Effect of normobaric hypoxic exercise on blood pressure in old individuals.

PURPOSE: To test the hypothesis that the combination of endurance training and hypoxia leads to greater improvements in resting and exercise blood pressure in old sedentary individuals compared to endurance training only. METHODS: We randomly assigned 29 old overweight participants (age: 62 ± 6 years, body mass index (BMI): 28.5 ± 0.5 kg/m2, 52% men) to single blind 8-week bicycle exercise in hypoxia (fraction of inspired oxygen (FIO2) = 0.15) or normoxia (FIO2 = 0.21). Brachial blood pressure was measured at rest, during maximal incremental exercise testing, and during a 30 min constant work rate test, at baseline and after the training period. RESULTS: Work rate, heart rate and perceived exertion during training were similar in both groups, with lower oxygen saturation for participants exercising under hypoxia (88.7 ± 1.5 vs. 96.2 ± 1.2%, t(27) = - 13.04, p < 0.001, |g|= 4.85). Office blood pressure and blood pressure during incremental exercise tests did not change significantly in either group after the training program. Systolic blood pressure during the constant work rate test was reduced after training in hypoxia (160 ± 18 vs. 151 ± 14 mmHg, t(13) = 2.44 p < 0.05, |d|= 0.55) but not normoxia (154 ± 22 vs. 150 ± 16 mmHg, t(14) = 0.75, p = 0.46, |d|= 0.18) with no difference between groups over time (F = 0.08, p = 0.77, η2 = 0.01). CONCLUSION: In old individuals hypoxia in addition to exercise does not have superior effects on office or exercise blood pressure compared to training in normoxia. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov No. NCT02196623 (registered 22 July 2014).

Iron-regulatory proteins secure iron availability in cardiomyocytes to prevent heart failure.

Aims: Iron deficiency (ID) is associated with adverse outcomes in heart failure (HF) but the underlying mechanisms are incompletely understood. Intracellular iron availability is secured by two mRNA-binding iron-regulatory proteins (IRPs), IRP1 and IRP2. We generated mice with a cardiomyocyte-targeted deletion of Irp1 and Irp2 to explore the functional implications of ID in the heart independent of systemic ID and anaemia. Methods and results: Iron content in cardiomyocytes was reduced in Irp-targeted mice. The animals were not anaemic and did not show a phenotype under baseline conditions. Irp-targeted mice, however, were unable to increase left ventricular (LV) systolic function in response to an acute dobutamine challenge. After myocardial infarction, Irp-targeted mice developed more severe LV dysfunction with increased HF mortality. Mechanistically, the activity of the iron-sulphur cluster-containing complex I of the mitochondrial electron transport chain was reduced in left ventricles from Irp-targeted mice. As demonstrated by extracellular flux analysis in vitro, mitochondrial respiration was preserved at baseline but failed to increase in response to dobutamine in Irp-targeted cardiomyocytes. As shown by 31P-magnetic resonance spectroscopy in vivo, LV phosphocreatine/ATP ratio declined during dobutamine stress in Irp-targeted mice but remained stable in control mice. Intravenous injection of ferric carboxymaltose replenished cardiac iron stores, restored mitochondrial respiratory capacity and inotropic reserve, and attenuated adverse remodelling after myocardial infarction in Irp-targeted mice but not in control mice. As shown by electrophoretic mobility shift assays, IRP activity was significantly reduced in LV tissue samples from patients with advanced HF and reduced LV tissue iron content. Conclusions: ID in cardiomyocytes impairs mitochondrial respiration and adaptation to acute and chronic increases in workload. Iron supplementation restores cardiac energy reserve and function in iron-deficient hearts.

ANGPTL8 (Betatrophin) is Expressed in Visceral Adipose Tissue and Relates to Human Hepatic Steatosis in Two Independent Clinical Collectives.

