|Year : 2022 | Volume
| Issue : 2 | Page : 119-124
Serum copper, zinc, and copper − zinc ratio in children with malaria
Belinda Onosen Jimoh1, Moyosoreoluwa Temiloluwa Fadipe2, Mathias Abiodun Emokpae1
1 Department of Medical Laboratory Science, School of Basic Medical Sciences, University of Benin, Benin City, Edo State, Nigeria
2 Department of Medical Laboratory Science, College of Basic Health Sciences, Achievers University, Owo, Ondo State, Nigeria
|Date of Submission||16-Jun-2022|
|Date of Decision||23-Jul-2022|
|Date of Acceptance||01-Aug-2022|
|Date of Web Publication||22-Aug-2022|
Mathias Abiodun Emokpae
Department of Medical Laboratory Science, School of Basic Medical Sciences, University of Benin, Benin City, Edo State
Source of Support: None, Conflict of Interest: None
Background: Nutrition is not only vital to reducing the risk of individual's susceptibility to malaria infection but enhances the prevention and treatment of disease. Nutrition can also modify the course of malaria infection, especially among children. Aims: The aim of this study was to determine the concentrations of copper, zinc, and copper − zinc ratio in children with malaria infection and correlate same with parasite density. Setting and Design: A cross-sectional study of malaria-infected children randomly recruited from two secondary health institutions in Benin City, Nigeria. Methods: A total of 200 malaria-infected children (age range 0.5–11 years, 113 (56.3%) males and 87 (43.7%) females were recruited in the study. Semi-structured questionnaire was used to collect the sociodemographic data. Blood sample was collected from each of the participants and malaria parasite density was determined using thick blood film. Serum zinc and copper were determined using atomic absorption spectrophotometry. Statistical Analysis Used: The categorical and continuous data were compared using the Chi-square, unpaired Student's-test, and analysis of variance, respectively. Results: Serum copper was significantly higher among malaria-infected children than nonmalaria infected children, while zinc was significantly lower in malaria-infected subjects than controls. The copper/zinc ratio was significantly higher in malaria infected than nonmalaria infected children. Serum copper and copper/zinc ratio correlated positively while zinc correlated negatively with malaria parasite density. Conclusion: The observed significantly higher copper and copper/zinc ratio and lower zinc level may indicate oxidative stress, inflammation, and lower immune status in malaria infection.
Keywords: Child, malaria, zinc
|How to cite this article:|
Jimoh BO, Fadipe MT, Emokpae MA. Serum copper, zinc, and copper − zinc ratio in children with malaria. Saudi J Health Sci 2022;11:119-24
| Introduction|| |
Nutrition is vital to reducing the susceptibility of individual to malaria infection, prevention, and treatment of the disease. Nutrition can also modify the course of malaria infection, especially among children. Some authors have suggested that malaria may increase the incidence and severity of malnutrition, while malnutrition may exacerbate the risk of malaria infection. Therefore, adequate medical and nutritional management are necessary to prevent adverse effects of malaria infection. Malaria is the sixth leading cause of death among children aged <5 years all over the world, and Nigeria accounted for almost 24% of all global malaria deaths. Despite the commitment and investment made by successive governments to control or eliminate malaria, these goals have not been completely achieved. There has been increased use of insecticide treated nets with artemisinin-based combination therapy, but malaria remains a major contributor to the burden of disease among young children. Some of the obstacles to the total success of these treatment options include a local weather condition that encourages malaria transmission all year round, and the risk of acute malnutrition in young children which increases their susceptibility to malaria infection and causing a huge economic burden., Because of the merger resources most countries in sub-Saharan Africa cannot sustain the distribution of free nets at the expense of the infrastructural needs in the region. Inadequate nutrition may predispose children to infection or make it more difficult to recover from infection. There is a need to scale up strategies which are geared toward understanding ways to reducing or preventing malaria-associated deaths. Periodic assessments of potential biomarkers of malnutrition and immune status in children may help prevent malaria infection and severity. Prevention they say is better than cure. It may be more beneficial to prevent malaria infection than treatment. Serum copper-to-zinc ratio is a more relevant diagnostic measure than the concentration of either metal alone in clinical practice.
