from a 2.0-ml Kampo. Auto- matic Pipetting. Syringe, Cornwall type (Elios Vanti- ni, Padova,. Italy). Centrifugation was carried out within 10 mm after the charcoal.
CLIN. CHEM.
20/3,
369-375
(1974)
ParathyroidHormone Assay inPrimaryHyperparathyroidism: Experiences with a RadioimmunoassayBased on CommerciallyAvailableReagents M. Kleerekoper, J. P. lngham, S. W. McCarthy, and S. Posen
A radioimmunoassay is described for parathyroid hormone in human serum, in which commercially available reagents are used almost exclusively. This
assay can be done by any laboratory with experience in radloimmunoassay. Thirty-two of thirty-three patients with surgically proven primary hyperparathyroidism had detectable concentrations of parathyroid hormone in their serum, and concentrations of the hormone exceeded the normal range in 24 of them. Significant positive correlations were found between
pre-operative
serum
calcium,
pre-operative
serum
parathyroid hormone, and the weight of parathyroid tissue removed at operation. These three parameters were also significantly correlated with severity of the skeletal changes as assessed by semiquantitative histological methods. Additional Keyphrases: indexes to hyperparathyroidism #{149} diagnostic aid #{149}calculous disease #{149}parathyroid adenoma e normal values Several variables are used to assess the severity of primary hyperparathyroidism: serum calcium (1, 2), serum immunoreactive parathyroid hormone (3, 4), quantitation of skeletal lesions (5, 6), measurement of urinary excretion of cyclic AMP (7), and estimation of plasma ionic calcium (8). We recently correlated the first three of these with one another and with the weight of abnormal parathyroid tissue removed from 33 patients at operation or autopsy. Our results are reported in this paper, and the value of pre-operative parathyroid hormone (PTH) assay and skeletal biopsy is assessed.
Patients and Methods Primary hyperparathyroidism was diagnosed in 33 patients by criteria previously outlined (9). Patients were included only if their pre-operative serum calcium was at least 10.8 mg/dl, if their serum creatinine was 3.0 mg or less per deciliter, and if at least 121 mg of parathyroid tissue were removed surgically or Departments of Medicine and Morbid Anatomy. Sydney Hospital, Sydney, N.S.W., Australia, 2000. Direct reprint requests to Solomon Posen, Department of Medicine, Sydney Hospital. Received Oct. 30, 1973; accepted Dec. 28, 1973.
at autopsy. Surgical patients were included only if their postoperative serum calcium was 10.0 mg or less per deciliter. There were 24 women and nine men, ranging in age from 24 to 72 years. A single enlarged parathyroid adenoma was found in all but one patient, in whom all four glands were enlarged. Fourteen of the patients have been included in a previous publication (9). Blood from the antecubital vein was allowed to clot, and the sera were stored at -20 #{176}C until required. Serum calcium was estimated by the automated fluorimetric method of Fingerhut et al. (10), serum creatinine by the automated picrate method of Chasson et al. (11), and serum PTH by radioimmunoassay. We used a commercially available guinea-pig anti-bovine PTH antiserum (AS 211/32; Wellcome Laboratories, Beckenham, Kent, U.K.). High purified bovine PTH (Lot No. 150545; Wilson Laboratories, Chicago, Ill.) was used to prepare iodinated tracers and for the standards in the assay system. lodination was carried out by the chloramine-T method of Greenwood et al. (12) and the iodinated PTH was purified with “QUSO G 32” (Philadelphia Quartz Co. Chester, Pa. 19106) according to the method of Yalow and Berson (13). Solutions used in the assay system are listed in Table 1; the protocol for the assay is given in Table 2. All reagents were prepared in standard laboratory glassware that had not been pre-treated. The assay was performed in untreated 80 x 10 mm polystyrene tubes (Australian Laboratory Services, Arncliffe, N.S.W., Australia, 2205). The reagents were added in the order listed in Table 2, and the tubes were momentarily agitated on a vortex-type mixer before the initial four-day incubation period. They were agitated again after the radioactive tracer was added. and a third time after charcoal suspension was added. Standard diluent, antiserum, and iodinated tracer were added from a 5.0-ml Hamilton gas-tight syringe fitted with a Chancy adapter (Hamilton Co., Reno, Nev. 89510) calibrated to dispense lOO-l aliquots. Bovine PTH standards and test sera were added with an Eppendorf microliter pipette (Eppendorf CLINICAL
CHEMISTRY,
Vol.
