Description of analytical method and clinical utility of measuring serum 7-alpha-hydroxy-4-cholesten-3-one (7aC4) by mass spectrometry

Clin Biochem. 2018 Feb:52:106-111. doi: 10.1016/j.clinbiochem.2017.10.008. Epub 2017 Oct 16.


Background: Imbalance of bile acids (BA) homeostasis in the gastrointestinal tract can lead to chronic diarrhea or constipation when BA in the colon are in excess or low, respectively. Since both disturbances of bowel function can result from other etiologies, identifying BA imbalance is important to tailor treatment strategies. Serum concentrations of 7-alpha-hydroxy-4-cholesten-3-one (7aC4), a precursor in bile acid synthesis, reflect BA homeostasis. Here we describe a method to accurately measure serum 7aC4 and evaluate the clinical utility in patients with diarrhea or constipation phenotypes.

Methods: Serum 7aC4 is measured after acetonitrile protein precipitation using C18 liquid chromatography, tandem mass spectrometry, and deuterium-labeled 7aC4 internal standard. Assay performance including linearity, precision, and accuracy was assessed using waste serum samples. The reference interval was established in healthy individuals without BA-altering conditions or medications. Clinical performance was assessed in patients with irritable bowel syndrome.

Results: The method precisely and accurately measured 7aC4 in human serum from 1.4-338ng/mL with no ion suppression or interference from related 7-keto-cholesterol. Central 95th percentile reference interval was 2.5-63.2ng/mL. Lower serum 7aC4 was found in patients with constipation with sensitivity/specificity of 79%/55% compared to healthy controls. Higher 7aC4 was found in patients with bile acid diarrhea (BAD) compared to those without BAD with sensitivity/specificity of 82%/53%.

Conclusions: We have developed a sensitive and precise assay for measuring the concentration of 7aC4 in serum. The assay can be used to screen for diarrhea caused by bile acid malabsorption.

MeSH terms

  • Bile Acids and Salts / analysis
  • Bile Acids and Salts / metabolism*
  • Cholestenones / analysis*
  • Cholestenones / blood
  • Cholestenones / metabolism
  • Chromatography, Liquid / methods
  • Constipation
  • Diarrhea / metabolism
  • Feces / chemistry
  • Humans
  • Mass Spectrometry / methods*
  • Sensitivity and Specificity
  • Serum
  • Steatorrhea / metabolism
  • Tandem Mass Spectrometry / methods


  • Bile Acids and Salts
  • Cholestenones
  • 7 alpha-hydroxy-4-cholesten-3-one
  • cholest-4-en-3-one

Supplementary concepts

  • Bile Acid Malabsorption, Primary