Vol. 68, No.3
Printed in U.S.A.
68:157-160, 1977
Copyright \0 1977 by The Williams & Wilkins Co.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY,
PHOTOPROTECTION IN ERYTHROPOIETIC PROTOPORPHYRIA: MECHANISM
OF PHOTOPROTECTION BY BETA CAROTENE
ALAN
N.
MOSHELL, M.D.* , A.ND LoRING BJORNSON , PH.D.t
Departments of Dermatology (ANMJ and Medicine (LBJ, New Y ork University School of Medicine ,
New York , New York , U . S. A .
Erythropoietic protoporphyria is a genetic disease caused by the accumulation of protoporphyrin IX. This molecule absorbs 400-nm light and its presence is at times associated with
severe cutaneous photosensitivity. The only effective treatment for this disease is oral
administration of beta carotene.
Several possible mechanisms of photoprotection by beta carotene were investigated using
the photohemolysis of red blood cells as an in vitro model. Additional studies were done in an
in vivo model which involves lethal hematoporphyrin photosensitization of white mice. The
photoprotective effects of beta carotene were compared with those of alpha tocopherol, an
agent which possesses some but not all the properties that have been implicated in explaining the known effectiveness of beta carotene. In the photohemolysis model, both compounds
demonstrated partial protection. In hematoporphyrin-photosensitized mice, tocopherol
showed some protection at high doses, while beta carotene showed greater protection at
lower concentrations.
Although these results suggest that photoprotection was due to free radical scavenging or
singlet oxygen qu enching, properties common to both agents, they do not rule out the
possible role of 400-nm light absorption, a property of beta carotene alone.
Erythropoietic protoporphyria \EPP) is a dominantly inherited defect in porphyrin metabolism
resulting in the accumulation of protoporphyrin
IX 11 ,2], a wate r-insoluble porphyrin demonstrable in the patients' erythrocytes that strongly absorbs light of wavelength 400 nm. Clinically, these
patients have severe photosensitivity characterized by marked burning, pruritus, and edema following brief exposure to intense 400-nrn light ll3]. Avoidance is very difficult and most topical
preparations are of little value in protecting patients with EPP, but oral beta carotene has proved
very effective.
Several mechanisms for this protection have
been postulated. Beta carotene strongly absorbs
400-nm light, and simple absorption of light by
beta carotene in the stratum corneum may prevent penetration and symptoms. Porphyrin-induced photosens itivity in laboratory models is an
oxygen-dependent process 14- 6) involving an exManuscript received March 13, 1975: accepted for
publication October 15, 1976.
This investigation was supported in part by USPHS
Grants ES01041 , HE 06481, and Dermatology Training
Grant. AM 05326 13.
* Present address: National Naval Medical Center,
Department of Dermatology, Bethesda, Maryland
20014.
t Present address: Long Island College Hospital,
Special Chemistry Laboratory, 340 Henry Street,
Brooklyn, New York 11201.
Reprint requests to: Dr. A. N. Moshell, National
Naval Medical Center, Department of Dermatology,
Bethesda, Maryland 20014.
cited singlet oxygen intermediate l7]. Free radicals are generated following photoexcitation of
various porphyrin de rivatives in vitro 18-11]. Beta
carotene is very efficient in trapping singlet excited oxygen and quenching free radicals [7. 12].
Either of these properties may explain beta carotene's efficacy. Finally, beta carotene may act in
some undefined way to prevent or repair the phototoxic damage. Alpha tocopherol also traps free
radicals and quenches singlet oxygen [13], but
does not absorb 400-nm light. Alpha tocopherol
and beta carotene were studied and compared using in vivo and in vitro techniques previously
described [5,6,9, 14-20] in an attempt to evaluate
the mechanisms involved in photoprotection.
MA TERlALS AND METHODS
Photohemolysis Studies
Protection against. in vitro photohemolysis of red
cells from patients with EPP, and of normal red cells
photosensitized with hematoporphyrin was st.udied, using techniques previously described, with minor modifications ]15.16.21 ].
