Acta physiol. scand. 1970. 80. 249-283 From the Institiite of Pharmacology and Toxicology, Royal Veterinary and Agricultural University,

Copenhagen

Acetplation of P-Aminohippuric Acid in the Kidney. Renal Clearance of P-Aminohippuric Acid and N4-Acetylated

P-Aminohippuric Acid in Pigs BY

N. GYKD-HANSEN and FOLKE RASMUSSEN

Received 26 February 1970

Abstract

GYKC-HANSEN, N. and F. RASMUSSEN. Acetylation o f p-aminohippuric acid in the kidney. Renal clearance of p-aminohippuric acid and JV4-acetylated p-aminohippuric acid in pigs. Ac ta physiol. scand. 1970. 80. 249-253.

20 pigs weighing 21-124 kg were used to determine the clearance of PAH, N4-acetylated PAH and PAHt,t,l, and for determination of acetylation of PAH and de-acetylation of N4-acetylated PAH in the kidney. By examining blood samples taken from a. renalis and v. renalis and urine collected simultaneously, hie acetylation and de-acetylation of PAH and N4-acetylated PAH in the kidney were determined on 9 pigs. 6 of these acetylated PAH (1.5-10 per cent), while 3 de-acetylated N4-acetylated PAH (2--21 per cent). The extraction percentage for PAH and N4-acetylated PAH were 91 & 1.4 (S.E.M.) and 84 & 2.1 respectively. The ratio between clearance of N4-acetylated PAH and PAH was found to be 1.08 0.07 and between clearance for PAHtotal and PAH 1.01 Jr 0.02.

Renal clearance of p-aminohippuric acid (PAH) can be used for determination of the effective renal plasma flow (RPFeffective) only if metabolism of PAH does not take place to any great extent during passage through the kidneys. However, Rennick, Hamilton and Evans (1961) and Setchell and Blanch (1961) showed that PAH is conjugated in vitro by kidney slices in many animal species. The conjugated derivative is probably N4-acetylated PAH, and Nielsen, Maaske and Booth (1966) stated that PAH cannot be used for determining RPFeffective in pigs because the kidney acetylates PAH too rapidly. The aim of the present study was to determine the extent of this acetylation and to assess its significance for determination of RPFeffertive in pigs.

Material and methods The experiments wzre carried out on 1 7 female and 3 (no. 1, 2, and 3) castrated male Danish Land- race pigs, weighing 21-124 kg. The animals were fed during the observation period with a nor- mal fodder mixture as described by Gyrd-Hansen (1968) and they were allowed free access to water.

249

250 N . GYRD-HANSEN AND FOLKE R A S M U S J E N

’The experiments on pigs no. 1-9 were done on animals anesthetized with phencyclidinum NFN (2 mgikg i.m.) combined with inhalation of halothane NFN. Details concerning the technique used are reported in Gyrd-Hansen’s study (1968) where the determination of the extraction per- centage of PAH is described. Urine was collected from the kidney for 5 periods of 20 min. through a plastic catheter placed in the ureter. Blood samples from a. renalis and v. renalis were taken a t intervals of 15 min. Clearance experiments were performed in 14 cases (pigs no. 10-20) on un- anesthetized pigs using the technique described by Gyrd-Hansen (1968). In all experiments PAH was given by continiuons intravenons infusion.

The concentration of PAH was determined in urine and plasma by the method describcd hy Bratton and Marshall (1 939) for sulphonamide determination. PAHt,t,l (PAH N4-acetylated PAH) was estimated on the same samples after hydrolysis with hydrochloric acid. All analyses were done in duplicate. The concentration of N4-acrtylated PAH was determined as the differ- ence betwren PAHt,t,l and PAH. PAH and PAHtotal were estimated with a standard deviation of 1.4 o/o in both plasma and urine. PAH and acetylated PAH could not be detected in blood plasma and urine sampled before the start of the experiment.

The extent of acetylation or de-acetylation in the kidney was calculated from the concentrations of PAH and N4-acetylated PAH in plasma from a. renalis and v. renalis and in urine.

