Serum Inorganic Phosphorus Concentrations Research Articles

Increase in Bone Components of Rats Orally Administered Isoflovone-containing Soybean Extract (Nijiru)

The effect of isoflavone-containing soybean extract (nijiru) administration on bone components in rats was investigated. Rats were orally administered nijiru solution (100mg/ml/100g body weight) twice a day for 7 and 14 d. Nijiru suspension contained saponin (66μg/ml), daidzin (77μg/ml), daidzein (0.6μg/ml), genistin (58μg/ml), and genistein (0.6μg/ml). The solution administered for both periods caused a significant elevation of calcium content, alkaline phosphatase activity, and deoxyribonucleic acid (DNA) content in the diaphyseal and metaphyseal tissues of the femur in rats, while administration for 14 d did not cause a significant alteration in body weight or serum calcium and inorganic phosphorus concentrations, indicating that the solution may not have a toxic effect. This study demonstrates that intake of isoflavone-containing nijiru has an anabolic effect on bone components in rats, suggesting its role in the prevention of osteoporosis.

Alterations in Ca2+-ATPase activity and calcium-binding protein regucalcin mRNA expression in the kidney cortex of rats with saline ingestion.

The alteration in calcium metabolism in rats ingested with saline was investigated. Rats were freely given saline as drinking water for 2 and 7 days. Calcium concentration in the serum was significantly elevated by saline ingestion for 2 and 7 days, while serum inorganic phosphorus concentration was not altered. Serum urea nitrogen concentration was significantly increased by saline ingestion for 7 days. Calcium content in the femoral-diaphyseal and metaphyseal tissues was not altered by saline ingestion for 7 days. Calcium content in the kidney cortex was significantly elevated by saline ingestion for 7 days. Ca2+-ATPase activity in the basolsateral membranes of kidney cortex was clearly increased by saline ingestion for 2 and 7 days. The enzyme activity was not altered by the addition of sodium chloride (10(-3) and 10(-2) M), parathyroid hormone (10(-7) and 10(-6) M), and calcitonin (3 x 10(-8) and 3 x 10(-7) M) in the enzyme reaction mixture. A calcium-binding protein regucalcin mRNA expression in the kidney cortex was markedly suppressed by saline ingestion for 7 days, although such a suppression was not seen for 2 days. These results suggest that saline ingestion causes the disturbance of calcium transport system in the kidney cortex of rats, and that the renal disorder may induce hypercalcemia.

Suppressed expression of calcium-binding protein regucalcin mRNA in the renal cortex of rats with chemically induced kidney damage.

The alteration of Ca(2+)-binding protein regucalcin mRNA expression in the kidney cortex of rats administered cisplatin and cephaloridine, which can induce kidney damage, was investigated. Cisplatin (0.25, 0.5 and 1.0 mg/100 g body weight) or cephaloridine (25, 50 and 100 mg/100 g) was intraperitoneally administered in rats, and 1, 2 and 3 days later they were sacrificed. The alteration in serum findings after the administration of cisplatin (1.0 mg/100 g) or cephaloridine (50 and 100 mg/100 g) demonstrated chemically induced kidney damage; blood urea nitrogen (BUN) concentration increased markedly and serum inorganic phosphorus or calcium concentration decreased significantly. Moreover, the administration of cisplatin (1.0 mg/100 g) or cephaloridine (100 mg/100 g) caused a remarkable increase of calcium content in the kidney cortex of rats, indicating kidney damage. The expression of regucalcin mRNA in the kidney cortex was markedly reduced by the administration of cisplatin or cephaloridine in rats, when the mRNA levels were analyzed by Northern blotting using rat liver regucalcin cDNA (0.9 kb). The mRNA decreases were seen with the used lowest dose of cisplatin or cephaloridine. The present study clearly demonstrates that the mRNA expression of Ca(2+)-binding protein regucalcin in the kidney cortex of rats is decreased by chemically induced kidney damage.

Effects of oral administration of a calcium-containing gel on serum calcium concentration in postparturient dairy cows

Summary: Various nutrious nutritional-supplement gels are being marketed for use in veterinary medicine. This study was designed to determine whether serum calcium, phosphorous, or magnesium concentrations were different between cows given a gel containing calcium chloride as its active ingredient (treated) and cows given inert carrier gel (control). The study revealed a significant (P < 0.01) increase in serum total calcium concentration within 5 minutes of administration of a calcium gel given to cows within 1 hour of parturition. Serum total calcium concentration had returned to baseline value by 24 hours after calcium gel administration. Serum inorganic phosphorus concentration also increased significantly (P < 0.05) after treatment. Significant changes in serum magnesium concentrations were not detected.

