Development and validation of a sensitive liquid chromatography tandem mass spectrometry assay for the simultaneous determination of ten kinase inhibitors in human serum and plasma.

A liquid chromatography tandem mass spectrometry method for the analysis of ten kinase inhibitors (afatinib, axitinib, bosutinib, cabozantinib, dabrafenib, lenvatinib, nilotinib, osimertinib, ruxolitinib, and trametinib) in human serum and plasma for the application in daily clinical routine has been developed and validated according to the US Food and Drug Administration and European Medicines Agency validation guidelines for bioanalytical methods. After protein precipitation of plasma samples with acetonitrile, chromatographic separation was performed at ambient temperature using a Waters XBridge® Phenyl 3.5 µm (2.1 × 50 mm) column. The mobile phases consisted of water-methanol (9:1, v/v) with 10 mM ammonium bicarbonate as phase A and methanol-water (9:1, v/v) with 10 mM ammonium bicarbonate as phase B. Gradient elution was applied at a flow rate of 400 µL/min. Analytes were detected and quantified using multiple reaction monitoring in electrospray ionization positive mode. Stable isotopically labeled compounds of each kinase inhibitor were used as internal standards. The acquisition time was 7.0 min per run. All analytes and internal standards eluted within 3.0 min. The calibration curves were linear over the range of 2-500 ng/mL for afatinib, axitinib, bosutinib, lenvatinib, ruxolitinib, and trametinib, and 6-1500 ng/mL for cabozantinib, dabrafenib, nilotinib, and osimertinib (coefficients of correlation ≥ 0.99). Validation assays for accuracy and precision, matrix effect, recovery, carryover, and stability were appropriate according to regulatory agencies. The rapid and sensitive assay ensures high throughput and was successfully applied to monitor concentrations of kinase inhibitors in patients. Graphical abstract.

Estrogen and estrogen receptors in cardiovascular oxidative stress.

The cardiovascular system of a premenopausal woman is prepared to adapt to the challenges of increased cardiac output and work load that accompany pregnancy. Thus, it is tempting to speculate whether enhanced adaptability of the female cardiovascular system might be advantageous under conditions that promote cardiovascular disease. In support of this concept, 17ß-estradiol as the major female sex hormone has been shown to confer protective cardiovascular effects in experimental studies. Mechanistically, these have been partially linked to the prevention and protection against oxidative stress. Current evidence indicates that estrogens attenuate oxidative stress at two levels: first, by preventing generation of reactive oxygen species (ROS) and, second, by scavenging ROS in the myocardium and in the vasculature. The purpose of this review is to give an overview on current concepts on conditions and mechanisms by which estrogens protect the cardiovascular system against ROS-mediated cellular injury.

In-Vivo Somatostatin-Receptor Expression in Small Cell Lung Cancer as a Prognostic Image Biomarker and Therapeutic Target.

BACKGROUND: Given the dismal prognosis of small cell lung cancer (SCLC), novel therapeutic targets are urgently needed. We aimed to evaluate whether SSTR expression, as assessed by positron emission tomography (PET), can be applied as a prognostic image biomarker and determined subjects eligible for peptide receptor radionuclide therapy (PRRT). METHODS: A total of 67 patients (26 females; age, 41-80 years) with advanced SCLC underwent SSTR-directed PET/computed tomography (somatostatin receptor imaging, SRI). SRI-avid tumor burden was quantified by maximum standardized uptake values (SUVmax) and tumor-to-liver ratios (T/L) of the most intense SCLC lesion. Scan findings were correlated with progression-free (PFS) and overall survival (OS). In addition, subjects eligible for SSTR-directed radioligand therapy were identified, and treatment outcome and toxicity profile were recorded. RESULTS: On a patient basis, 36/67 (53.7%) subjects presented with mainly SSTR-positive SCLC lesions (>50% lesions positive); in 10/67 patients (14.9%), all lesions were positive. The median SUVmax was found to be 8.5, while the median T/L was 1.12. SRI-uptake was not associated with PFS or OS, respectively (SUVmax vs. PFS, ρ = 0.13 with p = 0.30 and vs. OS, ρ = 0.00 with p = 0.97; T/L vs. PFS, ρ = 0.07 with p = 0.58 and vs. OS, ρ = -0.05 with p = 0.70). PRRT was performed in 14 patients. One patient succumbed to treatment-independent infectious complications immediately after PRRT. In the remaining 13 subjects, disease control was achieved in 5/13 (38.5%) with a single patient achieving a partial response (stable disease in the remainder). In the sub-group of responding patients, PFS and OS were 357 days and 480 days, respectively. CONCLUSIONS: SSTR expression as detected by SRI is not predictive of outcome in patients with advanced SCLC. However, it might serve as a therapeutic target in selected patients.

