EFFECT OF SHORT TERM DIETARY ZINC DEFICIENCY AND ZINC SUPPLEMENTATION ON WISTAR RAT ERYTHROCYTE: BIOCHEMICAL STUDIESHTML Full Text
EFFECT OF SHORT TERM DIETARY ZINC DEFICIENCY AND ZINC SUPPLEMENTATION ON WISTAR RAT ERYTHROCYTE: BIOCHEMICAL STUDIES
Neena Nair *, Pooja Sharma, Sunita Bedwal, Swati Singh, Deepa Kumari, Astha Saini and R. S. Bedwal
Department of Zoology, Centre for Advanced Studies, University of Rajasthan, Jaipur - 302004, Rajasthan, India.
ABSTRACT: Deficiency of zinc has been reported in developed and developing countries due to low zinc intake, which also accounts for crops grown in low zinc areas. 2 billion people still remain at risk of zinc deficiency affecting all age groups in spite of proven benefits of adequate zinc nutrition. The study evaluates the effect of dietary zinc deficiency on male Wistar rat erythrocytes for 2-, 4- and 6 weeks and 4 weeks zinc supplementation to the deficient groups. A significant decrease in the total protein of erythrocyte ghost membrane and erythrocyte cell suspension was recorded in zinc deficient groups. Increase (P< 0.05) in erythrocyte ghost membrane protein carbonyl, protein thiol, and lipid peroxidation (TBARS / induced) was observed. Erythrocyte cell suspension OH radical and hydroperoxide levels increased while α-tocopherol and ascorbic acid decreased in ZD groups. A decrease was observed in ghost membrane zinc concentration in zinc-deficient groups, while there was a gradual increase in copper level of ZD groups. Zinc supplementation studies revealed the same pattern of changes within its group /subgroups. There was an evident alteration in severity pattern when ZD group experiments were compared with dietary zinc supplementation experiments. Zinc deficiency from pre-pubertal age onwards revealed perturbations in the parameters indicative of radical generation and could be one factor contributing to oxidative stress which could lead to erythrocyte dysfunction. Zinc supplementation to zinc deficient group altered the severity to some extent (dose and duration dependent) indicating the essential role of zinc in maintaining the structural and functional integrity of erythrocytes.
Zinc deficiency, Zinc supplementation, Erythrocyte, Lipid peroxidation, Protein carbonyl & thiol, Hydroperoxides, OH•, α-tocopherol, Ascorbic acid, zinc & copper
INTRODUCTION: Trace element zinc is invol-ved in growth, differentiation, genomic stability, cell division, cell signaling, reproduction, apop-tosis, synthesis and secretion of hormones, antioxidant etc. 1- 10.
17.3% of the global population is at risk of developing zinc deficiency, with children <5 yrs contributing 1.7% of total deaths 11. Erythrocytes are susceptible to oxidative stress being rich in polyunsaturated membrane lipids and auto-oxidation of hemoglobin due to endogenous production of reactive oxygen species, which would affect the physicochemical properties 12.
Changes in the primary structure of protein due to lipid oxidation products would lead to changes in the secondary and tertiary structure of proteins. Protein carbonylation is involved in functional changes of various structural and enzymatic proteins 13 can be due to oxidative attack on cysteine/methionine/lysine/proline/threonine/histidine/ tryptophan /tyrosine etc. and by the secondary reaction of these residues with reactive carbonyl compounds 14, 15. Oxidation of protein sulfhydryls to mixed disulfides and their reduction back to sulfhydryls is an early cellular response to oxidative stress 16. Polyunsaturated fatty acid lipid peroxidation may be: (a) enzymatic generating peroxy radicals or (b) may be non-enzymatic requiring molecular oxygen and Fe2+ ions 17, 18. Hydroxyl radical with in-vivo half-life of approximately 10-9 s 19 usually reacts in its immediate surroundings and with DNA molecule 20. Under oxidative stress, the consumption of ascorbate can be high, and in order to restore ascorbate levels, systems exist that reduce AFR and/or DHA 21. Ascorbic acid, a water-soluble chain-breaking antioxidant, acts synergistically with vitamin E protecting plasma membranes from oxidative damage 22. α-tocopherol may probably protect the red cell from oxidative damage via a free radical scavenging mechanism and as a structural component of the cell membrane 23. The present study evaluates the effect of dietary zinc deficiency and zinc supplementation on erythrocytes of male Wistar rats.
MATERIALS AND METHOD: The synthetic experimental diet was prepared based on ICN Research Diet protocol (1999). Pre-pubertal (30-40 days of age) male Wistar rats (40-50 gm wt.) were divided into three groups (thirty animals each for 2 -, 4 - and 6 week) (i) Zinc control (ZC) group fed with diet containing 100 µg/gm zinc (ii) Pair fed (PF) group fed with 100 µg/gm zinc diet but the amount of feed given was equal to the feed consumed by zinc-deficient group during the previous day. This group was taken into account so as to study the starvation effects due to reduced intake of diet as well as stress effects. Demineralized water was provided ad libitum (iii) Zinc deficient (ZD) group were fed zinc-deficient (1.00 µg/gm) diet and provided demineralized water ad libitum. The experiment was carried out for 2-, 4- and 6 weeks. The animals were housed individually in polypropylene cages with stainless steel grills. The polypropylene cages, grills, and water bottles were washed with detergent solution, de-mineralized water, and finally rinsed in 1% EDTA solution prepared in de-mineralized water so as to avoid contamination and subsequent removal of zinc from cages, grills, and bottles. Supple-mentation of zinc was carried out for a period of 4 weeks after deficiency. Fifteen animals each from 2-, 4- and 6- weeks zinc-deficient experiments were supplemented with 100 µg /gm zinc diet (Control: ZC-S; Pair fed: PF-S; Zinc supplemented: ZD-S). The experiment design was approved by Institutional Animal Ethics Committee (IAEC), Department of Zoology, University of Rajasthan, Jaipur, India, and Committee for Control and Supervision of Experiments on Animals (CPCSEA) (No.1678/GO/Re/s/12/CPCSEA dated 16.06.17).
