The boy, almost 4 years old, could not speak or walk, eat or drink on his own. He was unable to fix his eyes on anything, and stool and urine habits were those of a baby.
Urine examination showed no protein or glucose. He then added some ferric chloride to the urines. Ferric chloride was used to detect ketones in the urine of diabetics. Therefore this was part of his thorough routine examination. On adding ferric chloride the color normally stays brownish, and when ketones are present it turns purple or burgundy.
Instead a deep green color developed in the urines. He had not seen this reaction before and to his knowledge it had not been described in the literature. This first important observation prompted the question: is the reaction reproducible and persistent? He told the mother to bring new urine samples after stopping all forms of medication.
The reaction was still there and he concluded that two mentally retarded children excreted a substance not found in normal urine. But which substance? The first task was to isolate and purify the substance.
However, this was before chromatography, isotope techniques, enzymatic methods, and immunochemistry. He had to rely on classical organic chemistry. He tried several extraction procedures. Through these he could detect the substance by the green color reaction. He then used chemical analysis to determine the nature of the substance. Using very accurate and precise methods, he determined that the material had a molecular formula of nine carbon atoms, eight hydrogen atoms and three oxygen atoms.
It was acidic. On mild oxidation it smelled of benzaldehyde. On stronger oxidation it produced oxalic acid which could be precipitated by calcium and benzoic acid. The latter was detected by distillation.
I remember well him telling about his joy and delight when he saw the characteristic crystals of benzoic acid gradually developing on the glass wall of the condensing pan of the distilling apparatus. Phenylpyruvic acid fit all of these observations. He then synthesized phenylpyruvic acid and compared its melting point with that of the crystals. They were identical. However, two different compounds may have the same melting point, but if you mix them, the melting point becomes lower.
Mixing of the two in this instance did not alter the melting point, proving the identity of the substance. Therefore he concluded that the two mentally retarded children, brother and sister, excreted phenylpyruvic acid in their urine.
Normal people did not. This was his main discovery. It was achieved over several months and large amounts of urine were carried from the children to his laboratory. It was of course tempting to speculate that there was a connection between the feeblemindedness and the acid.
He collected samples from patients in different institutions and found the green color in eight Most of the patients were of fair complexion, with a tendency to eczema, broad shoulders, a stooping figure and spastic gait. All were mentally retarded. He continued to work in this field, now with collaboration, but his later publications are less well known.
His next question was: Is the disease hereditary, and if so what is the pattern? The question was obvious. A recessive trait seemed most likely, because among the ten patients there were three pairs of siblings. Moreover, in three families the parents were close relatives, and two parents had seven and five children, respectively, in their second marriages, all healthy children.
He conducted a new investigation of 22 families of affected children where he found an additional 18 affected and 86 healthy siblings. The parents were normal.
This fit well with a recessive autosomal trait. But why do patients produce phenylpyruvic acid? On the basis of chemical similarity, in his first publication, he put forward the hypothesis that these patients were unable to metabolize phenylalanine normally. If so, one would expect high levels of this amino acid to accumulate in the blood. But at that time there was no method available to measure phenylalanine and he had to invent one.
He asked a friend who was a bacteriologist if he could find a bacterial strain that behaved like the patients so that it converted phenylalanine to phenylpyruvic acid. Proteus vulgaris was found to do the job and they then measured the product by the color reaction. To my knowledge this was the first time that bacteria were used as a tool in biochemical quantitation.
But why Proteus vulgaris? They had searched the bacteriological literature and found a description of a similar, but slightly different biochemical conversion by Proteus. They tried it and it was successful. Hence, their misreading had led to a fruitful discovery. Instead of measuring phenylpyruvic acid, they had actually measured phenylalanine. The discovery of phenylketonuria.
Acta Paediatrica ; Jervis GA. Phenylpyruvic oligophrenia: Introductory study of fifty cases of mental deficiency associated with excretion of phenylpyruvic acid.
Metabolic studies in phenylketonuria. Biochem J ; Bearn AG. Archibald Garrod and the individuality of man. Oxford: Oxford University Press; On detection of heterozygotes for phenylpyruvic oligophrenia. Scandinavian J Clin Lab Invest ; Influence of phenylalanine intake on phenylketonuria. Lancet ; Kaufman S. Overcoming a bad gene. The story of the discovery and successful treatment of Phenylketonuria, a genetic disease that causes mental retardation.
Bloomington, IN: AuthorHouse; Guthrie R, Susi R. A simple phenylalanine method for detecting phenylketonuria in large populations of newborn infants. Pediatrics ; Disorders of phenylalanine and tetrahydrobiopterin metabolism.
Heidelberg: Springer; Cloned human phenylalanine hydroxylase gene allows prenatal diagnosis and carrier detection of classical phenylketonuria. Nature ; Phenylketonuria: high plasma phenylalanine decreases cerebral protein synthesis. The substance was extracted through a series of reactions, the final product was tested for its ability to give the characteristic ferric chloride reaction purple color change.
After six recrystalizations, the melting point of the substance able to produce this reaction became constant, suggesting a single chemical present. Further chemical analysis determined the substance was organic 9C, 8H, 3O and contained a benzene ring. Along with additional reaction characteristics, it was determined the substance was most likely phenylpyruvic acid. The increased levels of phenylalanine and its metabolites ie, phenylpyruvic acid in PKU exert their toxicity exclusively on the central nervous system CNS.
This toxicity is believed to be a result of either: a direct neuronal damage by phenylalanine and its metabolites; or b indirect CNS damage by competing with neurotransmitter precursors such as tyrosine and tryptophan, resulting in lower dopamine, serotonin, epinephrine, and norepinephrine needed for brain function.
Until the mids, PKU was considered an unfortunate cause of mental retardation. In , the role of dietary phenylalanine on the severity of PKU was discovered. Efforts to screen newborns for PKU were initiated in the early s. He was also made an honorary member of the Norwegian Medical Society. He was honored with the first Joseph P.
His contributions to metabolic disease, genetics, and clinical chemistry make him a luminary in laboratory medicine. Folling A. The excretion of phenylpyruvic acid in the urine, an anomaly of metabolism in connection with imbecility.
Zeitschrift fur Physiologische Chemie. Google Scholar. Ann Hum Genet. Woolf LI. The heterozygote advantage in phenylketonuria. Am J Hum Genet. The discovery of phenylketonuria: The story of a young couple, two retarded children, and a scientist.
The story of its discovery. J Hist Med. Elgjo RF. Prog Clin Biol Res. Christ SE. J Hist Neurosci. The discovery of phenylketonuria. Acta Paediatr Suppl.
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