Physico-chemical and immunological studies of pathological serum macroglobulins

CLIMICA CHIMICA ACTA

PHYSICO-CHEMICAL OF

AND

PATHOLOGICAL

IMMUNOLOGICAL

SERUM

P. RATCLIFF, J. F. SOOTHILL Department of Experimental

Pathology,

AND

University

(Received February

STUDIES

MACROGLOBULINS D. R. STANWORTH of Birmingham (Great Britain)

Roth, 1962)

SUMMARY (I) Physico-chemical and quantitative gel-diffusion precipitin studies of 14sera containing pathological macroglobulins are reported. (2) The gel-diffusion precipitin technique, using a specific antiserum to the IgS component of y-globulin, was shown to be a satisfactory substitute for ultracentrifugation for identifying and estimating pathological macroglobulins. Other simple diagnostic tests studied were found to be of limited value. (3) It is suggested that the aggregation of pathological macroglobulins to form even larger components occurs in vivo above certain critical serum concentrations. (4) Acidification of 4 of the pathological sera (to pH 4.2) failed to dissociate their macroglobulin constituents although this was readily achieved by mercaptoethanol treatment.

In 194.4 WALDENSTR~M~ described a series of patients in whose serum ultracentrifugation revealed markedly increased amounts of components with sedimentation coefficients (Seo,w) greater than 15s and to whom he gave the diagnosis “macroglobulinaemia”. Since then there has been considerable interest in the nature of the pathological macroglobulins, both because of their possible role in the aetiology of the disease, and because their study may lead to a better understanding of the function and mode of production of normal serum macroglobulins. The IgS component of normal serum has been sub-fractionated by zone-electrophoresisZ into y,-globulin (termed “IgSy” in this report, and “pB~~” by BURTIN et ~1.~) and or,-globulin (subsequently referred to as “IgScr”) but it is difficult to determine the exact relative amounts of these 2 constituents in human sera4. The greater specificity and sensitivity of immunological techniques, however, offer a means of overcoming this difficulty and also provide an indication of the relationship of the pathological macroglobulins to normal serum globulins. For instance, by such methods it has been shown that the pathological macroglobulins are closely related antigenically to the normal IgSy, although the abnormal proteins appear to be deficient in some of the normal macroglobulin’s antigenic determinant groups6-8. A reaction of partial identity between the pathological macroglobuhns and normal 7Sy-globulin can also be demonstrated by gel-diffusion precipitin techniques9 y10. In the present study the macroglobulin constituents, and also the @-globulin Clin. Chim. Ada, 8 (1963) gI-ro8

P. RATCLIFF et al.

92

(called 7Sy hereafter) of a series of 14 macroglobulinaemic sera have been measured by both quantitative gel-diffusion precipitin and ultracentrifugal techniques. Other, simple, methods of estimating the abnormal serum proteins have been employed for comparison. In this way, and also by means of dissocia.tion studies using mercaptoethanol, the properties of a relatively large group of pathological serum macroglobulins have been compared. MATERIALS AND METHODS Pathological

sera

Fourteen sera containing pathological macroglobulins were studied, out of a total of 70 sera submitted for ultracentrifugal analysis as possible cases of Waldenstr6m’s macroglubulinaemia on the basis of suggestive clinical evidence and biochemical findings such as the presence of a sharp abnormal band (“M” band) in the “fast” y-globulin region on paper electrophoresis and-in most cases-a positive Sia test. All these 14 sera contained a higher concentration of macroglobulin than the arbitrarily chosen value of 460 mg/roo ml. This is twice the highest concehtration found in sera from a group of 6 healthy individuals. For comparison, results are presented from 6 other sera, submitted for ultracentrifugal analysis as possible cases of macroglobulinaemia, in which the macroglobulin concentration was found to be within normal limits but in which a marked increase in 7Sy was observed. All sera were stored at -_-IO’. Antisera The methods

ad

were described

gel-di@sion by GELLl’

preci@tin

techniques

and SOOTHILI.~~. Rabbit

antisera

were

used. The antiserum

to 7Sy u-as raised with a DEAE

column

preparation

human y-globulin. It did not require absorption, as it was virtually globulin. The antiserum to IqSy was raised with the washed cryoglobulin

of normal

specific to 7Syfrom the serum

of patient A. D. and absorbed with pure 7Sy and with a selected hypogammaglobulinaemia serum. The antiserum to 19% was raised with the purified protein (kindly supplied by Professor Schultze) and absorbed with the urine of a patient with moderately selective heavy proteinuria. For the gel-diffusion precipitin technique, the unknown is compared with a standard. For 7Sy a purified preparation of the protein was used, and so results are expressed as mg/roo ml. The 19Sy and the 19% were not obtainable pure, and so serum from a healthy adult male, stored frozen in small portions, was used as standard, and results are expressed as a percentage of the concentration in this serum. It is known to be normal for these proteins (by comparison with results of analysis of the sera of 20 healthy adult ma1es12), so results can be converted to mg/roo ml by using the best available assessment of the “normal” for these proteins. There is considerable doubt about this, but, for certain calculations, we use the assumed figures of 50 mg/roo ml for IgSy, and IOO mg/roo ml for 19% The error introduced by this would be systematic, involving all results in proportion to the concentration present. (See the Discussion, however.)

STUDIES

OF PATHOLOGICAL

SERUM

MACROGLOBULINS

93

Sia test One drop of serum was added to 300 ml of distilled water in a measuring cylinder, a positive result being recorded if precipitation occurred before the drop had stopped falling. Total The were

biuret

setam protein

and paper-electrophoretic

and electrophoresis techniques

described

by

HARDWICKE

13

employed. Serum viscosity Determinations were carried out at 25O by the method of (for viscosity 14, who gave a normal serum range of 1.62-1.74

