- Email: [email protected]
Changes in serum level of trace elements in pulmonary tuberculosis patients during anti-tuberculosis treatment
Accepted Manuscript Title: Changes in Serum Level of Trace Elements in Pulmonary Tuberculosis Patients during Anti-Tuberculosis Treatment Authors: Zahra Sepehri, Donya Arefi, Nima Mirzaei, Asma Afshari, Zohre Kiani, Alireza Sargazi, Abolfazl Panahi Mishkar, Hamid Owaysee Oskoee, MohammadReza Masjedi, Aliyeh Sargazi, Saeid Ghavami PII: DOI: Reference:
S0946-672X(17)30817-9 https://doi.org/10.1016/j.jtemb.2018.06.024 JTEMB 26179
To appear in: Received date: Revised date: Accepted date:
3-10-2017 17-6-2018 26-6-2018
Please cite this article as: Sepehri Z, Arefi D, Mirzaei N, Afshari A, Kiani Z, Sargazi A, Mishkar AP, Oskoee HO, Masjedi M, Sargazi A, Ghavami S, Changes in Serum Level of Trace Elements in Pulmonary Tuberculosis Patients during AntiTuberculosis Treatment, Journal of Trace Elements in Medicine and Biology (2018), https://doi.org/10.1016/j.jtemb.2018.06.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Changes in Serum Level of Trace Elements in Pulmonary Tuberculosis Patients during Anti-Tuberculosis Treatment Zahra Sepehri1, Donya Arefi2, Nima Mirzaei2,3 ,Asma Afshari4, Zohre Kiani5,6, Alireza Sargazi5, Abolfazl Panahi Mishkar7, Hamid Owaysee Oskoee8, MohammadReza Masjedi9, Aliyeh Sargazi5*, Saeid
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Ghavami10 1
Department of Internal Medicine, Zabol University of Medical Sciences, Zabol, Iran.
2
Zabol University of Medical Sciences, Zabol, Iran.
3
Department of Human Anatomy and Cell Science, Max Rady College of Medicine, Rady Faculty of Health
Sciences, University of Manitoba, Winnipeg, MB, R3E 3P4, Canada. 4
Department of Nutrition, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
5
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Medical student, Students Research Committee, Zabol University of Medical Sciences, Zabol, Iran.
6
Medical student, Students Research Committee, Kerman University of Medical Sciences, Kerman, Iran.
7
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Human Ecology MSc student, Kerman University of Medical Sciences, Kerman, Iran.
8
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Department of Infectious disease, Tabriz University of Medical Sciences, Tabriz, Iran.
9
Telemedicine Research Center, Shahid Beheshty University of Medical Sciences, Tehran, Iran.
10
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Department of Human Anatomy and Cell Science, Max Rady College of Medicine, Rady Faculty of
Health Sciences, University of Manitoba, Winnipeg, MB, R3E 3P4, Canada .
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*Corresponding Author
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Aliyeh Sargazi, Medical Student, Students Research Committee, Zabol University of Medical Sciences, Zabol, Iran. Add: Iran, Zabol, Shahid Rajaee Street, Zabol University of Medical Sciences. Phone no:+98-915-191-3504, Email: [email protected]
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Summary
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Elements during Anti-Tuberculosis Therapy
Introductions: Tuberculosis is spreading throughout the globe, while it is a crucial cause of death in developing countries. In this study, trace elements concentrations and their alterations were
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determined in TB patients during anti-tuberculosis treatment period. Materials and methods: We have collected blood samples from a total of 180 TB patients with pulmonary Tuberculosis, and 180 healthy controls in Sistan, Iran. The serum iron, copper, lead, calcium, arsenic and selenium concentrations were detected at the beginning of anti-TB chemotherapy, at the end of 2nd, 4th and 6thmonth after treatment initiation. Data were then analyzed using SPSS version 20. 1
Results and discussions: Although Ca, Pb, and As levels did not change during the treatment period, serum concentrations of Fe, Zn, Cu, and Se were diminished in TB patients significantly during treatment in comparison with controls (P<0.001).We also found that there was a significant difference in the
Cu
/Se and
Cu
/Zn ratios in tuberculosis patients in comparison with healthy
individuals (P<0.001).
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Conclusions: Trace elements serum concentrations are affected by TB infection and anti-TB therapy. Their serum levels were strongly perturbed during infection as well as anti-TB treatment. Keywords: Pulmonary tuberculosis; trace elements; graphite furnace atomic absorption spectrometry; Sistan. INTRODUCTION
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Trace elements like iron (Fe), copper (Cu), lead (Pb), arsenic (As), selenium (Se), calcium
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(Ca) and zinc (Zn) play important roles in many biological systems. These elements are accurately distributed between blood and tissues. In this way, any turbulent factors might cause imbalanced
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circulatory element level. Elevated serum copper and decreased serum zinc are the most common
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trace element imbalances in population. Copper is an essential element mostly found in liver, brain, kidneys, heart and muscles.[1] Zinc is mostly involved in enzymes structure, wound healing,
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protein and DNA synthesis and immune system to destroy microbes such as M. tuberculosis or Escherichia coli.[2-5] Moreover the
copper
/zinc ratio is a significant diagnosis marker in many
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statements such as in hepatitis, tuberculosis, brain, breast and colorectal cancers.[6-8] Iron is an essential element for a number of highly complex biological processes that continuously take place
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on a molecular level, and play a vital role in red blood cells, new cells, amino acids, hormones, and neurotransmitters production.[9] Arsenic (As) has been recognized as a compelling
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immunomodulatory mediator in numerous experimental and epidemiologic studies.[10] Arsenic exposure decreases immune response to viral and bacterial infections and increases chronic
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diseases risk.[11] Selenium in Selenoproteins associate with reactive oxygen species (ROS) regulation and strongly influence different immune responses such as inflammation.[12,13] Although these elements are involved in several biological processes, their serum concentrations vary during many infections or treatment processes. In active pulmonary tuberculosis, serum levels of Fe, Zn, and Se are considerably decreased.[14] The reduction in the Fe, Zn, or Se levels is
2
mainly a protective strategy to reduce micronutrient availability for mycobacterium metabolism and growth during infection.[15,1] Considering the critical role of these materials on immune system and importance of this system in fighting against M. tuberculosis, we aimed to evaluate serum level changes of trace elements during anti-TB chemotherapy.
