Originalarbeit Original Article
Forsch Komplementrmed Klass Naturheilkd 2003;10:1925
Effects of Zinc Histidine and Zinc Sulfate on Natural
Human Keratinocytes
A. Detersa, b E. Schnetzc M. Schmidtd
a Pharmazeutische Biotechnologie, Grental, Hochschule Wdenswil, Schweiz
b Institut
fr Pharmazeutische Biologie, Friedrich-Alexander-Universitt Erlangen Nrnberg, Erlangen
c Abteilung fr Dermatologie, Friedrich-Alexander-Universitt Erlangen Nrnberg, Erlangen
d Redinomedica AG, Mnchen
Key Words
Zinc histidine Zinc sulfate Keratinocytes
Cell physiology Proliferation Differentiation
Summary
Background: Zinc salts are widely used as food supple-
ments and medicinal mineral supplementation. Zinc defi-
ciency is associated with impaired skin conditions. The
influence of zinc on skin functionality has been proven in
clinical investigations. Objective: Within the following
study comparative in vitro experiments were performed
to study the influence of zinc sulfate (ZnSO4) and zinc his-
tidine (Zn(His)2) on the physiology of cultured natural
human keratinocytes. Method: Proliferation of natural
human keratinocytes was quantitatively assessed by
measurement of 5-bromo-2-deoxyuridine (BrdU) incor-
poration against an oligomeric procyanidin as positive
control. Differentiation was determined by monitoring in-
volucrin formation. Cell viability and cytotoxicity were
assessed by dimethylthiazolyldiphenyltetrazolium bro-
mide (MTT) testing and quantification of lactate dehydro-
genase. Results: Neither keratin synthesis as a late mark-
er of cell differentiation nor mitochondrial cell activity
were influenced by either zinc compound. The synthesis
of involucrin, an early marker of differentiation, was sig-
nificantly increased by both zinc salts, ZnSO4 being the
more potent stimulator. Both zinc salts significantly in-
creased cell proliferation, with the histidine complex
being more potent. Effects were in the range of the posi-
tive control. Necrotic cell toxicity decreased significantly
when Zn(His)2 was added to the cells. Conclusion: Under
in vitro conditions Zn(His)2 is a strong proliferation in-
ductor of keratinocytes with a better tolerability and a
lower induction of differentiation behavior than ZnSO4.
2003 S. Karger GmbH, Freiburg
Fax +49 761 4 52 07 14 Accessible online at:
E-mail Information@Karger.de www.karger.com/journals/fkm
www.karger.com
A. Hensela
Schlsselwrter
Zinkhistidin Zinksulfat Keratinozyten Zellphysiologie
Proliferation Differenzierung
Zusammenfassung
Hintergrund: Die Verwendung von Zinksalzen zur Nah-
rungsergnzung und therapeutischen Mineralsupple-
mentierung ist weit verbreitet. Insbesondere ein positiver
Einfluss von Zinksalzen auf die Funktionalitt der Haut ist
klinisch gut dokumentiert. Zielsetzung: Innerhalb der
vorliegenden Studie sollten die beiden Zinksalze Zinksul-
fat (ZnSO4) und Zinkhistidin (Zn(His)2) in vergleichenden
Untersuchungen hinsichtlich ihres Einflusses auf die
Physiologie humaner Keratinozyten untersucht werden.
