Claudia Borelli1, Kathrin Merk1, Martin Schaller1,2, Karl Jacob3, Michael Vogeser3, Günther Weindl1,2, Ursula Berger4 and Gerd Plewig1

1Department of Dermatology and Allergology, Ludwig-Maximilian-University of Munich, Munich, 2Department of Dermatology, University of Tuebingen, Tuebingen, 3Institute of Clinical Chemistry, Ludwig-Maximilian-University of Munich, Munich, and 4Institute of Statistics (StatBeCe), University of Bielefeld, Bielefeld, Germany

Propionibacterium acnes is often discussed as a contributing pathogenic factor in the aetiology of acne lesions. The aim of this study was to test which porphyrin patterns are synthesized by P. acnes in vivo in untreated acne patients and during standard acne regimens. These photosensitive compounds are potential targets for photodynamic therapy of acne and need to be better characterized in the skin. Using high-performance liquid chromatography coproporphyrin III was the main porphyrin identified in all patients. Coproporphyrin I and protoporphyrin were found at considerably lower concentrations. When the porphyrin concentration of individual patients receiving isotretinoin was analysed repeatedly over time, clinical improvement was associated with lowered levels of porphyrins. Statistical analysis demonstrated a significant reduction in the porphyrin fractions only in the isotretinoin group which was associated with clinical improvement 2 months after starting therapy. Key words: Propionibacterium acnes; coproporphyrin I; coproporphyrin III; protoporphyrin.

(Accepted February 20, 2006.)

Acta Derm Venereol 2006; 86: 316–319.

Claudia Borelli, Department of Dermatology and Allergology, Ludwig-Maximilian-University of Munich, Frauenlobstrasse 9–11, D-80337 Munich, Germany. E-mail: Claudia.Borelli@med.uni-muenchen.de

Acne is a chronic inflammatory disorder of the pilosebaceous follicles with a multifactorial aetiology. Androgen-induced seborrhoea, follicular hyperkeratinization, and the commensal bacterium Propionibacterium acnes appear to play a role in its pathophysiology (1). P. acnes not only colonizes sebaceous follicles, but there are also some abnormalities of the growth or metabolism of this organism in acne resulting in comedogenesis and the development of inflammatory acne lesions (2). In recently published papers the co-incubation of keratinocytes with viable, but not heat-killed, P. acnes modulated the cytokine response for interleukins (IL)-1a and IL-1b, tumour necrosis factor, granulocyte monocyte colony stimulating factor, and IL-8 (3–5), while other studies failed to demonstrate such cytokine modulations by P. acnes (6–8).

Intradermal injection of P. acnes into the ears of rats produced chronic inflammation with formation of acneiform lesions (9). After injecting bacterial suspensions into sterile keratin cysts, an inflammation was detected morphologically (10). The identity of these inflammatory factors has not been clarified conclusively, but various enzymes produced by P. acnes, such as lipases, hyaluronidases, proteinases, toxins and microbial allergens are suspected (11).

Porphyrins are further metabolic products of Propionibacteria. Their existence has been proved by coral-red fluorescence in the follicle openings by examining facial skin under Wood’s light (ultraviolet-A 320–400 nm) (12). Porphyrins might contribute to the perifollicular inflammatory reaction by their cytotoxic effect and by stimulating expression of keratinocyte-derived IL-8 (5). After rupture of the follicle epithelia, porphyrins secreted perifollicularly could also contribute to the inflammatory reaction of the follicle or its environment by favouring the development of cytotoxic substances such as squalene peroxide possibly via singlet oxygen (13).

Successful therapy of acne with visible light by using a blue-light high-energy lamp was first reported by Meffert et al. (14). Recently, improvement of acne was reported after blue light-mediated photo-inactivation of P. acnes by its endogenic porphyrins (15, 16). While the in vitro porphyrin pattern of P. acnes has been qualitatively and quantitatively analysed in several studies by high-performance liquid chromatography (HPLC) (15, 17, 18) there is still a lack of in vivo HPLC investigations. In addition, the effect of isotretinoin as the most effective acne therapy on the in vivo porphyrin pattern remains to be investigated.

