Radiolabeling Heme and Porphyrin with 14C-Glycine or 14C δ-Aminolevulinic Acid.

Radiolabeling enables the quantitation of newly synthesized heme and porphyrin, allowing us to distinguish heme synthesis rates from total cellular heme. Here, we describe a protocol for labeling heme with 14C-glycine or ALA and the sequential extraction of heme and porphyrin from the same samples for quantitation by liquid scintillation.

Methods for Cultivation of Bacteroides thetaiotaomicron and Analysis of Heme Metabolism by Mass Spectrometry and Spectroscopic Approaches.

Traditional studies of cellular metabolism have relied on the use of radioisotopes. These have clear disadvantages associated with safety and waste generation. Furthermore, detection of the labeled species by scintillation counting provides only a quantification of its presence or absence. The use of stable isotopes, by contrast, allows the application of powerful, orthogonal spectroscopic approaches such as nuclear magnetic resonance spectroscopy (NMR) and various mass spectrometric methods. Using stable isotope labeling to study heme metabolism requires integrating methods for (a) generating the heme in labeled forms, (b) cultivating and quantifying the organism of choice in chemically defined media, to which labeled compounds can be added, (c) recovering cellular components and/or spent growth media, and (d) analyzing these materials for the labeled species using spectroscopic and mass spectrometric methods. These methods are summarized here in the context of Bacteroides thetaiotaomicron, a generally nonpathogenic anaerobe and heme auxotroph.

Global protein turnover quantification in Escherichia coli reveals cytoplasmic recycling under nitrogen limitation.

Protein turnover is critical for proteostasis, but turnover quantification is challenging, and even in well-studied E. coli, proteome-wide measurements remain scarce. Here, we quantify the turnover rates of ~3200 E. coli proteins under 13 conditions by combining heavy isotope labeling with complement reporter ion quantification and find that cytoplasmic proteins are recycled when nitrogen is limited. We use knockout experiments to assign substrates to the known cytoplasmic ATP-dependent proteases. Surprisingly, none of these proteases are responsible for the observed cytoplasmic protein degradation in nitrogen limitation, suggesting that a major proteolysis pathway in E. coli remains to be discovered. Lastly, we show that protein degradation rates are generally independent of cell division rates. Thus, we present broadly applicable technology for protein turnover measurements and provide a rich resource for protein half-lives and protease substrates in E. coli, complementary to genomics data, that will allow researchers to study the control of proteostasis.

Direct Multi-Deuterium Labelling of Pirtobrutinib.

Herein, we demonstrate an efficient method for multi-deuterium labelling of pirtobrutinib-a Bruton's tyrosine kinase inhibitor recently approved by the FDA-using a straightforward hydrogen isotope exchange (HIE) reaction. A remarkably high level of deuterium incorporation was achieved using an excess of a Kerr-type iridium catalyst. The key factor in the significant deuterium labelling was the decision to employ a deuterium uniformly labelled solvent, chlorobenzene-d5, at an elevated temperature. Virtually, no d0-d3 species were detected, with only traces of d4-d5 isotopomers (< 5%) observable in the mass spectrum of pirtobrutinib-d8, fulfilling requirements for stable isotope-labelled internal standard. The labelled compound-mainly consisting of isotopomers d6-d9 at 82.4% of the total abundance-was isolated in a high yield (73%) and purity (99%). Noteworthy, fluorine group acting as a directing group was observed for the first time. Significant incorporation of deuterium in ortho-positions, exceeding 87%, was observed. Interestingly, chlorinated solvent used in the HIE reactions was non-specifically deuterated yielding up to 0.42 deuterium per chlorobenzene molecule even at an exceptionally low iridium catalyst loading of 4.17 × 10-2 mol%.

6-Plex Tandem Phosphorus Tags (TPT) for Accurate Quantitative Proteomics.

Isobaric chemical labeling is a widely used strategy for high-throughput quantitative proteomics based on mass spectrometry. However, commercially available reagents have high costs in applications as well as the sensitivity limitations for detection of the trace protein samples. Previously, we developed a 2-plex isobaric labeling strategy based on phosphorus chemistry for ultrasensitive proteome quantification with high accuracy. In this work, 6-plex tandem phosphorus tags (TPT) were developed with 3-fold increase in the multiplexing quantitative capacity compared to the 2-plex isobaric phosphorus reagents introduced previously. High isotope enrichment of 18O labeling was incorporated into the phosphoryl group with three exchangeable oxygen atoms by using commercially available H218O. The combinational incorporations of 18O atom in reporter ions and balance group set up the low-cost foundation for development of multiplex TPT reagents. The novel 6-plex TPT reagents could produce phosphoramidate as unique reporter ions with approximately 1 Da mass difference and thus enable 6-plex quantitative analysis in high-resolution ESI-MS/MS analysis. Using HeLa cell tryptic peptides, we concluded that 6-plex TPT reagents could facilitate large-scale accurate quantitative proteomics with very high labeling efficiency.

