3 research outputs found
In-Vial Dual Extraction for Direct LC-MS Analysis of Plasma for Comprehensive and Highly Reproducible Metabolic Fingerprinting.
Metabolic fingerprinting of biological tissues has become
an important
area of research, particularly in the biomarker discovery field. Methods
have inherent analytical variation, and new approaches are necessary
to ensure that the vast numbers of intact metabolites present in biofluids
are detected. Here, we describe an in-vial dual extraction (IVDE)
method and a direct injection method that shows the total number of
features recovered to be over 4500 from a single 20 μL plasma
aliquot. By applying a one-step extraction consisting of a lipophilic
and hydrophilic layer within a single vial insert, we showed that
analytical variation was decreased. This was achieved by reducing
sample preparation stages including procedures of drying and transfers.
The two phases in the vial, upper and lower, underwent HPLC-QTOF analysis
on individually customized LC gradients in both positive and negative
ionization modes. A 60 min lipid profiling HPLC-QTOF method for the
lipophilic phase was specifically developed, enabling the separation
and putative identification of fatty acids, glycerolipids, glycerophospholipids,
sphingolipids, and sterols. The aqueous phase of the extract underwent
direct injection onto a 45 min gradient, enabling the detection of
both polarities. The IVDE method was compared to two traditional extraction
methods. The first method was a two-step ether evaporation and IPA
resuspension, and the second method was a methanol precipitation typically
used in fingerprinting studies. The IVDE provided a 378% increase
in reproducible features when compared to evaporation and a 269% increase
when compared to the precipitate and inject method. As a proof of
concept, the method was applied to an animal model of diabetes. A
2-fold increase in discriminant metabolites was found when comparing
diabetic and control rats with IVDE. These discriminant metabolites
accounted for around 600 entities, out of which 388 were identified
in available databases
Rapid and Self-Administrable Capillary Blood Microsampling Demonstrates Statistical Equivalence with Standard Venous Collections in NMR-Based Lipoprotein Analysis
We investigated plasma and serum blood derivatives from
capillary
blood microsamples (500 μL, MiniCollect tubes) and corresponding
venous blood (10 mL vacutainers). Samples from 20 healthy participants
were analyzed by 1H NMR, and 112 lipoprotein subfraction
parameters; 3 supramolecular phospholipid composite (SPC) parameters
from SPC1, SPC2, and SPC3 subfractions;
2 N-acetyl signals from α-1-acid glycoprotein
(Glyc), GlycA, and GlycB; and 3 calculated parameters, SPC (total),
SPC3/SPC2, and Glyc (total) were assessed. Using
linear regression between capillary and venous collection sites, we
explained that agreement (Adj. R2 ≥
0.8, p < 0.001) was witnessed for 86% of plasma
parameters (103/120) and 88% of serum parameters (106/120), indicating
that capillary lipoprotein, SPC, and Glyc concentrations follow changes
in venous concentrations. These results indicate that capillary blood
microsamples are suitable for sampling in remote areas and for high-frequency
longitudinal sampling of the majority of lipoproteins, SPCs, and Glycs
Integrative Molecular Structure Elucidation and Construction of an Extended Metabolic Pathway Associated with an Ancient Innate Immune Response in COVID-19 Patients
We present compelling
evidence for the existence of an extended
innate viperin-dependent pathway, which provides crucial evidence
for an adaptive response to viral agents, such as SARS-CoV-2. We show
the in vivo biosynthesis of a family of novel endogenous cytosine
metabolites with potential antiviral activities. Two-dimensional nuclear
magnetic resonance (NMR) spectroscopy revealed a characteristic spin-system
motif, indicating the presence of an extended panel of urinary metabolites
during the acute viral replication phase. Mass spectrometry additionally
enabled the characterization and quantification of the most abundant
serum metabolites, showing the potential diagnostic value of the compounds
for viral infections. In total, we unveiled ten nucleoside (cytosine-
and uracil-based) analogue structures, eight of which were previously
unknown in humans allowing us to propose a new extended viperin
pathway for the innate production of antiviral compounds.
The molecular structures of the nucleoside analogues and their correlation
with an array of serum cytokines, including IFN-α2, IFN-γ,
and IL-10, suggest an association with the viperin enzyme contributing
to an ancient endogenous innate immune defense mechanism against viral
infection