LH, progesterone, and TSH can stimulate aldosterone in vitro: a study on normal adrenal cortex and aldosterone producing adenoma.

Endocrine factors different from ACTH or angiotensin II can stimulate aldosterone secretion and have a role in the pathophysiology of hyperaldosteronism. Aldosterone may increase in luteotropic/progestogenic and in hypothyroid states; LH and, occasionally, TSH receptors have been detected in normal adrenal cortex and aldosterone-producing adenoma. The aim of the study was to compare adrenal contents of LH and TSH receptors between normal cortex and aldosterone-producing adenoma and to evaluate the ability of LH, its product progesterone, and TSH to stimulate aldosterone secretion in vitro from primary adrenocortical cells. Surgical aldosterone-producing adenoma fragments from 19 patients and adrenal cortex fragments from 10 kidney donors were used for Western blotting and cell cultures. LH (n=26), TSH (n=19) and progesterone (n=8) receptor proteins were investigated; LH receptor-mRNA was also tested in 8 samples. Aldosterone responses in vitro to LH, progesterone, and TSH stimulation were assayed. LH and TSH receptors were more expressed in adenoma than normal cortex (p<0.01, p<0.05, respectively); progesterone receptor was observed in 6/8 samples. Aldosterone increased after in vitro stimulation with LH (5/12 adenoma, 1/7 normal cells), progesterone (4/5 adenoma, 5/6 normal cells), and TSH (3/5 adenoma and 3/5 normal cells). LH and TSH receptors were more expressed in aldosterone producing adenoma than normal adrenal cortex. LH, progesterone, and TSH can stimulate aldosterone in vitro. Similar mechanisms could participate in vivo in the aldosterone increase in lutheotropic, progestogenic, or hypothyroid states and may exist in both normal adrenal cortex and adenoma in responsive individuals.

Outflow modulation to target liver regeneration: something old, something new.

BACKGROUND: Stimulation of hepatic hypertrophy is a useful aid to accomplish hepatic resections when the future liver remnant (FLR) is small. Although inflow occlusion, especially through portal flow, has been extensively studied, the role of outflow modulation has not yet been described. METHODS: Description of outflow modulation to tailor hypertrophy of future liver remnant in the context of bilobar metastatic disease. A patient with small FLR (segments I and IV) was managed with a two-stage procedure. The first stage consisted of a right hepatectomy and modulation of the left hepatic vein outflow through reduction of its diameter, with macroscopic congestion of segments II-III. The second stage consisted of a left lateral sectionectomy six weeks later. Postoperative courses were uneventful without any sign of liver failure. RESULTS: Following the first stage procedure computed tomography revealed distinct hypertrophy rates between sections. The non-congested area had an increase of 156% in the volume of segment IV (from 137 to 351 cm(3)) and 100% in the volume of segment I (from 20 to 40 cm(3)). The congested area, segments II-III, increased only 24% (from 205 to 253 cm(3)). CONCLUSION: Modulation of liver outflow allows maintenance of function in the segments to be resected while avoiding their hypertrophy. This process prevents liver failure and optimizes regeneration of hepatic territories to be preserved.

Erythrocyte Na/K-ATPase is increased in subjects with subclinical hypothyroidism.

OBJECTIVE: In erythrocytes of patients with overt hyperthyroidism, the number of ouabain-binding sites and the activity of the Na(+)/K(+)-ATPase have been demonstrated to be decreased, whereas the opposite is true in patients with overt hypothyroidism. No information has been reported on the status of the Na(+)/K(+)-ATPase in subclinically hypothyroid (Sub Hypo) patients. DESIGN: We investigated the number of ouabain-binding sites and Na(+)/K(+)-ATPase activity in erythrocytes of chronic Sub Hypo subjects. PATIENTS AND METHODS: We measured (3)H-ouabain-binding sites in erythrocytes from 15 patients with subclinical hypothyroidism, and compared with those found in 17 normal subjects (N), seven with overt hypothyroidism (Hypo) and 10 with overt hyperthyroidism (Hyper). The activity of the sodium pump was assessed by measuring ouabain-sensitive (86)Rb uptake in a subpopulation of the same groups. RESULTS: The number of ouabain-binding sites in Sub Hypo patients (252 +/- 17; mean +/- SEM) was significantly higher (P < 0.02) than in Hyper (135 +/- 12) and N (203 +/- 10) groups, whereas it was not significant different from Hypo (293 +/- 31). There was a positive correlation between the number of ouabain-binding sites and TSH concentrations (P < 0.002) when Sub Hypo and N groups were considered together. There was a negative correlation between the number of ouabain-binding sites and free thyroxine (FT4; P < 0.0001) and free triiodothyronine (FT3) concentrations (P < 0.001) when all subjects were considered. Ouabain-sensitive (86)Rb uptake (picomoles (86)Rb/h 10(6) cells) in Sub Hypo was significantly higher (4.2 +/- 0.5) when compared with N (2.5 +/- 0.2, P < 0.01) and Hyper (2.5 +/- 0.5, P < 0.02). CONCLUSIONS: Erythrocytes of subclinically hypothyroid patients show a significant increase in the number of ouabain-binding sites and in ouabain-sensitive (86)Rb uptake. The state of erythrocyte Na(+)/K(+)-ATPase may therefore represent a biochemical marker of subclinical hypothyroidism.

