Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
835
HEAVY METALS IN SERA OF PATIENTS WITH HEPATITIS B
DETERMINATION AND ANALYSIS
Wahran M.Saod*,Tahseen .A. Zaidan *, Abdul Wahab .A. Alfaluji **
*Department of Chemistry, college of Science, University of Anbar, Ramadi, IRAQ
** Fallujah teaching hospital, Fallujah, IRAQ
Keywords: Hepatitis, B , Heavy Metals and ICP-MS.
Corresponding author Email:[email protected]
ABSTRACT
Chronic Hepatitis B is a major global health problem worldwide, However, two
Billion People could be infected with this disease Worldwide. Living organisms
require varying amounts of heavy metals. Such as manganese(Mn), cobalt(Co) and
zinc(Zn). Some heavy metals have bio-importance, but the bio toxic effects of many
of them in human biochemistry are of great concern. Heavy metals are essential
micronutrients involved in many metabolic processes. One hundred sixty patient
samples and thirty healthy individuals were investigated in this study. All patients
included in this research are tested positive for the HBSAg test and their specimens
were examined by profile test which includes (HBS Ab, HBe Ag, HBe Ab, HBc Ab
,(IgM-IgG), HCV and HIV) and also viral load (HBV PCR) with unit (copies/ml)
were carried to test the patients sera. Manganese, chromium, cobalt, and nickel have
levels were determined by using inductively Coupled plasma- Mass spectrometry
(ICP-MS). Results show a significant increase in the concentration of manganese,
chromium, cobalt, and nickel in serum of patients with chronic hepatitis B compared
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
836
to healthy individuals. We conclude that hepatitis B may affect the level of studied
elements in patients infected with HBV.
INTRODUCTION
Hepatitis B is an infectious disease caused by the hepatitis B virus (HBV) that
affects the liver. The clinical result of infection is related to immune response (8). The
disease may develop to liver carcinoma during infection. An estimated 350 million
people worldwide are chronically infected with HBV (19). In the United States, there
are about 1.25 million hepatitis B carriers and it is estimated that approximately 75%
of the hepatitis B carriers live in Asia and Africa (1). Many studies showed that the
suffusion of HBV infection has reduced from 4.1% to less than 1% in the period of
the form 1970 to 1990 In Iraq (5, 3).
Heavy metals could adversely affect the male reproductive system, either by
causing hypothalamic-pituitary axis disruption or by directly affecting
spermatogenesis, resulting in impair semen quality (27) . The main problem with
heavy metals in our bodies is their ability to bio-accumulate (21). The metals will not
be cleared by the system unless some type of intervention is used to chelate the metals
and flush them out. There are some heavy metal toxicities that are more prevalent
than others, like mercury from dental fillings and lead from lead pipes and paint (13).
Manganese is located mainly in the mitochondria. It is a constituent of many
important metalloenzymes like superoxide dismutase, pyruvate carboxylase, arginase,
and glycosyltransferase. It is absorbed in small intestine, then bound to albumin in
circulation and is transported to the liver and excreted in bile (6). Manganese is an
antioxidant nutrient and is important in the breakdown of amino acids and the
production of energy. It is essentially required for the metabolism of vitamin B1, C,
and E and for the activation of various enzymes which are important for proper
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
837
digestion and utilization of foods (25). Chromium is an essential nutrient required by
the human body to promote the action of insulin in body tissues, so that the body can
use sugars, proteins, and fats (22). In humans, systemic effects have been reported to
occur in the airways, cardiovascular system, kidneys and liver (16). Cobalt is required
as a constituent of vitamin B12 (22). In addition to its role in vitamin B12, Cobalt is
also a cofactor of enzymes involved in DNA biosynthesis and amino acid metabolism
(4). Nickel is an essential trace element for many species (2). nickel is found in blood
and tissues at consistent levels and is also associated with DNA and RNA in amounts
that suggest physiological significance. Nickel is required for normal growth and
reproduction in animals, and human beings as well. It has a role in the modulation of
the immune system and in development of the brain (10).
The aim of this study is: to compare the level of heavy metals in sera of patients
infected with HBV and healthy participants.
MATERIALS AND METHODS
. Materials
A 150 μL aliquot of serum samples were thawed at room temperature for 20
minutes and pipette into pre-cleaned polyethylene tubes. 150 μL of optima grade
nitric acid and 100 μL of trace select grade hydrogen peroxide were then added to the
samples. The tubes were tightly capped, centrifuged for 10 minutes at a speed of 4400
r/min in a centrifuge. Samples were then placed in a hot block digester and digested at
95 °C for 90 minutes with the tube caps loosened. Following digestion the samples
were diluted to 150 μL(12) .
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
838
.Procedure
Multi element working standards were prepared containing (Mn ,Cr, Co and Ni) by
diluting high purity 1000mg L-1 stock solutions with deionized water and nitric acid.
