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Barbiturate Concentrations in the Skin and Hair of Guinea Pigs1
BARBITURATE CONCENTRATIONS IN THE SKIN AND HAIR OF GUINEA PIGS* 1ST LT. RAYMOND W. GOLDBLUM, MC., LEO R. GOLDBAUM, PH.D. AND LT. COLONEL WILLIAM N. PIPER, M.C.
Barbiturates are known to frequently cause dermatitis medicamentosa. Soon after the introduction of barbital in 1903 and phenobarbital in 1911, cases of dermatitis were reported from the use of these drugs. The etiology of barbiturate
dermatitis remains unknown. Potter and Whitacre expressed the opinion that long acting barbiturates cause skin eruptions more frequently than short acting barbiturates. The greatest number of barbiturate dermatitides reported in the literature are produced by phenobarbital followed by barbital. To illustrate the incidence of dermatitis due to phenobarbital, Menniger (1), in 1928, reported that 1.2 per cent of 167 patients administered phenobarbital developed a dermatitis. Huddleston (2) observed 1000 cases of epileptics receiving phenobarbital and
reported that 22 cases or 2.2 per cent developed a dermatitis medicamentosa. Many cases of barbital dermatitis (3, 4, 5, 6,7,8,9) have also been reported. How-
ever, reports of dermatitis caused by short acting barbiturates are infrequent. Only isolated reports appear in the literature. One of the explanations for the greater number of cases of dermatitis medicamentosa from long-acting barbiturates than short-acting ones may be the difference between their distribution in and elimination from the skin. Since no data on skin levels of barbiturates have been reported in the literature, a study was performed on guinea pigs to determine the concentration of various barbiturates in the skin, the length of time that they remained and the relation between plasma and skin levels. Since phenobarbital is prescribed over long periods of time, the concentration of barbiturates in the hair was studied. EXPERThIENTAL
I. A comparison of plasma and skin barbiturate concentrations after administration of long acting and short acting barbiturates
Guinea pigs weighing 350 to 500 grams were injected intraperitoneally with aqueous solutions of phenobarbital, barbital, amytal, thiopental, and pentobarbital with doses ranging from 50 to 150 mg./kg. of body weight. The guinea pigs were sacrificed at varying intervals of time by fracturing the cervical spine. Three to 5 ml. of blood were drawn by intracardiac puncture and the blood was collected in oxalate tubes. The plasma was separated by centrifugation. Samples of shaved skin were excised from the middorsal region of the back, and the fascia and fat were carefully trimmed away. The skins were placed in weighing bottles and the weights of the samples were recorded. To prepare the skin samples for analyses, it was necessary to remove the water by placing the * From the Department of Dermatology and Syphilology, and the Department of Bio. chemistry, Army Medical Service Graduate School, Washington, D. C. Received for publication July 15, 1953. 121
122
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
TABLE I Barbital levels in skin and plasma NO.
DOSE
(NO.1KG.)
CONDITION
WET SKIN
I
rusE
PLASMA (MICRO.
(flOuRs)
GLUSS/ML.)
(MICROGRAMS/
92
45 53 37 42
.49 .53 .61 .41
47
.87 .92 .77 1.07 .75 .78
cus.)
WET SKIN /PLASMA
h-hour levels* 1
2 3 4
75 75 75 75
Light Light Light Light
100
I
60 102
2-hour Ievelsf 1
2 3 4 5 6
50 50 50 150 150 150
2 2 2 5 5 5
Light Light Light Deep Deep Deep
54 46 52
43 40 143
134 179
134 116
148
* Wet skin/plasma: Mean = .51 + or — .07. t Wet skin/plasma: Mean = .86 + or .04.
TABLE II Phenobarbilal levels in skin and plasma NO.
DOSE
(MG./KG.)
