A combined DHA-rich fish oil and cocoa flavanols intervention does not improve cognition or brain structure in older adults with memory complaints: results from the CANN randomized, controlled parallel-design study.

BACKGROUND: There is evidence that both omega-3 long-chain polyunsaturated fatty acids (PUFAs) (eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA]) and cocoa flavanols can improve cognitive performance in both healthy individuals and in those with memory complaints. However, their combined effect is unknown. OBJECTIVES: To investigate the combined effect of EPA/DHA and cocoa flavanols (OM3FLAV) on cognitive performance and brain structures in older adults with memory complaints. METHODS: A randomized placebo-controlled trial of DHA-rich fish oil (providing 1.1 g/d DHA and 0.4 g/d EPA) and a flavanol-rich dark chocolate (providing 500 mg/d flavan-3-ols) was conducted in 259 older adults with either subjective cognitive impairment or mild cognitive impairment. Participants underwent assessment at baseline, 3 mo, and 12 mo. The primary outcome was the number of false-positives on a picture recognition task from the Cognitive Drug Research computerized assessment battery. Secondary outcomes included other cognition and mood outcomes, plasma lipids, brain-derived neurotrophic factor (BDNF), and glucose levels. A subset of 110 participants underwent structural neuroimaging at baseline and at 12 mo. RESULTS: 197 participants completed the study. The combined intervention had no significant effect on any cognitive outcomes, with the exception of reaction time variability (P = 0.007), alertness (P < 0.001), and executive function (P < 0.001), with a decline in function observed in the OM3FLAV group (118.6 [SD 25.3] at baseline versus 113.3 [SD 25.4] at 12 mo for executive function) relative to the control, and an associated decrease in cortical volume (P = 0.039). Compared with the control group, OM3FLAV increased plasma HDL, total cholesterol ratio (P < 0.001), and glucose (P = 0.008) and reduced TG concentrations (P < 0.001) by 3 mo, which were sustained to 12 mo, with no effect on BDNF. Changes in plasma EPA and DHA and urinary flavonoid metabolite concentrations confirmed compliance to the intervention. CONCLUSIONS: These results suggest that cosupplementation with ω-3 PUFAs and cocoa flavanols for 12 mo does not improve cognitive outcomes in those with cognitive impairment. This trial was registered at clinicaltrials.gov as NCT02525198.

Cardiovascular Autonomic Neuropathy in Type 1 Diabetes Is Associated With Disturbances in TCA, Lipid, and Glucose Metabolism.

Introduction: Diabetic cardiovascular autonomic neuropathy (CAN) is associated with increased mortality and morbidity. To explore metabolic mechanisms associated with CAN we investigated associations between serum metabolites and CAN in persons with type 1 diabetes (T1D). Materials and Methods: Cardiovascular reflex tests (CARTs) (heart rate response to: deep breathing; lying-to-standing test; and the Valsalva maneuver) were used to diagnose CAN in 302 persons with T1D. More than one pathological CARTs defined the CAN diagnosis. Serum metabolomics and lipidomic profiles were analyzed with two complementary non-targeted mass-spectrometry methods. Cross-sectional associations between metabolites and CAN were assessed by linear regression models adjusted for relevant confounders. Results: Participants were median (IQR) aged 55(49, 63) years, 48% males with diabetes duration 39(32, 47) years, HbA1c 63(55,69) mmol/mol and 34% had CAN. A total of 75 metabolites and 106 lipids were analyzed. In crude models, the CAN diagnosis was associated with higher levels of hydroxy fatty acids (2,4- and 3,4-dihydroxybutanoic acids, 4-deoxytetronic acid), creatinine, sugar derivates (ribitol, ribonic acid, myo-inositol), citric acid, glycerol, phenols, phosphatidylcholines and lower levels of free fatty acids and the amino acid methionine (p<0.05). Upon adjustment, positive associations with the CAN diagnoses were retained for hydroxy fatty acids, tricarboxylic acid (TCA) cycle-based sugar derivates, citric acid, and phenols (P<0.05). Conclusion: Metabolic pathways, including the TCA cycle, hydroxy fatty acids, phosphatidylcholines and sugar derivatives are associated with the CAN diagnosis in T1D. These pathway may be part of the pathogeneses leading to CAN and may be modifiable risk factors for the complication.

