Characterization of D-amino acids in colostral, transitional, and mature preterm human milk.

D-Amino acids are regulatory molecules that affect biological processes. Therefore, being able to accurately detect and quantify these compounds is important for understanding their impact on nutrition and health. There is a paucity of information regarding D-amino acids in human milk. We developed a fast method for simultaneous analysis of amino acid enantiomers in human milk using liquid chromatography with tandem mass spectrometry. The method enables the separation of 41 amino acids without chemical derivatization. Our results revealed that human milk from mothers of preterm infants contains concentrations of D-amino acids that range from 0.5 to 45% that of their L-counterparts and that levels of most D-amino acids decrease as the milk production matures. Moreover, we found that Holder pasteurization of milk does not cause racemization of L-amino acids. To our knowledge, this is the first study to describe percentages of D-amino acid levels in human milk; changes in D-amino acid concentration as the milk matures; and the effect of Holder pasteurization on D- and L-amino acid concentrations in human milk.

Protective effects of NAMPT or MAPK inhibitors and NaR on Wallerian degeneration of mammalian axons.

Wallerian degeneration (WD) is a conserved axonal self-destruction program implicated in several neurological diseases. WD is driven by the degradation of the NAD+ synthesizing enzyme NMNAT2, the buildup of its substrate NMN, and the activation of the NAD+ degrading SARM1, eventually leading to axonal fragmentation. The regulation and amenability of these events to therapeutic interventions remain unclear. Here we explored pharmacological strategies that modulate NMN and NAD+ metabolism, namely the inhibition of the NMN-synthesizing enzyme NAMPT, activation of the nicotinic acid riboside (NaR) salvage pathway and inhibition of the NMNAT2-degrading DLK MAPK pathway in an axotomy model in vitro. Results show that NAMPT and DLK inhibition cause a significant but time-dependent delay of WD. These time-dependent effects are related to NMNAT2 degradation and changes in NMN and NAD+ levels. Supplementation of NAMPT inhibition with NaR has an enhanced effect that does not depend on timing of intervention and leads to robust protection up to 4 days. Additional DLK inhibition extends this even further to 6 days. Metabolite analyses reveal complex effects indicating that NAMPT and MAPK inhibition act by reducing NMN levels, ameliorating NAD+ loss and suppressing SARM1 activity. Finally, the axonal NAD+/NMN ratio is highly predictive of cADPR levels, extending previous cell-free evidence on the allosteric regulation of SARM1. Our findings establish a window of axon protection extending several hours following injury. Moreover, we show prolonged protection by mixed treatments combining MAPK and NAMPT inhibition that proceed via complex effects on NAD+ metabolism and inhibition of SARM1.

Neonatal encephalopathy plasma metabolites are associated with neurodevelopmental outcomes.

BACKGROUND: To investigate mechanisms of injury and recovery in neonatal encephalopathy (NE), we performed targeted metabolomic analysis of plasma using liquid chromatography with tandem mass spectrometry (LC/MS/MS) from healthy term neonates or neonates with NE. METHODS: Plasma samples from the NE (n = 45, day of life 0-1) or healthy neonatal (n = 30, ≥36 weeks gestation) cohorts had LC/MS/MS metabolomic profiling with a 193-plex targeted metabolite assay covering >366 metabolic pathways. Metabolite levels were compared to 2-year neurodevelopmental outcomes measured by the Bayley Scales of Infant and Toddler Development III (Bayley-III). RESULTS: Out of 193 metabolites, 57 met the pre-defined quality control criteria for analysis. Significant (after false discovery rate correction) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways included aminoacyl-tRNA biosynthesis, arginine biosynthesis, and metabolism of multiple amino acids. Significant disease pathways included seizures. In regression models, histidine and C6 sugar amine were significantly associated with cognitive, motor, and language and betaine with cognitive and motor Bayley-III composite scores. The addition of histidine, C6 sugar amine, and betaine to a Sarnat score-based clinical regression model significantly improved model performance (Akaike information criterion and adjusted r2) for Bayley-III cognitive, motor, and language scores. CONCLUSIONS: Plasma metabolites may help to predict neurological outcomes in neonatal brain injury and enhance current clinical predictors. IMPACT: Plasma metabolites may help to predict neurological outcomes in NE and supplement current clinical predictors. Current metabolomics research is limited in terms of clinical application and association with long-term outcomes. Our study presents novel associations of plasma metabolites from the first 24 h of life and 2-year neurodevelopmental outcomes for infants with NE. Our metabolomics discovery provides insight into possible disease mechanisms and methods to rescue and/or supplement metabolic pathways involved in NE. Our metabolomics discovery of metabolic pathway supplementations and/or rescue mechanisms may serve as adjunctive therapies for NE.

Metabolic effects of avocado/soy unsaponifiables on articular chondrocytes.

Avocado/soy unsaponifiable (ASU) components are reported to have a chondroprotective effect by virtue of anti-inflammatory and proanabolic effects on articular chondrocytes. The identity of the active component(s) remains unknown. In general, sterols, the major component of unsaponifiable plant material have been demonstrated to be anti-inflammatory in vitro and in animal models. These studies were designed to clarify whether the sterol content of ASU preparations were the primary contributors to biological activity in articular chondrocytes. ASU samples were analyzed by high pressure liquid chromatography (HPLC) and GC mass spectrometry. The sterol content was normalized between diverse samples prior to in vitro testing on bovine chondrocytes. Anabolic activity was monitored by uptake of 35-sulfate into proteoglycans and quantitation of labeled hydroxyproline and proline content after incubation with labeled proline. Anti-inflammatory activity was assayed by measuring reduction of interleukin-1 (IL-1)-induced synthesis of PGE2 and metalloproteases and release of label from tissue prelabeled with S-35.All ASU samples exerted a similar time-dependent up-regulation of 35-sulfate uptake in bovine cells reaching a maximum of greater than 100% after 72 h at sterol doses of 1-10 mug/ml. Non-collagenous protein (NCP) and collagen synthesis were similarly up-regulated. All ASU were equally effective in dose dependently inhibiting IL-1-induced MMP-3 activity (23-37%), labeled sulfate release (15-23%) and PGE2 synthesis (45-58%). Up-regulation of glycosaminoglycan and collagen synthesis and reduction of IL-1 effects in cartilage are consistent with chondroprotective activity. The similarity of activity of ASU from diverse sources when tested at equal sterol levels suggests sterols are important for biologic effects in articular chondrocytes.