Lipid Fingerprints Research Articles

Lipid Profiling in Cancer Diagnosis with Hand-Held Ambient Mass Spectrometry Probes: Addressing the Late-Stage Performance Concerns.

Untargeted lipid fingerprinting with hand-held ambient mass spectrometry (MS) probes without chromatographic separation has shown promise in the rapid characterization of cancers. As human cancers present significant molecular heterogeneities, careful molecular modeling and data validation strategies are required to minimize late-stage performance variations of these models across a large population. This review utilizes parallels from the pitfalls of conventional protein biomarkers in reaching bedside utility and provides recommendations for robust modeling as well as validation strategies that could enable the next logical steps in large scale assessment of the utility of ambient MS profiling for cancer diagnosis. Six recommendations are provided that range from careful initial determination of clinical added value to moving beyond just statistical associations to validate lipid involvements in disease processes mechanistically. Further guidelines for careful selection of suitable samples to capture expected and unexpected intragroup variance are provided and discussed in the context of demographic heterogeneities in the lipidome, further influenced by lifestyle factors, diet, and potential intersect with cancer lipid pathways probed in ambient mass spectrometry profiling studies.

Lipid fingerprinting of yellow mealwormTenebrio molitor by untargeted liquid chromatography-mass spectrometry

Insects such asTenebrio molitor have been considered an alternative source of nutrition for animals and have also been adopted as human food throughout history, especially in Asia and Africa. Lipids are the second most abundant component followed by proteins. However, studies focusing on comprehensive lipid composition analsysis of these widely reared species are limited. The untargeted lipidomic analysis of yellow mealworm larvae (T. molitor) led to the identification of several lipid molecular species from lipid classes such as: free fatty acids, sphingolipids, phospholipids, and triacylglycerols. The results revealed that polyunsaturated fatty acids (PUFAs) (45%) are the most abundant fatty acids, followed by monounsaturated fatty acids (MUFAs) (42%) and saturated fatty acids (13%). Fatty acids such as FA 18:1 and FA 18:2 are the most abundant fatty acids and are substantially enriched in other complex lipids in the form of esters. Moreover, functional lipids such as sphingomyelins, ceramides, cardiolipins, phosphatidylinositols, and phosphatidylethanolamines were characterised for the first time, with a large number of MUFAs and PUFAs as their main acyl chains. Overall, our data showed the occurrence of multiple structurally diverse lipids inT. molitor, suggesting that mealworms are not only enriched with proteins but also have several functional lipids, which are highly beneficial to human and animal health. Thus, the larvae ofT. molitor could serve as a promising candidate for the development of functional food and feed products.

An Improved Method for Rapid Detection of Mycobacterium abscessus Complex Based on Species-Specific Lipid Fingerprint by Routine MALDI-TOF.

Rapid diagnostics of bacterial infection is the key to successful recovery and eradication of the disease. Currently, identification of bacteria is based on the detection of highly abundant proteins, mainly ribosomal proteins, by routine MALDI-TOF mass spectrometry. However, relying solely on proteins is limited in subspecies typing for some pathogens. This is the case for, for example, the mycobacteria belonging to the Mycobacterium abscessus (MABS) complex, which is classified into three subspecies, namely, M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense. Being able to detect bacteria accurately and rapidly at the subspecies level could not only reliably identify the pathogen causing the disease but also enable better antibiotic stewardship. For instance, M. abscessus subsp. abscessus and M. abscessus subsp. bolletii possess a functional erm41 (erythromycin ribosomal methylation gene 41) gene, whilst M. abscessus subsp. massiliense does not, resulting in differences in macrolide antibiotic (e.g., clarithromycin and azithromycin) susceptibilities. This presents a challenge for physicians when designing an appropriate treatment regimen. To address this challenge, in addition to proteins, species-specific lipids have now been considered as a game changer in clinical microbiology diagnostics. However, their extraction can be time-consuming, and analysis requires the use of apolar toxic organic solvents (e.g., chloroform). Here, we present a new method to accurately detect species and subspecies, allowing the discrimination of the mycobacteria within the MABS complex and relying on the use of ethanol. We found that a combination of the matrix named super-DHB with 25% ethanol with a bacterial suspension at McFarland 20 gave robust and reproducible data, allowing the discrimination of the bacteria within the MABS complex strains tested in this study (n = 9). Further investigations have to be conducted to validate the method on a larger panel of strains for its use in diagnostic laboratories.

