Urinary Metabolite Profiling to Non-Invasively Monitor the Omega-3 Index: An Exploratory Secondary Analysis of a Randomized Clinical Trial in Young Adults.

The Omega-3 Index (O3I) reflects eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) content in erythrocytes. While the O3I is associated with numerous health outcomes, its widespread use is limited. We investigated whether urinary metabolites could be used to non-invasively monitor the O3I in an exploratory analysis of a previous placebo-controlled, parallel arm randomized clinical trial in males and females (n = 88) who consumed either ~3 g/d olive oil (OO; control), EPA, or DHA for 12 weeks. Fasted blood and first-void urine samples were collected at baseline and following supplementation, and they were analyzed via gas chromatography and multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS), respectively. We tentatively identified S-carboxypropylcysteamine (CPCA) as a novel urinary biomarker reflecting O3I status, which increased following both EPA and DHA (p < 0.001), but not OO supplementation, and was positively correlated to the O3I (R = 0.30, p < 0.001). Additionally, an unknown dianion increased following DHA supplementation, but not EPA or OO. In ROC curve analyses, CPCA outperformed all other urinary metabolites in distinguishing both between OO and EPA or DHA supplementation groups (AUC > 80.0%), whereas the unknown dianion performed best in discriminating OO from DHA alone (AUC = 93.6%). Candidate urinary biomarkers of the O3I were identified that lay the foundation for a non-invasive assessment of omega-3 status.

Lipidomic studies reveal two specific circulating phosphatidylcholines as surrogate biomarkers of the omega-3 index.

Optimal dietary intake of omega-3 long-chain polyunsaturated fatty acids (n3-LCPUFAs) is critical to human health across the lifespan. However, omega-3 index (O3I) determination is not routinely assessed due to complicated procedures for n3-LCPUFA analysis from the phospholipid (PL) fraction of erythrocytes. Herein, a high-throughput method for lipidomics based on multisegment injection-nonaqueous capillary electrophoresis-mass spectrometry was applied to identify circulating PLs as surrogate biomarkers of O3I in two randomized placebo-controlled trials. An untargeted lipidomic data workflow using a subgroup analysis of serum extracts from sunflower oil versus high-dose fish oil (FO)-supplemented participants revealed that ingested n3-LCPUFAs were primarily distributed as their phosphatidylcholines (PCs) relative to other PL classes. In both high-dose FO (5.0 g/day) and EPA-only trials (3.0 g/day), PC (16:0_20:5) was the most responsive PL, whereas PC (16:0_22:6) was selective to DHA-only supplementation. We also demonstrated that the sum concentration of both these PCs in fasting serum or plasma samples was positively correlated to the O3I following FO (r = 0.708, P = 1.02 × 10-11, n = 69) and EPA- or DHA-only supplementation (r = 0.768, P = 1.01 × 10-33, n = 167). Overall, DHA was more effective in improving the O3I (ΔO3I = 4.90 ± 1.33%) compared to EPA (ΔO3I = 2.99 ± 1.19%) in young Canadian adults who had a poor nutritional status with an O3I (3.50 ± 0.68%) at baseline. Our method enables the rapid assessment of the O3I by directly measuring two circulating PC species in small volumes of blood, which may facilitate screening applications for population and precision health.

Dynamic Metabolic Signatures of Choline and Carnitine across Healthy Pregnancy and in Cord Blood: Association with Maternal Dietary Protein.