TThSE
CONDITION
I
(HouRs) I
PLASMA
I
WET SKIN
(MIcROG&&us/ (WCR0GL&sss/ MI..) GM.) j
WET SKIN! PLASMA
s-hour levels5 1
2 3 4 5 6 7
100 100 100 150 150 150 150
Deep Deep Medium
Light Deep Deep Deep
'
111 111 104 75 160
101
98.5 80.5
.91 .81 .78 .73 .77 .76 .70
142
55 124 108
180
125
50
43
40 42
41
34
92 89 96 73 97 90
73 73
.78
87 57 72 72 93
.91
2- to 7-hour levelst 1 2 3
50 50 50
4 5
100 100 100 100 100 100 150 150 150
6
7 8 9 10 11 12
Light Light Light Deep Light Deep Light Medium Medium Deep Deep Deep
* Wet skin/plasma: Mean = t Wet skin/plasma: Mean =
.77 .82
2 2 2 4 4 7 7 7 7 5 5 5
+ or — .06. + or — .07.
125 125 112
113
81
.79
.97 .83 .82 .78 .74 .80 .74 .90 .73
TABLE III Amytal levels in skin and plasma NO.
DOSE
(No.1KG.)
I
ONDIflON
TIME
(HouRs)
PLASMA
I
WET SKIN
(sncRoGRAJSS/ I (MIcRoGcs/ o&.)
I I
WET SKIN! PLASMA
h-hour levels* 1 2 3
4 5
6 7
75 75 75 75 75 75 75
3'
Deep Deep Deep Deep Deep Deep Deep
3
3' 3'
53 67 63 56 58
36 62 51 52 42
.68 .93 .82 .92 .73
63
37
.59
60
52
.87
17 15
11 12
.65 .80
2-hour leveist 1
50
2
50
3
50 100
4
5 6 7
100 100 100
2 2
Light Light Light
2
15
10
.70
Deep Deep Deep Deep
2 2 2 2
56
33 43 24 30
.59
52
45 49
.78 .55 .60
* Wet skin/plasma: Mean = f
Wet skin/plasma:
.79 + or — .04. Mean = .67 + or .09.
TABLE IV Perdobarbital levels in skin and plasma NO.
DOSE
(Mo./Ko.)
CONorrION
TIME
(HouRs)
PLASMA (MICROGRAMS!
WET SKIN
(MIcRooRAMs/ GM.)
I
WET SKIN/ PLASMA
s-hour levels* 1
50
Deep
2
50
Deep
3 4 5 6 7 8 9
50 50 50 50 50 50 50 75 75 75
Deep Deep Deep Deep Deep Deep Deep Deep Deep Deep
10 11
12
3'
j 3'
',
39
30.4 31.0
.95 .80
47.5
21.0
.44
36
19.5
.54
35 32
24 27 24 22 27 27
.67
21
.30
60
20
.32
15 14
.68
19 21
15
.71
32
40 42
32 73 70
.83 .60 .53 .83
.36
2-hour leveist
2 2 2 2
22
50
Deep Deep Deep Deep
27
11
.70
50
Light
2
12
7
.58
1
50
2 3 4 5
50
50
* Wet skin/plasma: Mean .57 + or — — t Wet skin/plasma: Mean = .68 + or
.18. .05.
123
.71
124
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
weighing bottles with skin in a vacuum oven at 50°C. overnight. The dry weights were recorded, and the dry skins were powdered in a Wiley mifi. It was necessary to dry the skin before powdering in a Wiley mill, since wet skin cannot be milled. A sample of the powdered skin equivalent to 1 to 3 grams of wet skin was used
for analysis. The concentration of barbiturates in the skin was calculated by dividing the concentration of barbiturates in the dry skin by the weight of dry skin weight of wet skin
The barbiturate concentration in the plasma and skin were determined by the ultraviolet spectrophotometric method of Goldbaum (10). The results are shown in Tables I to IV. II. A comparison of the disappearance of barbiturates from plasma and skin Guinea pigs were injected intraperitoneally with anesthetic doses of representative long-acting and short-acting barbiturates. The doses were 150 mg./kg. for phenobarbital and barbital, 75 mg./kg. for amytal and 50 mg./kg. for thiopental and pentobarbital. Samples of blood and skin were collected 3' hour after the injection as indicated in Experiment I. The skin wounds were closed by skin clips. Five hours after the injection, samples of blood and skin were again collected. The h-hour and 5-hour specimens of skin and plasma were analyzed for barbiturates. The results are shown in Tables V and VI. TABLE V Thiopental levels in skin and plasma NO.
DOSE
(MG/KG.)