Multi-scale simulations of the T cell receptor reveal its lipid interactions, dynamics and the arrangement of its cytoplasmic region.

The T cell receptor (TCR-CD3) initiates T cell activation by binding to peptides of Major Histocompatibility Complexes (pMHC). The TCR-CD3 topology is well understood but the arrangement and dynamics of its cytoplasmic tails remains unknown, limiting our grasp of the signalling mechanism. Here, we use molecular dynamics simulations and modelling to investigate the entire TCR-CD3 embedded in a model membrane. Our study demonstrates conformational changes in the extracellular and transmembrane domains, and the arrangement of the TCR-CD3 cytoplasmic tails. The cytoplasmic tails formed highly interlaced structures while some tyrosines within the immunoreceptor tyrosine-based activation motifs (ITAMs) penetrated the hydrophobic core of the membrane. Interactions between the cytoplasmic tails and phosphatidylinositol phosphate lipids in the inner membrane leaflet led to the formation of a distinct anionic lipid fingerprint around the TCR-CD3. These results increase our understanding of the TCR-CD3 dynamics and the importance of membrane lipids in regulating T cell activation.

High-Resolution Human Kidney Molecular Histology by Imaging Mass Spectrometry of Lipids.

For many years, traditional histology has been the gold standard for the diagnosis of many diseases. However, alternative and powerful techniques have appeared in recent years that complement the information extracted from a tissue section. One of the most promising techniques is imaging mass spectrometry applied to lipidomics. Here, we demonstrate the capabilities of this technique to highlight the architectural features of the human kidney at a spatial resolution of 10 μm. Our data demonstrate that up to seven different segments of the nephron and the interstitial tissue can be readily identified in the sections according to their characteristic lipid fingerprints and that such fingerprints are maintained among different individuals (n = 32). These results set the foundation for further studies on the metabolic bases of the diseases affecting the human kidney.

Metabolomics and lipid profile analysis of Coccomyxa melkonianii SCCA 048

With an unsupervised GC–MS metabolomics approach, polar metabolite changes of the microalgae Coccomyxa melkonianii SCCA 048 grown under standard conditions for seven weeks were studied. C. melkonianii was sampled at the Rio Irvi River, in the mining site of Montevecchio-Ingurtosu (Sardinia, Italy), which is severely contaminated by heavy metals and shows high concentrations of sulfates. The partial-least-square (PLS) analysis of the GC–MS data indicated that growth of C. melkonianii was characterized by an increase of the levels of threonic acid, myo-inositol, malic acid, and fumaric acid. Furthermore, at the sixth week of exponential phase the lipid fingerprint of C. melkonianii was studied by LC-QTOF-MS. C. melkonianii lipid extract characterized through an iterative MS/MS analysis showed the following percent levels: 61.34 ± 0.60% for triacylglycerols (TAG); 11.55 ± 0.09% for diacylglyceryltrimethyl homoserines (DGTS), 11.34 ± 0.10% for sulfoquinovosyldiacylglycerols (SQDG) and, 5.29 ± 0.04% for lysodiacylglyceryltrimethyl homoserines (LDGTS). Noteworthy, we were able to annotate different fatty acid ester of hydroxyl fatty acid, such as FAHFA (18:1_20:3), FAHFA (18:2_20:4), FAHFA (18:0_20:2), and FAHFA (18:1_18:0), with relevant biological activity. These approaches can be useful to study the biochemistry of this extremophile algae in the view of its potential exploitation in the phycoremediation of polluted mining areas.

