[Practical aspects regarding sample size in clinical research]. / Aspectos prácticos sobre el tamanño de la muestra en 1a investigación clínica.

BACKGROUND: The knowledge of the right sample size let us to be sure if the published results in medical papers had a suitable design and a proper conclusion according to the statistics analysis. To estimate the sample size we must consider the type I error, type II error, variance, the size of the effect, significance and power of the test. To decide what kind of mathematics formula will be used, we must define what kind of study we have, it means if its a prevalence study, a means values one or a comparative one. In this paper we explain some basic topics of statistics and we describe four simple samples of estimation of sample size.

Clinical implications of renal cyst in primary aldosteronism.

The present study surveyed 69 patients with aldosteronoma to study the clinical implications of renal cysts demonstrated in computed tomography. Patients who had cysts (n = 16, 23.2%) were older and had a longer duration of hypertension and more severe hypokalemia than those without cysts (n = 53). Patients with cysts therefore had longer-term, more severe hypokalemia than those without cysts. Endogeneous creatinine clearance (Ccr), measured in 61 patients, was significantly lower in patients with cysts (58.4 +/- 7.1 ml/min, n = 16) than in those without cysts (77.3 +/- 7.1 ml/min, n = 45, P = 0.0039). This significant difference was observed even after adjusting for covariables (age, duration of hypertension, and serum potassium) between the two groups by analysis of covariance (ANCOVA). No significant difference was observed in gender, blood pressure, serum creatinine, plasma aldosterone, or PRA. Age, serum potassium levels, and systolic and diastolic blood pressure were the significant determinants in predicting Ccr in a backward stepwise multiple regression analysis (r = 0.505, n = 61, P = 0.0025). Cysts were graded into four classes on the basis of number and size. Cyst grading correlated negatively with Ccr at a Spearman rank correlation (rho = -0.33, n = 61, P = 0.0103). The incidence of chronic renal failure was significantly higher in patients with cysts (18.8%) than in patients without (0%) in a Fischer's exact probability test (P = 0.0107). Thus, both renal cysts and dysfunction arose and/or developed from common roots, i.e., the duration and severity of hypokalemia, in primary aldosteronism. In addition, we surveyed 27 patients with pheochromocytoma. Patients with renal cysts (n = 8) had a significantly longer duration of hypertension than those without cysts. No significant difference was observed in Ccr between patients with and those without cysts. Thus, a significant link between renal cysts and Ccr was a specific feature of primary aldosteronism, but not of pheochromocytoma. In summary, the renal cysts in primary aldosteronism should be recognized as a significant complication representing the extent of renal injury and dysfunction.

LH and testosterone modulate mercuric chloride-induced acute renal failure in male rats: the implication of stress-induced hypogonadism.

The significance of stress-induced hypogonadism remains unclear. Since plasma testosterone and LH have renotropic activity that is other than reproductive, we hypothesize that stress-induced hypogonadism is an adaptive response to protect the kidney. To examine this hypothesis, we prepared hypogonadal male rats with different levels of LH and testosterone through orchiectomy (castration), through chronic treatment with a slowly secreted form of gonadotropin-releasing hormone agonist (GnRHA; GnRHA pretreatment), or through both treatments concomitantly (castration with GnRHA pretreatment). Castrated rats had undetectable plasma testosterone and high plasma LH. GnRHA-pretreated rats had low plasma testosterone and normal plasma LH. Castrated rats with GnRHA pretreatment had undetectable plasma testosterone and normal plasma LH. We compared their sensitivity to HgCl2 nephrotoxicity and found that, when a low dose of HgCl2 (1.5 mg/kg body weight (BW)) was injected s.c. to induce acute renal failure, endogenous creatinine clearance (Ccr) decreased from 390 +/- 30 to 94 +/- 17 ml/h per kg BW in intact (unpretreated) rats. Such a decrease in Ccr was completely prevented in castrated rats (388 +/- 30 ml/h per kg BW) and partially prevented in GnRHA-pretreated rats (216 +/- 40 ml/h per kg BW). When a high dose of HgCl2 (2.25 mg/kg BW) was injected, half of the eight intact rats died but castrated rats and GnRHA-pretreated rats survived (P < 0.05). The elevated resistance in castrated rats was reduced when plasma LH was reduced with GnRHA pretreatment, but was restored by additional pretreatment with ovine LH (40 micrograms/day), as evidenced by changes in Ccr. Elevated resistance in castrated rats was also reduced by the administration of testosterone propionate. In conclusion, hypogonadism activated the preventive and defensive mechanisms that protect the kidney through both decreased plasma testosterone and high or even normal plasma LH.

