R.W. Retallack

SERUM FREE 1,25‐DIHYDROXYVITAMIN D AND THE FREE 1,25‐DIHYDROXYVITAMIN D INDEX DURING A LONGITUDINAL STUDY OF HUMAN PREGNANCY AND LACTATION

The changes in three different indices of 1,25‐dihydroxyvitamin D (1,25(OH)2D) biological activity were studied longitudinally in 35 women during late pregnancy and lactation and in 26 control women. Measurements were made of maternal serum total 1,25(OH)2D and free 1,25(OH)2D concentration (by centrifugal ultrafiltration) and the free 1,25(OH)2D index (the molar ratio of total 1,25(OH)2D and vitamin D binding protein (DBP)). During late pregnancy total 1,25(OH)2D concentrations were significantly elevated when compared to controls, as were free 1,25(OH)2D and DBP concentrations and the free 1,25(OH)2D index. Serum total 1,25(OH)2D, free 1,25(OH)2D and DBP concentrations all fell dramatically during the first 2 weeks of lactation with total 1,25(OH)2D and free 1,25(OH)2D concentrations falling to levels below those of controls. During the course of lactation both total I,25(OH)2D and free 1,25(OH)2D levels rose significantly although they were not different from controls at 18 weeks of lactation. In contrast, the free 1,25(OH)2D index fell during the first 2 weeks of lactation, but remained at this level, significantly lower than controls. Neither urinary calcium excretion nor dietary calcium intake correlated with total or free 1,25(OH)2D, DBP, or the free 1,25(OH)2D index. The disagreement in the results of free 1,25(OH)2D concentration and free 1,25(OH)2D index demonstrates that these two approaches to measuring biologically active 1,25(OH)2D are not equivalent. In attempting to account for the increased calcium requirements of human reproduction we conclude that in pregnancy any of the 1,25(OH)2D measurements may be appropriate. In lactation, however, either 1,25(OH)2D is not a major factor or 1,25(OH)2D biological activity is inadequately represented by any of the currently available methods.

Clinical, biochemical, hematologic, and radiographic responses in Paget's disease following intravenous pamidronate disodium: A 2-year study☆

An intravenous dosage schedule using pamidronate disodium, based on biochemical severity, was used to treat 71 patients with Pagets disease who had no previous bisphosphonate treatment. Disease severity was stratified by fasting hydroxyproline excretion (HypE): Group (Gp) I (mild disease; HypE < 5.0 mumol/LGF) received a total dose of 120 mg; Gp II (moderate; HypE 5.00-9.99) received 180 mg; and Gp III (severe; HypE > or = 10) received 240 mg. Within each group patients were randomly allocated to receive daily 30 mg or 60 mg infusions. Observations for 2 years included pain scores, indices of bone turnover, and radiology of lytic lesions. There was no difference in biochemical responses, or in the percentage of patients with early fever, between the 30 mg and 60 mg daily subgroups; for convenience, 60 mg infusions are recommended. Neutrophils and total white cell counts were both significantly below baseline 4 days after the first infusion; lymphocytes were significantly reduced by day 2; and all three measures had returned to within the reference range by day 6. Remission was assessed at 6 months, when both plasma alkaline phosphatase (ALP) and HypE had reached stable nadirs. Increasing severity was associated with increasing resistance to suppression of HypE at 6 months to within the reference range: Gp I, 87%; Gp II, 44%; and Gp III, 0% (p < 0.0001 by chi-square test). Biochemical relapse at 2 years (defined as ALP 50% above the 6 month level) was also dependent on initial disease severity (Gp I, 6%; GpII, 39%; Gp III, 62%; p < 0.0005 by chi-square test). There was no association between time to relapse and either initial dose or log dose. Radiologic lytic lesions (in 22 patients) were all in remission at 3 months; however, relapse rates at 2 years appeared to be severity-dependent: Gp I, 13%; Gp II, 43%; and Gp III, 57% (n.s. by chi-square test). Remission rates based on a fall to < 50% of pretreatment of either HypE or ALP were more in accord with lytic lesion remission rates than were rates based on HypE falling to within the reference range. Pamidronate produced a significant reduction from baseline in Pagetic bone, Pagetic joint, and unrelated musculoskeletal pain in the first 6 months (p < 0.0001). From 0 months to 2 years the maintenance of improvement in bone pain (p < 0.005) and joint pain (p < 0.05) was significantly better than in unrelated pain. Pamidronate is a safe, welltolerated, and effective treatment for Pagets disease. In spite of larger dosage in severe disease, increasing severity was associated with resistance to normalization of biochemistry and a higher incidence of biochemical and radiological relapse at 2 years. Our current dosage recommendation would be for two 60 mg infusions for mild disease (Gp I); and four 60 mg infusions for moderate disease (Gp II). Severe disease (Gp III) remains a challenge; regardless of dosage, the majority of patients will be in relapse 2 years after a single course of treatment.