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MATERIALS AND METHODS Patient group
This study was carried out on 360 subjects, and two groups were formed: patient and healthy. Of the total of 360 samples, there were 180 normal healthy individuals and 180 patients with active TB. All the TB samples were collected from confirmed pulmonary tuberculosis cases receiving anti-tuberculosis treatment in TB centers and hospitals in Sistan (SouthEast of Iran), Iran.
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Demographic factors such as age, gender, weight, height and body mass index (BMI) were
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measured and saved for each single subject. The subjects were matched for age and sex. Sampling was performed from each subject at the beginning of the anti-TB treatment, after 2, 4 and 6 months
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of treatment initiation. All of them received anti-tuberculosis treatment according to WHO
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guidelines under Sistan Tuberculosis Center. The study protocol was approved by Zabol University of Medical Sciences ethical committee with the ethical code of ZBMU.1.REC.1395.63.
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Healthy group
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Healthy individuals were selected using probability sampling method. First, Zabol city (center of Sistan area) was divided in six areas. Then, we picked two zones in each area (the largest and the smallest zones). We referred to the homes (based on the number of homes in the way that 5th home
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was our first point as well as other 5th doors from the right), and asked about previous exposure to TB or tuberculosis symptoms. All of the subjects were physically examined by a trained
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physician. They were screened for any underlying diseases and infections at the experiment initiation, 2nd, 4th and 6th months. These subjects were matched for age and sex too. Finally, we
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selected 15 subjects from each zone. Sampling such as Patient group was done for each subject at the start of the research, after two, four, six months of research. Sample preparation Blood samples were taken and stored into vacutainer tubes which were cleaned from any contamination. Each blood tube was centrifuged at 2500rpm for 15 min for cells and serum
3
separation. The sera were collected in polyethylene tubes and froze at -20ºC for the element analysis . Serum trace elements determination In order to determine Cu, Zn, Ca, Se, As, Pb, and Fe levels, 10ml of concentrated nitric acid (65%, Merck, Germany) was added to 1ml of serum in a beaker and heated gently below the boiling point
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for 3 hours, on a hot plate. When the volumes of the samples reduced to about one third, 5 ml of 30% hydrogen peroxide (30%, Merck, Germany) solution was included and heated almost to dryness at the same temperature. Finally, the residues were dissolved in 50ml of 1% nitric acid (65%, Merck, Germany) and were filtered.
The prepared solution was then added to 50ml polyethylene tubes for trace element analysis. To measure serum copper, zinc, calcium, lead and iron, Flame Atomic Absorption Spectrometry
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(FAAS) (model WFX-210, Rayleigh, China), and to measure selenium and Arsenic Hydride
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Generation Atomic Absorption Spectroscopy (HGAAS) (model WFX-210, Rayleigh, China) were used. The levels of elements were determined after 2, 4 and 6 months of anti-TB treatment
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levels of trace elements illustrated in Table 1.
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initiation in TB infected groups. The main parameters of instruments used for measuring serum
Statistical analysis
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Serum concentrations of trace metals were described by mean and standard deviation. Quantitative
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differences during the follow up time in both patients and healthy controls were determined according to repeated measure and Friedman tests adjusting for potential confounders such as baseline measurements as well as gender of the patients. SPSS software was applied for statistical
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analysis. We have used SPSS-20 software (SPSS Inc., Chicago, IL, USA) to analyze data with considering P value less than 0.001 significant statistically.
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Elements reference ranges: The normal reference ranges of blood materials were considered 55–160 µg/dL in men and40–155
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µg/dL in women for iron.[16] The normal range of Lead is <10μg/dL in children and <25μg/dL in adults.[17] The total and free serum copper are normally 63.7-140.12 μg/dL and 10-15μg/dL respectively.[18,19] The normal total and free serum calcium are 9.5-10.5mg/dl and 5.1-5.9mg/dl in children and 8.9-10.1mg/dL and 4.8-5.7mg/dL in adults respectively.[20] This range is 0-12 ng/mL for arsenic and 70 to 150 ng/mL for selenium and 66 to 110 μg/dL for zinc.[21,22] 4
Quality control:
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We had used certified reference materials in the same scale of our samples for quality control in our project. We had used BCR® which is a registered trademark of the European Commission from joint research center. The reference value BCR-637 was considered for zinc and selenium which were equal 81±7 µg/L and (1.11±0.22)*103 µg/L respectively. We had used BCR-635 for lead which was equal 210±24 µg/L and BCR-304 for calcium which was equal 2.201±0.019mmol/L in human serum. No certified references material was available for iron, copper and arsenic in The JRC-Geel (Joint Research Center) which is including IRMM, BCR and ERM.[23]
RESULTS
Totally 180 tuberculosis infected patients with the age ranged between 17 and 87 years were included in this study. Out of all TB patients, 182 cases (50.5%) were males and 178 cases (49.5%) kg
/m2 and 21.04
kg
/m2,
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were females. Mean BMI for patients and healthy group were 21.3
respectively. Tuberculosis patients were followed for six consecutive months.
