Steroid hormones sulfatase inactivation extends lifespan and ameliorates age-related diseases.

Aging and fertility are two interconnected processes. From invertebrates to mammals, absence of the germline increases longevity. Here we show that loss of function of sul-2, the Caenorhabditis elegans steroid sulfatase (STS), raises the pool of sulfated steroid hormones, increases longevity and ameliorates protein aggregation diseases. This increased longevity requires factors involved in germline-mediated longevity (daf-16, daf-12, kri-1, tcer-1 and daf-36 genes) although sul-2 mutations do not affect fertility. Interestingly, sul-2 is only expressed in sensory neurons, suggesting a regulation of sulfated hormones state by environmental cues. Treatment with the specific STS inhibitor STX64, as well as with testosterone-derived sulfated hormones reproduces the longevity phenotype of sul-2 mutants. Remarkably, those treatments ameliorate protein aggregation diseases in C. elegans, and STX64 also Alzheimer's disease in a mammalian model. These results open the possibility of reallocating steroid sulfatase inhibitors or derivates for the treatment of aging and aging related diseases.

Vanillic Acid Restores Coenzyme Q Biosynthesis and ATP Production in Human Cells Lacking COQ6.

Coenzyme Q (CoQ), a redox-active lipid, is comprised of a quinone group and a polyisoprenoid tail. It is an electron carrier in the mitochondrial respiratory chain, a cofactor of other mitochondrial dehydrogenases, and an essential antioxidant. CoQ requires a large set of enzymes for its biosynthesis; mutations in genes encoding these proteins cause primary CoQ deficiency, a clinically and genetically heterogeneous group of diseases. Patients with CoQ deficiency often respond to oral CoQ10 supplementation. Treatment is however problematic because of the low bioavailability of CoQ10 and the poor tissue delivery. In recent years, bypass therapy using analogues of the precursor of the aromatic ring of CoQ has been proposed as a promising alternative. We have previously shown using a yeast model that vanillic acid (VA) can bypass mutations of COQ6, a monooxygenase required for the hydroxylation of the C5 carbon of the ring. In this work, we have generated a human cell line lacking functional COQ6 using CRISPR/Cas9 technology. We show that these cells cannot synthesize CoQ and display severe ATP deficiency. Treatment with VA can recover CoQ biosynthesis and ATP production. Moreover, these cells display increased ROS production, which is only partially corrected by exogenous CoQ, while VA restores ROS to normal levels. Furthermore, we show that these cells accumulate 3-decaprenyl-1,4-benzoquinone, suggesting that in mammals, the decarboxylation and C1 hydroxylation reactions occur before or independently of the C5 hydroxylation. Finally, we show that COQ6 isoform c (transcript NM_182480) does not encode an active enzyme. VA can be produced in the liver by the oxidation of vanillin, a nontoxic compound commonly used as a food additive, and crosses the blood-brain barrier. These characteristics make it a promising compound for the treatment of patients with CoQ deficiency due to COQ6 mutations.

Non-fluorescent and yellow chlorophyll catabolites in Japanese plum fruits (Prunus salicina, Lindl.).

Although several chlorophyll metabolites have been shown to exert prominent benefits to human health when consumed, the battery of linear chlorophyll derivatives (phyllobilins) presents in fruits is poorly understood. Yellow chlorophyll catabolites (YCCs) are a new kind of phyllobilins recently identified in senescent leaves, probably arising from an oxidative process of the terminal chlorophyll catabolites, NCCs (non-fluorescent chlorophyll catabolites). This work deals with the characterization by first time of this kind of phytochemicals in edible fruits. Two YCCs have been identified in yellow Japanese plums, one (Ps-YCC1) previously described in the senescent leaves of Cercidiphyllum japonicum Siebold & Zucc. and Ps-YCC2, a chlorophyll catabolite structure described by first time in the edible parts of Japanese plum fruits. These YCCs were characterized by high-resolution MS/MS, describing the specific fragmentation (ring A) and the absence of the typical cleavage of phyllobilins (ring D), as a consequence of the unsaturated bond at C15-16 typical of YCCs, allowing the differentiation from NCCs. To the already known array of phenolic acids, anthocyanins and carotenoids, NCCs and YCCs may contribute to the antioxidant potential of these fruits, a potential that deserves attention and future research, considering the photochemical and photophysical behaviour of this group of tetrapyrrolic breakdown products.

