Abnormal Proteinuria in Malignant Diseases1

ABNORMAL PROTEINURIA IN MALIGNANT DISEASES1

W . Pruzanski and M. A . Ogryzlo Department of Medicine and the lmmunoprotein Research laboratory. University of Toronto Rheumatic Disease Unit. Toronto. Canada

. .

Introduction ........................................................ Detection of Urinary Proteins ......................................... 2.1. Quantitative Methods ........................................... 2.2. Qualitative Methods ............................................ 3. Proteins in Normal Urine ............................................. 3.1. Quantitative Analysis ........................................... 3.2. Qualitative Analysis ............................................ 4 . Malignant Diseases with Production of M components. . . . . . . . . . . . . . . . . . . 4.1. Classification. .................................................. 4.2. Definition of M Components (Immunoglobulins or Their Fragments) . 4.3. Classes of M Components....................................... 4.4. Synthesis of M Components..................................... 4.5. Bence Jones Globulin........................................... 4.6. Low-Molecular-Weight Bence Jones-Related Globulins.............. 5 . Multiple Myeloma., ................................................. 5.1. Bence Jones Proteinuria .................................... 5.2. Other M Components in the Urine. ............................... 5.3. Ligh t-Chain Disease ............................................ 5.4. Rare Types of Myeloma ......................................... 5.5. Biclonal IgG/IgM Gammopathy ................................. 5.6. Syndrome with Production of IgG Half-Molecules. . . . . . . . . . . . . . . . . . 6. Plasma Cell Leukemia ................................................ 7 . Macroglobulinemia ................................................... 7.1. Syndrome with Production of 7 S IgM ............................ 8. Heavy-Chain Diseases ................................................ 8.1. 7-Heavy-Chain Disease .......................................... 8.2. a-Heavy-Chain Disease .......................................... 8.3. p-Heavy-Chain Disease.......................................... 9. Lymphoma ......................................................... 10. Leukemias.......................................................... 11. Epithelial Malignancies ............................................... 12. Urinary M Components without Malignant Disease ...................... 13. Lysozymuria (Muramidasuria) in Mono- and Myelomonocytic Leukemia . . . References.............................................................. 1 2

336 337 337 337 338 338 339 340 340 341 342

344

344

349 351 352 355 355 358 358 359 359 360 361 362 362 363 363 364 364 365 367 367 370

'The study was supported by grants-in-aid from the Ontario Cancer Treatment and Research Foundation (No . 220) and the Canadian Arthritis and Rheumatism Society (No. 7.12669) . 335

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W. PRUZANSKI AND M. A. OGRYZLO

1.

Introduction

Neoplastic diseases originate as uncontrolled, pathological proliferations of one or more types of cells within the organism. However, every ceIl in the body serves as a complex physiological unit with a special function to perform. It is therefore not surprising to find that, in many instances, tumors arising from these cells may retain some of their original functional activity, depending on the degree of differentiation and maturity of the neoplasm. Neoplasms of the antibody-forming cells or of the endocrine organs, such as the ovary or the adrenal cortex, are good examples. It is of interest that other tumors, although arising from the same cell line and morphologically indistinguishable, may nevertheless produce different secretions. Medullary carcinoma of the thyroid is such an example, secreting in one instance calcitonin and in another, material having ADH activity. On occasion, one may observe tumors, particularly those in the malignant group, which may secrete substances not necessarily related to any known secretory activity of the parent cell line, which in turn may exercise profound effects on the general metabolism of the host. Oat cell carcinoma of the lung with secretion of ACTH-like peptide is an excellent example. All these substances, which can usually be detected in the circulation, are eventually either metabolized or excreted from the body, largely in the urine. Of the many metabolic products of tumors, polypeptides which belong to the family of immunoglobulins are perhaps the most readily recognized, and have consequently aroused the greatest interest. Secreted in varying amounts by the vast majority of plasmacytic and certain other neoplasms, these proteins can often be detected in the urine and have been the object of intensive investigation since the earliest days of protein chemistry (B15).The classical example of such a protein in the urine was discovered as early as 1848 by Bence Jones, and rightly bears his name (B5). However, it was not until forty years later that Kahler first related Bence Jones proteinuria to the presence of tumors of the bone marrow (K2). Thereafter, nearly a century elapsed before any significant advances were made in the study of these proteins. Tiselius in 1937 (TlO), and Grabar and Williams in 1953 (G2, G 3 ) , respectively, introduced the techniques of electrophoresis and immunoelectrophoresis of proteins in biological fluids, thereby initiating the modern era of protein analysis. During the past two decades, we have witnessed a remarkable increment of knowledge relating to protein chemistry, and in particular immunochemistry. Many new proteins have been discovered, a number of new diseases have been recognized, and there has evolved a better understanding of the physiological and pathological

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processes relating to protein metabolism. It is now apparent that numerous proteins associated with the metabolism of malignant cells are excreted in the urine, and, indeed, rather than containing merely “physiological garbage,” the urine has provided a source of “limitless treasure’’ for investigators in this field. 2.

Detection of Urinary Proteins

2.1. QUANTITATIVE METHODS Methods employed for the quantitative determination of proteins in the urine usually vary depending on the protein concentration. When there is gross proteinuria, precipitation with 207% sulfosalicylic acid (K4) and the biuret reaction (H2) will give a reasonably accurate estimation of the total protein content. Precipitation by ammonium sulfate or sodium sulfate, cold ethanol fractionation, dialysis and subsequent lyophilization, evaporation, ultrafiltration, and a variety of other methods have also been employed (G7, K1, M10). When the concentration of urinary protein is small, it is often necessary to concentrate the urine as much as 3000 times (B13) or more prior to quantitation or qualitative analysis. Dialysis of urine in seamless cellulose dialysis tubes against 2 U 5 % solution of polyvinylpyrrolidone or against other polymers, and positive or negative pressure dialysis, are widely used as methods of concentration ( K l , K5). A highly sensitive radial immunodiffusion method for the quantitation of various proteins, particularly when these are present in trace amounts, has been developed by Mancini (M3) * 2.2. QUALITATIVE METHODS The qualitative evaluation of urinary proteins usually requires a variety of procedures including (a) electrophoresis in various media such as filter paper, cellulose acetate, or starch gel (G2, K10, S13), (b) immunoelectrophoresis (G3) against poly- and monospecific antisera, (c) single or double immunodiffusion analysis (09, 010, 011) and (d) ultracentrifugation (529). Starch block or agar block electrophoresis (T11) provide a simple method for the isolation of small amounts of individual protein fractions. However, the isolation and purification of sizable amounts of certain proteins is best accomplished by means of chromatography using various cellulose ion exchangers such as CMcellulose or DEAE-cellulose, or by using Sephadex (54, P2, S17). Bence Jones globulin was the first abnormal protein recognized in the urine, being excreted as a by-product of certain forms of malignant disease. Its detection and quantitation are still considered to have im-

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W. PRUZANSKI AND M. A. OGRYZLO

portant diagnostic significance and, in some instances, may also be of value in prognosis. Many tests have been employed for its detection. The original procedure described by Bence Jones (B5) was based on the observation that the addition of a strong acid (HNO,) to the urine, caused precipitation of a protein which redissolved when the mixture was boiled. Since then, no fewer than twenty-four procedures have been described for the detection of Bence Jones globulin (N3). However, it has been noted that even when the heat precipitation test is performed under optimal conditions, occasional false negative and false positive results may be observed (B15, L5, P18). Rarely, the excretion of Bence Jones globulin, in amounts as high as 1.2g in 24 hours may not be detected by the heat precipitation test (L5).Comparison of the various methods for the detection and estimation of Bence Jones globulin have shown that a combination of electrophoresis and immunoelectrophoresis of concentrated urine is the most effective, especially when the Bence Jones globulin is heat-insoluble or is contained in a mixture along with other proteins (M13). Minute quantities of Bence Jones globulin are especially well detected by the double radial immunodiffusion method of Ouchterlony or by the single diffusion method of Oudin, using specific antisera against light chains absorbed with normal y-globulins (D3, L5). 3.

Proteins in Normal Urine

3.1. QUANTITATIVE ANALYSIS

In order to have a better understanding of the significance of abnormal proteins appearing in urine in neoplastic disorders, one must be aware of those proteins which are physiologically found in normal urine, so-called physiological proteinuria. MGrner in 1895 was the first to show that normal human urine contains small amounts of protein (M12). Available studies of total urinary protein excretion in healthy individuals indicate no general agreement as to the average or maximum normal values. The commonly reported range has varied from 40 to 80 mg per 24 hours, with a mean value of 50 mg (R2, S6). Some authors have reported higher normal values for total urinary protein, up to 193.8 & 53 mg per 24 hours (54, S l ) . The total protein excreted in the urine in normal subjects, as estimated by a combination of immunological techniques in our laboratory, has varied from 50 to 187 mg per 24 hours (mean 103 mg per 24 hours) (P9).Physical exercise increases the amount of protein excreted in the urine in healthy individuals as much as 6-fold (F12, P5).

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ABNORMAL PROTEINURIA I N MALXGNANT DISEASES

3.2. QUALITATIVE ANALYSIS Many of the proteins found in normal human urine appear to originate in the circulating plasma. However, normal urine also contains other proteins which have not as yet been detected in the blood. Normal urine has also been shown to contain most of the proteins which are present in the urine in various pathological conditions, with variations only in the amounts excreted (B8, R2, W4). Thus far, no fewer than 50 different proteins have been identified in urine (P3, P4), 27 of which have been detected in the plasma (W4). It is quite possible that a t least some of the others may arise from the postglomerular portion of the nephron. For example, so-called Tamm-Horsfall protein probably originates in the renal tubules (T3). At the same time, it would be reasonable to assume that certain proteins presently considered t o be urinary proteins of nonplasmatic origin probably have their origin in plasma, but because of their very low concentration are difficult to identify with the techniques currently available. Albumin (M.W., 70,000) usually constitutes a t least 25% of the total urinary protein (P5).The majority of other proteins in the urine originating from the plasma have a molecular weight of less than 200,000, probably because of the molecular-sieve effect of the glomerular filtering membranes (S6). Several immunoglobulins are known to be excreted in normal urine (Table 1 ) . IgG and IgA have frequently been encountered (B7, B11, F1, T17), and both types of IgA, namely serum and secretory type, have been observed (B18). However, no IgM, IgD ( B l l ) , or IgE have as yet been detected in the urine. Low molecular weight proteins related antigenically to immunoglobulins have likewise been observed in normal urine. These include the light polypeptide chains as well as Fab, Fc, and F’c fragments (B11, B17, T15, T16, V4), which are similar if not identical with those fragments produced experimentally

TABLE 1 QTJANTITATION OB IMMUNOGLOBULINS IN NORMALURINP ~

RWge

(mg/24 hours)

Mean (mg/24 hours)

1.2-6.5 0.7-2.7 1.2-7.2 0.1-0.4

3.2 1.4 3.4 0.2

IgG IgAb

+

A Light chains Fc fragment

K

a

Modified from Berggard and Peterson (B11).

* Both serum and excretory types.

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W. PRUZANSKI AND M. A. OGRYZLO

by the splitting of normal immunoglobulins with proteolytic enzymes (C5, C6). The majority of the low molecular weight proteins which have been identified in normal urine have closely resembled the light chains of the immunoglobulin molecules (B8, B9, B10, B13). Both types of light chains, kappa and lambda, have been encountered (Tl). On electrophoresis, they are located mainly in the gamma-beta regions, with trace amounts in the alpha-2 region (B11). The light chains may consist of monomers (M.W., 20,000-26,000, s&, = 2.5 S) dimers (M.W., 40,00046,000, s&, = 3.6s) or tetramers (M.W., 88,000, si0,, = 5.3s) (B11, B12, F13). The kappa type dimers consist of monomers that are either disulfide-linked or noncovalent bonded, whereas lambda type dimers consist of monomers that are predominantly disulfide-linked (B11). All these globulins exhibit physicochemical, thermosolubility, and other properties similar to those of Bence Jones proteins. For this reason, it has been suggested that the free light chains present in normal urine are the physiological counterparts of Bence Jones globulins (B10, B12, T1) . Normal urine also contains light-chain subunits of either kappa or lambda type having a lower molecular weight than the monomers of Bence Jones globulin (B11). Freedman observed a protein closely resembling the Fc fragment of IgG in postexercise urines (F12), an observation which has been confirmed by others (B17, V4). This Fc fragment has comprised approximately 15% of the IgG present in postexercise urines (V4). It is not yet certain whether this fragment originates from the degradation of IgG molecules by proteolytic enzymes (C16, V4), or whether it is excreted in the course of de novo synthesis of immunoglobulins (B9). Turner and Rowe and others have also shown that normal urine may contain globulin similar to the F’c fragment of IgG (T15, T16). The fragment was encountered in approximately 20% of normal urines (B17). 4.

Malignant Diseases with Production of M Components

4.1. CLASSIFICATION Multiple myeloma and macroglobulinemia are the best known examples of malignant diseases in which the proliferation of immunoglobulin-producing cells leads to an excessive secretion of homogeneous globulins. However, there have recently been described a number of new syndromes that also appear to belong to the same group of plasmacytic-lymphocytic disorders. The one characteristic common to this group is the observation that homogeneous globulins, either whole molecules or their fragments, produced in excess by the malignant cells,

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TABLE 2 CLASSIFICATION OF MALIGNANT DISEASES WITH M COMPONENTS Diagnosis 1. Multiple myeloma

2. Plasma cell leukemia 3. Biclonal IgG/IgM gammopathy 4. Syndrome with production of IgG half-molecules 5. Macroglobulinemia 6. Syndrome with production of 7 S IgM 7. 7-Heavy-chain disease 8. a-Heavy-chain disease 9. p-Heavy-chain disease 10. Lymphomas“ 11. Leukemias0 12. Epithelial neoplasms@

M Components found in the serum and/or urine IgG, IgA, IgD, IgM, IgE, Bence Jones globulin, low-molecular-weight Bence Jones related globulins, Fc, F’c, or Fab fragments, incomplete molecules Same as Group 1 IgG, IgM, and Bence Jones globulin IgG half-molecules IgM, 7 S IgM, Bence Jones globulin IgM related 7 S globulin 7-Heavy-chain related globulins a-Heavy-chain related globulins p-Heavy-chain related globulin, Bence Jones globulin Same as group 1 Same as group 1 Same as group 1

a It is not clear whether the M component is produced by the tumor cells or by simultaneously proliferating plasma cells.

appear in the serum and are frequently excreted in the urine. At the present time, the problem has become increasingly complex due to the variety of proteins that have been identified. Furthermore, no meaningful classification of these disorders, either from the standpoint of the predominant cell morphology or on the basis of the type of protein being synthesized, has gained general acceptance. I n the present state of our knowledge, this group might well be called “malignancies of the immunoglobulin-producing cells,’’ or “malignant diseases with production of M components’’ (Table 2 ) .

