unit 3 vbc full notes.pdf

I. Hormonal regulation of carbohydrate metabolism Why glucose concentration should be maintained or regulated? Glucose is supplied by intestinal absorption of dietary glucose or by hepatic glucose Production from its precursors, for example, carbohydrates (glycogen, fructose, galactose) and amino ac...

I. Hormonal regulation of carbohydrate metabolism Why glucose concentration should be maintained or regulated? Glucose is supplied by intestinal absorption of dietary glucose or by hepatic glucose Production from its precursors, for example, carbohydrates (glycogen, fructose, galactose) and amino acids (gluconeogenesis). The monosaccharides are completely absorbed through mucosa of small intestine occurs by sodium-independent active transport mechanism using GLUCOSE TRANSPORTERS (GLUT).  Muscle, on the other hand, does not contain G-6-Pase, so it cannot provide free glucose and is therefore primarily a glucose-utilizing tissue. The glucose transporter system (GLUT-4) across the membrane is rate limiting in peripheral tissues that are sensitive to insulin (muscle,fat).  In the liver, however, glucose moves freely across the plasma membrane through transporter system 2 (GLUT-2) that are not sensitive to insulin. The normal glucose concentration: 140 mg/dL (postprandial), 70 to 99 mg/dL (pre-prandial). A slight variation of this normal level leads to hyperglycaemia or hypoglycaemia. Thus, the level of glucose is regulated by various hormones. And this hormonal regulation of carbohydrate metabolism is mainly occurred by the internal chemical messengers. Hormones like insulin, glucagon, epinephrine, cortisol, TH and GH regulates this metabolism. PANCREAS The pancreas is an organ and a gland. Glands are organs that produce and release substances in the body. The pancreas performs two main functions: Exocrine function: Produces substances (enzymes) that help with digestion. Endocrine function: Sends out hormones that control the amount of sugar in your bloodstream.  Insulin: Insulin is a peptide hormone. Secreted by 𝛽 cells of islets of Langerhans from pancreas. Elevated blood glucose level leads to insulin secretion. The secreted insulin carries various anabolic functions. Thus, maintain high blood glucose in normal range. Effect of insulin: 1. Reduces blood glucose mainly by uptake of glucose by the cells through GLUT 4 glucose transporters. 2. In other tissues like Adipose tissue - increases fatty acid and triglyceride synthesis. - Decreases lipolysis. 3. Liver and muscle - increases glycogen synthesis. - Decreases glycogenolysis. (The action of insulin is opposed by the diabetogenic factors, growth hormone, glucagon, cortisol, and epinephrine.)  Glucagon: Glucagon is also a peptide hormone secreted by 𝛼 cells of islets of Langerhans from pancreas. It is an antagonist of insulin which shows the catabolic activities. It is secreted when there is fall in blood glucose level from normal range. Thus, it stops insulin secretion during low blood glucose level. It increases blood glucose mainly by breaking down of stored glycogen and triglycerides. Effect of glucagon: In liver: - 1. Increases glycogenolysis. 2. Increases gluconeogenesis. 3. Increases ketone body synthesis. 4. Decreases glycogen synthesis. In muscle: Increases protein degradation. Decreases protein synthesis. In adipose tissue: -Increases lipolysis. -Decreases triacylglycerol synthesis. The overall effect of glucagon is to stimulate glucose synthesis, use of ketone bodies as fuel for brain and other tissues. Epinephrine: This hormone is secreted by adrenal medulla. lt acts both on muscle and liver to bring about glycogenolysis by increasing phosphorylase activity. The end product is glucose in liver and lactate in muscle. The net outcome is that epinephrine increases blood glucose level. The net result is an increase in glucose uptake by the liver with increased glucose oxidation, glycogenesis, and hypoglycemia. The kinases (see in glycolysis) direct metabolism toward glucose utilization and the opposing enzymes reverse the direction so they are gluconeogenic. The effect of insulin tends to lower blood glucose, whereas the opposing effects of growth hormone, glucagon, and adrenal cortical hormones tend to raise it.  1. Disorders of Carbohydrate Metabolism The major function of dietary carbohydrate is to serve energy sources and their storage function. II. Diabetes Mellitus Is chronic heterogenous metabolic disorder which is characterized by elevated blood glucose level or Hyperglycemia, which results from abnormalities in either insulin secretion or insulin action or both. Diabetes mellitus has been reported in virtually all laboratory animals (gerbils, guinea pigs, hamsters, mice, rats, nonhuman primates) and in horses, cattle, sheep, and pigs, but it is most frequently found in dogs and cats. In the dog, it is frequently associated with obesity and it is now known that obesity is the single most important contributing factor to the development of diabetes. Little is known of the genetic aspects of diabetes in animals as compared to humans in which the hereditary predisposition is well known. TYPES OF DIABETES Diabetes mellitus is broadly divided into 2 groups, namely  insulin-dependent diabetes mellitus (IDDM)  non-insulin dependent diabetes mellitus (NIDDM). This classification is mainly based on the requirement of insulin for treatment. 1. Type-1 Diabetes/Insulin-Dependent Diabetes Mellitus (IDDM) is also known as Juvenile onset of Diabetes. It is also defined by development of ketoacidosis in absence of insulin therapy. Is characterized by Autoimmune destruction of the pancreatic 𝛽 cells i.e. by no or very low initial insulin level or no response to glucose load. The β-cell destruction may be caused by drugs, viruses or autoimmunity. Due to certain genetic variation, the β-cells are recognized as non-self and they are destroyed by immune mediated injury. Usually, the symptoms of diabetes appear when 80-90% of the β- cells have been destroyed. The pancreas ultimately fails to secrete insulin in response to glucose ingestion. There is a form of type 1 diabetes called idiopathic diabetes mellitus that is not autoimmune mediated but is strongly inherited. 2. Type-2 Diabetes/ Non-Insulin Dependent Diabetes Mellitus (NIDDM). Is characterized by Insulin resistance and insulin deficiency.  It is also characterized by persistent hyperglycemia but rarely leads to ketoacidosis. This is the most common variety worldwide (about 90 per cent of all diabetes mellitus cases).  Type 2 Diabetes generally manifests after a later age and therefore has the obsolete name of adult onset -type of diabetes. Type 2 Diabetes can result from genetic defects that cause insulin resistance and insulin deficiency. Onset is most usual during adult life; there is a familial tendency and an association with obesity. BIOCHEMISTRY OF DIABETES 1. GLUCOSE METABOLISM The uncontrolled diabetes leads to increased hepatic glucose output. This is firstly by mobilizing liver glycogen and initiating gluconeogenesis to produce glucose. The lack of insulin also impairs non-hepatic tissue utilization of glucose. REF:The insulin is responsible for glucose uptake by adipose tissue and skeletal muscle which is accomplished by insulin-mediated movement of glucose transporter proteins. Reduced glucose uptake by peripheral tissues in turn leads to reduced rate of glucose metabolisms This combination of increased hepatic glucose production and reduced peripheral tissue metabolism leads to elevated plasma glucose levels. When blood glucose levels reach beyond threshold capacity of the kidneys to absorbs glucose leads to GLUCOSURIA. Due to Osmotic effect more water accompanies glucose that leads to Polyuria. Thus polyuria (excessive urine excretion), intracellular and extracellular dehydration, and increased thirst (POLYDIPSIA) are classic symptoms of diabetes. The loss and ineffective utilization of glucose leads to breakdown of fat and protein which results in loss of weight When glucose level of extracellular fluid is increased, bacteria gets more nutrition for multiple recurrent infection.  