Clinical Endocrinology (1992) 37,565-569
Case report
Non-autonomy of parathyroid hormone secretion in acute primary hyperparathyroidism Pinhas P. Schachter', Mark D. Christy, ltamar S. Avigad*, Moshe Shabtay* and George S. Leight Jr Duke University Medical Center, Durham, North Carolina and 'Chaim Sheba Medical Center, Tel-Hashomer, Israel (Received 12 March 1992; returned for revision 15 May 1992; finally revised 17 June 1992; accepted 22 July 1992)
Summary A patient with acute primary hyperparathyroidismtreated with mithramycin preoperatively, underwent neck exploration and two enlarged parathyroidglands were excised one huge adenoma (69) and another smaller gland. Mithramycin was administered preoperatively to lower life-threatening hypercalcaemia, and parathyroid slices from the huge adenoma removed at surgery were submitted in vitro to various calcium concentrations in the media to determine the influence of calcium on parathyroid adenoma secretory pattern in acute primary hyperparathyroidism. Mithramycin induced a significant decline in calclum levels and significant elevations of calciotrophic hormones (intact PTH, mid-regionspecific PTH, calcitonin and calcitriol). Significant suppression in PTH output in vitro was achieved by increasing calcium levels in the media. These results exclude autonomous PTH secretion (non-calciumdependent) as a possible aetiology of acute primary hyperparathyroidism. We suggest that a sudden increase in the set-point of the diseased parathyroidcells in the presenceof a huge cell mass accounts, in large part, for both the marked hypercalcaemia and elevated PTH levels in this patient.
Acute primary hyperparathyroidism (APH) or parathyrotoxicosis is a rare, life-threatening disorder characterized by severe hypercalcaemia, high PTH levels, mental changes and coma (Payne & Fitchett, 1964; Fitzpatrick & Bilezikian, 1987). As a form of primary hyperparathyroidism with a fulminant course this condition may provide a unique insight into normal and abnormal regulatory mechanisms of the parathyroid gland. In-vitro studies with parathyroid slices and dispersed cells have shown a subtle but significant difference in the set-point Correspondence: Dr Pinhas P. Schachter, Department of Surgery B, Chaim Sheba Medical Center, Tel-Hashomer 52621, Israel.
for secretory suppression by calcium between normal and adenomatous parathyroid cells (Brown et al., 1978, 1979a, b; Dietel et al., 1984). A moderate elevation in this set-point in addition to an increase in parathyroid cell mass is probably responsible for the mild hypercalcaemia of primary hyperparathyroidism patients (Brown, 1983; Dietel et al., 1987). However, PTH secretion from adenoma cells maintains responsiveness to circulating calcium levels, with a particular sensitivity to declining calcium concentrations (Murray et al., 1972; Conlin et al., 1989). On the other hand, the high PTH levels in spite of severe hypercalcaemia, as recorded in APH patients, imply an autonomous PTH secretion pattern (Payne & Fitchett, 1964). We recently had the opportunity to study the secretory regulation by calcium with a parathyroid adenoma from a patient with acute primary hyperparathyroidism. An unusual and striking defect in the secretory control mechanism of calciotrophic hormones, in vitro as well as in vivo, was found. Case report and methods
An 82-year-old Indian female was referred to Duke University Medical Center with a 6-day history of progressive mental deterioration following a fall. Her medical history revealed weight loss, recurrent urinary tract infections, hypothyroidism and, recently, hypercalcaemia. On admission, she was disoriented, ill-looking, with poorly articulated speech and decreased memory. Laboratory data indicated chronic anaemia with thrombocytopaenia, total calcium levels of 3.54 mmol/l; ionized calcium 2.2 mmol/l(1~19-1~33 mmol/l)*; inorganic phosphorus 0.87 mmol/l (0.87-1.49); alkaline phosphatase 146 units/] (20-90); chloride 107 mmol/ l(97-108); and creatinine levels of 151 pmol/l (53.2-141.9). Initial attempts to reduce serum calcium that included i.v. administration of fluids and diuretics (frusemide) failed and high calcium concentrations persisted with further deterioration in the mental condition. Mithramycin 25 pg/kg body weight was administered for two consecutive days and discontinued due to hepatic toxicity and deteriorating renal function. Calcium levels declined to 2.59 mmol/l followed by a substantial improvement in the patient's mental status. Two enlarged parathyroid glands were resected at neck exploration; one huge gland weighing 6060 mg and a second weighing 350 nig. Histopathological examination defined the
* Reference range is given in parentheses. 565
566
Clinical Endocrinology (1992)37
P . P. Schachter et al.
