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Quantitation of hydrogen peroxide formed during UV-visible irradiation of protoporphyrin, coproporphyrin and uroporphyrin
CIinica Chimiea Acta, 186 (1989) 375-381 Elsevier
37s
CCA 04648
Quantitation of hydrogen peroxide formed during UV-visible irradiation of protoporphyrin, coproporphyrin and uroporphyrin 1. Aravind
Menon’, Mary Ann C. Becker 2, Suruj D. Persad 3 and Herbert F. Haberman 4
’ Departments of Medicine and Ophthalmology,
’ Department of Medicine, 3 Department of Ophthalmology, 4 Department of Medicine, University of Toronto, Toronto ON (Canada)
(Received
21 July 1989; revision received 25 September
Key words: Porphyrin;
Photosensitizer;
1989; accepted
Light; Hydrogen
peroxide;
30 September
1989)
Singlet oxygen
Summary Free porphyrins are strong photosensitizers. Previously reported findings indicate that the in vitro cell lysis induced by irradiation in the presence of coproporphyrin (CP) and uropo~hy~n (UP) is mediated by H202 and that induced by irradiation with protopo~hy~n (PP) is not mediated by H,02. In the present study the possible role of H,O, in the porphyrin photosensitization was investigated by direct measurement of the H,O, formed during the irradiation of PP, CP and UP. Our results show that the amount of H,O, formed decreased in the following order: UP, CP, PP. The amounts of H202 formed during irradiation of CP and PP were appro~mately 86% and 38% respectively in comparison to the H,02 formed during the irradiation of UP. The formation of H,O, was inhibited by sodium azide, a strong quencher of singlet oxygen. These observations are in good agreement with the previous report that the in vitro photolysis of Ehrlich ascites carcinoma cells by UP and CP, but not that by PP, was inhibited by catalase and clinical findings with patients with erythropoetic protoporphyria (EPP) and porphyria cutanea tarda (PCT). The patients with EPP, where the photosensiti~ty is due to PP, respond well to P-carotene while p-carotene does not protect against the photosensitivity in PCT, in which case the photosensitivity is due to uroporphyrin.
Correspondence Bldg., University
to: Dr. LA. Menon, Departments of Medicine of Toronto, Toronto, Ontario, Canada.
0009-8981/89/$03.50
0 1989 Elsevier Science Publishers
and OphthalmoIogy,
B.V. (Biomedical
Division)
Medical
Sciences
376
Introduction Porphyrins are well recognized to be natural metabolites in the mammalian organisms and are formed during the biosynthesis of heme and the cytochromes. Free porphyrins are strong photosensitizers; on the other hand, the Fe-containing species, viz. heme and cytochromes are devoid of the photosensitizing activity [l-3]. The disorders in the porphyrin metabolism, termed ‘porphyrias’ represent several types. The classification of the porphyrias varies depending upon the clinical symptoms, the organs involved or the biochemical manifestation. Thus one classification divides the porphyrias into the hepatic or erythropoetic types. From the clinical standpoint the porphyrias may be classified into those producing cutaneous manifestations and those causing systemic diseases. These are discussed in a large number of reviews and books [l-7]. There is abundant evidence to show that singlet oxygen is formed during the irradiation of the porphyrins, presumably by the transfer of the excess energy of the porphyrins in the excited states to oxygen. Several reports have indicated that singlet oxygen may be involved in the phototoxicity of the porphyrins [6-lo]. Two common porphyrias where photosensitivity is a major clinical manifestation are erythropoetic protoporphyria (EPP) and porphyria cutanea tarda (PCT). However the cutaneous manifestations of these two diseases differ. Similarly the porphyrins accumulated in the tissues and the porphyrins excreted in these disease states differ. EPP is characterized by the accumulation of abnormally high levels of protoporphyrin (PP) in red blood cells, whereas high levels of uroporphyrin (UP) and coproporphyrin (CP) in plasma and urine are characteristic of PCT [11,12]. The reasons why the symptoms differ in PCT and EPP are not clearly understood. It has been postulated that these differences may result from the differences in