2. ON LIPID METABOLISM Insulin normally has positive effect on glycogenesis in hepatocytes and skeletal muscle.IT also stimulates hepatocytes to synthesize triglycerides and storage of triglycerides. Ref: In presence of insulin, the levels of malonyl-CoA are high. That inhibit carnitine acyl transferase 1(CAT-1). This enzyme is required for transportation of fatty acyl CoA into mitochondria. In absence of insulin: malonyl CoA falls so increase in CAT-1 will be seen That leads to increase in transportation of fatty acyl CoA into mitochondria leads to Beta - oxidation. ↓ Generates acetyl Co-A further oxidized in TCA cycle. ↓ But excess acetyl CoA than body needs always metabolized to ketone body formation. In Diabetes availability of ketone bodies leads to decrease glucose utilization contributing to hyperglycemia. Excess of ketone bodies leads to ketoacidosis. Diagnosis : Biochemical tests for the detection of disturbances in carbohydrate metabolism: Glycosuria: The commonest cause of glucose excretion in urine (glycosuria) is diabetes mellitus. Therefore, glycosuria is the first line screening test for diabetes. Normally, glucose does not appear in urine until the plasma glucose concentration exceeds renal threshold (180 mg/dl). As age advances, renal threshold for glucose increases marginally. GLUCOSE TOLERANCE TEST (GTT): The diagnosis of diabetes can be made on the basis of individual’s response to oral glucose load, the oral glucose tolerance test (OGTT)/ Intravenous GTT. Glucose tolerance (GT) Tolerance is referred to the amount of glucose that could be ingested by an animal without producing a glycosuria, hence, tolerance for glucose. The ability to utilize carbohydrate can be determined by glucose Tolerance.  Initially fasting blood glucose is estimated.  A loading dose of glucose is given.  The blood glucose levels are estimated at regular intervals after glucose load.  In conditions of insulin deficiency, blood glucose levels get elevated due to impaired utilization of glucose. Because, in the normal animal, the absence of a glycosuria indicates only a limited rise in blood glucose where the renal threshold is not exceeded, GT now refers to the blood glucose curve following glucose administration. Glycated hemoglobin : The glucose molecule in the body has the capability to bind irreversibly to protein forming a product known as glycated proteins.  When the protein the complex is hemoglobin (Hb), the product is called hemoglobin A1C.  Among the glycated hemoglobin’s, the most abundant form is HbA1c. HbA1c is produced by the condensation of glucose with N-terminal valine of each β-chain of HbA. Diagnostic importance of HbA1c : reflects the average blood glucose level over the period of half-life of hemoglobin or RBCs. Thus, the concentration of HbA1c serves as an indication of the blood glucose concentration over a period, approximating to the half-life of RBC (hemoglobin) i.e. 6–8 weeks. A close correlation between blood glucose and HbA1c concentrations has been observed when simultaneously monitored for several months. Normally, HbA1c concentration is about 3–5% of the total hemoglobin. In diabetic patients, HbA1c is elevated (to as high as 15%). Determination of HbA1c is used for monitoring of diabetes control. HbA1c reflects the mean blood glucose level over 2 months period prior to its measurement. Fructosamine : Besides HbA1c, several other proteins in the blood are glycated. Glycated serum proteins (fructosamine) can also be measured in diabetics. As albumin is the most abundant plasma protein, glycated albumin largely contributes to plasma fructosamine measurements. Albumin has shorter half-life than Hb. Thus, glycated albumin represents glucose status over 3 weeks prior to its determination. III. KETOSIS Ketone Bodies are metabolic products that are produced in excess during excessive breakdown of fatty acids. Acetoacetate, B- Hydroxybutyrate and acetone are collectively known as Ketone bodies. Only Acetoacetate and acetone are true ketone bodies but B-hydroxy butaryte doesnot pocess keto group. Ketone bodies are water soluble and energy yielding.  The predominant source of ketones in healthy animals is long chain fatty acids (LCFA) released during lipolysis in adipose tissue.  The LCFA in liver can be used for either triacylglycerol synthesis or can go through β- oxidation to acetyl-CoA in the mitochondrion.  Mitochondrial acetyl-CoA can have a number of fates, but under circumstances that elevate plasma LCFA levels, the two main fates are combustion in the TCA cycle or conversion to ketones. Ketogenesis occurs in the liver during periods of low carbohydrate availability (Fasting condition) and ketones are an important fuel source for the brain and cardiac and skeletal muscle during starvation. Ketones also reduce the amount of glucose and protein utilised for energy production when glucose levels are deficient. Ketogenesis is controlled by three enzymes:  hormone-sensitive lipase (in adipocytes),  acetyl CoA carboxylase and  HMG CoA synthase (in the liver). When carbohydrate and glucose levels are low, fatty acid production is stimulated by the release of adrenaline and glucagon. Hormone-sensitive lipase is stimulated by glucagon release (and inhibited by insulin) and breaks down triglycerides into fatty acids, resulting in raised serum fatty acid levels which leads to the production of increased amounts of acetyl CoA, the substrate for ketone synthesis. Acetyl CoA is produced from β-oxidation of fatty acids and can enter the citric acid cycle. However, acetyl CoA has to combine with oxaloacetate in order to be able to enter the citric acid cycle. Oxaloacetate is produced from pyruvate during glycolysis and hence if glucose levels are low there is not enough oxaloacetate production and the remaining oxaloacetate is preferentially used for gluconeogenesis. The abundance of acetyl CoA that then remains forms ketone bodies. These ketone bodies are soluble, require no protein carrier, and diffuse (in their unionized form) or are transported rapidly through biological membranes including blood-brain barrier. KETOSIS: Excessive production of ketone bodies.  Causes: Excessive ketone bodies are produced mainly in two conditions  Starvation and  uncontrolled diabetes(impaired uptake of glucose by the peripheral tissues)  Clinical Significance: Both B-hydroxy bytyrate and acetoacetate are Organic acids and are released in protonated form, to lower the pH of the blood; the reduced pH affect the normal buffering mechanism leading to KETO-ACIDODIS.  Biochemical And Clinical Findings The important features of ketosis are ketonemia, ketonuria, acetone odour of breathe, metabolic acidosis and hyperkalemia. 