Gibson RS, Principles Book
of Nutritional
Assessment:
Zinc

3rd Edition
October 2020

Abstract

Multiple physio­logical functions are affected by zinc defi­ciency because of the involve­ment of zinc in numerous meta­bolic processes as a catalytic, structural, and regulatory ion. As a consequence, in zinc defi­ciency numerous nonspecific changes occur, including impairments in growth, immune function, appetite, taste acuity, and possibly neuro­behavioural function. Zinc homeo­stasis in the body is tightly controlled over a wide range of zinc intakes, primarily by adjusting losses of endogenous fecal zinc. Such strict homeo­static control leads to difficulties in the assessment of zinc insufficiency and excess. The BOND Expert Zinc Panel recommen­ded three indicators to identify popu­lations at elevated risk of zinc defi­ciency — the prevalence of usual zinc intakes below the estimated average require­ment, the percentage with low plasma / serum zinc concen­trations, and the percentage of children < 5y who are stunted (i.e., HAZ < –2SD). Reference limits for plasma / serum zinc (depending on age, sex, time of day, fasting status) are available to identify popu­lations at risk of zinc defi­ciency, provided the correct protocols are followed for the collection, separation, and analysis of plasma zinc. Moreover, in view of the poor specificity of impaired linear growth, assessment of this functional indicator should be made in com­bination with at least one of the other indicators. Assessment of marginal zinc defi­ciency in individuals is more difficult because of the absence of frank clinical signs and the lack of a reliable sensitive and specific biomarker. The recom­men­ded approach com­bines a medical history, qualitative dietary and clinical assessment, with plasma / serum zinc providing supple­mentary data. The BOND Expert Zinc Panel classified other potential zinc biomarkers into three groups: (1) potentially useful (hair zinc, urinary zinc, and neuro­behavioral function); (2) emerging biomarkers (nail zinc, zinc-dependent proteins, oxidative stress and DNA integrity; zinc kinetics, taste acuity), and (3) not useful (zinc-dependent enzymes, erythrocyte and leukocyte zinc); strengths and weaknesses of each are discussed.

CITE AS: Gibson RS. Principles of Nutritional Assessment. Zinc. https://nutritionalassessment.org/zinc/
Email: Rosalind.Gibson@Otago.AC.NZ
Licensed under CC-BY-SA-4.0

24c.1 Zinc

In an adult 70kg male there is approximately 1.5–2.5g of zinc, of which over 80% is found in the skeletal muscle and bone. Much smaller amounts are present in the liver, gastro­intestinal tract, skin, kidney, lung, prostate, and other organs, as shown in (Table 24c.1).
Table 24c.1 Zinc content of major organs and tissues in adult (70 kg) man. Adapted from Iyengar, Radiation Physics and Chemistry 51: 545–560, 1998.
Tissue Zn conc.
(mg/kg
wet wt)
Total
content
(mg)
Percent
of total
body
zinc
Skeletal muscle
Skeleton
—Bone
—Marrow
—Periarticular tissue
—Cartilage
Liver
Lung
Skin
Whole blood
Kidney
Brain
Teeth
Hair
Spleen
Lymph nodes
Gastrointestinal tract
Prostate
Other organs/tissues
Total
50

90
20
11
34
40
40
15
6
50
10
250
200
20
14
15
100
Variable

1400

450
60
11
30
72
40
39
33
15
14
11.5
4
3.6
3.5
1.8
1.6
50
2240
63

20
3
< 1
1
3
2
2
1
1
1
1
< 1
< 1
< 1
< 1
< 1
2
100

Unlike other trace elements such as iron and copper, or minerals such as calcium, there are no large, sequestered, readily mobilizable stores of zinc that can be rapidly released in response to variations in dietary intakes of zinc. Instead, there is a small pool of rapidly exchangeable zinc which is located in bone, liver, pancreas, kidney, spleen and plasma and accounts for about 10% of whole-body zinc (King et al., 2000). This pool turns over com­pletely about five times each day to provide zinc for zinc-dependent meta­bolic functions throughout the body. It is the loss of a critical but small amount of zinc from this pool that leads to the biochemical and clinical signs of zinc defi­ciency (King, 2018). Bone may also provide a passive reserve of zinc, especially during growth, so that when dietary zinc intakes are low, less of the zinc released during normal bone turnover, is re-deposited in the skeleton (Zhou et al., 1993). There is also some evidence that hepatic zinc, bound to metallo­thionein and accrued during gestation, can also be mobilized during early infancy to supple­ment the infant’s needs for zinc (Zlotkin and Cherian, 1988; Coni et al., 1996).

24c.1.1 Functions of zinc at the cellular level

At the cellular level, zinc has three major meta­bolic functions — catalytic, structural, and regulatory (King and Cousins, 2014). More than 300 metallo­enzymes require zinc as a catalyst for function or regulation. During zinc defi­ciency, their activity is decreased, although their structure does not change, and with the addition of zinc, enzyme activity is restored. Zinc metallo­enzymes are involved in nucleic acid meta­bolism and cellular proliferation, differ­entiation, and growth; examples are listed in (Table 24c.2).
Table 24c.2: Selected zinc metallo­enzymes.
Zinc metalloenzymes
Alcohol dehydrogenase
Alkaline phosphatase
Carbonic anhydrase
Carboxypeptidase
Deoxynucleotidyl transferase
DNA polymerase
Glutamic acid dehydrogenase
Malic acid dehydrogenase
Nucleoside phosphorylase
RNA polymerase
Tyrosine kinase
Zinc-copper superoxide dismutase

So far, no consistent changes in the activity of any zinc metallo­enzymes have been linked with signs and symptoms of zinc defi­ciency in experimental zinc depletion-repletion studies in humans (King et al., 2015).

Zinc also plays an essential structural role in the folding of some proteins and a lack of zinc results in protein misfolding and the loss of function. A finger-like structure, known as a zinc finger motif, stabilizes the structure of a variety of proteins, including those involved in cellular differ­entiation or proliferation, signal trans­duction, cellular adhesion, or trans­cription. Zinc fingers use cysteine and histidine to form a tetrahedral Zn2+ coordination com­plex. The extent to which the intake of dietary zinc affects the function of zinc finger proteins is unclear. Zinc also maintains the structure of certain enzymes, notably the antioxidant enzyme, copper-zinc superoxide dismutase (Cu,Zn‑SOD) (King, 2011).

Over 3% of all identified human genes contain zinc finger proteins that regulate gene expression by acting as trans­cription factors. Gene expression may also regulate the effects of zinc defi­ciency on lipid peroxidation, immune function, apoptosis, and neuronal function (King et al., 2015). Zinc is also involved in cell signaling through the metal response element-binding trans­cription factor 1 (MTF1). Whether MTF1 regulates genes negatively or positively appears to depend on the cellular zinc status. Zinc may also have a direct regulatory role controlling numerous cell-signaling pathways by modulating kinase and phosphorylase activities. For more details of the meta­bolic functions of zinc at the cellular level, see King and Cousins (2014).

Zinc-dependent cellular functions are coordinated by one or more of twenty four zinc trans­porters. Two classes of zinc trans­porters exist: ZIP proteins and ZnT proteins. It is the up- or down-regulation of these trans­porter genes in response to zinc intake that helps to maintain cellular zinc homeo­stasis. The ZnT proteins (n=10) generally trans­port zinc ions out of the cytosol, whereas ZIP (SLC 39) proteins (n=14) import the ions from the cellular com­partments or the extra­cellular space into the cytosol. Individual ZIP and ZnT trans­porters are located in specific cell types. For example, ZIP‑14 appears to be involved in the uptake of zinc by the liver in response to acute inflam­mation and infection, whereas ZnT3 is found in the synaptic vesicles of some types of neurons, and ZnT8 in the secretory granules of β‑pancreatic cells (King et al., 2015). However, the role of many of the twenty four zinc trans­porters and how their expression is altered to maintain cellular zinc homeo­stasis remains uncertain (King and Cousins, 2014).

24c.1.2 Physiological functions of zinc

Multiple physio­logical functions are affected by zinc defi­ciency due to the ubiquitous involve­ment of zinc in cellular meta­bolism. A modest deprivation in zinc intake over the long-term results in detectable changes in growth, immune function, and possibly neuro­behavioural function. Other signs of marginal zinc defi­ciency include poor appetite and impaired taste acuity (hypogeusia).

Impaired growth in zinc defi­ciency arises from the role of zinc in DNA trans­cription and gene expression, signal trans­duction pathways, and endocrine function (e.g., insulin-like growth factor); these effects are summarized in Figure 24c.1.
Figure 24c.1
Figure 24c.1. Effects of zinc deficiency on metabolic processes associated with growth. Redrawn from MacDonald R. S. (2000). The Journal of nutrition, 130(5S Suppl), 1500S–1508S.
Of the growth indices, height‑ or length-for‑age is considered the best functional bioindicator associated with the risk of zinc defi­ciency in popu­lations. Increased linear growth is the primary response to increased zinc intake, with an associated gain in weight. (de Benoist et al., 2007).

Zinc is also required for the synthesis and functions of immune regulatory proteins and for the maintenance of normal immune function. Consequently, zinc defi­ciency causes immune dysfunction. Both barrier function and cellular com­ponents of the innate immune responses (i.e., macrophage and neutrophil function) are com­promised as well as the acquired immune system, with the largest effect being on reducing CD4 T cell function (King et al., 2015). The decline in innate immunity with zinc defi­c­iency promotes systemic inflam­mation, charac­terized by elevated levels of proinflam­matory cytokines. Supplementation with zinc has been associated with decreased inflam­matory responses in children with zinc defi­ciency (Sandstead et al., 2008). With a reduced resistance to infection, the rate and duration of infections increases. Zinc defi­ciency also increases oxidative stress and DNA damage which, in turn, play a role in promoting the inflam­matory process (Wong and Ho, 2012). For more details of zinc and immune function, see Raiten et al. (2015).

The mechanism whereby zinc influences neuro­behavioral functioning is not well established. In the central nervous system, zinc is concen­trated in the presynaptic vesicles of zinc-containing neurons, which are found primarily in the forebrain and connect with other cerebral cortices and limbic structures. During synaptic events, zinc is released and passes into postsynaptic neurons, serving as neuro­trans­mitters. Zinc defi­ciency may interfere with these processes and thus com­promise neuro­behavioral functioning, especially during times of rapid growth and development, such as infancy, when demands for zinc are high (Black, 2003). Meta-analysis, however, has failed to show a significant overall effect of zinc intake on biomarkers of cognitive function in children, although the number of available studies in this review was small (n=6) (Warthon-Medina et al., 2015). There is some evidence that zinc defi­ciency may also be related to anxiety and depression. Possible mechanisms that warrant investigation include the involve­ment of zinc in the uptake of the neuro­trans­mitter serotonin (5‑HT) in the brain (Levenson, 2006). More research is also needed to clarify the mechanisms by which zinc affects taste acuity (Aliani et al, 2013). Some investigations suggest that reduced taste acuity may be linked with reductions in certain zinc metallo­proteins that act as olfactory receptors (King and Cousins, 2014).

24c.1.3 Absorption and meta­bolism of zinc

Zinc is mainly absorbed in the upper part of the small intestine by two mechanisms: a saturable, carrier-mediated process and, secondly, by a non-mediated, or passive process. Two families of zinc trans­porters control the trans­port of zinc on the enterocyte membrane and account for the saturable com­ponent. Once zinc is absorbed into the enterocyte, movement of zinc within the cell is influenced by metallo­thionein and the trans­porter ZnT7. Export of zinc from the intestinal epithelial cells into the portal vein is controlled by the ZnT1 trans­porter. About 30%–40% of the zinc in portal blood is exchanged with the liver, the main organ involved in zinc meta­bolism. From the liver, zinc is released into the systemic circulation for delivery to peripheral tissues bound mainly to albumin and α2‑macroglobulin (King and Cousins, 2014). Almost no zinc circulates in an unbound state.

Absorption of zinc by a nonsaturable or passive mechanism only occurs at high intestinal luminal zinc concen­trations generated through consumption of pharmacologic doses of zinc (King and Cousins, 2014). At such high doses (i.e., > 20mg/d Zn), fractional absorp­tion of zinc via the passive mechanism is very low, suggesting that a dose of supple­mental zinc above 20mg will have a limited influence on zinc nutrition (Tran et al., 2004).

Zinc homeo­stasis in the body is tightly controlled over a wide range of zinc intakes. This is achieved primarily by adjusting zinc excretion through the gastro­intestinal tract. Losses of zinc via the gastro­intestinal tract com­prise both unabsorbed dietary zinc and endogenous fecal zinc. The sources of endogenous fecal zinc are uncertain but probably include secretions from pancreatic and intestinal mucosal cells. When zinc intakes are very low, there is a decrease in zinc secretion into the intestinal lumen, and endogenous fecal zinc declines. With increasing zinc intakes, endogenous fecal zinc losses increase to maintain homeo­stasis. If the adult zinc intake is extremely low (e.g., < 2mg/day), then the reductions in losses of endogenous fecal zinc may be insufficient to re-establish zinc homeo­stasis. In these circumstances, additional meta­bolic adjustments occur to mobilize zinc from the small, exchangeable pool located in liver, pancreas, kidney, spleen, and possibly bone, to maintain zinc-dependent functions (King, 2011).

