Comparison of donkey hemogram using the LaserCyte hematology analyzer, an impedance system, and a manual method.

BACKGROUND: Donkeys are becoming increasingly important worldwide; therefore a reliable and accurate method of diagnosing disease is necessary. Flow cytometry-based hematologic analyzers are present in veterinary laboratories, but performance of LaserCyte has not been evaluated in donkeys. OBJECTIVES: The objective of the study was to compare the results of donkey blood obtained from the LaserCyte with impedance and manual methods. METHODS: Blood samples were collected from 84 healthy donkeys (1-20 years old) and measured with LaserCyte, Sysmex F-820 and manually. Agreement between methods was studied using Passing-Bablok test and Bland-Altman plots. Influence of blood abnormalities found on blood smears on LaserCyte counts was examined using Mann-Whitney or Kruskal-Wallis test. Intraassay precision was calculated. RESULTS: Hematologic variables obtained from the LaserCyte were significantly different from those obtained with impedance or manual methods; numerous values were flagged. Agreement between LaserCyte and manual method was poor for the majority of variables, but agreement between LaserCyte and impedance was only poor for HCT, MCH, and MCHC. LaserCyte had an intraassay precision < 10% for RBC and platelet variables, and > 10% for WBC variables. CONCLUSIONS: LaserCyte results were not interchangeable with results from other methods due to poor agreement. LaserCyte provided no additional hematologic variables or clinically relevant indices for donkey blood analysis. A large number of results were flagged, requiring the evaluation of blood smears. No benefits were found for the use of LaserCyte analyzer over the use of impedance or manual methods in this study. Specific software for LaserCyte for donkey blood would be beneficial.

In vascular smooth muscle cells paricalcitol prevents phosphate-induced Wnt/ß-catenin activation.

The present study investigates the differential effect of two vitamin D receptor agonists, calcitriol and paricalcitol, on human aortic smooth muscle cells calcification in vitro. Human vascular smooth muscle cells were incubated in a high phosphate (HP) medium alone or supplemented with either calcitriol 10(-8)M (HP + CTR) or paricalcitol 3·10(-8) M (HP + PC). HP medium induced calcification, which was associated with the upregulation of mRNA expression of osteogenic factors such as bone morphogenetic protein 2 (BMP2), Runx2/Cbfa1, Msx2, and osteocalcin. In these cells, activation of Wnt/ß-catenin signaling was evidenced by the translocation of ß-catenin into the nucleus and the increase in the expression of direct target genes as cyclin D1, axin 2, and VCAN/versican. Addition of calcitriol to HP medium (HP + CTR) further increased calcification and also enhanced the expression of osteogenic factors together with a significant elevation of nuclear ß-catenin levels and the expression of cyclin D1, axin 2, and VCAN. By contrast, the addition of paricalcitol (HP + PC) not only reduced calcification but also downregulated the expression of BMP2 and other osteoblastic phenotype markers as well as the levels of nuclear ß-catenin and the expression of its target genes. The role of Wnt/ß-catenin on phosphate- and calcitriol-induced calcification was further demonstrated by the inhibition of calcification after addition of Dickkopf-related protein 1 (DKK-1), a specific natural antagonist of the Wnt/ß-catenin signaling pathway. In conclusion, the differential effect of calcitriol and paricalcitol on vascular calcification appears to be mediated by a distinct regulation of the BMP and Wnt/ß-catenin signaling pathways.

FGF23 fails to inhibit uremic parathyroid glands.

