Serum vitamin D and calcium in tuberculous patients

415

Abstracts from the Calcified Tissues Workshop Pediatr. Nephrol. Assaf Harofe Med. Ctr., Metabolic Beilinson Med. Ctr., Israel

Unit

A new syndrome, H.H.R.H. was recently described in 8 members of one kindred. Studies of 49 additional asymptomatic members of this kindred revealed: 29 normal subjects (N.), while 20 others had hypercalciuria (I.H.) with no evidence of bone disease. The following indices distinguished the 3 different groups: 24h urinary calcium creatinine ratio (C&r.) 0.43 * 0.14 (mean 2 SD) in H.H.R.H., 0.34 ? 0.07 in I.H., and 0.14 + 0.05 in N. Serum phosphorus (Pi) and TmP/GFR as determined by age related means and expressed in SD units were -4.31 + 2.38 and -3.0 ? 1.24 in H.H.R.H., -1.11 f 0.98 and -1.13 ? 0.57 for I.H., +O.Ol ? 0.98 and +0.21 + 0.94 for N. Serum levels of 1.25(OH)*D were 303 + 208 pg/ml, 145 ? 99 and 84 2 36, in H.H.R.H., I.H. and N., respectively. Urinary CAMP excretion in H.H.R.H., I.H. and N. was 1.66 2 0.73, 2.69 -t- 1.05 and 3.28 ? 0.96 nmol/lOO ml.GF. respectively. A significant negative linear correlation was found between TmP/GFR, serum 1.25(OH)2D levels and urinary C&r in all subjects. We propose that the pivotal genetic defect in this kindred is a renal Pi leak resulting in hypophosphatemia and an appropriate elevation of 1.25(OH),D levels, which causes increased Ca absorption parathyroid suppression and hypercalciuria. We conclude 1. This kindred represents a new hereditary syndrome in which the affected site in the kidney is different from the known hypophosphatemic syndromes. 2. Pi is an important mediator in controlling 1.25(OH)2D in human. 3. The pathophysiological sequence operating in our I.H. subjects and H.H.R.H. patients is identical. 4. Quantitatively the abnormalities in I.H. are milder and the biochemical parameters are in between the values of H.H.R.H. and normals. 5. For the first time, the concept of idiopathic hypercalciuria as an expression of a specific inherited renal defect, is clearly illustrated.

INHIBITORY EFFECTS OF BISPHOSPHONATES DIURNAL VARIATIONS OF BLOOD 45CA AND 3H-TETRACYCLINE IN YOUNG DOGS

ON

Kam M. Wong, LeRoy Klein Dept. of Orthopaedics Case Western Reserve University, School of Medicine, Cleveland, Ohio, USA We have shown that diurnal variations of blood 45Ca, 3Htetracycline, and vitamin D metabolites occur in normal dogs, and these diurnal variations are completely eliminated in calcium-deficient or thyroparathyroidectomized dogs (Amer. J. Physiol. 246:R688-692, 1984). To further elucidate the mechanisms for this diurnal rhythm, the effects of the bisphosphonates, EHDP and CI,MDP, on diurnal variations in young growing dogs were studied. Labrador retriever dogs were extensively prelabelled with 45Ca and 3H-tetracycline. Groups of 3 dogs were injected daily with either EHDP (2.5 mgP/kg body wt/day) or CI,MDP (5.0 mgP/kg body wt/day) while 4 dogs served as controls. Sequential blood sampling at 8 am, 12 and 4 pm were performed daily. In normal intact dogs, blood 45Ca and 3H-tetracycline decreased continuously during the day to minima of 65 ? 3% and 67 ? 3% of baseline, respectively, at 4-8 pm, while blood calcium remained constant throughout the day. In the bisphosphonate-treated dogs, blood calcium remained in the normal range throughout the entire experimental period. The diurnal

variations of blood radioactivities, however, gradually decreased in amplitude and were completely eliminated in both EHDP and CI,MDP treated dogs after 3 and 4 weeks of treatment. The maximum daily change in blood radioactivity was 91 ? 3.5% of baseline. After treatment was terminated, the diurnal variation remained inhibited for up to two weeks and gradually returned to a normal rhythm. Data from this study show that the diurnal variations of blood 45Ca and 3H-tetracycline in extensively prelabelled dogs can be inhibited by EHDP and C12MDP, known inhibitors of bone resorption. These data further support the hypothesis that bone resorption changes reciprocally in response to daily fluctuations in dietary calcium intake, resulting in the rhythmic changes in blood radioactivities.

