The Discovery of PKU by Dr. Asbjørn Følling: Norway, 1934

(Quote reprinted with permission from:  Virginia Schuett of National PKU News USA)

This story is based on a talk given by Dr. Ivar Følling, son of the man who discovered PKU. It was presented at a meeting in Elsinore, Denmark, May 24-27,1994. The meeting was organized to celebrate the 60th anniversary of Dr. Asbjørn Følling's discovery of PKU in Norway, 1934. I was fortunate to be able to attend that meeting. I thought you would find the presentation as fascinating as did the rest of us there. It will give you a wonderful insight into the man whose discovery paved the way for early diagnosis and treatment of PKU. The talk, along with other talks at the symposium, were just published in a special supplement to the journal Acta Pediatrica (#407) and I print the story with permission from the publisher. I have made a few minor editorial changes, simplifying some technical aspects and shortening it slightly. --Virginia Schuett, National PKU News.

This story is a small chapter in the history of medicine. It tells about the discovery of phenylketonuria (PKU), which has a more general importance than merely benefit to PKU patients. The reason that I tell the story is that the discoverer was my father, and in Norway we often term it Følling's disease, characteristic of the modesty of my countrymen. 

The Discovery

The stage is set in 1934. A mother with two severely mentally retarded children came to see my father, and to ask for his advice. As usual in such cases she had asked many doctors for help, which none had been able to give. But this woman was unusually persistent and would not accept the situation without explanation. She had also noticed that a peculiar smell always clung to her children. Why did she choose my father as a source of advice? Today doctors usually have some knowledge of chemistry and biochemistry, whereas at that time this was more unusual. He was first trained as a chemist and later studied medicine. He held a professorship of nutrient research at the University Hospital in Norway. There was no laboratory of clinical chemistry in the hospital and so he worked in the attic of the medical ward. At that time there was an increasing awareness of the potential power of biochemistry in the field of medicine. Many doctors too optimistically believed that almost every problem could be solved by skilled biochemists. 

This woman was advised to seek help from my father. He of course had no real hope of being able to help her. But he did not want to reject her, and he agreed to examine the children. On clinical examination he found nothing of importance, except the feeblemindedness, which was beyond doubt. The girl, 6.5 years old, could say a few words, was fond of music, had a spastic gait and a whimsy way of moving about, apparently at random. At times she had an enormous appetite, at other times none. 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. 'Men he used procedures that resulted in obtaining neat crystals with a constant melting point, a probable sigh of purity. 

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. His friends in the medical ward at once renamed the substance "the idiot acid." Did other mentally retarded patients excrete the acid? He collected samples from 430 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 then wrote about his discovery and these 10 patients, in German, in the journal Hoppe Seylers Zeitschrift fur Physiologische Chemie, in 1934: "On excretion of phenylpyruvic acid in the urine as an anomaly of metabolism in connection with mental retardation."He suggested the name "oligophrenia phenylpyruvica." This is undoubtedly his main scientific contribution.

Further Elucidation

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. On rereading the article they realized that they had missed the word "not." Proteus was not able to do the conversion. Hence, their misreading had led to a fruitful discovery. Instead of measuring phenylpyruvic acid, they had actually measured phenylalanine. Now they were able to measure phenylalanine in blood and they showed that these patients had very high levels. Loading experiments in animals and humans indicated that the high phenylalanine levels were probably the source of phenylpyruvic acid. The hypothesis of a block in phenylalanine metabolism was strengthened. They also found large amounts of phenyllactic acid in urine. 

The last question that he addressed experimentally was: Is it possible to detect the carriers of the trait, and thereby provide genetic counseling? His line of reasoning was as follows: patients with two defective genes cannot metabolize phenylalanine normally, whereas with two normal genes metabolism is effective. Perhaps those with one normal gene lie somewhere in between and excrete phenylpyruvic acid if they are given a large dose of phenylalanine. He bought phenylalanine and took a dose. His urine turned green with ferric chloride. Was he a carrier? His assistant took a dose, with the same result, and so did several others. They could not all be carriers. It was far too unlikely. What went wrong? It turned out that phenylalanine was a mixture of the "L" and "D" forms of the amino acid. None of us can hydroxylate the "D" form and therefore it accumulates and turns into phenylpyruvic acid. Several years later, when they finally go hold of the pure "L" form, they were able to show that carriers had higher phenylalanine levels than non-carriers on loading. Therefore they concluded that genetic counseling is possible. In the meantime others had reached the same conclusion. 

Perspective

The discovery and elucidation of PKU is a medical advance because it has changed the lives of patients from one of disability to one of ability. However, to my mind there is a general aspect to the story which is more important and which represents the major step. 