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As illustrated in table 2, the iron level was in mid normal range in healthy individuals and was
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near lower limit of normal range in TB patients at treatment initiation whereas it got increased by
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the time during anti-TB treatment. Selenium level was near lower limit in health group but below normal range in TB infected patients. The selenium level had been increased during treatment till normal range at treatment final. Lead levels were normal in both groups but almost two times
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higher in TB infected group. Copper levels were normal in both groups. It was lower in healthy
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individuals in comparison with TB patients. Copper level got decreased in TB group during treatment. Zinc level was normal in healthy group and below normal in TB patients. It got
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increased by complete treatment to normal range. The calcium level was higher than normal range in both groups. Arsenic level was normal in both groups but higher overall in TB infected group.
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Moreover in the first and second measurement, the mean serum zinc, selenium and iron were lower significantly in tuberculosis patients compared to control group (P<0.001), in contrast serum arsenic, lead and copper were higher in the patients significantly(P<0.001).
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Serum concentrations of iron, selenium and zinc were increased significantly in six months after the treatment initiation in both groups (p<0.001) but this increase was higher among patients comparing healthy individuals significantly (p<0.001). In contrast lead serum level decreased after six months in both groups significantly (p<0.001). Yet this reduction was higher among TB patients in comparison with patients (p<0.001). Copper and arsenic were significantly dropped in patients while increased among healthy individuals (p<0.001). In both cases of Copper and arsenic, 5
the changes were significantly higher among patients. Finally, serum calcium concentration showed significant increase just in TB patients (p<0.001) (Fig. 1). We also observed that the
Cu
/Zn and
Cu
/Se ratios were significantly decreased (p<0.001) in TB
patients during six month treatment, while healthy individuals experienced significant increase in these ratios (p<0.05). After adjustment for the effects of baseline measurements of all elements,
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the observed effects were remained, except for iron which was similarly changed in both groups. (Fig. 1).
DISCUSSION
Tuberculosis is considered an important transmitted infectious disease in Iran and many other countries throughout the world.[24] In this study we have examined serum zinc, copper, arsenic,
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calcium, selenium, lead, and iron in both groups before treatment initiation. The serum concentrations of zinc, selenium and iron were lower and arsenic, lead and copper were higher in
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TB infected patients significantly before treatment initiation in comparison with healthy
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individuals.
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Numerous studies have revealed that zinc concentration in serum diminish extensively during the process of tuberculosis, and rise after anti-tuberculosis therapy initiation.[25]
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In a similar study Ciftci et al. demonstrated higher serum copper level, higher Cu/Zn ratio and lower serum zinc and selenium level in patients with active pulmonary tuberculosis in comparison with
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control group. They have also revealed that these elements level have been changed during treatment initiation.[26]
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Because of cell mediated immunity role in anti tuberculosis defense and the close association of zinc with this immune pathway, this metal plays a significant role against M.tuberculosis.[27,28]
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Lower serum zinc in TB patients could be related to its redistribution from plasma to tissues during infection.[29] Results of another study carried out by Arntsen et al. in Malaw showed lower serum selenium levels in tuberculosis patients compared with controls.[30] These results are in keeping
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with those observed in our study. The immune system and inflammatory responses are tightly associated with blood selenium levels. Sufficient body selenium causes resistance in front of many types of pathogens such as bacteria, parasite and fungi depending on involved pathogen variant.[15] Selenium is involved in selenoproteins structure, immune system regulation and oxidative response protection. The antioxidant activity of selenium can alter mycobacterium DNA. To avoid 6
DNA damage, M. tuberculosis has developed effective mechanisms to interrupt the activity of selenoproteins. The impaired selenoproteins can result in a significant selenium deficiency in TB patients.[31,32] Iron is a necessary nutrient which is extensively allocated in food. Like selenium and zinc, serum Fe levels were drastically lower in TB patients than healthy subjects. A possible explanation is that
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multiplication of M. tuberculosis within host macrophages depends on the presence of iron.[33] Hence, the iron deficiency that we observed in TB infected patients could be due to the mycobacterial iron consumption . Our findings showed that although iron levels were changed during treatment in TB patients, but its decrease pattern was same in both case and control groups. The immune system requires copper to perform several functions such as fighting microorganisms. In addition, copper is critical for nervous system function, Iron transport, cholesterol and glucose
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metabolism.[34,35] Because of essential role of copper in iron absorption, elevated copper level is
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seen in patients with active tuberculosis. Copper may interfere with iron absorption by binding to mucosal transferring.[36,37] Similarly, Frediani1et al. in Georgia measured serum iron level in
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adults with pulmonary tuberculosis, and found lower Fe concentrations.[38]
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Like copper, most of the patients had high serum levels of lead. In line with this, Afridi et al. indicated significantly higher levels of Pb in TB-HIV co-infected patients, compared with control
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subjects. Lead can interrupt antibody productions, and affects the lymphocytes.[39] Increased
CONCLUSION
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serum concentrations of lead may help Mycobacterium tuberculosises caping immune system.
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We found significant increases in the levels of Zn, Fe, calcium and Se, as well as decreases in the Cu, Pb, and As levels during the anti-TB treatment. There is a logical explanation for these
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alterations during anti-TB therapy. After starting chemotherapy, the number of active mycobacteria drops significantly. This causes considerable changes in serum concentrations of trace elements so that the values become closer and closer to those values of the healthy controls.