20. No.
3,
1974
369
Table 1. Solutions for Radioimmunoassay of PTH Standard serum
diluent: 10-fold diluted hypoparathyroid’ in barbital buffer (pFI 8.6, 50 mmol/liter).
Antiserum (AS211/32, Wellcome Laboratories): luted 100000-fold in standard diluent.
Di-
PTH standards (Wilson Laboratories): Di. luted in hypoparathyroid serum to contain 0, 12.5, 25, 50, 100, 200, 500, and 1000 pg/100 /hl. Bovine [“-51]PTH: Diluted in standard diluent to contain 500 000 cpm/ml. Activated charcoal (Merck, Darmstadt, Germany): 10 mg/mI in barbital buffer (pH 8.6, 20 mmol/liter). Bovine
‘Hypoparathyroidism was diagnosed concentration of 8.0 mg or IessLdl, three
thyroidectomyor evidence
parathyroidectomy,
by a serum calcium months or more after
of clinical
in the absence
of
malabsorption and retention (46). In addition,
in the absence of each “hypoparathyroid”
ni-
trogenous serum was tested to ensure that it produced no displacement of the radioactive tracer from the antiserum. Sera from normal subjects previously tested to ensure that they produced no displacement
of radioactive
be acceptable
as a source
but we have
not investigated
tracer
from
of protein this
the
antibody
may
in the standard
also
diluent,
in detail.
Table 2. Protocol for Radioimmunoassay of PTH Standard diluent Antiserum Bovine PTH standard or unknown serum Hypoparathyroid serum
100 100 100
50-100
0-50 Total
300
Incubate at 4#{176}C for 4 days Add bovine [‘#{176}I)PTH
100 400
Total Incubate at Add charcoal
4 ‘C
for 3 days 1000 Total
Centrifuge
1400
(3000 rpm, 4 #{176}C, 45 mm).
Count radioactivity in supernate (= “bound” [llJPTH) and in the precipitate (= “free” [1251J PTH). Geratbrau Netheler & Hinz GmbH, Hamburg, Germany) of appropriate capacity. The charcoal suspension was prepared at least 1 h
before being added
to the assay tubes.
It was stirred
continuously at 4 #{176}C during that time and while 1-ml aliquots were dispensed from a 2.0-ml Kampo Automatic Pipetting Syringe, Cornwall type (Elios Vantini, Padova, Italy). Centrifugation was carried out within 10 mm after the charcoal was added. Substitution of dextran-coated charcoal for the untreated charcoal suspension listed in Table 1 did not cause any significant alteration in the results obtained
(Kleerekoper
and Morris, unpublished).
All assays were performed in triplicate and all unknown sera were assayed in two dilutions. “Control tubes,” in which 100 l of antiserum were replaced by 100 l of standard diluent, were set up for the 370
CLINICAL
CHEMISTRY,
Vol. 20, No. 3, 1974
standards and for each of the unknown sera. A PTH assay of any serum specimen therefore requires nine tubes (three with 50 il, three with 100 l, and three with 100 il for the control tubes). This makes it possible to perform a PTH estimation on a serum sample of 1 ml and to test 10-15 unknown samples per assay. The radioactivity of the supernatant fluid (Table 2) of each control tube was subtracted from the radioactivity of the supernatant fluid of the corresponding tube containing antiserum (14), so that the percentage of [‘25I]PTH bound to antibody was calculated according to the formula (a - c/b - c) x 100, where a = “bound” [1251]PTH (Table 2), b = “bound” [1251]PTH plus “free” [‘251]PTH (Table 2), and c = radioactivity in the supernate of the appropriate control tube. Figure 1 shows a standard curve for bovine PTH, and also a curve obtained with serial dilutions of serum from a patient with primary hyperparathyroidism. Figure 2 shows that radioactive tracer is also displaced from this antiserum by the synthetic N-terminal fragment of bovine PTH (amino acids 1-34, lot No. 21013; Beckman Instruments, Inc., Fullerton, Calif. 92634), but that the curve does not parallel those obtained either with commercial bovine PTH or with sera from patients with primary hyperparathyroidism 1 Cancellous bone was obtained from the iliac crest as previously described (16). Decalcified sections, stained with haematoxylin and eosin, were used to semiquantitatively assess changes in the bone. All sections were examined on the same day by one of us (S. W. McCarthy) without any knowledge of the clinical and laboratory data. Severity of histological change was graded from 0 to 3 according to arbitrary criteria (Figure 3). A score of 0 was assigned to a specimen if there was no bone erosion or marrow fibrosis. A maximum score of 3 was assigned if there were more than two areas of bone erosion with adjacent fibrosis per high-power field (x320 magnification). Bone scores of 1 and 2 were assigned to specimens with intermediate histological changes. During neck exploration, all tissue thought to consist of enlarged parathyroid glands was removed and submitted for histological examination. Parathyroid glands that appeared macroscopically normal were left in place, even when our surgeon (Dr. E. M. Cortis) believed that these had been identified. Surgically removed parathyroid tissue was dissected free from fat, thyroid tissue, and other identifiable non-
I
Both
the commercial
bovine
standards
and
the synthetic
1-34
fragment were assayed with two antisera (GP1M and Ch 14) kindly donated by Dr. C. D. Arnaud of the Mayo Clinic. We have confirmed his finding that both these antisera recognize the presumably intact bovine PTH molecule but that only Ch 14 detects the synthetic N-terminal fragment. Under similar conditions, hyperparathyroid serum displaced iodinated tracers less readily from Ch 14 than from either GPIM or from BW AS 211/32. This finding is in keeping with the statement by Habener et al. (15) that the major molecular PTH species circulating in primary hyperparathyroid serum does not include the N-terminal fragment.
