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Comparative analysis of serum zinc, copper, magnesium, calcium and iron level in acute and chronic patients of visceral leishmaniasis
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ARTICLE IN PRESS Journal of Trace Elements in Medicine and Biology xxx (2012) xxx–xxx
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Biochemistry
Comparative analysis of serum zinc, copper, magnesium, calcium and iron level in acute and chronic patients of visceral leishmaniasis Chandra S. Lal a,∗ , Sanjay Kumar a , Alok Ranjan b , Vidya N. Rabidas c , Neena Verma d , Krishna Pandey c , Rakesh B. Verma b , Sushmita Das e , Dharmendra Singh e , Pradeep Das e a
Laboratory of Clinical Biochemistry, Division of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences (Indian Council of Medical Research), Patna, Bihar, India Division of Biostatistics, Rajendra Memorial Research Institute of Medical Sciences, Patna, Bihar, India c Division of Medicine, Rajendra Memorial Research Institute of Medical Sciences, Patna, Bihar, India d Division of Pathology, Rajendra Memorial Research Institute of Medical Sciences, Patna, Bihar, India e Division of Molecular Biology, Rajendra Memorial Research Institute of Medical Sciences, Patna, Bihar, India b
a r t i c l e
i n f o
Article history: Received 7 February 2012 Accepted 30 September 2012 Keywords: Trace elements Visceral leishmaniasis (VL) Acute VL Chronic VL
a b s t r a c t Project: Chronic visceral leishmaniasis (VL) is an increasingly common problem in disease endemic states of India. Identification of prognosis risk factor in patients with VL may lead to preventive actions, toward decreasing its mortality in chronic individuals. Though serum Zinc levels are decreased in patients of VL, limited information is available regarding trace elements status in acute and chronic VL patients. The present study was undertaken to compare serum trace elements concentrations in acute and chronic VL patients. Procedure: Acute (mean age = 28.64 years), chronic (mean age = 23.68 years) VL patients and healthy controls (mean age = 23.05 years) who agreed to provide blood specimens for laboratory investigations participated in this study. Serum zinc (Zn), copper (Cu), iron (Fe), magnesium (Mg) and calcium (Ca) were measured spectrophotometrically using chemistry analyzer. Results: Serum Zn concentration was comparatively much decreased in chronic VL than to acute ones (p = 0.007) while serum Mg was higher in chronic VL than acute (p = 0.002) ones. There was no statistically significant difference between acute and chronic VL in serum concentrations of Cu, Fe and Ca. Conclusions: Serum Zn levels were much decreased and serum Mg were increased in chronic VL as compared to acute cases. The serum concentrations of Fe and Ca did not show any difference between two groups. The serum Cu was increased in both groups but more in chronic ones. Serum Zn and Mg could be a potential prognosis factor for chronic VL patients. We hypothesize zinc supplementation as a chemo preventive agent for chronic VL cases, particularly in endemic areas. © 2012 Elsevier GmbH. All rights reserved.
Introduction Visceral leishmaniasis (VL), generally called kala-azar, occurs in several Mediterranean countries and Indian sub-continent, viz.: India, Nepal and Bangladesh. Protozoa of the species Leishmania donovani (L. donovani), which are responsible for the disease, are transmitted to humans by the sand fly, Phlebotomus argentipes. The incidence of kala-azar in India is among the highest in the world [1,2]. Bihar contributes as one of the major state in India for visceral leishmaniasis. At present, 28 of 37 districts of Bihar are endemic contributing about 90% of total Indian cases. Marked enlargement
∗ Corresponding author at: Laboratory of Clinical Biochemistry, Division of Biochemistry, RMRIMS (ICMR), Agamkuan, Patna 800 007, India. Tel.: +91 9835867492; fax: +91 6122634379. E-mail address: [email protected] (C.S. Lal).
of spleen and liver with moderate to severe anemia, pancytopenia are the presenting feature [3]. Leishmaniasis was selected by the World Health Organization for elimination by 2015, along with other neglected tropical diseases. Since there is no antileishmanial vaccine in clinical use, control of VL relies almost exclusively on chemotherapy. The use of conventional antileishmanials as well as new drugs is hampered mainly due to high costs and also due to concerns of toxicity and emergence of resistance. The chronic VL (CVL) patients are a serious health problem in disease endemic states of Bihar, India. The immune system of the body plays an important role during response to drug in any disease. Several enzymes that contribute to immune system responses require zinc and copper as trace elements for their activity which includes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) which contribute to immune system responses, require the mentioned trace elements for their activity [4]. The concentrations of serum iron (Fe), zinc (Zn), copper (Cu), and other
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Please cite this article in press as: Lal CS, et al. Comparative analysis of serum zinc, copper, magnesium, calcium and iron level in acute and chronic patients of visceral leishmaniasis. J Trace Elem Med Biol (2012), http://dx.doi.org/10.1016/j.jtemb.2012.09.007
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trace elements have been of interest to investigators for a number of years in many pathological conditions such as congestive heart failure, pneumonia, rheumatic heart diseases, bronchitis, and various infections, hemolytic anemia and psoriasis [5]. In cutaneous leishmaniasis Zn and Fe concentration are found lower while Cu has tendency to decrease [6]. Low serum zinc levels in an endemic area of visceral leishmaniasis in Bihar, India has also been reported [7]. Further, zinc deficiency has showed decreased Th1 but not Th2 immune response in experimental human leishmaniasis [8]. However, no information about assessment of serum concentrations of essential trace elements in acute and chronic VL patients is available from disease endemic areas. The indispensable feature of the most prominent of these trace elements rests on the functional role and structural components of crucial metallo-enzymes and metallo-proteins [9]. One of the mechanisms, the serum redistribution of essential trace elements Zn, Cu and Fe together with the increase in synthesis of acute-phase proteins (like ceruloplasmin), which takes part during the course of most infection, is well established [10]. The changes are part of defense strategies of organism and are induced by the hormone-like substances, such as interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-␣), and interleukin-6 (IL-6) [11,12]. These substances are immunocytokines liberated in a dose-dependent mode, mostly by activated macrophages, in response to several stimuli, including exercise, trauma, stress, or infection [13]. Zinc acts as an effective anti-inflammatory and antioxidant agent [14]. For example, Cu–Zn SOD is an important intracellular enzyme that requires both Cu and Zn for normal activity; Zn stabilizes the enzyme and Cu is necessary for catalysis. It acts as an antioxidant by oxidizing free-radical oxygen and it is the major Cu enzyme in erythrocyte [15]. Secondly, Zn deficiency has been shown to lead to a selective Th1 deficiency in human volunteers [16]. Besides this, Cu plays an important role in the biochemical pathway of mammals, and is a building block of many enzymes that have key functions in the metabolism [17,18]. Elevated copper is thought to induce pathological changes in tissues by stimulating the production of reactive oxygen species that damage multiple cells. Magnesium (Mg) is one of four bulk metals in the human and it is a co-factor for about 300 cellular enzymes and is required for the activity of many enzymes, particularly those utilizing an ATP [19]. Besides this, Mg2+ seemed to increase parasite proliferation in L. major and activate macrophage for establishment of infection [20,21]. Calcium (Ca) has several important functions in the formation of bone and dental tissues, in the releasing of hormones, contraction of muscles and glycogen metabolism [9]. Oxidative stress is known to be an important contributing factor in many chronic diseases. Hence, keeping in view the above observations, we considered the assessment of trace elements concentrations in serum of acute and chronic VL patients. The 3-fold purpose of this study includes: to investigate the serum levels of Zn, Cu, Fe, Ca and Mg; to determine any deficiency in these elements; to determine the differences in these two susceptible VL groups.