Both estrogen receptor subtypes, ERα and ERß, prevent aldosterone-induced oxidative stress in VSMC via increased NADPH bioavailability.

Activation of vascular mineralocorticoid (MR) or estrogen receptors (ER) exerts opposing effects on vascular remodeling. As we have previously shown, activation of either estrogen receptor subtype, ERα or ERß, is fully sufficient to attenuate vascular remodeling in aldosterone salt-treated rats. To further elucidate the underlying mechanism(s) we tested the hypothesis that ER and MR activation might differentially modulate vascular reactive oxygen species (ROS) generation. In support of this concept, aldosterone increased ROS generation in vascular smooth muscle cells as determined by quantitative dihydroethidium fluorescence microscopy. Co-treatment with the selective ERα agonist 16α-LE2, the selective ERß agonist 8ß-VE2 or the non-selective ER agonist 17ß-estradiol (E2) significantly reduced aldosterone-induced ROS generation. The pure ER antagonist ICI 182,780 completely blocked these salutary effects of E2, 16α-LE2 and 8ß-VE2. Activation of ERα or ERß fully blocked the reduction of intracellular nicotinamide adenine dinucleotide phosphate (NADPH) levels observed in aldosterone treated vascular smooth muscle cells. Intracellular NADPH levels were closely associated with expression and activity of the NADPH generating enzyme glucose-6-phosphate dehydrogenase. In conclusion, estrogens attenuate the detrimental vascular effects of excessive MR activation at least in part by preventing the depletion of intracellular NADPH levels.

Comparison of a newly developed high performance liquid chromatography method with diode array detection to a liquid chromatography tandem mass spectrometry method for the quantification of cabozantinib, dabrafenib, nilotinib and osimertinib in human serum - Application to therapeutic drug monitoring.

OBJECTIVE: Liquid chromatography tandem mass spectrometry (LC-MS/MS) is a highly selective and sensitive method for the quantification of kinase inhibitors, yet not widely available in clinical routine for therapeutic drug monitoring (TDM). To provide a more accessible alternative, a high-performance liquid chromatography method with ultraviolet/diode array detection (HPLC-UV/DAD) to quantify cabozantinib, dabrafenib, nilotinib and osimertinib, was developed and validated. Results were compared to LC-MS/MS. METHOD: After liquid-liquid-extraction and reconstitution of the residue in 20 mM potassium dihydrogen phosphate (KH2PO4) (pH4.6), acetonitrile and methanol (50:25:25,v/v/v), chromatographic separation was achieved in 20.0 min using a Luna® C18(2)-HST column (100 × 2 mm, 2.5 µm), protected by a C18 guard column (4 × 2 mm) (column temperature: 30 °C, autosampler: 10 °C). Mobile phase A and B consisted of 20 mM KH2PO4 (pH4.9) and acetonitrile (9:1,v/v) and acetonitrile:20 mM KH2PO4 (pH4.9) (7:3,v/v), respectively. Gradient elution was performed at 200 µL/min. Analytes were quantified at 250, 280 and 330 nm, using sorafenib as internal standard. RESULTS: Calibration curves were linear (35-2,000 ng/mL). Method validation assays met requirements by U.S. Food and Drug Administration and European Medicines Agency. Compared to the more sensitive and specific LC-MS/MS, HPLC-UV/DAD showed a good correlation and a strong positive association (Kendall's tau 0.811¬-0.963, p < 0.05). Bland-Altman-plots revealed 100% (cabozantinib), 98.6% (dabrafenib), 98.6% (nilotinib) and 96.2% (osimertinib) of relative differences inside the limits of agreement. Regulatory agency criteria for sample reanalysis and cross validation were met (±20%-criterion:100% (cabozantinib), 94.3% (dabrafenib), 92% (nilotinib) and 84.6% (osimertinib). CONCLUSION: The developed HPLC-UV/DAD method is "fit-for-TDM" in clinical routine and serves as a genuine alternative to LC-MS/MS.

