Dietary Treatment Of Phenylketonuria Research Articles

Louis Isaac Woolf (24 April 1919 – 7 February 2021)

Professor Emeritus Louis Woolf died aged 101 on Sunday, 7 February 2021, in Vancouver, British Columbia. He is best known for his pioneering work conceiving and developing the first dietary treatment for phenylketonuria (PKU) based on a phenylalanine – a depleted hydrolysate of milk protein. His idea came from a ‘light bulb’ moment at a biochemistry meeting.Louis joined the Biochemical Society in May 1949 and continued until his death. He was thereby one of the longest ever serving members of the Society.He was born in London, the third child of Jewish parents from northern Romania. In 1938 he gained scholarships to study for a degree in chemistry at University College London (UCL) achieving First Class Honours and then a PhD. In 1939, at the outbreak of World War II, he was evacuated to Aberystwyth in Wales, along with the rest of UCL, to complete his degrees. There he met his future wife Frances Mary Richards.In 1947 he secured a post at Great Ormond Street Hospital (GOSH) to research tyrosine metabolism in premature babies. His first publication in a 1950 issue of the Biochemical Journal described this. Included in his chromatography studies were patients with PKU, then considered ‘untreatable’. He had postulated that their severe learning difficulties arose either from their abnormally raised blood phenylalanine concentrations or its toxic metabolite phenylacetic acid. He presented his investigations on the latter at the 1951 Biochemical Society Annual General Meeting in London. He wondered, could PKU patients be treated by lowering their concentration of phenylalanine with an artificial, low phenylalanine diet with added nutrients to make it a complete food? A protein substitute of pure amino acids was then extremely expensive. Fortuitously he went to a Biochemical Society meeting at which an economical growth medium for the microbiological assay of amino acids in protein was described. Phenylalanine and aromatic amino acids were removed from an acid hydrolysate of protein by filtration through charcoal. Louis knew that an acid hydrolysate of the milk protein, casein, was available from Allen and Hanbury, where he had worked, and could provide the basis for his dietary concept. Louis was unable to persuade paediatricians at GOSH to trial this. More receptive was a German paediatrician, Horst Bickel, visiting from his post in Birmingham, who enquired whether Louis could think of a way to treat PKU. Whilst sceptical of Louis’ idea, Bickel successfully treated a 2-year-old girl with PKU in Birmingham utilizing Louis’ method for producing a liquid food low in phenylalanine content. This was a world first and a lasting legacy.Louis moved to Oxford in 1958 to continue research into PKU. It is less well known that, since his time at GOSH, Louis had been convinced that newborn screening for PKU would improve outcomes by enabling earlier dietary therapy. Urine testing was a simple and non-invasive way and he and Senior Medical Officer in Public Health, Dr Nancy Gibbs, trialled a urine screening programme aimed at detecting all cases of PKU in a cohort of babies in Cardiff, Wales, from 1958 to 1959 – another world first which was partially successful. By 1968, with experience from other trials in other UK countries, it was apparent that urine detection in new-borns was insufficiently reliable and blood analysis became the recommendation with the heel prick test we are now so familiar with.Louis’ published work and presentations had become known internationally. In 1968 Dr William Gibson offered him a post as associate professor at the University of British Columbia (UBC). He was promoted to professor 6 years later in 1974. He published extensively, with over 120 papers to his name, a book on renal tubular dysfunction and contributions to books, conferences and supervision of many research students. He retired in 1984 after a 16-year teaching and research career at UBC.A shy and modest man, he was devoted to his family. His wife Frances died in 1991 and Louis is survived by his only daughter Lesley, grandsons Benjamin and Oliver, two great-grandchildren, Graham and Willow, and his youngest brother, Henry. He was beautifully spoken in a refined, old-fashioned British way with an astonishing memory to the end. Generous to people who criticized him, he quietly stood his ground, recognizing that valid debate is integral to scientific discovery. And – like many another innovative scientist – it is in this way that he triumphed over sceptical adversity to leave a legacy in pioneering the successful treatment of PKU.

