Effect of age and the X-linked Hyp mutation on renal adaptation to vitamin D and calcium deficiency

The age (4 weeks vs 5 weeks vs 6.5 weeks) at which dietary restriction of vitamin D and calcium is initiated has a profound effect on the resulting concentration of serum calcium, urinary cAMP and on renal 25-hydroxyvitamin D3-1-hydroxylase (1-hydroxylase) activity in normal (+/Y) mice; no such age relationship is apparent in Hyp/Y littermates. After 40 days on the restrictive diet, it was found that the younger the +/Y mice at the time of diet initiation, the lower the resulting serum calcium (4 weeks less than 5 weeks less than 6.5 weeks) and the higher the urinary cAMP and 1-hydroxylase activity (4 weeks greater than 5 weeks greater than 6.5 weeks). Age on diet has no effect on serum phosphate and fractional excretion index of phosphate in +/Y and Hyp/Y littermates. Renal 1-hydroxylase activity is significantly lower than normal in the younger groups of Hyp/Y mice whereas 24-hydroxylase (25-hydroxyvitamin D3-24-hydroxylase) activity is higher than normal in all groups of Hyp/Y mice.     

Plasma levels of levonorgestrel, gestodene, norethisterone and cyproterone acetate on single-dose subcutaneous administration in oily solution in the rat, beagle and rhesus monkey

This report describes the pharmacokinetics of levonorgestrel, gestodene, norethisterone and cyproterone acetate following subcutaneous administration of oily solutions in the rat, beagle dog and rhesus monkey. The plasma levels of the progestogens were measured by means of specific radioimmunoassays. Half-lives calculated for the disposition process of a particular metabolically unchanged drug in plasma revealed marked differences in different animal species. Furthermore, comparison of the different progestogens showed large variations in this parameter in all the animal species. It became obvious that there are physico-chemical properties as well as metabolic rate limitations effecting the release and elimination of synthetic progestogens administered in oily solution. The results are compared with the half-lives of these progestogens administered intravenously as reported previously. A prolongation of half-life as a result of the depot effect of subcutaneous administration was demonstrated for all the progestogens in the rat, the beagle dog and the rhesus monkey.     

Evaluation of a spontaneous canine model of immunoglobulin E-mediated food hypersensitivity: dynamic changes in serum and fecal allergen-specific immunoglobulin E values relative to dietary change

The purpose of the pilot study reported here was to evaluate serum and fecal total and allergen-specific immunoglobulin E (IgE) responses to dietary change in five Maltese x beagle dogs with suspected food hypersensitivity, compared with those of five clinically normal dogs. Clinical parameters (pruritus, otitis, and diarrhea) improved in the Maltese x beagle dogs during feeding of a novel diet, and signs were exacerbated by oral allergen provocation. Relative concentrations of serum and fecal wheat-, corn-, and milk-specific IgE were determined by use of an ELISA. The onset of clinical signs of disease was accompanied by an increase in serum allergen-specific IgE concentrations. In contrast, changes in clinical signs of disease or allergen-specific IgE values were not seen in the control group undergoing the same regimen. Total serum IgE concentration was measured by use of the ELISA, and comparison with known quantities of a monoclonal IgE allowed absolute values to be reported. Values were high in the Maltese x beagle colony (7 to 34 microg/ml), compared with those in the control dogs (0.7 to 6 microg/ml). Total serum and total fecal IgE concentrations did not change in either group during the study. Although allergen-specific IgE was detected in the feces of both groups, significant interassay variability made interpretation of the results difficult. The authors concluded that these Maltese x beagle dogs satisfied the currently recognized clinical criteria for the diagnosis of canine food hypersensitivity. Furthermore, the clinical and serologic responses seen in these dogs in response to oral allergen provocation suggest that this may be a useful model for the study of spontaneous food hypersensitivity.     

Up-regulation of liver glucose-6-phosphatase in x-linked hypophosphatemic mice.

We previously showed that a phosphate-deficient diet resulting in hypophosphatemia upregulated the catalytic subunit p36 of rat liver glucose-6-phosphatase, which is responsible for hepatic glucose production. A possible association between phosphate and glucose homeostasis was now further evaluated in the Hyp mouse, a murine homologue of human X-linked hypophosphatemia. We found that in the Hyp mouse as in the dietary Pi deficiency model, serum insulin was reduced while glycemia was increased, and that liver glucose-6-phosphatase activity was enhanced as a consequence of increased mRNA and protein levels of p36. In contrast, the Hyp model had decreased mRNA and protein levels of the putative glucose-6-phosphate translocase p46 and liver cyclic AMP was not increased as in the phosphate-deficient diet rats. It is concluded that in genetic as in dietary hypophosphatemia, elevated glucose-6-phosphatase activity could be partially responsible for the impaired glucose metabolism albeit through distinct mechanisms.     

