Oxidation of PTH: in vivo feature or effect of preanalytical conditions?

BACKGROUND: Posttranslational oxidation of parathyroid hormone (PTH) modifies its biological activity. Measurement of non-oxidized PTH (n-oxPTH) could be an improvement in assessing PTH status, as intact PTH may rather reflect oxidative stress. However, it is debated whether oxidation of PTH occurs in vivo, or whether it is mainly an in vitro artifact. The aim of this study was to investigate the influence of different preanalytical conditions on the oxidation of PTH within a wide range of plasma PTH concentrations and oxidation propensity. METHODS: n-oxPTH was separated from its oxidized form using an affinity column capturing the oxidized PTH. n-oxPTH was measured in eluate using commercially available PTH assays. The study included ethylenediaminetetraacetic acid plasma samples from 17 patients undergoing hemodialysis and 32 healthy subjects. We determined effects of storage temperature, time until centrifugation and freeze-thaw cycles. PTH and n-oxPTH concentrations were measured in each sample using six different immunoassays. RESULTS: n-oxPTH concentrations remained unchanged up to 180 min until centrifugation, two freeze-thaw cycles or after storage at -20°C or -80°C up to 79 days. Various methods for n-oxPTH and PTH measurements yielded highly comparable results, apart from standardization differences between various PTH and n-oxPTH assays. CONCLUSIONS: n-oxPTH concentrations were stable under our study conditions, indicating negligible ex vivo oxidation of PTH. In addition, PTH immunoassays have a different sensitivity for n-oxPTH than for total PTH. For this reason, the n-oxPTH/total PTH ratio cannot be used in absence of a n-oxPTH standard. Clinical implications of determining n-oxPTH require additional study.

Mandometer treatment not superior to treatment as usual for anorexia nervosa.

OBJECTIVE: A comparison of the efficacy of a novel treatment method for anorexia nervosa (AN), the Mandometer treatment (MT), with treatment as usual (TAU). METHOD: During treatment data were collected to determine weight recovery and outcome as assessed by the Morgan Russell Outcome Assessment Schedule (MROAS). RESULTS: After treatment 63% of the MT group and 85% of the TAU group had reached a normal weight level and both MT and TAU showed a good outcome on the MROAS (75 and 71%, respectively). After two years more MT than TAU patients were still in treatment and more MT patients had relapsed. DISCUSSION: The outcome for both treatments in our study were similar and comparable with, if not better than outcome data of other AN studies. MT is not superior to TAU in outcome results and in relapse rate during the first two years following admission for AN treatment.

Defective Levothyroxine Response in a Patient with Dyshormonogenic Congenital Hypothyroidism Caused by a Concurrent Pathogenic Variant in Thyroid Hormone Receptor-ß.

Background: Pituitary resistance to thyroid hormone (PRTH) is often seen in congenital hypothyroidism (CH), presenting as elevated thyrotropin (TSH) values despite (high-)normal thyroid hormone (TH) values achieved by levothyroxine treatment. In this study, we describe a girl with CH who was referred because of difficulties interpreting thyroid function tests. She was thought to have PRTH associated with CH, but genetic studies discovered a pathogenic variant in THRB, causing resistance to TH (RTH-ß). Methods: Clinical, genetic, and biochemical data of the proband's family were collected. Results: The 3-year-old girl was diagnosed with CH due to a homozygous pathogenic c.470del p.(Asn157Thrfs*3) SLC5A5 variant in the neonatal period. She needed a notably high levothyroxine dose to normalize TSH, leading to high free thyroxine levels. There were no signs of hyperthyroidism. Sequencing identified a heterozygous pathogenic c.947G>A p.(Arg316His) THRB variant. Conclusions: To our knowledge, this is the first report of concomitant SLC5A5 and THRB variants causing CH and RTH-ß.

AgRP(83-132) and SHU9119 differently affect activity-based anorexia.

