Acta Med Scand 206: 283-287, 1979
Free Light Chains of Immunoglobulins in Amyloidosis J . S~llingand K. Sslling From Drpurtment of’Medicine C , Aurhus Kommunehospital, Aarhus, Denmark
ABSTRACT. Monomeric (M) and dimeric (D) forms of free light chains in serum have been measured by a sensitive radioimmunoassay in eight patients with amyloidosis without monoclonal proteins. Significantly elevated concentrations of D lambda chains were demonstrated in two of four patients with localized amyloidosis. The two patients had a significantly increased D/M ratio of both kappa and lambda chains. One patient with localized amyloidosis and normal concentration of monomeric and dimeric light chains had an abnormal fragment of light chains. A low kappa/lambda ratio was found in patients with localized amyloidosis. Four patients with secondary amyloidosis and renal insufficiency had increased amounts of free light chains. The concentration of light chains and the D/M ratio in this group corresponded to the impairment of renal function. Key words: immunoglobulin, light chains, amyloidosis. Acta Med Scand 206: 283, 1979.
Amyloid proteins may be both of immunoglobulin and non-immunoglobulin origin (1, 4, 15). Light chains of immunoglobulins are presumed to be one of the proteins constituting the amyloid fibrils. Bence Jones proteins have been shown to be the source of amyloid fibrils in a patient with plasma cell dyscrasia (14). The source of the amyloid fibrils in patients without monoclonal proteins is unknown. Kappa and lambda light chains of immunoglobulins or polymeric forms of light chains in serum may be of pathogenetic significance. In the present study we have used a sensitive radioimmunoassay for light chains to investigate the concentrations of light chains in eight patients with amyloidosis. STUDY POPULATION Four patients had localized amyloidosis limited to the respiratory tract and no Coexisting diseases (3, 6, 7). Four patients had secondary amyloidosis: three had
amyloidosis secondary to rheumatoid arthritis and one had amyloidosis secondary to tuberculosis (7). Patients with monoclonal proteins on immunoelectrophoresis (Scheidegger) were excluded; this method detects monoclonal proteins at concentrations above 1 g/l. All patients with primary amyloidosis had serum creatinine below 1.3 mg/lOO ml and no proteinuria. The patients with secondary amyloidosis had amyloid deposits in the kidneys with proteinuria and decreased creatinine clearance. As controls we investigated serum from 1I patients with rheumatoid arthritis (six seropositive, four seronegative and one juvenile arthritis), six patients with tuberculosis (three with active tuberculosis and three with previously treated tuberculosis) and six normal individuals.
METHODS The diagnosis of amyloidosis was made on biopsy material. The amyloid showed green birefringence with Congo red under polarized light. Polymeric forms and split products of free light chains were isolated by gel filtration using a Sephadex (3-100 S F column measuring 1 4 0 1.5 ~ cm. The concentrations of light chains in each fraction were measured by a sensitive radioimmunoassay as previously described (10, 11, 12). The total amount of monomeric and dimeric light chains was calculated by summation of the respective fractions. The molecular weight was estimated by markers of radio-labelled lambda chains. Non-absorbed broadly reactive antibodies against kappa and lambda Bence Jones proteins were used. This was possible because the regular immunoglobulins were separated from the light chains by gel filtration.
RESULTS On gel filtration on Sephadex G-100 the free light chains appeared after intact immunoglobulins in two peaks with molecular weight about 44000 and 22000, i.e. as dimers (D) and monomers (M) (Fig. I). Higher polymeric light chains (tri- or tetramers) were demonstrated as a small peak between intact immunoglobulins and dimeric light chains. The total Abbreviations: M=monomeric light chains, D=dimeric
light chains. Acta M i d
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amount of dimeric and monomeric kappa and lambda light chains in serum is shown in Table I. I n the group with localized amyloidosis, two of the four patients had elevated concentrations of D lambda chains, when compared to normals (t-distribution). All patients had normal concentrations of kappa chains and M lambda chains. The concentrations of the tri- or tetrameric chains could not be precisely quantitated due to contamination of intact immunoglobulins. Patient 4 with normal concentrations of M and D light chains had a small abnormal peak appearing after the M light chains (Fig. 2 ) . This peak represents fragments of kappa and lambda light chains.