Management of Phenylketonuria: Current and Future Perspectives

Phenylketonuria (PKU) is an inborn error of phenylalanine (phe) and tyrosine (tyr) metabolism. It is anautosomal recessive disease occurred due to deficiency of liver enzyme phenylalanine hydroxylase (PAH).Hence, phe is not converted to tyr and phe is accumulated in the body. Phe thus channeled to alternativeroutes of metabolism and forms Phenylketones excreted in urine. Early treatment is essential to preventmental retardation and other intellectual disabilities. Dietary treatment remains the main cornerstone tomanage PKU since last 3-4 decades. A diet low in Phe supplemented with special amino acids formulasmust be started soon after diagnosis within seven days of life. Inspite of good results obtained from dietarytreatment in PKU, still there are some issues with palatability of the dietary formulations. There are alsoissues of nutritional deficiencies of vitamins like calcitriol and cobalamin (B12). Poor cognitive and executivefunctions have been observed in patients who do not follow proper dietary treatment. Attempts have alsobeen made to increase the palatability of food under dietary management. Role of large neutral amino acids(LNAAs) and glycomacropeptides (GMP; found in bovine milk) as a newer dietary management have alsobeen explored. In recent era, advances occurred in terms of genetic therapy and enzyme replacement therapywhich opened a new door towards management of PKU. In this review, various treatment aspects of PKUare discussed and explored.

The Early History of PKU.

The story of phenylketonuria (PKU) started in 1934 with Asbjørn Følling’s examination of two mentally retarded siblings from a Norwegian family. However, if their mother had not been so persistent in her search for somebody who could give her a reason why both her children were retarded, Asbjørn Følling’s name might never have been associated with PKU and surely the history of PKU would have started differently. In the short review below, the authors give a partly personal and therefore rare account of the early history of PKU, its treatment and the start of neonatal screening. Prof. Woolf is a pioneer of both the dietary treatment of PKU and neonatal screening; Mr. Adams is a long-time advocate for PKU patient interests.

Biochemical Evaluation of Phenylalanine Ammonia Lyase from Endemic Plant Cyathobasis fruticulosa (Bunge) Aellen. for the Dietary Treatment of Phenylketonuria.

Enzyme substitution therapy with the phenylalanine ammonia lyase (PAL) is a new approach to the treatment of patients with phenylketonuria (PKU). This enzyme is responsible for the conversion of phenylalanine to trans-cinnamic acid. We assessed the PAL enzyme of the endemic plant Cyathobasis fruticulosa (Bunge) Aellen. for its possible role in the dietary treatment of PKU. The enzyme was found to have a high activity of (64.9±0.1) U/mg, with the optimum pH, temperature and buffer (Tris-HCl and l-phenylalanine) concentration levels of pH=8.8, 37 °C and 100 mM, respectively. Optimum enzyme activity was achieved at pH=4.0 and 7.5, corresponding to pH levels of gastric and intestinal juice, and NaCl concentration of 200 mM. The purification of the enzyme by 1.87-fold yielded an activity of 98.6 U/mg. PAL activities determined by HPLC analyses before and after purification were similar. Two protein bands, one at 70 and the other at 23 kDa, were determined by Western blot analysis of the enzyme. This enzyme is a potential candidate for serial production of dietary food and biotechnological products.

Intelligent quotient of late treated phenylketonuric patients: is brain damage still preventable?

Over the past 30 years and since the first trial to treat phenylketonuria (PKU) by Horst Bickel and his coworkers, the dietary treatment of PKU has produced a generation of adult women with PKU who have minimal cognitive deficits and are now of child-bearing age. If PKU is detected early enough in infancy, mental retardation can be prevented by giving a diet containing a restricted amount of phenylalanine. The principle of therapy is that all newborn babies with phenylalanine concentration of 360 µmol or above are to be treated as early as possible to achieve a higher mental function in terms of intelligence quotient, blood phenylalanine levels should be maintained in the range of 120 - 360 µmol/l. Subnormal as well as high phenylalanine levels are to be avoided. In the last few years there are increasing published articles on late treated PKU patients. In these patients, the mean IQ was 57. For those treated after 2 months of age, recent studies have suggested that approx. 23% of those children have normal IQ. In our hospital most of our cases were late treated (15 patients out of 22), the age at which treatment was started ranges from 2 weeks to 9 years. It is noted that those patients who were diagnosed before 2 years of age (6 children) and were on strict phenylalanine low diet with a phenylalanine maintained less than 4mg/dl have a normal IQ (75 or above) (6 patients), one child has difficulty in speech, 2 children had border line IQ (above 70 and less than 75), two children had moderate learning difficulties (IQ above 35 and below 50), 4 children had severe learning difficulties (IQ above 20 and less than 35 of which two of them diagnosed before 2 years of age.