Fibroblast growth factor 23 (FGF23) modulates mineral metabolism by promoting phosphaturia and decreasing the production of 1,25-dihydroxyvitamin D(3). FGF23 decreases parathyroid hormone (PTH) mRNA and secretion, but despite a marked elevation in FGF23 in uremia, PTH production increases. Here, we investigated the effect of FGF23 on parathyroid function in normal and uremic hyperplastic parathyroid glands in rats. In normal parathyroid glands, FGF23 decreased PTH production, increased expression of both the parathyroid calcium-sensing receptor and the vitamin D receptor, and reduced cell proliferation. Furthermore, FGF23 induced phosphorylation of extracellular signal-regulated kinase 1/2, which mediates the action of FGF23. In contrast, in hyperplastic parathyroid glands, FGF23 did not reduce PTH production, did not affect expression of the calcium-sensing receptor or vitamin D receptor, and did not affect cell proliferation. In addition, FGF23 failed to activate the extracellular signal-regulated kinase 1/2-mitogen-activated protein kinase pathway in hyperplastic parathyroid glands. We observed very low expression of the FGF23 receptor 1 and the co-receptor Klotho in uremic hyperplastic parathyroid glands, which may explain the lack of response to FGF23 in this tissue. In conclusion, in hyperparathyroidism secondary to renal failure, the parathyroid cells resist the inhibitory effects of FGF23, perhaps as a result of the low expression of FGF23 receptor 1 and Klotho in this condition.

Serum concentrations of calcium, phosphorus, magnesium and calciotropic hormones in donkeys.

OBJECTIVE: To provide reference values for serum biochemical variables that are used for evaluation of mineral metabolism in donkeys and compare values with those in horses. ANIMALS: 18 donkeys and 18 horses. PROCEDURES: Total calcium (tCa), total magnesium (tMg), and inorganic phosphorus (P) concentrations were measured in serum samples via spectrophotometry. Ionized calcium (iCa) and magnesium (iMg) concentrations were quantified with selective electrodes. By use of a micropartition system, tCa and tMg were fractionated to separate protein-bound (pCa, pMg) and ultrafiltrable fractions. Complexed calcium (cCa) and magnesium (cMg) concentrations were calculated by substracting ionized fractions from ultrafiltrable fractions. Parathyroid hormone (PTH) and calcitriol (CTR) concentrations were measured via radioimmunoassay. RESULTS: Serum tCa concentration in donkeys (3.37 +/- 0.21 mmol/L) was composed of pCa (1.59 +/- 0.21 mmol/L [47.0 +/- 4.2%]), iCa (1.69 +/- 0.04 mmol/L [50.4 +/- 3.0%]), and cCa (0.09 +/- 0.08 mmol/L [2.6 +/- 2.9%]). Serum tMg concentration (1.00 +/- 0.08 mmol/L) was fractioned in pMg (0.23 +/- 0.08 mmol/L [23.4 +/- 8.1%]), iMg (0.59 +/- 0.04 mmol/L [58.8 +/- 5.1%]), and cMg (0.18 +/- 0.08 mmol/L [17.8 +/- 7.2%]). Serum concentrations of P (1.14 +/- 0.30 mmol/L), PTH (20.4 +/- 21.2 pg/mL), and CTR (13.4 +/- 5.9 pg/mL) were determined. CONCLUSIONS AND CLINICAL RELEVANCE: Serum variables of mineral metabolism in donkeys were within reference ranges for horses. However, when compared with horses, donkeys had higher iCa, cMg, and CTR and lower pMg and PTH concentrations.

Effect of changes in ionized calcium concentration in arterial blood and metabolic acidosis on the arterial partial pressure of oxygen in dogs.

OBJECTIVE: To evaluate the effects of metabolic acidosis and changes in ionized calcium (Ca2+) concentration on PaO2 in dogs. ANIMALS: 33 anesthetized dogs receiving assisted ventilation. PROCEDURE: Normal acid-base status was maintained in 8 dogs (group I), and metabolic acidosis was induced in 25 dogs. For 60 minutes, normocalcemia was maintained in group I and 10 other dogs (group II), and 10 dogs were allowed to become hypercalcemic (group III); hypocalcemia was then induced in groups I and II. Groups II and IV (5 dogs) were treated identically except that, at 90 minutes, the latter underwent parathyroidectomy. At intervals, variables including PaO2, Ca2+ concentration, arterial blood pH (pHa), and systolic blood pressure were assessed. RESULTS: In group II, PaO2 increased from baseline value (96 +/- 2 mm Hg) within 10 minutes (pHa, 7.33 +/- 0.001); at 60 minutes (pHa, 7.21 +/- 0.02), PaO2 was 108 +/- 2 mm Hg. For the same pHa decrease, the PaO2 increase was less in group III. In group I, hypocalcemia caused PaO2 to progressively increase (from 95 +/- 2 mm Hg to 104 +/- 3 mm Hg), which correlated (r = -0.66) significantly with a decrease in systolic blood pressure (from 156 +/- 9 mm Hg to 118 +/- 10 mm Hg). Parathyroidectomy did not alter PaO2 values. CONCLUSIONS AND CLINICAL RELEVANCE: Induction of hypocalcemia and metabolic acidosis each increased PaO2 in anesthetized dogs, whereas acidosis-induced hypercalcemia attenuated that increase. In anesthetized dogs, development of metabolic acidosis or hypocalcemia is likely to affect ventilatory control.

