Amelioration of chronic fluoride toxicity by calcium and fluoride-free water in rats.

The study was undertaken to explore the amelioration of chronic fluoride (F) toxicity (with low and normal Ca) in rats. The study was conducted in two phases. In phase I (6 months), seventy-six Wistar, weanling male rats were assigned to four treatment groups: normal-Ca (0·5 %) diet (NCD), Ca+F - ; low-Ca (0·25 %) diet (LCD), Ca - F - ; NCD +100 parts per million (ppm) F water, Ca+F+; LCD +100 ppm F water, Ca - F+. In phase II (reversal experiment, 3 months), LCD was replaced with the NCD. Treatment groups Ca+F+ and Ca - F+ were divided into two subgroups to compare the effect of continuation v. discontinuation along with Ca supplementation on reversal of chronic F toxicity. In phase I, significantly reduced food efficiency ratio (FER), body weight gain (BWG), faecal F excretion, serum Ca and increased bone F deposition were observed in the treatment group Ca - F+. Reduced serum 25-hydroxy-vitamin D3, increased 1,25-dihydroxy-vitamin D3 and up-regulation of Ca-sensing receptor, vitamin D receptor and S100 Ca-binding protein G (S100G) were observed in treatment groups Ca - F - and Ca - F+. In phase II (reversal phase), FER, BWG and serum Ca in treatment groups Ca - F+/Ca+F - and Ca - F+/Ca+F+ were still lower, as compared with other groups. However, other variables were comparable. Down-regulation of S100G was observed in F-fed groups (Ca+F+/Ca+F+ and Ca - F+/Ca+F+) in phase II. It is concluded that low Ca aggravates F toxicity, which can be ameliorated after providing adequate Ca and F-free water. However, chronic F toxicity can interfere with Ca absorption by down-regulating S100G expression irrespective of Ca nutrition.

Parenchymal trafficking of pleural mesothelial cells in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterised by myofibroblast proliferation leading to architectural destruction. Neither the origin nor the continued proliferation of myofibroblasts is well understood. Explanted human IPF lungs were stained by immunohistochemistry for calretinin, a marker of pleural mesothelial cells (PMCs). Chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) lungs acted as controls. The number of PMCs per 100 nucleated cells and per photomicrograph was estimated along with the Ashcroft score of fibrosis. Mouse PMCs expressing green fluorescent protein (GFP) or labelled with nanoparticles were injected into the pleural space of mice given intranasal transforming growth factor (TGF)-ß1. Mouse lungs were lavaged and examined for the presence of GFP, smooth muscle α-actin (α-SMA) and calretinin. Calretinin-positive PMCs were found throughout IPF lungs, but not in COPD or CF lungs. The number of PMCs correlated with the Ashcroft score. In mice, nanoparticle-laden PMCs were recoverable by bronchoalveolar lavage, depending on the TGF-ß1 dose. Fluorescent staining showed α-SMA expression in GFP-expressing PMCs, with co-localisation of GFP and α-SMA. PMCs can traffic through the lung and show myofibroblast phenotypic markers. PMCs are present in IPF lungs, and their number correlates with IPF severity. Since IPF presumably begins subpleurally, PMCs could play a pathogenetic role via mesothelial-mesenchymal transition.

Development of an enzyme-linked immunosorbent assay for the detection of human calretinin in plasma and serum of mesothelioma patients.

