Bile Acid Precursors Research Articles

The importance of choosing the appropriate cholesterol quantification method: enzymatic assay versus gas chromatography

Cholesterol is a major lipid of the animal realm with many biological roles. It is an important component of cellular membranes and a precursor of steroid hormones and bile acids. It is particularly abundant in nervous tissues, and dysregulation of cholesterol metabolism has been associated with neurodegenerative diseases such as Alzheimer's and Huntington's diseases. Deciphering the pathophysiological mechanisms of these disorders often involves animal models such as mice and Drosophila. Accurate quantification of cholesterol levels in the chosen models is a critical point of these studies. In the present work, we compare two common methods, gas chromatography coupled to flame-ionization detection (GC/FID) and a cholesterol oxidase-based fluorometric assay to measure cholesterol in mouse brains and Drosophila heads. Cholesterol levels measured by the two methods were similar for the mouse brain, which presents a huge majority of cholesterol in its sterol profile. On the contrary, depending on the method, measured cholesterol levels were very different for Drosophila heads, which present a complex sterol profile with a minority of cholesterol. We showed that the enzyme-based assay is not specific for cholesterol and detects other sterols as well. This method is therefore not suited for cholesterol measurement in models such as Drosophila. Alternatively, chromatographic methods, such as GC/FID, offer the required specificity for cholesterol quantification. Understanding the limitations of the quantification techniques is essential for reliable interpretation of the results in cholesterol-related research.

Lemon Flavonoid Extract Eriomin Improves Pro/Antioxidant Status and Interferes with Cholesterol Metabolism without Affecting Serum Cholesterol Levels in Aged Rats.

This study aimed to assess the antioxidant capacity of lemon flavonoid extract Eriomin® (LE) and its impact on cholesterol metabolism in the context of healthy aging. We orally treated 24-month-old male Wistar rats with an LE (40 mg/kg) suspended in 0.3 mL of sunflower oil. At the same time, control groups received an equal volume of sunflower oil (CON) or remained untreated (ICON) daily for 4 weeks. We examined LE's effects on superoxide dismutase and catalase- and glutathione-related enzyme activities, the concentration of lipid peroxides and protein carbonyls, total oxidant status (TOS) and antioxidant status (TAS), and oxidative stress index (OSI) in the liver, jejunum, and ileum. We also measured total cholesterol, its biosynthetic precursors (lanosterol, lathosterol, desmosterol), its degradation products (bile acid precursors) in the serum, liver, jejunum, and ileum, and serum phytosterols (intestinal absorption markers). LE reduced TOS, TAS, and OSI (p < 0.05) compared with control values, indicating its consistent antioxidant action in all examined organs. LE lowered hepatic desmosterol (p < 0.05) while also reducing 7α- and 24-hydroxycholesterol levels in the liver and ileum (p < 0.01). Serum cholesterol, hepatic gene expression, and the immunostaining intensity of CYP7A1 were unchanged. In conclusion, LE exerted non-enzymatic antioxidant effects and reduced cholesterol degradation, reducing its biosynthesis products, thereby maintaining serum cholesterol levels.

Cholesterol uptake in the intestine is regulated by the LASP1-AKT-NPC1L1 signaling pathway.

Cholesterol is essential for the stability and architecture of the plasma membrane and a precursor of bile acids and steroid hormones in mammals. Excess dietary cholesterol uptake leads to hypercholesterolemia and atherosclerosis and plays a role in cancer development. The role of actin-binding scaffolding protein LIM and SH3 protein 1 (LASP1) in cholesterol trafficking has not been investigated previously. Cholesterol levels, its uptake, and excretion were studied in mice deficient for low-density lipoprotein receptor and Lasp1 (Ldlr-/-Lasp1-/- mice) upon feeding a high-fat diet, and in LASP1-knockdown, differentiated human intestinal epithelial CaCo-2 cells. When compared with diet-fed Ldlr-/- control mice, Ldlr-/-Lasp1-/- mice displayed a reduction in serum cholesterol levels. Mechanistically, we identified a new role of LASP1 in controlling the translocation of the intestinal cholesterol transporter Niemann-Pick C1-like 1 (NPC1L1) to the apical cell surface, which was limited in LASP1-knockdown human CaCo-2 enterocytes and in the intestine of Ldlr-/- Lasp1-/- compared with Ldlr-/- mice, linked to LASP1-pAKT signaling but not CDC42 activation. In line, a reduction in cholesterol reabsorption was noted in LASP1-knockdown CaCo-2 cells in vitro, and an enhanced cholesterol excretion via the feces was observed in Ldlr-/- Lasp1-/- mice. These data uncover a novel function of Lasp1 in cholesterol trafficking, promoting cholesterol reabsorption in the intestine. Targeting LASP1 locally could thus represent a novel targeting strategy to ameliorate hypercholesterolemia and associated diseases.NEW & NOTEWORTHY We here uncovered LASP1 as a novel regulator of the shuttling of the sterol transporter NPC1L1 to the cell surface in enterocytes to control cholesterol absorption. Accordingly, LASP1-deficient mice displayed lowered serum cholesterol levels under dietary cholesterol supplementation.

