An article by Lan Gao et al, at PTC Therapeutics in the United States published in the June 2023 edition of Clinical Biochemistry, described a validation and phase 2 clinical trial of a VAMS method to measure phenylalanine (Phe). The paper is entitled “Validation and application of volumetric absorptive microsampling (VAMS) dried blood method for phenylalanine measurement in patients with phenylketonuria.” The researchers applied Mitra® microsampling devices based on VAMS® technology to collect finger-stick blood samples for their study.
The researchers were able to report that concordant results were obtained using VAMS and plasma methods, which demonstrated that “VAMS is a reliable method for clinical applications to monitor blood Phe for PKU patients.”
PKU is an autosomal recessive disorder affecting both physical development and neurodevelopment, leading to premature death. From a biochemical perspective, the genetic disorder leads to a deficiency in an enzyme called phenylalanine hydroxylase, which functions to catalyze the conversion of phenylalanine (Phe) into tyrosine. In the absence of phenylalanine hydroxylase, the amino acid is converted into the brain toxin, phenylpyruvic acid by the action of a transaminase.
Up until the 1950s, the way to diagnose PKU was to measure the build-up phenylpyruvic acid in urine by reacting it with ferric chloride. Although this test was accurate, it was not sensitive enough to detect all cases in time for early intervention. For this reason, irreversible brain damage often occurred in those afflicted with the condition.
Early approaches to diagnosing PKU changed with a man named Robert Guthrie, MD, PhD. He was a medical doctor and research scientist who had earned his doctorate at the University of Minnesota. His initial research focus was on bacterial inhibition assays to study antibiotic sensitivity.
In 1947, his second son, John, was born with a neurodevelopmental disorder and, despite his best efforts, Dr. Guthrie was unable to find a diagnosis or treatment for his son. In 1957, his 15-month-old niece was diagnosed with the developmental condition called phenylketonuria (PKU), which led Dr. Guthrie to research PKU.
In 1958, after watching a lecture on children with developmental disorders by Dr. Robert Warner from the Children’s Rehabilitation Center at Buffalo Children’s Hospital, Dr Guthrie moved to Buffalo Children’s Hospital to work on a more sensitive assay for diagnosing PKU. Dr. Guthrie believed that earlier diagnosis would allow for earlier intervention using bacterial inhibition, his area of expertise. At the time, the only treatment for PKU was to put the patients on a low phenylalanine (Phe) diet, but this only worked if physiological damage had not yet occurred.
In 1960, Dr. Guthrie’s work led to a PKU screening trial using dried blood spot (DBS), which involved collecting dried capillary blood spot samples on disks of filter paper from PKU patients.
In 1961, Dr. Guthrie began to run routine neonatal dried blood spot testing for PKU. This was the beginning of what has since become a highly successful neonatal screening protocol in hospitals where up to 50 conditions can be screened in blood samples for inborn errors of metabolism.
Nowadays, in addition to a phenylalanine restricted diet, there are two other treatments for PKU. The first is to replace the defective phenylalanine hydroxylase with a treatment called Pegvaliase. The second is called sapropterin dihydrochloride which, in combination with a phenylalanine restricted diet, acts to lower levels of the amino acid.
One of the challenges to monitoring Phe levels using dried blood spots (DBS) is the inherent error observed with DBS technique and sample quality, stemming from a number of hematocrit effects. For example, one PKU study using DBS to measure Phe noted that 47.7% of the DBS samples would be rejected due to quality issues.
It is for this reason that Lan Gao et al at PTC Therapeutics decided to employ a more precise volumetric microsampling approach using Mitra® devices with VAMS® technology for a phase 2 randomized, crossover PKU study of sepiapterin. In their paper, the study authors noted that the VAMS technique negated some of the core disadvantages of DBS such as “the HCT effect, humidity, volume inaccuracy, and drying time, all of which can influence results.”
This phase 2 randomized, crossover PKU study of sepiapterin by the team at PTC Therapeutics shows promise for future PKU interventions and is a testament to the legacy of Dr. Guthrie.
It is interesting to speculate that if Robert Guthrie were alive today, he would be amazed to see the evolution of a technique that he invented back in the early 1960s, which is still going strong today. Moreover, that in the ensuing years, PKU, a disease for which there was no treatment prior to his genius idea to use dried blood spots, is now increasingly treatable. What’s more, he’d be surprised to see that research is still ongoing to lessen the burden of the disease and similar conditions.
The world owes Dr. Guthrie a debt of gratitude for chasing what was an incredibly simple but brilliant idea that has led to millions of lives being saved and improved by the monitoring a few small drops of blood.
This blog includes content curated from a published study paper that was summarized for our readers by James Rudge, PhD, Microsampling Technical Director. To learn more about the important research discussed here, please visit the original article in Clinical Biochemistry.