Fatty Acid Unsaturation Degree of Plasma Exosomes in Colorectal Cancer Patients: A Promising Biomarker.

Even though colorectal cancer (CRC) is one of the most preventable cancers, it is currently one of the deadliest. Worryingly, incidence in people <50 years has increased unexpectedly, and for unknown causes, despite the successful implementation of screening programs in the population aged >50 years. Thus, there is a need to improve early diagnosis detection strategies by identifying more precise biomarkers. In this scenario, the analysis of exosomes is given considerable attention. Previously, we demonstrated the exosome lipidome was able to classify CRC cell lines according to their malignancy. Herein, we investigated the use of the lipidome of plasma extracellular vesicles as a potential source of non-invasive biomarkers for CRC. A plasma exosome-enriched fraction was analyzed from patients undergoing colonoscopic procedure. Patients were divided into a healthy group and four pathological groups (patients with hyperplastic polyps; adenomatous polyps; invasive neoplasia (CRC patients); or hereditary non-polyposis CRC. The results showed a shift from 34:1- to 38:4-containing species in the pathological groups. We demonstrate that the ratio Σ34:1-containing species/Σ38:4-containing species has the potential to discriminate between healthy and pathological patients. Altogether, the results reinforce the utility of plasma exosome lipid fingerprint to provide new non-invasive biomarkers in a clinical context.

Metabolic fluxes for nutritional flexibility of Mycobacterium tuberculosis.

The co‐catabolism of multiple host‐derived carbon substrates is required by Mycobacterium tuberculosis (Mtb) to successfully sustain a tuberculosis infection. However, the metabolic plasticity of this pathogen and the complexity of the metabolic networks present a major obstacle in identifying those nodes most amenable to therapeutic interventions. It is therefore critical that we define the metabolic phenotypes of Mtb in different conditions. We applied metabolic flux analysis using stable isotopes and lipid fingerprinting to investigate the metabolic network of Mtb growing slowly in our steady‐state chemostat system. We demonstrate that Mtb efficiently co‐metabolises either cholesterol or glycerol, in combination with two‐carbon generating substrates without any compartmentalisation of metabolism. We discovered that partitioning of flux between the TCA cycle and the glyoxylate shunt combined with a reversible methyl citrate cycle is the critical metabolic nodes which underlie the nutritional flexibility of Mtb. These findings provide novel insights into the metabolic architecture that affords adaptability of bacteria to divergent carbon substrates and expand our fundamental knowledge about the methyl citrate cycle and the glyoxylate shunt.

A Drastic Shift in Lipid Adducts in Colon Cancer Detected by MALDI-IMS Exposes Alterations in Specific K+ Channels.

Simple SummaryColorectal cancer (CRC) is one of the most preventable yet deadliest cancers, one reason being that it involves very different lesions. Currently, there is a great international effort to improve CRC classification using as many molecular features as possible. A cutting-edge technique, imaging mass spectrometry, is used to enable the visualization of the bidimensional (2D) distribution of molecules across tissues in order to study how the composition of the cell membrane, in particular membrane lipids, changes in tumors. Our previous studies indicate that lipid composition is highly sensitive to cell alterations. Importantly, during the analysis, we are also able to establish changes in charged lipids, observations that can be misinterpreted. A close study of our results alongside information from public databases leads to the identification of gene coding for a potassium channel that could account for our observations and could represent a suitable target for drug development.Even though colorectal cancer (CRC) is one of the most preventable cancers, it is one of the deadliest, and recent data show that the incidence in people <50 years has unexpectedly increased. While new techniques for CRC molecular classification are emerging, no molecular feature is as yet firmly associated with prognosis. Imaging mass spectrometry (IMS) lipidomic analyses have demonstrated the specificity of the lipid fingerprint in differentiating pathological from healthy tissues. During IMS lipidomic analysis, the formation of ionic adducts is common. Of particular interest is the [Na+]/[K+] adduct ratio, which already functions as a biomarker for homeostatic alterations. Herein, we show a drastic shift of the [Na+]/[K+] adduct ratio in adenomatous colon mucosa compared to healthy mucosa, suggesting a robust increase in K+ levels. Interrogating public databases, a strong association was found between poor diagnosis and voltage-gated potassium channel subunit beta-2 (KCNAB2) overexpression. We found this overexpression in three CRC molecular subtypes defined by the CRC Subtyping Consortium, making KCNAB2 an interesting pharmacological target. Consistently, its pharmacological inhibition resulted in a dramatic halt in commercial CRC cell proliferation. Identification of potential pharmacologic targets using lipid adduct information emphasizes the great potential of IMS lipidomic techniques in the clinical field.

