The biologic bases for using hair and nail for analyses of trace elements

The Science of the Total Environment, 7 (1977) 71-89 © Elsevier Scientific Publishing Company, Amsterdam - - Printed in Belgium

THE BIOLOGIC BASES FOR USING HAIR AND NAIL FOR ANALYSES OF TRACE ELEMENTS*

HOWARD C. HOPPS

Department of Pathology, School oJ'Medicine, University of Missouri, Columbia, Mo. (U.S.A.) (Received April 5th, 1976)

ABSTRACT

Sampling of human beings for trace element content can be a complex and costly procedure. The use of hair and nails simplifies the process and it is for this reason that such samples are extensively used. The primary consideration, however, is not how easily the samples can be collected nor even how accurately trace elements can be measured in hair and nails; rather, it is what do the values mean, i.e., to what extent does the measured level reflect the concentration and/or activity of the element in other parts of the body? The structure and histogenesis of hair and nails are discussed in relation to mechanisms by which trace elements are incorporated into these tissues. Problems of contamination in vivo are also considered. A r6sum6 of current literature is presented, considering the trace elements that are appropriate for measurement in samples of human hair and nail and some of the problems involved in analysis.

INTRODUCTION

Schoenheimer4~ described body tissues as being in a state of dynamic flux. Hair and nail are exceptions. They are formed in a relatively short time, after which the finished structure is expelled from the skin surface to become isolated from the body's continuing metabolic activities. The endogenous components of a hair reflect only those metabolic events that occurred during the relatively short time of its formation----ordinarily several days. Moreover, the hard, relatively im* This is an extension of the paper presented at the 8th Annual Con.lerence on Trace Substances in Environmental Health, sponsored by the University of Missouri, Columbia (12 June 1974), which subsequently appeared in the Proceedings of that Trade Substam'es Cot~ference, Edited by Delbert D. Hemphill. It is reprinted with permission of the University of Missouri, Columbia.

71

permeable keratinous outer structure seals in the hair's constituents, holding them in place for a very much longer time than is true for most other tissues. Nail is comparable to hair in these respects, but the time of its formation is longer. Some observers have stated or implied that the trace element content of hair does not adequately reflect the tissue stores 34.42 but since the various tissue stores do not necessarily correlate with each other, use of the term "tissue stores" in this sense is confusing. Should one attempt to correlate hair and nail levels with blood, or with liver, or with kidney, or with brain or with bone? An impressive body of literature supports the view that trace element content of hair and nail reflects body intake, however, from which one can conclude that hair and nail are suitable samples for evaluating body stores 14,16,t7'20'29`37,3s'49 Accepting the view that the trace element content of hair and nails does reflect body content, it is clear that there is considerable variation according to age, sex, race and geographic l o c a t i o n 14'27'28'37'38''z. Moreover, other factors also significantly affect trace element content: the part of the body from which hair is removed, the part of the hair that is analyzed (especially its length and distance from the follicle) and the extent and amount of exposure of hair to exogenous materials--particularly shampoos, dyes and medications 2,14.39. STRUCTURE AND HISTOGENESIS OF HAIR AND NAIL

Hairs are thread-like epithelial fibers formed from a cluster of matrix cells that comprise the soft bulb-shaped follicle located in the dermis. During the growth phase of their cycle these matrix cells display intense metabolic activity, producing 0.2 to 0.5 mm of hair per day. At this rate, the period during which the developing hair is exposed to the metabolic milieu of the matrix cells, including circulating blood and lymph and extracellular fluids, is but several days. Then as the extruding hair approaches the skin surface, its outer layer become hardened and relatively impermeable, locking in, so to speak, the metabolic products accumulated during the period of the hair's formation. Each hair follicle, representing a skin appendage, is actually a miniature organ. In addition to the matrix, with its distal portion connected to dermal tissue and its proximal portion connected to the epidermis, the follicle has a connective tissue component that includes smooth muscle (the arrectores pilorum) and glandular components: a sebaceous gland, and in the axillary pubic and perineal areas of the body an apocrine gland. Although the production of hair is the most evidertt function of the hair follicle, glandular secretions are also important products. The follicle's internal roof sheath is an extension of the matrix and is intimately associated with the hair for approximately 2/3 of the distance from the bulb to the skin surface. It "... provides a strong link or connection between the newly formed hair and the living cells of the external root sheath until the hair is keratinized" (Stralle48). The external root sheath, on the other hand, is an extension of the epidermis and its function is mainly mechanical. It lines the upper 72

part of the canal through which the hair emerges to the skin surface and helps to hold the hair in place during the follicle's resting stage. The diameter of human hairs varies considerably with the region of the body. There is also quite a variation among hairs from the same region among different individuals and to lesser extent within the same region of the same individual. Sims and Knollmeyer44 have made a careful study of the size of scalp hairs in a group of young adult males. Diameters ranged from 0.05 to 0.125 ram, the median being slightly less than 1/10 mm (0.09). Key structures of the developing and fully formed hair are shown in Figs. 1--4. The sebaceous and sweat glands, which are intimately associated with the hair, are discussed under the heading Sources of trace elements in hair. The formation of nail has much in common with that of hair despite the fact that the nail is a plate-like structure, not a thread, and that the nail is in physical contact with body tissues (the nail bed) for a considerable time after its formation. (That part of the nail which overlies the nail bed is fully keratinized and virtually isolated from it in the metabolic sense; the nail slides over this bed in the course of its extrusive growth.) Major structures and their relationships are shown in Fig. 5. From a general viewpoint the formation of feathers is also analogous to that of hairs47; thus one could expect feathers to reflect unusual levels of expo-

