0021-972X/90/7006-1744$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright© 1990 by The Endocrine Society
Vol. 70, No. 6 Printed in U.S.A.
Comments
Spontaneously Occurring Antibodies to Parathyroid Hormone* MARGARET WILKINSON, AIDAN McELDUFF, JEREMY WILSON, PAUL HABER, ANTHONY FREEMAN, MALCOLM ROBERTSON, AND PHILLIP MATHEWS Department of Endocrinology, Royal North Shore Hospital (M. W., A.M.), St. Leonards, New South Wales; and the Departments of Gastroenterology, Cardiology, Nephrology, and Surgery, Prince of Wales Hospital, Sydney, New South Wales, Australia
ABSTRACT. A 76-yr-old female with acute pancreatitis and a normal/borderline elevated serum calcium level was found to have an elevated immunoreactive circulating PTH concentration using a C-terminal assay. This high PTH concentration misled the attending physicians and resulted, in retrospect, in an unnecessary neck exploration. When the patient's serum was examined it was found to contain a binding component that bound both C-terminal and PTH-(l-84). This binding compo-
T
HE OCCURRENCE of antibodies to circulating hormones is well described in the endocrine literature. These antibodies may produce various abnormalities or problems. They almost universally lead to errors in RIA estimations of the hormone concentration (1). They may cause hormone resistance by binding to the hormone and inactivating it (2). Alternatively, they may cause apparent hormone sensitivity by binding the hormone, delaying its clearance, and then releasing the hormone at inappropriate times, as has been described for spontaneously occurring antiinsulin antibodies (3). Most commonly, circulating antihormone antibodies are produced by the administration of exogenous hormone, frequently from a different species (2, 4), but the spontaneous development of such antibodies has been described (3, 5). Two well documented cases of antibodies directed against PTH have been associated with PTH resistance and followed treatment with PTH or PTH fragments (6, 7). Spontaneously occurring antiparathyroid antibodies have not previously been reported. We report their occurrence in a 76-yr-old woman. Case Report A 76-yr-old Caucasian female presented to the Emergency Room with several painful red nodules which had appeared on Received October 2, 1989. Address all correspondence and requests for reprints to: Dr. A. McElduff, Department of Endocrinology, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia.
nent was not retained on a Sep-Pak column and was precipitated by antiserum directed against human immunoglobulin M. The presence of circulating anti-PTH immunoglobulin M explains the apparently high PTH concentrations measured by RIA. The antibodies occurred spontaneously. To the best of our knowledge, this phenomenon has not previously been described. (J Clin Endocrinol Metab 70: 1744-1749, 1990)
her shins over the preceding days. Four months before admission a right mastectomy had been performed for infiltrating ductal carcinoma of the breast. Postoperatively, adjuvant tamoxifen (10 mg twice daily) had been commenced, although there was no obvious residual disease. On examination she appeared pale. Several raised erythematous tender mobile lesions were scattered over the anterior shins, ankles, and buttocks. They measured 1-2 cm and were not ulcerated. There was no lymphadenopathy. The temperature was 37.4 C, the blood pressure was 170/70 mm Hg, and pulse rate was 92 beats/min. The rhythm was totally irregular. An electrocardiograph confirmed atrial fibrillation. A grade 2/ 6 aortic ejection murmur was audible over the aortic outflow tract. There were no abdominal masses or tenderness. The remaining examination was normal. Histological examination of the lesions revealed enzymatic fat necrosis. Serum amylase was noted to be elevated at 2,730 (normal, 30-100 U/L) and was of pancreatic origin. Lipase was 345 IU/L (normal, 0-200), and urinary amylase was raised at 10,840 U/L. The hemoglobin was 8.3 g/dL, with a normal blood film. Serum ferritin, folate, and B12 concentrations were normal. An electrophoresis protein gel and immunoelectrophoresis protein gel showed no evidence of a monoclonal spike, but demonstrated a broad increase in 7- and a2-globulins. The anemia was corrected by transfusion, and the hemoglobin level remained normal. There was mild acute renal failure, with a raised serum urea of 12.4 mmol/L (normal, 2.8-7.6) and serum creatinine of 160 ^mol/L (normal, 60-110). Serum alkaline phosphatase was 147 IU/L (normal, 25-110), and other liver function tests were normal. The serum creatinine level fell to 120 /imol/L with
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COMMENTS rehydration. It was never higher than the initial value. Serum calcium was 2.46 mmol/L (normal, 2.05-2.55), and inorganic phosphate was 0.9 mmol/L (normal, 0.8-1.5). Serum albumin was 42 g/L (normal, 33-55). Serum triglycerides were 2.2 mmol/L (normal, 8.0, and 3.9 ng/ mL; normal, 0-0.5). A 24-h urine collection demonstrated calcium excretion of 0.32 mmol and normal inorganic phosphorus excretion of 14 mmol/L at a serum phosphate level of 1 mmol/L. Urinary cAMP excretion was 700 nmol/mmol creatinine (normal, 200890). The hospital course was protracted and complicated by two episodes of acute painful pancreatitis and advancing enzymatic fat necrosis. The skin lesions became ulcerated, and the patient's mobility was limited by acute synovitis involving several joints. Persistent pain in the left wrist was attributed to medullary fat necrosis of the ulna associated with a lytic lesion on plain x-ray and a "hot spot" on radionuclide bone scan. The serum PTH level remained raised in association with borderline hypercalcemia. In view of the adverse clinical course, neck exploration was undertaken. Two parathyroid glands of normal histology were removed. Two additional parathyroid glands were identified and appeared to be of normal size; no biopsies were taken of these two remaining glands. Postoperative serum PTH and calcium concentrations were unchanged. The patient's condition eventually settled on supportive measures only. After a 3-month period of hospitalization she was discharged home. She remains well with normal serum calcium (2.15 mmol/L; albumin, 36 g/L) and serum amylase, and no recurrence of the skin lesions 6 months later. The PTH concentration measured in the same C-terminal RIA remains elevated.