SQUIRE

HARDWICKE

relative

AND

to water

at 25”). Technique

of ultracentrifugation

Ultracentrifugal analyses were carried out at 20~ and 60,000 rev./min in a Spinco Model E machine. Solutions were dialysed for 16 h at 4O against I 1 of barbitone buffer (pH 8.6, I = o.o3), containing 0.15 M NaCI, prior to analysis. Szo,urvalues were obtained by making the usual correction for temperature, density and viscosity using a V value of 0.73. The concentration of each component was determined from the area under its peak averaged over the whole run (after “fitting” a solvent base-line), by applying the formula of PICKELS~~, assuming each protein to have the same refractive index increment (A Y) of 0.00188. The sedimentation coefficient (L,,) of each macroglobulin component was corrected for concentration dependence using values thus obtained for its own concentration and the concentrations of the more slowly sedimenting components. In correcting the sedimentation coefficient of the “19s” component by this method the K value of 0.007 ml per mg assigned by WALLENIUS et a1.2 to the slower sedimenting components (4.5s and 7s) was used in the relationship: .Sio = .Siob*. / Sedimentation

coefficients

at infinite

(I -:KjCj)

dilution,

[SO,,], were obtained

from

the

plots of sedimentation coefficient vs. concentration. Slopes of these curves for the “rgS”-macroglobulin components were averaged to obtain a K value (0.022 ml per mg) for this protein, which was used in correcting the sedimentation coefficient of the “2gS”-macroglobulin component. A K value of 0.132 ml per mg, obtained in a similar manner, nent

was

used

to correct

(“38s”). No correction

the S 20,W value

of the most

was made for the Johnston-Ogston

rapidly

sedimenting

compo-

effect.

RESULTS The

results

of quantitative

physico-chemical

and

immunological

measurements

the 14 macroglobulinaemic sera studied are given in Table I. IJltracentrifugal analysis revealed in most cases a pattern similar to that described initially by WALDENSTR~M’ as is shown in Fig. ra (comprising the pattern of the serum

on

Clin. Chim. Acta,

8 (1963) 91-108

2 5’ f 2

r

2

7.3

ro.4 9.4 7.3 -

L.R.

S.W.

1,800

5,840 4,800 2,400

4*6oo

2,100

7,800

3.2oo

4,300 6,900

5.7oo I*,300

3,451

2,130

6,4r2 1,488

1,036

1,810

‘8753

4,700 2,780 6,080

mg/roo

ml

Total macvogEobuEi?c concentration (ultracentrifugalj

._

I ANALYSES

‘7.5 100

3,200 2,400

100

37

12,800 3,200

50 100

75

37

75

44

I25

100

75

50

@a

6,400 19,200

5,600

6,400 5’,“00

9,600

4.800

38.400

38,400 16,000

‘9%

YO o.f standard**

960 2,560

750

800

240 II

1,280

345

590 250

504 2,016

534

330

260

952

910

282

1,280 960

545 180

3oo

_^^ 7S compordnt concenfrafion (ultracentrijugal) mg/Ioo mE

____

320

1,280

7.50

mgi 100 ml 7.v

Gel di@sion precipitin resulls

VARlOUS ----

TABLE OF ____

-

1.83

2.10

6.8~ -

3.22

1.81

7.03

2.17

5.18

3.12

II.85

4.63

r2,47

-..

Relative viscosity

.%a test

----

-_

CVYOglobulin

_

Isoagglutinin titres A& AtiF

* Paper electrophoresis revealed an “WI” band in all sera except that of patient A.D., but as this was superimposed on the diffuse normal y band no attempt has been made to estimate its concentration separately. ** These results can be converted to mg/roo ml for comparison purposes by assuming that the concentrations of the 19Sy and 19Sa in the standard serum were 50 mg/loo ml and 100 mg/roo ml respectively. Therefore the values for 19Sa can be read directly as mg/roo ml, and those for 19Sy should be halved.

_.-_--.. -_

E.A.

M.A.

6.7

9-r

J.B.

P.R.

7-3

G.M.

A.S.

12.2

A.D.

J.K.

7.55 9.1

9.2 rr.9

L.M.

J.W.

B.S.

L.S.

_-___

Patient

serum

phoresis protein y + M* components g/loo ml mglroo ml _ __~ ~~ 12.3 9,800

Total

Paperelectro-

__-.___...__

RESULTS

STUDIES

OF PATHOLOGICAL

SERUM

95

MACROGLOBULINS

of patient J. W.). Such sera contained a predominant macroglobulin component of the 19s class, and many contained additional components (29s and 38s) sedimenting still faster. Two other less usual patterns are also shown. Fig. rb shows the pattern obtained from the serum of patient J. I(. in which there is an additional macroglobulin component sedimenting slightly slower than the predominant 19s component. In the serum of patient S. W., abnormal low molecular weight components were observed sedimenting more slowly than the 4.5s component, in addition to the raised 19s peak (Fig. IC). The concentrations of each of the macroglobulin components in the various patients’ sera and, also, their total macroglobulin concentrations are given in Table I, together with the concentrations of 7S constituent. .

52000

112

min Normal serum

19ST globulin 44000. % standard (Gel-diffusion precipitin) 36000.

.

.

28000(a) J.W.

1 20000

. 1

.

(b) J K. 12000

. . 1 .

I

4000

_

L

- 60,000

rev/mln

Fig. I. Ultracentrifugal patterns of normal human serum and representative macroglobulinaemic sera (from patients J. W., J. K. and S.W.). The serawere diluted appropriately and analysed at 60,000 rev./min in barbitone buffer (pH 8.6,I = 0.05 M) containing 0.15 M NaCl.

0

l

,

1.0

l

,

,

,

,

,

20 3.0 5.0 Total macro lobulin g/lOOm P (ultracentrifugal)

Fig. z. Relationship between the total serum macroglobulin concentration as estimated ultracentrifugally and by quantitative gel-diffusion precipitin analysis.

The gel-diffusion precipitin results (given in Table I) show that the 19Sy was grossly raised in every case (range from 20 healthy adults: 1oo-2oo~~ of standard) whereas the r9Sa and 7Sy are normal or low. In Fig. z the ultracentrifugal determination of the total macroglobulin concentrations are plotted against immunologically determined results for 19s~. At the lower concentrations, there is good agreement with surprisingly little scatter in view of the errors of the techniques. At higher levels, the gel-diffusion precipitin results are high relative to the ultracentrifugal concentrations. This may reflect the limitation of applicability of this technique to the precise estimation of these qualitatively abnormal proteins but the satisfactory correlation shows that the method is of considerable value for estimating pathological macroglobulins, none the Clin. Chim. A&, 8 (1963)gx-108

IO.5

15.3 Il.5

8.4

8.4

Ii.7

IO.4

R.1,.

