Middle-aged offspring of nonagenarians, as compared to their spouses (controls), show a favorable lipid metabolism marked by larger LDL particle size in men and lower total triglyceride levels in women. To investigate which specific lipids associate with familial longevity, we explore the plasma lipidome by measuring 128 lipid species using liquid chromatography coupled to mass spectrometry in 1526 offspring of nonagenarians (59 years ± 6.6) and 675 (59 years ± 7.4) controls from the Leiden Longevity Study. In men, no significant differences were observed between offspring and controls. In women, however, 19 lipid species associated with familial longevity. Female offspring showed higher levels of ether phosphocholine (PC) and sphingomyelin (SM) species (3.5–8.7%) and lower levels of phosphoethanolamine PE (38:6) and long-chain triglycerides (TG) (9.4–12.4%). The association with familial longevity of two ether PC and four SM species was independent of total triglyceride levels. In addition, the longevity-associated lipid profile was characterized by a higher ratio of monounsaturated (MUFA) over polyunsaturated (PUFA) lipid species, suggesting that female offspring have a plasma lipidome less prone to oxidative stress. Ether PC and SM species were identified as novel longevity markers in females, independent of total triglycerides levels. Several longevity-associated lipids correlated with a lower risk of hypertension and diabetes in the Leiden Longevity Study cohort. This sex-specific lipid signature marks familial longevity and may suggest a plasma lipidome with a better antioxidant capacity, lower lipid peroxidation and inflammatory precursors, and an efficient beta-oxidation function.

Introduction: Slow channel congenital myasthenic syndrome is a dominant disorder characterized by prolonged acetylcholine receptor ion‐channel activation. Methods: Molecular genetic techniques, electrophysiology, and binding studies in human embryonic kidney (HEK) 293 cells determined mutant function and expression levels. Patient response to treatment was measured by quantitative myasthenic gravis and Medical Research Council grade strength scores. Results: We report an unusual case due to heteroallelic mutations in CHRNE. The slow channel mutation, p.εS278del, is accompanied by a severe low‐expression mutation, p.εR217L, on the second allele. Expression studies and cosegregation of p.εS278del with the disorder in the patient's offspring demonstrate robust expression of the p.εS278del mutation. The patient showed modest benefits from standard treatment with fluoxetine, but there was dramatic improvement when salbutamol was combined with fluoxetine. Conclusions: This case suggests that salbutamol, which is beneficial in some other congenital myasthenic syndromes, might also be considered in addition to fluoxetine in slow channel syndrome. Muscle Nerve, 2013

Liquid chromatography (LC)/mass spectrometry (MS) in selected-reactions-monitoring (SRM) mode provides a powerful tool for targeted protein quantification. However, efficient, high-throughput strategies for proper selection of signature peptides (SP) for protein quantification and accurate optimization of their SRM conditions remain elusive. Here we describe an on-the-fly, orthogonal array optimization (OAO) approach that enables rapid, comprehensive, and reproducible SRM optimization of a large number of candidate peptides in a single nanoflow-LC/MS run. With the optimized conditions, many peptide candidates can be evaluated in biological matrices for selection of the final SP. The OAO strategy employs a systematic experimental design that strategically varies product ions, de-clustering energy and collision energy in a cycle of 25 consecutive SRM trials, which accurately reveals the effects of these factors on the single-to-noise ratio of a candidate peptide, and optimizes each. As proof of concept, we developed a highly sensitive, accurate, and reproducible method for the quantification of carbonyl reductases CBR1 and CBR3 in human liver. Candidate peptides were identified by nano-LC/LTQ/Orbitrap, filtered using a stringent set of criteria, and subjected to OAO. After evaluating both sensitivity and stability of the candidates, two SP were selected for quantification of each protein. As a result of the accurate OAO of assay conditions, sensitivities of 80 and 110 amol were achieved for CBR1 and CBR3, respectively. The method was validated and used to quantify the CBRs in 33 human liver samples. The mean level of CBR1 was 93.4±49.7 (range: 26.2–241) ppm of total protein, and for CBR3 was 7.69±4.38 (range: 1.26–17.9) ppm. Key observations of this study are that: i) evaluation of peptide stability in the target matrix is essential for final selection of the SP; ii) utilization of two unique SP contributes to high reliability of target protein quantification; and iii) it is beneficial to construct calibration curves using standard proteins of verified concentrations to avoid severe biases that may result if synthesized peptides alone are used. Overall, the OAO method is versatile and adaptable to high-throughput quantification of validated biomarkers identified by proteomic discovery experiments.

ABSTRACT:Carbonyl reductase 1 (CBR1) reduces the anticancer drug doxorubicin into the cardiotoxic metabolite doxorubicinol. We documented the hepatic expression of CBR1 in samples from white and black donors. Concordance between ethnicity and geographical ancestry was examined with ancestry informative markers. Livers from blacks and whites showed similar CBR1 mRNA levels (CBR1 mRNA blacks ‫؍‬ 4.8 ؎ 4.3 relative -fold versus CBR1 mRNA whites ‫؍‬ 3.6 ؎ 3.6 relative -fold; p ‫؍‬ 0.217). CBR1 protein levels did not differ between both groups (CBR1 blacks

