Workflow and sampling
Step Procedure performed Equipment Data collected 1 urine collection daily for 1 wk microcentrifuge - 2 urine albumin and creatinine measured urine microalbumin and creatinine concentrations
Autoanalyzer Synchron CX5 Clinical Chemistry Analyzer (Beckman Coulter, Brea, CA)
- Eppendorf microcentrifuge tubes for urine collection
- Pasteur pipettes with rubber bulbs
- Mouse albumin standards, Kamiya Biomedical Company (Seattle, WA) and Sigma (St Louis, MO)
- Goat anti-human albumin antibody
- Picric acid
Acclimation to test conditions
Mice are allowed to acclimate to testing room over the weekend.
I. Urine collection in mice.
a. Spot urine samples are collected daily in the morning for 1 wk.
b. Urine samples from each mouse are pooled and at least 125 µL of urine is used for testing microalbumin (MA) and creatinine.
c. Each mouse is picked-up gently via the loose skin in the neck, behind the ears, and securing the head.
d. Male mouse are then positioned at approximately 60° angle over microcentrifuge tube, allowing the mouse to void urine freely.
e. Proper positioning of the mouse over the microcentrifuge tube is critical to ensure the maximum volume of urine. With a full bladder that is readily palpable, a mouse can be stimulated to void by gently massaging the inguinal area of the abdomen using the free index and middle fingers.
II. Measuring mouse urine microalbumin concentrations.
a. To validate the autoanalyzer method for quantifying mouse albumin concentrations, a commercial mouse albumin standard is used.
b. The series of mouse albumin standard concentrations are first measured using the Beckman Synchron CX5 Clinical Chemistry autoanalyzer.
Kamiya Mouse Standards Known microalbumin Measured microalbumin 0 0.00 1.5625 0.67 3.125 1.01 6.25 1.82 12.5 3.60 25 5.87
c. Microalbumin in urine samples is then detected with a goat anti-human albumin antibody to form antigen–antibody complexes increasing the turbidity of the sample.
d. The change in absorbency at 380 nm spectral wavelength is measured by the autoanalyzer, and the value obtained is proportional to the concentration of albumin in the urine sample.
e. Calculation: measured microalbumin sample (mg/dL) ÷ standard slope (0.249) = actual urine microalbumin concentration (mg/dL).
III. Measuring mouse urine creatinine concentrations.
Urinary creatinine concentrations are measured on the Beckman Synchron CX5 Clinical Chemistry autoanalyzer using the Jaffe method, where creatinine combines with picric acid to form a creatinine-picrate complex that changes the absorbency of the solution.
- urine microalbumin (MA)
- urine creatinine (CREA)
- urine albumin-creatinine ratio (ACR)