Nutrient composition of different energy-restricted diets determines plasma endocannabinoid profiles and adipose tissue DAGL-α expression; a 12-week randomized controlled trial in subjects with abdominal obesity.

The endocannabinoid system (ECS) is dysregulated during obesity and metabolic disorders. Weight loss favours the re-establishment of ECS homeostatic conditions, but also the fatty acid composition of the diet can modulate endocannabinoid profiles. However, the combined impact of nutrient quality and energy restriction on the ECS remains unclear. In this 12 weeks randomized controlled trial, men and women (40-70 years) with obesity (BMI: 31.3 ± 3.5 kg/ m2) followed either a low nutrient quality 25% energy-restricted (ER) diet (n=39) high in saturated fats and fructose, or a high nutrient quality ER diet (n=34) amongst others enriched in n-3 polyunsaturated fatty acids (PUFAs) or kept their habitual diet (controls). Profiles of plasma- and adipose N-acylethanolamines and mono-acyl glycerol esters were quantified using LC-MS/MS. Gene expression of ECS-related enzymes and receptors was determined in adipose tissue. Measurements were performed under fasting conditions before and after 12 weeks. Our results showed that plasma level of the DHA-derived compound docosahexaenoylethanolamide (DHEA) was decreased in the low nutrient quality ER diet (P<0.001) compared with the high nutrient quality ER diet, whereas anandamide (AEA) and arachidonoylglycerol (2-AG) levels were unaltered. However, adipose tissue gene expression of the 2-AG synthesizing enzyme diacylglycerol lipase alpha (DAGL-α) was increased following the low nutrient quality ER diet (P<.009) and differed upon intervention with both other diets. Concluding, nutrient quality of the diet affects N-acylethanolamine profiles and gene expression of ECS-related enzymes and receptors even under conditions of high energy restriction in abdominally obese humans. ClinicalTrials.gov NCT02194504.

Endocannabinoid levels in plasma and neurotransmitters in the brain: a preliminary report on patients with a psychotic disorder and healthy individuals.

BACKGROUND: Interactions between the endocannabinoid system (ECS) and neurotransmitter systems might mediate the risk of developing a schizophrenia spectrum disorder (SSD). Consequently, we investigated in patients with SSD and healthy controls (HC) the relations between (1) plasma concentrations of two prototypical endocannabinoids (N-arachidonoylethanolamine [anandamide] and 2-arachidonoylglycerol [2-AG]) and (2) striatal dopamine synthesis capacity (DSC), and glutamate and y-aminobutyric acid (GABA) levels in the anterior cingulate cortex (ACC). As anandamide and 2-AG might reduce the activity of these neurotransmitters, we hypothesized negative correlations between their plasma levels and the abovementioned neurotransmitters in both groups. METHODS: Blood samples were obtained from 18 patients and 16 HC to measure anandamide and 2-AG plasma concentrations. For all subjects, we acquired proton magnetic resonance spectroscopy scans to assess Glx (i.e. glutamate plus glutamine) and GABA + (i.e. GABA plus macromolecules) concentrations in the ACC. Ten patients and 14 HC also underwent [18F]F-DOPA positron emission tomography for assessment of striatal DSC. Multiple linear regression analyses were used to investigate the relations between the outcome measures. RESULTS: A negative association between 2-AG plasma concentration and ACC Glx concentration was found in patients (p = 0.008). We found no evidence of other significant relationships between 2-AG or anandamide plasma concentrations and dopaminergic, glutamatergic, or GABAergic measures in either group. CONCLUSIONS: Our preliminary results suggest an association between peripheral 2-AG and ACC Glx levels in patients.

Relative validity of habitual sugar and low/no-calorie sweetener consumption assessed by food frequency questionnaire, multiple 24-h dietary recalls and urinary biomarkers: an observational study within the SWEET project.