1. KETONEMIA : In ketosis, the plasma concentration of ketone bodies is well above normal limits. The condition is called Ketonemia 2. KETONURIA: When the concentration of ketone bodies significantly increased (above 70mg/dl) in plasma, they appear in urine. The condition is called ketonuria. 3. Acetone in Breathe: acetone is also excreted by the lungs and produces a characteristic odor in breathe acetone odor of breathe). 4. Metabolic acidosis: Metabolic acidosis is caused by excessive accumulation of B- hydroxybutyrate and acetoacetate. 5. Hyperkalemia: Acidosis results in the shift of potassium from intracellular to extracellular compartment  Biochemical Diagnosis  B-hydroxy butyrate in plasma  Acetoacetate in urine  Rothera’s test: Acetoacetic acid and acetone react with alkaline solution of sodium nitroprusside to form a purple colored complex. This method can detect above 1-5 mg/dl of acetoacetic acid and 10-20 mg/dl of acetone. Beta-hydroxybutyrate is not detected.  Management of Ketosis  Provision of glucose to the tissues Ketosis is supressed by restoring adequate level of carbohydrate meatbolisn.  Correction Of electrolyte imbalance by bicarbonate metabolism IV. BOVINE KETOSIS  Ketosis is an important metabolic disease in dairy cattle which occurred due to increase of ketone bodies.(especially B-hydroxy butyrate in plasma) It is characterized by ketonuria, loss of potassium in the urine, and a fruity odour of acetone on the breath. Untreated, ketosis may progress to ketoacidosis, coma, and death. This condition is seen in starvation, occasionally in pregnancy if the intake of protein and carbohydrates is inadequate, and most frequently in diabetes mellitus. Bovine Ketosis Introduction  Bovine ketosis occurs in the high producing dairy cows during the early stages of lactation, when the milk production is generally the highest.  Abnormally high levels of the ketone bodies, acetone, acetoacetic and beta-hydroxy butyric acid and also iso- propanol appear in blood, urine and in milk.  The alterations are accompanied by loss of appetite, weight loss, decrease in milk production and nervous disturbances.  Hypoglycemia (starvation) is a common finding in bovine ketosis and in ovine pregnancy toxemia.  In non-ruminants, liver is the sole source of ketone bodies.  In ruminants, the rumen epithelium and mammary gland are also sources of ketone bodies production.  Among the ketone bodies acetone does not ionize to the appreciable level, whereas, acetoacetic and b hydroxybutyric acids will ionize readily.  Acetoacetic and b-hydroxybutyric acid are more powerful acids than the volatile fatty acids. Ketosis in Lactation Ketosis is the most common metabolic disease in high performance dairy cows during the first 6-8 weeks of lactation. Ketosis is a lactational disorder usually associate with intense milk production and negative energy balance.  During lactation plasma glucose is drained for the synthesis of lactose by the mammary gland.   The two sources of plasma glucose are absorption from the gut and gluconeogenesis. In ruminanats little glucose is absorbed from the gut. Most of the glucose is synthesized in the liver and in the kidney. The chief substrates are propionate , which is produced in high grain diet.  When there is a mismatch between mammary drain of glucose for lactose synthesis and gluconeogenesis in liver, hypogycemia will result.  The condition leads to ketosis. Underfeeding Ketosis  This type of ketosis occurs when a dairy cow receives insufficient calories to meet the lactational demands plus body maintenance.  This type is further divided into Nutritional Underfeeding Ketosis and Secondary Ketosis. I. The Underfeeding Ketosis It occurs when the cow is given an insufficient quantity of feed or a diet with low metabolic energy densities.  II. The Secondary Ketosis it occurs in cows that has other disease like hypocalcemia, mastitis, or metritis, which suppresses appetite and prevents feed consumption. Alimentary Ketosis  This type of ketosis occurs when cattle have been fed spoiled silage that contains excessive amounts of butyric acid.  The rumen epithelium has a high capacity to activate butyrate to acetoacetate and betahydroxybutyrate.  When there is excess presence of butyrate, large quantity of beta hydroxbutyrate will be formed and released into the circulation with resulting ketosis. Spontaneous Ketosis This is the most common form of ketosis and occurs near peak of lactation, that have access to abundant high quality feed, and that have no other diseases.  The disease is not accompanied by severe acidosis and spontaneous recovery is common although there is a large decrease in milk production. Hypoglycemic Theory  The most widely accepted theory of bovine ketosis is the hypoglycemic theory.  During lactation mammary gland might withdraw glucose from the plasma more rapidly than the liver can supply it, which leads to hypoglycemia.  The hypoglycemia will lead to ketonemia as more of the LCFAs will reach the liver and oxidized. The net result of this is an increase in the level of ketone bodies. V. PREGNANCY TOXAEMIA Pregnancy toxemia in sheep and goats has also been called ketosis, lambing/kidding sickness, pregnancy disease and twin-lamb/ kid disease. The principal cause of pregnancy toxemia is low blood sugar (glucose). Onset of the disease is often triggered by one of several types of stress including nutritional or inclement weather. The disease is most prevalent in ewes and does carrying two or more lambs or kids. The disease also affects ewes and does that are extremely fat or excessively thin. Since glucose is essential for proper functioning of the brain, a deficiency of glucose will result in nervous dysfunction and eventually coma and death. Glucose is also required for the muscles during exercise, but one of its greatest uses is by the fetuses. The growing fetuses continually remove large quantities of glucose and amino acids for their growth and energy requirements which results in hypoglycemia, lipid mobilization, accmulation ketone bodies. Since glucose is essential for proper functioning of the brain, a deficiency of glucose will result in nervous dysfunction and eventually coma and death.  BLOOD BIOCHEMISTRY OF KETOSIS TREATMENT: 1. Glucose and Glucose precursors helps in ketosis treatment 2. Parenteral glucose provides immediate relieve of hypoglycemia. 3. Gluconeogenic precursors such as propylene glycol, glycerol and sodium pyruvate have been effective. VI. Hyperinsulinism The disease is due to a persistent hyperactivity of the pancreas as the result of insulin secreting islet cell tumors. Hyperinsulinism is characterized by a persistent hypoglycemia with periods of weakness, apathy, fainting, and during hypoglycemic crises, convulsions, and coma. Establishment of the diagnosis depends on finding a hypoglycemia of 3 mmol/l ( 55 mg/dl) at thetime of symptoms and a hyperinsulinemia, usually 20μU/ml. The symptoms are also relieved by glucose administration. An insulinoma is a malignant pancreatic tumor that inappropriately secretes excessive insulin, resulting in profound hypoglycaemia. Any breed of dog can be affected, but large breeds tend to be overrepresented. Diagnosis: 1. Blood Glucose A simple fasting blood glucose level of less than 40 mg/dL can suggest hyperinsulinemia/ 2. Insulin-to-Glucose Ratio: A positive insulin-to-glucose ratio demonstrates an inappropriately elevated insulin level in the setting of hypoglycaemia. 