28.6: Hair zinc concen­trations (24c.6)

Zinc is incorporated into the hair matrix when the hair is exposed to the blood supply during synthesis within the dermal papilla (Kempson et al., 2007). When the growing hair approaches the skin surface, the hair undergoes keratinization and the zinc accumulated during its formation becom­es sealed into keratin protein structures and isolated from meta­bolic processes. Hence, the zinc content of the hair shaft reflects the quantity of zinc available in the blood supply at the time of the hair growth, not at the time of sampling (Kempson et al., 2007). Consequently, positive correlations between hair and serum zinc concen­trations can only be expected in settings where zinc status is unchanged or chronic zinc defi­ciency exists. Assuming a normal rate of hair growth (i.e., about 1cm hair growth/month), the zinc concen­tration in the proximal 1–2cm of hair (i.e., the hair closest to the scalp) reflects the zinc uptake by the follicle 4–8 weeks before sample collection (Hopps, 1977). In cases where hair growth is arrested, as may occur in severe acute malnutrition (Erten et al., 1978) and acrodermatitis enteropathica (Bradfield and Hambidge, 1980), hair zinc should not be used. In such cases, hair zinc concen­trations may be normal or even high. The advantages of using hair as a biomarker are listed in Box 24c.6.

Box 24c.6 The advantages of using hair as a biomarker. From IZiNCG Technical Brief #8. (IZiNCG, 2018).

• Concentrations are higher than in serum or urine making their measure­ment easier.
• Specimen collection is relatively non-invasive and samples can be collected and stored at room temperature without deterioration and the need for speial prservatives.
• Concentrations are not sub­ject to the rapid fluctuations produced by a recent meal, diurnal or circadian variation, or infection, as seen in serum zinc,
• Concentrations reflect exposure over a longer retrospective time frame than serum or urine.

Nevertheless, several technical and biological factors may affect hair zinc concen­trations. Hair is exposed to exogenous surface contamination so all specimens must be washed using a recom­men­ded procedure. Biological factors may include age, possibly sex, season of the year, and rate of growth, as noted earlier (King et al., 2015; IZiNCG, 2018). Neither cosmetic treatments or hair color affect hair zinc concen­trations, provided appropriate washing methods are used (Kempson et al., 2007). The effects of all these possible confounding factors must be considered when interpreting hair zinc concen­trations. The BOND Zinc Expert Panel classified hair zinc as a “potential” biomarker that shows promise, but requires the establishment of specific cutoffs to indicate zinc inadequacy in popu­lations (King et al., 2015).

In part, the use of hair zinc concen­tration as a biomarker of zinc exposure has been hampered by the failure of com­mercial laboratories to adopt standardized methods for sampling and washing the hair samples, and to report the accuracy and precision of their analytical methods(Hambidge, 1982; King et al., 2015). Hair zinc concen­trations were shown to respond positively and significantly to supple­mental zinc in a systematic review and meta-analysis of three studies in healthy adults (Lowe et al., 2009), although their response to zinc depletion is uncertain(Lowe et al., 2009). In children, responses of hair zinc to supple­mental zinc have been inconsistent, although some associations between low hair zinc concen­trations and zinc-related functional outcom­es (e.g., impaired taste acuity, appetite, linear growth, recurrent respiratory tract infection) have been reported (Hambidge et al., 1972; Buzina et al., 1980; Chen et al., 1985; Smit-Vanderkooy and Gibson, 1987; Gibson et al., 1989a; Cavan et al., 1993a; Ferguson et al., 1993). In addition, some studies have shown significant relationships between hair zinc concen­trations and dietary zinc indices, most notably dietary phytate:zinc molar ratios, in individuals consuming predominantly plant-based diets (Ferguson et al., 1989; Gibson and Huddle, 1998).

24c.6.1 Interpretive criteria

There are no universally accepted reference values for hair zinc concen­trations (Mikulewicz et al., 2013), and, as a result, the use of hairr zinc as a biomarker for assessing risk of zinc defi­ciency in popu­lations has been limited. None of the reference values published to date have been com­piled from a nationally representative reference sample of well nourished, healthy individuals free from condi­tions known to affect zinc status, unlike the IZiNCG procedure used to define reference values for serum zinc (King et al., 2015). In addition, standardized procedures for the sample collection, washing, and chemical analysis of the specimens, have not been employed (Mikulewicz et al., 2013). Hence there is an urgent need to com­pile a set of universal reference values for hair zinc concen­trations.

Guidelines for establishing universal reference values are available in Mikulewicz et al. (2013). Reference values should be represented by the geometric mean ± CV (%) or median for hair zinc concen­trations, preferably by sex and life-stage group. Reference limits indicative of unusually low hair zinc concen­trations, represented by the 2.5^th percentiles for males and females for each life-stage group, should also be deter­mined. With these data, the feasibility of employing hair zinc to assess the likely risk of zinc defi­ciency in future national nutrition surveys could be explored.

Table 24c.7 presents the best available reference values for hair zinc (µg/g) derived from published studies for children ranging in age from 3 13y.Note data for both sexes are com­bined in five of the study groups in Table 24c.7, and that younger children tend to have lower hair zinc concen­trations (IZiNCG, 2018, Table 24c.7).

