Intestinal calcium-binding protein in animals fed normal and rachitogenic diets: I. Rat studies.

Measurements were made of duodenal calcium-binding protein (CaBP) on rats during development of rickets and, subsequently, following vitamin-D2 treatment. Results showed a poor inverse correlation between duodenal CaBP and rickets. In rats fed a phosphate-deficient rachitogenic diet, duodenal CaBP concentration finally fell below detectable limits, but CaBP was still readily measurable 2 weeks after rickets was clearly established. Following a massive dose of vitamin D2 (50 000 I.U.) to rachitic animals, CaBP was formed. However, a small dose of vitamin D2 (500 I.U. daily for 4 days) was insufficient to demonstrate CaBP synthesis than vitamin-D treatment alone. The rachitogenic diet supplemented with phosphate, which caused osteoporosis but not rickets, inhibited CaBP synthesis. The results suggest that nutritional deficiencies from the rachitogenic diet, in addition to vitamin-D deficiency, inhibited CaBP synthesis.     

D-glucose uptake in intestinal brush-border membrane vesicles of rachitic rats

We have previously reported the metabolic consequences of feeding rats Steenbock and Black's rickets-inducing diet, deficient in vitamin D and with an altered Ca/P ratio. Using isolated brush-border membrane vesicles prepared from the jejunum, ileum and duodenum of control and rachitic rats, we have demonstrated a marked decrease of Na+-dependent D-glucose uptake at jejunum-ileum level of rachitic rats. At duodenum level Na+-dependent D-glucose transport was not influenced by rickets. A lack of any significant difference between the two animal groups was observed studying the facilitated transport of D-glucose, the diffusion of L-glucose and the Na+-dependent uptake of phenylalanine and aspartate.     

Vitamin D3 and 1,25-dihydroxyvitamin D3 stimulate the skeletal muscle-calcium mobilization in rachitic chicks

The Ca content in skeletal muscle relative to vitamin D3 intake was studied in chicks. It was found that the Ca content in rachitic chick muscle was significantly higher than normal and it decreased with vitamin D3 treatment. In 4-week-old chicks fed a vitamin D-deficient diet, the Ca content in leg muscle reached 9.86 +/- 1.07 mg/100 g wet wt, although in chicks receiving vitamin D3 in doses of 100 and 500 IU/kg diet, it was 7.80 +/- 0.72 and 6.08 +/- 0.61 mg/100 g wet wt, respectively. A single i.m. dose of 0.50 micrograms of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) or vitamin D3 caused a dramatic decrease in the muscle Ca content by 3 to 6 h after the injection. A simultaneous rise in the Ca level in blood serum was observed. However, at this time the Ca binding protein content in duodenal mucosa and the stimulation of Ca absorption were negligible. These findings allow the conclusion that the vitamin D deficiency in chicks leads to a surplus Ca accumulation in skeletal muscle. The administration of vitamin D3 or its metabolites causes rapid Ca release during the first 6 h. This may be the source of the Ca level increase in blood serum. In this respect 1,25(OH)2D3 was much more effective than vitamin D3.     

Rickets and Osteomalacia in the Glasgow Pakistani Community, 1961-71

The prevalence of vitamin D deficiency was reassessed in April and May 1971, 10 years after the discovery of widespread late rickets and osteomalacia in the Glasgow Pakistani community. Evidence of vitamin D deficiency was found in 28 out of 115 adults and children examined (24%). Children at the age of puberty were most severely affected by rickets, whereas most infants and younger children in the survey were protected by vitamin D supplements. Mild biochemical osteomalacia was common in Pakistani women.A total of 21 Pakistani and Indian children with rickets were admitted to Glasgow hospitals during 1968-70. These comprised 10 children with infantile rickets and 11 with late rickets. Four of the latter group required osteotomy for severe rachitic deformity.Late rickets and osteomalacia in Pakistani and Indian immigrants are not primarily due to nutritional deficiency of vitamin D, though the high phytate content of their diet may be of aetiological importance. A combination of environmental, social, and endogenous factors, the relative importance of which is not at present clear, may also be involved. Advice on the prophylaxis of vitamin D deficiency should be given to all Pakistani and Indian communities in the United Kingdom.     

