Cholesterol and oxysterol sulfates: Pathophysiological roles and analytical challenges

Cholesterol and oxysterol sulfates are important regulators of lipid metabolism, inflammation, cell apoptosis, and cell survival. Among the sulfate‐based lipids, cholesterol sulfate (CS) is the most studied lipid both quantitatively and functionally. Despite the importance, very few studies have analysed and linked the actions of oxysterol sulfates to their physiological and pathophysiological roles. Overexpression of sulfotransferases confirmed the formation of a range of oxysterol sulfates and their antagonistic effects on liver X receptors (LXRs) prompting further investigations how are the changes to oxysterol/oxysterol sulfate homeostasis can contribute to LXR activity in the physiological milieu. Here, we aim to bring together for novel roles of oxysterol sulfates, the available techniques and the challenges associated with their analysis. Understanding the oxysterol/oxysterol sulfate levels and their pathophysiological mechanisms could lead to new therapeutic targets for metabolic diseases.


| INTRODUCTION
Sulfate-based lipids (SL) represent a wide range of lipid classes, from low to high molecular weight compounds  with key functions in many aspects of human health and disease (Hu et al., 2007;Merten, 2001;Suzuki et al., 2003). The biotransformation of lipids by sulfation and desulfation reactions is fundamental to many cellular pathways. SL represent a diverse class of lipids including sulfate-, sulfonate-, and thiol-or thioether-based lipids (Dias, Ferreira, et al., 2019). In humans, steroid sulfates represent a highly abundant and extensively studied lipid class among the other glycerol-, sphingosine-, or taurine-derived lipids (Mueller, Gilligan, Idkowiak, Arlt, & Foster, 2015). Steroid sulfates were traditionally viewed as inactive precursors as they require active transport into cells via organic anion transporters. However, recent research suggests that these derivatives have active roles. For example, cholesterol sulfate (CS) acts as a signalling molecule (Shi et al., 2014), pregnenolone sulfate (PregS) and dehydroepiandrosterone sulfate (DHEAS) are neuroactive and more membrane transporters have been uncovered for cellular uptake of sulfated sterols (Fietz et al., 2013). Among other sulfated sterols, CS is the most reported and ubiquitously distributed sterol in mammalian tissues (Strott & Higashi, 2003). In addition to sulfation by sulfotransferases, cholesterol and its precursors undergo enzymic or free radical driven oxidations, resulting in oxidized derivatives (oxysterols).
Recent research in to oxysterols has identified many biological targets (Griffiths & Wang, 2019) despite their abundance being $10to 1,000-fold lower when compared to cholesterol in cells and biological fluids van Meer, Voelker, & Feigenson, 2008). Some of these oxysterols have been reported to be sulfated, and new biological functions of oxysterol sulfates are emerging. In fact, research groups who have focused their attention on oxysterol sulfates found that these molecules are key mediators in the cellular processes, such as attenuation of the inflammatory response (L. Xu et al., 2012), and the regulation of lipid metabolism via SREBP (sterol regulatory element-binding protein-1; Bai et al., 2012;Ma et al., 2008;Ren et al., 2007). Oxysterol sulfates show dynamic ways of activating, inhibiting, or shuttling of cholesterol in biological systems. This review brings together current understanding of sulfated cholesterol and oxysterols and analytical challenges in measuring their biological levels.
The family of SULTs consist of membrane-related enzymes, mainly localized in the Golgi apparatus and cytosolic enzymes (Falany, 1997). The cytosolic SULTs have been associated with the metabolism of endobiotic and xenobiotic substrates while the membrane-bound enzymes are primarily involved in sulfation of tyrosyl protein residues (Nowell & Falany, 2006). So far, four families of human cytosolic SULTs have been identified: SULT1, SULT2, SULT4, and SULT6. As enzymes of the SULT2 family have been associated with the sulfation of oxysterols, this review will focus on this group (Lindsay, Wang, Li, & Zhou, 2008). Members of the SULT2 family are divided into two subfamilies, SULT2A and SULT2B, based on their amino acid sequence and encoded by the two corresponding genes, SULT2A1 and SULT2B1 (Gamage et al., 2006).

