Variable expressivity of familial medullary thyroid carcinoma (FMTC) due to a RET V804M (GTG-->ATG) mutation.

BACKGROUND: Multiple endocrine neoplasia type 2 (MEN 2) and familial medullary thyroid carcinoma (FMTC) are autosomal dominantly inherited cancer syndromes that predispose to C-cell hyperplasia and MTC. MEN 2A and FMTC are caused by mutations in the RET proto-oncogene. METHODS: We used a multiplex polymerase chain reaction-based assay to screen exons 10, 11, 13, and 14 of RET for mutations in 2 families with FMTC. We correlated mutation status with calcitonin and pathologic studies to determine genotype-phenotype correlations. RESULTS: We identified a mutation in codon 804 in exon 14 (GTG-->ATG; V804M) in both families. An 86-year-old person who was a gene carrier and other individuals over age 70 who were suspected by pedigree analysis to be gene carriers had no overt clinical evidence of MTC. Four of 21 patients who underwent a thyroidectomy also had papillary thyroid cancer. One individual in each family had metastatic MTC at age 30 and 32 years, and all 26 people having thyroidectomies had either MTC or C-cell hyperplasia, leading us to continue to recommend prophylactic thyroidectomy for all identified patients who were gene carriers. CONCLUSIONS: Because of active MTC in younger members of these families, including metastases, we have continued to advocate thyroid surgery in mutation-positive individuals. While DNA diagnosis of gene carriers and subsequent genetic counseling was relatively straightforward, the acceptance of surgical recommendations was more difficult for some individuals. These families demonstrate that the search for RET mutations should include exons 13, 14, 15, and 16 in patients whose studies in exons 10 and 11 are negative.

Effect of chronic vitamin D infusion upon in vivo glucose disposal.

We have previously demonstrated that parathyroid hormone (PTH) infusion decreases glucose disappearance rate (Kg) in vivo. Because in the rodent model used it was not possible to determine whether the PTH itself, the induced hypercalcemia, or both contributed to the glucose intolerance, we examined the effect of vitamin D infusion on insulin-mediated glucose disposal. In this model also hypercalcemia is induced but PTH levels are suppressed. Thirty male Sprague Dawley rats were continuously infused with vit D for 5 days using an Alzet miniosmotic pump, at a rate of 9.7 pmol/hour. Thirty controls were infused with the vehicle alone. On the 5th day, glucose 700 mg/kg and insulin 0.35 U/kg were given as a bolus through the left femoral vein and blood samples were obtained from the right femoral vein just prior to and at 2, 5, 10, and 20 minutes post-glucose/insulin infusion. At the end of 5 days, plasma calcium levels were higher in the vit D-infused rats than in the control rats (12.8 +/- 0.1 versus 10.0 +/- 0.1 mg/dL, P < 0.01) and rat PTH levels were suppressed (2.1 +/- 0.1 versus 62 +/- 12 pg/ml, P < 0. 01). Glucose levels were higher in the vit D animals only at 5 minutes following glucose/insulin bolus (375 +/- 7 versus 350 +/- 6 mg/dL, P < 0.01) but at no other time. There were no differences between serum insulin levels at any time. Unlike previous findings in PTH-infused rats, Kg (measured from 2 to 20 minutes following glucose/insulin bolus) was not different between groups (4.5 +/- 0.3 versus 4.7 +/- 0.2, P = 0.92.) A positive correlation between serum calcium and serum glucose was found only at 5 minutes (r = 0.55, P < 0.01) and only in the vit D animals. The areas under the glucose curves approached statistically significant differences (vit D-infused 5258 +/- 142 mg/dL/18 minutes versus control 4947 +/- 127, P = 0.06.) Analysis of serum glucose data by two-factor analysis of variance (ANOVA) suggests that the two groups differ slightly in glucose values (P = 0.03) but have parallel Kg. In order to define whether different effects of PTH (1-34) and vit D on intracellular calcium [Ca2+]i levels could partly explain the different effects of PTH and vit D infusion on glucose disposal, we investigated the effect of PTH and vit D infusions on basal and concanavalin A (con A)-stimulated changes in mononuclear [Ca+2]i levels. Following 5 days of PTH, vit D, or control infusion, peripheral mononuclear cells were incubated with 50 microgram/ml con A. Changes in [Ca+2]i over 5 minutes were calculated by flow cytometric measurement of the calcium sensitive fluo-3 AM dye. Despite achieving significant and comparable degrees of hypercalcemia in the PTH and vit D infused animals, there were no differences in basal or con A-stimulated [Ca+2]i levels from control. Consequently, we conclude that vit D-induced hypercalcemia associated with suppressed PTH levels has mild affects on glucose homeostasis but does not affect glucose disappearance rate in vivo (Kg) as does hypercalcemia induced by PTH infusion, and that neither chronic PTH infusion nor chronic vit D infusion are associated with long-standing changes in [Ca2+]i levels.

