Cervical cytology in adolescence

To further define the chemical structure of human endogenous digoxinlike immunoreactive factors (DLIF) we used human pleural effusions as a source of the substance. Digoxinlike immunoreactive factor activity was detected by radioimmunoassay in the pleural fluid of each of four patients; average concentration was 0.35 ng/mL. The chemical profile of DLIF was determined by initial extraction and concentration of DLIF by ion exchange chromatography followed by reverse phase-high-pressure liquid chromatography (RP-HPLC) separation and purification. Using high-pressure liquid chromatography cochromatography of DLIF, together with several radioactively marked glycosides, we observed a single peak of DLIF activity that was chromatographically identical to digoxin. The present study further supports the recent finding that DLIF is related structurally to the cardiac glycosides, and for the first time it has been proven that DLIF is present in pleural fluids.     

Deglycosylated products of endogenous digoxin-like immunoreactive factor in mammalian tissue

Digoxin-like immunoreactive factor (DLIF) from adrenal cortex is an endogenous molecule with structural features remarkably similar to those of digoxin, a plant-derived cardiac glycoside (Shaikh, I. M., Lau, B. W. C., Siegfried, B. A., and Valdes, R., Jr. (1991) J. Biol. Chem. 266, 13672-13678). Two characteristic structural and functional features of digoxin are a lactone ring and three digitoxose sugars attached to a steroid nucleus. Digoxin is known to undergo deglycosylation during metabolism in humans. We now demonstrate the existence of several naturally occurring deglycosylated components of DLIF in human serum. The components are identified as DLIF-genin, DLIF-mono, and DLIF-bis, corresponding to the aglycone, and the aglycone with one and two sugars, respectively. Similar components are produced by acid-induced deglycosylation of DLIF isolated from bovine adrenal cortex. The elution pattern and sequence of DLIF-deglycosylation was identical to that of digoxin suggesting identical sugar stoichiometry. However, analysis of these newly discovered congeners by reverse-phase chromatography, spectrophotometry, antibody reactivity, and kinetics of deglycosylation, demonstrates that subtle structural and physical differences do exist when compared to digoxin. DLIF was chromatographically distinct from digoxin, and interestingly, the mobility of the DLIF-genin was shifted toward increased polarity relative to digoxigenin. DLIF and DLIF-bis, -mono, and -genin congeners have absorbance maxima at 216 nm, whereas digoxin and its congeners absorb at 220 nm. Reaction with specific antibodies directed at the lactone portion of these molecules shows DLIF and its deglycosylated congeners to be 10(3)-fold less reactive than digoxin. Kinetics of sugar removal suggests that DLIF is 8-fold more susceptible to deglycosylation than is digoxin. Two less polar DLIF components produced from the DLIF-genin have lambdamax at 196 nm and are 4-fold less immunoreactive than DLIF. Our data suggest that subtle structural differences exist between DLIF and digoxin at or near the lactone ring as well as in the nature of the sugars. The presence of deglycosylated congeners of DLIF in human serum, including the less polar components, suggests in vivo deglycosylation of these factors. This is the first demonstration of the existence of naturally occurring deglycosylated derivatives of DLIF and establishes the likelihood of active metabolism of DLIF in mammals.     

Comparison of four digoxin immunoassays with respect to interference from digoxin-like immunoreactive factors

OBJECTIVES: Comparison of a new monoclonal digoxin assay with three polyclonal digoxin assays for their cross-reactivity to digoxin-like immunoreactive factors (DLIF) and digoxin metabolites. DESIGN AND METHODS: Sixty-six nondigitalized patient samples from 5 different groups: neonates, women in 3rd trimester pregnancy, and patients with liver or renal diseases, or undergoing organ transplants, and 139 samples from digoxin-treated patients of 4 categories (hospital sick, liver, renal, and outpatients) were compared in 4 different digoxin assays: (a) ACS Digoxin (ACS) developed for the automated chemiluminescent Ciba Corning ACS 180 system, (b) Baxter Stratus (Stratus, a fluoroimmunoassay), (c) Ciba-Corning Magic (Magic, a radioimmunoassay), and (d) an in-house radioimmunoassay (RIA). The ACS and RIA were also compared for their cross-reactivity to four principal digoxin metabolites. RESULTS AND CONCLUSION: Among the nondigitalized specimens, no significant DLIF interference was found for all 4 assays among the pregnant women or liver and transplant patients. However, the neonates registered high DLIF interference with Magic and RIA, but none for ACS or Stratus. DLIF interference in renal samples was highest in the Magic assay and lowest in RIA. Among the specimens with digoxin, a higher number of discrepant samples were found from the sick patients than from outpatients. In 75% of such discrepant samples, the ACS result was less than other assay results, suggesting DLIF as the probable cause. The two assays differed most in their cross-reactivity to the deglycated metabolites, digoxigenin and its mono-digitoxoside.     