Materials and Methods Materials Human PTH-(l-34) [hPTH-(l-34)], [Asn76]hPTH-(l-84), and [Nle818,Tyr34]hPTH-(l-34) were obtained from Peninsula
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Laboratories, Inc. (Belmont, CA). Bovine PTH-(1-84) [bPTH(1-84)] and [Tyr52]hPTH-(52-84) were obtained from Bachem Fine Chemicals (Torrance, CA). Sodium iodine for protein iodination was obtained from Amersham Radiopharmacuticals (Amersham, United Kingdom). Human immunoglobulin G (IgG), rabbit antihuman IgG, and rabbit antihuman IgA were obtained from Bioproducts Ltd. (Brussels, Belgium). Human IgM was obtained from Organon Technika (Turnhout, Belgium). Human IgA was obtained from Sigma Chemical Co. (St. Louis, MO). Goat antihuman IgM was obtained from Cambridge Medical Technologies (Cambridge, MA). Antihuman IgG (7-chain) for binding to Sepharose was obtained from Silenis Laboratories (Hawthorn, Victoria, Australia). CNBractivated Sepharose was obtained from Pharmacia Fine Chemicals (Uppsala, Sweden). Sac-Cel cellulose-coated antibody was obtained from INS (Washington, England). Methods RIA. The PTH-(l-84) RIA was performed as previously described (8), with modifications reported previously (9). Separation of bound from free [125I]bPTH-(l-84) was by Sac-Cel goat antiguinea pig-coated cellulose suspension, as per the manufacturer's recommendations. The PTH-(1-34) RIA was performed as previously described (9). Both assays are briefly outlined below. Charcoal separation. Charcoal separation of bound from free [125I]PTH was performed by the addition of 1 mL cold 1% activated Charcoal (Merck, Darmstadt, West Germany) in 0.02 M Veronal buffer, pH 8.6, and the tubes were centrifuged for 45 min at 4 C. The supernatant (free) was decanted into a separate tube. Both bound and free PTH were counted, and bound/free ratios (B/F ratio) were calculated. We have previously shown (9), using sera that do not contain PTH-binding components, that these two separation methods give similar results. Radiolabeled PTH and PTH fragments: binding to serum. [125I] bPTH-(l-84) and [125I]-[Nle8|18,Tyr34]hPTH-(l-34) were iodinated and purified as previously described (8, 9). [125I]-[Tyr52] hPTH-(53-84) was iodinated and purified by a method identical to that used for [125I]bPTH-(l-84). Two hundred microliters of 0.05 M Veronal buffer, pH 8.6, containing approximately 5000 cpm [125I]bPTH-(l-84), [125I][Nle818,Tyr34]hPTH-(l-34), or [125I]-[Tyr62]hPTH-(53-84) were added to 100 ^L of the patient's serum or serum from a control subject in 12 x 75-mm polystyrene tubes. The tubes were incubated for 20 h at 4 C. Separation of bound from free tracer was performed with charcoal. These conditions were identical to those of the corresponding RIA, except that 200 nL buffer were added to replace the 200 ^1 antibody-containing buffer, separation was with charcoal rather than second antibody, and no preincubation with radiolabel was performed. Serial dilutions of the patient's serum were made in assay buffer to give microliter equivalents of 100, 20, 10, or 5 in a total volume of 100 fiL. Binding studies with [125I]bPTH-(l84) and [125I]-[Tyr52]hPTH-(53-84) were then performed as described above. At the end of the incubation, 100 yiL appropriately diluted control serum were added to all tubes to bring
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COMMENTS
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the final serum concentration in each tube to 100 /xL undiluted serum. Displacement of radiolabeled PTH fragments by cold PTH frag-
ments. One hundred microliters of patient or control serum were incubated with 200 nh (-4000 cpm) [125I]bPTH-(l-84) or [125I]-[Tyr52]hPTH-(53-84) and 100 nL hPTH-(l-34), [Tyr52] hPTH-(53-84), or bPTH-(l-84) diluted in surgical hypoparathyroid serum to reach the concentration (final) shown. Sep-Pak separation. Patient serum or serum from a control subject in volumes of 2 mL (except the experiment reported in Table 2 where the volume used was 1 mL) was applied to 6-mL SPE Ci8 cartridges (Baker Chemical Co., Phillipsburg, NJ) under vacuum. The columns were washed with 2 mL phosphate buffer, followed by 20 mL 0.1% trifluoroacetic acid (TFA). The columns were then eluted with 3 mL 80% acetonitrile in 0.1% trifluoroacetic acid high pressure liquid chromatography (Spec-
trograde). The flow-through and phosphate buffer fractions were pooled. The pooled flow-through and the eluant fractions were lyophylized and reconstituted in 0.05% M Veronal buffer, pH 8.6, each to the volume of the original sample size. Under identical conditions these columns were able to extract 99% of IgM, IgG, and IgA from serum as measured directly on serum and flow-through by rate nephilometry on a Beckman assay nephilometer (Beckman Instruments, Gladesville, New South Wales, Australia). Recovery of binding activity from the columns, as assessed by the B/F ratio per 100 nh serum or serum equivalent, was approximately 90-95%. Radiolabeled PTH and PTH fragment binding to Sep-Pak C18 flow-through and eluant fractions. These experiments were performed as described previously, except that in some experiments separation of the bound from free tracer was carried out by the addition of 100 nL rabbit antihuman IgG, 50 /JL goat antihuman IgM or 50 nL rabbit antihuman IgA together with 100 nL (1.0 mg) of the appropriate Ig added as carrier. After a 2-h incubation at 4 C, 1.0 mL 0.05 M Veronal buffer, pH 8.6, was added to all tubes, and the tubes were centrifuged at 2230 X g for 45 min at 4 C. The supernatants were decanted into separate tubes, and both the precipitate (bound) and supernatant (free) tubes were counted. IgG column chromatography. Antihuman IgG (7-chains) was coupled to CNBr-activated Sepharose 4B according to the manufacturer's instructions. One half milliliter of the patient's serum was applied to 0.5 X 9-cm affinity chromatography column packed with the antihuman IgG-coupled Sepharose. The column was washed with 20 mL phosphate-buffered saline and eluted with 0.2 M acetic acid in 1.0-mL fractions, which were rapidly neutralized with ammonia solution. The fractions were then lyophylized and reconstituted in 0.02 M Veronal buffer, pH 8.6. The fractions were processed as described for the serum and Sep-Pak fractions.