3. Imaging: Radiography & Ultrasound Thoracic radiography and abdominal ultrasound are recommended to assess for the presence of a possible pancreatic mass and associated metastatic disease VII. Hypoglycemia of Baby Pigs: Hypoglycemia of baby pigs occurs during the first few days of life and is characterized by hypoglycemia of around 40 mg/dl, apathy, weakness, convulsions, coma, and finally death. At birth, the blood glucose level is around 110 mg/dl and, unless the pig is fed or suckles shortly after birth, its blood glucose drops rapidly to hypoglycemic levels within 24 to 36 hours. The liver glycogen, which is high at birth, is almost totally absent at death. In contrast, newborn lambs, calves, and foals are able to resist starvation hypoglycemia for more than a week. If the baby pig suckles, its ability to withstand starvation progressively increases from the day of birth. Gluconeogenic mechanisms are undeveloped in the newborn pig, which indicates that the gluconeogenic enzymes of the baby pig are inadequate at birth. Starvation of the newborn pig under natural conditions can occur because of factors relating to the sow (agalactia, metritis, etc.) or to the health of the baby pig (anemia, infections, etc.), either case resulting in inadequate food intake. The requirement for feeding to induce the hepatic gluconeogenic mechanisms in the newborn baby pig explains its inability to withstand starvation in contrast to the newborn lamb, calf, or foal, which is born with fully functioning hepatic gluconeogenesis. VIII. Plasma proteins  Blood proteins, also termed plasma proteins, are proteins present in blood plasma.  Total blood volume is about 4.5 to 5 liters in adult human being. If blood is mixed with an anticoagulant and centrifuged, the cell components (RBC and WBC) are precipitated. The supernatant is called plasma. About 55–60% of blood is made up of plasma.  If blood is withdrawn without anticoagulant and allowed to clot, after about 2 hours liquid portion is separated from the clot. This defibrinated plasma is called serum, which lacks coagulation factors including prothrombin and fibrinogen.  Total protein content of normal plasma is 6 to 8 g/100 mL.  The plasma proteins consist of albumin (3.5 to 5 g/dL), globulins (2.5 – 3.5 g/dL) and fibrinogen (200– 400 mg/dL). The albumin : globulin ratio is usually between 1.2:1 to 1.5:1.  Almost all plasma proteins, except immunoglobulins are synthesized in liver. Plasma proteins are generally synthesized on membrane-bound polyribosomes. Most plasma proteins are glycoproteins.  In laboratory, separation can be done by salts. Thus, fibrinogen is precipitated by 10% and globulins by 22% concentration of sodium sulfate. Ammonium sulfate will precipitate globulins at half saturation and albumin at full saturation. o Serum albumin accounts for 55% of blood proteins, is a major contributor to maintaining the oncotic pressure of plasma and assists, as a carrier, in the transport of lipids and steroid hormones. o Globulins make up 38% of blood proteins and transport ions, hormones, and lipids assisting in immune function. o Fibrinogen comprises 7% of blood proteins; conversion of fibrinogen to insoluble fibrin is essential for blood clotting. o The remainder of the plasma proteins (1%) are regulatory proteins, such as enzymes, proenzymes, and hormones. All blood proteins are synthesized in liver except for the gamma globulins. Functions of the Plasma Proteins:  Maintaining osmotic pressure (albumin).  Catalyzing biochemical reactions  buffering acid-base balance  blood coagulation (fibrinogen),  host defenses against pathogens (immunoglobulins, complement),  transport of metabolites (transferrin, albumin),111  regulation of cellular metabolism (hormones),  prevention of proteolysis (α1-antitrypsin),  provision of nitrogen balance for nutrition. ALBUMIN: Albumin is a major constituent of plasma proteins with a concentration of 3.5- 5.0g.dl. Synthesis of albumin- Bovine serum albumin is synthesized and secreted by the hepatocytes. Functions of Albumin: 1) Osmotic function- due to its high concentration and low molecular weight, albumin contribute to 75-80% of the total plasma osmotic pressure. It also play role in maintaining blood volume andbody fluid distribution. Decreased albumin conc. Results in a fall in osmotic pressure leading to enhanced fluid retention in tissue spaces causing edema. 2) Transport protein: Albumin is the carrier of various hydrophobic substances in the blood. Being a watery medium, blood cannot solubilize lipid components. i. Bilirubin and non-esterified fatty acids are specifically transported by albumin. ii. Drugs (sulfa, aspirin, salicylate, dicoumarol, phenytoin). iii. Hormones: Steroid hormones, thyroxine. iv. Metals: Albumin transports copper. Calcium and heavy metals are non-specifically carried by albumin. 3) Buffering Action: All proteins have buffering capacity. Because of its high concentration in blood, albumin has maximum buffering capacity GLOBULIN: They perform variety of function which includes transport and immunity. There are α globulins- α1-Anti trypsin and α2 –macroglobulin, Haptoglobin, ceruloplasmin. Theses globulin acts as acute phase proteins. Β-globulins: Transports lipids and iron and heme. Β- Lipoprotein, transports lipids. Transferrin- iron, Hemopexin- heme Γ- globulins: are immunoglobulins which performs antibody functions. Fibrinogen: participates in blood coagulation. CLINICAL SIGNIFICANCE OF PLAMS PROTEINS: Abnormal Protein Concentration The liver synthesizes of albumin, fibrinogen, prothrombin and most of the globulins particularly alpha and beta globulins. The gamma globulins are synthesized in the lymphoid organs.  The normal range of total protein levels in most of the animals ranges between 5 and 8 g/Dl  Edema develops when the total protein concentration in plasma falls below 5g/dL. Dysproteinemia- can be defined as an abnormality in the conten of blood proteins and also protein dyscrasia. Generally diagnosed by electrophoresis and by calculating albumin and globulin ratio(A:G ratio) Types of dysproteinemia: Hypoproteinemia: (decreased protein concentration) Hypoalbuminemia with hypoglobulinemia: It may be due to decreased concentrations of albumin, globulin or both.  Blood Loss- Due to proportional loss of all blood constituents, interstitial fluid moves into the circulatory system and dilutes the remaining blood causing a decrease in the level of albumin and globulin.  Over hydration  Oedema  Haemorrhage  Decreased protein synthesis/ Increased protein catabolism.  Protein losing entropy- During a variety of intestinal lesions both albumin and globulin leak from the intestinal wall into the intestinal lumen and then are digested or excreted.  Severe burns: These cause increased vascular permeability that can result in loss of both albumin and globulin.  Effusive disease: This results in the accumulation of body cavity fluids with high protein concentrations that can result in decreased albumin and globulin concentrations. Hyperproteinemia: (Increased Protein Concentration) Hyperalbuminemia and Hyperglobulinemia. Loss of water from the blood causes an increased concentrations of albumin and globulin.  