Hyperparathyroidism due to clear cell hyperplasia

Hyperparathyroidism Due to Clear Cell Hyperplasia Serial Determinations of Serum Ionized Calcium, Parathyroid Hormone and Calcitonin

ROGER

L. DAWKINS,

M.B.*

ARMEN

H. TASHJIAN,

Jr.,

BENJAMIN EDWARD

CASTLEMAN, W. MOORE,

M.D.t M.D:

M.D.3

Boston, Massachusetts

From the Metabolic and Gastrointestinal Research Unit, Lemuel Shattuck Hospital, Commonwealth of Massachusetts, Tufts University School of Medicine; the Pharmacology Deoartment. Harvard School of Dental Meditine and Department of Pharmacology, Harvard Medical School; and the Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts. This study was supported by U.S. Public Health Service Research Grants AM-07417, AM-13357 and AM10206, National Institute of Arthritis and Metabolic Diseases. Preliminary reports have appeared previously [1,2]. Requests for reprints should be addressed to Dr. Edward W. Moore. Manuscript received November 10, 1970. *Present address: Medical School, Victoria Square, Perth, Western Australia 6000. TRecipient of U.S. Public Health Service Research Career Development Award, K3AM-13,561, National Institute of Arthritis and Metabolic Diseases. $Recipient of U.S. Public Health Service Research Career Development Award, K3GM-l 1,386, National Institute of General Medical Sciences. Present address: Health Sciences Center, Medical College of Virginia, Richmond, Virginia 23219.

Serial determinations of serum ionized calcium (Ca++), serum total calcium (Ca), parathyroid hormone (PTH) and calcitonin levels were made in a patient with hyperparathyroidism due to clear cell hyperplasia. Although serum Ca declined to normal levels prior to parathyroidectomy, serum Ca++ and PTH levels remained elevated until after operation. This suggests that the calcium ion electrode may prove valuable in the diagnosis and management of patients with hyperparathyroidism, especially when they are normocalcemic. The excised parathyroid glands yielded very low PTH concentrations upon extraction, implying that in this form of hyperparathyroidism PTH secretion is excessive whereas storage is markedly reduced. CT levels were normal in both serum and excised glands. This finding is of interest in relation to the patient’s associated generalized neurofibromatosis and serves further to distinguish the present syndrome from that characterized by mucosal neuromas, hyperparathyroidism and medullary carcinoma of the thyroid. The diagnosis of primary hyperparathyroidism in cases without definite or constant hypercalcemia has proved to be difficult despite the introduction of many potential diagnostic refinements [3-71. The combined application of methods for measuring serum ionized calcium (Cat+) and parathyroid hormone (PTH) should lead to earlier and more definitive diagnosis while improving our understanding of the disease process. We report a case which illustrates the value of these two technics. Serum total calcium (Ca) was increased on admission but then spontaneously fell to within the normal range. On the other hand, Ca++ and PTH levels remained elevated until after parathyroidectomy. cised glands revealed

Histopathologic examination of the exclear cell hyperplasia. The hyperplastic

tissue was extracted and assayed for PTH. The concentration of hormone in the tissue was about 1,000 times less than that stored in normal human parathyroid glands. This case is of additional interest because of associated hereditary neurofibromatosis, an association apparently not recognized previously.

January

1973

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Volume 54

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CALCIUM

ION AND PARATHYROID

HORMONE

IN HYPERPARATHYROIDISM-DAWKINS

ET AL.