Effect of 25-hydroxyvitamin D3 on Na+-dependent D-glucose transport in rachitic rats

We have previously reported that feeding rats on Steenbock and Black's rickets-inducing diet, deficient in vitamin D and with an altered Ca/P ratio, leads to metabolic consequences and a marked decrease of Na+-dependent D-glucose uptake at the jejunum-ileum level. To clarify the relationship between experimental rickets and D-glucose uptake, 25-hydroxyvitamin D3 (25-OH-D3) was given to rats fed on the rickets inducing diet. In the jejunum-ileum of these animals Na+-dependent D-glucose uptake returned to the values of the controls while the decrease in D-glucose uptake in the brush-border membrane vesicles prepared from kidney cortex of rachitic animals was not corrected by the administration of 25-OH-D3.     

Comparative studies of type X collagen expression in normal and rachitic chicken epiphyseal cartilage

The levels of type X collagen in mineralizing normal chicken epiphyses and nonmineralizing rachitic chicken tibial epiphyses were measured and compared. Qualitative immunoperoxidase studies with anti-chick type X collagen monoclonal antibodies on sections from normal and rachitic cartilage demonstrated that the type X collagen levels in rachitic growth plates are reduced. Northern hybridization of mRNA and biosynthetic studies have confirmed that type X collagen synthesis in rickets is also decreased. In hypocalcemic rickets, the level of type X collagen mRNA is reduced by 80% whereas the level of type X collagen mRNA is only reduced by 50% in normocalcemic rickets. These observations provide additional evidence that type X collagen is involved in the process of cartilage mineralization and also suggest that the partial recovery of type X collagen synthesis in normocalcemic rickets may be related to the elevated plasma concentration of calcium. Calcium concentration may therefore play an important role in the control of type X collagen synthesis.     

Altered cathepsin D metabolism in PHEX antisense human osteoblast cells

X-linked hypophosphatemia (XLH), the most common form of hereditary rickets, is caused by loss-of-function mutations of PHEX gene in osteoblast cells, leading to rachitic bone disease and hypophosphatemia. Available evidence today indicates that the bone defect in XLH is caused not only by hypophosphatemia and altered vitamin D metabolism, but also by locally released osteoblastic mineralization inhibitory factor(s), referred to as minhibin. In our present study, we found that suppression of PHEX expression by PHEX antisense in human osteoblast cells caused an increase in cathepsin D expression at protein, but not mRNA, levels. This was associated with a decrease in cathepsin D degradation and an increased cathepsin D release into culture media. Our results also showed that lowering cathepsin D activity in antisense cell conditioned media abolished their inhibitory effect on osteoblast cell calcification, suggesting the involvement of cathepsin D in mediating the minhibin activity of the antisense cell conditioned media.     

Type X collagen alterations in rachitic chick epiphyseal growth cartilage

We examined collagens of both normal and vitamin D-deficient chick epiphyseal growth cartilage. Special emphasis was placed on the study of Type X collagen, a recently described product of hypertrophic chondrocytes. Scanning electron microscopy of the epiphyseal growth cartilage of vitamin D-deficient chickens showed an enlarged growth cartilage with a disorganized extracellular matrix. The cartilage collagens were solubilized by proteolytic digestion and disulfide bond reduction of both normal and rachitic growth tissues. Sequential extraction with neutral salt and acetic acid buffers followed by pepsin digestion at 4 degrees C solubilized about 12% of normal tissues and about 7% of collagen from rachitic growth cartilage. Treatment of the pepsin-resistant collagens with neutral salt-dithiothreitol buffer under nondenaturing conditions and a subsequent pepsin digestion increased the yield of solubilized collagen to greater than 95% of the total tissue collagen. Results of the biochemical studies showed a marked increase in the relative proportion of Type X collagen (from 5.6 to 27.9%), a corresponding decrease in the proportions of Types II and IX collagens, and a moderate increase in Type XI collagen in rachitic cartilage. Amino acid analysis indicated that there were no differences in the Types II and X collagens of normal and rachitic cartilage. However, an abnormality in the relative proportions of the CNBr peptides of Type X collagen was detected in the rachitic cartilage. We suggest that the increase in collagen in the rachitic state may reflect increased levels of Type X collagen synthesis by cells in the hypertrophic region. It is likely that in rickets the overproduction of Type X collagen may be a compensatory mechanism by which the hypertrophic chondrocyte attempts to provide a maximum area of calcifiable matrix for the calcium-depleted serum.     

