Assessment of Serum Copper Level among Sudanese Patients with vitiligo

Background: Vitiligo is a common skin disease of unknown etiology characterized clinically by depigmented patches, which can be localized or generalized; it usually runs a chronic course with an un predictable outcome and failure of complete cure in many affected individuals. Many communities consider it a contagious disease which leads to a great psychological and social stigma for patients; previous studies showed that copper might be associated with the pathogenesis of vitiligo. The aim of this study was to assess copper level in Sudanese vitiligo patients. 
Methods: This is a case-control study conducted in dermatology clinics in Khartoum state during the period from November 2018 to February 2019. Blood samples were obtained from 100 participants, 50 from vitiligo patients and 50 from non-vitiligo subjects representing a control  group. Serum copper was measured by mind-ray (automation). 
Results: A highly significant increase (P-value = 0.000) in the copper level was seen in vitiligo patients compared with the control group. Of the total number of patients, 17 (34%) were females and 33 (66%) were males. According to the duration of the disease, the copper level was significantly increased in patient group with a disease duration of > one year compared to the patient group six months–one year and patient group < 6 months; we found no significance of the family history, 18% of the case group had a family history while 42 (82%) had no family history. Discussion: The relationship between the serum level of copper and vitiligo has been assessed by many studies. Copper is one of the trace elements that was found to be important for tyrosinase enzyme that catalyzes the first steps in melanin synthesis in the skin. Some studies showed that the disease was associated with low serum levels of copper and since vitiligo is a disease that is characterized clinically by white areas of skin with no melanin, these studies seem to be logical. However, in this study, the serum level of copper was found to be high in vitiligo patients which might be justified by the release of copper from the destroyed melanocytes. Another justification might be a defect in the carrier protein for copper. 
Conclusion: The study found that the serum copper was significantly increased in vitiligo patients compared to the control and it is recommended that serum copper level and copper profile should be assessed routinely in vitiligo patients. 
Keywords: vitiligo, depigmentation, copper, Sudanese


Introduction
Sudan is the third largest country in Africa with a total population of around 40 million people [1]. It borders seven countries and its capital is Khartoum. Sudan is a miniature representation of the diversity found in most African countries [2,3].
The country is composed of 18 states; approximately 66% of the population lives in rural areas [4], and the percentage of poverty is around 46.5% [5]. The country suffers from a marked shortage in health workforce worsened by poor distribution over the Introduction Vitiligo is a chronic idiopathic skin disease, characterized by sharply marginated depigmented patches [1]. Lesions often start on sun-exposed areas [2]. Both genetic and environmental factors are believed to play a role in the pathogenesis of the disease [1,2].
The distribution of the disease can be unilateral segmental or non-segmental [1]. Vitiligo is estimated to account for 1% of the general population globally [3], however, in some populations, the incidence has reached 2-3% [4]. Patients who are stigmatized for the disease can experience depression and other psychological mood disorders [5].
The risk factors for developing vitiligo include family history and other autoimmune diseases [2]. The disease is usually diagnosed clinically. The diagnosis is confirmed by tissue biopsy and histopathological study [2]. Vitiligo should be differentiated from other skin conditions in which there is decrease in or loss of pigment, for example, tinea versicolor, piebaldism, idiopathic guttate hypomelanosis, etc. [6]. There is no cure for vitiligo [1], but it can be treated with the many available options, including topical steroids, sunscreens, phototherapy, etc. [1,2]. Melanin is the pigment majorly responsible for skin color; it is synthesized from amino acid tyrosine in the melanosomes that are found within epidermal melanocytes. Tyrosinase enzyme catalyzes the first two reactions in the pathway of melanogenesis [7]. It is found in animal and plant tissues responsible for the production of melanin as well as other pigments in these tissues [8]. Melanin is an effective absorbent of ultraviolet rays (UV) [10] and prevents the skin from its harmful effects. Some studies have shown that there is a reduced incidence of some types of skin cancers in individuals with dark skin compared to others, but the relationship between photo protection and the incidence of skin cancer is not clearly understood [11].
Tyrosinase is a copper-containing enzyme encoded by the TYR gene in humans [9].
Copper is a trace element needed in small amount in the diet, the proximal small intestine is recognized as the main site of dietary copper absorption in mammals. The transport of copper from the intestinal lumen into the intestinal mucosa is a carriermediated process involving a saturable transport component. The overall intestinal copper uptake is influenced by amino acids, ascorbic acid, and other dietary factors [12]. Once in mucosal cells, approximately 80% of the newly absorbed copper is in the cytosol, mainly bound to metallothionein (MTs). These are low-molecular weight-inducible proteins with many functions including homeostasis, storage, transport, and detoxification of metals.
The MTs bind to many metals, but in normal circumstances only Zn, Cu, and Cd binding is significant [13]. After passing through the enterocytes, copper enters the portal circulation where it is bound to carrier proteins (primarily albumin), peptides, and amino acids and is transported to the liver, with lesser amounts entering the kidneys.
Recently, many studies have been conducted to show that copper plays an important role in pigmentation, it accelerates the oxidation of dopa by skin extracts containing dopa-oxidase. Furthermore, the action of copper in pigmentation was not clear until Gorter [14], in 1935, conclusively demonstrated the fact that copper-free diets resulted in depigmentation of the hair of rats, rabbits, and cats and that this depigmentation disappeared following the administration of copper. On the other hand, no effect on melanogenesis was obtained if other minerals or vitamins were added to the diet [14].
In an old study submitted in Philadelphia 1931, Cunningham [15] made an observation relevant to this problem; he noted that the skin of black-coated animals contained more copper than the white-coated ones and that copper was concentrated mainly in the epidermis and "in vitro" experiments demonstrated the fact that copper accelerated the oxidation of dopa by skin extracts containing dopa-oxidase [15]. In the same year, Sarata [15] made the first definite attempt to correlate the copper content of skin with its degree of pigmentation. A study carried out in mottled dogs and cats showed that the copper content of pigmented hair was much higher than that of the nonpigmented hair of the same animal. Moreover, generally speaking, copper was found to be present in greater amounts in a skin covered by dark hair than the skin underlying the colorless hairs [15].
In 1934, Schroeder, Gruenberg, and Schade, quoted by Cornbleet [16], demonstrated that vitamin "C" inhibits the dopa reaction. Cornbleet [16] found that pigmentary precipitates can occur in a solution of dopa under the action of UV light alone, Buthe was able to accelerate this reaction considerably by adding copper to the solution. On the other hand, the addition of vitamin C tended to slow down the reaction or, in other words, to neutralize the catalytic effect of copper. Cornbleet concluded that the presence of these two substances, having antagonistic actions, so far as the oxidation rate of dopa is concerned, makes pigment formation susceptible to ready physiologic control [16].
Also, a study conducted at the Faculty of Medicine, Fayoum University showed that serum Zn levels were lower in different studied groups but it was much lower in the vitiligo group and that the serum Cu levels in the vitiligo group were insignificantly higher compared to the control group. Hence, serum Zn and Cu may have an effect on the vitiligo disease as Zn in combination with other micronutrients such as Cu, cobalt, nickel, iron, manganese, and Ca++ plays an important role in the process of melanogenesis [17].  A blood sample of 3 ml was collected in plain containers from each volunteer under optimum condition. The blood was centrifuged at 5000 r.p.m for 10 min which was stored in small aliquots and kept in a deep freezer (-20ºC) until analyzed. Data were collected from patients using questionnaire forms. After explaining the aim of the study, verbal consent was taken from all participants before sample collection.