BACKGROUND: Studies investigating associations between sweeteners and health yield inconsistent results, possibly due to subjective self-report dietary assessment methods. OBJECTIVES: We compared the performance of a food frequency questionnaire (FFQ), multiple 24-h dietary recalls (24hRs), and urinary biomarkers to estimate intake of sugars and low/no-calorie sweeteners (LNCSs). METHODS: Participants (n = 848, age 54 ± 12 y) from a 2-y observational study completed 1 semiquantitative FFQ and ≥ 3 nonconsecutive 24hRs. Both methods assessed intake of sugars (mono- and disaccharides, sucrose, fructose, free and added sugars) and sweetened foods and beverages (sugary foods, fruit juice, and sugar or LNCS-containing beverages [sugar-sweetened beverages and low/no-calorie sweetened beverages (LNCSBs)]); 24hRs also included LNCS-containing foods and tabletop sweeteners (low/no-calorie sweetened foods [LNCSFs]). Urinary excretion of sugars (fructose+sucrose) and LNCSs (acesulfame K+sucralose+steviol glucuronide+cyclamate+saccharin) were simultaneously assessed using ultrapressure liquid chromatography coupled to tandem mass spectrometry in 288 participants with 3 annual 24-h urine samples. Methods were compared using, amongst others, validity coefficients (correlations corrected for measurement error). RESULTS: Median (interquartile range) FFQ intakes ranged from 0 (0-7) g/d for LNCSBs to 94 (73-117) g/d for mono- and disaccharides. LNCSB use was reported by 32% of participants. Median LNCSB+LNCSF intake using 24hRs was 1 (0-50) g/d and reported by 58%. Total sugar excretions were detected in 100% of samples [56 (37-85) mg/d] and LNCSs in 99% of urine samples [3 (1-10) mg/d]. Comparing FFQ against 24hRs showed VCs ranging from 0.38 (fruit juice) to 0.74 (LNCSB). VCs for comparing FFQ with urinary excretions were 0.25 to 0.29 for sugars and 0.39 for LNCSBs; for 24hR they amounted to 0.31-0.38 for sugars, 0.37 for LNCSBs, and 0.45 for LNCSFs. CONCLUSIONS: The validity of the FFQ against 24hRs for the assessment of sugars and LNCSBs ranged from moderate to good. Comparing self-reports and urine excretions showed moderate agreement but highlighted an important underestimation of LNCS exposure using self-reports.

Changes in nutrient composition and diet quality in the first 6 months following bariatric surgery: An observational cohort study.

BACKGROUND: Bariatric surgery (BS) may result in inadequate nutrient intake and poor diet quality, which can lead to nutritional complications. The present study aimed to evaluate changes in macro- and micronutrient composition and diet quality in the first 6 months following BS. METHODS: One hundred seven participants undergoing BS (Roux-en-Y gastric bypass: n = 87, sleeve gastrectomy: n = 20) completed 3-day food records before and 6 months after surgery. Changes in energy, macronutrient (carbohydrates, protein, fat, dietary fibre) and micronutrient intake (folate, vitamin B12, vitamin D, calcium, iron) were evaluated. Diet quality was assessed by adherence to the Dutch food-based dietary guidelines. RESULTS: After BS, we observed a significant decrease in intake of energy and all macro- and micronutrients (p < 0.01 for all), except for calcium (-39.0 ± 404.6 mg; p = 0.32). Overall, nutrient composition slightly changed with an increase in the relative intake of protein (+1.1 ± 4.3 energy percentage [en%]; p = 0.01) and mono- and disaccharides (+4.2 ± 6.4 en%; p < 0.001) post-surgery. Consumption (median [Q1, Q3]) of vegetables (-50 [-120, 6] g day-1 ), wholegrain products (-38 [-81, -8] g day-1 ), liquid fats (-5 [-13, 2] g day-1 ), red meat (-3 [-30, 4] g day-1 ), processed meat (-32 [-55, 13] g day-1 ), sodium (-0.7 [-1.1, -0.2] g day-1 ) and unhealthy food choices (-2.4 [-5.0, 0.6] serves week-1 ) significantly decreased after BS (p < 0.01 for all). CONCLUSIONS: Our results demonstrate both favourable and unfavourable changes in macro- and micronutrient composition and diet quality in the first 6 months following BS. Insight into these changes can improve dietary counselling in this population. Future research into underlying causes, consequences and long-term changes in dietary intake is needed.

Association between Low Vitamin D Status, Serotonin, and Clinico-Biobehavioral Parameters in Alzheimer's Disease.