Animals from the deficient group were autopsied after 2-, 4- and 6- weeks under anesthesia, while supplementation groups were autopsied after 4 weeks. Blood samples from the animals of all the groups were collected by cardiac puncture using a heparinized syringe and processed.
Preparation of Erythrocyte Ghost Membrane and Erythrocyte Cell Suspension: Red blood cells were washed thrice with PBS (pH 7.4), and erythrocytes were separated from the blood, haemolysed in a hypotonic lysing buffer, and centrifuged at 27,000 g for 30 min at 4 ºC in Sigma high-speed centrifuge using 18015 rotors and membrane was processed by conventional method till free of hemoglobin 24, 25.
Biochemical Estimation: Total Protein 26, Protein carbonyl 27, Protein sulphydryl 28, Lipid Peroxidation 29, Hydroperoxides 30, Hydroxyl radical 31, α-Tocopherol 32, Ascorbic acid 33, 34 were estimated on Systronics spectrophotometer 169. Total zinc and copper were estimated at 213.9 and 324.8 nm, respectively, with 0.5 nm slit width with background correction and an integration time of 3 sec on GBC-902 double beam atomic absorption spectrophotometer (Australia).
Statistical Analysis: Data are expressed as mean ± SEM. Further, the analysis was carried using One Way Analysis of Variance (ANOVA), and if the difference was found significant posthoc test (Duncan’s Multiple Comparison Test) was carried out, and P<0.05 was considered significant. Sigma stat 3.5 software was utilized for analysis (Systat Software Asia Pacific Ltd., Bangalore, India).
RESULTS: After 2-, 4- and 6- weeks of dietary zinc deficiency significant (P< 0.05) decrease was observed in erythrocyte ghost membrane total protein in zinc-deficient group when compared with their respective pair-fed and control groups. Significant (P<0.05) decrease was evident when 4-and 6-week pair-fed groups were compared with their respective control groups Table 1.
Dietary zinc supplementation for a period of 4 weeks revealed an increase in comparison with their respective deficient group's Table 1 & Table 2. However, comparing supplementation groups (ZD-S) (2-4-and 6-weeks) with their respective control and pair-fed group decrease was still evident in Table 2.
TABLE 1: WISTAR RATS ERYTHROCYTE GHOST MEMBRANE TOTAL PROTEIN, PROTEIN CARBONYL, THIOL AND LIPID EROXIDATION AFTER 2, 4 AND 6 WEEKS OF ZINC DEFICIENCY (MEAN ± SEM)
|Groups||Total Protein (mg / gm )||Protein Carbonyl
(n Moles / mg Protein)
|Protein Thiol (-SH) Group (n Moles / mg Protein)||Lipid Peroxidation
(n Moles / mg Protein)
|2ZC||2.59 ± 0.02||0.071±0.002||0.413±0.007||0.0933±0.003||0.0615±0.0012|
|2PF||2.428±0.03||0.079±0.002 a*||0.269±0.005 a*||0.145±0.023 a*||0.0675±0.0041a*|
|2ZD||1.05±0.03 b*c*||0.126±0.002 b*c*||0.254±0.007 b*c*||0.266±0.0089 b*c*||0.0979±0.0046 b*c*|
|4ZC||3.041 ± 0.013||0.169 ± 0.002||0.438 ± 0.174||0.116 ± 0.0008||0.114 ± 0.0005|
|4PF||2.023 ± 0.048 a*||0.274 ± 0.004 a*||0.405 ± 0.002a*||0.133 ± 0.0003 a*||0.232 ± 0.002 a*|
|4ZD||0.349 ± 0.007 b*c*||0.476 ± 0.005b*c*||0.32 ± 0.002 b* c*||0.250 ± 0.0009 b*c*||0.348 ± 0.0005 b*c*|
|6ZC||6.018 ± 0.029||0.187 ± 0.004||0.612 ± 0.007||0.158 ± 0.0013||0.218 ± 0.0029|
|6PF||2.136 ± 0.169 a*||0.339 ± 0.005 a*||0.3392 ± 0.005 a*||0.180 ± 0.0013a*||0.421 ± 0.0045a*|
|6ZD||0.216 ± 0.023 b*c*||0.612 ± 0.007b*c*||0.1968 ± 0.005 b*c*||0.605 ± 0.0111 b*c*||0.669 ± 0.0107 b*c*|
Zc vs. pf = a significant (p<0.05) = * pf vs zd = b zc vs zd =c note: multiple comparison of means were performed separately for2-, 4- and 6 weeks sub groups
Significant (P<0.05) increase in erythrocyte ghost membrane protein carbonyl concentration was evident when ZD groups were compared with their respective pair-fed and control groups. Further, a comparison of pair-fed groups with their respective control groups revealed an increase in Table 1. Supplementation of dietary zinc to zinc-deficient experiments revealed a significant (P<0.05) increase on a comparison between the group's Table 2. Comparison of zinc supplementation experiment groups Table 2 with zinc deficient experiment groups Table 1 revealed decrease.
TABLE 2: ERYTHROCYTE GHOST MEMBRANE TOTAL PROTEIN, PROTEIN CARBONYL, THIOL AND LIPID PEROXIDATION AFTER 4 WEEKS OF ZINC SUPPLEMENTATION TO ZINC DEFICIENT WISTAR RATS (2-, 4- & 6 WEEKS) (MEAN ± SEM)
(mg / gm )
(n Moles / mg Protein)
|Protein Thiol (SH) Group (n Moles / mg Protein)||Lipid Peroxidation
(n Moles / mg Protein)
|2ZC-S||3.91± 0.06||0.073 ± 0.002||0. 554 ± 0.003||0.119 ± 0.006||0.029 ± 0.007|
|2PF-S||2.96 ± 0.03 a*||0.058 ± 0.004 a*||0.330±0.007 a*||0.135 ± 0.01 a*||0.054 ± 0.003 a*|
|2ZD-S||2.09 ± 0.05 b*c*||0.094 ±0.006 b*c*||0.276 ± 0.008 b*c*||0.196 ± 0.008 b*c*||0.076 ± 0.004 b*c*|
|4ZC -S||5.36 ±0.024||0.167±0.002||0.638 ± 0.005||0.131 ± 0.001||0.322 ± 0.134|
|4PF-S||3.18 ± 0.012 a*||0.233± 0.015 a*||0.596 ± 0.006||0.146 ± 0.003 a*||0.154 ± 0.005 a*|
|4ZD -S||1.96 ± 0.014 b*c*||0.238 ±0.008 c*||0.412 ± 0.005 b*c*||0.187 ± 0.002 b*c*||0.224 ± 0.004 b*c*|
|6ZC -S||7.18± 0.02||0.183± 0.0002||0.805 ± 0.003||0.159 ± 0.001||0.196 ± 0.013|
|6PF- S||3.98± 0.09 a*||0.284± 0.002 a*||0.483 ± 0.003||0.224 ± 0.007 a*||0.310 ± 0.002 a*|
|6ZD-S||1.76 ± 0.016 b*c*||0.407 ± 0.003 b * c*||0.331 ± 0.003 b*c*||0.402 ± 0.0005 b*c*||0.396 ± 0.016 b*c*|
Zc-s vs pf-s = a significant (p<0.05) = *pf-s vs zd –s = b zc –s vs zd-s = c note: multiple comparison of means were performed separately for 4 and 6 weeks sub groups.
