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The In Vitro Photochemistry of Urocanic Acid
Nicole Haralampus-Grynaviski, Carla Ransom, Kerry Hanson, John D. Simon
Department of Chemistry, Duke University, Durham, NC 27708
The epidermal chromophore urocanic acid (UA) has received considerable attention because of its immunomodulatory behavior.UA exists as its trans isomer (t-UA, approximately 30 mg/cm2) in the uppermost layer of the skin (stratum corneum). t-UA is formed as the cells of the second layer of skin become metabolically inactive.During this process, proteins and membranes degrade, histidine is released, and histidase (histidine ammonia lyase) catalyzes the deamination of histidine to form t-UA.10 t-UA accumulates in the epidermis until removal by either the monthly skin renewal cycle or sweat.Upon absorption of UV light, the naturally occurring t-UA isomerizes to its cis form, c-UA, (Scheme I), which absorbs in the UV as well. Because DNA lesions (e.g., pyrimidine dimers) in the lower epidermis can result from UV-B absorption, initial research proposed that t-UA acted as a natural sunscreen absorbing UV-B in the stratum corneum before the damaging rays could penetrate into lower epidermal zones. However, in 1983, Dr. Ed De Fabo and Dr. Frances Noonan showed that the action spectrum for immune suppression of contact hypersensitivity overlapped that of the absorption spectrum for t-UA. Since then, researchers have found that c-UA suppresses contact hypersensitivity and delayed hypersensitivity, reduces the Langerhans cell count in the epidermis, prolongs skin-graft survival time, and affects natural killer cell activity. c-UA is now postulated to act as a mediator for UV-induced immunosuppression. Photochemically, t-UA is an unusual molecule because the isomerization quantum yield for formation of c-UA and thereby the subsequent accumulation of c-UA is wavelength dependent. The isomerization efficiency peaks at 310 nm (the red edge of the absorption spectrum) with F = 0.49, and is reduced near the absorption maximum (at 266 nm) with F = 0.05. Because the accumulation of c-UA is of immunological concern, our work has focused on elucidating the mechanism for the wavelength-dependent isomerization of t-UA under representative physiological conditions (e.g., pH 7.2, the average pH of the cell, and pH 5.5, the average pH of sweat). We have definitively shown that the steady-state absorption spectrum of t-UA is comprised of overlapping absorption bands to different excited electronic states. In the course of our research, we discovered that t-UA exhibits a wavelength-dependent efficiency for sensitizing the formation of singlet molecular oxygen. This observation forms the foundation for our current studies on t-UA.
At the start of our work a major unsolved question concerning the basic photoreactivity of t-UA was why is the photochemistry of t-UA wavelength dependent? A second issue that interested us was whether there are photoreactions of UA that had not been determined. We undertook a series of spectroscopic and kinetic measurements to address these issues and the results of our studies are summarized below. We conducted experiments at pH 7.2 and pH 5.5, which represent the average pH of the cell and sweat, respectively. The pKa of the tertiary nitrogen atom on the imidazole (pKa = 5.8) lies within this range. This nitrogen is protonated at pH 5.5 and deprotonated at pH 7.0.
Excitation of either a pH 7.2 or a pH 5.5 t-UA solution at 266 nm produces
a long-lived triplet excited state. Using femtosecond time-resolved spectroscopy,
we were able to directly observe the intersystem crossing from the singlet-state
manifold to the triplet-state manifold (see Figure 1). This intersystem
crossing occurs with a half-life of 7 picoseconds, and explains why the
emission quantum yield from electronically excited t-UA is so small (on
the order of 0.00001). Using photoacoustic calorimetry we determined that
the lowest triplet state lies 230 kJ mol-1 above the ground state, in agreement
with the value determined by Morrison and coworkers from triplet sensitization
studies. Furthermore, the kinetic and calorimetic data indicated that the
triplet state of t-UA does not lead to isomerization.
