broadsheet no 144 july 1994 - from bmj and acpcalcaemia on renal function. hypercalcaemia causes...

J Clin Pathol 1994;47:579-584

ACP Broadsheet No 144

The investigation of hypercalcaemia

P L Selby, P H Adams

IntroductionHypercalcaemia occurs in up to 0-3% of ahealthy population and is even more pre-valent among hospital patients where it mayoccur in up to 3%. This makes hyper-calcaemia one of the most common metabolicdisorders encountered in clinical practice. Itis therefore important to have well developedstrategies for its investigation and manage-ment.

Aetiology and pathogenesisA large number of diseases can lead to hyper-calcaemia (table). In most instances theunderlying cause is either primary hyper-parathyroidism or malignant disease; themany other causes are rarely encounteredeven within specialist units. The prevalenceof primary hyperparathyroidism and hyper-calcaemia of malignancy varies with the pop-ulation sampled. In ambulant subjects in thecommunity who were found to be hypercal-caemic as part of a screening programme,86% were ultimately shown to have primaryhyperparathyroidism and only 2% malignantdisease.' The findings in hypercalcaemicinpatients are different: 58% of patients havemalignant disease and 33% primary hyper-parathyroidism. Indeed, primary hyper-parathyroidism is one of the most commonendocrine diseases with a prevalence ofaround 1-3 per thousand, exceeded only bydiabetes mellitus and thyroid disease.

This Broadsheet has beenprepared by the authors at theinvitation of the Association ofClinical Pathologists whoreserve the copyright. Furthercopies of this Broadsheet maybe obtainedfrom thePublishing Manager, J7ournalof Clinical Pathology, BMAHouse, Tavistock Square,London WClH 9JR

Department ofMedicine, ManchesterRoyal Infirmary,Oxford Road,Manchester M13 9WLP L SelbyP H AdamsAccepted for publication14 December 1993

Causes ofhypercalcaemia

Common:Primary hyperparathyroidismMalignant disease: Solid tumours

Myeloma and other haematologicaltumours

Less common:Sarcoidosis and other granulomatous diseasesThyrotoxicosisVitamin D intoxicationFamilial hypocalciuric hypercalcaemia

Rare:Addison's diseaseImmobilisationThiazide diureticsRecovery from renal failureFollowing renal transplantationMilk-alkali syndromeLithium treatmentHypothyroidismVitamin A toxicity

July 1994

In patients with primary hyperparathy-roidism hypercalcaemia is the direct result ofan increased circulating concentration ofparathyroid hormone (PTH) which leads tohypercalcaemia through a variety of differentmechanisms. In most cases the most impor-tant mechanism is an enhanced renal tubularreabsorption of calcium brought aboutthrough the direct effect of PTH on the distalconvoluted tubule. PTH also acts on theskeleton and stimulates bone resorption andthereby the movement of calcium into theextracellular space. PTH also stimulates therenal production of calcitriol, the activemetabolite of vitamin D, which in turn pro-motes the intestinal absorption of calciumand also bone resorption.Some malignant processes, especially

myelomatosis, lead to the local production ofcytokines which stimulate bone resorption. Inmost patients with malignancy and hypercal-caemia this is brought about by a humoralfactor with actions which closely resemblethose of PTH. This has been identified andcalled parathyroid hormone-related peptide(PTHrP).3 True "ectopic" PTH productionby malignant tumours is rare. PTHrP is con-siderably larger than PTH, but it shares aclose sequence homology at the amino termi-nal, which is responsible for much of the bio-logical activity of PTH. It is not surprising,therefore, that PTHrP can mimic the effectsof PTH, and it is now clear that many casesof hypercalcaemia of malignancy are relatedto the production of this compound bytumour cells. This is particularly the case insquamous cell carcinoma of the lung andbreast carcinoma, including those withmetastatic disease.4

Once hypercalcaemia is established, theserum calcium concentration can remainstable for many years and this is commonlythe case in many patients with mild primaryhyperparathyroidism. In other patients theserum calcium can rise progressively mainlyas a result of the deleterious effects of hyper-calcaemia on renal function. Hypercalcaemiacauses losses of sodium and water througheffects on the renal tubules which lead todecreased glomerular filtration and filtered