CASE REPORT This 34 year old white woman (R.K.) was referred to The Lemuel Shattuck Hospital in September 1968 for investigation of hypercalcemia. She had been well until June 1967 when she noticed numbness of lips and malar areas. These symptoms gradually disappeared. In November 1967 she was said to be asymptomatic and free of any objective abnormalities on physical examination. Later that month, however, she went to a psychiatric hospital because of increasing anxiety and anorexia. Schizophrenia was diagnosed and outpatient treatment with 400 mg chlorpromazine daily was begun. In January 1968 a nodule was felt in the left lobe of the thyroid. Investigation then revealed serum total calcium values of 13.6, 11.0, 11.4, 10.4, 11.0, 12.5, 12.0 mg/lOO ml and corresponding serum inorganic phosphorus values of 3.6, 1.8, 2.5, 2.8, 2.2, 2.5, 2.4 mg/lOO ml. Thyroid function tests were normal. Thereafter the patient was maintained on chlorpromazine. No further studies were undertaken until August 1968 when serum calcium values were 12.0 and 13.0 mg/lOO ml. She was then referred to The Lemuel Shattuck Hospital. Additional history obtained on admission included mild thirst, polyuria and constipation for one year. The patient had been aware of many scattered brown spots and pearly lumps on her skin for many years. Family history revealed a mother and nephew with generalized neurofibromatosis and a brother with muscular dystrophy. There was no family history of cancer, parathyroid, thyroid or adrenal disease. On physical examination, pulse rate was 9Z/minute, blood pressure 110/70 mm Hg and weight 97 pounds. There were many cafe-au-lait spots typical of generalized neurofibromatosis. In addition there were multiple neurofibromas over the trunk and limbs, but no mucosal neuromas were seen. A moderately firm nodule approximately 1.5 cm in diameter was felt at the lower pole of the left lobe of the thyroid. There was no clinical evidence of thyroid dysfunction. Neurologic examination was unremarkable except for slight facial asymmetry. There was no definite evidence of schizophrenia. Urinalysis and blood urea nitrogen, creatinine, uric acid, electrolyte and fasting blood sugar levels were normal. The hematocrit was 39 per cent, white blood cell count 6,050/mm3, protein-bound iodine 5.2 pg/ 100 ml, T3 resin uptake low normal, thyroid antibodies negative, alkaline phosphatase normal, albumin 4.1 gm/lOO ml, globulin 2.7 g/100 ml with normal electrophoresis, lumbar puncture normal (protein 41 mg/lOO ml, creatinine clearance 116 ml/minute. The 24-hour urinary excretion of 5-hydroxyindole acetic acid, vanillylmandelic acid and 17-keto and 17-hydroxysteroids was normal. Glucose and tolbutamide tolerance tests were normal. Serum calcium values and related data are presented later under principal studies. Chest roentgenogram was normal except for a 1.5 by 3 cm soft tissue density in the fifth left intercostal space in the mid-axillary line, presumed to be an inter-

I/

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1973

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costal neurofibroma. Review of chest roentgenograms obtained in January 1968 revealed no change in size. Barium swallow demonstrated slight displacement and irregularity of the esophagus consistent with a left thyroid or parathyroid mass. An x-ray series of the upper gastrointestinal tract with follow-through and barium enema were normal. Films of the skull (including internal auditory meati), hands, spine and pelvis were normal. There was no roentgenologic evidence of parathyroid or metastatic disease. Intravenous pyelogram and bilateral mammograms were normal. An electrocardiogram and pulmonary function tests were normal. Biopsy of a typical skin tumour revealed a benign neurofibroma. The patient remained essentially asymptomatic except for some anorexia. Her weight progressively fell and was 90 pounds at the time of discharge on November 18, 1968. Chlorpromazine dosage was gradually decreased and finally discontinued on October 27, 1968. Over the next week sedatives were necessary intermittently to control anxiety, but frank psychosis did not appear. By November 1, the hematocrit value had fallen to 31 per cent. Peripheral smear and bone marrow aspirate were not diagnostic; serum iron was 36 pg/lOO ml and iron binding capacity 184 pg/lOO ml. Two units of packed red blood cells were given on November 2. On November 4 the neck was explored. A lobulated, encapsulated, yellow tumour (2 by 1.5 cm) was found displacing the inferior pole of the left lobe of the thyroid. This was excised together with adjacent thyroid tissue. The right inferior parathyroid gland measured 0.5 by 0.7 cm and was also removed. The right superior gland appeared normal and was left intact. A left superior gland was not identified. Section of the encapsulated nodule at the lower pole of the left thyroid gland revealed a soft mass with a homogeneous, gray surface. The right inferior parathyroid was similar in appearance. Microscopic examination (Figure 1) of both glands revealed very large

HORMONE

IN HYPERPARATHYROIDISM-DAWKINS

ET AL.

1. Clear cell hyperplasia of parathyroid g/and. Original magnification X 270, reduced 30 per cent.

Figure

cells with clear cytoplasm and small chromatin-rich nuclei arranged in places in an acinar pattern-findings characteristic of primary clear cell hyperplasia. At the periphery of both masses were strands of chief cells without any intervening fat cells. These cells were not interpreted as normal but rather as hyperplastic chief cells. The postoperative course was uncomplicated except for mild hypocalcemia (Figure 2). Reinstitution of tranquilizers or sedatives was not considered necessary. Anorexia diminished somewhat, and the patient was discharged on November 18, 1968. She was then lost to follow-up until April 1969 when she returned after swallowing a razor blade with suicidal intent. There was no evidence of recurrent hyperparathyroidism. Serum ionized and total calcium concentrations were below normal (Table I). Serum PTH level was normal. Three weeks later she was killed by a subway train.

