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PubMed2005

Vitamin D deficiency and reduced bone mineral density in multiple sclerosis: effect of ambulatory status and functional capacity.

Multiple sclerosis (MS) is a chronic disease and a major cause of disability in young adults. The aims of this study were to assess bone mass in patients with MS in comparison to healthy age- and sex-matched controls, and to evaluate factors influencing bone mineral density (BMD), and the relationship of the pain threshold at peripheral and axial sites with BMD in MS. Thirty-one patients with MS and 30 matched healthy controls participated in the study. The Kurtzke expanded disability status scale (EDSS) and the functional independence measure (FIM) were used to scale disability, mobility, and functional status. Serum 25(OH) vitamin D levels were measured. BMD was measured using dual X-ray absorptiometry (DXA). MS patients had significantly lower BMD at the lumbar spine (L2-L4) and femur trochanter compared to the matched controls. BMD of the lumbar spine was nearly 1 SD lower in MS patients compared with the healthy reference population (Z scores). MS patients had significantly lower vitamin D levels (17.3 ng/ml vs 43.1 ng/ml; P < 0.001) compared to controls, and 19 patients (61%) had a serum level of vitamin D that was less than 20 ng/ml. EDSS scores in the patients were inversely correlated with proximal femur BMD but not with spinal BMD. There was a negative correlation with the cumulative steroid dose and BMD only for femur trochanter BMD. Total myalgia scores for paravertebral muscles correlated significantly with spinal BMD. In conclusion, BMD is significantly lower in MS patients than in healthy controls, vitamin D deficiency is prevalent in MS, and ambulatory status is a determinative factor for osteoporosis in MS. Patients should be encouraged to have adequate sunlight exposure and to increase their mobility. Specific strengthening exercises for hip and back muscles in MS patients would have a substantial impact on bone density, osteoporosis, fracture risk, and mobility.

Adult↗
PubMed2005

Serum 25-hydroxyvitamin D levels and activities of daily living in noninstitutionalized elderly Japanese requiring care.

To date, no study has investigated the nutritional status of vitamin D in frail elderly people living at home. The purposes of this study were to assess serum 25-hydroxyvitamin D (25[OH]D) levels and associated factors in noninstitutionalized elderly people who had various levels of physical disability, and to propose an adequate vitamin D nutritional status for the elderly by interpreting the serum 25(OH)D levels in relation to serum parathyroid hormone (PTH) levels in this population. Health examinations were conducted in the winter and summer of 2003. The subjects were 143 elderly people in the winter, and 120 elderly people in the summer, who all used the long-term care insurance system at home. Serum 25(OH)D concentrations were determined with a chemiluminescence protein-binding assay, and serum intact PTH concentrations were determined with an immunoradiometric assay. The subjects' disease histories and lifestyle information were obtained through an interview. Activities of daily living (ADL) levels were evaluated using the Barthel index, and grip strength was measured with a digital hand dynamometer. Average serum 25(OH)D levels in the winter and summer were 54.2 nmol/l (SD 29.0) and 53.3 nmol/l (SD 32.3), respectively, and intact PTH concentrations in the winter and summer were 4.2 pmol/l (SD 1.8) and 4.3 pmol/l (SD 1.8), respectively. The proportion of people who had a low 25(OH)D (<30 nmol/l) and high intact PTH levels (>6.9 pmol/l) were 15%-20% and 8%, respectively. Significant predictors of low serum 25(OH)D concentrations were low ADL levels, female sex, and low fish consumption in both seasons. Serum 25(OH)D concentrations of less than 50 nmol/l were associated with elevated serum intact PTH concentrations. In conclusion, elderly people requiring care at home are at high risk of hypovitaminosis D, and their low serum 25(OH)D levels are mainly associated with low ADL levels. In addition, maintenance of serum 25(OH)D concentrations above 50 nmol/l may prevent hypovitaminosis D-induced hyperparathyroidism.

Activities of Daily Living↗
PubMed2003

Long-term hospitalization during pregnancy is a risk factor for vitamin D deficiency in neonates.

