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The characterisation of urine protein by gel filtration
357
CLINICA CHIMICA ACTA
THE CHARACTERISATION
JENNIFER
S. DAVIS,
OF URINE PROTEIN
F. V. FLYNN
BY GEL FILTRATION
AND H. S. PLATl
Department of CfinicaE PathoEog?i, University College HospifaE, Londolz, W.C. I (U.K.] (Received
April 5. x968)
SUMMARY I. Urine protein from healthy adults and 29 patients with clinical proteinuria has been studied by gel filtration using Sephadex G-75, The resolution obtained of the lower molecular weight components was superior to that reported using G-100 and G-200 Sephadex. Six major fractions were recognised. 2. The proteinurias associated with glomerular and tubular malfunction and with “abnormal” light-chain excretion have been further characterised by this technique. 3. A simple thin-layer gel filtration technique is advocated for the distinction of tubular proteinuria from glomerular and Bence Jones types. 4. A close similarity between the proteins present in normal urine and in tubular proteinuria has been demonstrated. 5. Small quantities of low molecular weight protein similar to that characteristically found in tubular proteinuria have been demonstrated in the plasma of a patient with chronic cadmium poisoning by column gel filtration using Sephadex G-75. 6. The anomalous elution behaviour of Bence Jones proteins is discussed.
Previous work has made it increasingly clear that the predominant proteins in the different types of clinical proteinuria are of different molecular weight. The slight proteinuria which may accompany renal tubular disorders has been shown by Creeth et a1.l to have a high proportion of low molecular weight components and to differ in this respect from the gross proteinuria known to be due to the passage of increased amounts of serum protein through the glomerulus. The average molecular size of the light chain components found in Bence Jones proteinuria has similarly been shown to be smaller than most of the serum proteins, the median molecular weight being 44000 (ref. 2). It was a logical step therefore to employ Sephadex gel filtration in the further study of proteinuria as this separates proteins according to their molecular size. Other workers have examined urine proteins by gel filtration, but none have particularly set about comparing the fractionation obtained in the different types of proteinuria. The urine proteins excreted in health and in a variety of conditions associated with clinical proteinuria have been studied by gel filtration using Sephadex G-75 and C&in.Chim. Acta, 21 (1968)
357-376
“;:
2
a % P
I
SUBJECTS
32 25 5’ 66
M F M M
46 42 5’ 54
53 48
55
16 ‘7
18
--
RESULTS
syndrome
Chronic cadmium poisoning Chronic cadmium poisoning
M M
ICI Adult Fanconi (hereditary)
Balkan Balkan Chronic Chronic
M E M M
nephropathy nepbropathy cadmium poisoning cadmium poisoning
Nephrotic nephritis Nephrotic nephritis Nephrotic nephritis Nephrotic nephritis Amyloidosis Chronic nephritis
Z‘ltbulav fwoteinurias
12 13 ‘4 15
AND
Healthy adult Healthy adult Chronic cadmium poisoning Chronic cadmium poisoning
GEomeruLar proteinurias
I 2 3 4
.~
STUDIED
Sex Diagnosis
Izi
_ Plasma OYserum
AVO.
Spot. Age
...~__
a
DI.IIGSOSIS
5
TABLE
0 nl s (5
-_.
-___-
150 m,s PROTEIN
USING
- ..-_-_
___
A COLUMN
~
OF
0.33 0.50
0.38 0.25
0.22
0.29
0.37
0.2x*
0.61 I .03 0.37*
1.13%
1.or
2.29
r.26*
1.85 1.87
1.24 B2.00 0.87* 1.04 I.59 r.26* 1.17
>2.00
.>2.00
>2.00
~2.00
>2.00
0.74%
>2.00
>2.00
>z.ao
0.76 1.84 0.77 0.53 I.23
>2.00
>2.00
0.42
I.90
>2.00
1.60 1.68
>2.00
1.q
1.75 1.60
>2.00
1.62
0.43
1.00*
0.31
0.31* 0.40
0.27
0.42’
0.4s*
1.21
0.38 0.24’
0.64 0.29
0.97
0.43
0.44 0.77
0.45 0.47
0.26
0.08
trace 0.06* trace 0. IO” 0.07
0.17
0.07*
0.27
0.62*
o.‘p*
0.12
0.25
trace
0.10 trace
o.og*
0.87
trace* 0.05*
trace*
!Waxirnwn A reading al 280 nzp _.~_^__~~_ .Peak 3 Peak 3 Peak I Peak 2 Peaks --_____V,l fJ* I,‘,/ v0 Vel v, V,l v, l’el v/D vt?i v, [‘f?i v, E”e/v* I.621~75r.grI.00 r.ogX.17IA?& r.40I.60 I.72 I.90 2.04 1.09 I.27 I,34 ---l____l_-
OF FRACTIONXTINC
trace
0.20
0.17
0.16
0. I I
0.27
trace
0.10*
urea urea urea urea urea urea
protein l’eak 6 Some
Some protein Peak 6
Plasma Plasma Plasma Plasma Plasma Plasma
..I_-___-.
Coinnzcnis
G 75
V,l v, V,l v, Z.II2.232.34 2.17
Peak 6
SEPHADEX
eluted after
eluted after
ml ml ml ml ml ml
___._
-__
31 mg/roo 26 mg/roo 45 mg/roo 57 mg/roo 36 mg/roo 125 q/I00
._-
7
5 D
Multiple myeloma Multiple myeloma ~lacro~lol~ulinae~~~i~~
I’rimar-y hyperparathyroidism. nephrocalcinosis and renal calculi ILlkan ~qhrupatl~q-
my&ma
my&ma
Hnlkan
Multiple
Multiple
Alultiplc
I; F F
.\I
1:
11
F
I;
hI
11 Multiple
80 00
62
40
61
trace
= ‘-1 reading
53
5o
40
25 26 27
nt
less than 0.05
my&ma
my&ma
nephropathy
myeloma myeloma
Multiple Multiple
SI
56
67
23 24
Oculo-cerchro-renal
M
I8
22
3
on side ot adlaccnt
0.22
0.30
o.iO
0.71
o.j+*
0.20*
1.55
0.84*
1.59
cl..+0
0.18
o.11*
1.61
I.78
1.83
0.09
2.00
I.41
o.39*
1.72
0.99*
0.02
trace*
tract*
I.34
0.17
0.85
0.37’
0.45
peak.
0.83 I .05 I.15
1.64
>z.oo
>2.00
+ =
0.79%
0.62* 0.34
0.26
0.70* 0.23
>2.00
0.39 >2.00 I.42 >2.00 0.76*
0.28
>2.00
0.83
0.93
1.08
0.32* o.rR
1.01
>2.00
1.14* I.33
1.68
0.23
0.46
0.40
0.77
0.48
0.05 trace
tmcc*
0.34
0.17
=- shoulder
syndrome
syndrome
Oculo-ccrcbro-renal
14
21
Al
Adult
M
IO
20
F
Fanconi syndrome (hereditary) Fanconi syndrome (type 3 +)
56
19
0.07*
o.or*
0.43
0.41
0.34
0.05*
0.35 0.07
trace
0.45
of Dent”‘.
o.r5*
0.08*
0.26
0.14
0.70
O.jT
0.16
0.16
0.66 0.35
0.62
I.33
Classification
0.92
0.20*
0.36
0.24*
0.43
o.r(i*
0.49
0.79”
eluted
eluted
eluted
after
after
after
Mixed ,olomcrular and tubular proteinuria l’lasmacalcium I*.I mg/rooml Mixed $xnerulnr and tubular protcinuria Mixed glornerular and tubular proteinuria Mixed Hence Jones and tubular protcinuria Plasmacalcium 14.7mg/1ooml Mixed Bcnce Jones and tubular proteinuria Plasrnacalciu~n17.0mg/1ooml Mixed Rence Jones and glomcrulnr proteinuria Mixctl l
Some protein Peak 6 Some protein Peak ti Some protein Prak h
E Q
DAVIS et ai.
360
we report here the findings using both column and thin-layer techniques. Gel filtration has been found to differentiate clearly between the main types of proteinuria, and the simple thin-layer technique is advocated for routine use. Sephadex fractionation has also demonstrated a general similarity between the protein of normal urine and that of tubular proteinuria. 4 preliminary account of this work was given at the VIth International Congress of Clinical Chemistry3. MATERIALANDMETHODS Urine specifnens
Twenty-four-hour collections of urine were obtained from 29 selected patients with clinical proteinuria, the definitive diagnosis in each being shown in Table I. In addition, 15 1 of pooled normal urine was collected from 18 ad&t males aged 20-45 years, each of whom had been shown to be free of clinical proteinuria. The urine samples were collected into vessels containing I g sodium azide and more was added later to give a final concentration of I g/l. The specimens were stored at 4’ while awaiting processing. In some cases the urine was filtered through Oxoid cellulose acetate membranes, but debris was usually removed by centrifugation.
The total protein in the original urine samples was determined by a turbidometric method employing 5% sulphosalicylic acid and a nephelometer. The concentration of protein in the samples which were used for gel filtration was measured by a biuret method4. Preparation
of urine protein
concentrates
The urine protein was concentrated by ultrafiltration, using autoclaved a-inch Visking cellulose tubing without external support and a negative pressure of -400 mm Hg. Prior to use the Visking tubing was autoclaved for zo min at 15 lb/sq.in. to decrease the pore size and prevent the loss of low molecular weight protein which has been reported in studies by Berg&d5 and Piscatore. Under the conditions used for the concentration process, when cytochrome c (molecular weight 12270) was added to the urine it could not be detected in the ultrafiltrate. In most cases the urine protein was concentrated to a level well above 6 g/Ioo ml and after 48 h dialysis against IOO volumes of a phosphate-sodium chloride solution pH 7.3 (see below), was adjusted to exactly 6 g/roe ml by dilution with the same buffer.
Bloods$ecimerzs Venous blood specimens were collected from 2 healthy adults and from 2 patients with chronic cadmium poisoning; the patients’ blood was collected into heparin but serum was separated from the specimens taken from the healthy adults.
Two-dilnensional electrophoresis was carried out on the urine protein concentrates after the method of Poulik and Smithies7, using cellulose acetate and barbiturate buffer pH 8.6 for the first separation, and starch gel and the tris-citrate/borate discontinuous buffer system of Pouliks for the second. This procedure was used to Clin.
Chim.
Ada,
21
(1968) 357-376
GELPILTRATIOX
OF URINE
PROTEIN
36r
classify the proteinurias into glomerular, tubular, Bence Jones and mixed types, on the basis of earlier work by Flynn and Stowv, Butler and Flynnl” and Butler et d.ll It was also used to determine the content of the fractions obtained by column gel filtration.
Glass columns (Column I: 80 cm longx 4.0 cm in diameter. Column 2: roe cm Iongx 2.5 cm in diameter) were filled to within 4-7 cm of the top with the bead form of Sephadex G-75 cross-linked dextran gel (lot No. To 5x30), using the procedure described by Flodinlz. The fines were decanted 10 times and the gel particles allowed to swell for 3 days before packing The columns were packed and re-packed until a Blue I?extran solution was seen to migrate evenly through the gel. Phosphate-sodium chloride buffer pH 7.3 (0.035 M NazHP04, 0.006 M NaH,PO,, 0.15 X NaCl) containing I gjl sodium azide as preservative was used for both packing and eluting the columns. A 2.5~ml aliquot of urine concentrate, containing ISO mg protein, was applied to the top of the column by layering under the supernatant buffer, and elution was carried out at a flow rate of approximately 12 ml/h. The whole procedure was carried out at room temperature (rg-.z5”). Using syphons of 3.0 or 3.6 ml capacity in conjunction with a fraction collector, aliquots were collected continuously until elution of the protein was complete. An indication of protein concentration in the individual fractions was obtained by me~uring their optical density at 280 mp in a Unicam S.P, 500 spectrop~otometer, using a x-cm silica cuvette. The void volumes of the columns were determined repeatedly by noting the elution volume at which the maximum optical density reading at 620 m/t was obtained following the application of a Blue Dextran solution to the top of the column. A solution of 0.2 g Blue Dextran in IOO ml elution buffer was used for this purpose. To express the elution behaviour of the proteins in a manner that is independent of column size the elution volume of each peak (V,) was divided by the current void volume of the column (V,) as advocated by Whitaker’“.