( R.K. 34

mM

)

W 0

PARATHYROIO REMOVED

L-k+&-,;_____---_____ *

- - - -

95

Serum total calcium (Caj and ionized calcium (Ca++) before and after parathyroidectomy. 2.

I8

I 22

26

SW-t.

30

1973

_-A

1

5

6

7

24

40

72

NO”.

OCI.

HRS. POST-OP

January

Co

INTERVAL ----

-_-_-_-------

Figure

%

NORMAL

0

12

The American Journal of Medicine

Volume 54

96

121

CALCIUM

ION AND

.Ca

PARATHYROID

HORMONE

IN HYPERPARATHYROIDISM-DAWKINS

ET AL

( R.K. 34 W 9 1

co*+ I

mM

2.9-

1.6-

2.8-

l.5-

2.7-

1.4 -

2.6-

l.3A = Total Calcium 0 =Ionized ”

2.5-

1.2-

2.4-

I.1 -

95 Y NORMAL Co** CONFldENCE INTERVAL

Figure -30

0

+30

STUDIES

Methods. cium

90

(MINUTES 1

TIME

PRINCIPAL

60

Methods

have

been

for

the fractionation

described

of serum

in detail

elsewhere

cal[2,8].

calcium [Ca] in serum and ultrafiltrates was measured by ethylene diamine tetraacetate (EDTA) titration using Cal-Red as indicator (serum 95 per cent normal interval, mean f 2 standard deviations [SD] = 2.477 f 0.286 mM). Ionized calcium (Ca++) in Total

serum

and

through

Inc.

serum

1.26

mM

with

a

(Cambridge,

with

previously

subjects

f

cent

Serum

2 standard

serum

(CaR),

trafiltrate

Serum

biuret

method

Ca -

= 0.98

(normal [9]

and

(CaProt), Ca (normal

*

0.074

f

(TP)

Ca

2 SE = 0.34 was

albumin

mM).

complexes

as ultrafiltrate

mean

protein

obtained

as described

calcium

calcium

and

of -

f

measured

(Alb)

i.e.,

1.01 to

continuous

ultrafiltrate

[SE]

calculated

total

mM,

were

using

dioxide

of diffusible

was

Ca++

mM).

cell

carbon

errors

concentration

Orion

laboratory,

0.126

of serum

protein-bound

as serum

from

In our f

a flow-

lie in the range

dialysis

5 per

[2].

calculated

mean

Mass.).

Ultrafiltrates

with

obtained

2 SD is 1.136

high-pressure

gassing

The

f

of normal [2,8].

measured

electrode

Ca++

95 per cent

was

was

ion-exchange

Research mean

ultrafiltrates

ul0.04

by the

globulin

by

electrophoresis. Parathyroid tracted say

[lo-121.

equivalents roid tissue containing 1 ml/100 boiling

was

Results

mg fresh was

30 minutes used

(PTH)

or in tissue are

was

extracts

measured

in unex-

by radioimmunoas-

expressed

in nanogram

(ng)

of bovine PTH. The hyperplastic parathywas homogenized in O.lN hydrochloric acid cysteine hydrochloric acid (5 mg/ml) using

water

material for

hormone

serum

bath

tissue. for

removed

The

Similar

clear control

pared glands,

from normal hyperplastic

postmortem glands (either

122

January

The American

1973

was and

by centrifugation

at 4°C.

for assay.

The extract

15 minutes,

heated

the

at 10,000

supernatant extracts

in a

insoluble X g

solution were

pre-

human parathyroid from patients with

Journal

of Medicine

Volume

3.

EDTA

infusion.

Serum

total (Ca) and ionized calcium (Ca+f) fell during infusion. (Note: Ca’ i was measured using heparinized whole blood.)

chronic renal disease or chief cell hyperplasia) and parathyroid adenomas. Assays of serum or tissue extract for calcitonin were initially made by biologic assay [13]. Recently, however,

the

method

for

examine

some

development

human

calcitonin

samples

of

a

[14]

by this more

radioimmunoassay made

it possible

sensitive

to

method.