Genetically and clinically confirmed atypical cerebrotendinous xanthomatosis with normal cholestanol and marked elevations of bile acid precursors and bile alcohols

Cerebrotendinous xanthomatosis (CTX) is a rare autosomal recessive lipid disorder. Affected patients often remain undiagnosed until the age of 20-30 years, when they have already developed significant neurologic disease that may not be reversible. An elevated plasma cholestanol concentration has been accepted as a diagnostic criterion for CTX for decades. Full biochemical characterization was performed for three genetically and clinically confirmed atypical CTX cases with normal plasma cholestanol levels. Clinical assessment and genetic/biochemical testing for patients with CTX was performed by their physician providing routine standard of care. We report three new atypical CTX cases with large extensor tendon xanthomas but normal plasma cholestanol levels. All three cases had marked elevations of bile acid precursors and bile alcohols in plasma and urine that decreased on treatment with chenodeoxycholic acid. We also review eight published cases of atypical CTX with normal/near normal circulating cholestanol levels. The atypical biochemical presentation of these cases provides a diagnostic challenge for CTX, a disorder for which cholestanol has been believed to be a sensitive biomarker. These cases demonstrate measurements of plasma cholestanol alone are insufficient to exclude a diagnosis of CTX. The data presented is consistent with the concept that bile acid precursors and bile alcohols are sensitive biomarkers for atypical CTX with normal cholestanol, and that such testing is indicated, along with CYP27A1 gene analyses, in patients presenting with significant tendon and/or tuberous xanthomas and/or neurologic disease in early adulthood despite normal or near normal cholesterol and cholestanol levels.

Cholesterol is more readily oxidized than phospholipid linoleates in cell membranes to produce cholesterol hydroperoxides

Cholesterol is an essential component of cell membranes and serves as an important precursor of steroidal hormones and bile acids, but elevated levels of cholesterol and its oxidation products have been accepted as a risk factor for maintenance of health. The free and ester forms of cholesterol and fatty acids are the two major biological lipids. The aim of this hypothesis paper is to address the long-standing dogma that cholesterol is less susceptible to free radical peroxidation than polyunsaturated fatty acids (PUFAs). It has been observed that cholesterol is peroxidized much slower than PUFAs in plasma but that, contrary to expectations from chemical reactivity toward peroxyl radicals, cholesterol appears to be more readily autoxidized than linoleates in cell membranes. The levels of oxidation products of cholesterol and linoleates observed in humans support this notion. It is speculated that this discrepancy is ascribed to the fact that cholesterol and phospholipids bearing PUFAs are localized apart in raft and non-raft domains of cell membranes respectively and that the antioxidant vitamin E distributed predominantly in the non-raft domains cannot suppress the oxidation of cholesterol lying in raft domains which are relatively deficient in antioxidant.