Materials and methods Subjects The clinical and observational study included adult VL patients admitted to the indoor ward of Rajendra Memorial Research Institute of Medical Sciences, Patna, India for the anti-leishmanial treatment. All together, 66 subjects were selected for this study: 22 acute VL, 22 chronic VL and 22 healthy not exposed to
leishmania infection. Our inclusion criteria for acute VL group were (a) any age or sex, any degree of parasitic load (b) had not taken any anti-leishmanial treatment before admission to the institute and (c) the duration of illness up to 4 weeks. For chronic VL group, the inclusion criteria were (a) any age or sex, any degree of parasitic load (b) prior to admission in the institute had taken anti-leishmanial treatment (i.e.: sodium antimony gluconate) and did not respond to that drug and disease recurred and was referred to this institute and (c) the duration of illness more than 3 months. The control group was selected among healthy people who were not exposed to visceral leishmaniasis or any other diseases. Ethics approval was obtained from ethics committee of the institute and was in accordance with the 1975 Helsinki Declaration on Human Rights, as revised in Edinburgh 2000. Patients were included after written informed consent was obtained from each patient’s next of kin. Clinical evaluation The medical staff of the Department of Clinical Medicine evaluated the patients for clinical diagnosis for VL infection. Laboratory Laboratory diagnosis was parsitologically confirmed by the microscopic demonstration of leishmania parasite in the splenic aspirate smears taken from the patients and the parasite load was graded as per the standard quantization grading [22]. Blood samples were collected into vacutainer tubes without any anticoagulant for biochemical investigations. The blood was centrifuged at 3000 rpm for 5 min and the serum was extracted in plastic tubes until assayed. Biochemical assay All biochemical assays were standardized as per the described protocol. The reagents used in the study were of the analytical grade (FAR srl, Italy). All estimations were repeated 3 times and put in triplicates every time. Average of all 9 readings was taken as final value. The entire assays were carried out on semi-automated chemistry analyzer Merck Microlab-300 LX (Vital Scientific N.V., The Netherlands). Zinc assay: Serum zinc level was quantified by colorimetry as previously described. This method was previously shown to produce similar results in zinc quantification as atomic absorption spectrophotometry (AAS) with a positive correlation (r = 0.982) and approximately 4% difference in readings [23]. Copper assay: The sensitive direct colorometric assay for copper in serum was followed as previously described [24]. The result of this method correlated well with those determined by atomic absorption spectrophotometric techniques (r = 0.977). Magnesium assay: Serum magnesium was assayed following Calmagite method [25]. Calcium assay: Serum calcium was quantified according to Arsenazo III (1,8-dihydroxy-3,6-disulpho-2,7-naphthalene-bis(azo)dibenzenearsonic acid) method following protocol of commercial kit (FAR srl, Italy). Iron assay: Serum iron was quantified according to Ferrozine method following protocol of commercial kit (LABKIT, Spain). Statistical analysis We used one-way analysis of variance (ANOVA) for comparing means of physical characteristics (age and body weight) and mean levels of trace elements (Cu, Zn, Fe, Ca and Mg) among three groups
Please cite this article in press as: Lal CS, et al. Comparative analysis of serum zinc, copper, magnesium, calcium and iron level in acute and chronic patients of visceral leishmaniasis. J Trace Elem Med Biol (2012), http://dx.doi.org/10.1016/j.jtemb.2012.09.007
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C.S. Lal et al. / Journal of Trace Elements in Medicine and Biology xxx (2012) xxx–xxx Table 1 Sex wise distribution of VL patients and healthy controls. Sex
Groups
Total
Chronic VL
Acute VL
Healthy controls
Female Male
11 11
6 16
11 11
28 38
Total
22
22
22
66
Chi-square = 3.102 at 2 d.f., p = 0.212.