localization of these porphyrins. This effect can be expected to be a composite function of solubility and subcellular localization of the particular porphyrins concerned [13]. Certain photobiological investigations on leukocytes [14], red blood cells [15], fibroblasts [16] and mitochondria [17] have provided evidence in support of this mechanism. We have recently reported that the photosensitized lysis of Ehrlich ascites carcinoma cells induced by UP and CP is inhibited by catalase where as the cell lysis produced by PP is not affected by catalase [lS]. These findings indicate that the phototoxicity of CP and UP is mediated by H,O, and that of PP is not mediated by H,O,. The possible role of H,O, in the porphyrin photosensitization was investigated by direct measurement of the H,O, formed during the irradiation of PP, CP and UP. This paper describes a quantitative comparison of the formation H,O, during the irradiation of these porphyrins. Materials and methods Materials The porphyrins, PP, CP and UP were obtained as free acids from Porphyrin Products Inc. (Logan, UT, USA). Superoxide dismutase (SOD) (specific activity
3 570 U/mg protein) and catalase (spec act 46 200 U/mg protein), horse radish peroxidase (spec act 310 U/mg solid), 4-amino-antipyrine and p-carotene were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Sodium azide, H,O,, methanol and phenol were purchased from Fisher Scientific Limited (Fair Lawn, NJ, USA). 1,4’-Diaza-bicyclo[2.2.2]octane (DABCO) was obtained from Eastman Kodak Company (Rochester, NY, USA). 2,5-Dimethylfuran (DMF) was purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). Stock solutions of PP, CP and UP were made in 3.0 mol/l, 0.1 mol/l and 1.0 mol/l HCl, respectively. These solutions were brought to pH 7 with NaOH. Appropriate dilutions were made with 0.01 mol/l phosphate buffer pH 7.4; 0.35 ml was added to the irradiation medium. All porphyrin solutions were contained in opaque glassware and stored in the dark. Irradiation Various concentrations of PP, CP and UP, in the presence of SOD or other reagents as mentioned in each section, were irradiated for the specific periods, using a Westinghouse Mercury Vapour lamp. This source emits discontinuous radiation (320 to 700 nm), with peaks at 360,400, 430, 550 and 580 nm [19,20]. The irradiance was measured using either a narrow band filter (365 nm) or no filter. The irradiance with the filter was 23 k 2.98 Watts/sqM, and without filter 170 + 9.75 Watts/sqM. All solutions were irradiated without the filter. Determination of H202 H,O, was assayed according to the method of Freu et al. [21]. The reagent for H,O, assay consisted of 5.85 mg phenol, 2.50 mg 4-aminoantipyrine, 5.0 pg horseradish peroxidase and 1.25 ~1 10 mmol/l H,O, to stabilize the phenol-C aminoantipyrine complex in 0.5 ml 0.01 mol/l phosphate buffer, pH 7.4, in a total volume of 3.5 ml. The absorbance was read at 505 nm on a Gilson 2400 spectrophotometer. The amount of H,O, formed was determined using a standard curve. Analysis Each sample. similar
of results experiment was performed at least four times, using triplicates for each The mean and standard error of the mean (SEM) for each sample from all experiments were calculated.
Results Formation of H,O, during irradiation of different porphyrins for various periods of time Table I shows the formation of H,O, during irradiation of PP, CP and UP for various periods of time. A fixed concentration of PP, CP and UP was irradiated for 0, 10, 15, 30, 45 and 60 min in the presence of SOD and the reagents for H,Oz assay. It was found that the amount of H,O, formed during irradiation of PP increased with respect to time up to 60 min. While the amount of H,O, formed during irradiation of CP and UP also increased with respect to the irradiation period, the H,O, formation was not proportional to the period of irradiation. At 60
378
TABLE
I
Formation
of H,O,
during
irradiation
of different
porphyrins
for various
periods
of time
Time
H,O,
(mm)
PP
CP
UP
0 10 15 30 45 60
1.2kO.9 2.6kO.3 3.8*1.0 7.8 k 2.6 11.5*4.2 14.2 f 2.3
0.1 kO.1 18.6i1.3 20.7 + 1 .O 25.1 f 0.9 26.3 f 0.7 31.7*0.9
o.o+o.o 14.7 * 2.2 21.9k3.1 31.7*4.5 36.2i2.2 37.0 + 2.0
The final concentration
formed (mean f SEM)
of PP. CP and UP was 1000 nmol/l.
