Age Related Differences in Topical Zinc Absorption From Different Formulations

Zinc ion facilitates biochemical reactions that mediate the growth and remodeling of tissue. Because marginal zinc deficiency is so common, especially among the elderly, the epidermal absorption of zinc from four topical zinc preparations (Derma Sciences, Inc., Old Forge, PA), as well as from USP zinc oxide ointment, was assayed by means of atomic absorption spectroscopy in healthy volunteers (9 males, 6 females) ranging in age from 20 to 75 years. Baseline epidermal zinc levels declined as a function of age (p 0.0014). When baseline zinc levels were compared to those after exposure to the products, all treatment groups significantly enriched the skin (p < 0.001). Elderly subjects had significantly greater skin absorption than the young subjects after exposure to either a zinc-containing spray and ointment or a zinc-containing wound cleanser and hydrophilic bulk ointment. These findings are consistent with both the pharmacokinetics of topical preparations and the structure of senescent skin. The importance of topical nutrient supplementation with zinc is discussed in terms of aging, marginal zinc deficiency syndromes, and oxidative stress. Directions for future research are proposed.

Zinc is a ubiquitous micronutrient whose activity is so crucial to bodily function that it has been called a master hormone.(1) Processes facilitated by zinc include genetic transcription and other reactions leading to cell division; synthesis of the monomers required for protein, RNA, and DNA synthesis; and degradation of external peptides (hormones) and extracellular structures (collagen).(1) This range of activities has evolved under physiologic conditions as a result of zinc's properties. Zinc ion (Zn2+) forms ligands, from common substances such as sulfur from cysteine, nitrogen from histidine, and oxygen from glutamate, aspartate, and water. Zn2 does not readily oxidize or reduce, and easily undergoes ligand exchange reactions. Zn2 forms ligands between cysteine and histidine more tightly than Fe2+, Ni2+, and Co2+. These effects are so striking that organisms using only copper or iron in certain DNA- binding proteins would have disappeared long ago.(2)

It therefore is not surprising that marginal zinc deficiency is associated with problems ranging from impaired wound healing in general to reduced immune function in elderly patients.(3) It is for these reasons that we assayed the uptake of Zn2 into the epidermis of free-living young adult and elderly subjects. A number of products intended to make zinc ion available to both intact and breached skin were tested: USP zinc oxide ointment (Zn oxide), Dermagran® Spray and Dermagran® Ointment (DS + 0), Dermagran® Wound Cleanser and Dermagran® (bulk) Hydrophilic Ointment (DWC + B-O), Dermagran® Barrier Cream (DBC), and Dermagran® Hydrogel (DH). The results are discussed in light of the importance of topical zinc application to aging skin. There are precedents for addressing the health benefits of topical supplementation. To draw a limited analogy, the well-publicized use of topically applied glaucoma drugs, nicotine, and cardiac agents exemplify the utility of this phenomenon. The topical application of substances to dry or friable skin is also useful insofar as oral supplementation will not serve the dual purpose of supplying the skin with nutrients and rehydrating the skin.

Table 1.
Subject characteristics and baseline epidermal zinc concentrations
+/- standard deviations

Subject	Sex	Age	Baseline Zn (ygm/cm2)
1	M	20	0.732 +/- 0.047
2	F	21	0.761 +/- 0.017
3	F	23	0.825 +/- 0.019
4	M	25	1.04 +/- 0.040
5	M	25	0.840 +/- 0.026
6	M	28	0.887 +/- 0.037
7	F	32	0.887 +/- 0.028
8	M	40	0.800 +/- 0.025
9	F	43	0.853 +/- 0.028
10	F	49	0.535 +/- 0.019
11	M	55	0.725 +/- 0.019
12	M	65	0.917 +/- 0.023
13	F	67	0.611 +/- 0.033
14	M	71	0.648 +/- 0.016
15	M	75	0.539 +/- 0.023

Healthy, free-living college-age, middle-age, and elderly subjects gave informed consent for this study (n = 15). An indelible pen was used to inscribe seven 1" x 1" squares on the skin of both forearms--five sites for the products and two sites for duplicate blanks. The products were applied in aliquots of 50 yL of spray, 60 mgm of ointment, or both, as required by product-use protocols. Each site was covered with an adhesive bandage fitted with a 1" x 1" gauze pad that was left in place for 4 hours. The bandages were then taken off and the excess product removed with a dry Kimwipe® (Kimberly-Clark Corp., Roswell, GA). Trace product was removed by rubbing the sites twice with 95 percent ethanol and twice with water using cotton swabs (Hardwood Products Co, Guilford, ME) to apply the liquids. The sites were dried with a tissue and the treated skin stripped ten times with adhesive tape to remove the stratum corneum. The duplicate blanks were treated identically to product-treated sites.

