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Niger. J. Physiol. Sci. 31(December 2016) 171-176
www.njps.com.ng
Studies on Zinc and Copper Ion in Relation to Wound Healing
in Male and Female West African Dwarf Goats
1*A. K. Olaifa and 2S. T. Fadason
1Department of Veterinary Surgery and Reproduction, University of Ibadan, Ibadan, Nigeria.
2Department of Veterinary Surgery and Radiology. Ahmadu Bello University, Zaria, Nigeria.
Summary: Wound healing remains a challenging clinical problem for which precise and efficient management is essential
in order to curtail morbidity and mortality. Wound healing has been shown to depend upon the availability of appropriate
trace elements like copper and zinc which serve as enzyme cofactors and structural components in tissue repair. This study
aims at evaluating the distribution of zinc and copper found in the hair as well as skin during epidermal wound healing. Adult
and healthy West African dwarf (WAD) goats of both sexes fed with concentrate, grass, cassava peel and water ad libitum
were used. The animals were housed for three weeks before commencement of the experiments. Epidermal wounds were
created on the trunks of all the goats using cardboard template of 1cm². Progressive changes in wound contraction were
monitored grossly by placing clean and sterile venier calliper on the wound margin. Hair and skin elemental (copper and
zinc) analyses were done using atomic absorption spectroscopy (AAS). Significant increases in Cu level were observed in
the female hair compared with that of males. There were significant increases in the Zn levels of the females’ hair compared
with the males. The wound healed faster in female goat compared with the males. The ratio of copper to zinc is clinically
more important than the concentration of either of these trace metals. The pattern of distribution between zinc and copper
concentration in the skin and hair of the male and female goats observed in this study could be added factor responsible for
early wound healing in female. Therefore, our findings suggest that the distribution in the Cu and Zinc level in skin and hair
of both male and female goats could also be a factor for wound healing in the animals.
Keywords: Wound healing, Copper, Zinc, Hair, Skin.
©Physiological Society of Nigeria
*Address for correspondence: akolaifa@yahoo.com
Manuscript Accepted: December, 2016
INTRODUCTION
The skin is the largest organ that makes up
about10% of the whole body which is largely exposed
to physical and mechanical assaults daily. Loss of a
large portion of the skin integrity arising as a result of
injury or illness usually leads to disabilities or death
(Adam et al., 1999; Robson et al., 2001). In normal
skin, the epidermis and dermis exist in steady state of
equilibrium, forming a protective barrier against the
external environment. This can range from a simple
break in the epithelial integrity of the skin or it can be
deeper, extending into subcutaneous tissue with
damage to other structures such as tendons, muscles,
vessels, nerves, parenchymal organs and even bone
(Alonso et al., 1996).
Wounds can be grouped according to various criteria
(Robson et al., 2001) which can be clinically
categorized as acute and chronic with reference to their
time frame of healing (Bischoff et al., 1999). Other
criteria considered during wound classification include
aetiology, degree of contamination, morphological
characteristics and communication with hollow or
solid organs (Komarcevic et al., 2000). Wounds may
also be referred to as closed, where the underlying
tissue has been traumatized but the skin has not been
severed; or as open, where the skin layer has been
damaged with the underlying tissue exposed (Attinger
et al., 2006). Once the protective barrier is broken, the
normal physiological process of wound healing sets in
immediately (Nguyen et al., 2009).
Wound healing is a dynamic and highly regulated
process consisting of cellular, humoral and molecular
mechanisms (Reinke and Sorg, 2012). The healing
process consists of a sequence of overlapping events
including inflammatory responses, regeneration of the
epidermis, shrinkage of the wound and finally
connective tissue formation and remodelling
(Choucair and Phillips, 1997; Leaper and Harding,
1998).
Several factors are responsible for wound healing
many of which if interfered with might lead to
improper or impaired tissue repair (Guo and DiPietro,
2010). Skin wound healing depends upon the
availability of appropriate trace elements serving as
enzyme cofactors and structural components in tissue
repair (Lansdown et al., 1999).
