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BIOCHEMICAL SOCIETY TRANSACTIONS

production, increased with increasing initial phenylalanine concentration up to 5 mmol/ litre and then fell progressively. Although the rate of hydroxylation fell above this concentration, the initial rate of the phenylalanine removal increased over the same concentration range, being 0.6pmol/min per g at 5mmol/litre, and 1.5pmol/h per g at 30mmol/litre. These results suggest that at phenylalanine concentrations above 5 mmol/litre, alternative pathways of phenylalanine degradation are followed. Experiments in which 14C-labelledphenylalanine was incubated with isolated hepatocytes, and 14C02collected, showed that, at concentrations above 5 mmol/litre, there was a substantial decarboxylation of phenylalanine. The initial concentration of phenylalanine giving half-maximal rates of phenylalanine hydroxylation (‘apparent K,,,’) in the perfused liver was 1.50mmol/litre. This is similar to the K,,, for phenylalanine reported for purified rat liver phenylalanine hydroxylase assayed in the presence of dimethyltetrahydropterin[ 1.22-1.42mmol/litre (Gillam et al., 1974), 1.Ommol/litre (Kaufmann, 1969)l. In the perfused-liver experiment, no cofactor is added, thus the ‘apparent K,,,’ for phenylalanine is determined in the presence of the naturally occurring pterin cofactor present in the liver. In the presence of 1 mmol of phenylalanine/litre, the initial rate of phenylalanine hydroxylation, measured in the second hour of perfusion after the addition of a second load of phenylalanine (1 mmol/ litre) at 1h, is identical with that measured during the first hour. This suggests that the cofactor content of the liver is maintained over the 2h period. Intraperitoneal treatment of rats with p-chlorophenylalanine (300mg/kg for 2 days), a n inhibitor of phenylalanine hydroxylase, resulted in an almost complete inhibition of phenylalanine hydroxylation at phenylalanine concentrations below 5mmol/litre. However, under these conditions, phenylalanine removal still took place, suggesting that when the hydroxylation pathway is inhibited, phenylalanine is metabolized via alternative pathways under conditions where hydroxylation is normally the major reaction. Kaufmann (1971) has suggested that the inhibition of phenylalanine hydroxylase by its own substrate found in vitro is relevant to the regulation of the enzyme activity in vivo. The results presented above suggest that an alternative explanation of the decreased rate of hydroxylation at high concentrations of phenylalanine is the involvement of alternative reaction pathways having a K,,, for phenylalanine greater than that of phenylalanine hydroxylase. This explanation is also supported by the observation that at low phenylalanine concentrations, when the hydroxylase is inhibited by pchlorophenylalanine, phenylalanine metabolism still takes place. Ernbden, G. & Baldes, K. (1913) Biochem. Z . 55,301-322 Gillarn, S. S., Woo, S. L. & Woolf, L. I. (1974) Biochem. J. 139,731-739 Hems, R., Ross, B. D., Berry, M. N. & Krebs, H. A. (1966) Biochem. J. 101,284-292 Kaufrnan, S.(1969) Arch. Biochem. Biophys. 134,249-252 Kaufrnan, S . (1971) Ado. Enzymol. Relat. Areas Mol. Biol. 35,245-319 Krebs, H. A., Cornell, N. W., Lund, P. & Hems, R. (1974) Proc. Alfred Benzon Symp. 6th 726-750 Woods, H. F., Eggleston, L. V. & Krebs, H. A. (1970) Biochem. J. 119,501-510 Schirnassek,H. & Gerok, W. (1965) Biochem. Z . 343,407-415

The Metabolism of Some [l-’4C]Glycyl Dipeptides in Mice BARRY SAMPSON, BRIAN BARLOW and ANDREW WILKINSON Department of Paediatric Swgery, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, U.K. Peptides occur as an important component (130%) of the total amino acid content of the protein hydrolysates used for intravenous feeding. The fate of peptides administered intravenously is largely unknown, although many workers have reported urinary losses of up to 40% of the peptide input (Christensen et al., 1947; Wei et al., 1972). 1975

w

Table

Recovery of radioactivity in expired COz and in urinaryfractions

Results are expressed as a percentage of total injected radioactivity, f s . ~Additional . urinary radioactiv

,

.

coz

.

