Download RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

RAJIV GANDHI UNIVERSITY OF HEALTH
SCIENCES
BANGALORE, KARNATAKA
ANNEXURE – ΙΙ
PROFORMA FOR REGISTRATION OF SUBJECTS FOR
DISSERTATION
1 NAME OF THE CANDITATE DR. SHWETHA
ADDRESS
2 NAME OF THE INSTITUTE
3 COURSE OF STUDY AND
SUBJECT
4 DATE OF ADMISSION TO
COURSE
5 TITLE OF THE TOPIC
INTRODUCTION
#418, SHRINIKETAN 1ST MAIN
3RD STAGE BEML LAYOUT
RAJESHWARINAGAR,
BANGALORE – 560098
KARNATAKA
DR. B R AMBEDKAR
MEDICAL COLLEGE,
K G HALLI, BANGLAORE –
560045
M.D. BIOCHEMISTRY
28/07/2010
“ TRACE ELEMENTS
(MAGNESIUM, ZINC, COPPER)
IN CHRONIC ALCOHOLIC
LIVER DISEASE“
Alcohol is a chemical substance capable of producing pharmacological effects
consistent with narcotics including central nervous system and respiratory depression
and induction of euphoria, tolerance and addiction.1
Alcoholism is a repetitive pattern of drinking alcohol that has adverse effects on
personal, family, occupational, social or health status.
An alcoholic is a person who progressively consumes an amount of ethanol capable
of producing pathological changes and who exhibit a cumulative pattern of social
behaviors associated drinking including frequent intoxication, physical injuries, problem
with family, job and accidents while drinking under the influence.1
Alcohol dependence is a maladaptive pattern of alcohol use leading to clinically
significant impairment and distress. Dependence may be physical or psychological.
Physical and psychological dependence is characterized by presence of withdrawal
symptoms and tolerance. Withdrawal symptoms include sweating, hand tremors,
vomiting, insomnia, anxiety, psychomotor agitation and hallucination. Tolerance is
defined as need for markedly increased amounts of alcohol to achieve a desired effect.2
Percentage of alcohol in each drink
Alcohol Content in
Percentage
50-60%
Beverage
Rum, Liquor
Whisky, Gin, Brandy
Wine
Beer
40-45%
10-15%
4-8%
Alcoholic equivalents: 3
A Standard alcoholic drink is equivalent to:
30 ml of Whisky
=
10 gm
= 70 calories
100 ml of wine
=
10 gm
= 70 calories
250 ml of Beer
=
10 gm
= 70 calories
1 gm of alcohol yields 7 calories
Drinking patterns:
The average intake alcohol in a large group male alcoholic cirrhotics was 160 g / day
for 8 years. Quantity, quality, duration is important. The liver injury is related to type of
beverage and its alcohol content. Continued daily imbibing is more dangerous
intermittent consumption when the liver is given a chance to recover. For at least two
days in a week a person should not drink alcohol.3
Metabolism of alcohol: Ethanol is primarily an exogenous compound consumed in
alcoholic beverages and readily absorbed from entire gastrointestinal tract. Normal
absorption is complete within an hour but food intake can delay absorption by 4-6
hours. Oxidative processes, located primarily in the liver degrade most of the absorbed
ethanol by following steps:
20% MEOS
Acetaldehyde
Ethanol
Aldehyde
80%
Alcohol
Acetaldehyde
Dehydrogenase
Citric Acid cycle
Acetate
CO2
dehydrogenase
AcetylcoA
Fatty Acids
The most important pathway occurs in cell cytosol where ADH produces acetaldehyde
which is rapidly destroyed by ALDH in the cytosol and mitochondria. A second pathway
in the microsomes of the smooth endoplasmic reticulum ( the microsomal ethanol
oxidizing system) is responsible >= 10% of ethanol oxidation at high blood alcohol
concentration. 4
Biochemical and metabolic alterations in alcoholism :
In chronic alcoholics, there is an increase in MEOS activity that may result in an
increased metabolism of upto 72%.
