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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. Hensela
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 Schlsselwrter


Zinc histidine Zinc sulfate Keratinocytes Zinkhistidin Zinksulfat Keratinozyten Zellphysiologie
Cell physiology Proliferation Differentiation Proliferation Differenzierung

Summary Zusammenfassung
Background: Zinc salts are widely used as food supple- Hintergrund: Die Verwendung von Zinksalzen zur Nah-
ments and medicinal mineral supplementation. Zinc defi- rungsergnzung und therapeutischen Mineralsupple-
ciency is associated with impaired skin conditions. The mentierung ist weit verbreitet. Insbesondere ein positiver
influence of zinc on skin functionality has been proven in Einfluss von Zinksalzen auf die Funktionalitt der Haut ist
clinical investigations. Objective: Within the following klinisch gut dokumentiert. Zielsetzung: Innerhalb der
study comparative in vitro experiments were performed vorliegenden Studie sollten die beiden Zinksalze Zinksul-
to study the influence of zinc sulfate (ZnSO4) and zinc his- fat (ZnSO4) und Zinkhistidin (Zn(His)2) in vergleichenden
tidine (Zn(His)2) on the physiology of cultured natural Untersuchungen hinsichtlich ihres Einflusses auf die
human keratinocytes. Method: Proliferation of natural Physiologie humaner Keratinozyten untersucht werden.
human keratinocytes was quantitatively assessed by Methodik: Proliferationstestung an primren, humanen
measurement of 5-bromo-2-deoxyuridine (BrdU) incor- Keratinozyten erfolgte ber den Einbau von 5-Brom-2-
poration against an oligomeric procyanidin as positive desoxyuridin (BrdU) unter Verwendung eines oligome-
control. Differentiation was determined by monitoring in- ren Procyanidins als Positivkontrolle. Zelldifferenzierung
volucrin formation. Cell viability and cytotoxicity were wurde ber die Involucrintiter bestimmt, Zellvitalitt
assessed by dimethylthiazolyldiphenyltetrazolium bro- und Zytotoxizitt mittels Dimethylthiazoldiphenylte-
mide (MTT) testing and quantification of lactate dehydro- trazoliumbromid (MTT) und Quantifizierung der Lactatde-
genase. Results: Neither keratin synthesis as a late mark- hydrogenase. Ergebnisse: Weder die Keratinbildung als
er of cell differentiation nor mitochondrial cell activity spter Marker fr Differenzierungsvorgnge noch die
were influenced by either zinc compound. The synthesis mitochondriale Zellaktivitt wurde durch die beiden Zink-
of involucrin, an early marker of differentiation, was sig- salze beeinflusst. Die Bildung des frhen Differenzie-
nificantly increased by both zinc salts, ZnSO4 being the rungsmarkers Involucrin wurde signifikant durch ZnSO4
more potent stimulator. Both zinc salts significantly in- stimuliert. Die Zellproliferation wurde durch beide Zink-
creased cell proliferation, with the histidine complex salze gleichermassen stimuliert, wobei Zinkhistidin den
being more potent. Effects were in the range of the posi- strkeren Induktor darstellte. Die nekrotische Toxizitt auf
tive control. Necrotic cell toxicity decreased significantly die Zellen war bei Einsatz von Zinkhistidin signifikant
when Zn(His)2 was added to the cells. Conclusion: Under geringer als bei Verwendung von Zinksulfat. Schlussfol-
in vitro conditions Zn(His)2 is a strong proliferation in- gerung: Zinkhistidin stellt einen starken Induktor der
ductor of keratinocytes with a better tolerability and a Zellproliferation fr humane Keratinozyten dar, der die
lower induction of differentiation behavior than ZnSO4. zellulre Differenzierung weniger beeinflusst und eine
bessere Vertrglichkeit aufweist als Zinksulfat.

