Bioactive phytochemicals of tree nuts. Determination of the melatonin and sphingolipid content in almonds and pistachios

Abstract

Tree nuts are healthy foods rich in bioactive phytochemicals. Their regular, moderate consumption has been associated with a reduced risk of chronic-degenerative diseases, in the context of a healthy diet and lifestyle. This study aimed to investigate the phytochemical profile of almonds and pistachios, in order to add new elements in the complex scenario of nut chemistry. A LC-MS/MS method was developed to quantify melatonin and ceramides in almonds and pistachios. In general, pistachios were richer in melatonin (2609±3096 vs 1222±500 pg/g) and total ceramides (302±77 vs 165±21 pmol/g) than almonds. Among total ceramides fatty acyl homologs, the most represented was the C16:0 species, both for ceramides and dihydroceramides, and both in almonds (37-40%) and pistachios (51-74%). Overall, these results add a piece of information to elucidate the chemical composition of almonds and pistachios and provide a rationale for the nutraceutical potential of nuts in the Mediterranean diet.

Discovery of Unexpected Sphingolipids in Almonds and Pistachios with an Innovative Use of Triple Quadrupole Tandem Mass Spectrometry

“…This report furthers our previous one on the targeted analysis of ceramides (d18:0, d18:1) in almond and pistachio [9]. This technique exploits the performance of the triple quadrupole tandem mass spectrometer well beyond its traditional use by analytical chemists.…”

“…Miraliakbari and Shahidi et al [8] reported that almond and pistachio oils contain 240 and 330 mg/100 g of total sphingolipids, respectively, measured utilising TLC-FID as a non-specific method of quantification. Using 750 Da as a mean representative value for sphingolipid molecular masses, this value corresponds to approximately 500 nmol/100 g. By using LC-MS/MS, we found 200 pmol/g = 20 nmol/100 g of ceramide species with the "standard" (i.e., mammalian) sphingoid base, 1,3-dihydroxy-D4-C18 sphingosine [9]. Even accounting for inefficient extraction and unspecific measurement, this discrepancy suggests that there is a major pool of sphingolipids unaccounted for in nuts.…”

“…We modified our standard LC-MS-MS method used for targeted ceramide measurement [9] to allow the systematic identification (discovery) of ceramides and ceramide hexosides with different sphingosine and fatty acid components. Modifications include:…”

“…(a) The use of full scan Precursor Ion (PI scan) of five O" ceramide reporter fragments (m/z 262, 264, 266, 280, 282; Table 1) to highlight ceramides with modified sphingosines; (b) The use of a full scan Neutral Loss of C 6 H 12 O 6 hexose (180.2 Da) to ascertain whether the putatively highlighted ceramides have a hexose attached unit; (c) The use of a collision energy ramp synchronized to the scan of Q1 in the PI and NL modes (iso-energetic Precursor Ion, i-PI, and iso-energetic Neutral Loss, i-NL) to analyze all ceramides at the same value of effective collision energy (see Appendix A); (d) An extended isocratic step (in respect to the previously published conditions [9]) at full gradient strength during the UPLC analysis of sphingolipids (total analysis time 22 min) to investigate the possible presence of ceramides with much longer fatty acids. To verify whether any identified compound is really a ceramide hexoside (there is no possibility to ascertain hexose stereochemistry and configuration with this set of experiments), an iso-energetic Neutral Loss scan (i-NL) of the hexose fragment (C6H12O6, MW 180.2 Da) was paired to the Precursor Ion scan that yielded the putative ceramide hexoside.…”

“…The total lipid extracts were prepared as described elsewhere [9]. Briefly, after the addition of 10 µL of IS (Cer C12, gluCer C12 20 mM), the powder (250 mg) was extracted in a microtube with an O-ring seal screw with 500 µL of methanol, 100 µL water, and 250 µL chloroform [32].…”