Comparison of N-methyl-2-pyrrolidone (NMP) and the "date rape" drug GHB: behavioral toxicology in the mouse model.
Adjuvants, Anesthesia
/ toxicity
Animals
Dose-Response Relationship, Drug
Hypnotics and Sedatives
/ toxicity
Illicit Drugs
/ toxicity
Locomotion
/ drug effects
Male
Mice
Mice, Inbred ICR
Models, Animal
Psychomotor Performance
/ drug effects
Pyrrolidinones
/ toxicity
Rape
Reflex, Startle
/ drug effects
Respiratory Insufficiency
/ chemically induced
Sodium Oxybate
/ toxicity
Cardiorespiratory changes
Date-rape drug
Drug-facilitated crimes
Drug-facilitated sexual assault
GHB
Knock out drugs
NMP
New psychoactive substances
Sedative hypnotics
Journal
Psychopharmacology
ISSN: 1432-2072
Titre abrégé: Psychopharmacology (Berl)
Pays: Germany
ID NLM: 7608025
Informations de publication
Date de publication:
Aug 2021
Aug 2021
Historique:
received:
03
11
2020
accepted:
12
04
2021
pubmed:
22
4
2021
medline:
27
7
2021
entrez:
21
4
2021
Statut:
ppublish
Résumé
N-methyl-2-pyrrolidone (NMP) and γ-hydroxybutyrate acid (GHB) are synthetic solvents detected in the recreational drug market. GHB has sedative/hypnotic properties and is used for criminal purposes to compromise reaction ability and commit drug-facilitated sexual assaults and other crimes. NMP is a strong solubilizing solvent that has been used alone or mixed with GHB in case of abuse and robberies. The aim of this experimental study is to compare the acute pharmaco-toxicological effects of NMP and GHB on neurological signs (myoclonia, convulsions), sensorimotor (visual, acoustic, and overall tactile) responses, righting reflex, thermoregulation, and motor activity (bar, drag, and accelerod test) in CD-1 male mice. Moreover, since cardiorespiratory depression is one of the main adverse effects related to GHB intake, we investigated the effect of NMP and GHB on cardiorespiratory changes (heart rate, breath rate, oxygen saturation, and pulse distension) in mice. The present study demonstrates that NMP inhibited sensorimotor and motor responses and induced cardiorespiratory depression, with a lower potency and efficacy compared to GHB. These results suggest that NMP can hardly be used alone as a substance to perpetrate sexual assault or robberies.
Identifiants
pubmed: 33881584
doi: 10.1007/s00213-021-05852-5
pii: 10.1007/s00213-021-05852-5
doi:
Substances chimiques
Adjuvants, Anesthesia
0
Hypnotics and Sedatives
0
Illicit Drugs
0
Pyrrolidinones
0
Sodium Oxybate
7G33012534
N-methylpyrrolidone
JR9CE63FPM
Types de publication
Comparative Study
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
2275-2295Informations de copyright
© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.
Références
Al-Samarraie MS, Karinen R, Mørland J, Stokke Opdal M (2010) Blood GHB concentrations and results of medical examinations in 25 car drivers in Norway. Eur J Clin Pharmacol 66(10):987–998. https://doi.org/10.1007/s00228-010-0870-x
doi: 10.1007/s00228-010-0870-x
pubmed: 20717656
Andresen H, Sprys N, Schmoldt A, Mueller A, Iwersen-Bergmann S (2010) Gamma-hydroxybutyrate in urine and serum: additional data supporting current cut-off recommendations. Forensic Sci Int 200(1–3):93–99. https://doi.org/10.1016/j.forsciint.2010.03.035
