ANTIMICROBIAL AGENTs AND CHEMOTHERAPY, Aug. 1979, p. 221-224 0066-4804/79/08-0221/04$2.00/0

Vol. 16, No. 2

Spectrophotometric Assay for Amikacin Using Purified Kanamycin Acetyltransferase EMANUAL SCARBROUGH, JEFFREY W. WILLIAMS AND DEXTER B. NORTHROP* School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53706 Received for publication 4 June 1979

A rapid spectrophotometric assay has been developed for measuring the concentrations of amikacin and related antibiotics in serum. The assay uses a purified enzyme from R-factor E. coli which acetylates amikacin with the production of coenzyme A, the latter in turn being reacted with a sulfhydryl reagent to produce stoichiometric amounts of a sensitive chromophore, that is measured in the visible spectrum. The system complements an earlier assay for gentamicin-related antibiotics thereby facilitating the rapid measurement of the concentrations of all clinically important aminoglycosides in serum.

We previously described a spectrophotometric assay for the gentamicin series of aminoglycosides, based on gentamicin acetyltransferase, and demonstrated its advantages over microbiological and nonspectrophotometric enzymatic assays (10). This report describes an analogous spectrophotometric assay, based on kanamycin acetyltransferase [designated AAC(6')-4 by the plasmid group nomenclature (6)]. Because of the broad substrate specificity of these two enzymes (1, 12), blood serum levels of all the clinically important aminoglycosides may be readily deternined by using these spectrophotometric methods.

of MgCl2 usually produced a normal yield of enzyme. Nucleic acids were precipitated from shock fluids by the addition of 0.75% (wt/vol) of streptomycin sulfate and removed by centrifugation at 15,000 x g for 15 min. Proteins were precipitated by ammonium sulfate precipitation of 65% saturation, stirred for 1 h, collected by centrifugation at 30,000 x g for 15 min, and dialyzed for 6 to 8 h in 10 mM Tris buffer, pH 7.8. Kanamycin acetyltransferase was purified from these proteins by affinity chromatography on a neomycin-Sepharose column. The column resin was prepared by reacting 2 g of neomycin sulfate with 35 ml of settled Sepharose, activated by the cyanogen bromide method of Cuatrecasas et al. (2). The dialyzed enzyme was applied to the column and eluted with 0.1 M NaOH (Fig. 1). Active fractions were neutralized with 2 M Tris-hydrochloride (pH 7.8) and diluted 50% MATERIALS AND METHODS with glycerol to stabilize the enzyme. The yield, recovPurified amnikacin (free base potency = 870 pg/mg) ery, and specific activity of a typical enzyme preparawas provided by Kenneth Price of Bristol Laborato- tion are illustrated in Table 1. ries. Neomycin was a gift from Marvin Gorman of Eli The procedure for the spectrophotometric assay of Lilly & Co. Veterinary grade gentamicin was a gift amikacin is a modification of the method of Benveniste from Allen Waitz of Schering Corp. Human serum was and Davies (1). Kanamycin acetyltransferase catalyzes obtained from the University Hospitals of the Univer- the formation of 6'-N-acetylamikacin accompanied by sity of Wisconsin. Tris(hydroxymethyl)aminomethane the release of CoA, the sulfhydryl group of which is (Tris), ethylenediaminetetraacetic acid (EDTA), and reacted with DTNB to produce a stoichiometric 5,5'-dithiobis'(2,2)-nitrobenzoic acid (DTNB) were amount of a disulfide and thionitrobenzoic acid (3). purchased from Sigma Chemical Co. Acetyl coenzyme The production of the latter compound is monitored A (acetyl-CoA) was from P-L Biochemicals. Genta- by measuring the increase in absorbance at 412 nm micin acetyltransferase I was prepared as described with a Gilford model 240 spectrophotometer coupled to a Leeds and Northrup recorder equipped with varpreviously (11). Escherichia coli CH-15 were obtained from Julian iable scale deflections and a multispeed chart drive. Davies and grown for 24 h at 37°C as described pre- Full-scale optical densities (OD) of 0.4 and 1.0 and viously (11), but in a modified medium containing 8 g chart speed of 1 inch (ca. 25.4 mm) per min were used. of peptone per liter (Difco Laboratories), 5 g of yeast The temperature of the cuvette compartment was extract per liter, 10 ml of glycerol per liter, 6.8 g of maintained at 25°C. Assays were carried out in cuvettes having a 1-cm KH2PO4 per liter, and 7.8 g of K2HPO per liter. Isolation of enzyme-containing protein from bacterial light path and a total reaction volume of 2.5 ml. Each cells closely followed the procedure described for gen- cuvette contained (in order of addition): 12.5 ;&mol of tamicin acetyltransferase I (11). On occasion, little or Tris-hydrochloride, 0.25 umol of EDTA, 12.5 1mol of no enzyme was released by the normal osmotic shock- (NH4)2SO4 (added together as a pre-mix, adjusted to ing procedure, but storage of the remainder of un- pH 7.8), 0.05 IU of enzyme, 1 ml of sample, 0.175 ymol shocked cells at 4 to 6°C for 24 h before the addition of acetyl-CoA, and 0.625 junol of DTNB. Samples of 221