An accelerated increase of plasma adrenomedullin in acute asthma

A novel vasorelaxant peptide, adrenomedullin (AM), has been isolated from the acid extract of human pheochromocytoma. We have recently shown that AM inhibits histamine- and acetylcholine-induced bronchoconstriction in anesthetized guinea pigs in vivo, and this bronchodilatory effect is long-lasting. Here, we measured plasma AM concentrations in nine patients with an acute attack of bronchial asthma. The results were compared with values in 30 age-matched normal control subjects and seven age-matched stable asthmatic patients. The mean AM concentrations of patients with an acute asthma attack (98 +/- 22 pg/mL) were clearly higher than those of normal control subjects (18 +/- 2 pg/mL) and stable asthmatic patients (21 +/- 3 pg/mL). Reverse-phase high-performance liquid chromatography (HPLC) showed that the major component of plasma immunoreactive AM in patients with an asthma attack and in normal subjects equally corresponded to authentic human AM(1-52). Our results suggest that plasma AM is markedly increased in many of the patients during an acute attack of bronchial asthma, but it is not observed in stable asthmatic patients. Although this report is preliminary, the observed increase of circulating AM during an acute asthma attack may represent a compensatory mechanism against the bronchoconstriction, probably through its bronchodilatory action.     

High plasma proopiomelanocortin in aggressive adrenocorticotropin-secreting tumors

A specific propiomelanocortin (POMC) immunoradiometric assay was developed using antibodies directed against ACTH and beta-endorphin (beta end). Partially purified standard POMC was prepared from the human small cell lung carcinoma cell line DMS-79 culture medium. Ten units (U) POMC had the same displacement ability as one pg beta end in a C-terminal beta end radioimmunoassay and thus were close if not equal to 10 pg POMC. This POMC assay was used to investigate patients with ACTH-dependent Cushing's syndrome. Plasma POMC was undetectable (< 60 U/mL) in 17 normal controls and in 4 patients with Addison's disease (concomitant ACTH plasma levels between 362 and 1058 pg/mL). Forty-two patients with Cushing's disease were studied, either before (n = 25) or after (n = 17) bilateral adrenalectomy: 7 patients with highly invasive macroadenomas had high POMC plasma levels, between 240 and 4200 U/ml (concomitant ACTH plasma levels between 77 and 5730 pg/mL); 35 patients, including one with an invasive macroadenoma, had undetectable POMC plasma levels (concomitant ACTH plasma levels between 31 and 2820 pg/mL). Among 20 patients with histologically proven ectopic ACTH syndrome, 16 had high POMC plasma levels, between 80 and 8000 U/mL (concomitant ACTH plasma levels between 45 and 9265 pg/mL); all those tumors were malignant, and the highest POMC/ACTH plasma levels ratios (taken as an index of altered POMC processing) were observed in the 3 patients with small cell carcinomas of the lung; in one of these patients, ACTH and POMC plasma levels both decreased during the course of chemotherapy, in parallel with the reduction of the tumoral mass. Four patients with ectopic ACTH syndrome had undetectable POMC plasma levels (concomitant ACTH plasma levels between 78 and 335 pg/mL): they were all typical bronchial carcinoids. These data show that high POMC plasma level is neither specific for nor constant in ectopic ACTH syndrome. Rather it should be considered as a marker of tumor aggressivity, in pituitary- and non-pituitary tumors. Its diagnostic help appears limited for the most frequent cause of occult ectopic ACTH syndrome, the typical bronchial carcinoids.     

