Correlations between the euthanasia and physician-assisted suicide rates and the non-assisted suicide rates at the municipal level in the Netherlands.

In the Netherlands, rates of euthanasia and physician-assisted suicide (henceforth "EPAS") display substantial variation at the municipal level. If a similar variation can be found in non-assisted suicide (henceforth "suicide"), this may enable us to establish a possible correlation between these variations. This cross-sectional study assessed proportions of suicide in the years 2013-2017 in The Netherlands. Negative binomial regression analysis was performed to identify potential explanatory variables and to calculate adjusted proportions. The magnitude of variation was calculated by ratios between the highest and lowest municipality proportions. Outliers were detected by Funnel Plots. A possible correlation between suicide and EPAS was calculated. From 2013-2017 the suicide rate between Dutch municipalities varied by a factor 6.7. This variation could only be partially explained by gender, political orientation, the availability of voluntary workers, and mobility limitations. No correlation was found with the variation in EPAS. A "waterbed effect" between suicides and EPAS-cases could not be confirmed. Advice for further research is given.

[Why regulation of active life termination in children remains problematic]. / Waarom een regeling levensbeëindiging bij kinderen problematisch blijft.

The case description regards a nine-years old patient who, as a consequence of a very serious epilepsy syndrome, several forms of comorbidity, severe developmental impairments, and the absence of meaningful contact with relatives, is felt to be in very serious suffering. Parents and physicians decide to end the child's life by withholding nutrition and hydration. Based on this case description and on the parents' experiences, its authors argue in favour of a stately regulated procedure for active life termination. In this comment I argue that the regulation of life termination without a patient request should remain a no-go area. If we allow life termination in children, there is no reason why we should not also allow such requests on behalf of incompetent patients such as handicapped adults and elderly patients with advanced dementia who have not issued an advance directive. Let the very rare cases in which a doctor sees no other option than to terminate an infant's life, remain in the realm of the non-regulated.

Challenges in pastoral care practice in euthanasia: A concept mapping study among Dutch protestant pastors.

This study explores the various difficulties that protestant pastors experience in the context of care for parishioners with a wish for euthanasia. In a reproducible and non-subjective way, using the concept mapping method, we cluster and rank-order 600 elements that pastors had mentioned as "difficult in caring for parishioners with a death wish" in an earlier survey study. The three clusters of items ranked as most difficult are connected with inappropriate care, doubts whether euthanasia in specific circumstances is justified, and disagreements between pastor and parishioner, including the emotional burden arising from those disagreements.

Understanding regional variation in euthanasia using geomedical frameworks: a critical ethical reflection.

Considerable geographical variation in the uptake of euthanasia has been reported: some municipalities in the Netherlands have a 25 times higher euthanasia rate than other municipalities. Current 'geomedical' frameworks for interpreting practice variation in health care utilization seem inadequately tailored to understand regional variation in morally controversial procedures such as euthanasia. The aim of this conceptual article is threefold: i) to add relevant medical ethical principles to current frameworks; ii) to provide a four-step ethical-geomedical model for the interpretation of geographical differences in the utilization of health care in general and for ethically controversial treatments in specific; iii) to gain better understanding of the existing geographical variation in the incidence of euthanasia by using this framework in our analysis.

Simultaneous Quantification of the Concentration and Carbon Isotopologue Distribution of Polar Metabolites in a Single Analysis by Gas Chromatography and Mass Spectrometry.

