Dermatology Online Journal

The effect of clinical UVA/B exposures on urinary urocanic acid isomer levels in individuals with caucasian type (II/III) skin types
Chandan M Sastry¹, Susan E Whitmore², Patrick N Breysse¹, Warwick L Morison², Paul T Strickland¹
Dermatology Online Journal 11 (3): 1

Johns Hopkins University, Bloomberg School of Public Health¹, and Dermatology Department² School of Medicine, Baltimore, Maryland. sastrych@mail.nih.gov

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Abstract

Terrestrial ultraviolet radiation (UVR) exposure, consisting of ultraviolet A (320-40nm) and B (290-320 nm), results in the photoisomerizion of epidermal trans-urocanic acid (trans-UCA) to cis-urocanic acid (cis-UCA), a potential suppressor of local and systemic immune responses. This study examines urinary UCA isomers as biomarkers of UVA/B exposure. It presents results measuring both cis- and trans-UCA in human urine samples collected from a group of study subjects (skin types II/III) that underwent controlled UVA/B exposures similar to those administered in commercial suntanning parlors. The UCA isomers were purified from urine using C18 solid-phase extraction columns followed by high-performance liquid chromatography (HPLC) with UV absorbance (268 nm) detection. The UCA biomarker was expressed as the ratio of cis-UCA to trans-UCA (UCA ratio), or as cis-UCA concentration corrected for urine volume using creatinine (cis-UCA-Cr). The UCA ratio increased over baseline in the urine of individuals exposed to UVA/B. A single exposure to approximately 70 percent minimal erythema dose (MED) of UVR (95 % UVA/5 % UVB to approximately 90 % of skin area) produced a 4.75-fold increase in the UCA ratio (p< 0.001) relative to baseline. Repeated daily UV exposures of similar doses produced a minimal increase in UCA ratio above that of the single UV exposure. These findings indicate that UCA cis-trans ratio holds promise as a biomarker for recent solar UV exposure.

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Abbreviations: UCA, urocanic acid; UV, ultraviolet; UVR, ultraviolet radiation; LOD, limit of detection; MED, minimal erythema dose

Introduction

More than 700,000 non-melanoma and 32,000 melanoma skin cancer cases are diagnosed annually in the US [1, 2, 3]. Research indicates that skin cancer development is strongly associated with solar ultraviolet radiation (UV) exposure [4]. This project investigated the utility of urocanic acid (UCA) isomers as a urinary biomarker for UVA/B exposure. UCA, an amino acid derivative in the epidermis, converts from its natural trans isomeric state to a cis isomer upon UV absorbance [5]. Cis-UCA has been implicated in mediating UV immunosuppression, and is proposed to contribute to epidermal carcinogenesis by altering biological pathways related to the function of Langerhan cells and other antigen presenting cells (APCs) [6].

Previous studies of UV exposures in humans have quantitated UCA isomers in urine and documented a change in cis-to-trans UCA ratio following UV exposure [10]. In this study, we seek to clarify the biomarker response to UV further and present an alternative method for measuring UCA isomers in urine.

Materials and methods

UCA Analytical Method. The analytical method presented is a modification of methods used to examine UCA in both skin and urine [7, 8, 9, 10]. Sep-Pak C18 cartridges (Waters, Inc.) were used with a low-pressure vacuum manifold (Waters, Inc.) to purify 1 ml of urine prior to HPLC analysis. The interfering non-polar compounds in the urine are retained on the Sep-Pak C18 cartridges while UCA, a polar compound, elutes with polar solutions (i.e., water). Before addition of the urine, the cartridges were washed with 5 ml water (dH₂O), 5 ml of 70 percent methanol (MeOH), and 10 ml dH₂O. Next, 1 ml of urine was loaded onto the cartridge, and the cis and trans UCA isomers were eluted with 5 ml dH₂O. The total eluant was then filtered through a 0.22 µm filter (Millipore, Inc.). A previously published cation-exchange method for UCA purification was attempted but not utilized due to lower yields [10].

The conditions for high performance liquid chromatography (HPLC) were adapted from previously published methods [7, 10, 11]. The cis-UCA and trans-UCA isomers were eluted isocratically using a pH 2.9 phosphate buffer that contained 2 percent methanol (MeOH, J.T. Baker, Inc.), 10 mM potassium phosphate (KH₂PO₄, Fisher, Inc.), and 1 g/l sodium octanesulfonic acid (Fisher, Inc.). The HPLC system consisted of a Varian pump (Model 5000), a reverse-phase C18 column (HDS-M80, YMC, Inc.), a UV absorbance detector (Waters, Inc., Model 486), and a chart recorder (Hewlett-Packard, Model 3390A). Injection volumes were either 25 µl and 50 µl. Absorbance was measured at 268 nm; a typical chromatogram is provided in Figure 1. At a flow rate of 0.9 ml/min, the cis-UCA and trans-UCA isomers eluted at 34 and 40 min, respectively.