Due to dehydration  Increased protein synthesis  Inflammation  Infections viral, bacterial  hepatotoxicity, Nephrotoxicity Hyperglobulinemia: Increased gamma globulin concentration. It depends on the type of globulin that is increased: -Acute inflammation is the most common cause. - in acute inflammation Concentrations of several proteins in the globulin fraction ( e.g., Ceruloplasmin, haptoglobin, and alpha 2 macroglobulin) are increased. These proteins are collectively called as acute phase proteins. IX. AUTE-PHASE PROTEINS Proteins whose concentrations in blood plasma either increase (positive acute-phase proteins) or decrease (negative acute-phase proteins) in response to inflammation. APPs are blood proteins primarily synthesized by hepatocytes as part of acute phase response. Acute phase reaction is early defence which is triggered by different stimuli trauma, infection, stress, or inflammation.APR results in a complex systemic reaction with the goal of re- establishing homeostasis and healing. These APPs have wide range of activities that contribute to host defence mechanism by directly neutralizing inflammatory agents, helping to minimize the extent of local tissue damage and tissue repair and regeneration. The proteins participate in killings infectious agents and the clearance of foreign and host cell debris. Coagulation proteins including fibrinogen plays essential role in wound healing. TYPES OF APP 1. Positive Acute phase proteins – the liver responds by producing large number of acute phase reactants. Positive PPs are further classified into: Major, Moderate, Minor on their concentration. Major APPs – Show up to 100-1000fold Increase in its serum concentration in 1-2 days. Moderate APPs - Show up to 5-10 fold Increase in its serum concentration in 2-3 days. Minor in minimal quantity. 2. Negative phase proteins- at the same time some protein production is reduced. The functions APP: 1. Anti-protease: Inhibit proteases released by phagocytes and other cells of the immune system to minimize damage to normal tissues Eg., α1-antitrypsin, α2- macroglobulin. 2. Scavenging activities and bind metabolites released from cellular degradation so they can re- enter host metabolic processes rather than have be utilized by pathogen. Eg.,haptoglobin, Serum Amyloid A(SAA), C-Reactive protein(CRP). Antibacterial activity: eg. Alpha 1 acid glycoprotein (AGP), SAA, CRP. 1) C-Reactive Protein:  C-reactive protein (CRP) 20 kDa) was the first acute phase protein to be recognized. It is named from its ability to bind to C-polysaccharide of Gram bacteria. CRP binds to pathogen and activates the classical complement pathway leading to the opsonization of the bacteria.  In dog and pig, CRP is a major APP. CRP is increased in infection and implantation of the embryo in the endometrium of bitch. 2) Haptoglobin: Haptoglobin is a glycoprotein. The primary function of Hp is to bind free hemoglobin in the blood. And by removing from the circulation any free hemoglobin, which has inherent peroxidase activity.  Hp prevents it causing oxidative damage to tissues. The Hp- hemoglobin binding also reduces the availability of the heme residue and its iron from bacterial use, and therefore Hp has an indirect antibacterial activity.  In ruminants, it is a major APP. Haptoglobin has been the main APP studied in ruminants because of its reaction during the acute phase response and also because of its ease of analysis. Haptoglobin is a moderate APP in dogs and responds to inflammatory and infectious disease. 3. Serum Amyloid A:Serum amyloid A (SAA) is a small hydrophobic protein (9 to 14 kDa) that is found in serum associated with high density lipoprotein (HDL).  Function of SAA: Transports of cholesterol from dying cells to hepatocytes , inhibitory effect on fever, inhibitory effect on oxidative burst of neutrophilic granulocytes. In ruminants, horses, and cats, SAA assay has become a routine assessments of infection and inflammation. In the horse, SAA is a major APP and increased SAA concentrations have been observed in horses following surgery, with aseptic inflammation or arthritis, septicemia, enteritis, pneumonia, and diarrhea in colic. 4. α-1 Acid Glycoprotein: Alpha-1 acid glycoprotein (AGP) is one of the most highly glycosylated proteins in serum with a molecular mass of around 43kD.  Though the précis role of AGP is not clear, it does bind to a number of endogenous metabolites such as heparin, histamine, serotonin, steroids, and catecholamines. In most species, AGP is a moderate APP increasing more slowly but also remaining elevated for longer than the major APP such as canine CRP or bovine SAA and Hp. 5. Fibrinogen: Fibrinogen is a large protein of 340 kDa that constitutes nearly 5% of the total plasma protein.It is more consistently increased during inflammation in horses and cattle than it is in dogs and cats. Negative Acute Phase Proteins: Negative acute phase proteins are serum proteins that decrease in concentration by greater than 25% during the acute phase in response to infection, inflammation, and trauma.  Serum albumin is a negative acute phase protein, and the concentration of this protein falls gradually with the reduction in concentration being more noticeable in chronic inflammatory disease.  Transferrin, the iron transport protein of serum has also been described as a negative APP, but in chickens, ovatransferrin, is a positive APP.  X. LIPID PROFILE IN DISEASE DIAGNOSIS Lipid profile or lipid panel, is the collective term given to the estimation of, typically, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglycerides. An extended lipid profile may include very low-density lipoprotein. This is used to identify hyperlipidaemia (various disturbances of cholesterol and triglyceride levels), many forms of which are recognized risk factors for cardiovascular disease and sometimes pancreatitis Lipid Blood Tests : 1.) Cholesterol: directly linked to risk of heart and blood vessel disease. It evaluates risk of atherosclerosis , coronary heart diseases and a part of thyroid function, liver function, diabetes mellitus. Goal values: 75-169 mg/dL for those age 20 and younger 100-199 mg/dL for those over age 21 2.) High Density Lipoprotein (HDL) :“Good cholesterol” High levels linked to a reduced risk of heart and blood vessel disease. The higher your HDL level, the better. Goal value: Greater than 40 mg/dL HDL is a lipoprotein (a combination of fat and protein) found in the blood. It is called "good" cholesterol because it removes excess cholesterol from the blood and takes it to the liver. A high HDL level is related to lower risk of heart and blood vessel disease. 3.) Low Density Lipoprotein (LDL) “Bad cholesterol” High levels are linked to an increased risk of heart and blood vessel disease, including coronary artery disease, heart attack and death. Reducing LDL levels is a major treatment target for cholesterol-lowering medications. Goal values: Less than 70 mg/dL for those with heart or blood vessel disease and for other patients at very high risk of heart disease (those with metabolic syndrome) Less than 100 mg/dL for high risk patients (e.g., some patients who have multiple heart disease risk factors) Less than 130 mg/dL for individuals who are at low risk for coronary artery disease. 