E.R.

Ii. 1

K. z

J.K.

J.W.

14.3

ml

M.S.

g/r00

SerUm proteins

D.E.

Pafient

Total

~_

-

8,470

8,460

4,060

--.

5,340

I 1,000

-

Tl,260

-

--

r9.s

~~

r7. j’i

16.84

17.14

16.73

x6.61

‘7.7’

??‘Sl

_-

--

--

250

I20

270

140

120

‘4”

trace

8.24

il.90

a.80

.--

_-concn.

9!:0

94o

2so

IOU m1 ^I--

Eo?lC?t.

WI

Jo0 ml

.Lw

I_^

9.49

4=0

5.04

6.10

i>,zr

6.22

6.03

5.66

6.19

_ _,___~_ __. 7.60 b>PP 6.17

.hw

---.

concenlratiows --~

62570

G,tiOO

3.710

1,53o

6,080

12,440

3.670

L,T20

WI

-.

x00 ml

--._.. concn.

component

---

.--

.-

a?ld

co%lpone!:ts

-~-

“7s”

-

.-Ibnormal

-____

results

BUT

--_

*_

4.09

4.57

4.32

4.13

4,‘G

4.49 4.13

1.20

r,87o

4,570 4,650

6,650

2,740 5,280

5*2.30

5.160

700 m!

msl

concrt

compo+2ent

“4.55”

__-_

CONFIRMED

.%oIw

_-

CRYOGLOBULIN __ ~__

SUSPECTED SER.4 CONTAINED -.I_--.--_-

(Jltracentri&gal

OF THE _

Sedintcutatiox coeficients

NONE ~

~A~R~GL~~uL~~~~M~,~

m2gl .5po~w fql

_~

100 ml ---.

_____ roncii.

.-

wHux

compwzent

.__

77-7,p

mg~roow2~ S,,,,

GOm#OlZetitS

y + M*

phoresis

electro-

Paper

RESULTS OFANALYS~S OF 8 s~zz.4

-

.~_--_

%I of

_

11,000

IO0

100

300

30 300

IO0

400

25

75

75

150

100

75

100

50

19.%

standard** .___

ml

420

5,110

2,560

1,920

5,120

2,000

1,280

8,000

7.Sy

IO0

mgl

______I_ results ____

precipit&

Gd-diqzr5ion

19%

._

-~_

(PRE~ENTEDSIMILARLYTOTABL&

Sin test

Relative

viscosity

I)

STUDIES

OF PATHOLOGICAL

SERUM MACROGLOBULINS

97

less. The slope of the lower part of the curve suggests that the concentration of 19Sy in the standard is nearer 25 mg/roo ml than the figure of 50 mg/roo ml given in the caption of Table I, which was derived from previous worklB. As will be seen in Table I, the small quantity of 19% included in the ultracentrifugalresultsisnegligible in these sera in view of the very high concentration of 19s~.

Other hyfierglobulkaemic 6 of the sera submitted Strom’s macroglobulinaemia,

serum specimens

for ultracentrifugal analysis as possible cases of Waldenbut in which abnormal macroglobulins were not found,

were arbitrarily selected for studies comparable to those carried out on the macroglobulinaemic sera. Results of ultracentrifugal and immunological analyses on these specimens together with those on z kala azar sera are given in Table II. In each case the total macroglobulin concentration was found to be within normal limits with the exception of serum E.R., which showed a slightly raised value, well below the concentration of 460 mg/roo ml arbitrarily chosen as indicative of macroglobulinaemia. The concentration of 7s component was found to be raised above the normal in every case, varying from a 150% increase in serum E.R. to a 650% increase in serum J.R. In addition, 3 of the sera contained abnormal components with sedimentation coefficients ranging from 7.6s to 11.9s. Even if it had been possible to correct the SZO,wvalues of these components for concentration it is most unlikely that any would have exceeded 15 Svedberg units, which means that none of these abnormal components would be classified as “macroglobulins” by the current definition. Although it must be stressed that the gel-diffusion precipitin estimation of 7Sy in these sera containing abnormal y-globulin-like constituents must be interpreted with considerable caution, there is no doubt that the concentration of protein precipitated by specific anti-7Sy serum was raised above the normal level in all the sera listed in Table II with the exception of that from patient J.R. (in which it was reduced). The latter serum was of particular interest because it was found to contain a very high concentration of material reacting with the specific antiserum to 19Sy, although ultracentrifugal analysis had revealed no component with a sedimentation coefficient (.SzO,ur)greater than 8.2s. A possible explanation of this finding is that the abnormal 8.2s component and most of the 5.9s component is composed of material antigenically related to 19Sy (as will be discussed later). The 19Sy and 19% concentrations were not raised substantially above normal levels in the other sera containing abnormal constituents with sedimentation coefficients between 12s and 7.5s. The linear relationship demonstrated by plotting the combined concentration of paper electrophoretic components y and “M” against the total macroglobulin concentration obtained by ultracentrifugal analysis of the group of macroglobulinaemic sera (Fig. 3) indicated that electrophoresis provides a good alternative method of estimating macroglobulin concentration. Clearly, however, paper electrophoretic analysis fails to distinguish between sera containing pathological macroglobulin and those containing a high concentration of (7s) y-globulin from other causes. Other relatively simple tests which are sometimes used in an attempt to diagnose macroglobulinaemia were found to be of limited value, as is seen from the results in Tables I and II. The Sia test was not always positive for the 14 sera studied, while Clih. Chim. Ada,

8 (1963)

91-108

P. RATCLIFFet al.

98

6 of the 8 sera with high concentrations of 7Sy gave positive tests. These findings provide further evidence that this test is not of absolute diagnostic value, although a positive finding does raise a suspicion of the presence of macroglobulins. It is interesting to note that the z sera from patients with kala azar, K.I and K.2, gave positive Sia tests and had the expected high concentration of y-globulin, but both the ultracentrifugal and the immunological analyses showed no evidence of elevation of IgSy, but only of 7Sy. Only z of the macroglobulinaemic sera were found to contain cryoglobulins and one of these was inconspicuous in amount. As the 1gSy component of normal serum is known to include part of the ABO isoagglutinin activity, this was assayed in order to establish whether a raised titre accompanied a high macroglobulin concentration. No abnormally high values were shown by the sera of the patients with macroglobulinaemia (Table I).

