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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Spot urine glucose test
<ol>
	<li>Place a piece of clean Parafilm on a flat surface.</li>
	<li>Gently pick up a mouse by the tail and set it on the Parafilm.</li>
	<li>Restrain the mouse by the scruff using the thumb and index finger. Gently pick up the mouse while restrained at the scruff, causing the mouse to urinate as a stress response. Allow for a drop of urine to fall onto the parafilm surface. Return the mouse to its cage after collecting the urine drop.</li>
	<li>Dip the reagent end of a Diastix strip in the fresh urine drop collected on the Parafilm surface. Remove the strip immediately and lay it flat on a surface with the color side up. Wait at least 30 seconds for a color to develop on the strip fully. Compare the developed color with the reference chart provided on the Diastix container (Figure 1). 
	Tips: If the mouse does not urinate on the Parafilm, apply gentle pressure on the lower abdomen of the mouse (around the urinary bladder) using the index finger. This approach will help release urine drops onto the parafilm. For mice that don&#39;t urinate after following the procedure described above, wait 20 min and start the procedure again. Ensure all procedures are carried out while adhering to the IACUC guidelines.</li>
</ol>
2. Collection of 24 h urine using a mouse metabolic cage
<ol>
	<li>Assemble a Tecniplast metabolic cage (Figure 2) according to the manufacturer&#39;s instructions.</li>
	<li>Fill up the feed-assembly with powdered chow and then attach the feed assembly to the appropriate metal tab on the upper chamber.</li>
	<li>Fix the water bottle support onto the remaining metal tab on the upper chamber.</li>
	<li>Fill the empty water bottle and then slide it into the bottle support, ensuring that the sipper tube is in the hole.</li>
	<li>Put a mouse in the metabolic cage and then cover it with a lid.</li>
	<li>Ensure that the cage lid is fitted properly.</li>
	<li>Position a urine collection tube and a feces collection tube in their appropriate spots on the lower chamber floor.</li>
	<li>After 24 h, retrieve the urine collection tube and perform a glucose assay to determine urine glucose concentration. If the glucose assay is not performed on the same day, freeze the urine sample in the -80&#176;C freezer.</li>
</ol>
3. Determination of 24 h urine Glucose concentration
<ol>
	<li>If the urine sample is stored at -80&#176;C, let the sample thaw before performing a glucose assay.</li>
	<li>From 100mg/dl stock, prepare eight standards ranging from 25mg/dl to 0mg/dl according to the assay kit directions.</li>
	<li>Prepare standard wells and urine sample wells in a 96-well plate, all in duplicates, according to the kit manual.</li>
	<li>After initiating the reaction by adding the recommended amount of enzyme mixture to all wells, cover the plate and let it incubate for 10 min at 37&#176;C.</li>
	<li>Read absorbance at 500-520 nm using a plate reader.</li>
	<li>Analyze the data according to the assay kit instructions. Plot the absorbance of each standard vs the glucose concentration and use the graph to determine the glucose concentration of the 24-hour urine sample.</li>
</ol>

assays for qualitative and quantitative assessment of mouse urine glucose concentrations

This video demonstrates a procedure for collecting mouse urine and assessing its glucose levels. To evaluate the glucose level, immerse a test strip coated with enzymes and a chromogenic dye into the sample, initiating a color change that indicates the glucose levels. For quantifying urine glucose levels, introduce a urine sample to a multiwell plate with escalating glucose concentrations, add the reaction mixture, and incubate. Measure the absorbance to create a standard curve and compute the glucose levels based on the sample absorbance.

<img alt="Figure 1" src="/files/ftp_upload/22214/22214_Figure1.jpg" /> 
Figure 1. Spot urine glucose test. The mouse urine drops collected on Parafilm and the color reference chart provided on the Diastix container.
<img alt="Figure 2" src="/files/ftp_upload/22214/22214_Figure2.jpg" /> 
Figure 2. Tecniplast metabolic cage for 24 h mouse urine collection. A mouse is singly housed in this cage to collect 24 h urine.

Assays for Qualitative and Quantitative Assessment of Mouse Urine Glucose Concentrations

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Spot ...

Take a drop of fresh mouse urine and immerse a glucose detection strip containing glucose oxidase, peroxidase, and chromogen.
Glucose oxidase oxidizes urine glucose, forming gluconolactone and hydrogen peroxide.
Further, peroxidase utilizes hydrogen peroxide to catalyze the chromogen&#39;s oxidation, forming a colored product, with intensity proportional to urine glucose concentration.
Compare the color with the strip&#39;s reference chart for qualitative assessment of urine glucose.
For quantitative urine glucose measurement, take a multi-well plate with glucose concentration standards and the mouse urine sample collected over a defined period.
Add an assay mixture containing glucose oxidase, peroxidase, and indicator coupling reagents.
Glucose oxidase facilitates the oxidation of glucose, forming gluconolactone and hydrogen peroxide. Peroxidase catalyzes the reaction between coupling reagents and hydrogen peroxide, forming a pink-colored product.
Using a microplate reader, measure the absorbance of the colored product.
With the generated glucose standard curve, determine the glucose concentration in the urine.

