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\s=b\ Fluorescence is a feature of elastin and collagen, able to detect changes in the integument that result
both major compounds of human dermis that are altered from solar ultraviolet radiation exposure but do not
by age and photoexposure. We studied the intrinsic appear to relate to chronologic aging. Thus, skin
fluorescence of skin in vivo in 28 human volunteers to autofluorescence may serve as a marker for photoag¬
determine whether photoaging and chronologic aging of ing.
the skin could be evaluated by this noninvasive tech-
SUBJECTS AND METHODS
nique. We demonstrate that the excitation of skin auto-
fluorescence by laser ultraviolet radiation yields charac- Twenty-eight volunteers consented to participate in a
teristic tissue fluorescence spectra that are unrelated to study approved by the Human Studies Subcommittee of
age, pigmentation, or skin thickness. The differences in the Veterans Administration Medical Center, West Haven,
skin autofluorescence appear to be related to photoex- Conn. The subjects were chosen on the basis of age (Table
posure. Thus, laser-induced fluorimetry, a noninvasive 1) to represent three stages in the aging process. Group 1
consisted of children with a mean age of 3 years without a
technique, may be adaptable as a marker of photoag-
ing. history of excessive sun exposure. Group 2, with a mean
age of 30 years, represented young adults who have had
(Arch Dermatol 1988;124:1514-1518) routine exposure to sun in the northeastern United States
for most of their lives. Group 3, with a mean age of 63
years, included subjects with outdoor occupations who had
Fluorescence is natural phenomenon of many
a
compounds13 in which light is absorbed at one
clinical evidence of solar damage.
Patient pigmentation was categorized according to the
wavelength and emitted at a higher wavelength. criteria developed in the cooperative trials of psoralens
Individual chemical compounds have characteristic and ultraviolet A therapy.'3 The classification is summa¬
absorption and emission spectra but to date in vivo rized in Table 1. The study was conducted between the
months of November and March during which no subject
fluorimetry has had limited diagnostic use and has had significant tanning secondary to ultraviolet radiation
not been applied to the skin. The diagnostic potential
of the fluorescence of malignant tissue after treat¬ exposure.
All studies were performed in the Cardiac Laser
ment with hematoporphyrin derivative has been Research Laboratory at the Veterans Administration
reported.4 There have also been reports of fluores¬ Medical Center.
cence in dental caries5 and in atherosclerotic and Skin fluorescence was induced by a helium-cadmium
normal arteries.6-7 Fluorescent compounds in the skin laser (Omnichrome model 356-5MS) emitting radiation at
include elastin8 and collagen.9 In addition, it is known 325 nm (ultraviolet light in the B range [UVB]) at 0.5 to 5
that with age10 and exposure to ultraviolet radia¬ mW continuous-wave power (Fig 1). The laser was coupled
tion,11 these molecules may be altered. The mecha¬ to the central fiber of a coaxial fiberoptic probe (EOTec
nism by which ultraviolet radiation ages skin is Corporation, West Haven, Conn) and placed 3 mm from the
skin surface over the buttocks, axillae, left temple, fore¬
unknown, and, to a large extent, measurement of this head, and dorsa of the hands after wiping with alcohol.
effect has been limited to in vitro qualitative micro¬
Sun-protected sites included the buttocks and axillae.
scopic analysis.12 This study defines the fluorescent Sun-exposed areas included the left temple, forehead, and
properties of skin in vivo and determines whether dorsa of the hands. Each of these sites represented region¬
age or sun exposure altered the spectral pattern of al variation in dermal thickness.'4
fluorescence. By inducing fluorescence of skin and Tissue fluorescence was transmitted by the coaxial fiber
evaluating the fluorescence spectra, we have been bundle, focused into a spectrograph (Jarrell Ash Monospec
27), spectrally dispersed, and imaged onto an optical
multichannel analyzer (OMA, Princeton [NJ] Instruments
Inc). The OMA consisted of a linear diode array of 1024
Accepted for publication June 3, 1988. discrete elements coupled to a microchannel plate intensi¬
From the Departments of Dermatology (Drs Leffell and Mil- fier. The detector simultaneously detected the fluorescence
stone) and Medicine, Section of Cardiology (Dr Deckelbaum and intensity in the wavelength range 300 nm to 700 nm. Ten
Mr Stetz), Yale University School of Medicine, New Haven, and 33-ms scans per reading were averaged. The OMA system
the Veterans Administration Medical Center, West Haven,
was spectrally calibrated using a mercury vapor lamp and
Conn.
