Figure 1: Immunoblots of 2.5 µg native full length human cardiac troponin I (cTnI).

A: Human cardiac troponin I identified as a methylated protein by anti-mono and dimethyl arginine antibody (α-ArgMe).  B:  Human cardiac troponin I recognised by anti-human cardiac troponin antibody (α-cTnI). Images are representative of two experimental repeats

MALDI/MS identified multi methylation peaks in the synthetic peptide of the inhibitory region of cTnI MALDI analysis of the peptide showed a peak at 1785.097 corresponding to the peptide and another peak corresponding to the sodium adduct. When this peptide was incubated with S-adenosyl methionine and protein arginine methyl transferase 1 (PRMT1) for 6 hours and a MALDI analysis done, more peaks were obtained at 1799.132 corresponding a methylated product of the peptide. Another peak (although weak) was observed at 1813.140 potentially corresponding to the dimethyl derivative of the peptide. These are in addition to other peaks at 1807.099 and 1821.111 potentially corresponding to the sodium adducts of the peptide and sodium adduct of the methylated peptide,  See figure 2.

Figure 2: MALDI-TOF mass spectrum of wild type peptide at 1785.09 Da  Upper panel: GKFKRPTLRRVRISA + SAM showing peaks of unmodified peptide at 1785.116 Da and peptide + sodium adducts at 1807.008 Da.

Lower panel: GKFKRPTLRRVRISA + SAM + PRMT1. Showing unmodified peptide peak at 1785.097 Da, monomethylated peptide peak at 1799.132, dimethylated peptide at 1813.140, peptide + sodium adduct peak at 1807.099 Da and sodium adduct + monomethylated peptide at 1821.111 Da. Images are representative of at least three experimental repeats.

MALDI/MS/MS identified the arginine methylated groups on the peptide, an MS/MS of the peptide was residue in the synthetic peptide of the inhibitory region conducted as a base line to study the fragmentation of the of cTnI ions in the methylated products, the mass of the observed

In order to identify the position of the addition of the methyl   ions are shown below, see figure 3, table 1.

Figure 3: MS/MS of m/z 1785.097 = GKFKRPTLRRVRISA. Fragmented unmodified peptide showing the b & y-ion series. Images are representative of at least three experimental repeats.

Table 1. Mass of fragmented GKFKRPTLRRVRISA peptides at m/z 1785.09

Mass of peptides resulting from fragmentation of unmodified peptide with respect to a, b & y-ion series, Mascot score: 62; Expect: 9.7 E-06. Note only the mass of observed fragments is included. Empty cells mean that the corresponding ion was not observed.

When the peak at 1799.132 was fractionated into the component ions, it was possible to identify mono-methylation of arginine at position 10 (y6-ion) (GKFKRPTLRR*VRISA) in the MS/MS spectrum of peptide at m/z 1799.132, see figure 4, table 2. This arginine residue corresponds to R146 of cardiac troponin I protein. There was a +14 Da increase in mass of y6ion which was propagated through the rest of the spectrum, such that successive y-ions had +14 Da increases in the mass of fragments with respect to unmodified peptide in figure 3.

Figure 4: MS/MS of m/z 1799.11 = GKFKRPTLRRVRISA. Fragmented modified peptide showing the b & y-ion series. Images are representative of at least three experimental repeats

Onwuli, Donatus Onukwufor 

Table 2. Mass of fragmented GKFKRPTLRRVRISA peptides at m/z 1799.132 However,

Fragmented modified peptide at 1799.132 Da showing a, b & y-ion series. Note +14 Da shift in y-ion series from y6, compared to unmodified peptide in table1. Mascot score: 42; Expect: 0.00095. Note only the mass of corresponding observed fragments is included. Empty cells mean that the corresponding ion was not observed. Highlighted in bold signify diagnostic ions.

Figure 5. MS/MS of m/z 1813.140 = GKFKRPTLRRVRISA. Fragmented modified peptide showing the b & y-ion series. Note the weak b11-H2O diagnostic ion. Images are representative of at least three experimental repeats

Table 3. Mass of fragmented GKFKRPTLRRVRISA peptides at m/z 1813.140

 Fragmented modified peptide at 1813.140 Da showing a, b & y-ion series. Mascot score: 19, Expect: 0.014. Only the mass of corresponding observed fragments is  included. Empty cells mean that the corresponding ion was not observed. Highlighted  in bold signify diagnostic ions.

The post Human Cardiac Troponin I is Post Translationally Modified by Arginine Methylation. appeared first on Nigerian Biomedical Science Journal.

Adejumo BabatundeIshola Gabriel and Abhulimhen Godwin
Medical Laboratory Science Department, University of Benin, Benin City, Nigeria.

All Correspondences to: babatunde.adejumo@uniben.edu,bigadejumo@yahoo.com

ABSTRACT

Background: There are lots of documentary evidence to showthedeleterious effects occasioned by the inhalation of cement dust on human health. Aim: This work is aimed atevaluating the effects of occupational exposure of cement dust on the level of copper and zinc in selected male subjects exposed to cement dust and compare them with those of the nonexposed group in Benin City.  Methods: A total number of eighty-six (86) malescomprised of 46 exposed subjects and 40 unexposed subjects participated in this study. They were recruited for this study from various construction sites in Benin City. Serum level of zinc and copper were assayed using flame atomic absorption spectrophotometer. Results: Theresult indicates that the level of copper of the exposed group (126.24 µg/dl) was significantly higher than the level of the control group (71.72 µg/dl), (P<0.001). However, this was not the case with the other micronutrient, zinc, although, there was also a statistically significant difference in its level between the exposed group (116 µg/dl) and the control group (145.52 µg/dl) P-value of>0.005. Conclusion: In this study, higher level of copper was recorded among people who are exposed to cement dust compared to the controls. However, the serum level of zinc was lower in the exposed group compared to the controls. The use of hand gloves, face masks, protective foot wear and gowns will help in reducing the entrance of these micro elements into the body. Government should increase advocacy on the need to take micronutrient supplements among this group of people to boost their immunity.
Keywords : copper, zinc, cement dust, Benin city.

INTRODUCTION

Cement is the most widely used building material throughout the world. Unfortunately, cement industry is one of the 17 most polluting industries listed by the central pollution control board. The industry releases huge amounts of cement dust into the atmosphere which settle on the surrounding areas forming a hard crust and causes various adverse impacts (1). However, the destructive effects of exposure to cement dust constituents on human have been documented. Cement constituents include 60 -70%calcium oxide, 17 – 25% silicon oxide, and 3-5% aluminum oxide, with some iron oxide, chromium, potassium, sodium, sulphur and magnesium oxide(2)(3). Exposures to most of these metals have been associated with decreased lung function indicators (4)(5). Lipid peroxidation, oxidative damage, and immunological mechanisms are all pathological conditions cement dusts have induced their toxicities (6)(7). The effects of these toxicities can only be felt when their serum levels are altered from the normal physiological values (8).

Trace elements are inorganic compounds that are needed in minute amounts in our diets, for the performance and maintenance of the normal functions of the body. It has been documented that trace elements may play an important role in the diseases caused by viruses (9) (10). However, deficiency of these trace elements causes severe economic loss due to increased susceptibility to oxidative stress, growth retardation, anemia, decrease in feed efficiency and fertility, enhance the virulence of the infectious agent, and decrease immune system function (11).

Specifically, trace elements such as copper (Cu) and zinc (Zn), are essential in human nutrition and are needed in very small amounts for essential metabolic reactions in the body.  Copper is an essential trace metal which is a component of a wide range of intracellular metalloenzymes, including cytochrome oxide, superoxide dismutase, tyrosine, dopamine hydroxylase and lysyl oxidase; more than 75% of the copper is associated with specific copper binding protein like ceruloplasmin (12), moreover, 60% of copper in the blood is tightly bound to a copper-zinc-dependent enzyme known as superoxide dismutase (CuZnSOD) which is a powerful antioxidant. On the other hand, zinc is the second most abundant trace element in the human body. It is an essential element that is required in many very important biological processes when it is known to function as a cofactor in over 200 enzyme reactions and is known to be essential for the function of numerous transcription factors and nuclear regulatory elements(13). Zinc deficiency depresses the ability of the body to respond to infection, affecting both cell-mediated immune and humouralresponses (14). It has also been revealed that people of cement dust zone are badly affected by reproductive problems, respiratory Copper and Zinc Levels Among Occupationally…

problems, gastro intestinal diseases etc.  (15).In Benin City, Edo state, the exponential increase in demand for cement has resulted in the proliferation of cement factories, distribution outlets, blocks molding industries etc.

There are lots of documentary evidence on deleterious effects of metallic components of cement dust on human in Nigeria (3)(16)(17), but none has singled out the effects of pair of copper and zinc in workers who are exposed to cement dust in Benin City, hence the justification of this work.

2.1 Methodology

A total ofeighty-six(86) males within the ages of 18-60 years participated in this study, 46 are those who were exposed to cement dust, while 40 are control subjects.Participants who were unhealthy or have history of hypertension were excluded.They included cement distributors, bricklayers and block moulders.They were recruited from various construction sites at Ugbowo area ofBenin City. The controls are students and staff of University of Benin, Ugbowo campus who have never been exposed to cement dust.Their consents were sought after explaining the purpose of the research. Structured questionnaire was administered to each participant to obtaintheir demographic information. This study was approved by the ethical committee of Ministry of Health, Edo State and the leaders of all the construction sites.

2.2 Sample collection and processing

Five milliliters of blood was collected and dispensed into a plain container. The non-anticoagulated blood was allowed to clot for 1 hour, and thenspun at 1500rpm for 10minutes and the supernatant serum was separated into a separate sterile tubes. The serum was stored at -20⁰C for up to 2 weeks prior to analysis for copper and zinc using flame atomic absorption spectrophotometer (FAAS).

2.3 Data analysis

Data was expressed as mean and standard deviation. Comparative analysis was done using independent sample t-test and analysis of variance (ANOVA). Statistical significance was set at p < 0.05. All statistics were done using IBM/SPSS software (version 20.0).

RESULTS

Table 1 shows the demographic information of the participants. Table 1 shows that 30(65%) of the exposed participants are single while, 16(35%) are married. However, 20(50%) of the non-exposed are single, while

20(50%) are married. All the participants 86(100%) are Nigerians, with 5(11%), 10(22%), 30(65%), and 1(2%) represent Anambra, Delta, Edo, and other states of Nigeria respectively among the cement dust exposed participants. 10(25%), 29(73%), and 1(3%) are non-exposed from Delta, Edo and other states of Nigeria. 35(76%) of the exposed are frequent smokers while the rest 11(24%) are occasional smokers. However, 35(77%) of the controls are occasional smokers, while only 5(13%), are frequent smokers. 40(87%) and 6(13%) represent those who consume alcohol regularly and those who consume alcohol occasionally among those who are exposed to the cement dust, while 4(10%), consume alcohol frequently and 36(90%) occasionally among the controls. 40(87%), 32(70%), and 20(43%) experience body pains, chest pains, and weakness among the exposed group while none was recorded among the controls as part of their medical history. Also, there was no signs of nasal congestion, cough, and eye irritation in the control group, but, 41(89%), out of 46(100%) of the exposed group were currently experiencing cough, nasal and eye irritations respectively. All 46(100%) of the exposed group used pain relievers regularly, but none 0(0%) of them takes multivitamin supplements.6(13%), 2(4%) and 10(22%) wear face mask, hand gloves and foot wears, protective gowns respectively among the participants that are exposed to the cement dust

Table 2.Shows the mean serum levels of the copper and zinc among the participants. The result indicates that the levels of copper of the exposed group (126.24 ± 63.19 µg/dl) was significantly higher than the controls (71.72 µg/dl), (P<0.001). While serum zinc level is lower (116 ± 42.73 µg/dl) among the exposed group compared to the control group (145.52 ± 51.43 µg/dl), (P- value, >0.005).

