Unravelling new pathways of sterol metabolism: lessons learned from in-born errors and cancer

Purpose of review To update researchers of recently discovered metabolites of cholesterol and of its precursors and to suggest relevant metabolic pathways. Recent findings Patients suffering from inborn errors of sterol biosynthesis, transport and metabolism display unusual metabolic pathways, which may be major routes in the diseased state but minor in the healthy individual. Although quantitatively minor, these pathways may still be important in healthy individuals. Four inborn errors of metabolism, Smith-Lemli-Opitz syndrome, cerebrotendinous xanthomatosis and Niemann Pick disease types B (NPB) and C (NPC) result from mutations in different genes but can generate elevated levels of the same sterol metabolite, 7-oxocholesterol, in plasma. How this molecule is metabolized further is of great interest as its metabolites may have an important role in embryonic development. A second metabolite, abundant in NPC and NPB diseases, cholestane-3β,5α,6β-triol (3β,5α,6β-triol), has recently been shown to be metabolized to the corresponding bile acid, 3β,5α,6β-trihydroxycholanoic acid, providing a diagnostic marker in plasma. The origin of cholestane-3β,5α,6β-triol is likely to be 3β-hydroxycholestan-5,6-epoxide, which can alternatively be metabolized to the tumour suppressor dendrogenin A (DDA). In breast tumours, DDA levels are found to be decreased compared with normal tissues linking sterol metabolism to cancer. Summary Unusual sterol metabolites and pathways may not only provide markers of disease, but also clues towards cause and treatment.


INTRODUCTION
In vertebrates, cholesterol can be synthesized by all cells from acetyl-CoA. Following cyclization of squalene to lanosterol via squalene-2,3-epoxide the pathway divides into two main routes known as the Bloch and Kandustch-Russell pathways leading to desmosterol and 7-dehydrocholesterol (7-DHC), respectively, as the immediate precursors of cholesterol [1]. Alternatively, cholesterol can be taken up by cells as lipoproteins and have a dietary origin. Cholesterol is an essential molecule to maintain membrane structure and is the metabolic precursor of bile acids and steroid hormones. It has also been suggested to be a signalling molecule in its own right [2 && ]. 7-DHC is the precursor of 1a,25-dihydroxyvitamin D 3 , the biologically active form of vitamin D. Although the major pathways of cholesterol metabolism were delineated in the 20th century [3 & ], recent studies have revealed new metabolic pathways from cholesterol and 7-DHC, generating metabolites with unexpected biological activity.

7-OXOCHOLSTEROL
7-Oxocholesterol (7-OC), also known as 7-ketocholesterol, is a challenging sterol for biochemists to analyse as it may be formed by reaction of cholesterol with oxygen in air [4,5], but can also be formed endogenously via reaction of cholesterol with reactive oxygen species [6] or from 7-DHC enzymatically [7]. Many analytical scientists have been wary of reports of high levels of 7-OC in tissue and plasma, however, there is now convincing evidence that 7-OC is abundant in some disease states.
In agreement with earlier studies by Björkhem et al. [8], Pajares et al. [9] have reported elevated of 7-OC in plasma of patients suffering from cerebrotendinous xanthomatosis (CTX). They used a liquid chromatography (LC)-tandem mass spectrometry (MS/MS) method exploiting derivatization to N,Ndimethylglycine (DMG) esters and electrospray ionization (ESI). Levels of 7-OC in some CTX patients prior to treatment were as high as 1000 ng/ml (n ¼ 11, mean 830 ng/ml, range 137-529 ng/ml), compared with control values of about 10 ng/ml (adults n ¼ 75, median 9.8 ng/ml, range 5.3-22.8 ng/ml, 5th to 95th percentile; children n ¼ 32, median 13.8 ng/ml, range 8.3-34.5, 5th to 95th percentile). These values are for the free sterol as no hydrolysis step was carried out prior to analysis. CTX is an autosomal recessive disorder, where the enzyme cytochrome P450 (CYP) 27A1 is defective. People with CTX often develop neurological problems in early adulthood, which are thought to be caused by an abnormal accumulation of sterols and an increasing number of xanthomas in brain. In young patients CTX often present with liver disease. CYP27A1 is required for bile acid biosynthesis via the conventional pathways, introducing first an alcohol group and then a carboxylic acid to the terminal carbon of the sterol side-chain. We have also found 7-OC to be elevated in CTX plasma and speculate that this is a result of upregulation of CYP7A1, as consequence of reduced negative-feedback by primary bile acids and use of 7-DHC as the enzyme substrate [7,8] (Fig. 1).