The H,O,
formed
is expressed
as pmoI/I.
min, the amounts of H,O, formed during irradiation of CP and PP were approximately 86% and 38% respectively in comparison to the H,O, formed from UP. Formation of H202 during irradiation of various concentrations of different porphyrins A comparison of the amounts of H,O, formed during the irradiation of various concentrations of PP, CP and UP is shown in Table II. The amount of H,O, formed during the irradiation of each of these porphyrins was approximately proportional to the porphyrin concentration up to 1 mmol/l. The H,02 formation decreased in the order: UP, CP, PP. The amounts of H,O, formed from PP and CP at 1 ~mol/l were approximately 44% and 77% in comparison to that formed from UP. The possibility that the H,O, formation was mediated by superoxide was investigated, by omitting SOD from the system. Since the spontaneous dismutation (in the absence of SOD) of superoxide to produce H,O, is slow it was expected that omission of SOD would lower the amount of H202 formed in the system. As may
TABLE Formation Porphyrin (nmol/l)
II of H,O,
during
H,Oa
250 500 1000
0.0 3zo.o 5.8 + 2.4 8.9 k3.1 16.8 f 3.6
of various
concentrations
of different
porphyrins
formed (mean f SEM) CP
PP + SOD
0
irradiation
- SOD
UP
+ SOD
-SOD
+ SOD
- SOD
0.0 f 0.0 2.3 i-1.4 4.1 + 1.8 6.2 k1.7
0.0 + 0.0 7.3 k3.4 17.6 f 4.3 38.4 f 4.2
0.0 * 0.0 1.3 f 1.9 2.7 k1.6 7.0 *1.2
0.0
0.0
*o.o
*o.o
1.7 & 1.9 2.2 *1.9 6.2 f 1.1
8.7 f 3.0 16.8 f 3.4 29.7 + 4.0
The irradiation period was 60 min. The final concentration expressed as pmoI/l.
of SOD was 180 U/ml.
The H,O,
formed
is
379 TABLE
III
Effects of sodium
azide on the formation
Sodium
H ,O,
azide
(mmoI/I)
0 1 5 10
of H,O,
during
irradiation
of various
porphyrins
formed (mean k SEM)
PP
UP
CP
Light
Dark
Light
Dark
Light
Dark
15.0*1.4 14.6 f 1.4 12.8 f 0.7 10.7 + 1.2
5.9 f 1.0 5.6kl.O 5.6* 1.2 5.7+0.8
40.7+1.1 35.1 k 2.8 21.6 k 3.6 21.Ok3.9
3.8+0.9 4.1* 1.4 4.4 f 2.4 5.0+2.0
42.5 + 3.6 35.9k4.1 27.1 i 2.8 23.2 + 3.0
3.6+1.1 3.7 f 1.0 4.OkO.7 4.3 5 0.8
The final concentration of PP, CP and UP was 1000 nmol/l. H,O, formed is expressed as pmol/l.
