Gut microbiome remodeling and metabolomic profile improves in response to protein pacing with intermittent fasting versus continuous caloric restriction.

The gut microbiome (GM) modulates body weight/composition and gastrointestinal functioning; therefore, approaches targeting resident gut microbes have attracted considerable interest. Intermittent fasting (IF) and protein pacing (P) regimens are effective in facilitating weight loss (WL) and enhancing body composition. However, the interrelationships between IF- and P-induced WL and the GM are unknown. The current randomized controlled study describes distinct fecal microbial and plasma metabolomic signatures between combined IF-P (n = 21) versus a heart-healthy, calorie-restricted (CR, n = 20) diet matched for overall energy intake in free-living human participants (women = 27; men = 14) with overweight/obesity for 8 weeks. Gut symptomatology improves and abundance of Christensenellaceae microbes and circulating cytokines and amino acid metabolites favoring fat oxidation increase with IF-P (p < 0.05), whereas metabolites associated with a longevity-related metabolic pathway increase with CR (p < 0.05). Differences indicate GM and metabolomic factors play a role in WL maintenance and body composition. This novel work provides insight into the GM and metabolomic profile of participants following an IF-P or CR diet and highlights important differences in microbial assembly associated with WL and body composition responsiveness. These data may inform future GM-focused precision nutrition recommendations using larger sample sizes of longer duration. Trial registration, March 6, 2020 (ClinicalTrials.gov as NCT04327141), based on a previous randomized intervention trial.

Insulin signaling and pharmacology in humans and in corals.

Once thought to be a unique capability of the Langerhans islets in the pancreas of mammals, insulin (INS) signaling is now recognized as an evolutionarily ancient function going back to prokaryotes. INS is ubiquitously present not only in humans but also in unicellular eukaryotes, fungi, worms, and Drosophila. Remote homologue identification also supports the presence of INS and INS receptor in corals where the availability of glucose is largely dependent on the photosynthetic activity of the symbiotic algae. The cnidarian animal host of corals operates together with a 20,000-sized microbiome, in direct analogy to the human gut microbiome. In humans, aberrant INS signaling is the hallmark of metabolic disease, and is thought to play a major role in aging, and age-related diseases, such as Alzheimer's disease. We here would like to argue that a broader view of INS beyond its human homeostasis function may help us understand other organisms, and in turn, studying those non-model organisms may enable a novel view of the human INS signaling system. To this end, we here review INS signaling from a new angle, by drawing analogies between humans and corals at the molecular level.

Inflammation and the pathological progression of Alzheimer's disease are associated with low circulating choline levels.

Deficiency of dietary choline, an essential nutrient, is observed worldwide, with ~ 90% of Americans being deficient. Previous work highlights a relationship between decreased choline intake and an increased risk for cognitive decline and Alzheimer's disease (AD). The associations between blood circulating choline and the pathological progression in both mild cognitive impairment (MCI) and AD remain unknown. Here, we examined these associations in a cohort of patients with MCI with presence of either sparse or high neuritic plaque density and Braak stage and a second cohort with either moderate AD (moderate to frequent neuritic plaques, Braak stage = IV) or severe AD (frequent neuritic plaques, Braak stage = VI), compared to age-matched controls. Metabolomic analysis was performed on serum from the AD cohort. We then assessed the effects of dietary choline deficiency (Ch-) in 3xTg-AD mice and choline supplementation (Ch+) in APP/PS1 mice, two rodent models of AD. The levels of circulating choline were reduced while pro-inflammatory cytokine TNFα was elevated in serum of both MCI sparse and high pathology cases. Reduced choline and elevated TNFα correlated with higher neuritic plaque density and Braak stage. In AD patients, we found reductions in choline, its derivative acetylcholine (ACh), and elevated TNFα. Choline and ACh levels were negatively correlated with neuritic plaque load, Braak stage, and TNFα, but positively correlated with MMSE, and brain weight. Metabolites L-Valine, 4-Hydroxyphenylpyruvic, Methylmalonic, and Ferulic acids were significantly associated with circuiting choline levels. In 3xTg-AD mice, the Ch- diet increased amyloid-ß levels and tau phosphorylation in cortical tissue, and TNFα in both blood and cortical tissue, paralleling the severe human-AD profile. Conversely, the Ch+ diet increased choline and ACh while reducing amyloid-ß and TNFα levels in brains of APP/PS1 mice. Collectively, low circulating choline is associated with AD-neuropathological progression, illustrating the importance of adequate dietary choline intake to offset disease.