Angiopoietin-like protein 8 (ANGPTL8)/betatrophin expression in visceral adipose tissue and associations with circulating fatty acid profile have not yet been investigated.Forty subjects were included in a cross-sectional study, 57 in a dietary weight reduction intervention. Circulating Angiopoietin-like protein 8/betatrophin was measured in all subjects. Liver and adipose tissue were sampled and plasma fatty acids and tissue Angiopoietin-like protein 8/betatrophin expression were evaluated in the cross-sectional study. In the intervention study oral glucose testing and liver magnetic resonance scanning at baseline and after 6 months were performed. Angiopoietin-like protein 8/betatrophin mRNA was increased in visceral compared to subcutaneous adipose tissue (p<0.001). Circulating ANGPTL8/betatrophin correlated with liver steatosis (r=0.42, p=0.047), triacylglycerols (r=0.34, p=0.046), saturated (r=0.43, p=0.022), monounsaturated (r=0.51, p=0.007), and polyunsaturated fatty acids (r=-0.53, p=0.004). In the intervention study, baseline Angiopoietin-like protein 8/betatrophin correlated with age (r=0.32, p=0.010) and triacylglycerols (r=0.30, p=0.02) and was increased with hepatic steatosis (p=0.033). Weight loss reduced liver fat by 45% and circulating Angiopoietin-like protein 8/betatrophin by 11% (288±17 vs. 258±17 pg/ml; p=0.015). Angiopoietin-like protein 8/betatrophin is related to liver steatosis, while visceral adipose tissue represents an additional site of expression in humans.

Adipokine concentrations in lipoaspirates may have a role in wound healing.

OBJECTIVES: In addition to its use as a volume filler, fat grafting may have a potential role in wound healing based on the concentration of growth factors in the lipoaspirate. In this study, we compare the quantitative and qualitative concentration of the various growth factors and adipokines using the Shippert or the Coleman techniques to prepare the lipoaspirate. METHODS: We measured leptin, adiponectin and the growth factors, i.e., acidic fibroblast growth factor (aFGF), basic FGF (bFGF), keratinocyte growth factor (KGF), bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) by ELISA in solid and liquid fractions obtained with both techniques in human fat obtained with Coleman technique and Shippert technique. RESULTS: All of these peptides, except BMP-2, were detected in relevant quantities in the solid fraction. The Coleman but not the Shippert technique resulted in statistically higher adiponectin concentrations in the solid tissue fraction. The other four growth factors occurred in significantly higher concentrations in the solid fractions compared to the liquid fractions, independent of the processing technique. CONCLUSION: In summary, we demonstrated that KGF, aFGF, bFGF and VEGF, as well as leptin and adiponectin, are contained in fat suspensions obtained by liposuction and in the supernatant. Only the concentration of adiponectin was in the range reported to contribute to wound healing.

Plasma and tissue homoarginine concentrations in healthy and obese humans.

Increased cardiovascular risk associated with obesity cannot be fully explained by traditional risk markers. We therefore assessed plasma and interstitial concentrations of the novel cardiovascular risk biomarker homoarginine (hArg) in 18 individuals without signs of cardiovascular disease, including 4 morbidly obese subjects before and after bariatric surgery and subsequent weight reduction of 36 ± 7 kg. hArg concentrations were greater in skeletal muscle compared with adipose tissue. Plasma and tissue hArg concentrations did not correlate with BMI. Adipose tissue interstitial hArg concentrations were not affected by obesity, an oral glucose load, or dramatic weight loss. In conclusion, obesity seems not to have a major effect on hArg homeostasis, and hArg may not explain the added cardiovascular risk associated with obesity. Yet, given the small sample size of the study, the significance of hArg in obesity should be investigated in a larger population.

Temperamental factors in severe weight cycling. A cross-sectional study.