Fractionation of calcium and magnesium in equine serum.

OBJECTIVE: To establish reference values for protein-bound, ionized, and weak-acid complexed fractions of calcium and magnesium in equine serum and determine stability of ionized calcium (iCa) and ionized magnesium (iMg) in serum samples kept under various storage conditions. ANIMALS: 28 clinically normal horses. PROCEDURE: Total calcium (tCa) and magnesium (tMg) in equine serum were fractionated by use of a micropartition system that allows separation of protein-bound calcium (pCa) and magnesium (pMg) and ultrafiltrable calcium (microCa) and magnesium (microMg) fractions. Serum concentrations of iCa and iMg were measured in the ultrafiltrate by use of selective electrodes. Serum concentration of complexed calcium (cCa) or magnesium (cMg) was calculated by subtracting iCa or iMg from microCa or microMg, respectively. RESULTS: Mean +/-SE serum tCa concentration was 3.26 +/- 0.06 mmol/L. Calcium fractions were as follows: pCa, 1.55 +/- 0.03 mmol/L (47.4 +/- 0.9%); iCa, 1.58 +/- 0.03 mmol/L (48.5 +/- 0.7%); and cCa, 0.13 +/- 0.02 mmol/L (4.1 +/- 0.9%). Serum tMg concentration was 0.99 +/- 0.04 mmol/L. Magnesium fractions were as follows: pMg, 0.33 +/- 0.04 mmol/L (33.3 +/- 4.2%); iMg, 0.57 +/- 0.02 mmol/L (57.6 +/- 1.7%); and cMg, 0.09 +/- 0.02 mmol/L (9.1 +/- 1.9%). Refrigeration (4 degrees C) did not affect iCa values, whereas iMg declined by 8% after 120 hours. Neither iCa nor iMg was affected by freezing (-20 degrees C). CONCLUSIONS AND CLINICAL RELEVANCE: In equine serum, iMg is less stable than iCa; thus, when serum samples are not going to be analyzed promptly, freezing may be preferable to refrigeration for storage.

Calcium-sensing receptor expression and parathyroid hormone secretion in hyperplastic parathyroid glands from humans.

In uremic patients, severe parathyroid hyperplasia is associated with reduced parathyroid calcium-sensing receptor (CaR) expression. Thus, in these patients, a high serum Ca concentration may be required to inhibit parathyroid hormone (PTH) secretion. This study compares the magnitude of reduction in CaR expression and the degree of the abnormality in Ca-regulated PTH release in vitro. A total of 50 glands from 23 hemodialysis patients with refractory hyperparathyroidism were studied. Tissue slices were incubated in vitro to evaluate (1) the PTH secretory output in a normal Ca concentration (1.25 mM) and (2) the PTH secretory response to high (1.5 mM) and low (0.6 mM) Ca concentration. Tissue aliquots were processed for determination of CaRmRNA expression. The results showed that, corrected for DNA, parathyroid tissue with lowest CaR expression secreted more PTH than that with relatively high CaR expression (146 +/- 23 versus 60 +/- 2 pg/microg DNA; P < 0.01). Furthermore, glands with low CaR expression demonstrated a blunted PTH secretory response to both the inhibitory effect of high Ca and the stimulatory effect of low Ca. The study also showed that the larger the gland, the lower the CaRmRNA expression. Thus, large parathyroid glands produce a large amount of PTH not only as a result of the increased gland size but also because the parathyroid tissue secretory output is increased. These abnormalities in PTH regulation are related to low CaR expression.