Targeted metabolomics reveals plasma biomarkers and metabolic alterations of the aging process in healthy young and older adults.

With the exponential growth in the older population in the coming years, many studies have aimed to further investigate potential biomarkers associated with the aging process and its incumbent morbidities. Age is the largest risk factor for chronic disease, likely due to younger individuals possessing more competent adaptive metabolic networks that result in overall health and homeostasis. With aging, physiological alterations occur throughout the metabolic system that contribute to functional decline. In this cross-sectional analysis, a targeted metabolomic approach was applied to investigate the plasma metabolome of young (21-40y; n = 75) and older adults (65y + ; n = 76). A corrected general linear model (GLM) was generated, with covariates of gender, BMI, and chronic condition score (CCS), to compare the metabolome of the two populations. Among the 109 targeted metabolites, those associated with impaired fatty acid metabolism in the older population were found to be most significant: palmitic acid (p < 0.001), 3-hexenedioic acid (p < 0.001), stearic acid (p = 0.005), and decanoylcarnitine (p = 0.036). Derivatives of amino acid metabolism, 1-methlyhistidine (p = 0.035) and methylhistamine (p = 0.027), were found to be increased in the younger population and several novel metabolites were identified, such as cadaverine (p = 0.034) and 4-ethylbenzoic acid (p = 0.029). Principal component analysis was conducted and highlighted a shift in the metabolome for both groups. Receiver operating characteristic analyses of partial least squares-discriminant analysis models showed the candidate markers to be more powerful indicators of age than chronic disease. Pathway and enrichment analyses uncovered several pathways and enzymes predicted to underlie the aging process, and an integrated hypothesis describing functional characteristics of the aging process was synthesized. Compared to older participants, the young group displayed greater abundance of metabolites related to lipid and nucleotide synthesis; older participants displayed decreased fatty acid oxidation and reduced tryptophan metabolism, relative to the young group. As a result, we offer a better understanding of the aging metabolome and potentially reveal new biomarkers and predicted mechanisms for future study.

Low circulating choline, a modifiable dietary factor, is associated with the pathological progression and metabolome dysfunction in Alzheimer's disease.

Most Americans (∼90%) are deficient in dietary choline, an essential nutrient. Associations between circulating choline and pathological progression in Alzheimer's disease (AD) remain unknown. Here, we examined these associations and performed a metabolomic analysis in blood serum from severe AD, moderate AD, and healthy controls. Additionally, to gain mechanistic insight, we assessed the effects of dietary choline deficiency (Ch-) in 3xTg-AD mice and choline supplementation (Ch+) in APP/PS1 mice. In humans, we found AD-associated reductions in choline, it's derivative acetylcholine (ACh), and elevated pro-inflammatory cytokine TNFα. Choline and ACh were negatively correlated with Plaque density, Braak stage, and TNFα, but positively correlated with MMSE and brain weight. Metabolites L-Valine, 4-Hydroxyphenylpyruvic, Methylmalonic, and Ferulic acids were associated with choline levels. In mice, Ch-paralleled AD severe, but Ch+ was protective. In conclusion, low circulating choline is associated with AD-neuropathological progression, illustrating the importance of dietary choline consumption to offset disease.

An integrative cellular metabolomic study reveals downregulated tricarboxylic acid cycle and potential biomarkers induced by tetrabromobisphenol A in human lung A549 cells.