Preventive effect of beta-alanyl-L-histidinato zinc on the deterioration of bone metabolism in ovariectomized rats.

The preventive effect of beta-alanyl-L-histidinato zinc (AHZ) on the deterioration of bone metabolism was investigated in the femoral diaphysis of ovariectomized rats. AHZ (10, 30 and 100 mg/kg body weight/d) was orally administered to ovariectomized rats for 6 weeks. Ovariectomy produced a significant decrease in estradiol, calcitonin, calcium and inorganic phosphorus concentrations in the serum as compared with those from sham-operated rats. The dose of 30 and 100 mg AHZ/kg prevented any decrease in serum inorganic phosphorus concentration caused by ovariectomy. Alkaline phosphatase activity, deoxyribonucleic acid (DNA) and calcium contents in the femoral diaphysis of ovariectomized rats significantly decreased in comparison with those from sham-operated rats. These decreases were completely prevented by the dose of AHZ (10, 30 and 100 mg/kg). Electron microscopical analysis showed a rough alteration of bone matrix in the femoral diaphysis of ovariectomized rats. This alteration was clearly modified by the doses of AHZ (10, 30 and 100 mg/kg). Also, dosages of AHZ (30 and 100 mg/kg) restored the atrophy of osteoblasts and cartilage cells caused by ovariectomy. The present study suggests that oral administration of AHZ can prevent the deterioration of bone metabolism by ovariectomy. AHZ may have a therapeutic role in the treatment of osteoporosis.

Calcium and Inorganic Phosphorus Metabolism in Naked Mole Rats Heterocephalus glaber Is Only Indirectly Affected by Cholecalciferol

Naked mole rats seem to be naturally deficient in cholecalciferol yet exhibit net calcium and inorganic phosphorus absorption efficiencies that approach a physiological maximum (i.e., exceed 94%). Oral supplementation with cholecalciferol therefore does not markedly enhance the efficiency of gastrointestinal absorption of these minerals, but rather exerts indirect effects on mineral metabolism by increasing food intake (1.7 ×) from 3.6 ± 0.4 to 6.2 ± 0.3 g/100 g body wt day -1. This, in turn, results in a concomitant increase in the daily rate of calcium absorption from 2.9 ± 0.3 to 4.4 ± 0.4 (mmol/100 g body wt day -1) and inorganic phosphorus absorption from 4.5 ± 0.4 to 7.9 ± 0.4 (mmol/100 g body wt day -1) with cholecalciferol supplementation. Excretion of calcium decreases from 2.9 ± 0.5 to 1.2 ± 0.4 μmol/100 g body wt day -1 with cholecalciferol supplementation whereas inorganic phosphorus excretion is unchanged (4.3 ± 0.9 to 4.3 ± 1.2 μmol/100 g body wt day -1), to give a positive mineral balance, without any evident pathology. Indeed serum calcium (2.3 ± 0.1 vs 2.5 ± 0.1 mmol/liter) and inorganic phosphorus (1.5 ± 0.3 vs 1.6 ± 0.2 mmol/liter) concentrations remain tightly regulated irrespective of vitamin D 3 status. Mineral balance in naked mole rats is concluded not to he directly influenced by cholecalciferol, rather it may be affected by pleiotropic actions of cholecalciferol on gut function.

Beta-Alanyl-L-histidinato zinc prevents hydrocortisone-induced disorder of bone metabolism in rats.