Fig. I . Mean values of kappa and lambda free light chains after gel filtration on Sephadex (3-100 SF in four patients with localized amyloidosis (-) and six normal individuals (- - -). Vertical lines indicate standard error.
Two patients (nos. 2 and 3) had significantly elevated D/M ratio of both kappa and lambda chains. The mean D/M ratio of kappa chains in localized amyloidosis was 1.6k0.5 (S.D.) compared to 1.2f-0.2 (S.D.) in the six normal individuals; the mean D/M ratio of lambda chains was 3.9kl.S (S.D.) compared to 2.7k0.7 (S.D.) in the normal individuals. Both these differences are insignificant (Mann-Whitney rank sum test). The kappahambda ratio in localized amyloidosis was 0.87 compared to 1.05 in the six normal individuals. The difference is not significant (Mann-Whitney test). Patients with secondary amyloidosis had significantly increased mean values of kappa and lambda
Table I. MPri.siirrt?ients of'M und D ,fi-ee light c h i n s ( L c ) of lcirnhdu and kappcr type in 4 putients nYth loc~uli,-oduniyloidosis trritl in 4 putients ititli secondary amvloiclosis Pat. no.
Lambda Lc (pglml)
Kappa Lc (pg/ml)
M
D
M+L)
DIM
M
4.1 3.6 4.1 2.9
5.6 6.7 8.1 2.6
9.7 10.3 12.2 5.5
1.4 1.9* 2.0* 0.9
2.9 1.8 3 .O 1.1
Mean
3.7
S.D.
0.6
5.8 2.3
9.4 2.8
1.6 0.5
2.2 0.9
17.6* 9.7* 12.0* 6.6*
19.1*
10.1" 18.9* 8.7*
36.7* 19.8* 30.9* 15.3*
1.1 1.0 1.6 1.3
11.5* 4.6
14.2* 5.6
25.7* 9.8
3.7 0.9
4.5 1.4
8.2 2.3
Localized
1 L
3 4
Secondary
Mean
S.D. Mean of 6 normals S.D.
*
5 6 7 8
D 6.8 10.7% 13.3* 3.4
M+D
D/M
9.7 12.5 16.3* 4.5
2.4 5.9* 4.4* 3.1
8.6 4.4
10.8 5.0
3.9 I .5
2.9 I .4 2.8 1.6
17.4* 15.4* 33.4* 8.2
20.3* 16.8* 36.2* 9.8
6.0* I 1 .o* 11.9*
1.3 0.3
2.2 0.8
18.6* 10.6
20.8* 11.1
8.5*
1.2 0.2
2.1 0.7
5.7 1.6
7.8 2.1
2.7 0.7
5.1*
3.4
pio.05.
