Keywords: bolus i.v. injection, inulin clearance, kinetic analysis

Transcription

1 Br J Clin Pharmacol 1998; 46: Determination of inulin clearance by bolus intravenous injection in healthy subjects and ascitic patients: equivalence of systemic and renal clearances as glomerular filtration markers R. Orlando, 1 M. Floreani, 2 R. Padrini 2 & P. Palatini 2 1 Institute of Clinical Medicine and 2 Department of Pharmacology, University of Padova, Italy Aims Determination of systemic inulin clearance by the standard technique of constant intravenous infusion has long been accepted as a reliable method for measuring glomerular filtration rate (GFR) without urine collection, except in oedematous patients. However, recent studies using standard clearance techniques have claimed that systemic inulin clearance is significantly greater than renal clearance and therefore overestimates GFR. The main purpose of this investigation was to re-evaluate the relationship between systemic and renal inulin clearance using a different technical approach. A reassessment was also made of inulin disposition kinetics. Methods Systemic and renal inulin clearances were simultaneously evaluated, in healthy subjects and patients with oedema and ascites, by analysis of the total area under the plasma concentration-time curve (AUC) following bolus intravenous injection. Renal clearance was calculated as the ratio of the total amount recovered in the urine to the AUC, and systemic clearance as dose/auc. Results Inulin disposition kinetics were best described by a tri-exponential model. In healthy subjects the volume of the central compartment (mean (s.d.) value 3.86 (1.00) 70 kg 1 ) was slightly greater than the plasma volume; steady-state volume of distribution was (1.21) l 70 kg 1, in accordance with the tenet that the inulin space is somewhat smaller than the extracellular fluid volume. The values of systemic and renal inulin clearances were very similar (96.1 (10.0) and 94.6 (12.5) ml min 1 70 kg 1, respectively, in healthy subjects; (16.3) and (18.5) ml min 1 in ascitic patients). They were also highly correlated to each other in both healthy subjects (r=0.96, P<0.001) and patients with ascites (r=0.98, P<0.001). Conclusions The method described here constitutes a simpler and more precise technique for measuring renal inulin clearance than the standard method, which is based on constant infusion and timed collections of urine samples, since it avoids errors connected with short successive urine collections. By the present method we demonstrated that renal and systemic inulin clearances are virtually identical in both healthy subjects and patients with expanded extracellular fluid volume. Determination of systemic inulin clearance by the presently described technique is therefore a method of general validity for measuring GFR without urine collection. Keywords: bolus i.v. injection, inulin clearance, kinetic analysis Introduction is eliminated exclusively by renal excretion, Earle and coworkers [2, 3] proposed to determine systemic inulin Measurement of glomerular filtration rate (GFR) is necessary clearance in place of renal clearance. This technique, which in both clinical practice and research fields for assessing renal obviates the need for urine collection, has since been shown function. The renal clearance of inulin is accepted as one of to be a valid and convenient substitute for measurement of the reference standards for GFR estimation [1]. The classical renal clearance, except in patients with expanded extracellular method for determination of inulin clearance, which requires fluid volume because of oedema and/or ascites [1, 3]. This constant intravenous infusion and timed collections of urine practice has been questioned by three recent studies [4 6], samples, is impractical and particularly inconvenient for which found systemic inulin clearance values significantly patients. Therefore, alternative clearance techniques have higher (up to 20%) than those of renal clearance. According been developed. Since there is general consensus that inulin to van Acker et al. [6] this was attributable to extrarenal clearance or to storage in a compartment that is characterized Correspondence: Professor Pietro Palatini, Dipartimento di Farmacologia, Università by slow diffusion. In these studies, renal clearance was di Padova, Largo E. Meneghetti 2, Padova, Italy. measured by means of the classic UV/P method, i.e. from 1998 Blackwell Science Ltd 605

2 R. Orlando et al. the ratio of renal excretion rate to plasma inulin concentration, rate over 1 min by a precise volumetric infusion pump. following either constant intravenous (i.v.) infusion [4, 6] or Inulin vials used throughout the study were from the same bolus i.v. ( single-shot ) injection [5]. However, with either batch. Blood samples (4 ml) were taken from a cannula administration technique, this method has technical drawbacks. placed in an antecubital vein of the opposite arm at 0 If it is based on short, successive urine collections, ( predosing), 2, 5, 10, 15, 20, 30, 45, 60, 90, 120, 150, 180, variations in the completeness of bladder emptying produce 210 and 240 min after the end of the infusion. Blood was substantial errors in the evaluation of the renal excretion rate drawn into heparinized plastic tubes and centrifuged immediately. [1]. Bladder