A Polarographic Study on the Effects of Ultraviolet Light on Scales, Epidermis, Dermis and Serum*

A POLAROGRAPHIC STUDY ON THE EFFECTS OF ULTRAVIOLET LIGHT ON SCALES, EPIDERMIS, DERMIS AND SERUM* RYOHEI OGURA, M.D., PH.D., JOHN M. KNOX, M.D. AND MASAKAZLT KUSTJHARA, M.D.

When the skin is irradiated by ultraviolet nic. Scale, separated epidermis, dermis and light a number of different effects are known to serum were examined before and after irradiaoccur; however, the mechanism of action of tion to look for possible changes in the protein ultraviolet on the skin is not adequately under- wave pattern on the polarogram. stood. Several hypotheses have been advanced, MATERIALS AND METHODS yet very little is actually known. When a moleNormal skin specimens were obtained from the cule absorbs a photon of ultraviolet light it is either raised to a higher energy level or is dis- abdomen of autopsy material. Subcutaneous fat was trimmed from the dermis immediately upon associated. Molecules may dissipate this energy removal. Later the epidermis was separated from by fluorescence, collision with another molecule, the dermis by Baumberger's ammonium method or a reaction with other molecules at collision (4). After washing with cold ether three times at (1). Possibly other as yet unrecognized pathways room temperature, the defatted epidermis was are equally important. Proteins are known to allowed to stand for several minutes in a small

beaker immersed in dry ice and acetone. Following

absorb ultraviolet in the erythema producing

this procedure the material was lyophilized by

spectra and changes in proteins may be directly or indirectly responsible for one or more of the

the usual technic. The lyophilized epidermis was

refrigerated in a tightly sealed container under one atmosphere of nitrogen. The separated dermis was also lyophilized but instead of being pulverized it was scraped with a sharp scalpel. Psoriatic scales were collected from hospitalized patients.

harmful effects of ultraviolet irradiation. Included among the adverse effects of ultraviolet are sunburn, degeneration of connective tissue, carcinogenesis, and photosensitivity phenomena. Since ultraviolet wavelengths are absorbed by thin layers of material such as a sheet of paper it has long been thought that all or almost all

These scales were prepared and preserved as

described in a previous publication (5). The serum

used in these studies was obtained from horse blood. Samples of the above described solid materials

of the erythema producing wavelengths are were spread over the surface of a Petri dish and absorbed by superficial layers of the epidermis

irradiated at room temperature with a hot quartz

diffusible substance which produces vaso dila-

irradiated material was polarographed immedi-

(stratum corneum primarily) and that ultra- mercury vapor ultraviolet light source* at a disof 25 cm. The serum formed a layer about 1 violet erythema is caused by the release of a tance cm. thick in a Petri dish (diameter 2.8 cm.). The

tation and inflammation (2). The pathway ately after irradiation. Nonirradiated control whereby this substance is released and the samples were similarly studied. chemical nature of this agent have not been identified. Kirby-Smith, et al (3), have shown that only a small percentage of ultraviolet light in the erythema producing range (up to 10% with untanned skin) passes through the epidermis and penetrates into the dermis. However, a small percentage of a large amount can be significant.

Polarographic Procedure (6)

Following irradiation study samples were

immediately homogenized in a cold saline solution (immersed in ice water to prevent degeneration by the heat of grinding). Thirty mg. of epidermis were homogenized in 2 ml. of saline, 50 mg. of dermis in 3 ml., and 15 mg. of scale in 2 ml. The homogenate

was kept in the ice bath until it was pipetted

vessel. In this presentation the effects of ultraviolet into a polarographic 1. Brdicka's Total Protein Reaction irradiation on epidermal and dermal protein A sample of 0.1 g. of pepsin was dissolved in

100 ml. of 0.05 N HC1, and 5 ml. of the resulting

were studied by employing a polarographic tech-

solution was heated on a water bath to 40° C.