The phosphorylation of 5′-oligoadenylic acids by adenylate kinase and adenosine triphosphate

1. This paper reports studies on the metabolism of bone from normal chicks and from chicks with vitamin D-deficiency rickets. Both in vitro and in vivo there was an increased incorporation of [(14)C]proline into collagen hydroxyproline by rachitic bone. The proportion of the collagen that was soluble in cold salt solutions was greater with the rachitic bone. These results show that in rickets there is an increased synthesis of bone collagen, but they do not provide any evidence of a defect in the maturation of collagen. 2. Rachitic bone incubated aerobically in vitro consumed more glucose and released more lactate than normal bone. Bone from rachitic chicks treated with vitamin D 48hr. previously had rates of glycolysis that were nearly normal. Though we were unable to show any direct action of vitamin D in vitro, we consider that vitamin D probably has a direct action on bone, possibly related to matrix biosynthesis.     

Intestinal calcium-binding protein in animals fed normal and rachitogenic diets: II. Pig studies.

Experiments were carried out in pigs to investigate the relationship between concentration of intestinal calcium-binding protein (CaBP) and the presence of rickets. Pigs made rachitic by a diet deficient in calcium and in vitamin D had concentrations of intestinal CaBP no less than values obtained on control pigs. These experiments, together with earlier work on rats (Can. J. Physiol. Pharmacol. 1975. 53, 137--143), demonstrate a poor inverse correlation between levels of intestinal CaBP and rickets.     

The effect of citric acid and microbial phytase on mineral utilization in broiler chicks

An experiment was conducted to study the effect of microbial phytase (Natuphos^® 500) supplementation in chicks fed different levels of available phosphorus (AP) and citric acid (CA) on performance, mineral retention (Ca, P, Mg, and Zn), bone and plasma minerals (Ca, P, Mg, and Zn), plasma total protein (TP), and serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) activities. Data were analyzed as a 2×4×2 factorial arrangement with two levels of AP (3.5 and 2.5 g/kg), four levels of phytase (0, 200, 400 and 600 U/kg), and two levels of citric acid (0 and 20 g/kg). The low-AP diets reduced performance. Phytase supplementation increased weight gain (up to 7% quadratically) and feed consumption (up to 5%). This response was statistically maximized by 200 U/kg phytase. Feed to gain ratio was not affected by phytase addition. Growth response to phytase was negatively affected by citric acid. Decreasing AP content in the diet increased Ca, P, and Mg retention, and reduced Zn retention. Phytase supplementation linearly increased Ca, P, and Zn retention by 9, 10 and 16%, respectively. Citric acid addition also increased Ca, P, and Zn retention by 3, 3 and 4%, respectively. Likewise, the decrease in AP content in the diet caused a reduction of tibia ash and tibia Zn, and an increase in tibia Ca and P contents. Phytase supplementation increased tibia ash (up to 4%), tibia Ca (up to 2%), P (up to 1%) and Zn (up to 4%) contents, tibia weight (up to 9%), and relative tibia (up to 19%) and liver (up to 13%) weights. Citric acid increased tibia ash (2%), and tibia Ca (2%) and P (2%) contents. Finally, by decreasing AP levels in the diet, plasma Ca and Zn concentrations as well as AST, ALP, and LDH activities were increased. However, plasma P and TP content were reduced. Phytase supplementation increased linearly plasma Ca (up to 4%), P (up to 12%), Mg (up to 10%), Zn (up to 22%) and TP (up to 7%) content, and serum AST (up to 22%), ALT (up to 40%), and LDH (up to 17%) activities, and reduced linearly serum ALP (up to 34%) activity. Citric acid addition increased plasma Ca, Mg, and Zn content by 10, 4, and 5%, respectively, and reduced ALP activity by 13%. In conclusion, these results indicated that the addition of phytase to maize and soyabean meal low-AP diets improved the performance and increased Ca, P, and Zn utilization in chicks. However, the inclusion of citric acid depressed the performance and caused an increase in mineral utilization. Growth response to phytase was negatively affected by citric acid.     

