122 T. Marshall, 0. Vesterberg and K. M. Williams Electrophoresis 1984,5, 122- I28

Thomas Marshall Olof Vesterberg Katherine M. Williams

Effects of alcohol abuse on human serum proteins revealed by two-dimensional electrophoresis

Chemistry Division, Occupational Safety and Health, Solna

Alcohol-associated changes in human serum protein patterns have been detected by silver staining following high resolution two-dimensional electrophoresis and isoelectric focusing-multiple gel piece electrophoresis. In the sera of alcoholics the concentration of a,-acid glycoprotein was consistently increased and in a high proportion of sera elevated levels of IgA, a,-antichymotrypsin, haptoglobins and apo A-I lipoprotein were observed. Increased levels of additional unidentified polypep- tides were also detected. In contrast, reduced levels of antithrombin I11 were predo- minantly associated with the sera of alcoholics. The use of these polypeptides as potential clinical markers for alcohol abuse is discussed.

1 Introduction

Alcohol abuse is a serious and costly worldwide problem. In France, for example, it contributes towards 30 % of hospital admissions [ 11. Whilst alcohol-associated elevations in blood enzyme activities, particularly gamma-glutamyl transferase (GGT) [2-41, have been proposed as diagnostic markers, their specificity is limited [51. Consequently an urgent demand still exists for a reliable clinical marker. We are currently using high resolution two-dimensional electrophoresis [6-81 in con- junction with the methylamine-incorporating silver stain [9, 101 to detect serum protein modifications following exposure to organic solvents [81. Such modifications should prove to be valuable indicators of liver damage and altered hepatic func- tioning since synthesis, secretion and reabsorption of most blood proteins occurs mainly in the liver. In this report we de- scribe changes in human serum protein patterns arising from excessive alcohol abuse, the latter representing an extreme form of ethanol exposure. The advantage of the adopted ap- proach is that it allows simultaneous analysis of many hundreds of polypeptides and thereby provides a unique means of detecting possible clinical markers for human dis- orders 1111. To maximise the potential of this approach we have recently developed a modified version of the technique, isoelectric focusing-multiple gel piece electrophoresis (IEF- MGPE) [81, which is particularly suitable for clinical screen- ing since it provides an economical means of analysing a large number of samples with a high degree of reproducibility.

2 Materials and methods

2.1 Studied populations

The primary analysis was based upon comparison of sera from 60 different apparently healthy individuals (controls) and sera taken from 32 alcoholics on the first day of hos- pitalisation. In addition, sera were obtained from 8 of the al- coholics during the 2 week period following hospital admis- sion. The control group was of both sexes within the age range of 23-55 years. The alcoholics were predominantly of the same age range but 90 % were male. All alcoholics fulfilled the criteriaof alcohol dependence according to the W.H.O. [ 121.

Correspondence: Professor 0. Vesterberg, Chemistry Division, National Board of Occupational Safety and Health, S-17 1 84 Solna, Sweden

Abbreviations: IEF: Isoelectric focusing; SDS: Sodium dodecyl sulphate; M,: Molecular weight; pk Isoelectric point; MGPE: Multiple gel pieceelec- trophoresis; ZIA: Zone immunoelectrophoresis assay

2.2 Chemicals

Electrophoresis grade (Electran) acrylamide and N,N'- methylene-bisacrylamide were purchased from BDH (Poole, Dorset, UK) and Ampholines from LKB (Bromma, Sweden). For silver staining, formaldehyde solution (3 7 %), ammonia solution (25 %) and silver nitrate were purchased from Merck (Darmstadt, FRG) and methylamine solution (40 %) from Fluka AB (Switzerland). For identification purposes, human serum protein immunoglobulin fractions of antisera to human serum antigens were purchased from Dakopatts (Copen- hagen, Denmark). Purified albumin, transferrin and hapto- globin (mixed type) were obtained from Behringwerke (Mar- burg, FRG). Purified apo A-I and apo A-I1 lipoprotein frac- tions were kindly supplied by Dr. L. Holmquist, Karolinska Institute, Stockholm, Sweden.