Volumetric absorptive microsampling (VAMS) for the quantification of ten kinase inhibitors and determination of their in vitro VAMS-to-plasma ratio.

Personalized dosing of kinase inhibitors (KI) might be beneficial in oral anti-cancer therapy to overcome individual pharmacokinetic variability. Volumetric absorptive microsampling (VAMS) has emerged as an attractive alternative compared to conventional invasive sampling methods enabling remote and frequent specimen collection. Therefore, an LC-MS/MS VAMS method was developed and validated to monitor drug exposure of ten KI from 20 µL dried capillary blood. The assay includes the KI cabozantinib, dabrafenib, nilotinib, and osimertinib with a calibration range of 6-1500 ng/mL and afatinib, axitinib, bosutinib, lenvatinib, ruxolitinib and trametinib within a range of 2-500 ng/mL. Using acetonitrile containing isotope labelled internal standards (IS) as solid-liquid extraction solvent, analytes and IS were detected by multiple reaction monitoring (MRM) after electro-spray ionization (ESI) in positive ionization mode after chromatographic separation using a phenyl-hexyl column. The method was validated according to the FDA and EMA guidelines for bioanalytical method validation and in accordance with the guideline of the International Association for Therapeutic Drug Monitoring and Clinical Toxicology for dried blood spot-based methods. The calibration model was linear and reproducible for all KI (R2> 0.994). Furthermore, the validation demonstrated that the VAMS method is accurate, precise, and sensitive. The method fulfilled the acceptance criteria for matrix effects, recovery, carry over, selectivity as well as for the haematocrit effect and all substances proved to be stable in dried condition for at least six weeks at room temperature. In vitro experiments using spiked venous blood were conducted to establish a VAMS-to-plasma conversion factor for each analyte for comparison of VAMS and plasma concentrations. The method was successfully used in a real-life setting demonstrating its applicability in clinical routine. VAMS concentrations of afatinib, cabozantinib, dabrafenib, nilotinib, ruxolitinib and trametinib were assessed in capillary blood samples collected from either trained healthcare professionals or patients at home.

Medroxyprogesterone acetate aggravates oxidative stress and left ventricular dysfunction in rats with chronic myocardial infarction.

The role of estrogens during myocardial ischemia has been extensively studied. However, effects of a standard hormone replacement therapy including 17ß-estradiol (E2) combined with medroxyprogesterone acetate (MPA) have not been assessed, and this combination could have contributed to the negative outcomes of the clinical studies on hormone replacement. We hypothesized that adding MPA to an E2 treatment would aggravate chronic heart failure after experimental myocardial infarction (MI). To address this issue, we evaluated clinical signs of heart failure as well as left ventricular (LV) dysfunction and remodeling in ovariectomized rats subjected to chronic MI receiving E2 or E2 plus MPA. After eight weeks MI E2 showed no effects. Adding MPA to E2 aggravated LV remodeling and dysfunction as judged by increased heart weight, elevated myocyte cross-sectional areas, increased elevated left ventricle end diastolic pressure, and decreased LV fractional shortening. Impaired LV function in rats receiving MPA plus E2 was associated with increased cardiac reactive oxygen species generation and myocardial expression levels of NADPH oxidase subunits. These results support the interpretation that adding MPA to an E2 treatment complicates cardiovascular injury damage post-MI and therefore contributes to explain the adverse outcome of prospective clinical studies.

[Pulmonary and Cardiac Sarcoidosis - Diagnosis and Therapy]. / Diagnostik und Therapie der pulmonalen und kardialen Sarkoidose.