Zinc is an essential trace element for humans and other animals, for plant and for microorganisms. Zinc is necessary for the function of over 300 enzymes and 1000 transcription factors and is stored and transferred in metallothioneins. It is the second highest trace metal in humans after iron and is the only metal that is present in all enzyme classes. The distribution of zinc is about 4–5 g in the human body which are mostly found in the brain, muscle, bones, kidney, and liver, while the highest concentrations are found in the prostate and parts of the eye. Zinc homeostasis of the body is mainly controlled by the intestine due to ZIP4 and especially TRPM7. In humans, the biological roles of zinc include the metabolism of RNA and DNA, homeostasis, signal transduction, and gene expression. It is recognized that excessive copper reduces zinc absorption, and copper concentration in the blood plasma remains relatively constant regardless of zinc intake. The cells are able to use zinc signaling to communicate with other cells.
Copper is involved in the formation of red blood cells, the absorption and utilization of iron, the metabolism of cholesterol and glucose, and the synthesis and release of life-sustaining proteins and enzymes. These enzymes in turn produce cellular energy and regulate nerve transmission, blood clotting, and oxygen transport. Copper stimulates the immune system to fight infections, to repair injured tissues, and to promote healing. Copper also helps to neutralize free-radicals, which can cause severe damage to cells. Normal copper levels are necessary to carry out such metabolic functions as cellular respiration, melanin pigment and connective tissue synthesis, iron metabolism, free radical defense, gene expression, and the normal functioning of the heart and immune systems in infants. The aim of this study was to determine the concentrations of copper, zinc, and copper-to-zinc ratio in children suffering from malaria infection.
| Methods|| |
Study area and population
A total of 200 malaria-infected children and 100 nonmalaria infected children attending the outpatient departments of Central Hospital and Stella Obasanjo children and women hospital within Benin metropolis were recruited for this study. The age range between 6 months and 11 years, mean age: 38 months, 113 males, and 87 females. All subjects were evaluated for malaria parasite after clinical and physical examination. Those provisionally diagnosed for malaria infection were confirmed by microscopic examination of both thin and thick blood films for malaria parasite detection and count. The age and sex of children who visited the hospitals for medical check and vaccination and found to be negative for malaria parasitemia were recruited as controls.
All subjects who gave informed consent and met the inclusion criteria were enrolled into the study.
Sample size determination
The sample size was determined using the sample size determination formula, n = Z2 P(1−P)/d2. Where: n = minimum sample size, Z = Standard normal deviate that corresponds to 95% confidence limit (1.96), d = alpha level of significance (5%), P = Prevalence rate of malaria in children in Benin metropolis is 9.23%. The calculated sample size was 128, but for the purpose of the study, the sample size was increased to 200 subjects.
Only children with clinical signs/symptoms for malaria such as fever, cough, diarrhea, pallor, vomiting, between the ages of 6 months and 11 years and tested positive to malaria infection by microscopic examination and have not used anti-malaria drugs in the past 1–2 weeks will be used. Aged-matched asymptomatic healthy children that were on routine medical screening and vaccination who tested negative for malaria parasite were used as controls.
Children who are malnourished, with known history of sickle-cell anemia, HIV positive or any other ailment or disease that may contribute to the oxidative stress level of the participating subjects were excluded from the study. Furthermore, those that are on anti-malaria drugs were also excluded.
Clinical and anthropometric measurements
Measurements were done by the attending qualified nurses using standard scales for the height and weight. Height to age ratio was calculated. Other areas of special interest considered are body temperature, history of any of underlying ailment, and current medication regimen. Socioeconomic status and the general knowledge of malaria preventive measures were obtained using questionnaire answered by the child's parent or custodian.
Exactly 3 mL of blood was collected from the subject for the assay of zinc and copper, by venipuncture into properly prelabeled plain sterile bottles. The samples were allowed to properly retract within 1 h of collection and spun at 2000 rpm for 15 min to obtain the serum. Serum was then dispensed into a properly labeled, plain clean tube and stored at -20°C prior to analysis. Thick blood film will be prepared simultaneously in duplicates while collecting the blood into plain tubes for malaria parasite density count.
Thick film preparation and determination of parasite density count
Using a grease-free microscope slide, a large drop of blood about 15 mm was placed on the slide. Without delay, the end of a plastic bulb pipette was used to spread the drop of blood to make the thick smear to cover an area of 15 mm × 15 mm. The blood films were allowed to dry at the room temperature. The dried thick blood smear was stained and examined using Giemsa stain and light microscopy, respectively. Smears were examined using ×100 oil immersion ×100 (oil immersion) objectives to determine the parasite density (WHO, 2016). At least 100 high power fields were examined before a thick smear was declared negative. Plasmodium falciparum parasites were counted per 200 or 500 leukocytes, which were used to determine the parasite density per microliter of blood. Thin films were examined to confirm the species identification on the thick film. The malaria parasites were enumerated and reported thus: Low parasitemia, (<500 cells/μl of blood), moderate parasitemia (500–1000 cells/μl of blood), and high parasitemia (>1000 cells/μl of blood).
