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With an increasing awareness that vitamin D deficiency has far-reaching implications on human health, researchers have been actively investigating more effective methods for assessing vitamin D status. Vitamin D deficiency, which is relatively common in the United Kingdom (UK), not only increases the risk of osteoporosis but can also lead to rickets in children, and osteomalacia in adults. So what are some of the factors that contribute to vitamin D deficiency in the UK? There is actually a well observed seasonal cycle in population serum 25-hydroxyvitamin [25(OH)D] concentrations. Particularly during winter, the small amount of UVB in sunlight is insufficient to initiate synthesis of any biologically relevant quantities of vitamin D1. Although supplementation is a common element of the treatment plan for deficiency, the blood levels of vitamin D that warrant supplementation are under debate.
Laboratory tests are required to assess vitamin D status, since no physical test is available. Most assays provide a measure of total 25(OH)D [where total 25(OH)D is equal to the sum of all 'free' and protein-bound 25(OH)D]. However, the levels of vitamin D binding proteins can vary from one individual to the next, and can also vary under certain clinical situations, and therefore it has been postulated that a measurement of the total 25(OH)D concentration may not accurately reflect the amount of bioavailable vitamin D. The "free hormone hypothesis" proposes that there is more clinical utility in directly measuring the levels of "free" vitamin D.
Researchers at the University of Sheffield2, working in collaboration with researchers at Ghent University3, are currently investigating the 'free hormone hypothesis' for the measurement of 25(OH)D using human saliva samples. Laboratorians are increasingly looking towards alternative sample matrices, such as human saliva, which allow for less invasive sample collection procedures, and have the potential to be more cost-effective. Human saliva provides the additional advantage that it does not contain any vitamin D binding proteins, and therefore may be an ideal biological fluid for the direct measurement of free 25(OH)D levels. However, the 25(OH)D concentrations in saliva are relatively low and therefore an ultra-sensitive analytical technique is required.
The research team selected LC-MS/MS, a technique which is gaining traction in clinical research laboratories due to its increased sensitivity and specificity compared to traditional techniques. Using highly sensitive LC-MS/MS methods developed on the SCIEX Triple Quad™ 5500 system, the researchers are investigating the measurement 25(OH)D in saliva as a surrogate for 'free' 25(OH)D levels in plasma.
In order to further enhance the sensitivity of this methodology, the researchers have employed a derivatization strategy using the SCIEX Amplifex™ Diene reagent. Derivatization with this reagent boosts analytical sensitivity by increasing the ionization efficiency of the target molecule, providing a signal enhancement of up to 1000 fold. With a one-step, 30 minute derivatization at room temperature, Amplifex Diene reagent is positioned to be a key advantage in this research.
Speed and simplicity: Amplifex Diene derivatization is a one step, 30 minute reaction carried out at room temperature.
You asked for it, we designed it
Recently our customers told us they wanted an Amplifex Diene reagent with greater stability/ longer shelf life, so we changed the formulation and packaging to accomplish just that. We re-formulated from a liquid formulation to a more chemically stable solid pellet. In addition, the reagent is now packaged in glass vials which are more inert relative to the plastic vials used previously. The primary advantage of the reformulated and repackaged Amplifex Diene reagent is a greater than 30% increase in shelf life.
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