In order to examine the effects of long-term hospitalization during pregnancy on vitamin D metabolism in pregnant women and neonates, we measured the serum 25-hydroxyvitamin D (25OHD) levels in pregnant women, as well as measuring 25OHD levels in cord blood and breast milk. In pregnant women hospitalized for longer than 1 month, the serum 25OHD levels were decreased at delivery compared with those in control subjects (10.9 +/- 2.6 ng/l vs 19.5 +/- 4.9 ng/l; P < 0.01). Although the levels of 25OHD in the cord blood were not significantly different between the long-term hospitalized and control pregnant women in this study (9.36 +/- 1.7 ng/l vs 11.1 +/- 3.0 ng/l), the 25OHD concentrations in the cord blood were significantly lower than the maternal levels in both groups; the ratios of the levels in cord blood to sera in the long-term hospitalized women and control subjects were 82.1% and 60.3%, respectively. Long maternal hospitalization does not always cause neonatal vitamin D deficiency, but could be one of its major risk factors. Therefore, sufficient sunlight exposure and intake of sufficient vitamin D are considered to be important to prevent vitamin D deficiency in long-term hospitalized pregnant women as well as their babies.

Adult↗
PubMed2005

The oxidant and antioxidant effects of 25-hydroxyvitamin D3 in liver, kidney and heart tissues of diabetic rats.

Previous studies have implicated protective effects of vitamin D on insulin secretion and pancreas beta cell function. The goal of the present study is to determine if a combination therapy of 25-hydroxyvitamin D3 and insulin had any advantage over insulin therapy alone on lipid peroxidation and antioxidant activity in the streptozotocin (STZ)-induced diabetic rat. The lipid peroxidation product, thiobarbituric acid-reacting substances (TBARS), was measured to assess free radical activity in the heart, kidney and liver tissues. The enzymatic activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) were measured as indicators of antioxidation in these tissues. Sprague-Dawley rats were made diabetic with a single injection of STZ (75 mg/kg i.p.). Rats were separated into three groups, each containing 10 animals: Group 1, non-diabetic and no drug treatment was given; Group 2, diabetic rats were treated with 3 IU/day subcutaneous (s.c.) insulin; and Group 3, diabetic rats were treated with 3 IU/day (s.c.) insulin plus 1 mg/kg/day per oral (p.o.) 25-hydroxyvitamin D3 for a period of 4 weeks. At the end of the study, TBARS contents of the liver, kidney and heart tissues in Groups 2 and 3 were found to be significantly increased as compared to Group 1 (P<0.05) and kidney MDA levels in Group 3 were also significantly increased as compared to Group 2 (P<0.05). The SOD and CAT contents of the heart in Group 2 were significantly increased as compared to Groups 1 and 3 (P<0.05). GSH-Px activity was unaltered in all groups (P>0.05). We suggest that a combination of insulin with 25-hydroxyvitamin D3 treatment would not be more beneficial than the use of insulin alone in antioxidant defence of diabetic liver and kidney tissues.

Animals↗
PubMed2006

25(OH) vitamin D3 in patients with chronic kidney disease and those on dialysis: rediscovering its importance.

In terms of both exogenous sources (diet), and endogenous production (activation through exposure to ultraviolet light), vitamin D is unique. Few foods naturally contain vitamin D and only a few are fortified with vitamin D. Most people get more than 90% of their vitamin D requirements from exposure to sunlight. Those who protect their skin from ultraviolet-B radiation with clothing or sunscreen, the elderly, and dark-skinned individuals have limited capacity to produce vitamin D. Vitamin D deficiency is common in the general population and even more common in patients with chronic renal failure (CKD). Increased use of sun-blocking agents and decreased exposure to sunlight, to reduce the risk of skin cancer, attributed to exposure to UV radiation, may contribute to the increase in vitamin D deficiency in the population. These issues are particularly important in the dialysis population who are at particular risk because these, mostly elderly, individuals have an inactive life style and have reduced exposure to sunshine and UV light, thus limiting the actinic synthesis of vitamin D. The nephrology community seems to have overlooked the importance of vitamin D for overall health and well being in patients with CKD. Recently however, several authors have called attention to the role of plasma 25(OH)D3 levels in mineral metabolism dysregulation in patients with chronic kidney diseases, and those on dialysis. Vitamin D not only contributes to skeletal health but also plays a major role in the health of a wide variety of other organ systems. It seems that vitamin D supplementation is the most effective way of preventing vitamin D deficiency.

Aging↗
PubMed1997

Individual and combined effects of calciotropic hormones and growth factors on mineral metabolism in embryonic chick tibiae.

