Mechanisms of drug incorporation into hair

Forensic Science lnternational 63 (1993) 19-29

Forensic Science

Intermtiil

G.L. Henderson

Department of Medical Pharmacology and Toxicology. School of Medicine, University of Cuiiforniu.

Davis, CA 95616. USA

(Accepted IO December 1992)

Abstract

The model generally proposed to explain the incorporation of drugs into hair is one in which drugs enter hair only by passive diffusion from the blood stream into the growing cells at the base of the hair follicle. However, this model may be over-simplified. More recent ex- perimental findings suggest that drugs may enter hair from multiple sites, via multiple mechanisms, and at various times during the hair growth cycle. A more complex model is pro- posed in which drugs and metabolites are incorporated into hair during formation of the hair shaft (via diffusion from blood to the actively growing follicle), after formation (via secretions of the apocrine and sebaceous glands), and after hair has emerged from the skin (from the external environment). Further, drugs can be transferred to hair from multiple body compart- ments or pools located in tissues surrounding the hair follicle. These mechanisms could also be drug-specific. A more precise understanding of the mechanisms involved in the incorpora- tion of drugs into hair is critical for forensic scientists in order to interpret the results of hair analysis properly.

Key words: Hair analysis; Drugs; Mechanisms; Drug testing

1. Introduction

Over the last few decades there has been a steady accumulation of experimental data on the analysis of hair for trace elements and drugs. These data demonstrate rather convincingly that drugs and elements found in the body can be found in hair. However, the precise mechanism or mechanisms involved in the incorporation of chemicals into hair or the factors influencing incorporation have yet to be determined.

0379-0738/93/$06.00 0 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0379-0738(93)01397-A

20 G.L. Henderson/Forensic Sci. Inl. 63 (1993) 19-29

The mechanism generally proposed for drug incorporation into hair is one in which chemicals present in the blood move via passive diffusion into the rapidly growing cells of the hair follicle. As the cells die and fuse to form hair strands, the drug becomes trapped in this extremely stable structure. This mechanism seems intuitively reasonable since the base of the hair follicle is surrounded by a dense network of capillaries.

It has been suggested that passive diffusion may be augmented by drug binding to intracellular components of the hair cells such as the hair pigment, melanin [ 1,2]. This is an especially attractive mechanism for drugs like amphetamine and metham- phetamine which are chemically similar to tyrosine and DOPA, the precursors of melanin. However, this is probably not an important mechanism since drugs are incorporated into the hair of albino animals, which does not contain melanin [3]. Also, when melanin is present in hair, only a small amount of the drug found in hair is in the melanin fraction [ 1,2]. Another augmenting mechanism proposed is the bin- ding of drugs with sulfhydryl-containing amino acids present in hair. There is an abundance of amino acids such as cystine in hair which form cross-linking S-S bonds and stabilize the protein fiber network. Drugs diffusing into hair cells could be bound in this way. This mechanism seems more attractive, however, for divalent cations which can readily form stable directed covalent bonds.

2. Simple passive transfer model for drug incorporation into hair

Fig. 1 shows a diagram of the simplest model proposed for the incorporation of drugs into hair. In this model, drugs move by passive diffusion from the bloodstream into the growing hair cells at the base of the follicle and then become tightly bound in the interior of the hair shaft during subsequent keratogenesis. In this model, drug incorporation into hair is dependent on the drug concentration in blood which, in turn, is dependent on the dose of drug ingested.

This model also forms the scientific basis for segmental analysis in which suc- cessive hair segments are analyzed to determine the time-course of drug use. Because hair is assumed to grow at a constant rate, the position of drugs along the hair shaft can be correlated with the time the drug was present in the bloodstream. This has led to the claim that drug levels in hair reflect a permanent record of the drug content of the body during the growing phase of the hair and therefore segmental analysis can provide a ‘calendar’ of drug use for an individual. However, as techniques for hair analysis of drugs have become more accurate and precise, newer experimental findings suggest that the simple passive transfer model may be oversimplified or may not be correct.

