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Enzymes and Protein Markers |
1
Laboratoires de Biochimie A et B and
2
Laboratoire d'Immunologie, Hôpital St-Louis, 1 avenue Claude Vellefaux, 75475 Paris Cedex 10, France.
a Author for correspondence. Fax 33 1 42 49 92 47; e-mail Biochimie-a{at}chu-stlouis.fr.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Electrophoretic methods (agarose or cellulose acetate) are used routinely for analysis of urinary proteins on the basis of charge and molecular size. These methods require prior concentration of urine, usually by ultrafiltration, which is time-consuming and expensive and may lead to protein loss (5)(6). Some results are particularly difficult to interpret because Bence Jones proteins may comigrate with intact immunoglobulins and other proteins (such as transferrin) (7). Routine agarose electrophoresis (AGE) also shows poor sensitivity for detecting small concentrations of Bence Jones proteins (1).
Recently, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has been introduced for qualitative analysis of urinary proteins. SDS-PAGE separates proteins according to their molecular size (8), and free light chain monomers (polyclonal or monoclonal) are visualized as a band of Mr 25 000, easily distinguished from intact immunoglobulins (Mr 150 000). SDS-PAGE also differentiates proteinuria of glomerular (proteins with a Mr >66 000), tubular (Mr <66 000), or mixed origin. This method is thus a potentially powerful clinical tool for assessing kidney function, an important prognostic factor in MM (9)(10). SDS-PAGE, however, is not widely used in clinical laboratories because it is technically demanding, time-consuming, and expensive (11)(12)(13)(14).
In this study, we evaluated, for the first time, a new SDS-agarose gel
electrophoresis (SDS-AGE) for analysis of proteins in the urine of
patients with MM. Results were compared with those obtained by routine
(nondenaturing) AGE and IFE. Free and bound light chains (
,
),
IgG, and albumin were measured by immunonephelometry.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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urinary samples
Samples from 24-h urines were collected in 25-mL
Monovette® tubes without additive (Sarstedt) and
centrifuged for 5 min at 1500g (4 °C) before screening
for blood and protein (detection limit, 150 mg/L) by
Multistix® dipstick tests (Bayer Diagnostics). Samples
were then stored at -20 °C. Before analysis, urine was resuspended
and centrifuged as described above.
analysis
Routine AGE.
Before routine AGE, urine was concentrated
25-fold by ultrafiltration in Minicon® chambers (Amicon).
The time required for urine concentration by ultrafiltration varied
from 30 min to 4 h. Concentrated urine was analyzed using the
Hydragel® kit on an Hydrasis® automated gel
electrophoresis apparatus (Sebia). Briefly, 10 µL of concentrated
urine was applied in each well (15 wells/gel) and run at 272 V (68 mA)
at 20 °C for 7 min. After the gels were dried at 65 °C for 10
min, they were stained with 5 g/L amido schwarz in a 50 mL/L acetic
acid solution for 4 min and destained before a final drying at 75 °C
for 8 min. Total analysis time for electrophoresis was 45 min.
The gels were then scanned with a Preference®
densitometer (Sebia) using a yellow filter (570 nm) for determination
of five different fractions: albumin, 
1-globulin, 2-globulin,
ß-globulin, and 
-globulin. A serum protein control,
Precinorm® (Boehringer), was run in parallel with urine
specimens on each gel for appropriate separation of different
fractions. Abnormal fractions present in the - or ß-globulin zones
of the gel were individually integrated and expressed in
percentage of total proteinuria and in g/L.
SDS-AGE.
Urine was analyzed by SDS-AGE using the Hydragel
proteinurie® kit (Sebia), without prior concentration.
Urine containing >2 g protein/L was diluted with 9 g/L NaCl to 1 g/L
before analysis, according to the manufacturer's recommendations.