The technique of Johansson and Rymo14, and MorrisIS, was adopted with some modifications. Sixty grams of Superfine Sephadex G-75 was allowed to swell for 48 11 while continuously stirred in I 1 of a phosphate-sodium chloride buffer pH 7.2 jo.mci M Na,IIPO,, o.ao4 M ICH,l?O,, 0.2 *Q NaCl) containing I g/l sodium azide. Sufficient supernatant fluid was subsequently removed to give a slurry, the ideal consistency of which was found by trial and error. An excess of the slurry, i.e. 15 ml, was used to make a film approximately 0.5~mm thick on a thoroughly cleaned glass plate measuring IOX 16 cm, an even film being obtained by running a glass rod swiftly and smoothly along metal plates mounted on either side of the glass plate. The coated plates were placed horizontally within a closed tank to form a bridge between reservoirs containing the same buffer, and wicks of Whatman No. 3 MN filter paper, ro-cm wide, were applied over the last cm at each end of the gel and dipped into the buffer. The difference in level of the buffer in the two reservoirs was adjusted so that a flow rate of about z cm/h was obtained. Blue Dextran was used as a marker during these adjustments and usually a difference in height of about 1.5 cm was required between the buffer levels. After allowing the thin-layer plate to equiliClin. Chinz. ActC6,21 (1968) 357-375
DAVIS
362
al.
et
brate overnight, protein was applied to the surface of the gel on discs of Whatman No. 52 filter paper, 1.5 mm in diameter, cut with a paper tape punch. These discs were dipped in the protein or Blue Dextran solutions and blotted lightly before being laid gently on the surface of the gel at intervals of 1.5 cm along a line 2 cm from the top end of the plate. The buffer was allowed to continue flowing until a Blue Dcxtran marker had moved 10-12 cm, which usually required 5-O 11. The plate was then removed from the tank and totally covered by a sheet of Whatman No. 3 MRI filter paper which was rolled on in such a way as to avoid forming ridges or trapping air bubbles. The protein was taken up into the paper and fixed there by drying the covered plate in an oven at 80” for 30 min. Subsequently the protein was stained on the filter paper by immersion in 0.15?; Ponceau S in 37; trichloracetic acid for 30 min, excess dye being removed
by subsequent
washing in 2”,4 (v/v) acetic acid.
Exarvtinatiou of the protein fractions obtained 631column gel filtration When the optical density at 280 nip of each sample collected gel filtration
procedure
was plotted graphically,
from the column
it became possible to recognise major
peaks of eluted protein. Divisions were made at the points where undoubted troughs occurred and the samples constituting each main fraction were bulked. The fractions
Glomerular Protemunas TWO- dimenstonal Electrophoresls of Urme Proteins Glomerular
Proteinurla
Tubular
Elutlon vol ml
100
Proteinurla
I
VOID
VOLUME
2ooy
1.8
300
400
I
500
1
600
I
700
I
NEPHRlilS flypeni
1.5-
Bence
Jones
A at 28Oml
NORMALURINE
Proteinurla
lo0,5-
n
. Alb RENALAMt'LOIOOSIS
l’ig. 1. IXagrams showing two-dimensional electrophoretic separations of urine proteins in the different types of proteinuria. Electrophoresis was carried out on cellulose acetate from right to left and subsequently in starch gel from below upwards. The line of insertion of the ccllulosc acetate strip into the gel is indicated by the horizontal dotted line, and the relative concentration of the protein fractions by the density of shading. The whole of the arca cncloscd by the dotted line in the diagram of the normal urine pattern stained slightly darker than the background. Alb : albumin, ‘l‘r = transfcrrin. I?g. 2. Elution patterns glomerulsr proteinuria.
of urine protein
obtained
from
a column
of Sephadex
(i-75 in .3 cases of
GEL FILTRATION
OF URINE PROTEIK
363
so obtained were reconcentrated by ultrafiltration sional electrophoresis and thin-layer gel filtration.
and examined
by both two-dimen-
RESULTS
T-zmdiwaensional
electrophoretic analyses of urine protein concentrates Typical examples of two-dimensional patterns obtained in the different types of clinical proteinuria and in normal urine are illustrated in Fig. I. In glomerular proteinurias there were prominent albumin, a,-antitrypsin and transferrin zones with traces of other serum components including y-globulin. Tubular proteinurias were more complex and a series of protein zones unlike any seen in serum occupied various positions along an arc roughly parallel to, and on the anodic side of, a curve linking the albumin, cqantitrypsin and transferrin zones. They varied widely in relative amount from case to case, but three of them were particularly prominent and were found in nearly all cases. Bence Jones proteinurias characteristically showed a prominent group of parallel zones in close proximity; usually there were 3 and that nearest the cathode was the most conspicuous. These were situated in positions corresponding to somewhere between the p- and y-globulin regions in the first-dimensional separation and anywhere between the post-albumin and y-globulin positions in the starch gel separation. Concentrates of normal urine protein revealed albumin, traces of prealbumin, y-globulin and several tlr- and a,-globulins. Bence JonesProtemwas vc&
LUME 300 LOO
I
I
I
500
600
700
MULTIPLEMYELOMA
MULTIPLEMYELOMA
J' MULTIPLFMYELOMA + ren~lunpsirment
1.5 1.0 0.5 :I.
Tube No.
Fig. 3. tubular
Elution patterns proteinuria.
150
\
of urine protein
200 obtained
Fig. 4. Elution patterns of urine protein obtained multiple myeloma; upper 2 from uncomplicated mixed Bcnce Jones and glomerular proteinuria.
1
Tube No. from
,
50
a column of Sephadex
G-75 in 3 cases of
from a column of Sephadex G-75 in 3 cases of Bcnce Jones proteinurias, lowest one from a
CZi?z. Chim. Acta,
LI (1968)
357-376
DAVIS et al.
364 Colawan gd filtration
Examples of elution patterns obtained from Column I in the different types of proteinuria are shown in Figs. 2,~ and 4. As can be seen from these figures a variable number of major protein peaks was obtained, up to a maximum of 6, and these peaks eluted at very similar buffer volumes. The volume of elution of the Blue Dextran, i.e. the void volume of the columns (V,), varied slightly over the period of the experiments. The 6 major peaks that could be distinguished eluted from the columns as follows : Peak I: The elution volume of this peak was at or near the void volume and consequentlythevalueof V,,WO was at or close to 1.00. In some cases the peak eluted exactly at the void volume and in others it emerged a few ml later, the average V,/V, then being 1.06 (range 1.03 to 1.09).In 6 of the urine specimens the peak was clearly bifid with the highest points corresponding to the z usually seen separately; in 6 others there was a shoulder on one side of the peak indicating the presence of 2 components. Peak 2: In some cases the V,/V, value for this peak was close to 1.22 (range 1.17 to 1.27) and in others near to 1.31 (range 1.28 to r.34), and in 4 instances the peak was clearly bifid due to the presence of both components. Bind second peaks were onlv clearly seen when the specimens were fractionated on Column 2, the longer narrower column, but asymmetric peaks with definite shoulders, indicating the presence of z components, were sometimes found to elute from Column T. Peak 3: The average V,jV, value for this peak was 1.50. The range of values for V,,/V, when Bence Jones proteinurias were excluded was 1.45 to 1.56. The peak only appeared complex in the case of some Bence Jones proteinurias when I or z additional components produced subsidiary peaks or shoulders; the range of values for V,jV,, when these were included was x.40 to x.60. Peak 4 : This was not always seen as a real peak. Often it was overshadowed by taller adjacent peaks and appeared as a shoulder on the side of Peak 3, or occasionally Peak 5, and in these instances it was difficult to be precise about its elution volume. In most cases the V,jV, value for the peak fell within the range 1.75 to 1.90, the mean then being 1.81, but in some it eluted earlier, the mean V,/V, value then being 1.68 (range 1.62 to 1.72). In 6 specimens both components were present, and in 3 of these both were well defined. Peak 3: The average r/,/V, value for this peak was 1.99 (range 1.91 to 2.04). Peuk 6: In most cases this eluted with an average V,jV, value of 2.29 (range 2.23 to 2.34). In 3 instances of Bence- Jones proteinuria this peak emerged earlier, V,/V, then being 2.16, and in a further similar case this early component appeared together with the more usual Peak 6. The findings in all the specimens are summarized in Table I, which shows the maximum optical density readings at 280 m,u for the major discernible peaks. In all 6 glomerular proteinurias there was a very large early Peak 2, a sizeable Peak I and a small Peak 3. In addition a tiny Peak 4 was found in 5 of the cases and a minute Peak 5 was discerned in 3. In one patient with chronic nephritis, with a plasma urea of 125 mg/roo ml, a trace of Peak 6 was recognisable. In all but one of the 11 cases of tubular proteinuria, obvious protein fractions corresponding to all 6 major peaks were found. In 9 of the IX cases Peaks 2 and 3 Clin. Chim.
Acfa,
21
(1968)357-376
GEL FILTRATIOK
OF URINE
PROTEI1;
365
predominated, and their height tended to be similar. Peaks I, 4 and 5 were appreciably smaller, being about one third of the height of Peaks 2 and 3; Peak 5 was usually the tallest of the 3 and Peak 4 often appeared as a shoulder on the side of Peaks 3 or 5. In one exceptional case Peak 4 formed the tallest peak of all and in another the second tallest. Peak I was clearly bifid in 4 cases, as also was Peak 4 in 3 cases. Peak 6 was the smallest of the 6 major fractions, its mean height being about one third of the height of Peak 5 ; it was not discernible in one case. Compared with glomerular proteinurias, tubular proteinurias had a smaller Peak I, a much smaller Peak 2, a much larger Peak 3, obvious Peaks 4 and 5, and in nearly all cases a Peak 6. In addition to protein eluting in these major peaks, 5 of the tubular proteinurias had traces of protein eluting later than Peak 6, and a small residuum of urate was occasionally responsible for an even later peak. In the uncomplicated Bence Jones proteinurias a more variable pattern was seen but in all cases Peak 3 was the tallest fraction and usually a Peak z or a Peak 5 predominated over a small Peak I and/or a small Peak 4, An obvious Peak 5 was present in 3 of the 5 pure Pence Jones proteinurias and an early Peak 6 was found in one. Compared with glomerular and tubular proteinurias the characteristic feature of Bence Jones proteinurias was the great predominance of Peak 3. The contrasting features of the elution patterns in the different types of clinical proteinuria can be readily appreciated from Fig. 5, where they are shown alongside that obtained with normal urine protein. The pattern obtained from the pooled
15IO 05-
A at28Omy
,,,.,*? I T T
IENCE JBNI
Fig. 5. The elution pattern of normal urine protein obtained from a column of Sephadex G-75 compared with that of typical tubular, glomerular and Bence Jones proteinurias. Fig. 6. .4 Sephadex G-75 thin-layer gel filtration chromatogram of urine protein concentrates showing the clear differentiation of the 3 main types of clinical proteinuria and the similarity of the normal urine protein and tubular proteinuria patterns. Clin. Chim.
