The oxysterol 25-hydroxycholesterol is a widely used compound displaying an array of pharmacological actions in in vitro systems and cell based experimental systems. In spite of the frequent use of ...this compound over the last few decades and a large number of studies in vitro and in vivo, its mechanism of formation in vivo is still not well understood.
Cholesterol autoxidation does not seem to be an important contributor to in vivo formation of 25-hydroxycholesterol. A number of different cytochrome P450 enzymes such as CYP27A1 and CYP3A4 have been reported to catalyze the conversion of cholesterol to 25-hydroxycholesterol in vitro, but the importance of these reactions in vivo remains unclear. The dioxygenase enzyme cholesterol 25-hydroxylase has been shown to generate 25-hydroxycholesterol, but in cholesterol 25-hydroxylase knockout mice there are still significant levels of 25-hydroxycholesterol in several tissues. This suggests that cholesterol 25-hydroxylase is not the sole producer of 25-hydroxycholesterol. The relative importance of different mechanisms of formation of 25-hydroxycholesterol in vivo have still to be elucidated.
The maintenance of cholesterol homeostasis is of great importance to supply tissues with the appropriate amount of cholesterol and prevent accumulation that may affect health. Numerous articles mention 25-hydroxycholesterol as an important regulator of cholesterol metabolism. However, mice with a disruption of the cholesterol 25-hydroxylase gene regulate cholesterol metabolism normally and patients with highly elevated levels of 25-hydroxycholesterol also display normal cholesterol and bile acid levels. These reports challenge the hypothesis that 25-hydroxycholesterol is an important regulator of cholesterol metabolism. Recent reports suggest that 25-hydroxycholesterol and one of its metabolites may have functions in regulation of humoral immunity. Thus, 25-hydroxycholesterol may be more important as a regulator of immunity than as a regulator of cholesterol metabolism.
► 25-Hydroxycholesterol is formed by several different mechanisms. ► The relative importance of the different mechanisms of formation in vivo is unknown. ► Low nanomolar concentrations of 25-hydroxycholesterol are present in circulation. ► Regulation of cholesterol homeostasis by 25-hydroxycholesterol is controversial. ► 25-Hydroxycholesterol may be a regulator of immunity.
We have proposed that 4β‐hydroxycholesterol (4β‐OHC) may be used as an endogenous marker of CYP3A activity. The cholesterol metabolite 4β‐OHC is formed by CYP3A4. Treatment of patients with strong ...inducers of CYP3A enzymes, e.g. anti‐epileptic drugs, resulted in 10‐fold increased concentrations of plasma 4β‐OHC, while treatment with CYP3A inhibitors such as ritonavir or itraconazole resulted in decreased plasma concentrations. There was a relationship between the 4β‐OHC concentration and the number of active CYP3A5*1 alleles showing that 4β‐OHC was not only formed by CYP3A4, but also by CYP3A5. The concentration of 4β‐OHC was higher in women than in men, confirming previous studies indicating a gender difference in CYP3A4/5‐activity. The rate of elimination of 4β‐OHC is slow (half‐life 17 days) which results in stable plasma concentrations within individuals, but limits its use to study rapid changes in CYP3A activity. In short‐term studies exogenous markers such as midazolam or quinine may be superior, but in long‐term studies 4β‐OHC is a sensitive marker of CYP3A activity, especially to assess induction but also inhibition. Under conditions where the cholesterol concentration is changing, the ratio of 4β‐OHC : cholesterol may be used as an alternative to 4β‐OHC itself. The use of an endogenous CYP3A marker has obvious advantages and may be of value both during drug development and for monitoring CYP3A activity in patients.