Figure 1
Figure 1. Example chromatogram showing resolution of a 15ng (10 kJ/m²) test sample consisting of 4.05ng of cis-UCA and 9.75ng trans-UCA peaks.

A standard curve was constructed to define the relationship between the chromatographic peak height (mm) of UCA and mass (ng). Peak height was calculated in triplicate measurements for six levels of trans-UCA (Aldrich Chemical, Co.) standard: 0.625 ng, 1.25 ng, 2.5 ng, 5 ng, 10 ng and 15 ng. There was a linear relationship between peak height (mm) and amount of trans-urocanic acid (ng) injected into the HPLC. The best-fit regression line (r² = 0.999) was: peak height (mm) = 4.48* trans-urocanic acid (ng) + 0.94.

After generating the standard curve and standardizing HPLC conditions for trans-UCA, the quantification procedures for measuring cis-UCA were established. Since cis-UCA is not commercially available, it was generated from trans-UCA using a UVC lamp. Half a milligram (0.5 mg) of solid trans-UCA (Aldrich Chemical, Co.) was diluted in 10 ml of dH₂O to obtain a solution of 50 ng/ul. The solution was divided equally into five 2 ml aliquots. Each aliquot was placed in a separate well of a 3 cm tissue culture plate (Costar, Inc.) and irradiated for different time periods using a germicidal lamp (254 nm, 10 J/m²/sec output) resulting in five doses: 0 kJ/m², 1 kJ/m², 5 kJ/m², 10 kJ/m², 50 kJ/m². A stir bar was used to continuously mix the urocanic acid solution to reduce shielding and to allow for uniform irradiation of the entire volume in the well. Volume adjustment back to 2 ml was necessary for the 5 kJ/m², 10 kJ/m², and 50 kJ/m² standards due to evaporation of water during irradiation.

The method of standard additions was used to assess the yield of the assay. Urine samples (1 ml) were spiked with 5 ng, 10 ng, or 15 ng of the 10 kJ/m² UCA standard. Each urine sample, in addition to unspiked baseline urine, was analyzed using the purification and HPLC methods described previously. The baseline UCA levels (peak heights) from the unspiked urine were subtracted from the spiked samples. The resulting UCA levels were then compared to the 10-kJ/m² UCA standard amounts added to obtain percent yield for each spike level. Figure 2 displays the urinary UCA yields for the standard additions to the test sample. The mean yield for cis-UCA and trans-UCA was 92.6±3.2 percent and 94.4±6.2 percent, respectively.

Figure 2
Figure 2. Urinary UCA yields for 5ng (10kJ/m²) standard additions to a test sample. Each 5ng spike of 10kJ/m² standard consisted of 1.35 ng of cis-UCA, and 3.25 ng trans-UCA

The limit of detection (LOD) for the UCA assay used in this study was calculated by averaging the results from two different methods. The first method assigns the LOD to the level associated with a signal to noise ratio of 3:1. A mean baseline noise of 1.3 mm (peak height) was calculated from five chromatograms randomly taken from this study. The LOD was calculated as 3.9 mm, three times the average noise [12]. The second method sets the LOD to a level that is three times the standard deviation of a sample with a very low amount of UCA [10]. A low UCA sample was analyzed six consecutive times and the LOD was calculated to be 4.2 mm, three times the standard deviation of 1.4 mm. The average LOD from methods 1 and 2 is 4.05 mm. Based on the regression model developed for the standard curve, the LOD of 4.0 mm corresponds to 0.7 ng (5 pmol). For statistical purposes, values below the LOD were assigned the value of the LOD divided by the square root of 2 (√2). A diagnostic kit (Sigma, Inc. Cat. No 555) and standard procedure was utilized for measuring urinary creatinine.

Study Group. Twenty-two human subjects between the ages of 18 and 50 years old were recruited and provided informed consent. They were randomly assigned to one of two experimental groups: a UV exposed group and an un-exposed control group. Individuals that met the following criteria were excluded: (1) general poor health, (2) not skin type II or III, (3) pregnant, (4) history of malignancies, (5) past or present diagnosis of connective tissue diseases or any type of photosensitivity, (6) past or present treatment with photosensitizing medications, (7) prior sensitization to dinitrochlorobenzene (DNCB) or prior involvement in any studies using DNCB, (8) presently existent or previously excised dysplastic nevi, (9) exogenous or endogenous immunosuppression due to any potentially immunosuppressive medications or medical illnesses, and (10) outdoor suntanning or suntan parlor exposures within the prior 30 days. After recruitment, one control subject dropped out of the study after randomization, resulting in ten control subjects and eleven UV exposed subjects. Table 1 summarizes the age, gender, and skin type distributions of both groups.

