Charge-tagging liquid chromatography–mass spectrometry methodology targeting oxysterol diastereoisomers

The introduction of a hydroxy group to the cholesterol skeleton introduces not only the possibility for positional isomers but also diastereoisomers, where two or more isomers have different configurations at one or more of the stereocentres but are not mirror images. The differentiation of diastereoisomers is important as differing isomers can have differing biochemical properties and are formed via different biochemical pathways. Separation of diasterioisomers is not always easy by chromatographic methods. Here we demonstrate, by application of charge-tagging and derivatisation with the Girard P reagent, the separation and detection of biologically relevant diastereoisomers using liquid chromatography – mass spectrometry with multistage fragmentation

Developing an Enzyme-Assisted Derivatization Method for Analysis of C27 Bile Alcohols and Acids by Electrospray Ionization-Mass Spectrometry

Enzyme-assisted derivatization for sterol analysis (EADSA) is a technology designed to enhance sensitivity and specificity for sterol analysis using electrospray ionization–mass spectrometry. To date it has only been exploited on sterols with a 3β-hydroxy-5-ene or 3β-hydroxy-5α-hydrogen structure, using bacterial cholesterol oxidase enzyme to convert the 3β-hydroxy group to a 3-oxo group for subsequent derivatization with the positively charged Girard hydrazine reagents, or on substrates with a native oxo group. Here we describe an extension of the technology by substituting 3α-hydroxysteroid dehydrogenase (3α-HSD) for cholesterol oxidase, making the method applicable to sterols with a 3α-hydroxy-5β-hydrogen structure. The 3α-HSD enzyme works efficiently on bile alcohols and bile acids with this stereochemistry. However, as found by others, derivatization of the resultant 3-oxo group with a hydrazine reagent does not go to completion in the absence of a conjugating double bond in the sterol structure. Nevertheless, Girard P derivatives of bile alcohols and C27 acids give an intense molecular ion ([M]+) upon electrospray ionization and informative fragmentation spectra. The method shows promise for analysis of bile alcohols and 3α-hydroxy-5β-C27-acids, enhancing the range of sterols that can be analyzed at high sensitivity in sterolomic studies

Improving adherence in mental health service users with severe mental illness in South Africa: a pilot randomized controlled trial of a treatment partner and text message intervention vs. treatment as usual

Objectives: Medication non-adherence is a significant problem in treatment of severe mental disorders and is associated with poor clinical outcomes and increased demand on services. Task-shifting interventions incorporating mobile health may improve adherence in mental health service users in low- and middle-income countries. Seventy-seven participants were recruited from a psychiatric hospital in Cape Town, with 42 randomized to receive the intervention and 35 to treatment as usual. Intervention pairs underwent treatment-partner contracting and psychoeducation, and received monthly text message reminders of clinic appointments. Primary outcomes were intervention acceptability and feasibility. Secondary outcome for efficacy were adherence to clinic visit; relapse; quality of life; symptomatic relief and medication adherence. Results: Treatment partner and psychoeducation components were acceptable and feasible. The text message component was acceptable but not feasible in its current form. Efficacy outcomes favoured the intervention but did not reach statistical significance. A treatment-partner intervention is acceptable and feasible in a low- and middle-income setting. Work is needed to ensure that additional components of such interventions are tailored to the local context. Appropriately powered efficacy studies are needed. Trial Registration PACTR PACTR201610001830190, Registered 21 October 2016 (Retrospectively registered

Piloting a mental health training programme for community health workers in South Africa: an exploration of changes in knowledge, confidence and attitudes

Background There is a shortage of trained mental health workers in spite of the significant contribution of psychiatric disorders to the global disease burden. Task shifting, through the delegation of health care tasks to less specialised health workers such as community health workers (CHWs), is a promising approach to address the human resource shortage. CHWs in the Western Cape province of South Africa provide comprehensive chronic support which includes that for mental illness, but have thus far not received standardized mental health training. It is unknown whether a structured mental health training programme would be acceptable and feasible, and result improved knowledge, confidence and attitudes amongst CHWs. Methods We developed and piloted a mental health training programme for CHWs, in line with the UNESCO guidelines; the WHO Mental Health Gap Action Programme and the South African National framework for CHW training. In our quasi-experimental (before-after) cohort intervention study we measured outcomes at the start and end of training included: 1) Mental health knowledge, measured through the use of case vignettes and the Mental Health Knowledge Schedule; 2) confidence, measured with the Mental Health Nurse Clinical Confidence Scale; and 3) attitudes, measured with the Community Attitudes towards the Mentally Ill Scale. Knowledge measures were repeated 3 months later. Acceptability data were obtained from daily evaluation questionnaires and a training evaluation questionnaire, while feasibility was measured by participant attendance at training sessions. Results Fifty-eight CHWs received the training, with most (n = 56, 97.0%) attending at least 7 of the 8 sessions. Most participants (n = 29, 63.04%) demonstrated significant improvement in knowledge, which was sustained at 3-months. There was significant improvement in confidence, along with changes in attitude, indicating improved benevolence, reduced social restrictiveness, and increased tolerance to rehabilitation of the mentally ill in the community but there was no change in authoritarian attitudes. The training was acceptable and feasible. Conclusions Mental health training was successful in improving knowledge, confidence and attitudes amongst trained CHWs. The training was acceptable and feasible. Further controlled studies are required to evaluate the impact of such training on patient health outcomes. Trial registration PACTR PACTR201610001834198 , Registered 26 October 2016

