Plasma Vitamin D3 Research Articles

Simultaneous measurement of plasma vitamin D3 metabolites, including 4β,25-dihydroxyvitamin D3, using liquid chromatography–tandem mass spectrometry

Simultaneous and accurate measurement of circulating vitamin D metabolites is critical to studies of the metabolic regulation of vitamin D and its impact on health and disease. To that end, we have developed a specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method that permits the quantification of major circulating vitamin D3 metabolites in human plasma. Plasma samples were subjected to a protein precipitation, liquid–liquid extraction, and Diels–Alder derivatization procedure prior to LC–MS/MS analysis. Importantly, in all human plasma samples tested, we identified a significant dihydroxyvitamin D3 peak that could potentially interfere with the determination of 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] concentrations. This interfering metabolite has been identified as 4β,25-dihydroxyvitamin D3 [4β,25(OH)2D3] and was found at concentrations comparable to 1α,25(OH)2D3. Quantification of 1α,25(OH)2D3 in plasma required complete chromatographic separation of 1α,25(OH)2D3 from 4β,25(OH)2D3. An assay incorporating this feature was used to simultaneously determine the plasma concentrations of 25OHD3, 24R,25(OH)2D3, 1α,25(OH)2D3, and 4β,25(OH)2D3 in healthy individuals. The LC–MS/MS method developed and described here could result in considerable improvement in quantifying 1α,25(OH)2D3 as well as monitoring the newly identified circulating metabolite, 4β,25(OH)2D3.

Abstract 82: A comprehensive characterization of cholecalciferol supplementation induced changes in the vitamin D3 metabolic pathway in metastatic colorectal cancer patients

Abstract Epidemiological data suggest that vitamin D3 deficiency plays a role in the pathogenesis and progression of human colorectal adenomas and adenocarcinomas. We have characterized the changes in plasma vitamin D3 metabolites (25-D3, 24,25-D3 and 1,25-D3), PTH and vitamin D3 binding protein (VDBP) levels, and peripheral blood mononuclear cells (PBMC) CYP24A1 activity in metastatic colorectal cancer (CRC) patients receiving 2000 IU of oral cholecalciferol daily over a 12 month period. Plasma samples were collected on day 0, 14, 30, 60, 90, 180, 270 and 360. Serum 25-D3 and 24,25-D3 were measured by LC/MS/MS method; plasma PTH and 1,25-D3 levels were determined by RIA. Plasma VDBP levels were measured by ELISA. PBMC CYP24A1activity was determined by HPLC method. A total of 50 CRC patients completed the study. Baseline 25-D3 were significantly influenced by season (p= 0.0145) . Baseline plasma 25-D3 levels were <30ng/mL in 66% of patients. Baseline plasma 24,25-D3, 1,25-D3 and VDBP levels were within the normal range. (The normal ranges are 0- 5ng/mL for 24,25-D3, 16 −80pg/mL for 1,25-D3 and 200 - 550μg/mL for VDBP). Mean baseline plasma PTH levels in patients with plasma 25-D3 <20ng/mL and >30ng/mL were 74.82 ± 27.02 and 56.46 ± 18.10pg/mL, respectively (p>0.05). Baseline PBMC CYP24A1activity was not affected by baseline 25-D3 levels. The increase in serum 25-D3 and 24,25-D3 levels during cholecalciferol treatment was characterized by an initial phase of rapid increase (between day 1 and day 90) followed by a phase of stable plasma 25-D3 and 24,25-D3 levels. A linear relation between plasma 25-D3 and 24,25-D3 was observed at baseline and during cholecalciferol supplementation (r2 = 0.55). No consistent changes in PBMC CYP24A1 activity was observed after 90 days of cholecalciferol treatment. The plasma 24,25-D3/25-D3 ratio at day 60 and 90 of cholecalciferol treatment was weakly correlated (r2=0.03 and 0.06, respectively) to changes in plasma 25-D3 levels. No CRC patient subsets with disproportional increase in plasma 24,25-D3 levels relative to 25-D3 levels during cholecalciferol treatment were identified suggesting that that variable 25-D3 responses cannot be explained by increased 24-hydroxylation. The increase in 1,25-D3 was linear, remained below 100pg/mL and did not correlate with plasma 25-D3 levels. A decrease in plasma PTH levels from 62.83 ± 24.84 to 41.61 ± 18.42 pg/mL was observed after 2 months of cholecalciferol supplementation (p<0.0001). Plasma VDBP protein levels were inconsistent across the study duration; no specific patterns or correlations with 25-D3 or 1,25-D3 could be identified. In summary, vitamin D3 deficiency, defined as 25-D3 <30 ng/mL, is prevalent in metastatic CRC patients. Furthermore, CYP24A1-mediated catabolism of 25-D3 to 24,25-D3 does not appear to be a major determinant of systemic 25-D3 levels. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 82.