Comprehensive chlorophyll composition in the main edible seaweeds.

Natural chlorophylls present in seaweeds have been studied regarding their biological activities and health benefit effects. However, detailed studies regarding characterization of the complete chlorophyll profile either qualitatively and quantitatively are scarce. This work deals with the comprehensive spectrometric study of the chlorophyll derivatives present in the five main coloured edible seaweeds. The novel complete MS2 characterization of five chlorophyll derivatives: chlorophyll c2, chlorophyll c1, purpurin-18 a, pheophytin d and phytyl-purpurin-18 a has allowed to obtain fragmentation patterns associated with their different structural features. New chlorophyll derivatives have been identified and quantified by first time in red, green and brown seaweeds, including some oxidative structures. Quantitative data of the chlorophyll content comes to achieve significant information for food composition databases in bioactive compounds.

Chlorophyll catabolism in olive fruits (var. Arbequina and Hojiblanca) during maturation.

The central reaction of chlorophyll (chl) breakdown pathway occurring during olive fruits maturation is the cleavage of the macrocycle pheophorbide a to a primary fluorescent chl catabolite (pFCC) and it is catalyzed by two enzymes: pheophorbide a oxygenase (PaO) and red chl catabolite reductase (RCCR). In subsequent steps, pFCC is converted to different fluorescent chlorophyll catabolites (FCCs) and nonfluorescent chlorophyll catabolites (NCCs). This work demonstrated that RCCR activity of olive fruits is type II. During the study of evolution of PaO and RCCR activities through the olive fruits maturation in two varieties: Hojiblanca and Arbequina, a significant increase in PaO and RCCR activity was found in ripening stage. In addition, the profile and structure of NCCs present in epicarp of this fruit was studied using HPLC/ESI-TOF-MS. Five different NCCs were defined and for the first time the enzymatic reactions implied in chlorophyll degradations in olive fruits elucidated.

Extraction of phenolic compounds from virgin olive oil by deep eutectic solvents (DESs).

Deep eutectic solvents (DESs) are "green" solvents, applied in this study for the extraction of phenolic compounds from virgin olive oil (VOO). Different combinations of DES consisting of choline chloride (ChCl) in various mixing ratios with sugars, alcohols, organic acids, and urea, as well as a mixture of three sugars were used. The yields of the DES extractions were compared with those from conventional 80% (v/v) methanol/water. DES showed a good solubility of phenolic compounds with different polarities. The two most abundant secoiridoid derivatives in olive oil, oleacein and oleocanthal, extracted with ChCl/xylitol and ChCl/1,2-propanediol showed an increase of 20-33% and 67.9-68.3% with respect to conventional extraction, respectively. To our knowledge, this is the first time that phenolic compounds have been extracted from VOO oil using DES. Our results suggest that DES offers an efficient, safe, sustainable, and cost effective alternative to methanol for extraction of bioactive compounds from VOO.

Development of an accurate and high-throughput methodology for structural comprehension of chlorophylls derivatives. (II) Dephytylated derivatives.

