Simulation of Phenylketonuria in Rats by Extended p-Chlorophenylalanine Treatment

Abstract

The brain damage which occurs in untreated phenylketonuria probably results from the high plasma phenylalanine concentration prevailing in this condition, as treatment with a low-phenylalanine diet mitigates the situation. The actual cause of the brain damage is not known, but investigations have been carried out with high phenylalanine loads to simulate phenylketonuria and so elucidate the nature of the ensuing abnormalities. Hypotheses involving phenylalanine or its toxic metabolites in the impairment of vital metabolic processes have been tested (Agrawal et al., 1970; Weber et al., 1970). The results obtained, however, under short-term acute loads probably do not reflect the situation prevailing in phenylketonuria; although the plasma phenylalanine reaches phenylketonuria values for short periods, tyrosine also rises appreciably. A number of workers have used p-chlorophenylalanine as an inhibitor of phenylalanine hydroxylase in vivo (Lipton et al., 1967), hoping to produce a more sustained hyperphenylalaninaemia with a near normal tyrosine concentration, i.e. a more valid model phenylketonuria. However, we found that a single pretreatment of young rats with p-chlorophenylalanine does not significantly alter the response to a phenylalanine load. We have extended this by carrying out phenylalanine tolerance tests on weanling rats after treatment with p-chlorophenylalanine for up to 20 days. Four groups of weanling rats were treated daily, by intraperitoneal injection, with (a) 300mg of p-chlorophenylalanine/kg body wt., or (6) 300mg ofp-chlorophenylalanine/kg plus 200mg of L-phenylalanine/kg, or (c) 200mg of L-phenylalanine/kg, or ( d ) an equal volume of saline. Animals from each group were killed on different days and their liver phenylalanine hydroxylase activity was determined by using ~-[U-'~C]phenylalanine as substrate, separating the product chromatographically and counting the radioactivity in both substrate and product (Bender & Coulson, 1972). Liver phenylalanine hydroxylase activity reached a minimum between 5 and 7 days in groups (a) and (6) (about 15% of control values), whereas group (c) did not differ from control value. After 7 days of p-chlorophenylalanine treatment the activity gradually increased towards the control values and cessation of treatment resulted in a rapid return to control values (2 days). Phenylalanine tolerance tests were performed by injecting rats with 500mg of L-phenylalanine/kg, and removing blood at 30min intervals. Plasma phenylalanine and pchlorophenylalanine were determined by the method of McCaman & Robins (1962), after separation on t.1.c. (this eliminates the mutual interference between phenylalanine and p-chlorophenylalanine). Tyrosine was determined by the method of Wong et al. (1964). Figs. l(a) and l(6) show phenylalanine and phenylalanine/tyrosine ratios after phenylalanine loads in control animals and in rats after 1 and 7 days ofp-chlorophenylalanine treatment. Both are of the same order as those observed in untreated phenylketonuria children. The basal phenylalanine concentration (before injection of the phenylalanine load) was subtracted from each value, after the peak, and the net elevated values plotted against time on semi-logarithmic paper. The slope of the line gives the first-order rate constant for the disappearance of phenylalanine load ( k ) (Woolf et al., 1967). Fig. 2 shows phenylalanine tolerance curves for animals treated with p-chlorophenylalanine for different periods and for controls. Phenylalanine loads produce a sustained hyperphenylalaninaemia over several hours in the 5-7-day-treated animals. Further, there is a good correlation (r = 0.89; P<O.OOl) between k and the liver phenylalanine hydroxylase activity. This supports the view that the main metabolic pathway of phenylalanine in these animals is by liver hydroxylation to tyrosine. From these studies we concluded that extended treatment withp-chlorophenylalanine over 5-7 days causes a decrease in liver phenylalanine hydroxylase activity which consequently lowers phenylalanine tolerance so that loading doses of phenylalanine produce a simulated phenylketonuria, over a period of several hours.