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During or after beginning of anti-TB treatment the circulating Zn, Fe, and zinc start increasing in order to enhance the availability of these vital micronutrients for body. However serum concentrations of Cu, Pb, and As decreases after anti-TB therapy due to their toxicity to cells. One of the limitations of our study was that we did not consider a time period for measuring serum concentration of trace elements in healthy controls.
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In conclusion, our study revealed that anti-TB treatment not only removes the microbial agents, but also is effective in serum trace elements level control. Therefore using supplementary agents and controlling trace elements levels in blood during tuberculosis treatment could cause faster, more efficient and successful treatment. Further studies with various control groups should be done to detect the exact effects of elements in tuberculosis treatment.
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Declaration of Conflicting Interests Authors declare no potential conflicts of interest.
Acknowledgments
The authors would like to thank all who participated in this study and specially the Amiralmomenin Hospital of Zabol University of Medical Sciences, tuberculosis and addiction withdrawal centers of Zabol, Hirmand and Hamoun.
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Table1. The main instrument parameters
Flame atomic absorption spectrometry (FAAS) Wavelength(nm)
Slit
Lamp
Burner
Acetylene
Air flow
width
current
Height
flow (L/min)
(L/min)
(nm)
(mA)
(mm)
324.8
4
10
1.70
13.5
213.9
1.0
4
10
2.00
13.5
0.5
Fe
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Element
248.3
0.2
8
10
2.50
13.5
Pb
283.3
0.7
4
10
2.5
13.5
Cu
CC
Zn
A
Hydride Generation Atomic Absorption Spectroscopy(HGAAS)
NaBH4 concentration
0.5%
NaOH concentration
1M
NaOH/NaBH4 flow-rate
0.33 ml min-1
HCl concentration
5%
HCl flow-rate
0.33 ml min-1
10
Sample flow-rate
0.33 ml min-1
Wavelength(nm) for As
193.7 nm
Wavelength(nm) for Se
196.0 nm
Table 2: Serum levels of trace elements six months study among subjects with and without tuberculosis
s
Treatment
Healthy
phase
TB patients
changes during time between
Normal
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Element
individuals
two groups
Range
Adjustment
Adjustment for
for gender
all baseline
and baseline
elements
measure of the same
Before
78.80(3.31)
52.26(4.47) 3
79.29(3.29) 1
62.55(9.72) 13
months After four
79.56(3.23) 1
months
79.15(3.29) 1
Before
76.81(7.11)
42.38(3.81)3
76.96(6.32) 2
45.40(4.53) 13
m
months
After four
155(160)
80.07(3.17) 1 3
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After two
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Seleniu
<0.0001
40(55)-
72.65(8.33) 1 3
After six months
treatment
0.21
M
Iron
A
After two
N
treatment
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element
0.003
70-150
<0.001
<25
<0.0001
77.45(5.71) 1
63.33(7.04) 13
After six months
75.74(5.86) 1
70.61(7.33) 13
Before
10.71(0.90)
19.32(1.94) 3
9.55(0.89) 1
18.99(2.32)13
A
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months
Lead
treatment After two
<0.0001
months After four
9.81(0.88) 1
22.15(2.40) 13
9.42(0.59) 1
18.25(1.87) 13
months After six months
11
Before
91.03(5.84)
119.07(7.50) 3
93.06(5.61) 1
113.60(6.49) 1
treatment
Copper
months
92.44(5.57) 1
109.47(5.59) 1
months
3
After six months
93.70(5.54) 1
99.72(5.14) 1 3
Before
94.94(8.60)
58.48(5.72) 3
94.92(8.42)
63.78(6.39) 1 3
treatment After two
96.12(8.33) 1
76.03(9.45) 1 3
After six months
95.60(8.39) 1
86.86(6.78) 1 3
Before
13.07(0.92)
12.21(1.15) 3
After two
12.56(0.99) 1
0.004
8.9-10.1
<0.001
0-12
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12.05(1.19) 1 3 <0.0001
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months
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treatment
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months
After four
13.12(1.37)
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months
66-110
<0.0001
months After four
Calcium
<0.001
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Zinc
70-140
<0.0001
3
After four
0.05
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After two
13.02(1.27)
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After six months
Before
11.27(1.40) 1 3 13.64(1.65) 1 3
3.42(0.58)
8.35(0.43) 3
3.74(0.60) 1
7.66(0.48) 1 3
treatment
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After two
Arsenic
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After four
Cu/Zn
3.85(0.29) 1
6.12(0.45) 1 3
After six months
3.60(0.18) 1
4.72(0.43) 1 3
Before
0.97(0.11)
2.06(0.25) 3
months
treatment
12
<0.0001
months
<0.0001
After two
0.99(0.11) 1
2.06(0.25) 3
0.97(0.10) 2
1.80 (0.21) 1 3
After six months
0.99(0.11) 1
1.15(0.11) 1 3
Before
1.19 (0.12)
2.83 (0.31)
1.21(0.11) 1
2.53 (0.29) 1 3
<0.001
months After four
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months
treatment After two
Cu/Se
0.004
<0.0001
months After four
1.20 (0.11) 1
1.75 (0.22) 1 3
1.24 (0.12) 1
1.43 (0.16) 1 3
months After six months
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1: Significantly different from baseline in the same group (p<0.001)2: Significantly different from baseline in the same group (p<0.05) 3: Significantly different from healthy group in the same phase of treatment
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*The evaluation unites for elements are µg/dl for iron, lead, copper and zinc. It is ng/ml for selenium and arsenic. The unit of calcium concentration is mg/dl.
