Theriogenology 61 (2004) 1259–1272

Canine pyometra: a study of the urinary proteins

by SDS–PAGE and Western blot
C. Zaragozaa,*, R. Barreraa, F. Centenob,
J.A. Tapiac, M.C. Mañea
a
Department of Medicine and Animal Health, Faculty of Veterinary Sciences, University
of Extremadura, Avda. Universidad s/n, 10071 Caceres, Spain
b
Department of Biochemistry, Molecular Biology and Genetic, University of Extremadura,
Avda. Universidad s/n, 10071 Caceres, Spain
c
Digestive Diseases Branch, National Institute of Diabetes and Digestive and
Kidney Diseases, National Institutes of Health, Bethesda, MD, USA
Received 2 April 2002; accepted 10 July 2003

Abstract

Canine pyometra often causes glomerulonephritis by immune complex deposition in the

glomeruli. Proteinuria, ranging from moderate to severe, may be present secondary to renal
damage. To determine urinary protein excretion due to pyometra, sodium dodecyl sulphate–
polyacrylamide gel electrophoresis (SDS–PAGE) was conducted on urine from 15 bitches with
pyometra and 10 healthy bitches. To characterize urinary immunoglobin excretion, Western blot
analysis of the urine samples using antibodies to canine IgG and IgA was also performed. Nine
bands were detected by electrophoresis in bitches with pyometra, while only four were detected in
the healthy animals. The urinary proteins from bitches with pyometra were primarily of glomerular
origin; 58% were of medium-high molecular weight (MW), and the remainder were low MW. None
of the healthy dogs had IgG or IgA in their urine, whereas three bitches with pyometra had IgG in
their urine and another bitch with pyometra had both IgG and IgA. The low proportion of bitches
with urinary immunoglobins was probably be due to early diagnosis of the disease. Although only a
limited number of dogs was used, this study is apparently the first to characterize the electro-
phoretic pattern of urinary proteins and to quantify urinary excretion of IgG and IgA in bitches with
pyometra.
# 2003 Elsevier Inc. All rights reserved.

Keywords: Pyometra; Dog; Proteinuria; SDS–PAGE; Western blot

*
Corresponding author. Tel.: þ34-9-2725-7164; fax: þ34-9-2725-7110.
E-mail address: zaragoza@unex.es (C. Zaragoza).

0093-691X/$ – see front matter # 2003 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2003.07.019
1260 C. Zaragoza et al. / Theriogenology 61 (2004) 1259–1272

1. Introduction

Pyometra in the bitch is considered to result from bacterial and hormonal interaction [1–3],
with hormonal factors playing a predisposing role [1–4]. Renal disease and renal failure are
serious complications of canine pyometra [5]. Immune complex deposition in the glomeruli
causes glomerulonephritis [6,7]; leakage of plasma proteins into the glomerular filtrate leads
to proteinuria [8,9]. Proximal tubular damage develops after glomerular dysfunction and
precedes renal failure [5]. However, the renal lesion may be difficult to detect, since
laboratory tests are not consistently commensurate with renal damage. The diagnosis of renal
disease can be based on proteinuria (suggestive of a glomerular lesion) and a lack of
concentrating ability in the dehydrated bitch, indicating a distal tubular lesion [10].
Although proteinuria can be difficult to characterize, lesions at different kidney locations
result in particular patterns of urinary proteins according to their molecular weight (MW)
[11–13]. In that regard, sodium dodecyl sulphate–polyacrylamide gel electrophoresis
(SDS–PAGE) has been used to separate urinary proteins according to MW [11]. Urinary
proteins with a MW equal to or higher than albumin (69 kDa) are considered to be medium-
high MW, while those smaller than albumin are classified as low MW proteins [14].
Although analysis of urinary proteins can be useful in the diagnosis of renal disease
associated with pyometra [5,15–17], to date SDS–PAGE has not been applied to pyometra-
induced proteinuria.
Analysis of urinary proteins may hasten the diagnosis of renal disease and improve the
characterization of the renal lesion. Sorting proteins according to their MW would only
enable general characterization of the origin of the proteinuria (i.e. glomerular, tubular or
both). However, SDS–PAGE and Western blot analysis would result in identification of
some specific urinary proteins, and give more information regarding the type and severity
of the renal dysfunction [18].
The objective of the present study was to determine the MW of urinary proteins from
bitches with pyometra and to quantify the elimination of immunoglobulins IgG and IgA.
Urine samples from healthy bitches (Group I) and bitches with pyometra (Group II) were
analyzed by SDS–PAGE and Western blot and the results were related to the clinical
manifestations in bitches with pyometra.