Trace elements refer to “elements that occur in
natural and perturbed environments in small amounts
Niger. J. Physiol. Sci. 31 (2016): Olaifa and Fadason
Studies on Zinc and Copper in WAD goats 172
and that, when present in sufficient bioavailable
concentrations are toxic to living organisms.’’
Although these elements account for only 0.02% of the
total body weight, they play significant roles, such as,
being active centers of enzymes or as trace bioactive
substances (Wada, 2004). Some of the trace elements
control important biological processes by facilitating
the binding of molecules to their receptor sites on cell
membrane, alternating the structures or ionic nature of
membranes to prevent or thus allowing or obstructing
the entry of specific molecules into a cell thus inducing
gene expression leading to protein formation involved
in life processes (Nielsen, 1990).
Copper, as a trace element, plays a very important
role in body metabolism as it aids the functionality of
many critical enzymes (Harris, 2001). Copper has a
selected biochemical function in haemoglobin (Hb)
synthesis, connective tissue metabolism and bone
development (Turnlund, 1998). Copper metal in
contact with skin is purported to exert anti-
inflammatory properties, but the extent to which
copper penetrates the layers of the skin is a matter of
debate (Hostynek and Maibach, 2003). Scientists think
that introducing copper into wound dressings would
not only reduce the risk of contamination, but also
stimulate healing. Releasing copper from the dressings
directly onto the wound promotes skin regeneration
(Wilkinson and Hawke, 2000).
Several copper-dependent enzymes, mainly amine
oxidase increase during wound healing (Rea et al.,
1998). These copper-dependent enzymes are
important in the remodeling and healing of wounds.
The direct role of copper in facilitating angiogenesis
has been evident for two decades (Alessandri et al.,
1984).
Zinc stabilizes cell membranes, serves as an
essential cofactor for several metallo-enzymes,
participates in basal cell mitosis and differentiation
(Landsdown et al., 2007).The function of zinc in cells
and tissues is dependent on metalloproteinase and
these enzymes are associated with reproductive,
neurological, immune, dermatological systems as well
as gastrointestinal tract (GIT). Zinc is essential for
normal spermatogenesis and maturation of sperm,
genomic integrity of sperm, normal organogenesis,
proper functioning of neurotransmitters, development
of thymus, epithelialisation in wound healing, taste
sensation, secretion of pancreas and gastric enzymes
(Watson, 1998).
Zinc is also present in a number of zinc-dependent
metallo-enzymes in the skin, including matrix
metalloproteases (MMPs), superoxide dismutase
(SOD), alkaline phosphatase, and RNA/DNA
polymerases (Schwartz et al., 2005). Many of the
biochemical and molecular events in wound repair can
be expedited by addition of supplementary zinc ion
through up-regulation of metallothioneins (Lansdown,
2002) and zinc metallo-enzymes (Ravanti and Kahari,
2000). Zinc helps maintain the integrity of skin and
mucosal membranes (Wintergerst et al., 2007).
Patients with chronic leg ulcers have abnormal zinc
metabolism and low serum zinc levels (Lansdown et
al., 2007). However, the importance of copper and zinc
during wound healing in the hair and skin has not being
investigated especially in goats. There is also a dearth
of information on the distribution of these elements as
responsible for wound healing differences in male and
female goats, hence the need for this study.
MATERIALS AND METHODS
Experimental animals
Ten adult West African Dwarf goats of equal sexes
(between 1-1.5 years and weighing 10-12 kg), were
put in stalls. The animals were housed in individual
pens three weeks for stabilization before
commencement of the experiment. Well-balanced diet
consisting of concentrate, grass and cassava peels were
fed to the animals and water provided ad libitum. The
animals were dewormed with levamisole (10%) I/M at
the dose rate of 10mg/kg body weight and also given
penicillin-streptomycin pre-emptively to take care of
possible bacterial infections.
Epidermal wound creation
Using a square stencil of dimension 1cm by 1cm, the
portion of the epidermis to be surgically removed
which is the right lateral side of the animal just ventral
to the vertebrae column was marked using an ink
marker. Three mg/kg of 2% lignocaine was used in
caudal epidural block and local infiltration (inverted L-
Block) to desensitize the skin in order to ensure
complete desensitization of nerves that might escape
epidural block and provide the required anaesthesia.