4.2. DEFINITIONOF M COMPONENTS (IMMUNOGLOBULINS OR THEIRFRAGMENTS) The term M component (Myeloma, Macroglobulinemia, Malignant, Monoclonal) was first introduced by Gutman (G7, G8) and later revived by Riva (R4),to indicate the presence of an abnormal spike on free electrophoresis or an abnormal narrow band of protein on paper or cellul.ose acetate electrophoresis, of serum or other biological fluids. Almost invariably, these bands represent the accumulation of an anti-

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genically and physicochemically homogeneous protein originating from one abnormal proliferating line of immunoglobulin-producing cells. By definition, all molecules of such a protein have the same types of heavy and light chains and the same allelic specificities. Still, many M components will show multiple discrete bands (usually 4 or 5 ) on starch gel electrophoresis (F2, F3). An explanation for this heterogeneity has been offered by Askonas, who has shown that it may be related to differences in the electrical properties of various groups of molecules, occurring subsequent to the secretion of the synthesized homogeneous protein into the serum. Intracellularly, the newly synthesized myeloma protein is invariably homogeneous (A7, A8).

4.3. CLASSESOF M COMPONENTS The vast majority of the abnormal proteins that appear in the urine in malignant diseases belong to the family of immunoglobulins. During the past decade, the number and variety of these globulins or their fragments which have been discovered, have increased rapidly, (G5) emphasizing the need for an internationally recognized classification. All these globulins may appear in the serum and/or in the urine of patients with malignant disorders, constituting M components (Table 3 ) . To date, five major classes of human immunoglobulins have been recognized (IgG, IgA, IgM, IgD, and IgE). They have a constant fourchain structure consisting of two light and two heavy polypeptide chains, linked covalently by disulfide bonds. The individual classes while having distinct physicochemical, immunological, and biological properties are nevertheless heterogeneous (C9, C10). All immunoglobulins of the same class which have been analyzed thus far have been shown to have a unique amino acid sequence (C12). A given chain is determined by the amino acid sequence of its C-terminal (invariable or constant) half, varying almost invariably only allotypically. However, within a single type, there is a remarkable structural heterogeneity confined to the N-terminal (variable) half. This variability has been explained either by the existence of as many genes in the germ line as there are immunoglobulin chains, or by somatic mutations presupposing the existence of some mechanisms producing variability of amino acid sequences, coded by a few genes (C11). Light chains have been divided into two major types-kappa and lambda-and further subdivisions (allotypic and nonallotypic) have been proposed (see below). Within each class of heavy chain, several subclasses have also been detected (four for IgG, two for IgA, two for IgM) , which are further subdivided according to the allotypic specificities.

cLASf3ES OF

M

TABLE 3

CoMPONENTa

FOUNDIN

SERVM AND/OR URINE IN

Heavy-chain WPQ

Class IgG Incomplete IgG IgG half-molecules 7-Heavy-chain disease globulin

1, 2, 3, 4 1 ? 1 or3

Ig-4 a-Heavy-chain disease globulin IgA half-molecules in mice plasmacytoma

a-Major, a-Minor a-Related a-Related

IgM 7 S IgM p-Heavy-chain disease globulin

p-Major, N-Minor paelated p-Related

w

6

IgE Bence Jones globulin

Sedimentstion Light-chain constant type and subtype (S) or X

N

6.7-7.2 5.4

-

2.8-4.0

N

-

N

N

or X

K

or X

7-22 3.2 3.9

or or X

19 7

N

N

N

K

-

MALIQNANT DISEASES

or X or X

N

Approximate molecular weight

160,000 125,OOO 75,000 52,000-56,OOO 170,000-900,000or more 36,000-38,OOO

-

900,OOO

5.3

-

7 7.9 3.5-5.5

160,000 200,000 22,000-88,000

N2, N3 Go (+I GO (-1

Kll

Low-molecular-weight Bence Jones-rehted globulins -

*g

!s Z

1 34

D

X

05

2!

(4-1, oz (-1 (-1

St ( + I 1 St N or X

1.2-1.8

11,000-17,000

03

&

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W. PRUZANSHI AND M. A. OGRYZLO

4.4. SYNTHESIS OF M COMPONENTS Immunoglobulins are synthesized in lymphoid tissues, such m those of the spleen, lymph nodes, and bone marrow, as well as by lymphocytes and plasma cells of the thoracic duct lymph and peripheral blood (V2). Light and heavy chains appear to be formed on separate polyribosomes (W10). Each cell usually produces only one allellic allotypic specificity on the heavy chain and one on the light chain (A?). It would appear that the synthesis of light and heavy chains is closely balanced, although there is usually a small surplus pool of free light chains which act as intermediates in assembly (A6). The free light chains exist mainly as et pool maintained within the cell, in contrast to assembled immunoglobulin molecules which are continuously secreted from the cell (A7). In plasmacytic neoplasia, control of the synthesis of the polypeptide chains is frequently impaired, such that one chain may be produced in excess of, or to the exclusion of the other. I n the most common circumstances, there is excessive formation of light chains, resulting in Bence Jones proteinemia and proteinuria. In addition, these patients frequently have high concentrations of whole molecules of one, or rarely more, of the immunoglobulins in the serum, specifically produced by the abnormally proliferating cells. Such patients will usually show one or More abnormal peaks on electrophoresis of the serum, comprising homogeneous populations of whole immunoglobulin molecules, and also an abnormal peak in the urine comprising the Bence Jones protein. I n some patients, the synthesis or release of heavy chains appears to be suppressed so that only Bence Jones protein may be detected (light-chain disease) . I n these circumstances, electrophoresis of the urine will show an abnormal homogeneous peak comprising the Bence Jones protein, which appears in the urine because of the high clearance of the light chains, while the serum pattern may be normal or even show a reduced gamma fraction. Rarely, only abnormal fragments of heavy chains are synthesized such as in patients with heavy chain diseases. I n still others, parts of light and heavy chains may be deleted, leaving incomplete molecules. Absent links between two heavy chains may result in the production of half-molecule protein. 4.5. BENCBJONESGLOBULIN

On the basis of radial immunodiffusion analysis, Korngold and Lipari in 1956 divided Bence Jones globulins, myeloma proteins, and normal 7-globulins into two antigenic groups, A and B (K6, K7). Similarly, Fahey showed that all immunoglobulins, including the urinary y-microglobulins, could be divided antigenically into two types, namely

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ABNORMAL PROTEINURIA I N MALIGNANT DISEASES

types I and I1 (F4). It was subsequently shown that the antigenic determinants that identified groups B and A or types I and I1 were located on the light chains. Eventually the nomenclature of “kappa” and “lambda” was accepted for these two types. It was found that kappa type was present in 67-69% of myeloma proteins and lambda type in 31-33% (C3, H4). The recognition of two major antigenic types of Bence Jones globulins immediately stimulated an intensive study of structural differences between these proteins (El, M4, P14, P15, S7). Both types have an Nterminal half with a variable sequence of amino acids and a C-terminal half with an invariable sequence. I n analyses cf peptide maps of tryptic digests of kappa and lambda Bence Jones globulins, it was observed that a set of common peptides could be identified in various Bence Jones globulins of the same type. The common set of kappa type, however, was completely different from the common set of type lambda (B2). I n individual patients, the serum M component and the autologous urinary Bence Jones protein usually have the same antigenic determinants (M4). In most instances, however, they tend to have different electrophoretic mobilities, the urinary proteins migrating faster (more anodal) than those in the serum (C17, E6). In our experience, similar differences have been observed, with the exception of myelomas in which only Bence Jones proteins are produced (light-chain disease) (Fig. 1). I n the latter instances, the electrophoretic mobility tends to be the same.

-

4

ELECTROPHORETIC MOBILITY OF SERUM M COMPONENTS AND URINARY

30j

4

BENCE JONES PROTEINS I N VARIOUS TYPES OF MULTIPLE MYELOMA IgG (51)

.... **

B - J (31)

’ ’ .....‘ .. .... .......

MOBILITY 301

FIG.1. Electrophoretic mobility in each type of myeloma is divided into five segments: left to right-slow gamma, mid gamma, fast gamma, beta, and alpha-2. BJ =.Bence Jones myeloma = light-chain disease.

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PRUZANSKI AND M. A. OGRYZLO

Others have also noted identical electrophoretic mobility of the serum and urinary Bence Jones globulins in light chain disease (S18). I n rare instances, two Bence Jones globulins of different type have been found in the urine, whereas the serum has contained either a single M component (E4), or two M components of the same type of light chain (D4). Only two examples have been reported in which the light chains of serum M component and urinary Bence Jones protein were of different antigenic type. I n one, the serum M component was of the IgG/K type while the Bence Jones globulin in the urine was of the lambda type (M4), and in the second IgG/L was found in the serum and Bence Jones kappa type globulin in the urine (M2). Examples of two serum M components with different types of light chain and two types of Bence Jones globulin in the urine corresponding to the same light chains have also been recorded (D8, R5). Gutman in 1941 was the first to detect the presence of Bence Jones globulin in the serum (G8, M11). Except for some cases of light-chain disease, the identification of Bence Jones protein in the serum has been difficult. I n recent years, however, the use of modern immunological techniques and of specific strong antisera has greatly facilitated the detection of Bence Jones globulin in serum. I n one series, comprising of 46 patients with multiple myeloma, it was possible to identify 8 patients (17%) with Bence Jones globulin in the serum, in addition to the main M component (A9). About 25% of patients with IgD myeloma have demonstrable Bence Jones proteinemia (P10). However, in the majority of patients in whom Bence Jones can be demonstrated in the serum, the concentration is low (P10, S18, W8). On the other hand, the presence of Bence Jones globulin in the serum when it is absent in the urine must be rare indeed. Two such examples have recently been described, comprising a tetramer of Bence Jones type lambda globulin (M.W. 84,00&88,000). The large size of these molecules apparently accounted for their lack of filtration through the glomerular membrane (C2, G4). In another case observed by us, a tetramer of Bence Jones type lambda globulin was found in large amount in the serum but was also excreted excessively in the urine, along with albumin and other globulins. 4.5.1. Physicochemical Characteristics

Although it is now more than 100 years since the discovery of Bence Jones globulin, it is rather surprising that the rigid criteria for the definition of this protein for the most part, still remain valid. In 1964, Snapper stated that any urinary substance which did not precipitate a t a temperature between 40" and 60°C at pH 5.0, and did not redissolve on boiling

ABNORMAL PROTEINURIA IN MALIGNANT DISEASES

347

a t p H 3, was not a Bence Jones globulin (S14). Others have supported this view ( H l ) . However, this concept has required modification in the light of newer knowledge. First, it has been shown that heat precipitability and dissolution a t boiling are not obligatory prerequisites for the diagnosis of Bence Jones proteinuria, since not all Bence Jones globulins, as immunochemically defined, have the property of heat precipitation (B15, S21), and some precipitate only partially (521). Second, i t has also been observed that other proteins present in urine, may precipitate on heating and redissolve on boiling in the range of temperatures very close to that of Bence Jones globulin. Transferrin is perhaps the best example, precipitating at 59" and redissolving at 95°C (B15). The majority of Bence Jones globulins have ultracentrifugal sedimentation constants between 2.44 and 4.40, diffusion coefficient Dgo, 4.7-9.8 X cm2/sec, molecular weights between 24,000 and 9O,OOO, and isoelectric points between 4.6 and 6.7 (S12). As has been shown, Bence Jones globulins are remarkably heterogeneous (P16). For example, no two identical globulins were found among 102 Bence Jones proteins as analyzed by peptide maps of tryptic digests (Q2). Bernier and Putnam distinguished between two types of heterogeneity of Bence Jones globulins, namely polymerism and polymorphism (B15). Various authors = 2.0 S), monomer variants (s20,w= 2.3 S), have found monomers (szo,w dimers (s?~,, = 3.6 S) and tetramers (sz0,, = 5.5 S) of Bence Jones globulin in wines of myeloma and patients (B15, G1) . On the other hand, multiple components of Bence Jones globulin with different mobility on starch gel electrophoresis may be present in the urine, yet exhibit the same sedimentation rate and appear to be antigenically identical to the major component (B15). Another type of heterogeneity of Bence Jones globulins has been ascribed to variations of sialic acid content rather than to differences in amino acid sequence (M6). It has also been shown that monomers and noncovalently linked dimers of Bence Jones globulin are usually of type kappa whereas disulfide-linked dimers are usually of type lambda (M8, M9). 4.5.2. Bence Jones T y p e Kappa Globulin

This type of globulin comprises approximately 67-69% of the Bence Jones globulins found in the urine in multiple myeloma (C3). I n an extensive study of type kappa Bence Jones globulins, it has been shown that in all instances the C-terminal (invariable) half has been identical except for residue 189 in the peptide K9A (residues 189-192). This residue, being either valine or leucine, is determined by a genetic factor present in the Inv locus (B3, B4, Q2). Three Inv factors, namely 1, 2, and 3 aTe recognized (F10). In general, Bence Jones type kappa globulins

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W. PRUZANSKI AND M. A. OGRYZLO

are either Inv (1,2) or Inv (-1, -2), although a few Bence Jones globulins have been found to be Inv (1, -2). This specificity has not necessarily been located on the invariable part, but rather on a region which overlaps the variable and invariable halves (L6, Q2). Additional subtypes, independent of the Inv type of Bence Jones type kappa globulin, have been detected by the use of various techniques. Milstein succeeded in subdividing kappa type Bence Jones globulins into three subgroups, I, 11, and I11 (M9), which were defined by a characteristic sequence of the first 23 residues in the variable part. I n peptide maps, there were, on an average, 9 peptide differences between these types. One antiserum was able to distinguish between kappa type with Nterminal glutamyl residue and that with N-terminal aspartyl residue (522). Using this antiserum, it was possible to recognize three subtypes of kappa light chains, which were designated Kglu,K,,, 11, and KaspI. It was further shown that these three subtypes of kappa light chains were distributed unequally among different immunoglobulins. For example, 90% of IgM type kappa globulins were of Kglutype (522). However, it would appear that the classification of Milstein (B9) and that of Solomon and McLaughlin are interrelated. Still other subtypes, depending on a single peptide difference, have been detected by antisera against individual Bence Jones globulins (Nl, N2). An antiserum against Bence Jones “Go1’ differentiated 27 Bence Jones type kappa globulins into Go (+) and Go (-) subtypes. Thus far, however, specific Go (+) antigenic determinants have not been demonstrated on normal gamma globulins (T6). It has been estimated that Bence Jones type kappa globulins may have between 214 (T12) and 221 amino acid residues (H3). For more detail on this aspect of the subject, the interested reader is referred to the specific articles describing the amino acid sequences of kappa type Bence Jones globulins (P16, P17, T12, T13, T14, W6). 4.5.3. Bence Jones T y p e Lambda Globulin

This type comprises approximately 3133% of all Bence Jones proteins found in the urine of patients with multiple myeloma. Heterogeneity within this type of Bence Jones globulin has been revealed by a rabbit antiserum against Bence Jones “Oz” (A4, E2, Q l ) . It has been found that the difference between Oz (+) and Oz (-) globulins results from a variation in one amino acid located on the invariable part of the globulin a t residue 190, in which either arginine or lysine may be found (A4, E2, Q1, R8, T8). Other substitutions on the constant parts have recently been reported and have been summarized by Putnam (P16).