Methodik: Proliferationstestung an primren, humanen
Keratinozyten erfolgte ber den Einbau von 5-Brom-2-
desoxyuridin (BrdU) unter Verwendung eines oligome-
ren Procyanidins als Positivkontrolle. Zelldifferenzierung
wurde ber die Involucrintiter bestimmt, Zellvitalitt
und Zytotoxizitt mittels Dimethylthiazoldiphenylte-
trazoliumbromid (MTT) und Quantifizierung der Lactatde-
hydrogenase. Ergebnisse: Weder die Keratinbildung als
spter Marker fr Differenzierungsvorgnge noch die
mitochondriale Zellaktivitt wurde durch die beiden Zink-
salze beeinflusst. Die Bildung des frhen Differenzie-
rungsmarkers Involucrin wurde signifikant durch ZnSO4
stimuliert. Die Zellproliferation wurde durch beide Zink-
salze gleichermassen stimuliert, wobei Zinkhistidin den
strkeren Induktor darstellte. Die nekrotische Toxizitt auf
die Zellen war bei Einsatz von Zinkhistidin signifikant
geringer als bei Verwendung von Zinksulfat. Schlussfol-
gerung: Zinkhistidin stellt einen starken Induktor der
Zellproliferation fr humane Keratinozyten dar, der die
zellulre Differenzierung weniger beeinflusst und eine
bessere Vertrglichkeit aufweist als Zinksulfat.
Andreas Hensel
Hochschule Wdenswil
Postfach 335
CH-8820 Wdenswil
Tel. +41 17899670, Fax -950
E-mail a.hensel@hswzfh.ch
Introduction
Zinc is an 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 the degradation of external peptides and extracellular
structures like collagen [1]. Vitally important for skin integrity,
zinc is rapidly transported to the skin following intestinal ab-
sorption [2]. In a number of skin conditions and diseases a zinc
deficiency, inadequate zinc absorption or metabolic disorder
are discussed: e.g. atrophic and eczematous skin, ulcers and
bad sores, acne, dandruff, skin lesions, keratitis or dermatitis
[39]; especially acrodermatitis enteropathica reacts directly
to zinc substitution [10].
As a co-factor of metalloenzymes and of the DNA and RNA
polymerases required for protein synthesis, zinc has a consid-
erable effect on nearly all of the metabolic processes taking
place in any organ of the body, including the skin [11]. Al-
though the biological activity of zinc is commonly attributed
to its role in enzymes, zinc is also important for the structure
and function of biomembranes [3]. An adequate zinc level is
necessary during maturation of the epidermis and for main-
taining cutaneous integrity [3].
Of the total amount of zinc in the body, 6% is present in the
skin, with approximately three quarters of that amount found
in the epidermis [12]. The high epidermal zinc content sug-
gests that zinc plays an important role in the metabolism of
keratinocytes [4]. Keratinocytes undergo a progressive and
complex differentiation process resulting in the horny layer
composed of corneocytes, filled with fibrous keratins which
are plasticized with water [13]. Transglutaminase activity is in-
volved in the differentiation process leading to the formation
of the hydrophobic corneocyte envelope. Involucrin is consid-
ered to be an efficient substrate for transglutaminase and has
been shown to be incorporated in the corneocyte envelope.
Involucrin can thus serve as a parameter for the differentia-
tion activity of keratinocytes.
The differentiation of keratinocytes to corneocytes is a com-
plex process following an extensive proliferation phase, both
functions leading to the formation of the epidermal barrier
function. However, for a healthy skin both processes have to
be balanced. Impaired keratinocyte proliferation and differ-
entiation is expressed in an impaired water retention in the
stratum corneum, and in high rates of scaling as in various cu-
taneous diseases.
Zinc can induce both processes, differentiation and prolifera-
tion. However, the effects and the tolerability of zinc supple-
ments are strongly dependent of the form in which the metal
ions are substituted [14]. In comparison to ZnSO4, equivalent
zinc doses as Zn(His)2 display a threefold bioavailability [15,
16]. In addition, in in vitro experiments strong antioxidative
effects were found with Zn(His)2, but not with ZnSO4 [17]. In
20 Forsch Komplementrmed Klass Naturheilkd
2003;10:1925
vitro, histidine has been shown to be the preferred binding site
of zinc in the epidermis [2].
Relevant differences in the pharmacological behavior of zinc
supplements in the metabolism of the skin should reveal
themselves in the extent of the activation of the proliferation
and differentiation processes of keratinocytes. Such processes
can be tested by in vitro experiments with cultured ker-
atinocytes [18, 19].
