Falsely decreased thyroglobulin levels in a patient with differentiated thyroid carcinoma.

We describe a case of a patient with metastasized differentiated thyroid carcinoma who was treated with total thyroidectomy followed-up by radioactive iodine treatment. During treatment and follow-up the thyroglobulin levels were assayed which surprisingly did not match the clinical condition. An analytical flaw was suspected. Re-analysis in the laboratory showed the presence of a high dose hook effect (HDH), resulting in falsely low Tg levels. This case shows that HDH effects in immunoassays, like the thyroglobulin assay, still exist in daily practice. Discordance between laboratory results and clinical condition underlines the importance of short lines of communication between clinical chemists and medical doctors.

Effects of repeated freeze-thaw cycles on endocrine parameters in plasma and serum.

Background Repeated freezing and thawing of plasma (or serum) may influence the stability of plasma (or serum) constituents. Despite the alarming warnings from commercial manuals that freeze-thaw cycles affect the stability of hormones in plasma (or serum), surprisingly little, consistent information about this concept is available in literature. Methods We studied the stability of 15 endocrine parameters (adrenocorticotropic hormone, osteocalcin, plasma renin activity, α-subunits, cortisol binding globulin, glucagon, inhibin B, fT4, TT4, TT3, rT3, TBG, TSH, chromogranin A and thyroglobulin upon repeated freeze-thaw cycles in plasma (or serum) samples from 10 volunteers. Blood was collected by venipuncture and after centrifugation and aliquoting, all samples were frozen at -20℃. Aliquots were thawed up to four times and changes in concentrations of endocrine parameters were compared to baseline condition. Results Repeated freeze-thaw cycling resulted in significant and relevant increases of plasma renin activity and a small decrease of adrenocorticotropic hormone. Conclusions For most of the analysed endocrine parameters, we found no effects of multiple freeze-thaw cycles despite alarming notifications in assay manuals. Plasma renin activity was the only endocrine parameter that showed significant and relevant changes following repeated freeze-thaw cycling.

Validation of a liquid chromatographic method for the pharmaceutical quality control of products containing elacridar$ / 药物分析学报

Many anticancer drugs have an impaired bioavailability and poor brain penetration because they are sub-strates to drug efflux pumps such as P-glycoprotein and Breast Cancer Resistance Protein. Elacridar is a strong inhibitor of these two drug efflux pumps and therefore has great potential to improve oral absorption and brain penetration of many anticancer drugs. Currently, a clinical formulation of elacridar is unavailable and therefore the pharmaceutical development of a drug product is highly warranted. This also necessitates the availability of an analytical method for its quality control. A reverse-phase high-performance liquid chro-matographic method with ultraviolet detection was developed for the pharmaceutical quality control of products containing elacridar as the active pharmaceutical ingredient. The analytical method was validated for linearity, accuracy, precision, selectivity, carry-over, stability of stock and reference solutions, stability of the final extract, stability-indicating capability and impurity testing. We found that elacridar is unstable in aqueous solutions that are exposed to light because a hydroxylation product of elacridar is formed. Therefore, sample solutions with elacridar must be protected from light.

Changes in Farr radioimmunoassay and EliA fluorescence immunoassay anti-dsDNA in relation to exacerbation of SLE.

Measuring anti-dsDNA levels could support treatment adjustment during follow-up of patients with systemic lupus erythematosus (SLE). We investigated whether patients with exacerbations of SLE showed changes in anti-double-stranded DNA (anti-dsDNA) levels prior to the exacerbation using the Farr and EliA assay and examined which assay showed highest specificity and predictive value for exacerbations. Changes in anti-dsDNA of ≥ 25% prior to exacerbation were considered of clinical significance. Exacerbations were retrospectively abstracted from medical records. Eighteen of 48 patients showed one or more exacerbations. We found 22 exacerbations with complete lab work-up, all accompanied by ≥ 25% change in anti-dsDNA in one or both assays. Only 10 exacerbations showed concordant changes in anti-dsDNA in both assays. Changes in anti-dsDNA had a low predictive value for exacerbations of SLE, but the specificity of anti-dsDNA changes for patients with exacerbations was higher for EliA than Farr. We conclude that despite the limited relation between anti-dsDNA changes and exacerbations of SLE, anti-dsDNA testing could still support clinical decision making when used in the correct setting. We conclude that EliA is preferable over Farr for assaying anti-dsDNA during follow-up of patients with SLE because of higher specificity, less "hands-on" time and absence of radioactivity.

Neurobiology driving hyperactivity in activity-based anorexia.

