Ultraviolet A phototest positivity is associated with higher free erythrocyte protoporphyrin IX concentration and lower transferrin saturation values in erythropoietic protoporphyria

Abstract Background Erythropoietic protoporphyria (EPP) is a rare disorder of heme biosynthesis hallmarked by early‐onset photosensitivity and mainly due to defective ferrochelatase activity leading to increased erythrocyte protoporphyrin IX (PPIX) levels. Evidence regarding the relationship between erythrocyte PPIX concentration and photosensitivity is limited. Methods To investigate the relationship between free erythrocyte PPIX (FEP) concentration; routine laboratory tests, particularly iron metabolism biomarkers; and ultraviolet (UV) A/visible light phototesting findings, 20 genetically confirmed EPP and one XLPP treatment‐naive patients were included in our study. They underwent UVA and visible light phototesting. On the same day, blood samples were collected for measurement of FEP, serum iron, transferrin, transferrin saturation, and ferritin, 25‐hydroxyvitamin D, and liver enzyme levels. Results Median FEP concentration at the time of phototesting was 57.50 (IQR: 34.58‐102.70) μg/g of Hb. UVA and visible light phototesting were positive in 9 (42.9%) and 8 (38.1%) patients, respectively. Median FEP concentration was significantly higher in UVA phototest–positive patients than in those negative (64.37 [IQR: 57.45‐121.82] vs 45.35 [IQR: 24.53‐74.61] μg/g of Hb, respectively; P = .04486). Similarly, UVA photosensitive individuals had significantly lower median serum iron levels (61.5 [IQR: 33.5‐84] μg/dL vs 109 [IQR: 63.25‐154] μg/dL, respectively; P = .01862) and transferrin saturation values (15.005 [IQR: 7.0775‐18.41] % vs 29.645 [IQR: 17.8225‐34.3575] %; P = .0109) than those negative. Conclusions Our study demonstrates that UVA phototest positivity is associated with higher FEP concentration and lower transferrin saturation and serum iron concentration in EPP.

D (25OHD), aspartate transaminase, alanine transaminase, and gamma-glutamyl transferase. The secondary endpoint was to explore the correlation between FEP concentration, patient age, and iron metabolism biomarkers.

| Patients
This was a cross-sectional study conducted at the Dermatology

| Study protocol
In March 2019, each patient attended the Phototherapy Outpatient Service of our Dermatology Unit to undergo UVA and visible light phototesting. To prevent the potential biases ensuing from the priming phenomenon, phototesting was performed on non-sun-exposed areas, and patients were asked to avoid sun exposure in the preceding week.
On the same day, blood samples were collected for measurement of FEP concentration, serum iron, serum transferrin, serum ferritin, 25OHD, and liver function tests. Transferrin saturation was calculated using the following formula: (iron (μg/dL)/transferrin (mg/dl)) × 71. 24. Initial irradiation time was 15 minutes and then half of the said area was irradiated for 5 additional minutes. A photochemotherapy device (Waldmann PUVA 3001) was employed for UVA delivery, with an emission spectrum peaking at around 365 and 405 nm. The K E Y W O R D S erythropoietic protoporphyria, photosensitivity, phototesting, porphyria, protoporphyrin IX, UVA phototesting delivered UVA dosage was 7 J/cm 2 for the first 15 minutes and 9 J/ cm 2 for the entire duration of the phototest.
Visible light phototesting was carried out with a standard halogen lamp slide projector (NOVAMAT 820, 150 W; emission spectrum 400-1000 nm) on a 10 × 10-cm area on right gluteal skin. Initial irradiation lasted 15 minutes and then half of the area was irradiated for 5 additional minutes. The delivered visible light dosage was 1.79 J/ cm 2 for the first 15 minutes and 2.39 J/cm 2 for the entire duration of the phototest.
The distance between skin and light source was 30 cm for visible light phototesting and 20 cm for UVA phototesting. Although both artificial sources for phototesting produce heat, they were endowed with a ventilation system to reduce the heating effect on the skin.
Results of UVA and visible light phototesting were read immediately and after 15 minutes. Phototesting positivity was based on the latter readings. A cautionary additional reading was performed after 24 hours. Both objective and subjective clinical variables, such as erythema, edema, and burning or itching sensation, were recorded.
The severity of each manifestation was assessed using a threelevel scoring system (absent, 0; mild, +; intense, ++) adapted from the European Dermatology Guideline for the photodermatoses. 12 Finally, phototest positivity was defined based on the presence of at least one sign or symptom.