Figure 1: The transient-absorption dynamics of t-UA at 340 nm following
excitation at 266 nm. The probe wavelength of 340 nm detected the S1 to
SN absorption. The dynamics reflect the time-dependent population of the
first excited singlet state. The decay corresponds to the intersystem crossing
kinetics to the excited triplet state.
Excitation of a pH 7.2 solution of t-UA at 310 nm leads to isomerization.
Femtosecond time-resolved studies show that the entire isomerization event
(excited state isomerization, surface crossing back to the ground-state
potential, and vibrational relaxation of the ground state molecule) occurs
with a half-life of 10 picoseconds, see Figure 2. Assuming an isomerization
quantum yield of 0.5 at this excitation wavelength, photoacoustic calorimetry
data show that the cis isomer is less stable than the trans isomer by 40
Figure 2: The transient-absorption dynamics of t-UA at 310 nm (pH 7.2
solution) following excitation at 310 nm. The dynamics reveal the repopulation
of the ground electronic state of the molecule. The quantum yield for isomerization
is 0.5 at this excitation wavelength and so the recovery time of 10 picoseconds
is an upper bound for the time constant associated with the excited-state
Excitation of a pH 7.2 solution of t-UA at 355 nm causes both isomerization and the production of a long-lived triplet state. No ultrafast measurements have been performed at this wavelength but a complete set of photoacoustic calorimetry data have been recorded. We believe that this excitation wavelength is resonant with both the S0 to S1 transition and the forbidden S0 to T1 transition. We will return to this topic below.
In the presence of molecular oxygen at both pHs studied, the triplet
state of t-UA transfers its energy to molecular oxygen, thereby forming
singlet molecular oxygen. Figure 3 shows the effect of molecular oxygen
on the photoacoustic signal recorded following 355 nm excitation of a pH
7.2 solution of t-UA. We have confirmed that the species created is O2(1Dg),
by directly recording the emission kinetics of the 1Dg
transition. Similar results are observed for 266 nm excitation of t-UA.
This energy-transfer quenching reaction occurs significantly faster than
that predicted for a diffusion-controlled process. The quantum efficiency
for this quenching reaction determined from photoacoustic calorimetry experiments
is also higher than can be explained by a bimolecular diffusive reaction.
We conclude that t-UA forms a ground-state complex with molecular oxygen.
Figure 3: The photoacoustic signals following 355 nm excitation of a
pH 7.2 solution of a reference, of an argon saturated t-UA solution, and
of an oxygen saturated t-UA solution. The amplitude of the reference signal
corresponds to the complete release of the photon energy as heat. The t-UA
in argon releases less heat than the reference, reflecting the formation
of a long-lived triplet. In the presence of oxygen, t-UA releases more
heat than in the absence of oxygen, consistent with the transfer of energy
from the triplet state of t-UA to the lower-energy first excited singlet
state of O2. Similar results are observed at 266 nm
An important and contentious question is whether the different behavior observed at 266 nm and 310 nm are due to the photoreactions of differing rotamers of t-UA or if excitation at these wavelengths accesses different electronic states of t-UA. This has been argued extensively in the literature and has not been resolved. However, for the reasons discussed above, the answer is needed to fully understand the relevance of in vitro t-UA studies to its in vivo activity. To answer this question we conducted the following two-pulse photoacoustic measurement. The photoacoustic signals for a sample of t-UA (10-4 M, pH 7) was collected following excitation at 266 and 310 nm (individually). Then the same sample was excited at 266 nm and after a delay of 9 nanoseconds, the volume traversed by the 266 nm light pulse was exposed to a laser pulse at 310 nm. The resulting photoacoustic signal caused by the two-pulse excitation sequence was then collected. Now consider what type of data is expected from these measurement for the two models proposed for the wavelength-dependent behavior of t-UA. If the molecules absorbing at 266 nm and 310 nm are different rotamers of t-UA, then the amplitude of the photoacoustic signal for the two-pulse experiment would simply be the sum of the amplitudes of the one pulse experiments. However, if the excitations at 266 nm and 310 nm access different electronic states of the same population of molecules, then the photoacoustic signal for the two-pulse experiment would be less than the sum of the amplitudes of the one pulse experiments. This must be the case because we independently know that excitation at 266 nm produces a long-lived triplet that lasts significantly longer than 9 nanoseconds; therefore the molecules excited at 266 nm do not return to the ground state by the time the 310 nm pulse arrives and so the optical density of the sample at 310 nm would be reduced, leading to a decrease in the photoacoustic signal. This expected difference between the signal amplitude in the two-pulse experiment and the sum of the amplitudes of the one pulse experiments can be calculated given the laser spot size, sample concentration, and pulse intensities. For the conditions of our experiment we expected to observe a 11% difference if this later interpretation is correct. Experimentally we find that the signal amplitude in the two-pulse experiment is 12 ± 3% less than the sum of the amplitudes of the one pulse experiments. Thus, we can conclude that the wavelength-dependent behavior of t-UA arises from absorption to different electronic states.