Dephytylated chlorophylls (chlorophyllides and pheophorbides) are the starting point of the chlorophyll catabolism in green tissues, components of the chlorophyll pattern in storage/processed food vegetables, as well as the favoured structural arrangement for chlorophyll absorption. In addition, dephytylated native chlorophylls are prone to several modifications of their structure yielding pyro-, 13(2)-hydroxy- and 15(1)-hydroxy-lactone derivatives. Despite of these outstanding remarks only few of them have been analysed by MS(n). Besides new protocols for obtaining standards, we have developed a new high throughput methodology able to determine the fragmentation pathway of 16 dephytylated chlorophyll derivatives, elucidating the structures of the new product ions and new mechanisms of fragmentation. The new methodology combines, by first time, high resolution time-of-flight mass spectrometry and powerful post-processing software. Native chlorophyllides and pheophorbides mainly exhibit product ions that involve the fragmentation of D ring, as well as additional exclusive product ions. The introduction of an oxygenated function at E ring enhances the progress of fragmentation reactions through the ß-keto ester group, developing also exclusive product ions for 13(2)-hydroxy derivatives and for 15(1)-hydroxy-lactone ones. Consequently, while MS(2)-based reactions of phytylated chlorophyll derivatives point to fragmentations at the phytyl and propionic chains, dephytylated chlorophyll derivatives behave different as the absence of phytyl makes ß-keto ester group and E ring more prone to fragmentation. Proposals of the key reaction mechanisms underlying the origin of new product ions have been made.

Development of an accurate and high-throughput methodology for structural comprehension of chlorophylls derivatives. (I) Phytylated derivatives.

Phytylated chlorophyll derivatives undergo specific oxidative reactions through the natural metabolism or during food processing or storage, and consequently pyro-, 13(2)-hydroxy-, 15(1)-hydroxy-lactone chlorophylls, and pheophytins (a and b) are originated. New analytical procedures have been developed here to reproduce controlled oxidation reactions that specifically, and in reasonable amounts, produce those natural target standards. At the same time and under the same conditions, 16 natural chlorophyll derivatives have been analyzed by APCI-HPLC-hrMS(2) and most of them by the first time. The combination of the high-resolution MS mode with powerful post-processing software has allowed the identification of new fragmentation patterns, characterizing specific product ions for some particular standards. In addition, new hypotheses and reaction mechanisms for the established MS(2)-based reactions have been proposed. As a general rule, the main product ions involve the phytyl and the propionic chains but the introduction of oxygenated functional groups at the isocyclic ring produces new and specific productions and at the same time inhibits some particular fragmentations. It is noteworthy that all b derivatives, except 15(1)-hydroxy-lactone compounds, undergo specific CO losses. We propose a new reaction mechanism based in the structural configuration of a and b chlorophyll derivatives that explain the exclusive CO fragmentation in all b series except for 15(1)-hydroxy-lactone b and all a series compounds.

Systematic HPLC/ESI-High Resolution-qTOF-MS Methodology for Metabolomic Studies in Nonfluorescent Chlorophyll Catabolites Pathway.

Characterization of nonfluorescent chlorophyll catabolites (NCCs) and dioxobilane-type nonfluorescent chlorophyll catabolite (DNCC) in peel extracts of ripened lemon fruits (Citrus limon L.) was performed by HPLC/ESI-high resolution-qTOF-MS method. Compounds were identified in samples on the basis of measured accurate mass, isotopic pattern, and characteristic fragmentation profile with an implemented software postprocessing routine. Three NCC structures already identified in other vegetal tissues were present in the lemon fruit peels (Cl-NCC1; Cl-NCC2; Cl-NCC4) while a new structure not defined so far was characterized (Cl-NCC3). This catabolite exhibits an exceptional arrangement of the peripheral substituents, allowing concluding that the preferences for the NCC modifications could be a species-related matter.

Nonfluorescent chlorophyll catabolites in loquat fruits (Eriobotrya japonica Lindl.).