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Figures
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Fig. 1. Serum trace element levels in control and patient groups; copper (A), Calcium (B), Zinc (C), Selenium(D), Arsenic (E) ,Lead (F), Iron (G) as well as Serum Cu/Zn and Cu/Se ratios; (*)
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significantly different from baseline in the same group (p<0.001), (***)significantly different
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from healthy group in the same phase of treatment
13
14
D
TE
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CC
A
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A
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15
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S0946-672X(17)30817-9 https://doi.org/10.1016/j.jtemb.2018.06.024 JTEMB 26179
To appear in: Received date: Revised date: Accepted date:
3-10-2017 17-6-2018 26-6-2018
Please cite this article as: Sepehri Z, Arefi D, Mirzaei N, Afshari A, Kiani Z, Sargazi A, Mishkar AP, Oskoee HO, Masjedi M, Sargazi A, Ghavami S, Changes in Serum Level of Trace Elements in Pulmonary Tuberculosis Patients during AntiTuberculosis Treatment, Journal of Trace Elements in Medicine and Biology (2018), https://doi.org/10.1016/j.jtemb.2018.06.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Changes in Serum Level of Trace Elements in Pulmonary Tuberculosis Patients during Anti-Tuberculosis Treatment Zahra Sepehri1, Donya Arefi2, Nima Mirzaei2,3 ,Asma Afshari4, Zohre Kiani5,6, Alireza Sargazi5, Abolfazl Panahi Mishkar7, Hamid Owaysee Oskoee8, MohammadReza Masjedi9, Aliyeh Sargazi5*, Saeid
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Ghavami10 1
Department of Internal Medicine, Zabol University of Medical Sciences, Zabol, Iran.
2
Zabol University of Medical Sciences, Zabol, Iran.
3
Department of Human Anatomy and Cell Science, Max Rady College of Medicine, Rady Faculty of Health
Sciences, University of Manitoba, Winnipeg, MB, R3E 3P4, Canada. 4
Department of Nutrition, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
5
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Medical student, Students Research Committee, Zabol University of Medical Sciences, Zabol, Iran.
6
Medical student, Students Research Committee, Kerman University of Medical Sciences, Kerman, Iran.
7
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Human Ecology MSc student, Kerman University of Medical Sciences, Kerman, Iran.
8
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Department of Infectious disease, Tabriz University of Medical Sciences, Tabriz, Iran.
9
Telemedicine Research Center, Shahid Beheshty University of Medical Sciences, Tehran, Iran.
10
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Department of Human Anatomy and Cell Science, Max Rady College of Medicine, Rady Faculty of
Health Sciences, University of Manitoba, Winnipeg, MB, R3E 3P4, Canada .
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*Corresponding Author
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Aliyeh Sargazi, Medical Student, Students Research Committee, Zabol University of Medical Sciences, Zabol, Iran. Add: Iran, Zabol, Shahid Rajaee Street, Zabol University of Medical Sciences. Phone no:+98-915-191-3504, Email: [email protected]
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Summary
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Elements during Anti-Tuberculosis Therapy
Introductions: Tuberculosis is spreading throughout the globe, while it is a crucial cause of death in developing countries. In this study, trace elements concentrations and their alterations were
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determined in TB patients during anti-tuberculosis treatment period. Materials and methods: We have collected blood samples from a total of 180 TB patients with pulmonary Tuberculosis, and 180 healthy controls in Sistan, Iran. The serum iron, copper, lead, calcium, arsenic and selenium concentrations were detected at the beginning of anti-TB chemotherapy, at the end of 2nd, 4th and 6thmonth after treatment initiation. Data were then analyzed using SPSS version 20. 1
Results and discussions: Although Ca, Pb, and As levels did not change during the treatment period, serum concentrations of Fe, Zn, Cu, and Se were diminished in TB patients significantly during treatment in comparison with controls (P<0.001).We also found that there was a significant difference in the
Cu
/Se and
Cu
/Zn ratios in tuberculosis patients in comparison with healthy
individuals (P<0.001).
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Conclusions: Trace elements serum concentrations are affected by TB infection and anti-TB therapy. Their serum levels were strongly perturbed during infection as well as anti-TB treatment. Keywords: Pulmonary tuberculosis; trace elements; graphite furnace atomic absorption spectrometry; Sistan. INTRODUCTION
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Trace elements like iron (Fe), copper (Cu), lead (Pb), arsenic (As), selenium (Se), calcium
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(Ca) and zinc (Zn) play important roles in many biological systems. These elements are accurately distributed between blood and tissues. In this way, any turbulent factors might cause imbalanced
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circulatory element level. Elevated serum copper and decreased serum zinc are the most common
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trace element imbalances in population. Copper is an essential element mostly found in liver, brain, kidneys, heart and muscles.[1] Zinc is mostly involved in enzymes structure, wound healing,
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protein and DNA synthesis and immune system to destroy microbes such as M. tuberculosis or Escherichia coli.[2-5] Moreover the
copper
/zinc ratio is a significant diagnosis marker in many
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statements such as in hepatitis, tuberculosis, brain, breast and colorectal cancers.[6-8] Iron is an essential element for a number of highly complex biological processes that continuously take place
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on a molecular level, and play a vital role in red blood cells, new cells, amino acids, hormones, and neurotransmitters production.[9] Arsenic (As) has been recognized as a compelling
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immunomodulatory mediator in numerous experimental and epidemiologic studies.[10] Arsenic exposure decreases immune response to viral and bacterial infections and increases chronic
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diseases risk.[11] Selenium in Selenoproteins associate with reactive oxygen species (ROS) regulation and strongly influence different immune responses such as inflammation.[12,13] Although these elements are involved in several biological processes, their serum concentrations vary during many infections or treatment processes. In active pulmonary tuberculosis, serum levels of Fe, Zn, and Se are considerably decreased.[14] The reduction in the Fe, Zn, or Se levels is
2
mainly a protective strategy to reduce micronutrient availability for mycobacterium metabolism and growth during infection.[15,1] Considering the critical role of these materials on immune system and importance of this system in fighting against M. tuberculosis, we aimed to evaluate serum level changes of trace elements during anti-TB chemotherapy.