The preventive effect of beta-alanyl-L-histidinato zinc (AHZ) on osteopenia was investigated in rats treated with hydrocortisone. Rats received hydrocortisone (75 mg/kg body weight per day) s.c. for 30 days. The steroid treatment caused a significant increase in serum alkaline phosphatase activity and parathyroid hormone (PTH-c) level, while serum calcium, inorganic phosphorus, and zinc concentrations were not significantly altered. The femoral-diaphyseal alkaline phosphatase activity, deoxyribonucleic acid (DNA), and calcium contents were significantly decreased by the treatment of steroid, although the bone zinc content was not appreciably altered. When AHZ (10, 30, and 100 mg/kg per day) was administered p.o. for 30 days to rats giving the steroid, the dose of AHZ (30 and 100 mg/kg) completely prevented the increases in serum alkaline phosphatase activity and PTH-c level and the decreases in femoral-diaphyseal alkaline phosphatase activity, DNA, and calcium contents caused by the steroid treatment. The dose of AHZ (10, 30, and 100 mg/kg) significantly increased zinc content in the femoral diaphysis. Present results indicate that the dose of AHZ can prevent the disorder of bone metabolism caused by hydrocortisone treatment. AHZ may have a therapeutic role in the steroid-induced osteopenia.

Oral Administration of Calcium Hydroxide Stimulates Bone Metabolism in the Femoral Diaphysis of Rats with Skeletal Unloading.

The preventive effect of calcium hydroxide on the disorder of bone metabolism caused by skeletal unloading was investigated. Skeletal unloading was designed using the model of hindlimb hang in rats. Skeletal unloading for 7 d caused a significant decrease of inorganic phosphorus concentration in the serum and of alkaline phosphatase activity and deoxyribonucleic acid (DNA) content in the femoral diaphysis of rats. Oral administration of calcium hydroxide (16 and 24 mg Ca/kg) caused a significant increase in serum inorganic phosphorus concentration and femoral-diaphyseal calcium content and alkaline phosphatase activity of rats with skeletal unloading. Bone DNA content was significantly increased by the dose of 24 mg Ca/kg. These results clearly indicate that skeletal unloading-induced disorder of bone metabolism is partly prevented by oral administration of calcium hydroxide. Calcium ingestion may be useful as a therapeutic tool in the disorder of bone metabolism caused by skeletal unloading.

Episodic Fluctuation in Serum Intact Parathyroid Hormone Concentration in Men

To evaluate the temporal features of physiological fluctuation in serum PTH concentration, we sampled peripheral blood at 4-min intervals for 24 h from five normal men (32.8 yr; range, 26-40 yr) and measured serum PTH levels using a two-site immunoradiometric assay with the exquisite sensitivity and specificity for human PTH-(1-84) (intact PTH). The resultant 24-h time series of serum intact PTH levels were assessed by contemporary techniques in chronophysiology for rhythmic and episodic peak detection. Cosinor analysis disclosed a significant circadian rhythm in serum intact PTH concentrations in all five men, with the mean circadian amplitude and acrophase of 7.2 +/- 4.4 ng/L and 2305 +/- 401 h, respectively (mean +/- SD; n = 5). No apparent fixed ultradian periodicity was found by autocorrelation and spectral analyses. Evaluation of episodic intact PTH pulsatility by Cluster analysis revealed 23.0 +/- 4.4 discrete PTH pulses/24 h (P less than 0.01 vs. signal-free noise), which occurred at an interpulse interval of 61.6 +/- 11.1 min. The average duration of a serum intact PTH peak was 42.8 +/- 7.3 min, and its mean incremental amplitude was 12.6 +/- 1.3 ng/L, which corresponded to a 31.8 +/- 5.2% increase above the preceding nadir. Discrete PTH peaks were separated by nonpulsatile valleys which lasted for 17.9 +/- 4.4 min. Cross-correlation between the time series of serum intact PTH and whole blood ionized calcium (Ca2+) was at its maximum (-0.5) at concurrent time points in three subjects, while significant positive correlation between serum intact PTH and simultaneous serum inorganic phosphorus concentrations was observed in four of five subjects. There was no apparent correlation between the levels of serum intact PTH and serum magnesium. Our data show that serum levels of intact PTH, the only biologically active form of PTH in the blood, is characterized by a significant circadian periodicity, spontaneous episodic pulsatility with distinct peak properties, and a significant temporal coupling with Ca2+ and inorganic phosphorus concentrations. We conclude that PTH secretion, as judged by the temporal pattern of serum intact PTH levels, is pulsatile in normal men.

