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Primary Hyperaldosteronism in Essential Hypertensives: Prevalence, Biochemical Profile, and Molecular Biology¹

Departments of Endocrinology (C.E.F., L.M., P.C., L.G., M.P., E.O.), Internal Medicine (J.M.), Radiology (A.H.), and The Associated Unit of Clinical Laboratories (J.S., A.F.), Faculty of Medicine, Catholic University of Chile, Marcoleta 391, Santiago, Chile; and Endocrine Section (C.G.-S.), Harry S. Truman Memorial Hospital, Department of Internal Medicine, Columbia, Missouri 65201-5297

Address correspondence and requests for reprints to: Carlos E. Fardella, Department of Endocrinology, Faculty of Medicine, P. Universidad Católica de Chile, Marcoleta 391, Santiago, Chile.

	Abstract

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There is evidence that primary aldosteronism (PA) may be common in patients with essential hypertension (EH) when determinations of serum aldosterone (SA), plasma renin activity (PRA), and the SA/PRA ratio are used as screening. An inherited form of primary hyperaldosteronism is the glucocorticoid-remediable aldosteronism (GRA) caused by an unequal crossing over between the CYP11B1 and CYP11B2 genes that results in a chimeric gene, which has aldosterone synthase activity regulated by ACTH. The aim of this study was to evaluate the prevalence of PA and the GRA in 305 EH patients and 205 normotensive controls. We measured SA (1–16 ng/dL) and PRA (1–2.5 ng/mL·h) and calculated the SA/PRA ratio in all patients. A SA/PRA ratio level greater than 25 was defined as being elevated. PA was diagnosed in the presence of high SA levels (>16 ng/dL), low PRA levels (<0.5 ng/mL·h), and very high SA/PRA ratio (>50). Probable PA was diagnosed when the SA/PRA ratio was more than 25 but the other criteria were not present. A Fludrocortisone test was done to confirm the diagnosis. GRA was differentiated from other forms of PA by: the aldosterone suppression test with dexamethasone, the high levels of 18-hydroxycortisol, and the genetic detection of the chimeric gene. In EH patients, 29 of 305 (9.5%) had PA, 13 of 29 met all the criteria for PA, and 16 of 29 were initially diagnosed as having a probable PA and confirmed by the fludrocortisone test. Plasma potassium was normal in all patients. The dexamethasone suppression test was positive for GRA in 10 of 29 and 18-hydroxycortisol levels were high in 2 of 29 patients who had also a chimeric gene. In normotensive subjects, 3 of 205 (1.46%) had PA, and 1 of 205 had a GRA. In summary, we found a high frequency of normokalemic PA in EH patients. A high proportion of PA suppressed SA with dexamethasone, but only a few had a chimeric gene or high levels of 18-hydroxycortisol. These results emphasize the need to further investigate EH patients.

	Introduction

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HYPERTENSION is a common disease affecting about 20% of the adult population (1, 2, 3). Although a great deal is known about the regulation of blood pressure, a specific causal abnormality can be found in only a small percentage of patients. Thus, about 95% of patients with hypertension are said to have essential or idiopathic hypertension. A key mechanism in the regulation of blood pressure is the renin-angiotensin-system. Angiotensin II is the most potent stimulus to aldosterone synthesis, which, in turn, controls sodium and water excretion (4).

Low levels of plasma renin activity (PRA) are found frequently in essential hypertension (EH), but the significance of low-renin hypertension and the mechanisms involved remain controversial. A survey of 436 Japanese patients with EH showed that 12.4% had low PRA values with normal or high serum aldosterone (SA) levels, suggesting a persistent mineralocorticoid synthesis in the presence of minimal stimulation from the renin-angiotensin system (5). However, the quoted prevalence of primary aldosteronism (PA) is usually cited as being less than 1% for an unselected hypertensive population (6, 7, 8, 9). Recently, Gordon et al. (10) presented evidence that PA may not be uncommon and demonstrated an incidence up to 12% of PA when determinations of SA, PRA, and the SA/PRA ratio are used in the diagnosis. They also found that hypokalemia is present only in the more severe form of the disease and that most patients with PA are normokalemic.

