Pyoverdin-facilitated iron uptake in Pseudomonas aeruginosa: immunological characterization of the ferripyoverdin receptor.

A purified polyclonal antiserum directed against the isolated main 80 kD IROMP (iron-regulated outer-membrane protein) from Pseudomonas aeruginosa PAO1 detected only the 80 kD polypeptide of outer-membrane proteins from PAO1 cells grown in iron deficiency in Western blots. It was also shown to inhibit the uptake of 59Fe pyoverdin by PAO1 cells as well as its binding to purified outer membranes. Immunofluorescence experiments with intact PAO1 cells confirmed that the receptor is present only at the surface of cells grown under conditions of iron deficiency. All these data allow us to conclude that the 80 kD main IROMP of P. aeruginosa is indeed the receptor for the siderophore ferripyoverdin.

Evidence for different pyoverdine-mediated iron uptake systems among Pseudomonas aeruginosa strains.

Fourteen strains of Pseudomonas aeruginosa (P. aeruginosa ATCC 15692, P. aeruginosa ATCC 27853, and 12 clinical isolates) were checked for the production of pyoverdine and for pyoverdine-mediated iron uptake. Under iron restriction, two isolates produced undetectable amounts of pyoverdine, but all the other strains produced a compound with physicochemical properties identical or close to those of the pyoverdine of P. aeruginosa ATCC 15692 (strain PAO1). The pyoverdines were purified and tested for their growth-promoting activity and for their ability to facilitate 59Fe uptake in homologous experiments involving each pyoverdine and its producing strain, as well as in heterologous systems involving all the other strains. The results of both types of experiments suggested the existence of three specificity groups. This was confirmed by analysis of the amino acid composition of the pyoverdines, which differed for each group. A partially purified polyclonal antiserum raised against a major 80-kilodalton (kDa) iron-regulated outer membrane protein (IROMP) of P. aeruginosa PAO1 recognized the 80-kDa IROMPs from P. aeruginosa PAO1 and the clinical isolates belonging to the same group, whereas the IROMPs from the strains belonging to the two other groups were not detected. A second antiserum raised against the P. aeruginosa ATCC 27853 80-kDa IROMP gave similar results by reacting specifically with the 80-kDa IROMP from the strains belonging to this group. Thus, together with the already known pyoverdine from P. aeruginosa PAO1, two new types of pyoverdines produced by strains belonging to this species were characterized.

Cepabactin from Pseudomonas cepacia, a new type of siderophore.

In iron-deficient conditions of growth Pseudomonas cepacia ATCC 25416 excreted both pyochelin and a low-molecular-mass compound which strongly chelated iron(III), and facilitated iron translocation as demonstrated by growth and uptake experiments. The name cepabactin is proposed for this new siderophore. Comparisons of UV-visible spectra and chromatographic behaviour, together with 1H-NMR spectra, led to the conclusion that cepabactin is 1-hydroxy-5-methoxy-6-methyl-2(1H)-pyridinone, a compound which can be considered as a cyclic hydroxamate, but also as a heterocyclic analogue of catechol. This pyridinone has already been described by other workers as an antibiotic produced by Pseudomonas alcaligenes, and by a soil isolate closely related to Pseudomonas cepacia. Thus, cepabactin appears to act as a siderophore for more than one species of non-fluorescent pseudomonad.

Specificity of pyoverdine-mediated iron uptake among fluorescent Pseudomonas strains.