Studies on the effect of 5α-androstane-3α, 17α-diol in the canine prostate in vivo

Adult beagle dogs, castrated one month previously, were treated with 5α-androstane-3α, 17α-diol (total dose 300 mg) over a period of one month. Examination of the prostate after treatment showed no significant change in size, weight or histological appearance from the castrate dog prostate. Subsequent administration of 5α-androstane-3α, 17β-diol (300 mg) over the same period of time resulted in restoration of the prostate size, weight and histological appearance to that of the normal intact dog prostate. It is concluded that exogenously administered 5α-androstane-3α, 17α-diol does not promote prostatic growth in the castrate beagle dog.     

Investigation of total, free, peptide-bound, protein-bound, soluble and insoluble collagen hydroxyproline content in tissues from the Arabian camel (Camelus dromedarius)

This study was conducted to determine the concentration of total, free, peptide-bound, protein-bound, soluble and insoluble collagen hydroxyproline (Hyp) in tissues from the Arabian camel (Camelus dromedarius). Results indicated that there were significant differences in the concentration of total, free, peptide-bound, protein-bound, soluble and insoluble collagen Hyp in various tissues (P < 0.01). Camel kidney showed a significantly high concentration of total, free, peptide-bound and protein-bound Hyp and collagen content as compared to other tissues examined (P < 0.01). Kidney also showed a significantly high concentration of soluble collagen Hyp as compared to other tissues examined (P < 0.01). However, the concentration of insoluble collagen Hyp was significantly high in liver when compared to other tissues (P < 0.01). These variations may result from differences in the collagen structure and/or composition in this species.     

Breed differences in allele frequency of the dopamine receptor D4 gene in dogs.

We previously reported that the dog dopamine receptor D4 (DRD4) gene is polymorphic as observed in humans, and four alleles were identified based on the number and/or order of the 12 and 39 bp sequences located in the homologous region of human DRD4. To assess the diversity of the DRD4 gene in dogs we examined the allelic variations in four breeds (beagle, golden retriever, Shetland sheepdog, and shiba) employing the polymerase chain reaction (PCR). As a result, we found three novel alleles and determined the DNA sequences of these alleles. The beagle shared four alleles, including 396, 435, 447a, and 447b, with the 435 (52.6%) and 447a (39.5%) alleles being common. The golden retriever had the 435 and 447a alleles, and the 435 allele was frequent (73.3%). In the Shetland sheepdog, the 435, 447a, and 498 alleles were observed, of which the 447a allele was most frequent (82.5%). The shiba had five alleles-447a, 447b, 486, 498, and 549-and the 447b allele was most common (55.4%). These findings suggest that the allele frequency varied among the four dog breeds, and analysis of the DRD4 polymorphism may therefore be useful for elucidating the relationships among dog breeds.     

Development of a gradient reversed-phase HPLC method for the determination of sodium ferulate in beagle dog plasma

A gradient reversed-phase HPLC assay has been developed to determine sodium ferulate (SF) in beagle dog plasma with tinidazole as an internal standard. Chromatographic separation was made on a C(18) column using 0.5% acetic acid and acetonitrile (80:20, v/v) as mobile phase. UV detection was performed at 320 nm. The calibration curve for SF was linear in the range of 0.05-10 microg/ml, and the achieved limit of quantification (LOQ) was 51.4 ng/ml. The results of linearity, within- and between-day precision, and accuracy demonstrate that this method is reliable, sensitive and sufficient for in vivo beagle dog pharmacokinetic (PK) studies of SF.     