(see Table 1)

UV Treatments. Each participant in the exposed group received 10 days of UV treatment over a 2-week period. The control group received no UV treatments. The UV light exposures were administered using a bank of F72T12BLHO bulbs (UV Resources, Cleveland, OH). These bulbs are commonly used in suntan parlors and emit 95 percent UVA and 5 percent UVB. In a standing position, each subject received total body UV doses, starting at 70 percent of the MED determined in one individual with skin type II. This corresponded to a 6-minute UV exposure for both the front and backsides of the subject. The UV doses were planned to increase by 20 percent each day to a maximum 15-minute treatment. However, if an individual exhibited mild erythema (pinkish color) from the previous day's treatment, then the dosage was not increased. If the person experienced moderate or severe erythema (reddish skin color), then UV treatment was skipped on that day. As a result of these differences in skin sensitivity, the UV dosage increments varied as detailed in Table 2.

(see Table 2)

All of the subjects experienced at least one case of mild erythema due to UV treatments. In addition, subject 2 and 14 received altered doses on the tenth exposure (i.e., the final dose before the third urine collection). Subject 2 received only a 50 percent dose (15 minutes on back, 0 minutes on front) and Subject 14 did not receive a tenth treatment. These two subjects did, however, receive regular doses for 15 minutes (front and back) on the ninth exposure. Subject 1 also missed Exposure 8 due to moderate erythema from Exposure 7, but had normal ninth and tenth exposures. Subject 18 missed Exposure 4 due to illness.

In addition to detailing the exact exposure times administered, Table 2 also provides three urine collection dates. The first pre-exposure urine (Day 0 urine) was collected on the morning before each subject was exposed for the first time (Exposure 1). This first exposure was the same for all ten subjects. The following morning, after Exposure 1, each subject provided a second urine sample (Day 1 urine). The third urine sample (Day 10 urine) was collected the morning after the tenth UV exposure. The unexposed subjects provided only two samples, an initial urine (Day 0) and another after ten days (Day 10).

Results

The UCA measurements for the control and exposed subjects are categorized as follows:

• UE₀: Baseline UCA for unexposed subjects at Day 0
• UE₁₀: UCA for unexposed subjects at Day 10
• E₀: Baseline UCA for exposed subjects at Day 0
• E₁: UCA for exposed subjects at Day 1 (After initial UV exposure)
• E₁₀: UCA for exposed subjects at Day 10 (After tenth UV exposure)

Table 3 presents the statistical mean, median, and quartiles (25 %, and 75 %) for both the UCA ratio and cis-UCA-Cr levels in the control subjects (UE₀ and UE₁₀) and the exposed subjects (E₀, E₁, and E₁₀). Twenty-one of the total 58 samples had cis-UCA values below the limit of detection (LOD) of 5 pmol. For statistical purposes, these observations were assigned a value of 3.6 pmol (LOD/ (2). All of the samples below the LOD were from the three UV unexposed groups (UE₀, UE₁₀, E₀). None of the trans-UCA measurements were below the LOD.

(see Table 3)

UCA ratio

As shown in Figure 3, the urinary UCA ratio in the UV exposed subjects displays a clear increase after UV exposures (E₁ and E₁₀) compared to baseline (E₀) and the control subjects (UE₀ and UE₁₀).

Figure 3
Figure 3. UCA ratios for control group (UE0, UE10) and exposed group (E0, E1, and E10).

Most of the observations in the box plots are tightly centered on their median value. Individual paired t-tests were used to compare the mean UCA ratios of the five collection points. The unexposed subjects did not exhibit a statistically significant difference in mean UCA ratio levels between UE₀ and UE₁₀ (p=0.205). In contrast, the exposed subjects displayed a significant increase after E₁ (0.225 units) compared to baseline E₀ (p< 0.001). After E₁₀ the subjects showed a 0.309 increase in their average UCA ratio level compared to E₀ (p< 0.001). However, there was no significant difference in mean UCA ratio of E₁₀ compared to E₁ (p=0.114).

The outlying high UCA ratio measurements in E₀, E₁, and E₁₀ were not from the same subject. It is possible that these different subjects experienced undocumented UV exposures during the study. Removal of these three observations from the data set did not alter the results of any statistical tests examining the change in UCA ratio after UV exposure.