doi: 10.1016/j.forsciint.2010.03.035
Ansell JM, Fowler JA (1988) The acute oral toxicity and primary ocular and dermal irritation of selected N-alkyl-2-pyrrolidones. Food Chem Toxicol 26(5):475–479. https://doi.org/10.1016/0278-6915(88)90060-9
doi: 10.1016/0278-6915(88)90060-9
pubmed: 3391472
Bay T, Eghorn LF, Klein AB, Wellendorph P (2014) GHB receptor targets in the CNS: focus on high-affinity binding sites. Biochem Pharmacol 87(2):220–228. https://doi.org/10.1016/j.bcp.2013.10.028
doi: 10.1016/j.bcp.2013.10.028
pubmed: 24269284
Bilel S, Tirri M, Arfè R, Stopponi S, Soverchia L, Ciccocioppo R, Frisoni P, Strano-Rossi S, Miliano C, De-Giorgio F, Serpelloni G, Fantinati A, De Luca MA, Neri M, Marti M (2019) Pharmacological and behavioral effects of the synthetic cannabinoid AKB48 in rats. Front Neurosci 13:1163. https://doi.org/10.3389/fnins.2019.01163
doi: 10.3389/fnins.2019.01163
pubmed: 31736697
pmcid: 6831561
Bilel S, Azevedo NJ, Arfè R, Tirri M, Gregori A, Serpelloni G, De-Giorgio F, Frisoni P, Neri M, Calò G, Marti M (2020) In vitro and in vivo pharmacological characterization of the synthetic opioid MT-45. Neuropharmacology 171:108110. https://doi.org/10.1016/j.neuropharm.2020.108110
doi: 10.1016/j.neuropharm.2020.108110
pubmed: 32344007
Bosman IJ, Lusthof KJ (2003) Forensic cases involving the use of GHB in The Netherlands. Forensic Sci Int 133(1–2):17–21. https://doi.org/10.1016/s0379-0738(03)00044-6
doi: 10.1016/s0379-0738(03)00044-6
pubmed: 12742684
Callery PS, Geelhaar LA, Stogniew M (1978) 2-Pyrrolidinone—a cyclization product of gamma-aminobutyric acid detected in mouse brain. Biochem Pharmacol 27(16):2061–2063. https://doi.org/10.1016/0006-2952(78)90068-0
doi: 10.1016/0006-2952(78)90068-0
pubmed: 718729
Callery PS, Stogniew M, Geelhaar LA (1979) Detection of the in vivo conversion of 2-pyrrolidinone to gamma-aminobutyric acid in mouse brain. Biomed Mass Spectrom 6(1):23–26. https://doi.org/10.1002/bms.1200060106
doi: 10.1002/bms.1200060106
pubmed: 427258
Canazza I, Ossato A, Trapella C, Fantinati A, De Luca MA, Margiani G, Vincenzi F, Rimondo C, Di Rosa F, Gregori A, Varani K, Borea PA, Serpelloni G, Marti M (2016) Effect of the novel synthetic cannabinoids AKB48 and 5F-AKB48 on “tetrad”, sensorimotor, neurological and neurochemical responses in mice. In vitro and in vivo pharmacological studies. Psychopharmacology 233(21–22):3685–3709. https://doi.org/10.1007/s00213-016-4402-y
doi: 10.1007/s00213-016-4402-y
pubmed: 27527584
Carai MA, Lobina C, Maccioni P, Cabras C, Colombo G, Gessa GL (2008) Gamma-aminobutyric acidB (GABAB)-receptor mediation of different in vivo effects of gamma-butyrolactone. J Pharmacol Sci 106(2):199–207. https://doi.org/10.1254/jphs.fp0071487
doi: 10.1254/jphs.fp0071487
pubmed: 18270475
Carnerup MA, Saillenfait AM, Jönsson BA (2005) Concentrations of N-methyl-2-pyrrolidone (NMP) and its metabolites in plasma and urine following oral administration of NMP to rats. Food Chem Toxicol 43(9):1441–1447. https://doi.org/10.1016/j.fct.2005.04.007
doi: 10.1016/j.fct.2005.04.007
pubmed: 15951091
