Mass Spectrometric Confirmation of γ-Linolenic Acid Ester-Linked Ceramide 1 in the Epidermis of Borage Oil Fed Guinea Pigs.

Ceramide 1 (Cer1), a Cer species with eicosasphingenine (d20:1) amide-linked to two different ω-hydroxy fatty acids (C30wh:0:C32wh:1), which are, in turn, ester-linked to linoleic acid (LNA; 18:2n-6), plays a critical role in maintaining the structural integrity of the epidermal barrier. Prompted by the recovery of a disrupted epidermal barrier with dietary borage oil [BO: 36.5% LNA and 23.5% γ-linolenic acid (GLA; 18:3n-6)], in essential fatty acid (EFA)-deficient guinea pigs, we further investigated the effects of BO on the substitution of ester-linked GLA for LNA in these two epidermal Cer1 species by LC-MS in positive and negative modes. Dietary supplementation of BO for 2 weeks in EFA-deficient guinea pigs increased LNA ester-linked to C32wh:1/d20:1 and C30wh:0/d20:1 of Cer1. Moreover, GLA ester-linked to C32wh:1/d20:1, but not to C30wh:0/d20:1, of Cer1 was detected, which was further confirmed by the product ions of m/z 277.2 for ester-linked GLA and m/z 802.3 for the deprotonated C32wh:1/d20:1. C20-Metabolized fatty acids of LNA or GLA were not ester-linked to these Cer1 species. Dietary BO induced GLA ester-linked to C32wh:1/d20:1 of epidermal Cer1.

Quantification of 4-methylimidazole in carbonated beverages by ultra-performance liquid chromatography-tandem mass spectrometry.

2- and 4-methylimidazoles (2-MI and 4-MI) are undesired byproducts produced during the manufacture of caramel color used to darken food products such as carbonated beverages. The Office of Environmental Health Hazard Assessment in California listed 4-MI as carcinogen in January 2011 with a proposed no significant risk level at 29 µg per person per day. Thus, a quantitative analytical measurement for 2-MI and 4-MI is desired for reliable risk assessments for exposure. An ultra-performance liquid chromatography (UPLC) coupled tandem mass spectrometric (MS/MS) method was developed for the quantification of 4-MI in beverage samples. Chromatographic separation of 2-MI and 4-MI were achieved by using a PFP reversed-phase column and a stepwise gradient of methanol and distilled water containing 0.1 % formic acid. Identification and quantification of 2-MI and 4-MI were performed using electrospray ionization-tandem mass monitoring the precursor to product ion transitions for 2-MI at m/z 83.1 → 42.2 and 4-MI at m/z 83.1 → 56.1 with melamine at m/z 127.1 → 85.1 as the internal standard. The performance of the method was evaluated against validation parameters such as specificity, carryover, linearity and calibration, correlation of determination (r(2)), detection limit, precision, accuracy, and recovery. Calibration curves at 10-400 ng/mL were constructed by plotting concentration versus peak-area ratio (analyte/internal standard) and fitting the data with a weighted 1/x. The accuracy of the assay ranged from 93.58 to 110.53 % for all analytes. Intra-assay precision for 2-MI and 4-MI were below 7.28 (relative standard deviation/RSD %) at QC samples. Here we present a new and improved method using UPLC-MS/MS to significantly simplify sample preparation and decrease chromatographic run time. This method allows accurate and reproducible quantification of 4-MI in carbonated beverages as low as sub ng/mL (ppb) levels.

Ginsenoside compound K inhibits angiogenesis via regulation of sphingosine kinase-1 in human umbilical vein endothelial cells.