ABNORMAL PROTEINURIA I N MALIGNANT DISEASES

349

Another subdivision has been made possible using antiserum against Bence Jones “St.” The difference between St (+) and St (-) types was located on the N-terminal variable part. Among 18 Bence Jones type lambda globulins, four were S t (+) and the others St (-) (T6, T9). I n addition to the above, the existence of additional subtypes has been suggested by a study of the peptides of the invariable C-terminal portion of 18 Bence Jones type lambda globulins. A difference in two peptides was observed in one globulin as compared to the other 17 globulins (T8). 4.5.4. Atypical Bence Jones Globulins

There have been several descriptions of atypical Bence Jones globulins. For example, one globulin had two chains of unequal length and a molecular weight of 28,000 ( A l ) . Also two Bence Jones globulins with properties of cryoprecipitation have been reported (A3, K9). One Bence Jones type kappa globulin, having a sedimentation constant of szo,w= 3.5 S (K9) and another type lambda of s20,w = 3.4 S (A3), both had a tendency to produce aggregates in cold. Another urinary cryoprotein with s20,w= 3.6 S, although not defined immunologically, was probably also Bence Jones globulin (V3). BENCEJONES-RELATED GLOBULINS 4.6. LOW-MOLECULAR-WEIGHT In 1965, Deutsch (D6) described a patient whose urine contained a classical Bence Jones type kappa globulin and also a lower-molecularweight globulin related to the Bence Jones globulin. There were two spikes in the urine on electrophoresis. Both globulins were isolated in crystalline form. One had a molecular weight of 35,000 and sedimentation rate s20,w = 3.5 S, the other a molecular weight of 17,000 and a sedimentation constant s20,w = 1.85 S. More recently, similar globulins have been described in several additional reports. According to Kunkel, low molecular weight globulins antigenically related to Bence Jones globulins have been observed in 30% of urines of patients with multiple myeloma and Bence Jones proteinuria (K11, 523). Small molecular weight components related to Bence Jones globulins were detected in 9 of 24 urines with type kappa Bence Jones protein and in 7 of 22 urines with type lambda Bence Jones protein, The amount comprised approximately 15% of the total Bence Jones globulin in the urine (523). Some of these low-molecular-weight globulins are slower, while others are faster, in their electrophoretic mobility than the major Bence Jones component (B4) . On immunoelectrophoresis and starch gel electrophoresis, they are placed more cathodally than the major Bence Jones

350

W. PRUZANSKI AND M. A. OGRYZLO

component and produce separate bands (523). I n these instances, different antisera against Bence Jones proteins have often produced more than one precipitation line on immunoelectrophoresis. Some of the antisera have elicited precipitation lines which showed a reaction of identity with the line of the major Bence Jones component, while others showed a reaction of partial identity, implying that the low-molecular-weight substances were antigenically deficient as compared with the major Bence Jones component (K11, S25, W9). Various molecular weights have been reported for these substances, ranging from 11,OOO (B11, W9) to 17,000 (D6). In ultracentrifugation, their sedimentation constant has varied between szo,w = 1.2 and 1.8s (B11, 523, V1, W9).It has been suggested that these low-molecularweight fragments may be derived from the variable N-terminal part of Bence Jones globulin (B4, T4, V1, W9).Fingerprinting and amino acid analyses have supported this view, especially since no peptides corresponding to the invariable part of the Bence Jones globulin have been observed (B4). Other investigators have found low molecular weight Bence Jones-related fragments corresponding to the invariable C-terminal half (B11) , or even to both halves (520). It is still not clear whether these fragments are products of degradation, or whether they arise in the course of de novo synthesis of immunoglobulin molecules (C7). A significant contribution to understanding of the origin of these low molecular weight globulins has recently been made by Solomon and McLaughlin (521). These authors observed that in acid milieux (pH less than 5.8), normal urines and also the urines of patients with multiple myeloma possess proteolytic activity, and are capable of splitting Bence Jones globulim into variable and constant halves. Thus, they were able to identify one or both halves in addition to the whole molecules of Bence Jones globulins in urines of patients with myeloma, where examined under these conditions. In one patient a constant half was found in the serum in addition to the Bence Jones globulin. On electrophoresis and immunoelectrophoresis, it was shown that the variable half moved cathodally and the constant half anodally as compared to the whole molecule of the Bence Jones globulin. It was also observed that the variable part possessed heat precipitation properties similar to the intact Bence Jones globulin, whereas the constant part remained in solution a t all temperatures. The proteolytic cleavage in kappa type Bence Jones globulins occurs in position 107 (lysine) or 108 (arginine) whereas, in lambda type globulins, it occurs in the position 111 (lysine). Both halves are necessary for the antigenic expression of Inv. The authors concluded that these variable and constant halves in the urine were derived from both the catabolic activity of proteolytic

ABNORMAL PROTRTINURIA IN MALIGNANT DISEASES

351

urinary factors and also from de novo synthesis in the immunoglobulinproducing cells (521). I n the urine of one patient with multiple myeloma and a Bence Jonestype lambda proteinuria, Snyder found a polypeptide with a molecular weight of 3000 comprising 32 amino acid residues (S16). This polypeptide, which corresponded in sequence to a portion, 147 (valine) t o 178 (tyrosine), of the lambda type Bence Jones globulin molecule, constituted 25% of the total Bence Jones globulin excreted in the urine. It was assumed that the polypepetide was not a degradation product, but rather that it resulted from an abnormal synthesis due to malfunction in genetic transcription or in translation. Further study of these low molecular weight substances, such as the demonstration of their existence in the serum and their synthesis in tissue culture by immunoglobulin-producing cells, may eventually provide the answer as to their origin. 5.

Multiple Myeloma

Multiple myeloma was the first malignant disease in which abnormal urinary proteins were detected. The disease results from the malignant TABLE 4 CLINICAL STAGES OF PLASMA CELLNEOPLASIA‘ Stage 1. Early incipient disease. May be

asymptomatic

2. Overt disease. Usually symptomatic

3. Advanced disease

4. Fulminant disease 0

Clinical manifestations An M protein may be discovered during investigation of mild anemia, rouleaux formation, serum anticomplementary activity, elevated erythrocyte sedimentation rate, pneumonia, or other infections, or unexplained proteinuria. Occasionally, there may be a “solitary” plasmacytomaskeletal or extraskeletal M protein in serum and/or urine, proteinuria, marrow plasmacytosis, osteolytic l e sions, osteoporosis, elevated BUN, anemia, hypercalcemia, hyperuricemia, lymphadenopathy, splenomegaly Renal insufficiency, hypercalcemh reaktant to therapy with prednisone and adequate hydration, inability to maintain a hemoglobin concentration higher than 9.0 g/ 100 ml without transfusions, rapidy increasing numbers of plasma cells in peripheral blood. Hyperviscosity syndrome Plasma cell leukemia

Modified from Bergsagel and Pruzanski (B14).

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W. PRUZANSKI AND M. A. OGRYZLO

TABLE 5 BREAKDOWN OF PLASMACYTIC NEOPLASIA ACCORDING TO THE TYPEOF M COMPONENT ~~~

~

Type of M component IgG myeloma IgA myeloma Light-chain disease Other myeloma8 Macroglobulinemia Summary

Data compiled by Zawadzki and Edwards (Z2) 816 369 171 39 194 1589

Data on our patients“ Combined data 133 31 37 13 24 238

949 400 208 62 218 1827

Percent 52.0 21.9 11.4 2.8 11.9 100

Patients investigated in the Immunoproteins Research Laboratory of the University of Toronto Rheumatic Disease Unit. b Include IgD, biclonal, and “neabnormality” myelomas.

proliferation of more or less immature plasma cells, with the production of medullary osseous tumors and infiltration of internal organs associated with anemia, proteinuria, and a variety of other complications (B14, 012, 515) (Table 4). In the great majority of patients with multiple myeloma, an M component is usually found in the serum and/or in the urine. However, in about 1% of the patients, no M component may be apparent either in the serum or urine (H5, 07), while in another 276, more than one M component may be present (H5). The overall proportion of various types of myeloma is given in Table 5. It has recently been recognized that myelomas with different types of M components have been associated with varying clinical pictures. This phenomenon is most probably related to the variable biological behavior of plasmacytic tumors and to the different physicochemical properties of the M components produced by these tumors. I n general, patients with light-chain disease and with IgD myeloma are younger than those with IgG or IgA types, and their disease seems to be more severe (H5, P10). It has also been noted that severe azotemia occurs a t least twice as frequent in light-chain disease and in IgD myeloma as compared to IgG or IgA myelomas (H5, P10). 5.1. BENCEJONES PROTEINURIA

The excretion of Bence Jones protein in the urine is dependent on a number of factors, such as (1) the rate of synthesis, (2) plasma volume, (3) degradation rate, (4) renal catabolism, and (5) urinary volume (Hl, W11).

ABNORMAL PROTEINURIA IN MALIGNANT DISEASES

353

I n general the renal clearance of Bence Jones globulin is inversely related to the molecular size. When the mean molecular radii are 22-36 A, the renal clearance of Bence Jones is in the range of 940% of the creatinine clearance (Hl). The total amount of Bence Jones globulin which may be excreted in the urine also varies widely from trace amounts, detectable only by highly sensitive immunochemical techniques (D2, D3, L4, L5) , to as much as 22 g ( C l ) , 30 g (S18),or even 77 g in 24 hours ( M l ) . The frequency with which Bence Jones globulins have been demonstrated in the urine in multiple myeloma, has increased in parallel with the sensitivity of the technique employed. I n one series of patients with multiple myeloma reported in 1955,Bence Jones protein m tested by heat precipitation, was detected in only 45% of the cases ((34).Different reports have indicated a frequency varying from 41% (012) to 55% (Cl),whereas others claimed that Bence Jones globulin could be found in the urine in virtually all patients with multiple myeloma (C14). In our own experience in recent years, applying a combination of electrophoresis and immunoelectrophoresis, the frequency has been 70% (Table 6 ) . Others have reported a similar range, 62% for IgG myeloma and 70% for IgA myeloma (H5). However, when a highly sensitive technique was used for the detection of free light chains, 87% of the urines from patients with multiple myeloma were found to contain an increased amount of one antigenic type of light chain (D3). This method is based on the use of antiserum made specific for specific antigenic sites TABLE 6 URINARYPROTEINS IN VARIOUSCONDITIONS WITH SERUMM COMPONENTS~

Disease Multiple myeloma Macroglobulinemia Other neoplastic diseases Nonneoplastic diseases Summary a

Number of cases with Num- availber of able patients urine

Bence Jones globulin

Other No significant M compoiients proteinuria

214 24 22

157 15 12

110 (70.OgZ)* 6 (40%) 8 (67%)

11 (7%) 2 (13%) 2 (1757~

40 (25.5%) 8 (53%) 4 (33%)

45

23

7 (30.4%)

2 (8.7%)

16 (69.6%)

305

207

131 (63.3%)

17 (8.2%)

68 (32.8%)

Patients investigated in t,he Immiinoproteins Research Laboratory of the University

of Toronto Rheumatic Disease Unit. Percentage of the tot,al number of cases in which urine was tested.

354 W. PRUZANSKI AND M. A. OGRYZLO

FIG.2. Three examples of IgG myeloma: (A) Bence Jones type kappa in the urine i s of slower electrophoretic mobility than IgG M component in the serum, but migrates toward the mode on immunoelectruphoresis. (B) IgG M component in the serum and urine. Spontaneous partial split into Fc and F‘c fragments is observed. ( C ) Bence Jones type lambda in the urine is of faster electrophoretic mobility than IgG M component in the serum. Spontaneous partial split of IgG into Fc and F’c fragments is observed in the serum and urine. S = serum; U = urine; a-Fc = goat antihuman Fc fragment antiserum.