I will therefore turn to the background against which the discovery has to be viewed. The English scientist Archibald Garrod had coined the term "inborn errors of metabolism" in 1909, and he knew of four such conditions at that time: alcaptonuria, pentosuria, cystinuria and albinism. I found his article on alcaptonuria from 1902 stored in the attic of the library. It is a masterpiece, showing his great power of biological insight and imagination. He described this rare biochemical abnormality of excreting homogentisic acid as congenital, hereditary, widely harmless but perhaps slightly inferior to the normal way, with theoretical waste of energy. He foresaw that similar biochemical individualities cause such characteristics as obesity and hair color. He also suggested their Mendelian genetic pattern and that small biochemical steps are important in evolution and in creating biological diversity. Penetrating thoughts in 1902. However, none of the four inborn efforts affected the brain. PKU is obviously an inborn error of metabolism and it was the first to be shown to affect the mind. This was a completely new concept in medicine and to my mind this represents the major step. Today more than 200 such conditions are known and it is common to search for biochemical explanations in mental diseases. 

PKU has for many years served as a model in the field. The pattern of inheritance is illustrative. Screening is efficient and the logical treatment of the disease converts the fate of the patient from severe disability to good health. The problem of the pregnant patient, which once threatened to create as many retarded patients as had been prevented, is now virtually solved. The gene technology of PKU is an instructive lesson in the field: purifying the rat enzyme; raising antibodies; precipitating the enzymes together with its messenger RNA: using messenger RNA to make DNA: assuming similarity between rat and humans and using the DNA probe to find the human gene with its introns and exons; tracing the mutations of several types; and the concept of patients being compound heterozygotes. As far as I am aware, more than 100 different mutations have now been found, with corresponding enzyme activities ranging from zero to normality. Gene technology has led to detailed screening of families and prenatal diagnosis. It would be fitting if PKU also became an example of successful somatic gene therapy. We will see. 

Honor

I will now turn to some more circumstantial events connected to the story. After the discovery of PKU in 1934 only a small group of specialists in the field were interested. Its general importance was acknowledged much later. In 1962 my father received an invitation to attend a conference on mental retardation in Washington, DC. He was reluctant to go. He had retired and believed that only younger people should travel to conferences. After a second letter, with an urging plea that he should attend, he decided to. It turned out to be a major event. The late President John F. Kennedy had founded an award for scientists in the field of mental retardation to commemorate his brother who died as a pilot in the second world war, and because he had a mentally retarded sister. My father was the first prize winner, together with three other scientists and the president himself presented the award. 

My father rarely told about his scientific work at home. The story that I have told is therefore by no means his version. I have read his papers, made the review, and put it into a context. I hope that it is not too biased. 

Knowledge Leads to Humility

Finally, I would like to write a few words on a more personal note about my father. He was a kind and modest man, well-liked and many asked for his advice on personal matters. He never advised without being asked, out of respect for personal integrity. But when asked, he made an effort to give valuable answers. He was philosophically inclined as is evident from some of his writings. He pledged the importance of the equilibrium of nature and held that man should consume no more than will be regained. For example, today we are all concerned about pollution and exploitation of resources. He voiced that concern in the 1930's. I will quote parts of an essay that he wrote on this matter (translated).

"Twenty or thirty thousand years ago I- man- lived in a subtropical forest I made my living from fruit and roots, and I stole eggs from birds' nests because birds had not discovered what a predator man is, and therefore didn't hide their nests properly. I gathered oysters in the bay, and occasionally I killed a buck with a stone or a bat. What did my presence cost? Nothing. Nothing, even if I consumed my 3000 calories a day? Already before I was there the fruits grew by taking up water, potassium, phosphorus and nitrogen from the soil, and carbon dioxide back to the air. Each turn is only a loan of resources given back at the end of the cycle. Then I came. It meant no change. I dropped my urine and my stools on the ground, and when I died everything was returned. I gave back to the soil and to the atmosphere what belonged to them. The balance of nature was zero. I owed nothing." 

He proceeded by describing man's agricultural and technological development. He was not against it. He praised it, when it alleviated toil and suffering and provided cultural growth. But he was concerned when the equilibrium was disturbed too much, and when resources were irreversibly lost, as for example with the consumption of oil. It is a mere detail whether the exploitation takes 10 years, 100 or 1000, when man should live on earth for a million years. He would certainly have been deeply concerned about today's extinction of species at a rate of thousands a year, when evolution takes millions of years to recreate new ones. I believe that his ideas about the importance of equilibrium were based not only on his medical and chemical insight, but also on his background as a child on a farm. 

Finally, there is a saying that "knowledge leads to power." This idea was alien to him. He held "knowledge leads to humility." He also said that we are all tools in the hand of the Lord and he was grateful for having been a toot of some use. If I should select one word above others to characterize my father, it would be "insight," both professionally and privately. There is no difficulty in holding current views, and we are all clever in thinking the thoughts of fashion. But to create a new idea, and to foresee the development before time is ripe, that is insight. To see further than the obvious, and to put things into a wider context, is insight.