1. THE IMPORTANCE OF URINALYSIS AND
THE ROLE OF METABOLIC CAGES IN
ANIMAL STUDY
Presented by
Sachin Nayak
22PCM3636
Pharmacology and Toxicology
2. Flow of presentation
• Introduction to urinalysis
• Advances in urinalysis
• Urinary biomarkers
• Urine sample
• Urine collection methods
• Metabolic cage
• Common urine parameters
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3. Introduction to urinalysis
Basically urinalysis is a test of the urine to check for UTI, kidney problems, diabetes
etc Along with this it is also performed during the regular checkups (surgery,
pregnancy etc).
Physical characteristics: color, clarity, odor , specific gravity
Chemical analysis: glucose (indicative of diabetes), protein (a sign of kidney disease),
ketones (seen in conditions like diabetes or starvation), and bilirubin (a marker of liver
problems).
Microscopic examination- red blood cells, white blood cells, bacteria, crystals, or other
abnormal elements
Urinalysis offers several advantages compared to blood tests
Non invasive easy sample collection, cost effective, wide range of information,
screening tool(dope test)
Importance of urinalysis in rat preclinical studies
Kidney function test biomarker identification, toxicity monitoring, pharmacokinetic
studies and metabolic evaluation
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4. Advances in urinalysis
• Prostate Cancer: Prostate-specific antigen
(PSA) can be measured in urine, and
elevated levels may indicate prostate cancer.
• Metabolic Disorders: Metabolic disorders,
such as phenylketonuria (PKU), can be
detected through urine screening.
• Wilson's Disease
• Diabetic nephropathy etc
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5. Urinary biomarkers of diseases discovered using NMR based metabolomics
Emwas, Abdul-Hamid, et al. "Recommendations and standardization of biomarker quantification using NMR-based metabolomics with particular focus on urinary analysis." Journal of proteome research 15.2
(2016): 360-373.
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6. Urine sample
Spot urine
• First morning sample
• Easy to obtain
• Rapid transfer to laboratory that avoids degradation
• Feasibility
• Accuracy of collection
24 hr Urine
• Takes into account circadian fluctuations
• Help to diagnose kidney problems
• Defines Bence jones protein excretion(Multiple myeloma)
• Time consuming
• Affected by food intake, vigorous exercise, acute stress,
storage errors
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7. Urine collection methods
Metabolic cage method
• A metabolic cage for rats is a specialized housing
apparatus designed to monitor and analyze the
animal's metabolic activity, including urine and feces
production, water and food intake, and other related
measurements.
• Extended period of isolation
• Stressful, risk of injury is high
• Less floor space
• Single continuous session
• Cleaning and maintenance required
Hydrophobic sand method
• Hydrophobic sand (for example, Lab Sand,
Kit4Cat), a biodegradable material with a nontoxic
urine-repelling coating, replaces the bedding in a
normal cage during the urine collection period .
• Less stressful and no injury
• Maintain integrity of urine sample
• More floor space
• Collection can be repeated more easily over
multiple days in shorter session.
• No requirement of cleaning , easily discarded.
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11. Why metabolic cage is needed?
• Measurement of food and water intake
• Assessment of energy expenditure(amt of heat produced, gaseous exchange
• Collection and analysis of urine
• Study of physiological parameters(sleep pattern, locomotor activity etc)
• Behaviour studies etc
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12. Effects of Metabolic Cage Housing on Rat Behavior and Performance in
the Social Interaction Test
• Metabolic cage housing is widely used in pharmacokinetic and pharmacodynamic studies
require the use of grid flooring, absence of bedding substrate and enrichment, and social
isolation, and they are coupled with a smaller-than-usual living space area
(approximately one third),which elicit negative behavioral and physiological responses in
rodents.