| SULT2A1
In humans, SULT2A1 has been primarily linked to sulfation of DHEA, although it is also responsible for the sulfation of other steroid substrates such as pregnenolone, androgens, and bile acids (Gamage et al., 2006;Kong, Yang, Ma, Tao, & Bjornsson, 1992;Otterness et al., 1992). The SULT2A1 isoform is highly expressed in human liver, foetal adrenal glands, adult adrenal cortex, and small intestine (Nowell & Falany, 2006;Thomae, Eckloff, Freimuth, Wieben, & Weinshilboum, 2002). As a result, endogenous and orally administered steroids undergo sulfation by SULT2A1 as part of their metabolism. In particular, DHEAS obtained from DHEA by SULT2A1 serves as a precursor in the synthesis of androgens and oestrogens in human peripheral tissues (Mortola & Yen, 1990). The circulating endogenous levels of DHEAS are known to decrease with age and therefore associated with age-related diseases such as osteoporosis, muscle loss, vaginal atrophy, fat accumulation, hot flashes, skin atrophy, Type 2 diabetes, and cognitive deficits (Orentreich, Brind, Vogelman, Andres, & Baldwin, 1992). Observations by Thomae et al. (2002) suggested an ethnic-specific variation in the expression and activity of SULT2A1 among Caucasian and African American individuals that is likely to contribute to the high interindividual variability of DHEAS.

| SULT2B1a and SULT2B1b
The subfamily of SULT2B, including its two splice variants, namely, SULT2B1a and SULT2B1b, is widely distributed in human tissues and is able to metabolize sterol-like structures (Javitt et al., 2001). Both isoforms originate from the alternative splicing of the SULT2B1 gene localized to chromosome band 19q13.3, approximately 500 kb telomeric to the location of SULT2A1 (Her et al., 1998). In the gene for SULT2B1, exon 1A encodes a unique amino-terminal end for the B1a isoform and additional 48 amino acids, compared to the B1b spliced variant (H. Fuda, Lee, Shimizu, Javitt, & Strott, 2002). Javitt et al. (2001) reported that SULT2B1b is expressed to a greater extent F I G U R E 1 Structures of sterol and oxysterol sulfates than SULT2B1a, in tissues responsive to hormones. In fact, the B1b isoform preferentially acts on cholesterol, whereas the B1a isoform catalyses the sulfation of pregnenolone, but not cholesterol (H. Fuda et al., 2002). The expression of the isoform B1b is usually several-fold higher than the isoform B1a (Falany, He, Dumas, Frost, & Falany, 2006) and widely distributed in many tissues including human liver, trace amounts in brain, prostate, placenta, breast, lungs, platelets, and kidney Geese & Raftogianis, 2001;He, Meloche, Dumas, Frost, & Falany, 2004). Double knockout Sult2b1 −/− mice are viable and show significant decrease in their CS/cholesterol ratio compared with their wild-type counterparts (Wang, Beck-García, Zorzin, Schamel, & Davis, 2016), suggesting that low level of CS may be formed by other SULTs. CS-deficient mice displayed a heightened sensitivity to self-antigens (Wang et al., 2016). Systemic up-regulation of SULT2B1b inhibited lipogenesis by sulfonating and deactivating the liver X receptor (LXR)-activating oxysterols in LDLR −/− mice  and overexpression of hepatic SULT2B1b sensitized the mice to drug-induced liver damage  and inhibition of gluconeogenesis (Shi et al., 2014).

| METABOLISM OF STEROL SULFATES
The cleavage of the sulfate moiety of 3β-hydroxysteroid sulfate is catalysed by membrane-bound microsomal steroid sulfatase (STS; Conary, Nauerth, Burns, Hasilik, & von Figura, 1986). The gene encoding human STS is located on the distal short arm of the X-chromosome (Yen et al., 1988) and ubiquitously expressed in many human tissues including placenta, breast, skin, lungs, ovaries, adrenal glands, and brain (Reed, Purohit, Woo, Newman, & Potter, 2005). STS have been associated with high intra-tumour oestrogen and androgen levels and therefore linked to steroid hormone-dependent tumour growth (Nardi et al., 2009). Studies by Zaichuk, Ivancic, Scholtens, Schiller, and Khan (2007) showed that oestrogen regulates the transcription of STSs in breast carcinoma. X-linked ichthyosis, a disease clinically characterized by skin peeling localized in the anterior and posterior areas of upper and lower extremities, is caused by a mutation in the enzyme STS. Patients with recessive X-linked ichthyosis not only display a significant increase in CS in squamous keratinizing epithelia but also exhibit effects in overall lipid metabolism and mental retardation (Elias, Williams, Choi, & Feingold, 2014). In healthy epidermis, CS is produced by the action of SULT2B1b and desulfated in the outer epidermis, thus contributing to epidermal differentiation, maintenance of barrier function and desquamation. As a consequence of STS deficiency, CS levels could exceed 10% of the total lipid mass in epidermal cells (Rizner, 2016).