Tetrabromobisphenol A (TBBPA) is extensively utilized as a brominated flame retardant in numerous chemical products. As an environmental contaminant, the potential human toxicity of TBBPA has been attracting increasing attention. Nonetheless, the exact underlying mechanisms of toxicological effects caused by TBBPA remain uncertain. In this study, we investigated the potential mechanisms of TBBPA toxicity in vitro in the A549 cell line, one of the widely used type II pulmonary epithelial cell models in toxicology research. Cell viability was determined after treatment with varying concentrations of TBBPA. Liquid chromatography-mass spectrometry (LC-MS) metabolomics and metabolic flux approaches were utilized to evaluate metabolite and tricarboxylic acid (TCA) cycle oxidative flux changes. Our findings demonstrated that TBBPA significantly reduced the viability of cells and attenuated mitochondrial respiration in A549 cells. Additionally, LC-MS data showed significant reductions in TCA cycle metabolites including citrate, malate, fumarate, and alpha-ketoglutarate in 50 µM TBBPA-treated A549 cells. Metabolic flux analysis indicated reduced oxidative capacity in mitochondrial metabolism following TBBPA exposure. Moreover, diverse metabolic pathways, particularly alanine, aspartate, and glutamate metabolism and the TCA cycle, were found to be dysregulated. In total, 12 metabolites were significantly changed (p < .05) in response to 50 µM TBBPA exposure. Our results provide potential biomarkers of TBBPA toxicity in A549 cells and help elucidate the molecular mechanisms of pulmonary toxicity induced by TBBPA exposure.

Metabolomic profiles of metformin in breast cancer survivors: a pooled analysis of plasmas from two randomized placebo-controlled trials.

BACKGROUND: Obesity is a major health concern for breast cancer survivors, being associated with high recurrence and reduced efficacy during cancer treatment. Metformin treatment is associated with reduced breast cancer incidence, recurrence and mortality. To better understand the underlying mechanisms through which metformin may reduce recurrence, we aimed to conduct metabolic profiling of overweight/obese breast cancer survivors before and after metformin treatment. METHODS: Fasting plasma samples from 373 overweight or obese breast cancer survivors randomly assigned to metformin (n = 194) or placebo (n = 179) administration were collected at baseline, after 6 months (Reach For Health trial), and after 12 months (MetBreCS trial). Archival samples were concurrently analyzed using three complementary methods: untargeted LC-QTOF-MS metabolomics, targeted LC-MS metabolomics (AbsoluteIDQ p180, Biocrates), and gas chromatography phospholipid fatty acid assay. Multivariable linear regression models and family-wise error correction were used to identify metabolites that significantly changed after metformin treatment. RESULTS: Participants (n = 352) with both baseline and study end point samples available were included in the analysis. After adjusting for confounders such as study center, age, body mass index and false discovery rate, we found that metformin treatment was significantly associated with decreased levels of citrulline, arginine, tyrosine, caffeine, paraxanthine, and theophylline, and increased levels of leucine, isoleucine, proline, 3-methyl-2-oxovalerate, 4-methyl-2-oxovalerate, alanine and indoxyl-sulphate. Long-chain unsaturated phosphatidylcholines (PC ae C36:4, PC ae C38:5, PC ae C36:5 and PC ae C38:6) were significantly decreased with the metformin treatment, as were phospholipid-derived long-chain n-6 fatty acids. The metabolomic profiles of metformin treatment suggest change in specific biochemical pathways known to impair cancer cell growth including activation of CYP1A2, alterations in fatty acid desaturase activity, and altered metabolism of specific amino acids, including impaired branched chain amino acid catabolism. CONCLUSIONS: Our results in overweight breast cancer survivors identify new metabolic effects of metformin treatment that may mechanistically contribute to reduced risk of recurrence in this population and reduced obesity-related cancer risk reported in observational studies. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01302379 and EudraCT Protocol #: 2015-001001-14.

Exploratory analysis of one versus two-day intermittent fasting protocols on the gut microbiome and plasma metabolome in adults with overweight/obesity.

