Biochemistry

Cholesterol is oxygenated by a variety of cholesterol hydroxylases; oxysterols play diverse important roles in physiological and pathophysiological conditions by regulating several transcription factors and cell-surface receptors. Each oxysterol has distinct and overlapping functions. The expression of cholesterol hydroxylases is highly regulated, but their physiological and pathophysiological roles are not fully understood. Although the activity of cholesterol hydroxylases has been characterized biochemically using radiolabeled cholesterol as the substrate, their specificities remain to be comprehensively determined quantitatively. To better understand their roles, a highly sensitive method to measure the amount of various oxysterols synthesized by cholesterol hydroxylases in living mammalian cells is required. Our method described here, with gas chromatography coupled with tandem mass spectrometry (GC–MS/MS), can quantitatively determine a series of oxysterols endogenously synthesized by forced expression of one of the four major cholesterol hydroxylases—CH25H, CYP7A1, CYP27A1, and CYP46A1—or induction of CH25H expression by a physiological stimulus. This protocol can also simultaneously measure the amount of intermediate sterols, which serve as markers for cellular cholesterol synthesis activity.

Key features

• Allows measuring the amount of a variety of oxysterols synthesized endogenously by cholesterol hydroxylases using GC–MS/MS.

• Comprehensive and quantitative analysis of cholesterol hydroxylase specificities in living mammalian cells.

• Simultaneous quantification of intermediate sterols to assess cholesterol synthesis activity.

Graphical overview

Dietary saturated fatty acids (SFAs) are upregulated in the blood circulation following digestion. A variety of circulating lipid species have been implicated in metabolic and inflammatory diseases; however, due to the extreme variability in serum or plasma lipid concentrations found in human studies, established reference ranges are still lacking, in addition to lipid specificity and diagnostic biomarkers. Mass spectrometry is widely used for identification of lipid species in the plasma, and there are many differences in sample extraction methods within the literature. We used ultra-high performance liquid chromatography (UPLC) coupled to a high-resolution hybrid triple quadrupole-time-of-flight (QToF) mass spectrometry (MS) to compare relative peak abundance of specific lipid species within the following lipid classes: free fatty acids (FFAs), triglycerides (TAGs), phosphatidylcholines (PCs), and sphingolipids (SGs), in the plasma of mice fed a standard chow (SC; low in SFAs) or ketogenic diet (KD; high in SFAs) for two weeks. In this protocol, we used Principal Component Analysis (PCA) and R to visualize how individual mice clustered together according to their diet, and we found that KD-fed mice displayed unique blood profiles for many lipid species identified within each lipid class compared to SC-fed mice. We conclude that two weeks of KD feeding is sufficient to significantly alter circulating lipids, with PCs being the most altered lipid class, followed by SGs, TAGs, and FFAs, including palmitic acid (PA) and PA-saturated lipids. This protocol is needed to advance knowledge on the impact that SFA-enriched diets have on concentrations of specific lipids in the blood that are known to be associated with metabolic and inflammatory diseases.

Key features

• Analysis of relative plasma lipid concentrations from mice on different diets using R.

• Lipidomics data collected via ultra-high performance liquid chromatography (UPLC) coupled to a high-resolution hybrid triple quadrupole-time-of-flight (QToF) mass spectrometry (MS).

• Allows for a comprehensive comparison of diet-dependent plasma lipid profiles, including a variety of specific lipid species within several different lipid classes.

• Accumulation of certain free fatty acids, phosphatidylcholines, triglycerides, and sphingolipids are associated with metabolic and inflammatory diseases, and plasma concentrations may be clinically useful.

Graphical overview

Non-alcoholic steatohepatitis (NASH) is a condition characterized by inflammation and hepatic injury/fibrosis caused by the accumulation of ectopic fats in the liver. Recent advances in lipidomics have allowed the identification and characterization of lipid species and have revealed signature patterns of various diseases. Here, we describe a lipidomics workflow to assess the lipid profiles of liver homogenates taken from a NASH mouse model. The protocol described below was used to extract and analyze the metabolites from the livers of mice with NASH by liquid chromatography–mass spectrometry (LC-MS); however, it can be applied to other tissue homogenate samples. Using this method, over 1,000 species of lipids from five classes can be analyzed in a single run on the LC-MS. Also, partial elucidation of the identity of neutral lipid (triacylglycerides and diacylglycerides) aliphatic chains can be performed with this simple LC-MS setup.

Key features

• Over 1,000 lipid species (sphingolipids, cholesteryl esters, neutral lipids, phospholipids, fatty acids) are analyzed in one run.

• Analysis of liver lipids in non-alcoholic steatohepatitis (NASH) mouse model.

• Normal-phase chromatography coupled to a triple quadrupole mass spectrometer.

Graphical overview

Schematic procedure for the homogenization and extraction of mouse liver tissue in preparation for LC-MS analysis (Created with BioRender.com)

Lipid-conjugated pH sensors based on fluorophores coupled to lipids are a powerful tool for monitoring pH gradients in biological microcompartments and reconstituted membrane systems. This protocol describes the synthesis of pH sensors based on amine-reactive pHrodo esters and the amino phospholipid phosphatidylethanolamine. The major features of this sensor include efficient partitioning into membranes and strong fluorescence under acidic conditions. The protocol described here can be used as a template to couple other amine-reactive fluorophores to phosphatidylethanolamines.