In addition we can use the above spectroscopy and kinetic data to explain why different studies have reported varying action spectra for the immune suppression of photoexcited t-UA, and thereby also quantify the conditions which are optimum for in vivo measurements. This is a clear case where a quantitative understanding of the molecular photoreactions was necessary in order to understand measurements such as action spectra.
Does urocanic acid play a role in the photoaging of skin?
Most of the visible signs of aging result from chronic exposure of the
skin to ultraviolet radiation. Unlike chronologically aged skin that results
from a general atrophy and a gradual decline in the production of the dermal
matrix, UV-A (320-400 nm) photoaged skin is characterized by a gross increase
in the elastic fibers (elastin, fibrillin, desmosine) of the skin replacing
the collagenated dermal matrix (elastosis), an increase in glycosaminoglycans,
collagen cross-linking, epidermal thickening, and an increase in the number
of dermal cysts. The deep lines, leathered appearance, and the sagging
of the skin surface typically associated with "old age" are thought to
result from UV-induced photodamage to the skin and occur over the course
of a lifetime. Although obvious differences between photoaged and chronologically
aged skin exist, the anatomic basis of the visible signs of photoaging
is not fully understood. Absorption of UV-A must induce photobiologic effects
within the skin that lead to the visible and histological differences of
photoaged skin, and although the mechanisms by which UV-A induced photodamage
occur have not been completely determined, reactive oxygen species are
postulated to play a role. The natural shift toward a more prooxidant state
in chronologically aged skin could then be exacerbated by absorption of
UV-A radiation by endogenous chromophores like NADH/NADPH, tryptophan,
and riboflavin, which then sensitize the formation of reactive oxygen species.
Solar radiation has been shown both to reduce the antioxidant population
in the skin and to sensitize the production of reactive oxygen species
such as singlet oxygen, hydrogen peroxide, and the superoxide anion, increasing
the potential for reactions like the oxidation of lipids and proteins that
influence the degree of cross-linking between collagen and other proteins
within the skin. The first step towards identifying the chromophore(s)
responsible for physiological changes such as those seen in photoaged skin
is to match the action spectrum for the physiological change with the absorption
spectrum of the chromophore. Once such a comparison is made, the pathways
that lead to the physiological change can be unraveled. A quantitative
comparison between photoaging action spectra and the absorption spectra
of endogenous chromophores in the UV-A has remained elusive. Here, we focus
upon the UV-A absorption of t-UA between 310 nm and 351 nm and the role
of t-UA in the photoaging of the skin. Examination of Figure 4 shows that
this region corresponds to the tail of the absorption spectrum.
Figure 4. The absorption spectrum of naturally occurring trans-urocanic
acid, pH 7.2. The broad and structureless spectrum masks the complicated
wavelength-dependent photochemistry exhibited by the chromophore. Wavelength-dependent
isomerization between t-UA and photoinduced cis-urocanic acid results from
the presence of weakly coupled distinct electronic states between 266 nm
and 400 nm . In addition, as shown in this report, there is a weak absorption
band of t-UA from 320 to 360 nm. This region of the spectrum is enlarged
in the inset.