9.0

1 I

l

zo-

l

l

5.03.0-

. . .

*

*

l

l

l.O0

1.0

3.0 5.0 ZO Totalmacfoglobulh gllOOml (uitracentriffQal~

Fig. 3. Relationship between the total serum macroglobulinconcentration as estimated ultracentrifugaliy and by paper electrophoresis (together with normal 7~component).

HARDWICKE AND SQUIRE~~showed that the serum viscosity depended on the concentration of high molecular weight protein constituents. Measurement of this property, therefore, seemed likely to provide the basis of a possible diagnostic test for macroglobulinaemia. This was also suggested by the findings of JAHNKE et aE.17 relationship of macroglobuliand STEELED,who studied the viscosity-concentration naemic sera as well as of euglobulin preparations. As is shown in Table I the serum relative viscosity is very high in all the macroglobulinaemic sera studied but, as would be expected, some of the sera containing high concentrations of 7Sy gave values of similar magnitude (see Table II). Obviously, a single relative viscosity measurement (i.e. at only one serum concentration) would not differentiate a h~erglobulinaemic serum containing a high concentration of 7Sy from another containing an abnormal (though lower) concentration of rosy. On the other hand, the results shown in Fig. 4 demonstrate that it should be possible to distinguish between the two types of hyperglobulinaemic sera by comparing their Clin.Chirn. Acfa,

8

(rgh3)gx-108

STUDIES OF PATHOLOGICAL SERUM MACROGLOBULINS

99

viscosity-concentration relationships. In this figure the relative viscosities of the sera in each series are plotted against their y-globulin contents as measured by paper electrophoresis (both techniques being easily performed). As would be anticipated the relative viscosities of the macroglobulinaemic sera (plotted as circles) showed a greater concentration dependence than did those of the other sera with high concentrations of 7Sy molecules (plotted as triangles). Thus, below a concentration of about 5 g protein/roe ml there is no distinction between the 2 curves, but above this concentration the macroglobulinaemic sera do show higher viscosities than the sera containing similar concentrations of 7Sy. Above this total y-globulin concentration level the 2 types of sera can be distinguished by the ratio relative viscosity M + y concentration

(g/roe ml)

as is shown in Fig. 5. The number of sera studied here is very small, but the results suggest that the test would have application in clinical laboratories. Relative viscosity (at 259

.

.

11.0 9.01 ZO

5.0

Fig. 4. The relationship of the relative viscosity of sera containing high concentrations of IgSy (points shown as circles) and sera containing high concentrations of $y(points shown as triangles) to abnormal protein concentration measured by paper electrophoresis. (Extra values, obtained from measurement of high 7Sy sera not shown in Table II; are included.)

Ultracentrifugal

properties

3.0

1

.

.

I

1.0

w

-I 0

1.0

.

l

.

. .

-. t

3.0

5.0

zo

9.0

T+‘M’globulin.

11.0 13.0 gllOOml

(paper electrophoretic)

of some serum macroglobulin

constituents

Where practicable, sera were analysed at several dilutions and the sedimentation coefficients of each of the macroglobulin components were extrapolated to infinite dilution (thus providing the [S’&]values given in Table III). In the cases where it was only possible to obtain measurements at a single serum dilution the .SzO,, values are given together with the concentration of the solution analysed. The relatively wide ranges of the [S’J,,] values of the 3 classes of macroglobulin found in the various pathological sera reflect variations in the molecular properties of these abnormal proteins. Theaverage [Solo] valueswere: rg.S6S, 29.49s and 37.55s. In Fig. 6 the concentrations of the zgS and 38s components in the various macroglobulinaemic sera (given in Table III) have been plotted against the IgSy concentration. The linear relationships obtained suggest that the concentrations of the faster sedimenting components in the serum are dependent upon the IgS-macroglobulin concentration. It seems that additional macroglobulin peaks appear in the ultracentrifugal pattern of a macroglobulinaemic serum only when the IgS-component Clin. Chim

Acta, 8 (1963)

91-108

I’. RATCLIFF et

100 concentration

is above

critical concentrations for the 3%

a certain critical value. are about 400 mg/Ioo

al. From

Fig. 6 it will be seen that the

ml for the 29s peak, and

1,300mg/roo

ml

peak. Relative viscosity (at 25”) ‘M”+

,4

r

concentration

(gllooml)

12 10

6

i

4 2 0

High 75 t set-a

Fig. 5. Comparison

of ~ M + y

Concentration

(mgI

High 195 P

sera

relative viscosity

1

IOCO

3000 195

6000

4000

component concentration (mg J100 ml)

Fig. 6. Relationship of the concentration of zgS component (points shown as circles) and of 3% component (points shown as triangles) to the IgS-component concentration in the group of macroglobulinaemic sera.

Degradation studies on serum macroglobulin constituents 4 of the macroglobulinaemic mercaptoethanol This

involved

(pH 8.6, I =

degradation equilibrating

0.05) containing

sera from the group studied

treatment the

diluted

first employed sera by

dialysis

0.1 M a-mercaptoethanol.

were subjected

to the

by DEUTSCH AND MORTON~~. against

barbitone

The ultracentrifugal Clin. Chim.

Acta,

buffer compo-

8 (1963)

91-108

STUDIES

OF PATHOLOGICAL TABLE

ULTRACENTRIFUGAL

L.S. B.S. J.W. L.M.

i.5 A.S.

G.M. J.B.

OF

ABNORMAL

Macroglobulin

c__Patient

PROPERTIES

in swum mglxoo ml Gown.

(So,,)

39.00 32.37 37.34

26.10*

I90

140 460 440

(SO,“)

III COMPONENTS

OF

MACROGLOBULINAEMIC

Concn. in sewm mg/Ioo ml

(SO,,)

Concn. in sewm

29.58 31.90 28.35 32.95 28.41

138 144

19.73

1,325

22.10

1,920

3.430 2,250 4,680 1,588 1,558 910 5,160

ml

15.5-19s

<

15s

14

28

25

29

I2

(44) 28.21*

:z

(11)

L.R.

41.50

200

34.30

402

22.06

2,849

F.R.