| CHOLESTEROL-3-SULFATE
Besides being the most abundant steroidal sulfoconjugate present in human plasma, with an average concentration of 2 μM (Meng, Griffiths, Nazer, Yang, & Sjövall, 1997), CS has also been detected in other biological fluids, such as urine, bile, seminal plasma, and in many tissues, as described previously (Castellanos, Hernandez, Tomic-Canic, Jozic, & Fernandez-Lima, 2020;Drayer & Lieberman, 1967;Lopalco et al., 2019;Strott & Higashi, 2003). Even though CS is typically considered the hydrophilic excretion form of cholesterol, CS also represents a biosynthetic precursor of several bioactive steroids. In this context, the sulfoconjugation reaction may represent a key step in the formation of a readily available hydrophilic form of cholesterol. CS regulates cholesterol homeostasis, indirectly, by negative regulation of the key enzyme in cholesterol synthesis pathway, 3-hydroxy 3-methylglutaryl-CoA reductase (HMG-CoA reductase) (Williams, Hughes-Fulford, & Elias, 1985) and, directly, blocks the esterification of cholesterol by inhibiting the activity of lecithin-cholesterol acyltransferase enzyme (Nakagawa & Kojima, 1976). Indeed, CS can be subjected to several enzymic transformations carried out by microsomal cytochromes (e.g., CYP11A1, also referred to as cholesterol side-chain cleavage enzyme) in order to obtain sulfated precursors of sex hormones. During the last decades, the role of CS as a signalling molecule has been investigated (Sakurai et al., 2018;Shi et al., 2014;Wang et al., 2016), although many questions remain unanswered. Niemann-Pick disease type C2 protein (NPC2), a key protein involved in cholesterol transport from the lysosomal compartment after the endocytic uptake of low-density lipoproteins (Liou et al., 2006). The interaction between NPC2 and CS was demonstrated both by a chromatographic shift assay and by competition assay. It is noteworthy to mention that CS was unable to interact with the functional analogue Niemann-Pick disease type C1 protein (NPC1) according to a scintillation counting binding assay (Infante et al., 2008).

| CHOLESTEROL-3-SULFATE AND ITS RECEPTORS
As described above, recessive X-linked ichthyosis has been related to a deficiency in cholesterol sulfatase expression with subsequent accumulation of CS. Sato, Denda, Nakanishi, Nomura, and Koyama (1998) correlated this pathological condition with the ability of CS to inhibit serine proteases involved in cell dissociation, a key feature in skin development. As a matter of fact, Ito et al. demonstrated the direct inhibition of several hydrolytic enzymes by CS (e.g., pancreatic elastase, trypsin, chymotrypsin, thrombin, plasmin, and DNAse I) in the late '90s (Ito, Iwamori, Hanaoka, & Iwamori, 1998;Iwamori, Iwamori, & Ito, 1997;Iwamori, Suzuki, Kimura, & Iwamori, 2000). The inhibitory behaviour of CS towards these pancreatic enzymes has been related to its protective role at the gastrointestinal mucosa level. In addition, it is noteworthy to underline that the inhibition of these enzymes occurred in a non-specific fashion. In other words, the interaction between the two molecular partners is based only on the physicochemical properties of CS and the presence of an anion binding region on the tertiary structure of the target protein.
The ability of CS to inhibit serine proteases was extended by Iwamori, Iwamori, and Ito (1999) to thrombin and plasmin. As these two proteases are involved in blood clotting and fibrinolysis, respectively, CS can be considered an endogenous modulator of homeostasis of the blood clotting system within the vascular network by a presumably non-specific irreversible mechanism. Moreover, CS has been found to promote divalent cation-independent adhesion of both activated and inactivated platelets, although the mechanisms by which CS exert these prothrombotic activities are not clear (Merten, 2001).