OBJECTIVE: Weight cycling is a prevalent phenomenon in obese individuals. There is evidence that temperamental factors are associated with obesity and subgroups among the obese have been identified based on reactive and regulative aspects of temperament. METHODS: We aimed at investigating the association between reactive and regulative aspects of temperament and severe weight cycling in overweight and obese individuals of a representative German population sample (n = 923). Participants completed questionnaires assessing weight parameters including BMI and weight cycling, sensitivity to punishment and to reward (BIS/BAS scales), self-regulatory abilities (effortful control scale), depressive symptoms, and binge eating. RESULTS: Severe weight cycling was more common in women, and was associated with higher reward sensitivity, higher current and maximum-ever BMI, higher weight suppression, more depressive symptoms, and a higher prevalence of binge eating. In contrast, sensitivity to punishment and effortful control were not associated with severe weight cycling. Also, the interaction between sensitivity to reward and effortful control did not predict weight cycling. DISCUSSION: Higher reward sensitivity might not only render individuals vulnerable for weight regain but might also be associated with a higher frequency of weight loss attempts due to the putative rewarding properties of the initial success in weight loss at the early stages of a diet. Temperamental factors should be considered in the treatment of obesity.

Blood pressure effects of glucagon-like peptide 1 analogues and sodium glucose transporter 2 inhibitors.

PURPOSE OF REVIEW: The growing number of obese patients with a high risk of developing hypertension and type 2 diabetes mellitus requires several drugs to treat all the associated morbidities. Ideally, one drug would help to tackle several health problems at the same time. We review available information on the blood pressure-reducing effects of the new antidiabetic drug classes, glucagon-like peptide 1 analogues and sodium glucose transporter 2 inhibitors. RECENT FINDINGS: Blood pressure reduction with glucagon-like peptide 1 analogues or sodium glucose transporter 2 inhibitors ranges between 1 and 7 mmHg, both systolic and diastolic. As these drugs have not been sufficiently investigated in studies with office or ambulatory blood pressure as the primary efficacy measure or in prespecified hypertensive patient populations, their true efficacy in reducing blood pressure remains unclear. These studies are needed because the blood pressure-lowering effects of metabolic drugs may help to improve the clinical management of hypertensive patients with type 2 diabetes mellitus. SUMMARY: Obese patients with type 2 diabetes mellitus and difficult to control arterial hypertension represent a clinically important patient group at high cardiovascular risk that may profit from combined cardiovascular and metabolic actions of a drug.

Clinical evaluation of extracellular ADMA concentrations in human blood and adipose tissue.

Circulating asymmetrical dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthesis, has been proposed as a biomarker for clinical outcome. Dimethylarginine dimethylaminohydrolase (DDAH) is the main enzyme responsible for ADMA metabolism and elimination. Adipose tissue ADMA concentrations and DDAH activity and their role in diabetes and obesity have not yet been investigated. In this study, we evaluated clinical microdialysis in combination with a sensitive analytical method (GC-MS/MS) to measure ADMA concentrations in extracellular fluid. Adipose tissue ADMA concentrations were assessed before and during an oral glucose tolerance test in lean healthy subjects and subjects with diabetes (n = 4 each), and in morbidly obese subjects before and after weight loss of 30 kg (n = 7). DDAH activity was determined in subcutaneous and visceral adipose tissue obtained during laparoscopic surgery (n = 5 paired samples). Mean interstitial ADMA concentrations did not differ between study populations (healthy 0.17 ± 0.03 µM; diabetic 0.21 ± 0.03 µM; morbidly obese 0.16 ± 0.01 and 0.17 ± 0.01 µM before and after weight loss, respectively). We did not observe any response of interstitial ADMA concentrations to the oral glucose challenge. Adipose tissue DDAH activity was negligible compared to liver tissue. Thus, adipose tissue ADMA plays a minor role in NO-dependent regulation of adipose tissue blood flow and metabolism.

Impact of advanced age on the gastric emptying of water under fasted and fed state conditions.