Ginsenoside compound K (CK) is a metabolite of the protopanaxadiol-type saponins of Panax ginseng C.A. Meyer (Araliaceae), has long been used to treat against the development of cancer, inflammation, allergies, and diabetes. This study examined the anti-angiogenic properties of CK against sphingosine 1-phosphate (S1P)-induced cell migration via regulation of sphingosine kinase 1 (SPHK1) in human umbilical vein endothelial cells (HUVEC). Studies on S1P-induced cell migration, expression of SPHK1 and MMPs and analysis of sphingolipid metabolites by LC-MS/MS were examined after the treatment of CK (2.5, 5, 10 µg/mL) in HUVEC. S1P produced by SPHK1 is also involved in cell growth, migration, and protection of apoptosis; therefore, we sought to investigate whether ginsenosides are able to regulate SPHK1. For this purpose, we developed an inhibitory assay of SPHK1 activity and an analytical method for detection of S1P and other sphingolipid metabolites in HUVEC. Ginsenoside CK inhibited 100 nM S1P-induced cell migrations in a dose-dependent manner. Among tested ginsenosides, CK exclusively inhibited S1P production, SPHK1 activity and SPHK1 expression in HUVEC, whereas expression of the pro-apoptotic sphingolipids, sphingosine and ceramide, was increased in response to CK. The major subspecies of the increased ceramide was C24:0-ceramide. CK also disrupted the sphingolipid rheostat, which ultimately influences cell fate, and dose-dependently inhibited HUVEC migration by reducing expression of metalloproteinases (MMPs). Ginsenoside CK acts as a unique HUVEC migration inhibitor by regulating MMP expression, as well as the activity of SPHK1 and its related sphingolipid metabolites.

Terpene alcohols inhibit de novo sphingolipid biosynthesis.

The terpene alcohols geranyllinalool, phytol (diterpene alcohol), and farnesol (sesquiterpene alcohol) were newly found to inhibit sphingolipid de novo biosynthesis in LLC-PK1 cells (pig kidney epithelial cells). A simple chromatographic bioassay was established for the screening of inhibitory compounds able to reduce the amount of sphinganine, an intermediate metabolite of sphingolipid biosynthesis. The screening strategy was based on the degree of suppression of fumonisin B1 (FB1-induced sphinganine accumulation following co-treatment with selected terpene alcohols. L-cycloserine and ISP-1, specific serine palmitoyltransferase (SPT) inhibitors, were used as positive controls. Our results show that measuring reduced sphinganine levels after treatment with 2 µM FB1 in combination with the putative inhibitory compounds provides a useful screening bioassay for evaluating compounds causing sphingolipid depletion. Intracellular sphinganine concentrations were analyzed using the fluorescent peak areas of the O-phthalaldehyde (OPA) derivatives of sphinganine eluted with 87 % acetonitrile on a reversed-phase column. Geranyllinalool, phytol, and farnesol were identified as novel SPT inhibitors that reduce FB1-induced sphinganine accumulation and thus inhibit the first step of sphingolipid de novo synthesis.

Inhibition of sphingolipid metabolism enhances resveratrol chemotherapy in human gastric cancer cells.

Resveratrol, a chemopreventive agent, is rapidly metabolized in the intestine and liver via glucuronidation. Thus, the pharmacokinetics of resveratrol limits its efficacy. To improve efficacy, the activity of resveratrol was investigated in the context of sphingolipid metabolism in human gastric cancer cells. Diverse sphingolipid metabolites, including dihydroceramides (DHCer), were tested for their ability to induce resveratrol cytotoxicity. Exposure to resveratrol (100 µM) for 24 hr induced cell death and cell cycle arrest in gastric cancer cells. Exposure to the combination of resveratrol and dimethylsphingosine (DMS) increased cytotoxicity, demonstrating that sphingolipid metabolites intensify resveratrol activity. Specifically, DHCer accumulated in a resveratrol concentration-dependent manner in SNU-1 and HT-29 cells, but not in SNU-668 cells. LC-MS/MS analysis showed that specific DHCer species containing C24:0, C16:0, C24:1, and C22:0 fatty acids chain were increased by up to 30-fold by resveratrol, indicating that resveratrol may partially inhibit DHCer desaturase. Indeed, resveratrol mildly inhibited DHCer desaturase activity compared to the specific inhibitor GT-11 or to retinamide (4-HPR); however, in SNU-1 cells resveratrol alone exhibited a typical cell cycle arrest pattern, which GT-11 did not alter, indicating that inhibition of DHCer desaturase is not essential to the cytotoxicity induced by the combination of resveratrol and sphingolipid metabolites. Resveratrol-induced p53 expression strongly correlated with the enhancement of cytotoxicity observed upon combination of resveratrol with DMS or 4-HPR. Taken together, these results show that DHCer accumulation is a novel lipid biomarker of resveratrol-induced cytotoxicity in human gastric cancer cells.