Preparation of erythrocytes. A hexane solution of alpha tocopherol (1.5 mg) or beta carotene (0.5 mg) was
evaporated to dryness with nitrogen in a 25-ml Erlenmeyer flask. Five milliliters of whole blood was added
to the flask and incubated for 6 to 12 hr in a water bath
at 37°C with constant shaking. After incubation, red
blood cell CRBC) concentrations of alpha tocopherol and
beta carotene ranged from 20 to 60 and 0.2 to 0.8 J.Lg/ml
of packed RBC, respectively. The analytical procedures
for these determinations are described elsewhere 122J.
Control erythrocytes were incubated identically, but in
157
158
Vol . 68 , No.3
MOSHELL AND BJORNSON
the a bsence of the protective agent. All cells were then
washed 3 times in normal saline, and suspended at a
1:800 dilution in either isotonic phosphate-buffered saline, pH 7.4 <EPP cells), or in identical buffered solution
to which 0. 1 mg% hematoporphyrin had been added
(normal cells).
Photoprotection assay. Three milliliters of the RBC
suspensions were placed in a 3-ml quartz cuvette 2.5 em
in diameter with an optical pathway of 1 em. The cuvettes were then exposed to 400-nm radiation from a
bank of 3 General Electric 20-watt bluelight fluorescent
tubes (spectral range 380-520 run) at a distance of 10 em
(output 696 J.LW/cm2 ) for various time periods. Hemolysis was determined by means of direct cell counts in a
hemocytometer befor e, during, and after irradiation.
Additional control samples were kept in the dark, and
hemolysis determined at the conclusion of each photohemolysis experiment.
Animal Studies
H ematoporphyrin phototoxicity in mice. The approximate dose of hematoporphyrin intr aper itoneally administered required to kill half the exposed animals 24
hr after exposure (LD;,,/1 day) was determined as described previously !21], except that 2 hr of bluelight
fluorescent tube (see above) exposure replaced the
blacklight fluorescent tubes.
Toxicity studies ofalpha tocopherol and beta carotene.
Using identical exposure conditions (2 hr bluelight at
10 em ) mice were exposed 24 hr after the intraperitoneal
administration of either agent in various dosages adjusted to maintain the total intraperitoneal volume at
or below 1 mi. No toxicity was observed at any of the
dosages used.
Photoprotection studies using beta carotene and alpha
tocopherol. Two hundred and thirteen mice were irradiated in groups as summarized in the Table. Fifty-nine
animals received beta carotene, 0.2 mg/gm body weight
in Emulfor intraperitoneally 24 hr prior to irradiation,
and hematoporphyrin. 0.1 mg/gm intraperitoneally, 30
min before irradiation. Fifty animals received alpha
tocopherol. 1 mg/gm in Emulfor intraperitoneally <high
dose) 24 hr prior to irradiation, and hematoporphyrin.
0.1 mg/gm intraperitoneally 30 min before irradiation.
Thirty animals received alpha tocopherol. 0.5 mg/gm in
Emulfor intraperitoneally tlow dose) 24 hr prior to irradiation, and hematoporphyrin. 0.1 mg/gm intraperi toneally 30 min before irradiation. In all animals, the
total volume injected intraperitoneally 24 hr before irradiation was approximately 0.5 ml. Seventy-four animals served as controls and received the vehicle, Ernulfor alone, 0.5 ml intraperitoneally 24 hr before irradiation, and hematoporphyrin, 0.1 mg/gm 30 min before
TABLE. Photoprotectiue effects of intraperitoneal
administration of alpha tocopherol and beta carotene in
hematoporphyrin-photosensitized mice after 2 hr of light
exposure
Photoprotective agent
Experimental
#
Tocopherol (1 mg/gm)1'
Tocopherol (0.5 mg/gmJ
Carotene (0.2 mg/gm)''
%
18/50 36
10/30 33
32/59 54
irradiation. All animals were exposed to light in groups
of 5 or 6 on successive days, and in each case at least one
group of control animals was exposed simultaneously
with the experimental groups. No anesthesia was required during either the intraperitoneal administration
of the various agents or during the exposure to bluelight. During the light exposure, the animals were
individually restrained under half-inch wire mesh
holders. Viability was assessed after 24 and 48 hr.