I
COITION
TTh
(Ros)
PLASMA
WET SKIN
(ucRoGLcs/ (MICROGRAMS! J
GM.)
I
WET SKIN/ PLASMA
s-hour levels* 1
50
2 3 4 5 6 7
Deep
50
Deep
8
9
50 50 50 50 50 50 50
Deep Deep Deep Deep Deep
3-
3'
3
Deep Deep
42 36
25 28 26 30 39 27 33
25 15 11 15 15
.58 .41 .39 .52
20 20 14 17
.55 .63 .47 .48 .49
10
.42
10 12 11
.40 .50 .51
2-hour leveist 1 2 3 4
50 50 50
50
Medium Medium Medium Medium
2 2 2 2
* Wet skin/plasma: Mean = .50 + or — .07. t Wet skin/plasma: Mean .45 + or — .05.
23 23 23 20
BARBITURATE CONCENTRATIONS IN GUINEA PIGS
125
TABLE VI A comparison of the barbiturate levels in the plasma and skin at h-hour and 5-hour intervals* DOSE
(MG/KG.)
CONDITION
TIME
PLASMA
WET SKIN
(HouRs)
(MIcR0GRAMS/ML.)
(MICROGRAMS/GM.)
Phenobarhital 150 150 150
Light Light Deep Deep Deep Deep
5 5 5
75 125 160 125 142 112
55 93 124 113 109
157
139 211 179
85 143 186 134
223
152
148
116
23 9
7 5
36
20
7 35 12
3 24 7
42
22
10
7
25 21 30
11
81
Barbital
150 150
Light Deep Medium
Deep 150
5 5
Deep
Deep
5 Pentobarbital
50 50
Light Light Deep
Light 50
5
Deep
Light 50
5
5
Deep
Light
5 Thiopental
50 50
Medium Medium Deep Deep
50
Deep
50
Deep Deep Deep
3 4 4
16
6 20 13
27
14
13 14
10 17 11
58
42
20 53
36
33
4 Amytal
75
Deep Light
75
Deep Light
75
In all
5
Deep Light
*
5
Deep Light
75
5
5
6
15
3
68
37
4
5
60
52
6
8
cases, the same guinea pig was used for the j-hour and 5-hour specimens.
126
TIlE JOURNAL OF INVESTIGATIVE DERMATOLOGY
TABLE VII Comparison of plasma, skin and hair lrrels for phenobarbital NO.
DOSE
DAYS OF AD-
(OR/DAY)
SIINISTRAT1ON
1
12
2 3 4
]4
S
9 6 6 5 8
PLASMA (MICROGRAMS/
ML.)
53.9 37 33 280 51
SKIN (MICROGRASIS/ GM.)
HAIR
TIME (HOURS)
(MICROGRAMS/
SPECIMENS
GM.)
COLLECTED
47 35
4000 4760
25
295
252 48
620
12
24 24
159
12
24
TABLE VIII Phenoharbilat lerels in hair DOSE
NO.
1
(no/DAY) I
2 3
II
S
6
'ii
DAYS OF ADMINISTRA-
TiON
11 11 11 11 11 11
HAIR )MICR0GRAM5/ GM.)
CONCENTRATION IN HAIR ONE MONTH AFTER DISCONTINUING
1545
392 508 645 654 235
DRUG
0 0 0 0 0 0
PRES IOUSLY UNSHAS RD AREA ONE MONTH AFTRR DISCONTINUING DRUG MICROGRAMS/GM.)