Acta,
21 (1968) 357-370
DAvIS et al.
366
normal urine was strikingly similar to that of tubular proteinuria, except that there was a larger protein peak eluting at the void volume; characteristically Peak I was the tallest peak and the subsequent ones were progressively less tall. As in some tubular proteinurias, there was a suggestion of protein material eluting later than Peak 6. In all 4 plasma or serum specimens
large peaks corresponding
to Peaks
I
and z
dominated the patterns. In 3 of the sera a Peak 3 was just discernible, and in the serum from one of z patients with chronic cadmium poisoning (Specimen 3) a small but well-defined Peak 5 was detected. Thiwlayev
gel filtration
Examples of the patterns obtained when whole urine protein is subjected to thin-layer gel filtration are shown in Fig. 6, and the findings are those which might be expected from the results of the column gel filtration. In glomerular proteinuria there is a single compact protein zone situated a little behind the Blue Dextran marker. In Rence Jones proteinuria the bulk of the protein migrates slightly less far through the gel, though there may be a smear in front of the main zone. In tubular proteinuria there is a long trail of protein spreading from the position of the glomerular zone in front to more than half-way back towards the origin, well behind the main Bence Jones protein zone. The trail varies in density along its length, being made up of a series of overlapping spots, as can be appreciated from Fig. 7, where the 6 individual fractions obtained from a column separation have been run alongside each otlier on a thin-layer gel. The protein of normal urine behaves in a very similar way to that of tubular protcinuria (Fig. 6); however the trail is somewhat shorter and less obvious. Fig. 8 shows a comparison on a thin-layer gel of the main column fractions obtained from a normal urine and from the bulked low molecular weight fractions I\‘, \’ and VI of a case of tubular proteinuria; fraction IV from the normal urine in this instance
Nelecolar
wt
uffer Flow t
tow
Y
HIGH
Fig. 7. A Sephatlex C;-75 thin-layer gel filtration chromatogram of the h major urine protein fractions separated from a tubular proteinuria by column gel filtration. I;ig. 8. A Scphxlex (i-75 thin-layer gel filtration chromatogram of concentrates of urine protAn fractions separated from normal urine ant1 from tubular proteinuria hy column gel filtration. I~raction 11. of the normal urine protein comprised all protein eluting oft the column after l’eak 3.
GEL FILTRATIOh’
comprised molecular
OF URINE
all protein
PROTEIN
eluting
367
off the column
weight range to fractions
from this simultaneous filtration similar molecular weight range.
after
Peak
3 and it is equivalent
IV, V and VI of tubular
that these two specimens
Electrophoretic analyses of gel filtration fractions When the content of the individual major
fractions
proteinuria.
contain
obtained
protein
of a very
by column
filtration was analysed by two-dimensional electrophoresis the findings, which are illustrated in Figs. 9, IO, II and 12, were as follows:
in
It is clear
gel
some of
Fig. 9. Two-dimensional cellulose acetate/starch gel electrophoretic analyses of concentrates of urine protein fractions separated from a glomerular proteinuria (Specimen No. IO) by column gel filtration using Sephadcx G-75. (a) Fraction I (b) Fraction II (c) Fraction III. Direction of eleotrophoretic separations as in Fis. I. Clin.
Chim.
Acta,
21 (1968) 357-376
I’ig. IO. ‘l‘wo-dimensional cellulose acetate/starch gel electrophoretic analyses of concentrates urine protein fractions separated from a tubular proteinuria (Spccimcn No. 16) hy column filtration using Sephadex (G-75. (a) ITraction 1 (1)) ITraction II (c) Fraction III (tl) ITraction (c) Fraction \: (f) I;raction VI. Direction of electrophoretic separations as in Fig, I.
of gel I\-
Fig. I I. Two-dimensional cellulose acetate/starch gel electrophoretic analyses of concentrates of urine protein fractions separated from a mixed Bence Jones and tubular proteinuria (Specimen NO. 32) by column gel filtration using Sephadex G-75. (a) Fraction I (b) Fraction 11 (c) Fraction III (d) Fraction IV (e) Fraction V) (f) Fraction VI. Direction of electrophoretic separations as in Fig. I.
370
IlAT’IS
et al.
Fmctio?z I. In the glomerular proteinurias this peak showed a large number of the globulins usually seen in serum, particularly 1;-globulin, transferrin and haptoglobins; there was also a little albumin and albumin dimer. In the case of tubular proteinurias
and normal
urine, Peak
I
showed very little protein
on the starch
gel,
Fig. 12. A two-dimensional cellulose acetate/starch gel clcctrophorctic analysis of a conccntratc of normal urine protein Fraction IV separated by column gel filtration, usmg Sephadex (l-75; this fraction comprised all protein cluting off the column after Peak 3. Direction of electrophoretic separations as in Fig. 1,
but y-globulin the absence
was constantly
of the expected
present amount
and there were traces of cc and /?components; of protein
suggested
non-entry
of much of the
protein into the gel. Fvactioe II. In normal urine and in all the cases of clinical proteinuria
with the
exception of some Bence Jones proteinurias, Peak 2 showed well-marked zones attributable to thyroxine-binding pre-albumin, albumin, cr,-acid glycoprotein, a,-antitrypsin and transferrin ; in addition there were small amounts of other a- and Bglobulins and some y-globulin. In tubular proteinuria and normal urine the predom inance of albumin and transferrin over other fractions was less marked than in glomerular proteinuria. In some Bence Jones proteinurias Peak z consisted almost entirely of 2 or 3 Rence Jones components similar to those found on two-dimensional electrophoresis of the whole urine protein. Fmction III. In Peak 3 from normal urine the most prominent zone was a protein of albumin to a,-globulin mobility on cellulose acetate electrophoresis, which had such fast mobility on starch gel that it moved with the buffer front. This protein was also prominent in tubular proteinuria, in which the faster moving y-globulins were also much in evidence. In all cases there was also some albumin, al-acid glycoprotein and c+antitrypsin, presumably due to overlap of fraction 2, and these formed part of a continuous arc of protein spreading from the c(, components to a post-y protein. In all the Kence Jones proteinurias Peak 3 contained 3 prominent Bence Jones components similar to those found on electrophoresis of the whole urine protein; in most cases little else was visible.
GEL FILTRATION
OF URINE PROTEIK
371
Fraction IV. This showed a continuous the prominent
fast-moving
zone noted
series of protein zones on an arc linking
in Peak
3 to the faster-moving
y-globulin. Usually about IO definite zones were discernible, particularly well-defined. If protein &ted beyond the Peak teinuria, the content of the 4th peak was similar to that of normal urine. In Bence Jones proteinurias inevitably two or
end of the
the faster 3 or 4 being 3 in a glomerular protubular proteinuria and three Bence Jones com-
ponents
were dominant. V. This contained the most characteristic a,-globulins of tubular proteinuria, a series of 3 or 4 fast-moving CC,zones being present. When a Peak 6 was
Fraction
present, small amounts of the &globulin In uncomplicated
found in that fraction were usually detected.
Bence Jones proteinurias
Peak 5 contained
components and little else, but in one case of mixed Bence teinuria this peak contained only the a,-gobulins characteristic
prominent
Bence Jones
Jones and tubular proof tubular malfunction.
Fraction VI. This contained small amounts of the same a,-globulin zones seen in Peak 5, together with a large amount of the characteristic &globulin of tubular proteinuria, namely P-microglobulin. Sometimes seen in this fraction was a small amount of another globulin. In normal
/&globulin
moving half as fast in the starch
urine it was not possible
to examine
gel as the fl-micro-
fractions
corresponding
to
Peaks 4, 5 and 6 separately as the points of demarcation between them were too uncertain; when all the protein eluting beyond Peak 3 was bulked and analysed, however, it was possible to recognise most of the protein zones which were seen in the separate
peaks obtained
from tubular
proteinurias.
This is apparent
from Fig.
12.
DISCUSSION
Since Sephadex gel filtration and shape, it was to be anticipated
separates proteins on the basis of molecular size that this technique would differentiate between
the 3 main types of clinical proteinuria, earlier studies having shown that the predominant proteins in each was of different molecular weightly2. In practice, column gel filtration with Sephadex G-75 has achieved a clear-cut distinction between pure glomerular, tubular and Bence Jones proteinurias, and even when mixed types occur it is possible to recognise the characteristic elements of each. Most previous work in this field has been carried out with Sephadex G-100 or G-200, but we believe that G-75 is preferable as it gives a better resolution of those proteins in the lower molecular weight range which are of particular significance. Thin-layer gel filtration, though less precise, is far simpler to perform than column gel filtration and provides the quickest method of demonstrating the presence of low molecular weight urine proteins. It has additional advantages in that less protein concentrate is needed and several specimens can be analysed at the same time with markers run alongside. This technique can therefore be recommended for the routine diagnostic investigation of clinical proteinuria. The two-dimensional cellulose acetate/starch gel electrophoretic findings show conclusively that the gel filtration fractions corresponding to Peaks 5 and 6 contain the most characteristic proteins of tubular proteinuria. Sephadex gel filtration thus segregates the low molecular weight proteins of tubular proteinuria in a way which is not possible with electrophoresis and it makes practical their large-scale collection. Clin. Chim.
Acta,
21 (1968) 357-376
DAVIS el al.
372
The electrophoretic analyses of the gel filtration fractions also demonstrate the conplexity of the protein excretion in different proteinurias, all fractions having been shown to contain several different proteins. The heterogeneity of each fraction may partly major
explain the variation sometimes found in the elution peak; a shift in the value of V,/l’, might reasonably
alteration
of the proportions
Because proteinuria
of the constituent
some of the characteristic
elute later off a Sephadex
volumes of the same be expected with an
proteins.
low molecular
weight
proteins
of tubular
G-75 column than the major proteins
excreted
in the other main types of proteinuria, gel filtration provides a sensitive technique for demonstrating the presence of tubular malfunction in a case of multiple myeloma or of primary glomerular damage. A good example of this is provided by Specimen 31; the patient had a plasma calcium of 14.7 mg/roo ml complicating myelomatosis, and the sizeable Peak 6 can be attributed to a tubular proteinuria being superimposed on a Bence Jones proteinuria, as hypercalcaemia of some duration is known to be capable of causing tubular malfunction. Several of the patients with glomerular proteinuria had small amounts of protein eluting as Peaks 4 and j and one had a detectable Peak 6, but none had been judged from the two-dimensional electrophoretic analysis of the whole urine concentrate to have mixed glomerular and tubular proteinuria; electrophoresis of the individual fractions however revealed the typical tubular
components. Both column and thin-layer
in the molecular
gel filtration
have demonstrated
a great similarity
weight range of the protein of normal urine and of tubular
protein-
uria and the main difference seen in the elution patterns is the presence in normal urine of a relatively larger quantity of protein coming off at the void volume. Most of the protein in this first peak fails to enter a starch gel and is therefore thought to be uromucoid, the macromolecular urinary tract protein described by Tamm and HorsfalllG. The proportion of uromucoid would be expected to be higher in normal urine because of the smaller absolute contribution of protein from other sources. The demonstration by two-dimensional electrophoresis of a marked similarity between the constituent proteins of the low molecular weight fractions of normal urine and tubular proteinuria is of considerable importance. The finding fits in with the concept that tubular proteinuria is largely due to impaired renal tubular reabsorption of protein normally present in the glomerular filtrate. The clear demonstration of a small Peak 5 in the plasma of a patient with tubular proteinuria is of great interest. It provides more direct evidence that the characteristic low molecular weight proteins found in tubular proteinuria may be derived from the plasma. However, it was not possible to detect Peak 5 in the sera of z healthy adults when these were examined in identical fashion. The considerable variation in relative peak height noted in the elution patterns of the different cases of tubular proteinuria, even those with a common aetiology, requires explanation. There may be variation in the rate of production of low molecular weight proteins from patient to patient, or there may be some selectivity in protein reabsorption by the kidney tubules. Alternatively, it is possible that the protein content of the urine is modified by a variable contribution from damaged renal tubule cells, or that complexes are variably formed between basic and acidic proteins, or with mucopolysaccharides. Estimates of the average molecular weight of the proteins constituting the difClin.