2.1. Dose-concentration studies For some heavy metals, there are numerous reports in the forensic literature in

which heavy metal poisoning is accompanied by high levels of these elements in hair. For example, high levels of mercury were found in the hair of Iraqi peasants who ingested grain seed treated with a mercurial fungicide [4]. Similarly, a direct relation- ship was found between mercury levels in hair and the consumption of mercury- contaminated fish [5] and elevated lead levels have been found in the hair of children

G.L. Henderson/Forensic Sci. Int. 63 (1993) 19-29 21

Hair Shaft

skin Surface __________-----

Permanent Hair

Keratogenous Zone

____I

Zone of

Hair Synthesis

4 Blood

Fig. 1. Diagram of the simple passive diffusion model for drug incorporation into hair showing drugs transferring only from blood to the growing hair cells in the bulb.

with clinical signs of chronic lead poisoning [6]. However, the evidence for other elements is less convincing. There appears to be a poor correlation between the con- centrations of zinc in scalp or beard hair and the amount of zinc administered [7]. In controlled feeding studies with experimental animals, there was not a good cor- relation between zinc levels in rat hair and in plasma [8].

The poor correlation between the body burden of some elements and their resulting levels in hair has been attributed to inter-laboratory differences in methodology, and external contamination from air, water and cosmetic hair treatments [9]. Others have proposed a ‘multiple pool model’ in which elements are exchanged (perhaps through active transfer) between several pools in the body via multiple equilibria and steady states [7]. The exact form of the pool could vary with the element under consideration and a change in the exposure to the element is ultimately reflected in a change in hair concentration only after transfer from pool to pool via multiple equilibria and only after new steady-states have been establish- ed. As yet, this pool model has not been verified experimentally. Because of the large variability found in experimental data and the lack of a unifying theory describing trace metal incorporation into hair, the use of hair as a ‘biomarker’ for exposure to heavy metals and trace elements has not been universally accepted.

The body of literature on hair analysis drugs of abuse is considerably smaller than that for metallic elements, but it is increasing steadily. Baumgartner and coworkers

22 G. L. Henderson /Forensic Sri. Inr. 63 (1993) 19-29

[lo- 151 have reported a positive correlation between subject’s self-reported drug use history and the concentrations of either heroin, marijuana, cocaine, or PCP in their hair. However, these studies compared immunoassay data with self-reported drug intake. More recent studies have used specific methods like GC-MS and some have shown a positive correlation between dose and amount of drug in hair. Nakahara et al. [16] found a good correlation between the dose of methoxyamphetamine (a model compound for methamphetamine) and the concentration of drug in the hair of 5 subjects. Also, the drug moved along the hair shaft at a rate of 0.40-0.46 mm/day which is consistent with values reported for scalp hair growth. Similarly, Cone et al. [17] found a positive correlation between the dose of opiates and the resulting concentration in beard hair; however, only 2 subjects were involved.

On the other hand, Puschel et al. [18] analyzed hair obtained from carcinoma patients receiving therapeutic doses of morphine, and found a poor correlation between the dose and drug concentration in hair. In fact, the highest concentrations were found in the hair of those subjects who received the lowest dose. In controlled dose studies conducted in our laboratory, we found a relatively poor correlation between the dose of cocaine administered and the concentration in hair. In these studies, approximately 24 subjects were administered deuterated cocaine intraven- ously and a sensitive GC-MS method was used to quantitate drug and metabolites in their hair, blood, urine, and sweat. The data, shown in Fig. 2, suggest that, in general, cocaine concentrations in hair do increase with increasing doses; however, the inter-subject variability is so great that it is difficult to estimate the amount of drug administered based on the drug concentration in hair. Further, the variability could not be explained by differences in the plasma pharmacokinetics of the subjects. There were the expected intersubject differences in pharmacokinetic parameters such as peak plasma concentration, area under the curve, clearance and elimination rate constants; however, these differences were relatively small and not sufficient to ex- plain the very large differences found in the resulting drug concentrations found in hair. Thus, to date a clear correlation between drug intake and drug concentration in hair, as predicted by the passive diffusion model, has not been firmly established for all drugs.