RESULTS
Photohemolysis
The results of various photohemolysis experiments, using both normal red cells photosensitized
with hematoporphyrin and red cells from patients
with EPP, are summarized in Figures 1-4. These
results are expressed as percent hemolysis per
unit time. The hemolysis was determined by direct
cell counts. The time course of hemolysis of the
controls was adjusted by varying the total irradiation time so that the hemolysis could be conveniently observed. Control studies, using cells
treated in a manner identical to the experimental
groups but without irradiation, showed no significant hemolysis. Similarly , normal cells, in the
absence ofhematoporphyrin but in the presence of
beta carotene or alpha tocopherol and irradiation,
showed negligible hemolysis. As noted in Figure 1,
the protection afforded by beta carotene against
photohemolysis was demonstrated by the prolongation of t he time required for 100% hemolysis of
the red cells from 20 to 24 hr. No signif1cant hemolysis was observed during the first 10 hr in either
the experimental or control red cells. As shown in
Figure 2, essentially the same data were obtained
when alpha tocopherol was used as the protective
agent. In Figures 3 and 4, when the dose of radiation was increased by lengthening the exposure
time from 20 min io 1 hr, complete hemolysis was
noted in less than 8 hr. In both studies, the alpha
tocopherol-treated cells were protected, as indicated by the prolongation of the time required for
complete hemolysis.
Animal Studies
The resuJts of the photoprotection studies in
mice are summa rized in the Table and Figure 5.
Survival in control animals, given only hematoporphyr in and Emulfor 1 day after exposure to
100
75
Control"
II /
#
%
11/50 22 o/C
9/24 38
15/54 28
C
ontrol/
Aarotono
II
ll'--~18----~19----~2.----~2~1----2~
2 ----2)----~2~
HOURS
Control animal received only Emulfor vehicle and
hematoporphyrin.
b Significant at 0.05 > p > 0.025 level by chi square.
' Significant at 0.005 > p level by chi square.
a
Pre. 1. Photoprot.ection by beta carotene against photohemolysis of EPP red cells. Summary of 3 experiments each using at least 6 groups. Data obtained by
direct cell counts. Cells exposed to light for 20 min.
March 1977
PHOTOPROTECTION IN EPP
100
?5
"u;
!:;
~
so
X
...
25
II
I I
18
19
20
21
22
2J
HOURS
FIG. 2. Photoprotection by alpha tocopherol against
photohemolysis of EPP red cells. Summary of 4 experiments each using at least 6 exeerimental groups. Data
obtained by direct cell counts. Cells exposed to light for
20 min.
100
?S
;;;
"'
~ so
..
25
HOURS
FIG. 3. Photoprotection by alpha tocopherol against
photo hemolysis of EPP red cells. Summary of 4 exper iments each using at least 6 groups. Data obtained by
direct cell counts. Exposed to light for 1 hr.
159
tion of holes in the cell membrane under 10 A in
size 117.1.
The red cell photoprotective agents discovered
in the studies have infrequently been tested in
animal experimen ts. A recent report by Harber et
aJ 121) demonstrated that ruthiothreitol and glycerol act synergistically to protect hematoporphyrin-photosensitized mice and inhibit the photohemolysis of red cells. In addition. beta carotene
was shmvn to protect hematoprophyrin-photosensitized mice prior to its very successful use in
pa tients with EPP 125].
In the present study, we were able to evaluate
and compare beta car otene a nd al pha tocopherol
not only in vitro in red cell photohemolysis, but
also in hematoporphyr in-photosensitized mice
studies.
Beta carotene contains 11 conjugated double
bonds, a nd shows significant absorption at 400 run.
It is an active free radical t rap and singlet excited
oxygen q uencher 112]. Here and in previously presented data, beta carotene has been demonstrated
to be effective in preventing t he deleterious effects
of porphyrin derivatives plus 400-nm ligh t in red
cell photohemolysis models, photosensitized laboratory animals, and EPP patients [14,19,25). Alpha tocopherol contains only 3 conjugated double
bonds, does not absorb 400-nm light (it has no
significant a bsorption above 310 nml, less avidly
quenches singlet excited oxygen, but is an even
more active free radical trap t han is beta carotene
(A. A. Lamola, personal communication) 113].
The similarity in the results of the photohemolysis experiments using beta carotene and alpha
bluelight, r anged from 22 to 38%- of the animals.