General screening for celiac disease is advisable in children with type 1 diabetes.

The association between celiac disease (CD) and diabetes mellitus type 1 is well known. Only about one-third of all patients with CD are diagnosed in childhood as a result of typical gastrointestinal symptoms or growth retardation. To evaluate the feasibility of CD screening in diabetic children, we tested autoantibodies to tissue transglutaminase (tTGA) in all children with type 1 diabetes from our pediatric department during a 12-month period. In antibody-positive cases, we analyzed the clinical presentation and offered a duodenal biopsy to confirm the diagnosis and grade the severity of the inflammatory process. Of 205 children, 13 (6.3 %) were tTGA-positive. In seven of eight children who agreed to perform a biopsy, CD typical histological signs were detected (Marsh 1: n = 1, Marsh 3: n = 6). In three patients with confirmed disease, symptoms (iron deficiency, recurrent abdominal pain) remained undiscovered up to time of screening (latent form); in four, the disease was asymptomatic (silent form). Since clinical symptoms are mostly mild or absent in spite of severe signs of duodenal inflammation, we recommend tTGA screening in all diabetic children. This strategy may allow the identification of patients in an early stage in respect of prevention of long-term complications.     

Vitamin-D-deficient rickets in Manitoba, 1972-84.

Vitamin-D-deficient rickets still exists in children in Manitoba and adjacent areas. Between 1972 and 1984, 48 cases were documented at Winnipeg Children''s Hospital. The patients ranged in age from 1 to 49 months; 40 were Canadian natives (38 Indians and 2 Inuit), most of whom lived in the Island Lake area of northern Manitoba. Of the 48, 16 had clinical signs of rickets, 12 had tetany due to hypocalcemia and 38 had radiologic evidence of rickets. Hypocalcemia was found in 27, and hypophosphatemia in 19; hyperaminoaciduria was found in 7 of 20. All 48 had elevated serum alkaline phosphatase levels. In addition to rickets, 16 patients aged 12 months or more had evidence of malnutrition. Climate and lifestyle in northern areas of the Canadian midwest result in little or no biosynthesis of vitamin D by solar radiation; therefore, adequate dietary vitamin D intake is essential to prevent deficiency. The diets of pregnant women and infants in these areas are deficient in vitamin D. The authors recommend vitamin D supplements for all pregnant women and infants in areas of risk to eradicate this preventable disease.     

骨密度检测对婴幼儿佝偻病早期诊断的临床意义

目的:探讨骨密度检测在佝偻病早期诊断中的应用价值。方法:收集衡水市哈励逊国际和平医院门诊诊治的600例婴幼儿,以血清维生素D327.5 nmol/L为判定标准分为非佝偻病组和佝偻病组,比较两组婴幼儿维生素D3、骨碱性磷酸酶和骨密度,绘制维生素D3、骨碱性磷酸酶、骨密度诊断结果的ROC曲线图,对骨密度检测结果进行评价。结果:佝偻病组婴幼儿维生素D3和骨密度Z值明显低于非佝偻病组,骨碱性磷酸酶显著高于非佝偻病组(P0.05)。维生素D3诊断佝偻病的ROC曲线下面积为0.951,灵敏度为0.973,特异度为0.840;骨碱性磷酸酶诊断佝偻病的ROC曲线下面积为0.866,灵敏度为0.824,特异度为0.747;骨密度Z值诊断佝偻病的ROC曲线下面积为0.923,灵敏度为0.826,特异度为0.875,骨密度指标诊断佝偻病的曲线下面积和维生素D3比较无统计学意义(P0.05),但骨密度指标诊断佝偻病的曲线下面积大于骨碱性磷酸酶(P0.05)。结论:超声骨密度检测在婴幼儿佝偻病早期诊断中具有一定价值,其诊断敏感性、特异性、准确率与维生素D3诊断基本相当,且骨密度检测存在无创、可重复性高等优点。     

