Biological activities of 7-dehydrocholesterol-derived oxysterols: implications for Smith-Lemli-Opitz syndrome

Smith-Lemli-Opitz syndrome (SLOS) is a metabolic and developmental disorder caused by mutations in the gene encoding the enzyme 7-dehydrocholesterol reductase (Dhcr7). This reductase catalyzes the last step in cholesterol biosynthesis, and levels of 7-dehydrocholesterol (7-DHC), the substrate for this enzyme, are elevated in SLOS patients as a result of this defect. Our group has previously shown that 7-DHC is extremely prone to free radical autoxidation, and we identified about a dozen different oxysterols formed from oxidation of 7-DHC. We report here that 7-DHC-derived oxysterols reduce cell viability in a dose- and time-dependent manner, some of the compounds showing activity at sub-micromolar concentrations. The reduction of cell survival is caused by a combination of reduced proliferation and induced differentiation of the Neuro2a cells. The complex 7-DHC oxysterol mixture added to control Neuro2a cells also triggers the gene expression changes that were previously identified in Dhcr7-deficient Neuro2a cells. Based on the identification of overlapping gene expression changes in Dhcr7-deficient and 7-DHC oxysterol-treated Neuro2a cells, we hypothesize that some of the pathophysiological findings in the mouse SLOS model and SLOS patients might be due to accumulated 7-DHC oxysterols.     

7-Dehydrocholesterol-derived oxysterols and retinal degeneration in a rat model of Smith–Lemli–Opitz syndrome

Smith–Lemli–Opitz syndrome (SLOS) is a recessive disease characterized by markedly elevated levels of 7-dehydrocholesterol (7-DHC) and reduced levels of cholesterol in tissues and fluids of affected individuals, due to defective 3β-hydroxysterol-Δ⁷-reductase (Dhcr7). Treatment of Sprague Dawley rats with AY9944 (an inhibitor of Dhcr7) leads to similar biochemical features as observed in SLOS. Eighteen oxysterols previously have been identified as oxidation products of 7-DHC (most of them distinct from cholesterol (Chol)-derived oxysterols) in solution, in cells, and in brains obtained from Dhcr7-KO mice and AY9944-treated rats, formed either via free radical oxidation (peroxidation) or P450-catalyzed enzymatic oxidation. We report here the identification of five 7-DHC-derived oxysterols, including 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), 4α- and 4β-hydroxy-7-DHC, 24-hydroxy-7-DHC and 7-ketocholesterol (7-kChol, an oxysterol that is normally derived from Chol), in the retinas of AY9944-treated rats by comparing the retention times and mass spectrometric characteristics with corresponding synthetic standards in HPLC-MS analysis. Levels of 4α- and 4β-hydroxy-7-DHC, DHCEO, and 7-kChol were quantified using d₇-DHCEO as an internal standard. Among the five oxysterols identified, only 7-kChol was observed in retinas of control rats, but the levels of 7-kChol in retinas of AY9944-rats were 30-fold higher. Intravitreal injection of 7-kChol (0.25 μmol) into a normal rat eye induced panretinal degeneration within one week; by comparison, contralateral (control) eyes injected with vehicle alone exhibited normal histology. These findings are discussed in the context of the potential involvement of 7-DHC-derived oxysterols in the retinal degeneration associated with the SLOS rat model and in SLOS patients.     

