Matthew D. Teegarden

Chromatographic separation of PTAD-derivatized 25-hydroxyvitamin D3 and its C-3 epimer from human serum and murine skin.

The detection of 25-hydroxyvitamin D at low levels in biological samples is facilitated by the use of chemical derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) in concert with liquid chromatography-tandem mass spectrometry (LC-MS/MS). This mode of analysis is notably hampered by chromatographic co-elution of 25-hydroxyvitamin D3 (25OHD3) and its C-3 epimer (C3epi). The objective of this work was to improve upon current LC-MS/MS methods used for the analysis of PTAD-derivatized 25-hydroxyvitamin D3 by resolving it from C3epi. Additionally, the applicability of this method in human serum and murine skin was investigated. C18 columns of increasing length and varying particle sizes were assessed for performance using a mixed standard of PTAD-derivatized 25OHD3 and C3epi. Serum samples were processed using solid phase extraction, and skin was powdered and extracted for lipophilic compounds. The samples were derivatized with PTAD and subsequently analyzed using isotope dilution LC-MS/MS with atmospheric pressure chemical ionization operated in positive mode. Near baseline resolution of PTAD-25OHD3 from PTAD-C3epi was achieved on a 250mm C18 column with 3μm sized particles. This separation allowed for detection and quantification of both metabolites in serum and skin samples. PTAD-C3epi represented a significant confounding analyte in all samples, and comprised up to 20% of the status measurement in skin. This method is a significant improvement on the chromatography of PTAD-derivatized vitamin D metabolites that could greatly influence the assessment of vitamin D status and C3epi biology in low abundance samples.

Chemical Characterization and Antioxidant Potential of Wild Ganoderma Species from Ghana

The chemical characterization and antioxidant potential of twelve wild strains of Ganoderma sp. from Ghana, nine (LS1–LS9) of which were found growing wild simultaneously on the same dying Delonix regia tree, were evaluated. Parameters evaluated included the nutritional value, composition in sugars, fatty acids, phenolic and other organic compounds and some vitamins and vitamin precursors. Antioxidant potential was evaluated by investigating reducing power, radical scavenging activity and lipid peroxidation inhibition using five in vitro assays. Protein, carbohydrate, fat, ash and energy contents ranged between 15.7–24.5 g/100 g·dw, 73.31–81.90 g/100 g, 0.48–1.40 g/100 g, 0.68–2.12 g/100 g ash and 396.1–402.02 kcal/100 g, respectively. Fatty acids such as linoleic, oleic and palmitic acids were relatively abundant. Free sugars included rhamnose, fructose, mannitol, sucrose and trehalose. Total tocopherols, organic acids and phenolic compounds’ content ranged between 741–3191 µg/100 g, 77–1003 mg/100 g and 7.6–489 µg/100 g, respectively. There were variations in the β-glucans, ergosterol and vitamin D2 contents. The three major minerals in decreasing order were K > P > S. Ganoderma sp. strain AM1 showed the highest antioxidant activity. This study reveals, for the first time, chemical characteristics of Ganoderma spp. which grew simultaneously on the same tree.

Tomatoes protect against development of UV-induced keratinocyte carcinoma via metabolomic alterations

Prolonged tomato consumption can mitigate ultraviolet (UV) light induced sunburn via unknown mechanisms. Dietary carotenoids distributed to skin are hypothesized to protect skin against UV-induced damage, although other phytochemicals may play a role. We hypothesize that tomato consumption would protect against skin cancer. SKH-1 hairless and immunocompetent mice (n = 180) were fed AIN-93G or AIN-93G + 10% tangerine or red tomato powder for 35 weeks. From weeks 11–20, mice (n = 120) were exposed to 2240 J/m2 UV-B light, 3x/week, and tumors were tracked weekly. Control mice were fed the same diets but not exposed to UV. Tumor number was significantly lower in male mice consuming red tomato diets (1.73 ± 0.50, P = 0.015) or pooled tomato diets (2.03 ± 0.45, P = 0.017) compared to controls (4.04 ± 0.65). Carotenoid levels in plasma and skin were quantitated, with total lycopene higher in skin of tangerine fed animals despite a lower dose. Metabolomic analyses elucidated compounds derived from tomato glycoalkaloids (including tomatidine and hydroxylated-tomatidine) as significantly different metabolites in skin after tomato exposure. Here, we describe that tomato consumption can modulate risk for keratinocyte carcinomas; however, the role of the newly identified specific phytochemicals possibly responsible for this action require further investigation.