Although older people are the main users of oral medications, few studies are reported on the influence of advanced age on gastric emptying rate of non-caloric liquids. This study aimed at evaluating the gastric emptying of 240 ml water in healthy older and young adults in fasted and fed state conditions using the established method of salivary caffeine kinetics. The gastric emptying of water was evaluated in 12 healthy older volunteers (mean age: 73 ± 6 years) and 12 healthy younger volunteers (mean age: 25 ± 2 years) with the ingestion of a rapid disintegrating tablet containing 20 mg of 13C3-caffeine. The gastric emptying of water was assessed indirectly by calculating the AUC ratios of salivary caffeine concentrations in specific time segments. Comparison of the AUC ratios showed no statistically significant difference between young and older volunteers in both fasted and fed state conditions (p > 0.05). Advanced age itself seems to have no relevant effect on gastric emptying of water in either fasted or fed state conditions and the phenomenon of Magenstrasse appears to follow a similar pattern in healthy older adults as in healthy younger adults.

Prednisolone Versus Colchicine for Acute Gout in Primary Care: statistical analysis plan for the pragmatic, multicenter, randomized, and double-blinded COPAGO non-inferiority trial.

BACKGROUND: To date, colchicine and prednisolone are two effective therapies for the treatment of acute gout but have never been compared directly in a randomized clinical trial. In addition, in previous trials of treating acute gout patients with concomitant comorbidities were often excluded due to contraindications to naproxen. STUDY DESIGN: This pragmatic, prospective, double-blind, double-dummy, parallel-group, randomized, non-inferiority trial compares prednisolone with colchicine in terms of non-inferiority in patients with acute gout. Patients presenting to their general practitioner with acute gout can be included if the gout attack has occurred within the last 2 days. A total of 60 practices in the vicinity of three university medical centers (Greifswald, Göttingen, and Würzburg) participate in the study. The intervention group receives 30 mg prednisolone for 5 days, while the group of standard care receives low-dose colchicine (day 1: 1.5 mg; days 2-5: 1 mg). The first dose of treatment is provided at day 0 when patients present to the general practitioner due to an acute gout attack. From day 0 to day 6, patients will be asked to complete a study diary on daily basis regarding pain quantification. For safety reasons, potential side effects and the course of systolic blood pressure are also assessed. STATISTICAL ANALYSIS PLAN: N = 314 patients have to be recruited to compensate for 10% of dropout and to allow for showing non-inferiority of prednisolone compared to colchicine with a power of 90%. We use permuted block randomization with block sizes of 2, 4, and 6 to avoid imbalanced treatment arms in this multi-center study; patients are randomized in a 1:1 ratio. The absolute level of pain on day 3 (in the last 24 h) is the primary outcome and measured on a numerical rating scale (NRS: 0-10). Using a multiple linear regression model adjusted for age, sex, and pain at baseline, prednisolone is considered non-inferior if the effect estimate including the confidence intervals is lower than a margin of 1 unit on the NRS. Average response to treatment, joint swelling and tenderness, physical function of the joint, and patients' global assessment of treatment success are secondary outcomes. DISCUSSION: The trial will provide evidence from a direct comparison of colchicine and prednisolone regarding their efficacy of pain reduction in acute gout patients of primary care and to indicate possible safety signals. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT05698680 first posted on January 26, 2023 (retrospectively registered).

Apoptotic pathways in adipose tissue.

To treat the ever growing number of obese patients, reduction of adipocyte number by apoptosis may complement other therapeutic options. On the other hand in free fat grafts, apoptosis along with necrosis is responsible for long term volume reduction. To ensure successful soft tissue reconstruction it is mandatory to keep apoptosis on a low level in adipocytes, adipose-derived stromal cells and others cells of the fat graft. Apoptotic pathways have been sufficiently studied in various tissues, but the knowledge about apoptotic pathways in adipocytes is surprisingly scarce. Current knowledge about apoptotic pathways in adipose tissue is elaborately reflected in this review as well as the association of cancer with obesity. Possibilities to induce and reduce adipose tissue apoptosis in animal models are discussed as well as clinical implications of fat cell apoptosis. Mechanisms of apoptosis induction have been studied in animal models and suggest that a tight control of apoptosis induction is necessary because otherwise detrimental metabolic effects of fat mass loss will occur that may mimic lipodystrophic diseases. At present, targeted induction of adipocyte apoptosis appears to be of some concern related to increased blood lipid concentrations, ectopic lipid storage and other detrimental metabolic effects. Treatment of autologous adipocytes used for lipofilling procedures with appropriate substances may result in more satisfactory long-term outcomes as well as stimulation of stem cell differentiation in a strictly local manner.

