1. Animal Ethics
Before performing the long duration hyperthermia on mice make sure that the procedure has been approved by Institutional Animal Care and Use Committee. All the procedures discussed in this paper and shown in the accompanying video were approved by the Institutional Animal Ethics Committee of the National Institute of Immunology.
2. Understanding Hyperthermia
Whole Body Hyperthermia (WBH) is delivered by placing the animals in prewarmed cages that are placed in incubator maintained at 39.5&deg;C. During the standardization of protocol, a temperature sensor was surgically implanted in the mice and their body temperature was monitored in different experimental conditions. It was observed that upon placing the animals in heated environment, their temperature gradually increased from 37 &deg;C to up to 42 &deg;C over the duration of 100 min (Figure 1). The resulting "spike" in temperature is intriguing as the internal body temperature shot even higher than the external environmental temperature. 
When the body temperature exceeded 40 &deg;C, the animal would show signs of stress and dehydration such as wetting around the snout. Increase the body temperature beyond 40 &deg;C, will lead to wetting and irreversible stress or even death of animals due to heat stress. So it is important to prevent the temperature spikes during the course of hyperthermia. 
This phenomenon of high temperature spikes is associated with the physical hyperactivity of animal during hyperthermia exposure. Similar temperature spikes were observed by other groups too and were associated with the physical hyperactivity of the animal upon being introduced to environment having higher temperatures 10. 
In our protocol, mice are immobilized during the hyperthermia exposure. As shown in Figure 2, immobilization of animal prevents the temperature spikes.
We have used 8-12 weeks old BALB/Cj mice for demonstrating this protocol. The protocol is not restricted to a particular type/strain of the mice.
3. Preparing for the Experiment
3.1 Incubator
This experiment can be performed in a normal CO2 incubator as they have a good temperature control. Disconnect CO2 gas supply and remove water holding tray of the CO2 incubator. In some models of incubators it is possible to switch off the CO2 controller. Arrange shelves of the incubator so that a mice cage could be placed conveniently inside. Set the temperature of the incubator at 39.5 &deg;C.
3.2 Cages
Typically, the hyperthermia experiment is performed on 4 mice at a time. Two cages are required; one is placed inside the incubator and the other is placed outside at room temperature. Bedding of cages is avoided, as the patches of bedding become wet with time which results in non-uniform temperature inside the cage. The outside cage should have food pellets and water bottle. 
3.3 Mice immobilization tubes
During the course of first 4 cycles of hyperthermia mice are immobilized in perforated tubes. These immobilization tubes can be made from 50 ml 'centrifuge' tubes by piercing 3 mm holes all over its body and four of such tubes will be required. The 50ml tube can conveniently immobilize BALB/Cj mice which are older than 8 weeks. Tubes of appropriate sizes may be required for some unusually different strain of mice or for mice which are very young or older than 50 weeks.
Place immobilization tubes and a cage inside the incubator maintained at 39.5 &deg;C. Wait for at least 2 hour so that the cage and immobilization tubes attain the temperature of the incubator. 
4. First cycle of the Hyperthermia
Mice may experience dehydration during the hyperthermia exposure. Therefore before beginning the experiment, administer 0.5 ml normal saline intraperitoneally to every mouse. Use a 26 G needle with 1 ml syringe.
Quickly take out the cage placed in the incubator, place one animal in each immobilization tubes and place the cage back in the incubator. Record the time. It is essential to keep the cage at the same place in the incubator every time.
After 90 min, take out the cage from the incubator. Release animals from the immobilization tubes and place them in the cage at normal temperature with easy access to food and water. Place empty immobilization tubes back in the incubator. 
After 15 min, the second cycle of hyperthermia is started by delivering 0.5 ml saline intraperitoneally to every mouse. 
They are then immobilized in 50 ml perforated immobilization tubes and placed in the incubator. 
5. Subsequent Hyperthermia Cycles
For first 4 cycles, it is essential to immobilize mice during the hyperthermia exposure and for the first two cycles, saline injection is given before beginning the hyperthermia period. 
After these cycles of hyperthermia followed by rest are completed, mice get acclimatize and do not become hyperactive during the hyperthermia exposure. The intraperitoneal injection of saline may not be necessary if mice readily take food and water during the resting time.
6. After All the Cycles Completed, the Final Step is to Examine the Animals
There should not be any death during the hyperthermia cycles. Every animal has to be watched very carefully in the first few cycles. Rapidly look for any wetting around the snout. This symptom is a signature of dehydration. Mice with such symptoms should immediately be given 0.5 ml normal saline i.p and rest at room temperature. Most of the immobilized mice will not require any additional care and will successfully bear the stress the hyperthermia. 
After completing required cycles, animals may look weak and dehydrated but they will quickly recover within few hours. 
<img src="/files/ftp_upload/3801/3801fig1.jpg" alt="Figure 1" /> 
Figure 1. Variation of the core body temperature of Mice when the cage was placed in an incubator at 39.5 &deg;C. After 90 min the cage was removed from the incubator and placed at 25 &deg;C. Red circles indicate high temperature spikes.
<img src="/files/ftp_upload/3801/3801fig2.jpg" alt="Figure 2" /> 
Figure 2. Variation of the core body temperature of an immobilized Mice during the hyperthermia cycles. After immobilizing the animal, high temperatures spikes were not observe when the cage was placed at 39.5 &deg;C.