ABNORMAL PROTEINURIA IN MALIGNANT DISEASES

355

of light chains and is capable of detecting amounts as small as 2 mg/100 ml of free light chains (D3). 5.2. OTHERM COMPONENTS IN THE URJNE

M components other than Bence Jones globulin may also be found in the urine, comprising either whole molecules of the serum immunoglobulins or their fragments. In a series of 30 patients with multiple myeloma, the urine contained Fc fragment in three instances and a larger component related to the heavy chain of IgG in another ( 0 3 ) . In our series of 157 patients with multiple myeloma, M components other than Bence Jones globulin were found in the urines of 11 patients (7%) (Table 6) (Figs. 2 and 3). One patient with multiple myeloma and a serum M component of IgG type of s.&, = 6.6S, had two components in the urine, one consisting of disulfide-linked heavy chains of s&, = 5.8 S and the other, an Fc fragment bound to lambda type light chains (54). Another, very unusual, M component of IgG-1/K type present in the serum of one patient, had an si0,, = 5.4 S and a molecular weight of 125,000. The same protein was also found in the urine (L2). Preliminary studies of this protein showed that there was partial deletion of the heavy and also of the light chain (C15). Half-molecules of IgA, composed presumably of one light and one heavy chain of sz0,, = 3.9 S, have been found in the serum of MOPC 47A mice with plasma cell tumors (P6). Examining these “half”molecules by peptide mapping, others have found a considerable deletion of some 120-130 residues in the heavy chain (L3, S8). Sirniliar “half”-molecules of IgA have been found in the urine of BALB/c mice with plasma cell tumors (LA). 5.3. LIGHT-CHAIN DISEASE This term is applied to circumstances in which the abnormally proliferating plasmaeytes excrete homogeneous light chains only. Frequently no M component is seen in the serum on electrophoresis in such patients (Fig. 4) although Bence Jones globulin is detected in the urine. There is usually heavy proteinuria (B16, S5, S18), and quantities varying from 4 to 3 2 g per 24 hours have frequently been recorded (Sl8). However, small quantities of Bence Jones globulin varying in concentration from 0.3 to 1.8 g/lOO ml (SlS), can usually be detected in the serum as well, by means of immunoelectrophoresis and other techniques. Even when there is no immunoelectrophoretically detectable homogeneous Bence Jones protein in the serum, neverthelem, the presence of free light chains, identical in type to the urinary Bence Jones globulin, may be detected by more sensitive methods (W8). However, it is possi-

356 W. PRUZANSKI AND M. A. OGRYZLO

a

0

8* N

r

0

FIG. Fm.3. Three examples of IgA myeloma: (A) Bence Jones globulin in the urine of slower electrophoretic mobility than the IgA M component. (B) The urine contains, in in addition to the Bence Jones globulin, also IgA M component. Both are of the same electrophoretic mobility. (C) The urine contains, in addition to the Bence Jones globulin, also IgA M component of different Merent electrophoretic mobility. S = serum; serum ; U = urine; urine ; a-IgA a-Id = rabbit antihuman IgA antiserum. mtkn~n.

ABNORMAL PROTEINURIA IN MALIGNANT DISEASES

357

FIG.4. Four examples of light-chain disease: (A and C) “Normal” electrophoretic pattern of the serum and urine. Immunoelectrophoresis identified Bence Jones type lambda of alpha-2 mobility in both fluids. (B) “Almost normal” electrophoretic pattern of the serum and urine. Bence Jones globulin identified in both immunoelectrophoretically. (D and E) Small spikes of M component in the serum were identified as a Bence Jones globulin. S = serum; U = urine; a-BJ/K and a-BJ/L = rabbit anti-Bence Jones type K and type L antiserum, respectively.

358

W. PRUZANSKI AND M. A. OGRYZLO

ble that some of the patients with light-chain disease reported thus far, may well have been examples of IgD or I g E myelomas, since the M components of these types are often of low concentration and frequently do not produce spikes. Until recently, specific antisera have not been available for their detection. 5.4. RARETYPEX OF MYELOMA

At the present time, there are 50 reported examples of IgD myeloma in the world literature (PlO). The mean age of these patients has been 57 years as compared to 62.1 years of IgG myeloma, 63.7 years for IgA myeloma and 55.5 years for light-chain disease. More than 80% of the patients with IgD myeloma have shown evidence of renal impairment exceeding the incidence in light chain disease. Bence Jones globulin has been demonstrated in the urine in more than 90% of the cases of which 90% were of type lambda (P10). I n one patient only, the IgD M component was also found in the urine (S24), as well as in the serum. Similarly, there have been 25 reported instances of multiple myeloma with IgM M components, all of whom excreted Bence Jones globulin in the urine. One subsequently developed plasma cell leukemia (B20, H5, W5). The only two patients described with an IgE M component thus far, also suffered from plasma cell leukemia. Bence Jones-type lambda globulin was present in the serum and in the urine in both patients (B6,52, 01). Fewer than 0.1% of patients with multiple myeloma are associated with M components in serum or urine which are composed of incomplete molecules of immunoglobulins (H5,L2). 5.5. BICLONAL IgG/IgM GAMMOPATHY

Three patients with both myeloma (IgG) and macroglobulinemia (IgM) M components in the serum have recently been described (53). One had, in addition, an IgA M component. It was noted that the clinical picture differed from that of classical multiple myeloma. There was lymphocytic-plasmacytic infiltration of the bone marrow and/or of the lymph nodes, as well as a monocytosis in the peripheral blood. No osteolytic lesions were noted. One had a plasmacytoma of the tongue. Thrombocytopenia and recurrent infections were frequent. All three patients exhibited a single peak in the serum electrophoresis, but multiple M components were observed on immunoelectrophoresis. I n each individual patient, the light chains were of the same type in all of the M components. However, the light chains of lambda type were of differ-

ABNORMAL PROTEINURIA I N MALIGNANT DISEASES

359

ent Oz subtype, while those of kappa type were of different Inv. One patient excreted Bence Jones protein in the urine. 5.6.

SYNDROME WITH PRODUCTION O F

IgG HALF-MOLECULES

I n two patients, M components of IgG/K antigenic type were found to have a low molecular weight of approximately 75,000 (H5,H6). One of the patients described in detail was a 76-year-old man with lymphadenopathy and hepatosplenomegaly. There was a leukocytosis of 14,700/cmm with monocytosis of 13% in the peripheral blood. The bone marrow and lymph nodes were infiltrated by lymphoid and plasma cells, but there was no evidence of bone rarefaction or osteolytic lesions. Physicochemical analysis of the IgG/K M component, which was present in both the serum and urine, revealed that it was composed of one heavy and one light chain. Further study will be required for a more detailed characterization of these molecules. 6.

Plama Cell leukemia

The acute leukemic form of immunocytic dyscrasia is very rare and almost without exception is a fatal disease. The clinical picture comprises a combination of the symptoms of multiple myeloma, such as back pain, along with those of acute leukemia, including bleeding phenomena, rapid weight loss and general emaciation. Hepatosplenomegaly and lymphadenopathy appear with the same frequency as in adults with acute leukemia ( D l ) . This form of plasma cell dyscrasia, which behaves much like other acute leukemias, has given rise to controversial opinions in regard to its relationship to multiple myeloma, the degree of maturity of proliferating plasmacytes and the production of M components. According to several workers, M components, such as those which are commonly found in myeloma, have rarely been observed in plasma cell leukemia (E5,M14, R6, T5),although Osserman (04) stated that abnormal serum globulins occurred with the same frequency as in myeloma. It should be emphasized that, in older publications, the conclusion that M components were not produced in plasma cell leukemia was based on routine laboratory procedures that were less sensitive than the newer techniques for the demonstration of M components. More recent observations have shown that M components, antigenically and structurally indistinguishable from myeloma proteins, are frequently encountered and comparable to those in myeloma patients. In a review of 50 published cases (P12) proteinuria occurred in 38 patients, the Bence Jones heat test being positive in 30. No protein was detected qualitatively in 12 patients. A correlation of the serum and urinary proteins showed that among 37 patients with M component in

360

W. PRUZANSICI AND M. A. OGRYZLO

the serum, 24 gave a positive reaction to the test for Bence Jones protein in the urine, whereas in 7 others, only proteinuria was recorded. Bence Jones protein was also found in the urine of 6 other patients without spikes in the serum. This proportion is comparable to that in multiple myeloma. Nevertheless, it seems that occasionally the function of globulin synthesis is not retained by the neoplastic plasmacytes and, as in myeloma, so also in plasma cell leukemia, M components may not be synthesized. 7. Macroglobulinemia

Macroglobulinemia of Waldenstrom is a malignant disease predominantly of the lymphoid elements. Lymphadenopathy, hepatosplenomegaly, anemia, and various complications related to the physicochemical properties of the IgM M components (hyperviscosity, cryoprecipitation, etc.) are the characteristic clinical features (M5). The serum protein abnormalities in macroglobulinemia, comprising in the main a homogeneous increase in 19 S IgM and rarely in 7 S IgM M component, have been investigated in great detail (525). However, insufficient study has been made of the urinary abnormalities. Some authors have reported a complete absence of Bence Jones globulin in the urine of patients with macroglobulinemia (C8), while others have recorded its presence in 1045% of cases, indicating a wide disparity in its detection (A5, B1, R3). I n a number of individual case reports of macroglobulinemia, the presence of Bence Jones globulin in the urine has been recorded (D3, G6, 527, W12). However, with the use of modern immunological techniques, it has been shown that the incidence of Bence Jones proteinuria may be as high as 60% (D2), while in an additional 2076, an excess of free light chains may be demonstrated (3D). I n our experience, Bence Jones globulin was found in the urine in 40% of cases studied (Table 6 ; Fig. 5 ) . One example of a macroglobulinemia with detectable Bence Jones globulin in the serum, in addition to the major IgM M component, and the same type of Bence Jones globulin in the urine, has also been described ( G 6 ) . It has been found that the urinary Bence Jones globulin and the light chains of the serum IgM M component have similiar thermosolubility properties and sedimentation constants on ultracentrifugation, as well as identical peptide maps ( G 6 ) . Urinary M components, other than Bence Jones globulin, have also been found in macroglobulinemia. In one patient, a urinary protein with gamma mobility, when isolated by DEAE-cellulose chromatography, was found to consist of two components; one of s:,,~ = 5.0s

ABNORMAL PROTEINURIA I N MALIGNANT DISEASES

361

FIQ.5. Two examples of mwroglobulinemia: (A) Urinary Bence Jones type kappa is of slower mobility than serum IgM M component on electrophoresis and immunoelectrophoresis. (B) Urinary Bence Jones type lambda and serum IgM M component are of almost identical electrophoretic and immunoelectrophoretic mobility. S = serum; U = urine; a-IgM, a-BJ/K, a-BJ/L = rabbit antihuman IgM, Bence Jones kappa, and Bence Jones lambda antisera, respectively.

had both kappa and pantigenic determinants and gave a reaction of partial identity with 1 9 s IgM. The author postulated that this component may have represented a Fab-like fragment of IgM (D5,D7). In our experience, two of 15 patients with macroglobulinemia excreted IgM globulins in the urine.

7.1. SYNDROME WITH PRODUCTION OF 7 5 IgM A serum M component of IgM type lambda having a low molecular weight and sedimentation constant of s ~ , , ,= ~ 6.5 S has been observed in the serum of a patient with malignant lymphocytic plasmacytic disease (S19). This 67-year-old woman presented with a fever, lymphadenopathy, hepatosplenomegaly, and a secondary anemia. There was 1+ proteinuria, but the Bence Jones heat test was negative. Unfortunately, no further information regarding the urinary excretion of the serum M-component was provided.

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W. PRUZANSKI AND M. A. OGRYZLO

8.

Heavy-Chain Diseases

8.1. Y-HEAVY-CHAIN DISEASE

In 1964, the first five cases of gamma heavy chain disease were described in detail (F11, 08). The clinical picture included susceptibility to infection, lymphadenopathy, hepatosplenomegaly, palatal swelling, and anemia. Five additional cases have since been reported (E3, L1, W1, 23) (Table 7), in one of which there was peripheral blood leukocytosis of 22,00O/cmm with 19% atypical lymphocytes (E3). Histological studies in these patients have shown a malignant proliferation of plasmacytes, lymphocytes, and reticular elements (Ll, 08) in the bone marrow and lymphatic tissues. Three more patients are currently under investigation (FQ). In almost all cases, electrophoresis of the serum and urine revealed broad-based spikes of beta mobility. However, the urinary proteins have not had the characteristics of Bence Jones globulin. Immunochemical analyses have shown that the abnormal components are devoid of light chains and are closely related to the Fc fragment of the heavy chain of IgG (F11, 08, T2). The molecular weights of these components have ranged from 52,000 t o 56,000 (F11, Ll), while the sedimentation constants have varied from 2.8 to 4.0 (E3, L1, 08, W1, 23). They have TABLE 7

PATIENTS WITH 7-HEAVY-CHAIN DISEASP Seruni Urine

Amount of No. Age 1 2 3 4 5 6 7 8 9 10

p-

M

component Sex Spike (g/lOO ml)

4 4 M 6 9 M 7 1 M 4 9 M 6 6 M 6 0 F 7 6 M 5 0 M 6 5 F 63 M

+ + + + + + + + ?

34 0.3-0.6 2.4 3.5 3.34.3 ? ?

2.3 Present Present

pSpike

+ + + + + + + + + +

Proteinuria 15-20 g/24 hr 3+ 15-20 g/24 hr 2+ 4-6 g/24 hr 0,35/liter 40 mg/24 hr 1 . 4 g/24 hr

+ +

Bence Subtype Jones of heavy globulin chainb Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. ? ?

1 (Cr.) 1 1 1 3 (Xu)

3 3 3 ? ?

a Ellman and Bloch (E3), Franklin et al. ( F l l ) , Lebreton et al. (Ll), Osserman and Takatsuki (OS),Wager et al. (Wl), and Zawadaki et al. (23). WHO Nomenclature, Kunkel et al. (K12).

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363

been shown to be rich in carbohydrates comprising up to 21% of the molecule (F11, W l ) . It has been suggested that the abnormal protein of y-heavy-chain disease is synthesized de novo by malignant cells and is not a catabolic product of IgG ( F l l ) ,an observation which is supported by amino acid sequence studiea. It has also been shown that the heavy-chain disease globulins have both the N-terminal and C-terminal parts of heavy chains, but that there are very large deletions of the mid portion of the molecule (F8, PS). 8.2. a-HEAVY-CHAIN DISEASE I n 1968, the first three patients with a-heavy-chain disease were reported (S9, S10). Now, more than 9 patients are under study (S11). Clinically, they have had an abdominal lymphoma with manifestations of severe malabsorption, associated with a diffuse and predominantly plasmacytic infiltration of the small intestine. I n the first patient, an IgA related globulin devoid of light chains was found in the urine, serum, saliva, and proliferating cells. For want of a better term, it was called TL protein (patients’ initials) . Serum electrophoresis showed a broad band of beta mobility comprising approximately 40% of the total protein (4 g/100 ml) . Using anti-IgA antiserum, immunoelectrophoresis showed an abnormal precipitation line extending from%he alpha2 to the beta-2 region. The absence of light chains in this globulin and in the other four studied, was demonstrated by a variety of imrnunochemical techniques. Ultracentrifugation analysis showed an szo,w = 3.2S globulin with a tendency to polymerize, giving szo,wvalues from 4 to 11. Molecular weight ranged from 36,000-38,OOO.The globulin had a high proportion of carbohydrate, constituting as much as 16.6% of the molecule. Proteinuria varied from 5 to 180 mg/100 ml and consisted of the same IgA related globulin. I n two patients, the urine showed a broad abnormal band of a-p mobility. In all patients studied, this protein has been shown to belong to I&-1 subclass as examined by specific antisera (S10, S l l ) . 8.3. ~-HEAVY-CHAIN DISEASE The first case of p-heavy chain disease was reported in 1969 by Forte et al. (F7). The patient suffered from chronic lymphocytic leukemia and amyloidosis. Bone marrow examination revealed an infiltration with over 50% lymphocytes and 33% plasma cells, the latter containing large vacuoles. Immunochemical studies showed reduced IgG and IgA in the serum as well as Bence Jones type kappa proteinemia and

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proteinuria. Low-molecular-weight globulin of s & , = ~ 5.3 S, related to the pheavy chain was found in the serum and in the urine of the patient. 9.