Characteristics of aldosterone-producing adenoma responsive to upright posture.

A small subgroup of primary aldosteronism due to aldosteronoma, named aldosterone-producing renin-responsive adenoma (AP-RA), has been reported to masquerade as idiopathic hyperaldosteronism (IHA) because of the responsiveness of the plasma aldosterone concentration (PAC) to upright posture (UP). We found two patients with AP-RA in 19 patients with aldosteronoma who were examined by UP stimulation and were treated surgically. In 17 patients with typical aldosterone-producing adenoma (APA), PAC decreased or increased only slightly (less than 200% of the basal level); in contrast, it increased to over 300% of the basal level in two patients with AP-RA. The two groups were comparatively studied as to their hormonal levels, adrenal computed tomography (CT) scan and histological findings in order to clarify the characteristics of AP-RA. Basal PAC was within the normal range (11.1 and 13.0 ng/dl) in AP-RA but in APA it ranged from 14.8 to 58.1 ng/dl with a mean of 32.3 +/- 2.7 ng/dl. The diameters of the adenoma in AP-RA were apparently smaller (6 and 9 mm) than those in APA ranged from 10 to 25 mm with a mean of 15.5 +/- 1.1 mm. After a contrast medium was injected at CT scan, the density of the normal adrenal gland adjacent to the adenoma increased but that of the adenoma did not in APA, making a clear distinction between the adenoma and the gland. On the other hand, the density of the adenoma and gland increased to almost the same degree in AP-RA.(ABSTRACT TRUNCATED AT 250 WORDS)

Appropriate intravenous doses of L-thyroxine and magnesium in a thyroidectomized patient with thyroid and parathyroid carcinomas receiving total parenteral nutrition during acute necrotizing pancreatitis.

A totally thyroidectomized patient with thyroid and parathyroid carcinomas, which had developed after neck irradiation in childhood, became hypercalcemic due to pulmonary metastases. The hypercalcemia was ameliorated by intermittent iv administration of bisphosphonate for 3.5 years, but this gradually became refractory to the bisphosphonate treatment. After right thoracotomy for resection of pulmonary metastases, acute necrotizing pancreatitis developed. The patient was therefore placed on total parenteral nutrition supplemented with T4 and a restricted dose of magnesium. Thyroxine(T4) (30 micrograms/day, iv) was not sufficient to maintain euthyroidism, but a higher dose (60 micrograms/day) elicited mild hyperthyroidism to the same extent as that elicited by an oral dose of 100 micrograms/day. The present case showed that the appropriate iv dose of T4 in this thyroidectomized patient with acute pancreatitis was about 60% of the oral dose. Furthermore, bisphosphonates (pamidronate and alendronate) and magnesium depletion were very effective in controlling the hypercalcemia.

Progressively increased serum 1,25-dihydroxyvitamin D2 concentration in a hypoparathyroid patient with protracted hypercalcemia due to vitamin D2 intoxication.

A 76-year-old female patient who had been taking vitamin D2 100,000 U/day for more than 14 years due to hypoparathyroidism following total throidectomy was admitted because of protracted hypercalcemia. On admission, the levels of serum vitamin D2 (99.8 ng/ml) and 25-OHD2 (356 ng/ml) were very high, and 1,25-(OH)2D2 was low (4.0-18.7 pg/ml). Serum D3' 25-OHD3 and 1,25-(OH)2D3 were below the normal range. Despite intensive hydration with saline, intravenous hyperalimentation with phosphate- and calcium-free nutrients, and administration of glucocorticoid and calcitonin, the hypercalcemia persisted, accompanied by hypoproteinemia, edema, pleural effusion and congestive heart failure. The serum D2 and 25-OHD2 concentrations remained high and were accompanied by a gradual increase in 1,25-(OH)2D2 (121 pg/ml), which further increased after the administration of bisphosphonate (pamidronate) to 183 pg/ml. Seventeen months later, serum calcium and 1,25-(OH)2D2 were normalized but serum D2 and 25-OHD2 remained high. The serum 24,25-(OH)2D2/25-OHD2 ratio was relatively constant throughout her clinical course, whereas the low serum 1,25-(OH)2D2/25-OHD2 ratio at admission gradually increased during admission, suggesting that the increase in serum 1,25-(OH)2D2 is due to increased production rather than decreased degradation. The administration of pamidronate further increased serum 1,25-(OH)2D2. These features of the clinical course demonstrate that the 1,25-dihydroxyvitamin D concentration in hypercalcemic patients with protracted vitamin D intoxication may be decreased, normal or increased. Possible factors responsible for a protracted increase in serum 1,25-(OH)2D2 are body weight loss, hypoproteinemia, and phosphate depletion. In addition, some bisphosphonates would certainly promote PTH-independent production of 1,25-(OH)2D2.