Protein thiol (-SH) concentration of erythrocyte ghost membrane decreased significantly (P<0.05) when ZD groups of 2-, 4-and 6-weeks were compared with their respective pair-fed and control groups. Further, a comparison of pair-fed groups with the control group’s revealed a significant decrease Table 1. Dietary supplementation of zinc for 4 weeks although revealed an increase when supple-mentation experiment groups (ZD-S) were compared with zinc deficient experiment groups Tables 1 and 2 but comparison of ZD-S groups with ZC-S and PF-S till 6 weeks there was still a significant (P<0.05) decrease Table 2. Lipid per oxidation (TBARS and Induced) of erythrocyte ghost membrane increased significantly (P<0.05) after 2-, 4- and 6-weeks of zinc deficiency when ZD groups were compared with their respective control and pair-fed groups as well as when pair-fed groups were compared with respective control groups Table 1. Supplementation of dietary zinc still revealed a significant increase in all groups Table 2, but in comparison with ZD experiment groups Table 1 there was a decline. After 2-, 4- and 6-weeks of dietary zinc deficiency erythrocyte cell suspension, total protein concentration decreased significantly (P<0.05) when ZD groups were compared with their respective pair-fed and control groups. Furthermore, a significant decrease was observed when pair-fed groups were compared with respective controls, except the decrease was non-significant after 2 weeks Table 3.
TABLE 3: BIOCHEMISTRY OF WISTAR ERYTHROCYTE CELL SUSPENSION AFTER 2, 4 AND 6 WEEKS OF ZINC DEFICIENCY (MEAN ± SEM)
|Groups||Total Protein(mg/gm)||OH radical
(n Moles / mg Protein)
|Hydroperoxides (FOX) (µM Hydroperoxides /gm)||α – Tocopherol
(µg /mg Protein)
(µg /mg Protein)
|2ZC||4.476 ±0.04||0.245±0.014||2.357±0.048||4.512 ± 0.072||2.318 ± 0.037|
|2PF||4.44± 0.04||0.384±0.002 a *||2.863±0.053 a *||3.09 ± 0.09 a *||2.307 ± 0.051|
|2ZD||3.44±0.162 b*c*||0.444±0.007 b*c*||4.331±0.144 b*c*||2.109 ± 0.062 b*c*||2.156 ± 0.034 b*c*|
|4ZC||4.208 ± 0.023||0.268 ± 0.003||2.061 ± 0.037||3.251 ± 0.046||2.522 ± 0.074|
|4PF||3.160 ± 0.063 a*||0.426 ± 0.003 a*||4.188 ± 0.056 a*||2.708 ± 0.045 a*||1.506 ± 0.014 a*|
|4ZD||1.895 ± 0.042 b*c*||0.615 ± 0.0112
|8.233 ± 0.071b*c*||2.062 ± 0.025 b* c*||0.946 ± 0.016 b* c*|
|6ZC||5.594 ± 0.085||0.362 ± 0.142||3.086 ± 0.059||4.020 ± 0.034||3.799 ± 0.098|
|6PF||2.644 ± 0.034 a*||0.584 ± 0.007 a*||6.111 ± 0.055 a*||1.598 ± 0.023 a*||1.206 ± 0.018 a*|
|12.156 ± 0.043
|0.848 ± 0.019
|0.706 ± 0.015
Zc vs pf = a significant (p<0.05) = * pf vs zd = b zc vs zd = c note: multiple comparison of means were performed separately for 2-,4 -and 6- weeks sub groups.
Supplementation of dietary zinc for a period of 4 weeks still revealed a significant decrease when ZD-S groups were compared with their respective pair-fed (PF-S) and control groups (ZC-S) Table 4, but on comparison with zinc-deficient experiment groups Table 3 an increase was evident.
TABLE 4: BIOCHEMISTRY OF ERYTHROCYTE CELL SUSPENSION AFTER 4 WEEKS OF ZINC SUPPLE-MENTATION TO ZINC DEFICIENT (2-, 4- & 6 WEEKS) WISTAR RATS (MEAN ± SEM)
|Groups||Total Protein (mg/gm)||OH Radical
(n Moles / mg Protein)
|Hyderoperoxides (FOX) (µM Hydroperoxides /gm)||α – Tocopherol
(µg /mg Protein)
(µg /mg Protein)
|2ZPF-S||5.09±0.16||0.310±0.001a*||2.573±0.026 a*||3.73±0.028 a*||2.832±0.036 a*|
|2ZD-S||4.32± 0.11b* c*||0.350±0.009b*c*||3.187±0.132b*c*||2.98±0.04 b*c*||2.481±0.011b*c*|
|4ZC -S||6.015 ± 0.025||0.221 ± 0.004||2.013 ± 0.041||4.087 ± 0.047||3.695 ± 0.023|
|4PF-S||3.986 ± 0.045 a*||0.345 ± 0.003 a*||2.621 ± 0.031 a*||2.879 ± 0.019 a*||1.921 ± 0.017 a*|
|4ZD -S||2.524 ± 0.111 b*c*||0.565 ± 0.006 b*c*||5.608 ± 0.168 b*c*||2.294 ± 0.012 b*c*||1.262 ± 0.007 b*c*|
|6ZC -S||6.808 ± 0.026||0.237 ± 0.004||2.844 ± 0.059||6.564 ± 0.062||4.194 ± 0.053|
|6PF- S||4.153 ± 0.055 a*||0.468 ± 0.004 a*||4.093 ± 0.044 a*||5.634 ± 0.040 a*||3.088 ± 0.036 a*|
|6ZD-S||2.765 ± 0.121 b*c*||0.742 ± 0.005 b*c*||8.532 ± 0.154 b*c*||1.667 ± 0.027 b*c*||2.643 ± 0.147 b*c*|
ZC-S Vs PF-S = a Significant (P<0.05) = *PF-S Vs ZD –S = b ZC –S Vs ZD-S = c Note: Multiple comparisons of means were performed separately for 4 and 6 weeks subgroups.
