8 research outputs found
Development of a Rapid Microbore Metabolic Profiling Ultraperformance Liquid Chromatography–Mass Spectrometry Approach for High-Throughput Phenotyping Studies
A rapid
gradient microbore ultraperformance liquid chromatography–mass
spectrometry (UPLC–MS) method has been developed to provide
a high-throughput analytical platform for the metabolic phenotyping
of urine from large sample cohorts. The rapid microbore metabolic
profiling (RAMMP) approach was based on scaling a conventional reversed-phase
UPLC–MS method for urinary profiling from 2.1 mm × 100
mm columns to 1 mm × 50 mm columns, increasing the linear velocity
of the solvent, and decreasing the gradient time to provide an analysis
time of 2.5 min/sample. Comparison showed that conventional UPLC–MS
and rapid gradient approaches provided peak capacities of 150 and
50, respectively, with the conventional method detecting approximately
19 000 features compared to the ∼6 000 found
using the rapid gradient method. Similar levels of repeatability were
seen for both methods. Despite the reduced peak capacity and the reduction
in ions detected, the RAMMP method was able to achieve similar levels
of group discrimination as conventional UPLC–MS when applied
to rat urine samples obtained from investigative studies on the effects
of acute 2-bromophenol and chronic acetaminophen administration. When
compared to a direct infusion MS method of similar analysis time the
RAMMP method provided superior selectivity. The RAMMP approach provides
a robust and sensitive method that is well suited to high-throughput
metabonomic analysis of complex mixtures such as urine combined with
a 5-fold reduction in analysis time compared with the conventional
UPLC–MS method
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
High-Speed Quantitative UPLC-MS Analysis of Multiple Amines in Human Plasma and Serum via Precolumn Derivatization with 6‑Aminoquinolyl‑<i>N</i>‑hydroxysuccinimidyl Carbamate: Application to Acetaminophen-Induced Liver Failure
A targeted reversed-phase gradient
UPLC-MS/MS assay has been developed
for the quantification /monitoring of 66 amino acids and amino-containing
compounds in human plasma and serum using precolumn derivatization
with 6-aminoquinolyl-<i>N</i>-hydroxysuccinimidyl carbamate
(AccQTag Ultra). Derivatization of the target amines required minimal
sample preparation and resulted in analytes with excellent chromatographic
and mass spectrometric detection properties. The resulting method,
which requires only 10 μL of sample, provides the reproducible
and robust separation of 66 analytes in 7.5 min, including baseline
resolution of isomers such as leucine and isoleucine. The assay has
been validated for the quantification of 33 amino compounds (predominantly
amino acids) over a concentration range from 2 to 20 and 800 μM.
Intra- and interday accuracy of between 0.05 and 15.6 and 0.78–13.7%
and precision between 0.91 and 16.9% and 2.12–15.9% were obtained.
A further 33 biogenic amines can be monitored in samples for relative
changes in concentration rather than quantification. Application of
the assay to samples derived from healthy controls and patients suffering
from acetaminophen (APAP, paracetamol)-induced acute liver failure
(ALF) showed significant differences in the amounts of aromatic and
branched chain amino acids between the groups as well as a number
of other analytes, including the novel observation of increased concentrations
of sarcosine in ALF patients. The properties of the developed assay,
including short analysis time, make it suitable for high-throughput
targeted UPLC-ESI-MS/MS metabonomic analysis in clinical and epidemiological
environments
iNOS activity in LPS-activated mouse macrophages treated in plasma from healthy donors and patients with sepsis.
<p><b>(A)</b> iNOS activity in plasma from healthy vs sepsis patients at 0, 24 and 72 hours. Correlations in ADMA:L-arginine ratio with iNOS activity at <b>(B)</b> 0–24 hours and <b>(C)</b> 24–72 hours post diagnosis. Data are mean ± SEM for n = 21 healthy donors and n = 38 patients with sepsis. Data was analysed by <b>(A)</b> Kruskal-Wallis one-way ANOVA with Dunn’s post-hoc test and <b>(B-C)</b> linear regression. *p<0.05 when compared to healthy.</p
Targeted metabolic profiling of amines and methylarginines in human plasma from healthy donors and patients with sepsis.
<p>Amine and methylarginine levels were measured using <b>(A)</b> UHPLC-MS/MS, LC-MS/MS and/or ELISA in the plasma of healthy donors and patients with sepsis at diagnosis (0 hours), 24 hours and 72 hours post diagnosis. Comparisons between levels of L-arginine from <b>(B)</b> UHPLC-MS/MS, LC-MS/MS and ELISA and <b>(C)</b> ADMA from LC-MS/MS and ELISA are shown at diagnosis (0 hours), 24 hours and 72 hours, post diagnosis. <b>(D)</b> Canonical pathways extracted from IPA software were based on an input of read outs comparing plasma levels of amines in healthy donors and patients with sepsis as a ratio. Data are ± SEM for n = 21 healthy donors and n = 38 patients with sepsis. Data was analysed by one-way ANOVA with Dunnett’s post-hoc test and Benjamini-Hochberg test with a false discovery rate of 0.05; *p<0.05.</p
iNOS activity in LPS-activated mouse macrophages treated in culture media.
<p>iNOS activity in macrophages was measured in cells cultured in the presence of <b>(A,B)</b> 50μM or <b>(C,D)</b> 80μM L-arginine. Data are ± SEM for n = 4–6. Data was analysed by <b>(A,C)</b> paired t-test and <b>(B,D)</b> one-way ANOVA with Dunnett’s post-hoc test. *p<0.05.</p
ADMA:L-arginine ratio in healthy donors vs patients with sepsis.
<p>ADMA and L-arginine were measured from plasma of healthy donors and patients with sepsis at diagnosis (0 hours), 24 and 72 hours post diagnosis using ELISA. Data are ± SEM for n = 21 healthy donors and n = 38 patients with sepsis. Data was analysed by one-way ANOVA with Dunnett’s post-hoc test. *p<0.05.</p
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