Dietary treatment in phenylketonuria does not lead to increased risk of obesity or metabolic syndrome

BackgroundLittle is known about the consequences of the special energy enriched diet used to treat patients with phenylketonuria (PKU) in terms of obesity and metabolic syndrome (MetSyn) development. ObjectiveTo investigate the prevalence of overweight and obesity, and its consequences in terms of body composition and MetSyn in early treated patients with PKU compared to controls. DesignA sample of 89 patients with PKU (3–30y; 14.4±6.6y) and 79 controls (3–47y; 16.3±7.9y) were studied. In the fasted state, anthropometric, body composition, blood pressure and analytical parameters [amino acids, glucose, insulin, total and HDL-cholesterol (HDL-c), triglycerides (TG), high sensitivity c-reactive protein and uric acid] were performed. Data on dietary intake was collected. BMI was classified using WHO criteria, while the definition from International Diabetes Federation (IDF) was used for MetSyn. ResultsPrevalence of overweight and obesity (32.6% vs. 24.1%; p=0.293), body fat percentage (22% vs. 23.1%, p=0.581) and central obesity (36.9% vs. 36.4%, p=0.999) were comparable to controls. Patients revealed a higher TG/HDL-c (p<0.001). The prevalence of MetSyn was 1.5% and 6.1% in patients and controls, respectively. Patients and not controls with central obesity revealed a further significant increase in TG/HDL-c compared with those without central obesity (p=0.023). ConclusionPatients and controls were similar in terms of overweight and obesity, body composition and MetSyn. However, the dyslipidemia in patients with PKU in relation to overweight and obesity may help us trying to understand the course and the etiology of MetSyn not only in PKU but also in the general population.

Dietary treatment of phenylketonuria: the effect of phenylalanine on reaction time

There is no evidence that high phenylalanine (Phe) levels have irreversible effects on the adult brain. Many adults with phenylketonuria (PKU) no longer follow a protein-restricted diet. Neuropsychological studies have shown that reaction time in adults with PKU is slower than controls. There are no data to show that this is directly related to Phe levels. Another way to assess reaction time is to measure saccadic latency. We have used a portable, head-mounted saccadometer to measure latency in the outpatient setting. Patients with PKU were split into three groups: off-diet (Phe >1,200 μmol/l), on-diet (Phe < 800 μmol/l) and maternal diet (Phe 100-400 μmol/l ). Reciprocal median latency (RML) was compared between groups. Latency was significantly slower in patients who were off-diet than in patients on-diet, on a maternal diet or in normal controls. Reaction times in both diet-treated groups were not significantly different from normal controls. In 16 women planning pregnancy we obtained values before and after they commenced the maternal diet. Stricter control of Phe levels resulted in a significant improvement in reaction times. We conclude that saccadometry is useful in monitoring PKU patients. Adult patients with PKU not on a protein-restricted diet have significantly slower reaction times than controls. In addition, off-diet patients have significantly slower reaction times than on-diet. Paired data show that effects of Phe levels on reaction time are reversible.

Sapropterin Dihydrochloride (6R-BH4) and Maternal Phenylketonuria Two Case Studies

The dietary treatment of phenylketonuria (PKU) is a success story. However, the Maternal PKU Collaborative Study reported microcephaly, congenital heart defects, facial dysmorphology, and intrauterine and postnatal growth retardation in the offspring of women with blood phenylalanine (phe) concentrations &gt;600 µ mol/L. Dietary control of phe during pregnancy is extremely difficult for many women because of hyper-emesis gravidarum, intolerance of the medical food products, and inability/ refusal to follow dietary recommendations. The authors report 2 case studies using 6R-BH4 along with the phere-stricted diet. Two individuals received 6R-BH4 shortly before conception and throughout the pregnancy. A pherestricted diet was followed with blood phe analysis obtained weekly. Interim plasma amino acids, complete metabolic panels, complete blood counts, and analyses for nutritional deficiencies were also obtained during the pregnancy. Both individuals maintained blood phe concentrations within the recommended range (120-360 µ mol/L) nearly all through the pregnancy with a small percentage outside of recommendations. One participant showed deficiencies in vitamin D and zinc and received supplementation. Both were able to increase natural protein as the pregnancy progressed. Ingestion of the 6R-BH4 was well tolerated. Both gave birth to healthy normal infants. In these case studies, both babies’ birth measurements were in the normal range, and the 6R-BH4 had no adverse effects.