PLASMA 1aOHD3, 1,25(OH),D AND CALCIUM IN CALVES AND IN DAMS TREATED PREPARTUM WITH 1aOHD, R. Perlman, M. Sachs, A. Bar ARO, The Volcani Center, Bet-Dagan,

Israel

1ahydroxyvitamin D, (1CXOHD,) has been used to prevent bovine Parturient Paresis in cows, which is the result of a severe hypocalcemia. Following an IM injection of laOHD,, the D-derivative appeared in plasma after 12 h, reaching a peak 24 h after the injection. The disappearance rate of laOHD, from the blood was 0.330 d-l (biological half-life of 2.1 d). Plasma 1 ,25(OH)2D increased as early as 6 h and peaked between 24 to 48 h after the laOHD, injection. Plasma calcium increased after 6 h and remained high for at least 8 d. At parturition, plasma laOHD3 in calves was higher than that of their IaOHD,treated mothers. Plasma Ca was always higher and 1,25(OH),D always lower in the plasma of calves than in their maternal plasma. Plasma 1,25(OH),D was higher in cows treated with IaOHD, and in their offspring than in their respective controls. The results provide additional evidence that in cattle, neonate plasma 1,25(OH),D is not correlated with the maternal plasma concentration. The high plasma 1,25(OH)*D concentration observed in calves of laOHD, treated cows could result from its 25-hydroxylation in the calf liver rather than from its placental transfer. Elevated leves of vitamin D metabolites in the plasma of calves of laOHD,-treated mothers, did not result in any change in plasma Ca concentration.

SERUM VITAMIN D AND CALCIUM TUBERCULOUS PATIENTS

IN

P.D.O. Davies,’ R.C. Brown2 H.A. Church,2 J.S. Woodhead,‘J. M. Grange3 ‘Dept. of Medicine, Llandough Hospital, Cardiff, 2Dept. of Biochemistry, University Hosp. of Wales, 3Cardiothoracic Institute, Brompton Hospital, Wales It has been widely reported that, in common with other granulomatous conditions, tuberculosis is implicated in hypercalcaemia. It is believed that granuloma macrophages convert 25-(0H)D to l-25(OH),D, resulting in hypercalcaemia. We have studied two groups of tuberculous patients together with matched healthy controls: a UK and an African group. In 50 patients and healthy controls resident in the UK (Cardiff, South Wales): median 25(OH)D (6.4 ng/ml) was significantly lower than controls (10.9

416

ng/ml) (p < 0.005). l-25(OH)>D showed no significant dtfference (median 35.7 pg/ml and 32.3 pg/ml respectively). Parathyroid hormone levels were also similar. Uncorrected serum calcium was significantly lower in the patient group (2.23 mmol/l compared with 2.37 mmolil) (p < 0.001) but when corrected for albumin levels became virtually identical. In 15 patients from equatorial Africa (Nairobi, Kenya): serum median 25-(0H)D was significantly lower in patients (15.9 and 26.2 ng/ml) (p < 0.05). Median serum calcium however when corrected for albumin was significantly higher in patients than controls (2.57 and 2.47 mmol/l respectively) (p < 0.001). Both African and British tuberculous patients therefore showed significant differences to controls with respect to serum 25-(0H)D. 1-25(OH)2D and parathyroid hormone levels were similar to controls for both groups of patients. Corrected calcium however was significantly higher in the African patients. It is possible that higher serum 25-(0H)D present in tuberculous patients dwelling in equatorial Africa where sunlight is plentiful may give rise toa higher turnover of I-25(OH),D resulting in hypercalcaemia. INTERACTIONS BETWEEN PHOSPHATURIC PEPTIDE HORMONES AND VITAMIN D METABOLITES: A MODEL FOR A BALANCED HORMONAL REGULATION Mordecai M. Popovtzer, Hanna Wald, Michael M. Friedlaender, Dvora Rubinger Nephrology and Hypertension Services, Hadassah University Hospital, Jerusalem, Israel In vitamin D-replete rats, phosphaturic responses to peptide hormones; PTH, calcitonin and glucagon are modified by derivatives of vitamin D. In contrast, phosphaturia induced by non-hormonal factors is not altered by derivatives of vitamin D. Inhibition of hormone-induced stimulation of adenylate cyclase is the common mechanism for the specific action of vitamin D derivatives, not only in the kidney but also in other cellular systems, as demonstrated both in vivo and in vitro systems by us and by others. Additional features of this biologic interaction are: 1. Vitamin D derivatives inhibit the hormone-induced formation of cyclic AMP, however, they do not interfere with the physiological response to cyclic AMP. 2. Cycloheximide but not colchicine suppresses this effect, indicating dependence of this interaction on post-transcriptional protein synthesis but not on the integrity of cytoplasmic mrcrotubules. 3. The nature of the mediating protein IS unknown but in vitro studies suggest that calmodulin may be the key factor. 4. There is a distinct specificity of the molecular species of vitamin D regarding the biological activity. Thus vitamin D, and Ia(O lack activity while 25(OH)D,, 1 ,25(OH)*D3 and 24,25(OH)2D3 are active, suggesting the importance of 25-hydroxyl group for biological expression. These experiments cast new light on the role of metabolites of vitamin D in modifying the responses to peptide hormones through interaction at the level of adenylate cyclase activation, both in renal and other tissues. THE POTENTIAL OF DIFFERENT CELL TYPES TO PRODUCE CALCIFIED TISSUES IN DIFFUSION CHAMBERS IN VIVO I. Bab Division of Oral Pathology, Hebrew Un/vers/ty-Hadassah School of Dental Medicine, Jerusalem, lsrael