2.3 Sample preparation

Sera from both controls and alcoholics were either prepared fresh (without storage) or stored at -70 "C prior to use. Con- trol experiments showed that storage did not affect the pro- teins of relevance to this study. Ten microlitres of human serum were mixed with 590 p1 of sample denaturing solution (2 % w/v sodium dodecyl sulfate (SDS), 5 % v/v 2-mercap- toethanol in 0.0625 M Tris-HC1, pH 6.8) and heated at 95 "C for 5 min. On cooling, the sample was saturated with urea by adding 0.55 g of solid urea. Routinely, 7.5 pl of the final mix- ture was analysed (equivalent to 0.075 pl of neat serum). Purified human serum proteins were dissolved in sample denaturing solution at a concentration of 1 mg/ml and pre- pared for electrophoresis as described above. Each protein (0.5-2.0 pg) was analysed individually and by coelectro- phoresis with 0.075 pl of serum.

2.4 Two-dimensional electrophoresis

Simplified high resolution two-dimensional electrophoresis was performed essentially as previously described [71.

(a) First dimension: IEF was carried out in 4 % w/v poly- acrylamide gel cylinders containing 9.2 M urea, 2 % w/v Non- idet P-40 (NP-40) and 2.1 % w/v Ampholine (pH ranges, 2.5 4.0,3.5- 10.0 and 9-1 1,1:9 : 1, v/v/v) [ 7,8 1. Focusing was continuedfor 17 hat 330 V witha90 KC2 resistorinseries [71. The IEF gels were subsequently equilibrated for 2 h in a solu- tionof2.3 %w/vSDSand 10 %~/~glycer0lin0.0625 MTris- HCl, pH 6.8 [ 131.

0 173-0835/84/0204-0 122 $02.50/0 0 Verlag Chemie GmbH, D-6940 Weinheim, 1984

Electrophoresis 1984,5, 122-128 Effects of alcohol abuse on human serum proteins 123

(b) Second dimension: SDS-polyacrylamide gel electrophore- sis (SDS-PAGE) was performed on 4-20 % w/v polyacryl- amide gradient gels (7.5 x 7.5 x 0.3 cm) at 25 mA/gel in a solution of 0.192 M glycine, 0.1 % w/v SDS in 0.0625 M Tris [7-91. The molecular weights of the polypeptides were esti- mated as previously described 17, 81 using Low Molecular Weight (LMW) calibration proteins (Pharmacia, Uppsala, Sweden).

2.5 IEF-MGPE

This is essentially a modification and improvement of the two- dimensional electrophoresis procedure described above [81. Briefly, eighteen sera (9 controls, 9 alcoholics) were simul- taneously focused in the first dimension. Subsequently the anode ends of the IEF gels, pH 4-4.7, (0.5 cm) were isolated and simultaneously electrophoresed (9 per gel) on two second- dimension gels. A further 2.2 cm gel piece (pH range 4.8-7.0) from the anode end ofeach IEF gel was isolated and three such gel pieces electrophoresed simultaneously on each of six sec- ond-dimension gels. The complete two-dimensional proce- dure was repeated on two subsequent days to give a com- parison of 54 different sera (27 controls, 27 alcoholics). The six second-dimension gels containing electrophoresed IEF anode end (0.5 cm) gel pieces were silver stained in one batch. The remaining 18 second-dimension gels containing electro- phoresed IEF gel pieces, pH range 4.8-7.0, were then silver stained, again in a single batch.

2.6 Measurement of pH gradient

Eleven equal sections (0.55 cm) of a first-dimension gel, fo- cused without sample, were each equilibrated overnight at 4 "C in sealed polypropylene microcentrifuge tubes contain- ing 0.665 ml of boiled deionised water and the pH values (25 "C) were subsequently measured using a pH meter, Type PM, with a microelectrode, Type GK 242 1 C (Radiometer, Copenhagen, Denmark) 181.

2.7 Silver staining

Polypeptides were detected with the methylamine-incor- porating silver stain [91 using the improved destaining proce- dure [lo, 131.