Chim.
Acta,
2I
(r9hX) 357-376
GEL FILTRATIOS
OF URINE PROTEIN
373.
ferent peaks can be derived from the experimental data of AndrewsIT. After working out V,/V, ratios for the different proteins that Andrews examined on a column of Sephadex G-75, we were able to produce a plot relating V e/ V Oto log molecular weight, similar to that given by Whitaker 13. Using the linear portion of the line relating the two, we obtained the following estimates for the average molecular weight of the proteins contained in Peaks 3 to 6: Peak 3 Peak 4 early component Peak 4 late component
31000 23500 rg 000
Peak 5 Peak 6 early component Peak 6 late component
‘5 000 11500 9400
The estimates of molecular weight of the proteins contained in the earlier peaks are less certain because their V,jV, values lie in the region where the correlation between the logarithm of molecular weight and elution value ceases to be linear; however, all the proteins in Peak I can be assumed to have molecular weights greater than 80000 and probably the average molecular weight of the protein in the early and late components of Peak 2 are 53 ooo and 44000 respectively. All these estimates of molecular weight must be considered at best to have an accuracy of ~IoD;’ because of the experimental error in measuring elution volumes, and because WhitakeF and Andrew@ have shown that anomalous results may be obtained with glycoproteins containing more than 5% of carbollydrate, with proteins of non-spherical shape or unusual density, or with proteins which form a complex with the gel or are liable to dissociate into subunjts. Comparison of our results with those of other workers is difficult because most reports in the literature describe the use of G-ZOO or G-100 Sephadex. Hardwickels, and Maclean and PetrieZo studied the urine of patients with heavy proteinuria for the purpose of investigating the selectivity of the protein clearance. Using columns of Sephadex G-zoo they obtained 3 peaks, but the first two represented fractions of high molecular weight globulins and would be equivalent to the z components in our Peak I, and the third, containing albumin and transferrin, would be equivalent to our Peak z; with this gel, however, one would not expect to achieve a separation of small quantities of protein of around 30000 molecular weight in the presence of a large amount of albumin and cc,-antitrypsin. Gel filtration has been used by a number of workers to study Bence Jones that it was possible to obtain some proteinuria. Snapper and Tillema 21 demonstrated separation of albumin and Bence Jones protein by passing an appropriate mixture of urine proteins through a column of Sephadex G-75, but others have carried out more detailed studies. Bernier and PutnamZz investigated 4 Bence Jones proteinurias using G-100 Sephadex and obtained multiple components which they attributed to variable poly~nerization of monomer Bence Jones molecules, Bence Jones variants and other proteins such as albumin, transferrin, y-globulin and low molecular weight glycoproteins. In the course of studying renal clearances of protein in II patients with myeloma and one with macroglobulinaemia, all of whom were excreting ‘abnormal’ light chain protein, Harrison et nLZ3 investigated the behaviour of the urine proteins by gel filtration using G-200 and G-100 Sephadex. They found that with one exception the Bence Jones proteins fell into z main groups and the K,, values they obtained suggest that they usually behaved as proteins with a molecular weight of around 53000 and 32000 ; these would correspond to proteins present in our Peaks 2 and 3. They did not regularly find minor components of lower molecular weight but in the Clin. Chim. Acta, 21 (1968) 357-376
DAVIS et al.
374 2
cases in whom a mixed tubular
and Bence Jones proteinuria
was detected
on paper
electrophoresis they did find appreciable quantities of protein of smaller molecular size. The elution behaviour of the Bence Jones components illustrated in the paper of Bernier
and Putnam22 appears to be as much at variance
the molecular weights of different Bence Jones components
with current
estimates
of
as that found by Harrison
ct (~1.~~ and by us. The major components in the Bence Jones proteinurias eluted as though the molecules had molecular weights of around 53000
we examined and/or 31 ooo
and/or rgooo. The dimer and monomer molecules are believed, however, to have molecular weights of 44000 and zzooo respectivelyz2. This anomalous elution behaviour of Bence Jones proteins cannot be attributed to the molecules being of nonspherical shape2 or containing carbohydrate a4. Drawing an analogy with the explanation advanced filtration,
by Andrews *? to account
we suggest
that
the elution
for the
of Bence
behaviour Jones
of haemoglobin
proteins
as though
on gel they had
molecular weights of around 53000 or 31000 may be due to dissociation of tetramer or dimer molecules occurring while the protein is passing through the column. The Wence Jones components eluting as a protein of around 15 ooo molecular weight may represent a 1.2 S fragment of the monomer molecule such as has been recognised b! Solomon et aLz5 and Van Eyk and Myszkowska WJ. , the fact that we find more of this component than others may be due to the greater reliability of our ultrafiltration procedure to prevent all loss of such molecules. Traeger
et &.a7 using G-ZOO Sephadex
to fractionate
urine proteins,
noted that
in tubular proteinuria a peak eluting later than the peak which mainly contains albumin is characteristically obtained. Piscatore studied the serum and urine proteins of an unspecified number of healthy individuals and workers exposed to cadmium compounds by gel filtration using G-100 Sephadex; he also performed gel filtration using G-75 Sephadex on fractions separated by ion exchange chromatography. In all cases the fractionation obtained appears similar to ours. Harrison and Northam’” also investigated
the excretion
of low molecular
weight proteins by normal adult men and
by patients with evidence of renal tubular damage, using Sephadex G-loo. In most cases they used a thin-layer technique, but they studied normal urine protein and the protein excreted by two patients with evidence of renal tubular malfunction by column gel filtration. They also studied the protein excretion by a hybrid thin-layer gel filtration and electrophoretic technique, and showed that the low molecular weight protein pattern of normal urine differed slightly from that associated with renal tubular disease. Their gel filtration findings are very similar to ours but like Piscator they have poorer resolution in the low molecular weight range and do not seem to have encountered an equivalent to our Peak 4. They suggested that the 3 low molecular weight peaks they found in 2 tubular proteinurias represented proteins of 44000, 16000 and IIOOO molecular weight. The last two would correspond reasonably well therefore with our Peaks 5 and 6. Their finding of increased quantities of low molecular weight protein in the urine of all patients with creatinine clearances of less than 20 ml/min fits well with our finding of small Peaks 4 and 5 in patients with glomerular proteinuria and elevated plasma urea Ievels. Walravens et a1.29*3@have studied normal urine protein and the tubular proteinuria of 15 patients with renal transplants and a case of the Fanconi syndrome by gel filtration. They used columns of Sephadex G-zoo but re-filtered portions of the eluate subsequently through G-75 columns. They also obtained only 5 peaks of protein, but Clin. Chim. Acta, :!I
(1908) 357-376
GEL FILTRATION
OF URIXE
like us they found some differences and a smaller
proportion
375
PROTEIN
from one case of tubular
of low molecular
weight
proteins
proteinuria in normal
to another urine.
Their
detailed immunoelectrophoretic analyses of the gel filtration fractions are not easily related to our findings by two-dimensional electrophoresis, but their finding of low molecular weight q-proteins and fi-microglobulin in the last peak correlates exactly with our findings in tubular proteinuria. The demonstration by immunoelectrophoresis of such proteins as /-microglobulin and post-l) protein in a low molecular weight gel filtration fraction of normal serum also correlates with our finding of a Peak 5 in a specimen of plasma. The theoretical implications of these findings in relation to the mechanism
of production
of tubular
proteinuria
will be discussed
elsewhere.
ACKSOWLEDGMEh-TS
This work was supported
by a grant from the Medical Research
Council.
We
also wish to thank the physicians at University College Hospital, particularly Professor C. E. Dent, for providing us with many of the specimens and Drs. P.W. Hall, J. R. Hobbs and G. Kazantzis for sending us others. We also wish to thank Mr. Y. K. Asta for the diagrams and Mr. A. C. Lees and his staff for the photographs.
I J. 11. CREETH, R. A. I
>I.D. POuLIK, NatUW,180 (1957) 1477. 1;.v. FLY??& AND E. A. STOW, J. C~?lz. Path&., II (1958) 334. E. A. BUTLER AND F. v. FLYNN. I.Clilz. Pathol.. 14 (1961) 172 E. .-\. BUTLER, F. V. FLYNN, H. HARRIS AND E. B. iio&oN, blin. Clzznz. 4cfa, 7 (1962) 34. 12 P. FLO~IN, Upx(ran (Xs and their Applicatiors in Gel Filtvation, A. H. Pharmacia, l’ppsala, 8 9 IO II
1962. 13 J, R. \VHITAKER, <4nal.Chem.,
35 (1963) 1950. B, JOHANSSON AND L. RYMO, .4cta Chrm. .Sca?zd., 18 (1964) 217. C. J. 0. R. RIORRIS,,I.Chromafog., 16 (1964) 167. I.TAX&I AND F. L. HORSFALL, J. Expfl. Med., 95 (1952) 71. I?.ANDRI?WS, Biochpm. J., 91 (1964) 222. P. ANDREWS, B~OC/ZPI~Z. J.. 96 (1965) 595. J, HARDWICKE, Clin. Chim. Acta, IL (1965) 89. 20 P. I<. M.~cLE.~N .~ND 1. 1. B. PETRIE. Clan. Chzm. Acta. Ed (1966) 167. 2I 1. SNAPPER AKD A. vi~"O. TILLEMA, Am. J. Med., 38 (19b;)‘40;.’
I4 I5 IO 17 18 I9
2~ G. hf. BERNIER AND F. \I’. P1’TNAM, B&him. Riophys. Acta, 86 (1964) 295. 23 J. F. HARRISON, J. D. BLAIP~EY, J. HARDWICKB, D. S. ROWF. AND J. F. SOOTHILL,CZ~%. SC%., .3I(1966)95. J. R. CLA~IP, G. RI.BERNIER AND F. W. PUTNAM, Biochim.Biophys. Acta, 86 (1964) 149. A. SOLOMOK, J. KILLANDER, H. &I.GREY AND H. G. KUNKEL, S&we, r51 (1966) 1237. H. G. VAN EYK AND K. MYSZKOWSKA, Clin.Chinz.Acta, 18 (1967) IOI. J. TRAEGER, R. FRAN~OIS, R. CREYSSEL, J.-P.REVILLARD, Y. MANUEL, M. T. FREYCON ANU W. SITE, Pathol. Biol. Semazm Hot)., 14 (1966) 5, 28 J. F. HARRISON ASD B. E. NORT&I, ?li+zy ChLm. Acta. 14 (1966) 679. 24 25 26 27
Clilz. Chim.
Acta,
21 (1968)
357-376
r)Aws
376 29 I'H.WALRAVENS,
E. C. LATERRE,
335. 30 PH. WALRAVEKS, JZ.C. LATERRC 31 C. E. DENT, ,I.Bonp Joint Swg., Clin. Chiwz. Acfa,
z I (1968)
357-37f1
et al.
A. EST.&S AND J. 1;.~~EREMAIUS, c&la. Chiun. .dcfa, 18 (1967) AND J. I'.HBRERIINS, QPJ (1952) 266.
Clix. Chim.
ilcfa,
19 (1968)
107.