Material and Methods
General
Heat sterilisation was performed either at 121 C / 1 h in an autoclave or
at 175 C / 2 h in an oven.
Statistical analysis was done using an unpaired Student t-test.
Material
Zinc bishistidinate dihydrate and zinc sulfate heptahydrate were obtained
from Redinomedica (Mnchen, Germany). Proanthocyanidin (patent
pending) was obtained under the trade name ProcyanoPlus by Dermo-
Plus-Hensel (Wdenswil, Switzerland) [22].
DMEM medium and keratinocyte-SFM were purchased from Gibco
(Karlsruhe, Germany), ionophor A 23187 from ICN (Eschwege, Ger-
many), Triton-X100 from Sigma-Aldrich (Taufkirchen, Germany), ECL
solution 1 and 2 from Amersham PharmaciaBiotech (Freiburg, Ger-
many).
Monoclonal mouse anti-human-involucrin-antibodies and goat anti-
mouse-IgG-antibodies were ordered from Sigma (Taufkirchen, Ger-
many), monoclonal mouse anti-human-keratin-antibodies from Dako
(Hamburg, Germany).
Tissue well plates were manufactured by Greiner (Frickenhausen, Ger-
many), kits for 5-bromo-2-deoxyuridine (BrdU) cell proliferation and
LDH cytotoxicity detection by Roche (Mannheim, Germany).
Submerged Keratinocyte Culture
Keratinocytes were isolated from human skin by surgical resection from
Caucasian subjects with no history of atopic eczema or other skin diseases.
The skin was decontaminated using a solution of Fungizone (0.2%), gen-
tamycin (5.9 mg/ml), and sodium hydrogen carbonate (7.5%) in DMEM
medium [19]. Tissue was dissected mechanically, and after incubation of
the tissue in dispase solution dermis and epidermis were separated. The
epidermis was trypsinated for 1 h at 36 C with trypsin 0.05% and EDTA
sodium 0.53 mM. Keratinocytes were centrifuged at 463 g for 5 min and
resuspended in defined keratinocyte-SFM. Cell culture was performed in
96-well-tissue plates or, in case of determination of involucrin and keratin
concentrations, 24-well-tissue plates at 36 C in a humidified 5% CO2 at-
mosphere.
After 10 days of culture the keratinocytes were incubated with 14 M of
either Zn(His)2 or ZnSO4, dissolved in defined keratinocyte-SFM and cul-
tivated for further 9 days. Positive controls were obtained by incubation
with 13 g/ml ionophor A 23187, negative controls were cultured in medi-
um without incubation.
Characterization of Keratinocyte Physiology
For determination of mitochondrial dehydrogenase activity cells were
washed with PBS and incubated with 5 mg/ml dimethylthiazolyldiphenyl-
tetrazolium bromide (MTT) in defined keratinocyte-SFM. Formazan crys-
tals were dissolved in DMSO and the resulting absorbance was deter-
mined in an ELISA reader at 492 nm.
DNA synthesis was determined by Cell Proliferation ELISA kit, BrdU
(colorimetric) using the methodology described by Porstmann et al. [24].
Deters/Schnetz/Schmidt/Hensel

regard to the control (control = 1)
Relative DNA synthesis rate in
2.00
1.50
Fig. 1. Effects of zinc salts on cell proliferation
of normal human keratinocytes after 9 days of
incubation. Results, obtained by BrdU incorpo- 1.00
ration in cell DNA, are the mean SD (n = 30).
= Significant to zinc sulfate and to the control
with p < 0.01; ** = significant to the control with 0.50
p < 0.01.
Relative involucrin concentration in
regard to the control (control = 1)
Fig. 2. Half-quantitative results of the testing
of the effect of Zn(His)2 and ZnSO4 on cell dif-
ferentiation (involucrin induction) of NHK over
a 9-day incubation period (mean SD; n = 20). 1.2
Involucrin content was determined by dot blot-
ting. ++ = Significant to the control with p < 0.01;
** = significant to the control and ionophor
with p < 0.01 and to zinc histidine with p < 0.05.