Figure 1.Shows the bar chat of duration of exposure and the level of copper and zinc of the exposed group; indicating no statistical significance (P-values >0.05). Data show that the concentrations of zinc and copper rose sharply after first year of exposure. However, the concentrations remain constant between the two metals within 5 years of exposure. Furthermore, within 20 years of exposure, zinc level rose higher than copper. Meanwhile, the concentration of copper surpasses that of zinc after 20 years of exposure.

DISCUSSION

The deleterious effects of exposure to constituents of cement dust on human organs system have been well described. Based on this fact, there is need to investigate the effects of this dust on human total wellbeing. Serum level of copper and zinc of group of young men who were exposed to cement dust were estimated in this study. Table 1 shows the demographic information of the exposed participants, 30(65%) of the participants are single while, 16(35%) are married. However, 20(50%) of the nonexposed aresingle, while 20(50%) are married. All the Copper and Zinc Levels Among Occupationally…

participants 86(100%) are Nigerians, with 5(11%), 10(22%), 30(65%), and 1(2%) represent Anambra, Delta, Edo, and other states of Nigeria respectively among the cement dust exposed participants. 10(25%), 29(73%), and 1(3%) are non-exposed from Delta, Edo and other states of Nigeria. 35(76%) of the exposed are frequent smokers, while the rest 11(24%)  are occasional smokers. However, 35(77%) of the controls are occasional smokers, while only 5(13%), are frequent smokers. 40(87%) and 6(13%) represent those who consume  alcohol regularly and those who consume alcoholoccasionally among those who are exposed to the cement dust, while 4(10%),  consume alcohol frequently and 36(90%) occasionally among the controls. 40(87%), 32(70%), and 20(43%) experience body pains, chest pains, and weakness among the exposed group while none was   recorded among the controls as part of their medical history. Also, there was no signs of nasal congestion,cough, and eye irritation in the control group, but, 41(89%), out of 46(100%) of the exposed group were currently experiencing cough, nasal and eye irritations respectively. All 46(100%) ofthe exposed group used pain relievers regularly, but none 0(0%) of them takes multivitamin supplements.6(13%), 2(4%) and 10(22%) wear face mask, hand gloves and foot wears, protective gowns respectively among the participants that are exposed to the cement dust. This agrees with some workers (18) who reported the association of cement dust and respiratory discomforts such as cough, phlegm, and chest tightness and lung function indicators.

Figure 1 show the duration of exposure to the cement dust by the participants. Serum copper level increases with increasing time of exposure and reach the peak at over 20years of exposure while, highest serum zinc level was recorded after 6- 20 years of exposure. However, both metals recorded their lowest serum level at 2-5years of exposure. It is interesting to note that the serum levels of both metals rose sharply at the beginning of exposure (below 1 year).Continuous and prolong exposure to cement dust has been linked with decreased peak expiratory flow as reported by some authorities(19) (20) (21) (22).

In this study the level of copper was higher among the exposed subjects compared to the controls, while lower serum level of zinc was recorded among the exposed group compared to the controls (Table 2). This agrees with the work of Richard et al., 2016(23) in Mfamosing, Cross river state, Nigeria where they worked on cement factory workers. Increased level of copper has also been reported by other researchers among cement factory workers and the increment has been attributed to their exposure to cement dust (7) (24). The toxic effects of most of these metals depend on the absorption, concentration and continuous persistence at the action site. These metals react with endogenous target organs such as receptors, enzymes, DNA, proteins and lipids and altered their biological functional changes that results in toxic damage (25). Zinc and copper are the components of antioxidant enzyme superoxide dismutase (Cu – Zn SOD).  The lower zinc level among the exposed subjects may be due to low level of awareness on the need to regularly take multivitamin supplements and increased demand on the antioxidant system to buffer the deleterious effect of heavy metals. This may have accounted for lower zinc levels and a compensatory increase in copper level as seen among the exposed group compared to unexposed controls. The body in turn may want to conserve copper to combat heavy antioxidant demands (6).

CONCLUSION

In this study, higher level of copper was recorded among people who are exposed to cement dust compared to the controls. However, the serum value of zinc was lower in the exposed group compared to the controls. The use of hand gloves, face masks, protective foot wear and gowns will help in reducing the entrance of these micro elements into the body, which will in turn reduce organ damage occasioned by the absorption of these metals. Government should also increase level of awareness on the need to take multivitamin supplements among the exposed subjects to boost their immunity.

REFERENCES

1. Mehraj, S.S. Cement Factories, Air Pollution and Consequences. Indian Journal of the Department of Environmental Science & Centre of research for development. Universityof Kashmir, Jammu and Kashmir,2013; 190006: Pp. 6-39.
2. K. M. Fell, T. R. Thomassen, P. Kristensen, T. Egeland, and J. Kongerud, “Respiratory symptoms and ventilatory function in workers exposed to Portland cement dust,” Journal of Occupational and Environmental Medicine, vol. 45, no. 9, pp. 1008–1014, 2003.
3. M. Gbadebo and O. D. Bankole, “Analysis of potentially toxic metals in airborne cement dust around Sagamu, Southwestern Nigeria,” Journal of Applied Sciences, vol. 7, no. 1, pp. 35–40, 2007.
4. A. Baccarelli, Y. Zheng, X. Zhang et al., “Air pollution exposure and lung function in highly exposed subjects in Beijing, China: repeated-measure study,” Particle and Fibre Toxicology, vol. 11, pp. 51–60, 2014.
5. K. Zeleke, B. E. Moen, and M. Bråtveit, “Cement dust exposure and acute lung function: a cross shift study,” BMC Pulmonary Medicine, vol. 10, article 19, 2010.
6. Zawilla, F. Taha, and Y. Ibrahim, “Liver functions in silica-exposed workers in Egypt: possible role of matrix remodeling and immunological factors,” International Journal of Occupational and Environmental Health, vol. 20, no. 2, pp. 146–156, 2014.

7. O. Ogunbileje and O. M. Akinosun, “Biochemical and haematological profile in Nigerian cement factory workers,” Research Journal of Environmental Toxicology, vol. 5, no. 2, pp. 133–140, 2011.inorganic compounds,” HERAG fact sheet 12007,

8. Health Risk Assessment Guidiance for Metals (HERAG), “Assessment of occupational dermal exposure and dermal absorption for metals and 2007.
9. Mayes, P. A. and Bender, D. A. Overview of Metabolism. In: Murray R. K., Granner, D. K., Mayes P. A. and Rodwell, V. W: Harper’s Illustrated Biochemistry. 27th Edition.Lange Medical Publications.

10. A. Lameed, “Environmental impact assessment of cement factory production on biodiversity: a case study of UNICEM, Calabar Nigeria,” World Journal of Biological Research, vol. 1, pp. 1–7, 2008.
11. Oke, K. O. Socioeconomic and Environmental Sustainability Through Recycling of Chemical Wastes. Research Journal of AppliedScience, 2010;5(1): 13-19.
12. Gaw, A., Cowan R. A., O’Relly, D. J., Stewart M. J. and Shepherd J. Core Biochemistry. In: An Illustrated Colour Text Clinical Biochemistry. 2nd Edition, 1999, Published by Churchill Livingstone.
13. Koolman, J. and Roehm, K. Color Atlas of Biochemistry: Minerals and trace elements. 2nd Edition, 2005, Thieme. Stuttgart. New York. 108. 262- 368.
14. Tuormaa, T. E. Adverse Effects of Zinc Deficiency: A Review from the Literature. Journal of Orthomology Medical, 1995;10(3/4): 149-165.
15. Adak, M. D., Adak, S. and Purohit, K. M. Ambient air quality and health hazards near mining-cement plants. Pollution Research, 2007;26(3): 361- 364.
16. O. Ogunbileje, O. M. Akinosun, O. G. Arinola, and P. Akinduti, “Immunological classes (IgG, IgA, IgM and IgE) and liver function tests in Nigerian cement factory workers,” Researcher, vol. 2, no. 4, pp. 55–58, 2010.
17. Alakija, V. I. Iyawe, L. N. Jarikre, and J. C. Chiwuzie, “Ventilatory function of workers at Okpella cement factory in Nigeria,” West African Journal of Medicine, vol. 9, no. 3, pp. 187–192, 1990.
18. Noor, C. L. Yap, O. Zolkepli, and M. Faridah, “Effect of exposure to dust on lung function of cement factory workers,” Medical Journal of Malaysia, vol. 55, no. 1, pp. 51–58, 2000.
19. Shaikh, A. A. Nafees, V. Khetpal, A. A. Jamali, A. M. Arain, and A. Yousuf, “Respiratory symptoms and illnesses among brick kiln workers: a cross sectional study from rural districts of Pakistan,” BMC Public Health, vol. 12, article 999, 2012.
20. Mandal (Majee) and R. Majumder, “Assessment of pulmonary function of cement industry workers from,” Progress in Health Sciences, vol. 3, no. 1, pp. 65–71, 2013.
21. G. Akanbi, O. Ismaila, W. Olaoniye, K. T. Oriolowo, and A. Odusote, “Assessment of post-work peak expiratory flow rate of workers in cement company,” Sigurnost, vol. 56, no. 4, pp. 315–322, 2014. View at Google Scholar
22. A. Meo, A. M. Al-Drees, A. A. Al Masri, F. Al Rouq, and M. A. Azeem, “Effect of duration of exposure to cement dust on respiratory function of non-smoking cement mill workers,” International Journal of Environmental Research and Public Health, vol. 10, no. 1, pp. 390–398, 2013.
23. Egbe Edmund Richard, Nsonwu-Anyanwu Augusta Chinyere, Offor Sunday Jeremaiah, Usoro Chinyere Adanna Opara, EtukudoMaiseHenrieta, and Egbe Deborah Ifunanya. Cement Dust Exposure and Perturbations in Some Elements and Lung and Liver Functions of Cement Factory Workers. Journal of ToxicologyVolume 2016 page 1-7.
24. O. Ogunbilege, O. M. Akinosun, P. A. Akinduti, L. A. Nwaobi, and O. A. Ejilude, “Serum levels of some trace metals and leucocyte differential count in Nigeria cement factory workers; Possible toxicity implications,” Florida Int. J., vol. 2, pp. 55–58, 2010.
25. J. Langman and B. M. Kapur, “Toxicology: then and now,” Clinical Biochemistry, vol. 39, no. 5, pp. 498–510, 2006.

The post Copper and Zinc Levels Among Occupationally Exposed Male Workers to Cement Dust in Benin City, Edo State, Nigeria appeared first on Nigerian Biomedical Science Journal.

Animoku Abdulrazaq A., Suleiman, Muritala O. and Iliyasu Musa O.
Department of Anatomy, Kogi State University, Anyigba-Nigeria

Suleiman Haruna O
Department of Physiology, Kogi State University, Anyigba-Nigeria

Mesole Samuel Bolaji
Department of Human Anatomy, University of Gitwe, Gitwe-Rwanda

Yusuf Uthman Ademola
School of Medicine and Health Sciences, Mulungushi University-Zambia

Okpanachi Alfred Omachonu
Department of Physiology Kampala International University-Uganda

Animoku Abdulrazaq A., Ibrahim Z. Muhammad and Maliki Joseph Simpa
Department of Anatomy, Ahmadu Bello University Zaria-Nigeria.

