Diminished thyroxine-binding globulin in pubertal diabetic children.

OBJECTIVE: To determine the effect of diabetes on thyroid hormone and thyroxine-binding globulin (TBG) concentrations during puberty. RESEARCH DESIGN AND METHODS: Total thyroxine (TT4), free thyroxine (FT4), and TBG levels of 171 thyroid microsomal antibody-negative subjects with normal thyroid-stimulating hormone (TSH) levels were measured and compared with those of nondiabetic adolescents. A random subset of 68 diabetic patients (40 boys and 28 girls) and 51 control subjects (24 boys and 27 girls) were analyzed for puberty-related changes. RESULTS: Most TT4 levels of diabetic subjects (80% of girls and 63% of boys) were below the 50th percentile for the normal range. TT4 increased with age in girls (r = 0.25, P < 0.04) but not in boys. FT4 was within normal limits in both sexes. TBG measurements were below the 50th percentile and 20% were below the 95% CI for both sexes; TT4 correlated with TBG in boys (r = 0.54, P < 0.001) and in girls (r = 0.58, P < 0.001). Duration of diabetes had no effect, whereas TT4 and FT4 levels were higher in girls with the lowest HbA1 levels (r = -0.29, P < 0.01 and r = -0.45, P < 0.01). Levels of TBG were reduced for all male pubertal stages (P < 0.01) and for early and late female pubertal stages (P < 0.01). There was no direct relationship between glucose control or the duration of diabetes and levels of TBG. CONCLUSIONS: Because TT4 levels are low and correlate with the low levels of TBG, it is important to measure free thyroid hormone and TSH levels in diabetic adolescents to establish euthyroidism.

An endothelin-1 mediated autocrine growth loop involved in human renal tubular regeneration.

Renal tubules have the capacity to regenerate following injury. We have investigated the possibility that tubular-derived endothelins, acting as autocrine growth factors, may be involved in this response in human kidney. ET-1 immunoreactivity was demonstrated by immunohistochemical staining in proximal tubules, distal cortical tubules and medullary collecting ducts of human kidney. In cultured human renal proximal tubular cells, RNAase protection assays demonstrated the expression of ET-1 and ET-2 mRNA's, and radioimmunoassay, following separation of conditioned medium by reverse phase HPLC, showed immunoreactive material which co-eluted with ET-1 and ET-2. Competition binding studies revealed the presence of at least two types of endothelin receptor: one with high and one with low affinity for ET-3 relative to ET-1. Analysis of cellular RNA by RT-PCR demonstrated expression of mRNA's for both ETA and ETB receptor subtypes. Combined blockade of ETA and ETB receptors (by PD-145065) but not that of ETA receptors alone (by BQ-123) blocked the mitogenic effect of exogenous or endogenous ET-1 and also profoundly suppressed endogenous ET-1 synthesis. By contrast, incubation with the ETB receptor agonist, BQ-3020, stimulated endogenous ET-1 synthesis. Exposure of the cells to hypoxia (1% O2 for 16 to 24 hr) resulted in specific up-regulation of ET-1 but not ET-2 gene expression. These findings reveal the existence of a hypoxia-inducible, autocrine growth system in human proximal tubular cells, which is mediated by ET-1 through the ETB receptor, and which could function in vivo as an autoregenerative system for restoring tubular integrity after injury. The widespread distribution of ET-1 peptide in different tubular segment suggests that ET-1 mediated tubular regeneration may also occur in other nephron segments.

Human high density lipoproteins stimulate endothelin-1 release by cultured human renal proximal tubular cells.