13C-isotope tracing is a frequently employed approach to study metabolic pathway activity. When combined with the subsequent quantification of absolute metabolite concentrations, this enables detailed characterization of the metabolome in biological specimens and facilitates computational time-resolved flux quantification. Classically, a 13C-isotopically labeled sample is required to quantify 13C-isotope enrichments and a second unlabeled sample for the quantification of metabolite concentrations. The rationale for a second unlabeled sample is that the current methods for metabolite quantification rely mostly on isotope dilution mass spectrometry (IDMS) and thus isotopically labeled internal standards are added to the unlabeled sample. This excludes the absolute quantification of metabolite concentrations in 13C-isotopically labeled samples. To address this issue, we have developed and validated a new strategy using an unlabeled internal standard to simultaneously quantify metabolite concentrations and 13C-isotope enrichments in a single 13C-labeled sample based on gas chromatography-mass spectrometry (GC/MS). The method was optimized for amino acids and citric acid cycle intermediates and was shown to have high analytical precision and accuracy. Metabolite concentrations could be quantified in small tissue samples (≥20 mg). Also, we applied the method on 13C-isotopically labeled mammalian cells treated with and without a metabolic inhibitor. We proved that we can quantify absolute metabolite concentrations and 13C-isotope enrichments in a single 13C-isotopically labeled sample.

A toolbox for the comprehensive analysis of small volume human intestinal samples that can be used with gastrointestinal sampling capsules.

Detailed knowledge on the fate of dietary components inside the human intestinal tract is lacking. Access to this inner world of digestion is now possible through novel human gastrointestinal sampling capsules. Due to the novelty of such devices, no methodology has been published to stabilise and analyse the resulting samples. A complicating factor is that excretion of such capsules in faeces may take days, while degradation of the dietary components continues. Therefore a stabilising reagent should be pre-loaded in the capsule to ensure the measurement of a representative sample. Considering the small volume of recovered samples, analytical methods must be optimized to collect as many data as possible from little material. We present a complete workflow for stabilising and analysing the fermentation status of dietary fibres in such samples, including microbiota, fibre degradation, and short chain fatty acids. The final quenching reagent was designed based on safety and effectiveness to inhibit fructo- and galacto-oligosaccharides degradation and short chain fatty acids production by human ileostomy microbiota, and subsequently validated in faecal samples. The final composition of the stock quenching reagent is 175 mM Tris, 525 mM NaCl, 35 mM EDTA, 12% SDS, and 8 M urea at pH 8.5.

Euthanasia in the Netherlands: a claims data cross-sectional study of geographical variation.

BACKGROUND: The annual incidence of euthanasia in the Netherlands as a percentage of all deaths rose from 1.9% in 1990 to 4.4% in 2017. Scarce literature on regional patterns calls for more detailed insight into the geographical variation in euthanasia and its possible explanations. OBJECTIVES: This paper (1) shows the geographical variation in the incidence of euthanasia over time (2013-2017); (2) identifies the associations with demographic, socioeconomic, preferential and health-related factors; and (3) shows the remaining variation after adjustment and discusses its meaning. DESIGN, SETTING AND METHODS: This cross-sectional study used national claims data, covering all healthcare claims during 12 months preceding the death of Dutch insured inhabitants who died between 2013 and 2017. From these claims all euthanasia procedures by general practitioners were selected (85% of all euthanasia cases). Rates were calculated and compared at three levels: 90 regions, 388 municipalities and 196 districts in the three largest Dutch cities. Data on possibly associated variables were retrieved from national data sets. Negative binomial regression analysis was performed to identify factors associated with geographical variation in euthanasia. RESULTS: There is considerable variation in euthanasia ratio. Throughout the years (2013-2017) the ratio in the three municipalities with the highest incidence was 25 times higher than in the three municipalities with the lowest incidence. Associated factors are age, church attendance, political orientation, income, self-experienced health and availability of voluntary workers. After adjustment for these characteristics a considerable amount of geographical variation remains (factor score of 7), which calls for further exploration. CONCLUSION: The Netherlands, with 28 years of legal euthanasia, experiences large-scale unexplained geographical variation in the incidence of euthanasia. Other countries that have legalised physician-assisted dying or are in the process of doing so may encounter similar patterns. The unexplained part of the variation may include the possibility that part of the euthanasia practice may have to be understood in terms of underuse, overuse or misuse.

Urinary 3-hydroxyisovaleryl carnitine excretion, protein energy malnutrition and risk of all-cause mortality in kidney transplant recipients: Results from the TransplantLines cohort studies.