ELOVL5 Participates in Embryonic Lipid Determination of Cellular Membranes and Cytoplasmic Droplets.

Embryonic lipids are crucial for the formation of cellular membranes and dynamically participate in metabolic pathways. Cells can synthesize simple fatty acids, and the elongation of fatty acids facilitates the formation of complex lipids. The aim of this work was to investigate the involvement of the elongation of very long chain fatty acid enzyme 5 (ELOVL5) in embryonic development and lipid determination. Bovine embryos were produced in vitro using a standard protocol and randomly divided to receive one of three treatments at Day 4: morpholino (Mo) gene expression knockdown assay for ELOVL5 (ELOVL5-Mo), Mo antisense oligonucleotides for the thalassemic β-globulin human mRNA (technical control Mo), and placebo (biological control). The phenotypes of embryonic development, cell number, ELOVL5 protein abundance, lipid droplet deposits, and lipid fingerprint were investigated. No detrimental effects (p > 0.05) were observed on embryo development in terms of cleavage (59.4 ± 3.5%, 63.6 ± 4.1%, and 65.4 ± 2.2%), blastocyst production (31.3 ± 4.2%, 28.1 ± 4.9%, and 36.1 ± 2.1%), and blastocyst cell number (99.6 ± 7.7, 100.2 ± 6.2, 86.8 ± 5.6), respectively, for biological control, technical control Mo, and ELOVL5-Mo. ELOVL5 protein abundance and cytoplasmic lipid droplet deposition were increased (p < 0.05) in ELOVL5-Mo–derived blastocysts compared with the controls. However, seven lipid species, including phosphatidylcholines, phosphatidylethanolamines, and triacylglycerol, were downregulated in the ELOVL5-Mo–derived blastocysts compared with the biological control. Therefore, ELOVL5 is involved in the determination of embryonic lipid content and composition. Transient translational blockage of ELOVL5 reduced the expression of specific lipid species and promoted increased cytoplasmic lipid droplet deposition, but with no apparent deleterious effect on embryonic development and blastocyst cell number.

Shotgun Bacterial Lipid A Analysis Using Routine MALDI-TOF Mass Spectrometry.

Detection of bacterial lipids and particularly the lipid A, the lipid anchor of the lipopolysaccharide, can be very challenging and requires a certain level of expertise. Here, this chapter describes a straightforward and simple method for the analysis of bacterial lipid A. In addition, such approach, lipid fingerprint, has the potential to be applied to other bacteria such as mycobacteria.

Investigating the lipid fingerprint of SLC6 neurotransmitter transporters: a comparison of dDAT, hDAT, hSERT, and GlyT2

The local lipid annulus, or "fingerprint", of four SLC6 transporters (dDAT, hDAT, hSERT, and GlyT2) embedded in a complex neuronal membrane were compared and characterised using molecular dynamics. Our analysis included the development of new tools to improve membrane leaflet detection and the analysis of leaflet-dependent properties. Overall, the lipid fingerprints of the four transporters are comprised of similar lipids when grouped by headgroup or tail saturation. The enrichment and depletion of specific lipids, including sites of cholesterol contacts, varies between transporters. The subtle differences in lipid fingerprints results in varying membrane biophysical properties near the transporter. Our results highlight that the lipid-fingerprint of SLC6 transporters in complex membranes is highly dependent on membrane composition. Our results further characterize how the presence and identity of membrane proteins affects the complex interplay of lipid-protein interactions, influencing the local lipid environment and membrane biophysical properties.