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load of calcium. These combine to reduce therenal excretion of calcium and exacerbate thehypercalcaemia still further. This situation of"disequilibrium hypercalcaemia" is a poten-tial threat to life.5

Clinical featuresThe clinical features of hypercalcaemia arelargely non-specific and it is seldom, if ever,possible to be confident of the diagnosis onclinical grounds alone.6 When the serum con-centration of calcium is less than 3 0 mmol/lsymptoms are rare; above this value there isan increasing likelihood of symptoms-notably thirst and polyuria, constipation, nau-sea and anorexia; as the concentration risesfurther (above 3-5 mmol/l) impaired con-sciousness, coma, and even death may occur.

In some patients with malignant disease itcan be difficult to decide on clinical groundswhether the presence of these symptomsrelates to the malignancy itself (or its treat-ment) or signals the onset of hypercalcaemia.In these circumstances the laboratory clearlyhas an important role in diagnosis and man-agement. Patients can also present with thesymptoms of conditions known to be causeddirectly or indirectly by the hypercalcaemia-examples of these include abdominal painassociated with acute pancreatitis, uretericcolic secondary to renal stones, or pseudo-gout associated with chondrocalcinosis.

Investigation ofhypercalcaemiaCONFIRMATION OF DIAGNOSISThe laboratory has a central role in establish-ing the diagnosis of hypercalcaemia. Althoughthe "gold standard" method for the estima-tion of plasma calcium concentration isatomic absorption spectrophotometry, thecresolphthalein complexone based colorimet-ric methods, used by multichannel and dis-crete analysers, give results which are just asreliable for clinical purposes. In both casesthe between batch precision is similar at lessthan 0Q5%.7 As the variation within subjectsin plasma calcium concentration is also smallit can be estimated that a change in plasmacalcium concentration of 5% is likely to beclinically important.8 In the past it has fre-quently been stated that blood samples forthe estimation of calcium concentrationshould be taken in the fasting state and with-out the use of a tourniquet. These conditionsminimise the risk of spurious hypercalcaemia,but the clinical importance of both has beengrossly overstated. There is no evidence thatfasting has any relevant effect on measuredplasma calcium concentrations and even pro-longed venous stasis only increases it by 0-15mmol/l at the most, an effect largely offset bycorrecting for albumin concentration.9 Thuswhile it is advisable to avoid prolongedvenous stasis, the failure to observe either ofthese precautions is unlikely to perturb themeasured concentration of calcium to anyclinically important degree.About 45% of the calcium in serum is

bound to protein (albumin and globulins)and hence unavailable for biological activity.Relatively recently ion specific electrodeshave been developed which measure theactual activity of the calcium ion in biologicalfluids. Although these techniques provide ameasure which is much more closely relatedto the biological activity of calcium, theirclinical usefulness is limited in the routinelaboratory. Measurement of the plasma con-centration of ionised calcium may be of valuein the investigation of the occasional patientwith mild or intermittent hypercalcaemia, andin patients with abnormal plasma proteinswho may develop spurious hypercalcaemia.In most clinical situations measurement ofthe total plasma calcium is much simpler andprovides a reasonable estimate of the ionisedfraction. However, it is usually recommendedthat the measured calcium concentration beadjusted for the prevailing concentration ofplasma proteins although this is unlikely to beof major clinical consequence. An appropriateformula to apply is:

adjusted calcium = measured calcium +0-02* (mean normal albumin-measured albumin)[calcium measured in mmol/l and albumin in g/l]

It is important to emphasise that thisadjustment should be carried out to the meanof the normal range for albumin in the labo-ratory concerned using the normal methodfor plasma albumin rather than a standardalbumin concentration of 40 g/l as previouslysuggested. Calcium is also bound to plasmaglobulins although with only a quarter of theaffinity of the albumin binding so that, inpatients with abnormal globulin concentra-tions (such as myeloma'0), a correction forthis should be considered.1'

Establishing the cause ofhypercalcaemiaHaving established that the plasma calciumconcentration is raised, it is important todetermine the underlying cause. In mostinstances this will be either primary hyper-parathyroidism or malignant disease, but theother rarer causes of hypercalcaemia must beborne in mind (table). Although we will con-centrate on the laboratory procedures whichmay be helpful in this situation, it is impor-tant to highlight those areas of the clinicalhistory and examination which can shed lighton the diagnosis.

