Quantitative Metabolomic Analysis of Urinary Citrulline and Calcitroic Acid in Mice after Exposure to Various Types of Ionizing Radiation.

With the safety of existing nuclear power plants being brought into question after the Fukushima disaster and the increased level of concern over terrorism-sponsored use of improvised nuclear devices, it is more crucial to develop well-defined radiation injury markers in easily accessible biofluids to help emergency-responders with injury assessment during patient triage. Here, we focused on utilizing ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to identify and quantitate the unique changes in the urinary excretion of two metabolite markers, calcitroic acid and citrulline, in mice induced by different forms of irradiation; external γ irradiation at a low dose rate (LDR) of 3.0 mGy/min and a high dose rate (HDR) of 1.1 Gy/min, and internal exposure to Cesium-137 ((137)Cs) and Strontium-90 ((90)Sr). The multiple reaction monitoring analysis showed that, while exposure to (137)Cs and (90)Sr induced a statistically significant and persistent decrease, similar doses of external γ beam at the HDR had the opposite effect, and the LDR had no effect on the urinary levels of these two metabolites. This suggests that the source of exposure and the dose rate strongly modulate the in vivo metabolomic injury responses, which may have utility in clinical biodosimetry assays for the assessment of exposure in an affected population. This study complements our previous investigations into the metabolomic profile of urine from mice internally exposed to (90)Sr and (137)Cs and to external γ beam radiation.

Evidence in favor of a severely impaired net intestinal calcium absorption in patients with (early-stage) chronic kidney disease.

INTRODUCTION: Calcium and phosphorus are essential to many vital physiological processes. Little is known about the net and fractional intestinal absorption of calcium and phosphorus in patients with chronic kidney disease (CKD) and their clinical and hormonal determinants. METHODS: Blood and 24-hour urine samples were collected in 20 healthy volunteers (HV) and 72 stable CKD stage 1-4 patients and analyzed for parameters of mineral metabolism including calcidiol, calcitriol, and parathyroid hormone (PTH). Dietary intake was assessed by dietary history. RESULTS: The 24-hour urinary calcium excretion, as opposed to the phosphorus excretion, showed a stepwise decrease across CKD stages (median of 219, 84, 40, and 22 mg/day in HV and patients with CKD stages 1-2, 3 and 4, respectively). Younger age, high serum calcitriol, and high estimated GFR were associated with a high 24-hour urinary calcium excretion. High serum calcitriol levels and dietary phosphorus intake were associated with a high 24-hour urinary phosphorus excretion. The fractional intestinal calcium absorption, as estimated by the urinary-to-ingested calcium ratio, decreased across CKD stages. CONCLUSIONS: The 24-hour urinary excretion of calcium, as opposed to phosphorus, is markedly decreased in CKD, even in early-stage disease. This is partly explained by low calcitriol levels and older age. Assuming a neutral calcium balance at the time of urine collection, we infer that net intestinal calcium absorption may be severely impaired in CKD.

Plasma 25-hydroxycholecalciferol and 1,25-dihydroxycholecalciferol concentrations are decreased in hind limb unloaded Dahl salt-sensitive female rats.

Plasma 1,25-dihydroxyvitamin D (1,25-(OH)(2)D) concentration was shown to decrease during bed rest in several studies when baseline plasma 25-hydroxyvitamin D (25-OHD) concentration was sub-optimal. Dahl salt-sensitive female (S) rats, but not Dahl salt-resistant female (R) rats, demonstrated a 50% decrease in plasma 1,25-dihydroxycholecalciferol (1,25-(OH)(2)D(3)) concentration after 28 days of hind limb unloading (HU, disuse model) during low salt intake (0.3%). We tested the vitamin D endocrine system response of female S rats to hind limb unloading during high salt intake (2%, twice that of standard rat chow to mimic salt intake in the USA). Hind limb unloading resulted in lower plasma 25-OHD(3) concentrations in S-HU rats than in R-HU rats (P<0.05) and greater urinary loss of 25-OHD(3) by S-HU rats than by S rats (P<0.05). Plasma 1,25-(OH)(2)D(3) concentration of S-HU rats was half that of S rats, but was unchanged in R-HU rats. The association of low plasma 25-OHD concentration with decrease in plasma 1,25-(OH)(2)D concentration of hind limb unloaded rats and of bed rest participants (published studies) suggests that low vitamin D status might be a risk factor for decrease in plasma vitamin D hormone concentration during long-term immobilization or bed rest.