catheterization, that would ensure more precise Plasma samples were stored at 40 C until assayed. determinations of the excretion rate, may cause bladder Urine was collected before and from 0 4 and 4 12 h after infections and is not considered acceptable in volunteers. If dosing. Serial determinations in the urine of the first four observations are performed over an extended period of time, subjects (two healthy subjects and two ascitic patients) had wide fluctuations (up to 50%) may occur in plasma inulin shown that inulin was no longer detectable or present in concentration, probably due to the circadian rhythm of renal traces after 12 h. Mean urinary recovery was 98 [5]% of the function [6]. Therefore, the customary determination of a injected dose. Before starting the determination of inulin single plasma concentration at the mid-point of the urine clearance, all subjects received oral hydration with collection period may either grossly overestimate or underestimate 10 ml kg 1 of tap water. To maintain diuresis, urinary losses the average plasma concentration. The UV/P method were replaced by equal amounts of water. afterbolusi.v.injectionhasrecentlybeenshowntoprovide a poor estimate of inulin clearance [7]. The main aim of this study was to compare systemic and Assay method renal inulin clearances in healthy subjects and patients with Inulin in plasma and urine was determined by means of the ascites. Accordingly, both clearance parameters were deteranthrone method, after removal of glucose, essentially as mined by the area method [8], after bolus i.v. injection. described by Jung et al. [10]. Briefly, 0.3 ml of plasma or The disposition kinetics of inulin was also reassessed, since urine, appropriately diluted, were mixed with 0.05 ml of discrepant results regarding the distribution characteristics of the glucose-removing reagent (300 IU of glucose oxidase inulin have been obtained by two recent studies [7, 9]. (Sigma, St Louis, MO, USA) and IU of catalase (Boehringer, Mannheim, FRG) in 1 ml of 100 mm Methods triethanolamine-hcl buffer, ph 7.0) and incubated at 37 C for 4 h. Control experiments showed that the Subjects addition of glucose up to 10 mm (about twice the physiological concentration) to inulin samples had no effect Sixteen healthy male volunteers (mean (s.d.) age 54 (7) years, on the determinations. After incubation was completed, weight 73 (8) kg, height 170 (6) (cm) and eight male patients 0.35 ml of 10% trichloroacetic acid were added and the with decompensated liver cirrhosis (mean (s.d.) age 50 (7) mixture centrifuged at g for 10 min. One ml of years, weight 77 (10) kg, height 173 (5) cm) gave their 5.15 mm anthrone (Sigma) in sulphuric acid was then informed written consent to participate in this study, which added to 0.4 ml of supernatant and the mixture incubated was approved by the local Ethics Committee. Healthy subjects at 37 C for 60 min. All samples were run in duplicate. were recruited from outpatients attending the hospital for Absorbance was read at 623 nm. For each assay, inulin routine laboratory tests. Criteria for their selection were that concentration was calculated from a calibration curve they did not require any regular medication and had no obtained by dissolving inulin in water. Preliminary history of allergy to drugs. They were diagnosed as being experiments had shown that identical calibration curves healthy by means of a thorough clinical examination, were obtained if inulin was dissolved in plasma or urine. including medical history, physical examination and standard The assay was linear (r 2 >0.99) up to 250 mg l 1. The clinical laboratory tests. All cirrhotic patients had ascites and detection limit in plasma and urine was 2 mg l 1. The oedema of the lower extremities, and could be categorized as intra- and inter-assay coefficients of variation (n=10), Child s class C. Apart from the biochemical indices of liver determined at 20 and 200 mg l 1, were below 7 and 5%, function, all patients had normal laboratory test values, respectively. including glycaemia. Patients were excluded from this study if they had a recent history of gastrointestinal bleeding, severe encephalopathy or refractory ascites. Creatinine clearance of healthy subjects and patients ranged from and Pharmacokinetic analysis ml min 1, respectively. During the period of investi- The data were modelled by using the GraphPad Prism 2.0 gation all participants abstained from alcohol and tobacco and software. The inulin decay curve of each subject was took no drugs, apart from those used for the treatment of analysed by using a bi- or a tri-exponential equation. Initial cirrhosis (spironolactone or canrenone and vitamin estimates of the coefficients and exponents of the equations supplements). were obtained by a numerical technique. These estimates were then refined by iterative nonlinear regression analysis Protocol with a weighting factor of 1/C 2. Comparison between At h, after an overnight fast, 5 g of inulin (Inutest R, Laevosan GmHB, Linz, Austria) were infused at a constant competing models was made by means of the F-test. The total area under the plasma concentration-time curve (AUC) was calculated from the coefficients and exponents Blackwell Science Ltd Br J Clin Pharmacol, 46,

