gentamicin, netilmicin, or amikacin at two different dosages. The groups of animals assigned to gentamicin and netilmicin received doses of 10 mg/kg or 60 ...
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 1984, 0066-4804/84/060783-02$02.00/0 Copyright ©D 1984, American Society for Microbiology
p.
Vol. 25, No. 6
783-784
Improved Procedure for Extracting Aminoglycosides from Renal Cortical Tissue RUBEN A.
GIULIANO,'
GERT A. VERPOOTEN,1 DIRK E. POLLET,' LUDO VERBIST,2 SIMON L. SCHARPE,3 AND MARC E. DE BROEl* Departments of Nephrology-Hypertension' and Medical Biochemistry,3 University Hospital Antwerp, University of Antwerp, Antwerp, and Diagnostic Microbiological Laboratory, St. Rafael University Hospital, Leuven,2 Belgium Received 26 March 1984/Accepted 27 March 1984
An efficient and reproducible procedure was developed for extracting aminoglycosides from renal cortical tissue. It involves a double homogenization and two rinsings with trichloroacetic acid. A higher recovery is obtained compared with that of other previously reported methods.
and the single-step homogenization and trichloroacetic acid precipitation (4) (procedure B) were performed as previously described. In the first procedure, polypropylene containers were used, and the elution lasted for 3 months; in the latter the homogenization step was carried out in glass containers. Our methods consisted of a four-step extraction procedure in which the cortex sample was first thoroughly homogenized (with a Potter Elvehjem model Potter S, including a borosilicate glass homogenizer and a glass piston; B. Braun, Melsungen AG, Melsungen, Federal Republic of Germany) in 5 ml of a 10 mM phosphate buffer (pH 7.4). The piston speed was 1,000 rpm. The homogenate was centrifuged in a polypropylene tube at 1,200 x g for 5 min; the supernatant was collected in a polystyrene flask. The pellet was resuspended in 5 ml of buffer and transferred to the Potter S vessel for rehomogenization. The second homogenate was pooled with the previous supernatant. The homogenizer vessel was washed twice with 5 ml of buffer, each time with ca. 15 strokes. The washing liquids were added to the homogenate. Two milliliters of 50% trichloroacetic acid was added to the final homogenate, which was allowed to stand for 15 min for protein precipitation (procedure C). An alternative was tried by adding the trichloroacetic acid during the washing steps (1 ml of 50% trichloroacetic acid along with the buffer in each washing) (procedure D). After gentle agitation the homogenate was transferred to two polypropylene tubes and centrifuged at 1,200 x g for 20 min. The pellets were discarded because reextraction did not yield further antibiotic. The acid supernatant was titrated to a pH of 7.8 with 10% NaOH. The final volume was accurately measured in a plastic or chlortrimethylsilane-treated glass measuring cylinder. The aminoglycosides concentrations in all tissue homogenates and supernatants were determined by microbiological assay by using an agar well method in antibiotic medium no. 2 (Oxoid CM335) adjusted to pH 7.8 with 1 N NaOH and Bacillus subtilis ATCC 6633 as the test organism. The melted medium was cooled to 56°C; B. subtilis spore suspension (1 ml/liter; Difco 0453-36) was added, and 18-ml amounts were poured into plastic petri dishes (9 cm in diameter). After solidification, the plates were cooled at 4°C for at least 2 h. Four to six wells (4 mm in diameter) were cut out of the agar at equal distances and filled with 25 Ill of samples or standards. Appropriate standard concentrations (ranging from 64 to 0.25 ,ug/ml) of the aminoglycosides were prepared in homogenates of blank tissues for each extraction procedure. Tissue samples were diluted in their own blank homogenates. Samples were determined in duplicate and standards were determined in quadruplate. After incubation for 18 h at