Perhaps the most important information tobe obtained is to establish whether or notthere has been any evidence of hypercal-caemia in the past. Unfortunately the symp-toms of hypercalcaemia (thirst, polyuria,constipation, tiredness, mental changes, etc.)are non-specific and do not usually allow thisdiagnosis to be made. However, a carefulreview of the case notes will often reveal araised plasma calcium concentration someyears before which has passed apparentlyunnoticed. Such a finding going back overseveral years is a very strong indicator that theunderlying diagnosis is primary hyperparathy-roidism. A similar inference can also be

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drawn from a long history of renal stonesespecially if the hypercalcaemia was stableand of moderate degree. Conversely, therecent development of hypercalcaemia in apatient with the clinical features of malignantdisease would strongly suggest that the hyper-calcaemia was the result of that process. Adetailed drug history is also important.Thiazide diuretics, although said to producemild hypercalcaemia in their own right, aremore likely to unmask hypercalcaemia fromother causes such as primary hyperparathy-roidism."2 Vitamin D intoxication is morelikely to produce severe hypercalcaemia; inmany instances the offending agent has notbeen prescribed but taken as a proprietarymedication available over the counter forconditions such as chilblains. In order to pro-duce clinically important hypercalcaemia it isnecessary to consume at least 250 ,ug (10 000IU) of calciferol daily, unless an associateddisorder of vitamin D metabolism, such assarcoidosis, is also present.

Hypercalcaemia does not lead to anypathognomonic clinical signs, but in severecases the patient is invariably salt and waterdepleted and will have the clinical features ofthis. Specific searches should also be madefor any clinical pointers to the underlyingcause of hypercalcaemia. If such features arefound they are likely to point towards malig-nant disease. On the other hand, it is impor-tant to remember that certain benign causesof hypercalcaemia, such as thyrotoxicosis, canproduce distinctive clinical features.A variety of different investigations are

likely to be helpful at this stage. As the pri-mary differential diagnosis is between hyper-parathyroidism and malignancy the mostimportant single investigation is estimation ofthe serum PTH concentration which shouldbe suppressed in the face of non-parathyroiddriven hypercalcaemia. On the other hand,there are a variety of other investigationswhich should be undertaken at this stage.These fall into two groups: those aimed atdetermining the cause of the hypercalcaemia;and those aimed at monitoring the state ofthe patient and providing a baseline fromwhich to judge response to treatment. Theformer include thyroid function tests, liverfunction tests, angiotensin converting enzymeactivity, serum and urine protein elec-trophoresis, and, in male patients, acid phos-phatase or prostate specific antigen (PSA).Other investigations which may also be con-sidered in this category include chest x raypicture, skeletal radiology, isotope bone scan,abdominal ultrasonography, blood film and,in selected cases, bone marrow examination.The latter group include renal function andserum electrolytes, blood count and markersof bone turnover such as alkaline phosphataseactivity.

Parathyroid hormone assayAs the major curable cause of hypercalcaemiais parathyroid overactivity it is imperative thatthis be sought in all cases of hypercalcaemia.

The early assays for PTH were immunoassaysdirected at the carboxyl terminal of the mole-cule. This is well separated from the majorarea of biological activity and these assaysdetected many biologically inactive frag-ments, especially in patients with impairedrenal function. This explains the considerableoverlap between the results obtained inpatients with hyperparathyroidism and thosewith hypercalcaemia from other causes, par-ticularly malignant disease.'3 These assayswere also very time consuming and requiredlong preincubation and reaction times. Theywere therefore rarely available outside special-ist centres, and if they were not performedthere were notoriously unreliable.