Sarcoidosis occurs predominantly in younger adults and may involve multiple organ systems. Although classical features such as bihilar lymphadenopathy, low grade fever, fatigue, pulmonary opacities, ocular lesions and arthritis occur frequently it must be kept in mind that sarcoidosis is able to mimic virtually any other disease. A thorough and systematic diagnostic strategy is warranted since even the histological hallmark of non-caseating granulomas occurs in a variety of other granulomatous diseases and even malignancies. A firm diagnosis is based on a match of clinical features, imaging results and histopathological findings. The Scadding classification describes four different types, not stages, of thoracic sarcoidosis based on mediastinal lymph node and lung parenchyma involvement. Medical treatment of pulmonary sarcoidosis is indicated only in patients exhibiting progressive disease and/or organ function impairment. Cardiac sarcoidosis manifests itself by clinical signs of heart failure, impaired cardiac function and arrhythmias including ventricular tachycardia, ventricular fibrillation and AV-conduction abnormalities. Patients with symptomatic or suspected cardiac sarcoidosis require rapid and elaborate diagnostic testing including cardiac MRI and PET imaging. Referral to a specialized center should be considered to establish a firm diagnosis and to initiate medical treatment and eventual device implantation. Oral corticosteroids, as the initial medical treatment of choice, carry side effects that must be weighed carefully against clinical benefits. Immunosuppressive therapy with methotrexate, azathioprine or TNF-blockers is usually reserved for patients that are either not responsive or intolerant to systemic steroids or that require steroid maintenance therapy above the cushing threshold. Diagnosed early and treated correctly, pulmonary and cardiac sarcoidosis mostly carry a favorable prognosis.

Estrogen receptor alpha interacts with 17beta-hydroxysteroid dehydrogenase type 10 in mitochondria.

Estrogen receptor alpha (ERalpha) is present in the nucleus, the cytosol and in mitochondria. The rat ERalpha ligand binding domain was employed as bait in a bacterial two-hybrid screening of a human heart cDNA library to detect novel protein-protein interaction partners of ERalpha in the heart. 17beta-Hydroxysteroid dehydrogenase type 10 (17beta-HSD10), which converts potent (17beta-estradiol) to less potent estrogens (estrone), co-localized with 17beta-HSD10 in the mitochondria of rat cardiac myocytes. GST pull-down experiments confirmed the interaction of ERalpha and 17beta-HSD10. These findings suggest that the ERalpha estrogen receptor might be involved in regulating intracellular estrogen levels by modulating 17beta-HSD10 activity.

Differential effects of 17beta-estradiol and of synthetic progestins on aldosterone-salt-induced kidney disease.

UNLABELLED: Elevated mineralocorticoid levels and female sex hormones have been shown to confer opposing effects on renal injury, but their combined effects are still unknown. OBJECTIVE: Identify the function of estrogens and of different synthetic progestins on aldosterone salt-mediated renal disease. METHODS: The role of 17beta-estradiol, medroxyprogesterone acetate (MPA), and drospirenone during renal injury was studied in Wistar rats subjected to uni-nephrectomy plus aldosterone salt treatment. RESULTS: Aldo-salt treatment of intact, ovariectomized, and estradiol-treated female rats resulted in remnant kidney hypertrophy without structural damage. Co-treatment with MPA, but not with drospirenone, increased kidney hypertrophy, fluid turnover, sodium retention, and potassium excretion. Medroxyprogesterone acetate also caused glomerular, vascular, tubular, and interstitial lesions that were accompanied by increased blood pressure and enhanced NADPH oxidase (p67phox) and sodium channel (alpha-ENaC) expression. Drospirenone, a progestin with anti-mineralocorticoid function, and spironolactone prevented kidney hypertrophy, hypertension, and sodium retention. Drospirenone and spironolactone also increased renal angiotensin II type 2 receptor expression and relieved aldosterone-induced suppression of serum angiotensin II levels. CONCLUSION: The choice of specific synthetic progestins has profound implications on the development of kidney injury and renal gene expression under conditions of elevated aldosterone serum levels and salt intake.

Ligand-dependent activation of ER{beta} lowers blood pressure and attenuates cardiac hypertrophy in ovariectomized spontaneously hypertensive rats.