INTRODUCTION: Studies suggest a role of vitamin D in the progression and symptomatology of Alzheimer's disease (AD), with few in vitro studies pointing to effects on serotonergic and amyloidogenic turnover. However, limited data exist in AD patients on the potential association with cognition and behavioral and psychological signs and symptoms of dementia (BPSD). In this retrospective cross-sectional study, we, therefore, explored potential correlations of serum 25-hydroxyvitamin D3 (25(OH)D3) concentrations, indicative of vitamin D status, with serum serotonin (5-hydroxytryptamine, 5-HT) levels, cognitive/BPSD scorings, and cerebrospinal fluid (CSF) biomarker levels. METHODS: Frozen serum samples of 25 well-characterized AD subjects as part of a previous BPSD cohort were analyzed, of which 15 had a neuropathologically confirmed diagnosis. Serum 25(OH)D3 levels were analyzed by means of LC-MS/MS, whereas 5-HT concentrations were quantified by competitive ELISA. RESULTS: Among AD patients, vitamin D deficiency was highly prevalent, defined as levels below 50 nmol/L. Regression analyses, adjusted for age, gender, and psychotropic medications, revealed that serum 25(OH)D3 and 5-HT levels were positively associated (p = 0.012). Furthermore, serum 25(OH)D3 concentrations correlated inversely with CSF amyloid-beta (Aß1-42) levels (p = 0.006), and serum 5-HT levels correlated positively with aggressiveness (p = 0.001), frontal behavior (p = 0.001), depression (p = 0.004), and partly with cognitive performance (p < 0.005). Lastly, AD patients on cholinesterase inhibitors had higher serum 25(OH)D3 (p = 0.030) and lower serum 5-HT (p = 0.012) levels. CONCLUSIONS: The molecular associations between low vitamin D status, serum 5-HT, and CSF Aß1-42 levels are highly remarkable, warranting further mechanistic and intervention studies to disclose potential involvement in the clinico-biobehavioral pathophysiology of AD.

Efficacy and utility of a tool for both measurement of vitamin B6, B12, D, folate and iron status and assessment of diet quality in athletes.

NutriProfiel® is a tool to measure micronutrient status and to assess diet quality. It consists of measurement of micronutrient status in blood and a short food frequency questionnaire (FFQ) ('Eetscore-FFQ'). Based on the results, individuals receive a dietary advice. In this study, we evaluated the application of NutriProfiel in athletes ('NutriProfiel-Sport') by assessing the coverage of nutrient intake of the Eetscore-FFQ (part 1) and by evaluating athlete's dietary behaviour after using NutriProfiel-Sport and their satisfaction with this tool (part 2). For part 1, data of 419 athletes were used. We evaluated the coverage of nutrient intake of the Eetscore-FFQ using first and second MOMents (MOM1 and MOM2) sum scores of food items in the questionnaire. Forty-eight athletes were involved in part 2. They gave blood samples for micronutrient status measurement and were asked to complete the Eetscore-FFQ at baseline and after 3 months, as well as a questionnaire on their satisfaction with NutriProfiel-Sport. Results showed that for most nutrients, MOM1 and MOM2 scores were above 80 %, meaning that nutrient intake was sufficiently covered by the Eetscore-FFQ. No difference in diet quality was observed between baseline and after 3 months. Nevertheless, a majority of athletes reported the NutriProfiel-Sport results and advice as useful. On a scale from 0 to 10, NutriProfiel-Sport was graded with a mean (±sd) score of 7⋅6 (±0⋅8). In conclusion, NutriProfiel-Sport is a potential valuable and appreciated tool for athletes and the Eetscore-FFQ as part of this tool sufficiently covers nutrient intake in athletes.

Effects of γ-aminobutyric acid supplementation on glucose control in adults with prediabetes: A double-blind, randomized, placebo-controlled trial.

BACKGROUND: Gamma-aminobutyric acid (GABA) is mainly known as an endogenously produced neurotransmitter. However, GABA intake from dietary sources like tomatoes and fermented foods can be considerable. Studies in rodent models have shown beneficial effects of oral GABA supplementation on glucose homeostasis and cardiovascular health. Still, it is currently unknown whether oral GABA supplementation produces cardiometabolic benefits in humans. OBJECTIVES: This study aimed to investigate whether oral GABA supplementation can improve glucose homeostasis in individuals at risk of developing type 2 diabetes. METHODS: In a randomized, placebo-controlled, double-blind, parallel-arm trial, 52 individuals with prediabetes (classified by impaired glucose tolerance and/or impaired fasting glucose), aged 50 to 70 y with a body mass index ≥25 kg/m2 received either 500 mg GABA 3 times daily or a placebo for 95 days. The primary outcome was the effect of the intervention on glucose response after an OGTT. As exploratory secondary outcomes, markers of glycemic control (glycated hemoglobin, insulin, glucagon, mean amplitude of glycemic excursions, and standard deviation as measured with flash glucose monitoring), cardiovascular health (blood pressure, 24-h blood pressure, circulating triglycerides, cholesterol), and self-reported sleep quality were measured before and after the intervention. RESULTS: Compared with placebo, GABA supplementation for 95 days did not change the postprandial glucose response (0.21 mmol/L; 95% confidence interval: -0.252, 0.674; P = 0.364). After correction for the false discovery rate, all other outcomes (including fasting plasma GABA concentration) showed no significant effects from GABA intervention at a group level. CONCLUSIONS: GABA supplementation does not change the postprandial glucose response in individuals at risk of developing type 2 diabetes. However, based on findings in secondary outcome measures, further research is warranted in other study populations. Research could focus on the effects of GABA in individuals with advanced diabetes or other cardiometabolic disorders. This trial was registered at www. CLINICALTRIALS: gov as NCT04303468.

