Evaluation of Plasma Aldosterone to Plasma Renin Activity Ratio in Patients with Primary Aldosteronism

ORIGINAL ARTICLE Evaluation of plasma aldosterone to plasma renin activity ratio in patients with primary aldosteronism

H Ignatowska-S´ witalska, J Chodakowska, W Januszewicz, T Feltynowski, M Adamczyk and J Lewandowski Department of Hypertension and Angiology, Academy of Medicine, Warsaw, Poland

The plasma aldosterone to renin activity ratio (A/PRA) ratios in patients with primary aldosteronism were asso- was assessed retrospectively in 103 patients with pri- ciated with unsuppressed plasma renin activity (PRA). mary aldosteronism including 74 patients with surgi- Although the mean values of the A/PRA ratio differed cally proven adrenal cortical adenoma (APA) and 29 significantly between the groups, complete separation patients with idiopathic adrenal cortical hyperplasia was not obtained. The serum potassium level at time of (IHA). The results were compared with those obtained testing did not influence the diagnostic value of the in 31 patients with essential hypertension (EH) and 45 A/PRA ratio, although an inverse correlation between healthy normotensive controls. The upper limit of nor- serum potassium and the A/PRA ratio was found in the mal A/PRA ratio, as obtained in the controls was 17.8. patients with APA. This study supports the high sensi- This value was exceeded in 89% of patients with APA; tivity of the A/PRA ratio in diagnosis of primary aldos- postoperatively it decreased in 97% of the APA group, teronism, however, a single determination with a normal and returned to normal in 81%. In the IHA group the result may not be sufficient for exclusion of the disease. A/PRA was elevated in 70% of patients. The normal