15N metabolic labeling-TMT multiplexing approach to facilitate the quantitation of glycopeptides derived from cell lines.

The study of glycoproteomics presents a set of unique challenges, primarily due to the low abundance of glycopeptides and their intricate heterogeneity, which is specific to each site. Glycoproteins play a crucial role in numerous biological functions, including cell signaling, adhesion, and intercellular communication, and are increasingly recognized as vital markers in the diagnosis and study of various diseases. Consequently, a quantitative approach to glycopeptide research is essential. One effective strategy to address this need is the use of multiplex glycopeptide labeling. By harnessing the synergies of 15N metabolic labeling via the isotopic detection of amino sugars with glutamine (IDAWG) technique for glycan parts and tandem mass tag (TMT)pro labeling for peptide backbones, we have developed a method that allows for the accurate quantification and comparison of multiple samples simultaneously. The adoption of the liquid chromatography-synchronous precursor selection (LC-SPS-MS3) technique minimizes fragmentation interference, enhancing data reliability, as shown by a 97% TMT labeling efficiency. This method allows for detailed, high-throughput analysis of 32 diverse samples from 231BR cell lines, using both 14N and 15N glycopeptides at a 1:1 ratio. A key component of our methodology was the precise correction for isotope and TMTpro distortions, significantly improving quantification accuracy to less than 5% distortion. This breakthrough enhances the efficiency and accuracy of glycoproteomic studies, increasing our understanding of glycoproteins in health and disease. Its applicability to various cancer cell types sets a new standard in quantitative glycoproteomics, enabling deeper investigation into glycopeptide profiles.

Hetero-Diels-Alder and CuAAC Click Reactions for Fluorine-18 Labeling of Peptides: Automation and Comparative Study of the Two Methods.

Radiolabeled peptides are valuable tools for diagnosis or therapies; they are often radiofluorinated using an indirect approach based on an F-18 prosthetic group. Herein, we are reporting our results on the F-18 radiolabeling of three peptides using two different methods based on click reactions. The first one used the well-known CuAAC reaction, and the second one is based on our recently reported hetero-Diels-Alder (HDA) using a dithioesters (thia-Diels-Alder) reaction. Both methods have been automated, and the 18F-peptides were obtained in similar yields and synthesis time (37-39% decay corrected yields by both methods in 120-140 min). However, to obtain similar yields, the CuAAC needs a large amount of copper along with many additives, while the HDA is a catalyst and metal-free reaction necessitating only an appropriate ratio of water/ethanol. The HDA can therefore be considered as a minimalist method offering easy access to fluorine-18 labeled peptides and making it a valuable additional tool for the indirect and site-specific labeling of peptides or biomolecules.

In Vivo PET Imaging of 89Zr-Labeled Natural Killer Cells and the Modulating Effects of a Therapeutic Antibody.