A survey of the phosphorus and calcium contents of pastures and the serum inorganic phosphorus and calcium contents of cows on four Manawatu dairy farms

Serum inorganic phosphorus (Pi) and calcium (Ca) concentrations were assessed in 20 cows on each of four Manawatu factory supply dairy farms. Blood was taken from each cow before calving and at six-week intervals during lactation. Bleeding coincided with herd testing. Herds of Friesian or Friesian X cows and Jersey or Jersey X cows were compared on adjacent farms on a Central Yellow-brown Sand and on adjacent farms on a Peat Loam overlying a Central Yellow-brown Earth soil. Pasture mass and composition were estimated to grazing height in the next two paddocks to be grazed in the rotation. Mean serum Pi concentration was higher in cows on sandy soils (1.55 mmol Pi/l than in cows on the peat loam (1.34 mmol Pi/l (P<0.001). Concentrations were highest before calving (1.69 mmol Pi/l) but fell to low levels at peak lactation (1.17mmol Pi/l when 70% of cows were below the minima of the 'normal range', and during the drought (1.29 mmol Pi/l. Pasture phosphorus (P) concentrations were adequate to support cow nutrition for lactation (>0.33% DM, ad lib. feeding) until the summer drought when low herbage mass would have restricted milk production. Serum Ca was adequate for lactating cows and changed little between months or between cows (mean 2.12 mmol Ca/l). No metabolic disorders relating to mineral deficiencies were observed. It appears that serum Pi in a high proportion of cows falls below the normal range during peak lactation without cows displaying clinical deficiency symptoms or a depression in butterfat production.

Simulated weightlessness and bone metabolism: decrease of alkaline phosphatase activity in the femoral diaphysis of rats.

The effect of simulated weightlessness on bone metabolism was investigated in the skeletal unloading for up to 9 days. The skeletal unloading was designed by using the model of hindlimb hang in rats. The food ingestion of rats with the skeletal unloading was not altered in comparison to that of normal rats. Calcium concentration in the serum was not significantly altered by the skeletal unloading for 9 days, while the serum inorganic phosphorus concentration was significantly decreased at 6 and 9 days. Calcium content in the femoral diaphysis was not altered by the skeletal unloading for 9 days. However, the activities of alkaline and acid phosphatases in the femoral diaphysis were markedly decreased by the skeletal unloading. The decrease in bone alkaline phosphatase activity was seen at 2 days with the skeletal unloading. The present results demonstrate that the skeletal unloading with hindlimb hang can induce the disorder of bone metabolism. This model is useful for studying the effects of simulated weightlessness on bone metabolism.

In vivo effects of 1,25-dihydroxyvitamin D 3 and minerals on synthesis of leukotriene B 4 and 5-hydroxyeicosatetraenoic acid by rat peritoneal neutrophils

We investigated the in vivo effects of 1,25-dihydroxyvitamin D 3, and serum calcium and inorganic phosphorus concentrations on productions of leukotriene B 4 and 5-hydroxyeicosatetraenoic acid by glycogen stimulated peritoneal neutrophils of the rat. Neutrophils were incubated with ionophore A23187 for 5 min and the incubation medium was subjected to a reverse-phase high performance liquid chromatography for quantitation. Productions of these lipoxygenase metabolites by neutrophils from vitamin D depleted rats were decreased compared to the vitamin D sufficient levels. This difference was not observed when arachidonic acid was added to the incubation medium. When serum calcium concentrations of rats were altered by dietary calcium levels but 1,25-dihydroxyvitamin D 3 was administered to those rats, the difference in serum calcium concentration did not show any effect on the production of leukotriene B 4. In contrast, neutriphils from 1,25-dihydroxyvitamin D 3 sufficient but phosphorus deficient rats still demonstrated decreased leukotriene B 4 production. This reduction was corrected by exogenous arachidonic acid. The data suggest that 1,25-dihydroxyvitamin D 3 or cellular calcium translocation mediated by the hormone, and serum inorganic phosphorus independent of 1,25-dihydroxyvitamin D 3 affect arachidonic acid release, thus productions of leukotriene B 4 and 5-hydroxyeicosatetraenoic acid.

The role of 1,25-dihydroxyvitamin D in the inhibition of bone formation induced by skeletal unloading.