Generation of OH radical and hydroperoxides after 2-, 4- and 6-weeks of dietary zinc deficiency in erythrocyte cell suspension was significant (P<0.05). Significance was evident when ZD groups were compared with their respective control and pair-fed groups Table 3. Increase (P<0.05) was still evident after supplementation of dietary zinc for 4 weeks Table 4, but comparison between supple-mentation groups with zinc-deficient experiment groups decrease in OH radical concentration was evident. α - Tocopherol and ascorbic acid of Wistar rat erythrocyte cell suspension decreased significantly (P<0.05) when ZD groups (2-,4- and 6-weeks ) were compared with their respective pair-fed and control groups as well as when pair-fed groups was compared with their control groups Table 3. The decrease was still evident in - groups when ZD-S was compared with PF-S and ZC-S groups Table 4, but in comparison with ZD group Table 3 an increase was observed.
TABLE 5: WISTAR RATS ERYTHROCYTE GHOST MEMBRANE ZINC AND COPPER (µG / MG PROTEIN) AFTER 2, 4 AND 6 WEEKS OF ZINC DEFICIENCY (MEAN ± SEM)
|Groups||Zinc (µg / gm protein)||Copper (µg / gm protein)|
|2ZC||58.67 ± 0.66||22.19±0.09|
|2PF||50.78 ± 0.34||29.67±0.45|
|2ZD||48.56±0.22 b*c*||38.52±0.28 b*c*|
|4ZC||61.66 ± 0.66||34.166 ± 0.31|
|4PF||54.50 ± 0.76 a*||42.50 ± 0.43 a*|
|4ZD||41.33 ± 0.55 b*c*||58.50 ± 0.22 b*c*|
|6ZC||73.50 ± 0.67||45.16 ± 0.31|
|6PF||46.00 ± 0.52 a*||64.66 ± 0.71 a*|
|6ZD||33.83 ± 0.60 b*c*||68.83 ± 0.31 b*c*|
Zc vs pf = a * p < 0.05 significance level pf vs zd =b; zc vs zd = c note: multiple comparison of means were performed separately for 2-, 4- and 6 weeks sub groups.
Erythrocyte ghost membrane trace element zinc decreased significantly (P<0.05) after 2-, 4- and 6-weeks of dietary zinc deficiency when ZD groups were compared with respective control and pair-fed groups. Copper, another trace element, increased significantly (P<0.05) Table 5. Supplementation of dietary zinc to zinc-deficient experiment groups still revealed a decline in zinc concentration when ZD experiment groups were compared with ZC-S groups Table 6, but comparison with zinc-deficient experiment groups Table 5 an increase was evident. Copper concentration remained enhanced even after dietary zinc supplementation for 4 weeks Table 6, but a comparison of supplementation experiment groups with zinc-deficient experiment groups revealed a decline in Tables 5 & 6.
TABLE 6: WISTAR RATS ERYTHROCYTE GHOST MEMBRANE ZINC AND COPPER (µG / MG PROTEIN) AFTER 4 WEEKS OF ZINC SUPPLEMENTATION TO ZINC DEFICIENT (2-, 4 - & 6 WEEKS) WISTAR RATS (MEAN ± SEM)
|Groups||Zinc (µg / gm Protein)||Copper (µg / gm Protein)|
|4ZC -S||70.015± 0.306||38.257 ± 0.394|
|4PF-S||58.247 ± 0.359 a*||43.540 ±0.447 a*|
|4ZD -S||52.042 ± 0.354 c*||46.387 ± 0.388 b* c*|
|6ZC -S||82.424±0.420||47.832 ± 0.285|
|6PF- S||50.445 ± 0.187 a*||50.505 ± 1.206|
|6ZD-S||41.422 ± 0.254 b* c *||53.358 ± 0.178 b* c *|
Zc-s vs pf-s = a significant (p<0.05) = * pf-s vs zd –s = b; zc –s vs zd-s = c note: multiple comparison of means were performed separately for 4 and 6 weeks sub groups.
DISCUSSION: Erythrocyte's structural integrity and its other physiological properties are dependent on its membrane. Erythrocyte ghost membrane total protein, cell suspension total protein showed a significant reduction in their values in zinc-deficient animals when compared to their respective pair-fed and control groups. Zinc deficiency markedly affects protein to varying degrees Marreiro et al., 35 in their review reported altered expression of zinc transporters Zip-7 and ZnT-7 found in several organelles - endoplasmic reticulum, Golgi apparatus etc. rendering them non-functional during zinc deficiency. Erythrocyte membrane proteins due to oxy radicals are covalently modified by lipid peroxidation products which enhance its hemolysis – a phenomenon observed during zinc deficiency 8. RBC structural shape is affected by alteration in the structure of lipids, band 3 as well as spectrin 36, which is associated with erythrocyte properties. Studies on haemolysis in hemoglobinopathy patients reported membrane damage due to combined spectrin, band 3 with denatured hemoglobin 37. The reduction in total protein content appears as zinc has a prominent role to play in protein synthesis and its deficiency lowers the transcriptional and translational capacity leading ultimately to low levels of protein synthesis 38. Increased protein levels in PF groups compared to ZD groups probably accounts for upregulation of stress genes. Similar increase has been reported in organs 5, 7.