Nutritional interventions are a promising therapeutic option for addressing obesity and cardiometabolic dysfunction. One such option, intermittent fasting (IF), has emerged as a viable alternative to daily caloric restriction and may beneficially modulate body weight regulation and alter the gut microbiome (GM) and plasma metabolome. This secondary analysis of a larger, registered trial (ClinicalTrials.gov ID: NCT04327141) examined the effect of a four-week intervention comparing one vs. two-consecutive days of IF in combination with protein pacing (IF-P; 4-5 meals/day, >30% protein/day) on the GM, the plasma metabolome, and associated clinical outcomes in overweight and obese adults. Participants (n = 20) were randomly assigned to either a diet consisting of one fasting day (total of 36 h) and six low-calorie P days per week (IF1-P, n = 10) or two fasting days (60 h total) and five low-calorie P days per week (IF2-P, n = 10). The fecal microbiome, clinical outcomes, and plasma metabolome were analyzed at baseline (week 0) and after four weeks. There were no significant time or interaction effects for alpha diversity; however, baseline alpha diversity was negatively correlated with percent body fat change after the four-week intervention (p = 0.030). In addition, beta-diversity for both IF groups was altered significantly by time (p = 0.001), with no significant differences between groups. The IF1-P group had a significant increase in abundance of Ruminococcaceae Incertae Sedis and Eubacterium fissicatena group (q ≤ 0.007), while the IF2-P group had a significant increase in abundance of Ruminococcaceae Incertae Sedis and a decrease in Eubacterium ventriosum group (q ≤ 0.005). The plasma metabolite profile of IF2-P participants displayed significant increases in serine, trimethylamine oxide (TMAO), levulinic acid, 3-aminobutyric acid, citrate, isocitrate, and glucuronic acid (q ≤ 0.049) compared to IF1-P. Fecal short-chain fatty acid concentrations did not differ significantly by time or between groups (p ≥ 0.126). Interestingly, gastrointestinal symptoms were significantly reduced for the IF2-P group but not for the IF1-P group. Our results demonstrate that short-term IF modestly influenced the GM community structure and the plasma metabolome, suggesting these protocols could be viable for certain nutritional intervention strategies.

Longitudinal Comparison of Neutralizing Antibody Responses to COVID-19 mRNA Vaccines after Second and Third Doses.

COVID-19 mRNA vaccines protect against severe disease and hospitalization. Neutralizing antibodies (NAbs) are a first-line defense mechanism, but protective NAb responses are variable. Currently, NAb testing is not widely available. This study employed a lateral flow assay for monitoring NAb levels postvaccination and natural infection, using a finger-stick drop of blood. We report longitudinal NAb data from BNT162b2 (Pfizer) and mRNA-1273 (Moderna) recipients after second and third doses. Results demonstrate a third dose of mRNA vaccine elicits higher and more durable NAb titers than the second dose, independent of manufacturer, sex, and age. Our analyses also revealed that vaccinated individuals could be categorized as strong, moderate, and poorly neutralizing responders. After the second dose, 34% of subjects were classified as strong responders, compared to 79% after the third dose. The final months of this study coincided with the emergence of the SARS-CoV-2 Omicron variant and symptomatic breakthrough infections within our study population. Lastly, we show that NAb levels sufficient for protection from symptomatic infection with early SARS-CoV-2 variants were not protective against Omicron infection and disease. This work highlights the need for accessible vaccine response monitoring for use in healthcare, such that individuals, particularly those in vulnerable populations, can make informed vaccination decisions.

Association of food insecurity on gut microbiome and metabolome profiles in a diverse college-based sample.