APOE ε4 alters associations between docosahexaenoic acid and preclinical markers of Alzheimer's disease.

Docosahexaenoic acid is the main long-chain omega-3 polyunsaturated fatty acids in the brain and accounts for 30-40% of fatty acids in the grey matter of the human cortex. Although the influence of docosahexaenoic acid on memory function is widely researched, its association with brain volumes is under investigated and its association with spatial navigation is virtually unknown. This is despite the fact that spatial navigation deficits are a new cognitive fingerprint for symptomatic and asymptomatic Alzheimer's disease. We investigated the cross-sectional relationship between docosahexaenoic acid levels and the major structural and cognitive markers of preclinical Alzheimer's disease, namely hippocampal volume, entorhinal volume and spatial navigation ability. Fifty-three cognitively normal adults underwent volumetric magnetic resonance imaging, measurements of serum docosahexaenoic acid (DHA, including lysophosphatidylcholine DHA) and APOE ε4 genotyping. Relative regional brain volumes were calculated and linear regression models were fitted to examine DHA associations with brain volume. APOE genotype modulated serum DHA associations with entorhinal cortex volume and hippocampal volume. Linear models showed that greater serum DHA was associated with increased entorhinal cortex volume, but not hippocampal volume, in non APOΕ Îµ4 carriers. APOE also interacted with serum lysophosphatidylcholine DHA to predict hippocampal volume. After testing interactions between DHA and APOE on brain volume, we investigated whether DHA and APOE interact to predict spatial navigation performance on a novel virtual reality diagnostic test for Alzheimer's disease in an independent population of APOE genotyped adults (n = 46). APOE genotype modulated DHA associations with spatial navigation performance, showing that DHA was inversely associated with path integration in APOE ε4 carriers only. This exploratory analysis suggests that interventions aiming to increase DHA blood levels to protect against cognitive decline should consider APOE ε4 carrier status. Future work should focus on replicating our initial findings and establishing whether a specific dose of supplementary DHA, at a particular time in the preclinical disease course can have a positive impact on Alzheimer's disease progression in APOE ε4 carriers.

Oxyresveratrol exerts ATF4- and Grp78-mediated neuroprotection against endoplasmic reticulum stress in experimental Parkinson's disease.

Objectives: Endoplasmic reticulum (ER) stress is one of the key mechanisms contributing to Parkinson's disease (PD) pathology. Pathways triggered by ER stress are protective at early stages and initiate apoptosis when the damage is extensive. Methods: We have previously reported that oxyresveratrol rescues cells from oxidative stress and apoptosis in a cell culture model of PD. The aim of this study was to investigate whether the neuroprotective mechanism of oxyresveratrol extends to PD-associated ER stress. For this purpose, we employed two cellular models; to induce severe ER stress, Mes23.5 cells were treated with 6-hydroxydopamine (6-OHDA) and for ER stress driven by chaperones, human neuroblastoma cells were stably transfected to overexpress familial mutants of α-synuclein (α-syn). Results: Our results indicate that oxyresveratrol exhibits distinct modes of protection in both models. In the 6-OHDA model, it inhibited the transcription of activating transcription factor 4 (ATF4), which controls the fate of pro-apoptotic proteins. On the other hand, in the α-syn model, oxyresveratrol suppressed mutant A30P oligomer formation, thereby facilitating a reduction of the ER-chaperone, 78-kDa glucose-regulated protein (Grp78). Discussion: In summary, oxyresveratrol is protective against ER stress induced by two different triggers of PD. Owing to its wide range of defense mechanisms, oxyresveratrol is an ideal candidate for a multifactorial disease like PD.

Metabolomics reveals immunomodulation as a possible mechanism for the antibiotic effect of Persicaria capitata (Buch.-Ham. ex D. Don) H.Gross.