Hyperactivity in anorexia nervosa is difficult to control and negatively impacts outcome. Hyperactivity is a key driving force to starvation in an animal model named activity-based anorexia (ABA). Recent research has started unraveling what mechanisms underlie this hyperactivity. Besides a general increase in locomotor activity that may be an expression of foraging behavior and involves frontal brain regions, the increased locomotor activity expressed before food is presented (food anticipatory behavior or FAA) involves hypothalamic neural circuits. Ghrelin plays a role in FAA, whereas decreased leptin signaling is involved in both aspects of increased locomotor activity. We hypothesize that increased ghrelin and decreased leptin signaling drive the activity of dopamine neurons in the ventral tegmental area. In anorexia nervosa patients, this altered activity of the dopamine system may be involved not only in hyperactivity but also in aberrant cognitive processing related to food.

Hepatic-portal vein infusions of glucagon-like peptide-1 reduce meal size and increase c-Fos expression in the nucleus tractus solitarii, area postrema and central nucleus of the amygdala in rats.

We recently reported that brief, remotely controlled intrameal hepatic-portal vein infusions of glucagon-like peptide-1 (GLP-1) reduced spontaneous meal size in rats. To investigate the neurobehavioural correlates of this effect, we equipped male Sprague-Dawley rats with hepatic-portal vein catheters and assessed (i) the effect on eating of remotely triggered infusions of GLP-1 (1 nmol/kg, 5 min) or vehicle during the first nocturnal meal after 3 h of food deprivation and (ii) the effect of identical infusions performed at dark onset on c-Fos expression in several brain areas involved in the control of eating. GLP-1 reduced (P < 0.05) the size of the first nocturnal meal and increased its satiety ratio. Also, GLP-1 increased (P < 0.05) the number of c-Fos-expressing cells in the nucleus tractus solitarii, the area postrema and the central nucleus of the amygdala, but not in the arcuate or paraventricular hypothalamic nuclei. These data suggest that the nucleus tractus solitarii, the area postrema and the central nucleus of the amygdala play a role in the eating-inhibitory actions of GLP-1 infused into the hepatic-portal vein; it remains to be established whether activation of these brain nuclei reflect satiation, aversion, or both.

Leptin's effect on hyperactivity: potential downstream effector mechanisms.

Up to 80% of patients with Anorexia Nervosa (AN) demonstrate hyperactivity. Hyperactivity counteracts weight gain during treatment and is associated with poor outcome of the disease. We hypothesized that hyperactivity in AN patients has a neurobiological basis and used an animal model-based translational approach to gain insight in mechanisms underlying this hyperactivity. Previously we and others showed that leptin treatment attenuates hyperactivity in the rat activity-based anorexia (ABA) model. The mechanisms involved in this process are, however, unknown. Here we describe potential downstream effector mechanisms involved in the attenuation of hyperactivity by leptin treatment in ABA rats.

The impact of hyperactivity and leptin on recovery from anorexia nervosa.

In anorexia nervosa (AN), hyperactivity is observed in about 80% of patients and has been associated with low leptin levels in the acute stage of AN and in anorexia animal models. To further understand the importance of this correlation in AN, we investigated the relationship between hypoleptinaemia and hyperactivity in AN patients longitudinally and assessed their predictive value for recovery. Body weight, activity levels, and serum leptin levels were assessed in adolescents and adult AN patient groups at the start and during treatment, up to a year. In the adolescent group, initial leptin and activity levels were correlated. This negative correlation changes over time into a positive correlation with physiological recovery. Treatment outcome in both groups could be predicted by initial BMI and leptin levels but not by activity levels. No major relationship of activity with the course of recovery was detected, suggesting that in contrast to the acute stage of the disease, leptin and activity levels during the recovery process are dissociated.

The MC4 receptor and control of appetite.

Mutations in the human melanocortin (MC)4 receptor have been associated with obesity, which underscores the relevance of this receptor as a drug target to treat obesity. Infusion of MC4R agonists decreases food intake, whereas inhibition of MC receptor activity by infusion of an MC receptor antagonist or with the inverse agonist AgRP results in increased food intake. This review addresses the role of the MC system in different aspects of feeding behaviour. MC4R activity affects meal size and meal choice, but not meal frequency, and the type of diet affects the efficacy of MC4R agonists to reduce food intake. The central sites involved in the different aspects of feeding behaviour that are affected by MC4R signalling are being unravelled. The paraventricular nucleus plays an important role in food intake per se, whereas MC signalling in the lateral hypothalamus is associated with the response to a high fat diet. MC4R signalling in the brainstem has been shown to affect meal size. Further genetic, behavioural and brain-region specific studies need to clarify how the MC4R agonists affect feeding behaviour in order to determine which obese individuals would benefit most from treatment with these drugs. Application of MCR agonists in humans has already revealed side effects, such as penile erections, which may complicate introduction of these drugs in the treatment of obesity.