Irradiation throughout this spectral region has recently been
shown to induce photoisomerization of t-UA to its cis isomer (c-UA). Excitation
of a pH 7.2 solution of t-UA at 310 nm results in photoisomerization where
the quantum yield for the conversion of t-UA to c-UA at this wavelength
is approximately 0.49. The above described pulsed laser photoacoustic spectroscopic
results revealed that essentially all of the incident photon energy is
released as heat on the subnanosecond time-scale at this excitation wavelength.
That result is consistent with time-resolved absorption studies that indicate
that the photoisomerization reaction takes place within the singlet manifold
of electronic states in less than 100 ps. Intersystem crossing from the
initially populated excited singlet state to the triplet-state manifold
does not compete with isomerization to c-UA following irradiation at 310
nm. We now use photoacoustic spectroscopy to study the UV-A portion of
the t-UA absorption spectrum from 310 nm to 351 nm. We find that the photoacoustic
signal in this region of the spectrum is wavelength dependent. Starting
at 320 nm, the amount of energy retained by the molecule increases with
increasing excitation wavelength. The energy retained reaches a maximum
around 340 nm and then decreases with increasing excitation wavelength.
Figure 5 shows the efficiency spectra for triplet formation or heat
retained as measured by photoacoustics from 310 to 350nm. Both isomers,
trans and cis urocanic acid, were irradiated at each wavelength under argon
and oxygen saturated solution in pH 7.2 buffer and showed similar behavior.
The irradiation wavelengths were generated using a Spectra Physics millenium/tsunami/spitfire/opa
femtosecond laser system (1kHz, 5mW).The 10-3 M t-UA samples had an optical
density of between 0.05-0.1, and at each irradiation wavelength the t-UA
optical density was matched to within 3% of the optical density of the
standard bromocresol purple.
Because of the low extinction coefficient for t-UA for wavelengths greater than 370 nm, the photoacoustic technique could not be used to determine if energy is retained by t-UA following excitation in this region. Recent studies establish that photoisomerization occurs following excitation of t-UA in the UV-A (320 nm - 400 nm). The data obtained in the present study reveal that the energy retained following excitation at 340 nm is almost three times the energy difference between the two isomers of UA (110 kJ mol-1 vs. 40 kJ mol-1). Consequently, we can conclude that photoisomerization is not the sole photochemical process that is initiated by UV-A exposure. To account for this observed energy storage, a long-lived intermediate must be formed. The absence of any time delays between the sample and reference photoacoustic waves indicate that this intermediate is formed on the subnanosecond time scale and that its lifetime is longer than the instrument response time, hundreds of nanoseconds. Therefore its decay kinetics do not contribute to the photoacoustic signal. The absence of any photochemistry except isomerization contributing to the photoacoustic signal requires that this intermediate be a long-lived excited state of the molecule, and so it is reasonable to conclude that this state is an excited triplet state of t-UA, vide infra. Because excitation at 310 nm accesses an electronic state that only leads to isomerization, it is also reasonable to conclude that excitation between 320 nm and 351 nm populates two distinct but overlapping excited electronic states: the same state that leads to isomerization at 310 nm and an electronic state that either directly or by intersystem crossing populates the long-lived excited triplet state. As a result, the wavelength-dependent photoacoustic measurements determine the action spectrum for triplet state formation for excitation of t-UA in the UV-A. Figure 6A shows this action spectrum as determined from these photoacoustic measurements. This action spectrum is in excellent agreement with the action spectrum for photoinduced sagging of mouse skin, Figure 6B (reproduced from the work of Bissett, D. L.; Hannon, D. P.; Orr, T. V. Photochem. Photobiol. 1989, 57, 763).