The role of classical lipids in aging diseases and human longevity has been widely acknowledged. Triglyceride and cholesterol concentrations are clinically assessed to infer the risk of cardiovascular disease while larger lipoprotein particle size and low triglyceride levels have been identified as markers of human longevity. The rise of lipidomics as a branch of metabolomics has provided an additional layer of accuracy to pinpoint specific lipids and its association with aging diseases and longevity. The molecular composition and concentration of lipid species determine their cellular localization, metabolism, and consequently, their impact in disease and health. For example, low density lipoproteins are the main carriers of sphingomyelins and ceramides, while high density lipoproteins are mostly loaded with ether phosphocholines, partly explaining their opposing roles in atherogenesis. Moreover, the identification of specific lipid species in aging diseases and longevity would aid to clarify how these lipids alter health and influence longevity. For instance, ether phosphocholines PC (O-34:1) and PC (O-34:3) have been positively associated with longevity and negatively with diabetes, and hypertension, but other species of phosphocholines show no effect or an opposite association with these traits confirming the relevance of the identification of molecular lipid species to tackle our understanding of healthy aging and disease. Up-to-date, a minor fraction of the human plasma lipidome has been associated to healthy aging and longevity, further research would pinpoint toward specific lipidomic profiles as potential markers of healthy aging and metabolic diseases.

ABSTRACT:Human carbonyl reductase 1 (CBR1) metabolizes endogenous and xenobiotic substrates such as the fever mediator, prostaglandin E2 (PGE 2 ), and the anticancer anthracycline drug, daunorubicin. We screened 33 CBR1 full-length cDNA samples from white and black liver donors and performed database analyses to identify genetic determinants of CBR1 activity. We pinpointed a single nucleotide polymorphism on CBR1 (CBR1 V88I) that encodes for a valine-toisoleucine substitution for further characterization. Human carbonyl reductase 1 (CBR1) is a monomeric cytosolic enzyme that catalyzes the two-electron reduction of biologically and pharmacologically active substrates by using NADPH as cofactor . For example, CBR1 converts the potent fever mediator prostaglandin E 2 (PGE 2 ) into the less active metabolite PGF 2␣ . Recent studies have shown that the transcription of CBR1 in lung and liver is down-regulated to suppress the catabolism of PGE 2 during the course of the febrile response (Ivanov et al., 2003;Ivanov and Romanovsky, 2004). CBR1 also plays a predominant role in the metabolism of the anticancer anthracyclines doxorubicin and daunorubicin by catalyzing the formation of their corresponding C-13 alcohol metabolites (doxorubicinol and daunorubicinol). Anthracycline C-13 alcohol metabolites circulate in plasma, have half-lives similar to those of the parent compounds, and are devoid of significant tumor cell-killing activity. The use of anthracyclines for cancer treatment is hampered by the development of clinical cardiotoxicity in some patients (Wouters et al., 2005). The pathogenesis of anthracycline-related cardiotoxicity is complex and appears to be mediated by a combination of oxidative stress and intracardiac metabolic perturbations induced by C-13 anthracycline alcohol metabolites (Minotti et al., 2004). In human myocardium, cytosolic carbonyl reductases (CBRs), and aldo/keto reductases catalyze the two-electron reduction of the anthracycline side chain C-13 carbonyl group to form cardiotoxic alcohol metabolites (e.g., doxorubicinol, daunorubicinol). CBR activity is the main source of quinone detoxification in humans, and biochemical studies showed that aldo/keto reductases have 7-to 18-fold lower catalytic efficiencies (V max /K m ) for the reduction of anthracycline substrates than CBRs (Wermuth et al., 1986;Ohara et al., 1995;Rosemond and Walsh, 2004;Covarrubias et al., 2006). The key role of CBR1 during the pathogenesis of anthracycline-related cardiotoxicity has been established in biochemical studies and in experiments with murine models (Mordente et al., 2001). Mice overexpressing human CBR1 in heart showed high intracardiac levels of doxorubicinol and increased signs of myocardial damage after doxorubicin administration . Conversely, mice with a

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Human carbonyl reductase 1 (CBR1) metabolizes a variety of substrates, including the anticancer doxorubicin and the antipsychotic haloperidol. The transcriptional regulation of CBR1 has been largely unexplored. Therefore, we first investigated the promoter activities of progressive gene-reporter constructs encompassing up to 2.4 kilobases upstream of the translation start site of CBR1. Next, we investigated whether CBR1 mRNA levels were altered in cells incubated with prototypical receptor activators (e.g., dexamethasone and rifampicin). CBR1 mRNA levels were significantly induced (5-fold) by the ligand of the aryl hydrocarbon receptor (AHR) ␤-naphthoflavone. DNA sequence analysis revealed two xenobiotic response elements ( Ϫ122 XRE and Ϫ5783 XRE) with potential regulatory functions. CBR1 promoter constructs lacking the Ϫ122 XRE showed diminished (9-fold) promoter activity in AHR-proficient cells incubated with ␤-naphthoflavone. Fusion of Ϫ5783 XRE to the Ϫ2485 CBR1 reporter construct enhanced its promoter activity after incubations with ␤-naphthoflavone by 5-fold. Furthermore, we tested whether the potent AHR ligand 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) induced Cbr1 expression in Ahr ϩ/Ϫ and Ahr Ϫ/Ϫ mice. TCDD induced hepatic Cbr1 mRNA (TCDD, 2-fold) and Cbr1 protein levels (TCDD, 2-fold) in Ahr ϩ/Ϫ mice compared with vehicle-injected controls. In contrast, no significant Cbr1 mRNA and Cbr1 protein induction was detected in livers from Ahr Ϫ/Ϫ mice treated with TCDD. These studies provide the first insights on the functional characteristics of the human CBR1 gene promoter. Our data indicate that the AHR pathway contributes to the transcriptional regulation of CBR1.