Restricted use of cationic germline V(H) gene segments in human Rh(D) red cell antibodies

The human red cell Rh(D) antigen elicits the production of high-affinity IgG antibodies, which can prevent blood transfusion and cause hemolytic disease of the newborn. It has been known for 20 years that Rh(D) antibodies are among the most positively charged human serum IgGs. Analysis by IEF of 9 human anti-Rh(D) monoclonal antibodies showed that their isoelectric points (pI) (8.3 to 8.6) were also significantly higher than the average pI of serum IgGs (7.0 to 8.5). Sequencing of the anti-Rh(D) H and L chains cDNAs showed a preferential use of V(H)1, V(H)3, J(H)6, and V(kappa)1 gene segments. The high pIs in IEF were correlated with a higher number of cationic amino acid residues in the H chain V regions without clustering in the complementary determining region. Computer analysis indicated that the germline V(H) used in anti-Rh(D) was selected among the most cationic segments available in the human V(H) repertoire or expressed in normal B cells. These results indicate that the selection of cationic V(H) segments may be an important early step in the formation of clinically relevant anti-Rh(D) and other red cell antibodies, possibly to facilitate epitope binding in the negatively charged red cell membrane environment.     

A study of cis-diamminedichloroplatinum(II) suppositories for the treatment of rabbit uterine endometrial carcinoma

OBJECTIVE: To determine the effects of continuous ambulatory peritoneal dialysis (CAPD) and hemodialysis (HD) on endothelin-1 (ET-1) levels in patients with end-stage renal disease (ESRD) and to assess the relationship between plasma ET-1 levels and selected patient parameters. DESIGN: Prospective, nonrandomized comparison study. SETTING: Outpatient CAPD and HD units of a university medical center. PARTICIPANTS: Twelve ESRD patients (6 on CAPD and 6 on HD) and 5 healthy normotensive subjects. INTERVENTIONS: CAPD patients had blood and peritoneal dialysate samples collected and measurements made following an overnight exchange. HD patients had blood collected and measurements made at 0 hours (basal) and again at 3 hours during a midweek HD session. Blood samples were also collected from normal subjects and served as ET-1 controls. MEASUREMENTS: ET-1 and patient parameters (creatinine, peritoneal dialysate volume, blood pressure, body weight, age, and treatment duration) were determined. Data are reported as the mean +/- one standard deviation. RESULTS: Plasma and dialysate ET-1 levels in the CAPD group were 19.5 +/- 4.2 pg/mL and 9.2 +/- 4.2 pg/mL, respectively. The control group plasma and unused dialysate contained no detectable ET-1 (< 3.0 pg/mL, the limit of detection). The peritoneal clearance of ET-1 was less than that of creatinine (2.29 +/- 0.69 mL/minute vs 4.22 +/- 0.66 mL/minute, p = 0.005). The basal (0 hour) plasma ET-1 level in the HD group (16.5 +/- 7.8 pg/mL) did not differ from that of the CAPD group, p = 0.423. Furthermore, no differences in patient parameters were detected between the CAPD and basal HD groups. Although the mean arterial pressure (MAP) decreased during HD, the plasma ET-1 level at 3 hours (13.5 +/- 5.4 pg/mL) remained unchanged from the basal level, p = 0.307. An analysis of pooled data from the CAPD and HD groups revealed no significant correlation between plasma ET-1 and MAP, body weight, creatinine, or treatment duration. There was, however, a positive correlation between plasma ET-1 and age (r = 0.643, p = 0.024).     

