Antiphosphaturic action of 25 (OH) vitamin D3 in experimental Fanconi syndrome.

Renal handling of phosphorus was studied in the following groups of parathyroidectomized rats with maleate-induced Fanconi syndrome: 1) 6 rats receiving intravenous parathyroid hormone, 2) 6 rats receiving intravenous dibutyryl cyclic AMP (DBcAMP), 3) 6 rats undergoing volume expansion with saline, 4) 12 rats receiving intravenous 25 (OH)vitamin D3, 5) 12 rats with acute hypercalcemia induced by intravenous CaCl2, 6) 6 rats with phosphate deprivation, and 7) 6 rats receiving intravenous calcitonin. Parathyroid hormone and calcitonin failed to increase the urinary excretion of both cAMP and phosphorus. Likewise, DBcAMP failed to increase the urinary excretion of phosphorus. Extracellular volume expansion and hypercalcemia (serum calcium 12.9 +/- 0.7 mg/100 ml) did not alter the tubular reabsorption of phosphorus. In phosphate-deprived animals, the fractional excretion 0.16 +/- 0.05 (mean +/- SE) was lower than that in the control animals (maleate-treated without phosphate depletion), 0.46 +/- 0.04 (P less than 0.001). 25 (OH)vitamin D3 decreased the fractional excretion of phosphorus from 0.39 +/- 0.03 in the control (maleate-treated not receiving 25 (OH)vitamin D3) to 0.23 +/- 0.02 (P less than 0.001) in the experimental animals. The present study demonstrated an antiphosphaturic effect of 25(OH)vitamin D3 in experimental Fanconi syndrome; the mechanism of this action is not well understood.     

Emotional disturbance following childbirth: clinical findings and urinary excretion of cyclic AMP (adenosine 3'5'cyclic monophosphate).

Emotional disturbance was assessed in a group of women in the first few days following childbirth and again 2 months and 1 year following childbirth; the clinical features are described. Variables such as social class, age and parity were not related to the level of emotional disturbance, but a history of marital problems, sexual difficulties, poor relationships with immediate family or disrupted family relationships in childhood were so related. Twenty-four hour urinary excretion of cyclic AMP (adenosine 3'5' cyclic monophosphate) was estimated in the same group of women on 2 occasions in the week following childbirth and again 2-3 months later in approximately one third of the original sample. Excretion of cyclic AMP in the few days following delivery was elevated compared with excretion 2-3 months later, and there was a significant rise in urinary excretion of cyclic AMP between the 1st and 2nd urine collections. Those women showing most emotional disturbance on the 3rd day after delivery and women indicating most mood change in the direction of becoming elated had the highest levels of cyclic AMP in the 2nd urine collection.     

Evidence for interference of 25 (OH) vitamin D3 with phosphaturic action of calcitonin.

The present study evaluated the effect of 25(OH)vitamin D3[25(OH)vit D3] on the phosphaturic action of calcitonin in anesthetized parathyroidectomized rats. In group 1, calcitonin was given intravenously over six clearance periods. In group 2, after three periods of calcitonin given intravenously, 25(OH)vit D3 was added and given together with calcitonin for three additional periods. During calcitonin infusion, Cp/CIn 0.18 +/- 0.02 (mean +/- SE) in group 1 was not different from the corresponding Cp/CIn 0.18 +/- 0.03 in group 2; but when 25(OH)vit D3 was added, Cp/CIn 0.12 +/- 0.01 in group 2 was lower (P less than 0.001) than the corresponding Cp/CIn 0.26 +/- 0.02 in group 1. With intravenous calcitonin the urinary excretion of cycle AMP (UcAMP) 97 +/- 29 in group 1 did not differ from the corresponding UcAMP 86 +/- 27 pmol/min in group 2, but when 25(OH)vit D3 was added UcAMP 41 +/- 12 in group 2 was lower (P less than 0.001) than the corresponding UcAMP 131 +/- 14 pmol/min in group 1. This study demonstrated that 25(OH)vit D3 blocks the phosphaturic action of calcitonin. The associated fall in Uctamp suggests that25 (OV)vit D3 acts possibly by inhibiting the calcitonin-induced activation of adenylate cyclase in the kidney. However, other alternative mechanisms of action have not been excluded.     