Unlike iron, absorp­tion of zinc does not change in response to alterations in whole-body zinc homeo­stasis or status. Instead, zinc absorp­tion is influenced by current zinc intake, not the past or long-term zinc intakes, or status (Chung et al., 2008; King, 2010). Figure 24c.2.
Figure 24c.2
Figure 24c.2 Effect of dietary zinc on fractional (FZA)and total zinc absorption (TAZ}. With permission from King (2010) International Journal of Vitamin and Nutrition Research 80 (4–5): 300–306.
Absorption of zinc can be measured / defi­ned in two ways: (1) the fraction (or percent) of zinc ingested that is absorbed (Fractional Zinc Absorption or FZA), determined by the dividing the amount absorbed by the amount digested, and (2) the total quantity of absorbed zinc (TAZ) over the whole day. The fraction of zinc absorbed depends on the bioavailability of the ingested zinc, whereas the total quantity absorbed is dependent mainly on the amount of zinc consumed and the bioavailability of the zinc in the diet. Intake of dietary zinc affects fractional zinc absorp­tion and the total quantity of absorbed zinc differ­ently. With increasing zinc intakes, the total amount of absorbed zinc increases while the percent absorbed declines (King, 2010, Figure 24c.2). These adjustments in the efficiency of zinc absorp­tion with changes in zinc intake are controlled by the up-regulation and down-regulation of zinc trans­porters and possibly other proteins involved in the trans­port of zinc (King and Cousins, 2014; King et al., 2015).

In addition to the amount of the dietary zinc intake, the food sources of zinc may also affect zinc absorp­tion. The dietary factor with the greatest effect on zinc absorp­tion is phytic acid (myoinositol hexakisphosphoric acid (InsP6) and its associated magnesium, potassium, and calcium salts — termed phytates. Phytic acid and its salts are found in high concen­trations in unrefined cereals, legumes, nuts and oil seeds. Phytate binds zinc in the intestinal lumen and forms an insoluble com­plex that cannot be digested or absorbed by humans because of the absence of the intestinal enzyme phytase (Iqbal et al., 1994). This inhibitory effect on zinc absorp­tion can be sub­stantial. If the habitual diet is rich in phytate, adults cannot adapt to increase zinc absorp­tion (Hunt et al., 2008) or to enhance reabsorp­tion of endogenous zinc (Hambidge et al., 2010). Whether phytate has an inhibitory effect on zinc absorp­tion in young children is uncertain. Miller et al. (2015) failed to detect a negative effect of phytate on zinc absorp­tion in their isotope studies of infants and young children. In contrast to phytate, the effect of dietary intakes of protein, calcium, iron and organic acids on zinc absorp­tion appears limited or non-existent (IZiNCG, 2004; Miller et al., 2013).

Absorption and/or utilization of zinc may also be influenced by physio­logical, host-related, and contextual factors over and above that of dietary factors (Krebs et al., 2014), Figure 24c.3. Studies have shown an increase in zinc absorp­tion in late pregnancy and lactation, most notably when dietary zinc intake is low (Fung et al., 1997; Donangelo et al., 2005; Donangelo and King, 2012), and possibly a decline with aging (Turnlund et al., 1986).
Figure 24c.3
Figure 24c.3. Conceptual diagram to illustrate determinants of zinc status, the gastro-intestinal homeostatic response, and the features associated with normal and abnormal zinc status. In the condition of impaired zinc status, the thick arrows reflect bidirectional, co-existing efects between determinants and zinc deficiency. FAZ: fractional absorption of zinc; TAZ: total absorbed zinc; SRM: saturation response model; EE: environmental enteropathy; EZP: exchangeable zinc pool. From (Krebs et al., 2014).
Not surprisingly, given the dominant role of the gastro­intestinal tract in zinc homeo­stasis, malabsorp­tive disorders that alter the integrity of the mucosal cells, reduce zinc absorp­tion, although the magnitude of their effect is uncertain. An exception are patients with celiac disease in whom impairments in absorp­tion and increases in intestinal endogenous zinc losses have been quantified through stable isotope studies (Crofton et al., 1990; Tran et al., 2011). Accumulating evidence suggests that these same disturbances in zinc absorp­tion occur in environmental enteric dysfunction (EED), a chronic inflam­matory condition linked to chronic, sub­clinical exposure to fecal pathogens (Syed et al., 2016). However, again, the magnitude of the effect of EED has yet to be quantified (Manary et al., 2010; Lindenmayer et al., 2014).

24c.1.4 Zinc defi­ciency in humans

When homeo­static mechanisms fail to ensure that require­ments for zinc are met, clinical symptoms of zinc defi­ciency ensue. The first cases of dietary zinc defi­ciency in humans were described in the 1960s in male dwarfs from the Middle East (Prasad et al., 1963). Typical clinical features, which were corrected by zinc supple­mentation, included growth retardation, delayed secondary sexual maturation (hypogonadism), poor appetite, mental lethargy, and skin changes. In North America and New Zealand, overt and severe nutritional zinc defi­ciency was first recognized in hospital patients who received either parenteral nutrition or enteral feedings without zinc supple­ments (Kay and Tasman-Jones, 1975; Arakawa et al., 1976).

Two genetic disorders are known to induce zinc defi­ciency: acrodermatitis enteropathica and sickle cell disease (King et al., 2015).

Secondary zinc defi­ciency has been documented in the presence of Crohn disease, inflam­matory bowel disease, ulcerative colitis, and malabsorp­tion syndromes. A variety of other diseases — including renal and liver diseases, diabetes, acquired immuno-defi­ciency syndrome, and alcoholism — also induce secondary zinc defi­ciency. In such cases, zinc defi­ciency may arise from either increased urinary excretion of zinc (hyper­zincuria) or excess endogenous fecal zinc losses (Aggett and Comerford, 1995).

Treatment with certain drugs has also been associated with secondary zinc defi­ciency. For example, long-term treatment with penicillamine for Wilson’s disease (Smolarek and Stremmel, 1999) diethylenetriamine pentaacetate for the treatment of iron overload in thalassemia and sickle cell anemia (King and Cousins, 2014), and the administration of chlorthiazide (used to treat edema) and glucagon (used to control blood glucose levels) (Prasad and Oberleas, 1976) have all been implicated as iatrogenic causes of zinc defi­ciency.

In some industrialized countries marginal zinc defi­ciency, charac­terized by slow physical growth, has been identified in apparently healthy infants and children (Hambidge et al., 1972a; Walravens and Hambidge, 1976; Walravens et al., 1983; Smit-Vanderkooy and Gibson, 1987; Gibson et al., 1989a). More recent double-blind preventative zinc supple­mentation studies in both industrialized and low-incom­e countries have confirmed an increasing range of other functional impairments associated with zinc defi­ciency in children, in addition to impaired growth (Brown et al., 2009). In many of these studies, abnormalities of the immune system and increased risk of some com­mon childhood infections have been reported. In settings where zinc defi­ciency is likely to be a problem, reductions in the incidence of diarrhea following preventive zinc supple­mentation have been observed, although the impact on lower respiratory infections, including pneumonia, has been less consistent (Haider and Bhutta, 2009; Brown et al., 2009; Lassi et al., 2010; Roth et al., 2010; Mayo-Wilson et al., 2014; Liu et al., 2018), and no clear effect on the incidence malaria (Brown et al., 2009; Mayo-Wilson et al., 2014). Therapeutic zinc supple­mentation has been shown to reduce the duration of acute and persistent diarrhea (Bhutta et al., 2000; Haider and Bhutta, 2009) , and is now recom­men­ded by World Health Organization to be included in diarrhea control programs (WHO/UNICF, 2004).

Reports of zinc-related neuro­behavioral abnormalities in children and/or adults include delays in child development, anorexia, dysfunction of smell and taste, irritability, mood and depression. However, studies of zinc defi­ciency or zinc supple­mentation on neuro­behavioral function, have produced inconsistent results, possibly due in part to the differ­ences in the measure­ment methods used (Warthon-Medina et al., 2015). In a meta-analysis of trials in infants and young children, for example, analysis of a small subset of randomized controlled trials (n=6) showed no significant effect of zinc on cognitive function or motor skills (Warthon-Medina et al., 2015). In contrast, results of a randomized controlled trial on Peruvian infants suggested that supple­mental zinc may support normative neuro-development in infants consuming low zinc diets (Colombo et al., 2014).

Several preventive zinc supple­mentation trials have also been conducted during pregnancy. The results have been inconsistent; only some have reported a positive effect of supple­mental zinc on pregnancy outcom­e measures. In 2007, a meta-analysis showed a 14% reduction in infants born prematurely among zinc supple­mented women, but no impact on infant birth­weight, although there was a suggestion of a positive effect on birth­weight in a subset of the women who were underweight or zinc-defi­cient (Hess and King, 2009). Two sub­sequent meta-analysis, one spanning five continents, of randomized controlled trials in which zinc supple­ments were given together with other micronutrient supple­ments, again indicated that only the effect on risk of preterm birth was significant, with no evidence of any effect on any parameter of fetal growth (low-birth­weight, birth­weight, length at birth, head circumference at birth) (Chaffee and King, 2012; Ota et al., 2015). More recently, a meta-analysis of studies of preventive zinc supple­mentation during pregnancy for three months or longer, reported no significant increase in birth­weight and no decrease in the risk of low birth­weight (Liu et al., 2018).

Zinc status is sometimes com­promised in the elderly, probably because of low zinc intakes and age-related changes in physio­logical function (de Jong et al., 2001). As well, the presence of hypochlorhydria, malabsorp­tion syndromes, and the use of certain medications may have exacerbating roles (Aggett and Comerford, 1995). There are several reports of improvements in some aspects of immune function in older adults, including nursing home elderly, following zinc supple­mentation (Bogden et al., 1994; Barnett et al., 2016). Other possible zinc-related degenerative changes in the elderly that warrant further study include hypogeusia, delayed wound healing, anorexia, deterioration of glucose tolerance, and depression (Marcellini et al., 2006; Levenson, 2006; Lin et al., 2017). For example, correlations between zinc status (based on serum zinc concen­trations) and symptoms of depression have been observed in observational studies in elderly women (Marcellini et al., 2006). Improvements in patients with depression given supple­mental zinc have also been reported, especially when the supple­mental zinc is used in com­bination with antidepressant drug therapy (Ranjbar et al., 2014).

24c.1.5 Food sources and dietary intakes

There is no functional reserve or body store of zinc, as noted earlier, so an adequate regular supply of readily absorbable zinc is required. Hence, individuals consuming a usual diet low in zinc and/or with poor zinc bioavailability may be nutritionally zinc defi­cient. Food sources of readily absorbable zinc include organ meats, red meat, poultry, fish, and shellfish; oysters are one of the richest food sources of absorbable zinc. In general, the amount of zinc is higher in dark red meat than in white meat. Starchy roots, tubers, fruits, and vegetables have a low zinc content. Zinc is less readily available in whole grain cereals, nuts and legumes: their zinc content varies, depending on the zinc content of the soil or fertilizer treatment, growing location, and processing methods (e.g., milling of cereals). Loss of zinc from most foods during cooking is minimal.

Several dietary com­ponents have been investigated as potential modifiers of zinc bioavailability. A systematic review, however, concluded that for many of these potential modifiers, data were insufficient to accurately determine their impact on zinc absorp­tion (Bel-Serrat et al., 2014). A notable exception is phytic acid (myoinositol hexakisphosphoric acid (InsP6) and its associated magnesium, potassium, and calcium salts — termed “phytates” — potent inhibitors of zinc absorp­tion, as noted earlier. In general, phytic acid and its salts are found in high concen­trations in unrefined cereals, legumes, nuts and oil seeds. Concentations vary widely depending on the botanical variety, environmental or climatic growing conditions, the use of phosphate fertilizers, and the stage of maturation. The highest levels are reached at seed maturity. During some food-processing, preparation and cooking practices, such as milling / pounding, soaking, germination / malting, mixtamalization, or fermentation, phytate is dephosphorylated to the lower myo-inositol phosphate forms (IP1 to IP4) that don't inhibit zinc absorp­tion (Gibson et al., 2018).

The inhibiting effect of phytate on zinc absorp­tion follows a dose-dependent response. Hambidge and colleagues have developed a trivariant model to predict the inhibitory effect of differ­ent levels of phytate on zinc absorp­tion as a function of zinc intake (Miller et al., 2007). Development of this model led the European Food Safety Authority (EFSA) to generate dietary zinc require­ments for adults based on four levels of dietary phytate (EFSA, 2014). When daily phytate intakes in adults reach 1200mg/d, a level surpassed in some low incom­e countries with staple diets of unfermented cereals and/or legumes, the average zinc require­ment for adults, com­piled by EFSA (2014) nearly doubles (Gibson et al., 2019). Values for the phytate content of raw and processed plant-based staples are available in the FAO / INFOODS / IZiNCG Global Food Composition Database for Phytate.

