Urinary megalin in association with progression factors of diabetic nephropathy.

AIM: The aim of this study is to evaluate the association between urinary megalin, renal function, blood pressure, lipid profile, vitamin D and glycemic control in patients with type 2 diabetes mellitus (T2DM). METHODS: . This was a cross-sectional study which recruited 209 patients with T2DM. Urinary megalin was positively associated with systolic blood pressure (SBP) (r=0.218, p=0.04) but negatively with glomerular filtration rate (GFR) (r=-0.16, p=0.023). The levels of urinary albumin, triglycerides (TGs) and glycosylated hemoglobin (HbA1c) were higher in the "high-megalin" group, compared to those in "low-megalin" group. Moreover, there was a significant inverse association between vitamin D3 levels and megalin levels in urine (OR=0.281, p=0.047). CONCLUSION: Our study showed for the first time that megalin is associated with progression factors of diabetic nephropathy as well as vitamin D deficiency (Tab. 3, Fig. 1, Ref. 15).

Identification of conjugation positions of urinary glucuronidated vitamin D3 metabolites by LC/ESI-MS/MS after conversion to MS/MS-fragmentable derivatives.

A liquid chromatography/electrospray ionization-tandem mass spectrometry-based method was developed for the identification of the conjugation positions of the monoglucuronides of 25-hydroxyvitamin D3 [25(OH)D3 ] and 24,25-dihydroxyvitamin D3 [24,25(OH)2 D3 ] in human urine. The method employed derivatization with 4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione to convert the glucuronides into fragmentable derivatives, which provided useful product ions for identifying the conjugation positions during the MS/MS. The derivatization also enhanced the assay sensitivity and specificity for urine sample analysis. The positional isomeric monoglucuronides, 25(OH)D3 -3- and -25-glucuronides, or 24,25(OH)2 D3 -3-, -24- and -25-glucuronides, were completely separated from each other under the optimized LC conditions. Using this method, the conjugation positions were successfully determined to be the C3 and C24 positions for the glucuronidated 25(OH)D3 and 24,25(OH)2 D3 , respectively. The 3-glucuronide was not present for 24,25(OH)2 D3 , unlike 25(OH)D3 , thus we found that selective glucuronidation occurs at the C24-hydroxy group for 24,25(OH)2 D3 .

Maternal vitamin D supplementation to improve the vitamin D status of breast-fed infants: a randomized controlled trial.

OBJECTIVE: To determine whether a single monthly supplement is as effective as a daily maternal supplement in increasing breast milk vitamin D to achieve vitamin D sufficiency in their infants. PATIENTS AND METHODS: Forty mothers with exclusively breast-fed infants were randomized to receive oral cholecalciferol (vitamin D3) 5000 IU/d for 28 days or 150,000 IU once. Maternal serum, breast milk, and urine were collected on days 0, 1, 3, 7, 14, and 28; infant serum was obtained on days 0 and 28. Enrollment occurred between January 7, 2011, and July 29, 2011. RESULTS: In mothers given daily cholecalciferol, concentrations of serum and breast milk cholecalciferol attained steady levels of 18 and 8 ng/mL, respectively, from day 3 through 28. In mothers given the single dose, serum and breast milk cholecalciferol peaked at 160 and 40 ng/mL, respectively, at day 1 before rapidly declining. Maternal milk and serum cholecalciferol concentrations were related (r=0.87). Infant mean serum 25-hydroxyvitamin D concentration increased from 17±13 to 39±6 ng/mL in the single-dose group and from 16±12 to 39±12 ng/mL in the daily-dose group (P=.88). All infants achieved serum 25-hydroxyvitamin D concentrations of more than 20 ng/mL. CONCLUSION: Either single-dose or daily-dose cholecalciferol supplementation of mothers provided breast milk concentrations that result in vitamin D sufficiency in breast-fed infants. CLINICAL TRIAL REGISTRATION: clinicaltrials.gov NCT01240265.

The relative influence of calcium intake and vitamin D status on serum parathyroid hormone and bone turnover biomarkers in a double-blind, placebo-controlled parallel group, longitudinal factorial design.