We have investigated single and combined effects of calciotropic hormones and growth factors on the regulation of alkaline phosphatase (ALP) activity and calcium metabolism in an optimized serum-free bone organ culture system of embryonic chick tibiae. Parathyroid hormone PTH(1-34) alone mobilized calcium from bone tissue time- and dose-dependently and inhibited ALP activity. Both the bisphosphonate (BM 21.0955) and to a lesser extent salmon calcitonin alone slightly increased calcium uptake and inhibited the stimulation of bone resorption by PTH(1-34). 1,25(OH)2D3 mobilized calcium and inhibited ALP activity in contrast to 24,25(OH)2D3 which inhibited ALP activity but had no significant effect on calcium metabolism. Interestingly the combination of PTH(1-34) with 1,25(OH)2D3 but not 24,25(OH)2D3 reduced calcium mobilization. The combination of the midregional fragment PTH(28-48), which by itself has no effect on calcium metabolism, with 1,25(OH)2D3 reduced calcium mobilization more efficiently. Several PTH-regulated mediators have been assayed in this system. Of the tested growth factors, IGF-I at high concentrations caused bone resorption with no effect on ALP activity. TGF-beta 1 (transforming growth factor beta) and BMP-2 had no significant effect on calcium metabolism; however, ALP activity was inhibited by TGF-beta 1 and induced dose dependently by BMP-2. Of the other factors known to be present in bone, platelet-derived growth factor (PDGFA/B) and epidermal growth factor (EGF) had a small effect on calcium mobilization but had no effect on ALP activity. bFGF reduced ALP activity slightly without an effect on calcium metabolism. Our results show that this in vitro system can mimic some interactions of calciotropic hormones in vivo and allows the assaying of mediators in terms of regulation of ALP activity and of calcium metabolism.

Alkaline Phosphatase↗
PubMed1989

Changes in plasma vitamin levels in the first 48 hours after onset of acute myocardial infarction.

To establish whether plasma vitamin measurements made after acute myocardial infarction (AMI) can be used in case-control studies of coronary artery disease, the short-term effect of AMI on plasma concentrations of 25-hydroxyvitamin D3, beta-carotene, vitamin E and retinol was investigated. Sequential measures of these vitamins were made during the first 48 hours after AMI in 13 patients admitted to the hospital within 4 hours after the onset of symptoms. Plasma levels of 25-hydroxyvitamin D did not change significantly during the first 12 hours after onset of symptoms. Beta-carotene levels increased significantly (p less than 0.05) during the first 12 hours and then decreased, whereas levels of vitamin E and retinol progressively decreased during the first 48 hours by 26 and 25%, respectively. These results suggest that, of these vitamins, only plasma measurements of 25-hydroxyvitamin D3 collected within 12 hours of onset of symptoms may provide reliable information for case-control studies of AMI.

Aged↗
PubMed1984

Oncogenous osteomalacia. Review of the world literature of 42 cases and report of two new cases.

The clinical and biochemical data in 42 reported cases of oncogenous osteomalacia are reviewed, and data in two previously unreported cases are recorded. It is likely that the syndrome is more common than suggested by the paucity of reports and may account for a substantial fraction of nonfamilial, adult-onset "idiopathic" osteomalacia. Tumors associated with the syndrome are characteristically benign, of mesenchymal origin, highly vascular, and composed principally of giant and spindle cells. Complete excision of tumors results in cure of the osteomalacia in the majority of patients. The syndrome presumably represents an instance of humor-induced phosphaturia but supporting experimental data are scanty. Plasma levels of 1,25-dihydroxycholecalciferol are uniformly low, and treatment with this metabolite is generally very effective; however, abnormal vitamin D metabolism cannot by itself account for the syndrome.

Adolescent↗
PubMed1987

Neoplastic hypercalcemia: physiologic response to intravenous etidronate disodium.

Following a four-day control period during which an elevated serum calcium level either stabilized or continued to rise despite maximally tolerated saline diuresis, 12 patients with neoplastic hypercalcemia were treated with intravenous etidronate disodium (etidronate) 7.5 mg/kg/day for up to seven days. Serum calcium reverted to normal levels in all patients, with the mean pretreatment serum calcium level of 12.5 +/- 0.4 mg/dl dropping to 9.2 +/- 0.2 mg/dl (p less than 0.01) by Day 7. Elevated urinary calcium (1,107 +/- 134 mg/g creatinine) and hydroxyproline levels (154 +/- 16 mg/g creatinine) declined to 245 +/- 52 mg/g creatinine and 75 +/- 14 mg/g creatinine, respectively, suggesting a marked reduction in bone resorption following treatment. Serum phosphorus levels were unchanged, but urinary phosphorus levels dropped rapidly from 1,181 +/- 125 mg/g creatinine before treatment to 723 +/- 94 mg/g creatinine after two days. Serum parathyroid hormone levels (mid-molecule assay) were suppressed before treatment (64 +/- 16 pg/ml), but rose rapidly to 223 +/- 68 pg/ml by Day 7 of treatment. The value of serum 1,25-dihydroxyvitamin D was initially below normal (16 +/- 3 pg/ml), but rose rapidly with treatment to 42 +/- 12 pg/ml by Day 7. Symptoms of hypercalcemia and bone pain improved with treatment, and no serious adverse reactions to treatment were encountered. Intravenous etidronate is apparently an effective and safe treatment for neoplastic hypercalcemia.