Parathyroid hormone decreases in vivo insulin effect on glucose utilization.

Hyperparathyroidism is associated with impaired glucose tolerance, and parathyroidectomy may improve carbohydrate homeostasis. It has been suggested that parathyroid hormone (PTH) suppresses insulin secretion but it is unclear whether it also interferes with the peripheral action of insulin. To evaluate in vivo effects of PTH on insulin-mediated glucose utilization, 15 male Sprague Dawley rats were continuously infused with rat PTH (1-34) using an Alzet miniosmotic pump at a rate of 0.03 nm/hour. Controls were infused with the vehicle alone. Following 5 days of PTH infusion, plasma calcium (Ca) levels were higher in the PTH-infused rats (12.3 +/- 0.2 versus 9.9 +/- 0.1 mg/dl, P < 0.01). On the 5th day, glucose (700 mg/kg) and insulin (0.175 U/kg) were given as a bolus infusion through the left femoral vein, blood samples were obtained from the right femoral vein, and plasma glucose and insulin were measured at basal (0 minutes) and at 2, 5, 10, and 20 minutes postinfusion. Basal, nonfasting glucose levels were higher (166 +/- 4 versus 155 +/- 4 mg/dL, P < 0.04) in the PTH-infused rats but their insulin levels were similar to those of controls (6.5 +/- 0.6 versus 5.6 +/- 0.5 ng/ml). Postinfusions and maximal (2 minutes) glucose and insulin levels were similar in both groups. However, although insulin levels were similar in both groups at all measured time points, glucose levels at 20 minutes were higher in the PTH-treated rats (205 +/- 13 versus 173 +/- 9; P < 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of estrogen and progesterone receptors in abnormal human parathyroid tissue.

Estrogen and/or progestin administration to postmenopausal women with primary hyperparathyroidism lowers serum calcium. We measured cytosolic estrogen receptors (ER) and progesterone receptors (PR) by classical hormone-receptor binding techniques in parathyroid tissue removed from 10 men and 20 women, and ER by immunocytochemistry in tissue from an additional one man and seven women in order to ascertain whether these agents might exert a direct effect upon tissue responsible for hyperparathyroidism. ER were negative (< 3.1 fmol bound estradiol/10 mg tissue) in all 8 adenomas and 4 of 5 secondary hyperplasias removed from men, and from women in 19 of 22 adenomas, 2 of 3 secondary hyperplasias, and 3 of 4 primary hyperplasias. PR were negative (< 10.1 fmol bound progesterone/10 mg tissue) in 7 of 8 adenomas and all 5 secondary hyperplasias removed from men, and from women in 20 of 22 adenomas, all 3 secondary hyperplasias, and all 4 primary hyperplasias. For immunocytochemical studies, quick-frozen specimens were analyzed with a monoclonal antibody (Abbott Laboratory) directed at nuclear ER. All eight samples--five adenoma and three primary hyperplasia--were negative. We conclude that abnormal human parathyroid tissues have nondetectable levels of ER and PR. It is unlikely that estrogen and progesterone exert a direct, ER, or PR-mediated effect upon parathyroid tissue.

Lithium increases tritiated thymidine uptake by abnormal human parathyroid tissue.

