Quantitative determination of lysophosphatidic acids (LPAs) in human saliva and gingival crevicular fluid (GCF) by LC–MS/MS

Abstract

Lysophosphatidic acid (LPA) is a phospholipid mediator that plays multiple cellular functions by acting through G protein-coupled LPA receptors.LPAs are known to be key mediators in inflammation,and several lines of evidence suggest a role for LPAs in inflammatory periodontal diseases. A simple and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method has been developed and validated to quantify LPA species (LPA 18:0, LPA 16:0, LPA 18:1 and LPA 20:4) in human saliva and gingival crevicular fluid (GCF). LPA 17:0 was used as an internal standard and the LPA species were extracted from saliva by liquid-liquid extraction using butanol. Chromatography was performed using a Macherey-Nagel NUCLEODUR® C8 Gravity Column (125mm × 2.0mm ID) with a mixture of methanol/water: 75/25 (v/v) containing 0.5% formic acid and 5mM ammonium formate (mobile Phase A) and methanol/water : 99/0.5 (v/v) containing 0.5% formic acid and 5mM ammonium formate (mobile phase B) at a flow rate of 0.5 mL/min. LPAs were detected by a linear ion trap-triple quadrupole mass spectrometer with a total run time of 8.5 min. The limit of quantification (LOQ) in saliva was 1 ng/mL for all LPA species and the method was validatedover the range of 1-200 ng/mL. The method was validated in GCF over the ranges of 10-500 ng/mL for LPA 18:0 and LPA 16:0, and 5-500 ng/mL for LPA 18:1 and LPA 20:4. This sensitive LC-MS/MS assay was successfully applied to obtain quantitative data of individual LPA levels from control subjects and patients with various periodontal diseases. All four LPA species were consistently elevated in samples obtained from periodontal diseases, which supports a role of LPAs in thepathogenesis of periodontal diseases.

Quantitative analysis of endogenous compounds

“…For example, our group quantified bile acids in mouse liver, bile, plasma and urine, as well as human plasma and urine using charcoal stripping after demonstrating similar extraction recoveries from stripped matrices and original matrices for two stable-labeled isotope internal standards [8,10]. Similarly, we used charcoal-stripped human saliva for the quantification of four lysophosphatidic acids in saliva and gingival cervical fluids [82].…”

“…Various approaches ( Figure 2) are followed to address the lack of blank matrices for the quantification of endogenous compounds by LC-MS/MS including, background subtraction [26-31], the standard addition method [24,25,[32][33][34][35][36][37][38][39][40][41][42][43][44], neat solutions [45][46][47][48][49][50][51][52], artificial matrices of biological fluids [23,, stripped matrices [8,10,11,[74][75][76][77][78][79][80][81][82][83][84][85][86][87][88][89][90][91], and surrogate analytes [66,[92][93][94][95][96][97]…”

A Major Human Oral Lysophosphatidic Acid Species, LPA 18:1, Regulates Novel Genes in Human Gingival Fibroblasts

“…LPA is present in all normal bodily fluids but contributes to pathology in different systems when levels are elevated 8 . The present authors have shown that LPAs 18:1, 18:0, and 16:0 are 10‐fold increased over normal (to pharmacologic levels) in the saliva and gingival crevicular fluid (GCF) of patients with moderate to severe periodontitis 9 …”

“…The instrument specifications, software and setting conditions, mobile phase, and method validation have been reported previously; LPA calibration curves were prepared in serum‐free media as described 9 …”

Lysophosphatidic Acid-Induced TWIST1 and Slug Expression in Oral Cancer Cell Invasion

“…is present in saliva, and becomes active in the tumor microenvironment 9) . The effects of LPA are mediated by several LPA receptors, which belong to the G protein-couple receptor class 10,11) .…”