Molar ratios of phytate-to-zinc of individual foods or whole diets can be used to estimate the likely proportion of zinc absorbed. Diets with a phytate-to-zinc molar ratio of more than about 15 have poor zinc bioavailability, whereas those with ratios between 5 and 15 are said to have medium bioavailability, and those with ratios less than 5 have good bioavailability (WHO/FAO, 2004).

In the past, millimolar ratios of phytate × calcium : zinc were also used to predict zinc absorp­tion because dietary calcium was said to influence the inhibitory effect of phytate on zinc bioavailability. The use of this ratio has now been discontinued in the absence of any demonstrated effect of calcium on zinc absorp­tion from diets adequate in zinc, irrespective of whether intakes of dietary phytate were high or low (Hunt and Beiseigel, 2009). Whether calcium has an adverse effect in phytate-containing diets low in zinc is uncertain (King et al., 2015). Earlier, the amount and type of dietary protein was said to enhance zinc absorp­tion but more recent data have not confirmed this (IZiNCG, 2004; Miller et al., 2013).

A com­petitive interaction between iron and zinc may occur, depending on the form of the iron, as well as the conditions and levels under which both the iron and zinc are given (Lönnerdal, 2000). A decrease in zinc absorp­tion may occur when the ratio of iron to zinc is very high (i.e., > 25:1) and when the iron is administered in water and fed under fasting conditions. Hence, women taking high-dose prenatal iron supple­ments (60mg elemental iron/day) may require additional zinc (Caulfield et al., 1999). For example, supple­mentation with 60mg elemental iron in a multiple micronutrient supple­ment blunted the increase in serum zinc observed in Cambodian women who received the same supple­ment but with no iron (Holmes et al., 2019). However, this interaction is less apparent when the iron is provided in a com­plex food matrix (Olivares et al., 2012; Esamai et al., 2014).

In general, in omnivorous adult diets in affluent countries, meat and meat products supply the greatest amount of zinc, followed by cereals and dairy products (Briefel and Johnson, 2004; EFSA, 2014). However, interest in vegetarian diets has increased in these countries with high-phytate diets of cereals, legumes and nuts replacing meat and meat products (American Dietetic Association, 2003). Increasingly zinc fortified beverages, fortified cereals, and supple­ments are becom­ing important sources of dietary zinc, especially among young children in North America (Arsenault and Brown, 2003; Butte et al., 2010).

24c.1.6 Effects of high intakes of zinc

There are no reported cases of toxicity from excess intakes of dietary zinc, although toxicity arising from short-term exposure to very high levels of contaminant zinc (> 300ppm), from the improper storage of food or beverages in galvanized vessels, has been reported (Brown et al., 1964). Signs and symptoms include dehydration, vomiting, electrolyte imbalance, abdominal pain, nausea, lethargy, dizziness, and muscular incoordination (Fosmire, 1990).

Adverse effects of excess zinc on copper meta­bolism have been described (Prasad et al., 1978a; Fischer et al.,1984; Samman and Roberts, 1988; Yadrick et al., 1989) with a decrease in the activity of erythrocyte Cu,Zn‑SOD being the most consistent finding (Fischer et al.,1984). However, the clinical significance of a decrease in erythrocyte SOD activity is unknown.

High intakes of zinc have also been associated with reductions in serum HDL cholesterol levels in some studies (Hooper et al., 1980),. and detrimental effects on the immune system (Chandra, 1984).. Hence, excessive self-supple­mentation with zinc may have an adverse effect on health, although doses of 25–35mg/day zinc in adults do not appear to pose a health hazard (Smith, 1994). In Europe the zinc content of the most com­mon single nutrient supple­ment is 30mg per capsule (range 15–50mg), and 10–15mg (range 2–20mg) for multi-nutrient supple­ments (EFSA, 2002).

The Tolerable Upper Intake Level (UL) for zinc set by the U.S. Food and Nutrition Board for adults and pregnant and lactating women > 19y, is 40mg/d, a level based on the adverse effects of high doses of supple­mental zinc (50mg Zn/d) on copper status biomarkers, notably reductions in the activity of erythrocyte Cu,Zn‑SOD. For infants the corresponding UL is 4mg, with adjustments for children based on body weight (IOM, 2001). Concern has been raised that the UL set by U.S. Food and Nutrition Board for preschool children (i.e., 7mg/day) in the US and Canada may be too low as many have dietary zinc intakes that exceed the UL due to their consumption of zinc-fortified beverages, fortified ready-to‑eat breakfast cereals, and zinc supple­ments (Arsenault and Brown, 2003; Butte et al., 2010). Sensitive biomarkers of copper status were reportedly unchanged by supple­mentation with 5, 10, or 15mg of zinc daily for four months in healthy boys in a double-blind, placebo-controlled randomized trial (Bertinato et al 2013). EFSA (2002) has set a lower level for the UL for adults and pregnant and lactating women (25mg Zn/d), although their UL for preschoolers 1–3y is the same as that of U.S. Food and Nutrition Board (i.e., 7mg/d).

24c.1.7 Biomarkers of zinc status

Zinc is classified as a type II nutrient (Golden, 1995) because it is essential for multiple meta­bolic functions, as discussed earlier. As a consequence, in zinc defi­ciency the physio­logical effects that arise are associated with a number of diverse biochemical changes, making it difficult to identify biomarkers of zinc nutrition (King, 2011). In contrast, reductions in tissue nutrient concen­trations of type I nutrients, such as iron, iodine, and vitamin A, arising from defi­cits in dietary intakes, cause a decline in one or more specific functions which can be readily identified using selected biomarkers (King, 2011).

To identify popu­lations at elevated risk of zinc defi­ciency, three indicators were recom­men­ded by WHO / UNICF / IAEA / IZiNCG: (a) prevalence of usual zinc intakes below the estimated average require­ment, (b) percentage with low plasma/serum zinc concen­trations, and (c) percentage of children aged < 5y who are stunted (i.e., HAZ < –2SD) ; (de Benoist et. al., 2007), These recom­men­dations were endorsed by the BOND Expert Zinc Panel (King et al., 2015). For details see IZiNCG Technical Brief No. 1 (2007).

For each indicator, a “trigger level” is given for the prevalence considered indicative of elevated risk and of public health concern, at which level an intervention to improve popu­lation zinc status is warranted. Reference limits for plasma/serum zinc (depending on age, sex, time of day, fasting status) are available to identify popu­lations at risk of zinc defi­ciency, provided the correct protocols are followed for the collection, separation, and analysis of plasma zinc. Assessment of the functional indicator — impaired linear growth — should be made in com­bination with at least one of the other indicators in view of its poor specificity (King et al., 2015). In the future, a model com­prising key meta­bolic indicators, serum albumin, and a biomarker of inflam­mation may be developed for popu­lation zinc assessment (King, 2018).

Assessment of zinc status at the individual level is particularly difficult because serum or plasma zinc concen­trations are homeo­statically controlled and do not decline with marginal zinc intakes. Hence, serum or plasma zinc is only useful to measure zinc status in individuals with either a very low or high supply of dietary zinc. Instead, the recom­men­ded approach for individuals com­bines a medical history, dietary and clinical assessment, with plasma or serum zinc providing only supple­mentary data (King et al., 2015).

The BOND Zinc Expert Panel classified the other possible zinc biomarkers into three groups: (1) potentially useful (2) emerging biomarkers and (3) those deemed not useful. These are shown below.

Box 24c.1 Possible Zinc Biomarkers. From (King et al., 2015).

24c.2 Dietary Zinc Intake

Assessment of dietary zinc intakes is the best method for estimating zinc exposure in individuals and popu­lations. To determine dietary zinc intakes, a quantitative dietary method such as a weighed or estimated food record or a validated 24-h recall must be used. Details of the procedures to estimate dietary zinc intakes using a modified 24-hr recall specifically for use in lower incom­e countries are available in a Dietary Technical Monograph (#8) (Gibson and Ferguson, 2008).

Information on dietary zinc intakes can be used for several purposes. At the popu­lation level, data can be used to: estimate the prevalence of inadequate zinc intakes, classify sub­popu­lations at elevated risk, design and monitor zinc intervention programs, and identify dietary patterns that contribute to inadequate zinc intakes. At the individual level, those at risk of inadequate intakes can be identified for dietary counseling. Tools have been developed to facilitate the collection of dietary zinc intakes and analyze the data to assess dietary adequacy (IZiNCG Technical Briefs) No.3 and No.7.

Inadequate intakes of dietary zinc can arise from low intakes of zinc per se, poor bioavailability, or a com­bination of these dietary factors, as noted earlier. In low incom­e countries, where unrefined cereals and legumes containing high levels of phytate are often the major source of energy and zinc, inadequate intakes of bioavailable forms of zinc are the most likely cause of zinc defi­ciency. Other diets based on starchy roots or tubers have a low total zinc and phytate content. Values for the phytate content of raw and processed plant-based staples are available in the FAO / INFOODS / IZiNCG Global Food Composition Database (Infoods Phytate Database). When collecting dietary intake data for zinc, intakes of dietary phytate should also be determined, whenever possible.

Data on both zinc and phytate intakes permit the calculation of phytate-to-zinc molar ratios from which an estimate of the bioavailability of zinc can be obtained, as noted in Section 24c.1.5

24c.2.1 Measurement of zinc intake for popu­lations

The recom­men­ded dietary indicator to identify a popu­lation or popu­lation sub­group at elevated risk of zinc defi­ciency is the prevalence of usual zinc intakes below the estimated average require­ment (King et al., 2015). Because information on usual zinc intakes is required, food intakes must be measured on two non-consecutive days or three consecutive days. It is preferable that multiple days of intake data are collected for all individuals in the popu­lation. However, if this is not possible, then at least two non-consecutive days of dietary intake data from a sub-sample (40–50) of individuals per stratum should be collected. Figure 24c.4
Figure 24c.4
Figure 24c.4 Estimates of usual intake distribution for zinc for New Zealand adults obtained from 24h recall data and adjusted with replicate intake data using the refined NRC method. The y-axis (frequency of intake) shows the likelihood of each level of intake in the population. EAR, Estimated Average Requirement. With permission. from Gibson RS et al., (2003) The Risk of Inadequate Zinc Intake in United States and New Zealand Adults. Nutrition Today. 2003 Mar-Apr;38(2): 63–70.
doi: 10.1097/00017285-200303000-00010.
These data permit an estimate of the day-to-day variation in zinc intake within one individual (i.e., within-person variation) to be estimated, allowing a distribution of usual zinc intakes for the popu­lation group to be generated using specialized software. Finally, the Estimated Average Requirements (EARs) for zinc, specific for age, sex, physio­logical status, and in some cases the phytate-to-zinc molar ratio of the diet, are needed to calculate the prevalence of inadequate intakes. If quantitative data on phytate intakes are not available, then the phytate-to-zinc molar ratio can be estimated based on the diet type. Figure 24c.4 shows an example com­paring the adjusted distributions of usual zinc intakes with the observed one-day zinc intakes for New Zealand adult females aged 19–50y, using the program developed by Iowa State (ISU) and implements the ISU method (Nusser et al., 1996) for the adjustment. The adjustment process used yields a distribution with reduced variability that preserves the shape of the original observed distribution (Gibson et al., 2003). The example also shows that in this particular case, adjusting the distribution significantly reduces the proportion of individuals considered to have intakes below the EAR. An elevated risk of zinc defi­ciency in the popu­lation is said to exist when 25% or more of the popu­lation have zinc intakes less than the EAR.

The five main steps required to calculate this recom­men­ded dietary indicator are summarized in Box 24c.2

Box 24c.2 Steps to determine the dietary intake at the popu­lation level

24c.2.2 Interpretation of zinc intakes for popu­lations

Several expert groups have set dietary zinc recom­men­dations that include EARs for zinc, although discrepancies exist, depending on the sources of the data, the concepts and methods used, as described in Gibson et al. (2016). For example, the EARs for zinc set by the United States and Canada are based on a fixed adjustment for zinc absorp­tion from habitual diets for both children and adults (Food and Nutrition Board, 2001). In contrast, the European Food Safety Authority (EFSA, 2014) have generated dietary zinc require­ments based on four levels of dietary phytate intake for adults (>  18y) and said to cover the average phytate intakes in European popu­lations: (300mg/d; 600mg/d; 900mg/d; 1200mg/d). In view of the uncertainty about whether recom­men­dations for dietary zinc based on phytate intakes can also be made for young children, the EFSA EARs for infants and children are not adjusted for phytate.

The prevalence of usual zinc intakes below the EAR is simply estimated by calculating the percentage of individuals within a specific life-stage group with usual intakes below the respective EAR. An elevated risk of zinc defi­ciency in the popu­lation is said to exist when 25% or more of the popu­lation have zinc intakes less than the EAR. However, to assist with the interpretation of the risk of zinc defi­ciency, it is recom­men­ded that estimates of the adequacy of zinc intakes be com­bined with biochemical data on serum zinc concen­trations (IZiNCG Technical Brief) No.3.