The appetite suppressant d-fenfluramine reduces water intake, but not food intake, in activity-based anorexia.

Biochemical, genetic and imaging studies support the involvement of the serotonin (5-HT) system in anorexia nervosa. Activity-based anorexia (ABA) is considered an animal model of anorexia nervosa, and combines scheduled feeding with voluntary running wheel activity (RWA). We investigated the effect of d-fenfluramine (d-FEN) treatment on development and propagation of ABA. d-FEN is an appetite suppressant and acts on 5-HT(2C) receptors that are located on pro-opiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus. Since stimulation activation of the melanocortin system stimulates ABA, we hypothesized that d-FEN treatment enhances the development and propagation of ABA. Rats were exposed to the ABA model and chronically infused with d-FEN. Unexpectedly, d-FEN-treated ABA rats did not reduce food intake or increase wheel running as compared with vehicle-treated ABA rats. Furthermore d-FEN treatment did not affect body weight loss, hypothalamus-pituitary-adrenal axis activation, or starvation-induced hypothermia in ABA rats. POMC mRNA levels in d-FEN-treated rats were not different from vehicle-treated rats after one week of exposure to the ABA paradigm. However, d-FEN-treated ABA rats showed hypodypsia and increased plasma osmolality and arginine-vasopressin expression levels in the hypothalamus. We conclude that d-FEN treatment does not enhance ABA under the experimental conditions of this study, but strongly reduces water intake in ABA rats.

Hypothalamic neuropeptide expression following chronic food restriction in sedentary and wheel-running rats.

When rats are given access to a running-wheel in combination with food restriction, they will become hyperactive and decrease their food intake, a paradoxical phenomenon known as activity-based anorexia (ABA). Little is known about the regulation of the hypothalamic neuropeptides that are involved in the regulation of food intake and energy balance during the development of ABA. Therefore, rats were killed during the development of ABA, before they entered a state of severe starvation. Neuropeptide mRNA expression levels were analysed using quantitative real-time PCR on punches of separate hypothalamic nuclei. As is expected in a state of negative energy balance, expression levels of agouti-related protein (AgRP) and neuropeptide Y (NPY) were increased 5-fold in the arcuate nucleus (ARC) of food-restricted running ABA rats vs 2-fold in sedentary food-restricted controls. The co-regulated expression of AgRP and NPY strongly correlated with relative body weight and white adipose tissue mass. Arcuate expression of pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) was reduced 2-fold in the ABA group. In second-order neurons of the lateral hypothalamic area (LHA), melanin-concentrating hormone (MCH) mRNA expression was upregulated 2-fold in food-restricted running rats, but not in food-restricted sedentary controls. Prepro-orexin, CART and corticotropin-releasing hormone expression levels in the LHA and the paraventricular nucleus (PVN) were unchanged in both food-restricted groups. From this study it was concluded that during the development of ABA, neuropeptides in first-order neurons in the ARC and MCH in the LHA are regulated in an adequate response to negative energy balance, whereas expression levels of the other studied neuropeptides in secondary neurons of the LHA and PVN are unchanged and are probably regulated by factors other than energy status alone.

[Endovascular aneurysm treatment: Radiological and macropathological findings of the endoluminal surface of modular stent grafts]. / Endovaskuläre Aneurysmatherapie: Radiologisch-makropathologische Befundkorrelation der endoluminalen Oberfläche einer modularen Stentprothese.

PURPOSE: Characterization of the endoluminal surface of a modular stent graft and correlation of macropathological findings with results of radiological methods. METHODS: Aneurysms of the infrarenal aorta were created in 36 mongrel FBI dogs using autologous material. Endovascular treatment was performed with modular stent grafts using two polyester-covered nitinol stents connected with overlap within the aneurysm. Follow-up was 1 and 6 weeks, and 6 months for 12 animals, respectively. Results were documented using sonography, intravascular ultrasound (IVUS), spiral CT, MRI, and digital subtraction angiography (DSA). After fixation, the aorta was prepared and incised lengthwise. Before histopathological work-up, the different segments were macropathologically characterized and correlated with the respective findings of the radiological methods. RESULTS: 4 cases showed high grade stenosis within the stent grafts and graft occlusion occurred in two cases. The connection sites of these modular stent grafts showed steps and partial separation of the graft components was causative in 5 of these cases. Migration of graft components occurred in three cases, one of them with complete disconnection of the modular device. Irregulartities and deposits on the endoluminal surface were systematically underestimated with all radiological techniques used. IVUS showed moderate concordance concerning deployment and unfolding of the stent graft, however, irregularities of the endoluminal surface were systematically underestimated. Concordance of MRI, sonography, and CT was worse. Contrast-enhanced T1-w MRI detected endoluminal deposits with moderate concordance. However, the thickness of deposits was underestimated. DSA, IVUS, and CT showed only poor concordance with macropathological findings. CONCLUSIONS: The connection site of modular stent grafts predisposes to stenosis, occlusion, and disconnection of the modular device. Stent deployment and unfolding of the membrane might be examined with IVUS. MRI is useful for detecting endoluminal deposits and stenosis. However, radiological methods will underrate irregularities and deposits of the endoluminal surface.

