Darya O. Mishchuk

Dietary pyrroloquinoline quinone (PQQ) alters indicators of inflammation and mitochondrial-related metabolism in human subjects

Pyrroloquinoline quinone (PQQ) influences energy-related metabolism and neurologic functions in animals. The mechanism of action involves interactions with cell signaling pathways and mitochondrial function. However, little is known about the response to PQQ in humans. Using a crossover study design, 10 subjects (5 females, 5 males) ingested PQQ added to a fruit-flavored drink in two separate studies. In study 1, PQQ was given in a single dose (0.2 mg PQQ/kg). Multiple measurements of plasma and urine PQQ levels and changes in antioxidant potential [based on total peroxyl radical-trapping potential and thiobarbituric acid reactive product (TBAR) assays] were made throughout the period of 48 h. In study 2, PQQ was administered as a daily dose (0.3 mg PQQ/kg). After 76 h, measurements included indices of inflammation [plasma C-reactive protein, interleukin (IL)-6 levels], standard clinical indices (e.g., cholesterol, glucose, high-density lipoprotein, low-density lipoprotein, triglycerides, etc.) and (1)H-nuclear magnetic resonance estimates of urinary metabolites related in part to oxidative metabolism. The standard clinical indices were normal and not altered by PQQ supplementation. However, dietary PQQ exposure (Study 1) resulted in apparent changes in antioxidant potential based on malonaldehyde-related TBAR assessments. In Study 2, PQQ supplementation resulted in significant decreases in the levels of plasma C-reactive protein, IL-6 and urinary methylated amines such as trimethylamine N-oxide, and changes in urinary metabolites consistent with enhanced mitochondria-related functions. The data are among the first to link systemic effects of PQQ in animals to corresponding effects in humans.

Metabolomics of Cerebrospinal Fluid from Humans Treated for Rabies

Rabies is a rapidly progressive lyssavirus encephalitis that is statistically 100% fatal. There are no clinically effective antiviral drugs for rabies. An immunologically naïve teenager survived rabies in 2004 through improvised supportive care; since then, 5 additional survivors have been associated with use of the so-called Milwaukee Protocol (MP). The MP applies critical care focused on the altered metabolic and physiologic states associated with rabies. The aim of this study was to examine the metabolic profile of cerebrospinal fluid (CSF) from rabies patients during clinical progression of rabies encephalitis in survivors and nonsurvivors and to compare these samples with control CSF samples. Unsupervised clustering algorithms distinguished three stages of rabies disease and identified several metabolites that differentiated rabies survivors from those who subsequently died, in particular, metabolites related to energy metabolism and cell volume control. Moreover, for those patients who survived, the trajectory of their metabolic profile tracked toward the control profile and away from the rabies profile. NMR metabolomics of human rabies CSF provide new insights into the mechanisms of rabies pathogenesis, which may guide future therapy of this disease.

Elevation, rootstock, and soil depth affect the nutritional quality of mandarin oranges.

The effects of elevation, rootstock, and soil depth on the nutritional quality of mandarin oranges from 11 groves in California were investigated by nuclear magnetic resonance (NMR) spectroscopy by quantifying 29 compounds and applying multivariate statistical data analysis. A comparison of the juice from oranges in groves with deeper soil and trifoliate rootstock versus those with shallow soil and C-35 rootstock revealed differences in the concentrations of 4-aminobutyrate, ethanol, phenylalanine, succinate, and isoleucine. A comparison of fruit from trees grown at higher versus lower elevation revealed that those at higher elevation had higher concentrations of amino acids, succinate, and 4-aminobutyrate and lower concentrations of sugars and limonin glucoside. Such differences indicate that rootstock, soil depth, and differences in elevation influence the fruit nutrient composition. This study highlights how metabolomics coupled with multivariate statistical analysis can illuminate the metabolic characteristics of citrus, thereby aiding in the determination of the grove identity and fruit quality during orange production.