Development and validation of a UPLC-MS/MS method for the quantification of sugars and non-nutritive sweeteners in human urine.

High-intensity sweeteners ('sweeteners'), such as sucralose, saccharine, acesulfame, cyclamate and steviol, are replacing sugars in many food products, but biomarker-based data on their population-wide exposure, as well as analytical methods that can quantify urinary concentrations of sugars and sweeteners simultaneously, are lacking. Here, we developed and validated an ultra-pressure liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) method to quantify glucose, sucrose, fructose, sucralose, saccharine, acesulfame, cyclamate and steviol glucuronide in human urine. Urine samples were prepared by a simple dilution step containing the internal standards in water and methanol. Separation was achieved on a Shodex Asahipak NH2P-40 hydrophilic interaction liquid chromatography (HILIC) column using gradient elution. The analytes were detected using electrospray ionization in negative ion mode, and selective reaction monitoring was optimized using the [M-H]- ions. Calibration curves ranged between 34 and 19,230 ng/mL for glucose and fructose, and 1.8 to 1,026 ng/mL for sucrose and the sweeteners. The method has acceptable accuracy and precision, which depends on the application of appropriate internal standards. Storage of urine samples in lithium monophosphate gives the best overall analytical performance, and storage at room temperature without any preservatives should be avoided since this leads to reduced glucose and fructose concentrations. With the exception of fructose, all analytes were stable throughout 3 freeze-thaw cycles. The validated method was applied to human urine samples, demonstrating quantifiable concentrations of the analytes which were in the expected range. It is concluded that the method has acceptable performance to quantitatively determine dietary sugars and sweeteners in human urine.

Postdiagnostic intake of a more proinflammatory diet is associated with a higher risk of recurrence and all-cause mortality in colorectal cancer survivors.

BACKGROUND: The inflammatory potential of the diet has been associated with colorectal cancer (CRC) risk, but its association with CRC prognosis is unclear. OBJECTIVE: To investigate the inflammatory potential of the diet in relation to recurrence and all-cause mortality among persons diagnosed with stage I to III CRC. METHODS: Data of the COLON study, a prospective cohort among CRC survivors were used. Dietary intake, 6 mo after diagnosis, was assessed by using a food frequency questionnaire and was available for 1631 individuals. The empirical dietary inflammatory pattern (EDIP) score was used as a proxy for the inflammatory potential of the diet. The EDIP score was created by using reduced rank regression and stepwise linear regression to identify food groups that explained most of the variations in plasma inflammatory markers (IL6, IL8, C-reactive protein, and tumor necrosis factor-α) measured in a subgroup of survivors (n = 421). Multivariable Cox proportional hazard models with restricted cubic splines were used to investigate the relation between the EDIP score and CRC recurrence and all-cause mortality. Models were adjusted for age, sex, BMI, PAL, smoking status, stage of disease, and tumor location. RESULTS: The median follow-up time was 2.6 y (IQR: 2.1) for recurrence and 5.6 y (IQR: 3.0) for all-cause mortality, during which 154 and 239 events occurred, respectively. A nonlinear positive association between the EDIP score and recurrence and all-cause mortality was observed. For example, a more proinflammatory diet (EDIP score +0.75) compared with the median (EDIP score 0) was associated with a higher risk of CRC recurrence (HR: 1.15; 95% CI: 1.03, 1.29) and all-cause mortality (HR: 1.23; 95% CI: 1.12, 1.35). CONCLUSIONS: A more proinflammatory diet was associated with a higher risk of recurrence and all-cause mortality in CRC survivors. Further intervention studies should investigate whether a switch to a more anti-inflammatory diet improves CRC prognosis.

Analysis of Omega-3 Fatty Acid-Derived N-Acylethanolamines in Biological Matrices.

