Laparoscopic adrenalectomy.

Using the transperitoneal, laparoscopic approach, we performed 67 successful adrenalectomies between June 1993 and July 1995 at Greenslopes Hospital, Brisbane. There were 30 women and 37 men. Syndromes of primary adrenal hormone overproduction--primary aldosteronism (n = 52), pheochromocytoma (n = 6), and hypercortisolism (n = 1)--were present in 59 patients and apparently nonfunctioning adrenal tumors (of which one was malignant) in 8 patients. There was a significant difference in the time of operation between patients weighing < 80 kg and those weighing > 80 kg. Operations on males were slower than those on females, possibly explained by males being significantly heavier. Left-sided tumors outnumbered right-sided tumors; removal of right-sided adrenals took, on average, longer, but this difference was not significant.

PCR-SSCP analysis of the p53 gene in tumours of the adrenal gland.

1. Mutations of the p53 tumour suppressor gene are relatively common in the aetiology of a wide spectrum of tumour types, both sporadic and familial. 2. The majority of mutations of the p53 gene are reported to be in the highly conserved region of exons 5-8. 3. Alterations in exons 4, 5 and 7 of the p53 gene in patients with functional adrenal tumours, including aldosterone-producing adenomas, have recently been described. 4. In the present study PCR-SSCP was used to examine the exons 4-9 of the p53 gene in paired peripheral blood leucocyte and tumour DNA in a variety of adrenal tumours, including aldosterone-producing carcinoma and adenoma (both familial and sporadic), phaeochromocytoma and incidentaloma. 5. No evidence was found for mutations in exons 4-9 of the p53 gene in these varieties of adrenal tumours.

PCR-SSCP analysis of the promoter region of the renin gene in patients with aldosterone-producing adenomas.

1. Aldosterone-producing adenomas (APA) of the adrenal gland may be responsive or un-responsive to the renin-angiotensin system. 2. We have described increased expression of renin mRNA in angiotensin-responsive aldosterone-producing adenomas (AII-R-APA) compared with angiotensin-un-responsive aldosterone-producing adenomas (AII-U-APA) and significantly different allelic frequencies of the BglI, TaqI and HinfI restriction fragment length polymorphisms of the renin gene between the two groups. 3. An area including the 5' flanking region -500 bp from exon 1, exon 1 and intron A contained no gross insertions or deletions when studied by a long polymerase chain reaction technique. 4. In the present study, polymerase chain reaction-single strand conformation polymorphism analysis (PCR-SSCP) revealed no single base pair alteration in the proximal promoter region (-600 bp to transcription start) of the renin gene in patients with APA (either AII-U-APA or AII-R-APA) when compared with normal subjects. 5. Therefore, mutations in this regulatory region do not appear to explain the different levels of renin gene expression observed in these two subtypes of APA.

Clinical, biochemical and genetic approaches to the detection of familial hyperaldosteronism type I.

AIM: Since detection of familial hyperaldosteronism type I (glucocorticoid-suppressible hyperaldosteronism) allows specific treatment of hypertension with dexamethasone, we compared clinical, biochemical and genetic approaches to detection. PATIENTS AND METHODS: We studied 22 affected patients, 21 from a single, large family and an additional adopted male. Plasma aldosterone, plasma renin activity and urinary 18-oxo-cortisol were measured by radioimmunoassay. The hybrid gene was demonstrated using either Southern blotting or a long polymerase chain reaction technique. RESULTS: Thirteen out of 22 (59%) patients with familial hyperaldosteronism type I, but only four out of 12 (33%) under 20 years of age, were hypertensive. Plasma potassium and aldosterone were each normal in 20 out of 22 (91%), and unhelpful in diagnosis. Plasma renin activity, the aldosterone: plasma renin activity ratio and 18-oxo-cortisol were more sensitive, being abnormal in 20 out of 22 (91%), 19 out of 22 (86%) and 20 out of 20 (100%) patients, respectively. Aldosterone was unresponsive (<50% rise) to 2 h of upright posture following overnight recumbency in 15 out of 15 (100%) patients studied, and to angiotensin II infusion (2 ng/kg per min for 1 h) in 14 out of 14 patients (100%). Whereas all the abovementioned abnormalities are also characteristic of angiotensin II-unresponsive aldosterone-producing adenoma, marked aldosterone suppression following 4 days of dexamethasone (0.5 mg every 6 h) was sensitive and specific for familial hyperaldosteronism type I (n = 11). The hybrid gene was detectable in peripheral blood leucocyte DNA in all 22 affected patients by Southern blotting, and by a faster, long polymerase chain reaction method developed in our laboratory, both methods requiring only a single blood collection. CONCLUSIONS: Should studies in other families confirm its universal applicability, long polymerase chain reaction should prove to be the most practical means of detecting familial hyperaldosteronism type I in laboratories equipped with this technique.