The irradiation
period
was 60 min. The
be seen from the results in Table II, when SOD was omitted from the irradiation mixture, the formation of H,O, was decreased. Thus at 1 pmol/l concentration of the porphyrins, the quantities of H,O, formed from PP, CP and UP were decreased by 63%, 79% and 82% when SOD was omitted. Effects of singlet oxygen scavengers
upon the formation
of H,O,
during the irradiation
of UP
The effects of various scavengers of singlet oxygen on the formation of H,O, were investigated. PP, CP and UP were irradiated in the presence of each of the following scavengers: DABCO (1 mmol/l), sodium azide (10 mmol/l), P-carotene (1 mmol/l), DMF/methanol (1 mmol/l and 50 mmol/l, respectively). Only sodium azide did not react with the reagents for the H,O, assay. Therefore only this scavenger could be employed for this purpose. The effects of sodium azide at 0, 1, 5 and 10 mmol/l are shown in Table III. These findings show that 10 mmol/l tide decreased the H,O, formation during irradiation of PP, CP and UP by approximately 29%, 48% and 458, respectively. Discussion Our comparison of the formation of H,O, during the irradiation of the three porphyrins showed that UP produced the largest amount, PP was the least effective and CP was intermediate between UP and PP. It is obvious that the H,O, formation is parallel to the lipid solubility of porphyrins and inversely related to their solubility in water. The H,O, formation is also inversely related to the photolytic efficacy of these porphyrins, where the potency decreased in the following order PP, CP, UP [18]. Previous reports have shown that the PP-induced photolysis of Ehrlich ascites carcinoma cells was not inhibited by SOD or catalase, indicating that the cell lysis was not mediated by either superoxide or H,O, [18]. On the other hand, the photolysis produced by CP and UP was slightly enhanced by SOD and was markedly inhibited by catalase. It was suggested from these results that the cell lysis
380
due to UP might be mediated by H,O,, where as that due to PP may be mediated by singiet oxygen [18]. The present results demonstrating that more H,O, was formed during irradiation of UP and CP compared to PP provide more direct evidence for the above postulated mechanism. It was observed (Table III) that the formation of H,Oz was inhibited by sodium azide, a strong quencher of singlet oxygen [22,23]. Two other singlet oxygen quenchers, viz. DABCO and DMF, were found to interfere with the assay for H,O,; hence these could not be employed in these experiments. The inhibition of H,O, formation by sodium azide indicates that the formation of H,O, during irradiation of the porphyrins was mediated by singlet oxygen. The in vitro observations described in this paper are in good agreement with the clinical findings with patients with EPP and PCT. Several reports show that the patients with EPP, where the photosensitivity is due to PP [11,12], respond well to p-carotene, which is a good singlet oxygen scavenger [22-251, while P-carotene does not protect against the photosensitivity in PCT, in which case the photosensitivity is due to uroporphyrin. This is understandable if the photosensitivity due to UP is mainly mediated by H,O,. Acknowledgements This work was supported by a grant from Canada. H.F.H. is an Associate of Ontario Foundation.
the Medical Research Council of Cancer Treatment and Research
References 1 Harber LC, Baer RL, Bickens DR. Fundamental and clinical aspects of the porphyrias. In: Pathak MA, Harber LC, Seiji M, Kukita A. Sunlight and the man. Tokyo: University of Tokyo Press, 1972;631-653. 2 Slater TF. Free radical mechanisms in tissue injury. London: Pion Ltd., 1972. 3 Valemeno DP. Photomodification of biological membranes with emphasis on singlet oxygen mechanisms. Photochem Photobiol 1987;46:147-160. 4 Lipson RL, Baldes ET. The photodynamic properties of a particular hematoporphyrin derivative. Arch Dermatol 1960;82:508-516. 5 Dougherty TJ. Photosensitizers: therapy and detection of malignant tumors. Photochem Photo&o1 1987;45:879-889. 6 Volden G, Christensen T, Moan, J. Photodyna~c membrane damage of hematopo~hy~n derivative treated NHIK cells in vitro. Photobiochem Photobiophys 1981;3:105-111. 7 Girotti AW. Mechanisms of photosensitization. Photochem Photobiol 1983;38:745-751. 8 Moan J, Pettersen EO, Christensen T. The mechanism of photodynamic inactivation of human cells in vitro in the presence of hematoporphyrin. Br J Cancer 1979;39:398-407. 9 Valenzeno DP. Photomodification of biological membranes with emphasis on singlet oxygen mechanisms. Photochem Photobiol 1987;46:147-160. 10 Spikes JD. Photosensitization in mammalian cells. In: Parrish JA, Kripke ML, Morison WL, eds. Photoimmunology. New York: Plenum Medical Book Co., 1983;23-49. 11 Eales L. Clinical chemistry of the porphyrias. In: Dolphin D, ed. The porphyrins. Vol. VI. New York: Academic Press, 1979;663-804. 12 Hindmarsh JT. The porphyrias. Clin Biochem 1986;32:1255-1263.