* From the Department of Medical Chemistry, Kurume University School of Medicine, Kurume, Japan, and the Departments of Dermatology and

The 0.2 ml. of serum or 0.5 ml. of skin homogenate being analyzed, was added and heated at the same

temperature for 30 minutes. The heated solution

Biochemistry, Baylor University College of Medicine, Houston, Texas. Received for publication March 14, 1962.

* Acme 37

Mercury Lamp, Japan 200 watts

38

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TABLE 1 Water evaporation by irradiation

5.0 ml. (Petri dish)

Serum

25.0 cm.

Distance Room Temperature

23.8°C. Weight of Serum

Time of Irradiation

Temperature

Loss of Weight

of Serum

After Irrad.

Before Irrad.

0

15 minutes 30 minutes

45 minutes 60 minutes

24.2 27.0

28.0

28.2

28.8

—

—

5.097 g 5.102

5.052 g

—0.045 g

5.052

—0.050

5.068

5.024

5.055 5.062

4.992 4.999

5.063

4.995

5.062

4.988

5.067

4.994

5.061

4.987

5.064

5.002

5.059

4.990

—0.044 —0.063 —0.063 —0.068 —0.074 —0.073 —0.074 —0.062 —0.069

5.063

5.007

—0.056

(0.2 ml.) was diluted with 10 ml. of cobaltous solution (0.001 M CoC12, 0.1 N NH4CI, 0.1 N NH3) and

polarographed* from —1.0 volt against the saturated calomel electrode. 2. Brdicka's Filtrate Reaction To the serum or skin homogenate 0.1 N KOH

was added (in the amount specified below) at room temperature. After allowing the solution to stand for 45 minutes 20% sulfosalicylic acid was added and the solution was shaken vigorously in order to obtain a homogenous mixture. After 10 minutes, the precipitated proteins were separated

— —0.88% —0.98% —0.87% —1.25% —1.24% —1.34% —1.46% —1.44% —1.46% —1.22% —1.36% —1.11%

irradiation was 24° C. and following irradiation was 28° C. On solid skin samples it is not necessary to consider water evaporation; however, on serum

it is necessary to determine whether water evapo-

ration has taken place. Table 1 illustrates that

the loss of water from serum was approximately 0.9 to 1.5% and not great enough to significantly alter results. RESULTS

The catalytic protein wave of irradiated serum was increased when compared to the The filtrate (0.5 ml.—1.0 ml.) was added to 5 ml. protein wave of control serum. This was true of cobaltic solution (0.001 M Co(NH3)eCls, 0.1 N NH4C1, 1 N NH3), and after mixing, polaro- when serum was irradiated directly and also graphed from —0.8 volt against the saturated after precipitation with sulfosalicylic acid (see calomel electrode. Table 2). Sixty minutes of irradiation gave a from solution by filtering through filter paper.

lower value than 15 minutes in the total protein KOH

20%

2

0.1 N

Serum Epidermis 30 mg/2m1

Dermis

IN

Filtrate

for o Sulfosali- Polarcylic Acid ography

0.5 ml 1.0 — — 1.0 ml — 0.1 0.4

1.0 0.5 ml 1.0 0.5 ml

1.0 ml — 0.1 0.4 1.0

0.7 ml

reaction, but the highest value in the filtrate reaction.

For scale, the catalytic wave in both reactions was decreased by ultraviolet irradiation and it appears that the height is lowered gradually with increasing amounts of ultraviolet irradiation. However, with statistical calculation

(a = 5%), there is no significant difference between the three irradiation times in the total protein reaction, but 30 minutes gave a significantly lower value than 15 minutes in the filSamples were irradiated in open air at room trate reaction. The results are given in Table 3. temperature. The temperature of serum before Table 4 shows the height of the protein wave * for epidermis before and after irradiation. There Yanagimoto Polarograph, Japan, PA 102 50 mg/3m! Scale 0.5 ml — 0.1 — l5mg/2m1

2.0

1.0 ml

39

ULTRAVIOLET EFFECT ON SCALES, EPIDERMIS, DERMIS AND SERUM

TABLE 2 Height

of the protein wave following ultraviolet irradiation of serum Total Protein Reaction

Specimen No.

