E. Sinagowitz, K. Pelz, A. Burgert, W. Kaczkowski

Concentrations of Cefazolin in Human Skeletal Muscle Summary: Tissue concentrations of cefazolin were measured in 18 patients undergoing urological operations. Specimens of skeletal muscle and blood were taken simultaneously at predetermined intervals. The concentrations in serum and homogenized skeletal muscle were determined by means of the agar well diffusion method. A comparison of standard curves obtained with phosphate buffer solution and the supernatant of muscle homogenate revealed no substantial binding of cefazolin to the supernatant of muscle homogenate. Because of methodological difficulties in determining the different compartments of a tissue specimen concentrations in tissue were not corrected. Within two hours after a short infusion (25 min.) of 2 g of Cefazolin in muscle tissue a peak concentration of 20 ~g/g was reached. A tissue level above 10 %g/g was maintained for three hours. This concentration is high enough to inhibit nearly all strains of E. coli, Klebsiella, Salmonella, Shigella arid a large part of Proteus mirabilis, whereas nearly all gram-positive bacteria are inhibited at a lower concentration.

Zusammen/assung: Konzentrationen von Cefazolin in der menschlichen Skelettmuskulatur. Bei 18 urologischen Patienten wurden intraoperativ Gewebekonzentrationen von Cefazolin (CEZ) bestimmt. Proben von Skelettmuskulatur u n d Blur wurden gleichzeitig in vorbestimmten Intervallen entnommen. Die Konzentration yon C E Z im Serum und Muskelhomogenat wurde biologisch mit dem Agar-Diffusionstest bestimmt. Ein Vergleich der Standardkurven mit PBS u n d Oberstand von antibiotikafreiem Muskelhomogenat zeigte, dab fiir C E Z keine wesentliche Bindung an das Muskelhomogenat nachzuweisen ist. Da die verschiedenen Gewebekompartimente in einer einzelnen Gewebeprobe noch nicht genau erfagt werden k6nnen, wurde auf die Korrektur der gemessenen Gewebekonzentrationen verzichtet. 2 Stunden nach Beginn einer Kurzinfusion (25 Min.) von 2 g CEZ wurde in der Muskulatur ein Spitzenspiegel von 20 #g/g erreicht. Eine Gewebekonzentration fiber 10 #g/g wurde ffir 3 Stunden aufrechterhalten. Diese Konzentration ist hoch genug, u m fast alle StSmme von E. coli, Klebsiella, Salmonella, Shigella und einen groBen Tell yon Proteus mirabilis therapeutisch zu erfassen, wghrend grampositive Bakterien schon bei niedrigeren Konzentrationen erreicht werden.

Introduction

specimens were dipped with a swab to remove additionally adhering blood from the surrounding tissue. Muscle was homogenized with phosphate buffer saline (PBS) in a 1 : 1 ratio by means of an homogenator (Ultra-Turrax, IKA-Werk, Staufen, GFR). The homogenate was centrifuged with 13000 g for 20 min. To obtain measureable concentrations the supernatant of the muscle homogenate was diluted with phosphate buffer saline, and the test serum was diluted with serum obtained from the same patient before application of cefazolin. T h e concentrations were determined biologically by means of the agar well diffusion method according to Klein (7) in the modification as described by V6mel a. Hoffmann (22). The medium was Bacto Antibiotic Medium 1 (Penassay Seed Agar from Difco) inoculated at a temperature of 70 ° C with a spore suspension of Bacillus subtilis A T C C 6633 (dilution 1 : 1000). After an incubation time of 2I hours at 37 ° C the diameters of the inhibiting zones were measured by means of vernier callipers by two persons in order to avoid reading errors. Finally the concentrations were determined graphically using the standard curves obtained in the same assay. F o r each serum sample and tissue specimen the assay was done twice. In addition, methodological experiments were carried out: a. Standard curve for tissue measurements: Poolserum, PBS, and skeletal muscle (homogenized as described) from five patients free of antibiotics were compounded with predetermined amounts of CEZ. The diameters of the inhibiting zones, representing the antimicrobial activity of C E Z in the three fluids, were determined by the agar well diffusion method, as described.

