AJAC  Vol.3 No.12 , December 2012
The Detection of 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol and Ethyl Glucuronide in Human Umbilical Cord
Abstract: In utero exposure to ethanol continues to be a significant public health issue and neonatal healthcare professionals are in need of objective means to identify exposed newborns. The aim of this study was to fully validate two methods for the detection of two direct alcohol biomarkers, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol (POPE) and ethyl glucuronide (EtG), in umbilical cord and apply the assays to a group of authentic specimens. The limits of detections were 2 and 1 ng/g for POPE and ETG and the limits of quantitation were 4 and 3 ng/g, respectively. Inter and intra-day precision and accuracy measurements were within 15%. The assays were applied to 308 authentic specimens where we detected POPE in five (1.6%) specimens and EtG in twelve (3.9%) specimens. The mean concentrations were 11.4 ng/g ± 9.4 ng/g and 127.2 ± 227.7 ng/g for POPE and EtG, respectively. This study suggested that umbilical cord was a suitable specimen type for the identification of newborns exposed to ethanol in the womb and the prevalence of POPE and EtG detected in umbilical cord were consistent with the prevalence of self-reported binge drinking reported by the National Birth Defect Prevention Study (NBDPS) and Behavioral Risk Factor Surveillance System (BRFSS). Further studies are required to fully describe the association between the observed concentrations of POPE and EtG in umbilical cord to the level of maternal consumption of ethanol.
Cite this paper: J. Jones, M. Jones, C. Plate and D. Lewis, "The Detection of 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol and Ethyl Glucuronide in Human Umbilical Cord," American Journal of Analytical Chemistry, Vol. 3 No. 12, 2012, pp. 800-810. doi: 10.4236/ajac.2012.312106.

[1]   Healthy People 2020, MICH-25, Retrieved October 8, 2011.

[2]   J. Bertrand, R. Floyd, M. Weber, M. O’Connor, E. Riley, K. Johnson, D. Cohen and the National Task Force on FAS/FAE, “Fetal Alcohol Syndrome: Guidelines for Referral and Diagnosis,” Centers for Disease Control and Prevention, Atlanta, 2004.

[3]   S. Gahagan, T. Sharpe, M. Brimacombe, Y. Fry-Johnson, R. Levine, M. Mengel, M. O’Connor, B. Paley, S. Adubato and G. Brenneman, “Pediatrician’s Knowledge, Training, and Experience in the Care of Children with Fetal Alcohol Syndrome,” Pediatrics, Vol. 118, No. 3, 2006, pp. e657-e668. doi:10.1542/peds.2005-0516

[4]   M. Ethen, T. Ramadhani, A. Scheuerle, M. Canfield, D. Wyszynski, C. Druschel, P. Romitti and the National Birth Defects Prevention Study, “Alcohol Consumption by Women Before and During Pregnancy,” Maternal and Child Health Journal, Vol. 13, No. 2, 2009, pp. 274-285. doi:10.1007/s10995-008-0328-2

[5]   C. Marchetta, C. Denny, L. Floyd, N. Cheal and J. Sniezek, “Alcohol Use and Binge Drinking among Women of Childbearing Age—United States, 2006-2010,” Morbidity and Mortality Weekly Report, Vol. 61, No. 28, 2012, pp. 534-538.

[6]   F. Del Boca and J. Darkes, “The Validity of Self Reports of Alcohol Consumption: State of the Science and Challenges for Research,” Addiction, Vol. 98, No. S2, 2003, pp. 1-12. doi:10.1046/j.1359-6357.2003.00586.x

[7]   S. Stewart, A. Reuben, W. Brzezinski, D. Koch, J. Basile, P. Randall and P. Miller, “Preliminary Evaluation of Phosphatidylethanol and Alcohol Consumption in Patients with Liver Disease and Hypertension,” Alcohol and Alcoholism, Vol. 44, No. 5, 2009, pp. 464-467. doi:10.1093/alcalc/agp039

[8]   P. Marques, S. Tippetts, J. Allen, M. Javors, C. Alling, M. Yegles, G. Pragst and F. Wurst, “Estimating Driver Risk Using Alcohol Biomarkers, Interlock Blood Alcohol Concentration Tests and Psychometric Assessments: Initial Descriptives,” Addiction, Vol. 105, 2009, pp. 226-239. doi:10.1111/j.1360-0443.2009.02738.x