Reduced renal extraction of atrial natriuretic peptide in primary aldosteronism.

We investigated renal and peripheral forearm extraction of atrial natriuretic peptide in patients with primary aldosteronism to determine whether alterations in extraction may contribute to the elevated levels of circulating atrial natriuretic peptide observed in primary aldosteronism. We obtained simultaneous venous blood samples from the left renal vein and a peripheral vein and from the radial artery in 28 patients with primary aldosteronism and 10 patients with essential hypertension. Renal extraction of atrial natriuretic peptide was significantly (P < .001) reduced (40 +/- 2%) in primary aldosteronism compared with essential hypertensive patients (62 +/- 3%). Peripheral forearm extraction was also reduced (P < .01) in primary aldosteronism compared with essential hypertensive patients (24 +/- 3% versus 38 +/- 4%). These findings are consistent with widespread downregulation of atrial natriuretic peptide receptors in primary aldosteronism. Consistent with reports that marked reduction in glomerular filtration rate is required before the renal extraction of atrial natriuretic peptide is reduced, no significant relationship between renal extraction of atrial natriuretic peptide and plasma creatinine was seen in primary aldosteronism or essential hypertension. Although the major regulators of atrial natriuretic peptide secretion in primary aldosteronism are presumably alterations in arterial blood pressure and plasma volume, reduced renal and peripheral extraction of atrial natriuretic peptide in primary aldosteronism may also contribute significantly to the elevated circulating levels observed.

Long-PCR of the ANP gene and PCR-SSCP analysis of the proximal promoter region of the ANP gene in patients with aldosterone producing adenoma.

Previous studies have shown a significant association between allelic frequencies at the ANP gene locus and aldosterone responsiveness to angiotensin in aldosterone-producing adenoma (APA). We searched for any gross insertions or deletions in the ANP gene in APA and any associations between allelic frequencies at the Hpa II and Sca I RFLP sites within the ANP gene and angiotensin-responsive and unresponsive APA and normal subjects. We also searched for possible point mutations in the promoter region of the ANP gene (-595 to transcription start site) in peripheral blood and tumor DNA from 59 patients with APA and in peripheral blood DNA from 39 normal subjects by polymerase chain reaction and single strand conformation polymorphism (PCR-SSCP) analysis. No large alterations in the ANP gene were observed, and no difference in allelic frequencies at the RFLP sites were seen between the two tumor subtypes, angiotensin-responsive and angiotensin-unresponsive APA, or between the APA group and normal subjects. SSCP analysis, however, did reveal mutations in the promoter region of the ANP gene (-375 to -595) in both peripheral blood and tumor DNA from 8 of 59 (14%) patients with APA, compared with only one of 39 normal controls (2.6%). This study suggests that alterations in the proximal promoter region of the ANP gene in APA may be important in the regulation of ANP transcription and may be involved in the underlying pathophysiology of aldosterone-producing adenoma in at least some patients.

Effects of felodipine, metoprolol and their combination on blood pressure at rest and during exercise and on volume regulatory hormones in hypertensive patients.