The renal cortical concentration of aminoglycosides is commonly used as an index of nephrotoxicity (6, 7, 9). For this reason several rigorous procedures have been described for the extraction of these antibiotics from the kidney cortex before quantitative assay. Schentag et al. (8) reported a method based on a prolonged elution of the homogenized or minced tissue by repeated soakings with a low molarity phosphate buffer solution (pH 7.4). Only partial recovery was achieved by a single homogenization and determination of the concentration in a tissue homogenate, indicating that aminoglycosides have a strong affinity for tissue (5). Four to five soakings at 14- to 30-day intervals were required to completely recover known amounts of aminoglycosides added to tissue homogenates (8). Kornguth and Kunin (4) described a single homogenization-extraction of tissues in 3 volumes of buffer solution. They compared filtration through cheesecloth versus a trichloroacetic acid deproteinization of the homogenate. Higher values of aminoglycosides accumulated in cortex were reported when the trichloroacetic acid method was used. A substantial adsorption of these cationic antibiotics occurs when glass containers are used for homogenization (3). The extent of adsorption is dependent on factors like pH, ionic strength, duration of contact, etc. We studied whether rinsing the glass homogenizer vessel with concentrated trichloroacetic acid before deproteinization and repetitive homogenization would increase the ability to recover aminoglycosides from the renal cortex. Female Wistar rats, 230 to 250 g in weight, received gentamicin, netilmicin, or amikacin at two different dosages. The groups of animals assigned to gentamicin and netilmicin received doses of 10 mg/kg or 60 mg/kg, respectively, intraperitoneally once daily for 4 days. The groups of amikacin-treated rats received doses of 37.5 mg/kg or 225 mg/kg on the same schedule. There were seven rats in each group. The animals were sacrificed 24 h after the last injection. Th'e cortices of both kidneys were carefully removed and divided in four portions of ca. 100 to 200 mg each. Immediately after sampling, the tissues were weighed, tightly wrapped in Parafilm (American Can Co., Greenwich, Conn.) to minimize water loss, and stored at -20°C. The four cortex samples obtained from each animal were each extracted by a different method. The above-mentioned prolonged elution by repeated soakings (8) (procedure A) *
Corresponding author. 783
784
NOTES
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. Comparison of four procedures for extraction of gentamicin, netilmicin, and amikacin from rat kidney cortical tissue after in vivo administrationa Antibiotic Fg of antibiotic/g of wet tissue (mean ± SD) extracted byb:
(mg/kcg per Procedure A Procedure B Procedure C day) Gentamicin 10 110 ± 23** 162 ± 22** 155 ± 24** 60 309 ± 71** 404 ± 56** 438 62**
Netilmicin 10 111 ± 19** 60 297 ± 58**
167 ± 50* 432 ± 59*
166 38* 391 ± 39**
Procedure D 203 ± 31 560 ± 93
214 ± 66 524 ± 84
Amikacin 37.5 225
126 ± 66** 139 ± 81* 137 ± 31* 184 ± 52 453 ± 102** 869 ± 236* 856 155** 1,173 ± 155 a Procedure: A, prolonged elution by repeated soakings (8); B, single-step homogenization and trichloroacetic acid deproteinization (4); C, four-step extraction (homogenization and rinsing) and trichloroacetic acid deproteinization; D, four-step extraction (homogenization and rinsing of the homogenization vessel with trichloroacetic acid in the last two steps) and trichloroacetic acid deproteinization. b *, P < 0.01; **, P < 0.05. Paired t test between the proposed procedure D and the three other procedures.
