The Making of the Oxysterols Targeted Panel

Posted In: Blog
Oxysterols

Meet the brains behind the people responsible for the design and validation of our latest targeted panel.

Metabolon’s Oxysterols Targeted Panel was released on October 25, 2022, offering a reproducible solution for routine oxysterol quantification in human plasma samples. Deirdre Hauser, Principal Research Scientist at Metabolon, led the design and validation of the panel. In her own words, this is the making of the Oxysterols Targeted Panel.

Why were oxysterols chosen to develop into a targeted metabolomics panel?

Oxysterols, which are oxidized forms of cholesterol, are intermediates in the synthesis of other metabolites, such as vitamin D and bile acids. Additionally, many oxysterols are bioactive molecules that regulate or modulate certain pathways and responses, such as sterol synthesis, inflammatory and immune responses, as well as developmental processes. Because of their bioactivity, oxysterols have been associated with disease pathogenesis, such as cancer, atherosclerosis, and neurodegenerative diseases; however, the mechanism of action of many oxysterols is not well understood. This, in addition to the fact that oxysterols are present at very low concentrations, which makes them analytically difficult compounds to measure, is the reason why we wanted to develop the Oxysterols Targeted Panel, to help further research endeavors in this class of compounds.

For whom is the Oxysterols Targeted Panel designed? Which applications or research projects?

The Oxysterols Targeted Panel is designed for researchers interested in diseases with an inflammatory, immune, or oxidative stress association. Additionally, researchers investigating cholesterol metabolism, bile acid metabolism, vitamin D metabolism, or disorders associated with these may find oxysterols to be a new avenue for their research.

What metabolites are included in the panel?

Currently, there are 12 sterol and oxysterol metabolites in the panel. The metabolites that are included are cholesterol, lathosterol, lanosterol, desmosterol, 24-hydroxycholesterol, 27-hydroxycholesterol, 4β-hydroxycholesterol, 7-dehydrocholesterol, 8-dehydrocholesterol, 5α, 6α-epoxycholesterol, 5α, 6β-dihydroxycholestenal, and 7α, 27-dihydroxycholesterol. In this panel, we are measuring the hydrolyzed sterol or oxysterol metabolites, which means that we convert the conjugated sterol and oxysterol esters to their free form prior to measurement.

Can you take us through your validation process when developing the panel?

Typically, when developing a new panel, we first determine which metabolites might be of interest to our customers, and then we purchase reference standards for those analytes that are commercially available. We also obtain isotopically labeled Internal Standards for each analyte, which allows us to ensure selectivity and consistent quantitation. If we can’t find the isotopically labeled material, we also have the capability to synthesize these standards in some cases. We then optimize mass spectrometric conditions to ensure that we can measure the metabolites. Next, we develop an extraction procedure to separate the compounds of interest from the matrix material, without changing or degrading the metabolites that we want to measure. This can include a simple protein precipitation, or if additional sensitivity or selectivity is needed, we may also use more specialized techniques including solid-phase or liquid-liquid extraction, or derivatization. We make calibration standards at known concentrations and run those through the same extraction procedure along with the samples to determine the exact concentrations of the analytes in the panel. We make Quality Control (QC) samples in the same matrix and run those at three different levels in each batch.

Prior to running the validation, a validation plan is prepared and approved, which details the experiments and the acceptance criteria for all components. These experiments are defined in our targeted assay method validation standard operating procedure (SOP). The validation includes the determination of linearity, sensitivity, accuracy, precision, stability, and recovery of the analytes in the matrix. During the validation of the panel, the performance of the procedure is evaluated across analyte concentration ranges determined from measuring several lots of the appropriate matrix. The validation of each panel consists of at least three different runs with accuracy and precision data for Calibration Standards and QC samples, which must meet the acceptance criteria set forth in the validation plan. Samples are tested for stability of the analytes by subjecting them to different temperatures and freeze-thaw conditions. Relative recovery is assessed by over-spiking matrix QC samples with known concentrations of the metabolites and comparing back to the nominal concentration. Once all planned experiments have been conducted, the data is presented in tables, and all the analytes that have passed the acceptance criteria are considered validated.

What applications are you most excited about seeing this panel utilized for?

There are many applications for the analysis of cholesterol and oxysterols for neurological diseases. There are very high levels of cholesterol in the brain, it’s the organ with the highest levels, and it’s locally synthesized. Thus, oxysterol levels in the blood can be used as biomarkers for neurodegenerative diseases, such as Alzheimer’s or Huntington’s disease.

Cholesterol and oxysterols can alter the action of cell membrane receptors, and some neurological diseases are caused by the effects of aberrant cholesterol and oxysterol metabolism on cell membrane receptors, such as with Smith-Lemli-Optiz syndrome.

Neurodegeneration can be assessed systemically with sterol or oxysterol biomarkers, but additionally, mechanistic insights into certain neurological disorders associated with deranged cholesterol and oxysterol metabolism may be gained due to their membrane receptor modulating activities.

What’s next? Are there any additional metabolites you are validating or will include at a later stage?

There were some hydroxycholesterols that didn’t perform as expected during our method development and validation, and therefore could not be included in this current iteration of the panel.  We would like to be able to offer 7α-hydroxycholesterol, 7β-hydroxycholesterol, and 5β, 6β-epoxycholesterol in the future, and possibly even 7-ketocholesterol as well, but these will require more method development time as these analytes tend to be less stable.

If someone wants to find out more information about this product, who can they contact?

All of the Metabolon team are incredibly helpful and will be able to direct your question to the best possible person. If you visit the Oxysterols Targeted Panel page and submit an inquiry or use our Contact Us page, any requests for more information will be followed up within a couple of business days!

Ready to see what new insights oxysterols can help your research reveal?
Contact us today to discuss your project or study.

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