3 Short report Table 1 Mean (s.d.) pharmacokinetic parameters of inulin. Values of clearance and distribution volumes are normalized to 70 kg of body weight. V C V SS t 1/2 CL R CL CL R /CL% ( l) ( l) (h) (ml min 1 ) (ml min 1 ) (95%CI) Healthy subjects Mean a 98.5 (s.d.) (1.00) (1.21) (0.13) (12.5) (10.0) ( ) Ascitic patients Mean b 98.1 (s.d.) (1.21) (1.39) (0.23) (18.5) (16.3) ( ) P-value <0.02 <0.005 NS NS NS (healthy vs ascitic subjects) V C, apparent volume of the central compartment; V SS, apparent volume of distribution at steady-state; t 1/2, elimination half-life; CL R, renal clearance (calculated as Ae (2)/AUC); CL, systemic clearance. a P=0.48 and b P=0.26 vs CL R. of the equation which better fitted the data. Renal clearance (CL R ) was then calculated either as Ae (2)/AUC, i.e. from the ratio of the total amount recovered in the urine to total AUC, or as Ae (0 4)/AUC (0 4), (that is from the ratio of the amount excreted during the first 4 h to the corresponding AUC). Systemic clearance was calculated as dose/auc. Systemic clearance was also obtained from a limited number of data points (at 2, 15, 30, 60, 120 and 240 min) using the logarithmic trapezoidal rule with extrapolation to infinity. The other pharmacokinetic parameters, defined in Table 1, were calculated from standard equations [11]. Statistical analysis Plasma concentration (mgl 1 ) Time (min) A power analysis (CSS power assessment procedure; CSS,Statsoft Inc, Tulsa OK, USA, 1991) based on the coefficients of variation obtained by Buclin et al. [7] for renal clearance of inulin indicated that the sample sizes used in this study should be sufficient to detect differences of 10%, with a significance level (a) of 0.05 and a power (1- b) of Intragroup differences in inulin clearances were evaluated by means of Student s two-tailed paired t-test, whereas Student s nonpaired t-test was used for comparison of pharmacokinetic parameters between different study groups. Correlations were examined by linear regression analysis. Probability values <0.05 were considered statisti- cally significant. Figure 1 Plasma decay curves of inulin in a healthy subject ($) and an ascitic patient (#). The solid lines are the curves obtained by nonlinear least-squares regression analysis of the data. Results renal and systemic inulin clearances in both healthy and ascitic subjects. The correlation between the two parameters Representative examples of the plasma decay of inulin in was highly significant in both groups (r=0.96, P<0.001 healthy and ascitic subjects are shown in Figure 1. With the and r=0.98, P<0.001, respectively). Determination of renal exception of two ascitic patients, the best agreement between clearance as Ae (0,4 h)/auc (0,4 h) yielded values very theoretical curves and experimental data points was always similar and highly correlated to those calculated from total obtained using a tri-exponential equation. The pharmacokinetic urinary recovery (97.0 (12.4) and (18.6) ml min 1 for parameters characterizing inulin disposition are healthy and ascitic subjects, respectively; r>0.98 and shown in Table 1. The values of the volume of the central P<0.001 in either case). Systemic clearance estimates compartment and the steady-state volume of distribution in obtained by application of the logarithmic trapezoidal rule healthy volunteers were very similar to those observed in to six points (see Methods) were in close agreement with healthy subjects by Odeh et al. (3.90 and l 70 kg 1, those obtained from pharmacokinetic modelling of the respectively; [9]) and significantly lower than those observed decay curves (93.5 (9.2) and (19.6) ml min 1 for in ascitic patients. Very similar values were obtained for healthy and ascitic subjects, respectively) and also showed a 1998 Blackwell Science Ltd Br J Clin Pharmacol, 46,

4 R. Orlando et al. close correlation with renal clearance values (r=0.99, longer in patients with expanded extracellular fluid volume. P<0.001; r=0.97, P<0.001, respectively). The variability The method described in the present study requires no of the clearance estimates was similar with all four methods assumption regarding equilibrium and is therefore of more used. Intersubject coefficients of variation ranged from 10 general applicability. By this method, GFR can be accurately to 13% for healthy subjects and from 15 to 19% for and easily estimated also in oedematous patients from plasma ascitic patients. data alone over a relatively short period of time. When, as in routine clinical practice, the above described approach, Discussion requiring numerous blood samples for pharmacokinetic modelling, is not feasible, GFR can be estimated by a Our results regarding healthy subjects are very similar to simplified sampling protocol, without appreciable loss of those reported by Odeh et al. [9]. Like these authors, we precision. We have in fact shown that application of the found that inulin disposition kinetics are best described by a logarithmic trapezoidal rule to only six data points yields tri-exponential model. This underlines the inadequacy of the essentially equal results, with very similar coefficients of one- or two-exponential models often used to calculate inulin variation. clearance from plasma concentration data (reviewed in 1). In conclusion, this study has shown that determination of The values of V C and V SS found in this study are also in renal