All correspondence to: Animoku AA email: animokuaa@gmail.com

ABSTRACT

Mercury is a heavy metal contaminant of known toxicity with potential for global mobilization following its give off through air, soil, water and food  Aim: The present study was aimed at investigating the role of ascorbic acid on mercury induced alterations in cerebellum and body weights of Adult Wistar rats.  Methods: Twenty five (25) Adult Wistar rats with average weight 185 g were randomly divided into five groups of five rats per group (n=5); The animals in Group I (Control) were administered with normal saline, Group II received 49.8mg/kg body weight of mercuric chloride. Animals in Groups III and IV were treated with 49.8mg/kg body weight of mercuric chloride and distilled water, and 49.8mg/kg body weight of mercuric chloride and 595mg/kg body weight of ascorbic acid respectively while Group V animals were administered with 49.8mg/kg body weight of mercuric chloride and 1,190mg/kg body weight of ascorbic acid. All administration was carried out orally for a period of three to six weeks and the animals were humanely sacrificed.  Results: Results revealed alterations in cerebellum histoarchitecture involving necrosis, neuronal vacuolation, neuronal degeneration, cytoplasminc shrinkage and reduction in the number of Purkinje cells (p<0.05) in HgCl₂ intoxicated groups. However, ascorbic acid administration significantly ameliorated the induced degenerative changes in the cerebellum caused by mercury exposure in Wistar rats suggestive of its neuroprotective potentials against HgCl₂. The results also revealed significant decrease (p<0.05) in weight gain by mercury intoxicated rats when compared to weights gain by rats treated with ascorbic acid and rats in the control groups. Conclusion: The administration of ascorbic acid remarkably ameliorated HgCl₂ induced changes in the cerebellum histoarchitecture and body weights alterations in Wistar rats.

Key words: Cerebellum, Mercuric Chloride, Ascorbic acid, Body Weight, Wistar rats.

INTRODUCTION

Mercury intoxication has been a public health problem for many decades (Berlin, 2007; Burger et al., 2011). Consideration of the role of environmental factors in determining the susceptibility to mercury has recently been renewed by evidence from epidemiological studies (Wang, et al., 2007). Many populations Worldwide have been exposed to mercury through the consumption of fishes and sea foods (European Commision, 2005), dental amalgam and mining of gold, silver in industries (WHO, 2007). There are many reported cases of mercury food poisoning in Sweden, Mexico, USA and the Minamata Bay incidence that led to the poisoning of over 800 people (WHO, 2005). Mercury readily crosses the blood-brain barrier due to limited lipid solubility and can result in neurological symptoms; mental retardation, seizures, vision and hearing loss, delayed development, language disorders and memory loss (WHO, 2003, 2007; Animoku et al., 2018). It is a potential factor in brain damage (Ibegbu et al., 2014), mental impairment, behavioral anomalies (Farina et al., 2011), impaired cognitive functions and coma (Flora et al., 2007) while, mild subclinical signs of central nervous system toxicity can be seen in workers exposed to an elemental mercury level in the air (WHO, 2007). Mercuric salts can be absorbed through the skin of animals (Altmann et al., 2008) and distribute to all tissues reaching peak levels within hours or days (WHO, 2003). The urine and feces are the main excretory pathways of mercury compounds in humans (WHO, 2005; 2007). In Nigeria, Tilapia fishes from Lagos Lagoon and the use of “Kohl” a traditional cosmetic had been reported as an agent of mercury toxicity (Onyeike et al., 2002). Presenting symptoms include depression, headache, dizziness, itching, burning, irritability, excitability, restlessness, irrational outburst of temper, profuse sweating, tachycardia, frequent urination, increased salivation, and hypertension (Grant and Lipman, 2009; ATDRS,  2011). Ascorbic acid is an essential nutrient for humans and some other animal species. Vitamin C functions as an antioxidant that scavenges free radicals (Padayatty et al., 2003), prevents scurvy (WHO, 2001), pneumonia (Hemila and Louhiala, 2007) and may be useful in lowering the incidence of gout (Choi et al., 2009). Antioxidants are substances that nullify the effect of free radical by either inhibiting the initial production or inhibiting the preparative phase of free radicals (Sujatha et al., 2011). Vitamin C is found in high concentration in immune cells and is consumed quickly during infections (Preedy et al., 2010). Examples of antioxidants are Ascorbic acid (Vitamin C), Vitamin E and Vitamin A (Vasudevan and Sreekumari, 2007). These anti-oxides are generally regarded as primary first-line protective agent that nullifies free radicals by donating a single electron to yield dehydroascorbic acid (UKFSA, 2007; Gemma et al., 2010). The aim of the study was to determine the role of ascorbic acid on mercury induced cerebellum and body weight impairments in Wistar rats.

MATERIALS AND METHODS

Twenty five (25) Adult Male Wistar rats of average weight 185g were used for this study. After acclimatization in the Animal House of the Department of Human Anatomy, Ahmadu Bello University, Zaria, the animals were grouped into five groups of five animals each (n = 5). Mercuric chloride (X-N202, May and Bakers, England) was utilized at LD₅₀ of 166 mg/kg body weight as adopted from ATSDR (2011).While; the LD₅₀ of ascorbic acid (S42238, Sam Pharmaceuticals, Nigeria) was adopted from MSDS (2008) as 11,900 mg/kg body weight. The mercury chloride was the approved laboratory grade chemical by Standard Organization of Nigeria, marketed and sold in Nigeria, while the ascorbic acid tablets was approved by National Agency for Food and Drug Administration and Control to be marketed and used in Nigeria. Before the commencement of the study, ethical approval was sort and obtained from the Ahmadu Bello University Zaria Ethical and Animal Use Committee, Faculty of Veterinary Medicine with reference Number ABU/FVM/ EAUC/2015/12. The animals were dosed as follows: control group was administered with normal saline, group II with 30% mercuric chloride (HgCl2, 49.8 mg/kg) only, group III received HgCl₂ with distilled water only, group IV received HgCl₂ with 5% low dose ascorbic acid (595 mg/kg), while group V received HgCl₂ with 10% high dose ascorbic acid (1,190 mg/kg). However, administrations of distilled water and ascorbic acid from weeks 3-6 were done in order to observe for any possible natural recovery and possible ameliorative potentials of ascorbic acid respectively (Table 1). The administration was by oral route daily and lasted for 3-6 weeks, while animal feed and water were allowed ad libitum. Table 1: Animal grouping, number of rats, treatment and duration of administration of mercuric chloride and ascorbic acid

Animal Sacrifice

After the administration, the animals were weighed and anaesthetized by inhalation of chloroform in the sacrificing chamber. The skull was opened with the aid of brain opener through a mid sagittal incision while brain tissues were removed and fixed in Bouin’s fluid. The tissues were routinely processed for paraffin embedded histology and stained using H&E and Cresyl violet staining methods.

Tissue Processing Procedure

The fixed tissues were removed from the Bouin’s fluid and dehydrated using ascending grades of alcohol. This method involved dehydration of tissues in two (2) changes of 70% alcohol and two (2) changes of 90% alcohol, three (3) changes of 95% alcohol and three (3) changes of absolute alcohol, each of which lasted for 30 minutes. The dehydrated tissues were further cleared in two (2) changes of chloroform for two (2) hours each. The cleared tissues were infiltrated by immersion into molten paraffin wax. The embedded tissues were blocked in rectangular blocks, while tissues were sectioned coronally using the rotary microtome at 5 µm per section. The tissue sections were allowed to float in water bath at 30°C to help the spreading of the paraffin ribbons. The clean slides were used to pick the tissues from the warm water bath. The slides were left to dry and later stained using H&E and Cresyl violet solutions.

Cresyl violet staining method

The tissue sections were deparaffinized and hydrated to distilled water and then stained for 5 minutes in Cresyl
violet solution. The stained sections were rinsed in two changes of distilled water and placed in 95% alcohol for 30 seconds. Sections were transferred to absolute alcohol for 30 seconds and then placed in xylene for 1 minute and 2 minutes sequentially. Differentiations were made in absolute alcohol, two changes for 10 and 30 seconds each. The sections were then taken through several changes of xylene and mounted with synthetic resin. Digital photomicrographs were made from all the experimental groups with the aid of MD900 Amscope microscope digital camera.

Cell Count Analysis

Purkinje cells involving the cerebellum was counted using Digimizer image analysis software.

Photomicrographs of cerebellar cortical regions were uploaded into the image area of the software. This was followed by the utilization of marker tools to mark and count cells in the aforementioned regions. The numbers of counted cells were automatically indicated on the statistics area of the software, while results obtained were further subjected to statistical analysis.

Statistical Analysis

All the results were analyzed using the Statistical package for Social Scientist (SPSS version 20) and the results were expressed as Mean ± SEM. The Statistical significance between means were analyzed using one-way analysis of variance (ANOVA) followed by post HOC test; Tukey’s multiple comparison test was utilized to test for significant difference between control and

experimental groups. A p-value < 0.05 was considered significant.

RESULTS:

Physical observation of the animals

On physical observation of the animals, the control group animals were very active and behaviorally stable while mercury treated animals were observed to be ataxic,

apathetic, agitated, distressed, with diarrhea for the first 3 weeks of administration. However, there were improvements in activity, agility, and behavioral stability as observed in animals treated with ascorbic acid in the last 3 weeks of administration.

Histological observation of the cerebellum

The results revealed normal histoarchitecture and cellular layers of the cerebellum in the control group (Fig.1A and 2A), while animals in the HgCl₂ (49.8 mg/kg) only and HgCl₂ with distilled water groups revealed necrosis of cells, disorientation of Purkinje cells, vacuolation and congestion of cells (Fig.1B, 1C, 2B and 2C). However, HgCl₂ with low dose vitamin C (595 mg/kg) and HgCl₂ with high dose vitamin C (1,190 mg/kg) groups showed minimal

Figure 1: Photomicrographs of the Cerebellar cortices (H&E × 250)

1. Control Group showing normal histoarchitechture of the Molecular layer (ML), Purkinje cell layer (PCL), Purkinje cell (PC) and Granular layer (GL)
2. Group II (48.9mg/kg mercuric chloride) showing the Molecular layer (ML), Separated Purkinje cells (SPC) and Granular layer (GL).
3. Group III (48.9mg/kg mercuric chloride and Distilled Water) showing the Molecular layer (ML), Vacuolated Purkinje cell (VPC) and Granular layer (GL).
4. Group IV (48.9mg/kg mercuric chloride and 595mg/kg ascorbic acid) showing the Molecular layer (ML), Separated Purkinje Cell (SPC), some Normal Purkinje cells (NPC) and Granular layer (GL).
5. Group V (48.9mg/kg mercuric chloride and 1,190mg/kg ascorbic acid) showing the Molecular layer (ML), Degenerating Purkinje Cells (DPC), some normal Purkinje cells (PC) and Granular layer (GL).

cellular degeneration with some normal cerebellar cortical cells (Fig. 1D, 1E, 2D and 2E).

Figure 2: Photomicrographs of the Cerebellar cortices (Cresyl violet × 250)

1. Control Group showing normal histoarchitechture of the Molecular layer (ML), Purkinje cell layer (PCL), Purkinje cell (PC) and Granular layer (GL).
2. Group II (48.9mg/kg mercuric chloride) showing the Molecular layer (ML), Separated Purkinje cells (SPC), Degenerating Purkinje cells (DPC) and Granular layer (GL).
3. Group III (48.9mg/kg mercuric chloride and Distilled Water) showing the Molecular layer (ML), Degenerating Purkinje cell (DPC) and Granular layer (GL).
4. Group IV (48.9mg/kg mercuric chloride and 595mg/kg ascorbic acid) showing the Molecular layer (ML), Granular layer (GL), Normal Purkinje Cells (NPC) with some evidence of Degenerating Cells (DC).
5. Group V (48.9mg/kg mercuric chloride and 1,190mg/kg ascorbic acid) showing the Molecular layer (ML), Granular layer (GL), Numerous Purkinje Cells (NPC) with some evidence of Degenerating Cells (DC).

Cell Count Analysis

Number of Purkinje cells in the Cerebellar cortex There was significant decrease (p<0.05) in the number of cerebellar cortical Purkinje cells in HgCl₂ (49.8mg/kg) and HgCl₂ (49.8mg/kg) with distilled water groups compared to the control, HgCl₂ (49.8 mg/kg) with low dose vitamin C (595 mg/kg) and HgCl₂ (49.8 mg/kg) with high dose vitamin C (1,190 mg/kg) groups. This decrease (p<0.05) in the number of cerebellar Purkinje cells was also observed in HgCl₂ (49.8 mg/kg) with low dose vitamin C (595 mg/kg) compared to the control group (Table 2).

