THE EFFECTS OF ALCOHOL ADMINISTRATION ON SERUM LIPID PROFILE TOTAL PROTEIN AND LIVER ENZYMES IN RATS

ABSTRACT

Alcohol  has many biological actions,  and adverse  effects on lipid metabolism.  The  main objective of this present study was designed to study the effects of alcohol administration on serum   lipid profile, total protein, liver enzymes and histopathological effect in rats. A total twenty five male wistar rats (190-200g) were divided into five groups of five animals each. The animals were grouped so that the mean difference in the various groups would not vary significantly. Group one served as the control whereas groups two, three, four and five were made up of ethanol treated rats. The ethanol was administered intraperitoneally (I.P) and the treatment was carried out for three months and analysis of the parameters done on a four week basis. After .the last dose of every four weeks, blood was collected, centrifuged to form the  serum.  Ethanol  administration  on  rats  produced  a  marked  and  significant  (p<0.05) increase in lipid profile when compared to the normal. It caused an initial increase in total cholesterol in first four weeks of the treatment and decline. Ethanol administration made a significant   increase   in   high   density   lipoprotein   cholesterol,   low   density   lipoprotein cholesterol  and   triacylglycerol.   Furthermore  ethanol  administration  enhanced  the  liver enzymes by  increasing their activities. The activities of alanine aminotransferase,  alkaline phosphatase and aspartate aminotransferase increased significantly (p<0.05) when compared to control. Serum total protein levels indicated a significant increase (p<0.05) with ethanol administration. Alcoholism  is a progressive disease. Liver disease is the most common complication from ethanol abuse. Alcoholic fatty liver may progress to alcoholic hepatitis and finally to cirrhosis and liver failure

CHAPTER ONE

INTRODUCTION

Alcoholism  leads to fat accumulation in the liver, hyperlipidemia,  and ultimately cirrhosis (Murray et al., 2006). The fatty liver is caused  by a combination  of  impaired fatty acid oxidation and increased lipogenesis, which is thought to be due to changes in the [NADH]/ [NAD+]  redox  balance  in  the  liver.  Lipid  homeostasis  is  altered  by  chronic  ethanol

consumption leading to the development of a fatty liver as well as lipid alterations in other organs (Carrasco et al., 2002). Liver disease is the most common complication from ethanol abuse (Mello et al., 2007). It is estimated that 15 to 30% of chronic heavy drinkers eventually develop severe liver diseases. Alcoholic fatty liver may progress to  alcoholic hepatitis and finally to cirrhosis and liver failure (Reuben, 2008). In the USA, chronic alcohol abuse is the leading cause of liver cirrhosis and the need for liver transplantation (Masters, 2001). On the other hand, it has been shown that alcohol consumption may protect against severe coronary atherosclerosis,  but the mechanism  through which alcohol might exert its protective effect remains unclear (Dai and  Miller, 1997). High-density lipoprotein-cholesterol  (HDL-C) like other  lipids  shows  a  dose-dependent  relationship  to  alcohol  intake.  Because  HDL-C  is thought to play an important role in preventing atherosclerosis (Seppa et al., 1992), it has been  proposed  that  alcohol  protection  occurs  via  increasing  HDL-C.  Sillanaukee  et  al., (2000)  showed  that all lipid  values,  except  low density lipoprotein  cholesterol  (LDL-C), positively correlated with reported alcohol consumption.

1.1.      Alcohols

Alcohols  are  compounds  that  have  hydroxyl  groups  bonded  to  saturated  carbon  atoms. Alcohols can be thought of as organic derivative of water in which one of the water hydrogen is replaced by an organic group (Fig. 1).

H-O-H versus R-O-H.

Fig. 1: Formular of alcohol.

Alcohols  occur  widely  in  nature  and  have  a  great  many  industrial  and  pharmaceutical applications.  Ethanol,  for  instance  is one of the  simplest  yet  best  known  of all organic substances, usually used as a fuel additive, an industrial solvent and a beverage.

