Hair Analysis Panel Discussion:
Section: Appendix C, Michael Kosnett

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Appendix C
Pre-Meeting Comments

Hair Analysis: Exploring the State of the Science

Michael Kosnett

June 4, 2001

The following are preliminary comments regarding some topics that constitute the charge to the panel. However, I am still in the process of reviewing some relevant studies and therefore may revise or amend this material in a subsequent submission.

Topic #1: Analytical methods

The key analytical methods currently used by clinical laboratories to measure trace elements in hair appear to be inductively coupled plasma atomic emission spectrometry, and inductively coupled plasma mass spectrometry (Miekeley et al, 1998; Seidel et al, 2001). Graphite furnace atomic absorption spectrometry has been used to measure arsenic in hair, with reported limits of detection of 0.005 to 0.01 µg/g (Rebel et al, 1998; Hewlett et al, 1995). Total and inorganic mercury in hair has been determined by cold vapor atomic absorption (Boischio and Cernichiari, 1998; NRC, 2000), and the difference between total and inorganic Hg yielded by this method has been used as a surrogate for the methyl mercury hair content. Methyl mercury in hair has also been determined directly by gas chromatography using a tritium foil electron capture detector (Smith et al, 1997). Selenium in hair has been measured fluorometrically after complexation with 2,3-diaminonaphthalene (Yoshinaga et al, 1990). The preceding methods appear to have generally required a hair specimen size on the order of 50 mg or more. Although commercial laboratories commonly measure the submitted hair sample in bulk, the methodology is sufficiently sensitive to allow investigators to yield segmental analysis (³ 1 cm) on bundles of hair for which information on the alignment and distance from the root has been preserved. Segmental analysis may potentially offer information on the temporal pattern of exposure to the element in question that is of value in epidemiological and forensic investigations.

Neutron activation analysis (NAA) has been used in forensic investigations and occasionally in epidemiological or clinical studies for the sensitive determination of certain trace elements in minute quantities of hair. For example, neutron activation analysis has been used to measure arsenic in 2 mm segments of an individual hair, each segment weighing approximately 3 µg (Smith, 1964; Curry and Pounds, 1977). NAA has also been used to measure the hair content of Zn, Au, Cu, Mn, Hg, Sb, and Th (Jervis, 1968; Cornelius, 1973). The distribution of mercury in 2 mm segments along the length of a single strand of hair may be determined by nondestructive x-ray fluorescence (Cox et al, 1989, cited by NRC, 2000). Proton induced x-ray emission has been used to measure the spatial distribution of multiple elements in 10 micron increments across axial cross section of a single shaft of hair (Cookson and Pilling, 1975; Hindmarsh et al, 1999).

A multitude of factors influence the quality control of laboratory hair analysis. These include the finite limitations of the assay method (ideal method recovery and precision), and the variability associated with within-run and day to day operation of the assay (actual method recovery and precision). Although not necessarily reflective of a systematic review of the literature, a few references may be cited as offering examples of operational precision in research investigations. Using NAA to measure 7 elements in a single specimen of hair, the coefficient of variation ranged from 5.92% in the case of Mn (mean concentration 1.65 ppm) to 15.7% in the case of Sb (mean concentration 0.18 ppm) (Cornelius, 1973). Wilhelm et al (1989) reported a day to day coefficient of variation of approximately 6% for atomic absorption measurement of Zn, Pb, Cu and Cd in hair. The issue of inter-laboratory variability of multi-element hair analysis for trace elements provided by commercial laboratories using ICP-AES and ICP-MS has recently been addressed by Miekeley et al (1998) and Seidel et al (2001), both of whom obtained widely discrepant results from split samples sent to 4 to 6 different commercial laboratories.

Topic #2: Factors Influencing the Interpretation of Analytical Results

One of the most fundamental factors impacting the potential utility of hair analysis as an exposure assessment tool in public health evaluations is the limited capacity of such measurements to distinguish external contamination from internal incorporation. In particular, multiple studies have noted that toxic metals may become incorporated into hair following external contact with metal containing dust, soil, water or hair care products. There is no reliable analytical approach that can distinguish this external contamination from elevations in hair metal content that result from metal ingestion or inhalation (Chittleborough, 1980). Although pre-analysis washing or rinsing methods are often used in an attempt to selectively remove external contamination, there is no standardized approach that has been shown to achieve the desired result.