Nonfluorescent chlorophyll catabolites (NCCs) and nonfluorescent dioxobilane chlorophyll catabolites (NDCCs) are the terminal compounds of the chlorophyll degradation pathway that may display beneficial properties to human health related to their antioxidant properties, which were recently shown. A profile of NCCs/NDCC of the loquat fruit Eriobotrya japonica Lindl. is described. From the 13 known different NCC structures described to date, three have been identified in loquats. Two new structures not defined so far were characterized in loquat fruits: Ej-NCC2, which corresponds to the methyl ester at C13(2) of Bn-NCC1 and in very low amount Ej-NDCC1, the only NDCC found in loquats. Keto-enol tautomerism at the C13(1) position in NCCs is described for the first time as a regular process in chlorophyll catabolism, probably through a nonspecific mechanism since almost all the chlorophyll catabolites structures detected in fruits of loquat present keto and enol tautomers. The results obtained have been possible through a high-performance liquid chromatography coupled with electrospray ionization ion trap and quadropole time-of-flight mass spectrometry fitted with a powerful postprocessing software.

A new high-performance liquid chromatographic method with evaporative light scattering detector for the analysis of phospholipids. Application to Iberian pig subcutaneous fat.

A new method for the analysis of phospholipids by normal-phase HPLC is described using a silica column. Addition of ammonia and triethylamine to a gradient based on chloroform/methanol/water promoted a good and rapid separation of phospholipid classes (20 min run). The use of an evaporative light scattering detector permitted an accurate analysis of a mixture of phospholipids. Calibration curves were linear within different range for each phospholipid class. The LOD and LOQ obtained were below 0.03 and 0.05 mg kg⁻¹ for all cases, respectively. Besides, a new method for the separation of phospholipids from total lipids before HPLC analysis by a solid-phase extraction (SPE) with Si cartridges has been developed. This methodology gave a good recovery ranging from 97 to 117%. The method was validated with a standard mixture of phospholipids. This method has been applied to characterize the phospholipid fraction of subcutaneous fat from Iberian pig. Cardiolipin, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, and sphingomyelin have been described for first time in these samples. The fatty acid composition of the different phospholipid classes and their HPLC electrospray ionization mass spectrometry have been used for characterizing the molecular species present in each one.

Determination of ent-kaurene in subcutaneous fat of Iberian pigs by gas chromatography multi-stage mass spectrometry with the aim to differentiate between intensive and extensive fattening systems.

This work presents a gas chromatography multi-stage mass spectrometry (GC-MS(3)) method for the determination of ent-kaurene in subcutaneous fat of Iberian pig, present in adipose tissue of animals due to pasture ingestion (extensive fattening system). The method comprises a saponification and a liquid-liquid extraction of the unsaponifiable fraction, followed by an isolation of the hydrocarbon fraction by high performance liquid chromatography (HPLC) and analysis by GC-MS(3) (ion trap) with electronic ionization. The GC-MS(3) analysis allows the isolation and characterization of specific fragments from the original (MS(1)) molecular structure, and particularly, those fragments originated from the precursor ion (m/z=229) characteristic of ent-kaurene. The MS/MS product fragment m/z=213 is used as a further precursor fragment giving rise to a MS(3) spectrum specific for ent-kaurene. The limit of detection of the MS(3) technique is lower than 0.2 microg kg(-1) and a linear regression has been found between 0.2 and 112 microg kg(-1). This method is applicable for the determination of the fattening system of the Iberian pig.

Changes in phenolic composition induced by Pseudomonas savastanoi pv. Savastanoi infection in olive tree: presence of large amounts of verbascoside in nodules of tuberculosis disease.

A study was carried out to determine the changes in phenolic composition induced by tuberculosis infection in olive trees. Four ethanolic extracts were compared: olive leaf from shoots affected by Pseudomonas savastanoi pv. Savastanoi, nodules induced by this bacteria, leaf from healthy (asymptomatic) shoots, and shoots. Among the differences found, the presence of a phenolic compound in nodules was significant in much larger quantities than in leaf or shoots. Mass spectrometric analysis showed this compound to be verbascoside. The enhancement of its biosynthesis could be related to the defense mechanisms of the tree in the nodules induced by P. savastanoi and suggests the possibility of exploration of natural and biotechnological sources of this compound.