Effect of supplementing high levels of vitamin D3 on calcium homeostasis of steers fed barley-based finishing diets

The objective of this study was to evaluate the effect of supplementing high levels of vitamin D3 to steers fed barley-grain-based finishing diets on Ca metabolism. Fifteen Hereford steers (607 ± 12 kg) were individually penned and fed at 0800 and 1600 with a ration consisting of 90% barley-grain-based concentrate and 10% barley silage [dry matter (DM) basis]. Steers were randomly assigned to one of three treatments: 0, 2.5 or 5 million IU (MIU) of vitamin D3 steer-1 d-1 for 7 d. Daily blood samples were obtained for measurement of serum for total and ionized Ca, parathyroid hormone (PTH) and calcitonin and plasma for vitamin D3 and 25(OH)D3. Data were analyzed by repeated measures analysis and single degree of freedom contrasts. Feed intake was depressed relative to control animals by 18 and 37% for the 2.5 and 5 MIU D3 treatments, respectively. Relative to controls, total and ionized Ca increased (P < 0.05) by 8 and 19% and 6 and 18% for the 2.5 and 5 MIU D3 treatments, respectively. Serum PTH concentrations were reduced (P < 0.05) by vitamin D3 feeding with the greatest reduction seen with animals fed the 5 MIU treatment. Calcitonin values were not (P > 0.05) affected. Relative to controls, plasma vitamin D3 concentrations increased (P < 0.05) in a quadratic fashion over time with vitamin D3 supplementation, while that of 25(OH)D3 increased (P < 0.05) in a linear fashion. It was concluded that the increase in serum calcium was stimulated by a rise in plasma vitamin D3 and the resulting increased 25(OH)D3 concentrations, which have been shown to influence calcium absorption either directly or via induced synthesis of 1,25(OH)2D3. Key words: Calcium homeostasis, vitamin D3, cattle, beef tenderness

Improving pork quality by feeding supranutritional concentrations of vitamin D3.

Duroc-cross pigs (n = 25) were assigned to one of three experimental finishing diets containing 0 (control), 40,000 (40), or 80,000 (80) IU of supplemental vitamin D3/kg of feed (as-fed basis)to test the effects of vitamin D3 on pork quality traits. Experimental diets were fed for 44 or 51 d before slaughter, and days on feed were blocked in the experimental design. A trend existed for pigs receiving the highest concentration of vitamin D3 supplementation to have a lower (P = 0.08) ADG (0.77 kg/d) compared with pigs fed either the 40-diet (0.88 kg/d) or control (0.92 kg/d). Diet did not (P > 0.10) affect backfat thickness measured along the midline, 10th-rib fat depth, longissimus muscle area, muscle score, or hot carcass weights. Longissimus pH, measured at 0.5, 1, 2, 3, 4, and 24 h postmortem, was higher (P < 0.05) for pigs on the 80-diet than those fed the control diet. Longissimus muscle color, measured at 24 h postmortem, from pigs fed either the 40- or 80-diet were darker (lower L* values; P < 0.05) than those fed the control diet. Objective longissimus color scores were higher (P < 0.01), and firmness/wetness scores lower (P < 0.05), for pigs on the 80-diet as compared to those on the 40-diet or control diet. The diet had no (P > 0.10) effect on Warner-Bratzler shear force values; percentage of cook loss; or trained sensory panel evaluations for tenderness, juiciness, and flavor. Feeding the 80-diet increased (P < 0.05) plasma vitamin D3 and calcium after 2, 4, and 6 wk on feed compared with the control diet. Vitamin D3 and 25-hydroxy vitamin D3 concentrations in the longissimus muscle increased (P = 0.001) with increasing vitamin D3 levels in the diet; however, muscle calcium concentrations and fiber type were not (P > 0.30) affected by diet. These results indicate that feeding supranutritional levels of vitamin D3 for at least 44 d improves pork color and increases pH, but may retard growth if fed at 80,000 IU/kg of feed.

