J.

:jet.

Pharmacol. Therap. 15,416420, 1992.

Effect of dietary nitrogen intake on gentamicin disposition in sheep M. OUKES S OU* & P. L. T O U T A I N t

*Department of Physiology and Therapeutics, Hassan I1 Agronomic and Veterinary Institute, Rabat, Morocco and t Ecole Nationale Veterinaire, Department of Physiopathology, 23 chemin des Capelles, 3 1076 Toulouse Cedex, France Oukessou, M., Toutain, P.L. Effect of dietary nitrogen intake o n gentamicin disposition in sheep. J. vet. Phannacol. Tlierap. 15, 4 1 6 4 2 0 . T h e effect on gentamicin pharmacokinetics of a diet high (HP) (120 g/day) o r low (LP) (25 glday) in digestible proteins was studied in sheep. Gentamicin sulphate ( 4 mglkg) and inulin (40 mglkg) were administered by the intravenous route to six ewes of local Moroccan breed. T h e serum gentamicin concentrations were consistently higher in ewes that received a LP diet. Clearance was 0.93 2 0.13 mYmm/kg in the LP group and 1.64 k 0.40 ml/mm/kg in the HP group. T h e volume of distribution at steady state (Vss)was lower in the LP group ( 1 1 % of body weight) than in the HP group (2 1.8% of body weight). These diet-linked variations in pharmacokinetic parameters were also obtained in the disposition of inulin following the intravenous administration of a single dose. This subp 7 ests that the protein content of the diet modifies the distribution of body water and kidney function. T h e therapeutic, toxicological and hygienic implications of these modifications are discussed.

P . L. Toulain, ENVT, Department o/ Pliysiopnlliology, 23 chewin des Capdlrs, 3 1076 Torilowe Cedex, Fmnce.

INTRODUCTION The effects of nitrogen intake on kidney

function have been widely investigated in domestic animals and man (Rabinowitz et al.. 1973; Eriksson & Valtonen, 1982; Kitt ct al., 1988; Faix el a / . , 1988). In sheep a reduction in dietary protein content leads to a reduction in glomerular filtration rate (GFR) and renal plasma flow of approximately 40% (Ergene & Pickering, 1978). Under Moroccan husbandry conditions, shrep are subjected to fluctuations in the quantity and quality of food supply througho u t the year. Food is abundant and nitrogenrich in spring and summer, becoming scarce a n d l o w in nitrogen during autumn and winter. As the kidney represents a major route of elimination for numerous drugs, 416

particularly antibiotics, it is important l o determine the effect of dietary nitrogen on the disposition of antibiotics that are eliniinated mainly by the kidney. T h e objective of the present study was to determine the disposition parameters for gentamicin in sheep fed on two different types of diet: one rich in proteins (HP), the other low in proteins (LP) and to correlate the pharmacokinetic parameters obtained with the GFR based on inulin clearance. T h e same six Sardi an Dman ewes (A to F) weighing between 29 and 56 kg were used in both phases of this study. T h e same aninials were kept in individual metabolism capes. T h e H P diet consisted of alfalfa hay ( 1 kg) and barley (200 g) and the LP diet of straw (500 g) and bariey (400 g). Water was available ad libifurn. Both diets provided approximately

Gentamicin in sheep 417 one McaVday and the protein supply amounted to 120 g (HP) and 25 g (LP) of digestible proteindday. T h e animals received the HP diet for at least 5 weeks before receiving the first administration of gentamicin and inulin. They were then put on the LP diet for 3 weeks before receiving a second administration of gentamicin and inulin, Gentamicin sulphate in aqueous solution (Septigen, Rigaux, Galena, France) containing 40 g of gentamicin base/ml of solution was administered at a dose of 4 mg/kg. Inulin (Sigma, St Louis, MO, USA) in aqueous solution (20%) was administered at a dose.of 40 mg/kg. Both substances were administered simultaneously by the intravenous route via a catheter inserted in the jugular vein. Blood samples (6-8 ml) were taken from the other jugular vein before and at 2,4,8, 15,30,60, 120, 180,240, 300, 360 and 420 min after the injection. T h e blood was collected in dry vacutainers (Becton Dickinsson vacutainer system, Becton Dickinsson Co., France). T h e serum was separated by centrifugation, dispensed into several aliquot fractions and stored at -25°C until analysed. T h e quantitation of serum gentamicin concentration was carried out by m icrobiological method using an agar diffusion technique with Bacillus subtilis ATCC 6633 as test microorganism (Edberg, 1986). Each sample was determined twice. T he limit of sensitivity was 0.01 pg/ml. T h e inulin was determined by a method described by Clarke el al. (1985). The individual data were analysed with a non-linear least squares regression programme (Yamaoka et d.,1981). T h e serum concentrations of gentamicin and inulin werE fitted to the following biexponential equation:

