Martijn van Faassen

Measuring residual estrogen receptor availability during fulvestrant therapy in patients with metastatic breast cancer

UNLABELLED It is unknown whether the current dose of fulvestrant, an estrogen receptor (ER) antagonist, is sufficient for maximal ER downregulation in patients with metastatic breast cancer. We performed a feasibility study to assess ER availability before and during fulvestrant. Sixteen patients with ER-positive metastatic breast cancer underwent positron emission tomography/computed tomography (PET/CT) at baseline (scan 1), day 28 (scan 2), and day 84 (scan 3) to monitor tumor [(18)F]fluoroestradiol (FES) uptake. Incomplete reduction in ER availability was predefined as <75% decrease in median tumor FES uptake and a residual standardized uptake value (SUVmax) of ≥1.5. In total, 131 FES-positive lesions were identified (median SUVmax of 2.9; range, 1.7-6.5). The median change in patients during fulvestrant treatment was -85% at scan 2, but varied widely (-99% to +60%). Fulvestrant reduced tumor FES uptake incompletely at scan 2 in 6 (38%) of the 16 patients, which was associated with early progression. SIGNIFICANCE Serial imaging of tumor estrogen uptake by FES-PET can give insight into the dose needed for ER antagonists to completely abolish ER. FES-PET showed significant residual ER availability in tumors during fulvestrant therapy in 38% of patients, which was associated with early progression.

Intra- and inter-individual variability of longitudinal daytime melatonin secretion patterns in depressed and non-depressed individuals

Disrupted melatonin secretion is regarded as a link between circadian rhythm and major depression, but results have been contradictory. We hypothesize that this might be due to averaging across individuals and too short measurements periods. In this study, pair-matched depressed and non-depressed individuals sampled their saliva three times a day, 30 days, in their natural environment. The depressed group showed significantly more variance and higher melatonin levels (p < 0.05). Substantial interindividual heterogeneity and day-to-day variability was found. The individual time-series approach allowed us to reveal this variability. Important information remains unnoticed when analyzing melatonin only at the group level.

Cortisol and α-Amylase Secretion Patterns between and within Depressed and Non-Depressed Individuals

Objectives Associations between biological stress markers and depression are inconsistent across studies. We assessed whether inter- and intra-individual variability explain these inconsistencies. Methods Pair-matched depressed and non-depressed participants (N = 30) collected saliva thrice a day for 30 days, resulting in 90 measurements per individual. The relationships between measures of stress-system function and depression were examined at the group level by means of mixed model analyses, and at the individual level by means of pair-matched comparisons. The analyses were repeated after adjusting for time-varying lifestyle factors by means of time-series regression analyses. Results Cortisol and α-amylase levels were higher, the α-amylase/cortisol ratio larger, and the daily cortisol slope steeper in the depressed compared to the non-depressed group. Adjusting for lifestyle factors and antidepressant use reduced the associations under study. In 40%–60% of the matched comparisons, depressed individuals had higher cortisol and α-amylase levels, a larger α-amylase/cortisol ratio, and a steeper daily slope than their non-depressed match, regardless of adjustment. Conclusions Our group-level findings were mostly in line with the literature but generalization to individuals appeared troublesome. Findings of studies on this topic should be interpreted with care, because in clinical practice the focus is on individuals instead of groups.

Large Neutral Amino Acid Supplementation Exerts Its Effect through Three Synergistic Mechanisms: Proof of Principle in Phenylketonuria Mice

Background Phenylketonuria (PKU) was the first disorder in which severe neurocognitive dysfunction could be prevented by dietary treatment. However, despite this effect, neuropsychological outcome in PKU still remains suboptimal and the phenylalanine-restricted diet is very demanding. To improve neuropsychological outcome and relieve the dietary restrictions for PKU patients, supplementation of large neutral amino acids (LNAA) is suggested as alternative treatment strategy that might correct all brain biochemical disturbances caused by high blood phenylalanine, and thereby improve neurocognitive functioning. Objective As a proof-of-principle, this study aimed to investigate all hypothesized biochemical treatment objectives of LNAA supplementation (normalizing brain phenylalanine, non-phenylalanine LNAA, and monoaminergic neurotransmitter concentrations) in PKU mice. Methods C57Bl/6 Pah-enu2 (PKU) mice and wild-type mice received a LNAA supplemented diet, an isonitrogenic/isocaloric high-protein control diet, or normal chow. After six weeks of dietary treatment, blood and brain amino acid and monoaminergic neurotransmitter concentrations were assessed. Results In PKU mice, the investigated LNAA supplementation regimen significantly reduced blood and brain phenylalanine concentrations by 33% and 26%, respectively, compared to normal chow (p<0.01), while alleviating brain deficiencies of some but not all supplemented LNAA. Moreover, LNAA supplementation in PKU mice significantly increased brain serotonin and norepinephrine concentrations from 35% to 71% and from 57% to 86% of wild-type concentrations (p<0.01), respectively, but not brain dopamine concentrations (p = 0.307). Conclusions This study shows that LNAA supplementation without dietary phenylalanine restriction in PKU mice improves brain biochemistry through all three hypothesized biochemical mechanisms. Thereby, these data provide proof-of-concept for LNAA supplementation as a valuable alternative dietary treatment strategy in PKU. Based on these results, LNAA treatment should be further optimized for clinical application with regard to the composition and dose of the LNAA supplement, taking into account all three working mechanisms of LNAA treatment.

