Bone and parathyroid inhibitory effects of S-2(3-aminopropylamino)ethylphosphorothioic acid. Studies in experimental animals and cultured bone cells.

S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR 2721) is a radio- and chemoprotective agent which produces hypocalcemia in humans. Intravenous injection of 30 mg/kg WR 2721 in rats and 15 mg/kg in dogs lowers serum calcium by 19 and 25%, respectively. Hypocalcemia in dogs is associated with a fall in serum immunoreactive parathyroid hormone (PTH), which suggests that the mechanism of its hypocalcemic effect is acute hypoparathyroidism. Despite this effect on PTH, in eight chronically parathyroidectomized rats on a low phosphate diet, WR 2721 reduced serum calcium from 9.4 +/- 0.6 to 7.7 +/- 0.5 mg/dl (P less than 0.01) at 3 h. Furthermore, in dogs rendered hypercalcemic by continuous infusion of PTH, WR 2721 reduced serum calcium from 11.0 +/- 0.5 to 10.6 +/- 0.5 mg/dl (P less than 0.01). To determine whether WR 2721 causes hypocalcemia by enhancing the exit of calcium from the circulation or inhibiting its entry, the drug was infused 3 h after administration of 45Ca to rats. WR 2721 did not significantly increase the rate of disappearance of 45Ca from the circulation even though serum calcium fell by 19%. However, serum 45Ca specific activity was higher at 1.5 h (P less than 0.01) and 3 h (P less than 0.05) in rats given WR 2721 than in rats given vehicle alone, which suggests that WR 2721 blocks the entry of nonradioactive calcium into the circulation, presumably from bone. In incubations with fetal rat long bone labeled in utero with 45Ca, 10(-3) M WR 2721 inhibited PTH-stimulated, but not base-line release of 45Ca. Bone resorption by primary culture of chick osteoclasts was inhibited by WR 2721 at concentrations as low as 10(-4) M in the absence of hormonal stimulation. These studies suggest that WR 2721 lowers serum calcium predominantly by blocking calcium release from bone. This acute hypocalcemic effect is at least in part independent of its effect on the parathyroid glands, and is most likely a direct effect of the agent on bone resorption.

Exogenous hyperthyroidism with osteoporosis.

Symptomatic osteopenia accompanied by subclinical hyperthyroidism developed in three women who were receiving excess thyroid hormone medication. Excessive thyroid replacement therapy resulted in mild hypercalcemia, hyperphosphatemia, and hyperphosphatasemia associated with diffuse skeletal demineralization and multiple fractures. Nondecalcified sections of double tetracycline-labeled iliac crest bone showed an accelerated rate of bone turnover with marked osteoclastosis and resorption of the cortical as well as the trabecular bone, typical of endogenous hyperthyroidism. Since thyroid hormones are among the most frequently prescribed medications, bone loss caused by exogenous hyperthyroidism may be more common than previously recognized.

Osteoporosis in young men: a syndrome of hypercalciuria and accelerated bone turnover.

Five young men had a similar syndrome of osteopenia and hypercalciuria, probably resorptive and absorptive, with histomorphometric data suggesting decreased bone mass with increased rate of bone formation. A search for causes of osteopenia, either primary or secondary, was unrewarding.

WR-2721 reduces bone loss after hindlimb tenotomy in rats.

WR-2721 is a thiophosphate analog of cysteamine that produces hypocalcemia in vivo. Previous studies suggest that WR-2721 produces hypocalcemia by independent inhibitory effects on parathyroid hormone (PTH) secretion, osteoclastic bone resorption, and tubular reabsorption of calcium. We sought to determine if WR-2721 would decrease bone loss in an animal model of disuse osteoporosis produced by unilateral knee tenotomy in the rat. Tenotomy significantly increased osteoclast number in tibias on the side of the procedure compared with tibias on the opposite side which had not undergone the procedure at 3 and 14 days. Femoral weight of tenotomized limbs were also reduced significantly compared with the contralateral limb at 3 and 14 days. WR-2721 treatment (240 mg/kg daily) prevented 26% of the loss of femoral dry weight and 29% of the loss of femoral ashed weight produced 14 days after tenotomy. In addition, WR-2721 treated (240 mg/kg daily) animals had fewer osteoclasts in tenotomized tibias than control animals at 3 days (6.6 +/- 0.7/mm versus 10.3 +/- 0.9/mm, p less than 0.02) and at 14 days (5.8 +/- 0.3/mm versus 8.7 +/- 0.4/mm, p less than 0.02). These data suggest that WR-2721 decreases bone loss in this model by decreasing osteoclastic bone resorption.