Nutritional secondary hyperparathyroidism in rabbits.

The present study was designed to document the effect of a low (0.6%) calcium-high (1.2%) phosphorus (LCaHP) diet on the development of parathyroid gland hyperplasia in rabbits and to describe the dynamics of parathyroid function (PTH-Ca2+ curves) in rabbits with nutritional secondary hyperparathyroidism (N2HPT). Parathyroid gland weight, parathyroid cell proliferation (measured as percentage of cells in S-phase), and parathyroid calcium (CaRmRNA) and Vitamin D (VDRmRNA) receptor expression were measured in normal rabbits and in rabbits with N2HPT. The PTH-Ca2+ curve was studied in normal rabbits (Group I) and in rabbits with N2HPT at two stages: 2-3 weeks (Group IIA) and 5-6 weeks (Group IIB) after being fed LCaHP diet. An increase in parathyroid gland weight and percentage of cells in S-phase was detected in the course of N2HPT. After receiving a LCaHP diet for 6 weeks rabbits had decreased levels of CaRmRNA but VDRmRNA remained unchanged. A progressive increase in the concentrations of plasma PTH (Group IIA=167+/-14 pg/ml and Group IIB=377+/-54 pg/ml, P<0.05 versus Group I=27+/-3 pg/ml) was detected in the rabbits fed a LCaHP diet. This was accompanied by an increase in maximal and minimal PTH, reductions in plasma Ca2+ and calcitriol and elevations in plasma phosphate and creatinine. In conclusion, feeding a LCaHPD results in a rapid induction of N2HPT in rabbits. After 6 weeks on the LCaHPD rabbits develop parathyroid hyperplasia characterized by increases in PTH secretion, glandular weight and proliferation and by a decrease in CaRmRNA.

Dynamics of secretion and metabolism of PTH during hypo- and hypercalcaemia in the dog as determined by the 'intact' and 'whole' PTH assays.

BACKGROUND: Recent evidence has shown that the assay for 'intact' parathyroid hormone (I-PTH) not only reacts with 1-84 PTH but also with large non-1-84 PTH fragments, most of which is probably 7-84 PTH. As a result, an assay specific for 1-84 PTH named 'whole' PTH (W-PTH) has been developed. The present study was designed: (i) to determine whether the W-PTH assay reliably measures PTH values in the dog; (ii) to evaluate differences between the W-PTH and I-PTH assays during hypo- and hypercalcaemia; and (iii) to assess the peripheral metabolism of W-PTH and I-PTH. METHODS: In normal dogs, hypocalcaemia was induced by EDTA infusion and was followed with a 90 min hypocalcaemic clamp. Hypercalcaemia was induced with a calcium infusion. RESULTS: I-PTH and W-PTH values increased from 36+/-8 and 13+/-3 pg/ml (P=0.01) at baseline to a maximum of 158+/-40 and 62+/-15 pg/ml (P=0.02 vs I-PTH) during hypocalcaemia. The W-PTH/I-PTH ratio, 38+/-4% at baseline, did not change during the induction of hypocalcaemia, but sustained hypocalcaemia increased (P<0.05) this ratio. During hypercalcaemia, maximal suppression for I-PTH was 2.0+/-0.5 and only 5.7+/-0.6 pg/ml for W-PTH, due to a decreased sensitivity of the W-PTH assay at values <5 pg/ml. The disappearance rate of PTH was determined in five additional dogs which underwent a parathyroidectomy (PTX). At 2.5 min after PTX, W-PTH was metabolized more rapidly, with a value of 25+/-2% of the pre-PTX value vs 30+/-3% for I-PTH (P<0.05). CONCLUSIONS: (i) The W-PTH/I-PTH ratio is less in the normal dog than in the normal human, suggesting that the percentage of non-1-84 PTH measured with the I-PTH assay is greater in normal dogs than in normal humans; (ii) the lack of change in the W-PTH/I-PTH ratio during acute hypocalcaemia is different from the situation observed in humans; and (iii) the dog appears to be a good model to study I-PTH and W-PTH assays during hypocalcaemia.