Citations

Long‐chain acyl‐coenzymes A (acyl‐CoAs) (LCACoA) are the activated forms of long‐chain fatty acids and serve as key lipid metabolites. Excess accumulation of intracellular LCACoA, diacylglycerols (DAGs) and ceramides may create insulin resistance with respect to glucose metabolism. We present a new method to measure LCACoA concentrations and isotopic enrichment of palmitoyl‐CoA ([U‐ 13 C]16‐CoA) and oleoyl‐CoA ([U‐ 13 C]18:1‐CoA) using ultra‐performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) to quantitate seven different LCACoA (C14‐CoA, C16‐CoA, C16:1‐CoA, C18‐CoA, C18:1‐CoA, C18:2‐CoA, C20‐CoA). The molecules are separated on a reversed‐phase UPLC column using a binary gradient with ammonium hydroxide (NH 4 OH) in water and NH 4 OH in acetonitrile (ACN). The LCACoA are quantified using selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer in positive electrospray ionization (ESI) mode. All LCACoA ions except enriched palmitate enrichment of palmitoyl‐CoA ([U −13 C]16‐CoA) and oleoyl‐CoA ([U −13 C]18:1‐CoA) using ultra‐performance liquid chromatography/mass spectrometry (UPLC/MS/MS) to quantitate seven different LCACoA (C14‐CoA, C16‐CoA, C16:1‐CoA, C18‐CoA, C18:1‐CoA, C18:2‐CoA, C20‐CoA). The molecules are separated on a reversed‐phase UPLC column using a binary gradient with ammonium hydroxide (NH 4 OH) in water and NH 4 OH in acetonitrile. The LCACoA are quantified using selected reaction monitoring (SRM) on a triple quadrupolemass spectrometer in positive electrospray ionization (ESI) mode. All LCACoA ions except enriched palmitate and oleate were monitored as [M + 2 + H] + and [U 13 C]16‐CoA and [U 13 C]18:1‐CoA were monitored as [M + 16 + H] + and [M + 18 + H] + , respectively. The method is simple, sensitive and efficient (run time as short as 5 min) and allowed us to measure the concentration and detect enrichment in intramyocellular [U 13 C]16‐CoA and [U 13 C]18:1‐CoA during a low dose intravenous infusion of [U 13 C]palmitate and [U 13 C]oleate in adults undergoing either a saline control experiment or an insulin/glucose infusion experiment. This technique should allow investigators to measure the trafficking of extracellular fatty acids to the intracellular LCACoA pool. Copyright © 2011 John Wiley & Sons, Ltd.

OBJECTIVE Preferential upper-body fat gain, a typical male pattern, is associated with a greater cardiometabolic risk. Regional differences in lipolysis and meal fat storage cannot explain sex differences in body fat distribution. We examined the potential role of the novel free fatty acid (FFA) storage pathway in determining body fat distribution in postabsorptive humans and whether adipocyte lipogenic proteins (CD36, acyl-CoA synthetases, and diacylglycerol acyltransferase) predict differences in FFA storage. RESEARCH DESIGN AND METHODS Rates of postabsorptive FFA (palmitate) storage into upper-body subcutaneous (UBSQ) and lower-body subcutaneous (LBSQ) fat were measured in 28 men and 53 premenopausal women. Stable and radiolabeled palmitate tracers were intravenously infused followed by subcutaneous fat biopsies. Body composition was assessed with a combination of dual-energy X-ray absorptiometry and computed tomography. RESULTS Women had greater FFA (palmitate) storage than men in both UBSQ (0.37 ± 0.15 vs. 0.27 ± 0.18 μmol · kg−1 · min−1, P = 0.0001) and LBSQ (0.42 ± 0.19 vs. 0.22 ± 0.11 μmol · kg−1 · min−1, P < 0.0001) fat. Palmitate storage rates were significantly greater in LBSQ than UBSQ fat in women, whereas the opposite was true in men. Plasma palmitate concentration positively predicted palmitate storage in both depots and sexes. Adipocyte CD36 content predicted UBSQ palmitate storage and sex-predicted storage in LBSQ fat. Palmitate storage rates per kilogram fat did not decrease as a function of fat mass, whereas lipolysis did. CONCLUSIONS The FFA storage pathway, which had remained undetected in postabsorptive humans until recently, can have considerable, long-term, and sex-specific effects on body fat distribution. It can also offer a way of protecting the body from excessive circulating FFA in obesity.

