Binding Of Gentamicin Research Articles

A chemical method of gentamicin bonding to gelatine-sealed prosthetic vascular grafts

Our aim was to develop a new method of chemical binding of gentamicin to vascular prostheses made of poly(ethylene terephthalate) (PET) fibres and covered with pig gelatine. We estimated (with the HPLC method) the immobilization yield, which equalled 76 or 8% depending on the concentration of the antibiotic used and the amount of gentamicin bound to the prosthesis (1.08–20.6 mg/g of prosthesis). The antibiotic was coupled in two modes: stable covalent binding or weak adhesion. The results confirmed that only a small quantity of the antibiotic (1.03–3.09%) was bound by adsorption. The modification of the prosthesis surface with immobilized gentamicin was visualized with a scanning microscope (SEM). Bacteriostatic properties of bound gentamicin were verified against different concentrations (cfu) of Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus strains. We have found lack of growth of these pathogen strains in Luria–Bertani (LB) medium containing pieces of gentamicin-coupled prosthesis during at least 28 days of the experiment. Contrary to that, a control medium containing pieces of prosthesis only soaked with gentamicin allowed a constant growth of bacteria.

Gentamicin binds to the lectin site of calreticulin and inhibits its chaperone activity

Recently, it became clear that aminoglycoside antibiotics affect protein–protein interactions involving protein disulfide isomerase as well as protein synthesis in the endoplasmic reticulum. In this study, we used affinity column chromatography to screen gentamicin-binding proteins in microsomes derived from bovine kidney in order to learn about the possible mechanisms of gentamicin-associated nephrotoxicity. One of the gentamicin-binding proteins was identified as calreticulin (CRT) by N-terminal amino acid sequence analysis. Interestingly, gentamicin inhibited the chaperone and oxidative refolding activities of CRT when N-glycosylated substrates such as α1-antitrypsin and α-mannosidase were used as substrates, but it did not inhibit the chaperone activity of CRT when unglycosylated citrate synthase was used. Moreover, CRT suppressed the aggregation of deglycosylated and denatured α-mannosidase, but gentamicin did not inhibit its chaperone activity. Experiments with domain mutants suggest that the lectin site of CRT is the main target for gentamicin binding and that binding of gentamicin to this site inhibits the chaperone activity of CRT.

Gentamicin supplementation of polyvinylidenfluoride mesh materials for infection prophylaxis

Hernia repair evolved from pure tissue repair to mesh repair due to decreased recurrence rates. However, concern exists about mesh-related infections occurring even several years after initial operation. Therefore, a polyvinylidenfluoride (PVDF) mesh material was constructed and surface modified by plasma-induced graft polymerization of acrylic acid (PVDF+PAAc). Antimicrobial treatment was sought by binding of gentamicin (PVDF+PAAc+Gentamicin). In vitro efficacy and cytotoxicity was measured by agar diffusion test, L929 cytotoxicity testing and by analyzing the amount of gentamicin release from the mesh surface. In vivo biocompatibility was evaluated in 45 Sprague–Dawley rats. 7, 21 and 90 days after mesh implantation the amount of inflammatory and connective tissue as well as the percentage of proliferating (Ki67) and apoptotic cells (TUNEL) were analyzed at the perifilamentary region. Agar diffusion tests showed sufficient local antimicrobiotic effects against the bacteria tested after 24 h of incubation. No signs of cytotoxicity could be identified by L929 testing. Furthermore, surface modification did not affect the in vivo biocompatibility. At the end of the observation period, no significant differences were found for the perifilamentary amount of inflammatory cells and connective tissue and the percentage of Ki67 and TUNEL positive stained cells. The presented data confirm that an antibiotic surface modification of PVDF mesh samples is feasible. By analyzing cytotoxicity in vitro as well as biocompatibility in vivo no side effects were observed.

Gentamicin and tobramycin binding to human serum in vitro.