Alcohols are classified as primary (1), secondary (2) or tertiary (3) depending on the number of the organic groups bonded to the hydroxyl bearing carbon and ethanol is a primary alcohol (Garrett and Grisham, 2005).

Ethanol was one of the first organic chemicals to be prepared and purified. Its production by fermentation of grains and sugar has been carried out for millennia and its purification by distillation. Only about five percent (5%) of the ethanol produced  industrially comes from fermentation.

Most ethanol is currently obtained by acid-catalyzed hydration of ethylene (Fig. 2).

H2C=CH2 + H2O ——–CH3CH2OH

Fig. 2: Equation for ethanol formation.

1.2.      Lipids

Fats absorbed from the diet and lipids synthesized by the liver and adipose tissue must be transported between the various tissues and organs for utilization and storage. Since lipids are insoluble in water, the problem of how to transport them in the aqueous  blood plasma is solved by associating non-polar lipids with amphipathic lipids and  proteins to make water miscible lipoproteins. Lipids are transported in the plasma as lipoproteins.

1.2.1.   Plasma Lipids

Plasma lipids consist of triacylglycerols, phospholipids, cholesterol and cholesteryl esters and a much smaller fraction of unesterified long chain fatty acids. These are metabolically the most  active  of the plasma  acids.  Because  fat  is less dense  than  water,  the  density  of a lipoprotein decreases as the proportion of lipid to protein  increases (Murray et al., 2006). Four major groups of lipoproteins have been identified that are physiologically important in clinical diagnosis. These are:

(a)       Chylomicrons, derived from intestinal absorption of triacylglycerol and other lipids. (b)       Very  low  density  lipoproteins  (VLDL)  derived  from  the  liver  for  the  export  of triacylglycerols.

(c)       Low density lipoproteins representing a final stage in the catabolism of VLDL.

(d)       High  density  lipoproteins,  involved  in  cholesterol  transport  and  also  in  VLDL metabolism. Triacyglycerols are the predominant lipids in chylomicrons and VLDL, whereas cholesterol and phospholipids are the predominant lipids in LDL and HDL.

1.3.     Liver’s central role in lipid transport and metabolism

The liver carries out the following major functions in lipid metabolism.

(a)   It facilitates  the digestion  and absorption  of lipids by the production  of bile,  which contains cholesterol and bile salts synthesized  within the liver de novo or from  uptake of lipoprotein cholesterol.

(b) It actively synthesizes and oxidizes fatty acids and also synthesizes triacylglycerol and

phospholipids.

(c)  It converts fatty acids to ketone bodies.

(d) It plays an integral part in the synthesis and metabolism of plasma lipoprotein (Murray et al., 2006).

1.4.     The effects of alcohol on the liver

Alcohol  is metabolized  by  alcohol  dehydrogenase  (ADH)  into  acetaldehyde,  then  further metabolized by  aldehyde dehydrogenase (ALDH) into  acetic acid, which is finally oxidized into   carbon  dioxide  (CO2)  and  water  (Inaba  and  Cohen  2004).    This  process  generates NADH,  and increases the  NADPH/NADP+ ratio. A higher NADH  concentration  induces fatty acid synthesis while a decreased  NAD level results in decreased fatty acid oxidation

(Murray et al., 2006).

Alcohol Dehydrogenase

CH3-CH2-OH                                    CH3-CHO – – – – – – — – – (Fig.3) Ethanol                                             Acetaldehyde

NAD+                  NADH + H+

Fig. 3: Enzymic oxidation of ethanol.

Fatty liver or steatosis is the accumulation of fatty acids in liver cells. Alcoholism  causes development of large fatty globules (macro vesicular steatosis) throughout the liver and can begin to occur after a few days of heavy drinking. Subsequently, the  higher levels of fatty acids  signal  the  liver  cells  to  compound  it  to  glycerol  to  form  triacylglycerols.  These triacylglycerols accumulate, resulting in fatty liver.

