Liquid chromatography quadrupole time-of-flight mass spectrometry quantification and screening of organophosphate compounds in sludge.

For the first time, we assess the performance of liquid chromatography (LC) quadrupole time-of-flight (QTOF) mass spectrometry (MS) for the selective quantification of eight organophosphate compounds (OPs), used as plasticizers and flame retardants additives, in sludge from urban sewage treatment plants (STPs). Moreover, the usefulness of accurate, full scan MS and MS/MS spectra to screen and to confirm the presence of additional OPs, without using reference standards, in sludge samples is discussed. Matrix solid-phase dispersion (MSPD) was used as a sample preparation technique. Under optimized conditions, MSPD provided quantitative recoveries for the group of targeted analytes, requiring just 15 mL of solvent and integrating extraction and clean-up processes in the same step. For these species, the achieved limits of quantification (LOQs) varied between 2 and 50 ng g(-1) and the efficiency of electrospray ionization (ESI) did not change significantly between pure standards and sludge extracts. Among targeted OPs, tri(chloroisopropyl) phosphate (TCPP), tributoxyethyl phosphate (TBEP) and triphenyl phosphate (TPP) were ubiquitous in sludge. The average concentrations of TCPP and TBEP stayed above 700 ng g(-1), whereas the mean value for TPP was 67 ng g(-1). Full scan, accurate spectra provided relevant clues for the screening of additional OPs, using a database containing just their empirical formulae and exact molecular weights; however, the occurrence of in-source fragmentation processes hampered the detection and correct identification of those species which did not render the expected [M+H](+) molecular ion, as was the case of 2-ethylhexyl-diphenyl phosphate (EHDPP).

Anti-HIV activity of dibenzylbutyrolactone-type lignans from Phenax species endemic in Costa Rica.

Previously, we isolated two new dibenzylbutyrolactone-type lignans, named phenaxolactones and, from the leaves of Phenax angustifolius Wedd. (Urticaceae). In this investigation three new dibenzylbutyrolactone lignans (phenaxolactones), together with phenaxolactone, and flavones vitexin, isovitexin, were isolated from Phenax rugosus Wedd. leaves collected in Santa Ana, Costa Rica. The structures were elucidated using 1D and 2D NMR spectroscopy as well as mass spectrometry. Phenaxolactones and flavones and were evaluated for their inhibitory activity against HIV-1MN in infected C8166 cells. The most promising compound was phenaxolactone with an EC50 value of 3.0 microM, no cytotoxicity at 112 microM and a therapeutic index value of 37.3.

[Recovery of urinary incontinence after retropubic radical prostatectomy. Results in 100 patients]. / Ripresa della continenza urinaria dopo prostatectomia radicale retropubica. Risultati in 100 pazienti.

AIM OF THE STUDY: To shorten the time to recovery of full urinary continence after radical retropubic prostatectomy and to increase the postoperative continence rate. MATERIALS AND METHODS: One hundred and five consecutive patients were submitted to radical retropubic prostatectomy for prostate cancer, with curative intent, focusing our attention on three steps of the operation. First, to achieve a complete control of bleeding from the dorsal vein complex and to perform a minimal touch dissection of the urethral stump; second, to perform a conservative dissection of the bladder neck, and, third, to implement a vascular type, watertight, vesico urethral anastomosis. Continence was assessed 24 hours after catheter removal and at monthly follow up visits until full recovery and graded as dry if no dropping was visible and the patient was able to interrupt the urinary stream during micturition; stress incontinence, if any dropping was observed during abdominal strain between micturitions; and wet, if uncontrollable dropping occurred, and the number of pads needed per day recorded. RESULTS: At a median follow-up time of 18 months, range 6 to 30, a total of 87 of 100 evaluable patients (87%) resulted as dry; 10 patients (10%) resulted as having a variable degree of stress incontinence needing one to three pads per day, and 3 patients (3%) resulted as wet. In 41 of the 87 dry patients (47.1%) continence was achieved within the first day from catheter removal, and in a median time of 4 weeks, range 2 to 16 in the remaining 46 patients (52.9%). CONCLUSIONS: The results of total continence rate of the present study seem to compare to the recent literature except for the time to full recovery which is shorter; it is difficult to identify the contribution of each single surgical step.