chains in serum (Mann-Whitney rank sum test). The concentrations of kappa and lambda chains in serum were inversely correlated to the creatinine clearance. Both M and D kappa chains were elevated, whereas only D lambda chains were elevated in three of four patients. The D/M ratios of kappa and lambda chains were I .3*0.3 (S.D.) and 8.5k3.4 (S.D.). The corresponding values in anephric patients were 1.0k0.3 and 6.9k3.4 (S.D.) (10, 1 1 ) . These differences are insignificant. The kappa/ lambda ratio was 1.24 and did not differ significantly from normal. The mean value of free kappa light chains in 1 1 patients with rheumatoid arthritis was 10.2k4.2 (S.D.) pg/ml serum and of lambda chains 6.6k2.3 (S.D.) yglml. One patient with rheumatoid arthritis had increased values of both kappa and lambda chains, and one patient had increased values of only kappa chains. All patients with tuberculosis had normal values. Mean value of kappa light chains: 10.1k3.5 (S.D.) pg/ml serum and of lambda chains 7.1 k 1.3 (S.D.) yg/ml. None of the mean values differed significantly from normal values (Mann-Whitney rank sum test). DISCUSSION In this study we have demonstrated elevated concentrations of lambda light chains in two of four
patients with localized amyloidosis. None of the patients had monoclonal immunoglobulins detectable by immunoelectrophoresis (Scheidegger). Chemical studies have shown that the main protein component of purified amyloid fibrils in primary amyloidosis and localized amyloidosis is a whole light chain o r the amino-terminal, variable fragment of a light chain (4, 5, 7 , 9). Thus circulating light chains of immunoglobulins may be of pathogenetic significance in the deposition of amyloid fibrils in primary and localized amyloidosis. Free light chains have, however, never before been measured in serum from patients with primary and localized amyloidosis, probably due to the rather insensitive methods generally used for measurements of light chains. Glenner and Page (4) isolated from a patient with primary amyloidosis a serum protein with antigenic determinants in common with both amyloid fibrils and lambda Bence Jones protein. The concentration of this serum protein was estimated to be less than 0.1 mg/ml and the molecular weight about 90000. The demonstration of such small amounts requires a sensitive radioimmunoassay. The detection limit for our assay is 0.002 mg/l. On gel filtration we found that the lambda and kappa chains were eluted corresponding t o intact free light chains with molecular weights of about 22000 and 44000. Fragments of light chains were detected in only one patient (no. 4). In the deposits, however, intact light
286
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chains as well as fragments of light chains have been demonstrated (14). In patients with localized amyloidosis the mean D/M ratio of kappa chains was 1.6 compared to 1.2 in normal sera. For lambda it was 3.9 compared to 2.7 in normal sera. The values are not significantly different from normal, probably due to the small number of patients. Two patients (nos. 2 and 3), however, had increased D/M ratios of both kappa and lambda chains. This is suggestive of an increased polymerization rate in localized arnyloidosis (Fig. 1). The amount of tetrameric lambda light chains was accentuated in patients with localized amyloidosis but precise quantitation Ai
/
Free Light Chains of Immunoglobulins in Amyloidosis J . S~llingand K. Sslling From Drpurtment of’Medicine C , Aurhus Kommunehospital, Aarhus, Denmark
ABSTRACT. Monomeric (M) and dimeric (D) forms of free light chains in serum have been measured by a sensitive radioimmunoassay in eight patients with amyloidosis without monoclonal proteins. Significantly elevated concentrations of D lambda chains were demonstrated in two of four patients with localized amyloidosis. The two patients had a significantly increased D/M ratio of both kappa and lambda chains. One patient with localized amyloidosis and normal concentration of monomeric and dimeric light chains had an abnormal fragment of light chains. A low kappa/lambda ratio was found in patients with localized amyloidosis. Four patients with secondary amyloidosis and renal insufficiency had increased amounts of free light chains. The concentration of light chains and the D/M ratio in this group corresponded to the impairment of renal function. Key words: immunoglobulin, light chains, amyloidosis. Acta Med Scand 206: 283, 1979.
Amyloid proteins may be both of immunoglobulin and non-immunoglobulin origin (1, 4, 15). Light chains of immunoglobulins are presumed to be one of the proteins constituting the amyloid fibrils. Bence Jones proteins have been shown to be the source of amyloid fibrils in a patient with plasma cell dyscrasia (14). The source of the amyloid fibrils in patients without monoclonal proteins is unknown. Kappa and lambda light chains of immunoglobulins or polymeric forms of light chains in serum may be of pathogenetic significance. In the present study we have used a sensitive radioimmunoassay for light chains to investigate the concentrations of light chains in eight patients with amyloidosis. STUDY POPULATION Four patients had localized amyloidosis limited to the respiratory tract and no Coexisting diseases (3, 6, 7). Four patients had secondary amyloidosis: three had
amyloidosis secondary to rheumatoid arthritis and one had amyloidosis secondary to tuberculosis (7). Patients with monoclonal proteins on immunoelectrophoresis (Scheidegger) were excluded; this method detects monoclonal proteins at concentrations above 1 g/l. All patients with primary amyloidosis had serum creatinine below 1.3 mg/lOO ml and no proteinuria. The patients with secondary amyloidosis had amyloid deposits in the kidneys with proteinuria and decreased creatinine clearance. As controls we investigated serum from 1I patients with rheumatoid arthritis (six seropositive, four seronegative and one juvenile arthritis), six patients with tuberculosis (three with active tuberculosis and three with previously treated tuberculosis) and six normal individuals.