Fig. I. Diagram of skin to show the generalized anatomy of a hair follicle and associated skin structuras. Reproduced by permission from A Textbook of Histology, by J. C. Fineny and E. V. Cowdry. published by Lea and Febiger.

73

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Fig. 2. Diagram of a hair follicle,showing the distribution of soft and hard keratin and the kcratogenous zone in which hard keratin is produced. (Based on C. P. Leblond, Ann. N.Y. Acad. Sci.,53 (1951) 464.) Reproduced by permission from Ham's Histology, J. B. Lippincott Co., Philadelphia.

sure to trace elements, and there is evidence for this view. Hanson and Jones have determined levels of Mg, Mn, Si, Al and B in feathers from wild geese and were able to relate differences among two populations of geese to their habitat2L But Jones et al. were unable to correlate differences in K, Ca, Na and Mg values from feathers of pheasants in two geographic regions of Illinois with levels found locally in soil and in several species of plants that served as major food sources 26. KERAT1NIZATION

The hardening of hair and nails (keratinization) is not completely understood although much is known about it. Jarrett's discussion 24 is an important basic reference and much of the material in this paragraph is from this source. First, it should be noted that keratin is not a specific chemical substance but a category of substances. Jarrett defines it as ".., a highly stable, fibrous protein (or group 74

Fig. 3. Schematic view of magnif~d oblique section of hair follicle cut at level indicated in upper left corner. Reproduced by permission from Ham's Histology, J. B. Lippincott Co., Philadelphia.

of proteins) which contains disulphide bonds and which is remarkably resistant to enzymatic digestion and all but the strongest chemicals". A typical keratin molecule from mammalian skin * and hair is a two (or three) stranded cable of highly oriented polypeptide chains wound into a helix, with secondary folds or distortions associated with a relatively unorganized matrix. Of the several types of bonds that stabilize the keratin molecule the most important is sulfur to sulfur, and it is this characteristic- S-S-linkage (formed by two cystine residues contained in adjacent polypeptide chains) that is unique for keratin among the fibrous proteins found in the skin. Moreover, it is this disulfide bond that is mainly responsible for keratin's resistance to enzyme digestion and to hydrolysis. The as~ i a t i o n of lipids with hair keratin is important in that it affects incorporation

The entire outer layer of body skin is keratinized but this is "soft keratin" which is much less resistant than the hard keratin of hair and nails.

75

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Fig. 4. Diagram of a longitudinal section (low-power) cut through the nail groove and the root of a growing nail. Reproduced by permission from Ham's Histology, J. B. Lippincott Co., Philadelphia.

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Fig. 5. Schematic view of a mature hair. The cuticular cells are arranged in a very thin layer and overlap each other much as shingles on a roof. They tightly encase and provide considerable protection to the underlying cortex. (The thickness of the cuticle is considerably exaggerated in the cross-section shown above.) The cortex provides the bulk of the hair substance and contains the most resistant keratin. The medulla is usually absent in fine hairs and is often discontinuous even in coarser hairs, such as those of the scalp. Color is contributed largely by fine granules of melanin, which are more concentrated in the medulla but present also in the cortex. Variations in color depend upon chemical variations of the melanin itself,the frequency of the granules and their size and distribution. Intensely red hair contains special Fe-containing pigment•

76

into hair of constituents from sebum, sweat and exogenous materials. Phospholipids are a major component of hair lipid and are thought to be chemically linked to keratin through their fatty acid side chains. This will be discussed further under Exogenous sources of trace elements. The actual process of keratinization of hair is very complex. The following description by Greep and Weiss is gives some insight as to the nature of the intracellular events that accomplish this transformation: "The differentiating conical cells, which are the structural cells of the hair, develop many 60 to 80 A filaments which are assembled into heavy bundles. As these cells assume a spindle shape and form the cortex of the emerging hair fiber, they become filled with filaments between which an amorphous matrix is deposited". The reader is referred to the general references listed for further information on this important aspect of hair formation/structure. GROWTH OF HAIR AND NAIL