Effects of cysteamine, a somatostatin depleter, on the renin-angiotensin-aldosterone axis and glomerulosa cell growth in rats.

Cysteamine, a specific somatostatin depleter, was given to male rats to clarify its role in relation to the renin-angiotensin-aldosterone (RAA) axis and glomerulosa cell growth. Rats received seven daily sc injections of cysteamine at doses of 50 or 150 mg/kg body weight (BW). Their adrenal weights and whole cortical thickness increased, but zona glomerulosa thickness decreased dose-responsively. Plasma renin activity (PRA) and aldosterone concentration (PAC) decreased. Similar results were observed in rats on a low or high salt diet and receiving daily doses of 150 mg/kg BW of cysteamine. In hypophysectomized rats, however, cysteamine given for seven days at daily doses of 100 mg/kg BW did not change either PRA or PAC. Adrenal weight did not change either too. Our results indicate that cysteamine suppresses the RAA axis and glomerulosa cell growth, probably through pituitary factors.

Increased 1,25-(OH)2D2 concentration in a patient with malignancy-associated hypercalcemia receiving intravenous hyperalimentation inadvertently supplemented with vitamin D2.

A 55-year-old patient with hypercalcemic crisis due to gastric carcinoma with bone marrow metastasis was treated with bisphosphonate (pamidronate) and calcitonin. Urinary excretion of parathyroid hormone-related protein (PTHrP) was increased. When normocalcemia had been attained, intravenous hyperalimentation was started, in which 1,000 U vitamin D2 was inadvertently supplemented on days 5-18, On days 15-18, hypercalcemia rapidly recurred, accompanied by markedly increased serum levels of 25-OHD2 (9.1 ng/dl) and 1,25-(OH)2D2 (161 pg/ml). This clinical course suggests that PTHrP, like PTH, stimulated 1 alpha-hydroxylase activity and produced excessive 1,25-(OH)2D2. Vitamin D should not be administered to patients with malignancy-associated hypercalcemia, particularly that due to PTHrP-producing tumors.

Clinical significance of adrenal computed tomography in Addison's disease.

Adrenal computed tomographic (CT) scanning was conducted in twelve patients with Addison's disease during the clinical course. In tuberculous Addison's disease (n = 8), three of four patients examined during the first two years after disease onset had bilaterally enlarged adrenals, while one of four had a unilaterally enlarged one. At least one adrenal gland was enlarged after onset in all six patients examined during the first four years. Thereafter, the adrenal glands may atrophy bilaterally, in contrast to adrenal glands in idiopathic Addison's disease, which atrophy bilaterally from disease onset (n = 2). Adrenal calcification was a less sensitive clue in tracing pathogenesis, i.e., adrenal calcification was observed in five of eight patients with tuberculous Addison's disease, but not in idiopathic patients. Thus, adrenal CT scanning could show the etiology of Addison's disease (infection or autoimmunity) and the phase of Addison's disease secondary to tuberculosis, which may be clinically important for initiating antituberculous treatment.

Plasma aldosterone response to upright posture and angiotensin II infusion in aldosterone-producing adenoma.