Serum bone gla protein and the vitamin D endocrine system in the oophorectomized rat.

Because of the importance of estrogen in osteoporosis, the effects of decreased estrogen production using sensitive measurements of bone mineral metabolism were studied in oophorectomized rats. Serum levels of ionized calcium, bone gla protein (BGP), vitamin D metabolites (25-hydroxyvitamin D and 1,25-dihydroxyvitamin D), and estradiol were measured before and serially for 6 weeks after oophorectomy in the rat. In addition, static and dynamic indices of bone histomorphometry were determined after double tetracycline labeling. Fifty Sprague-Dawley female rats (approximately 250 g) were studied. Twenty-five rats underwent oophorectomy (O), while the remaining rats were sham operated. Estrogen deficiency was noted in the O group within a week after surgery (estradiol, 2.45 +/- 0.78 vs. 27.9 +/- 4.15 pg/ml; P less than 0.05). Serum ionized calcium levels, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, and PTH levels did not differ between the two groups during the length of the study. Serum BGP levels were the same in both groups until the second week postoophorectomy, after which BGP remained significantly elevated in the O animals (121.7 +/- 5.95 vs. 76.7 +/- 3.87; P less than 0.001). Bone histomorphometry revealed increased osteoid volume (4.4 +/- 0.9% vs. 2.3 +/- 0.7%), osteoblast surface (26.5 +/- 2.4% vs. 3.2 +/- 1.2%), tetracycline surface (18.9 +/- 4.1% vs. 6.8 +/- 2.2%), as well as osteoclast surface (8.2 +/- 1.4% vs. 2.5 +/- 2%) in all O animals compared with those in the sham-operated group. These data indicate that oophorectomy and decreased estrogen result in increased bone turnover with elevated BGP levels. The marked BGP elevation within 2 weeks postoophorectomy suggests that estrogen withdrawal results in rapid altered bone mineral metabolism. The lack of concomitant increase in circulating PTH levels suggests that other factors may be mediating the bone loss following surgical oophorectomy.

1,25-Dihydroxyvitamin D3 modulates glucocorticoid-induced alteration in serum bone Gla protein and bone histomorphometry.

Glucocorticoid excess is associated with alterations in the vitamin D endocrine system. The aim of this study was to assess change in serum bone Gla protein (BGP) after low and high dose cortisone acetate treatment and to assess whether these alterations are altered or attenuated by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] administration. Five groups of rats were studied and compared to a control group [cortisone acetate in doses of 0.2, 3.3, and 5.0 mg/100 g BW; 1,25-(OH)2D3 in a dose of 100 ng/100 g BW; and a combination of 1,25-(OH)2D3 (100 ng/100 g BW) plus cortisone acetate (3.3 mg/100 g BW)]. Each animal received daily sc injections for 27 days. BGP decreased significantly by day 7 in the two groups receiving high doses of cortisone acetate compared to control group values (65.20 +/- 4.38 vs. 150.18 +/- 6.13 ng/ml in the intermediate dose group and 91.57 +/- 5.30 vs. 150.18 +/- 6.13 ng/ml in the high dose group; P less than 0.01); this effect persisted until day 28. Histomorphometry revealed decreased formation and resorption in the two high dose cortisone acetate groups, whereas low dose cortisone acetate produced no histological change. The combination therapy lessens any change in BGP until day 28 when BGP was lower than the control value (P less than 0.01); histomorphometry showed that combination therapy prevents the effect of cortisone acetate by increasing bone formation and resorption. The data demonstrate that high doses of cortisone acetate suppress bone formation and that this is reflected in the low serum BGP values. Thus, BGP may be a marker of glucocorticoid-induced bone disease. 1,25-(OH)2D3 protects against glucocorticoid-induced bone disease and the normal BGP level reflects this.

Effects of semistarvation on skeletal homeostasis.