Skeletal unloading results in osteopenia. To examine the involvement of vitamin D in this process, the rear limbs of growing rats were unloaded, and alterations in bone calcium and bone histology were related to changes in serum calcium (Ca), inorganic phosphorus, 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D [24,25-(OH)2D], and 1,25-dihydroxyvitamin D [1,25-(OH)2D]. Acute skeletal unloading induced a transitory inhibition of Ca accumulation in unloaded bones. This was accompanied by a transitory rise in serum Ca, a 21% decrease in longitudinal bone growth (P less than 0.01), a 32% decrease in bone surface lined with osteoblasts (P less than 0.05), no change in bone surface lined with osteoclasts, and a decrease in circulating 1,25-(OH)2D from 130 +/- 10 to 53 +/- 11 pg/ml. No significant changes in the serum concentrations of inorganic phosphorus, 25-hydroxyvitamin D, or 24,25-(OH)2D were observed. After 2 weeks of unloading, bone Ca stabilized at approximately 70% of control values, and serum Ca and 1,25-(OH)2D returned to control values. Maintenance of a constant serum 1,25-(OH)2D concentration by chronic infusion of 1,25-(OH)2D (Alza osmotic minipump) throughout the study period did not prevent the bone changes induced by acute unloading. These results suggest that acute skeletal unloading in the growing rat produces a transitory inhibition of bone formation, which, in turn, produces a transitory hypercalcemia, leading to a temporary decrease in serum 1,25-(OH)2D. No evidence could be found for a direct involvement of 1,25-(OH)2D in the bone changes induced by skeletal unloading.

A survey of the phosphorus content of pastures and the serum inorganic phosphorus content of dairy cows

Serum inorganic phosphorus (Pi) concentration of 20 cows in each of ten factory supply dairy herds was assessed at monthly or two-monthly intervals during the 198243 lactation. Pasture on offer was ranked low, medium or high and the phosphorus (P) content assessed monthly on all farms from the two paddocks to be grazed next in the rotation. The mean serum Pi concentration was high (1.98 mmol/l) prior to calving but fell to low levels at peak lactation (1.28 mmol/l) and again during the drought (1.28-1.38 mmol/l) in January, February and March. Individual cows had Pi levels as low as 0.32 mmol/l. Herds on Northern Yellow-brown Earths had higher Pi levels than herds predominantly on Brown Granular Loams (P<0.01). There were differences between cows within herds (P<0.01) and Pi levels declined with cow age (P<0.01). Pasture P content was above minimum requirements for lactating cows (0.33% DM; with ad lib. feeding) from July through October but below requirements in most pastures from December through April, when pasture availability also limited production. The P content in pasture was unrelated to either its grass or legume content, but was higher in pastures given a medium or high DM ranking (P<0.05). The possibility of increasing dairy production with P supplementation in spring is discussed.

Bovine post-parturient haemoglobinuria: two distinct entities in New Zealand

Haematological and biochemical features of sixteen cases of post-parturient haemoglobinuria (PPH) from 15 farms in the Waikato and Bay of Plenty regions are described. Characteristically there was a marked regenerative anaemia as well as spherocytosis and haemoglobinaemia. Heinz body formation occurred in 140% of erythrocytes depending on the case. Nine of 11 cases examined had subnormal serum inorganic phosphorus concentrations. On two of eight farms there was widespread anaemia in clinically healthy herd mates and in one of these the anaemia was associated with large numbers of Heinz bodies. Hypophosphataemia was widespread in four of ten herds and individual cows were hypophosphataemic in all ten. Six of eight herds had a low selenium status as determined by glutathione peroxidase activity in whole blood. Hypocupraemia and hypomagnesaemia were not consistent findings. It is concluded that there are two distinct entities of PPH in the region. On one farm the disease was typical of that seen in Northland, New Zealand. The clinical case was a young cow and there was widespread subclinical Heinz body anaemia in herd mates. All but one of the cows sampled in that herd had normal serum inorganic phosphorus levels. On most other farms PPH closely resembled that described from North America. Affected cases were miltiparous, high producing and had low serum inorganic phosphorus levels. The possible pathogenesis of these two entities is discussed.

Serum parathyroid hormone levels in active pulmonary tuberculosis.