25-Hydroxyvitamin D3 and its C-3 epimer are elevated in the skin and serum of Skh-1 mice supplemented with dietary vitamin D3

SCOPE UV exposure is a risk factor for keratinocyte carcinoma (KC) while critical for endogenous vitamin D production. We investigated dietary modulation of skin and serum 25-hydroxyvitamin D3 (25OHD3 ) and its C-3 epimer (C3epi) in a mouse model of KC. C3epi is an under-investigated metabolite of vitamin D with respect to its biological implications. METHODS AND RESULTS Male and female Skh-1 mice were supplemented with 25, 150 or 1000 IU/kg diet vitamin D3 for 25 weeks, with some exposed to UV light. Skin and serum vitamin D metabolites were quantitated using HPLC-MS/MS (n = 3 per dose/sex/UV treatment). Serum and skin 25OHD3 and C3epi significantly increased with dose (P<0.0001), but with different response patterns. UV exposure significantly attenuated serum, but not skin, levels of both metabolites (P<0.001, P = 0.0287), while up-regulating expression of renal Cyp24a1 (P < 0.01). A dose by sex interaction trended toward significance with serum and skin levels of C3epi, wherein male mice attained higher levels of C3epi with higher dietary vitamin D3 . This reflected a similar, but non-significant pattern in average tumor size. CONCLUSION The complex relationship between vitamin D and KC requires further investigation. This study provides insight into modulation of local and systemic vitamin D status with dietary supplementation.

Profiling the impact of thermal processing on black raspberry phytochemicals using untargeted metabolomics

Clinical and laboratory studies have implicated black raspberries (BRBs) and their associated phytochemicals in the modulation of several chronic diseases. Most research on the health benefits of BRBs is conducted using freeze-dried or otherwise minimally processed products, yet BRBs are typically consumed as thermally processed goods like jams and syrups. The objective of this work was to profile the chemical changes that result from thermal processing of BRB powder into a nectar beverage. Using an untargeted UHPLC-QTOF-MS metabolomics approach, key degradation products of anthocyanins were identified along with several other proposed phenolic degradants. The effects of processing on other key BRB compound groups, including ellagitannins, are also discussed. This work demonstrates the utility of an untargeted metabolomics approach in describing the chemistry of complex food systems and provides a foundation for future research on the impact of processing on BRB product bioactivity.

Modulating conversion of isoflavone glycosides to aglycones using crude beta-glycosidase extracts from almonds and processed soy

Food processing alters the physicochemical state of soy which can enhance chemical and enzymatic conversion of isoflavones to their aglycone forms. This study investigated the role of β-glycosidase from processed soy-ingredient mixture (SIM) or almonds, and examined the impact of isoflavone composition in mediating conversion to aglycones. β-Glycosidase activity was quantified using p-nitrophenol-β-d-glucopyranoside and SIM isoflavone extracts. Almond β-glycosidase activity was significantly (p<0.001) reduced after roasting (99% reduction) or steaming (97% reduction) compared to raw almonds. SIM β-glycosidase activity, however, increased, with steaming by 66% (p<0.001) and with roasting by 52% (p=0.022), compared to raw SIM. After incubation with β-glycosidase, percentage of aglycone (total aglycone/total isoflavones) in SIM isoflavone extracts increased significantly in raw (35%), fermented (48%), roasted (88%) and steamed (91%) SIM, compared to their initial (∼5%) compositions. Manipulation of β-glycosidase activity and isoflavone composition can be used to modulate aglycone content in soy food products.

Abstract LB-295: Food-based approach to cancer prevention: A phase I assessment of amorphous confections in modulating exposure and metabolism of black raspberry compounds in the oral cavity

Background: Oral cancer remains a major global malignancy, largely due to persistence of tobacco use worldwide. Preventive strategies focusing upon high risk individuals due to long term tobacco use or presence of pre-malignancy are needed. Based upon strong epidemiologic and laboratory evidence we have developed a black raspberry food-based product targeting this population and have tested variations in a phase I trial. Objective: 1. To test three amorphous solids (glassy, elastic, and viscous) containing a bioactive rich black raspberry (BRB) fraction in a confectionary system for compliance. 2. To investigate the impact of amorphous structure in modulating oral exposure and metabolism of black raspberry compounds in the oral cavity as measured by differences in duration of black raspberry exposure and profile of black raspberry metabolites from saliva. Methods: A four week, phase I, randomized, parallel study design with 60 non-smoking adults was used to investigate three amorphous forms having different release rates (prolonged, intermediate, and rapid), each at two doses (4 or 8 g BRB/day). Salivary kinetics and changes in black raspberry composition among and within individuals were assessed. HPLC with Tandem MS/MS was used to quantify and characterize black raspberry compounds from the confections and profile them in the saliva. Results: All confections were well accepted having excellent adherence (94 ± 2%). Toxicities were limited to a grade I toxicity (NIH criteria). Oral residence time was 74% longer and saliva volume was 42% greater with glassy than the other two amorphous forms. Anthocyanin (cyanidin-3-glucoside) and ellagitannins were prevalent compounds in the saliva. Conclusions: Three amorphous confectionary forms of a BRB food-based delivery vehicle demonstrated excellent compliance, with the glassy form showing potential for greater oral distribution, suggesting the potential for greater bioactivity. Food science and technology provides many strategies to develop novel and fully characterized food products for cancer prevention trials. Citation Format: Jennifer H. Ahn-Jarvis, Steven K. Clinton, Elizabeth M. Grainger, Christina Simpson, Junnan Gu, Matthew D. Teegarden, Kenneth M. Riedl, Thomas J. Knobloch, Christopher M. Weghorst, Steven J. Schwartz, Yael Vodovotz. Food-based approach to cancer prevention: A phase I assessment of amorphous confections in modulating exposure and metabolism of black raspberry compounds in the oral cavity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-295.