一种常用免疫分析系统对25(OH)D3和25(OH)D2回收率的评价研究

目的 以液相色谱串联质谱(LC-MS/MS)为参考方法,评价临床常用的索灵(DiaSorin)全自动免疫分析系统对25-羟基维生素D3[25(OH)D3]和25-羟基维生素D2[25(OH)D2]的回收率是否满足临床检测的要求.方法 参考实验室定值的DEQAS 5个水平质控品,用以评价索灵检测25(OH)D的准确度.索灵自带高低值质控品每日4次重复测试,连续5d检测用以评价索灵检测高低值25(OH)D的精密度.解放军总医院健康查体剩余血清样本810例,分别由索灵及LC-MS/MS检测25(OH)D.Passing-Bablok回归、组内相关系数(intraclass correlation coefficient,ICC)分析两种检验方法的相关性和一致性.所有样本根据25(OH)D3及25(OH)D2占总25(OH)D的百分含量由低到高分别分组,计算各组检测偏差,分析索灵检测偏差随25(OH)D3、25(OH)D2百分含量增加的变化趋势.结果 索灵免疫法检测25(OH)D的精密度较好(CV<10％),检测结果与LC-MS/MS相比相关性和一致性均较好,但仍然存在一定的检测偏差.810例样本平均检测偏差为-4.65ng/mL(-16.55％).索灵检测偏差与25(OH)D3或25(OH)D2百分含量不具有线性相关关系;25(OH)D3或25(OH)D2百分含量的增加不增加索灵的检测偏差.结论 索灵全自动免疫分析系统检测维生素D与LC-MS/MS一致性较好,该免疫法对25(OH)D3和25(OH)D2回收率满足临床检测的要求.     

Na,K-ATPase activity and 25(OH)vitamin D3 hydroxylation in rat proximal tubules

The proximal tubule is the target site for parathyroid hormone (PTH), and conversion of 25(OH)vitamin D₃ hormones which impinge on calcium (Ca) homeostatis, as well as a major site for sodium (Na) reabsorption. The effect of changes in PTH and vitamin D status on Na,K-ATPase activity, as a measure of Na transport, were studied in the proximal tubules of adult rat kidneys where Na and Ca reabsorption rates are in parallel. Na,K-ATPase activity and 25(OH)D₃ metabolism were determined in cortical and juxtamedullary proximal tubule segments from normal, parathyroidectomized (PTX), and vitamin D-deficient (-D) rats. Na,K-ATPase activity was highest in cortical segments. PTX led to a decrease in activity in convoluted segments but increased activity in straight segments. In-D rats, Na,K-ATPase activity decreased in cortical segments but increased in juxtamedullary segments. 25(OH)D₃ was metabolized more to 24,25(OH)₂D₃ than to 1,25(OH)₂D₃ in all normal segments. Juxtamedullary segments were more sensitive to PTX and-D conditions. These findings suggest that cortical and juxtamedullary nephrons are inherently different in basal Na,K-ATPase activity, in conversion of 25(OH)D₃ to active metabolites, and in response to altered PTH and vitamin D₃ status.     

Influence of estrogen on renal vitamin D hydroxylases and serum 1alpha,25-(OH)2D3 in chicks.

The influence of estrogen on the metabolism of 25-hydroxyvitamin D3 was studied in 2- to 5-wk-old chicks. Single injections of at least 500 microgram diethylstibestrol (DES) increased the conversion of 25-hydroxyvitamin D3 to 1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3) and suppressed the production of 24,25-dihydroxyvitamin D3 in chick kidney homogenates. Acute (one 5-mg) injections of testosterone or progesterone did not enhance the 25-hydroxyvitamin D3-1alpha-hydroxylase, indicating specificity. However, chronic pretreatment with DES appeared to allow the potentiation of previously unstimulatory steroids such as progesterone and testosterone. In addition, the hormonal metabolite of vitamin D3, 1alpha,25-(OH)2D3, was measured in 4- to 6-wk chick plasma after steroid treatment. Greater than 1 mg DES per day for 5 days was necessary to enhance the circulating level of 1alpha,25-(OH)2D3; testosterone alone had no effect. This elevation was rapid, occurring within 12--24 h after injection. These data suggest that estrogen (as evidenced by DES treatment) is a modulator of vitamin D metabolism along with other known regulators such as parathyroid hormone, phosphate, and 1alpha,25-(OH)2D3. The mechanism of the regulation is as yet unknown.     