The binding of gentamicin and tobramycin to human serum was studied in vitro using equilibrium dialysis of pooled human serum supplemented to various concentrations of either drug. Only minimal and variable non-specific binding was noted for each drug: gentamicin, less than 15% and tobramycin, less than 30%. Conventional Scatchard analysis conducted over an array of drug concentrations failed to identify any specific binding proteins.

Targeted prevention of renal accumulation and toxicity of gentamicin by aminoglycoside binding receptor antagonists

Receptor-mediated endocytosis plays an important role in accumulation of aminoglycosides in renal proximal tubule. To prevent aminoglycoside-induced nephrotoxicity following concentrated accumulation of gentamicin in the kidney, effect of cationic proteins and their peptide fragments, which could inhibit gentamicin binding to its binding receptor(s), was investigated. Among several substrates for megalin, an endocytic receptor responsible for renal accumulation of aminoglycosides, cytochrome c potently inhibited gentamicin accumulation in renal cortex. Concentration-dependent inhibition by cytochrome c on gentamicin uptake was also observed in OK kidney epithelial cells expressing megalin. In addition, gentamicin-induced increase in urinary excretion of N-acetyl-β- d-glucosaminidase (NAG), a marker of renal tubular damage, was significantly reduced by cytochrome c. We next attempted to find a peptide fragment with lower molecular size showing inhibitory effect on gentamicin uptake. Cyto79-88 inhibited gentamicin uptake in OK cells, but had little effect on renal accumulation of gentamicin in mice in vivo. On one hand, a peptide fragment of neural Wiskott–Aldrich syndrome protein (N-WASP), which interacts with acidic phospholipids like aminoglycosides, inhibited gentamicin accumulation not only in OK cells but also in mouse kidney. These results show that substrates and/or their peptide fragments for aminoglycoside binding receptor such as megalin might be useful for preventing aminoglycoside-induced nephrotoxicity.

P1F140 - Adverse drug reaction (ADR) monitoring of aminoglycosides (AG) in rajavithi hospital

Receptor-mediated endocytosis plays an important role in accumulation of aminoglycosides in renal proximal tubule. To prevent aminoglycoside-induced nephrotoxicity following concentrated accumulation of gentamicin in the kidney, effect of cationic proteins and their peptide fragments, which could inhibit gentamicin binding to its binding receptor(s), was investigated. Among several substrates for megalin, an endocytic receptor responsible for renal accumulation of aminoglycosides, cytochrome c potently inhibited gentamicin accumulation in renal cortex. Concentration-dependent inhibition by cytochrome c on gentamicin uptake was also observed in OK kidney epithelial cells expressing megalin. In addition, gentamicin-induced increase in urinary excretion of N-acetyl-β-d-glucosaminidase (NAG), a marker of renal tubular damage, was significantly reduced by cytochrome c. We next attempted to find a peptide fragment with lower molecular size showing inhibitory effect on gentamicin uptake. Cyto79-88 inhibited gentamicin uptake in OK cells, but had little effect on renal accumulation of gentamicin in mice in vivo. On one hand, a peptide fragment of neural Wiskott–Aldrich syndrome protein (N-WASP), which interacts with acidic phospholipids like aminoglycosides, inhibited gentamicin accumulation not only in OK cells but also in mouse kidney. These results show that substrates and/or their peptide fragments for aminoglycoside binding receptor such as megalin might be useful for preventing aminoglycoside-induced nephrotoxicity.

NMR studies of iron-gentamicin complexes and the implications for aminoglycoside toxicity