The effects on blood pressure (BP) and heart rate (HR), at rest and during bicycle exercise, of the vascular selective calcium antagonist felodipine, the cardio-selective beta-blocker metoprolol, and of the two drugs in combination, were assessed in a double-blind, three-way cross-over study comprising 23 patients with essential, mild to moderate hypertension. All three treatment regimens were given to each patient in randomised order for 4 weeks after a 4 week placebo run-in period. Felodipine 10-20 mg daily, metoprolol 100-200 mg daily and the combination of felodipine 10-20 mg plus metoprolol 100 mg daily were all effective antihypertensive treatments both at rest and during exercise. The two drugs seemed to have additive effects and the effect on BP of the combination was greater than that of either drug given as monotherapy. The mean sitting BP was 148/103 mmHg at randomisation, after 4 weeks of placebo treatment, and 134/88, 134/94 and 121/84 mmHg, respectively, after 4 weeks' treatment with felodipine, metoprolol and the combination. Maximal exercise capacity was similar irrespective of treatment regimen, and the normal response to exercise BP and HR was maintained during all active treatments. Changes observed in volume regulatory hormones (PRA, aldosterone and ANP) were consistent with a direct tubular natriuretic-diuretic effect of felodipine and of beta-blocker attenuated release of renin. All treatment regimens were well tolerated and adverse events reported were usually mild and transient.

Hybrid gene or hybrid steroids in the detection and screening for familial hyperaldosteronism type I.

1. Early diagnosis of Familial Hyperaldosteronism Type I (FH-I, glucocorticoid-suppressible hyperaldosteronism) in asymptomatic, affected individuals is essential if death from stroke is to be prevented. 2. In 21 patients with FH-I (presence of the causative hybrid 11 beta-hydroxylase/aldosterone synthase gene confirmed by Southern blot testing), various biochemical parameters were compared as possible screening tests. Hypokalaemia and elevated plasma aldosterone each detected only two (10%) of the affected individuals. 3. Plasma renin activity 19 (90%) and aldosterone/renin ratio 18 (86%) were more reliable but not free from false negatives. 4. Levels of the urinary 'hybrid' steroid, 18-oxocortisol, were elevated (P < 0.01) in all 15 patients tested (138.2 +/- 17.4 micrograms/g creatinine, range 41.6 +/- 281.0 micrograms/g) with no overlap when compared with 11 normals (9.7 +/- 1.3 micrograms/g, range 2.8-17.4 micrograms/g). 5. We conclude that measurement of urinary 'hybrid' steroids is probably the most rapid and reliable biochemical screening test currently available for FH-I, with confirmation dependent on demonstration of the hybrid gene by genetic techniques.

PCR-SSCP analysis of the angiotensin II type 1 receptor gene in patients with aldosterone-producing adenomas.

1. In patients with primary aldosteronism due to angiotensin-responsive and angiotensin-unresponsive aldosterone-producing adenomas, no differences in the coding region of the angiotensin II type 1 (AT1) receptor gene were observed compared to normal subjects in peripheral blood leucocyte DNA. 2. Furthermore, no differences in the AT1 receptor gene were observed in DNA extracted from tumour tissue of either subgroup. 3. Genotypic and allelic frequencies for an RFLP detected in the coding region of the AT1 receptor gene were not significantly different between normal subjects and patients with aldosterone-producing adenomas as a group, nor between normal subjects and patients of either subgroup when compared with each other. 4. In those patients heterozygous in peripheral blood at the RFLP site, tumour DNA showed the same allelic pattern. 5. In patients with aldosterone-producing adenomas either responsive or unresponsive to the renin-angiotensin system, no differences were detected using SSCP analysis in the coding region of the AT1 receptor gene in peripheral blood or tumour tissue.

Plasma aldosterone response to ACTH in subtypes of primary aldosteronism.

1. Aldosterone responsiveness to ACTH was compared in eleven patients with angiotensin-unresponsive (AII-U) aldosterone-producing adenomas (APA), 16 with AII-responsive (AII-R) APA and 19 with bilateral adrenal hyperplasia (BAH). 2. After overnight recumbency, aldosterone levels were highest in AII-U APA and lowest in BAH. Following 2 h of upright posture, however, levels were similar among the three groups. 3. During ACTH infusion, aldosterone levels in AII-U and AII-R APA were similar, and higher than those in BAH. Because of the higher basal level, the percentage rise in aldosterone was lower in AII-U APA compared with the other groups, as was the ratio of per cent aldosterone rise to per cent cortisol rise. 4. Slightly but significantly reduced plasma cortisol levels observed in the AII-R APA group may reflect secretion by AII-R APA of a cortisol-like substance that is capable of suppressing ACTH and thus adrenal cortisol production. 5. The tendency of aldosterone to follow the diurnal rhythm of ACTH in AII-U APA may thus represent an unmasking of the normal ability of ACTH to regulate aldosterone, secondary to the loss of AII responsiveness, rather than an enhancement of ACTH effect.

