Increase of the Reduced Glutathione Level in Kidneys of Old Rats - Research Paper Example

 Does organosulfur, Diallyl Trisulfide (DATS), of garlic increase the reduced Glutathione level (GSH) in kidneys of old rats? Abstract This study aims at investigating the possible reduction in Glutathione level (GSH) in kidneys of old rats after being treated with Diallyll Trisulfide (DATs) contained in Garlic. It will involve 20 randomly selected old rats aged 1.5 to 2 years. The control group of 10 rats will be treated with distilled water while the second group will receive oral DAT Through enzymatic method that follows other laboratory procedures, the level of activity in samples will depict the amount of Glutathione level (GSH) in rats’ kidneys. It is expected that the study will confidently indicate the possible impact of organosulfur, DAT in the amount of Glutathione level (GSH) in the rats’ kidney. Key Word: Glutathione level (GSH), Diallyll Trisulfide (DATs), Garlic, kidney, rat Introduction From the beginning of civilization, plants have formed a fundamental part of society of humans. In most cultures, garlic (Allium sativum L.) is widely used as condiment and as spice in many dishes. A number of cultures use garlic as medicine. There are also numerous publications concerning the pharmacological characteristics of garlic including their benefits on health (Rahman, 2007; Kojuri et al., 2007). Garlic is used as a medicinal herb and as a spice. Majority of the recent researches on garlic have utilized garlic in various forms such as tablets, raw, flesh, dried, boiled, and cooked (Gorinstein et al., 2006). Commercially, garlic is available in form of garlic powder, garlic oil, and pills and is widely used for therapeutic purposes (Elkayam et al., 2003). Garlic contains sulfuric and non-sulfuric compounds. Many studies focus on the benefit of the oil soluble sulfuric compounds such as diallyl sulfide (DAS), diallyl disulfide (DADS), diallyl trisulfide, allyl methyl trisulfide, dithiins, and ajoene. Some other research focus on the water soluble sulfuric compounds such as S-allyl cysteine (SAC), S-allyl mercaptocysteine (SAMC) and S-methyl cysteine, and gamma-glutamyl cysteine derivatives (Velmurugan, Subapriya, Nagini, 2003). Figure 1 Diallyl Trisulfide structure Organosulfur compounds in garlic have been found to contain antioxidant properties. A study showed that the use of garlic oil supplements by hypertensive adults decreased a biomarker of in vivo fat oxidation in a controlled trial (Falcioni, et al., 2010). However, it is not clear yet whether organosulfur compounds in garlic exhibit antioxidant effects in vivo. GSH is the key regulator of cell proliferation, metabolism, pathways, cell signal transmission into the nucleus (Pallardo, 2009). Moreover, its nuclear concentration is the most important player in preventing oxidation (Kulinskii, 2010). Besides, decreased level of GSH has been linked to different diseases. Several studies have been carried out in relation to glutathione including more than 99, 000 peer-reviewed papers. One of the reviews revealed that about 80% of patients with chronic ailments had glutathione deficiency. In fact, low glutathione levels are involved in every disease state. Glutathione is the key detoxifier and antioxidant in the body, thus increase in glutathione levels enable cells to detoxify and function more effectively and efficiently (Pallardó, et al., 2009). The diseases, illnesses and symptoms linked to low levels of glutathione include Alzheimer’s disease, dermatitis, HIV/Aids, alcoholism, bronchitis, bird flu, cancer, obesity, hepatitis, coronary diseases, diabetes, osteoporosis, depression, headaches among others. Diallyl trisulfide (DATS) is a fat-soluble organosulfur compound which is believed to be having stimulating effects on glutathione synthesis, hence a vital intracellular antioxidant Animal and cell culture studies provide evidence linking diallyl trisulfide (DATS) in garlic to increased intracellular concentrations of glutathione. Like the genes of most phase II enzymes, the genetic makeup of an essential enzyme in the synthesis of glutathione also has an ARE. Therefore, organosulfur diallyl trisulfide (DATS) may increase cellular synthesis of glutathione through promoting nuclear translocation as well as binding of Nrf2 transcription-factor to the genes with AREs (Diaz, et al., 2010). Compounds of garlic have been studied extensively. Many of these studies test the outcome of garlic related to range of diseases