Lymphoma

Although the presence of M components in the serum of patients with lymphomatous diseases has been well documented (K8), no systematic studies of the urinary proteins have been undertaken. I n one patient with lymphosarcoma and an IgG/L M component in the serum, there was heavy proteinuria with the excretion of a Bence Jones type kappa protein (M2). Since the type of light chains of the serum M component and of the urinary Bence Jones globulin were different, this condition was called asynchronous biclonal gammopathy. In another patient with lymphosarcoma, two M components, IgG/K and IgG/L, were found in the serum along with Bence Jones type kappa protein in the urine (A2). I n our own material, two of seven patients with malignant lymphomatous diseases and serum M components, excreted Bence Jones globulin in the urine, one of whom also excreted IgG/L, Fc, and F’c fragments. Lindstrom reported an increase in free light-chain excretion in 7 of 30 patients with lymphoma and in 3 of 23 patients with Hodgkin’s disease. However, the author pointed out that these proteins were much more heterogeneous than Bence Jones proteins in multiple myeloma (L5).Heavy proteinuria has been reported in patients with lymphoma complicated by amyloidosis involving the kidneys and producing the clinical manifestations of a nephrotio syndrome (A10, K3). 10.

leukemias

It is well known that serum M components may be observed in patients with various leukemias, although here also no systematic study of the urine has been undertaken. Bence Jones globulin has been found in the urine in a few patients with chronic lymphatic leukemia with serum M components (Jl, R9).I n one example of an acute blast cell leukemia in a 2-year-old child, IgG-1/L M component was found in the serum and a Bence Jones type lambda protein in the urine (526). Lindstrom et al. (L4)have observed an increased excretion of heterogeneous free light chains in acute and chronic myelogeneous leukemia and in monoand myelomonocytic leukemia. Poulik et al. (P7) have reported an example of monocytic leukemia with IgG/K M component in the serum and IgG, Bence Jones type kappa globulin, Fab, Fc, and F’c fragments in the urine. In our experience, the urinary protein in 3 of 27 patients with proteinuria associated with mono- and myelomonocytic leukemia exhibited the thermal solubility properties of Bence Jones protein. One patient excreted Bence Jones type kappa globulin in the urine for 3 months, after which this globulin disappeared from the urine (Fig. 6).

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FIQ. 6. Cellulose acetate electrophoresis of the concentrated urine from a patient with monocytic leukemia with transient excretion of Bence Jones globulin. (A) No Bence Jones globulin. (B) Benee Jones globulin ( B J ) of fast gamma mobility. L = lysozyme; 1 = unidentified substance of postgamma mobility; ALB = albumin.

1 1.

Epithelial Malignancies

There have been no systematic studies of urinary proteins in malignancies of epithelial origin. However, an increase in the total polypeptide: total carbohydrate ratio in the urine has been noted in various forms of cancer ( R l ) . In one report, a substantial excretion of glycoproteins in the urine was observed in 31 patients with bronchial carcinoma (52). In another study, 27 of 56 patients with various forms of carcinoma excreted in the urine as much as eight times the amount of small molecular-weight peptides (M.W., 5000-8000) than a group of controls (R7).Normal human urine contained 30-120 mg per 24 hours

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of a group of four or more polypeptides of this molecular weight. They were rich in glycine, proline, and alanine and were devoid of hydroxyproline. Some of them were rich in hexose and hexosamine. Nine patients also excreted proteins of molecular weight 12,000-50,000. These various polypeptides were found especially in the late stages of the disease, usually during the last 6 months of life. The significance of these observations is still not clear. Proteinuria has been reported in a variety of carcinomas (N4), especially when amyloidosis complicates the clinical course (A10). Also, serum M components, which cannot be distinguished from those occurring

FIO.7. Cellulose acetate electrophoresis of serum and urine of a patient with disseminated cancer. No plasmacytic neoplasia was found. Serum = IgG/K M component; Urine = IgG/K M component and Bence Jones type kappa globulin.

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in multiple myeloma, have been recorded (M7, W3). For example, among 56 individuals with serum M components, revealed in the course of screening a general population, 27 had carcinomas. No urinary findings were reported (M7). Bence Jones protein has been observed in the urine in one case of bronchial carcinoma in which autopsy failed to reveal any evidence of myeloma (H7). In our experience, 3 of 7 patients with various forms of carcinoma and serum M components excreted Bence Jones globulin in the urine. One also excreted an IgG/K M component (Fig. 7). 12.

Urinary M Components without Malignant Disease

It should be stressed that abnormal proteins identical to those found in malignant plasmacytic-lymphocytic disorders, may also appear in the urine in other than malignant conditions. One patient with a gamma spike in the serum and a beta spike in the urine, but with a normal bone marrow and a completely normal radiological survey, came to autopsy for unrelated reasons, and no malignancy was found (W2). IgG/K M components have been found in the serum and urine of one patient with amyloidosis and also in a patient with diabetes ( 0 2 ) . It has been found that many patients with “primary” generalized amyloidosis excrete Bence Jones globulin in the urine and frequently also have M components in the serum (04). Bence Jones proteinuria has been reported in 4 of 18 patients with serum M components, but without evidence of multiple myeloma, macroglobulinemia, or other malignant disease (21). We have encountered 21 patients with serum M components unassociated with any demonstrable neoplastic disease. Bence Jones globulin was present in the urine in 5 (25%). Two additional patients with primary amyloidosis excreted Bence Jones protein in the urine along with an IgA/L M component. In one series of 42 patients with “benign” monoclonal gammopathy, a combination of electrophoresis and immunoelectrophoresis of concentrated urines revealed Bence Jones protein in 24% of the cases (R7).However, i t has been noted that in “benign” monoclonal gammopathy the amount of Bence Jones globulin usually does not exceed 60 mg/l. I n most instances, it is lower than in multiple myeloma or macroglobulinemia (D2). 13.

lysozymuria (Muramidasuria) in Mono- and Myelomonocytic leukemia

Lysozyme is a bacteriolytic enzyme which was first discovered by Fleming and Allison in 1922 (F6). It is present in normal serum as well as in other biological fluids and tissues (F6). Intracellularly, it is found in high concentration in the lysosomal fraction of monocytes and,

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FIG.8. Cellulose acetate electrophoresis of the concentrated urine from a patient with monocytic leukemia. L = lysozyme constituted 97% of urinary protein.

to a lesser extent, in polymorphonuclears (B19, C13). The enzyme is present in high concentration in normal renal cortex (S28) and is usually excreted in the urine in amounts not exceeding 2 pg/ml (P11). Lysoeyme (muramidase) is a basic protein (isoelectric point approximately 10.5) having sedimentation constant of s2,,,,., = 1.8 S and molecular weight of 14,000-15,000 (06). Tryptic digests of lysozyme show 18 to 19 ninhydrin-positive peptides (T7). The enzyme is readily crystallized (05). Cellulose acetate electrophoresis of urines with hi& concentrations of lysozyme show a band which migrates far beyond the application point, in the postgamma region (Figs. 8 and 9). The detection of lysozyme is based on this electrophoretic mobility and also on the observation that this enzyme lyses the walls of certain bacteria, a strain of grampositive cocci known as Micrococcus lysodeikticus being especially susceptible. The lysoplate method of Osserman and Lawlor employs heat killed M . Zysodeikticus homogeneously suspended in agar in a turbid mixture. The turbidity clears in areas surrounding wells containing lyso-

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zyme. Purified human lysozyme, isolated from the urine of patients with monocytic leukemia is usually used as a standard (06). Although increased levels of lysozyme in the serum of patients with certain forms of leukemias have been recognized for a long time (F5,J3), it is only recently that Osserman and Lawlor have demonstrated that this enzyme was excreted in large quantities in the urine, particularly in patients with monocytic and myelomonocytic leukemia (M15, 0 6 ) . Subsequently, screening and quantitative procedures were developed for the detection of lysozyme, and the enzyme has been purified in large quantities (06). Values as high as 200-1800 pg/ml were found in urines of 15 consecutive cases of monocytic and monomyelomonocytic leukemia, while the total daily excretion of lysozyme ranged from 0.4 to 2.6g (05, 0 6 ) . Normal values in the urine vary from 0.005 to 2 pg/ml with

FIG.9. Cellulose acetate electrophoresis of the concentrated urine from a patient with monocytic leukemia. Lysozyme is excreted along with albumin and various globulins. L = lysozyme.

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24-hour excretions up to 2.9 mg (P11). Frequently, other proteins have also been present, in one instance, a marked transferrinuria being observed (06). Comparable excretions of lysozyme in the urine (0.2 to 2.8 g/24 hours) have been observed by others in 8 of 11 patients with monocytic and myelomonocytic leukemia and also in 4 of 7 patients with granulocytic ’eukemia (Pl). No renal failure was evident in any of these cases. Wiernik and Serpick (W7) observed a very significant excretion of lysozyme in 10 patients with monocytic leukemia, ranging from 62 to 211 pg/ml, and in 5 of 9 patients with myelomonocytic leukemia, the urinary lysozyme reaching levels as high as 87 pg/ml. Lysozyme activity was increased in only 3 of 30 cases with myelocytic leukemia, but was never higher than 32 pg/ml. I n our group of 18 patients with monocytic and myelomonocytic leukemia, excretion was high prior to drug-induced remissions. The level reached 1480 pg/ml with daily excretion as high as 2.0 g per 24 hours. Lysozyme constituted up to 97% of the total protein excreted in the urine (P9). ACKNOWLEDGMENTS The authors wish to thank Mrs. E. M. Marsland, our Chief Librarian, and her

staff, and the MEDLARS Search Section of the National Library of Medicine, Bethesda, Maryland, for their assistance in search of the literature. Our thanks, also, to Miss M. Bliss from the Department of Medical Photography, The Wellesley Hospital, for the photographic reproductions. The technical assistance of Mrs. S. Saito and Miss S. Champion is gratefully acknowledged.

REFERENCES Al. Adams-Mayne, M. E., and Jirgnsons, B., Purification and characterization of four Bence Jones proteins. Arch. Biochem. Biophys. 113, 575-583 (1966). A2. Alami, S. Y., Reese, M. H., Bowman, R. O., and Race, G. J., Lymphosarcoma with two serum IgG M-components of different L chain types. Am. J . CZin. Pathol. 61, 185-193 (1969). A3. Alper, C. A., Cryoglobulinuria: Studies of a cryo-Bence Jones protein. A d a Med. Scand. Suppl. 446, 200-205 (1966). A4. Appella, E., and Ein, D., Two type8 of lambda polypept,ide chains in human immunoglobulins based on an amino acid substitution a t position 190. Proc. Natl. A d . Sn’. U.S. 679 1449-1454 (1967). A5. Argani, I., and Kipkie, G. F., Macroglobulinemic nephropathy. Acute renal failure in macroglobulinemia of Waldenstrom. Am. J . Med. 36, 151-157 (1964). A6. Askonas, B. A., and Williamson, A. R., Biosynthesis of immunoglobulins. Free light chain as an intermediate in the assembly of YG-molecules. Nature 211, 369372 (1966). A7. Askonas, B. A., and Williamson, A. R., Balanced heavy and light chain synthesis in immune tissue and disulphide bond formation in IgG assembly. I n “Gamma Globulins,” Nobel Symp. 3 (J. Killander, ed.), pp. 369-383. Wiley (Interscience), New York and Almqvist & Wiksell, Stockholm, 1967. A8. Awdeh, 2. L., Askonas, B. A., and Williamson, A. R., The homogeneous-gamma-

ABNORMAL PROTEINURIA IN MALIGNANT DISEASES

A9. A10. B1.

B2. B3. B4. B5. B6.

B7. B8. B9. B10. B11. B12. B13. B14. B15. B16.

B17.

371

G-immunoglobulin produced by mouse plasmacytoma 5563 and its subsequent heterogeneity in serum. Biochem. J. lOa, 548-553 (1967). Axelsson, U., The plasma cells of the bone marrow in myelomatosis treated with Alkeran (Melphlan). Scund. J . Haemutol. 3, 123-135 (1966). Azzopardi, J. G., and Lehner, T., Systemic amyloidosis and malignant disease. J . Clin. Pathol. 19, 539-548 (1966). Baars, H., Imhof, J. W., and de Wael, J., Serum and urinary proteins in macroglobulinemia of Waldenstrom. An investigation of 7 cases treated by the authors and a review of 107 cases from the literature. Protides Bwl. Fluids Proc. Colloq. 6, 131-135 (1959). Baglioni, G., and Cioli, D., A study of immunoglobulin structure. 11. The comparison of Bence Jones proteins by peptide mapping. J. Exptl. Med. 184,307-330 (1966). Baglioni, C., Zonta, L. A., Cioli, D., and Carbonara, A., Allelic antigenic factor Inv (a) of the light chains of human immunoglobulins: Chemical basis. Science 162, 1517-1519 (1966). Baglioni, C., Cioli, D., Gorini, G., Ruffilli, A., and Alescio-Zonta, L., Studies on fragments of light chains of human immunoglobulins: Genetic and biochemical implications. Cold Spring Harbor Symp. Quant. Biol. 32, 147-159 (1967). Bence Jones, H., On a new substance occurring in the urine of a patient with “mollities ossium.” Phil. Trans. Roy. SOC.London 138, 55-62 (1848). Bennich, H., and Johansson, S. G. O., Studies on a new class of human immunoglobulins. 11. Chemical and physical properties. In “Gamma Globulins,” Nobel Symp. 3 (J. Killander, ed.), pp. 199-205. Wiley (Interscience), New York and Almqvist & Wiksell, Stockholm, 1967. Berger, R., Ainbender, E., Hodes, H. L., Zepp, H. D., and Hevizy, M. M., Demonstration of IgA polioantibody in saliva, duodenal fluid and urine. Nature 214, 420422 (1967). Berggard, I., Studies on the plasma proteins in normal human urine. Clin. Chirn. Acta 6, 413429 (1961). Berggard, I.,and Bennich, H., Fc fragment of immunoglobulin Gin normal human plasma and urine. Nature 214, 697-699 (1967). Berggard, I., and Edelman, G. M. Normal counterparts to Bence Jones proteins: Natl. A d . Sci. U.S. 4% Free L polypeptide chains of human y-globulin. PTOC. 330-337 (1963). Berggard, I., and Peterson, P., Immunoglobulin components in normal human urine. In “Gamma Globulins,” Nobel Symp. 3 (J. Killander, ed.), pp. 71-80. Wiley (Interscience), New York and Almqvist & Wiksell, Stockholm, 1967. Berggard, I., and Peterson, P. A,, Polymeric forms of free normal K and h chains of human immunoglobulin. J. Bwl. Chem. 244, 42994307 (1969). Berggard, I., Clem, H., and Bearn, A. G., The excretion of five plasma proteins previously unidentified in normal human urine. Clin. Chim. Acta 10, 1-11 (1964). Bergsagel, D. E., and Pruzanski, W., Recognizing and treating plasma Cell neoplasia. Postgrad. Med. 45, 200-209 (1968). Bernier, G. M., and Putnam, F. W., Polymerism, polymorphism and impurities in Bence Jones proteins. Biochim. Biophys. Acta 86, 295-308 (1964). Berrod, J., Larrat, J., Sandor, G., and Sureau, B., A case of micromolecular myelomatosis and demonstration of immunological identity between the micromolecule of the serum and the urinary Bence Jones protein of the same patient. Nature 2Oa, 407408 (1964). Bienenstock, J., Urinary Fc and F’cfragments. J . Immunol. lOO,280-285 (1968).