– acute VL patients, chronic VL patients and healthy controls. We also used multiple comparisons using Tuckey’s test for inter-groups comparison. Chi-square statistic was used to compare sex distribution among three groups. All statistical analyses were performed using Statistical Package for Social Sciences (SPSS) program version 15 (SPSS, Inc., North Carolina, USA). Results Out of 66 subjects, 22 were in chronic VL group (Mean age 23.68 years), 22 in acute VL group (Mean age 28.64 years) and 22 were healthy controls (Mean age 23.05 years). There was no statistically significant difference of sex composition (p = 0.212) among these three groups (Table 1). We did not observe any statistically significant difference in mean values of age (p = 0.382) and body weight (p = 0.066) among the groups, and also in between the groups when compared with each other (Table 2) showing similar distribution in the groups without any confounding factor with respect to sex, age and body weight. Table 3 presents comparison of trace elements’ levels among three groups. The mean level of Cu was significantly different among three groups (p = 0.001) without significant difference between chronic and acute VL patients (p = 0.114). It was significantly higher in both chronic and acute VL patients when compared with healthy controls (p = 0.001) indicating increased Cu level in both chronic VL and acute VL patients. We observed a statistically
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significant difference of Zn level among three groups (p = 0.001) and also significantly different when compared with each other. The level of Zn was found decreased significantly in both chronic and acute VL patients as compared to healthy controls (p = 0.001). There was a significant indication of more decreasing trend of Zn levels as the disease becomes chronic. There was significantly lower mean level of Fe in both chronic (p = 0.001) and acute (p = 0.001) VL patients as compared to healthy controls. But we did not observe any statistically significant difference of mean Fe level between chronic and acute VL patients (p = 0.817), almost having the same level of Fe. The mean level of Ca was almost similar in three groups showing no statistically significant difference between chronic and acute VL patients (p = 0.238). There was statistically significant difference of Mg mean level among three groups (p = 0.001), and also between chronic and acute VL patients (p = 0.002). The level of Mg was significantly higher in chronic VL patients as compared to healthy individuals (p = 0.006), but the level was almost similar in acute VL patients as compared to healthy individuals (p = 0.928). No correlation was observed between parasitic load and serum concentrations of trace elements. Discussion Disorders of essential trace elements arise from inadequate intake, genetic defects, excessive exposure, or impaired elimination. Severe deficiency is rare, but specific symptoms and reduced protection against risk factors have been ascribed to suboptimal intake of trace elements. Besides this, secondary changes in the trace elements occur as a result of diseases and these changes are still unnoticed in visceral leishmaniasis cases in Bihar, India and which might be playing some significant role. Though, the antileishmanial treatment by sodium antimony gluconate (pentavalent antimonial compound) affects trace elements in cutaneous leishmaniasis during the treatment, these changes return to normal levels by unknown mechanisms [6].
Table 2 Physical characteristics of patients with chronic and acute visceral leishmaniasis and control group. Characteristics
Chronic VL (1) patients (n = 22)
Acute VL (2) patients (n = 22)
Healthy controls (3) (n = 22)
Age (year) Mean (95% CI)
23.68 (17.31–30.05)
28.64 (22.47–34.80)
23.05 (16.27–29.82)
0.382
0.499 (1 vs. 2) 0.988 (1 vs. 3) 0.414 (2 vs. 3)
Body weight (kg) Mean (95% CI)
33.05 (26.11–39.98)
33.41 (28.07–38.75)
43.68 (34.21–53.15)
0.066
0.998 (1 vs. 2) 0.098 (1 vs. 3) 0.114 (2 vs. 3)
p-Value for ANOVA
p-Value for Tuckey’s test
Table 3 Comparison of levels of trace elements of chronic and acute visceral leishmaniasis patients and control group. Variables
Chronic VL (1) patients (n = 22)
Acute VL (2) patients (n = 22)
Healthy controls (3) (n = 22)
p-Value for ANOVA
p-Value for Tuckey’s test
Cu (g/dL) Mean (95% CI)
384.94 (348.39–421.49)
332.80 (279.21–386.40)
111.41 (101.96–120.86)
0.001
0.114 (1 vs. 2) 0.001 (1 vs. 3) 0.001 (2 vs. 3)
Zn (g/dL) Mean (95% CI)
44.51 (42–47)
56.27 (48.83–63.71)
78.04 (72.63–83.45)
0.001
0.007 (1 vs. 2) 0.001 (1 vs. 3) 0.001 (2 vs. 3)
Fe (g/dL) Mean (95% CI)
42.86 (39.20–46.52)
40.61 (37.33–43.88)
93.62 (85.53–101.72)
0.001
0.817 (1 vs. 2) 0.001 (1 vs. 3) 0.001 (2 vs. 3)
Ca (mg/dL) Mean (95% CI)
9.64 (9.14–10.15)
9.11 (8.62–9.58)
9.79 (9.33–10.25)
0.096
0.238 (1 vs. 2) 0.889 (1 vs. 3) 0.098 (2 vs. 3)
Mg (mg/dL) Mean (95% CI)
2.30 (2.14–2.47)
1.96 (1.82–2.10)
1.99 (1.87–2.12)
0.001
0.002 (1 vs. 2) 0.006 (1 vs. 3) 0.928 (2 vs. 3)
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In the present study, we investigated trace elements such as Cu, Zn, Fe, Ca and Mg in acute and chronic VL cases and demonstrated that serum Zn and Fe concentrations were lower in both acute and chronic VL patients (p = 0.001 and p = 0.001), respectively, than healthy control group but there was a significant indication of more decreased Zn level in chronic VL (p = 0.007) in comparison to acute VL. Zn is crucial for immune functioning of the body. The role of certain inflammatory products in the regulation of the Zn balance has been well documented [10]. Thus, leukocyte-endogenous mediators (interleukins), released from activated phagocytic cells, induce hypozincemia in experimental animals by the redistribution of Zn from plasma to the liver [26]. Decreasing serum Zn concentrations apparently results from the synthesis of methallothionein in liver and other tissues. Methallothionein binds 7 g atoms of Zn/mol and serves to draw Zn away from free-circulating pools; it was induced by IL-1 in vivo [27]. Decreased Zn enhances humoral response (Th2) which results in decrease of IFN-␥ production. This could contribute to the chronic state of the disease. However, serum Cu concentrations were significantly raised in both acute and chronic VL cases (p = 0.001 and p = 0.001) than healthy control group but increased level had been observed in