Biochemical phenotyping unravels novel metabolic abnormalities and potential biomarkers associated with treatment of GLUT1 deficiency with ketogenic diet

<div><p>Global metabolomic profiling offers novel opportunities for the discovery of biomarkers and for the elucidation of pathogenic mechanisms that might lead to the development of novel therapies. GLUT1 deficiency syndrome (GLUT1-DS) is an inborn error of metabolism due to reduced function of glucose transporter type 1. Clinical presentation of GLUT1-DS is heterogeneous and the disorder mirrors patients with epilepsy, movement disorders, or any paroxysmal events or unexplained neurological manifestation triggered by exercise or fasting. The diagnostic biochemical hallmark of the disease is a reduced cerebrospinal fluid (CSF)/blood glucose ratio and the only available treatment is ketogenic diet. This study aimed at advancing our understanding of the biochemical perturbations in GLUT1-DS pathogenesis through biochemical phenotyping and the treatment of GLUT1-DS with a ketogenic diet. Metabolomic analysis of three CSF samples from GLUT1-DS patients not on ketogenic diet was feasible inasmuch as CSF sampling was used for diagnosis before to start with ketogenic diet. The analysis of plasma and urine samples obtained from GLUT1-DS patients treated with a ketogenic diet showed alterations in lipid and amino acid profiles. While subtle, these were consistent findings across the patients with GLUT1-DS on ketogenic diet, suggesting impacts on mitochondrial physiology. Moreover, low levels of free carnitine were present suggesting its consumption in GLUT1-DS on ketogenic diet. 3-hydroxybutyrate, 3-hydroxybutyrylcarnitine, 3-methyladipate, and N-acetylglycine were identified as potential biomarkers of GLUT1-DS on ketogenic diet. This is the first study to identify CSF, plasma, and urine metabolites associated with GLUT1-DS, as well as biochemical changes impacted by a ketogenic diet. Potential biomarkers and metabolic insights deserve further investigation.</p></div

Z-scores of carbohydrate levels in CSF from patients diagnosed with GLUT1-DS not being treated with a KD.

<p>Z-scores of carbohydrate levels in CSF from patients diagnosed with GLUT1-DS not being treated with a KD.</p

Significant free carnitine and carnitine derivatives for 6 patients with GLUT1-DS patients.

<p>Significant free carnitine and carnitine derivatives for 6 patients with GLUT1-DS patients.</p

Plasma biochemical profiles of GLUT1-DS patients on KD.

<p>A) Low plasma free carnitine in GLUT1-DS patients on ketogenic diet. Box-plot of free and grouped bound-carnitine compounds z-scores for 6 patients with GLUT1-DS on ketogenic diet. Low levels of free carnitine were detected, while all carnitine conjugates showed higher levels in GLUT1-DS patients on ketogenic diet. B) Carnitine-bound metabolites are elevated in plasma of patients on ketogenic diet. Box-plot of free carnitine and specific carnitine-derived compound for 6 patients with GLUT1-DS on ketogenic diet. Higher levels of 3-hydroxybutyrylcarnitine is detected in face of low levels of other carnitine derived compounds (isovalerylcarnitine. 2-methylbutyrylcarnitine. 2-methylmalonyl carnitine. propionylcarnitine. succinylcarnitine) in GLUT1-DS patients on ketogenic diet.</p

Z-scores for consistently elevated compounds in plasma and urine from GLUT1-DS on KD diet.

<p>Z-scores for consistently elevated compounds in plasma and urine from GLUT1-DS on KD diet.</p

Metabolomic profiling identifies significantly altered levels of molecules in cerebrospinal fluid.

<p>Analytes significantly outside of the normal range are reported (relative to the general population. Z-scores <-2 or >2 are considered significant. A boxplot demonstrating analytes from the affected patient (represented by red circle) identified in the metabolomics profile with significant deviation from the normal range are shown. Elevations of glutamine (Z-score +3.98) and inosine (Z-score +3.91), along with reductions of glucose (Z-score -3.2), fructose (Z-score -2.8), and mannose (Z-score -2.86), are indicated. A complete listing of significantly altered analytes identified in the metabolomics analysis is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184022#pone.0184022.s001" target="_blank">S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184022#pone.0184022.s002" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184022#pone.0184022.s003" target="_blank">S3</a> Tables.</p

Clinical features of patients with GLUT1-DS.

<p>Clinical features of patients with GLUT1-DS.</p

Carbohydrate levels in plasma from patients diagnosed with GLUT1-DS and taking a KD.

<p>Carbohydrate levels in plasma from patients diagnosed with GLUT1-DS and taking a KD.</p