Nineteen patients with primary aldosteronism due to surgically confirmed aldosterone-producing adenoma (APA) were examined to evaluate the response of aldosterone to upright posture and angiotensin II infusion. Upright posture reportedly decreases the plasma aldosterone concentration (PAC) in APA but raises it in idiopathic hyperaldosteronism. However, our findings showed the opposite result, in that the upright posture did not change or raised PAC in 15 of 19 cases (79%). Angiotensin II was infused i.v. at doses from 0.5-2 ng/min.kg body weight in six patients in whom the upright posture raised PAC, but did not raise PAC in all cases. This result supports the assumption that APA is functionally insensitive to angiotensin II. A concomitant rise of ACTH, pretreatment with calcium channel blockade, and other modulating factors may be involved in this PAC rise. Whatever the reason, such a high frequency of patients with increased PAC in APA raises some question about the clinical value of the upright posture test. We believe, then, there is reason to check any interpretation concerning increased PAC in the case of the upright posture test in distinguishing between APA and idiopathic hyperaldosteronism.

Downward regulation of plasma LH by LHRH agonist, leuprolide acetate, resulting in inhibited renal growth and function in the castrated male rat.

We previously reported that ovine and porcine luteinizing hormone (LH) stimulated kidney growth in castrated hypophysectomized rats. Our present study focuses on the physiological role of the renotropic activity of LH isoforms. Plasma LH levels were decreased to 10% of that of castrated control rats by injections of a slow-releasing LHRH agonist, leuprolide acetate, from microcapsules. Compared to controls, which were injected with microcapsules only, the kidney weight in leuprolide-treated castrated rats decreased 12%. Renal protein and DNA contents decreased significantly. Body, liver and spleen weights were not changed by the treatment, however. This effect on the kidney was not observed in castrated hypophysectomized rats, suggesting that leuprolide affected the kidneys indirectly, rather than directly, by suppressing LH secretion. In leuprolide-treated castrated rats, urinary fractional excretion of sodium (FENa) increased, indicating suppressed renal function at the proximal tubules. We concluded that the secretion of renotropically active LH isoforms was regulated at least partially by LHRH and played a physiological role in growth and the function of the proximal tubules.

Renotropic and gonadotropic activity in homologous and heterologous hybrids of ovine luteinizing hormone and human chorionic gonadotropin subunits.

We previously demonstrated the renotropic activity of ovine and porcine LH, i.e. the stimulation of [3H]thymidine incorporation into the renal DNA of castrated hypophysectomized rats. We conducted this study to determine which condition is required to promote renotropic activity. We prepared homologous (oLH alpha.oLH beta, hCG alpha.hCG beta) and heterologous (oLH alpha.hCG beta, hCG alpha.oLH beta) hybrids from isolated oLH- and/or hCG-derived subunits. Native oLH and all hybrids showed gonadotropic activity when examined for cyclic AMP generation in cultured Leydig tumour cells (MA-10). Native oLH and the hybrids which showed renotropic activity were all found to contain at least one of the oLH subunits. Our findings point out the important, special role of oLH subunits in promoting renotropic activity. The condition required is suggested to be presence of a common structure in the two oLH subunits. Such a structure should consequently not be present in hCG. A carbohydrate moiety of oLH containing a sulphate group is proposed as a candidate, but the assumption remains to be demonstrated.

Uninephrectomy inducing quantitative and qualitative changes in LH isoforms in the male rat.

Recent findings that LH stimulates renal growth have prompted us to examine whether LH exercises renotropic effects on the growing kidney following uninephrectomy. Intact (not castrated) male rats were used for sham uninephrectomy to induce surgical stress suppressing plasma LH and testosterone levels. Compared to sham surgery, uninephrectomy caused a transient 240% increase in plasma LH with a transient increase in plasma testosterone (80%) and in prostate weight on day 2. Although pituitary LH content did not change, chromatofocusing of pituitary extract revealed changed distribution patterns among seven LH isoforms, suggesting selective synthesis or secretion of LH isoforms. Castrated rats were then used so that surgical stress did not affect LH. Uninephrectomy caused sustained increases in plasma LH levels (60-110% increase) for up to six days. Three days after uninephrectomy, pituitary LH content increased significantly (30% increase) with a change in chromatofocusing profiles of LH isoforms. Plasma and pituitary FSH levels did not change. In summary, uninephrectomy changed LH isoforms quantitatively and qualitatively. These changes imply a change in gonadotropic and renotropic activities of LH after uninephrectomy.