BACKGROUND: In pregnancy, choline is deemed an essential nutrient and carnitine needs are increased, but amounts remain undefined. OBJECTIVES: We aimed to measure choline and total dietary protein and dairy protein intake from food and supplements across pregnancy and the response to intake by profiling choline and carnitine metabolites across pregnancy and in cord blood. METHODS: An exploratory analysis of choline and protein intake from 3-d diet records and measures of 36 serum choline and carnitine metabolites in early (12-17 wk) and late (36-38 wk) pregnancy was conducted in participants from the Be Healthy in Pregnancy study randomized to high dairy protein+walking exercise or usual care. Metabolites were measured in fasted maternal and cord serum using multisegment injection-capillary electrophoresis-mass spectrometry. Mixed ANOVA adjusted for body mass index was performed for comparison of metabolites across pregnancy and between intervention and control. RESULTS: In 104 participants, the median (Q1, Q3) total choline intake was 347 (263, 427) mg/d in early and 322 (270, 437) mg/d in late pregnancy. Only ∼20% of participants achieved the recommended adequate intake (450 mg/d) and ∼10% consumed supplemental choline (8-200 mg/d). Serum-free choline (µmol/L) was higher in late compared with early pregnancy [12.9 (11.4, 15.1) compared with 9.68 (8.25, 10.61), P < 0.001], but choline downstream metabolites were similar across pregnancy. Serum carnitine [10.3 (9.01, 12.2) compared with 15.9 (14.1, 17.9) µmol/L, P < 0.001] and acetylcarnitine [2.35 (1.92, 2.68) compared with 3.0 (2.56, 3.59), P < 0.001] were significantly lower in late pregnancy. High cord:maternal serum metabolite ratios were found in most measured metabolites. CONCLUSIONS: Despite inadequate choline intake, serum-free choline was elevated in late pregnancy and enriched in cord blood compared with maternal serum. Serum carnitine declined in late pregnancy despite a high protein diet. The higher cord:maternal concentrations in choline and carnitine metabolites suggest active uptake in late pregnancy, reflecting the importance of these circulating metabolites in fetal development. This trial was registered at clinicaltrials.gov as NCT01689961.

The Prevalence and Risk Factors Associated with Iodine Deficiency in Canadian Adults.

Iodine is a trace micronutrient that is critical for normal thyroid function and human health. Inadequate dietary intake is associated with cognitive impairment, infertility, growth retardation and iodine deficiency disorders in affected populations. Herein, we examined the prevalence of iodine deficiency in adults (median age of 61 years) based on the analysis of 24 h urine samples collected from 800 participants in four clinical sites across Canada in the Prospective Urban and Rural Epidemiological (PURE) study. Urinary iodide together with thiocyanate and nitrate were measured using a validated capillary electrophoresis assay. Protective/risk factors associated with iodine deficiency were identified using a binary logistic regression model, whereas daily urinary iodine concentration (24 h UIC, µg/L) and urinary iodine excretion (24 h UIE, µg/day) were compared using complementary statistical methods with covariate adjustments. Overall, our Canadian adult cohort had adequate iodine status as reflected by a median UIC of 111 µg/L with 11.9% of the population <50 µg/L categorized as having moderate to severe iodine deficiency. Iodine adequacy was also evident with a median 24 h UIE of 226 µg/day as a more robust metric of iodine status with an estimated average requirement (EAR) of 7.1% (< 95 µg/day) and a tolerable upper level (UL) of 1.8% (≥1100 µg/day) based on Canadian dietary reference intake values. Participants taking iodine supplements (OR = 0.18; p = 6.35 × 10−5), had greater 24 h urine volume (OR = 0.69; p = 4.07 × 10−4), excreted higher daily urinary sodium (OR = 0.71; p = 3.03 × 10−5), and/or were prescribed thyroxine (OR = 0.33; p = 1.20 × 10−2) had lower risk for iodine deficiency. Self-reported intake of dairy products was most strongly associated with iodine status (r = 0.24; p = 2.38 × 10−9) after excluding for iodine supplementation and T4 use. Participants residing in Quebec City (OR = 2.58; p = 1.74 × 10−4) and Vancouver (OR = 2.54; p = 3.57 × 10−4) were more susceptible to iodine deficiency than Hamilton or Ottawa. Also, greater exposure to abundant iodine uptake inhibitors from tobacco smoking and intake of specific goitrogenic foods corresponded to elevated urinary thiocyanate and nitrate, which were found for residents from Quebec City as compared to other clinical sites. Recent public health policies that advocate for salt restriction and lower dairy intake may inadvertently reduce iodine nutrition of Canadians, and further exacerbate regional variations in iodine deficiency risk.

A high-throughput platform for the rapid screening of vitamin D status by direct infusion-MS/MS.