The biosynthesis of aldosterone is controlled by P450c11AS (aldosterone synthase), which has all three activities needed to convert 11-deoxycorticosterone to aldosterone, i.e. 11ß-hydroxylase, 18-hydroxylase, and 18-oxidase activities (11, 12, 13, 14). This enzyme is encoded by the CYP11B2 gene and is regulated by angiotensin II and potassium via protein kinase C. This enzyme is different from P450c11ß (11ß-hydroxylase), the product of the CYP11B1 gene, which is expressed in the zona fasciculata and reticularis, and converts 11-deoxycortisol to cortisol. P450c11ß is regulated by ACTH via cAMP and protein kinase A. Human CYP11B1 and CYP11B2 have 90% nucleotide sequence identity in the introns and 95% in the exons and lie on chromosome 8q (12, 15). Both CYP11B1 and CYP11B2 have been implicated in the genesis of arterial hypertension through an increase in the aldosterone synthesis, as occurs in the glucocorticoid-remediable aldosteronism (GRA) (16, 17). Moreover, in vitro studies have demonstrated that specific mutations in CYP11B2 or CYP11B1 should also explain some forms of hyperaldosteronism (18, 19, 20).

GRA is an autosomal dominant disorder characterized by hyperaldosteronism (21, 22) and high levels of the abnormal adrenal steroids 18-oxocortisol and 18-hydroxycortisol, which are all under control of ACTH and suppressible by glucocorticoid (23, 24, 25, 26). The GRA is caused by an unequal crossing-over between the CYP11B1 and CYP11B2 genes that results in a chimeric gene that has aldosterone synthase activity but is regulated by ACTH rather than angiotensin II (16, 17). In vitro studies have shown that breakpoints through exon 4 (codon 247) result in a hybrid enzyme (11ß-hydroxylase/aldosterone synthase) that could be expected to lead to full GRA (17). Moreover, Pascoe et al. (20) have demonstrated that the product of CYP11B1 may acquire the aldosterone synthase activity if serine-288 and valine-320 are replaced by the corresponding CYP11B2 residues, glycine and alanine. Thus, the amino acid substitution serine-288-glicine (S288G) in CYP11B1 confers on it the 18-hydroxylase activity, and the substitution valine-320-alanine (V320A) confers the 18-oxidase activity required for aldosterone synthesis.

The aim of this study was to establish the prevalence of PA and GRA in a Chilean population of 305 EH patients and 205 normotensive controls, through the routine determinations of SA, PRA, and the SA/PRA ratio. The fludrocortisone suppression test was used to confirm the diagnosis of PA. The prevalence of GRA was studied using an aldosterone suppression test with dexamethasone, determinations of 18-hydroxycortisol, and genetic studies. Genetic studies were done to demonstrate the presence of the chimeric gene and the amino acid substitutions S288G and V320A in the CYP11B1 gene.

	Subjects and Methods

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Patients were recruited from the Primary Outpatient Clinic Hypertension Program associated with the Universidad Católica de Chile in Santiago. They were considered hypertensives if their diastolic blood pressure was more than 90 mm Hg and the systolic blood pressure more than 140 mm Hg on at least 3 occasions on different days and without taking any antihypertensive medication or estrogen replacement (3). All individuals with clinical evidence of secondary hypertension, diabetes, or renal disease were excluded. Using these criteria, we selected 305 EH patients. A group of 204 normotensive subjects of similar age and gender was included as control. The normotensive subjects were selected from the same primary clinics, according to the criteria that they had a diastolic blood pressure less than 85 mm Hg and a systolic blood pressure less than 140 mm Hg, determined at least on 2 occasions. The normotensives had no family history of hypertension, at least in first-degree relatives. Blood pressure was measured with a mercury sphygmomanometer, by a health care professional, after the patients were seated for 10 min. The first and fifth Korotkoff sounds were used to designate systolic and diastolic blood pressure. Informed consent was obtained from all participants, according to the guidelines of the Declaration of Helsinki, and the protocol was approved by the Research Commission of the School of Medicine at the Catholic University of Chile.