Results RIA results The C-terminal PTH assay gave consistently elevated estimations for the PTH concentration. Serial dilutions
JCE & M • 1990 Vol70«No6
(1, 0.5, and 0.25) in this assay produced values of more than 8.0, 7, and 7.2 ng/mL, respectively. This was interpreted as parallel to the standard curve. This was the only PTH assay performed preoperatively. Postoperatively, serum was assayed in our routine PTH-(1-84) assay (8, 9), which preferentially sees the complete 1-84 molecule (9). The PTH concentration estimated in this assay was also elevated. However, the nonspecific binding or blank tube was lower than normal (see below). Nonspecific binding is defined as binding occurring in the absence of the anti-PTH antibody routinely added in the RIA. The low nonspecific binding caused the value to be rejected. Further investigations of the sample, as outlined below, were undertaken. Parenthetically, serial dilutions to 0.25 read at values above the highest standard. When the patient's serum was assayed in a PTH-(134) assay (9), there was no difference between the nonspecific binding of the patient's serum and that of serum from other patients regardless of whether the separation of bound and free PTH was performed with second antibody or charcoal. The hPTH-(l-34) result was 30 pg/mL (reference range, up to 130 pg/mL; limit of detection, 16 pg/mL). This value was too low for reliable serial dilution studies. Nonspecific binding [125I]bPTH-(l-84) was incubated with the patient's serum. When bound and free PTH were separated with our antiguinea pig second antibody (8, 9), nonspecific binding was low, with a B/F ratio of 0.015 compared with a control B/F of 0.032-0.050 (n = 30). Low nonspecific binding suggests a binding component in the patient's serum not precipitated by the second antibody. When separated with charcoal, the B/F ratio was high (0.967) in the patient's serum compared to that (0.067) in control samples (Fig. 1). This is again consistent with a binding agent in the patient's serum. Studies of radiolabel binding to serum and serum fractions To determine the presence and specificity of the possible binding agent from the patient's serum, radiolabeled bPTH-(l-84), [Nle8-18,Tyr34]hPTH-(l-34) and [Tyr52] hPTH-(53-84) were incubated with the serum. As seen in Fig. 1, serum from the patient bound both radiolabeled bPTH-(l-84) and [Tyr52]hPTH-(53-84) to a significantly greater degree than the control serum. [125I][Nle8-18,Tyr34]hPTH-(l-34) had equally low binding in both patient and control serum. As shown in Fig. 2, increasing concentrations of the patient's serum resulted in increasing binding of both and [125I]-[Tyr52] hPTH-(53-84). [125I]bPTH-(l-84) Binding to serum from a control subject did not vary
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COMMENTS 125
125 I PTH 1 - 8 4
I PTH: BINDING TO SERUM
1.0
on L
o m
0.90.80.70.60.50.40.30.2 0.1-I 0.0
LJ
a: o m
1-34
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1-84
53-84
1.1 1.0, 0.9' 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0
1
10
100
PTH (nmolar)
RADIOLABEL
125
FIG. 1. The binding of various radiolabeled PTH fragments to serum. One hundred microliters of serum from either the patient (•) or a control (•) were incubated with 200 nL radiolabeled PTH fragment (5000 cpm; identified at the base of each set of columns) in 0.05 M Veronal buffer. Charcoal was used to separate bound from free PTH. The B/F ratio for each radiolabel is shown. Each column represents the mean ± 1 SD of triplicate estimations.
I PTH 53-84
B
u. 125
I PTH: BINDING TO SERUM
O,» 1-84 A,A 53-84
1
10
100
PTH (nmolar)
O m
1
10
100
SERUM ADDED (/zl) FIG. 2. The binding of various radiolabeled PTH fragments to varying concentrations of serum. One hundred microliters of either serum or the various amounts of serum (as shown) made up to 100 JUL with buffer were incubated with 200 nh radiolabeled PTH fragment, identified on the figure, in 0.05 M Veronal buffer. Charcoal was used to separate bound from free PTH. Each point is the mean ± 1 SD of triplicate estimations. O or A, The patient; • or A, the control.
with the amount of serum added (Fig. 2, • ) . The ability of unlabeled PTH and PTH fragments to compete for [125I]PTH-(l-84) or [125I]-[Tyr52]hPTH(53-84) binding to the PTH-binding agent is shown in Fig. 3. Nanomolar concentrations of unlabeled hPTH(1-84) or hPTH-(53-84) produced significant displacement. To further characterize this apparent binding agent, we separated the serum by Sep-Pak extraction. The ability of the flow-through and eluant fractions to bind
FIG. 3. A, The ability of various PTH fragments to compete with [125I] PTH-(l-84) for binding to the serum. One hundred microliters of serum from the patient were incubated with 300 ^L [12BI]bPTH-(l-84) (5000 cpm) in 0.05 M Veronal buffer with or without PTH or PTH fragments at the final concentrations shown. Charcoal was used to separate bound from free PTH. Each point is the mean ± 1 SD of duplicate estimations. The various unlabeled PTHs are identified. B, The ability of various PTH fragments to compete with [126I]-[Tyr62] PTH-(53-84). The experiments were performed as described in A, except that the radiolabel was [126I]-[Tyr62]PTH-(53-84). The displacement with hPTH-(l-34) was performed separately and in duplicate, thus explaining the different basal B/F ratio. The other data points are the mean ± SD of triplicate estimations.
the various radiolabeled PTH fragments is shown in Table 1. The PTH-binding component was not retained on the Sep-Pak cartridges, since the flow-through bound 51.6% of the added [125I]bPTH-(l-84) or 51.9% of the added [125I]-[Tyr52]hPTH-(53-84), whereas the eluant bound only 16.5% and 17.4%, respectively. This suggests that the binding component is of high mol wt (>50,000). Studies with anti-Ig antibodies The PTH-binding activity present in the whole serum was not retained on an anti-IgG column (data not
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COMMENTS
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TABLE 1. Binding of various radiolabeled PTH fragments to Sep-Pak separated serum fractions Sep-Pak serum fraction
125
I-Radiolabeled PTH fragment
Flow-through
Eluant
1-84 53-84° 1-34
1.069 ± 0.081* 1.079 ± 0.028 0.09 ± 0.005
0.199 ± 0.005 0.211 ± 0.011 0.089 ± 0.005
Values are the B/F ratio (mean ± SD; n = 3). ° 53-84 denotes [Tyr62]hPTH-(53-84). TABLE 2. The ability of various anti-Ig antisera to precipitate 125Iradiolabeled PTH fragments bound to the Sep-Pak flow-through fraction of the patient's serum Ig fraction against which antiserum directed IgA IgG IgM
126
I-Radiolabeled PTH fragment 1-84
53-84"
0.039 ± 0.002 0.054 ± 0.0006 0.287 ± 0.024*
0.034 ± 0.001 0.053 ± 0.007 0.300 ± 0.009
Values are the B/F ratio (mean ± SD; n = 3). "Denotes [Tyr52]hPTH-(53-84). 6 Charcoal separation gave a B/F ratio of 0.315 in equivalent fractions. The B/F ratios in all combinations of radiolabeled fragments bound to the eluant fraction were less than 0.064. shown).