The tapes were ashed for 4 hours at 450 C and the residue dissolved in 10 mL of 3 percent HCl. The residue was then heated to 60 C. The mixtures were vortexed before and after heating, and then centrifuged. Zinc concentrations in the resulting supernatants were assayed by means of atomic absorption spectroscopy calibrated against zinc standards. The concentration of zinc in the stratum corneum after exposure to the products was calculated by subtracting the average concentration of zinc in the blanks from the zinc concentration in the investigational supernatants. The percentage of zinc absorbed from each product was calculated from the amount of zinc applied to the skin (based on the product's label) and the amount found in the stratum corneum as corrected by the blanks. The products were compared statistically by means of t-tests for normal distributions and Mann-Whitney rank sum tests for non-normal distributions. A p-value less than 0.05 was considered significant. Correlations were calculated using a Pearson's correlation.

Fifteen subjects (9 male, 6 female) were enrolled and ranged in age from 20 to 75 years. Subjects' age, gender, and baseline epidermal zinc levels, as computed from the blanks, appear in Table 1. Table 2 is derived from the data in Table 1 and gives average baseline epidermal zinc levels as related to age. From the 15 subjects, the overall average reproducibility of the tape-stripping data was 3.8 percent. When baseline zinc levels were compared to zinc levels after treatment, all groups were significantly enriched (p < 0.001). When subjects were divided into two groups based on age (20 to 43 years, n = 9 and 44 to 75 years, n = 6), the average baseline zinc levels were 0.85 + 0.03 ygm/cm2 and 0.61 + 0.07 ygm / cm2, respectively. This age-based difference was highly significant (p = 0.0014). There were no significant sex-related differences.

Table 3 shows the percent change in epidermal zinc after 4 hours. In general, there was a highly significant correlation between the dose of zinc applied and the average percent changes in epidermal zinc (p = 0.0006). Elderly subjects had significantly greater skin absorption than young subjects with DH, DS + 0, and DWC + B-O. The age groups were statistically indistinguishable for Zn oxide. Young subjects absorbed significantly more zinc from DBC than did the elderly subjects (p = 0.029). Table 4 shows the average percent of zinc absorbed from the products. There was no statistical correlation between the dose of zinc applied and the average percent of zinc absorbed from the products. A greater percentage of zinc was absorbed from the hydrogel than from the other formulations (p < 0.001). A significantly greater percentage of zinc was absorbed from DS + 0 than from DBC and Zn oxide (p = 0.001). Elderly subjects had significantly smaller percentages of zinc absorbed from the DBC and Zn oxide than the young subjects (p = 0.013 and 0.018, respectively).

These results are best understood by remembering that the performance of the products was analyzed in two ways: (1) by computing the percent changes in epidermal zinc resulting from product application (Table 3); and (2) by computing the percent of zinc absorbed from a given product. (Table 4) Put another way, in the first instance we measure how much the epidermal zinc levels change, and in the second instance we measure how much zinc is absorbed from a given product. In effect, measures of skin absorption versus product characteristics are provided.

Within this context, note that Table 4 shows an approximately inverse relation between the amount of zinc applied and the percent of zinc that the epidermis absorbs. This is not a strict statistical correlation but a qualitative observation supporting a well-known pharmacokinetic phenomenon: the lower the dose of a substance applied, the greater the percent available for absorption. On the other hand, a comparison of Tables 3 and 4 shows an approximate relation between the amount of zinc applied to a unit area of the epidermis and the percent increase in epidermal zinc. Put another way, the more zinc applied to the skin, the greater the increase in epidermal zinc levels. As a result, all of the products significantly enriched the epidermal zinc content relative to baseline (p < 0.001).

These two sets of observations seem to contradict each other, but do not. The data in Table 3 provide absolute measures of changes in epidermal zinc. Table 4 yields more relative measures of product performance. These analyses provide two ways of analyzing the same phenomena. These results are to be expected on the basis of pharmacokinetics and formulation. What becomes relevant to gerontology is the finding that the percent of zinc absorbed from the products is significantly greater in the elderly subjects versus the young subjects for both DWC + B-O and DS + 0, and marginally greater for DH. The opposite is true for DBC and Zn oxide. In the former case, the wound cleanser and spray may have breached the thinning, friable stratum corneum of the older subjects more effectively than the comparatively intact tissue of the younger subjects. Similar considerations may have applied to the heavily hydrated vehicle of the DH. However, such statements must be made cautiously. The amount of stratum corneum removed from one site on the forearm relative to another can vary by several cell layers, which renders tentative our interpretation of age-related differences. The latter should be confirmed through a larger study.