Patients were admitted to our Metabolic Ward between 0800 and 0900 h, after a 12-h fast. All subjects consumed a normal diet, with no attempt to control sodium intake. Weight and height were measured at the time of admission. Upon admission, a catheter was placed in an antecubital vein; and after sitting for 15 min, free-flowing blood was withdrawn to measure sodium, potassium, calcium, albumin, blood urea nitrogen, creatinine, aldosterone, and PRA. Twenty-four-hour urinary sodium was measured in all subjects.

SA was measured by RIA using a commercial kit from Diagnostic Products (Los Angeles, CA). The intra- and interassay coefficients of variation for SA were 5.1% and 7.1%, respectively, and the normal range was 1–16 ng/dL. The PRA was determined as previously described by Menard (27). The intra- and interassay coefficients of variation for PRA were 6.1% and 8.2%, respectively, and the normal range was 1–2.5 ng/mL/h (28). The lower limit of PRA determination was 0.1 ng/mL·h (29). PRA values less than 0.5 ng/mL/h were considered as low renin. A SA/PRA ratio more than 25 was considered as high; and a ratio more than 50, as very high (30, 31, 32). The cut point for SA/PRA ratio in 25 was validated in our population of normotensive controls (see Results). Serum 18-hydroxycortisol was measured using a biotine-avidine enzyme-linked immunoassay, as described by Gómez-Sanchez et al. (33, 34). The normal 18-hydroxycortisol value in our population was 2.67 ± 1.44 nmol/L (unpublished data). A citrated blood sample was also obtained to analyze the genome.

The diagnosis was PA if the patients met the following criteria: high SA levels (>16 ng/dL), low levels of PRA (<0.5 ng/mL·h), and a very high SA/PRA ratio (>50) in at least two determinations. The diagnosis was probable PA if the SA levels were normal but the SA/PRA ratio was more than 25 or when the SA levels were high but the SA/PRA ratio was less than 50. The cut points to define probable PA were arbitrarily established considering patients that did not meet all the classical criteria for PA but had a high SA/PRA ratio in two different determinations. A fludrocortisone test was done to confirm the diagnosis of PA in all patients. In patients with PA, the administration of a sodium-retaining steroid should not suppress SA, indicating the autonomous overproduction of aldosterone. For the fludrocortisone test, supine SA levels were measured under baseline conditions and after 4 days of fludrocortisone (0.4 mg/day, orally; 0.1 mg every 6 h), during a dietary supplementation with 110 mmol of sodium per day (35, 36). Blood samples were taken on the fifth day at 0800 h. A fludrocortisone test was considered positive when SA levels failed to suppress under 5ng/dL (36, 37, 38). In patients with confirmed PA, a dexamethasone suppression test was done. For this test, supine SA and cortisol were measured under baseline conditions and after 2 days of dexamethasone (2 mg/day, orally; 0.5 mg every 6 h). Blood samples were taken on the third day at 0800 h. Suppression of aldosterone by dexamethasone was considered positive if the SA levels were below 4 ng/dL (39, 40). Plasma cortisol suppression (under 2.5 ug/dL) was assumed as an index of the dexamethasone effect. Serum 18-hydroxycortisol measurements and a computed tomography (CT) scan of the adrenal were performed in patients with PA. Although there are no strict measurements of normal adrenal size, the CT scan was considered abnormal when any area thicker than 10 mm was detected (41).