2003 S. Karger GmbH, Freiburg Andreas Hensel


Hochschule Wdenswil
Fax +49 761 4 52 07 14 Accessible online at: Postfach 335
E-mail Information@Karger.de www.karger.com/journals/fkm CH-8820 Wdenswil
www.karger.com Tel. +41 17899670, Fax -950
E-mail a.hensel@hswzfh.ch
Introduction vitro, histidine has been shown to be the preferred binding site
of zinc in the epidermis [2].
Zinc is an ubiquitous micronutrient whose activity is so crucial Relevant differences in the pharmacological behavior of zinc
to bodily function that it has been called a master hormone supplements in the metabolism of the skin should reveal
[1]. Processes facilitated by zinc include genetic transcription themselves in the extent of the activation of the proliferation
and other reactions leading to cell division, synthesis of the and differentiation processes of keratinocytes. Such processes
monomers required for protein, RNA, and DNA synthesis, can be tested by in vitro experiments with cultured ker-
and the degradation of external peptides and extracellular atinocytes [18, 19].
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 Material and Methods
deficiency, inadequate zinc absorption or metabolic disorder
are discussed: e.g. atrophic and eczematous skin, ulcers and General
bad sores, acne, dandruff, skin lesions, keratitis or dermatitis Heat sterilisation was performed either at 121 C / 1 h in an autoclave or
[39]; especially acrodermatitis enteropathica reacts directly at 175 C / 2 h in an oven.
Statistical analysis was done using an unpaired Student t-test.
to zinc substitution [10].
As a co-factor of metalloenzymes and of the DNA and RNA Material
polymerases required for protein synthesis, zinc has a consid- Zinc bishistidinate dihydrate and zinc sulfate heptahydrate were obtained
erable effect on nearly all of the metabolic processes taking from Redinomedica (Mnchen, Germany). Proanthocyanidin (patent
place in any organ of the body, including the skin [11]. Al- pending) was obtained under the trade name ProcyanoPlus by Dermo-
Plus-Hensel (Wdenswil, Switzerland) [22].
though the biological activity of zinc is commonly attributed
DMEM medium and keratinocyte-SFM were purchased from Gibco
to its role in enzymes, zinc is also important for the structure (Karlsruhe, Germany), ionophor A 23187 from ICN (Eschwege, Ger-
and function of biomembranes [3]. An adequate zinc level is many), Triton-X100 from Sigma-Aldrich (Taufkirchen, Germany), ECL
necessary during maturation of the epidermis and for main- solution 1 and 2 from Amersham PharmaciaBiotech (Freiburg, Ger-
taining cutaneous integrity [3]. many).
Monoclonal mouse anti-human-involucrin-antibodies and goat anti-
Of the total amount of zinc in the body, 6% is present in the
mouse-IgG-antibodies were ordered from Sigma (Taufkirchen, Ger-
skin, with approximately three quarters of that amount found many), monoclonal mouse anti-human-keratin-antibodies from Dako
in the epidermis [12]. The high epidermal zinc content sug- (Hamburg, Germany).
gests that zinc plays an important role in the metabolism of Tissue well plates were manufactured by Greiner (Frickenhausen, Ger-
keratinocytes [4]. Keratinocytes undergo a progressive and many), kits for 5-bromo-2-deoxyuridine (BrdU) cell proliferation and
LDH cytotoxicity detection by Roche (Mannheim, Germany).
complex differentiation process resulting in the horny layer
composed of corneocytes, filled with fibrous keratins which Submerged Keratinocyte Culture
are plasticized with water [13]. Transglutaminase activity is in- Keratinocytes were isolated from human skin by surgical resection from
volved in the differentiation process leading to the formation Caucasian subjects with no history of atopic eczema or other skin diseases.
of the hydrophobic corneocyte envelope. Involucrin is consid- 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
ered to be an efficient substrate for transglutaminase and has
medium [19]. Tissue was dissected mechanically, and after incubation of
been shown to be incorporated in the corneocyte envelope. the tissue in dispase solution dermis and epidermis were separated. The
Involucrin can thus serve as a parameter for the differentia- epidermis was trypsinated for 1 h at 36 C with trypsin 0.05% and EDTA
tion activity of keratinocytes. sodium 0.53 mM. Keratinocytes were centrifuged at 463 g for 5 min and
The differentiation of keratinocytes to corneocytes is a com- resuspended in defined keratinocyte-SFM. Cell culture was performed in
96-well-tissue plates or, in case of determination of involucrin and keratin
plex process following an extensive proliferation phase, both
concentrations, 24-well-tissue plates at 36 C in a humidified 5% CO2 at-
functions leading to the formation of the epidermal barrier mosphere.
function. However, for a healthy skin both processes have to After 10 days of culture the keratinocytes were incubated with 14 M of
be balanced. Impaired keratinocyte proliferation and differ- either Zn(His)2 or ZnSO4, dissolved in defined keratinocyte-SFM and cul-
entiation is expressed in an impaired water retention in the tivated for further 9 days. Positive controls were obtained by incubation
with 13 g/ml ionophor A 23187, negative controls were cultured in medi-
stratum corneum, and in high rates of scaling as in various cu-
um without incubation.
taneous diseases.
Zinc can induce both processes, differentiation and prolifera- Characterization of Keratinocyte Physiology
tion. However, the effects and the tolerability of zinc supple- For determination of mitochondrial dehydrogenase activity cells were
ments are strongly dependent of the form in which the metal washed with PBS and incubated with 5 mg/ml dimethylthiazolyldiphenyl-
tetrazolium bromide (MTT) in defined keratinocyte-SFM. Formazan crys-
ions are substituted [14]. In comparison to ZnSO4, equivalent
tals were dissolved in DMSO and the resulting absorbance was deter-
zinc doses as Zn(His)2 display a threefold bioavailability [15, mined in an ELISA reader at 492 nm.
16]. In addition, in in vitro experiments strong antioxidative DNA synthesis was determined by Cell Proliferation ELISA kit, BrdU
effects were found with Zn(His)2, but not with ZnSO4 [17]. In (colorimetric) using the methodology described by Porstmann et al. [24].

