Indonesian Journal of
Medical Laboratory Science and Technology
Volume 2 Number 1, April 2020
EDITORIAL TEAM Editor in Chief
Maharani Pertiwi K
(Biomolecular, Universitas Nahdlatul Ulama Surabaya, Indoneisa)
Associate Editor
Samsul Bahari Shamsudin
(Environmental and occupational health, Universiti Malaysia Sabah, Malaysia)
Journal Editor
Achmad Syafiuddin
(Toxicology, Universiti Teknologi Malaysia, Malaysia) Fildzah Karunia Putri
(Clinical Nutrition, Universitas Nahdlatul Ulama Surabaya, Indonesia) Agusniar Furkani Listyawati
(Microbiology, Universitas Wijaya Kusuma Surabaya, Indonesia)
Peer Reviewers
Win Darmanto
(Human fisiology, Airlangga University, Indonesia) Ika Nindya Kadariswantiningsih
(Microbiology, Airlangga University, Indonesia) Hotimah Masdan Salim
(Cell biology, Universitas Nahdlatul Ulama Surabaya, Indonesia) Firdaus Hayati
(Surgery, Universiti Sabah Malaysia, Malaysia) Umi Hamidah
(Microbiology and Biotechnology, Indonesian Institute of Sciences, Indonesia) Budi Santosa
(Hematology and Immunohematology, Universitas Muhammadiyah Semarang, Indonesia) Dewi Noor Hidayati
(Virology, Farma Veterinary Center, Indonesia)
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Gilang Nugraha
(Medical Laboratory, Nahdlatul Ulama University of Surabaya, Indoneisa)
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E-mail: [email protected] Website: http://journal2.unusa.ac.id/index.php/IJMLST
p-ISSN 2684-6748 e-ISSN 2656-9825
Content
Indonesian Journal of Medical Laboratory Science and Technology Volume 2 Number 1, April 2020
A Powerful ELISA Technique to Test The Potential of Extra Virgin Olive Oil in Reducing TNF-α Level and Edema Volume in Male Rattus norvegicus Exposed to Carrageenan
Andina Putri Aulia, Suprapto Maat, Aryati
1 – 10
Blood Lead Concentrations and The Neuropsychology Scores of Pregnant Women in Klang Valley, Malaysia
Shamsul Bahari Shamsudin, Jamal Hisham Hashim, Nik Nasri Nik Ismail, A. Jamal A. Rahman, Maharani Pertiwi Koentjoro
11 – 20
Cytotoxicity Assay Using Brine Shrimp Lethality Test on Collagen-Chitosan Wond Dressing Sterilized by Ultraviolet Light
Ary Andini, Endah Prayekti, Devyana Dyah Wulandari, Ersalina Nidianti
21 – 26
Invitro Citotoxicity Assays of Seagrass (Enhalus acoroides) Methanol Extract from Soropia Coastal Waters Southeast Sulawesi Regency
Theosobia Grace Orno, Agnes Rantesalu
27 – 33
Laboratory Trial of Protein Determination in Urine Using Different pH Values of Acetic Acid and Acetate Buffer Method
Dinar Rahayu, Tuti Rustiana
34 – 41
The Effect of Environmental Factor and Use of Personal Protective Equipment on Symptoms of Acute Respiratory Tract Infections in Workers Furniture Industry
Merry Sunaryo
42 – 49
p-ISSN 2684-6748 e-ISSN 2656-9825
1
A Powerful ELISA Technique to Test The Potential of Extra Virgin Olive Oil in Reducing TNF-α Level and Edema Volume in Male Rattus norvegicus Exposed to Carrageenan
Andina Putri Aulia1, Suprapto Maat2, Aryati3
1Department of Clinical Pathology, Faculty of Medicine, Universitas Islam Sultan Agung, Semarang, Indonesia
2Department of Medical Laboratory Technology, Faculty of Health, Universitas Nahdlatul Ulama Surabaya, Surabaya, Indonesia
3Department of Clinical Pathology, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia Correspondence:
Andina Putri Aulia,
Jl. Kaligawe Raya Km.4 Semarang, Central Java, Indonesia
Zip Code : 50112
Email: [email protected] Received: November 30, 2020 Revised: March 3, 2020 Accepted: March 31, 2020
Abstract
Extra virgin Olive oil is extracted from fruit that can be used as anti-inflammatory agent. This research aimed to test the potential of extra virgin olive oil in reducing edema volume and TNF-α plasma in carrageenan-induced rats. This research was purely experimental research with the post test control group design. A total of twenty eight Wistar rats were divided randomly into four treatment groups. Group I was a control negative group, while the group II, III, and IV were orally administered with extra virgin Olive oil at the dose of 0.9 ; 1.8 ; 2.7 mL/day, respectively. Paw edema was measured one hour before the rats was induced to carrageenan and every hour until four hours after it was induced to carrageenan. TNF-α plasma was measured at four hours. Analysis of the data was done by calculating the presentation of edema inhibition in every group, then the data was statistically analyzed by Anova, Repeated Anova, LSD and Kruskal Wallis test with 95% confidence interval.
The result showed that extra virgin olive oil has an anti- inflammatory effect. The highest decrease in edema volume percentage was in group III (14.21%). There was a significant difference in the edema volume of all treatment groups at each time of the experiment with TNF-α value (p
< 0.05). In conclusion, the administration of extra virgin olive oil can lower the volume of carrageenan-induced edema in rats depend on the dose. Also, the administration of extra virgin olive oil can be dose-dependent in reducing the levels of TNF-α in carrageenan-induced edema in rats.
Keywords
Anti inflammation, extra virgin olive oil, edema volume, TNF-α, carrageenan
INTRODUCTION
Inflammation is a physiological response to various stimuli such as infection, irritation or tissue injury. Inflammation is also known as a type of non-specific immune response,
and it involves many mediators (1,2).
Inflammation is a beneficial response but it can be detrimental to the host because it contributes to numerous pathogenesis of diseases including allergic, autoimmune,
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infectious, heart disease, arthritis, osteoporosis, diabetes, myopathy and cancer (3,4).