Metabolism of 25-hydroxyvitamin D3 in renal slices from the X-linked hypophosphatemic (Hyp) mouse: abnormal response to fall in serum calcium

The effect of the X-linked Hyp mutation on 25-hydroxyvitamin D3 (25-OH-D3) metabolism in mouse renal cortical slices was investigated. Vitamin D replete normal mice and Hyp littermates fed the control diet synthesized primarily 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3); only minimal synthesis of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) was detected in both genotypes and 1,25-(OH)2D3 formation was not significantly greater in Hyp mice relative to normal littermates, despite hypophosphatemia and hypocalcemia in the mutants. Calcium-deficient diet fed to normal mice reduced serum calcium (p less than 0.01), increased renal 25-hydroxyvitamin D3-1-hydroxylase (1-OHase) activity (p less than 0.05), and decreased 25-hydroxyvitamin D3-24-hydroxylase (24-OHase) activity (p less than 0.05). In contrast, Hyp littermates on the calcium-deficient diet had decreased serum calcium (p less than 0.01), without significant changes in the renal metabolism of 25-OH-D3. Both normal and Hyp mice responded to the vitamin D-deficient diet with a fall in serum calcium (p less than 0.01), significantly increased renal 1-OHase, and significantly decreased renal 24-OHase activities. In Hyp mice, the fall in serum calcium on the vitamin D-deficient diet was significantly greater than that observed on the calcium-deficient diet. Therefore the ability of Hyp mice to increase renal 1-OHase activity when fed the vitamin D-deficient diet and their failure to do so on the calcium-deficient diet may be related to the resulting degree of hypocalcemia. The results suggest that although Hyp mice can respond to a disturbance of calcium homeostasis, the in vivo signal for the stimulation of renal 1-OHase activity may be set at a different threshold in the Hyp mouse; i.e. a lower serum calcium concentration is necessary for Hyp mice to initiate increased synthesis of 1,25(-OH)2D3.     

Stochastic resonance is applied to quantitative analysis for weak chromatographic signal of roxithromycin in beagle dog plasma

Based on the theory of stochastic resonance, the signal to noise ratio (SNR) of HPLC/UV chromatographic signal of roxithromycin is enhanced by cooperation of signal, noise and nonlinear system. A simple new method for the determination of low concentration of roxithromycin in beagle dog plasma is presented. Using signal enhancement by stochastic resonance, this method extends the limit of quantitation from the reported 0.5 to 0.1 microg/ml. During validation of the new method, HPLC/MS was used as a comparison technique. The results indicate that the recovery and low concentrations of roxithromycin in beagle dog plasma were equivalent between the two methods (P>0.05). Stochastic resonance may be a promising tool for improving detection limits in trace analysis.     

Effect of mercuric chloride on various hydroxyproline fractions in rat serum

Mercuric chloride (HgCl₂) disturbs the collagen metabolism in the body which is reflected by altered hydroxyproline fractions in the serum. The aim of the present investigation was to study the effect of HgCl₂ treatment on various hydroxyproline (Hyp) fractions in rat serum and the effect of 2,3-dimercapto-1-propane sulfonic acid (DMPS) treatment on serum Hyp fractions in HgCl₂ treated rats. Other parameters studied included body weight, food intake, water intake and kidney weight. Doses of HgCl₂ used were 0.1, 0.5, 1.0, 2.0, 3.0 mg/kg body weight and that of DMPS was 100 mg DMPS/kg body weight. All the doses of HgCl₂ used caused significant (p < 0.01) alterations in free, peptide-bound and protein-bound Hyp in the serum when compared with control rats but a dose of 2 mg/kg body weight caused significant (p < 0.001) alteration even in the total serum Hyp when compared to control rats. Administration of DMPS prior HgCl₂ treatment of rats sacrificed 24 h after the treatment caused a significant decrease of 52% (p < 0.01) in free Hyp when compared to similar HgCl₂ treated rats. DMPS treatment with HgCl₂ also caused an increase of 61% (p < 0.001) and 114% (p < 0. 001) in peptide- and protein-bound Hyp respectively, when compared to HgCl₂ treated rats sacrificed 24 h after mercuric chloride and DMPS treatment. Administration of DMPS followed by HgCl₂ to rats which were sacrificed 48 h later caused no significant change in the total and free Hyp when compared to HgCl₂ treated rats which were sacrificed 48 h after the treatment. But there was a significant decrease of 40% (p < 0.001) in peptide-bound Hyp and an increase in of 77% (p < 0.001) in protein-bound Hyp when compared to HgCl₂ treated rats sacrificed 48 h after the treatment. The present study shows that HgCl₂ treatment caused significant alterations in serum Hyp fractions reflecting disturbed composition of connective tissues which were not reversed by DMPS treatment. (Mol Cell Biochem 271: 159–165, 2005)     