Table 2: Number of Cerebellar Purkinje cells counted

n= number of cells counted.
SEM:Standard Error of Mean.
HgCl₂: Mercuric Chloride.
Vit. C: Vitamin C

*p<0.05 indicates significant difference compared to Group I (Control).

*^a indicates significant difference between Group V and Group II.

*^b indicates significant difference between Group V and Group III. *^c indicates significant difference between Group IV and Group II *^d indicates significant difference between Group IV and Group III.

Body weight assessment of Animals

The results of body weight assessment showed that there was progressive increase in the mean body weights of the animals in Groups I-V throughout the duration of the experiment. However, the degree of weight gain by the animals in Groups II and III decreased significantly (p<0.05) when compared to the weights gain by animals in Groups I (Control), IV and V as shown in Table 3 and Figure 3.

Table 3: Body weight assessment following administration of mercury and ascorbic acid.

*p<0.05 indicates significant difference compared to Group I (Normal saline). g = mean weight in grams. SEM: Standard Error of Mean *^a indicates significant difference between Group V and Group II.   *^b indicates significant difference between Group V and Group III.

*^c indicates significant difference between Group IV and Group II     *^d indicates significant difference between Group IV and Group III.

GI= Control,      GII=HgCl₂ (alone),      GIII =HgCl₂ and Distilled H₂O,     GIV =HgCl₂ and Vit.C_595mg/kg, GV=HgCl₂ and Vit.C_1,190mg/kg.

Figure 3: The % body weight change of animals throughout the experiment.

GI= Control,        GII=HgCl₂ (alone),    GIII=HgCl₂ and Distilled H₂O,

GIV=HgCl₂ and Vit.C_595mg/kg,     GV=HgCl₂ and Vit.C_1,190mg/kg.

*p<0.05 indicates significant difference compared to Group I (Control).

*^a indicates significant difference between Group V and Group II.

*^b indicates significant difference between Group V and Group III. *^c indicates significant difference between Group IV and Group II              *^d indicates significant difference between Group IV and Group III.

DISCUSSION

Mercury is a heavy metal contaminant with potential for global mobilization following its give off through air, soil, water and food from anthropogenic activities or natural processes (Gochfeld, 2003). The present study revealed histological changes ranging from degeneration of Purkinje cells, necrotic features, clumping and disorientation of cerebellar cortical cells in HgCl₂ exposed rats with or without ascorbic acid treatment while, the control group showed normal histology. These neurodegenerative alterations could actively affect the cerebellum resulting in cerebellar syndromes which include: muscular hypotonia, tremor, nystagmus, scanning speech and ataxic gait (Farina et al., 2011). In addition, degeneration and decrease in the number of Purkinje cells which result from exposure to HgCl₂ implies that activities associated with the cerebellum in fine movement, coordination posture, equilibrium will be impaired while motor learning function could also be lost (Fine et al., 2002; Ibegbu et al., 2014).

This study agree with the findings of several authors who reported that many heavy metals such as mercury, lead, cadmium and other organic compounds have the capacity to damage nervous system (Mahmoud, 2007; Ibegbu et al., 2014) because this system is very sensitive to mercury and permanent damage to the brain can occur from exposure to sufficiently high levels of mercury (ATSDR ,1999). After crossing into the brain, mercury may affect many different areas of the brain and their associated function, resulting in a variety of symptoms. These include personality changes, irritability, shyness, nervousness, tremors, changes in vision, deafness, muscle incoordination, loss of sensation and difficulties with memory (ATSDR,1999; WHO, 2003) while the most sensitive elements of the cerebellar cortex to HgCl₂ are the Purkinje cells which react to this heavy metal by undergoing degeneration and as such disappear from their relative positions in the Purkinje cell layer (Farina et al., 2011) resulting in cerebellar dysfunction (Wolf et al., 2009). The present study also revealed progressive increase in the mean body weights of the animals throughout the period of administration of mercuric chloride and ascorbic acid. However, the degree of weight gain by the animals treated with mercuric chloride alone throughout the experiment was less than that of the weight gain by animals treated with ascorbic acids and animals in the control groups. This difference in decreased body weight gain was significant (p<0.05). This agrees with the findings of Mohammad, (2009) who reported that the body weight of mercuric chloride treated rats was less than their controls significantly during the 8 weeks of exposure while Thomas et al., (2001) observed that exposure of rats, rabbits and dogs to metallic mercury vapour for 7h/day, 5 days/week for 72-83 weeks, resulted significantly in weight decrease. However, the present study showed that animals administered with ascorbic acid both at low and high doses showed significant improvements (p<0.05) when compared with animals intoxicated with mercury alone and this agrees to the fact that ascorbic acid can improve the oxidative stress effect of mercury substances (Bernhoft, 2012) which can result in reduced superoxide dismutase, catalase, glutathione and increased Lipid peroxidase levels (Farina et al., 2013). Hence, ascorbic acid as an antioxidant plays significant ameliorative role (p<0.05) in the reversion (to certain level) of mercury induced cerebellum impairment possibly by forming inert complexes and inhibiting their toxicity (Burger et al. 2011; Ibegbu et al.

2014).

Conclusion

The findings from the present study justify the ameliorative effect of ascorbic acid against mercury induced temporal lobe neurotoxicity and hence populations exposed to mercury poison should consume foods rich in ascorbic acid (vitamin C) along with other antioxidants.

Conflict of Interest None declared.

Reference:

1. Agency for Toxic Substances and Disease Registry (ATSDR). (1999). Toxicological Profile for mercury. US Department of Health and Human Services. Atlanta, US.
2. Agency for Toxic Substances and Disease Registry (ATSDR). (2011) Exposure to hazardous substances and reproductive health. American Family Physician 48(8):1441-1448.
3. Altmann, L., Sveinsson, K., Kraemer, U. (2008). Visual functions in 6 year old children in relation to lead and mercury levels. Neurotoxicology Teratology 20(1)9-17.
4. Animoku AA, Buraimoh AA, Hamman WO, Ibegbu AO, Samuel SB, Uthman AY, Joseph SM, Akpulu, P. (2018). Ameliorative effect of ascorbic acid (vitamin c) on mercury induced temporal lobe damage in rat. Nigerian Journal of Neuroscience. 9(1):9-15.
5. Berlin, M., Zalups, R.K. and Fowler, B.A. (2007). “Mercury,” in Handbook on the Toxicology of Metals, G.F. Nordberg, A. Fowler, M. Nordberg, and L.T. Friberg, Eds., Chapter 33, Elsevier, New York, NY, USA, 3^rd edition.
6. Bernhoft, R.A. (2012) Mercury Toxicity and Treatment. A Review of the Literature. Journal of Environmental and Public Health, Hindawi Publishing Corporation, Article ID 460508.

7. Burger, J., Jeitne, C. and Gochfeld, M. (2011) Locational differences in mercury and selenium levels in18 species of saltwater fish from New Jersey. Journal of Total Environmental Health 74(13):863-874.
8. Choi, M. D., Gao, X., Curhan, G., Xiang Gao, M. D. and Gary Curhan, M. D. (2009)Vitamin C Intake and the Risk of Gout in Men. Archives of Internal Medicine. 169 (5): 502–507.
9. European Commission. (2005) 101 Communication from the Comamission to the Council and the European Parliament on Community Strategy Concerning Mercury Extended Impact Assessment 101:20 final 28:12.
10. Farina, M., Rocha, J.B. and Aschner, M. (2011) Mechanisms of methylmercury-induced neurotoxicity: Evidence from experimental studies. Journal of Life Sciences (89):555–563.
11. Farina, M., Avila, D.S., da Rocha, J.B. and Aschner, M. (2013) Metals, oxidative stress and neurodegeneration: a focus on iron, manganese and mercury. Neurochemical International. 62(5): 575-594.
12. Fine, EJ, Ionita, CC and Lohr, L. (2002). The history of the development of the cerebellar examination. Semin. Neurol. 22 (4): 375–384.
13. Flora, S. J., Saxena, G. and Mehta, A. (2007). Reversal of lead-induced neuronal apoptosis by chelation treatment in rats: Role of ROS and intracellular Ca+. Journal of Pharmacological Experiment, 322:108173.
14. Gemma, C., Bachstetter, A.D. and Bickford, P.C. (2010) Neuron-Microglia dialogue and hippocampal neurogenesis in the aged brain. Aging and Disease. 1(3):232-244.
15. Gochfeld, M. (2003) Cases of mercury exposure, bioavailability, and absorption. Ecotoxicol Environ Saf 2003; 56 : 174-9.
16. Grant, L.D. and Lipman, M. (2009) Human exposure and their effect. Environmental toxicant; 3^rded 108112.
17. Hemila, H. and Louhiala, P. (2007). Vitamin C for preventing and treating pneumonia.Cochrane Database System Rev (1).
18. Ibegbu, A.O., Animoku, A.A., Ayuba, M., Brosu, D., Adamu, S.A., Akpulu, P., Hamman, W.O., Umana, U.E. and Musa, S.A. (2014) Effect of Ascorbic Acid on Mercuric Chloride-Induced Changes on the Histomorphology of the Cerebellum of Wistar Rats. African Journal of Cellular Pathology. 3:9-15.

19. Mahmoud, A., Mokhtar, M.M. and Hanaa, G.A. (2017) Neurodegenerative Disorders Associated with Mercuric Chloride Toxicity in Mice and the Role of Some Antioxidant. International Journal of Science and Research (IJSR) 6:(1253-1260).
20. Material Safety Data Sheet (MSDS). (2008) Safety data for Mercuric Chloride Physical and Chemical Properties. Janssen Pharmaceutical 3a. 2440 Geel, Belgium.
21. Mohammad, A.M. (2009). Effects of Mercury Exposure on Blood Chemistry and Liver Histopathology of Male Rats. Journal of Pharmacology and Toxicology, 4: 126-131.

22. Onyeike, E.N., Obbuja, S.I. and Nwinuka, N.M. (2002) Inorganic ion levels of soils and streams in some area of Ogoni land, Nigeria as affected by crude oil spillage. Journal of Environmental Monitoring Assessments. 73:191-205.
23. Padayatty, S.J., Katz, A., Wang, Y., Eck, P., Kwon, O., Lee, J., Chen, S. and Corpe, C. (2003) Vitamin C as an antioxidant: evaluation of its role in disease prevention. Journal of the American College of Nutrition. 22(1):18–35.
24. Preedy, V. R., Watson, R. R. and Sherma, Z. (2010). Dietary Components and Immune  Function (Nutrition and Health). Totowa, NJ: Humana Press: 36-52.

25. Sujatha, K., Srilatha, C., Anjaneyulu, Y. and Amaravathi, P. (2011) Lead Acetate Induced Nephrotoxicity in Wister Albino Rats. A pathological immunohistochemical and Ultra-structural Studies.International Journal of Pharmaceutical and Biosciences. 2:2-11.

26. Thomas, C.J., Chen, Y., Buck, D.J. and Davis, R.L. (2001). Chronic inorganic mercury exposure induces sex specific changes in central TNFα expression: importance in autism Neuroscience letters, 44:2-6.
27. United Kingdom Food Standards Agency (UKFSA). (2007). Vitamin C risk assessment Available from: http://www.food.gov.uk/multimedia/pdfs/evm_c.pdf. [Accessed: 2012].
28. Vasudevan, D.M. and Sreekumari, S. (2007) Textbook of Biochemistry For Medical Students 5th ed. Jaypee Brothers Medical Publishers (p) Ltd. New Delhi.143223.
29. Wang, JS., Huang, PM., Liaw, WK. (2007). Kinetics of the desorption of mercury from selected fresh water sediment as Influenced by mercury chloride. Water, Air, Soil Pollution 12(56) 533-542.
30. Wolf U, Rapoport MJ and Schweizer TA (2009). Evaluating the affective component of the cerebellar cognitive affective syndrome. Journal of Neuropsychiatry Clinical. Neurosciences, 21 (3): 245–53.
31. World Health Organization. (2001). Area of work: nutrition. Progress report 2000 (PDF). Archived from the original.
32. World Health Organization (WHO) (2003). Elemental mercury and inorganic mercury compounds: Human health aspects. Concise International Chemical Assessment Document. CICAD 50. Geneva.
33. World Health Organisation (WHO). (2005) Mercury Training Module. WHO Training package for Health Sector. Geneva, World Health Organization.
34. World Health Organisation (WHO). (2007). Preventing disease through healthy environments; exposure to mercury, a major public health concern. Geneva, Switzerland.