The vasoconstrictive and mitogenic actions of endothelins have been implicated in the pathogenesis of progressive renal disease. In the present study, we have assessed whether plasma high density lipoproteins (HDL), the major filtered urinary lipoprotein in nephrotic states, can influence endothelin-1 (ET-1) production by cultured human renal proximal tubular cells. Human HDL was found to stimulate ET-1 secretion up to fourfold in a dose- and time-dependent manner; the effect was greater in subconfluent cultures than in confluent ones. There was little difference between the stimulatory effect of HDL2 and the major HDL subclass, HDL3. Preincubation of the cells with albumin did not abolish the HDL effect, while partially- or fully-delipidated HDL3 largely reproduced the effect of whole HDL3. These findings suggest that stimulation of ET-1 secretion was not simply due to protein or lipid repletion of the cells. Rather, the effect was mediated by HDL apolipoproteins, although binding to the HDL receptors involved in cellular cholesterol homeostasis was not required as tetranitromethane-modified HDL3 was an equally effective agonist of ET-1 release. Apolipoprotein (apo) A-I was indirectly implicated in the process since modified HDL3 in which apoA-II largely replaced apoA-I was less potent than HDL3. A one hour exposure of the cells to HDL3 was sufficient to activate ET-1 production for the following 12 hours, although maximum activation required six hours.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of cyclosporin A on endothelin synthesis by cultured human renal cortical epithelial cells.

We report here for the first time that human renal proximal tubular cells secrete endothelin, clear evidence of de-novo endothelin synthesis by these cells and the effect of cyclosporin A (CsA) on endothelin synthesis both in short-term (24 h) and medium-term (5-day) culture. Human renal cortical epithelial cells were cultured and shown to possess proximal tubular characteristics. These cells produced endothelin in culture in a time-dependent manner, as measured by radioimmunoassay (291.6 +/- 51.4 pg/well/24 h). Furthermore, endothelin production by these cells was significantly decreased by up to 80% by cycloheximide (1051.8 +/- 54.9 pg/mg cell protein/24 h versus 253.2 +/- 12.6 pg/mg cell protein/24 h), showing that these cells actively synthesize endothelin. In short-term culture (24 h), CsA significantly inhibited endothelin synthesis at a medium concentration of 10,000 micrograms/l. No change in endothelin synthesis was seen at lower CsA concentrations. In contrast, over a 5-day period, a non-significant increase in endothelin synthesis was observed at CsA concentrations of 2000 micrograms/l (152.5 +/- 20.4%); however, cell growth was significantly decreased at this concentration (71.33 +/- 6.39%). Using a newly developed two-site immunoradiometric assay specific for endothelin-1 (ET-1), we demonstrate that ET-1 is the major endothelin isoform produced by human renal proximal tubular cells.

A longitudinal study of total and free thyroid hormones and thyroxine binding globulin during normal puberty.

Thyroid hormones are essential for normal pubertal growth, yet the changes in total and, especially, free thyroid hormones and thyroxine-binding globulin during puberty have not been adequately defined. Serum from 39 normal children (20 girls, 19 boys) between the ages of 10 and 15 years were assayed for total T4, free T4, free T3 and thyroxine-binding globulin at 6-monthly intervals; the free hormone assays were valid, non-analogue methodologies. In the girls, free T4 levels fell from 15.7 +/- 0.6 pmol/l at 10 years to 13.0 +/- 0.6 (p less than 0.001) at 12.5 years before rising to 15.9 +/- 0.7 at 15 years; this nadir occurred at puberty stages 3-4. Changes in total T4 followed a similar pattern with a slight delay in the nadir (13 years, puberty stage 4). In the boys, free T4 fell from 16.3 +/- 0.6 pmol/l at 10 years to 14.3 +/- 0.3 at 13.5 years, then rising to 15.4 +/- 0.5 at 15 years; the nadir again occurred at puberty stages 3-4. The corresponding nadir in total T4 which occurred at puberty stages 4-5 was not apparent by age analysis. Thyroxine-binding globulin concentrations remained unchanged in the girls, but fell slightly in the boys during later puberty. Free T3 concentrations in the girls showed a progressive fall after 12.5 years which was significant by the age of 14 when most had been in puberty stage 5 for more than 1 year. The boys showed no change of free T3 concentration throughout the study.(ABSTRACT TRUNCATED AT 250 WORDS)

Perinatal transmission of hepatitis B virus in Kenya: its relation to the presence of serum HBV-DNA and anti-HBe in the mother.