Hypercalcaemia in active pulmonary tuberculosis - more often encountered during treatment - is a well known laboratory finding (Abbasi et al. 1979, Need et al. 1980, Kitrou et al. 1982). However the mechanism (s) of this hypercalcaemia has not been elucidated. It has been suggested (Abbasi et al. 1979, Kitrou et al. 1982) that vitamin D overactivity might be involved, as in sarcoidosis (Papapoulos et al. 1979). The finding that serum inorganic phosphorus concentration is increased parallel to calcium (Abbasi et al. 1979, Kitrou et al. 1982) favours this hypothesis. It was found, however, that intestinal calcium absorption decreases significantly during treatment of tuberculosis (Singhellakis et al. 1983). In view of this finding the hypothesis of vitamin D overactivity seems highly improbable, since this condition would be expected to be associated with an increase in intestinal calcium absorption. Although the increase in serum inorganic phosphorus rendered improbable the involvement of parathyroid hormone

Synthetic (Asu1,7) eel calcitonin increases phosphorus content in the hepatic bile of intact and nephrectomized rats.

The effect of synthetic [Asu1,7] eel calcitonin (CT) on phosphorus content in the hepatic bile was investigated in intact and nephrectomized rats. The subcutaneous administration of CT (80 MRC mU/100 g BW) produced a significant fall in the serum inorganic phosphorus concentration and a corresponding increase in the amount of phosphorus in the liver and kidney. These changes were observed even at the lowest dose of CT (40 MRC mU/100 g BW). The phosphorus contents in the spleen, heart, lungs and muscle were not significantly changed by CT administration. CT also provoked a marked elevation of phosphorus excretion into the bile. In nephrectomized rats, CT (80 MRC mU/100 g BW) caused a significant decrease in serum inorganic phosphorus concentration and a remarkable increase in liver phosphorus content. CT markedly increased phosphorus excretion into the bile of nephrectomized rats. These results support the theory that the hypophosphatemic effect of CT may be, at least partly, based on its action on liver in addition to kidney. This effect of CT on the liver may be the cause of increased excretion of phosphorus into the bile.

Low results for inorganic phosphorus with the SMAC continuous-flow analyzer.

Serum inorganic phosphorus concentrations as measured with the SMAC are lower than those found with other methods. To resolve this problem we analyzed patients' specimens and performed analytical recovery studies with four different systems (SMAC, AutoAnalyzer II, aca, and the Fiske--SubbaRow method). With the SMAC, results for patients' specimens are significantly lower (p less than 0.0001) than with any of the other three methods. The SMAC recovered only about 87% of the added inorganic phosphorus. The value assigned to SMAC Reference I for inorganic phosphorus was 0.876 of the value obtained when the material was analyzed by the reference method. Thus there is a significant systematic error in the SMAC method for inorganic phosphorus determination, attributable to an erroneous inorganic phosphorus concentration assigned the SMAC calibration material by the supplier (Technicon).

Selective phosphorus deficiency in the hyperalimented hypophosphatemic dog and phosphorylation potentials in the muscle cell.

Administration of nutrients after underfeeding is associated with a decline of serum inorganic phosphorus concentration (1). When nutrients are administered in excess and especially with inadequate quantities of phosphorus, hypophosphatemia is apt to become pronounced and is invariably associated with excretion of only trace quantities of phosphorus in the urine. The decline of serum phosphorus concentration and its simultaneous disappearance from the urine suggests that phosphorus is being taken up by cells. The site of phosphorus uptake with regard to specific tissues has not been determined. We have examined the effects of hypophosphatemia induced by hyperalimentation of the underfed dog. The results show that phosphorus deficiency appears rapidly in skeletal muscle during hyperalimentation. Significant deficiencies of total phosphorus content were also observed in bone, parathyroid glands and the liver. Phosphorus content did not change significantly in other tissues.

Effect of adding superphosphate to the drinking water on the fertility of dairy cows.

Mean serum inorganic phosphorus concentrations of cows in an infertile dairy herd of 170 cows in the Hunter Valley increased from less than 4.5 mg/100 ml to greater than 5.8 mg/100 ml after defluorinated superphosphate was added to their drinking water at the rate of 2.5 kg/450 1 once weekly from September, 1976. The first service pregnancy rate increased from 36.5% to 63.2%, the mean calving to conception interval decreased from 109 days to 85 days and the number of cows culled each year for infertility fell from 15 to 5. The pasture fed to the milking cows contained on average greater than 17% crude protein, greater than or equal to 0.39% phosphorus, greater than or equal to 9 ppm copper and 38--46 ppm manganese according to the dominant species. The Ca/P ratio ranged from 2 to 5.5:1. Of these, only the Ca/P ratio and the manganese concentrations appeared to be abnormal.