The growth of (human) hairs in utero begins early during the third month. The resultant very fine hair is called lanugo, also primary hair. Replacement with a coarser hair begins during the eight month in utero and continues until at least the sixth month of age. The growth of nails begins late in the third month. The growth of individual hairs of most animals including man is strikingly cyclic; periods of growth alternate with periods of rest in an on/off manner. The length of the cycle and the ratio of rest/growth vary considerably depending upon the species and, particularly in man, the region of the body. On the chest, for example, the growth period of several weeks is matched or even exceeded by the length of the rest period. But on the scalp, growth continues for years and this growth period is on the average nine times longer than the rest period. There is variation also among different follicles within the same region of the body but this is not so great. When the growth phase (anagen) of the cycle ends, cells of the matrix rapidly degenerate (catagen phase) and the follicle shrinks down, leaving a small focus of relatively undifferentiated multipotential cells from which the new follicle forms when next growth phase begins. During the catagen phase, actually a transition period between anagen (growth phase) and telogen (resting phase), that part of the follicle surrounding the base of the hair is lost, the hair develops a club shaped proximal swelling and in a sense dies. It is held in place by only thin keratinized strands and the mechanical pressure of its tight fitting external sheathma somewhat tenuous attachment. During the follicle's resting phase, the so-called telogen or club hair is rather easily dislodged and often falls out. If it remains in place throughout the resting phase it will probably be pushed out as the new hair develops and emerges. Occassionally, however, the telogen hair is pushed aside by its replacement but not dislodged, so that both the old and the new hair project from the same canal*. * From microscopic examination of a plucked or dislodged hair, one can readily determine whether the follicle was in anagen or telogen phase.

77

In most animals, excluding man (also the guinea pig and the cat), hair follicles are in synchmny, and there is a coordinated periodicity of growth/rest resuiting in seasonal moults. In many animals, waves of hair growth sweep posteriorly and dorsally, proceeding in an orderly fashion from one end of the body to the other. In human beings, however, hair follicles have independent cycles of growth, referred to as a mosaic pattern. Thus, in any given region one finds hair follicles in all stages of activity. The duration of different phases of the human hair cycle in various regions of the body has been measured repeatedly by different workers with variable resuits. Table 1 presents data for the growth cycle of scalp hair, identifying the various phases of the cycle. Perhaps the best information on the rate of human hair growth in various regions is that published by Saitoh et al.~o. Figure 6 presents some of their data. The subjects studied were young adults and measurements were made on 8-12 individuals for each of the body areas listed. They did not include axillary or pubic hair in their measurements. The growth role of axillary hair has been measured at 0.3--0.42 ram/day, comparable to that of the scalp and bearded region ~3. Pubic hair grows more slowly--on the order of 0.2 ram/day 'a. Figure 7 presents data ~9contrasting growth of beard by Caucasian and Japanese populations, also variations among individuals of each group. Growth/rest phases for the beard have been reported over the range of 4-11 mo/10-75 days. Growth of the nail has been reported as 0.1 mm/day and toenail 1/4 this much. Considering for a moment other mammalian species, most animals have two types of hair in a given region: an undercoat of fine hairs that provides the REGIONAL DIFFERENCES IN HAIR GROWTH 3.0

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78

TABLE 1 GROWTH/REST CYCLE OF A SCALP HAIR The average cycle of growth/rest for a scalp hair is approximately 1000 days and the ratio is 9/1. 900 days ±

Anagen I-Ill--growth of follicle Anagen IV-VI--production of hair

(Several days)

Catagen----degeneration/destruction of lower follicle

100 days _+

Telogen--resting stage

Other determinations of anagen/teiogen range from 22 weeks-six weeks/8 weeks--several months. At least a part of this discrepancy relates to race; generally, shorter time periods have been measured for Japanese and longer periods of Caucasians.

major mechanical protection and insulation, and an outercoat of longer "guard hairs" that serve as tactile organs. In many species the two kinds of fibers are produced within the same compound follicle. Most adult (furbearing) animals have two seasonal moults per year but some have three (e.g., the mountain hare in Scotlandt°), others one (e.g., horses---in Britain at any rate3), and still others none (e.g., sheep and angora rabbits~°). The sheep has been studied quite extensively with respect to hair growth. Unfortunately, it is hazardous to extrapolate from sheep to other species because sheep are atypical. One of the most striking atypical features is that their hair is in a continuous growth phase, i.e., there is no resting stage*. The rate of growth does vary with respect to seasons, however, largely influenced by length of day ~. And unlike most other mammals, growth of wool is markedly hormone-dependent 9.12. Cattle ordinarily shed their coats twice a year and individual follicles produce two or three hairs per year with a resting stage between each. During the time the winter coat is maintained, most follicles are in a resting stage 22. According to Blackburn 3, in British horses: "The coat is shed in the spring and the new hairs appear to go through a period of slow growth in the summer followed by a period of rapid growth in the autumn". There is also considerable information about those laboratory animals commonly used in experimental studies of hair growth, principally mice, rats, guinea pigs and rabbits. Unfortunately, rather little is known about growth rates and durations of the growth/rest cycles in most wild animals. Because of this, when one uses the hair of wild animals to determine the level of their environmental exposure to trace elements and other substances, caution is the better part of valor in concluding that variations in different portions of the hair, or hair removed at different times, indicate high or low level exposure during a specific time period.

* Exceptions do occur: e.g. Wiltshire sheep regularly moult in the spring in relation to a definite growth/rest cycle4s.