Vitamin D is an important fat-soluble prohormone with pleiotropic effects on human health, such as immunomodulation of the innate and adaptive immune system. There is an unmet clinical need for a rapid screening platform for 25-hydroxyvitamin D (25OH-D) determination without chromatographic separation that offers better precision and accuracy than immunoassays. Here, we introduce a high-throughput method for assessing vitamin D status from blood specimens based on direct infusion-MS/MS (DI-MS/MS) following click derivatization using 2-nitrosopyridine. We developed an optimized liquid-phase extraction protocol to minimize ion suppression when directly infusing serum or plasma extracts via a capillary electrophoresis system for quantitative determination of 25OH-D. Acceptable reproducibility (mean coefficient of variation = 10.9%, n = 412), recovery (mean = 102% at 15, 30, and 45 nmol/l), and linearity (R2 > 0.998) were achieved for 25OH-D with lower detection limits (limit of detection ∼1.2 nmol/l, S/N ∼ 3), greater throughput (∼3 min/sample), and less bias than a commercial chemiluminescence immunoassay prone to batch effects. There was mutual agreement in 25OH-D concentrations from reference blood samples measured by DI-MS/MS as compared with LC-MS/MS (mean bias = 7.8%, n = 18). We also demonstrate that this method could reduce immunoassay misclassification of vitamin D deficiency in a cohort of critically ill children (n = 30). In conclusion, DI-MS/MS offers a viable alternative to LC-MS/MS for assessment of vitamin D status in support of large-scale studies in nutritional epidemiology as well as clinical trials to rapidly screen individual patients who may benefit from vitamin D supplementation.

Effect of Exclusive Enteral Nutrition and Corticosteroid Induction Therapy on the Gut Microbiota of Pediatric Patients with Inflammatory Bowel Disease.

INTRODUCTION: Exclusive enteral nutrition (EEN) and corticosteroids (CS) are effective induction therapies for pediatric Crohn's Disease (CD). CS are also therapy for ulcerative colitis (UC). Host-microbe interactions may be able to explain the effectiveness of these treatments. This is the first prospective study to longitudinally characterize compositional changes in the bacterial community structure of pediatric UC and CD patients receiving EEN or CS induction therapy. METHODS: Patients with diagnoses of CD or UC were recruited from McMaster Children's Hospital (Hamilton, Canada). Fecal samples were collected from participants aged 5-18 years old undergoing 8 weeks of induction therapy with EEN or CS. Fecal samples were submitted for 16S rRNA sequencing. The Shannon diversity index and the relative abundance of specific bacterial taxa were compared using a linear mixed model. RESULTS: The clustering of microbiota was the highest between patients who achieved remission compared to patients still showing active disease (p = 0.029); this effect was independent of the diagnosis or treatment type. All patients showed a significant increase in Shannon diversity over the 8 weeks of treatment. By week 2, a significant difference was seen in Shannon diversity between patients who would go on to achieve remission and those who would not. CONCLUSION: The gut microbiota of pediatric UC and CD patients was most influenced by patients' success or failure to achieve remission and was largely independent of the choice of treatment or disease type. Significant differences in Shannon diversity indices occurred as early as week 2 between patients who went on to achieve remission and those who continued to have active disease.

Serum nonesterified fatty acids have utility as dietary biomarkers of fat intake from fish, fish oil, and dairy in women.

Nutritional studies rely on various biological specimens for FA determination, yet it is unclear how levels of serum NEFAs correlate with other circulating lipid pools. Here, we used a high-throughput method (<4 min/sample) based on multisegment injection-nonaqueous capillary electrophoresis-mass spectrometry (MSI-NACE-MS) to investigate whether specific serum NEFAs have utility as biomarkers of dietary fat intake in women. We first identified circulating NEFAs correlated with long-term/habitual food intake among pregnant women with contrasting dietary patterns (n = 50). Acute changes in serum NEFA trajectories were also studied in nonpregnant women (n = 18) following high-dose (5 g/day) fish oil (FO) supplementation or isoenergetic sunflower oil placebo over 56 days. In the cross-sectional study, serum ω-3 FAs correlated with self-reported total ω-3 daily intake, notably EPA as its NEFA (r = 0.46; P = 0.001), whereas pentadecanoic acid was associated with full-fat dairy intake (r = 0.43; P = 0.002), outcomes consistent with results from total FA serum hydrolysates. In the intervention cohort, serum ω-3 NEFAs increased 2.5-fold from baseline within 28 days following FO supplementation, and this increase was most pronounced for EPA (P = 0.0004). Unlike for DHA, circulating EPA as its NEFA also strongly correlated to EPA concentrations measured from erythrocyte phospholipid hydrolysates (r = 0.66; P = 4.6 × 10-10) and was better suited to detect dietary nonadherence. We conclude that MSI-NACE-MS offers a rapid method to quantify serum NEFAs and objectively monitor dietary fat intake in women that is complementary to food-frequency questionnaires.