Analysis of the renin gene in patients with aldosterone-producing adenomas by polymerase chain reaction-single stranded conformational polymorphisms and long polymerase chain reaction.

1. Angiotensin-responsive aldosterone-producing adenomas (AII-R-APA) have increased expression of renin mRNA compared with angiotensin-unresponsive aldosterone-producing adenomas (AII-U-APA) or normal adrenals. 2. Further, significant associations between the BglI, TaqI and HinfI RFLP and aldosterone responsiveness to the renin-angiotensin system of the two subgroups of patients have been reported. 3. Using the polymerase chain reaction based technique single stranded conformational polymorphism, we detected no alterations in exon 1 of the renin gene in peripheral blood leucocyte DNA from normal AII-U-APA and AII-R-APA subjects. 4. Using long-PCR, we amplified a fragment of the renin gene consisting of a region covering 500 bp upstream of exon 1, exon 1 and intron A. No gross changes in this area of the renin gene were found in the three groups of subjects studied. However this does not exclude small alterations in this area.

Laparoscopic adrenalectomy for adrenal tumours causing hypertension and for 'incidentalomas' of the adrenal on computerized tomography scanning.

1. In a 19 month period from June 1993 to December 1994, 60 patients (mean age 54.8 +/- 1.5 years s.e.m.; 32 males, 28 females) underwent unilateral laparoscopic adrenalectomy by one of us (JCR) for the treatment of hypertension due to primary aldosteronism (n = 48), phaeochromocytoma (n = 3) and cortisol-producing adenoma (n = 1) or to remove adrenal massess incidentally discovered on abdominal computerized tomography scanning ('incidentaloma') performed for other reasons (seven adenomas without biochemical evidence of excessive steroid hormone or catecholamine secretion and one carcinoma autonomously producing cortisol). 2. Compared with conventional open procedures, laparoscopic adrenalectomy was associated with reduced recovery time and a low complication rate (one pulmonary embolus and one port site incisional hernia). 3. Operation time with experience approximates that of open procedures (60 min), but is significantly longer in obese than in non-obese patients, and in males than in females. 4. Patients with adrenal causes of hypertension were cured or significantly improved by laparoscopic unilateral adrenalectomy. 5. Because of our concern regarding malignant potential of incidentalomas and high patient acceptance of laparoscopic techniques, we have reduced our size criteria for removal of incidentalomas.

A new genetic test for familial hyperaldosteronism type I aids in the detection of curable hypertension.

In Familial Hyperaldosteronism Type I (FH-I, glucocorticoid-suppressible hyperaldosteronism), a curable form of hypertension inherited in an autosomal dominant fashion, the underlying genetic defect is a "hybrid gene" in which 11 beta-hydroxylase gene regulatory elements are fused to the coding region of the aldosterone synthase gene. The detection of this hybrid gene by Southern blotting is time consuming and involves the use of radioactive isotopes. We describe a new, long polymerase chain reaction-based method for detecting the hybrid gene which greatly reduces the time required to obtain a result, avoids exposure of laboratory workers to radioactive materials, and will thereby facilitate the screening of patients for the presence of FH-I.

Primary aldosteronism--some genetic, morphological, and biochemical aspects of subtypes.