[9]   R. Floyd, M. Sobell, M. Velasquez, K. Ingersoll, M. Nettleman, L. Sobell, P. Mullen and Project CHOICES Efficacy Study Group, “Preventing Alcohol-Exposed Pregnancies: A Randomized Controlled Trial,” American Journal of Preventive Medicine, Vol. 32, No. 1, 2007, pp. 1-10. doi:10.1016/j.amepre.2006.08.028

[10]   C. Moore, J. Jones, D. Lewis and K. Buchi, “Prevalence of Fatty Acid Ethyl Esters in Meconium Specimens,” Clinical Chemistry, Vol. 49, No. 1, 2003, pp. 133-136. doi:10.1373/49.1.133

[11]   C. Bearer, J. Jacobson, S. Jacobson, D. Barr, J. Croxfor, C. Molteno and A. Cwik, “Validation of a New Biomarker of Fatal Exposure to Alcohol,” The Journal of Pediatrics, Vol. 143, No. 4, 2003, pp. 463-469. doi:10.1067/S0022-3476(03)00442-6

[12]   D. Chan, J. Klein, T. Karaskov and G. Koren, “Fetal Exposure to Alcohol as Evidenced by Fatty Acid Ethyl Esters in Meconium in the Absence of Maternal Drinking History in Pregnancy,” Therapeutic Drug Monitoring, Vol. 26, No. 5, 2004, pp. 474-481. doi:10.1097/00007691-200410000-00003

[13]   E. Ostrea, J. Hernandez, D. Bielawski, J. Kan, G. Leonardo, M. Abela and R. Sokol, “Fatty Acid Ethyl Esters in Meconium: Are They Biomarkers of Fetal Alcohol Exposure and Effect?” Alcoholism: Clinical and Experimental Research, Vol. 30, No. 7, 2006, pp. 1152-1159. doi:10.1111/j.1530-0277.2006.00131.x

[14]   Y. Goh, J. Hutson, L. Lum, H. Roukema, J. Gareri, H. Lynn and G. Koren, “Rates of Fetal Alcohol Exposure Among Newborns in a High-Risk Obstetric Unit,” Alcohol, Vol. 44, No. 7-8, 2010, pp. 629-634. doi:10.1016/j.alcohol.2010.02.008

[15]   A. Bakdash, P. Burger, T. Goecke, P. Fasching, U. Reulback, S. Bleich and H. Kornhuber, “Quantification of Fatty Acid Ethyl Esters (FAEE) and Ethyl Glucuronide (EtG) in Meconium from Newborns for Detection of Alcohol Abuse in a Maternal Health Evaluation Study,” Analytical and Bioanalytical Chemistry, Vol. 396, No. 7, 2010, pp. 2469-2477. doi:10.1007/s00216-010-3474-5

[16]   R. Arendt, L. Singer, S. Minnes and A. Salvator, “Accuracy in Detecting Prenatal Drug Exposure,” Journal of Drug Issues, Vol. 29, No. 2, 1999, pp. 203-214.

[17]   E. Ostrea, D. Knapp, L. Tannenbaum, A. Ostrea, A. Romero, V. Salari and J. Ager, “Estimates of Illicit Drug Use during Pregnancy by Maternal Interview, Hair Analysis, and Meconium Analysis,” Pediatrics, Vol. 138, No. 3, 2001, pp. 344-348. doi:10.1067/mpd.2001.111429

[18]   B. Lester, M. ElSohly, L. Wright, V. Smeriglio, J. Verter, C. Bauer, S. Shankaran, H. Bada, C. Walls, M. Huestis, L. Finnegan and P. Maza, “The Maternal Lifestyle Study: Drug Use by Meconium Toxicology and Maternal Self-Report,” Pediatrics, Vol. 107, No. 2, 2001, pp. 309-317. doi:10.1542/peds.107.2.309

[19]   C. Derauf, A. Katz and D. Easa, “Agreement between Maternal Self-reported Ethanol Intake and Tobacco Use during Pregnancy and Meconium Assays for Fatty Acid Ethyl Esters and Cotinine,” American Journal Epidemiology, Vol. 158, No. 7, 2003, pp. 705-709. doi:10.1093/aje/kwg215