157
57
III. Concentrations of phenobarbital in hair of guinea pigs One group of five guinea pigs was injected intraperitoneally with grain of phenobarbital daily for five to nine days. The animals were sacrificed by frac-
turing the spine and samples of blood, shaved hair and shaved skin were collected and analyzed for phenobarbital concentrations. In two animals the collections were made 12 hour after the last injection, and in three animals the collections were made 24 hours after the last injection. Results are shown in Table VII. In another group of six guinea pigs, the hair on the back was shaved with an Oster hair clipper. One-half grain of phenobarbital was injected intraperitoneally daily for 11 days. An area of the 11-day growth of hair on the back was shaved and analyzed for phenobarbital. One month after discontinuing the phenobarbital, a sample of hair was collected again from the same area that contained the 30-day growth after discontinuing phenobarbital, and other samples from another
area which included the 11-day growth plus the 30-day growth after the drug was discontinued. These samples were analyzed for phenobarbital. Results shown
in Table VIII. RESULTS AND DISCUSSION
All the five barbiturates were detected in the skin and plasma of guinea pigs as early as thirty minutes after intraperitoneal administration. After administering the same dose, the long-acting barbiturates (barbital and phenobarbital) attained higher levels in the skin and plasma than the short-acting barbiturates
BARBITURATE CONCENTRATIONS IN GUINEA PIGS
127
(pentobarbital, thiopental and amytal). In all instances plasma levels were greater than skin levels. Varying doses did not alter significantly the wet skin to plasma ratios. With the exception of barbital, the plasma/skin ratios for hour and two to seven hours were approximately equal for all the barbiturates. With barbital there was a significant difference between the h-hour and 2-hour concentrations.
This probably indicates that equilibrium between plasma and skin was not reached in } hour. The plasma levels reflect the skin levels since they increased with an increase in the plasma levels and decreased correspondingly with no tendency for the barbiturates to be bound in the skia. Phenobarbital was found to enter the hair and was deposited there when the level was maintained for six or more days. There was no correlation between concentrations in plasma, skin and hair. Thirty days after discontinuing the drug, the phenobarbital could no longer be detected in the 30-day growth of hair in the area previously shaved, but it could be found in other areas of the same guinea pig, which had not been shaved.
These results indicate that if the plasma phenoharbital concentration is not maintained long enough for the hair cells to leave the hair bulb, the barbiturates will be deposited in such low concentrations that they cannot be detected. These experiments also indicate that there is an equilibrium between the cells of the germinating layer of the hair bulb and the plasma. CONCLU5ION5
As stated in the introduction, long-acting barbiturates, especially phenobarbital and barbital, are responsible for the greatest number of barbiturate dermatitides. The results of these experiments reveal that phenobarbital and barbital have higher plasma and skin concentrations than short-acting barbiturates after
similar doses are administered. After five hours, the long-acting barbiturates maintained high plasma and skin concentrations while the plasma and skin concentrations for the short-acting barbiturates \vere markedly decreased. It is known that phenobarbital and barbital remain in the body of humans for long periods of time. An estimated rate of fall in plasma concentration is 20 per cent (11) every 24 hours. From our studies one may speculate that the same skin/ plasma ratios that are found in guinea pigs are present in humans. The shortacting barbiturates in humans will fall approximately 35 per cent to 60 per cent (12) every 24 hours. Thus, the long-acting barbiturates may be present for longer periods of time in the plasma and skin, and would be more likely to sensitize.
Further work on this problem is indicated. It would be interesting to separate the dermis and epidermis in humans to determine the barbiturate concentration in lesions of dermatitis medicamentosa. In addition, the detection of barbiturates in hair may be valuable in toxicologic studies. SUMMARY
Barbiturate levels in the plasma and skin for phenobarbital, barbital, amytal, pentobarbital and thiopental were determined by spectrophotometric methods,
128
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
and their significance discussed. This may serve as a base line study in determining the etiology of dermatitis medicamentosa. Phenobarbital levels were also determined in the hair of guinea pigs. REFERENCES 1. MENNINOER, W. C.: Skin eruption with phenobarbital. J. A. M. A., 41: 14, 1928. 2. IITJDDLESTON, J. H.: Non-dermatological disturbances during continual treatment
with phenobarbital. J. A. M. A., 93: 1637, 1929. P. W.: Rash following barbitone-sodium. Brit. M. J., 323, 1926. 4. ARIAS, B. R.: Erythrodermia produced by use of veronal. Arch. de Med., cir. y. espec., 3. MATHEW,
26: 773, 1927. Pathogenesis of skin lesions from barbital and its compounds. Bull. et med. Soc. Med. d. hop. de Paris, 49: 1483, 1925. 6. CAUTIER, P.: Poisoning from veronal. Rev. med. de la Suisse Rom., 38: 641, 1918. 7. VALLERY-RADOT, P., AND BLAMONTIER, P.: Effects on skin and mucosa from barbital and its compounds, Bull. et med. Soc. Med. d. hop. de Paris, 49: 1382, 1925. 5.