The adequate quantification of endocannabinoids and related N-acylethanolamines can be complex due to their low endogenous levels, structural diversity, and metabolism. Therefore, advanced analytical approaches, involving LC-MS, are required to quantify these molecules in plasma, tissues, and other matrices. It has been shown that endocannabinoid congeners synthesized from n-3 poly-unsaturated fatty acids (n-3 PUFAs), such as docosahexaenoylethanolamide (DHEA) and eicosapentaenoylethanolamide (EPEA), have interesting immunomodulatory and tumor-inhibiting properties. Recent work has shown that DHEA and EPEA can be further enzymatically metabolized by cyclo-oxygenase 2 (COX-2), forming oxygenated metabolites. Here, an LC-MS-based method for the quantification of the n-3 PUFA-derived endocannabinoid congeners DHEA and EPEA is described, which is also suited to measure a wider spectrum of endocannabinoids. The chapter contains a step-by-step protocol for the analysis of (n-3) endocannabinoids in plasma, including sample collection and solid phase extraction, LC-MS analysis, and data processing. In addition, protocol modifications are provided to allow quantification of n-3 PUFA-derived endocannabinoids and their COX-2 metabolites in tissues and cell culture media. Finally, conditions that alter endocannabinoid concentrations are briefly discussed.

Relative validity of a short screener to assess diet quality in patients with severe obesity before and after bariatric surgery.

OBJECTIVE: To determine the relative validity and reproducibility of the Eetscore FFQ, a short screener for assessing diet quality, in patients with (severe) obesity before and after bariatric surgery (BS). DESIGN: The Eetscore FFQ was evaluated against 3-d food records (3d-FR) before (T0) and 6 months after BS (T6) by comparing index scores of the Dutch Healthy Diet index 2015 (DHD2015-index). Relative validity was assessed using paired t tests, Kendall's tau-b correlation coefficients (τb), cross-classification by tertiles, weighted kappa values (k w ) and Bland-Altman plots. Reproducibility of the Eetscore FFQ was assessed using intraclass correlation coefficients (ICC). SETTING: Regional hospital, the Netherlands. PARTICIPANTS: Hundred and forty participants with obesity who were scheduled for BS. RESULTS: At T0, mean total DHD2015-index score derived from the Eetscore FFQ was 10·2 points higher than the food record-derived score (P < 0·001) and showed an acceptable correlation (τb = 0·42, 95 % CI: 0·27, 0·55). There was a fair agreement with a correct classification of 50 % (k w = 0·37, 95 % CI: 0·25, 0·49). Correlation coefficients of the individual DHD components varied from 0·01-0·54. Similar results were observed at T6 (τb = 0·31, 95 % CI: 0·12, 0·48, correct classification of 43·7 %; k w = 0·25, 95 % CI: 0·11, 0·40). Reproducibility of the Eetscore FFQ was good (ICC = 0·78, 95 % CI: 0·69, 0·84). CONCLUSION: The Eetscore FFQ showed to be acceptably correlated with the DHD2015-index derived from 3d-FR, but absolute agreement was poor. Considering the need for dietary assessment methods that reduce the burden for patients, practitioners and researchers, the Eetscore FFQ can be used for ranking according to diet quality and for monitoring changes over time.

PUFA-Derived N-Acylethanolamide Probes Identify Peroxiredoxins and Small GTPases as Molecular Targets in LPS-Stimulated RAW264.7 Macrophages.

We studied the mechanistic and biological origins of anti-inflammatory poly-unsaturated fatty acid-derived N-acylethanolamines using synthetic bifunctional chemical probes of docosahexaenoyl ethanolamide (DHEA) and arachidonoyl ethanolamide (AEA) in RAW264.7 macrophages stimulated with 1.0 µg mL-1 lipopolysaccharide. Using a photoreactive diazirine, probes were covalently attached to their target proteins, which were further studied by introducing a fluorescent probe or biotin-based affinity purification. Fluorescence confocal microscopy showed DHEA and AEA probes localized in cytosol, specifically in structures that point toward the endoplasmic reticulum and in membrane vesicles. Affinity purification followed by proteomic analysis revealed peroxiredoxin-1 (Prdx1) as the most significant binding interactor of both DHEA and AEA probes. In addition, Prdx4, endosomal related proteins, small GTPase signaling proteins, and prostaglandin synthase 2 (Ptgs2, also known as cyclooxygenase 2 or COX-2) were identified. Lastly, confocal fluorescence microscopy revealed the colocalization of Ptgs2 and Rac1 with DHEA and AEA probes. These data identified new molecular targets suggesting that DHEA and AEA may be involved in reactive oxidation species regulation, cell migration, cytoskeletal remodeling, and endosomal trafficking and support endocytosis as an uptake mechanism.

The influence of a tomato food matrix on the bioavailability and plasma kinetics of oral gamma-aminobutyric acid (GABA) and its precursor glutamate in healthy men.