Dietary P regulates phosphate transporter expression, phosphatase activity, and effluent P partitioning in trout culture.

Phosphate utilization by fish is an important issue because of its critical roles in fish growth and aquatic environmental pollution. High dietary phosphorus (P) levels typically decrease the efficiency of P utilization, thereby increasing the amount of P excreted as metabolic waste in effluents emanating from rainbow trout aquaculture. In mammals, vitamin D3 is a known regulator of P utilization but in fish, its regulatory role is unclear. Moreover, the effects of dietary P and vitamin D3 on expression of enzymatic and transport systems potentially involved in phosphate utilization are little known. We therefore monitored production of effluent P, levels of plasma vitamin D3 metabolites, as well as expression of phosphatases and the sodium phosphate cotransporter (NaPi2) in trout fed semipu diets that varied in dietary P and vitamin D3 levels. Mean soluble P concentrations varied markedly with dietary P but not with vitamin D3, and constituted 40-70% of total effluent P production by trout. Particulate P concentrations accounted for 25-50% of effluent P production, but did not vary with dietary P or vitamin D3. P in settleable wastes accounted for <10% of effluent P. The stronger effect of dietary P on effluent P levels is paralleled by its striking effects on phosphatases and NaPi2. The mRNA abundance of the intestinal and renal sodium phosphate transporters increased in fish fed low dietary P; vitamin D3 had no effect. Low-P diets reduced plasma phosphate concentrations. Intracellular phytase activity increased but brushborder alkaline phosphatase activity decreased in the intestine, pyloric caeca, and gills of trout fed diets containing low dietary P. Vitamin D3 had no effect on enzyme activities. Moreover, plasma concentrations of 25-hydroxyvitamin D3 and of 1,25-dihydroxyvitamin D3 were unaffected by dietary P and vitamin D3 levels. The major regulator of P metabolism, and ultimately of levels of P in the effluent from trout culture, is dietary P.

Plasma Vitamin D and 25OHD Responses of Young and Old Men to Supplementation with Vitamin D3

Objectives: This study was conducted to determine whether there are age differences in the plasma parent vitamin D and 25-hydroxyvitamin D (25OHD) responses to eight weeks of supplementation with 20 μg/day of vitamin D3.Methods: Twenty-five healthy young men (age 18–35) and 25 healthy older men (62–79) were randomly assigned to supplementation with 20 μg/day of vitamin D3 or to no intervention and followed for eight weeks. Plasma vitamin D3 was measured by high performance liquid chromatography and 25OHD was measured by competitive protein binding.Results: Both young and old men in the supplemented group had pronounced, rapid and similar increases in plasma vitamin D3, whereas vitamin D3 concentrations were stable in the control group. By the end of the eight-week adaptation period, plasma vitamin D3 of young and old men had increased by 4.3 and 6.2 nmol/L respectively. In the supplemented group, mean 25OHD concentrations of both the young and old men increased during the study, and the magnitude of the change after eight weeks was nearly identical in the two age groups (22.5 and 22.1 nmol/L in the young and the old men, respectively). In the control group there was a modest decrease in 25OHD of both the young and old men.Conclusions: There appears to be no age-related impairment among men in the absorption or metabolism of 20 μg/day of vitamin D3 taken orally for at least eight weeks.