4.) Triglycerides (TG) Elevated in obese or diabetic patients. Level increases from eating simple sugars or drinking alcohol. Associated with heart and blood vessel disease. Goal value: Less than 150 mg/dl. Triglycerides are a type of fat found in the blood. The blood level of this type of fat is most affected by the foods you eat (such as sugar, fat or alcohol) but can also be high due to being overweight, having thyroid or liver disease and genetic conditions. High levels of triglycerides are related to a higher risk of heart and blood vessels. XI. CLINICAL ENZYMOLOGY Diagnostic importance of non-functional diagnosis. Clinical enzymology is the discipline that studies and tests enzyme activity in serum, plasma, urine, or other body fluids for the purpose of helping to establish the diagnosis and prognosis of disease and to screen for abnormal organ function. Enzymes are classified into two groups based upon their clinical importance as  “functional plasma enzymes” i.e. those enzymes present in the plasma in considerable high amounts and are functional in the plasma due to the presence of their substrate in the plasma. Ex. Serum lipase, blood clotting enzymes  “non-functional plasma enzymes” i.e. those enzymes that are present in the plasma in negligible amounts and has no function in the plasma due to the absence of their substrate in the plasma. Non-functional plasma enzymes are of diagnostic importance. Functional plasma enzymes:  Present in plasma at higher concentration than tissues  They function in plasma.  Mostly synthesized by the liver  Usually decreased in disease conditions Eg. Clotting enzymes, lipoprotein lipase Non-functional plasma enzymes:  Present in plasma at lower concentration than tissues  Do not have any function in plasma  Mostly synthesized by liver, skeletal muscle, heart, brain etc  Usually increased in disease conditions Eg. Creatine kinase, Alanine transaminase etc Gross damage to the cells or abnormal membrane permeability, overproduction of the enzymes or abnormal high cellular proliferation may allow their leakage in abnormally high amount into plasma and other body fluids. Measurement of these enzymes in plasma can be used to assess cell damage and proliferation i.e. diagnosis of disease. A well-known example is renal tubular gamma glutamyltransferase (GGT) located on the luminal surface of renal tubular epithelial cells. Injury to these cells results in release of the gamma GT into urine but not into blood. Similarly, alkaline phosphatase (ALP) located on the luminal surface of enterocytes is lost into the gut lumen rather than blood with enterocyte injury. Hepatocellular ALP, however, with activity over both bile canalicular and sinusoidal surfaces, can be increased in both bile and blood. ISOENZYMES: Isoenzymes (also known as isozymes) are enzymes that differ in amino acid sequence but catalyze the same chemical reaction. They all have independent action E.g. Lactate dehydrogenase have 5 isoenzymes (LDH1, LDH2, LDH3, LDH4 & LDH5) They can be used to identify the specific affected tissues. Specific Enzymes 1. CREATINE KINASE: Its predominant physiologic function occurs in muscle cells, where it is involved in the storage of high-energy creatine phosphate. Every contraction cycle of muscle results in creatine phosphate use, with the production of ATP. Tissue Source : CK is widely distributed in tissue, with highest activities found in skeletal muscle, heart muscle, and brain tissue. CK is present in much smaller quantities in other tissue sources, including the bladder, placenta, gastrointestinal tract, thyroid, uterus, kidney, lung, prostate, spleen, liver, and pancreas. Diagnostic Significance :  Because of the high concentrations of CK in muscle tissue, CK levels are frequently elevated in disorders of cardiac and skeletal muscle.  The CK level is considered a sensitive indicator of acute myocardial infarction (AMI) and muscular dystrophy  CPK also exists in various isoenzyme forms. It has two polypeptides ‘B’ & ‘M’ that forms dimmers in the following combinations to give rise to three isoenzymes of CPK.  BM – Predominant in cardiac muscle  B2 – Predominant in brain  M2 – Predominant in skeletal muscle Thus estimation of the isoenzyme BM is indicative of heart attack. CPK maintains a higher concentration in the plasma for 1-2 days. The concentration of CPK after the first attack is 10 times more than the normal and if another attack occurs within a day or two the concentration further increases to 100 fold and a third attack within a short span of time raises the level of CPK to 300 fold which is lethal concentration. 2. LACTATE DEHYDROGENASE LDH is widely distributed in the body. High activities are found in the heart, liver, skeletal muscle, kidney, and erythrocytes , lesser amounts are found in the lung, smooth muscle, and brain.  Because of its widespread activity in numerous body tissues, LDH is elevated in a variety of disorders.  LDH catalyzes the inter conversion of pyruvate to lactate, a very important reaction of anaerobic glycolysis.  LDH is present in each and every cell of the body. Therefore damage to any of the tissues of the body results in release of LDH into the plasma. Hence it becomes a difficult task to trace out the organ from which it has been leaked.  3. ASPARTATE AMINOTRANSFERASE Aspartate aminotransferase (AST) is an enzyme belonging to the class of transferases. It is commonly referred to as a transaminase and is involved in the transfer of an amino group between aspartate and α-keto acids. o The older terminology, serum glutamic-oxaloacetic transaminase (SGOT, or GOT), may also be used. Pyridoxal phosphate functions as a coenzyme. o The reaction proceeds according to the following equation: The transamination reaction is important in intermediary metabolism because of its function in the synthesis and degradation of amino acids. The keto-acids formed by the reaction are ultimately oxidized by the tricarboxylic acid cycle to provide a source of energy. Tissue Source: AST is widely distributed in human tissue. The highest concentrations are found in cardiac tissue, liver, and skeletal muscle, with smaller amounts found in the kidney, pancreas, and erythrocytes Diagnostic Significance:The clinical use of AST is limited mainly to the evaluation of hepatocellular disorders and skeletal muscle involvement.  AST elevations are frequently seen in pulmonary embolism. AST activity is relatively high and in similar amounts in liver and in skeletal and cardiac muscle, but it varies between species. It is routinely used in equine and food animal medicine as a screening test for injury to both organs. Reference Range  AST, 5 to 30 U/L (37°C). 4. ALANINE AMINOTRANSFERASE Alanine aminotransferase (ALT) is a transferase with enzymatic activity similar to that of AST. Specifically, it catalyzes the transfer of an amino group from alanine to α-ketoglutarate with the formation of glutamate and pyruvate.  The older terminology was serum glutamic-pyruvic transaminase (SGPT, or GPT).  The following equation indicates the transferase reaction.  Pyridoxal phosphate acts as the coenzyme. Tissue Source: ALT is distributed in many tissues, with comparatively high concentrations in the liver. It is considered the more liver-specific enzyme of the transferases. Diagnostic Significance:  Clinical applications of ALT assays are confined mainly to evaluation of hepatic disorders.  Higher elevations are found in hepatocellular disorders than in extra hepatic or intrahepatic obstructive disorders.  In acute inflammatory conditions of the liver, ALT elevations are frequently higher than those of AST and tend to remain elevated longer as a result of the longer half-life of ALT in serum (16 and 24 hours, respectfully.  Cardiac tissue contains a small amount of ALT activity.  ALT levels have historically been compared with levels of AST to help determine the source of an elevated AST level and to detect liver involvement concurrent with myocardial injury. Reference Range  ALT, 6–37 U/L (37°C).  