During the inflammatory process, pro- inflammatory and cytokines are secreted. The vascular reactions cause fluids, blood elements, leukocytes, mediators and pro- inflammatory cytokines to accumulate at the site of injury to remove harmful agents and to repair damaged tissue. The cytokines are immune system proteins regulating interactions between cells and stimulating immune reactivity, either in a specific or non- specific immunity (2,5). Inflammatory cytokine is a small peptide secreted primarily by macrophages and lymphocytes that will activated the tissue in response to trauma stimuli, such as endotoxin, immune complexes, physical and chemical trauma (3,6). The pro-inflammatory mediators respond to various stimuli, including bacterial lipopolysaccharide (LPS), cytokines, and UV radiation that will further induces the activation of Nuclear factor- kappa B (NF-kB) and activator protein-1 (AP-1). The NF-kB activates a number of molecules involved in the inflammatory response (proinflammatory cytokines), including iNOS, COX-2, TNF-α, IL-1β, and IL-6 (7,8). Meanwhile, tumor Necrosis Factor-α (TNF-α) is a cytokine that has a different reaction in different cells. It involved in all process of inflammations and it can be used as an indicator of oxidative
stress, apoptosis or necrosis that occur in the cells. This cytokine induces acute phase reaction and activates the vascular endothelium, leukocytes, platelets and fibroblasts, so the cascade of inflammatory process is initiated by vascular, cellular and humoral immune system (6,9). The vascular changes due to pro inflammatory cytokines induction that will cause the movement of fluid to interstitial tissue called edema, one of the cardinal signs of inflammation (3).
The prevalence of inflammation is associated with place, race and disease. Non- steroidal anti-Inflammatory drugs (NSAIDs) is one of the most commonly prescribed for the treatment of inflammation (10). Thirty million tablets of non-steroidal anti- inflammatory drugs are sold in the United States annually. This number reflects the dependency on anti-inflammatory drugs (4).
Additionally, the incidence of inflammatory disease such as osteoarthritis and gout disease has increased. More than fifty percent of NSAIDs are administrated to patients over 60 years old, so it leads to the increase in the side effect of NSAIDs.
There are several ways to prevent or slow down the progress of inflammation, either by using drugs or medicinal plants. Up to now, it is estimated that the Indonesian people still use a variety of plants for an alternative therapy. The use of plants as drugs are expected to have relatively low side effects compared to anti-inflammatory drugs. The
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long term use of NSAIDs can cause erosion and bleeding in the lower gastrointestinal tract. It was reported that NSAIDs cause injury and affect the integrity of the mucous membrane of the gastrointestinal tract (10).
One of the plants widely used as a drug is olive’s fruit. Olive can be processed into olive oil, virgin olive oil and extra virgin olive oil. Extra virgin olive oil has different characteristic from other types of olive oil because of its refining process and its composition (11). Olive oil is used as a dietary component by the Mediterranean community to reduce the risk of illness and death. The Mediterranean people highly value the high oleic acid in olive oil as well as its minor components; the phenolic compounds (12). Phenolic compounds in extra virgin olive oil have been shown to have anti-inflammatory and anti-oxidant properties, as well as anti-microbial activity.
Various phenolic compounds in extra virgin olive oil play an anti-inflammatory role in decarboxy methyl ligstroside aglycone (oleocanthal), hydroxytyrosol, flavonoid, and oleoropein (13). The phenol compound of extra virgin olive oil has been shown to decrease the concentration of Interleukin-6 (IL-6), a pro-inflammatory cytokine secreted by response to trauma. Other studies have shown that phenolic compounds in olive oil can inhibit the cyclooxygenase-2 (COX-2) activity. Impellizzer et al. also reported that oleuropein could reduce TNF-α, IL-1β and
NO in carrageenan-induced pleura in rats (8,14). Based on the afore mentioned reasons, the researcher wanted to analyzed the effects of extra virgin olive oil on carrageenan- induced paw edema volume and levels of TNF-α in rats.
MATERIALS AND METHODS This research method is an experimental with post-test only design only randomized control group design. The population in this study were 28 male Wistar rats (Rattus norvegistus) aged 2 - 3 months, weighing about 200 grams. Animals were acclimatized for 7 days and were divided into 4 groups.
Group I was the control group. Group II, III and IV were orally administrated with a single dose of extra virgin olive oil at 0.9 mL/day, 1.8 mL/day, and 2.7 mL/day, respectively. All groups were given 2% of carrageenan injection (0.1 mL).
One hour before the injection of carrageenan, all labolatory animals were subjected to paw volume evaluation. The Extra virgin olive oil was produced in Italy with the brand Bertolli. It was administrated 30 minutes after carrageenan injection, and was followed by edema paw volume measurement at h1 (after 1 hour), h2 (after 2 hour), h3 (after 3 hour) and h4 (after 4 hour) after injection. In h4, all groups were sacrificed under ether anesthesia. Blood samples were taken through the heart after surgery. The blood samples were centrifuged
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at 1500 rpm for 30 minutes to obtain serum for TNF-α levels analysis. This research has been approved by the Research Ethics committee of the Faculty of Veterinary Medicine, Airlangga Animal Care and Use Committee (ACUC) with the Ethical Clearance Number 576-KE.
All data was analyzed using Saphiro wilk normality test (p > 0.05) and homogeneity of variance Levene's (p > 0.05). The differences between unpaired groups were analyzed using ANOVA, REPEATED ANOVA and LSD test for rat paw edema volume, and one- way ANOVA (p < 0.05) and LSD (p < 0.05) test for the TNF-α. All statistical tests were performed with SPSS program.
RESULTS
The increased volume of edema (Table 1) showed that the control group experienced an increase in edema volume during the first,
second and third hours after induction of carrageenan. Afterwards, there was a decrease in edema volume at the h4. In Group II and III, edema volume increased up to 1 hour after injection of carrageenan, and it began to decrease in the second hour after the injection of carrageenan (Figure 1). The groups were sacrificed by anesthetizing using ether, and the blood samples were taken through the heart after surgery. Blood samples were centrifuged at 1500 rpm for 30 min to obtain serum for TNF-α levels analysis.