The post Histomorphological Studies of the Cerebellum in Mercury Exposed Rats and the Role of Ascorbic Acid (Vitamin C) appeared first on Nigerian Biomedical Science Journal.

INTRODUCTION

The total Laboratory testing process includes three phases namely preanalytical, analytical and postanalytical. Several studies have shown that most errors occur in the preanalytical phase [1,2]. A major emphasis is currently placed on this initial phase of the total testing cycle because the preanalytical variables is believed to exert a significant influence on laboratory test results [3]

Besides the variation due to preparation of patient and blood collection such as physical activity, the fasting state, the blood collection technique [4,5], and tourniquets application time [6,7] there are additional preanalytical variables that might influence the reliability of laboratory testing. In particular variations and errors due to preparation of the specimen prior to the measurement of blood analytes such as specimen interferences. Processing of specimen and storing of specimen before the measurement causing leakage of intracellular components and producing significant biological and analytical interference [8].

Inherent in any laboratory test results on patients are influences of biological variation, inherent analytical error, preanalytical and postanalytical sources of variation and possible pathophysiological alterations. To be able to reduce the errors and for proper interpretation of test results, one must have a good understanding of these preanaltycial variables.

Generally, laboratory users need only limited knowledge of the technical details of the laboratory tests. However, they should understand that the appropriate collection of patient specimens can affect results and they should therefore work with the laboratory in its effort to produce reliable test results rapidly and accurately and identifiable with the relevant patient [9]. Preanalytical variables can be grouped into four categories which include: Physiologic specimen collection, handling and interference variables [10]

In its review, we focus on the preventable preanalytical variables which include specimen type selection, blood collection, blood collection equipment and factors interfering biochemical tests.

Physiological Variables

Biological influences – Heredity, gender, age and race can affect individual lab results and these are non-controllable variables.

Age has a notable effect on reference internals; typical changes occur in serum composition at extremes of ages. In general, individuals are considered in four groups – newborn, the older child to puberty, the sexually mature adult and the elderly adult. One must always look at the age of the patient when evaluating laboratory results. Paediatric values vary in many lab tests.

Gender can be a factor because of hormonal variations that occur with each gender. After puberty the serum activities of ALP, ALT, AST, CK are greater in men than in women. The concentrations or Albumin, Calcium and Magnesium arealso higher in men than to women. Differentiation or the effects of race from those of socio-economic conditions is often difficult. Nevertheless, the serum total protein concentration is known to be higher in blacks than in whites. Carbohydrates and Lipid metabolism differ in blacks and whites [11].Glucose tolerance is less in blacks (Table 1)

Diurnal Variation

Many constituents of body fluids exhibit cyclical variations, throughout the day Factorscontributing to such variations include posture, activity, food ingestion, stress, daylight or darkness and sleep or wakefulness. These cyclical variation, maybe quite large and therefore the drawing of the specimen must be strictly controlled. For example, the concentration of serum iron and cortisol may change by as much as 50% between 0800 and 1600. Serum potassium has been reported to decline from 5.4 mnol/L at 0800 to 4.3 mnol/L at1400 [11]

Exercise

Strenuous exercise can increase the bilirubin, creatine kinase (CK), asparticaminotransferase (AST), high density lipo protein (HDL), cholesterol, lactate, lactate

dehydrogenase (LD) and uric acid [4]

Lifestyle

Diet, caffeine, smoking and alcohol intake can have an effect on some chemical analytes. High protein diets increase levels of uric acid, urea, and ammonia in blood compared with vegetarians. Caffeine can decrease pH, increase ionic calcium and catecholamine levels. Smoking can increase glucose, triglyceride Cholesterol and LDL Cholesterol. Short-term effects of ethanol include a decrease in glucose, increase in plasma lactate and an increase in uric acid and triglyceride. Moderate intake of alcohol increases the HDL cholesterol. Long term effects of alcohol include an increase in gamma glutamyl transferase (GGT), AST and ALT [12,13]

Fasting Status

Certain specimens are required to be fasting. They include fasting glucose and lipid profile. A non-fastiing glucose will be increased compared to a fasting sample. The triglyceride and low-density lipo protein (LDL) cholesterol will be increased in a non-fasting sample compared to a fasting sample. [14]

Prolonged fasting can decrease transthyretin (prealbumin) glucose, albumin, LD, HDL, cholesterol and insulin. Dehydration causes hemo concentration, which can result in the false elevation of some chemical analytes including iron, calcium, sodium, and enzymes. An elevated haematocrit or protein can be an indication of dehydration and the patient should be rehydrated before reassessing the chemical analytes

Posture

Substantial changes take place with a change from lying to the sitting position or from standing to a supine or sitting position [14]

A decrease in albumin, alkaline phosphate, ALT,bilirubin, calcium, cholesterol, total protein and triglyceride is noted when going for a standing position to a supine position. Accurate and legibly written information about the patient is essential, although electronic requesting symptoms are now available. This information includes the patient’s hospital case, number, name, date of birth. The requesting doctor must sign the form legibly.

3. Specimens collection variables.

Specimen collection variables include requisition errors, patient identification errors, tourniquet time variability improper cleansing agents, improper collection time, and intravenous or drug medication interference with the sample.

• Tourniquet a application time- A tourniquets that is kept on too long (>3 minutes) will increase the total protein, iron, AST, bilirubin and total lipids (Table2) A total cholesterol level may increase 5% at 2mins and 15% at 5 minutes. Repeated fist clinching can increase the potassium by 1 to 2 mmo1/L. [15, 16]
• Intravenous infusion site – Drawing from above an intravenous infusion (iv) site should be avoided if at all possible. Drawing from below the iv site after turning off the iv for 2 to 5 minutes and discarding the first is 5ml seems to be preferable. Some laboratories may draw from above the IV as a last resort. If the IV has been shut off for 10minutes. [17]
• Cleansing Agents – Providone – iodine (Betadine) used as a cleansing agent falsely elevates phosphorus, uric acid, and potassium. (Table 2)

Isopropyl (70%) alcohol should not be used for medical or legal ethanol levels. The site for venepuncture is to be cleaned with 70% ethanol. The type of collection sample, capillary or venous serum, or serum versus plasma sample can cause variances in the analyte measurement. Glucose capillary values are 1.4% higher than venous serum samples and potassium capillary samples are 0.9% higher than venous samples. Capillary samples of bilirubin calcium chloride, sodium and total protein are lower than venous serum samples (18) plasma values of potassium, phosphorus and glucose are lower than serum values. Plasma values of total protein LD, and Calcium are higher than serum values A cholesterol, triglyceride and HDL cholesterol measured with EDTA plasma should be multiplied by 1.03 to be equivalent to a serum sample. [19, 20].

• Anticoagulants – most chemistry analytes are run are on serum or heparinized plasma samples depending on the analyte and methodology used to analyse it. Some anticoagulants cannot be used for certain tests. Anticoagulants containing fluoride (an inhibitor of erythrocyte glycolysis) may be used for glucose testing but will interfere with electrolyte studies by altering blood cell membrane permeability. Potassium should be estimated on plasma from heparinised blood rather than serum –potassium released from cells especially platelets during clotting serum potassium concentrations are usually higher than those of plasma by a variable amount. Marked difference may be found in patients with leukaemia- [21]
• Gel versus Non-Gel tubes –serum or plasma separator tubes may be unacceptable for some analytes, for example, therapeutic drugs. The manufacturer of the separator tube should provide documentation of analytes that have been shown to give comparable results in serum of plasma obtained from tubes containing gels versus nongel tubes.
• Order of Draw- Clinical and Laboratory Standard Institute CLSI (formerly NCCLS) recommendations for vacutainer or syringe order of the draw of filling tubes is the following. Blood culture tubes, non additive or serum tubes, citrate or coagulation tubes, gel separator tubes, heparin, EDTA, and fluoride tubes. Filling the tubes out of this order may cause some cross contamination in the tube leading to interference in testing the analytes [22 23]

4. Handling

(I)                 Light and Temperature- bilirubin, vitamin B₁₂,

Vitamin A and Carotere are affected by light Temperature labile analytes include ammonia, blood gases, lactate, pyruvate. Specimens can be chilled by placing in ice water if blood gases are collected in plastic syringes and run within 20minutes, they do not need to be iced. [24]

(ii) Specimen processing – A clot –tube specimen should be allowed to clot for 30 to 60 minutes and nolonger than 2 hours before it is centrifuged and the serum separated from the clot.  Analytes that increase on standing include CK, lactate, LD, phosphate and ammonia.Analytes that may decrease on standing include glucose and bicarbonate [25]

If plasma is not separated from blood cells within a few hours, the effect on plasma concentrations will be similar to that resulting from haemolysis.

The refrigeration of whole blood has the effect of raising the plasma potassium concentration probably by reducing the activity of ATPase Pump. Therefore, blood specimens must be centrifuged and the plasma separated from the cells before storing, for example overnight.

5.  Interfering substances

The presence of lipemia, haemolysis and fibrin strands can be evaluated in the specimen before the analytical rum.

Haemolysis. Is one of the most frequently occurring interferences in analysis in the clinical laboratory.

Haemolysis results from mechanical factors in specimen collection and processing.

Haemolysis interferes, with the analysis in two ways

1. Haemolysis absorbs at 431 and 555mm, if the analyte is measured spectrophotometricallyat or around 430 and 555mm, the absorbance of haemoglobin can cause false elevated results [26]
2. The concentrations of various blood constituents differ in erythrocyte and plasma.

Several constituents such as potassium, LDH, acid phosphatase, AST are present in very large amounts in red cells in comparison with plasma. Consequently, haemolysed plasma gives higher values for this test. On the other hand, sodium and chloride which are present in small amounts in erythrocytes, tend to grow lower values If the serum is haemolysed [27]

It is also important to separate the serum from the clot or the plasma from the cell as soon as possible after collection to avoid the free exchange of analytes between the cell and the serum or plasma

CONCLUSION

Blood and urine samples constitute over 95% of all specimens analysed in Clinical Laboratories, the remaining 5% are cytological and surgical specimens. Blood can be considered as the major biological fluid on which quantitative analyses are performed. Consequently, specimen requirements are most rigid for blood samples. Serum is used for most of the chemical analyses. Before performing the actual analysis, one should be sure of the quality of the specimen. The analytical results on bad specimens are not only useless but also misleading and sometimes even dangerous to the patients involved. Generally speaking, analytical methodology and instrumentation do not make any adjustment s or compensations for interfering factors. Therefore, it is the responsibility of the analyst to see that the samples analysed are free from any interference or deterioration.

