Optimization of LC​/MS (APCI)​+ Methods for the Determination of Possible Lutein Oxidation Products in Plasma and Tissues of Adult Rats

In spite of lutein and its isomer zeaxanthin being richly available in natural sources, the role of these components on redn. of age-​related macular degeneration, cancer, and cardiovascular disorders suggested that an update of the anal. procedure is required to det. the oxidative products and to understand their nutritional significance. In the present study, we have standardized and developed an improved method to obtain characteristic ions of lutein, zeaxanthin, and its major oxidative products in vivo (rats) using LC-​MS (APCI)​+. In addn., lutein and zeaxanthin isomer were sepd. on a C30 column with shorter run time with high resoln. and calibrated on the basis of picomolar concn. on HPLC (DAD)​, with the lower detection limit of 0.125 for lutein and 0.128 pmol for zeaxanthin. Characteristic mass spectral ion for lutein is m​/z 568.7 [M]​+ and 551.5 [M + H-​H2O]​+ and for zeaxanthin isomer is m​/z 568.8 [M]​+, 569.8 [M + H]​+. Further, optimized conditions produced structurally characteristic fragmented ions under standardized MS (APCI)​+ conditions. Total ionic chromatogram together with fine UV-​Visible and mass spectra were used to differentiate lutein isomers and its oxidative products, such as 523 [M+ + H+-​3CH3]​, 479 [M+ + H+-​6CH3]​, 551 [M+ + H+-​H2O]​, 276.43 [M+-​C22H19O]​, di-​epoxides and 3'-​oxolutein. The APCI mass spectral characteristics of major oxidative products of lutein in adult rat tissues are reported here for the first time, to our knowledge. These findings could provide new insights into lutein bioavailability and bioconversions with respect to health benefits

Biofunctionality of Carotenoid Metabolites: An Insight into Qualitative and Quantitative Analysis

Epidemiological and clinical studies have shown that dietary intake of carotenoid-rich fruits and vegetables is positively correlated with reduction in age-related eye diseases, atherosclerosis, certain cancers and chronic diseases. Carotenoids consist of unique chemical characteristics and are highly vulnerable to structural modifications, leading to the formation of various derivatives under physiological conditions. The identification of these molecules is necessary before addressing their biological functions. Carotenoid metabolomics is believed to be highly complex to fingerprint due to instability and interference with complex biological matrices. Noteworthy, progress has been made in understanding carotenoid metabolism or its biotransformation in biological samples. In this regard, the chapter highlights the concept of metabolomics and their related bio-analytical techniques pertaining to the detection of carotenoids and their derived products to elucidate their bio-transformation on targeted biological functions. Further, this chapter highlights the various hyphenated analytical tools and their optimization

Enhanced cytotoxic and apoptosis inducing activity of lycopene oxidation products in different cancer cell lines

Currently, upon understanding the metabolomics of carotenoids, it is important to address the key role of carotenoid derived products. In this regard, aim of the study was to elucidate and explore the role of lycopene (LYC) oxidative products generated through autoxidation (AOL) or chemical (KMnO4) oxidation (COL) against proliferation of selected cancer cells. Preliminary, we investigated the effect of LYC on cell viability of various cancer cell lines (PC-3, MCF-7, A431, HepG2, HeLa and A549). Based on the results of LYC treatment on cell cytotoxicity levels, MCF-7, PC-3 and HeLa cell lines were further tested with AOL and COL products. The decreased cell viability with depleted GSH and increased MDA levels were observed when treated with COL products than control, LYC and AOL. In addition, COL products increased ROS levels and percent apoptosis. The typical morphological changes and nuclear condensations showed that COL products have anti-proliferation and apoptosis inducing activity. Based on results, we hypothesized that ROS generation by LYC oxidation products may be one of intermediate step involved in apoptosis. The redox status and therapeutic approach of COL products in modulating ROS and induction of apoptosis in cancer cells were reported for the first time, to our knowledge. To conclude, COL products involves in cancer growth inhibition efficiently than intact LYC and AOL. Hence, there is a great potential for synthesizing or producing such carotenoid oxidation products to augment cancer complication

Sirtuin 6 inhibition protects against glucocorticoid-induced skeletal muscle atrophy by regulating IGF/PI3K/AKT signaling