41.1 f 4.1 53.7 f 7.3 43.8 f 1.5 47.1 f 4.5 33.8 f 3.2

27.5 f 3.1 27.1 f 4.1 25.8 f 2.0 29.1 f 3.5 1.86 f 1.6

24h

2h

Radioactivity (% of total injected) U c

2h c

GlY Gly-Gly Gly-L-Leu Gly-L-Phe G~Y-L-P~o

-

-

0.83 f 0.03 0.9 f 0.67 0.3 f 0.18 0.76 f 0.08 2.5 f 0.12

Peptides

Amino acids

2.3 rf: 1.6 1.2 f 0.3 0.8 rf: 0.3 8.8 f 4.4

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BIOCHEMICAL SOCIETY TRANSACTIONS

There have been few studies on the metabolism of peptides, apart from the recent 'avalanche' of data on peptide transport and intestinal absorption. Hoberman & Stone (1952) showed that 'SN-labelled glycylglycine, given intradermally, was metabolized by the rat to the same urinary products as are obtained from 'SN-labelled glycine. Similarly, a-~-aspartyl-[l-'~C]glycine,given intravenously, was shown to be metabolized, by man to COz, to the same extent as [l-'4C]glycine, whereas the corresponding 8-aspartyl peptide was recovered unchanged in the urine (Buchanan et al., 1962). There have been (Weiss & Klein, 1969); E-(N-y-L-glutamy1)-L-lysine studies [L-prolyl-L-hydroxyproline (Waibel &Carpenter, 1972)] on a few other small peptides, and indeed there is currently a debate over the metabolism of one (L-prolyl-L-leucylglycineamide) of the sinallcr peptide hormones (Walter et al., 1975). The metabolic fate of several [l-14C]glycyldipeptides has been studied, and compared with that of [l-'4C]glycine. The labelled dipeptide was diluted with unlabelled peptide to a specific radioactivity of 20-50mCi/mmol and dissolved in 0.9 % NaCl to a concentration of 0.1 M. A 0.2ml portion (containing 20pmol of dipeptide) was injected into the tail vein of adult A2G mice. A minimum of four animals was used per experiment. The mice were placed in a totally enclosed metabolism cage for the collection of urine and expired COz. The latter was absorbed into ethanolamine-2-methoxyethanol(1 :2, v/v) by means of a forced air flow. At the end of the experiment, the animals were killed by cervical fracture, and various tissues were removed, as indicated below. Protein was isolated by homogenizing the tissues in 3 vol. of 10% (w/v) trichloroacetic acid, and the precipitate extracted with ether and acetone. Urinary peptides were separated from amino acids by chromatography of their Cu(I1) complexes on DEAE-Sephadex A-25 (Giliberti & Niederwiesser, 1972). The peptides studied in this way were glycylglycire, glycyl-L-leucine, glycyl-L-phenylalanine and glycyl-L-proline. The percentage of total administered radioactivity recovered in the urine fractions and as COz from each of the peptides at various times after administration is shown in Table 1. The urinary radioactivity shown is only that in the peptide and amino acid fractions. This is about half of the total urinary radioactivity, the remainder being lost as organic acids etc. The only peptide to differ significantly from glycine is glycylproline (P< 0.05 for all results). Studies on the rate of expiration of labelled COz show very similar results for glycine and glycylglycine. The amount of radioactivity bound to the protein of various tissues from labelled glycine and glycylglycine is shown in Table 2. Significant differences between the peptide and amino acid occur only in the intestine and liver, after 24h (P< 0.01, P < 0.001 respectively). The time-variations observed are to be expected from a single pulse dose, namely, an initial rise in activity, followed by a decline, owing to catabolism of labelled protein and redistribution of the label in the body.

Table 2. Radioactivity in protein-bound tissue fraction Results are expressed as percentage of total administered radioactivity, ~ s . D .Muscle radioactivity is an estimate based on the assumption that muscle is 40% of total body weight. Radioactivity (% of total administered) GlY-dY c

Intestine Kidney Liver Muscle

.