| Statistical analysis
The normality of distribution of continuous variables was assessed by the Shapiro-Wilk test. Continuous variables with normal distribution were presented as mean ± standard deviation (SD); nonnormal variables were reported as median (interquartile range The Kruskal-Wallis test was used for comparison between groups. Pearson's r was used to assess the correlation between continuous variables. A P-value <.05 was considered statistically significant. Statistical software SAS (release 9.4; SAS Institute, Inc) was used to perform all the statistical analyses.

| RE SULTS
Age, genetic data, laboratory findings, and phototesting results of the 20 EPP and 1 XLPP patients included in our study are summarized in Tables 1 and 2      .3029
Bold P values are statistically significant.
The clinical relevance of different erythrocyte PPIX levels in predicting cutaneous photosensitivity is not entirely clear. Although erythrocyte PPIX levels have been reported to be significantly related to photosensitivity, 8 no convincing evidence of a simple correlation has been documented. 14 Previous reports failed to demonstrate a correlation between total erythrocyte porphyrin levels and time to symptom onset. 5 Heerfordt and Wulf 8 found that skin PPIX was significantly associated with erythrocyte PPIX, skin erythema, and symptoms, namely, stinging or pain, during controlled illumination.
However, another study by the same authors documented that increasing FEP concentration correlated neither with tolerable daily light dose nor with percentages of days with symptoms. 6 Moreover, cutaneous ultra-weak photoemissions, a by-product of PPIX-induced phototoxic reactions, were also shown to correlate with erythrocyte PPIX levels. 15 In the present study, significantly higher FEP concentration was documented in individuals with a positive UVA phototest. Moreover, no statistically significant difference in terms of FEP concentration was found between patients with a positive visible light phototest and those with a negative one.
However, differential expression of erythrocyte membrane transporter ABCG2 could explain the lack of a linear relation be- Curiously, slightly higher levels of ALT were measured in UVA photosensitive individuals than those with a negative UVA phototest. ALT levels were well within normal ranges in both groups, and none of the subjects had a history of hepatopathy. Although this finding could be linked to both disease severity and risk of future liver disease, its interpretation remains uncertain.
The main limitation of the present study is the scarce numerosity of our cohort mainly due to the rarity of EPP, and its major strength resides in the use of phototesting as objective means for the assessment of acute photosensitivity and related symptoms. Other noteworthy limitations include (a) the propensity of patients to be more aware of symptoms and to report them in a clinical setting, rather than in everyday life; (b) day-to-day variability of photosensitivity in EPP; (c) differences in photosensitivity at different body sites due to discrepancies in dermal thickness and vascular density 6,35 ; (d) the lack of 7-hour readings, 36 which were not performed due to practical reasons; (e) although UVA radiation accounts for more than 95% of total emissions of our UVA source, a small, negligible percentage of radiations was represented by blue light-skewed visible light.
In conclusion, the present study provides novel insights into the relationship between FEP concentration, laboratory findings, and phototesting results in EPP patients. Although higher FEP levels, lower serum iron concentrations, and transferrin saturation values were found in patients with positive UVA phototesting results, photosensitivity to both UVA and visible light was documented in the majority of those with at least a positive phototest result, indicating the viability of both techniques in the evaluation of EPP. Iron metabolism imbalance may be an epiphenomenon of disease severity, which also translates into higher photosensitivity. From a practical perspective, we believe that these findings may provide guidance in the management of the condition, counseling patients so that they may avoid unintended UVA exposure, especially if the laboratory examinations, such as low transferrin saturation, lower serum iron, and higher FEP levels, suggest greater proneness to UVA photosensitivity. Further research on larger samples will be required to confirm our findings.