Twenty microliters of a 10 g/L SDS solution (with bromphenol blue as a
marker) were added to 80 µL of urine and briefly vortex-mixed at room
temperature. Five microliters of the treated samples were added to each
well (5 wells/gel) and allowed to diffuse for 10 min. A constant
voltage of 60 V (10 mA) was applied to the gel for 60 min in an
SDS–imidazole (1.0 g/L of SDS and 3.4 g/L imidazole) buffer (150
mL/compartment) by use of a MG 500 power source (Sebia). After the gel
was completely dried at 80 °C for 20 min, it was immersed for 30 min
in the following aqueous staining solution: 1.33 g/L coomassie blue,
250 mL/L methanol, and 200 mL/L acetic acid. The gel was destained in
two successive aqueous baths of 150 mL/L acetic acid before it was
immersed in a 150 mL/L glycerol aqueous solution and dried at 80 °C
(15 min). Total analysis time was 3.5 h.
On the gel, we detected immunoglobulins (Mr
850 000–150 000), haptoglobin (Mr 86 000),
transferrin (Mr 76 000), albumin
(Mr 66 000), 1-microglobulin
(Mr 30 000), light chain monomers
(Mr 25 000), retinol-binding protein
(Mr 21 000), and ß2-microglobulin
(Mr 12 000). Polyclonal light chains could not
be distinguished from monoclonal light chains. SDS-AGE differentiated
physiological proteinuria (<150 mg/24 h, mainly albumin) and
proteinuria from renal (glomerular, tubular, or mixed) or prerenal
origin (Table 1
). Five selected SDS-AGE patterns are presented in Fig. 1
. Monoclonal light chains (BJP) appear as a broad intense band
at Mr 25 000 and can be found in the absence
(pure prerenal BJP) or in the presence of renal proteinuria.
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Urine containing polymeric forms of light chains was pretreated with ß-mercaptoethanol (ß-ME) before electrophoresis. Five microliters of 100 mL/L ß-ME were added to 100 µL of urine and briefly vortex-mixed at room temperature. A 5-µL sample was loaded onto the gel and allowed to diffuse for 10 min before electrophoresis. After electrophoresis was complete, the gels were scanned, and the results were expressed as a percentage of total proteinuria and in g/L. The stated detection limit for a band was 15 mg/L (manufacturer's data).
A protein Mr marker LMW® (Pharmacia Biotech) suitable for SDS-PAGE was run on each gel. Band relative mobility (Rf) compared with lactalbumin (Mr 14 000) was calculated for each LMW protein (phosphorylase b, Mr 94 000; albumin, Mr 67 000; ovalbumin, Mr 43 000; carbonic anhydrase, Mr 30 000; and trypsin inhibitor, Mr 20 000) in nine different gels on separate days to evaluate the reproducibility of electrophoretic separations. Two urine samples containing high (pure prerenal BJP; light chains, 500 mg/L) and low (tubular proteinuria; light chains, 50 mg/L) concentrations of light chains were selected for estimation of the precision (within-run and day-to-day CV, n = 9 for each). The high control was treated with ß-ME before electrophoresis.
Other biochemical analyses.
Total protein was determined on a
Hitachi 747® analyzer (Boehringer) by an SDS-pyrogallol
red-molybdate technique (Merck-Clevenot)
(15)(16). The concentrations of urinary albumin,
immunoglobulin G (IgG), and free and bound light chains kappa (L)
and lambda (L) were measured by laser immunonephelometry on a
BNII® analyzer (Behring) (17)(18)(19). The urinary
L/L ratio was calculated (usual values, 1.0 < ratio <
5.2); urinary samples with ratios outside this reference are likely to
contain monoclonal light chains (Bence Jones proteins). IFE was
performed using the Paragon® IFE kit (Beckman). Samples
with total proteinuria <300 mg/L were concentrated up to 100-fold by
ultrafiltration in Minicon chambers before IFE.