Fibroblast growth factor 23 is increased in calcium nephrolithiasis with hypophosphatemia and renal phosphate leak.

CONTEXT: Nephrolithiasis affects about 10% of the population in industrialized countries, with calcium salts composing more than 80% of renal stones. A significant percentage of patients with calcium nephrolithiasis and normal parathyroid function show hypophosphatemia and reduced renal phosphate reabsorption (i.e. a renal phosphate leak). OBJECTIVES: The objective of the study was to compare serum levels of fibroblast growth factor 23 (FGF23), a regulator of phosphate homeostasis, in 110 recurrent stone formers with or without renal phosphate leak, six patients affected by X-linked hypophosphatemic rickets, five patients affected by oncogenic osteomalacia, and 60 unrelated healthy controls. DESIGN: This was a prospective interventional study. METHODS: Renal phosphate leak was identified based on the occurrence of idiopathic hypophosphatemia [serum phosphate concentration < 2.50 mg/dl (<0.80 mmol/liter)] and reduced renal threshold phosphate concentration [<2.2 mg/liter (<0.70 mmol/liter)]. RESULTS: In 22 stone formers with renal phosphate leak, serum FGF23 concentration was significantly higher as compared with 88 stone formers without renal phosphate leak and with controls [83.3 (65.6-101.1) vs. 32.1 (26.8-37.4) and 24.5 (19.8-29.1) reference units (RU)/ml, respectively]. Stone formers with renal phosphate leak showed lower FGF23, compared with patients with oncogenic osteomalacia and X-linked hypophosphatemic rickets [572.3 (235.9-908.7) RU/ml]. Among stone formers and controls, serum FGF23 concentration displayed a strong inverse association with serum phosphate (r = -0.784, P = 0.009) and the rate of tubular phosphate reabsorption (r = -0.791, P = 0.008). CONCLUSIONS: In our study population, renal phosphate leak affected 20% of stone formers and was strongly associated with increased serum FGF23 concentration.

Effects of thiazide- and loop-diuretics, alone or in combination, on calcitropic hormones and biochemical bone markers: a randomized controlled study.

OBJECTIVE: Diuretics are commonly used drugs that in addition to their effect on the cardiovascular system also affect calcium homeostasis and bone metabolism. We evaluated the effects of loop diuretics (LD) and thiazide diuretics (TD) on calcitropic hormones and biochemical bone markers. DESIGN: A total of 50 postmenopausal women were randomized to 7 days of treatment with either the TD bendroflumethiazide, the LD bumetanide, bendroflumethiazide plus bumetanide, or placebo. Blood and urine (24 h) were sampled on each day. Statistical inferences were made versus the concomitant changes in the placebo group. RESULTS: Bendroflumethiazide increased the tubular reabsorption of calcium (TRCa) (+0.46 +/- 0.11%, P=0.009), plasma levels of parathyroid hormones (PTH) (+24 +/- 10%, P=0.06), and 1,25(OH)2D (+12 +/- 6%, P=0.03). Bumetanide decreased the TRCa (-0.5 +/- 0.1%, P=0.01) and increased plasma PTH and 1,25(OH)2D levels (+27 +/- 9%, P=0.02 and +36 +/- 12%, P=0.006, respectively). Treatment with either of the drugs did not alter plasma calcium, osteocalcin, bone alkaline phosphatase (bone-ALP) or urinary NTx/creatinine ratio. However, treatment with both drugs caused an increased plasma calcium level (+2.7 +/- 1.0%, P=0.007) and decreased plasma levels of bone-ALP (-21 +/- 3%, P=0.001), osteocalcin (-6 +/- 3%, P=0.03), and urinary NTx/creatinine ratio (-39 +/- 6%, P=0.001). CONCLUSION: Calcium homeostasis and bone metabolism are to a major degree influenced by diuretic treatment. Surprisingly, LD and TD exerted a similar effect on calcitropic hormones despite their opposite effects on the renal calcium excretion. In clinical practice, treatment with diuretics has to be considered as a cause of parathyroid stimulation.

Effect of parathyroid hormone (hPTH[1-34]) infusion on serum 1,25-dihydroxyvitamin D and parathyroid hormone in normal women.