Necrotic cell death was determined using the Cytotoxicity Detection Kit
(LDH). For positive control some cell cultures were incubated with 10%
Triton-X100 on the 8th day of the incubation period. Tests were per-
formed with 5 independent samples from different donors at different
times.
As markers of early differentiation, involucrin and keratin K1, K10, K5,
and K14 were detected by dot blotting [25]. Cells were transferred into
extraction buffer (50 mM NaPP pH 6.8, 5% SDS, 40 mM DTT, 5 mM
EDTA, 5 mM EGTA, 15% glycerol) and sonicated 3 times for 5 min. In-
soluble material was removed by centrifugation at 7000 g. Supernatants
were directly applied to nitrocellulose for dot blotting. After blocking
with 3.5% powdered milk in PBS, nitrocellulose membranes were incu-
bated for 60 min at 30 C with a monoclonal mouse anti-human-involu-
crin-antibody and monoclonal mouse anti-human-keratin-antibody re-
spectively diluted 1:1000 in PBS. Following 3 washing steps (10 min with
PBS), the membranes were incubated with POD-conjugated goat anti-
mouse-IgG-antibody diluted 1:1000 in PBS at 30 C for 60 min. After
washing 3 times with PBS a mixture of ECL solution 1 and 2 was added
and the system was developed.
Results
Influence of Zinc Salts on Proliferation of Normal Human
Keratinocytes
Zn(His)2 and ZnSO4 were tested on their influence on the
physiology of normal human keratinocytes (NHK) in concen-
trations of 14 according to the average serum zinc concen-
tration [20, 21] against the untreated negative control and the
positive control, treated with 10 M ProcyanoPlus [22].
Effects of Zinc Histidine and Zinc Sulfate
on NHK
**
**
Zinc histidine Zinc sulfate ProcyanoPlus Control
**
3.2
2.7 ++
2.2
++
1.7
0.7
Zinc histidine Zinc sulfate Control Ionophor
After 9 days of incubation the Zn(His)2- and ZnSO4-treated
cells showed enhanced proliferation on microscopic examina-
tion compared to the untreated cells from the negative con-
trol. Treatment of keratinocytes with zinc salts resulted in the
formation of typical cell monolayers which differed from the
untreated control groups by the degree of confluence. While
the zinc-treated groups formed complete monolayers, the con-
trol groups displayed only about 4050% confluence at the
same time. Quantitative evaluation of proliferation rates by
BrdU incorporation showed a significant increase of prolifera-
tion of cells treated with zinc in comparison to cells from the
control groups (fig. 1). Significant differences were found be-
tween the two zinc salts, with Zn(His)2 being a significantly
more potent inductor of proliferation than ZnSO4. Results
comparable to these data were found in additional experi-
ments performed on HaCat cell line: again Zn(His)2 showed
significant stimulatory effects on cell proliferation.
Effects of Zinc Salts on Differentiation of NHK
Differentiation behavior of keratinocytes under the influence
of zinc salts was evaluated by monitoring involucrin in NHK
by dot blotting of protein extracts with a mouse anti-human
involucrin antibody. The keratinocytes used within the test
were positively stimulated as shown by treatment with
ionophor A23187 as a positive control (fig. 2). Compared to
the untreated control, significantly higher involucrin titers re-
sulted under the influence of both zinc salts, with the effect of
Forsch Komplementrmed Klass Naturheilkd 21
2003;10:1925
 regard to the control (control = 1)
Relative keratin concentration in
Fig. 3. Effects of zinc salts (14 M) on cell dif-
ferentiation of NHK after 9 days of incubation.
Results, determined by quantification of keratin
K1, K10, K5, and K14 concentrations by dot
blotting and Western blotting (n = 20, data not
shown), are not significant to the untreated con-
trol group and the ionophor-treated cells.