High apolipoprotein B, low apolipoprotein A-1, and improvement in the prediction of fatal myocardial infarction (AMORIS study): a prospective study

Determination of vitamin D levels in human biological specimens has gained a high relevance over the last decades, essentially because low levels have been associated with several biological disorders. In fact, vitamin D deficiency has become a worldwide health concern covering all ages and genders. The storage of biofluids has to be considered for determination of vitamin D and metabolites in order to fully preserve matrices status. This study attempts to evaluate lyophilization of serum and plasma as a pre-processing step for sample storage prior to quantitative analysis of vitamin D3 and its main hydroxylated metabolites −25(OH)D3, 24,25(OH)2D3 and 1,25(OH)2D3. The protocol including sample lyophilization was characterized in terms of analytical features and compared to the same method, based on SPE–LC–MS/MS, without lyophilization. Sensitivity, precision and accuracy were not affected when we operated with lyophilized serum and plasma and results provided by a set of twenty-four serum samples from DEQAS (Vitamin D External Quality Assessment Scheme) were in agreement with reported concentrations for 25(OH)D3 and 1,25(OH)2D3. A stability study programmed for 9 months allowed ensuring that the concentration of vitamin D3 and metabolites in lyophilized serum and plasma stored at room temperature was not affected during this period. This research has demonstrated that the quantitation of target metabolites is not under the influence of lyophilization. Therefore, including lyophilization prior to analysis could reduce shipment and storage costs, avoid delays of sample processing, and increase the stability of the target analytes due to an effective quenching process.

Probable genetic origin for a large number of cataracts among captive-bred vervet monkeys (Chlorocebus aethiops).

During a period of 6 years, uni- or bilateral cataracts occurred in 55 captive-bred vervet monkeys, representing 27.6% of all offspring produced in that time. Fifty-eight percent of all cases were detectable only by ophthalmoscopy. A total of 30% of cases were offspring of wild-caught parents, 40% from first-generation captive-bred parents, 4% from second-generation captive-bred parents, and 26% from parents of which one was wild caught. Cataracts that could be observed macroscopically appeared at about 6-8 mo of age. A sample of juvenile vervet monkeys with cataracts and their parents had no antibodies against rubella virus, herpes simplex virus, or toxoplasma gondi, and all except two wild-caught breeding females had no antibodies against cytomegalovirus. Plasma vitamin D3 (25 [OH] D), calcium, phosphorus, and glucose concentrations of six cataract cases were comparable to a control group of healthy individuals. The exclusion of these extraneous factors, as well as the fact that the majority of all cases were related, was considered evidence of hereditary etiology. Males and females that had either produced offspring with cataracts or were related to individuals with cataracts were replaced. To date, 35 babies have been born, and no new cases have since occurred.

Determination of vitamins D2, D3, K1 and K3 and some hydroxy metabolites of vitamin D3 in plasma using a continuous clean-up-preconcentration procedure coupled on-line with liquid chromatography-UV detection.

A semi-automatic procedure for the continuous clean-up and concentration of several fat-soluble vitamins prior to their separation by HPLC and UV detection is reported. The procedure is based on the use of a minicolumn packed with aminopropylsilica as sorbent located prior to the chromatographic detection system. The overall process was developed and applied to the main liposoluble vitamins (A, D2, D3, E, K1, K3) and several hydroxy metabolites of vitamin D3 [25-(OH)-D3,24,25-(OH)2-D3 and 1,25-(OH)2-D3]. All the analytes were monitored at a compromise wavelength of 270 nm. Calibration graphs were constructed between 0.01 and 100 ng ml-1 for vitamin D2 and D3 and their hydroxy metabolites, between 0.1 and 100 ng ml-1 for vitamin A, K1 and K3 and between 1 and 100 ng ml-1 for vitamin E, with excellent regression coefficients (> or = 0.9901) in all cases. The precision was established at two concentration levels with acceptable RSDs in all instances (between 3.6 and 8.7%). The method was appropriate for the determination of vitamin D2, D3, K1 and K3 and the 24,25-dihydroxy and 25-hydroxy metabolites of vitamin D3 in human plasma. The method was applied to plasma samples spiked with the target analytes and the recoveries ranged between 78 and 109%.