Booster injections of up to one-half of the initial dose
were administered as needed in order to ensure that the
goats were pain-free during the skin excision
procedure. Each marked portion was blocked
individually before surgery was done.
Epidermal wounds were created on the trunk of all
the goats. A sharp sterilized scalpel was used and
bleeding reduced by the use of pressure gauze and
shortening of surgery duration. The full thickness of
the skin within the incision was then carefully stripped
away by sharp dissection from its underlying muscle.
All excisions were made using scalpel blade and
forceps with particular care taken that wound edges
were sharply defined (Olaifa, 2016)
Measurement of wound contraction
Each wound was measured (in centimetre2) daily using
the length of the mid-horizontal and mid-vertical sides
of the wound with the aid of a vernier calliper. Error
due to parallax was reduced by ensuring that wounds
were measured under adequate illumination using the
same blind observer all through the experiment. The
length (L) and breadth (B) were then used to calculate
the wound area in cm2 (Olaifa, 2016)
Niger. J. Physiol. Sci. 31 (2016): Olaifa and Fadason
Studies on Zinc and Copper in WAD goats 173
Elemental analysis
Skin biopsies and hair shavings were taken on the day
of wound creation and submitted to I.A.R&T
laboratory Ibadan, to check for copper and zinc levels
in the skin and hair of the trunk. This was done using
Atomic absorption spectroscopy (AAS) which is a
spectroanalytical procedure for the quantitative
determination of chemical elements using the
absorption of optical radiation (light) by free atoms in
the gaseous state. The skin and hair were weighed, and
0.5g of each was put in the digestion tube. Five (5) ml
perchloric: nitric acid mixture in the ratio 1:3 was
dispensed into digestion tube containing each sample,
put in microwave oven for 5 minutes, removed after
and put in cupboard to cool. After cooling, 45mls of
distilled water was dispensed, mixed together for
homogeneity and the portion needed for analysis was
decanted. Copper and zinc were read from the decant
of both skin and hair samples in the atomic absorption
spectrophotometer (AAS) from their individual
fluorescent lamps (Siraj and Kitte, 2013).
Statistical Analysis
Statistical analysis was performed using SPSS
software for windows (version 16.0). Results for
reaction time and paw oedema (Mean ± SEM) were
analyzed using One way ANOVA followed by
Tuckey’s post Hoc test to identify significance
between groups. Result for pain score were analyzed
using Kruskal Wallis non-parametric test. The
differences were considered significant at P < 0.05.
RESULTS
On days 20 and 25 post creation of wounds, the healing
rate of the wound in the female was significantly
higher than that of the male goat (Fig. 1). The wounds
in the male did not heal completely until after day 30.
Fig 1: Wound healing in the male and female goats
Fig 2: Average concentration of Cu on the skin of male and
female West African Dwarf goats in µg/g (ppb)
Fig 3: Average concentration of Zn on the skin of male and
female West African Dwarf goats in µg/g (ppb)
Evaluation of the rate of wound healing continued
until day 35 when all the wounds in the goats were
healed.
The copper analysis (fig 2) shows that there was an
insignificant increase in copper concentration in the
skin of the male than the female. While the skin of the
male goats had a mean copper distribution of 197.81
µg/g (ppb), the female had 197.26 µg/g(ppb).
However, there was less copper on the hair of the male
goats than the female goats. The hair of the male goats
had a mean copper concentration of 91.35 µg/g (ppb),
while the female had 100.62 µg/g (ppb). The result of
the zinc analysis (fig 3) shows that there was more zinc
concentration in the skin of the male than the female.
While the skin of the male goats had a mean zinc
distribution of 127.78 µg/g (ppb), the female had 99.82
µg/g (ppb). However, there was less zinc on the hair of
the male goats than the female goats. The hair of the
male goats had a mean zinc distribution of 45.26 µg/g
(ppb) while the female had 69.01µg/g (ppb).