24.83*

144

20.14* (74) 22.1g* (9)

444

14.23’

**

S.W.

mglroo

mg/Ioo ml

18.46 16.82 19.44 22.15 21.05 20.94 15.85

SERA

Concentration of other abnormal components

components

1,080 390 940 165 252 126 812

26.75 24.92 28.24

101

SERUM WACROGLOBULINS

(24)

-

iK.4.

-

E.i\.

-

45 60

* %lmJvalues, the concentration (mg/~oo being given in brackets. ** Ultracentrifugal peak not resolved.

14.30* (122) 16.32: (93) 16.og* (120)

245 (3.6s) I IO (2.6s) 465

ml) of the component

in the solution

analysed

sitions of the 4 sera thus treated and, also, of the same sera after removal of the reducing agent are given in Table IV. In each case the major part of the macroglobulin constituents were degraded to material sedimenting as a 7s component. Gel-diffusion precipitin analysis results showed that the 19% was relatively little affected by the mercaptoethanol, so it is likely that the traces of 19 Smaterial detected ultracentrifugally were a,-macroglobulin. This is in agreement with the findings of HITZIG AND ISLIKER~~, who showed that an cc,-macroglobulin preparation remained unchanged by cysteamine treatment. Attempted quantitative gel-diffusion analyses of the degradation products were of limited value because of the formation of multiple precipitin lines with antisera to both 7Sy and 19Sy, an effect which persisted after removal of the mercaptoethanol. This could no doubt be explained by the observations of KORNGOLD AND VAN LEEUWEN$ and others on the antigenic properties of dissociated macroglobulins. As reported by DEUTSCH AND MORTONI9 and other investigators, the ultracentrifugal patterns of macroglobulinaemic sera after removal of mercaptoethanol revealed re-aggregation into macroglobulins of differing size from the original high molecular weight constituents. It was noticed, too, that in each case the sedimentation coefficient of the “7s” component increased significantly after removal of the reducing agent as compared with its value in the original untreated parent serum. A similar observation has been made by REISNER AND FRANKLIN~~. The results of acid treatment, which involved equilibration of the macroglobuClin. Chim. Acta, 8 (1963) 91-108

E ._ 0 ‘1

2

0) -

c 2 p b$

I in 5

Mercaptcethanol Mercaptoethanol 5.0

I in to

Mercaptoethanol Mercaptoethsnol

on thcsc minor components. ** Comprised 3 minor components.

24.9

3.0

22.0 0.8

5.1

29.8

11.6

I.0

(10s)

(10s)

(Id)

(10s)

(10s) (IIS)

2.1

I.9

I.2

5.9

3.3

0.8

ICI.50 I.4 (IIS) 4,’

10.23

because of the difficulty of making accurate sedimentation

5.2

17.20 25.60

T in 6

_%cid

‘7.37

12.57 16.45

I in 6

2.1

4.4 1.8**

16.71

2 .3

28.5

1g.00

56.9

11.60

15.2

31.3

26.8

0.7 20.4

0.3 ‘4.4

15.71

14.1”

‘5.57

15.30

13.82

20.05

‘4.19 14.21

‘9.97 18.67

29.62

(z5)* (26)

5.7

2.7

7.5

31.7

13.6

2.0

1.8

2.7

SERA

Ultracentrifugal composition -_ .~ ____.~ “19s” “1O.S”

I in 6

Trace

18.40

24.04

22.33

14.90

2Xd$0

21.66

16.11

19.22

%wJ

“29.s” component

MACROGLOBULINAEMIC

Lintreated

I 2

IV SELECTED

1Mercaptoethanol added Mercaptaethanol removed

26.

1 in 7

Mercaptcethanol added ~~erca~toethan~i removed

I in 7

(3>24s)

1 in 7

~‘ntreated 0.5

I.0

28.84

r in 10

Acid

2.1.9

16.82

added removed

Trace

Xcid

I in 5

0.7

T in ro Lb.&

24.44

__

t!ntrcated

added removed

1 in 5

lrntreated

--__

ON

TABLE TREATMENTS

“38,s” component ~___.. __ % s %W t&d

DEGRhnATION

of treated sertbm andysed

Dihfi0n

OF

* The S,,,,” valnes gi\,en in bl-ackets arc only approsimatc

1.X.

J .K.

J,\Y,

I3.S.

EFFECTS

15.1

15.1

38.1

36.8

'4.3

10.4

37.5

59.0

12.5

9.2

12.2

67.6

10.1

21.5

40.6 16.3

“4.5s"

4.56

4.20

4.04

4,49

4.50

4.42 4.50

4.45

4.64

4.37 4.81

4.75

4.43

4.03 4.12

4.18

52.6

57.0

62.4

60.4

42.6

38.7 4r.1

50.3

24.7

3=-4 19.0

40.0

24.7

58.7 61.5

65.8

component --~ % ‘S L %w total

coefficient determinations

7.17

7.42

6.15

7.08

7.00

7.82

6.20

6.90

7.35

7.46

6.27

6.60

6.65

6.03 7.50

6.07

“7.5”

STUDIES

OF PATHOLOGICAL

SERUM

103

~_4CROGLO~ULI~S

linaemic sera with acetate buffer (PH 4.2, I = 0.01) are also included in Table IV for comparison. In none of the 4 cases studied by this technique was there any evidence of dissociation of the macroglobulins to material of $5 sedimentation coefficient, but the 3% and 29s components of I serum (from patient J.W.) appeared to be degraded to 19s component. TABLE V INCIDENCE

OF ABNORMALITY

OF CERTAIN

CLINICAL

Patient

Age

Sex

Anosmia

Pathological haemorrhage

L.S. B.S.

74 50

F F

+ +

+ +

J.W.

63

141

L.M.

49

E.

PATHOLOGICAL

MACROGLOBULINS

-_--

-_--...

AND

Generalised Eympltade~ao~a~~y

Splenomegaly

TESTS

IN

PATIENTS

I4

WITH

---~-

..~

Excess of Excess of abnormal abnormal mononuclear ~0non~c~eaY cells in bEood bo~~l~~~~o~

A bnormality of optic fmdi

I_--t +

+

-k -

+

+

-

-

F

I-

f

+

i-

32 73

M

:

J.S. G.M.

60 8rM

M

i-

L”R

49

M

+

F:R:

63 59

I; M

1

S.W. M.A. EA.