Table 24c.7 Reference values for hair zinc (µg/g) based on healthy volunteer children from urban areas. From IZiNCG Technical Brief #8 (IZiNCG, 2018).

Country Reference	Age (y)	Sex	n	Mean (SD)	Median
Italy (Senofonte et al., 2000)	3–6	F/M	58	101 (67)	92
Korea (Park et al., 2007)	3–6	F/M	655	70 (50)	66
Italy (Senofonte et al., 2000)	6–10	F/M	96	157 (50)	152
Italy (Senofonte et al., 2000)	10–13	F/M	258	158 (41)	155
Italy (Dongarra et al., 2011)	11–13	F/M	130	189 (59)	179
Belgium (Vanaelst et al., 2012)	6–10	F	218		216

A cutoff point of 70µg/g (1.07µmol/g) has been established for hair zinc in young children that appears to indicate risk of zinc defi­ciency in both clinical and popu­lation studies. Cutoff points, unlike reference limits, are generally based on data from individuals with either clinical or functional manifestations of a nutrient defi­ciency. Zinc-related adverse health outcom­es such as impairments in linear growth, appetite, and taste acuity (Hambidge et al., 1972; Smit-Vanderkooy and Gibson, 1987; Gibson et al., 1989a) have been associated with hair zinc concen­trations less than 70µg/g (< 1.07µmol/g). A higher cut‑off (< 110µg/g or < 1.68µmol/g) has been used in some studies for hair samples from children collected in the Autumn / Winter months to take into account the possible effect of season on hair zinc concen­trations (Gibson et al., 1989b).

An additional application for hair zinc concen­trations includes their use as a longer-term, retrospective measure of zinc exposure in case-control studies. Of note, is the existence of withon-person variability for hair zinc which has the potential to attenuate estimates of association in case-control or prospective cohort studies, as well as popu­lation prevalence estimates for risk of defi­ciency (Park et al., 2016). Such attenuation can be reduced by obtaining several replicate hair samples from each individual in the group and applying the mean value to represent true exposure.

24c.6.2 Measurement of hair zinc

Hair specimens should be collected from a representative sample of the target popu­lation or sub­groups of interest during the same season of the year. Samples (at least 50mg) should be cut at skin level from the occipital region of the scalp (i.e., across the back of the head in a line between the top of the ears) with stainless steel scissors. Only the proximal (i.e., closest to the scalp) 1.0–2.0cm of the hair strands should be retained. These specimens will reflect the zinc uptake by the follicles 4–8 weeks prior to sample collection provided the rate of hair growth has been normal. Hair samples should be placed in labeled trace-element-free polyethylene bags for storage; any remaining hair strands should be discarded. Before washing the specimens to remove exogenous contaminants such as atmospheric pollutants, water, and sweat, any nits and lice should be removed if necessary using a microscope or magnifying glass and Teflon-coated tweezers. For each specimen, details of the ethnicity, age, gender, hair color, height, weight, date of collection, presence of malnutrition (where relevant), and use of dandruff shampoos or cosmetic treatments should always be recorded to aid in the interpretation of the data.

Washing with nonionic detergents (e.g., Triton X‑100) with or without acetone is preferred for hair as these detergents are less likely to leach bound zinc from the hair and yet are effective in removing superficial adsorbed zinc. Washing with chelating agents such as EDTA should be avoided because they remove some of the tightly bound zinc that is an integral part of the hair sample (Shapcott, 1978). After washing and rinsing, the hair samples must be vacuum- or oven-dried depending on the chosen analytical method, and stored in a desiccator prior to laboratory analysis. For a detailed guide for measuring hair zinc concen­trations the reader is advised to consult IZiNCG (Technical Brief No.8).

Several laboratory methods can be used to measure hair zinc concen­trations in the washed specimens, depending on the instruments available. Washed specimens can be be prepared for analysis using microwave digestion when available, or wet or dry ashing, although the use of tetramethylammonium hydroxide (TMAH) to solubilize hair at room temperature warrants investigation. The latter method eliminates time-consuming ashing or wet digestion. Traditional analytical methods include flame AAS or multi-element ICP‑MS, although non-destructive instrumental neutron activation analysis (INAA) can also be used. For INAA the washed hair specimens are placed in small, weighed trace-element-free polyethylene bags or tubes and oven-dried for 22 hours at 55°C. After cooling in a desiccator, the packaged specimens are sealed and weighed, prior to irradiation in a nuclear reactor.

Certified reference materials (CRM)s should always be used to assess the accuracy and precision of the chosen analytical method for hair zinc. Unsatisfactory quality control for hair trace element analysis among laboratories is a widespread problem (Hambidge, 1982; Mikulewicz et al., 2013). A CRM for human hair is available (e.g., Community Bureau of Reference, Certified Reference Material no. 397) from the Institute for Reference Materials and Measurements, Retieseweg, B‑2440 Geel, Belgium. In-house controls prepared from a homogenous sample of finely cut digested hair (or powdered for INAA) should also be prepared and spiked with several differ­ent known quantities of zinc and the recoveries measured. The in-house controls should be analyzed in conjunction with a CRM periodically to monitor assay variations in the instrument and digestion procedures.