Immunoassay of digoxin in hair

Digoxin analysis in blood is an essential tool for therapeutic drug monitoring in cardiology because compliance with the treatment is a critical issue for the patient. Unfortunately, in postmortem cases blood digoxin concentration is of poor quality because there is a possible drug redistribution in the corpse and because of digoxin-like factors present in some people's blood. On the other hand, no biological fluid can be obtained at the autopsy. The aim of the present study was to evaluate the ability of an immunological method to determine digoxin in hair, in order to confirm that hair analysis can provide information on digoxin use before death. We studied 35 elderly patients who had been taking digoxin (60-250 micrograms/day) for 1-5 years. Two decontamination procedures were tested: washing by dichloromethane or by water and methanol. Three extraction procedures were compared: crushing in a ball mill and chloroform/acetone: crushing and methanol; enzymatic digestion. Immunoassays were performed by a microparticulate enzyme immunoassay. Serum digoxin levels were also assayed when sampling hair. The best results were obtained after decontamination with water and methanol followed by enzymatic digestion. Hair digoxin concentrations range from 3.6 to 11.4 pg/mg. Those very low concentrations are probably due to low and narrow range serum digoxin levels (0.3-1.4 ng/ml). No correlation was found between hair and blood digoxin. A forensic case is presented with 5 pg/mg digoxin in hair.     

Daily variations of platelet aggregation in relation to blood and plasma serotonin in diabetes

INTRODUCTION: An increase in digitalis-like substances has been reported in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. We hypothesized that the role of saline and unilateral nephrectomy in DOCA hypertension may be due to stimulation of endogenous digitalis-like substances. METHODS: We investigated the effects of digoxin and DOCA alone and in combination in intact rats drinking water. Forty male Sprague-Dawley rats were used (body weight 223-298 g). RESULTS: Neither digoxin (40 micrograms/kg per day, by gavage, for 35 days, n = 10) nor DOCA (30 mg/kg twice a week, subcutaneously, for 5 weeks, n = 10) caused a consistent increase in blood pressure in intact rats drinking water. In contrast, combined digoxin and DOCA administration (n = 10) increased systolic blood pressure from day 18 of treatment onwards, to a maximum at day 34 compared with sham-treated rats (n = 10). There were no consistent changes in water intake, urine volume, urinary sodium or potassium excretion, or plasma sodium or potassium concentration with digoxin treatment. DOCA increased water intake and urine volume, and caused an initial decrease in urinary sodium excretion, but no change in urinary potassium excretion or plasma sodium concentration. Plasma potassium excretion was lower in DOCA- than sham-treated rats. CONCLUSION: Combined digoxin and DOCA administration in intact rats drinking water increased blood pressure significantly compared with either drug alone, raising the possibility that the mechanism by which nephrectomy and salt loading contribute to DOCA hypertension in the rat might be through stimulation of endogenous digitalis-like substances.     

A specific binding protein for cardiac glycosides exists in bovine serum

Searching for a binding protein in blood, which may be involved in the specific transport of cardiac glycosides to their receptor sites on the sodium pump, we isolated a cardiac glycoside-binding protein (CGBG) of 26 kDa from the globulin fraction of bovine serum by affinity chromatography and on a ouabain-Sepharose 4B column by a purification factor of 5000. The cardiac glycoside-binding globulin was labeled specifically and covalently by the protein-reactive digoxigenin derivative HDMA (N-hydroxysuccimidyldigoxigenin-3-O-methylcarbonyl-epsilon-+ ++aminocapro ate). Even very high concentrations of other steroids, such as estrogen, testosterone, progesterone, and cortisone, did not prevent HDMA-labeling (at 5 and 100 nM) of CGBG, but the cardenolides ouabain and digoxin or the bufadienolide proscillaridin A did so. CGBG is a homodimer of two 26-kDa subunits forming disulfide bonds, since HDMA labeling of a protein of 53 kDa was observed in SDS-polyacrylamide gel electrophoresis when beta-mercaptoethanol was absent during SDS denaturation. The N-terminal amino acid sequence K-D-V-Y-R-A-P-D-G-T-Q-S-A showed no sequence similarity with proteins recorded in gene and protein sequence data banks. A 90-kDa cytosolic CGBG exists in bovine kidneys and reacts with antibodies against CGBG. Binding of ouabain to the cardiac glycoside-binding globulin was monitored by quenching of intrinsic tryptophan fluorescence. Such studies reveal two negatively cooperative ouabain binding sites with Kd' of 1.52 nM and Kd' = 75 nM and with an interaction factor of 50 using a Koshland-Némethy-Filmer model. The demonstration of a cardiac glycoside-binding globulin in plasma is consistent with the recent finding of endogenous cardiac glycosides in mammals.     