Analysis of genomic DNA

All patients with confirmed PA were studied for chimeric 11ß- hydroxylase/aldosterone synthase genes and the CYP11B1 gene conversions S-288-G and V320A. Genomic DNA was prepared from the citrate-treated blood of 305 EH patients and 205 normotensive controls, as previously described (42). The presence of the chimeric 11ß-hydroxylase/aldosterone synthase gene was studied using the long PCR technique described by Jonsson et al. (43). This technique uses two amplification reactions in which two sense primers and one antisense primer performed concurrently. In the first reaction, the sense primer was specific for the 5' untranslated region of the aldosterone synthase gene; whereas, in the second, the sense primer was specific for the untranslated region of the 11ß-hydroxylase gene. For both reactions, the antisense primer was specific for the intron-E region of the aldosterone synthase gene. The amplification reactions were carried out using the XL PCR Kit from Perkin-Elmer Corp. (Branchburg, NJ).

The detection of the gene conversion S288G and V320A was performed using two PCR reactions (nested PCR). The first reaction amplified the CYP11B1 gene using primers to amplify exons 3–5 and 6–8, as previously described (44). The second reaction used the product of the first PCR as template. The mutant S288 allele was recognized using a modified sense primer that creates a restriction site for BsaOI (5'-AAC TGG CCT TCA GCC GCC CTC AAC AGT CGA CC-3') in the presence of the same antisense primer. The mutant V320 allele was recognized using the same sense primer and a new antisense primers (5'-GGG CCG CAC GCA GCA A-3'), and the product was digested with a BglI enzyme. Positive controls for 288G and 320A mutant were artificially constructed and used to verify the specificity of the restriction enzyme digestion. The restriction fragments were visualized on nondenaturing polyacrylamide gels at 7.5%, stained with ethidium bromide.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
We studied 305 EH patients and 205 normotensive controls of similar age and gender. The hypertensive patients showed higher levels of SA and SA/PRA ratio than the normotensives. The levels of urinary sodium excretion were similar in hypertensives and normotensives (Table 1). In normotensive subjects, the mean SA/PRA ratio plus 2 SD was 24.0, and we considered it a high ratio when it was more than 25.

Diagnosis of probable PA was made in 31 EH patients; 26 of 31 cases (83.9%) had normal aldosterone levels (<16 ng/dL), but SA/PRA ratios more than 25. In the other 5 (16.1%) patients, the diagnosis of probable PA was made because they had high SA levels but an SA/PRA ratio less than 50. The fludrocortisone test confirmed the diagnosis of PA in 16 of 31 patients (51.6%); 13 had normal- and 3 had high aldosterone levels. The fludrocortisone test was always negative in patients with aldosterone levels less than 9 ng/dL, independent of the magnitude of the SA/PRA ratio. The SA/PRA ratios were not significantly different between patients with positive and negative fludrocortisone test results (Table 2). Of those patients with very high ratios (>50) but SA levels less than 16 ng/dL, 4 of 9 were confirmed as having PA. The biochemical characteristics of patients with positive and negative fludrocortisone tests are shown in Table 2.

All 29 patients with PA responded to the dexamethasone suppression test with a decrease in SA from 17.4 ± 7.4 ng/dL to 6.5 ± 5.0 ng/dL, P < 0.05 (Fig. 1); and the serum cortisol, from 13.6 ± 4.1 ug/dL to 0.75 ± 0.35 ug/dL. The dexamethasone test was positive, as defined by the suppression of aldosterone to less than 4 ng/dL, in 10 of 29 (34.5%) patients. Serum 18-hydroxycortisol level measured in these 29 patients was 4.64 ± 5.67 nmol/L, and 2 of 29 showed significantly elevated levels (19.4 and 26.7 nmol/L).

View larger version (13K): [in this window] [in a new window]   Figure 1. SA levels before and after dexamethasone (Dx) suppression test. The horizontal bars correspond to means. The dotted line indicates the cut-off for aldosterone suppression of 4 ng/dL.