huge gland as adenoma and the second as ‘enlarged parathyroid’ whilc a biopsy from a third gland was consistent with normal parthyroid tissue. An uneventful post-operative course followed; the patient was asymptomatic with clear mental functions and a serum calcium that stabilized within normal range after a nadir of 2.05 mmol/l. In-vitro experiment
The large parathyroid adenoma resected at neck exploration was divided into two specimens. One was submitted for histopathological examination, while the other was placed in chilled saline for the in-vitro experiment. The tissue was divided into ten similar 2 x 1 x 1 mm slices each suspended in a separate flask with Hank’s MEM (Minimal Essential Media) with 2.2 g/1 NaHC03, 25 mM HEPES, L-glutamine (Sigma Chemical Comp. St. Louis, MO), 1 Yo human serum and variable CaCI, (1.13 mmol/l or 1.68 mmol/l) at a pH of 7.42. Incubations were performed in a shaking water-bath at 37°C. Two groups of five slices each were defined according to the ionized calcium concentration in the media (group 1 and group 2). Following a 20-minute period of stabilization within the specific media, each parathyroid slice was rinsed and redispersed into fresh media containing the same ionized calcium concentration for a 15-minute period. A sample of medium was collected from each flask for PTH determination at the end of the 15-minuteincubation period. The same procedure was repeated changing the calcium concentration in the medium for each group of parathyroid slices, so that each parathyroid piece was exposed to both hypocalcaemic and hypercalcaemic media.
spectrophotometric reaction using the Kodak Ektachem DT-60 Analyzer (Eastman Kodak Co. Rochester NY). Serum and medium ionized calcium were measured with an ion selective electrode using the ICAjl Ionized Calcium Analyzer (Copenhagen, Denmark). Two PTH assays were employed: a double antibody immunoradiometric method (Nichols Institute, San Juan Capistrano, CA) was used to quantify intact (1-84) human PTH (i-PTH). Intra-assay and interassay coefficients of variation for this procedure average 5.7 and 18.3% respectively. The second RIA (Cambridge Medical Diagnostics, Billerica, Mass.) recognizes human PTH species that contain the 53-68 amino acid sequence (mid-molecule PTH (mPTH)). Coefficients of variation are 6.1 and 11.8% respectively. A double antibody sequential competitive RIA was employed to determine calcitonin levels (Diagnostic Products Corp. Los Angeles, CA). Intra and inter-assay coefficients for this assay are 6-8and 7.6%. Serum calcitriol(l,25dihydroxyvitamin D) quantitation was performed with a radioreceptor assay (Incstar Corp. Stillwater, MN). The assay includes preliminary extraction and purification of vitamin D metabolites from serum and quantitation using a non-equilibrium competitive protein binding assay. Intra and inter-assay coefficients for this assay are 5.4 and 19% respectively. Statistical methods
Data are summarized as mean & standard error of the means (SEM). The statistical significance of differences was determined by paired t-test. Exact P-values are reported. Results
Analytical methods
Serum levels of total calcium, phosphorus, chloride, alkaline phosphatase and creatinine were determined by a reflectance
Levels were determined before administration of mithramycin and at 12-hour intervals after the drug was started, as well as post-operatively (Table 1). Concomitant levels of calcio-
Table 1 Biochemical results
Total Ca (mmol/l) (2.17-2.54) Ionized Ca (mmol/lf (1.19-1.31) Phosphorus (mmol/l) (0.87- 1-49) Creatinine (pmol/l) (53.2- 141.9) Chloride (mmol/l) (97-108) Alk. phosph. (Ujl) (20-96) Reference range is given in parentheses.
Premithramycin adminis.
12h Postadminis.
24 h Postadminis.
72 h Postadminis.
3.54 2.20 0.87 150.7 107 146
3.44 2.05 0.87 141.9 112 150
3.44 1.98 0.94 159.6 I10 145
2.59 1.49 1.1 195.1 1 I4 151
PostOP.
levels 2.45 1.43 0.8 1
159.6 112 148
PTH secretory pattern
Clinical Endocrinology (1992) 37
567
Table 2 Serum level of calcium related hormones
Premithramycin adminis. Intact PTH (ng/l) (1 3-64) Mid-Mol. PTH (nmolil) (0.04-0.15) Calcitonin (ng/l) (0-33) Calcitriol @mol/l) (36-120)
105 2.96 39 14.4
12h
24 h
Postadminis.
Postadminis.
12 h Postadminis.