[Precancerous lesions of the penis]. / Le lesioni pre-cancerose del pene.

Premalignant penile lesions have been associated to squamous cancer, although the true incidence of progression of these entities to squamous cell carcinoma is unknown. These lesions may coexist with or may antecede the occurrence of carcinoma. For this reason close follow-up of the patient is necessary to detect early evidence of malignant degeneration.

[Conventional surgical treatment of penile tumors]. / La terapia chirurgica tradizionale del tumore del pene.

The surgical treatment of primary lesion in Carcinoma of the penis is the total excision of the carcinoma with tumor free margins. Distal preputial carcinomas without deep infiltration are often treated by extensive circumcision. Partial or total amputation of the penis is required by the location and the extent of the tumor. The evaluation and the treatment of the lymph nodes in penile cancer are still unclear.

Chromatin organization during mouse oocyte growth.

We investigated the changes in the organization of oocyte nuclear chromatin and nucleolar-associated chromatin throughout folliculogenesis. Zona-free oocytes were isolated from ovaries, grouped into seven classes according to size and chromatin organization, and analyzed after staining with Hoechst 33342. We show that oocyte differentiation from the dictyate stage to the conclusion of maturation is associated with either of two chromatin configurations. Initially, all oocytes are in the NSN configuration (nonsurrounded nucleolus oocytes; characterized by a Hoechst positive-chromatin pattern of small clumps forming a network on the nuclear surface, with a nucleolus nonsurrounded by chromatin). While growing some of these NSN oocytes continue their development in the NSN configuration, whereas others shift (from class IV on) into the SN configuration (surrounded nucleolus oocytes; characterized by a threadlike chromatin organization that may partially surround the nucleolus or project towards the nuclear periphery). The percentage of SN oocytes increases both with increasing size of the oocyte (class I-III, 10-40 microns in diameter: 100% NSN vs. 0% SN; class VII 70-80 microns in diameter: 47.3% NSN vs. 52.3 SN, in 4-6-week-old females), and with aging (class VII: 94.1% NSN vs. 5.9% SN in 2-week-old females; 11.8% NSN vs. 8.2% SN in 56-week-old females). Further, we suggest as a working hypothesis that those oocytes that switch to the SN chromatin organization early in maturation may not be ovulated, even though this particular chromatin structure normally occurs just prior to ovulation.

Development and loss of the ability of mouse oolemma to fuse with spermatozoa.

To further our knowledge on the mechanisms and molecules involved in mouse sperm-oocyte plasma membrane interaction, experiments were carried out to determine the stage during oogenesis at which an oocyte acquires the capacity to fuse with acrosome-reacted sperm. Zona-free oocytes 10 microns in diameter do not fuse with sperm. Oolemma fusibility is first acquired when the oocyte reaches about 20 microns in diameter. Fusibility is maintained even after fertilisation has occurred and is lost completely by the 4-cell stage.

Dystrophin abnormalities in Duchenne and Becker dystrophy carriers: correlation with cytoskeletal proteins and myosins.

Characterization with a panel of six antibodies revealed abnormal dystrophin expression in 6 of 20 Duchenne muscular dystrophy (DMD) carriers examined, and in 5 of 12 Becker muscular dystrophy (BMD) carriers examined. The immunocytochemistry of muscle fibres was normal with five of the antibodies in two BMD carriers, but some muscle fibres were negative to the antibody directed against a portion of the dystrophin rod domain. Mosaicism was detected with all six antibodies in the other three BMD (but in only a small number of fibres) and in all DMD carriers muscles. Spectrin, vinculin and talin were immunolocalized in the same muscle specimens in order to assess membrane cytoskeletal integrity and to correlate their expression with that of dystrophin. These proteins, including vinculin, which was previously reported to be reduced in DMD patient muscles, were normally present on the surface of all dystrophin-deficient fibres. Muscle fibre types were characterized using monoclonal antibodies against fetal myosin and adult fast and adult slow myosin heavy chains. In both the DMD and BMD carriers, a significant reduction in type 2B fibres, as well as an increase in type 2C and fetal myosin-containing fibres was found - as has also been reported in DMD patients. Altered dystrophin expression was observed more frequently in type 2 than type 1 fibres. Dystrophin deficiency was found in a high percentage of type 2C fibres as well as in all fibres expressing fetal myosin; this suggests that dystrophin-deficient fibres are more susceptible to degeneration, leading to regeneration.

