Primary aldosteronism (PAL) is gaining recognition as an important cause of hypertension, and attention is focusing more critically on the reliability of screening tests for PAL. However, less attention has focused on reliable tests to establish a firm diagnosis, or on tests to differentiate the surgically correctable form, unilateral aldosterone-producing adenoma (APA), associated with a ‘normal’ contralateral adrenal, from other forms.
In this issue of the journal, Ferrari et al.  make a small but significant contribution to these difficult but important questions by showing that immunoreactive renin (irR) can probably be substituted for the theoretically more variable (i.e. because of widely differing conditions of assay) plasma renin activity (PRA), which incorporates assessment of renin substrate (angiotensinogen), and which has contributed so much to our early understanding of renin–angiotensin–aldosterone physiology. More reproducible assays are essential at this critical time for the management of PAL because, due to the wide availability of commercial ‘kits', the measurement of aldosterone to renin ratio (ARR) has moved from the basic research or Specialized Unit laboratory, with meticulous quality control, clinical feedback and long experience, to the busy shrinking budget-driven general hospital laboratory and to the profit-driven private laboratory. Although clearly preferable, the smaller laboratories possibly could not cope with the heavy load of widely based screening. It might reasonably be expected that there is less laboratory to laboratory variability with irR (with an international standard for renin) than with PRA, permitting comparison and even pooling of results in patients from different clinical units. This might strengthen our combined ability to resolve some of the important issues regarding screening for PAL, together with its firm diagnosis and optimal treatment.
By confining the question to a direct comparison of PRA and irR measured simultaneously in the same sample in order to determine ARR, Ferrari et al.  have effectively avoided becoming bogged down in the many other more complex, important, practical issues such as posture, time of day, methods for measuring plasma aldosterone concentration (PAC) and medications, although they briefly refer to some of them in their introduction and discussion, and of necessity, make their choice of sampling conditions for the study. Within that restricted context and despite the small number of patients involved (36 in all), their study delivers a relatively clear answer. Ideally, this should be confirmed in a larger study. As expected, there was an excellent correlation between irR and PRA values, although admittedly not quite as robust at the important lower end of the range, but hopefully the assay is accurate enough at these levels for meaningful discrimination. Because the same value for PAC was used in each case, there was also an excellent correlation between ARRs measured using these two techniques for estimating renin.
Differences between what is actually measured by PRA and by irR are briefly discussed. The authors reasonably conclude that irR may be as useful as PRA to assay renin in clinical practice in screening for PAL. However, one could question how Table 1, which usefully converts the cut-off point for ARR from only one study  into the various units used by different authors (mass or SI units), takes into account the different methods used in the literature, as suggested by the authors.
While the aldosterone level is in fact a less powerful determinant of ARR than renin, which seems to move physiologically on a logarithmic scale, nevertheless the radioimmunoassay of PAC using commercial kits is not free from problems, especially if the long-utilized antibody component is exhausted and replaced by another by the manufacturer, without adequate examination of the effects of this on the assay. The problem of reproducibility of the aldosterone assay, when performed in different laboratories, is not addressed by Ferrari et al. ; it would be very useful to have this information. Perhaps future approaches to the measurement of aldosterone which more directly estimate mass will help to resolve it. In the meantime, the same difficulties of comparison of results from different laboratories employing different kits remains for aldosterone as for renin.
The wider and more general scientific problem which this study briefly addresses is the possibly unanswerable question of a precise cut-off point for ARR in screening for PAL. This question is of very great importance now that it is widely recognized that PAL is not uncommon among the hypertensive population, indeed it is common enough and specifically treatable enough to make failure to detect its presence a significant clinical oversight. Some of the evidence for PAL as a not infrequent cause of hypertension is cited by Ferrari et al. . Reference is not made to the unfortunate consequences of longstanding hypertension, which is increasingly difficult to control, and which is eventually (perhaps when hypokalemia finally emerges) diagnosed as PAL too late to prevent irreversible organ damage. The hypertension could have been much better controlled, or possibly cured, years earlier if effective screening had been performed. This scenario should become less common.