Metabolomic Insights into the Pathogenesis and Prognostic Potential of Adult Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) comprises hematological malignancies affecting B or T cell precursors (BCP-ALL or T-ALL). Despite considerable progress made over the past three decades in the treatment of childhood ALL [5-year disease survival rate (5-y DFS): 90%], the prognosis of adult ALL remains relatively poor (5-y DFS: 50-70%). Hence, gaining a deeper understanding of the pathogenic factors is of timely importance. Recent advancements in next-generation genome sequencing technologies have facilitated the refinement of disease diagnosis and prognosis. However, due to the inherent genetic diversity among patients, additional tools are needed to unravel the complex etiological basis and improve risk stratification for personalized therapeutic interventions, particularly in adult ALL. Metabolomics offers a powerful approach that enables the comprehensive exploration of cellular states by analyzing metabolites quantitatively and qualitatively, which reflects altered enzyme kinetic activities and changes in metabolic reactions. It was previously reported that most bone marrow metabolites could be detected in plasma in BCP-ALL (81%). In this study, we reported an integrated analysis of serum metabolites using liquid chromatography/mass spectrometry as well as the metabolic gene expression profiles from RNA-sequencing data of bone marrow blasts, generating a comprehensive metabolic information dataset (CMID) in 211 adult ALL patients (BCP-ALL: n=160; T-ALL: n=51) who participated in our recent clinical trial (ChiCTR-ONRC-14004968). The graphical abstract is depicted in Figure 1A. Serum metabolomic landscape of adult ALL was obtained at various time points during treatment, including samples at diagnosis (Day 0, D0, n=198), during the induction phase (D14, n=177 and D28, n=147) and the early consolidation phase (Cons, n=58). These samples were subject to comparison to those from 164 age/sex-matched healthy controls (HC). Using CMID, we further subtyped BCP-ALL (clusters BC1 and BC2, and a special cluster of Ph + ALL) and T-ALL (clusters TC1 and TC2), revealing that BC2 and TC1 were associated with relatively favorable outcome, compared respectively to BC1 (OS: P&amp;lt;0.05) and TC2 (OS: P&amp;lt;0.05). Regarding serum metabolomic profiles over the treatment course, we found a number of featured disturbances ( Figure 1B). Aberrant lipid-related metabolisms, such as steroid metabolism, were detectable throughout different phases. In contrast, the metabolism of amino acids and a number of metabolites pumped by ATP-binding cassette transporter were severely impacted by therapeutic agents during the induction phase (on D14). Notably, at the time of diagnosis, primary bile acid biosynthesis was enriched, consistent with a previous study on pediatric ALL. In our functional experiment, inhibiting the synthesis of bile acid precursors such as cholesterol with Simvastatin suppressed leukemic cell viability in 10 ALL cell lines, indicating a potential role of this pathway in leukemogenesis. In addition, we identified arachidonic acid (AA) metabolism enrichment in the favorable BC2 subgroup. Coincidentally, glycerophospholipid metabolism, which provides substrates for AA synthesis, was also enriched in the TC1 subgroup, together with an active AA metabolism. Increasing data suggests that AA metabolites act as immunoregulators in cancer, thus we hypothesize that they could play a role in ALL prognosis by regulating the immune reaction against leukemic cells in the hematopoietic microenvironment. In our further efforts to address the correlation between CMID-based subtypes and known genetic events in Ph --BCP-ALL and T-ALL leukemogenesis, we discovered that some favorable genetic markers (ZNF-384/362 fusions and high ploidy) tended to fall into BC2, while NRAS mutations were only observed in TC1. On the other hand, both CMID-based subtypes and genetic events-based ones stand as independent factors in multivariate Cox analysis for risk stratification of BCP-ALL, demonstrating an added value of the CMID subtypes in the prognosis of adult BCP-ALL. Our work has thus highlighted the significance of altered metabolome in ALL pathogenesis, proved its clinical relevance for prognosis stratification, and identified its potential vulnerabilities for improving tailored therapy.

Characterization of Postprandial Bile Acid Profiles and Glucose Metabolism in Cerebrotendinous Xanthomatosis.