)
Relative dehydrogenase activity in
regard to the control (control = 1)
(
Fig. 4. Effects of 14 M Zn(His)2 and ZnSO4
on mitochondrial dehydrogenase activity of
NHK after 9 days of incubation. Results, deter-
mined by MTT cleavage to a colored formazan,
are presented as the mean SD (n = 20) and
are not significant to the untreated control
g
groups.
ZnSO4 being significantly more pronounced than the involu-
crin induction by Zn(His)2 (fig. 2).
When a monoclonal mouse anti-human-keratin antibody was
used, no deviating differentiation behavior was found after in-
cubation with Zn(His)2, ZnSO4, and ionophor A 23187 com-
pared with untreated NHK (fig. 3). On treatment of samples
of each keratinocyte group with the same monoclonal mouse-
anti-human-keratin antibody, Western blotting yielded a pat-
tern of 4 keratins with a molecular weight of 50, 54, 60, and
66 kDa (table 1). The 66 kDa keratin was predominant in all
samples, while the amounts of the other keratins differed:
keratinocytes incubated with Zn(His)2 produced mainly the
54 kDa keratin, minor amounts of keratin K 14 (50 kDa) and
only very small amounts of the 60 kDa keratin. ZnSO4-treated
keratinocytes were characterized by both the 60 and 54 kDa
keratins, with minor amounts of the 50 kDa. A pattern similar
to the ZnSO4-induced keratin fingerprints was detected in
keratinocytes of both the positive and negative control groups.
Influence of Zinc Salts on Cell Viability and Cytotoxicity in
NHK
Neither zinc-treated keratinocytes nor untreated control
groups exhibited any positive or negative influence on the mi-
tochondrial activity of the cells as determined by MTT testing
[23] (fig. 4).
In contrast, the evaluation of direct cytotoxicity of the zinc
salts by determination of lactate dehydrogenase (LDH) as a
22 Forsch Komplementrmed Klass Naturheilkd
2003;10:1925
1.8
1.3
0.8
0.3
Zinc histidine Zinc sulfate Control Ionophor
1.30
1.10
0.90
0.70
0.50
0.30
Zinc histidine Zinc sulfate Control
marker enzyme for necrotic processes yielded not only slightly
lower LDH levels for the Zn(His)2-treated cells compared to
the untreated cells of the negative control (fig. 5), but also a
lower cytotoxicity in comparison to ZnSO4. The histidine
complex is well tolerated by the cells, and under the condi-
tions of the testing a better supplement to cell culture media
than ZnSO4.
Discussion
Keratinocytes count among the most important functional cell
types of the human skin barrier. During maturation, they un-
dergo various states of differentiation in a complex system of
cellular components embedded in an extracellular matrix
composed of lipids. The biosynthesis of these lipids takes place
in the keratinocytes. In addition, the keratinocytes undergo
morphological changes, being constantly renewed by prolifer-
ation processes in the stratum basale as undifferentiated cells,
and then moving upwards to the horny layer, at the same time
flattening and growing in size as well as increasing the number
of mitochondria. These morphological changes are paralleled
by changes in the biosynthesis of keratins. In the course of the
differentiation, the production of keratins of low molecular
weight decreases, while the production of keratins of high
molecular weight increases. The latter are considered to be
typical markers of differentiation (K1 and K10).
Deters/Schnetz/Schmidt/Hensel
 **
3.50

control (absorption 492 nm)

Cytotoxicity in regard to the
3.00

2.50
*
2.00
Fig. 5. Effects of Zn(His)2 and ZnSO4 on
necrotic cell death of NHK. Results were deter- 1.50
mined by quantification of LDH in the cell
1.00
medium (n = 39), Triton-treated cells served as
a positive control. The viability of Zn(His)2- 0.50
treated cells was significantly better than in the
0.00
untreated cells. Significant to the control with * Zinc histidine Zinc sulfate Control Triton-X100
= p < 0.05 and ** = p < 0.01.