112 2.16
109 2.12 28 12.5
618 8.99 151 21.6
-
PostOP. levels
5 0.23
I1 11.8
~~
Reference range is given in parentheses
Table 3 Calcium dependent PTH secretion in uitro
m-PTH (nmolil)
m-PTH (nmolil) Hypocalcaemia (1.13 mmol/l) Hypercalcemia (1.68 mmol/l) P-value
39,4+ 5.9 21.5 2.3 0.03 1
0,3_+0.06 0.16 & 0.23 0.055*
28.9 3.4 14.3k 4 . 5 0.006 1
0.26 0.05 0.08+0.01 0.021
* Not statistically significant. Values are expressed as mean k SEM. trophic hormones is shown in Table 2. Mithramycin induced a significant decrease in calcium levels by the third day; however, a parallel sharp increment in PTH levels is evident: i-PTH increased from 105 to 618 ng/l, and m-PTH from 2.96 to 8.99 nmol/l. Calcitonin and calcitriol levels also increased significantly (39 to 151 ng/l and 14.4 to 21.6 pmolll, respectively). Creatinine and inorganic phosphorus levels increased while no significant alteration in the chloride or alkaline phosphatase concentrations was recorded. Surgery resulted in a sharp decline towards the normal range in both calcium and calcium-related hormones. In-vitro PTH secretion from parathyroid slices is depicted in Table 3. A significant decrease in i-PTH and m-PTH secretion by both groups of tissue slices is achieved with higher calcium concentration in the media ( P = 0.006; 0.031 for i-PTH and P=0.021; 0.055 for m-PTH; Fig. 1). Discussion
The differential diagnosis of acute, life-threatening hypercalcaemia includes acute primary hyperparathyroidism. The exacerbation of the relatively benign primary hyperparathyroidism is invariably accompanied by marked signs and symptoms of hypercalcaemia (Fitzpatrick & Bilezikian, 1987; Schweitzer et al., 1978). Usually, patients with APH reveal a history of mild, asymptomatic hypercalcaemia with
involvement of the skeleton and/or renal disease. These characteristic manifestations of primary hyperparathyroidism have evolved over the prolonged duration of hypercalcaemia. This implies that the acute state is a superimposed aggravation of the mild primary hyperparathyroidism. The sudden development of hypercalcaemic crisis with remarkable elevation in circulating PTH levels (20 times normal values) was attributed to autonomous PTH secretion from the diseased gland (Fitzpatrick & Bilezikian, 1987). PTH secretion generally shows a sigmoidal relation to ambient calcium concentrations, a curve characterized by four parameters: ( 1 ) suppressibility; (2) maximal release; (3) set-point (calcium level that induces 50% of maximal suppressibility); and (4)slope at set-point (Brown, 1983; Marx et al., 1986). In-vitro experiments defined an increased set-point for adenoma cells and abnormal calcium-regulated PTH release from uninvolved parathyroid glands of primary hyperparathyroidism patients (Brown et aE., 1981; Pocotte et nl., 1991). However, the set-point for adenoma cells is only slightly higher than that of normal parathyroid cells (Marx et al., 1986; Brown et al., 1979). Even a moderate elevation in the set-point combined with an increased mass of secreting parathyroid tissue could make an important contribution to the aetiology of hypercalcaemia in primary hyperparathyroidism (Marx et al., 1986). Most APH patients reveal a huge parathyroid adenoma, but such enlargement of the
568
Clinical Endocrinology (1992) 37
P. P. Schachter et a / .
P.0.02
Group I
Groilp 2
Fig. 1 a, Mid-region specific PTH and b, intact PTH secretion in v i m at calcium concentrations in the media of a, 1 . 1 3; 0 . 1.68
mmol/l. A significant suppression by high calcium levels is evident for both groups of parathyroid tissue slices.
tissue mass is a prolonged process. Therefore, it is reasonable to attribute the development of acute hypercalcaemia and high PTH levels to a sudden and significant increase in the set-point of the parathyroid adenoma cells, or to complete disruption of calcium-regulated PTH release rendering autonomous PTH secretion from the parathyroid adenoma. The in-vitro experiment with parathyroid slices from this APH patient indicates a definite response in PTH secretion to alterations in the calcium concentrations in the media. It is impossible to calculate a precise set-point using this method; however, a significant change in the output of intact-PTH is evident ( P = 0.03 1; P = 0.006). Both groups of tissue slices responded in an adequate manner to different calcium levels in the media, excluding the possibility of autonomous (noncalcium dependent) PTH secretion. Therefore, since autonomous PTH secretion is excluded and an abrupt change in the adenoma mass is very unlikely, the only acceptable explana-
