A Pilot Data Analysis of a Metabolomic Liquid Chromatography-Tandem Mass Spectrometry (LC/MS/MS) Based Study of Patients with Sickle Cell/Beta Thalassemia

Abstract Background: The complex pathophysiology of Sickle Cell Disease (SCD) makes unlikely that a single therapeutic agent will prevent or reverse all SCD complications. Metabolomic analysis might help in the characterization of the endogenous and exogenous effects of potential new treatments. Metabolites are small molecules that are chemically transformed during metabolism and provide a functional readout of cellular state. Metabolites serve as direct signatures of biochemical activity and are therefore easier to correlate with phenotype. The metabolome is typically defined as the collection of small molecules produced by cells and offers a window for investigating how mechanistic biochemistry relates to cellular phenotype. There are very few reports associated with SCD providing comprehensive measurements of metabolites present in blood. Low arginine bioavailability has been associated with a clinical phenotype of increased hemolytic rate, pulmonary hypertension risk and early mortality. Recently, the FDA approved the use of L-glutamine for the treatment of adults and children with SCD, on the basis of the results of randomized phase 3 clinical trials1, while L-arginine's involvement is under investigation2,3. In this context we aimed to quantify targeted metabolites' abnormalities in patients with Sickle Cell/beta thalassemia (HbS/βThal), to identify pathways that might be of interest to prevent disease complications. Patients and Methods: Thirty adult Caucasian patients with HbS/βThal aged 45.6±10.9y, (43% male), at steady-state were enrolled in the study, while 20 age-matched healthy individuals (45% male) served as controls. Along with measurements of hematologic and blood chemistry parameters, targeted metabolome analyses for 13 aminoacids and 2 aminoacid's derivatives were performed after extraction from dry blood spots (DBSs) on filter paper using LC/MS/MS, with derivatization (AB SCIEX 5500 triple quadrupole QTRAP® LC/MS/MS Systems, Framingham, MA, USA). Results: Multiple metabolite differences are identified in HbS/βThal vs. Controls (Figure1). From metabolites involved in the biosynthesis of glutathione, only L-glutamine's levels were lower in patients with HbS/βThal compared to controls, while 5-oxyproline levels, a catabolic product of glutathione metabolism, were markedly increased in patients with HbS/βThal compared to controls. Urea cycle amino acids, also involved in the production of nitric oxide, L-arginine and L-ornithine concentrations were significantly lower in patients with HbS/βThal compared to controls, with a trend towards lower L-citrulline in patients with HbS/βThal p=0.06). Finally, amino acids involved in catecholamines (dopamine, nor-epinephrine and epinephrine) biosynthesis, such as L-phenylalanine and L-tyrosine and its metabolite succinylacetone levels were significantly lower in patients with HbS/βThal compared to controls. No significant correlations were found between any metabolites and markers of hemolysis, HbF levels or iron burden. Conclusions: This study confirms prior observations concerning aberrations in multiple blood specific amino acid levels in patients with SCD compared to controls, but this is the first report on Caucasian patients with HbS/βThal and on whole blood from DBS. We also identified for the first time in Caucasian patients with HbS/βThal important metabolic abnormalities of glutathione and nitric oxide biosynthesis pathways associated with altered concentrations of the metabolites serving of substrates in these cycles. Importantly, we also demonstrate low levels of L-phenylanine and L-tyrosine, which are essential sources for multiple neurotransmitters biosynthesis and neurobehavioral health. The latter novel observation should be confirmed in larger studies, while measurements of urinary amino acid clearances are necessary to evaluate for potential etiologies of the deficiencies through urinary losses vs. low substrate availability, increased utilization or abnormal metabolism. Patients with SCD are in a precarious state with respect to many amino acid deficiencies, all of which may have clinical consequences warranting further investigation. 1N Engl J Med. 2018 Jul 19;379(3):226-235. 2Anesth Analg. 2017 Apr;124(4):1369-1370. 3Haematologica. 2013 Sep;98(9):1375-82. Figure 1. Figure 1. Disclosures Morris: Pfizer: Consultancy; Calithera: Consultancy; MAST Therapeutics: Research Funding; UCSF-Benioff Children's Hospital Oakland: Patents & Royalties: For nutritional supplement licensed to Lifetrients; FDA: Research Funding; NIH/NHLBI: Research Funding. Voskaridou:Celgene Corp: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acceleron: Membership on an entity's Board of Directors or advisory committees, Research Funding.