BACKGROUND: Calretinin is one of the well-established immunohistochemical markers in the diagnostics of malignant mesothelioma (MM). Its utility as a diagnostic tool in human blood, however, is scarcely investigated. The aim of this study was to develop an enzyme-linked immunosorbent assay (ELISA) for human calretinin in blood and to assess its usefulness as a potential minimally invasive diagnostic marker for MM. METHODS: Initially, attempts were made to establish an assay using commercially available antibodies and to optimize it by including a biotin-streptavidin complex into the assay protocol. Subsequently, a novel ELISA based on polyclonal antibodies raised in rabbit immunized with human recombinant calretinin was developed. The assay performance in human serum and plasma (EDTA/heparin) and the influence of calcium concentrations on antibody recognition were studied. Stability of spiked-in calretinin in EDTA plasma under different storage conditions was also examined. In preliminary studies serum and plasma samples from 97 healthy volunteers, 35 asbestos-exposed workers, and 42 MM patients were analyzed. RESULTS: The mean detection range of the new ELISA was 0.12 to 8.97 ng/ml calretinin. The assay demonstrated markedly lower background and significantly higher sensitivity compared to the initially contrived assay that used commercial antibodies. Recovery rate experiments confirmed dependence of calretinin antibody recognition on calcium concentration. Calcium adjustment is necessary for calretinin measurement in EDTA plasma. Spiked-in calretinin revealed high stability in EDTA plasma when stored at room temperature, 4 degrees C, or after repeated freeze/thaw cycles. Median calretinin values in healthy volunteers, asbestos workers, and MM patients were 0.20, 0.33, and 0.84 ng/ml, respectively (p < 0.0001 for healthy vs. MM, p = 0.0036 for healthy vs. asbestos-exposed, p < 0.0001 for asbestos-exposed vs. MM). Median values in patients with epithelioid and biphasic MM were similar. No influence of age, gender, smoking status, or type of medium (plasma/serum) on calretinin values was found. CONCLUSIONS: The novel assay is highly sensitive and applicable to human serum and plasma. Calretinin appears to be a promising marker for the blood-based detection of MM and might complement other markers. However, further studies are required to prove its usefulness in the diagnosis of MM patients.

Aluminum effects upon calbindin D9k-linked duodenal calcium transport in diabetic male rats.

In order to elucidate if the inhibition mechanisms of Aluminum (Al) on intestinal calcium flux involve some possible action on calbindin-D9k, a series of in vivo and in vitro experiments were carried out in normal and in streptozotocin-induced diabetic male rats. The dose-response curves obtained from the in vitro studies indicate that, in the diabetic group (which has a lower content of calbindin-D9k), the effect of Al on JCa(ms) has a small dependence on rising Al concentration (0-10 microM). The parameters obtained from those curves: Emax (maximum reduction percentage of JCa(ms)) and ED50 (Al concentration that produces half of the highest inhibition) were significantly diminished in this group compared to control. Both s.c. injections of calcitriol (D3) at doses of 0.08 and 0.40 microg/kg body wt. per day and insulin (10 IU/kg body wt. per day), increase the inhibitory effect of Al to levels that did not differ from controls. In vivo gavage of 60 mg/kg body wt. per day of aluminum chloride for 1 week reveals that the degree of reduction of intestinal CaBP9k by Al is directly correlated to duodenal content of this protein (r2 = 0.683, P = 0.022).

The calcium-binding protein calretinin-22k is detectable in the serum and specific cells of cancer patients.

BACKGROUND: Calretinin-22k (CR-22k), an alternatively spliced form of calretinin (CR) belongs to the EF-hand family of calcium-binding proteins and is expressed in several colon adenocarcinoma cell lines (e.g. WiDr, HT-29). MATERIALS AND METHODS: Serum samples of cancer patients were screened with a sandwich ELISA technique using the CR-specific antiserum 7696. Highly positive samples were analyzed by Western blots and immunohistochemistry. RESULTS: CR-22k was detected in the serum of several patients and values were as high as 0.19 microgram/ml. Western blot analysis confirmed the identity of the bound protein as CR-22k. The highest concentrations were detected in patients with colon or breast cancer, but also in a patient with ischemic necrosis of the gut. CR immunoreactivity was localized to epithelial cells, nerve fibres, cells of the connective tissue and to mesothelial cells. CONCLUSIONS: Our results establish that CR-22k is detectable in the serum of cancer patients under specific pathological conditions.

Significant elevation of urinary 28-kD calbindin-D and N-acetyl-beta-D-glucosaminidase levels in patients undergoing extracorporeal shock wave lithotripsy.

Calbindin-D, a vitamin D-dependent calcium binding protein with a molecular mass of 28 kD, is found predominantly in distal renal tubules and central nervous system tissues in man. We have developed a highly sensitive enzyme immunoassay for human 28-kD calbindin-D and demonstrated its advantages as a new marker for damage to distal renal tubules. Urinary N-acetyl-beta-D-glucosaminidase (NAG), a lysosomal enzyme of the proximal renal tubules, is another segment-specific indicator of renal damage. To clarify whether both proximal and distal renal tubules are similarly affected by extracorporeal shock wave lithotripsy (SWL) treatment, urinary 28-kD calbindin-D and NAG were measured before, then immediately, 2 and 24 hours after SWL in 17 renal lithiasis patients. Levels of urinary calbindin-D were markedly elevated immediately and 2 hours after SWL and then decreased. In sera, levels of calbindin-D also increased, closely correlated with the changes in urinary values. Levels of urinary NAG were also significantly elevated immediately after SWL and then decreased. The results indicate that damage to both proximal and distal renal tubules occurs simultaneously with SWL and that the two markers can be applied as sensitive indicators of such side effects and their alleviation with protective agents.

