Paper Electrophoretic Analysis of Serum Glycoproteins in Dermatitis Herpetiformis*

PAPER ELECTROPHORETIC ANALYSIS OF SERUM GLYCOPROTEINS IN DERMATITIS HERPETIFORMIS* J. O'D. ALEXANDER, M.B., Crs.B., F.R.F.P.S. (GLAsGow)

In recent years several papers have appeared describing the analysis of serum proteins in bullous diseases. Most of the work has been carried out on serum from cases of pemphigus. Lever et al. (1) showed that in pemphigus vulgaris there was a decrease of serum albumin and an increase in aj , al , fi and y globulins. They indicated that the increase in aj and a globulins could be attributed to increase in glycoproteins.

by dapsone therapy. Note that some of the active cases from whom serum was obtained were ren-

dered active by withdrawing treatment temporarily. Serum was also taken from one case of senile dermatitis herpetiformis (bullous pcmphigoid) and from 4 cases controlled by steroid

therapy. As controls, serum was obtained from 10 normal persons and 9 patients with eczema. The sera was stored in the deep freeze at —60° C. Estimation of total protein. The Biuret method

was used, the results being read in a Spekker

There was a tendency after treatment with absorptiometer at 520 mi with green filter (Kingssteroids for the albumin fraction to increase and

ley) (5).

Electrophoretic method. The sera were fracfor the aj and a2 fractions to return towards tionated by paper electrophoresis using Whatnormal although still remaining slightly elevated. Lever ci al. (2) analyzed serum from five cases

man's 3MM papS strips 4 em wide. For normal protein analysis 0.01 ml of serum was applied to of dermatitis herpetiformis. One, aged 73, had each strip, while for glycoproteins 0.05 ml of was applied. Electrophoresis was carried slight hypoproteinemia but the rest had normal serum out for 16 hours in a barbiturate buffer at pH 8.6 protein values. The electrophoretic patterns and ionic strength 0.1 M at 2 Ma per strip. The were all within normal limits. Lever and his col- strips were then dried and fixed for 10—15 minutes leagues quoted Robert (3), who analyzed serum at 100° C. Staining methods, a) Normal protein fractionafrom two cases of dermatitis herpetiformis and tion. The dried paper strips were stained for 10 found the total protein values normal but there minutes in 0.3% Lissamine Green SF150 (I.C.I.) was a slight decrease in albumin and a slight in- in 15% acetic acid. They were then washed for 20 crease in a globulins. The results in seventeen minutes in four successive rinses of 2% acetic acid,

other cases examined by four other groups of until the background was white (Gorringe (6)). b) Glycoproteins. The procedure used was that workers, were also mentioned by Lever. Their recommended by Bj Ufnesj 6 (7). findings were either normal or similar to those Reagents. Periodic acid. 3.6 g periodic acid and

of Robert but in a few cases there was a distinct 1.21 g sodium acetate (NaO'OC'C113'31120) are dissolved in 135 ml distilled water and then 300 ml hypoproteinemia. Apart from Lever's (1) mention of an increase absolute alcohol are added. The solution can be for up to 2 weeks if stored in the dark. in glycoproteins in the a and a2 fractions, no used Reducing solution. 15 g potassium iodide and reference has been found to the glycoproteins in 15 g sodium thiosulfate are dissolved in 300 ml dermatitis herpetiformis. In view of the findings of distilled water. Fuchsin sulphite solution. 2 g basic fuchsin are reported elsewhere (Alexander (4)) concerning the effects of serum from this disease on the dissolved in 400 ml of boiling water (distilled), spreading properties of hyaluronidase it was cooled to 50° C and filtered. To the filtrate add

decided to carry out paper electrophoretic analysis on the serum glycoproteins in cases of this skin disorder, to ascertain if there were any detectable abnormality. MATERIALS AND METHODS

Sera. Serum for analysis was taken from 16 patients with adult type of dermatitis herpetiformis and from seventeen patients under control * From

the Department of Dermatology,

Glasgow Royal Infirmary, Glasgow, Scotland.