Characteristic MALDI-MS lipid profiles of Gir, Holstein and crossbred (Gir x Holstein) oocytes recovered by ovum pick-up

The differential expression of many productive and reproductive traits has already been associated with the genotype effect observed between Bos taurus indicus (Bos indicus) and Bos taurus taurus (Bos taurus) breeds. Lipids have a crucial role during oocyte development. The aim of this work was to characterize the lipid profiles of oocytes recovered from dairy breeds: Gir (Bos indicus), Holstein (Bos taurus) and their crossbred (Gir x Holstein). Cumulus-oocyte complexes were collected by ovum pick-up from Gir (n=21), Holstein (n=36), and crossbreed (n=33) cows and submitted to MALDI-MS lipid fingerprint analysis. Multi- and univariate analyses were used to characterize lipid profiles of oocytes derived from each breed. Third lipid profiles (Gir n=7; Holstein n=12; and crossbred n=11) were evaluated and allowed characterization of oocyte's lipids among breeds. The lipid profiles from Gir and Holstein were observed to be the most distinct, whereas Gir and crossbred oocytes presented greater similarities. Overall, a wide-range oocyte lipid profile characterization from distinctive bovine dairy breeds recovered by OPU were provided. We have identified two distinguishable lipid profile patterns among groups and revealed that phospholipids and triacylglycerol were the predominantly lipid assigned in Gir (Bos indicus) and Holstein (Bos taurus) oocytes, respectively.

Comparative lipidomic analysis of mammalian retinal ganglion cells and M\xfcller glia in situ and in vitro using High-Resolution Imaging Mass Spectrometry

In order to better understand retinal physiology, alterations to which underlie some ocular diseases, we set out to establish the lipid signature of two fundamental cell types in the retina, Müller Glia and Retinal Ganglion Cells (RGCs). Moreover, we compared the lipid signature of these cells in sections (in situ), as well as after culturing the cells and isolating their cell membranes (in vitro). The lipidome of Müller glia and RGCs was analyzed in porcine retinal sections using Matrix Assisted Laser Desorption Ionization Imaging Mass Spectrometry (MALDI-IMS). Isolated membranes, as well as whole cells from primary cell cultures of RGCs and Müller glia, were printed onto glass slides using a non-contact microarrayer (Nano Plotter), and a LTQ-Orbitrap XL analyzer was used to scan the samples in negative ion mode, thereafter identifying the RGCs and Müller cells immunohistochemically. The spectra acquired were aligned and normalized against the total ion current, and a statistical analysis was carried out to select the lipids specific to each cell type in the retinal sections and microarrays. The peaks of interest were identified by MS/MS analysis. A cluster analysis of the MS spectra obtained from the retinal sections identified regions containing RGCs and Müller glia, as confirmed by immunohistochemistry in the same sections. The relative density of certain lipids differed significantly (p-value ≤ 0.05) between the areas containing Müller glia and RGCs. Likewise, different densities of lipids were evident between the RGC and Müller glia cultures in vitro. Finally, a comparative analysis of the lipid profiles in the retinal sections and microarrays identified six peaks that corresponded to a collection of 10 lipids characteristic of retinal cells. These lipids were identified by MS/MS. The analyses performed on the RGC layer of the retina, on RGCs in culture and using cell membrane microarrays of RGCs indicate that the lipid composition of the retina detected in sections is preserved in primary cell cultures. Specific lipid species were found in RGCs and Müller glia, allowing both cell types to be identified by a lipid fingerprint. Further studies into these specific lipids and of their behavior in pathological conditions may well help identify novel therapeutic targets for ocular diseases.

Integration of 3D-printing for a desorption electrospray ionization source for mass spectrometry.