Effect of canrenone on the digitalis site of Na+/K(+)-ATPase in human placental membranes and in erythrocytes.

It has been reported that canrenone, which is used in hypertensive therapy as an antialdosteronic drug, may also act as a blocker of ouabain effects. Several studies suggest that human plasma contains an endogenous ouabain-like factor similar to ouabain, which may be increased in hypertension, in pregnancy, and in the neonatal state. This study evaluated (1) the effect of canrenone on Na+/K(+)-ATPase in relation to ouabain in human placental membranes and erythrocytes by 3H-ouabain binding assay; (2) the capacity of canrenone (10 microM) to reverse the inhibition of Na+/K(+)-ATPase by ouabain and by ouabain-like factor (from umbilical cord plasma) in human erythrocytes employing a 86Rb uptake assay. Increasing concentrations of canrenone (0-350 microM) partially competed with 3H-ouabain binding in placental membrane (40%) and erythrocytes (60%). Scatchard plot from radioreceptor assay in placental membrane showed that ouabain and canrenone compete for the same binding site. In erythrocytes, canrenone completely reversed the inhibition caused by ouabain (5 x 10(-9) M) and ouabain-like factor (2 x 10(-9) M ouabain equivalents). A reduction of inhibition of about 50% was observed with ouabain and ouabain-like factor respectively at a concentration of 5 x 10(-8) M and 2 x 10(-8) M (ouabain equivalents). Our results thus provide evidence that canrenone, at therapeutical concentrations, is a partial competitive agonist of ouabain and of ouabain-like factor in human placental membranes and erythrocytes.

Rabbit tissue distribution of 3H-ouabain by digital radioautography.

3H-ouabain is useful to evaluate the tissue localization of Na,K-ATPase. In this work we determined the distribution of 3H-ouabain in rabbit tissue by digital radioautography. Using this method, we were able to obtain a comparison of various organs in a relatively short time (6.5 days), while with traditional radioautography, only the kidney was detectable after seven months of film exposure. The kidney has the highest intensity (concentration of 3H-ouabain), followed by the heart, liver, muscle, lung, spleen and finally brain, which was almost undetectable. In kidney the activity was higher in the cortex, while the other tissues displayed a more uniform intensity, suggesting that Na,K-ATPase was evenly distributed.

Posttraumatic splenic cysts and partial splenectomy: report of a case.

Nonparasitic splenic cysts are uncommon, with only around 800 cases described in the literature. Posttraumatic splenic pseudocysts constitute most such cases and require surgical treatment when symptomatic or voluminous. Recent studies have provided a better understanding of splenic tissue function and the consequent risks of complete resection of the spleen. Hence surgeons should make every possible effort to preserve splenic tissue. Several spleen-conserving surgical treatments have been proposed, especially for treatment of splenic posttraumatic pseudocysts. The authors report the case of a 13-year-old girl who had a posttraumatic splenic cyst with progressive growth. The diameter of the cyst at surgery was 15 cm, and partial splenectomy was performed. The most common spleen-conserving surgical techniques are briefly reviewed.

Selective inhibition of human erythrocyte Na+/K+ ATPase by cardiac glycosides and by a mammalian digitalis like factor.