In Group IV, there was an increase in edema volume occurred shortly after the injection of carrageenan (t0) and there was a decrease in edema volume at 1 hour after injection of carrageenan (t1). Table 1 shows the percentage of a decline in edema volume in each group. The percentage of reduction in edema volume in the group IV was 14.21%.
Table 1. Mean and Standard Deviation of Rat’s Paw Edema Volume (mL)
Group h-1 h0 h1 h2 h3 h4 ΔVolume
(%) K* 2.63 ± 0.40 3.34 ± 0.46 3.42 ± 0.45 3.48 ± 0.44 3.6 ± 0.40 3.29 ± 0.54 10,25 ± 5,6 P1* 2.53 ± 0.38 3.14 ± 0.29 3.17 ± 0.36 2.99 ± 0.33 2.88 ± 0.44 2.87 ± 0.37 11,8 ± 11,6 P2* 2.47 ± 0.22 3.13 ± 0.23 3.22 ± 0.27 3.06 ± 0.23 2.94 ± 0.2 2.8 ± 0.25 13,28 ± 6,0 P3* 2.59 ± 0.31 3.23 ± 0.30 3.16 ± 0.33 3.16 ± 0.29 3.08 ± 0.33 2.85 ± 0.28 14,21 ± 4,5
*Anova test : p < 0.05
Fig 1. Mean Increase in Edem Volume (mL)
2,00 2,50 3,00 3,50 4,00
t before t0 t1 t2 t3 t4
CONTROL KP1 KP2 KP3
Interval time of measurement (hour) Mean edema volume (mL)
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The ANOVA test showed a significant difference in all groups. Thus, there was a significant difference among the time of administration in each group. LSD test of edema volume variables showed that there was much difference in edema volume in the time between h-1 and h3 (p = 0.001). In the Group I, the most significant difference in edema volume was found between h-1 and h3 (p = 0.001). Meanwhile, in the treatment group III, the most different in edema volume was the time between h-1 and h1 (p = 0.001), and time between h-1 and h1 (p = 0.001).
Furthermore, In the treatment group IV, the most different in edema volume was the time between h-1 and h0 (p = 0.001).
A repeated ANOVA test was used to determine whether there were mean differences in the repeated measurements.
The results showed an overall significant difference between the time of measurement (p = 0.001) and a significant difference in the mean of edema volume between the groups based on the time of measurement (p = 0.013).
The LSD test based on the time of measurement that showed a significant difference were: the time group between h-1 and h0 (p = 0.001), h-1 and h1 (p = 0.001), h- 1 and h2 (p = 0.001), h-1 and h3 = 0.001), h- 1 and h4 (p = 0.001), h0 and h4 (p = 0.001), h1 and h3 (p = 0.019), h1 and h4 (p = 0.001), h2 and h4 (p = 0.001) and h2 and h4 (p = 0.001). There was a significant difference
between h-1 and h0 (p = 0.001). Therefore, it can be concluded that all groups has edema in the rat paw after injection of carrageenan.
The LSD results also showed that there were significant differences in edema volume based on the time of repeated measurement which was found between Group I and II (p
= 0.037), and Group I and III (p = 0.042).
The percentage of reduction in edema volume has a normal distribution yet the variance was not homogeneous, so that the different test used was Kruskal Wallis test.
The different test results of the percentage of reduction in edema volume after administration of extra virgin olive oil showed that there was no significant difference (p = 0.268) among the groups (Table 1). Thus, there was no difference in the percentage of reduction in edema volume in all of groups.
In the variable of TNF-α level (Figure 2), the highest mean was found in the control group (2,736.6 ± 1,535.2 pg/mL) while the lowest one was found in the group P3 (380.64
± 90.0 pg/mL) ANOVA test result P = 0.001).
It indicated that there were significant differences between groups. The result of LSD test showed that there was a significant difference between Group I and Group III (p
= 0.003), Group I and Group IV (p = 0.001), Group II and Group III (p = 0.033), Group II and Group IV (p = 0.001), as well as Group III and Group IV (p = 0.012). The most
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significant difference was between Group I and Group IV, along with Group II and IV.
Fig 2. Difference in The Mean of TNF-α
DISCUSSION
Anti-Inflammation Test of Olive Oil to reduce Edema Volume
Based on the Table 1, we can analyse the change of rat paw volume in each treatment group. In h0 (approximately 15 minutes after injection of 2% of carrageenan), there was an inflammation triggered by 2% of carrageenan as shown by the increasing volume of rat paw in all of groups. The control group showed a difference in the increase of edema volume of 0.71 mL compared to the previous leg volume at the time h-1. Group II, Group III and Group IV showed a difference in the increase of edema volume of 0.61 mL, 0.66 mL, and 0.63 mL, respectively. This research is in accordance with the research conducted by Hidayati et al. (5), showing that there was an increase in edema volume at 15 minutes after the injection of carrageenan (5).
Table 1 also showed that the largest dose of 2.7 mL of extra virgin olive oil administered to the group IV has the most rapid anti-inflammatory effect compared to that of the other treatment groups and has the smallest percentage in the increase of edema volume. The highest decrease in edema volume was the treatment in the group IV (14.21%).
Based on result in Table 1, there was a significant difference in edema volume between the measurement time in all of groups. This research is supported by Fezai et al (14), which stating that there was a significant effect on the volume of rat paw edema injected with extra virgin olive oil due to the olive oil phenolic compounds that can lower the prostaglandin level by inhibiting the Cyclooxygenase.
The inhibition of edema volume by extra virgin olive oil was due to the inhibition of
0 500 1000 1500 2000 2500 3000
K P1 P2 P3
TNF-α(pg/mL)
Group
TNF-α
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COX-2 which play an important role in the conversion of arachidonic acid to prostaglandin formation, so that the inflammation will not occur (15). During the inflammation, various inflammatory mediators including 5-hydrocytriptamine (5HT), chemotactic factors, bradykinin, leukotrien and prostaglandin are released.
