Georges Riss

Rapid determination of 25-hydroxy vitamin D3 in swine tissue using an isotope dilution HPLC-MS assay.

A rapid method for quantification of 25-hydroxy vitamin D3 in different swine tissues based on isotope dilution HPLC-MS has been developed and validated. Six times deuterated analyte is used as internal standard. The method is fast and can be performed with only 1g sample. Sample preparation for kidney, liver, muscle and spleen requires only homogenisation and extraction with methanol. An additional enzymatic digest is required for skin, and clean-up of the extract by solid-phase extraction (SPE) is used for adipose tissue and skin. The lower limit of detection varies from 1 ng/g (muscle) to 5 ng/g (adipose and skin). The method has been successfully applied to various tissue samples of pigs fed for 119 days either 2000 IU of vitamin D3 or 50 microg of 25-hydroxy vitamin D3 per kg feed. For animals ingesting 25-OH-D3 supplements the highest tissue contents were observed in the skin (24.8+/-3.5 ng/g), followed by kidney (14.2+/-1.5 ng/g), liver and muscle (5.7+/-0.6 ng/g). The 25-OH-D3 content in the skin was significantly higher in animals ingesting 2000 IU/kg of vitamin D3 (39.5+/-13.4 ng/g). Levels in selected tissues of some animals were below the lower limit of quantification. No measurable amounts of 25-OH-D3 were found in spleen, abdominal fat and subcutaneous fat of the animals of both groups as well as in the liver, kidney and muscle of the animals ingesting 2000 IU/kg of vitamin D3.

The Natural Compound Ascorbigen Modulates NADPH-Quinone Oxidoreductase (NQO1) mRNA and Enzyme Activity Levels in Cultured Liver Cells and in Laboratory Rats

Ascorbigen (ABG) is a natural compound that represents a breakdown product of the glucosinolates that are present in Brassica vegetables. It is postulated that ABG may have anticarcinogenic activity; however, the underlying molecular and cellular mechanisms are largely unknown. In the present study we investigated the effect of ABG on the mRNA and enzyme activity levels of NADPH-quinone oxidoreductase (NQO1), which is centrally involved in the detoxification of xenobiotics, in cultured liver cells and in rats. The mRNA levels of NQO1 showed an increase of up to 100% in cultured liver cells (HepG2) following incubation with different concentrations of ABG (3–100 µmol/l) compared to control cells. Furthermore, NQO1 activity was elevated (up to 20%) by ABG treatment. The in vitro results were confirmed in rats who received either 5 mg/day ABG or vehicle for 7 days. Significantly higher mRNA (a 90% increase) and enzyme activity levels (a 40% increase) of NQO1 were detected in the liver of ABG-treated rats as compared to control animals. Current data indicate that ABG is a moderate inducer of the phase II enzyme NQO1, both in cultured hepatocytes and in vivo.

β-Carotene interference with UVA-induced gene expression by multiple pathways

UVA exposure causes skin photoaging by singlet oxygen (1O2)-mediated induction of matrix metalloproteases (MMPs). We assessed whether β-carotene, a carotenoid known as 1O2 quencher and retinoic acid (RA) precursor, interferes with UVA-induced gene regulation and prevents UVA-induced gene regulation in HaCaT human keratinocytes. HaCaT cells accumulated β-carotene in a time- and dose-dependent manner. UVA irradiation massively reduced the cellular β-carotene contents. β-Carotene suppressed UVA induction of MMP-1, MMP-3, and MMP-10 - three major MMPs involved in photoaging. HaCaT cells produced weak retinoid activity from β-carotene, as demonstrated by mild up-regulation of retinoid receptor RARβ and activation of an RARE-dependent reporter gene. Of the 568 UVA-regulated genes, β-carotene reduced the UVA effect for 143, enhanced it for 180, and did not interact with UVA for 245 genes. The pathways regulated β-carotene in interaction with UVA were characterized by genes involved in growth factor signaling, stress response, apoptosis, cell cycle, extracellular matrix (ECM) degradation, tanning, and inflammation. In conclusion, β-carotene at physiological concentrations interacted with UVA effects by multiple mechanisms that included, but were not restricted to, 1O2 quenching. With our results, we provide a mechanistic basis for the long-known and clinically established photoprotective effects of β-carotene in human skin.