Activity-based anorexia (ABA) mimics starvation and hyperactivity of anorexia nervosa patients in rats. Activation of the melanocortin (MC) system leads to hypophagia and increased energy expenditure in ad libitum fed rats. Therefore, activation of the MC system might underlie the development and propagation of ABA. Pro-opiomelanocortin (POMC) gene expression is normally decreased during negative energy balance. Strikingly, we found a transient up-regulation of POMC mRNA levels in the arcuate nucleus during the development of ABA, indicating a hyperactive MC system. However, wheel running and food intake were not influenced by treating ABA rats with the competitive antagonist SHU9119. This suggests that agonism of MC receptors by endogenous alpha-melanocyte-stimulating hormone (alpha-MSH) levels does not underlie ABA. Instead, treatment with the inverse agonist AgRP(83-132) did ameliorate signs of ABA. This implies that modulation of constitutive MC receptor activity rather than antagonizing putative alpha-MSH release contributes to the development and propagation of ABA.

To eat or not to eat; regulation by the melanocortin system.

The central melanocortin (MC) system is one of the best-characterized neuropeptidergic systems involved in the regulation of energy balance. This short review describes the role of the central MC system in feeding behavior. Pharmacological, anatomical and genetic studies show that activation of the MC system reduces meal size, whereas de-activation of the MC system increases meal size. Several brain regions, including distinct hypothalamic nuclei and the hindbrain, are involved in this process. Further dissection of MC pathways in feeding behavior is the subject of recent and probably future studies. As the MC system is involved in animal models of obesity and (possibly) anorexia, it appears that this is a target system for development of drugs for the treatment of disturbed human eating behavior.

Leptin treatment in activity-based anorexia.

BACKGROUND: Activity-based anorexia (ABA) is considered an animal model of anorexia nervosa (AN). In ABA, scheduled feeding together with voluntary access to a running wheel results in increased running wheel activity (RWA), hypophagia, and body weight loss. Previously it was shown that leptin treatment reduced semi-starvation-induced hyperactivity in rats. The present study was performed to confirm and extend this finding, to evaluate leptin's effect on energy balance in ABA. METHODS: The effects of chronic leptin treatment (intracerebroventricular, 4 microg/day) in ABA rats, ad libitum-fed running rats, and sedentary rats exposed to ad libitum feeding or scheduled feeding were investigated. RESULTS: Leptin treatment decreased RWA in ABA rats. Additionally, leptin treatment reduced food intake and increased energy expenditure by thermogenesis in ABA rats. Ad libitum-fed running/sedentary rats or food-restricted sedentary rats did not reduce activity after leptin treatment, whereas all leptin-treated rats showed hypophagia. Body temperature was slightly increased in leptin-treated food-restricted sedentary rats. CONCLUSIONS: Although leptin treatment reduced RWA in ABA rats, it also prevented hypothermia and decreased food intake. Altogether, this resulted in a stronger negative energy balance and body weight loss in leptin-treated ABA rats.

Olanzapine reduces physical activity in rats exposed to activity-based anorexia: possible implications for treatment of anorexia nervosa?

BACKGROUND: Anorexia nervosa (AN) patients often show extreme hypophagia and excessive physical activity. Activity-based anorexia (ABA) is considered an animal model of AN and mimics food restriction and hyperactivity in rats. This study investigated whether treatment with olanzapine (Zyprexa) reduces the development of ABA in rats. The effect of olanzapine treatment in AN patients was also evaluated in a small open-label study. METHODS: Rats were chronically (1 week) infused with olanzapine (7.5 mg/kg) and exposed to the ABA model or ad libitum feeding. Hyperactive AN patients were followed for up to 3 months of olanzapine treatment (5 mg/kg). RESULTS: Olanzapine treatment reduced development of ABA in rats by reducing running wheel activity, starvation-induced hypothermia and activation of the hypothalamus-pituitary-adrenal axis. Olanzapine treatment reduced activity levels of AN patients compared with untreated AN patients, without affecting body weight and plasma leptin levels. CONCLUSIONS: Olanzapine treatment reduced wheel running and thereby diminished development of ABA in rats. Olanzapine treatment also reduced physical activity in hyperactive AN patients in a small open-label study. These data support the need for controlled studies investigating the putative beneficial effects of olanzapine treatment in AN patients.

alpha-MSH enhances activity-based anorexia.

Activity-based anorexia (ABA) is considered an animal model of anorexia nervosa (AN). In ABA, scheduled feeding in combination with voluntary access to running wheels, results in hyperactivity, hypophagia, body weight loss and activation of the HPA axis. Since stimulation of the melanocortin (MC) system has similar effects, this system is a candidate system involved in ABA. Here it is shown that chronic alpha-MSH treatment enhances ABA by increasing running wheel activity (RWA), decreasing food intake and increasing HPA axis activation.