Keywords: plasma aldosterone/PRA ratio; primary aldosteronism; screening test

Introduction cating the need for further diagnostic work-up for suspected primary aldosteronism. Primary aldosteronism has been hitherto considered However, reports from several centres have dem- a rare cause of hypertension. Its occurrence has been onstrated a limited effectiveness of this test, since generally estimated at less than 2% of the total in a sizeable proportion of patients the plasma pot- hypertensive population. Hypertension and bio- assium may be normal.2,3 In 1981 Hiramatsu et al4 chemical abnormalities result from excessive proposed a simple test, practicable in an outpatient secretion of aldosterone from the adrenal cortex setting, using a calculated ratio of plasma aldo- which is due to the presence of an adenoma, or to sterone concentration to plasma renin activity idiopathic or primary adrenal cortical hyperplasia. (A/PRA). Subsequently, several authors reported Familial forms of primary aldosteronism, aldos- A/PRA ratio in patients with primary aldosteronism teronism caused by adrenal carcinoma, or extra- and demonstrated its usefulness for screening pur- 1 adrenal ectopic aldosterone hypersecretion are rare. poses.4–7 Establishing the aetiology of primary aldosteronism In the present study, to further support the diag- is important for therapeutic decisions; cortical aden- nostic value of the A/PRA ratio, we analyse retro- omas are best treated surgically, while conservative spectively a large group of patients with primary treatment is usually advocated for non-tumorous aldosteronism treated in our institution between forms. 1974 and 1992. The retrospectively assessed A/PRA Recurrent hypokalaemia in a hypertensive patient ratios of patients with tumorous and non-tumorous is the typical feature pointing to a possible diagnosis primary aldosteronism are compared to those of of primary aldosteronism. The demonstration of a patients with essential hypertension and of healthy lowered plasma potassium concentration in the face normotensive controls. of an increased urinary potassium excretion has long been considered the basic screening test, indi- Patients and methods Three groups of patients were included in the retro- Correspondence: Professor W Januszewicz, Department of Hyper- spective analysis of clinical and biochemical rec- tension and Angiology, Academy of Medicine, Banacha 1a, 02- ords: 74 patients (18 men and 56 women), aged 22– 0097 Warsaw, Poland ± Received 8 September 1996; revised 10 February 1997; accepted 66 years (mean age 45.4 1.1 years) with primary 25 February 1997 aldosteronism and surgically and histologically Plasma aldosterone/PRA ratio in primary aldosteronism H Ignatowska-S´witalska et al 374 proven adrenal cortical adenomas, 29 patients (11 plasma aldosterone concentration was normal in all men and 18 women) aged 17–60 years (mean age cases. Secondary forms of hypertension had been 45.2 ± 1.6 years) with primary aldosteronism with- excluded involving the same procedures as in out discernible adrenal tumours, classified as adre- patients with primary aldosteronism. None of the nal cortical hyperplasia, and 31 patients (12 men patients had diabetes, elevated serum creatinine or and 19 women) aged 19–74 years (mean age 40.2 ± overt hepatic dysfunction. 2.4 years) with essential hypertension. The control group included clinically healthy nor- The control group consisted of 45 healthy normo- motensive volunteers recruited similarly to the EH tensive volunteers (23 men and 22 women) aged 25– group. 50 years (mean age 35.9 ± 1.1 years). All tests were done during hospitalisation, after As a rule, patients with primary aldosteronism diuretics including spironolactone had been with- were referred to our institution because of recurrent held for at least 4 weeks, and any other drugs poss- hypokalaemia and hypertension diagnosed else- ibly interfering with plasma aldosterone and renin where. The only exceptions concerned three activity, for at least 2 weeks. None of the patients patients referred with normal plasma potassium had been previously receiving angiotensin-con- concentration but with suggestive clinical features verting enzyme (ACE) inhibitors. In a few cases (each patient exhibiting at least three of the follow- where hypotensive therapy was necessary, calcium ing: muscular weakness, polyuria with nocturia, channel blockers were given, but omitted on the day polydipsia, parestesia) and one normokalaemic of the study. The subjects had not been subject to patient with a previously demonstrated adrenal sodium restriction during the preceding months or tumour. Other causes of secondary hypertension at the time of the study and remained on normal had been excluded on the basis of clinical findings sodium intake as evidenced by 24-h urinary sodium and an array of laboratory tests, including urinary excretion of 120–160 mmol. Most patients with pri- free corticoids, urinary catecholamines and metab- mary aldosteronism received potassium salts p.o. olites, renal ultrasonography, and in some