Gamma-aminobutyric acid (GABA) and its precursor glutamate play signaling roles in a range of tissues. Both function as neurotransmitters in the central nervous system, but they also modulate pancreatic and immune functioning, for example. Besides endogenous production, both compounds are found in food products, reaching relatively high levels in tomatoes. Recent studies in rodents suggest beneficial effects of oral GABA on glucose homeostasis and blood pressure. However, the bioavailability from food remains unknown. We studied the bioavailability of GABA and glutamate from tomatoes relative to a solution in water. After a fasting blood sample was taken, eleven healthy men randomly received 1 liter of 4 different drinks in a cross-over design with a one-week interval. The drinks were a solution of 888 mg L-1 GABA, a solution of 3673 mg L-1 glutamate, pureed fresh tomatoes and plain water as the control. Following intake, 18 blood samples were taken at intervals for 24 hours. Plasma GABA and glutamate concentrations were determined by ultra-pressure liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). Fasting plasma GABA and glutamate concentrations were found to be 16.71 (SD 2.18) ng mL-1 and 4626 (SD 1666) ng mL-1, respectively. Fasting GABA levels were constant (5.8 CV%) between individuals, while fasting glutamate levels varied considerably (23.5 CV%). GABA from pureed tomatoes showed similar bioavailability to that of a solution in water. For glutamate, the absorption from pureed tomatoes occurred more slowly as seen from a longer tmax (0.98 ± 0.14 h vs. 0.41 ± 0.04 h, P = 0.003) and lower Cmax (7815 ± 627 ng mL-1vs. 16 420 ± 2778 ng mL-1, P = 0.006). These data suggest that GABA is bioavailable from tomatoes, and that food products containing GABA could potentially induce health effects similar to those claimed for GABA supplements. The results merit further studies on the bioavailability of GABA from other food products and the health effects of GABA-rich diets. The clinical trial registry number is NCT04086108 (https://clinicaltrials.gov/ct2/show/NCT04303468).

Prevalence of Micronutrient Deficiencies and Relationship with Clinical and Patient-Related Outcomes in Pulmonary Hypertension Types I and IV.

BACKGROUND: Pulmonary hypertension (PH) is a rare progressive and lethal disease affecting pulmonary arteries and heart function. The disease may compromise the nutritional status of the patient, which impairs their physical performance. This study aimed to determine the prevalence of micronutrient deficiencies in pulmonary arterial hypertension (PAH) and chronic thrombo-embolic pulmonary hypertension (CTEPH) patients. METHODS: Eighty-one blood samples from a prospective observational cohort study were analyzed for concentrations of micronutrients and inflammation-related factors. The samples consisted of newly diagnosed (treatment-naive) PAH and CTEPH patients and patients treated for 1.5 years according to ERS/ESC guidelines. RESULTS: In the newly diagnosed group, 42% of PAH patients and 21% of CTEPH patients were iron deficient compared to 29% of PAH patients and 20% of CTEPH patients in the treatment group. Vitamin D deficiency occurred in 42% of the newly diagnosed PAH patients, 71% of the newly diagnosed CTEPH patients, 68% of the treated PAH patients, and 70% of the treated CTEPH patients. Iron levels correlated with the 6 min walking distance (6MWD). CONCLUSIONS: Iron and vitamin D deficiencies are highly prevalent in PAH and CTEPH patients, underlining the need for monitoring their status. Studies evaluating the effects of supplementation strategies for iron and vitamin D are necessary.

Pitfalls in interpreting red blood cell parameters in elite high-altitude and sea-level athletes: A unique case series.

Standard routine hematological measurements are commonly used to investigate differences in blood parameters between high-altitude athletes (HAA) and sea-level athletes (SLA), and to monitor the effect of high-altitude training. In this way, red blood cell (RBC) parameters are usually expressed as relative parameters (concentration) rather than absolute parameters (total amount). In this unique case series of elite HAA and SLA, we describe how different ways of parameter expression can affect the interpretation of blood tests. In a group of 42 elite athletes, relative and absolute RBC parameters were compared between HAA and SLA. Absolute parameters were calculated by multiplying relative values with formula-based estimated blood volume (BV-e). Additionally, in two individual athletes, one HAA and one SLA, absolute parameters were also calculated with blood volume (BV) obtained by measurement with a dilution method (BV-m). In men, HAA had a significantly higher hemoglobin (Hb) concentration (+7.8%; p = 0.001) and total Hb mass per kg body weight (BW) (+12.0%; p = 0.002). When not corrected for BW, HAA had a lower, non-significant, total Hb mass (-7.8%; p = 0.055). In women, no significant differences between HLA and SLA were observed. The two individual athletes showed that, based on BV-m, in the HAA, total Hb mass and total Hb mass per kg BW were respectively 14.1% and 31.0% higher than in the SLA, whereas based on BV-e, in the HAA, total Hb mass was 20.8% lower and total Hb mass per kg BW was only 2.4% higher. Similar inconsistencies were observed for total RBC count. Thus, different ways of parameter expression, and different methods of BV assessment for the calculation of absolute parameter values, influence the interpretation of blood tests in athletes, which may lead to misinterpretation and incorrect conclusions.