Supplementation studies for 4 weeks exhibited an increase in the total protein content when compared with 2-, 4- and 6- weeks zinc-deficient groups suggesting that deficiency-induced changes for the protein machinery were not severe and irreversible and the protein status could be brought back to the normal physiological range indicating: (a) homeostatic balance was attempted to obviate the injury caused by zinc deficiency and (b) duration of zinc deficiency has a significant impact as it may be a critical point for the onset of events that encompasses zinc deficiency. Proteins are sensitive to reactive oxygen species and rapidly undergo oxidation. The accumulation of oxidized protein and protein carbonyl is complex involving ROS and /or secondary oxidation products of the macromolecules that would impair the cells and affects the balance between pro-oxidant and antioxidant activities 39.
Increase in reactive carbonyl compounds as a result of oxidative and non-oxidative reactions lead to increased chemical modification of proteins, defect in detoxification of carbonyl compounds, and consequently oxidative stress generation 40, 41. Decreased protein concentration, i.e., inhibition of protein synthesis, would enhance the possibility of oxidized protein to induce oxidative stress. The enhanced level of protein carbonyl in the present study contributes to oxidative stress leading to cellular dysfunction via impacts on cellular metabolism and may even lead to toxicity. This finding supports the change in morphology as well as enhanced osmotic fragility in erythrocytes reported from our laboratory 8. Supplementation studies revealed a decrease in protein carbonyl content on comparison with zinc-deficient groups (2-, 4- & 6- weeks). Maintaining low carbonyl content, the erythrocyte would be able to maintain sufficient protein synthesis essential for regulating cell structure as well as enzymatic processes. Similar decline in protein oxidation after zinc supplementation has been reported 20.
Under physiological and pathophysiological conditions, cysteines (-SH group) have an essential role in protein function and are susceptible to irreversible damage by oxidative conditions. Oxidation of cysteine would lead to misfolding of protein, affecting protein function 42. In the present study, the decline in –SH concentration after zinc deficiency (2-, 4- and 6-weeks) was observed which reflects a decrease in binding affinity of zinc to membrane –SH group as against enhanced affinity towards cysteine. Decline in erythrocyte SH concentration (band 3 protein) from 75 nmol/mg protein to 68 nmol/mg protein was observed after 21 days of zinc depletion 43 . Even pair-fed groups showed a decrease in comparison to their respective control groups.
The SH concentration of membrane, which decreased after zinc deficiency reversed after zinc supplementation, suggests that zinc plays a major role in protecting membrane –SH group oxidation. Lipid peroxidation and induced lipid peroxidation activities of the erythrocyte ghost membrane revealed a significant increase. Erythrocyte membrane has band 3 transmembrane protein which not only interacts with phospholipids at its surface but has a significant role in anion transport. Due to accumulation of malondialdehyde: (a) anion transport as well as function of band 3 associated enzymes, namely glyceraldehyde-3-phosphate dehydrogenase and phosphofructokinase, are affected and (b) polyunsaturated fatty acids of the cellular membrane are degraded disrupting the membrane integrity ultimately leading to cell membrane damage 44, 45.
Enhanced lipid peroxidation due to oxidative damage of membrane lipids and consequent cross-linking on account of free radical formation due to impaired detoxification function would account for the change in morphology and increased osmotic fragility as reported earlier 8 as well altered glutathione concentration, catalase, superoxide dismutase, glutathione peroxidase, glutathione-s transferase, and glutathione reductase activities 46 which could be an important cause of destruction of the cell membrane. Erythrocytes of zinc-deficient rats appear to be susceptible to oxidative stress. Supplementation studies for a period of 4 weeks revealed a significant increase between various groups indicating that lipid peroxidation production was relatively high, although decrease was observed compared with zinc-deficient experiment.
Hydroperoxides are the major primary product formed during the process of lipid peroxidation. Hydro-peroxides concentration in erythrocyte cell suspension revealed a significant increase after 2, 4, and 6 weeks of zinc deficiency. Comparison of data after zinc supplementation revealed a significant increase in both the deficient groups (4- and 6- weeks), although the concentration decreased when the supplementation group was compared with the deficient (4- and 6-weeks) group. Hydroperoxides may disastrously affect the cell membrane directly through (a) degradation to highly toxic hydroxyl radical and also (b) by the formation of stable aldehydes 18, 47.
Enhancement of hydroperoxides is indicative of an increase in oxidative stress. Hydroxyl radical, a potent reactive oxygen species in the biological system, increased significantly in the erythrocyte cell suspension after zinc deficiency. The increase contributes significantly to alterations in membrane integrity as well as may account for an increase in oxidative stress, which leads to pathologic states. Supplementation of zinc to zinc-deficient groups (4- & 6- weeks) still exhibited an insignificant increase but on comparing the data with zinc-deficient groups (4- & 6- weeks), there was a decline. Chen 48 reported that high levels of zinc decreased O2.-and OH· formation in the erythrocytes. Ascorbic acid, considered the strongest reductant for free radicals, decreased significantly after zinc deficiency. Aqueous peroxyl radicals attack the erythrocyte and induce oxidation of proteins as well as lipids which is detrimental to the cell. Subsequently, supplementation studies although revealed decrease but on comparison with deficient (2-, 4- and 6-weeks) groups, there was an evident increase which could be due to an efflux probably from neutrophils, lymphocytes, monocytes, and platelets which has mill molar concentration of ascorbate49 enhancing the oxidative defense mechanism by probably reducing the ascorbate radical generated not only during oxidative stress as well as by reduction of α-tocopheroxyl radical during lipid peroxidation. Ascorbate can act on catalytic metal ions reducing Fe 3+ to Fe 2+ (Fenton reaction), and itself gets oxidized to ascorbate radical. The ascorbate radical so generated could serve as a source for regeneration of extracellular ascorbate from transmembrane ascorbate radical reductase in erythrocyte using either NADH/intracellular ascorbate 50.