Effects and interactions of 24R,25(OH)2D3 and 1,25(OH)2D3 on bone

The effects of various combinations of therapy with 1 alpha,25-dihydroxycholecalciferol (1,25(OH)2D3) and 24R,25-dihydroxycholecalciferol (24R,25(OH)2D3) on structural and dynamic parameters of bone were evaluated in 40 chicks raised on a vitamin D-deficient diet from time of hatching and supplemented with the dihydroxylated metabolites. The results showed that: 1) the maintenance of volumetric density of bone is dependent on the presence of 1,25(OH)2D3, 2) lack of 1,25(OH)2D3 is associated with an increase in the number of osteocytes per unit volume of bone, most probably due to decreased amounts of bone formed by each osteoblast before becoming an osteocyte, 3) adequate quantities of either 24R,25(OH)2D3 or 1,25(OH)2D3 are needed to prevent accumulation of osteoid or the production of endosteal fibrosis, and 4) maintenance of normal tetracycline label width requires both hydroxylated compounds with one of them in sufficient amounts. The data of this study demonstrate that the integrity of certain parameters of bone structure could be maintained only with 1,25(OH)2D3, others with either dihydroxylated metabolites, and still others with a combination of both. These data underscore the biological activity of 24R,25(OH)2D3 by demonstrating its effectiveness on bone.     

25(OH)D3与糖尿病视网膜病变的关系研究

目的 探讨25(OH)D3与糖尿病视网膜病变之间的关系。方法 回顾性分析2016年1月~2017年3月我科收治的2型糖尿病患者83例,根据患者是否合并视网膜病变,分为观察组43例和对照组40例。所有患者检测并比较血糖（FBG）水平、总胆固醇（TC）、甘油三酯（TG）、高密度脂蛋白（HDLC）、低密度脂蛋白（LDL-C）、糖化血红蛋白（HbA1c）以及25(OH)D3的水平。结果 两组患者在TG（t=5.572,P=0.037）、HDLC（t=5.548,P=0.037）、HbA1c（t=6.627,P=0.012）、25（OH）D3（t=9.738,P=0.000）等临床生化资料比较中差异均具有统计学意义（P＜0.05）。对25(OH)D3的Pearson进行相关性分析后发现,25(OH)D3与T2DM病程（r=0.163,P=0.027）、TC（r=0.170,P=0.025）、HDLC（r=0.177,P=0.023）、LDLC（r=0.185,P=0.015）具有高度相关性;偏相关显示,25(OH)D3与TC、HDLC、LDLC具有相关性。结论 25(OH)D3水平能够反映糖尿病视网膜病变患者的疾病发展状态,在诊断及评估中具有一定的参考价值。     

Influence of cyclic 3',5'-adenosine monophosphate and adenosine on vascular reactivity.

cAMP, 5'-AMP and adenosine in doses of 1, 2 and 5 mg/kg i.v. in rats diminish vascular resistance in the hind limbs in vivo and in isolated limbs perfused with nutrient fluid containing adrenaline (A) (10(-3) M), slowing action of the heart and lowering blood pressure. After administration of cAMP, 5'-AMP and adenosine (5 mg/kg), vasoconstricting action of noradrenaline (NA) and A was depressed, and the vasodilating action of isoprenaline (I) was enhanced. The changes in blood pressure observed after administration of the tested adenosine compounds were not blocked by phentolamine, but after I were blocked by propranolol. cAMP, 5'-AMP and adenosine had no influence on the drop in blood pressure elicited by acetylcholine. The results indicate that cAMP, 5'-AMP and adenosine increase reactivity of beta-receptors of the sympathetic system in the blood vessels and modify the action of catecholamines on blood vessels.     

Age associated changes in intracellular cyclic adenosine monophosphate.

Cyclic adenosine monophosphate (cAMP) levels of isolated human peripheral blood lymphocytes show age associated changes. These changes are apparent in basal cAMP levels as well as in cAMP levels after trypsin treatment. Both cord blood lymphocytes and lymphocytes isolated from the blood of old people exhibit lower basal levels of cAMP and diminished increase in cAMP by trypsin treatment, in comparison to lymphocytes isolated from the other age groups. The results seem to indicate a low number of reactive cells and a low reactivity of the cells in the case of cord blood lymphocytes, and a decrease in number of the reactive cells without a decrease in specific reactivity of isolated lymphocytes of old people.     

Enzyme immunoassays for the estimation of adenosine 3',5' cyclic monophosphate and guanosine 3',5' cyclic monophosphate in biological fluids.