Attempts have been made to obviate theseproblems by developing assays directed at thebioactive amino terminal of PTH, the middleof the PTH molecule, and most recently theintact molecule.Amino terminal PTH assays make use of

antibodies directed at the 1-34 portion of themolecule which is responsible for its biologi-cal activity. It is believed that the plasma halflife of this moiety is so short that the assaysreally measure intact PTH concentration.Although good discrimination betweenhyperparathyroid patients and others can beachieved using such an assay,14 their use haslargely been superseded by intact PTHassays.

Mid-molecule assays are directed at a vari-ety of sites in the 44-68 amino acid range ofthe molecule. This is biologically inactive buthas a relatively long plasma half life, allowingits concentration to be used as an index ofPTH secretion. These assays have beenreported to have varying success in discrimi-nating between primary hyperparathyroidismand other causes of hypercalcaemia.'5'6 Inany case the use of such assays, like that ofN-terminal PTH, has been rendered obsolete bythe development of reliable intact PTHassays.Measurement of intact PTH is undertaken

using immunoradiometric assays (IRMAs) inwhich PTH is immobilised by an antibodydirected at a site close to one end of themolecule and then detected by the use of anantibody directed at an epitope close to theopposite end. These assays are both sensitiveand specific'7-19 and some of the more recentmethods use a chemiluminometric method ofdetection rather than radioiodine which is ofconsiderable benefit.'920 Some of these assaysare now available as commercial kits and offera reasonably robust and sensitive option forgeneral use. Typical PTH values obtainedusing these assays give a normal range of 1-6pmol/l (10-60 pg/ml).

Measurement of PTH using one of thesespecific methods is the single most importantinvestigation in the determination of thecause of hypercalcaemia. It has been theusual practice to obtain blood for the estima-tion of PTH concentration on a fasting speci-men in the early morning; using modernassays for intact PTH, a circadian rhythm forPTH secretion can be shown. This suggests

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that the best time to sample to discriminatebetween normal and hyperparathyroidpatients is the late morning or early after-noon.21 Whether this theoretical considera-tion has any major relevance for clinicalpractice is yet to be determined.

PTHrP ASSAYThe precise value of PTHrP measurementin the evaluation of patients with hyper-calcaemia is still under investigation.Measurement of PTHrP is expensive andvery careful sample collection and prepara-

tion is required. Although kits for the estima-tion of PTHrP concentrations are available, itis not yet possible to recommend their use inthe routine investigation of hypercalcaemicpatients. No doubt this will become clearer inthe near future as experience with the use ofthese assays increases. Early reports indicat-ing that the circulating concentration ofPTHrP is increased in many patients withhypercalcaemia and malignant disease, insome with primary hyperparathyroidism, andin patients with renal insufficiency have beenreviewed.22 More recently highly sensitiveimmunoradiometric assays of PTH andPTHrP have been applied to the investigationof patients with hypercalcaemia.2'24 In onesuch study PTHrP was undetectable in mostpatients (92%) with primary hyperparathy-roidism. In contrast, most patients (88%)with solid tumours had detectable plasmaPTHrP, and in the absence of coexisting pri-mary hyperparathyroidism, undetectable or

measurable (subnormal or normal) serum

concentrations of PTH.4 In some patientswith hypercalcaemia the finding of detectablePTHrP preceded the discovery of malignantdisease and was an important clue to theunderlying diagnosis.

