Effect of circadian rhythm on NAD and other metabolites in human brain.

Nicotinamide Adenine Dinucleotide (NAD) plays a central role in the master circadian clock of the brain (the suprachiasmatic nuclei, SCN) as demonstrated in many model organisms. NAD acts as an enzyme co-factor and substrate and its modulation was found to be tightly regulated to the periodicity of the cycles. However, in human brain, the effect of the circadian rhythm (CR) on the metabolism of the SCN and other brain regions is poorly understood. We conducted a magnetic resonance spectroscopy (MRS) study at a high magnetic field, measuring the occipital brain NAD levels and other metabolites in two different morning and afternoon diurnal states in 25 healthy participants. Salivary cortisol levels were determined to confirm that the experiment was done in two chronologically different physiological conditions, and a behavioral test of risk-taking propensity was administered. Overall, we found that the CR did not significantly affect NAD levels in the occipital brain region. The other brain metabolites measured, including lactate, were not significantly affected by the CR either, except for taurine. The CR did impact risk-taking behavior and salivary cortisol level, confirming that the participants were in two circadian different behavioral and physiological states in the morning and in the afternoon. Measurement of the CR effect on NAD and taurine levels in other brain regions might provide stronger effects.

Cardiorenal ketone metabolism: a positron emission tomography study in healthy humans.

Ketones are alternative energy substrates for the heart and kidney but no studies have investigated their metabolism simultaneously in both organs in humans. The present double tracer positron emission tomography (PET) study evaluated the organ distribution and basal kinetic rates of the radiolabeled ketone, 11C-acetoacetate (11C-AcAc), in the heart and kidney compared to 11C-acetate (11C-Ac), which is a well-validated metabolic radiotracer. Both tracers were highly metabolized by the left ventricle and the renal cortex. In the heart, kinetic rates were similar for both tracers. But in the renal cortex, uptake of 11C-Ac was higher compared to 11C-AcAc, while the reverse was observed for the clearance. Interestingly, infusion of 11C-AcAc led to a significantly delayed release of radioactivity in the renal medulla and pelvis, a phenomenon not observed with 11C-Ac. This suggests an equilibrium of 11C-AcAc with the other ketone, 11C-D-beta-hydroxybutyrate, and a different clearance profile. Overall, this suggests that in the kidney, the absorption and metabolism of 11C-AcAc is different compared to 11C-Ac. This dual tracer PET protocol provides the opportunity to explore the relative importance of ketone metabolism in cardiac and renal diseases, and to improve our mechanistic understanding of new metabolic interventions targeting these two organs.

Effect of an enteral amino acid blend on muscle and gut functionality in critically ill patients: a proof-of-concept randomized controlled trial.

BACKGROUND: A defining feature of prolonged critical illness is muscle wasting, leading to impaired recovery. Supplementation with a tailored blend of amino acids may bolster the innate gut defence, promote intestinal mucosa repair and limit muscle loss. METHODS: This was a monocentric, randomized, double-blind, placebo-controlled study that included patients with sepsis or acute respiratory distress syndrome. Patients received a specific combination of five amino acids or placebo mixed with enteral feeding for 21 days. Markers of renal function, gut barrier structure and functionality were collected at baseline and 1, 2, 3 and 8 weeks after randomization. Muscle structure and function were assessed through MRI measurements of the anterior quadriceps volume and by twitch airway pressure. Data were compared between groups relative to the baseline. RESULTS: Thirty-five critically ill patients were randomized. The amino acid blend did not impair urine output, blood creatinine levels or creatinine clearance. Plasma citrulline levels increased significantly along the treatment period in the amino acid group (difference in means [95% CI] 5.86 [1.72; 10.00] nmol/mL P = 0.007). Alanine aminotransferase and alkaline phosphatase concentrations were lower in the amino acid group than in the placebo group at one week (ratio of means 0.5 [0.29; 0.86] (P = 0.015) and 0.73 [0.57; 0.94] (P = 0.015), respectively). Twitch airway pressure and volume of the anterior quadriceps were greater in the amino acid group than in the placebo group 3 weeks after randomization (difference in means 10.6 [0.99; 20.20] cmH20 (P = 0.035) and 3.12 [0.5; 5.73] cm3/kg (P = 0.022), respectively). CONCLUSIONS: Amino acid supplementation increased plasma citrulline levels, reduced alanine aminotransferase and alkaline phosphatase levels, and improved twitch airway pressure and anterior quadriceps volume. Trial registration ClinicalTrials.gov, NCT02968836. Registered November 21, 2016.