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MATERIALS AND METHODS Patient group
This study was carried out on 360 subjects, and two groups were formed: patient and healthy. Of the total of 360 samples, there were 180 normal healthy individuals and 180 patients with active TB. All the TB samples were collected from confirmed pulmonary tuberculosis cases receiving anti-tuberculosis treatment in TB centers and hospitals in Sistan (SouthEast of Iran), Iran.
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Demographic factors such as age, gender, weight, height and body mass index (BMI) were
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measured and saved for each single subject. The subjects were matched for age and sex. Sampling was performed from each subject at the beginning of the anti-TB treatment, after 2, 4 and 6 months
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of treatment initiation. All of them received anti-tuberculosis treatment according to WHO
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guidelines under Sistan Tuberculosis Center. The study protocol was approved by Zabol University of Medical Sciences ethical committee with the ethical code of ZBMU.1.REC.1395.63.
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Healthy group
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Healthy individuals were selected using probability sampling method. First, Zabol city (center of Sistan area) was divided in six areas. Then, we picked two zones in each area (the largest and the smallest zones). We referred to the homes (based on the number of homes in the way that 5th home
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was our first point as well as other 5th doors from the right), and asked about previous exposure to TB or tuberculosis symptoms. All of the subjects were physically examined by a trained
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physician. They were screened for any underlying diseases and infections at the experiment initiation, 2nd, 4th and 6th months. These subjects were matched for age and sex too. Finally, we
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selected 15 subjects from each zone. Sampling such as Patient group was done for each subject at the start of the research, after two, four, six months of research. Sample preparation Blood samples were taken and stored into vacutainer tubes which were cleaned from any contamination. Each blood tube was centrifuged at 2500rpm for 15 min for cells and serum
3
separation. The sera were collected in polyethylene tubes and froze at -20ºC for the element analysis . Serum trace elements determination In order to determine Cu, Zn, Ca, Se, As, Pb, and Fe levels, 10ml of concentrated nitric acid (65%, Merck, Germany) was added to 1ml of serum in a beaker and heated gently below the boiling point
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for 3 hours, on a hot plate. When the volumes of the samples reduced to about one third, 5 ml of 30% hydrogen peroxide (30%, Merck, Germany) solution was included and heated almost to dryness at the same temperature. Finally, the residues were dissolved in 50ml of 1% nitric acid (65%, Merck, Germany) and were filtered.
The prepared solution was then added to 50ml polyethylene tubes for trace element analysis. To measure serum copper, zinc, calcium, lead and iron, Flame Atomic Absorption Spectrometry
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(FAAS) (model WFX-210, Rayleigh, China), and to measure selenium and Arsenic Hydride
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Generation Atomic Absorption Spectroscopy (HGAAS) (model WFX-210, Rayleigh, China) were used. The levels of elements were determined after 2, 4 and 6 months of anti-TB treatment
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levels of trace elements illustrated in Table 1.
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initiation in TB infected groups. The main parameters of instruments used for measuring serum
Statistical analysis
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Serum concentrations of trace metals were described by mean and standard deviation. Quantitative
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differences during the follow up time in both patients and healthy controls were determined according to repeated measure and Friedman tests adjusting for potential confounders such as baseline measurements as well as gender of the patients. SPSS software was applied for statistical
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analysis. We have used SPSS-20 software (SPSS Inc., Chicago, IL, USA) to analyze data with considering P value less than 0.001 significant statistically.
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Elements reference ranges: The normal reference ranges of blood materials were considered 55–160 µg/dL in men and40–155
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µg/dL in women for iron.[16] The normal range of Lead is <10μg/dL in children and <25μg/dL in adults.[17] The total and free serum copper are normally 63.7-140.12 μg/dL and 10-15μg/dL respectively.[18,19] The normal total and free serum calcium are 9.5-10.5mg/dl and 5.1-5.9mg/dl in children and 8.9-10.1mg/dL and 4.8-5.7mg/dL in adults respectively.[20] This range is 0-12 ng/mL for arsenic and 70 to 150 ng/mL for selenium and 66 to 110 μg/dL for zinc.[21,22] 4
Quality control:
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We had used certified reference materials in the same scale of our samples for quality control in our project. We had used BCR® which is a registered trademark of the European Commission from joint research center. The reference value BCR-637 was considered for zinc and selenium which were equal 81±7 µg/L and (1.11±0.22)*103 µg/L respectively. We had used BCR-635 for lead which was equal 210±24 µg/L and BCR-304 for calcium which was equal 2.201±0.019mmol/L in human serum. No certified references material was available for iron, copper and arsenic in The JRC-Geel (Joint Research Center) which is including IRMM, BCR and ERM.[23]
RESULTS
Totally 180 tuberculosis infected patients with the age ranged between 17 and 87 years were included in this study. Out of all TB patients, 182 cases (50.5%) were males and 178 cases (49.5%) kg
/m2 and 21.04
kg
/m2,
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were females. Mean BMI for patients and healthy group were 21.3
respectively. Tuberculosis patients were followed for six consecutive months.