Materials and Methods
Serum copper level was analyzed through spectrophotometric method using Mindray.
The precision and accuracy of the methods used in this study were checked each time using a control material.

Results
The study enrolled 50 vitiligo patients and 50 healthy subjects as a control group with matched age and sex. It was conducted at different dermatology clinics in Khartoum city, Khartoum state. The aim was to assess the serum copper level and to compare the results of the case and control groups of the study populations.        Table 2 shows the mean comparison of study parameter in case versus control group. The copper level in the case and control groups was 21.69 ±5 .17 mμol/l and 18.05 ± 3.51 mμol/l, respectively). The serum copper was significantly increased in vitiligo patients compared to the control group (p value = 0.000).  Table 3 shows mean comparison of study parameter across gender. There is no difference between males and females.  Table 4 shows mean comparison of study parameter across family history. The copper levels in family history were 22.48 ± 5.53 mμol/l and 22.48 ± 5.53 mμol/l, respectively, there were insignificant differences with p value = 0.612.  Table 5 shows the mean comparison of study parameter across the duration of vitiligo disease. There was no significant difference of serum copper level regarding the duration of the disease.  Figure 4 shows the correlations between the age and the serum copper level. There was a negative correlation between age and serum copper level (r = 0.230, p = 0.108).

Discussion
Vitiligo is a chronic skin disease that is considered as a stigma to patients in certain communities, it is wrongly believed to be contagious. were fed with only milk, which is a copper-deficient diet while being studied for milk anemia [1]. Cunningham in the same year found that the white-coated animals have lower copper levels in their skin than the black-coated ones, he also found that copper is concentrated mainly in the epidermis [2]. This observation is very important. The first which is similar to that reported by McBurney [19] but in disagreement with Lu et al. [20] who reported that vitiligo is distributed equally in men and women. showed no significant differences, disagreeing with a Chinese study that reported 20% of the patients with positive family history in first-degree relatives [18].

Conclusions
This study concluded that serum copper was highly and significantly increased in Sudanese vitiligo patients studied compared to the control group.

Recommendation
In our study, we recommend encouragement of healthy diets containing copper and all other trace elements important for enzymatic actions to prevent serious diseases, for example, vitiligo, measurement of copper profile, routine investigations for copper, and other trace elements for vitiligo patients, and further studies regarding the role of other trace elements need to be performed in vitiligo patients because the etiopathogenesis of the disease is still unclear and also the treatment of vitiligo still represents a great challenge.