Saliva Sampling Methods Optimized Using UHPLC-MS

Scientists from the Institute of Analytical Chemistry of the Czech Academy of Sciences and Masaryk University in Brno, Czech Republic recently tested various saliva sampling methods to optimize bile acid analysis. Their findings were published in the Journal of Chromatography A (1).

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Bile acids (BAs) are a large family of molecules that have a steroidal structure and are synthesized from cholesterol in the liver, which are actively secreted along with cholesterol and phospholipids into the bile (2). The primary BAs, cholic acid (CA) and chenodeoxycholic acid (CDCA), are synthesized in hepatocytes from cholesterol, which are typically conjugated with glycine or taurine. Secondary BAs, which are formed within the intestine, creating a diverse BA pool with varying properties. These molecules have many purposes, including facilitating digestion and lipid absorption in the small intestine, in addition to regulating cholesterol homeostasis. Furthermore, through activating various signaling pathways, BAs not only regulate their own synthesis and enterohepatic circulation, but also triglyceride, cholesterol, glucose, and energy homeostasis (3).

Despite its usefulness, BA profile analysis in various human fluids has been under scrutiny lately, especially for evaluating the severity of gastroesophageal reflux disease and duodenogastroesophageal reflux (DGER). BAs have been shown to cause damage and permanent changes to the esophageal lining (mucosa) during reflux episodes in patients with DGER, where refluxate contains both gastric acid and BAs. These changes can also increase the risk of developing Barrett’s esophagus, a condition that can potentially cause esophageal cancer. That said, BA exposure does not show typical reflux symptoms, like heartburn and regurgitation; as such early detection of risk for Barrett's esophagus or esophageal cancer susceptibility is vital, with BA profiling playing a crucial role. Barrett’s esophagus is a condition where tissue similar to one’s intestine lining replaces the tissue lining the esophagus (4). However, despite its notoriety, scientists do not know the exact cause of this conditions.

In this study, the scientists investigated methods for sampling BAs in saliva, a potential non-invasive diagnostic biofluid. Three saliva collection methods were compared: spitting, Salivette swabs, and Salivette Cortisol swabs. Overall, spitting was proven to be the superior method with the highest recoveries and the least interference; this likely stems from Salivette swabs retaining BAs or introducing unknown interferences. Each saliva sample was analyzed using ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), specifically with the Zorbax RRHD Eclipse Plus C18 column (3 × 50 mm, 1.8 µm) in gradient elution of 0.1 % formic acid in water and methanol. The saliva samples’ stability was assessed over 14 days, thus reflecting typical storage times. Detected BA levels were stable through the measured period (RSD ≤ 22%) with no degradation being observed. BA levels in saliva fluctuated throughout the day, with the most notable changes being found in glycine-conjugated BAs after meals.

According to the researchers, saliva collection via spitting overnight fasting is recommended for future studies to minimize sampling variability. Furthermore, their findings, which proved applicable for standardized bile acid sampling, are currently being applied in a large clinical study evaluating BAs as potential susceptibility markers for Barrett’s esophagus diagnostics.

References

(1) Dosedělová, V.; Laštovičková, M.; Konečný, Š.; Dolina, J.; Kubáň, P. Optimizatoin of Saliva Sampling Methods for Analysis of Bile Acids by UHPLC-MS. J. Chromatogr. A 2024, 1736, 465354. DOI: 10.1016/j.chroma.2024.465354

(2) LiverTox: Clinical and Research Information on Drug-Induced Liver Injury [Internet]. Bethesda, Maryland). National Institute of Diabetes and Digestive and Kidney Diseases; 2012-. Bile Acids. Updated 2017-09-25. https://www.ncbi.nlm.nih.gov/books/NBK548626/

(3) Staels, B.; Fonseca, V. A. Bile Acids and Metabolic Regulation. Diabetes Care 2009, 32 (Suppl 2), S237–S245. DOI: 10.2337/dc09-S355

(4) Barrett’s Esophagus. National Institute of Diabetes and Digestive and Kidney Diseases 2024. https://www.niddk.nih.gov/health-information/digestive-diseases/barretts-esophagus (accessed 2024-10-22)