Changes in cortisol and other hormones during pregnancy may alter CYP3A enzymes activity, but data from sub-Saharan Africa are sparse. We investigated the effect of pregnancy and CYP3A5 genotypes on ...CYP3A enzymes activity using the plasma 4β-hydroxycholesterol (4β-OHC)/cholesterol (Chol) ratio, a known endogenous biomarker. Tanzanian pregnant women (n = 110) and non-pregnant women (n = 59) controls were enrolled. Plasma 4β-OHC and Chol were determined in the second and third trimesters for pregnant women and once for non-pregnant women using gas chromatography−mass spectrometry. Genotyping for CYP3A5 (*3, *6, *7) was performed. Wilcoxon Signed-Rank Test and Mann−Whitney U test were used to compare the median 4β-OHC/Chol ratio between trimesters in pregnant women and between pregnant and non-pregnant women. Repeated-measure ANOVA was used to evaluate the effect of the CYP3A5 genotypes on the 4β-OHC/Chol ratio in pregnant women. No significant effect of the pregnancy status or the CYP3A5 genotype on the cholesterol level was observed. The plasma 4β-OHC/Chol ratio significantly increased by 7.3% from the second trimester to the third trimester (p = 0.02). Pregnant women had a significantly higher mean 4β-OHC/Chol ratio than non-pregnant women, (p < 0.001). In non-pregnant women, the mean 4β-OHC/Chol ratio was significantly lower in carriers of defective CYP3A5 alleles (*3, *6 or *7) as compared to women with the CYP3A5*1/*1 genotypes (p = 0.002). Pregnancy increases CYP3A enzymes activity in a gestational-stage manner. The CYP3A5 genotype predicts CYP3A enzymes activity in the black Tanzanian population, but not during pregnancy-mediated CYP3A enzyme induction.
During screening of genes upregulated by lipopolysaccharide (LPS; endotoxin) treatment of bone marrow-derived mouse macrophages, it was unexpectedly found that cholesterol 25-hydroxylase (Ch25h) was ...strongly upregulated. Treatment of macrophages with 10 ng/ml of LPS for 2 h resulted in a 35-fold increase in the expression of Ch25h. In contrast, LPS treatment did not increase the expression of Cyp27a1 or Cyp7b1. The increased Ch25h expression was found to be independent of Myeloid differentiation protein 88 signaling but dependent on Toll-like receptor 4 signaling. LPS treatment of macrophages caused a 6- to 7-fold increase in cellular 25-hydroxycholesterol concentration. When macrophages were treated with increasing concentrations of 25-hydroxycholesterol, a dose-dependent release of CCL5 into the culture medium was observed. Intravenous injection of LPS in eight healthy volunteers resulted in an increase in plasma 25-hydroxycholesterol concentration. The possibility is discussed that 25-hydroxycholesterol may have a role in the inflammatory response, in addition to its more established role in the regulation of cholesterol homeostasis.
Side chain oxidizedoxysterols have a unique ability to traverse lipophilic membranes. We tested thehypothesis that there is a net flux of 27-hydroxycholesterol from the circulationinto the brain ...using plasma samples collected from the internal jugular vein andan artery of healthy male volunteers. Two independent studies were performed, onein which total levels of 27-hydroxycholesterol were measured and one in which thefree fraction of 27-hydroxycholesterol was measured. In the majority of subjectsstudied, the level of 27-hydroxycholesterol was higher in the artery than in thevein, and uptake from the circulation was calculated to be about 5 mg/24h.
