Investigations into the Etiology of Dermatitis Herpetiformis

Vol. 45, No. 2

Ton JOUENAI. OF INVESTIOAT1VE DERMATOLOOY

Copyright

1965 by The Williams & Wilkins Co.

Printed in U.S.A.

INVESTIGATIONS INTO THE ETIOLOGY OF DERMATITIS HERPETIFORMIS II. THE EFFECTS OF SERUM FROM DERMATITIS HERPETITORMIS CASES AND OF CERTAIN DRUGS ON THE VISCOSITY RED UCT[OY PROPERTY OF HYALURONIDASE *

J. O'D. ALEXANDER, M.B., Css.B.

In a previous paper (1) it was shown that of 3.0 ml and a "falling time" for distilled water serum from eases of dermatitis herpetiformis

of about 200 seconds. Tubes With similar falling

should be used or results may vary con(D.H.) had the property of accelerating the times siderably.

spreading effect of hyaluronidase and that this action was inhibited by treatment with Dapsone. The reduction of viscosity of solutions of hyaluronic acid or its sodium or potassium salts is another means of assessing hyaluronidase activity. Normal human serum and many other substances such as heparin, magnesium

and phosphate ions, ACTH, cortisone and high concentrations of some antihistamines (2) inhibit this action of hyaluronidase. The effect of human serum can itself be inhibited by some of these substances in weak concentration; and certain substances, notably halo-

Two stop-watches.

A thermostatically controlled water bath with mechanical stirrer is essential. The viscometer is charged by means of a Pasteur pipette sufficiently long in the capillary portion to reach the lower storage bulb and thus avoid air locks. The test solutions are forced from the storage arm of the U tube to the testing arm by positive pressure. Test Solutions. Substrate-buffer mixture. (subsequently referred to as substrate): Four volumes of 0.5% commercial hyaluronic acid in distilled water

are mixed with one volume of molar sodium citrate at pH 4.6 (this buffer contains approximately one volume of molar citric acid and 2

volumes of molar sodium hydroxide). The hyalu-

acid was extracted from tumor tissue and gen ions, have an accelerating effect on ronic was obtained from Light and Co. Ltd. It gave

hyaluronidase viscosity reduction at specific consistent readings with different batches. Ten mg concentrations. Glick and Gollan (3) and Grais and Glick (4, 5) have shown that this hyaluronidase activity is markedly inhibited by serum in certain acute infections such as poliomyelitis,

was found sufficient for 12 tests. Fresh substrate must be made for each day's tests. Enryme solution: Commercial testicular hyaluronidase (fly-

alase-Benger) is dissolved in water so that 0.1 ml of the solution contains 2 units of enzyme. subacute bacterial endocarditis and primary Less than 2 units resulted in too slow a reaction

atypical pneumonia and in several skin dis- and 5 or 10 units was too fast except when testing orders such as pemphigus vulgaris, acute lupus for strong inhibitory power (see Fig. 2). Test Sera. Serum was obtained from normal erythematosus and erythema multiforme. In all persons, from patients with netive and controlled these conditions inhibition was most marked at D.H. and from patients said to be in the febrile

the height of the infection and returned to stage of pneumonia. The latter scm were obtained normal as the condition subsided. In the few sera of D.H. which they tested the effects were normal. Pyrexia did not increase the inhibitor effect of normal serum.

through the courtesy of Dr. R. G. Sommerville, Virologist to Belvidere Hospital for Infectious Diseases, Glasgow. All sera were diluted at the time of testing so that 0.05 ml of solution con-

tained 0.01 ml of serum. Therapeutic Agents. Dapsone, liquor arsenicalis the D.H. sera findings and to ascertain whether and other substances were suitably diluted to the any drugs used in the treatment of this disease concentrations indicated in Table III. Sulfapyridine could not be tested because it precipitated from influences hyaluronidase activity. solution at pH 4.6 Method. The tests were carried out in the water MATERIALS AND METHOD bath at 35.5°C. Between tests the viscometer was Viscometer. A simple vertical U-tube Ostwald cleaned by successive rinsing with warm water, viscometer (Fig. 1) was used. It had a capacity absolute alcohol and ether. The reaction mixtures were prepared as follows: Received for publication April 17, 1963. a) Substrate 1.75 ml plus enzyme solution 02 * From the Department of Dermatology, Glasml plus distilled water 0.05 ml. gow Royal Infirmary, Glasgow, Scotland.