Presented in part at the American Society for Dermatologic radiometrically calibrated using a National Bureau of
Surgery Annual Meeting, Monterey, Calif, April 15, 1988. Standards traceable calibrated white light source (Uptron-
Reprint requests to Department of Dermatology, Yale Universi- ics Model 245A). The latter calibration corrects for any
ty School of Medicine, 333 Cedar St, New Haven, CT 06510 (Dr nonuniformity in the spectral sensitivity of the system.
Leffell). The fluorescence spectrum was digitized using the diode
Coaxial Fiberoptic
Catheter
Fiber Bundle
CRT Display
Achromatic Lens
/\ Spectrograph
Controller
Computer
Fig 1.—Laser apparatus for induction of skin fluorescence (see "Subjects and Methods" section for detailed
explanation).
UV indicates ultraviolet light and CRT, cathode ray tube.
exposed skin has a different inducible fluorescence those patients with darker pigmentation. The pres¬
pattern than that of solar-protected skin. The simi¬ ence of melanin in the epidermis may attenuate the
larity of solar-exposed and solar-protected skin spec¬ fluorescence intensity at 390 nm thus altering the
tra in the youngest age group is consistent with the R390/429. Experimentally, the pattern of the fluo¬
fact that these children have not yet had much sun rescence spectra of those patients with skin types III
exposure. Conversely, subjects in groups 2 and 3 and IV were similar to those of patients with less
showed significant differences in the fluorescence of melanin although, as expected, the overall intensity
buttocks and axillae when compared with the temple of the curves was diminished. The observation of
or other solar-exposed areas. That the differences R390/429 typical for solar-exposed and solar-pro¬
observed in groups 2 and 3 were not simply a tected in in vitro skin lacking melanin suggests that
function of regional variation in skin thickness is this system may be sufficiently sensitive to detect
confirmed by the fact that spectra of the buttocks solar-induced changes despite the potential reab-
were similar to those of the axillae—another sun- sorption artifact related to this pigment.
protected region of the body with different morphol¬ The anatomic source of the observed in vivo fluo¬
ogy.14 In addition, the fluorescence spectra of skin rescence remains to be determined. Studies1' with
from the shoulders were similar to those of the human cadaveric aorta suggest that fluorescence,
forehead despite the consistently thicker dermis of when induced by ultraviolet radiation, appears to
the former sites.16 derive from tissue at a depth of 150 nm to 200 nm.
The fact that the greatest difference in fluores¬ While aorta has a different composition than human
cence exists between groups 1 and 2, when clinically skin and the fluorescence spectrum is different, this
one might have expected it to occur between groups 2 observed depth of fluorescence appears to correspond
and 3 suggests that the changes in dermal composi¬ with preliminary studies performed by us in human
tion reflected by fluorescence activity may be sub- mastectomy skin in vitro. Since human epidermis is
clinical, occurring early in the evolution of photoag¬ 75 to 100 nm thick in all areas except the palms and
ing. soles,18 and the dermis varies in thickness from 700 to
The majority of our subjects were of skin types I 1500 nm,"' it is possible that the in vivo induced
and II and tended not to tan. They therefore may fluorescence in human skin derives from excited
have had more severe photoaging compared with compounds in the papillary dermis. Interestingly, it
2000-
3
sí
6000 •Desmosine
---Collagen
is in the papillary dermis that important photo- 5000 -Elastin
induced changes in elastin and collagen are noted.19
The quantitative fluorescence data reported here 4000
appear to relate more to photoinduced changes than
3
>í
to chronologic aging but the precise source of the « 3000-
fluorescence that we have observed remains to be c
Sic
elucidated. In light microscopy, it is often the colla¬
2000
gen that is thought to autofluoresce. Importantly,
collagen, despite its slow turnover rate in adults does
change with age.2021 With photoexposure, the amount 1000
of mature collagen decreases.22 Elastic tissue in skin12
and aorta23 changes with age and in the skin with 0
300 350 400 450 500 550 600 650
exposure to ultraviolet radiation.24 The changes in 700
elastic tissue in skin due to ultraviolet radiation are Wavelength, nm
different from those due to chronologic age alone.12 Fig 4.—In vitro fluorescence spectra of elastin, desmosine, and
Since it is known that ultraviolet-induced changes collagen. All compounds were studied in solid, nonsolubilized
in elastin are different from those due to age alone, it state.
is interesting to consider that components of elastic
tissue may be responsible for the observed changes
in the fluorescence pattern. While photodamaged therefore deserve further study. LaBella and Lind¬
skin may contain more elastin than sun-protected say23 suggested that the major fluorophore in elastin
skin, the biochemical nature of this elastotic materi¬ was probably a cross-linking agent because fluores¬
al is different from elastin itself.24 The possibility cence was greatest in those peptides resistant to
that changes in fluorescence pattern due to sun degradation by elastase. One of the major cross-
exposure relate to elastin in one form or another may linking amino acids of elastic tissue, desmosine, has