2. Materials and methods

2.1. Animals

In total, 25 adult bitches of various breeds, including 10 healthy bitches (Group I) and 15
bitches with pyometra (Group II). The Group II bitches ranged in age from 26 months to 14
years and were presented to the Veterinary Teaching Hospital of Extremadura (Spain) with
symptoms compatible with pyometra (Table 1) [7,19,20]. History, physical examination,
serum and urine tests, and radiological and/or sonographic examinations were done to
confirm the diagnosis. Physical examination as well as hematological, biochemical and
urinary tests were also performed in the healthy bitches to confirm the absence of pyometra
(and other diseases).
 C. Zaragoza et al. / Theriogenology 61 (2004) 1259–1272 1261

Table 1
Clinical findings in 15 bitches with pyometra

Dog Anorexia, Weight Fever Dehydration Mucous Vomiting Diarrhea Polyuria Vaginal
lethargy loss (%) membranesa polydipsia discharge

1 þ þ þ 5 B    –
2 þ þ þ 5 A þ  þ Sanguineous
3 þ    A    Purulent
4     A   þ Mucopurulent
5 þ þ þ 5 C  þ þ Purulent
6 þ  þ  B  þ þ Sanguineous
7 þ þ   C þ  þ –
8 þ þ þ  C   þ Sanguineous
9 þ þ  5 A   þ –
10   þ  A   þ Purulent
11 þ  þ  A þ  þ –
12 þ    A   þ Mucopurulent
13 þ    B   þ –
14 þ  þ  A þ  þ Purulent
15 þ þ  7 A þ þ þ Sanguineous

þ/: Presence/absence of clinical manifestation.

a
A: normal pink; B: pale; C: congested.

2.2. Blood and urine tests

Leukocytes, erythrocytes and platelets counts, packed cell volume, mean cell volume
(MCV), mean cell hemoglobin (MCH), and mean cell hemoglobin concentration (MCHC)
were performed using the Sysmex F-800 semiautomated hematology analyzer (Toa
Medical Electronics, Hamburg, Germany). A visual differential count of leukocytes
was also performed. Serum biochemistry (blood urea nitrogen, creatinine, calcium,
phosphorus, cholesterol, total proteins, and albumin) was determined by a spectrophoto-
metric method using the Clima Plus analyzer (RAL, Madrid, Spain) with the corresponding
reagents (Human, Wiesbaden, Germany). Urine was obtained by aseptic cystocentesis
(using ultrasound guidance) to reduce the risk of penetrating the uterus. An aliquot of urine
was used for routine urinalysis and bacteriological culture (to rule out urinary tract
infection). The remainder of the urine sample was centrifuged for 5 min at 200  g; the
sediment was examined, and a portion of the supernatant was frozen at 80 8C until further
analysis by SDS–PAGE and Western blot. The rest of the supernatant was immediately
used to determine protein concentration [21] using the Shimadzu UV-160 spectrophot-
ometer (PACISA, Sevilla, Spain) and creatinine, to calculate the urine protein/creatinine
ratio (U P/C) [22].

2.3. SDS–PAGE analysis

SDS–PAGE was carried out using a 10.4% resolving gel following the procedure
described by Laemmli [23]. Three electrophoresis procedures were performed for each
urine sample, using the Mini Protean II Cell system (Bio-Rad, Hercules, CA, USA). One of
them was used to study the MW of the bands obtained, and the remaining gels to perform
1262 C. Zaragoza et al. / Theriogenology 61 (2004) 1259–1272

the Western blot analysis. In each electrophoresis (10 wells), a MW marker was
included (a-lactalbumin: 14.4 kDa; inhibitor of trypsin: 20.1 kDa; carbonic anhydrase:
30 kDa; ovalbumin: 43 kDa; albumin: 67 kDa; phosphorylase b: 94 kDa; all markers
were from Amersham-Pharmacia Biotech, Piscataway, NJ, USA), along with one urine
sample from a healthy bitch and eight from bitches with pyometra. The amount of
proteins loaded was 5 mg per sample. Gels were stained using Coomassie brilliant blue
R-250 (Bio-Rad) and scanned with a LKB UltroScan XL laser densitometer (Pharmacia
LKB Biotechnology, Inc., Piscataway, NJ, USA). The densitometric scans and MW of
the bands for each lane were obtained using the Ultroscan GSX software (Pharmacia
LKB Biotechnology, Inc.).