Introduction Each year, the US Antarctic Program rapidly transports scientists and support personnel from sea level (SL) to the South Pole (SP, 2835 m) providing a unique natural laboratory to quantify the incidence of acute mountain sickness (AMS), patterns of altitude related symptoms and the field effectiveness of acetazolamide in a highly controlled setting. We hypothesized that the combination of rapid ascent (3 hr), accentuated hypobarism (relative to altitude), cold, and immediate exertion would increase altitude illness risk. Methods Medically screened adults (N = 246, age = 37 ± 11 yr, 30% female, BMI = 26 ± 4 kg/m 2 ) were recruited. All underwent SL and SP physiological evaluation, completed Lake Louise symptom questionnaires (LLSQ, to define AMS), and answered additional symptom related questions (eg, exertional dyspnea, mental status, cough, edema and general health), during the 1st week at altitude. Acetazolamide, while not mandatory, was used by 40% of participants. Results At SP, the barometric pressure resulted in physiological altitudes that approached 3400 m, while T ° C averaged -42, humidity 0.03%. Arterial oxygen saturation averaged 89% ± 3%. Overall, 52% developed LLSQ defined AMS. The most common symptoms reported were exertional dyspnea-(87%), sleeping difficulty-(74%), headache-(66%), fatigue-(65%), and dizziness/lightheadedness-(46%). Symptom severity peaked on days 1-2, yet in >20% exertional dyspnea, fatigue and sleep problems persisted through day 7. AMS incidence was similar between those using acetazolamide and those abstaining (51 vs. 52%, P = 0.87). Those who used acetazolamide tended to be older, have less altitude experience, worse symptoms on previous exposures, and less SP experience. Conclusion The incidence of AMS at SP tended to be higher than previously reports in other geographic locations at similar altitudes. Thus, the SP constitutes a more intense altitude exposure than might be expected considering physical altitude alone. Many symptoms persist, possibly due to extremely cold, arid conditions and the benefits of acetazolamide appeared negligible, though it may have prevented more severe symptoms in higher risk subjects.

Caveolae are specialized membrane microdomains expressing the scaffolding protein caveolin-1. We recently demonstrated the presence of caveolae in human airway smooth muscle (ASM) and the contribution of caveolin-1 to intracellular calcium ([Ca2+]i) regulation. In the present study, we tested the hypothesis that caveolin-1 regulates ASM contractility. We examined the role of caveolins in force regulation of porcine ASM under control conditions as well as TNF-α-induced airway inflammation. In porcine ASM strips, exposure to 10 mM methyl-β-cyclodextrin (CD) or 5 μM of the caveolin-1 specific scaffolding domain inhibitor peptide (CSD) resulted in time-dependent decrease in force responses to 1 μM ACh. Overnight exposure to the cytokine TNF-α (50 ng/ml) accelerated and increased caveolin-1 expression and enhanced force responses to ACh. Suppression of caveolin-1 with small interfering RNA mimicked the effects of CD or CSD. Regarding mechanisms by which caveolae contribute to contractile changes, inhibition of MAP kinase with 10 μM PD98059 did not alter control or TNF-α-induced increases in force responses to ACh. However, inhibiting RhoA with 100 μM fasudil or 10 μM Y27632 resulted in significant decreases in force responses, with lesser effects in TNF-α exposed samples. Furthermore, Ca2+sensitivity for force generation was substantially reduced by fasudil or Y27632, an effect even more enhanced in the absence of caveolin-1 signaling. Overall, these results indicate that caveolin-1 is a critical player in enhanced ASM contractility with airway inflammation.

