Department of Surgery, Lund University, Lund, Sweden
(Received November 13, 1992/March 11, 1993)
Summary: The introduction of assays for the intact parathyrin (parathyroid hormone) has dramaticallyimproved the diagnosis and follow-up of patients with primary hyperparathyroidism. However, in somepatients with mild or intermittent hypercalcaemia, when plasma concentrations of intact parathyrin may bewithin the normal reference concentrations, the diagnosis of primary hyperparathyroidism may still beproblematic. In these patients, the EDTA-infusion test is of potential value, as it also might be in patientswith equivocal operative findings.
Definition and Diagnosisof Primary Hyperparathyroidism
Primary hyperparathyroidism is characterized by in-creased secretion of parathyrin (parathyroid hor-mone) in combination with increased parathyroid par-enchymal cell mass. In primary hyperparathyroidismthe secretion of parathyrin is higher than that requiredfor the maintenance of nörmocalcaemia, which leadsto manifest hypercalcaemia. The biochemical diag-nosis of primary hyperparathyroidism, therefore, con-sists in demonstrating hypercalcaemia in combinationwith a relative or absolute increase in plasma concen-trations of parathyrin.
Hypocalcaemia Provocation Tests
The demonstration of increased plasma concentrationof parathyrin is vital for the differential diagnosis ofprimary hyperparathyroidism from hypercalcaemiadue to other diseases. This used to be a clinical prob-lem, since the radioimmunological assays used for thedetermination of parathyrin also measured fragmentsof the hormone, the concentration of which exceedthat of the intact hormone (1). Other tests were there-fore introduced and initially held to be of value in the
diagnosis of primary hyperparathyroidism — e.g.,determination of urinary excretion of calcium, hy-droxyproline or cyclic AMP, measurement of the uri-nary chloride to phosphate ratio or of the circulatingconcentrations of vitamin D metabolites or osteocal-cin. These tests have failed, however, to contributeany useful diagnostic information (2). Similarly, ve-nous catheterization for measurement of the localconcentration of parathyrin has not been found to beof diagnostic value, although such a procedure mightbe of use in the preoperative localization of the en-larged parathyroid gland (3). To improve the diag-nosis of primary hyperparathyroidism other testsseemed to be required, and hypocalcaemia provoca-·tion tests were developed. In these tests, hypocalcae-mia is induced, which in turn stimulates parathyrinsecretion. The hypocalcaemia was induced by calci-tonin or EDTA (ethylenediamine tetraacetate) (4 — 6).
The theoretical basis for the hypocalcaemia provo-cation tests is the inverse S-shaped relationship be-tween the extracellular concentration of calcium andparathyrin secretion (fig. 1 (7)). The basis for thisrelation is the activation by extracellular calcium ofa membrane-bound receptor in the parathyroid cells(fig. 2). This leads to opening of receptor-operated
Fig. 1. The relation between the extracellular concentration ofionized calcium and the secretion of parathyrin. Theinverse, S-shaped relation is mathematically describedby a 4-quantity relation involving the quantities A, B,D and the "set point". For a detailed explanation seethe text.
membrane-bound calcium channels, which leads inturn to uptake of extracellular calcium. Activation ofthe calcium receptor also initiates phosphoinositidehydrolysis, resulting in formation of inositol-1,4,5-trisphosphate, with the subsequent liberation of cal-cium from intracellular calcium storage sites. Boththese processes increase the cytoplasmic concentrationof calcium, which is followed by inhibition of theparathyrin secretion. Conversely, during hypocalcae-mia the intracellular calcium concentration is reduced,which stimulates parathyrin secretion.