| Role of CS in inflammation and the immune system
Recent research found that CS play a significant role in the control of inflammation by modulating key targets (Aleksandrov et al., 2006).
Inflammation is a complex multistep biological response of body tissues to harmful stimulations which typically involves a multitude of mediators and many different cell types. 5-Lipoxygenase (5-LO) is involved in the production of leukotrienes (LTs), soluble mediators of the inflammatory state, and immune system functionality. In particular, LTs play a pivotal role in asthma and bronchitis. When a Ca 2+ influx takes place, 5-LO binds to the nuclear membrane where it can convert arachidonic acid into the bioactive leukotrienes. As a constituent of cell membranes, CS can modulate the function of several proteins, including 5-LO, directly interacting at the membrane level. Aleksandrov et al. (2006), demonstrated the inhibitory behaviour of CS towards 5-LO in a cell-free assay. Here, CS decreased 5-LO interaction with the nuclear membrane in a cell-based assay upon stimulation, thus decreasing LT biosynthesis. Wang et al. (2016) showed CS to act as a modulator of T-cell receptor (TCR) functionality. The TCR is a multisubunit membrane receptor which includes an antigen-recognition domain composed of the TCRα and β (or γ and δ) heterodimer and a signalling domain, typically three CD3 dimers. Although TCR binds its corresponding peptide -MHC ligands -with extremely weak affinity, it is wellknown that a single molecule of its ligand is able to activate the T cell. The low affinity and the high sensitivity of this receptor have been related to the nanoclustering of several TCRs. Cholesterol is able to interact with TCRβ, thus promoting TCR nanoclustering.
Conversely, CS can disrupt TCR clusters by interfering in the cholesterol-TCRβ interaction. Interestingly, the cholesterol/CS ratio is a variable parameter during T cell development and differentiation (Wang et al., 2016).
The protein, dedicator of cytokinesis 2 (DOCK2), is a guanine nucleotide exchange factor which plays a key role in immune surveillance and immune responses by regulating the chemotaxis and the activation of leukocytes. Sakurai et al. (2018) demonstrated that CS is highly expressed in Harderian gland, an orbital gland that produces the lipids that form the oily layer of the tear film in the eye of Sult2b1 +/+ mice, was able to inhibit the action of DOCK2. In particular, a direct interaction between CS and DOCK2 has been confirmed by a cell-free surface plasmon resonance binding assay (Sakurai et al., 2018). Human tear film also contains a high level of CS (Lam et al., 2014), and it is possible that CS limit ocular surface inflammation by inhibiting DOCK2.
CS has been also reported as an endogenous ligand of macrophage inducible Ca 2+ -dependent lectin receptor (Mincle), an innate immune receptor involved in skin allergic inflammation (Kostarnoy et al., 2017). In the studies reported above, the specific interaction of CS with the corresponding target protein was not proven, and in most cases, the observed activity of CS was attributed to its amphiphilic nature without identifying a proper binding pocket/site on the polypeptidic counterpart. Kallen, Schlaeppi, Bitsch, Delhon, and Fournier (2004)  Indeed, even if the activation of this nuclear receptor occurs upon stimulation with CS, the latter is considered so far only a putative RORα endogenous ligand (Han et al., 2014;Kim et al., 2008;Zenri et al., 2012).

| Role of CS as a ligand in signalling pathways
CS has an important role in the substrate specificity of PI3K (Woscholski, Kodaki, Palmer, Waterfield, & Parker, 1995).
Phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ), produced by the action of PI3K, is associated with the signalling pathway of several growth factors, and it is considered a secondary messenger.
Phosphatidyinositol bisphosphate (PIP 2 ) is the preferred substrate of PI3K in vivo, inside the cell. Conversely, phosphatidyinositol monophosphate and phosphatidylinositol are the preferred substrates of PI3K in cell-free systems. Woscholski et al. (1995) demonstrated that the characteristic substrate specificity of this enzyme in vivo could be restored in the presence of CS, pointing out its potential relevance as an interacting partner inside the cell.

