5-Hydroxyindoles in Phenylketonuric and Nonphenylketonuric Mental Defectives

5-Hydroxyindoles in Phenylketonuric and Nonphenylketonuric Mental Defectives C. M. B. PARE St. Bartholomew’s Hospital, London, England

Phenyl ketonuria Phenylketonuria is an inborn error of metabolism. Fundamentally there is a failure of hydroxylation of phenylalanine to tyrosine in the liver. This results in plasma concentrations of phenylalanine 2 0 4 0 times greater than normal and the presence of abnormal metabolites in the urine, of which phenylpyruvic acid is the best known. Clinically phenylketonuric patients are severely mentally retarded with a defective myelination of the brain. These children are of normal intelligence a t birth and if the plasma phenylalanine concentrations can be maintained a t normal levels with a phenylalanine-restricted diet, the children retain a relatively normal I.&. In 1954, Armstrong and Robinson reported the finding of an increased excretion of indoleacetic (IAA), indolelactic (ILA) and indolepyruvic acids and indican in the urine of phenylketonurics and in 1957 we demonstrated decreased concentrations of 5-HT in the serum and of 5-HIAA in the urine of affected patients (Pare et al., 1957, 1959). Inquiry into the cause of these changes has been helped considerably by the study of animals fed on diets high in phenylalanine. A biochemical state similar to that of phenylketonuria is induced and similar abnormalities in indole metabolism occur, including a decreased concentration of 5-HT in brain (Yuwiler and Louttit, 1961). There are three main ways in which an abnormality of tryptophan metabolism can occur and these have been well reviewed by Berlet (1965). OF TRYPTOPHAN TO 5-HTP HYDROXYLATION

Baldridge et al. (1959) and Perry et al. (1964) found that following an oral load of tryptophan the excretion of 5-HT and 5-HIAA in the urine of phenylketonurics is only slightly increased and they suggested a defect in the hydroxylation of tryptophan similar to that of phenylalanine. Freedland et al. (1961) in fact demonstrated that phenylalanine and tryptophan hydroxylase activity in the liver depend on one and the same enzyme though tryptophan has a much poorer affinity for the system. Renson et al. 159

160

C. M. B. PARE

(1962) confirmed this but suggested first, that this system has little physiological significance for tryptophan metabolism compared with extrahepatic systems. Second, they pointed out that a lack of hydroxylation of tryptophan by the liver could not be the cause of the tryptophan abnormalities in phenylketonuria as these abnormalities are reversible by dietary regimes (Pare et al., 1958; Baldridge et al., 1959). Yet again, the fact that these abnormalities of tryptophan metabolism can be produced in animals with an intact phenylalanine hydroxylase system (Huang et al., 1961; Yuwiler and Louttit, 1961; Boggs et al., 1963a,b) suggests that other mechanisms are more important and the demonstration by Lovcnbcrg et al. (this symposium) of an inhibition of tryptophan hydroxylase in brain by phenylalanine is obviously rclevant. 5-HTP DECARBOXYLASE McKean et al. (1962) found that 5-HTP failed to increase brain 5-HT when given to animals pretreated with phenylalanine compared with untreated controls. This finding might be due to interference either with uptake of 5-HTP by brain or with 5-HTP decarboxylase activity. Davison and Sandler (1958) had previously shown that in witro phenylalanine metabolites were inhibitors of 5-HTP decarboxylase activity and one of these a t least, phenylacetic acid, has a similar action in Viwo (Sandler and Close, 1959; Sandler ct al., 1959). On the basis of a somewhat retarded urinary excretion of 5-HIAA following intravenous administration of 5-HTP in phenylketonurics, Pare et al. (1958) suggested that such an inhibition of decarboxylase activity might be the cause of the decreased concentrations of 5-HT and 5-HIAA, a deficiency which would be reversible by dietary measures. However, this has not been confirmed by animal experiments. As mentioned above, animals fed on a diet high in phenylalanine developed high concentrations of senini phenylalanine, low levels of 5-HT and 5-HIAA in serum and urine respectively, and a decreased concentration of 5-HT in brain. This decreased brain concentration of 5-HT is, however, not accompanied by any decrease in 5-HTP decarboxylase activity (Hsia et al., 1963). DEFECTSIN TRANSPORT OF TRYPTOPHAN AND 5-HTP Defects in transport of tryptophan and 5-HTP now seem more important than enzymic defects. Boggs et al. (1963a) showed that raising the blood phenylalanine concentration of experimental rats would cause a decrease in liver 5-HT which they thought might be due to an inhibition of absorption of tryptophan from the intestine. Conversely, McKean et al. (1962)showed that such an elevation of phenylalanine plasma levels could be prevented by the addition of 5% tryptophan to a high phenylalanine-tyrosine diet. Last year, Yarbro and Anderson (1966) in an important paper demonstrated a transport