When antihuman IgG, antihuman IgM, or antihuman IgA was used to precipitate the radiolabeled PTH fragments incubated with the Sep-Pak fractions, only the IgM antiserum precipitated the [125I]bPTH-(l-84) and [125I]-[Tyr52]hPTH-(53-84). Neither antihuman IgG nor IgA caused significant precipitation (Table 2). The amount of radiolabel precipitated by antihuman Ig antiserum in extracts from control subjects is regarded as baseline trapping. These results suggest that the circulating binding component from the patient is an IgM. Follow-up These findings were reproduced on serum obtained from the patient 6 months after her discharge from hospital, when her serum calcium level was clearly normal. Discussion The data demonstrated that the patient' s serum contains an IgM that binds hPTH-(l-84) and -(53-84), but not hPTH-(l-34). Unlabeled bPTH-(53-84) competes with the antibody for radiolabeled hPTH-(l-84). It seems reasonable to conclude that the anti-PTH antibodies are directed against the C-terminal portion of PTH. This domain of the molecule would appear to be more immunogenic than other domains, since when PTH-(1-84) is injected in animals, antisera directed
JCE & M • 1990 Vol 70 • No 6
against the C-terminal domain are frequently produced (Wilkinson, M., and A. McElduff, unpublished observations). The anti-PTH IgM in the patient's serum would account for the reported high PTH concentrations in the C-terminal RIA. The IgM would bind radiolabel, thus reducing the quantities of radiolabel that would interact with the added anti-PTH antiserum from the RIA kit. Second antibody precipitation specific for the added antiPTH antiserum precipitates only that serum; hence, the counts precipitated would be artefactually low in an assay system using second antibody separation. This would be interpreted as unlabeled PTH competing with radiolabeled PTH for the antibody-binding sites in the RIA. The apparent high PTH values are, thus, artefactual. Similarly, in the nonspecific binding tubes the nonspecific immunoprecipitation by the second antibody precipitates fewer counts, since some are specifically bound to the anti-PTH IgM antibody. This method of detecting unsuspected binding components in serum is relatively insensitive, hence the use of charcoal separation in subsequent studies once a binding component is suspected. Charcoal is more sensitive, as it traps free hormone, and the bound fraction is more readily approximated. Since the C-terminal PTH fragments cannot be measured in the presence of the anti-C-terminal antibody a contribution to the apparently elevated concentration from circulating C-terminal fragments cannot be excluded. The patient had a normal or borderline elevated (when corrected for albumin concentration) serum calcium level. This suggests a number of points. Firstly, the circulating PTH had normal (or slightly increased) bioactivity either because free levels of PTH, either 184 or 1-34, were sufficient to maintain normal calcium homeostasis or the amino-terminal end of PTH bound to the C-terminal-directed antibodies was free to interact with PTH receptors or the enzyme system that cleaves PTH-(1-34) from the 1-84 parent molecule, and this maintained serum calcium. These explanations are clearly not mutually exclusive. The one elevated measured serum calcium value (2.61 mmol/L) together with the borderline corrected serum calcium may or may not indicate hypercalcemia. If it indicates hypercalcemia, how can this be explained? Possibly, the adjuvant tamoxifen therapy produced hypercalcemia, albeit no metastatic bone disease was obvious even on bone scan examination. Alternatively, the anti-PTH antibodies could have delayed PTH clearance from the circulation, and this could have produced hypercalcemia that was intermittent in nature, as PTH secretion would be readily inhibited by the hypercalcemia. However, given the normal serum calcium values when the patient became well, in the presence of persistent anti-PTH antibodies it seems most likely that true
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COMMENTS
hypercalcemia was not present, and the values are an artefact of the correction method used. However, we have not excluded familial benign hypocalciuric hypercalcemia, although there is no family history to support this diagnosis. Regardless of the presence or cause of the possible hypercalcemia which cannot be identified retrospectively, the presence of spontaneous anti-PTH antibodies is clearly documented, and these persist when the patient recovered and had a clearly normal serum calcium level. The two previously reported patients with anti-PTH antibodies were treated with PTH. One received bPTH(1-84), and the other received hPTH-(l-34). The antibodies were detected when the patients became hypocalcemic. Possibly, other patients developed antibodies without this blocking activity but were not detected as hypocalcemia did not develop. As mentioned previously, the 1-34 domain of the molecules is less antigenic than the C-terminal domain, which might explain in part the relative infrequency of this occurrence. The patient had never received PTH, either as therapy or as a diagnostic test. We can be reasonably confident of this, since PTH in Australia is restricted in availability. The artefactually elevated PTH concentration contributed to the clinical decision-making process that resulted in this woman undergoing neck exploration. In summary, we report, we believe for the first time, spontaneously occurring anti-PTH antibodies in a patient with pancreatitis. The apparent high PTH values
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together with high normal serum calcium concentrations misled her clinical attendants.
Acknowledgment Mrs. K. Garlan provided excellent secretarial assistance.
References 1. Ingbar SH. The thyroid gland. In: Wilson JD, Foster DW, eds. Williams' textbook of endocrinology. Philadelphia: Saunders; 1985;726. 2. Underwood LE, Voina SJ, Van Wyk JJ. Restoration of growth by human growth hormone (Roos) in hypopituitary dwarfs immunized by other human growth hormone preparations: clinical and immunological studies. J Clin Endocrinol Metab. 1974;38:288-97. 3. Goldman J, Baldwin D, Rubenstein AH, et al. Characterization of circulating insulin and pro-insulin-binding antibodies in autoimmune hypoglycemia. J Clin Invest. 1979;63:1050-9. 4. Berson SA, Yalow RS, Bauman A, Rothschild MA, Newerly K. Insulin-I131 metabolism in human subjects: demonstration of insulin binding globulin in the circulation of insulin treated subjects. J Clin Invest. 1950;35:170-90. 5. Robbins J, Rail JE, Rawson RW. An unusual instance of thyroxine binding by human serum gamma-globulin. J Clin Endocrinol Metab. 1956;16:573-9. 6. Melick RA, Gill JR, Berson SA, et al. Antibodies and clinical resistance to parathyroid hormone. N Engl J Med. 1967;276:1447. 7. Audran M, Basle M-F, Defontaine A, et al. Transient hypoparathyroidism induced by synthetic human parathyroid hormone (134) treatment. J Clin Endocrinol Metab. 1987;64:937-43. 8. Kleerekoper M, Ingham JP, McCarthy SW, Posen S. Parathyroid hormone assay in primary hyperparathyroidism: experiences with a radioimmunoassay based on commercially available reagents. Clin Chem. 1974;20:369-75. 9. McElduff A, Wilkinson M, Lackmann M, et al. Familial hypoparathyroidism due to an abnormal parathyroid hormone molecule. Aust NZ J Med 1989;19:22-30.