Adult↗
PubMed1993

Noninvasive testing in the diagnosis of osteomalacia.

PURPOSE: Osteomalacia is associated with many clinical, radiographic, and biochemical abnormalities. Unfortunately, none of these are pathognomonic of the disorder, and histologic examination of a bone biopsy specimen is often necessary to confirm the diagnosis. Noninvasive methods of diagnosis would be preferable to decrease patient morbidity and increase cost-effectiveness. Previous studies addressing the adequacy of these methods were performed prior to the widespread availability of 1,25-dihydroxycalcitriol (1,25-(OH)2D3) and parathyroid hormone (PTH) levels. Therefore, we reviewed our experience with patients with biopsy-proven osteomalacia to determine if PTH or 1,25(OH)2D3 levels would serve a useful role in establishing the diagnosis of osteomalacia. METHODS: We retrospectively studied 17 patients who had biopsy-proven osteomalacia (defined as an osteoid volume greater than 10% and an osteoid width greater than 15 microns) in order to ascertain if their clinical presentation, biochemical profile, and radiographic features were sufficient to establish a diagnosis of osteomalacia. RESULTS: We found that 94% of our patients exhibited symptoms of osteomalacia, and all patients had at least one demonstrable sign of osteomalacia on examination. Biochemically, the patients presented a heterogeneous picture: 94% had an elevated alkaline phosphatase level; 47% had either a low serum calcium or phosphate (12% had both) level; urinary calcium excretion was low in 18%; and 25-(OH)D3 concentration was low in 29%. Levels of 1,25-(OH)2D3 were measured in eight patients; three had low values despite normal 25-(OH)D3 levels. PTH levels were elevated in 41% of patients in the absence of histologic evidence of hyperparathyroidism; however, these patients were noted to have multiple other abnormal clinical, biochemical, or radiograph features. Radiographically, 18% of patients had pseudofractures; the rest had nonspecific findings suggestive of osteomalacia. In summary, all patients had at least two of the following abnormalities: low calcium, low phosphate, elevated alkaline phosphatase, or a radiographic finding suggestive of osteomalacia. CONCLUSION: We conclude: (1) a careful history and physical examination remain important in the evaluation of potential osteomalacia patients; (2) PTH offered no apparent benefit as a screening test in our patients; (3) 1,25-(OH)2D3 was low in three patients with normal levels of 25-(OH)D3; (4) decreased urinary calcium excretion had low sensitivity for detecting osteomalacia; and (5) serum calcium, phosphate, alkaline phosphatase, and radiographic examination may be adequate screening tests in patients who have a clinical presentation suggestive of osteomalacia.

Alkaline Phosphatase↗
PubMed1984

Transfer of 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 across the perfused human placenta.

The rates of placental transfer of 25-hydroxyvitamin D3 [25-(OH)D3] and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] were determined in an in vitro perfusion system. Antipyrine was included in each perfusion, and the data are expressed as clearance index, the ratio of hydroxyvitamin D3 to antipyrine clearance. In most experiments, serum, 0.2%, was added to the perfusates as a source of vitamin D3-binding protein. Binding as measured by dextran-coated charcoal assay for 25-(OH)D3 was over 90%, for 1,25-(OH)2D3, only 25% to 50%. The clearance index from the maternal to fetal circulation averaged 0.02 and 0.26 for 25-(OH)D3 and 1,25-(OH)2D3, respectively. When vitamin D3-binding protein was omitted from the perfusate, the clearance indices of 25-(OH)D3 were 0.12 and 0.46 in two experiments. Binding to vitamin D3-binding protein is a major determining factor for the transfer rates of 25-(OH)D3 and 1,25-(OH)2D3.

