SYNOPSIS

RajivGandhiUniversity of Health Sciences, Karnataka, Bangalore

“A COMPARATIVE STUDY OF SALIVARY AND BLOOD SIALIC ACID, LIPID PEROXIDATION AND ANTIOXIDANT STATUS IN CHRONIC ALCOHOLICS”

Name of the candidate:Dr.SUSANNA.T.Y.

Guide: Dr.SHIVASHANKARA.A.R

Course and Subject :M.D.(BIOCHEMISTRY)

Department of Biochemistry,

FatherMullerMedicalCollege,

Kankanady, Mangalore – 575002.

2009

RajivGandhiUniversity of Health Sciences Karnataka,

Bangalore.

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. / Name of the candidate
And address
(in block letters) / Dr. SUSANNA.T.Y.
P.G. RESIDENT
DEPARTMENT OF BIOCHEMISTRY
FATHERMULLERMEDICALCOLLEGE
MANGALORE.
2 / Name of the Institution / FATHER MULLER’S MEDICALCOLLEGE
3 / Course of study and
Subject / M.D. (Biochemistry)
4 / Date of admission to course
5 / Title of the Topic
A COMPARATIVE STUDY OF SALIVARY AND BLOOD SIALIC ACID, LIPID PEROXIDATION AND ANTIOXIDANTSTATUS IN CHRONIC ALCOHOLICS
6 / BRIEF RESUME OF THE INTENDED WORK:
6.1 Need for the study
Chronic alcohol consumption leads to multiorgan failure. Many biochemical parameters in blood and urine have been proposed as markers of alcoholism . Assay of these biomarkers is critical for detection of alcohol abusers and also complications of alcoholism, so that prompt clinical intervention can be made, which could help in improving the quality of life of alcoholics.
The most commonly used laboratory diagnostic procedures employ analyses of blood constituents. Use of saliva as a diagnostic tool, is gaining increased attention in recent years. At the international level, multiple studies have been done to evaluate the changes in salivary parameters in various diseases. In India, salivary analysis is not given due thrust. Saliva as a diagnostic fluid offers distinctive advantages. Whole saliva can be collected non-invasively, and without the help of skilled technician and special equipments, it is stress free, easy and frequent collections are possible. Analysis of saliva may provide a cost-effective approach for the screening of large populations.
There is paucity of studies on salivary biomarkers of alcoholism. Studies are needed to evaluate the biological effects of alcohol by assays of biochemical constituents of saliva. The present study aims to evaluate the effect of chronic alcoholism on sialic acid and oxidant-antioxidant status in blood and saliva. The study also aims to compare the changes in biochemical parameters of saliva with the changes in blood, hence making a prompt attempt to develop saliva as a tool in detection, diagnosis and management of alcoholism and alcohol-related complications.
7. / 6.2 Review of Literature
Chronic consumption of alcohol is highly prevalent in our modern society. Alcoholism not only causes adverse effects on the health and socio-economic aspects of the individual, but also affects the family and society as a whole. Alcohol is metabolized in the body mainly by the liver. The brain, pancreas, and stomach also metabolize alcohol.Most of the ethanol in the body is broken down in the liver by an enzyme called alcohol dehydrogenase (ADH), which transforms ethanol into a toxic compound called acetaldehyde (CH3CHO). However, acetaldehyde is generally short-lived; it is quickly broken down to a less toxic compound called acetate (CH3COO-) by another enzyme called aldehyde dehydrogenase (ALDH). Acetate then is broken down to carbon dioxide and water, mainly in tissues other than the liver. Acetaldehyde is known to form adducts with biomolecules like proteins and nucleic acids. Alcohol metabolism also generates free radicals which impair the structure and function of vital biomolecules. The enzymes cytochrome P450 2E1 (CYP2E1) and catalase also break down alcohol to acetaldehyde. Small amounts of alcohol also are removed by interacting with fatty acids to form compounds called fatty acid ethyl esters (FAEEs). These compounds have been shown to contribute to damage to vital organs of the body. Through various metabolic processes, alcohol becomes toxic for the body.1
Alcohol biomarkers are physiological indicators of alcohol exposure or ingestion and may reflect the presence of an alcohol use disorder. Most readily measurable biomarkers are indirectly correlated with alcohol problems, such as alcohol dependence or chronic heavy alcohol consumption.