This may have been possible due to alteration in osmotic fragility and morphology of erythrocyte after zinc supplementation 8. Change in osmotic fragility and lowered lipid peroxidation after zinc supplementation can be associated with the increase in ascorbic acid concentration in the present study. Canadan et al., 51 observed the same phenomena wherein supplemented zinc and ascorbic acid improved osmotic fragility and decreased plasma lipid peroxides. α-tocopherol a major lipid-soluble chain-breaking antioxidant, readily reacts with superoxide.
It protects the membrane from damage by trapping the lipid peroxyl radicals 52. Studies carried out on plasma levels of vitamin E of the adult population in the west revealed normal range from 5 µg/ml – 15 µg/ ml 53. The decrease in the present study can possibly account for: (a) increased consumption to destroy oxygen free radicals (b) reduction in regeneration of α-tocopherol from tocopheroxyl radical as for its biological function it requires constant regulation (c) decreased concentration of ascorbate and /or (d) ring breakage of tocopherol quinine leading to loss of α-tocopherol. Synergistic action of zinc and α- tocopherol has been reported54. Both at low levels appear to cause damage to the cell membrane. Supplementation of zinc for a period of 4-weeks revealed decreased levels when groups were compared, but when these groups were compared with deficient experiment groups, an increase was observed. This can be correlated with decreased lipid peroxidation owing to suppression of free radical formation. The decrease of both vitamins suggests that deficiency of these two vitamins contributes to enhanced susceptibility of erythrocytes to oxidation injury. It is also apparent that ascorbic acid decreased first in comparison to α-tocopherol. Zinc concentration when observed in the erythrocyte ghost membrane during the experiment decreased significantly after 4- and 6- weeks of zinc deficiency when ZD groups were compared with their respective ZC and PF groups. The decrease was significant even when PF groups were compared with respective ZC groups. The decline probably reflects: (a) reduced expression of Zn2+ transporters - ZIP which mediates the influx of Zn2+, and (b) decreased metallothionein level, which maintains the homeostatic balance. Deficiency of zinc has been reported to induce oxidative stress in organs 1.
One of the factors involved for altered zinc homeostasis has been reported to be due to nitrosative stress 55 as oxidative or nitrosative stress have been known to release zinc from proteins containing zinc-fingers and cluster motifs and its re-distribution56. Zinc supplementation to deficient group (2-,4- and 6- weeks) still showed an evident decrease when ZD groups were compared to respective ZC and PF groups as well as when PF group was compared with respective ZC group. Moreover, in comparison between zinc supple-mentation and deficient experiments, an increase was observed in supplementation groups. Supplementation studies emphasize the role of zinc as an antioxidant. Decreased NF-kβ activity causing a decline in the gene expression of TNF-α and IL β has been reported after zinc supplementation in HUT [Tho (T helper) cell line] cell line 57. Affect the involvement of zinc in reducing oxidative stress. Further, it also decreases the formation of free radicals through its antagonism to redox-active transition metals.
Copper, a structural requirement for the efficient functioning of various critical enzymes, functions both as an antioxidant and pro-oxidant. Although normally bound to proteins, copper may be released and become free to catalyze the formation of highly reactive hydroxyl radical. Authors 17, 18 reported that intracellular concentration of redox-active transition metal to be low with free iron in the range of 0.2-0.5 µM and free Cu2+ about a single ion/cell. In the present study, a high level of transition metal copper seems to have initiated the Fenton reaction, which may also have been one of the mechanisms to promote lipid peroxidation. Data obtained from in-vitro and culture studies largely indicate the capacity of copper to initiate oxidative damage and interfere with important cellular events 58. Further, copper cannot exist in ionic form and is readily taken up by metallothionein (MT), forming Cu-MT, which in excess is prone to oxidative stress 59. The present study revealed an increase in copper concentration which corroborates well with damage to the membrane as evident by increased protein oxidation, lipid peroxidation, hydroperoxide generation, OH radical formation, etc. Under situations of zinc deficiency, the balance maintained between both zinc and copper trace elements is affected. Zinc supplementation to zinc-deficient experiment groups declined the concentration of copper, achieving a balance between zinc and copper, probably a subsequent increase in stability of copper bound proteins and decrease in Cu-MT levels.
CONCLUSION: Dietary zinc deficiency after 2-, 4- and 6 weeks affected erythrocyte ghost membrane - total protein, protein carbonyl, protein SH group, lipid peroxidation, trace element - total zinc and copper. Erythrocyte cell suspension –total protein was affected with subsequent generation of OH radicals and hydroperoxides along with a disturbance in the physiological concentration of α-tocopherol and ascorbic acid. Supplementation of dietary zinc to deficient groups altered the profile, which was found to duration dependent. These observations provide evidence that a low level of zinc affects the integrity of erythrocytes which could lead to dysfunction.
ACKNOWLEDGEMENT: Authors acknow-ledge Department of Science & Technology, New Delhi -Dr. Pooja Sharma for the award of Inspire fellowship (No. DST/INSPIRE Fellowship /2011/ ); Council Scientific & Industrial Research, New Delhi - Ms. Swati Singh for the award of Junior Research Fellowship (No.18-12/2011(ii) EU-V); University Grants Commission, New Delhi - Ms Astha Saini for the award of Junior Research fellowship (No. 22/06/2014 (1) EV-V) and Senior Fellowship and University Grants Commission, New Delhi - Dr. Deepa Kumari for the award of post-doctoral fellowship (No. F.15-1/2011-12/PDFWM-2011-12-GE-RAJ-3826 [SA-II]). Authors thank Department of Zoology, Centre for Advanced Studies, the University of Rajasthan for providing the necessary facilities.
CONFLICTS OF INTEREST: There is no conflict of interest between the authors.
- Nair N, Bedwal S, Prasad S, Saini MR and Bedwal RS: Short term zinc deficiency in diet induces increased oxidative stress in testes and epididymis of rats. Indian Journal of Experimental Biology 2005; 43: 786-94.
- Krèzel A, Hao Q and Maret W: The zinc/thiolate redox biochemistry of metallothionein and the control of zinc ion fluctuations in cell signaling. Archives Biochemistry Biophysics 2007; 463: 188-200.