AIMS: The biological effects of oestrogens are mediated by two different oestrogen receptor (ER) subtypes, ERalpha and ERbeta, which might play different, redundant, or opposing roles in cardiovascular disease. Previously, we have shown that the selective ERalpha agonist 16alpha-LE2 improves vascular relaxation, attenuates cardiac hypertrophy, and increases cardiac output without lowering elevated blood pressure in spontaneously hypertensive rats (SHR). Because ERbeta-deficient mice exhibit elevated blood pressure and since the ERbeta agonist 8beta-VE2 attenuated hypertension in aldosterone-salt-treated rats, we have now tested the hypothesis that the isotype-selective ERbeta agonist 8beta-VE2 might be capable of lowering elevated blood pressure in ovariectomized SHR. METHODS AND RESULTS: Treatment of ovariectomized SHR with 8beta-VE2 for 12 weeks conferred no uterotrophic effects but lowered elevated systolic blood pressure (-38 +/- 5 mmHg, n = 31, P < 0.001 vs. placebo) as well as peripheral vascular resistance (-31.3 +/- 4.6%, P < 0.001 vs. placebo). 8beta-VE2 enhanced aortic ERbeta expression (+75.7 +/- 7.1%, P < 0.01 vs. placebo), improved NO-dependent vasorelaxation, augmented phosphorylation of the vasodilator-stimulated phosphoprotein in isolated aortic rings (P < 0.05 vs. placebo), increased cardiac output (+20.4 +/- 2.5%, P < 0.01 vs. placebo), and attenuated cardiac hypertrophy (-22.2 +/- 3.2%, p < 0.01 vs. placebo). 8beta-VE2, in contrast to oestradiol, did not enhance cardiac alpha-myosin heavy chain expression. CONCLUSION: Ligand-dependent activation of ERbeta confers blood pressure lowering effects in SHR that are superior to those of 17beta-estradiol or the ERalpha agonist 16alpha-LE2 and attenuates cardiac hypertrophy primarily by a reduction of cardiac afterload without promoting uterine growth.

Genetic and pharmacologic strategies to determine the function of estrogen receptor alpha and estrogen receptor beta in cardiovascular system.

BACKGROUND: The biological functions of estrogens extend beyond the female and male reproductive tract, affecting the cardiovascular and renal systems. Traditional views on the role of postmenopausal hormone therapy (HT) in protecting against heart disease, which were challenged by clinical end point studies that found adverse effects of combined HT, are now being replaced by more differentiated concepts suggesting a beneficial role of early and unopposed HT that does not include a progestin. OBJECTIVE: We reviewed recent insights, concepts, and research results on the biology of both estrogen receptor (ER) subtypes, ERalpha and ERbeta, in cardiac and vascular tissues. Knowledge of these ER subtypes is crucial to understanding gender and estrogen effects and to developing novel, exciting strategies that may have a profound clinical impact. METHODS: This review focuses on in vivo studies and includes data presented at the August 2007 meeting of the American Physiological Society as well as data from a search of the MEDLINE and Ovid databases from January 1986 to November 2007. Search results were restricted to English-language publications, using the following search terms: estrogen, estrogen receptor alpha, estrogen receptor beta, estrogen receptor alpha agonist, estrogen receptor alpha antagonist, estrogen receptor beta agonist, estrogen receptor beta antagonist, PPT, DPN, heart, vasculature, ERKO mice, BERKO mice, transgenic mice, and knockout mice. RESULTS: Genetic mouse models and pharmacologic studies that employed selective as well as nonselective ER agonists support the concept that both ER subtypes confer protective effects in experimental models of human heart disease, including hypertension, cardiac hypertrophy, and chronic heart failure. CONCLUSIONS: Genetic models and novel ligands hold the promise of further improving our understanding of estrogen action in multiple tissues and organs. These efforts will ultimately enhance the safety and efficacy of HT and may also result in new applications for synthetic female sex hormone analogues.

Peptide receptor radionuclide therapy as a new tool in treatment-refractory sarcoidosis - initial experience in two patients.

Sarcoidosis is a multisystem granulomatous disorder of unknown etiology that can involve virtually all organ systems. Whereas most patients present without symptoms, progressive and disabling organ failure can occur in up to 10% of subjects. Somatostatin receptor (SSTR)-directed peptide receptor radionuclide therapy (PRRT) has recently received market authorization for treatment of SSTR-positive neuroendocrine tumors. Methods: We describe the first case series comprising two patients with refractory multi-organ involvement of sarcoidosis who received 4 cycles of PRRT. Results: PRRT was well-tolerated without any acute adverse effects. No relevant toxicities could be recorded during follow-up. Therapy resulted in partial response accompanied by a pronounced reduction in pain (patient #1) and stable disease regarding morphology as well as disease activity (patient #2), respectively. Conclusion: Peptide receptor radionuclide therapy in sarcoidosis is feasible and might be a new valuable tool in patients with otherwise treatment-refractory disease. Given the long experience with and good tolerability of PRRT, further evaluation of this new treatment option for otherwise treatment-refractory sarcoidosis in larger patient cohorts is warranted.