such as cancer, and Alzheimer. Scholars show a correlation between garlic intakes and GSH level (Ahmad & Ahmed, 2006). Nevertheless, I did not find a study linking the intake of garlic with nuclear level of GSH in the kidney of old rats. Furthermore, which compounds among the organo-sulfur of garlic have the most significant effect on the GSH level? In this regard, this study is going to determine whether Organosulfur Diallyll Trisulfide (DATs) of garlic increases the Glutathione level (GSH) in kidneys of old rats. This study hypothesizes that organo-sulfur, Diallyll Trisulfide (DATs), of garlic increase GSH level in kidneys of old rats (Herman-Antosiewicz, 2007). The significant of this study should reject the null hypothesis with p-value less than alpha at 0.05. This research proposal will address the methodology, ethical consideration and the expected outcome of the study. Motivation One thousand and four hundred years ago, garlic was mentioned in Qur’an: “…And when you said: O Musa! We cannot bear with one food, therefore prays Lord on our behalf to bring forth for us out of what the earth grows, of its herbs and its cumbers and its garlic and its lentils and its onions...” (Al-Baqara, Verse 61). Garlic is very famous in Arabic and Islamic culture. They use it in their cocked food and treating many diseases such as common cold, kidney pain, judies, influenza, sore throat, and many others. Actually, we use garlic for every single disease! My mother, for example, eats row garlic with yogurt when she has common cold or feels malaise. Personally, I never use garlic as a treatment. Nevertheless when I aimed to write a paper about garlic, I decided to try it. At that time, I had a sore throat and running nose. Accordingly, I chopped a bulb, about ten cloves, of garlic and swallow water. I did that every eight hours. Although, the chipped garlic had little burning on my tongue, I kept using it on the same frequency. At the first and second times, I did not vomit. On the third time, I had nausea. Fortunately, I didn’t vomit. It was a scary moment because vomiting may burn my esophagus. Ironically, I told myself, “If I didn’t feel better within three days, I will quit this topic and find another one.” Surprisingly, I did feel better within the first twenty four hours! Yes, this is not an academic experiment, but it gave me another reason to do the research proposal about garlic. Research Question Does organosulfur , Diallyll Trisulfide (DATs), of garlic increase the reduced Glutathione level (GSH) in kidneys of old rats? Null hypothesis Organosulfur Diallyll Trisulfide (DATs) of garlic does not increase Glutathione (GSH) level in kidneys of older rats. Justification There many reasons for choosing aged kidney. First, having longer life with less disease is part of the answer for choosing aged rat. Second, I came from a culture with high respect to elder people. Third, when I searched aged kidney, I found tens of researches focus on the end stage renal disease (ESRD) associated with aging (Zhou, 2008). Fourth, I had graduate course about renal and I did a research about diabetic nephropathy. I found that End stage kidney disease (ESKD) develops primarily from diabetes type two. And ageing is worsening the kidney problem because many elder people develop hypertension, type two diabetic and other disease that affect renal, (Figure 1). Figure 2Complex factors along with aging leading to renal insufficiency, (Zhou, 2008) GSH is the key regulator of cell proliferation, metabolism, pathways, cell signal transmission into the nucleus (Jose Vina, 2009). Moreover, its nuclear concentration is the most important player in preventing oxidation (Kulinskii, 2010). Besides, decreased level of GSH has been linked to different diseases. Different compounds of Garlic have been studied extensively. Many of these studies test the outcome of garlic related to range of diseases such as cancer, and Alzheimer. Scholars show a correlation between garlic intakes and GSH level (Giblin, 2000). Nevertheless, I did not find a study linking the intake of garlic with nuclear level of GSH in the kidney of old rats. Furthermore, which compounds among the organo-sulfur of garlic have the most significant effect on the GSH level? Materials and Methods Material and Animals 20 Wistar rats of weight between 51-75 grams will be purchased from Charles River, UK. Several studies have identified rats to be having an average life span of two years. Therefore, those of age between 1 and a half years and two