381 13 Sandberg S, Romslo I, Hovding G, Bjomdal T. Porphyrin-induced photodamage as related to the subcellular localizing of the porphyrins. Acta Dermatovenerol Suppl. 1982;100:75-80. 14 Sandberg S, Glette J, Hoppen G, Solberg CO, Romslo I. Porphyrin-induced photodamage to isolated human neutrophils. Photochem Photobiol 1981;34:471-475. 15 Sandberg S, Romslo I. Porphyrin-induced photodamage at the cellular and the subcellular level as related to the solubility of the porphyrin. Clin Chim Acta 1981;109:193-201. 16 Wakulchik SD, Schiltz IR, Bickers DR. Photolysis of protoporphyrin-treated human fibroblasts in vitro: studies on the mechanism. J Lab Clin Med 1980;96:158-167. 17 Sandberg S, Romslo I. Porphyrin-sensitized photodynamic damage of isolated rat liver mitochondria. Biochim Biophys Acta 180;593:187-195. 18 Menon IA, Persad S, Haberman HF. A comparison of the phototoxicity of protoporphyrin, coproporphyrin and uroporphyrin using a cellular system in vitro. Clin Biochem 1989;22:197-200. 19 Menon IA, Persad S, Ranadive NS, Haberman HF. Effects of ultravioletvisible irradiation in the presence of melanin isolated from human black or red hair upon Ehrlich ascites carcinoma cells. Cancer Res 1983;43:3165-3169. 20 Persad S, Menon IA, Haberman HF. Comparison of the effects of UV-visible irradiation of melanins and melanin-hematoporphyrin complexes from human black and red hair. Photochem Photobiol 1983;37:63-68. 21 Frew JE, Jones P, Scholes P. Spectrophotometric determination of hydrogen peroxide and organic hydroperoxides at low concentrations in aqueous solutions. Anal Chim Acta 1983;155:139-150. 22 Burton GW, Ingold KU. B-Carotene: An unusual type of antioxidant. Science 1984;224:569-573. 23 Mathews-Roth MM. Porphyrin photosensitization and carotenoid protection in mice: In vitro and in vivo studies. Photochem Photobiol 1984;40:63-67. 24 Krinsky NI. Photobiology of carotenoid protection. In: Regan JD, Parrish JA, eds. The science of photomedicine. 1982;397-407. 25 Mathews-Roth MM. Photobiology of carotenoid protection. In: Regan JD, Parrish JA, eds. The science of photomedicine. 1982;409-410.
37s
CCA 04648
Quantitation of hydrogen peroxide formed during UV-visible irradiation of protoporphyrin, coproporphyrin and uroporphyrin 1. Aravind
Menon’, Mary Ann C. Becker 2, Suruj D. Persad 3 and Herbert F. Haberman 4
’ Departments of Medicine and Ophthalmology,
’ Department of Medicine, 3 Department of Ophthalmology, 4 Department of Medicine, University of Toronto, Toronto ON (Canada)
(Received
21 July 1989; revision received 25 September
Key words: Porphyrin;
Photosensitizer;
1989; accepted
Light; Hydrogen
peroxide;
30 September
1989)
Singlet oxygen
Summary Free porphyrins are strong photosensitizers. Previously reported findings indicate that the in vitro cell lysis induced by irradiation in the presence of coproporphyrin (CP) and uropo~hy~n (UP) is mediated by H202 and that induced by irradiation with protopo~hy~n (PP) is not mediated by H,02. In the present study the possible role of H,O, in the porphyrin photosensitization was investigated by direct measurement of the H,O, formed during the irradiation of PP, CP and UP. Our results show that the amount of H,O, formed decreased in the following order: UP, CP, PP. The amounts of H202 formed during irradiation of CP and PP were appro~mately 86% and 38% respectively in comparison to the H,02 formed during the irradiation of UP. The formation of H,O, was inhibited by sodium azide, a strong quencher of singlet oxygen. These observations are in good agreement with the previous report that the in vitro photolysis of Ehrlich ascites carcinoma cells by UP and CP, but not that by PP, was inhibited by catalase and clinical findings with patients with erythropoetic protoporphyria (EPP) and porphyria cutanea tarda (PCT). The patients with EPP, where the photosensiti~ty is due to PP, respond well to P-carotene while p-carotene does not protect against the photosensitivity in PCT, in which case the photosensitivity is due to uroporphyrin.