Voluntary caloric restriction (e.g., eating disorders) often results in alterations in the gut microbiota composition and function. However, these findings may not translate to food insecurity, where an individual experiences inconsistent access to healthy food options. In this study we compared the fecal microbiome and metabolome of racially and ethnically diverse first year college students (n = 60) experiencing different levels of food access. Students were dichotomized into food secure (FS) and food insecure (FI) groups using a validated, 2-question screener assessing food security status over the previous 30 days. Fecal samples were collected up to 5 days post survey-completion. Gut microbiome and metabolome were established using 16S rRNA amplicon sequencing, targeted liquid chromatography-tandem mass spectrometry, and gas chromatography-mass spectrometry. FI students experienced significantly greater microbial diversity with increased abundance of Enterobacteriaceae and Eisenbergiella, while FS students had greater abundance of Megasphaera and Holdemanella. Metabolites related to energy transfer and gut-brain-axis communication (picolinic acid, phosphocreatine, 2-pyrrolidinone) were elevated in FI students (q < 0.05). These findings suggest that food insecurity is associated with differential gut microbial and metabolite composition for which the future implications are unknown. Further work is needed to elucidate the longitudinal metabolic effects of food insecurity and how gut microbes influence metabolic outcomes.

Ischemic Stroke and Dietary Vitamin B12 Deficiency in Old-Aged Females: Impaired Motor Function, Increased Ischemic Damage Size, and Changed Metabolite Profiles in Brain and Cecum Tissue.

A vitamin B12 deficiency (vit. B12 def.) is common in the elderly, because of changes in metabolism. Clinical studies have reported that a vit. B12 def. results in worse outcome after stroke, and the mechanisms through which a vit. B12 def. changes the brain requires further investigation. This study investigated the role of vit. B12 def. on stroke outcome and mechanisms using aged female mice. Eighteen-month-old females were put on a control or vit. B12 def. diet for 4 weeks, after which an ischemic stroke was induced in the sensorimotor cortex. After damage, motor function was measured, the animals were euthanized, and tissues were collected for analysis. Vit. B12 def. animals had increased levels of total homocysteine in plasma and liver, and choline levels were also increased in the liver. Vit. B12 def. animals had larger damage volume in brain tissue and more apoptosis. The cecum tissue pathway analysis showed dysfunction in B12 transport. The analysis of mitochondrial metabolomics in brain tissue showed reduced levels of metabolites involved in the TCA cycle in vit. B12 def. animals. Motor function after stroke was impaired in vit. B12 def. animals. A dietary vit. B12 def. impairs motor function through increased apoptosis and changes in mitochondrial metabolism in brain tissue.

Third COVID-19 vaccine dose boosts neutralizing antibodies in poor responders.

Background: While evaluating COVID-19 vaccine responses using a rapid neutralizing antibody (NAb) test, we observed that 25% of mRNA vaccine recipients did not neutralize >50%. We termed this group "vaccine poor responders" (VPRs). The objective of this study was to determine if individuals who neutralized <50% would remain VPRs, or if a third dose would elicit high levels of NAbs. Methods: 269 healthy individuals ranging in age from 19 to 80 (Average age = 51; 165 females and 104 males) who received either BNT162b2 (Pfizer) or mRNA-1273 (Moderna) vaccines were evaluated. NAb levels were measured: (i) 2-4 weeks after a second vaccine dose, (ii) 2-4 months after the second dose, (iii) within 1-2 weeks prior to a third dose and (iv) 2-4 weeks after a third mRNA vaccine dose. Results: Analysis of vaccine recipients reveals that 25% did not neutralize above 50% (Median neutralization = 21%, titers <1:80) within a month after their second dose. Twenty-three of these VPRs obtained a third dose of either BNT162b2 or mRNA-1273 vaccine 1-8 months (average = 5 months) after their second dose. Within a month after their third dose, VPRs show an average 5.4-fold increase in NAb levels (range: 46-99%). Conclusions: The results suggest that VPRs are not permanently poor responders; they can generate high NAb levels with an additional vaccine dose. Although it is not known what levels of NAbs protect from infection or disease, those in high-risk professions may wish to keep peripheral NAb levels high, limiting infection, and potential transmission.

Lipopolysaccharide and the gut microbiota: considering structural variation.

