NMR metabolomics profiling of blood plasma mimics shows that medium- and long-chain fatty acids differently release metabolites from human serum albumin.

Metabolite profiling by NMR of body fluids is increasingly used to successfully differentiate patients from healthy individuals. Metabolites and their concentrations are direct reporters of body biochemistry. However, in blood plasma the NMR-detected free-metabolite concentrations are also strongly affected by interactions with the abundant plasma proteins, which have as of yet not been considered much in metabolic profiling. We previously reported that many of the common NMR-detected metabolites in blood plasma bind to human serum albumin (HSA) and many are released by fatty acids present in fatted HSA. HSA is the most abundant plasma protein and main transporter of endogenous and exogenous metabolites. Here, we show by NMR how the two most common fatty acids (FAs) in blood plasma - the long-chain FA, stearate (C18:0) and medium-chain FA, myristate (C14:0) - affect metabolite-HSA interaction. Of the set of 18 common NMR-detected metabolites, many are released by stearate and/or myristate, lactate appearing the most strongly affected. Myristate, but not stearate, reduces HSA-binding of phenylalanine and pyruvate. Citrate signals were NMR invisible in the presence of HSA. Only at high myristate-HSA mole ratios 11:1, is citrate sufficiently released to be detected. Finally, we find that limited dilution of blood-plasma mimics releases HSA-bound metabolites, a finding confirmed in real blood plasma samples. Based on these findings, we provide recommendations for NMR experiments for quantitative metabolite profiling.

(1)H NMR signal at 2.10 ppm in the spectrum of KMnO(4)-bleached heparin sodium: identification of the chemical origin using an NMR-only approach.

The recently revised European Pharmacopeia and US Pharmacopeia heparin sodium monographs include nuclear magnetic resonance (NMR) tests on both identity and purity. In KMnO(4)-bleached heparin, an unidentified NMR signal is present at 2.10 ppm at a level of 15-20% of the mean of signal height of the major glucosamine (GlcNAc/GlcNS,6S) anomeric proton signal at 5.42 ppm and of the major iduronic acid (IdoA2S) anomeric proton signal at 5.21 ppm. According to the new monographs, no unidentified signals greater than 4% should be detected at that position. Thus, the material did not meet the acceptance criterion. The signal at 2.10 ppm has been present at the same level in all released MSD KMnO(4)-bleached heparin sodium batches analyzed over the past 10 years. The signal is a result of the KMnO(4) bleaching. No (oversulfated) chondroitin sulfate or dermatan sulfate was detected in this material. A comprehensive NMR study using long-range heteronuclear 2D techniques identifies this signal at 2.10 ppm as originating from the acetyl methyl group of (6-sulfated) 2-N-acetyl-2-deoxy-glucono-1,5-lactone. This modified monosaccharide is formed by the KMnO(4) oxidation of the reducing end of a terminal N-acetylglucosamine.

1H, 13C and 15N NMR assignments of Duck HBV primer loop of the encapsidation signal epsilon.

The replication of the hepatitis B virus is initiated by binding of the viral reverse transcriptase protein complex to the apical stem loop of the epsilon element to place it next to the primer loop, from which a four nucleotide DNA primer is subsequently synthesized. Here, we present the (1)H/(13)C/(15)N NMR assignments of the bases and sugars of the 37 residues primer loop of Duck HBV epsilon (BMRB-entry 15786).

1H, 13C and 15N NMR assignments of Duck HBV apical stem loop of the epsilon encapsidation signal.

The replication of Hepatitis B virus is initiated by binding of its reverse transcriptase to the apical stem loop and primer loop of epsilon. Here, we present the (1)H/(13)C/(15)N NMR assignments of the bases and sugars of the 29 residues apical stem loop of Duck HBV epsilon.