Figure 6. Graph A. The line shape of action spectrum for triplet formation
(and singlet oxygen generation) for trans-urocanic acid in a deoxygenated
pH 7.2 solution (solid line). The action spectra is obtained by multiplying
the effciency spectra times the urocanic acid absorbance.The line-shape
was determined by fitting a Gaussian line-shape function to the data collected
from 310 nm to 360 nm (data points) Graph B. The measured in vivo action
spectrum for the photosagging of mouse skin taken from the results of Bissett
and coworkers. The in vivo action spectrum mimics the action spectrum shown
in Graph A.
Given the similarity between the two spectra shown in Figure 6, we undertook in vitro experiments to elucidate the photochemical event that mediates this physiological response. Specifically, pulsed laser photoacoustic data following UV-A excitation of t-UA were collected both in deoxygenated and oxygen-saturated solutions. In the presence of oxygen, the identical line shape is observed as seen in Figure 5. However, when compared to the deoxygenated sample, the oxygen-saturated solution retained significantly less heat. These data showed that oxygen quenches the long-lived state of t-UA generated from UV-A irradiation. Quenching reactions of triplet states of organic molecules by O2 are common, and energy transfer from an excited triplet state to O2 leads to the formation of O2(1Dg). We have confirmed that O2(1Dg) is generated following the UV-A excitation of t-UA at 351 nm by measuring the "forbidden" 1Dg to 3Sg emission of O2 (31). This result confirms the above assignment that the long-lived excited state of t-UA that is formed upon UV-A irradiation is an excited triplet state of the molecule. From the similarity of the spectra shown in Figure 5, we propose that the generation of O2(1Dg) that results from the UV-A excitation of t-UA initiates chemical processes that lead to the physiological responses characteristic of UV-A photoaged skin. It is important to emphasize that we are not comparing the in vivo action spectrum of photosagging with the overall absorption spectrum of t-UA. Rather, the in vivo action spectrum is being compared with a weak transition that contributes to the photochemically complicated, yet structureless absorption spectrum of trans-urocanic acid. At first glance, the t-UA absorption spectrum would appear to be completely unrelated to any physiological response in the UV-A. However, the present work shows that one must consider the possible role of weak transitions of endogenous absorbing chromophores in initiating physiological responses. This includes chromophores like NADH/NADPH, tryptophan, and riboflavin that have been postulated to play a role in photoaging. Additional action spectra (skin fold thickening, glycosaminoglycan production, collagen damage, cellularity increase and elastosis in mice models) also reflect a UV-A component that have a similar line shape as the triplet state action spectrum of t-UA. It would not be surprising if the generation of O2(1Dg) by energy transfer from the excited triplet state of t-UA in the UV-A also contributes to these physiological responses. O2(1Dg) is not a ÒselectiveÓ reactant, and it initiates a wide range of physiological responses. Recent reports of the UV-A irradiation of fibroblasts indicate that singlet oxygen is both an early intermediate in the signaling pathway of interstitial colleganase induction, preceding the synthesis of the proinflamatory cytokines IL-1 and IL-6, and can activate JNKs (stress-activated kinases), which can affect gene expression. Correlation between UV-A and singlet oxygen have also bee discussed for the induced synthesis of mRNA heme oxygenase-1, colleganase, and ICAM-1. Whether UA is a major contributing source of the singlet oxygen that causes these responses requires more research.
Our work on the photochemistry of urocanic acid has been supported by the National Institutes of Health and the Office of Naval Research through the Medical Free Electron Laser Program.
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2. Li, Bulang, Hanson, Kerry M., Simon, John D., Primary Processes of the Electronic Excited States of trans-Urocanic Acid, Journal of Physical Chemistry A, 101, 969 (1997).
3. Hanson, Kerry M., Simon, John D., The Photochemical Isomerization Kinetics of Urocanic Acid and Their Effects Upon the In Vitro and In Vivo Photoisomerization Action Spectra, Photochemistry and Photobiology, 66, 817 (1997).
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7. Simon, John D. Spectroscopic and Dynamic Studies of the Epidermal Chromophores trans-Urocanic Acid and Eumelanin, Accounts of Chemical Research, 33, 307-313 (2000).