Correspondence Bldg., University
to: Dr. LA. Menon, Departments of Medicine of Toronto, Toronto, Ontario, Canada.
0009-8981/89/$03.50
0 1989 Elsevier Science Publishers
and OphthalmoIogy,
B.V. (Biomedical
Division)
Medical
Sciences
376
Introduction Porphyrins are well recognized to be natural metabolites in the mammalian organisms and are formed during the biosynthesis of heme and the cytochromes. Free porphyrins are strong photosensitizers; on the other hand, the Fe-containing species, viz. heme and cytochromes are devoid of the photosensitizing activity [l-3]. The disorders in the porphyrin metabolism, termed ‘porphyrias’ represent several types. The classification of the porphyrias varies depending upon the clinical symptoms, the organs involved or the biochemical manifestation. Thus one classification divides the porphyrias into the hepatic or erythropoetic types. From the clinical standpoint the porphyrias may be classified into those producing cutaneous manifestations and those causing systemic diseases. These are discussed in a large number of reviews and books [l-7]. There is abundant evidence to show that singlet oxygen is formed during the irradiation of the porphyrins, presumably by the transfer of the excess energy of the porphyrins in the excited states to oxygen. Several reports have indicated that singlet oxygen may be involved in the phototoxicity of the porphyrins [6-lo]. Two common porphyrias where photosensitivity is a major clinical manifestation are erythropoetic protoporphyria (EPP) and porphyria cutanea tarda (PCT). However the cutaneous manifestations of these two diseases differ. Similarly the porphyrins accumulated in the tissues and the porphyrins excreted in these disease states differ. EPP is characterized by the accumulation of abnormally high levels of protoporphyrin (PP) in red blood cells, whereas high levels of uroporphyrin (UP) and coproporphyrin (CP) in plasma and urine are characteristic of PCT [11,12]. The reasons why the symptoms differ in PCT and EPP are not clearly understood. It has been postulated that these differences may result from the differences in localization of these porphyrins. This effect can be expected to be a composite function of solubility and subcellular localization of the particular porphyrins concerned [13]. Certain photobiological investigations on leukocytes [14], red blood cells [15], fibroblasts [16] and mitochondria [17] have provided evidence in support of this mechanism. We have recently reported that the photosensitized lysis of Ehrlich ascites carcinoma cells induced by UP and CP is inhibited by catalase where as the cell lysis produced by PP is not affected by catalase [lS]. These findings indicate that the phototoxicity of CP and UP is mediated by H,O, and that of PP is not mediated by H,O,. The possible role of H,O, in the porphyrin photosensitization was investigated by direct measurement of the H,O, formed during the irradiation of PP, CP and UP. This paper describes a quantitative comparison of the formation H,O, during the irradiation of these porphyrins. Materials and methods Materials The porphyrins, PP, CP and UP were obtained as free acids from Porphyrin Products Inc. (Logan, UT, USA). Superoxide dismutase (SOD) (specific activity
3 570 U/mg protein) and catalase (spec act 46 200 U/mg protein), horse radish peroxidase (spec act 310 U/mg solid), 4-amino-antipyrine and p-carotene were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Sodium azide, H,O,, methanol and phenol were purchased from Fisher Scientific Limited (Fair Lawn, NJ, USA). 1,4’-Diaza-bicyclo[2.2.2]octane (DABCO) was obtained from Eastman Kodak Company (Rochester, NY, USA). 2,5-Dimethylfuran (DMF) was purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). Stock solutions of PP, CP and UP were made in 3.0 mol/l, 0.1 mol/l and 1.0 mol/l HCl, respectively. These solutions were brought to pH 7 with NaOH. Appropriate dilutions were made with 0.01 mol/l phosphate buffer pH 7.4; 0.35 ml was added to the irradiation medium. All porphyrin solutions were contained in opaque glassware and stored in the dark. Irradiation Various concentrations of PP, CP and UP, in the presence of SOD or other reagents as mentioned in each section, were irradiated for the specific periods, using a Westinghouse Mercury Vapour lamp. This source emits discontinuous radiation (320 to 700 nm), with peaks at 360,400, 430, 550 and 580 nm [19,20]. The irradiance was measured using either a narrow band filter (365 nm) or no filter. The irradiance with the filter was 23 k 2.98 Watts/sqM, and without filter 170 + 9.75 Watts/sqM. All solutions were irradiated without the filter. Determination of H202 H,O, was assayed according to the method of Freu et al. [21]. The reagent for H,O, assay consisted of 5.85 mg phenol, 2.50 mg 4-aminoantipyrine, 5.0 pg horseradish peroxidase and 1.25 ~1 10 mmol/l H,O, to stabilize the phenol-C aminoantipyrine complex in 0.5 ml 0.01 mol/l phosphate buffer, pH 7.4, in a total volume of 3.5 ml. The absorbance was read at 505 nm on a Gilson 2400 spectrophotometer. The amount of H,O, formed was determined using a standard curve. Analysis Each sample. similar
of results experiment was performed at least four times, using triplicates for each The mean and standard error of the mean (SEM) for each sample from all experiments were calculated.