372

W. PRUZANSKI A N D M. A. OGRYZLO

B18. Bienenstock, J., and Tomasi, T. B., Jr., Secretory ?A in urine. Clin. Res. 15, 292 (1967). B19. Briggs, R. S., Perillie, P. E., and Finch, S. C., Lysoryme in bone marrow and peripheral blast cells. J . H i s t o c h . Cytochem. 14, 167-170 (1966). €320. Bureau, Y., Senelar, R., Barriere, H., Bureau, B., Litoux, P., and Bray, B., Macroglobulinemie au cours d’une maladie de Kahler. Presse Med. 76, 961-964 (1968). C1. Caggiano, V., Cuttner, J., and Solomon, A., Myeloma proteins, Bence Jones proteins and normal immunoglobulins in multiple myeloma. Blood SO, 265-287 (1967). c2. Caggiano, V., Dominguer, G., Opfell, R. W., Kochwa, S., and Wasserman, L. R., IgG myeloma with closed tetrameric Bence Jones proteinemia. Am. J . Med. 47, 978-985 (1968). c3. Carbone, P. P., Kellerhouse, L. E., and Gehan, E. A., Plasmacytic myeloma. A study of the relationship of survival to various clinical manifestations and anomalous protein type in 112 patients. Am. J . Med. 42, 937-948 (1967). c4. Carson, C. P., Ackerman, L. V., and Maltby, J. D., Plasma cell myeloma. A clinical, pathologic and roentgenologic review of 90 cases. Am. J. Clin. Pathl. 26, 849-888 (1955). c5. Ceppellini, R., Dray, S., Edelman, G., Fahey, J., FranBk, F., Franklin, E., Goodman, H. C., Grabar, P., Gurvich, A. E., Heremans, J. F., Karush, F., Press, E., and Trnka, Z., Nomenclature for human immunoglobulins. Bull. World Health Organ. SO, 447450 (1964). C6. Ceppellini, R., Dray, S., Fahey, J. L., Franklin, E. C., Fudenberg, H., Gell, P. G. H., Goodman, H. C., Grubb, R., Harboe, M., Kirk, R. L., Oudin, J., Ropartr, C., Smithies, O., Steinberg, A. G., and Trkna, Z., Notation for genetic factors of human immunoglobulins. Nature 209, 653-655 (1966). C7. Cioli, D., and Baglioni, C., Origin of structural variation in Bence Jones proteins. J . MoZ. Biol. 16, 385-388 (1966). C8. Cohen, R. J., Bohannon, R. A., and Wttllentein, R. O., Waldenstrom’s macroglobulinemia. A study of ten cases. Am. J . Med. 41, 274-284 (1966). C9. Cohen, S., Properties of the peptide chains of normal and pathological human 7-globulins. Biochem. J . 89, 334-341 (1963). C10. Cohen, S., The nature of myeloma proteins. Brit. J . Haenzatol. 16, 211-215 (1968). C11. Cohen, S., and Milstein, C., Structure and biological properties of immuncglobulins. Advan. Immunol. 7 , 1-89 (1967). C12. Cohn, M., The molecular biology of expectation. In “Nucleic Acids in Immunology” (0.J. Plescia and W. Braun, ed.), pp. 671-715. Springer Verlag, New York, 1968. C13. Cohn, Z. A., and Hinch, J. G., The isolation and properties of the specific cytoplasmic granules of rabbit polymorphonuclear leukocytes. J . Ezptl. Med. 112, 983-1004 (1960). C14. Collier, F. C., and Jackson, P., The precipitin test for Bence Jones protein. Further studies of clinical evaluation of the test employing antibody prepared by the adjuvant technic. New Engl. J . Med. 248, 409414 (1953). C15. Connell, G. E., personal communication (1969). (316. Connell, G. E., and Painter, R. H., Fragmentation of immunoglobulin during storage. Can. J. Bwchem. 44,371-379 (1966). C17. Creyssel, R., Site, J., and Richard, G. B., ktude comparative des mobiliffi 6lectrophor6tiques des prot6ines mydomateuses s6riques et urinaires. Rev. Franc. Etudes CZin. Biol. 11, 290-293 (1966).

ABNORMAL PROTEINURIA IN MALLGNANT DISEASES

373

D1. Dameshek, W., and Gunz, F., “Leukemia.” Grune & Stratton, New York, 1958. D2. Dammacco, F., and Waldenstrom, J., Bence Jones proteinuria in benign monoclonal gammapathies. Acta Med. Scahd. 184, 403409 (1968). D3. D a m c c o , F., and Waldenstrom, J., Serum and urine light chain levels in benign monoclonal gammapathies, multiple myeloma and Waldenstrom’s macroglobulinemia. Clin. Exptl. Immunol. 3, 911-921 (1968). D4. Dammacco, F., Trizio, D., and Bonomo, L., A case of IgA K-myelomatosis with two urinary Bence Jones proteins (BJK and BJL) and multiple chromosomal abnormalities. Ada Haematol. 41, 309-320 (1969). D5. Deutsch, H. F., Crystalline low molecular weight 7-globulin from a human urine. Science 141, 435-436 (1963). D6. Deutsch, H. F., Relationships of two light chain immunoglobulins isolated from the same human source (low molecular weight immunoglobulins). Imumchemistry 2, 207-218 (1965). D7. Deutsch, H. F., Subunit structure of gamma M-globulins. In “Gamma Globulins,” Nobel Symp. 3 (J.Killander, ed.), pp. 153-167. Wiley (Interscience),New Yorkand Almqvist I% Wiksell, Stockholm, 1967. D8. Dittmar, K., Kochwa, S., Zucker-Franklin, D., and Wasserman, L. R., Coexistence of polycythemia Vera and bialonal gammopathy (7GK and 7AL) with two Bence Jones proteins (BJK and BJL). Blood 31, 81-92 (1968). El. Edelman, G. M., and Gally, J. A., The nature of Bence Jones proteins. Chemical similarities to polypeptide chains of myeloma globulins and normal 7-globulins. J. Ezptl. Med. 116, 207-227 (1962). E2. Ein, D., and Fahey, J. I..,Two types of lambda polypeptide chains in human immunoglobulins. Science 166, 947-948 (1967). E3. Ellman, L. L., and Bloch, K. J., Heavy-chain disease. Report of a seventh case. New Engl. J. Med. 278, 1195-1201 (1968). E4. Engle, R. L., Jr., and Nachman, R. L., Two Bence Jones proteins of different immunologic types in the same patient with multiple myeloma. Blood 27, 7 4 7 7 (1966). E5. Engle, R. L., Jr., and Wallis, L. A., Paraimmunoglobulinopathies. In “TieeHarvey Practice of Medicine” (J. C. Harvey, ed.) Vol. 1, pp. 301-364. Prior, Hagerstown, Maryland, 1965. E6. Engle, R. L., Jr., Woods, K. R., Castillo, G. B., and Pert, J. H., Starch gel electrophoresis of serum proteins and urinary proteins from patients with multiple myeloma, macroglobulinemia and ot,her forms of dysproteinemia. J . Lab. Clin. Med. 68, 1-22 (1961). F1. Fagelman, D., McGhee, B., and Chaplin, H., Jr., The stability of yG-globulin and yG related fragments in normal human urine. J. Lab. CZin. Med. 68, 445454 (1966). F2. Fahey, J. L., Heterogeneity of 7-globulins. Aduan. Immunol. 2 , 41-109 (1962). F3. Fahey, J. L., Heterogeneity of myeloma proteins. J. Clin. Invest. 42, 111-123 (1963). F4. Fahey, J. L., Two types of 6.6 S 7-globulins, j3zA-globulins and 18 S 71-macroglobulins in normal serum and 7-microglobulins in normal urine. J . Immunol. 91, 438-447 (1963). F5. Finch, S. C., Gnabasic, F. J., and Rogoway, W., Lysozyme and leukopoiesis. Proc. 3rd Symp. Intern. Lisozima Fleming. Scuola Arti Grafiche, 0. 8. F., Cesano Boscone, Milan, 1964. F6. Fleming, A., and Allison, V. D., Observations on a bacteriolytic substance (“lysozyme”) found in secretions and tissues. Brit. J. Exptl. PathoZ. 13, 252-260 (1922).

374

W. PRUZANSKI AND M. A. OGRYZLO

IT.Forte, F. A., PreUi, F., Yount, W., Kochwa, S., Franklin, E. C., and Kunkel, H.,

Heavy chain disease of the p type: Report of the first case. Blood 84, 831 (1969). F8. Frangione, R., and Milstein, C., Partial deletion in the heavy chain disease protein ZUC. Nature 224, 597-599 (1969). Fg. Franklin, E. C., personal communication (1969). F ~ oFranklin, . E. C , Fudenberg, H., Meltzer, M., and Stanworth, D. R., The structural basis for genetic variations of normal human 7-globulins. Proc. Natl. A d . Sci. U.S. 48, 914-922 (1962). ~ 1 1Franklin, , E. C., Lowenstein, J., Bigelow, B., and Meltzer, M., Heavy chain disease-A new disorder of serum y globulins. Am. J . Med. 37, 332-350 (1964). F12. Freedman, M. H., and Connell, G. E., The heterogeneity of gamma-globulin in post-exercise urine. Can. J. Bioehem. 42, 1065-1097 (1964). M. H., and Connell, G. E., Monomer and dimer forms of gamma~ 1 3 Freedman, , globulin polypeptides in normal urine. Can. J . Biochem. 42, 1815-1823 (1964). GI, Gally, J . A., and Edelman, G. M., Protein-protein interactions among L polypeptide chains of Bence Jones proteins and human gamma-globulins. J. Ezptl. Med. llB, 817-836 (1964). G2. Grabar, P., and Williams, C. A., MBthode permettant l’dtude conjuguee des proprid& dlectrophordtiques et immunochimiques d’un melange de protdines. Application au s&um sanguin. Biochirn. Biophys. Acta 10, 193-194 (1953). G3. Grabar, P., and Williams, C. A., Jr., Mdthode immuno4ectrophoretique d’analyse de melanges de substances antigkniques. Biochim. Biophys. Acta 17, 67-74 (1955). (34. Grey, H. M., and Kohler, P. F., A case of tetramer Bence Jones proteinemia. Clin. Exptl. Immunol. 3, 277-285 (1968). (35. Grey, H. M., and Kunkel, H. G., H-chain subgroups of myeloma proteins and normal gamma globulin. J . Exptl. Med. 120, 253-266 (1964). G6. Gross, D., and Epstein, W. V., Macroglobulinemia with Bence Jones proteinuria: Comparison of urinary protein and L chain of serum proteins. J. Clin. Invest. 43, 83-93 (1964). G7. Gutman, A. B., The plasma proteins in disease. Advan. Protein Chem. 4, 155-250 (1948). G8. Gutman, A. B., Moore, D. H., Gutman, E. B., McClellan, V., and Kabat, E. A., Fractionation of serum proteins in hyperproteinemia with special reference to multiple myeloma. J. Clin. Invest. 20, 765-783 (1941). HI. Harrison, J. F., Blainey, 6. D., Hardwicke, J., Rowe, D. S., and Soothill, J. F. Proteinuria in multiple myeloma. Clin. Sci. 31, 95-110 (1966). H2. Hiller, A., Greig, R. L., and Beckman, W. W., Determination of protein in urine by Biuret method. J . Bwl. Chem. 176, 1421-1429 (1948). H3. Hilschmann, N., The structure of the immunoglobulins (L-chains) and the antibody problem. In “Gamma Globulins,” Nobel Symp. 3 (J. Killander, ed.), pp. 33-44. Wiley (Interscience), New York and Almqvist & Wiksell, Stockholm, 1967. H4. Hobbs, J. R., Monoclonal immunoglobulins from random mutations. Brit. J. Cancer 22, 717-719 (1968). H5. Hobbs, J. R., Immunochemical classes of myelomatosis including data from a therapeutic trial conducted by a Medical Research Council working party. Brit. J. Haematol. 16, 599406 (1969). H6. Hobbs, J. R., and Jacobs, A., A half-molecule GK plasmacytoma. Clin. Exptl. Immunol. 6, 199-207 (1969). H7. Hosley, H. F., M-proteins, plasmacytosis and cancer. Cancer 20, 295-307 (1967). J1. Jaeger, M., and Lapp, R., Une leucemie lymphoide chronique avec syndrome de