(a) Fatty liver

Alcoholic  liver  disease  is a term that encompasses  the  hepatic manifestations  of  alcohol overconsumption, including fatty liver, alcoholic hepatitis, and chronic hepatitis with hepatic

fibrosis or cirrhosis (O shea et al.,2010). It is the major cause of liver disease in Western countries.  Although steatosis  (fatty liver) will develop  in any individual who  consumes a large quantity of alcoholic beverages over a long period of time, this process is transient and reversible  (Menon  et  al.,2001).  Of  all  chronic  heavy  drinkers,  only  15–20%  develops hepatitis or cirrhosis, which occurs in succession (Menon et al.,2001).

How alcohol damages the liver is not completely understood. 80% of alcohol passes through

the liver to be detoxified. Chronic consumption of alcohol results in the secretion of  pro- inflammatory cytokines, oxidative stress, lipid peroxidation, and acetaldehyde toxicity. These factors cause inflammation, apoptosis and eventually fibrosis of liver cells. Additionally, the liver has tremendous capacity to regenerate and even when 75% of hepatocytes are dead, it continues to function as normal (Longstreth et al.2009).

Pathophysiology

Fig. 4: Pathogenesis of alcohol induced liver injury [htt: //www Wikipedia, 2010]. (b) Alcoholic hepatitis

Alcoholic hepatitis is characterized  by the inflammation of hepatocytes. Between 10% and

35% of heavy drinkers develop alcoholic hepatitis. While development of hepatitis is  not directly related to the dose of alcohol, some people seem more prone to this reaction than others. This is called alcoholic steato necrosis and the inflammation appears to predispose to liver  fibrosis.  Inflammatory  cytokines  are  thought  to  be  essential  in  the  initiation  and perpetuation of liver injury by inducing apoptosis and necrosis. One possible mechanism for the increased activity of TNF-α is the increased intestinal permeability due to liver disease. This facilitates the absorption of the gut-produced endotoxin into the portal circulation. The

Kupffer cells of the liver then phagocytose endotoxin, stimulates the release of TNF-α. TNF- α then triggers apoptotic pathways through the activation of caspases, resulting in cell death (Menon et al., 2001).

(c)  Cirrhosis

Cirrhosis is a late stage of liver disease marked by inflammation (swelling), fibrosis (cellular hardening)  and  damaged  membranes  preventing  detoxification  of chemicals  in  the body, ending in scarring and necrosis (cell death). Between 10% to 20% of  heavy drinkers will develop cirrhosis of the liver. Acetaldehyd e may be responsible for alcohol-induced fibrosis by stimulating  collagen  deposition  by  hepatic  stellate  cells  The  production  of  oxidants derived  from  NADPH  oxidase  and/or  cytochrome   P-450   2E1  and  the  formation  of acetaldehyde-protein adducts damage the cell membrane (Menon et al.,2001).

Symptoms  include  jaundice  (yellowing),  liver enlargement,  pain and tenderness  from  the structural changes in damaged liver architecture. Continued alcohol use, will eventually lead to liver failure. Late complications  of cirrhosis or liver failure include  portal hypertensio n (high blood  pressure  in the portal vein due to the increased  flow  resistance  through the damaged liver), coagulation disorders (due to impaired  production of coagulation factors), ascites  (heavy  abdominal  swelling  due  to  build  up  of  fluids  in  the  tissues)  and  other complications, including hepatic encephalopathy and the hepatorenal syndrome.

Cirrhosis can also result from other causes than alcohol abuse, such as viral hepatitis  and heavy exposure to toxins other than alcohol. This phenomenon is termed the “final common pathway” for the disease.

Fatty change and alcoholic hepatitis with abstinence can be reversible. The later stages  of fibrosis and cirrhosis tend to be irreversible, but can usually be contained with abstinence for long periods of time.

Lipids, mainly as triacylglycerol accumulate in the liver. Extensive accumulation is regarded as a pathological  condition.  When  accumulation  of lipids  in the  liver  becomes  chronic, fibrotic changes occur in the cells that progress to cirrhosis and impaired liver function (Plate

Fatty liver falls into two main categories. The first type is associated with raised levels of plasma free fatty acids resulting from hydrolysis of lipoprotein triacyglycerols by lipoprotein lipase in extra hepatic tissues. The production of very low density lipoprotein (VLDL) does not  keep  pace  with  the  increasing  influx  and   esterification   of  fatty  acids,  allowing triacylglycerol to accumulate, causing fatty liver (Murray et al., 2006).