Novel oxysterols observed in tissues and fluids of AY9944-treated rats: a model for Smith-Lemli-Opitz syndrome

Treatment of Sprague-Dawley rats with AY9944, an inhibitor of 3β-hydroxysterol-Δ(7)-reductase (Dhcr7), leads to elevated levels of 7-dehydrocholesterol (7-DHC) and reduced levels of cholesterol in all biological tissues, mimicking the key biochemical hallmark of Smith-Lemli-Opitz syndrome (SLOS). Fourteen 7-DHC-derived oxysterols previously have been identified as products of free radical oxidation in vitro; one of these oxysterols, 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), was recently identified in Dhcr7-deficient cells and in brain tissues of Dhcr7-null mouse. We report here the isolation and characterization of three novel 7-DHC-derived oxysterols (4α- and 4β-hydroxy-7-DHC and 24-hydroxy-7-DHC) in addition to DHCEO and 7-ketocholesterol (7-kChol) from the brain tissues of AY9944-treated rats. The identities of these five oxysterols were elucidated by HPLC-ultraviolet (UV), HPLC-MS, and 1D- and 2D-NMR. Quantification of 4α- and 4β-hydroxy-7-DHC, DHCEO, and 7-kChol in rat brain, liver, and serum were carried out by HPLC-MS using d(7)-DHCEO as an internal standard. With the exception of 7-kChol, these oxysterols were present only in tissues of AY9944-treated, but not control rats, and 7-kChol levels were markedly (>10-fold) higher in treated versus control rats. These findings are discussed in the context of the potential involvement of 7-DHC-derived oxysterols in the pathogenesis of SLOS.     

Metabolism of oxysterols derived from nonenzymatic oxidation of 7-dehydrocholesterol in cells

Recent studies suggest that 7-dehydrocholesterol (7-DHC)-derived oxysterols play important roles in the pathophysiology of Smith-Lemli-Opitz syndrome (SLOS), a metabolic disorder that is caused by defective 3β-hydroxysterol-Δ⁷-reductase (DHCR7). Although 14 oxysterols have been identified as the primary products of 7-DHC autoxidation in organic solution, the metabolic fate of these oxysterols in a biological environment has not yet been elucidated. Therefore, we incubated these primary 7-DHC oxysterols in control Neuro2a and control human fibroblast cells and identified metabolites of these oxysterols by HPLC-MS. We also incubated Dhcr7-deficient Neuro2a cells and fibroblasts from SLOS patients with isotopically labeled 7-DHC (d₇-7-DHC). The observation of matching d₀- and d₇ peaks in HPLC-MS confirmed the presence of true metabolites of 7-DHC after excluding the possibility of ex vivo oxidation. The metabolites of primary 7-DHC oxysterols were found to contribute to the majority of the metabolic profile of 7-DHC in cells. Furthermore, based on this new data, we identified three new 7-DHC-derived metabolites in the brain of Dhcr7-KO mice. Our studies suggest that 7-DHC peroxidation is a major source of oxysterols observed in cells and in vivo and that the stable metabolites of primary 7-DHC oxysterols can be used as markers of 7-DHC peroxidation in these biological systems.     

Assays of plasma dehydrocholesteryl esters and oxysterols from Smith-Lemli-Opitz syndrome patients

Smith-Lemli-Opitz syndrome (SLOS) is caused by mutations in the gene encoding 3β-hydroxysterol-Δ⁷-reductase and as a result of this defect, 7-dehydrocholesterol (7-DHC) and 8-dehydrocholesterol (8-DHC) accumulate in the fluids and tissues of patients with this syndrome. Both 7- and 8-DHC are susceptible to peroxidation reactions, and several biologically active DHC oxysterols are found in cell and animal models of SLOS. Ex vivo oxidation of DHCs can be a confounding factor in the analysis of these sterols and their esters, and we developed HPLC/MS methods that permit the direct analysis of cholesterol, 7-DHC, 8-DHC, and their esters in human plasma, thus avoiding ex vivo oxidation. In addition, three oxysterols were classified as endogenously formed products by the use of an isotopically-labeled 7-DHC (d₇-7-DHC) added to the sample before workup, followed by MS analysis of products formed. Analysis of 17 SLOS plasma samples shows that 8-DHC linoleate correlates better with the SLOS severity score of the patients than other sterols or metabolites, including cholesterol and 7-DHC. Levels of 7-ketocholesterol also correlate with the SLOS severity score. 8-DHC esters should have utility as surrogate markers of severity in SLOS for prognostication and as endpoints in clinical trials.     