Chronic activation of stress hormones such as glucocorticoids leads to skeletal muscle wasting in mammals. However, the molecular events that mediate glucocorticoid-induced muscle wasting are not well understood. Here, we show that SIRT6, a chromatin-associated deacetylase indirectly regulates glucocorticoid-induced muscle wasting by modulating IGF/PI3K/AKT signaling. Our results show that SIRT6 levels are increased during glucocorticoid-induced reduction of myotube size and during skeletal muscle atrophy in mice. Notably, overexpression of SIRT6 spontaneously decreases the size of primary myotubes in a cell-autonomous manner. On the other hand, SIRT6 depletion increases the diameter of myotubes and protects them against glucocorticoid-induced reduction in myotube size, which is associated with enhanced protein synthesis and repression of atrogenes. In line with this, we find that muscle-specific SIRT6 deficient mice are resistant to glucocorticoid-induced muscle wasting. Mechanistically, we find that SIRT6 deficiency hyperactivates IGF/PI3K/AKT signaling through c-Jun transcription factor-mediated increase in IGF2 expression. The increased activation, in turn, leads to nuclear exclusion and transcriptional repression of the FoxO transcription factor, a key activator of muscle atrophy. Further, we find that pharmacological inhibition of SIRT6 protects against glucocorticoid-induced muscle wasting in mice by regulating IGF/PI3K/AKT signaling implicating the role of SIRT6 in glucocorticoid-induced muscle atrophy.Fil: Mishra, Sneha. No especifíca;Fil: Cosentino, Claudia. Harvard Medical School; Estados UnidosFil: Tamta, Ankit Kumar. No especifíca;Fil: Khan, Danish. No especifíca;Fil: Srinivasan, Shalini. No especifíca;Fil: Ravi, Venkatraman. No especifíca;Fil: Abbotto, Elena. Università degli Studi di Genova; ItaliaFil: Arathi, Bangalore Prabhashankar. No especifíca;Fil: Kumar, Shweta. No especifíca;Fil: Jain, Aditi. No especifíca;Fil: Ramaian, Anand S.. No especifíca;Fil: Kizkekra, Shruti M.. No especifíca;Fil: Rajagopal, Raksha. No especifíca;Fil: Rao, Swathi. No especifíca;Fil: Krishna, Swati. No especifíca;Fil: Asirvatham Jeyaraj, Ninitha. Indian Institute of Technology; IndiaFil: Haggerty, Elizabeth R.. Harvard Medical School; Estados UnidosFil: Silberman, Dafne Magalí. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Kurland, Irwin J.. No especifíca;Fil: Veeranna, Ravindra P.. No especifíca;Fil: Jayavelu, Tamilselvan. No especifíca;Fil: Bruzzone, Santina. Università degli Studi di Genova; ItaliaFil: Mostoslavsky, Raul. Harvard Medical School; Estados UnidosFil: Sundaresan, Nagalingam R.. No especifíca

Low-dose doxorubicin with carotenoids selectively alters redox status and upregulates oxidative stress-mediated apoptosis in breast cancer cells

The combination of carotenoids and doxorubicin (DOX) selectively alters oxidative stress-mediated apoptosis in breast cancer cells. Primarily, cytotoxic efficiency of carotenoids (β-carotene, BC; lutein, LUT; astaxanthin, AST; or fucoxanthin, FUCO) either with or without a minimal cytotoxic dose of DOX was evaluated in MCF-7 (0.12 μM) and MDA-MB-231 cells (0.28 μM). The higher cell growth inhibition of BC and/or LUT with DOX was selected for testing in further cell-based assays. Low-dose DOX significantly enhanced cytotoxicity in carotenoid (1 μM) or carotenoid (20 μM) treatment alone. Depleted glutathione, increased lipid peroxides and increased ROS levels in cells confirmed the cytotoxic effect. Furthermore, mitochondrial dysfunction, cell growth arrest at G0/G1 phase and caspase cascades as well as up- and down-regulated expression levels of related proteins (p21, p27, Bax, p53, Bcl-2, and cyclin D1) revealed the synergistic effect of carotenoid and DOX treatment on ROS-mediated apoptosis. These observations demonstrated increased apoptosis in BC + DOX/LUT + DOX-treated cells due to the pronounced pro-oxidant action. Interestingly, normal breast epithelial cells (MCF 10A) exposed to similar treatments resulted in non-significant cytotoxicity. These newly observed mechanistic differences of anticancer drugs on the mitigation of toxicity with carotenoids may provide insight into the targeting of cancer therapy

Fractionation and Characterization of Lycopene-Oxidation Products by LC-MS/MS (ESI)(+): Elucidation of the Chemopreventative Potency of Oxidized Lycopene in Breast-Cancer Cell Lines

Lycopene (LYC) has been correlated with the reduction of certain cancers and chronic diseases. However, the existence and biofunctionality of degraded, oxidized, and biotransformed LYC products in vivo have not been revealed. Therefore, this study aimed to screen and elucidate the potential bioactive lycopene-derived products in breast-cancer and noncancerous cells. LYC-oxidation or -cleavage products were generated using KMnO4. These oxidation products were separated as fractions I-III by silica column chromatography using gradient solvent systems. Further, LC-MS/MS (ESI)(+) was used to elucidate their possible fragmentation patterns and structures. Fraction II showed higher cytotoxicity (IC50 value of 64.5 mu M), cellular uptake, and apoptosis-inducing activity in MCF-7 cells. This fraction consists of major peak m/z 323, identified as apo-8,6'-carotendial. The cytotoxicity-inducing activity may be due to partial ROS generation with mitochondrial dysfunction. Further, the role of apo-8,6'-carotendial in the induction of apoptosis is demonstrated for the first time. These results illustrated that LYC-oxidation derivatives or metabolites are involved in growth inhibition of cancer cells. Exploration of specific oxidized-carotenoid products will give further insight into the field of nutritional biochemistry