statistical analysis
Results are expressed as mean and SE. Because population
distribution was not gaussian, differences between groups were assessed
by the Kruskal–Wallis ANOVA, followed by the Dunn's procedure for
multiple comparisons. Correlations between techniques and biological
parameters were evaluated by linear regression and ANOVA. Statistical
significance was set at P <0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/L ratio
within the normal reference interval; 20 had a ratio >5.2; and 18 had
a ratio <1. Intact IgG was found in 66% of urine specimens (range,
4–187 mg/L). Urinary BJP was detected by IFE in 38 of 47 urinary
samples (L, n = 20; L, n = 18).
routine age
Albumin values obtained by electrophoresis were significantly
higher than those measured by immunonephelometry (187 ± 33 vs
145 ± 26 mg/L, P <0.05); the two techniques
correlated significantly for this parameter (r = 0.958,
P <0.0001). In ~75% of cases, poor resolution was
obtained in the 1-globulin, 2-globulin, and ß-globulin zones of
the gel. An abnormal fraction, which suggested BJP, was detected in the
ß- or -globulin zones of the gel in 33 of 47 urine samples (70%
of cases) and represented 4–95% of the total proteinuria. A
significant correlation (r = 0.950, P
<0.0001) was noted between this fraction (662 ± 188 mg/L) and
light chains measured by immunonephelometry (L and L, 953
± 345 mg/L). Samples with an abnormal fraction detected by routine
electrophoresis were all positive for BJP by IFE (no false positives).
Detailed analysis of 14 negative MM patients by routine electrophoresis
is presented in Table 2
.In four specimens, protein was <150 mg/L. For the remaining
10 samples, protein concentrations ranged from 180 to 2080 mg/L and
consisted of a complex mixture of albumin, 1-globulin,
2-globulin, ß-globulin, and -globulin. In 10 cases, light
chains measured by immunonephelometry were <50 mg/L; one of these had
no detectable light chains. BJP was identified by IFE in five cases
(five false negatives for routine electrophoresis); therefore, the
calculated sensitivity for BJP detection was 87% (33 of 38 patients).
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sds-age
The migration of the molecular weight markers and a urine sample
with mixed proteinuria and BJP (identified by IFE) is presented in Fig. 2
. The mean CV for the Rf of the reference
proteins (LMW control) was 2.7%, ranging from 1.3% (trypsin
inhibitor) to 4.0% (albumin). The within-run CV for light chain
quantitation was 1% for the high control and 4% for the low
control (n = 9). The day-to-day CV was 2% and 5% for high and
low controls, respectively (9 days).
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Fifty percent of the patients from the control group had a mixed
proteinuria, according to their SDS-AGE pattern (Table 1
). The mean
proteinuria in this group was 2630 ± 1190 mg protein/24 h. Eight
samples were identified as glomerular proteinuria, either selective
(n = 5) or not selective (n = 3); the total protein was
390 ± 88 and 4500 ± 133 mg/24 h, respectively. There was
one case of tubular proteinuria (540 mg protein/24 h) and six cases of
physiological proteinuria (<150 mg protein/24 h). Polyclonal free
light chain monomers were detected in all urine samples with mixed and
tubular proteinuria (n = 16), but there was no evidence of BJP in
the control group. This was confirmed by IFE (no false positives).
In MM patients, SDS-AGE patterns were glomerular (n = 13), tubular
(n = 9), mixed (n = 13), or prerenal BJP (n = 12),
according to criteria summarized in Table 1
. Patients with proteinuria
of mixed origin excreted significantly (P <0.05) more
albumin (453 ± 120 mg/24 h) than those with tubular (76
± 26 mg/24 h) or prerenal proteinuria (74 ± 19 mg/24 h), as
determined by immunonephelometry. A band suggesting BJP was detected in
40 of 47 MM patients (85%), including 8 that were negative in routine
electrophoresis (Table 2
). IFE detected BJP in 37 of these 40 samples
(3 false positives). Among negative SDS-PAGE samples (n = 7), IFE
detected BJP in one (one false negative). Selected gels from
urinary samples that were difficult to interpret by routine
electrophoresis are presented in Fig. 3
. Specimens with polymeric forms of light chains (47% of cases)
were treated with ß-ME before quantification (Fig. 4
). A significant correlation (r = 0.963,
P <0.0001) was found between light chains detected by
SDS-AGE (661 ± 204 mg/L) and those measured by
immunonephelometry. The two electrophoresis techniques also correlated
significantly for light chains (in mg/L): SDS-agarose =
0.9682 x routine agarose - 74.696, r =
0.994, P <0.0001.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/ ratio for BJP identification (17).