Systemic inflammation is associated with chronic disease and is purported to be a main pathogenic mechanism underlying metabolic conditions. Microbes harbored in the host gastrointestinal tract release signaling byproducts from their cell wall, such as lipopolysaccharides (LPS), which can act locally and, after crossing the gut barrier and entering circulation, also systemically. Defined as metabolic endotoxemia, elevated concentrations of LPS in circulation are associated with metabolic conditions and chronic disease. As such, measurement of LPS is highly prevalent in animal and human research investigating these states. Indeed, LPS can be a potent stimulant of host immunity, but this response depends on the microbial species' origin, a parameter often overlooked in both preclinical and clinical investigations. Indeed, the lipid A portion of LPS is mutable and comprises the main virulence and endotoxic component, thus contributing to the structural and functional diversity among LPSs from microbial species. In this review, we discuss how such structural differences in LPS can induce differential immunological responses in the host.

Microbiome and metabolome profiles of high screen time in a cohort of healthy college students.

As screens are increasingly integrated into every facet of modern life, there is growing concern over the potential effects of high screen time. Previous studies have largely utilized self-report data on mood and behavioral aspects of screen time, and no molecular theory has yet been developed. In this study, we explored the fecal microbiome and metabolome of a diverse group of 60 college students, classified by high (≥ 75 min/day) or low (0-75 min/day) self-reported screen time using 16S rRNA amplicon sequencing, targeted liquid chromatography-tandem mass spectrometry, and targeted detection of short-chain fatty acids using gas chromatography-mass spectrometry. Several key taxa and metabolites were significantly altered between groups and found to be highly co-occurrent. Results of pathway and enzyme enrichment analyses were synthesized to articulate an integrated hypothesis indicating widespread mitochondrial dysfunction and aberrant amino acid metabolism. High screen time was also predicted to be significantly associated with type I diabetes, obesity, chronic fatigue syndrome, and various manifestations of inflammatory bowel. This is the first-ever study to report the effects of high screen time at the molecular level, and these results provide a data-driven hypothesis for future experimental research.

Development of a rapid point-of-care test that measures neutralizing antibodies to SARS-CoV-2.

BACKGROUND: After receiving a COVID-19 vaccine, most recipients want to know if they are protected from infection and for how long. Since neutralizing antibodies are a correlate of protection, we developed a lateral flow assay (LFA) that measures levels of neutralizing antibodies from a drop of blood. The LFA is based on the principle that neutralizing antibodies block binding of the receptor-binding domain (RBD) to angiotensin-converting enzyme 2 (ACE2). METHODS: The ability of the LFA was assessed to correctly measure neutralization of sera, plasma or whole blood from patients with COVID-19 using SARS-CoV-2 microneutralization assays. We also determined if the LFA distinguished patients with seasonal respiratory viruses from patients with COVID-19. To demonstrate the usefulness of the LFA, we tested previously infected and non-infected COVID-19 vaccine recipients at baseline and after first and second vaccine doses. RESULTS: The LFA compared favorably with SARS-CoV-2 microneutralization assays with an area under the ROC curve of 98%. Sera obtained from patients with seasonal coronaviruses did not show neutralizing activity in the LFA. After a single mRNA vaccine dose, 87% of previously infected individuals demonstrated high levels of neutralizing antibodies. However, if individuals were not previously infected, only 24% demonstrated high levels of neutralizing antibodies after one vaccine dose. A second dose boosted neutralizing antibody levels just 8% higher in previously infected individuals, but over 63% higher in non-infected individuals. CONCLUSIONS: A rapid, semi-quantitative, highly portable and inexpensive neutralizing antibody test might be useful for monitoring rise and fall in vaccine-induced neutralizing antibodies to COVID-19.

Daily Vinegar Ingestion Improves Depression Scores and Alters the Metabolome in Healthy Adults: A Randomized Controlled Trial.