• Behavioural measures-The nylon chew toy method. (But no significant difference)
• Home-cage behavior recording In general, there was a significantly increased number
of active, attentive, grooming, and ingestion behaviors expressed during the dark period
compared with the light period, consequently, inactive behaviors such as lying decreased
in open-top cages, but there was less distinction between behavioral frequencies in both
cycle phases for the metabolic cage-housed animals. Clearly, behaviors categorized as
inactive occupied a significant portion of rat time in both housing treatments: 53.43% in
open-top cages versus 77.73% in metabolic cages . Overall, rats in metabolic cages spent
less time moving, manipulating enrichment, and carrying out rearing behaviors. As a
consequence, the portion of time spent carrying out active and attentive behaviors
decreased, with a corresponding shift toward inactivity.
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Whittaker, Alexandra L., Kerry A. Lymn, and Gordon S. Howarth. "Effects of metabolic cage housing on rat behavior and performance in the social
interaction test." Journal of applied animal welfare science 19.4 (2016): 363-374.
13. • Social interaction test
• Proportion of time spent engaging in drinking behavior also decreased in metabolic-cage housing
• Metabolic-cage housing has been determined to be mildly stressful to rats based on reduced levels of
IgA secretion in saliva (Eriksson et al., 2004). A reduction in Immunoglobulin A (IgA) concentration has
previously been shown to be a reliable indicator of stress in rats (Guhad & Hau, 1996; Royo, Bjork,
Carlsson, Mayo, & Hau, 2004).
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14. Some of the common tests that are performed
in urine to analyse nephropathy
15. Albumin estimation
• The BCG (Bromocresol Green) albumin assay kit is designed to measure albumin
directly without any pretreatment of samples, such as serum, plasma, urine, and
biological preparations.
{ Bromocresol green (an anionic dye) + the protein at acid pH produce a color change}
• A dipstick test performed on a urine sample can detect the presence of albumin in
the urine. A strip of chemically treated paper, into the urine changes color if albumin
is present in the urine.
• This Enzyme-Linked Immunosorbent Assay (ELISA) is a two-step assay for the
ultra-sensitive determination of human albumin in urine. A polyclonal rabbit antibody
specific for human albumin is immobilized on a microtiter plate and a second anti-
albumin antibody is conjugated to peroxidase
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16. Creatinine test
• High cr in blood and low in urine indicates kidney disease
• Diazyme's Creatinine Liquid Reagents Assay is a quick, easy to use enzymatic procedure involves a series of coupled
enzymatic reactions including creatininase enzymatic conversion of creatinine into the product creatine which itself is
converted to sarcosine by creatine amidohydrolase (creatinase), followed by oxidation of sarcosine by sarcosine oxidase
(SOD) producing hydrogen peroxide. In the presence of peroxidase (POD) the hydrogen peroxide is quantified at 550 nm by
the formation of a colored dye. Any endogenous creatine present in the sample is removed by creatinase and sarcosine
oxidase during pre-incubation.
• Briefly, ClCr(creatinine clearance) in rat had been determined with the following formula: ClCr = UF24h × uCr/pCr, where
UF24h is the volume of urine collected in 24 h, pCr is the plasma concentration of creatinine, and uCr is the urinary
concentration of creatinine.
• Usually, rat serum creatinine levels are in the range of 0.4–0.8 mg/dL
• Urine albumin to creatinine ratio (ACR), also known as urine microalbumin, helps identify kidney disease that can occur
as a complication of diabetes.
• ACR is calculated by dividing albumin concentration in milligrams by creatinine concentration in grams.
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17. BUN AND GFR
• Blood urea nitrogen (BUN) levels can be affected. BUN is a measure of the amount of nitrogen in the blood that comes
from the waste product urea, which is produced when proteins are broken down. The kidneys play a crucial role in filtering
and removing urea from the bloodstream, so when the kidneys are not functioning properly, BUN levels can rise.
• GFR quantifies the rate at which blood is filtered by the glomeruli, which are tiny blood vessels in the kidneys responsible
for filtering waste products and excess fluids from the bloodstream. GFR is considered one of the most reliable indicators
of kidney function.
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