REFERENCES

1. T Assessment of Extra-analytical phase: improving Laboratory service and patient safety. JAMMR 2018,26(3) 1-5
2. Akande T. Quality Management of the pre-Analytical phase of total laboratory testing process. Monitoring and control (JAST.2018 29(5) 1-8
3. Lippi G, Franchini M, Schena F, Guidi G, Comparison of serum creatinine, uric acid, albumin and glucose in male professional endurance athletes compared with healthy controls. Clin chem lab med 2004, 42:644-7
4. Lippi G, Guich GC, Effect of specimen collection on routine coagulation assays and D-dimer measurement. Clin Chem 2004; 50:2150-2
5. Lipp G, Salvagno GL, Guidi GC, No influence of a butter-fly device on routine coagulation assays and Ddimer measurement. J thromb Haemost 2005, 3:389-91

6. Lippi,G, Salvagno GL, Montagana M, Brocco G, Guidi GC. Influence of short-term venous stasis on clinical chemistry testing. Clin Chem Med 2005; 43:869-75
7. Lippi G, Salvagno GL, Montagnana M, Guidi GC, Short term venous stasis influences routine coagulation testing Blood Coagul Fibrinolysis 2005;16;453-8
8. Carraro P, Servidio G, Plebani M. Hemolysed specimens a reason for rejection or a clinical challenge? Clinical Chem 2000:46:306-7
9. Crook MA Clinical Chemistry and Metabolic Medicine 7^th ed Hodder Arnold; 2006 p387-392

10. Coleman M. Method Evaluation and pre-analytical variable in Clinical Chemistry Concepts and Applications.The McGraw-Hill Companies; 2006 p65-79
11. Haverstick DM, Grosz bach AR. Specimen collection and processing in: Burtis CA, Ashwood ER, Bruns DE editors. TietzTextbook of Clinical Chemistry and molecular diagnotics. 5^th ed Philadelphia: NB Sanders company; 2012 p 145-56
12. Sacks DB, Bruns DE. Goldstern DE, Maclaren NK, McDonald JM, Parrott M, Guidelines and recommendations for laboratory analysis in the diagnosis and management of diabeties mellitus. Clin Chem 2002 Mar 48 (3): 436-72
13. Schrot RJ, Patel KT, Foulis P. Evaluation of accuracies in the measurement of glycemia in the laboratory by glucose meters, and through measurement of haemoglobin A1_c. Clin Diabetes 2007; 25 (2): 43-9
14. Ginsberg BH. Factors affecting blood glucose monitoring: Sources of errors in measurement. J Diabetes Sci Technol 2009 July 3 (4): 903-13
15. Stankoric AK, Smith S. Elevated serum potassium values: The role of pre-analytic Variables. Am J Clin pathol. 2004 Jun 121 (suppl); 105-12
16. Cuhadar S. Pre- analytical Variables and factors that interfere with the biochemical parameters; a review OA Biotechnology 2013 Jun 01,2, (2) 19: 1-7
17. Lippi G, Salvagno GL, Montagana M, Brocco G, Guidi GC, influence of hemolysis on routine Clinical Chemistry testing Clin Chem Lab Med 2006; 44 (3) ;311-316
18. Fatas M. Franquelo P, Franquelo R. Anomalous flotation of separator gel: density or Viscosity? Clin Chem 2008 Apr; 54 (4): 771-2
19. Egidi MG, DÁlessandro A, Mandarella G. Zolla L. Trouble shouting in platelet storage temperature and new perspectives through proteomics. Blood Transfus. 2010 June 8 (suppl 3): 73-81
20. Naraganan S. The pre-analytical phase. An important Tichy M, Fredecky B, Budina M, Maisnar V,component of laboratory medicine Am J Clin pathol 2000 mar; 113 (3); 429-52
21. BucklerT. Holekova M et al. interference of 1gm- Lambda paraprotein with biuret type quantification for total serum protein qualification. Clin Chem Lab Med 2009; 47 (2): 235-6

The post The Effects of Preanalytical Variables on Some Biochemical Parameters: A Review appeared first on Nigerian Biomedical Science Journal.

INTRODUCTION

Lassa fever is an acute viral illness that occurs in West Africa. The illness was discovered in 1969 in Lassa Village in Borno State in Nigeria among missionary nurses [1-3]. The illness was named after the town where the first cases originated.  The causative organism of Lassa fever is a single stranded RNA enveloped virus. Lassa virus infection causes Lassa haemorrhagic fever (LHF) [4]. The virus is transmitted to humans through contact with urine or excreta from infected Mastomys rats.  The disease occurs throughout the year, but more cases are recorded during dry season. Lassa virus survives better in humid conditions especially during the rainy season.  In raining seasons rats are more often contaminated as a result of their frequent movements, for mating or dispersing into the surrounding fields [4]. Viral aerosol is higher when the humidity is lower especially during the dry season [4]. Currently, there is no approved Vaccine for Lassa fever [5].Lassa Fever cases are more frequent in hospitals between November and early April. Lassa fever is a significant cause of morbidity and mortality. About 80% of people infected with the Lassa fever have mild or no observable symptoms while 20% of those infected with Lassa virus develop severe symptoms/multisystem disease. Globally, Lassa fever causes around 5,000 deaths per year Sierra Leone, Liberia, Guinea and Nigeria are worst affected by Lassa fever. Lassa fever can cause multisystem and multi-organ failure [1-3]. Clinical Diagnosis of Lassa fever is difficult due to of its wide array of symptoms. Currently, there is no vaccine for Lassa fever. A common complication of Lassa fever is deafness, occurring in around one third of cases which could be permanent or temporary and does not correlate with the severity of the infection. Multiple organ failure can lead to Death within 2 weeks after the onset of symptoms. About 15%-20% of Lassa fever hospitalizations end in death, although, in total, only 1% of infections end in fatality. Lassa virus infects all ages, races and gender. Lassa fever can cause foetal, neonatal (>85%) and maternal death (>30%) especially during the third trimester of pregnancy [1-3].

The transmitting agent of Lassa virus ismultimammate rat (Mastomysnatalensis) that are populous in the savannahs and forests of West Africa. They live in human homes and locations where food is stored. The rats breed frequently and produce large numbers of offspring Which makes them a factor contributing to spread the virus. Once a rat is infected with Lassa virus, it excretes the virus through A Situational Analysis of Lassa…

urine and faeces for a long period of timeand even for the rest of its life. The virus can also be transmitted to humans through the inhalation or ingestion of dust particles carrying the virus [1-3].

Spread of Lassa Virus Infections Rat to Human Lassa virus is spread from rats to human through direct exposure or contact with urine, faeces, saliva or blood of infected Mastomys rats or by eating food or taking drinks contaminated with urine, faeces, saliva or blood of infected Mastomys rats. Man can also be infected through contact with blood, urine, saliva, throat secretion or semen of an infected person. Touching of floors, beddings and household materials contaminated with urine, faeces, saliva or blood of rats or an infected person [1-3].

There is also an evidence of multiple, independent introductions of different viruses and viruses similar to previously circulating lineages identified in Nigeria. The main mode of transmission is through spill over from the rodent population, and limited human to human transmission [1,3,6].

Pathophysiology/Pathogenesis OF LASSA VIRUS Upon invasion, Lassa virus targets antigen-presenting cells, (mainly dendritic cells) and endothelial cells. Lassa virus multiplies intracellular using an L-polymerase enzyme and nucleocapsid protein (NP), which synthesize ribonucleoprotein (RNP) that produces mRNA and antigenomic RNA required for transcription. The Lassa virus uses nucleocapsid protein (NP) to evade the host immune system. Just after the transcription there will be vascular dysfunction resulting in the development of clinical manifestations of Lassa fever. The common target for Lassa virus is the liver which causes, inflammation, liver dysfunction such as reduction in the synthesis of coagulation factors and albumin which could result into bleeding disorders, furthermore, Lassa virus infection also results into thrombocytopenia,inhibition of platelet function, complement activation and Disseminated intravascular coagulation (DIC). All these are the causes of bleeding disorder experience by an infected individual almost at the end stage of the infection. The virus can also infect the adrenal-cortical cells causing impaired synthesis of steroid-synthesizing enzymes [5].

The Lassa virus infection can generate inflammatory process leading to fatal hyper-release of pro and antiinflammatory mediators (TNF-alpha, IL-10, IL-1Ra etc.) in response to stimulation of T cells and macrophages by Lassa virus and immune insults which could result into fever, malaise and fatigue. Pro-inflammatory cytokine such as TNF-alpha worsen the cause of the infection of the diseases [5].

The inflammatory process multi-organ failure,

multisystem failure, adrenal cortex and liver dysfunctions can lead to Hypotension, hypertension, shock, circulatory collapse, impaired innate immune response, Purulent Pharyngitis accompanied by headache, fever, myalgia, back or abdominal pain, vomiting, and diarrhoea [5]. Despite the severe signs and symptoms most patients recover spontaneously while some patients deteriorate rapidly, developing facial and neck oedema, respiratory distress, oliguria or anuria, and finally hypovolemic shock that responds poorly to fluid replacement [5].

Specific Symptoms Lassa fever

1. Gastrointestinal tract: Nausea, Vomiting (bloody), Diarrhoea (bloody), Stomach ache, Constipation, difficulty swallowing and Hepatitis.
2. Cardiovascular system: Pericarditis,

Hypertension, Low blood pressure and High heart rate.

3. Respiratory tract: Cough, Chest pain, Dyspnoea, Pharyngitis and Pleuritis.
4. Nervous system: Encephalitis, Meningitis,

Unilateral or bilateral hearing loss, observed in up to one third of adults, which becomes permanent in two thirds and Seizures [7].

LABORATORY DIAGNOSIS OF LASSA FEVER

Currently, three laboratories (Abuja, Irrua and Lagos) are operational at testing samples for Lassa fever by polymerase chain reaction (PCR) which do not provide adequate accessibility to laboratory tests for Lassa fever [8].

Clinical diagnosis often difficult [6].

Laboratory diagnostic methods include:

1. ELISA (Enzyme Linked Immunosorbent Assay) for antigen, IgM and IgG
2. IgM ELISA in a patient’s serum indicates recent infection, or in a neonate’s serum indicates intrauterine infection. IgM is an antibody produced during the primary immune response
3. IgG ELISA-The predominant antibody produced during a secondary immune response is immunoglobulin G (IgG). It indicates previous infection
4. Reverse Transcription Polymerase Chain Reaction(RT-PCR)
5. Virus isolation
6. Immunohistochemistry performed on formalinfixed tissue specimen for post-mortem diagnosis [29].

Reverse transcription polymerase chain reaction (RTPCR)

Reverse transcription polymerase chain reaction (RTPCR), a variant of polymerase chain reaction (PCR), Is a technique commonly used in molecular biology to detect RNA expression. RT-PCR is used to qualitatively detect gene expression through the creation of complementary DNA (cDNA) transcripts from RNA. RT-PCR is used to clone expressed genes by reverse transcribing the RNA of interest into its DNA complement through the use of reverse transcriptase. Subsequently, the newly synthesized cDNA is amplified using traditional PCR. This method is currently used in Nigeria to confirm Lassa virus infection [2][9].

Antibody Enzyme-linked-immunosorbent assay(ELISA)

ELISAbegin with a coating step, where the first layer, either an antigen or an antibody, is adsorbed to a well in a plate. Coating is followed by blocking and detection steps. Since the assay uses surface binding for separation, several washes are repeated between each ELISA step to remove unbound materials. During this process it is essential that excess liquid is removed in order to prevent the dilution of the solutions added in the next stage. For greatest consistency specialized plate washers are used [2][9].

Antigen detection tests

Antigen detection(ELISA) is particularly useful in providing early diagnosis as well as prognostic information. Level of antigenemia varied inversely with survival. The high sensitivity and specificity, capability for early diagnosis, and prognostic value of the ELISAs make them the diagnostic tests of choice for the detection of Lassa fever [2][9].

Virus isolation by cell culture

Cells from primary cultures can often be transferred serially a number of times. The cells may then continue to multiply at a constant rate over many successive transfers. Eventually, after a number of transfers, the cells undergo culture senescence and cannot be transferred any longer. For human diploid cell cultures, the growth rate declines after about 50 duplications. During the multiplication of the cell strain, some cells become altered in that they acquire a different morphology, grow faster, and become able to start a cell culture from a smaller number of cells. These cells are immortalized and have an unlimited lifespan. However, they retain contact inhibition [2,3,9].

Cell cultures are separated into 3 types:

Primary cells – prepared directly from animal or human tissues and can be sub cultured only once or twice e.g. primary monkey or baboon kidney.

Semi-continuous diploid cells – which are derived from human foetal tissue and can be sub cultured 20 to 50 times e.g. human diploid fibroblasts such as MRC-5 Continuous cells – derived from tumours of human or animal tissue.