[20]   F. Eylera, M. Behnkea, K. Wobiea, C. Garvanb and I. Tebb, “Relative Ability of Biologic Specimens and Interviews to Detect Prenatal Cocaine Use,” Neurotoxicology and Teratology, Vol. 27, No. 4, 2005, pp. 677-687. doi:10.1016/

[21]   T. Gray, R. Magri, D. Shakleya and M. Huestis, “Meconium Nicotine and Metabolites by Liquid Chromatography-Tandem Mass Spectrometry: Differentiation of Passive and Nonexposure and Correlation with Neonatal Outcome Measures,” Clinical Chemistry, Vol. 54, No. 12, 2008, pp. 2018-2027. doi:10.1373/clinchem.2008.109173

[22]   T. Gray, R. Eiden, K. Leonard, G. Connors, S. Shisler and M. Huestis. “Identifying Prenatal Cannabis Exposure and Effects of Concurrent Tobacco Exposure on Neonatal Growth,” Clinical Chemistry, Vol. 56, No. 9, 2010, pp. 1442-1450. doi:10.1373/clinchem.2010.147876

[23]   D. Montgomery, C. Plate, S. Alder, M. Jones, J. Jones and R. Christensen, “Testing for Fetal Exposure to Illicit Drugs Using Umbilical Cord Tissue vs Meconium,” Journal of Perinatology, Vol. 26, No. 1, 2006, pp. 11-14. doi:10.1038/

[24]   D. Montgomery, C. Plate, M. Jones, J. Jones, R. Rios, K. Lambert, N. Schumtz and R. Christensen, “Using Umbilical Cord Tissue to Detect Fetal Exposure to Illicit Drugs: A Multicentered Study in Utah and New Jersey,” Journal of Perinatology, Vol. 28, No. 11, 2008, pp. 750-753. doi:10.1038/jp.2008.97

[25]   C. Moore, S. Brown, A. Negrusz, I. Tebbett, W. Meyer and L. Jain, “Determination of Cocaine and its Major Metabolite, Benzoylecgonine, in Amniotic Fluid, Umbilical Cord Blood, Umbilical Cord Tissue, and Neonatal Urine: A Case Study,” Journal of Analytical Toxicology, Vol. 17, No. 1, 1993, p. 62.

[26]   State v. McKnight, 352 S.C. 635, 576 S.E.2d 168 (2003).

[27]   McKnight v. State, No. 26484 (SC, 2008).

[28]   J. Jones, R. Rios, M. Jones, D. Lewis and C. Plate, “Determination of Amphetamine and Methamphetamine in Umbilical Cord Using Liquid Chromatography-Tandem Mass Spectrometry,” Journal of Chromatography B, Vol. 877, No. 27, 2009, pp. 3701-3706. doi:10.1016/j.jchromb.2009.09.021

[29]   A. Castro, M. Concheiro, D. Shakleya and M. Huestis, “Development and Validation of a Liquid Chromatography Mass Spectrometry Assay for the Simultaneous Quantification of Methadone, Cocaine, Opiates and Metabolites in Human Umbilical Cord,” Journal of Chromatography B, Vol. 877, No. 27, 2009, pp. 3065-3071. doi:10.1016/j.jchromb.2009.07.028

[30]   M. Conchiero, D. Shakleya and M. Huestis, “Simultaneous Quantification of Buprenorphine, Norbuprenorphine, Buprenorphine-Glucuronide and Norbuprenorphine-Glucuronide in Human Umbilical Cord by Liquid Chromatography Tandem Mass Spectrometry,” Forensic Science International, Vol. 188, No. 1, 2009, pp. 144-151. doi:10.1016/j.forsciint.2009.04.005

[31]   M. Conchiero, H. Jones, R. Johnson, R. Choo, D. Shakleya and M. Huestis, “Umbilical Cord Monitoring of In Utero Drug Exposure to Buprenorphine and Correlation with Maternal Dose and Neonatal Outcomes,” Journal of Analytical Toxicology, Vol. 34, No. 8, 2010, pp. 498-505.

[32]   H. Miguez, R. Magri, M. Suarez, H. Suarez, J. Jones and P. Putti, “Embarzo y Alcholizacion Social,” Acta Psiquiatrica y Psicologica de America Latina, Vol. 56, No. 3, 2010, pp. 163-167.