chronic VL category. Increased serum Cu is associated with ceruloplasmin and induced by IL-1 [28]. It was reported in one of the study [29] that IL-1, not TNF-␣, induces hypercupremia when injected into the preoptic anterior hypothalamus. However, the high production of IL-1 in cutaneous leishmaniasis is in contrast to visceral leishmaniasis, where infection of mice with L. donovani resulted in the suppression of the IL-1 response [30]. These findings may reflect the distinct pathology and immune responses caused by the two species of Leishmania. In our study, we observed that Cu levels were significantly higher in patient’s sera than that of healthy subjects. Increased Cu may be attributable to inflammation associated with the disease and increased Cu level might increase resistance to leishmaniasis [4]. Under stress or attack (i.e., infection) serum Cu increases more than normal to facilitate vital processes such as Cu-ATPases, which is crucial for: (i) central nervous system development, (ii) liver function (VL has to do with liver), (iii) connective tissues and (iv) many other physiological processes. Plasma Cu in VL patients increased to levels which have been shown to be toxic in vitro [31] and our result trend in Cu is also suggestive for the chronicity of the disease. These characteristic changes in the trace element metabolism are an integral part of the acute-phase response. These changes are usually reflected in decreased serum Zn and Fe and increased serum Cu concentrations [32]. Further, it had been reported that Cu/Zn imbalance might serve as a marker for decreased Th1 response and immunodeficiency in leishmaniasis, being more pronounced in its most severe and possibly fatal visceral form [33,34]. With Cu raised and Zn decreased, humoral response (Th2) is enhanced, thus a decrease of IFN-␥ production. This could contribute to the chronic state of the disease. We observed significantly raised Mg value (p = 0.006) in only chronic VL cases than healthy controls, while it was not raised in acute cases (p = 0.928). In case of Zn deficiency in chronic VL patients, the defensive mechanism of the body is to raise Mg levels. Mg is necessary for the synthesis of various compounds that have energy-rich bonds of any type (e.g., phosphoric anhydride bond found mainly in ATP and others). Thus allows cellular respiration to be used to store energy in the form of high energy phosphate. This indicates that elevated Mg could be contributory to the chronic state of the disease. It is reported that establishment of infection by Leishmania depends on the transformation of the invading metacyclic promastigotes into the obligatory intracellular amastigotes, and their subsequent survival in the macrophage phagolysosome, which is low in magnesium [35]. Mg2+ deficiency enhances NO production via iNos by alveolar macrophages isolated from rats [36]. Nitric oxide has been identified as a key molecule for leishmanicidal
function of macrophages [37]. It has been suggested that L. major uses a Mg2+ dependent pathway to proliferate within macrophage and evade their microbicidal activity [38]. Our study suggests that increased Mg levels may down-regulate NO production and thus, affecting parasite clearance in chronic VL cases. This may also contribute to the chronic state of the disease. In addition, it has been demonstrated that Mg2+ dependent ecto-ATPase activity in L. tropica may play a crucial role in enabling the parasite to avoid the microbicidal activity of macrophages [21]. Hence, the decreased level of Zn as well as increased level of Mg in only chronic VL patients may be predictive for clinical evolution or susceptibility to chronic VL. It has therefore been proposed that trace metal levels should be taken into account in vaccine strategies for leishmaniasis, because of the importance of Zn in a protective Th1 response and because of the possible deleterious effect of Cu [15]. Two recent large-scale vaccination trials for cutaneous and visceral leishmaniasis were carried out in regions where Zn deficiency is prevalent, namely Iran and Sudan, which might have contributed in part to low protection ratio observed in both trials [39,40]. The variation in concentrations of essential trace elements has been observed to be associated with susceptibility or resistance in L. major infected Balb/c and C57bl/6 mice [4]. Our study revealed the more decreased Zn levels and increased Mg levels only in CVL, which could have contributed to the chronic state of the disease. We detected comparatively decreased zinc concentration in serum of CVL cases and suggest that therapeutic administration of Zn would be useful for treating this form of leishmaniasis. Conclusions Despite the low number of patients included in the present study, lower levels of Zn and higher levels of Mg could be associated with chronicity in patients with visceral leishmaniasis. This finding may represent only more advanced grades of the disease or it can mean an independent prognostic factor. Conflict of interest None disclosed. Acknowledgements Authors thank Mrs. Manju Shree Roy, Technical assistant and Mr. Sudarshan Prasad, Technician, for their excellent help in the laboratory. All authors have made a significant intellectual contribution to the manuscript. References [1] Bora D. Epidemiology of visceral leishmaniasis in India. Natl Med J India 1999;12:62–8. [2] Desjeux P. Human leishmaniasis: epidemiology and public health aspects. World Health Stat Q 1992;45:267–75. [3] Bhattacharya SK, Sur D, Karbwang J. Childhood visceral leishmaniasis. Indian J Med Res 2006;123:353–6. [4] Amini M, Nahrevian H, Khatmi S, Farahmand M, Mirkhani F, Javadian S. Biochemical association between essential trace elements and susceptibility to Leishmania major in BALB/c and C57BL/6 mice. Braz Infect Dis 2009;13(April (2)):83–5. [5] El-Khoyl MS, Gas Allah MA, el-Shimi S, el-Baz F, el-Tayeb H, Abdel-Hamit MS. Zinc and copper status in children with bronchial asthma and atopic dermatitis. J Egypt Public Health Assoc 1990;65(5–6):657–68. [6] Kocyigit A, Erel O, Seyrek A, Gurel MS, Aktepe N, Avci S, et al. Effects of antimonial therapy on serum zinc, copper and iron concentrations in patients with cutaneous leishmaniasis. Eastern J Med 1998;3(2):58–61. [7] Mishra J, Carpenter S, Singh S. Low serum zinc levels in an endemic area of visceral leishmaniasis in Bihar, India. Indian J Med Res 2010;131:793–8.