Increased interleukin-6 production during the acute phase of the syndrome of episodic angioedema and hypereosinophilia

BACKGROUND: The Gleich syndrome is rare and associates recurrent angioedema, urticaria, fever, weight gain and blood hypereosinophilia, underlying systemic and local inflammation. The pathogenesis of those symptoms remains unclear. OBJECTIVE: We wanted to address the possible implication of Interleukin-6 (IL-6) in the development of those clinical features, and to identify the cells involved in its production. METHODS: A 26-year-old man suffering of this disease was referred in hospital. During an acute attack with weight gain, fever and a diffuse oedema, a marked increase in eosinophils count (42700/mm3 was observed. Serum ECP was elevated at 47 microg/L (normal less than 16). Corticosteroid therapy administrated on the 7th day was followed by a rapid remission. Blood samples were collected (before, during the attack and under corticosteroid therapy) for measurements of serum IL-6 (ELISA, Immunotech, Marseille, France) and plasma histamine (RIA, Immunotech, Marseille, France). Blood monocytes and eosinophils were isolated and a skin biopsy was performed during the attack. RESULTS: The plasma histamine level was within normal range. The level of IL-6 in sera peaked to 74 pg/mL, concomitant with the peak of eosinophilia at the acute phase phase of the attack. Under corticosteroids, we observed a drop in the IL-6 serum level to 29 pg/mL, concomitant with the clinical remission. During the attack, an increase in IL-6 production was observed in 24 h blood monocyte supernatants (11.10(3) pg/mL compared with 2.4+/-0.8.10(3) pg/mL for BM from controls) as well as in skin endothelial cells but not in the blood and skin eosinophils. In vitro, when endothelial cells were incubated in eosinophils supernatants of the patient, liberation of IL-6 was observed (3.3 10(3) pg/mL compared with controls: 2.1 10(3) pg/mL) CONCLUSION: Serum IL-6 elevation may be related to an increased production by blood monocytes and endothelial cells, possibly stimulated by eosinophil mediator during the acute phase of the disease, and might participate in the inflammatory reaction of this syndrome.     

Heterogeneity of antibodies reactive with the dominant antigen of Actinobacillus actinomycetemcomitans

The serotype b-specific carbohydrate antigen (SbAg) of Actinobacillus actinomycetemcomitans Y4 is reported to be the O antigen of lipopolysaccharide, and the highest titers of serum antibody reactive with A. actinomycetemcomitans in early-onset periodontitis (EOP) patients bind SbAg. These high titers of serum antibody reactive with SbAg are associated with a lesser extent and severity of periodontal disease. The aim of this study was to determine if a limited number of genes code for anti-SbAg antibodies as has been shown for immunoglobulin G (IgG) reactive with the type b polysaccharide from Haemophilus influenzae. Serum IgG reactive with the SbAg was prepared from 20 high-titer EOP patients by affinity chromatography. The IgG subclass concentrations were determined, and heterogeneity was analyzed by isoelectric focusing (IEF). IgG2 was the dominant subclass (83% of total IgG) in the anti-SbAg IgG fraction and represented an average of 1.33% of total serum IgG2. The IgG2 reactive with SbAg was isolated from the affinity-purified IgG fraction by affinity chromatography with protein A and subclass-specific monoclonal antibodies. On IEF gels, only 4 to 20 bands were observed in the anti-SbAg IgG fractions, indicating limited heterogeneity. N-terminal amino acid sequence analysis of eight representative anti-SbAg IgG2 preparations indicated that variable heavy and light chains consisted largely of V(H)III and V(kappa)II, respectively. However, a significant fraction of anti-SbAg may use V(H) and V(lambda) genes with blocked N termini. In short, these findings indicate that IgG reactive with SbAg is very much like the antibody reactive with H. influenzae type b polysaccharide. Similarities include IgG2 dominance, limited bands on IEF gels, supporting an oligoclonal response, and use of genes from V(H)III and V(kappa)II regions.     