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As illustrated in table 2, the iron level was in mid normal range in healthy individuals and was
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near lower limit of normal range in TB patients at treatment initiation whereas it got increased by
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the time during anti-TB treatment. Selenium level was near lower limit in health group but below normal range in TB infected patients. The selenium level had been increased during treatment till normal range at treatment final. Lead levels were normal in both groups but almost two times
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higher in TB infected group. Copper levels were normal in both groups. It was lower in healthy
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individuals in comparison with TB patients. Copper level got decreased in TB group during treatment. Zinc level was normal in healthy group and below normal in TB patients. It got
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increased by complete treatment to normal range. The calcium level was higher than normal range in both groups. Arsenic level was normal in both groups but higher overall in TB infected group.
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Moreover in the first and second measurement, the mean serum zinc, selenium and iron were lower significantly in tuberculosis patients compared to control group (P<0.001), in contrast serum arsenic, lead and copper were higher in the patients significantly(P<0.001).
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Serum concentrations of iron, selenium and zinc were increased significantly in six months after the treatment initiation in both groups (p<0.001) but this increase was higher among patients comparing healthy individuals significantly (p<0.001). In contrast lead serum level decreased after six months in both groups significantly (p<0.001). Yet this reduction was higher among TB patients in comparison with patients (p<0.001). Copper and arsenic were significantly dropped in patients while increased among healthy individuals (p<0.001). In both cases of Copper and arsenic, 5
the changes were significantly higher among patients. Finally, serum calcium concentration showed significant increase just in TB patients (p<0.001) (Fig. 1). We also observed that the
Cu
/Zn and
Cu
/Se ratios were significantly decreased (p<0.001) in TB
patients during six month treatment, while healthy individuals experienced significant increase in these ratios (p<0.05). After adjustment for the effects of baseline measurements of all elements,
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the observed effects were remained, except for iron which was similarly changed in both groups. (Fig. 1).
DISCUSSION
Tuberculosis is considered an important transmitted infectious disease in Iran and many other countries throughout the world.[24] In this study we have examined serum zinc, copper, arsenic,
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calcium, selenium, lead, and iron in both groups before treatment initiation. The serum concentrations of zinc, selenium and iron were lower and arsenic, lead and copper were higher in
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TB infected patients significantly before treatment initiation in comparison with healthy
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individuals.
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Numerous studies have revealed that zinc concentration in serum diminish extensively during the process of tuberculosis, and rise after anti-tuberculosis therapy initiation.[25]
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In a similar study Ciftci et al. demonstrated higher serum copper level, higher Cu/Zn ratio and lower serum zinc and selenium level in patients with active pulmonary tuberculosis in comparison with
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control group. They have also revealed that these elements level have been changed during treatment initiation.[26]
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Because of cell mediated immunity role in anti tuberculosis defense and the close association of zinc with this immune pathway, this metal plays a significant role against M.tuberculosis.[27,28]
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Lower serum zinc in TB patients could be related to its redistribution from plasma to tissues during infection.[29] Results of another study carried out by Arntsen et al. in Malaw showed lower serum selenium levels in tuberculosis patients compared with controls.[30] These results are in keeping
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with those observed in our study. The immune system and inflammatory responses are tightly associated with blood selenium levels. Sufficient body selenium causes resistance in front of many types of pathogens such as bacteria, parasite and fungi depending on involved pathogen variant.[15] Selenium is involved in selenoproteins structure, immune system regulation and oxidative response protection. The antioxidant activity of selenium can alter mycobacterium DNA. To avoid 6
DNA damage, M. tuberculosis has developed effective mechanisms to interrupt the activity of selenoproteins. The impaired selenoproteins can result in a significant selenium deficiency in TB patients.[31,32] Iron is a necessary nutrient which is extensively allocated in food. Like selenium and zinc, serum Fe levels were drastically lower in TB patients than healthy subjects. A possible explanation is that
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multiplication of M. tuberculosis within host macrophages depends on the presence of iron.[33] Hence, the iron deficiency that we observed in TB infected patients could be due to the mycobacterial iron consumption . Our findings showed that although iron levels were changed during treatment in TB patients, but its decrease pattern was same in both case and control groups. The immune system requires copper to perform several functions such as fighting microorganisms. In addition, copper is critical for nervous system function, Iron transport, cholesterol and glucose
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metabolism.[34,35] Because of essential role of copper in iron absorption, elevated copper level is
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seen in patients with active tuberculosis. Copper may interfere with iron absorption by binding to mucosal transferring.[36,37] Similarly, Frediani1et al. in Georgia measured serum iron level in
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adults with pulmonary tuberculosis, and found lower Fe concentrations.[38]
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Like copper, most of the patients had high serum levels of lead. In line with this, Afridi et al. indicated significantly higher levels of Pb in TB-HIV co-infected patients, compared with control
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subjects. Lead can interrupt antibody productions, and affects the lymphocytes.[39] Increased
CONCLUSION
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serum concentrations of lead may help Mycobacterium tuberculosises caping immune system.
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We found significant increases in the levels of Zn, Fe, calcium and Se, as well as decreases in the Cu, Pb, and As levels during the anti-TB treatment. There is a logical explanation for these
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alterations during anti-TB therapy. After starting chemotherapy, the number of active mycobacteria drops significantly. This causes considerable changes in serum concentrations of trace elements so that the values become closer and closer to those values of the healthy controls.
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During or after beginning of anti-TB treatment the circulating Zn, Fe, and zinc start increasing in order to enhance the availability of these vital micronutrients for body. However serum concentrations of Cu, Pb, and As decreases after anti-TB therapy due to their toxicity to cells. One of the limitations of our study was that we did not consider a time period for measuring serum concentration of trace elements in healthy controls.
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In conclusion, our study revealed that anti-TB treatment not only removes the microbial agents, but also is effective in serum trace elements level control. Therefore using supplementary agents and controlling trace elements levels in blood during tuberculosis treatment could cause faster, more efficient and successful treatment. Further studies with various control groups should be done to detect the exact effects of elements in tuberculosis treatment.