2.8 Identification of major serum polypeptides [ 141

For each of the major serum proteins the titre volume equiv- alent of the appropriate antibody (Dakopatts, technical data) was diluted to 175 pl by addition of aqueous 0.1 M sodium chloride and then mixed with 25 pl of human serum. Follow- ing an overnight incubation at 4 "C each mixture was ul- tracentrifuged (Airfuge, Beckmann) at 100 000 g for 10 min. The supernatants (immunodeleted sera) were carefully re- moved and the incubation tubes and surface of the remaining immunoprecipitates rinsed by addition and subsequent removal of 200 pl of aqueous 0.1 M sodium chloride. The im- munodeleted sera were each prepared for two-dimensional analysis by mixing with 9 volumes of sample denaturing solu- tion, heatinq at 95 "C for 5 min, and subsequently saturating with urea. The immunoprecipitates were each resuspended in 100 pl ofsample denaturing solution, allowed to stand at room temperature for 2 hand then heated at 95 "C for 5 min prior to

saturation with urea. The volume of immunodeleted serum or resuspended immunoprecipitate analysed was equivalent to that recovered from 0.1 pl of the original serum.

2.9 Quantification of al-acid glycoprotein

The a,-acid glycoprotein concentration of each unfractio- nated serum was determined with zone immunoelectropho- resis assay (ZIA) [ 15 I using antibodies from Dakopatts. This method has proven to be extremely reliable [151.

3 Results

The two-dimensional pattern of a typical normal human serum (0.075 pl) as revealed by silver staining is shown in Fig. 1 A. A composite of the polypeptide positions of proteins we have identified (either by immunoprecipitation from serum with specific antiserum or co-electrophoresis of the serum with a small amount of the purified proteins) is shown in Fig. 1B. The positions correspond closely to those ofthe same proteins in the more comprehensive patterns determined by others using theISO-DALT two-dimensional system [ 14,161. Consequently, this was the basis for a tentative identification ofthe positions ofq-antichymotrypsin and antithrombin 111.

Fig. 2 shows the two-dimensional pattern of a normal human serum (Fig. 2A) as compared to that of serum of an alcoholic (Fig. 2B) taken on the first day of admission to an alcoholic clinic. The polypeptides indicated by arrows demonstrate quantitative variations associated with alcohol abuse as as- sessed by detailed visual comparison of the two-dimensional patterns from 60 different apparently healthy individuals and 32 different alcoholics taken on the first day ofhospitalisation (similar trends were evident in a further 16 sera takenfrom 8 of these alcoholics on days following the admission). However, both the degree of quantitative variation of each individual polypeptide and the incidence of simultaneous variation ofthe indicated polypeptides varied widely within the alcoholic group. This was confirmed by IEF-MGPE (Fig. 3,4) which gave a more reliable group comparison than the standard (wide pH range) two-dimensional procedure by allowing simultaneous electrophoresis in parallel, silver staining and visual comparison of a large number of samples.

The alcohol-associated increase in al-acid glycoprotein (M, 43 000, Fig. 3) was consistently accompanied by a corres- ponding increase in the intensity of a relatively high molecular weight component (M, 90 000) of similar isoelectric point (- pH 4.3-4.7). Immunoprecipitation of al-acid glycoprotein did not result in co-precipitation of this high molecular weight polypeptide. Visual comparison of the staining intensities of both the a,-acid glycoprotein and the high molecular weight polypeptide (Fig. 3) indicated elevated levels of both in ap- proximately 70 % of the alcoholics relative to the controls. Quantitative determination of a,-acid glycoprotein in the un- fractionated sera using ZIA gave a mean value of 1.52 g/L in the alcoholics as compared to 0.98 g/L in the control group. The latter were all within the upper limit of the normal serum range (0.5-1.4 g/L, Behringwerke, technical data) whereas 50 % ofthe alcoholics were above the upper limit of this range.