In cases where the amount of zinc likely to be absorbed is calculated for non-pregnant and non-lactating adults ≥ 19y using the updated trivariate saturation response model of Miller et al. (2007), then the prevalence of absorbable zinc intakes below the appropriate physio­logical require­ment for absorbed zinc should be calculated. For IZiNCG, these require­ments are 1.86mg/d for adult women and 2.69mg/d for adult men ≥ 19y of age. For further details of this calculation see (IZiNCG Technical Brief) No.3.

24c.2.3 Measurement and interpretation of usual zinc intakes for individuals

Usual intakes of dietary zinc at the individual level can also be determined using quantitative dietary methods such as a weighed or estimated food records or validated 24-h recalls, provided a large number of measure­ment days per individual are conducted. Details on how to defi­ne the desired number of replicate days per individual are available in Gibson and Ferguson (2008). Alternatively, a dietary history or validated semi-quantitative food frequency questionnaire can be used to provide retrospective information about usual food consumption patterns, preferably over more than one month; details are available in Thompson et al. (2015). Assessment of the intake of zinc-rich foods (e.g., meat, poultry, fish), zinc-fortified foods, and high-phytate foods (e.g., unrefined cereals, nuts, and legumes) can be used to predict the intake of absorbable zinc (King et al., 2015). Inferences can be made about the adequacy of the zinc intake of an individual by com­paring the differ­ence between the estimate of the usual zinc intake of the individual and the respective values for the EAR and Recom­mended Nutrient Intake (RNI) for zinc. If the usual intake of the individual is greater than the RNI, then there is a high level of confidence that the usual intake is adequate, whereas if the usual intake is between the EAR and the RNI, the usual zinc intake probably needs to be improved, and if the usual intake is less than the EAR, then the usual intake very likely needs to be improved. Details on the guidelines for the qualitative interpretation of individual intakes are available from the Institute of Medicine (2000).

24c.3 Serum zinc

Serum and plasma zinc concen­trations can be used interchangeably because differ­ences between these two sources of circulating zinc are very small (English and Hambidge, 1988). Fasting serum zinc concen­trations are homeo­statically controlled within fairly narrow limits (about 80–100µg/dL (12–15µmol/L). Only 0.1% of the total body zinc is present in the serum, whereas 3% and 63% are in the liver and skeletal muscle, respectively (Table 24c.1). Of the zinc in whole blood, from 12% to 22% is in the serum; the remainder is within the erythrocytes. Zinc is trans­ported in the serum bound principally to albumin (70%), so conditions that alter serum albumin levels will, in turn, affect serum zinc concen­trations. The remaining zinc in serum is tightly bound to α2‑macroglobulin (18%), and the rest is bound to other proteins such as trans­ferrin and ceruloplasmin and to amino acids, primarily histidine and cysteine (Cousins, 1985).

Serum zinc concen­tration is considered the best available biomarker of zinc exposure, status, and risk of zinc defi­ciency at the popu­lation level (de Benoist et. al., 2007; Lowe et al., 2009; Hess et al., 2007). Evidence for this recom­men­dation is derived from investigations on the following: (1) the effect of dietary zinc restrictions and repletion on serum zinc concen­trations; (2) the effects of zinc supple­mentation on serum zinc; (3) the association between serum zinc and clinical signs of zinc defi­ciency; and (4) a com­parison of initial serum zinc concen­trations between individuals who do or do not show a functional response to the correction of zinc defi­ciency (King et al., 2015).

In brief, in severe dietary zinc restriction (i.e., < 1mg/d) in previously healthy adults, there is a sharp decrease in serum zinc concen­trations, with values returning to baseline levels within 1–2 weeks of zinc supple­mentation Figure 24c.5.
Figure 24c.5
Figure 24c.5. Changes in plasma zinc (mean±SD) at the end of basal, depletion, and repletion periods. The shaded area represents the normal range. From Baer and King, American Journal of Clinical Nutrition 39: 556–570, 1984 © oup.com
In more moderate dietary zinc restrictions (3–5mg/d), however, reductions in serum zinc only occur after prolonged periods of a restricted diet or when accom­panied by high phytate intakes (Gibson et al., 2008).

Supplemental zinc consistently and rapidly increases serum zinc in both children and adults regardless of initial serum zinc concen­trations (Hess et al., 2007; Wessells et al., 2020). Of interest is the finding that serum zinc responds less to additional zinc provided in food than to additional zinc supplied as a supple­ment administered between meals. It is possible that absorbed zinc may be meta­bolized differ­ently when consumed in food or as a supple­ment (King et al., 2015).

Low serum/plasma zinc has been related to clinical signs of zinc defi­ciency based on data from experimental zinc-depletion / repletion studies and case reports of acrodermatitis enteropathica. From these data, the sensitivity and specificity of a plasma zinc below 50µg/dL (7.65µmol/L) for detecting clinical signs of defi­ciency was 82% and 92%, respectively (Wessells et al., 2014).

Finally, if the initial serum zinc is very low, a functional response to zinc supple­mentation may occur. Functional responses may include increases in growth (including linear growth, weight gain, and accrual of fat-free mass) and decreased morbidity from diarrhea and respiratory infection. Of these responses, IZiNCG has selected linear growth outcom­e as the preferred functional response based on height- or length-for‑age (de Benoist et. al., 2007).

Serum or plasma zinc has been used to assess the risk of zinc defi­ciency in several national surveys. The popu­lation is considered to have an elevated risk of zinc defi­ciency of public health importance when a particular popu­lation has more than 20% of individuals with a serum zinc concen­tration below the age and sex-specific cutoffs. In a review of national surveys in twenty low‑ or middle-incom­e countries the prevalence of low plasma zinc was more than 20% in 68% of the children and 93% of the women (Hess, 2017), com­pared to the US NHANES 2011–2014 survey where only 3.8% of children (6– < 10y) and 8.2% of females (≥ 10y) had low serum zinc concen­trations (Hennigar et al., 2018).

In general, national surveys (Gibson et al., 2011; Engle-Stone et al., 2014; Hennigar et al., 2018). have not shown a positive relationship between individual dietary zinc intakes and serum zinc concen­trations. Meta-analyses conducted on adults (Lowe et al., 2012), pregnant and lactating women (Moran et al., 2012a), and children (Moran et al., 2012b). showed only small changes in serum zinc when dietary intakes were doubled. These findings are not unexpected in view of the strong homeo­static mechanisms that prevent changes in serum zinc when zinc levels in the diet fluctuate. King (2018) has emphasized that doubling dietary zinc intake in adults only results in a 6% change in serum zinc concen­trations, a differ­ence within the margin of error in measuring serum zinc. Among some popu­lation groups in whom high-phytate cereals and legumes replace animal source foods as the major source of dietary zinc (e.g., vegetarians), significant inverse relationships between serum zinc and dietary phytate : zinc molar ratios have been observed (Donovan and Gibson, 1995; Cantoral et al., 2015).

For an individual, serum zinc concen­trations are not a reliable indicator of zinc status due to both the strong homeo­static control mechanisms and the many factors other than zinc intake that can independently influence serum zinc concen­trations (see below). This means that serum zinc concen­trations may be within the normal range despite the presence of mild zinc defi­ciency generating growth retardation, loss of appetite, or impaired immune function. Currently for individuals the recom­men­ded approach com­bines a thorough medical history of risk factors for zinc defi­ciency, a qualitative dietary assessment to identify an inadequate dietary intake, and a clinical assessment, with plasma or serum zinc providing only supple­mentary data (King et al., 2015).

In individuals with more severe zinc defi­ciency, with manifestations such as skin lesions, hair loss, diarrhea, delayed sexual maturation, impotence, hypogonadism in males, and eye lesions, serum zinc concen­trations are usually low. For example, markedly low serum zinc concen­trations are present in individuals with the genetic disorder acrodematitis enteropathica (Wessells et al., 2014), in patients receiving TPN unsupple­mented with zinc (Arakawa et al., 1976), and in those individuals who have developed clinical signs during experimentally-induced zinc defi­ciency (King et al., 2015).

24c.3.1 Factors affecting serum zinc

Age-related changes in serum zinc concen­trations (Table 24c.3)
Table 24c.3 Median ±SE serum zinc levels of males and females in the United States by age. Data from the US NHANES 2011–2014 (Hennigar et al., 2018 J Nutr. 2018 Aug 1;148(8) :1341–1351.) (For conversion to µmol/L, divide by 6.536)
Age (years) Male serum
zinc (μg/dL)
Female serum
zinc (μg/dL)
6–8 80.8±1.4 77.7±0.9
9–13 81.7±1.2 81.1±1.6
14–18 85.5±2.3 81.3±1.9
19–30 84.9±1.4 77.9±1.3
31–50 84.4±0.7 79.6±0.7
51–70 82.4±1.9 79.8±1.5
≥71 78.7±2.3 80.5±1.5
may be apparent, with levels increasing with age in some (Pilch and Senti, 1984; Villalpando et al., 2003; Gibson et al., 2011), but not all (Hennigar et al., 2018) national surveys and in some smaller studies (Rükgauer et al., 1997). In the 2011–2014 US NHANES III survey, where serum zinc was determined in males and females aged ≥ 6y, only the serum zinc concen­trations for the males (not the females) increased from the ages of 6–30y, declining thereafter

Gender may influence serum zinc concen­trations. During infancy and early childhood, boys tend to have lower serum zinc levels than girls (Smit-Vanderkooy and Gibson, 1987; Cavan et al., 1993a; Gibson et al., 2011). In the US NHANES III 2011–2014 survey males from 6y to adults ≥ 71y had higher serum zinc concen­trations than females (Table 24c.3), a relationship that may be related to differ­ences in body size and lean body mass.

Diurnal variation in serum zinc concen­trations has been reported. Levels are higher in serum in the morning, regardless of fasting status, com­pared to the afternoon (Pilch and Senti, 1984). In the US NHANES III 2011–2014 survey, serum zinc concen­trations were 9% lower in the blood samples drawn in the afternoon (Hennigar et al., 2018),(Table 24c.4)

Fasting status also markedly affects serum zinc concen­trations. During a usual overnight fast of ≥ 8h, serum zinc levels increase, generally reaching a peak just before breakfast. Fluctuations in serum zinc also occur throughout the day, mainly in response to the consumption of meals. Serum zinc levels decline progressively for several hours after each meal, before rising prior to the next meal (King et al., 1994). Hence, in addition to the time of day, fasting status and meal status should also be carefully controlled during the collection of blood samples for serum / plasma zinc to avoid confounding the results. Alternatively, the time of day and the time elapsed since the previous meal can be recorded and taken into account statistically during analysis (Diana et al., 2017) (Table 24c.4).

Table 24c.4 Median serum zinc for different collection times from NHANES III. Data from the US NHANES 2011–2014 (Hennigar et al., 2018 J Nutr. 2018 Aug 1;148(8) :1341–1351.) (For conversion to µmol/L, divide by 6.536)
All participants
Time/fasting status
n Median serum
zinc (µg/dL ± SE)
Morning, fasting142386.8 ± 0.8
Morning, nonfasting8985.0 ± 3.1
Afternoon112476.9 ± 0.9
Evening48973.8 ± 0.9

Hemolysis either in vivo or in vitro has the potential to increase serum zinc concen­trations because the concen­tration of zinc in erythrocytes is about 10–20 times higher than in serum or plasma. Hemolysis may be particularly important in cases of zinc defi­ciency, when red cell fragility increases (Bettger et al., 1978). Killilea et al. (2017) suggest that a 5% increase in plasma or serum zinc concen­tration occurs with each 1g hemoglobin/L in plasma or serum, a concen­tration that can be identified by chemical hemoglobin assays or by matching to a color scale. A threshold of 1g hemoglobin/L is recom­men­ded for measure­ments of plasma or serum zinc to avoid significant increases in zinc caused by hemolysis.

Storage of blood samples prior to separation affects serum zinc concen­trations. Long intervals prior to separation are associated with progressively increasing serum zinc concen­trations as zinc is released from platelets (English and Hambidge, 1988). To avoid this increase, serum or plasma should be separated rapidly from the red blood cells within 30–40 minutes after collection. If this is not feasible, the blood should be held at 2–10°C for no more than 24 hours to limit movement of cellular zinc into the plasma or serum (IZiNCG Technical Brief No.2, 2007).

Prolonged use of a tourniquet may result in an increase in serum or plasma zinc levels. This occurs as a result of increased intravascular pressure caused by venous occlusion, which may cause movement of fluid into the interstitial space, thus increasing the zinc concen­tration (Juswigg et al., 1982). Hence, tourniquet occlusion should only be used for about one minute (King et al., 2015).

Acute and chronic infection and systemic inflam­mation leads to serum or plasma zinc values that are spuriously low because of the transfer of zinc from the blood to the liver. This redistribution of zinc is caused by hepatic synthesis of metallo­thionein activated by the release of cytokines during the acute phase response (Raiten et al., 2015). Both the stage and severity of the infection influence the change in plasma zinc, but these changes do not correspond to a change in zinc nutrition. To adjust for the presence of systemic inflam­mation, two inflam­matory biomarkers (C‑reactive protein (CRP) and α‑1‑acid glycoprotein (AGP)) should be measured along with serum or plasma zinc, allowing a regression-based correction to be applied (McDonald et al., 2020).