Ion-pair reverse-phase high performance liquid chromatography method for determination of Huperzine-A in beagle dog serum

Huperzine-A (Hup-A), a biologically potent, reversible acetylcholinesterase inhibitor for the treatment of Alzheimer disease (AD) in China, has very low blood concentration. In order to study the pharmacokinetics of newly developed Hup-A transdermal patches in animal, a rapid and sensitive ion-pair reverse-phase high performance liquid chromatography (RP-HPLC) method for the determination of Hup-A in beagle dog serum using mebendazole as internal standard has been developed and validated. The analyte and internal standard were extracted from serum using chloroform-isopropanol (95:5, v/v), analyzed on a C (18) column (5 microm, 150 mm x 4.6 mm i.d.) with a mobile phase consisting of methanol-water-glacial acetic acid (50:48.5:1.5, v/v/v), using sodium dodecylsulfonate as an ion-pair reagent, and detected with UV detector at 313 nm. The chromatographic run time was within 15 min. The assay was linear over the concentration range of 1-12 ng/ml and intra- and inter-day precision over this range was not more than 12.8%. The limit of quantification in serum was 1 ng/ml. The method was successfully applied to characterize the Hup-A concentration-time profiles and study the single and multiple doses phamacokinetics of Hup-A transdermal patches in beagle dogs. The pharmacokinetic study results showed that Hup-A patches has the characteristic of sustained or controlled drug release in vivo.     

Hydroxyproline concentrations in ocular tissues of Arabian camel (Camelus dromedarius Linn.)

Hydroxyproline (Hyp) concentrations (total, free, peptide-bound and protein-bound) in camel eye tissues were determined. Total Hyp concentration was highest in iris, followed by ciliary body, sclera, cornea, lens and retina; the difference between total Hyp concentration of iris and sclera (P < 0.05) and cornea, lens and retina (P < 0.001) was statistically significant. Cornea had the highest concentration of free Hyp, followed by ciliary body, retina, iris, sclera and lens (P < 0.001). Peptide-bound Hyp concentration was highest in iris, followed by lens, cornea, ciliary body, retina and sclera (P < 0.001). Iris also had the highest concentration of protein-bound Hyp, followed by ciliary body, sclera, cornea, retina and lens; the difference in the protein-bound Hyp concentration between iris and sclera (P < 0.05) and cornea, retina and lens (P < 0.001) was statistically significant. Iris was also found to have the highest concentration of collagen, followed by ciliary body, sclera, cornea, lens and retina; the difference between the collagen concentration of iris and sclera (P < 0.05) and cornea, lens and retina (P < 0.001) was statistically significant. These variations may result from differences in the collagen structure and/or composition in these tissues.     

Comparison of histomorphometric values in iliac trabecular bone of beagle dogs raised under different breeding systems.

In order to clarify individual differences in bone metabolism among colony-raised beagle dogs, histomorphometric values of iliac trabecular bone and values of serum biochemical constituents related to bone were examined in 10 and 17 beagle dogs raised, respectively, under our two breeding systems in which differences in factors such as exercise, ultraviolet rays, and mineral content of the diet affect bone metabolism. At the age of 14 months, all dogs were injected with tetracycline hydrochloride and calcein twice for double bone labeling in order to measure dynamic as well as static parameters by bone histomorphometry and the ilium was later biopsied. The measurement on cancellous bone areas of undecalcified iliac sections was performed with a semiautomatic image analyser. Values of total calcium, phosphorus, alkalinephosphatase activity, parathyroid hormone and calcitonin in serum were also determined. The results showed that there were no significant differences between the two groups in histomorphometric values, except for the osteoid volume (p less than 0.05) and osteoid surface/trabecular surface ratio (p less than 0.01) in females, or in serum biochemical constituents, except for alkalinephosphatase activity (p less than 0.001) in males, indicating there were virtually no individual differences in bone metabolism in normal colony raised beagle dogs.     

SD大鼠和Beagle犬大唾液腺的形态学观察

目的-研究及观察SD大鼠和Beagle犬大唾液腺正常比较组织学.方法-SD大鼠和Beagle犬三对大唾液腺剖取后进行石蜡切片、HE染色和PAS染色,光学显微镜观察.结果-SD大鼠腮腺是纯浆液腺,Beagle犬腮腺属混合腺,以浆液性腺泡为主,偶见小的粘液细胞群.SD大鼠的下颌下腺属于以浆液腺泡为主的混合腺,Beagle犬的下颌下腺属于以粘液腺泡为主的混合腺.SD大鼠与Beagle犬的舌下腺均为粘液性腺泡为主的混合腺.Beagle犬的眶腺亦是以纯粘液性腺泡为主的混合腺结构.     