Rats were semistarved over a 7-week period, resulting in a loss of 28.2 +/- 1.6% (SEM) of their initial body weights, while ad libitum fed controls gained 15.1 +/- 1.8% (SEM). Bone loss occurred and skeletal turnover was markedly reduced in the semistarved rats, as evidenced by a paucity of osteoid and osteoclasts, failure of the bone to assume a tetracycline label, and reduced urinary hydroxyproline excretion. Despite these changes, there were no alterations of serum or bone alkaline phosphatase activity with semistarvation, and analysis of tibial mineral content revealed reductions only in magnesium and sodium. The malnourished animals, however, were hypercalciuric and hypophosphatemic. Semistarvation had no effect on circulating levels of immunoreactive parathyroid hormone or 25-hydroxyvitamin D, but did result in reduced serum levels of corticosterone, insulin, and 1,25-dihydroxyvitamin D. Therefore, it appears that the effects of semistarvation on the rat skeleton are osteoporotic rather than osteomalacic, and that the defect is the consequence of reduced bone turnover. The contribution which the abnormalities of bone-regulating hormones play in the genesis of this skeletal lesion remains to be determined.

The effect of cyclosporin A administration and its withdrawal on bone mineral metabolism in the rat.

The in vivo administration of cyclosporin A (CsA) has been associated with significant bone loss and increased bone remodeling. To observe whether these changes are reversible, we have investigated the consequences of the administration and withdrawal of CsA immunotherapy on bone mineral metabolism. Three groups of Sprague-Dawley rats were studied for 28 days. Group A received vehicle, and group B received CsA (15 mg/kg BW) for 28 days, while group C received CsA (15 mg/kg BW) for 14 days and then vehicle from days 15-28 by daily gavage. Serum ionized calcium (Ca2+), PTH, bone gla protein, blood urea nitrogen, creatinine, magnesium, phosphorus, and 1.25-dihydroxyvitamin D were measured weekly. Bone histomorphometry was analyzed on days 14 and 28 in groups A and B and on day 28 in group C. CsA administration resulted in reversible hypomagnesemia and mild transient elevation in circulating 1,25-dihydroxyvitamin D levels with no change in Ca2+, PTH, blood urea nitrogen, or phosphorus. Serum bone gla protein levels were significantly increased (P less than 0.002) during CsA therapy, but tended to return to control values after CsA withdrawal. Enhanced bone remodeling and significant trabecular bone loss accompanied CsA administration. Withdrawal of CsA resulted in all of the histomorphometric parameters, except for bone volume, returning to control values within 2 weeks. Incomplete restoration of bone volume occurred 5 weeks after CsA withdrawal. This limited restoration of bone volume despite CsA withdrawal may have important clinical implications.

Does strict phosphorus control precipitate renal osteomalacia?

Hyperphosphatemia leads to the development of osteitis fibrosa in patients with chronic renal failure. In contrast, crippling osteomalacia may appear in uremic patients who are hypophosphatemic or aluminum intoxicated or who undergo total or subtotal parathyroidectomy. Thus, strict phosphorus control by use of aluminum-containing gels may ameliorate renal osteitis fibrosa, but may potentiate the development of osteomalacia. To evaluate this possibility, we compared the bone histologies of 10 chronic renal hemodialysis patients who consistently maintained predialysis phosphorus levels between 4-5 mg/dl (Strict-P) to those of 46 randomly selected dialysis patients (Random-P). We found that the Strict-P group had lower circulating immunoreactive PTH (P less than 0.02) and alkaline phosphatase (P less than 0.05) levels and, as expected, less evidence of hyperparathyroid bone disease. On the other hand, the Strict-P patients had osteomalacia, as evidenced by moderate osteoid accumulation and reduced capacity of bone to assume a fluorescent tetracycline label. Furthermore, all Strict-P patients had histological evidence of bone aluminum accumulation. We conclude that maintenance of normal serum P levels with aluminum-containing gels in hemodialysis patients prevents severe hyperparathyroid bone disease. Such treatment, however, is also attended by a moderate degree of aluminum-associated osteomalacia.

Hypophosphatasia: clinicopathologic comparison of the infantile, childhood, and adult forms.