2.4. Western blot analysis

Western blot was performed as described previously [24,25]. Briefly, after fractionation
by SDS–PAGE, proteins were transferred to nitrocellulose membranes. Membranes were
blocked for 30 min using 10% non-fat dried milk in a PBS-Tween 20 solution: 0.08 M
K2HPO4, 0.02 M KH2PO4, 0.10 M NaCl and 0.2% (v/v) Tween 20. Blots were washed
twice for 1 min with PBS-Tween 20 solution, and incubated for 1 h at 25 8C with
0.24 mg ml1 of rabbit anti-IgG polyclonal antibody or with 0.15 mg ml1 of rabbit
anti-IgA polyclonal antibody (ICN Biomedicals, Aurora, OH, USA). After the incubation
with the primary antibody, membranes were washed twice for 1 min and once for 10 min
with PBS-Tween 20, and incubated at 25 8C for 30 min with 0.40 mg ml1 of goat anti-
rabbit IgG-horseradish peroxidase conjugate (ICN Biomedicals). After incubation with the
secondary antibody, membranes were washed once for 1 min and twice for 10 min with
PBS-Tween 20. Finally, developing and detection of bands were done by chemilumines-
cence [26]. Briefly, the membranes were incubated with enhanced chemiluminescence
detection reagents (Pierce, Rockford, IL, USA) for 5 min, and exposed to Kodak X-OMAT
film (Sigma–Aldrich, Inc., St. Louis, MO, USA) for 1 min.

2.5. Statistical analysis

Means and standard deviations of urine and serum parameters and MW were
performed using the statistical software SPSS 2000 (SPSS, Inc., Chicago, IL, USA).
A Fisher’s exact test was used to compare, between the two groups, the proportion of
bitches with specific bands. A Student’s t-test was performed to compare U P/C ratios
between the two groups.

3. Results

3.1. Clinical manifestations, blood and urine tests

For Group II bitches, clinical manifestations and data from analyses of blood and urine
are shown in Tables 1–3. The U P/C ratio in urine was higher (P ¼ 0:013) in Group I versus
Group II dogs (mean  S:D:: 0:43  0:15 and 1:03  0:97, respectively).
Table 2
Hematology data from 15 bitches with pyometra

Dog RBC PCV MCV MCH MCHC PLTC WBC(103 ml1)g Band Seg Eosin Lymph Monoc
(106 ml1)a (%)b (fl)c (pg)d (g dl1)e (103 ml1)f ( ml1)h ( ml1)i ( ml1)j ( ml1)k ( ml1)l

1 5.18 37.60 72.60 24.73 34.00 155 13.30 5586 2926 133 3591 1064
2 7.96 48.00 60.70 22.60 37.30 89 44.60 8920 28990 1338 4460 892
3 6.39 43.20 67.60 23.80 35.20 464 27.40 4932 18084 1096 3288 0

C. Zaragoza et al. / Theriogenology 61 (2004) 1259–1272

4 5.46 41.80 76.60 25.50 33.30 481 15.60 156 13572 1248 468 156
5 5.70 36.70 64.40 23.20 36.00 143 24.90 0 19920 498 2988 1494
6 5.04 36.20 71.80 24.20 33.70 134 27.00 6480 15660 0 3240 1620
7 3.93 28.40 72.30 26.70 37.00 130 26.10 6525 15660 261 3132 522
8 6.21 42.50 68.40 23.50 34.40 150 20.50 410 13325 0 4715 2050
9 6.02 39.60 65.80 20.30 30.80 199 82.20 33702 45210 0 822 2466
10 4.01 28.50 71.10 23.90 33.70 163 12.70 508 8509 127 2667 889
11 6.55 41.40 63.20 23.10 36.50 497 9.30 1581 7254 0 372 93
12 4.87 35.00 71.30 23.80 33.40 266 6.40 320 3904 704 1344 128
13 4.35 28.70 66.00 19.10 28.90 150 52.40 8384 41396 524 524 1572
14 4.20 29.70 70.70 21.20 30.00 270 11.40 798 9348 228 570 456
15 5.27 36.80 69.80 24.30 34.80 130 48.70 2922 42856 0 974 1948
Bitches that had bands in the ranges of 30–60 and 80–90 kDa are marked with asterisk (*) (not detected in healthy bitches).
a
Red blood cells.
b
Packed cell volume.
c
Mean cell volume.
d
Mean cell hemoglobin.
e
Mean cell hemoglobin concentration.
f
Platelets.
g
White blood cells.
h
Band neutrophils.
i
Segmented neutrophils.
j
Eosinophils.
k
Lymphocytes.
l
Monocytes.