Natural killer (NK) cells can kill cancer cells via antibody-dependent cell-mediated cytotoxicity (ADCC): a tumor-associated IgG antibody binds to the Fcγ receptor CD16 on NK cells via the antibody Fc region and activates the cytotoxic functions of the NK cell. Here, we used PET imaging to assess NK cell migration to human epidermal growth factor receptor 2 (HER2)-positive HCC1954 breast tumors, examining the influence of HER2-targeted trastuzumab antibody treatment on NK cell tumor accumulation. Methods: Human NK cells from healthy donors were expanded ex vivo and labeled with [89Zr]Zr-oxine. In vitro experiments compared the phenotypic markers, viability, proliferation, migration, degranulation, and ADCC behaviors of both labeled (89Zr-NK) and unlabeled NK cells. Female mice bearing orthotopic human breast HCC1954 tumors were administered 89Zr-NK cells alongside trastuzumab treatment or a sham treatment and then scanned using PET/CT imaging over 7 d. Flow cytometry and γ-counting were used to analyze the presence of 89Zr-NK cells in liver and spleen tissues. Results: 89Zr cell radiolabeling yields measured 42.2% ± 8.0%. At an average specific activity of 16.7 ± 4.7 kBq/106 cells, 89Zr-NK cells retained phenotypic and functional characteristics including CD56 and CD16 expression, viability, migration, degranulation, and ADCC capabilities. In vivo PET/CT studies indicated predominant accumulation of 89Zr-NK cells in the liver and spleen. Ex vivo analyses of liver and spleen tissues indicated that the administered human 89Zr-NK cells retained their radioactivity in vivo and that 89Zr did not transfer to cells of murine soft tissues, thus validating this 89Zr PET method for NK cell tracking. Notably, 89Zr-NK cells migrated to HER2-positive tumors, both with and without trastuzumab treatment. Trastuzumab treatment was associated with an increased 89Zr-NK cell signal at days 1 and 3 after injection. Conclusion: In vitro, 89Zr-NK cells maintained key cellular and cytotoxic functions. In vivo, 89Zr-NK cells trafficked to HER2-postive tumors, with trastuzumab treatment correlating with enhanced 89Zr-NK infiltration. This study demonstrates the feasibility of using PET to image 89Zr-NK cell infiltration into solid tumors.

Tracing isotopically labeled selenium nanoparticles in plants via single-particle ICP-mass spectrometry.

Agronomic biofortification using selenium nanoparticles (SeNPs) shows potential for addressing selenium deficiency but further research on SeNPs-plants interaction is required before it can be effectively used to improve nutritional quality. In this work, single-particle inductively coupled plasma-mass spectrometry (SP-ICP-MS) was used for tracing isotopically labeled SeNPs (82SeNPs) in Oryza sativa L. tissues. For this purpose, SeNPs with natural isotopic abundance and 82SeNPs were synthesized by a chemical method. The NPs characterization by transmission electron microscopy (TEM) confirmed that enriched NPs maintained the basic properties of unlabeled NPs, showing spherical shape, monodispersity, and sizes in the nano-range (82.8 ± 6.6 nm and 73.2 ± 4.4 nm for SeNPs and 82SeNPs, respectively). The use of 82SeNPs resulted in an 11-fold enhancement in the detection power for ICP-MS analysis, accompanied by an improvement in the signal-to-background ratio and a reduction of the size limits of detection from 89.9 to 39.9 nm in SP-ICP-MS analysis. This enabled 82SeNPs to be tracked in O. sativa L. plants cultivated under foliar application of 82SeNPs. Tracing studies combining SP-ICP-MS and TEM-energy-dispersive X-ray spectroscopy data confirmed the uptake of intact 82SeNPs by rice leaves, with most NPs remaining in the leaves and very few particles translocated to shoots and roots. Translocation of Se from leaves to roots and shoots was found to be lower when applied as NPs compared to selenite application. From the size distributions, as obtained by SP-ICP-MS, it can be concluded that a fraction of the 82SeNPs remained within the same size range as that of the applied NP suspension, while other fraction underwent an agglomeration process in the leaves, as confirmed by TEM images. This illustrates the potential of SP-ICP-MS analysis of isotopically enriched 82SeNPs for tracing NPs in the presence of background elements within complex plant matrices, providing important information about the uptake, accumulation, and biotransformation of SeNPs in rice plants.

Quantification of gut microbiome metabolites using chemical isotope derivatization strategy combined with LC-MS/MS: Application in neonatal hypoxic-ischemic encephalopathy rat model.

The gut microbiome plays pivotal roles in various physiological and pathological processes, with key metabolites including short chain fatty acids (SCFAs), bile acids (BAs), and tryptophan (TRP) derivatives gaining significant attention for their diverse physiological roles. However, quantifying these metabolites presents challenges due to structural similarity, low abundance, and inherent technical limitations in traditional detection methods. In this study, we developed a precise and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method utilizing a chemical isotope derivatization technique employing 4-(aminomethyl)-N,N-dimethylaniline-d0/d6 (4-AND-d0/d6) reagents to quantify 37 typical gut microbiome-derived metabolites. This method achieved an impressive 1500-fold enhancement in sensitivity for detecting metabolites, compared to methods using non-derivatized, intact molecules. Moreover, the quantitative accuracy of our chemical isotope derivatization strategy proved comparable to the stable isotope labeled internal standards (SIL-IS) method. Subsequently, we successfully applied this newly developed method to quantify target metabolites in plasma, brain, and fecal samples obtained from a neonatal hypoxic-ischemic encephalopathy (HIE) rat model. The aim was to identify crucial metabolites associated with the progression of HIE. Overall, our sensitive and reliable quantification method holds promise in elucidating the role of gut microbiome metabolites in the pathogenesis of various diseases.