RESULTS

Preoperative Serum Ca and Ca+ +. Ca and Ca++ values are summarized in Figure 2. On admission, Ca was 3.01 mM (12 mg/lOO ml) or i-3.7 SD, and Ca++ was 1.56 mM or +6.7 SD. In terms of standard deviations, the elevation of Ca++ was about twice as great as that of Ca and remained so during the 45 days prior to surgery. Both Ca++ and Ca declined during the preoperative period, however, and after the first 27 days Ca was either normal or borderline. In contrast, Ca++ remained consistently well above the upper limit of the normal range until surgery. Other Preoperative Data. Serum phosphorus varied from 2.1 to 2.8 mg/lOO ml. Urinary calcium (after 200 mg calcium diet for seven days and while the patient was receiving chlorpromazine) was 520 mg/24 hours and urinary hydroxyproline (after gelatine-free diet for two days) was 44 mg/24 hours (normal 20 to 30 mg/24 hours). Tubular reabsorption of phosphate was 68 per cent on one occasion and 79 per cent on another. Cerebrospinal fluid Ca and Caf+ were 1.30 and 0.94 mM when corresponding serum values were 2.68 and 1.54 mM. In an effort to stimulate the release of PTH, an intravenous infusion of di-sodium EDTA (ethylene diamine tetraacetate) was given and Caf+ was

54

CALCIUM

ION AND

PARATHYROID

monitored with the flow-through electrode at 15 minute intervals, using heparinized whole blood.” To our knowledge, these data (Figure 3) represent the first measurements of Ca++ upon administration of EDTA in vivo. The infusion consisted of 20 mg/kg EDTA in isotonic sodium chloride during the first half hour and 40 mg/kg in the second half hour; blood was obtained from an indwelling catheter in the opposite arm. There was a prompt and parallel decrease in both Ca++ and Ca concentrations. Ca measured by atomic absorption spectroscopy (AAS) but not shown in Figure 3, remained quite constant; the difference between AAS and EDTA methods reflected calcium chelated by administered EDTA. A glucagon infusion (1.2 mg in 150 cc isotonic sodium chloride over two hours) produced the expected glucose response but did not significantly affect Ca or Ca++ and did not lead to the appearance of measurable calcitonin in serum as measured by bioassay. Postoperative Studies.

Serum

Ca+tf

and

Ca de-

clined after parathyroidectomy (Figure 2 and Table I). Both were below the respective normal confidence intervals one day postoperatively. In the five months postoperatively, values were consistently normal or low. Serum phosphorus gradually increased to 4.3 mg/lOO ml 12 days post-

HORMONE

TABLE

II

IN HYPERPARATHYROIDISM-DAWKINS

ET AL.

Parathyroid Hormone Concentrations man Parathyroid Tissue

in Hu-

Parathyroid Hormone*

(ng/mg fresh tissue)

No. of

Specimen

Samples

Patient R.K. Normal glands Hyperplastic glands Adenomas

1 8 7 9

_

* Expressed

as equivalents

Mean

Range

0.13 155 323 109 ~_--~._~~-__~-~ of bovine PTH.

... 72-360 114-480 15-260

50.7 and 45.0 per cent and for CaProt of 33.0 and 30.0 per cent. The relative contribution of CaR was somewhat increased postoperatively. Total ultrafiltrable calcium, representing the sum of Ca++ and CaR, did not change significantly; prior to the operation an average of 66.8 per cent of the serum Ca was ultrafiltrable whereas after the operation it was 69.6 per cent. Previous studies [2] in normal serum have shown that (1) about 80 per cent of CaProt is bound to albumin and the remaining 20 per cent to globulin(s), (2) the over-all dissociation constant for CaAlb pi Ca++ + Albis about 6.55 X 1O-3 (pK’ = 2.18) and (3) that about 8.4 negative sites per albumin molecule are available for calcium-binding. In any given serum sample, K’ may therefore

be evaluated:

operatively.

Ca+’ [nAlb - CaAlbl K’ = -~~-~-CaAlb

The extract of the hyperplastic parathyroid tissue contained only 0.13 ng PTH/mg fresh tissue, a value which was many times below that found in other types of human parathyroid tissue (Table II). No calcitonin was detected when this extract was assayed biologically at a dose level equivalent to 50 mg fresh Calcium Fractions

tissue per assay rat. of Serum. The three

I .6

calcium

.