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Vol. 70, No. 6 Printed in U.S.A.
Comments
Spontaneously Occurring Antibodies to Parathyroid Hormone* MARGARET WILKINSON, AIDAN McELDUFF, JEREMY WILSON, PAUL HABER, ANTHONY FREEMAN, MALCOLM ROBERTSON, AND PHILLIP MATHEWS Department of Endocrinology, Royal North Shore Hospital (M. W., A.M.), St. Leonards, New South Wales; and the Departments of Gastroenterology, Cardiology, Nephrology, and Surgery, Prince of Wales Hospital, Sydney, New South Wales, Australia
ABSTRACT. A 76-yr-old female with acute pancreatitis and a normal/borderline elevated serum calcium level was found to have an elevated immunoreactive circulating PTH concentration using a C-terminal assay. This high PTH concentration misled the attending physicians and resulted, in retrospect, in an unnecessary neck exploration. When the patient's serum was examined it was found to contain a binding component that bound both C-terminal and PTH-(l-84). This binding compo-
T
HE OCCURRENCE of antibodies to circulating hormones is well described in the endocrine literature. These antibodies may produce various abnormalities or problems. They almost universally lead to errors in RIA estimations of the hormone concentration (1). They may cause hormone resistance by binding to the hormone and inactivating it (2). Alternatively, they may cause apparent hormone sensitivity by binding the hormone, delaying its clearance, and then releasing the hormone at inappropriate times, as has been described for spontaneously occurring antiinsulin antibodies (3). Most commonly, circulating antihormone antibodies are produced by the administration of exogenous hormone, frequently from a different species (2, 4), but the spontaneous development of such antibodies has been described (3, 5). Two well documented cases of antibodies directed against PTH have been associated with PTH resistance and followed treatment with PTH or PTH fragments (6, 7). Spontaneously occurring antiparathyroid antibodies have not previously been reported. We report their occurrence in a 76-yr-old woman. Case Report A 76-yr-old Caucasian female presented to the Emergency Room with several painful red nodules which had appeared on Received October 2, 1989. Address all correspondence and requests for reprints to: Dr. A. McElduff, Department of Endocrinology, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia.
nent was not retained on a Sep-Pak column and was precipitated by antiserum directed against human immunoglobulin M. The presence of circulating anti-PTH immunoglobulin M explains the apparently high PTH concentrations measured by RIA. The antibodies occurred spontaneously. To the best of our knowledge, this phenomenon has not previously been described. (J Clin Endocrinol Metab 70: 1744-1749, 1990)
her shins over the preceding days. Four months before admission a right mastectomy had been performed for infiltrating ductal carcinoma of the breast. Postoperatively, adjuvant tamoxifen (10 mg twice daily) had been commenced, although there was no obvious residual disease. On examination she appeared pale. Several raised erythematous tender mobile lesions were scattered over the anterior shins, ankles, and buttocks. They measured 1-2 cm and were not ulcerated. There was no lymphadenopathy. The temperature was 37.4 C, the blood pressure was 170/70 mm Hg, and pulse rate was 92 beats/min. The rhythm was totally irregular. An electrocardiograph confirmed atrial fibrillation. A grade 2/ 6 aortic ejection murmur was audible over the aortic outflow tract. There were no abdominal masses or tenderness. The remaining examination was normal. Histological examination of the lesions revealed enzymatic fat necrosis. Serum amylase was noted to be elevated at 2,730 (normal, 30-100 U/L) and was of pancreatic origin. Lipase was 345 IU/L (normal, 0-200), and urinary amylase was raised at 10,840 U/L. The hemoglobin was 8.3 g/dL, with a normal blood film. Serum ferritin, folate, and B12 concentrations were normal. An electrophoresis protein gel and immunoelectrophoresis protein gel showed no evidence of a monoclonal spike, but demonstrated a broad increase in 7- and a2-globulins. The anemia was corrected by transfusion, and the hemoglobin level remained normal. There was mild acute renal failure, with a raised serum urea of 12.4 mmol/L (normal, 2.8-7.6) and serum creatinine of 160 ^mol/L (normal, 60-110). Serum alkaline phosphatase was 147 IU/L (normal, 25-110), and other liver function tests were normal. The serum creatinine level fell to 120 /imol/L with
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COMMENTS rehydration. It was never higher than the initial value. Serum calcium was 2.46 mmol/L (normal, 2.05-2.55), and inorganic phosphate was 0.9 mmol/L (normal, 0.8-1.5). Serum albumin was 42 g/L (normal, 33-55). Serum triglycerides were 2.2 mmol/L (normal, 8.0, and 3.9 ng/ mL; normal, 0-0.5). A 24-h urine collection demonstrated calcium excretion of 0.32 mmol and normal inorganic phosphorus excretion of 14 mmol/L at a serum phosphate level of 1 mmol/L. Urinary cAMP excretion was 700 nmol/mmol creatinine (normal, 200890). The hospital course was protracted and complicated by two episodes of acute painful pancreatitis and advancing enzymatic fat necrosis. The skin lesions became ulcerated, and the patient's mobility was limited by acute synovitis involving several joints. Persistent pain in the left wrist was attributed to medullary fat necrosis of the ulna associated with a lytic lesion on plain x-ray and a "hot spot" on radionuclide bone scan. The serum PTH level remained raised in association with borderline hypercalcemia. In view of the adverse clinical course, neck exploration was undertaken. Two parathyroid glands of normal histology were removed. Two additional parathyroid glands were identified and appeared to be of normal size; no biopsies were taken of these two remaining glands. Postoperative serum PTH and calcium concentrations were unchanged. The patient's condition eventually settled on supportive measures only. After a 3-month period of hospitalization she was discharged home. She remains well with normal serum calcium (2.15 mmol/L; albumin, 36 g/L) and serum amylase, and no recurrence of the skin lesions 6 months later. The PTH concentration measured in the same C-terminal RIA remains elevated.