1263
 1264
Table 3
Serum biochemistry and urine data in 15 bitches with pyometra

Dog BUN Creatinine Calcium Phosphorus Cholesterol TP (g dl1)b Albumin Specific U P/Cc Igd type

C. Zaragoza et al. / Theriogenology 61 (2004) 1259–1272

(mg dl1)a (mg dl1) (mg dl1) (mg dl1) (mg dl1) (mg dl1) gravity

1 37 1.2 10.3 4.9 300 8.5 2.9 1045 1.09

2 48 1.3 9.9 5.8 437 9.8 2.7 1021 1.40 IgG
3 60 1.3 10.3 4.0 203 8.8 2.5 1025 0.43
4 30 1.2 10.1 3.7 239 7.1 2.4 1015 0.27
5 80 1.4 9.6 5.5 448 8.0 1.8 1015 1.62
6 30 1.1 10.2 3.8 332 8.7 1.8 1006 1.19
7 24 1.1 9.5 5.2 223 6.8 2.0 1020 0.41
8 58 1.7 9.8 6.8 94 11.0 2.1 1017 0.30 IgG, IgA
9 252 2.3 10.3 8.7 335 9.0 2.2 1015 2.41 IgG
10 34 1.4 10.3 4.3 314 7.1 2.8 1019 0.77
11 17 1.0 9.4 5.9 342 7.6 3.3 1018 0.86
12 49 1.6 10.2 4.3 221 7.1 2.9 1020 2.50
13 26 0.9 9.9 4.5 320 8.0 2.6 1012 3.29 IgG
14 20 1.0 9.0 3.1 350 8.1 3.3 1039 0.32
15 28 1.0 9.8 5.2 390 7.9 2.7 1009 0.14
Bitches that had bands in the ranges of 30–60 and 80–90 kDa (marked with *), that were absent in healthy bitches.
a
Blood urea nitrogen.
b
Total proteins.
c
Urine protein/creatinine ratio.
d
Type of immunoglobin detected in the urine.
 C. Zaragoza et al. / Theriogenology 61 (2004) 1259–1272 1265

3.2. SDS–PAGE

Fig. 1 shows the SDS–PAGE electrophoretic patterns of urine from nine healthy bitches
(Group I), where four different bands were observed. These patterns were characterized by
two bands observed in most dogs and located in the MW ranges of 100–110 (n ¼ 10) and
70–80 kDa (n ¼ 9; Fig. 1 and Table 4). A third band, in the range of 20–30 kDa, was
detected in three dogs, and the last band (in the 10–20 kDa range) was only observed in one
dog (Fig. 1 and Table 4).

Fig. 1. SDS–PAGE of urine from nine healthy bitches. (A) Bands observed following electrophoresis. MW:
marker of molecular weight. Lanes 1–9 each contain a urine sample from one healthy bitch. (B) Densitometric
scan of the electrophoresis from one healthy bitch (with four bands).
1266 C. Zaragoza et al. / Theriogenology 61 (2004) 1259–1272

Table 4
Classification of bands detected by SDS–PAGE analysis

MW Group I-healthy bitches (n ¼ 10) Group II-bitches with pyometra (n ¼ 15)

rangesa
MW No. of bitches MW No. of bitches Probabilityb
with bands (mean  S.D.) with bands
(% of total) (% of total)

130–140 – 0 130.00 1 (7) NS

120–130 – 0 – 0 –
110–120 – 0 – 0 –
100–110 105.34  4.13 10 (100) 106.92  1.88 12 (80) 0.25
90–100 – 0 – 0 –
80–90 – 0 83.49  2.77 10 (67), 0.002
70–80 72.40  3.55 9 (90) 70.29  4.75 14 (93), NS
60–70 – 0 – 0 –
50–60 – 0 54.16  1.13 7 (47) 0.020
40–50 – 0 42.62  1.46 7 (47) 0.020
30–40 – 0 35.19  1.88 11 (73) 0.001
20–30 29.37  0.75 3 (30) 27.02  2.02 10 (67) 0.111
10–20 13.30 1 (10) 15.23  1.97 10 (67) 0.012
Mean  S:D:, molecular weight (MW; kDa) and frequency of appearance of the bands in each range of MW are
shown.
a
One band was only observed in the ranges of 130–140 and 10–20 kDa.
b
Probability of a difference between Groups I and II (NS: not significant).