The aim of this study was to analyse both qualitatively and quantitatively using a highly sensitive HPLC, the porphyrins produced by P. acnes in vivo before and after oral isotretinoin therapy, in comparison with other acne treatments.

Materials and Methods

Patients and collection of samples

Fifty-eight patients (32 women and 26 men) with acne papulopustulosa and conglobata were examined in a randomized trial after evaluation with a standardized questionnaire. Studies were carried out following informed consent and in accordance with the ethical standards of the responsible committee on human experimentation and with the Helsinki Declaration of 1975, as revised in 1983. The patients’ age ranged from 13 to 37 years, with a median of 20 years. Porphyrins were measured in skin samples, both in 16 patients before treatment and another 16 patients 2 months after starting oral isotretinoin therapy with doses ranging from 0.2 mg/kg/day to 0.5 mg/kg/day. In 9 additional patients the porphyrin levels were analysed intra-individually before isotretinoin treatment and 2 months after having started therapy. Also investigated were samples of 17 patients receiving oral minocycline 50–100 mg/day combined with topical benzoyl peroxide for 2 months. In all treated groups the samples were taken 2 months after having started acne therapy.

After cleansing of the acne lesions with 70% ethanol the contents of 3 lesions were squeezed out and collected using a comedo extractor. The weight of the samples was measured by high-precision laboratory scale (Sartorius, Goettingen, Germany).

Microbiological analysis

Part of the material described above was used to isolate Propionibacterium spp. under micro-aerophilic and dark conditions on brain-heart infusion agar at pH 7.1 and 37°C. The jars contained a mixture of hydrogen and carbon dioxide (95:5, v/v) and the oxygen content was less than 5%. Using the biochemical identification system API 20 A (BioMérieux, Lyon, France), all P. acnes isolates could be characterized as P. acnes serotype I.

Qualitative and quantitative porphyrin analysis

The samples were suspended in 2 ml 0.9% NaCl solution and homogenized in a mortar. The homogenizate was transferred to Eppendorf tubes (Eppendorf, Hamburg, Germany) washed with 1 ml 0.9% NaCl solution and centrifuged for 4 min at 2000 rpm in a 5415°C Eppendorf centrifuge. The residue was disposed of and 50 µl Celite® (Fluka, Paesel, Germany), a filtration aid consisting of kieselguhr of varied granular size and 250 µl ethylacetate/ethanoic acid (4:1) were added. Finally, the suspension was mixed for 10 sec in a vortexer and then centrifuged for 1 min at 1500 rpm. The residue was transferred via a pipette into another Eppendorf tube and 250 µl 1.5 M hydrochloric acid (HCl) was added. The solution was mixed for 10 sec in the vortexer. Finally, approximately 300 µl of the lower phase, which contains the porphyrins, was transferred via a pipette to another Eppendorf tube.

Because of the high photosensitivity of porphyrins, the extracted samples were kept in the dark at 4°C and then reprocessed for HPLC after adding HCl not more than 2 h later. Prior to HPLC analysis the porphyrin containing extract was mixed with 2 ng mesoporphyrin as an internal standard (SIGMA, Deisenhofen, Germany) and loaded onto a C18(e) solid phase extraction column (Separtis, Grenzach-Wyhlen, Germany). After washing with water, the porphyrins were eluted with acetone/methanol (1:1) containing 1% triethylamine; the content of specific porphyrins was quantified as described by Jacob & Luppa (19). The amounts of porphyrins analysed were related to the weight of comedones processed (ng porphyrin/mg tissue).

Statistical analysis

The results were evaluated statistically with the “SPSS 10.0 for Windows” statistics program. The relationship between porphyrin concentrations from untreated patients and after therapy was tested using the Mann-Whitney U test, a non-parametric test for unassociated random samples. A p value of 0.05 or less was considered significant.