S.
9.
Csiissrin, C., TAEDIEU, A., AND LEJARD, C.:
TARDJEU, A., AND BLONDEL, A.: Skin eruptions from poisoning with veronal and derivatives of barbituric acid. Rev. de Med., 42: 255, 1925. VANIER,
101:
J.: Erythemas due to barbital and its derivatives. J. de Med. et Chir. Prat.,
509, 1930.
10. GOLDBAUM, L. R.: Determination of barbiturates. Analyt. Chem., 24: 1604, 1952. 11. SHJDEMAN, F. E., COULD, T. C., WINTERS, W. D., PETERSON, R. C., AND WILNER,
W. K.:
and
The distribution
and in vivo rate of metabolism of thiopental. Jour. of Pharm.
Exptl. Therap., 107: 368, 1953.
R. S., WALKER, J. T., AND PLUMMER, B. S.: Quantitative estimation of barbiturates in blood by ultraviolet spectrophotometry. Am. J. of Clin. Path., 18: 462,
12. FISHER,
1948.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
94
linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.
Barbiturates are known to frequently cause dermatitis medicamentosa. Soon after the introduction of barbital in 1903 and phenobarbital in 1911, cases of dermatitis were reported from the use of these drugs. The etiology of barbiturate
dermatitis remains unknown. Potter and Whitacre expressed the opinion that long acting barbiturates cause skin eruptions more frequently than short acting barbiturates. The greatest number of barbiturate dermatitides reported in the literature are produced by phenobarbital followed by barbital. To illustrate the incidence of dermatitis due to phenobarbital, Menniger (1), in 1928, reported that 1.2 per cent of 167 patients administered phenobarbital developed a dermatitis. Huddleston (2) observed 1000 cases of epileptics receiving phenobarbital and
reported that 22 cases or 2.2 per cent developed a dermatitis medicamentosa. Many cases of barbital dermatitis (3, 4, 5, 6,7,8,9) have also been reported. How-
ever, reports of dermatitis caused by short acting barbiturates are infrequent. Only isolated reports appear in the literature. One of the explanations for the greater number of cases of dermatitis medicamentosa from long-acting barbiturates than short-acting ones may be the difference between their distribution in and elimination from the skin. Since no data on skin levels of barbiturates have been reported in the literature, a study was performed on guinea pigs to determine the concentration of various barbiturates in the skin, the length of time that they remained and the relation between plasma and skin levels. Since phenobarbital is prescribed over long periods of time, the concentration of barbiturates in the hair was studied. EXPERThIENTAL
I. A comparison of plasma and skin barbiturate concentrations after administration of long acting and short acting barbiturates
Guinea pigs weighing 350 to 500 grams were injected intraperitoneally with aqueous solutions of phenobarbital, barbital, amytal, thiopental, and pentobarbital with doses ranging from 50 to 150 mg./kg. of body weight. The guinea pigs were sacrificed at varying intervals of time by fracturing the cervical spine. Three to 5 ml. of blood were drawn by intracardiac puncture and the blood was collected in oxalate tubes. The plasma was separated by centrifugation. Samples of shaved skin were excised from the middorsal region of the back, and the fascia and fat were carefully trimmed away. The skins were placed in weighing bottles and the weights of the samples were recorded. To prepare the skin samples for analyses, it was necessary to remove the water by placing the * From the Department of Dermatology and Syphilology, and the Department of Bio. chemistry, Army Medical Service Graduate School, Washington, D. C. Received for publication July 15, 1953. 121
122
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
TABLE I Barbital levels in skin and plasma NO.
DOSE
(NO.1KG.)
CONDITION
WET SKIN
I
rusE
PLASMA (MICRO.
(flOuRs)
GLUSS/ML.)
(MICROGRAMS/
92
45 53 37 42
.49 .53 .61 .41
47
.87 .92 .77 1.07 .75 .78
cus.)
WET SKIN /PLASMA
h-hour levels* 1
2 3 4
75 75 75 75
Light Light Light Light
100
I
60 102
2-hour Ievelsf 1
2 3 4 5 6
50 50 50 150 150 150
2 2 2 5 5 5
Light Light Light Deep Deep Deep
54 46 52
43 40 143
134 179
134 116
148
* Wet skin/plasma: Mean = .51 + or — .07. t Wet skin/plasma: Mean = .86 + or .04.
