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Hydroxycholesterols Analysis Service

Hydroxycholesterols are mono-oxygenated derivatives of cholesterol that comprise a family of polycyclic compounds that contain a second oxygen atom as a hydroxyl group on the skeleton of cholesterol. In vivo, hydroxycholesterols are present as unesterified (biologically active) and esterified forms (largely biologically inert). Hydroxycholesterols are formed through enzymatic conversion of cholesterol or by free radical autoxidation, and exhibit a short half life relative to cholesterol.

Hydroxycholesterols are important intermediates in a number of catabolic pathways that regulate a variety of biological effects. Hydroxycholesterols are critical for cholesterol transport from the periphery to the liver, regulate the expression of sterol sensitive genes which are involved in lipid and sterol biosynthesis, act as substrates for the formation of bile salts, serve as ligands that activate nuclear liver X receptors-α and -β, and are involved in the regulation of cholesterol and lipid metabolism and homeostasis. In the central nervous system, hydroxycholesterols regulate arachidonic acid release, voltage-gated calcium channels, synaptic plasticity, induce IL-8, promote neurogenesis and induce apoptosis. Consistent with these critical roles for modulating biological functions, content of free hydroxycholesterols are extremely low and tightly controlled, with the majority of hydroxycholesterols maintained in esterified forms.

Hydroxycholesterols QuantifiedFigure. Structural representation of cholesterol and related products.

Many cell types have the ability to oxygenate cholesterol by mechanisms that involve the cytochrome P450 family of oxidases (CYP). Cell type specific expression CYP subtypes results in the tissue-specific production of particular oxysterol species. Several CYP are present in the central nervous system including 24S-hydroxycholesterolhydroxylase (CYP46), a P450 family member that is expressed in neurons, glia and in endothelial cells of the blood–brain barrier. 24S-hydroxycholesterol (Cholest-5-en-3β, 24(S)-diol) is the most abundant hydroxycholesterol in brain and is the primary transport form of cholesterol from the central nervous system into the blood, with smaller amounts eliminated through cerebrospinal fluid. It has been suggested by several studies that serum or plasma levels of 24S-hydroxycholesterol may reflect brain developmental and neuropathological changes associated with Alzheimer’s disease (AD), Huntington’s disease and Multiple Sclerosis. 24S-hydroxycholesterol is often expressed as a ratio to 27-hydroxycholesterol (25R-Cholest-5-en-3β, 26-diol). 27-hydroxycholesterol is formed primarily in the periphery by the P450 enzyme sterol 27-hydroxylase (CYP27). CYP27 is expressed in arterial endothelium, macrophages and to lesser extents in other tissues such as cortex, spleen, liver, kidney, adrenal gland and heart. 27-hydroxycholesterol can function as a ligand for nuclear receptors, liver X receptors (LXR) and farnesoid X-activated receptors (FXRs). 27-hydroxycholesterol can also regulate hydrocymethylglutaryl-CoA reductase, and enhances cholesterol efflux from the vascular endothelium. Macrophages have the highest capacity to convert cholesterol to 27-hydroxycholesterol, which is then transported in blood to the liver where it is converted to bile acids.

Hydroxycholesterol detection and quantification has been accomplished in a variety of tissues by isotope-dilution gas-chromatography-mass spectrometry (GC-MS), gas- and high-performance liquid chromatography (HPLC)/mass spectrometry, HPLC with UV detection of cholesterol oxidation products in tissues, as ∆4- 3-ketone derivatives by HPLC and as derivatives of GP hydrazones. Because “free” hydroxycholesterol levels are below the detection limits of many instruments, saponification and/or solid phase extraction techniques have typically been used to extract “total” hydroxycholesterols. The primary advantage of these methods is a high yield of hydroxycholesterols. The disadvantages include lengthy sample preparation times, sample loss, inconsistent yields, and the inability to discriminate between free and esterified hydroxycholesterols. Here Creative Proteomics developed a simple and direct extraction protocol and sensitive LC/MS/MS method for separation and quantitative determination of hydroxycholesterols.




  • Identification and quantification of hydroxycholesterols

Sample Requirement

  • Normal Volume: 200 uL plasma, 100 mg tissue, (2E7) cells, (80 mg–human; 40 mg-rodent) feces.
  • Minimal Volume: 100uL plasma, 50 mg tissue, (5E6) cells, Feces (50 mg –human; 30 mg-rodent) feces.


  • A detailed technical report will be provided at the end of the whole project, including the experiment procedure, MS/MS instrument parameters.
  • Analytes are reported as uM or ug/mg (tissue), and CV's are generally<10%.
  • The name of the analytes, abbreviation, formula, molecular weight and CAS# would also be included in the report.
Hydroxycholesterols Quantified in This Service

With integrated set of separation, characterization, identification and quantification systems featured with excellent robustness & reproducibility, high and ultra-sensitivity, Creative Proteomics provides reliable, rapid and cost-effective Hydroxycholesterols targeted metabolomics services.

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* For Research Use Only. Not for use in diagnostic procedures.
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