Evidence of a genetic marker associated with early onset in Friedreich's ataxia.

We evaluated the association between age at onset of Friedreich's ataxia and alleles of two restriction fragment length polymorphisms (RFLP) at D9S15 and D9S5 in the 9q13-9q21.1 region. We studied 65 Italian patients from 49 families. Age at onset was not normally distributed in our patients, suggesting allelic heterogeneity. Patients homozygous for allele 1 of MspI RFLP detected by probe MCT112 at D9S15 (M1) had an earlier onset (mean 9.3, SD 3.4 years) than patients homozygous for allele 2 (M2; mean 12.1, SD 4.3). Heterozygotes had an onset age similar to that of the M2 homozygotes. These findings suggest that the M1 allele might be a marker of one allelic early-onset Friedreich's ataxia mutation.

The acute effect of FK506 and cyclosporine on endothelial cell function and renal vascular resistance.

We have previously documented that cyclosporine exerts a direct cytotoxic effect on endothelial cells and causes an increase in renal vascular resistance (RVR) in the rat. In the present study we investigated whether FK506, a novel immunosuppressive agent thought to be less nephrotoxic than CsA, impairs endothelial cell function in vitro and affects RVR in vivo. In vitro eicosanoid release and endothelin release were measured in bovine aortic endothelial cells in culture exposed for 1, 6, and 24 hr to increasing concentrations of FK506 (1 nM to 10 microM) or CsA (0.5, 10 microM). No significant changes in TxB2 and 6-keto-PGF1 alpha (the stable breakdown products of TxA2 and PGI2, respectively) and endothelin release were found after 1 and 6 hr of incubation with all the concentrations of FK506 and CsA considered. FK506 did not affect endothelin release even after 24 hr of incubation. In contrast, cell exposure to CsA was associated with a dose-dependent increase in TxB2, 6-keto-PGF1 alpha, and endothelin release that reached statistical significance after incubation with 10 microM CsA. FK506 did not induce cell detachment or lysis at any concentration and time considered, while 10 microM CsA induced a significant reduction in cell count accompanied by cell lysis. In vivo studies showed that a single i.v. injection of FK506 to rats within a broad range of doses (28 ng/kg to 2.8 micrograms/kg) did not modify RVR. This was true even for a dose as high as 20 mg/kg, while 20 mg/kg CsA caused a dramatic increase in RVR. We conclude that FK506, unlike CsA, does not induce endothelial cell injury in vitro. Whether this explains the differences in renovascular resistance observed in vivo after acute injection of FK506 and CsA is an attractive possibility that needs to be further explored.

Atrial natriuretic peptide and prostacyclin synergistically mediate hyperfiltration and hyperperfusion of diabetic rats.

The relative contribution of atrial natriuretic peptide (ANP) and vasodilatory prostaglandins to hyperfiltration in Wistar rats with experimental diabetes was studied 6-8 wk after streptozocin injection. Plasma levels of immunoreactive ANP were significantly higher (P less than 0.01) in hyperglycemic diabetic (72.9 +/- 11.7 pg/ml) than in normoglycemic diabetic (44.8 +/- 8.6 pg/ml) or nondiabetic (40.0 +/- 6.8 pg/ml) rats. Blocking endogenous ANP by specific ANP-antiserum infusion reduced significantly (P less than 0.01) glomerular filtration rate (GFR) and renal plasma flow (RPF) of hyperglycemic rats compared with preinfusion values (1.23 +/- 0.06-1.02 +/- 0.04; 2.87 +/- 0.25-2.40 +/- 0.10 ml.min-1.100 g-1, respectively). However, correction of hyperfiltration and hyperperfusion was only partial (nondiabetic rats GFR 0.85 +/- 0.07; RPF 2.27 +/- 0.13 ml.min-1.100 g-1). Because diabetic rats with hyperglycemia also had an increased urinary excretion of prostacyclin metabolite 6-keto-prostaglandin F1 alpha (220.6 +/- 62.8 ng/24 h) compared with nondiabetic rats (51.2 +/- 2.7 ng/24 h), we wondered whether excessive prostacyclin formation contributed to hyperfiltration and hyperperfusion in this setting. Indomethacin infusion partially reduced GFR (1.25 +/- 0.07 to 1.06 +/- 0.07 ml.min-1.100 g-1, P less than 0.05) and RPF (2.85 +/- 0.11 to 2.46 +/- 0.12 ml.min-1.100 g-1, P less than 0.01) in diabetic rats. The combined infusion of ANP antiserum and indomethacin normalized GFR and RPF in diabetic rats with hyperglycemia (1.27 +/- 0.05 to 0.88 +/- 0.05 and 2.84 +/- 0.10 to 2.22 +/- 0.06 ml.min-1.100 g-1, respectively; P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Membrane vesicles in the study of transport processes: a critical analysis of the experimental procedure.