The alcohol-associated trends of the remaining polypeptides (Fiq. 2,4) were more difficult to correlate because ofwideintra

124 T. Marshall, 0. Vesterberg and K. M. Williams

1A

Electrophoresis 1984,5, 122-128

18 PH L L 5 6 6.1 1.9 8.9

10 5 0 6 1 13 8 3 9.1

I I I I 1 I I I I I I

MG

A A

I 6

0 - A P O A-ll

Mr

V - 9L

- 67

- 13

- 30

- 20

- 1L

Figure 1. Two-dimensional pattern of 0.075 @ of normal human serum (A) as revealed by ultrasensitive silver staining. The anode of the IEF gel was to the left and electrophoresis performed from top to bottom. The composite of the polypeptide positons of the major serum proteins (B) was determined using either imrnunoprecipitation or co-electrophoresis of purified protein preparations. The identified proteins are a,-macroglobulin (MG), transferrin (Tr), al- bumin (Alb), a,-antitrypsin (AT), al-acid glycoprotein (AA), haptoglobins (Hapt), apo A-I lipoprotein (APO A-l), apo A-I1 lipoprotein(AP0 A-II) and the heavy chains of the IgM, IgA and IgG immunoglobulins (IgM H, IgA H and IgG H, respectively). The immunoglobulin light chains are positioned in the zones indicated IgG L. In each case all spots within the scope of the bracket correspond to the protein indicated. M, is the molecular weight x lo3.

Figure 2. Two-dimensional pattern of a normal human serum (A, 0.1 fl) as compared to that of an alcoholic immediately following hospitalisation (B, 0.1 pl). In each case, the anode of the IEF gel was to the left and electrophoresis performed from top to bottom. The polypeptides indicated demonstrated alcohol-associated changes as detailed in the text. Of those indicated, the following were positively identified (c.f. Fig. 1); a,-acid glycoprotein (2), the IgA heavy chain (4), the main haptoglobin cluster (7) and apo A-Ilipoprotein (9). Polypeptides (3) and(5) were alsotentatively identified ass,-antichymotrypsin and antithrombin 111, respectively, by comparison with polypeptide patterns determined by others [ 14, 161.

Electrophoresis 1984,5, 122-128 Effects of alcohol abuse on human serum proteins 125

Figure 3. IEF-MGPE of the anode end gel pieces (0.5 cm, pH range 4.0-4.7) obtained following simultaneous IEF of 9 sera from different controls (A. 0.075 pl) and 9 sera from different alcoholics (B, 0.075 d). Polypeptide 2 (M, 43 000) was identified as a,-acid glycoprotein by immunoprecipitation. Polypeptide 1 (M, 90 000) remains unidentified.

group variations within both the control and alcoholic group. The polypeptide patterns (of 27 controls and 27 alcoholics with IEF-MGPE) were each assigned a random code number (to allow unbiased evaluation) and then arranged in order of polypeptide intensity, repeating the comparison for each polypeptide investigated. Upon completion ofthe comparison the identity of each pattern was revealed and the results ex- pressed as the percentage of alcoholics comprising the total number of sera in each of three classes (Table 1). Although these classes were defined on an arbitrary basis (class l-low- comprising 25 % of the total sample number, class 2-interme- diate-comprising 50 % and class 3-high-comprising 25 %), similar results were obtained when the total sample number was divided into 2, 3 ,4 or 5 classes of approximately equal number. Within the limits of this analysis it was evident (Table 1) that the sera from alcoholics demonstrate a trait towards elevated levels of the haptoglobins, IgA, al-an- tichymotrypsin and the unidentified polypeptides denoted 6,8 and 10 (in Fig. 2 and 4) as well as reduced levels of antithrom- bin 111. When the criteria for demonstration of each alcohol- associated trait was defined as the frequency of occurrence of each sera in either class 1 (for antithrombin 111) or class 3 (for the remaining polypeptides), then 90 % of the alcoholics (as compared to only 44 % of the controls) demonstrated at least one trait, 63 % of the alcoholics (as compared to only 18 % of the controls) demonstrated at least three traits and 48 % ofthe alcoholics (as compared to none of the controls) demonstrate at least four traits. However, some ofthese traits are presumed to be unreliable since they correspond to proteins which are known to demonstrate a wide normal concentration range in serum (Behringwerke technical data). These include IgA (0.9-4.5 g/L serum), the haptoglobins (0.5-2.2 g/L) and the high density lipoproteins (2.9-7.7 g/L) which include apo A-I lipoprotein. If these components (in addition to polypeptide 10, closely associated with the apo A-I lipoprotein) are omit- ted from the above analysis, then almost 90 % of the al-