tion for the appearance of APH in this patient is a sudden, major rise in set-point of the diseased gland. Severe hypercalcaemia due to a high set-point has also been reported in neonatal primary hyperparathyroidism (Marx et al., 1986). Moreover, despite a marked abnormality in PTH secretion in this patient, mithramycin induced a significant increase in PTH levels. This drug reduces serum calcium levels by inhibiting bone resorption even in the presence of high PTH or PTH-like protein, without any direct influence on parathyroid secretory functions (Yarbro ef al., 1966; Singer et al., 1970; Perlia et al., 1970). Following administration of mithramycin to our patient i-PTH levels increased sixfold (from 105 to 618 ng/l) while m-PTH levels increased only threefold (from 2.96 to 8.99 nmol/l). Calcium also regulates the intra-glandular degradation of intact-PTH, thereby controlling the relative proportion of bioactive hormone released from the gland (Habener et a/., 1975; Fuleihan et al., 1989; Mayer et al., 1979; Hanley & Ayer, 1986). The mechanism responsible for this additional regulation is unclear. However, decreasing the ambient calcium concentration triggers an increase in the relative proportion of i-PTH secreted by the parathyroid gland (Conlin et al., 1989;Mallette, 1991).Preservation of this adequate response in our APH patient is shown by the magnitude of i-PTH versus m-PTH secretion caused by declining serum calcium levels. This observation further emphasizes non-autonomous PTH secretion from the parathyroid adenoma of APH patients. The significant increase in serum intact-PTH is not reflected by the modest increase in calcitriol levels (from 14.4 to 21.6 pmol/l). This poor response to PTH can be attributed to initial vitamin D deficiency, even though, on admission, the level of 25-hydroxyvitamin D was 54.9 nmol/l (reference range 37.4-200), or down-regulation of PTH receptors (Abou-Samra et nl., 1989;Yamamoto et al., 1988; Ajlouni & Theil, 1975).Hepatic and renal toxicity caused by mithramycin might interfere with 1,25-dihydroxyvitamin D production since there was no change in 25-hydroxyvitarnin D level (60 nmol/l) after the significant decline in calcium levels and huge increase in PTH levels caused by mithramycin. The unexpected increase in serum calcitonin levels (from 39 to 151 ng/l) despite decreasing calcium concentrations might be explained by competitive binding of mithramycin to the same receptors on the osteoclast (Martin, 1980). Another possible reason is that mithramycin induced hepatic injury delaying calcitonin degradation, thereby increasing the circulating levels of this hormone. In conclusion, the data obtained from this interesting patient indicate that acute primary hyperparathyroidism is probably caused by a sudden change in the set-point of parathyroid adenoma cells and is not due to autonomous PTH secretion.
Clinical Endocrinology (1992) 37
References Abou-Samra, A.B., Jueppner, H., Potts, J.T. & Segre, G.V. (1989) Inactivation of pertussis toxin-sensitive guanyl nucleotide-binding proteins increase parathyroid hormone receptors and reverse agonist-induced receptor down-regulation in ROS 17/2.8 cells. Endocrinology, 125,2594-2599. Ajlouni, K . & Theil, GB. (1975) Mithramycin effects on calcium, phosphorus and parathyroid hormone in osseous Paget’s disease. American Journal of Medical Science, 269, 13-18. Brown, E.M. (1983) Four-parameter model of the sigmoidal relationship between parathyroid hormone release and extracellular calcium concentration in normal and abnormal parathyroid tissue. Journal of Clinical Endocrinology and Metabolism, 56,572581. Brown, E.M., Brennan, M.F., Hurwitz, S., Windek, R., Marx, S.J., Spiegel, A.M., Koehler, J.H., Gardner, D.G. & Aurbach, G.D. (1978) Dispersed cells prepared from human parathyroid glands: distinct calcium sensitivity of adenoma us primary hyperplasia. Journal of Clinical Endocrinology and Metabolism, 46, 267-276. Brown, E.M., Broadus, A.E. & Brennan, M.F. (1979a) Direct comparison in vivo and in vitro of suppressibility of parathyroid function by calcium in primary hyperparathyroidism. Journal of Clinical Endocrinology and Metabolism, 48,606-610. Brown, E.M., Gardner, D.G., Brennan, M.F., Marx, S.J., Spiegel, A.M., Attie, M.F., Downs, R.W. Jr, Doppman, J.L. &Aurbach, G.D. (1979b) Calcium regulated parathyroid hormone release in primary hyperparathyroidism. Studies in vitro with dispersed parathyroid cells. American Journal of Medicine, 66,923-928. Brown, E.M., Wilson, R.E.,Thatcher, J.G. &Marynick, S.P. (1981) Abnormal calcium-regulated PTH release in normal parathyroid tissue from patients with adenoma. American Journal of Medicine, 71, 565-570. Conlin, P.R., Fajtova, V.T., Mortensen, R.M., LeBoff, M.S. & Brown, E.M. (1989) Hysteresis in the relationship between serum ionized calcium and intact parathyroid hormone during recovery from induced hyper and hypocalcemia in normal humans. Journal of Clinical Endocrinology and Metabolism, 69, 593-599. Dietel, M., Arps, H., Niedorf, A,, Schafer, H.J. & Holtzel, F. (1987) Abnormal calcium distribution in human parathyroid adenomas as possible cause of primary hyperparathyroidism. Hormone and Metabolism Research, 19, 177-181. Dietel, M., Holzel, F., Arps, H. & Bressel, M. (1984) Differential calcium response of normal and adenomatous parathyroid glands. Acta Endocrinologica, 107, 375-38 1. Fitzpatrick, L.A. & Bilezikian,J.P. (1987) Acute primary hyperparathyroidism. American Journal of Medicine, 82,275-282. Fuleihan, G.E., Chen, C.J., Rivkees, S.A., Marynick, S.P., Stock, J., Pallota, J.A. &Brown, E.M. (1989) Calcium-dependent release of N-terminal fragments and intact immunoreactive PTH by human