Primary aldosteronism is the commonest cause of potentially curable hypertension when diagnosed in both florid and less florid forms. Genetic screening, so far available only for glucocorticoid-suppressible hyperaldosteronism, permits diagnosis from birth, before any biochemical or clinical abnormalities appear. Biochemical screening using the aldosterone-to-renin ratio permits diagnosis in the absence of raised aldosterone or of hypokalemia. Primary aldosteronism occurs in several familial forms. As well as the variety described in 1966 which is ACTH-dependent and glucocorticoid-suppressible, and not so far associated with tumors, another variety described in 1991 is not glucocorticoid-suppressible and is frequently associated with aldosterone-producing adenomas (APAs). Primary aldosteronism due to adrenocortical hyperplasia, adenoma, or carcinoma can also occur as part of the multiple endocrine neoplasia syndromes, where normoplasia, hyperplasia, benign neoplasia, and malignant neoplasia can exist in the same patient in the same endocrine gland(s) at the same time. The morphology of adrenocortical hyperplasia causing primary aldosteronism ranges from glomerulosa-like (idiopathic hyperplasia of the adrenals) to fasciculata-like (glucocorticoid-suppressible hyperaldosteronism). The morphology of adrenocortical neoplasia causing primary aldosteronism can also be either predominantly glomerulosa-like or fasciculata-like, in our experience equally often. Varying morphology of APAs is associated with varying responses of aldosterone to angiotensin II. Tumors predominantly fasciculata-like are unresponsive to angiotensin II, whereas those predominantly glomerulosa-like are responsive to angiotensin II. Both subtypes can be seen in a single family. Primary aldosteronism represents a spectrum of genetic disorders resulting in hyperplasia or neoplasia, but all are associated with some degree of autonomy of aldosterone production, independent of the renin-angiotensin system.

The renin gene and aldosterone-producing adenomas.

Approximately one half of the aldosterone-producing adenomas (APA) removed from patients with primary aldosteronism in the Hypertension Unit at Greenslopes Hospital belong to a subgroup in which aldosterone levels are responsive to the renin-angiotensin system (angiotensin-responsive APA; AII-R-APA), unlike classical APAs in which aldosterone is unresponsive (AII-U-APA). Renin mRNA levels in AII-R-APA were elevated when compared with those in AII-U-APA or normal adrenal cortices. Renin mRNA levels in some adrenal cortices surrounding AII-R-APA (but never in AII-U-APA) were raised, suggesting that a genetic defect is not confined to the tumor. Renin gene RFLP analysis in peripheral blood DNA revealed a significant difference in allelic frequencies between patients with AII-R-APA and AII-U-APA, suggesting an association between an alteration in the renin gene and aldosterone responsiveness to the renin-angiotensin system in patients with APAs.

Association of restriction fragment length polymorphism at the atrial natriuretic peptide gene locus with aldosterone responsiveness to angiotensin in aldosterone-producing adenoma.

Primary aldosteronism is an important, potentially curable, form of hypertension. We examined the possible association between restriction fragment length polymorphisms in the atrial natriuretic peptide (ANP) gene and responsiveness of aldosterone to angiotensin II in 59 patients with primary aldosteronism due to aldosterone-producing adenoma (APA). Significant differences in the allelic frequencies of the BglI, TaqI and XhoI polymorphic sites at the ANP gene locus (chromosome 1; 1p36) between angiotensin II-unresponsive and angiotensin II-responsive tumors were observed. Variation in the ANP gene between the two groups may result in altered expression of ANP within the adrenal gland, and may contribute to the biochemical regulation of aldosterone production of these two subgroups of patients with APA.

Genetics of primary aldosteronism.

1. In 1991 we described a familial variety of primary hyperaldosteronism which was not glucocorticoid-suppressible and was associated with adenoma formation, and called it familial hyperaldosteronism type II (FH-II) in order to distinguish it from the glucocorticoid-suppressible variety described in 1966, familial hyperaldosteronism type I (FH-I). 2. In 1992 the genetic basis of FH-I was clarified by description of a hybrid gene. 3. Primary aldosteronism due to bilateral adrenocortical hyperplasia or to aldosterone-producing tumour can be part of the multiple endocrine neoplasia type I syndrome (MEN I), in which loss of heterozygosity has been described on chromosome 11q13. Loss of heterozygosity at the MEN I locus was found in five of 26 aldosterone-producing tumours from our series (by Japanese collaborators). These included two with adrenal cancer and two with FH-II. 4. We recently described an association of aldosterone responsiveness of aldosterone-producing adenomas with renin gene restriction fragment length polymorphisms, suggesting a possible role for renin genotype and intra-adrenal renin gene expression in the development and biochemical expression of some aldosterone-producing tumours. 5. We found abnormal karyotypes in 13 of 32 benign aldosterone-producing adenomas.