cases, and had high urinary potassium excretion. In the aortonephrography. The diagnosis of primary aldo- remaining groups potassium intake was normal steronism was based on increased plasma aldo- (urinary excretion 20–40 mmol/24 h). sterone concentration and/or increased urinary After overnight fasting and 30 min rest lying aldosterone excretion in the face of low PRA unre- down, blood samples were taken without stasis from sponsive to stimulation by furosemide and 4-h an antecubital vein, placed in the test tubes contain- upright posture. However, in three hypokalaemic ing sodium EDTA and immediately centrifuged at patients with eventually visualised adrenal 4°C for 10 min, whereupon plasma was separated, tumours, the requisite of suppressed PRA was omit- frozen and stored at −20°C until analyses were per- ted. formed within 2 weeks of freezing. Blood for serum The visualisation techniques used, depending on potassium concentration was collected in separate the progress of their availability, included adrenal test tubes and analysed within a few hours. In 63 of phlebography with adrenal venous sampling, scin- the patients with primary aldosteronism, identical tigraphy, ultrasonography, computerised tomogra- studies were repeated 2–12 weeks postoperatively. phy, and magnetic resonance of the adrenals. Dem- Plasma aldosterone concentration was determined onstration of an adrenal tumour or a unilateral by radioimmunoassay (RIA), according to the aldosterone hypersecretion usually by more than method of Ignatowska-S´witalska8 with a modifi- one of these techniques provided the diagnosis of an cation which omitted thin layer chromatography of aldosterone-producing tumour in all the 74 patients the dichloromethane extract. Initially we used the brought to surgery. This subsequently provided his- anti-aldosterone antibodies (088) obtained from NIH tological confirmation of the diagnosis of an aden- (Bethesda, MD, USA), replaced later by the Calbi- oma of the adrenal cortex (APA). ochem antibodies. Two millilitres of plasma were In the 29 patients with primary hyperaldosteron- used for dichloromethane extraction. Recovery for ism in whom at least two techniques had failed to the extraction procedure was 90 ± 8% (n = 40). The demonstrate an adrenal tumour, the diagnosis of extract was evaporated and dissolved in RIA buffer idiopathic adrenal cortical hyperplasia (IHA) was (0.1 M borate, pH = 8.0, bovine albumin 0.5%). We assumed with the ensuing indication for conserva- used the standard in the range 20.8–693.5 fmol (7.5– tive treatment. 250 pg) per test tube. Working dilutions of the anti- Usually the case histories included the results of body were adjusted to obtain zero binding at 30– more than one determination of PRA and plasma 50%. The standard or samples in presence of the aldosterone concentration; for the purpose of this antibody were incubated overnight at 4°C with study we selected those which were synchronous, 10 000 cpm of 3H-aldosterone (Amersham TRK 196) preferably with a corresponding serum potassium as the labelled tracer antigen. Bound antibody was determination, and conformed to the basal con- separated from free antigen with charcoal/dextran ditions at the time of sample collection. As a rule, suspension and 10 min incubation. After centrifug- these were not the same determinations as those ation at 4°C, the supernatant was decanted into scin- serving for the initial diagnosis of primary aldos- tillation cocktail and radioactivity of the antibody- teronism. bound fraction was measured in the Packard beta The essential hypertension (EH) group consisted scintillation counter. Aldosterone concentration of patients with mild to moderate hypertension was interpolated from the standard curve. Cross- recruited for various previous clinical studies. Basal reactivity with cortisol, corticosterone and deoxy- Plasma aldosterone/PRA ratio in primary aldosteronism H Ignatowska-S´witalska et al 375 corticosterone of both these antibodies was less than had unsuppressed PRA at the initial selection, two 0.001%, with intra- and inter-assay coefficient of had an elevated A/PRA ratio (97 and 261 variation of 8%, for a mean value of 263 mol/l respectively); and the third patient had a normal (9.5 ng%) and 15%, for a mean value of 336 pmol/l ratio (8.2), but the removal of his adrenal adenoma (12.1 ng%), respectively. The normal values of was followed by normalisation of plasma and uri- plasma aldosterone for our laboratory, as determ- nary aldosterone, disappearance of hypokalaemia, ined by this method, with either of the antibodies polydipsia, polyuria and muscular weakness, and used ranged between 83 pmol/l (3 ng%) and 416 improved control of his residual hypertension. pmol/l (15 ng%). The A/PRA ratio of patients with adrenal cortical PRA was determined using Renk kit (Cea Ire hyperplasia ranged from 3.9 to 736, median 31.2, Sorin, Italy), replaced later with the RJ 131 kit with the mean value of 74.9 ± 25.3. In this group (Immunotech, Czechoslovakia). Angiotensin I was nine (30%) patients displayed normal