Isotopic labelings for mechanistic studies.

The intricate mechanisms in the biosynthesis of terpenes belong to the most challenging problems in natural product chemistry. Methods to address these problems include the structure-based site-directed mutagenesis of terpene synthases, computational approaches, and isotopic labeling experiments. The latter approach has a long tradition in biosynthesis studies and has recently experienced a revival, after genome sequencing enabled rapid access to biosynthetic genes and enzymes. Today, this allows for a combined approach in which isotopically labeled substrates can be incubated with recombinant terpene synthases. These clearly defined reaction setups can give detailed mechanistic insights into the reactions catalyzed by terpene synthases, and recent developments have substantially deepened our understanding of terpene biosynthesis. This chapter will discuss the state of the art and introduce some of the most important methods that make use of isotopic labelings in mechanistic studies on terpene synthases.

Metabolic Analysis of Intracellular Pathogenic Bacteria Using NMR.

Pathogen proliferation and virulence depend on available nutrients, and these vary when the pathogen moves from outside of the host cell (extracellular) to the inside of the host cell (intracellular). Nuclear Magnetic Resonance (NMR) is a versatile analytical method, which lends itself for metabolic studies. In this chapter, we describe how 1H NMR can be combined with a cellular infection model to study the metabolic crosstalk between a bacterial pathogen and its host both in the extracellular and intracellular compartments. Central carbon metabolism is highlighted by using glucose labeled with the stable isotope 13C.

A Sample Preparation Procedure for Isobaric Labeling-Based Single-Cell Proteomics.

Mass spectrometry-based proteomics has traditionally been limited by the amount of input material for analysis. Single-cell proteomics has emerged as a challenging discipline due to the ultra-high sensitivity required. Isobaric labeling-based multiplex strategies with a carrier proteome offer an approach to overcome the sensitivity limitations. Following this as the basic strategy, we show here the general workflow for preparing cells for single-cell mass spectrometry-based proteomics. This protocol can also be applied to manually isolated cells when large cells, such as cardiomyocytes, are difficult to isolate properly with conventional fluorescence-activated cell sorting (FACS) sorter methods.

Wideband PRM: Highly Accurate and Sensitive Method for High-Throughput Targeted Proteomics.

Targeted mass spectrometry (MS) approaches, which are powerful methods for uniquely and confidently quantifying a specific panel of proteins in complex biological samples, play a crucial role in validating and clinically translating protein biomarkers discovered through global proteomic profiling. Common targeted MS methods, such as multiple reaction monitoring (MRM) and parallel-reaction monitoring (PRM), employ specific mass spectrometric technologies to quantify protein levels by comparing the transitions of surrogate endogenous (ENDO) peptides with those of stable isotope-labeled (SIL) peptide counterparts. These methods utilizing amino acid analyzed (AAA) SIL peptides warrant sensitive and precise measurements required for targeted MS assays. Compared with MRM, PRM provides higher experimental throughput by simultaneously acquiring all transitions of the target peptides and thereby compensates for different ion suppressions among transitions of a target peptide. However, PRM still suffers different ion suppressions between ENDO and SIL peptides due to spray instability, as the ENDO and SIL peptides were monitored at different liquid chromatography (LC) retention times. Here we introduce a new targeted MS method, termed wideband PRM (WBPRM), that is designed for high-throughput targeted MS analysis. WBPRM employs a wide isolation window for simultaneous fragmentation of both ENDO and SIL peptides along with multiplexed single ion monitoring (SIM) scans for enhanced MS sensitivity of the target peptides. Compared with PRM, WBPRM was demonstrated to provide increased sensitivity, precision, and reproducibility of quantitative measurements of target peptides with increased throughput, allowing more target peptide measurements in a shortened experiment time. WBPRM is a straightforward adaptation to a manufacturer-provided MS method, making it an easily implementable technique, particularly in complex biological samples where the demand for higher precision, sensitivity, and efficiency is paramount.

Microbial consortium assembly and functional analysis via isotope labelling and single-cell manipulation of polycyclic aromatic hydrocarbon degraders.