CLINICA CHIMICA ACTA
THE CHARACTERISATION
JENNIFER
S. DAVIS,
OF URINE PROTEIN
F. V. FLYNN
BY GEL FILTRATION
AND H. S. PLATl
Department of CfinicaE PathoEog?i, University College HospifaE, Londolz, W.C. I (U.K.] (Received
April 5. x968)
SUMMARY I. Urine protein from healthy adults and 29 patients with clinical proteinuria has been studied by gel filtration using Sephadex G-75, The resolution obtained of the lower molecular weight components was superior to that reported using G-100 and G-200 Sephadex. Six major fractions were recognised. 2. The proteinurias associated with glomerular and tubular malfunction and with “abnormal” light-chain excretion have been further characterised by this technique. 3. A simple thin-layer gel filtration technique is advocated for the distinction of tubular proteinuria from glomerular and Bence Jones types. 4. A close similarity between the proteins present in normal urine and in tubular proteinuria has been demonstrated. 5. Small quantities of low molecular weight protein similar to that characteristically found in tubular proteinuria have been demonstrated in the plasma of a patient with chronic cadmium poisoning by column gel filtration using Sephadex G-75. 6. The anomalous elution behaviour of Bence Jones proteins is discussed.
Previous work has made it increasingly clear that the predominant proteins in the different types of clinical proteinuria are of different molecular weight. The slight proteinuria which may accompany renal tubular disorders has been shown by Creeth et a1.l to have a high proportion of low molecular weight components and to differ in this respect from the gross proteinuria known to be due to the passage of increased amounts of serum protein through the glomerulus. The average molecular size of the light chain components found in Bence Jones proteinuria has similarly been shown to be smaller than most of the serum proteins, the median molecular weight being 44000 (ref. 2). It was a logical step therefore to employ Sephadex gel filtration in the further study of proteinuria as this separates proteins according to their molecular size. Other workers have examined urine proteins by gel filtration, but none have particularly set about comparing the fractionation obtained in the different types of proteinuria. The urine proteins excreted in health and in a variety of conditions associated with clinical proteinuria have been studied by gel filtration using Sephadex G-75 and C&in.Chim. Acta, 21 (1968)
357-376
“;:
2
a % P
I
SUBJECTS
32 25 5’ 66
M F M M
46 42 5’ 54
53 48
55
16 ‘7
18
--
RESULTS
syndrome
Chronic cadmium poisoning Chronic cadmium poisoning
M M
ICI Adult Fanconi (hereditary)
Balkan Balkan Chronic Chronic
M E M M
nephropathy nepbropathy cadmium poisoning cadmium poisoning
Nephrotic nephritis Nephrotic nephritis Nephrotic nephritis Nephrotic nephritis Amyloidosis Chronic nephritis
Z‘ltbulav fwoteinurias
12 13 ‘4 15
AND
Healthy adult Healthy adult Chronic cadmium poisoning Chronic cadmium poisoning
GEomeruLar proteinurias
I 2 3 4
.~
STUDIED
Sex Diagnosis
Izi
_ Plasma OYserum
AVO.
Spot. Age
...~__
a
DI.IIGSOSIS
5
TABLE
0 nl s (5
-_.
-___-
150 m,s PROTEIN
USING
- ..-_-_
___
A COLUMN
~
OF
0.33 0.50
0.38 0.25
0.22
0.29
0.37
0.2x*
0.61 I .03 0.37*
1.13%
1.or
2.29
r.26*
1.85 1.87
1.24 B2.00 0.87* 1.04 I.59 r.26* 1.17
>2.00
.>2.00
>2.00
~2.00
>2.00
0.74%
>2.00
>2.00
>z.ao
0.76 1.84 0.77 0.53 I.23
>2.00
>2.00
0.42
I.90
>2.00
1.60 1.68
>2.00
1.q
1.75 1.60
>2.00
1.62
0.43
1.00*
0.31
0.31* 0.40
0.27
0.42’
0.4s*
1.21
0.38 0.24’
0.64 0.29
0.97
0.43
0.44 0.77
0.45 0.47
0.26
0.08
trace 0.06* trace 0. IO” 0.07
0.17
0.07*
0.27
0.62*
o.‘p*
0.12
0.25
trace
0.10 trace
o.og*
0.87
trace* 0.05*
trace*
!Waxirnwn A reading al 280 nzp _.~_^__~~_ .Peak 3 Peak 3 Peak I Peak 2 Peaks --_____V,l fJ* I,‘,/ v0 Vel v, V,l v, l’el v/D vt?i v, [‘f?i v, E”e/v* I.621~75r.grI.00 r.ogX.17IA?& r.40I.60 I.72 I.90 2.04 1.09 I.27 I,34 ---l____l_-
OF FRACTIONXTINC
trace
0.20
0.17
0.16
0. I I
0.27
trace
0.10*
urea urea urea urea urea urea
protein l’eak 6 Some
Some protein Peak 6
Plasma Plasma Plasma Plasma Plasma Plasma
..I_-___-.
Coinnzcnis
G 75
V,l v, V,l v, Z.II2.232.34 2.17
Peak 6
SEPHADEX
eluted after
eluted after
ml ml ml ml ml ml
___._
-__
31 mg/roo 26 mg/roo 45 mg/roo 57 mg/roo 36 mg/roo 125 q/I00
._-
7
5 D
Multiple myeloma Multiple myeloma ~lacro~lol~ulinae~~~i~~
I’rimar-y hyperparathyroidism. nephrocalcinosis and renal calculi ILlkan ~qhrupatl~q-
my&ma
my&ma
Hnlkan
Multiple
Multiple
Alultiplc
I; F F
.\I
1:
11
F
I;
hI
11 Multiple
80 00
62
40
61
trace
= ‘-1 reading
53
5o
40
25 26 27
nt
less than 0.05
my&ma
my&ma
nephropathy
myeloma myeloma
Multiple Multiple
SI
56
67
23 24
Oculo-cerchro-renal
M
I8
22
3
on side ot adlaccnt
0.22
0.30
o.iO
0.71
o.j+*
0.20*
1.55
0.84*
1.59
cl..+0
0.18
o.11*
1.61
I.78
1.83
0.09
2.00
I.41
o.39*
1.72
0.99*
0.02
trace*
tract*
I.34
0.17
0.85
0.37’
0.45
peak.
0.83 I .05 I.15
1.64
>z.oo
>2.00
+ =
0.79%
0.62* 0.34
0.26
0.70* 0.23
>2.00
0.39 >2.00 I.42 >2.00 0.76*
0.28
>2.00
0.83
0.93
1.08
0.32* o.rR
1.01
>2.00
1.14* I.33
1.68
0.23
0.46
0.40
0.77
0.48
0.05 trace
tmcc*
0.34
0.17
=- shoulder
syndrome
syndrome
Oculo-ccrcbro-renal
14
21
Al
Adult
M
IO
20
F
Fanconi syndrome (hereditary) Fanconi syndrome (type 3 +)
56
19
0.07*
o.or*
0.43
0.41
0.34
0.05*
0.35 0.07
trace
0.45
of Dent”‘.
o.r5*
0.08*
0.26
0.14
0.70
O.jT
0.16
0.16
0.66 0.35
0.62
I.33
Classification
0.92
0.20*
0.36
0.24*
0.43
o.r(i*
0.49
0.79”
eluted
eluted
eluted
after
after
after
Mixed ,olomcrular and tubular proteinuria l’lasmacalcium I*.I mg/rooml Mixed $xnerulnr and tubular protcinuria Mixed glornerular and tubular proteinuria Mixed Hence Jones and tubular protcinuria Plasmacalcium 14.7mg/1ooml Mixed Bcnce Jones and tubular proteinuria Plasrnacalciu~n17.0mg/1ooml Mixed Rence Jones and glomcrulnr proteinuria Mixctl l
Some protein Peak 6 Some protein Peak ti Some protein Prak h
E Q
DAVIS et ai.
360
we report here the findings using both column and thin-layer techniques. Gel filtration has been found to differentiate clearly between the main types of proteinuria, and the simple thin-layer technique is advocated for routine use. Sephadex fractionation has also demonstrated a general similarity between the protein of normal urine and that of tubular proteinuria. 4 preliminary account of this work was given at the VIth International Congress of Clinical Chemistry3. MATERIALANDMETHODS Urine specifnens
Twenty-four-hour collections of urine were obtained from 29 selected patients with clinical proteinuria, the definitive diagnosis in each being shown in Table I. In addition, 15 1 of pooled normal urine was collected from 18 ad&t males aged 20-45 years, each of whom had been shown to be free of clinical proteinuria. The urine samples were collected into vessels containing I g sodium azide and more was added later to give a final concentration of I g/l. The specimens were stored at 4’ while awaiting processing. In some cases the urine was filtered through Oxoid cellulose acetate membranes, but debris was usually removed by centrifugation.
The total protein in the original urine samples was determined by a turbidometric method employing 5% sulphosalicylic acid and a nephelometer. The concentration of protein in the samples which were used for gel filtration was measured by a biuret method4. Preparation
of urine protein
concentrates
The urine protein was concentrated by ultrafiltration, using autoclaved a-inch Visking cellulose tubing without external support and a negative pressure of -400 mm Hg. Prior to use the Visking tubing was autoclaved for zo min at 15 lb/sq.in. to decrease the pore size and prevent the loss of low molecular weight protein which has been reported in studies by Berg&d5 and Piscatore. Under the conditions used for the concentration process, when cytochrome c (molecular weight 12270) was added to the urine it could not be detected in the ultrafiltrate. In most cases the urine protein was concentrated to a level well above 6 g/Ioo ml and after 48 h dialysis against IOO volumes of a phosphate-sodium chloride solution pH 7.3 (see below), was adjusted to exactly 6 g/roe ml by dilution with the same buffer.
Bloods$ecimerzs Venous blood specimens were collected from 2 healthy adults and from 2 patients with chronic cadmium poisoning; the patients’ blood was collected into heparin but serum was separated from the specimens taken from the healthy adults.
Two-dilnensional electrophoresis was carried out on the urine protein concentrates after the method of Poulik and Smithies7, using cellulose acetate and barbiturate buffer pH 8.6 for the first separation, and starch gel and the tris-citrate/borate discontinuous buffer system of Pouliks for the second. This procedure was used to Clin.
Chim.
Ada,
21
(1968) 357-376
GELPILTRATIOX
OF URINE
PROTEIN
36r
classify the proteinurias into glomerular, tubular, Bence Jones and mixed types, on the basis of earlier work by Flynn and Stowv, Butler and Flynnl” and Butler et d.ll It was also used to determine the content of the fractions obtained by column gel filtration.
Glass columns (Column I: 80 cm longx 4.0 cm in diameter. Column 2: roe cm Iongx 2.5 cm in diameter) were filled to within 4-7 cm of the top with the bead form of Sephadex G-75 cross-linked dextran gel (lot No. To 5x30), using the procedure described by Flodinlz. The fines were decanted 10 times and the gel particles allowed to swell for 3 days before packing The columns were packed and re-packed until a Blue I?extran solution was seen to migrate evenly through the gel. Phosphate-sodium chloride buffer pH 7.3 (0.035 M NazHP04, 0.006 M NaH,PO,, 0.15 X NaCl) containing I gjl sodium azide as preservative was used for both packing and eluting the columns. A 2.5~ml aliquot of urine concentrate, containing ISO mg protein, was applied to the top of the column by layering under the supernatant buffer, and elution was carried out at a flow rate of approximately 12 ml/h. The whole procedure was carried out at room temperature (rg-.z5”). Using syphons of 3.0 or 3.6 ml capacity in conjunction with a fraction collector, aliquots were collected continuously until elution of the protein was complete. An indication of protein concentration in the individual fractions was obtained by me~uring their optical density at 280 mp in a Unicam S.P, 500 spectrop~otometer, using a x-cm silica cuvette. The void volumes of the columns were determined repeatedly by noting the elution volume at which the maximum optical density reading at 620 m/t was obtained following the application of a Blue Dextran solution to the top of the column. A solution of 0.2 g Blue Dextran in IOO ml elution buffer was used for this purpose. To express the elution behaviour of the proteins in a manner that is independent of column size the elution volume of each peak (V,) was divided by the current void volume of the column (V,) as advocated by Whitaker’“.