High incidence of primary aldosteronism in 199 patients referred with hypertension.

1. This study sought to assess the incidence of primary aldosteronism in 199 hypertensives who were normokalaemic and in whom the question of primary aldosteronism had never been raised. 2. The screening test applied was the aldosterone to renin ratio in plasma, which was raised in 40 and normal in 159 patients. A second ratio was normal in 14 of these 40. 3. Twenty-two patients with two further raised ratios required fludrocortisone suppression testing. This has been completed in 17, and failure to suppress led to a diagnosis of primary aldosteronism in all. 4. A dexamethasone suppression test (DST) excluded ACTH-dependent hyperaldosteronism and laterality of aldosterone production was determined by adrenal vein sampling. 5. Unilaterality in five patients led to adrenalectomy in four and spironolactone in one. Bilaterality in six patients led to spironolactone. 6. This study so far provides a proven (minimum) incidence for primary aldosteronism of 8.5%, a probable incidence of 12.0% (including two raised ratios) and a possible (maximum) incidence of 13.0% (leaving out those with second ratio normal). Exclusion of hypokalaemic hypertensives will lead to an underestimation of the true incidence of primary aldosteronism. 7. Based on this and other evidence, it is estimated that the incidence of primary aldosteronism in the 'essential hypertensive' population is between 5 and 15%, and is probably around 10%.

Response to unilateral adrenalectomy for aldosterone-producing adenoma: effect of potassium levels and angiotensin responsiveness.

1. Normokalaemic primary aldosteronism (PA) masquerades as 'essential hypertension', and 50% of patients with aldosterone-producing adenoma (APA) are normokalaemic at presentation to this unit. 2. Angiotensin-responsive (AII-R) APA is as common as angiotensin-unresponsive (AII-U) APA, and requires adrenal venous sampling for differentiation from bilateral adrenal hyperplasia (BAH). 3. From 1981 to 1992, 55 patients with APA underwent unilateral adrenalectomy and were followed up for at least 12 months postoperatively. Hypertension was cured in 55% and improved in the remainder. 4. Cure rate was lower (P < 0.001) in males (11/32, 34%) vs females (19/23, 83%), lower (P < 0.005) in patients over 45 years of age (13/33, 39%) vs those 45 years or younger (17/22, 77%), lower (P < 0.05) in AII-R APA (11/28, 39%) vs AII-U APA (19/27, 70%) and tended to be lower (not significant) in normokalaemic APA (7/17, 41%) vs hypokalaemic APA (23/38, 61%). 5. A higher proportion (P <0.001) of AII-R APA patients were males (23/28, 82%) vs AII-U APA (9/27, 33%), and a higher proportion were from the older age group AII-U APA 13/27, 48%; P < 0.05). Females with AII-U APA who were hypokalaemic had a very high cure rate (16/17, 94%). 6. Since unilateral adrenalectomy cures or improves blood pressure in normokalaemic and AII-R as well as in hypokalaemic and AII-U patients, all hypertensives should be screened for PA, and AII-R APA differentiated from BAH in proven PA.

Renal extraction of atrial natriuretic peptide in unilateral renal artery stenosis.

1. Elevated peripheral atrial natriuretic peptide (ANP) levels were observed in 12 patients with unilateral renal artery stenosis (U-RAS). 2. Renal extraction of ANP was higher across the affected than the unaffected kidney in U-RAS, provided the glomerular filtration rate in the affected kidney was not severely reduced (> 12 mL/min). As ANP is a high clearance compound, reduced flow on the affected side may result in increased renal extraction of ANP. 3. When glomerular filtration rate (GFR) in the affected kidney was severely reduced (< 12 mL/min), renal extraction of ANP was also reduced, possibly contributing to increased circulating ANP levels in this subgroup. 4. Overall, renal extraction of ANP was inversely correlated to peripheral ANP levels in patients with U-RAS. This might be explained by progressive sodium retention as GFR falls leading to volume expansion and increased ANP secretion.