7-Dehydrocholesterol-derived oxysterols and retinal degeneration in a rat model of Smith-Lemli-Opitz syndrome

Smith-Lemli-Opitz syndrome (SLOS) is a recessive disease characterized by markedly elevated levels of 7-dehydrocholesterol (7-DHC) and reduced levels of cholesterol in tissues and fluids of affected individuals, due to defective 3β-hydroxysterol-Δ(7)-reductase (Dhcr7). Treatment of Sprague Dawley rats with AY9944 (an inhibitor of Dhcr7) leads to similar biochemical features as observed in SLOS. Eighteen oxysterols previously have been identified as oxidation products of 7-DHC (most of them distinct from cholesterol (Chol)-derived oxysterols) in solution, in cells, and in brains obtained from Dhcr7-KO mice and AY9944-treated rats, formed either via free radical oxidation (peroxidation) or P450-catalyzed enzymatic oxidation. We report here the identification of five 7-DHC-derived oxysterols, including 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), 4α- and 4β-hydroxy-7-DHC, 24-hydroxy-7-DHC and 7-ketocholesterol (7-kChol, an oxysterol that is normally derived from Chol), in the retinas of AY9944-treated rats by comparing the retention times and mass spectrometric characteristics with corresponding synthetic standards in HPLC-MS analysis. Levels of 4α- and 4β-hydroxy-7-DHC, DHCEO, and 7-kChol were quantified using d(7)-DHCEO as an internal standard. Among the five oxysterols identified, only 7-kChol was observed in retinas of control rats, but the levels of 7-kChol in retinas of AY9944-rats were 30-fold higher. Intravitreal injection of 7-kChol (0.25μmol) into a normal rat eye induced panretinal degeneration within one week; by comparison, contralateral (control) eyes injected with vehicle alone exhibited normal histology. These findings are discussed in the context of the potential involvement of 7-DHC-derived oxysterols in the retinal degeneration associated with the SLOS rat model and in SLOS patients.     

An oxysterol biomarker for 7-dehydrocholesterol oxidation in cell/mouse models for Smith-Lemli-Opitz syndrome

The level of 7-dehydrocholesterol (7-DHC) is elevated in tissues and fluids of Smith-Lemli-Opitz syndrome (SLOS) patients due to defective 7-DHC reductase. Although over a dozen oxysterols have been identified from 7-DHC free radical oxidation in solution, oxysterol profiles in SLOS cells and tissues have never been studied. We report here the identification and complete characterization of a novel oxysterol, 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), as a biomarker for 7-DHC oxidation in fibroblasts from SLOS patients and brain tissue from a SLOS mouse model. Deuterated (d₇)-standards of 7-DHC and DHCEO were synthesized from d₇-cholesterol. The presence of DHCEO in SLOS samples was supported by chemical derivatization in the presence of d₇-DHCEO standard followed by HPLC-MS or GC-MS analysis. Quantification of cholesterol, 7-DHC, and DHCEO was carried out by isotope dilution MS with the d₇-standards. The level of DHCEO was high and correlated well with the level of 7-DHC in all samples examined (R = 0.9851). Based on our in vitro studies in two different cell lines, the mechanism of formation of DHCEO that involves 5α,6α-epoxycholest-7-en-3β-ol, a primary free radical oxidation product of 7-DHC, and 7-cholesten-3β,5α,6β-triol is proposed. In a preliminary test, a pyrimidinol antioxidant was found to effectively suppress the formation of DHCEO in SLOS fibroblasts.     