Evaluation of zinc intervention programs requires scheduling the end-line blood collection before the end of the intervention because serum zinc concen­trations rapidly return to baseline levels after the withdrawal of supple­mentation (King et al., 2015).

Pregnancy is associated with decreases in serum zinc concen­trations irrespective of zinc intake Donangelo and King, 2012). The decline is evident at two months of gestation and at this early stage is attributed to hormonal changes, whereas the later decline is linked to plasma volume expansion (King, 2018). At the end of pregnancy, serum zinc concen­trations are about 20–25% lower than pre-pregnancy values. (Hennigar et al., 2018). In the US NHANES III 2011–2014 survey, the geometric mean ± SD serum zinc for pregnant women (n=34) was 69.1±l2.0µg/dL vs. 79.1±1.9µg/dL (10.6±0.3 vs. 12.1±0.3µmol/L) for the age-matched non-pregnant women popu­lation; data by month of pregnancy are not available. The median serum zinc concen­trations by month of pregnancy, drawn from the NHANES II data is shown in Figure 24c.6.

Figure 24c.6
Figure 24c.6. Median serum zinc concentrations during pregnancy at 1-mo intervals with the line of linear regression. From Brown et al., (2004) Food and Nutrition Bulletin 25 (1 supp. 2) : S94–S203. © SAGE Publishing

Hypoalbuminemia (serum albumin ≤ 3.5g/dL) is associated with an increased risk of low serum zinc concen­trations because about 60–70% of the serum zinc is bound to albumin (Cousins, 1989). Conditions such as inflam­mation and infection, and disease states such as alcoholic cirrhosis and protein-energy malnutrition, result in a decline in serum zinc concen­trations concurrent with hypoalbuminemia which are probably independent of an individual’s zinc status (King, 2018). Not surprisingly, in the US NHANES III 2011–2014 survey, low serum albumen concen­trations were associated with an increased risk of low serum zinc (Hennigar et al., 2018).

Estrogen-containing preparations, such as oral contraceptive agents and other hormones, when used, may lead to markedly lower serum zinc oncentrations (Swanson and King, 1982), depending on the hormone dosage (Kamp et al., 2011). (Table 24c.5).

Table 24c.5 The effect of oral contraceptive (OC) use on serum zinc and alkaline phosphatase concentrations. aAfter cor­rection for age. Data from Gibson et al., British Journal of Nutrition 86: 71–80, 2001.
Participants (n) Mean serum
zinc (µmol/L)
Mean serum
alkaline phos-
phatase (U/L)
Non-users (202) 12.1122.8
OC Users (128) 11.81 19.5
Probability of
significant diff. a
0.05 0.01
In the US NHANES III survey, however, there were no differ­ences in serum zinc concen­trations between oral contraceptive users and nonusers, perhaps because of the now decreased hormone dosage (Hennigar et al., 2018). The IZiNCG Technical Brief No.2 (2007) advises the collection of data on the use of oral contraceptive agents and other hormones in studies measuring serum zinc concen­trations.

Rapid synthesis of tissues during growth may lead to a fall in serum or plasma zinc. This effect has been reported in children during the anabolic phases of recovery from malnutrition (Golden and Golden, 1981) and during the rapid growth that occurs in preterm infants (Altigani et al., 1989). The decline in plasma zinc probably arises from the increased uptake of zinc from the exchangeable zinc pool in the plasma, induced by the increased demands for zinc for growth (Aggett and Comerford, 1995).

Weight loss or starvation releases zinc from muscle breakdown, resulting in transient increases in serum zinc concen­trations (Henry and Elmes, 1975; IZiNCG Technical Brief No.2, 2007).

Anemia has been associated with low serum zinc concen­trations in a few studies in children in both high-resource (Cole et al., 2010; Houghton et al., 2016) and low resource (Wieringa et al., 2016) settings, in pregnant women in rural Ethiopia (Gibson et al., 2008), and in females in the 2011–2014 US  NHANES III survey (Hennigar et al., 2018). Several mechanisms may explain the association of serum zinc and hemoglobin concen­trations (King, 2018): zinc-dependent enzymes are needed for heme synthesis, and zinc may also stabilize the erythrocyte membrane, thereby protecting the membrane from degradation during oxidative stress (O’Dell, 2000).

Malabsorp­tion syndromes and inflam­matory bowel diseases result in low serum or plasma zinc concen­trations arising from alterations in the integrity of the mucosal cells and a reduction in zinc absorp­tion (King and Cousins, 2014).

Environmental enteric dysfunction is known to impair zinc absorp­tion and increase intestinal endogenous zinc losses (Lindenmayer et al., 2014) that together result in low serum or plasma zinc concen­trations (Manary et al., 2010).

Sickle cell disease has been associated with low plasma zinc concen­trations in children and young adults (Abshire et al., 1988). This has been attributed to increased urinary zinc excretion, possibly mediated by increased zinc mobilization from bones due to recurrent bone ischemia (Schimmel et al., 2016).

High levels of supple­mental iron beyond those usually consumed in the diet may reduce serum zinc concen­trations due to a decrease in zinc absorp­tion. The mechanism is uncertain. Iron and zinc are thought to com­pete for intestinal absorp­tion via the shared divalent metal trans­porter 1 (DMT1) and/or another com­mon pathway in the apical membrane of the intestinal cell (Gunshin et al., 1997). The adverse effect on zinc absorp­tion, however, is less when lower amounts (≤ 10mg) of iron supple­ments are given (Fischer Walker et al., 2005) or when both the iron and zinc are provided in a com­plex food matrix (Olivares et al., 2012; Esamai et al., 2014).

24c.3.2 Interpretive criteria

Table 24c.4 (shown above) presents the medium serum zinc by fasting status and time of sampling for all NHANES III participants.

The International Zinc Consultative Group (IZiNCG) reanalyzed serum zinc data from NHANES II. In this reanalysis, data for participants with conditions significantly affecting serum zinc concen­trations were excluded. These were individuals: with low serum albumin (< 3.5g/dL); with an elevated white blood cell count (> 11.500 cells per µL); using oral contraceptive agents, hormones or steroids; or experiencing diarrhea. The IZiNCG also took age, gender, fasting status (i.e., > 8h since the last meal), and time of day of the sample collection into account in the reanalysis; details are given in (Hotz et al., 2003) and (IZiNCG, 2004).

From these reference data on healthy individuals, IZiNCG defined statistically derived reference limits based on the 2.5th percentile of serum zinc concen­tration for males and females aged < 10y and ≥ 10y (by fasting status) and time of sampling. These are given in (Table 24c.6) for children < 3y. IZiNCG suggests that the cutoffs for children < 10y also be applied to children < 3y, until appropriate reference data are available (IZiNCG, 2004).

Table 24c.6 Suggested lower reference limits (2.5th percentiles) (µmol/L) for serum zinc concentration based on age group, sex, fasting status, and time of day collection, derived from the NHANES II data. For conversion to µg/dL, divide by 0.1530. Adult reference limits based on data from subjects 20y and older only. NA, not available. Data from Brown et al., (2004) Food and Nutrition Bulletin 25 (1 supp. 2): S94–S203.
Collection
time
Children
< 10y
Males
≥ 10y
Females
≥ 10y
AM fasting NA 11.310.7
AM other 9.910.710.3
PM 8.7 9.39.0

Only a small number of pregnant women were examined during NHANES II (n=61). The 2.5th percentile values for the first and second plus third trimesters of pregnancy were calculated by IZiNCG and are 8.6 and 7.6µmol/L, respectively. No estimate for the 2.5th percentile serum zinc value for lactating women can be derived from the NHANES II survey results because of the limited sample size. Instead, at present the cutoff derived for nonpregnant women should be used for lactating women (IZiNCG, 2004). Further reference data are required to confirm and develop lower cutoffs for pregnant and lactating women and infants and children < 3y.

In addition to recording age group and sex, data on both fasting status (including the time interval since the last meal) and the time of day of the blood collection, should also be collected. The IZiNCG Technical Brief No.2 (2007) also recom­mends measuring an acute-phase protein indicative of infection or inflam­mation to assist in the interpretation of the serum zinc results. Ideally the two acute phase proteins recom­men­ded by WHO (2014) — C‑reactive protein (CRP) and α1‑acid glycoprotein (AGP) — should be measured as elevated levels could indicate underlying inflam­mation, which reduces serum/plasma zinc concen­trations (Raiten et al., 2015). Hence, before applying the appropriate IZiNCG reference limits, serum or plasma zinc concen­trations may need to be adjusted statistically for the time of day, the time interval since the previous meal (Arsenault et al., 2011), and inflam­mation (Macdonald et al., 2020), in order to provide a true estimate of the prevalence of low serum zinc concen­trations. To ascertain whether adjustment for inflam­mation is warranted, correlation and decile analysis between plasma / serum zinc concen­trations and CRP or AGP should be conducted. If the correlation is negative and significant, a visual inspection of the decile analysis should be undertaken to confirm whether there is an increasing prevalence of zinc defi­ciency by CRP or AGP decile (McDonald et al., 2020). If either of the latter is observed, then the regression correction approach recom­men­ded by the Biomarkers Reflecting Inflammation and Nutritional Determinants of Anemia (BRINDA) Project should be used to adjust the serum / plasma zinc concen­trations affected by inflam­mation. In this approach the inflam­matory biomarkers (CRP and AGP) are treated as continuous variables so that greater corrections can be applied when the inflam­matory biomarkers indicate severe inflam­mation (Namaste et al., 2017; McDonald et al., 2020).

IZiNCG (Technical Brief No.2, 2007) has recom­men­ded that if more than 20% of the popu­lation (or popu­lation sub-group) has a serum zinc concen­tration (after appropriate adjustment) below the relevant reference limit (based on age, sex, time of day of the blood collection, and fasting status), the whole popu­lation (or sub-group) should be considered to have an elevated risk of defi­ciency of public health importance. This “trigger level”, however, can be modified depending on the available resources within a country to control zinc defi­ciency (King et al., 2015).

Not surprisingly, the cutoff associated with overt clinical signs of zinc defi­ciency (i.e. 50µg/L, 7.6µmol/L) (Wessells et al., 2014) is higher than the statistically derived IZiNCG reference limits presented in Table 24c.6 that are based on reference data from healthy individuals. As such, these IZiNCG reference limits provide a greater margin of safety for assessing an increased risk of zinc defi­ciency at the popu­lation level before the appearance of clinical signs (King et al., 2015).

24c.3.3 Measurement of serum zinc

Blood samples for serum or plasma zinc should be taken under carefully controlled, standardized conditions. Contamination from various sources such as preservatives, evacuated tubes, lubricants, anticoagulants, water, and rubber stoppers must be avoided. The reader is advised to consult the IZiNCG website for practical tips on the collection of blood samples for the measure­ment of serum or plasma zinc (IZiNCG Technical Brief No.2, 2007). For venipuncture blood samples, trace-element-free evacuated tubes with siliconized rather than rubber stoppers must always be used. Stainless steel or siliconized needles and Teflon or polypropylene catheters can all be used. For capillary blood samples, the use of polyethylene serum separators with polyethylene stoppers and olefin-oligomer is recom­men­ded (Iyengar et al., 1998). As noted earlier, fasting status, time of day of blood collection, time elapsed since the previous meal and since the blood sample was centrifuged, and presence of inflam­mation should all be recorded so that serum zinc values can be adjusted statistically as needed (Arsenault et al., 2011) prior to selecting the appropriate IZiNCG reference limits.

Box 24c.3 shows pre-collection considerations recom­men­ded by IZiNCG (2007) to minimize contamination during the collection of samples for serum / plasma zinc:

Box 24c.3 Pre-collection considerations
Once the blood has been collected, blood samples should be placed in a refrigerator or on ice and allowed to clot for 30–40 minutes, and then centrifuged to separate the serum or plasma. This approach minimizes any transfer of zinc from the cells to the serum or plasma. Alternatively, if centrifugation on site is not feasible, then the blood should be placed in a cool box or refrigerator immediately after collection where it can be held at 2–10°C for up to 24 hours prior to being centrifuged and the serum or plasma separated. After separation, the serum or plasma should be trans­ferred to a trace-element free screw-top vial for storage either in a refrigerator (for up to several days) or frozen until analyses. Any obviously hemolyzed samples (i.e., > 1g hemoglobin/L) should be discarded (Killilea et al., 2017; IZiNCG Technical Brief No.xx, 2020).

Some investigators prefer to use plasma because it is readily separated, less susceptible to platelet contamination, and not subject to contamination from a reaming instrument (Smith et al., 1985). For plasma samples, the recom­men­ded anticoagulant is zinc-free heparin. Before using any other anticoagulant it should be screened for contaminant zinc. For more details, see King et al. (2015).