A ketogenic supplement improves white matter energy supply and processing speed in mild cognitive impairment.

INTRODUCTION: White matter (WM) energy supply is crucial for axonal function and myelin maintenance. An exogenous source of ketones, the brain's alternative fuel to glucose, bypasses the brain's glucose-specific energy deficit and improves cognitive outcomes in mild cognitive impairment (MCI). How an additional supply of ketones affects glucose or ketone uptake in specific WM fascicles in MCI has not previously been reported. METHODS: This 6-month interventional study included MCI participants randomized to a placebo (n = 16) or ketogenic medium chain triglyceride (kMCT; n = 17) drink. A neurocognitive battery and brain imaging were performed pre- and post-intervention. WM fascicle uptake of ketone and glucose and structural properties were assessed using positron emission tomography and diffusion imaging, respectively. RESULTS: Ketone uptake was increased in the kMCT group by 2.5- to 3.2-fold in all nine WM fascicles of interest (P < .001), an effect seen both in deep WM and in fascicle cortical endpoints. Improvement in processing speed was positively associated with WM ketone uptake globally and in individual fascicles, most importantly the fornix (r = +0.61; P = .014). DISCUSSION: A 6-month kMCT supplement improved WM energy supply in MCI by increasing ketone uptake in WM fascicles. The significant positive association with processing speed suggests that ketones may have a role in myelin integrity in MCI.

A ketogenic drink improves cognition in mild cognitive impairment: Results of a 6-month RCT.

INTRODUCTION: Counteracting impaired brain glucose metabolism with ketones may improve cognition in mild cognitive impairment (MCI). METHODS: Cognition, plasma ketone response, and metabolic profile were assessed before and 6 months after supplementation with a ketogenic drink containing medium chain triglyceride (ketogenic medium chain triglyceride [kMCT]; 15 g twice/day; n = 39) or placebo (n = 44). RESULTS: Free and cued recall (Trial 1; P = .047), verbal fluency (categories; P = .024), Boston Naming Test (total correct answers; P = .033), and the Trail-Making Test (total errors; P = .017) improved significantly in the kMCT group compared to placebo (analysis of covariance; pre-intervention score, sex, age, education, and apolipoprotein E4 as covariates). Some cognitive outcomes also correlated positively with plasma ketones. Plasma metabolic profile and ketone response were unchanged. CONCLUSIONS: This kMCT drink improved cognitive outcomes in MCI, at least in part by increasing blood ketone level. These data support further assessment of MCI progression to Alzheimer's disease.

Monoacylglycerol Form of Omega-3s Improves Its Bioavailability in Humans Compared to Other Forms.

Numerous benefits are attributed to omega-3 fatty acids (OM3) especially in cardiovascular health. However, bioavailability and clinical efficacy depend on numerous factors, including OM3 form, food matrix effects (especially the lipid content of the diet), and metabolic capacity. Here, we show in humans that a "pre-digested" OM3-sn-1(3)-monoacylglycerol lipid structure (OM3-MAG) has a significantly greater absorption at high therapeutic doses (2.9 g/day) than the most commonly OM3-ethyl ester (3.1 g/day) form (used for the treatment of hypertriglyceridemia), and a comparable profile to other pre-digested OM3 free fatty acids (OM3-FFA) structure (3.2 g/day). Nutritional supplement doses of MAG resulted in similar increases in OM3 blood level, compared to OM3 triacylglycerols (OM3-TAG) supplements in obese subjects (1.2 g/day) under low fat diet, and in children with cystic fibrosis (1.0 g/day). These results suggest that both forms of pre-digested OM3-MAG and OM3-FFA are effectively absorbed and re-incorporated effectively into triacylglycerols inside the enterocytes, before being exported into the chylomicrons lipid transport system. The pre-digested OM3-MAG might provide a more effective therapy in severe cardiovascular conditions where high doses of OM3 are required and a low-fat diet is indicated, which limited digestive lipase activity.