Many studies have been done to establish biomarkers of alcohol abuse. Enzymes gamma-glutamyl transferase, alanine aminotransferase, aspartate amino transferase, and erythrocyte mean corpuscular volume are conventionally used as markers of alcohol toxicity. Carbohydrate-deficient transferrin, a glycoprotein of human serum, is the only marker approved by FDA for the identification of heavy alcohol use. Sialic acid, sialic acid index of apolipoprotein J, betahexosaminidase, acetaldehyde adducts are the other biomarkers of alcoholism. Urinary ratio of 5-hydroxytryptophol and 5-hydroxyindoleacetic acid is also used as a marker of alcoholism. The biochemical and hematological changes in alcoholics, make the above suggested biomarkers to be useful in the evaluation of alcoholism and associated toxicity manifestations. (2)
Alcohol consumption is associated with changes in the oxidant-antioxidant system. Enhanced generation of free radicals and reactive species, enhanced oxidation of biomolecules, and depletion of antioxidants in blood, have been reported in the studies on alcoholics.(2)
Saliva as a tool for diagnosis of diseases, has gained attention in recent three decades. Since 1982, many researchers have worked on use of saliva, gingival crevicular fluid and mucosal transudates for the detection and management of oral and systemic diseases. Whole saliva has been analyzed in systemic diseases, autoimmune disorders, malignancy, infectious diseases, drug monitoring, and hormonal imbalances. Researchers have assayed salivary parameters such as flow rate, enzymes, interleukins, tumor markers, immunoglobulins, and oxidative stress markers in various diseases.(3) Saliva was used for the detection and monitoring the levels of ethanol, opioids, barbiturates, diazepins and amphetamines.(3) In India, there have been few studies reporting altered chemical composition of saliva in cancer, periodontal diseases, infectious conditions such as HIV infection and hepatitis, and also in follow up of treatment of these diseases. The salivary parameters evaluated in these studies include, sialic acid, glycoproteins, oxidative stress markers, antioxidants, antigens, and immunoglobulins. (4)
Oxidative stress is a phenomenon in which there is increased production of free radicals or depletion of antioxidants, or both. Oxidative stress is implicated in the etiopathogenesis of a variety of human diseases, and in the toxic manifestations of various exogenous compounds.
Saliva is equipped with antioxidant mechanisms such as superoxide dismutase, peroxidase, uric acid, glutathione and vitamin C.(5) Salivary glycoconjugates such as mucins, proline-rich glycoproteins and defensins are considered very important for the protective role of saliva. Glycoconjugates in general, have sialic acid at their termini. Sialic acid is released into the circulation through increased turn over, secretion and shedding from the cells. Sialic acid as a constituent of mucins of saliva, was considered as a scavenger of hydroxyl radicals. (6) Elevated levels of salivary sialic acid in periodontal diseases, was attributed to its role as an antioxidant. (6)Serum and salivary sialic acid levels were found to be significantly higher in alcoholics. (7)
6.3 Objectives of the study
  1. To estimate sialic acid levels in blood and saliva of chronic alcoholics and compare the values with those of healthy controls
  2. To assess the oxidative stress in blood and saliva of chronic alcoholics by assay of malondialdehyde, the marker of lipid peroxidation, and compare the values with those of healthy controls
  3. To assess the antioxidant status in blood and saliva of chronic alcoholics by assays of protein thiols, uric acid and superoxide dismutase, and compare the values with those of healthy controls.
  4. To compare and correlate the changes in sialic acid, lipid peroxidation and antioxidants in saliva and blood of chronic alcoholics.
Materials and Methods
7.1. Source of Data :
I. Fifty chronic alcoholics admitted for treatment or deaddiction, to the in-patient wards at FatherMullerMedicalCollege hospital, form subjects of the study. Detailed history of alcohol intake ,clinical complications if any, habits in particular smoking and tobacco chewing will be collected from subjects of the study , by giving them a questionnaire. Clinical diagnosis of the patient will also be taken into consideration.