If confirmed, our finding that the products in this study significantly enriched the zinc content of senescent epidermis has interesting implications for the care of aging skin. These implications become clear when one considers that substances absorbed in the stratum corneum form a reservoir that reflects the levels of those substances deeper in the body.(4)

Product	Average amount of zinc applied per dose (ygm)	Percent change - all subjects
DBC Dermagran Barrier Cream	11,505 +/- 1090	211 +/- 128
Zn Oxide USP Zinc Oxide Ointment	9,900 +/- 1041	168 +/- 120
DWC + B-O Dermagran Wound Cleanser and Dermagran (bulk) Hydrophilic Ointment	252 +/- 38	53.5 +/- 64.8
DS + O Dermagran Spray and Dermagran Ointment	18.3 +/- 0.03	8.2 +/- 3.5
DH Dermagran Hydrogel	0.495 +/- 0.016	4.9 +/- 1.8

		Average % of zinc absorbed
Product	Average Amount of Zinc Applied Per Dose (ygm)	All subjects	Young (Y) vs. Elderly (E)	Comparison of young and elderly
DH Dermagran Hydrogel	0.495 +/- 0.016	56.1 +/- 20.3	Y = 60.0 +/- 22.0 E = 50.0 +/- 18.0	p = 0.065 (NS)
DS + O Dermagran Spray and Dermagran Ointment	18.3 +/- 0.03	2.1 +/- 0.7	Y = 1.8 +/- 0.6 E = 2.4 +/- 0.5	E > Y p = 0.007
DWC + B-O Dermagran Wound Cleanser and Dermagran (bulk) Hydrophilic Ointment	252 +/- 38	0.89 +/- 1.02	Y = 0.31 +/- 0.22 E = 1.79 +/- 1.05	E > Y p = 0.004
DBC Dermagran Barrier Cream	11,505 +/- 1090	0.11 +/- 0.08	Y = 0.15 +/- 0.08 E = 0.05 +/- 0.02	Y > E p = 0.013
Zn Oxide USP Zinc Oxide Ointment	9,900 +/- 1041	0.10 +/- 0.08	Y = 0.14 +/- 0.08 E = 0.05 +/- 0.01	Y > E p = 0.018

To better appreciate these statements, it is useful to consider the epidermis as a potentially starved tissue. It has no direct blood supply but depends on nutrients provided by the diffusion of substances across their concentration gradients from underlying dermal capillary tufts. With age, these dermal papillae flatten and the blood supply correspondingly diminishes, thus further starving the epidermis. The integrity of the stratum corneum also diminishes, which may facilitate the uptake of topically applied substances.(5) Within this context, it makes sense to supplement the skin from the outside, thus taking advantage of the liabilities of age (which in this study involved greater percentage changes in skin zinc in the elderly), and creating an optimum nutrient environment for the maintenance of healthy skin.

Because senescent skin may be proportionally supplemented with zinc ion to a greater degree than young skin with some zinc formulations, it is important to consider some of the processes facilitated by zinc, which may contribute to the health of senescent skin. Such speculation suggests themes for future research.

It has been suggested that chronic, marginal zinc deficiency is strongly associated with the deleterious effects of aging.(6) This hypothesis is supported by data indicating marginal zinc deficiency in elderly patients,(3) as well as signs of presumably normal aging that actually resemble zinc deficiency syndromes. These include skin changes such as reduced integrity of the stratum corneum, rough or dry skin, impaired wound healing, and alopecia. Acrodermatitis enteropathica is an autosomal recessive disease with symptoms and treatment that support this hypothesis. The disease has catastrophic multisystemic effects, including some of the skin symptoms mentioned. All of these symptoms are resolved with zinc supplementation.

Another function of zinc not commonly appreciated is its antioxidant activity. Two known mechanisms for this activity are zinc's ability to protect sulfhydryl groups and inhibit the metal ion-induced formation of reactive oxygen species, which are chronically toxic.(7) The antioxidant effects of zinc have been observed in many experimental systems, including human skin fibroblasts that were stressed with either hydrogen peroxide, ultraviolet-A light, or ultraviolet-B light.(6,9) Zinc also decreased the cytotoxicity of these stressors to skin cells, and increased the viability of unstressed cells.

The amount of zinc suitable for various skin afflictions is, unfortunately, not well understood, although the advent of atomic absorption spectroscopy has facilitated the study of zinc metabolism. The therapeutic issues arising in connection with these ongoing challenges include the way in which zinc should be presented -- as an aqueous paste, cream in an amphiphilic vehicle, emollient, or bandage.(10)

As mentioned above, an immediate extension of the work presented here would be a larger study addressing directly the changes in epidermal zinc as a function of age. Subsequent to this, an autoradiographic study of topical zinc supplementation would provide insight as to where zinc localizes within tissues. Other issues in the care of aging skin would be clarified by elucidating zinc's antioxidant effects in the appropriate experimental systems.

Age (years)	Number	Baseline Zn (ygm/cm2)
20-31	6	0.847 +/- 0.11
32-43	3	0.846 +/- 0.04
44-55	2	0.630 +/- 0.16
56-75	4	0.604 +/- 0.05

Age Related Differences in Topical Zinc Absorption From Different Formulations: Implications for Health and Disease

Age Related Differences in Topical Zinc
Absorption From Different Formulations:
Implications for Health and Disease