The following symbols were used in the calculations: ~-

; I p . k ~ = Concentration of PAH in plasma from a. renalis .-Ia.~p.,H = Concentration of N4-acetylated PAH in plasma from a. renalis VpAH = Concentration of PAH in plasma from v. renalis Va.p.\~ == Concentration of N4-acetylated PAH in plasma from v. rcnalis U1’.kE = Conrentration of PAH in urine U,-P.\H = Concentration of N4-acetylated PAH in urine

The ratio U, P~H/UP. \H i s designated k. D = Urine flow F .= Renal plasma flow

The total amount of PAH plus Nkcetylated PAH excreted in the urine must correspond to the amount of PAH plus N4-acetylated PAH removed by the passage of blood through the kidney, thereforc : ( 1 ) (CPAH 7. U ~ - P A H ) D = ( . ~ P . ~ H - V P A H ’ A ~ PAH-V~-I,.~H)F Substitution of k x c i p . ~ ~ instead of Ua PAH gives the following:

(2 ) ! G P A H + k S U f A H ) L ) = (-4P1H-VPZHIAa-P.XH-Va P . ~ H ) F

From f 1) and ( 3 ) the following is obtained:

(4) c a P.ZH

The acetylation percentage is calculated as percentage of the amount of PAH delivered to the kid- ney by the renal artery:

F ( AP.~H-\’P.~H + -ha-PAH-\ .a-PAH ~.. F (.%FA ti -\.Pr\H i ;\a PaH--Vs P.i H

D ( 1 +k)D

The placing of (4) in (5) and reduction gives:

A negative acetylation prccentage indicates de-acetylation.

the mean (S .E.M.) . and p were determined by standard techniques (Freund 1967). Analysis of variance and statistical calculations of standard deviation (S.D.). standard error o l

Results Acetylation and de-acetylation

9 expts., each with 5 observations, were carried out in 9 pigs in order to determine the degree of acetylation of PAH in the kidney. In Table I the mean values of the 5 observations for each animal are shown. It is seen that the acetylation percentage

PAH CLEARANCE I N PIGS 251 TABLE I. Acetylation and de-acetylation in the kidney*

Animal Renal artery no. -

PAH N4-a-PAH Flglml Flglml

Renal vein Urine Acetylated in the kid- ney as percentage of

PAH N4-a-PAH N4-a-PAH/PAH the amount of PAH Flg/ml Flg/ml delivered to the kidney

1 45 11 2 40 13 3 23 8.5 4 70 33 5 43 18 6 74 14 7 13 3.9 8 94 10 9 110 18 Mean kS.E.M.

2.0 1.9 0.35 10.1 3.3 2.7 0.39 6.3 0.9 0.9 0.44 4.0 5.6 2.6 0.2 1 -21.1 7.3 4.1 0.29 - 6.3 7.5 4.0 0.22 4.7 1.8 0.5 0.33 1.5 5.5 1.5 0.09 - 2.1

13.8 2.4 0.19 2.0 - 0.1 i 3 . 1

* The figures are average values of 5 observations.

varies between + 10.1 and -21.1, with an average of -0.1 0/6. This means that acetylation of PAH was found in 6 of the pigs, while there was de-acetylation of N4- acetylated PAH in the last 3 ones. By analysis of variance was shown that there was a significant difference among the animals (p<O.OOl) while there were no signifi- cant differences within the periods (p>0.50). This means that the analytical error itself can not explain the variations in acetylation percentage.

Extraction percentages

x 100 for PAH and N4-acetylated PAH The extraction percentage

were calculated from the concentration of PAH and N4-acetylated PAH in plasma from a. renalis and v. renalis (Table I). The arterial concentrations varied between 13 and 110 pg PAH/ml and 4-33 pg N4-acetylated PAH/ml, and the average ex- traction percentages were 91 per cent for PAH and 84 cent per for N4-acetylated PAH (Table 11). The arterial blood pressure was between 90/75 and 165/105 mm Hg during the experiments.

1 Aconc.-Vconc.

Ace,,.

