Tatyana Karaskov

A novel method using hair for determining hormonal levels in wildlife

ormones influence behaviour, and are also influ-enced by behaviour. Monitoring their levels cantherefore provide insights into the mechanistic aspects ofbehaviour. In male mammals for example, elevated levelsof testosterone are associated with increased aggressionand dominance (Creel et al. 1993, 1997; Mazur & Booth1998) and in social mammals, levels of stress hormones(e.g. corticosterone, glucocorticoid and cortisol) areassociated with rank (Sapolsky 1985; Creel et al. 1996,1997). Research has associated hormone levels with dif-ferent behaviours such as sexual, reproductive, courtship,parental, aggressive and feeding behaviours. Comparativetools for hormonal analysis provide insights into evolu-tionary theories based on behavioural aspects, such asreproductive suppression and the ‘challenge hypothesis’(e.g. Creel et al. 1993).In field studies, hormones are usually extracted fromblood samples, or noninvasively from saliva, urine andfaeces (Creel et al. 1992; Cavigelli 1999; Hirschenhauseret al. 1999; von Engelhardt et al. 2000). Samples derivedfrom trapped or handled animals are problematicalbecause stress may alter blood and urine hormonal levels(Creel et al. 1992). Additional problems with bloodsamples are that they are not always available, theamount that can be taken at a given time is limited, andvarious safety and ethical issues exist. Furthermore, bloodand saliva must be transported cold or frozen, conditionsthat are sometimes difficult to obtain in the field (Yang etal. 1998). Urine and faeces samples are sometimes diffi-cult to obtain from free-ranging animals that cannot becontinuously observed, or from species that deposit incommon latrines.An alternative source for hormones may be found inhair, which can be collected noninvasively, and is alreadyused to extract DNA (Woodruff 1993; Morin et al. 1994),trace metals, naturally occurring compounds and drugs(Wheeler et al. 1998). Hair is safe, readily available, andeasy to store and transport. Hair sampling does notinvolve pain or possible infection, and the analysis isunaffected by the momentary stress of capture (Yang et al.1998). Hair analysis may allow one to monitor hormonalchanges over weeks or months (between moults; Maurelet al. 1986) by shaving off a patch of hair and resamplingthe newly grown hair. Hormonal hair analysis offersonly a long-term profile, however, and is not suitable formonitoring hourly or daily (short-term) fluctuations inhormonal levels. It provides the resolution needed forstudies of main behavioural trends, especially in stablehierarchical social systems. Hair has already been used todiagnose early pregnancy in cows by detection of proges-terone (Liu et al. 1988), to detect oestradiol and testoster-one in cattle (Gleixner & Meyer 1997) and anabolicsteroid and corticosteroid abuse in athletes (Bowers S Hold et al. 1999; Kintz et al. 1999; Cirimeleet al. 2000). In humans, the levels of steroid hormones inhair do not vary significantly between different regions ofthe scalp (Wheeler et al. 1998). Oestradiol, progesteroneand testosterone levels measured in healthy humanadults’ hair correlate significantly with the levelsmeasured in their serum (Yang et al. 1998).As an example of the utility of this method, we usedata from our long-term study on rock hyrax,

Comparison of meconium and neonatal hair analysis for detection of gestational exposure to drugs of abuse.

Background: Meconium and hair are two biological markers of in utero exposure to illicit drugs. Objective: To compare the sensitivity of the two tests for different drugs. Setting: Motherisk laboratory which tests in utero drug exposure in Toronto. Methods: Cocaine, benzoylecgonine, opiates, cannabis, benzodiazepines, methadone, and barbiturates were measured in pairs of hair and meconium samples from the same neonates. Results: Meconium was marginally more sensitive than neonatal hair for detection of cocaine and cannabis, possibly because it may detect second trimester exposure whereas hair grows only during the third trimester of pregnancy. There was a significant correlation between hair and meconium concentrations of cocaine, cannabis, and opiates. Conclusion: In cases of clinical suspicion and a negative neonatal urine test, both meconium and hair are effective biological markers of in utero illicit drug exposure. Meconium may be more sensitive, but neonatal hair is available for three months whereas meconium is available for only one or two days. In contrast, the use of meconium, being a discarded material, is more acceptable to some parents than hair testing, which entails cutting scalp hair from the newborn.

Fatty acid ethyl esters: a novel biologic marker for heavy in utero ethanol exposure: a case report.

The authors report testing the meconium of a newborn for the presence of FAEE. Meconium from a newborn of a woman who acknowledged drinking beer throughout pregnancy was tested. The authors also tested the meconiums of 3 newborns whose mothers did not drink at all while pregnant. The FAEE were extracted from the meconium samples using solid phase extraction (SPE), and were identified and quantitated by gas chromatography with flame ionization detection (FID). For assignment of retention times and determination of individual concentrations, authentic mixtures of FAEE were injected. The total FAEE concentration in the meconium of the alcohol-exposed infant was 13126 ng/g compared to a mean of 410 ng/g in the control meconiums. Also, in this case, palmitic, linoleic, and stearic ethyl esters were found in the alcohol-exposed infants meconium while they were not found in the unexposed infants meconium. In a parallel experiment, the authors spiked increasing amounts of ethyl alcohol (0-40mM) into the meconium from a newborn that was not exposed to ethanol in utero. The spiked samples were incubated for 4 hours at 37 degrees C and subsequently assayed for the presence of ethyl linoleate. In these experiments, they document for the first time that FAEE is produced in meconium. If confirmed by large studies, FAEE may become the first neonatal biologic marker for babies at risk for alcohol-related birth defects.