Cerebrotendinous xanthomatosis (CTX) is a rare inherited disease characterized by sterol 27-hydroxylase (CYP27A1) deficiency and, thus, a lack of bile acid synthesis with a marked accumulation of 7α-hydroxylated bile acid precursors. In addition to their renowned lipid-emulgating role, bile acids have been shown to stimulate secretion of the glucose-lowering and satiety-promoting gut hormone glucagon-like peptide 1 (GLP-1). In this paper, we examined postprandial bile acid, glucose, insulin, GLP-1 and fibroblast growth factor 19 (FGF19) plasma profiles in patients with CTX and matched healthy controls. Seven patients and seven age, gender and body mass index matched controls were included and subjected to a 4 h mixed meal test with regular blood sampling. CTX patients withdrew from chenodeoxycholic acid (CDCA) and statin therapy three weeks prior to the test. Postprandial levels of total bile acids were significantly lower in CTX patients and consisted of residual CDCA with low amounts of ursodeoxycholic acid (UDCA). The postprandial plasma glucose peak concentration occurred later in CTX patients compared to controls, and patients' insulin levels remained elevated for a longer time. Postprandial GLP-1 levels were slightly higher in CTX subjects whereas postprandial FGF19 levels were lower in CTX subjects. This novel characterization of CTX patients reveals very low circulating bile acid levels and FGF19 levels, aberrant postprandial glucose and insulin profiles, and elevated postprandial GLP-1 responses.

Cholesterol metabolism in T-cell aging: Accomplices or victims.

Aging has a significant impact on the function and metabolism of T cells. Cholesterol, the most important sterol in mammals, is known as the "gold of the body" because it maintains membrane fluidity, rigidity, and signal transduction while also serving as a precursor of oxysterols, bile acids, and steroid hormones. Cholesterol homeostasis is primarily controlled by uptake, biosynthesis, efflux, and regulatory mechanisms. Previous studies have suggested that there are reciprocal interactions between cholesterol metabolism and T lymphocytes. Here, we will summarize the most recent advances in the effects of cholesterol and its derivatives on T-cell aging. We will furthermore discuss interventions that might be used to help older individuals with immune deficiencies or diminishing immune competence.

Differences in Cholesterol Metabolism, Hepato-Intestinal Aging, and Hepatic Endocrine Milieu in Rats as Affected by the Sex and Age.

Age and sex influence serum cholesterol levels, but the underlying mechanisms remain unclear. To investigate further, we measured cholesterol, precursors (surrogate synthesis markers), degradation products (oxysterols and bile acid precursors) in serum, the liver, jejunum, and ileum, as well as serum plant sterols (intestinal absorption markers) in male and female Wistar rats (4 and 24 months old). The analysis of histomorphometric and oxidative stress parameters (superoxide dismutase, catalase, glutathione-related enzyme activities, lipid peroxide, and protein carbonyl concentrations) in the liver and jejunum offered further insights into the age- and sex-related differences. The hepatic gene expression analysis included AR, ERα, and sex-specific growth hormone-regulated (Cyp2c11 and Cyp2c12) and thyroid-responsive (Dio1, Tbg, and Spot 14) genes by qPCR. We observed age-related changes in both sexes, with greater prominence in females. Aged females had significantly higher serum cholesterol (p < 0.05), jejunum cholesterol (p < 0.05), and serum plant sterols (p < 0.05). They exhibited poorer hepato-intestinal health compared with males, which was characterized by mild liver dysfunction (hydropic degeneration, increased serum ALT, p < 0.05, and decreased activity of some antioxidant defense enzymes, p < 0.05), mononuclear inflammation in the jejunal lamina propria, and age-related decreases in jejunal catalase and glutathione peroxidase activity (p < 0.05). Aged females showed increased levels of 27-hydroxycholesterol (p < 0.05) and upregulated ERα gene expression (p < 0.05) in the liver. Our study suggests that the more significant age-related increase in serum cholesterol in females is associated with poorer hepato-intestinal health and increased jejunal cholesterol absorption. The local increase in 27-hydroxycholesterol during aging might reduce the hepatoprotective effects of endogenous estrogen in the female liver.

Altered gut microbiota–host bile acid metabolism in IBS-D patients with liver depression and spleen deficiency pattern