The most frequently used method for the analysis of serum zinc is flame atomic absorp­tion spectrom­etry (AAS), using a direct method in which the samples are diluted by 5 to 10-fold in solvents such as 6% aqueous butanol or 10% aqueous propanol (King et al., 2015). Dilution of the serum reduces the viscosity of the sample, which minimizes both the matrix effect on the rate of aspiration and the tendency for the flame atomic absorp­tion burner-head to becom­e blocked. This may be especially a problem with plasma because of precipitates that form in these samples. Sometimes the serum or plasma samples are ashed at low temperatures prior to analysis by flame AAS. For very small samples, flameless AAS can be used. Increasingly, inductively coupled plasma (ICP) atomic emission spectrom­etry or ICP mass spectrom­etry are being used.. For all methods, in‑house quality controls and standard reference materials (SRMs) such as bovine serum (SRM 1598) from the National Institute of Standards and Technology should be used. A coefficient of variation of < 5% is attainable for zinc using flame AAS. Use of a trichloroacetic acid deproteinization technique is not recom­men­ded.

Box 24c.4 shows the optimal procedures for the collection and analysis of serum or plasma samples to avoid adventitious trace element contamination. as recom­men­ded by IZiNCG (2004).

Box 24c.4 Optimal collection and analysis procedures

Practical tips for implementing these procedures are provided in IZiNCG Technical Brief No.2 (2012). Serum or plasma samples for zinc analyses can be refrigerated (4°C) for 2–3 weeks prior to analysis. For longer storage periods, samples should be frozen at −25°C or below. To prevent dehydration during long-term storage, especially if frost-free freezers are used, serum or plasma samples should be stored together with ice cubes in sealed plastic bags.

24c.4 Linear growth

The limited sensitivity and specificity of linear growth and other similar “bioindicators” means linear growth must be measured alongside other biomarkers of zinc (i.e., plasma or serum zinc) and other growth-limiting nutrients to establish the role of zinc in poor growth. There is no pharmacological effect of zinc on growth in zinc-replete individuals and linear growth is considered the best functional bioindicator associated with the risk of zinc defi­ciency in popu­lations (King et al., 2015). Other functional bioindicators known to be responsive to zinc supple­mentation (e.g., diarrheal episodes) are difficult to defi­ne in a standardized manner. Justification for the selection of linear growth as the zinc functional bioindicator is summarized below (Fischer -Walker and Black, 2005).

Box 24c.5
In a study by Wessells and Brown (2012), the prevalence of inadequate zinc intakes in 138 low- and middle-incom­e countries was estimated from food balance sheet data. In these countries the prevalence of stunting in children less than 5y was positively correlated with the estimated prevalence of inadequate zinc intake (r=0.48. P=0.0001) as shown in Figure 24c.7.
Figure 24c.7
Figure 24c.7 Relationship between the estimated prevalence of inadequate zinc intake and the prevalence of childhood stunting. Stunting data (low height-for-age) are for children less than 5y in138 low- and middle-income countries. The solid line represents the line of identity (intercept=0, slope=1). The dashed line represents the best-fit regression line. Dotted lines demarcate countries with a high risk of inadequate zinc intake and where the prevalence of stunting is > 20% From Wessells KR, Brown KH. PLoS One. 2012;7(11):e50568.
The figure also defines an at-risk group of 32 countries where the estimated prevalence of inadequate zinc intake is > 25%, and the prevalence of stunting is > 20%. Nevertheless, both inadequate intakes of zinc and stunting only provide suggestive evidence of zinc defi­ciency, and the multi-factorial causes of childhood stunting may be responsible, at least in part, for the marked variability around the “best-fit” regression (dashed) line.

24c.4.1 Interpretive criteria

The percentage of children < 5y with height- or length-for‑age less than −2SDs below the age-specific median of the WHO Multicentre Growth Reference Study (MGRS) popu­lation is recom­men­ded for assessing the zinc status of popu­lations (WHO, 2006). Risk of zinc defi­ciency is considered to be of public health concern when the prevalence of low height‑ or length-for‑age Z-scores among children aged less than 5y is ≥ 20% (de Benoist et. al., 2007. The prevalence of low height- or length-for-age Z-scores (HAZ) for children 0–5y can be calculated from the WHO Child Growth Standard (WHO , 2006) and the com­puter program WHO (AnthroPlus). Note that in a healthy popu­lation of children the mean Z-score will be about 0.0 and the SD of the Z-score about 1.0; 2.5% of all the childen will have an HAZ-score < –2.

24c.4.2 Measurement of height or length

For infants and children ≤ 85cm (i.e. ≤ 2y), recumbent length is the recom­men­ded measure, preferably with the use of an infantometer with a range of 30–110cm, equipped with a digital counter reader. Recumbent length should be recorded to the nearest millimeter, or even more precisely (i.e., 0.1mm) when possible. Wooden or acrylic length measuring boards can be used, but they are rarely fitted with digital counters so are less reliable. Note that recumbent length for a child of about 2y is about 5mm greater than standing height for the same child (Haschke and van’t Hof, 2000).

Children > 85cm and adults should be measured in the standing position, preferably using a free-standing stadiometer (range 65–206cm), again equipped with a digital counter reader capable of measuring stature to 0.1mm. Platform scales with movable measuring rods should not be used as they are less accurate. Clothing should be minimal when height measure­ments are taken so that posture can be clearly seen. Shoes and socks should not be worn. The timing of the measure­ment should be recorded; diurnal variations in height occur due to com­pression of the spine as the day progresses (Buckler, 1978). Consequently, in popu­lation studies, standing height should always be measured at the same time of day, preferably in the afternoon.

When measuring recumbent length or standing height, attempts should be made to minimize measure­ment errors. In longitudinal studies involving sequential measure­ments on the same group of individuals, one person should conduct all of the measure­ments throughout the study to eliminate between-examiner errors. This is especially critical when growth velocity is estimated; growth increments are generally small and are associated with two error terms, one for each measure­ment occasion. Recom­mendations of the minimal intervals necessary to provide reliable data on growth increments during infancy and early childhood are available (de Onis et al., 2004). In the WHO MGRS, the minimal interval recom­men­ded for reliable data on length measure­ments was every two weeks for infants from 2–6 weeks of age, monthly for ages 2–12 months, and bimonthly in the second year. During adolescence, increments measured over 6 months are the minimum interval recom­men­ded (WHO, 1997). For shorter intervals, the com­bined errors may be too large in relation to the expected mean increment.

In large regional surveys, several well-trained anthro­pometrists are often needed to rotate among the participants to reduce the effect of measure­ment bias. Regular standardization sessions to assess both within‑ and between-examiner reliability should be conducted throughout the data collection period to maintain the quality of the measure­ments and to identify and correct systematic errors in the measure­ments; details of the procedures used in the WHO Multicenter Growth Reference Study (MGRS) are given in de Onis et al. (2004). An anthro­pometric training video prepared for the WHO MGRS is also available on request from WHO.

The WHO MGRS recom­mends that the maximum allowable differ­ence in length for acceptable precision between measure­ments by two anthro­pometrists is 7.0mm. (de Onis et al., 2004). Details of the measure­ment techniques and standardization protocols for both recumbent length and stature are given. Statistical methods exist for removing anthro­pometric measure­ment errors from cross-sectional anthro­pometric data; details are given in Ulijaszek and Lourie (1994).

24c.5 Urine zinc

Daily excretion of zinc in the urine is typically 0.3–0.6mg. A systematic review and meta-analysis examining the response of urinary zinc excretion to changes in dietary intake was conducted by Lowe and co-workers (2009). They concluded that urinary zinc is a useful biomarker of increases in exposure to zinc supple­ments (i.e. > 15mg Zn/d) in adults with adequate zinc status at baseline. However, only when dietary zinc intakes are very low (< 1mg dietary Zn/d) is a decrease in urinary zinc excretion in adults apparent (Baer and King, 1984; Johnson et al., 1993; King et al., 2000). Comparable studies on infants and children have not been conducted.

Urinary zinc excretion is confounded by a variety of clinical conditions that induce hyper­zincuria. These include sickle cell disease, alcoholism and liver disease, certain renal diseases and infections, injury, and burns. Starvation, strenuous physical exercise, diabetes, and trauma may also increase urinary zinc excretion (King et al., 2015). Hyper­tensive patients on long-term therapy with chlorothiazide also exhibit hyper­zincuria (Prasad, 1983). Certain physio­logical conditions also influence urinary zinc excretion. Increases have been reported during pregnancy with concen­trations returning to prepregnant levels after delivery (Fung et al., 1997). Whether changes occur during lactation is uncertain (King et al., 2015). Consequently, the overall health and diet of an individual must always be evaluated when using urinary zinc as a biomarker of zinc exposure.

The BOND Zinc Expert Panel classified urinary zinc as a “potential” biomarker that shows promise (King et al., 2015), based on its usefulness as a marker of com­pliance with zinc supple­mentation programs when at least 15mg Zn/d was given (Lowe et al., 2009). They emphasize, however, that the need to collect a 24‑hour urine sample and the lack of established cutoffs limit the usefulness of urinary zinc as a biomarker. Moreover, because urinary zinc only responds to very low dietary zinc intakes, its use as a biomarker of dietary zinc intake among free-living popu­lations is limited.

24c.5.1 Measurement of urinary zinc excretion

The amounts of zinc excreted in the urine usually range from 300 to 600µg/d. A 24-h urine collection is preferred because diurnal variation in urinary zinc excretion may be significant. Measurements of urinary zinc are usually performed by flame AAS.

24c.6 Hair zinc concen­trations

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.

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

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 2.5th 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)
3–6F/M65570
(50)
66
Italy
(Senofonte et al., 2000)
6–10F/M96157
(50)
152
Italy
(Senofonte et al., 2000)
10–13F/M258158
(41)
155
Italy
(Dongarra et al., 2011)
11–13F/M130189
(59)
179
Belgium
(Vanaelst et al., 2012)
6–10F218 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.

24c.7 Nail zinc

Fewer studies have examined nail zinc as a biomarker of zinc exposure than hair zinc even though the validity of nail selenium as a biomarker of selenium exposure in adults has been confirmed by several reports (Hunter et al., 1990). Nails, like hair, also incorporate zinc into their matrix when they are exposed to the blood supply within the germinal layer of the nail matrix, reflecting the quantity of zinc available in the blood supply at the time of the nail synthesis (He, 2011). The normal rate of growth for nails ranges from 1.6mm per month for toenails to about 3.5mm per month for fingernails (He, 2011). In cases where nail growth is arrested, as may occur in onychophagia (com­pulsive nail biting), nail zinc should not be used (He, 2011).

In healthy individuals not exposed to high levels of zinc in the atmosphere, nail zinc levels are similar to those in hair, with concen­trations ranging from 80 to 200µg/g (1.22 to 3.06µmol/g). For example, in German school children aged 3–7years, the average toenail zinc concen­tration was about 130µg/g (1.99µmol/g) (Wilhelm et al., 1991), although nail zinc concen­trations as high as 2000µg/g (30.6µmol/g)have been reported in galvanizers with a high exposure to zinc in the atmosphere (King et al., 2015). In a recent study of young children in Laos, nail zinc concen­trations were higher at endline in those children receiving a daily preventive zinc supple­ment (7–10mg Zn/d) for 32–40 weeks com­pared to those being given only a therapeutic zinc dose (20g) for 10 days (geometric mean, 95%CI) (115.8, 111.6–119.9 vs. 110.4, 106–104.8µg/g.; p=0.055) (Wessells et al., 2014). Nail zinc concen­trations can also be used as a longer-term retrospective measure of zinc exposure in case-control studies. For example, in a prospective study of US urban adults, toenail zinc was assessed in relation to the incidence of diabetes but no significant longitudinal association was found (Park et al., 2016).

Nails are exposed to exogenous surface contaminants so all specimens must always be washed using a recom­men­ded procedure. Chemicals introduced by nail polish can be removed by washing (He, 2011). Biological factors also affect nail zinc concen­trations and include age, possibly sex, rate of growth, and onychophagia (com­pulsive nail biting).

The BOND Zinc Expert Panel classified nail zinc as an “emerging” biomarker with a theoretical association with zinc intake or status, although the sensitivity and specificity to any changes in zinc nutrition need further study (King et al., 2015). No universally accepted reference values exist for fingernail or toenail zinc concen­trations exist and the reader is referred to Mikulewicz et al. (2013) for guidelines on their com­pilation.

24c.7.1 Measurement of nail zinc

Nails from all fingers or toes (at least 50mg) should be cleaned before clipping, and then stored in labeled T‑E free polyethylene bags prior to washing. Bank et al. (1981) recom­mend using an aqueous nonionic detergent, followed by vacuum drying for traditional analytical techniques. For non-destructive analytical methods such as INAA and the newer technique involving laser-induced breakdown spectroscopy (LIBS), cleaning fingernail clippings with acetone (analytical grade) in an ultrasonic bath for 10 minutes followed by drying in air for 20–30 minutes is recom­men­ded (Riberdy et al., 2017). All dried specimens should be stored in a desiccator after weighing, prior to analysis. The wet and dry ashing methods and the analytical techniques outlined above for hair zinc concen­trations can also be used for nail zinc. Alternatively, tetramethylammonium hydroxide (TMAH) can be used to solubilize nails at room temperature (Batista et al., 2008).