Results Formation of H,O, during irradiation of different porphyrins for various periods of time Table I shows the formation of H,O, during irradiation of PP, CP and UP for various periods of time. A fixed concentration of PP, CP and UP was irradiated for 0, 10, 15, 30, 45 and 60 min in the presence of SOD and the reagents for H,Oz assay. It was found that the amount of H,O, formed during irradiation of PP increased with respect to time up to 60 min. While the amount of H,O, formed during irradiation of CP and UP also increased with respect to the irradiation period, the H,O, formation was not proportional to the period of irradiation. At 60
378
TABLE
I
Formation
of H,O,
during
irradiation
of different
porphyrins
for various
periods
of time
Time
H,O,
(mm)
PP
CP
UP
0 10 15 30 45 60
1.2kO.9 2.6kO.3 3.8*1.0 7.8 k 2.6 11.5*4.2 14.2 f 2.3
0.1 kO.1 18.6i1.3 20.7 + 1 .O 25.1 f 0.9 26.3 f 0.7 31.7*0.9
o.o+o.o 14.7 * 2.2 21.9k3.1 31.7*4.5 36.2i2.2 37.0 + 2.0
The final concentration
formed (mean f SEM)
of PP. CP and UP was 1000 nmol/l.
The H,O,
formed
is expressed
as pmoI/I.
min, the amounts of H,O, formed during irradiation of CP and PP were approximately 86% and 38% respectively in comparison to the H,O, formed from UP. Formation of H202 during irradiation of various concentrations of different porphyrins A comparison of the amounts of H,O, formed during the irradiation of various concentrations of PP, CP and UP is shown in Table II. The amount of H,O, formed during the irradiation of each of these porphyrins was approximately proportional to the porphyrin concentration up to 1 mmol/l. The H,02 formation decreased in the order: UP, CP, PP. The amounts of H,O, formed from PP and CP at 1 ~mol/l were approximately 44% and 77% in comparison to that formed from UP. The possibility that the H,O, formation was mediated by superoxide was investigated, by omitting SOD from the system. Since the spontaneous dismutation (in the absence of SOD) of superoxide to produce H,O, is slow it was expected that omission of SOD would lower the amount of H202 formed in the system. As may
TABLE Formation Porphyrin (nmol/l)
II of H,O,
during
H,Oa
250 500 1000
0.0 3zo.o 5.8 + 2.4 8.9 k3.1 16.8 f 3.6
of various
concentrations
of different
porphyrins
formed (mean f SEM) CP
PP + SOD
0
irradiation
- SOD
UP
+ SOD
-SOD
+ SOD
- SOD
0.0 f 0.0 2.3 i-1.4 4.1 + 1.8 6.2 k1.7
0.0 + 0.0 7.3 k3.4 17.6 f 4.3 38.4 f 4.2
0.0 * 0.0 1.3 f 1.9 2.7 k1.6 7.0 *1.2
0.0
0.0
*o.o
*o.o
1.7 & 1.9 2.2 *1.9 6.2 f 1.1
8.7 f 3.0 16.8 f 3.4 29.7 + 4.0
The irradiation period was 60 min. The final concentration expressed as pmoI/l.
of SOD was 180 U/ml.