Altered lipid, protein and carbohydrate biochemistry: Low to moderate use of ethanol
(< 20 gm / day) is associated with an increase in HDL and lowers the risk of coronary
artery disease. When it is > 80gm / day causes excessive serum triglycerides, VLDL
and chylomicrons.
+water
w
As seen with poor nutritional status of many alcoholics, the rate of protein degradation
exceeds the rate of synthesis resulting in alcoholic myopathy, osteopathy and intestinal
atrophy.
Gluconeogenesis is impaired by ethanol by variety of mechanisms. Results in the fall in
the amount of glucose produced from glycogen and also increase in lactate
production.1
Alteration in mineral metabolism occurs in chronic alcoholics and also in alcoholic
liver disease. Minerals which are markedly decreased are magnesium and zinc and
which are markedly increased are copper.
In this study magnesium, zinc, copper are taken into consideration.
Magnesium is the fourth most abundant cation in the body. The body contains 21gm
of magnesium of which 60% is in bone, 20% in the skeletal muscles, 19% in the cells
and 1% in extracellular fluids.
Normal level – 18 to 29 mg/ litre
There are two major roles for magnesium in biological system.
1) It can compete with calcium for binding sites on membrane.
2) It can form chelates with important intracellular anionic ligands notably
adenosine triphosphate
Magnesium catalyses or activates more than 300 enzymes in the body. Magnesium act
as an cofactor for enzymes concerned with cell respiration, glycolysis and
transmembrane transport of other cation such as calcium and sodium. It affects enzyme
activity by binding with active site of enzyme( pyruvate kinase, enolase) by ligand
binding ( ATP requiring enzyme ) by causing changes during catalytic process ( Na – K
– ATPase ) and by promoting aggregation of multienzyme complexes. Reduced
extracellular magnesium concentration increases membrane excitability.
Metabolism : Only 20 – 30% of ingested magnesium is absorbed. High intake of fat,
phosphate, calcium, alcohol diminish absorption. Absorption is mainly at small intestine.
Absorption begins with an hour of ingestion and continues at a rate of 2 – 8 hours. By
this time 80% of the absorption is completed. Magnesium is excreted in faeces & the
remaining 1/3 rd in urine.
In alcoholic liver disease, there will be hypomagnesemia due to poor dietary intake and
increased diuersis that is a direct affect of alcohol, defects in renal tubular reabsorption,
starvation and vomiting.
Hypomagnesemia manifests with increased muscle tone, exaggerated tendon reflexes,
hyperirritability & tetany. It is associated with hypokalemia, hypocalcemia,
hypoparathyroidism. Hypomagnesemia occurs in 30% of alcohol abuse. Severe
depletion of magnesium affects thiamine indirectly leading to wernicks-Korsakoff’s
syndrome.1
Zinc: Zinc is widely distributed in the tissues of the body. 20% is present in skin, 80%
is present in prostrate, epididymis in bones, teeth in kidneys, muscles, brain & lungs
Normal Level 100-1200 µg/L.
Zinc is
essential for normal growth & reproduction. It has a beneficial effect on tissue repair &
wound healing. It is a component of a number of enzymes present in animal tissues
including ADH, Alkaline Phosphatase, carbonic anhydrase, procarboxypeptidase,
cytosolic superoxide dismutase. Zinc stabilizes membrane stabilizes membrane
structure and gives protection at the cellular level by preventing lipid peroxidation &
reducing free radical formation
Metabolism:- Only a small percentage of dietary zinc is absorbed mainly from
deodenum and ileum. After absorption it is taken up by liver & redistributed to other
tissues primarily bone & muscle. Zinc is lost in faeces & some percentage in urine.
In alcoholic liver disease, there will be hypozincemia due to poor dietary intake,
increased diuresis, destruction of zinc metalloenzymes. Zinc deficiency manifest with
loss of appetite, dwarfism, hypogonadism, hepatosplenomegaly , anaemia , dermatitis
& delayed wound healing.
