Measurement of deuterium-labeled phylloquinone in plasma by high-performance liquid chromatography/mass spectrometry.

Phylloquinone (vitamin K(1)) is a lipophilic compound present in plasma at low concentrations, which presents technical challenges for determining its bioavailability or metabolic fate using stable isotopes. We developed a method to simultaneously measure unlabeled and deuterium-labeled phylloquinone concentrations in plasma specimens using high-performance liquid chromatography/mass spectrometry with atmospheric pressure chemical ionization (LC-APCI/MS). Phylloquinone was extracted from plasma using hexane, further purified by solid-phase extraction, and then quantified using high-performance liquid chromatography with an APCI/MS as a detector. Plotting the expected versus the measured amount of serial dilutions of either unlabeled or labeled phylloquinone gave correlation coefficients (R) of 0.999 for both compounds. The minimum detectable concentrations of unlabeled and labeled phylloquinone were 0.05 and 0.08 pmol/injection, respectively. Pooled plasma samples spiked with between 0.5 and 32 nmol phylloquinone/L gave average recoveries of 96.7% with 5.4% relative standard deviation (RSD) for unlabeled phylloquinone and 96.2% with 6.6% RSD for labeled phylloquinone. Plasma phylloquinone concentrations determined by LC-fluorescence and LC-APCI/MS methods from healthy subjects (n = 17) were not statistically different (P = 0.13). The LC-APCI/MS method is a sensitive technique for simultaneous determination of both unlabeled and labeled phylloquinone and can be applied to bioavailability studies.

Significant correlation of species longevity with DNA double strand break recognition but not with telomere length.

The identification of the cellular mechanisms responsible for the wide differences in species lifespan remains one of the major unsolved problems of the biology of aging. We measured the capacity of nuclear protein to recognize DNA double strand breaks (DSBs) and telomere length of skin fibroblasts derived from mammalian species that exhibit wide differences in longevity. Our results indicate DNA DSB recognition increases exponentially with longevity. Further, an analysis of the level of Ku80 protein in human, cow, and mouse suggests that Ku levels vary dramatically between species and these levels are strongly correlated with longevity. In contrast mean telomere length appears to decrease with increasing longevity of the species, although not significantly. These findings suggest that an enhanced ability to bind to DNA ends may be important for longevity. A number of possible roles for increased levels of Ku and DNA-PKcs are discussed.