ratios; in generated in vitro within 1 h (or 2 h for lower these nine cases the basal PRA ranged from 0.04 to expected PRA) of enzymatic reaction at 37°C and pH 0.7 pmol/l/s (0.2–3.4 ng/ml/h, mean 1.6 ± 0.3 6.0. For valid assay the concentration of free circul- ng/ml/h)). ating angiotensin (blank) was not higher than 0.02 The EH group displayed A/PRA ratios ranging pmol/l/s (0.1 ng/ml/h). Intra- and inter-assay vari- between 0.5 and 37.5, median 7.0 (mean 11.8 ± 2.0). ation coefficient with both these methods was 6% In 22.6% of patients in this group the ratios were and 8% for a mean value of 0.4 pmol/l/s elevated. (1.6 ng/ml/h), respectively, and the normal basal The differences between the mean values of the PRA for our laboratory ranged between 0.1 and 0.9 A/PRA ratio of the APA group and those of the pmol/l/s (0.5 and 4.2 ng/ml/h) with either of the remaining groups were highly statistically signifi- kits. cant (P Ͻ 0.05). Likewise, the IHA group differed The A/PRA ratio was calculated from plasma significantly from the EH group and the control aldosterone concentration in ng% and PRA in group. ng/ml/h, and was expressed as an absolute number. The assessment of the relationships of the A/PRA Serum potassium concentration was estimated by ratio to plasma aldosterone concentration and PRA flame photometry (normal range 3.5–5.0 mmol/l). by the Spearman’s rank test demonstrated that in Statistical analysis of the results was done using both the APA and IHA groups the A/PRA correlated the PC-Super-Stat system. The results were with plasma A (r = 0.540, P Ͻ 0.001 for APA, and r expressed as ranges; medians and arithmetic means = 0.372, P Ͻ 0.05 for IHA), but stronger significant with standard errors, depending on the distribution negative correlations were disclosed between of the parameters. After an initial appraisal of the A/PRA and PRA (r =−0.837 for APA and r =−0.887 distribution of results, the unpaired and paired for IHA, P Ͻ 0.0001 in both groups). group differences of the A/PRA ratios were com- The changes in the A/PRA ratios following surgi- pared using the Wilcoxon test. For sets of para- cal removal of adrenal adenoma are presented in meters characterised by normal distribution and Figure 2. Postoperatively, the A/PRA ratio decreased equal variance, Student’s t-test for unpaired samples in 61 (97%) out of 63 analysed cases of APA, and was applied. The interrelations between parameters in 51 (81%) it attained a normal value. Thus in two were calculated using the Spearman rank test. A P- patients the elevated A/PRA ratio did not fall post- level of less than 0.05 was regarded as statistically operatively. In one of them a second aldosterone- significant. producing adenoma in the contralateral adrenal has been subsequently demonstrated. In the second case Results a repeated postoperative testing disclosed a normal ratio. The mean group A/PRA value decreased sig- The results of synchronous plasma aldosterone, PRA nificantly from 221.1 ± 37.7 preoperatively to 14.9 ± and serum potassium measurements as well as the 2.8 postoperatively (P Ͻ 0.01). calculated A/PRA ratios are presented in Table 1. Serum K concentration synchronous with A/PRA The individual A/PRA ratios in the four investigated ratio was available in 80 patients with primary aldo- groups are depicted in Figure 1. steronism, including 58 with APA and 22 with IHA. In healthy normotensive controls, the A/PRA ratio In the APA group 27 patients (46%) had normal ranged between 1 and 25, median 4.8, with the mean serum K (у3.5 mmol/l). Out of these, 23 (85%) had value of 6.4 ± 0.8 (s.d. = 5.7). The upper limit of an elevated A/PRA ratio. In the IHA group, normal normal was set at 2 standard deviations above the serum K was found in 16 (73%) patients, including mean, which corresponded to 17.8. The A/PRA ratio 10 (62.5%) with elevated A/PRA ratio. exceeded this limit in three subjects (6.6%) of the A statistically signicant inverse correlation control group. between the A/PRA value and serum K concen- In patients with adrenal cortical adenoma, the tration was found in the APA group (r =−307, P Ͻ A/PRA ratio ranged from 6.6 to 1710, median 125.1, 0.05), but not so in the IHA group (r =−0.177, P Ͼ with the mean value of 223.8 ± 34.2. In eight (11%) 0.5). patients of this group the ratios were within the nor- In 51 patients (26 with APA and 25 with IHA), the mal limits; these patients were characterised by nor- records permitted repeated calculations of A/PRA mal basal PRA ranging from 0.2 to 0.9 pmol/l/s (0.9– ratio in basal condition. The comparison of the dou- 4.2 ng/ml/h), mean 0.5 ± 0.3 pmol/l/s (2.4 ± 1.2 ble sets of results (Figure 3) did not disclose signifi- ng/ml/h). Out of three patients with APA who had cant difference of the mean values (P Ͼ 0.5). The Plasma aldosterone/PRA ratio in primary aldosteronism H Ignatowska-S´witalska et al 376 Table 1 Synchronous plasma aldosterone (A), plasma renin activity (PRA), A/PRA ratio and serum potassium (K) in patients with adenoma (APA), hyperplasia (IHA), essential hypertension (EH) and controls (C)