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+mild+whole-body+hyperthermia+protocols+using+human+breast,+ovarian,+and+colon+tumors+grown+in+severe+combined+immunodeficient+mice.">Characterization of mild whole-body hyperthermia protocols using human breast, ovarian, and colon tumors grown in severe combined immunodeficient mice.</a> Infect. Dis. Obstet. Gynecol. 7, 19 (1999).</li><li>Willnow, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+otherwise+incurable+tumor+diseases+in+childhood+using+whole-body+hyperthermia+and+chemotherapy.">Treatment of otherwise incurable tumor diseases in childhood using whole-body hyperthermia and chemotherapy.</a> Dtsch. Med. Wochenschr. 114, 208 (1989).</li></ol>

We have nothing to disclose.

As far back as 5,000 B.C., Egyptian doctors treated tumors with heat. The Greeks recognized the value of heat in some medical treatments. The most ancient texts of the Law of Moses mention hot springs (Genesis 36:24) to therapeutically elevate body temperature.
The earlier techniques employed either the use of pyrogenic microbial extracts for induction of fever or hot water baths for induction of hyperthermia 1. However none of these approaches could be easily monitored or controlle...

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 <td>Thermistor</td>
 <td>RS components</td>
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 <td>CO2 Incubator</td>
 <td>Heraeus</td>
 <td>B5060 EK-CO2</td>
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 <td>Mice cages</td>
 <td>Citizen industries ltd.</td>
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 <td>Type B 365X207X190 cm</td>
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 <td>Provimi India</td>
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 <td>Water bottle for cage</td>
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 <td>Solderon, India</td>
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 <td>50 ml Centrifuge tubes</td>
 <td>BD Biosciences</td>
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</table>

protocol for long duration whole body hyperthermia in mice

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.

Watch this Scientific Journal Video about Protocol for Long Duration Whole Body Hyperthermia in Mice at JoVE.com

Protocol for Long Duration Whole Body Hyperthermia in Mice

This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

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11.5K Views.  National Institute of Immunology. This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

Video: Protocol for Long Duration Whole Body Hyperthermia in Mice

Parabiosis in Mice: A Detailed Protocol

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of microvasculature at the site of inflammation. Parabiotic partners share their circulating antigens and thus are free of adverse immune reaction. First described by Paul Bert in 18641, the parabiosis surgery was refined by Bunster and Meyer in 1933 to improve animal survival2. In the current protocol, two mice are surgically joined following a modification of the Bunster and Meyer technique. Animals are connected through the elbow and knee joints followed by attachment of the skin allowing firm support that prevents strain on the sutured skin. Herein, we describe in detail the parabiotic joining of a ubiquitous GFP expressing mouse to a wild type (WT) mouse. Two weeks after the procedure, the pair is separated and GFP positive cells can be detected by flow cytometric analysis in the blood circulation of the WT mouse. The blood chimerism allows one to examine the contribution of the circulating cells from one animal in the other.

Parabiotic joining of two organisms leads to the development of a shared circulatory system. In this protocol, we describe the surgical steps to form a parabiotic connection between a wild-type mouse and a constitutive GFP-expressing mouse.