Pyoverdine-mediated iron transport was determined for seven fluorescent Pseudomonas strains belonging to different species. For all strains, cell or cell outer membrane and iron(III)-pyoverdine combinations were compared with their homologous counterparts in uptake, binding, and cross-feeding experiments. For four strains (Pseudomonas putida ATCC 12633, Pseudomonas fluorescens W, P. fluorescens ATCC 17400, and Pseudomonas tolaasii NCPPB 2192), the pyoverdine-mediated iron transport appeared to be strictly strain specific; pyoverdine-facilitated iron uptake by iron-starved cells and binding of ferripyoverdine to the purified outer membranes of such cells were efficient only in the case of the homologous systems. Cross-feeding assays, in liquid or solid cultures, resulted, however, especially for P. fluorescens ATCC 17400, in some discrepancies compared with uptake and binding assays, suggesting that growth experiments are the least likely to yield correct information on specificity of the pyoverdine-mediated iron transport. For the three other strains (P. fluorescens ATCC 13525, P. chlororaphis ATCC 9446, and P. aeruginosa ATCC 15692), cross-reactivity was demonstrated by the uptake, binding, and cross-feeding experiments. In an attempt to determine which parts of the iron transport system were responsible for the specificity, the differences in amino acid composition of the pyoverdines, together with the differences observed at the level of the iron-sensitive outer membrane protein pattern of the seven strains, are discussed.

Unconjugated estriol in serum as measured by liquid chromatography with electrochemical detection, compared with radioimmunoassay.

Concentrations of unconjugated estriol in maternal serum or plasma increase throughout pregnancy, particularly during the third trimester. We present and discuss the results of a comparative study of unconjugated estriol as measured by a new "high-performance" liquid-chromatographic assay and by a conventional semiautomated radioimmunoassay procedure. In the new method, serum is extracted and chromatographed on a reversed-phase mu Bondapak-C8 column under radial compression. The estriol peak is detected with a glassy-carbon electrochemical detector. The chromatographic results (y) correlated well with the RIA results (x) for 105 samples from 45 pregnant women in their third trimester (y = 1.07x - 0.55 micrograms/L, r = 0.933), with no significant difference between the means of the two methods.

Decreased ionized calcium during therapeutic plasma exchange pheresis and platelet pheresis.

Marked reductions in ionized calcium were observed in normal donors undergoing platelet pheresis using acid-citrate dextrose formula A (ACD)-A) anticoagulant, and in patients undergoing therapeutic plasma exchange pheresis using citrate phosphate dextrose (CPD)-ABO compatible plasma. Donors showed a greater tendency to symptoms of hypocalcemia, although patients receiving CPD plasma demonstrated lower values of ionized calcium. It is suggested that there may be important differences in the availability of calcium from plasma containing these two anticoagulants.

Effect of calcium and antacids on phenytoin bioavailability.

The effects of bihourly administration of gluconate calcium and tow magnesium-aluminum-containing acids on the bioavailability of a single capsule dose of phenytoin sodium were determined in two normal volunteers. Neither the rate nor the extent of phenytoin absorption were altered by the treatments, in spite of indirect evidence from other studies that might suggest such an interaction. No interaction between calcium or magnesium and either unionized or anionic phenytoin could be demonstrated in vitro using solubility and spectral techniques.

Liquid-chromatographic determination of acetaminophen in serum.

We describe a sensitive and precise "high-pressure" liquid-chromatographic method for determining acetaminophen in serum. The 2-acetaminophenol and 3-acetaminophenol structural isomers of acetaminophen are used as internal standards. The method, which involves solvent extraction and adsorption chromatography on silica, provides excellent sensitivity, accuracy, and selectivity. The standard curve is linear over the range of acetaminophen concentrations of 0.5 to 300 mg/L, which makes the method useful for both pharmacokinetic studies and overdose monitoring. Analytical recovery is 97% for acetaminophen concentrations ranging from 5 to 300 mg/L. Many commonly used drugs were tested and found not to interfere. The procedure has been successfully adapted as a microscale method requiring only 50 microL of sample. The microscale method is particularly useful for pediatric and neonatal patients for whom sample size is a major concern.

Peritoneal dialysis in rabbits. A study of transperitoneal theophylline flux and peritoneal permeability.