In normotensive controls, a PA was diagnosed in 3 of 205 (1.46%) cases. Only 1 of them met all the criteria for PA. The other 2 subjects were initially diagnosed as having a probable PA and confirmed by a fludrocortisone test. GRA was diagnosed in 1 of 3 normotensives with PA.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The results of this study show a high prevalence of normokalemic PA in an unselected population of EH Chilean patients. We also found a high proportion of patients with PA that suppress the SA levels with dexamethasone. However, the presence of a chimeric 11ß-hydroxylase/aldosterone synthase gene, explaining this suppression, is found only in few cases.

The frequency of PA found in our study is clearly higher than that classically described (6, 7, 8, 9), and it confirms reports of a significant high incidence of PA in patients with EH when determinations of SA, PRA, and SA/PRA ratio are used as screening (5, 10, 32, 45, 46, 47, 48). Screening patients with hypertension for PA has usually been limited to those who presented with unprovoked or easily induced hypokalemia (3, 8); our patients with PA were normokalemic and could not have been diagnosed if the screening was done using these criteria. These results support the hypothesis that normokalemic PA constitutes the most common presentation of the disease, and the hypokalemic variant probably represents only the most severe cases (31, 49). It is also interesting to note that CT scanning revealed only one nodule in 29 patients, suggesting that the hyperplasia variant is the most common.

The SA/PRA ratio has been considered a useful tool in the screening for PA (30, 31, 32). In our study, the calculation of SA/PRA ratio was useful in identifying patients with PA who had normal SA levels, but the predictive value was low. The fludrocortisone test confirmed the diagnoses of PA only in 50% of patients with probable PA, and the test was always negative in patients with SA levels less than 9 ng/dL. This fact could be explained because normal or low-normal SA levels were highly amplified by very low PRA levels, given a high SA/PA ratio and then a wrong suspicion of PA. In this work, the SA/PRA ratio predictive value improved from 50% to 65% (without losing any positive patients with PA) when the cut-off of the lower limit of PRA was moved from 0.1 to 0.35 ng/mL·h. For those reasons, we propose that the lower limit for PRA should be not less than 0.3 ng/mL·h, and confirmatory testing must be done when SA levels are not clearly elevated. In patients with high SA levels and a SA/PRA ratio more than 50, the PA was always confirmed, suggesting that SA/PRA ratios more than 50 are highly specific in patients with high PA levels, independent of the cut-off used for the lower limit of PRA.

The dexamethasone suppression test was positive in 10 of 29 patients with PA, but only 2 of these patients had a chimeric 11ß-hydroxylase/aldosterone synthase gene and elevated levels of 18-hydroxycortisol. Similar results were reported recently by Mulatero et al. (49) in 6 patients that showed suppression of SA to less than 2 ng/dL, but none of their patients were positive for the chimeric gene, suggesting than the dexamethasone suppression test can be misleading in identifying GRA. The measurement of 18-hydroxycortisol constituted the most reliable biochemical determination in the diagnosis of GRA in this study, as well as others (50). The existence of other genetic defects responsible for the suppression with dexamethasone is unknown. We were unable to demonstrate the conversions S288G and V320A in the CYP11B1 gene that should also explain a GRA (12).

It is interesting to note that one of the two patients having a chimeric gene had normal SA levels, exemplifying the wide spectrum of SA levels that can be found in patients with GRA. Moreover, we detected one normotensive subject having a chimeric gene, demonstrating that the GRA is also present in a wide spectrum of blood pressure. The presence of GRA in normotensives had been previously demonstrated and probably reflect a prehypertensive state (39).

In summary, we found a high frequency of normokalemic PA in a group of Chilean EH patients. The measurement of the SA/PRA ratio constitutes only a screening test that is especially useful in patients with normal SA levels. Dexamethasone suppresses SA in many patients with PA and, thus, is very unspecific in the identification of patients with chimeric gene. We suggest the routine determination of SA and PRA in all EH patients and serial determinations of 18-hydroxycortisol levels in the positive patients.

	Footnotes

 
¹ This work was supported by grants from the Chilean Fondecyt (1980999) and the Chilean Society of Endocrinology (97.2.05).

Received August 31, 1999.

Revised December 12, 1999.

Accepted February 7, 2000.

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