BACKGROUND: Leucine is an essential amino acid and a potent stimulator of muscle protein synthesis. Since muscle wasting is a major risk factor for mortality in kidney transplant recipients (KTR), dietary leucine intake might be linked to long-term mortality. Urinary 3-hydroxyisovaleryl carnitine (3-HIC) excretion, a functional marker of marginal biotin deficiency, may also serve as a marker for dietary leucine intake. OBJECTIVE: In this study we aimed to investigate the cross-sectional determinants of urinary 3-HIC excretion and to prospectively investigate the association of urinary 3-HIC excretion with all-cause mortality in KTR. DESIGN: Urinary 3-HIC excretion and plasma biotin were measured in a longitudinal cohort of 694 stable KTR. Cross-sectional and prospective analyses were performed using ordinary least squares linear regression analyses and Cox regression analyses, respectively. RESULTS: In KTR (57% male, 53 ± 13 years, estimated glomerular filtration rate 45 ± 19 mL/min/1.73 m2), urinary 3-HIC excretion (0.80 [0.57-1.16] µmol/24 h) was significantly associated with plasma biotin (std. ß = -0.17; P < 0.001). Subsequent adjustment for potential covariates revealed urinary creatinine excretion (std. ß = 0.24; P < 0.001) and urinary urea excretion (std. ß = 0.53; P < 0.001) as the primary determinant of urinary 3-HIC excretion. Whereas plasma biotin explained only 1% of the variance in urinary 3-HIC excretion, urinary urea excretion explained >45%. During median follow-up for 5.4 [4.8-6.1] years, 150 (22%) patients died. Log2-transformed urinary 3-HIC excretion was inversely associated with all-cause mortality (HR: 0.52 [0.43-0.63]; P < 0.001). This association was independent of potential confounders. CONCLUSIONS: Urinary 3-HIC excretion more strongly serves as a marker of leucine intake than of biotin status. A higher urinary 3-HIC excretion is associated with a lower risk of all-cause mortality. Future studies are warranted to explore the underlying mechanism. TRIAL REGISTRATION ID: NCT02811835. TRIAL REGISTRATION URL: https://clinicaltrials.gov/ct2/show/NCT02811835.

A human-like bile acid pool induced by deletion of hepatic Cyp2c70 modulates effects of FXR activation in mice.

Bile acids (BAs) facilitate intestinal absorption of lipid-soluble nutrients and modulate various metabolic pathways through the farnesoid X receptor (FXR) and Takeda G-protein-coupled receptor 5. These receptors are targets for therapy in cholestatic and metabolic diseases. However, dissimilarities in BA metabolism between humans and mice complicate translation of preclinical data. Cytochrome P450 family 2 subfamily c polypeptide 70 (CYP2C70) was recently proposed to catalyze the formation of rodent-specific muricholic acids (MCAs). With CRISPR/Cas9-mediated somatic genome editing, we generated an acute hepatic Cyp2c70 knockout mouse model (Cyp2c70ako) to clarify the role of CYP2C70 in BA metabolism in vivo and evaluate whether its activity modulates effects of pharmacologic FXR activation on cholesterol homeostasis. In Cyp2c70ako mice, chenodeoxycholic acid (CDCA) increased at the expense of ßMCA, resulting in a more hydrophobic human-like BA pool. Tracer studies demonstrated that, in vivo, CYP2C70 catalyzes the formation of ßMCA primarily by sequential 6ß-hydroxylation and C7-epimerization of CDCA, generating αMCA as an intermediate metabolite. Physiologically, the humanized BA composition in Cyp2c70ako mice blunted the stimulation of fecal cholesterol disposal in response to FXR activation compared with WT mice, predominantly due to reduced stimulation of transintestinal cholesterol excretion. Thus, deletion of hepatic Cyp2c70 in adult mice translates into a human-like BA pool composition and impacts the response to pharmacologic FXR activation. This Cyp2c70ako mouse model may be a useful tool for future studies of BA signaling and metabolism that informs human disease development and treatment.