Filtrate Reaction Irradiation Time

Irradiation Time(Min.)

Control

Control

mm

15

30

45

60

mm

15

30

45

60

1

74

78

75

75

75

39

46

50

47

52

2

74

77

81

81

77

70

74

70

76

79

3 4

59

63

66

59

63

68

61

61 54

61

63

63

64

61

66

56

70

71

54

59

63 55

66 58

70

71

73 71

63 54

68

6 7

62 62 65 72

59

5

67 66 66

55

57

59

60

62

Mean

57

66.4

63

67 72 72

70.9

(Average Deviation from Control)

73

70.4

70.0

1.06

1.07

68.9

55.6

60.1

1.04

1.05

61.0

61.1

1.09

1.11

1.11

—

64.1

1.16

0.2 eA:0.06 ,A

Sensitivity:

Parallel capacitance: 600 MF:400 eF

TABLE 3 Height of the protein wave following ultraviolet irradiation of scale Total Protein Reaction Specimen No.

Filtrate Reaction

Irradiation Time (Mm)

Irradiation Time

Control

1

60 mm

2

48 42 55 52 55

3 4

5 6

Mean

52.0

Deviation (Average from Control)

Control 15

30

60

90

98 90 89

89 86 87

108

102

90

88 88 83 93 87

98

91

15

30

60

54 44

55 41

100

38 52

39 51 44

51 40 41

42 43 50

105 107

48 51

51

47.8

46.8

0.92

44.5

0.90

Sensitivity:

mm

91

100.2

94.5

0.86

0.95

88.3

0.89

100 84 99 90.8

0.91

0.04 pA:0.06 MA

Parallel capacitance: 200 pF:200 MF

was no significant change in either the total Analysis of the data revealed that there were no protein reaction or the filtrate reaction.

significant differences between the control and 15 minutes, or between the 30 and 60 minute dermis are presented in Table 5. With the total value. The filtrate reaction was not appreciably protein reaction, the height of the protein wave altered by ultraviolet irradiation. The above data was studied by an analysis of at 30 and 60 minutes was lowered by irradiation.

Results obtained following irradiation of

40

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TABLE 4 Height of the protein wave following ultraviolet irradiation of epidermis Filtrate Reaction

Total Protein Reaction

Irradiation Time

Irradiation Time (Mm)

Specimen No.

Control

Control 15

30

60

31

35

38 44 53 53

38 41

54 54

1

34mm

2

35

3

41

34 37 45

4 5

55

56

52

55

Mean

45.4

43.4

Derivation (Average from Control)

44.4

43.8

1.05

15

30

60

86mm

87

88

84

75

65

90

68

66 70

73

72

81

88

88

84

83

98

93

85

78.6

1.04

1.02

1.01

83.0

81.4

81.8

1.06

1.03

0.04 MA:0.06 MF Sensitivity: Parallel Capacitance: 200 rF:200 viF

TABLE 5 Height of the protein wave following ultraviolet irradiation of dermis Filtrate Reaction

Total Protein Reaction

Irradiation Time

Irradiation Time (Mm)

Specimen No.

Control

1

2

3

4 5 Mean

30 31 35 34 36

Control

mm

33.2

Deviation from Control)

15

30

60

40 30 29 36 41

20 26 23 33 35

22 26

27.4

35.2

(Average

1.07

30

60

100 110 114 110

107

110 123 108

114 102

105 120 113 105

98

95

90

99

104 mm

21

29 35

26.6

0.82

-

15

108.6

1.058

0.82

117

106.0

0.98

0.98

108.4

1.00

0.04 ,A:0.06 rA Sensitivity: Parallel Capacitance: 200 MF:200 ,F

variants technic to determine its statistical

DISCUSSION

significance (a = 5%). Figures 1 and 2 graphically illustrate this data with deviations from the control for the total protein reaction being plotted on Figure 1 and deviations for the filtrate reaction being shown in Figure 2.

typical double wave on the polarograph. Brdicka, who has extensively studied normal and patho-

Complex compounds such as proteins give a

logic sara with the polarograph, concludes that in the total protein reaction alterations may be

ULTRAVIOLET EFFECT ON SCALES, EPIDERMIS, DERMIS AND SERUM

41

Deviation

1.10

07

1 •

Serum

1 •

00

Time of Irradiation 45

30

60

0.90

Scale

0.