Calcium absorption declines with age. Because 1,25-dihydroxyvitamin D (1,25(OH)2D) is the major hormone controlling calcium absorption, changes in vitamin D metabolism may account for the malabsorption of aging. Serum levels of 1,25(OH)2D have been reported to either decrease or remain unchanged with age. To assess the effect of aging on renal production of 1,25(OH)2D, we evaluated the response of renal 25OHD 1 alpha hydroxylase to human parathyroid hormone (hPTH(1-34) stimulation in 119 women ages 25-83 years. In this population, baseline serum 25OHD and 1,25(OH)2D values did not significantly change with age, but serum iPTH (r = 0.44; p < 0.001) and serum creatinine (r = 0.31; p < 0.01) increased with age. However, the stimulatory activity of hPTH(1-34) on the renal production of 1,25(OH)2D declined with age (r3 = -0.36; p < 0.001) and was most apparent after age 75, being 50% less than that of younger women. Besides age, the production of 1,25(OH)2D was found to be dependent on baseline serum iPTH (r = -0.31; p < 0.0001). Administration of hPTH(1-34) led to suppression of endogenous PTH, and suppressibility of endogenous PTH declined with age (r = 0.53; p < 0.0001). The increase in serum PTH and decreased suppressibility of PTH with age could be due to mild secondary hyperparathyroidism. The increase in PTH with age is probably responsible for maintaining normal serum 1,25(OH)2D levels in elderly subjects; however, decreased metabolism of 1,25(OH)2D in the elderly could also maintain normal serum 1,25(OH)2D levels.

The effect of estrogen deficiency on bone mineral density, renal calcium and phosphorus handling and calcitropic hormones in the rat.

The oophorectomized (OOX) rat has been proposed as a good model of postmenopausal osteoporosis in women. The aim of this study was to compare the effect of OOX in 6-month-old rats to the effects of menopause in women with respect to bone mass, the renal handling of calcium and phosphorus, and calcitropic hormones. To more closely replicate the human situation the rats were pair fed a 0.1% calcium diet. Thirty four, 6-month-old rats were randomized to sham operation or OOX. Whole body and regional bone density was performed at baseline and 6 weeks postoperation. Blood and 24-hour urine samples were obtained at baseline, 1, 3, and 6 weeks and assayed for various biochemical variables, parathyroid hormone (PTH), and calcitriol. The OOX rats lost significantly more bone than the sham-operated rats (change in global bone mineral density, sham -1.7 +/- 2.0%, OOX -3.9 +/- 2.6%, P < 0.001). In the OOX animals, an increase in the 24-hour urine calcium was observed at 1 and 3 weeks, which had returned to sham-operated levels by 6 weeks. In the whole group, the increase in urine calcium at 1 week was negatively correlated with the change in bone mass at 6 weeks (r = -0.39, P = 0. 029). OOX resulted in an increased filtered load of calcium and phosphorus. There was an increase in the maximal renal tubular reabsorption of phosphorus (TmP-GFR) but no clear change in renal calcium handling. Neither calcitriol nor parathyroid hormone showed a significant change as a result of OOX. As in postmenopausal women, following oophorectomy in the rat, there was significant generalized bone loss and a negative calcium balance. This was associated with an initial rise in urine calcium due to a rise in the filtered calcium load; plasma phosphorus and TmP-GFR also rose. The rat model may differ from postmenopausal bone loss in that the initial rise in urine calcium was not present at later time points as occurs in natural menopause in women. Calcitropic hormone levels did not change. This study has shown that the 6-month-old OOX rat fed a 0.1% calcium diet has many similarities of calcium and phosphorus homeostasis to that seen at menopause in women.

Calcitriol production in hypercalcemic and normocalcemic patients with non-Hodgkin lymphoma.