The present tests were intended to verify

73

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

74

t

.t

F

1. Fm. 1. Apparatus for determination of viscosity, arranged to show the various components but not in the normal working manner as the latter is inconvenient for photography. knurled screws at the corners of the base A. Electronic relay connected to board B. Heating element and controlled by H. Thermometer C. Toluene regulator set to 35.5° C. I. Basket containing reaction ingredients subD. Mechanical stirrer merged below the water level indicated by E. Upright arm of stand, set exactly at right the pencil line on the tank. angles to the base. J. Ostwald viscometer. (It is in the wrong clamp in the picture because otherwise it obscures F. Horizontal arm exactly at right angles to the the other components. It is consequently not upright arm quite vertical.) G. Two-dimensional spirit level adjusted by

b) As for a) but the enzyme first boiled for the value for reaction mixture a). If the inhibitory activity of the test substances is denoted by A

30 minutes.

c) As for a) but 0.5 ml of test solution substi- then tuted for distilled water. d) As for c) but the enzyme first boiled for 30 minutes.

— A = F. F0

F0

The ingredients were kept at 35.5° C for 10 The curves from which the value of F may be minutes before preparing these mixtures. Because the inhibitory effect of the test substances ascertained are obtained from the test readings might be delnyed the enzyme and test substance were mixed 10 minutes before adding to the substrate and testing. Test. The reaction is timed by stop-watch from the moment the mixture is completed. It is trans-

taken at times designated to 12

= I + 1i

ferred at once to the viscometer and serial readings where I is the time on the first stop-watch at of "falling time" are taken. In the case of mixture which the reading is started and to is the reading a) the readings are taken until they are constant. on the second stop-watch for each reading. Thus, = 60 seconds and the falling time = 290 seconds, The falling time is plotted against reaction time and the "half-viscosity" time determined. There- then

after readings for the other mixtures are taken until the half-viscosity level is reached. Celculotions. The time, in seconds, taken to reach half-viscosity level is designated R. F8 is

the value for F of the test substance and F0

12 = 60

+= 290

205.

t is plotted as ordinate and t as abscissa.

75

HYALURONIDASE IN DERMATITIS HERPETIFORMIS RESULTS

325

The results of the viscosity reduction tests have been expressed as values of the factor A. Examples of the different reaction curves

Th00

ton at 275

r>

325 250

w 300 225

'5, z,

Falling ti,ne of co,npl.fe ronation.

200

4 '4.

175

5

II

15

20

25

35

40

RE.TION TIME (mins.)

Fic. 4. Viscosity diagram for Dapsone. At very

weak concentration it has a slight accelerating effect but at a concentration of 0.001 mg per test there was a strong inhibitory effect. At higher concentrations the inhibitory action was complete. REACIIcSJ TIME (mats,)

X = Substrate + enzyme + water; • = Sub-

substrate with different amounts of hyaluronidase.

strate + enzyme + Dapsone, 0.0001 mg; Q = Substrate + enzyme + Dapsone, 0.001 mg.

nated the relative half-viscosity time (R). It can be seen from the graph that with 5 or 10

Results of viscosity tests for cases of dermatitis herpetiformis The figures are for values of A (see text). There

Fic. 2. Graph showing the fall in viscosity of

The "half-viscosity" level lies midway between the falling time of the inactive reaction mixture and that of the completed reaction. The point at which the various curves cross this line is desig-

TABLE I

units per ml. the value for R is very small and consequently any small changes, would be very is no statistical difference between the two difficult to detect and measure. With 1 unit per ml the time is too long and for this reason the amount of 2 units per ml was chosen as the most suitable for the tests.

0 = 1 unit of hyaluronidase per ml; + = 2 units of hyaluronidase per ml; X = 5 units of = 10 units of hyaluhyaluronidase per ml; ronidase per ml.

groups.

Case

Active state

Controlled state

H. C. I. F. F. MeG. T. A. D. M. R. H. J. R. F. B. F. B. J. S. A. H.

—0.50

+2.25 +0.39

Mean

+0.26 +2.04 +0.33 +0.41 +0.70

—0.26

+0.43 +0.45 +0.10

—0.03

—0.04

+0.09 +1.30 +0.96 +1.19

+0.02

+0.61

+0.42

are given in figures 2 to 4 and the values for A in Tables I to IV. The results were analyzed 35 REACTION

40 35 TIME (mins.)

statistically using "Student's" 't' test. Comparing the figures for active and controlled

Viscosity diagram for a serum from a dermatitis herpetiformis in Table I with those case of dermatitis herpetiformis. The serum has a for normal sara in Table I, active serum was slightly inhibiting influence on the reaction. The less inhibitory than normal serum but just not FIG. 3.

value for A is +0.26.

X = Substrate + enzyme + water; 0 Sub- significantly so at P = 0.05. After treatment, however, the dermatitis herpetiformis serum

strate + enzyme + serum.