Beta carotene showed the best protection (54t.k- vs
27o/c) at a dose !0.2 mglgm) that was significantly
lower than either a lpha tocopherol dose tested.
Low doses !0.5 mg/gml of alpha tocopherol showed
no photoproteclion f33o/c) . High doses (1 mg/gml,
however, showed some protection (36%- vs 22o/cl.
There is an apparent discrepancy in the number of
control animals as listed in the Table and F igure 5
and as stated in Materials and M ethods. This
resulted from the processing of several experimental groups simuhaneou sly with a single control
group. Thus, the same animals may have served
as con trols for several but not all exper iments.
F1a. 4. Photoprotection by alpha tocopherol against
Animals wer e tabulated as controls only when photohemolysis of hematoporphyrin-photosensitized
processed simultaneously with the corresponding red cells. Summary of 4 experiments each using 6
Data obtained by direct cell counts. Cells exexper imental animals. The solubilizer (Emulfor) groups.
posed to light for 1 hr.
a lone h ad no protective effect. In the absence of
hematoporphyrin, none of th e animals s uccumbed.
Similarly, in the absence of exposure to 400-nm
light, no animals succumbed.
..
DISCUSSION
Previous investiga tions have demonstrated the
value of red blood cells from EPP patients and
photosensitized normal red cells in the study of the
mechanism of cell membrane damage from 400-nm
radiation 14,8,14,16- 18,20.23). This photohemolysis is oxygen dependent 14-6], associated with
free radical l8-11) and per oxide !6,8,24) formation,
and th e observed cell destruction is due to colloid
osmotic hemolysis 114,17] initiated by the forma-
SURVIVAL
)0
20
10
AW'!IA
TOCOPI!£ROL
l. 0 ..y,
AlJ'IIA
TOCOI'l!EIIOL
o.s ..Uc
.F~a. 5. _
P hotoprotective effects of intraperitoneal adm1mstrat10n of alpha tocopherol and beta carotene in
hematoporphyrin-photosensitized mice after 2 hr of
light exposure.
160
Vol . 68, No.3
MOSHELL AND BJORNSON
tocopherol as protective agents would suggest that
free radicals do take part in the reaction and that
quenching t hese free radicals can inhibit membrane damage [21). These results also suggest that
photon absorption of 400-nm light is not the only
mechanism by which beta carotene may exert a
protective effect.
The animal experiments demonstrate beta carotene to be a more effective photoprotective agent
than alpha tocopherol. This implies that free radical scavenging is not the only mechanism of beta
carotene's photoprotection , for were this the case,
one would expect alpha tocopherol, the more avid
free radical scavenger, to be superior . However,
there are several other possible explanations for
this result. In this animal model, the tested agents
were administered intraperitoneally. Active drug
must reach the site of photo toxic damage in order
to be protective. Major differences in absorption,
distribution. or metabolism could alter the concentration of active drug at this site. overshadowing
any differences in free radical scavenging or singlet oxygen quenching. Some observations as to
the differences in transport and storage of these
two agents in man a ppears in another report by
our group l22].
The available data are insufficient to comment
on the effect of drug localization on photoprotection, although the drug must be in close proximity
to any short-lived free radicals or excited singlet
oxygen generated in the phototoxic reaction in
order to prevent irreversible damage. Simple a bsorption of 400-nm light by beta carotene seems an
unlikely explanation for the prime mechanism of
photoprotection, as the patients are ra rely grossly
carotenemic at doses that prove protective, and
topical application is totally ineffective as it is
rapidly bleached upon exposure to intense light
l25]. Thus, it seems likely that both free radical
trapping and singlet excited oxygen quenching are
involved in beta carotene's photoprotection in
EPP, a lthough the relative importance of these
two properties cannot be evaluated at present, and
th e simple absorption of 400-nm light. a lthough
unlikely, cannot be ruled out.