50 70 73

I: F M

: +

y. Incidence

-I,

100

+ T

+

+

-t

-

+ +

-

+ -

+ -t -

-

_-i

-

i

_L

-

+

-

+ -

I__

+ -

‘3

69

r.5

66

_!_

Mi.XelEaneous

- -+ -

+ -

69

PATHOLOGICAL

i-

i -

? Lymphosarcoma Swelling of lacrimal and salivary glands

Duodenal ulcer Myeioblastic leukaemia Gastric ulcer Ljmphosarcoma

+ -

42

DISCUSSION

The sera studied were sent from many parts of the country for confirmation of the clinically suspected diagnosis of macroglobulinaemia by ultracentrifugation. We did not see the patients personally but their physicians kindly forwarded to us their findings (the incidence of some of which are given in Table V). They are consistent with the previously reported clinical findings in patients with macroglobuIinaemia2~-z~ ; that is, anaemia, splenomegaly and abnormal bleeding were common whilst abnormal cells in the bone marrow and abnormality of the optic fundi were often of diagnostic value when present. These features were shared, however, by many other patients in whom the condition was suspected clinically but not confirmed by study of the serum protein. The practical value of making the diagnosis, at present, is one of prognosis which, in most patients, is distinctly better than that of diseases with which it may be conClin. Chim. A&, 8 (1963) gr-108

104

P. RATCLLFFet @I.

fused such as leukaemia and myelomatosis (although KAPPELER et aLz3 have described a rapidly progressive form). Hence frequent estimations of the concentration of abnormal serum protein may well be required which, in the past, has necessitated ultracentrifugal analysis. Results obtained in the present study, however, suggest that it is possible to employ quantitative gel-diffusion precipitin analysis as a satisfactory (and much simpler) alternative method of measuring pathological serum macroglobulins. As was shown (in Fig. 2) the correlation between the ultracentrifugal and gel-diffusion estimations was very good, particularly in the sera with macroglobulin concentrations of less than 5.0 g/Ioo ml. The apparent “over-estimation” of the macroglobulin concentration by the imm~lnological technique suggests that the tentative conversion factor of 50 mg rgSy/roo ml of normal serum (the lowest figure suggested in the literature) is far too high. Our results indicate that a factor of half this value would be more appropriate but we are reluctant to attach too much. significance to this as a means of estimating the rgSy concentration in normal. serum, in view of the fact that the results were obtained with qualitatively abnormal proteins. On the other hand, the gel-di~usion precipitin technique has the advantage of greater specificity and sensitivity. This could be useful in differentiating WaldenStrom’s macroglobulinaemia from other conditions involving increases in concentration of the high molecular weight proteins of serum such as nephrotic syndrome (in which both the rgSy and the 19Sa arc increased) and chronic infections and liver disease (in which the rgSy is increased). At present the diagnosis of macroglobulinaemia depends on ultracentrifugal detection of the substantial increase in serum macroglobulin concentration (often accompanied by the appearance of the faster 2gS and 3% components) together with the general clinical picture. The rgSy has to be raised markedly in concentration before the total 19Sy peak is considered abnormal and it seems likely that some cases, perhaps early or less severe, may be missed because of this. The specificity of the quantitative gel-diffusion technique, however, permits detection of much smaller increases in 19Sy concentration and it seems likely that the disease must be redefined in terms of an abnormal concentration of IgSy, determined in this way. The present data are not suitable for providing such a definition, as only those sera in which macroglobulinaemia was established ultracentrifugally were studied by the immunological technique. The presence of a large excess of 19Sy can also be demonstrated non-quantitati~~ely by immunoelectrophoresisz~. It is interesting to find that the quantitative gel-diffusion precipitin technique provides a reasonable estimation of proteins which are probably qualitatively abnormal, when a normal LgSy is used as standard. The antiserum used was raised by immunisation with a pathological macroglobulin (from the serum of patient A.D.) and was rendered specific by absorption with both 7Sy and with the serum of a patient with hypogammaglobulinaemia which contained no detectable 19s~‘~. Ouchterlony analysis revealed reactions of identity between all the macroglobulinaemic sera and normal serum, using the antiserum. Similar results were obtained with a relatively weak antiserum raised against the I$$ of normal serum, similarly absorbed. The absorption with purified 7Sy would be expected to eliminate the antibodies directed against antigens with determinant groups common to 7Sy and r&Is. Similar cross-reactions have been observed between 19$ and ~~~-globulin26. Our absorbed antiserum, however, did not react with the /&-globulin in normal serum on immunoelectrophoresis. Nevertheless, it is possible that the abnormal proteins in serum J.R.,

STUDIES OF PATHOLOGICALSERUM MACROGLOBULINS which were antigenically

related

components,

to b,,-globulin

were related

to IgSy

but which sedimented as are the /?Z~myeloma

as 8.24s

105 and 5.94s

proteinsae.

Unfor-

tunately we had no satisfactory anti+A antiserum at that time to investigate this possibility, but the abnormal components of the serum of patient J.R. may have been low molecular weight “IgSy-like” proteins. The other simple tests studied as possible means of diagnosing the presence of a pathological macroglobulin were of limited value. Although paper electrophoresis is capable of estimating macroglobulin concentration (as is shown in Fig. 3) it is not possible, of course, to use this technique to distinguish between sera containing pathological macroglobulins and those containing a high concentration of 7Sy-globulin from other causes. It should be mentioned that this work was planned before the publication by BUTLER et uLz7 of the method of demonstrating macroglobulinaemia by starch gel electrophoresis of serum before and after mercaptoethanol treatment; the intact macroglobulin fails to move in the starch but, after it is degraded, an abnormal band is shown. We understand this method to be valuable in detecting significant macroglobulinaemia, though we have not used it. Such a failure of the macroglobulin to diffuse in agar gel has been detected in one serum studied since this work was completed. Ultracentrifugal analysis was typical of macroglobulinaemia, and the serum contained a protein which was precipitated by the anti-IgSy at extremely high dilution in “ring” tests, but no line of precipitate was obtained in agar gel, at various strengths of agar and with various buffers. There was obviously, however, a high concentration of protein precipitated around the antigen cup. Because of this no diagnostic difficulty would arise, though the macroglobulin could not be quantitated by the gel-diffusion technique (JONES, JOSEPHS AND SooTHILL,-unpublished data). STANWORTH et aL2” have also shown recently that zone-centrifugation can be used to differentiate between myeloma and macroglobulinaemic sera, providing their concentrations of abnormal globulin are sufficiently high. Similarly, the results presented here show that a combination of serum relative viscosity and paper-electrophoretic measurement permits confirmation of macroglobulinaemia in high concentration (6.g. greater than 5 g protein/Ioo ml). In using viscosity measurements it would appear necessary to compare the viscosity value with the concentration of serum macroglobulin in order to obtain an informative index. (See also JAHNKE et al.ly and WALDENSTR~M ,,.) Another possibility the detection of abnormal macroglobulins

