|
Gli esami
delle urine, relativi a questo lavoro scientifico |
|
Gli esami
delle urine, relativi a questo lavoro scientifico |
|
QUALITATIVE
DETERMINATION OF PROTEINURIA BY SDS-AGE IN THE HEALTHY DOG |
|
The
determination of proteinuria by
Sodium Dodecyl Sulphate Polyacrilamide Gradient Gel electrophoresis
(SDS-AGE) makes it possible to evidence in the urine protein with a
molecular weight (MW), from 9000 (b2
microglobulin) to 900,000 (a2
macroblobulin). The method has a sensitivity for each protein fraction of
20 mg/l. The proteins identifiable with SDS-AGE ARE:
b2
microglobulin, lysozyme, retinol binding protein, light chains (k
and
l),
a1
microprotein, dimers of light chains, albumin, transferrin, IgG, IgA,
haptoglobulin and
a2
macroglobulin. The method is based on the migration of each protein
fraction according to its mass (not charge). In this way it is possible to
eliminate interference by high-molecular-weight proteins on the migration
of the low-molecular-weight proteins. Distinction of the individual
protein bands makes it possible to identify proteins of tubular origin (MW
<70,000) and proteins of glomerular origin (MW >70,000). The scope of the
study was the qualitative determination of physiological protein in the
dog. Fourtheen dogs aged 24 to 48 months (6 whole males, 1 castrated male,
and 7 females), with a body surface of 0.60 to 0.92 m2, were
evaluated preventively to exclude extrarenal causes of proteinuria. After
a period of dietetic pretreatment and restricted physical activity of 4
months, each dog was sedated and subjected to echo-assisted renal biopsy
with an 18-gauge Tru-cut. The biopsy samples were preserved in 10%
buffered formalin for conventional optical microscope examination and in
liquid nitrogen, after immersion in the appropriate medium, for
immunofluorescence examination. Conventional optical microscope
examination was done on samples stained with hematoxylin-eosin, periodic
acid-Schiff, trichrome according to Goldner, methenamine, and Congo red.
Immunofluorescence examination was performed by testing the samples with
IgG, IgA, C3 complement fraction and fibrinogen. At the time of
the biopsy, a pre-established volume (10 ml) of urine was collected by
centesis and kept (after the addition of 1% sodium azide equal to 1
ml/ml
of urine) at 4-8°C. The following analyses were performed on the urine
samples: physico-chemical, sediment, proteinuria/creatinuria ratio,
determination of proteinuria by a quantitative (staining with pyrogallol)
and a qualitative method (SDS-AGE). All the dogs with inert sediment and
with normal conventional optical microscopic, immunohistochemical and
immunofluorescence examinations at the qualitative study of proteinuria
showed exclusively a minimum band of albumin or the complete absence of
protein bands. |