The distribution of 27-hydroxycholesterol in human brain was found to beconsistent with an extracerebral origin, with a concentration gradient from thewhite to the gray matter
Reduction of hydroxylamines and amidoximes is important for drug activation and detoxification of aromatic and heterocyclic amines. Such a reductase system was previously found to be of high activity ...in adipose tissue and liver, and furthermore, in vitro studies using recombinant truncated and purified enzymes suggested the participation of cytochrome b5 reductase (CYB5R), cytochrome b5 (CYB5), and molybdenum cofactor sulfurase C-terminal containing 1 and 2 (MOSC1 and -2). Here, we show that purified rat liver outer mitochondrial membrane contains high amidoxime reductase activity and that MOSC2 is exclusively localized to these membranes. Moreover, using the same membrane fraction, we could show direct binding of a radiolabeled benzamidoxime substrate to MOSC2. Following differentiation of murine 3T3-L1 cells into mature adipocytes, the MOSC2 levels as well as the amidoxime reductase activity were increased, indicating that the enzyme is highly regulated under lipogenic conditions. siRNA-mediated down-regulation of MOSC2 and the mitochondrial form of cytochrome b5 type B (CYB5B) significantly inhibited the reductase activity in the differentiated adipocytes, whereas down-regulation of MOSC1, cytochrome b5 type A (CYB5A), CYB5R1, CYB5R2, or CYB5R3 had no effect. Down-regulation of MOSC2 caused impaired lipid synthesis. These results demonstrate for the first time the direct involvement of MOSC2 and CYB5B in the amidoxime reductase activity in an intact cell system. We postulate the presence of a novel reductive enzyme system of importance for lipid synthesis that is exclusively localized to the outer mitochondrial membrane and is composed of CYB5B, MOSC2, and a third unknown component (a CYB5B reductase).
High amidoxime reductase activity is found in liver and fat tissue, although its composition in cells is unknown.
Amidoxime reductase is up-regulated during adipogenesis and requires MOSC2 and CYB5B.
MOSC2 and CYB5B are essential components of the mitochondrial amidoxime reductase, and MOSC2 is important for lipogenesis.
We suggest a role of MOSC2 in adipogenesis and fatty acid synthesis and as a potential novel drug target.
The hepatic predominating cytochrome P450, CYP3A4, plays an essential role in the detoxification of bile acids and is important in pathological conditions such as cholestasis where CYP3A4 is ...adaptively up-regulated. However, the mechanism that triggers the up-regulation of CYP3A4 is still not clear. In this study, using recombinant CYP3A4 and human liver microsomes, we demonstrate that CYP3A4 can metabolise lithocholic acid into 3-dehydrolithocholic acid, a potent activator of the nuclear receptors, pregnane X receptor and 1,25-dihydroxy vitamin D3 receptor, which are known to regulate the expression of CYP3A4. This process thus provides a feed-forward metabolism of toxic bile acid that may be of importance in maintaining bile acid homeostasis. We also provide evidence for a novel CYP3A4-mediated metabolic pathway of the secondary bile acid deoxycholic acid. Patients treated with the antiepileptic drug carbamazepine, a CYP3A4 inducer, had markedly elevated urinary excretion of 1β-hydroxydeoxycholic acid compared to healthy controls. The importance of CYP3A4 in this process was verified by incubations with recombinant CYP3A4 and human liver microsomes, both of which efficiently converted deoxycholic acid into 1β-hydroxydeoxycholic acid. Interestingly, CYP3A4 was also found to be active against the secondary bile acid ursodeoxycholic acid.
Strong association between malnutrition, inflammation, and atherosclerosis in chronic renal failure.
Atherosclerotic cardiovascular disease and malnutrition are widely recognized as leading causes of ...the increased morbidity and mortality observed in uremic patients. C-reactive protein (CRP), an acute-phase protein, is a predictor of cardiovascular mortality in nonrenal patient populations. In chronic renal failure (CRF), the prevalence of an acute-phase response has been associated with an increased mortality.
One hundred and nine predialysis patients (age 52±1years) with terminal CRF (glomerular filtration rate 7±1ml/min) were studied. By using noninvasive B-mode ultrasonography, the cross-sectional carotid intima-media area was calculated, and the presence or absence of carotid plaques was determined. Nutritional status was assessed by subjective global assessment (SGA), dual-energy x-ray absorptiometry (DXA), serum albumin, serum creatinine, serum urea, and 24-hour urine urea excretion. The presence of an inflammatory reaction was assessed by CRP, fibrinogen (N = 46), and tumor necrosis factor-α (TNF-α; N = 87). Lipid parameters, including Lp(a) and apo(a)-isoforms, as well as markers of oxidative stress (autoantibodies against oxidized low-density lipoprotein and vitamin E), were also determined.