Although the discovery of PTHrP was as a

result of a biological assay system, this isunlikely to be of routine clinical use. A varietyof immunoassays have accordingly beendeveloped. Most of these are directedtowards the amino terminal where the majorbiological activity resides. As this is alsowhere there is most homology with PTH it isimportant that such assays are capable of dif-ferentiating between these two compounds.Currently available amino terminal assaysinclude a radioimmunoassay which usedcomponents very similar to those in theIncstar kit23; this gave good discriminationbetween normal subjects in whom 37 out of38 had undetectable (< 2 pmol/l) PTHrP andpatients with humoral hypercalcaemia ofmalignancy whose values ranged from 2-8 to51-2 pmol/l. However, the kit itself still awaitsformal independent assessment. Immuno-radiometric assays have also been developed,the use of which will tend to obviate any wor-ries there may be as to the seat of biological

activity within the molecule. Such assays are

capable of good discrimination between nor-

mal subjects and those with hypercalcaemiaof malignancy.2526 One of these has recentlybeen developed as a commercial kit27 which isproduced by the Nichols Institute. An assess-

ment of this28 failed to give as good separationbetween the diagnostic groups as had beenseen in earlier studies. At present the use ofassays for PTHrP must still be considered tobe experimental, but once the problems withassay methodology and sample collectionhave been overcome the combination of sen-sitive PTHrP and PTH assays is likely tobecome extremely valuable in the diagnosisand management of patients with hypercal-caemia.4

INDIRECT MARKERS OF PARATHYROID ACTIVITYBefore the more specific assays for PTH weredeveloped and became readily available, con-siderable time was devoted to establishingother indicators of PTH activity which mighthelp to discriminate between hyperparathy-roidism and other causes of hypercalcaemia.Among the most popular of these measureswere the renal production of cyclic adenosinemonophosphate (nephrogenous cAMP) andthe renal tubular reabsorption of phosphate(most commonly expressed as per cent TRPor as a theoretical tubular maximum for re-absorption-TmP/GFR). As it is now knownthat many cases of hypercalcaemia of malig-nancy are mediated by PTHrP, which hassimilar renal effects to PTH, it is not surpris-ing that none of these measures has any greatdiscriminant power.29 These indirect mea-sures of parathyroid activity still have theiruse in a research setting, but they are unnec-essary for the routine investigation of hyper-calcaemia.

Similarly, biological assays of parathyroidactivity do not discriminate between PTHand PTHrP, and so have little value in theinvestigation of hypercalcaemia.

OTHER HORMONE ASSAYSAlthough uncommon, vitamin D intoxicationis an important treatable cause of hypercal-caemia. If vitamin D intoxication is suspectedin patients not receiving large doses of vita-min D for therapeutic purposes the concen-tration of 25 hydroxyvitamin D (250HD)should be measured; grossly increased con-centrations would support the diagnosis.Measurement of 250HD in the serum can bemeasured by a variety of different techniquesincluding competitive protein binding,'0radioimmunoassay,3" and high performanceliquid chromatography (HPLC).'2 Widelydiffering results have been reported betweenlaboratories."'4 Serum 250HD concentra-tions of up to 160 pmol/l have been reportedas the result of sunlight exposure35 and henceconcentrations below this are unlikely to be ofclinical importance. In general, patients withvitamin D intoxication have 250HD concen-trations in excess of 500 nmol/l.'6

Intoxication with the active metabolites ofvitamin D (calcitriol and alfacalcidol) is notassociated with increased concentrations of250HD but with increased concentrations of1,25 dihydroxyvitamin D (1,250HD2; cal-citriol). Measurement of 1,250HD2 can alsobe of use in the investigation of hypercal-caemia because of granulomatous diseases,

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particularly sarcoidosis, in which there isautonomous production of this metabolitein the granulomata.37 1,25OHD2 can bemeasured by either radioreceptor assay38 orradioimmunoassay,39 although in each caseconsiderable sample preparation is necessary.The former technique is utilised in thecommercially available kits for the measure-ment of 1,25OHD,. A recent study suggeststhat these methods compare favourably withthe more time consuming competitive bind-ing assay following HPLC purification.40Although radioimmunoassay following HPLCpurification permits quantification of thevitamin D2 and vitamin D3 components sepa-rately, this is seldom if ever required on aclinical basis and so this technique is largelyconfined to research applications.