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Fig. 7. Mean weight of beard grown per day at successiveages by normal males in randomly selected Caucasian Japanesepopulations Each Vertical line represents +S.D. from the mean of that age group. Reproduced by permission, from W. Montasna and R. A. Ellis (Eds.), in The Biology of Hair Growth, A c a d e m i c Press, N e w Y o r k , Ch. 16, by J. B. H a m i l t o n .

Much work has been done on mechanisms that act to increase or decrease the rate of hair growth. The plucking of hairs is one of the most effective ways to increase hairgrowth; shaving or cutting hair (in the absence of pulling) seems to have little effect. Certain chemical irritants stimulate hair growth, including some dekeratinizing depilatory agents, e.g., barium sulfide. At the borders of wounds hair growth is stimulated. In all of the above instances (discussed by ChaseS), increased hair growth comes from shortening the resting stage; the rate of growth, once established, is not increased. Hormones have relatively little effect on hair growth in human beings and most other animals, excluding instances of marked endocrine abnormality--except sex hormones, which are necessary for the growth of axillary and pubic hair and in men hair of the bearded area and chest. Sheep are an exception, as has been mentioned. Comaish 8 presents a good overview of metabolic disorders as they affect human hair growth, considering effects of malnutrition, genetic disorders, endocrine dysfunctions and drugs. SOURCES OF TRACE ELEMENTS IN HAIR Figure 8 is a schematized view of a growing hair as related to six possible sources of trace elements. Source 1, the matrix, has already been discussed in the section on histogenesis and will not be considered further. Endogenous sources 80

labeled 2, 3, 4 and 5 deserve much more attention than they usually receive and will be discussed separately. Sebum

The quantity of sebaceous secretion is quite significant. Kirk, as cited by Carruthers 4, measured the total lipid component from 10 cm 2 areas of forehead skin in a series of human beings and found that this amounted to 0.7-2.4 mg during a 4-h period. The quantity decreased in women after the age of 50, but not in men. (One presumes that the amount of sebum secreted in the scalp is significantly greater than that of the forearm.) The secretion of sebaceous glands varies somewhat in composition, depending upon the area in which it is secreted. In a comprehensive study of forearm sebum by korincz 32 roughly one third of the nonaqueous material was free fatty acid, one third combined fatty acids and one third unsaponifiable matter including squalene, cholesterol and waxes. Table 2 presents data on the lipid composition of sebum. Eccrine sweat

Both the quantity and concentration of eccrine sweat varies enormously depending upon the individual and his environmental circumstances. Water and salts of Na and K are the principal ingredients although there is considerable urea (30--60 mg urea N/100 ml), amino acids, latic and pyruvic acids. Other elements present in small but significant amounts include: N, Ca, P and the trace elements Cu, Mn, Mg and Fe. Table 3 presents data on the electrolyte composition of eccrine sweat. Apocrine seat

This material has not been extensively studied in man. It contains a variety of volatile substances and mucopolysaccharides. In human beings apocrine glands are largely confined to axillary and perineal regions. Eccrine sweat is a quantitatively important source of trace elements that may become incorporated in hair after its formation. Sebaceous secretion and desquamated epithelium (apocrine sweat in public and axillary hair) are also important, but principally because of their iipids and waxes, which may provide the physical/chemical means to incorporate trace elements into the hair substance in such a way that they cannot be washed off or extracted without also removing some of the truly endogenous trace elements. The extent to which sebum and desquamated epithelium function in the manner just described is unknown and experiments are sorely needed to resolve this important question. In any event, many of the experiments that have been carded out to measure uptake by hair of specific (exogenous) trace substances in vitro, using simple aqueous solutions of an inorganic salt, are suspect because they certainly do not reflect natural, i.e., physiologic, conditions. This may be a major factor in the continuing confusions as to the extent that exogenous sources contribute to trace element content of

81

TABLE 2 LIPID CONSTITUENTS OF SEBUM* Fatty acids Combined**

Triglycerides Waxes (including cholesterol esters) Free

Unsaponifiable matter Total Aliphatic alcohols Straight-chain

Branched-chain Cholesterol Dihydrocholesterol Hydrocarbons Phosphatides

Squalene

Forearm Scalp Forearm Forehead Scalp Forearm Forehead Scalp Forearm Scalp Forearm Scalp Forearm Scalp Forearm Scalp Forearm Forehead Scalp Forearm Forearm Scalp Forehead

For~rm Sclap

* Data reproduced by permission, from Metabolism, published by Fed. Am. Soc. Exp. Biol., Bethesda, 1968. ** As triglycerides, waxes and other esters.

hair and the extent to which these exogenous materials can be dissolved or washed away from the hair fibers. Looking at specific trace elements, Renshaw et al)9 measured lead in 1 cm portions of hairs from the same (female) head and found that the average value for the hair 1-10 cm from the scalp surface was approximately the same, 3 ppm. However, there was an almost linear increase in Pb in that portion of hair l l - 3 0 c m from the scalp, reaching an average maximum of approximately 24 ppm. This was despite refluxing the hair samples in diethyl ether in a Soxhlet apparatus. Hambidge et al. 17 studying chromium in hair found variations in different portions of the same individual's hair, which they thought reflected variations in intake. Another report ~6 on babies one year old or less disclosed higher values during the first few months than subsequently, and this was consistent with results in older children when proximal parts were compared with distal parts of the same hair. 82