Measurement of vitamin D3 metabolites in smelter workers exposed to lead and cadmium.

OBJECTIVES: To investigate the effects of lead and cadmium on the metabolic pathway of vitamin D3. METHODS: Blood and urinary cadmium and urinary total proteins were measured in 59 smelter...

Determination of 25-hydroxyvitamin D3 in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection.

A method for the determination of 25-hydroxyvitamin D3, the major metabolite of vitamin D3 in human plasma, using a non-radioactive internal standard and reversed-phase high-performance liquid chromatography with UV detection (265 nm) has been developed. The method was applied to the determination of the metabolite in plasma from healthy subjects (n = 25) and from patients with chronic renal failure (n = 12). 25-Hydroxyvitamin D3 3-sulfate, a major conjugated metabolite of 25-hydroxyvitamin D3, was also determined and the correlation between the concentrations of these metabolites was examined. The study showed that almost equal amounts of both compounds were detected in the plasma of healthy subjects, however, in two subjects, the amount of sulfate in the free form was found to be about twice as high as normally detected. In contrast, the free form was predominant in the plasma of patients with chronic renal failure and the sulfate was not detected in four patients.

Osteodystrophy in liver cirrhosis: Detection and treatment evaluation using 99Tc m methylene diphosphonate bone scintigraphy

To investigate osteodystrophy in liver cirrhosis, 99Tcm MDP (methylene diphosphonate) bone scintigraphy was performed on 36 patients with liver cirrhosis. Abnormal lesions were detected in 17 out of 36 scans (47.2%). On the other hand, areas of increased uptake were uncommon in patients with chronic active hepatitis (1/11 cases). Plasma vitamin D3 fractions [25(OH)D3, 24.25(OH)2D3 and 1.25(OH)2D3] were decreased. Statistically significant depletion of 1.25(OH)2D3 was observed in cases with positive bone scintigraphy. 1 alpha(OH)D3 (1-2 micrograms/day) was administered for 6 months to nine patients having abnormal bone scans. Six of them showed improvement without any apparent side-effects. We conclude that hepatic cirrhotic osteodystrophy can be diagnosed positively by 99Tcm MDP bone scintigraphy and can be treated accordingly.

Thin-layer chromatography as an efficient alternative to high-performance liquid chromatography in the assay of plasma vitamin D3 metabolites

Thin-layer chromatography as an efficient alternative to high-performance liquid chromatography in the assay of plasma vitamin D3 metabolites

Effects of large amounts of vitamin D3 injection on plasma vitamin D3 metabolites in lactating cows.

Effects of injection of large amounts of vitamin D3 (VD3) on the concentrations of plasma 25-hydroxyvitamin D (25OHD), 1α, 25-dihydroxyvitamin D (1, 25 (OH)2D), calcium (Ca) and inorganic phosphorus (Pi) in the lactating Holstein-Friesian cows were examined. The cows were divided into two groups, an untreated control group consisting of 4 cows and a treatment group (VD3 group) consisting of 5 cows. Each cow in the VD3 group received 10 million I.U. of VD3 intramuscularly at 7 days after parturition. Blood and milk samples were taken from 0 to 15 days after the VD3 injection. The plasma 25OHD concentration was significantly higher in the VD3 group than that in the control group throughout the experimental period, whereas the plasma 1, 25 (OH)2D concentration rose only transiently after the VD3 injection. The plasma Ca concentration was also significantly higher in the VD3 group than that in the control group throughout the experiment, while the plasma Pi concentration was higher in the VD3 group only from the 3rd to 6th day of the experiment. These results suggest that the initial increase in the plasma Ca concentration may be due to the increase in the 1, 25 (OH)2D level whereas the substained increase in the Ca concentration may be due to the increase in the plasma 25OHD concentration. Further, it seems likely that the plasma 1, 25 (OH)2D concentration is suppressed by the increase in the plasma Ca concentration in the later stage of experiment and also by the negative feedback of plasma 1, 25 (OH)2D concentration.