INTRODUCTION: In spite of advances in antibiotics, urinary tract infection (UTI) is still among the most common reasons for antibiotic medication worldwide. Persicaria capitata (Buch.-Ham. ex D. Don) H.Gross (P. capitata) is a herbal medicine used by the Miao people in China to treat UTI. However studies of its mechanism are challenging, owing to the complexity of P. capitata with multiple constituents acting on multiple metabolic pathways. OBJECTIVE: The objective of this study was to explore the working mechanism of P. capitata on urinary tract infection. METHODS: Relinqing® granule, which is solely made from aqueous extracts of the whole P. capitata plant, was used in this study. Urine metabolomics based on gas chromatography-mass spectroscopy was employed to assess the metabolic changes caused by administration of Relinqing® granule in a UTI mouse model. Female specific-pathogen-free Kunming mice were divided into control group (mock infection, saline treatment), model group (E.coli infection, saline treatment), Relinqing® group (E.coli infection, Relinqing® granule treatment), ciprofloxacin group (E.coli infection, ciprofloxacin treatment), and sham-Relinqing® group (no surgery, Relinqing® granule treatment). RESULTS: The results showed that after the treatments, urine levels of itaconic acid in Relinqing® group increased by 4.9 fold and 11.3 fold compared with model and ciprofloxacin groups respectively. Itaconic acid is an endogenous antibacterial metabolite produced by macrophages, which also functions as a checkpoint for metabolic reprogramming of macrophage. CONCLUSION: Our findings suggest that this herbal medicine can cure urinary tract infection through modulation of immune system.

Transient receptor potential canonical 5 channels plays an essential role in hepatic dyslipidemia associated with cholestasis.

Transient receptor potential canonical 5 (TRPC5), a calcium-permeable, non-selective cation channel is expressed in the periphery, but there is limited knowledge of its regulatory roles in vivo. Endogenous modulators of TRPC5 include a range of phospholipids that have an established role in liver disease, including lysophosphatidylcholine (LPC). Cholestasis is characterized by impairment of excretion of bile acids, leading to elevation of hepatic bile acids. We investigated the contribution of TRPC5 in a murine model of cholestasis. Wild-type (WT) and TRPC5 knock-out (KO) mice were fed a diet supplemented with 0.5% cholic acid (CA) for 21 days. CA-diet supplementation resulted in enlargement of the liver in WT mice, which was ameliorated in TRPC5 KO mice. Hepatic bile acid and lipid content was elevated in WT mice, with a reduction observed in TRPC5 KO mice. Consistently, liver enzymes were significantly increased in cholestatic WT mice and significantly blunted in TRPC5 KO mice. Localized dyslipidaemia, secondary to cholestasis, was investigated utilizing a selected lipid analysis. This revealed significant perturbations in the lipid profile following CA-diet feeding, with increased cholesterol, triglycerides and phospholipids, in WT, but not TRPC5 KO mice. Our results suggest that activation of TRPC5 contributes to the development of cholestasis and associated dyslipidemia. Modulation of TRPC5 activity may present as a novel therapeutic target for liver disease.

Assessment of Polygonum capitatum Buch.-Ham. ex D.Don by metabolomics based on gas chromatography with mass spectrometry.

Polygonum capitatum is widely used in southwest China. It has considerable therapeutic efficacy for urinary tract infections. P. capitatum contains multiple components and quality assessment can be achieved by means of metabolic fingerprinting. In this paper, a new strategy for P. capitatum quality determination was developed. Eleven batches of P. capitatum were collected from five geographical areas in China including a standard batch regulated by Good Agriculture Practice. Gas chromatography with mass spectrometry was used to generate fingerprints from triplicate extractions to each batch (n = 33). Hierarchical clustering analysis was applied to assess similarities among the ten batches to the standard batches. Orthogonal projection to latent structures discriminate analysis, cross-validated with permutation tests, was performed to investigate discriminating metabolites. Results demonstrated that the overall evaluation hierarchical clustering analysis clustered two batches with distance > 3. Orthogonal projection to latent structures discriminate analysis (R(2) Y (cum) = 0.997, Q(2) (cum) = 0.97, CV-ANOVA = 8.48 × 10(-11) ) indicated that several sugars contributed to batch classification. This method is a rational approach that can classify against a regulated plant standard and distinguishes samples from different origins or processing time in a holistic manner and metabolites driving any differences can be easily identified.