C e f a z o i i n ( C E Z ) is k n o w n as a c e p h a l o s p o r i n C derivate with a wide r a n g e of activity against gram-positive a n d g r a m - n e g a t i v e m i c r o o r g a n i s m s . As c o m p a r e d to cephalotin its activity against E. coli, Klebsiella, a n d S a l m o n e l l a is h i g h e r (5, 8, 14, 15). A n e l i m i n a t i o n h a l f - t i m e of a b o u t t w o h o u r s p r o v i d e s h i g h s e r u m levels a n d t h e r e f o r e allows longer dosage intervals as c o m p a r e d with clinically used c e p h a l o s p o r i n e s (1.4, 16). It is assumed t h a t the high protein binding rate of 7 0 - - 8 6 % (14, 16) leads to lower cefazolin levels in p l a s m a water, the latter being considered the only effective f r a c t i o n (1, 18). H o w e v e r , in order to evaluate t h e antimicrobial activity of cefazolin at the site of infection we m e a s u r e d its c o n c e n t r a t i o n in h m n a n skeletal muscle, a n d d e t e r m i n e d t i m e - d e p e n d e n t changes in tissue levels. M o r e o v e r , the influence of a) a suitable solv e n t used in p r e p a r i n g the s t a n d a r d curve, a n d b) the b l o o d c o n t e n t of the muscle s p e c i m e n o n the tissue c o n c e n t r a t i o n of C E Z was tested in m e t h o d o l o g i c a l experiments.

Material and Methods 18 patients undergoing urological operations were given CEZ in different modes of application: four patients received I g C E Z by i. v. injection, three patients received 2 g C E Z in a 2 hour infusion, and 11 patients received 2 g C E Z in a short infusion of 25 rain. Blood samples and muscle tissue specimens were taken simultaneously at predetermined intervals of 30 minutes. Only muscle tissue which macroscopically looked well perfused was removed in small pieces from the operation wound and the psoas muscle. The average weight was 1 to 3 g. All muscle

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Received: 30 July 1975 Dr. E. Sinagowitz, A. Burgert, W. Kaczkowski, Urologische Klinik im Zentrum ffir Chirurgie, Universitiit Freiburg, Hugstetterstral3e 55, D-7800 Freiburg; Dr. K. Pelz, Zentrum ffir Hygiene, Universit~it Freiburg, Hermann-Herder-Strage 11, D-7800 Freiburg.

E. Sinagowitz, K. Pelz, A. Burgert/ W. Kaczkowski: Concentrations of Cefazolin in Human Skeletal Muscle

b. Blood content of tissue specimen: In four patients the hemoglobin concentrations in the blood and in the supernatant of the homogenate were determined according to the hemoglobin-cyanide method. The hemoglobin content in the blood was corrected for serum by using the venous hematocrit, so that the tissue: serum ratio of hemoglobin could be determined. Thus the relative serum content in the muscle specimen could be evaluated.

Results The first two figures show the results of the methodological experiments. Figure 1 show the comparison of standard curves obtained with different solvents. The curves obtained with the supernatant of muscle homogenate and phosphate buffer saline correspond closely. However, the position of the serum curve indicates that higher tissue concentrations would be determined by using it than actu,ally exist. Thus the values of tissue concentration would be falsely elevated by 150 to 2000/0. Further, it is demonstrated that there seems to be no significant binding of CEZ to proteins in the muscle homogenate. .......... serum . . . . muscle