Simple, rapid and sensitive competitive enzyme immunoassays for the estimation of adenosine 3',5' cyclic monophosphate (cAMP) and guanosine 3',5' cyclic monophosphate (cGMP) in human plasma and urine are described. Specific antisera to each nucleotide were raised in rabbits by immunization with succinyl cyclic nucleotide--human serum albumin conjugates. For the assay, specific antibodies were incubated with a mixture of succinyl cyclic nucleotide labelled with horseradish peroxidase together with unlabelled standard or sample. The antibody-bound enzyme conjugate was separated from free hapten by anti-rabbit (IgG) sera immobilized to a microtitre plate. Activity of the bound enzyme conjugate was determined with tetramethylbenzidine. The assays were capable of detecting levels as low as 2 fmol of cAMP and cGMP. Good correlations were obtained between values generated by enzyme immunoassay and radioimmunoassay.     

Double antibody enzyme immunoassay for the quantitation of adenosine 3', 5' -cyclic monophosphate (cyclic AMP) and guanosine 3', 5'-cyclic monophosphate (cyclic GMP) in tissue and plasma

A sensitive double antibody enzyme immunoassay for the quantitation of cyclic AMP and cyclic GMP is presented. Specific antisera to each nucleotide were raised in rabbits by immunization with succinyl cyclic nucleotide-human serum albumin conjugates. For competitive reaction, antibodies were incubated with a mixture of succinyl cyclic nucleotide labelled with beta-D-galactosidase and unlabelled succinylated standard or sample cyclic nucleotides. The antibody-bound enzyme-hapten was separated from free hapten by anti-rabbit IgG immobilized to a polystyrene ball. Activity of the enzyme on the solid phase was fluorometrically determined. The assay system made it possible to ascertain values as low as 5 fmole of cyclic AMP or cyclic GMP. Cyclic nucleotides in plasma could be accurately determined by this method without requiring a deproteinizing reagent as the first step of assay.     

The in vivo and in vitro effect of calmodulin antagonists on the renal actions of 25(OH) vitamin D3 in the rat

Previous work from this laboratory has demonstrated that 25(OH) vitamin D₃ [25(OH)D₃] acutely suppresses the phosphaturic action of parathyroid hormone (PTH) and interferes with the PTH-induced activation of adenylate cyclase (AC). Calmodulin inhibitors block vitamin D-induced Ca²⁺ transport in the gut and phosphorus uptake in renal BBMV's. We have examined whether calmodulin antagonists affect the renal action of 25(OH)D₃. Acute clearance experiments were performed in PTH-infused parathyroidectomized rats receiving 25(OH)D₃ after pretreatment with trifluoperazine (TFP) or promethazine (P). In vitro PTH-induced activation of renal AC was also studied in membrane preparations from pretreated rats in the presence of 25(OH)D₃. 25(OH)D₃ reduced the PTH-stimulated increase in fractional excretion of phosphorus (CP/CIn) from 0.292±0.024 to 0.195±0.018 (p<0.005) and urinary cAMP from 149.3±20.3 to 78.1±10.4 pmol/min (p<0.01) and also blunted AC activation in vitro. TFP but not P abolished the effects of 25(OH)D₃ both in vivo and in vitro. R 24571 also abolished the in vitro effect of 25(OH)D₃. Thus, (1) TFP abolishes both the antiphosphaturic and the AC/cAMP-related actions of 25(OH)D₃, (2) P does not have these effects, and (3) R 24571 abolishes the in vitro effect of 25(OH)D₃. These results suggest that the antiphosphaturic effect of 25(OH)D₃ acting via the AC/cAMP system may be calmodulin dependent.     

Soluble ST2 regulation by rhinovirus and 25(OH)‐vitamin D3 in the blood of asthmatic children