Fig. 8. Sources of trace elements found in hair. 1 = The matrix and connective tissue papilla (with its blood and lymph vessels) ate the source of those trace elements that become incorporated in the hair during its formation; 2 = the sebaceous gland is a source of trace elements derived from body tissues to which the hair is continually exposed; 3 = eccrine sweat glands secrete a predominantly aqueous solution, i.e., sweat, which, as with the sebum produced by sebaceous glands, is a continuing source of trace elements derived from bodily tissues; 4 = apocrine glands represent a special type of sweat gland that secretes a milky fluid, rich in lipids. In human beings they are found principally in the axillary, pubic and perianai areas; they are not found in the scalp (except in a rudimentary state). They, too, are a continuing source of endogenous materials; 5 = the epidermis, since it is continually desquamating and, when incorporated in sebum and/or secretions from sweat glands, may come into intimate contact with the hair, is considered a potential (mino0 source of trace elements derived from bodily tissue to which the hair is continually exposed; 6 = exogenous materials containing trace elements to which the hair is or may be exposed include: (a) air and water, (b) soaps, shampoos, oils, lacquers, dyes, etc, and (c) medications.

Nishiyama and Nordberg 3s found great variation in the amount of cadmium absorbed by hair, which they attribute to acidity of hair. Using Cd ~°9 to differentiate exogenous from endogenous sources, they found that various treatments could remove Cd from hair but they found not treatment that would differentially separate exogenous from endogenous Cd. 83

TABLE 3 ELECTROLYTES IN ECCRINE SWEAT* Calcium Chloride Copper Iodine Iron Magnesium Manganese Phosphorus Potussiu m Sodium Sulfur Zinc Bicarbonate Sulfate

(I.0--24) nag/100 ml (36--468) nag/100 ml 0.005 mg/100 ml 0.9 (0.5-1.2)Itg/ 100 ml 0.027 (0.002--0.045)mg/I00 ml (0.04--0.286) nag/100 ml 0.006 (0.004-0.07) rag/100 ml (0.009--0.043) nag/100 ml (2 I- 126) nag/100 ml (24--312) mg/100 ml (0.7-7.4) mg/100 ml 93 +_26pg/100 ml (I.6--18.6) vol % (4.0--6) mg/100 ml

* Data reprinted, by permission, from Metabolism, published by Fed. Am. Soc. Exp. Biol., Bethesda, 1968.

Flesch ~3 points out that keratin has a greater affinity for arsenic than any other tissue and this is one of the reasons that As is among the elements considered most suitable for analysis in hairs. But there are problems even here. Young and Rice s° were able to detect As in hair within two days after it had been injected subcutaneously into guinea pigs. When they soaked such hair in sodium arsenite, the hair absorbed a great deal of the As and they were not able to differentiate this exogenous As from the endogenous As; both were removed to considerable extent by prolonged soaking in water. Shapiro 43 made careful evaluation of the As content of hair and nails in an individual who died from As poisoning. He was able to demonstrate quite good correlation in values from different segments of hair and nails as related to the time of four different levels of As intake during the course of a seven-month period. Lander et al. s° were not able to make such differentiation in the series of patients that they studied. More recent studies using more sensitive analytical methods (neutron activation analysis) have shown that As appears in sweat soon after it is administered, quickly leading to contamination (by sweat) of the extruded portion of individual hairs u,31. Erickson H found, however, that: "There is a much higher internal deposition of arsenic in sections of growing hair than in corresponding sections of non-growing hairs. In acute poisonings, no significant levels of arsenic were found, other than that of the root sections". Gordus j4 has observed that persons who swam often in a pool where the water was brominated developed bromine levels in scalp hair 1-13 times normal, and copper hair levels were elevated in those who frequented swimming pools where Cu salts were added to the water. He also reported that application of seleniumcontaining scalp medication increased the level of Se in scalp hair 20--40 x. Obviously, hair of other parts of the body is also subjected to particular types of pre84

parations that contain varying amounts of different trace elements. The bearded area is often treated with pre-shave and after-shave lotions, colognes, powders and emollient creams and lotions; the axiilary (and perineal) areas with deodorants containing large amounts of AI or Zn and sometimes Zr; the body as a whole with talcum powders, some of which contain Mg or Zn, etc. Unfortunately, the composition of most of these preparations is not easily obtained. Human hair samples for analysis of trace substances are usually taken from the scalp. Recently, Baumslag et al? have suggested that public hair may offer adv.antages over scalp hair in that it is not usually subjected to the application of cosmetic preparations or shampoos that contain heavy metals. They analyzed pubic and scalp hair from patients on the delivery ward; mean values for pubic hair were slightly higher for Zn, and somewhat lower for Cu, Fe and Pb. They also compared values of maternal hair with hair of the newborn offspring and reported good correlation for the four trace elements measured. Pubic hair would seem to be less suitable than scalp hairs as a sampling source for the following reasons. It grows more slowly than scalp hair, as has been mentioned. Moreover, it has a much longer rest period; growth/rest has been reported as 11-18 mo./12-17 mo., which means that in a given sample, approximately 50 % of the pubic hair would be in the resting cycle as compared with 10 % of scalp hair. Another disadvantage comes from the fact that pubic region contains apocrine glands, which secrete a lipid rich fluid and this represents a little understood source of trace elements that may become incorporated in the formed hair. (See discussion under Sweat glands.) NORMAL VALUES FOR TRACE ELEMENTS 1N HUMAN HAIR

