39 research outputs found
Whole Blood Metabolomics by <sup>1</sup>H NMR Spectroscopy Provides a New Opportunity To Evaluate Coenzymes and Antioxidants
Conventional
human blood metabolomics employs serum or plasma and
provides a wealth of metabolic information therein. However, this
approach lacks the ability to measure and evaluate important metabolites
such as coenzymes and antioxidants that are present at high concentrations
in red blood cells. As an important alternative to serum/plasma metabolomics,
we show here that a simple <sup>1</sup>H NMR experiment can simultaneously
measure coenzymes and antioxidants in extracts of whole human blood,
in addition to the nearly 70 metabolites that were shown to be quantitated
in serum/plasma recently [Anal. Chem. 2015, 87, 706−715]. Coenzymes of redox
reactions: oxidized/reduced nicotinamide adenine dinucleotide (NAD<sup>+</sup> and NADH) and nicotinamide adenine dinucleotide phosphate
(NADP<sup>+</sup> and NADPH); coenzymes of energy including adenosine
triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate
(AMP); and antioxidants, the sum of oxidized and reduced glutathione
(GSSG and GSH) can be measured with essentially no additional effort.
A new method was developed for detecting many of these unstable species
without affecting other blood/blood plasma metabolites. The identities
of coenzymes and antioxidants in blood NMR spectra were established
combining 1D/2D NMR techniques, chemical shift databases, pH measurements
and, finally, spiking with authentic compounds. This is the first
study to report identification of major coenzymes and antioxidants
and quantify them, simultaneously, with the large pool of other metabolites
in human blood using NMR spectroscopy. Considering that the levels
of coenzymes and antioxidants represent a sensitive measure of cellular
functions in health and numerous diseases, the NMR method presented
here potentially opens a new chapter in the metabolomics of blood
Quantitating Metabolites in Protein Precipitated Serum Using NMR Spectroscopy
Quantitative NMR-based metabolite
profiling is challenged by the
deleterious effects of abundant proteins in the intact blood plasma/serum,
which underscores the need for alternative approaches. Protein removal
by ultrafiltration using low molecular weight cutoff filters thus
represents an important step. However, protein precipitation, an alternative
and simple approach for protein removal, lacks detailed quantitative
assessment for use in NMR based metabolomics. In this study, we have
comprehensively evaluated the performance of protein precipitation
using methanol, acetonitrile, perchloric acid, and trichloroacetic
acid and ultrafiltration approaches using 1D and 2D NMR, based on
the identification and absolute quantitation of 44 human blood metabolites,
including a few identified for the first time in the NMR spectra of
human serum. We also investigated the use of a “smart isotope
tag,” <sup>15</sup>N-cholamine for further resolution enhancement,
which resulted in the detection of a number of additional metabolites. <sup>1</sup>H NMR of both protein precipitated and ultrafiltered serum
detected all 44 metabolites with comparable reproducibility (average
CV, 3.7% for precipitation; 3.6% for filtration). However, nearly
half of the quantified metabolites in ultrafiltered serum exhibited
10–74% lower concentrations; specifically, tryptophan, benzoate,
and 2-oxoisocaproate showed much lower concentrations compared to
protein precipitated serum. These results indicate that protein precipitation
using methanol offers a reliable approach for routine NMR-based metabolomics
of human blood serum/plasma and should be considered as an alternative
to ultrafiltration. Importantly, protein precipitation, which is commonly
used by mass spectrometry (MS), promises avenues for direct comparison
and correlation of metabolite data obtained from the two analytical
platforms to exploit their combined strength in the metabolomics of
blood
Massive Glutamine Cyclization to Pyroglutamic Acid in Human Serum Discovered Using NMR Spectroscopy
Glutamine is one of the most abundant
metabolites in blood and
is a precursor as well as end product central to numerous important
metabolic pathways. A number of surprising and unexpected roles for