An Adjunct to Dietary Treatment for PKU

Genetic inability to convert phenylalanine to tyrosine increases blood levels of neurotoxic phenylalanine in patients with phenylketonuria (PKU).

Nutrition, physical growth, and bone density in treated phenylketonuria.

Dietary treatment of phenylketonuria is well established to be safe and to prevent developmental and mental impairment in patients with low or absent phenylalanine hydroxylase activity. The use of semi-synthetic diets necessitates careful and longitudinal control not only of physical and intellectual development, which are both near normal in well treated patients, but also of potential diet inherent insufficiencies of essential nutrients. Concern has been raised by some reports on growth retardation in young patients on strict diets and on decreased bone density in older phenylketonuric children. The clinical significance of these findings is not known. Changes have been found, although inconsistently, in connection with selenium, zinc, iron, retinol and polyunsaturated fatty acid status in dietetically treated patients with phenylketonuria. Both the mechanism and significance of these changes is doubtful at present.

Executive function and psychosocial adjustment in children with early treated phenylketonuria: correlation with historical and concurrent phenylalanine levels.

The issue of what level of phenylalanine (phe) constitutes a safe upper limit for the therapeutic range in dietary treatment of phenylketonuria (PKU) remains unsettled. It has been proposed that the previous guideline figure of 600 mumol l-1 may result in specific impairment of executive functions such as attention, planning and set maintenance. The executive dysfunction theory was investigated by correlation historical and concurrent phe with executive, non-executive and personality tests in a group of early and continuously treated children, aged 10-13 years, with classical PKU, whose average phe levels of 355 mumol l-1 (SD = +/-144) for the pre-school period and 480 mumol l-1 (SD +/-193) for the primary school period corresponded to the upper limits presently recommended in the UK of 360 and 480 mumol l-1 for these age ranges. No clear associations were found between historical or concurrent phe levels and any of the neuropsychological or personality measures, thereby weakening the case for the emergence of executive deficits, at least when average phe levels remain close to the upper limits now considered safe. Furthermore no significant differences were found on psychological variables where comparison with population norms was possible.

Phenylketonuria: treatment in adolescence and adult life.

In our clinic the decision on whether to continue with dietary treatment of phenylketonuria or not is left to each adolescent and adult patient after the advantages and disadvantages, as discussed in this paper, of continuing diet have been presented to them. As a result 61 of 132 patients have stopped diet or declined to restart and only 4 of them have phenylalanine values below 1000 mumol/l. Seventy-one patients have remained on diet or started again with phenylalanine values below 1000 mumol/l in 58 of them. This series of 132 excludes women who returned to diet to conceive.

74 LONGTERM INTELLECTUAL DEVELOPMENT IN 86 CHILDREN WITH PHENYLKETONURIA (PKU)

Dietary treatment in phenylketonuria (PKU) prevents severe impairment of psychomotor development. However, mere are unequivocal recommendations in regards to diet liberlization. Aim of this retrospective sfudy was to examine intellectual development in eighty-six individuals with “classical” PKU, who were treated early in life. Intellectual development was recorded using the Kramer, Hawik & Hawle test at the age of 4, 6, 8, and 12years. Intellectual development was not influenced by the time dietary treatment was started, probably due to the fact that treatment in all patients was introduced within the first two months (mean±SD: 23±8 days, range: 6 to 61 days). Compared with their non-PKU brothers and sisters PKU children showed significantly lower IQ at the age of 6 years (105±14 vs 114±14, P< 0.001). IQ of the parents did not differ from IQ of the PKU children at the age of 12 years. A significant association was observed between the quality of diet control (measured by Guthrie-test) and intellectual outcome: IQ at 6.8. and 12 years significantly correlated with qualify of diet control during all observation periods (r= 0.388. P<0.001). Children with “strict” dietary control - already at the age of 2 years-showed significantly higher IQ than those with “poor” dietary control. These observations underline the importance of a strict adherance of PKU patients to the diet to acchieve adequate intellectual development.