Gentamicin, an aminoglycoside antibiotic with known adverse side effects to the kidney and the inner ear, forms detectable complexes with both ferric and ferrous ions. The ferrous-gentamicin species promotes lipid peroxidation and produces oxygen-derived radicals, suggestive of a possible mechanism for these toxicities. The electronic spin states of the iron-gentamicin complexes were inferred from a combination of the longitudinal relaxation times of the 1H NMR resonances (ferric complex) and an Evans experiment (ferrous and ferric complexes). The magnetic susceptibility of the Fe(III)-gentamicin sample is intermediate for high spin Fe(III), S=5/2, and low spin Fe(III), S=3/2, suggestive of the presence of more than one species in solution. For Fe(II), the electronic spin state and the binding affinity of gentamicin were calculated from NMR magnetic susceptibility data (Evans experiment) which yielded two binding constants of 0.02 M 2 and 0.2 M 2. From the dependence of the complex formation on gentamicin, a gentamicin to iron ratio of 2:1 for the low spin Fe(II) complex was determined. Based on these data, a gentamicin binding site for iron is postulated. These results explain the previously reported biphasic nature of gentamicin-dependent lipid peroxidation, suggesting that a 1:1 gentamicin:iron complex is active and a 2:1 complex is inert with respect to peroxidation.

Cellular mechanism of aminoglycoside tolerance in long-term gentamicin treatment.

In the rat, nephrotoxicity results from uptake of gentamicin at the apical membrane of proximal tubule (PT) cells. However, during continuous gentamicin treatment, the PT epithelium has been shown to recover. The mechanism(s) of cellular recovery and development of tolerance remains unknown. Therefore, we undertook studies designed to characterize cellular adaptations that occur during long-term gentamicin (LTG) treatment. After 19 days of gentamicin treatment, electron microscopy morphological evaluation revealed cellular recovery with an apparent mild decrease in height and number of microvilli. Enzymatic analysis of LTG PT membranes showed that apical and basolateral membranes had essentially returned to normal. Analysis of apical membrane lipid content revealed persistent statistically significant (P < 0.01) elevations in phosphatidylinositol (PI). In vivo immunogold morphological studies and biochemical studies in LTG rats revealed that endocytosis of gentamicin was selectively reduced, whereas the markers of fluid-phase (horseradish peroxidase) and receptor-mediated (beta2-microglobulin) endocytoses were unaffected or increased. Biochemical analysis showed that, although gentamicin binding to apical membranes isolated from LTG rats increased greater than twofold (P < 0.05) over membranes from untreated rats, in vivo cellular uptake, quantified with [3H]gentamicin, was reduced. Western blot analysis of LTG apical membranes and immunofluorescent staining of perfusion-fixed LTG kidneys showed no change in megalin levels or its apical membrane localization. These data imply that recovery of PT cells from and tolerance to LTG treatment involve a selective inhibition of gentamicin uptake across the apical membrane. They indicate that the mediators of gentamicin endocytosis were affected differently: PI levels increased, whereas megalin levels did not change. We conclude that selective inhibition of gentamicin uptake during LTG treatment is not affected by a reduction in PI or megalin levels. We postulate that trafficking of gentamicin and/or gentamicin-containing endocytic structures is reduced in LTG rats, allowing cells to develop tolerance to gentamicin.

Binding of antibiotics to glycoproteins of the vitelline and fertilization envelopes of cherry salmon eggs.

The binding of antibiotics (gentamicin, oleandomycin and chloramphenicol) to vitelline and fertilization envelopes and their extracts was investigated by immunohistochemical and immunocytochemical techniques and immunoblot analysis using mature and artificially activated eggs of the fish Oncorhynchus masou. Binding of antibiotics was detected in the vitelline and fertilization envelope outermost layers, the fertilization envelope inner surface and cortical alveolus exudates, with differences in immunoreactive intensity and deposition. The fertilization envelope outermost layer had the capacity to bind much greater amounts of the antibiotics than the vitelline envelope outermost layer. The greater capacity was caused by the deposition of cortical alveolus exudates, which were known to be responsible for functional roles of protection against bacteria, fungi and noxious materials. Treatment of the vitelline and fertilization envelopes with neuraminidase markedly reduced the binding of gentamicin and chloramphenicol but slightly increased that of oleandomycin; binding of the latter to the vitelline and fertilization envelope outermost layers was considerably reduced after treatment with alpha-fucosidase. Treatment of the two envelopes with alpha-mannosidase, beta-galactosidase or beta-D-glucosaminidase did not cause any alteration in immunoreactive intensity or number of immunoreactive deposits. Immunoblot analysis of the vitelline or fertilization envelope extracts indicated that many of the antibiotic-binding substances were glycoproteins, and several major bands were bound by all three antibiotics. These results suggest that the vitelline or fertilization envelopes may have the ability to protect the egg itself, or the embryo, respectively, by trapping antibiotics, and the trapping may be related to the presence of carbohydrate moieties, such as sialyl or fucosyl residues.