DISCUSSION
In the skin, oestrogens and androgens are involved in
proliferation and differentiation of epithelial cells as
well as activities of fibroblasts and skin immune cells
all play important roles in wound healing (Strudwick
Niger. J. Physiol. Sci. 31 (2016): Olaifa and Fadason
Studies on Zinc and Copper in WAD goats 174
et al., 2006). The faster rate of wound healing in the
females than the males agrees with earlier works in
humans (Jorgensen et al., 2002; Ashcroft and
Ashworth 2003) which might be due to the
involvement of sex hormone in the animals. There is
striking evidence from animal studies dating back to
1962 that oestrogens play a crucial role in cutaneous
wound healing. Repair is significantly delayed in its
absence, an event characterized by profound leukocyte
recruitment during the initial stages of injury and
tissue destruction (Jorgensen and Schmidt, 1962;
Calvin et al., 1998; Ashcroft et al., 1999). Oestrogen
could also modulate the local wound-healing response
and downregulate inflammation through oestrogen
receptor-mediated inhibition of macrophage migration
inhibitory factor (MIF). In the absence of oestrogen,
local increase in MIF expression occurs unchecked,
resulting in excessive inflammation and delayed
healing (Ashcroft et al., 2003).
There was no appreciable difference in the average
concentration of copper in the skin of both male and
female WAD goats; while the average copper
concentration in the female hair was elevated than in
the male hair which may probably be a mechanism by
which the wounds healed faster in the female. Some
researchers have also measured copper and zinc in the
skin and hair of animal (Onwuka et al., 2000;
Filistowicz et al., 2012). The healing promoting
activities of copper has also been reported when
applied together with other treatments (Pereira and
Felcman, 1998; Somayaji et al., 1995). Copper metal
in contact with skin is purported to exert anti-
inflammatory properties; however, the extent to which
copper penetrates the layers of the skin is a matter of
debate (Hostynek and Maibach 2003). Several copper-
dependent enzymes, mainly amine oxidases, are
known to be increased during wound healing (Rea et
al., 1998). These copper-dependent enzymes are
important in the remodelling and healing of wounds.
For example, lysyl oxidase catalyses the formation of
aldehyde cross-links and acts primarily on collagen
and elastin during wound healing (Kobayashi et al.,
1994).
However, average zinc concentration was elevated
in the skin of the males than the females while it was
higher in the hair of the female than the male.
Although this pattern of difference in zinc distribution
on the skin and hair of male and female could not be
easily explained except by natural consistency.
Meanwhile, the importance of zinc to wound healing
has earlier been highlighted (McCarthy et al., 1992).
Zinc is the only metal that appears in all enzyme
classes (Broadley et al., 2007) and it is involved in
numerous aspects of cellular metabolism (Classen et
al., 2011). It has been estimated that 6 % of body zinc
is located in the skin (King et al., 2000).
Many of the biochemical and molecular events in
wound repair can be expedited by addition of
supplementary zinc ion through up-regulation of
Metallothioneins (Lansdown, 2002) and zinc
metalloenzymes (Ravanti and Kahari, 2000). Zinc acts
as a co-factor for enzymes involved in wound healing,
most notable lysyl oxidase, which catalyzes the cross-
linkage of collagen (Levenson and Demetriou, 1992).
The importance of cross- linking for the mechanical
strength of wounds was demonstrated by topical
administration of ß-aminopropionitril, which
irreversibly inhibits lysyl oxidase (Ågren and Franzen,
1990). Besides, any defect in the expression of zinc
finger transcription factors in mRNA coding of growth
factors is consistent with impaired wound healing
(Sum et al., 2005). The pattern of distribution between
zinc and copper concentration in the skin and hair of
the male and female goats observed in this study could
be an added factor responsible for early wound healing
in female aside the oestrogen factor which has been
sufficiently established in both human and animals.
The ratio and distribution of copper to zinc is clinically
more important than the concentration of either of
these trace metals (Osredkar and Suster, 2011).
Therefore, imbalance in zinc and copper ratio in the
hair and skin as a result of deficiency could alter
wound healing. So, normalising the ratio below toxic
level in wound therapy either through topical or oral
route will be beneficial in accelerating wound healing
in animals and humans. More studies are on-going in
determining the probable mechanism responsible for
these observations.
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