Na+/K+ATPase is a transport membrane protein which contains the functional receptor for digitalis compounds. In this work we compare the inhibition curves of Na+/K+ATPase measured by the inhibition of 86Rb uptake in human red blood cells by cardiac glycosides and by an endogenous digitalis like factor (EDLF) extracted from human newborn cord blood. The curves of Na+/K+TPase inhibition show a monophasic shape for ouabain, strophantidin, digitoxin, proscillaridin and EDLF whereas a biphasic shape for ouabagenin, digoxin, digoxigenin and digitoxigenin. All the drugs are potent inhibitors of erythrocyte Na+/K+ATPase with an IC50 ranging from 1.8 x 10(-9) M to 1.4 x 10(-11) M for the higher affinity binding site and from 1.8 x 10(-6) M to 5.5 x 10(-9) M for the lower affinity site. Digitoxigenin is the most active showing the higher active site at 1.4 x 10(-11) M. Ouabain and digoxin have higher affinity compared with their corresponding genins, while digitoxigenin shows a binding site with higher affinity than the respective cardiac glycosides. The increased affinity of the drugs to Na+/K+ATPase may be related to a lipophilic region in correspondence of the carbons 10, 9, 11, 12, 13 of the steroid nucleus, situated in the opposite side with respect of the C-OH-14. The comparison of the inhibition curves and the HPLC profile of newborn EDLF and of the investigated cardenolides suggest that EDLF may be a compound identical or very similar to ouabain.

Risk of major liver resection in patients with underlying chronic liver disease: a reappraisal.

OBJECTIVE: To explore the relation of patient age, status of liver parenchyma, presence of markers of active hepatitis, and blood loss to subsequent death and complications in patients undergoing a similar major hepatectomy for the same disease using a standardized technique. SUMMARY BACKGROUND DATA: Major liver resection carries a high risk of postoperative liver failure in patients with chronic liver disease. However, this underlying liver disease may comprise a wide range of pathologic changes that have, in the past, not been well defined. METHODS: The nontumorous liver of 55 patients undergoing a right hepatectomy for hepatocellular carcinoma was classified according to a semiquantitative grading of fibrosis. The authors analyzed the influence of this pathologic feature and of other preoperative variables on the risk of postoperative death and complications. RESULTS: Serum bilirubin and prothrombin time increased on postoperative day 1, and their speed of recovery was influenced by the severity of fibrosis. Incidence of death from liver failure was 32% in patients with grade 4 fibrosis (cirrhosis) and 0% in patients with grade 0 to 3 fibrosis. The preoperative serum aspartate transaminase (ASAT) level ranged from 68 to 207 IU/l in patients with cirrhosis who died, compared with 20 to 62 in patients with cirrhosis who survived. CONCLUSION: A major liver resection such as a right hepatectomy may be safely performed in patients with underlying liver disease, provided no additional risk factors are present. Patients with a preoperative increase in ASAT should undergo a liver biopsy to rule out the presence of grade 4 fibrosis, which should contraindicate this resection.

Detection of digitalis compounds using a surface plasmon resonance-based biosensor.

An automated surface plasmon resonance-based biosensor system has been used to detect endogenous and exogenous digitalis-like factors (EDLF) in the pmolar range in real time. EDLF was purified from umbilical cord blood. EDLF has been suggested to play a role in hypertension and in perinatal adaptation. Highly specific polyclonal anti-ouabain antibodies showed a high affinity binding capacity for ouabain, ouabagenin and strophantidin with an IC50 value of 5 x 10(-10) M, 7.0 x 10(-10) M and 2 x 10(-8) M, respectively. EDLF cross-reacted with antibodies and its concentration in plasma at IC50 was around 50 pmol ouabain equivalent. This study shows the potential usefulness of the biosensor technology for biomolecular interaction analysis. The features of this technology (fully automated, measured in real time, sharpened response) offer several advantages compared with a traditional immunoassay like radioimmunoassay (RIA) in the detection of digitalis compounds in human fluids.

Inhibition of rat Na+/K+-ATPase isoforms by endogenous digitalis extracts from neonatal human plasma.

An unique endogenous digitalis-like factor (EDLF) has been previously purified from human newborn cord plasma and its differential effects tested on the three well defined functional isoforms (alpha1, alpha2 and alpha3) of the alpha subunits of Na+/K+-ATPase in rat. EDLF specifically inhibits the enzymatic activity. It differs from ouabain by three criteria: a preincubation with the membranes is required for full activity, no effect on the rat cerebral alpha3 isoform and a steep dose-response curve with the same apparent potency for rat alpha2 and alpha1 isoforms of high (10(-7) M) and low affinity (3 x 10(-5) M) for ouabain. These results indicate that the Na+/K+-ATPase inhibitor involved in the regulation of sodium and body fluid volume and present in neonate and adult human plasmas is distinct from ouabain.