A critical analysis of the use of membrane vesicles in the study of cotransport processes is presented. Transport experiments were simulated according to two different models, stressing those conditions that seemed more relevant in affecting the measurements. In particular, we observed that the experimental Vmax values were underestimated. This underevaluation depended on the incubation time employed to measure the initial uptake rate and on the time necessary to wash the vesicles. Also the temperature and the composition of the washing solution, together with the Q10 of the transport process taken into consideration, had a consistent influence on the uptake. All the above mentioned effects were affected by the vesicle volume: the smaller the volume, the greater the underestimate of the uptake. This theoretical analysis underlines, on the one side, that the experimental data should be interpreted with some caution, on the other, that the examined procedure allows an internal check of its validity by adopting suitable simulations of the experiments. The use of the presented models as a tool for the planning and the critical analysis of the experimental results is suggested.

[Intra- and postoperative changes in blood viscosity]. / Variazioni intra e postoperatorie della viscosità ematica.

Blood and plasma viscosity has been controlled in a group of patients undergone to aorto-iliac reconstruction and in a group of control after thyroidectomy, cholecystectomy, and hernioplasty. The hemodilution induced by intraoperative infusion in the vascular reconstruction produced an important decrease of hematic and plasmatic viscosity which lasted for several days after the operation. Removing the hemodilution effect by a mathematical correction of the viscosity measured values to a standard haematocrit, it has demonstrated as surgical operation, apart from its entity, promoted an increase of the viscosity which persisted long in the postoperative course. For what it concerns the risk of postoperative thrombosis from one side protective effect of hemodilution is confirmed, from the other, in absence of the hemodilution, it would be useful to continue the antithrombotic prophylaxis longer the perioperative time as usual.

Preliminary report: renal thromboxane A2 synthesis in children with frequent relapsing nephrotic syndrome.

An evaluation was made of the possible relation between renal thromboxane (Tx)A2 synthesis (measured as urinary excretion of TxB2) and the loss of glomerular permeability to proteins, in 5 children with seven episodes of minimal change nephrotic syndrome. Urinary TxB2 excretion was significantly higher in children with minimal change nephrotic syndrome than in 14 healthy controls, and reached its maximum at the time of peak proteinuria. During remission of nephrotic syndrome urinary excretion of TxB2 was still significantly higher than in healthy controls. A significant positive correlation between urinary excretion of TxB2 and proteinuria was observed in 3 patients. The results suggest that renal TxA2 could be regarded as one of the possible mediators of the altered glomerular permeability to proteins in minimal change nephrotic syndrome.

Time course analysis of cotransport in membrane vesicles: solutes and tracers.

A theoretical analysis of the time course of a ternary cotransport system in membrane vesicles has been developed by extending previous work (Weiss, S.D. et al. (1981) J. Theor. Biol. 93, 597-608; Heinz, E. and Weinstein, M. (1984) Biochim. Biophys. Acta 776, 83-91). It has been assumed that the translocation of the carrier is the rate-limiting step of the transport process. Our approach includes, in particular, the presence of isotope tracer fluxes and the generalization to the case when many solutes share the same carrier. The situation when the tracer and the solute behave differently, as in the countertransport case, is stressed. Also, the interaction of two different solutes, internal and external to vesicles, is considered. Other points regard the analysis of the solute binding to the membrane vesicles, the influence of water permeability and the possible asymmetry of the transport system. In the Appendix, the assumption of no net translocation of all carrier species is discussed.

Endothelin and eicosanoid synthesis in cultured mesangial cells.