PTH secretory pattern
569
pathological parathyroid tissue in vitro. Journal of Clinical Endocrinology and Metabolism, 69, 860-867. Habener, J.F., Kemper, B. & Potts, J.T. Jr. (1975) Calciumdependent intracellular degradation of parathyroid hormone: A possible mechanism for regulation of hormone stores. Endocrinology, 97,43 1-44 1. Hanky, D.A. & Ayer, L.M. (1986) Calcium-dependent release of carboxyl-terminal fragments of parathyroid hormone by hyperplastic human parathyroid tissue in vitro. Journal of Clinical Endocrinology and Metabolism, 63, 1075-1079. Mallette, L.E. (1991) The parathyroid polyhormones: New concepts in the spectrum of peptide hormone action. Endocrinology Reviews, 12, 110-1 17. Martin, T.J. (1980) Actions of calcitonin and mithramycin. Arthritis and Rheumatism, 23, 1131-1138. Marx, S.J., Lasker, R.D., Brown, E.M., Fitzpatrick, L.A., Sweeny, N.B., Goldbloom, R.B., Gillis, D.A. & Cole, D.E.C. (1986) Secretory dysfunction in parathyroid cells from a neonate with severe primary hyperparathyroidism. Journal of Clinical Endocrinology and Metabolism, 62,445-449. Mayer, G.P., Keaton, J.A., Hurst, J.G. & Habener, J.F. (1979) Effects of plasma calcium concentration on the relative proportion of hormone and carboxyl fragments in parathyroid venous blood. Endocrinology, 104, 1778-1 784. Murray, T.M., Peacock, M., Powell, D., Monchik, J.M. & Potts, J.T. Jr. (1972) Non-autonomy of hormone secretion in primary hyperpara thyroidism. Clinical Endocrinology, 1,235-246. Payne, R.L. Jr. & Fitchett, C.W. (1964) Hyperparathyroid crisis: survey of the literature and a report of two additional cases. Annals of Surgery, 161, 737-745. Perlia, C.P., Gubisch, N.J., Wolter, J., Edelberg, D., Dederick, M.M. & Taylor, S.G. 111. (1970) Mithramycin treatment of hypercalcemia. Cancer, 25, 389-394. Pocotte, S.L., Ehrenstein, G. & Fitzpatrick, L.A. (1991) Regulation of parathyroid hormone secretion. Endocrinology Reviews, 12, 291 -301. Schweitzer, V.G., Thompson, N.W., Harness, J.K. & Nishiyama, R.H. (1978) Management of severe hypercalcemia caused by primary hyperparathyroidism. Archives ofsurgery, 113,373-381. Singer, F., Neer, R. & Murray, T. (1970) Mithramycin treatment of intractable hypercalcemia due to parathyroid carcinoma. New England Journal of Medicine, 1283, 634-636. Yamamoto, I., Shigeno, C., Potts, J.T. & Segre, G.V. (1988) Characterization and agonist-induced down-regulation of parathyroid hormone receptors in clonal rat osteosarcoma cells. Endocrinology, 122, 1208-1217. Yarbro, J.W., Kennedy, B.J. & Barnum, C.P. (1966) Mithramycin inhibition of ribonucleic acid synthesis. Cancer Research, 26, 3639.
Case report
Non-autonomy of parathyroid hormone secretion in acute primary hyperparathyroidism Pinhas P. Schachter', Mark D. Christy, ltamar S. Avigad*, Moshe Shabtay* and George S. Leight Jr Duke University Medical Center, Durham, North Carolina and 'Chaim Sheba Medical Center, Tel-Hashomer, Israel (Received 12 March 1992; returned for revision 15 May 1992; finally revised 17 June 1992; accepted 22 July 1992)
Summary A patient with acute primary hyperparathyroidismtreated with mithramycin preoperatively, underwent neck exploration and two enlarged parathyroidglands were excised one huge adenoma (69) and another smaller gland. Mithramycin was administered preoperatively to lower life-threatening hypercalcaemia, and parathyroid slices from the huge adenoma removed at surgery were submitted in vitro to various calcium concentrations in the media to determine the influence of calcium on parathyroid adenoma secretory pattern in acute primary hyperparathyroidism. Mithramycin induced a significant decline in calclum levels and significant elevations of calciotrophic hormones (intact PTH, mid-regionspecific PTH, calcitonin and calcitriol). Significant suppression in PTH output in vitro was achieved by increasing calcium levels in the media. These results exclude autonomous PTH secretion (non-calciumdependent) as a possible aetiology of acute primary hyperparathyroidism. We suggest that a sudden increase in the set-point of the diseased parathyroidcells in the presenceof a huge cell mass accounts, in large part, for both the marked hypercalcaemia and elevated PTH levels in this patient.