Histochemical and direct enzyme analysis of osseous tissue from 23 patients with hypophosphatasia revealed that all clinical forms of this inherited metabolic bone disease are characterized by deficiency of alkaline phosphatase (ALP) activity in bone. The severe infantile form has the most profound deficiency, infantile form has the most profound deficiency, yet the cellular source of this enzyme--osteoblasts and their matrix vesicles--are normal by routine light and electron microscopy. Despite radiographic changes in bone metaphyses consistent with rickets, iliac crest biopsy of one affected child revealed no abnormalities; the other had evidence of a mineralization defect, but not as severe as that in affected infants. In this child and several affected adults with osteomalacia, osteoblasts appeared flat and metabolically inactive. Although these histological changes suggested a different pathogenetic mechanism for adult and childhood hypophosphatasia, these changes are most likely secondary to the underlying osteomalacia. Our findings are most consistent with evidence that childhood and adult hypophosphatasia often represent clinical expression of the heterozygous state for ALP deficiency which, when homozygous, results in the clinically severe, recessive, infantile form. Histochemical and direct analysis of bone tissue from controls and patients with hypophosphatasia demonstrated that the severe infantile form is associated with the most severe ALP deficiency. In the milder clinical forms, ALP deficiency in bone is not as profound. In general, the severity of the clinical expression of hypophosphatasia reflects the magnitude of the deficiency of ALP in bone. This is the expected finding for this inborn error of metabolism, which illustrates the major role bone ALP activity has in the process of normal skeletal mineralization.

Sclerosteosis: neurogenetic and pathophysiologic analysis of an American kinship.

We studied an American kinship with sclerosteosis, an autosomal-recessive disorder of bone remodeling and bone overgrowth of the calvaria, skull base, and tubular bones. Unlike osteopetrosis, which is attributed to abnormal immune and osteoclast function as well as bone resorption, sclerosteosis appears to be primarily a disorder of osteoblast (bone formation) hyperactivity. Related to cranial vascular and neural foraminal narrowing and reduced intracranial volume, affected patients with sclerosteosis demonstrate frequent seventh nerve palsy, progressive optic and cranial neuropathies, mixed hearing loss, brainstem compression, intracranial hypertension with increased elastance, and sudden, premature death. Management should involve early childhood identification of homozygotes, monitoring and aggressive treatment of intracranial hypertension, and extensive bone removal from skull, posterior fossa, and cervical spine.

An improved method for the retrieval of cultured macrophages.

We report a simple procedure for the maintenance and retrieval of rodent peritoneal macrophages on dialysis membrane supports. Culture units are easily constructed from plastic petri dishes and dialysis membranes. Macrophages adhere readily but are detached easily from the dialysis membrane by mild mechanical agitation. Cell viability remains high and the morphologic and functional characteristics of the macrophages are maintained.

Adult hypophosphatasia with chondrocalcinosis and arthropathy. Variable penetrance of hypophosphatasemia in a large Oklahoma kindred.

Three sisters, each with chondrocalcinosis/arthropathy, are described who have the clinical, laboratory and pathologic findings characteristic of the adult form of hypophosphatasia. Premature loss of adult teeth, arthralgias and pain from bilateral femoral pseudo-fractures were associated with subnormal circulating alkaline phosphatase levels, phosphoethanolaminuria and osteomalacia diagnosed by iliac crest biopsy. Assay of alkaline phosphatase activity in the blood of kindred members revealed hypophosphatasemia in one of two younger brothers. Several subjects in subsequent generations also had suspiciously low alkaline phosphatase activity, but did not have histories of significant dental, bone or joint disease. Review of the medical records of the sisters' parents, aunts and uncles revealed normal alkaline phosphatase levels in their father and five of his siblings, but consistently low levels in their mother and two of her siblings. Despite hypophosphatasemia, the sisters' mother and her siblings lived to old age without clinical or radiographic evidence of bone disease. Our findings suggest that although adult hypophosphatasia can be transmitted as a dominant trait in some kindreds, there is considerable variation in the clinical expression of the biochemical defect. One person, generation or family may manifest clinical bone disease and arthropathy whereas the biochemical defect may be present but remain asymptomatic in others. Furthermore, in some cases, the adult form of hypophosphatasia may represent a developmental disorder with hypophosphatasemia appearing during adulthood.

Controlled study of renal osteodystrophy in patients undergoing dialysis. Improved response to continuous ambulatory peritoneal dialysis compared with hemodialysis.