Metabolism of Exogenous D-Beta-Hydroxybutyrate, an Energy Substrate Avidly Consumed by the Heart and Kidney.

There is growing interest in the metabolism of ketones owing to their reported benefits in neurological and more recently in cardiovascular and renal diseases. As an alternative to a very high fat ketogenic diet, ketones precursors for oral intake are being developed to achieve ketosis without the need for dietary carbohydrate restriction. Here we report that an oral D-beta-hydroxybutyrate (D-BHB) supplement is rapidly absorbed and metabolized in humans and increases blood ketones to millimolar levels. At the same dose, D-BHB is significantly more ketogenic and provides fewer calories than a racemic mixture of BHB or medium chain triglyceride. In a whole body ketone positron emission tomography pilot study, we observed that after D-BHB consumption, the ketone tracer 11C-acetoacetate is rapidly metabolized, mostly by the heart and the kidneys. Beyond brain energy rescue, this opens additional opportunities for therapeutic exploration of D-BHB supplements as a "super fuel" in cardiac and chronic kidney diseases.

Medium Chain Triglycerides Modulate the Ketogenic Effect of a Metabolic Switch.

Ketones provide an alternative brain fuel and may be neuroprotective in older people. Little is known of how to optimize the ketogenic effect of C8:0-C10:0 medium chain triglyceride supplement (kMCT). Metabolic switching (MS) from glucose to ketones as a fuel may have metabolic benefits but has not been extensively studied in humans. The objective of the present study was to use an 8 h metabolic study day protocol to assess the influence of typical components of MS, including a kMCT supplement, low-carbohydrate meal and meal timing, on blood ketones, glucose, insulin and free fatty acids (FFA). In one test, the effect of age was also investigated. Over the 8 h metabolic study day, two 10 g doses of the kMCT increased the plasma ketone response by 19% while reducing overall glycemia by 12% without altering insulin or FFA levels. Moreover, a single early meal (breakfast but no lunch) potentiated the ketogenic effect of MS over 8 h, compared to a single delayed meal (lunch but no breakfast). Age and the low carbohydrate meal did not affect the ketones response. We conclude that an 8-h test period can be used to assess metabolic changes during short-term MS. kMCT provide a robust short-term increase in ketones and might enhance the metabolic effectiveness of short-term or intermittent fasting as a component of MS.

Brain NAD Is Associated With ATP Energy Production and Membrane Phospholipid Turnover in Humans.

The brain requires a large amount of energy, mostly derived from the metabolism of glucose, which decreases substantially with age and neurological diseases. While mounting evidence in model organisms illustrates the central role of brain nicotinamide adenine dinucleotide (NAD) for maintaining energy homeostasis, similar data are sparse in humans. This study explores the correlations between brain NAD, energy production and membrane phospholipid metabolism by 31-phosphorous magnetic resonance spectroscopy (31P-MRS) across 50 healthy participants including a young (mean age 27.1-year-old) and middle-aged (mean age 56.4-year-old) group. The analysis revealed that brain NAD level and NAD+/NADH redox ratio were positively associated with ATP level and the rate of energy production, respectively. Moreover, a metabolic network linking NAD with membrane phospholipid metabolism, energy production, and aging was identified. An inverted trend between age and NAD level was detected. These results pave the way for the use of 31P-MRS as a powerful non-invasive tool to support the development of new therapeutic interventions targeting NAD associated phospho-metabolic pathways in brain aging and neurological diseases.

Modulation of cerebral ketone metabolism following traumatic brain injury in humans.