A Randomized Trial of Sodium Fluoride (60 mg) ± Estrogen in Postmenopausal Osteoporotic Vertebral Fractures: Increased Vertebral Fractures and Peripheral Bone Loss with Sodium Fluoride; Concurrent Estrogen Prevents Peripheral Loss, But Not Vertebral Fractures

Abstract: Postmenopausal Caucasian women aged less than 80 years (n= 99) with one or more atraumatic vertebral fracture and no hip fractures, were treated by cyclical administration of enteric coated sodium fluoride (NaF) or no NaF for 27 months, with precautions to prevent excessive stimulation of bone turnover. In the first study 65 women, unexposed to estrogen (–E study), age 70.8 ± 0.8 years (mean ± SEM) were all treated with calcium (Ca) 1.0–1.2 g daily and ergocalciferol (D) 0.25 mg per 25 kg once weekly and were randomly assigned to cyclical NaF (6 months on, 3 months off, initial dose 60 mg/day; group F CaD, n= 34) or no NaF (group CaD, n= 31). In the second study 34 patients, age 65.5 ± 1.2 years, on hormone replacement therapy (E) at baseline, had this standardized, and were all treated with Ca and D and similarly randomized (FE CaD, n= 17; E CaD, n= 17) (+E study). The patients were stratified according to E status and subsequently assigned randomly to ± NaF. Seventy-five patients completed the trial. Both groups treated with NaF showed an increase in lumbar spinal density (by DXA) above baseline by 27 months: FE CaD + 16.2% and F CaD +9.3% (both p= 0.0001). In neither group CaD nor E CaD did lumbar spinal density increase. Peripheral bone loss occurred at most sites in the F CaD group at 27 months: tibia/fibula shaft –7.3% (p= 0.005); femoral shaft –7.1% (p= 0.004); distal forearm –4.0% (p = 0.004); total hip –4.1% (p = 0.003); and femoral neck –3.5% (p= 0.006). No significant loss occurred in group FE CaD. Differences between the two NaF groups were greatest at the total hip at 27 months but were not significant [p<0.05; in view of the multiple bone mineral density (BMD) sites, an alpha of 0.01 was employed to denote significance in BMD changes throughout this paper]. Using Cox’s proportional hazards model, in the –E study there were significantly more patients with first fresh vertebral fractures in those treated with NaF than in those not so treated (RR = 24.2, p= 0.008, 95% CI 2.3–255). Patients developing first fresh fractures in the first 9 months were markedly different between groups: –23% of F CaD, 0 of CaD, 29% of FE CaD and 0 of E CaD. The incidence of incomplete (stress) fractures was similar in the two NaF-treated groups. Complete nonvertebral fractures did not occur in the two +E groups; there were no differences between groups F CaD and CaD. Baseline BMD (spine and femoral neck) was related to incident vertebral fractures in the control groups (no NaF), but not in the two NaF groups. Our results and a literature review indicate that fluoride salts, if used, should be at low dosage, with pretreatment and co-treatment with a bone resorption inhibitor.

Effect of alcohol on renal vitamin D metabolism in chickens.

Abstract Intraperitoneal administration of ethanol to young chickens (both vitamin D-replete and vitamin D-deficient) produced a significant impairment of renal 25 hydroxyvitamin D 3 1α-hydroxylase (EC 1.14.13.13) activity with no significant change in serum calcium or phosphorus. In ethanol treated D-replete chicks the renal 25 hydroxyvitamin D 3 24-hydroxylase activity was enhanced, and serum 25 hydroxyvitamin D 3 was significantly increased. The alkaline phosphatase levels in the D-deficient ethanol treated chicks were significantly less than the controls. Our data suggest that the impairment of the metabolic effects of vitamin D due to ethanol occurs chiefly via a renal, rather than a hepatic mechanism. Furthermore, 1α -hydroxylated metabolites of vitamin D would appear to be the logical treatment of choice for the bone disease of alcoholism.

Physiological hyperparathyroidism in human lactation.