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Biochemistry
Comparative analysis of serum zinc, copper, magnesium, calcium and iron level in acute and chronic patients of visceral leishmaniasis Chandra S. Lal a,∗ , Sanjay Kumar a , Alok Ranjan b , Vidya N. Rabidas c , Neena Verma d , Krishna Pandey c , Rakesh B. Verma b , Sushmita Das e , Dharmendra Singh e , Pradeep Das e a
Laboratory of Clinical Biochemistry, Division of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences (Indian Council of Medical Research), Patna, Bihar, India Division of Biostatistics, Rajendra Memorial Research Institute of Medical Sciences, Patna, Bihar, India c Division of Medicine, Rajendra Memorial Research Institute of Medical Sciences, Patna, Bihar, India d Division of Pathology, Rajendra Memorial Research Institute of Medical Sciences, Patna, Bihar, India e Division of Molecular Biology, Rajendra Memorial Research Institute of Medical Sciences, Patna, Bihar, India b
a r t i c l e
i n f o
Article history: Received 7 February 2012 Accepted 30 September 2012 Keywords: Trace elements Visceral leishmaniasis (VL) Acute VL Chronic VL
a b s t r a c t Project: Chronic visceral leishmaniasis (VL) is an increasingly common problem in disease endemic states of India. Identification of prognosis risk factor in patients with VL may lead to preventive actions, toward decreasing its mortality in chronic individuals. Though serum Zinc levels are decreased in patients of VL, limited information is available regarding trace elements status in acute and chronic VL patients. The present study was undertaken to compare serum trace elements concentrations in acute and chronic VL patients. Procedure: Acute (mean age = 28.64 years), chronic (mean age = 23.68 years) VL patients and healthy controls (mean age = 23.05 years) who agreed to provide blood specimens for laboratory investigations participated in this study. Serum zinc (Zn), copper (Cu), iron (Fe), magnesium (Mg) and calcium (Ca) were measured spectrophotometrically using chemistry analyzer. Results: Serum Zn concentration was comparatively much decreased in chronic VL than to acute ones (p = 0.007) while serum Mg was higher in chronic VL than acute (p = 0.002) ones. There was no statistically significant difference between acute and chronic VL in serum concentrations of Cu, Fe and Ca. Conclusions: Serum Zn levels were much decreased and serum Mg were increased in chronic VL as compared to acute cases. The serum concentrations of Fe and Ca did not show any difference between two groups. The serum Cu was increased in both groups but more in chronic ones. Serum Zn and Mg could be a potential prognosis factor for chronic VL patients. We hypothesize zinc supplementation as a chemo preventive agent for chronic VL cases, particularly in endemic areas. © 2012 Elsevier GmbH. All rights reserved.
Introduction Visceral leishmaniasis (VL), generally called kala-azar, occurs in several Mediterranean countries and Indian sub-continent, viz.: India, Nepal and Bangladesh. Protozoa of the species Leishmania donovani (L. donovani), which are responsible for the disease, are transmitted to humans by the sand fly, Phlebotomus argentipes. The incidence of kala-azar in India is among the highest in the world [1,2]. Bihar contributes as one of the major state in India for visceral leishmaniasis. At present, 28 of 37 districts of Bihar are endemic contributing about 90% of total Indian cases. Marked enlargement
∗ Corresponding author at: Laboratory of Clinical Biochemistry, Division of Biochemistry, RMRIMS (ICMR), Agamkuan, Patna 800 007, India. Tel.: +91 9835867492; fax: +91 6122634379. E-mail address: [email protected] (C.S. Lal).
of spleen and liver with moderate to severe anemia, pancytopenia are the presenting feature [3]. Leishmaniasis was selected by the World Health Organization for elimination by 2015, along with other neglected tropical diseases. Since there is no antileishmanial vaccine in clinical use, control of VL relies almost exclusively on chemotherapy. The use of conventional antileishmanials as well as new drugs is hampered mainly due to high costs and also due to concerns of toxicity and emergence of resistance. The chronic VL (CVL) patients are a serious health problem in disease endemic states of Bihar, India. The immune system of the body plays an important role during response to drug in any disease. Several enzymes that contribute to immune system responses require zinc and copper as trace elements for their activity which includes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) which contribute to immune system responses, require the mentioned trace elements for their activity [4]. The concentrations of serum iron (Fe), zinc (Zn), copper (Cu), and other
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Please cite this article in press as: Lal CS, et al. Comparative analysis of serum zinc, copper, magnesium, calcium and iron level in acute and chronic patients of visceral leishmaniasis. J Trace Elem Med Biol (2012), http://dx.doi.org/10.1016/j.jtemb.2012.09.007
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trace elements have been of interest to investigators for a number of years in many pathological conditions such as congestive heart failure, pneumonia, rheumatic heart diseases, bronchitis, and various infections, hemolytic anemia and psoriasis [5]. In cutaneous leishmaniasis Zn and Fe concentration are found lower while Cu has tendency to decrease [6]. Low serum zinc levels in an endemic area of visceral leishmaniasis in Bihar, India has also been reported [7]. Further, zinc deficiency has showed decreased Th1 but not Th2 immune response in experimental human leishmaniasis [8]. However, no information about assessment of serum concentrations of essential trace elements in acute and chronic VL patients is available from disease endemic areas. The indispensable feature of the most prominent of these trace elements rests on the functional role and structural components of crucial metallo-enzymes and metallo-proteins [9]. One of the mechanisms, the serum redistribution of essential trace elements Zn, Cu and Fe together with the increase in synthesis of acute-phase proteins (like ceruloplasmin), which takes part during the course of most infection, is well established [10]. The changes are part of defense strategies of organism and are induced by the hormone-like substances, such as interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-␣), and interleukin-6 (IL-6) [11,12]. These substances are immunocytokines liberated in a dose-dependent mode, mostly by activated macrophages, in response to several stimuli, including exercise, trauma, stress, or infection [13]. Zinc acts as an effective anti-inflammatory and antioxidant agent [14]. For example, Cu–Zn SOD is an important intracellular enzyme that requires both Cu and Zn for normal activity; Zn stabilizes the enzyme and Cu is necessary for catalysis. It acts as an antioxidant by oxidizing free-radical oxygen and it is the major Cu enzyme in erythrocyte [15]. Secondly, Zn deficiency has been shown to lead to a selective Th1 deficiency in human volunteers [16]. Besides this, Cu plays an important role in the biochemical pathway of mammals, and is a building block of many enzymes that have key functions in the metabolism [17,18]. Elevated copper is thought to induce pathological changes in tissues by stimulating the production of reactive oxygen species that damage multiple cells. Magnesium (Mg) is one of four bulk metals in the human and it is a co-factor for about 300 cellular enzymes and is required for the activity of many enzymes, particularly those utilizing an ATP [19]. Besides this, Mg2+ seemed to increase parasite proliferation in L. major and activate macrophage for establishment of infection [20,21]. Calcium (Ca) has several important functions in the formation of bone and dental tissues, in the releasing of hormones, contraction of muscles and glycogen metabolism [9]. Oxidative stress is known to be an important contributing factor in many chronic diseases. Hence, keeping in view the above observations, we considered the assessment of trace elements concentrations in serum of acute and chronic VL patients. The 3-fold purpose of this study includes: to investigate the serum levels of Zn, Cu, Fe, Ca and Mg; to determine any deficiency in these elements; to determine the differences in these two susceptible VL groups.