Antipyrine↗
PubMed1985

Calcium metabolism in pregnancy and the perinatal period: a review.

Calcium homeostasis is a complex process involving calcium, other involved ions, and three calcitropic hormones, parathyroid hormone, calcitonin, and 1,25-dihydroxyvitamin D3. The principal maternal adjustment during pregnancy is an increasing parathyroid hormone secretion which maintains the serum calcium concentration in the face of a falling albumin level, an expanding extracellular fluid volume, an increasing renal excretion, and placental calcium transfer. The placenta transports calcium ions actively, making the fetus hypercalcemic relative to its mother, which in turn stimulates calcitonin release and perhaps suppresses parathyroid hormone secretion by the fetus. A unique extrarenal system for 1 alpha-hydroxylation of 25-hydroxyvitamin D3 exists in the placenta and/or decidua, providing a source of 1,25-dihydroxyvitamin D3 for the fetus. With the abrupt cessation of the placental source of calcium at birth, the neonate's serum calcium level falls for 24 to 48 hours, then stabilizes and rises slightly. Hyperparathyroidism during pregnancy causes complications in both mother and infant and should usually be treated surgically as soon as diagnosed. Maternal hypoparathyroidism can be treated satisfactorily with high doses of supplemental calcium and vitamin D. Osteopenia accompanying long-term heparin administration may respond to 1,25-dihydroxyvitamin D3 (calcitriol) therapy. Diabetes in pregnancy is associated with disturbed neonatal calcium homeostasis, perhaps due to chronic hypomagnesemia. A possible etiologic role of calcium deficiency in pregnancy-related hypertension has been suggested. Dietary deficiency of calcium and/or vitamin D during gestation may lead to several adverse effects in the newborn infant.

Absorption↗
PubMed1986

Purification of human serum vitamin D-binding protein by 25-hydroxyvitamin D3-Sepharose chromatography.

25-Hydroxyvitamin D3-Sepharose was prepared by coupling 25-hydroxyvitamin D3-3 beta-(1,2-epoxypropyl)-ether to thio-activated Sepharose CL-6B, forming a protease-resistant linkage between the sterol and the matrix. Vitamin D-binding protein from human plasma was obtained 85-92% pure after ligand affinity chromatography. Subsequent hydroxylapatite chromatography provided homogeneous protein. The purified vitamin D-binding protein was fully active in regard to 25-hydroxyvitamin D3 and actin binding capabilities.

Calcifediol↗
PubMed1987

A new tritium-release assay for 25-hydroxyvitamin D-1 alpha-hydroxylase.

A new, rapid assay for 1 alpha-hydroxylase has been developed using 25-hydroxy-[1 alpha-3H]vitamin D3 as the substrate. Using the solubilized and reconstituted chick 1 alpha-hydroxylase, conversion of this substrate to 1,25-dihydroxyvitamin D3 causes the release of tritium into the aqueous medium. This 3H2O can be easily separated from the labeled substrate by passing the reaction mixture through a reverse-phase silica cartridge. The release of tritium is stereospecific as evidenced by the lack of 3H2O formed when 25-hydroxy-[1 beta-3H]vitamin D3 is used as the substrate. In parallel reactions containing the 25-hydroxy-[26,27-3H]vitamin D3 substrate, production of labeled 1,25-dihydroxyvitamin D3 was assessed by extraction and high-performance liquid chromatography and found to agree very closely with the amount of 3H2O produced from 25-hydroxy-[1 alpha-3H]vitamin D3, validating the accuracy of the new assay. Finally, a major advantage of the tritium-release assay for 1 alpha-hydroxylase is that the results are not affected by further metabolism of the 1,25-dihydroxyvitamin D formed in the incubations.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed1990

A rapid assay for 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D 24-hydroxylase.

A rapid method for the measurement of the 24-hydroxylated metabolites of 25-hydroxy[26,27-3H]vitamin D3 and 1,25-dihydroxy[26,27-3H]vitamin D3 has been developed. This measurement has, in turn, made possible a rapid assay for the 24-hydroxylases of the vitamin D system. The assay involves the use of 26,27-3H-labeled 1,25-dihydroxyvitamin D3 or 25-hydroxyvitamin D3 as the substrate and treatment of the enzyme reaction mixture with sodium periodate, which specifically cleaves the 24-hydroxylated products between carbons 24 and 25, releasing tritiated acetone. The acetone is measured after its separation from the labeled substrate by using a reversed-phase cartridge. The results obtained with this assay were validated by comparison with the results obtained with a well-established high-performance liquid chromatography assay. The activity of the enzyme determined by both methods was equal. This assay has been successfully used for the rapid screening of column fractions during purification of the enzyme and in the screening for monoclonal antibodies to the 24-hydroxylase.