Urinary 28-kD calbindin-D as a new marker for damage to distal renal tubules caused by cisplatin-based chemotherapy.

Calbindin-D, a vitamin D-dependent calcium-binding protein of 28 kD, is found predominantly in the distal tubules of the kidney and central nervous system tissues in humans. To evaluate damage to the renal tubules caused by cisplatin-based chemotherapy, levels of urinary and serum calbindin-D were determined in patients treated with cisplatin- or carboplatin-based chemotherapies using a highly sensitive enzyme immunoassay system developed in our laboratory. Levels of urinary 28-kD calbindin-D were also determined in patients with benign and malignant urological diseases. The mean urinary calbindin-D level was 2.44 + or - 0.31 (mean + or - SE) ng/mg creatinine in 40 healthy subjects. Urinary calbindin-D levels were elevated (>10 ng/mg creatinine) in 2 of 33 patients (6%) with benign and 1 of 50 (2%) with malignant urological diseases. Urinary calbindin-D levels were significantly increased after cisplatin-based chemotherapy in 14 patients, with peaks (71.8 + or - 13.5 ng/mg creatinine) being found 8 days after administration of cisplatin, and then a gradual return to the baseline. On the other hand, 7 patients receiving carboplatin-based chemotherapy demonstrated no significant elevation (highest level 7.7 + or - 2.5 ng/mg creatinine). In 7 patients treated with cisplatin-based chemotherapy the serum calbindin-D level was also raised after treatment, with a good correlation to urinary values. These findings suggest that urinary and serum calbindin-D may be kidney-derived and that 28-kDa calbindin-D is a useful marker for damage to the distal renal tubules associated with cisplatin-based chemotherapy.

Dietary calcium levels and treatment interval determine the effects of lead ingestion on plasma 1,25-dihydroxyvitamin D concentration in chicks.

The combined effects of dietary calcium level and lead level on several indices of vitamin D endocrine function were examined in young, growing chicks. Day-old animals fed a nutritionally adequate diet for 2 wk were fed diets either adequate (1.2%) or low (0.1%) in calcium, and containing 0, 0.2 or 0.8% lead for an additional 1 or 2 wk. In the calcium-adequate group, lead ingestion significantly elevated intestinal calbindin-D28k protein and mRNA levels as well as plasma 1,25-dihydroxyvitamin D concentration compared with the control animals fed a lead-free diet. The effect was apparent after 1 wk of treatment and continued through wk 2. In the calcium-deficient group, the early (1 wk) increases in plasma 1,25-dihydroxyvitamin D and calbindin-D28k protein and mRNA were significantly reversed by lead ingestion over the 2-wk trial period in a dose-dependent fashion. In these circumstances, vitamin D endocrine function is severely compromised. Therefore, lead ingestion may result in either enhanced or diminished circulating 1,25-dihydroxyvitamin D concentrations and ensuing intestinal responses, depending of dietary calcium level and the duration of lead intake. These results provide possible explanations for several apparently conflicting sets of observations regarding lead-calcium interactions.

Neural tissue-related proteins (NSE, G0 alpha, 28-kDa calbindin-D, S100b and CK-BB) in serum and cerebrospinal fluid after cardiac arrest.