Materials and methods Subjects The clinical and observational study included adult VL patients admitted to the indoor ward of Rajendra Memorial Research Institute of Medical Sciences, Patna, India for the anti-leishmanial treatment. All together, 66 subjects were selected for this study: 22 acute VL, 22 chronic VL and 22 healthy not exposed to
leishmania infection. Our inclusion criteria for acute VL group were (a) any age or sex, any degree of parasitic load (b) had not taken any anti-leishmanial treatment before admission to the institute and (c) the duration of illness up to 4 weeks. For chronic VL group, the inclusion criteria were (a) any age or sex, any degree of parasitic load (b) prior to admission in the institute had taken anti-leishmanial treatment (i.e.: sodium antimony gluconate) and did not respond to that drug and disease recurred and was referred to this institute and (c) the duration of illness more than 3 months. The control group was selected among healthy people who were not exposed to visceral leishmaniasis or any other diseases. Ethics approval was obtained from ethics committee of the institute and was in accordance with the 1975 Helsinki Declaration on Human Rights, as revised in Edinburgh 2000. Patients were included after written informed consent was obtained from each patient’s next of kin. Clinical evaluation The medical staff of the Department of Clinical Medicine evaluated the patients for clinical diagnosis for VL infection. Laboratory Laboratory diagnosis was parsitologically confirmed by the microscopic demonstration of leishmania parasite in the splenic aspirate smears taken from the patients and the parasite load was graded as per the standard quantization grading [22]. Blood samples were collected into vacutainer tubes without any anticoagulant for biochemical investigations. The blood was centrifuged at 3000 rpm for 5 min and the serum was extracted in plastic tubes until assayed. Biochemical assay All biochemical assays were standardized as per the described protocol. The reagents used in the study were of the analytical grade (FAR srl, Italy). All estimations were repeated 3 times and put in triplicates every time. Average of all 9 readings was taken as final value. The entire assays were carried out on semi-automated chemistry analyzer Merck Microlab-300 LX (Vital Scientific N.V., The Netherlands). Zinc assay: Serum zinc level was quantified by colorimetry as previously described. This method was previously shown to produce similar results in zinc quantification as atomic absorption spectrophotometry (AAS) with a positive correlation (r = 0.982) and approximately 4% difference in readings [23]. Copper assay: The sensitive direct colorometric assay for copper in serum was followed as previously described [24]. The result of this method correlated well with those determined by atomic absorption spectrophotometric techniques (r = 0.977). Magnesium assay: Serum magnesium was assayed following Calmagite method [25]. Calcium assay: Serum calcium was quantified according to Arsenazo III (1,8-dihydroxy-3,6-disulpho-2,7-naphthalene-bis(azo)dibenzenearsonic acid) method following protocol of commercial kit (FAR srl, Italy). Iron assay: Serum iron was quantified according to Ferrozine method following protocol of commercial kit (LABKIT, Spain). Statistical analysis We used one-way analysis of variance (ANOVA) for comparing means of physical characteristics (age and body weight) and mean levels of trace elements (Cu, Zn, Fe, Ca and Mg) among three groups
Please cite this article in press as: Lal CS, et al. Comparative analysis of serum zinc, copper, magnesium, calcium and iron level in acute and chronic patients of visceral leishmaniasis. J Trace Elem Med Biol (2012), http://dx.doi.org/10.1016/j.jtemb.2012.09.007
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C.S. Lal et al. / Journal of Trace Elements in Medicine and Biology xxx (2012) xxx–xxx Table 1 Sex wise distribution of VL patients and healthy controls. Sex
Groups
Total
Chronic VL
Acute VL
Healthy controls
Female Male
11 11
6 16
11 11
28 38
Total
22
22
22
66
Chi-square = 3.102 at 2 d.f., p = 0.212.