The two subjects with altered E₁₀ exposures displayed UCA responses proportionate to their corresponding modified UV treatments. Subject 2 received a 50 percent dose on E₁₀ (15 min. on back, 0 min. on front) and displayed approximately half the UCA ratio level for E₁₀ compared to the entire group average. Subject 14 did not receive the tenth dose and did not exhibit an increase in the UCA ratio from E₁ to E₁₀ as seen in the other participants.

Figure 4 displays the distributions of the creatinine adjusted cis-UCA results (cis-UCA-Cr). The cis-UCA-Cr results were similar to those of the UCA ratio. After E₁ and E₁₀ the exposed group showed significant increases in their mean cis-UCA-Cr levels compared to baseline E₀ (p< 0.001 for both). The mean cis-UCA-Cr levels were not significantly different between E₁ and E₁₀ (p=0.110). Similarly, the control subjects showed no difference in average cis-UCA-Cr levels at UE₀ and UE₁₀ (p=0.360).

Figure 4
Figure 4. Distribution of the cis-UCA-Cr (pmol /µmol Cr) corrected for creatinine for the clinical study control group (UE0 and UE10) and exposed group (E0, E1, and E10).

Discussion

The overall increase in the UCA ratio seen in the exposed subjects indicates that recent UV exposure influences the urinary UCA ratio. The first UV exposure (E₁) dramatically increased the UCA ratio by almost five fold (0.060 to 0.285) within 24 hours. In contrast, the subjects exhibited little increase in biomarker levels after nine additional UV exposures (E₁₀). Although statistically different, the change in the mean UCA levels between the E₁ and E₁₀ exposures was only 0.084 (1.2 fold increase). This minimal increase and the presence of broad overlap in UCA ratio distributions for E₁ and E₁₀, suggest a rapid elimination effect, in which the UCA biomarker is substantially excreted between repeated UV exposures. After adjusting for volume, the cis-UCA levels (cis-UCA-Cr) for E₁ and E₁₀ were statistically indistinguishable.

The interpretation of the UCA ratios for E₁₀, however, is complicated by the dosing regimen given to the subjects. As shown in Table 2, the UV doses varied according to the erythemal response of each subject. In addition, no treatments were given on the weekend days (Saturday, Sunday) over the 2-week period. The ninth and tenth UV exposures were administered on Monday and Tuesday after a weekend. Given that the urinary UCA ratio levels drop to baseline levels within 2-3 days (unpublished findings), a minimal increase in the UCA ratio and lack of increase in cis-UCA-Cr levels between E₁ and E₁₀ appears reasonable.

The subjects in this study showed a wide range of UCA ratios in response to UV for both their E₁ and E₁₀ measurements. The E₁ findings represent inter-individual variation in the biomarker with respect to the same UV dose. The E₁₀ variation is most likely attributed to the variation in the dosing regimens between subjects. Another explanation for biomarker variation is that genetic factors may influence the enzymes necessary for elimination of urocanic acid [13]. Also, since L-histidine is a component of many foods and is a direct precursor of trans-UCA and histidine, dietary differences may possibly increase variation. The rates of cis-UCA and trans-UCA absorption into the systemic circulation as well as their elimination in the urine may vary between people. Larger epidemiological studies are needed to determine if different rates of UCA metabolism (i.e., high, low) exist. In addition, further toxicological research is necessary to elucidate the health effects of cis-UCA. Although immunosuppressive effects of UV have been associated with cis-UCA, its role in skin cancer needs clarification. If a threshold level of cis-UCA is necessary for detrimental effects, then the cis-UCA-Cr biomarker may be a more appropriate health index than the UCA ratio.

There are several incentives for developing UCA biomarkers for UV exposure. First, they appear to relate to UV exposure, a known factor in promoting skin cancer. Secondly, these biomarkers may provide a sensitive indicator of suberythemal UV doses. Finally, they are quantitative measures of UV response, in comparison to the minimal erythemal dose (MED), which is somewhat qualitative. Upon additional validation, the UCA biomarkers could: 1) allow for the evaluation of current UV exposure assessment methods; 2) provide a means to more accurately classify current exposure groups in research studies; 3) be used to clarify the exposure-disease relationship between solar UV radiation, cancer, immunosuppression, and other outcomes; and 4) provide a means to evaluate skin cancer prevention methods (i.e., sunscreen, clothing).

Conclusion

The analytical method developed in this study provides an alternative means for quantitation of UCA in human urine. The urinary UCA biomarker shows a rapid increase after a single UVA/B exposure. Repeated UVA/B exposures suggest a transient effect in which the biomarker displays a minimal increase. UCA ratio levels below 0.10 are characteristic of UV unexposed individuals. Lastly, the creatinine adjusted cis-UCA results were similar to those of the UCA ratio.

Acknowledgments - This project was supported in part by DHHS Grant # P30 ES03819 (Center for Urban Environmental Health).

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