Table 1. Keratin fingerprints in NHK treated

with 14 mol Zn(His)2, ZnSO4, and ionophor Treatment 50 kDa 54 kDa 60 kDa 66 kDa
A23187 (positive control) versus untreated ne- (keratin K 14) (keratin K 10) (keratin K 5) (keratin K 1)
gative control. Detection of the keratins was
performed by Western blotting with monoclo- Negative control ++ ++(+) ++
nal mouse-anti-human keratin antibody Ionophor A23187 + ++ ++ ++++
Zn(His)2 ++ ++ + ++
ZnSO4 + ++ + ++++

+ = Minor amounts; ++ = higher amounts; +++ = very high amounts.

When a certain concentration of keratins is reached, the cells
are no longer viable and are embedded as corneocytes in the
extracellular lipid matrix, contributing to the barrier function
of the horny layer.
Thus, the barrier function of the skin is regulated by two
processes: proliferation of keratinocytes for a constant renew-
al of the skin and differentiation for regeneration of the epi-
dermal barrier function. In dermatic disorders, proliferation
and/or differentiation of keratinocytes may be impaired. Both
processes can be influenced by nutritional factors or drugs.
The influence of zinc in wound healing and the treatment of
diseases such as psoriasis or neurodermitis is well known [4].
Significantly lowered plasma zinc concentrations were found
in patients with psoriasis, other dermatoses, and venous leg ul-
ceration [26, 27].
In wound treatment and dermatic disorders, zinc is often ap-
plied topically [3]. However, the zinc content of keratinocytes
is normally supplied by the dermis which obtains its zinc from
the bloodstream. Oral supplementation of zinc can thus sup-
port the healing process in diseases of the skin. These effects
can be attributed to effects on the differentiation and prolifer-
ation of keratinocytes. In addition, in inflammatory cutaneous
diseases zinc ions exert a direct effect on keratinocytes by re-
ducing the activation state of cells through the ICAM-1 ex-
pression and TNF- production [28].
Different zinc salts can display different efficacy. Agren [3]
found a positive effect of topically applied zinc on the healing
of leg ulcers with zinc oxide ointments, but not with zinc sul-
fate. In clinical studies, a threefold bioavailability was demon-
strated for orally given zinc histidine when compared with zinc
sulfate [15, 16]. In an in vitro study, Yeomans et al. [17] found
distinct antioxidant properties for zinc histidine in the model
of copper-induced oxidation of low-density lipoproteins,
whereas zinc sulfate was ineffective.
In this study, the effects of Zn(His)2 and ZnSO4 on prolifera-
tion and differentiation of cellular components of the skin
were studied in an in vitro model with primary cultured
human keratinocytes. According to our experience, freshly
isolated primary cultivated keratinocytes yield more repro-
ducible results in proliferation assays than the utilization of
commercially available cultures. In previous experimentation,
we observed a distinct tendency to high proliferation rates in
commercially available cell cultures, leading to an exponential
cell proliferation on induction which does not allow a repro-
ducible observation of significant differences in proliferation
enhancement by the tested substances.
Determination of Proliferation Rate
The testing of proliferation is based on cells in the S-phase
which display DNA synthesis. Typically, the assay is based on
the quantification of the incorporation of radioactively labeled
thymidine into the DNA, alternatively on utilization of BrdU
instead of labeled thymidine [24]. As an analogon of thymi-
dine, BrdU is incorporated into the DNA and can subsequent-
ly be detected with the aid of a peroxidase-labeled antibody.