Soil microbial flora constitutes a highly diverse and complex microbiome on Earth, often challenging to cultivation, with unclear metabolic mechanisms in situ. Here, we present a pioneering concept for the in situ construction of functional microbial consortia (FMCs) and introduce an innovative method for creating FMCs by utilizing phenanthrene as a model compound to elucidate their in situ biodegradation mechanisms. Our methodology involves single-cell identification, sorting, and culture of functional microorganisms, resulting in the formation of a precise in situ FMC. Through Raman-activated cell sorting-stable-isotope probing, we identified and isolated phenanthrene-degrading bacterial cells from Achromobacter sp. and Pseudomonas sp., achieving precise and controllable in situ consortia based on genome-guided cultivation. Our in situ FMC outperformed conventionally designed functional flora when tested in real soil, indicating its superior phenanthrene degradation capacity. We revealed that microorganisms with high degradation efficiency isolated through conventional methods may exhibit pollutant tolerance but lack actual degradation ability in natural environments. This finding highlights the potential to construct FMCs based on thorough elucidation of in situ functional degraders, thereby achieving sustained and efficient pollutant degradation. Single-cell sequencing linked degraders with their genes and metabolic pathways, providing insights regarding the construction of in situ FMCs. The consortium in situ comprising microorganisms with diverse phenanthrene metabolic pathways might offer distinct advantages for enhancing phenanthrene degradation efficiency, such as the division of labour and cooperation or communication among microbial species. Our approach underscores the importance of in situ, single-cell precision identification, isolation, and cultivation for comprehensive bacterial functional analysis and resource exploration, which can extend to investigate MFCs in archaea and fungi, clarifying FMC construction methods for element recycling and pollutant transformation in complex real-world ecosystems.

Elevated Temperature Effects on Protein Turnover Dynamics in Arabidopsis thaliana Seedlings Revealed by 15N-Stable Isotope Labeling and ProteinTurnover Algorithm.

Global warming poses a threat to plant survival, impacting growth and agricultural yield. Protein turnover, a critical regulatory mechanism balancing protein synthesis and degradation, is crucial for the cellular response to environmental changes. We investigated the effects of elevated temperature on proteome dynamics in Arabidopsis thaliana seedlings using 15N-stable isotope labeling and ultra-performance liquid chromatography-high resolution mass spectrometry, coupled with the ProteinTurnover algorithm. Analyzing different cellular fractions from plants grown under 22 °C and 30 °C growth conditions, we found significant changes in the turnover rates of 571 proteins, with a median 1.4-fold increase, indicating accelerated protein dynamics under thermal stress. Notably, soluble root fraction proteins exhibited smaller turnover changes, suggesting tissue-specific adaptations. Significant turnover alterations occurred with redox signaling, stress response, protein folding, secondary metabolism, and photorespiration, indicating complex responses enhancing plant thermal resilience. Conversely, proteins involved in carbohydrate metabolism and mitochondrial ATP synthesis showed minimal changes, highlighting their stability. This analysis highlights the intricate balance between proteome stability and adaptability, advancing our understanding of plant responses to heat stress and supporting the development of improved thermotolerant crops.

A Mass Spectrometry Strategy for Protein Quantification Based on the Differential Alkylation of Cysteines Using Iodoacetamide and Acrylamide.

Mass spectrometry has become the most prominent yet evolving technology in quantitative proteomics. Today, a number of label-free and label-based approaches are available for the relative and absolute quantification of proteins and peptides. However, the label-based methods rely solely on the employment of stable isotopes, which are expensive and often limited in availability. Here we propose a label-based quantification strategy, where the mass difference is identified by the differential alkylation of cysteines using iodoacetamide and acrylamide. The alkylation reactions were performed under identical experimental conditions; therefore, the method can be easily integrated into standard proteomic workflows. Using high-resolution mass spectrometry, the feasibility of this approach was assessed with a set of tryptic peptides of human serum albumin. Several critical questions, such as the efficiency of labeling and the effect of the differential alkylation on the peptide retention and fragmentation, were addressed. The concentration of the quality control samples calculated against the calibration curves were within the ±20% acceptance range. It was also demonstrated that heavy labeled peptides exhibit a similar extraction recovery and matrix effect to light ones. Consequently, the approach presented here may be a viable and cost-effective alternative of stable isotope labeling strategies for the quantification of cysteine-containing proteins.

Unexpected Intermolecular C-H···O Hydrogen Bonds and 1H NMR Chemical Shifts in a Key Linker for Fluorine-18 Labeling of Dimeric Drugs.