Fertilisation and pesticides affect mandarin orange nutrient composition

The effects of the application of foliar fertilisation and pesticide on nutritional quality of mandarin orange juices were evaluated using (1)H NMR metabolomics. Significant differences between the use of fertiliser and pesticides during fruit formation were observed, and included changes in sugar, amino acid and organic acid composition. To determine whether the difference in sugar concentration was enough for the consumer to detect, a sensory experiment was performed in which two orange juice samples were prepared to resemble the sweet/sour taste balance of juice from mandarin oranges in which foliar fertilisation was either applied or not. In a test using non-trained individuals, 68% could correctly identify which juice had a sourer, or less sweet, taste. The implications of this study could impact citrus growers, and ultimately aid in development of fruit with superior sensory quality.

Cross-talk between E. coli strains and a human colorectal adenocarcinoma-derived cell line

Although there is great interest in the specific mechanisms of how gut microbiota modulate the biological processes of the human host, the extent of host-microbe interactions and the bacteria-specific metabolic activities for survival in the co-evolved gastrointestinal environment remain unclear. Here, we demonstrate a comprehensive comparison of the host epithelial response induced by either a pathogenic or commensal strain of Escherichia coli using a multi-omics approach. We show that Caco-2 cells incubated with E. coli display an activation of defense response genes associated with oxidative stress. Indeed, in the bacteria co-culture system, the host cells experience an altered environment compared with the germ-free system that includes reduced pH, depletion of major energy substrates, and accumulation of fermentation by-products. Measurement of intracellular Caco-2 cell metabolites revealed a significantly increased lactate concentration, as well as changes in TCA cycle intermediates. Our results will lead to a deeper understanding of acute microbial-host interactions.

An NMR Metabolomic Study on the Effect of Alendronate in Ovariectomized Mice

Alendronate sodium (Fosamax) is most widely used for the prevention and treatment of osteoporosis. It is a type of anti-resorptive agent that reduces the risk of fractures by changing bone turnover and bone mineral density. We investigated the effect of Fosamax on a mouse model of osteoporosis. Twenty-seven female C57BL/6JNarl mice were divided into three groups: sham, ovariectomized (OVX) and OVX + Fosamax (Fosamax). After 23 weeks, bone density of femurs was analyzed using microcomputed tomography (micro-CT), and serum was analyzed for osteoblast and osteoclast activity, as well as metabolites using nuclear magnetic resonance (NMR) spectroscopy. Fosamax increased bone mineral density and cortical bone thickness, and decreased osteoblast activity slightly. Fosamax did not significantly change osteoclast activity. Serum metabolomics revealed that Fosamax had profound effects on overall metabolism, as significantly higher concentrations of metabolites associated with energy metabolism (including TCA-cycle intermediates and glucose), 3-hydroxybutyrate, taurine, allantoin, acetate, and ethanol, as well as lower concentrations of aspartate were observed in the Fosamax-treated mice compared with the OVX mice. These results suggest that alendronate may work by increasing bone density through altered metabolic activity.

Metabolomics reveals differences between three daidzein metabolizing phenotypes in adults with cardiometabolic risk factors.

SCOPE The soy isoflavone, daidzein, is metabolized by gut microbiota to O-desmethylangolensin (ODMA) and/or equol. Producing equol is postulated as a contributing factor for the beneficial effects of soy. METHODS AND RESULTS This randomized, controlled, cross-over design used an untargeted metabolomic approach to assess the metabolic profile of different daidzein metabolizers. Adults (n = 17) with cardiometabolic risk factors received soy nuts or control food for 4 weeks, separated by a 2-week washout. No significant differences were detected pre- and postintervention and between interventions. Examination of the ability to metabolize daidzein revealed three groups: ODMA only producers (n = 4), equol + ODMA producers (n = 8), and nonproducers (n = 5). Analysis of the serum metabolome revealed nonproducers could be distinguished from ODMA-only and equol + ODMA producers. Differences between these phenotypes were related to obesity and metabolic risk (methionine, asparagine, and trimethylamine) with equol + ODMA producers having lower concentrations, yet paradoxically higher pro-inflammatory cytokines. In urine, nonproducers clustered with ODMA producers and were distinct from equol + ODMA producers. Urinary metabolite profiles revealed significantly higher excretion of fumarate and 2-oxoglutarate, as well as pyroglutamate, alanine, and the gut microbial metabolite dimethylamine in equol + ODMA producers. CONCLUSION These results emphasize that the serum and urine metabolomes are distinct based on the ability to metabolize isoflavones.