10 ml of 2NHCL and 4 g potassium metabisulphite. The solution is kept in a well stoppered bottle in the dark at about 2° C overnight. On the following day add 1 g decolorizing carbon, mix and filter immediately. Add about 10 ml 2N .HCL in small amounts until after the last addition the

mixture does not turn violet-red when drying spontaneously at room temperature. Keep the solution in a cool dark place.

Sulfite rinse solution. 4 g of potassium metabisulphite are dissolved in 1000 . ml of distilled water and 10 ml of concentrated HCL are then added.

Received for publication December 17, 1960.

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TilE JOURNAL OF INVESTIGATIVE DERMATOLOGY

256

Staining procedure. 1) Fix in absolute alcohol for 5—10 minutes.

RESULTS

Table 1 shows the results of the examinations

2) Place in periodic acid solution for 5 minutes.

condensed to give the mean reading for each fraction, together with the standard deviation ethanol. 4) Place for 5 minutes in the following solution for both routine protein and glycoprotein fracimmediately after it is prepared: To 30 ml tionation. It will be observed that in addition 3) Rinse for 2—3 minutes in two changes of 70%

of the reducing solution above add 45 ml

of absolute ethanol in small amounts

stirring constantly to clear and then add 0.7 ml 2NHCL. It is essential for success-

to giving percentage figures for the albumin and a fractions separately, these are also given as a

combined figure. This was done because in ful staining that this solution be made several cases it was not possible, with the technic freshly for each batch of strips and used immediately it is made.

used, to separate these fractions clearly as shown

in figures 1—4. The results were analyzed sta-

5) Rinse for 2—3 minutes in two further changes tistically by Dr. S. D. Silvey of the Mathematics of 70% ethanol.

6) Place in fuchsin solution for about 40 minutes, until the maximum color develops. 7) Rinse 4 or 5 times, for 4—5 minutes each, in successive changes of sulfite rinse solution. Rinsing should be continued as long

as fuchsin sulfite can be washed out of

the strips. 8) Dehydrate in absolute alcohol.

Department of Glasgow University. The method used was the analysis of variance (F tests) and the results are given in Table 2. This shows that

there were significant differences between the groups in the a2 fractions of both protein and glycoprotein and it the -y fraction of the glyco-

proteins. Detailed analysis of the individual

This staining procedure is best carried out in groups of patients shows that those with signifishallow white enamel trays. It is important at each stage to see that the strips are fully immersed in cant differences from normal are the a2 protein fractions in the treated adult and senile dermathe solutions. After drying, the strips stained by both methods titis herpetiformis groups; the az glycoprotein were scanned in a reflection scanner and the pro- fractions of both active and treated dermatitis portions of each fraction traced on graph paper. herpetiformis; and the y globulins of active It was then easy to calculate the percentage of each fraction. As the total glycoproteins were not adult dermatitis herpetiformis and treated senile extracted chemically the amount in g is not given dermatitis herpetiformis. hut only the percentage. In normal sera, the values for total proteins TABLE 1

Showing the condensed results of paper electrophoretic analysis of protein and glyco proteins in various groups of cases Paper Electrophoretic Analysis

Eczema (9)

ul

4.8 4.62 1.28

54.1

6.57

50.95 5.05

55.3

0.35

6.41 0.83

6.3

52.3

5.9

0.55

5.1

1.8

5.3

TreatedD.H. 6.88 0.5 (18) Treated 6.46

54.3

Senile

0.52

14.4 5.1

12.3 1.93

16.1

12.15 16.2

10.2

13.9

2.6

2.03

3.27

2.89

4.98 59.3 8.8 13.4 6.58 1.8 5.28 2.13 2.9

46.2 6.7 7.78 0.78

ai

14.3 16.8 2.13 2.5

57.5 11.4

7.08 0.71*

6.84

D.H.

(17)

AIhu-

u2

5.3 58.3

Active

Glycoprntein percentage

(g/100 ml) Ath

Normals (9)

-_ —_ —

Protein percentage

.