Manganese alters mitochodrial integrity in the hearts of swine marginally deficient in magnesium

It was previously reported that pigs marginally deficient in magnesium (Mg) and fed diets high in manganese (Mn) died suddenly with signs of sudden cardiac death. Manganese, which has properties similar to Mg, may exacerbate Mg-deficiency and be accumulated by mitochondria resulting in ultrastructural damage. The objective of this study was to determine whether deaths of the type previously observed were mediated by adverse interactions of Mn and Mg resulting in ultrastructural damage to the myocardium, alterations in electrocardiographic recordings and tissue retention of Mn, Mg and calcium (Ca). Forty-eight pigs were fed one of six diets in a 2 X 3 factorial arrangement of Mg (100 or 1000 mg Mg/kg) and Mn (5, 50 or 500 mg Mn/kg) for 8 weeks. Left ventricle muscle samples were collected for examination by transmission electron microscopy. No differences in heart muscle ultrastructure were observed between pigs fed low and adequate dietary Mg. However, marked myocardial necrosis and mitochondrial swelling were observed in pigs fed high dietary Mn when combined with low Mg. Feeding low dietary Mg elevated minimum (P < 0.01), maximum (P < 0.05) and average (P < 0.001) heart rates. Low dietary Mg resulted in a 55% probability of a ventricular beat being recorded (P = 0.05) and lower Mg (P < 0.02) and Ca (P < 0.04) contents in heart atria and ventricles. These results suggest that high Mn, when fed in combination with low Mg, disrupts mitochondrial ultrastructure and is associated with the sudden deaths previously reported.     

Prevention of rickets in Asian children: assessment of the Glasgow campaign.

In March 1979 the Greater Glasgow Health Board launched a campaign to reduce the high prevalence of rickets in Asian children in the city. A precampaign survey had shown that voluntary low dose vitamin D supplementation would reduce the prevalence of rickets in Asian children. A survey carried out two and three years after the launch of the official campaign also showed a reduction in the prevalence of rickets in children taking low dose supplements equivalent to about 2.5 micrograms (100 IU) vitamin D daily. There was a considerable reduction in the total prevalence of rickets in this survey compared with the precampaign survey. Hospital discharges of Asian children with rickets declined rapidly after the start of the campaign.     

Dietary magnesium increases calcium absorption of ovine small intestine in vivo and in vitro

The purpose of this study was to investigate whether or not an increase in dietary Mg intake increases Ca absorption in the ovine gastrointestinal tract. In an in vivo experiment, an increase in the infused MgCl2 level (0.0, 25.0 and 75.0 mg Mg x kg BW(-1) x day(-1) with 75.0 mg Ca x kg BW(-1) x day(-1) as CaCl2) into the rumen for ten days significantly decreased fecal excretion but increased urinary excretion (P < 0.05) of Ca in five castrated male sheep. Apparent Ca absorption tended to increase (P = 0.067) whilst the retention and plasma concentration of Ca were not changed. In an in vitro experiment with isolated segments from the rumen, upper jejunum, cecum and upper colon under the presence of an electrochemical gradient, the mucosal to serosal Ca flux rate was significantly greater in the presence of 60.0 mM as compared with 1.2 mM MgCl2 (P < 0.05). From these results, we conclude that the mucosal Mg has the ability to increase the Ca absorption in the gastrointestinal tract in sheep when the dietary Mg level is raised.     