Prostaglandins and prostacyclin cause erythema and vasodilatation as well as increase the local blood flow in vitro.
Histamine and bradykinin also play a role in the increase of vascular permeability yet the vasodilation effect is not as much as that in the prostaglandins. There is an inflammation in the initial phase due to the release of histamine, serotonin and other similar substances. Then, in the next phase, there is an activation of prostaglandins, protease, lysosomes and other quinine substances (16).
This research is also supported by Hidayati (5) study showing that flavonoids can decrease the volume of inflammatory edema- induced rat. Another study conducted by Favacho et al. (17) also said that oleic acid in Euterpe oleracea Martmay’s fruit has decrease the volume of rat paw edema injected with carrageenan (17).
The Repeated ANOVA test result shows overall significant differences between the measurement time (p = 0.001). There were significant differences in mean between repeated measurement edema volume period (p = 0.013) among the groups. The LSD test
results showed that there was a significant difference (p = 0.001) between the time group h-1 the time group h0 (shortly after the injection of carrageenan), indicating that there was a significant increase in rat edema volume (edema) after carrageenan injection.
The result of LSD test among all treatment groups based on repetition of measurement showed that there was a significant difference between group I and group II treated with olive oil at the dose of 0.9 mL. Furthermore, a significant difference was also obtained between group I and group III treated with olive oil at the dose of 1.8 mL while those in groups I and group IV treated with olive oil at 2.7 mL showed no significant difference. This result is consistent with the previous research elaborated by Fezai et al., (14) showing that the largest dose of extra virgin oil has no statistically significant difference. It is possible that the relation between the volume of carrageenan injected was slightly different in each animal so that the volume of edema was not similar. The results of different tests indicated the percentage of reduction in edema volume p = 0.480 (p > 0.05), meaning that there was no significant difference in the effect of extra virgin olive oil on the percentage of reduction of edema volume among the groups.
However, the clinical percentage of reduction in edema volume was greater along with the increasing dose. It is likely due to the absorption, distribution, metabolism and
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excretion of the active compounds in extra virgin olive oil will vary depending on the body. Therefore, the dose of olive oil in this study can not be used as a reference of effective dose of olive oil as an anti- inflammatory agent.
Anti-Inflammation Test of Olive Oil on TNF-α
The carrageenan injected in rat's paw involves several mediators. In the early phases of inflammation, the first detected mediators are histamine, serotonin and bradykinin. In the next phase, the detected mediator is prostaglandin, a mediator that causes an increase in the vascular permeability. Additionally, local or systemic inflammation due to carrageenan was associated with the increase in pro- inflammatory cytokines such as TNF-α, IL-1 and IL-6 (18). The administration of extra virgin olive oil containing phenolic compounds such as hydroxytyrosol, oleochantal and flavonoids can lower TNF-α levels through the blocking of IKK phosphorylation resulting in inhibition of the degradation of IKB proteosomal, thus preventing the activation of NF-kB (19). The level of TNF-α increased four hour after injection, it is based on the research conducted by Ogata et al. (20) showing that elevated levels of the highest plasma TNF-α present at the fourth hour in rats injected by carrageenan intraperitonially.
Furthermore, the results showed that the highest level of TNF-α was in the control group, and there was a decrease along with an increasing dose. This is in line with the research conducted by Amijaya et al. (21) which suggested that flavonoids may lower the levels of TNF-α in inflammatory-induced mice. The research conducted by Hardyanto et al. (22) also showed that flavonoids can decrease TNF-α in rats induced by urolithiasis.
The result of LSD test showed that there was a significant difference between control group and P2 group administrated with olive oil at the dose of 1.8 mL. Additionally, there was a significant difference between control group and P3 group treated with 2.7 mL olive oil. This statistic difference was not similar from clinically decreased levels of TNF-α.
The highest dose of olive oil has the highest anti-inflammatory effect in lowering plasma TNF-α levels. It is possible that the absorption, distribution, metabolism and excretion of active compounds of olive oil in the body vary depending on the microenvironment.
In a study conducted by Nugraheni (11), the percentage of oleic acid in olive oil was 77.478%. Oleic acid contained in olive oil acts as an anti-inflammatory agent by inhibiting COX-2 regulation. At 30-50 g of olive oil is consumed, with a concentration of 180 mg/kg of phenol compounds, it is estimated that 8 mg of phenolic compounds
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such as hydroxytyrosol, tyrosol, oleocanthal and flavonoids are absorbed (23).
The molecular mechanism of phenolic compounds as anti-inflammatory agents includes enzyme inhibition of pro- inflammatory, such as cyclooxygenase (COX-2) and lipoxygenase (LOX) and inducible nitric oxide synthase (iNOS). It involves the activation of the peroxisome proliferator activated receptor gamma (PPAR γ) and inhibition of nuclear factor-kappa B (NF-kB) (24).
The result of volume edema showed significant differences between Group I and group II, Group I and group III. Also, TNF-α levels showed significant differences between group I and group III as well as group I and group IV. This is presumably related to the capture point difference of each active compounds of extra virgin olive oil, causing differences in the effectiveness of extra virgin olive oil in various dosage levels of the edema volume and serum TNF-α.
Therefore, this study could not show the
effective dose of anti inflammatory response of extra virgin olive oil.
CONCLUSIONS
This study concludes that extra virgin olive oil can reduce the volume edema in rats according to the dose and it can be dose- dependent in reducing the levels of TNF-α in carrageenan-induced edema in rats.
Further studies are needed to determine the efectiveness and toxicity of the extra virgin olive oil dose that has an anti inflammatory activity. In addition, the purification, identification, and quantification of the active subtance in the extra virgin oil that has an anti inflammatory activity is also necessary to be analysed.