Group Examined

16.6 3.71 17.4

7.3 10.4

3.74 5.8

19.0 31.8 3.75 3.6

152

/3

27.2 23.7

17.1

2.82 2.76 2.77

29.4 28.8 25.8 17.1 2.08 1.62 3.18 3.67 20.75 30.8 23.7 23.4 21.5 7.1

5.03 8.99 4.17

5.7

4.5

18.6 14.1 3.45 7.0

18.9 33.0 23.0 23.6 20.6 5.19 6.7 4.15 5.99 4.65

52.9 15.3 14.8 17.0 11.5 7.09 1.88 3.03 2.87 4.3

23.6 33.5 30.3 24.3 11.9 2.86 3:46 3.45 3.13 3.51

DR. (4)

* The figures in italics represent the standard deviations for the different groups for each fraction.

257

SERUM GLYCOPEOTEINS IN DERMATITIS HERPETIFORMIS

only significantly so after treatment with dapsone. However, these values are much the same as those given for normal persons by Neuhaus and Letzring (8). The glycoprotein values for normal persons

are much the same as those found by other workers (10, 11, 12) using similar technics

t"— O% I

(Table 3). In the adult eases of dermatitis herpetiformis there is a somewhat higher level of glycoprotein in the 'y globulin fraction than is P

seen in normal persons and in the untreated cases this is significantly higher. However, although the average value in the treated cases is lower than in the active eases, the results for 10

cases before and after treatment showed that while there generally tended to be a fall in the FIG. 1 shows the appearance of a paper strip

a FIGURES 1 TO 4

stained with Lissamine Green for protein and the

'y glycoprotein fraction after treatment, this

scanning diagram with the percentages of each was not invariably the case. This same tendency fraction written below. In this figure there is a clear definition of both the albumin and a, fractions.

FIG. 2 shows a similar strip in which the al-

Fru. 3 shows the appearance of a paper strip stained for glycoproteins and a similar scanning diagram as in Fig. 1. In this figure the glycoproteins of the albumin and a, fractions have separated fairly well.

bumin and a, fractions have not separated clearly.

and the percentage values of the various protein

fractions conform to the generally accepted values for normal persons. The results for the cases of eczema show essentially similar findings. In active adult cases of dermatitis herpetiformis,

there was a slightly higher percentage level of cr1

globulins

and also in the cases of treated

senile dermatitis herpetiformis and one ease of the same condition before treatment (this case is not in the table). These differences, however, were not significant. The levels of as globulins in both active and treated cases of adult dermatitis

.4

361,

FIG. 4 shows a similar strip to that in Fig. 3 but

in which the albumin and a, fractions have not herpetiformis were lower than normal though separated clearly.

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TABLE 2 Showing the statistical analysis of the various groups

using the analysis of variance (F test) Protein Fraction

Degrees

Albumin + as...

phoresis. In some eases the separation was fairly clear-cut, while in others there was no separation. For the purposes of this paper the term glyeoprotein is used to include all carbohydrate compounds such as the hexoses, hexosamine, sialie

F

1.67

4,42

0.56

1.47

4,42 4,52 4,42 4,42 4,52

1.14

4,35 4,35

0.50

4,50

acid and fucose, which are combined with

6.8*.*

0.35

4,48 4,48

5.81'1'

4,50

protein. According to Winzler (9) these hexoses are distributed throughout the various protein fractions, the globulins being especially rich in

Freedom

1.89 943*1' 1.44 0.74

a2

Degrees of Freedom

culty in obtaining clear separation of the albumin and aj glycoprotein by paper eleetro-

of

F.

Albumin

Glycoprotein

proteins in the albumin fraction. This was confirmed by the individual results in the present series. A partial explanation may be the diffi-

Those groups marked with ** show a significant

variation among the different groups at the 1% level.