glutamine, including cancer cell glutamine addiction discovered recently,
stress the importance of accurate analysis of glutamine concentrations
for understanding its role in health and numerous diseases. Utilizing
a recently developed NMR approach that offers access to an unprecedented
number of quantifiable blood metabolites, we have identified a surprising
glutamine cyclization to pyroglutamic acid that occurs during protein
removal. Intact, ultrafiltered and protein precipitated samples from
the same pool of human serum were comprehensively investigated using <sup>1</sup>H NMR spectroscopy at 800 MHz to detect and quantitatively
evaluate the phenomenon. Interestingly, although glutamine cyclization
occurs in both ultrafiltered and protein precipitated serum, the cyclization
was not detected in intact serum. Strikingly, due to cyclization,
the apparent serum glutamine level drops by up to 75% and, concomitantly,
the pyroglutamic acid level increases proportionately. Further, virtually
under identical conditions, the magnitude of cyclization is vastly
different for different portions of samples from the same pool of
human serum. However, the sum of glutamine and pyroglutamic acid concentrations
in each sample remains the same for all portions. These unexpected
findings indicate the importance of considering the sum of apparent
glutamine and pyroglutamic acid levels, obtained from the contemporary
analytical methods, as the actual blood glutamine level for biomarker
discovery and biological interpretations
Expanding the Limits of Human Blood Metabolite Quantitation Using NMR Spectroscopy
A current challenge in metabolomics
is the reliable quantitation
of many metabolites. Limited resolution and sensitivity combined with
the challenges associated with unknown metabolite identification have
restricted both the number and the quantitative accuracy of blood
metabolites. Focused on alleviating this bottleneck in NMR-based metabolomics,
investigations of pooled human serum combining an array of 1D/2D NMR
experiments at 800 MHz, database searches, and spiking with authentic
compounds enabled the identification of 67 blood metabolites. Many
of these (∼1/3) are new compared with those reported previously
as a part of the Human Serum Metabolome Database. In addition, considering
both the high reproducibility and quantitative nature of NMR as well
as the sensitivity of NMR chemical shifts to altered sample conditions,
experimental protocols and comprehensive peak annotations are provided
here as a guide for identification and quantitation of the new pool
of blood metabolites for routine applications. Further, investigations
focused on the evaluation of quantitation using organic solvents revealed
a surprisingly poor performance for protein precipitation using acetonitrile.
One-third of the detected metabolites were attenuated by 10–67%
compared with methanol precipitation at the same solvent-to-serum
ratio of 2:1 (v/v). Nearly 2/3 of the metabolites were further attenuated
by up to 65% upon increasing the acetonitrile-to-serum ratio to 4:1
(v/v). These results, combined with the newly established identity
for many unknown metabolites in the NMR spectrum, offer new avenues
for human serum/plasma-based metabolomics. Further, the ability to
quantitatively evaluate nearly 70 blood metabolites that represent
numerous classes, including amino acids, organic acids, carbohydrates,
and heterocyclic compounds, using a simple and highly reproducible
analytical method such as NMR may potentially guide the evaluation
of samples for analysis using mass spectrometry
Ethanol induces cell damage in dog PDEC.
<p>(A) Cells were treated with the indicated ethanol concentrations for 1, 4, or 24 h, respectively. After the treatment, cell viability was determined with MTS assay. The values were calculated relative to the control group. The results are mean ± SEM and representative of three independent experiments (<i>n</i> = 4 - 6 for each condition). (B) After 1, 4, or 24 h exposure to the indicated ethanol, LDH activity in extracellular culture medium was measured. The values are presented as relative to LDH release by 0.5% Triton-X 100. </p
Ethanol evokes ROS generation and mitochondrial depolarization, which are blocked by an antioxidant, NAC.