Left Ventricular Diastolic Dysfunction in a Rat Model of Diabetic Cardiomyopathy using ECG-gated 18F-FDG PET.

In diabetic cardiomyopathy, left ventricular (LV) diastolic dysfunction is one of the earliest signs of cardiac involvement prior to the definitive development of heart failure (HF). We aimed to explore the LV diastolic function using electrocardiography (ECG)-gated 18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET) imaging beyond the assessment of cardiac glucose utilization in a diabetic rat model. ECG-gated 18F-FDG PET imaging was performed in a rat model of type 2 diabetes (ZDF fa/fa) and ZL control rats at age of 13 weeks (n = 6, respectively). Under hyperinsulinemic-euglycemic clamp to enhance cardiac activity, 18F-FDG was administered and subsequently, list-mode imaging using a dedicated small animal PET system with ECG signal recording was performed. List-mode data were sorted and reconstructed into tomographic images of 16 frames per cardiac cycle. Left ventricular functional parameters (systolic: LV ejection fraction (EF), heart rate (HR) vs. diastolic: peak filling rate (PFR)) were obtained using an automatic ventricular edge detection software. No significant difference in systolic function could be obtained (ZL controls vs. ZDF rats: LVEF, 62.5 ± 4.2 vs. 59.4 ± 4.5%; HR: 331 ± 35 vs. 309 ± 24 bpm; n.s., respectively). On the contrary, ECG-gated PET imaging showed a mild but significant decrease of PFR in the diabetic rats (ZL controls vs. ZDF rats: 12.1 ± 0.8 vs. 10.2 ± 1 Enddiastolic Volume/sec, P < 0.01). Investigating a diabetic rat model, ECG-gated 18F-FDG PET imaging detected LV diastolic dysfunction while systolic function was still preserved. This might open avenues for an early detection of HF onset in high-risk type 2 diabetes before cardiac symptoms become apparent.

Targeting CXCR4 with [68Ga]Pentixafor: a suitable theranostic approach in pleural mesothelioma?

C-X-C motif chemokine receptor 4 (CXCR4) is a key factor for tumor growth and metastasis in several types of human cancer. This study investigated the feasibility of CXCR4-directed imaging with positron emission tomography/computed tomography (PET/CT) using [68Ga]Pentixafor in malignant pleural mesothelioma. Six patients with pleural mesothelioma underwent [68Ga]Pentixafor-PET/CT. 2'-[18F]fluoro-2'-deoxy-D-glucose ([18F]FDG)-PET/CT (4/6 patients) and immunohistochemistry obtained from biopsy or surgery (all) served as standards of reference. Additionally, 9 surgical mesothelioma samples were available for histological work-up. Whereas [18F]FDG-PET depicted active lesions in all patients, [68Ga]Pentixafor-PET/CT recorded physiologic tracer distribution and none of the 6 patients presented [68Ga]Pentixafor-positive lesions. This finding paralleled results of immunohistochemistry which also could not identify relevant CXCR4 surface expression in the samples analyzed. In contrast to past reports, our data suggest widely absence of CXCR4 expression in pleural mesothelioma. Hence, robust cell surface expression should be confirmed prior to targeting this chemokine receptor for diagnosis and/or therapy.

Impact of tissue photon attenuation in small animal cardiac PET imaging.