TABLE II Phenobarbilal levels in skin and plasma NO.
DOSE
(MG./KG.)
TThSE
CONDITION
I
(HouRs) I
PLASMA
I
WET SKIN
(MIcROG&&us/ (WCR0GL&sss/ MI..) GM.) j
WET SKIN! PLASMA
s-hour levels5 1
2 3 4 5 6 7
100 100 100 150 150 150 150
Deep Deep Medium
Light Deep Deep Deep
'
111 111 104 75 160
101
98.5 80.5
.91 .81 .78 .73 .77 .76 .70
142
55 124 108
180
125
50
43
40 42
41
34
92 89 96 73 97 90
73 73
.78
87 57 72 72 93
.91
2- to 7-hour levelst 1 2 3
50 50 50
4 5
100 100 100 100 100 100 150 150 150
6
7 8 9 10 11 12
Light Light Light Deep Light Deep Light Medium Medium Deep Deep Deep
* Wet skin/plasma: Mean = t Wet skin/plasma: Mean =
.77 .82
2 2 2 4 4 7 7 7 7 5 5 5
+ or — .06. + or — .07.
125 125 112
113
81
.79
.97 .83 .82 .78 .74 .80 .74 .90 .73
TABLE III Amytal levels in skin and plasma NO.
DOSE
(No.1KG.)
I
ONDIflON
TIME
(HouRs)
PLASMA
I
WET SKIN
(sncRoGRAJSS/ I (MIcRoGcs/ o&.)
I I
WET SKIN! PLASMA
h-hour levels* 1 2 3
4 5
6 7
75 75 75 75 75 75 75
3'
Deep Deep Deep Deep Deep Deep Deep
3
3' 3'
53 67 63 56 58
36 62 51 52 42
.68 .93 .82 .92 .73
63
37
.59
60
52
.87
17 15
11 12
.65 .80
2-hour leveist 1
50
2
50
3
50 100
4
5 6 7
100 100 100
2 2
Light Light Light
2
15
10
.70
Deep Deep Deep Deep
2 2 2 2
56
33 43 24 30
.59
52
45 49
.78 .55 .60
* Wet skin/plasma: Mean = f
Wet skin/plasma:
.79 + or — .04. Mean = .67 + or .09.
TABLE IV Perdobarbital levels in skin and plasma NO.
DOSE
(Mo./Ko.)
CONorrION
TIME
(HouRs)
PLASMA (MICROGRAMS!
WET SKIN
(MIcRooRAMs/ GM.)
I
WET SKIN/ PLASMA
s-hour levels* 1
50
Deep
2
50
Deep
3 4 5 6 7 8 9
50 50 50 50 50 50 50 75 75 75
Deep Deep Deep Deep Deep Deep Deep Deep Deep Deep
10 11
12
3'
j 3'
',
39
30.4 31.0
.95 .80
47.5
21.0
.44
36
19.5
.54
35 32
24 27 24 22 27 27
.67
21
.30
60
20
.32
15 14
.68
19 21
15
.71
32
40 42
32 73 70
.83 .60 .53 .83
.36
2-hour leveist
2 2 2 2
22
50
Deep Deep Deep Deep
27
11
.70
50
Light
2
12
7
.58
1
50
2 3 4 5
50
50
* Wet skin/plasma: Mean .57 + or — — t Wet skin/plasma: Mean = .68 + or
.18. .05.