Novel metabolic drugs and blood pressure: implications for the treatment of obese hypertensive patients?

Hypertension and obesity often coexist, exposing patients to cardiovascular and metabolic risks, particularly type 2 diabetes mellitus. Moreover, obesity may render hypertensive patients treatment resistant. We review how drugs recently approved for obesity or type 2 diabetes mellitus treatment affect blood pressure. The weight-reducing drug lorcaserin induces modest reductions in body weight while slightly improving blood pressure. The fixed low-dose topiramate/phentermine combinations elicit larger reductions in body weight and blood pressure. Concomitant improvements in glucose metabolism, adiposity, and blood pressure differentiate the first clinically available SGLT2 inhibitor dapagliflozin from other oral antidiabetic drugs. Yet, the mechanisms through which metabolic drugs affect blood pressure and their interaction with antihypertensive drugs are poorly understood. Blood pressure-lowering effects of metabolic drugs could be exploited in the clinical management of obese hypertensive patients with and without type 2 diabetes mellitus, particularly in patients with difficult to control arterial hypertension.

Alterations of endocannabinoids in cerebrospinal fluid of dogs with epileptic seizure disorder.

BACKGROUND: Epilepsy is one of the most common chronic neurological disorders in dogs characterized by recurrent seizures. The endocannabinoid (EC) system plays a central role in suppressing pathologic neuronal excitability and in controlling the spread of activity in an epileptic network. Endocannabinoids are released on demand and their dysregulation has been described in several pathological conditions. Recurrent seizures may lead to an adverse reorganization of the EC system and impairment of its protective effect. In the current study, we tested the hypothesis that cerebrospinal fluid (CSF) concentrations of the endocannabinoids anandamide (AEA) and 2-arachidonoyl glycerol (2AG) are altered in epileptic dogs. Concentrations of AEA and total AG (sum of 2AG and 1AG) were measured in 40 dogs with idiopathic epilepsy and in 16 unaffected, healthy control dogs using liquid chromatography combined with tandem mass spectrometry. RESULTS: AEA and total AG were measured at 4.94 (3.18 - 9.17) pM and 1.43 (0.90 - 1.92) nM in epileptic dogs and at 3.19 (2.04 - 4.28) pM and 1.76 (1.08 - 2.69) nM in the control group, respectively (median, 25 - 75% percentiles in brackets). The AEA difference between epileptic and healthy dogs was statistically significant (p < 0.05). Values correlated with seizure severity and duration of seizure activity. Dogs with cluster seizures and/or status epilepticus and with seizure activity for more than six months displayed the highest EC concentrations. CONCLUSION: In conclusion, we present the first endocannabinoid measurements in canine CSF and confirm the hypothesis that the EC system is altered in canine idiopathic epilepsy.

Intragastric Carbon Dioxide Release Prolongs the Gastric Residence Time of Postprandially Administered Caffeine.

Sparkling water is said to increase gastric motility by the release of carbon dioxide, thereby potentially affecting the pharmacokinetics of orally administered drugs. The hypothesis of the present work was that the induction of gastric motility by intragastric release of carbon dioxide from effervescent granules could promote the mixing of drugs into the chyme under postprandial conditions, resulting in a prolonged drug absorption. For this purpose, an effervescent and a non-effervescent granule formulation of caffeine as a marker for gastric emptying were developed. In a three-way crossover study with twelve healthy volunteers, the salivary caffeine pharmacokinetics, after administration of the effervescent granules with still water and the administration of the non-effervescent granules with still and sparkling water, were investigated after intake of a standard meal. While the administration of the effervescent granules with 240 mL of still water led to a significantly prolonged gastric residence of the substance compared to the administration of the non-effervescent granules with 240 mL still water, the application of the non-effervescent granules with 240 mL sparkling water did not prolong gastric residence via mixing into caloric chyme. Overall, the mixing of caffeine into the chyme following the administration of the effervescent granules did not seem to be a motility mediated process.