The color intensity detected in the ELISA reading correlates
with the rate of DNA biosynthesis of the cells, and thus with
the proliferation rate. Zn(His)2 and ZnSO4 both stimulated

Effects of Zinc Histidine and Zinc Sulfate Forsch Komplementrmed Klass Naturheilkd 23
on NHK 2003;10:1925
proliferation of keratinocytes, with Zn(His)2 displaying a sig-
nificantly more potent effect. The proliferation-enhancing ef-
fect is highly welcome in all cases where an enhanced skin re-
generation may contribute to an amelioration of the dermatic
disorder. For orally and topically applied zinc supplements, a
different pharmacological behavior of zinc sulfate and zinc
histidine would have to be expected for pharmacokinetical
reasons. In comparison to zinc sulfate, zinc histidine displays a
threefold bioavailability [15]. In addition, the physiological
transport form of zinc in vivo is a complex with amino acids,
mainly histidine or cystein [20], and the transport from the
bloodstream into the cells has been shown to be an active and
L-histidine-dependent mechanism [2931]. Within the skin,
zinc is primarily bound to histidine [2]. The more pronounced
effect of zinc histidine on keratinocyte proliferation might be
advantageous in the treatment of skin diseases.
Determination of Differentiation Processes
During differentiation the spectrum of proteins produced by
the cell changes. In keratinocytes, the appearance of involu-
crin is specific for the early phase of differentiation, whereas
the composition of keratins can serve as a marker for later
phases [32]. The low-molecular-weight keratins K5 and K14
are produced by non-differentiated cells in the stratum basale,
whereas the higher-molecular-weight keratins K1 and K10 ap-
pear in differentiating keratinocytes. Both compounds can be
determined with semiquantitative methods using antibodies in
dot blot experiments. As expected, both zinc salts increased
keratinocyte differentiation, but the effects were more pro-
nounced with zinc sulfate. The involucrin content of zinc-sul-
fate-treated cells was higher than that of zinc-histidine-treated
cells, and the pattern of the keratins displayed a tendency to-
wards higher-molecular-weight forms. The overall effect of
zinc histidine seems to favor proliferation processes, whereas
with zinc sulfate an earlier differentiation of the keratinocytes
is achieved. These findings speak in favor of the treatment of
desquamation and scaling skin diseases with zinc histidine,
where there is already a fast differentiation of keratinocytes
and where an increase of proliferative processes would be
more adequate.
Determination of Cytotoxicity
MTT is transformed to a colored compound by mitochondrial
dehydrogenase. The color intensity is directly correlated to the
number of cells with active metabolic turnover. The MTT test-
ing can thus be used for the determination of cytoprotective or
cytotoxic activity of substances incubated with the cell culture
[18, 23]. The lack of any response in the test did not only con-
firm the non-toxicity of zinc in physiological concentrations,
but also led to the conclusion that the effect of zinc salts on
proliferation and differentiation of keratinocytes follows an
indirect mechanism of action.
Another well-established test of cytotoxicity is the LDH test.
Cell damage or necrotic cell death lead to a disrupture of the
cell membrane integrity, leading to a leaking of cytoplasmatic
enzymes like LDH into the medium. The activity of LDH in
the medium correlates to the extent of cell destruction and cy-
totoxicity of incubated substances. In the LDH test zinc histi-
dine was not only less toxic than zinc sulfate, but in addition
had a cytoprotective effect in comparison to untreated nega-
tive controls.
Conclusions
Zinc histidine and zinc sulfate both activate under in vitro
conditions proliferation and differentiation processes in pri-
mary cultured human keratinocytes. The enhancement of ker-
atinocyte proliferation was significantly higher with zinc histi-
dine in comparison to zinc sulfate and control, whereas the
overall effect of zinc sulfate was more pronounced for differ-
entiation. In addition, zinc histidine was significantly less cyto-
toxic than zinc sulfate. Regarded from a clinical point of view,
the enhancement of keratinocyte proliferation by zinc histi-
dine is highly interesting in the treatment of diverse dermatic
disorders, where the efficacy of zinc supplements was already
proven in clinical studies. Nevertheless, the adjustment be-
tween in vitro an in vivo effects has to be investigated in fur-
ther clinical studies.

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