ABNORMAL PROTEINURIA IN MALIGNANT DISEASES

375

Waldenstrom ii double paraprotkine et syndrome de carence en anticorps. Schweiz. Med. Wochschr. 97, 1461-1462 (1967). J2. Johansson, S. G. O., and Bennich, H., Immunological studies of an atypical (myeloma) immunoglobulin. Immunology 13, 381-394 (1967). 53. Jolles, P., Sternberg, M., and Mathe, G., The relationship between semmlysozyme levels and the blood leukocytes. Israel J . Med. Sci. 1, 445-447 (1965). 54. Jnrrgensen, M. B., A gel filtration method for the determination of protein in normal urine with some observations on normal renal protein excretion. Acta Med. Scund. 181, 153-162 (19671. K1. Kabat, E. A., and Mayer, M. M., “Experimental Immunochemistry,” 2nd Ed. Thomas, Springfield, Illinois, 1964. m.Kahler, O., Zur Symptomatologie des multipen Myelomas; Beobachtung von Albumosuria. Prager. Med. Wochschr. 14, 3 3 4 5 (1889). K3. Kiely, J. M., Wagoner, R. D., and Holley, K. E., Renal complications of lymphoma. Ann. Internal. Med. 71, 1159-1175 (1969). K4. Kingsbury, F. B., Clark, C. P., Williams, G., and Post, A. L., Rapid determination of albumin in urine. J . Lab. Clin. Med. 11, 981-989 (1926). K5. Kohn, J., Small-scale membrane filter electrophoresis and immuno-electrophoresis. Clin. Chim. Acta 3, 450454 (1958). K6. Korngold, L., and Lipari, R., Multiple myeloma proteins: I. Immunological studies. Cancer 9, 183-192 (1956). K7. Korngold, L., and Lipari, R., Multiple myeloma proteins. III. The antigenic relationship of Bence Jones proteins to normal gamma-globulin and multiple myeloma serum proteins. Cancer 9, 262-272 (1956). K8. Krauss, S., and Sokal, J. E., Paraproteinemia in the lymphomas. Am. J . Med. 40, 400-413 (1966). K9. Kritsman, J., Fudenberg, H. H., and Liss, M., A Bence Jones cryoglobulin: Chemical, physical and immunologic properties. Clin. Exptl. Immunol. 2, 467-475 (1967). B10. Kunkel, H. G., and Tiselius, A., Electrophoresis of proteins on filter paper. J . Gen. Phy~iol.36, 89-118 (1951). K l l . Kunkel, H. G., Killander, J., and Mannik, M., Current trends in immune globulin research. In “Gamma Globulins,” Nobel Symp. 3 (J. Killander, ed.), pp. 63-73. Wiley (Interscience), New York and Almqvist & Wiksell, Stockholm, 1967. K12. Kunkel, H. G., Fahey, J. L., Franklin, E. G., Osserman, E. F., and Terry, W. D., Notation for human immunoglobulin subclasses. Bull. World Health Organ. 36, 953 (1966). L1. Lebreton, J.-P., Rivat, C., Rivat, L., Guillemot, L., and Roparts, C., Une immunoglobulinopathie m6connue: La maladie des chafnes lourdes. Presse Med. 76, 22512254 (1967). L2. Lewis, A. F., Bergsagel, D. E., Bruce-Robertaon, A., Schachter, R. K., and Connell, G. E., An atypical immunoglobulin. Blood 32, 189-204 (1968). L3. Lieberman, R., Mushinski, J. F., and Potter, M., %Chain immunoglobulin A molecules: Abnormal or normal intermediates in synthesis. Science 169, 1355-1357 (1968). L4. Lindstrom, F. D., Williams, R. C., Jr., and Theologides, A., Urinary light chain excretion in leukemia and lymphoma. Clin. Ezptl. Immunol. 6, 83-90 (1969). L5. Lindstrom, F. D., Williams, R. C., Jr., Swaim, W. R., and Freier, E. F., Urinary lightchain excretion in myeloma and other disorders-an evaluation of the Bence Jones test. J . Lab. Clin. Med. 71, 812-825 (1968).

376

W. PRUEANSKI AND M. A. OQRYZLO

L6. Litwin, S. D., and Kunkel, H. G., l'he relationship between the Inv (1) and (2) genetic antigens of kappa human light chains. J. Immunol. 99, 603-609 (1967). MI. Magnus-Levy, A., Multiple myelome (XII). Acta Med. Scand. 96,218-280 (1938). M2. Maldonado, J. E., Silverstein, M. N., William, R. C., Jr., and Harrison, E. G., Jr., Lymphosarcoma wiU Bence Jones proteinuria (type K) and rG serum M-component (type L) : An asynchronous gammopathy. Abstracts of the simultaneous sessions. Proc. 19th Congr. Intern. SOC.Hematol., New York p. 43 (1968). M3. Mancini, G., Carbonara, A. O., and Heremans, J. F., Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochiatry 2, 235-254 (1965). M4. Mannik, M., and Kunkel, E. G., Classification of myeloma proteins, Bence Jones proteins and macroglobulins into two groups on the basis of common antigenic characters. J. Ezptl. Med. 116, 859-877 (1962). M5. Martin, N. H., Macroglobulinemia. A clinical and pathological study. Quart. J. Med. 29, 179-196 (1960). Me. Melchers, F., Lennox, E. S., and Facon, M., A carbohydrate containing mouse light chain-protein. Bwchem. Biophys. Res. Commun. 24, 244-251 (1966). M7. Migliore, P. J., and Alexanian, R., Monoclonal gammopathy in human neoplasia. Cancer 21, 1127-1131 (1968). M8. Milstein, C., Interchain disulphide bridge in Bence Jones proteins and in gamma globulin B chains. Nature 206, 1171-1173 (1965). Mg. Milstein, C., Linked groups of residues in immunoglobulin K chains. Nature 216, 330-332 (1967). M10. Miyasato, F., and Pollak, V. E., Serum proteins in urine: An examinat,ion of the effects of some methods used to concentrate the urine. J. Lab. Clin. Med. 67, 1036-1043 (1966). M11. Moore, D. R., &bat, E. A., and Gutman, A. B., Bence Jones proteinemia in multiple myeloma. J. Clin. Invest. 22, 67-75 (1943). M12. Morner, I(.A. H., Untersuchungen iiber die Proteinstoffe und die Eiweissfallenden substawen des Normalen Menschenharns. Skand. Arch. Physwl. 6, 33-37 (1895). M13. Morstad, K. S., On the demonstration of Bence Jones proteinuria. Acta Med. Scand. 182, 457463 (1967). M14. Moss, W. T., and Ackerman, L. V., Plaama cell leukemia. Blood 1,396-406 (1946). M15. Muggia, F. M., Heinemann, H. O., Farhangi, M., and Osserman, E. F., Lysosymuria and renal tubular dysfunction in monocytic and myelomonocytic leukemia. Am. J. Med. 47, 351366 (1969). N1. Nachman, R. L., Engle, R. L., Jr., and Stein, S., Subtypes of Bence-Jones proteins. J. Zmmunol. 96, 295-299 (1965). N2. Nachman, R. L., Engle, R. L., Jr., and Copeland, L., Correlation of immunologic and structural heterogeneity of Bence Jones proteins. J . Zmmunol. 97, 356-362 (1966). N3. Naumann, H. N., Differentiation of Bence Jones protein from uroglobulins. A new test based on differential extraction of uroproteins dried on filter paper. Am. J. sin. Pathol. 44,413-425 (1965). N4. Nettleship, A., Strother, J. A., and Smith, H. S., Relationship of serum and urinary proteins in neoplastic states: A preliminary survey. Clin. C h . 9, 608414 (1963). 01. Ogawa, M., Kochwa, S., Smith, C., Ishizaka, K., and MoIntyre, 0. R., Clinical aspects of IgE myeloma. New Engl. J . Med. 281, 1217-1220 (1969).

ABNORMAL PROTDINURIA IN MALIGNANT DISEASES

377

02. Ooi, B. S., Pesce, A. J., Gaizutis, M., and Pollak, V. E., Monoclonal gammopathy and protein transport in the kidney. Clin. Res. 17, 442 (1969). 03. Ortolani, C., Cattaneo, R., and Massarotti, G., Eliminaeione urinaria di frammenti di catene pesanti immunglobuliniche oltre alla proteins. di Bence Jones in tre casi di mieloma. Boll. Ist.Skoterap. Milan. 46, 596-601 (1967). 04. Osserman, E. F., Plasma cell dyscrasia. In “Cecil-Loeb Textbook of Medicine” (P. B. Beeson and W. McDermott, eds.), 12th Ed., pp. 1101-1116. Saunders, Philadelphia, Pennsylvania, 1967. 05. Osserman, E. F., Crystallization of human lysozyme. Science 166, 1536-1537 (1967). 06. Osserman, E. F., and Lawlor, D. P., Serum and urinary lysozyme (muramidase) in monocytic and monomyelocytic leukemia. J. Exptl. Med. 124, 921-952 (1966). 07. Osserman, E. F., and Takatsuki, K., Plasma celI myeloma: Gemma globulin synthesis and structure. A review of biochemical and clinical data, with the description of a newly recognized and related syndrome, “ H y k h a i n (Franklin’s) disease.” Medicine 43, 357-384 (1963). 08. Osserman, E. F., and Takatsuki, K., Clinical and immunochemical studiea of four cases of heavy (HT*)chain disease. Am. J. Med. 37, 351-373 (1964). 09. Ouchterlony, O., In vitro method for testing toxin-producing capacity of diphtheria bacteria. Acta Pathol. Microbwl. Scund. 26, 186-191 (1948). 010. Ouchterlony, O., Antigen-antibody reactions in gels. A d a Pathol. Microbiol. Scand. 26, 507-515 (1949). 011. Oudin, J., L’analyse immunochimique du serum de cheval par prkipitation specifique en milieu g6lifib. Premiers r6sultats. Bull. 80c. Clin. Bwl. 2& 140-149 (1947). 012. Owen, D. M., Multiple myeloma. A review based on 98 cases. Geriatrics 20, 10481064 (1965). PI. Perillie, P. E., Kaplan, S. S., Lefkowitz, E., Rogaway, W., and Finch, S. C., Studies of muramidase (lysozyme) in leukemia. J. Am. Med. Assoc. 203, 317322 (1968). P2. Peterson, E. A., and Sober, H. A., Chromatography of proteins. I. Cellulose Ionexchange adsorbenb. J . Am. Chem. SOC.78, 751-755 (1956). P3. Piscator, M., Proteinuria in chronic cadmium poisoning. 111. Electrophoretic and immunoelectrophoreticstudies on urinary proteins from cadmium workers, with special reference to the excretion of low molecular weight proteins. Arch. Environ. Health 12, 335-344 (1966). P4. Piscator, M., Proteinuria in chronic cadmium poisoning. IV. Gel filtration and ion exchange chromatography of urinary proteins from cadmium workers. Arch. Environ. Health 12, 345-359 (1966). P5. Poortmans, J., and Jeanloz, R. W., Quantitative immunological determination of 12 plasma proteins excreted in human urine collected before and after exercise. J . Clin. Invest. 47, 386-393 (1968). P6. Potter, M., and Kuff, E. L., Disorders in the differentiation of protein secretion in neoplastic plasma cells. J . MoZ. Biol. 9, 537-544 (1964). W .Poulik, M. D., Berman, L., and Prasad, A. S., “Myeloma protein” in a patient with monocytic leukemia. Blood 33, 746-758 (1969). P8. Pwhl, J. W., N- and C-terminal sequences of a heavy chain disease protein and its genetic implications. Nature 216, 1386-1387 (1967). P9. Pruzanski, W., Proteins, muramidase and renal tubular dysfunction in mono- and myelomonocytic leukemia. Ann. Roy. Coll. Phye. Surq. Can. 3, 28 (1970).

378

W. PRUZANSKI AND M. A. OGRYZLO

P10. Pruzanslri, W., and Rother, I., IgD plasma cell neoplasia-clinical manifestations and characteristic features. Can. Med. Assoc. J . 102, 1061-1065 (1970). P11. Pruzanski, W., and Saito, S. G., The diagnostic value of lysozyme (muramidase) estimation in biological fluids. Am. J . Med. Sci. 268, 405-415 (1969). P12. Pruzanski, W., Platts, M. E., and Ogryzlo, M. A., Leukemic form of imrnunocytic dyscrasia (plasma cell leukemia). A study of ten cases and a review of the literature. Am. J . Med. 47, 60-74 (1969). p13. Putnam, F., Aberrations of protein metabolism in multiple myeloma. Interrelationships of abnormal serum globulins and Bence-Jones proteins. Physwl. Rev. 37, 512-538 (1957). P14. Putnam, F. W., Structural relationships among normal ?globulin, myeIoma globulins, and Bence Jones proteins. Biochim. Biophys. Acta 63, 539-541 (1962). F. W., Structural evolution of kappa and lambda light chains. In ~ 1 5 Putnam, . “Gamma Globulins,” Nobel Symp. 3 (J. Killander, ed.), pp. 45-70. Wiley (Interscience), New York and Almqvist & Wiksell, Stockholm, 1967. P16. Putnam, F. W., Immunoglobulin structure: Variability and homology. Amino acid sequences of immunoglobulins reflect evolutionary change and may explain antibody variability. Science 165,633-644 (1969). P17. Putnam, F. W., Titani, K., and Whitley, E., Jr., Chemical structure of light chains: Amino acid sequence of type I( Bence Jones proteins. Proc. Roy. SOC. (London) B166, 12.1-137 (1966). P18. Putnam, F. W., Easley, C. W., Lynn, L. T., Ritchie, A. E., and Phelps, R. A., The heat precipitation of Bence Jones proteins. I. Optimum condition. Arch. Bwchem. B i ~ p h y s83, . 116-130 (1959). Q1. Quattrocchi, R., Cioli, D., and Baglioni, G., Distribution of an arginine-lysine interchange in the invariable half of human Gtype Bence Jones proteins. Nature 216, 56-57 (1967). Q2. Quattrocchi, R., Cioli, D., and Baglioni, G., A study of immunoglobulin st-ructure. 111. An estimate of the variability of human light chains. J. Exptl. Med. 130, 401415 (1969). R1. Raventos, E. B., Leyton, G. R., Simon, S. H., Contreras, E. G., and Barriga, B. R., Chemistry and immunology of urinary proteoses in cancer. Acta Unw Intern. Contra Cancrum 20, 1141-1145 (1964). R2. Rigas, D. A., and Heller, C. G., The amount and natAureof urinary proteins in normal human subjects. J. Clin. Invest. 30, 853-861 (1951). R3. Ritzmann, S. E., Thurm, R. H., Truax, W. E., and Levin, W. C., The syndrome of macroglobulinemia. Review of the literature and a report of two case9 of macrocryogelglobulinemia. Arch. Internal Med. 106,939-965 (1960). R4. Riva, G., The macroglobulinemiaof Waldenstrom. Helv. Med. Acta 26, 1-2 (1958). R5. Rosen, B. J., Smith, T. W., and Bloch, I(.J., Multiple myeloma associated with two serum M-componenta, yG type K and r A type L. New Engl. J. Med. 277, 902-907 (1967). R6. Rubinstein, M. A., Multiple myeloma as a fofm of leukemia. Blood 4, 1049-1067 (1949). R7. Rudman, D., Del Rio, A., Akgun, S., and Frumin, E., Novel proteins and peptides in the urine of patients with advanced neoplastic disease. Am. J .Med. 46, 174-187 (1969). R8. Ruffilli, A., and Baglioni, G., Subgroups of L type Bence Jones proteins. J . Immunol. 98, 874-880 (1967). R9. Rundles, R. W., Coonrad, E. V., and Arends, T., Serum proteins in leukemia. Am. J . Med. 16, 842-853 (1954).