Levels of Inflammation Markers Are Associated with the Risk of Recurrence and All-Cause Mortality in Patients with Colorectal Cancer.

BACKGROUND: We investigated whether preoperative and postoperative levels of inflammation markers, which have mechanistically been linked to colorectal cancer progression, were associated with recurrence and all-cause mortality in patients with colorectal cancer. METHODS: Data of two prospective cohort studies were used. For the current analysis, patients with stage I to III colorectal cancer were considered. Data on inflammation [IL6, IL8, IL10, TNFα, high-sensitivity C-reactive protein (hsCRP), and a combined inflammatory z-score] were available for 747 patients before surgery and for 614 patients after surgery. The associations between inflammation marker levels and colorectal cancer recurrence and all-cause mortality were examined using multivariable Cox proportional hazard regression models, considering patient characteristics and clinical and lifestyle factors. RESULTS: Higher preoperative and postoperative hsCRP levels were associated with a higher risk of recurrence [HRper doubling (95% CI), 1.15 (1.02-1.30) and 1.34 (1.16-1.55)] and all-cause mortality [HRper doubling (95% CI) 1.13 (1.01-1.28) and 1.15 (0.98-1.35)]. A doubling in IL8 levels (preoperative levels HR = 1.23; 95% CI, 1.00-1.53 and postoperative levels HR = 1.61; 95% CI, 1.23-2.12) and a higher combined inflammatory z-score (preoperative HRper doubling = 1.39; 95% CI, 1.03-1.89 and postoperative HRper doubling = 1.56; 95% CI, 1.06-2.28) were associated with a higher risk of all-cause mortality, but not recurrence. No associations between IL6, IL10, and TNFα and recurrence or all-cause mortality were observed. CONCLUSIONS: Preoperative and postoperative levels of specific inflammation markers were associated with recurrence and/or all-cause mortality. IMPACT: The complex role of inflammation in cancer recurrence merits further elucidation by investigating local inflammation at the tumor site.

Immunomodulating effects of 13- and 16-hydroxylated docosahexaenoyl ethanolamide in LPS stimulated RAW264.7 macrophages.

Docosahexaenoyl ethanolamide (DHEA), the ethanolamine conjugate of the n-3 long chain polyunsaturated fatty acid docosahexaenoic acid, is endogenously present in the human circulation and in tissues. Its immunomodulating properties have been (partly) attributed to an interaction with the cyclooxygenase-2 (COX-2) enzyme. Recently, we discovered that COX-2 converts DHEA into two oxygenated metabolites, 13- and 16-hydroxylated-DHEA (13- and 16-HDHEA, respectively). It remained unclear whether these oxygenated metabolites also display immunomodulating properties like their parent DHEA. In the current study we investigated the immunomodulating properties of 13- and 16-HDHEA in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. The compounds reduced production of tumor necrosis factor alpha (TNFα), interleukin (IL)-1ß and IL-1Ra, but did not affect nitric oxide (NO) and IL-6 release. Transcriptome analysis showed that the compounds inhibited the LPS-mediated induction of pro-inflammatory genes (InhbA, Ifit1) and suggested potential inhibition of regulators such as toll-like receptor 4 (TLR4), MyD88, and interferon regulatory factor 3 (IRF3), whereas anti-inflammatory genes (SerpinB2) and potential regulators IL-10, sirtuin 1 (Sirt-1), fluticasone propionate were induced. Additionally, transcriptome analysis of 13-HDHEA suggests a potential anti-angiogenic role. In contrast to the known oxylipin-lowering effects of DHEA, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analyses revealed that 13- and 16-HDHEA did not affect oxylipin formation. Overall, the anti-inflammatory effects of 13-HDHEA and 16-HDHEA are less pronounced compared to their parent molecule DHEA. Therefore, we propose that COX-2 metabolism of DHEA acts as a regulatory mechanism to limit the anti-inflammatory properties of DHEA.

Development and validation of a UPLC-MS/MS method for the simultaneous determination of gamma-aminobutyric acid and glutamic acid in human plasma.