BackgroundDysregulation of gut microbiota–host bile acid (BA) co-metabolism is a critical pathogenic factor of diarrhea-predominant irritable bowel syndrome (IBS-D). Traditional Chinese Medicine (TCM), instructed by pattern differentiation, is effective in treating IBS-D, in which liver depression and spleen deficiency (LDSD) is the most prevalent pattern. Still, it is unclear the linkage between the LDSD pattern and the BA metabolic phenotype.PurposeThis study aimed to uncover the biological basis of the LDSD pattern from the BA metabolic perspective.MethodsPatients with IBS-D completed questionnaires regarding the irritable bowel severity scoring system (IBS-SSS), stool frequency, Stool Bristol scale, and Self-Rating Scales of mental health. Fasting blood and morning feces were collected to analyze the gut metagenome and BA-related indices/metabolites.ResultsIBS-D patients with LDSD had a higher incidence of BA overexcretion (41% vs. 23% non-LDSD) with significant elevations in fecal total BAs and serum BA precursor 7α-hydroxy-4-cholesten-3-one levels. Compared to controls or non-LDSD patients, LDSD patients had a featured fecal BA profile, with higher proportions of deoxycholic acid (DCA), 7-ketodeoxycholic acid, and lithocholic acid. It is consistent with the BA-metabolizing genomic changes in the LDSD gut microbiota characterized by overabundances of 7-dehydroxylating bacteria and BA-inducible genes (baiCD/E/H). The score of bowel symptoms (stool frequency and abdominal pain) showing greater severity in the LDSD pattern were positively correlated with bai-expressing bacterial abundances and fecal DCA levels separately.ConclusionWe clarified a differed BA metabolic phenotype in IBS patients with LDSD, which closely correlates with the severity of bowel symptoms. It demonstrates that gut microbiota and host co-metabolism of BAs would provide crucial insight into the biology of the LDSD pattern and its internal relationship with IBS progression.

PREPARATION CHOLESTEROL AND EFFECTS OF CHOLESTEROL PARADOX: A REVIEW

Cholesterol is the most significant sterol in mammals, which contributes to preserving plasma membrane fluidity and serves as a precursor of oxysterols, bile acids, and steroid hormones. Plasma membranes contain large amounts of cholesterol, which interacts with other molecules in the membrane in various ways. Cholesterol, one of the most significant structural elements, is regarded as a lipid-type molecule. It plays a crucial role in life, primarily in cell membranes and as a precursor to the biosynthesis of numerous steroid hormones in cell membranes. Cholesterol absorption is linked to recurrent cardiovascular events, and patients with higher cholesterol absorption experienced less of a statin's favourable effects. One of the essential and prevalent conditions that affect people's health worldwide is coronary artery disease (CAD), and its incidence has grown over time. The best predictor and moderator of coronary artery disease is the low-density lipoprotein cholesterol plasma level. Higher than lower HDL cholesterol levels were associated with a lower risk of major cardiovascular events. Contrarily in cholesterol paradox states, individuals with advanced HF often have low cholesterol, associated with a poor prognosis and hypercholesterolemia, a key risk factor for CAD. On average, patients with a total cholesterol level of 232 mg/dl had a 25% higher survival rate than those with a total cholesterol level of 193 mg/dl who were suffering from heart failure. A total cholesterol level under 200 mg/dl is generally preferred.

Synthesis, function, and regulation of sterol and nonsterol isoprenoids.

Cholesterol, the bulk end-product of the mevalonate pathway, is a key component of cellular membranes and lipoproteins that transport lipids throughout the body. It is also a precursor of steroid hormones, vitamin D, and bile acids. In addition to cholesterol, the mevalonate pathway yields a variety of nonsterol isoprenoids that are essential to cell survival. Flux through the mevalonate pathway is tightly controlled to ensure cells continuously synthesize nonsterol isoprenoids but avoid overproducing cholesterol and other sterols. Endoplasmic reticulum (ER)-localized 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase (HMGCR), the rate limiting enzyme in the mevalonate pathway, is the focus of a complex feedback regulatory system governed by sterol and nonsterol isoprenoids. This review highlights transcriptional and post-translational regulation of HMGCR. Transcriptional regulation of HMGCR is mediated by the Scap-SREBP pathway. Post-translational control is initiated by the intracellular accumulation of sterols, which causes HMGCR to become ubiquitinated and subjected to proteasome-mediated ER-associated degradation (ERAD). Sterols also cause a subfraction of HMGCR molecules to bind the vitamin K2 synthetic enzyme, UbiA prenyltransferase domain-containing protein-1 (UBIAD1). This binding inhibits ERAD of HMGCR, which allows cells to continuously synthesize nonsterol isoprenoids such as geranylgeranyl pyrophosphate (GGPP), even when sterols are abundant. Recent studies reveal that UBIAD1 is a GGPP sensor, dissociating from HMGCR when GGPP thresholds are met to allow maximal ERAD. Animal studies using genetically manipulated mice disclose the physiological significance of the HMGCR regulatory system and we describe how dysregulation of these pathways contributes to disease.