111/ No Statistically

significant Difference from Control

FIG. 1

present in constituents containing the cystine

Both Stricks (7) and Wenig and Jirovec (8)

group. With the filtrate reaction, important found that the catalytic double wave height for changes have been found to occur in mucoproteins containing hexosamine and polysaccharide. Brdicka has demonstrated significant differences between serum from patients with cancer, other pathological states, and normal sera. The most specific changes were thought to occur in the filtrate reaction (6).

serum was increased following ultraviolet light

irradiation. In this study irradiated serum revealed changes similar to those reported by these authors. The height of the curve was decreased with scale in both the total protein and filtrate reaction, and decreased with dermis in only the total protein reaction. Interestingly,

42

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1.20

Serum

1.10

Time of Irradiation

1.00 Dermis

Scale

0.90

No Statistically significant Difference from Control

FIG. 2

liberated on denaturation and the change in the epidermis even after 60 minutes of con- structure of the denatured protein molecule tinuous irradiation. However, it must be em- becomes evident from the change in shape of the phasized that this is an in vitro study and that catalytic double wave. The effect of ultraviolet radiation upon prooniy powdered samples were irradiated. Disulfide or sulfhydryl groups are liberated teins was investigated polarographically by when a protein molecule is denatured. In most Wenig and Jirovec (8) and these authors rethere were no statistically significant changes in

cases the polarographic examination of protein ported an increase in both of the double protein solutions during the denaturation process shows waves. Greater increase was found in the second an increase in the catalytic protein waves. Po- wave and the ratio of the two reached a maximum larographically active groups are apparently after a certain period of irradiation. The protein

ULTRAVIOLET EFFECT ON SCALES, EPIDERMIS, DERMIS AND SERUM

started to precipitate after this maximum had been reached and at this point both waves then

43

SUMMARY

1. The effect of ultraviolet irradiation on the

decreased although the ratio remained the same. According to Schmidt (9) the effect of ultraviolet light on protein solutions increases as the concentration of the protein is decreased. Undiluted

protein of powdered scale, epidermis, and dermis and on serum protein was studied by employing a polarographic technic.

bumins have been found to be effected more by

3. Epidermis manifested noteworthy stability;

2. Scales, serum and dermis showed striking serum did not show an irradiation effect. Al- alterations.

irradiation than globulins. The presence or no significant changes were found following absence of oxygen does not appear to alter the intensive ultraviolet irradiation. effect of ultraviolet irradiation on albumin solu4. It is possible that although only a small tions, as determined by the polarograph (10). percentage of the ultraviolet from sunlight X-rays do not produce similar polarographic reaches the dermis, it is in this area that the changes for Heeren and Seuberling (11) ir- most significant biological changes occur. radiated protein solutions with 100,000 r. and REFERENCES detected no appreciable effect on the height or 1. BLUM, H. F.: Photodynamic Action and shape of the catalytic waves. This result is Diseases Caused by Light. American Publishing Corporation, 1941. somewhat surprising since ultraviolet and x-rays 2. BLUM, H. F.: Carcinogenesis by Ultraviolet are quite similar in their biological action. Light: An Essay in Quantitative Biology,

Magnus (12) placed psoriatic scales and scales from patients with chronic dermatitis in a boiling water bath for ten minutes. The mean sulfhydryl values for psoriatic scale was reduced to half by boiling; whereas, the sulfhydryl values in chronic

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The effect of heat on protein is generally recognized as producing two types of change; either denaturation or a recoiling of the protein molecule. The findings in this experiment indicate that ultraviolet may also be capable of producing denaturation or a recoiling of protein molecules within the skin.

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