[33]   J. Jones, R. Magri, R. Rios, M. Jones and D. Lewis, “The Detection of Caffeine and Cotinine in Umbilical Cord Tissue Using Liquid Chromatography-Tandem Mass Spectrometry,” Analytical Methods, Vol. 3, No. 6, 2011, pp. 1310-1315. doi:10.1039/c0ay00636j

[34]   S. Marin, R. Christensen, V. Baer, C. Clark and G. Mc- Millin, “Nicotine and Metabolites in Paired Umbilical Cord Tissue and Meconium Specimens,” Therapeutic Drug Monitoring, Vol. 33, No. 1, 2011, pp. 80-85. doi:10.1097/FTD.0b013e3182055f14

[35]   L. Gustavsson and C. Alling, “Formation of Phosphatidylethanol in Rat Brain by Phospholipase D,” Biochemical and Biophysical Research Communications, Vol. 142, No. 3, 1987, pp. 958-963. doi:10.1016/0006-291X(87)91507-5

[36]   T. Gunnerson, A. Karlsson, P. Hansson, G. Johnson, C. Alling and G. Oddham, “Determination of Phosphatidylethanol in Blood from Alcoholic Males using High-Performance Liquid Chromatography and Evaporative Light Scattering or Electrospray Mass Spectrometric Detection,” Journal of Chromatography B, Vol. 705, No. 2, 1998, pp. 243-249. doi:10.1016/S0378-4347(97)00541-0

[37]   Helander and Y. Zheng, “Molecular Species of the Alcohol Biomarker Phosphatidylethanol in Human Blood Measured by LC-MS,” Clinical Chemistry, Vol. 55, No. 7, 2009, pp. 1395-1405. doi:10.1373/clinchem.2008.120923

[38]   A. Varga, P. Hansson, G. Johnson and C. Alling, “Normalization Rate and Cellular Localization of Phosphatidylethanol in Whole Blood from Chronic Alcoholics,” Clinica Chimica Acta, Vol. 299, No. 1-2, 2000, pp. 141-150. doi:10.1016/S0009-8981(00)00291-6

[39]   F. Wurst, C. Kempter, S. Seidl and A. Alt, “Ethyl Glucuronide—A Marker of Alcohol Consumption and a Relapse Marker with Clinical and Forensic Implication,” Alcohol and Alcoholism, Vol. 34, No. 1, 1999, pp. 71-77. doi:10.1093/alcalc/34.1.71

[40]   L. Morini, E. Marchei, M. Pellegrini, A. Groppi, C. Stramese, F. Vagnarelli, O. Garcia-Algar, R. Pacifici and S. Pichini, “Liquid Chromatography with Tandem Mass Spectrometric Detection for the Measurement of Ethyl Glucuronide and Ethyl Sulfate in Meconium: New Biomarkers of Gestational Ethanol Exposure,” Therapeutic Drug Monitoring, Vol. 30, No. 6, 2008, pp. 725-735. doi:10.1097/FTD.0b013e31818b2fd9

[41]   L. Morini, E. Marchei, F. Vagnarelli, O. Garcia-Algar, A. Groppi, L. Mastrobattista and S. Pichini, “Ethyl-Glucuronide and Ethyl-Sulfate in Meconium and Hair: Potential Biomarkers for Intrauterine Exposure to Ethanol,” Forensic Science International, Vol. 196, 2010, pp. 74-77. doi:10.1016/j.forsciint.2009.12.035

[42]   L. Morini, A. Groppi, E. Marchei, F. Vagnarelli, O. Garcia-Alga, P. Zuccaro and S. Pichini, “Population Baseline of Meconium Ethyl-Glucuronide and Ethyl-Sulfate Concentrations in Newborns of Nondrinking Women in 2 Mediterranean Cohorts,” Therapeutic Drug Monitoring, Vol. 32, No. 3, 2010, pp. 359-363. doi:10.1097/FTD.0b013e3181d5f14a

[43]   L. Morini, M. Falcon, S. Pichini, O. Garcia-Algar, P. Danesino, A. Groppi and A. Luna, “Ethyl-glucuronide and Ethyl-sulfate in Placental and Fetal Tissues by Liquid Chromatography Coupled with Tandem Mass Spectrometry,” Analytical Biochemistry, Vol. 418, No. 1, 2011, pp. 30-36. doi:10.1016/j.ab.2011.06.038

[44]   S. Pichini, L. Morini, E. Marchei, I. Palmi, M. Rotolo, F. Vagnarelli, O. Garcia-Algar, O. Vall and P. Zuccaro, “Ethylglucuronide and Ethylsulfate in Meconium to Assess Gestational Ethanol Exposure: Preliminary Results in Two Mediterranean Cohorts,” Canadian Journal of Clinical Pharmacology, Vol. 16, No. 2, 2009, pp. 370-375.