The field of ambient ionization mass spectrometry has witnessed the development of many novel and capable methods for the analysis and imaging of surfaces, with desorption electrospray ionization being a prominent technique that has been commercialized. The adaptation of this technique to existing mass spectrometry platforms requires a laboratory-built solution manufactured with the capability of fine, stable adjustments of the electrospray emitter for liquid or solid sampling purposes. The development, fabrication, and machining require tens of hours of labor for many custom solutions. Herein described is a highly modifiable alternative approach for the fabrication of a desorption electrospray ionization source, using computer-aided design and fused deposition modeling to three-dimensionally print a source platform that utilizes standard accessories of a commercial Bruker Daltonics mass spectrometer. Three-dimensional printing allows for the inexpensive, rapid development of highly modifiable plastic parts, with the total printing time of the apparatus requiring a singular day and only a few dollars of material using a consumer grade printer. To demonstrate the utility of this printed desorption electrospray ionization source, it was fitted on an unmodified Fourier transform ion cyclotron resonance mass spectrometer for a lipid fingerprint analysis in serial sections of rat brain tissue, with the acquisition of line scans of dye-coated slides for the demonstration of serial acquisition.

MALDI-TOF Mass Spectrometry Revealed Significant Lipid Variations in Follicular Fluid and Somatic Follicular Cells but Not in Enclosed Oocytes between the Large Dominant and Small Subordinate Follicles in Bovine Ovary.

Lipid metabolism in ovarian follicular cells supports the preparation of an enclosed oocyte to ovulation. We aimed to compare lipid composition of a dominant large follicle (LF) and subordinated small follicles (SFs) within the same ovaries. Mass spectrometry imaging displayed the differences in the distribution of several lipid features between the different follicles. Comparison of lipid fingerprints between LF and SF by Matrix Assisted Laser Desorption/Ionisation Time-Of-Flight (MALDI-TOF) mass spectrometry revealed that in the oocytes, only 8 out of 468 detected lipids (1.7%) significantly changed their abundance (p < 0.05, fold change > 2). In contrast, follicular fluid (FF), granulosa, theca and cumulus cells demonstrated 55.5%, 14.9%, 5.3% and 9.8% of significantly varied features between LF and SF, respectively. In total, 25.2% of differential lipids were identified and indicated potential changes in membrane and signaling lipids. Tremendous changes in FF lipid composition were likely due to the stage specific secretions from somatic follicular cells that was in line with the differences observed from FF extracellular vesicles and gene expression of candidate genes in granulosa and theca cells between LF and SF. In addition, lipid storage in granulosa and theca cells varied in relation to follicular size and atresia. Differences in follicular cells lipid profiles between LF and SF may probably reflect follicle atresia degree and/or accumulation of appropriate lipids for post-ovulation processes as formation of corpus luteum. In contrast, the enclosed oocyte seems to be protected during final follicular growth, likely due in part to significant lipid transformations in surrounding cumulus cells. Therefore, the enclosed oocyte could likely keep lipid building blocks and energy resources to support further maturation and early embryo development.

Lipid Fingerprinting of Different Material Sources by UPLC-Q-Exactive Orbitrap/MS Approach and Their Zebrafish-Based Activities Comparison.

Lipids are considered to be critical contributors to nutrition, structural function, metabolic function, and other biological roles. They vary from one biological source to another. Here, the lipids from three common sources (soybean, egg yolk, and shrimp head) were comprehensively compared and characterized using the lipidomics approach, together with the UPLC-Q-Exactive Orbitrap/MS method. A total of 3027 lipid structures containing 778 fatty acids, 750 glycerolipids, 1283 glycerophospholipids, 200 sphingolipids, and 16 sterol lipids were first identified and quantified in these resources. The characteristic lipid species with significant differences among groups were determined by lipidomics analysis. Besides, the antithrombotic, antioxidant, and anti-inflammatory activities were evaluated based on the zebrafish model. The correlation between differential lipids and activities was also analyzed. Our comprehensive lipidomics profiling and bioactivities of lipids from different sources in vivo can provide evidence for their future applications.

