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Sisters, Brothers and an Orphan Disease

“Researchers explore how NMR technology might help people with metabolic disorders”

When the mother of a little boy and girl with severe intellectual disabilities turned to Norwegian physician Asbjörn Fölling in 1934, she suspected a link between the children’s deficits and the musty smell of their urine. That suspicion led to the identification of Phenylketonuria (PKU), a genetic metabolic disorder.

About PKU

For most people, protein is a healthy part of a balanced diet and a crucial building block of life. For those whose bodies fail to produce or don’t produce enough of the key enzyme phenylalanine hydroxylase (PAH), the crucial conversion of the essential amino acid phenylalanine into tyrosine fails. The metabolite of unconverted phenylalanine severely damages the nervous system and, within weeks of birth, can cause devastating brain damage.

Around the world, the incidence of PKU varies greatly as do screening practices. PKU is most common in European Caucasians and their descendants. The highest incidence is in Turkey where 1 in 2,600 have PKU. In Ireland, PKU occurs in 1 in 4,500 people. But in Finland, the incidence of PKU is so low, approximately 1:100,000, that newborns are not screened for it.

The United States, Australia, Canada and New Zealand began newborn screening for PKU in the early- to mid-60s, and virtually all newborns are screened. In other countries, testing began much later. For instance, Kazakstan began testing in 2006. In China, where testing began in 1981, some regions test most newborns while other regions test very few.

PKU under the spotlight sparks newborn screening

Almost 30 years after PKU was identified, John F. Kennedy was elected president of the United States. With a soft spot in his heart for children with intellectual disabilities – the president’s sister Rosemary was considered “mentally retarded” – President Kennedy welcomed the McGrath sisters of Colorado to the White House.”

Both McGrath girls were clad in dainty skirts. Both sported white socks and black Mary Jane’s. Both had PKU. Older sister, Sheila, had profound and permanent brain damage. Younger sister, Kammy, had been tested for and diagnosed with PKU at birth and was fed a special diet that protected her brain from the ravages of PKU.

That White House meeting and subsequent articles in the Saturday Evening Post and Life put a spotlight on PKU and set the stage for screening all newborns in the United States for PKU. Today, testing is mandatory in all 50 states for PKU and dozens of other diseases, many treatable.

PKU treatment

The primary treatment for PKU is a restricted diet that almost eliminates protein. Since protein is necessary for growth and health, patients compensate by consuming manufactured protein drinks that exclude phenylalanine. The costly shakes are often dreaded for their awful taste and sickening smell. All other foods must be measured and weighed as part of a cumbersome process of calculating and monitoring phenylalanine intake. For children under five, proper monitoring means the difference between healthy development and profound intellectual disability. Adults who stray from a low-Phe diet often experience negative neurological and behavioral effects.

In addition to a restricted diet and special foods, the medication sapropterin helps some patients control Phe levels. Sapropterin (Kuvan) is a form of BH4, the cofactor of the enzyme that metabolizes phenylalanine. For patients with a type of PKU in which some enzyme is produced, the addition of BH4 can increase the healthy metabolism of Phe into tyrosine. Another treatment option is large neutral amino acid supplements. LNAAs block Phe from getting into the gut or crossing the blood-brain barrier.

Monitoring Phe levels

Most patients go to a physician’s office every few weeks to have blood drawn to measure the level of Phe. Not only is that process time-consuming, it fails to provide timely feedback about diet and phenylalanine levels.

Much the way people with diabetes do better and limit damage to their bodies by precisely monitoring glucose levels and altering diet and medication when glucose levels get too high, patients with PKU might benefit from frequent monitoring of Phe levels.

Using NMR to understand the urinary metabolome

Recently, a team from Universitas Klinikum Freiburg/University of Heidelberg in Germany, and the Kennedy Centre for PKU in Glostrup, Denmark, set out to see if it is possible to monitor Phe levels in urine, not by odor, but by using NMR. They shared their findings in a scientific poster, “Investigating the urinary metabolome for monitoring PKU patients under treatment using high resolution nuclear magnetic resonance spectroscopy (NMR).” Their methods may serve as a model of how NMR can be used to monitor PKU and other metabolic diseases.

The pilot study included 60 patients (age 1-40 years) who either followed a restricted diet (43), took sapropterin (12) or did not follow the diet but did have large neutral amino acid supplementation (5).

Patient samples were evaluated using a Bruker Avance IVDr System at 600 MHz. Statistical analyses against a reference of healthy children (n=43) using PCA-LDA were performed. Blood phenylalanine was measured according to local routine methods by tandem mass spectrometry in blood spots.

The researchers established normal profiles by analyzing the urine of PKU patients undergoing treatment and compared those profiles to healthy matched controls. They illustrated that it is possible to separate the metabolic profiles of patients and controls.

Further, individual profiles demonstrated the effect of the treatments based on Phe intake and Phe control. This difference can most clearly be seen when viewing the blood Phe levels in patients following a restricted, low-Phe diet and patients treated with the medication sapropterin (Kuvan) in Table 1.

table1
Table 1 Overview of patient characteristics with treatment group, blood phenylalanine and phenylalanine intake

Figure 2 shows the spectral differences coming from the different treatment. (on the top) Individual profiles of the 5 non treated (but supplemented with large neutral amino acids) show in Fig 2a a high phenylalanine excretion in urine. (on the bottom) The Fig 2b shows that the NMR profiles from the patients treated with sapropterin (Kuvan) are between the patients treated with amino acid supplement and the controls.

figure2Afigure2BImplications
The researchers believe that future studies could clarify spectrum differences that might be caused by natural protein intake in the restricted diet together with a Phe control in the target range.

Improved options to monitor Phe levels in a timely way might help people with PKU follow and adjust their diets to keep Phe levels low. Tightly controlling Phe levels reduces the long- and short-term side effects of Phe.

In addition, using NMR to analyze metabolomes could improve understanding and monitoring of other metabolic disorders.