20 Forsch Komplementrmed Klass Naturheilkd Deters/Schnetz/Schmidt/Hensel


2003;10:1925

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. Zinc histidine Zinc sulfate ProcyanoPlus Control

**
Relative involucrin concentration in
regard to the control (control = 1)

3.2

2.7 ++

2.2
Fig. 2. Half-quantitative results of the testing ++
of the effect of Zn(His)2 and ZnSO4 on cell dif-
1.7
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; 0.7
** = significant to the control and ionophor Zinc histidine Zinc sulfate Control Ionophor
with p < 0.01 and to zinc histidine with p < 0.05.

Necrotic cell death was determined using the Cytotoxicity Detection Kit After 9 days of incubation the Zn(His)2- and ZnSO4-treated
(LDH). For positive control some cell cultures were incubated with 10% cells showed enhanced proliferation on microscopic examina-
Triton-X100 on the 8th day of the incubation period. Tests were per-
tion compared to the untreated cells from the negative con-
formed with 5 independent samples from different donors at different
times. trol. Treatment of keratinocytes with zinc salts resulted in the
As markers of early differentiation, involucrin and keratin K1, K10, K5, formation of typical cell monolayers which differed from the
and K14 were detected by dot blotting [25]. Cells were transferred into untreated control groups by the degree of confluence. While
extraction buffer (50 mM NaPP pH 6.8, 5% SDS, 40 mM DTT, 5 mM the zinc-treated groups formed complete monolayers, the con-
EDTA, 5 mM EGTA, 15% glycerol) and sonicated 3 times for 5 min. In-
trol groups displayed only about 4050% confluence at the
soluble material was removed by centrifugation at 7000 g. Supernatants
were directly applied to nitrocellulose for dot blotting. After blocking same time. Quantitative evaluation of proliferation rates by
with 3.5% powdered milk in PBS, nitrocellulose membranes were incu- BrdU incorporation showed a significant increase of prolifera-
bated for 60 min at 30 C with a monoclonal mouse anti-human-involu- tion of cells treated with zinc in comparison to cells from the
crin-antibody and monoclonal mouse anti-human-keratin-antibody re- control groups (fig. 1). Significant differences were found be-
spectively diluted 1:1000 in PBS. Following 3 washing steps (10 min with
tween the two zinc salts, with Zn(His)2 being a significantly
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 more potent inductor of proliferation than ZnSO4. Results
washing 3 times with PBS a mixture of ECL solution 1 and 2 was added comparable to these data were found in additional experi-
and the system was developed. ments performed on HaCat cell line: again Zn(His)2 showed
significant stimulatory effects on cell proliferation.