Investigating sub-2 µm particle stationary phase supercritical fluid chromatography coupled to mass spectrometry for chemical profiling of chamomile extracts.

Roman and German chamomile are widely used throughout the world. Chamomiles contain a wide variety of active constituents including sesquiterpene lactones. Various extraction techniques were performed on these two types of chamomile. A packed-column supercritical fluid chromatography-mass spectrometry method was designed for the identification of sesquiterpenes and other constituents from chamomile extracts with no derivatization step prior to analysis. Mass spectrometry detection was achieved by using electrospray ionization. All of the compounds of interest were separated within 15 min. The chamomile extracts were analyzed and compared for similarities and distinct differences. Multivariate statistical analysis including principal component analysis and orthogonal partial least squares-discriminant analysis (OPLS-DA) were used to differentiate between the chamomile samples. German chamomile samples confirmed the presence of cis- and trans-tonghaosu, chrysosplenols, apigenin diglucoside whereas Roman chamomile samples confirmed the presence of apigenin, nobilin, 1,10-epioxynobilin, and hydroxyisonobilin.

Evaluation of Chinese medicinal herbs fingerprinting by HPLC-DAD for the detection of toxic aristolochic acids.

Aristolochic acids are known to contribute to various renal disorders; therefore, expanding the availability of analytical methodology to detect these compounds is important in order to assess the quality of Chinese herbal medicines in which they can be found. Twelve medicinal herbal samples were procured from various sources and extracted in duplicate prior to a "fingerprint" analysis using conventional HPLC-DAD. Multivariate analysis was performed on the entire chromatographed fingerprints. The resulting output was a partial least-square discriminant analysis model, which was able to evaluate the potential presence of aristolochic acids I and II as well as providing an individual herbal "fingerprint". The results of this study provide evidence that the presence of aristolochic acids contained within certain herbal extractions could be detected using a simple method, although some limitations apply to this method for quality control, since newly detected samples for aristolochic acid (positives) will need further confirmation with purity checks or MS hyphenation.

Liquid chromatography-mass spectrometry methods for urinary biomarker detection in metabonomic studies with application to nutritional studies.

The effects of sample preparation and chromatographic method differences on the classification and recovery of metabolic biomarkers from UPLC-MS measurements on urine samples of humans exposed to different dietary interventions have been investigated. Eight volunteers consumed three high-fat meals (rich in saturated, monounsaturated and polyunsaturated fatty acids, respectively) in randomized order with a washout period in between. For each participant, urine samples were obtained prior to and at three timed intervals after each meal. Samples were processed either by dilution (1 : 4) or by liquid-liquid extraction and then run under two different gradient conditions. For each analysis method, a total of 96 observations (eight participants, four time points, three diets) were measured. The total ion count chromatograms were analyzed using partial-least-squares discriminant analysis. All three dietary classes could be discriminated irrespective of sample preparation and chromatographic method. However, the main discriminating metabolites varied according to sample preparation, indicating that sample treatment and chromatographic conditions influence the ability to extract biomolecular information. Diluted samples showed higher m/z compounds (ca 400 u) while liquid-liquid extraction samples showed low m/z at the same retention time span. Optimized methods for metabolite identification (e.g. organic acids) were statistically inferior to global screening for mixed compound identification, confirming that multiple compound class-based metabolic profiles are likely to give superior metabonomic (diagnostic) classification, although great care has to be taken in the interpretation in relation to matrix effects.

Identification of metabolites in human hepatic bile using 800 MHz 1H NMR spectroscopy, HPLC-NMR/MS and UPLC-MS.