years old may be considered old and suitable for this inquiry. The selected rats will have to be acclimatized to adapt to the conditions of the laboratory after being removed from their locations. The acclimatization takes one week after which the rats will remain caged in a steady controlled room temperature of 23oC (Noori, Azmat, & Mahboob, 2012). During this time, the rats will have unlimited access to standard rat diet and water. Diallyl Trisulfide, DATS compound to be used in this experiment will be purchased from Cayman Chemical, Ann Arbor, USA. The preceding and following phases of this study will be conducted with according to the conventional presets for laboratory animals’ investigations (National Research Council, 2011). Study Design The twenty rats will be randomly divided into two experimental groups where each group will have 10 participants (n=10). Prior to further investigations, the selected group of rats for the experiment will be weighed. They will then be subjected to various treatments. One of the two groups (Group One) of rats will act as a control experiment and will not receive any treatment. Instead, it will maintain the standard rat diet and water like before. The second lot (Group Two) of experimental rats will be administered orally with Diallyl Trisulfide, DATS (70 mg/kg body weight) thrice a week for 6 weeks (Wu, et al., 2001). Sample Collection At the end of the treatment period, the rats will be allowed to rest for 48 hours after which the sample will be collected (Noori, Azmat, & Mahboob, 2012). This period allow for maximum, possible absorption of the ingested nutrients. Upon the expiry of this time, all animals will be beheaded and blood sampled from head wounds. To safeguard the integrity of collected blood samples, the blood samples will be collected in lithium heparin coated tubules (Wu, et al., 2001). Kidneys will be excised from the carcasses and trimmed of their connective tissues. The obtained kidney parts will be rinsed in saline to remove any traces of blood contamination and dried through filter paper and weights determined. The prepared tissues will then be kept in the laboratory freezer while awaiting analysis. Preparation of Kidney Homogenate The kidney homogenates will be obtained through tissue homogenator of any model as long as it meets the prerequisites of scientific laboratory. The process will have to be carried out at a maintained ambient temperature of 4oC. The 1:10 w/v kidney homogenate will be prepared from a 100mmol KCL buffer with a pH of 7 and having EDTA 0.3 mmol. The centrifuge of all homogenates will be done at 600 g for one hour at a constant ambient temperature of 4oC. This is in consideration of the fact that elevated temperatures inactivate alliinase enzyme (Noori, Azmat, & Mahboob, 2012). Tissue Lipid peroxides Assessment 10μl of BHT, which is equivalent to 0.5M in acetonitrile, will be added to the homogenate to prevent it from oxidation and stored at freezing temperature of -70oC as it awaits 4-HNE and MDA analysis (Noori, Azmat, & Mahboob, 2012). Estimation of GSH The estimation of GSH in the kidney samples will involve enzymatic method of DTNB/GSSG reductase recycling assay (Anderson, 1985). The recycling assay is highly responsive to certain enzymatic procedures. The assay solution will have 10% BSA, 2mM NADPH, 50nM Phosphate buffer of pH 7.6, and 20mM GSSG. While maintain the temperatures at 25oC, the absorbance will be recorded. The level of activity will be used to establish the molar coefficient of NADPH which is given in U/gm of kidney tissue. A sample considered to be lacking GSH and obtained from old rats subjected to standard diet and water is the reference point of background rate of reaction. The values of reactivity level will be given in GSH equivalents comprising of μmol per gram for each tissue sample (Wu, et al., 2001). Tentative Time Table Oct Nov Dec Jan Feb. Mar. Apr. General literature search Proposal Development Approval by the instructor Carry out in-depth reading on the topic Laboratory Inquiry (Experiments) Compile and analyze lab results Make general inferences Revise literature review according to emerging issues Compile draft report Write final draft and submit Statistical analysis The results of the aforementioned procedure will be analyzed statistically through initial representation of findings in measures of central tendencies. The evaluation statistical significance and disparities will be done through student’s t-test. Statistical probabilities of p Read More