Sample preparation and analysis by atomic absorption spectrometry
Precisely 1000 μl of the serum was pipetted using Thermo Finnpipette into 10.0 mL centrifuge tube and 4.0 mL of 0.5 N HCL made by British Drug House was added and left for 24 h to remove the protein, break the bond and make the elements of interest mobile for assay. After 24 h, the mixture was centrifuged at 4000 rpm for 5 min. The supernatant was aspirated into the atomic absorption spectrometry (AAS) equipment. Blank was also prepared in the same way, using de-ionized water as the sample. Portion of this solutions was analyzed for Cu and Zn. The measurements were taken twice and the average was calculated. Standard solutions were prepared and used to calibrate the AAS instrument for all the analytes and also to prepare their calibration curves. The multielement standards manufactured by Sigma Aldrich were used to prepare the standard of various concentrations.
Principle of atomic absorption spectrometry
AAS is based on the principle that free atoms in an atomizer can absorb radiation at specific wavelength or frequency. Radiation from a hollow cathode lamp (in which the cathode is made from the element of interest) is modulated and passed through a flame into which is sprayed on the solution for the analysis. The radiation then passes to a monochromator where the particular resonance line required is isolated, and any absorption due to the atomic vapor in the flame is measured by means of photomultiplier. The photocurrent is amplified, demodulated and fed either directly to a meter or to a logarithmic converter. The calibration curve obtained by plotting the response or absorbance of an analyte against concentration helps to determine the concentration of analyte in an unknown sample and by comparing the unknown to a set of standard samples of known concentration. The correlation coefficients of the standards prepared were between 0.95 and 0.999.
Data were analyzed using the SPSS software for Windows version 21.0.(Chicago, IL, USA). The values are represented as mean ± standard deviation. Student's t-test and analysis of variance were used to compare the means between the groups while correlations between continuous variables were assessed by the Spearman–rank test. P < 0.05 was considered statistically significant.
| Results|| |
A total number of 300 subjects comprising 200 malaria infected children with age mean 3 yrs 2 months, age range 6m-11yrs, male 113 (56.3%), female 87 (43.7%) weight (kg) 99.4±19.2 height (cm) 13.6±4.6 temperature (oC) 38.7±1.2 and 100 non malaria infected children with mean age 3 yrs 9 months, age range 6m-11yrs, male 63 (63%), female 37 (37%) weight (Kg) 100±20.1 height (cm) 13.9±4.2 temperature (oC) 37.3±1.1 were evaluated [Table 1]. [Table 2] indicates the comparison of mean levels of copper, zinc, and copper-to-zinc ratio in malaria infected and nonmalaria infected children. Serum copper (104.18 ± 2.81) was significantly higher (P < 0.001) among malaria-infected children than nonmalaria-infected children (95.66 ± 2.81) while zinc (93.36 ± 2.84) was significantly lower (P < 0.001) among malaria infected than nonmalaria-infected children (105.62 ± 2.62). The Cu/Zn ratio was significantly higher (P < 0.001) among malaria-infected children (1.16 ± 0.02) than nonmalaria-infected children (0.90 ± 0.02). The comparison of the mean levels of copper, zinc, and copper-to-zinc ratio in malaria-infected children based on parasite density shows that serum copper levels increased with increasing malaria parasite density while serum zinc and copper-to-zinc ratio decreased with increasing malaria parasite density [Table 3].
|Table 2: Comparison of mean levels of copper, zinc, and copper-to-zinc ratio in malaria infected and nonmalaria infected children (mean±standard deviation)|
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|Table 3: Comparison of the mean levels of copper, zinc, and copper-to-zinc ratio in malaria-infected children based on parasite density|
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[Table 4] shows the correlation between serum copper, zinc, copper-to-zinc ratio, and parasite density among malaria-infected children. Serum copper correlated positively (R = 0.198, P < 0.05), while zinc (R = −0.281, P < 0.005) and copper-to-zinc ratio (R =−0.289, P < 0.005) correlated negatively with malaria parasite density among children with malaria infection.
|Table 4: Correlation between measured parameters and parasite density in the cases of malaria-infected children|
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| Discussion|| |
Malaria is a life-threatening disease caused by parasites that are transmitted to people through the bites of infected female Anopheles mosquitoes. Despite considerable breakthroughs in the treatment and management of malaria in the last decade, there remains an urgent need to understand the roles certain biomarkers play in the pathogenesis of malaria. In this study, it was observed that more male than female subjects were infected with malaria. This suggests that the incidence of malaria was higher among males than females. This finding is consistent with previous study in the same geo-political zone of Nigeria.