ABNORMAL PROTEINURIA IN MALIGNANT DISEASE5

379

S1. Saifer, A., and Gerstenfeld, S., Photometric determination of urinary proteins. Clin. Chem. 10,321-334 (1964). 52. Saint-Cyr, C. V., Cleve, H., and Hermann, G.,Etude de quelques protdinuries de malades atteints de cancer bronchique. Rev. Franc Etudes Clin. Biol. 8, 4-89 (1963). S3. Sanders, J. H., Fahey, J. L., Finegold, I., Ein, D., Reisfeld, R., and Berard, C., Multiple anomalous immunoglobulins. Clinical, structural and cellular studies in three patients. A m . J. Med. 47, 43-59 (1969). 54. Sargent, A. U.,Rose, B., Saha, A., and Kendall, A. G., A case of multiple myeloma associated with urinary excretion of a unique heavy chain distinct from that described in heavy chain disease. An abstract. Am. SOC.Hemutol., 9th Ann. Meeting, New Orleans p. 30 (1966). S5. Rchenrlen, P. G., Multiple myeloma with several anomalous proteins. Ger. Med. Monthly 9, 138-141 (1964). S6. Schultze, H. E.,and Heremans, J. F., “Molecular Biology of Human Proteins with Special Reference to Plasma Proteins,” Vol. 1, p. 673.Elsevier, Amsterdam, 1966. and Edelman, G. M., Comparisons of Bence Jones proteins and L 57. Schwarts, J. H., polypeptide chains of myeloma globulins after hydrolysis with trypsin. J . Ezptl. Med. 118,41-53 (1963) S8. Seki, T., Appella, E., and Itano, H. A., Chain models of 6.6s and 3.95 mouse myeloma yA immunoglobulinmolecules. Proc. Natl. Acud. Sci. U.S.61,1071-1078 (1968). S9. Seligmann, M.,and Basch, A., The clinical significance of pathological immunoglobulins. Proc. i%th Congr. Intern. SOC.Hematol., Plenary Session Papers, New York p. 21 (1968). S10. Seligmann, M.,Danon, F., Hurez, D., Mihaesco, E., and Preud’homme, J.-L., Alpha-chain disease: A new immunoglobulin abnormality. Science 162, 1396-1397 (1968). Sll. Seligmann, M.,Mihaesco, E., Hurez, D., Mihaesco, C., Preud’homme, J.-L., and Rambaud, J.-C., Immunochemical studies in four cases of alpha chain disease. J. Clin. Invest. 48, 2374-2389 (1969). 512. Seppala, P.,Nikkari, T., and Nanto, V., Physical chemical properties of a urinary protein in a case of multiple myeloma. Suznd. J . Clin. Lab. Inuest. 18, 666-667 (1961). 513. Smithies, O.,Zone electrophoresis in starch gels: Group variations in the serum proteins of normal human adults. Biochem. J. 61,629-641 (1955). 514. Snapper, I., and Kahn, A. I., Multiple myeloma. Seminars Hematol. 1, 87-143 (1964). S15. Snapper, I., Turner, L. B., and Moscovitz, H. L., “Multiple Myeloma.” Grune & Stratton, New York, 1953. S16. Snyder, H.,and Schults, J., A polypeptide found in urine containing Bence Jones protein has identical primary structure with 32 residue segment of A-chain. Federation Proc. 28,496 (1969). S17. Sober, H.A., Gutter, F. J., Wyckoff, M. M., and Peterson, E. A., Chromatography of proteins. 11. Fractionation of serum protein on anion-exchange cellulose. J. Am. Chem. SOC.78, 756-763 (1956). 918. Solomon, A., and Fahey, J. L., Bence Jones protehemia. Am. J. Med. 37,206-222 (1964). S19. Solomon, A., and Kunkcl, H. G., A “monoclonal” type, low molecular weight protein related to VM-macroglobulins. Am. J . Med. 4!2, 95-67 (1967).

380

W. PRUZANSKI AND M. A. OGRYZLO

520. Solomon, A., and McLaughlin, C. L., Production of Bence Jones protein subunits. Proc. 19th Congr. Intern. Sac. Hematol., Abstr. Simultaneous SessWns, New York p. 140 (1968). S21. Solomon, A., and McLaughlin, C. L., Bence Jones proteins and light chains of immunoglobulins. I. Formation and characterization of amino-terminal (variant) and carboxyl-terminal (constant) halves. J . Bwl. Chem. 244, 3393-3404 (1969). 522. Solomon, A., and McLaughlin, C. L., Bence Jones proteins and light chains of immunoglobulins. 11. Immunochemical differentiation and classification of kappa-chains. J . Ezptl. Med. 130, 1295-1311 (1969). 623. Solomon, A., Killander, J., Grey, H. M., and Kunkel, H. G., Low-molecular-weight proteins related to Bence Jones proteins in multiple myeloma. Science 161, 12371239 (1966). 8%. Spengler, G. A., Butler, R., Pflugshaupt, It., Lopez, V., and Barandun, S., 7Dparaproteiniimie. Kasuistischer Beitrag an Hand von Zwei Beobachtungen. Schweiz. Med. Wochschr. 97, 170-178 (1967). 525. Stobo, J. D., and Tomasi, T. B., Jr., A low molecular weight immunoglobulin antigenically related to 19s IgM. J. Clin. Invest. 46, 1329-1337 (1967). ,926. Stoop, J. W., Zegers, B. J. M., Van’ Der Heiden, C., and Ballieux, R. E., Monoclonal pmmopathy in a child with leukemia. Blood 32, 774-786 (196s). s27. Studer-Wobmann, E., and Koller, F., Paraproteinemia, Bence Jones proteinuria and amyloidosis. Acta Med. Scand. Suppl. 446, 147-153 (1966). 8%. Sussman, M., Asscher, A. W., and Jenkins, J. A. S., The intrarenal distribution of lysozyme (muramidase). Invest. Urol. 6, 148-152 (1968). 5%. Svedberg, T., and Pedersen, K. O., “The Ultracentrifuge.” Oxford Univ. Press (Clarendon), London and New York, 1940. TI. Takatsuki, X., and Osserman, E. F., Demonstration of two types of low molecular weight 7-globulins in normal human urine. J . Immunol. 92, 100-107 (1964). T2. Takatsuki, K., and Osserman, E. F., Structural differences between two types of “heavy chain” disease proteins and myeloma globulins of corresponding types. Science 146, 499-500 (1904). T3. Tamm, I., and Horsfall, F. L., Jr., A mucoprotein derived from human urine which reacts with influenza, mumps and Newcastle disease viruses. J . Ezptl. Med. 961 71-97 (1952). T4. Tan, M., and Epstein, W., Antigenic analysis of an N-terminal fragment of a type K Bence Jones protein. J . Immunol. 98, 568-575 (1967). T5. Thorling, E. B., Leukaemic myelomatosis (plasma-cellleukaemia). Acta Haematol. 28, 222-229 (1962). T6. Tischendorf, F. W., Two types of human K and X Bence Jones proteins (BJP). PTOC.18th Congr. Intern. Sac. Hematol., Abstr. Simultaneous Sessions, New York p. 141 (1968). T7. Tischendorf, F. W., and Osserman, E. F., Tryptic peptides (TP’s) of human lysozyme (LZM) from urine of monocytic leukemics. Federation Proc. 26, 845 (1967). T8. Tischendorf, F. W., and Osserman, E. F., Unterschiede im Konstanten (Cterminalen) peptidsatz menschlicher Typ L Bence-Jones - Proteine. 2. Naturforsch. 22b, 1337-1339 (1967). T9. Tischendorf, F. W., and Osserman, E. F., Two antigenic subtypes of human lambda immunoglobulin chains. J . Immunol. 102, 172-178 (1969). T10. Tiselius, A., Electrophormis of serum globulin. 11. Electrophoretic analysis of normal and immune sera. Biochm. J . 81, 1464-1477 (1937).

-

ABNORMAL PROTEINURIA IN MALIGNANT DISEASES

381

T11. Tiselius, A., and Flodin, P., Zone electrophoresis. Advan. Protein Chem. 8, 461486 (1953). T12. Titani, K., Shinoda, T., and Putnam, F. W., The amino acid sequence of a K type Bence-Jones protein. 111.The complete sequence and the location of the &sulfide bridges. J. Bhl. Chem. 244, 3550-3560 (1969). T13. Titani, K., Whitley, E., Jr., and Putnam, F. W., Immunoglobulin structure: Variation in the sequence of Bence Jones proteins. Science 162, 1513-1516 (1966). T14. Titani, K., Whitley, E. J., Jr., and Putnam, F. W., The amino acid sequence of a K type Bence Jones protein. I. Tryptic peptides. J . Bwl. Chem. 244, 35214536 (1968). T15. Turner, M. W., and Berggard, I., Characteriaation of the fragment of immunoglobulin G in normal human urine. Nature 224, 912-913 (1969). T16. Turner, M. W., and Rowe, D. S., A naturally occurring fragment related to the heavy chains of immunoglobulin G in normal human urine. Nature 210, 130-132 (1966). T17. Turner, M. W., and Rowe, D. S., Antibodies of IgA and IgG class in normal human urine. Immunology 12, 689-699 (1967). V1. Van Eyk, H. G., and Myszkowska, K., On the localisation of antigenic determinants in a Bence Jones protein. Clin. Chim. Acta 18, 101-106 (1967). V2. Van Furth, R., Schuit, H. R. E , and Hijmans, W., In vitro synthesis and immunofluorescent studies of monoclonal IgG in non-myeloma bone marrow. In “Gamma Globulins,” Nobel Symp. 3 (J. Killander, ed.), pp. 443453. Wiley (Interscience), New York and Almqvist & Wiksell, Stockholm, 1967. V3. Varriale, P., Ginsberg, D. M., and Sass, M. D., A urinary cryoprotein in multiple myeloma. Ann. Internal Med. 67, 819-823 (1962). V4. Vaughan, J. H., Jacox, R. F., and Gray, B. A., Light and heavy chain components of gamma globulins in urine of normal persons and patients with agammaglobulinemia. J . Clin. Invest. 46, 266-279 (1967). W1. Wager, O., Riisanen, J. A., Lindeberg, L., and Makela, V., Two cases of IgG heavy-chain disease. Acta Pathol. Microbiol. Smnd. 76, 350-352 (1969). W2. Waldenstrom, J., The occurrence of benign, essential monoclonal (M Type), non-macromolecular hyperglobulinemia and its differential diagnosis. IV. Studies in the gammopathies. Acta Med. Scand. 176, 345-365 (1964). W3. Waidenstrom, 3. G., “Monoclonal and Polyclonal Kypergammaglobulinemia. Clinical and Biological Significance.” Vanderbilt Univ. Press, Nashville, Tennessee, 1968. W4. Walravens, P., Laterre, E. C., and Heremans, J. F., Studies on tubular proteinuria. Clin. Chim. Acta 19, 107-120 (1968). W5. Welton, J., Walker, S. R., Sharp, G. C., Herzenberg, L. A., Wistar, R., Jr., and Creger, W. P., Macroglobulinemia with bone destruction. Am. J . Med. 44,280-288 (1968). W6. Whitley, E. J., Jr., Titani, K., and Putnam, F. W., The amino acid sequence of a K type Bence Jones protein. 11. Chymotryptic peptides. J . Biol. Chem. 244, 3537-3549 (19691. W7. Wiernik, P. H., and Serpick, A. A., Clinical significance of serum and urinary muramidase activity in leukemia and other hematologic malignancies. Am. J. Med. 46, 330-343 (1969). W8. Williams, R. C., Jr., Brunning, R. D., and Wollheim, F. A., Light-chain disease. An abortive variant of multiple myeloma. Ann. Internal Med. 66, 471486 (1966). W9. William, R . C., Jr., Pinnell, S. R., and Bratt, G. T., Low molecular weight

382

W10. W11. W12. Z1. 22. 23.

W. PRUZANSKI AND M. A. OGRYZLO

Zrohain components related to Bence Jones proteins. J . Lab. Clin. Med. 68, 81-89 (1966). Williamson, A. R., and Askonas, B. A., Biosynthesis of immunoglobulins: The separate classes of polyribosomes synthesizing heavy and light chains. J . Mo2. Biol. 28, 201-216 (1967). Wochner, R.D., Strober, W., and Waldmann, T. A., The role of the kidney in the catabolism of Bence Jones proteins and immunoglobulinfragments. J . Ezptl. Med. 126, 207-221 (1967). Wood, T. A,, and Frenkel, E. P., An unusual case of macroglobulinemia. Arch. Internal Med. 119, 631-637 (1967). Zawadzki, Z. A., and Edwards, G. A., Dysimmunoglobuliiemiain the absence of clinical features of multiple myeloma and macroglobulinemia. Am. J . Med. 42, 67-88 (1967). Zawadzki, Z. A., and Edwards, G. A., M-components in immunoproliferative disorders. Electrophoretic and immunologic analysis of 200 cases. Am. J. Clin. Pathol. 48, 418430 (1967). Zawadzki, Z. A., Benedek, T. G., Em, D., and Easton, J. M., Rheumatoid arthritis terminating in heavy-chain disease. Ann. Internal Med. 70, 335-347 (1969).