Adaptive metabolic response to injury includes the utilization of alternative energy substrates - such as ketone bodies (KB) - to protect the brain against further damage. Here, we examined cerebral ketone metabolism in patients with traumatic brain injury (TBI; n = 34 subjects) monitored with cerebral microdialysis to measure total brain interstitial tissue KB levels (acetoacetate and ß-hydroxybutyrate). Nutrition - from fasting vs. stable nutrition state - was associated with a significant decrease of brain KB (34.7 [10th-90th percentiles 10.7-189] µmol/L vs. 13.1 [6.5-64.3] µmol/L, p < 0.001) and blood KB (668 [168.4-3824.9] vs. 129.4 [82.6-1033.8] µmol/L, p < 0.01). Blood KB correlated with brain KB (Spearman's rho 0.56, p = 0.0013). Continuous feeding with medium-chain triglycerides-enriched enteral nutrition did not increase blood KB, and provided a modest increase in blood and brain free medium chain fatty acids. Higher brain KB at the acute TBI phase correlated with age and brain lactate, pyruvate and glutamate, but not brain glucose. These novel findings suggest that nutritional ketosis was the main determinant of cerebral KB metabolism following TBI. Age and cerebral metabolic distress contributed to brain KB supporting the hypothesis that ketones might act as alternative energy substrates to glucose. Further studies testing KB supplementation after TBI are warranted.

Discovery and validation of temporal patterns involved in human brain ketometabolism in cerebral microdialysis fluids of traumatic brain injury patients.

BACKGROUND: Traumatic brain injury (TBI) is recognized as a metabolic disease, characterized by acute cerebral glucose hypo-metabolism. Adaptive metabolic responses to TBI involve the utilization of alternative energy substrates, such as ketone bodies. Cerebral microdialysis (CMD) has evolved as an accurate technique allowing continuous sampling of brain extracellular fluid and assessment of regional cerebral metabolism. We present the successful application of a combined hypothesis- and data-driven metabolomics approach using repeated CMD sampling obtained routinely at patient bedside. Investigating two patient cohorts (n = 26 and n = 12), we identified clinically relevant metabolic patterns at the acute post-TBI critical care phase. METHODS: Clinical and CMD metabolomics data were integrated and analysed using in silico and data modelling approaches. We used both unsupervised and supervised multivariate analysis techniques to investigate structures within the time series and associations with patient outcome. FINDINGS: The multivariate metabolite time series exhibited two characteristic brain metabolic states that were attributed to changes in key metabolites: valine, 4-methyl-2-oxovaleric acid (4-MOV), isobeta-hydroxybutyrate (iso-bHB), tyrosyine, and 2-ketoisovaleric acid (2-KIV). These identified cerebral metabolic states differed significantly with respect to standard clinical values. We validated our findings in a second cohort using a classification model trained on the cerebral metabolic states. We demonstrated that short-term (therapeutic intensity level (TIL)) and mid-term patient outcome (6-month Glasgow Outcome Score (GOS)) can be predicted from the time series characteristics. INTERPRETATION: We identified two specific cerebral metabolic patterns that are closely linked to ketometabolism and were associated with both TIL and GOS. Our findings support the view that advanced metabolomics approaches combined with CMD may be applied in real-time to predict short-term treatment intensity and long-term patient outcome.

Effects of interesterified lipid design on the short/medium chain fatty acid hydrolysis rate and extent (in vitro).

Short/medium chain fatty acids have well known health effects such as gut immune regulation and ketogenesis. The ability to realise these health effects is potentially limited by their rapid gastro-intestinal lipolysis. It was proposed that synthesising novel interesterified lipids via an interesterification reaction to generate a combination of short/medium and long chain fatty acids would modulate their gastrointestinal digestion. Using in vitro gastric and gastro-intestinal digestion models, the effect of the fatty acid chain length and interesterification on the rate and extent of lipolysis was analysed. Overall, "pure" (consisting of a single fatty acid) lipids of ≤C8 underwent rapid lipolysis releasing three fatty acids after intestinal hydrolysis while lipids of ≥C10 released two fatty acids after intestinal hydrolysis. The most interesting observation is that the extent of gastric lipolysis of C4 fatty acids was much lower when they were interesterified with longer chain fatty acids compared to that with the pure C4 triglyceride. Tributyrin underwent ∼60% lipolysis by gastric lipase as indicated by a decrease in total fatty acid release during SIF lipolysis after pre-exposure to rabbit gastric lipase (RGL) in SGF. In comparison, the C4-C8 interesterified lipid exhibited only a 18.1% decrease, and the C4-C18:1 interesterified lipid a 6.1% decrease in total fatty acid release in SGF-SIF. These results suggest that interesterification modulates the digestion of butyric acid from within the stomach to later in the intestine. This study reveals that the design of interesterified lipids alters the timing, but not the extent of short chain fatty acid delivery in the gastrointestinal tract. Such understanding has likely benefits for designing novel interesterified lipids which may have unique applications in various dietary and therapeutic modalities.

