.
Therefore, patients with the latter complication or with
hypercalcemia due to sarcoidosis might benefit from sup-
pression of unneeded vitamin D formation by the topical
application of sunscreening agents on the sun-exposed skin
areasz9 A prospective controlled evaluation of this low-
cost, low-risk therapeutic intervention might then be advis-
able; perhaps such a trial could be extended to include even
unselected patients with chronic hypercalcemia.
To conclude, the carrier mechanism transporting newly
synthesized vitamin D3 from the basal layers of the epider-
mis to the dermal capillaries remains unaffected at circulat-
ing vitamin D concentrations that far exceed the physiologic
range.
CUTANEOUS RELEASE OF VITAMIN 03 1259

* = p<o.o1

** = p<o.os
22 ** 70
n = UVB

!
20
= 60 7
g 18
,E
2 16
z 50
‘; 14
a
c 12 D2 O” 40
._ .E:
f 10 E
2 30
5- 8 t
5

5 6 B 20
I n
5 4 +G
a 10
2
0
A Basal 12 hrs 24 hrs 36 hrs
0
6 Bay 36 hrs

26
1 _ 50
=
E E
Gl 24 &
5. ,o 47
0
.s 22
5
.Z
: 20
::
4
I” 16

N”

?
E 16

12 hrs 24 hrs 36 hrs

12 hrs 24 hrs 36 hrs

-375-
-f?
& 370-
3
365
.E
$j 360-

p’ 355-
!?
< 350-

; 345.

E 340.

‘i 335-
z
5 330-

Fig 1. Plasma response of healthy subjects to irradiation with UVB administered alone or in combination with a single oral dose of vitamin D2
[50,000 IU). The experimental protocol is outlined in panel A. Significant differences between groups were found only in serum vitamin Ds and Dz
concentrations (P < .Ol by ANOVA); location of significant differences by post-hoc, paired t tests is marked with asterisks. The ranges for the SEM
of analyte concentrations not differing significantly throughout the study period were as follows: serum 25-hydroxyvitamin D, 2 to 3 ng/mL;
serum 1,25-dihydroxyvitamin D, 1 to 5 pg /mL; and serum DBP, 15 to 30 pg /mL.
1260
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