The standard solutions were kept at 4 C0 in dark room to reduce the risk of
contamination. All works were carried out under clear room conditions. Ultra-pure
de-ionized water was used for sample preparations. Standard solutions were prepared
freshly from the stocks, with dilute nitric acid (5% v/v). In order to obtain appropriate
. ICP-MS responses, the experiments were performed using different concentration
levels.
. Samples analysis
The digested serum samples were analyzed for (Mn, Cr, Co and Ni) using
Inductively Coupled plasma- Mass spectrometry (Agilent Technology¸ Japan). The
analytical calibration method was accomplished with aqueous standards in 0.5% (v/v)
HNO3. Fresh calibrations were made each time before analysis.
.Statistical Analysis
The Pearson correlation test was used to correlate between different variables among
the studied groups (26) .
RESULTS AND DISCUSSION
. Prevalence of Chronic Hepatitis B
The record of patients and healthy group shows a difference in disease incidence in
rural and urban regions. There is an increase in disease’s incidence in rural areas (114
patients) as compared to the urban regions (76 patients) as shown in (Table 1).
This finding can be attributed to lack of the health awareness and because the patients
have not been vaccinated with hepatitis B vaccine. details of patients and health
groups are shown in (Table 2).
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
839
Table (1) List of Symbols and details for study groups .
Table( 2) gender and residence distributions of different groups for HBV patient
and healthy individuals
Manganese
Serum concentration of manganese was significantly higher (0.94±0.12μg\dl) in
chronic hepatitis B patients in comparison to controls (0.77±0.08 μg/dl), as in
fig.(1A). Serum concentration of manganese was significantly higher
(0.94±0.12μg\dl) in chronic hepatitis B patients in comparison to controls (0.77±0.08
μg/dl), as in fig. (1A). study concurs with (23) who reported that serum manganese
concentration increased in patients during the active phase of acute hepatitis, chronic
Symbols Details Numbers
1A Patients HBeAg (+ve)normal liver function PCR= (>100000) 36
2A Patients, HBeAg (+ve)abnormal liver function PCR= (>100000) 28
1B Patients HBeAg (-ve) PCR= (>100000) 30
2B Patients HBeAg (-ve) PCR=4000-100000 36
3B Patients HBeAg (-ve) PCR=N0N 30
C healthy individuals 30
Parameters 1A 2A 1B 2B 3B C Total
Male
20 15 15 17 17 21 105
Female
16 13 15 19 13 9 85
Urban
9(25%) 8(28.5%) 9(30%) 18(50%) 16(53.3%) 16(53.3%) 76
Rural
27(75%) 20(71.4%) 21(70%) 18(50%) 14(46.6%) 14(46.6%) 114
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
840
hepatitis and post-hepatitis cirrhosis and the study by (20) who reported that serum
manganese may be caused from two reasons, necrosis of liver parenchyma, leading to
an important release of hepatic Manganese or decreased hepatobiliary excretion(7).
.Chromium, cobalt and nickel
The results showed that the mean serum chromium level were significantly
higher(28.11±2.68 μg\l) in hepatitis patient than in healthy individuals (22.56 +2.54
μg\l) , fig. (1B) This denotes that risk of exposure to chromium was associated with
all liver diseases, but fatty liver cases were the ones that were more exposed. These
results may be in part due to high initial levels and in another part to liver affection by
hepatitis or schistosomiasis. It was claimed to be hepatotoxic (9).
The mean of serum Cobalt level in hepatitis patients was significantly
higher(1.81±0.34 μg\l) than in healthy individuals1(11±0.17 μg\l) as in fig. (1C).
Cobalt is a metal that is required by the body for blood formation. It is an integral
part of vitamin B12, this vitamin needful for producing red blood cells and
maintaining the nervous system. Cobalt activates many metabolic enzymes (17). our
study agreement with (15) who studied cobalt in serum patients and control group
and found that the mean concentration of serum cobalt in control group was less than
patients
The mean of serum nickel level (5.35±0.94 μg\l ) showed significantly higher in
hepatitis patients than in the healthy individuals (4.18±0.54 μg\l) as in fig.(1D).Our
result disagreement with (17) result is very high when compared to our findings.
Nickel is a trace element, required in minute quantities by the human body. Enzymes
containing Nickel have not been found, though Nickel functions to activate or inhibit
enzymes containing other elements. The study (24) found that level of nickel higher
in early and advanced stages of hepatic cirrhosis. The Study (18) suggested that the
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
841
diminution of serum nickel in hepatic cirrhosis may reflect diminished concentrations
of serum nickel oplamin and albumin. nickel oplamin and albumin may play an
essential physiological role that includes a Nickel-containing protein ‘‘Nickel
oplasmin’’inhuman serum (14).