The effects of medications on the ARR (mainly through effects on renin) are briefly addressed. Significantly, the bibliography listed in Ferrari et al.  includes studies in which all medications have been continued, studies in which only beta-blocking drugs and diuretics have been discontinued, and studies in which all medications have been ceased (from 7 days to 1 month previously), clearly indicating the difficulty of comparing results. If renin was the sole regulator of aldosterone production, rather than the main regulator (as indicated by persisting low PAC levels after removal of a long-present APA), diagnosing PAL would be much easier. Either because it is not [potassium and adrenocorticotropic hormone (ACTH) also being very important], or for reasons not currently understood, aldosterone and renin do not move in a parallel, predictable, inter-dependent fashion in response to physiological or medication-induced changes. For example, the usually profound suppression of renin by beta-adrenoceptor blocking antihypertensive medications is not accompanied by an equally profound suppression of aldosterone, and the ARR is usually raised above any arbitrary cut-off point for PAL, giving results which are most often false positive. In a somewhat half-hearted attempt to address the medication issue, the authors ceased antihypertensive medications for 7–10 days in an attempt to minimize, not eradicate, such effects on endocrine function. They probably chose this compromise because it was used in the study which provided their chosen cut-off point  If any of their patients were receiving diuretics, the effects on renin (through volume) and aldosterone (through potassium) would have only partially disappeared after 1 week; the effects of prolonged beta-blockade or angiotensin-blockade might also not have completely disappeared. Other medications, such as non-steroidal anti-inflammatory drugs (available without prescription in some countries) also affect renin levels, in this case suppressing them, with resultant false-positive ARR (Table 1). Cessation of these drugs for at least 1 week is essential if ARR is to be interpretable, but this depends on first discovering that the patient has been taking them.
The authors draw on one of the best studies to have addressed this issue so far, carried out at the University of Torino, Italy . In that study, ARR was measured after a 1-month washout period off all medications, and again after at least 2 months of monotherapy with either a beta-blocker (atenolol, 100 mg daily), an alpha-blocker (doxazosin, 8 mg daily), a calcium channel blocker (amlodipine 10 mg daily), an angiotensin-converting enzyme inhibitor (fosinopril 20 mg daily) or an angiotensin-blocker (irbesartan 300 mg daily). These are top-of-the-range doses, and the drugs were chosen because of their well-established effects on renin; reduction by atenolol, elevation by amlodipine, fosinopril and irbesartan, and little change effected by doxazosin. Severely hypertensive patients (three medications or more) were excluded because of the risk of a ‘washout’ period; PAL might have been well represented in this subgroup and lost to the study. A large number of 2160 hypertensives were screened by ARR after washout, and 230 with an ARR greater than 50 (PAC ng/100 ml divided by PRA ng/ml per h) then had saline infusion (2 l of 0.9% NaC1 infused over 4 h) confirming PAL in 154 patients according to the criteria of Holland et al. . Only these patients entered the study, and thus it was a study of the effects of medication on the ARR in patients with established PAL, and not in the vast majority of patients (generally accepted to be 90% or so) who do not have PAL and, accordingly, the results should be interpreted in this context. The study showed that atenolol in this dose increased the ratio by an average 62%, and predictably would have increased the ratio by a greater percentage in patients who did not already have the suppressed renin characteristic of PAL, creating many false positives in the general hypertensive population. Clearly, atenolol cannot create a false positive in a patient already positive, and so the authors dismissed it as a problem: ‘beta-blockers do not interfere with the diagnosis of primary aldosteronism’ . However, they conceded that ‘they can be responsible for an increased rate of false-positive ARRs', which, it could be argued, interferes in the diagnosis (screening stage) of PAL. They confirmed that doxazosin had little effect on the ratio (average –5%) at least in patients with PAL, but, importantly, amlodipine (average –17%), fosinopril (average –30%), and irbesartan (–43%)  were capable of causing false-negative results in patients with PAL established by suppression testing.