To estimate brain damage after cardiac arrest, the concentrations of neuron specific enolase (NSE), GTP-binding protein (G0 alpha), 28 kDa calbindin-D, S100b protein, and creatine kinase BB (CK-BB) in serum and cerebrospinal fluid (CSF) were determined by enzyme immunoassays. Ten mongrel dogs were subjected to 30 min of circulatory arrest at normal body temperature and serial CSF and blood samples were taken during the first 18 h after reperfusion. The NSE concentration in CSF increased significantly after reperfusion, reaching a 15-fold increase (243 +/- 107 ng/ml, p < 0.01) 18 h later, however, it did not increased significantly in serum (8.1 +/- 3.3 ng/ml vs. 23.5 +/- 7.0 ng/ml). G0 alpha concentration in CSF increased sharply between the 2nd and 4th h after reperfusion and peaked 18 h after reperfusion (428 +/- 195 pg/ml, p < 0.01), however, it did not increase significantly in serum. Calbindin-D concentration in CSF increased between the 1st and 6th h after reperfusion, and reached a plateau thereafter (621 +/- 235 ng/ml, a 23-fold increase, p < 0.05) and also increased significantly in serum (p < 0.05). The S100b concentration in CSF also increased dramatically after the 4th h of reperfusion and reached a plateau at the 8th h after reperfusion (16.0 +/- 9.3 ng/ml, a 50-fold increase, p < 0.01), however, it in serum was below the detection threshold. The CK-BB concentration in CSF peaked 4 h after reperfusion (113 +/- 69 ng/ml, a 19-fold increase, p < 0.01) and it in serum increased 4-fold (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Altered levels of erythrocyte calcium-binding proteins in essential hypertensives with genetic predisposition: correlation with ambulatory blood pressure.

OBJECTIVE: To examine whether changes in calcium-binding proteins, one of the components of the calcium ion handling mechanism, occur in humans with essential hypertension. DESIGN: We measured the levels of cytosolic calcium-binding proteins purified from human erythrocytes using a felodipine fluorescence assay, and examined the correlation between this parameter and the ambulatory blood pressure (ABP). We divided 127 subjects into four age-matched groups according to their mean ABP levels and whether they had a family history of both hypertension and stroke [group A hypertensives with a positive family history (n = 30), group B hypertensives with no family history (n = 31), group C normotensives with a family history (n = 31) and group D normotensives with no family history (n = 35) of hypertension and stroke]. RESULTS: The erythrocyte cytosolic level of calcium-binding proteins in group A was significantly lower than that in group B, as was that in group C compared with group D. There was no significant correlation between the erythrocyte level of calcium-binding proteins and casual blood pressure values in any group. However, in group A significant negative correlations between the erythrocyte level of calcium-binding proteins and systolic and mean ABP were observed (r = -0.34, P < 0.05 and r = -0.39, P < 0.05, respectively). No significant correlations between the ABP and erythrocyte levels of calcium-binding proteins were observed in the other groups. When each group was subdivided according to sex, there were significant negative correlations between the erythrocyte level of calcium-binding proteins and the systolic and mean ABP in the males of groups A and C, but no correlations were found in any of the female subgroups or the males of groups B and D. Reducing the blood pressure by antihypertensive drug therapy did not affect the erythrocyte calcium-binding proteins level in 13 patients from groups A and B. Analysis using anion-exchange fast-performance liquid chromatography on a Mono-Q column and sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that the calcium-binding proteins in human erythrocytes, the levels of which were low in group A, formed a single protein band with a molecular weight of 17,000, which was assumed to be a calmodulin. CONCLUSIONS: These results suggest that there are subgroups of hypertensive patients with low erythrocyte cytosolic levels of calcium-binding proteins, which are genetically determined. Furthermore, our data suggest that the erythrocyte level of calcium-binding proteins and ABP in male subjects with hypertension and normotensives with a genetic predisposition are correlated strongly, whereas no such correlation was observed in any female subgroup. This indicates that the regulatory mechanism or mechanisms involved in the control of blood pressure in men and women may be different.

Intestinal calcium and lead absorption: effects of dietary lead and calcium.

The combined effects of dietary calcium (Ca) and lead (Pb) status on intestinal Ca and Pb absorption and related parameters were investigated in young growing chicks. Dietary Pb intake resulted in two remarkable, apparently independent and essentially opposite effects on intestinal Ca and Pb absorption, depending on dietary Ca and Pb levels and duration of treatment. The initial response (1 week) to Ca deficiency was stimulated Ca absorption and calbindin-D level, regardless of dietary Pb intake. The later response (2 weeks) was a reversal, by Pb, of the early phase stimulation. Intestinal Pb absorption was similarly enhanced by Ca deficiency initially, and this response was also inhibited by prolonged dietary Pb intake. Ingestion of Pb by chicks fed adequate Ca resulted in generally elevated intestinal Ca absorption and calbindin-D levels after both 1 and 2 weeks. Intestinal Pb absorption was also increased in the adequate Ca situation, but only after 2 weeks at the lower levels of dietary Pb. The results underscore the complicated nature of Pb-Ca interactions and demonstrate the importance of thorough characterization of the animal model system.