C(,)= Y I exp ( - A l l )

+ Y2 exp (-A2

t)

in which C(,) is the serum concentration at time 1, Y , and Y2 are preexponential coefficients, and A, and A2 are exponents. T h e disposition parameters for gentamicin and inulin were calculated with the usual equations (Baggot, 1977). Body clearance (Cl) was calculated.with equation 2: Dose

c1 =

(2) AuC

( M ~ a s t )

in which AUC is the area under the curve of the concentrations obtained by the arithmetic trapezoidal method without extrapolation to infinity i.e. up until the last measured concentration (Cbdst). T h e steady state volume of distribution (Vss) was calculated with equation 3:

vss= vc

[I

+$I

(3)

in which V , represents the volume of the central compartment, and K12 and KPI are transfer constants between the central and peripheral compartments. T h e mean residence time (MRT) was calculated by the arithmetic trapezoidal method without extrapolation to infinity. T h e pharmacokinetic parameters for the two diets were compared using the Student’s t-test for for paired data. The null hypothesis was rejected for P < 0.05. The effect of the dietary protein content on gentamicin is shown in Fig. 1. The serum concentrations of gentamicin were consistently higher in the LP group than in the HP group with an AVC 1.7 times higher for the LP group. T he mean (+ SD) values of pharmacokinetic parameters are given in Table I. T he body clearance of gentamicin was significantly lower in the LP group (0.93 f. 0.13 nil/mn/kg) than in the HP group (1.64 f. 0.40 ml/mn/kg) (P < 0.001). A similar result was reported for tetracycline in man (Raghuram & Krishnaswamy, 1982). Parallel variations were observed for GFR, calculated from inulin clearance (Table 11). As a result the ratio between gentamicin and inulin clearance values (filtered gentamicin fraction) was not modified by the protein diet. Whatever the diet, gentamicin clearance was about 52-55% of inulin clearance (Table 11). For the data as a whole, the correlation between gentamicin clearance and inulin clearance was very highly significant (r = 0.94, P < 0.00 1). T h e increase in clearance explains that the MRT, i.e., the mean time a molecule of gentamicin persists within the body was shorter with the H P diet (77.1 k 32.5 vs. 91.1 f 5.9 mn) although the difference between the two diets in MRT was not statistically significant (P > 0.05). T h e volume of distribution at steady state (VSJ of gentamicin was higher in the HP

418

M. OuRcsJou &3 P . L. ToUtain

c

0

60

120

180

240

300

360

420

Time (min) FIG. 1. T h e semilogarithmic plot of mean serum concentration (pg/ml) vs. time (min) for gentamicin after intravenous administration of gentamicin at a dose rate of 4 mglkg in six sheep fed with a high protein (0) or a low protein diet ( 0 ) .

T A B L E 1. Selected pharmacokinetic parameters describing the disposition of gentamicin after the intravenous administration of gentamicin (4 mgkg) to six sheep for a low and or high protein diet (mean f SD) Diets Parameters

(units)

Low-protein

High-protein

97.7 5 9.4 91.1 f 5.9

117.0 f 67.7 77.1 f 32.5 0.035 f 0.029 0.218 +- 0.1 12 1.64 f 0.40 2532.6 +- 500.7

0.030 f 0.004 0.1 I0 5 0.009 0.93 k 0. I3 4382.8 +- 599.5

Statistical significance (paired &test)

NS

NS NS ( P < 0.05) (P < 0.001) ( P < 0.001)

:,,lt (Ad: serum half-time; MRT: mean residence time; V,: volume of the central compartment Vrs: steady sfate volume of distribution; C1: plasma clearance. A U C : area under the serum concentration-time curve. NS: not significant ( P > 0.05). MRT, Cl and A U C were calculated using trapezoidal rule from time 0 to the last observed concentration (Clr,,).