The BAX gene, the promoter of apoptosis, is mutated in genetically unstable cancers of the colorectum, stomach, and endometrium

OBJECTIVE: The study's objective was to determine whether there is a difference in the plasma concentration of adrenomedullin, a hypotensive peptide, between arterial and venous umbilical cord blood of uncomplicated gestations with vaginal delivery. STUDY DESIGN: Arterial and venous umbilical cord blood was obtained immediately after vaginal delivery of 44 term infants with uncomplicated antepartum and intrapartum courses. Radioimmunoassay was performed to assess adrenomedullin concentrations in the plasma. The paired t test was used to compare arterial and venous concentrations. Significance was set at P < .05. RESULTS: Mean +/- SE adrenomedullin concentrations were 178.7 +/- 4.7 pg/mL and 190.6 +/- 6.3 pg/mL for arterial and venous cord plasma, respectively. The difference between the 2 concentrations was not significant (11.8 pg/mL, P = .09). CONCLUSION: Arterial and venous umbilical plasma concentrations of adrenomedullin do not differ significantly in uncomplicated gestations terminating with uncomplicated vaginal deliveries. This suggests that in the normal state there is neither net production nor net clearance of adrenomedullin in the placenta.     

Comparative study of the effects of ouabain and digoxin on blood pressure of rats

OBJECTIVE: To compare the effect of ouabain on the blood pressure of rats with that of digoxin to find the evidences of the relationship between endogenous ouabain (EO) and development of hypertension. METHODS: Sprague-Dawley rats, which were divided into 3 groups, were infused with ouabain (23 x 75 micrograms.kg-1/day, i.p.), digoxin (36 x 84 micrograms.kg-1/day, i.p.) and normal saline (NS) once a day respectively. Systolic blood pressure and body weight were recorded weekly. Five weeks later, rats of ouabain group were randomly assigned to three infusion subgroups: Oc group, continued with ouabain infusion; Od group, added digoxin (73 x 68 micrograms.kg-1/day, i.p.) and Os group, stopped administration of ouabain. Another week later, direct blood pressure was recorded in aorta. Systolic and diastolic cardiac function, plasma renin activity and aldosterone levels of all the rats were measured. RESULTS: After a latent period of one week, blood pressure of Ouabain group increased significantly [95.4 +/- 11.8 mmHg (1 mmHg = 0.133 kPa) at the beginning of the experiment vs 122.5 +/- 16.9 mmHg at the end of week 6, P < 0.05] with normal plasma renin activity and higher aldosterone (1.28 +/- 0.45 ng/ml vs 0.69 +/- 0.27 ng/ml, P < 0.05). The blood pressure decreased after either withdrawal of ouabain or addition of digoxin (116.3 +/- 14.4 mmHg vs 100 +/- 10.7 mmHg, P < 0.05; 123.9 +/- 13.9 vs 103.3 +/- 10.5 mmHg, P < 0.05, respectively). No difference of blood pressure was found between the digoxin and NS group. CONCLUSIONS: Our results suggested that EO might be one of the causes of the development of hypertension. Aldosterone might play some role in the mechanism of ouabain-induced hypertension. Digoxin can not induce hypertension. There is a great difference between the effect of ouabain and digoxin on the blood pressure. Moreover, digoxin can reverse the hypertension induced by ouabain.     

The short-term effects of digoxin in patients with right ventricular dysfunction from pulmonary hypertension

OBJECTIVE: Studies on the effects of digoxin in patients with right ventricular failure and normal left ventricular function have not been performed. We evaluated the short-term effects of digoxin administration in patients with primary pulmonary hypertension on hemodynamics, neurohormones, and baroreceptor responsiveness. DESIGN: This was a prospective study with patients serving as their own controls. SETTING: University Hospital Intensive Care Unit with central monitoring. PATIENTS: Seventeen patients with primary pulmonary hypertension and symptomatic heart failure were enrolled. INTERVENTIONS: Following baseline hemodynamics, neurohormonal samples were drawn and the heart rate response to change in blood pressure following a challenge of phenylephrine and nitroprusside were recorded. One mg of intravenous digoxin was given and the measurements repeated after 2 hours. RESULTS: Following digoxin there was a significant increase in cardiac output (3.49+/-1.2 to 3.81+/-1.2 L/min., p=0.028), a significant fall in norepinephrine (680+/-89 to 580+/-85 pg/ml, p=.013), and a significant increase in atrial natriuretic peptide (311+/-44 to 421+/-9 pg/ml, p=0.01). All of the patients had changes in heart rate and blood pressure following phenylephrine and nitroprusside challenge, but there was no significant difference in the change in heart rate response to change in blood pressure when rechallenged after digoxin treatment. CONCLUSION: Digoxin produces a modest increase in cardiac output in patients with pulmonary hypertension and right ventricular failure, as well as a significant reduction in circulating norepinephrine. No detectable effects of digoxin on baroreceptor responsiveness were apparent. The use of digoxin in pulmonary hypertension is warranted.     