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SCARBROUGH, WILLLAMS, AND NORTHROP

serum were prepared by adding dilutions of amikacin standards to 1 ml of normal serum and incubating the mixture at 25°C for 10 min. Control assays contained serum similarly incubated but lacking amikacin. The latter were conducted parallel to each determination and yielded significant background rates. These rates were similar to those obtained in the absence of enzyme and are presumably due to a reaction between serum and DTNB (3). Initial attempts to apply the spectrophotometric assay were unsuccessful because of the protein impurities in crude extracts which reacted with DTNB to produce background rates greatly in excess of the enzymatic rate. A partially purified preparation of kanamycin acetyltransferase is a minimal requirement for the assay; a highly purified preparation is necessary for a maximum precision. The regression analysis of the data was done by the Madison Academic Computing Center Program REAGAN 2.

RESULTS Assays conducted in the absence of serum were complete in less than 5 min, and the absorbance of thionitrobenzoate at 412 nm was stable for at least 15 min. Figure 2 shows the dependence of the absorbance upon the amount of amikacin present. The plot is linear from 0 to 60 jig of amikacin with a slope of 0.0099 ± 0.0001 OD/,ug.

ANTimICROB. AGENTS CHEMOTHIRR.

In contrast, assays conducted in the presence of serum required more than 15 min to reach completion, and the absorbance at 412 nm continued to increase after the added amikacin was depleted. Figure 3 shows the time course of the reactions in the presence and absence of serum, and illustrates the decreased rate of development of both amikacin-dependent and background absorbance. Nevertheless, Fig. 4 shows

20

30

50

H20 FIG. 2. The relationship between the absorbance ofthionitrobenzoate and amikacin in the spectrophotometric assay. Assays were performed in duplicate in the absence of serum and read after 7 mnn of reaction time. OD412, Optical density at 412 nm.

2.0

I0

AMIKACIN(_g)

0

0

a

140

pig AIKACNfWML

B-Za

a

0i6I

.2

0

*I

/c

I0.1.85

4

o

L4!i 40

s0

*_

-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~1.4 .6 -j 0.6

-

120

ISO

2m0

240

210

320

VOLME(ML)

FIG. 1. Affinity chromatography of kanamycin acetyltransferase on neomycin-Sepharose. A 340-mg amount ofprotein obtained from a dialyzed ammonium sulfate precipitate was added to a column (1 by 30 cm) equilibrated with 10 mM Tris-hydrochloride (pH 7.8). Non-specifically bound proteins were eluted with 2 M ammonium sulfate (volume of 160 to 200). Enzymatically active protein was eluted with 0.1 N NaOH (volume of 220 to 240). OLo,, Optical density at 280 nm.

/@

0.2C

0

2

4

6

10

8

TIME(min) FIG. 3. The time course of the spectropnotometric assay in the presence (@- *) and absence (----0) of serum, at different levels of amikacin. OD412, Optical density at 412 nm.

TABLE 1. Preparation summary of kanamycin acetyltransferase from 10 liters of E. coli culture Protein (mg/mi)

(U/mi)

Sp act (U/mg)

Total Ua

598 8.83 0.042 Shock fluid 11.1 30.89 1.7 Ammonium sulfate 9.0 1.2 1.08 Neomycin column a Units are defined as micromoles of product formed per minute at 250C.