37°C, the inhibition zones were measured twice with a Fisher-Lilly zone reader. The concentrations of aminoglycosides were calculated from the mean zone diameters according to the regression line of the respective standards. Results were expressed as amount of drug per gram of wet cortical tissue. In addition, gentamicin concentrations were determined by radioimmunoassay (Gamma Coat [12511 Gentamicin radioimmunoassay kit; Clinical Assays, Div. Travenol Laboratories, Inc., Cambridge, Mass.). The dilution of the samples and the construction of the standard curve was performed according to the instructions of the kit. The limit of sensitivity for both assays was 0.1 to 0.2 ,ug/ml of homogenate. A good correlation was observed for the gentamicin cortical concentrations measured by radioimmunoassay and by microbiological assay (r = 0.899, P < 0.01, y = 0.799 x + 0.628). A comparative recovery study between the four procedures for extraction of the three aminoglycosides from renal cortical tissue is presented in Table 1. A paired t test was used to evaluate the extraction recoveries obtained with the different techniques. The highest recoveries were obtained in all cases by procedure D. The main difference between procedure D and procedures B and C was the use of trichloroacetic acid in the last two rinsing steps. In this way, the antibiotic was not only liberated from the tissue into the protein-free supematant (4), as in procedures B and C, but its adsorption to the glass
homogenizer vessel was minimized as well (3). Aminoglycoside concentrations measured after extraction with methods B and C were not significantly different, indicating that repetitious homogenization did not enhance recovery. Extraction procedure D was further evaluated by comparing gentamicin concentrations in randomly extracted right and left kidneys from rats treated according to several experimental protocols. An excellent correlation was found between the concentrations in both kidneys of each individual animal (r = 0.979, P < 0.01, y = 1.046 x + 0.126, n = 250), and the mean coefficient of variation was 5.6%. Extraction procedure D has been applied to comparative cortical accumulation studies of different aminoglycosides in humans (1). Via its use it was possible to distinguish among cortical concentrations of gentamicin after administration of an equal daily dose according to two dosage regimens over a period of 8 days (2). We thank R. Verkerk and D. Bauwens for their expert technical assistance. LITERATURE CITED 1. De Broe, M. E., G. J. Paulus, G. A. Verpooten, R. A. Gluliano, F. Roels, and P. M. Tulkens. 1983. Early toxicity of aminoglycosides in human kidney: a prospective, comparative study of amikacin, gentamicin, netilmicine and tobramycin, p. 11-23. In K. H. Spitzy and K. Karrer (ed.), Proceedings of the 13th International Congress of Chemotherapy: side effects of antibiotics, SE 8.4/1, part 86. Vienna, Austria. 2. Giuliano, R. A., D. E. Polet, G. A. Verpooten, S. L. Scharpd, G. J. Paulus, and M. E. De Broe. 1982. Influence of dose regimen on renal accumulation of aminoglycosides. Arch. Int. Pharmacodyn. Ther. 260:277-279. 3. Josephson, R., P. Hoube, and M. Haggerty. 1979. Stability of dilute solutions of gentamicin and tobramycin. Clin. Chem. 25:298-300. 4. Kornguth, M. L., and C. M. Kunin. 1977. Distribution of gentamicin and amikacin in rabbit tissues. Antimicrob. Agents Chemother. 11:974-977. 5. Kun, C. M. 1970. Binding of antibiotics to tissue homogenates. J. Infect. Dis. 121:55-64. 6. Lult, F. C., R. Block, R. S. Sloan, M. N. Yum, R. Costello, and D. R. Maxwell. 1978. Comparative nephrotoxicity of aminoglycoside antibiotics in rat. J. Infect. Dis. 138:541-545. 7. Reiner, N. E., D. D. Bloxham, and W. L. Thompson. 1978. Nephrotoxicity of gentamicin and tobramycin given once daily or continuously in dogs. J. Antimicrob. Chemother. 4:(Suppl. A):85-101. 8. Schentag, J. J., W. J. Jusko, J. W. Vance, T. J. Cumbo, E. Abrutyn, M. DeLattre, and L. M. Gerbracht. 1977. Gentamicin disposition and tissue accumulation on multiple dosing. J. Pharmacokinet. Biopharm. 5:559-577. 9. Soberon, L., R. L. Bowman, E. Pastoriza-Munoz, and G. J. Kaloyanldes. 1979. Comparative nephrotoxicities of gentamicin, netilmicine and tobramycin in the rat. J. Pharmacol. Exp. Ther. 210:334-343.