METHODS The diagnosis of amyloidosis was made on biopsy material. The amyloid showed green birefringence with Congo red under polarized light. Polymeric forms and split products of free light chains were isolated by gel filtration using a Sephadex (3-100 S F column measuring 1 4 0 1.5 ~ cm. The concentrations of light chains in each fraction were measured by a sensitive radioimmunoassay as previously described (10, 11, 12). The total amount of monomeric and dimeric light chains was calculated by summation of the respective fractions. The molecular weight was estimated by markers of radio-labelled lambda chains. Non-absorbed broadly reactive antibodies against kappa and lambda Bence Jones proteins were used. This was possible because the regular immunoglobulins were separated from the light chains by gel filtration.
RESULTS On gel filtration on Sephadex G-100 the free light chains appeared after intact immunoglobulins in two peaks with molecular weight about 44000 and 22000, i.e. as dimers (D) and monomers (M) (Fig. I). Higher polymeric light chains (tri- or tetramers) were demonstrated as a small peak between intact immunoglobulins and dimeric light chains. The total Abbreviations: M=monomeric light chains, D=dimeric
light chains. Acta M i d
Scnnd 206
284
I
I
I
J . S d l i n g cind K . S d l i n g 0.41
Effluent
Effluent
ml
ml
amount of dimeric and monomeric kappa and lambda light chains in serum is shown in Table I. I n the group with localized amyloidosis, two of the four patients had elevated concentrations of D lambda chains, when compared to normals (t-distribution). All patients had normal concentrations of kappa chains and M lambda chains. The concentrations of the tri- or tetrameric chains could not be precisely quantitated due to contamination of intact immunoglobulins. Patient 4 with normal concentrations of M and D light chains had a small abnormal peak appearing after the M light chains (Fig. 2 ) . This peak represents fragments of kappa and lambda light chains.
Fig. I . Mean values of kappa and lambda free light chains after gel filtration on Sephadex (3-100 SF in four patients with localized amyloidosis (-) and six normal individuals (- - -). Vertical lines indicate standard error.
Two patients (nos. 2 and 3) had significantly elevated D/M ratio of both kappa and lambda chains. The mean D/M ratio of kappa chains in localized amyloidosis was 1.6k0.5 (S.D.) compared to 1.2f-0.2 (S.D.) in the six normal individuals; the mean D/M ratio of lambda chains was 3.9kl.S (S.D.) compared to 2.7k0.7 (S.D.) in the normal individuals. Both these differences are insignificant (Mann-Whitney rank sum test). The kappahambda ratio in localized amyloidosis was 0.87 compared to 1.05 in the six normal individuals. The difference is not significant (Mann-Whitney test). Patients with secondary amyloidosis had significantly increased mean values of kappa and lambda
Table I. MPri.siirrt?ients of'M und D ,fi-ee light c h i n s ( L c ) of lcirnhdu and kappcr type in 4 putients nYth loc~uli,-oduniyloidosis trritl in 4 putients ititli secondary amvloiclosis Pat. no.
Lambda Lc (pglml)
Kappa Lc (pg/ml)
M
D
M+L)
DIM
M
4.1 3.6 4.1 2.9
5.6 6.7 8.1 2.6
9.7 10.3 12.2 5.5
1.4 1.9* 2.0* 0.9
2.9 1.8 3 .O 1.1
Mean
3.7
S.D.