KIT is required for hepatic function during mouse post-natal development

Background

Targeted disruption of the murine 3β-hydroxysterol-Δ7-reductase gene (Dhcr7), an animal model of Smith-Lemli-Opitz syndrome, leads to loss of cholesterol synthesis and neonatal death that can be partially rescued by transgenic replacement of DHCR7 expression in brain during embryogenesis. To gain further insight into the role of non-brain tissue cholesterol deficiency in the pathophysiology, we tested whether the lethal phenotype could be abrogated by selective transgenic complementation with DHCR7 expression in the liver.

Results

We generated mice that carried a liver-specific human DHCR7 transgene whose expression was driven by the human apolipoprotein E (ApoE) promoter and its associated liver-specific enhancer. These mice were then crossed with Dhcr7+/- mutants to generate Dhcr7-/- mice bearing a human DHCR7 transgene. Robust hepatic transgene expression resulted in significant improvement of cholesterol homeostasis with cholesterol concentrations increasing to 80~90 % of normal levels in liver and lung. Significantly, cholesterol deficiency in brain was not altered. Although late gestational lung sacculation defect reported previously was significantly improved, there was no parallel increase in postnatal survival in the transgenic mutant mice.

Conclusion

The reconstitution of DHCR7 function selectively in liver induced a significant improvement of cholesterol homeostasis in non-brain tissues, but failed to rescue the neonatal lethality of Dhcr7 null mice. These results provided further evidence that CNS defects caused by Dhcr7 null likely play a major role in the lethal pathogenesis of Dhcr7 ^-/- mice, with the peripheral organs contributing the morbidity.     

Cholesterol biosynthesis from birth to adulthood in a mouse model for 7-dehydrosterol reductase deficiency (Smith-Lemli-Opitz syndrome)

Smith-Lemli-Opitz syndrome (SLOS) is caused by deficiency in the terminal step of cholesterol biosynthesis, which is catalyzed by 7-dehydrocholesterol reductase (DHCR7). The disorder exhibits several phenotypic traits including dysmorphia and mental retardation with a broad range of severity. Pathogenesis of SLOS is complex due to multiple roles of cholesterol and may be further complicated by unknown effects of aberrant metabolites that arise when 7-dehydrocholesterol (7-DHC), the substrate for DHCR7, accumulates. A viable mouse model for SLOS has recently been developed, and here we characterize cholesterol metabolism in this model with emphasis on changes during the first few weeks of postnatal development. Cholesterol and 7-DHC were measured in "SLOS" mice and compared with measurements in normal mice. SLOS mice had measurable levels of 7-DHC at all ages tested (up to 1 year), while 7-DHC was below the threshold for detection in normal mice. In perinatal to weaning age SLOS mice, cholesterol and 7-DHC levels changed dramatically. Changes in brain and liver were independent; in brain cholesterol increased several fold while 7-DHC remained relatively constant, but in liver cholesterol first increased then decreased again while 7-DHC first decreased then increased. In older SLOS animals the ratio of 7-DHC/cholesterol, which is an index of biochemical severity, tended to approach, but not reach, normal. While these mice provide the best available genetic animal model for the study of SLOS pathogenesis and treatment, they probably will be most useful at early ages when the metabolic effects of the mutations are most dramatic. To correlate any experimental treatment with improved sterol metabolism will require age-matched controls. Finally, determining the mechanism by which these "SLOS" mice tend to normalize may provide insight into the future development of therapy.     

