Carbohydrate content of anomalous seroglobulin and bence-jones protein in myeloma a comparative study

CLINICA

CARBOHYDRATE AND

CONTENT BENCE-JONES

CHIMICA

R.

MAIORCA

ofMedical

PROTEIN

SEROGLOBULIN

IN MYELOMA

STUDY

AND L. SCARPIONI

Pathology,

(Received

403

OF ANOMALOUS

A COMPARATIVE

Institute

ACT.4

University

October

z5th,

of Parma

(Italy)

*

1960)

The determination of the glucidic content of serum and urine paraprotein of myeloma has usually been carried out by staining with periodic acid-fuchsin (Schiff staining) of the electrophoretic strips 1- 5. Although these methods are not specific, they would reveal a marked difference in glucide content between myelomatous proteins of serum and urine. In addition to these studies, a few others exist concerning qualitative and quantitative analysis of carbohydrates bound to pathological proteins. As to qualitative composition, no difference seems to exist in respect of normal serum glycoproteins5+. The carbohydrate content is nearly equal to that of normal y-7 S-globulins for y-myelomas, and somewhat higher for ,&myelomas. SCHRADE et aL7 found a 1.7o-5.oo~/~ hexose and 0.14-0.64~/~ hexosamine content in Bence-Jones proteins isolated and purified by means of several precipitations in ammonium sulphate. This conflicts with OSSERMAN’Sfinding 3, that out of II cases observed, IO were Schiff-negative. Completely different glucide contents of serum and urine pathologic proteins in the same case may show a different constitution of the two proteins. This could provide a valuable argument in support of the hypothesis about their different origin and proves the usefulness of further studies on this subject. In order to clarify this problem, we studied comparatively, case by case, the glucide composition of serum and urine paraproteins which were collected, isolated and purified at the same time. In this way, individual variations depending on many uncontrollable factors and possible alterations in the myelomatous protein synthesis during the several stages of the disease were avoided so that every case becomes significant in itself. MATERIALSANDMETHODS Our investigation was carried out on the serum and urine of two patients affected by myeloma. They both showed a plasmocyte metaplasia of bone marrow, with bone injuries of myelomatous type, evident dysproteinemia (respectively 10.6 and 9.1 g% of total proteinemia) with the presence of an electrophoretically homogenous y-globulin fraction, equal to 659/O(case I) and 51% (case Z) of total proteins. Bence-Jones proteinuria occurred in both cases. Pathological proteins were isolated from sera and urines by continuous flow electrophoresis (Spinco-Beckman apparatus mod. CP, veronal-veronal-sodium, pH 8.6 *

Head:

Prof. L. MIGONE. Clin. Chim. Acta, 6 (196x) 403-406

404

R. MAIORCA,L. SCARPIONI

buffer, p = 0.02; current range 70 mA, power supply 700 V; sample feed rate = 0.4 ml/h; migration on Schleicher and Schiill paper 470). The urines had been previously dialyzed against running water and against buffer solution, then concentrated against polyvinyl pyrrolidone. Sera were dialyzed for 12 h against buffer solution (dialysis of sera and urines was carried out with cellulose dialysis tubing from the Arthur H. Thomas Co). Isolated pathological fractions, spotted by staining the migration sheet, were concentrated against polyvinyl pyrrolidone and then examined by means of paper electrophoresis in order to check their purity. Urinary proteins were also checked, according to PUTNAM”,for the thermal behaviour typical of Bence-Jones proteins. The migration speed of serum and urine pathological fractions was compared by mixing the two isolated protein solutions in suitable proportions, and by running them electrophoretically along the same strip. Then the polysaccharide staining (periodic acid-fuchsin) was done on electropherograms of serum and urines irt toto. On most of the isolated and concentrated pathological fractions the protein content was precipitated with ethanol (I : IO), then washed with ethanol and eventually dissolved in 0.1 N sodium hydroxide. The following determinations were performed: proteins : biuret ; total hexoses : Soerensen and Haugaard; hexosamines : Elson and Morgan: sialic acid : Warren ; fucose: Dische and Shettles. The actual techniques used were the modifications proposed by AZZOLINIet al. !‘. RESULTS Serum and urine myelomatous proteins had an identical electrophoretic migration speed, in one of the two cases; in the other (case I), the Bence-Jones protein had a higher speed than the corresponding serum paraprotein (see Figs. I and 2). The staining of electropherograms with periodic acid-fuchsin was positive for serum myelomatous proteins, and negative for both Bence- Jones proteins. The results of the determination of protide-bound carbohydrates are shown in Table I. Determinations of total pentoses, hexuronic acids and heptoses (see AZZOLINI et aLs) have also been carried out on isolated pathological protein fractions. These calorimetric reactions revealed no pentose and no hexuronic acid contents either in serum or urine paraproteins. On the other hand, heptose appeared to be entirely positive for serum paraproteins and negative for urinary paraproteins. The glucide composition of serum myelomatous paraproteins showed no significant differences from that found in three y-globulins isolated from normal sera. However, the composition of urinary paraproteins was different. In both cases, Bence-Jones protein had a glucide content clearly lower than the serum protein content: it equalled 18% in case I, and 38% in case 2. Quantitative ratios between single glucides were quite different in each of the four protein fractions; they are better expressed, in Table I, as molar ratios (fucose = I). DISCUSSION The above data show that, in the cases we studied, a clear difference existed in the glucide composition of the two myelomatous proteins of serum and urine. Clin. Chim.Acta,6 (x961)403-406