The second type of fatty liver is usually due to metabolic block in the production of plasma lipoproteins  thus allowing triacylglycerol  to accumulate.  This may   be due to  one of the following;

(a) A block in apolipoprotein synthesis.

(b) A block in the synthesis of the lipoprotein from lipid and apolipoprotein. (c)  A failure in provision of phospholipids that are found in lipoprotein.

(d)  A failure in the secretory mechanism itself.

Plate  1:  A  cross  section  of  the  Normal  Liver,  Fatty  Liver  and  Cirrhosis;  [htt://www

Wikipedia, 2010].

1.5        Cholesterol

Cholesterol is an amphipathic lipid and essential structural component of membranes and of the outer layer of plasma  lipoproteins.  Cholesterol   is present   in   plasma either   as  free cholesterol  or as a storage form, combined  with a  long-chain  fatty  acid as cholestery  ester. In plasma, both forms are transported in lipoproteins.  It is synthesized in many tissues from acetyl-CoA and is the precursor of all other steroids in the body. Cholesterol occurs in foods of animal origin such as egg yolk, meat, liver and brain.  Cholesterol is a major constituent of

gallstones.  However,  its chief role in pathologic  process is as a factor  in the genesis  of atherosclerosis  of vital arteries, causing cerebrovascular,  coronary, and peripheral  vascular disease (Murray et al., 2006).

Cholesterol  cannot travel alone through the blood stream,  it has to combine with  certain proteins.  These  proteins  act  like trucks,  picking  up the cholesterol  and  transporting  it to different parts of the body. When this happens, the cholesterol and protein form a lipoprotein. The two most important types of lipoproteins are high-density lipoproteins (HDL) and low- density  lipoproteins  (LDL).  People  call  LDL   cholesterol   “bad  cholesterol”  and  HDL cholesterol  “good  cholesterol”  because  of  their  very different  effects  on the  body.  Most cholesterol in the body are found in LDL, and this is the kind that is most likely to clog the blood vessels, keeping blood from flowing through the body the way it should (Murray et al., 2006). On the other hand, HDL cholesterol removes cholesterol from the blood vessels and carries it back to the liver, where it can be processed and sent out of the body.

1.5.1. Biosynthesis

All animal cells manufacture cholesterol with relative production rates varying by cell type and organ function. About 20–25% of total daily cholesterol production occurs in the liver; other sites of higher synthesis rates include the intestines, adrenal glands, and reproductive organs. Synthesis within the body starts with one molecule of acetyl CoA and one molecule of acetoacetyl-CoA,  which are hydrated  to form  3-hydroxy-3-methylglutaryl  CoA (HMG- CoA). This molecule is then reduced to  mevalonate by the enzyme HMG-CoA reductase. This is the regulated, rate-limiting and irreversible step in cholesterol synthesis and is the site of action for the statin drugs (HMG-CoA reductase competitive inhibitors).

Mevalonate is then converted to 3-isopentenyl pyrophosphate in three reactions that require ATP. Mevalonate is decarboxylated to isopentenyl pyrophosphate, which is a key metabolite for various biological reactions. Three molecules of isopentenyl pyrophosphate condense to form farnesyl pyrophosphate through the action of geranyl transferase. Two molecules of f arnesyl pyrophosphate then condense to form squalene by the action of squalene synthase in the endoplasmic reticulum. Oxidosqualene cyclase then cyclizes squalene to form lanosterol. Finally, lanosterol is converted to cholesterol through a 19-step process (Rhodes et al.,1995).

1.5.2.   Functions of cholesterol

Cholesterol has several functions. These include;

It  builds  and  maintains   cell  membranes   (outer  layer),  it  prevents   crystallization   of hydrocarbons in the membrane.

It is essential for determining which molecules can pass into the cell and which cannot (cell membrane permeability).

It is involved in the production of sex hormones (androgens and estrogens).