Figure 1 shows that the steepest part of the relationbetween extracellular calcium and parathyrin secre-tion is close to the calcium concentration that inhibitsthe parathyrin secretion by 50°^o. This calcium valueis called the "set point", and it is close to the concen-tration of plasma ionized calcium that is normallyseen (8). The curvilinear relation between calcium andparathyrin has been characterized by four differentquantities, as shown in figure 1. Quantity A is themaximal parathyrin secretion obtained during hypo-calcaemia, whereas quantity D is the minimal secre-tion seen after maximal hypercalcaemia. Quantity Cis the "set point" and quantity B is a quantity relatedto the slope of the curve at the "set point". Thesecretion of parathyrin (i. e., Y) is dependent on theextracellular calcium (i.e., X) according to the relationΥ = A^D/[1 + (X/C)B] + D (7).
Changes in the Calcium-Parathyrin-Relation in Pri-mary Hyperparathyroidism
The normal relation between extracellular calciumand parathyrin secretion is altered in patients withprimary hyperparathyroidism, since calcium does notnormally inhibit parathyrin secretion. There existsevidence that this defective sensitivity to calcium isdue to a down-regulation of the calcium receptor inthe parathyroid cell (9). Thus, to obtain a sufficiently
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high intracellular calcium concentration to induce in-hibition of parathyrin secretion, a higher extracellularconcentration of calcium is required in primary hy-perparathyroidism than under normal conditions.Consequently, a patient with primary hyperparathy-roidism has a right-shifted relation between extracel-lular calcium and parathyrin secretion when com-pared to a healthy individual, i.e., primary hyperpara-thyroidism is associated with an increased "set point"(fig. 3).
0.00.10 1.20 1.30 UO 1.50 1.60
Ionized calcium [mmol/fj
Fig. 3. The right-shifted inverse S-shape relationship betweenextracellular calcium and the parathyrin secretion inpatients with primary hyperparathyroidism (right curve)compared with healthy subjects (left curve).
By reducing the extracellular calcium concentration,by means of calcitonin of EDTA, and by measuringthe plasma parathyrin concentrations, the highestconcentration of calcium that stimulates the para-thyrin secretion can be measured: in primary hyper-parathyroidism this occurs at calcium concentrationsexceeding the upper reference concentration for nor-mality. In contrast, in patients with hypercalcaemiadue to other causes, parathyrin secretion is not stim-ulated until the calcium concentration has droppedto concentrations within the normal -range. The hy-pocalcaemia provocation tests, therefore, are able todistinguish hypercalcaemic patients with primary hy-perparathyroidism from those with malignancy, forexample (4—6).
Intact Parathyrin
In the late 1980s new immunoradiometric and im-munochemilunometric assays for the determinationof parathyrin were introduced. These assays measureintact parathyrin without interfering with inactive
parathyrin fragments (10). With these assays an al-most complete separation is established between hy-percalcaemic patients with primary hyperparathyroid-ism and those with malignancy (10). By the use ofthese new assays for intact parathyrin, the need forhypocalcaemia-provocation tests in the diagnosis ofprimary hyperparathyroidism initially seemed to beeliminated. However, it has been established that thenew assays for intact parathyrin can not completelyseparate patients with primary hyperparathyroidismfrom normal individuals (10). This has been explainedby the high sensitivity of the tests, since parathyrinsecretion under normal conditions is pulsatile with acircadian variation (11). Therefore, the concentrationof intact parathyrin might occassionally be normal ina patient with primary hyperparathyroidism. Thismight present a particular diagnostic problem in pa-tients with mild or intermittent hypercalcaemia, sincethere is a correlation between the calcium concentra-tion and the intact parathyrin concentration (12). Insuch cases, a hypocalcaemia provocation test mightbe useful.
EDTA-Infusion Test
We undertake the EDTA-infusion test followed anovernight fast with the patient placed in a recumbentposition. An antecubital catheter is inserted on bothsides. From one catheter blood samples are takenregularly, whereas the infusions are given through theother catheter. Six g Na2EDTA are dissolved in 250ml saline and infused intravenously with a constantinfusion rate of 24 mg/h · kg body weight, i. e., by 1.0ml/h · kg. During the infusion, local discomfort orpain might be felt in the arm, sometimes radiating tothe shoulder. This pain is reduced by infusing lidocain.We dissolve 200 mg lidocain in 250 ml saline andinfuse up to 20 ml/hour. We have found that the localdiscomfort is usually prevented by preinfusing lido-cain at the rate of 10 ml/h our before starting theEDTA infusion.