in employing viscosity measurement in is to determine the viscosity before and

after mercaptoethanol treatment of the serumIs. A positive Sia test or the presence of a cryoglobulin (sometimes found subsequently to be associated exclusively with a raised 7Sy-globulin component of serum) can only be considered suggestive of macroglobulinaemia. Both kala azar sera studied (see Table II), containing normal rgSy concentrations but grossly raised 7Sy concentrations, gave positive Sia tests, as did several of the other sera in which macroglobulinaemia was suspected but not later confirmed. It is customary to carry out physico-chemical measurements of a protein on a purified preparation. There are, however, advantages in studying proteins in as near a native state as possible and thereby avoiding any alterations created by the isolation procedure. This is particularly so in studying the sedimentation properties of pathological macroglobulins, where the effect of the concentrations of the low molecular Clin. Chim.

Ada.

8 (1963) gr-Io.5

106

P. RATCLIFF et d.

weight serum components

(7s and 4.5s) can be taken into account. In this connection recently shown that up to a concentration of about 2 g of added albumin and 7Syglobulin per IOO ml, the sedimentation rate of macroglobulin was retarded by a factor identical to that expressing the retardation of the added substance from its own infinite dilution value. It was interesting to find appreciable differences in the sedimentation coefficients of our macroglobulins, even after malting such allowance for the retarding effect of the slower sedimenting components and, also, after correction for concentration dependence (to obtain [Solo] values). MULLER-EBERHART AND KUNKEL~~ have attributed similar variations observed by other investigators to the failure to study isolated macroglobulins and neglect of extrapolating S 20,Wvalues to infinite dilution. ALBERT

AND JOHNSON~~ have

In contrast,

AND KUNKEL~~ found the macroglobulin

MULLER-EBERHART

of their 4 purified preparations

RELATIVE

to have highly similar ultracentrifugal

COMPOSITION

OF

9,,

Patient

L.S. B.S. J.“. L.M. J.K. x.n. ,l.s. G.M. J.B. L.R. F.R. S.\\‘. M.A. I?.‘%.

THE

MACROGLOBULIN

of total swum

constituents properties,

the

COMPONENTS

macroglobulin*

38.~

29s

19s

4.0 5.0 7.6 0.0 0.0 0.0 6.9 1.7 3.1 5.8

23.0 14.0 ‘5.5 9.4 13.9 12.2 12.7

73.0 81.0 76.9 90.6 86.1 87.8 80.4 89.0 90. L

0.0 0.0 0.0

6.9 0.0 9.X

9.3 b.8 11.7

8L.j

Sot resolved 93.7 100.0 90.9

~___~.__~ * Calculated from the macroglobulin concentrations given itl Table III.

mean [SzO] values being: rg.zS, 29.3s and 38.2s. The averaging of the [So,,] values of our macroglobulins gave very similar mean values (namely, 19.9S, 29.5s and 37.6S), in spite of their appreciably wider scatter (shown in Table III). KUNKEL~ interpreted the constancy of the ratios of the concentrations of the 3 macroglobulin constituents in his 4 purified preparations (namely: rgS: 767/o; zgS: 16.794 and 3%: 5.5%) as indicating that the zgS and 38s components are “real entities and not polymeric forms in equilibrium with the IgS component”. It would have been interesting, had sufficient serum been available, to compare the relative compositions of the high molecular weight components of purified macroglobulins isolated from the 14 macroglobulinaemic sera used in the present study. As will be seen, however, from the relative concentrations of our macroglobulin components-estimated by analysis of the whole serum (Table VI)-there was no evidence of constancy in the ratio of rgS : 2gS : 3% component concentrations. The ranges of the relative concentrations of the macroglobulin components, in the 7 seracontaining all 3, were: rgS: 73.0-90.1~~; zgS: 6.8-23.0~~~; 38s: 1.7-7.6yb as compared with the narrower ranges (i.e. rgS: 7r-81%; zgS: 12-2196; 3%: 5-6%) C2in.Ckim.

Acta, 8 (1963) go-x08

STUDIES OF PATHOLOGICALSERUM MACROGLOBULINS shown by the relative concentrations of the macroglobulin EBERHART’S AND KUNKEL’S~~ 4 preparations.

components

107 in MULLER-

As was shown (Fig. 6), however, the concentrations of the minor, faster sedimenting macroglobulin components are related to the level of the IgS-macroglobulin in the serum. This would seem to suggest that the higher molecular weight aggregates are produced in vivo probably by disulphide bond formation in the manner proposed by PUTNAM”. There was no evidence to suggest that the 2gS and 38s molecules resulted from ilz vitro reversible aggregations of the type occurring in some protein systems during ultracentrifugation32. Failure to dissociate the macroglobulins in 4 of the pathological sera by adjusting the pH to 4.2 with acetate buffer indicated that the high molecular weight proteins (with the probable exception of the 29s and 38s components of serum J.W.) are not bound together by secondary valency forces as results reported by REES AND RESNER~~ would imply. The small differences in the sedimentation coefficients of the serum macroglobulins when run at pH 4.2 as compared to pH 8.6 can be attributed to charge effects. The results obtained from mercaptoethanol treatment of the macroglobulin constituents of 4 of the pathological sera were similar to those previously reported by DEUTSCH AND MORTONlg and others. The increase in the sedimentation coefficient of the 7s component after removal of the mercaptoethanol in every case was an interesting observation, particularly as this did not appear to be due to the presence of degraded macroglobulin which had failed to re-aggregate. Such a finding deserves further investigation. The normal isoagglutinin titres of the macroglobulinaemic sera contrast with the finding of raised values in many sera from patients with hypogamma-globulinaemia associated with a high concentration of y-macroglobulin33.