Neuropeptides, food intake and body weight regulation: a hypothalamic focus.

Energy homeostasis is controlled by a complex neuroendocrine system consisting of peripheral signals like leptin and central signals, in particular, neuropeptides. Several neuropeptides with anorexigenic (POMC, CART, and CRH) as well as orexigenic (NPY, AgRP, and MCH) actions are involved in this complex (partly redundant) controlling system. Starvation as well as overfeeding lead to changes in expression levels of these neuropeptides, which act downstream of leptin, resulting in a physiological response. In this review the role of several anorexigenic and orexigenic (hypothalamic) neuropeptides on food intake and body weight regulation is summarized.

Excessive alcohol consumption and drinking expectations among clients in methadone maintenance.

Excessive alcohol consumption and related problems are common among clients in methadone maintenance treatment (MMT), yet relatively little is known about the psychological and social determinants of alcohol-related attitudes and behaviors during treatment. This study reports on the prevalence of alcohol dependence, patterns of alcohol consumption and preliminary findings about clients' beliefs that they will change their drinking behavior in the future. Data were gathered from personal interviews with 66 clients attending a MMT program in South London (some 80.5% of the eligible caseload). Forty-one percent of the overall sample met DSM-IV criteria for alcohol dependence in the past 12 months. Among clients who reported drinking in the past month (n = 50), 54% were classified as dependent, and these clients reported consuming an average of 23.5 UK standard units of absolute alcohol (188g/6.58 ounces) on a typical drinking day in the past month. Exploratory analyses suggested that expectations to change drinking behavior were predicted by subjective norms (social pressures), perceived functions of alcohol use, past drinking levels and current dose of methadone. Clinicians engaged in alcohol problems assessment and counseling during MMT could usefully examine these influences to strengthen treatment provision.

New perspectives on the manipulation of opiate urges and the assessment of cognitive effort associated with opiate urges.

Behavioral models of drug urges assume that conditioned urges are strongly associated with drug consumption. An alternative, cognitive model assumes that urges represent the operation of cognitively demanding processes devoted to either supporting or blocking the automatized drug-use behavior. In Study 1, the effect of verbal drug cues and mood induction on self-reported opiate urges were examined. Twenty-four opiate addicts were either instructed to listen to verbal drug cures or neutral cues. Negative mood induction was applied on 12 addicts. Study 2 examined the cognitive processes underlying these urges. In a dual task paradigm, participants responded to a probe stimulus and listened simultaneously to an imagery script. It was predicted that response times to the probe should increase to the extent that urge-related nonautomatic processing is invoked. Although negative affect was not associated with nonautomatic processing, the findings suggest that drug urges can be activated by drug cues and that cue-related urges are supported by nonautomatic cognitive processes.

Characterization of nicotine-induced desensitization of evoked dopamine release from rat striatal synaptosomes.

Nicotine has been shown to stimulate neurotransmitter release from brain tissue by acting on presynaptic receptors. In this study, the ability of nicotine pretreatment to produce functional desensitization was investigated in rat striatal synaptosomes in which the release of [3H]dopamine was measured with an in vitro superfusion system. Pretreatment of synaptosomes with low concentrations of L-nicotine resulted in a decrease in the ability of a subsequent nicotine challenge to evoke [3H]dopamine release. The IC50 for nicotine-induced desensitization was found to be 12 nM with a maximum inhibition of > 90% at 300 nM. Nicotine pretreatment did not affect the release evoked by amphetamine, veratridine, or 15 mM K+. The onset of nicotine-induced desensitization occurred with a t1/2 of 43 s at 30 nM nicotine. The temperature dependence of onset yielded a Q10 of 1.2. Recovery from desensitization was slower (t 1/2 = 4.33 min), and both the onset and recovery appeared to follow a single first-order process. Several intermittent schedules of nicotine treatment were found to be effective at inducing and maintaining desensitization. The results of this study show that nonstimulating concentrations of nicotine can produce a complete functional desensitization of subsequent nicotine-induced neurotransmitter release.