Immunohistochemistry (IHC) involves the process of selectively imaging antigens (proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. IHC takes its name from the roots “immuno”, in reference to antibodies used in the procedure, and “histo,” meaning tissue (compare to immunocytochemistry). Albert Coons conceptualized and first implemented the procedure in 1941[2,3,9].

Immunohistochemical Staining

Immunohistochemical staining can be used for the diagnosis of Lassa fever in post-mortem samples. Immunohistochemistry is also widely used in basic research to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a biological tissue. In involves visualizingof Lassa virus antibody-antigen interaction which can be achieved in a number of ways especially by conjugating Lassa virus antibody to an enzyme which include peroxidase, that can catalyse a colour-producing reaction (immune-peroxidase staining) or the Lassa virus antibody can also be conjugated to a fluorophore, such as fluorescein or rhodamine (immunofluorescence) [2,3,9].

SITUATIONAL ANALYSIS OF LASSA FEVER IN NIGERIA

According to the reports of World Health Organization [3] Centre for Disease Control and Prevention [2] and Nigeria Centre for Disease Control [6] on Lassa fever in Nigeria;

1. Between 1^st of January and 4^th of February 2018, about 450 suspected cases were reported out of which 132 were confirmed by laboratory test (RTPCR). Of these, 43 deaths out of 450 suspected cases were reported while 37 of the 43 death were confirmed by Laboratory test as Lassa virus infection/Fever.

2. As at 13th February 2018, – The World Health Organization reported that the outbreak of Lassa fever has spread to 17 states and may have infected up to 450 people in less than five weeks.
3. Since the onset of the 2018 outbreak: there have been 134 deaths;95 in positive-confirmed cases, 9 in probable cases, 30 were negative to Lassa fever and the Case Fatality Rate in confirmed cases was 24.1%.
4. The Lassa haemorrhagic fever is endemic in Nigeria but more in southern states of Edo, Ondo and Ebonyi.
5. Among those infected are health workers some of whom have died from 1^st of January through 18^th of March, 2018; 17 health care workers in six states (Benue, Ebonyi, Edo, Kogi, Nasarawa, and Ondo) have been infected, four of whom have died.
6. 19 states have recorded at least one confirmed case across 56 Local Government Areas (Edo, Ondo, Bauchi, Nasarawa, Ebonyi, Anambra, Benue, Kogi, Imo, Plateau, Lagos, Taraba, Delta, Osun, Rivers, FCT, Gombe, Ekiti and Kaduna) between January and April 2018 in Nigeria.

7. Seven states have been reported to exit the active phase of the outbreak while 12 States still remain active as at April, 2018.
8. Between 19^th and 25^th of March, 2018: 18 new confirmed cases were recorded from 10 States as listed below: Edo (5), Ondo (2), Bauchi (1), Ebonyi (2), Taraba (3), Plateau (1), Kogi (1), Osun (1) FCT (1) and Gombe (1).6 new deaths in confirmed cases within the period were reported from; Edo (1), Taraba (2), Ebonyi (1), Plateau (1) and Gombe (1)
9. Generally, between 1st January to 25th March 2018, a total of 1613 suspected cases were reported of these include; 394 confirmed positive, 9 were probable, 1198 were negative (not a case) and 12 were awaiting laboratory results (pending).
10. However, since mid-February, there has been a downward trend in the weekly reported number of Lassa fever[2]
11. Though Nigeria witnessed the largest Lassa fever outbreak at the beginning of 2018: but after more than 100 deaths and almost 400 confirmed infections, Nigeria in April, 2018 recorded a sharp decline in the spread of Lassa fever as reported by Nigeria Centre for Disease Control.Only five new confirmed cases of Lassa fever were reported in the week that ended on the 15th of April 2018, according to NCDC [6].

FACTORS CONTRIBUTING TO THE TIDE OF LASSA FEVER IN NIGERIA

1. Inadequate facilities for Laboratory Diagnosis [8]
2. Poor healthcare services

A Situational Analysis of Lassa…

3. Inadequate Personal Protective Equipment and Hospital Infrastructure
4. Poor inter-professional relationship among healthcare professionals
5. Political instabilities
6. Insurgencies
7. Poor Interest in Rodent Control
8. Poor health policy plans and implementation as it affects the control of infectious diseases
9. Uneven distribution of healthcare resources
10. Being Ignorant of the fact that the Virus is also transmitted from Human to Human
11. The problem of knowledge gaps in Health

Workers[2, 3, 9].

Public Health Response

1. Activation of National Lassa fever Emergency Operations Centre (EOC) that coordinate response activities in collaboration with WHO and other partners.
2. Training of Health Care Professionals on the diagnosis and treatment of Lassa fever by World Health Organization, Nigeria Centre for Disease Control, Federal Ministry of Agriculture and Rural Development, Irrua Specialist Teaching Hospital, African Field Epidemiology Network, US Centres for Disease Control, University of Maryland Baltimore (UMB) and Alliance for International Medical Action (ALIMA)

3. Collaborative response between World Health Organization, Nigeria Centre for Disease Control, Federal Ministry of Agriculture and Rural Development, Irrua Specialist Teaching Hospital, African Field Epidemiology Network, US Centres for Disease Control, University of Maryland Baltimore (UMB) and Alliance for International Medical Action (ALIMA) on Lassa fever preventive Health Care.

4. NCDC is collaborating with the World Health Organization (WHO), Federal Ministry of Agriculture and Rural Development, Irrua Specialist Teaching Hospital, African Field Epidemiology Network, US Centres for Disease Control, University of Maryland Baltimore (UMB), Alliance for International Medical Action (ALIMA) and other agencies, in supporting the response in the affected States

5. NCDC has generated a comprehensive incident action plan to guide response activities and inform priority areas for collaboration with partners and resource mobilization.
6. WHO is supporting the Ministry of Health to reduce the outbreak by: Finding new cases, quickly so that they can be isolated, treated and stop further spread, procuring medical supplies and equipment and Supporting public health education campaigns
7. NCDC is collaborating with a non-governmental organization, the Alliance for International Medical Action (ALIMA), to support the treatment centres in Owo and Irrua; and with Médecins Sans Frontières (MSF) to support IPC interventions (Personal Protective Equipment (PPE) and training) in Abakaliki. WHO case management/IPC team has provided training to medical staff at Abakaliki and Irrrua.
8. NCDC, with WHO support, continues to supply PPE to all Lassa fever treatment centres.
9. Staffs from Irrua Specialist Teaching Hospital are providing clinical case management advice to other hospitals with suspected cases, and a 24hour Lassa fever case management call line has been established. A Lassa fever committee has been established in Abakaliki to improve the care of patients affected by Lassa fever.
10. NCDC has deployed risk communication and community engagement teams to Edo, Ondo and Ebonyi to promote personal and community hygiene, and appropriate health seeking behaviour. Mechanisms are being set up to better understand and respond to community concerns.

CONCLUSION

Lassa fever is a deadly haemorrhagic fever caused by Lassa virus transmitted by rats with persistent outbreaks in Nigeria of recent especially during the dry season. Government at various levels, communities, organizations and individuals should generate policies and habits targeted at reducing the tide of the infection and make the Laboratory tests for Lassa fever including other haemorrhagic fevers accessible to the populace

RECOMMENDATIONS

1. Intensive awareness campaign for the eradication of Lassa fever
2. Accessibility to Laboratory tests

Adequately equipped screening and confirmatory laboratories for Lassa fever including other haemorrhagic fevers should be established by the government in each of the local governments of Nigeria. Provision for Free Laboratory tests by Government and donor agents

3. Reconstitution of Nigeria Centre for Disease Control

(NCDC)

The Nigeria Centre for Disease Control must be restructured to accommodate more of Medical Laboratory services and Scientists.

REFERENCES

1. Donaldson, Ross, I. (2009). The Lassa Ward:One Man’s Fight Against One of the World’s Deadliest Diseases. St. Martin’s Press. ISBN 0-312-37700-2. ISBN 978-0-312-37700-7.
2. Centres for Disease Control and Prevention (2018). Lassa Fever in Nigeria. https://wwwnc.cdc. gov/travel/notices/watch/lassa-fever-nigeria
3. World Health Organization(2018) Lassa Fever – Nigeria Disease outbreak news http://www. who.int/csr/don/01-march-2018-lassa-fevernigeria/en/
4. Sogoba, N., Feldmann, H.,Safronetz, D. (2012). “Lassa Fever in West Africa: Evidence for an Expanded Region of Endemicity”. Zoonoses & Public Health. 59 (59): 43–47. doi:10.1111/j.18632378.2012.01469.
5. Yun, N., Walker, D. (2012). “Pathogenesis of Lassa Fever”. Viruses. 4: 2031–2048. doi:10.3390/ v4102031. PMC 3497040? Freely accessible. PMID 23202452.
6. Nigeria Centre for Disease Control NCDC) (2018) Lassa fever outbreak achieves http://ncdc.gov.ng/
7. David G., Barbara K., Eric, J. D. (2018). What Paediatricians Should Know About Lassa Virus. JAMA Pediatr. 172(5):407-408. doi:10.1001/ jamapediatrics.2017.5223
8. Olaniyan M.F and Afolabi, T. (2017)Accessibility to laboratory diagnosis of Lassa fever in Nigeria: a possible threat to the control of infectious diseases International Journal of Current Medical and Pharmaceutical Research, Vol. 3, Issue, 08, pp.21112115.
9. Asogun, D. A., et al. (2012). Bausch, Daniel G, ed. “Molecular Diagnostics for Lassa Fever at Irrua Specialist Teaching Hospital, Nigeria: Lessons Learnt from Two Years of Laboratory Operation”. PLoS Neglected Tropical Diseases. 6 (9): e1839. doi:10.1371/journal.pntd.0001839. PMC3459880? Freely accessible. PMID 23029594.

10. Federal Ministry of Health (2018), Nigeria

The post A Situational Analysis of Lassa Fever In Nigeria appeared first on Nigerian Biomedical Science Journal.

Akor-Dewu M. B., Wada, H.U.,and Abdulkareem,J.
Department of Human Physiology, Faculty of Basic Medical Sciences,  College of Health Sciences, Ahmadu Bello University, Zaria, Kaduna State, Nigeria.

 Ciroma,F.L.
Department of Human Physiology, Faculty of Basic Medical Sciences,College of Medicine, Kaduna State University, Kaduna State, Nigeria.

All correspondence to: E-mail: maryamdewu02@gmail.com;

ABSTRACT

The aim of this study is to investigate the effects of cough syrup containing codeine on lipid peroxidation, some oxidative stress biomarkers (catalase (CAT) activity, superoxide dismutase (SOD) activity, glutathione (GSH) concentration) and haematological indices (red blood cell count(RBC), white blood cell count (WBC), packed cell volume (PCV) and haemoglobin concentration (Hb)) in female Wistar rats. Twenty female Wistar rats were grouped into four groups, with five animals in each group. Group 1 (control) received 10ml of normal saline/kg body weight, whilegroups 2, 3 and 4 received 10.9 5 mg/kg, 21.90 mg/kg and 43.80mg/kg Benylin® with codeine respectively for 14 days, all administrations were given via oral gavage.After 14 days of administration the animals were sacrificed, 5mls of blood was collected by cardiac puncture and serum collected by centrifugation wasanalysed for antioxidant enzymes by colorimetric enzymatic methods. Whole-blood collected was used to manually analyse for some haematological parameters. The result showed no significant difference (p > 0.05) in MDA, SOD, GSH, RBC, WBC, PCV and Hb concentration between the groups, but catalase activity was significantly (p < 0.05) increased in group 3 (8.028±0.213u/mg) and group 4 (7.668±0.390u/mg) when compared to control (3.834±0.750u/mg). In conclusion, increase in catalase activity was observed at higher concentrations (21.9mg/kg and 43.8mg/kg) of Benylin® with codeine.

Keywords: oxidative stress, haematology, cough syrup, codeine.