Table l . Frequency of occurrence of sera from alcoholics in relation to the total sera in three arbitrarily defined classesa)

1 Classb)

2 3

Antithrombin HIc) Polypeptide sd) Polypeptide zone sd) u I-antichymotrypsinC) Apo A-I lipoprotein Polypeptide lod) Haptoglobins IgA

92 31 0

31 23 23 23 54

36 42 39 100 61 77 39 92 46 85 54 69 54 69 44 62

a) Number of sera from alcoholics expressed as a % of total number

b) Class 1 comprised the least intense 25 % of the samples, class 2

c) Tentatively identified from previous two-dimensional patterns

d) Two-dimensional positions indicated in Fig. 2 and 4.

of sera in each class.

the intermediate 50 % and class 3 the most intense 25 %.

[14, 161.

coholics (as compared to only 18 % of the controls) demon- strate at least one of the remaining traits and 55 % of the alcoholics (as compared to none of the controls) demonstrate at least two. Quantitative determination (ZIA) of al-anti- chymotrypsin indicates a mean value of 0.55 g/L in the con- trol group as compared to 0.78 g/L in the alcoholics, with 63 % of the latter above the upper limit of the normal serum range (0.4-0.6 g/L, Behringwerke technical data). Prelim- inary quantitative findings (ZIA) also confirm the trend to- wards reduced levels of antithrombin I11 in the sera of alco- holics but definitive values must await more accurate standar- disation.

The above analyses were obtained by comparing sera from a control group with those obtained from alcoholics immediate-

126 T. Marshall, 0. Vesterberg and K. M. Williams Electrophoresis 1984,5, 122-128

Figure 4. IEF-MGPE of IEF gel pieces (PH range 4.8-7.0) following simultaneous IEF of 3 sera from different controls (A, 0.075 jd) and 3 sera from dif- ferent alcoholics (B, 0.075 jd). The polypeptides indicated demonstrate alcohol-associated changes. The polypeptides denoted 4,7 and 9 were identified as IgA heavy chains, haptoglobins (c.f. Fig. 1B) and apo A-I lipoprotein, respectively. Polypeptides (3) and (5) were also tentatively identified as a,-anti- chymotrypsin and antithrombin 111, by comparison with polypeptide patterns determined by others [ 14, 161.

ly following hospitalisation. In a limited number of cases it was possible to obtain sera from the same alcoholics over a2-week time period following hospital admission in order to assess the effect of alcohol withdrawal, improved diet and medication, (c.f. Fig. 5) . In two such cases a progressive decrease was observed in the intensity of the alcohol-associated, diffusely- staining region (polypeptide zone 8, Fig. 2,4) to the acidic side

5 l a lb

of the immunoglobulin light chains (Fig. 5). In one person this was accompanied by a progressive increase in the intensity of antithrombin 111. The remaining alcohol-associated polypep- tides did not appear to be affected by alcohol withdrawal although an increase in the intensity of other polypeptides was observed, notably the haptoglobins and the polypeptides in- dicated by arrows (Fig. 5).

1C Id

Figures. Response of serum protein patterns oftwo different alcoholics ( la-d, 2a-d) to alcohol withdrawal and hospitalisation over a two-week period. The time course is indicated by (a), t = 0 (day of hospitalisation); (b), t = +3 days; (c) t = +7 days and (d), t = + 14 days and the area of each two-dimensional gel shown corresponds to that indicated in Fig. 4B. A progressive decrease was observed in the intensity of the diffusely-staining region denoted 8 (see also Fig. 2 and 4). Alcohol withdrawal and hospitalisation were also associated with elevation in the levels of polypeptides indicated by arrows. (7) refers to the complete haptoglobin cluster (Fig. 1 B). Both alcoholics obtained daily tranquilizers (Pentymalum (barbiturate), alcoholic 1 and Oxazepam (bensodizepin), alcoholic 2) with Antabus (disulfiram) being administered daily after t = +7 days.