Vitamin D3 and D3 Metabolites in Young Goats Fed Varying Amounts of Calcium and Vitamin D3

Twenty-four male goats, 2 to 4 wk of age, were allotted to four dietary treatments in a 2×2 factorial design and, for 20 wk, were fed a milk diet at 12.5% body weight. Treatments varied in amounts of supplemental calcium and vitamin D3. Daily allowances per kilogram body weight were: 9.4 IU vitamin D3 (basal), 9.4 IU vitamin D3 plus 406mg calcium carbonate (basal plus Ca), 940 IU vitamin D3 (basal plus D3), and 940 IU vitamin D3 plus 406mg calcium carbonate (basal plus Ca plus D3). At the end of wk 7, a corn supplement was added to all diets at 1% body weight daily. Addition of vitamin D3 to the diet resulted in a dramatic increase in plasma concentrations of vitamin D3. Goats in the basal plus D3 and basal plus Ca plus D3 groups had nearly 100× greater concentrations of vitamin D3 than goats in the basal and basal plus Ca groups. When greater amounts of vitamin D3 were fed, dietary calcium interacted to decrease plasma vitamin D3 concentrations. Plasma concentrations of 25-hydroxyvitamin D3 were unaffected by additional dietary calcium but were increased by dietary vitamin D3, increasing sixfold to seven-fold in the basal plus D3 and basal plus Ca plus D3 groups. Supplemental calcium resulted in decreased plasma 1,25-dihydroxyvitamin D3. No signs of vitamin D toxicity were noted. The physiological responses reported implicate the goat as a potential animal model for vitamin D research in dairy cattle.

Effect of kallikrein-kinin system on calcium-vitamin D3 metabolism.

Kallikrein has been reported to stimulate callus formation and cell proliferation. It is well-known that vitamin D3 play an role on the metabolism of calcium. However, the relation between vitamin D3 and kallikrein are still poorly understood. We have studied the effect of kallikrein on calcium and vitamin D3 with canine kidney. Mongrel dogs were anesthetized with sodium pentobarbital and prepared by the method of Nakanishi et al. Drugs were infused into renal artery for 60 minutes under three different serum calcium concentrations (Exp. I, Exp. II, Exp. III). Blood samples were collected at the midpoint of the 60 minutes urine collection period. Plasma vitamin D3 concentrations were measured by the metabolites of 25-hydroxyvitamin D3 and 1 alpha,25-dihydroxyvitamin D3. 25-hydroxyvitamin D3 was estimated by competitive protein binding assay and 1 alpha,25-dihydroxyvitamin D3 was determined by radioreceptor assay after separated using HPLC. Electrolytes of sodium, potassium, chloride, calcium and phosphorus in serum and urine, plasma cyclic AMP, glomerular filtration rate, renal blood flow and blood pressure were also measured respectively. Plasma 1 alpha,25-dihydroxyvitamin D3 concentration was found to be decreased by the infusion of kallikrein (0.02 KU/kg/min) in three experimental conditions, especially in Exp. II. Bradykinin (0.02 microgram/kg/min) also caused to kallikrein-like changes of vitamin D3. It is suggested that kallikrein-kinin system is related to inhibit the mechanism of vitamin D3 activation system on kidney.

Effect of Dietary Calcium Stress on Plasma Vitamin D3 Metabolites in the Egg-Laying Japanese Quail

Experiments were performed to ascertain whether circulating levels of 25-hydroxy-vitamin D3 and 1,25-dihydroxyvitamin D3 varied in a cyclic manner during the 24-hr ovulatory period in Japanese quail hens and to assess the effects of dietary calcium restriction upon levels of the plasma vitamin D3 metabolites during that period. Birds fed a marginally-deficient calcium diet (1.7%) had significantly elevated plasma 1,25-dihydroxyvitamin D3 (.71 ± .11 ng/ml, mean ± SEM) compared with those fed high (3.5%) calcium (.35 ± .04; P<.01), although these values did not reveal any significant cyclic variation or correlation with known diurnal pulses in circulating 17β-estradiol. The magnitude of the plasma 1,25-dihydroxyvitamin D3 response to dietary calcium stress in some individuals was as great as 2.2 ng/ml, which are certainly among the highest values reported in any avian or mammalian species to date. These observations suggest that the total circulating level of plasma 1,25-dihydroxyvitamin D3 is not important in the direct cyclic regulation of laying hen calcium metabolism. The levels of free plasma hormone, target cell membrane permeability of the hormone, or regulation of cytosolic/nuclear 1,25-dihydroxyvitamin D3 receptor activity are potential aternate levels of the control of vitamin D3 mediated calcium metabolism in the quail hen.