Consumption of vitamin D2 enhanced mushrooms is associated with improved bone health

Mushrooms are the best nonanimal food source of vitamin D2. Pulsed irradiation can enhance vitamin D2 in mushrooms quickly. We investigated the effect of supplementing high vitamin D2Pleurotus ferulae mushrooms in a mouse model of osteoporosis. Thirty-two female C57BL/6JNarl mice were divided into four groups including sham, ovariectomized (OVX), OVX+nonpulsed mushroom (NPM) and OVX+pulsed mushroom (PM). After 23 weeks of treatment, serum samples were analyzed for osteoblast and osteoclast indicators, as well as metabolites using NMR spectroscopy. To examine bone density, femurs were analyzed using micro-computed tomography. The NPM and PM treatment mice showed increased bone density in comparison with OVX mice. In addition, the PM mice showed higher osteoblast and lower osteoclast indicators in comparison with OVX mice. Serum metabolomics analysis indicated several metabolites that were different in PM mice, some of which could be correlated with bone health. Taken together, these results suggest that pulsed irradiated mushrooms are able to increase bone density in osteoporotic mice possibly through enhanced bone metabolism. Further studies in humans are needed to show their efficacy in preventing osteoporosis.

Microbiota, metabolome, and immune alterations in obese mice fed a high fat diet containing type 2 resistant starch

SCOPE We examined the intestinal and systemic responses to incorporating a type 2 resistant starch (RS) into a high fat diet fed to obese mice. METHODS AND RESULTS Diet-induced obese, C57BL/6J male mice were fed an HF diet without or with 20% (by weight) high-amylose maize resistant starch (HF-RS) for 6 weeks. Serum adiponectin levels were higher with RS consumption, but there were no differences in weight gain and adiposity. With HF-RS, the expression levels of ileal TLR2 and Reg3g and cecal occludin, TLR2, TLR4, NOD1 and NOD2 were induced; whereas colonic concentrations of the inflammatory cytokine IL-17A declined. The intestinal, serum, liver, and urinary metabolomes were also altered. HF-RS resulted in lower amino acid concentrations, including lower serum branched chain amino acids, and increased quantities of urinary di/trimethylamine, 3-indoxylsulfate, and phenylacetylglycine. Corresponding to these changes were enrichments in Bacteroidetes (S24-7 family) and certain Firmicutes taxa (Lactobacillales and Erysipelotrichaceae) with the HF-RS diet. Parabacteroides and S24-7 positively associated with cecal maltose concentrations. These taxa and Erysipelotrichaceae, Allobaculum, and Bifidobacterium were directly correlated with uremic metabolites. CONCLUSION Consumption of RS modified the intestinal microbiota, stimulated intestinal immunity and endocrine-responses, and modified systemic metabolomes in obese mice consuming an otherwise obesogenic diet.

Dextran Sulfate Sodium Inhibits Alanine Synthesis in Caco-2 Cells

To understand and characterize the pathogenic mechanisms of inflammatory bowel disease, dextran sulfate sodium (DSS) has been used to induce acute and chronic colitis in animal models by causing intestinal epithelium damage. The mechanism of action of DSS in producing this outcome is not well understood. In an effort to understand how DSS might impact epithelial cell metabolism, we studied the intestinal epithelial cell line Caco-2 incubated with 1% DSS over 56 hours using 1H NMR spectroscopy. We observed no difference in cell viability as compared to control cultures, and an approximately 1.5-fold increase in IL-6 production upon incubation with 1% DSS. The effect on Caco-2 cell metabolism as measured through changes in the concentration of metabolites in the cell supernatant included a three-fold decrease in the concentration of alanine. Given that the concentrations of other amino acids in the cell culture supernatant were not different between treated and control cultures over 56 hours suggest that DSS inhibits alanine synthesis, specifically alanine aminotransferase, without affecting other key metabolic pathways. The importance of alanine aminotransferase in inflammatory bowel disease is discussed.