To assess the effect of different dialysis modalities on renal osteodystrophy, a controlled study was performed in six patients undergoing continuous ambulatory peritoneal dialysis and six hemodialysis-treated patients. All patients were enrolled at the initiation of dialysis, and age, sex, cause of renal failure, prior treatment of renal osteodystrophy, and baseline serum and bone histologic variables were similar in the two groups. After initial blood samples and bone biopsy specimens (with double-tetracycline labels) were obtained, renal osteodystrophy in both groups received comparable treatment with aluminum hydroxide to maintain serum phosphorus levels between 3.5 and 5.5 mg/dl, and with calcium carbonate and calcitriol to maintain total serum calcium levels between 10 and 11 mg/dl. Blood and bone samples were obtained again after nine months. All patients were asymptomatic at the beginning and end of the study. Phosphorus values were well controlled, and total calcium increased similarly in both groups. Although ionized calcium levels increased in both groups, the final level was higher in hemodialysis-treated patients than in patients undergoing continuous ambulatory peritoneal dialysis (2.82 +/- 0.07 meq/liter and 2.5 +/- 0.05 meq/liter, respectively; p = 0.005). Amino-terminal parathyroid hormone levels normalized in both groups, and histologic improvement of osteitis fibrosa occurred in a similar proportion of patients in both groups; however, quantitative improvement was greater in the hemodialysis-treated patients. Osteomalacia, assessed qualitatively and by dynamic histomorphometric measurements, was ameliorated to a much greater degree in patients undergoing continuous ambulatory peritoneal dialysis compared with hemodialysis-treated patients. Bone aluminum staining was absent in all biopsy specimens. Overall, bone histologic findings improved to a greater degree in patients undergoing continuous ambulatory peritoneal dialysis. When patients undergoing continuous ambulatory peritoneal dialysis or hemodialysis and receiving similar treatment for renal osteodystrophy were compared, patients treated with continuous ambulatory peritoneal dialysis appeared to have a greater improvement in their metabolic bone disease.

Osteomalacia and osteitis fibrosa in a man ingesting aluminum hydroxide antacid.

A 53-year-old man with a history of long-term aluminum hydroxide antacid ingestion reported diffuse bone pain and multiple stress fractures over a two-year period. An undecalcified transiliac bone biopsy specimen revealed osteomalacia with osteitis fibrosa; plasma parathyroid hormone and cyclic AMP levels were normal. Following withdrawal of antacids and treatment with calcium and phosphorus, an initially elevated plasma, 1,25-dihydroxyvitamin D level fell to within the normal range, accompanied by decreased bone pain, healed stress fractures, and increased axial bone mineral content as determined by computed tomography of lumbar trabecular bone. Phosphate deprivation and 1,25-dihydroxyvitamin D excess may contribute to the poor mineralization and exaggerated resorption of bone observed in this syndrome. The clinical, biochemical, radiologic, and histologic features of previously reported cases are reviewed. Early recognition of this syndrome is important, since appropriate therapy promotes skeletal remineralization and prevents morbidity.

Axial osteomalacia. Clinical, laboratory and genetic investigation of an affected mother and son.

Axial osteomalacia--a rare osteosclerotic bone disorder characterized by axial skeleton pain, coarsening of the trabecular bone pattern on radiographs of the axial but not appendicular skeleton, and osteomalacia on biopsy of a rib or iliac crest--has been reported in 10 apparently sporadic cases, all of which were in middle-aged or elderly Caucasian men. The etiology is unknown but has been postulated to be a bone cell defect. We describe the clinical, laboratory, pathologic and family study of a black mother and son with axial osteomalacia associated with polycystic liver and kidney disease. Investigation of the son suggested that radiographic osteosclerosis can be detected in early adulthood. Limited skeletal survey of his three children revealed no abnormalities. Examination of undecalcified iliac crest bone after in vivo tetracycline labeling revealed severe osteomalacia in the son despite normal circulating calcium, inorganic phosphate and vitamin D metabolite levels and persistently elevated alkaline phosphatase activity. Although osteoblasts appeared flat and inactive, histochemical studies showed intense alkaline phosphatase activity in the osteoblasts along most trabecular bone surfaces. Electron microscopy revealed intact matrix vesicles within unmineralized osteoid. The presence also of unexplained myopathy in the son--characterized by proximal muscle weakness, persistently elevated circulating creatine phosphokinase levels and pathogenic changes of myopathy on biopsy of quadriceps muscle--together with impaired bone mineralization, suggests that a disorder of vitamin D action may be involved in the pathogenesis of this unusual condition. Axial osteomalacia affects blacks as well as Caucasians, women as well as men, may be familial, and may perhaps be a developmental abnormality inherited in association with polycystic kidney and liver disease.