Clinical applications of hair testing for drugs of abuse--the Canadian experience.

During the last 2 decades there has been a substantial increase in illicit drug consumption in North America. It has been repeatedly shown that the personal history of drug use is far from being accurate. Fearing legal consequences and embarrassment of admitted illicit substance use, most users tend to deny or, to under-report illicit drug consumption. These facts have stressed an urgent need for a biological marker which does not lose its sensitivity within a few days after the end of exposure and which may yield a cumulative reflection of long term exposure to illicit drugs. Hair analysis has emerged as such a marker. A variety of illicit and medicinal compounds have been shown to be incorporated into hair including trace metals, barbiturates, amphetamines, opiates, phencyclidine, cocaine, nicotine and cannabis. Hair analysis for drugs of abuse provides long-term information on an individuals drug use; its window of detection is limited only by the length of the hair and typically, ranges from a week to several months. After establishing and validating several hair tests during the last decade, we have analyzed over 1000 hair samples for different drugs of abuse. We used RIA for screening and GC-MS for confirmation of positive results. The aim of this report is to illustrate the diagnostic usefulness of hair testing in different age groups (newborns, children, adults) and circumstances: (criminal cases, athletes, child custody cases, etc.).

Fetal exposure to alcohol as evidenced by fatty acid ethyl esters in meconium in the absence of maternal drinking history in pregnancy.

The detection of fatty acid ethyl esters (FAEE) in neonatal meconium has been proposed as a novel screening method for intrauterine exposure to alcohol. We investigated the potential use of meconium FAEE screening in a high-risk neonatal population in the absence of maternal drinking history. One hundred forty-two meconium samples of neonates suspected of intrauterine illicit substance exposure and referred to the Motherisk Laboratory were analyzed for the existence of drugs by enzyme-linked immunosorbent assay (ELISA) and FAEE by gas chromatography-flame ionization detection (GC-FID). A positive FAEE test was previously defined as a cumulative measurement of 7 individual FAEE ≥2 nmol/g. Seventy-one percent of the samples tested positive for at least 1 illicit drug, with cannabis being the most prevalent (52.3%). Fourteen percent of all samples tested positive for prenatal alcohol exposure, as evidenced by cumulative meconium FAEE ≥2 nmol/g. Ethyl oleate, linoleate, palmitate, and arachidonate were detected most often and at the highest levels. At least 3 individual FAEE were detected in 95% of all positive samples, and none could be identified by the use of 1 selected FAEE. Significantly elevated levels of FAEE above the baseline and the presence of multiple FAEE species in meconium are exclusive to neonates who have likely been exposed to excessive amounts of alcohol in utero. Babies born to mothers who are suspected to use illicit drugs in pregnancy are at elevated risk for exposure also to alcohol in utero. Meconium FAEE are emerging biologic markers that can potentially facilitate earlier diagnosis and intervention for less apparent forms of alcohol-related disabilities that cannot be confirmed in the absence of maternal drinking history.

Solid-phase microextraction for the detection of codeine, morphine and 6-monoacetylmorphine in human hair by gas chromatography-mass spectrometry.

INTRODUCTION Opiate hair analysis continues to prove difficult due to the scarcity of hair sample and low drug concentrations. For this reason, we developed a sensitive method utilizing headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) for the detection of three principle opiates; codeine, morphine, and 6-acetylmorphine. METHODS Experimental conditions for HS-SPME and GC-MS were systematically optimized to produce the sensitive analytical method reported. Briefly, opiates were extracted from adult hair with methanol under agitation. The methanolic extract was then decanted into SPME autosampler vials, where deuterated standards of each of the 3 opiates were added at a concentration of 2 ng/mg. Samples were dried under N(2), derivatized, and subjected to HS-SPME coupled with GC/MS for analysis. RESULTS Preliminary datum for this study indicates detection limits for these 3 opiates are superior to that reported in the literature; an LOQ of 0.01 ng/mg for morphine and 6-acetylmorphine and 0.005 ng/mg for codeine. Linearity was evident between 0.01 ng/mg and 5 ng/mg for each opiate, with R(2) above 0.992. The robustness of the method was demonstrated to be acceptable as inter-day and intra-day precision fell below 15% for each opiate analyzed. CONCLUSION Compared with conventional methods, this method of detection for opiates is fast, simple, and accurate, with the sensitivity and specificity required in forensic and clinical toxicology.