Nutrient pattern analysis in critically ill patients using Omics technology (NAChO) - Study protocol for a prospective observational study.

INTRODUCTION: Intensive care unit-acquired weakness (ICU-AW) is often observed in critically ill patients with prolonged intensive care unit (ICU) stay. We hypothesized that evolving metabolic abnormalities during prolonged ICU stay are reflected by changing nutrient patterns in blood, urine and skeletal muscle, and that these patterns differ in patients with/without ICU-AW and between patients with/without sepsis. METHODS: In a prospective single-center observational trial, we aim to recruit 100 critically ill patients (ICU length of stay ≥ 5 days) with severe sepsis/septic shock ("sepsis group", n = 50) or severe head trauma/intracerebral hemorrhage ("CNS group", n = 50). Patients will be sub-grouped for presence or absence of ICU-AW as determined by the Medical Research Council sum score. Blood and urine samples will be collected and subjected to comprehensive nutrient analysis at different time points by targeted quantitative mass spectrometric methods. In addition, changes in muscular tissue (biopsy, when available), muscular architecture (ultrasound), electrophysiology, body composition analyses (bioimpedance, cerebral magnetic resonance imaging), along with clinical status will be assessed. Patients will be followed-up for 180 and 360 days including assessment of quality of life. DISCUSSION: Key objective of this trial is to assess changes in nutrient pattern in blood and urine over time in critically ill patients with/without ICU-AW by using quantitative nutrient analysis techniques. Peer-reviewed published NAChO data will allow for a better understanding of metabolic changes in critically ill patients on standard liquid enteral nutrition and will likely open up new avenues for future therapeutic and nutritional interventions.

Nutritional Ketosis Increases NAD+/NADH Ratio in Healthy Human Brain: An in Vivo Study by 31P-MRS.

Ketones represent an important alternative fuel for the brain under glucose hypo-metabolic conditions induced by neurological diseases or aging, however their metabolic consequences in healthy brain remain unclear. Here we report that ketones can increase the redox NAD+/NADH ratio in the resting brain of healthy young adults. As NAD is an important energetic and signaling metabolic modulator, these results provide mechanistic clues on how nutritional ketosis might contribute to the preservation of brain health.

Development of isoform selective PI3-kinase inhibitors as pharmacological tools for elucidating the PI3K pathway.

Using a parallel synthesis approach to target a non-conserved region of the PI3K catalytic domain a pan-PI3K inhibitor 1 was elaborated to provide alpha, delta and gamma isoform selective Class I PI3K inhibitors 21, 24, 26 and 27. The compounds had good cellular activity and were selective against protein kinases and other members of the PI3K superfamily including mTOR and DNA-PK.

An investigation into the structure-activity relationships associated with the systematic modification of the ß(2)-adrenoceptor agonist indacaterol.

The synthesis of a series of indacaterol analogues in which each of the three structural regions of indacaterol are modified in a systematic manner is described. Evaluation of the affinity of these analogues for the ß(2)-adrenoceptor identified the 3,4-dihydroquinolinone and 5-n-butylindanyl analogues to demonstrate the most similar profiles to indacaterol. An α-methyl aminoindane analogue was discovered to be 25-fold more potent than indacaterol, and functional studies revealed an atypical ß(2)-adrenoceptor activation profile for this compound consistent with that of a slowly dissociating 'super agonist'.