Graphical overview

Synthesis of lipid-conjugated pH sensors based on amine-reactive fluorophore esters and the aminophospholipid phosphoethanolamine (PE)

Glycerol-3-phosphate (G3P) is a conserved precursor of glycerolipids that also plays an important role in plant defense. Its levels and/or metabolism are also associated with many human disorders including insulin resistance, diabetes, obesity, and cancer, among others. In plants, G3P accumulates upon pathogen infection and is a critical component of systemic acquired resistance, which confers broad spectrum disease resistance against secondary infections. G3P also plays an important role in root-shoot-root signaling in soybean that regulates incompatible interactions with nitrogen-fixing bacteria. Thus, accurate quantification of G3P is key to drawing a valid conclusion regarding its role in diverse processes ranging from lipid biosynthesis to defense. G3P quantification is further compounded by its rapid degradation in extracts prepared at room temperature.

Here, we describe a simplified procedure for accurate quantitative analysis of G3P from plant tissues. G3P was extracted along with the internal standard ribitol, derivatized with N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and analyzed by gas chromatography–coupled mass spectrometry using selective ion mode. This procedure is simple, economical, and efficient, and does not involve isotopic internal standards or multiple-step derivatizations.

Redox status assessments are time-consuming, require a large volume of samples and great reagent amounts, and are not adequately described for methodological reproducibility. Here, the objective was to standardize redox balance determination, based on previously described spectrophotometric tests in pregnant rats, to improve precision, time dispensed, and the volume of samples and reagents, while maintaining accuracy and adequate cost benefits. This protocol summarizes oxidative stress markers, which focus on spectrophotometric tests for the assessment of thiobarbituric acid–reactive substances, reduced thiol groups, and hydrogen peroxide, as well as the antioxidant activity of superoxide dismutase, glutathione peroxidase, and catalase in washed erythrocyte and serum samples from full-term pregnant rats. For non-pregnant rats and other species, it is necessary to standardize these determinations, especially the sample volume. All measurements were normalized by the estimated protein concentrations in each sample. To establish optimum conditions for the reproducibility of the proposed methods, we describe all changes made in each assay’s steps based on the reference method reassessed for the new standardizations. Furthermore, the calculations of the concentrations or activities of each marker are presented. Thus, we demonstrate that the analysis of serum samples is easier and faster, but it is impossible to detect catalase activity. Furthermore, the proposed methods can be applied for redox balance determination, especially using smaller reagent amounts and lower sample volumes in lesser time without losing accuracy, as is required in obtaining samples during rat pregnancy.

The ability to stain lipid stores in vivo allows for the facile assessment of metabolic status in individuals of a population following genetic and environmental manipulation or pharmacological treatment. In the animal model Caenorhabditis elegans, lipids are stored in and mobilized from intracellular lipid droplets in the intestinal and hypodermal tissues. The abundance, size, and distribution of these lipids can be readily assessed by two staining methods for neutral lipids: Oil Red O (ORO) and Nile Red (NR). ORO and NR can be used to quantitatively measure lipid droplet abundance, while ORO can also define tissue distribution and lipid droplet size. C. elegans are a useful animal model in studying pathways relating to aging, fat storage, and metabolism, as their transparent nature allows for easy microscopic assessment of lipid droplets. This is done by fixation and permeabilization, staining with NR or ORO, image capture on a microscope, and computational identification and quantification of lipid droplets in individuals within a cohort. To ensure reproducibility in lipid measurements, we provide a detailed protocol to measure intracellular lipid dynamics in C. elegans.

Graphic abstract:

Flow chart depicting the preparation of C. elegans for fat staining protocols.

The nematode Caenorhabditis elegans has emerged as a popular model system for studying the regulation of lipid metabolism. Therefore, it is critical to develop a method for determining fat storage in individual worms. Oil Red O (ORO) staining has been validated as an accurate assessment for major fat storage in C. elegans. Here, we describe an optimized protocol for ORO staining of C. elegans and provide detailed instructions for quantifying the intensity of ORO signal in images acquired by light microscopy.

Lipid droplets store triacylglycerols (triglycerides) and sterol esters to regulate lipid and energy homeostasis. Triacylglycerol measurement is often performed during the investigation of lipid droplet formation and growth. This protocol describes a reliable method using a fluorometric lipid quantification kit to measure triacylglycerols extracted from HeLa cells, which were treated with oleic acid to trigger the formation of lipid droplets. The lipid quantification kit employs a lipid-binding molecule that emits bright fluorescence only when bound to extracted triacylglycerols, whose content can be quantified by a simple fluorescence readout.

Several studies suggest an important role of lipid metabolism in regulating longevity of Caenorhabditis elegans. Therefore, assays to quantify lipids have enormous value in understanding aging and pathologies associated with it. Approximately 70% of lipid metabolism genes in the nematode have orthologs in humans. Amenability of C. elegans to genetic manipulations has allowed investigations into the role of specific genetic factors in lipid metabolism. Here, we describe a protocol to quantify total triglycerides in C. elegans, which can be extended to studies of the effects of altered environmental and genetic factors on stored fats. This protocol quantifies the picomoles of the triglycerides, in whole worm lysate. Due to the sensitivity of the assay, it could help in identifying subtle changes in the total stored fat which are not discernible with microscopy techniques.