APA IHA EH C

No. of cases 74 29 31 45 A pmol/l (ng%)

Range 205–4743 80–1123 27.7–360 50–416 (7.4–171) (2.9–40.5) (1.0–13.0) (1.8–15) Mean ± s.e. 1156 ± 97 519 ± 44 197 ± 16.6 138.7 ± 11.1 (41.7 ± 3.5) (18.7 ± 1.6) (7.1 ± 0.6) (5.0 ± 0.4)

PRA pmol/l/s (ng/ml/h) Range 0.02–0.9 0.02–0.7 0.02–1.1 0.02–0.8 (0.1–4.2) (0.1–3.4) (0.1–5.2) (0.1–3.8) Mean ± s.e. 0.1 ± 0.02 0.17 ± 0.02 0.3 ± 0.06 0.3 ± 0.04 (0.5 ± 0.09) (0.8 ± 0.1) (1.5 ± 0.3) (1.4 ± 0.2)

A/PRA Range 6.6–1710 3.9–736 0.5–37.5 1–25 Mean ± s.e. 223.8 ± 34.2 74.9 ± 25.3 11.8 ± 2.0 6.4 ± 0.8

K mmol/l Range 1.7–3.9 1.8–3.6 3.6–4.5 3.8–4.7 Mean ± s.e. 2.7 ± 0.06 2.9 ± 0.08 4 ± 0.06 4 ± 0.08

Figure 2 The A/PRA ratio before and after surgical removal of adenoma in 63 patients with APA (mean ± s.e.).

Figure 1 Individual A/PRA ratio in patients with adenoma (APA), hyperplasia (IHA), essential hypertension (EH) and con- Discussion trols (C). The results of our retrospective study indicate that patients with primary aldosteronism are character- individual values of the A/PRA ratios elevated dur- ised by an elevated A/PRA ratio as compared to ing the study were normal at another preoperative those with essential hypertension and to healthy examination in two patients with APA, and in four normotensive controls. Moreover, in the majority of patients with IHA, while the reverse sequence cases the A/PRA ratio may be indicative of either the occurred in two patients each with APA and IHA. tumorous or the non-tumorous form of the disease. Thus in 10 patients out of 51 the repeat test would Our results are in keeping with those of other change the initial classiﬁcation of patients, albeit the workers,4–7,9–12 although the design of our study dif- differences in the ratios were usually small. fers from other reports, which may have a bearing Plasma aldosterone/PRA ratio in primary aldosteronism H Ignatowska-S´witalska et al 377