| OXYSTEROLS SULFATES AND THEIR RECEPTORS
Oxysterols are bioactive lipids which share the 27-carbon skeleton with cholesterol and differ from the latter by the presence of extra oxygenated functional groups, apart from the 3β-hydroxyl group. In addition to being biosynthetic precursors of bile acids and sex hormones, they serve as selective ligands towards several targets (e.g., GPCRs, enzymes, nuclear receptors, and other membrane and cytosolic proteins). Similarly, their sulfoconjugates have been found to act as modulators of different targets. Traditionally, oxysterol sulfates have been viewed as detoxification derivatives of oxysterols that are synthesized for excretion. However, recent work has proposed that oxysterol sulfates were bioactive molecules that act as selective ligands with biological outcomes. Table 1  Oxysterol sulfoconjugation occurs mainly by the cytosolic PAPSdependent enzyme SULT2B1b, also referred to as hydroxysteroid sulfotransferase. This metabolic transformation is generally reversible, as the enzymic activity of STS is able to provide the parent oxysterol in its active form. Song et al. (2001) demonstrated that 5α,6αepoxycholesterol-3-sulfate (5,6αECS) and 7-ketocholesterol-3-sulfate (7KCS) bind to both nuclear receptors LXRα and LXRβ, inhibiting their activation and acting as antagonists. It is noteworthy that in addition to a cell-based gene transactivation assay, the authors also performed a cell-free coactivator peptide recruitment binding assay in order to demonstrate the direct interaction of 5,6α-ECS and 7KCS with the receptors. Moreover, a structure-dependant ligand recognition mechanism was sought, by testing two closely related sulfated oxysterols, 5β,6β-epoxycholesterol-3-sulfate (5,6βECS) and 6-ketocholestanol-3-sulfate, in the same assays. As both of the latter compounds failed to modulate LXR activation, the authors speculated that the antagonistic behaviour of 5,6α-ECS and 7KCS towards LXRs was independent of their physiochemical properties (e.g., amphiphilicity).  Table. 5,6αECS, 5α,6α-epoxycholesterol-3-sulfate; 7ketoC, 7-ketocholesterol; 7KCS, 7-ketocholesterol-3-sulfate; 24HC3S, 24(S)-hydroxycholesterol-3-sulfate; 24HCDS, 24(S)-hydroxycholesterol-3,24-disulfate; 25HC3S, 25-hydroxycholesterol-3-sulfate be considered a negative feedback mechanism to control the activation of LXRs.