Extracellular dextran-induced p-nitrophenyl-alpha-D-glucoside-hydrolyzing enzyme of Bacillus circulans KA-304: a producer of Schizophyllum commune-lytic enzyme

We previously reported that centrally administered neuropeptide FF (NPFF) inhibited arginine vasopressin (AVP) release. In this study, immunoneutralization of central NPFF was performed to evaluate the role of endogenous NPFF in the regulation of AVP release. Intracerebroventricular (ICV) injection of antiserum against NPFF (Anti-NPFF) significantly augmented the plasma AVP increase induced by hyperosmolality [intraperitoneal injection of hypertonic saline (600 mOsm/kg, 2% BW)] at 60 min after ICV injection compared with normal rabbit serum (NRS) (NRS: 4.20+/-0.30 pg/ml, Anti-NPFF: 5.83+/-0.46 pg/ml, p < 0.01). Anti-NPFF did not cause significant change in plasma osmolality, plasma volume or arterial blood pressure. This evidence indicates that endogenous NPFF might be physiologically involved in osmoregulation of the plasma AVP level through its inhibitory action.     

Development of a highly sensitive enzyme-linked immunosorbent assay based on polyclonal antibodies for the detection of polychlorinated dibenzo-p-dioxins

The development of an enzyme-linked immunosorbent assay (ELISA) based on polyclonal antibodies for the polychlorinated dibenzo-p-dioxins is described. We previously reported the synthesis of haptens and generation of antibodies for detection of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Antisera were screened with seven different coating antigens (hapten-protein conjugates), including trans-3-(7,8-dichlorodibenzo-p-dioxin-2-yl)-cis-2-methylpropeno ic acid (VII) and 5-(3,7,8-trichlorodibenzo-p-dioxin-2-yl)penta-trans,trans-2,4-dien oic acid (X). All inhibition screening and optimization studies were conducted using a less toxic surrogate standard for TCDD [2,3,7-trichloro-8-methyl-dibenzo-p-dioxin (TMDD; XVII)] which responded similarly to 2,3,7,8-TCDD in the ELISA. The most sensitive assay from the screening studies [coating antigen VII-BSA, 0.1 microgram/mL, and antiserum 7598 (anti-X-LPH), 1:10,000] was further optimized and characterized. It exhibited an IC50 value of 12 pg/well (240 pg/mL), with working range from 2 to 240 pg/well (40 to 4800 pg/mL). The influence of various physical and chemical factors (time, solvent, detergent) was investigated. The optimized assay was then used to assess cross-reactivity by congeners of halogenated dioxins and related structures. DMSO up to concentrations of 37.5% decreased the IC50 value in the assay, whereas methanol to concentrations of 30% did not lead to improved IC50 values.     

Low picogram determination of Ro 48-6791 and its major metabolite, Ro 48-6792, in plasma with column-switching microbore high-performance liquid chromatography coupled to ion spray tandem mass spectrometry

A coupled liquid chromatography/tandem mass spectrometry assay was developed for simultaneous determination of Ro 48-6791 and its secondary amine metabolite in human plasma samples with a quantification limit for both compounds of 1 pg/mL using a 1 mL plasma aliquot. The method exploits the enhanced mass sensitivity of a microbore (300 microns i.d.) reversed-phase capillary column coupled to an ion spray probe combined with tandem mass spectrometry. A straightforward column-switching system was utilized to focus the analytes onto a microbore trapping column following solid-phase extraction of a 50 microL plasma sample extract from liquid/liquid extraction. Backflushing of the retained analytes from the trapping column onto the microbore capillary column provided the requisite high peak concentration for high sensitivity. The inter-assay precision and accuracy for Ro 48-6791 and its metabolite, at 10 pg/mL, were found to be 3.4%, and 105%, and 9.1%, and 99.9%, respectively. The calibration curves were linear over the range 1 to 1000 pg/mL. The method proved to be sufficiently rugged for analysis of samples.     