Patients receiving lithium for the management of manic depressive disorders appear to be at increased risk for development of hypercalcemia. Some (but not all) clinical studies and several in vitro studies suggest that lithium alters release of parathyroid hormone. Because hypercalcemia may result from an increase in the mass of parathyroid tissue, we studied the in vitro effect of lithium on tritiated thymidine (3H-TdR) incorporation as a measure of DNA synthesis. Dispersed cells from previously cryopreserved tissue from 18 patients undergoing surgery for single-gland hyperparathyroidism (adenoma) and five patients with secondary hyperparathyroidism were incubated with graded concentrations of lithium chloride and, after a 5-day incubation, were pulsed with 3H-TdR. Adenoma cells exposed to 2.0 mmol/L lithium (therapeutic level is approximately 0.8 to 2.0 mmol/L) demonstrated increased 3H-TdR incorporation compared with cells not exposed to lithium (average increase 56%). Secondary hyperplasia cells exhibited a similar but less striking response. There was no lithium-induced 3H-TdR incorporation in four preparations with normal bovine parathyroid cells. We conclude that lithium in therapeutic doses increases 3H-TdR incorporation into adenoma cells, may serve as a mitogen for human parathyroid adenoma, and could promote or accelerate hyperparathyroidism.

Characterization of a simplified method of cryopreserving human parathyroid tissue.

Cryopreservation of human parathyroid tissue plays an important role in managing difficult parathyroid disease. It also can permit investigators to conduct experiments without dependence on the operating room schedule. Availability of cryopreservation has been limited by the perceived need for expensive, complex equipment. We adapted a simple method of freezing cell suspensions to freezing human parathyroid tissue. Vials containing human parathyroid in culture media, dimethylsulfoxide, and patient serum were placed in a plastic rack in a metal pan containing prechilled (4 degrees C) ethanol and placed in a -70 degrees C freezer. We compared viability (trypan blue dye exclusion by collagenase dispersed cells) of tissue frozen in this manner to that of tissue frozen in a programmable liquid nitrogen freezer at 1 degrees C per minute, a cooling rate recommended for human parathyroid tissue. The viability of 30 patients' samples cooled in liquid nitrogen (average length of storage 5 months) was 74% +/- 15% and that of 64 patients' samples cooled in ethanol (average length of storage 26 months) was 71% +/- 15%. Viability of 19 samples of fresh tissue was 79% +/- 10%. Neither method had a statistically significant correlation between length of storage and viability. Successful cryopreservation with simplified technology may expand the availability of parathyroid tissue to meet both clinical and investigative requirements.

Hyperparathyroidism and cellular mechanisms of gastric acid secretion.

Patients with hyperparathyroidism appear to be a particular risk for peptic ulcer disease. To test the hypothesis that hypercalcemia or parathyroid hormone plays a role in promoting ulcer disease, we studied the effect of varying concentrations of extracellular calcium on acid secretion using in vitro isolated rabbit gastric glands. Acid secretion was assessed by the accumulation of carbon 14-labeled aminopyrine (14C-AP). Glands were incubated with varying calcium concentrations in the unstimulated state and with histamine or carbachol (10(-7) to 10(-4) mol/L) in 1 or 2 mmol/L calcium medium. The effect of parathyroid hormone was also examined under identical conditions. Compared to 1 mmol/L standard calcium medium, unstimulated 14C-AP accumulation was significantly inhibited (p less than 0.05) at both lower (0.33 mmol/L) and higher (2 and 2.5 mmol/L) calcium concentrations. Accumulation of 14C-AP in response to histamine stimulation was unaffected by alteration of extracellular calcium (p greater than 0.2). Carbachol-stimulated 14C-AP accumulation was significantly augmented (p less than 0.01) by an increase in calcium concentration from 1 to 2 mmol/L. The addition of parathyroid hormone (10(-7) to 10(-4) mmol/L) alone or in combination with carbachol or histamine (10(-6) mmol/L) incubation did not alter 14C-AP accumulation. These data suggest that elevations in extracellular calcium play an active role in the potentiation of cholinergic-mediated gastric gland acid secretion and may thereby play a role in hyperparathyroid-related ulcer disease.

In vitro assessment of parathyroid immunogenicity: the effect of cryopreservation.