Electrophoresis 1984,5, 122-128 Effects of alcohol abuse on human serum proteins 127

4 Discussion

The potential of high resolution two-dimensional electro- phoresis as a means of detecting changes in serum proteinpat- terns resulting from alcohol abuse has been investigated. The results are encouraging, particularly when the alcohol-as- sociated traits are used in combination. Alcohol-associated trends in human blood proteins have been reported including increased levels of IgA [171 and high density lipoproteins [3, 5 1. Furthermore, increased levels of orosomucoid and al-an- tichymotrypsin have been detected in the plasma of chronic alcoholics and plasma antithrombin I11 levels shown to be ab- normal in 40 % ofchronic alcoholics (G. Skude, The Hospital (Sjukhuset) Kalmar, Sweden). However, the advantage ofthe two-dimensional electrophoresis approach is that it demon- strates these changes simultaneously together with variations in additional unidentified polypeptides. Nevertheless, the fre- quency ofour findings should be interpreted with caution since they are based upon classification ofthe two-dimensional pat- terns into three arbitrarily defined classes. Even if such classes could be accurately defined for a single population of alco- holics it seems likely that the frequency of occurrence of each individual trait might be expected to vary within other popula- tions according to age, sex, history of abuse, degree of cir- rhosis and other factors. The limited number of analysed samples constrains detailed investigations of these factors. For example, the results of the present study apply particular- ly to male alcoholics since only 10 % of the total alcoholic samples could be obtained from females. However, the latter did demonstrate the same alcohol-associated traits. Similar discrepancies may be expected to arise in the control group, since it is impossible to define a typical normal human serum. The resolving power of the two-dimensional technique is such that it reveals not only a diversity of polypeptide patterns be- tween different samples of apparentlynormal serum but also a wide range of quantitative variations in the polypeptides. No other assay procedure is capable of achieving this with such clarity. In this context, it is relavent to note that the control group used in this study was chosen almost entirely at random (apparently healthy and without excessive alcohol intake) and no attempt was made to eliminate alcohol users with a limited history of abuse. Consequently, by more careful selection of appropriate controls combined with detailed analysis of medical case histories, it should be possible to increase further the clinical potential of the observed changes in polypeptide patterns. For example, a recent comparison between the two- dimensional patterns obtained with pooled sera from (a) 24 male alcoholics and (b) 24 male controls confirmed the quantitative protein differences illustrated in Fig. 1, thereby indicating they do not arise from the higher proportion of females in the original control group.

A major contributing factor towards successful detection of these alcohol-associated polypeptides is the current repro- ducibility of our silver staining and two-dimensional elec- trophoretic procedures in combination with the application of IEF-MGPE IS]. This latter approach allows the appropriate polypeptide constituents of up to 24 different sera to be analys- ed simultaneously with a high degree of reproducibility. By decreasing the length of the IEF gel piece, the number of samples can be increased proportionally (Fig. 2). High sample turnover is essential for future application of the technique in many research projects and in clinical laboratories. Further- more, whilst visual comparison of quantitative differences be-

tween polypeptide patterns is not optimal, more sophisticated computerised approaches 118, 191 are unlikely to become available to the majority of clinical laboratories within the foreseeable future. The diagnostic potential of these tech- niques has to remain within the technical and financial scope of such laboratories. In this respect it is interesting to note that a laboratory worker totally inexperienced in comparison of two-dimensional patterns was able to reproduce our fre- quency findings when given the coded two-dimensional pat- terns.

Whilst identification of the alcohol-associated polypeptides may not be essential for clinical diagnosis (providing they can be reproducibly located on the two-dimensional pattern) a positive identification has three major advantages. First, it may provide insight into the biochemical nature of the dis- order, offering the potential for new and perhaps more suc- cessful approaches to treatment. Secondly, it may substan- tiate previous findings based upon more traditional en- zymatic, biochemical and immunological methods. Thirdly, it may initiate the application of such methods to give a simple, quantitative assay applicable to the unfractionated sera. The increasing availability of antibody preparations specific to defined human serum proteins offers some potential for polypeptide identification either by immunodeletion from un- fractionated serum 141 or application of immunoblotting techniques [20l following two-dimensional analysis. Howev- er, the detection sensitivity of silver staining [2 1,221 combined with high resolution two-dimensional electrophoresis now reveals a previously unknown complexity of serum poly- peptides, for example > 150 polypeptides in the limited gel area shown in Fig. 5. We have recently confirmed this by detailed two-dimensional analysis of serum using a variety of narrow pH gradients in the first dimension and estimate that we can separate well over 1000 polypeptides (manuscript in preparation). Suitable specific antibody preparations are available for identification and quantitation of only a fraction of these polypeptides.