Evaluation of cytotoxic effects of 7-dehydrocholesterol on melanoma cells

Ultraviolet radiation is the main cause of skin cancers, and melanoma is the most serious form of tumor. There is no therapy for advanced-stage melanoma and its metastasis because of their high resistance to various anticancer therapies. Human skin is an important metabolic organ in which occurs photoinduced synthesis of vitamin D3 from 7-dehydrocholesterol (7-DHC). 7-DHC, the precursor of cholesterol biosynthesis, is highly reactive and easily modifiable to produce 7-DHC-derived compounds. The intracellular levels of 7-DHC or its derivatives can have deleterious effects on cellular functionality and viability. In this study we evaluated the effects on melanoma cell lines of 7-DHC as such and for this aim we used much care to minimize 7-DHC modifications. We found that from 12 to 72 h of treatment 82–86% of 7-DHC entered the cells, and the levels of 7-DHC-derived compounds were not significant. Simultaneously, reactive oxygen species production was significantly increased already after 2 h. After 24 h and up to 72 h, 7-DHC-treated melanoma cells showed a reduction in cell growth and viability. The cytotoxic effect of 7-DHC was associated with an increase in Bax levels, decrease in Bcl-2/Bax ratio, reduction of mitochondrial membrane potential, increase in apoptosis-inducing factor levels, unchanged caspase-3 activity, and absence of cleavage of PARP-1. These findings could explain the mechanism through which 7-DHC exerts its cytotoxic effects. This is the first report in which the biological effects found in melanoma cells are mainly attributable to 7-DHC as such.     

Sterol balance in the Smith-Lemli-Opitz syndrome. Reduction in whole body cholesterol synthesis and normal bile acid production

The Smith-Lemli-Opitz syndrome (SLOS) is a multiple malformation/mental retardation syndrome caused by a deficiency of the enzyme 7-dehydrocholesterol Delta(7)-reductase. This enzyme converts 7-dehydrocholesterol (7-DHC) to cholesterol in the last step in cholesterol biosynthesis. The pathology of this condition may result from two different factors: the deficiency of cholesterol itself and/or the accumulation of precursor sterols such as 7-DHC. Although cholesterol synthesis is defective in cultured SLOS cells, to date there has been no evidence of decreased whole body cholesterol synthesis in SLOS and only incomplete information on the synthesis of 7-DHC and bile acids. In this first report of the sterol balance in SLOS, we measured the synthesis of cholesterol, other sterols, and bile acids in eight SLOS subjects and six normal children. The diets were very low in cholesterol content and precisely controlled. Cholesterol synthesis in SLOS subjects was significantly reduced when compared with control subjects (8.6 vs. 19.6 mg/kg per day, respectively, P < 0.002). Cholesterol precursors 7-DHC, 8-DHC, and 19-nor-cholestatrienol were synthesized in SLOS subjects (7-DHC synthesis was 1.66 +/- 1.15 mg/kg per day), but not in control subjects. Total sterol synthesis was also reduced in SLOS subjects (12 vs. 20 mg/kg per day, P < 0.022). Bile acid synthesis in SLOS subjects (3.5 mg/kg per day) did not differ significantly from control subjects (4.6 mg/kg per day) and was within the range reported previously in normals. Normal primary and secondary bile acids were identified.This study provides direct evidence that whole body cholesterol synthesis is reduced in patients with SLOS and that the synthesis of 7-DHC and other cholesterol precursors is profoundly increased. It is also the first reported measure of daily bile acid synthesis in SLOS and provides evidence that bile acid supplementation is not likely to be necessary for treatment. These sterol balance studies provide basic information about the biochemical defect in SLOS and strengthen the rationale for the use of dietary cholesterol in its treatment.     

7-Dehydrocholesterol enhances ultraviolet A-induced oxidative stress in keratinocytes: roles of NADPH oxidase, mitochondria, and lipid rafts

Long wavelength solar UVA radiation stimulates formation of reactive oxygen species (ROS) and prostaglandin E(2) (PGE(2)), which are involved in skin photosensitivity and tumor promotion. High levels of 7-dehydrocholesterol (7-DHC), the precursor to cholesterol, cause exaggerated photosensitivity to UVA in patients with Smith-Lemli-Opitz syndrome (SLOS). Partially replacing cholesterol with 7-DHC in keratinocytes rapidly (<5 min) increased UVA-induced ROS, intracellular calcium, phospholipase A(2) activity, PGE(2), and NADPH oxidase activity. UVA-induced ROS and PGE(2) production were inhibited in these cells by depleting the Nox1 subunit of NADPH oxidase using siRNA or using a mitochondrial radical quencher, MitoQ. Partial replacement of cholesterol with 7-DHC also disrupted membrane lipid raft domains, although depletion of cholesterol, which also disrupts lipid rafts, did not affect UVA-induced increases in ROS and PGE(2). Phospholipid liposomes containing 7-DHC were more rapidly oxidized by a free radical mechanism than those containing cholesterol. These results indicate that 7-DHC enhances rapid UVA-induced ROS and PGE(2) formation by enhancing free radical-mediated membrane lipid oxidation and suggests that this mechanism might underlie the UVA photosensitivity in SLOS.     