2.2. Drug-metabolite ratios Perhaps the greatest challenge to the passive transfer model is the relatively recent

finding of very high parent drug to metabolite ratios in hair. Goldberger et al. [19] have found highly metabolically labile drugs like heroin and short-lived metabolites like 6-monoacetylmorphine present in hair. Similarly, cocaine has been identified as the primary analyte in the hair of all individuals ingesting cocaine. Surprisingly, even though cocaine has a very short plasma half-life, it is present in hair at concentra- tions approximately B-fold greater than its primary metabolite, benzoylecgonine, and at approximately IO-fold higher concentrations than ecgonine methyl ester [20-221. Even when cocaine is consumed daily and the plasma BE concentrations are orders of magnitude higher than those of the parent drug, cocaine is still the predominant analyte found in hair [23-241. These findings are difficult to explain by the passive transfer model.

G.L. Henderson /Forensic Sci. ht. 63 (1993) 19-29 23

5

4

3

2

1

0

I 8 o= IV

I= IN

0 0 0

I 0 n

Ii!. Ym 0 1 2 3 4 5

Dose (mg/kg)

Fig. 2. Correlation between dose of deuterated cocaine administered and amount of drug found in hair. Open circles represent intravenous administration and dark squares represent intranasal administration.

Intranasal administration doses are corrected for bioavailability, calculated to be 30%.

2.3. Time course of drug incorporation The use of sectional analysis to determine drug use patterns is based on 2 assump-

tions: (i) hair grows at a constant rate of approximately 1 cm/month, and (ii) there is no diffusion of drug along the hair shaft. Some investigators have demonstrated that the presence of drug along the hair shaft does correspond to the time of drug ingestion [ 16,171. However, there is also experimental evidence that the distribution of drugs or elements along the hair shaft does not always correlate well with the time of exposure. Hair obtained from a cocaine overdose victim who survived 24 days was found to contain high cocaine concentrations distributed over segments correspon- ding to 2.5 month’s growth [25]. Similarly, we found that when a single dose of deuterated cocaine was administered to human volunteers the position of d5- cocaine along the hair shaft did not always correlate with the time of drug ingestion. Table 1 shows the results of segmental analysis from 2 subjects. Scalp hair from Sub- ject A obtained at 1 and 2 months after drug administration had the d5-cocaine confined to segments corresponding to l-2 month’s growth. Given the variability

24 G. L. Hrnderson / Forensic Sci. Int. 63 i I9931 I Y-29

Table I

Segmental analysis of hair samples from two subjects following a single dose of deuterated cocaine”

Segment Subject A

‘r/-Cocaine dose = 0.6 mgikg i.v.

I 2 3 4 5

1 mo 0.19 0 0

2 mo 0.13 0.13 0 0

3 mo 0 0 0 0.10

Segment Subject B

‘d-cocaine dose = I.2 mg/kg

I 2 3 4 5

I mo 27 day 0.1 0.44 I .07 0.60

2mo2lday 0 0.1 I 0.65 2.53 0.53

“Values shown are expressed as ng/mg hair. Each segment was I cm, measured from the scalp end, and

was analyzed by CC-MS with limit of quantitation of 0.1 ngimg.

in sample collecting and sectioning, these results are those predicted by the simple passive diffusion model. However, similar samples from Subject B had d5-cocaine distributed over four l-cm segments, which corresponds to 4 month’s growth. In other subjects, we were able to detect &-cocaine in hair as early as 8 h post-drug (data not shown). These latter findings are difficult to reconcile using a simple passive transfer model.

3. A more complex multi-compartment model

In the view of this author, there is a growing body of experimental data to suggest that a more complex model may be necessary to explain how drugs get into hair. Any new model would have to explain findings such as why drug and metabolite ratios in hair are quite different from those found in blood, why the time for drugs to appear in hair varies considerably between subjects, why the distribution of drug along the hair shaft is not always consistent with that predicted from hair growth rates, and why drug and metabolite concentrations in hair differ markedly between subjects receiving the same dose.