VITMAIN D3 RESPONSE IN SHEEP TO ORAL VERSUS PARENTERAL ADMINISTRATION AND TO INTRAMUSCULAR DOSE LEVELS OF VITAMIN D3

Plasma vitamin D3 concentration changes were assessed in 10 crossbred wethers following oral or intramuscular administration of a single massive dose (5 × 106 IU) of vitamin D3. Blood plasma was taken at different times during a 3-wk experimental period. The intramuscular response was higher than the oral. In another experiment 15 ewes were administered 5 × 106 IU of vitamin D3 orally, intramuscularly, or intravenously. Blood plasma was taken at different times during a 3-wk experimental period for analyses of plasma vitamin D3 and 25-hydroxyvitamin D3. All animals were killed at the end of the experiment and vitamin D3 determinations were carried out on various tissues. Vitamin D3 given to ewes parenterally had a greater and more lasting effect on their plasma concentrations of vitamin D3 and 25-hydroxyvitamin D3 than did oral administration. While plasma vitamin D3 concentrations of the two parenteral groups were approximately equal, the plasma 25-hydroxyvitamin D3 concentrations were substantially higher in the intravenously treated group than in the intramuscularly treated group. Tissue vitamin D3 concentrations in the parenteral group were higher than in the oral group, and the adipose tissue contained higher concentrations of vitamin D3 than did other tissues. In a third experiment crossbred wethers were administered intramuscularly various amounts (5 × 106, 5 × 105, and 5 × 104 IU) of vitamin D3 in a single dose. Blood plasma was taken at different times during the subsequent 2-wk period. There was proportionality between the amount of vitamin D3 injected and plasma vitamin D3 concentration. The results suggest that parenteral administration induces both higher tissue levels and higher circulating levels of vitamin D3 than does oral administration, and that intravenous injection produces higher circulating levels of 25-hydroxyvitamin D3 than does intramuscular injection. Key words: Vitamin D3 status, sheep, oral, parenteral

Liquid Chromatographic Cleanup and Determination of Low Levels of Vitamin D3 in Sheep Plasma

A liquid chromatographic (LC) method is described for the determination of vitamin D3 in sheep plasma. Samples are extracted by one of 2 different methods, depending on the concentration of vitamin D3. The samples are purified by using either a Sep-Pak silica cartridge or a small alumina column, followed by additional cleanup on a Metalsorb LC column. Final analysis was carried out on a 5 micron C18 column using a radial compression separation system with an acetonitrile-methanol solvent system. Vitamin D3 was completely resolved from any interfering compounds in the plasma; total run time was less than 15 min, using a variable wavelength detector set at 264 nm. The method was successfully applied to samples at levels of 1-10 ng added vitamin D3 mL sheep plasma, with recoveries in the range 90-97%.

Analysis of 25-Hydroxy Vitamin D3 in Plasma by High-Performance Liquid Chromatography

No AccessJournal of Urology1 Feb 1984Analysis of 25-Hydroxy Vitamin D3 in Plasma by High-Performance Liquid Chromatography J.C.K. Loo and R. Brien J.C.K. LooJ.C.K. Loo More articles by this author and R. BrienR. Brien More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(17)50431-0AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "Analysis of 25-Hydroxy Vitamin D3 in Plasma by High-Performance Liquid Chromatography." The Journal of Urology, 131(2), p. 418 © 1984 by The American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 131Issue 2February 1984Page: 418 Advertisement Copyright & Permissions© 1984 by The American Urological Association Education and Research, Inc.MetricsAuthor Information J.C.K. Loo More articles by this author R. Brien More articles by this author Expand All Advertisement PDF downloadLoading ...