Materials and Methods Materials Human PTH-(l-34) [hPTH-(l-34)], [Asn76]hPTH-(l-84), and [Nle818,Tyr34]hPTH-(l-34) were obtained from Peninsula
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Laboratories, Inc. (Belmont, CA). Bovine PTH-(1-84) [bPTH(1-84)] and [Tyr52]hPTH-(52-84) were obtained from Bachem Fine Chemicals (Torrance, CA). Sodium iodine for protein iodination was obtained from Amersham Radiopharmacuticals (Amersham, United Kingdom). Human immunoglobulin G (IgG), rabbit antihuman IgG, and rabbit antihuman IgA were obtained from Bioproducts Ltd. (Brussels, Belgium). Human IgM was obtained from Organon Technika (Turnhout, Belgium). Human IgA was obtained from Sigma Chemical Co. (St. Louis, MO). Goat antihuman IgM was obtained from Cambridge Medical Technologies (Cambridge, MA). Antihuman IgG (7-chain) for binding to Sepharose was obtained from Silenis Laboratories (Hawthorn, Victoria, Australia). CNBractivated Sepharose was obtained from Pharmacia Fine Chemicals (Uppsala, Sweden). Sac-Cel cellulose-coated antibody was obtained from INS (Washington, England). Methods RIA. The PTH-(l-84) RIA was performed as previously described (8), with modifications reported previously (9). Separation of bound from free [125I]bPTH-(l-84) was by Sac-Cel goat antiguinea pig-coated cellulose suspension, as per the manufacturer's recommendations. The PTH-(1-34) RIA was performed as previously described (9). Both assays are briefly outlined below. Charcoal separation. Charcoal separation of bound from free [125I]PTH was performed by the addition of 1 mL cold 1% activated Charcoal (Merck, Darmstadt, West Germany) in 0.02 M Veronal buffer, pH 8.6, and the tubes were centrifuged for 45 min at 4 C. The supernatant (free) was decanted into a separate tube. Both bound and free PTH were counted, and bound/free ratios (B/F ratio) were calculated. We have previously shown (9), using sera that do not contain PTH-binding components, that these two separation methods give similar results. Radiolabeled PTH and PTH fragments: binding to serum. [125I] bPTH-(l-84) and [125I]-[Nle8|18,Tyr34]hPTH-(l-34) were iodinated and purified as previously described (8, 9). [125I]-[Tyr52] hPTH-(53-84) was iodinated and purified by a method identical to that used for [125I]bPTH-(l-84). Two hundred microliters of 0.05 M Veronal buffer, pH 8.6, containing approximately 5000 cpm [125I]bPTH-(l-84), [125I][Nle818,Tyr34]hPTH-(l-34), or [125I]-[Tyr62]hPTH-(53-84) were added to 100 ^L of the patient's serum or serum from a control subject in 12 x 75-mm polystyrene tubes. The tubes were incubated for 20 h at 4 C. Separation of bound from free tracer was performed with charcoal. These conditions were identical to those of the corresponding RIA, except that 200 nL buffer were added to replace the 200 ^1 antibody-containing buffer, separation was with charcoal rather than second antibody, and no preincubation with radiolabel was performed. Serial dilutions of the patient's serum were made in assay buffer to give microliter equivalents of 100, 20, 10, or 5 in a total volume of 100 fiL. Binding studies with [125I]bPTH-(l84) and [125I]-[Tyr52]hPTH-(53-84) were then performed as described above. At the end of the incubation, 100 yiL appropriately diluted control serum were added to all tubes to bring
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COMMENTS
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the final serum concentration in each tube to 100 /xL undiluted serum. Displacement of radiolabeled PTH fragments by cold PTH frag-
ments. One hundred microliters of patient or control serum were incubated with 200 nh (-4000 cpm) [125I]bPTH-(l-84) or [125I]-[Tyr52]hPTH-(53-84) and 100 nL hPTH-(l-34), [Tyr52] hPTH-(53-84), or bPTH-(l-84) diluted in surgical hypoparathyroid serum to reach the concentration (final) shown. Sep-Pak separation. Patient serum or serum from a control subject in volumes of 2 mL (except the experiment reported in Table 2 where the volume used was 1 mL) was applied to 6-mL SPE Ci8 cartridges (Baker Chemical Co., Phillipsburg, NJ) under vacuum. The columns were washed with 2 mL phosphate buffer, followed by 20 mL 0.1% trifluoroacetic acid (TFA). The columns were then eluted with 3 mL 80% acetonitrile in 0.1% trifluoroacetic acid high pressure liquid chromatography (Spec-
trograde). The flow-through and phosphate buffer fractions were pooled. The pooled flow-through and the eluant fractions were lyophylized and reconstituted in 0.05% M Veronal buffer, pH 8.6, each to the volume of the original sample size. Under identical conditions these columns were able to extract 99% of IgM, IgG, and IgA from serum as measured directly on serum and flow-through by rate nephilometry on a Beckman assay nephilometer (Beckman Instruments, Gladesville, New South Wales, Australia). Recovery of binding activity from the columns, as assessed by the B/F ratio per 100 nh serum or serum equivalent, was approximately 90-95%. Radiolabeled PTH and PTH fragment binding to Sep-Pak C18 flow-through and eluant fractions. These experiments were performed as described previously, except that in some experiments separation of the bound from free tracer was carried out by the addition of 100 nL rabbit antihuman IgG, 50 /JL goat antihuman IgM or 50 nL rabbit antihuman IgA together with 100 nL (1.0 mg) of the appropriate Ig added as carrier. After a 2-h incubation at 4 C, 1.0 mL 0.05 M Veronal buffer, pH 8.6, was added to all tubes, and the tubes were centrifuged at 2230 X g for 45 min at 4 C. The supernatants were decanted into separate tubes, and both the precipitate (bound) and supernatant (free) tubes were counted. IgG column chromatography. Antihuman IgG (7-chains) was coupled to CNBr-activated Sepharose 4B according to the manufacturer's instructions. One half milliliter of the patient's serum was applied to 0.5 X 9-cm affinity chromatography column packed with the antihuman IgG-coupled Sepharose. The column was washed with 20 mL phosphate-buffered saline and eluted with 0.2 M acetic acid in 1.0-mL fractions, which were rapidly neutralized with ammonia solution. The fractions were then lyophylized and reconstituted in 0.02 M Veronal buffer, pH 8.6. The fractions were processed as described for the serum and Sep-Pak fractions.