Since vasodilators can restore toward normal the decreased peritoneal clearances associated with vascular disease, the influence of aminophylline on peritoneal solute transport was studied in unanesthetized rabbits. Mean control creatinine clearance was 0.56 ml/kg/min and urea clearance 0.80 ml/kg/min. Neither intraperitoneal nor intravenous aminophylline increased peritoneal clearances, nor did the ratio creatinine clearance/urea clearance change from the control value, 0.70. Bidirectional flux of theophylline occurred at clearances of 0.70 ml/kg/min efflux and 0.64 ml/min influx. The removal rate of theophylline was 0.05% min, allowing therapeutic removal of excess aminophylline and warranting supplemental therapy during dialysis if therapeutic theophylline concentrations are required. As the intraperitoneal aminophylline was well tolerated, this route can be considered for therapeutic administration.

Effect of intraperitoneal diuretics on solute transport during hypertonic dialysis.

Hypertonic periotoneal dialysis in New Zealand white rabbits results in increased dialyzate volume, but the sodium content of net ultrafiltrate is 109.5 MEq/l, less than extracellular fluid sodium concentration. With intraperitoneal furosemide, mean net ultrafilrate sodium concentration increased significantly to 121.2 mE1/l while ethacrynic acid had no such effect and both drugs affected dialyzate volume very slightly. Hypertonic peritoneal dialysis increased urea clearance significantly above isotonic dialysis and the addition of ethacrynic acid increased clearances further (P LESS THAN.02). Added furosemide decreased urea clearances suggesting that the effect on sodium transport is not an overall permeability change. During isotonic peritoneal dialysis, furosemide increased peritoneal permeability, i.e. urea and creatinine clearances, but a significantly higher urea clearance resulted from intraperitoneal ethacrynic acid. Furosemide influx clearance average 0.31 ml/kg/min, a mean of 27 percent of the urea clearance. The data suggest that furosemide may be useful to prevent the hypernatremia that may complicate hypertonic peritoneal dialysis, but is not as efficacious as other vasoactive drugs in enhancing peritoneal permeability.

Isoproterenol enhancement of peritoneal permeability.

As peritoneal dialysis is inefficient enouth to be time-consuming and sometimes clinically ineffective, we have evaluated pharmacologic enhancement of peritoneal permeability. Peritoneal dialyses were performed in New Zealand white rabbits by instillation of 50 ml/Kg of isotonic dialysis solution of standard composition. Mean peritoneal clearance of creatinine was 0.60 ml/Kg/min and urea was 0.80 ml/Kg/min, each decreasing as intraperitoneal dwell was prolonged (by .011 ml/Kg/min or less). With 0.04 micrometer/Kg of isoproterenol administered intraperitoneally, clearances increased to 0.91 and 1.30 ml/Kg/min (p less than 0.01). When isoproterenol was added to the dialysis solution one hour or more before instillation, the increment in clearances was less. Instillation of dialysis solution 24 hours after addition of a higher dose of isoproterenol (0.2 micrometer/Kg) did not increase clearances above control. No effect of isoproterenol on bulk flow of water, associated with the osmotic effect of dextrose, was demonstrated. As peritoneal clearances increased, the ratio creatinine clearance: urea clearance did not decrease, consistent with increased peritoneal permeability as well as blood flow.

Modified method for analysis of serum iron.

An improved method for analysis of serum iron is described which is simple, rapid, precise and convenient for routine use in clinical laboratories. Serum proteins are precipitated with trichloroacetic acid-hydrochloric acid solution, with simultaneous release of Fe(III) from transferrin. Fe(III) is reduced to Fe(II) by sodium ascorbate, and Fe(II) is reacted with ferrozine to form a lavender complex, which is measured by spectrophotometry at 562 nm. Measurements of iron in 183 serum samples by this method were compared with measurements by a "direct" spectrophotometric method without without deproteinization, as previously described. Close agreement was obtained in 171 of these 183 pairs of analyses (93.5 percent). Discrepancies (greater than 12 mug per dl) were noted in the remaining 12 serums, which were attributed to interference in direct spectrophotometric analyses of iron, owing to (1) hemolysis, (2) lipemia, (3) jaundice, (4) protracted storage or (5) repeated freezing and thawing of the serums.