Post-parathyroidectomy hypoparathyroidism, although fortunately uncommon, is a disorder of major inconvenience and potential morbidity. Attempts at modifying parathyroid tissue to facilitate allotransplantation without host immunosuppression are warranted. Cryopreservation has been reported to improve survival of canine parathyroid allografts. We employed a modification of the mixed lymphocyte culture to study the effect in vitro of cryopreservation on human parathyroid tissue. Dispersed parathyroid cells from fresh and previously cryopreserved tissue from 10 patients were incubated with unrelated mononuclear cells for 6 days, and incorporation of tritiated thymidine was measured after a 1-day pulse. Studies with irradiated mononuclear cells and parathyroid cells confirmed the model as a one-way test in which mononuclear cells respond to parathyroid cells but not vice versa. An antigenicity index was computed to express mononuclear cell tritiated thymidine incorporation for similar numbers of viable parathyroid cells. Although absolute values of the antigenicity index varied from patient to patient, there were no consistent differences in the antigenicity index of patients' fresh compared with cryopreserved tissue. In an attempt to identify the cells responsible for immunogenicity, we incubated cytocentrifuged parathyroid cell suspensions with antiserum directed at leukocyte common antigen, a marker of lymphoid tissue. Cell suspensions of parathyroid tissue demonstrated leukocyte common antigen-positive cells (median, 2.7% positive; range, 0% to 16%). There were no consistent differences in the number of leukocyte common antigen-positive cells in fresh compared with cryopreserved tissue, and the number of leukocyte common antigen-positive cells did not correlate with the antigenicity index.

Thyroid and parathyroid surgery performed with patient under regional anesthesia.

Thyroid and parathyroid surgery is usually performed with the patient under general anesthesia; however, for selected patients regional anesthesia may be preferable. Between September 1977 and March 1986 regional anesthesia was used successfully as the sole anesthetic technique in 17 patients who underwent thyroid surgery and two patients who underwent parathyroid surgery. Procedures included two total thyroidectomies, 14 lobectomies or lobectomies with isthmusectomies, and one isthmusectomy. These 17 operations represent approximately 5% of the thyroid operations performed by the senior surgeon over the corresponding time. One patient underwent combined completion thyroidectomy and parathyroidectomy, and another patient underwent successful parathyroidectomy under regional anesthesia. In two additional patients, procedures could not be completed under regional anesthesia alone. In one of these two patients regional anesthesia appeared to effect a transient recurrent nerve paralysis. The indications for use of regional anesthesia have been primarily patient preference and associated cardiac or pulmonary disease. We now consider as contraindications to regional anesthesia patient apprehension about the technique, deafness, high spinal cord injury, recurrent laryngeal or phrenic nerve palsy, and allergy to local anesthesia. During this period, from 1977 to 1986, our administration of regional anesthesia has evolved from bilateral deep and superficial cervical plexus blocks to bilateral superficial blocks alone using bupivacaine with epinephrine, 1:200,000.

The effect of phorbol ester on in vitro release of parathyroid hormone from abnormal human parathyroid cells.

Intracellular events that regulate parathyroid hormone (PTH) release are not well understood. Cyclic AMP (cAMP) and cAMP-dependent protein kinases play a role in the regulation of release due to several agonists, but these factors do not fully explain PTH release that is mediated by extracellular ionized calcium (Ca++). A calcium-phospholipid-dependent (non-cAMP-dependent) protein kinase can be activated by 12-O-tetradecanoylphorbol-13-acetate (TPA). To determine whether this protein kinase regulates PTH release, we examined the effect of TPA on PTH release from human parathyroid tissue. Cell suspensions of abnormal parathyroid tissue removed at surgery were prepared by enzymatic dispersion and incubated for several hours with and without 10(-7) mol/L TPA at low and high calcium levels. In ten preparations in the absence of TPA, increasing Ca++ from 0.25 to 2.5 mmol/L reduced PTH release to an average of 39% of maximal release (range, 11% to 67%). The effect on TPA on Ca++-regulated PTH release appeared biphasic. At low (0.25 mmol/L) Ca++ level, TPA suppressed PTH release to an average of 78% of maximal release without TPA (95% confidence interval, 67% to 88%) (p less than 0.01 compared to cells incubated without TPA). At high (2.25 mmol/L) Ca++ level, TPA augmented PTH release from an average of 39% of maximal release without TPA to 62% of maximal release without TPA (95% confidence level, 48% to 78%), an average augmentation of 22% (95% confidence level, 9% to 36%) (p less than 0.01 compared with cells incubated without TPA). TPA appeared to make PTH release independent of Ca++. Both inhibitory and stimulatory effects were dose dependent. Incubations with TPA demonstrated no toxicity as judged by trypan blue dye exclusion, linearity of PTH release, and cellular incorporation of tritiated leucine. TPA had no effect on the radioimmunoassay for PTH. We conclude that a calcium/phospholipid-dependent, non-cAMP-dependent protein kinase may play a role in mediating Ca++-regulated PTH release from abnormal human parathyroid cells. Its site of action and integration with other regulatory pathways remain to be determined.