0.0047 0.055 0.90

24.9 18.8 9.7

Preparative step

Vol (MI)

Enzyme

SPECTROPHOTOMETRIC ASSAY FOR AMIKACIN

VOL. 16, 1979

that the amikacin-dependent absorbance in the presence of serum is also linear from 0 to 60 ,ug of amikacin, with a slope of 0.0078 ± 0.0002 OD/

IgL

A combination of kanamycin acetyltransferase and gentamicin acetyltransferase was tested in an attempt to broaden the antibiotic specificity of a single assay system and to increase the sensitivity of the method by multiple acylations of antibiotics serving as substances for both enzymes. The two enzymes were combined both simultaneously and sequentially during assays of veterinary grade gentamicin, composed of 31% gentamicin Cla, 33% gentamicin Ci., and 36% gentamicin C2. The results are shown in Table 2. Kanamycin acetyltransferase produced approximately two-thirds of the absorbance cnange produced by gentamicin acetyltransfer0.6

90.4 _-/ 02 0

0

10

20 30 40 50 pg AMKACIN/ML SERUM

60

l'IG. 4. The relationship between the absorbance of thionitrobenzoate and amikacin in the spectrophotometric assay, conducted in the presence of serum. Assays were performed in triplicate, read after 7min of reaction time, and corrected for background reactions between serum and DTNB. OD412, Optical density at 412 nm. TABLE 2. Combination spectrophotometric enzyme assays of gentamicin complexa Enzyme Assay system 1st addition 2nd addition A B C D E

AAC(6')-4 AAC(3)-1

AAC(3)-1 AAC(6')-4

AAC(6')-4

AAC(3)-1

0.060 0.094 0.098 0.152

AAC(3)-1 +

0.103

AAC(6')-4 aAssays were performed on a 9.8-jg/ml sample of veterinary grade gentamicin, using 0.018 U/assay of AAC(6')-4 (kanamycin acetyltransferase), 0.032 U/assay of AAC(3)-1 (gentamicin acetyltransferase), or both. Second additions of enzyme were made after 5 min of reaction time with the first. OD412, Optical density at 412 nm.

223

ase (assay A versus B), consistent with the known substrate specificity of the enzymes: the former acts only on gentamicin C1I and C2, while the latter acts on all gentamicin components (4, 5). Subsequent addition of kanamycin acetyltransferase to a completed assay with gentamicin acetyltransferase produced a negligible increase in absorbance (assay C), indicating that the 3-N acetylated product is not a substrate for the 6'-N acetylating enzyme. The converse, however, produces an absorbance change equal to the sum of the individual reactions (assay D), indicating that the 6'-N acetylated product will serve as a substrate for the 3-N acetylating enzyme. Simultaneous addition of both enzymes produced an intermediate absorbance change (assay E), dependent upon the relative amounts of enzymes added.

DISCUSSION The results obtained with this spectrophotometric assay of amikacin are virtually identical with the results of a spectrophotometric assay of gentamicin described previously (10). Both assays are based on the same principle and conducted under identical conditions, but employ different enzymes to detect aminoglycoside antibiotics. Hence, the two assay systems share many characteristics and advantages, and differ only in their antibiotic specificities. Notable among these characteristics is the apparent lag in the time course of the assays in the presence of serum as compared to controls (Fig. 3). The lag does not decrease with increased amounts of enzyme or DTNB and most likely reflects a relatively slow release of bound antibiotic from serum proteins. It was suggested in the earlier report that this lag phenomenon could be exploited to provide a measurement of the dynamic functions of binding between antibiotics and serum proteins, to supplement and complement the more prevalent equilibrium binding measurements which are static functions (10). Notable among the advantages are speed, cost, precision, accuracy, and convenience. Results of antibiotic blood levels can be obtained in about 15 min versus 1 to 3 h for radioimmunoassays and radioenzymatic assays, and upwards to 24 h for microbiological assays (5, 79). The time required by the assay is very important if dosage schedules are to be adjusted to achieve optimal therapeutic levels yet avoid toxicity. The cost of conducting spectrophotometric assays appears to be significantly lower than radioactivity assays, because the high cost of radioisotopes and liquid scintillation spectrophotometry are avoided, but exact cost comparisons are difficult. The reader is referred to cost