In both the study by Ferrari et al.  and the study of Mulatero et al.  to which the former authors refer, the danger of cessation of medication in non-hospitalized patients to achieve wash-out is stressed. Although this can be achieved safely in mildly hypertensive patients who are seen frequently, more often the introduction of a relatively renin-neutral drug such as doxazosin or prazosin, or hydralazine or verapamil slow release, alone or in combination, is required.
The question of variability of the ARR from day to day despite identical time of day, posture and medications (or lack of them) has not been addressed by Ferrari et al.  or Mulatero et al. , with single samples being utilized in both studies. There are issues of practicality, but in clinical practice it is wise to be cognisant of possible significant variability and to repeat the ratio at least once before going on to a more definitive diagnostic test, which invariably involves salt-loading, and therefore is not risk-free in severely hypertensive or elderly patients with compromised cardiac function.
The circadian rhythm for renin in continuously recumbent patients peaks between midnight and 0800 h and has a nadir in the afternoon and early evening . The stimulatory effect of upright posture on renin and aldosterone is greater in the early morning than it is in the afternoon . Hence, levels obtained upright (seated) during a morning hypertension clinic will be higher, on average, than those obtained from the same patients during an afternoon clinic. The increase in PAC following the change from recumbency to upright posture is thought to result partly from the increase in renin and partly from reduced metabolic clearance of aldosterone due to reduced hepatic blood flow. As noted out by Ferrari et al. , the timing of the increase in PRA and PAC has been studied [5,6] but, to my knowledge, the decline in PRA and PRC following resumption of supine posture mid-morning after a period of ambulation has not. As noted by Ferrari et al. , many investigators have utilized a mid-morning upright sample, usually while seated for 5–15 min. Others have chosen a mid-morning sample after a period (such as 1 h) of recumbency. Ferrari et al.  utilize such a mid-morning recumbent sample in their study, yet employ the cut-off point taken from a study  utilizing mid-morning upright samples (seated for 10 min). Clearly, it is more practical to collect mid-morning upright samples rather than have to provide recumbent conditions for 1 h. However, once a research group has established its protocol, it is unlikely to change, and this represents a major area of difficulty in comparing, or pooling laboratory or clinical results from different groups. It is possible that samples collected recumbent might be more sensitive in detecting angiotensin-unresponsive forms of PAL, such as classic, fasciculata-cell-type APAs, and glucocorticoid-remediable hyperaldosteronism (familial hyperaldosteronism type I, FH I); upright samples, on the other hand, might be best for detecting angiotensin-responsive forms of PAL such as angiotensin-responsive APAs [7,8] and bilateral adrenal hyperplasia.
Other sampling conditions can also be critical. Hypokalemia lowers PAC and can lead to a false-negative ARR. A percentage of patients with PAL are hypokalemic, especially the long-standing, severe forms most urgently in need of diagnosis, where a false-negative screening test would be very unfortunate. It has long been known that venepuncture with stasis (tourniquet) or fist-clenching can mask hypokalemia  and the modern use of vacuum-tubes or any difficult venepuncture can cause sufficient haemolysis to mask hypokalemia. That aspect was not mentioned by Ferrari et al. , except that blood was collected into ‘vacutainers'. Mulatero et al.  repeated sampling after correction of hypokalemia in individuals with potassium levels less than 3.6 mmol/l, but attention to this potential problem has been very variable across the most often quoted literature on PAL, although it is not too difficult to address.
With diuretics so widely used in the treatment of hypertension, and increasingly advocated despite their ability to precipitate diabetes during long-term treatment, it is extremely important to consider their effects on ARR. Through reduction in total body potassium lowering PAC, and simultaneous elevation of renin through hypovolemia, they are a potent cause of false-negative ARR. Their effects subside slowly, and cessation for 7–10 days, as in the study under discussion, is insufficient. Thus, this comprises another argument for not making a clinical decision based on a single ratio.