SERUM

fractions of serum (1) ionized (Ca++), (2) complexed (CaR) and (3) protein-bound calcium (CaProt) were measured in three samples preoperatively and in two samples postoperatively. Parathyroidectomy resulted in significant reduction in Ca and in each of the three calcium fractions of serum (Table I). In contrast to these absolute changes, the relative proportions (Table I) of Ca++ and CaProt were similar pre- and postoperatively, with respective mean values for Ca++ of

co++

1.2

mM I.0

0.8

0

I

( mMg :“As noted elsewhere 12,8]. Ca++ lower in heparinized whole blood than rum, technic Caf+,

owing

to formation

oi

is about 3 per in corresponding

a calcium-heparin

complex.

is quite satisfactory, however, for following change as in this case, provided the heparin concentration

successive

samples

cent seThe in in

3

2 SERUM

4

PTH

/ml

)

Comparison of serum ionized calcium Figure 4. (Ca++) and parathyroid hormone (PTH) in five preoperative and two postoperative samples. The horizontal shading indicates the 95 per cent normal confidence interval for Cat + and vertical shading the normal range for PTH.

is constant.

January

1973

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123

CALCIUM

TABLE

ION AND

III

PARATHYROID

Calcitonin

HORMONE

IN HYPERPARATHYROIDISM-DAWKINS

Levels in Serum Ca++

Time ofSample Preoperative 46day 7day 1/2hour Postoperative lhour lday 4days 156 days Normal

range

Calcitonin

$I,

(mW

3.01 2.65 2.55

1.56 1.42 1.31

0.18 0.18 0.20

2.51 2.03 2.11 2.17

1.18 0.90 1.04 0.94

0.30 0.29 0.27 0.18

2.19-2.76

1.01-1.26

0.02-0.35

@s/ml)

where Alb is the molar albumin concentration and n = 8.4. Assuming that the molecular weight of albumin is 69,000 and CaAlb = 0.80 CaProt, calculated K’ values are given in Table I. The mean K’ value preoperatively was 8.25 X 10d3 (pK’ = 2.08) which did not differ significantly from the mean postoperative value of 7.80 X 10M3 (pK’ = 2.11). Ca++ and PTH. Serum Ca++ and PTH levels were measured in five serum samples preoperatively and in two samples postoperatively. Results are shown in Figure 4, in which the normal limits for each of the two measurements are also given. With the exception of one point, there appeared to be a relationship between Ca++ and PTH. Thus, in the five preoperative specimens the Caf+ level was elevated in all, whereas the PTH level was elevated in four of the five specimens. In the two postoperative samples, PTH was within normal limits, Caf+ was normal in one and slightly decreased in the other. Calcitonin in Serum. Preoperative serum was assayed biologically for calcitonin content, and no hypocalcemic activity was detected at dose levels of 0.5 to 0.6 ml/assay rat. Recently, a number of samples which had been stored at -70°C were examined by the more sensitive radioimmunoassay method. The results are given in Table I I I. All values were within the normal range (0.02 to 0.35 ng/ml). A small, transient increase in serum calcitonin occurred immediately after operation, possibly due to release from the thyroid gland which was manipulated extensively during surgery. COMMENTS This case is of particular interest for two reasons: (1) it is the first case of hyperparathyroidism of which we are aware in which Ca++, PTH and

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ET AL.