The technique of Johansson and Rymo14, and MorrisIS, was adopted with some modifications. Sixty grams of Superfine Sephadex G-75 was allowed to swell for 48 11 while continuously stirred in I 1 of a phosphate-sodium chloride buffer pH 7.2 jo.mci M Na,IIPO,, o.ao4 M ICH,l?O,, 0.2 *Q NaCl) containing I g/l sodium azide. Sufficient supernatant fluid was subsequently removed to give a slurry, the ideal consistency of which was found by trial and error. An excess of the slurry, i.e. 15 ml, was used to make a film approximately 0.5~mm thick on a thoroughly cleaned glass plate measuring IOX 16 cm, an even film being obtained by running a glass rod swiftly and smoothly along metal plates mounted on either side of the glass plate. The coated plates were placed horizontally within a closed tank to form a bridge between reservoirs containing the same buffer, and wicks of Whatman No. 3 MN filter paper, ro-cm wide, were applied over the last cm at each end of the gel and dipped into the buffer. The difference in level of the buffer in the two reservoirs was adjusted so that a flow rate of about z cm/h was obtained. Blue Dextran was used as a marker during these adjustments and usually a difference in height of about 1.5 cm was required between the buffer levels. After allowing the thin-layer plate to equiliClin. Chinz. ActC6,21 (1968) 357-375
DAVIS
362
al.
et
brate overnight, protein was applied to the surface of the gel on discs of Whatman No. 52 filter paper, 1.5 mm in diameter, cut with a paper tape punch. These discs were dipped in the protein or Blue Dextran solutions and blotted lightly before being laid gently on the surface of the gel at intervals of 1.5 cm along a line 2 cm from the top end of the plate. The buffer was allowed to continue flowing until a Blue Dcxtran marker had moved 10-12 cm, which usually required 5-O 11. The plate was then removed from the tank and totally covered by a sheet of Whatman No. 3 MRI filter paper which was rolled on in such a way as to avoid forming ridges or trapping air bubbles. The protein was taken up into the paper and fixed there by drying the covered plate in an oven at 80” for 30 min. Subsequently the protein was stained on the filter paper by immersion in 0.15?; Ponceau S in 37; trichloracetic acid for 30 min, excess dye being removed
by subsequent
washing in 2”,4 (v/v) acetic acid.
Exarvtinatiou of the protein fractions obtained 631column gel filtration When the optical density at 280 nip of each sample collected gel filtration
procedure
was plotted graphically,
from the column
it became possible to recognise major
peaks of eluted protein. Divisions were made at the points where undoubted troughs occurred and the samples constituting each main fraction were bulked. The fractions
Glomerular Protemunas TWO- dimenstonal Electrophoresls of Urme Proteins Glomerular
Proteinurla
Tubular
Elutlon vol ml
100
Proteinurla
I
VOID
VOLUME
2ooy
1.8
300
400
I
500
1
600
I
700
I
NEPHRlilS flypeni
1.5-
Bence
Jones
A at 28Oml
NORMALURINE
Proteinurla
lo0,5-
n
. Alb RENALAMt'LOIOOSIS
l’ig. 1. IXagrams showing two-dimensional electrophoretic separations of urine proteins in the different types of proteinuria. Electrophoresis was carried out on cellulose acetate from right to left and subsequently in starch gel from below upwards. The line of insertion of the ccllulosc acetate strip into the gel is indicated by the horizontal dotted line, and the relative concentration of the protein fractions by the density of shading. The whole of the arca cncloscd by the dotted line in the diagram of the normal urine pattern stained slightly darker than the background. Alb : albumin, ‘l‘r = transfcrrin. I?g. 2. Elution patterns glomerulsr proteinuria.
of urine protein
obtained
from
a column
of Sephadex
(i-75 in .3 cases of
GEL FILTRATION
OF URINE PROTEIK
363
so obtained were reconcentrated by ultrafiltration sional electrophoresis and thin-layer gel filtration.
and examined
by both two-dimen-
RESULTS
T-zmdiwaensional
electrophoretic analyses of urine protein concentrates Typical examples of two-dimensional patterns obtained in the different types of clinical proteinuria and in normal urine are illustrated in Fig. I. In glomerular proteinurias there were prominent albumin, a,-antitrypsin and transferrin zones with traces of other serum components including y-globulin. Tubular proteinurias were more complex and a series of protein zones unlike any seen in serum occupied various positions along an arc roughly parallel to, and on the anodic side of, a curve linking the albumin, cqantitrypsin and transferrin zones. They varied widely in relative amount from case to case, but three of them were particularly prominent and were found in nearly all cases. Bence Jones proteinurias characteristically showed a prominent group of parallel zones in close proximity; usually there were 3 and that nearest the cathode was the most conspicuous. These were situated in positions corresponding to somewhere between the p- and y-globulin regions in the first-dimensional separation and anywhere between the post-albumin and y-globulin positions in the starch gel separation. Concentrates of normal urine protein revealed albumin, traces of prealbumin, y-globulin and several tlr- and a,-globulins. Bence JonesProtemwas vc&
LUME 300 LOO
I
I
I
500
600
700
MULTIPLEMYELOMA
MULTIPLEMYELOMA
J' MULTIPLFMYELOMA + ren~lunpsirment
1.5 1.0 0.5 :I.
Tube No.
Fig. 3. tubular
Elution patterns proteinuria.
150
\
of urine protein
200 obtained
Fig. 4. Elution patterns of urine protein obtained multiple myeloma; upper 2 from uncomplicated mixed Bcnce Jones and glomerular proteinuria.
1
Tube No. from
,
50
a column of Sephadex
G-75 in 3 cases of
from a column of Sephadex G-75 in 3 cases of Bcnce Jones proteinurias, lowest one from a
CZi?z. Chim. Acta,
LI (1968)
357-376
DAVIS et al.
364 Colawan gd filtration
Examples of elution patterns obtained from Column I in the different types of proteinuria are shown in Figs. 2,~ and 4. As can be seen from these figures a variable number of major protein peaks was obtained, up to a maximum of 6, and these peaks eluted at very similar buffer volumes. The volume of elution of the Blue Dextran, i.e. the void volume of the columns (V,), varied slightly over the period of the experiments. The 6 major peaks that could be distinguished eluted from the columns as follows : Peak I: The elution volume of this peak was at or near the void volume and consequentlythevalueof V,,WO was at or close to 1.00. In some cases the peak eluted exactly at the void volume and in others it emerged a few ml later, the average V,/V, then being 1.06 (range 1.03 to 1.09).In 6 of the urine specimens the peak was clearly bifid with the highest points corresponding to the z usually seen separately; in 6 others there was a shoulder on one side of the peak indicating the presence of 2 components. Peak 2: In some cases the V,/V, value for this peak was close to 1.22 (range 1.17 to 1.27) and in others near to 1.31 (range 1.28 to r.34), and in 4 instances the peak was clearly bifid due to the presence of both components. Bind second peaks were onlv clearly seen when the specimens were fractionated on Column 2, the longer narrower column, but asymmetric peaks with definite shoulders, indicating the presence of z components, were sometimes found to elute from Column T. Peak 3: The average V,jV, value for this peak was 1.50. The range of values for V,,/V, when Bence Jones proteinurias were excluded was 1.45 to 1.56. The peak only appeared complex in the case of some Bence Jones proteinurias when I or z additional components produced subsidiary peaks or shoulders; the range of values for V,jV,, when these were included was x.40 to x.60. Peak 4 : This was not always seen as a real peak. Often it was overshadowed by taller adjacent peaks and appeared as a shoulder on the side of Peak 3, or occasionally Peak 5, and in these instances it was difficult to be precise about its elution volume. In most cases the V,jV, value for the peak fell within the range 1.75 to 1.90, the mean then being 1.81, but in some it eluted earlier, the mean V,/V, value then being 1.68 (range 1.62 to 1.72). In 6 specimens both components were present, and in 3 of these both were well defined. Peak 3: The average r/,/V, value for this peak was 1.99 (range 1.91 to 2.04). Peuk 6: In most cases this eluted with an average V,jV, value of 2.29 (range 2.23 to 2.34). In 3 instances of Bence- Jones proteinuria this peak emerged earlier, V,/V, then being 2.16, and in a further similar case this early component appeared together with the more usual Peak 6. The findings in all the specimens are summarized in Table I, which shows the maximum optical density readings at 280 m,u for the major discernible peaks. In all 6 glomerular proteinurias there was a very large early Peak 2, a sizeable Peak I and a small Peak 3. In addition a tiny Peak 4 was found in 5 of the cases and a minute Peak 5 was discerned in 3. In one patient with chronic nephritis, with a plasma urea of 125 mg/roo ml, a trace of Peak 6 was recognisable. In all but one of the 11 cases of tubular proteinuria, obvious protein fractions corresponding to all 6 major peaks were found. In 9 of the IX cases Peaks 2 and 3 Clin. Chim.
Acfa,
21
(1968)357-376
GEL FILTRATIOK
OF URINE
PROTEI1;
365
predominated, and their height tended to be similar. Peaks I, 4 and 5 were appreciably smaller, being about one third of the height of Peaks 2 and 3; Peak 5 was usually the tallest of the 3 and Peak 4 often appeared as a shoulder on the side of Peaks 3 or 5. In one exceptional case Peak 4 formed the tallest peak of all and in another the second tallest. Peak I was clearly bifid in 4 cases, as also was Peak 4 in 3 cases. Peak 6 was the smallest of the 6 major fractions, its mean height being about one third of the height of Peak 5 ; it was not discernible in one case. Compared with glomerular proteinurias, tubular proteinurias had a smaller Peak I, a much smaller Peak 2, a much larger Peak 3, obvious Peaks 4 and 5, and in nearly all cases a Peak 6. In addition to protein eluting in these major peaks, 5 of the tubular proteinurias had traces of protein eluting later than Peak 6, and a small residuum of urate was occasionally responsible for an even later peak. In the uncomplicated Bence Jones proteinurias a more variable pattern was seen but in all cases Peak 3 was the tallest fraction and usually a Peak z or a Peak 5 predominated over a small Peak I and/or a small Peak 4, An obvious Peak 5 was present in 3 of the 5 pure Pence Jones proteinurias and an early Peak 6 was found in one. Compared with glomerular and tubular proteinurias the characteristic feature of Bence Jones proteinurias was the great predominance of Peak 3. The contrasting features of the elution patterns in the different types of clinical proteinuria can be readily appreciated from Fig. 5, where they are shown alongside that obtained with normal urine protein. The pattern obtained from the pooled
15IO 05-
A at28Omy
,,,.,*? I T T
IENCE JBNI
Fig. 5. The elution pattern of normal urine protein obtained from a column of Sephadex G-75 compared with that of typical tubular, glomerular and Bence Jones proteinurias. Fig. 6. .4 Sephadex G-75 thin-layer gel filtration chromatogram of urine protein concentrates showing the clear differentiation of the 3 main types of clinical proteinuria and the similarity of the normal urine protein and tubular proteinuria patterns. Clin. Chim.
