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

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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

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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.

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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,

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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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