Gentamicin in sheep 419 T A B L E 11. Selected pharmacokineticparameters describing the disposition of inulin after the intravenous administration of 40 mgkg of inulin to six sheep for low or high protein diets

Diets Parameters vc

v,,

c1 (O-ClarJ C1 GentlC1Inulin

(units)

Low-protein

High-protein

(wd

0.059 & 0.012 0.132 f 0.037 1.89 f 0.50 0.52 f 0.11

0.072 f 0.016 0.180 f 0.109 3.05 f 0.72 0.55 f 0.05

Wkg) (mYminlkg)

Statistical significance (paired &test) NS NS

P

= 0.001

NS

Vc: volume of the central compartment Vs,: steady state volume of distribution; CI: plasma clearance calcdated without extrapolation to infinity; Cl GedCI Inulin: Ratio of gentamicin/inulin clearance.

group (21.8% of the body weight) than in the LP group (11% of the body weight) (P < 0.05). T h e lower value in the latter group may be explained by the fact that the distribution of gentamicin was limited to the extracellular water space which is reduced by a low-protein diet. This hypothesis is suggested by the reduction, albeit non-significant (P = 0.19). in the volume of distribution of inulin, considered as a marker of extracellular fluids. T h e volume of the central compartment for gentamicin was not changed by protein nutrition which implies that the observed variations in V,, were principally d u e to those of the peripheral compartment. T h e lower volume of distribution of gentamicin in LP animals compared with H P animals could' be explained by a reduction in tissue binding of gentamicin in animals in the LP group, as has been suggested for tetracycline in man (Raghuram lk Krishnaswamy, 1982). It should be noted that the half-life time was greater (non-significantly) in the H P group. This may be explained by the fact that the half-life is a hybrid parameter dependent on both clearance (which was increased) and volume of distribution (which was increased). Gentamicin is an antibiotic exhibiting bactericidal activity against numerous gram negative bacteria and certain gram positive ones. T h e minimal inhibiting concentrations (MIC) are generally between 2 and 4 pg/ml for sensitive strains (Prescott & Bagott, 1988). I n the present study, serum concentrations were maintained above 4 pg/ml for approximately 2 h in the HP group and 5 h in the LP group. This could have therapeutic, toxicological and

food safety implications. From the therapeutic point of view, the relationships between plasma concentrations, efficacy and toxicity have been the subject of contradictory statements (John, 1988). Gentamicin is currently considered to belong to the group of antibiotics whose activity is dose (concentration)dependent, i.e., whose efficacy is linked to peak plasma concentration rather than to the maintenance of a minimal inhibitory concen-. tration (Garraffo el al., 1990). In consequence it could be suggested that a LP diet would tend to increase the therapeutic activity of gentamicin especially if the reduction in V,, was due to a reduction in binding to tissue proteins (see above). From a toxicological point of view, toxic effects are acknowledged to be directly linked to the maintenance of relatively high plasma concentrations for a prolonged period (Bowles & Schentag, 1986). In the present study, it is not established whether that the increase in plasma gentamicin concentration increases the risk of nephrotoxicity. In order to he nephrotoxic the gentamicin must, in fact, be filtered by the glomerulus so that it can then act on tubule function (Bowles & Schentag, 1986). With an LP diet, glomerular filtration is reduced and it is not certain that an increase in plasma concentration results in a concurrent increase in tubule exposure to gentamicin (as would be the case with an increase in plasma concentration resulting from a simple increase in dosage). From a tissue residue point of view, the accumulation of gentamicin in the kidney cortex is linked to the rate of glomerular filtration so that a diet low in protein would

420

M . Oukessou k? P . L. Toutain

not necessarily increase the residue level in the kidney or require a lengthening of the withdrawal time. In conclusion, a LP diet increases gentamicin serum concentration by decreasing the volume of distribution at steady state and the glomerular filtration rate. Such a modification must be taken into account when selecting a dosage regimen for a standard dose of 4 mgl kg, it could be anticipated that gentamicin would have a greater antibacterial effect in sheep under a LP diet than a HP diet. Effect of LP on the presence of gentamicin kidney residue merits further experimental investigation.