The experience with arsenic, a toxic metalloid that is often encountered through environmental exposures, is a case in point. In vitro studies have demonstrated that hair incorporates appreciable amounts of arsenate and arsenite from aqueous solutions, and that the extent of absorption increases with duration of contact time and moderate decrements in pH (e.g. pH 3 to 5) (Atalla et al, 1965; Bate, 1966; Van den Berg et al, 1967; Fergusson et al, 1983). Adsorption of arsenic to hair may also be substantial following contact with arsenic containing dust (Atalla et al, 1965). The extent of adsorption may vary significantly along the length of a single hair (Maes and Pate, 1977). Adsorption-desorption experiments demonstrate that externally deposited arsenic cannot be completely removed from hair by a variety of washing and rinsing techniques (Smith, 1964; Atalla et al, 1965; Van den Berg et al, 1968). Moreover, washing may complicate interpretation further by partially removing arsenic present in hair as a result of internal incorporation (Atalla et al, 1965; Van den Berg et al, 1968; Young and Rice, 1944). Studies with other metals have reported similar findings with respect to adsorption onto hair from external contamination, and variable removal of both internal and externally derived traces by washing regimens (Chittleborough, 1980; Fergusson et al, 1983; Wilhelm et al, 1989).

The problems posed by this inability to distinguish external adsorption from internal incorporation places substantial constraints on what can be learned from the results of hair analysis for an environmental toxin where the suspected route of human exposure is via contact with contaminated dust, soil, airborne particulate, or tap water. Although these routes of exposure might result in ingestion or inhalation of an environmental toxin and its subsequent appearance in hair through incorporation at the hair follicle, they also create ample opportunity for the agent to become externally adsorbed onto hair via airborne deposition, hand to hair contact, or bathing. In such settings (which are probably characteristic of the majority of sites subject to ATSDR health assessments), the finding of elevated levels of a environmental toxin in the hair of a given subject or a study population is limited at best to establishing the potential for that subject or population to have come into contact with the agent in a manner that may have resulted in ingestion or inhalation. In addition to being a test of low specificity, the information on potential exposure gleaned from an elevated hair level in such settings is likely to be qualitative in nature. That is because with the notable exception of methyl mercury, quantitative information on the relationship between ingestion or inhalation of a environmental toxin and its concentration in hair is limited, and appears to be subject to considerable inter-subject and inter-population variability.

Again, an example derived from the measurement of arsenic in hair is instructive. Although several epidemiological studies have noted a correlation between levels of arsenic in hair and arsenic in dust, soil, or water, (e.g. Bencko and Symon, 1977; Hartwell et al, 1983; Valentine et al, 1979), the hair arsenic levels may not correlate with levels of arsenic in urine (Harrington et al, 1978; Hewlett et al, 1995). For example, Harrington et al (1978) studied hair and urine arsenic levels in a community near Fairbanks, Alaska, where the arsenic concentration of water obtained from domestic wells averaged 224 µg/L (range 1.0 to 2450 µg/L). A subset of subjects whose wells contained arsenic averaging 345 µg/L consumed only bottled water. Although they had relatively low arsenic levels in urine (average 43 µg/L), the arsenic concentration of their hair was high, averaging 5.74 ppm. Subjects consuming water from domestic wells with the lowest levels of arsenic (less than 50 µg/L in water) had hair arsenic concentrations averaging 0.46 ppm, and urine arsenic levels averaging 38 µg/L. Thus, the arsenic level in hair varied by 14-fold, despite similar levels of arsenic in urine. The authors noted the likely implication that the elevated hair arsenic levels were probably due to external contamination derived from bathing in, but not drinking, the high arsenic well water.

Topic #3 To what extent may hair analysis be used to predict adverse health outcomes? and Topic #5, Under what scenarios may hair analysis be appropriate for evaluating exposures to environmental contaminants?

From a medical standpoint, there appears to be no disease or illness caused by an environmental toxin for which there is a general medical consensus that the results of hair analysis would form the basis for specific medical treatment.

In the case of methyl mercury, segmental maternal hair analysis may have diagnostic value as a biomarker of fetal exposure to levels of this neurotoxin that are associated with a postnatal risk of adverse neurobehavioral development (NRC, 2000). Some data suggest that the level of hair methylmercury in children and adults may also be a biomarker of exposure associated with adverse effects on neurological function and other health endpoints (NRC, 2000). Because most contemporary exposure to methylmercury is confined to ingestion via seafood, there is little potential for high hair levels of methylmercury to be a result of external contamination. In most populations whose level of seafood ingestion is of a sufficient magnitude to pose a potential health risk from methylmercury, measurement of total mercury in hair may be an acceptable surrogate for measurement of methylmercury in hair.

In certain settings, segmental hair measurement of arsenic (and potentially other toxins such as thallium) may be of diagnostic and/or forensic value in identifying or confirming a high dose toxic exposure or poisoning that terminated months (but not years) in the past. For example, segmental analysis of a sufficiently long hair might help to confirm a suspicion that an episode or outbreak of severe gastroenteritis followed by peripheral neuropathy that occurred 8 to 10 months in the past was likely to have been the consequence of acute arsenic or thallium poisoning. Months after the exposure ended, levels of arsenic or thallium in the urine may have fallen to normal values, and high peak levels in the hair (or nails) may offer the only remaining confirmatory forensic evidence. It should be noted that although the hair measurements in such scenarios might conceivably be of value in confirming past poisoning, the epidemiological database on hair analysis is insufficient to use these measurements to predict the risk of latent diseases such as cancer.