[45]   B. Pansky, “Dynamic Anatomy and Physiology,” Macmillan Publishing Company, New York, 1975, p. 619.

[46]   P. Williams, R. Warwick, M. Dyson and L. Bannister, Eds., “Gray’s Anatomy,” 37th Edition, Churchill Livingstone, New York, 1989, p. 143

[47]   C. Blakemore and S. Jennett, “The Oxford Companion to the Body, 1st Edition,” Oxford University Press, New York, 2001, p. 700.

[48]   Food and Drug Administration, Center for Drug Evaluation Research and Center for Veterinary Medicine, “Guidance for Industry: Bioanalytical Method Validation,” 2001.

[49]   Official journal of the European communities L, Vol. 221, 2002, 8.

[50]   B. Matuszewski, M. Constanzer and C. Chavez-Eng, “Strategies for the Assessment of Matrix Effect in Quantitative Bioanalytical Methods Based on HPLC-MS/MS,” Analytical Chemistry, Vol. 75, No. 13, 2003, pp. 3019- 3030. doi:10.1021/ac020361s

[51]   B. Matuszewski, “Standard Line Slopes as a Measure of a Relative Matrix Effect in Quantitative HPLC-MS Bioanalysis,” Journal of Chromatography B, Vol. 830, No. 2, 2006, pp. 293-300. doi:10.1016/j.jchromb.2005.11.009

[52]   A. Helander, I. Olsson and H. Dahl, “Postcollection Synthesis of Ethyl Glucuronide by Bacteria in Urine May Cause False Identification of Alcohol Consumption,” Clinical Chemistry, Vol. 53, No. 10, 2007, p. 1855. doi:10.1373/clinchem.2007.089482

[53]   J. Klein, T. Karaskov and G. Koren, “Fatty Acid Ethyl Esters: A Novel Biologic Marker for Heavy In Utero Ethanol Exposure: A Case Report,” Therapeutic Drug Monitoring, Vol. 21, No. 6, 1999, pp. 644-646. doi:10.1097/00007691-199912000-00011

[54]   S. Aradottir, K. Moller and C. Alling, “Phosphatidylethanol Formation and Degradation in Human and Rat Blood,” Alcohol and Alcoholism, Vol. 39, No. 1, 2004, pp. 8-13. doi:10.1093/alcalc/agh003

[55]   S. Aradottir, S. Seidl, F. Wurst, B. Jonnsson and C. Alling, “Phosphatidylethanol in Human Organs and Blood: A Study on Autopsy Material and Influences by Storage Conditions,” Alcoholism: Clinical & Experimental Research, Vol. 28, No. 11, 2004, pp. 1718-1723. doi:10.1097/01.ALC.0000145687.41646.E5

[56]   J. Jones, M. Jones, C. Plate and D. Lewis, “The Detection of 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol in Human Dried Blood Spots,” Analytical Methods, Vol. 3, No. 2011, pp. 1101-1106. doi:10.1039/c0ay00636j

[57]   Faller, B. Richter, M. Kluge, P. Koenig, H. Seitz, A, Thierauf, H. Gnann, M. Winkler, R. Mattern and G. Skopp, “LC-MS/MS Analysis of Phosphatidylethanol in Dried Blood Spots Versus Conventional Blood Specimens,” Analytical and Bioanalytical Chemistry, Vol. 401, No. 4, 2011, pp. 1163-1166. doi:10.1007/s00216-011-5221-y

[58]   A.U. Schmid, “Metabolites of Ethanol as markers of alcohol abuse: Glutathione consumption and methionine kinetics in humans,” Ph.D. Dissertation, University of Basel, Bern, 2004.

[59]   H. Schloegl, S. Dresen, K. Spaczynski, M. Stoertzel, F. Wurst and W. Weinmann, “Stability of Ethyl Glucuronide in Urine, Post-Mortem Tissue and Blood Samples,” International Journal of Legal Medicine, Vol. 120, No. 2, 2006, pp. 83-88. doi:10.1007/s00414-005-0012-7