Results

Microbiological analysis

P. acnes (90%) was the predominant Propionibacterium spp. isolated from the acne lesions. We could only very rarely isolate P. granulosum (8%) or P. avidum (2%). Comparative analysis of in vivo porphyrin patterns produced by P. acnes and P. granulosum revealed no differences (not shown). We therefore decided to focus our studies on P. acnes.

Qualitative and quantitative analysis of porphyrins before and after acne therapy

Qualitative HPLC analysis demonstrated a uniform porphyrin profile in all investigated patient samples from comedones or inflammatory acne lesions (results not shown). In each case, coproporphyrin III accounted for the largest fraction. Coproporphyrin I and protoporphyrin were present in lower concentrations, whereas polar porphyrins were not detected. In general, the amount of coproporphyrin III correlated highly (correlation coefficient = 0.952) with the number of colony forming units of Propionibacteria isolated from the blackheads (Fig. 1).

In contrast to the group of untreated control patients the coproporphyrin I, III and protoporphyrin values for patients treated with isotretinoin for 2 months were significantly lower (Table I).

In 9 patients porphyrins were analysed during the course of isotretinoin treatment. Six of them also showed decreased porphyrin values 2 months after starting isotretinoin therapy, which correlated clinically with the improvement of the skin conditions. Interestingly in 3 patients with unimproved acne lesion the coproporphyrin levels were unchanged or increasing (Fig. 2).

Coproporphyrin III values for patients treated with oral minocycline plus topical benzoyl peroxide for 2 months were insignificantly increased compared with untreated patients, while coproporphyrin I and protoporphyrin levels were slightly decreased (see Table I).

Discussion

The porphyrins in the present study were separated by HPLC. The advantage of this technique over other chromatographic methods lies in the high-resolution power, enabling separation of porphyrin isomers even at the picogram level. Mesoporphyrin was added to the samples as internal standard. The mean mesoporphyrin recovery rate of 97% showed that only non-significant amounts of porphyrin were lost during sample preparation.

In the present study P. acnes (90%) was the predominant Propionibacterium spp. isolated from the acne lesions, while P. granulosum (8%) or P. avidum (2%) were only rarely isolated. As comparative porphyrin analyses were similar in all investigated samples, it is tempting to speculate that porphyrins are in vivo mainly produced by P. acnes. Contamination of our in vivo samples with keratinocyte-derived coproporphyrin III can be excluded, as microspectrofluorometric emission spectra has failed to demonstrate this porphyrin fraction in diseased as well as in normal skin (20).

HPLC analysis of acne extractions revealed a qualitatively uniform porphyrin profile, with coproporphyrin III as the major porphyrin fraction, whereas coproporphyrin I was present in low concentrations, and protoporphyrin hardly at all. This result matches the porphyrin formation of previously published in vivo (12, 21, 22) and in vitro studies (23–25).

The considerable inter-individual variations in the porphyrin concentration observed in the present study have also been seen in previous investigations (21, 23, 24). Similar porphyrin patterns in comedones and inflammatory acne lesions implicate that inflammation is not caused by modulation of the porphyrin profile.

A collective comparison of the porphyrin composition in untreated patients and patients treated with isotretinoin or minocycline plus benzoyl peroxide revealed no qualitative differences. These results suggest that the porphyrin profile secreted by P. acnes is not modulated by these standard therapies. Concentrations of all three porphyrin fractions (coproporphyrin I, coproporphyrin III, protoporphyrin), however, were significantly reduced in the isotretinoin treated group compared with an untreated group of acne patients. The reduction in porphyrin values was confirmed in repeated, vertical measurements in single patients before and 2 months after having started isotretinoin therapy and correlated with an improvement of acne. In contrast, in patients receiving oral minocycline therapy in combination with topical benzoyl peroxide, HPLC analyses demonstrated no reduction in porphyrin concentrations. Inadequate patient compliance and an increasing tendency towards resistance to minocycline of P. acnes are two possible reasons for this finding (26).