The effect of high compared with low dairy consumption on glucose metabolism, insulin sensitivity, and metabolic flexibility in overweight adults: a randomized crossover trial.

BACKGROUND: Dairy products contain many nutritious components that may benefit metabolic health. There are indications that glucose metabolism and insulin sensitivity, which are generally disturbed in overweight and obese individuals, may improve by increased dairy intake. This may also affect one's metabolic flexibility. OBJECTIVE: The aim of this study was to investigate the effects of high compared with low dairy intake on glucose metabolism, insulin sensitivity, and metabolic flexibility in overweight adults (aged 45-65 y). METHODS: In this randomized intervention study, subjects consumed a high- and a low-dairy diet [HDD (5-6 dairy portions) and LDD (≤1 dairy portion), respectively] for 6 wk in a crossover design, with a washout period of 4 wk. Dairy portions were 200 g semi-skimmed yoghurt, 30 g reduced-fat (30+) cheese, and 250 mL semiskimmed milk and buttermilk. After 6 wk, a 75-g oral-glucose-tolerance test (13C-labeled) and a subsequent fasting challenge were performed. Metabolic flexibility was studied by determining the respiratory quotient (RQ) using indirect calorimetry. Fasting and postprandial plasma concentrations of glucose and insulin were analyzed. The dual isotope technique enabled calculation of glucose kinetics. RESULTS: The study was completed by 45 overweight men and postmenopausal women [age 58.9 ± 4.3 y, BMI 27.9 ± 1.9 kg/m2 (mean ± SD)]. Fasting RQ and ΔRQ, reflecting metabolic flexibility, did not differ after both diets. Fasting glucose concentrations were similar, whereas fasting insulin concentrations were lower after the LDD (LDD: 8.1 ± 2.8 mU/L; HDD: 8.9 ± 3.3 mU/L; P = 0.024). This resulted in a higher HOMA-IR after the HDD (P = 0.027). Postprandial glucose and insulin responses as well as glucose kinetics were similar after both diets. CONCLUSIONS: The amount of dairy intake during a 6-wk period had a neutral effect on metabolic flexibility or postprandial glucose metabolism in middle-aged overweight subjects. More trials are needed to study the effects of specific dairy types and to differentiate between metabolic subgroups. This trial was registered at trialregister.nl as NTR4899.

Advance care planning conversations with palliative patients: looking through the GP's eyes.

BACKGROUND: Although it is often recommended that general practitioners (GPs) initiate advance care planning (ACP), little is known about their experiences with ACP. This study aimed to identify GP experiences when conducting ACP conversations with palliative patients, and what factors influence these experiences. METHODS: Dutch GPs (N = 17) who had participated in a training on timely ACP were interviewed. Data from these interviews were analysed using direct content analysis. RESULTS: Four themes were identified: ACP and society, the GP's perceived role in ACP, initiating ACP and tailor-made ACP. ACP was regarded as a 'hot topic'. At the same time, a tendency towards a society in which death is not a natural part of life was recognized, making it difficult to start ACP discussions. Interviewees perceived having ACP discussions as a typical GP task. They found initiating and timing ACP easier with proactive patients, e.g. who are anxious of losing capacity, and much more challenging when it concerned patients with COPD or heart failure. Patients still being treated in hospital posed another difficulty, because they often times are not open to discussion. Furthermore, interviewees emphasized that taking into account changing wishes and the fact that not everything can be anticipated, is of the utmost importance. Moreover, when patients are not open to ACP, at a certain point it should be granted that choosing not to know, for example about where things are going or what possible ways of care planning might be, is also a form of autonomy. CONCLUSIONS: ACP currently is a hot topic, which has favourable as well as unfavourable effects. As GPs experience difficulties in initiating ACP if patients are being treated in the hospital, future research could focus on a multidisciplinary ACP approach and the role of medical specialists in ACP. Furthermore, when starting ACP with palliative patients, we recommend starting with current issues. In doing so, a start can be made with future issues kept in view. Although the tension between ACP's focus on the patient's direction and the right not to know can be difficult, ACP has to be tailored to each individual patient.