OBJECTIVES: Tissue photon attenuation is one of the essential artifacts requiring correction in clinical cardiac positron emission tomography (PET) imaging. However, due to small body size its impact on diagnostic accuracy in small rodents is considered to be limited or even ignorable. The present cardiac PET study compares lean and obese rats to determine the influence of tissue attenuation on quantitative assessment as well as regional tracer distribution. METHODS: A dedicated small animal PET system equipped with a 57Co rotating source for transmission was used. To assess the impact of tissue attenuation in rats with different body sizes, cardiac 18F-FDG -PET studies for Zucker diabetic fatty rats (obese rats) and Zucker lean rats (lean rats) were performed. The radiotracer activity reduction by attenuation was compared between the two groups. Regional tracer distribution calculated with and without attenuation correction was also assessed. RESULTS: The chest diameter was significantly longer in obese than in lean rats (5.6±0.3cm in obese and 4.5±0.2cm in lean rats, p<0.0001). Whereas the activity reduction by attenuation was significantly greater in obese than in lean rats (44.1±2.5% and 5.1±3.1%, p<0.0001), the regional variation of tissue attenuation among the ventricular walls was minimal in both lean (p=0.73) and obese rats (p=0.65). CONCLUSION: Attenuation correction is indispensable for accurate comparison of cardiac tracer activity between animals with different body size, whereas it can be omitted for evaluation of regional tracer distribution.

Whitening and Impaired Glucose Utilization of Brown Adipose Tissue in a Rat Model of Type 2 Diabetes Mellitus.

Brown adipose tissue (BAT) is an attractive therapeutic target to combat diabetes and obesity due to its ability to increase glucose expenditure. In a genetic rat model (ZDF fa/fa) of type-2 diabetes and obesity, we aimed to investigate glucose utilization of BAT by 18F-FDG PET imaging. Male Zucker diabetic fatty (ZDF) and Male Zucker lean (ZL) control rats were studied at 13 weeks. Three weeks prior to imaging, ZDF rats were randomized into a no-restriction (ZDF-ND) and a mild calorie restriction (ZDF-CR) group. Dynamic 18F-FDG PET using a dedicated small animal PET system was performed under hyperinsulinemic-euglycemic clamp. 18F-FDG PET identified intense inter-scapular BAT glucose uptake in all ZL control rats, while no focally increased 18F-FDG uptake was detected in all ZDF-ND rats. Mild but significant improved BAT tracer uptake was identified after calorie restriction in diabetic rats (ZDF-CR). The weight of BAT tissue and fat deposits were significantly increased in ZDF-CR and ZDF-ND rats as compared to ZL controls, while UCP-1 and mitochondrial concentrations were significantly decreased. Whitening and severely impaired insulin-stimulated glucose uptake in BAT was confirmed in a rat model of type-2 diabetes. Additionally, calorie restriction partially restored the impaired BAT glucose uptake.

Increased mortality and aggravation of heart failure in estrogen receptor-beta knockout mice after myocardial infarction.

BACKGROUND: Lower mortality rates among women with chronic heart failure than among men may depend in part on the action of female sex hormones, especially estrogens. The biological effects of estrogens are mediated by 2 distinct estrogen receptor (ER) subtypes (ERalpha and ERbeta). The present study was undertaken to determine the role of ERbeta in the development of chronic heart failure after experimental myocardial infarction (MI). METHODS AND RESULTS: Female ERbeta null mice (BERKO(Chapel Hill)) and wild-type littermates (WT) were ovariectomized, given 17beta-estradiol, and subjected to chronic anterior MI (MI; BERKO n=31, WT n=30) or sham operation (sham; BERKO n=14, WT n=14). At 8 weeks after MI, both genotypes revealed left ventricular remodeling and impaired contractile function at similar average infarct size (BERKO-MI 32.9+/-5% versus WT-MI 33.0+/-4%); however, BERKO mice showed increased mortality (BERKO-MI 42% versus WT-MI 23%), increased body weight and fluid retention (P<0.01), higher ventricular pro-ANP expression (BERKO-MI 27.9-fold versus sham, WT-MI 5.2-fold versus sham; BERKO-MI versus WT-MI P<0.001), higher atrial natriuretic peptide serum levels, and increased phospholamban expression (P<0.05) compared with WT mice. CONCLUSIONS: Systemic deletion of ERbeta in female mice increases mortality, aggravates clinical and biochemical markers of heart failure, and contributes to impaired expression of Ca(2+)-handling proteins in chronic heart failure after MI. Further studies are required to delineate the relative importance of cardiac and vascular effects of ERbeta and the role of ERalpha in the development of heart failure.