Acute primary hyperparathyroidism (APH) or parathyrotoxicosis is a rare, life-threatening disorder characterized by severe hypercalcaemia, high PTH levels, mental changes and coma (Payne & Fitchett, 1964; Fitzpatrick & Bilezikian, 1987). As a form of primary hyperparathyroidism with a fulminant course this condition may provide a unique insight into normal and abnormal regulatory mechanisms of the parathyroid gland. In-vitro studies with parathyroid slices and dispersed cells have shown a subtle but significant difference in the set-point Correspondence: Dr Pinhas P. Schachter, Department of Surgery B, Chaim Sheba Medical Center, Tel-Hashomer 52621, Israel.
for secretory suppression by calcium between normal and adenomatous parathyroid cells (Brown et al., 1978, 1979a, b; Dietel et al., 1984). A moderate elevation in this set-point in addition to an increase in parathyroid cell mass is probably responsible for the mild hypercalcaemia of primary hyperparathyroidism patients (Brown, 1983; Dietel et al., 1987). However, PTH secretion from adenoma cells maintains responsiveness to circulating calcium levels, with a particular sensitivity to declining calcium concentrations (Murray et al., 1972; Conlin et al., 1989). On the other hand, the high PTH levels in spite of severe hypercalcaemia, as recorded in APH patients, imply an autonomous PTH secretion pattern (Payne & Fitchett, 1964). We recently had the opportunity to study the secretory regulation by calcium with a parathyroid adenoma from a patient with acute primary hyperparathyroidism. An unusual and striking defect in the secretory control mechanism of calciotrophic hormones, in vitro as well as in vivo, was found. Case report and methods
An 82-year-old Indian female was referred to Duke University Medical Center with a 6-day history of progressive mental deterioration following a fall. Her medical history revealed weight loss, recurrent urinary tract infections, hypothyroidism and, recently, hypercalcaemia. On admission, she was disoriented, ill-looking, with poorly articulated speech and decreased memory. Laboratory data indicated chronic anaemia with thrombocytopaenia, total calcium levels of 3.54 mmol/l; ionized calcium 2.2 mmol/l(1~19-1~33 mmol/l)*; inorganic phosphorus 0.87 mmol/l (0.87-1.49); alkaline phosphatase 146 units/] (20-90); chloride 107 mmol/ l(97-108); and creatinine levels of 151 pmol/l (53.2-141.9). Initial attempts to reduce serum calcium that included i.v. administration of fluids and diuretics (frusemide) failed and high calcium concentrations persisted with further deterioration in the mental condition. Mithramycin 25 pg/kg body weight was administered for two consecutive days and discontinued due to hepatic toxicity and deteriorating renal function. Calcium levels declined to 2.59 mmol/l followed by a substantial improvement in the patient's mental status. Two enlarged parathyroid glands were resected at neck exploration; one huge gland weighing 6060 mg and a second weighing 350 nig. Histopathological examination defined the
* Reference range is given in parentheses. 565
566
Clinical Endocrinology (1992)37
P . P. Schachter et al.
huge gland as adenoma and the second as ‘enlarged parathyroid’ whilc a biopsy from a third gland was consistent with normal parthyroid tissue. An uneventful post-operative course followed; the patient was asymptomatic with clear mental functions and a serum calcium that stabilized within normal range after a nadir of 2.05 mmol/l. In-vitro experiment
The large parathyroid adenoma resected at neck exploration was divided into two specimens. One was submitted for histopathological examination, while the other was placed in chilled saline for the in-vitro experiment. The tissue was divided into ten similar 2 x 1 x 1 mm slices each suspended in a separate flask with Hank’s MEM (Minimal Essential Media) with 2.2 g/1 NaHC03, 25 mM HEPES, L-glutamine (Sigma Chemical Comp. St. Louis, MO), 1 Yo human serum and variable CaCI, (1.13 mmol/l or 1.68 mmol/l) at a pH of 7.42. Incubations were performed in a shaking water-bath at 37°C. Two groups of five slices each were defined according to the ionized calcium concentration in the media (group 1 and group 2). Following a 20-minute period of stabilization within the specific media, each parathyroid slice was rinsed and redispersed into fresh media containing the same ionized calcium concentration for a 15-minute period. A sample of medium was collected from each flask for PTH determination at the end of the 15-minuteincubation period. The same procedure was repeated changing the calcium concentration in the medium for each group of parathyroid slices, so that each parathyroid piece was exposed to both hypocalcaemic and hypercalcaemic media.