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Long-term Blood Pressure Measurement in Freely Moving Mice Using Telemetry

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. Indeed, research focusing on the discovery of new potential therapeutic targets using genetically altered mice requires a reliable, long-term assessment of the systemic arterial pressure variation. Currently, the gold standard for obtaining long-term measurements of blood pressure in ambulatory mice uses implantable radio-transmitters, which require&#160;artery cannulation. This technique eliminates the need for tethering, restraining, or anesthetizing the animals which introduce stress and artifacts during data sampling. However, arterial blood pressure monitoring in mice via catheterization can be rather challenging due to the small size of the arteries. Here we present a step-by-step guide to illustrate the crucial key passages for a successful subcutaneous implantation of radio-transmitters and carotid artery cannulation in mice. We also include examples of long-term blood pressure activity taken from freely moving mice after a period of post-surgery recovery. Following this procedure will allow reliable direct blood pressure recordings from multiple animals simultaneously.

The goal of this protocol is to assess systemic blood pressure in conscious freely moving mice using implantable radio-telemetry devices.

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. ...

Body Composition and Metabolic Caging Analysis in High Fat Fed Mice

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate utilization, and behaviors such as food intake and physical activity can provide important information regarding the underlying mechanisms of disease. Given the importance of body composition and metabolism to the development of obesity and its subsequent sequelae, it is necessary to make accurate measures of these parameters in the pre-clinical research setting. Advances in technology over the past few decades have made it possible to derive these measures in rodent models in a non-invasive and longitudinal fashion. Consequently, these metabolic measures have proven useful when assessing the response of genetic manipulations (for example knockout or transgenic mice, viral knock-down or overexpression of genes), experimental drug/compound screening and dietary, behavioral or physical activity interventions. Herein, we describe the protocols used to measure body composition and metabolic parameters using an animal monitoring system in chow-fed and high fat diet-fed mice.

This protocol describes the use of a body composition analyzer and metabolic animal monitoring system to characterize body composition and metabolic parameters in mice. An obesity model induced by high-fat feeding is used as an example for the application of these techniques.

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate ...

Protocol to Create Chronic Wounds in Diabetic Mice

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ~6.5M people and cost ~$40B/year in the US alone. Although a significant effort has been invested in understanding how chronic wounds develop in humans, fundamental questions remain unanswered. Recently, we developed a novel mouse model for diabetic chronic wounds that have many characteristics of human chronic wounds. Using db/db-/- mice, we can generate chronic wounds by inducing high levels of oxidative stress (OS) in the wound tissue immediately after wounding, using a one-time treatment with inhibitors specific to the antioxidant enzymes catalase and glutathione peroxidase. These wounds have high levels of OS, develop biofilm naturally, become fully chronic within 20 days after treatment and can remain open more for more than 60 days. This novel model has many features of diabetic chronic wounds in humans and therefore can contribute significantly to advancing fundamental understanding of how wounds become chronic. This is a major breakthrough because chronic wounds in humans cause significant pain and distress to patients and result in amputation if unresolved. Moreover, these wounds are very expensive and time-consuming to treat, and lead to significant loss of personal income to patients. Advancements in this field of study through the use of our chronic wound model can significantly improve health care for millions who suffer under this debilitating condition. In this protocol, we describe in great detail the procedure to cause acute wounds to become chronic, which has not been done before.

Chronic wounds are developed from acute wounds on a diabetic mouse model by inducing high levels of oxidative stress after a full-thickness cutaneous wound. The wound is treated with inhibitors for catalase and glutathione peroxidase, resulting in impaired healing and biofilm development by bacteria present in the skin microbiome.

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ...