In vitro mechanisms and models of gentamicin nephrotoxicity

Nephrotoxicity is the dose limiting feature of gentamicin adminstration. The nephrotoxic potential of gentamicin was investigated in primary cultures of rat renal proximal tubular cells and in the established renal epithelial cell lines, LLC-PK 1 cells and MDCK cells. Polyaspartic acids have been reported to ameliorate the nephrotoxicity of gentamicin in vivo. However, these compounds have been reported to exert other toxic side effects. We investigated the role of magnesium- l-aspartate-hydrochloride in nephroprotection against gentamicin in these models of nephrotoxicity. Gentamicin admistration resulted in a reduction in the DNA and protein content of the primary proximal tubular cells and an impaired calcium uptake into these cells. Magnesium- l-aspartate-hydrochloride significantly reduced the extent of [ 3H]gentamicin binding to these cells. Transepithelial resistance determination in the established renal cell lines revealed that gentamicin disrupted intercellular communications, while magnesium- l-aspartate-hydrochloride abolished this gentamicin-induced alteration. The actions of gentamicin were shown to occur prior to any loss of cell viability as assessed by flow cytometry. It is concluded that these systems provide useful models for the investigation of gentamicin nephrotoxicity and that magnesium- l-aspartate-hydrochloride may be useful in ameliorating gentamicin nephrotoxicity in clinical conditions.

Mechanism of ischemia-enhanced aminoglycoside binding and uptake by proximal tubule cells.

Preceding ischemia or concurrent hypotension is known to enhance aminoglycoside nephrotoxicity; however, the underlying mechanisms responsible have not been determined. To investigate the effect of preceding mild ischemia on cellular gentamicin handling, brush-border membrane vesicle binding and in vivo cellular gentamicin uptake were quantified using [3H]gentamicin as a tracer. Fifteen minutes of ischemia resulted in a marked increase in apical membrane gentamicin binding (2.8 +/- 0.4 vs. 4.9 +/- 0.8 nmol/mg protein, P < 0.01). This increase was associated with an increased number of binding sites (3.7 +/- 0.3 vs. 9.1 +/- 2.3 nmol/mg protein, P < 0.01) and a reduced binding affinity (11.8 +/- 2.2 vs. 27.7 +/- 10.4 microM, P < 0.01). This increase in gentamicin binding was accompanied by alterations in apical membrane phospholipids including a doubling of phosphatidylinositol (PI) levels (13.8 +/- 0.4 vs. 27.5 +/- 3.1 nmol/mg protein, P < 0.01). Furthermore, treatment of apical membrane vesicles with PI-specific phospholipase C markedly reduced the difference in gentamicin binding between paired control and ischemic membrane fractions. Increased gentamicin binding was associated with increased in vivo uptake of gentamicin by S1/S2 and S3 cells. Outer cortical uptake of gentamicin increased from 2.18 +/- 0.39 to 2.68 +/- 0.27 nmol/mg protein (P < 0.01) after 15 min of ischemia and 4 h of reperfusion. Juxtamedullary uptake also increased from 1.39 +/- 0.31 to 1.75 +/- 0.12 nmol/mg protein (P < 0.01). Immunocytochemical techniques, utilizing immunogold labeling, showed gentamicin was taken up via the receptor-mediated endocytic pathway by S1/S2 and S3 cells. After ischemic injury gentamicin was localized in abnormal intracellular accumulations in S3 but not S1 or S2 cells. Taken together, these data indicate ischemia results in a marked increase in apical gentamicin binding due to increases in apical PI content. This is associated with increased internalization by S1/S2 and S3 cells and abnormal intracellular compartmentalization of gentamicin within S3 cells.