24,25-Dihydroxyvitamin D 3↗
PubMed1991

Determination of the affinity of vitamin D metabolites to serum vitamin D binding protein using assay employing lipid-coated polystyrene beads.

We have developed an assay to measure the affinity of serum vitamin D binding protein for 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3, and vitamin D3, using uniform diameter (6.4 microns) polystyrene beads coated with phosphatidylcholine and vitamin D metabolites as the vitamin D donor. The lipid metabolite coated beads have a solid core, and thus all of the vitamin D metabolites are on the bead surface from which transfer to protein occurs. After incubating these beads in neutral buffer for 3 h, essentially no 3H-labeled vitamin D metabolites desorb from this surface. Phosphatidylcholine/vitamin D metabolite-coated beads (1 microM vitamin D metabolite) were incubated with varying concentrations of serum vitamin D binding protein under conditions in which the bead surfaces were saturated with protein, but most of the protein was free in solution. After incubation, beads were rapidly centrifuged without disturbing the equilibrium of binding and vitamin D metabolite bound to sDBP in solution was assayed in the supernatant. All three vitamin D metabolites became bound to serum vitamin D binding protein, and after 10 min of incubation the transfer of the metabolites to serum vitamin D binding protein was time independent. The transfer followed a Langmuir isotherm, and the Kd for each metabolite binding to serum vitamin D binding protein was derived by nonlinear least-squares fit analysis. From this analysis the following values for the Kd were obtained: 5.59 x 10(-6) M, 25-hydroxyvitamin D; 9.45 x 10(-6) M, 1,25-dihydroxyvitamin D; and 9.17 x 10(-5) M, vitamin D. In conclusion, we have developed a method which avoids problems encountered in previous assays and allows the precise and convenient determination of binding affinities of vitamin D metabolites and serum vitamin D binding protein.

Animals↗
PubMed1991

Synthesis of a reagent for fluorescence-labeling of vitamin D and its use in assaying vitamin D metabolites.

The fluorogenic dienophile 1,2,4-triazoline-3,5-dione with a highly fluorescent quinoxalinone group at the 4-position (DMEQ-TAD) was synthesized and exploited as a reagent to assay vitamin D metabolites. 25-Hydroxyvitamin D3, 1 alpha,25-dihydroxyvitamin D3, and 24(R),25-dihydroxyvitamin D3 reacted quantitatively with DMEQ-TAD when the two substrates were mixed in dichloromethane at room temperature to yield the corresponding 6,19-cycloadduct. The reaction was very fast so that 1 alpha,25-dihydroxyvitamin D3 at a concentration as low as 10(-8) M could be quantitatively labeled with the fluorescent reagent within 30 min at room temperature. With this reagent, down to 10 fmol of vitamin D metabolites could be quantified linearly. The detection limit of the labeled vitamin D using high-performance liquid chromatography was usually about 1 fmol. Thus, it was shown in a model system that the fluorometric method using the new reagent (DMEQ-TAD) can be applied to the assay of the three major vitamin D metabolites in 1 ml of plasma. This is the first practical fluorometric method for assaying the active vitamin D metabolite.

24,25-Dihydroxyvitamin D 3↗
PubMed1992

Fluorometric assay of 25-hydroxyvitamin D3 and 24R,25-dihydroxyvitamin D3 in plasma.

The first practical fluorometric assay of plasma 25-hydroxyvitamin D3 (25-OH-D3) and 24R,25-dihydroxyvitamin D3 (24,25-(OH)2D3) is described. The method uses a highly fluorescent dienophile, 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1, 2,4- triazoline-3,5-dione (DMEQ-TAD), to fluorescence-label vitamin D. Vitamin D metabolites were roughly purified with a short cartridge column followed by HPLC, labeled with DMEQ-TAD, and the product was analyzed on HPLC. In the assay of 25-OH-D3 the new fluorometric method was compared with the HPLC-uv method and was confirmed to be as accurate and reliable (CV, 4-5%) as the HPLC-uv method. Plasma 24,25-(OH)2D3 was accurately assayed by the HPLC-FL method, where the standard addition method was successfully used to calculate the overall recovery.

24,25-Dihydroxyvitamin D 3↗