Copper:- Adult human body contains 100-150 mg of copper which is distributed in
muscles, bone and in liver.
Normal level - 0.01to 0.2 µgm per ml
Copper forms a integral part of enzymes like cytochrome oxidase, tyrosinase , MAO,
lysyl oxidase, catalase, ascorbic acid oxidase, uric case and superoxide, dismutase.It
plays an important role in haemoglobin synthesis, melanin formation, phospholipid
synthesis, collagen synthesis, bone formation and maintaining the integrity of myelin
sheath.
Metabolism: - About 32% of dietary copper is absorbed mainly at the duodenum. After
absorption enters the plasma where it is bound to amino acid particularly histidine and
serum albumin. About 96% of serum copper is as cerruloplasmin. Under normal
condition 85-99% is excreted in faeces via bile and remaining in urine.
In Alcoholic liver disease copper level increases due to the failure of excretion of
copper. Toxicity causes renal failure, liver failure and coma.
Alcoholic liver disease
Chronic and excessive alcohol ingestion is one of the major causes of liver disease.
Only 15% of alcoholics develop alcoholic liver disease. Alcoholic liver disease is among
the 10 most common causes of death world wide.
Quantity, quality and duration of alcohol intake are the most important risk factors of
alcoholic liver disease. In men, 40-80gm per day of ethanol produces fatty liver 160 gm
per day for 10-20 years causes hepatitis & cirrhosis.
Pathogenesis
Liver is the primary target organ for alcohol induced damage. The production of
acetaldehyde causes a variety of metabolic responses like increase in lipid
peroxidation, inhibition of nuclear repair, interference with mitochondrial electron
transport change and microtubule function, increase in collagen synthesis and
superoxide formation.
Stages of Alcoholic liver disease
1) Fatty Liver : It is characterized by accumulation of fat within the perivenular
hepatocytes along with hepatic mitochondrial DNA deletion. Usually patients are
asymptomatic. Occasionally present with upper quadrant discomfort, tender
hepatomegaly, nausea and jaundice
2) Alcoholic Hepatitis: It is characterized by ballooning degeneration with sclerosing
hyaline Necrosis. Mallory bodies are seen in florid case. Patient presents with
fever, spider nevi, jaundice and abdominal pain.
3) Alcoholic Cirrhosis: It is also called as Laennec’s cirrhosis. Classically, alcoholic
cirrhosis is micro nodular and no normal architecture can be identified. The
formation of nodule is often slow because of inhibitory effect of alcohol on
hepatic regeneration. With continuing Necrosis and fibrosis the cirrhosis may
progress from micro nodular to macro nodular pattern. Only liver biopsy
specimen will provide an accurate diagnosis of cirrhosis.
Patient presents with symptoms such as vague right upper quadrant pain,
Nausea, Vomiting, diarrhea, anorexia and Malaise. On examination,
Hepatosplenomegaly with liver edge being firm and nodular, scleral icterus,
palmar erythema, spider angiomas, Parotid enlargement, digital clubbing,
Muscle wasting, Gynaecomastia and testicular atrophy due to hormonal
abnormalities.4
Risk factors in alcoholic liver disease
Female gender, obesity, genetic predisposition, malnutrition, co-infection with hepatitis
B or C virus, Iron overload, other co-morbid condition, Hepatotoxic drug intake.
Laboratory diagnosis of Alcoholic liver disease
For Alcoholic fatty liver & hepatitis 4
Test
Comment
Increased two to seven fold, <400 U/L, greater than ALT
AST
ALT
AST/ALT
Bilirubin
Increased two to seven fold, <400 U/L
Usually > 1
May be markedly increased in alcoholic hepatitis despite
modest elevation in alkaline phosphatase
Cirrhosis:- Laboratory tests may be completely normal in patients with early
compensated alcoholic cirrhosis. Alternatively in advanced cases, many abnormalities
are usually present.