The changes in human maternal calcium metabolism due to foetal skeletal demands, are now well documented [5, 9, 16, 19]. Little is known, however, about maternal calcium metabolism, calcium-related hormone levels and their interactions during lactation. We are interested in this subject for the following reasons: Firstly, in rats there is a profound increase in bone turnover during lactation [10, 14, 18]. We have confirmed this observation and have found that the degree of bone turnover is enhanced by increasing the phosphate (P) content of the calcium replete diet of the animals [11]. Furthermore, there is a pronounced loss of bone mineral which may be demonstrated both radiologically and chemically during 20 days of lactation [12]. Once again, an increased dietary P profoundly increases the bone loss. Secondly, in human lactation a progressive loss of bone mineral has been demonstrated using the photon absorption technique [1] and a further study using the same technique [7] found that 20to 59-year-old women, who had lactated, had poorly mineralised bone compared with nulliparous women of similar age. Thirdly, an acute form of osteoporosis occurring during lactation has been described in humans [6]. In this study, our primary aim was to define the basal physiological changes that occur in human lactation by studying healthy volunteers serially. A preliminary account of this work has been published in the New Zealand Journal of Medicine 84, 161 (1976).

Calcium Absorption in Postmenopausal Osteoporosis: Benefit of HRT Plus Calcitriol, but not HRT Alone, in both Malabsorbers and Normal Absorbers

Abstract: In a randomized trial involving 71 postmenopausal osteoporotic women with vertebral compression fractures, radiocalcium absorption studies using the 45Ca single isotope method (α) were performed at baseline and after 8 months of treatment with either continuous combined hormone replacement therapy (HRT, as piperazine estrone sulfate 0.625–0.937mg daily ± medroxyprogesterone acetate 2.5 mg daily depending on uterine status) or HRT plus calcitriol 0.25 μg twice daily. A calcium supplement of 600 mg nocte was given to only those women who had a daily calcium intake of less than 1 g per day at baseline, as assessed by recalled dietary intake. There was a significant decrease [0.74 (± 0.35 SD) to 0.58 (± 0.22), Dα=−0.17 (± 0.26), p<0.0005] in α at 8 months compared with baseline in the HRT-treated group, but a significant increase [0.68 (± 0.31) to 0.84 (± 0.27), Dα=+0.16 (± 0.30), p<0.003] in the HRT-plus-calcitriol treated patients, resulting in α being significantly higher after 8 months in the latter group than in the HRT-only group. Although 72% of the patients had been supplemented with calcium between the first and second studies, separate analyses revealed that the change in calcium intake had not affected the result. Further breakdown of the groups into baseline ‘normal’ absorbers (α≥0.55) and ‘malabsorbers’ (α <0.55) revealed that α decreased with HRT treatment only in the normal absorbers, and remained stable in the malabsorbers. Conversely, following HRT plus calcitriol treatment, α increased only in the malabsorbers, the normal absorbers in this group remaining unchanged. In conclusion, our data show that HRT, of the type and dose used in this study, did not produce an increase in absorption efficiency; it was in fact associated with a fall. Increased absorption efficiency cannot be achieved unless calcitriol is used concurrently, and then only in patients with malabsorption. Calcitriol also had a significant effect in normal absorbers in that it prevented the decline in α seen with HRT alone, and thus should be considered in all patients with postmenopausal osteoporosis treated with HRT.

Calcium, phosphate and citrate in human milk at initiation of lactation

The onset of copious milk secretion (lactogenesis II) in women occurs between 1 and 3 d after birth, and during this period the composition of breast milk changes. During the first 5 d of lactation we measured the concentrations of total, diffusible and ionized Ca (Catot, Cad, Ca2+), diffusible phosphate (Pid), diffusible citrate (Citd) and lactose in the breast milk. On day 1 after birth the concentrations (mean +/- SEM) were Catot, 5.71 +/- 0.30 mM; Cad, 2.66 +/- 0.19 mM; Ca2+, 2.90 +/- 0.18 mM; Pid, 0.26 +/- 0.16 mM; Citd, 0.25 +/- 0.03 mM and lactose, 76 +/- 11 mM. Between day 1 and day 4 the concentration of Catot increased 1.7-fold to 9.56 +/- 0.39 mM, Cad increased 1.8-fold to 4.75 +/- 0.26 mM, Ca2+ decreased by 20% to 2.33 +/- 0.13 mM, Pid increased 6.6-fold to 1.69 +/- 0.11 mM, Citd increased 20-fold to 5.06 +/- 0.21 mM, and lactose increased 2.3-fold to 173 +/- 4 mM. A high correlation has been found between [Cad] and [Citd] in the milk of both ruminant and non-ruminant species, which show a wide range in concentrations of [Cad] and [Citd], and the data fit a simple physicochemical model of ion equilibria in the aqueous phase of milk. The results of the present study confirm the relationship between [Cad] and [Citd] in human milk, even during lactogenesis II when the composition of the milk is changing very rapidly.