Results RIA results The C-terminal PTH assay gave consistently elevated estimations for the PTH concentration. Serial dilutions
JCE & M • 1990 Vol70«No6
(1, 0.5, and 0.25) in this assay produced values of more than 8.0, 7, and 7.2 ng/mL, respectively. This was interpreted as parallel to the standard curve. This was the only PTH assay performed preoperatively. Postoperatively, serum was assayed in our routine PTH-(1-84) assay (8, 9), which preferentially sees the complete 1-84 molecule (9). The PTH concentration estimated in this assay was also elevated. However, the nonspecific binding or blank tube was lower than normal (see below). Nonspecific binding is defined as binding occurring in the absence of the anti-PTH antibody routinely added in the RIA. The low nonspecific binding caused the value to be rejected. Further investigations of the sample, as outlined below, were undertaken. Parenthetically, serial dilutions to 0.25 read at values above the highest standard. When the patient's serum was assayed in a PTH-(134) assay (9), there was no difference between the nonspecific binding of the patient's serum and that of serum from other patients regardless of whether the separation of bound and free PTH was performed with second antibody or charcoal. The hPTH-(l-34) result was 30 pg/mL (reference range, up to 130 pg/mL; limit of detection, 16 pg/mL). This value was too low for reliable serial dilution studies. Nonspecific binding [125I]bPTH-(l-84) was incubated with the patient's serum. When bound and free PTH were separated with our antiguinea pig second antibody (8, 9), nonspecific binding was low, with a B/F ratio of 0.015 compared with a control B/F of 0.032-0.050 (n = 30). Low nonspecific binding suggests a binding component in the patient's serum not precipitated by the second antibody. When separated with charcoal, the B/F ratio was high (0.967) in the patient's serum compared to that (0.067) in control samples (Fig. 1). This is again consistent with a binding agent in the patient's serum. Studies of radiolabel binding to serum and serum fractions To determine the presence and specificity of the possible binding agent from the patient's serum, radiolabeled bPTH-(l-84), [Nle8-18,Tyr34]hPTH-(l-34) and [Tyr52] hPTH-(53-84) were incubated with the serum. As seen in Fig. 1, serum from the patient bound both radiolabeled bPTH-(l-84) and [Tyr52]hPTH-(53-84) to a significantly greater degree than the control serum. [125I][Nle8-18,Tyr34]hPTH-(l-34) had equally low binding in both patient and control serum. As shown in Fig. 2, increasing concentrations of the patient's serum resulted in increasing binding of both and [125I]-[Tyr52] hPTH-(53-84). [125I]bPTH-(l-84) Binding to serum from a control subject did not vary
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COMMENTS 125
125 I PTH 1 - 8 4
I PTH: BINDING TO SERUM
1.0
on L
o m
0.90.80.70.60.50.40.30.2 0.1-I 0.0
LJ
a: o m
1-34
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1-84
53-84
1.1 1.0, 0.9' 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0
1
10
100
PTH (nmolar)
RADIOLABEL
125
FIG. 1. The binding of various radiolabeled PTH fragments to serum. One hundred microliters of serum from either the patient (•) or a control (•) were incubated with 200 nL radiolabeled PTH fragment (5000 cpm; identified at the base of each set of columns) in 0.05 M Veronal buffer. Charcoal was used to separate bound from free PTH. The B/F ratio for each radiolabel is shown. Each column represents the mean ± 1 SD of triplicate estimations.
I PTH 53-84
B
u. 125
I PTH: BINDING TO SERUM
O,» 1-84 A,A 53-84
1
10
100
PTH (nmolar)
O m
1
10
100
SERUM ADDED (/zl) FIG. 2. The binding of various radiolabeled PTH fragments to varying concentrations of serum. One hundred microliters of either serum or the various amounts of serum (as shown) made up to 100 JUL with buffer were incubated with 200 nh radiolabeled PTH fragment, identified on the figure, in 0.05 M Veronal buffer. Charcoal was used to separate bound from free PTH. Each point is the mean ± 1 SD of triplicate estimations. O or A, The patient; • or A, the control.
with the amount of serum added (Fig. 2, • ) . The ability of unlabeled PTH and PTH fragments to compete for [125I]PTH-(l-84) or [125I]-[Tyr52]hPTH(53-84) binding to the PTH-binding agent is shown in Fig. 3. Nanomolar concentrations of unlabeled hPTH(1-84) or hPTH-(53-84) produced significant displacement. To further characterize this apparent binding agent, we separated the serum by Sep-Pak extraction. The ability of the flow-through and eluant fractions to bind
FIG. 3. A, The ability of various PTH fragments to compete with [125I] PTH-(l-84) for binding to the serum. One hundred microliters of serum from the patient were incubated with 300 ^L [12BI]bPTH-(l-84) (5000 cpm) in 0.05 M Veronal buffer with or without PTH or PTH fragments at the final concentrations shown. Charcoal was used to separate bound from free PTH. Each point is the mean ± 1 SD of duplicate estimations. The various unlabeled PTHs are identified. B, The ability of various PTH fragments to compete with [126I]-[Tyr62] PTH-(53-84). The experiments were performed as described in A, except that the radiolabel was [126I]-[Tyr62]PTH-(53-84). The displacement with hPTH-(l-34) was performed separately and in duplicate, thus explaining the different basal B/F ratio. The other data points are the mean ± SD of triplicate estimations.
the various radiolabeled PTH fragments is shown in Table 1. The PTH-binding component was not retained on the Sep-Pak cartridges, since the flow-through bound 51.6% of the added [125I]bPTH-(l-84) or 51.9% of the added [125I]-[Tyr52]hPTH-(53-84), whereas the eluant bound only 16.5% and 17.4%, respectively. This suggests that the binding component is of high mol wt (>50,000). Studies with anti-Ig antibodies The PTH-binding activity present in the whole serum was not retained on an anti-IgG column (data not
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COMMENTS
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TABLE 1. Binding of various radiolabeled PTH fragments to Sep-Pak separated serum fractions Sep-Pak serum fraction
125
I-Radiolabeled PTH fragment
Flow-through
Eluant
1-84 53-84° 1-34
1.069 ± 0.081* 1.079 ± 0.028 0.09 ± 0.005
0.199 ± 0.005 0.211 ± 0.011 0.089 ± 0.005
Values are the B/F ratio (mean ± SD; n = 3). ° 53-84 denotes [Tyr62]hPTH-(53-84). TABLE 2. The ability of various anti-Ig antisera to precipitate 125Iradiolabeled PTH fragments bound to the Sep-Pak flow-through fraction of the patient's serum Ig fraction against which antiserum directed IgA IgG IgM
126
I-Radiolabeled PTH fragment 1-84
53-84"
0.039 ± 0.002 0.054 ± 0.0006 0.287 ± 0.024*
0.034 ± 0.001 0.053 ± 0.007 0.300 ± 0.009
Values are the B/F ratio (mean ± SD; n = 3). "Denotes [Tyr52]hPTH-(53-84). 6 Charcoal separation gave a B/F ratio of 0.315 in equivalent fractions. The B/F ratios in all combinations of radiolabeled fragments bound to the eluant fraction were less than 0.064. shown).