KEY POINTS
New pathways of sterol metabolism provide biomarkers for inborn errors of metabolism.
Newly discovered metabolites have unpredicted biological properties.
New sterols provide novel routes to cancer diagnosis and perhaps treatment. β FIGURE 1. Enzymatic or nonenzymatic formation of 7-oxocholesterol, 3b,5a-dihydroxycholest-7-en-6-one and 3bhydroxycholestan-5,6-epoxide. The pathways prevalent in SLOS are depicted in the red (upper) and blue (central) boxes, in CTX in the red (upper) box and in the lysosomal storage diseases NPB, NPC and LAL deficiency in the green (lower) box. The defective enzymatic step in SLOS is indicated by a horizontal T. ROO . , peroxy radical; RO . , alkoxy radical. patients with Niemann Pick type (NPB) and NPC, LAL deficiency and SLOS using LC-ESI-MS/MS, with derivatization to dimethylaminobutyric acid (DMAB) esters, again in the absence of saponification. Control levels of 7-OC were found to be 3.8-39.8 ng/ml (2.5th to 97.5th percentile) with a median of 16.1 ng/ml (n ¼ 135), in NPC the median was 86 ng/ml (n ¼ 16, range 21.9-963 ng/ ml), in NPB only two patients were analysed where 7-OC was 62.8-383 ng/ml, in two LAL deficiency patients 7-OC was 35.5-103 ng/ml and in SLOS patients, the median was 139 ng/ml (n ¼ 4, range 76.4-337 ng/ml).
SLOS is a congenital disease resulting from a defect in 7-dehydrocholesterol reductase (DHCR7), the final enzyme in the Kandustch-Russell pathway of cholesterol biosynthesis, resulting in elevated levels of 7-DHC in plasma and tissues. Patients with SLOS present with a broad phenotype ranging from autistic behaviour in mildly affected individuals to abnormalities in multiple organs, dysmorphology and failure to thrive in more severe cases [11]. In SLOS, elevated 7-OC can be explained by enzymatic conversion from abundant 7-DHC by CYP7A1 ( Fig. 1) [7,8,12]. NPC, NPB and LAL deficiency are all lysosomal storage diseases [13 && ]. In NPC and NPB, and perhaps LAL deficiency also, cholesterol accumulates in lysosomes. NPC has a variable age of onset, with a range of nonspecific neurological and psychiatric clinical features, it results from a defect in either NPC1 or NPC2 proteins required for the transport of nonesterified cholesterol from lysosomes [13 && ]. NPB, also known as acid sphingomyelinase deficiencies (ASMDs), caused by mutations in the SMPD1 gene, is believed to result from affected cholesterol transfer by NPC2 protein and presents with enlarged liver and spleen or spleen alone in early childhood. LAL deficiency results from defective LAL, the enzyme which hydrolyses cholesterol esters and triglycerides. Whenever LAL deficiency occurs in infants, it usually leads to death before 6 months of age; however, enzyme replacement therapy is now available [14 & ]. Using LC-MS/MS, DMG derivatization and atmospheric pressure chemical ionization (APCI), Romanello [16,17]. In NPC formation of 7-OC is likely to be by in vivo free radical oxidation [18 && ] (Fig. 1), this is probably true for NPB and LAL deficiency also. A concern for the analytical chemist whenever measuring 7-OC is that it can also be formed from cholesterol ex vivo [5], this may explain some of the variation in control values in the three studies highlighted above. A better diagnostic would be an enzymatically formed metabolite of 7-OC that could only be formed endogenously.
Mazzacuva et al. [18 && ] found 3b-hydroxy-7b-Nacetylglucosaminylchol-5-enoic acid (3b,7b-diH-D 5 -BA 7b-GlcNAc) to be elevated in NPC plasma and suggested its formation from 7-OC via 7b-hydroxycholesterol (7b-HC) (Fig. 2). We also suggested a pathway for the formation of this unusual bile acid involving conversion of 7-OC and its 7-oxo metabolites to 7b-hydroxy compounds by hydroxysteroid dehydrogenase (HSD) 11B1 and ultimate conjugation of the 7b-hydroxy group with N-acetylglucosamine (GlcNAc), a reaction known to be specific for the 7b stereochemistry (Fig. 2) [19]. Many years earlier we had found 3b-sulphated,7b-GlcNAc conjugated D 5 -bile acids modified with glycine or taurine in NPC urine and current studies in our laboratory indicate their formation in SLOS patients also [20]. 3b,7b-diH-D 5 -BA 7b-GlcNAc, and further conjugated forms, have potential as a biomarker for NPC and other disease states wherever 7-OC is elevated. However, Mazzacuva et al. [18 && ] found a common mutation inactivating the GlcNAc transferase enzyme necessary for the formation of the GlcNAc conjugate. About 20% of Asian and Caucasian populations carry this mutation and fail to produce GlcNAc conjugates, hence, if these bile acids were to be used as a biomarker, many cases would be missed for NPC and also NPB, LAL deficiency and SLOS. However, the suggested biosynthetic pathway for 3b,7b-diH-D 5 -BA 7b-GlcNAc, particularly with respect to SLOS, does introduce some interesting metabolites [19]. One such metabolite is (25R)26-hydroxy-7-oxocholesterol (26H,7-OC), also called 27-hydroxy-7-ketocholesterol (Fig. 2). 26H,7-OC has been shown to bind to and activate the G protein-coupled receptor (GPCR) smoothened (SMO), which transmits signal across the plasma membrane in the Hedgehog (Hh) signalling pathway. Significantly, SLOS phenocopies dysregulated Hh signalling, and defective Hh signalling has been implicated in dysmorphology associated with SLOS [21 & ]. In addition to 26H,7O-C, 3b,5adihydroxycholest-7-en-6-one (DHCEO) is a product of metabolism of 7-DHC in SLOS, in this case via the intermediate 7-dehydrocholesterol-5a,6a-epoxide ( Fig. 1) [4]. DHCEO is an inhibitor of Hh signalling and we suggest that dysregulated formation of Hh-signalling pathway modulatory sterols during development is the cause of some of the phenotypical features of SLOS [19].
As is the situation with 7-OC, ex vivo oxidation of cholesterol can lead to the formation of 5,6-EC, which may be subsequently hydrolysed to 3b,5a,6btriol during sample handling procedures. Hence, elevated 3b,5a,6b-triol may be a consequence of sample handling and storage. Whenever 3b,5a,6btriol is formed in vivo, it is likely to be metabolized further to a bile acid. In 2016, Mazzacuva et al. [18 && ] and Jiang et al. [25 && ] both reported the identification of elevated levels of the unusual bile acid 3b,5a,6b-trihydroxycholanoylglycine (3b,5a,6b-triHBA 24-G) in plasma of NPC patients. Mazzacuva et al. found the levels of this bile acid (median 118 ng/ml, n ¼ 73) to be more than 10-fold higher than in controls (9.3 ng/ml, n ¼ 84). Jiang et al. found levels of both the unconjugated and glycine-conjugated bile acid to be elevated in NPC plasma. They reported reference ranges for the glycine conjugate for controls of less than 5-5.34 ng/ ml (n ¼ 1013), NPC1 carriers of less than 5-12.5 ng/ ml (n ¼ 130) and NPC1 patients 5.45-294 ng/ml (n ¼ 25). We have performed a similar study and find that the unconjugated bile acid 3b,5a,6b-trihydroxycholanoic acid (3b,5a,6b-triHBA) is also elevated in plasma from patients with NPB and LAL deficiency [26]. In our study, we identify 3b,5a,6btrihydroxycholestanoic acid (3b,5a,6b-triHCa) and speculate that this acid is further metabolized to 3b,5a,6b-triHBA in the peroxisome (Fig. 3).