EDTA is infused for 70 minutes. Before, during, andafter the infusion, blood samples are taken every 10minutes. One sample is taken anaerobically in pre-heparinised tubes for the determination of ionizedcalcium, whereas another sample is taken for theanalyses of intact parathyrin. Before the test, thekidney function needs to be estimated, since in thecase of kidney damage, the infusion of EDTA mightbe hazardous. We therefore routinely analyse the se-rum concentration of creatinine in all cases before theEDTA infusion test. No other precautions are re-quired, however.
We have shown in a previous paper (13) that, whenusing an EDTA-infusion test in the diagnosis of pri-mary hyperparathyroidism, determination of intactparathyrin is superior to that of the mid-molecule C-terminal parathyrin. We showed that in 15 patientswith primary hyperparathyroidism the concentrationof intact parathyrin rose by 240% during the EDTAinfusion test, whereas the concentration of mid-mol-ecule C-terminal parathyrin rose by only 65%(P < 0.01). Furthermore, in four patients with pri-mary hyperparathyroidism, the concentration of mid-molecule C-terminal parathyrin did not rise at allduring the EDTA infusion, while the concentrationof intact parathyrin rose significantly in these patients.Intact parathyrin should, therefore, be determinedwhen using EDTA-infusion tests.
Figure 4 shows the result of the EDTA-infusion testin a healthy subject compared with that in a patientwith primary hyperparathyroidism. As is seen in theupper panel of the figure, the concentration of ionizedcalcium is reduced by approximately 0.15 mmol/1during the 70 min EDTA-infusion test, and the middlepanel of the figure shows that the concentration of
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0 20 /»OTime [min]
1.0 1.20 UO 1.60Ionized calcium (mmol/[]
Fig. 4. Serum concentration of ionized calcium (upper panel)and plasma concentration of intact parathyrin (middlepanel) before, during and after the 70 min EDTA-infusion test in a patient with primary hyperparathy-roidism (· —·) and in a healthy subject (o—o). Thelower panel shows the values transformed into a cal-cium-parathyrin-relation-curve.
intact parathyrin is increased in both individuals. Inthe patient with primary hyperparathyroidism, theplasma concentration of intact parathyrin alreadystarts to rise when the concentration of ionized cal-cium approaches 1.35 mmol/1 (the upper referencevalue is 1.29 mmol/1), whereas the corresponding cal-cium value in the healthy subject is 1.21 mmol/1. Thisdifference illustrates the increased "set point" in pa-tients with primary hyperparathyroidism and is di-agnostic for the disease. The lower panel of the figureshows the values in these individuals transformed toa calcium^parathyrin^relation-curve. The right-shiftedrelation in the patient with primary hyperparathy-roidism compared with the healthy individual isclearly illustrated.
The interpretation of the EDTA-infusion test is moredifficult in patients with mild hypercalcaemia withonly a slightly increased "set point". Figure 5 showssuch a patient. The figure shows the concentration ofintact parathyrin in relation to the concentration ofionized calcium during and immediately after theEDTA-infusion test. It can be seen that when theplasma concentration of intact parathyrin starts torise the concentration of ionized calcium is within thenormal reference values. It is, however, known thatwhen the calcium concentration is changed veryslowly or not at all, as is the case in the sample takenimmediately after the end of the EDTA-infusion test,the concentration of intact parathyrin shows its clos-est correlation to the absolute calcium concentration(14). Thus, the diagnostic efficiency is highest whenthe concentration of intact parathyrin is interpretedin the samples taken immediately after the end of theinfusion of EDTA: if intact parathyrin concentrationsthen are raised and the calcium concentration is nor-mal, the patient has primary hyperparathyroidism,whereas if intact parathyrin is within normal concen-trations, the patient does not have primary hyperpara-thyroidism (12). The patient in figure 5, therefore, has
1.10 1.20 1.30 1.40Ionized calcium ljpmol/ϊ]
Fig. 5. The relation between the plasma concentration of intactparathyrin and the serum cqncentration of calcium dur-ing (·-·) and immediately after (o—o) the EDTA-infusion test in a patient with mild primary hyperpara-thyroidism.