Metabolic Trajectories Following Contrasting Prudent and Western Diets from Food Provisions: Identifying Robust Biomarkers of Short-Term Changes in Habitual Diet.

A large body of evidence has linked unhealthy eating patterns with an alarming increase in obesity and chronic disease worldwide. However, existing methods of assessing dietary intake in nutritional epidemiology rely on food frequency questionnaires or dietary records that are prone to bias and selective reporting. Herein, metabolic phenotyping was performed on 42 healthy participants from the Diet and Gene Intervention (DIGEST) pilot study, a parallel two-arm randomized clinical trial that provided complete diets to all participants. Matching single-spot urine and fasting plasma specimens were collected at baseline, and then following two weeks of either a Prudent or Western diet with a weight-maintaining menu plan designed by a dietician. Targeted and nontargeted metabolite profiling was conducted using three complementary analytical platforms, where 80 plasma metabolites and 84 creatinine-normalized urinary metabolites were reliably measured (CV < 30%) in the majority of participants (>75%) after implementing a rigorous data workflow for metabolite authentication with stringent quality control. We classified a panel of metabolites with distinctive trajectories following two weeks of food provisions when using complementary univariate and multivariate statistical models. Unknown metabolites associated with contrasting dietary patterns were identified with high-resolution MS/MS, as well as co-elution after spiking with authentic standards if available. Overall, 3-methylhistidine and proline betaine concentrations increased in both plasma and urine samples after participants were assigned a Prudent diet (q < 0.05) with a corresponding decrease in the Western diet group. Similarly, creatinine-normalized urinary imidazole propionate, hydroxypipecolic acid, dihydroxybenzoic acid, and enterolactone glucuronide, as well as plasma ketoleucine and ketovaline increased with a Prudent diet (p < 0.05) after adjustments for age, sex, and BMI. In contrast, plasma myristic acid, linoelaidic acid, linoleic acid, α-linoleic acid, pentadecanoic acid, alanine, proline, carnitine, and deoxycarnitine, as well as urinary acesulfame K increased among participants following a Western diet. Most metabolites were also correlated (r > ± 0.30, p < 0.05) to changes in the average intake of specific nutrients from self-reported diet records reflecting good adherence to assigned food provisions. Our study revealed robust biomarkers sensitive to short-term changes in habitual diet, which is needed for accurate monitoring of healthy eating patterns in free-living populations, and evidence-based public health policies for chronic disease prevention.

Metabolomics for improved treatment monitoring of phenylketonuria: urinary biomarkers for non-invasive assessment of dietary adherence and nutritional deficiencies.

Management of phenylketonuria (PKU) requires lifelong restriction of phenylalanine (Phe) intake using specialized medical foods to prevent neurocognitive impairment in affected patients. However, dietary adherence is challenging to maintain while ensuring adequate nutrition, which can lead to sub-optimal clinical outcomes. Metabolomics offers a systematic approach to identify new biomarkers of disease progression in PKU when using urine as a surrogate for blood specimens that is more accurate than self-reported diet records. Herein, the plasma and urine metabolome of a cohort of classic PKU patients (median age = 11 years; n = 22) mainly prescribed (78%) a Phe-restricted diet were characterized using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Overall, there was good mutual agreement between plasma Phe and tyrosine (Tyr) concentrations measured from PKU patients when using an amino acid analyzer based on UPLC-UV as compared to MSI-CE-MS with a mean bias of 12% (n = 82). Longitudinal measurements of recently diagnosed PKU infants (n = 3) revealed good long-term regulation of blood Phe with dietary management, and only occasional episodes exceeding the recommended therapeutic range (>360 µM) unlike older PKU patients. Plasma metabolomic studies demonstrated that non-adherent PKU patients had lower circulating concentrations of Tyr, arginine, 2-aminobutyric acid, and propionylcarnitine (q < 0.05, FDR) that were inversely correlated to Phe (r ≈ -0.600 to -0.830). Nontargeted metabolite profiling also revealed urinary biomarkers associated with poor dietary adherence among PKU patients, including elevated concentrations of catabolites indicative of Phe intoxication (e.g., phenylpyruvic acid, phenylacetylglutamine, hydroxyphenylacetic acid). Additionally, PKU patients with poor blood Phe control had lower excretion of urinary compounds derived from co-metabolism of Tyr due to microbiota activity (e.g., cresol sulfate, phenylsulfate), as well as several metabolites associated with inadequate nutrient intake, including low carnitine and B vitamin status (e.g., folic acid, vitamin B12). Interestingly, an unknown urinary metabolite was strongly correlated with Phe excretion in PKU patients (r = 0.861), which was subsequently identified as imidazole lactic acid when using high resolution MS/MS. Overall, urine profiling offers a non-invasive approach for better treatment monitoring of individual PKU patients, which can also guide the design of novel therapies that improve adherence to Phe-restricted diets without acquired nutritional deficiencies.