In Kenya hepatitis B virus (HBV) infection and its sequelae are common. We followed up 49 hepatitis B surface antigen (HBsAg)- positive mothers and their newborn infants for 9 months to determine the importance of perinatal transmission in the African and to relate this to the HBe and HBV-DNA status of the mother. Our study shows that perinatal transmission is relatively unimportant in Kenya and that this may be a consequence of the low levels of circulating HBV-DNA in the maternal plasma. These results imply that vaccination without hyperimmune globulin may be adequate to control HBV infection in Kenya.

Identification of a dominant immunogenic epitope of the nucleocapsid (HBc) of the hepatitis B virus.

Four monoclonal antibodies to hepatitis B core antigen are described. The antibodies bind to the same or a very closely related epitope. Antibodies to this dominant epitope are present in the sera of patients with either acute or chronic hepatitis B virus (HBV) infection. A high percentage of inhibition of the binding of these antibodies to the core antigen by these four monoclonal antibodies suggests that the core antigen has a restricted antigenicity in man. Radiolabeled or peroxidase labeled forms of these monoclonal antibodies can be used to assay IgM and total anticore in serum.

Chronic hepatitis B virus infection. Viral replication and patterns of inflammatory activity: serological, clinical and histological correlations.

We have studied serum and tissue markers of viral replication in 39 patients with chronic hepatitis B virus (HBV) infection and correlated these with periportal and lobular activity in liver biopsies. HBV DNA positivity correlated with the presence of hepatitis B e antigen (HBeAg, P less than 0.001) and aspartate transaminase (AST) levels (P less than 0.005). The lobular but not the periportal inflammatory activity was significantly associated with the presence of HBV DNA (P less than 0.02) and HBeAg (P less than 0.001) and with higher AST levels. The periportal activity correlated with the periportal and lobular display of beta 2-microglobulin on hepatocytes (P less than 0.001 and P less than 0.002, respectively). In patients with chronic HBV infection therefore, the lobular rather than the periportal component of activity was related to viral replication. The association of display of beta 2-microglobulin on hepatocytes with the inflammatory process, in patients with active viral replication, is consistent with the hypothesis that increased display of HLA type I enhances recognition of hepatocytes bearing viral proteins and allows lysis of immune cells.

Studies of HBV replication during acute hepatitis followed by recovery and acute hepatitis progressing to chronic disease.

The serologic and viral profiles of 24 patients who presented with acute hepatitis B virus (HBV) infection were studied. Although in rare cases, HBV-DNA was detectable before hepatitis B surface antigen (HBsAg) and e antigen (HBeAg), in the majority the viral proteins appeared first. In acute hepatitis followed by recovery, as IgM anti-HBc (hepatitis B core antigen) titres rose, the level of HBV replication fell and serum transaminases became elevated. In patients progressing to chronic HBV infection, IgM anti-HBc titres rose early, viral replication was initially low but continued to rise as the serum transaminase levels became elevated. 7S IgM anti-HBc, although present in the phase of established chronic HBV infection, was not found in the early phase of the chronic infection. Thus this antibody appears to be a consequence of, rather than a causative factor in, chronic HBV infection.

Aetiology of acute sporadic hepatitis in adults in Kenya.

Markers for acute hepatitis A virus (HAV), hepatitis B virus (HBV), and hepatitis non-A, non-B (HNANB) infections were examined in the sera of 94 patients presenting with acute hepatitis in Kenya. Hepatitis B virus was responsible for 70% of cases, HNANB for 18%, and HAV for only 12%. The use of an IgM anti-HBc assay increased the rate of diagnosis of acute HBV infection, thereby reducing the proportion of cases designated as NANB.

Aldosterone 21-sulphate in man.