BACKGROUND: Adequate calcium and vitamin D are needed to maintain calcium balance. OBJECTIVE: Our objective was to examine the influence of calcium intake and vitamin D exposure separately and their interaction on biomarkers of calcium sufficiency. DESIGN: Healthy men and women, age 20-80 yr, were randomly allocated to four groups: 1) double placebo, 2) calcium (1200 mg daily) plus placebo, 3) vitamin D(3) (100 microg) plus placebo, and 4) vitamin D(3) and calcium. Fasting serum and urine as well as serum and urine 2 h after a calcium load (600 mg of calcium carbonate) were obtained at baseline and 3 months. RESULTS: Ninety-nine participants were randomized; 78 completed the study. Baseline demographics, protein intake and laboratory studies did not differ among the four groups. Study medication compliance was 90%. Fasting bone turnover markers declined after 3 months only in the two groups given calcium supplements and increased in the vitamin D(3) plus placebo calcium group. The calcium load resulted in a decrease in PTH and in bone turnover markers that did not differ among groups. Urinary calcium excretion increased in the combined group. Mean serum 25-hydroxyvitamin D increased from a baseline of 67 (18 sd) nmol/liter to 111 (30 sd) nmol/liter after vitamin D supplementation. CONCLUSION: Increased habitual calcium intake lowered markers of bone turnover. Acute ingestion of a calcium load lowered PTH and bone turnover markers. Additional intake of 100 microg/d vitamin D(3) did not lower PTH or markers of bone turnover.

High dietary cholecalciferol increases plasma 25-hydroxycholecalciferol concentration, but does not attenuate the hypertension of Dahl salt-sensitive rats fed a high salt diet.

The Dahl salt-sensitive rat, a model for salt-induced hypertension, develops hypovitaminosis D during high salt intake, which is caused by loss of protein-bound vitamin D metabolites into urine. We tested the hypothesis that high dietary cholecalciferol (5- and 10-fold standard) would increase plasma 25-hydroxycholecalciferol (25-OHD(3)) concentration (indicator of vitamin D status) of salt-sensitive rats during high salt intake. Salt-sensitive rats were fed 0.3% salt (low salt, LS), 3% salt (HS), 3% salt and 7.5 microg cholecalciferol/d (HS-D5), or 3% salt and 15 microg cholecalciferol/d (HS-D10) and sacrificed at week 4. Plasma 25-OHD(3) concentrations of the two groups of HS-D rats were similar to that of LS rats and more than twice that of HS rats. Urinary cholecalciferol metabolite content of HS-D rats was more than seven times that of HS rats. Systolic blood pressures of the hypertensive HS and HS-D rats did not significantly differ, whereas LS rats were not hypertensive. We conclude that high dietary cholecalciferol increases plasma 25-OHD(3) concentration, but does not attenuate the hypertension of salt-sensitive rats during high salt intake. Low salt intake may be necessary to both maintain optimal vitamin D status and prevent hypertension in salt-sensitive individuals.

Characterization of urinary metabolites of vitamin D(3) in man under physiological conditions using liquid chromatography-tandem mass spectrometry.

The characterization of the urinary metabolites of vitamin D(3) in man under physiological conditions was performed using liquid chromatography-tandem mass spectrometry (LC-MS-MS). The urine specimens obtained from healthy volunteers were treated with beta-glucuronidase, purified with disposal solid-phase extraction cartridges, derivatized with a Cookson-type reagent, 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione, and subjected to LC-MS-MS. The derivatization was employed to increase the ionization efficiencies of the vitamin D(3) metabolites, which enabled detection of the metabolites in the picogram range. The identification of the genin parts of the metabolites was done by comparison with authentic samples based on their LC-MS-MS data. The glucuronides of 23S,25-dihydroxyvitamin D(3) and 24R,25-dihydroxyvitamin D(3) were obtained as the main metabolites from the urine in almost equal amounts. In contrast to the fact that the plasma/serum concentration of the former is much lower than that of the latter, the hydroxylation at the C-23 position was considered to be the important side-chain modification of 25(OH)D(3) to excrete the excess vitamin D(3) in man. In addition, 23S,25-dihydroxy-24-oxovitamin D(3) occurred as its glucuronide in most of the urine, which suggested that this metabolite also plays a part in the excretion of vitamin D(3) in man.

Use of pamidronate to reverse vitamin D3-induced toxicosis in dogs.