II. Fifty age and sex-matched clinically apparently healthy individuals will be included as controls.
Inclusion criteria : chronic alcoholics with alcohol abuse for more than five years, and with or without clinical complications, will be included in group-I.
Exclusion Criteria : Alcoholics with smoking and occasional drinkers will be excluded from group-I. Individuals with any systemic illness and smokers will be excluded from the control group.
7.2. Method of Collection of Data :
Study type: Case control study.
Sample and sampling technique: Blood and saliva samples of alcoholics and controls will be collected and processed. 5 ml. unstimulated whole saliva collected according to the method of Navazesh,(8)between 9-11am,centrifuged. 5 ml. blood collected in EDTA-vacutainers, centrifuged.
Following parameters will be estimated –
  1. Protein-bound sialic and free sialic acid levels in saliva and serum – estimated by the method of Yao et al . (9) Proteins are precipitated with ethanol, sialic acid contents of precipitate and supernatant are assayed which give the values of protein-bound sialic acid and free sialic acid respectively.
  2. Malondialdehyde, as a marker of lipid peroxidation - assayed as thiobarbituric acid-reactive substances(TBARS), in saliva and red blood cell hemolysates by the method of Ohkawa et al(10)
  3. Protein thiols in plasma and saliva- measured by a spectrophotometric method using 5,5 dithio,bis-2- nitrobenzoic acid (11)
  4. Superoxide dismutase (SOD) activity in saliva and red blood cell hemolysates,based on inhibition of auto oxidation of pyrogallol by SOD.(12)
  5. Uric acid levels in serum and saliva – estimated by enzymatic spectrophotometric method(13)
Statistical Analysis : The collected data will be presented as mean with standard deviation, and analyzed by employing unpaired t test and correlation analysis.
7.3Does the study require any investigation/intervention to be conducted on patients/other humans/animals? If so,please describe briefly.
Yes, blood samples are collected with aseptic precaution. Informed consent from subjects will be obtained before collection of blood and saliva samples.
7.4Has ethical clearance been obtained from your institution in case of 7.3
Yes.
8. / LIST OF REFERENCES
1.National Institute on alcohol abuse and alcoholism. Alcohol alert : alcohol metabolism update. US Department of Health and Human Services, No.72; July 2007.
2.Das SK, Dhanya L, Vasudevan DM. Biomarkers of alcoholism : an updated review. Scand J Clin Lab Invest 2008; 68: 81-92.
3.Kaufman E, Lamster IB. The diagnostic applications of saliva –a review. Crit Rev Oral Biol Med 2002; 13: 197-212.
4.Rai B, Kharab S, Anand SC. Saliva as a diagnostic tool in medicine : a review study. Adv Med Dent Sci 2008; 2: 9-12.
5.Nagler RM, Klein I, Zharzevsky N, Drigues N, Reznick AZ. Characterization of the differentiated antioxidant profile of human saliva. Free Radic Biol Med 2002; 32: 268-277.
6.Ogasawara Y, Namai T, Yoshino F, Lee MC, Ishi K. Sialic acid is an essential moiety of mucin as a hydroxyl radical scavenger. FEBS Lett 2007; 581: 2473-2477.
7.Ponnio M, Alho H, Heinnala P, Nikkari ST, Sillanaukee P. Serum and saliva levels of sialic acid are elevated in alcoholics. Alcohol Clin Exp Res 2006; 23: 1060-1064.
8.Navazesh M. Methods for collecting saliva. Ann Ny Acad Sci 1993; 20: 72.
9.Yao K, Ubuka T, Masuoka N, Kinuta M, Ikeda T. Direct determination of bound sialic acids in sialoglycoproteins by acidic ninhydrin reaction. Anal Biochem 1989; 179:332-335.
10.Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979; 95: 351-358.
11.Motchnik AP, Frei B, Ames NB. Measurement of antioxidants in human blood plasma :protein thiols. In :. Oxygen radicals in biological systems, Methods in Enzymology, volume 234 (Packer L, ed). New York : Academic Press, 1994, pp.273-274.
12.Marklund S,Marklund E.Assay of SOD activity in animal tissue. J Biochem,1988;13:305-315.
13.BurtisCA,Ashwood ER,Bruns DE.Tietz textbook of clinical chemistry and molecular diagnostics.4th ed. New delhi (India),Reed Elsevier India private limited;2006,p.807.