2h

24h

0.5 h

2h

24h

2.63 ? 0.46 0.83 f 0.21 4.21 f 0.41 4.84 zk 0.22

3.08 k 0.37 0.56 ? 0.08 2.44 f 0.25 6.31 f 1.65

0.90 k 0.20 0.47 f 0.05 3.07 f 0.57 1.24 k 0.53

3.04 k 0.77 1.28 k 0.17 3.56 ? 0.63 3.33 f 1.06

1.87 k 0.22 0.63 k 0.06 3.47 f 0.58 4.99 k 0.84 1975

557th MEETING, LIVERPOOL

687

The data presented here suggest that some dipeptides are as efficient as glycine in energy metabolism. The urinary concentrations of these dipeptides are low, and the losses will be of little nutritional importance. Dipeptides are also readily utilizable for protein synthesis after hydrolysis. The nature of the differences observed is obscure and the interpretation of these preliminary results is difficult. Not all dipeptide homologues are used to the same degree. This will be attributable to C-terminal peptidase specificity. Lack of utilization of glycyl-L-proline may be predicted from the abundance of imino peptides, derived from collagen breakdown, in normal urine (Meilman et al., 1963). It is suggested that the bulk of the non-metabolizable peptides of hydrolysates will be imino and B-aspartyl peptides. Buchanan, D. E.,Haley, E. E., Marin, R. T. & Peterson, A. A. (1962) Biochemistry 1,620-623 Christensen, H. N., Lynch, E. L., Decker, D. G. & Powers, J. H. (1947) J. Clin. Invest. 26, 849-852

Giliberti, P. & Niederwiesser, A. (1972) J. Chromatog. 66,261-267 Hoberman, D. H. & Stone, D. (1953) J. Biol. Chem. 194,383-391 Meilman, E., Urivetsky, M. N. & Rapoport, C. M. (1963) J. Clin. Znuest. 42,40-50 Waibel, P. E. & Carpenter, K. J. (1972) Br. J. Nutr. 27, 509-515 Walter, N., Neidle, A. & Marks, N. (1975) Proc. SOC.Exp. Biol. Med. 148,98-103 Wei, P., Hamilton, J. N. & IeBlanc, A. E. (1972) J. Cun. Med. Ass. 106,969-974 Weiss, P. H. & Klein, C. (1969) J . Clin.Inuest. 48, 1-10

Metabolism of ''C-Ring-Labelled Paracetamol in Humans BARRY H. THOMAS, LAWRENCE T. WONG, IVO HYNIE and WALTER ZEITZ Drug Research Laboratories, Health Protection Branch, Health and Werfare Canada, Tunney's Pasture, Ottawa, Ontario K1A OL2, Canada

Paracetamol(4-hydroxyacetanilide) is a widely used analgesic that in large doses causes hepatotoxicity (Clark et al., 1973). It has now been shown in experimental animals that metabolism in the liver is responsible for hepatic necrosis (Mitchell et al., 1973). In the present paper we report a detailed study of the excretion and metabolism of paracetamol in humans. [14C]Paracetamol (Mallinckrodt, St. Louis, Mo., U S A . ) , specific radioactivity 17.24pCi/mg, was mixed with unlabelled paracetamol so that a 650mg dose contained

Table 1. Excretion of paracetamol and its metabolites in urine [14C]Paracetamol (650mg) was administered orally to five human females. Paracetamol and its glucuronide, mercapturate and sulphate derivatives were separated by paper chromatography, and the cysteine derivative was separated by column chromatography on Sephadex G-10. The data are expressed as the percentage of the dose of paracetamol excreted (mean f s.E.M.) Percentage of dose excreted Time after dosing (h) Glucuronide Mercapturate Sulphate Paracetamol Cysteine derivative Vol. 3

...

0-3

21.46 f 5.29 0.42 f 0.15 11.84 f 1.24 3.30 f 0.86 0.56 f 1.14

0-6

45.10 f 7.29 1.06 f 0.15 20.09 f 1.49 4.47 f 1.22 1.33 f 0.27

0-12 62.63 f 7.59 1.65 f 0.30 27.48 2.73 6.81 f 2.90 1.87 f 0.40