– acute VL patients, chronic VL patients and healthy controls. We also used multiple comparisons using Tuckey’s test for inter-groups comparison. Chi-square statistic was used to compare sex distribution among three groups. All statistical analyses were performed using Statistical Package for Social Sciences (SPSS) program version 15 (SPSS, Inc., North Carolina, USA). Results Out of 66 subjects, 22 were in chronic VL group (Mean age 23.68 years), 22 in acute VL group (Mean age 28.64 years) and 22 were healthy controls (Mean age 23.05 years). There was no statistically significant difference of sex composition (p = 0.212) among these three groups (Table 1). We did not observe any statistically significant difference in mean values of age (p = 0.382) and body weight (p = 0.066) among the groups, and also in between the groups when compared with each other (Table 2) showing similar distribution in the groups without any confounding factor with respect to sex, age and body weight. Table 3 presents comparison of trace elements’ levels among three groups. The mean level of Cu was significantly different among three groups (p = 0.001) without significant difference between chronic and acute VL patients (p = 0.114). It was significantly higher in both chronic and acute VL patients when compared with healthy controls (p = 0.001) indicating increased Cu level in both chronic VL and acute VL patients. We observed a statistically
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significant difference of Zn level among three groups (p = 0.001) and also significantly different when compared with each other. The level of Zn was found decreased significantly in both chronic and acute VL patients as compared to healthy controls (p = 0.001). There was a significant indication of more decreasing trend of Zn levels as the disease becomes chronic. There was significantly lower mean level of Fe in both chronic (p = 0.001) and acute (p = 0.001) VL patients as compared to healthy controls. But we did not observe any statistically significant difference of mean Fe level between chronic and acute VL patients (p = 0.817), almost having the same level of Fe. The mean level of Ca was almost similar in three groups showing no statistically significant difference between chronic and acute VL patients (p = 0.238). There was statistically significant difference of Mg mean level among three groups (p = 0.001), and also between chronic and acute VL patients (p = 0.002). The level of Mg was significantly higher in chronic VL patients as compared to healthy individuals (p = 0.006), but the level was almost similar in acute VL patients as compared to healthy individuals (p = 0.928). No correlation was observed between parasitic load and serum concentrations of trace elements. Discussion Disorders of essential trace elements arise from inadequate intake, genetic defects, excessive exposure, or impaired elimination. Severe deficiency is rare, but specific symptoms and reduced protection against risk factors have been ascribed to suboptimal intake of trace elements. Besides this, secondary changes in the trace elements occur as a result of diseases and these changes are still unnoticed in visceral leishmaniasis cases in Bihar, India and which might be playing some significant role. Though, the antileishmanial treatment by sodium antimony gluconate (pentavalent antimonial compound) affects trace elements in cutaneous leishmaniasis during the treatment, these changes return to normal levels by unknown mechanisms [6].
Table 2 Physical characteristics of patients with chronic and acute visceral leishmaniasis and control group. Characteristics
Chronic VL (1) patients (n = 22)
Acute VL (2) patients (n = 22)
Healthy controls (3) (n = 22)
Age (year) Mean (95% CI)
23.68 (17.31–30.05)
28.64 (22.47–34.80)
23.05 (16.27–29.82)
0.382
0.499 (1 vs. 2) 0.988 (1 vs. 3) 0.414 (2 vs. 3)
Body weight (kg) Mean (95% CI)
33.05 (26.11–39.98)
33.41 (28.07–38.75)
43.68 (34.21–53.15)
0.066
0.998 (1 vs. 2) 0.098 (1 vs. 3) 0.114 (2 vs. 3)
p-Value for ANOVA
p-Value for Tuckey’s test
Table 3 Comparison of levels of trace elements of chronic and acute visceral leishmaniasis patients and control group. Variables
Chronic VL (1) patients (n = 22)
Acute VL (2) patients (n = 22)
Healthy controls (3) (n = 22)
p-Value for ANOVA
p-Value for Tuckey’s test
Cu (g/dL) Mean (95% CI)
384.94 (348.39–421.49)
332.80 (279.21–386.40)
111.41 (101.96–120.86)
0.001
0.114 (1 vs. 2) 0.001 (1 vs. 3) 0.001 (2 vs. 3)
Zn (g/dL) Mean (95% CI)
44.51 (42–47)
56.27 (48.83–63.71)
78.04 (72.63–83.45)
0.001
0.007 (1 vs. 2) 0.001 (1 vs. 3) 0.001 (2 vs. 3)
Fe (g/dL) Mean (95% CI)
42.86 (39.20–46.52)
40.61 (37.33–43.88)
93.62 (85.53–101.72)
0.001
0.817 (1 vs. 2) 0.001 (1 vs. 3) 0.001 (2 vs. 3)
Ca (mg/dL) Mean (95% CI)
9.64 (9.14–10.15)
9.11 (8.62–9.58)
9.79 (9.33–10.25)
0.096
0.238 (1 vs. 2) 0.889 (1 vs. 3) 0.098 (2 vs. 3)
Mg (mg/dL) Mean (95% CI)
2.30 (2.14–2.47)
1.96 (1.82–2.10)
1.99 (1.87–2.12)
0.001
0.002 (1 vs. 2) 0.006 (1 vs. 3) 0.928 (2 vs. 3)
Please cite this article in press as: Lal CS, et al. Comparative analysis of serum zinc, copper, magnesium, calcium and iron level in acute and chronic patients of visceral leishmaniasis. J Trace Elem Med Biol (2012), http://dx.doi.org/10.1016/j.jtemb.2012.09.007
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In the present study, we investigated trace elements such as Cu, Zn, Fe, Ca and Mg in acute and chronic VL cases and demonstrated that serum Zn and Fe concentrations were lower in both acute and chronic VL patients (p = 0.001 and p = 0.001), respectively, than healthy control group but there was a significant indication of more decreased Zn level in chronic VL (p = 0.007) in comparison to acute VL. Zn is crucial for immune functioning of the body. The role of certain inflammatory products in the regulation of the Zn balance has been well documented [10]. Thus, leukocyte-endogenous mediators (interleukins), released from activated phagocytic cells, induce hypozincemia in experimental animals by the redistribution of Zn from plasma to the liver [26]. Decreasing serum Zn concentrations apparently results from the synthesis of methallothionein in liver and other tissues. Methallothionein binds 7 g atoms of Zn/mol and serves to draw Zn away from free-circulating pools; it was induced by IL-1 in vivo [27]. Decreased Zn enhances humoral response (Th2) which results in decrease of IFN-␥ production. This could contribute to the chronic state of the disease. However, serum Cu concentrations were significantly raised in both acute and chronic VL cases (p = 0.001 and p = 0.001) than healthy control group but increased level had been observed in chronic VL category. Increased serum Cu is associated with ceruloplasmin and induced by IL-1 [28]. It was reported in one of the study [29] that IL-1, not