From the previous consideration of sources of trace elements in hair, it is evident that at best "normal" covers a wide range. Perhaps the most reliable normal values for a large number of elements have been determined by Gordus ~4 and some of his data are shown in Table 4. Numerous other studies have focused on one or several elements and many of these have already been referred to. Mean concentrations and standard deviations for multiple elements is a technique that is being used by forensic scientists to identify hairs found at the scene of a crime as belonging or not belonging to a given suspect, and valuable normal data on multiple elements are available as a result of these studies ~,7,~. In a review of the extensive experiences of two forensic laboratories in Canada, Jervis 25 states that the most characteristic trace elements in human hair with respect to "individualization" are: Mn, Ni, Co, Ag, Rb, Zn, Ga, Sr, Se, Sc, Cu, and Au--in that order. METHODOLOGY OF SAMPLE PREPARATION AND ANALYSIS

One of the great advantages of some of the new methods for analysis of trace elements is that they give values for many elements simultaneously. But

85

TABLE 4 RANGE OF MEAN VALUES FOR GROUPS OF YOUNG MEN FROM AIR FORCE AND NAVAL ACADEMIES Values are expressed in ppm except where indicated by *; there, in ppb. Mg AI Ca Sc Ti V Cr

53-135 4.4-5.5 360-850 1.7-6.8" 2.6-4.I 20-41" 1.3-1.7

Se Ag Cd Sb I Hg Pb

0.58--0.76 0.15--0.33 0.47 (1 group) 73-200* 0.45-1.3 1.7-1.9 4.1 (I group)

Mn Fe Co Ni Cu Zn As

0.14--0.45 26-32 30-45* 2.8-3.2 15-17 150--190 0.12-0.17

* Data of Gorclus14.

this is also a disadvantage in that it is not feasible to vary preparative procedures to suit the "needs" of each of the many individual elements. Thus choice of a best preparative method is influenced somewhat by which element one is most interested in. Preparation of hair and nail for analysis ordinarily includes washing and extraction from (at least) the surface layers. Washing is usually accomplished by aqueous solutions that contain detergents. This in itself achieves limited extraction. Chelating agents may also be used. Sometimes lipid solvents are used with the intention of extracting materials absorbed to the surface of the sample. Ideally, such preparative procedures would remove all exogenous (contaminating) substances, leaving all endogenous ones. From evidence presented previously, this ideal is not attainable. Many studies have been done on sample preparation and there are several reasonably good general methods. Two of these are described in detail by their primary advocates, Petering et al.3~ and Gordus ~(. Clarke and Wilson 6 have considered special problems in analysis for Fb. These papers provide additional references. In view of the very extensive literature dealing with analytical procedures for trace elements and the fact that this paper is primarily concerned with biologic characteristics of hair and nail, it seems inappropriate even to attempt a comprehensive listing of the most appropriate references. Two that may be of special interest, however, which deal specifically with hair and nail are Mahler et al. 33and Jervis 25. CONCLUSIONS

Hair and nail samples are easy to get and easy to store. Trace element analysis of these materials is precise, accurate and reproducible. However, there are many (non-analytical) variables that must be taken into account if we are to evaluate the data in an adequate manner and avoid serious errors of interpretation. We need much more information about the biological phenomena responsible for t h e s e variations in order to make the most of trace element analysis of hair and nail--and we need to use more effectively the information that we already have. 86