New Advances in Amino Acid Profiling in Biological Samples by Capillary Electrophoresis-Mass Spectrometry.

Capillary electrophoresis-mass spectrometry (CE-MS) offers a high efficiency microseparation platform for amino acid profiling when analyzing volume-restricted biological samples, such as a dried blood spot punch. Direct analysis of amino acids and their analogs is routinely achieved using strongly acidic buffer conditions under positive-ion mode detection with a coaxial sheath liquid interface for electrospray ionization (ESI). New advances in online sample preconcentration, pre-column chemical derivatization, and/or low flow/sheathless CE-MS interface designs can further improve sensitivity while allowing for resolution of amino acid stereoisomers and labile aminothiols with low nanomolar detection limits. Additionally, multiplexed separations in CE-MS based on serial injection of seven or more samples within a single run greatly boosts sample throughput (<2-3 min/sample) without added infrastructure costs while allowing for stringent quality control and signal batch correction. Accurate prediction of the electromigration behavior of amino acids and their analogs offers a convenient approach for structural elucidation that is complementary to high-resolution MS and MS/MS. Simultaneous analysis of amino acids together with other classes of ionic metabolites by CE-MS allows for comprehensive metabolomic screening as required for new advances in clinical medicine, nutritional sciences, and population health.

Characterization of the Human Skeletal Muscle Metabolome for Elucidating the Mechanisms of Bicarbonate Ingestion on Strenuous Interval Exercise.

Bicarbonate has long been touted as a putative ergogenic aid that improves exercise performance and blood buffering capacity during strenuous exercise. However, the underlying mechanisms of action of bicarbonate intake on skeletal muscle metabolism have yet to be fully elucidated. Herein, we apply two orthogonal analytical platforms for nontargeted profiling of metabolites and targeted analysis of electrolytes from mass-limited muscle tissue biopsies (∼2 mg dried mass) when multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS) and CE with indirect UV detection are used, respectively. Seven untrained men performed a standardized bout of high-intensity interval exercise trial following either bicarbonate (0.40 g/kg) or placebo ingestion in a double-blinded, placebo-controlled, crossover study design, where paired skeletal muscle tissue and plasma specimens were collected at three time intervals at rest, postexercise, and recovery. Optimization of a quantitative microextraction procedure was first developed for lyophilized tissue prior to characterization of the human muscle metabolome, which resulted in the identification and quantification of more than 80 polar/ionic metabolites reliably (CV < 30%) detected in a majority (>75%) of samples with quality control. Complementary univariate and multivariate statistical methods were used to identify biomarkers associated with strenuous exercise and/or bicarbonate treatment responses, whereas structural elucidation of biologically significant intramuscular metabolites was performed using high-resolution MS/MS. Importantly, bicarbonate ingestion prior to strenuous interval exercise was found to elicit a modest treatment effect ( p < 0.05) in comparison to placebo on metabolic pathways associated with ionic homeostasis (potassium), purine degradation (uric acid), and oxidative stress as regulated by glutathione metabolism (oxidized mixed glutathione disulfide) and histidine-containing dipeptides (anserine) within muscle tissue that was distinctive from dynamic metabolic changes measured in circulation. This work provides deeper biochemical insights into the effect of acute alkalosis in preserving contracting muscle function during high-intensity exercise, which is also applicable to the study of muscle-related pathologies relevant to human health and aging.

Functional screening of pharmacological chaperones via restoration of enzyme activity upon denaturation.

Pharmacological chaperones (PCs) are small molecules that stabilize and promote protein folding. Enzyme inhibition is widely used for PC selection; however, it does not accurately reflect chaperone activity. We introduce a functional assay for characterization of PCs based on their capacity to restore enzyme activity that is abolished upon chemical denaturation. Dose-dependent activity curves were performed as a function of urea to assess the chaperone potency of various ligands to ß-glucocerebrosidase as a model system. Restoration of enzyme activity upon denaturation allows direct screening of PCs for treatment of genetic disorders associated with protein deficiency, such as Gaucher disease.