Paediatric asthma exacerbations are often caused by rhinovirus (RV). Moreover, 25(OH)‐vitamin D3 (VitD3) deficiency during infancy was found associated with asthma. Here, we investigated the innate immune responses to RV and their possible modulation by 25(OH)‐VitD3 serum levels in a preschool cohort of children with and without asthma. The innate lymphoid cell type 2 (ILC2)‐associated marker, ST2, was found up‐regulated in the blood cells of asthmatic children with low serum levels of 25(OH)‐VitD3 in the absence of RV in their airways. Furthermore, in blood cells from control and asthmatic children with RV in their airways, soluble (s) ST2 (sST2) protein was found reduced. Asthmatic children with low 25(OH)‐VitD3 in serum and with RV in vivo in their airways at the time of the analysis had the lowest sST2 protein levels in the peripheral blood compared to control children without RV and high levels of 25(OH)‐VitD3. Amphiregulin (AREG), another ILC2‐associated marker, was found induced in the control children with RV in their airways and low serum levels of 25(OH)‐VitD3. In conclusion, the anti‐inflammatory soluble form of ST2, also known as sST2, in serum correlated directly with interleukin (IL)‐33 in the airways of asthmatic children. Furthermore, RV colonization in the airways and low serum levels of 25(OH)‐VitD3 were found to be associated with down‐regulation of sST2 in serum in paediatric asthma. These data indicate a counter‐regulatory role of 25(OH)‐VitD3 on RV‐induced down‐regulation of serum sST2 in paediatric asthma, which is relevant for the therapy of this disease.     

Urinary cyclic adenosine monophosphate in young adults and elderly subjects.

The 24-hour urinary excretion of cyclic 3',5'-adenosine monophosphate (cAMP) was measured by a protein-binding assay in 55 healthy volunteers (aged 20-35 yr) and in 30 hospitalized elderly subjects (aged 70-93 yr). In the older subjects the mean 24-hour cAMP excretion was significantly lower; the correlation between cAMP excretion and age demonstrated a progressive decrease from the age of 70 to the tenth decade. Many different factors could account for the reduced urinary cAMP excretion in elderly subjects: a decline in the reactivity of the adenyl cyclase-cAMP system related to physiological ageing; reduced physical activity; a reduction in the glomerular filtration rate or decreased production of cAMP by tubular cells in the senile kidney.     

Immune dysfunction in hypophosphatemic vitamin D-resistant rickets: immunoregulatory reaction of 1 alpha(OH) vitamin D3

We investigated immunologic function in six cases with hypophosphatemic vitamin D-resistant rickets (VDRR) before and after treatment with 1 alpha-hydroxycholecalciferol (1 alpha(OH) vitamin D3). All cases suffered frequent episodes of infection, which tended to be more severe in the older patients. OKT9-, OKT10-, and OKM1-positive cells and adenosine deaminase (ADA) were significantly increased, whereas numbers and activity of natural killer (NK) cells were lower than normal before treatment. After administration of 1 alpha(OH) vitamin D3, however, the susceptibility to infection apparently decreased, and NK cell number and activity increased in all patients. ADA was also significantly decreased and remained in the normal range after treatment. These results suggest that vitamin D plays a role in the impaired immunoregulatory functions of NK cells in VDRR. Furthermore, ADA may be one parameter reflecting this immunologic impairment.     

Effect of vitamin K deficiency on urinary gamma-carboxyglutamic acid excretion in rats

Since gamma-carboxyglutamic acid (Gla) in Gla-containing proteins is stoichiometrically excreted into urine as free Gla, urinary Gla excretion is believed to reflect the rate of synthesis and degradation of vitamin K-dependent proteins and the utilization of vitamin K in body. We studied the daily changes in urinary Gla excretion and plasma vitamin K-dependent clotting factor levels in rats fed vitamin K-deficient diets followed by subcutaneous injection of vitamin K1 or after the oral administration of Warfarin. Urinary Gla excretion in normal rats fed a standard diet that contained about 500 ng of vitamin K1 per gram of diet was 2.35 +/- 0.25 mumoles/day, but the level in rats fed a markedly vitamin K-deficient diet (less than 5 ng/g) decreased to 1.40 +/- 0.14 mumoles/day. When rats were fed a moderately vitamin K-deficient diet (20-50 ng/g), plasma vitamin K-dependent clotting factor levels decreased significantly, but urinary Gla excretion did not decrease. Warfarin, a vitamin K antagonist, caused a significant decrease in urinary Gla excretion and plasma clotting factor levels. When vitamin K, (200 micrograms/kg) was injected subcutaneously in rats fed a markedly vitamin K-deficient diet, the plasma vitamin K-dependent clotting factor levels recovered quickly to normal, but urinary Gla excretion showed only a partial recovery to 1.74 +/- 0.15 mumoles/day. These results indicate that urinary Gla excretion decreases in vitamin K deficiency, but changes in urinary Gla excretion do not reflect vitamin K deficiency in rats as sensitively as changes in the prothrombin time and plasma K-dependent clotting factor levels.