Figure 3 Repeated determination of A/PRA ratio in 26 patients with APA (preoperatively) and in 25 patients with IHA. (I, ratio analysed in the study; II, repeat ratio) on the results. In our material, the A/PRA ratio had and the two primary aldosteronism groups would not been used for screening purposes and had not obviously increase. influenced the diagnostic process, indeed it was cal- It had to be expected that the A/PRA correlated culated from laboratory data retrospectively. positively with plasma A and inversely with PRA. The diagnostic criteria of primary aldosteronism However in our material the correlation with PRA used in our study may have not conformed to rigid was stronger and the majority of normal A/PRA ratio requirements in that in our selection of patients’ rec- concerned the APA patients with normal PRA. ords we omitted the requisite of an aldosterone sup- Although the normal PRA in these APA patients is pression test. Therefore, our IHA group may have not consistent with the usual diagnostic criteria of included some patients with insufficiently docu- primary aldosteronism, there seems little doubt that mentd primary aldosteronism. The criteria derived the histologically proven diagnosis in these cases from the visualisation procedures cannot be helpful was correct. One has to accept the possibility of nor- in this regard, since in most instances they were mal PRA in some patients and the probable failure based on exclusion of an adrenal tumour and not on of a single A/PRA ratio determination to screen out the confirmation of a bilateral non-tumorous such cases. It is noteworthy that leading authorities enlargement of the adrenal glands. Perhaps these advocate repetitive determinations for screening reservations may account for some of the normal purposes.10 We have not taken into account a con- A/PRA ratio results in the IHA group, but they do ceivable histological diversity of the resected aden- not seem to apply to the APA group. The few elev- omas and resulting possible differences in the mech- ated A/PRA ratio values found in the EH group may anisms regulating their hormonal activity. The represent low-renin essential hypertension or undi- probability of these factors influencing the A/PRA is agnosed normokalaemic primary aldosteronism a matter of conjecture. The present study confirms although normal plasma aldosterone was present in that the A/PRA ratio may be helpful in screening out all subjects of this group. patients with normokalaemic APA, since synchron- We set the upper limit of normal at 2 standard ously determined serum potassium concentration deviations above the mean value of the control was normal in over half of the 80 cases for whom group, which corresponded to an A/PRA ratio of data were available. However it should be recalled 17.8. With a higher arbitrarily selected cut-off point here that hypokalaemia had been present on referral of 30 or 50, as proposed by other authors,9,10 nearly in nearly all our patients who were subsequently all our patients with EH and all controls would fall treated with potassium salts, which increased serum into the normal range, but the overlap between them potassium at the time of A/PRA determination. On Plasma aldosterone/PRA ratio in primary aldosteronism H Ignatowska-S´witalska et al 378 the other hand it has been claimed that falsely low terone-producing adenoma by measuring plasma renin A/PRA ratios may be expected in the face of hypoka- activity. Results in hypertensive patients. Arch Intern laemia.3,10 Med 1981; 141: 1589–1593. Inferring practical conclusions from our study, we 5 Hamlet SM et al. Is the aldosterone/renin ratio useful support the recommendation that all patients with to screen a hypertensive population for primary aldosteronism? Clin Exp Pharmacol Physiol 1985; 12: an elevated A/PRA ratio should undergo further 249–252. diagnostic study for suspected primary aldosteron- 6 Lins PE, Adamson U. Plasma aldosterone-plasma renin ism. However, a normal A/PRA ratio does not activity ratio. Acta Endocrinol 1986; 113: 564–569. exclude the diagnosis and repetitive testing is 7 McKenna TJ et al. Diagnosis under random conditions advised. of all disorders of the renin-angiotensine-aldosterone axis, including primary hyperaldosteronism. J Clin Endocrinol Metab 1991; 73: 952–957. Acknowledgements 8 Ignatowska-S´ witalska H. Radioimmunologiczna We gratefully acknowledge the generous gift of aldo- metoda oznaczania aldosteronu w moczu (Radio- sterone antibody (088) kindly supplied by Dr RW immunological method for aldosterone determination in urine). Pol Arch Med Wewn 1976; 56: 23–34. Bates (NIH, Bethesda, MD, USA). 9 Blumenfed JD et al. Diagnosis and treatment of primary hyperaldosteronism. Ann Intern Med 1994; 121: References 877–885. 10 Gordon RD et al. Primary aldosteronism and other 1 Biglieri EG, Irony I, Kater CE. Identification and impli- forms of mineralocorticoid hypertension. In: Swales JD cation of key types of mineralocorticoid hypertension. (ed). Textbook of Hypertension. Blackwell Scientific J Steroid Biochem 1989; 329: 199–204. Publications: Oxford, 1994, pp 865–892. 2 Bravo EL, Tarazi RC, Dustan HP. The changing clinical 11 Weinberger MH, Fineberg NS. The diagnosis of pri- spectrum of primary aldosteronism. Am J Med 1983; mary aldosteronism and separation of two major sub- 74: 641–651. types. Arch Intern Med 1993; 153: 2125–2129. 3 Gordon RD et al. Primary aldosteronism: hypertension 12 Weinberger MH. Mineralocorticoids and blood press- with genetic basis. Lancet 1992; 340: 159–161. ure. Curr Opinion Nephrol and Hypertens 1994; 3: 4 Hiramatsu K et al. A screening test to identify aldos- 550–554.