ESTIMATION OF 1,25 DIHYDROXYVITAMIN D BY CYTORECEPTOR AND COMPETITIVE PROTEIN BINDING ASSAYS WITHOUT HIGH PRESSURE LIQUID CHROMATOGRAPHY

Using two different cultured rat osteosarcoma cell lines (UMR 106 and ROS 17/2·8) we have investigated the recently described cytoreceptor assay for 1,25‐dihydroxyvitamin D (1,25‐(OH)2D). The assay method is relatively simple and sensitive to 2·4 fmole per tube. Using either cell line, assay of serum samples, whose only preparation consisted of extraction and purification on a disposable diatomaceous earth column, produced variable values for serum 1,25‐(OH)2D. Additional purification, using a disposable silicic acid minicolumn to remove other vitamin D metabolites resulted in consistent values and additional HPLC resulted in no further decrease in the values obtained. Our results show that a single two stage non‐HPLC column can purify serum samples for assay in the cytoreceptor assay. The method is also applicable to the competitive protein binding assay employing calf thymus cytosol and the correlation between values obtained by both methods is highly significant. It is a sensitive, simple, and accurate method with technical advantages which allow greater sample throughput than other 1,25‐(OH)2D assays.

Vitamin D in lactation. I. The localization, specific binding and biological effect of 1,25-dihydroxyvitamin D3 in mammary tissue of lactating rats.

Abstract Radiolabelled 1, 25-dihydroxyvitamin D3 was found to accumulate in mammary tissue of lactating rats, to bind to a specific high affinity binding component in mammary cytosol and to associate with chromatin in vivo. 1,25-dihydroxyvitamin D3 was also shown to have a direct effect on milk calcium concentration in vivo. The cytosolic binding protein was found to sediment at 3.2S on sucrose gradients and to have a dissociation constant of 2.5 × 10−10 M. Localization of 1, 25-dihydroxyvitamin D3 in mammary gland and other tissues of lactating rats was compared. These results provide evidence that the lactating mammary gland is a target tissue for 1,25-dihydroxyvitamin D3.

Prevention of appendicular bone loss in Paget’s disease following treatment with intravenous pamidronate disodium

It has been shown previously that intravenous pamidronate treatment for severe Pagets disease is associated with appendicular bone loss. This 2 year study was designed to determine whether cotreatment with calcitriol and a calcium supplement would prevent this. Intravenous pamidronate was used to treat 49 patients with symptomatic Pagets disease. Patients were stratified into two groups of differing biochemical severity based on hydroxyproline excretion (HypE) expressed as micromoles per liter of glomerular filtrate (GF): (1) a severe group with HypE > 10 micromol/L GF; and (2) a moderate group with HypE 5-10 micromol/L GF. Within each group, patients were randomly allocated to receive supplements of calcium and calcitriol (supplemented) or no supplements (unsupplemented) after initiation of pamidronate therapy. The severe group received 360 mg of pamidronate as six doses of 60 mg once weekly and the moderate group received 240 mg as four weekly doses of 60 mg. Patients were followed for 24 months following treatment and had serial bone densitometry of the forearm measured as well as urine and plasma biochemistry. When the groups were combined, the unsupplemented patients showed a decrease in bone mineral density (BMD) at the ultradistal forearm site, which persisted to 24 months. Those supplemented with calcium and calcitriol showed an increase in BMD and the difference between the two groups was significant at all times posttreatment (p < 0.03). When the groups were analyzed separately, those with moderate disease again showed significant differences in BMD between supplemented and unsupplemented patients at all timepoints. In the severe group, the differences did not reach statistical significance due to smaller patient numbers. Similar changes in BMD were also observed at the forearm shaft site. When serial parathyroid hormone (PTH) levels (with the moderate and severe groups combined) were plotted against time since treatment the rise in PTH in the supplemented patients was less than the rise in the unsupplemented patients (p < 0.04). These results suggest that forearm bone loss after intravenous pamidronate treatment for moderate-to-severe Pagets disease can largely be prevented by administration of calcium and calcitriol. The mechanism may be a blunting of the secondary hyperparathyroidism that occurs after intravenous pamidronate. These findings may have wider application in moderate-to-severe Pagets disease treated with other bisphosphonates.