Electrophoresis and densitometric scanning of the gel is the best
available method for both screening and a quantitative approach for
measurement of urinary BJP. Routine (nondenaturing) AGE, however,
cannot differentiate Bence Jones proteins from intact Ig and shows poor
sensitivity for BJP detection. A prior concentration step is required,
but there is no agreement on the ideal concentration that should be
obtained for BJP screening (22)(23). In our
experience, concentrating urine more than 25-fold leads to excessive
background staining. Concentrating urine up to 100-fold or more is
time-consuming and not necessarily associated with a gain in
sensitivity for BJP detection, with the exception of glomerular
proteinuria and pure BJP (Table 2
, patients 1 and 5). In addition,
prior concentration of urine by ultrafiltration is associated with a
loss of low Mr proteins (24), leading
to a 30% overestimation of the albumin fraction, compared with
immunonephelometry.
SDS-AGE separates proteins according to their molecular mass and does
not require prior urine concentration, a substantial improvement over
routine electrophoresis. We obtained excellent reproducibility of the
Rf (CV <5%, n = 9) of identical proteins (LMW
calibrator). These results compare favorably with those obtained with
SDS-PAGE (8) or AGE (25). Quantitation of light
chains also showed excellent within-run and day-to-day precision (CV
<5%) and correlated well (P <0.001) with other methods.
In MM patients, BJP results from tubular overload because of excessive
synthesis. Under reducing conditions, Bence Jones proteins appear as a
single dense band at Mr 25 000, and were not
confused with polyclonal light chains in a large majority of cases
(three false positives). In terms of sensitivity, SDS-AGE is clearly
superior to routine electrophoresis for BJP detection (97% vs 87%)
with a sensitivity close to IFE. Interestingly, one false positive
(Table 2
, patient 6) with glomerular proteinuria (total protein, 80
mg/L; 40% as light chains) had a / ratio outside the reference
interval (0.33), strongly suggesting BJP. In some cases, SDS-AGE might
show higher sensitivity than IFE for detecting Bence Jones proteins at
low concentrations.
SDS-PAGE is a powerful tool for monitoring kidney function (8). For example, SDS-PAGE protein patterns can help elucidate the predominant renal lesion when a biopsy is not possible, such as in children or the elderly (13). In our group of MM patients, SDS-AGE easily differentiated glomerular (selective or not selective), from tubular or mixed proteinuria. Only 26% of MM patients had no signs of renal impairment. Renal failure (acute or chronic) is one of the major complications of MM (26). Renal impairment is observed in ~50% of MM patients during the course of their disease and has been associated with a poor prognosis. Interestingly, most of the severe cases of renal impairment (plasma creatinine >200 µmol/L) were found in patients with mixed proteinuria (43%), rather than in those with glomerular (15%) or tubular (20%) proteinuria or prerenal BJP (7%), identified by SDS-AGE. Light chain excretion has been described as an essential factor contributing to renal failure in MM (26). However, we did not find any relation between light chain proteinuria (determined by SDS-AGE) and renal failure.
A few limitations of the SDS-AGE technique should be emphasized. The
most important is that SDS-AGE has no definitive way to distinguish
between monoclonal and polyclonal proteins. In the absence of tubular
damage (pure BJP or glomerular proteinuria, 50% of our MM samples), we
found that a band at Mr 25 000 is always BJP.