Carter LP, Flores LR, Wu H, Chen W, Unzeitig AW, Coop A, France CP (2003) The role of GABAB receptors in the discriminative stimulus effects of gamma-hydroxybutyrate in rats: time course and antagonism studies. J Pharmacol Exp Ther 305(2):668–674. https://doi.org/10.1124/jpet.102.047860
doi: 10.1124/jpet.102.047860
pubmed: 12606639
Carter LP, Chen W, Wu H, Mehta AK, Hernandez RJ, Ticku MK, Coop A, Koek W, France CP (2005) Comparison of the behavioral effects of gamma-hydroxybutyric acid (GHB) and its 4-methyl-substituted analog, gamma-hydroxyvaleric acid (GHV). Drug Alcohol Depend 78(1):91–99. https://doi.org/10.1016/j.drugalcdep.2004.10.002
doi: 10.1016/j.drugalcdep.2004.10.002
pubmed: 15769562
Centola C, Giorgetti A, Zaami S, Giorgetti R (2018) Effects of GHB on psychomotor and driving performance. Curr Drug Metab 19(13):1065–1072. https://doi.org/10.2174/1389200219666180124113802
doi: 10.2174/1389200219666180124113802
pubmed: 29366411
Chanimov M, Bahar M, Cohen ML, Brenner R, Koifman I, Grinshpon Y (1999) Spinal anaesthesia with gamma hydroxybutyrate. A study in a rat model. Eur J Anaesthesiol 16(5):330–338. https://doi.org/10.1046/j.1365-2346.1999.00481.x
doi: 10.1046/j.1365-2346.1999.00481.x
pubmed: 10390669
Chin RL, Sporer KA, Cullison B, Dyer JE, Wu TD (1998) Clinical course of gamma-hydroxybutyrate overdose. Ann Emerg Med 31(6):716–722
doi: 10.1016/S0196-0644(98)70230-6
CICAD (2001) Concise International Chemical Assessment Document 35. N-Methyl-2-pyrrolidone. World Health Organization, Geneva
De-Giorgio F, Vetrugno G, Galluzzo F (2011) Droghe da stupro (DFSA): gestione clinica delle vittime e risvolti processuali tra consenso al prelievo di materiali biologici ed obbligo di denuncia da parte del sanitario. Diritto penale e processo, 12, 1505–1512 https://shop.wki.it/periodici/diritto-penale-e-processo-s13058/
De-Giorgio F, Bilel S, Tirri M, Arfè R, Trapella C, Camuto C, Foti F, Frisoni P, Neri M, Botrè F, Marti M (2020) Methiopropamine and its acute behavioral effects in mice: is there a gray zone in new psychoactive substances users? Int J Legal Med. https://doi.org/10.1007/s00414-020-02302-3 . Advance online publication
doi: 10.1007/s00414-020-02302-3
pubmed: 33219398
pmcid: 8036180
EMCDDA (2002) Report on the risk assessment of GHB in the framework of the joint action on new synthetic drugs, Lisbon. https://www.emcdda.europa.eu/publications/risk-assessment/ghb_en
Fantinati A, Ossato A, Bianco S, Canazza I, De Giorgio F, Trapella C, Marti M (2017) 1-cyclohexyl-x-methoxybenzene derivatives, novel psychoactive substances seized on the internet market Synthesis and in vivo pharmacological studies in mice. Hum Psychopharmacol 32(3). https://doi.org/10.1002/hup.2560
Feigenbaum JJ, Howard SG (1996) Naloxone reverses the inhibitory effect of gamma-hydroxybutyrate on central DA release in vivo in awake animals: a microdialysis study. Neurosci Lett 218(1):5–8. https://doi.org/10.1016/0304-3940(96)13032-9
doi: 10.1016/0304-3940(96)13032-9
pubmed: 8939467
Franks NP (2006) Molecular targets underlying general anaesthesia. Br J Pharmacol 147(Suppl 1):S72–S81. https://doi.org/10.1038/sj.bjp.0706441
doi: 10.1038/sj.bjp.0706441
pubmed: 16402123
pmcid: 1760740