It is possible that drugs are incorporated into hair via a more complex model in which drugs are transferred from the body to hair from multiple pools and during various times in the hair’s life cycle. Such a model is shown diagramatically in Fig. 3. In this model, it is suggested that drugs may be incorporated into hair: (i) from the blood during formation; (ii) from sweat and sebum after formation; and (iii) from the external environment after formation and after the hair has emerged from the skin. In addition, drugs and metabolites may be transferred from multiple body compartments or from pools in the tissues that surround the hair follicle as well.

G.L. Henderson/Forensic Sci. ht. 63 (1993) 19-29 25

Hair Shaft

Skin Surface ________________.

Permanent Hair

---mm

Keratogenous Zone

-----

Zone of

Hair Synthesis

External Contamination

Apocrlne and

Sebaceous Glands I- B

I

0

0

d

Fig. 3. Diagram of a proposed multi-compartment model for drug incorporation into hair showing drug transferring into hair during 3 phases of growth (during formation, after formation, and after exiting from skin) and from multiple sources (blood, glandular secretions, skin tissue, and external contamination).

3.1. Transfer of drugs to hair via sweat, sebaceous and apocrine gland secretions Nearly all drugs of abuse including alcohol [26], amphetamines [27], cocaine [28],

PCP [29], and methadone [30] have been found in sweat, often in concentrations greater than in blood. Studies on the time-course of excretion of cocaine in sweat have been conducted in our laboratory. Fig. 4 shows the concentrations of d5- cocaine and d5-BE in the sweat of a subject who received a single 2-mg/kg intra- nasal dose of deuterated cocaine. Cocaine is the primary analyte and can be detected in very significant levels (greater than 100 ng/ml) for up to 72 h after a single intra- nasal dose of drug. The values shown in this figure are typical of those found in the 6 other subjects studied. In another study, we administered 2 mg/kg d5-cocaine intranasally. Two hours later, the subjects held drug free, control hair in their hand for 30 min. These hair samples were analyzed by CC-MS and found to contain d5- cocaine in significant levels (range 0.28-58.49 ng/mg before washing, data not shown). After washing, the cocaine concentrations in hair ranged from 0 (drug was not detected in 2 of 7 samples) to 11.55 ng/mg. Both the high concentrations of drug in hair and the variability between subjects were surprising. These data suggest that

26 G. L. Henderson /Forensic Sci. ht. 63 ( 1993) 19-29

A 5d-Cocaine

0 5d-BE

LAA- 1 24 48 72

Time After Drug VW Fig. 4. Concentration of ‘d-cocaine and ‘d-BE (benzoylecgonine) in the sweat of a subject who received

a single 2 mg/kg dose of ‘d-cocaine intranasally.

for some individuals, sweat and sebum may be important vehicles for the transfer of drugs like cocaine to hair.

We were not able to separate sweat from sebaceous secretions in our studies and thus do not know the relative importance of sweat versus sebum as vehicles for cocaine excretion. Nevertheless, individual variability in these secretions could explain, in part, the variability in hair drug concentrations in subjects receiving the same dose. Also, excretion in sweat may be why the drug is sometimes dispersed over a large area of the hair shaft. Finally, because drugs transferred via these secretions are transferred to hair after it has formed, they may be bound less tightly and thus more easily removed by washing. If glandular secretions are significant routes for drug incorporation, then interlaboratory variability in washing and extraction pro- cedures could result in variability in their findings.

3.2. Transfer of drugs to hair from the external environment External contamination from air, water, and cosmetic hair treatments has been

suggested a a source for some of the trace elements present in hair and a possible reason for the difficulty in establishing acceptable or baseline concentrations for trace elements in hair. External contamination could be a potential route of entry into hair for drugs that are smoked such as amphetamine, cocaine, heroin, and mari- juana, In fact, hair analysis cannot distinguish cigarette smokers from non-smokers

G.L. Henderson/Forensic Sri. Int. 63 (1993) 19-29 ?I

because of external contamination from secondary smoke [31]. At present there is debate over the probability of evidentiary false positive hair tests resulting from ex- ternal contamination. Although there is general agreement that hair exposed to co- caine vapor will adsorb very large amounts of cocaine, there is not good agreement as to how effective various washing procedures are in removing this drug [22,32].