Laser-induced breakdown spectroscopy (LIBS) can be used and field-portable hand-held instruments operated from a battery have been developed. Investigations for measuring fingernail zinc in situ are underway (Riberdy et al., 2017). Preliminary results suggest that the in situ measure­ment of fingernail zinc by LIBS has potential as a non-invasive, convenient screening tool for identifying zinc defi­ciency in popu­lations but may lack the precision required to generate absolute concen­trations to charac­terize individuals (Riberdy et al., 2017). Research is ongoing to enhance the precision of this in situ method, and confirm its sensitivity and specificity to changes in zinc nutrition.

Unlike for hair, no Certified Reference Material exists for nail trace element analysis. Instead, in-house controls prepared from homogenous samples of powdered fingernails and toenails should be prepared, digested if necessary, and spiked with several differ­ent known quantities of zinc and the recoveries measured. Alternatively, an aliquot of the in-house control can be sent to an external quality laboratory and the results com­pared.

24c.8 Metallothionein concen­trations in circulating blood cell types

Metallothionein is a small, cysteine-rich, metal-binding protein that readily binds to zinc and heavy metals. Up to seven zinc molecules can bind to one metallo­thionein molecule, that may serve as a small zinc reserve for cells. The highest concen­trations of metallo­thionein are found in the liver, kidney, pancreas, and intestine. Concentrations in the intestine and pancreas respond to changes in dietary zinc intake, suggesting that metallo­thionein assists in the maintenance of zinc homeo­stasis in these tissues (King, 2011). Metallothionein also has a role in the acute phase response associated with inflam­mation when hepatic synthesis of metallo­thionein is activated, causing the redistribution of zinc from the plasma to the liver (Raiten et al., 2015).

Smaller quantities of metallo­thionein are also present in all tissue cells, and concen­trations in serum, erythrocytes, and monocytes have been investigated as potential biomarkers of human zinc status. Serum metallo­thionein concen­trations reflect changes in hepatic metallo­thionein, so any increase in hepatic levels in response to stress, inflam­mation etc., will result in concom­itantly elevated levels in serum, thus limiting the use of serum metallo­thionein as a biomarker of zinc status.

In contrast to serum, erythrocyte metallo­thionein is not responsive to stress and appears to be sensitive to severe and moderate restrictions in dietary zinc intake, based on results of depletion-repletion studies (Grider et al., 1990; Thomas et al., 1992). However, because most of the metallo­thionein in erythrocytes is concen­trated in the reticulocyte fraction, changes in erythropoiesis, as may occur with poor iron status, influence total erythrocyte metallo­thionein levels. With the advent of com­mercially available immunoassays for metallo­thionein, more research on the response of metallo­thionein concen­trations in erythrocytes and other blood cell types to changes in dietary zinc intake or status in com­munity-based studies is now possible. However, reference material for metallo­thionein concen­trations is also needed to allow com­parison of metallo­thionein concen­trations using differ­ent immunoassays across studies (Hennigar et al., 2016). At present, the sensitivity and specificity of metallo­thionein concen­trations in erythrocytes and other blood cell types and their usefulness in com­munity-based studies remains to be established. As a consequence, metallo­thionein concen­tration in circulating blood cell types was classified as an “emerging” biomarker of zinc exposure and status by the BOND Zinc Expert Panel (King et al., 2015). (King et al., 2015).

24c.9 Metallothionein mRNA expression in circulating blood cell types

Expression of the metallo­thionein gene is induced by zinc through the binding of zinc to metal-binding-regulatory trans­cription factor 1 (MTF-1). This trans­cription factor is known to be sensitive to cellular zinc concen­trations, with metallo­thionein synthesis increasing as cellular zinc increases. Reverse trans­criptase (RT) polymerase chain reaction (PCR) assays can be used to measure the response of metallo­thionein expression in various blood cell types to changes in dietary zinc. Hennigar et al. (2016), conducted a systematic review of sixteen experimentally controlled studies to determine the reliability and sensitivity of metallo­thionein expression to changes in dietary zinc in a variety of blood cell types. The studies were classified into three groups depending on the amount of zinc consumed per day (< 5, 15–22, and 50mg Zn/d). Only studies on healthy adults (> 18y) with no pre-existing health conditions, and that included baseline measure­ments or a placebo, were included. In all the studies, a depletion phase (mostly about 10 days) followed by repletion or a supple­mentation phase (ranging from 10 to 84 days) were included, although some also had a post-supple­mentation phase during which participants received a placebo tablet. Figure 24c.8
Figure 24c.8
Figure 24c.8 Metallothionein expression in leukocytes. Values are percentage changes from baseline±SEs for all studies that examined metallothionein expression in leukocytes. In studies that determined metallothionein expression at multiple time points (19–21), the day with the greatest percentage change was used. Data are grouped based on the amount of dietary zinc consumed per day. From Hennigar et.al. (2016) Advances in nutrition (Bethesda, Md.), 7(4), 735–746. © oup.com
presents the estimated effect of varying concen­trations of supple­mental zinc on metallo­thionein expression in leukocytes based on the percentage change from baseline. There was a 39% decrease from baseline in metallo­thionein expression in leukocytes in participants consuming < 5mg Zn/d, and increases from baseline of 135% and 267% for those receiving 15–22mg Zn/d and 50mg Zn/d, respectively. Hence these findings indicate that metallo­thionein expression in leukocytes is sensitive to changes in dietary zinc intake, decreasing in zinc depletion, and increasing in response to zinc supple­mentation in a dose-dependent manner. However, these changes were not associated with consistent changes in plasma zinc, which suggests that metallo­thionein expression in leukocytes may be a more sensitive biomarker than plasma zinc for determining zinc exposure. Of interest was the lack of sensitivity of metallo­thionein expression in erythrocytes to changes in dietary zinc.

Clearly, further work is needed to investigate whether metallo­thionein expression in leukocytes can be used as a biomarker of zinc status among other age groups and free-living volunteers. The effect of potential confounders (e.g., inflam­matory agents, free radicals, glucocorticoids, and pharmacologic agents)needs to be explored. In addition, more research is needed on whether the sensitivity to zinc supple­mentation and depletion is affected by variations in the proportions of blood cell sub­types among individuals. The abundance of metallo­thionein trans­cript in monocytes is three times that of granulocytes, and twice that found in T lymphocytes (Aydemir et al., 2006). In view of these uncertainties, metallo­thionein expression in various blood cell types was classified as an “emerging biomarker” by the BOND Zinc Expert Panel (King et al., 2015).

24c.10 Zinc trans­porter mRNA expression in circulating blood cell types

The discovery of shifts in the expression of the two families of zinc trans­porters: ZIP 1-14 (zinc “importers”) and ZnT 1-10 (“zinc exporters”) with changes in cellular zinc has stimulated investigations on the usefulness of zinc trans­porter mRNA expression in circulating blood cell types as biomarkers of zinc status (King et al., 2015). Hennigar et al. (2016), in their systematic review also investigated the response of the expression of several ZIP and ZnT zinc trans­porter proteins to zinc supple­mentation and depletion in various blood cell types isolated from healthy adults. Of the trans­porters, ZIP1 and ZnT1 were the most com­monly measured proteins in the review. Inconsistent changes in the zinc trans­porter expression levels in response to zinc supple­mentation and depletion across studies were reported. Consequently, more studies are needed to assess the usefulness of zinc trans­porter expression in circulating blood cell types as a biomarker of zinc exposure or status, especially under free-living conditions when a more typical range of zinc intakes is consumed. (Hennigar et al. 2016) Selection of peripheral blood mononuclear cells as the tissue source for such investigations may be an advantage given the suggestion that immune calls may be the first to respond to a change in zinc status even before plasma zinc falls below the normal range (Prasad, 1998).

24c.11 Oxidative stress and DNA integrity

Oxidative stress is caused by an imbalance between the increased production of multiple reactive oxygen species (ROS) (superoxide, hydrogen peroxide, and hydroxyl radicals) and the decreased protective action of antioxidants that are responsible for the neutralization and removal of ROS. Oxidative stress arising from this imbalance has the potential to damage proteins, deoxyribonucleic acid (DNA), and lipids, and may serve as a potential predictor in the development of differ­ent ROS-dependent diseases (e.g.,vascular diseases). Zinc plays an important role in antioxidant defence and the maintenance of cellular DNA integrity. As zinc itself is redox inert, it does not function as an antioxidant per se, instead functioning indirectly as an antioxidant and as a “pro-oxidant” over a limited range of zinc concen­trations. Consequently, the term “ pro-antioxidant” is used to describe the indirect functions of zinc as an antioxidant. In contrast, outside this range, zinc is a pro-oxidant. Hence, an increase in cellular oxidative stress and DNA damage may be an early sign of both reductions in cellular zinc as well as zinc overload. For more details see Kloubert and Rink (2015).

Several human studies have investigated whether intracellular DNA strand breaks, assayed using the com­et assay, could serve as a functional biomarker of zinc status. In an experimentally-controlled depletion-repletion study in adult men with low intakes of dietary zinc for six weeks (i.e., 0.6mg Zn/d for one week followed by 4mg Zn/d for five weeks), the number of leukocytic DNA strand breaks increased, but declined when dietary zinc intakes were increased to 11mg Zn/d for four weeks (Song et al., 2009). Another study of adult men in which dietary zinc intakes containing 6mg Zn/d (with added phytate) for two weeks were increased to 10mg Zn/d for 4 weeks, also showed improvements in the repair of DNA strand breaks with increased intakes of dietary zinc. Serum protein concen­trations associated with the DNA repair process also increased, despite no change in plasma zinc (Zyba et al., 2017). In a field setting in Ethiopia a decrease in DNA strand breaks was observed after supple­menting women with 20mg zinc as zinc sulfate or placebo daily for 17 days, again despite no significant changes in plasma zinc.

Taken together, these findings indicate that even modest changes in dietary zinc appear to modulate DNA damage, primarily by reducing cellular oxidative stress, and confirm the sensitivity of the com­et assay; see Singh et al. (1988) for assay details. Of note, however, despite modest increases in dietary zinc, there were no measured changes in plasma zinc concen­trations, exchangeable zinc pool, erythrocyte or leukocyte zinc concen­trations and metallo­thionein in leukocytes. These findings indicate that zinc attenuates oxidant stress over the range of usual zinc intakes, and in that capacity zinc modulates DNA damage. Nevertheless, many other conditions alter the redox state, so markers of oxidative stress, including DNA strand breaks, are not specific biomarkers for zinc nutrition. Hence, oxidative stress and DNA integrity were classified as “emerging” biomarkers by the BOND Zinc Expert Panel (King et al., 2015).

24c.12 Kinetic markers: pool sizes and turnover rates

Isotopic tracer studies in conjunction with a model-based com­partmental analysis have identified and measured a relatively small whole-body exchangeable pool of zinc (EZP). This pool com­prises the most meta­bolically active forms of zinc in the plasma, extra­cellular fluid, liver, pancreas, kidney, and intestine that make up about 10% of the whole-body zinc. The EZP provides zinc for zinc-dependent functions throughout the body and has a turnover rate of about 12.5 days. The size of EZP is estimated to contain 150–200mg zinc in adults, although varying markedly on a per kilogram body weight basis between adults and infants: 2.5mg Zn/kg in adults vs. 4.5mg Zn/kg in infants (King et al., 2001; Krebs et al., 2003).

Some but not all studies have shown a relation between dietary zinc and the size of the EZP. For example, the size of the EZP was significantly reduced in adults by severe dietary zinc restrictions (i.e, < 1mg Zn/d for 4–5 weeks) (King et al., 2001) or by chronically low habitual zinc intakes (i.e., average 5.2mg Zn/d) (Sian et al., 1996), but not by modest short-term changes in zinc intake (i.e., 4.6mg Zn/d for 10 weeks) (Pinna et al., 2001). Long-term (6 months) moderate zinc supple­mentation in male adults was shown to increase the size of EZP, which was reported to correlate positively with total zinc intake (Feillet-Coudray et al., 2005).

Of interest is the finding that during the study of severe restriction of dietary zinc (i.e., < 1mg Zn/d for 4–5 weeks) (King et al., 2001), the decline in EZP was nearly com­parable to that of plasma zinc (i.e., 60% vs. 65%). This finding suggests that the EZP is part of the vulnerable pool susceptible to zinc depletion, although not more sensitive than plasma zinc concen­trations. Whether the size of EZP is reduced by the meta­bolic redistribution of zinc due to inflam­mation and stress remains uncertain, although such a decrease is likely since plasma zinc is a com­ponent of the EZP (King, 2011). Other factors shown to influence the size of the EZP besides the level of dietary zinc include zinc absorp­tion, age, and sex, with men having a larger pool size than women (Pinna et al., 2001; Krebs et al., 2003; Ruz et al., 2011).

Use of the EZP size as a biomarker of zinc nutrition remains limited, in part because of uncertainties about age and sex-dependent cutoffs for zinc insufficiency, the limited sensitivity of EZP to small changes in dietary zinc intake, and the relationship between EZP size and zinc function. In addition, the measure­ment of EZP necessitates the use of stable isotopes and a mass spectrom­eter for analysis (King, 2011; King et al., 2015).