One- vs two-phase extraction: re-evaluation of sample preparation procedures for untargeted lipidomics in plasma samples.

Lipidomics is a rapidly developing field in modern biomedical research. While LC-MS systems are able to detect most of the known lipid classes in a biological matrix, there is no single technique able to extract all of them simultaneously. In comparison with two-phase extractions, one-phase extraction systems are of particular interest, since they decrease the complexity of the experimental procedure. By using an untargeted lipidomics approach, we explored the differences/similarities between the most commonly used two-phase extraction systems (Folch, Bligh and Dyer, and MTBE) and one of the more recently introduced one-phase extraction systems for lipid analysis based on the MMC solvent mixture (MeOH/MTBE/CHCl3). The four extraction methods were evaluated and thoroughly compared against a pooled extract that qualitatively and quantitatively represents the average of the combined extractions. Our results show that the lipid profile obtained with the MMC system displayed the highest similarity to the pooled extract, indicating that it was most representative of the lipidome in the original sample. Furthermore, it showed better extraction efficiencies for moderate and highly apolar lipid species in comparison with the Folch, Bligh and Dyer, and MTBE extraction systems. Finally, the technical simplicity of the MMC procedure makes this solvent system highly suitable for automated, untargeted lipidomics analysis.

Acetyl-4'-phosphopantetheine is stable in serum and prevents phenotypes induced by pantothenate kinase deficiency.

Coenzyme A is an essential metabolite known for its central role in over one hundred cellular metabolic reactions. In cells, Coenzyme A is synthesized de novo in five enzymatic steps with vitamin B5 as the starting metabolite, phosphorylated by pantothenate kinase. Mutations in the pantothenate kinase 2 gene cause a severe form of neurodegeneration for which no treatment is available. One therapeutic strategy is to generate Coenzyme A precursors downstream of the defective step in the pathway. Here we describe the synthesis, characteristics and in vivo rescue potential of the acetyl-Coenzyme A precursor S-acetyl-4'-phosphopantetheine as a possible treatment for neurodegeneration associated with pantothenate kinase deficiency.

Transintestinal Cholesterol Transport Is Active in Mice and Humans and Controls Ezetimibe-Induced Fecal Neutral Sterol Excretion.

Except for conversion to bile salts, there is no major cholesterol degradation pathway in mammals. Efficient excretion from the body is therefore a crucial element in cholesterol homeostasis. Yet, the existence and importance of cholesterol degradation pathways in humans is a matter of debate. We quantified cholesterol fluxes in 15 male volunteers using a cholesterol balance approach. Ten participants repeated the protocol after 4 weeks of treatment with ezetimibe, an inhibitor of intestinal and biliary cholesterol absorption. Under basal conditions, about 65% of daily fecal neutral sterol excretion was bile derived, with the remainder being contributed by direct transintestinal cholesterol excretion (TICE). Surprisingly, ezetimibe induced a 4-fold increase in cholesterol elimination via TICE. Mouse studies revealed that most of ezetimibe-induced TICE flux is mediated by the cholesterol transporter Abcg5/Abcg8. In conclusion, TICE is active in humans and may serve as a novel target to stimulate cholesterol elimination in patients at risk for cardiovascular disease.

Identifying 24 h variation in the pharmacokinetics of levofloxacin: a population pharmacokinetic approach.