Ouabain-like factor quantification in mammalian tissues and plasma: comparison of two independent assays.

The resolution of controversies that concern the detectability of an endogenous ouabain-like factor (OLF) in mammalian tissues and plasma was approached by the application of a standardized method for its extraction and quantification. Two independent assays were used to quantify the OLF: (1) a radioimmunoassay, which used a polyclonal anti-ouabain antiserum, and (2) a radioenzymatic assay based on the inhibition of dog kidney Na+,K+-ATPase. Plasma and tissues were obtained from the Milan hypertensive strain (MHS) and the Milan normotensive strain (MNS) of rats and from healthy human volunteers. Results indicate that (1) a single high-performance liquid chromatography (HPLC) fraction identical to that of ouabain was identified by both assay methods in the rat hypothalamus and hypophysis and in both rat and human plasma; (2) dilution curves of OLF and standard ouabain were parallel and with a similar Kd, both in radioimmunoassay (3 nmol/L) and ATPase assay (14 nmol/L); (3) after HPLC, OLF was similarly quantified by the two methods in the hypothalamus, hypophysis, adrenals, and plasma of rats and in human plasma; (4) OLF was present in larger amounts in the hypothalamus, hypophysis, and plasma of MHS rats than that of MNS rats; (5) the HPLC fraction of human plasma was quantified similarly by both assays (range, 60 to 150 pmol/L); (6) recovery of standard ouabain in pre-HPLC plasma extracts was approximately 90%; and (7) pre-HPLC OLF concentrations in human plasma ranged between 0.05 and 0.75 nmol/L. Rat cerebral tissues and both rat and human plasma contained measurable amounts of OLF, which were quantified similarly by radioimmunoassay and ATPase assay, both before and after HPLC fractionation. The increased MHS tissue and plasma levels of OLF are in keeping with the pathogenetic role of this factor in MHS hypertension.

Further evidence for an endogenous digitalis-like compound in newborn and adult plasma detected by anti-ouabain antiserum.

It is widely but not unanimously accepted that one or more endogenous digitalis-like factors (EDLF) circulate in human plasma. In this paper we provide confirmatory evidence that newborn plasma contains a factor with immunological and biological properties similar to ouabain and demonstrate that this factor may be present also in the adult. In fact, we obtained in newborn and adult plasma extracts, identical HPLC elution profiles of ouabain-like immunoreactivity and 86Rb erythrocyte uptake inhibitory activity with a major peak corresponding to the retention time of ouabain. The fact that immunoreactivity and biological digitalis-like activity in the peak are due to an identical substance is strongly supported by the correlation between RIA and 86Rb uptake inhibitory values observed in the purified fractions. Finally, the strong correlation between immunoreactivity observed in plasma samples after simple SepPak C18 extraction and after additional HPLC suggests that less purified samples may be assayed for screening purposes. However, for a more quantitative assessment, this simple extraction method needs a subsequent HPLC purification for eliminating an overestimation of values due to cross-reacting impurities.

Different methods for measuring endogenous digitalis-like factor(s).

It is well recognized that one of the difficulties in the search for endogenous ligand(s) with digitalis-like properties (endogenous digitalis-like factor(s), EDLF) in mammals has been the lack of a unique, specific method for the accurate measurement of EDLF. Using C18 solid-phase extracts of plasma from normal adults and various patient groups, and purified extracts from umbilical cord plasma by affinity resin chromatography and HPLC, different methods to measure EDLF were evaluated. These were: (a) a human placenta radioreceptor assay (RRA) developed on the premise that competition for cardiac glycoside receptors was an absolute requirement for EDLF; (b) the inhibition of 86Rb uptake in human erythrocytes to estimate the potassium transport by the sodium pump; (c) an enzyme immunoassay specific for ouabain recently introduced in the market (DuPont Ouabain EIA Reagent Pack). The human placenta RRA was found to have the same ease of application as immunoassay, but could have major advantages in detecting active molecules, being "biologically more meaningful". Ouabain immunoreactivity correlated with EDLF values obtained by RRA, but in some instances the two assays were completely unrelated. Moreover, the high specificity of the DuPont antibody for ouabain (< 3% cross reactivity with digoxin) could be disadvantageous to detect EDLF not strictly resembling ouabain. The 86Rb uptake inhibition method correlated with RRA for EDLF purified by HPLC. It tested the complete enzymatic cycle and could therefore better reflect the in-vivo inhibitory activity of EDLF. However, it appeared not suitable for the routine EDLF evaluation in clinical studies since it was susceptible to sample osmolarity and required daily isolation of human erythrocytes possibly from the same donor. Results of the present study demonstrate that every assay has its limitations, and would suggest the use of multiple assays for EDLF detection.