The association of clinical improvement with a significant reduction in the porphyrin concentration implicates a decreased number of P. acnes as a result of isotretinoin therapy, and this has also been shown repeatedly by others. In corollary, we suggest that oral isotretinoin is more effective in reducing P. acnes and suppressing porphyrin production than oral minocycline/topical benzoyl peroxide therapy. The role of porphyrins as a factor contributing to the pathogenesis of acne is still unclear. Coproporphyrin III can induce a keratinocytic IL-8 response and might via this mechanism contribute to the development of inflammation (5). Knowledge of the relatively large amounts of porphyrins in acne skin and the identification of the secretion pattern of porphyrins by P. acnes in vivo might be helpful to improve the efficacy of photodynamic destruction of these bacteria (27). Treating acne patients with phototherapy is possible without externally applied photosensitizers because of the presence of endogenous porphyrins. This modality is receiving increased attention because of the increasing tendency for antibiotic resistance of P. acnes (26). Initial studies with blue light reported encouraging results (15, 16, 28).

AcknowledgementS

This work was supported by the Hans-Fischer-Gesellschaft (Munich, Germany). We wish to thank Mr E. Egeler, Institute of Clinical Chemistry/University of Munich, for his skilful technical assistance and Dr W. Burgdorf, Department of Dermatology and Allergology, University of Munich, for critical reading of the manuscript.

References

1. Bojar RA, Holland KT. Acne and Propionibacterium acnes. Clin Dermatol 2004; 22: 375–379.

2. Farrar MD, Ingham E. Acne: inflammation. Clin Dermatol 2004; 22: 380–384.

3. Graham GM, Farrar MD, Cruse-Sawyer JE, Holland KT, Ingham E. Proinflammatory cytokine production by human keratinocytes stimulated with Propionibacterium acnes and P. acnes GroEL. Br J Dermatol 2004; 150: 421–428.

4. Nagy I, Pivarcsi A, Koreck A, Szell M, Urban E, Kemeny L. Distinct strains of Propionibacterium acnes induce selective human beta-defensin-2 and interleukin-8 expression in human keratinocytes through toll-like receptors. J Invest Dermatol 2005; 124: 931–938.

5. Schaller M, Loewenstein M, Borelli C, Jacob K, Vogeser M, Burgdorf W, et al. Induction of a chemoattractive proinflammatory cytokine response after stimulation of keratinocytes with Propionibacterium acnes and coproporphyrin III. Br J Dermatol 2005; 153: 66–71.

6. Chen Q, Koga T, Uchi H, Hara H, Terao H, Moroi Y, et al. Propionibacterium acnes-induced IL-8 production may be mediated by NF-kappa B activation in human monocytes. J Dermatol Sci 2002; 29: 97–103.

7. Vowels BR, Yang S, Leyden JJ. Induction of proinflammatory cytokines by a soluble factor of Propionibacterium acnes: implications for chronic inflammatory acne. Infect Immun 1995; 63: 3158–3165.

8. Walters CE, Ingham E, Eady EA, Cove JH, Kearney JN, Cunliffe WJ. In vitro modulation of keratinocyte-derived interleukin-1 alpha (IL-1 alpha) and peripheral blood mononuclear cell-derived IL-1 beta release in response to cutaneous commensal microorganisms. Infect Immun 1995; 63: 1223–1228.

9. De Young LM, Spires DA, Ballaron SJ, Cummins CS, Young JM, Allison AC. Acne-like chronic inflammatory activity of Propionibacterium acnes preparations in an animal model: correlation with ability to stimulate the reticuloendothelial system. J Invest Dermatol 1985; 85: 255–258.

10. Kirschbaum JO, Kligman AM. The pathogenic role of Corynebacterium acnes in acne vulgaris. Arch Dermatol 1963; 88: 832–833.