When antihuman IgG, antihuman IgM, or antihuman IgA was used to precipitate the radiolabeled PTH fragments incubated with the Sep-Pak fractions, only the IgM antiserum precipitated the [125I]bPTH-(l-84) and [125I]-[Tyr52]hPTH-(53-84). Neither antihuman IgG nor IgA caused significant precipitation (Table 2). The amount of radiolabel precipitated by antihuman Ig antiserum in extracts from control subjects is regarded as baseline trapping. These results suggest that the circulating binding component from the patient is an IgM. Follow-up These findings were reproduced on serum obtained from the patient 6 months after her discharge from hospital, when her serum calcium level was clearly normal. Discussion The data demonstrated that the patient' s serum contains an IgM that binds hPTH-(l-84) and -(53-84), but not hPTH-(l-34). Unlabeled bPTH-(53-84) competes with the antibody for radiolabeled hPTH-(l-84). It seems reasonable to conclude that the anti-PTH antibodies are directed against the C-terminal portion of PTH. This domain of the molecule would appear to be more immunogenic than other domains, since when PTH-(1-84) is injected in animals, antisera directed
JCE & M • 1990 Vol 70 • No 6
against the C-terminal domain are frequently produced (Wilkinson, M., and A. McElduff, unpublished observations). The anti-PTH IgM in the patient's serum would account for the reported high PTH concentrations in the C-terminal RIA. The IgM would bind radiolabel, thus reducing the quantities of radiolabel that would interact with the added anti-PTH antiserum from the RIA kit. Second antibody precipitation specific for the added antiPTH antiserum precipitates only that serum; hence, the counts precipitated would be artefactually low in an assay system using second antibody separation. This would be interpreted as unlabeled PTH competing with radiolabeled PTH for the antibody-binding sites in the RIA. The apparent high PTH values are, thus, artefactual. Similarly, in the nonspecific binding tubes the nonspecific immunoprecipitation by the second antibody precipitates fewer counts, since some are specifically bound to the anti-PTH IgM antibody. This method of detecting unsuspected binding components in serum is relatively insensitive, hence the use of charcoal separation in subsequent studies once a binding component is suspected. Charcoal is more sensitive, as it traps free hormone, and the bound fraction is more readily approximated. Since the C-terminal PTH fragments cannot be measured in the presence of the anti-C-terminal antibody a contribution to the apparently elevated concentration from circulating C-terminal fragments cannot be excluded. The patient had a normal or borderline elevated (when corrected for albumin concentration) serum calcium level. This suggests a number of points. Firstly, the circulating PTH had normal (or slightly increased) bioactivity either because free levels of PTH, either 184 or 1-34, were sufficient to maintain normal calcium homeostasis or the amino-terminal end of PTH bound to the C-terminal-directed antibodies was free to interact with PTH receptors or the enzyme system that cleaves PTH-(1-34) from the 1-84 parent molecule, and this maintained serum calcium. These explanations are clearly not mutually exclusive. The one elevated measured serum calcium value (2.61 mmol/L) together with the borderline corrected serum calcium may or may not indicate hypercalcemia. If it indicates hypercalcemia, how can this be explained? Possibly, the adjuvant tamoxifen therapy produced hypercalcemia, albeit no metastatic bone disease was obvious even on bone scan examination. Alternatively, the anti-PTH antibodies could have delayed PTH clearance from the circulation, and this could have produced hypercalcemia that was intermittent in nature, as PTH secretion would be readily inhibited by the hypercalcemia. However, given the normal serum calcium values when the patient became well, in the presence of persistent anti-PTH antibodies it seems most likely that true
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COMMENTS
hypercalcemia was not present, and the values are an artefact of the correction method used. However, we have not excluded familial benign hypocalciuric hypercalcemia, although there is no family history to support this diagnosis. Regardless of the presence or cause of the possible hypercalcemia which cannot be identified retrospectively, the presence of spontaneous anti-PTH antibodies is clearly documented, and these persist when the patient recovered and had a clearly normal serum calcium level. The two previously reported patients with anti-PTH antibodies were treated with PTH. One received bPTH(1-84), and the other received hPTH-(l-34). The antibodies were detected when the patients became hypocalcemic. Possibly, other patients developed antibodies without this blocking activity but were not detected as hypocalcemia did not develop. As mentioned previously, the 1-34 domain of the molecules is less antigenic than the C-terminal domain, which might explain in part the relative infrequency of this occurrence. The patient had never received PTH, either as therapy or as a diagnostic test. We can be reasonably confident of this, since PTH in Australia is restricted in availability. The artefactually elevated PTH concentration contributed to the clinical decision-making process that resulted in this woman undergoing neck exploration. In summary, we report, we believe for the first time, spontaneously occurring anti-PTH antibodies in a patient with pancreatitis. The apparent high PTH values
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together with high normal serum calcium concentrations misled her clinical attendants.
Acknowledgment Mrs. K. Garlan provided excellent secretarial assistance.
References 1. Ingbar SH. The thyroid gland. In: Wilson JD, Foster DW, eds. Williams' textbook of endocrinology. Philadelphia: Saunders; 1985;726. 2. Underwood LE, Voina SJ, Van Wyk JJ. Restoration of growth by human growth hormone (Roos) in hypopituitary dwarfs immunized by other human growth hormone preparations: clinical and immunological studies. J Clin Endocrinol Metab. 1974;38:288-97. 3. Goldman J, Baldwin D, Rubenstein AH, et al. Characterization of circulating insulin and pro-insulin-binding antibodies in autoimmune hypoglycemia. J Clin Invest. 1979;63:1050-9. 4. Berson SA, Yalow RS, Bauman A, Rothschild MA, Newerly K. Insulin-I131 metabolism in human subjects: demonstration of insulin binding globulin in the circulation of insulin treated subjects. J Clin Invest. 1950;35:170-90. 5. Robbins J, Rail JE, Rawson RW. An unusual instance of thyroxine binding by human serum gamma-globulin. J Clin Endocrinol Metab. 1956;16:573-9. 6. Melick RA, Gill JR, Berson SA, et al. Antibodies and clinical resistance to parathyroid hormone. N Engl J Med. 1967;276:1447. 7. Audran M, Basle M-F, Defontaine A, et al. Transient hypoparathyroidism induced by synthetic human parathyroid hormone (134) treatment. J Clin Endocrinol Metab. 1987;64:937-43. 8. Kleerekoper M, Ingham JP, McCarthy SW, Posen S. Parathyroid hormone assay in primary hyperparathyroidism: experiences with a radioimmunoassay based on commercially available reagents. Clin Chem. 1974;20:369-75. 9. McElduff A, Wilkinson M, Lackmann M, et al. Familial hypoparathyroidism due to an abnormal parathyroid hormone molecule. Aust NZ J Med 1989;19:22-30.
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