Can the volume of abnormal parathyroid tissue be predicted by preoperative biochemical measurement?

A strong correlation between the biochemical manifestations of hyperparathyroidism and the volume of abnormal parathyroid tissue could be used to guide the extent of surgical exploration and parathyroid gland resection (i.e., finding a small "adenoma" in a patient with marked hypercalcemia would dictate further exploration). We examined this relationship in patients for whom data were collected prospectively (n = 14) and retrospectively (n = 27). We considered only patients cured after excision of a single gland and glands for which a three-dimensional description or volume of water displacement was available. To exclude artifactually elevated serum concentrations of parathyroid hormone (PTH), PTH values were used only from patients with levels of serum creatinine less than 2 mg/dl. To accommodate different assays, highest preoperative PTH, ionized calcium, and alkaline phosphatase (AP) values were expressed as percent above upper normal limit. There was excellent agreement (r = 0.93, p less than 0.05) between measured and calculated gland volume. In the prospective study (but not in the retrospective study) there was a significant (p less than 0.05) correlation (r = 0.61) between gland volume and highest preoperative total calcium value; however, there was considerable variation in gland size in patients with similar calcium levels. In neither study was there a significant correlation between gland volume and any of the following: calcium, ionized calcium, midregional PTH, carboxyterminal PTH, or intact PTH, alkaline phosphatase, and urine cyclic adenosine monophosphate (AMP). In the prospective study there was a tendency for urine cyclic AMP, ionized calcium, and AP to increase with increasing gland volume (r = 0.42, 0.45, and 0.51, respectively). Preoperative measurements of calcium, PTH, urine cyclic AMP, and AP are too inconsistent to rely on for determining the extent of parathyroid gland resection.

Effect of bilirubin on the spectrophotometric and radionuclide assay for serum angiotensin-converting enzyme.

The effect of bilirubin on serum angiotensin-converting enzyme (ACE) activity was studied with spectrophotometric and radionuclide assays. In the spectrophotometric assay addition of bilirubin to normal serum from dog, mouse, and human produced a dose-related inhibition of ACE activity. A 50% decrease in human ACE activity was produced by the addition of approximately 250 mg/L in vitro. Serum from icteric patients with elevated bilirubin was also associated with a reduction in ACE activity in the spectrophotometric assay. A 50% decrease in ACE activity in these samples was associated with a serum bilirubin of approximately 220 mg/L. In the radionuclide assay, however, addition of bilirubin to normal human serum failed to reduce measured ACE activity. The use of a radionuclide assay for serum ACE in clinical samples offers the advantage of less interference from serum bilirubin.

Morphologic and functional studies of a rat hypercalcemia-associated testicular tumor maintained in cell culture.

The Rice H500 tumor is a transplantable nonmetastasizing testicular tumor of Fischer rats associated with hypercalcemia and increased urine cyclic adenosine monophosphate (AMP) excretion, features similar to those of the clinical syndrome of humoral hypercalcemia of malignancy. Tumor cells can be maintained in tissue culture; one million cells grown in culture reinoculated in Fischer rats reproduce the syndrome of tumor growth and lethal hypercalcemia. Infusion of concentrated, serum-free cell culture supernatant into parathyroidectomized rats produced an increase in urine cyclic AMP similar to that produced by an infusion of bovine parathyroid hormone. Light and electron microscopic appearance of the H500 tumor in vivo and in vitro is similar to previous descriptions of a hypercalcemia-associated rat testicular tumor believed to be of Leydig cell origin. Ultrastructural characteristics of microvilli, intracellular glandlike lumina, and cell-cell attachments, however, suggest an epithelial origin. Absence of smooth endoplasmic reticulum typical of steroid-secreting Leydig cells suggest these cells are not actively involved in steroid synthesis and secretion. The ultrastructure of this tumor is sufficiently different from that of normal Leydig cells that the cell of origin is unclear. Nonetheless, this tumor provides a useful model of hormonally mediated tumor-associated hypercalcemia.

Autotransplantation of adrenal cortical tissue: a rodent model.