123
.71
124
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
weighing bottles with skin in a vacuum oven at 50°C. overnight. The dry weights were recorded, and the dry skins were powdered in a Wiley mifi. It was necessary to dry the skin before powdering in a Wiley mill, since wet skin cannot be milled. A sample of the powdered skin equivalent to 1 to 3 grams of wet skin was used
for analysis. The concentration of barbiturates in the skin was calculated by dividing the concentration of barbiturates in the dry skin by the weight of dry skin weight of wet skin
The barbiturate concentration in the plasma and skin were determined by the ultraviolet spectrophotometric method of Goldbaum (10). The results are shown in Tables I to IV. II. A comparison of the disappearance of barbiturates from plasma and skin Guinea pigs were injected intraperitoneally with anesthetic doses of representative long-acting and short-acting barbiturates. The doses were 150 mg./kg. for phenobarbital and barbital, 75 mg./kg. for amytal and 50 mg./kg. for thiopental and pentobarbital. Samples of blood and skin were collected 3' hour after the injection as indicated in Experiment I. The skin wounds were closed by skin clips. Five hours after the injection, samples of blood and skin were again collected. The h-hour and 5-hour specimens of skin and plasma were analyzed for barbiturates. The results are shown in Tables V and VI. TABLE V Thiopental levels in skin and plasma NO.
DOSE
(MG/KG.)
I
COITION
TTh
(Ros)
PLASMA
WET SKIN
(ucRoGLcs/ (MICROGRAMS! J
GM.)
I
WET SKIN/ PLASMA
s-hour levels* 1
50
2 3 4 5 6 7
Deep
50
Deep
8
9
50 50 50 50 50 50 50
Deep Deep Deep Deep Deep
3-
3'
3
Deep Deep
42 36
25 28 26 30 39 27 33
25 15 11 15 15
.58 .41 .39 .52
20 20 14 17
.55 .63 .47 .48 .49
10
.42
10 12 11
.40 .50 .51
2-hour leveist 1 2 3 4
50 50 50
50
Medium Medium Medium Medium
2 2 2 2
* Wet skin/plasma: Mean = .50 + or — .07. t Wet skin/plasma: Mean .45 + or — .05.
23 23 23 20
BARBITURATE CONCENTRATIONS IN GUINEA PIGS
125
TABLE VI A comparison of the barbiturate levels in the plasma and skin at h-hour and 5-hour intervals* DOSE
(MG/KG.)
CONDITION
TIME
PLASMA
WET SKIN
(HouRs)
(MIcR0GRAMS/ML.)
(MICROGRAMS/GM.)
Phenobarhital 150 150 150
Light Light Deep Deep Deep Deep
5 5 5
75 125 160 125 142 112
55 93 124 113 109
157
139 211 179
85 143 186 134
223
152
148
116
23 9
7 5
36
20
7 35 12
3 24 7
42
22
10
7
25 21 30
11
81
Barbital
150 150
Light Deep Medium
Deep 150
5 5
Deep
Deep
5 Pentobarbital
50 50
Light Light Deep
Light 50
5
Deep
Light 50
5
5
Deep
Light
5 Thiopental
50 50
Medium Medium Deep Deep
50
Deep
50
Deep Deep Deep
3 4 4
16
6 20 13
27
14
13 14
10 17 11
58
42
20 53
36
33
4 Amytal
75
Deep Light
75
Deep Light
75
In all
5
Deep Light
*
5
Deep Light
75
5
5
6
15
3
68
37
4
5
60
52
6
8
cases, the same guinea pig was used for the j-hour and 5-hour specimens.
126
TIlE JOURNAL OF INVESTIGATIVE DERMATOLOGY
TABLE VII Comparison of plasma, skin and hair lrrels for phenobarbital NO.
DOSE
DAYS OF AD-
(OR/DAY)
SIINISTRAT1ON
1
12
2 3 4
]4
S
9 6 6 5 8
PLASMA (MICROGRAMS/
ML.)
53.9 37 33 280 51
SKIN (MICROGRASIS/ GM.)
HAIR
TIME (HOURS)
(MICROGRAMS/
SPECIMENS
GM.)
COLLECTED
47 35
4000 4760
25
295
252 48
620
12
24 24
159
12
24
TABLE VIII Phenoharbilat lerels in hair DOSE
NO.
1
(no/DAY) I
2 3
II
S
6
'ii
DAYS OF ADMINISTRA-
TiON
11 11 11 11 11 11
HAIR )MICR0GRAM5/ GM.)
CONCENTRATION IN HAIR ONE MONTH AFTER DISCONTINUING
1545
392 508 645 654 235
DRUG
0 0 0 0 0 0
PRES IOUSLY UNSHAS RD AREA ONE MONTH AFTRR DISCONTINUING DRUG MICROGRAMS/GM.)