Osteopetrosis, renal tubular acidosis and basal ganglia calcification in three sisters.

Three adult sisters with osteopetrosis in infancy had spontaneous resolution of bone modeling defects and osteosclerosis. During adolescence, basal ganglia calcification developed in two. Renal tubular acidosis (type I) was diagnosed in each during early adulthood. The disorder was transmitted apparently as a recessive trait--the same mode of inheritance as for the "malignant" form of osteopetrosis which is usually fatal during childhood. Electron microscopy of bone suggested that osteoclasts failed to form "ruffled membranes" characteristic of active bone resorbing cells. Chronic systemic acidosis may have ameliorated the skeletal manifestations of this new syndrome.

Meningeal osteomas and chronic renal failure.

Osteomas, although usually found in the frontal and ethmoid sinuses or the mandible, may be located on the inner table of the skull. We report the incidence, distribution, histologic features, and clinical correlates of intracranial osteomas arising in the dura mater and the falx cerebri in 200 consecutive adult autopsies. Ten patients (5 per cent of autopsies) were found to have meningeal osteomas. The tumors were usually located at the dural-falx junction at the superior longitudinal sinus. Histologically, they resembled osteomas arising from other sites, but undecalcified sections generally demonstrated histologic features of active bone remodeling--namely, the presence of abundant osteoid and numerous osteoclasts, and, in some instances, osteitis fibrosa. The age and sex distributions were similar among autopsied patients with meningeal osteomas (mean age, 66 years; 60 per cent female) and those without (mean age, 64 years; 58 per cent female). Although the incidence of uremia in the general autopsy population was only 13 per cent, 60 per cent of patients with osteomas died with concomitant renal failure. The authors consider these cases of meningeal ossifications and reports of meningeal calcifications to represent osteomas arising from the dura mater and the falx cerebri. Thus, intracranial osteomas may be more common than was previously recognized. In some instances, the abnormal biochemical state accompanying chronic renal failure may stimulate new bone formation in the osteogenic tissue of the dura mater.

Systemic mastocytosis associated with generalized osteopenia. Histopathological characterization of the skeletal lesion using undecalcified bone from two patients.

Although mast cell proliferation in the bone marrow frequently occurs in systemic mastocytosis and is often associated with radiographically detectable bone lesions, the pathologic correlates of the skeletal abnormalities are poorly characterized. We therefore examined three nondecalcified transiliac crest biopsy specimens from two patients with systemic mastocytosis and diffuse osteopenia with vertebral crush fractures. Marrow involvement included unusual mast cell aggregates, as revealed by metachromatic staining, that mimicked granulomas. Histomorphometric analysis or trabecular bone revealed accelerated bone remodeling or "turn over" characterized by osteoidosis, peritrabecular fibrosis, increased numbers of osteoblasts and osteoclasts, and an increase in osteoclastic resorbing surfaces. Our observations and review of the literature suggest that with the recent development of techniques for assessing undecalcified bone biopsy specimens, mastocytosis will probably be shown to be a more common etiology in "osteoporosis" than previously recognized. Metachromatic staining of the biopsy specimen should be a routine procedure in the investigation of any patient who undergoes diagnostic bone biopsy.

Cystic parathyroid gland hyperplasia--hyperparathyroidism presenting as a neck mass.

Hyperparathyroidism may occasionally present as a neck mass resulting from cystic change in a parathyroid adenoma. We report an unusual instance of hyperparathyroidism presenting as a palpable, but asymptomatic hyperplastic parathyroid gland cyst. We conclude that hyperplastic parathyroid glands may undergo cystic degeneration resulting in a palpable neck mass; and that all functional parathyroid gland cysts are not necessarily adenomas. Therefore, during parathyroidectomy procedures, biopsy material from at least one additional parathyroid gland should be obtained.