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(SEM). Figure 2 shows the tissue: serum ratio of hemoglobin and CEZ in one representative patient. Whereas the relative hemoglobin content remained nearly stable in a range from 4--7°/0, the tissue: serum ratio of cefazolin showed marked differences with a peak of 4 5 % at 2 hours. Due to the mode of application different concentrations of CEZ in serum and tissue were measured. In four patients given an i. v. injection of 1 g CEZ the concentration of CEZ in serum was found after 120 min to range from 23.7 to 53.8 pg/mt. At the same time the tissue concentration ranged from 5.7 to 17.2 ,ug/g. After 6 hours the serum concentration ranged from 5.7 to 30.0/~g/ml. Three patients received 2 g CEZ in an infusion time of 120 min. At this time the serum concentration was between 92 and 114 Ng/ml. After 6 hours it ranged from 23.8 to 35.2 /~g/ml. After 3 hours a maximal tissue concentration of 22 pg/g was measured. 11 patients were given 2 g CEZ in a short infusion of 25 minutes. The serum level was 171 ktg/ml _+ 17.5 (SEM) 30 minutes after start of the infusion (Figure 3). It decreased in 6 hours to 22.5 #g/ml _+ 2.6. The highest concentration in muscle was reached after 2 hours with 20.0/~g/g _+ 4.8, after 3 hours it was still 9.6 +_ 2.4.

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Figure 1: Standard curves o[ ce~azolin determined with human pool serum, supernatant o[ homogenized human skeletal muscle and phosphate bu[[er solution.

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Figure 3: Concentrations o[ C E Z in serum and skeletal muscl e a/ter a short in[usion of 2 g C E Z /or a 25 minutes period (11 patients, ,7 + SEM).

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Figure 2: The tissue : serum ratio o[ hemoglobin and ce[azolin in human skeletal muscle (a representative patient). The mean hemoglobin content of the muscle specimen was 1.53 g/100 ml + 0.075 (SEM). The mean tissue: serum ratio of hemoglobin in muscle was 6.3°/0 + 0.3

Among the literature several studies on the pharmacokinetics of CEZ in humans are available (1, 11, 14, 19, 22). But with the exception of one paper (19) only serum levels were determined. These investigations were carried out in healthy volunteers, and with different modes of application. Thus they are not exactly comparable with our results, which were obtained in patients under operation, which together with anesthesia can cause impaired renal function (4, ]2). Our investigations on serum and tissue levels after application of 1 and 2 g CEZ by an i.v. injection and by a 2 hour infusion respectively should be regarded

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E. Sinagowitz, K. Pelz, A. Burgert, W. Kaczkowski: Concentrations of Cefazolin in Human Skeletal Muscle

only as orientating studies. Because of the small number of measurements under these conditions calculations of mean values would not be correct. However, the range of our values correspond to those of the cited literature. Since no marked differences in the serum and tissue levels were observed we finally chose the short infusion of 2 g. Another important pragmatic reason for this preference was that a short infusion may be more easily supervised than a 2 hour infusion, especially in the case when an infusion pump is not available. Most bacterial infections take place in the interstitial space. Thus it is of importance to obtain and maintain a high concentration of antimicrobial agents at the site of infection. Different attempts have been made to determine concentrations of antibiotics in tissue. C h i s h o l m et al. (3) regarded the fluid collected in "tissue cages" as interstitial fluid. Tan et al. (21) used a skin window for sampling interstitial fluid, and S i m o n et at. (19) used the fluid in an artificial skin blister for evaluation of tissue concentrations. But as yet it is not quite clear to what extent these fluids really represent interstitial fluid. A more reliable method seems to be to measure the concentration of an antimicrobial agent in the lymph as done by N a b e r et al. (13) (renal lymph of the dog). However, preparing and cannulating tiny lymph vessels in human patients seem to prolong the time of operation to an extent which presumably could endanger the patient. Thus, because of the technical difficulties in sampling human lymph and the questionable equation of the fluid in arteficial spaces with the interstitial fluid, we regard determinations of tissue concentrations of antimicrobial agents at least in homogenates of human muscle to be helpful evaluating its antimicrobial efficacy. Further, measurements of concentrations in muscle homogenates are simple, and muscle can be repeatedly obtained during operation in order to make possible the determination of a time course of tissue concentrations. However, when measuring concentrations in tissue homogenates possible methodological errors must be considered. In accordance with the necessity of diluting the solvent of an antibiotic drug only with the same or at least with a similar solution (7), for measuring serum concentrations we used serum of the same patient, which was obtained before the application of CEZ. For determinations of concentrations in the supernatant of muscle homogenate the supernatant of CEZ free muscle homogenate should be used. Since such amounts of CEZ free muscle as needed were not available, we searched for a suitable substitute which was found to be phosphate buffer saline. As demonstrated in Figure 1 the position of the curves for homogenate and PBS reveal no marked differences, thus making it possible to use PBS for dilution as well as for preparing standard curves for muscle homogenate. Such a comparison should be made for each antibiotic which is to be tested. As we had shown with cephradin (20) the difference between PBS and serum .and homogenate and serum was not so marked due to the tow protein binding rate.

