My "words of wisdom are below" - HA - :)
Denise
Denise I. Bounous, DVM, PhD, Diplomate ACVP
Bristol-Myers Squibb Co.
Group Director, Discovery Toxicology
Clinical Pathology - LVL
609-252-3247 (work)
908-328-7471 (cell)
denise.bounous@bms.com
From: American Society for Veterinary Clinical Pathology List
[mailto:asvcp_list@asvcp.org] On Behalf Of Sandra McConkey
Sent: Monday, October 03, 2011 4:08 PM
To: American Society for Veterinary Clinical Pathology List
Subject: [ASVCP List ] mouse urine protein
Hi,
I have no experience with mouse urine but have been asked to help with
a
research project. I am hoping that someone can help me with information
regarding protein measurement in mouse urine. Our group is examining a
possible
knockout model for glomerular disease. We have found that there is a
large
discrepancy between whether the protein is measured by our microprotein
method
(pyrogallol red method) or our usual total protein method that we use
on blood
(uses a reaction with Cu). SS seems to agree with the total protein
method. For
example, mice with 30-37 g/L protein using a total protein method may
have
supposedly have ~.02 g/L using the micro method. The micro method is
not
flagging to indicate that it requires dilutions.
So my questions would be:
1)
What is the best method for measuring protein in mouse urine?
Does
anyone know what is likely interfering with the results of our micro
method?
Pyrogallol red is fine for total protein, but for albumin, we use the
immunoturbidometric method on our instrument and make it species
specific by
running rat albumin as the calibrator. We are in the process of
validating for
mouse also.
2)
Does it matter if we use urine from a metabolic cage or clean
urine
(cystocentesis following euthanasia)? You just have to be consistent
with the
collection, length of time, time of day, over ice, etc.
3)
What is the normal protein concentration in healthy wildtype
mice? We
don't have any reference intervals for mice.
4)
Do old mice typically develop renal disease as they age and
therefore
normally have an increase of proteinuria as they age and if so by what
age? Not
sure - but not like male rats as far as I know. But would have to do
some
checking to confirm.
5)
We are also doing fractional excretions but some electrolytes
vary
between the metabolic cage urine and cysto. urine, possibly because of
the
marked crystaluria. Is there too much crystaluria (triple phosphates)
to
accurately measure urine electrolytes?
Theoretically, you have to
acidify
urine for calcium and phosphorus measurement. This dissolves crystals
so all
are measured. You can read the method sheets for your instruments.
5)
Any other words of wisdom?
There are metabolism cages
specifically
for mice. If you use the ones for rats, the urine collection volume is
reduced
- likely due to evaporation. We collect all our urines in refrigerated
conditions too. And any analyte that is measured in urine should be
normalized
to urine creatinine or to urine volume in order to compare across
animals.
Any and all help would be appreciated, thank you.
Sandra McConkey, DVM, PhD, Diplomate ACVP
Associate Professor of Clinical Pharmacology
Atlantic Veterinary College, Dept. of Biomedical Sciences
University of Prince Edward Island
550 University Avenue, C1A 4P3
Charlottetown, PE
Phone: 902-566-0814, Fax:902-566-0832
There was an interesting poster on this issue presented by Hala Willis
and
Holly Jordan at the 2008 ACVP/ASVCP meeting with some data. Here is the
abstract published by Vet Clin Pathol.
URINE EVALUATION IN NORMAL ADULT CD-1 MICE. H. Willis, H. Jordan.
Safety
Assessment Dept., GlaxoSmithKline, Research Triangle Park, NC, USA.
There is limited information regarding mouse urinalysis. Our goal was
to obtain
select urine data from healthy untreated CD-1 mice. Urine was collected
from
non-fasted, adult male (10) and female (10) mice. Mice were housed
individually
in metabolism cages and urine collected at room temperature overnight.
Samples
were not pooled prior to analysis. Volume, specific gravity and pH were
measured by manual methods. Samples were centrifuged (3000 rpm, 10 min)
and
supernatant stored at -80 degrees C. Thawed supernatant was analyzed on
the
Olympus AU640e chemistry analyzer using routine methods for urine
protein,
urine creatinine, and urine glucose. Volumes from 6 males and 7 females
were
sufficient for determination of all or some analytes. Five samples were
rejected due to contamination from automatic waterers. Result ranges:
urine
volume= 0.6-1.4mL for males and 0.4-4.0mL for females; specific gravity
=
1.022-1.057 for males and 1.006-1.055 for females; pH = 7.0-8.0 for
males and
females; total protein concentration = 9593-10550 mg/L for males and
571-3107
mg/L for females; total protein excreted per collection period (cp) =
2.1-8.4
mg/cp for males and 0.6-3.3 mg/cp for females; protein/creatinine ratio
=
11.3-25.0 for males and 3.2-9.0 for females; creatinine concentration =
1.15-4.72 mmol/L for males and 0.56-2.66 mmol/L for females; total
creatinine
excreted per collection period = 2-3 umol/cp for males and 1-5 umol/cp
for
females; glucose concentration = 1.20-1.91 mmol/L for males and 0.392.59
mmol/L for females; and total glucose excreted per collection period =
1-2
umol/cp for males and 1-4 umol/cp for females. This pilot study
established
preliminary ranges for select urine analytes in CD-1 mice. Obtaining
quality
urine specimens from individual mice is technically challenging and
insufficient volume and water contamination were common limitations.
Compared
with females, male mice had smaller urine volumes and higher protein
and
creatinine levels.
Mehrdad
Mehrdad Ameri, DVM, MS, PhD, Diplomate ACVP
Pathologist Director
Department of Pathology
Comparative Biology & Safety Sciences
Amgen Inc.
One Amgen Center Dr.
MS 29-2-A
Thousand Oaks, CA 91320