Is vitamin D necessary for skeletal integrity in the elderly?

Serum 1.25 dihydroxyvitamin D concentrations were reduced in elderly patients with femoral neck fractures, irrespective of the presence of osteomalacia. This reduction was not attributable to a decrease in vitamin D binding protein. The low rate of bone turnover in these elderly patients might reduce the requirement for vitamin D and protect against the development of osteomalacia. Serum vitamin D metabolite concentration cannot be used as a screening test for osteomalacia in these patients.

Calcium-dependent translocation of calbindin-D28k from intestine to blood.

Calbindin-D (vitamin D-induced calcium-binding protein; CaBP) is known to be present in blood at concentrations which vary directly with levels in the intestinal mucosa. Employing a sensitive radioimmunoassay and sampling mesentery venous blood, the present experiments demonstrated a direct relationship between intestinal calcium absorption and serum CaBP. Solutions containing 150 mM NaCl and 45Ca-labeled calcium chloride (5 or 20 mM) were placed in the lumen of ligated duodenal preparations in situ and mesentery venous blood sampled with time. The concentration of absorbed 45Ca in serum was maximal at 5 min, followed by a significant increase in mesentery CaBP maximizing at 15-20 min. Elevation of serum CaBP was not observed when calcium in the dosing solution was omitted or replaced by either glucose or glycine. The possible transfer of absorbed calcium from the enterocyte to the circulation as a CaBP complex was ruled out by calculations revealing that considerably more calcium was transferred than could be accounted for by the low and high affinity binding sites on the protein. It is proposed that vitamin D-dependent enhanced transcellular calcium transport constitutes a stimulus for the increased release of intestinal CaBP into the circulation.

Effect of 1,25-dihydroxyvitamin D3 on plasma concentrations of calcium-binding protein in normal and rachitic (vitamin D-dependent rickets type I) pigs.

An i.v. injection of calcitriol (1,25-(OH)2D3) had no effect within 2.5 h on plasma concentrations of calbindin-D9K (vitamin D-induced calcium-binding protein; CaBP) in hypocalcaemic pigs with inherited vitamin D-dependent rickets type I or in their normocalcaemic siblings or half-siblings. Three days later the plasma concentration of CaBP had doubled in the hypocalcaemic pigs, but was unaltered in the normocalcaemic siblings and half-siblings. Following daily i.v. injections of 1,25-(OH)2D3 for a further 5 days (days 4-8) plasma concentrations of CaBP increased in both the hypocalcaemic (days 4-8) and normocalcaemic (day 8) pigs, the effect being more rapid and greater in the hypocalcaemic 1,25-(OH)2D3-deficient animals. An i.v. injection of 1,25-(OH)2D3 to pure Yucatan pigs also had no effect on plasma concentrations of CaBP within 1.5 h, but in the following 1 h there was some indication of an increase in plasma CaBP levels. In contrast to the normal pigs, insulin-induced hypoglycaemia did not lead to a peak in plasma CaBP concentrations in the hypocalcaemic pigs. There was also no change in the plasma concentrations of 1,25-(OH)2D3 associated with the peak in plasma CaBP following insulin-induced hypoglycaemia in normocalcaemic pigs. These results suggest that changes in plasma concentrations of 1,25-(OH)2D3 are not directly involved in mediating the increase in plasma CaBP which follows hypoglycaemia induced by insulin in normal pigs, although 1,25-(OH)2D3 probably plays a permissive role.

Endocrine control of plasma concentrations of calcium-binding protein in the pig.