Characteristics of weekly pulmonary hypersensitivity responses elicited in the guinea pig by inhalation of ovalbumin aerosols.

We have reported the development of a guinea pig animal model for pulmonary hypersensitivity to inhaled chemicals in which respirations of animals are monitored continuously for 24 hr permitting detection of immediate- (IAR) and late-onset (LAR) airway reactions. Additionally implanted temperature transmitters allow determination of accompanying febrile responses. The current study investigated the relationship between severity of IAR and occurrence of LAR in individual animals. To quantify severity, a grading system was devised which took into account time to response, increase in breathing rate, and occurrence of airway constriction (ACR). Guinea pigs were sensitized by ip injection with 1 mg ovalbumin (OA). On Day 14, inhalation challenge with 12 mg/m3 OA resulted in severe IAR in all animals. No LAR were detected. Subsequent weekly inhalation challenges with OA resulted in less severe IARs and occasionally in absence of response. Febrile reactions were not detected. On one occasion a LAR was observed. It occurred in the animal demonstrating the most severe IARs and having the highest titer of specific homocytotropic antibody. These results are consistent with the mechanism of IAR involving release of spasmogenic mediators from mast cells as a result of antigen crosslinking of surface antibody. The mechanism of the LAR is addressed in the accompanying paper in which LARs are more consistently produced in OA-sensitized guinea pigs.

Experimental sensitization to subtilisin. II. Production of specific antibodies following inhalation exposure of guinea pigs.

We have previously reported the development of pulmonary sensitivity in guinea pigs exposed for 5 consecutive days to bacterial subtilisin in concentrations ranging from 1.9 to 15.0 mg proteolytic enzyme/m3. Animals exposed to lesser concentrations of 0.0083 and 0.041 mg/m3 failed to demonstrate this sensitivity. In the same study, animals exposed for 11 weeks to a subtilisin concentration of 0.00068 mg/m3 followed by 6 weeks to 0.0051 mg/m3 failed to demonstrate pulmonary sensitivity. We report here the antibody response in these animals. Both an enzyme-linked immunosorbent assay and a latex agglutination assay were developed for antibody detection. A concentration-related antibody response was detected in animals which had received acute subtilisin exposures of 0.0083 to 1.9 mg/m3. Antibody titers were not increased further upon exposure to higher subtilisin concentrations. A single 3-hr exposure to 1.9 mg/m3 resulted in the same antibody response as that produced following 5 days of exposure to the same concentration. The cumulative 17-week exposure of 1.12 mg/m3 X hr subtilisin resulted in 36% (9 of 25) of the animals producing significant levels of IgG or IgM antibodies. None of these animals demonstrated pulmonary sensitivity. These results indicate that the circulating antibody titer reflected antigen exposure levels and occurred at exposures below those necessary for production of pulmonary sensitivity.

Contact sensitivity by diisocyanates: potencies and cross-reactivities.

The dermal sensitizing potencies of four diisocyanates were investigated using the mouse ear-swelling test (MEST) with BALB/cBy mice. Mice were administered topical doses of diisocyanates to the abdomen and were challenged on the ear 4 days later with a nonirritating dose of chemical. The increase in ear thickness at 24 hr postchallenge indicated the extent of contact sensitivity. Ear thickness increase plotted against the log of the dose of diisocyanate indicated three regions of effects: a no-effect region, a dose-response region, and a region of reduced response at highest dosages. The potencies of the diisocyanates expressed as the SD50 (dose required to sensitize 50% of the animals in each group) were hexamethylene diisocyanate, 0.088 mg/kg; diphenylmethane-4,4'-diisocyanate, 0.73 mg/kg; and toluene diisocyanate, 5.3 mg/kg. For dicyclohexylmethane diisocyanate, the steep dose-response curve suggested a threshold-type response occurring at 0.24 mg/kg. The specificity of sensitization was evaluated by challenging sensitized animals with heterologous diisocyanates. The homologous reactions were always the most extensive. However, cross-reactions were noted between aryl and alkyl diisocyanates. Toluene diisocyanate, the weakest sensitizer, elicited the fewest cross-reactions. The MEST proved to be a simple and effective method for assessing delayed-type hypersensitivity without need of adjuvants, occlusive patches, abrasions, or other procedures which fail to mimic industrial exposures. The observation that very high exposures resulted in reduced response indicates that protocols which generate dose-response data must be utilized to accurately assess the sensitizing potencies of industrial chemicals.