Abstract Introduction: Despite significant progress over the last decade in the development of therapeutics for metastatic cancers including breast cancer (MBC), non small cell lung cancer (NSCLC), and renal cell carcinoma (RCC), most patients will ultimately succumb to their malignancy; more effective therapeutics are sorely needed. The microtubule (MT) is a well-validated target for anti-cancer therapeutics. Ixabepilone (Ixa), a MT stabilizer is approved for MBC and has demonstrated efficacy against other solid tumors. mTOR is another validated target for anti-cancer therapeutics. Temsirolimus (Tem) inhibits mTOR and is approved for advanced RCC. We tested the hypothesis that the addition of Ixa to Tem might augment its anti-tumor activity in vitro. Methods: The cell lines, A498 (RCC), MDA-MB-231 (MBC), and A549 (NSCLC) were treated with Tem alone, Ixa alone, or the combination. All cell lines were also treated with the combination of Tem and Ixa in a fixed ratio of 1:20; Tem doses ranged from 0.125 [nM] to 5 [nM] and Ixa doses ranged from 2.5 [nM] to 100 [nM]. The effects of the treatments were evaluated with clonogenic assays. Synergism was evaluated using CalucSyn 2.0 in accord with the isobologram method of Chou and Talalay. Results: Both Tem and Ixa inhibited cell growth in all tested cell lines as single agents in dose-dependent fashions. Moreover, the combination of Tem and Ixa synergistically enhanced cytotoxicity in all tested lines (see table below).Cell LineED50 [nM]CI (lowest value)A4980.449280.404A5490.285440.263MDA-MB-2310.483550.373 Conclusion: The combination of Ixa and Tem displayed promising activity in all tested cell lines. These findings support the further evaluation of this combination in a phase I clinical trial. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4480.

Background— Multiple B-type natriuretic peptide (BNP) fragments circulate in patients with heart failure (HF) but the types and relative quantities, particularly in relation to bioactive BNP 1-32, remain poorly defined. The purpose of the study was to relate clinically available BNP values with quantitative information on the concentration of pre-secretion and post-processed fragments of BNP detected by mass spectrometry. Methods and Results— Seventy Class I–IV patients were prospectively enrolled with blood drawn into tubes containing a preservative to protect against BNP degradation. Samples were analyzed by quantitative mass spectrometry (MS) immunoassay for intact BNP 1-32 and its fragments. Clinical BNP 1-2 was measured by standard clinical laboratory methods. ProBNP 1-108, corin, and clinically measured BNP levels were elevated, but MS BNP 1-32 levels were low and differed from clinical BNP ( P =0.01). Intact MS BNP 1-32 correlated modestly with clinical BNP ( r =0.46, P <0.001). MS BNP fragments 3-32, 4-32, and 5-32 demonstrated the best associations with clinical BNP; fragment 5-32 with a correlation coefficient of r =0.81 ( P <0.001). Conclusions— ProBNP 1-108 is measured by clinical BNP assays and contributes to the cumulative results of the BNP assay. However, the observation that clinically measured BNP correlates best with MS degradation fragments and relatively poorly with MS BNP 1-32 suggests that a significant component of circulating clinical BNP is composed of such fragments that are known to demonstrate little biological activity. There appear to be multiple pathways involved in the dysregulation of proBNP in HF, and both the processing of proBNP and the downstream degradation to BNP 1-32 appear to be critical.

Abstract Background: Adenoid cystic carcinoma of the breast is a rare type of breast cancer accounting for approximately 0.1% of all breast cancer and less than 200 cases are reported in the literature. Hence, data for treatment and outcome are limited. This retrospective review reports the Mayo Clinic's experience.Methods: We identified 30 patients between 1971 and 2009 who were diagnosed with adenoid cystic breast cancer at Mayo Clinic Rochester, Arizona or Florida. Median follow-up was 62 months with a range of 1 to 424 months. Data regarding demographics (age, gender, and race), tumor characteristics (size, grade, and margin status), biological features (Estrogen receptor (ER), Progesterone receptor (PR) status, HER2 status) and clinical course (recurrence, secondary treatment) were collected. Disease free survival was calculated using Kaplan Meier estimates.Results: The median age of all patients was 58 (range, 22 – 78 years). All were female. Median tumor size was 1.3 cm (range, 0.6 – 8.5cm). Axillary lymph node sampling (sentinel or complete dissection) was performed in 15 patients. No patient had lymph node positive disease on presentation. The majority of patients had hormone receptor negative disease. Only 3 patients (10%) had weak hormone receptor (1-10%) positivity. No tumors were HER2 positive. Only two patients had high grade disease. Initial surgical data only available in twenty one patients: fifteen (50%) were treated with lumpectomy and adjuvant radiation, and 6 (20%) were treated with mastectomy. There were no local recurrences. Only one patient was treated with adjuvant chemotherapy (with cyclophosphamide, methotrexate and fluorouracil (CMF) followed by tamoxifen) but still suffered a distant recurrence within 5 years. There were a total of five patients that recurred. The 5-year disease free survival (DFS) rate for the entire cohort was 90%, and 10 year DFS was 83%. Median time to recurrence was 47.5 months (Range, 23-77 months). Recurrence occurred almost exclusively in patients with ≥ T2 disease. The primary site of metastases was lung (80%) and bone (20%) but subsequent liver and brain metastases were also seen. Treatments at relapse included surgical resection, radiosurgery and chemotherapy.Conclusion: Adenoid cystic carcinomas of the breast are rare but have overall good prognosis. This review confirms that the majority of these patients present with node negative disease, low to intermediate histologic grade and endocrine unresponsive tumors. Primary treatment is surgery (mastectomy or lumpectomy plus radiation) and there seems to be no role of any adjuvant therapy at the present time. In general, recurrences (80%) occurred with tumors larger than 2 cm and adjuvant chemotherapy and/or targeted therapy could be explored for selected patients in the context of a clinical trial. Overall mortality after recurrence was 50% but median overall survival was variable in years (1-14 years). Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2055.