Acta,
21 (1968) 357-370
DAvIS et al.
366
normal urine was strikingly similar to that of tubular proteinuria, except that there was a larger protein peak eluting at the void volume; characteristically Peak I was the tallest peak and the subsequent ones were progressively less tall. As in some tubular proteinurias, there was a suggestion of protein material eluting later than Peak 6. In all 4 plasma or serum specimens
large peaks corresponding
to Peaks
I
and z
dominated the patterns. In 3 of the sera a Peak 3 was just discernible, and in the serum from one of z patients with chronic cadmium poisoning (Specimen 3) a small but well-defined Peak 5 was detected. Thiwlayev
gel filtration
Examples of the patterns obtained when whole urine protein is subjected to thin-layer gel filtration are shown in Fig. 6, and the findings are those which might be expected from the results of the column gel filtration. In glomerular proteinuria there is a single compact protein zone situated a little behind the Blue Dextran marker. In Rence Jones proteinuria the bulk of the protein migrates slightly less far through the gel, though there may be a smear in front of the main zone. In tubular proteinuria there is a long trail of protein spreading from the position of the glomerular zone in front to more than half-way back towards the origin, well behind the main Bence Jones protein zone. The trail varies in density along its length, being made up of a series of overlapping spots, as can be appreciated from Fig. 7, where the 6 individual fractions obtained from a column separation have been run alongside each otlier on a thin-layer gel. The protein of normal urine behaves in a very similar way to that of tubular protcinuria (Fig. 6); however the trail is somewhat shorter and less obvious. Fig. 8 shows a comparison on a thin-layer gel of the main column fractions obtained from a normal urine and from the bulked low molecular weight fractions I\‘, \’ and VI of a case of tubular proteinuria; fraction IV from the normal urine in this instance
Nelecolar
wt
uffer Flow t
tow
Y
HIGH
Fig. 7. A Sephatlex C;-75 thin-layer gel filtration chromatogram of the h major urine protein fractions separated from a tubular proteinuria by column gel filtration. I;ig. 8. A Scphxlex (i-75 thin-layer gel filtration chromatogram of concentrates of urine protAn fractions separated from normal urine ant1 from tubular proteinuria hy column gel filtration. I~raction 11. of the normal urine protein comprised all protein eluting oft the column after l’eak 3.
GEL FILTRATIOh’
comprised molecular
OF URINE
all protein
PROTEIN
eluting
367
off the column
weight range to fractions
from this simultaneous filtration similar molecular weight range.
after
Peak
3 and it is equivalent
IV, V and VI of tubular
that these two specimens
Electrophoretic analyses of gel filtration fractions When the content of the individual major
fractions
proteinuria.
contain
obtained
protein
of a very
by column
filtration was analysed by two-dimensional electrophoresis the findings, which are illustrated in Figs. 9, IO, II and 12, were as follows:
in
It is clear
gel
some of
Fig. 9. Two-dimensional cellulose acetate/starch gel electrophoretic analyses of concentrates of urine protein fractions separated from a glomerular proteinuria (Specimen No. IO) by column gel filtration using Sephadcx G-75. (a) Fraction I (b) Fraction II (c) Fraction III. Direction of eleotrophoretic separations as in Fis. I. Clin.
Chim.
Acta,
21 (1968) 357-376
I’ig. IO. ‘l‘wo-dimensional cellulose acetate/starch gel electrophoretic analyses of concentrates urine protein fractions separated from a tubular proteinuria (Spccimcn No. 16) hy column filtration using Sephadex (G-75. (a) ITraction 1 (1)) ITraction II (c) Fraction III (tl) ITraction (c) Fraction \: (f) I;raction VI. Direction of electrophoretic separations as in Fig, I.
of gel I\-
Fig. I I. Two-dimensional cellulose acetate/starch gel electrophoretic analyses of concentrates of urine protein fractions separated from a mixed Bence Jones and tubular proteinuria (Specimen NO. 32) by column gel filtration using Sephadex G-75. (a) Fraction I (b) Fraction 11 (c) Fraction III (d) Fraction IV (e) Fraction V) (f) Fraction VI. Direction of electrophoretic separations as in Fig. I.
370
IlAT’IS
et al.
Fmctio?z I. In the glomerular proteinurias this peak showed a large number of the globulins usually seen in serum, particularly 1;-globulin, transferrin and haptoglobins; there was also a little albumin and albumin dimer. In the case of tubular proteinurias
and normal
urine, Peak
I
showed very little protein
on the starch
gel,
Fig. 12. A two-dimensional cellulose acetate/starch gel clcctrophorctic analysis of a conccntratc of normal urine protein Fraction IV separated by column gel filtration, usmg Sephadex (l-75; this fraction comprised all protein cluting off the column after Peak 3. Direction of electrophoretic separations as in Fig. 1,
but y-globulin the absence
was constantly
of the expected
present amount
and there were traces of cc and /?components; of protein
suggested
non-entry
of much of the
protein into the gel. Fvactioe II. In normal urine and in all the cases of clinical proteinuria
with the
exception of some Bence Jones proteinurias, Peak 2 showed well-marked zones attributable to thyroxine-binding pre-albumin, albumin, cr,-acid glycoprotein, a,-antitrypsin and transferrin ; in addition there were small amounts of other a- and Bglobulins and some y-globulin. In tubular proteinuria and normal urine the predom inance of albumin and transferrin over other fractions was less marked than in glomerular proteinuria. In some Bence Jones proteinurias Peak z consisted almost entirely of 2 or 3 Rence Jones components similar to those found on two-dimensional electrophoresis of the whole urine protein. Fmction III. In Peak 3 from normal urine the most prominent zone was a protein of albumin to a,-globulin mobility on cellulose acetate electrophoresis, which had such fast mobility on starch gel that it moved with the buffer front. This protein was also prominent in tubular proteinuria, in which the faster moving y-globulins were also much in evidence. In all cases there was also some albumin, al-acid glycoprotein and c+antitrypsin, presumably due to overlap of fraction 2, and these formed part of a continuous arc of protein spreading from the c(, components to a post-y protein. In all the Kence Jones proteinurias Peak 3 contained 3 prominent Bence Jones components similar to those found on electrophoresis of the whole urine protein; in most cases little else was visible.
GEL FILTRATION
OF URINE PROTEIK
371
Fraction IV. This showed a continuous the prominent
fast-moving
zone noted
series of protein zones on an arc linking
in Peak
3 to the faster-moving
y-globulin. Usually about IO definite zones were discernible, particularly well-defined. If protein &ted beyond the Peak teinuria, the content of the 4th peak was similar to that of normal urine. In Bence Jones proteinurias inevitably two or
end of the
the faster 3 or 4 being 3 in a glomerular protubular proteinuria and three Bence Jones com-
ponents
were dominant. V. This contained the most characteristic a,-globulins of tubular proteinuria, a series of 3 or 4 fast-moving CC,zones being present. When a Peak 6 was
Fraction
present, small amounts of the &globulin In uncomplicated
found in that fraction were usually detected.
Bence Jones proteinurias
Peak 5 contained
components and little else, but in one case of mixed Bence teinuria this peak contained only the a,-gobulins characteristic
prominent
Bence Jones
Jones and tubular proof tubular malfunction.
Fraction VI. This contained small amounts of the same a,-globulin zones seen in Peak 5, together with a large amount of the characteristic &globulin of tubular proteinuria, namely P-microglobulin. Sometimes seen in this fraction was a small amount of another globulin. In normal
/&globulin
moving half as fast in the starch
urine it was not possible
to examine
gel as the fl-micro-
fractions
corresponding
to
Peaks 4, 5 and 6 separately as the points of demarcation between them were too uncertain; when all the protein eluting beyond Peak 3 was bulked and analysed, however, it was possible to recognise most of the protein zones which were seen in the separate
peaks obtained
from tubular
proteinurias.
This is apparent
from Fig.
12.
DISCUSSION
Since Sephadex gel filtration and shape, it was to be anticipated
separates proteins on the basis of molecular size that this technique would differentiate between
the 3 main types of clinical proteinuria, earlier studies having shown that the predominant proteins in each was of different molecular weightly2. In practice, column gel filtration with Sephadex G-75 has achieved a clear-cut distinction between pure glomerular, tubular and Bence Jones proteinurias, and even when mixed types occur it is possible to recognise the characteristic elements of each. Most previous work in this field has been carried out with Sephadex G-100 or G-200, but we believe that G-75 is preferable as it gives a better resolution of those proteins in the lower molecular weight range which are of particular significance. Thin-layer gel filtration, though less precise, is far simpler to perform than column gel filtration and provides the quickest method of demonstrating the presence of low molecular weight urine proteins. It has additional advantages in that less protein concentrate is needed and several specimens can be analysed at the same time with markers run alongside. This technique can therefore be recommended for the routine diagnostic investigation of clinical proteinuria. The two-dimensional cellulose acetate/starch gel electrophoretic findings show conclusively that the gel filtration fractions corresponding to Peaks 5 and 6 contain the most characteristic proteins of tubular proteinuria. Sephadex gel filtration thus segregates the low molecular weight proteins of tubular proteinuria in a way which is not possible with electrophoresis and it makes practical their large-scale collection. Clin. Chim.
Acta,
21 (1968) 357-376
DAVIS el al.
372
The electrophoretic analyses of the gel filtration fractions also demonstrate the conplexity of the protein excretion in different proteinurias, all fractions having been shown to contain several different proteins. The heterogeneity of each fraction may partly major
explain the variation sometimes found in the elution peak; a shift in the value of V,/l’, might reasonably
alteration
of the proportions
Because proteinuria
of the constituent
some of the characteristic
elute later off a Sephadex
volumes of the same be expected with an
proteins.
low molecular
weight
proteins
of tubular
G-75 column than the major proteins
excreted
in the other main types of proteinuria, gel filtration provides a sensitive technique for demonstrating the presence of tubular malfunction in a case of multiple myeloma or of primary glomerular damage. A good example of this is provided by Specimen 31; the patient had a plasma calcium of 14.7 mg/roo ml complicating myelomatosis, and the sizeable Peak 6 can be attributed to a tubular proteinuria being superimposed on a Bence Jones proteinuria, as hypercalcaemia of some duration is known to be capable of causing tubular malfunction. Several of the patients with glomerular proteinuria had small amounts of protein eluting as Peaks 4 and j and one had a detectable Peak 6, but none had been judged from the two-dimensional electrophoretic analysis of the whole urine concentrate to have mixed glomerular and tubular proteinuria; electrophoresis of the individual fractions however revealed the typical tubular
components. Both column and thin-layer
in the molecular
gel filtration
have demonstrated
a great similarity
weight range of the protein of normal urine and of tubular
protein-
uria and the main difference seen in the elution patterns is the presence in normal urine of a relatively larger quantity of protein coming off at the void volume. Most of the protein in this first peak fails to enter a starch gel and is therefore thought to be uromucoid, the macromolecular urinary tract protein described by Tamm and HorsfalllG. The proportion of uromucoid would be expected to be higher in normal urine because of the smaller absolute contribution of protein from other sources. The demonstration by two-dimensional electrophoresis of a marked similarity between the constituent proteins of the low molecular weight fractions of normal urine and tubular proteinuria is of considerable importance. The finding fits in with the concept that tubular proteinuria is largely due to impaired renal tubular reabsorption of protein normally present in the glomerular filtrate. The clear demonstration of a small Peak 5 in the plasma of a patient with tubular proteinuria is of great interest. It provides more direct evidence that the characteristic low molecular weight proteins found in tubular proteinuria may be derived from the plasma. However, it was not possible to detect Peak 5 in the sera of z healthy adults when these were examined in identical fashion. The considerable variation in relative peak height noted in the elution patterns of the different cases of tubular proteinuria, even those with a common aetiology, requires explanation. There may be variation in the rate of production of low molecular weight proteins from patient to patient, or there may be some selectivity in protein reabsorption by the kidney tubules. Alternatively, it is possible that the protein content of the urine is modified by a variable contribution from damaged renal tubule cells, or that complexes are variably formed between basic and acidic proteins, or with mucopolysaccharides. Estimates of the average molecular weight of the proteins constituting the difClin.