The compound 2-{[(trifluoromethyl)sulfonyl]oxy}propane-1,3-diyl bis(4-methylbenzenesulfonate) (TPB) is a crucial intermediate in the synthesis of 18F-radiolabeled cromolyn derivatives. In this work, we combine 1H NMR spectroscopy, X-ray crystallography, ab initio molecular dynamics, and NMR calculations to examine the structure, interactions, and solvation dynamics of the TPB molecule. In CDCl3, the CH2 groups within its glyceryl-derived linker exhibit a single set of proton signals in the 1H NMR measurements. However, when TPB is dissolved in DMSO-d6, distinct splitting patterns emerge despite its seemingly symmetric chemical structure. Crystallographic analysis further unveils the absence of overall symmetry in its three-dimensional arrangement. To elucidate these unique NMR features, we carry out ab initio molecular dynamics simulations and characterize the solvation structures and dynamics of TPB in CHCl3 and DMSO solutions. In contrast to the predominantly nonpolar nature of the CHCl3 solvents, DMSO directly participates in C-H···O hydrogen-bonding interactions with the solute molecule, leading to the splitting of its -CH2 chemical shifts into two distinct distributions. The comprehensive understanding of the structure and solvation interactions of TPB provides essential insights into its application in the radiofluorination reactions of cromolyn derivatives and holds promise for the future development of radiolabeled dimeric drugs.

Efficient Segmental Isotope Labeling of Integral Membrane Proteins for High-Resolution NMR Studies.

High-resolution structural NMR analyses of membrane proteins are challenging due to their large size, resulting in broad resonances and strong signal overlap. Among the isotope labeling methods that can remedy this situation, segmental isotope labeling is a suitable strategy to simplify NMR spectra and retain high-resolution structural information. However, protein ligation within integral membrane proteins is complicated since the hydrophobic protein fragments are insoluble, and the removal of ligation side-products is elaborate. Here, we show that a stabilized split-intein system can be used for rapid and high-yield protein trans-splicing of integral membrane proteins under denaturing conditions. This setup enables segmental isotope labeling experiments within folded protein domains for NMR studies. We show that high-quality NMR spectra of markedly reduced complexity can be obtained in detergent micelles and lipid nanodiscs. Of note, the nanodisc insertion step specifically selects for the ligated and correctly folded membrane protein and simultaneously removes ligation byproducts. Using this tailored workflow, we show that high-resolution NMR structure determination is strongly facilitated with just two segmentally isotope-labeled membrane protein samples. The presented method will be broadly applicable to structural and dynamical investigations of (membrane-) proteins and their complexes by solution and solid-state NMR but also other structural methods where segmental labeling is beneficial.

Intestinal absorption of sphingosine: new insights on generated ceramide species using stable isotope tracing in vitro.

Dietary sphingomyelin (SM) has been reported to favorably modulate postprandial lipemia. Mechanisms underlying these beneficial effects on cardiovascular risk markers are not fully elucidated. Rodent studies showed that tritiated SM was hydrolyzed in the intestinal lumen into ceramides (Cer) and further to sphingosine (SPH) and fatty acids (FA) that were absorbed by the intestine. Our objective was to investigate the uptake and metabolism of SPH and/or tricosanoic acid (C23:0), the main FA of milk SM, as well as lipid secretion in Caco-2/TC7 cells cultured on semipermeable inserts. Mixed micelles (MM) consisting of different digested lipids and taurocholate were prepared without or with SPH, SPH and C23:0 (SPH+C23:0), or C23:0. Triglycerides (TG) were quantified in the basolateral medium, and sphingolipids were analyzed by tandem mass spectrometry. TG secretion increased 11-fold in all MM-incubated cells compared with lipid-free medium. Apical supply of SPH-enriched MM led to increased concentrations of total Cer in cells, and coaddition of C23:0 in SPH-enriched MM led to a preferential increase of C23:0 Cer and C23:0 SM. Complementary experiments using deuterated SPH demonstrated that SPH-d9 was partly converted to sphingosine-1-phosphate-d9, Cer-d9, and SM-d9 within cells incubated with SPH-enriched MM. A few Cer-d9 (2% of added SPH-d9) was recovered in the basolateral medium of (MM+SPH)-incubated cells, especially C23:0 Cer-d9 in (MM+SPH+C23:0)-enriched cells. In conclusion, present results indicate that MM enriched with (SPH+C23:0), such as found in postprandial micelles formed after milk SM ingestion, directly impacts sphingolipid endogenous metabolism in enterocytes, resulting in the secretion of TG-rich particles enriched with C23:0 Cer.