Using a rigorous extraction procedure and radioimmunoassay we have established that aldosterone 21-sulphate concentrations in adrenal vein specimens are higher than peripheral concentrations. Incubations of homogenates of adrenal glands and adenomata in vitro also produced aldosterone 21-sulphate. Peripheral plasma concentrations in normal and hypertensive subjects are about one-tenth those of aldosterone. Mineralocorticoid activity, as assessed in adrenalectomized rats, is less than 1% of that of aldosterone. [3H]-Aldosterone 21-sulphate administered to normal and hypertensive subjects is excreted in an irregular manner, and in four out of six subjects the major metabolite was an unidentified less polar compound. We have characterized this compound chromatographically and discussed the mechanism which may be involved in its production.

Renin and aldosterone at high altitude in man.

Measurements have been made of hormonal changes relevant to salt and water balance during prolonged exposure to hypoxia to improve our understanding of the syndrome of acute mountain sickness. We have attempted to delineate the detailed inter-relationships between the renin-aldosterone and the vasopressin systems by a metabolically controlled study, involving an orthostatic stress (45 degrees head-up tilt) and an injection of a standard dose of ACTH to test adrenal responsiveness. Three Caucasian medical students underwent a 7-day equilibration at 150 m (Lima, Peru), followed by a 6-day sojourn at 4350 m (Cerro de Pasco, Peru) and a final 7 days at 150 m. Measurements were made of sodium and potassium balance, body weight and the 24-h renal excretion of vasopressin, cortisol and aldosterone 18-glucuronide. These variables showed little change, except for that of aldosterone 18-glucuronide, which fell sharply at altitude and rebounded even more sharply on return to sea level. At altitude, basal plasma levels of renin activity and aldosterone fell, and the response to orthostasis was attenuated, but the fall of plasma renin activity, as compared to plasma aldosterone, was delayed; on return to sea level this dissociation was exacerbated with the return of normal renin responsiveness lagging behind that of aldosterone. We suggest that unknown factors which dissociate the orthodox renin-aldosterone relationship, other than the activity of the angiotensin I-converting enzyme, are operative on exposure to hypoxia.

Factors relating to aldosterone secretion rate, the excretions of aldosterone 18-glucuronide, and the plasma aldosterone concentration in cirrhosis.

In a group of eight patients with cirrhosis the rate of renal excretion of the 18-glucuronide metabolite of aldosterone (U Aldo V) was found to be closely related to the aldosterone secretion rate (ASR). U Aldo V was therefore used as an index of ASR in a further group of fifty patients in order to evaluate the possible importance of factors known to regulate aldosterone secretion. U Aldo V showed statistically significant relationships to both plasma renin activity (PRA) and the plasma sodium concentration (P Na), but not to the plasma potassium concentration (PK) or the renal excretion of cortisol (U Cort V), the latter sued as an index of adrencorticotrophic hormone activity. The plasma aldosterone concentration (P Aldo) was determined in fifty-eight patients and also found to show statistically significant relationships to PRA and P Na. P Aldo showed a weak, though statistically significant, relationship to PK, but not to U Cort V. These findings are in keeping with a role for the renin-angiotensin system in the control of aldosterone secretion in cirrhosis although evidence from other studies suggest other factors to be involved also. Whether P Na was another determinant of ASR, or whether aldosterone was a determinant of P Na through regulating sodium reabsorption by the proximal tubule of the nephron, is uncertain.

The diagnosis of primary hyperaldosteronism.

An aldosterone-suppression test based on a simple method of extracellular-fluid volume expansion over three days reliably discriminated between patients with aldosterone-producing adenomas, idiopathic adrenal hyperplasia, and essential benign hypertension. In patients with primary hyperaldosteronism adrenal-vein plasma aldosterone/cortisol concentration ratios successfully lateralised all 21 adenomas. In patients with an adenoma the contralateral adrenal gland was always suppressed, as indicated by a ratio which was less than that seen in the lower inferior vena cava, whereas in patients with hyperplasia the adrenal-vein aldosterone/cortisol concentration ratio from each adrenal was always greater than that seen in the lower inferior vena cava. Thus adrenal-vein sampling not only lateralises solitary adenomas but also discriminates between patients with an adenoma or hyperplasia. However, in view of the diagnostic reliability of the suppression test, it is suggested that adrenal-vein sampling is unnecessary in hyperaldosteronism due to adrenal hyperplasia.