Handheld Metal Detector Screening for Metallic Foreign Body Ingestion in Children

Coins are the most common ingested metallic foreign bodies among children. The goal of this protocol is to assess the accuracy and feasibility of using a handheld metal detector to detect ingested metallic foreign bodies in children. We propose that by introducing handheld metal detector screening early in the triage process of children with high suspicion of metallic foreign body ingestion, the number of radiographs being ordered to localize the metallic foreign body can be reduced in this radio-sensitive population. The study protocol requires the screening of the participants for history of foreign body ingestion and exclusion of patients with respiratory distress or metallic implants. The patient changes to hospital gown and items that could contain metal like eyeglasses, earrings, pendants, and ornaments are removed. The patient is positioned in the center of the room away from other metallic interferences. The working status of the handheld metal detector is first confirmed by eliciting a positive audio-visual signal. Then the screening is done in an erect position with head in extension to expose the neck, from the level of the chin to the level of the hip joint, to cover the anatomical areas from neck to pelvis in a zig-zag manner both anteriorly and posteriorly. A positive audio-visual signal is carefully noted during the scanning for the presence of metallic foreign body. Relevant radiographs are ordered as per the area detected on the metal detector screening. The handheld metal detector was able to precisely identify all the coins among the ingested metallic foreign bodies in our study. The handheld metal detector could not consistently detect non-coin metallic foreign bodies. This protocol demonstrates the accuracy of handheld metal detector in the identification and localization of coins and coin like metallic foreign bodies.

Here, we present a protocol to use the handheld metal detector, to screen for the presence of ingested metallic foreign bodies in children who present to the pediatric emergency medicine department with a history of foreign body ingestion.

Coins are the most common ingested metallic foreign bodies among children. The goal of this protocol is to assess the accuracy and feasibility of using a ...

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to expend energy has made it a potential target for novel therapies to ameliorate obesity and associated metabolic disorders in humans. Though this tissue has been well studied in small animals, BAT&#39;s thermogenic capacity in humans remains largely unknown due to the difficulties of measuring its volume, activity, and distribution. Identifying and quantifying active human BAT is commonly performed using 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography and computed tomography (PET/CT) scans following cold-exposure or pharmacological activation. Here we describe a detailed image-analysis approach to quantify total-body human BAT from 18F-FDG PET/CT scans using an open-source software. We demonstrate the drawing of user-specified regions of interest to identify metabolically active adipose tissue while avoiding common non-BAT tissues, to measure BAT volume and activity, and to further characterize its anatomical distribution. Although this rigorous approach is time-consuming, we believe it will ultimately provide a foundation to develop future automated BAT quantification algorithms.

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

Using free, open-source software, we have developed an analytical approach to quantify total and regional brown adipose tissue (BAT) volume and metabolic activity of BAT using 18F-FDG PET/CT.

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to ...

A Whole Body Dosimetry Protocol for Peptide-Receptor Radionuclide Therapy (PRRT): 2D Planar Image and Hybrid 2D+3D SPECT/CT Image Methods

Peptide-receptor-radionuclide-therapy (PPRT) is a targeted therapy that combines a short-range energy radionuclide with a substrate with high specificity for cancer cell receptors. After injection, the radiotracer is distributed throughout the entire body, with a higher uptake in tissues where targeted receptors are overexpressed. The use of beta/gamma radionuclide emitters enables therapy imaging (beta-emission) and post-therapy imaging (gamma-emission) to be performed at the same time. Post-treatment sequential images permit absorbed dose calculation based on local uptake and wash-in/wash-out kinetics. We implemented a hybrid method that combines information derived from both 2D and 3D images. Serial whole-body images and blood samples are acquired to estimate the absorbed dose to different organs at risk and to lesions disseminated throughout the body. A single 3D-SPECT/CT image, limited to the abdominal region, overcomes projection overlap on planar images of different structures such as the intestines and kidneys. The hybrid 2D+3D-SPECT/CT method combines the effective half-life information derived from 2D planar images with the local uptake distribution derived from 3D images. We implemented this methodology to estimate the absorbed dose for patients undergoing PRRT with 177Lu-PSMA-617. The methodology could, however, be implemented with other beta-gamma radiotracers. To date, 10 patients have been enrolled into the dosimetry study with 177Lu-PSMA-617 combined with drug protectors for kidneys and salivary glands (mannitol and glutamate tablets, respectively). The median ratio between kidney uptake at 24 h evaluated on planar images and 3D-SPECT/CT is 0.45 (range:0.32-1.23). The comparison between hybrid and full 3D approach has been tested on one patient, resulting in a 1.6% underestimation with respect to full 3D (2D: 0.829 mGy/MBq, hybrid: 0.315 mGy/MBq, 3D: 0.320 mGy/MBq). Treatment safety has been confirmed, with a mean absorbed dose of 0.73 mGy/MBq (range:0.26-1.07) for kidneys, 0.56 mGy/MBq (0.33-2.63) for the parotid glands and 0.63 mGy/MBq (0.23-1.20) for submandibular glands, values in accordance with previously published data.