The progressive decrease in the intensity ofpolypeptide zone 8 (Fig. 5) observed in sera of two different alcoholics upon se- quential sampling following hospitalisation indicates a poten- tial clinical marker for assessing the effects of alcohol withdrawal, improved diet and subsequent medication. How- ever, additional samples and supportive clinical data will be necessary to confirm this potential. Likewise, the accompany- ing elevations in other polypeptides (Fig. 5 ) merit further in- vestigation. If, for example, they indicate a direct response of human serum protein patterns to drug therapy, then the value of high resolution two-dimensional electrophoresis in tox- icology and pharmacology could prove immense.

This work was supported by a grant from the Swedish Work Environment Fund. We wish to thank Ms. H . Saranius for technical assistance with ZIA. We also wish to thank Dr. D . Schmidt and the personnel involved of the Clinical Depart- ment of Alcohol and Drug Research, Karolinska Hospital, Stockholm, Sweden, for providing the sera from alcohofics.

Received December 7. 1983

5 References

[ 11 Babor, T. F., Treffardier, M., Weill, J., Feguer, L. and Ferrent, J. F.,J. Studies on Alcohol 1983,44, 600-6 16.

128 T. Marshall, 0. Vesterberg and K. M. Williams Electrophoresis 1984,5, 122-128

[21 Kristenson, H., Trell, E., Fex, G. and Hood, B., Prev. Med. 1980,9, 108-1 19.

131 Fex, G., Kristenson, H. and Trell, E., Ann. Clin. Biochem. 1982,19, 345-349.

(41 Peterson, B., Trell, E., Kristenson, H., Fex, F., Yettra, M. and Hood, B., Scand. J. Clin. Lab. Invest. 1983,43, 141-149.

[51 Batsakis, J. G., Ann. Clin. Lab. Sci. 1974,4,255-266. I61 O'Farrell, P. H., J. B i d Chem. 1975,250,4007-4021. [71 Latner, A. L., Marshall, T. and Gambie, M., Clin. Chim. Acta 1980,

[81 Marshall,T. andvesterberg, 0,Electrophoresis 1983,4,363-366. [9l Marshall, T. and Latner, A. L., Electrophoresis 1981,2,228-235.

[ l O l Marshall, T., Electrophoresis 1983,4, 269-272. [ill Anderson,N. G.andAnderson,N.L.,MedicalLaboratory 1982,11,

I121 Technical Report Series, 1964,273, 3.

103,51-59.

15-94.

[13] Marshall, T. and Latner, A. L., Electrophoresis 1983,4,354-358. [14] Anderson, N. L. and Anderson, N. G., Proc. Natl. Acad. Sci. USA

[ 151 Vesterberg, O., in: Tschesche, H. (Ed.), Modern Methods in Protein

[ 161 Tracy, R. P., Currie, R. M. and Young, D. S. , Clin. Chem. 1982,28

I171 Editorial, The Lancet 1983, (ii). 605-606. [ 181 Schneider, W. and Klose, J., Electrophoresis 1983,4,284-291. [191 Taylor, J., Anderson, N. L. and Anderson, N. G., Electrophoresis

[201 Gershoni, J. M. and Palade, G. E.,Anal.Biochem. 1983,131,l-15. [2 11 Switzer, R. C., Merril, C. R. and Shifrin, S.,Anal. Biochem. 1979,98,

[221 Merril, C. R., Goldman, D. and Van Keuren, M. L., Electrophoresis

1977,74,5421-5425.

Chemistry Walter de Gruyter, Berlin 1983, pp. 187-206.

890-899.

1983,4,338-346.

231-237.

1982,3,17-23.