Gamma-aminobutyric acid (GABA) and its precursor glutamic acid are important neurotransmitters. Both are also present in peripheral tissues and the circulation, where abnormal plasma concentrations have been linked to specific mental disorders. In addition to endogenous synthesis, GABA and glutamic acid can be obtained from dietary sources. An increasing number of studies suggest beneficial cardio-metabolic effects of GABA intake, and therefore GABA is being marketed as a food supplement. The need for further research into their health effects merits accurate and sensitive methods to analyze GABA and glutamic acid in plasma. To this end, an ultra-pressure liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the quantification of GABA and glutamic acid in human plasma. Samples were prepared by a protein precipitation step and subsequent solid phase extraction using acetonitrile. Chromatographic separation was achieved on an Acquity UPLC HSS reversed phase C18 column using gradient elution. Analytes were detected using electrospray ionization and selective reaction monitoring. Standard curve concentrations for GABA ranged from 3.4 to 2500 ng/mL and for glutamic acid from 30.9 ng/mL to 22,500 ng/mL. Within- and between-day accuracy and precision were <10% in quality control samples at low, medium and high concentrations for both GABA and glutamic acid. GABA and glutamic acid were found to be stable in plasma after freeze-thaw cycles and up to 12 months of storage. The validated method was applied to human plasma from 17 volunteers. The observed concentrations ranged between 11.5 and 20.0 ng/ml and 2269 and 7625 ng/ml for respectively GABA and glutamic acid. The reported method is well suited for the measurement of plasma GABA and glutamic acid in pre-clinical or clinical studies.

In vitro dissolution behaviour and absorption in humans of a novel mixed l-lysine salt formulation of EPA and DHA.

INTRODUCTION: Supplements with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are generally oil-based formulations containing their triacylglycerols, phospholipids or ethyl-esters (EE). Recently, a l-lysine salt of carboxylic EPA and DHA became available (Lys-FFA), which necessitated to study its oral absorption and plasma kinetics in humans. OBJECTIVES: The in vitro dissolution characteristics, oral bioavailability and 48 h plasma profiles of EPA and DHA (as triacylglycerides) of Lys-FFA, relative to a commercially available oil-based EE supplement. METHODS: Dissociation of the lysine from the FFAs was studied in vitro applying simulated gastric (12 h) and intestinal (3 h) conditions. In an open label, randomized, two-way cross-over design, oral administration of Lys-FFA (500 mg EPA plus 302 mg DHA) versus EE (504 mg EPA plus 378 mg DHA) was studied over 48 h, in eight female volunteers. Plasma profiles of EPA and DHA were described by Area Under the Curve (AUC; 0-12 h), Cmax and Tmax. RESULTS: Dissolution studies with Lys-FFA showed complete dissociation under both conditions. In volunteers Lys-FFA showed rapid absorption and high bioavailability indicated by significant differences in both the AUC0-12hr and Cmax when compared to the EE comparator (p<0.001), with AUC0-12hr which was for EPA 5 times higher with Lys-FFA than with the EE formulation. CONCLUSION: This first-in-man study of Lys-FFA demonstrated rapid absorption of EPA and DHA and a considerably higher bioavailability compared to an EE supplement under fasting conditions. The release and absorption characteristics from this solid form offer several new options in terms of formulation technology and dosing.

Inflammation Is a Mediating Factor in the Association between Lifestyle and Fatigue in Colorectal Cancer Patients.

Fatigue is very common among colorectal cancer (CRC) patients. We examined the association between adherence to the World Cancer Research Fund/American Institute for Cancer Research (WCRF/AICR) lifestyle recommendations and fatigue among stage I-III CRC patients, and whether inflammation mediated this association. Data from two prospective cohort studies were used. Adherence to the WCRF/AICR recommendations was expressed as a score ranging from 0-7, and assessed shortly after diagnosis. Six months post-diagnosis, fatigue was assessed with the European Organization for Research and Treatment of Cancer quality of life questionnaire C30 (EORTC QLQ-C30), and in a subpopulation, the plasma levels of inflammation markers (IL6, IL8, TNFα, and hsCRP) were assessed. Multiple linear regression analyses were performed to investigate the association between adherence to the WCRF/AICR recommendations and fatigue. To test mediation by inflammation, the PROCESS analytic tool developed by Hayes was used. A higher WCRF/AICR adherence score was associated with less fatigue six months after diagnosis (n = 1417, ß -2.22, 95%CI -3.65; -0.78). In the population of analysis for the mediation analyses (n = 551), the total association between lifestyle and fatigue was (ß -2.17, 95% CI -4.60; 0.25). A statistically significant indirect association via inflammation was observed (ß -0.97, 95% CI -1.92; -0.21), explaining 45% of the total association between lifestyle and fatigue (-0.97/-2.17 × 100). Thus, inflammation is probably one of the underlying mechanisms linking lifestyle to fatigue.