Results Effects of Zinc Salts on Differentiation of NHK


Differentiation behavior of keratinocytes under the influence
Influence of Zinc Salts on Proliferation of Normal Human of zinc salts was evaluated by monitoring involucrin in NHK
Keratinocytes by dot blotting of protein extracts with a mouse anti-human
Zn(His)2 and ZnSO4 were tested on their influence on the involucrin antibody. The keratinocytes used within the test
physiology of normal human keratinocytes (NHK) in concen- were positively stimulated as shown by treatment with
trations of 14 according to the average serum zinc concen- ionophor A23187 as a positive control (fig. 2). Compared to
tration [20, 21] against the untreated negative control and the the untreated control, significantly higher involucrin titers re-
positive control, treated with 10 M ProcyanoPlus [22]. sulted under the influence of both zinc salts, with the effect of

Effects of Zinc Histidine and Zinc Sulfate Forsch Komplementrmed Klass Naturheilkd 21
on NHK 2003;10:1925
regard to the control (control = 1)
Relative keratin concentration in
1.8

1.3

Fig. 3. Effects of zinc salts (14 M) on cell dif-


ferentiation of NHK after 9 days of incubation. 0.8
Results, determined by quantification of keratin
K1, K10, K5, and K14 concentrations by dot
blotting and Western blotting (n = 20, data not 0.3
shown), are not significant to the untreated con- Zinc histidine Zinc sulfate Control Ionophor
trol group and the ionophor-treated cells.
)
Relative dehydrogenase activity in
regard to the control (control = 1)
1.30

1.10
(

0.90

Fig. 4. Effects of 14 M Zn(His)2 and ZnSO4


0.70
on mitochondrial dehydrogenase activity of
NHK after 9 days of incubation. Results, deter- 0.50
mined by MTT cleavage to a colored formazan,
are presented as the mean SD (n = 20) and 0.30
are not significant to the untreated control Zinc histidine Zinc sulfate Control
g

groups.