OBJECTIVES: To determine whether pamidronate disodium can reduce vitamin D3-induced hypercalcemia in dogs and whether combination treatment with calcitonin is more effective than treatment with pamidronate alone. ANIMALS: 20 clinically normal male Beagles. PROCEDURE: All dogs were given 8 mg of cholecalciferol (CCF)/kg of body weight once orally, then were assigned randomly to 4 groups of 5 dogs each. Dogs were given 0.9% NaCl solution IV (group 1), calcitonin SC and 0.9% NaCl solution IV (group 2), pamidronate and 0.9% NaCl solution IV (group 3), or a combination of all 3 agents (group 4). Dogs were observed for 28 days, and serial blood and urine samples were collected for determination of serum biochemical, electrolyte, and 25(OH)D3 values, CBC, and urine mineral excretion. Samples of kidney, stomach, lung, aorta, liver, duodenum, and brain were evaluated by light microscopy and quantitative mineral analysis. RESULTS: Two dogs in group 1 were euthanatized 4 days after CCF administration because of severe clinical signs of disease. Dogs in group 3 lost less weight and had significantly lower serum phosphorus, total and ionized calcium, and urinary zinc concentrations, compared with dogs in group 1. On day 4, serum urea nitrogen concentration was significantly lower in dogs of groups 3 and 4, compared with dogs in group 1. Mild to moderate mineralization of kidneys and stomach were observed in the 2 group-1 dogs euthanatized on day 4. CONCLUSIONS: Pamidronate administration effectively prevents CCF-induced hypercalcemia and mineralization of soft tissues. CLINICAL RELEVANCE: Pamidronate is a potentially useful antidote against CCF toxicosis in dogs.

The kinetics of D3-3H metabolism in tropical sprue.

The metabolism of vitamin D3-3H was studied in a small group of controls and subjects with tropical sprue after the oral or intravenous administration of 8 to 10 microCi of D3-3H. The biological half life of D3-3H upon the administration of the isotope by the intravenous route was normal in 2 controls, very low in a subject with tropical sprue who had steatorrhea, and decreased in a subject with tropical sprue who did not present steatorrhea. After the administration of the isotope by the oral route, the biological half life was 35 hours in the control and no radioactivity could be detected in the plasma of the subject with tropical sprue who had steatorrhea. Twenty four hours after the intravenous dose the percentage of radioactivity in the plasma as HCC-3H was two times higher in the tropical sprue subjects than in the controls. When the dose was given orally the net absorption was 50.5% in the subject with tropical sprue and steatorrhea and 86.8% in the subject with tropical sprue who was partially treated. These results showed rapid clearance of the D3-3H in the subject with tropical sprue and steatorrhea indicating depletion of vitamin D stores in the tissues and decrease in the net absorption of the dose when given orally. The presence of a higher percentage of the dose in the plasma as HCC-3H after the intravenous and oral administrations in the tropical sprue subjects when compared to controls indicates that the diseased state does not alter vitamin D3 metabolism.

An evaluation of the use of Sep-Pak C18 cartridges for the extraction of vitamin D3 and some of its metabolites from plasma and urine.

The use of Sep-Pak C18 cartridges for the extraction of vitamin D and some of its metabolites from plasma and urine has been evaluated by studying the recovery of added tritiated secosteroids. The preparation of the cartridges, recoveries, extraction and elution with a number of solvents, effect of varying flow rates for application and elution, and the effect of increasing volumes of plasma and urine have been investigated. Two methods for the application of secosteroids present in plasma to Sep-Pak C18 cartridges have been examined, using methyl cyanide extracts removing precipitated protein by centrifugation, and using acidified methanolic plasma. Methyl cyanide extracts applied to Sep-Pak C18 cartridges and eluted with methanol or methyl cyanide gave the cleanest extracts suitable for direct HPLC. Acidified methanolic plasma, applied to Sep-Pak C18 cartridges and eluted with methanol or methyl cyanide gave extracts which could not be applied directly to an HPLC--further fractionation using Sep-Pak SIL cartridges was necessary. Recoveries of added tritiated secosteroids using both methods were greater than 80% with the exception of vitamin D itself which was poorly recovered--methyl cyanide extraction giving only 30% recovery and use of acidified methanolic plasma giving 66% recovery.

Adult coeliac disease, osteomalacia, hyperparathyroidism and acromegaly. Evidence for urinary losses of hydrosoluble metabolites of vitamin D3.