Prednisolone Versus Colchicine for Acute Gout in Primary Care (COPAGO): protocol for a two-arm multicentre, pragmatic, prospective, randomized, double-blind, controlled clinical trial of prednisolone and colchicine for non-inferiority with a parallel group design.

BACKGROUND: Gout is the most common form of rheumatic disease in which monosodium urate crystals are deposited in the joints followed by acute inflammatory reactions. There are various approved drugs that can be prescribed for pain relief during an acute gout attack. However, to date, no direct comparison of efficacy of colchicine and prednisolone for the treatment of acute gout attacks has been investigated. Furthermore, the majority of previous research studies were not only conducted in tertiary centres but also excluded patients with common comorbidities due to contraindications to naproxen. METHODS: This pragmatic, prospective, double-blind, double-dummy, parallel-group, randomized, non-inferiority trial investigates whether prednisolone (intervention) is non-inferior to treatment with colchicine (active control) in patients with acute gout. Adult patients presenting with acute gout to their general practitioners in 60 practices across 3 university sites (Greifswald, Göttingen, and Würzburg) are eligible to participate in the study. Participants in the intervention group receive 30 mg prednisolone for 5 days. Those in the control group receive low-dose colchicine (day 1: 1.5 mg; days 2-5: 1 mg). The primary outcome is the absolute level of the most severe pain on day 3 (in the last 24 h) measured with an 11-item numerical rating scale. Day 0 is the day patients take their study medication for the first time. They are then asked to fill out a study diary the same time each day for pain quantification. Pain scores are used for comparison between the two medications. Secondary outcomes are average response to treatment, swelling, tenderness and physical function of the joint, patients' global assessment of treatment success, use of additional pain medication and non-pharmacological pain therapies. For safety reasons, potential side effects and course of systolic blood pressure are assessed. DISCUSSION: This trial will provide evidence on the effectiveness of pain reduction and side effects of colchicine and prednisolone in acute gout in primary care. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT05698680 first posted on January 26, 2023 (retrospectively registered). URL of trial registry record: https://clinicaltrials.gov/study/NCT05698680.

Quantification of endocannabinoids in biological systems by chromatography and mass spectrometry: a comprehensive review from an analytical and biological perspective.

The endocannabinoids anandamide (arachidonoyl ethanolamide, AEA) and 2-arachidonoyl glycerol (2AG) are physiologically occurring, biologically active compounds on CB(1) and CB(2) receptors with multiple physiological functions. AEA and 2AG have been identified and quantified in many mammalian biological fluids and tissues, such as human plasma, adipocytes, tissues and tissue microdialysates, at concentrations in the picomolar-to-nanomolar range under basal conditions. In this article, recently published chromatographic and mass spectrometric analytical methods, i.e., HPLC with fluorescence or ultraviolet detection, LC-MS, LC-MS/MS, GC-MS and GC-MS/MS, are reviewed and discussed, notably from the quantitative point of view. We focus on and emphasize the particular importance of blood sampling, sample storage and work-up including solvent and solid-phase extraction and derivatization procedures, matrix-effects, and stability of analytes. As 2AG spontaneously isomerizes to its CB(1)/CB(2) receptors biologically inactive 1-arachidonoyl glycerol (1AG) by acyl migration, this phenomenon and its particular importance for accurate quantification of 2AG are discussed in detail. Due to the electrical neutrality of AEA and 2AG their solvent extraction by toluene offers the least matrix-effect and minimum isomerization. LC-MS/MS is the most frequently used analytical technique for AEA and 2AG. At present, the utility of the GC-MS/MS methodology seems to be limited to AEA measurement in human plasma, bronchoalveolar liquid (BAL) and microdialysate samples. Despite great instrumental advances in the LC-MS/MS methodology, sampling and sample treatment remains one of the most crucial analytical steps in 2AG analysis. Extension of the LC-MS/MS methodology, for instance to microdialysate and BAL samples from clinical studies, is a big analytical challenge in endocannabinoid analysis in clinical settings. Currently available LC-MS/MS and GC-MS/MS methods should be useful to investigate the metabolism of AEA and 2AG beyond hydrolysis, i.e., by ß- and ω-oxidation pathways.