calcitonin levels have been serially monitored both pre- and postoperatively; and (2) the patient, while under observation, showed a progressive decrease in serum Ca to within the normal range while both Ca++ and PTH levels remained elevated. The diagnosis of primary hyperparathyrpidism was established by the demonstration of elevated Caf+ levels, hypophosphatemia, hypercalciuria, hyperhydroxyprolinuria, decreased tubular reabsorption of phosphate and an elevated level of circulating PTH. The sharp decrease in Ca++, Ca and PTH after removal of two hyperplastic parathyroid glands provides confirmation. Such cases of “normocalcemic primary hyperparathyroidism” have been frequently reported [3] but preoperative proof of the diagnosis has always been difficult. The measurement of Ca++ has been recommended in this situation [15-171 but experience has been limited because of the complexity of previously available methods. The flowthrough ion-exchange calcium electrode allows the rapid direct determination of Ca++ in serum, analogous to a blood pH measurement. The measurement of PTH by radioimmunoassay has also been recommended [5-7, 181. However, since the method depends on immunologic rather than on biologic activity [19], we believe that a sensitive and direct measure of the result of hormonal activity should be estimated simultaneously. Serum Ca++ appears to be the most satisfactory measure available. The decline in serum Ca preoperatively appeared to be due to reductions in both Ca++ and CaProt. Although Ca+f levels remained elevated above the normal confidence interval they did show a downward trend (Figure 2). (Table I) Alb also declined preoperatively. Since CaProt is dependent on both Ca++ and Alb we presume that CaProt fell. The measurements of CaProt did not reflect this change, but ultrafiltration was not performed on the occasions when Alb was lowest (Table I). Since Ca is largely determined by the sum of Ca+f and CaProt we believe that the measured reduction in Ca++ and the deduced reduction in CaProt adequately account for the observed decline in Ca. Lloyd and Rose [15] suggested that PTH decreases the affinity of the serum proteins for calcium. In patients with hyperparathyroidism, but not in other hypercalcemic states, Lloyd, Rose and Smeenk [16] found that observed CaProt levels were lower than those predicted by the masslaw equation at given total protein levels. Data in the present case do not suggest an appreciable

Volume 54

CALCIUM

ION AND PARATHYROID

effect of PTH on protein binding and therefore support the findings of Walser [20]. Calculated K’ values were similar pre- and postoperatively, with mean pKs of 2.08 and 2.11, respectively. These values are similar to the value of 2.18 obtained in normal serum with the electrode [2], 2.22 with the frog-heart and 2.18 by ultracentrifugation [21], Our case is of additional interest in terms of the finding of clear cell hyperplasia of two parathyroids. This pathologic variant was first described in 1934 [22]. Since that time many additional cases have been reported [23]. Recent evidence suggests that the clinical behaviour in such cases may differ from that associated with the more common chief cell hyperplasia and with adenoma. For example, the hypercalcemia appears to be less pronounced [24,25]. Our finding of an extremely low concentration of PTH in the parathyroid gland is consistent with the electron microscopic data: although secretory granules are seen in normal and adenomatous glands they were not found in two cases of primary clear cell hyperplas,ia [26,27], The combination of high circulating PTH and low gland concentration suggests that the hormone was being excessively secreted but not stored in the gland. That the serum calcitonin levels in this patient were normal preoperatively when she was hyper-

HORMONE

IN HYPERPARATHYROIDISM-DAWKINS

ET AL.

calcemic may appear surprising. There is little doubt that acute hypercalcemia can stimulate calcitonin release [28]. However, we have noted consistently normaltcalcitonin levels in 17 patients with chronic hypercalcemia and hyperparathyroidism [14]. Apparently, the thyroid C cells in man do not continue to secrete large amounts of calcitonin in the face of prolonged hypercalcemia. Finally, the association with hereditary neurofibromatosis deserves comment. The syndrome of hyperparathyroidism, mucosal neuromas and calcitonin-secreting medullary carcinoma of the thyroid is now well known [lo, 121. In our case there was no evidence of either mucosal neuromas or medullary carcinoma. We are not aware of previously published evidence suggesting that there may be a significant association between hyperparathyroidism and hereditary generalized neurofibromatosis. ACKNOWLEDGMENT We wish to thank Dr. G. Curtis for providing the histopathologic sections of the parathyroid glands; Dr. E. Sion who performed the surgery; Dr. R. Neer for advice on the use of EDTA infusion; Drs. C. Guzzo and L. Arango for clinical assistance, and E. F. Voelkel, S. Lipchus, C. Benjamin and D. W. Bottomley for their able technical assistance.

REFERENCES 1.

2.

3.

4.

5. 6.

7.

8.

RL, Moore EW: A case of hyperparathyroidism with serial determinations of ionized calcium (abstract). Clin Res 17: 283, 1969. Moore EW: Studies with ion-exchange calcium electrodes in biological fluids: some applications in biomedical research and clinical medicine. Ion-Selective Electrodes (Durst R, ed), National Bureau of Standards Special Publications No. 316, Washington, D.C., 1969. Wills MR, Pak CYC, Hammond WG, Bartter FC: Normocalcemic. primary hyperparathyroidism. Amer J Med 47: 384, 1969. Strott CA, Nugent CA: Laboratory tests in the diagnosis of hyperparathyroidism in hypercalcemic patients. Ann Intern Med 68: 188. 1968. Avioli LV: The diagnosis of primary hyperparathyroidism. Med Clin N Amer 52: 451, 1968. Reitz RE, Pollard JJ, Wang C, Fleischli DJ, Cope 0, Murray TM, Deftos LJ, Potts, JT: Localization of parathyroid adenomas by selective venous catheterization and radioimmunoassay. New Eng J Med 281: 348, 1969. Reiss E, Canterbury JM: Primary hyperparathyroidism: application of radioimmunoassay to differentiation of adenoma and hyperplasia and to preoperative locafization of hyperfunctioning parathyroid glands. New Eng J Med 280: 1381,1969. Moore EW: Ionized calcium in normal serum. ultrafilbates and whole blood determined by ion-exchange Dawkins