TNF-α tissue examination and immunohistochemistry examination of rat’s paw expression are also needed.
CONFLICT OF INTEREST There are no conflicts of interest.
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11
Blood Lead Concentrations and The Neuropsychology Scores of Pregnant Women in Klang Valley, Malaysia
Shamsul Bahari Shamsudin
1, Jamal Hisham Hashim
2, Nik Nasri Nik Ismail
3, A. Jamal A. Rahman
3, Maharani Pertiwi Koentjoro
41Department of Community and Family Medicine, Faculty of Medicine & Health Sciences, Universiti Malaysia Sabah, Sabah, Malaysia
2United Nations University – International Institute for Global Health (UNU–IIGH) Building, UKM Medical Centre, Kuala Lumpur, Malasyia
3Universiti Kebangsaan Malaysia Medical Centre, Kuala Lumpur, Wilayah Persekutuan, Malasyia
4Department of Medical Laboratory Technology, Faculty of Health, Universitas Nahdlatul Ulama Surabaya, Surabaya, Indonesia Correspondence:
Shamsul Bahari Shamsudin, Faculty of Medical and Health Sciences, Universiti Malaysia Sabah, Kota Kinabalu, Sabah.
Malasyia Zip Code : 88400
Email: [email protected] Received: March 13, 2020 Revised: March 16, 2020 Accepted: April 4, 2020
Abstract
Pregnant women with high blood lead posed high risk to their fetus as placental transfer can occurs to the fetus. The objective of this study was to identify the relationship between blood lead and the neuropsychological score of women who were in their 3rd trimester of pregnancy. These respondents were undergoing a routine antenatal checkup at a teaching hospital located in Klang Valley areas. Blood lead concentrations were analyzed using graphite furnace Atomic Absorption Spectrophotometer (AAS). The neuropsychological scores were measured with WHO Neurobehavioral Core Test battery (NCTB). The test consists of 7 items, which made up of the Digit Symbol, Trail Making, Digit Span, Benton Visual Retention Test, Pursuit Aiming, Santa Ana Manual Dexterity, Reaction Time and Movement Time tests. The mean blood lead was 7.78±4.77 µg/dL. The mean score for the total NCTB test was 50.00±5.24. Statistical analysis showed blood lead concentrations were inversely correlated with the total NCTB score (r= –0.462, p≤0.01). The correlation was about 21.3%. The General Linear Model (GLM) showed that age (β= –0.15, p = 0.017), weight (β = 2.67, p = 0.05) and height (β= –1.97, p = 0.05) also influence the total neuropsychological scores. In conclusion, blood lead reduces the total neuropsychological scores. The scores for each of the 7 items were inversely and significantly correlated with blood lead concentrations except for the Trail Making and Santa Ana Manual Dexterity tests.
Keywords
Neuropsychological scores, blood lead concentrations, pregnant mothers
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INTRODUCTION
Lead could be produced by human activities such as industrial, mining and agricultural activities that can spread by air, water and soil (1). Lead route of entry to human body through the inhalation, ingestion and skin contact. Lead has no function in human body. Previous study suggested lead has a negative effect on human health even though at very low level. Lead is a neurotoxin and its toxicity affect the function of the central nervous system and peripheral nerve.
For adults, the main effects are peripheral neuropathy whereby the impulse conduction in the nerve is slowed down. Peripheral neuropathies influence the motor and sensory nerve. The effects are the wrist and the ankle drop that are the result of the defect in the radial and peroneal nerves (1).
Pregnant woman is at very high risk to the lead toxicity because pregnant mothers need high level of nutrient such as calcium as well as iron to support the growth and the development of the fetus. Lead could replace the function of calcium and iron. Tests on the biochemical and physiological process found that the concentration of calcium and iron in the blood are inversely proportional to the lead concentrations (2).
Study shows blood lead levels (BBLs) in pregnant women at 20.8 μg/dL can cause maternal disorders, when BLLs reaches 2.57 μg/dL, it can cause stress and fatigue levels in pregnant women (4). BLLs <5 μg/dL may
cause pregnant women to have pre–
eclampsia and endanger the mother’s kidney, while BLLs <10 μg/dL can increase blood pressure or hypertension (4). Another research done by Bayat et al, 2016 showed there was an increase in systolic blood pressure (0.014 mmHg) and diastolic (0.013 mmHg) following the increase of 1 μg/dL of BLLs (p = 0.04) (5).
This study aims to investigate the association of BLLs with neuropsychology of pregnant women in Klang Valley, Malaysia.
Klang Valley area was an ex-mining erea in Peninsular Malaysia. Active mining activity was in 1950-1970. The residue of lead and others heavy metal was still high in air, water and soil. The Neurobehavioral Core Test battery (NCTB) can detect early signs of disturbance or failure on the functions of nervous system. WHO was introduced NCTB identifying the nervous dysfunction among workers who are working with the neurotoxin chemicals (3).
MATERIALS AND METHODS
The study population is women in their 3rd trimester of pregnancy who reside in the Klang Valley areas. These pregnant women were attending their routine antenatal checkup at a hospital in Kuala Lumpur. Total 202 respondents were selected through purposive sampling based on specific criteria, that she must be Malaysian citizen, in the 3rd trimester pregnancy and have
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signed consent to participate. Their pregnancies were categorized as “high–risk”
because they were referral cases from private or government clinics within the areas. These women were either in their 1st or 5th pregnancies, had hypertension, diabetes, anemia or a history of previous delivery complication.
Questionnaire interviews were carried out to collect information on the demographic as well as socioeconomic background and their health status. The questionnaire consists of the questions on their age, ethnicity, number of children, educational level, occupation and areas of residential. While the questions on health status include of number of deliveries, medical history, smoking habit, alcohol intake and medication during pregnancy.