REFERENCES 1 Bate, L. C. and F. F. Dyer, in V. P. Guinn (Ed.), Proc First int. Conj'. on Forensic Activation Analysis. Gulf General Atomic, San Die8o~ 1967, pp. 247-259. 2 Baumsla8o N., D. Yseger, L. Levin and H. H. Petering, Environ. Health, (1974) 186. 3 Blackburn, P. S., in A. J. Rook and G. S. Walton (Eds.), Comparative Physiology and Pathology oJ the Skin, F. A. Davis, 1965, pp. 201-210. 4. Carruthers, C., in C. Carruthers (Ed.), Biochemistry oJSkin in Health and Disease, Charles C. Thomas, Springfield, 1962, pp. 73-99. 5 Chase, H. B., in W. Montagna and R. A. Ellis (Eds.), The Biology of Hair Growth, Academic Press, New York, 1958, pp. 435--440. 6 Clarke, Ann N. and D. J. Wilson, Arch. Environ. Health, 28 (1974) 292. 7 Coleman, R. F., F. H. Cripps, A. Stinson and H. D. Scott, in V. P. Guinn (Ed.), Proc. First Int. Conj'. on Forensic Activation Analysis, Gulf General Atomic, San Diego, 1967, pp. 203--219. 8 Comaish, S., Br. J. Dermatol., 84, (1971) 83. 9 Downes, A. M. and A. L. C. Wallace, in A. G. Lyne and B. F. Short (Eds.), Biology of the Skin and Hair Growth, Elsevier, New York, 1965, pp. 679-703. 10 Ebling, F. J., in A. J. Rook and G. S. Walton (Eds.), Comparative Physiology and Pathology oJ the Skin, F. A. Davis, Philadelvhia, 1965, pp. 87-102. 11 Erickson, N. E., in V. P. Guinn (Ed.), Proc. First Int. CoJ~. on Forensic Activation Analysis, Gulf General Atomic, San Diego, 1967, pp. 279-286. 12 Ferguson, K. A., A. L. C. Wallace and H. R. Linder, in A. G. Lyne and B. F. Short (Eds.), Biology of the Skin and Hair Growth, Elsevier, New York, 1965, pp. 655--678. 13 Fiesch, P., in S. Rothman (Ed.), Physiology and Biochemistry of the Skin, University of Chicago, Chicaso, 1954, pp. 601--661. 14 Gordus, A., J. Radional. Chem., 15 (1973) 229. 15 Greep, R. O. and L. Weiss, Histology, Mc Graw-Hill, New York, 3rd ed., 1973, p. 493. 16 Hambidge, K. M. and J. D. Baum, Am. j. C/in. Nutr., 25 (1972) 376. 17 Hambidge, K. M., M. L. Franklin and M. A. Jacobs, Am. J. C/in. Nutr., 25 (1972) 380. 18 Hambidl~ K. M., M. L. Franklin and M. A. Jacobs, Am. J. Clin, Nutr., 25 (1972) 384. 19 Hamilton J. B., in W. Montagna and R. A. Ellis (Eds.), The Biology of Hair Growth, Academic Press, New York, 1958, pp. 400-434. 20 Hammer, D. 1., J. F. Finkles, R. H. Hendricks, C. M. Shy and R. J. M. Horton, Am. J. Epidemiol., 69 (1971) 84. 21 Hanson, H. C. and R. L. Jones, Use of Feather Minerals as Biological Tracers to Determine the Breeding and Molting Grounds of Wild Geese, BiologicalNotes 60, Illinois Natural History Survey, Urbana, 1968, 8pp. 22 Hayman, R. H., in A. G. Lyne and B. F. Short (Eds.), Biology of the Skin and Hair Growth, Elsevier, New York, 1965, pp. 575-590. 23 Hutchinson, J. C. D., in A. G. Lyne and B. F. Short (Eds.), Biology o/the Skin and Hair Growth, Elsevier, New York, 1965, pp. 565-574. 24 Jarrett, A., in A. Jarrett (Ed.), The Physiology and Pathophysiology o./the Skin, V. 1, The Epidermis, A__,~:te_~micPress, New York, 1973, pp. 145-160. 25 Jervis, R. E., in V. P. Guinn (Ed.), Proc. First Int. C o ~ on Forensic Activation Analysis, Gulf General Atomic, San Diego, 1967, pp. 287-294. 26 Jones, R. L., R. F. Labisky and Win. L. Anderson, Sek,cted Minerals in Soils, Plants, and Pheasants: An Ecosystem Approach to Understanding Pheasant Distribution in Illinois, Biological Notes 63, Illinois Natural History Survey, Urbena, 1968, 8 pp. 27 Klevay, L, M., Am. J. Clin. Nutr.o 23 (1970) 284. 28 Kievay, L. M., Arrh. Environ. Health., 26 (1973) 169. 29 Kopito, L., R. K. Byers and H. Schwachman, New Eng. J. Med., 276 (1967) 949. 30 Lander, H., P. R. Hodge and C. S. Crisp, J. Foren~'c Med., 12 (1965) 52. 31 Lima, F, W., in V. P. Guinn (Ed.), Proc. First Int. Co~/~ on Forensic Activation Analysis, Gulf General Atomic, San Diego, 1967, pp. 261-278. 32 Lorincz, A. L., in S. Rothman (Ed.), The Human inu~gument, Am. Ass. for the Advancement of Science, Publ. No. 54, Washington, 1959, pp. 127-150. 33 Mahler, D, J.0 A. F. Scott, J. R. Welsh and G. Haynie, J, Nucl. Med., Ii (1970) 739. 34 McBean, L. D., M. Mahloudji, J. G. Reinhold and J. A. Halsted, Am. J. ('/in. Nutr., 24 (1971) 506.