Randomized comparison of reduced fat and reduced carbohydrate hypocaloric diets on intrahepatic fat in overweight and obese human subjects.

UNLABELLED: Obesity-related hepatic steatosis is a major risk factor for metabolic and cardiovascular disease. Fat reduced hypocaloric diets are able to relieve the liver from ectopically stored lipids. We hypothesized that the widely used low carbohydrate hypocaloric diets are similarly effective in this regard. A total of 170 overweight and obese, otherwise healthy subjects were randomized to either reduced carbohydrate (n = 84) or reduced fat (n = 86), total energy restricted diet (-30% of energy intake before diet) for 6 months. Body composition was estimated by bioimpedance analyses and abdominal fat distribution by magnetic resonance tomography. Subjects were also submitted to fat spectroscopy of liver and oral glucose tolerance testing. In all, 102 subjects completed the diet intervention with measurements of intrahepatic lipid content. Both hypocaloric diets decreased body weight, total body fat, visceral fat, and intrahepatic lipid content. Subjects with high baseline intrahepatic lipids (>5.56%) lost ≈7-fold more intrahepatic lipids compared with those with low baseline values (<5.56%) irrespective of diet composition. In contrast, changes in visceral fat mass and insulin sensitivity were similar between subgroups, with low and high baseline intrahepatic lipids. CONCLUSION: A prolonged hypocaloric diet low in carbohydrates and high in fat has the same beneficial effects on intrahepatic lipid accumulation as the traditional low-fat hypocaloric diet. The decrease in intrahepatic lipids appears to be independent of visceral fat loss and is not tightly coupled with changes in whole body insulin sensitivity during 6 months of an energy restricted diet.

Stable isotope liquid chromatography-tandem mass spectrometry assay for fatty acid amide hydrolase activity.

Fatty acid amide hydrolase (FAAH) is the main enzyme responsible for the hydrolysis of the endocannabinoid anandamide (arachidonoyl ethanolamide, AEA) to arachidonic acid (AA) and ethanolamine (EA). Published FAAH activity assays mostly employ radiolabeled anandamide or synthetic fluorogenic substrates. We report a stable isotope liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for specific, sensitive, and high-throughput capable FAAH activity measurements. The assay uses AEA labeled with deuterium on the EA moiety (d4-AEA) as substrate and measures the specific reaction product tetradeutero-EA (d4-EA) and the internal standard ¹³C2-EA. Selected reaction monitoring of m/z 66→m/z 48 (d4-EA) and m/z 64→m/z 46 (¹³C2-EA) in the positive electrospray ionization mode after liquid chromatographic separation on a HILIC (hydrophilic interaction liquid chromatography) column is performed. The assay was developed and thoroughly validated using recombinant human FAAH (rhFAAH) and then was applied to human blood and dog liver samples. rhFAAH-catalyzed d4-AEA hydrolysis obeyed Michaelis-Menten kinetics (K(M)=12.3 µM, V(max)=27.6 nmol/min mg). Oleoyl oxazolopyridine (oloxa) was a potent, partial noncompetitive inhibitor of rhFAAH (IC50=24.3 nM). Substrate specificity of other fatty acid ethanolamides decreased with decreasing length, number of double bonds, and lipophilicity of the fatty acid skeleton. In human whole blood, we detected FAAH activity that was inhibited by oloxa.

Changes of blood endocannabinoids during anaesthesia: a special case for fatty acid amide hydrolase inhibition by propofol?