OBJECTIVE: To determine the following: a reference range for serum calcitriol during hypercalcemia in a control group of patients with myeloma in whom calcitriol production is known to be appropriately suppressed; the incidence of elevated serum calcitriol levels in hypercalcemic patients with non-Hodgkin lymphoma according to this derived reference range; and the incidence of abnormal calcium metabolism in normocalcemic patients with non-Hodgkin lymphoma. DESIGN: Prospective clinical study. SETTING: Referral cancer center. PATIENTS: 2 groups of hypercalcemic patients: 16 control patients with myeloma and 22 patients with non-Hodgkin lymphoma divided into those with elevated or normal serum calcitriol levels; 1 group of 22 normocalcemic patients with non-Hodgkin lymphoma. MEASUREMENTS: Serum chemistries and intact parathyroid hormone, calcitriol, parathyroid hormone-related protein, and urinary electrolyte levels. RESULTS: On the basis of the mean serum calcitriol level of the control group plus 3 standard deviations, the reference range for serum calcitriol during hypercalcemia was defined as less than 42 pg/mL. Although serum calcium and parathyroid hormone levels in the study patients were similar to those in controls, 12 of the 22 hypercalcemic patients with non-Hodgkin lymphoma (55%) had serum calcitriol levels greater than 42 pg/mL (range, 51 to 170 pg/mL). No features distinguished the patients with elevated serum calcitriol levels from those with normal levels. Seventy-one percent of normocalcemic patients with non-Hodgkin lymphoma were hypercalciuric, and 18% had serum calcitriol levels greater than the normocalcemic reference range (20 to 76 pg/mL). CONCLUSIONS: Serum calcitriol levels are elevated in most hypercalcemic patients with non-Hodgkin lymphoma in the absence of elevated serum levels of parathyroid hormone, which implicates extrarenal calcitriol production in the pathogenesis of this syndrome. Abnormal calcium metabolism, hypercalciuria, and dysregulated calcitriol production are also common in normocalcemic patients with non-Hodgkin lymphoma.

Effects of estrogen on response to edetic acid infusion in postmenopausal osteoporotic women.

We and others have hypothesized that estrogen helps preserve bone mass by affecting the PTH/vitamin D regulation of skeletal metabolism. To evaluate this theory, we tested the effect of estrogen administration on parathyroid sensitivity to hypocalcemic challenge. Subjects were postmenopausal osteoporotic women recruited from a tertiary care clinic (9 untreated and 12 receiving hormone replacement therapy at the time of the investigation). After baseline serum and urine testing, edetic acid (50 mg/kg) was infused over a 2-h period. Serum and urine samples were obtained over 5 h and 24 h after beginning the infusion. Serum ionized calcium dropped equally in both groups of women. There were overall group differences in PTH-(1-84) secretion (P < 0.02), with a greater peak (P < 0.04) and a longer period of elevation (P < 0.01) in the untreated than in the hormone-treated osteoporotic women. Serum 1,25-dihydroxyvitamin D [1,25-(OH)2D] and phosphorus as well as urinary phosphate and cAMP responded similarly in the two groups of women. Estrogenized osteoporotic women demonstrate a smaller PTH increment to hypocalcemia, indicating that the parathyroid has reduced sensitivity under the influence of estrogen. Despite the smaller PTH increase in estrogenized individuals, renal responses to PTH were the same as those in untreated osteoporotic women, implying an estrogen-mediated increase in the sensitivity of the kidney to PTH.

Demonstration of a difference in urinary calcium, not calcium absorption, in black and white adolescents.

Bone mineral density (BMD) of the forearm, lumbar spine, and femoral neck is greater in black than in white children. Studies were performed to determine whether differences in intestinal absorption of calcium or urinary calcium or both account for an assumed more positive calcium balance and greater bone mass in black children. Normal black and white boys and girls were admitted to a metabolic ward and given a constant daily diet containing 1000 mg calcium, 60% as calcium carbonate, for 2 1/2 days (study I) or 3 1/2 days (study II). Fasting blood and 24 h urine collections were obtained, and in study II, unidirectional fractional absorption of calcium (alpha) was determined with stable isotopes of calcium. It was found that (1) serum 25-hydroxyvitamin D (25-OHD) and urinary calcium were lower and serum 1,25-dihydroxyvitamin D [1,25-(OH)2D] was higher in black than in white children, and (2) alpha was higher in boys than in girls with no racial difference, and (3) there were significant positive correlations between alpha and urinary calcium in the blacks and in the black and white children together. It is concluded that (1) alpha is higher in boys than in girls and (2) a lower urinary calcium, not increased intestinal absorption of calcium, is the means for a more positive calcium balance in blacks that accounts for the racial difference in BMD.

Evidence suggesting hyperoxaluria as a cause of nephrocalcinosis in phosphate-treated hypophosphataemic rickets.