Gobaille S, Schmidt C, Cupo A, Herbrecht F, Maitre M (1994) Characterization of methionine-enkephalin release in the rat striatum by in vivo dialysis: effects of gamma-hydroxybutyrate on cellular and extracellular methionine-enkephalin levels. Neuroscience 60(3):637–648. https://doi.org/10.1016/0306-4522(94)90492-8
doi: 10.1016/0306-4522(94)90492-8
pubmed: 7936191
Gould GG, Mehta AK, Frazer A, Ticku MK (2003) Quantitative autoradiographic analysis of the new radioligand [(3)H](2E)-(5-hydroxy-5,7,8,9-tetrahydro-6H-benzo[a][7]annulen-6-ylidene) ethanoic acid ([(3)H]NCS-382) at gamma-hydroxybutyric acid (GHB) binding sites in rat brain. Brain Res 979(1–2):51–56. https://doi.org/10.1016/s0006-8993(03)02865-8
doi: 10.1016/s0006-8993(03)02865-8
pubmed: 12850570
Hechler V, Gobaille S, Maitre M (1992) Selective distribution pattern of gamma-hydroxybutyrate receptors in the rat forebrain and midbrain as revealed by quantitative autoradiography. Brain Res 572(1–2):345–348. https://doi.org/10.1016/0006-8993(92)90498-x
doi: 10.1016/0006-8993(92)90498-x
pubmed: 1319274
Jones AW, Kugelberg FC, Holmgren A, Ahlner J (2008) Occurrence of ethanol and other drugs in blood and urine specimens from female victims of alleged sexual assault. Forensic Sci Int 181(1–3):40–46. https://doi.org/10.1016/j.forsciint.2008.08.010
doi: 10.1016/j.forsciint.2008.08.010
pubmed: 18922656
Jouyban A, Fakhree MA, Shayanfar A (2010) Review of pharmaceutical applications of N-methyl-2-pyrrolidone. J Pharm Pharmaceut Sci 13(4):524–535. https://doi.org/10.18433/j3p306
doi: 10.18433/j3p306
Kaupmann K, Cryan JF, Wellendorph P, Mombereau C, Sansig G, Klebs K, Schmutz M, Froestl W, van der Putten H, Mosbacher J, Bräuner-Osborne H, Waldmeier P, Bettler B (2003) Specific gamma-hydroxybutyrate-binding sites but loss of pharmacological effects of gamma-hydroxybutyrate in GABA(B)(1)-deficient mice. Eur J Neurosci 18(10):2722–2730. https://doi.org/10.1111/j.1460-9568.2003.03013.x
doi: 10.1111/j.1460-9568.2003.03013.x
pubmed: 14656321
Kietzerow J, Otto B, Wilke N, Rohde H, Iwersen-Bergmann S, Andresen-Streichert H (2020) The challenge of post-mortem GHB analysis: storage conditions and specimen types are both important. Int J Legal Med 134(1):205–215. https://doi.org/10.1007/s00414-019-02150-w
doi: 10.1007/s00414-019-02150-w
pubmed: 31598775
Kim H, Lee DH, Go A, Park M, Choe S, In S, Kim E, Lee H, Shin KH, Han E (2019) Differentiation of endogenous and exogenous γ-Hydroxybutyrate in rat and human urine by GC/C/IRMS. Int J Legal Med 133(6):1785–1794. https://doi.org/10.1007/s00414-019-02052-x
doi: 10.1007/s00414-019-02052-x
pubmed: 30937594
Koch M (1999) The neurobiology of startle. ProgNeurobiol 59(2):107–128
Laborit H (1964) SODIUM 4-HYDROXYBUTYRATE. Int J Neuropharmacol 3:433–451. https://doi.org/10.1016/0028-3908(64)90074-7
doi: 10.1016/0028-3908(64)90074-7
pubmed: 14334876
Lasoń W, Przewłocka B, Przewłocki R (1983) The effect of gamma-hydroxybutyrate and anticonvulsants on opioid peptide content in the rat brain. Life Sci 33(Suppl 1):599–602. https://doi.org/10.1016/0024-3205(83)90574-x
doi: 10.1016/0024-3205(83)90574-x
pubmed: 6141502
LeBeau M, Mozayani A (2001) Drug-facilitated sexual assault: a forensic handbook. Academic, San Diego. 326pp.
