Skip to main content
NCBI home page
Antimicrobial Agents and Chemotherapy logoLink to Antimicrobial Agents and Chemotherapy
. 1977 Nov;12(5):597–601. doi: 10.1128/aac.12.5.597

Inhibition of Acetylating Activity in an Enzymatic Assay for Aminoglycoside Antibiotics

Barbara H Minshew 1,2,3, Susan R Swanzy 1,2,3, Fritz D Schoenknecht 1,2,3
PMCID: PMC429982  PMID: 335965

Abstract

The radioenzymatic technique using aminoglycoside-6′-acetyltransferase is suitable for determining serum concentrations of aminoglycoside antibiotics that have a 6′-amino group. Standard curves constructed for each drug in normal human serum are generally satisfactory, but we encountered a pool of human serum (designated IN pool) that inhibited the acetylating activity of the assay. Standard curves for amikacin, kanamycin, gentamicin, and tobramycin in the acetylating assay were all abnormal with standards prepared in the IN pool. Acetylating activity was also inhibited with amikacin standards prepared in a filtrate of this serum pool. Heat inactivation (56°C for 30 min) of the IN pool did not eliminate the problem. When the IN pool was diluted 1:10 in normal human serum, the standard curve obtained with amikacin was comparable to the curve with standards prepared in normal human serum. With amikacin standards prepared in a 1:2 dilution of the IN pool in normal human serum, an intermediate level of acetylating activity was observed. When this IN pool was used for the preparation of gentamicin and amikacin standards in a bioassay with Klebsiella pneumoniae ATCC 27799 as the test strain, valid standard curves and concentrations were obtained. Furthermore, no differences in the biological activity of amikacin were observed with Pseudomonas aeruginosa or Bacillus subtilis as the test strain. Excellent agreement between the microbial assay and the enzymatic assay was obtained with serum specimens tested for gentamicin (r = 0.89), tobramycin (r = 0.96), and amikacin (r = 0.96). The results obtained with the IN pool illustrate the need for regular use of check samples of known performance, independent from the standards, when determining antibiotic levels in serum.

Full text

PDF
597

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bennett J. V., Brodie J. L., Benner E. J., Kirby W. M. Simplified, accurate method for antibiotic assay of clinical specimens. Appl Microbiol. 1966 Mar;14(2):170–177. doi: 10.1128/am.14.2.170-177.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Benveniste R., Davies J. Enzymatic acetylation of aminoglycoside antibiotics by Escherichia coli carrying an R factor. Biochemistry. 1971 May 11;10(10):1787–1796. doi: 10.1021/bi00786a009. [DOI] [PubMed] [Google Scholar]
  3. Broughall J. M., Reeves D. S. Properties of the gentamicin acetyltransferase enzyme and application to the assay of aminoglycoside antibiotics. Antimicrob Agents Chemother. 1975 Aug;8(2):222–223. doi: 10.1128/aac.8.2.222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Broughall J. M., Reeves D. S. The acetyltransferase enzyme method for the assay of serum gentamicon concentrations and a comparison with other methods. J Clin Pathol. 1975 Feb;28(2):140–145. doi: 10.1136/jcp.28.2.140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Haas M. J., Davies J. Enzymatic acetylation as a means of determining serum aminoglycoside concentrations. Antimicrob Agents Chemother. 1973 Oct;4(4):497–499. doi: 10.1128/aac.4.4.497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Holmes R. K., Sanford J. P. Enzymatic assay for gentamicin and related aminoglycoside antibiotics. J Infect Dis. 1974 May;129(5):519–527. doi: 10.1093/infdis/129.5.519. [DOI] [PubMed] [Google Scholar]
  7. Lewis J. E., Nelson J. C., Elder H. A. Amikacin: a rapid and sensitive radioimmunoassay. Antimicrob Agents Chemother. 1975 Jan;7(1):42–45. doi: 10.1128/aac.7.1.42. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Lund M. E., Blazevic D. J., Matsen J. M. Rapid gentamicin bioassay using a multiple-antibiotic-resistant strain of Klebsiella pneumoniae. Antimicrob Agents Chemother. 1973 Nov;4(5):569–573. doi: 10.1128/aac.4.5.569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Minshew B. H., Holmes R. K., Baxter C. R. Comparison of a radioimmunoassay with an enzymatic assay for gentamicin. Antimicrob Agents Chemother. 1975 Jan;7(1):107–109. doi: 10.1128/aac.7.1.107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Nossal N. G., Heppel L. A. The release of enzymes by osmotic shock from Escherichia coli in exponential phase. J Biol Chem. 1966 Jul 10;241(13):3055–3062. [PubMed] [Google Scholar]
  11. Plantier J., Forrey A. W., O'Neill M. A., Blair A. D., Christopher T. G., Cutler R. E. Pharmacokinetics of amikacin in patients with normal or impaired renal function: radioenzymatic acetylation assay. J Infect Dis. 1976 Nov;134(Suppl):S323–S330. doi: 10.1093/infdis/135.supplement_2.s323. [DOI] [PubMed] [Google Scholar]
  12. Smith A. L., Smith D. H. Gentamicin:adenine mononucleotide transferase: partial purification, characterization, and use in the clinical quantitation of gentamicin. J Infect Dis. 1974 Apr;129(4):391–401. doi: 10.1093/infdis/129.4.391. [DOI] [PubMed] [Google Scholar]
  13. Stevens P., Young L. S., Hewitt W. L. Improved acetylating radioenzymatic assay of amikacin, tobramycin, and sisomicin in serum. Antimicrob Agents Chemother. 1975 Mar;7(3):374–376. doi: 10.1128/aac.7.3.374. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Stevens P., Young L. S., Hewitt W. L. Radioimmunoassay, acetylating radio-enzymatic assay, and microbioassay of gentamicin: a comparative study. J Lab Clin Med. 1975 Aug;86(2):349–359. [PubMed] [Google Scholar]
  15. Trudeau D. L., Freier E. F. Determination of calcium in urine and serum by atomic absorption spectrophotometry (AAS). Clin Chem. 1967 Feb;13(2):101–114. [PubMed] [Google Scholar]

Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES