Quantification of ethyl glucuronide, ethyl sulfate, nicotine, and its metabolites in human fetal liver and placenta

Abstract

Tobacco and alcohol use during pregnancy are serious public health concerns and result in adverse developmental outcomes. Identifying in utero exposure is often achieved through meconium analysis or via maternal self-report. In this study, we analyzed fetal liver and placenta to examine second trimester alcohol and smoking exposure. Methods A validated liquid chromatography-tandem mass spectrometry method for simultaneous analysis of nicotine and its metabolites and alcohol markers (ethyl glucuronide: EtG and ethyl sulfate: EtS) was employed to analyze 193 fetal liver and 48 placenta (n=47 paired) samples from electively-terminated pregnancies. Results EtG, EtS and nicotine markers' limits of detection were 0.7-20 ng/g in fetal samples. Ninety-eight fetal liver and 23 placenta samples were EtG/EtS-positive, while 137 liver and 25 placenta samples were positive for tobacco exposure. When both alcohol markers were present in samples, EtG/EtS ratios were 1.6-11.1 in 17 livers and 2.5-31.1 in 10 placentas. Median [range] summed tobacco marker concentrations were 422 [1.0-2776] and 154 [1.6-1621] ng/g in livers and placentas. Median EtG and nicotine marker concentrations were higher in liver than placenta in paired samples. Strong evidence of exposure occurred in 11 and 22 pairs, respectively, when both samples were positive for alcohol and/or tobacco markers. Conclusions These paired fetal liver and placenta alcohol and tobacco data provided a unique means for examining the effects of in utero exposure, a critical first step in selecting fetal samples for proteomic and RNA-sequencing studies that could provide mechanisms for adverse developmental outcomes.

Recent Developments in the Determination of Biomarkers of Tobacco Smoke Exposure in Biological Specimens: A Review

“…Prior to sample loading, 0.25% ammonium hydroxide in methanol was added to increase pH and Isolute SLE was used to proceed to nicotine and metabolites quantification with dichloromethane/isopropanol (95:5, v/v) as elution solvent. This strategy lead to a method capable of reaching the cut-offs observed in other studies [15]. Liquid-liquid microextraction technique made with chloroform was explored by Gallart-Mateu et al [90] for nicotine quantification in oral fluid by gas chromatography coupled to mass spectrometry (GC-MS) in comparison with IONSCAN-LS IMS with good recoveries (104%).…”

“…Both nicotine and cotinine, as well as their glucuronides can be detected and measured in biological fluids [7]. Other compounds are formed in the metabolization process, such as trans-3′-hydroxycotinine, norcotinine, nornicotine, and the glucuronized compounds, among others [12][13][14][15]. Cotinine has a half-life of about 18 h in the organism and persists in biological matrices due to its poor lipid solubility, facilitating its identification in several tissues and biological fluids, including hair, blood, oral fluid, urine, and breast milk, making it an efficient and widely used biomarker [16][17][18][19][20].…”

“…Both nicotine and cotinine, as well as their glucuronides can be detected and measured in biological fluids [ 7 ]. Other compounds are formed in the metabolization process, such as trans -3′-hydroxycotinine, norcotinine, nornicotine, and the glucuronized compounds, among others [ 12 , 13 , 14 , 15 ]. Figure 1 resumes the metabolic profile of nicotine.…”

“…Regarding the publications, it is possible to use other biological samples such as liver [ 15 ], semen [ 78 ], or nasal fluid [ 79 ] to determine these biomarkers.…”

“…For r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (PheT), a PAHs, quantification, a urine sample suffers enzymatic hydrolysis made with a solution of β-glucuronidase and arylsulfatase mix with 0.5 M sodium acetate buffer (pH5), being extracted with Strata SDB-L plates, using methanol/water (1:1, v / v ) as elution solvent [ 35 ]. An SLE extraction method for liver and placenta by Swortwood et al [ 15 ] used metal spheres to be able to crush both samples, then using 0.01% formic acid in methanol for homogenization. Prior to sample loading, 0.25% ammonium hydroxide in methanol was added to increase pH and Isolute SLE was used to proceed to nicotine and metabolites quantification with dichloromethane/isopropanol (95:5, v / v ) as elution solvent.…”