Isotopic tracer studies have also been used to investigate changes in plasma zinc turnover rates as a biomarker of zinc status. When the total body zinc content is reduced, plasma zinc turnover rates are increased to meet tissue needs. Normally, total plasma zinc turns over about 150 times per day to provide zinc for numerous functions throughout the body. Increases in the plasma turnover rate from about 150–200 times per day have been reported in healthy men as a result of acute, severe zinc depletion (i.e., 0.23mg Zn/d) (King et al., 2001), but not when zinc depletion was more modest (4.6mg Zn/d for 10 weeks) (Pinna et al., 2001). In a study in premenopausal women (n=33), lower intakes of beef, a good source of absorbable zinc, were associated with a reduction in the plasma zinc turnover rate and a reduction in taste acuity (Yokoi et al., 2003). More research is required to establish the validity of plasma zinc turnover rate as a biomarker of zinc status and to standardize the conditions for making the measure­ments.

The BOND Zinc Expert Group classified the kinetic markers based on pool sizes and plasma zinc turnover rates as “emerging” biomarkers as they require more research to establish their sensitivity and specificity to changes in zinc nutrition (King et al., 2015). (Allan et al., 2000).

24c.13 Taste acuity

Diminished taste acuity (hypogeusia) is a non-specific charac­teristic of marginal zinc defi­ciency and has been investigated as a functional bioindicator of zinc status in both cross-sectional and experimentally controlled studies in children and adults (Hambidge et al., 1972a; Buzina et al., 1980; Gibson et al., 1989a; Wright et al., 1981). For example, in a study of boys 5–7y with low height percentiles and low hair zinc concen­trations, impaired taste acuity was reported (Gibson et al., 1989a), but in a zinc depletion study of young men (0.25mg Zn/d for 4–9 weeks), there was a significant decline in their ability to discriminate differ­ences in saltiness com­pared with baseline levels. This change was unrelated to changes in zinc concen­trations in parotid saliva or plasma (Wright et al., 1981).

An age-related decline in taste acuity is well established in the elderly, but whether it is associated with a decrease in zinc status is uncertain. Some cross-sectional studies have reported positive associations between taste acuity for salt and erythrocyte zinc in the elderly (Stewart-Knox et al., 2005). Moreover, taste acuity for salt (but not for sweet, sour, or bitter taste) reportedly increased in older adults from Grenoble but not from Rome in response to 30mg Zn/d com­pared to placebo during a six-month double-blind randomized controlled trial (Stewart-Knox et al., 2008). Loss of taste during irradiation or chemotherapy has been restored with zinc supple­mentation in some (Yamagata et al., 2003; Najafizade et al., 2013). but not all (Halyard et al., 2007; Khan et al., 2019) studies.

Measurement of taste acuity

Several methods for testing taste acuity are available. Usually, evaluation is based on assessment of both the detection and recognition thresholds for each of four taste qualities (e.g., sweet, salt, sour, bitter), and sometimes umami (monosodium glutamate). The detection threshold is defined as “the lowest concen­tration at which a taste can just be detected”; the recognition threshold is “the lowest concen­tration at which the quality of the taste stimulus can be recognized”. The two thresholds are each determined for one taste quality before proceeding to the next. Several investigators have used this technique on young children (Hambidge et al., 1972a; Buzina et al., 1980).

An alternative technique that assesses only recognition thresholds and is better suited to children who are easily distracted and have short attention spans, has been developed (Desor and Maller, 1975). In some of these studies on children, recognition thresholds for only one taste quality (often salt) have been determined. Salt is selected because taste perception to moderately salty solutions changed significantly during a zinc-depletion study of young adult men (Wright et al., 1981). Recognition thresholds for salt have been measured in a study of Canadian school boys with low height percentiles and in Guatemalan school children. In these two investigations, school children with low hair zinc concen­trations had higher recognition thresholds for salt than children with hair zinc concen­trations indicative of “normal” zinc status (Gibson et al., 1989a; Cavan et al., 1993a). Sodium chloride concen­trations used for testing recognition thresholds in these school children were 10, 15, 20, and 25mmol/L (Gibson et al., 1989a). Participants rinse a small amount (10mL) of each solution around in their mouths, expectorate it, and are then asked to identify the presented sample as salty or plain water. Salt solutions of increasing or decreasing concen­trations are used, where appropriate, until the sub­jects correctly identify salt at one concen­tration and fail to do so at the next lower concen­tration. Only 2 out of 10 judgments are allowed to be incorrect before moving to the next higher or lower salt concen­tration. The midpoint between these two concen­trations is used as the recognition threshold for salt for each participant.

Some studies have used an electrogustometer to measure taste thresholds by applying a weak electric current to the tongue (Prosser et al., 2010). A threshold value is determined by alternately lowering and raising the current todetermine the smallest stimulus that can be discriminated correctly (Grant et al., 1987). For a review of the inherent strengths and limitations of electrogustometry, see Stillman et al. (2003). Automated-com­puter-controlled electrogustometry can now be used to estimate taste detection thresholds (Stillman et al., 2000), although so far, this method has not been applied in studies of zinc status. For all taste acuity methods, it is preferable to perform the tests midmorning, at least 2 hours after a meal, with the same person administering the test on each occasion. Many other factors affect taste function. Hence, taste acuity should be used in conjunction with other biomarkers to assess zinc status. The BOND Zinc Expert Panel classified taste acuity as an “emerging” zinc biomarker.

24c.14 Zinc-dependent enzymes

Although zinc serves in a catalytic role for over three hundred zinc metallo­enzymes, no single zinc-dependent enzyme has gained widespread acceptance as a biomarker of zinc status in humans. Their response to zinc defi­ciency, even in experimental studies of zinc depletion-repletion, has been very equivocal, and none have proved to be a consistent biomarker of zinc status. The physio­logical symptoms of zinc defi­ciency probably reflect, a series of biochemical changes (King, 2011).

Several zinc metallo-enzymes have been investigated as possible biomarkers of zinc status in plasma, erythrocytes, erythrocyte membranes or in specific cell types. Examples include d-amino-levulinic acid dehydratase, angiotensin-1-converting enzyme, α-D-manno­sidase, extra­cellular super­oxide dismutase, nucleoside phosphorylase, carbonic anhydrases, ecto purine 5′nucleo­tidase, and alkaline phosphatase. Of these zinc metallo-enzymes, the activity of alkaline phosphatase (EC 3.1.3.1) in serum, erythrocytes, or erythrocyte membranes has been most frequently studied. In a systematic review of biomarkers of zinc status that included seven differ­ent enzymes (Lowe et al., 2009). six studies investigated the response of plasma alkaline phosphatase. However, no significant effect of zinc intakes on overall plasma alkaline phosphatase was found after com­bining the data from zinc depletion and supple­mentation trials.

Alkaline phosphatase in the circulation consists of a mixture of three differ­ent isozymes (intestinal, placental, and liver/kidney/bone) and several isoforms. It is possible that assessment of their individual responses to changes in zinc intakes may provide more sensitive and consistent data (King et al., 2015). For the other six enzymes included in the systematic review, the number of studies was too limited to assess their effectiveness as biomarkers of zinc status (Lowe et al., 2009). Hence, based on the available evidence the BOND Zinc Expert Panel classified the activity of zinc-dependent enzymes as “not useful” as a biomarker of zinc exposure or status (King et al., 2015).

24c.15 Erythrocyte zinc

The concen­tration of zinc in erythrocytes is approximately ten times higher than in plasma, and normally 8–14µg/g wet weight or 10–11µg/1010cells. Zinc is present mainly as carbonic anhydrase (EC 4.2.1.1) (80–88%), with a small amount bound to Cu,Zn-SOD (EC 1.15.1.1) (~5%). Approximately 2%–3% is bound to low-molecular-weight-binding ligands. The life span of erythrocytes is 120d.

The BOND Zinc Expert Panel classified erythrocyte zinc or erythrocyte membrane zinc as “not useful” based on the inconsistent response to changes in zinc intakes or plasma zinc concen­trations (King et al., 2015). For example, in a 49d zinc depletion study, no changes in erythrocyte zinc were seen, despite evidence of impaired taste acuity and immune function, two potential bio-indicators of functional zinc depletion (Ruz et al., 1992). In addition, no response was reported in zinc supple­mentation studies of 30d and 90d duration, even when a dose of 50mg of zinc/day was used (Thomas et al., 1992; Davis et al., 2000). Taken together, the results emphasize the low sensitivity of erythrocyte zinc.

There are no standardized units for the expression of zinc concen­trations in erythrocytes, making com­parisons among studies difficult. The units can be based on hemoglobin, volume of packed cells, and per cell. There are presently no accepted standards for conversion among these units. No interpretive criteria have been established for erythrocyte zinc.

24c.16 Leukocyte zinc

Leukocytes contain up to twenty-five times more zinc than erythrocytes, and have a very much shorter lifespan than the 120-day lifespan of erythrocytes. Concentrations of zinc in leukocytes vary according to the cell type, with monocytes and lymphocytes having the highest concen­trations, followed by neutrophils. Mixed white blood cells generally contain 75µg zinc/1010 cells.

The BOND Zinc Expert Panel classified concen­trations of zinc in leukocytes and leukocyte sub­popu­lations as “not useful” as biomarkers of zinc nutrition in view of the inconsistent results observed in studies of both experimental zinc depletion – repletion (Ruz et al., 1992) and prolonged high zinc supple­mentation (e.g., 50mg Zn/d) (King et al., 2015).

The separation of leukocytes and other specific cell types is difficult, and some of the inconsistencies reported for leukocyte zinc in zinc depletion-repletion studies may result from analyzing mixtures of cell types (e.g., neutrophils and lymphocytes), with differ­ent half-lives and zinc concen­trations. Further, leukocytes and other specific cellular types are prone to contamination from exogenous sources of zinc and from platelets. Data are also difficult to interpret because no consensus exists for the method used to express zinc concen­trations in leukocytes or specific cell types. Results can be expressed on a mg/kg dry weight basis to eliminate the effects of variable cell water content, but results expressed as ng/106 cells are generally preferred (Nishi, 1980; Milne et al., 1985). Additional factors limiting the use of leukocyte zinc or the zinc content of sub­popu­lations is the relatively large volumes of blood (i.e., about 5mL) required for the analysis, and the lack of established interpretive criteria (King et al., 2015).

24c.17 Salivary zinc

Concentrations of zinc in saliva have been investigated as a measure of zinc status because zinc appears to be a com­ponent of gustin, an essential protein involved in taste acuity, that may be impaired by marginal zinc defi­ciency (Henkin, 1984). Zinc concen­trations in mixed saliva, parotid saliva, salivary sediment, and salivary supernatant have all been investigated, but their use as biomarkers of zinc status is equivocal (Greger and Sickles, 1979; Freeland-Graves et al., 1981; Baer and King, 1984). As well, although the collection of saliva samples is quick and relatively noninvasive, the rate of flow and stimulation of saliva is difficult to control, and diurnal variation in zinc levels may occur (Warren et al., 1981). In view of these problems, and the contradictory results observed, the use of salivary zinc as a biomarker of zinc status has been abandoned.

24c.18 Multiple biomarkers

The efficient regulation of zinc homeo­stasis com­plicates the assessment of zinc defi­ciency and excess, and currently there is no single, sensitive, specific biomarker of zinc status. At the popu­lation level, three biomarkers have been recom­men­ded by WHO / UNICEF / IAEA /IZiNCG (de Benoist et. al., 2007) and endorsed by the BOND Zinc Expert Panel (King et al., 2015) to assess zinc status and/or evaluate nutrition interventions to com­bat zinc defi­ciency. They are itemized in Section 24c.1.7. Details on the measure­ment and interpretation of these biomarkers are available from IZiNCG (Technical Briefs No.2 and No.3) and a table listing the relative strengths and weaknesses of the biomarkers is summarized in (King et al., 2015). For each biomarker, an indicator has been developed to estimate risk of zinc defi­ciency at the popu­lation level. These are: (a) the prevalence of usual zinc intakes below the estimated average require­ment (EAR), (b) the percentage of the popu­lation with low serum zinc concen­trations below the appropriate reference limit, and (c) the percentage of children less than 5y with length- or height-for-age less than −2SD below the age-specific median of the reference popu­lation (WHO, 2006). For each indicator, a “trigger level” for the prevalence considered indicative of elevated risk and of public health concern is given, at which level an intervention to improve popu­lation zinc status is warranted. Ideally, all three indicators should be included in a survey to strengthen the conclusion regarding the zinc status of the popu­lation and the need for an intervention, although in practice, this is not always possible. de Benoist et. al., 2007 suggest selecting indicators according to the level of available resources: (Box 24.7)

Box 24c.7.

For individuals, unless zinc defi­ciency is severe, moderate or mild zinc defi­ciency can be present in the absence of abnormal zinc biomarkers, including serum zinc, despite the presence of disturbances such as growth retardation, loss of appetite, and impaired immune function. Hence, for assessing the zinc status of individuals, the recom­men­ded approach is based on a medical history, a qualitative assessment of the diet, and a clinical assessment, with plasma or serum zinc providing only supple­mentary data (King et al., 2015). as noted earlier.

Acknowledgements

RSG would like to thank past zinc collaborators, particularly my former graduate students, and is grateful to Michael Jory for the HTML design and his tireless work in directing the trans­lation to this HTML version.