New advances in amino acid profiling by capillary electrophoresis-electrospray ionization-mass spectrometry.

Capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) offers a selective, sensitive yet robust approach for amino acid profiling in complex biological samples with minimal sample pretreatment. Direct analysis of amino acids and their analogs is routinely performed using strongly acidic buffer conditions under positive-ion mode ESI-MS with a coaxial sheath liquid interface. New advances in online sample preconcentration, chemical derivatization, and/or ESI interface designs can further improve assay performance allowing for resolution of amino acid stereoisomers and labile aminothiols with low nanomolar detection limits. Accurate prediction of the electromigration behavior of amino acids offers a convenient approach for their qualitative identification complementary to ESI-MS. Simultaneous analysis of amino acids together with other classes of cationic metabolites can be realized by CE-ESI-MS for comprehensive metabolite profiling applications relevant to disease prognosis, drug efficacy, and food safety/quality control.

Inhibitor screening of pharmacological chaperones for lysosomal ß-glucocerebrosidase by capillary electrophoresis.

Pharmacological chaperones (PCs) represent a promising therapeutic strategy for treatment of lysosomal storage disorders based on enhanced stabilization and trafficking of mutant protein upon orthosteric and/or allosteric binding. Herein, we introduce a simple yet reliable enzyme assay using capillary electrophoresis (CE) for inhibitor screening of PCs that target the lysosomal enzyme, ß-glucocerebrosidase (GCase). The rate of GCase-catalyzed hydrolysis of the synthetic substrate, 4-methylumbelliferyl-ß-D: -glucopyranoside was performed using different classes of PCs by CE with UV detection under standardized conditions. The pH and surfactant dependence of inhibitor binding on recombinant GCase activity was also examined. Enzyme inhibition studies were investigated for five putative PCs including isofagomine (IFG), ambroxol, bromhexine, diltiazem, and fluphenazine. IFG was confirmed as a potent competitive inhibitor of recombinant GCase with half-maximal inhibitory concentration (IC(50)) of 47.5 ± 0.1 and 4.6 ± 1.4 nM at pH 5.2 and pH 7.2, respectively. In contrast, the four other non-carbohydrate amines were demonstrated to function as mixed-type inhibitors with high micromolar activity at neutral pH relative to acidic pH conditions reflective of the lysosome. CE offers a convenient platform for characterization of PCs as a way to accelerate the clinical translation of previously approved drugs for oral treatment of rare genetic disorders, such as Gaucher disease.

Differential metabolomics for quantitative assessment of oxidative stress with strenuous exercise and nutritional intervention: thiol-specific regulation of cellular metabolism with N-acetyl-L-cysteine pretreatment.

Despite several decades of active research, the success of large-scale clinical trials involving antioxidants remains equivocal given the complex biological interactions of reactive oxygen/nitrogen species in human health. Herein, we outline a differential metabolomics strategy by capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) to assess the efficacy of nutritional intervention to attenuate oxidative stress induced by strenuous exercise. A healthy volunteer was recruited to perform a submaximal prolonged ergometer cycling trial until volitional exhaustion with frequent blood collection over a 6 h time interval, which included pre-, during, and postexercise periods while at rest. A follow-up study was subsequently performed by the same subject after high-dose oral intake of N-acetyl-L-cysteine (NAC) prior to performing the same exercise protocol under standardized conditions. Time-dependent changes in global metabolism of filtered red blood cell lysates by CE-ESI-MS were measured to reveal a significant attenuation of cellular oxidation associated with high-dose oral NAC intake relative to a control. Untargeted metabolite profiling allowed for the identification and quantification of several putative early- and late-stage biomarkers that reflected oxidative stress inhibition due to nutritional intervention, including oxidized glutathione (GSSG), reduced glutathione (GSH), 3-methylhistidine (3-MeHis), L-carnitine (C0), O-acetyl-L-carnitine (C2), and creatine (Cre). Our work demonstrates the proof-of-principle that NAC pretreatment is effective at dampening acute episodes of oxidative stress by reversible perturbations in global metabolism that can provide deeper insight into the mechanisms of thiol-specific protein inhibition relevant to its successful translation as a prophylaxis in clinical medicine.