The aetiology of the rise in plasma calbindin-D9K (vitamin D-induced calcium-binding protein; CaBP), following insulin-induced hypoglycaemia, was studied in the pig. ACTH led to a rise in plasma concentrations of both CaBP and cortisol. Metyrapone, which blocks cortisol synthesis, abolished the increases in plasma concentrations of CaBP and cortisol normally observed in response to insulin-induced hypoglycaemia. However, there was no significant rise in plasma concentrations of CaBP in response to pharmacological or physiological doses of cortisol. Injection of clonidine, an alpha 2-adrenergic agonist, led to a rise in plasma concentrations of CaBP, whereas phenylephrine, an alpha 1-adrenergic agonist, tended to exert an inhibitory effect. Also, administration of phentolamine (an alpha-adrenergic blocker) before injection of insulin abolished the usual increase in plasma concentrations of CaBP, whereas propranolol (a beta-adrenergic blocker) enhanced the normal increase in plasma concentrations of CaBP in response to insulin-induced hypoglycaemia. Isoproterenol, a beta-adrenergic agonist, was without effect on plasma CaBP. Neither GH nor glucagon appear to be involved in the rise in plasma CaBP following insulin-induced hypoglycaemia. Although atropine abolished the effect of acute hypoglycaemia on plasma CaBP, carbamylcholine was without effect on plasma CaBP concentration. It is concluded that the increases in plasma CaBP induced by either ACTH or alpha 2-adrenergic stimulation may be interrelated since the administration of ACTH can lead to raised plasma concentrations of catecholamines.

Raised levels of calcium-binding protein in plasma following insulin-induced hypoglycaemia in the pig.

Insulin-induced hypoglycaemia in the pig elicited sharp increases in the plasma concentrations of vitamin D-dependent calcium-binding protein (CaBP) and cortisol and a decrease in plasma inorganic phosphate. Glucose infusion following insulin administration abolished the increases in plasma CaBP and cortisol in response to insulin and reduced the hypophosphataemia. The percentage increases in plasma CaBP and cortisol in response to insulin-induced hypoglycaemia were reduced when the pigs were fed a low-calcium diet, but the hypophosphataemic response was similar. We conclude that insulin-induced hypoglycaemia leads to increased plasma CaBP in pigs fed a normal calcium diet, which is associated with the hypoglycaemia rather than being a direct effect of insulin. We therefore suggest that plasma CaBP may represent more than a mere uncontrolled leak from its sites of storage.

The effects of betamethasone on plasma and intestinal calcium binding protein and on 25-hydroxyvitamin D3 metabolism in the pig.

Betamethasone (50 micrograms/kg body weight/day) given to young pigs reduced calcium absorption, growth and plasma vitamin D dependent calcium binding protein (CaBP) concentration. No changes occurred in plasma 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and intestinal CaBP concentrations. 1,25(OH)2D3 (0.1 microgram/kg body weight/day) given with betamethasone increased calcium absorption although growth and plasma CaBP concentrations remained low. Intestinal CaBP levels remained unchanged. Plasma CaBP concentrations were not consistently related to intestinal CaBP or calcium absorption in the presence of betamethasone. We conclude that betamethasone-induced depression of calcium absorption was not mediated by alterations in intestinal CaBP, but the mechanism remains obscure.

Studies of calcium-binding protein in cattle.

We have developed and validated a double-antibody radioimmunoassay for quantifying bovine calcium-binding protein (CaBP). Cross-reactivity between the antiserum and microgram quantities of thyrocalcitonin, calmodulin, gastrin, cholecystokinin, vasoactive intestinal polypeptide, serum albumin and concentrated extract of bovine pituitary gland was insignificant. Slight cross-reactivity (6%) of the antiserum with parathyroid hormone was demonstrated. Assay sensitivity was .25 ng/ml and intraassay and interassay coefficients of variation ranged from 4 to 11% and 10 to 24%, respectively. The CaBP immunoreactivity was not affected by endogenous Ca concentrations. Plasma and serum concentrations of immunoreactive CaBP were similar. The CaBP concentrations were unaffected when coagulated and anticoagulated blood samples were stored at 4 or 22 C for up to 72 h and when serum was stored at -20, 4 or 22 C for 8 d. Serum CaBP concentrations in cattle were not affected by gonadal steroids, but may have been influenced by age. Treatment with 500 mg of vitamin D3, but not 50 mg of dihydrotachysterol, significantly increased serum Ca and CaBP concentrations in Holstein heifers after a lag period of 7 to 10 d. Serum Ca and CaBP concentrations began to increase in serum at approximately the same time and both exhibited parallel responses to treatment with vitamin D3. Serum Ca concentrations were positively correlated (r = .81) with CaBP concentrations and this relationship was described by the equation, Y = 6.85 + 1.01X - .03X2. Serum Ca and CaBP concentrations were still elevated in heifers 75 d after initial treatment with vitamin D3. The radioimmunoassay we describe provides an opportunity to investigate the role of CaBP in Ca homeostasis during growth, pregnancy, lactation, parturient paresis and other physiological and pathological states in cattle.