Certain endocrine disorders can result inhypercalcaemia and these need to be consid-ered if there is any clinical suspicion of theirpresence or that the hypercalcaemia is notPTH dependent. Of these, the most impor-tant is thyrotoxicosis but adrenal insufficiencyand, very rarely hypothyroidism, can also leadto clinically important hypercalcaemia. Measure-ment of thyroxine and thyroid stimulatinghormone (TSH) concentrations will usuallybe sufficient to confirm or refute the diagno-sis of thyroid dysfunction. If Addison's dis-ease is seriously being considered a shortadrenocorticotropic hormone stimulation testshould be performed at the same time thatblood is drawn for cortisol estimation.Sometimes endocrine diseases (particularlyhyperthyroidism) and sarcoidosis co-existwith primary hyperparathyroidism. The sameis true for primary hyperparathyroidism andmalignant disease.

Components ofhypercalcaemiaCalcium infusion studies in normal subjectshave clearly shown that there is a relativelyconstant relation between the plasma calciumconcentration (or more precisely the ultrafil-trable calcium concentration) and theamount of calcium excreted in the urine,expressed as calcium excreted per litre ofglomerular filtrate (CaE)41 calculated as:

Urine calcium* plasma creatinineurine creatinine

This association can be examined for anypatient by plotting their plasma calcium andCaE against the normal curve and derivingcomponents of hypercalcaemia.42 In order tosimplify this approach a variety of nomo-grams and computer programs to ease thecalculations have been produced. Althoughclinically interesting, this approach cannotdistinguish between the hypercalcaemiainduced by PTH or PTHrP and is of onlylimited clinical usefulness, especially with theadvent of more discriminating PTH assays.None the less this analysis of the componentsof hypercalcaemia can still prove helpful inthe investigation of "difficult" cases of hyper-calcaemia, and may provide useful clues as tothe best approach to treatment.

Measurement of urine calcium excretion isvaluable in the diagnosis of hypocalciurichypercalcaemia. This condition "benignfamilial hypercalcaemia" is inherited as anautosomal dominant.43 The hypercalcaemia isusually mild and arises through the avid reab-sorption of calcium from the glomerularfiltrate which leads to very low urine calciumexcretion (CaE < 0-015 mmolJl glomerularfiltrate). It is imperative that patients are notreceiving thiazide diuretics while this investi-gation is performed because these drugs pro-duce an identical biochemical picture.Familial hypocalciuric hypercalcaemia isimportant to recognise because it seldomcauses symptoms and calls for no specifictreatment; parathyroid surgery is generallycontraindicated.

DISCRIMINANT FUNCTION ANALYSISSeveral authors have developed a variety ofso-called discriminant functions in which theresults of different investigations are weightedand combined to yield a value which providesan indication of the cause of hypercalcaemia.On the whole such approaches have not beenparticularly successful and their use has beenrendered obsolete by the newer PTH assays.

STEROID SUPPRESSION TESTHydrocortisone 40 mg given eight hourly for10 days was initially described as a means ofdifferentiating primary hyperparathyroidism,in which there is little change in calcium con-centration, from other causes of hypercal-caemia, in which the calcium concentrationmoves towards normal.44 Since its introduc-tion it has been the subject of much contro-versy, although many of its detractors havegiven insufficient corticosteroid for too littletime in their modification of the test. Thesteroid suppression test is rarely needed thesedays. It may occasionally be a useful investi-gation in the diagnosis of those difficult casesof hypercalcaemia where the PTH and otherassays have given equivocal results. If the testshows a positive response, even if no specificcause for the hypercalcaemia is determined, itdoes point to the possible use of steroids as atherapeutic measure.

OTHER INVESTIGATIONSThe investigation of hypercalcaemia is notconfined to the biochemistry laboratory and itmust be emphasised that successful manage-ment of this potentially life threatening condi-tion calls for a multidisciplinary approach inwhich there is full cooperation between clini-cians and the full range of clinical supportservices. Although it is likely that biochemicalstudies will give the most useful information,it will be apparent from the foregoing discus-sion that haematological and radiological ser-vices also have an important role in theestablishment of underlying diagnosis.

The authors are grateful to Dr E B Mawer for her helpfuldiscussions and criticism of the manuscript.

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