Blood Sampling and Analysis
Venous blood was sampled during or after the 28 weeks of pregnancy. Five (5) ml of the blood samples were put into a special vacuum container tube, which contained heparin (anticoagulant). All sample of blood lead were analyzed using Graphite Furnace Atomic Absorption Spectrophotometer model HITACHI Z5700 with polarized Zeeman. Blood sample were diluted with matrix modifier solution in a ratio of 1:5. The modified matrix solution also acts as antifreeze agent during storage. The modified matrix solution consists of 10 mL Triton–X, 10% 0.3 g ethylene diamine tetra acetic acid
(EDTA) and 5 g ammonium dehydrogenate phosphate in 1–liter deionizer distilled water.
Lypocheck sample were used as a reference sample for quality control procedure during the analysis.
Neuropsychological Assessment
A neuropsychological test conducted in this study were Neuropsychological Core Test Battery (NCTB) and it is commonly used by WHO (3). It was an instrument used to detect early nervous systems failure because of the exposure of neurotoxin agents.
This instrument is very sensitive and can detect early sign of nerve disturbance at a very low level of exposure. It consists of the Benton Visual Retention, Digit Span, Digit Symbol, Pursuit Aiming, Reaction Time, Santa Ana Manual Dexterity and Profile of Mood States (POMS) tests. However, the POMS test was not be conducted in this study, instead it was replaced by two more tests, Movement Time and Trail Making tests. The description and functional domain of the tests are shown in Table 1.
Standard Score Calculation
The raw score obtained from the neurobehavioral tests, was transformed into standard scores. From these transformed data or standard scores, the mean value is 50 and the standard deviation is 10. The calculation for the standard score is shown:
Standard score = [(raw data – mean) / standard deviation] x 10 + 50.
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Table 1. Descriptions of Neuropsychological (NCTB) Tests Test Functional domain
tested Description
Reaction/Movement time
Attention/Response
& movement speed
It measures how fast a person reacts/moves. It requires sustained attention by the subject.
Digit span Auditory memory It is a test of immediate (short–term) auditory memory that requires focused attention.
Santa Ana Manual Dexterity
Manual dexterity It requires rapid eye–hand coordination movements.
Digit symbol Perceptual–motor speed
It is a test of perceptual motor speed that also requires learning of associations.
Benton visual retention
Visual
perception/memory
It is a short–term visual memory that requires focused attention. It also measures the ability to organize geometrical patterns in space and memorize them.
Pursuit aiming Motor steadiness It measures the ability to make quick and accurate movements with the hand.
Trail making Motor tracking It measures visual motor tracking and visual scanning. It requires an attention.
Source: WHO. 1983. Operational Guide for The WHO Neurobehavioral Core Test Battery. World Health Organization, Geneva. 15 Februari (14).
Ethical Clearance
This research was approved by the Research Ethics Committee of Hospital Universiti Kebangsaan Malaysia (UKM/EC711002105465-1). Involvement of respondents is based on a written agreement with filling a consent form. Respondents may withdraw at any time if they do not agree or are not satisfied with any study procedures.
RESULTS
Table 2 shows the demography and socioeconomic background information of the respondents. The mean age for mothers was 29.6 years old and their mean gestation
period was 31.6 weeks. The ethnic and religious distributions of the respondents are shown in Table 2. The Malays made up 58.4
% of the respondents. About 35.6%
respondents are full time housewives; while others work as general worker, operate a business, self–employed or part–time wage earner. The average household income is RM 2884.7 (Table 2). The mean blood lead level was 7.78 with standard deviation of 4.77 µg/dL.
Figure 1 shows the data distribution of blood lead for the respondents. Kolmogorov–
Smirnov Normality Test shows that the distribution was normal (p > 0.05).
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Venous blood lead conc. (ug/dL)
24.0 22.0 20.0 18.0 16.0 14.0 12.0 10.0 8.0 6.0 4.0 2.0 0.0
Frequency
30
20
10
0
Fig 1. Distributions of Blood Lead Concentrations (µg/dL)
Table 2. Respondents’ Demographic and Socioeconomic Background
Variable Range Mean Std. Dev.
Mother age (Year) 19 – 44 29.6 5.57
Mother Education (Year) 6 – 19 12.1 2.08
Father’s education (Year) 6 – 22 12.8 2.64
Total Household Income (RM) 750 – 8500 2884.7 1383.98
Mother’s height (m) 1.43 – 1.72 1.57 7.12
Mother’s weight (kg) 50.0 – 92.1 68.3 7.68
Pregnancy duration (weeks) 28 – 36 31.6 2.13
Number of pregnancies 1 – 6 1.7 1.07
Number of children 1 – 6 1.6 1.04
N = 202
Table 3 shows the scores for neuropsychological test (NCTB) that was carried out on the respondents. It took about 30–45 minutes to complete all the 7 types of NCTB test. Standard Score Index Values
were calculated from the raw scores and used for all statistical analysis.
Figure 2 shows the standardized NCTB score test distributions, which were normal (p
> 0.05).
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NCTB average score
68.0 66.0 64.0 62.0 60.0 58.0 56.0 54.0 52.0 50.0 48.0 46.0 44.0 42.0 40.0 38.0 36.0
Frequency
40
30
20
10
0
Fig 2. Distributions of Neuropsychological Scores Table 3. Neuropsychological Scores (NCTB) of Pregnant Mothers
Neuropsychological scores (NCTB) Range Mean Std. Dev.
Digit Symbol 29.0 – 78.3 50.0 10.00
Pursuit Aiming 24.5 – 80.0 50.0 10.00
Trail Making 26.4 – 73.1 50.0 10.00
Digit Span 35.8 – 85.2 50.0 10.00
Benton Visual Retention Test 24.8 – 67.3 50.0 10.00
Santa Ana Manual Dexterity 23.1 – 70.8 50.0 10.00
Reaction Time 23.5 – 72.0 50.0 10.00
Total Score Total Score 36.3 – 67.6 50.0 5.54
N = 202
Table 4 shows the correlation between blood lead and each NCTB test. The total NCTB scores also showed inversely significant correlation with the respondents’
blood lead except for the Pursuit Aiming and Santa Ana Manual Dexterity tests. The linear model shows that about 21.3% of the variations in blood lead contributed to the
neuropsychological scores. Table 5 shows that the total NCTB scores were influenced by the respondents’ blood lead, age, weight and height after all the confounding factors were adjusted. This model shows that these factors contribute 27% of the variations in the NCTB scores.