5-HYDROXYINDOLES IN MENTAL DEFECTIVES

161

defect in the absorption of tryptophan from the gut of phenylketonuric subjects. Six patients were studied before and after a low phenylalanine diet. Hyperphenylalaninemia was associated with a striking limitation in absorption of tryptophan and tyrosine as shown by low fasting tryptophan values, limitation in the peak plasma tryptophan response to an oral load of tryptophan, increased fecal tryptophan and tyrosine, and increased fecal and urinary indoles. With a low phenylalanine diet all these values returned to normal (Fig. 1). Such a defect in intestinal absorption of tryptophan would

FIG.1 . Mean plasma tryptophan responses to oral L-tryptophan load in normal and in phenylketonuric children before and after dietary treatment. L-tryptophan load: 100 mg per kilogram (from Yarbro and Anderson, Journal of Pediatrics 68, 895-904, 1966, The C. V. hlosby Company, St. Louis, Missouri).

explain the low concentration of 5-HT in serum and of 5-HIAA in urine; the intestinal absorption of indole degradation products, arising from the bacterial decomposition of tryptophan, would account for the increased urinary concentration of IAA, ILA, and indican, the latter possibly setting up a vicious circle as suggested by De Laey et al. (1964).Such a defect in transport would account for the finding of only a slight increase in urinary 5-HIAA following oral administration of tryptophan (Baldridge et al., 1959; Perry et al., 1964). However, oral 5-HTP was found to produce a rapid rise in urinary 5-HT, suggesting that intestinal transport of 5-HTP is normal or a t any rate much less affected than that of tryptophan (Perry et al., 1964).

162

C. M. €4.

PARE

A transport defect similar to that in gut may occur for tryptophan or 5-HTP transfer into brain. Thus McKean et al. (1962) and Schanberg (1963) noted an active transport system for 5-HTP in brain slices and an inhibition of 5-HTP uptake into brain in vivo by phenylalanine to 50% of control levels. Transport defects thus seem to be the most important factor in causing the abnormalities of tryptophan metabolism found in phenylketonuria. The possibility of disturbances in enzyme function, particularly in the immature brain, remains important although there is little evidence to support it. A possible relationship between an abnormality of tryptophan metabolism and the mental deficiency in phenylketonuria is likewise unsubstantiated. No correlation could be found between performance of animals with experimental phenylketonuria in various tests and brain concentration of 5-HT (Yuwiler and Louttit, 1961). Nor was there any correlation between serum 5-HT and urinary 5-HIAA and I.&. in a series of 26 phenylketonuric children. (Pare et al., 1959). Furthermore, measures designed to increase brain 5-HT have failed to improve mental function. Thus Louttit restored the concentration of brain 5-HT to normal in experimental phenylketonuric animals with a MA0 inhibitor, yet failed to improve performance. I n humans we attempted to correct any such deficiency first by administering 5-HTP to three children for 6 months and in another series of patients by the use of iproniazid for 6 months (Kirman and Pare, 1961). Neither group showed any change compared to controls.