<p>(A and B) ROS measured with CM-H<sub>2</sub>DCFDA. The areas under the curves and above the 1.0 level were calculated during drug treatments (3 to 15 min) and normalized to that of the control. (C and D) Mitochondrial membrane potential (MMP) measured with JC-1 dye. Decrease of ratio (F561/F488) indicates MMP depolarization. For the bar graph (D), MMP was evaluated as averages during drug treatments. Note that 3 mM NAC reversed ROS generation and MMP depolarization induced by ethanol. <i>N</i> = 7 - 23 for each condition, <sup>## </sup><i>P</i> < 0.01 and <sup>### </sup><i>P</i> < 0.001 compared to the control. ** <i>P</i> < 0.01 compared to the ethanol-treated group.</p
Apoptotic Damage of Pancreatic Ductal Epithelia by Alcohol and Its Rescue by an Antioxidant
<div><p>Alcohol abuse is a major cause of pancreatitis. However alcohol toxicity has not been fully elucidated in the pancreas and little is known about the effect of alcohol on pancreatic ducts. We report the molecular mechanisms of ethanol-induced damage of pancreatic duct epithelial cells (PDEC). Ethanol treatment for 1, 4, and 24 h resulted in cell death in a dose-dependent manner. The ethanol-induced cell damage was mainly apoptosis due to generation of reactive oxygen species (ROS), depolarization of mitochondrial membrane potential (MMP), and activation of caspase-3 enzyme. The antioxidant N-acetylcysteine (NAC) attenuated these cellular responses and reduced cell death significantly, suggesting a critical role for ROS. Acetaldehyde, a metabolic product of alcohol dehydrogenase, induced significant cell death, depolarization of MMP, and caspase-3 activation as ethanol and this damage was also averted by NAC. Reverse transcription-polymerase chain reaction revealed the expression of several subtypes of alcohol dehydrogenase and acetaldehyde dehydrogenase. Nuclear magnetic resonance spectroscopy data confirmed the accumulation of acetaldehyde in ethanol-treated cells, suggesting that acetaldehyde formation can contribute to alcohol toxicity in PDEC. Finally, ethanol increased the leakage of PDEC monolayer which was again attenuated by NAC. In conclusion, ethanol induces apoptosis of PDEC and thereby may contribute to the development of alcohol-induced pancreatitis.</p> </div
Ethanol induces apoptosis.
<p>(A) Cells were treated with the indicated ethanol concentrations for 4 h, and then stained with annexin V-FITC (green, apoptotic cells), ethidium homodimer III (red), and Hoechst 33342 (blue, nucleus staining) for 15 min. Staurosporine was a positive control to trigger apoptosis. Bar is 20 μm. (B) Percentage of apoptotic cells from two independent experiments. (C) Cells treated with 500 or 750 mM ethanol for 4 h were analyzed by Western blot to detect activated caspase-3 and actin proteins. (D) Activated caspase-3 was calibrated by actin level of the samples and presented as relative to the control. Two experiments. ** <i>P</i> < 0.01 and *** <i>P</i> < 0.001 compared to control group.</p
Ethanol is oxidized in PDEC and its metabolite, acetaldehyde, is involved in ethanol-induced cell damage.
<p>(A) RT-PCR analysis on expression of oxidative metabolic genes of ethanol in control dog PDEC. Representative gels from at least three independent experiments. (B) Cells were treated with 500 mM ethanol for 4 h with or without 30 min pretreatment with disulfiram (ALDH1 inhibitor), daidzin (ALDH2 inhibitor), or both. n = 6 - 18 for each condition, <sup># </sup><i>P</i> < 0.05 compared to the control. ** <i>P</i> < 0.01 compared to the cells treated ethanol alone. Submaximal 500 mM ethanol was used in this experiment to test the further damage by acetaldehyde accumulation. (C) NMR analysis for the detection of cellular acetaldehyde production. Arrows indicate two peaks for acetaldehyde. Estimated acetaldehyde concentrations in the samples are 0, 1.7, 2.1, and 48.4 μM for control cells, cells treated with ethanol, cells treated with ethanol plus ALDH inhibitors, and standard solution including ethanol and acetaldehyde, respectively. </p
Acetaldehyde induces cell damage that is inhibited by NAC.
<p>(A) PDEC were treated with indicated concentrations of acetaldehyde for 4 h, and then cell viability was measured. (B) PDEC were treated with 1 mM acetaldehyde for 4 h with or without 30 min pretreatment of 3 or 10 mM NAC. n = 4 - 13 for each condition, <sup>### </sup><i>P</i> < 0.001 compared to control. ** <i>P</i> < 0.01 and *** <i>P</i> < 0.001 compared to the cells treated acetaldehyde alone. (C and D) MMP measured with JC-1 dye. For the bar graph, MMP was evaluated as averages during drug treatments. n = 5 - 6 for each condition, <sup>## </sup><i>P</i> < 0.01 compared to control and *** <i>P</i> < 0.001 compared to the cells treated with acetaldehyde alone. (E and F) Caspase-3 activation estimated with Western blot analysis. Cells treated with 1 mM acetaldehyde for 4 h were analyzed to detect activated caspase-3. For the summary bar graph, activated caspase-3 was calibrated by actin level of the samples and presented as relative to untreated control. n = 3 for each condition, *<i>P</i> < 0.05 compared to control.</p