Measurement of plasma vitamin K1 (phylloquinone) and K2 (menaquinones-4 and -7) using HPLC-tandem mass spectrometry.

Abstract Background: Given the growing interest in the health benefits of vitamin K, there is great need for development of new high-throughput methods for quantitative determination of vitamin K in plasma. We describe a simple and rapid method for measurement of plasma vitamin K1 (phylloquinone [PK]) and K2 (menaquinones [MK]-4 and -7). Furthermore, we investigated the association of fasting plasma vitamin K with functional vitamin K insufficiency in renal transplant recipients (RTR). Methods: We used HPLC-tandem mass spectrometry with atmospheric pressure chemical ionization for measurement of plasma PK, MK-4, and MK-7. Solid-phase extraction was used for sample clean-up. Mass spectrometric detection was performed in multiple reaction monitoring mode. Functional vitamin K insufficiency was defined as plasma desphospho-uncarboxylated matrix Gla protein (dp-ucMGP) >500 pmol/L. Results: Lower limits of quantitation were 0.14 nmol/L for PK and MK-4 and 4.40 nmol/L for MK-7. Linearity up to 15 nmol/L was excellent. Mean recoveries were >92%. Fasting plasma PK concentration was associated with recent PK intake (ρ=0.41, p=0.002) and with plasma MK-4 (ρ=0.49, p<0.001). Plasma PK (ρ=0.38, p=0.003) and MK-4 (ρ=0.46, p<0.001) were strongly correlated with plasma triglyceride concentrations. Furthermore, we found that MK-4-triglyceride ratio, but not PK-triglyceride ratio, was significantly associated with functional vitamin K insufficiency (OR 0.22 [0.07–0.70], p=0.01) in RTR. Conclusions: The developed rapid and easy-to-use LC-MS/MS method for quantitative determination of PK, MK-4, and MK-7 in human plasma may be a good alternative for the labor-intensive and time-consuming LC-MS/MS methods and enables a higher sample throughput.

Evidence against a role of serotonin in liver regeneration in humans

committees according to criteria outlined by the National Academy of Sciences (BMWF-66.010/0045-II/10b/2010 and GZ-BMWF-66. 010/0012-II/3b/2014). Potential conflict of interest: Prof. Trauner consults for, advises, is on the speakers’ bureau of, and received grants from Falk. He consults for and advises Albireo, Genfit, Intercept, and Phenex. He is on the speakers’ bureaus of Gilead, MSD, and Roche. He received grants from Intercept and Albireo.

The Behavioral Consequence of Phenylketonuria in Mice Depends on the Genetic Background.

To unravel the role of gene mutations in the healthy and the diseased state, countless studies have tried to link genotype with phenotype. However, over the years, it became clear that the strain of mice can influence these results. Nevertheless, identical gene mutations in different strains are often still considered equals. An example of this, is the research done in phenylketonuria (PKU), an inheritable metabolic disorder. In this field, a PKU mouse model (either on a BTBR or C57Bl/6 background) is often used to examine underlying mechanisms of the disease and/or new treatment strategies. Both strains have a point mutation in the gene coding for the enzyme phenylalanine hydroxylase which causes toxic concentrations of the amino acid phenylalanine in blood and brain, as found in PKU patients. Although the mutation is identical and therefore assumed to equally affect physiology and behavior in both strains, no studies directly compared the two genetic backgrounds to test this assumption. Therefore, this study compared the BTBR and C57Bl/6 wild-type and PKU mice on PKU-relevant amino acid- and neurotransmitter-levels and at a behavioral level. The behavioral paradigms were selected from previous literature on the PKU mouse model and address four domains, namely (1) activity levels, (2) motor performance, (3) anxiety and/or depression-like behavior, and (4) learning and memory. The results of this study showed comparable biochemical changes in phenylalanine and neurotransmitter concentrations. In contrast, clear differences in behavioral outcome between the strains in all four above-mentioned domains were found, most notably in the learning and memory domain. The outcome in this domain seem to be primarily due to factors inherent to the genetic background of the mouse and much less by differences in PKU-specific biochemical parameters in blood and brain. The difference in behavioral outcome between PKU of both strains emphasizes that the consequence of the PAH mutation is influenced by other factors than Phe levels alone. Therefore, future research should consider these differences when choosing one of the genetic strains to investigate the pathophysiological mechanism underlying PKU-related behavior, especially when combined with new treatment strategies.