Deciding on a specific cut-off point for the ARR and assessing the validity of more definitive tests for PAL (such as oral salt-loading, saline infusion or oral salt-loading plus fludrocortisone) when choosing the ultimate or gold standard criterion for a final diagnosis of PAL, both represent important problems that continue to complicate any interpretation of the literature on PAL. The degree of difficulty depends on the subtype. For FH-I, it is solved by demonstration of the hybrid gene [10,11] or, less unequivocally, the demonstration that PAL is glucocorticoid-suppressible, driven by ACTH; but this is a very rare form of PAL. For APA, the only proof of diagnosis is the removal of an adrenal shown to contain an adenoma, resulting in cure of hypertension and hypokalemia. Unfortunately, there are often other forces leading towards hypertension, which is present in 33% of individuals aged over 50 years in many Western countries. This can result in patients in whom the diagnosis of PAL was correct, but for whom hypertension was not completely ‘cured’ by removal of an APA, and this is why we routinely repeat a (fludrocortisone) suppression test 2–6 months after removal of an APA  to see whether or not we have fully corrected the biochemical abnormality of excessive, autonomous aldosterone production. We assess the correctness of our diagnostic criteria against this result, rather than blood pressure response. It also helps us to decide whether or not any residual hypertension might logically be best treated with a small dose of aldosterone-blocker. What criteria for APA are used by Ferrari et al. ? If an adrenal has not been removed, shown to contain a ‘tumour’ (however small), and followed by cure or marked improvement in hypertension, are there lesser criteria acceptable for a diagnosis of APA? What about aldosterone (PAC or a urinary aldosterone metabolite) not suppressible by salt-loading plus a unilateral abnormality on computed tomography ? Unfortunately, adrenals can be ‘lumpy', especially in patients aged over 50 years, with no recognizable hormone-excess syndrome such as PAL, and incidentally discovered adrenal masses (incidentalomas) are increasingly commonly discovered, and usually non-functional on endocrine testing. Unwilling to risk removing the wrong adrenal, we routinely perform adrenal venous sampling (AVS) according to a strict protocol before considering unilateral adrenalectomy [7,11–13]. This is invasive, and requires a skilled and experienced radiologist along with very careful interpretation of the results . Not surprisingly, it is not widely practised, and when it is, it is often reserved for problem patients .
What then is the gold standard for screening for, and then establishing a diagnosis of, the APA form of PAL? Even more difficult is the establishment of a gold standard for diagnosis of undifferentiated PAL in all its forms; two-thirds may be due to varieties other than APA. How did the study under examination address these issues? Patients with ARR higher than the selected cut-off point and with an adrenal mass were considered to have APAs (nine in all). At the time of publication, APAs had been removed with characteristic findings on gross and microscopic examination in six, and the remaining three were awaiting surgery. Neither aldosterone suppression studies nor adrenal venous sampling were performed in those nine subjects considered to have APAs. Nor were they performed on three patients with discordant results (raised ARR using one but not the other method of estimating renin). Might there have been others with PAL (even APAs) whose single estimation did not reach the arbitrary level for ARR, or whose PAC did not reach the arbitrary level of 600 pmol/l? In their discussion, the authors concede that, in their wider experience, they have encountered patients with an ARR (irR) greater than 150 (their chosen cut-off point) who had a PAC less than their usual cut-off point of 600 pmol/l, and who achieved control of hypertension after unilateral adrenalectomy. This serves to underline the arbitrariness of the criteria within which we must all work, utilizing our own experience and our knowledge of our assays and their performance in the wide population of hypertensives under a wide variety of conditions. It also underscores the need for us to be circumspect before completely ruling in, or excluding, the diagnosis of ‘primary aldosteronism'.
The study by Ferrari et al.  might have better been entitled ‘Immunroreactive renin versus plasma renin activity to define aldosterone-to-renin ratio for ‘aldosterone-producing adenoma', since it does not address the issue of PAL due to bilateral disease, which is possibly twice as common. However, the study can rightly be regarded as a straightforward attempt by workers with a reputation for good laboratory quality control to answer a simple but important question, and possibly open the way to better laboratory-to-laboratory comparability in utilizing the most popular screening test for an ever-increasingly recognized, important cause of hypertension. It represents a small step forward, and helps to bring into focus the remaining more complex problems and questions that face all who work in this area and, ultimately, all those who hope to provide the best care for hypertensive patients.
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