A Whole Body Dosimetry Protocol for Peptide-Receptor Radionuclide Therapy PRRT: 2D Planar Image and Hybrid 2D+3D SPECT/CT Image Methods

This method estimates the absorbed dose of different structures for peptide-receptor-radionuclide-therapy (PRRT) with the possibility of avoiding organ overlap on 2D-projections. Serial whole-body planar images permit estimation of mean absorbed doses along the whole body, while the hybrid approach, combining planar images and 3D-SPECT/CT image, overcomes the limitations of structure overlapping.

Peptide-receptor-radionuclide-therapy (PPRT) is a targeted therapy that combines a short-range energy radionuclide with a substrate with high specificity ...

In Vitro and In Vivo Delivery of Magnetic Nanoparticle Hyperthermia Using a Custom-Built Delivery System

Hyperthermia has long been used in the treatment of cancer. Techniques have varied from the intra-tumoral insertion of hot iron rods, to systemically delivered tumor antibody-targeted magnetic nanoparticles, at temperatures from 39 &#730;C (fever-level) to 1,000 &#730;C (electrocautery) and treatment times from seconds to hours. The temperature-time relationship (thermal dose) dictates the effect with high thermal doses resulting in the tissue ablation and lower thermal doses resulting in sublethal effects such as increased blood flow, accumulation of drugs and immune stimulation. One of the most promising current medical therapies is magnetic nanoparticle hyperthermia&#160; (mNPH). This technique involves activating magnetic nanoparticles, that can be delivered systemically or intratumorally, with a non-invasive, non-toxic alternating magnetic field. The size, construct and association of the magnetic nanoparticles and the frequency and field strength of the magnetic field are major heating determinants. We have developed sophisticated instrumentation and techniques for delivering reproducible magnetic nanoparticle hyperthermia in large and small animal models and cultured cells. This approach, using continuous, real time temperature monitoring in multiple locations, allows for the delivery of well-defined thermal doses to the target tissue (tumor) or cells while limiting non-target tissue heating. Precise control and monitoring of temperature, in multiple sites, and use of the industry standard algorithm (cumulative equivalent minutes at 43 &#730;C /CEM43), allows for an accurate determination and quantification of thermal dose. Our system, which allows for a wide variety of temperatures, thermal doses, and biological effects, was developed through a combination of commercial acquisitions and inhouse engineering and biology developments. This system has been optimized in a manner that allows for the rapid conversion between ex vivo, in vitro, and in vivo techniques. The goal of this protocol is to demonstrate how to design, develop and implement an effective technique and system for delivering reproducible and accurate magnetic nanoparticle therapy (mNP) hyperthermia.

This protocol presents techniques and methodology necessary for the accurate delivery of magnetic nanoparticle hyperthermia using a sophisticated delivery and monitoring system.

Hyperthermia has long been used in the treatment of cancer. Techniques have varied from the intra-tumoral insertion of hot iron rods, to systemically ...

Assessing Whole-Body Lipid-Handling Capacity in Mice

Assessing lipid metabolism is a cornerstone of evaluating metabolic function, and it is considered essential for in vivo metabolism studies. Lipids are a class of many different molecules with many pathways involved in their synthesis and metabolism. A starting point for evaluating lipid hemostasis for nutrition and obesity research is needed. This paper describes three easy and accessible methods that require little expertise or practice to master, and that can be adapted by most labs to screen for lipid-metabolism abnormalities in mice. These methods are (1) measuring several fasting serum lipid molecules using commercial kits (2) assaying for dietary lipid-handling capability through an oral intralipid tolerance test, and (3) evaluating the response to a pharmaceutical compound, CL 316,243, in mice. Together, these methods will provide a high-level overview of lipid handling capability in mice.

This paper provides three easy and accessible assays for assessing lipid metabolism in mice.

Assessing lipid metabolism is a cornerstone of evaluating metabolic function, and it is considered essential for in vivo metabolism studies. Lipids are a ...