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At the same time it is demonstrated that the rate of protein binding in the supernatant of muscle homogenate seems to be of no importance since the curves of PBS and supernatant show a very close proximity. This assumption is supported by the fact that in muscle the concentration of albumin is approximately tenfold lower than in serum (6). In unpublished experiments on rabbits with nearly the same protein binding rate in serum similar results with protein binding in tissue were obtained. Furthermore, K u n i n (10) could not find any binding of cephalexin in homogenates of different organs including muscle (with the reservation that cephalexin does not represent CEZ). Another methodological error factor involved in measurements in tissue homogenates is that, as yet, the real portion of the different compartments in a tissue specimen can only be estimated. One possible method for determining the amount of retained blood in a tissue specimen is to measure the hemoglobin content in the homogenate. Corrected for serum the tissue : serum ratio is relatively stable, between 4 and 7%. Using this figure as a correction factor the tissue concentration will be lower, especially at high serum levels. This correction may be useful in determining tissue concentrations in bone as shown by R o s i n et aL (17). However, in muscle this seems to be more difficult. Since in the method used here the portion of myoglobin cannot be eliminated due to similar spectra (2), the measured amount of hemoglobin may be overestimated. Moreover, the real hematocrit in a muscle specimen ist not accurately predictable since the conditions of microcirculatory perfusion may vary. The real distribution of CEZ in interstitial and intracellular space is not known. The relatively low distribution volume taken to equal the extracelhdar space of the human body leads one to assume that CEZ does not penetrate the cell. This seems to be supported by K o r n g u t h a. K u n i n (9), who did not find any substantial concentrations of cephalexin and cephalotin in human erythrocytes. On the other h~and CEZ is partly secreted in the renal tubules which indicates at least a temporary uptake in renal cells. Taking all these possible correction factors into account, such as the amount of retained blood, and the distribution of CEZ in interstitial and intracellular space, we did not correct the measured values of tissue concentration in the homogenized human skeletal muscle. Comparing our results with those of S i m o n et al. (19) the different mode of application should be considered as it does not allow a comparison of the absolute values. Moreover, as mentioned above, the composition of the fluid in the artificial skin blister is not known exactly. However, the values of the mean tissue : serum ratio (28%) and the blister : blood ratio (340/o) seem to be similar. Comparing the serum and tissue levels with the MICvalues of the main gram-negative bacteria according to K n o t h e (8) (Figure 4) it becomes evident that CEZ given in a dosage of 2 g for a 25 minutes period will reach nearly all strains of E. coli, KlebsieUa, Salmonella, Shigella and the main part of Proteus mirabilis. In addition, the

E. Sinagowitz, K. Pelz, A. Burgert, W. Kaczkowski: Concentrations of Cefazolin in Human Skeletal Muscle 7. Klein, P.: Bakteriologische Grundlagen der chemotherapeu-

Cefazolin Serum and Tissue Levels

MIC Gram-Negative Bacteda

tischen Laboratoriums-Praxis, Springer, Heidelberg 1957.

~S

1

/ag/ml

pg/g

(1974) Suppl. l, 1--5.

9. Kornguth, M.L., Kunin, C.M.: Uptake of antibiotics by

300 200! 100

8. Knothe, H.: The in vitro activity of cefazolin. Infection 2

human erythrocytes. J. Infect. Dis. 133 (1976) 175--185.