We investigated the effect of endothelin on the generation of eicosanoids, which are known to regulate basal and stimulated mesangial cell tone. The results showed that endothelin is a potent stimulus of prostaglandin E2 (PGE2), prostacyclin (PGI2), and thromboxane A2 (TxA2) synthesis by bovine mesangial cells. Percentage increases in eicosanoid synthesis induced by endothelin (10(-10) to 10(-6) M), were 50 to 275% for PGE2, 28 to 168% for PGI2 and 42 to 111% for TxA2, respectively. Endothelin-induced eicosanoid synthesis in mesangial cells was concentration, but not time dependent. Aspirin (500 microM) completely prevented endothelin-induced eicosanoid synthesis. The calcium entry blocker nitrendipine (10(-8) M) failed to inhibit endothelin-induced eicosanoid synthesis. These data suggest that endothelin-induced changes in renal circulation and glomerular function in normal and disease conditions may be modulated by the concomitant stimulation of mesangial eicosanoid synthesis.

Quantitative profiling of 6-ketoprostaglandin F1 alpha, 2,3-dinor-6-ketoprostaglandin F1 alpha, thromboxane B2 and 2,3-dinor-thromboxane B2 in human and rat urine by immunoaffinity extraction with gas chromatography-mass spectrometry.

A rapid and simple method based on immunoaffinity extraction, stable isotope dilution and gas chromatography-mass spectrometry has been developed for profiling urinary metabolites of prostacyclin and thromboxane. 6-Ketoprostaglandin F1 alpha (6-keto-PGF1 alpha), 2,3-dinor-6-ketoprostaglandin F1 alpha (2,3-dinor-6-keto-PGF1 alpha), thromboxane B2 (TXB2) and 2,3-dinor-thromboxane B2 (2,3-dinor-TXB2) were quantitatively extracted from human or rat urine spiked with deuterated internal standards using mixed-bed columns containing immobilized anti-6-keto-PGF1 alpha and anti-TXB2 antibodies (cross-reacting with 2,3-dinor-6-keto-PGF1 alpha and 2,3-dinor-TXB2, respectively). The extract was directly derivatized to form pentafluorobenzyl ester, methyloxime, trimethylsilyl ether derivatives. Quantitation was performed by stable isotope dilution assay and high-resolution gas chromatography-negative ion chemical ionization mass spectrometry, by monitoring the carboxylate anions (M-181) of the derivatized metabolites. The method was applied to evaluate the urinary excretion of 6-keto PGF1 alpha, 2,3-dinor-6-keto-PGF1 alpha, TXB2 and 2,3-dinor-TXB2 in humans and rats. Results were in accordance with previously reported data obtained by other methods. Novel data on the urinary excretion of 2,3-dinor-6-keto-PGF1 alpha in rats under basal conditions are presented. This sensitive and selective method represents a significant advance in terms of rapidity and simplicity over other immunoaffinity-gas chromatography-mass spectrometry methods for measuring single prostanoids, such as 6-keto-PGF1 alpha or TXB2, since it allows profiling of a group of metabolites whose balance is important in several physiopathological conditions.

Effect of low-dose aspirin on fetal and maternal generation of thromboxane by platelets in women at risk for pregnancy-induced hypertension.

There is evidence that aspirin in low doses favorably influences the course of pregnancy-induced hypertension, but the mechanism, although assumed to involve suppression of the production of thromboxane by platelets, has not been established. We performed a randomized study of the effect of the long-term daily administration of 60 mg of aspirin (n = 17) or placebo (n = 16) on platelet thromboxane A2 and vascular prostacyclin in women at risk for pregnancy-induced hypertension. Low doses of aspirin were associated with a longer pregnancy and increased weight of newborns. Serum levels of thromboxane B2, a stable product of thromboxane A2, were almost completely (greater than 90 percent) inhibited by low doses of aspirin. The urinary excretion of immunoreactive thromboxane B2 was significantly reduced without changes in the level of 6-keto-prostaglandin F1 alpha, a product of prostacyclin. Mass spectrometric analysis showed that aspirin reduced the excretion of the 2,3-dinor-thromboxane B2 metabolite--mainly of platelet origin--by 81 percent and of thromboxane B2, probably chiefly of renal origin, by 59 percent. The urinary excretion of 6-keto-prostaglandin F1 alpha and of its metabolite 2,3-dinor-6-keto-prostaglandin F1 alpha was not affected. Low doses of aspirin only partially (63 percent) reduced neonatal serum thromboxane B2. No hemorrhagic complications were observed in the newborns. Thus, in women at risk for pregnancy-induced hypertension, low doses of aspirin selectively suppressed maternal platelet thromboxane B2 while sparing vascular prostacyclin, but only partially suppressed neonatal platelet thromboxane B2, allowing hemostatic competence in the fetus and newborn.

Increased urinary excretion of thromboxane B2 and 2,3-dinor-TxB2 in cyclosporin A nephrotoxicity.

Cyclosporin A (CsA) administration to rats is associated with a selective increase in urinary excretion of immunoreactive thromboxane B2 (i-TxB2), the stable breakdown product of TxA2. The exaggerated synthesis of TxA2 may play a role in the reduction of glomerular filtration rate (GFR) observed both in animals and humans undergoing CsA treatment. The present study was designed to get further insight into the origin of the abnormal i-TxB2 urinary excretion. Rats given orally CsA (50 mg/kg/day) for 30 days had a significant increase in the urinary excretion of both 2,3-dinor-TxB2 and TxB2 measured by technique of capillary column gas chromatography-negative ion chemical ionization mass spectrometry (HRGC-NICIMS). Urinary TxB2 is more likely to reflect the renal synthesis of the parent compound, whereas 2,3-dinor-TxB2 is considered to reflect the amount of TxB2 formed in the circulation. Experiments in isolated perfused kidney (IPK) taken from animals given CsA for 30 days showed a lower percentage increase in urinary TxB2 over vehicle treated animals. Moreover in IPK the ratio 2,3-dinor-TxB2/TxB2 was lower than in vivo. The amount of i-TxB2 detectable in serum of animals given CsA was not different from that of control animals. In contrast, isolated glomeruli taken from rats given CsA had an increase in their TxA2 synthesis measured as i-TxB2 in the supernatants. Ultrastructural studies on kidney specimens from animals given CsA showed a focal glomerular endothelial damage together with a marked infiltration of blood borne cells of monocyte-macrophage type in the glomerular tuft. In contrast, kidney specimens taken from IPK preparations were devoid of inflammatory cells. In vitro CsA did not interfere with platelet arachidonic acid (AA) metabolism as shown by a normal i-TxB2 generation in vitro by rat platelet-rich plasma (PRP) exposed to CsA and then challenged with AA or ADP. Similarly isolated glomeruli and isolated proximal tubules from normal rats when challenged with CsA in vitro converted AA into TxA2 normally. It is suggested that the cause of the increased urinary excretion of 2,3-dinor-TxB2 is the consequence of intrarenal platelet and macrophage activation, probably triggered by the endothelial damage. The parallel increase in the urinary excretion of unmetabolized TxB2 is likely to reflect a concomitant activation of resident renal cell AA metabolism induced by CsA.

Antibody-mediated extraction/negative-ion chemical ionization mass spectrometric measurement of thromboxane B2 and 2,3-dinor-thromboxane B2 in human and rat urine.

An antibody-mediated extraction method for gas chromatographic-mass spectrometric analysis of thromboxane A2 (TXA2) urinary metabolites is reported. An antibody (Ab) raised against thromboxane B2 (TXB2) (35% cross-reacting with 2,3-dinor-TXB2) was coupled to CNBr-activated Sepharose 4B (Se) and used as stationary phase for simultaneous extraction of both compounds from urine. After addition of deuterium-labeled TXB2 as internal standard, rat or human urine was percolated through a small Ab-Se column. After being washed, the eluate was directly derivatized to the pentafluorobenzyl ester, methyloxime, and trimethylsilyl ether. Quantitation was performed by high-resolution gas chromatography-negative-ion chemical ionization mass spectrometry, monitoring the carboxylate anions. This method was applied to evaluate the urinary excretion of TXB2 and 2,3-dinor-TXB2 in humans and rats. We report on the excretion of 2,3-dinor-TXB2 in the rat. This novel approach to the extraction of urinary thromboxanes is more convenient than currently available methods in terms of simplicity, rapidity, and recovery. This method could be extended to any other prostanoid for which an antibody could be obtained.