REFERENCES
1. Magnus lA, Jarret A, Prankerd TAJ. Rimington D:
Erythr opoietic protoporphyria: new porphyria
syndrome with solar urticaria due to protoporphyrinaemia. La ncet 2:448-451, 1961
2. Magnus IA: Photobiological aspects of porphyria.
Proc R Soc Med 61:196-198, 1968
3. Harber LC: Photosensitivity associated with disorders of porphy rin metabolism. Med Clin North
Am 49:581-592, 1965
4. Suurmond D, Van Steveninck J , Went LN: Some
clinical and fundamental aspects of erythropoietic protoporphyria. Br J Dermatol 82:323-328,
1970
5. Goldstein BD, Hsu J , Harber LC: Photoh emolysis
in erythropoietic protoporphyria (abstr). Blood
34:856, 1969
6. Hsu J , Goldstein BD, Harber LC: Photoreactions
associated with in vitro hemolysis in erythropoietic protoporphyria. Photochem Photobiol 13:6777' 1971
7. Fugimori E. Tavla M: Light-induced electron trans-
8.
9.
10.
11.
12.
13.
14.
15.
16.
fer between chlorophyll and hydroxyquinone and
the effect of oxygen a nd beta-carotene. Photochem Photobiol 5:887, 1966
Ludwig GD, Bilheimer D, Iverson L: Mechanism of
photohemolysis in erythropoietic protoporphyrin
and r elationship to tocopherol (vitamin E) deficiency (abstr). Clin Res 15:284, 1967
Goldstein BD, Harber LC: Erythropoietic protoporphyria: lipid per oxidat ion and red cell membrane
damage associated with photohemolysis. J Clin
Invest 51:892-902, 1972
Einstein KK, Wang JH: Conver sion of light to
ch emical free energy. I. Porphyrin-sensitized
photoreduction of ferredoxin by glutathione. J
Bioi Ch ern 244:1720-1728, 1969
Mauzerall D, Feher G: A study of the photoinduced
porphyrin free radical by electron spin resonance. Biochim Biophys Acta 79:430-4.32. 1964
Foote CS. Denny RW: Chemistry of singlet oxygen.
VII. Quenching by beta-carotene. J Am Chern
Soc 90:6233-6235, 1968
Tappe! AL: Vitamin E as the biologic lipid antioxidant. Vitam Horm 20:493-510, 1962
Schothorst AA. Van Steveninck J, Wen t LN, Suur mond D: Photoporphyrin-induced photohemolysis in protoporphyria and in normal red blood
cells. Clin Chim Acta 28:41-49, 1970
Harber LC, Fleischer A, Baer RL: Photoh emolysis
associated with protoporphyrinaemia. J lnvest
Dermatol 42: 483-485, 1964
Ha rber LC. Fleischer AS. Baer RL: Erythropoietic
protoporphy ria and photohemoly!\i!\. .1 AM A
189:191-194, 1964
17. Fleischer AS, Ha rber LC, Cook JS. Baer RL: Mech-
anism of in vitro photohemolysis in erythropoietic protoporphy ria <EPPl. J Invest Dermatol
46:505-509, 1966
18. Peterka ES, Runge WJ, Fusaro RM: Erythropoietic
protoporphyria. ffi. Photohemolysis. Arch Dermatol 94:282-285, 1966
19. Mathews MM: Effect of beta-carotene against lethal photosensitization by hematoporphyrin. Nature (Lond) 203:1092, 1964
20. Kahn G, Curry MC: Ultra violet light protection by
several new compounds. Arch Dermatol 109:510517, 1974
21. Harber LC, Hsu J , Hsu H , Goldstein BD: Studies of
photoprotection against prophy r in photosens itization using ditniothreitol and glycerol. J Invest
Derma to! 58:373-380, 1972
22. Bjornson L. Kayden H, Miller S. Mosh ell AN: The
transport of alpha tocopherol and beta-carotene
in human blood. J Lipid Res 17:343-352, 1976
23. Kaplowitz N , Javitt N , Harber LC: Isolation of
erythrocytes with normal protopor2_hyrin levels
in erythropoietic protophyria . N Eng! J Med
278:1077-1081, 1968
24. Lamola AA , Yamane T, Trozzolo AM: Cholesterol
hydroperoxide formation in red cell membranes
and photohemolysis in erythropoietic protoporphyna. Scie nce 179:1131-1133 , 1973
25. Mathews-Rot h MM , Pa thak MA , Fitzpatrick TB,
Harber LC, Kass EH: Beta-carotene as a photoprotective agent in erythropoietic protoporphyria. Trans Assoc Am Physicians 83:176- 184,
1970