Bile acid biosynthesis in Smith-Lemli-Opitz syndrome bypassing cholesterol: Potential importance of pathway intermediates

Bile acids are the end products of cholesterol metabolism secreted into bile. They are essential for the absorption of lipids and lipid soluble compounds from the intestine. Here we have identified a series of unusual Δ5-unsaturated bile acids in plasma and urine of patients with Smith-Lemli-Opitz syndrome (SLOS), a defect in cholesterol biosynthesis resulting in elevated levels of 7-dehydrocholesterol (7-DHC), an immediate precursor of cholesterol. Using liquid chromatography – mass spectrometry (LC-MS) we have uncovered a pathway of bile acid biosynthesis in SLOS avoiding cholesterol starting with 7-DHC and proceeding through 7-oxo and 7β-hydroxy intermediates. This pathway also occurs to a minor extent in healthy humans, but elevated levels of pathway intermediates could be responsible for some of the features SLOS. The pathway is also active in SLOS affected pregnancies as revealed by analysis of amniotic fluid. Importantly, intermediates in the pathway, 25-hydroxy-7-oxocholesterol, (25R)26-hydroxy-7-oxocholesterol, 3β-hydroxy-7-oxocholest-5-en-(25R)26-oic acid and the analogous 7β-hydroxysterols are modulators of the activity of Smoothened (Smo), an oncoprotein that mediates Hedgehog (Hh) signalling across membranes during embryogenesis and in the regeneration of postembryonic tissue. Computational docking of the 7-oxo and 7β-hydroxy compounds to the extracellular cysteine rich domain of Smo reveals that they bind in the same groove as both 20S-hydroxycholesterol and cholesterol, known activators of the Hh pathway

Mining for Oxysterols in Cyp7b1−/− Mouse Brain and Plasma: Relevance to Spastic Paraplegia Type 5

Deficiency in cytochrome P450 (CYP) 7B1, also known as oxysterol 7α-hydroxylase, in humans leads to hereditary spastic paraplegia type 5 (SPG5) and in some cases in infants to liver disease. SPG5 is medically characterized by loss of motor neurons in the corticospinal tract. In an effort to gain a better understanding of the fundamental biochemistry of this disorder, we have extended our previous profiling of the oxysterol content of brain and plasma of Cyp7b1 knockout (-/-) mice to include, amongst other sterols, 25-hydroxylated cholesterol metabolites. Although brain cholesterol levels do not differ between wild-type (wt) and knockout mice, we find, using a charge-tagging methodology in combination with liquid chromatography–mass spectrometry (LC–MS) and multistage fragmentation (MSn), that there is a build-up of the CYP7B1 substrate 25-hydroxycholesterol (25-HC) in Cyp7b1-/- mouse brain and plasma. As reported earlier, levels of (25R)26-hydroxycholesterol (26-HC), 3β-hydroxycholest-5-en-(25R)26-oic acid and 24S,25-epoxycholesterol (24S,25-EC) are similarly elevated in brain and plasma. Side-chain oxysterols including 25-HC, 26-HC and 24S,25-EC are known to bind to INSIG (insulin-induced gene) and inhibit the processing of SREBP-2 (sterol regulatory element-binding protein-2) to its active form as a master regulator of cholesterol biosynthesis. We suggest the concentration of cholesterol in brain of the Cyp7b1-/- mouse is maintained by balancing reduced metabolism, as a consequence of a loss in CYP7B1, with reduced biosynthesis. The Cyp7b1-/- mouse does not show a motor defect; whether the defect in humans is a consequence of less efficient homeostasis of cholesterol in brain has yet to be uncovered