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35 Nishiyama, K. and G. F. Nordberg, Arch. Envhon. Health, 25 (1972) 92. 36 Perkons, A. K., in V. P. Guinn (Ed.), Proc. First Int. C'on]~ on Forensic'Activation Analysis, Gulf General Atomic, San Diego, 1967, pp. 221-235. 37 Petering, H. G., D. W. Yeager and S. O. Witherup, Arc'h. Environ. Health, 23 (1971.) 202. 38 Petering, H. G., D. W. Yaeger and S. O. Witherup, Arch. Environ. Health, 27 (1973) 327. 39 Renshaw, G. D., C. A. Pounds and E. F. Pearson, Nature, 238 (1972) 162. 40 Saitoh, M., M. Uzuka, M. Sakamoto and T. Kobori, in W. Montagna and R. L. Dobson (Eds.), Advances in Biology oJ Skin, V. IX, Hair Growth, Pergamon Press, New York, 196/, pp. 183-202. 41 Schoenheimer, R., The Dynamic' State o/Body Constituents, Harvard University Press, Cambridge, 1942, 78 pp. 42 Schroeder, H. A. and A. P. Nason, J. Invest. Dermatol.. 53 (1969) 71. 43 Shapiro, H. A., J. Forensic"Med., 14 (1967) 65. 44 Sims, R. T. and H. H. F. Knollmeyer, Br. J. Dermatol., 83 (1970) 200. 45 Slee, J., in A. G. Lyne and B. F. Short (Eds.), Biology ojthe Skin and Hair Growth, Elsevier, New York, 1965, pp. 545-564. 46 Sorenson, J. R. J., L. S. Levin and H. G. Petering, Interlace, 2 (1973) 17. 47 Spearman, R. I. C., The integument--A textbook o/Skin Biology, Part I, ComparativeMorphology, University Press, Cambridge, 1973, pp. 95-100. 48 Straile, W. E., in A. G. Lyne and B. F. Short (Eds.), Biology olthe Skin and Hair Growth, Elsevier, New York, 1965, p. 52. 49 Strain, Wm. H., W. J. Pories, A. Flynn and O. A. Hill, Jr., in Delbert D. Hemphill, (Ed.), Trace Substances in Environmental Health-V, University of Missouri, Columbia, 1972, pp. 383-397. 50 Young, E. G. and F. A. H. Pdce, J. Lab. Clin. Med., 29 (1944) 439.

RECOMMENDED GENERAL REFERENCES DEALING WITH STRUCTURE AND HISTOGENESIS OF HAIR AND NAIL 1 Carruthers, C., in C. Carruthers (Ed.), Biochemistry of Skin in Health and Disease, Charles C. Thomas, Springfield, 1962, pp. 198-216. 2 Hamilton J. B. and A. E. Light,Ann. N. Y. Acad. Sc., 530951)461-752. 3 Jarrett, A. (Ed.), The Physiology and Pathophysiology of the Skin, V.I, The Epidermis, Academic Press, New York, 1973, 343 pp. 4 Lyne, A. G. and B. F. Short (Eds.), Biology o./'the Skin and Hair Growth, Elsevier, New York, 1965, 806 pp. 5 Montagna, W. and R. L. Dobson (Eds.), Advances in Biology oJSkm, Pergamon Press, New York, 1967, 585 pp. 6 Montagna, W. and R. A. Ellis (Eds.), The Biology of Hair Growth, Academic Press, New York, 1958, 520 pp. 7 Pinkus. H. and A. H Mehregan, A Guide to Dermatohistopatbology, Appleton-Century-Crofts, New York, 1969, 546 pp. 8 Rook, A. J. and G. S. Walton (Eds.), Comparative Physiology and Pathology oJthe Skin, F. A. Davis, Philadelphia, 1965, 794 pp. 9 Rothman, S., Physiology and Biochemistry oJ the Skin. University of Chicago, Chicago, 1954, 741 pp. 10 Spearman, R. I. C., The Integument-A Textbook oJSkin Biology, The University Press, Cambridge, 1973, 208 pp. 11 Fraser, R. D. B., T. P. MacRae and G. E. Rogers, Keratins, Their Composition, Structure and Biosynthesis, Charles C. Thomas, Springfield, 1972, 304 pp. After preparation of this manuscript, the following excellent article was found: Obrusnik, I., J. Gislason, D. K. McMillan, J. D'Auria and B. D. Pate, J. Forensic Sci.. 17 (1972) 426. DISCUSSION Inquirer: Brian R. Moyer, University of California, Berkeley, California. Q. Public hair seems to be a better indicator than scalp. Can you relate levels in either to metabolic rate?

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A. To begin with, 1 doubt that pubic hair is a better indicator than scalp hair. A discussion of this point is included in my manuscript, but 1 did not have time to include it in my talk. Briefly, the points against pubic hair include: (1) slower rate of growth (approximately half that of scalp hair); (2) a resting phase that is much longer (5096 vs. 1096 for scalp hair); (3) the presence of apocrine glands in the aubic region, which serve as an indeterminate source of materials that contaminate the Ibrmed pubic hair. It has been suggested that pubic hair is less apt to be contaminated by materials ordinarily applied to the scalp, but deodorants, body powders, etc. are applied to the pubic region, so that one merely exchanges one set of exogenous contaminants for another.

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