Vitamin D-deficient osteomalacia with pronounced secondary hyperparathyroidism was observed in an acromegalic patient suffering from adult celiac disease. Two oral tests with tritiated vitamin D3 showed a nearly normal absorption coefficient, contrasting with a marked steatorrhea mainly due to functional and reversible pancreatic insufficiency. The urinary excretion of the radioactive label was strikingly increased and accounted for the completely flat curve of plasma radioactivity. This urinary loss of mainly water-soluble metabolite(s) of vitamin D3 represents a unique and presently unexplained feature.

Absorption, hydroxylation, and excretion of vitamin D3 in primary biliary cirrhosis.

Oral vitamin D3 was poorly absorbed by 4 out of 6 patients with primary biliary cirrhosis; absorption was negatively correlated with faecal fat excretion. 25-hydroxylation of vitamin D3 given by mouth or intravenously was not impaired in the patients compared with controls of similar vitamin-D nutritional status. Urinary radioactivity derived from the intravenous dose of vitamin D3 was significantly greater in patients than in controls and was positively correlated with the serum-bilirubin concentration. Excretion in the urine may lead to loss of administered and endogenous vitamin D and thus contribute to vitamin-D deficiency in patients with primary biliary cirrhosis.

The metabolic fate of vitamin D3-3H in chronic renal failure.

The absorption and metabolism of vitamin D(3)-(3)H was studied in eight patients with chronic renal failure. Although the intestinal absorption of vitamin D(3)-(3)H was normal, the metabolic fate of the vitamin was abnormal as characterized by a twofold increase in fractional turnover rate, an abnormal accumulation of biologically inactive lipid-soluble metabolites, and the urinary excretion of both vitamin D(3)-(3)H and biologically inactive metabolites. Neither alterations in water-soluble vitamin D(3) metabolites nor qualitative abnormalities in protein-binding of vitamin D(3) were observed in the uremic subjects. Although hemodialysis proved ineffectual in reversing the observed abnormalities in vitamin D(3) metabolism and excretion, renal homotransplantation was completely successful in this regard. These experiments support the conclusion that the resistance to therapeutic doses of vitamin D often seen in patients with chronic renal failure and renal osteodystrophy results from an acquired defect in the metabolism and excretion of vitamin D.

Metabolism of vitamin D3-3H in vitamin D-resistant rickets and familial hypophosphatemia.

The fate of an intravenous dose of tritiated vitamin D(3) was studied in seven normal subjects, four children with vitamin D-resistant rickets, and four adults with a familial history of vitamin D-resistant rickets and persistent hypophosphatemia. An abnormal metabolism of vitamin D in vitamin D-resistant rickets was defined and characterized by a decrease in the plasma fractional turnover rate, a marked increase in plasma water-soluble metabolites, and a relative decrease in the conversion of vitamin D to a polar, biologically active metabolite. Alterations in vitamin D metabolism in the adults with persistent hypophosphatemia were similar but less severe than those of affected children with vitamin D-resistant rickets. It is tentatively concluded that the abnormalities in vitamin D metabolism documented in patients with vitamin D-resistant rickets and familial hypophosphatemia may account for the observed osseous and biochemical changes.

Metabolism of vitamin D3-3H in human subjects: distribution in blood, bile, feces, and urine.

Vitamin D(3)-(3)H has been administered intravenously to seven normal subjects, three patients with biliary fistulas, and four patients with cirrhosis. Plasma D(3)-(3)H half-times normally ranged from 20 to 30 hours. in vivo evidence that a metabolic transformation of vitamin D occurs was obtained, and a polar biologically active vitamin D metabolite was isolated from plasma. Urinary radioactivity averaged 2.4% of the administered dose for the 48-hour period after infusion, and all the excreted radioactivity represented chemically altered metabolites of vitamin D. The metabolites in urine were mainly water-soluble, with 26% in conjugated form. From 3 to 6% of the injected radioactivity was excreted in the bile of subjects with T-tube drainage and 5% in the feces of patients having no T-tube. The pattern of fecal and biliary radioactivity suggested that the passage of vitamin D and its metabolites from bile into the intestine represents an essential stage for the fecal excretion of vitamin D metabolites in man. Abnormally slow plasma disappearance of vitamin D(3)-(3)H in patients with cirrhosis was associated with a significant decrease in the quantity and rate of glucuronide metabolite excretion in the urine.