Feedback inhibition of the cholesterol biosynthetic pathway in patients with Smith-Lemli-Opitz syndrome as demonstrated by urinary mevalonate excretion

Smith-Lemli-Opitz syndrome (SLOS) is a genetic disorder characterized by low plasma cholesterol and high 7-dehydrocholesterol (7-DHC). Synthesis of cholesterol and 7-DHC and its metabolites is regulated by HMG-CoA reductase, whose activity can be measured by 24-h excretion of its product mevalonate. We devised a simple, non-invasive method for collecting 24-h urine in our subjects. With a background of a very low cholesterol diet, mean mevalonate excretion did not differ between controls and SLOS children, indicating that SLOS subjects have normal HMG-CoA reductase activity. In a short term feeding study, the effects of a high cholesterol diet in SLOS subjects include a significant 55% increase in plasma cholesterol levels and 39% decrease in mevalonate excretion and no change in plasma 7-DHC levels. However, in four SLOS subjects, fed a high cholesterol diet for 2-3 years, plasma cholesterol levels continued to increase, urinary mevalonate excretion remained low and total 7-DHC decreased significantly, likely from decreased total sterol synthesis. Thus, in SLOS subjects, HMG-CoA reductase activity was normal and was subject to normal cholesterol induced feedback inhibition. However, total sterol synthesis in SLOS may still be decreased because of increased diversion of mevalonate into the shunt pathway away from sterol synthesis.     

A novel mutation of the human 7-dehydrocholesterol reductase gene reduces enzyme activity in patients with holoprosencephaly

Defects in cholesterol biosynthesis genes are recognized as a leading cause for holoprosencephaly (HPE). Previous reports suggest that mutations of human 7-dehydrocholesterol reductase (Dhcr7), which catalyzes the final step of cholesterol biosynthesis, may cause HPE [Clin. Genet. 53 (1998) 155]. To determine whether Dhcr7 mutations are involved in HPE pathogenesis, we analyzed the sequence of exon 9, which contains both a catalytic domain and a mutational hot spot. We examined 36 prematurely terminated fetuses with HPE at their gestation ages in the range from 21 to 33 weeks by single strand conformation polymorphism analysis and DNA sequencing. A novel missense mutation was identified: G344D. Dhcr7 enzyme assays using overexpressed recombinant mutant proteins revealed altered enzyme activity. Mutant G344D harbored less than 50% of enzyme activity compared with the control. Two previously reported mutations, R404C and G410S, abolished enzyme activity. These results suggest that mutation of the Dhcr7 gene is involved in HPE pathogenesis.     

Probing lipid-protein adduction with alkynyl surrogates: application to Smith-Lemli-Opitz syndrome