Serum total bilirubin can be normal or mildly elevated with advanced disease. Serum
amino transferases ( ALT, AST) are typical elevated with AST levels living higher than
ALT levels, usually by a 2:1 ratio.
6 Brief resume of the intended work
6.1 Need for Study
Alcohol is a common cause of cirrhosis all over the world including India.
An alcoholic liver disease is among the ten most common cause of death
worldwide. The consumption of alcohol has been steadily increasing in India
during last decade due to socioeconomic status.
Mineral abnormalities occur in Alcoholic liver disease. Many of the nutrients are
essential for body function and a deficient state can cause alarming
consequences.
Hypomagnesemia is not only a laboratory symptom of fatty liver, but due
to its connection with increased oxidative stress it might be a risk factor in the
progression of the disease and also to know whether severity is related to the
amount duration of alcohol intake and the type of alcoholic beverage. it is known
that deficiency increases the susceptibility of plasma triacylglycerol- rich in
lipoproteins and tissues to lipoperoxidation, but the precise mechanism by which
magnesium can potentiate oxidative injury remains to be determined. Hence
further studies have to be taken up.1
Alcoholic liver disease is associated with decreased zinc levels, studies
using animal models have shown than Zinc supplementation prevents alcohol
induced liver damaged under both acute and chronic alcohol exposure. Since
few studies have been done and there is a definitive changes in the zinc levels,
further study has to be taken up.
Ethanol consumption/Liver damage may alter the liver content of the
several trace elements like zinc and copper. Copper level is increased in
alcoholic liver diseases. Liver copper and urinary zinc and copper excretion
seem to be related with severity of alcoholic disease.8 Hence for the studies are
required to understand the changes in the trace elements in alcoholic liver
diseases.
6.2 Review of literature:
it is not surprising that magnesium levels were decreased in patients with alcoholic
fatty liver because the connection of hypomagnesemia with alcoholism has been known
for long time.5
Hypomagnesemia is not only a laboratory symptom of fatty liver but due to its
connection with increased oxidative stress it might be a risk factor in the progression of
fatty liver to steatohepatitis.5
Hypomagnesemic patients more frequently had other acid base and electrolyte
abnormalities, such as hypophasphatemia, hypokalemia, hypocalcemia and respiratory
alkalosis, as compared with the normomagnesemic patients.6
About 25% of alcoholics are chronically hypomagnesemic because of the combination
of poor nutritional intake and increased renal loss.7
Ethanol consumption and or liver damage may alter liver content of several trace
elements as iron, zinc, copper and manganese. This alteration may play a role on
ongoing fibrogenesis.8
Alcoholic liver disease is associated with decreased zinc and its major binding protein,
metallothionein.9
Zinc deficiency affects RNA and DNA synthesis, enzymatic process ( ADH ), vitamin
metabolism ( vit A ) and contributes to hypogonadism.10,11
Severe depletion of magnesium affects thiamine indirectly leading to wernicke –
korsakoff syndrome.12
6.3 Objectives:To study the level of trace elements ( Magnesium, Zinc and Copper) in chronic
Alcoholic liver disease.
7.Materials and Methods:
7.1 Source of data:
Test Subjects: 30 patients who are diagnosed with chronic alcoholic liver
disease
Between the age group of 30-60 years from Dr.BRAMC
Control Subjects: 30 controls with no history of chronic alcoholic liver disease
Between the age group of 30-60 years from Dr.BRAMC
Study period: January 2011 to January 2012
Exclusion criteria:

Patients with clinical evidence of hypertension, Diabetes, Mellitus,
Pancreatitis, Renal failure and malnutrition

Individuals belonging to other age group .