~

10. Kunin, C.M.: Binding of antibiotics to tissue homogenates. J. Infect. Dis. 12t (1970) 55--64.

5O

11. Lode, H., Gebert, S., Hendrisehek, A.: Comparative pharmacokinetics and clinical experience with a new cephalosporin derivate: cefazolin. Chemotherapy 21 (1975) 19--32. 12. Mazze, R. 1., Schwartz, F. D., Slocum, H. C., Barry, K. G.: Renal function during anesthesia and surgery. Anesthesiology 24 (1963) 279--284. 0,t25

1 2

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Figure 4: MIC-values of gram-negative bacteria (8) as com-

pared to serum and tissue levels o/ ce/azolin. fact that a tissue level above 10/~g/g is maintained for at least three hours means that C E Z can be effective during several generation times.

Literature i. Bergeron, M.G., Bruseh, J.L., Barza M., Weinstein, L.: Bactericidal activity and pharmacology of cefazolin. Antimicrob. Ag. Chemother. 4 (1973) 396--401.

2. Boulton, F.E., Huntsman, R.G.: The detection of myoglobin in urine and its distinction from normal and variant hemoglobins. J. olin. Path. 24 (1971) 816--821. 3. Chisholm, G.D., Waterworth, P., Calnan, J.S., Garrod, L.P.: Concentration of antibacterial agents in interstitial tissue

13. Naber, K., Madsen, P. 0., Bichler, K.H., Sauerwein, D.: Renal tissue levels of antibiotics. Infection 1 (t973) 208--213. 14. Naumann, P., Reintjens, E.: Antibacterial activity and pharmacokinetic behaviour of cefazolin as compared with five other cephalosporin antibiotics. Infection 2 (1974) 19 24. 15. Nishida, M., Matsubara, T., Murakawa, T., Mine, Y., Yokota, Y., Kuwahara, S., Goto, S.: In vitro and in vivo evatution of cefazolin, a new cephalosporin C derivate. Antimicrob. Ag. Chemother. (1969) 236--243. 16. Regamay C., Gordon, R. C., Kirby, W. M. M.: Cefazolin vs cephalotin and cephaloridine. A comparison of their clinical pharmacology. Arch. intern. Med. 133 (1974) 407---410. 17. Rosin, H., Rosin, A.M., Kriimer, J.: Determination of antibiotic levels in human bone: I. Gentamicin levels in bone. Infection 2 (1974) 3--6. 18. Scholtan, W.: Die Bindung der Penicilline an die EiweiBk6rper des Plasmas und der Gewebsfliissigkeit. Antibiot. Chemother. 14 (1968) 53--93. 19. Simon, C., Malerczyk, V., Brahmstaedt, E., Toeller, W.: Cefazolin, ein neues Breitspektrum-Antibiotikum. Dtsch. med. Wschr. 98 (1973) 2448--2450.

fluid. Brit. med. J. 10 (1973) 569--573.

20. Sinagowitz, E., Petz, K., Burgert, A., Kaczkowski, W., Sommerkamp, H.: Cephalosporinkonzentrationen im mensch-

4. Eigler, F. W.: Pathophysiologie der Niere im Rahmen chirurgiseher Erkrankungen, Vortfiige aus der praktischen Chirurgie, Heft 79. Enke, Stuttgart 1968.

lichen Gewebe. In: Asepsis und Antisepsis in der Urologic (Ed.: P. Porpaczy), pp. 179--184. Egermann, Wien 1975.

5. HoNmann, R., V6mel, W.: Investigations on the efficacy of the new cephalosporin antibiotic cefazolin in vitro and in vivo. Infection 2 (1974) Suppl. 1, 6--17.

6. Katz, 1., Bonorris, G., Sellers, A. L., Extravascular albumin in human tissues. Clin. Sci. Mol. Med. 39 (1970) 725--729.

21. Tan, ]. S., Trott, A., Phair, J.P.: A method for measurement of antibiotics in human interstitial fluid. J. Infect. Dis. 126 (1972) 492--497. 22. V6mel, W., Hoffmann, R.: Basis for the evaluation of the chemotherapeutical effect ef cefazelin in man. Infection 2 (1974) 40---48.

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Concentrations of cefazolin in human skeletal muscle.

Tissue concentrations of cefazolin were measured in 18 patients undergoing urological operations. Specimens of skeletal muscle and blood were taken si...
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