DENDROGENIN A
Cholesterol-5,6-epoxide hydrolase (ChEH) will transform 5,6-EC to 3b,5a,6b-triol. Interestingly, the enzyme is made up of two subunits, 3b-hydroxysteroid-D 8,7 -isomerase (D8D7I) and DHCR7, and is identical to the microsomal protein complex antiestrogen-binding site (AEBS), which binds to tamoxifen, the anticancer drug, with high affinity. Inhibition of ChEH activity by tamoxifen binding induced cancer cell-differentiation through accumulation of 5,6-EC [22 && ]. These findings lead Poirot and colleagues to search for a metabolite of 5,6-EC, other than those generated from 3b,5a,6b-triol, that may be display anticancer properties. They discovered dendrogenin A (DDA), a 6b-histamine adduct of 5a,6a-EC (Fig. 3) [22 && ]. DDA was found to display anticancer properties in vitro and in vivo. DDA is found in mammalian tissues and at significantly lower concentrations in patients with breast tumours than normal matched tissue. Analysis of DDA is challenging. Its polar nature dictates analysis by LC-MS/MS rather than GC-MS, however, Noguer et al. [27], using LC-MS/MS, have experienced serious problems of carryover between chromatographic runs. This, however, can be solved by addition of heptafluorobutyric acid to the mobile phase. This breakthrough should now allow the discovery of downstream metabolites

CONCLUSION
In recent years the biological significance of nonenzymatically derived sterols has been realised. How they are metabolized is an area of great interest as newly discovered sterol metabolites may have unexpected biological activity.