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SCARBROUGH, WILLIAMS, AND NORTHROP

analyses of Stevens and Young (8) regarding radioactivity methods. The major expense in the spectrophotometric assay is the preparation of purified kanamycin acetyltransferase, which in our laboratory is prepared at a direct cost of $11.00 per unit. This translates to approximately $0.20 per assay for enzyme and less than $0.40 per assay in total reagent costs. The cost can be reduced up to a factor of 10 by using smaller assay volumes and self-masking semimicro cuvettes, which yield comparable results. The precision of the method is similar to the gentanicin spectrophotometric assay, as evidenced by the standard errors of the slopes of Fig. 2 and 4, which were earlier shown to be an improvement over radioenzymatic and microbiological assays (9). Because of the extreme sensitivity of radioimmunoassays, highly dilute solutions of sera and standards are required which lead to errors due to adsorption of aminoglycosides to the glass walLs of containers (4). Regarding convenience, both radioenzymatic and radioimmunoassay methods require technical skill in performing a separation step not present in a direct spectrophotometric assay. A particularly attractive feature of the enzymatic assays is the immediate applicability of the system to determine other aminoglycoside antibiotics, due to the broad specificity of the inactivating enzymes. Whereas radioimmunoassays require preparation of specific antisera for each aminoglycoside (5), use of the 3-N and 6'N acetylating enzymes can be applied to determination of the kanamycins, neomycin, butirosin, sisomicin, netilmicin, tobramycin, and ribostamycin in addition to gentamicin and amikacin (1, 12). The results of Table 2 argue against combining the enzymes simultaneously in an attempt to form a single universal system; however, using the enzymes sequentially could be exploited to double the sensitivity of determination of selected antibiotics.

ANTIMICROB. AGENTS CHEMOTHM ACKNOWLEDGMENITS

This investigation was supported by a grant from Bristol Laboratories and Public Health Service grants A111603 and GM00254 from the National Institutes of Health. J.W.W. is an Edwin Leigh Newcomb Memorial Fellow of The American Foundation for Pharmaceutical Education.

LITERATURE CITED 1. Benveniste, R., and J. Davies 1971. Enzymatic acetylation of aminoglycoside antibiotics by Escherichia coli carrying an R-factor. Biochemistry 10:1787-1796. 2. Cuatreasas, P., M. Wilchek, and C. B. Anflnsen. 1968. Selective enzyme purification by affinity chromatography. Proc. Natl. Acad. Sci. U.S.A. 61:636-643. 3. Ellman, G. L 1959. Tissue suiflfydryl groups. Arch. Biochem. Biophys. 82:70-77. 4. Josephson, L, P. Houle, and KL Haggerty. 1979. Stability of dilute solutions of gentamicin and tobramycin. Clin. Chem 25:298-300. 5. Lewis, J. E., J. C. Nelson, and H. A. Elder. 1975. Amikacin: a rapid and sensitive radioimmunoassay. Antimicrob. Agents Chemother. 7:42-45. 6. Mitsuhashi, S. 1975. Proposal for a rational nomenclature for phenotype, genotype, and aminoglycoside-aminocyclitol modifying enzymes, p. 269. In S. Mitsuhashi (ed.), Drug action and drug resistance in bacteria. University of Tokyo Press, Japan. 7. Smith, A. L, J. A. Waitz, D. H. Smith, E. M. Oden, and B. B. Emerson. 1974. Comparison of enzymatic and microbiological gentamicin assays. Antimicrob. Agents Chemother. 6:316-319. 8. Stevens, P., and L. S. Young. 1975. Rapid assay of aminoglycosides by radioenzymatic techniques, p. 6472. In D. Schlessinger (ed.), Microbiology-1975. American Society for Microbiology, Washington, D.C. 9. Stevens, P., L S. Young, and W. L Hewitt. 1976. 121radioimmunoassay of amikacin and comparison with microbioassay. J. Antibiot. 29:829-832. 10. Williams, J. W., J. S. Langer, and D. B. Northrop. 1975. A spectrophotometric assay for gentamicin. J. Antibiot. 28:982-987. 11. Williams, J. W., and D. B. Northrop. 1976. Purification and properties of gentamicin acetyltransferase . Biochem. 15:125-131. 12. Williams, J. W., and D. B. Northrop. 1978. Substrate specificity and structure-activity relationships of gentamicin acetyltransferase. I. The dependence of antibiotic resistance upon substrate Vmx/Km values. J. Biol. Chem. 253:5908-5914.

Spectrophotometric assay for amikacin using purified kanamycin acetyltransferase.

ANTIMICROBIAL AGENTs AND CHEMOTHERAPY, Aug. 1979, p. 221-224 0066-4804/79/08-0221/04$2.00/0 Vol. 16, No. 2 Spectrophotometric Assay for Amikacin Usi...
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