ACKNOWLEDGMENTS This work was carried out with financial aid from the FIS (BI1110).

REFERENCES Baggot. J.D. (1977) Principles of Drug Disposition in Domestic Animals. The Basis of Veterinary Clinical Pharmacology. W. B. Saunders, Philadelphia. Bowles, S.K. & Schentag, J.J. (1988) Aminoglycoside commentary. In Eds Evans, W.E., Schentag, J.J.. Jusko, W.J. & Harrison. H. Appfied Phannacokinetics, Principle of Therapeutic drug monitoring, Applied Therapeutics Inc.. Spokane, Washington. Clarke, C.R., Short,C.R.. Hsu, R.-C. & Bagg0t.J.D. (1985) Pharmdcokinetics of gentamicin in the calf: Developmental changes. American Journal uf VeteriiraT Research, 46, 246 1-2466. Edberg, S.C. (1986)T h e measurement of antibiotics in human body fluids: Techniques and signifi-

cance. In Antibiotics in Luborato~y Medicine. Ed Lorian, V., pp. 381-476, Williams & Wilkins, Los An geles. Ergene, N. & Pickering, E.C. (1978) T h e effects of reducing dietary nitrogen and of increasing sodium chloride intake on urea excretion and reabsorption and urine osmolality in sheep. Quarterly Journal of Experimenfal Pliysiolu@, 6 3 , 6776. Ericksson, L.E. & Valtonen, M. (1982) Renal urea handling in goats fed high and low protein diets. Journal of Dairj Science, 65, 385-389. Faix, S., Leng, L., Szanyiova, M. & Boda, K. (1988) Creatinine and inulin clearance at the different nitrogen and energy iniake in sheep. Conrparative Biochemistrj and PliyJiologv, 91A, 689-69 I . Garaffo, R., Dellamonica, P., Drugeon, H.B., Etesse, H. & Lapalus, P. (1990) A new approach to optimal antibiotic dosage regimen by coupling pharmacokinetics and killing curve parameters. Methods Findings i n E x p e ~ t ~ n t aClinical i Phnrmncoloa, 5 , 325-332. John, J.F. (1988) What price success? The continuous saga of the toxic: therapeutic ratio in the use of aminoglycoside antibiotics. The joztrtrul 01 Infectious Diseases, 158. 1-6. Kitt, T.M., Park, G.D., Spector, R., Lawton, W. & Tsalikian, E. (1988) Renal clearance of oxypurinol and inulin on an isocaloric, low protein diet. Clinical Pltarnincolu~ and Tlrernlwutics, 4 3 , 68 I687. Prescott, J.F. & Baggot, J.D. (1988) Antimicrobial Therapy in Veterinary Medicine. Blmkurll Scienf ific P ublicutions , O x for& Rabinowitz. L., Gunther, K.A.. Shqji, E.S.. Freeclland. R.A. & Avery. E.A. (1973) Effects or high and low protein diets on slieep retial function and metabolism. Kidnqv Internnfiottd, 4. 188-207. Raghurani. T.C. (L Krishnaswamy, K. (1982) Pharniacokinetics of tetracycline in nutritional oedema. Cheniotherapy. 28, 428-433. Yamaoka. K.,Tanigwara, Y., Yakagawa, T . & Uno, T. (1981) A pharmacokinetic analysis program (MULTI) for microcomputer. Jounral of Pllarmacobio-Dynamics, 4, 879-885.

Effect of dietary nitrogen intake on gentamicin disposition in sheep.

The effect on gentamicin pharmacokinetics of a diet high (HP) (120 g/day) or low (LP) (25 g/day) in digestible proteins was studied in sheep. Gentamic...
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