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Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

343 Views.  This video demonstrates a procedure for collecting mouse urine and assessing its glucose levels. To evaluate the glucose level, immerse a test strip coated with enzymes and a chromogenic dye into the sample, initiating a color change that indicates the glucose levels. For quantifying urine glucose levels, introduce a urine sample to a multiwell plate with escalating glucose concentrations, add the reaction mixture, and incubate. Measure the absorbance to create a standard curve and compute the...

Video: Assays for Qualitative and Quantitative Assessment of Mouse Urine Glucose Concentrations - Concept

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese individuals worldwide by 2030, thus representing a growing socioeconomic burden. Simultaneously, obesity-related comorbidities, including diabetes as well as heart and chronic lung diseases, are continuously on the rise. Although obesity has been associated with increased risk for asthma exacerbations, worsening of respiratory symptoms, and poor control, the functional role of obesity and perturbed metabolism in the pathogenesis of chronic lung disease is often underestimated, and underlying molecular mechanisms remain elusive. This article aims to present methods to assess the effect of obesity on metabolism, as well as lung structure and function. Here, we describe three techniques for mice studies: (1) assessment of intraperitoneal glucose tolerance (ipGTT) to analyze the effect of obesity on glucose metabolism; (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn) to analyze the effect of obesity on lung function; and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. Obesity-related lung diseases are probably multifactorial, stemming from systemic inflammatory and metabolic dysregulation that potentially adversely influence lung function and the response to therapy. Therefore, a standardized methodology to study molecular mechanisms and the effect of novel treatments is essential.

The incidence of obesity is rising and increases the risk of chronic lung diseases. To establish the underlying mechanisms and preventive strategies, well-defined animal models are needed. Here, we provide three methods (glucose-tolerance-test, body plethysmography, and lung fixation) to study the effect of obesity on pulmonary outcomes in mice.

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese ...

JoVE Journal

Immunology and Infection

Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT

This paper describes the use of 18F-FDG and micro-PET/CT imaging to determine in vivo glucose metabolism kinetics in mice (and is transferable to rats). Impaired uptake and metabolism of glucose in multiple organ systems due to insulin resistance is a hallmark of type 2 diabetes. The ability of this technique to extract an image-derived input function from the vena cava using an iterative deconvolution method eliminates the requirement of the collection of arterial blood samples. Fitting of tissue and vena cava time activity curves to a two-tissue, three compartment model permits the estimation of kinetic micro-parameters related to the 18F-FDG uptake from the plasma to the intracellular space, the rate of transport from intracellular space to plasma and the rate of 18F-FDG phosphorylation. This methodology allows for multiple measures of glucose uptake and metabolism kinetics in the context of longitudinal studies and also provides insights into the efficacy of therapeutic interventions.

Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT

This study describes a protocol that uses 18F-FDG and positron emission tomography/computed tomography (PET/CT) imaging, together with kinetic modelling, to quantify the in vivo, real-time uptake of 18F-FDG into tissues.

This paper describes the use of 18F-FDG and micro-PET/CT imaging to determine in vivo glucose metabolism kinetics in mice (and is transferable to rats). ...

Medicine

Using a Combination of Indirect Calorimetry, Infrared Thermography, and Blood Glucose Levels to Measure Brown Adipose Tissue Thermogenesis in Humans

In mammals, brown adipose tissue (BAT) is activated rapidly in response to cold in order to maintain body temperature. Although BAT has been studied greatly in small animals, it is difficult to measure the activity of BAT in humans. Therefore, little is known about the heat-generating capacity and physiological significance of BAT in humans, including the degree to which components of the diet can activate BAT. This is due to the limitations in the currently most used method to assess the activation of BAT-radiolabeled glucose (fluorodeoxyglucose or 18FDG) measured by positron emission tomography-computerized tomography (PET-CT).
This method is usually performed in fasted subjects, as feeding induces glucose uptake by the muscles, which can mask the glucose uptake into the BAT. This paper describes a detailed protocol for quantifying total-body human energy expenditure and substrate utilization from BAT thermogenesis by combining indirect calorimetry, infrared thermography, and blood glucose monitoring in carbohydrate-loaded adult males. To characterize the physiological significance of BAT, measures of the impact of BAT activity on human health are critical. We demonstrate a protocol to achieve this by combining carbohydrate loading and indirect calorimetry with measurements of supraclavicular changes in temperature. This novel approach will help to understand the physiology and pharmacology of BAT thermogenesis in humans.

Here, we present a protocol to quantify the physiological significance of the impact of brown adipose tissue (BAT) activity on human metabolism. This is achieved by combining carbohydrate loading and indirect calorimetry with measurements of supraclavicular changes in temperature. This novel approach can help develop a pharmacological target for BAT thermogenesis in humans.

In mammals, brown adipose tissue (BAT) is activated rapidly in response to cold in order to maintain body temperature. Although BAT has been studied ...

Assays for Qualitative and Quantitative Assessment of Mouse Urine Glucose Concentrations

Transcript

Assays for Qualitative and Quantitative Assessment of Mouse Urine Glucose Concentrations

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

Determining Glucose Metabolism Kinetics Using ¹⁸F-FDG Micro-PET/CT

Using a Combination of Indirect Calorimetry, Infrared Thermography, and Blood Glucose Levels to Measure Brown Adipose Tissue Thermogenesis in Humans