Autotransplantation of human adrenal tissue has been attempted often, but results have been difficult to evaluate and success has been infrequent. Factors that may affect success include: volume of transplanted tissue, recipient site, inclusion of cortical capsule with the autograft, systemic or local growth factors, and the timing of evaluation. We have evaluated a model of autotransplantation in rats that will permit examination of these factors. Male Sprague-Dawley rats were assigned to (1) bilateral adrenalectomy (ADX), (2) bilateral adrenalectomy with immediate autotransplantation to a flank muscle pocket of one third of a single adrenal gland with its capsule attached (TX), or (3) sham operation. Animals were provided with 0.9% saline solution ad lib. At 2, 4, 6, and 12 weeks after surgery animals were stressed by brief exposure to ether and 15 minutes later had blood collected for determination of corticosterone concentration (C). ADX animals consistently weighed less than either TX or sham-operated animals; weights of sham-operated and TX animals were similar. Sham-operated animals uniformly had C levels higher than ADX or TX animals. At 2 and 4 weeks after surgery, C was similar in ADX and TX; but at 6 and 12 weeks, TX animals had higher values than had ADX animals. With this model, graft function can be demonstrated at 2 weeks by comparing body weight and at 6 weeks by comparing postether levels of C in TX animals to ADX animals. ADX animals can be maintained without steroid replacement on a regular diet with 0.9% saline solution ad lib. This model will permit examination of technical and physiologic influences on transplant success with both fresh and cryopreserved tissue and may lend itself to radionuclide or nuclear magnetic resonance assessment of graft function.

The role of the pathologist in the surgical treatment of hyperparathyroidism.

Appropriately extensive surgical treatment of hyperparathyroidism depends upon accurate assessment of the extent of disease. We have believed that such assessement is the responsibility of the surgeon because at random biopsy with light microscopy the pathologist may not be able to differentiate adenoma from hyperplasia or even normal from abnormal glands. To test this hypothesis, three pathologists reviewed 50 unlabelled slides of parathyroid tissue and attempted to correlate them with clinical diagnoses which were based upon widely accepted criteria. They were asked to identify each slide as adenoma or hyperplasia, or both, or normal using whatever criteria they wished. A specific diagnosis of adenoma was correct in 35 and 83 per cent of interpretations and of hyperplasia in 38 and 60 per cent of interpretations. The less specific diagnosis of adenoma or hyperplasia (that is, abnormal tissue) was correct in 78 to 100 per cent. A diagnosis of normal was correct in 71 to 78 per cent. Adenoma was most likely confused with hyperplasia; hyperplasia was equally mistaken for adenoma or normal. We conclude that with random, subtotal specimens taken at biopsy (simulating intraoperative conditions) differentiation of adenoma from hyperplasia of the parathyroid gland is poor. Differentiation of normal from abnormal parathyroid tissue also is unreliable. Because the consequences of misdiagnosis are severe, pathologists should not be asked to make specific diagnoses intraoperatively but only to distinguish the parathyroid tissue from the nonparathyroid tissue.

Angiogenesis of human parathyroid tissue.

The etiology of parathyroid disease remains obscure. The terms "adenoma" and "hyperplasia" imply neoplastic and nonneoplastic processes, respectively. Angiogenesis, the ability of transplanted tissue to evoke new blood vessel formation by the host, is a property generally attributed to neoplastic tissue. We tested human parathyroid tissues from six patients with clinical diagnoses of adenoma and four patients with hyperplasia for their ability to evoke angiogenesis in an established in vivo model. Six to 12 small pieces of parathyroid tissue from each patient were placed on the iris of New Zealand white rabbits, three pieces per eye. Macroscopic assessment of angiogenesis was performed on days 2 through 5 after implantation by a noninformed observer. Microscopic assessment of implant viability was made after the animal was killed on day 5. Some implants were lost to infection, necrosis, and failure to attach to the iris. Both adenomatous and hyperplastic human parathyroid tissue demonstrated angiogenesis, and with approximately the same frequency. However, there was considerable variation in response of tissue from the same patient and between patients with the same clinical diagnosis. The ability of human parathyroid tissue to evoke angiogenesis may help explain its relative ease of transplantation.

Implantable infusion pump in the therapy of canine adrenal insufficiency.