MALDI MS Analysis to Investigate the Lipid Composition of Sperm

Background: The sperm plasma membrane meets the requirements of sperm transit through the female genital tract and subsequent fertilization. Commonly, the (phospho)lipid composition of sperm is characterized by tremendous amounts of highly unsaturated fatty acyl residues such as docosahexaenoic and docosapentaenoic acid. While human sperm contain almost exclusively diacyl lipids, many animal sperm additionally contain significant amounts of ether lipids such as alkylacyl- and alkenyl-acyl lipids (plasmalogens). Hypothesis/Objective: It is suggested that deviations from the typical lipid composition are indicative of pathological changes. Therefore, simple methods to elucidate the sperm lipid composition are essential. Method: Matrix-assisted laser desorption and ionization (MALDI) mass spectrometry (MS) is a fast and simple method. Since the selection of the most suitable matrix is a crucial step in MALDI MS, this topic will be highlighted. It will also be shown that MALDI MS can be easily combined with thin-layer chromatography to overcome ion suppression effects. Results: The lipid composition of sperm from different species can be elucidated by MALDI MS. However, different matrix compounds have to be used to record positive and negative ion mass spectra. Since some sperm (glyco)lipids are characterized by the presence of sulfate residues which suppress the detection of less acidic lipids in the negative ion mode, previous separation is often necessary. It will be also emphasized that plasmalogens can be easily identified by either enzymatic digestion or treatment with acids. Conclusion: MALDI MS is a reliable method to obtain sperm lipid fingerprints in a simple and convenient way.

A Novel Lipidomics-Based Approach to Evaluating the Risk of Clinical Hepatotoxicity Potential of Drugs in 3D Human Microtissues.

The importance of adsorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis is expected to grow substantially due to recent failures in detecting severe toxicity issues of new chemical entities during preclinical/clinical development. Traditionally, safety risk assessment studies for humans have been conducted in animals during advanced preclinical or clinical phase of drug development. However, potential drug toxicity in humans now needs to be detected in the drug discovery process as soon as possible without reliance on animal studies. The "omics", such as genomics, proteomics, and metabolomics, have recently entered pharmaceutical research in both drug discovery and drug development, but to the best of our knowledge, no applications in high-throughput safety risk assessment have been attempted so far. This paper reports an innovative method to anticipate adverse drug effects in an early discovery phase based on lipid fingerprints using human three-dimensional microtissues. The risk of clinical hepatotoxicity potential was evaluated for a data set of 22 drugs belonging to five different therapeutic chemical classes and with various drug-induced liver injury effect. The treatment of microtissues with repeated doses of each drug allowed collecting lipid fingerprints for five time points (2, 4, 7, 9, and 11 days), and multivariate statistical analysis was applied to search for correlations with the hepatotoxic effect. The method allowed clustering of the drugs based on their hepatotoxic effect, and the observed lipid impairments for a number of drugs was confirmed by literature sources. Compared to traditional screening methods, here multiple interconnected variables (lipids) are measured simultaneously, providing a snapshot of the cellular status from the lipid perspective at a molecular level. Applied here to hepatotoxicity, the proposed workflow can be applied to several tissues, being tridimensional microtissues from various origins.

Microarray and Mass Spectrometry-Based Methodology for Lipid Profiling of Tissues and Cell Cultures.

The recent developments in mass spectrometry have revealed the importance of lipids as biomarkers in the context of different diseases and as indicators of the cell's homeostasis. However, further advances are required to unveil the complex relationships between lipid classes and lipid species with proteins. Here, we present a new methodology that combines microarrays with mass spectrometry to obtain the lipid fingerprint of samples of a different nature in a standardized and fast way, with minimal sample consumption. As a proof of concept, we use the methodology to obtain the lipid fingerprint of 20 rat tissues and to create a lipid library for tissue classification. Then, we combine those results with immunohistochemistry and enzymatic assays to unveil the relationship between some lipid species and two enzymes. Finally, we demonstrate the performance of the methodology to explore changes in lipid composition of the nucleus accumbens from mice subjected to two lipid diets.