5. ALKALINE PHOSPHATASE ALP activity is present on cell surfaces in most human tissue. The highest concentrations are found in the intestine, liver, bone, spleen, placenta, and kidney.  In the liver, the enzyme is located on both sinusoidal and bile canalicular membranes;  Activity in bone is confined to the osteoblasts, those cells involved in the production of bone matrix. Diagnostic Significance  Elevations of ALP are of most diagnostic significance in the evaluation of hepatobiliary and bone disorders.  In hepatobiliary disorders, elevations are more predominant in obstructive conditions than in hepatocellular disorders.  In bone disorders, elevations are observed when there is involvement of osteoblasts.  The highest elevations of ALP activity occur in Paget’s disease (osteitis deformans).  Other bone disorders include osteomalacia, rickets, hyperparathyroidism, osteogenic sarcoma, during healing bone fractures and during periods of physiologic bone growth.  In normal pregnancy, increased ALP activity can be detected between weeks 16 and 20. 6. ACID PHOSPHATASE Acid phosphatase is present in lysosomes, which are organelles present in all cells with the possible exception of erythrocytes. The prostate is the richest source, with many times the activity found in other tissue. Diagnostic Significance :  Historically, ACP measurement has been used as an aid in the detection of prostatic carcinoma, particularly metastatic carcinoma of the prostate.  Newer markers, such as prostate-specific antigen (PSA), are more useful screening and diagnostic tools. XII. Liver Function test Liver is a versatile organ which is the central organ of body metabolism and independently involves in many other biochemical functions. Liver perform several diversified functions enumerated below: 1. Metabolic function: Liver is the key organ and the principal site where the metabolism of carbohydrate, lipids, protein, minerals and vitamins take place. 2. Secretory function: Liver is responsible for the formation and secretion of bile in the intestinee. Bile pigment bilirubin formed from heme catabolism is conjugated in liver cells and secreted in the bile. Cholesterol and bile salts are also secreted in the bile into the intestine. 3. Excretory function: Exogenous dye BSP (bromosulphthalein) and Rose Bengal dye are excreted through liver cells. 4. Detoxification and Protective function: Ammonia is detoxified to urea. Liver cells can detoxified drugs, hormones and convert them into less toxic substances for excretion. Kuffer cells of liver perform phagocytosis to eliminate foreign compounds. 5. Storage function: Liver stored glycogen, trace mineral iron and vitamin A, D and B12. 6. Hematological function: Liver participates in the formation of blood particularly in the embryo (adults in some abnormal states), synthesis of plasma proteins and blood clotting factors and destruction of erythrocytes The major liver function tests may be classified as follows 1. Tests based on excretory function— Measurement of bile pigments, bile salts, bromosulphthalein. 2. Tests based on serum enzymes derived from liver—Determination of transaminases, alkaline phosphatase, , γ-glutamyltranspeptidase. 3. Tests based on metabolic capacity— Galactose tolerance, antipyrine clearance. 4. Tests based on synthetic functions— Prothrombin time, serum albumin. 5. Tests based on detoxification—Hippuric acid synthesis. Tests based on abnormalities of bile pigment metabolism: 1. VD Bergh reaction and Serum Bilirubin: Bilirubin is estimated by VD Bergh reaction involving Diazo reagent. Van Den Bergh reaction consists of two parts, the direct and indirect reactions. A. Direct reacton: In immediate development of violet colour in 10-30 seconds and delayed direct reaction in which colour appears from 5-30 minutes and then develops slowly to a maximum. B. Indirect reaction: Indirect reaction is a essentially method for the quantitative estimation of serum bilirubin. Serum diluted with distilled water and methanol added in an amount insufficient to precipitate the proteins, yet sufficient to permit all the bilirubin to react with the diazo-reagent. Bilirubin as such is insoluble in water while the conjugated bilirubin is soluble and gives direct positive Van den Bergh reaction. Unconjugated bilirubin gives indirect positive Van den Bergh reaction. If the serum contains both unconjugated and conjugated bilirubin in high concentration, the purple colour is produced immediately (direct positive) which is further intensify by the addition of alcohol (indirect positive). This type of reaction is known as “biphasic”. Interpretation: Normal serum bilirubin level is 0.2-0.6 mg/dl. Response of VD Bergh reaction can differentiate the jaundice as follow:-  In hemolytic/pre-hepatic jaundice un-conjugated bilirubin increased Hense indirect positive reaction obtained. Occasionally it may be a delayed direct reaction.  In obstructive/post-hepatic jaundice conjugated bilirubin is increased hence an immediate direct positive reaction obtained.  In hepatic jaundice either or both may be present (biphasic reaction). Tests based excretory function of liver:  Bromosulphthalein test (BSP): A measured amount of dye is injected intravenously. The liver removes the dye rapidly and excretes in the bile. If the liver function is impaired, the excretion is delayed and larger proportion of dye remains in the serum.  It is very sensitive test and is most useful in liver cell damage without jaundice, in cirrhosis and chronic hepatitis.  In healthy adults not more than 5% dye should be remain in blood but the bulk of dye is removed in 25 minutes. In hepatic diseases, cirrhosis, 40-50 % of dye retension takes place.  Also abnormal retention of dye in hepato-cellular or obstructive jaundice takes place Tests based metabolic capacity of liver: 1. Galactose tolerance test: LFT can be assessed by measuring the utilization of galactose. This is known as galactose tolerance test. 2. Serum protein, Albumin and A/G ratio: Serum protein estimation yields most useful information in chronic liver disease. 3. Estimation of total and esterified cholesterol: Liver synthesizes esterified cholesterol and excretes into bile. So cholesterol level is the marker for liver disease. 4. Prothrombin time: The time required for clotting to take place in citrated plasma to which optimum amounts of thromboplastin and calcium has been added. Prothrombin is formed by the liver cells, vitamin K. When bile salts are not present in the intestine, the absorption of vitamin K form the intestine is impaired. In jaundice and liver disease the prothrombin time is prolonged. Tests based on serum enzymes derived from liver: 1. Aspartate aminotransferase (AST) Normal range: 8 – 20 U/L A marker of hepatocellular damage High serum levels are observed in: Chronic hepatitis, cirrhosis and liver cancer 2. Alanine aminotransferase (ALT) More liver-specific than AST Normal range (U/L): ▫ Male: 13-35 ▫ Female: 10-30 High serum levels are observed in: Acute hepatitis, Alcoholic hepatitis. XIII. Kidney Function Tests (KFT)/ Biochemical Tests for Renal Function Kidney plays an important role in the maintenance of water volume, electrolyte and acidbase balance in the body. Kidney serves an important function of excretion of products of metabolism and other harmful substances. Renal/ Kidney function tests are done to assess the functional capacity of kidney (Blood flow to the kidney, glomerular filtration and tubular function). The aim of renal function