Methamphetamine detection in maternal and neonatal hair: implications for fetal safety

Background: Methamphetamine misuse is a serious health problem of epidemic proportions. Use of this drug, particularly during pregnancy, is difficult to ascertain. Sparse information is available on gestational exposure. Objectives: To quantify methamphetamine accumulation in hair, identify the use of methamphetamine with other drugs of abuse and characterise correlations between concentrations of methamphetamine in maternal and neonatal hair. Subjects and methods: Motherisk laboratory at the Hospital for Sick Children routinely carries out analysis of methamphetamine in hair. Mothers and infants with positive results for methamphetamine in hair were identified. Drugs present in hair were analysed by ELISA and positive results were confirmed by gas chromatgraphy/mass spectrometry. Results: 396 people positive for methamphetamine in their hair were identified from our database. Almost 85% of them were positive for at least one other drug of abuse, mostly cocaine. Eleven mother–baby pairs with hair positive for methamphetamine were identified. Methamphetamine levels in hair ranged between 0.13 and 51.97 ng/mg in the mothers and between 0 and 22.73 ng/mg in the neonates. Methamphetamine levels in mothers and neonates correlated significantly. One (9%) neonate was negative for methamphetamine even though the mother was positive. Conclusion: To our knowledge, this is the first report on fetal exposure to methamphetamine during pregnancy, showing transplacental transfer of the drug, with accumulation in fetal hair. Hair measurement for methamphetamine in neonates is a useful screening method to detect intra-uterine exposure to the drug. The data also indicate that positive exposure to methamphetamine strongly suggests that the person is a polydrug user, which may have important implications for fetal safety.

Pharmacokinetics of disappearance of cocaine from hair after discontinuation of drug use

UNLABELLED Methods that employ detection of drugs of abuse in hair are important for monitoring compliance with drug abstinence. Understanding the mechanisms and timeline of drug disappearance from hair is critical for clinical and forensic application of hair testing. We aimed to evaluate the kinetics of disappearance of cocaine and its metabolite, benzoylecgonine (BE), from hair after discontinuation of drug use. METHODS The Motherisk laboratory at the Hospital for Sick Children in Toronto routinely receives hair samples for toxicology analysis. Cocaine and BE hair results were obtained from the Motherisk Database for calculation of half-life of these compounds in hair. Subjects were included in the study if they had gradually decreasing concentrations of cocaine and/or BE in sequential hair samples, with higher levels in the 1-3 cm distal segments (i.e. earlier in time) and low or non-measurable levels in the segment closest to the scalp (i.e. closer to the date of sampling). Elimination half-life of cocaine and BE in hair was calculated using standard kinetics calculations. The study was anonymous, and received ethics approval by the Ethics Review Board of our institution. RESULTS 137 subjects met the inclusion criteria for the study. The median half-life of cocaine in hair was 1.5 months (95% CI 1.2-1.8) in females and 1.5 months (95% CI 1.1-1.8) in males. The median half-life of BE was 1.5 months (95% CI 1.1-2) in females and 1.5 months (95% CI 0.8-1.8) in males. Half lives of cocaine or BE were not statistically different between males and females (Mann-Whitney U-test; P=0.93 for cocaine, P=0.99 for BE). Half lives of cocaine and BE were strongly correlated (Spearman rank rho=0.73; P<0.001). CONCLUSION Cocaine and BE could be detected in hair of former drug users for several months after abstinence. The calculated half-life of over 1 month for cocaine implies that, assuming first order elimination, approximately 3-4 months have to pass for hair testing to become negative in the segment proximal to the scalp. This finding should be incorporated in interpreting compliance with abstinence of former drug users, and suggests that caution has to be exerted when evaluating potential breaches of abstinence.

Chronic Cocaine Exposure in a Toddler Revealed by Hair Test

Since then, hair assay has been widely used to detect intrauterine exposure to different substances, as well as exposure in adults. Although the length of hair available limits the detection window, this test can provide critical information during longer periods of time than a blood or urine test. We describe a case in which a hair test allowed diagnosis of chronic cocaine exposure in a toddler that was missed by an initial urine toxicology screen after an acute presentation.

Opioid detection in maternal and neonatal hair and meconium: characterization of an at-risk population and implications to fetal toxicology.

Identification of maternal opioid abuse in pregnancy is often difficult to ascertain in the absence of a reliable self-report. We aimed to characterize an at-risk neonatal population for opioid exposures as well as other drugs of abuse and alcohol. From June 2007 to January 2009, 563 neonatal hair and 1318 meconium specimens were assessed for opioids and were positive in 11.4% and 17.0%, respectively. Neonates testing positive for opioids in hair or meconium analysis were also more likely to test positive for other licit and illicit substances (odds ratiohair, 1.75; 95% confidence interval, 1.03-2.97; odds ratiomeconium, 1.61; 95% confidence interval, 1.16-2.22). Specifically, a positive neonatal hair test for opioids also predicted a positive result for oxycodone. In addition, a positive meconium test result for opioids was associated with positive results for cocaine, oxycodone, methadone, benzodiazepines, and fatty acid ethyl esters (alcohol). Finally, there was a significant correlation between maternal and neonatal hair test results for opioids (Spearman rank rho = 0.657, P = 0.03). Understanding the addiction profiles of these women may lead to better clinical and social management and may largely benefit an at-risk population.