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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

TABLE II

nlscussloN

Results of viscosity tests with febrile pneumonia sera and normal sera

The determination of viscosity of biological

fluids is set about by many complicating The figures are for values of A (see text). The factors among which arc PH, ionic concentra-

febrile sara were significantly less inhibitory of hyalurouidase activity than normal sera. Test Serum Febeile Sera

Value foe A

Test Serum Normal Sera

Value foe A

1

+0.47 +0.34 +1.30

a

+1.51 +2.72 +1.02 +0.82 +0.74 +1.20

2 3 4 5 6

Mean

b C

d e

—0.17

+0.25

f

—0.03

+0.36

tion of salts, optimal temperature, the choice

of substrate and even of viscometer. The

presence of small amounts of sodium chloride

may make a very great difference to the results. In low concentration it has an accelerating effect on the reaction (6). For this reason the substrate in these tests has been dissolved

in water rather than in weak solution of

+1.335

TABLE III Values of A with varying amounts of drugs tested

sodium chloride as used by Madinaveitia and Quibdll (7). The pH of 4.6 has been shown to be optimal for hyalurnnidase activity (8.6), although there is still considerable activity for a wide margin on either side of this figure. The choice of substrate is important. The commercially prepared sodium salt of hyaluronic acid described here gave easily reproducible results.

The amounts indicated were the actual quanThe results detailed reveal one or two intertities of drug present in 2 ml of reaction mixture. esting findings. As far as the various sera are Inhibitor

Amouot of Inhibitor io the Test Mixture

concerned Value of A

evidence

there is little comment to make. The

confirms the findings of Glick at al.

concerning serum of D.H. The sera of the

mg

Daps one

1.0

Complete

in-

hibitinn

0.01 0.001 0.0001

Potassium iodide

0.1

+ 1.45

+0.45 —0.12 Complete

+0.96 +0.28

As2Os as liquor arsenicalis

0.001 0.0001

+0.72 +0.68

Hydrocortisone acetate

0.01

+ 1.57

Phenergan

0.01

+0.99

Heparin

12.5 units

Test Substances

in1 2 6

+0.22 —0.15 —0.20

Normal serum a

+0.08

b

+0.08

Active serum H. C.

J. 17. Complete

in-

j

hibition

Dapsnne, 0.001 mg

was significantly less inhibitory thau normal serum (P lay between 0.05 and 0.02). A possible explanation of tbis anomaly is given below.

Values of A

Febrile serum

hibition

0.001 0.0001

TABLE IV Values for A when the test substances were tested against 10 units of hyaluronidase per ml of substrate mixture

Potassium iodide, 0.001

mg Heparin, 12.5 units

—0.20 —0.15

+0.22 Complete inhibition

HYALURONIDASE IN DERMATITIS HERPETIFORMIS

patients with pneumonia did not give the resuits anticipated but this may be because the exact time during the disease at which the sera were taken was not accurately specified. Heparin was confirmed as a potent inhibitor of hyaluronidase activity. Dapsone was also shown to be powerfully inhibitory, even at

77

hyaluronidase of solutions of hyaluronic acid. The inhibitory effect is rather less than that of normal serum but does not tally exactly with the findings in skin spreading (1). Dapsone, which is an excellent therapeutic weapon in D.H., is also a powerful hyaluronidase inhibitor.

Added to the previous finding that Dapsone weak concentration. At a concentration of inhibits the accelerating effect of DII. serum 0.0001 mg per test, however, there was slight on the spreading property of hyaluronidase, accelerating activity (Fig. 5). A clinical dose of this may have some significance on the mecha100 mg Dapsone completely absorbed into a nism of action of Dapsone in D.H. blood stream with a volume of 5 liters would SUMMARY result in a plasma concentration approximately Examination by viseometry had shown that equivalent to 0.07 mg in 2 ml (the volume of the reaction mixture). At approximately 15% serum from dermatitis herpetiformis has an inhibitory effect slightly less than that of norof this strength (0.01 mg) Dapsone was mal serum on the viscosity-reducing property strongly inhibitory. On the other hand 0.01 ml of such serum, which is the amount of of hyaluronidase. Dapsone, used for the treat-

serum used in the test, would contain about ment of dermatitis herpetiformis, is strongly 0.00035 mg. This is the maximum probable inhibitory at levels likely to be achieved by amount in any of the reaction mixtures used therapeutic doses. in these tests, many of which would contain REFERENCES only 0.0001 mg. This has a slight accelerating effect on viscosity reduction and might explain why serum from Dapsone-treated patients has a less inhibitory effect than that from untreated patients. Of the other substances tested the most interesting was potassium iodide a well known

cause of acute exacerbation of D.H. when administered orally. It had an inhibitory action on viscosity reduction which varied with concentration. These apparent contradictions may

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