- Fukada T, Yamasaki S, Nishida K, Murakami M and Hirano T: Zinc homeostasis and signaling in health and diseases. Journal of Biological Inorganic Chemistry 2011; 16(7): 1123-34.
- Kumari D, Nair N and Bedwal RS: Effect of dietary zinc deficiency on testes of Wistar rats: Morphometric and cell quantification studies. Journal of Trace Element in Medicine and Biology 2011; 25: 47-53.
- Kumari D, Nair N and Bedwal RS: Testicular apoptosis after dietary zinc deficiency: Ultrastructure and TUNEL studies. System Biology in Reproductive Medicine 201; 57(5): 233-43.
- Joshi S, Nair N and Bedwal RS: Dietary zinc deficiency affects dorso-lateral and ventral prostate of Wistar rats: Histological, biochemical and trace element study. Biological Trace Element Research 2014; 161(1): 91-100.
- Joshi S, Nair N and Bedwal RS: Stereological studies in prostate of dietary zinc deficient Wistar rats. Indian Journal of Applied Research 2014; 4: 566-69.
- Sharma P, Nair N, Kumari D and Bedwal RS: Morphology and osmotic fragility in Wistar rat erythrocytes after zinc deficiency and supplementation. Indian Journal of Applied Research 2016; 6(10): 314-18.
- Kuldeep A, Nair N and Bedwal RS: Impact of zinc deficiency on hormones secreted by anterior pituitary of male Wistar rats. IJCR 2016; 8(12): 43514-518.
- Kuldeep A, Nair N and Bedwal RS: Tracing of zinc nanocrystals in the anterior pituitary of zinc deficient Wistar rats. Biological Trace Element Research 2017; 177(2): 316-22.
- Black RE, Victoria CG, Walker SP, Bhutta ZA, Christian P, De Onis M, Ezzati M, Grantham–McGregor S, Katz J, Martorell R and Uauy R: Maternal and child under nutrition and overweight in low-income and middle income countries. The Lancet 2013; 382: 427-51.
- Mohanty JG, Nagababu E and Rifkind JM: Red blood cell oxidative stress impairs oxygen delivery and induces red blood cell aging. Frontiers in Physiology 2014; 5 Doi: 10.3389/fphys2014-00084.
- Suzuki YJ: Oxidant - mediated protein amino acid conversion. Antioxidants 2019; 8: 50-57.
- Davis MJ: Protein oxidation and peroxidation. Biochemical Journal 2016; 473: 805-25.
- Madian AG and Regneir FE: Proteomic identification of carbonylated proteins and their oxidative sites. Journal Proteome Research 2010; 9: 3766-80.
- Thomas, JA, Poland B and Honzatko R: Protein sulfhydryls and their role in the antioxidant function of protein S-thiolation. Archives of Biochemistry and Biophysics 1995; 319: 1-9.
- Repetto MG, Ferrarotti NF and Boveris A: The invol-vement of transition metal ions on iron dependent lipid peroxidation. Archives of Toxicology 2010; 84: 255- 62.
- Repetto MG and Boveris A: Transition metals: Bioinorganic and redox reactions in biological systems. In: A. Mishra (Ed.) Transition metals: Characteristics, properties and uses (Nova Science Publishers Inc. New York USA 2012; 349-70.
- Pastor N, Weinstein H, Jamison E and Brenowitz M: A detailed interpretation of OH radical footprints in a TBA DNA complex reveals the role of dynamics in the mechanism of sequence-specific binding. Journal of Molecular Biology 2000; 304: 55- 68.
- Stadman ER: Role of oxidant species in aging. Current Medicinal Chemistry 2004; 11: 1105- 1112.
- Tupe R and Agte V: Interactions of water soluble vitamins in Zn transport of Caco -2 cells and their implications under oxidative stress conditions. European Journal of Nutrition 2010; 49(1): 53-61.
- Sinbad OO, Folorunsho AA, Olabisi OL, Ayoola OA and Temitope EJ: Vitamins as Antioxidants. Journal of Food Science and Nutrition Research 2019; 2 (3): 214-35.
- Chitra S and Shyamaladevi CS: Modulatory action of α-tocopherol on erythrocyte membrane adenosine triphosphatase against radiation damage in oral cancer. The Journal of Membrane Biology 2011; 240(2): 83- 88.
- Dodge JT, Mitchel C and Hanahan DJ: The preparation and chemical characteristics of haemoglobin-free ghosts of human erythrocytes. Archives of Biochemistry and Biophysics 1963; 100: 119-30.
- Steck TL and Kant JA: Preparation of impermeable ghosts and inside-out vesicles from human erythrocyte membranes. Methods Enzymology 1974; 31:172-80.
- Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 1976; 72: 248-54.
- Levine RL, Garland D, Oliver CN, Amici A, Climent I, Lenz AG, Ahn BW, Shaltiel S and Stadtman ER: Determination of carbonyl content in oxidatively modified proteins. Methods Enzymology 1990; 186: 464-78.
- Habeeb AF: Reaction of protein sulfhydryl groups with Ellman’s reagent. Methods Enzymolog 1972; 25: 457- 64.
- Ohkhawa H, Ohishi N and Yagi K: Assay for lipid peroxides in animal tissues by thiobarbituric acid reactions. Analytical Biochemistry 1979; 95: 351-58.
- Jiang ZY, Hunt JV and Wolf SP: Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxides in low density lipoprotein. Analytical Biochemistry 1992; 202: 384-89.
- Aruoma OI: Nutrition and health aspects of free radicals and antioxidants. Food and Chem Toxi 1994; 62: 671-83.
- Desai I: Vitamin E analysis methods for animal tissues. Methods for animal tissues. Methods Enzymology 1984; 105: 138-47.
- Roe JH and Kuther CA: Detection of ascorbic acid in whole blood and urine through the 2, 4 DNPH derivative of dehydroascorbic acid. Journal of Biological Chemistry 1943; 147: 399-401.
- Omaya ST, Turnbul JD and Sauberlich HE: Selected methods for the determination of ascorbic acid in animal cells, tissues and fluids. Methods Enzymol 1979; 62: 3-11.
- Marreiro D do N, Cruz KJC, Morais JBS, Beserra JB, Severo JS and de Oliveira ARS: Zinc and oxidative stress: Current mechanisms. Antiox Basel 2017; 6(2): 24- 32.