11. Holland KT, Aldana O, Bojar RA, Cunliffe WJ, Eady EA, Holland DB, et al. Propionibacterium acnes and acne. Dermatology 1998; 196: 67–68.

12. Cornelius CE 3rd, Ludwig GD. Red fluorescence of comedones: production of porphyrins by Corynebacterium acnes. J Invest Dermatol 1967; 49: 368–370.

13. Saint-Leger D, Bague A, Cohen E, Chivot M. A possible role for squalene in the pathogenesis of acne. I. In vitro study of squalene oxidation. Br J Dermatol 1986; 114: 535–542.

14. Meffert H, Gaunitz K, Gutewort T, Amlong UJ. Aknetherapie mit sichtbarem Licht. Verkürzung der Bestrahlungszeit durch Verwendung eines Hochdruckstrahlers vom Blaulichttyp. Dermatol Monatsschr 1990; 176: 597–603.

15. Ashkenazi H, Malik Z, Harth Y, Nitzan Y. Eradication of Propionibacterium acnes by its endogenic porphyrins after illumination with high intensity blue light. FEMS Immunol Med Microbiol 2003; 35: 17–24.

16. Kawada A, Aragane Y, Kameyama H, Sangen Y, Tezuka T. Acne phototherapy with a high-intensity, enhanced, narrow-band, blue light source: an open study and in vitro investigation. J Dermatol Sci 2002; 30: 129–135.

17. Romiti R, Schaller M, Jacob K, Plewig G. High-performance liquid chromatography analysis of porphyrins in Propionibacterium acnes. Arch Dermatol Res 2000; 292: 320–322.

18. Kjeldstad B, Johnsson A, Sandberg S. Influence of pH on porphyrin production in Propionibacterium acnes. Arch Dermatol Res 1984; 276: 396–400.

19. Jacob K, Luppa P. Application of ion-pair high performance liquid chromatography to the analysis of porphyrins in clinical samples. Biomed Chromatogr 1991; 5: 122–127.

20. Bissonnette R, Zeng H, McLean DI, Schreiber WE, Roscoe DL, Lui H. Psoriatic plaques exhibit red autofluorescence that is due to protoporphyrin IX. J Invest Dermatol 1998; 111: 586–591.

21. Johnsson A, Kjeldstad B, Melo TB. Fluorescence from pilosebaceous follicles. Arch Dermatol Res 1987; 279: 190–193.

22. McGinley KJ, Webster GF, Ruggieri MR, Leyden JJ. Regional variations in density of cutaneous propionibacteria: correlation of Propionibacterium acnes populations with sebaceous secretion. J Clin Microbiol 1980; 12: 672–675.

23. Fanta D, Formanek I, Poitschek C, Thurner J. Die Porphyrinproduktion des Propionibacterium acnes bei Akne und Seborrhoe. Arch Dermatol Res 1978; 261: 175–179.

24. Formanek I, Fanta D, Poitschek C, Thurner J. Porphyrinproduktion des Propionibacterium acnes. Arch Dermatol Res 1977; 259: 169–176.

25. Lee WL, Shalita AR, Poh-Fitzpatrick MB. Comparative studies of porphyrin production in Propionibacterium acnes and Propionibacterium granulosum. J Bacteriol 1978; 133: 811–815.

26. Ross JI, Snelling AM, Eady EA, Cove JH, Cunliffe WJ, Leyden JJ, et al. Phenotypic and genotypic characterization of antibiotic-resistant Propionibacterium acnes isolated from acne patients attending dermatology clinics in Europe, the U.S.A., Japan and Australia. Br J Dermatol 2001; 144: 339–346.

27. Konig K, Schneckenburger H, Ruck A, Steiner R. In vivo photoproduct formation during PDT with ALA-induced endogenous porphyrins. J Photochem Photobiol B 1993; 18: 287–290.

28. Elman M, Lask G. The role of pulsed light and heat energy (LHE) in acne clearance. J Cosmet Laser Ther 2004; 6: 91–95.