Daily vinegar ingestion has been linked to improved glycemic control, but recent data suggest a separate unexplored role for vinegar in mental health. Utilizing a placebo-controlled, parallel arm study design, this 4-week trial examined the impact of daily vinegar ingestion on mood states and urinary metabolites in healthy college students. Participants were randomized to the vinegar group (VIN: n = 14; 1.5 g acetic acid/day as liquid vinegar) or the control group (CON: n = 11; 0.015 g acetic acid/day as a pill) with no change to customary diet or physical activity. At baseline and at study week four, participants completed the Profile of Mood States (POMS) and the Center for Epidemiological Studies-Depression (CES-D) questionnaires and provided a first-morning urine sample for targeted metabolomics analyses. The change in both POMS depression scores and CES-D scores differed significantly between groups favoring improved affect in the VIN versus CON participants after four weeks. Metabolomics analyses pre and post-intervention suggested metabolite alterations associated with vinegar ingestion that are consistent for improved mood, including enzymatic dysfunction in the hexosamine pathway as well as significant increases in glycine, serine, and threonine metabolism. These data warrant continued investigation of vinegar as a possible agent to improve mood state.

Metabolic Profiling of Neocortical Tissue Discriminates Alzheimer's Disease from Mild Cognitive Impairment, High Pathology Controls, and Normal Controls.

Alzheimer's disease (AD) is the most common cause of dementia, accounting for an estimated 60-80% of cases, and is the sixth-leading cause of death in the United States. While considerable advancements have been made in the clinical care of AD, it remains a complicated disorder that can be difficult to identify definitively in its earliest stages. Recently, mass spectrometry (MS)-based metabolomics has shown significant potential for elucidation of disease mechanisms and identification of therapeutic targets as well diagnostic and prognostic markers that may be useful in resolving some of the difficulties affecting clinical AD studies, such as effective stratification. In this study, complementary gas chromatography- and liquid chromatography-MS platforms were used to detect and monitor 2080 metabolites and features in 48 postmortem tissue samples harvested from the superior frontal gyrus of male and female subjects. Samples were taken from four groups: 12 normal control (NC) patients, 12 cognitively normal subjects characterized as high pathology controls (HPC), 12 subjects with nonspecific mild cognitive impairment (MCI), and 12 subjects with AD. Multivariate statistics informed the construction and cross-validation (p < 0.01) of partial least squares-discriminant analysis (PLS-DA) models defined by a nine-metabolite panel of disease markers (lauric acid, stearic acid, myristic acid, palmitic acid, palmitoleic acid, and four unidentified mass spectral features). Receiver operating characteristic analysis showed high predictive accuracy of the resulting PLS-DA models for discrimination of NC (97%), HPC (92%), MCI (∼96%), and AD (∼96%) groups. Pathway analysis revealed significant disturbances in lysine degradation, fatty acid metabolism, and the degradation of branched-chain amino acids. Network analysis showed significant enrichment of 11 enzymes, predominantly within the mitochondria. The results expand basic knowledge of the metabolome related to AD and reveal pathways that can be targeted therapeutically. This study also provides a promising basis for the development of larger multisite projects to validate these candidate markers in readily available biospecimens such as blood to enable the effective screening, rapid diagnosis, accurate surveillance, and therapeutic monitoring of AD. All raw mass spectrometry data have been deposited to MassIVE (data set identifier MSV000087165).

Enhanced Detection of Short-Chain Fatty Acids Using Gas Chromatography Mass Spectrometry.

Short-chain fatty acids (SCFAs) are produced mainly by intestinal microbiota and play an important role in many host biological processes such as immune system development, glucose and energy homeostasis, and regulation of immune response and inflammation. In addition, they participate in the regulation of anorectic hormones, which have a role in appetite control, tumor suppression, and regulating the central and peripheral nervous systems. As such, there is great interest in monitoring levels of SCFAs in various biological samples. Due to the highly hydrophilic and volatile characteristics of SCFAs, optimizing extraction and sample preparation procedures is often a central component to further improve SCFA quantification. Here, we describe a rapid and highly sensitive analytical method for measuring SCFAs in human serum and feces. Briefly, SCFAs are protected by adding sodium hydroxide, followed by a one-step extraction (pH > 7). Then, SCFAs are quantified by gas chromatography coupled to mass spectrometry (GC-MS) after derivatization with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA). This method demonstrates excellent sensitivity, linearity, and derivatization efficiency for simultaneous determination of 14 different SCFAs. Further, this validated method can be successfully applied to quantify SCFAs in micro-scale biological samples. In summary, we describe efficient and advanced sample preparation and detection procedures that are critically needed for monitoring SCFA concentrations in human biological samples. © 2021 Wiley Periodicals LLC. Basic Protocol: SCFA extraction and detection from fecal and serum samples with gas chromatography-mass spectrometry.