157
57
III. Concentrations of phenobarbital in hair of guinea pigs One group of five guinea pigs was injected intraperitoneally with grain of phenobarbital daily for five to nine days. The animals were sacrificed by frac-
turing the spine and samples of blood, shaved hair and shaved skin were collected and analyzed for phenobarbital concentrations. In two animals the collections were made 12 hour after the last injection, and in three animals the collections were made 24 hours after the last injection. Results are shown in Table VII. In another group of six guinea pigs, the hair on the back was shaved with an Oster hair clipper. One-half grain of phenobarbital was injected intraperitoneally daily for 11 days. An area of the 11-day growth of hair on the back was shaved and analyzed for phenobarbital. One month after discontinuing the phenobarbital, a sample of hair was collected again from the same area that contained the 30-day growth after discontinuing phenobarbital, and other samples from another
area which included the 11-day growth plus the 30-day growth after the drug was discontinued. These samples were analyzed for phenobarbital. Results shown
in Table VIII. RESULTS AND DISCUSSION
All the five barbiturates were detected in the skin and plasma of guinea pigs as early as thirty minutes after intraperitoneal administration. After administering the same dose, the long-acting barbiturates (barbital and phenobarbital) attained higher levels in the skin and plasma than the short-acting barbiturates
BARBITURATE CONCENTRATIONS IN GUINEA PIGS
127
(pentobarbital, thiopental and amytal). In all instances plasma levels were greater than skin levels. Varying doses did not alter significantly the wet skin to plasma ratios. With the exception of barbital, the plasma/skin ratios for hour and two to seven hours were approximately equal for all the barbiturates. With barbital there was a significant difference between the h-hour and 2-hour concentrations.
This probably indicates that equilibrium between plasma and skin was not reached in } hour. The plasma levels reflect the skin levels since they increased with an increase in the plasma levels and decreased correspondingly with no tendency for the barbiturates to be bound in the skia. Phenobarbital was found to enter the hair and was deposited there when the level was maintained for six or more days. There was no correlation between concentrations in plasma, skin and hair. Thirty days after discontinuing the drug, the phenobarbital could no longer be detected in the 30-day growth of hair in the area previously shaved, but it could be found in other areas of the same guinea pig, which had not been shaved.
These results indicate that if the plasma phenoharbital concentration is not maintained long enough for the hair cells to leave the hair bulb, the barbiturates will be deposited in such low concentrations that they cannot be detected. These experiments also indicate that there is an equilibrium between the cells of the germinating layer of the hair bulb and the plasma. CONCLU5ION5
As stated in the introduction, long-acting barbiturates, especially phenobarbital and barbital, are responsible for the greatest number of barbiturate dermatitides. The results of these experiments reveal that phenobarbital and barbital have higher plasma and skin concentrations than short-acting barbiturates after
similar doses are administered. After five hours, the long-acting barbiturates maintained high plasma and skin concentrations while the plasma and skin concentrations for the short-acting barbiturates \vere markedly decreased. It is known that phenobarbital and barbital remain in the body of humans for long periods of time. An estimated rate of fall in plasma concentration is 20 per cent (11) every 24 hours. From our studies one may speculate that the same skin/ plasma ratios that are found in guinea pigs are present in humans. The shortacting barbiturates in humans will fall approximately 35 per cent to 60 per cent (12) every 24 hours. Thus, the long-acting barbiturates may be present for longer periods of time in the plasma and skin, and would be more likely to sensitize.
Further work on this problem is indicated. It would be interesting to separate the dermis and epidermis in humans to determine the barbiturate concentration in lesions of dermatitis medicamentosa. In addition, the detection of barbiturates in hair may be valuable in toxicologic studies. SUMMARY
Barbiturate levels in the plasma and skin for phenobarbital, barbital, amytal, pentobarbital and thiopental were determined by spectrophotometric methods,
128
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
and their significance discussed. This may serve as a base line study in determining the etiology of dermatitis medicamentosa. Phenobarbital levels were also determined in the hair of guinea pigs. REFERENCES 1. MENNINOER, W. C.: Skin eruption with phenobarbital. J. A. M. A., 41: 14, 1928. 2. IITJDDLESTON, J. H.: Non-dermatological disturbances during continual treatment
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