Clearance of PAlY and JV4-acetylated PAH The concentra5on of N4-acetylated PAH in plasma was in average 28 per cent of the concentration of PAHtotal (Table 111). The ratio between the clearance of

TABLE 11. Renal 'extraction of p-aminohippuric acid and N4-acetylated p-aminohippuric acid.

Extraction per- Animal no. centage of Mean +S.E.M.

1 2 3 4 5 6 7 8 9

PAH 95 92 96 92 83 90 86 94 88 91&1.4 N4-a-PAH 83 80 89 92 77 72 87 86 87 84+2.1

2.52 N. GYKD-HANSEN 4ND FOLKE R4SZIt 'SbEN

I \BLF 111. Rrnal clearance of p-aminohippuric acid and N4-acetylated p-aminohippuric acid

Concentrations in plasma

.4nimal Body PAH W-a-PAH PAHt,t,, N4-a-PAH no. weight kg pgiml ficg'ml Pdm1 In plasma

4-32 17 -128 1 0--- 3 3 1 7 -41 20-- 44

XIcan .-. S.E..\I. '8 Z ~ 1 . 7

1-9 47---93 13--109 10 -20 21- -12-1 12-28 i -17

N3-acetylated PAH and PAH varied between 0.40 and 2.09, mean 1.08 = 0.07 'S.E.M.). The ratio between the clearance of PAH+,t,l and PAH varied from 0.79 to 1.23, with a mean of 1.01 0.02 (S.E.M.).

Discussion

I t was shown by the work of Setchell and Blanch (1961) that kidney tissue from pigs acetylates PXH in aitro. In the present study, acetylation of P-4H was demonstrated in oiiv in the kidneys of 6 pigs, while in 3 animals there was de-acetylation of 3 4 -

acetylated PAH in the kidneys. The percentage acetylation and de-acetylation in the kidneys involves a corresponding error in the PAH clearance and consequently the RPFpffectivp. This means that in six cases the clearance of PAH and RPFeffectivp bvould be determined 1.5-10 per cent lower and in three cases 2-21 per cent higher than if metabolism of PAH and XI-acetylated PAH did not take place in the kidneys. The error caused by metabolism of PAH and N4-acet)-lated PAH in the kidneys can be avoided if PAHt,t,l is used for calculation of PAH clearance and

'The large variations in the ratio between clearance of n'l-acetylated PAH and PAW ll 'able 111) may partly be due to acetylation and de-acetylation in the kidney and partl?- to the low concentration of N*-acetylated PAH in plasma estimated as the difference between PAHtotal and PAH. A comparison between the clearance of PAHtotal and PAH (Table 111) shows that the mean of the ratio between C' "PAH total

and C ~ A H is 1.01 -i 0.02 (S.E.M.) with variations fironi 0.79 to 1.23. The analytical crror on these clearance ratios is 2.8 ';, {S.D.) and is obviousl>- small in comparison ivith the variations in clearance ratios. This means that an acrtylation and de- acetylation has taken place in the kidneys.

It'hether use of PRH clearance is unsuitable for calculating RPF,ffe,ti,, in pigs, as reported by Nielsen et al. 119661, depends partly on the number of animals and on the purpose of the RPFeffeCtive determination. If the number of animals is small, Pr\Htocal is preferable in determining absolute KPVeffective, since in individual de- terminations there could be an error of up to 20 per cent if PBH were used (last colurnn of 'Table 111). However, if the mean value for a large number of animals is required, clearance of PAH and P.4Htotal will give identical KPFpffpctivp values, since the average ratio between clearance of PAHtotal and PAH is 1.01.

KPJ;effecti\.e.

P A H CLEARANCE IN PIGS 253

Clearance of Clearance ratio

PAH N4-a-PAH PAHtotat N4-a- PAH/ PAH PAHtotatiPAH ml/min/10 kg ml/min/lO kg ml/min/10 kg

16-43 17-44 18-34 0.40- 1.44 0.79-1.12 53-106 42-161 58-105 0.66-2.09 0.90-1.23

1.08*0.07 1.01 50.02

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