ACKNOWLEDGEMENTS We wish to thank Professor J. R. SQUIRE and Dr. J. HAKDWICKE for their advice and help and Dr. HARDWICKE for the total protein and paper electrophoresis results. We are indebted to the many physicians, surgeons, pathologists and biochemists who have sent us sera, and clinical data from the patients. Particular mention must be made of Professor H. W. FULLERTON and Dr. A. A. DAWSON of the University of Aberdeen, Dr. G. OWEN of the National Blood Transfusion Service in Sheffield, and Dr. B. E. NORTHAM of the Birmingham General Hospital. The kala azar sera were kindly supplied by Professor S. N. DE (Department of Pathology, Medical College, Calcutta, India). The ultracentrifugal analyses were carried out on a machine kindly donated by the Rockefeller Foundation (New York). REFERENCES 1 J. WALDENSTR~M, Acta Med. stand., 117 (1944) 216. 2 G.WALLENIUS,R.TRAUTMAN, H.G. KUNKELAND E.FRANKLIN, J.Biol.Chfm., 225 (1957)253. 3 P.BURTIN,L.HARTMANN,J.HEREMANS,J. J.SCHEIDEGGER,F.WESTENDORP-BOERMA, R.WIEME,C. WUNDERLY,R. FAUVERT AND P. GRABAR, Rev. fraq. &de clin. et biol., 2 (1959) 161. 4 H. G. KUNKEL in: F. W. PUTMAN (Ed.),The Plasma Proteins, Vol. I, Academic Press, New York and London, 1960, p. 279. Clin.Chim. Acta, 8 (1963) 91-108

P. RATCLIFF

108

d ial.

5 E. C. FRANKLIN AXII G. H. KUNKEL, J. Immunol., 78 (1957) II. @ L. KORNGULIJ AND G.VAN LEEUWEW,,]. Exptl. Me&, 1a6 (1957) 467. 7 H. I;.DEUTSCH AND J. I. MORTON, J.BioE. Chem, 231 (1958) 1107. 1119. * J. J. SCHEIDEGGER, R. WEBER AND A. HASSIG, Helu. Med. Acta, 25 (1958) 'j. 0 L. K~RNGoLD AND G.VAN L,EEUWEN,J. Exptl. Med., 110(x959) I. 10 E. C. FRANKLIN, J. Imnzunol.,85 (1960) 138. 11 F’. G. H. GELL, f.#Sn. PuthoE., IO (1957) 07. I2 J. F. SOOTHILL, J. Lab. Clin. Med., 59 (rg62) 859. I3 J. HARDWICKE, Biochem. J,, 57 (1954) 166. 14 J. HARDWICKE AND J. R. SQUIRE, CE& Sci.,II (1952) 333. I5 E. G. PTCKELS, Methods in Med. Research, 5 (1953) 107. 10 F. W. PWTNAM, Arch. Biochem. Biophys., 7g (1959) 67. 1’ I<. JAHNRE, W. SCHOLTAN AXIJ Ii.HEINZLER, He&. Med. ilcta, 25 (r9gSj 2. I8 A. E. STEEL, Glin. Citim.. Acta, J (1959) 503. I@ H. F. DEUTSCH AND J, I. MORTON, Science, rzg (1957) 600. 20 11’. H. HITZIG AND II. C. ISLIKER in: H. PEETERS, Proceedings of the 7th Colloquium on Pvotides of the Biological Flldids, Bruges, 1959, Elsevier, 1960, p. 368. 21 C. zi.REISNER AND E. C. FRANKLIN,.!. ~~rn~~a~.,87 (r961) 654. 2% J. \VALDENSTR~M, Acta haematol., 20 (1958) 33. 23 R. KAPPELER, .4.KREBS AND G. RIVA, N&J. &fed. A&, ~5 (1g58) 54, IOI. ?A S. H. MARTIN, Quart. /. Med., rg (1960) 179. *5 .1.HASSIG, E. GUGLER AND J. J. SCHEIDEGGER~~: 1".GRABAR AND f'.BURTIN (Eds.), Analyse Tlrz~~nurzo-dlectrophor~t~q~e, Masson, l'aris, 1960, p. 173. 26 J. HEREX~NS, Les Globtllimzs SPviques du Systime Gamma, Arscia, l&uxelles, 1960, p. 1 rg. 27 E. A. BUTLER, F. V. FLYNN, H. HARRIS AND E. R. ROBSON, Lnltcet, ii(1961) 2%. ?* D. R. STANWORTH, K. JAYES AND J. R. SQUIRE, Awz~. Biochcm., L (1961) 324. 29 J. WALDENSTR~~M, Advances in Interm Med., =j (1952) 398. 30 A. Ar..mmr AND P. JOHNSON, Bioclaem.J., 81 (1961) 658. %* I-I. J. MIJLLER-EBERHART AXD H. G. KUNKEL,C~~PZ. Chim. .4ctu, 1 (Iqsgj .zgr. S2 G. A. GILBERT, Nnlttre,186 (1960) 582. 33 E. D. REES 4x-31) R. RESNER, Clin.Cisi~~z. Acta, 4 (1959) 272. 34 J. F. SOOTHILL, Clix. Sri.,21 (1962) in the press.

INVESTIGATIONS FROM

OF THE

INFLUENCE

PREGNANT

WOMEN

CULTURES

IN CONJUNCTION

OCCURRENCE J. REJTEK,T.

ON THE

OF ABNORMAI~

BEDNARI-K,

OF SERA

GROWTH WITH

OF CELL

THE

~~-LIPOPROTEIN

E. RERABKOV~~AND

A. nomiar.

Institute of Haematology and Blood Transfusion*, Second G~~~ec~log~cal artd Obstetric Clinic**, Prague ~C~eckoslo~lak~a~ (Received

March 3rd, 1962)

The influence of sera from different stages of pregnancy on the proliferation of HeLa cells ifz vitro has been studied and correlated with the occurrence of an abnormal immuno-electro~horetic picture of a,-lipoprotein. Sera from early pregnancy, which * Head: Prof. Dr. J. Hofej& ** Head: Prof. Dr. J. LukA1. Clin. f’lriin. Acta, 8 (1963) 108 -115