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Declaration of Conflicting Interests Authors declare no potential conflicts of interest.
Acknowledgments
The authors would like to thank all who participated in this study and specially the Amiralmomenin Hospital of Zabol University of Medical Sciences, tuberculosis and addiction withdrawal centers of Zabol, Hirmand and Hamoun.
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Table1. The main instrument parameters
Flame atomic absorption spectrometry (FAAS) Wavelength(nm)
Slit
Lamp
Burner
Acetylene
Air flow
width
current
Height
flow (L/min)
(L/min)
(nm)
(mA)
(mm)
324.8
4
10
1.70
13.5
213.9
1.0
4
10
2.00
13.5
0.5
Fe
EP
TE
Element
248.3
0.2
8
10
2.50
13.5
Pb
283.3
0.7
4
10
2.5
13.5
Cu
CC
Zn
A
Hydride Generation Atomic Absorption Spectroscopy(HGAAS)
NaBH4 concentration
0.5%
NaOH concentration
1M
NaOH/NaBH4 flow-rate
0.33 ml min-1
HCl concentration
5%
HCl flow-rate
0.33 ml min-1
10
Sample flow-rate
0.33 ml min-1
Wavelength(nm) for As
193.7 nm
Wavelength(nm) for Se
196.0 nm
Table 2: Serum levels of trace elements six months study among subjects with and without tuberculosis
s
Treatment
Healthy
phase
TB patients
changes during time between
Normal
SC RI PT
Element
individuals
two groups
Range
Adjustment
Adjustment for
for gender
all baseline
and baseline
elements
measure of the same
Before
78.80(3.31)
52.26(4.47) 3
79.29(3.29) 1
62.55(9.72) 13
months After four
79.56(3.23) 1
months
79.15(3.29) 1
Before
76.81(7.11)
42.38(3.81)3
76.96(6.32) 2
45.40(4.53) 13
m
months
After four
155(160)
80.07(3.17) 1 3
D
TE
After two
EP
Seleniu
<0.0001
40(55)-
72.65(8.33) 1 3
After six months
treatment
0.21
M
Iron
A
After two
N
treatment
U
element
0.003
70-150
<0.001
<25
<0.0001
77.45(5.71) 1
63.33(7.04) 13
After six months
75.74(5.86) 1
70.61(7.33) 13
Before
10.71(0.90)
19.32(1.94) 3
9.55(0.89) 1
18.99(2.32)13
A
CC
months
Lead
treatment After two
<0.0001
months After four
9.81(0.88) 1
22.15(2.40) 13
9.42(0.59) 1
18.25(1.87) 13
months After six months
11
Before
91.03(5.84)
119.07(7.50) 3
93.06(5.61) 1
113.60(6.49) 1
treatment
Copper
months
92.44(5.57) 1
109.47(5.59) 1
months
3
After six months
93.70(5.54) 1
99.72(5.14) 1 3
Before
94.94(8.60)
58.48(5.72) 3
94.92(8.42)
63.78(6.39) 1 3
treatment After two
96.12(8.33) 1
76.03(9.45) 1 3
After six months
95.60(8.39) 1
86.86(6.78) 1 3
Before
13.07(0.92)
12.21(1.15) 3
After two
12.56(0.99) 1
0.004
8.9-10.1
<0.001
0-12
A
12.05(1.19) 1 3 <0.0001
D
months
M
treatment
N
months
After four
13.12(1.37)
TE
months
66-110
<0.0001
months After four
Calcium
<0.001
U
Zinc
70-140
<0.0001
3
After four
0.05
SC RI PT
After two
13.02(1.27)
EP
After six months
Before
11.27(1.40) 1 3 13.64(1.65) 1 3
3.42(0.58)
8.35(0.43) 3
3.74(0.60) 1
7.66(0.48) 1 3
treatment
CC
After two
Arsenic
A
After four
Cu/Zn
3.85(0.29) 1
6.12(0.45) 1 3
After six months
3.60(0.18) 1
4.72(0.43) 1 3
Before
0.97(0.11)
2.06(0.25) 3
months
treatment
12
<0.0001
months
<0.0001
After two
0.99(0.11) 1
2.06(0.25) 3
0.97(0.10) 2
1.80 (0.21) 1 3
After six months
0.99(0.11) 1
1.15(0.11) 1 3
Before
1.19 (0.12)
2.83 (0.31)
1.21(0.11) 1
2.53 (0.29) 1 3
<0.001
months After four
SC RI PT
months
treatment After two
Cu/Se
0.004
<0.0001
months After four
1.20 (0.11) 1
1.75 (0.22) 1 3
1.24 (0.12) 1
1.43 (0.16) 1 3
months After six months
U
1: Significantly different from baseline in the same group (p<0.001)2: Significantly different from baseline in the same group (p<0.05) 3: Significantly different from healthy group in the same phase of treatment
N
*The evaluation unites for elements are µg/dl for iron, lead, copper and zinc. It is ng/ml for selenium and arsenic. The unit of calcium concentration is mg/dl.
A
Figures
M
Fig. 1. Serum trace element levels in control and patient groups; copper (A), Calcium (B), Zinc (C), Selenium(D), Arsenic (E) ,Lead (F), Iron (G) as well as Serum Cu/Zn and Cu/Se ratios; (*)
D
significantly different from baseline in the same group (p<0.001), (***)significantly different
A
CC
EP
TE
from healthy group in the same phase of treatment
13
14
D
TE
EP
CC
A
SC RI PT
U
N
A
M
15
D
TE
EP
CC
A
SC RI PT
U
N
A
M