primary hyperparathyroidism, since intact parathyrinin the last sample is 7.4 pmol/1 (upper reference value5.0 pmol/1), whereas the ionized calcium concentra-tion is normal (1.22 mmol/1).
The Use of the EDTA-infusion Test in the Diagnosisof Primary Hyperparathyroidism
To distinguish hypercalcaemic patients with primaryhyperparathyroidism from those with malignancy, de-termination of the basal concentration of intact par-athyrin is usually sufficient (10). The clinical problemtoday is distinguishing patients with mild primaryhyperparathyroidism from normal individuals. Thesepatients have mild, or even only intermittent, hyper-calcaemia, but the basal concentration of intact par-athyrin is within the normal limits. They may or maynot be symptomatic. Recent studies have suggestedthat this patient group is not small (15). The EDTA-infusion test might be diagnostic in these patients.Hence, even though the introduction of assays forintact parathyrin has markedly improved the diag-nostic accuracy for primary hyperparathyroidism,there is still a need for hypocalcaemia provocationtests in a subgroup of patients with primary hyper-parathyroidism.
We recently examined the value of intact parathyrinin 26 consecutive patients with hypercalcaemia andsuspected primary hyperparathyroidism, of whom allexcept one at operation also were demonstrated tohave the disease (12). We used a commercially avail-able immunoradiometric assay for intact parathyrinwith an inter-assay variation of < 10% and an intra-assay variation of < 6%, when expressed as coeffi-cients of variation. We found that in six of the 25cases shown to have primary hyperparathyroidism,the intact parathyrin concentration was within thenormal range. This high incidence of patients withnormal concentrations of intact parathyrin is in agree-ment with a recent study (10), but in contrast withearlier reports where the incidence of primary hyper-parathyroidism with normal intact parathyrin con-
centrations was lower. A likely explanation for thisdifferences would be different selections of patients indifferent studies. In five of our cases with normalvalues of intact parathyrin we performed the EDTA-infusion test. The test was positive, i.e., diagnosticfor primary hyperparathyroidism, in all five patients,who at operation were shown to have the disease.Thus, our experience is that the EDTA-infusion testis of diagnostic value in this subset of patients with'suspected primary hyperparathyroidism.
The EDTA-infusion Test in the Follow-up of Patientswith Primary Hyperparathyroidism
Both molecular biological (16) and functional (17)studies have presented evidence that parathyroid ad-enomas are of monoclonal origin and that, therefore,the non-adenomatous glands are normal. This indi-cates that the "set point" should not be raised post-operatively in patients with adenomas, as we havedemonstrated as well (17). Hence, in patients operatedon for primary hyperparathyroidism but who haveequivocal operative findings, a postoperative EDTA-infusion test could be of clinical value. If the post-operative uset point" is not raised, it can be concludedthat the operation has been a success.
Conclusion
Our review has shown that, despite the introductionof sensitive assays for intact parathyrin, there is aneed for an EDTA-infusion test in a subset of patientswith suspected primary hyperparathyroidism, mainlyin those with mild or intermittent hypercalcaemia anda normal basal concentration of intact parathyrin.
AcknowledgementThe studies by the authors have been supported by the SwedishMedical Research Council, the Greta and Johan Kocks Foun-dation, and the Faculty of Medicine, Lund University.
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Dr. Bo AhrenDepartment of SurgeryLund UniversityS-221 85 LundSweden
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