Compared with healthy controls, CRF patients had an increased mean carotid intima-media area (18.3±0.6 vs. 13.2±0.7mm2, P < 0.0001) and a higher prevalence of carotid plaques (72 vs. 32%, P = 0.001). The prevalence of malnutrition (SGA 2 to 4) was 44%, and 32% of all patients had an acute-phase response (CRP ≥ 10mg/liter). Malnourished patients had higher CRP levels (23±3 vs. 13±2mg/liter, P < 0.01), elevated calculated intima-media area (20.2±0.8 vs. 16.9±0.7mm2, P < 0.01) and a higher prevalence of carotid plaques (90 vs. 60%, P < 0.0001) compared with well-nourished patients. During stepwise multivariate analysis adjusting for age and gender, vitamin E (P < 0.05) and CRP (P < 0.05) remained associated with an increased intima-media area. The presence of carotid plaques was significantly associated with age (P < 0.001), log oxidized low-density lipoprotein (oxLDL; P < 0.01), and small apo(a) isoform size (P < 0.05) in a multivariate logistic regression model.
These results indicate that the rapidly developing atherosclerosis in advanced CRF appears to be caused by a synergism of different mechanisms, such as malnutrition, inflammation, oxidative stress, and genetic components. Apart from classic risk factors, low vitamin E levels and elevated CRP levels are associated with an increased intima-media area, whereas small molecular weight apo(a) isoforms and increased levels of oxLDL are associated with the presence of carotid plaques.
24S-hydroxycholesterol is a side-chain oxidized oxysterol formed in the brain that is continuously crossing the blood-brain barrier to reach the circulation. There may be an opposite flux of ...27-hydroxycholesterol, which is formed to a lower extent in the brain than in most other organs. Here we measured cholesterol, lathosterol, 24S- and 27-hydroxycholesterol, and plant sterols in four different brain areas of deceased Alzheimer's disease (AD) patients and controls. 24S-hydroxycholesterol was decreased and 27-hydroxycholesterol increased in all the brain samples from the AD patients. The difference was statistically significant in four of the eight comparisons. The ratio of 27-hydroxycholesterol to 24S-hydroxycholesterol was significantly increased in all brain areas of the AD patients and also in the brains of aged mice expressing the Swedish Alzheimer mutation APP751. Cholesterol 24S-hydroxylase and 27-hydroxylase protein was not significantly different between AD patients and controls. A high correlation was observed between the levels of 24S-hydroxycholesterol and lathosterol in the frontal cortex of the AD patients but not in the controls. Most probably the high levels of 27-hydroxycholesterol are due to increased influx of this steroid over the blood-brain barrier and the lower levels of 24S-hydroxycholesterol to decreased production.
The high correlation between lathosterol and 24-hydroxycholesterol is consistent with a close coupling between synthesis and metabolism of cholesterol in the frontal cortex of the AD brain.
Cholesterol oxidation products, or oxysterols, have gained increased attention since it was suggested that they participate in cell signaling as ligands for the nuclear receptors liver X receptor ...alpha and beta. In addition, oxysterols serve as important intermediates in bile acid biosynthesis and are also involved in cholesterol transport. Several studies have suggested that certain oxysterols may be used as markers for oxidative stress, and still other oxysterols may be of use in diagnosing neurological diseases. This broad spectrum of functions in health and disease has created a demand for accurate and reliable methods to measure oxysterols in complex biological matrixes. At present, the most reliable and sensitive method for oxysterol determination in biological materials is isotope-dilution gas chromatography-mass spectrometry using deuterium-labeled internal standards. With this technique, the major oxysterols in human blood plasma and atherosclerotic plaques have been carefully determined. The knowledge of oxysterol contents in other tissues and organs is still very scarce. As oxysterols are found to participate in an increasing number of cellular events, it is obvious that improved methods are needed for their analysis to understand their roles in the living cell.
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