INTRODUCTION

Drug misuse refers to the use of drugsor prescription medicationfor alternative purposes from its original prescription, stimulating effects such as mood alteration or intoxication (Powell, 2011). Drug abuse may be defined as the “arbitrary” overdependence or misuse of drugs with or without a prior medical diagnosis from qualified health practitioners (Lakhanpal and Agnihotri, 2007).  Substances of abuse are categorized as: stimulants, hallucinogens, narcotics, depressants (sedatives and tranquilizers) cannabis, inhalants and anabolic steroids (Bassiet al., 2017).

The most commonly abused cough syrups are those containing Codeine. Codeine is an opiate used for its analgesic, antitussive and antidiarrheal properties, acting centrally to elicit its pharmacological effects (Uthmanet al., 2016). Despite its clinical benefits, dependence on codeine when it is abused or misused is a potential problem,according to Lawan and Adie (2012),“the abuse of cough syrups in Nigeria,especially among women of youthful and adult age is quietlyspreading like wild fire.” Addiction to codeine syrup is turning the otherwise conservative girls and women of the north into social miscreants and rebellious housewives. It is increasingly becoming common to see girls and women at ceremonies and social activities where they drink codeine allowing them to forget about all inhibitions.Women mix these drugs in drinks during ceremonies and share it among themselves, the goal is to get tipsy and have courage to dance and talk vulgarly. The major drug abused by girls and women is cough syrup containing codeine, which they take in large quantities to make them tipsy and almost impervious to pain, just the way narcotics do (Lawan and Adie, 2012; Uthmanet al., 2017).The potential for overuse and misuse of codeine containing medications is not only detrimental to a person’s health but has economic and social implications (Feinberg, 2006).

The misuse, abuse and dependence on codeine products present in a range of over the counter medicines that are dispensed to the public without prescription in now public health challenge throughout the world (Cooper, 2013).Therefore the aim of this study is to investigate the effects of cough syrup containing codeine on lipid peroxidation, some oxidative stress biomarkers and haematological indices. 

METHODOLOGY

Twenty (20) female Wistar rats weighing 150-200g were purchased from the National Veterinary Research Institute, Vom, Jos, PlateauState. The animals were housed in the Animal House of the Departmentof Human Physiology,

Faculty of Medicine, Ahmadu Bello University, Zaria and The Effect of Sub-acute Administration of Cough…

were acclimatized for 2 weeks before the commencement of the experiment. The animals weremaintained on standard diet (vital feeds) and water. Experiments were carried out between 9:00am and 12:00 pm daily in accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the National Institutes of Health.Weighing of the animals were done continuously each day before administration of the drug.Commercial Benylin® with Codeine was donatedfor this research from a licenced Pharmacy in Zaria, Kaduna State Nigeria.

Experimental Design

The animals were divided into four groups each weighing 150 ± 50 gram. Their weights were taken every day for the period of two (2) weeks before administration. The drug were administered orally to the rats by carefully inserting the cannula into the oral cavity of the rats. Animals of group 1 were treated with 1ml/kg normal saline which served as the control, while animals in group 2,3 and 4 received single daily dose of 10.95mg/kg, 21.90mg/kg and 43.8mg/kg of Benylin^® with Codeine Syrup adopted from Tijjaniet al.,(2012). At the last day of administration, the animals were sacrificed using ketamine hydrochloride and blood samples (5mls) were collected from each subjectby cardiac puncture. An average of2.5ml of each blood sample wasstored in plain tubes for 20 minutes, then centrifuged to collect the serum which was usedfor evaluation of biomarkers oxidative stress.The remaining and 2.5mls of each blood sample was stored in EDTA bottles for RBC, WBC, PCV and Hb estimation.

METHODS

Biomarkers of oxidative stress were evaluated by colorimetric enzymatic methods as follows:

Malondialdehyde by Okhawaet al.(1979); catalase activity by Aebiet al. (1974); superoxide dismutase activity by Maetinet al. (1987); glutathione concentration by Rajagopalan et al. (2004). Haematological parameters were measured by methods described by Lewis et al.(2006).

Statistical Analysis

Data obtained were processed and One Way analysis of variance (ANOVA) was used to check for significant differences. Tukey’s post-hoc test was implored to determine the level of significance. Results are expressed as mean ± SEM. Values of p<0.05 were considered significant.Graphpad prism 6.was used for the analysis

RESULTS

There was a significant increase for catalase concentration in group 3 and 4 (table 1) when compared to the control, while SOD, MDA, GSH and TP showed no significant difference in their concentration between the groups. There was no significant difference (table 2) in blood parameters investigated.

DISCUSSION

Results obtained from the present study showed no significant difference in the level of activities of MDA,SOD and GSH when the experimental groups were compared with control. This may indicate that the antioxidant enzymes MDA,SOD and GSH may not be affected by codeine administration in rats, in this study. However, a significant increase was observed in catalase activity, and this may suggest that catalase may be a more sensitive antioxidant enzyme when codeine is being administered. Again, the finding may alsopropose that catalase is a better tool to monitor in patients administered with codeine.Chattopadhyay et al.,(2007) reported that an increase in some antioxidant enzymes activities such as SOD and CAT, may be indicative of the failure of compensation in oxidative stress. These enzymes scavenge excess O₂^– , H₂O₂, and superoxide anion radical to H₂O₂, the resultinghydrogen peroxide in turn is decomposed by the enzymes Gpx and CAT leading to functional changes induced by radical over- production and an increase in the biosynthesis of antioxidant enzymes (Vendittiet al., 2003). There was no significance in haematological parameters when compared to control but a sedating and calming effect was noted in the rats during the period of administration. This behaviour can be compared to that of Tijjaniet al., (2012) where they reported decreased activity

(hypoactivity) in mice treated with single doses of

21.90mg/kg and 43.80mg/kg of Benylin^® with Codeine.Sacerdoteet al. (1997) reported that codeine possesses a weak antinociceptive effect and limited immunosuppressive activity, this could reflect the result of no significance in haematological parameters

The insignificant change in haematological parameters caused by Benylin with Codeine suggests that the immune system might not have been compromised, eventhough other immune responses were investigated.

Conclusion

Higher concentrations of Benylin with Codeine administration elicited a rise in the activity of catalase, but not with malondialdehyde, superoxide dismutase, or glutathione in rats. In addition, values of RBC, WBC, PCV and Hb were not significantlyaffected by administration of Benylin with Codeine in this study.

REFERENCES

1. Aebi, H., Wyss, S. R., Scherz, B. and Skvaril, F. (1974). Heterogeneity of erythrocyte catalase II. European Journal of Biochemistry, 48: 137-145.
2. Antai-Otong, D. (2008). Psychiatric Nursing:

The Effect of Sub-acute Administration of Cough…

Biological and Behavioral Concepts. Canada: Thompson Delmar Learning 2^ndEdition  p10.

3. Bassi, A. P., Idoko, L., Ogundeko, T. O., Ramyil, M.S.C., Abisoye-Ogunniyan, A., Ogbole, E. A.,

Thilza, S. A., Uredo’ojo, S., Ante, E., Baba, A., Bassey, I. and Chimbuoyim, I. N.(2017) Substance Abuse and its Prevalence Among Secondary School Adolescents in Kagoro, Kaduna State, Nigeria. World Journal of Research and Review, 5(1): 11-16.

4. Barrett, S.P., Meisner, J.R., and Stewart, S.H. (2008). What constitutes prescription drug misuse Problems and pitfalls of current conceptualizations. Current Drug Abuse Review,1 (3): 255–62. doi:10.2174/ 1874473710801030255.
5. Cooper, R.J. (2013). Over-the-counter medicine abuse: a review of the literature. Journal of Substance Use, 18(2), 82–107. doi:10.1016/j.drugalcdep. 2005.10.020
6. Chattopadhyay, S., Sahoo, D.K., Subudhi, U., and Chainy, G. B. (2007). Different expression profiles of antioxidant enzymes and glutathione redox status in hyperthyroid rats: a temporal analysis.

Comprehensive Biochemistry of physiology, 146:383391.

7. Lewis S.M., Bain,B. J., and Bates I. (2006).Dacie and Lewis Practical Haematology.10th Edition, Elsevier, Philadelphia, pp. 26-54
8. Dankani I. M. (2012). Abuse of cough syrups: a new trend in drug abuse in north-western Nigerian states of kano, Sokoto, Katsina, Zamfara and Kebbi. International Journal of Physical and Social Sciences. 2(8): 199-213.
9. De Gruchy, G.C (1976). Clinical haematology in Medical Practice. Blackwell Scientific Publication. Oxford, London Pp. 33-57.
10. Feinberg, D. T. (2006). The cost of over-the-counter substance abuse. Journal of Child & Adolescent Psychopharmacology, 16(6), 801-802.
11. Lakhanpal, P. and Agnihotri, A. K. (2007). Drug

Abuse an International Problem: A short Review with Special reference to African Continent. Journal of Medicine and Toxicology, 1(1), 1-11

12. Lawan, D. A. K. and Adie, V. O. (2012). Nigeria: Codeine Abuse Spreads Among Kano Women. Daily Trust. 21 July 2012. Daily Trust (Abuja)
13. Martin, J.P., Dailey, M. and Sugarman, E. (1987). Negative and Positive Assays of Superoxide Dismutase Based on Hematoxylin Autoxidation,

Archives ofBiochemistry and Biophysics 255:329-336

14. McCabe, S.E., Boyd, C.J., and Teter, C.J. (2009). Subtypes of nonmedical prescription. Drug Alcohol Dependence102 (1–3): 63–70. doi:10.1016/j. drugalcdep.2009.01.007.
15. Nielsen, S., Cameron, J., and Pahoki, S. (2010). Over the Counter Codeine Dependence. Melbourne,

Australia: Turning Point Drug and Alcohol Centre.

16. Ohkawa, H., Ohishi, N. and Yagi, K. (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry, 95(2):351-8.
17. Powell, J. (2011). Drug misuse and addiction. Available at: https://www.netdoctor.co.uk/healthyliving/wellbeing/a5013/drug-misuse-and-addiction/ [accessed 2018]
18. Rajagopalan, R., Kode, A., Penumatha, S. V., Kallikat, N. R. and Venugopal, P. M. (2004). Comparative effects of curcumin and an analog of curcumin on alcohol and PUFA induced oxidative stress. Journal of Pharmacy and Pharmaceutical Sciences, 83:27472752.
19. Tijani, A.Y., Salawu, O.A., John-Africa, L.B., Sadiq, A., and Chindo, B.A (2012). Behavioural effects of Benylin-Codein in mic. Department of Pharmacology and Toxicology National Institutefor Pharmaceutical Research and Development Idu industrial Area Abuja, Nigeria. Nature and Science, 10(4).
20. Umar, L. Y., Waziri, A. G. and Musa, A. Drug Abuse among Youths In Nigeria: Implications To National Development. retrieved from www.unimaid.edu. ng/social/general on Wednesday 15th June, 2018.
21. Uthman S. G., Zakama G. S., Modu B. M. and Usama, B. U. (2017) A Survey of the Usage and Users of Codeine Containing Cough Syrups in Maiduguri Nigeria. Advances in Psychology and Neuroscience, 2(2-1): 42-46.
22. Uthman, G. S., Danjuma, C. U., Usama, U. B. and Jidda, M. S. (2016). A Study of The Utilization Of

Cough Syrups Containing Codeine Among Drug

Abusers In AskiraUba Local Government Area Of Borno State Nigeria. Kanem Journal ofMedical Sciences, (10)2: 73-82.

23. Venditti, P., De Rhosa, R., and Di Meo, S.(2003). Effect of thyroid state on H₂O₂ production by rat liver mitochondria. Molecular Cell Endocrinology, 205:185-192.
24. World Health Orgnization (2006) Lexicon of Alcohol and Drug Terms Published by the World Health Organization. Available at: http://www.who.int/ substance_abuse/terminology/wh_lexicon/en/ [accessed 2018].

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