The first application of high field NMR spectroscopy (800 MHz for (1)H observation) to human hepatic bile (as opposed to gall bladder bile) is reported. The bile sample used for detailed investigation was from a donor liver with mild fat infiltration, collected during organ retrieval prior to transplantation. In addition, to focus on the detection of bile acids in particular, a bile extract was analysed by 800 MHz (1)H NMR spectroscopy, HPLC-NMR/MS and UPLC-MS. In the whole bile sample, 40 compounds have been assigned with the aid of two-dimensional (1)H-(1)H TOCSY and (1)H-(13)C HSQC spectra. These include phosphatidylcholine, 14 amino acids, 10 organic acids, 4 carbohydrates and polyols (glucose, glucuronate, glycerol and myo-inositol), choline, phosphocholine, betaine, trimethylamine-N-oxide and other small molecules. An initial NMR-based assessment of the concentration range of some key metabolites has been made. Some observed chemical shifts differ from expected database values, probably due to a difference in bulk diamagnetic susceptibility. The NMR spectra of the whole extract gave identification of the major bile acids (cholic, deoxycholic and chenodeoxycholic), but the glycine and taurine conjugates of a given bile acid could not be distinguished. However, this was achieved by HPLC-NMR/MS, which enabled the separation and identification of ten conjugated bile acids with relative abundances varying from approximately 0.1% (taurolithocholic acid) to 34.0% (glycocholic acid), of which, only the five most abundant acids could be detected by NMR, including the isomers glycodeoxycholic acid and glycochenodeoxycholic acid, which are difficult to distinguish by conventional LC-MS analysis. In a separate experiment, the use of UPLC-MS allowed the detection and identification of 13 bile acids. This work has shown the complementary potential of NMR spectroscopy, MS and hyphenated NMR/MS for elucidating the complex metabolic profile of human hepatic bile. This will be useful baseline information in ongoing studies of liver excretory function and organ transplantation.

Fingerprinting of human bile during liver transplantation by capillary electrophoresis.

Increasing rates of success in liver transplantation have increased the number of cases considered. However, liver post-transplant graft dysfunction of liver transplants (TXs) is not fully understood and by applying holistic approaches we can investigate metabolic change deriving from confounding factors such as liver fat content, ischaemia time, donor age, recipient's health, etc. Twenty-six hepatic bile samples taken from liver donors and recipients were retrieved from a total of six TXs, from these one recipient underwent post-graft dysfunction. CE was employed to fingerprint bile collected at 10 min increments in the donors and in the recipients. The electropherograms of these samples were aligned and normalised using correlation optimised warping algorithms and modelled with multivariate techniques. The resulting metabolic signatures were compared; in general donors and recipients showed distinct fingerprints and clustered separately. When a partial least square discriminant analysis (PLS-DA) model was constructed between donor and recipient's samples, a recipient of a 32 year old liver with normal steatosis, and shortest cold ischaemia time showed as the observation nearest to its donor observation, denoting minimal metabolic change. This study proposes CE fingerprinting of human bile as a promising technique to help unravel the complex metabolic pathways involved during transplantation.

Assessment of Chinese medicinal herb metabolite profiles by UPLC-MS-based methodology for the detection of aristolochic acids.

In this study, aristolochic acid in different herbal medicines containing a mixture of varying herb species was identified through fingerprint pattern similarities. Aristolochic acid I and II are nephrotoxic compounds naturally present in the Aristolochia plant species that are commonly used in Chinese herbal medicines. Twenty-four commercially available herbal formulations were extracted into an aqueous solution and injected into a UPLC-MS system. All the samples were analysed by multiple reaction monitoring (MRM) to check for the presence of aristolochic acids I and II. The same samples were then fingerprinted using two different gradient methods and the chromatograms deconvoluted into retention time (RT) and masses for the chemicals present taking concentration into account. Statistical analysis of this data revealed that samples were highly heterogeneous, and that the main differences between the preparations were concentrations of polar compounds. A model was constructed where the samples could be separated into two groups differentiated by the presence of the two forms of aristolochic acid.