Voluntary access to a warm plate reduces hyperactivity in activity-based anorexia.

Activity-based anorexia (ABA) is considered an animal model of anorexia nervosa. In ABA, scheduled feeding in combination with voluntary wheel running leads to hyperactivity, reduced food intake, severe body weight loss and hypothermia. In this study it was investigated whether hyperactivity in ABA could be reduced by introducing a warm plate (which was voluntary accessible and did not influence ambient temperature) into a part of the cage. In ad libitum fed rats, the presence of the warm plate did not influence body temperature, running wheel activity (RWA), body weight or food intake. During ABA, however, rats preferred the warm plate and hypothermia was prevented, while hyperactivity and body weight loss were significantly reduced when compared to ABA rats without a plate. Correlation analysis revealed a significant association between basal body temperature and RWA during the light phase in ABA rats. However, there was no evidence that initiation of light phase RWA was a result of hypothermia. These data suggest that ABA rats prefer to prevent hypothermia passively by choosing a warm plate rather than actively regulating body temperature by hyperactivity.

Melanocortin system and eating disorders.

The melanocortin (MC) system is involved in the regulation of energy balance and in the development of obesity. Here we briefly review why we became interested in investigating whether the MC system - more particularly, the increased activity of the MC system - is also involved in disorders of negative energy balance. We provide evidence that suppression of increased MC receptor activity by treatment with the inverse agonist agouti-related peptide (AgRP) (83-132) rescues rats exposed to an animal model known as activity-based anorexia. Furthermore, we found a polymorphism, Ala67Thr AgRP, that was observed more frequently in anorexia nervosa.

[Specific IgE testing in food allergies]. / Specifiek-IgE-bepaling bij voedselallergie.

The interpretation of specific IgE test results in patients with suspected food allergies is not always straightforward. In fact, specific IgE test results for food allergens can provide physicians with more questions than answers. Based on the description of two patient cases, we discuss the interpretation of specific IgE test results for food allergens; the description includes the diagnostic process, cross-reactivity and component-resolved diagnostics.

Acute and chronic suppression of the central ghrelin signaling system reveals a role in food anticipatory activity.

Using the rodent activity-based anorexia (ABA) model that mimics clinical features of anorexia nervosa that include food restriction-induced hyperlocomotion, we found that plasma ghrelin levels are highly associated with food anticipatory behaviour, measured by running wheel activity in rats. Furthermore, we showed that ghrelin receptor (GHS-R1A) knockout mice do not anticipate food when exposed to the ABA model, unlike their wild type littermate controls. Likewise, food anticipatory activity in the ABA model was suppressed by a GHS-R1A antagonist administered either by acute central (ICV) injection to rats or by chronic peripheral treatment to mice. Interestingly, the GHS-R1A antagonist did not alter food intake in any of these models. Therefore, we hypothesize that suppression of the central ghrelin signaling system via GHS-R1A provides an interesting therapeutic target to treat hyperactivity in patients suffering from anorexia nervosa.

Do leptin and insulin signal adiposity?

The physiological regulation of adiposity is supposed to depend on endocrine 'adiposity signals' that inform the brain about the mass of the adipose tissue. Basal levels of insulin and leptin are widely accepted to be adiposity signals, and amylin, ghrelin and peptide YY have been hypothesized to be. Support for these ideas comes from associations between basal hormone levels and levels of adiposity, from demonstrations of receptors for these hormones in neural circuits supposed to regulate energy homeostasis, from neuropharmacological manipulations of the hormones' actions on eating and energy expenditure, and from the effects on energy balance in animals or people bearing mutations in these endocrine signaling pathways. This chapter focuses on only the first of these four types of evidence and only on insulin and leptin. We ask whether circulating levels of either hormone indeed encodes the necessary information to act as an adiposity signal. In considering this question, we emphasize the distinction between regulation of AT mass in steady versus dynamic states. We argue that the best experimental designs for identifying potentially effective adiposity signals involve situations in which the level of adiposity is changing as the organism responds to imposed perturbations. Traditionally, this is the type of design that most convincingly supports the idea that adiposity is actively regulated. Unfortunately, there are few of such studies for any of the hypothesized endocrine adiposity signals, and the evidence that is available does not strongly support the hypotheses. Therefore, we conclude that the question of how adiposity is signaled to the brain remains an open frontier in the physiology of energy homeostasis.