Interaction of gentamicin with the A band and B band lipopolysaccharides of Pseudomonas aeruginosa and its possible lethal effect.

The lipopolysaccharide (LPS) of Pseudomonas aeruginosa PAO1 possesses two distinct types of O polysaccharide, A and B band LPSs, but the majority of clinical isolates from cystic fibrosis patients who are infected with the organism possess only the A band as the major LPS antigen. The initial step in a series of events during the uptake of aminoglycoside antibiotics such as gentamicin is the ionic binding of the molecule to the cell surface. In an attempt to elucidate the role of A and B band LPSs of P. aeruginosa in this passive ionic binding of gentamicin to the outer membrane and its possible lethal effects, strains PAO1 (A+B+) and LPS isogenic derivatives (A+B-,A-B+,A-B-) were treated with the antibiotic. Ionic binding of gentamicin appeared to be subtly different in PAO1 and its LPS derivatives; a lethal dose of drug was bound to all strains, although the degree of binding varied with each strain. The outer membrane affinity for gentamicin was higher in strains possessing the B band than in strains with A band LPS, and these B band strains were more prone to antibiotic-induced killing. Strains with both A and B band LPSs bound the most gentamicin of all strains, and this binding caused an almost 50% loss in viability. Ionic binding of aminoglycoside antibiotucs to the outer membrane of cell surfaces must not only weaken th cell surface (R. E. W. Hancock, Annu. Rev. Microbiol. 38:237-264, 1984; N. L. Martin and T. J. Beveridge, Antimicrob. Agents Chemother. 29:1079-1087, 1986; S. G. Walker and T. J. Beveridge, Can. J. Microbiol. 34:12-18, 1988) but it must also be more important in cell death than was originally thought.

A new model of systemic drug rescue based on permeability characteristics of the blood-brain barrier in intracerebral abscess-bearing rats

✓The authors report the results of their investigation of a systemic drug rescue method using antibody to a therapeutic drug based on the differential permeability of the blood-brain barrier to low- and high-molecular-weight compounds. Rats bearing intracerebral abscesses initially received systemic [125I]-gentamicin (Mr 462) followed 60 minutes later by specific gentamicin antiserum (immune group) or normal rabbit serum (nonimmune group). Animals receiving antigentamicin immunoglobulin G (IgG, Mr 150,000) demonstrated 90% binding of serum gentamicin. Immunoprecipitation of serum samples with protein A-Sepharose demonstrated that the increased binding of gentamicin in immune animals was due to antigen binding with antigentamicin IgG. By contrast, the percent of gentamicin bound to antibody within the brain did not differ between immune and nonimmune groups, implying that high-molecular-weight IgG was excluded from the brain and brain lesion. Significant differences in gentamicin deliveries were noted in the abscess, brain around the abscess, brain distant from the abscess, and normal brain areas. In contrast, analysis of comparable brain areas demonstrated no significant differences in gentamicin delivery between immune and nonimmune animals, indicating that the systemic presence of gentamicin antibody did not alter central nervous system delivery of unbound drug. These findings suggest the possibility of a drug rescue method using antidrug IgG, based on the differential permeability of the blood-brain barrier to drug versus antibody. While the present work was developed in a brain abscess model, the drug rescue method may also be applicable in the management of intracerebral tumors.