The H,O,
formed
is
379 TABLE
III
Effects of sodium
azide on the formation
Sodium
H ,O,
azide
(mmoI/I)
0 1 5 10
of H,O,
during
irradiation
of various
porphyrins
formed (mean k SEM)
PP
UP
CP
Light
Dark
Light
Dark
Light
Dark
15.0*1.4 14.6 f 1.4 12.8 f 0.7 10.7 + 1.2
5.9 f 1.0 5.6kl.O 5.6* 1.2 5.7+0.8
40.7+1.1 35.1 k 2.8 21.6 k 3.6 21.Ok3.9
3.8+0.9 4.1* 1.4 4.4 f 2.4 5.0+2.0
42.5 + 3.6 35.9k4.1 27.1 i 2.8 23.2 + 3.0
3.6+1.1 3.7 f 1.0 4.OkO.7 4.3 5 0.8
The final concentration of PP, CP and UP was 1000 nmol/l. H,O, formed is expressed as pmol/l.
The irradiation
period
was 60 min. The
be seen from the results in Table II, when SOD was omitted from the irradiation mixture, the formation of H,O, was decreased. Thus at 1 pmol/l concentration of the porphyrins, the quantities of H,O, formed from PP, CP and UP were decreased by 63%, 79% and 82% when SOD was omitted. Effects of singlet oxygen scavengers
upon the formation
of H,O,
during the irradiation
of UP
The effects of various scavengers of singlet oxygen on the formation of H,O, were investigated. PP, CP and UP were irradiated in the presence of each of the following scavengers: DABCO (1 mmol/l), sodium azide (10 mmol/l), P-carotene (1 mmol/l), DMF/methanol (1 mmol/l and 50 mmol/l, respectively). Only sodium azide did not react with the reagents for the H,O, assay. Therefore only this scavenger could be employed for this purpose. The effects of sodium azide at 0, 1, 5 and 10 mmol/l are shown in Table III. These findings show that 10 mmol/l tide decreased the H,O, formation during irradiation of PP, CP and UP by approximately 29%, 48% and 458, respectively. Discussion Our comparison of the formation of H,O, during the irradiation of the three porphyrins showed that UP produced the largest amount, PP was the least effective and CP was intermediate between UP and PP. It is obvious that the H,O, formation is parallel to the lipid solubility of porphyrins and inversely related to their solubility in water. The H,O, formation is also inversely related to the photolytic efficacy of these porphyrins, where the potency decreased in the following order PP, CP, UP [18]. Previous reports have shown that the PP-induced photolysis of Ehrlich ascites carcinoma cells was not inhibited by SOD or catalase, indicating that the cell lysis was not mediated by either superoxide or H,O, [18]. On the other hand, the photolysis produced by CP and UP was slightly enhanced by SOD and was markedly inhibited by catalase. It was suggested from these results that the cell lysis
380
due to UP might be mediated by H,O,, where as that due to PP may be mediated by singiet oxygen [18]. The present results demonstrating that more H,O, was formed during irradiation of UP and CP compared to PP provide more direct evidence for the above postulated mechanism. It was observed (Table III) that the formation of H,Oz was inhibited by sodium azide, a strong quencher of singlet oxygen [22,23]. Two other singlet oxygen quenchers, viz. DABCO and DMF, were found to interfere with the assay for H,O,; hence these could not be employed in these experiments. The inhibition of H,O, formation by sodium azide indicates that the formation of H,O, during irradiation of the porphyrins was mediated by singlet oxygen. The in vitro observations described in this paper are in good agreement with the clinical findings with patients with EPP and PCT. Several reports show that the patients with EPP, where the photosensitivity is due to PP [11,12], respond well to p-carotene, which is a good singlet oxygen scavenger [22-251, while P-carotene does not protect against the photosensitivity in PCT, in which case the photosensitivity is due to uroporphyrin. This is understandable if the photosensitivity due to UP is mainly mediated by H,O,. Acknowledgements This work was supported by a grant from Canada. H.F.H. is an Associate of Ontario Foundation.
the Medical Research Council of Cancer Treatment and Research
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