Intelligence and quality of dietary treatment in phenylketonuria.

The records of the Phenylketonuria Register were examined to determine the factors associated with early intellectual progress in children who had received a diet low in phenylalanine from soon after birth. A total of 1031 children were born between 1964 and 1980 and started treatment before they were 4 months of age, and 808 of them were followed up prospectively. In 263 children born between 1964 and 1971 (cohort 1), the mean intelligence quotient (IQ) at 4 years rose with year of birth from 24 points below revised IQ norms to 10 points below; in 545 born between 1972 and 1980 (cohort 2) there was no further rise in IQ and the mean remained eight points below the norms. IQ fell progressively by roughly four points for each four weeks' delay in starting treatment, for each 300 mumol/l rise in mean phenylalanine concentrations during treatment, and for each five months during the first two years during which phenylalanine concentrations were below 120 mumol/l. Forty six children in cohort 2 had the most favourable treatment characteristics. Their mean IQ, after standardising for social class, was 112.6, and similar to the mean for the same group estimated by multiple regression, and to revised population IQ norms for the period during which the children were tested. The data suggest that many children who are treated early continue to suffer a mild degree of neurological impairment because of the difficulties in fully controlling the metabolic abnormality.

TECHNIQUES AND EFFICACY OF SCREENING

The story of newborn screening for inborn errors of metabolism begins with the ideas expressed by Archibald Garrod in 1902. He published a paper called "The Incidence of Alkaptonuria, A Study in Chemical Individuality," in which he showed insight into the inheritance of specific chemical defects in metabolism. This was before any knowledge of enzymes or metabolism existed and was soon after the discovery of Mendel's laws of inheritance. The application of Mendel's laws of inheritance in the human organism was pioneered by Garrod. He originated the phrase "inborn errors of metabolism" that is still used today. Forty years later, Beedle and Tatum performed their Nobel prize-winning work with the mold Neurospora to demonstrate the "one gene-one enzyme" hypothesis. One of their associates, the late Dr David Bonner, later published a series of papers concerning the case of one auxotroph of Neurospora with two amino acid requirements for isoleucine and valine. This double requirement was found to be due to "internal inhibition": the accumulation of the precursor to one branch-chained amino acid, isoleucine, blocked the conversion of another branch-chained precursor to its amino acid, valine. This phenomenon of "internal inhibition" demonstrated in Neurospora was a prelude to more recent insight into human errors of metabolism, such as phenylketonuria (PKU). Bickel's publication in 1953 of a dietary treatment for PKU led to a great deal of interest in early treatment of PKU. There were many attempts to set up urine screening programs using the ferric chloride reagent that Folling used in discovering the disease in 1934.

Computer model of the metabolism of phenylalanine in normal subjects and in patients with phenylketonuria

The techniques of systems analysis have been applied to the metabolism of phenylalanine in healthy subjects and in phenylketonuria to derive a computer model. The model consists of seven compartments and four biological functions. Enzyme reactions are assumed to obey Michaelis-Menten kinetics. The computer program is written in FORTRAN. A biological validation of the model has been performed. The computer model has potential value for estimating concentrations of key metabolites in human organs which are not accessible to direct analysis from measurement of metabolites in blood and urine and phenylalanine intake. Such estimates would be helpful in clinical decisions concerning modification or termination of dietary treatment in phenylketonuria.

Transport inhibition in the pathology of phenylketonuria and other inherited metabolic diseases