Abstract Abstract #12 Background: Meta-analyses (EBCTCG, Lancet 2005; 365:2687) have established in estrogen receptor (ER) positive breast cancer (BC) that 5 yrs of adjuvant tamoxifen (Tam) substantially reduces recurrence and mortality. The aromatase inhibitors (AIs) anastrozole, exemestane and letrozole have been studied in prospective trials in postmenopausal patients (pts) in comparison with Tam either as initial monotherapy (Cohort 1) or after 2 to 3 years of Tam in a switching strategy (Cohort 2); both to a total of 5 years (yrs) of endocrine therapy.&#x2028; Methods: Outcomes data submitted to EBCTCG were used in separate meta-analyses of Cohort 1 (trials: ATAC, BIG 1-98/IBCSG 18-98) and of Cohort 2 (trials: ABCSG 8, ARNO 95, IES/BIG 2-97, ITA). The primary analysis involved pts with tumors reported to be ER positive with endpoints of BC recurrence (all, local only, contralateral BC only, distant), death with and without recurrence, any death. Subgroup analyses included progesterone receptor (PgR) status, age, tumor grade and nodal status. All p-values were 2-sided.&#x2028; Results: Highlights include: Cohort 1: 9,856 pts with 50,000 woman-years of follow-up. At 5 yrs, AI therapy was associated with an absolute 2.7% (standard error [SE] 0.7) decrease in BC recurrence (10.7% vs 13.4%, relative decrease 20% [SE 5], p=0.00004). There appeared to be greater proportional decreases in isolated local recurrence (30% [SE 10], p=0.003) and in contralateral disease (38% [SE 12], p=0.003) than in distant recurrence (12% [SE 6], p= 0.04). AIs yielded an absolute 1.0% (SE 0.5) decrease in BC mortality (5.5% vs 6.5%, relative decrease 7% [SE 7], p=0.28). Cohort 2: 9,015 pts with 33,000 woman-years of follow-up. At 6 yrs from treatment divergence (i.e., 8-9 yrs from diagnosis), AI therapy was associated with an absolute 3.5% (SE 1.1) decrease in BC recurrence (12.6% vs 16.1%, relative decrease 29% [SE 6], p&lt;0.00001). There appeared to be greater proportional reductions in isolated local recurrence (40% [SE 13], p=0.002) and in contralateral disease (35% [SE 16], p=0.03) than in distant recurrence (24% [SE 7], p= 0.001). AIs yielded an absolute 1.6% (SE 0.8) decrease in BC mortality (6.3% vs 8.0%, relative decrease 22% [SE 9], p=0.02). Subset analyses with respect to PgR status, age, tumor grade and nodal status revealed no apparent heterogeneity between the proportional reductions in recurrence and no indication of an increase or decrease in non-breast deaths with AIs in either cohort 1 or 2.&#x2028; Conclusions: In 2 separate meta-analyses considering both the initial monotherapy setting and the switching setting after 2 to 3 yrs of Tam, AIs produced significantly lower BC recurrence rates compared with Tam. Additional follow-up data will provide important information on long-term survival. Meta-analyses can be helpful even in the era of relatively large clinical trials. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 12.