0.6
5.8 2.3
9.4 2.8
1.6 0.5
2.2 0.9
17.6* 9.7* 12.0* 6.6*
19.1*
10.1" 18.9* 8.7*
36.7* 19.8* 30.9* 15.3*
1.1 1.0 1.6 1.3
11.5* 4.6
14.2* 5.6
25.7* 9.8
3.7 0.9
4.5 1.4
8.2 2.3
Localized
1 L
3 4
Secondary
Mean
S.D. Mean of 6 normals S.D.
*
5 6 7 8
D 6.8 10.7% 13.3* 3.4
M+D
D/M
9.7 12.5 16.3* 4.5
2.4 5.9* 4.4* 3.1
8.6 4.4
10.8 5.0
3.9 I .5
2.9 I .4 2.8 1.6
17.4* 15.4* 33.4* 8.2
20.3* 16.8* 36.2* 9.8
6.0* I 1 .o* 11.9*
1.3 0.3
2.2 0.8
18.6* 10.6
20.8* 11.1
8.5*
1.2 0.2
2.1 0.7
5.7 1.6
7.8 2.1
2.7 0.7
5.1*
3.4
pio.05.
chains in serum (Mann-Whitney rank sum test). The concentrations of kappa and lambda chains in serum were inversely correlated to the creatinine clearance. Both M and D kappa chains were elevated, whereas only D lambda chains were elevated in three of four patients. The D/M ratios of kappa and lambda chains were I .3*0.3 (S.D.) and 8.5k3.4 (S.D.). The corresponding values in anephric patients were 1.0k0.3 and 6.9k3.4 (S.D.) (10, 1 1 ) . These differences are insignificant. The kappa/ lambda ratio was 1.24 and did not differ significantly from normal. The mean value of free kappa light chains in 1 1 patients with rheumatoid arthritis was 10.2k4.2 (S.D.) pg/ml serum and of lambda chains 6.6k2.3 (S.D.) yglml. One patient with rheumatoid arthritis had increased values of both kappa and lambda chains, and one patient had increased values of only kappa chains. All patients with tuberculosis had normal values. Mean value of kappa light chains: 10.1k3.5 (S.D.) pg/ml serum and of lambda chains 7.1 k 1.3 (S.D.) yg/ml. None of the mean values differed significantly from normal values (Mann-Whitney rank sum test). DISCUSSION In this study we have demonstrated elevated concentrations of lambda light chains in two of four
patients with localized amyloidosis. None of the patients had monoclonal immunoglobulins detectable by immunoelectrophoresis (Scheidegger). Chemical studies have shown that the main protein component of purified amyloid fibrils in primary amyloidosis and localized amyloidosis is a whole light chain o r the amino-terminal, variable fragment of a light chain (4, 5, 7 , 9). Thus circulating light chains of immunoglobulins may be of pathogenetic significance in the deposition of amyloid fibrils in primary and localized amyloidosis. Free light chains have, however, never before been measured in serum from patients with primary and localized amyloidosis, probably due to the rather insensitive methods generally used for measurements of light chains. Glenner and Page (4) isolated from a patient with primary amyloidosis a serum protein with antigenic determinants in common with both amyloid fibrils and lambda Bence Jones protein. The concentration of this serum protein was estimated to be less than 0.1 mg/ml and the molecular weight about 90000. The demonstration of such small amounts requires a sensitive radioimmunoassay. The detection limit for our assay is 0.002 mg/l. On gel filtration we found that the lambda and kappa chains were eluted corresponding t o intact free light chains with molecular weights of about 22000 and 44000. Fragments of light chains were detected in only one patient (no. 4). In the deposits, however, intact light
286
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chains as well as fragments of light chains have been demonstrated (14). In patients with localized amyloidosis the mean D/M ratio of kappa chains was 1.6 compared to 1.2 in normal sera. For lambda it was 3.9 compared to 2.7 in normal sera. The values are not significantly different from normal, probably due to the small number of patients. Two patients (nos. 2 and 3), however, had increased D/M ratios of both kappa and lambda chains. This is suggestive of an increased polymerization rate in localized arnyloidosis (Fig. 1). The amount of tetrameric lambda light chains was accentuated in patients with localized amyloidosis but precise quantitation Ai
/