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Venous blood lead conc. (ug/dL)
30 20
10 0
-10
NCTB average score
70
60
50
40
30 Rsq = 0.2131
Fig 3. Correlation Between Blood Lead Concentrations and Neuropsychological Scores
Table 4. Correlation Blood Lead and Neuropsychological Scores Neuropsychological test
(NCTB)
Blood lead (µg /dL)
r p value
Digit Symbol –0.347 < 0.001**
Pursuit Aiming – 0.021 0.762
Trail Making – 0.254 < 0.001**
Digit Span – 0.447 < 0.001**
Benton Visual Retention Test – 0.256 < 0.001**
Santa Ana Manual Dexterity – 0.016 0.817
Reaction Time – 0.450 < 0.001**
Total score –0.462 < 0.001**
N = 202
** Significant at p < 0.01
Table 5. Correlations of Blood Lead on The Neuropsychological Scores After Adjustment of Confounders
Dependant variable
(Mean Neuropsychological score)
Regression coefficient
β
Statistict p value
(Constant) c 61.74 7.42 < 0.001**
Blood lead (µg /dL) – 0.47 – 7.51 < 0.001**
Age (years) – 0.15 – 2.40 0.017*
Weight (kg) 0.12 2.67 0.008**
Height (m) – 0.10 – 1.61 0.110
Educational level (years) 0.08 1.18 0.239
Household income (RM) – 0.13 – 1.79 0.075
N = 202
** Significant at p ≤ 0.01 F value = 12.787, p < 0.001
* Significant at p ≤ 0.05 r = 0.531, R2 = 0.282
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DISCUSSION
The mean blood lead of the respondents was 7.78 µg/dL (Figure 2), which is more than the blood lead of electronic industries soldering workers (6.10 µg/L) (4). Pregnant women need an optimum nutrient such as Ca2+ and Fe2+ or Fe3+, for the growth of the babies (5,6). The body tends to absorb lead if the calcium and iron intake were insufficient in the diet. Lead in the mothers’ blood systems has a potential of being transferred to the fetus through the placenta and this process began 2–3 months at an early stage of pregnancies. Blood lead concentrations for pregnant mothers should not exceed 10 µg/dL (7). However, almost 27% of the respondents have blood lead concentrations of more than 10 µg/dL. Studies (8) in African reported that mean blood lead for pregnant mothers who live in the city and the rural ranged from 0.83 to 99 µg/dL. The difference of those data was significantly difference.
This proves that environment can influence lead concentrations in the blood. There was no significant relationship between blood lead with age and the gestation period.
However, the mean blood lead in previous study was higher than this study.
Another study (9), on pregnant women reported that the mean blood lead concentration was 8.59 ± 4.45 µg/dL and about 27.8% respondents had blood lead concentrations higher than 10 µg/dL. The study found that housewives had higher
blood lead (9.55±5.5 µg/dL) than those working in offices (7.44±2.77 µg/dL), factories (8.61±3.39 µg/dL) and shops (7.01±3.13 µg/dL). However, the difference was not statistically significant. Study by (10) reported that the mean blood lead for women in the Avon are of the UK was range 0.41–
19.14 µg/dL. Studies at Port Pirie, Australia (11) reported that 646 women who are 30 to 36 weeks pregnant had an almost similar mean blood lead level of 7.2 µg/dL and 28%
had blood lead of more than 10 µg/dL.
In general, neuropsychological scores were calculated from the 7 tests in the NCTB.
Correlation tests indicated that blood lead for these pregnant mothers had an inverse significant correlation with Digit Symbol, Digit Span, Trail Making, Benton Visual Retention Test and Reaction Time (Table 4).
Only Pursuit Aiming and Santa Ana Manual Dexterity scores were not significantly correlated with blood lead (Table 4). The NCTB test result shows that pregnant mother with high blood lead have difficulties in concentrating and have short–
term hearing and visual abilities (Digit Span Test, Benton Visual Retention Test). They have slow motor speed through vision (Digit Symbol Test) slow reaction toward visual stimulation (Reaction Time Test) and low attention ability, visual scanning and visual motor trailing (Trail Making Test).
In Figure 3 Shown ultiple regression test result demonstrated that mean
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neuropsychological score for pregnant mothers were influenced by blood lead (β = –0.56, p < 0.001), age (β = 0.12, p < 0.017), weight (β = 0.12, p = 0.08) and height (β = – 0.01, p = 0.050). The model was significant
(F = 18.23, p < 0.001) with the R2 value, which shows that 27% of the variations in the NCTB scores were influenced by the variables shown below:
NCTB mean score = 65.71 – 0.56 (blood lead) – 0.15 (age) – 0.10 (height) + 0.12 (weight)
Previous studies showed that blood lead among female workers had the abilities to lower the NCTB score (12,13). Studies (12) showed that 140 female factory operators had a mean blood lead concentration of 30.77 µg/dL and it was inversely correlated only with the Reaction Time test (r = –0.201, p = 0.017). Meanwhile (4) found that mean blood lead for female soldering workers at an electronic factory was 6.1 µg/dL, and the comparative group was 4.6 µg/dL. The respondents' blood lead had significant relationships with Digit Span Test (p = 0.003), Santa Ana Manual Dexterity Test (p
= 0.007) and total NCTB scores (p = 0.001).
The exposed workers had significantly lower mean score compared to unexposed workers for Digit Symbol Test, Trail Making Test and Pursuit Aiming Test.
Pregnant mothers with high blood lead had problems with the ability to concentrate and have poor short–term auditory and visual memory and have the inability to organize geometry patterns in space and memorizing them. They have poor perceptual motor speed and fail to match between symbol and digits.
They also had slow reaction to stimulation due to poor ability to pay attention and low attention ability, which cause poor visual motor tracking as well as poor visual scanning.