Pharmacokinetics of gentamicin in newborn to 30-day-old foals

SUMMARY Gentamicin sulfate, equivalent to 4 mg of gentamicin base/kg of body weight, was administered iv to 6 Thoroughbred foals on day 1 (12 to 24 hours of age) and at 5, 10, 15, and 30 days after birth. On day 40 after parturition, gentamicin was given to the mares at a dosage similar to that used in foals. Decay of serum gentamicin concentrations was best described by a 2-compartment model. Among foals, the overall elimination rate constant at 30 days of age was significantly (P &lt; 0.05) greater than at days 1, 10, and 15. There was, however, no difference in the overall elimination rate constant between foals and mares. The volume of distribution (Vd), determined on the basis of total area under the disposition curve, did not change between day 1 and day 30. Mean values of Vd of foals were between 1.5 and 2.5 times higher than the mean Vd of the mares; however, only values from the foals at days 5 and 10 were significantly greater. Both age and interindividual differences were reflected in the total body clearance (ClB) of gentamicin. Total body clearance of gentamicin of foals on day 1 was less than that of foals on days 5, 10, and 30. Additionally, ClB of gentamicin on day 15 was less than that on day 30. There was no significant difference between ClB of foals and mares except for the day-30 group, which had a higher clearance rate than did the adults. Protein binding of gentamicin was &lt; 30% in all groups, and there were no apparent age-related differences.

Intracellular distribution of gentamicin within the rat kidney cortex: A cell fractionation study

The present study demonstrates that during the first 1.5–3 min after a single intraperitoneal administration of [ 3H]gentamicin to rats, most of the radioactivity in the kidney cortex is recovered in the cytosolic and microsomal fractions upon subcellular fractionation. Subsequently, the level of radioactivity recovered in the cytosolic fraction decreases markedly, whereas this level remains relatively unchanged in microsomes and increases somewhat in the nuclear and mitochondrial fractions. A steady state is apparently reached 13 hr after the injection. The high initial concentration of gentamicin in the cytosol may indicate that this substance is taken up to a large extent by diffusion. Such uptake is somewhat surprising, because of the polar nature of gentamicin. The small size of this drug may, however, allow it to diffuse through so-called pores and/or interaction with negatively charged phospholipids may be involved in the uptake of gentamicin. The initial total level of radioactivity recovered in microsomes after in vivo administration of [ 3H]gentamicin was considerably higher than in the nuclear and mitochondrial-lysosomal fractions. Furthermore, when gentamicin was added directly to kidney homogenate prepared from untreated rats, instead of being administered in vivo, this substance was still recovered in highest amounts in the total microsomal fraction. This observation may indicate that enrichment of gentamicin in the endoplasmic reticulum (or fragments thereof) reflects a special affinity of this drug for these membranes and is probably not the result of a particular in vivo process. There was no difference in the levels of radioactivity recovered in smooth and rough microsomes. In our experiments the greatest enrichment of [ 3H]gentamicin after in vivo administration for 3 days was seen in isolated myelin bodies, an observation which may be relevant to the mechanism by which these structures are formed. For instance, binding of gentamicin to specific phospholipids and/or membranes may prevent their breakdown and, thus, lead to the accumulation of membrane aggregates.

Pharmacokinetics and plasma protein binding of gentamicin in Bubalus bubalis calves

Pharmacokinetics and plasma protein binding of gentamicin in <i>Bubalus bubalis</i> calves

Influence of Membrane Surface Potential and of Net Charge on Aminoglycoside Binding to the Organ of Corti of Guinea Pigs

Gentamicin binding to homogenates of the organ of Corti of guinea pigs was investigated by altering the magnitude of the membrane surface potential and by testing the potencies of various polyamines and polyamino acids with different numbers of amino groups and different net charge to inhibit or displace bound gentamicin. Diminishing or increasing the ionic strength of the milieu resulted in an increase and a decrease in drug binding, respectively. This observation may be accounted for by the negativity of the membrane surface potential which was enhanced or reduced, when the concentration of cations in the milieu was decreased or increased, respectively. Polyamines and polyamino acids displaced bound gentamicin and inhibited gentamicin binding as a function of the number of amino groups present within the molecule and of their cationic nature. These results point out the importance of considering the surface potential of biological membranes and the net positive charge of the drug in estimating its affinity for the binding site.