ZnSO4 being significantly more pronounced than the involu- marker enzyme for necrotic processes yielded not only slightly
crin induction by Zn(His)2 (fig. 2). lower LDH levels for the Zn(His)2-treated cells compared to
When a monoclonal mouse anti-human-keratin antibody was the untreated cells of the negative control (fig. 5), but also a
used, no deviating differentiation behavior was found after in- lower cytotoxicity in comparison to ZnSO4. The histidine
cubation with Zn(His)2, ZnSO4, and ionophor A 23187 com- complex is well tolerated by the cells, and under the condi-
pared with untreated NHK (fig. 3). On treatment of samples tions of the testing a better supplement to cell culture media
of each keratinocyte group with the same monoclonal mouse- than ZnSO4.
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 Discussion
samples, while the amounts of the other keratins differed:
keratinocytes incubated with Zn(His)2 produced mainly the Keratinocytes count among the most important functional cell
54 kDa keratin, minor amounts of keratin K 14 (50 kDa) and types of the human skin barrier. During maturation, they un-
only very small amounts of the 60 kDa keratin. ZnSO4-treated dergo various states of differentiation in a complex system of
keratinocytes were characterized by both the 60 and 54 kDa cellular components embedded in an extracellular matrix
keratins, with minor amounts of the 50 kDa. A pattern similar composed of lipids. The biosynthesis of these lipids takes place
to the ZnSO4-induced keratin fingerprints was detected in in the keratinocytes. In addition, the keratinocytes undergo
keratinocytes of both the positive and negative control groups. morphological changes, being constantly renewed by prolifer-
ation processes in the stratum basale as undifferentiated cells,
Influence of Zinc Salts on Cell Viability and Cytotoxicity in and then moving upwards to the horny layer, at the same time
NHK flattening and growing in size as well as increasing the number
Neither zinc-treated keratinocytes nor untreated control of mitochondria. These morphological changes are paralleled
groups exhibited any positive or negative influence on the mi- by changes in the biosynthesis of keratins. In the course of the
tochondrial activity of the cells as determined by MTT testing differentiation, the production of keratins of low molecular
[23] (fig. 4). weight decreases, while the production of keratins of high
In contrast, the evaluation of direct cytotoxicity of the zinc molecular weight increases. The latter are considered to be
salts by determination of lactate dehydrogenase (LDH) as a typical markers of differentiation (K1 and K10).