Prenylation Defects and Oxidative Stress Trigger the Main Consequences of Neuroinflammation Linked to Mevalonate Pathway Deregulation.

The cholesterol biosynthesis represents a crucial metabolic pathway for cellular homeostasis. The end products of this pathway are sterols, such as cholesterol, which are essential components of cell membranes, precursors of steroid hormones, bile acids, and other molecules such as ubiquinone. Furthermore, some intermediates of this metabolic system perform biological activity in specific cellular compartments, such as isoprenoid molecules that can modulate different signal proteins through the prenylation process. The defects of prenylation represent one of the main causes that promote the activation of inflammation. In particular, this mechanism, in association with oxidative stress, induces a dysfunction of the mitochondrial activity. The purpose of this review is to describe the pleiotropic role of prenylation in neuroinflammation and to highlight the consequence of the defects of prenylation.

Central nervous system cholesterol metabolism in health and disease.

Cholesterol is a ubiquitous and essential component of cellular membranes, as it regulates membrane structure and fluidity. Furthermore, cholesterol serves as a precursor for steroid hormones, oxysterol, and bile acids, that are essential for maintaining many of the body's metabolic processes. The biosynthesis and excretion of cholesterol is tightly regulated in order to maintain homeostasis. Although virtually all cells have the capacity to make cholesterol, the liver and brain are the two main organs producing cholesterol in mammals. Once produced, cholesterol is transported in the form of lipoprotein particles to other cell types and tissues. Upon formation of the blood-brain barrier (BBB) during embryonic development, lipoproteins cannot move between the central nervous system (CNS) and the rest of the body. As such, cholesterol biosynthesis and metabolism in the CNS operate autonomously without input from the circulation system in normal physiological conditions. Nevertheless, similar regulatory mechanisms for maintaining cholesterol homeostasis are utilized in both the CNS and peripheral systems. Here, we discuss the functions and metabolism of cholesterol in the CNS. We further focus on how different CNS cell types contribute to cholesterol metabolism, and how ApoE, the major CNS apolipoprotein, is involved in normal and pathophysiological functions. Understanding these basic mechanisms will aid our ability to elucidate how CNS cholesterol dysmetabolism contributes to neurogenerative diseases.

Biochemical characterization of the bile acid synthetic enzyme 3β‐hydroxy‐Δ5‐C27 steroid dehydrogenase/isomerase type VII

Cholesterol is a key biological molecule that is a vital precursor of steroid molecule biosynthesis, including steroid hormones, bile acids and vitamin D3.These processes require 3β‐hydroxysteroid dehydrogenase (3β‐HSD) enzymes, which catalyze dehydrogenation of hydroxysteroids. This family of enzymes catalyzes a wide variety of reaction mechanisms, carrying out a combination of oxidation, isomerization and decarboxylase reactions that result in the formation of cholesterol, steroid hormones and bile acids. 3β‐hydroxy‐Δ5‐C27 steroid dehydrogenase/isomerase type VII (HSD3B7) is responsible for the NAD+ dependent conversion of 7α‐hydroxycholesterol (7‐OHC) to 7α‐hydroxy‐3‐oxo‐4‐cholestenoate (7‐HOC), the second step of the bile acid synthesis pathway, which is vital for nutrient absorption and excretion of excess lipids. This oxidation reaction is one of many steps that increases the solubility of cholesterol for either storage or secretion. HSD3B7 shows substantial functional differences from other family members leaving significant gaps in our understanding of its mode of catalysis and substrate specificity. To this end, I have characterized HSD3B7 using Michaelis‐Menten kinetics, thermal shift assays, and begun x‐ray crystallographic studies. These studies reveal that HSD3B7 displays activity towards multiple bile acid precursors with differing hydrocarbon tail oxidation, with Km values in the low micromolar range. HSD3B7 retains its catalytic efficiency towards these substrates, suggesting that its substrate binding pocket can withstand changes in polarity upon alterations to this hydrocarbon tail. Lastly, HSD3B7 can be crystallized after modification to its membrane associated region, which will result in one of the first structures of a 3β‐HSD enzyme. Together these studies provide key insight into the mechanism of substrate specificity of HSD3B7 and appears amenable for X‐ray crystal structure determination.