spectrophotometric reaction using the Kodak Ektachem DT-60 Analyzer (Eastman Kodak Co. Rochester NY). Serum and medium ionized calcium were measured with an ion selective electrode using the ICAjl Ionized Calcium Analyzer (Copenhagen, Denmark). Two PTH assays were employed: a double antibody immunoradiometric method (Nichols Institute, San Juan Capistrano, CA) was used to quantify intact (1-84) human PTH (i-PTH). Intra-assay and interassay coefficients of variation for this procedure average 5.7 and 18.3% respectively. The second RIA (Cambridge Medical Diagnostics, Billerica, Mass.) recognizes human PTH species that contain the 53-68 amino acid sequence (mid-molecule PTH (mPTH)). Coefficients of variation are 6.1 and 11.8% respectively. A double antibody sequential competitive RIA was employed to determine calcitonin levels (Diagnostic Products Corp. Los Angeles, CA). Intra and inter-assay coefficients for this assay are 6-8and 7.6%. Serum calcitriol(l,25dihydroxyvitamin D) quantitation was performed with a radioreceptor assay (Incstar Corp. Stillwater, MN). The assay includes preliminary extraction and purification of vitamin D metabolites from serum and quantitation using a non-equilibrium competitive protein binding assay. Intra and inter-assay coefficients for this assay are 5.4 and 19% respectively. Statistical methods
Data are summarized as mean & standard error of the means (SEM). The statistical significance of differences was determined by paired t-test. Exact P-values are reported. Results
Analytical methods
Serum levels of total calcium, phosphorus, chloride, alkaline phosphatase and creatinine were determined by a reflectance
Levels were determined before administration of mithramycin and at 12-hour intervals after the drug was started, as well as post-operatively (Table 1). Concomitant levels of calcio-
Table 1 Biochemical results
Total Ca (mmol/l) (2.17-2.54) Ionized Ca (mmol/lf (1.19-1.31) Phosphorus (mmol/l) (0.87- 1-49) Creatinine (pmol/l) (53.2- 141.9) Chloride (mmol/l) (97-108) Alk. phosph. (Ujl) (20-96) Reference range is given in parentheses.
Premithramycin adminis.
12h Postadminis.
24 h Postadminis.
72 h Postadminis.
3.54 2.20 0.87 150.7 107 146
3.44 2.05 0.87 141.9 112 150
3.44 1.98 0.94 159.6 I10 145
2.59 1.49 1.1 195.1 1 I4 151
PostOP.
levels 2.45 1.43 0.8 1
159.6 112 148
PTH secretory pattern
Clinical Endocrinology (1992) 37
567
Table 2 Serum level of calcium related hormones
Premithramycin adminis. Intact PTH (ng/l) (1 3-64) Mid-Mol. PTH (nmolil) (0.04-0.15) Calcitonin (ng/l) (0-33) Calcitriol @mol/l) (36-120)
105 2.96 39 14.4
12h
24 h
Postadminis.
Postadminis.
12 h Postadminis.
112 2.16
109 2.12 28 12.5
618 8.99 151 21.6
-
PostOP. levels
5 0.23
I1 11.8
~~
Reference range is given in parentheses
Table 3 Calcium dependent PTH secretion in uitro
m-PTH (nmolil)
m-PTH (nmolil) Hypocalcaemia (1.13 mmol/l) Hypercalcemia (1.68 mmol/l) P-value
39,4+ 5.9 21.5 2.3 0.03 1
0,3_+0.06 0.16 & 0.23 0.055*
28.9 3.4 14.3k 4 . 5 0.006 1
0.26 0.05 0.08+0.01 0.021
* Not statistically significant. Values are expressed as mean k SEM. trophic hormones is shown in Table 2. Mithramycin induced a significant decrease in calcium levels by the third day; however, a parallel sharp increment in PTH levels is evident: i-PTH increased from 105 to 618 ng/l, and m-PTH from 2.96 to 8.99 nmol/l. Calcitonin and calcitriol levels also increased significantly (39 to 151 ng/l and 14.4 to 21.6 pmolll, respectively). Creatinine and inorganic phosphorus levels increased while no significant alteration in the chloride or alkaline phosphatase concentrations was recorded. Surgery resulted in a sharp decline towards the normal range in both calcium and calcium-related hormones. In-vitro PTH secretion from parathyroid slices is depicted in Table 3. A significant decrease in i-PTH and m-PTH secretion by both groups of tissue slices is achieved with higher calcium concentration in the media ( P = 0.006; 0.031 for i-PTH and P=0.021; 0.055 for m-PTH; Fig. 1). Discussion
The differential diagnosis of acute, life-threatening hypercalcaemia includes acute primary hyperparathyroidism. The exacerbation of the relatively benign primary hyperparathyroidism is invariably accompanied by marked signs and symptoms of hypercalcaemia (Fitzpatrick & Bilezikian, 1987; Schweitzer et al., 1978). Usually, patients with APH reveal a history of mild, asymptomatic hypercalcaemia with
involvement of the skeleton and/or renal disease. These characteristic manifestations of primary hyperparathyroidism have evolved over the prolonged duration of hypercalcaemia. This implies that the acute state is a superimposed aggravation of the mild primary hyperparathyroidism. The sudden development of hypercalcaemic crisis with remarkable elevation in circulating PTH levels (20 times normal values) was attributed to autonomous PTH secretion from the diseased gland (Fitzpatrick & Bilezikian, 1987). PTH secretion generally shows a sigmoidal relation to ambient calcium concentrations, a curve characterized by four parameters: ( 1 ) suppressibility; (2) maximal release; (3) set-point (calcium level that induces 50% of maximal suppressibility); and (4)slope at set-point (Brown, 1983; Marx et al., 1986). In-vitro experiments defined an increased set-point for adenoma cells and abnormal calcium-regulated PTH release from uninvolved parathyroid glands of primary hyperparathyroidism patients (Brown et aE., 1981; Pocotte et nl., 1991). However, the set-point for adenoma cells is only slightly higher than that of normal parathyroid cells (Marx et al., 1986; Brown et al., 1979). Even a moderate elevation in the set-point combined with an increased mass of secreting parathyroid tissue could make an important contribution to the aetiology of hypercalcaemia in primary hyperparathyroidism (Marx et al., 1986). Most APH patients reveal a huge parathyroid adenoma, but such enlargement of the