DOWN’SSYNDROME (Mongolism) There has been some controversy about whether the levels of serum 5-HT are decreased in this condition. We reported them as normal (Pare et al., 1960) but since then the consensus of opinion is that the serum 5-HT is definitely low (Tu and Zellweger, 1965; Rosner et al., 1965; Berman et al., 1967). Rosner et al. (1965) found a difference between trisomic Down’s syndrome and that due to translocation of the chromosome, the latter having a normal serum 5-HT. However, Berman et al. (1967) could not confirm this. The cause of these low levels is unknown. Tu and Zellweger (1965) studied the effect of tryptophan feeding on serum 5-HT levels. Whereas controls showed a rise of approximately 15%, patients with Down’s syndrome showed no rise in serum 5-HT unless pyridoxine was given simultaneously. This comparatively small difference might suggest a defective absorption of tryptophan from the intestine. However, the levels of serum tryptophan were normal and although O’Brien and Groschek (1962) reported a deficient rise following an oral tryptophan load compared with normal, this difference is so slight as to be unconvincing.

5-HYDROXYINDOLESI N

163

MENTAL DEFECTIVES

OTHERTYPESOF SUBNORMALITY When we started investigating phenylketonuria in 1955, we were struck by the very high serum 5-HT in the subnormal patients we used as controls. We eventually investigated 73 such patients, the majority of whom had high serum 5-HT, sometimes as high as three times normal values (Table I) TABLE I

MEAN Smtrrx 5 - H T A N D URINE 5-HIAA IN NORMAL SUBJECTS, IN MENTALDEFECTIVES, A N D IN VARIOUS OTHERGROUPS

ng 5-HT/ml serum

m g 5-HIAA per gm urine creatinine

~~

Mean (No.) Normals Adults Children Cerebral palsy (normal intelligence) Cerebral palsy (I.&.i50) Mongolism Epiloia (tuberous sclerosis) Phenylketonuria Maternal rubella Unclassified

145(16) 130(7) 157(9) 136(12) 351(28) 146(10) 215(6) 79(26) 391(4) 297(30)

+ SE f 13

+ 16 + 15 f 12

-+ 26

+ 18 + 18

* 10 i 66 f 24

Mean (No.) 3.9(40) 3.9( 18) 3.9(22) 3.5(9) 7.7(25) 7.0(10) 6.15(6) 2.6(26) 6.9(4) 6.8(29)

5 SE

i 0.12 & 0.40 i 0.46 + 0.47 f 0.73 f 1.2 + 1.4 f 0.23 2.18 -+ 0.47

+

(Pare et ul., 1960). Significantly high values were found in groups with mental defect of both genetic and environmental origin. There was no particular drug ingestion pattern in any of these groups and conversely, a small number of epileptics of normal I.Q., who were taking large doses of phenytoin, had normal serum 5-HT levels. This finding has since been confirmed (Schain and Freedman, 1961; Paasonen and Kivalo, 1962; Berman et ul., 1967). The abnormality is not a hospital phenomenon, for high values were also noted in similar patients who were a t home; nor did it appear to be related directly to the associated physical disabilities, as equally disabled cases of cerebral palsy but with average intelligence had normal 5-HT levels (Pare et al., 1960). The rise in serum 5-HT can be partially explained by a significant increase in platelet numbers which was present in the affected group. However, these platelets also contained about twice the concentration of 5-HT as those from a normal control population. The platelets themselves seemed to be normal. There was no increase in adenosine triphosphate concentration. When the platelets were saturated by incubation with excess 5 -HT, their mean content

164

C. M. B. PARE

did not differ from that of normal platelets; more recently, Paasonen and Kivalo (1962) and Paasonen et at. (1964) have shown that these platelets release 5-HT normally with tetrabenazine and inactivate it to the same extent as platelets from patients with normal serum 5-HT. They also demonstrate normal MA0 activity in the homogenized platelets.

.

-

I6 I5 E 14-

:.

Q

f 13-

12II.10-

5

b 9-

$

8-

70

6-

!.

5

;

= 2

-

0 0

o 0.o

8

0"

100 200 300 400 Mean serum 5-HT (ng./ml.)

500

FIG. 2. Mean serum 5-HT and urine 5-HIAA creatinine ratio of groups in Table I. The area of the rings is roughly proportional to the number of cases in the group; solid spots indicate single cases.