jit Human Serum
#{149}-.
51020
1)
100 80
z 60 -J
4
I-
z
40 20
(a)
:.‘
10
100
500
1.000
b PTH(pgh.-s
Fig. 1. trations from a (patient
Standard curves obtained with increasing concenof highly purified bovine PTH (X-X) and serum patient with primary hyperparathyroidism (I-#{149}) No. 30, Table 3)
initial bound’ was assigned to the assay tubes containing no added bovine PTH in which about 27% of the radioactive tracer was antibody-bound (B:F ratio, about 0.4). The difference between this “zero A value ‘100%
standard” and the highest bovine tube of supernate (Table 2)
a z
PTH standard
was about 800 cpm per
Fig. 3. Photomicrographs of decalcified cellous bone stained with haematoxylin nal magnification X 320)
80
4
(a) Patient
No. 15 (Table 3)..gaded
sections of canand eosin (origi-
1 on a scale of 0-3: (b) Patient
No.
33 (Table 3), graded 3 on a scale of 0-3.
10 20 Extracted
b PTH
b PTH(pg)
Fig. 2. Standard curves obtained with increasing concenof highly purified bovine PTH (X_X) and the synthetic N-terminal fragment (amino acids 1-34) of the bovine PTH molecule (#{149}-#{149})
trations
parathyroid material. It was then weighed (wet) before histological examination. Levels of significance and correlation between the different variables were determined by standard statistical methods (17).
Results The
interassay
variability
was
determined
over
a
10-week period. The mean bound to free (B:F) ratio obtained with “zero standards” (serum from different hypoparathyroid patients) was 0.37 (coefficient of variation, 9.7%). The coefficient of variation for a pooled hyperparathyroid serum, assayed to contain 5.6 ng/ml, was 12.9% when 25-A’ aliquots were assayed, 10.0% when 50-id aliquots were assayed. The intra-assay coefficient of variation of zero standards in 60 consecutive assays ranged from 0.5% to 4.6% (median, 2.1%). The intra-assay variation of triplicate determinations performed on pooled hyperparathyroid serum in 40 consecutive assays ranged from 0.7% to 10.3% (median, 4.5%) when 25-sl aliquots were assayed and from 0.1% to 23.9% (median, 6.3%) when 50-l aliquots were assayed.
Variations among PTH concentrations of serum specimens taken from the same patient at different times were relatively slight. Values ranging from 0.7 to 1.1 ng/ml were obtained for one patient (No. 23, Table 3) over an 8-h period after an overnight fast (18). Six serum samples (patient fasting) taken during a five-month period from patient No. 23 (Table 3) and tested in different assays, showed PTH concentrations ranging from 0.7 to 0.9 ng/ml. In our best assays, the lower limit of detectability of PTH was 125 pg/mi. Only 20% of normal fasting subjects had detectable serum PTH concentrations, rang#{227}ng from 125-400 pg/ml. Table 3 shows parathyroid gland weights, serum immunoreactive PTH concentrations, semiquantitative histological data, and serum calcium concentrations in 33 patients with primary hyperparathyroidism. Of these, 32 had detectable PTH concentrations, and their values were above our normal range in 24. Twenty-seven patients had demonstrable histological bone changes and all six patients in whom skeletal changes were not found had PTH concentrations of