CARBOHYDRATE

CONTENT

OF ANOMALOUS

SEROGLOBULIN

405

Fig. I. CaseI. Serum and urine electropherograms : amidoschwarz staining. From the top downwan 3s: total se:roproteins; total urine ; isolated serum paraprotein ; isolated Bence- Jones p*-0tein ; serum and urine paraproteins, isolated and mixed.

Fig. z. Case

2.

As in the former.

Clin. C/aim.Acta, 6 (1961) 403-406

R. MAIORCA,

406

L.

TABLE

SCAKPIONI I Case 2

Case I Urine

Serum

paraprotein mg _ pmol molar y0 % ratio

%=

q101

%

paraprotcin

paraprotein

_paraprotein mg

r ‘rinu

Serum

molar ratio

nZg

% = pyOol $g

yt

= i’y;l

;;t!;

Hexoses

0.96

5.34

2.42

O.Ij

0.83

3.41

1.81

10.06

5.49

0.42

2.33 3.82

Hexosamines

I.II

6.20

2.81

0.20

I.12

4.59

I.84

10.29

5.62

0.44

2.40 4.0.3

Sialic acid Fucose

O.Ij

0.49 2.20

0.22 I

0.08

0.26

0.23

0.04

0.24

I.06 I

0.74 I.83

0.61 O.IO

I.98 3.24 o.61 I

0.36

0.30

0.41

I

Therefore, our investigation confirms previous studies in which different methods were used (see PUTNAM lo), and according to which the two molecules are not identical and there is no evidence that Bence- Jones protein originates from a fragmentation of serum paraprotein. Furthermore, our data indicate that it is impossible that Bence-Jones protein should constitute a believe that in this unaltered. Concerning the we obviously cannot

starting agent in the synthesis of serum paraprotein. In fact, we case the molar ratio between the various glucides should be kept total quantity of glucides to be found in the Bence-Jones protein, exclude (on the basis of the significant individual variations that

we found) that in rarer cases the quantity is comparatively high, and becomes evident on Schiff staining of electropherograms. Nevertheless, no relation seems to exist between electrophoretic mobility and glucide content, and no relation is likely to be found between the sialic acid content (the only protide-bound carbohydrate which can modify the isoelectric point of the protein molecule, owing to its acidic groups) and electrophoretic mobility. Summing up, we think that this study offers further arguments in support of a complete independence of the synthesis of serum and urine pathological proteins which are often to be found, but not always together, in multiple myeloma. SUMMARY

In two cases of y-myeloma, the carbohydrate content of myelomatous globulins, isolated by continuous flow electrophoresis, was considerably higher than that of the corresponding Bence-Jones protein. The presence of an entirely different glucide molar ratio in the respective serum and urine paraproteins suggests that their synthesis proceeds quite independently. REFERENCES I 2 3 * 5 6 7 8 9 III

R. DI GUGLIELMO, I plasmocitomi, Abruzzini, Roma, 1955. E. F. OSSERMAN, Am. J. Med., 23 (1957) 283. E. F. OSSERMAN AND D. P. LAWLOR, Science, IZO (1954) 715. J. SONNET, Les glycoprote’ins se’riques b Z’Ctat normal et pathologique, Ed. Arscia, Bruxelles, 1956. J, SONNET, L. LOUIS AND J. HEREMANS, Acta Haematol, I4 (1955) 193. H. J. MUELLER-EBERHARD AND H. G. KUNICEL, J. Exptl. Med., 104 (1956) Zj3. W. SCHRADE, E. BGHLE, R. BIEGLER AND H. BRUCH, Klin. Wochschr., 35 (19.57) 76j. F. W. PUTNA~L~AND P. STELOS, J. Biol. Chem., 203 (1953) 347. G. AZZOLINI, R. MAIORCA AXD L. SCARPIONI, Rass. fiszopatol. rlin. E terap., 31 (1959) 1003. F. W. PUTNAM, Physiol. Rev.. 37 (1957) 4. C‘ZilI.Chim. A&,

6 (1961) 403-406