Background: Phenylketonuria (PKU; OMIM#261600) is a rare metabolic disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene resulting in high phenylalanine (Phe) in blood and brain. If not treated early this results in intellectual disability, behavioral and psychiatric problems, microcephaly, motor deficits, eczematous rash, autism, seizures, and developmental problems. There is a controversial discussion of whether patients with PKU have an additional risk for atherosclerosis due to interference of Phe with cholesterol synthesis and LDL-cholesterol regulation. Since cholesterol also plays a role in membrane structure and myelination, better insight into the clinical significance of the impact of Phe on lipoprotein metabolism is desirable. In 22 treated PKU patients (mean age 38.7 years) and 14 healthy controls (mean age 35.2 years), we investigated plasma with NMR spectroscopy and quantified 105 lipoprotein parameters (including lipoprotein subclasses) and 24 low molecular weight parameters. Analysis was performed on a 600 MHz Bruker AVANCE IVDr spectrometer as previously described. Results: Concurrent plasma Phe in PKU patients showed a wide range with a mean of 899 μmol/L (50–1318 μmol/L). Total cholesterol and LDL-cholesterol were significantly lower in PKU patients versus controls: 179.4 versus 200.9 mg/dL (p < 0.02) and 79.5 versus 104.1 mg/dL (p < 0.0038), respectively. PKU patients also had lower levels of 22 LDL subclasses with the greatest differences in LDL2 Apo-B, LDL2 Particle Number, LDL2-phospholipids, and LDL2-cholesterol (p < 0.0001). There was a slight negative correlation of total cholesterol and LDL-cholesterol with concurrent Phe level. VLDL5-free cholesterol, VLDL5-cholesterol, VLDL5-phospholipids, and VLDL4-free cholesterol showed a significant (p < 0.05) negative correlation with concurrent Phe level. There was no difference in HDL and their subclasses between PKU patients and controls. Tyrosine, glutamine, and creatinine were significantly lower in PKU patients compared to controls, while citric and glutamic acids were significantly higher. Conclusions: Using NMR spectroscopy, a unique lipoprotein profile in PKU patients can be demonstrated which mimics a non-atherogenic profile as seen in patients treated by statins.

Phenylketonuria (PKU) is a rare metabolic disorder caused by mutations in the phenylalanine hydroxylase gene. Depending on the severity of the genetic mutation, medical treatment, and patient dietary management, elevated phenylalanine (Phe) may occur in blood and brain tissues. Research has recently shown that high Phe not only impacts the central nervous system, but also other organ systems (e.g., heart and microbiome). This study used ex vivo proton nuclear magnetic resonance (¹H-NMR) analysis of urine samples from PKU patients (mean 14.9 ± 9.2 years, n = 51) to identify the impact of elevated blood Phe and PKU treatment on metabolic profiles. Our results found that 24 out of 98 urinary metabolites showed a significant difference (p < 0.05) for PKU patients compared to age-matched healthy controls (n = 51) based on an analysis of urinary metabolome. These altered urinary metabolites were related to Phe metabolism, dysbiosis, creatine synthesis or intake, the tricarboxylic acid (TCA) cycle, end products of nicotinamide-adenine dinucleotide degradation, and metabolites associated with a low Phe diet. There was an excellent correlation between the metabolome and genotype of PKU patients and healthy controls of 96.7% in a confusion matrix model. Metabolomic investigations may contribute to a better understanding of PKU pathophysiology.

Monitoring phenylalanine (Phe) concentrations is critical for the management of phenylketonuria (PKU). This can be done in dried blood spots (DBS) or in EDTA plasma derived from capillary or venous blood. Different techniques are used to measure Phe, the most common being flow-injection analysis tandem mass spectrometry (FIA-MS-MS) and ion exchange chromatography (IEC). Significant differences have been reported between Phe concentrations in various sample types measured by different techniques, the cause of which is not yet understood. We measured Phe concentrations in 240 venous blood samples from 199 patients with hyperphenylalaninemia in dried blood spots, EDTA plasma and erythrocytes by FIA-MS-MS and IEC. Phe concentrations were significantly lower in erythrocytes than in plasma leading to about 19% lower Phe DBS concentrations compared with plasma independent from the method used for quantification. As most therapy recommendations for PKU patients are based on plasma concentrations reliable conversion of DBS into plasma concentrations is necessary. Variances of Phe concentrations in plasma and DBS are not linear but increases with higher concentrations indicating heteroscedasticity. We therefore suggest the slope of the 75th percentile from quantile regression as a correction factor.

Despite numerous studies in human patients and animal models for phenylketonuria (PKU; OMIM#261600), the pathophysiology of PKU and the underlying causes of brain dysfunction and cognitive problems in PKU patients are not well understood. In this study, lumbar cerebral spinal fluid (CSF) was obtained immediately after blood sampling from early-treated adult PKU patients who had fasted overnight. Metabolite and amino acid concentrations in the CSF of PKU patients were compared with those of non-PKU controls. The CSF concentrations and CSF/plasma ratios for glucose and lactate were found to be below normal, similar to what has been reported for glucose transporter1 (GLUT1) deficiency patients who exhibit many of the same clinical symptoms as untreated PKU patients. CSF glucose and lactate levels were negatively correlated with CSF phenylalanine (Phe), while CSF glutamine and glutamate levels were positively correlated with CSF Phe levels. Plasma glucose levels were negatively correlated with plasma Phe concentrations in PKU subjects, which partly explains the reduced CSF glucose concentrations. Although brain glucose concentrations are unlikely to be low enough to impair brain glucose utilization, it is possible that the metabolism of Phe in the brain to produce phenyllactate, which can be transported across the blood-brain barrier to the blood, may consume glucose and/or lactate to generate the carbon backbone for glutamate. This glutamate is then converted to glutamine and carries the Phe-derived ammonia from the brain to the blood. While this mechanism remains to be tested, it may explain the correlations of CSF glutamine, glucose, and lactate concentrations with CSF Phe.