25HC3S was identified by
One of the first studies focused on the screening of oxysterol sulfates in biological fluids described the presence of elevated levels of a compound compatible with the presence of a glucuronidated cholestenediol sulfate in serum and urine samples of children with severe cholestatic liver disease (Meng et al., 1997). The authors were able, after extensive sample handling and derivatisation steps, to identify and characterize it as the glucuronidated form of the 24HC3S, by fast atom bombardment mass spectrometry using glycerol as a matrix compound (Meng et al., 1997). The authors also reported the occurrence of oxysterol glycine and taurine conjugates, though sulfation seemed to be the main detoxification route in cholestatic liver disease and with potential prognostic value during clinical evaluation (Meng et al., 1997). Later, Acimovic et al. (2013) suggested that sulfation could act as a protective mechanism against the accumulation of Despite the evidence for a wider panel of oxysterol sulfates in circulation provided by these exploratory studies (Meng et al., 1997;Sánchez-Guijo, Oji, Hartmann, Schuppe, et al., 2015), very little is known about the predominant oxysterol sulfates present in body fluids and accumulated in cells and tissues, their basal levels, and any variations introduced with age, gender, and ethnicity in health and disease, despite the common knowledge that SL gather at the surface of lipid-raft domains (Weerachatyanukul, Probodh, Kongmanas, Tanphaichitr, & Johnston, 2007) and contribute to cell-cell communication processes (Honke, 2017;Strott & Higashi, 2003). On the other hand, structurally related compounds such as oxysterols are widely studied, and the oxysterol signature in normolipidaemic and normoglycaemic conditions and their basal levels are known (Dias et al., 2018;Grayaa et al., 2018;McDonald, Smith, Stiles, & Russell, 2012;Murakami, Tamasawa, Matsui, Yasujima, & Suda, 2000;Narayanaswamy et al., 2015). Oxysterols are predominantly found esterified to fatty acids (Dzeletovic, Breuer, Lund, & Diczfalusy, 1995) and are thought to be substrates for sulfotransferases (Fuda et al., 2007), leading the formation of oxysterol sulfates.  (Table 2) reveals a diversity of sample pretreatment strategies (e.g., anticoagulant), extraction solvent system used, and analytical methodology has been largely overlooked.
As shown in Table 2 Another aspect that is often ignored is the freeze-thaw cycle often required for biochemical and chemical analysis. Whereas this appeared not to affect the levels of CS , the number of freeze-thaw cycles decreased the level of oxysterols (Helmschrodt et al., 2013).. Storage up to 3 months did not affect levels of CS (Hautajärvi et al., 2018;Helmschrodt et al., 2013;. One other aspect that has been largely overlooked is the method of extraction. Extraction of steroid-related compounds is typically conducted by liquid-liquid extraction (LLE) protocols followed by fractionation in solid-phase extraction (SPE) cartridges (Table 2). In fact, LLE protocols remain the most popular method of choice due to their simplicity, cost, sample volume required, extraction efficiency, reproducibility, repeatability, lipidome coverage, and potential for automation, where the overall performance of LLE protocols is very similar in F I G U R E 2 Chromatographic separation of oxysterol sulfates in serum samples from patients with recessive X-linked ichthyosis (RLXI) (a) and healthy control subject (b) using targeted multiple reaction monitoring (MRM) detection mode. These data have been taken from Sánchez-Guijo, Oji, Hartmann, Schuppe, et al., (2015), with permission T A B L E 2 Analytical strategies employed in the collection, extraction, and analytical approach in the detection and quantification of cholesterol sulfate and oxysterols in human plasma samples Biological matrix (collection tube) Extraction approach (method and solvent system) Analytical approach and method performance Ref.
While the presence of hydroxy groups affects the hydrophobicity of the oxysterol moiety and may affect the performance of the extraction, the presence of the sulfate and hydroxy groups in oxysterols sulfates also affects the detection methods that can be used to quantify oxysterol sulfates. In terms of mass spectrometric assays, the oxysterols are usually detected in the positive ion detection mode (Dias et al., 2018;Hautajärvi et al., 2018;Helmschrodt et al., 2013;Mendiara et al., 2018;Murakami et al., 2000), whereas the presence of the sulfate group facilitates the detection of oxysterol sulfates in the negative ion mode. Because oxysterols sulfates occur in residual levels in biological samples, detection of oxysterols sulfates is often F I G U R E 3 Overview of cholesterol sulfate and oxysterol sulfate analysis achieved by targeted detection methods such as multiple reaction monitoring (MRM). Due to the specificity of the transitions in MRM approaches, these display an increased sensitivity in the detection step with the advantage of eliminating the contribution of the other sulfated metabolites that may contribute to the overall plasma sulfometabolome and already observed by targeted approaches (Dias, Ferreira, et al., 2019). Previous work by Sanchez-Guijo and colleagues established 1 ngÁml −1 as the limit of detection of oxysterol sulfates, using MRM detection (Sánchez-Guijo, Oji, Hartmann, Schuppe, et al., 2015).
Contrarily, the presence of the hydroxy group has no influence on the efficacy of ionization and hence on the detection step. As ionization of oxysterol sulfate occurs by removal of a hydrogen atom at the sulfate group, the ionization efficiency of oxysterol sulfates is similar to that of CS. This was confirmed by the injection of an equimolar mixture of oxysterol sulfates and CS and detection under reversephase elution conditions in the negative ion mode (unpublished results).
Regardless of the collection, extraction, and analytical strategy adopted in the analysis of oxysterol sulfate, the values reported (Acimovic et al., 2013;Meng et al., 1997;Sánchez-Guijo, Oji, Hartmann, Schuppe, et al., 2015) show that these are well below the micromolar range generally used in the biological assessment of oxysterol sulfates in cells and tissue (Ren et al., 2007;Leyuan Xu et al., 2010;. Based on the published values, oxysterols that are structurally related compounds of oxysterol sulfates account for less than 1% of total cholesterol in hyperlipidaemia (Björkhem et al., 2001;Dias et al., 2018;Reinicke et al., 2018) while oxysterol sulfates (24HC3S and 26HC3S) account for less than 15% of total oxysterols (Acimovic et al., 2013). This could explain why oxysterol sulfates have been largely overlooked by the scientific community.

| CONCLUDING REMARKS
In summary, it is clear that CS and oxysterol sulfates act as key players of many biological pathways influencing human health and disease.
While CS has been extensively studied, only a handful of papers have