During tubular or mixed proteinuria, polyclonal light chains are
excreted in the urine because of impairment of tubular reabsorption.
Because MM can cause tubular damage, a complex mixture of monoclonal
and polyclonal light chains may be excreted. We found that a band at
Mr 25 000 of greater intensity than other small
proteins (1-microglobulin, retinol-binding protein, and
ß2-microglobulin) strongly suggests BJP. In patients with
tubular or mixed proteinuria, we obtained only two false positives with
SDS-AGE, compared with both IFE and immunonephelometry. The presence of
substantial quantities of polyclonal light chains in these specimens,
however, likely leads to an overestimation of the amount of BJP, as
underscored by others (27). In addition, in our IFE negative
control group we verified that the SDS-AGE protein pattern produced by
polyclonal light chains observed during tubular or mixed proteinuria is
unlikely to be confused with monoclonal components (no false
positives).
Other difficulties encountered with SDS-AGE are associated with BJP
quantitation. Polymeric forms of light chains were present as high
Mr bands (
300 000), smears, or dimers
(Mr 50 000) in 50% of our MM urine specimens.
Comigration of polymers with albumin on the gel can be suspected by
comparing electrophoretic protein patterns with the results of
albuminuria (by immunonephelometry) and/or dipstick tests (light chains
do not react with tetrabromophenol). Frequencies of 80% for dimers,
30% for tetramers, 20% for higher polymers, and 25% for fragments of
Bence Jones proteins have been reported (27). If a large
number of MM patients need to be evaluated, we recommend reducing all
urinary samples with ß-ME before SDS-AGE, with a depolymerization
marker run in parallel. We verified that there is no impact of ß-ME
treatment on the Rf of other proteins. Finally, it should
be recalled that there is no reference method for estimation of total
urinary protein: the biuret method lacks sufficient sensitivity for
most urine samples (2). We used an improved SDS-pyrogallol
red-molybdate technique, equally sensitive to albumin and -globulin
(15)(16). Alternatively, results might be
expressed in percentage of total proteinuria, instead of in mg/L.
In conclusion, SDS-AGE appears to be a clinical tool for the laboratory follow-up of MM patients. SDS-AGE has high resolution, sensitivity, and reproducibility. In a specialized laboratory like ours, it is useful for monitoring patient progress through the semiquantitation of BJP. Compared with routine electrophoresis, SDS-AGE combines no need for prior urine concentration with increased sensitivity for BJP detection. Glomerular, tubular, and mixed patterns were easily identified on the gel. Interpreting SDS-AGE protein patterns requires, however, awareness of some pitfalls, especially with regard to light chain polymerization and the presence of mono- and/or polyclonal light chains in urine specimens from MM patients.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  M. Gai, V. Cantaluppi, C. Fenocchio, D. Motta, S. Masini, A. Pacitti, and G. Lanfranco Presence of Protein Fragments in Urine of Critically Ill Patients with Acute Renal Failure: A Nephrologic Enigma Clin. Chem., October 1, 2004; 50(10): 1822 - 1824. [Full Text] [PDF]
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M. Salomo, P. Gimsing, and L. B. Nielsen Simple Method for Quantification of Bence Jones Proteins Clin. Chem., December 1, 2002; 48(12): 2202 - 2207. [Abstract] [Full Text] [PDF]
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K. Matsuda, N. Hiratsuka, T. Koyama, Y. Kurihara, O. Hotta, Y. Itoh, and K. Shiba Sensitive Method for Detection and Semiquantification of Bence Jones Protein by Cellulose Acetate Membrane Electrophoresis Using Colloidal Silver Staining Clin. Chem., April 1, 2001; 47(4): 763 - 766. [Full Text] [PDF]
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D. F. Keren Detection and Characterization of Monoclonal Componentsin Serum and Urine Clin. Chem., June 1, 1998; 44(6): 1143 - 1145. [Full Text] [PDF]
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