Early Breast Cancer Detection Using Untargeted and Targeted Metabolomics.

Breast cancer (BC) is a common cause of morbidity and mortality, particularly in women. Moreover, the discovery of diagnostic biomarkers for early BC remains a challenging task. Previously, we [Jasbi et al. J. Chromatogr. B. 2019, 1105, 26-37] demonstrated a targeted metabolic profiling approach capable of identifying metabolite marker candidates that could enable highly sensitive and specific detection of BC. However, the coverage of this targeted method was limited and exhibited suboptimal classification of early BC (EBC). To expand the metabolome coverage and articulate a better panel of metabolites or mass spectral features for classification of EBC, we evaluated untargeted liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) data, both individually as well as in conjunction with previously published targeted LC-triple quadruple (QQQ)-MS data. Variable importance in projection scores were used to refine the biomarker panel, whereas orthogonal partial least squares-discriminant analysis was used to operationalize the enhanced biomarker panel for early diagnosis. In this approach, 33 altered metabolites/features were detected by LC-QTOF-MS from 124 BC patients and 86 healthy controls. For EBC diagnosis, significance testing and analysis of the area under receiver operating characteristic (AUROC) curve identified six metabolites/features [ethyl (R)-3-hydroxyhexanoate; caprylic acid; hypoxanthine; and m/z 358.0018, 354.0053, and 356.0037] with p < 0.05 and AUROC > 0.7. These metabolites informed the construction of EBC diagnostic models; evaluation of model performance for the prediction of EBC showed an AUROC = 0.938 (95% CI: 0.895-0.975), with sensitivity = 0.90 when specificity = 0.90. Using the combined untargeted and targeted data set, eight metabolic pathways of potential biological relevance were indicated to be significantly altered as a result of EBC. Metabolic pathway analysis showed fatty acid and aminoacyl-tRNA biosynthesis as well as inositol phosphate metabolism to be most impacted in response to the disease. The combination of untargeted and targeted metabolomics platforms has provided a highly predictive and accurate method for BC and EBC diagnosis from plasma samples. Furthermore, such a complementary approach yielded critical information regarding potential pathogenic mechanisms underlying EBC that, although critical to improved prognosis and enhanced survival, are understudied in the current literature. All mass spectrometry data and deidentified subject metadata analyzed in this study have been deposited to Mendeley Data and are publicly available (DOI: 10.17632/kcjg8ybk45.1).

Hypoxia promotes erythroid differentiation through the development of progenitors and proerythroblasts.

Oxygen is a critical noncellular component of the bone marrow microenvironment that plays an important role in the development of hematopoietic cell lineages. In this study, we investigated the impact of low oxygen (hypoxia) on ex vivo myeloerythroid differentiation of human cord blood-derived CD34+ hematopoietic stem and progenitor cells. We characterized the culture conditions to demonstrate that low oxygen inhibits cell proliferation and causes a metabolic shift in the stem and progenitor populations. We found that hypoxia promotes erythroid differentiation by supporting the development of progenitor populations. Hypoxia also increases the megakaryoerythroid potential of the common myeloid progenitors and the erythroid potential of megakaryoerythroid progenitors and significantly accelerates maturation of erythroid cells. Specifically, we determined that hypoxia promotes the loss of CD71 and the appearance of the erythroid markers CD235a and CD239. Further, evaluation of erythroid populations revealed a hypoxia-induced increase in proerythroblasts and in enucleation of CD235a+ cells. These results reveal the extensive role of hypoxia at multiple steps during erythroid development. Overall, our work establishes a valuable model for further investigations into the relationship between erythroid progenitors and/or erythroblast populations and their hypoxic microenvironment.