AIM: The objective of this study was to investigate whether the pharmacokinetics of orally administered levofloxacin show 24 h variation. Levofloxacin was used as a model compound for solubility and permeability independent absorption and passive renal elimination. METHODS: In this single centre, crossover, open label study, 12 healthy subjects received an oral dose of 1000 mg levofloxacin at six different time points equally divided over the 24 h period. Population pharmacokinetic modelling was used to identify potential 24 h variation in the pharmacokinetic parameters of this drug. RESULTS: The pharmacokinetics of levofloxacin could be described by a one compartment model with first order clearance and a transit compartment to describe drug absorption. The fit of the model was significantly improved when the absorption rate constant was described as a cosine function with a fixed period of 24 h, a relative amplitude of 47% and a peak around 08.00 h in the morning. Despite this variation in absorption rate constant, simulations of a once daily dosing regimen showed that tmax , Cmax and the area under the curve at steady-state were not affected by the time of drug administration. CONCLUSION: The finding that the absorption rate constant showed considerable 24 h variation may be relevant for drugs with similar physicochemical properties as levofloxacin that have a narrower therapeutic index. Levofloxacin, however, can be dosed without taking into account the time of day, at least in terms of its pharmacokinetics.

[The fringes of the euthanasia law]. / De rafelranden van de euthanasiewet.

A survey published in the Dutch Journal of Medicine (NTvG) shows that doctors asked to assess the suffering of patients making a request for euthanasia may give very different opinions. In this paper, I argue that this stems from the subjective character of the term 'unbearable' and that differences are bound to occur even more frequently in 'borderline' cases, such as those presented here. In my opinion, such differences do not justify a plea to abandon the assessment of unbearable suffering altogether. Rather, differences in assessment may serve as indications that the boundaries of the Dutch law on euthanasia and physician-assisted suicide are within sight. Moreover, it is argued that advance directives can only be seen as euthanasia requests in a legal sense if, at the time of issue of the directive, the patient was informed about his or her diagnosis and had discussed different treatment options with the physician. Such a directive may never serve as a generic request which is valid under hypothetical conditions.

Repeat analysis and incurred sample reanalysis: recommendation for best practices and harmonization from the global bioanalysis consortium harmonization team.

The A7 harmonization team (A7 HT), a part of the Global Bioanalysis Consortium (GBC), focused on reviewing best practices for repeat analysis and incurred sample reanalysis (ISR) as applied during regulated bioanalysis. With international representation from Europe, Latin America, North America, and the Asia Pacific region, the team first collated common practices and guidance recommendations and assessed their suitability from both a scientific and logistical perspective. Subsequently, team members developed best practice recommendations and refined them through discussions and presentations with industry experts at scientific meetings. This review summarizes the team findings and best practice recommendations. The few topics where no consensus could be reached are also discussed. The A7 HT recommendations, together with those from the other GBC teams, provide the basis for future international harmonization of regulated bioanalytical practices.

Peculiar observations in measuring testosterone in women treated with oral contraceptives supplemented with dehydroepiandrosterone (DHEA).

Total testosterone is considered to be decreased during the use of combined oral contraceptives. There is, however, considerable concern about the quality of testosterone assays, especially at low levels. We aimed to confirm testosterone levels measured by direct radioimmunoassay in a recent clinical trial with a state-of-the-art LC-MSMS method. Surplus specimens with known testosterone levels collected during the study (Clinical Trial Registration number ISRCTN06414473) were reanalyzed with an LC-MSMS method. This method was compared to another LC-MSMS method that had shown to concur excellently to a reference method. Follow-up experiments were designed to explain the results. In contrast to our expectation, LC-MSMS measurements did not corroborate the data obtained by radioimmunoassay. Subsequent experiments showed that this could be attributed to a strong dependency of the radioimmunoassay on SHBG. Testosterone results (n = 198) obtained by direct radioimmunoassay showed a negative correlation to SHBG levels (r = -0.676; p<0.001). By contrast, testosterone results obtained by LC-MSMS were not related to SHBG (r = 0.100; NS). In conclusion, our results indicate that total testosterone measurements during oral contraceptive use are unreliable when performed with assays sensitive to the SHBG concentration. The discrepancy with the literature can most likely be explained by the sensitivity of the immunoassay used to SHBG. Given the sharp increase in SHBG during the use of many oral contraceptives, total testosterone may not decrease, whereas its bioavailability, estimated by free testosterone levels, will be diminished. Studies aiming at restoration of testosterone homeostasis during oral contraception need to take this into account.