Chim.
Acta,
2I
(r9hX) 357-376
GEL FILTRATIOS
OF URINE PROTEIN
373.
ferent peaks can be derived from the experimental data of AndrewsIT. After working out V,/V, ratios for the different proteins that Andrews examined on a column of Sephadex G-75, we were able to produce a plot relating V e/ V Oto log molecular weight, similar to that given by Whitaker 13. Using the linear portion of the line relating the two, we obtained the following estimates for the average molecular weight of the proteins contained in Peaks 3 to 6: Peak 3 Peak 4 early component Peak 4 late component
31000 23500 rg 000
Peak 5 Peak 6 early component Peak 6 late component
‘5 000 11500 9400
The estimates of molecular weight of the proteins contained in the earlier peaks are less certain because their V,jV, values lie in the region where the correlation between the logarithm of molecular weight and elution value ceases to be linear; however, all the proteins in Peak I can be assumed to have molecular weights greater than 80000 and probably the average molecular weight of the protein in the early and late components of Peak 2 are 53 ooo and 44000 respectively. All these estimates of molecular weight must be considered at best to have an accuracy of ~IoD;’ because of the experimental error in measuring elution volumes, and because WhitakeF and Andrew@ have shown that anomalous results may be obtained with glycoproteins containing more than 5% of carbollydrate, with proteins of non-spherical shape or unusual density, or with proteins which form a complex with the gel or are liable to dissociate into subunjts. Comparison of our results with those of other workers is difficult because most reports in the literature describe the use of G-ZOO or G-100 Sephadex. Hardwickels, and Maclean and PetrieZo studied the urine of patients with heavy proteinuria for the purpose of investigating the selectivity of the protein clearance. Using columns of Sephadex G-zoo they obtained 3 peaks, but the first two represented fractions of high molecular weight globulins and would be equivalent to the z components in our Peak I, and the third, containing albumin and transferrin, would be equivalent to our Peak z; with this gel, however, one would not expect to achieve a separation of small quantities of protein of around 30000 molecular weight in the presence of a large amount of albumin and cc,-antitrypsin. Gel filtration has been used by a number of workers to study Bence Jones that it was possible to obtain some proteinuria. Snapper and Tillema 21 demonstrated separation of albumin and Bence Jones protein by passing an appropriate mixture of urine proteins through a column of Sephadex G-75, but others have carried out more detailed studies. Bernier and PutnamZz investigated 4 Bence Jones proteinurias using G-100 Sephadex and obtained multiple components which they attributed to variable poly~nerization of monomer Bence Jones molecules, Bence Jones variants and other proteins such as albumin, transferrin, y-globulin and low molecular weight glycoproteins. In the course of studying renal clearances of protein in II patients with myeloma and one with macroglobulinaemia, all of whom were excreting ‘abnormal’ light chain protein, Harrison et nLZ3 investigated the behaviour of the urine proteins by gel filtration using G-200 and G-100 Sephadex. They found that with one exception the Bence Jones proteins fell into z main groups and the K,, values they obtained suggest that they usually behaved as proteins with a molecular weight of around 53000 and 32000 ; these would correspond to proteins present in our Peaks 2 and 3. They did not regularly find minor components of lower molecular weight but in the Clin. Chim. Acta, 21 (1968) 357-376
DAVIS et al.
374 2
cases in whom a mixed tubular
and Bence Jones proteinuria
was detected
on paper
electrophoresis they did find appreciable quantities of protein of smaller molecular size. The elution behaviour of the Bence Jones components illustrated in the paper of Bernier
and Putnam22 appears to be as much at variance
the molecular weights of different Bence Jones components
with current
estimates
of
as that found by Harrison
ct (~1.~~ and by us. The major components in the Bence Jones proteinurias eluted as though the molecules had molecular weights of around 53000
we examined and/or 31 ooo
and/or rgooo. The dimer and monomer molecules are believed, however, to have molecular weights of 44000 and zzooo respectivelyz2. This anomalous elution behaviour of Bence Jones proteins cannot be attributed to the molecules being of nonspherical shape2 or containing carbohydrate a4. Drawing an analogy with the explanation advanced filtration,
by Andrews *? to account
we suggest
that
the elution
for the
of Bence
behaviour Jones
of haemoglobin
proteins
as though
on gel they had
molecular weights of around 53000 or 31000 may be due to dissociation of tetramer or dimer molecules occurring while the protein is passing through the column. The Wence Jones components eluting as a protein of around 15 ooo molecular weight may represent a 1.2 S fragment of the monomer molecule such as has been recognised b! Solomon et aLz5 and Van Eyk and Myszkowska WJ. , the fact that we find more of this component than others may be due to the greater reliability of our ultrafiltration procedure to prevent all loss of such molecules. Traeger
et &.a7 using G-ZOO Sephadex
to fractionate
urine proteins,
noted that
in tubular proteinuria a peak eluting later than the peak which mainly contains albumin is characteristically obtained. Piscatore studied the serum and urine proteins of an unspecified number of healthy individuals and workers exposed to cadmium compounds by gel filtration using G-100 Sephadex; he also performed gel filtration using G-75 Sephadex on fractions separated by ion exchange chromatography. In all cases the fractionation obtained appears similar to ours. Harrison and Northam’” also investigated
the excretion
of low molecular
weight proteins by normal adult men and
by patients with evidence of renal tubular damage, using Sephadex G-loo. In most cases they used a thin-layer technique, but they studied normal urine protein and the protein excreted by two patients with evidence of renal tubular malfunction by column gel filtration. They also studied the protein excretion by a hybrid thin-layer gel filtration and electrophoretic technique, and showed that the low molecular weight protein pattern of normal urine differed slightly from that associated with renal tubular disease. Their gel filtration findings are very similar to ours but like Piscator they have poorer resolution in the low molecular weight range and do not seem to have encountered an equivalent to our Peak 4. They suggested that the 3 low molecular weight peaks they found in 2 tubular proteinurias represented proteins of 44000, 16000 and IIOOO molecular weight. The last two would correspond reasonably well therefore with our Peaks 5 and 6. Their finding of increased quantities of low molecular weight protein in the urine of all patients with creatinine clearances of less than 20 ml/min fits well with our finding of small Peaks 4 and 5 in patients with glomerular proteinuria and elevated plasma urea Ievels. Walravens et a1.29*3@have studied normal urine protein and the tubular proteinuria of 15 patients with renal transplants and a case of the Fanconi syndrome by gel filtration. They used columns of Sephadex G-zoo but re-filtered portions of the eluate subsequently through G-75 columns. They also obtained only 5 peaks of protein, but Clin. Chim. Acta, :!I
(1908) 357-376
GEL FILTRATION
OF URIXE
like us they found some differences and a smaller
proportion
375
PROTEIN
from one case of tubular
of low molecular
weight
proteins
proteinuria in normal
to another urine.
Their
detailed immunoelectrophoretic analyses of the gel filtration fractions are not easily related to our findings by two-dimensional electrophoresis, but their finding of low molecular weight q-proteins and fi-microglobulin in the last peak correlates exactly with our findings in tubular proteinuria. The demonstration by immunoelectrophoresis of such proteins as /-microglobulin and post-l) protein in a low molecular weight gel filtration fraction of normal serum also correlates with our finding of a Peak 5 in a specimen of plasma. The theoretical implications of these findings in relation to the mechanism
of production
of tubular
proteinuria
will be discussed
elsewhere.
ACKSOWLEDGMEh-TS
This work was supported
by a grant from the Medical Research
Council.
We
also wish to thank the physicians at University College Hospital, particularly Professor C. E. Dent, for providing us with many of the specimens and Drs. P.W. Hall, J. R. Hobbs and G. Kazantzis for sending us others. We also wish to thank Mr. Y. K. Asta for the diagrams and Mr. A. C. Lees and his staff for the photographs.
I J. 11. CREETH, R. A. I
>I.D. POuLIK, NatUW,180 (1957) 1477. 1;.v. FLY??& AND E. A. STOW, J. C~?lz. Path&., II (1958) 334. E. A. BUTLER AND F. v. FLYNN. I.Clilz. Pathol.. 14 (1961) 172 E. .-\. BUTLER, F. V. FLYNN, H. HARRIS AND E. B. iio&oN, blin. Clzznz. 4cfa, 7 (1962) 34. 12 P. FLO~IN, Upx(ran (Xs and their Applicatiors in Gel Filtvation, A. H. Pharmacia, l’ppsala, 8 9 IO II
1962. 13 J, R. \VHITAKER, <4nal.Chem.,
35 (1963) 1950. B, JOHANSSON AND L. RYMO, .4cta Chrm. .Sca?zd., 18 (1964) 217. C. J. 0. R. RIORRIS,,I.Chromafog., 16 (1964) 167. I.TAX&I AND F. L. HORSFALL, J. Expfl. Med., 95 (1952) 71. I?.ANDRI?WS, Biochpm. J., 91 (1964) 222. P. ANDREWS, B~OC/ZPI~Z. J.. 96 (1965) 595. J, HARDWICKE, Clin. Chim. Acta, IL (1965) 89. 20 P. I<. M.~cLE.~N .~ND 1. 1. B. PETRIE. Clan. Chzm. Acta. Ed (1966) 167. 2I 1. SNAPPER AKD A. vi~"O. TILLEMA, Am. J. Med., 38 (19b;)‘40;.’
I4 I5 IO 17 18 I9
2~ G. hf. BERNIER AND F. \I’. P1’TNAM, B&him. Riophys. Acta, 86 (1964) 295. 23 J. F. HARRISON, J. D. BLAIP~EY, J. HARDWICKB, D. S. ROWF. AND J. F. SOOTHILL,CZ~%. SC%., .3I(1966)95. J. R. CLA~IP, G. RI.BERNIER AND F. W. PUTNAM, Biochim.Biophys. Acta, 86 (1964) 149. A. SOLOMOK, J. KILLANDER, H. &I.GREY AND H. G. KUNKEL, S&we, r51 (1966) 1237. H. G. VAN EYK AND K. MYSZKOWSKA, Clin.Chinz.Acta, 18 (1967) IOI. J. TRAEGER, R. FRAN~OIS, R. CREYSSEL, J.-P.REVILLARD, Y. MANUEL, M. T. FREYCON ANU W. SITE, Pathol. Biol. Semazm Hot)., 14 (1966) 5, 28 J. F. HARRISON ASD B. E. NORT&I, ?li+zy ChLm. Acta. 14 (1966) 679. 24 25 26 27
Clilz. Chim.
Acta,
21 (1968)
357-376
r)Aws
376 29 I'H.WALRAVENS,
E. C. LATERRE,
335. 30 PH. WALRAVEKS, JZ.C. LATERRC 31 C. E. DENT, ,I.Bonp Joint Swg., Clin. Chiwz. Acfa,
z I (1968)
357-37f1
et al.
A. EST.&S AND J. 1;.~~EREMAIUS, c&la. Chiun. .dcfa, 18 (1967) AND J. I'.HBRERIINS, QPJ (1952) 266.
Clix. Chim.
ilcfa,
19 (1968)
107.