AbstractKinetic analysis of the rates of transport of amino acids and of sugars across the blood‐brain barrier shows how severe is the competition which commonly occurs between groups of chemically related substances in the blood plasma for one of a number of shared transport carriers. In view of the possible importance of such competition in causing brain damage in inherited metabolic diseases an example is given of the use of these kinetic data to work out the best way of ensuring an adequate supply of amino acids to the brain during the dietary treatment of phenylketonuria. Indications are obtained of the degree to which the supply of amino acids other than phenylalanine to the brain may be reduced and of the conditions which are likely to prove most dangerous. It is concluded that the supplementation of the diet with tyrosine alone cannot be expected to provide an effective alternative to phenylalanine restriction. The high level of phenylalanine found commonly in untreated phenylketonuria carries a risk of reducing the supply to the brain of up to eleven other amino acids which the brain cells need. A lack of any one of these may interfere with brain growth and development. The implications are considered for possible improvements in the treatment of phenylketonuria or other inherited metabolic diseases. It is concluded that there may well be some advantage in increasing the intake of amino acids other than the restricted phenylalanine which the brain has to obtain from the circulation above the amounts currently given in the treatment of phenylketonuria (or of analogous compensatory nutrition in the treatment of other metabolic defects by dietary restriction).

Termination of dietary treatment in phenylketonuria.

The aim of this study was to evaluate the influence of early termination of dietary treatment in phenylketonuria by following the patients' mental development. Three groups of patients have been evaluated: Group I — 22 classical PKU cases with a mean treatment period of 4 years and 8 months; Group II — 10 cases of classical PKU with a mean treatment period of 2 years 4 months; Group III — 5 PKU variants treated on average for 3 years and 8 months. The mean period of observation after dietary discontinuation was form 4 to 6 years. Evaluation of development and further progress of PKU patients after terminating the diet was based on the following parameters: clinical symptoms, selected biochemical examinations, scores of mental development, behaviour and school achievements, and EEG examination. Detailed analysis of the data revealed a direct relationship between the long-term results of treatment and the age of starting treatment, even in the range of the first three months of life, and also the quality of dietary control and the period of treatment. Three significant changes emerged after discontinuation of the diet: 1) Decreasing intelligence quotients in most patients with classical PKU. 2) Difficulties in adaptation and school achievements in these patients. 3) Large number of EEG abnormalities after termination of the diet. These findings in classical PKU patients after discontinuation of the diet reveal that 5 year's dietary treatment is too short for effective therapy in classical PKU. Normal mental development and good school achievements in PKU variants (hyperphenylalaninaemia) with a treatment period of 31/2 years suggest that in this condition the period of dietary treatment can possibly be shorter than in classical phenylketonuria.

Carrier Detection and Assessment of Dietary Treatment in Phenylketonuria

SynopsisThis paper describes a recently started programme for the biochemical and psychological assessment of phenylketonurics and their families, done as an extension of the diagnosis and management already undertaken over the past 14 years at the Irrabeena Centre.Carrier detection was done on 73 members, plus one fiance, of 15 families. The tolerance tests used were based on those of Cunningham et al in U.S.A. (1969). A loading dose of phenylalanine was given and blood tests taken at intervals. The curves obtained differentiated the heterozygotes or carriers from the normals and confirmed the condition in the homozygotes.Of 25 parents 84% were typical heterozygotesOf 33 siblings 24% were normal36% heterozygotes18% the figures overlappedThis gives us a 60% definite result and 82% probably correct result, and in 18% it was impossible to say.The Assessment of Dietary TreatmentPKUs diagnosed by screening of newborns or siblings of the known cases, were rated by their latest I.Q. and adequacy of dietary treatment. The adequacy of dietary treatment being defined by percentage of blood phenylalanine levels over 10 mg/100 ml and the age of onset of treatment. The results indicated a significant positive correlation between latest I.Q. and adequacy of treatment.Similarly, the results of all PKU cases, including those diagnosed because of retardation, showed a significant correlation between adequacy of treatment and latest I.Q. There was also a significant correlation between adequacy of treatment and the parameter I.Q.pku—I.Q. , where I.Q.pku represents the latest I.Q. of the PKU case and I.Q.slb the I.Q. of an unaffected sibling closest in age.The results of this study were limited by procedural inadequacies. Recommendations are made about further programmes for similar studies to be undertaken.

Biological evaluation of a whey protein fraction, with special reference to its use as a phenylalanine-low protein source in the dietary treatment of PKU.

A biological evaluation of the nutritive value of a whey protein concentrate, as well as of a specific whey protein fraction low in phenylalanine and tyrosine, has been performed by means of an NPU assay. The nutritive values were found to be high, higher, in fact, than what could be expected from the theoretical calculations based on chemical analyses and calculation of chemical scores. The use of whey proteins, low in phenylalanine, in dietary treatment of phenylketonuria is proposed.