Urinary excretion of oxalate and phosphate was measured in twelve vitamin-D-treated, phosphate-supplemented patients with X-linked hypophosphataemia (XLH; four children, eight adolescents and adults) to investigate possible causative factors of nephrocalcinosis other than calcium. Oxalate excretion correlated highly with urinary phosphate excretion and with intake of phosphate supplements corrected for body surface area. Young children received the highest relative doses of phosphate (range 2.27-10.8 g/1.73 m2 daily) and their urinary oxalate excretion was very high (0.94-3.38 mmol/1.73 m2 daily). The urinary oxalate excretion of untreated adults with XLH was within normal limits. Six patients had evidence of nephrocalcinosis on ultrasound. The high urinary oxalate excretion in phosphate-supplemented XLH may be seen as a special type of enteric hyperoxaluria, in which the conditions of calcium-oxalate crystal precipitation could be reached even at normal levels of urinary calcium excretion. Urinary excretion of both calcium and oxalate should therefore be monitored during treatment in young XLH patients.

Effects of infusion of human parathyroid hormone-related protein-(1-40) in nude mice: histomorphometric and biochemical investigations.

Female nude mice were infused with 5.0, 3.0, or 1.0 micrograms/day of synthetic human parathyroid hormone-related protein (PTHrP) or control diluent with subcutaneous Alzet miniosmotic pumps for 7 days. Serum calcium was increased (p less than 0.01) on days 3 (13.9 mg/dl), 5 (13.6 mg/dl), and 7 (12.9 mg/dl) in mice infused with PTHrP at 5.0 micrograms/day compared with control nude mice (8.8 mg/dl). Serum calcium was significantly increased to a lesser degree in mice infused with 1.0 micrograms/day PTHrP (day 3) or 3.0 micrograms/day (days 3 and 7). Serum phosphorus was decreased (p less than 0.01) in all three groups of mice infused with PTHrP (4.6 mg/dl, 5.0 micrograms/day; 6.7 mg/dl, 3.0 micrograms/day; and 6.4 mg/dl, 1.0 micrograms/day) compared with controls (8.5 mg/dl). Serum 1,25-dihydroxycholecalciferol was increased (2.4-fold) in mice infused with PTHrP (5.0 and 3.0 micrograms/day). The urinary calcium-creatinine ratio (0.74 compared with 0.034 in controls) was increased (p less than 0.03) in mice infused with PTHrP (5.0 micrograms/day), but the urinary phosphorus-creatinine ratio was not different from that in controls. The urinary cAMP-creatinine ratio was increased (1.6-fold) in mice infused with PTHrP (5.0 micrograms/day). Static bone histomorphometry revealed increased (p less than 0.01) trabecular bone area, osteoblast perimeter, osteoid perimeter, osteoid width, wall width, osteoclast area, number of osteoclasts, and osteoclast perimeter in trabecular bone of lumbar vertebrae from mice infused with PTHrP. Dynamic bone histomorphometry demonstrated increased (p less than 0.01) double-labeled perimeter, mineralizing perimeter, and bone formation rate. The results of this study indicated that PTHrP increased serum calcium and 1,25-dihydroxycholecalciferol, decreased serum phosphorus, increased urinary excretion of calcium, phosphorus, and cAMP, and increased both bone resorption and formation in nude mice. PTHrP mimics the action of native PTH in vivo and is likely to be an important protein in the pathogenesis of humoral hypercalcemia of malignancy.

Effects of low-calcium diet on urine calcium excretion, parathyroid function and serum 1,25(OH)2D3 levels in patients with idiopathic hypercalciuria and in normal subjects.

We have used a low-calcium diet providing only 2 mg/kg (body weight) per 24 hours of calcium to distinguish between "renal" and "absorptive" idiopathic hypercalciuria. Sixteen of 27 hypercalciuric subjects excreted calcium in excess of intake during days seven, eight and nine of he diet, suggesting some element of renal hypercalciuria; however, all patients had low or normal serum PTH and urine cAMP levels. In general, fasting urine calcium was elevated in these 16 subjects and normal in the remaining 11, who conserved calcium more normally. SErum 1,25(OH)2D3 levels were the same in patients and normal subjects, even though PTH levels of the patients were below those of he normal subjects. Urine magnesium excretion and phosphorus excretion were both increased in the patients who excreted calcium in excess of intake. Our findings suggest that renal and absorptive hypercalciuria may not be distinct entities but rather the two extremes of a continuum of behavior. A uniform elevation of intestinal calcium absorption and a variable defect of renal calcium reabsorption could explain our results far better than the hypothesis of distinct absorptive and renal forms of hypercalciuria.