January

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15.

16.

1973

calcium electrodes. J Clin Invest 49: 318, 1970. Gornall AG, Bardawill CJ, David MM: Determination of serum proteins by means of the biuret reaction. J Biol Chem 177: 751, 1949. Melvin KEW, Tashjian AH Jr: The syndrome of excessive thyrocalcitonin produced by medullary carcinoma of the thyroid. Proc Nat Acad Sci USA 59: 1216, 1968. Tashjian AH Jr, Frantz AG, Lee JB: Pseudohypoparathyroidism: assay of parathyroid hormone and thyrocalcitonin. Proc Nat Acad Sci USA 56: 1138, 1966. Tashjian AH Jr, Melvin KEW: Medullary carcinoma of the thyroid gland. New Eng J Med 279: 279, 1968. Cooper CW, Hirsch PF. Toverud SU, Munson PL: Improved method for biological assay of thyrocalcitonin. Endocrinology 81: 610, 1967. Tashjian AH Jr, Howland BG. Melvin KEW, Hill CS Jr: Immunoassay of human calcitonin: clinical measurement, relationship to serum calcium and studies with patients with medullary carcinoma. New Eng J Med 283: 593, 1970. Lloyd HM, Rose GA: lonrzed, protein-bound and complexed calcium in the plasma in primary hyperparathyroidism. Lancet 2: 1258, 1958. Lloyd HM, Rose GA, Smeenk D: The ability of plasma proteins to bind calcium in normal subjects, in patients with primary hyperparathyroidism both preand postoperatively and in other hypercalcemic conditions. Clin Sci 22: 353. 1962.

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17.

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21.

22.

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Fanconi A, Rose GA: The ionized, complexed and protein-bound fractions of calcium in plasma. Quart J Med 27: 463.1956. Reiss E, Canterbury JM, Egdahl RH: Experience with radioimmunoassay of parathyroid hormone in human sera. Trans Ass Amer Physicians 61: 104, 1966. Berson SA, Yalow RS: lmmunochemical heterogeneity of parathyroid hormone in plasma. J Clin Endocr 26: 1037,1968. Walser M: The separate effects of hyperparathyroidism, hypercalcemia of malignancy, renal failure and acidosis on the state of calcium, phosphate and other ions in plasma. J Clin Invest 41: 1454, 1962. Loken HF, Have1 RJ, Gordan GS, Whittington SL: Ultracentifugal analysis of protein-bound and free calcium in human serum. J Biol Chem 235: 3654, 1960. Albright F, Bloomberg E, Castleman B, Churchill ED: Hyperparathyroidism due to diffuse hyperplasia of all parathyroid glands rather than adenoma of one: clini-

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23.

24. 25.

26.

27. 26.

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ET AL

cal studies on three such cases. Arch Intern Med (Chicago) 54: 315, 1934. Castleman B, Cope 0: Primary parathyroid hypertrophy and hyperplasia. A review of 11 cases at the Massachusetts General Hospital. Bull Hosp Joint Dis 12: 368,195l. Strauss FH, Paloyan E: The pathology of hyperparathyroidism. Surg Clin N Amer 49: 27, 1969. Werdelin 0: The parathyroids in primary hyperparathyroidism. A histological study of 35 cases. Acta Path Microbial Stand 61: 211. 1964. Holzmann VK, Lange R: Zur Zytologie der glandular Parathyreoidea des Menschen. Weitere Untersuchugen an Epithelkdrperadenomen. Zellforsch 56: 759, 1963. Sheldon H: On the water-clear cell in the human parathyroid gland. J Ultrastruct Res 10: 377, 1964. Hirsch PF, Munson PL: Thyrocalcitonin. Physiol Rev 49: 548, 1969.