The stimulatory effect on human erythrocyte rubidium-86 uptake by anti-cardiac-glycoside antibodies.

Considerable, but as yet still controversial evidence indicates the presence, in mammalian tissues of endogenous digitalis-like factors (EDLFs) which inhibit cell membrane Na+, K(+)-adenosine triphosphatase (Na+, K(+)-ATPase) and which may cross-react with anti-digitalis antibodies. The aim of this study was to evaluate the effect of antibodies against cardiac glycosides on Na+, K(+)-ATPase in human erythrocytes. For this purpose, we measured the effect of antibodies against two different cardiac glycosides (anti-ouabain rabbit antiserum and anti-digoxin Fab fragments) on the activity of the Na+, K(+)-ATPase, as measured by erythrocyte rubidium-86 (86Rb) uptake, in subjects who had never come into contact with exogenous cardiac glycosides, and compared these results with the effect of two control rabbit sera: a normal serum and an antiserum to a non-related antigen. Anti-ouabain rabbit antiserum and anti-digoxin Fab fragments induced a significantly greater percentage change in 86Rb uptake in the erythrocytes than the two control sera (ANOVA followed by multiple comparison by the Games-Howell test). The average percentage change was +11.8 +/- 16.3% (n = 19) (mean +/- SD) for anti-ouabain antiserum +10.8 +/- 15.6% (n = 23) for anti-digoxin Fab fragments, -1.68 +/- 11.2% (n = 11) for anti-rhGM-CSF antiserum, and -5.8 +/- 11.7 (n = 10) for normal control serum. In a subgroup of ten subjects in whom the 3 antisera were tested simultaneously, the stimulation of erythrocyte 86Rb uptake induced by the two antidigitalis antibodies correlated significantly (r = 0.906, p = 0.001, n = 10).(ABSTRACT TRUNCATED AT 250 WORDS)

Endogenous digitalis-like activity in the newborn.

Elevated levels of endogenous digoxin-like immunoreactivity have been reported in the body fluids of premature and full-term infants as well as in term pregnancy, in the amniotic fluid, and in human milk. Several lines of evidence suggest that these factors could also have biological properties in common with digitalis: i.e., they could represent truly endogenous digitalis-like factor(s). In recent years we succeeded in partially purifying this factor from umbilical cord blood, which represents an easily available source of this factor. The inhibitory activity of this factor on 86Rb uptake could be neutralized by antidigoxin antibodies (Fab fragments) and provided, for the first time, direct evidence of an association between digoxin-like immunoreactivity and biological digitalis-like activity. In addition, these antibodies could be used for immunoaffine chromatography as a purification step before separation by high-performance liquid chromatography. Preliminary experiments suggest that this endogenous compound has both a tissue and an isoenzyme selectivity and is not a well-known steroid (testosterone, progesterone, 17-OH progesterone, cortisol, dehydroepiandrosterone sulfate, and estradiol).

Antidigoxin antibodies neutralize the effect of newborn endogenous digitalis-like factor on erythrocyte 86Rb uptake.

Increasing evidence indicates the existence of endogenous digitalis like factor(s) (EDLF). We recently reported on the partial purification of an EDLF from newborn (cord) blood which possesses both digoxin-like immunoreactivity and the ability to inhibit the cell membrane sodium pump measured as the inhibitory activity on erythrocyte 86Rb uptake. We here report that high affinity digoxin-binding antibodies (Fab fragments; Digibind, Burroughs Wellcome Co.) are capable of neutralizing the inhibitory activity on erythrocyte Rb uptake not only of digoxin but also of ouabain and of partially purified newborn EDLF. These results provide, to our knowledge for the first time, direct evidence that antidigitalis antibodies may cross-react with one or more circulating substances which share antigenic determinants with digoxin and ouabain and possess endogenous digitalis-like properties, strongly suggesting that these antibodies may be useful tools both for the assay of EDLF and for the study of its biological effects.