A physical properties based approach for the exploration of a 4-hydroxybenzothiazolone series of beta2-adrenoceptor agonists as inhaled long-acting bronchodilators.

The chiral synthesis of a 4-hydroxybenzothiazolone based series of beta(2)-adrenoceptor agonists is described. Using this methodology a library of N-substituted analogues were prepared for the rapid identification of leads with the potential to be fast onset and long-acting inhaled bronchodilators with improved therapeutic margins. The design of the library to achieve the targeted profile was based upon lipophilicity and metabolism based hypotheses. This approach identified beta-phenethyl, alpha-substituted cyclopentyl and monoterpene N-substituents to be of particular interest for further evaluation, as exemplified by structures 19, 29 and 33, respectively.

The identification of indacaterol as an ultralong-acting inhaled beta2-adrenoceptor agonist.

Following a lipophilicity-based hypothesis, an 8-hydroxyquinolinone 2-aminoindan derived series of beta(2)-adrenoceptor agonists have been prepared and evaluated for their potential as inhaled ultralong-acting bronchodilators. Determination of their activities at the human beta(2)-adrenoceptor receptor showed symmetrical substitution of the 2-aminoindan moiety at the 5- and 6-positions delivered the targeted intermediate potency and intrinsic-efficacy profiles relative to a series of clinical reference beta(2)-adrenoceptor agonists. Further assessment with an in vitro superfused electrically stimulated guinea-pig tracheal-strip assay established the onset and duration of action time courses, which could be rationalized by considering the lipophilicity, potency, and intrinsic efficacy of the compounds. From these studies the 5,6-diethylindan analogue indacaterol 1c was shown to possess a unique profile of combining a rapid onset of action with a long duration of action. Further in vivo profiling of 1c supported the long duration of action and a wide therapeutic index following administration to the lung, which led to the compound being selected as a development candidate.

Lipid membrane interactions of indacaterol and salmeterol: do they influence their pharmacological properties?

This study compares the lipid membrane interactions of indacaterol, an ultra long acting beta-2 agonist that is given once a day, to salmeterol, a twice a day beta-2 agonist, in order to elucidate the potential mechanisms leading to their different pharmacological properties. Salmeterol but not indacaterol perturbed dimyristoyl-phosphatidylcholine membranes. While the liposome partitioning of the two compounds was similar, independent of the lipid composition, the membrane affinity of indacaterol was two-fold greater than that of salmeterol when rafts, i.e. detergent-insoluble membrane domains, were used as the partition phase. The observed association kinetics with immobilized liposomes at physiological pH were two times faster for indacaterol than for salmeterol. A new model to explain the relationships between the drug/membrane interactions and drug's pharmacological properties considering multiple factors is proposed. The synergy between the higher partitioning of indacaterol into the raft micro domains and the faster membrane permeation of indacaterol could explain the faster onset and longer duration of therapeutic effect of indacaterol. The higher fluidizing effect of salmeterol on membrane fluidity may contribute to its lower intrinsic efficacy compared to indacaterol.

Interaction kinetics of salmeterol with egg phosphatidylcholine liposomes by surface plasmon resonance.

Surface plasmon resonance (SPR) Biacore and equilibrium dialysis were applied to investigate the membrane affinities of salmeterol and propranolol and the kinetic interactions of salmeterol with egg phosphatidylcholine liposomes. The two methods revealed similar affinity values; however, they were dependent on the investigated drug concentrations. The kinetic experiments with salmeterol were optimized to obtain pseudo-first-order kinetics that were independent of the drug concentration. The adsorption and desorption phases followed biexponential functions up to pH 8.8 and mono or biexponential functions at higher pH values (i.e., between the two pK(a) values). The apparent rate constants of the faster phases of the biexponential functions were beyond the time resolution of the instrument in most measurements. The apparent rate constants of the slower phases ranged from 0.01 to 0.03 s(-1) and were pH independent between pH 5.0 and pH 8.0. The rates of the monoexponential kinetics were between 0.08 and 0.12 s(-1). We conclude that the biexponential kinetics at physiological pH reflect the partitioning into the outer lipid leaflet and "flip-flop," respectively, of the cationic species.