568
Clinical Endocrinology (1992) 37
P. P. Schachter et a / .
P.0.02
Group I
Groilp 2
Fig. 1 a, Mid-region specific PTH and b, intact PTH secretion in v i m at calcium concentrations in the media of a, 1 . 1 3; 0 . 1.68
mmol/l. A significant suppression by high calcium levels is evident for both groups of parathyroid tissue slices.
tissue mass is a prolonged process. Therefore, it is reasonable to attribute the development of acute hypercalcaemia and high PTH levels to a sudden and significant increase in the set-point of the parathyroid adenoma cells, or to complete disruption of calcium-regulated PTH release rendering autonomous PTH secretion from the parathyroid adenoma. The in-vitro experiment with parathyroid slices from this APH patient indicates a definite response in PTH secretion to alterations in the calcium concentrations in the media. It is impossible to calculate a precise set-point using this method; however, a significant change in the output of intact-PTH is evident ( P = 0.03 1; P = 0.006). Both groups of tissue slices responded in an adequate manner to different calcium levels in the media, excluding the possibility of autonomous (noncalcium dependent) PTH secretion. Therefore, since autonomous PTH secretion is excluded and an abrupt change in the adenoma mass is very unlikely, the only acceptable explana-
tion for the appearance of APH in this patient is a sudden, major rise in set-point of the diseased gland. Severe hypercalcaemia due to a high set-point has also been reported in neonatal primary hyperparathyroidism (Marx et al., 1986). Moreover, despite a marked abnormality in PTH secretion in this patient, mithramycin induced a significant increase in PTH levels. This drug reduces serum calcium levels by inhibiting bone resorption even in the presence of high PTH or PTH-like protein, without any direct influence on parathyroid secretory functions (Yarbro ef al., 1966; Singer et al., 1970; Perlia et al., 1970). Following administration of mithramycin to our patient i-PTH levels increased sixfold (from 105 to 618 ng/l) while m-PTH levels increased only threefold (from 2.96 to 8.99 nmol/l). Calcium also regulates the intra-glandular degradation of intact-PTH, thereby controlling the relative proportion of bioactive hormone released from the gland (Habener et a/., 1975; Fuleihan et al., 1989; Mayer et al., 1979; Hanley & Ayer, 1986). The mechanism responsible for this additional regulation is unclear. However, decreasing the ambient calcium concentration triggers an increase in the relative proportion of i-PTH secreted by the parathyroid gland (Conlin et al., 1989;Mallette, 1991).Preservation of this adequate response in our APH patient is shown by the magnitude of i-PTH versus m-PTH secretion caused by declining serum calcium levels. This observation further emphasizes non-autonomous PTH secretion from the parathyroid adenoma of APH patients. The significant increase in serum intact-PTH is not reflected by the modest increase in calcitriol levels (from 14.4 to 21.6 pmol/l). This poor response to PTH can be attributed to initial vitamin D deficiency, even though, on admission, the level of 25-hydroxyvitamin D was 54.9 nmol/l (reference range 37.4-200), or down-regulation of PTH receptors (Abou-Samra et nl., 1989;Yamamoto et al., 1988; Ajlouni & Theil, 1975).Hepatic and renal toxicity caused by mithramycin might interfere with 1,25-dihydroxyvitamin D production since there was no change in 25-hydroxyvitarnin D level (60 nmol/l) after the significant decline in calcium levels and huge increase in PTH levels caused by mithramycin. The unexpected increase in serum calcitonin levels (from 39 to 151 ng/l) despite decreasing calcium concentrations might be explained by competitive binding of mithramycin to the same receptors on the osteoclast (Martin, 1980). Another possible reason is that mithramycin induced hepatic injury delaying calcitonin degradation, thereby increasing the circulating levels of this hormone. In conclusion, the data obtained from this interesting patient indicate that acute primary hyperparathyroidism is probably caused by a sudden change in the set-point of parathyroid adenoma cells and is not due to autonomous PTH secretion.
Clinical Endocrinology (1992) 37
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