Abstracts

from the Calcified Tissues Workshop

To study their potential to produce a mineralizing matrix, the following cell types were cultured in diffusion chambers (DC) incubated intraperitonealy in athymic mice: human and rabbit marrow cells (HMC and RMC, respectively), human dental papilla (HDP) cells and osteoblastic rat osteosarcoma cells (ROS 1712). The DC content was examined after different time periods by light and electron microscopy. Bone, mineralizing cartilage and fibrous tissue appeared in RMC and HMC after 3-4 weeks. The tissues were aligned alongside the DC membrane filters. The center of the DC was occupied by a fibrin clot. lngrowth of blood vessels, hemopoiesis and remodelling of cartilage and bone were seen only in DC with violated membrane filters. HDP chambers showed many cells with odontoblastic features. The matrix contained banded and unbanded collagen. Mineral deposits were absent even in 8 week DC. 4 week ROS cell DC showed cell-rich tissue with pleomorphism and mitotic figures. The amount of extracellular matrix increased after 8 weeks and foci with tiny mineralized trabeculae were present. The overall appearance was that of osteosarcomatous bone. It is concluded that a) The osteogenic and part of the odontogenic expression of stromal cells is independent of interactions with other cell types and tissues but may be modified by such processes. b) Odontogenic epithelium is probably essential for the complete odontoblastic expression. c) Malignent cells show an intrinsic modification of the osteogenic phenotype. DISTRIBUTION OF EXTRACELLULAR MATRIX VESICLES IN HEALING RAT TIBIAL BONE D. Amir, Z. Schwartz,

H. Weinberg,

J. Sela

Departments of Orthopedics, Periodontics and Oral Pathology, Hebrew University-Hadassah Medical and Dental Schools, Jerusalem, Israel Matrrx vesicles are secreted by the forming cell to load calcium and phosphate to form an hydroxyapatite crystal. The crystal grows, ruptures the vesicle membrane to form calcospheritic structures and incorporates into the collagen and calcified fronts. This is the accepted sequence of calcification via matrix vesicles. However, direct evidence of a step by step propagation of this process is still lacking. The present study is a morphometric-electronmicroscopic study of rat tibia1 bone healing after marrow removal. A comparison of the behaviour of 4 types of vesicles was carried out 1 week after injury. The observations summarize the differences in the distribution, diameter and the distance from the calcified front of the 4 types of vesicles: a. Vesicles devoid of content-“empty.” b. Vesicles with amorphous electron-densities-“amorphic.” c. Vesicles with crystalline structures-“crystal.” d. Vesicles with ruptured membranes and crystals-“rupture.” 1 Distribution of all vesicles studied according to diameters manfested a normal pattern, characterized by concentration of more than 98% of vesicles in the diameter range of 0.07-0.22 pm. 2. The distribution of vesicles as to the distance from the front showed that 95% of vesicles were less than 3 t.r,rnfrom the front and that there was a highly significant inverse relation between their number and their distance from the front. 3. No correlation could be recorded between the diameters and the distance from the front. The formation of each vesicle is independent with regard to propagation of calcified front. 4. Content of most vesicles was “empty” of crystals mainly “amorphous.” 5. Highly significant difference was recorded for the mean