inulin clearance from the ratio of the amount excreted very close agreement with those obtained by Odeh et al. [9]. in the urine to the AUC over the corresponding period of On the contrary, significantly higher estimates were reported time, constitues a simpler and more precise clearance by Buclin et al. [7]: 7.4 and 13.4 l for V C and V SS, technique than the classic UV/P method, since it avoids respectively ( P<0.001 and P<0.01 compared to our errors connected with frequent urine collections. By this estimates). These discrepancies are most likely due to the fact method, we have demonstrated that renal and systemic that these authors used a bi-exponential equation to describe inulin clearances are virtually identical in both normal their data. We verified that using a bi-exponential instead of subjects and oedematous patients. Determination of systemic a tri-exponential model yields considerably lower values of inulin clearance by the presently described technique C(0) and, consequently, higher V C estimates. Atkinson and represents therefore a method of general applicability for coworkers [9, 12] interpreted the value of 3.90 l for V C as measuring GFR without the need for urine collection. indicating that the central compartment corresponds to the plasma space and proposed that transcapillary exchange is the rate-limiting step in inulin distribution. However, the This work was supported by a grant from MURST observed V (Ministero per l Università e la Ricerca Scientifica e C value is about 30% larger than the generally accepted value for plasma volume (3l 70 kg 1 ; [13]). This Tecnologica). The technical assistance of Mr P. Favero is suggests that part of the extracellular fluid is in rapid acknowledged. equilibrium with plasma. Consistent with this conclusion is the finding that the volume of the central compartment is about twice the plasma volume in ascitic patients. The value References of the steady-state volume of distribution obtained in this 1 Levinski NG, Lieberthal W. Clearance techniques. In study is in agreement with various previous estimates (see 6, Handbook of Physiology, section 8, volume 1, ed. Windhager 9 and references therein) and also in accordance with the EE. Oxford: Oxford University Press, 1992: tenet that the inulin space is rather smaller than the 2 Earle DP, Berliner RW. A simplified clinical procedure for extracellular fluid volume, averaging 13l 70 kg 1 [13]. measurement of glomerular filtration rate and renal plasma Our clearance measurements have shown that, contrary flow. Proc Soc Exp Biol Med 1946; 62: to the continuous i.v. infusion technique, the method based 3 Berger EY, Farber SJ, Earle DP. Comparison of the constant on the analysis of the area under the concentration-time infusion and urine collection techniques for the measurement of renal function. J Clin Invest 1948; 27: curve following bolus i.v. injection yields virtually identical 4 Hellerstein S, Berembom M, Alon U, Warady BA. The renal values of systemic and renal inulin clearances, in both clearance and infusion clearance of inulin are similar but not healthy subjects and patients with expanded extracellular identical. Kidney Int 1993; 44: fluid volume. In addition, the coefficients of variation of 5 Florijn KW, Barendregt JNM, Lentjes EGWM, et al. the two clearance parameters proved to be quite similar. Glomerular filtration rate measurement by single shot This contrasts with the results obtained by means of the injection of inulin. Kidney Int 1994; 45: UV/P method, which yielded renal clearance estimates 6 van Acker B, Koomen GCM, Arisz L. Drawbacks of the much more variable than those of total body clearance [5, 7]. constant-infusion technique for measurement of renal A major problem in the determination of systemic inulin function. Am J Physiol 1995; 268: F543 F552. clearance by the constant infusion technique is the attainment 7 Buclin T, Pechère-Bertschi A, Séchaud R, et al. Sinistrin of a true steady state. Van Acker et al. [6] showed that, 6 h clearance for determination of glomerular filtration rate: a reappraisal of various approaches using a new analytical after administration of a priming dose and onset of method. J Clin Pharmacol 1997; 37: continuous infusion, a steady-state concentration had still 8 Tucker GT. Measurement of the renal clearance of drugs. not been reached. In two patients they observed a continuous Br J Clin Pharmacol 1981; 12: increase in plasma inulin concentration throughout their 9 Odeh YK, Wang Z, Ruo TI, et al. Simultaneous analysis of 30 h study period. According to Hellerstein et al. 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5 Short report low concentrations of inulin in serum. Nephron 1990; 54: multicompartmental models of drug distribution. Trends Pharmacol Sci 1991; 12: Wagner JG. Linear pharmacokinetic equations allowing direct 13 Lang F. The body compartments and dynamic of water and calculation of many needed parameters from the coefficients electrolytes. In Comprehensive Human Physiology. Eds Greger and exponents of polyexponential equations which have R, Windhorst U, Berlin: Springer Verlag 1996: been fitted to the data. J Pharmacokin Biopharm 1976; 4: ( Received 12 March 1998, 12 Atkinson AJ, Ruo TI, Frederiksen MC. Physiological basis of accepted 2 July 1998) 1998 Blackwell Science Ltd Br J Clin Pharmacol, 46,