- Buys AV, Van Rooy MJ, Soma P, Van Papendorp D, Lipinski B and Pretorious E: Changes in red blood cell membrane structure in type 2 diabetes: A scanning electron and atomic force microscopy study. Cardio-vascular Diabetology 2013; 12: 25-30.
- Shibuya A, Kawashima H and Tanaka M: Analysis of erythrocyte membrane proteins in patients with hereditary spherocytosis and other types of haemolytic anaemia. Hematology 2018; 23(9): 669-75.
- Tamás MJ, Sandeep K, Sharma SK, Ibstedt S, Jacobson T and Christen P: Heavy metals and metalloids as a cause for protein misfolding and aggregation. Biomolecules 2014; 4(1): 252-67.
- Dukan S, Farewell A, Ballestros M, Taddei F, Radman M and Nyström T: Protein oxidation in response to increased transcriptional or translational errors. Proceedings of National Academy of Sciences USA 2000; 97: 5746-49.
- Celi P and Gabai G: Oxidant/antioxidant balance in animal nutrition and health: The role of protein oxidation. Frontiers in Veterinary Science 2015; 26.
- Claeyssen R, Andriollo – Sanchez M, Arnaud J, Touvard L, Alonso A, Chancerelle Y, Roussel AM and Agay D: Burn-induced oxidative stress is altered by a low zinc status: Kinetic study in burned rats fed a low zinc diet. Biological Trace Element Research 2008; 126: 80-96.
- Zhang K and Kaufman RJ: The unfolded protein response: A stress signaling pathway critical for health and disease. Neurology 2006; 66: 102-109.
- Xia J, Browning JD and O’Dell BL: Decreased plasma membrane thiol concentration is associated with increased osmotic fragility of erythrocytes in zinc-deficient rats. Journal of Nutrition 1999; 129: 814-19.
- Steck TL: The band 3 protein of the human red cell membrane: A review. Journal of Supramolecular Structure 1978; 8(3): 311-24.
- Harvey J, Kliman S and Steck TL: Association of Glyceraldehyde-3-phosphate dehydrogenase with the human red cell membrane. A kinetic analysis. Journal of Biological Chemistry 1980; 255(13): 6314-21.
- Nair N, Bedwal S, Sharma P, Saini A, Kumari D and Bedwal RS: Non enzymatic and enzymatic antioxidant status of Wistar rat erythrocyte after dietary zinc deficiency. International Journal of Pharmaceutical Sciences and Research 2019; 10(2): 763-68.
- Ayala A, Muñoz MF and Argüelles S: Lipid peroxidation: production, metabolism and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. Oxidative Medicine and Cellular Longevity 2014; http://dx.doi.org/10.1155/2014/360438.
- Chen M: Absent effect of zinc deficiency on the oxidative stress of erythrocytes in chronic uremic rats. Chinese Journal of Physiology 2002; 45(1): 9-18.
- Du J, Cullen JJ and Buettner GR: Ascorbic acid: Chemistry, biology and the treatment of cancer. Biochimica ET Biophysica Acta 2012; 1826(2): 443-57.
- May JM, Qu Z and Cobb CE: Recycling of the ascorbate free radical by human erythrocyte membranes. Free Radical Biology Medicine 2001; 31: 117-24.
- Canadan F, Gultekin F and Dandau F: Effect of vitamin C and zinc on osmotic fragility and lipid peroxidation in zinc deficient hemodialysis patients. Cell Biochemistry and Function 2002; 20: 95-98.
- Agarwal A and Prabakaran S: Mechanism, measurement and prevention of oxidative stress in male reproductive physiology. Indian Journal of Experimental Biology 2005; 43: 963-74.
- McBurney MI, Yu EA, Ciappio ED, Bird JK, Eggersdorfer M and Mehta S: Suboptimal serum α-tocopherol concentrations observed among younger adults and those depending exclusively upon food sources. PLOS One 2015; 10(8):e0135510- e0135513.
- Wasowicz W, Reszka E, Gromadzinska J and Rydzynski K: The role of essential elements in oxidative stress. Comments on Toxicology 2003; 9: 39- 48.
- Weisman DA, Wells SM, Wilham J, Hubbard M, Welker JE and Stephan MB: Endothelial response to stress from exogenous Zn2+ resembles that of NO-mediated nitrosative stress, and is protected by MT-1 overexpression. American Journal of Physiology Cell Physiology 2005; 291: C555-68.
- Ruttkay-Nedecky B, Nejdl L, Jaromir Gumulec J, Zitka O, Masarik M, Eckschlager T, Stiborova M, Adam V and Kizek R: The role of metallothionein in oxidative stress. International. Journal of Molecular Science 2013; 14: 6044-66.
- Prasad AS: Zinc in human health: Effect of zinc on immune cells. Molecular Medicine 2008; 14(5-6): 353-57.
- Slivkova J, Popelkova M, Massanyi P, Toporcerova S, Stawarz R, Formicki G, Lukac N, Putała A and Guzik M: Concentration of trace elements in human semen and relation to spermatozoa quality. Journal Environmental Science Health A 2009; 4(4): 370-75.
- Suntres ZE and Liu EMK: Pro oxidative effect of copper- metallothionein in the acute cytotoxicity of hydrogen peroxides in Ehrlich ascites tumour cells. Toxicology 2006; 217: 155- 68.
How to cite this article:
Nair N, Sharma P, Bedwal S, Singh S, Kumari D, Saini A and Bedwal RS: Effect of short term dietary zinc deficiency and zinc supplementation on Wistar rat erythrocyte: biochemical studies. Int J Pharm Sci & Res 2021; 12(3): 1713-22. doi: 10.13040/IJPSR.0975-8232.12(3).1713-22.
All © 2013 are reserved by the International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
N. Nair *, P. Sharma, S. Bedwal, S. Singh, D. Kumari, A. Saini and R. S. Bedwal
Department of Zoology, Centre for Advanced Studies, University of Rajasthan, Jaipur, Rajasthan, India.
23 March 2020
21 October 2020
17 November 2020
01 March 2021