Sterols and oxysterols in plasma from Smith-Lemli-Opitz syndrome patients

Smith-Lemli-Opitz syndrome (SLOS) is a severe autosomal recessive disorder resulting from defects in the cholesterol synthesising enzyme 7-dehydrocholesterol reductase (Δ7-sterol reductase, DHCR7, EC 1.3.1.21) leading to a build-up of the cholesterol precursor 7-dehydrocholesterol (7-DHC) in tissues and blood plasma. Although the underling enzyme deficiency associated with SLOS is clear there are likely to be multiple mechanisms responsible for SLOS pathology. In an effort to learn more of the aetiology of SLOS we have analysed plasma from SLOS patients to search for metabolites derived from 7-DHC which may be responsible for some of the pathology. We have identified a novel hydroxy-8-dehydrocholesterol, which is either 24- or 25-hydroxy-8-dehydrocholesterol and also the known metabolites 26-hydroxy-8-dehydrocholesterol, 4-hydroxy-7-dehydrocholesterol, 3β,5α-dihydroxycholest-7-en-6-one and 7α,8α-epoxycholesterol. None of these metabolites are detected in control plasma at quantifiable levels (0.5 ng/mL)

New methods for analysis of oxysterols and related compounds by LC–MS

Oxysterols are oxygenated forms of cholesterol or its precursors. They are formed enzymatically and via reactive oxygen species. Oxysterols are intermediates in bile acid and steroid hormone biosynthetic pathways and are also bioactive molecules in their own right, being ligands to nuclear receptors and also regulators of the processing of steroid regulatory element-binding proteins (SREBPs) to their active forms as transcription factors regulating cholesterol and fatty acid biosynthesis. Oxysterols are implicated in the pathogenesis of multiple disease states ranging from atherosclerosis and cancer to multiple sclerosis and other neurodegenerative diseases including Alzheimer’s and Parkinson’s disease. Analysis of oxysterols is challenging on account of their low abundance in biological systems in comparison to cholesterol, and due to the propensity of cholesterol to undergo oxidation in air to generate oxysterols with the same structures as those present endogenously. In this article we review the mass spectrometry-based methods for oxysterol analysis paying particular attention to analysis by liquid chromatography – mass spectrometry (LC-MS)

Reduced Plasma Levels of 25-Hydroxycholesterol and Increased Cerebrospinal Fluid Levels of Bile Acid Precursors in Multiple Sclerosis Patients

Multiple sclerosis (MS) is an autoimmune, inflammatory disease of the central nervous system (CNS). We have measured the levels of over 20 non-esterified sterols in plasma and cerebrospinal fluid (CSF) from patients suffering from MS, inflammatory CNS disease, neurodegenerative disease and control patients. Analysis was performed following enzyme-assisted derivatisation by liquid chromatography-mass spectrometry (LC-MS) exploiting multistage fragmentation (MS n ). We found increased concentrations of bile acid precursors in CSF from each of the disease states and that patients with inflammatory CNS disease classified as suspected autoimmune disease or of unknown aetiology also showed elevated concentrations of 25-hydroxycholestertol (25-HC, P < 0.05) in CSF. Cholesterol concentrations in CSF were not changed except for patients diagnosed with amyotrophic lateral sclerosis (P < 0.01) or pathogen-based infections of the CNS (P < 0.05) where they were elevated. In plasma, we found that 25-HC (P < 0.01), (25R)26-hydroxycholesterol ((25R)26-HC, P < 0.05) and 7α-hydroxy-3-oxocholest-4-enoic acid (7αH,3O-CA, P < 0.05) were reduced in relapsing-remitting MS (RRMS) patients compared to controls. The pattern of reduced plasma levels of 25-HC, (25R)26-HC and 7αH,3O-CA was unique to RRMS. In summary, in plasma, we find that the concentration of 25-HC in RRMS patients is significantly lower than in controls. This is consistent with the hypothesis that a lower propensity of macrophages to synthesise 25-HC will result in reduced negative feedback by 25-HC on IL-1 family cytokine production and exacerbated MS. In CSF, we find that the dominating metabolites reflect the acidic pathway of bile acid biosynthesis and the elevated levels of these in CNS disease is likely to reflect cholesterol release as a result of demyelination or neuronal death. 25-HC is elevated in patients with inflammatory CNS disease probably as a consequence of up-regulation of the type 1 interferon-stimulated gene cholesterol 25-hydroxylase in macrophage

A proposal for a coordinated effort for the determination of brainwide neuroanatomical connectivity in model organisms at a mesoscopic scale

In this era of complete genomes, our knowledge of neuroanatomical circuitry remains surprisingly sparse. Such knowledge is however critical both for basic and clinical research into brain function. Here we advocate for a concerted effort to fill this gap, through systematic, experimental mapping of neural circuits at a mesoscopic scale of resolution suitable for comprehensive, brain-wide coverage, using injections of tracers or viral vectors. We detail the scientific and medical rationale and briefly review existing knowledge and experimental techniques. We define a set of desiderata, including brain-wide coverage; validated and extensible experimental techniques suitable for standardization and automation; centralized, open access data repository; compatibility with existing resources, and tractability with current informatics technology. We discuss a hypothetical but tractable plan for mouse, additional efforts for the macaque, and technique development for human. We estimate that the mouse connectivity project could be completed within five years with a comparatively modest budget.Comment: 41 page