Features of bile acid metabolism in fish

The fish liver is an important organ involved in the complex metabolism of bile and bile acids. The biochemical reactions that occur during this process directly depend on the functional state of the digestive system and fish nutrition. Knowledge of the features of bile acid metabolism will allow designing the cheapest and at the same time effective drugs for the pharmacological correction of hepatopathy. The composition of bile acids depends on the type of food, so in fish such as pike, perch, carp, cholic and deoxycholic acids conjugated with taurine are more common. In predatory fish, cholic acid predominates, in contrast to benthivorous fish. Substances secreted with bile do not participate in the general metabolism of tissues, so that hepatocytes do not need to constantly secrete large volumes of bile.The metabolism of bile acids is carried out by the liver and is called the enterohepatic circulation. It all starts with the biological precursor of bile acids - cholesterol. Cholesterol in the body is formed during the absorption of lipids in the intestine. Two-thirds of exogenous cholesterol is excreted from the body in the form of bile acids. As a result of transformation by hepatocytes, primary bile acids are formed - cholic and chenodioxycholic. Then they are cojugated with taurine or glycine in the C-24 region of the carboxyl group and are excreted from hepatocytes continuously through the bile ducts to the gallbladder and further to the intestine. In the intestine, under the influence of microbial metabolites, they are hydroxylated and converted into secondary bile acids. Cholic becomes deoxycholic, and chenodisoxycholic becomes lithocholic. A small amount of bile acids are in a free state and form paired compounds. 90% of bile acids in the intestine form complexes of choleic acids in combination with fatty acids, which are absorbed through the wall of enterocytes. In enterocytes, these complexes again disintegrate and bile acids again enter the liver through the portal vein. Thus the circle of metabolism is closed.

Synthesis of new bile acid-fused tetrazoles using the Schmidt reaction.

A practical and high-yielding Schmidt reaction for the synthesis of fused tetrazoles from bile acid precursors was developed. Mild reaction conditions using TMSN3 instead of hydrazoic acid as an azide source produced good yields of the desired tetrazoles. These conditions could be applied to other steroidal precursors. Additionally, an improved methodology for the synthesis of different ketone and enone precursors from cholic acid, deoxycholic acid, and chenodeoxycholic acid was established. Newly obtained tetrazole derivatives were characterized by NMR and X-ray diffraction spectroscopy. In a number of cases, preliminary antiproliferative tests of new compounds showed strong and selective activity towards certain tumor cell lines.

Cholesterol dyshomeostasis in amyotrophic lateral sclerosis: cause, consequence, or epiphenomenon?

Amyotrophic lateral sclerosis (ALS), the most common adult-onset motor neuron disease, is characterized by the selective degeneration of motor neurons leading to paralysis and eventual death. Multiple pathogenic mechanisms, including systemic dysmetabolism, have been proposed to contribute to ALS. Among them, dyslipidemia, i.e., abnormal level of cholesterol and other lipids in the circulation and central nervous system (CNS), has been reported in ALS patients, but without a consensus. Cholesterol is a constituent of cellular membranes and a precursor of steroid hormones, oxysterols, and bile acids. Consequently, optimal cholesterol levels are essential for health. Due to the blood-brain barrier (BBB), cholesterol cannot move between the CNS and the rest of the body. As such, cholesterol metabolism in the CNS is proposed to operate autonomously. Despite its importance, it remains elusive how cholesterol dyshomeostasis may contribute to ALS. In this review, we aim to describe the current state of cholesterol metabolism research in ALS, identify unresolved issues, and provide potential directions.

An LC-MS/MS Method for Analysis of Vitamin D Metabolites and C3 Epimers in Mice Serum: Oral Supplementation Compared to UV Irradiation.