Blood lead concentrations, age, weight and height influenced 27% of the variations in neuropsychological score after adjusting for confounders. About 1 µg/dL increment in blood lead will reduce 0.4 of the neuropsychological mean score of these pregnant mothers.
CONCLUSIONS
In conclusion, even though the blood lead concentration is quite low, it affects the neuropsychological ability of the respondents. Digit Symbol, Digit Span, Trail Making, Benton Visual Retention and Reaction Time test scores had significant inverse correlations with the blood lead.
ACKNOWLADGEMENTS
Appreciation goes to all respondents and Management of Environmental Health Laboratory, Hospital Universiti Kebangsaan
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Malaysia who gave the commitment that this study can be implemented successfully.
CONFLICT OF INTEREST
There are no conflicts of interest.
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Cytotoxicity Assay Using Brine Shrimp Lethality Test on Collagen- Chitosan Wond Dressing Sterilized by Ultraviolet Light
Ary Andini1, Endah Prayekti1, Devyana Dyah Wulandari1, Ersalina Nidianti1
1Department of Medical Laboratory Technology, Faculty of Health, Universitas Nahdlatul Ulama Surabaya, Surabaya, Indonesia Correspondence:
Ary Andini,
Jl. Jemursari No. 51-57, Surabaya, East Java, Indonesia
Zip Code : 60237
Email: [email protected] Received: February 14, 2020 Revised: February 17, 2020 Accepted: March 23, 2020
Abstract
Collagen gives a moist state on the wound area to accelerate the wound healing process. Chitosan is a polymer known as non-toxic, antibacterial, antifungal, biodegradable, and biocompatible material. Combination of collagen and chitosan is expected to be the best biomaterials as a wound dressing for the healing process. The study aimed to determine the cytotoxicity assay on collagen-chitosan wound dressing sterilized by ultraviolet (UV) Light using Brine Shrimp Lethality Test (BSLT) method. The test groups were divided into K0, K1, K2, and K3 groups. K0 contained pure chitosan as a control group, K1 contained collagen 25%-chitosan 75%, K2 contained collagen 50%- chitosan 50%, K3 contained collagen 75%-Chitosan 25%.
Collagen was extracted from the skin and scalp of snakehead fish (Channa striata), then it was mixed with chitosan until collagen-chitosan wound dressing formed. This study used Brine Shrimp Level Test (BSLT) method with solution concentration: 10, 50, 100, 250, 500, 750 and 1000 ppm.
Based on the results, it showed that K0, K1, K2, and K3 group had LC50 > 1000, proved that collagen-chitosan wound dressing was non-toxic material. The conclusion of the study explain that composite wound dressing based on collagen-chitosan in all groups that was sterilized under UV- Light for 15 minutes was not toxic. Also, it showed LC50 >
1000 based on Brine Shrimp Lethality Test.
Keywords
Collagen, chitosan, BSLT, wound dressing, ultraviolet
INTRODUCTION
There are two kinds of wound dressing, such as traditional wound dressing including gauze, plasters, lint, bandages, cotton wool, and modern wound dressing including films, hydrogel, hydrocolloids, foam and composites (1,2). Wound dressing aplication
has main roles in healing the wound and preventing infection on wound area. Best wound dressing with good quality could accelerate the wound healing, prevent the wound from infection of bacteria and fungal, and reduce pains (2). The particular wound dressing can promote a newly epithelium
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layer that may not be damaged during the dressing removal around the wound (3). The composite dressing is made from a combination of any kind of materials for dressing. It is suitable to burn wounds, surgical wounds, infectious wound and refractory chronic wound (2).
Collagen and chitosan are known as biomaterials that have bioproperties including biocompatible and biodegradable (4). Therefore, it can be developed into composite dressing for wound healing.
However, its application is limited due to clinical test before using for human. Few steps were needed before clinical test of a wound dressing which are toxicity (5), quality (6), in vitro and in vivo evaluation test (7).
Based on Ariyadi and Dewi (8) study, it showed that dry sterilization using ultraviolet (UV) irradiation for 10 and 15 minutes would not be contaminated by Bacillus subtilis colony (8). Therefore, this research used composite collagen-chitosan extracted from Channa striata collagen that was sterilized under ultraviolet (UV) light for 15 minutes to determine LC50 using Brine Shrimp Lethally Test (BSLT). Previously, the brine shrimp was utilized in various bioassay such as analysis of pesticide residues, stream pollutant, anesthetics, dinoflagellate toxins, mycotoxins, cocarcinogenecity of phorbol esters, and toxicants in marine (9).
MATERIALS AND METHODS Skin and scales of sneakhead fish were collected from Srikandi fishmonger at Banjar Asri Village, Tanggulangin Sub-District, Sidoarjo, East Java Province, Indonesia. The 100 mesh Chitosan powder (food and medical grade) was made from black tiger shrimp shells and it was obtained from Monodon (Marine Natural Product). The cytotoxicity assay was conducted by using Artemia salina (Golden West Artemia, Supreme Plus).
The collagen was extracted from the skin and scales of sneakhead fish by macerating it in HCl 2% for 48 hours and it was neutralized with NaOH 1M. Chitosan powder was dissolved in CH3COOH 1% (4). Collagen- chitosan composite was synthesized by mixing collagen 25% : chitosan 75% as K1 group; collagen 50% : chitosan 50% as K2 group; collagen 75% and chitosan 25% as K3 group; and pure chitosan as a control group (K0).
The cytotoxicity assay was carried out with the Brine Shrimp Lethally Test (BSLT) by using A. salina. Each collagen-chitosan composite of K0, K1, K2 and K3 was dissolved with DMSO 1 % then was divided into 10 ppm, 50 ppm, 100 ppm, 250 ppm, 500 ppm, 750 ppm and 1000 ppm. As much as 5 mL of each concentration was added into petri dish then it was added with 5 ml of seawater contained in ten A. salina.
Hereafter, it was incubated for 24 hours (10).