Augmented exercise plasma noradrenaline with impaired chronotropic responsiveness in patients with hypertrophic cardiomyopathy

1. There is controversy regarding plasma catecholamine levels in patients with hypertrophic cardiomyopathy (HCM) and few data exist on serial plasma catecholamine measurements during exercise. The present study determined whether cardiovascular and plasma catecholamine responses to exercise were altered in patients with HCM. 2. Plasma noradrenaline (NA) and adrenaline were measured at rest, at the end of each stage during exercise and immediately and 5 min after submaximal treadmill exercise in 15 patients with non-obstructive HCM (13 males, two females; mean (+/- SEM) age 54 +/- 3 years) and in 15 age- and sex-matched controls. The ratio of the increment in heart rate (HR) divided by the increment in plasma NA during exercise (delta HR/delta NA) was used as an index of chronotropic sympathetic responsiveness to exercise. 3. Exercise duration was shorter (11.2 +/- 0.6 vs 8.7 +/- 0.6 min for control vs HCM, respectively; P < 0.01) and diastolic blood pressure was significantly higher at stages I and II of modified Bruce protocol HCM. 4. Resting plasma NA levels (149 +/- 17 vs 167 +/- 28 pg/mL for control vs HCM, respectively; NS) were not different, but plasma NA levels at stages I and II were significantly higher in HCM than in controls (243 +/- 26 vs 399 +/- 69 pg/mL (P < 0.05) and 308 +/- 30 vs 548 +/- 110 pg/mL (P < 0.05), respectively). 5. Peak plasma NA levels were not significantly higher in HCM than in controls (578 +/- 59 vs 918 +/- 184 pg/mL, respectively; NS). 6. The ratio delta HR/delta NA was significantly lower in HCM compared with control at stages I and II (0.49 +/- 0.10 vs 0.21 +/- 0.05 (P < 0.05) and 0.38 +/- 0.06 vs 0.20 +/- 0.05 (P < 0.05), respectively). There were no differences in plasma adrenaline responses during exercise between the two groups. 7. Patients with HCM had augmented plasma NA levels during submaximal exercise with a higher diastolic blood pressure response. Chronotropic sympathetic responsiveness was impaired during the early stages of exercise in patients with HCM.     

Routine measurement of plasma calcitonin in nodular thyroid diseases

In a prospective study, plasma concentrations of human calcitonin (hCT) were determined in 1062 consecutive patients with thyroid nodular disease. Basal plasma hCT was above the normal range (>6 pg/mL) in 55 patients and was elevated up to more than 100 pg/mL (range, 127-5459) in 3 of these 55 patients. A pentagastrin-induced rise in hCT up to more than 100 pg/mL was observed in only 1 of 38 patients with a basal concentration of hCT between 5-10 pg/mL, but was found in 10 of 31 patients with basal hCT ranging from 10-100 pg/mL. Histologically, 7 of the 14 patients with either basal or stimulated plasma concentrations of hCT above 100 pg/mL presented C cell hyperplasia, which in one case showed histological transition into a small (diameter, 3 mm) medullary thyroid carcinoma (MTC). Including this patient, MTC was found in 6 of the 12 patients. We conclude that the routine determination of hCT in all patients with thyroid nodular disease should be supplemented by pentagastrin-stimulation when the basal hCT concentration exceeds 10 pg/mL. Patients with basal and/or stimulated plasma CT concentrations of more than 100 pg/mL should be operated on because they run a substantial risk to suffer either MTC or C cell hyperplasia, a potentially precancerous condition. This will increase the chance of a timely diagnosis of MTC and provide the chance of curative surgery.