bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2021.04.16.440128

Validation and Application of a Protocol for the Extraction and Quantitative Analysis of Sphingomyelin in Erythrocyte Membranes of Patients with NAFLD

Papadopoulos, C.·Mimidis, K.·Tentes, I.·Anagnostopoulos, K.
View original source ↗Export metadata ↓

Abstract

A set of constituents of the erythrocyte membrane lipidome has been proposed to serve as biomarkers for liver disease and acute coronary syndrome. In erythrocytes, sphingomyelin hydrolysis provides ceramide, a signaling lipid necessary for phosphatidylserine exposure and eryptosis. Phosphatidylserine exposure further amplifies hepatic inflammation and fibrosis during non-alcoholic fatty liver disease (NAFLD). In this study, we developed and applied a quantitative TLC for erythrocyte membrane sphingomyelin of NAFLD patients. We also compared 10 extraction methods for the isolation of sphingomyelin from erythrocytes. For quantitative TLC, lipids were separated in Silica gel 60 F254 using a mixture of chloroform/methanol/acetic acid/water (60/50/1/4) (v/v/v/v). The separated lipids were stained in a chamber containing iodine, and the intensity of each of the primary colors (red, green, blue) and the sum of the Red plus Green colors (R+G) was analyzed. The method was linear over a wide range of concentrations, presented acceptable precision (inter-day CV(%) 0.34, 0.006 and 0.44 for 2.5, 5.0 and 10 g, respectively), good accuracy (recovery range 85.2-97.1%), and excellent limit of detection (0.137 g/spot) and limit of quantification (0.41 g/spot). Using this quantitation method, we compared various lipid extraction methods and found that lipid extraction with methanol led to higher yield of erythrocyte sphingomyelin (135.35{+/-}1.04% recovery, compared to the Folch method). Application of these methods showed that erythrocytes from NAFLD patients (9 men, 15 women, 57.95{+/-}11.11 years old) contained statistically significantly less sphingomyelin (829.82{+/-}511.60 vs 1892.08{+/-}606.25 g/ml of packed erythrocytes) compared to healthy controls (4 men, 6 women, 39.3{+/-}15.55 years old).

Source connections

Explore related subjects

biochemistryPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Papadopoulos, C., Mimidis, K., Tentes, I., Anagnostopoulos, K.. 2021-04-16. Validation and Application of a Protocol for the Extraction and Quantitative Analysis of Sphingomyelin in Erythrocyte Membranes of Patients with NAFLD. https://doi.org/10.1101/2021.04.16.440128

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Modular assembly of mitochondrial complex III from a dynamic matrix domain pool

Mitochondrial complex III is a central component of the cellular energy metabolism. It is an obligate dimer of catalytic and accessory subunits that are encoded by two genomes. How its large matrix domain, formed by the core proteins Qcr1 and Qcr2, is built and joined to the membrane part and during assembly is not known. Here we show that Qcr1 and Qcr2 preassemble independently of complex III into matrix domain modules. Cryo-EM structures from a cardiolipin-deficient yeast strain and of recombinant protein resolved single, double and triple matrix domains, which are linked by the C-terminal tail of Qcr1. In the mature complex III, the same tail binds cytochrome b and cardiolipin. The modules form a dynamic pool, in which assembly species interconvert and exchange subunits. Mutations at the module interfaces, including mimics of human disease variants, reshape this pool and deletion of the Qcr1 tail impairs respiratory growth. Substitution of a tyrosine residue linked to parkinsonism exposes a conserved cysteine residue to oxidation. A cryo-EM structure of an assembly intermediate shows a cap of two matrix domains bound to a monomeric membrane module of cytochrome b, Qcr7, Qcr8 and cytochrome c1. We propose that complex III receives its matrix domain from a dynamic pool of prebuilt modules, which organizes the dimerization of the complex.

biochemistry↗
bioRxiv2026

Vasopressin V2 receptor phosphopeptide reshapes the conformational landscape of arrestin-2 beyond C-tail detachment

Binding of phosphorylated GPCR tails influences arrestin conformation, but the contribution of this interaction to the interdomain rotation associated with activation remains incompletely characterised. In particular, it remains unclear whether displacement of the arrestin C-tail is sufficient to favor larger interdomain rotations or whether the bound phosphotail exerts an additional effect on the conformational landscape. Here, we used enhanced-sampling simulations to characterise the conformational free-energy landscapes of basal (unbound) and V2Rpp-bound arrestin-2. We find that V2Rpp binding broadened the range of energetically accessible interdomain rotation angles relative to basal arrestin-2. However, the pronounced rotation observed in the experimental structures, while accessible, was not strongly favored. Strikingly, C-tail displacement alone does not reproduce this effect, as arrestin-2 retains a strong preference for small interdomain rotation angles in the absence of V2Rpp. Moreover, the C-tail-detached state is energetically unfavorable in the absence of V2Rpp. Together, these findings quantify the effects of V2Rpp binding on the interdomain rotation of arrestin-2 and disentangle the effects of phosphopeptide binding from those of C-tail detachment.

biochemistry↗
bioRxiv2026

Interventional breath analysis reveals oral microbial conversion of 1,2-Propanediol into Propanal and 1-Propanol

Background: Orally exhaled breath contains volatile organic compounds (VOCs) that may reflect local microbial metabolism as well as systemic processes. Consequently, the breath sampling route can influence VOC profiles and complicate biomarker interpretation. Previous studies have shown that 1-propanol is substantially enriched in orally compared with nasally exhaled breath. Objective: To determine whether a controlled oral challenge with 1,2-propanediol (1,2-PD) induces sampling route-dependent changes in breath 1-propanol concentrations. Methods: Ten healthy volunteers received 0.1 mL 1,2-PD diluted in approximately 50 mL water. The solution was used as an oral rinse for approximately 30 s and then expectorated. Matched oral and nasal end-tidal breath samples were collected before administration, immediately afterwards, and at 15-min intervals. Propanal, 1-propanol, acetol, acetone, and isoprene were analysed using gas chromatograph-ion mobility spectrometer (GC-IMS). Results: Oral 1,2-PD exposure produced a rapid and transient increase in exhaled propanal and 1-propanol signals, with the largest responses immediately after administration and at the second post-intervention measurement. In contrast, nasal signals remained comparatively stable. Acetone and isoprene showed no consistent intervention-related changes, regardless of the sampling route. No reproducible increase in acetol was detected, although interpretation was limited by analytical interference. Conclusion: A controlled oral 1,2-PD challenge elicited a route-specific propanal and 1-propanol response, consistent with local microbial metabolism of 1,2-PD within the oral cavity. Both fucose and 1,2-PD have been previously reported in human saliva, including elevated concentrations in selected disease-associated contexts. Bacteria can degrade salivary mucin glycans, releasing sugars including fucose. Together, these observations motivate investigation of a proposed pathway linking mucin-derived fucose to 1,2-PD and subsequent propanal and 1-propanol formation in the human oral cavity.

biochemistry↗