Advances and challenges in the measurement of 1,25-dihydroxyvitamin D: a comprehensive review.

Vitamin D has received significant attention from clinical societies, researchers, and the general population in recent years. While 25-hydroxyvitamin D (25(OH)D) is the most commonly-used biomarker of vitamin D status, 1α,25-dihydroxyvitamin D (1,25(OH)2D), its bioactive form, plays a critical role in regulating calcium and phosphorus homeostasis and is also involved in the immune system and cellular differentiation. Consequently, accurate measurements of 1,25(OH)2D can aid in the differential diagnosis of calcium-related disorders such as hypocalcemia in vitamin D-dependent rickets and hypercalcemia due to inappropriate increase of serum 1,25(OH)2D in granulomatous diseases. However, due to its lipophilicity and very low circulating concentration, the measurement of 1,25(OH)2D is particularly challenging. Over the past several decades, numerous efforts have been made to develop sensitive, specific, and practical laboratory methods for measuring 1,25(OH)2D. Methods using radioreceptor assay, radioimmunoassay, enzyme immunoassay, enzyme-linked immunosorbent assay, automated chemiluminescent immunoassay, and liquid chromatography-tandem mass spectrometry have been described. Each of these methods has unique advantages and limitations, and some are no longer used. Despite the sophisticated methods in use today, substantial variations between methods still exist. A concerted effort toward standardization of 1,25(OH)2D measurement is needed to ensure accurate and reliable results across laboratories and methods.

Effects of saw palmetto extract on the vanilloid receptor TRPV1.

OBJECTIVE: Transient receptor potential vanilloid subtype 1 (TRPV1) may play a significant role in the pathophysiology of the bladder. The present study investigated the effects of the herbal product, saw palmetto extract (SPE) on TRPV1-mediated Ca2+ influx and specific [3 H]resiniferatoxin ([3 H]RTX) binding to TRPV1 in HEK293 cells expressing TRPV1 (HEK293VR11 cells). METHODS: Ca2+ influx induced by and the direct binding activity of TRPV1 were measured using a method with Fura 2-AM, a cytoplasmic calcium indicator, and a radioligand binding assay using a [3 H]RTX, respectively. RESULTS: SPE did not markedly affect Ca2+ influx in HEK293VR11 cells; however, it significantly inhibited capsaicin-induced increases in Ca2+ influx in these cells. The specific binding of [3 H]RTX in HEK293VR11 cells was saturable with Kd value of 120 ± 7 pM and Bmax of 1.07 ± 0.10 fmol/mg protein, and was inhibited by low concentrations of non-labeled RTX with Ki of 60.1 ± 7.6 nM. These results confirmed the pharmacological specificity of specific binding sites of [3 H]RTX to TRPV1 in HEK293VR11 cells. SPE inhibited the specific binding of [3 H]RTX in a concentration-dependent manner, with Ki of 24.2 ± 1.4 µg/mL. CONCLUSIONS: The present study demonstrated for the first time, that SPE inhibited capsaicin-induced Ca2+ influx with binding to TRPV1 in HEL293VR11 cells. These results will contribute to a more detailed understanding of the pharmacological effects of SPE on urinary dysfunction.

A distinct subfraction of Aß is responsible for the high-affinity Pittsburgh compound B-binding site in Alzheimer's disease brain.

The positron emission tomography (PET) ligand (11) C-labeled Pittsburgh compound B (PIB) is used to image ß-amyloid (Aß) deposits in the brains of living subjects with the intent of detecting early stages of Alzheimer's disease (AD). However, deposits of human-sequence Aß in amyloid precursor protein transgenic mice and non-human primates bind very little PIB. The high stoichiometry of PIB:Aß binding in human AD suggests that the PIB-binding site may represent a particularly pathogenic entity and/or report local pathologic conditions. In this study, (3) H-PIB was employed to track purification of the PIB-binding site in > 90% yield from frontal cortical tissue of autopsy-diagnosed AD subjects. The purified PIB-binding site comprises a distinct, highly insoluble subfraction of the Aß in AD brain with low buoyant density because of the sodium dodecyl sulfate-resistant association with a limited subset of brain proteins and lipids with physical properties similar to lipid rafts and to a ganglioside:Aß complex in AD and Down syndrome brain. Both the protein and lipid components are required for PIB binding. Elucidation of human-specific biological components and pathways will be important in guiding improvement of the animal models for AD and in identifying new potential therapeutic avenues. A lipid-associated subpopulation of Aß accounts for the high-affinity binding of Pittsburgh compound B (PIB) in Alzheimer's disease brain. Mass spectrometry of the isolated PIB-binding site from frontal cortex identified Aß peptides and a set of plaque-associated proteins in AD but not age-matched normal brain. The PIB-binding site may represent a particularly pathogenic entity and/or report local pathologic conditions.