In bitches with pyometra (Group II), nine different bands were observed by SDS–PAGE
(Fig. 2 and Table 4). For several bands, there were significant differences between the two
groups in the proportion of bitches in which the bands were detected (Table 4). The most
frequently visualized bands were those in the ranges of 70–80 (n ¼ 14), 100–110 (n ¼ 12)
and 30–40 kDa (n ¼ 11; Fig. 2A and Table 4). Furthermore, other bands in the ranges of
80–90, 20–30 and 10–20 kDa were also observed in 10 of them (Fig. 2A and Table 4).
Finally, bands in the ranges of 50–60 and 40–50 kDa were present in seven dogs. Only one
bitch had a band within the range of 130–140 kDa (Table 4).

3.3. Western blot

There was no IgG and/or IgA detected in the urine of any Group I bitch (Fig. 3).
However, both IgG and IgA antibodies recognized non-specifically two of the proteins of
the MW marker, i.e. carbonic anhydrase (30 kDa) and albumin (67 kDa), as well as one
protein of about 70 kDa present in the urine of Group I and Group II bitches (Figs. 3 and 4).
In Group II bitches, two intense bands were observed in four dogs (Nos. 2, 8, 9, and 13)
when an anti-IgG antibody was used (Fig. 4A and Table 4); the high and low MW bands
were 44:52  2:31 kDa and 25:70  1:20 kDa, respectively. When an anti-IgA antibody
was used, two bands (43.13 and 26.30 kDa, respectively; Fig. 4B) were detected in one of
the positive dogs for anti-IgG antibody (No. 8; Table 3). Furthermore, two bitches had a
band located in the range of 20–30 kDa (Fig. 4B) and another one had a band in the range of
40–50 kDa (Fig. 4B).
 C. Zaragoza et al. / Theriogenology 61 (2004) 1259–1272 1267

Fig. 2. SDS–PAGE of urine from seven bitches with pyometra. (A) Bands observed following electrophoresis.
MW: marker of molecular weight. Lane 1 represents a urine sample from one healthy bitch. Lanes 2–8 represent
urine samples from bitches with pyometra. (B) Densitometric scan of the electrophoresis from one bitch with
pyometra (nine bands).

4. Discussion

Renal disease and ultimately renal failure are severe complications of canine pyometra
[5]. This is apparently the first study to characterize the electrophoretic pattern of urinary
proteins and to quantify urinary excretion of IgG and IgA in bitches with pyometra.
Nine bands were observed in the urine of bitches with pyometra by SDS–PAGE, whereas
only four were found in the healthy bitches. Notably, bands detected in the majority of
1268 C. Zaragoza et al. / Theriogenology 61 (2004) 1259–1272

Fig. 3. Western blot of urine from nine healthy bitches. MW: marker of molecular weight. Lanes 1–9 represent
urine samples from healthy dogs. Non-specific signals in the marker of molecular weight (67 and 30 kDa) and in
the range of 70–80 kDa (Lanes 1–9). (A) Bands observed when using anti-IgG. (B) Bands observed when using
anti-IgA.

healthy and sick bitches were located in the ranges of 100–110 and 70–80 kDa; these bands
might correspond to transferrin and albumin, respectively, according to previous studies
that describe its excretion in the urine from healthy dogs [27]. Bands in the ranges of 20–30
and 10–20 kDa were also observed in both groups, but were more common in bitches with
pyometra. The high MW band might correspond to the a1-microglobulin (27 kDa) [22],
and the second band to b2-microglobulin (12 kDa) or lysozyme (14.40 kDa) [13,28].
Increased urinary excretion of low MW proteins such as b2-microglobulin, a1-micro-
globulin and lysozyme, related to tubular damage, have been reported [29–32]. Moreover,
proximal tubular damage has been described in dog with pyometra [5,33,34]. There were
low MW proteins (from 30 to 60 kDa) in bitches with pyometra that were absent in healthy
bitches, suggesting tubular injury in the former.
In six bitches with pyometra, four bands located in the ranges of 30–60 and 80–90 kDa
were detected by SDS–PAGE. Moreover, four of these dogs (Nos. 5, 8, 9 and 12) were the
only bitches that displayed signs of renal failure. Therefore, the presence of these bands
only in the urine from bitches with pyometra was consistent with other laboratory data.
Finally, the band located in the 130–140 kDa range was only observed in one dog (No. 15),
that did not display laboratory abnormalities severe enough to indicate a greater renal
injury than the remaining sick bitches.
As urinary proteins in dogs with glomerular dysfunction consist mainly of albumin and
high MW proteins like immunoglobulins [22,35,36], Western blot was carried out to
 C. Zaragoza et al. / Theriogenology 61 (2004) 1259–1272 1269

