BLOC-1 deficiency causes alterations in amino acid profile and in phospholipid and adenosine metabolism in the postnatal mouse hippocampus.

Biogenesis of lysosome-related organelles complex-1 (BLOC-1) is a protein complex involved in the formation of endosomal tubular structures that mediates the sorting of protein cargoes to specialised compartments. In this study, we present insights into the metabolic consequences caused by BLOC-1 deficiency in pallid mice, which carry a null mutation in the Bloc1s6 gene encoding an essential component of this complex. The metabolome of the hippocampus of pallid mice was analysed using an untargeted, liquid chromatography-coupled mass spectrometric approach. After data pre-treatment, statistical analysis and pathway enrichment, we have identified 28 metabolites that showed statistically significant changes between pallid and wild-type control. These metabolites included amino acids, nucleobase-containing compounds and lysophospholipids. Interestingly, pallid mice displayed increased hippocampal levels of the neurotransmitters glutamate and N-acetyl-aspartyl-glutamic acid (NAAG) and their precursor glutamine. Expression of the sodium-coupled neutral amino acid transporter 1 (SNAT1), which transports glutamine into neurons, was also upregulated. Conversely, levels of the neurotransmitter precursors phenylalanine and tryptophan were decreased. Interestingly, many of these changes could be mapped to overlapping metabolic pathways. The observed metabolic alterations are likely to affect neurotransmission and neuronal homeostasis and in turn could mediate the memory and behavioural impairments observed in BLOC-1-deficient mice.

On-line formation, separation, and estrogen receptor affinity screening of cytochrome P450-derived metabolites of selective estrogen receptor modulators.

We have developed a fully automated bioreactor coupled to an on-line receptor affinity detection system. This analytical system provides detailed information on pharmacologically active metabolites of selective estrogen receptor modulators (SERMs) generated by cytochromes P450 (P450s). We demonstrated this novel concept by investigating the metabolic activation of tamoxifen and raloxifene by P450-containing pig and rat liver microsomes. The high resolution screening (HRS) system is based on the coupling of a P450-bioreactor to an HPLC-based estrogen receptor alpha (ERalpha) affinity assay. P450-derived metabolites of the SERMs were generated in the bioreactor, subsequently trapped on-line with solid phase extraction, and finally separated with gradient HPLC. Upon elution, the metabolites were screened on affinity for ERalpha with an on-line HRS assay. With this HRS system, we were able to follow, in a time-dependent manner, the formation of ERalpha-binding metabolites of tamoxifen and raloxifene. By analyzing the bioaffinity chromatograms with liquid chromatography-tandem mass spectrometry, structural information of the pharmacologically active metabolites was obtained as well. For tamoxifen, 15 active and 6 nonactive metabolites were observed, of which 5 were of primary, 10 of secondary, and 6 of an as yet unknown order of metabolism. Raloxifene was biotransformed in three primary and three secondary metabolites. MS/MS analysis revealed that three of the observed active metabolites of raloxifene were not described before. The present automated on-line HRS system coupled to a P450-containing bioreactor and an ERalpha-affinity detector proved very efficient, sensitive, and selective in metabolic profiling of SERMs.