Subject on drugs affecting mineral metabolism ( gentamicin, Cisplatin)

Other causes of hepatitis
7.2 Method of collection of Data ( Including Sampling procedures)
Test subjects are patients with chronic alcoholic liver disease b/w age of 30-60
years ; from Dr. BRAMC
Control subjects are healthy individuals with no history of alcoholic liver disease b/w the
age of 30-60 years from Dr. BRAMC
The Blood pressure, height, weight, BMI are recorded in both tests subjects & control
subjects.
The Diagnosis of chronic alcoholic liver disease is based on history of chronic
significant alcohol abuse ( alcohol drinking history is assessed by interview and patient
questionnaire. Data from questionnaire are used to establish the type of alcoholic
beverages, frequency, pattern and duration of use), clinical signs of liver disease and
supporting laboratory tests which are ALT, AST and Bilirubin levels and GGT.
5 ml of venous blood sample is collected following an overnight fasting and centrifuged.
Serum is collected and analyzed by following methods:
Estimation of serum magnesium by Titan Yellow Method
Estimation of serum zinc by Dithizone method
Estimation of serum copper by Diethyl Dithio carbamate method
7.3 Does the study require any investigation or intervention to be conducted on patient
or animal?
Yes, Investigations are required .
No, Interventions are not required.
7.4 Has ethical clearance been obtained from your institution in case 7.3 ?
yes
8. List of References:
1. Lawrence A. Kaplan textbook of Clinical Chemistry Theory, Analysis, Correlation
4th edition, Alcoholism, 641 – 642.
2 American Psychiatric Association. 1994. Diagnostic and Statistical Manual of
Mental Disorders (4th edition) (DSM - IV).
3 Sheila Sherlock and James Dooley. Diseases of the liver and biliary system. 11 th
edition. Alcohol and the Liver. 381-383
4 Harrison’s Principles of Internal Medicine, Volume II, 17th Edition, Alcoholic Liver
Diseases, Page No.: 1969-1971
5 Turecky L, Kupcova V et al Serum Magnesium levels in patients with alcoholic
and non alcoholic fatty liver. Bratisl Listy 2006; 107(3) 58-61
6 Elisaf M, Merkouropoulos M et al, pathogenetic mechanism of hypomegnesemia
in alcoholic patients. J Trace elem Med Biol. 1995 Dec; 9(4): 210-4.
7 Lee Goldman, Devvis Ausiello , Cecil’s textbook of Medicine,23 rd edition
Disorders of magnesium and Phosphorus. 859
8 F.Rodriguez-Morenoa, E.Gonzalez – Reimers a, et al, Zinc, Copper, Manganese
and iron in chronic alcoholic liver disease. Alcohol. 1997 Jan-Feb;14(1):39-44.
9 Kang YJ, Zhou Z, Zinc prevention and treatment of alcoholic liver disease. Mol
Aspects Med. 2005 Aug-Oct;26(4-5):391-404
10 Devgun MS et al; vitamin and mineral nutrition in chronic alcoholics including
patients with korsakoff’s Psychosis. P Br J Nutr 45:469, 1981
11 Thomson AD, Majumdar SK: The influence of ethanol on intestinal absorption
and utilization of nutrients, clin Gastroenterol 10:263, 1981
12 Smith JC, Jr, et al: Zinc: a trace element essential in vitamin A metabolism,
Science 181:954, 1973
SIGNATURE OF THE CANDIDATE
9
10
REMARKS OF THE GUIDE
11
NAME AND DESIGNATION
a. GUIDE
DR. HEMALATHA. MD.
PROFESSOR AND HOD
OF BIOCHEMISTRY,
DR. BRAMC,
BANGALORE
b. SIGNATURE
c. CO-GUIDE
d. SIGNATURE
e. HEAD OF THE DEPARTMENT
f. SIGNATURE
12
12.1 REMARKS OF THE CHAIRMAN AND
PRINCIPAL
12.2 SIGNATURE
DR. HEMALATHA. MD.
PROFESSOR AND HOD
OF BIOCHEMISTRY,
DR. BRAMC,
BANGALORE