To test the feasibility of a commercially available infusion pump in the treatment of human adrenal insufficiency, we employed the pumps initially in dogs. Two dogs underwent total adrenalectomy and placement of an Infusaid Model 400 infusion pump. These pumps have a drug reservoir of 45 ml and are refilled by percutaneous injection approximately every 2 weeks. Hydrocortisone phosphate was used as replacement glucocorticoid. Serum cortisol values correlated with the amount of drug delivered, and daily administration of 10 to 50 mg/day maintained the plasma cortisol level within the normal range (1 to 6 micrograms/dl). Mineralocorticoid replacement initially consisted of fludrocortisone by mouth and subsequently aldosterone via infusion pump. Serum electrolytes fluctuated with the amount of drug delivered, and doses of approximately 100 to 400 micrograms administered orally per day and 70 to 100 micrograms/day per pump maintained serum electrolytes within the normal range. Dependence upon and effectiveness of pump administration of steroids for long-term survival was demonstrated by: (1) ACTH-stimulation tests after operation and approximately 1 year later, (2) autopsy studies demonstrating total adrenalectomy, (3) repeatedly undetectable plasma cortisol levels during periods of no glucocorticoid supplementation, and (4) repeated hyperkalemia and hyponatremia during periods of no mineralocorticoid supplementation. This study demonstrates that glucocorticoid and mineralocorticoid preparations can remain bioactive for 2 to 3 weeks at 37 degrees C and can be delivered in amounts adequate to sustain a totally adrenalectomized dog via a commercially available infusion pump. In the adrenalectomized dog hydrocortisone sufficient to maintain plasma cortisol values in the normal range does not provide adequate mineralocorticoid replacement.

Intraoperative urinary cyclic adenosine monophosphate as a guide to successful reoperative parathyroidectomy.

Sixty patients with persistent or recurrent primary hyperparathyroidism underwent reexploration during which urinary cyclic adenosine monophosphate (UcAMP) levels were determined at half-hour intervals by radioimmunoassay. Retrospective analysis of the data allowed us to develop UcAMP criteria for surgical success. Following removal of parathyroid tissue, if an individual UcAMP level dropped 50% from the median baseline level, or if elevated levels dropped to less than 4.0 nmol/dl glomerular filtrate, surgery was predicted to be successful. Eight unsuccessful procedures in seven patients produced no decline in UcAMP, and the intraoperative results accurately predicted surgical failure. Fifty-three patients underwent successful procedures and in every case UcAMP fell. Ninety-eight per cent of these successful procedures were predicted by our criteria. Levels of UcAMP fell 1.5 +/- 0.5 hours (means +/- SD) following abnormal parathyroidectomy. In 19 of 36 successful cases diagnosed before surgery as adenoma, the operative procedure was terminated before a significant drop in UcAMP. In 16 of 17 successful cases diagnosed before surgery as hyperplasia or uncertain histology, UcAMP fell during the operation. Intraoperative determination of UcAMP is helpful in reoperative parathyroid surgery. The criteria established allow intraoperative prediction of success with remarkable accuracy. Urinary cyclic AMP is especially helpful in reoperation for multigland disease; when enough pathologic tissue has been removed, the criteria will be met and the procedure may be terminated with confidence.

Humoral hypercalcemia caused by a rat Leydig-cell tumor is associated with suppressed parathyroid hormone secretion and increased urinary cAMP excretion.

We studied the effect of a transplantable Leydig-cell tumor (Rice H-500) on serum calcium, parathyroid hormone (PTH), and urinary cAMP in intact Fischer-344 rats. The tumor caused rapid and severe hypercalcemia (control = 10.5 +/- 0.1 mg/dl [mean +/- S.E.] vs. 14.6 +/- 0.9 at day 12 post tumor inoculation) without evidence of metastasis. Progressive renal impairment and death generally occurred within 15 days of tumor inoculation. Serum PTH declined from control values before hypercalcemia occurred and was significantly reduced in tumor-bearing hypercalcemic rats (mean = 60 +/- 8% of control values). Urinary cAMP excretion was increased in tumor-bearing rats (mean at day 12 post inoculation = 12.2 +/- 1.4 nmol/dl creatinine clearance vs. control = 6.2 +/- 0.2) and correlated positively with serum calcium. The Rice H-500 Leydig-cell tumor appears to secrete a humoral factor capable of causing hypercalcemia. This factor may also increase urinary cAMP excretion in a manner analogous to PTH, but it is not detected by PTH radioimmunoassay.