Quantitative Determination of <em>De Novo</em> Fatty Acid Synthesis in Brown Adipose Tissue Using Deuterium Oxide

Rory Turner; Rajib Mukherjee; Martina Wallace; Joan Sanchez-Gurmaches

doi:10.3791/64219

Détermination quantitative de la synthèse d’acides gras de novo dans le tissu adipeux br...

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Quantitative Determination of De Novo Fatty Acid Synthesis in Brown Adipose Tissue Using Deuterium Oxide

Détermination quantitative de la synthèse d’acides gras de novo dans le tissu adipeux brun à l’aide d’oxyde de deutérium

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07:34 min

May 12, 2023

DOI: 10.3791/64219-v

Rory Turner*¹, Rajib Mukherjee*², Martina Wallace¹, Joan Sanchez-Gurmaches^2,3,4

¹UCD Conway Institute and UCD Institute of Food and Health, School of Agriculture and Food Science,University College Dublin, ²Division of Endocrinology,Cincinnati Children’s Hospital Medical Center, Cincinnati, ³Division of Developmental Biology,Cincinnati Children’s Hospital Medical Center, Cincinnati, ⁴Department of Pediatrics,University of Cincinnati College of Medicine

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Overview

This study presents a novel quantitative method using deuterium oxide and gas chromatography mass spectrometry (GCMS) to analyze fatty acid de novo lipogenesis in brown adipose tissue in vivo. The method offers a non-invasive approach to accurately assess fatty acid synthesis flux, providing insights into metabolic status.

Key Study Components

Research Area

Metabolic pathways
Fatty acid synthesis
Brown adipose tissue analysis

Background

Fatty acid synthesis is under-studied compared to glucose disposal.
Existing methods may not accurately reflect synthesis flux due to impacts from upstream glucose handling.
This method is applicable across different mouse models and various tissues.

Methods Used

Sample preparation and extraction techniques using deuterium oxide.
In vivo analysis of brown adipose tissue in mouse models.
Gas chromatography mass spectrometry (GCMS) for measurement of fatty acid methyl esters (FAMEs).

Main Results

Documented deuterium enrichment levels in body water and fatty acids.
Observed differences in fatty acid enrichment based on temperature conditions.
Findings validate the efficiency of the method in assessing total fatty acidity synthesis in brown adipose tissue.

Conclusions

The study successfully demonstrates an effective method for evaluating fatty acid synthesis in vivo.
It contributes significantly to the understanding of metabolic processes and has potential applications in further biological research.

Frequently Asked Questions

What is the significance of using deuterium oxide in this method?

Deuterium oxide allows for the precise measurement of fatty acid synthesis by incorporating deuterium into fatty acids during metabolic processes.

How does this method improve upon traditional techniques?

This method provides a non-invasive approach that reflects fatty acid flux more accurately than techniques relying on isotope-labeled glucose.

Can this method be used in other tissues apart from brown adipose tissue?

Yes, the method is potentially adaptable for studying fatty acid synthesis in various tissues in different mouse models.

What was observed regarding temperature's effect on fatty acid enrichment?

The study found higher fatty acid enrichment at thermoneutral conditions compared to lower temperatures.

What are the potential implications of this research?

Understanding fatty acid synthesis's role in metabolism could inform research on obesity and metabolic diseases.

Is the method suitable for analyzing all fatty acids?

Yes, the method analyzes total fatty acids across multiple lipid classes, although additional separation techniques may be needed for specific studies.

What precautions should be taken during GCMS analysis?

It is essential to avoid overloading samples and to check for background peaks to ensure accurate results.

Nous présentons ici une méthode quantitative peu coûteuse utilisant l’oxyde de deutérium et la chromatographie en phase gazeuse par spectrométrie de masse (GCMS) pour l’analyse de la lipogenèse de novo des acides gras totaux dans le tissu adipeux brun in vivo.

Contrairement à d’autres voies métaboliques comme l’élimination du glucose, la synthèse des acides gras n’est pas systématiquement évaluée, ce qui conduit à des interprétations incomplètes de l’état métabolique. Les principaux avantages de cette méthode sont qu’elle est relativement non invasive et qu’elle reflète probablement mieux le flux de synthèse des acides gras que l’utilisation de glucose marqué aux isotopes, où les changements dans la manipulation du glucose en amont peuvent avoir un impact sur les résultats. Ici, nous nous concentrons sur le tissu adipeux brun, mais cette méthode est potentiellement utile pour étudier n’importe quel tissu dans n’importe quel modèle de souris.

Pour commencer, placez les échantillons sur de la glace carbonique. Prenez un tube de microcentrifugation pré-étiqueté, placez-le sur une balance d’analyse et tarez la balance. Placez ensuite une pince à épiler et un scalpel ou une lame de rasoir en acier sur de la glace carbonique pendant 10 à 20 secondes pour refroidir.

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