TABLE 3 Showing the results of other workers of the electro-

phoretic analysis of glyco proteins in normal persons and comparing them with the results in

the carbohydrate moiety. Winzler also states that the amount of free acid mueopolysaeeharides in serum is very small amounting to about

6 mg per 100 mls. The latter also stain with P.A.S., but as the quantity is very small it is unlikely that they have influenced the results of the present tests. DISCUSSION

the present series

The method of staining strips for glyeoproteins Glycoproteins (percentage)

as recommended by BjOrnesjo is very satis-

Author Albumin

Bjornesjo Magalini Grassi Shetlar

al

a2

fi

7

17.9 27.9 23.8 17.2 9.85 15.06 27.27 33.30 14.52 25.0 15.0 21.0 20.0 19.0 9.8 14.6 29.3 26.8 19.5 13.1

Present series

average

14.4

19.0

27.2

23.6

17.1

Note that the scanning process in the other

workers technic was a densitometric one, whereas in the present series the scanning was done in a reflection scanner.

factory providing there is strict adherence to the procedure outlined above. Apart from the diffi-

culty referred to above with regard to the albumin and en fractions the separation achieved is usually well marked.

The results show that in senile dermatitis herpetiformis (bullous pemphigoid) there is a significant increase of eel protein compared to normal. There is also a considerably lower -y glyeoprotein

level than normal after steroid

treatment. In the one ease examined before and after tion

treatment a

considerable fall in this frac-

was observed.

In adult dermatitis herpetiformis there is a slightly lower average eel protein level than treatment also applies to the other fractions. normal and after treatment with dapsone the In the steroid treated cases of senile dermatitis average falls to a level significantly below normal. herpetiformis the y glycoprotein fractions were The levels of a glyeoprotein in this type of significantly lower than normal but the other dermatitis herpetiformis are significantly below for a rise in some cases and a fall in others after

glycoprotein fractions in this group did not differ normal before and after treatment and the significantly from normal. The a glycoproteins -y glyeoproteins are also above normal but only in adult dermatitis herpetiformis were signifi- significantly so in the active state. Treatment of cantly lower than normal—a finding which was adult dermatitis herpetiformis results in a fall of more or less parallel with the a2 protein frac- eel protein, a1 glyeoprotein and -y glycoprotein. In the senile variety there is a fall in 'y globulin tions of the same group. Table 3 emphasizes the point that there is a and a corresponding rise in the albumin fraction

great variation in the percentages of the glyco- after treatment

with steroids and there is also a

SERUM GLYCOPROTEINS IN DERMATITIS HERPETIFORMIS

259

fall in the y glycoprotein. The changes in the y globulin and albumin fractions are similar to those found by Lever et cii. (1) in pemphigus. The author is well aware that paper electrophoretic analysis is a somewhat crude method and that there is an overall inherent error of up to 10%. Nevertheless the results here are of considerable interest and may have some bearing on the hyaluronidase accelerating properties of serum from active cases of dermatitis herpetiformis as reported earlier (Alexander (4)). Because of the apparent abnormalities reported here, a more detailed and accurate analysis by extraction, following electrophoresis either in column or on starch gel is indicated. This would enable the differences reported here to be more

I am indebted to Dr. J. C. Eaton for facilities

accurately assessed. In particular it will be necessary to determine whether treatment by dapsone

Dermatologica (Supp), 97: 89, 1948. 4. ALEXANDER, J. O'D.: The role of hyaluronidase in the aetiology of dermatitis herpetiformis. 1. The effects of serum from derma-

or steroids really does cause alterations in the a and y glycoproteins. The investigation has, therefore, been of value in that it has suggested a

possible abnormality and indicates further research is required. SUMMARY

Paper electrophoretic analysis of sera from normal persons, from cases of eczema, of active and treated cases of adult and senile dermatitis

to carry out the work in the biochemistry labora-

tory of the Glasgow Royal Infirmary. I am particularly grateful to Mr. C. J. Scotland for much technical advice. REFERENCES 1. LEVER, W. F., HURLEY, N. A. AND BLANEY,

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