Lipid modifications aid in regulating (and misregulating) protein function and localization. However, efficient methods to screen for a lipid''s ability to modify proteins are not readily available. We present a strategy to identify protein-reactive lipids and apply it to a neurodevelopmental disorder, Smith-Lemli-Opitz syndrome (SLOS). Alkynyl surrogates were synthesized for polyunsaturated fatty acids, phospholipids, cholesterol, 7-dehydrocholesterol (7-DHC), and a 7-DHC-derived oxysterol. To probe for protein-reactive lipids, we used click chemistry to biotinylate the alkynyl tag and detected the lipid-adducted proteins with streptavidin Western blotting. In Neuro2a cells, the trend in amount of protein adduction followed known rates of lipid peroxidation (7-DHC >> arachidonic acid > linoleic acid >> cholesterol), with alkynyl-7-DHC producing the most adduction among alkynyl lipids. 7-DHC reductase-deficient cells, which cannot properly metabolize 7-DHC, exhibited significantly more alkynyl-7-DHC-protein adduction than control cells. Model studies demonstrated that a 7-DHC peroxidation product covalently modifies proteins. We hypothesize that 7-DHC generates electrophiles that can modify the proteome, contributing to SLOS''s complex pathology. These probes and methods would allow for analysis of lipid-modified proteomes in SLOS and other disorders exhibiting 7-DHC accumulation. More broadly, the alkynyl lipid library would facilitate exploration of lipid peroxidation''s role in specific biological processes in numerous diseases.     

The interactions of amphotericin B with various sterols in relation to its possible use in anticancer therapy.

Amphotericin B (AmB) is still the most common anti-fungal agent used to treat systemic fungal infections. It is known that this antibiotic acts by forming pores with the ergosterol contained in the membranes of fungi, but it also interacts with the cholesterol contained in the membranes of eukaryotic cells, hence its toxicity. AmB may also interact with the most common oxidation products of cholesterol found in vivo, together with interacting with biosynthetic precursors of cholesterol, namely, lanosterol and 7-dehydrocholesterol (7-DHC). The purpose of the present work was to study the interactions in solution between AmB and these various sterols, the techniques used being UV-Vis spectroscopy and differential scanning calorimetry. The results are globally interpreted in terms of the structural differences between the sterols. We show that AmB selectively interacts with 7-DHC which, according to a recent hypothesis proposed in the literature, has been identified in connexion with a therapeutic strategy against hepatocellular carcinomas. We find that the affinity of AmB towards 7-DHC is even greater than the affinity of the antibiotic towards ergosterol. We also find that AmB selectively interacts with the principal oxidation product of cholesterol, 7-ketocholesterol, a situation that has to be taken into account when AmB is administered.     

Associations of cholesterol and vitamin D metabolites with the risk for development of high grade colorectal cancer

BackgroundVitamin D deficiency is repeatedly reported in colorectal cancer (CRC). Since cholesterol and vitamin D share common precursor 7-dehydrocholesterol (7-DHC), it would be important to explore the associations of key vitamin D metabolites and serum lipid parameters in patients with high and low grade CRC. The aim of this study was to analyze relationships between serum 25(OH)D3, 24,25(OH)2D3 and 7-DHC levels and serum lipids in patients with CRC, and to evaluate their potential for prediction of risk for development of high grade CRC.MethodsWe recruited 82 patients CRC and 77 controls. 7-DHC, 25(OH)D3 and 24,25(OH)2D3 were quantified by LC-MS/MS methods.Results7-DHC, 25(OH)D3 and vitamin D metabolic ratio (VDMR) were significantly lower in CRC patients than in control group (P<0.001, P<0.010, P<0.050 and P<0.050, respectively). 25(OH)D3 levels were higher in patients with grade I CRC when compared to grade II (P<0.050). All vitamin D metabolites positively correlated with total cholesterol (TC) concentration in CRC patients. 25(OH)D3 was significant predictor of increased CRC risk (P<0.010). After adjustment for TC concentration, 25(OH)D3 lost its predictive abilities. However, 25(OH)D3 remained significant predictor of poorly differentiated type of cancer (P<0.050).ConclusionsWe found significant positive association between vitamin D status and serum total cholesterol. Although low 25(OH)D3 was found to be a significant risk factor for CRC development, the obtained results primarily suggest profound impact of cholesterol level on vitamin D status in CRC. However, our results suggest that low 25(OH)D3 might independently contribute to development of poorly differentiated tumor.