A radioimmunoassay for the measurement of tetrahydroaldosterone 3-glucosiduronic acid in human plasma.

A simple, reliable and specific radioimmunoassay procedure for the measurement of the principal aldosterone metabolite, tetrahydroaldosterone 3-glucosiduronic acid, in human plasma is described. The glucosiduronate is extracted from plasma (generally 200 microliter) by the use of Amberlite XAD-2 resin, eluted with acidic, aqueous ethanol, separated from unconjugated steroids by partition and estimated using a specific antibody. A disequilibrium system is used, but with a conventional charcoal separation; the advantages of such a system are discussed. Data obtained by incorporating chromatographic separations into the assay procedure is presented as evidence for the specificity of the method. Data concerning the long-term storage of plasma samples is given, together with plasma concentrations of the glucosiduronate in both normal people and in people with abnormalities of aldosterone secretion.

Renal sodium retention in cirrhosis: relation to aldosterone and nephron site.

1. In a group of patients with cirrhosis who showed a wide range of values for the rate of renal sodium excretion, the latter was found to be inversely related to both the plasma concentration and rate of renal excretion of aldosterone. However, for a given sodium excretion the values for aldosterone were significantly lower in the patients than for a group of healthy control subjects. These findings suggest either an increased renal tubular sensitivity to aldosterone or the participation of other factors in the pathogenesis of the sodium retention. 2. Based on measurements of the rate of urine flow and the clearances of free water and inulin during a maximal water diuresis, the fractional reabsorption of sodium by the 'proximal', 'diluting segment' and 'distal' segments of the nephron was estimated. For patients retaining sodium the enhanced reabsorption occurred at both proximal and distal sites, the latter being quantitatively more important. There was no significantly enhanced sodium reabsorption in the diluting segment.

Development of radioimmunoassays for the measurement of aldosterone in unprocessed plasma and simple plasma extracts.

Inexpensive and rapid radioimmunoassay techniques for the measurement of aldosterone in unprocessed plasma and simple plasma extracts are described. The use of low pH (pH 5.0) and merthiolate to minimise plasma protein binding and the use of aldosterone-free plasma in the standards allows the measurement of aldosterone in 50 microliter of unprocessed plasma, which has been found useful in the diagnostic screening and classification of hyperaldosteronism. Despite quantitative recovery of added (+)-aldosterone and high specificity, the aldosterone content of unprocessed plasma is overestimated, probably by the presence of a water-soluble compound which closely resembles aldosterone. The use of a simple preliminary dichloromethane extraction procedure gives an excellent correlation with values obtained after chromatography. Values are given for chosen normal people and people with benign essential hypertension, using both assay procedures in three different physiological contexts.

Effect of beta adrenergic blocking drugs on the renin-aldosterone system, sodium excretion, and renal hemodynamics in cirrhosis with ascites.

As a result of effective beta adrenergic blockade with either propranolol or practolol, plasma renin activity was suppressed in all of 11 patients with cirrhosis and ascites. In contrast, the effect on the rate of renal excretion of aldosterone was variable, suggesting that factors other than the renin-angiotension system are responsible for the control of aldosterone secretion in cirrhosis. The changes in aldosterone could not be explained on the basis of changes in the plasma concentrations of potassium or sodium. The renal sodium excretion was inversely related to the values for aldosterone both before and after beta adrenergic blockade, indicating a major role for aldosterone in regulating sodium excretion. A number of patients had an abnormal intrarenal distribution of plasma flow with a relative hypoperfusion of the renin-secreting outer cortical nephrons. Plasma renin activity was inversely related to outer cortical plasma flow, suggesting that the reduced outer cortical flow may be a stimulus to increased renin secretion. Because the abnormal intrarenal hemodynamic pattern was not corrected by suppression of plasma renin activity, and presumably angiotension II concentrations, it is unlikely that it is attributable to the known renal vasonconstrictor effects of angiotensin II.