Introduction: The most common forms of vitamin D in human and mouse serum are vitamin D3 and vitamin D2 and their metabolites. The aim of this study is to determine whether diet and sunlight directly affect the circulating concentrations of vitamin D metabolites in a mouse model. We investigated the serum concentrations of eight vitamin D metabolites—vitamin D (vitamin D3 + vitamin D2), 25OHD (25OHD3 + 25OHD2), 1α25(OH)2D (1α25(OH)2D2, and 1α25(OH)2D3)—including their epimer, 3-epi-25OHD (3-epi-25OHD3 and 3-epi-25OHD2), and a bile acid precursor 7alpha-hydroxy-4-cholesten-3-one (7αC4), which is known to cause interference in liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Method: The LC-MS/MS method was validated according to FDA-US guidelines. The validated method was used for the analysis of mouse serum samples. Forty blood samples from mice were collected and divided into three groups. The first group, the DDD mice, were fed a vitamin D-deficient diet (25 IU VD3/kg of diet) and kept in the dark; the second group, the SDD mice, were maintained on a standard-vitamin D diet (1000 IU VD3) and kept in the dark; and the third group, SDL, were fed a standard-vitamin D diet (1000 IU VD3) but kept on a normal light/dark cycle. LC-MS/MS was used for the efficient separation and quantitation of all the analytes. Results: The validated method showed good linearity and specificity. The intraday and interday precision were both <16%, and the accuracy across the assay range was within 100 ± 15%. The recoveries ranged between 75 and 95%. The stability results showed that vitamin D metabolites are not very stable when exposed to continuous freeze–thaw cycles; the variations in concentrations of vitamin D metabolites ranged between 15 and 60%. The overlapping peaks of vitamin D, its epimers, and its isobar (7αC4) were resolved using chromatographic separation. There were significant differences in the concentrations of all metabolites of vitamin D between the DDD and SDL mice. Between the groups SDD (control) and SDL, a significant difference in the concentrations of 3-epi-25OHD was noted, where C3 epimer was about 30% higher in SDL group while no significant differences were noted in the concentrations of vitamin D, 25OHD, 1α25(OH)2D, and 7αC4 between SDD and SDL group. Conclusions: A validated method, combined with a simple extraction technique, for the sensitive LC-MS/MS determination of vitamin D metabolites is described here. The method can eliminate the interferences in LC-MS/MS analysis caused by the overlapping epimer and isobar due to them having the same molecular weights as 25OHD. The validated method was applied to mouse serum samples. It was concluded that a standard-vitamin D diet causes an increase in the proportion of all the vitamin D metabolites and C3 epimers and isobar, while UV light has no pronounced effect on the concentrations of the majority of the vitamin D metabolites except 3-epi-25OHD. Further studies are required to confirm this observation in humans and to investigate the biochemical pathways related to vitamin D’s metabolites and their epimers.

Seladelpar improved measures of pruritus, sleep, and fatigue and decreased serum bile acids in patients with primary biliary cholangitis.

Primary biliary cholangitis (PBC) can result in life-altering cholestatic pruritus and fatigue, but treatment options are limited. Seladelpar, a peroxisome proliferator-activated receptor-delta (PPARδ) agonist, has demonstrated potent anti-cholestatic effects in clinical studies. This open-label, uncontrolled phase 2 study in PBC patients evaluated the effects of 1-year of seladelpar treatment on measures of pruritus and quality of life. Self-reported experiences of 101 PBC patients were collected at baseline and after 1year of seladelpar treatment using the pruritus visual analog scale (VAS), 5D-itch scale, and PBC-40 questionnaires along with bile acid profiles. In patients with moderate-to-severe pruritus, substantial improvement in pruritus was seen in 58% and 93% of patients in 5/10mg and 10mg treatment groups, respectively. After 1year, patients reporting improvement substantially outnumbered those who worsened in the total 5-D itch (including individual domains) and PBC-40 (itch and fatigue domains) questionnaires. Improvement in sleep disturbance at 1-year was reported in 81% (5/10mg) and 78% (10mg) of the patients with baseline itch-related sleep disturbance by 5-D itch score with similar results using the PBC-40 sleep questionnaire. Seladelpar-treated patients had significant reductions of 46% (5/10mg) and 31% (10mg) in the serum bile acid precursor C4 and reductions of up to 38% in serum bile acids. Seladelpar treatment for 1year led to consistent improvement in both symptom burden and biochemical response, suggesting its potential as a single agent to address two key unmet needs in PBC patients.