Another possible mechanism for entry of drugs into hair from the external en- vironment is by intradermal transfer to hair. This could be a possible mechanism for very lipid-soluble drugs. For example, when an ethanolic solution of radiolabeled THC was applied to the skin of rats, very little of the drug was absorbed into the systemic circulation. Instead the drug was found concentrated in the stratum cor- neum, upper epidermis, and over the shaft and epithelium of hair follicles [33]. In fact, the autoradiographs suggested the main route of THC entry into the rat skin was the pilosebaceous system. Thus, skin could act as a trap for some lipophilic drugs and retain the drug for subsequent transfer to the hair follicle.

3.3. Transfer of drugs to hair from deep compartments in skin One possible explanation for the unusual elimination of cocaine and its

metabolites in hair is that transfer of drug to hair occurs via a ‘deep compartment’ or via binding sites in the skin. Skin has been shown to act as a reservoir or sink for certain drugs, especially lipophilic drugs [34-351. More specifically it is 2 layers of this organ, the epidermis and the hypodermis, or subcutaneous layer, that can ac- cumulate drugs. Studies have shown that when drugs penetrate from the external en- vironment across the skin they are retained by the lipid layers of the stratum corneum (the outer most layer of the epidermis) and are released very slowly into the bloodstream. Conversely, when drugs move from the systemic circulation to the surface of the skin they are retained primarily by the adipose tissue of the hypoder- mis and secondarily by the stratum corneum [34]. Anatomically, there is good evidence for deep compartments in the skin. Both the lipid rich stratum corneum and the hypodermis are in intimate contact with the hair follicle and its associated glands and the capillaries surrounding the hair bulb (and the sweat glands) could maintain an equilibrium between these structures and any pool or deep compartment in the adjacent tissues. Interestingly, studies with radiolabeled chemicals have shown that their incorporation into hair may occur primarily at the suprabulbar level rather than at the papilla, the site of rapid cell growth [36]. However, the pharmacokinetics of skin are quite complex and lipid solubility seems to be only one of many factors which determine a drug’s mobility through the various layers of skin tissue [37]. Finally, although the binding of drugs to melanin present in hair does not seem to be an important mechanism for drug incorporation, drug binding to melanin or to melanin binding sites in skin has not been evaluated. The potential for drug-melanin binding in skin to produce interindividual differences in drug uptake into hair is obvious.

4. Conclusions

In conclusion, although it is generally assumed that drugs diffuse into the hair roots from the blood stream and are incorporated into the protein structure of the hair matrix, there is experimental data to suggest that drugs may enter hair from

28 G. L. Henderson I’ Forensic Sci. Int. 63 (1993) 19-29

other sites. A more precise understanding of these mechanisms is of more than academic interest. It is critical for the proper interpretation of hair analysis results. For example, drugs entering hair after it has formed could be more easily removed by washing; thus, there could be considerable inter-subject variability due to dif- ferences in hair hygiene and considerable inter-laboratory variation depending upon their sample preparation techniques. The rate of sweating and the amount of apocrine and sebaceous gland secretions vary considerably between individuals and could result in considerable inter-subject variability in the amount of drug in the hair of subjects receiving the same dose of drug. If drugs are transferred to hair via glan- dular secretions, then the movement of drugs along the hair shaft may be different from that predicted by hair growth rates; that is, drugs could appear in hair shortly after ingestion and could spread along the hair shaft by wicking action. More should be known about these mechanisms and the factors influencing drug incorporation into hair before forensic scientists attempt to make precise correlations between the concentration and location of drugs found in hair and the drug use history of an in- dividual.

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