LeBeau M, Andollo W, Hearn WL, Baselt R, Cone E, Finkle B, Fraser D, Jenkins A, Mayer J, Negrusz A, Poklis A, Walls HC, Raymon L, Robertson M, Saady J (1999) Recommendations for toxicological investigations of drug-facilitated sexual assaults. J Forensic Sci 44(1):227–230
doi: 10.1520/JFS14442J
Ludes B, Geraut A, Väli M, Cusack D, Ferrara D, Keller E, Mangin P, Vieira DN (2018) Guidelines examination of victims of sexual assault harmonization of forensic and medico-legal examination of persons. Int J Legal Med 132(6):1671–1674. https://doi.org/10.1007/s00414-018-1791-y
doi: 10.1007/s00414-018-1791-y
pubmed: 29468380
Lukasiewicz PD, Eggers ED, Sagdullaev BT, McCall MA (2004) GABAC receptor-mediated inhibition in the retina. Vision Res 44(28):3289–3296. https://doi.org/10.1016/j.visres.2004.07.023
doi: 10.1016/j.visres.2004.07.023
pubmed: 15535996
Maitre M (1997) The gamma-hydroxybutyratesignalling system in brain: organization and functional implications. Prog Neurobiol 51(3):337–361. https://doi.org/10.1016/s0301-0082(96)00064-0
doi: 10.1016/s0301-0082(96)00064-0
pubmed: 9089792
Malek DE, Malley LA, Slone TW, Elliott GS, Kennedy GL, Mellert W, Deckardt K, Gembardt C, Hildebrand B, Murphy SR, Bower DB, Wright GA (1997) Repeated dose toxicity study (28 days) in rats and mice with N-methylpyrrolidone (NMP). Drug Chem Toxicol 20(1–2):63–77. https://doi.org/10.3109/01480549709011079
doi: 10.3109/01480549709011079
pubmed: 9183563
Mamelak M (1989) Gammahydroxybutyrate: an endogenous regulator of energy metabolism. Neurosci Biobehav Rev 13(4):187–198. https://doi.org/10.1016/s0149-7634(89)80053-3
doi: 10.1016/s0149-7634(89)80053-3
pubmed: 2691926
Mamelak M, Escriu JM, Stokan O (1977) The effects of gamma-hydroxybutyrate on sleep. Biol Psychiat 12(2):273–288
pubmed: 192353
Marinetti L, Montgomery MA (2010) The use of GHB to facilitate sexual assault. Forensic Sci Rev 22(1):41–59
pubmed: 26242455
Marinetti LJ, Leavell BJ, Jones CM, Hepler BR, Isenschmid DS, Commissaris RL (2012) Gamma butyrolactone (GBL) and gamma valerolactone (GVL): similarities and differences in their effects on the acoustic startle reflex and the conditioned enhancement of startle in the rat. Pharmacol Biochem Behav 101(4):602–608. https://doi.org/10.1016/j.pbb.2012.01.023
doi: 10.1016/j.pbb.2012.01.023
pubmed: 22349589
Morse BL, Vijay N, Morris ME (2012) γ-Hydroxybutyrate (GHB)-induced respiratory depression: combined receptor-transporter inhibition therapy for treatment in GHB overdose. Mol Pharmacol 82(2):226–235. https://doi.org/10.1124/mol.112.078154
doi: 10.1124/mol.112.078154
pubmed: 22561075
pmcid: 3400846
National Early Warning System (2013) National action plan for the prevention of distribution of New Psychoactive Substances and demand on the internet. http://www.politicheantidroga.gov.it/media/1292/312_altro.pdf
Ossato A, Vigolo A, Trapella C, Seri C, Rimondo C, Serpelloni G, Marti M (2015) JWH-018 impairs sensorimotor functions in mice. Neuroscience 300:174–188. https://doi.org/10.1016/j.neuroscience.2015.05.021
doi: 10.1016/j.neuroscience.2015.05.021
pubmed: 25987201
Ossato A, Bilel S, Gregori A, Talarico A, Trapella C, Gaudio RM, De-Giorgio F, Tagliaro F, Neri M, Fattore L, Marti M (2018) Neurological, sensorimotor and cardiorespiratory alterations induced by methoxetamine, ketamine and phencyclidine in mice. Neuropharmacology 141:167–180. https://doi.org/10.1016/j.neuropharm.2018.08.017
doi: 10.1016/j.neuropharm.2018.08.017
pubmed: 30165078
Quéva C, Bremner-Danielsen M, Edlund A, Ekstrand AJ, Elg S, Erickson S, Johansson T, Lehmann A, Mattsson JP (2003) Effects of GABA agonists on body temperature regulation in GABA(B(1))-/- mice. Br J Pharmacol 140(2):315–322. https://doi.org/10.1038/sj.bjp.0705447