FIGURE 1A. level of manganese concentrations in hepatitis B patients and control
group. 1B. Serum chromium concentration in in hepatitis B patients and control
group. 1C. Serum Cobalt concentration in in hepatitis B patients and control group.
1D. Serum Nickel concentration in in hepatitis B patients and control group.
A B
D E
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
842
CONCLUSIONS|
our study was elucidated that levels of manganese, chromium, cobalt, and nickel
were higher in chronic hepatitis B patients as compared to control group. ICP-MS
method is an efficient technique in the detection of heavy metals concentration.
تقدیر وتحلیل B المعادن الثقیلھ فی مرضى التھاب الکبد الفایروسی
وھران منعم سعود* ،تحسین علی زیدان *،عبد الوھاب الفلوجی**
*قسم الکیمیاء،کلیھ العلوم ،جامعة الانبار ، ألانبار ،العراق.
**مستشفى الفلوجھ التعلیمی ،الانبار ، العراق.
الخلاصة
المزمن مشکلة صحیة عالمیة، حیث أن ھناک ملیارین أنسان عرضة B یمثل مرض التھاب الکبد الفایروسی
للاصابة . تحتاج الکائنات الحیة الى کمیات متغیرة من العناصر الثقیلة مثل المنغنیز والکوبالت والزنک. تمتلک
بعض الفلزات الثقیلة أھمیة حیویة ولکن العدید منھا ذات تأثیرات سمیة مقلقة جدا على الأنسان . تکون
العناصر الثقیلة مغذیات دقیقة تدخل فی العدید من العملیات الأیضیة. مئة وستون مصابا وثلاثون فردا غیر
مصابا استخدمت کعینة فی ھذه الدراسة.
وتم فحص عیناتھم من خلال (HBSAg) تم اختبار جمیع المرضى المشمولین فی البحث بإیجابیة لفحص
(HBS Ab , HBe Ag , HBe Ab , HBc Ab , (IgM-IgG) , HCV,HIV) الاختبارات المتضمنة
بوحدة (نسخ / مل) التی اجریت لجمیع المرضى. (HBV PCR) وکذلک الحمل الفایروسی
تم قیاس مستویات المنغنیز والکروم والکوبالت والنیکل بواسطة مطیاف الکتلة-البلازما المزدوج. تظھر النتائج
زیادة مھمة فی ترکیز المنغنیز والکروم والکوبالت والنیکل فی مصل دم المرضى المصابین بالتھاب الکبد
ربما یؤثر على مستوى B مقارنة مع الأفراد الأصحاء. نستنتج أن مرض التھاب الکبد المزمن B المزمن
.HBV العناصر الثقیلة فی المرضى المصابین ب
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
843
REFERENCE
1. Alavian, SM.(2006). Immunization: an important strategy to control hepatitis B.
Hepat Mon . vol. 6 (1), pp.; 3 – 5.
2. Alexandrovn, R., Costisor, O. and Patron, L. (2006) . Nickel. Pathology and
parasitology . vol. 911, pp ; 64 – 74.
3. Al-Juboury, F., Salih, A., Al-Assadi, K. (2010) . Seroprevalance of hepatitis B
and C among blood donors in Babylon Governorate- Iraq. Med J Babylon.vol.7:1–2.
4. Arinola, OG. (2008) . Essential Trace Elements and Metal Binding Proteins in
Nigerian Consumers of Alcoholic Beverages . Pak. J. Nutr . vol, 7(6), pp ; 763 -765.
5. Ataallah, M., Hanan, A., Maysoun, S. (2011) . Prevalence of hepatitis B and C
among blood donors attending the National Blood Transfusion Center in Baghdad,
Iraq from 2006–2009. Saudi Med. J; 32:1046–50 .
6. Berdanier, D. (1998) .Trace Minerals. In: Wolinsky I and Hickson JF eds.
Advanced Nutrition. Micronutrients New York. CRC Press, pp ; 224-225.
7. Chaturvedi, UC., Shrivastava, R. and Upreti, RK . (2004) .Viral Infections
and Trace Elements: Acomplex Interaction .CurrSci, vol. 87(11), pp:54 – 1536,.
8. Das, A. and Maini, K .(2010) . Innate and Adaptive Iimmune Responses in
Hepatitis B Virus Infection. Dig Dis Sci , Vol. 28 (1) , p: 32 - 126.
9. Das, P., Santos, M ., Pereira, L ., Jesus, P. (2005) . Comparative histological
studies on liver of mice exposed to Cr(VI) and Cr(V) compounds. Hum Exp Toxicol;
21(7): 365-9(2002). Public Health Association (JEPHAss.), 80:.3-4.