Validation of computed tomographic estimates of intra-abdominal and subcutaneous adipose tissue in rats and mice.

We characterized the accuracy, sensitivity, and reliability of computed tomographic (CT) estimates of intra-abdominal (IA) and subcutaneous (S) adipose tissue (AT) in rats and mice using the Aloka rodent CT. Here, we present the first comparisons of CT estimates of the weights of AT samples ex vivo to balance weights of the same samples, of CT estimates of AT weights in vivo to the weights of resected whole-body AT, and of CT estimates of the weights of pieces of AT inserted IA or S in vivo to the weights of the same pieces ex vivo. CT underestimated AT weight ex vivo by approximately 10%, and correction of the automated categorization of IAAT and SAT by Aloka software was required. After these adjustments, correlations (r) of CT estimates and balance weights of resected AT were > or =0.99 in rats and > or =0.92 in mice. CT was impressively sensitive: the 95% probability range of CT estimates of 10,000 mg AT inserts into rats was +/-780 mg and of 500 mg inserts into mice, +/-20 mg. Scans limited to the abdominal region correlated well (r > 0.90) with whole-body scan measures of IAAT and SAT in rats and with IAAT, but not SAT (r < 0.80), in mice. Sums of IAAT and SAT correlated well with body weight in rats (r > 0.90), but not in mice (r < 0.80). Coefficients of variance (CVs) of duplicate scans were <5%. We conclude that CT is a valid tool for studies of AT weight in rats and mice, especially when rapid throughput or longitudinal measures are desired.

Dopamine antagonism inhibits anorectic behavior in an animal model for anorexia nervosa.

Excessive physical activity is commonly described as symptom of Anorexia Nervosa (AN). Activity-based anorexia (ABA) is considered an animal model for AN. The ABA model mimics severe body weight loss and increased physical activity. Suppression of hyperactivity by olanzapine in anorectic patients as well as in ABA rats suggested a role of dopamine and/or serotonin in this trait. Here, we investigated the effect of a non-selective dopamine antagonist in the ABA model. A dose-response curve of chronic treatment with the non-selective dopaminergic antagonist cis-flupenthixol was determined in the ABA model. Treatment reduced activity levels in both ad libitum fed and food-restricted rats. Treated ABA rats reduced body weight loss and increased food intake. These data support a role for dopamine in anorexia associated hyperactivity. Interestingly, in contrast to leptin treatment, food-anticipatory activity still persists in treated ABA rats.

Mu-opioid receptor knockout mice show diminished food-anticipatory activity.

We have previously suggested that during or prior to activation of anticipatory behaviour to a coming reward, mu-opioid receptors are activated. To test this hypothesis schedule induced food-anticipatory activity in mu-opioid receptor knockout mice was measured using running wheels. We hypothesized that mu-knockout mice show little food-anticipatory activity. In wildtype mice we observed that food-anticipatory activity increased proportional to reduced food intake levels during daily scheduled food access, and thus reflects the animal's physiological need for food. mu-Knockout mice do not adjust their schedule induced running wheel behaviour prior to and during feeding time in the same way as wildtype mice; rather than showing more running wheel activity before than during feeding, they showed an equal amount of activity before and during feeding. As food-anticipatory activity is dependent on the mesolimbic dopamine system and mu-opioid receptors regulate dopaminergic activity, these data suggest a change in the dopamine system's activity in mu-knockout mice. As we observed that mu-knockout mice tended to show a stronger locomotor activity response than wildtype mice to the indirect dopamine agonist d-amphetamine, it appears that the dopaminergic system per se is intact and sensitive to activation. We found no differences in the expression of pro-opiomelanocortin, a precursor of endogenous endorphin, in the arcuate nucleus between mu-knockout mice and wildtype mice during restricted feeding, showing that the mu-opioid receptor does not regulate endogenous endorphin levels. These data overall suggest a role for mu-opioid receptors in adapting reward related behaviour to the requirements of the environment.