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: • Available data from animal studies suggest that the narcotic drug propofol interacts with the endocannabinoid system. Inhibition of enzymatic degradation of anandamide could explain some of the characteristics of propofol. Direct measurements have not been reported yet in humans. WHAT THIS STUDY ADDS: • Propofol does not change the time course of anandamide plasma concentrations during anaesthesia. Furthermore, propofol does not inhibit fatty acid amide hydrolase activity ex vivo or in vitro. Thus, specific characteristics of the narcotic drug propofol cannot be explained by peripheral inhibition of anandamide degradation in humans. AIMS: The aim of our study was to describe the time course of endocannabinoids during different anaesthesia protocols in more detail, and to challenge the hypothesis that propofol acts as a FAAH inhibitor. METHODS: Endocannabinoids were measured during the first hour of anaesthesia in 14 women and 14 men undergoing general anaesthesia with propofol and in 14 women and 14 men receiving thiopental/sevoflurane. We also incubated whole human blood samples ex vivo with propofol and the known FAAH inhibitor oloxa and determined FAAH enzyme kinetics. RESULTS: Plasma anandamide decreased similarly with propofol and thiopental/sevoflurane anaesthesia, and reached a nadir after 10 min. Areas under the curve for anandamide (mean and 95% CI) were 53.3 (47.4, 59.2) nmol l(-1) 60 min with propofol and 48.5 (43.1, 53.8) nmol l(-1) 60 min with thiopental/sevoflurane (P= NS). Anandamide and propofol plasma concentrations were not correlated at any time point. Ex vivo FAAH activity was not inhibited by propofol. Enzyme kinetics (mean ± SD) of recombinant human FAAH were K(m) = 16.9 ± 8.8 µmol l(-1) and V(max) = 44.6 ± 15.8 nmol mg(-1) min(-1) FAAH without, and K(m) = 16.6 ± 4.0 µmol l(-1) and V(max) = 44.0 ± 7.6 nmol mg( 1 ) min(-1) FAAH with 50 µmol l(-1) propofol (P= NS for both). CONCLUSIONS: Our findings challenge the idea that propofol anaesthesia and also propofol addiction are directly mediated by FAAH inhibition, but we cannot exclude other indirect actions on cannabinoid receptors.

Peripheral endocannabinoid microdialysis: in vitro characterization and proof-of-concept in human subjects.

In vivo endocannabinoid (EC) microdialysis has only seldom been performed, mostly in rodent brain tissue. Low solubility in aqueous media, adsorption to surfaces, and instability with co-present human serum albumin (HSA) are the major obstacles in EC microdialysis. The addition of hydroxypropyl-ß-cyclodextrine (HPCD) to the perfusion fluid has been previously described to facilitate lipid microdialysis, but the general biophysical properties of HPCD, especially with respect to peripheral EC microdialysis, have not been described before. We report on the characterization of EC microdialysis using an in vitro system using Ringer's solution with 10% HPCD as the perfusion fluid and with fatty acid-free HSA as the matrix fluid. The endocannabinoids anandamide (AEA) and 2-arachidonoyl glycerol (2AG) were measured using LC-MS/MS. AEA was stable in the perfusion and matrix fluids, whereas 2AG was only stable in the perfusion fluid. In the matrix fluid, 2AG underwent rapid isomerization to 1-arachidonoyl glycerol. A relative recovery of 3.5% for AEA was found with 10% HPCD in the perfusion fluid and a flow rate of 1 µL/min. For 2AG, a similar relative recovery of 3.5% was estimated. Since 2AG was found unstable in the matrix fluid, a reliable calculation of the relative recovery rates was not possible. Delivery and recovery experiments revealed unequal inward and outward EC transport across the microdialysis membrane. Contrary to usual microdialysis findings, we observed increasing recovery rates for AEA with increasing flow rates. Long equilibration times of several hours were necessary to obtain constant relative recovery rates. In a proof-of-concept study in humans, we collected AEA from subcutaneous abdominal adipose tissue employing the described methodology. Our study suggests that the microdialysis technique is not suitable for the exact quantification of tissue EC concentrations, but it allows for their rough estimation.