Simultaneous determination of oxymatrine and its active metabolite matrine in dog plasma by liquid chromatography-mass spectrometry and its application to pharmacokinetic studies

A simple, rapid and reliable method was developed for the quantification of oxymatrine (OMT) and its metabolite matrine (MT) in beagle dog plasma using a liquid-liquid extraction procedure followed by liquid chromatography-electrospray ionization mass spectrometric (LC-ESI-MS) analysis. Extend-C18 column (2.1 mm i.d. x 50 mm, 5 microm) with a C18 guard column (2.1 mm i.d. x 12.5 mm) was used as the analytical column. Linear detection responses were obtained for OMT concentration ranging from 5 to 4000 ng/ml and for MT concentration ranging from 5 to 2000 ng/ml. The precision and accuracy data, based on intra- and inter-day variations over 5 days, were lower than 5%. The limit of quantitation for OMT and MT were 2 and 1 ng/ml, respectively, and their recoveries were greater than 90%. Pharmacokinetic data of OMT and its active metabolite MT obtained with this method following a single oral dose of 300 mg OMT capsules to six beagle dogs was also reported for the first time.     

Hypoxanthine enhances hydroxyurea-induced sister-chromatid exchanges in Chinese hamster ovary cells

Treatment with 1 mM hydroxyurea (HU) for 12 h induced sister-chromatid exchange (SCE) in CHO-K1 cells. The induced SCE frequency was always higher in cells grown in Ham's F12 medium than in those grown in RPMI 1640 medium. It was shown that hypoxanthine (Hyp), a component of Ham's F12, was to a great extent responsible for producing a higher level of HU-induced SCEs were synergistically enhanced when Hyp was added to RPMI 1640 medium supplemented with dialyzed fetal bovine serum at a concentration of 30 μM, which is the concentration in Ham's F12 medium.

The radioactivity of [¹⁴C]Hyp was readily incorporated into DNA in either the presence or the absence of HU. The greater part was in the forms of dGMP and dAMP. It was not clear whether Hyp was incorported in the form of dIMP or not. Deoxyguanosine (dGuo), but not deoxyadenosine (dAdo) reversed both the incorporation of radioactivity into DNA and the SCE-enhancing effect of Hyp. Our results indicate that incorporation of Hyp into the dNTP pools and into the DNA, together with perturbation of dGuo metabolism under abnormal conditions during and after HU treatment, is involved in the enhancement by Hyp of HU-induced SCEs.     

Exercise training attenuates diet-induced reduction in metabolic rate

The combined influence of exercise training and dietary restriction on daily energy expenditure was evaluated by exposing 48 male Sprague-Dawley rats to one of three food intake conditions [ad libitum (AL), moderately restricted (MR), or severely restricted (SR)] and to one of two exercise conditions [treadmill exercised (E) or cage confined (CC)]. After 10 wk of exercise and dietary restriction, the MR-CC and MR-E rats weighed 84 and 86%, respectively, of AL-CC, whereas the SR-CC and SR-E rats weighed 66 and 68% of AL-CC. Dietary restriction and subsequent weight loss produced significant reductions in both total and resting daily energy expenditure. Exercise partially reversed this effect, but the extent of this reversal diminished as the severity of dietary restriction was increased. These results raise the distinct possibility that inconsistencies in the current literature concerning the effects of exercise on whole body metabolism during periods of dietary restriction might be reconciled by an appreciation and an understanding of the influence that duration of exercise training and severity of food restriction have on this measure.     

SD大鼠、Beagle犬和新西兰白兔主泪腺的解剖学特点及形态学观察

目的观察及比较SD大鼠、Beagle犬、新西兰白兔主泪腺的解剖学和形态学特点.方法 SD大鼠、Bea-gle犬和新西兰白兔的主泪腺剖取并用12%福尔马林固定,进行石蜡切片、HE染色和PAS染色,光学显微镜观察.结果SD大鼠的眶外泪腺和眶内泪腺均为管泡状的浆液性腺;Beagle犬的主泪腺属于管泡状混合性腺,腺组织被结缔组...