Supplemental comments from Michael J. Kosnett, MD, MPH (submitted June 21, 2001)

A key factor to be addressed prior to ATSDR's use or interpretation of hair testing is the predictive value of a positive or negative test with respect to detecting an exposure and/or internally absorbed dose of a toxic substance of sufficient magnitude to be of pathological or public health significance.
One of the inherent limitations of hair analysis arises from the fact that hair represents a matrix that is in direct contact with the external environment and as such may be subject to greater contamination than other analytes traditionally used in biological monitoring, such as blood, urine, or even expired air.

Supplemental references submitted by Michael J. Kosnett, MD, MPH (June 21, 2001)

Atalla L, Silva CM, Lima FW. Activation Analysis of Arsenic in Human Hair—Some Observations on the Problems of External Contamination. Ann. Acad. Bras. Cien., 1965, 37:432-441.
Bate LC. Adsorption and Elution of Trace Elements on Human Hair. Int. J. Appl. Rad. Isot., 1966, 17:417-423.
Bencko V, Symon K. Health Aspects of Burning Coal with a High Arsenic Content. I. Arsenic in Hair, Urine, and Blood in Children Residing in a Polluted Area. Environ. Res., 1977, 13:378-385.
Chittleborough G. A Chemist's View of the Analysis of Human Hair for Trace Elements. Sci. Tot. Envir. 1980, 14:-75.
Cornelis R. Neutron Activation Analysis of Hair: Failure of a Mission. J. Radioanal. Chem., 1973, 15:305-316.
Cox C, Clarkson TW, Marsh DO, Amin-Zaki L, Tikriti S, and Myers GG. 1989. Dose-response analysis of infants prenatally exposed to methyl mercury: An application of a single compartment model to single-strand hair analysis. Environ. Res. 49(2):318-322.
Curry AS, Pounds CA. Arsenic in Hair. J. For. Sci. Soc., 1977, 17:37-44.
Ferguson JE, Holzbecher J, Ryan DE. The Sorption of Copper [II], Manganese [II], Zinc [II], and Arsenic [III] into Human Hair and Their Desorption. Sci. Tot. Envir., 1983, 26:121-135.
Gebel TW, Suchenwirth RHR, Bolten C et al. Human biomonitoring of arsenic and antimony in case of an elevated geogenic exposure. Environ Health Persp 1998; 106:33-39
Harrington JM, Middaugh JP, Morse DL et al. A Survey of a Population Exposed to High Concentrations of Arsenic in Well Water in Fairbanks, Alaska. Am. J. Epid., 1978, 108(5):377-385.
Hartwell TD, Handy RW, Harris BS et al. Heavy Metal Exposure in Populations Living Around Zinc and Copper Smelters. Arch. Environ. Health, 1983, 38(5):284-295.
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Houtman, JPW, de Bruin M., de Goeij JIM: Arsenic Levels of Human Hair as an Indicator for Environmental Exposure, in Nuclear Activation Techniques in the Life Sciences, Vienna, IAEA, 1978, pp. 599-614.
Jervis RE, Present Status of Activation Analysis Applications in Criminalistics. Isot. Rad. Tech., 1968, 6(1):57-70.
Leslie ACD, Smith H. Napoleon Bonaparte's Exposure to Arsenic During 1816. Arch. Toxicol., 1978, 41:163-167.
Maes D, Pate BD.: The Absorption of Arsenic into Single Human Head Hairs. J. For. Sci., 1977. 22:89-99.
National Research Council. Toxicological Effects of Methyl Mercury. National Academy Press: Washington, DC, 2000
Pounds CA, Pearson EF, Turner TD. Arsenic in Fingernails. J. For. Sci. Soc., 1979, 19:165-173.
Smith H. The Interpretation of the Arsenic Content of Human Hair. J. For. Sci. Soc., 1964, 4:192-199.
Valentine JL, King HK, Spivey G. Arsenic Levels in Human Blood, Urine, and Hair in Response to Exposure via Drinking Water. Environ. Res., 1979, 20:24-32.

Van den Berg AJ, de Geoij JJM, Houtman JPW et al. Arsenic Content of Human Hair After Washing as Determined by Activation Analysis, in DeVoe JR [ed.] Modern Trends in Activation Analysis, Vol. I. Washington, DC: NBS, 1968, pp. 272-282.
Wilhelm M, Ohnesorge FK, Lombeck I et al. Uptake of aluminum, cadmium, copper, lead, and zinc by human scalp hair and elution of the absorbed dose. J Anal Toxicol 1989; 13:17-21
Young EG, Rice FAH. On the Occurrence of Arsenic in Human Hair and Its Medicological Significance. J. Lab. Clin. Med., 1944, 29:439-446.