It  is  essential  for  the  production  of  hormones  released  by the  adrenal  glands  (cortisol, corticosterone, aldosterone, etc).

It aids in the production of bile.

It converts sunshine to vitamin D.

It is important for the metabolism of fat soluble vitamins, including vitamins A, D, E, and K. It insulates nerve fibers [htt://www Wikipedia, 2010].

Cholesterol cannot travel alone through the blood stream; it has to combine with  certain proteins.  These  proteins  act  like trucks, picking  up the cholesterol  and  transporting  it to different parts of the body. When this happens, the cholesterol and protein form a lipoprotein. The two most important types of lipoproteins are high-density lipoproteins (HDL) and low- density lipoproteins (LDL). Most cholesterol in the body are found in LDL, and this is the kind that is most likely to clog the blood vessels, keeping blood from flowing through the body the way it should (Murray et al., 2006).

On the other hand, HDL cholesterol removes cholesterol from the blood vessels and carries it back to the liver, where it can be processed and sent out of the body.

1.6      High-Density Lipoprotein

High-density lipoprotein (HDL) is one of the five major groups of lipoproteins  which,  in order of sizes, largest to smallest,  are chylomicrons,  VLDL,  IDL, LDL and HDL,  which enable lipids like cholesterol and triacylglycerols  to be transported within the  water-based bloodstream. In healthy individuals, about thirty percent of blood  cholesterol is carried by HDL (Kwiterovich, 2000).

Blood tests typically report HDL-C level, i.e., the amount of cholesterol contained in HDL particles. It is often contrasted  with low density lipoprotein or LDL-cholesterol  (LDL-C). HDL particles are able to remove cholesterol from within artery atheroma and  transport it back to the liver for excretion or re-utilization, which is the main reason why the cholesterol carried within HDL particles (HDL-C) is sometimes called “good cholesterol”. Those with

higher levels of HDL-C seem to have fewer problems with cardiovascular  diseases,  while those with low HDL-C cholesterol levels have increased rates for heart disease (Barter et al.,

2007).

1.6.1    Structure and Functions of High Density Lipoprotein

HDL is the smallest of the lipoprotein particles. They are the most dense because they contain the highest proportion of protein and cholesterol. The most abundant apolipoproteins are apo A-I and apo A-II (Lin et al., 1998). The liver synthesizes these lipoproteins as complexes of apolipoproteins   and   phospholipid,   which   resemble   cholesterol-free   flattened   spherical lipoprotein particles. They are capable of picking up cholesterol, carried internally, from cells by interaction with the ATP-binding cassette  transporter A1 (ABCA1). A plasma enzyme called   lecithin-cholesterol   acyltransferase   (LCAT)   converts   the   free   cholesterol   into cholesteryl ester (a more hydrophobic form of cholesterol), which is then sequestered into the core of the lipoprotein particle,  eventually making the newly synthesized  HDL spherical. They  increase  in  size  as  they  circulate  through  the  bloodstream  and  incorporate  more cholesterol and phospholipid molecules from cells and other lipoproteins.

HDL transports cholesterol mostly to the liver or steroidogenic organs such as adrenals, ovary and testes by direct  and indirect  pathways.  HDL  is removed  by HDL receptors  such  as scavenger receptor BI (SR-BI), which mediate the selective uptake of cholesterol from HDL. In humans, probably the most relevant pathway is the indirect one, which is mediated by cholesteryl ester transfer protein (CETP). This protein exchanges triacylglycerols of VLDL against cholesteryl esters of HDL. As the result, VLDLs are  processed to LDL, which are removed  from the circulation  by the LDL receptor  pathway.  The triacylglycerols  are not stable in HDL, but degraded by hepatic lipase so  that finally small HDL particles are left, which restart the uptake of cholesterol from cells.

The cholesterol delivered  to the liver is excreted into the bile and, hence intestine,  either directly  or  indirectly  after  conversion  into  bile  acids.  Delivery  of  HDL  cholesterol  to adrenals,   ovaries,   and   testes   is   important   for   the   synthesis   of   steroid   hormones 2010].

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