Serotonin-2C antagonism augments the effect of citalopram on serotonin and dopamine levels in the ventral tegmental area and nucleus accumbens.

Many patients with major depression do not respond to selective serotonin reuptake inhibitors (SSRIs). Lack of response could be due to inhibition of dopamine (DA) release by serotonin (5-HT) through 5-HT2C receptors. Combining an SSRI with a 5-HT2C antagonist may result in improved efficacy by causing simultaneous increases of 5-HT and DA. In order to test this augmentation strategy, male Wistar rats were treated (s.c.) with an acute dose of the SSRI citalopram (Cit, 5 mg/kg), the 5-HT2C antagonist SB 242084 (SB, 2 mg/kg), or Cit + SB, and the effect on 5-HT and DA release in the nucleus accumbens (NAcc) was assessed by microdialysis. In a separate experiment, animals were treated with vehicle, Cit (20 mg/kg/d), SB (2 mg/kg/d) or Cit + SB for a period of 2 days (s.c.), and the impact on the release of 5-HT and DA in the ventral tegmental area (VTA) and NAcc was studied. On the day of microdialysis, 5-HT2C receptor sensitivity was assessed with an SB challenge. Acutely administered Cit + SB increased 5-HT release in the NAcc more than Cit alone. SB alone increased DA release in the NAcc (not in the VTA), but when administered together with Cit, this effect was abolished. A 2-day treatment with Cit or Cit + SB increased 5-HT release in both VTA and NAcc. Combining Cit with SB augmented the effect of Cit in the VTA. DA release in VTA and NAcc was only significantly increased after 2-days of treatment with Cit + SB. In conclusion, Cit + SB had synergistic effects on 5-HT and DA release after 2-days of treatment, probably related to a decreased tonic inhibition of DA release via 5-HT2C receptors. Regional differences occur and future studies should elucidate if this augmentation strategy is beneficial at the behavioral level.

Relationship between plasma and salivary melatonin and cortisol investigated by LC-MS/MS

Abstract Background: Disturbance of the circadian rhythm has been associated with disease states, such as metabolic disorders, depression and cancer. Quantification of the circadian markers such as melatonin and cortisol critically depend on reliable and reproducible analytical methods. Previously, melatonin and cortisol were primarily analyzed separately, mainly using immunoassays. Methods: Here we describe the validation and application of a high-throughput liquid chromatography in combination with mass spectrometry (LC-MS/MS) method for the combined analysis of melatonin and cortisol in plasma and saliva. The LC-MS/MS method was validated according to international validation guidelines. We used this method to analyze total plasma, free plasma (as obtained by equilibrium dialysis) and saliva melatonin and cortisol in healthy adults. Results: Validation results for plasma and saliva melatonin and cortisol were well within the international validation criteria. We observed no difference between saliva collected by passive drooling or Salivette. Moreover, we noted a significant difference in saliva vs. free plasma melatonin. We observed on average 36% (95% CI: 4%–60%) higher salivary melatonin levels in comparison to free plasma melatonin, suggestive of local production of melatonin in the salivary glands. Conclusions: The novel outcome of this study is probably due to the high precision of our LC-MS/MS assay. These outcomes illustrate the added value of accurate and sensitive mass spectrometry based methods for the quantification of neuroendocrine biomarkers.

Identification of an evolutionary conserved structural loop that is required for the enzymatic and biological function of tryptophan 2,3-dioxygenase

The enzyme TDO (tryptophan 2,3-dioxygenase; TDO-2 in Caenorhabditis elegans) is a potential therapeutic target to cancer but is also thought to regulate proteotoxic events seen in the progression of neurodegenerative diseases. To better understand its function and develop specific compounds that target TDO we need to understand the structure of this molecule. In C. elegans we compared multiple different CRISPR/Cas9-induced tdo-2 deletion mutants and identified a motif of three amino acids (PLD) that is required for the enzymatic conversion of tryptophan to N-formylkynurenine. Loss of TDO-2’s enzymatic activity in PDL deletion mutants was accompanied by an increase in motility during aging and a prolonged lifespan, which is in line with the previously observed phenotypes induced by a knockdown of the full enzyme. Comparison of sequence structures suggests that blocking this motif might interfere with haem binding, which is essential for the enzyme’s activity. The fact that these three residues are situated in an evolutionary conserved structural loop of the enzyme suggests that the findings can be translated to humans. The identification of this specific loop region in TDO-2–essential for its catalytic function–will aid in the design of novel inhibitors to treat diseases in which the TDO enzyme is overexpressed or hyperactive.