Pharmacokinetic properties of intramuscular gentamicin in Chinese subjects

The pharmacokinetics and relative bioavailability of two different formulated injectable gentamicin products were examined i.m. in 12 healthy young Chinese men at intervals of 1 week in a randomized cross-over design. Serum concentrations of gentamicin were measured by a fluorescence polarization immunoassay (FPIA) method sensitive to 0.01 μg/ml. The urine concentration of gentamicin was measured by the USP bioassay method. The serum protein binding of gentamicin was determined by an ultracentrifuge method and the free drug concentration was also determined by FPIA. No differences were found in bioavailability between gentamicin products of two manufacturers (Schering and Veterans). However, longer half-life, larger volume of distribution, slower distribution rate and much higher percentage of gentamicin excreted in the urine were observed in Chinese subjects than in Caucasians. Despite the longer half-life which was observed in Chinese, the total body clearance of the antibiotic in Chinese was about the same as that of Caucasians, therefore, the same gentamicin dosage may be needed for Chinese to achieve the desired serum level.

Penetration of antibiotics into the surgical wound in a canine model.

The dose and timing of antimicrobial agents given for surgical wound prophylaxis should be based on the concentration-time profile of the drug in tissue at the site of contamination. However, concentrations of antimicrobial agents in surgical wounds are difficult to determine accurately. Since a surgical wound is a unique extravascular compartment with increased vascular permeability and a high surface area/volume ratio, antibiotic concentrations in sera and surgical wounds should be similar. To test this hypothesis, the pharmacokinetics of single intravenous doses of cefazolin (40 mg/kg) and gentamicin (4 mg/kg) in sera and surgical wounds in a clinically relevant surgical model using dogs were compared. Drug concentrations were determined in interstitial fluid in muscle biopsies taken randomly from wound surfaces and serial wound fluid samples collected after the incisions were closed. Protein binding of cefazolin and gentamicin in sera and wound fluids was low (less than or equal to 29 +/- 9%) in this canine model. Cefazolin and gentamicin equilibrated rapidly (less than or equal to 30 min) between serum and the surgical wound, and concentrations in the two sites declined in parallel. Values for the area under the concentration-time curve, mean residence time, and terminal half-life in serum and the surgical site for each drug were similar. Cefazolin concentrations in serum underestimated the time during which concentrations in surgical wounds exceeded the susceptibility breakpoint MIC for important pathogens by an average of 58 min (range, 26 to 109 min; P = 0.036); for gentamicin, the underestimation averaged 30 min (range, 10 to 60 min; P = 0.036). These data support the concept that the concentration-time profiles of antimicrobial agents in serum may prove valuable clinically as guides to determining the and timing of antibiotic administration necessary for effective antimicrobial prophylaxis in surgery. Further studies are needed to determine the surgical wound pharmacokinetics of highly protein-bound antibodies.

Aminoglycoside binding sites in the inner ears of guinea pigs.

With [125I]gentamicin as radioligand, the presence of aminoglycoside binding sites and kinetics of gentamicin binding to homogenates of organs of Corti, vestibular maculae, livers, spleens, and hearts of guinea pigs were investigated. The effects of temperature, osmolarity, 2,4-dinitrophenol, and digitonin on gentamicin binding were assessed. The affinities of several aminoglycosides for binding sites were tested. Gentamicin bound to cochlear and vestibular structures in a rapid and saturable fashion at a single class of noninteracting binding sites with Kds of 1.2.10(-6) and 3.10(-7) M and maximal binding capacities of 1.3 nmol and 43 pmol/mg of protein, respectively. In the liver, spleen, and heart, binding remained low and appeared to be nonspecific. In the organ of Corti, gentamicin uptake was unaffected by alterations in temperature or medium osmolarity or by 2,4-dinitrophenol, indicating that the uptake represented binding and not active transport. Digitonin at 10 nM increased markedly the uptake at 37 and 4 degrees C, suggesting the presence of internal binding sites. Various aminoglycosides compete for a common binding site.