A Retrospective Metabolomics Analysis of Gamma-Hydroxybutyrate in Humans: New Potential Markers and Changes in Metabolism Related to GHB Consumption.

GHB is an endogenous short-chain organic acid presumably also widely applied as a rape and knock out drug in cases of drug-facilitated crimes or sexual assaults (DFSA). Due to the endogenous nature of GHB and its fast metabolism in vivo, the detection window of exogenous GHB is however narrow, making it challenging to prove use of GHB in DFSA cases. Alternative markers of GHB intake have recently appeared though none has hitherto been validated for forensic use. UHPLC-HRMS based screening of blood samples for drugs of abuse is routinely performed in several forensic laboratories which leaves an enormous amount of unexploited data. Recently we devised a novel metabolomics approach to use archived data from such routine screenings for elucidating both direct metabolites from exogenous compounds, but potentially also regulation of endogenous metabolism and metabolites. In this paper we used UHPLC-HRMS data acquired over a 6-year period from whole blood analysis of 51 drivers driving under the influence of GHB as well as a matched control group. The data were analyzed using a metabolomics approach applying a range of advanced analytical methods such as OPLS-DA, LASSO, random forest, and Pearson correlation to examine the data in depth and demonstrate the feasibility and potential power of the approach. This was done by initially detecting a range of potential biomarkers of GHB consumption, some that previously have been found in controlled GHB studies, as well as several new potential markers not hitherto known. Furthermore, we investigate the impact of GHB intake on human metabolism. In aggregate, we demonstrate the feasibility to extract meaningful information from archived data here exemplified using GHB cases. Hereby we hope to pave the way for more general use of the principle to elucidate human metabolites of e.g. new legal or illegal drugs as well as for applications in more global and large scale metabolomics studies in the future.

The effect of mycophenolate mofetil on platelet function.

: Mycophenolate mofetil (MMF) raises platelet counts in patients with primary immune thrombocytopenia. However, studies indicate that MMF inhibits collagen-induced platelet aggregation, potentially increasing bleeding risk following MMF therapy. The study evaluates the in-vitro effect of MMF on platelet function. Blood samples (n = 6) from healthy donors were incubated with vehicle, MMF or mycophenolic acid (MPA) at clinically relevant concentrations. Platelet aggregation was measured with flow cytometry and 96-well light transmission aggregometry (LTA). Using flow cytometry, we measured the expression of platelet CD49b, CD42b, CD42a, CD61 and CD41. Platelet activation was measured as the expression of P-selectin and the active form of the GPIIb/IIIa receptor following agonist stimulation. Agonists were: ADP, thrombin receptor-activating peptide, collagen, collagen-related peptide and U46619. The Platelet Function Analyzer-200 was used to measure global platelet function. MMF and MPA did not change platelet aggregation regardless of the agonist used. An exception was a significant, but minor decrease in collagen-induced platelet aggregation in samples with MMF (6 ±â€Š3%, P = 0.02) and MPA (8 ±â€Š4%, P = 0.01) compared with vehicle (22 ±â€Š11%). However, this was not observed using the lesser sensitive LTA method. Compared with vehicle, MPA led to a significantly lower relative disposition of the surface collagen-receptor GPVI (7.8 ±â€Š1.8 versus 8.8 ±â€Š2.1 mean fluorescence intensity, P < 0.001). In all other platelet-related tests, neither MMF nor MPA showed any effect. In conclusion, MMF and MPA only had a minor effect on collagen-induced platelet aggregation, with MPA reducing the relative disposition of surface GPVI receptors.

Untargeted Metabolomics Analysis of ABCC6-Deficient Mice Discloses an Altered Metabolic Liver Profile.

Loss-of-function mutations in the transmembrane ABCC6 transport protein cause pseudoxanthoma elasticum (PXE), an ectopic, metabolic mineralization disorder that affects the skin, eye, and vessels. ABCC6 is assumed to mediate efflux of one or several small molecule compounds from the liver cytosol to the circulation. Untargeted metabolomics using liquid chromatography-mass spectrometry was employed to inspect liver cytosolic extracts from mice with targeted disruption of the Abcc6 gene. Absence of the ABCC6 protein induced an altered profile of metabolites in the liver causing accumulation of compounds as more features were upregulated than downregulated in ABCC6-deficient mice. However, no differences of the identified metabolites in liver could be detected in plasma, whereas urine reflected some of the changes. Of note, N-acetylated amino acids and pantothenic acid (vitamin B5), which is involved in acetylation reactions, were accumulated in the liver. None of the identified metabolites seems to explain mineralization in extrahepatic tissues, but the present study now shows that abrogated ABCC6 function does cause alterations in the metabolic profile of the liver in accordance with PXE being a metabolic disease originating from liver disturbance. Further studies of these changes and the further identification of yet unknown metabolites may help to clarify the liver-related pathomechanism of PXE.