In this present study, it was observed that serum copper and Cu/Zn ratio were significantly higher, while serum zinc was significantly lower among malaria-infected children than nonmalaria-infected children. The finding is consistent with previous study from Eastern Sudan. Serum zinc and copper levels in the body are very important as they contribute to the immune response of the host to the antigenic challenge by malaria parasites since both elements are important for normal immune function. The higher copper and lower zinc levels in malaria infection may be due either to an attempt of the body to respond to malaria parasite infection as immune response or the inhibition of zinc absorption from the intestine. It is recognized that excessive copper reduces zinc absorption, and copper concentration in the blood plasma may remain relatively constant regardless of zinc intake. Copper acts by stimulating the immune system to fight infections, to repair injured tissues, and to promote healing. Furthermore, the significantly higher copper level may be attributed to an attempt by the body to neutralize free-radicals which are generated during infection since malaria is an inflammatory and oxidative disease. The generation of free radicals by host phagocytes is an essential component of the host response to Plasmodium infection. There is a close relationship between inflammation and oxidative stress during infection. It is well recognized that innate immune cells respond to pathogens by enhancing inflammatory responses. Innate immune cells engulfed pathogens and attempt to eliminate them by exacerbating the generation of free radicals in their phagocytic cells in a mechanism referred to as oxidative or respiratory burst. Free radicals generated during the respiratory burst are released from the cells into the blood, thereby contributing to the increase of the oxidative state in the infected host. The exact mechanism by which oxidative stress increase with severity in malaria infection is not known, but it is possible that oxidative stress contributes to disease severity through decreased antioxidants enzymes functions or through the increase in the inflammatory response. The higher copper level may have occurred in an attempt to increase antioxidant enzymes activity to mob up increased free radicals generated during malaria infection. Copper is known to act in a bidirectional ways, as high concentrations of Cu may cause increased oxidative damage to lipids, proteins, and DNA. Conversely, Cu is essential for optimal antioxidant defense, since Cu deficiency has been reported to cause oxidative stress. Copper also plays some roles in protecting the body from the potential negative effects of free radicals by enhancing enzyme functions such as superoxide dismutase.
In humans, the biological roles of zinc include the metabolism of RNA and DNA, homeostasis, signal transduction, and gene expression. The significantly lower level of zinc may be due to decreased absorption or partly due to the utilization of zinc by Plasmodium to fight the oxidant flow generated in course of infection. This finding is also similar to previous report., The critically low level of serum zinc in children with malaria may be due redistribution of zinc from plasma to lymphocytes and liver during the acute-phase response. Significantly lower zinc levels may be an adaption process in malaria-infected children, since reduction of zinc in circulation also reduces the zinc available for the metabolism of microorganisms during infection.,
In this study, the serum Cu/Zn ratio was statistically higher in malaria-infected children than nonmalaria-infected children. The finding also aligns with previous studies., A higher value of Cu/Zn ratio among malaria-infected children than controls (P < 0.001) was reported in Abidjan, Côte d'Ivoire. This reported higher Cu/Zn ratio in malaria-infected children than the normal values (<1), is an indication of higher oxidative stress in malaria-infected children.
Copper, zinc, and copper-to-zinc ratio in malaria-infected children correlated with parasite density. The finding was partly similar with previous study,, who reported that mean serum zinc level correlated negatively with severity/complicated malaria. There was a negative correlation between zinc levels and malaria parasite density. Consequently, higher Cu/Zn ratio than healthy subjects may indicate an inflammatory state and a high risk of zinc deficiency in malaria infected children.
| Conclusion|| |
serum level of copper-to-zinc ratio was significantly higher among malaria-infected children than nonmalaria infected children and may indicate oxidative stress, inflammation, and low immune status.
We are grateful to the clinicians, nurses, medical laboratory scientists, and other supporting staff of Central Hospital and Stella Obasanjo Children and Women Hospital, Benin City toward the successful completion of this study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]