doi: 10.1038/sj.bjp.0705447
pubmed: 12970075
pmcid: 1574040
Salomone A, Gerace E, Di Corcia D, Martra G, Petrarulo M, Vincenti M (2012) Hair analysis of drugs involved in drug-facilitated sexual assault and detection of zolpidem in a suspected case. Int J Legal Med 126(3):451–459. https://doi.org/10.1007/s00414-011-0597-y
doi: 10.1007/s00414-011-0597-y
pubmed: 21751027
SCCS (2011) Scientific Committee on Consumer Safety: opinion on N-Methyl-2-pyrrolidone(NMP). https://ec.europa.eu/health/sites/health/files/scientific_committees/consumer_safety/docs/sccs_o_050.pdf
Schröck A, Hari Y, König S, Auwärter V, Schürch S, Weinmann W (2014) Pharmacokinetics of GHB and detection window in serum and urine after single uptake of a low dose of GBL - an experiment with two volunteers. Drug Test Anal 6(4):363–366. https://doi.org/10.1002/dta.1498
doi: 10.1002/dta.1498
pubmed: 23733593
Scott-Ham M, Burton FC (2005) Toxicological findings in cases of alleged drug-facilitated sexual assault in the United Kingdom over a 3-year period. J Clin Forensic Med 12(4):175–186. https://doi.org/10.1016/j.jcfm.2005.03.009
doi: 10.1016/j.jcfm.2005.03.009
pubmed: 16054005
Snead OC 3rd, Bearden LJ (1980) Naloxone overcomes the dopaminergic, EEG, and behavioral effects of gamma-hydroxybutyrate. Neurology 30(8):832–838. https://doi.org/10.1212/wnl.30.8.832
doi: 10.1212/wnl.30.8.832
pubmed: 7191065
Snead OC 3rd, Yu RK, Huttenlocher PR (1976) Gamma hydroxybutyrate. Correlation of serum and cerebrospinal fluid levels with electroencephalographic and behavioral effects. Neurology 26(1):51–56. https://doi.org/10.1212/wnl.26.1.51
doi: 10.1212/wnl.26.1.51
pubmed: 942770
Stephens BG, Baselt RC (1994) Driving under the influence of GHB? J Anal Toxicol 18(6):357–358. https://doi.org/10.1093/jat/18.6.357
doi: 10.1093/jat/18.6.357
pubmed: 7823545
U.S. Department of Justice, Drug Enforcement Administration, Office of Forensic Sciences, Microgram Bulletin. Vol XXXVII, No 3 March 2004; N-Methylpyrrolidinone in a gamma-hydroxybutyrate solution in Copley, Ohio. http://www.justice.gov/dea/pr/micrograms/2004/mg0304.pdf
United Nation Office on Drugs and Crime, Guidelines for the Forensic Analysis of Drug Facilitating Sexual Assault, DFSA, and other Criminal Acts (2011) https://www.unodc.org/documents/scientific/forensic_analys_of_drugs_facilitating_sexual_assault_and_other_criminal_acts.pdf
Van Amsterdam JG, Brunt TM, McMaster MT, Niesink R, van Noorden MS, van den Brink W (2012) Cognitieveschade door intensiefgebruiken overdoses van GHB [Cognitive impairment due to intensive use and overdoses of gammahydroxybutyric acid (GHB)]. Tijdschr Psychiatr 54(12):1001–1010
pubmed: 23250641
Vigolo A, Ossato A, Trapella C, Vincenzi F, Rimondo C, Seri C, Varani K, Serpelloni G, Marti M (2015) Novel halogenated derivates of JWH-018: behavioral and binding studies in mice. Neuropharmacology 95:68–82. https://doi.org/10.1016/j.neuropharm.2015.02.008
doi: 10.1016/j.neuropharm.2015.02.008
pubmed: 25769232
WHO (2012) Gamma-hydroxybutyric acid (GHB). Critical Review Report-Expert Committee on Drug Dependence Thirty-fifth Meeting Hammamet, Tunisia. https://www.who.int/medicines/areas/quality_safety/4.1GHBcritical_review.pdf
Wu Y, Ali S, Ahmadian G, Liu CC, Wang YT, Gibson KM, Calver AR, Francis J, Pangalos MN, Carter Snead O 3rd (2004) Gamma-hydroxybutyric acid (GHB) and gamma-aminobutyric acidB receptor (GABABR) binding sites are distinctive from one another: molecular evidence. Neuropharmacology 47(8):1146–1156. https://doi.org/10.1016/j.neuropharm.2004.08.019
doi: 10.1016/j.neuropharm.2004.08.019
pubmed: 15567424