Alterations of the gamma-carboxyglutamic acid and osteocalcin concentrations in vitamin D-deficient chick bone

The content of osteocalcin and protein bound gamma-carboxyglutamic acid (Gla) was studied as a function of bone maturation and mineralization in normal and vitamin D-deficient, rachitic chickens. The Gla/Ca2+ ratio was elevated in rachitic bone, particularly in the most undermineralized regions. For example, there is a 10- to 20-fold elevation in Gla/Ca2+ in the newly synthesized, least mineralized rachitic bone fraction, which progressively decreases to a 1.5-fold elevation in the most highly mineralized areas of rachitic tissue. Osteocalcin, which is the principal Gla-containing protein of mature bone, was quantitated by radioimmunoassay using specific antiserum to the 5670-dalton chicken protein. Surprisingly, the osteocalcin concentration is decreased 50% in vitamin D-deficient bone. From this we infer that accumulated Gla-containing protein in vitamin D-deficient and poorly mineralized bone may possibly represent a precursor of osteocalcin.     

Skeletal manifestations of rickets in infants and young children in a historic population from England

Gross and radiographic changes characteristic of inadequate bone mineralization due to rickets are described in 21 immature skeletons from a 19th century urban population from Birmingham, England. The aims of the study are as follows: to evaluate and if possible augment existing dry-bone criteria for the recognition of rickets in immature skeletal remains; to investigate the value of radiography for the paleopathological diagnosis of rickets; and to compare and contrast the expression of rickets in this group with that previously documented for a rural agrarian population from Wharram Percy, England. Some gross skeletal signs of rickets which were not previously well-documented in paleopathological studies are noted. The worth of radiography for evaluating structural changes to both cortical and trabecular bone in the disease is demonstrated, and features useful for the interpretation of vitamin D deficiency are discussed. The pattern of skeletal elements affected and the severity of changes differs in the Birmingham group from that seen in the comparative rural population. It is emphasized that a variety of factors may influence the expression of rickets in paleopathological material, including rate of skeletal growth, age cohort affected, and intensity of vitamin D deficiency. Nevertheless, careful analysis, not only of the frequency of rickets but also of the degree of severity of lesions and the patterning with respect to skeletal elements affected, may enable more nuanced understanding of the biocultural context of the disease in earlier populations.     

Characterization of a monoclonal antibody B1 that recognizes phosphorylated Ser-158 in the activation peptide region of human coagulation factor IX

Blood coagulation factor IX (FIX) undergoes various post-translational modifications such as gamma-carboxylation and glycosylation. Non-phosphorylated recombinant FIX has been reported to rapidly disappear from plasma, indicating that phosphorylation of FIX plays an important role in the physiological activity of this coagulation factor. In this study, we characterized the human FIX activation peptide (AP) using a monoclonal antibody that recognizes phosphorylated Ser-158 in the AP region. Murine monoclonal antibody B1 against human FIX recognized FIX with an apparent K(d) value of 5 nm in the presence of Ca(2+) (EC(50) = 0.58 mm). B1 bound to the isolated AP of FIX and retained the Ca(2+) dependence of binding to the isolated AP. The deglycosylation of AP did not affect the binding of B1 to AP, while B1 failed to bind to recombinant AP expressed in Escherichia coli. MALDI-TOF mass spectrometry showed that the m/z of plasma-derived deglycosylated AP is 82.54 Da greater than that of recombinant AP. The binding ability of B1 to AP was lost by the dephosphorylation of plasma-derived AP. B1 bound to synthetic peptide AP-(5-19), including phosphoserine-13, but not to the non-phosphorylated AP-(5-19) in the presence of Ca(2+). These data provide direct evidence that Ser-13 of the plasma-derived FIX AP region (Ser-158 of FIX) is phosphorylated and that B1 recognizes the epitope, which includes Ca(2+)-bound phosphoserine-158. B1 should be useful in the quality control of biologically active recombinant FIX containing phosphoserine-158.