22 Forsch Komplementrmed Klass Naturheilkd Deters/Schnetz/Schmidt/Hensel


2003;10:1925
**
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 strated for orally given zinc histidine when compared with zinc
are no longer viable and are embedded as corneocytes in the sulfate [15, 16]. In an in vitro study, Yeomans et al. [17] found
extracellular lipid matrix, contributing to the barrier function distinct antioxidant properties for zinc histidine in the model
of the horny layer. of copper-induced oxidation of low-density lipoproteins,
Thus, the barrier function of the skin is regulated by two whereas zinc sulfate was ineffective.
processes: proliferation of keratinocytes for a constant renew- In this study, the effects of Zn(His)2 and ZnSO4 on prolifera-
al of the skin and differentiation for regeneration of the epi- tion and differentiation of cellular components of the skin
dermal barrier function. In dermatic disorders, proliferation were studied in an in vitro model with primary cultured
and/or differentiation of keratinocytes may be impaired. Both human keratinocytes. According to our experience, freshly
processes can be influenced by nutritional factors or drugs. isolated primary cultivated keratinocytes yield more repro-
The influence of zinc in wound healing and the treatment of ducible results in proliferation assays than the utilization of
diseases such as psoriasis or neurodermitis is well known [4]. commercially available cultures. In previous experimentation,
Significantly lowered plasma zinc concentrations were found we observed a distinct tendency to high proliferation rates in
in patients with psoriasis, other dermatoses, and venous leg ul- commercially available cell cultures, leading to an exponential
ceration [26, 27]. cell proliferation on induction which does not allow a repro-
In wound treatment and dermatic disorders, zinc is often ap- ducible observation of significant differences in proliferation
plied topically [3]. However, the zinc content of keratinocytes enhancement by the tested substances.
is normally supplied by the dermis which obtains its zinc from
the bloodstream. Oral supplementation of zinc can thus sup- Determination of Proliferation Rate
port the healing process in diseases of the skin. These effects The testing of proliferation is based on cells in the S-phase
can be attributed to effects on the differentiation and prolifer- which display DNA synthesis. Typically, the assay is based on
ation of keratinocytes. In addition, in inflammatory cutaneous the quantification of the incorporation of radioactively labeled
diseases zinc ions exert a direct effect on keratinocytes by re- thymidine into the DNA, alternatively on utilization of BrdU
ducing the activation state of cells through the ICAM-1 ex- instead of labeled thymidine [24]. As an analogon of thymi-
pression and TNF- production [28]. dine, BrdU is incorporated into the DNA and can subsequent-
Different zinc salts can display different efficacy. Agren [3] ly be detected with the aid of a peroxidase-labeled antibody.
found a positive effect of topically applied zinc on the healing The color intensity detected in the ELISA reading correlates
of leg ulcers with zinc oxide ointments, but not with zinc sul- with the rate of DNA biosynthesis of the cells, and thus with
fate. In clinical studies, a threefold bioavailability was demon- 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- Determination of Cytotoxicity
nificantly more potent effect. The proliferation-enhancing ef- MTT is transformed to a colored compound by mitochondrial
fect is highly welcome in all cases where an enhanced skin re- dehydrogenase. The color intensity is directly correlated to the
generation may contribute to an amelioration of the dermatic number of cells with active metabolic turnover. The MTT test-
disorder. For orally and topically applied zinc supplements, a ing can thus be used for the determination of cytoprotective or
different pharmacological behavior of zinc sulfate and zinc cytotoxic activity of substances incubated with the cell culture
histidine would have to be expected for pharmacokinetical [18, 23]. The lack of any response in the test did not only con-
reasons. In comparison to zinc sulfate, zinc histidine displays a firm the non-toxicity of zinc in physiological concentrations,
threefold bioavailability [15]. In addition, the physiological but also led to the conclusion that the effect of zinc salts on
transport form of zinc in vivo is a complex with amino acids, proliferation and differentiation of keratinocytes follows an
mainly histidine or cystein [20], and the transport from the indirect mechanism of action.
bloodstream into the cells has been shown to be an active and Another well-established test of cytotoxicity is the LDH test.
L-histidine-dependent mechanism [2931]. Within the skin, Cell damage or necrotic cell death lead to a disrupture of the
zinc is primarily bound to histidine [2]. The more pronounced cell membrane integrity, leading to a leaking of cytoplasmatic
effect of zinc histidine on keratinocyte proliferation might be enzymes like LDH into the medium. The activity of LDH in
advantageous in the treatment of skin diseases. the medium correlates to the extent of cell destruction and cy-
totoxicity of incubated substances. In the LDH test zinc histi-
Determination of Differentiation Processes dine was not only less toxic than zinc sulfate, but in addition
During differentiation the spectrum of proteins produced by had a cytoprotective effect in comparison to untreated nega-
the cell changes. In keratinocytes, the appearance of involu- tive controls.
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 Conclusions
are produced by non-differentiated cells in the stratum basale,
whereas the higher-molecular-weight keratins K1 and K10 ap- Zinc histidine and zinc sulfate both activate under in vitro
pear in differentiating keratinocytes. Both compounds can be conditions proliferation and differentiation processes in pri-
determined with semiquantitative methods using antibodies in mary cultured human keratinocytes. The enhancement of ker-
dot blot experiments. As expected, both zinc salts increased atinocyte proliferation was significantly higher with zinc histi-
keratinocyte differentiation, but the effects were more pro- dine in comparison to zinc sulfate and control, whereas the
nounced with zinc sulfate. The involucrin content of zinc-sul- overall effect of zinc sulfate was more pronounced for differ-
fate-treated cells was higher than that of zinc-histidine-treated entiation. In addition, zinc histidine was significantly less cyto-
cells, and the pattern of the keratins displayed a tendency to- toxic than zinc sulfate. Regarded from a clinical point of view,
wards higher-molecular-weight forms. The overall effect of the enhancement of keratinocyte proliferation by zinc histi-
zinc histidine seems to favor proliferation processes, whereas dine is highly interesting in the treatment of diverse dermatic
with zinc sulfate an earlier differentiation of the keratinocytes disorders, where the efficacy of zinc supplements was already
is achieved. These findings speak in favor of the treatment of proven in clinical studies. Nevertheless, the adjustment be-
desquamation and scaling skin diseases with zinc histidine, tween in vitro an in vivo effects has to be investigated in fur-
where there is already a fast differentiation of keratinocytes ther clinical studies.
and where an increase of proliferative processes would be
more adequate.

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