The cause of these high values of serum 5-HT is unknown but the fact that the urinary 5-HIAA excretion appeared abnormally high and that there was highly significant between-group correlation for serum 5-HT and urinary 5-HIAA suggests the possibility of an increased 5-hydroxyindole production (Fig. 2). We viewed these findings with some caution because it was impracticable t o obtain 24-hour specimens of urine, and 5-HIAA had to be measured as milligrams per gram creatinine. It was also not possible to keep as close a check on food intake as we should have liked, to exclude dietary sources of 5-hydroxyindoles. However, a recent paper by Tissot et al. (1966) describes similar high levels of urinary 5-HIAA in a group of nonphenylketonuric, nonmongoloid mental defectives. REFERENCES Armstrong, M. D., and Robinson, K. S. (1954). Arch. Biochem. 52, 287. Haldridgc, R. C., Borofsky, L., Baird, H . , Reichle, F., and Bullock, D. (1959). Proc. SOC. Exptl. Biol. M s d . 100, 529.

~ H Y D R O X Y I N D O L E SIN MENTAL DEFECTIVES

165

Berlet, H. H. (1965). P r o p . BmiiL Res. 16, 184. Berman, J., Hulten, M., and Lindsten, J. (1967). Lancet 1, 730. Boggs, D. E., Rosenberg, It., and Waisman, H. A. (1963a). Proc. SOC.Exptl. Biol. Med. 114, 356. Boggs, D. E., McLay, D., Kappy, M., and Waisman, H. A. (1963b). Nature 200, 76. Davison, A. N., and Sandler, M. (1958). Nature 181, 186. Do Laoy, P., Hooft, C., Timmermans, J., and Snoeck, J. (1964). Ann. Puediat. 202, 145, 253, 321. Freedland, R. A., Wadzinski, I. M., and Waisman, H. A. (1961). Biochem. Biophys. Res. Commun. 5, 94. Hsia, D. Y.-Y., Nishimura, K., and Brenchley, Y. (1963). Nature 200, 578. Huang, I., Tannenbaum, S., Blume, L., and Hsia, D. Y.-Y. (1961). Proc. SOC.Exptl. Biol. Med. 106, 533. Kirman, B. H., and Pare, C. M. B. (1961). Lancet 1, 117. McKean, C., Schanberg, S. M., and Giarman, N. J. (1962). Science 137, 604. O’Brien, D., and Groschek, A. (1962). Arch. Diseases Childhood 37, 17. Paasonen, M. K., and Kivalo, E. (1962). Pqchophnmcologia 3, 188. Paasonen, M. K., Solatunturi, E., and Kivalo, E. (1964). Psychopharmacologia 6, 120. Pare, C. M. B., Sandler, M., and Stacey, R. S. (1957). Lanced 1, 551. Pare, C. M. B., Sandler, M., and Stacey, R. S. (1958). Lancet 2, 1099. Pare, C. M. B., Sandler, M., and Stacey, R. S. (1969). Arch. Diaease Childhood 34, 422. Pare, C. M. B., Sandler, M., and Stacey, R. S. (1960).J. Neurol. Neurosurg. Paychiat. 23, 341. Perry, T. L., Hansen, S., Tischler, B., and Hestrin, M. (1964). Proc. SOC.Exptl. Biol. Meal. 115, 118. Renson, J., Weiasbach, H., and Udenfriend, S. (1962). J . Biol. Chem. 237, 2261. Rosner, F., Ong, B. H., Paine, R. S., and Mahanand, D. (1965). Lancet 1, 1191. Sandler, M., and Close, H. G. (1959). Lancet 2, 318. Sandler, M., Davies, A., and Rimington, C. (1959). Lancet 2, 318. Schain, R. J., and Freedman, D. X. (1961). J. Pediat. 58, 315. Schanberg, S. M. (1963). J. P h a m c o l . Exptl. Therap. 139, 191. Tissot, R., Guggisberg, M., Constantinidis, J., and Bettschart, W. (1966). Pathol. Biol. Semczine Hop. 14, 312. Tu, J., and Zellweger, H. (1965). Lancet 2, 715. Yarbro, M. T., and Anderson, J. A. (1966). J. Pediat. 68, 896. Yuwiler, A., and Louttit, R. T. (1961). Science 134, 831.