10. Dunnick, K., EIwell, R., Eadovsky, E., Benson, .M., Hahn, .F., NikuIa, .J.,
Barr, B, and Hobbs, H. (1995) . Comparative Carcinogenic Effects of Nickel
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
844
Subsulfide, Nickel Oxide, or Nickel Sulphate Hexahydrate on Chronic Exposures in
The Lung. AACR. 55: 5251-5256.
11. Farell, J. (2010). Digestion and Absorption of Nutrients and Vitamins.
Gastrointestinal and Liver Disease. 9:7 - 423.
12. Gang L., John. B., Shih,. L., Christian A., Lance S., David R. J. ( 2012)
.Measurement of the Trace Elements Cu, Zn, Fe, and Mg and the Ultratrace Elements
Cd, Co, Mn, and Pb in Limited Quantity Human Plasma and Serum Samples by
Inductively Coupled Plasma-Mass Spectrometry. American Journal of Analytical
Chemistry. 3, 646-650.
13. Janine, B. (2005) . Heavy Metal Toxicity-An Unsuspected Illness.
http://www.janinebowring.com.
14. Himmelhoch, R ., Sober, A., Vallee, L.,Peterson, A .and Fuwa, K.(1966).
Spectrographic and Chromatographic Resolution of Metalloproteins in Human Serum
. Biochemistry.5: 2523.
15. Khan, H. and Qayyum, K .(2002) .Determination of Trace Amounts of Iron,
Copper ,Nickel ,Cadmium and Lead in Human Blood by Atomic Absorption
Spectrometry .PakJBiol Sci . 5(10): 7- 1104.
16. Langard, S., Friberg, L. and Norseth, T. (1986) . Chromium. In: ed.
Handbook on The Toxicology of Metals, 2nd ed . Elsevier/North-Holland Biomedical
Press . 2:185–210,
17. Lin.1., Huang, F., Tsai, Y. and Huang, L. (2006). Selenium, Iron, Copper, and
Zinc Levels and Copper-to-Zinc Ratios in Serum of Patients at Different Stages of
Viral Hepatic Diseases . Biol Trace Elem Res. 109(1):15-24.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
845
18. Mcneely, D., Sunderman, W., Nechay, W. and Levine, H . (1971). Abnormal
Concentrations of Nickel in Serum in Case of My Ocardialinfection, Stroke, Burns,
Hepatic Cirrhosis an Duremia . ClinChem. 17(11) : 8 - 1123.
19. McQuillan, M., Coleman, J., Kruszon, D., Moyer, A. and Lambert, B.
Margolis, S.(1999).Prevalence of Hepatitis B Virus Infection in The United States.
Am J Public Health.89:14-18.
20. Rashed, N., Mohamed, A., Farouk , A. and Mahmoud, S. (2010) . Trends in
Speciation Analysis of Some Heavy Metals in Serum of Patients With Chronic
Hepatitis C and Chronic Hepatitis B Using Differential Pulse Adsorptive Tripping
Voltammetric Measurement and Atomic Absorption Spectrophotometry . J Trace
Elem Med Biol . 24: 45 -138.
21. Sharma, K . and Agrawal, M . (2005) . Biological Effects of Heavy Metals : An
Overview . J. Environ Biol. 26: 301-13.
22. Shrivastava, R., Upreti, K., Seth, K. and Chaturvedi, C . (2002). Effects of
Chromium on The Immune System . Med. Microbiol, 34:1-7.
23. Versieck, J ., Barbier F., Speeke, A . and Hoste J. Mangaese .(1974)
.Copper and Zinc Concentrationsin Serum and Packed Blood Cells During Acute
Hepatitis, Chronic Hepatitis and Posthepatitic Cirrhosis .ClinChem . vol. 20(9), pp : 5
– 1141,.
24. Volini, F ., dalaHuerga, J ., Kent, G . (1968) . Trace Metal Studies in Liver
Disease Using Atomic Absorption Spectrometry in Laboratory Diagnosis of Liver
Diseases In: Sunderman ,W., SundermanJr, W. Laboratory diagnosis of liver
diseases. St. Louis, MO: Warren H. Green, Inc.; p. 199.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
846
25. Wang, M., Howell ,J. M., Libbey,E., Tainer, A.and Fujinami,S. (2003).
Manganese Superoxide Dismutase Induction During Measles Virus Infection . J. Med.
Virol. vol. 70, pp ; 470–474.
26. Williams, B. (1993) .Biostatistics Concepts and Applications for Biologists. first
ed., Chapman & Hall/CRC, London.
27. Wyrobek, J .,Schrader, M., Perreault , D., Fenster, L., Huszar, G ., Katz, F. ,
Osorio, M ., Sublet,V . and Evenson . (2010). Assessment of Reproductive
Disorders and Birth Defects in Communities Near Hazardous Chemical Sites. Fertil
Steril. 93:130-140.