TNF-␣, induces hypercupremia when injected into the preoptic anterior hypothalamus. However, the high production of IL-1 in cutaneous leishmaniasis is in contrast to visceral leishmaniasis, where infection of mice with L. donovani resulted in the suppression of the IL-1 response [30]. These findings may reflect the distinct pathology and immune responses caused by the two species of Leishmania. In our study, we observed that Cu levels were significantly higher in patient’s sera than that of healthy subjects. Increased Cu may be attributable to inflammation associated with the disease and increased Cu level might increase resistance to leishmaniasis [4]. Under stress or attack (i.e., infection) serum Cu increases more than normal to facilitate vital processes such as Cu-ATPases, which is crucial for: (i) central nervous system development, (ii) liver function (VL has to do with liver), (iii) connective tissues and (iv) many other physiological processes. Plasma Cu in VL patients increased to levels which have been shown to be toxic in vitro [31] and our result trend in Cu is also suggestive for the chronicity of the disease. These characteristic changes in the trace element metabolism are an integral part of the acute-phase response. These changes are usually reflected in decreased serum Zn and Fe and increased serum Cu concentrations [32]. Further, it had been reported that Cu/Zn imbalance might serve as a marker for decreased Th1 response and immunodeficiency in leishmaniasis, being more pronounced in its most severe and possibly fatal visceral form [33,34]. With Cu raised and Zn decreased, humoral response (Th2) is enhanced, thus a decrease of IFN-␥ production. This could contribute to the chronic state of the disease. We observed significantly raised Mg value (p = 0.006) in only chronic VL cases than healthy controls, while it was not raised in acute cases (p = 0.928). In case of Zn deficiency in chronic VL patients, the defensive mechanism of the body is to raise Mg levels. Mg is necessary for the synthesis of various compounds that have energy-rich bonds of any type (e.g., phosphoric anhydride bond found mainly in ATP and others). Thus allows cellular respiration to be used to store energy in the form of high energy phosphate. This indicates that elevated Mg could be contributory to the chronic state of the disease. It is reported that establishment of infection by Leishmania depends on the transformation of the invading metacyclic promastigotes into the obligatory intracellular amastigotes, and their subsequent survival in the macrophage phagolysosome, which is low in magnesium [35]. Mg2+ deficiency enhances NO production via iNos by alveolar macrophages isolated from rats [36]. Nitric oxide has been identified as a key molecule for leishmanicidal
function of macrophages [37]. It has been suggested that L. major uses a Mg2+ dependent pathway to proliferate within macrophage and evade their microbicidal activity [38]. Our study suggests that increased Mg levels may down-regulate NO production and thus, affecting parasite clearance in chronic VL cases. This may also contribute to the chronic state of the disease. In addition, it has been demonstrated that Mg2+ dependent ecto-ATPase activity in L. tropica may play a crucial role in enabling the parasite to avoid the microbicidal activity of macrophages [21]. Hence, the decreased level of Zn as well as increased level of Mg in only chronic VL patients may be predictive for clinical evolution or susceptibility to chronic VL. It has therefore been proposed that trace metal levels should be taken into account in vaccine strategies for leishmaniasis, because of the importance of Zn in a protective Th1 response and because of the possible deleterious effect of Cu [15]. Two recent large-scale vaccination trials for cutaneous and visceral leishmaniasis were carried out in regions where Zn deficiency is prevalent, namely Iran and Sudan, which might have contributed in part to low protection ratio observed in both trials [39,40]. The variation in concentrations of essential trace elements has been observed to be associated with susceptibility or resistance in L. major infected Balb/c and C57bl/6 mice [4]. Our study revealed the more decreased Zn levels and increased Mg levels only in CVL, which could have contributed to the chronic state of the disease. We detected comparatively decreased zinc concentration in serum of CVL cases and suggest that therapeutic administration of Zn would be useful for treating this form of leishmaniasis. Conclusions Despite the low number of patients included in the present study, lower levels of Zn and higher levels of Mg could be associated with chronicity in patients with visceral leishmaniasis. This finding may represent only more advanced grades of the disease or it can mean an independent prognostic factor. Conflict of interest None disclosed. Acknowledgements Authors thank Mrs. Manju Shree Roy, Technical assistant and Mr. Sudarshan Prasad, Technician, for their excellent help in the laboratory. All authors have made a significant intellectual contribution to the manuscript. References [1] Bora D. Epidemiology of visceral leishmaniasis in India. Natl Med J India 1999;12:62–8. [2] Desjeux P. Human leishmaniasis: epidemiology and public health aspects. World Health Stat Q 1992;45:267–75. [3] Bhattacharya SK, Sur D, Karbwang J. Childhood visceral leishmaniasis. Indian J Med Res 2006;123:353–6. [4] Amini M, Nahrevian H, Khatmi S, Farahmand M, Mirkhani F, Javadian S. Biochemical association between essential trace elements and susceptibility to Leishmania major in BALB/c and C57BL/6 mice. Braz Infect Dis 2009;13(April (2)):83–5. [5] El-Khoyl MS, Gas Allah MA, el-Shimi S, el-Baz F, el-Tayeb H, Abdel-Hamit MS. Zinc and copper status in children with bronchial asthma and atopic dermatitis. J Egypt Public Health Assoc 1990;65(5–6):657–68. [6] Kocyigit A, Erel O, Seyrek A, Gurel MS, Aktepe N, Avci S, et al. Effects of antimonial therapy on serum zinc, copper and iron concentrations in patients with cutaneous leishmaniasis. Eastern J Med 1998;3(2):58–61. [7] Mishra J, Carpenter S, Singh S. Low serum zinc levels in an endemic area of visceral leishmaniasis in Bihar, India. Indian J Med Res 2010;131:793–8.
Please cite this article in press as: Lal CS, et al. Comparative analysis of serum zinc, copper, magnesium, calcium and iron level in acute and chronic patients of visceral leishmaniasis. J Trace Elem Med Biol (2012), http://dx.doi.org/10.1016/j.jtemb.2012.09.007
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Please cite this article in press as: Lal CS, et al. Comparative analysis of serum zinc, copper, magnesium, calcium and iron level in acute and chronic patients of visceral leishmaniasis. J Trace Elem Med Biol (2012), http://dx.doi.org/10.1016/j.jtemb.2012.09.007