Fig. 4. Western blot of urine from bitches with pyometra. MW: marker of molecular weight. Non-specific
signals of the antibodies in the marker of molecular weight (67 and 30 kDa). (A) Bands observed when using
anti-IgG. Lane 1: urine from one healthy bitch. Lanes 2 and 3 represent urine from two bitches with pyometra.
Non-specific signals in the range of 70–80 kDa (Lanes 1–3). Heavy (hc) and light (lc) chains of IgG are showed
in Lane 3. (B) Bands observed when using anti-IgA. Lane 1 represents urine from one healthy bitch, whereas
Lanes 2–4 represent urine from three bitches with pyometra. Non-specific signals in the ranges of 70–80 kDa
(Lanes 1–3) and 40–50 kDa (Lane 4). Heavy (hc) and light (lc) chains of IgA are shown in Lane 3.

confirm the excretion of immunoglobulins in bitches with pyometra. One protein

(around 70 kDa) was recognized by both anti-IgG and anti-IgA antibodies in the urine
samples of healthy and sick bitches; this band could correspond to a non-specific
recognition of albumin; its MW was estimated as 69 kDa [8,14] and it can constitute
from 40 to 60% of the normal urinary proteins in dogs [37]. Neither IgG nor IgA was
detected in any of the urine samples from health bitches. However, IgA can be present
in urine from clinically health bitches, produced presumably by lymphoid tissues of
the urinary tract walls [38]. In four bitches with pyometra (Nos. 2, 8, 9 and 13), two
bands were detected when the anti-IgG antibody was used. The high MW band
(44:52  2:31 kDa) might correspond to IgG heavy chains, with a estimated molecular
size of 45 kDa [39]. The second band (25:70  1:20 kDa) might be IgG light chains, with
a MW around 25 kDa [39]. Three bitches (Nos. 2, 8 and 9) had four bands (detected by
SDS–PAGE) that were absent in healthy bitches. Moreover, two dogs (Nos. 8 and 9) had
signs of renal failure, consistent with the passage of IgG across the glomerular filtration
barrier. Finally, IgG was also detected in the urine from dog No. 13, which displayed
1270 C. Zaragoza et al. / Theriogenology 61 (2004) 1259–1272

isosthenuric urine (specific gravity ¼ 1012) and the highest U P/C ratio (3.29) in the
group of bitches with pyometra.
Using an anti-IgA antibody, two bands were observed only in one bitch (No. 8).
Notably, this dog also had IgG in its urine and signs of renal failure. The band of higher
MW (43.13 kDa) might correspond to IgA heavy chains, while the other band
(26.30 kDa) could be related to IgA light chains. Although IgA was detected in fewer
dogs than IgG, IgA is present in the serum in lower concentrations than IgG and it has a
higher MW [40]; therefore, urinary concentrations of IgA are expected to be lower than
IgG, even with severe glomerular damage. Overall, the relatively low excretion of
immunoglobulins in bitches with pyometra could due to the early diagnosis of the
disease. Finally, two bands with a MW similar to immunoglobulin light chains and
another band with a MW similar to immunoglobulin heavy chains were found in three
samples when anti-IgA was used. The three bands could correspond to an unspecific
recognition of antibody.
In conclusion, SDS–PAGE analysis of urine from bitches with pyometra had a
glomerular protein pattern with medium-high MW proteins [27,36,40–43] such as
immunoglobulins. In fact, IgG was detected in a 26.6% of sick bitches. Although there
was increased loss of low MW proteins, 67% of bitches with pyometra had bands in range
of 80–90 kDa, that were absent in healthy dogs. The relatively low excretion of immu-
noglobulins in bitches with pyometra could due to the early diagnosis of the disease.
Although only a limited number of dogs was used, this study is apparently the first to
characterize the electrophoretic pattern of urinary proteins and to quantify urinary
excretion of IgG and IgA in bitches with pyometra. Further studies are needed to
corroborate and extend these findings.

Acknowledgements

This work was supported by the ‘‘Junta de Extremadura’’ (grant IPR98A050) and
European Social Fund. The authors would like to thank Dr. Beatriz Hernández for critical
comments and English review of the manuscript.

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