tests is to detect impairment of renal function as early as possible. The kidney function can be assessed by examination of blood and urine. The following are commonly used kidney function tests:- A) Urine examination: Simple routine examination of urine for Volume, pH, Concentration test / specific gravity test, Osmolality and presence of certain abnormal constituents (Proteins, ketone bodies, blood, glucose etc.). (B) Blood/serum analysis: Estimation of blood urea nitrogen, serum creatinine, protein and electrolyte. (C) Glomerular function tests: Clearance test (Urea, inulin, creatinine)  Inulin clearance test: This test is done to find the glomerular filtration rate (GFR). Inulin is filterated by the glomerulus but it is neither secreted nor absorbed by tubules. Inulin is given subcutaneously or by intravenous infusion. The amount of inulin excreted in each minutes is equal to the amount filtered by the glomeruli. Normal rate is 110 to 150 ml per minute. (D)Tubular function tests: Urine concentration or dilution test, urine acidification test. Other important renal function tests:  Phenol Sulfonaphthalein (PSP) test: It indicates a general loss of nephron function. A measured amount of phenol Sulfonaphthalein is injected intravenously and then urine is collected at intervals of 40 to 60 minutes. The rate of disappearance of the dye from the plasma can be determined from blood sample taken prior to and then at regular intervals of 30 minutes following its injection. The dye clearance time is prolonged in kidney disease.  Methylene blue excretion test: Methylene blue (2% @0.4 ml/kg) is injected intramuscularly and examined for clearance of dye. In normal condition the excretion of dye reaches its maximum after 1 hour and the clearance is complete within 24 hours. Delay in time indicates kidney dysfunction. The choice of kidney function tests starts with routine urine examination, followed by serum creatinine and/or other blood urea estimation and finally the specific tests to measure the tubular and glomerular functions (Clearance tests). Estimation and clinical significance of creatinine Creatinine, in a protein-free filtrate, is determined by its reaction with alkaline picrate to form a yellow- red tautomer of creatinine picrate, the Jaffc's reaction. The intensity of the colour is proportional to the optical density which is measured at 520 nm. Clinical Significance: - Clinically insignificant at lower values. It is higher in males since it is related to body size. Increased values: - Increased serum levels are seen in renal failure and other renal diseases in a manner similar to urea. - Creatinine, however, does not increase with age, dehydration and catabolic states (eg fever, sepsis, haemorrhage) to the same extent as urea. - It is also not affected by diet. -But the Jaffe's reaction for measuring serum creatinine is not as sensitive and reliable as method for urea. It is interfered with by Ketone bodies and glucose and hence false high values may be obtained in diabetes ketoacidosis. - serum creatinine is not significant. It is associated with muscle wasting diseases. - The creatinine production depends on the modification of the muscular mass, and it varies little and the levels usually are very stable ESTIMATION OF BUN (Blood Urea Nitrogen) (DIACETYL MONOXIME PROCEDURE Urea is synthesized in the liver from ammonia released by the oxidation of amino acids. Thus urea is a water soluble, non toxic, excreatable form of toxic ammonia that is released into the blood and is maintained at the concentration of 15 – 40 mg/100 ml of the blood under normal conditions. Simultaneously it is excreted in urine varying from 15 – 40 gms /day. Conditions causing variation in the blood urea level. A decrease in the blood urea level indicates liver disorders/dysfunction. Whereas an increase in the blood urea level, termed as ureamia may be classified into three major classes. 1. Pre-renal 2. Renal 3. Post-renal.  Pre-renal ureamia: Dehydration due to vomiting (pyloric obstruction) or chronic intestinal obstruction. During dehydration the blood volume decreases leading to hemoconcentration as a result of which the blood urea values seems to be elevated. Due to low blood pressure the glomerular filtration ratewill be lowered leading to increased urea levels. Diarrhea can cause ureamia.  Renal ureamia:- An increase in the blood urea level above 300 mg/dl due to kidney disorders is termed as renal ureamia. This may be due to-  Nephritis  Acute glomerulonephritis.  Total renal failure  Post-renal ureamia:- Causes beyond kidney leading to increased blood urea level are mainly due to obstruction in the urinary tract arising from urolithiasis,urethral tumors urinary bladder tumors. Methodology —  Kjeldahl – a classical method for determining urea concentration by measuring the amount of nitrogen present — -  Berthelot reaction - Good manual method - that measures ammonia uses an enzyme (urease ) to split off the ammonia —  Diacetyl monoxide ( or monoxime) Popular method but not well suited for manual methods. Because uses strong acids and oxidizing chemicals.  DISTURBANCES IN ACID BASE BALANCE AND ITS DIAGNOSIS. Hydrogen ions (H+) are present in all body compartments. Maintenance of appropriate concentration of hydrogen ion (H+) is critical to normal cellular function. ACIDS AND BASES Acids are substances that are capable of donating protons and bases are those that accept protons. Acids are proton donors and bases are proton acceptors. Buffers are solutions which can resist changes in pH when acid or alkali is added. Composition of a Buffer Buffers are of two types: a. Mixtures of weak acids with their salt with a strong base or b. Mixtures of weak bases with their salt with a strong acid. A few examples are given below: i. H2 CO3 /NaHCO3 (Bicarbonate buffer) (Carbonic acid and sodium bicarbonate) ii. ii. CH3 COOH/CH3 COO Na (Acetate buffer) (Acetic acid and sodium acetate) iii. iii. Na2 HPO4/NaH2 PO4 (Phosphate buffer) An example: when hydrochloric acid is added to the acetate buffer, the salt reacts with the acid forming the weak acid, acetic acid and its salt. Similarly when a base is added, the acid reacts with it forming salt and water. Thus changes in the pH are minimized. ACID-BASE BALANCE Normal pH : The pH of plasma is 7.4 (average hydrogen ion concentration of 40 nmol/L). In normal life, the variation of plasma pH is very small. The pH of plasma is maintained within a narrow range of 7.38 to 7.42. The pH of the interstitial fluid is generally 0.5 units below that of the plasma. Acidosis: If the pH is below 7.38, it is called acidosis.  Life is threatened when the pH is lowered below 7.25.  Acidosis leads to CNS depression and coma.  Death occurs when pH is below 7.0. Alkalosis: When the pH is more than 7.42, it is alkalosis.  It is very dangerous if pH is increased above 7.55.  Alkalosis induces neuromuscular hyperexcitability and tetany.  Death occurs when the pH is above 7.6. Acid – Base is primarily concerned with two ions:   Hydrogen (H+)  Bicarbonate (HCO3) Acid bas balance is maintained in the extracellular fluids by three mechanisms: 1. Chemical Buffers/ blood buffer: React very rapidly (Less than a Second) - First line of defence. 2. Respiratory Regulation: Reacts rapidly (Seconds to minutes)-Second line of defence 3. Renal Regulation: Reacts Slowly (Minutes to hours)- Third line of defence BUFFERS OF THE BODY FLUIDS. The body has developed three lines of defense to regulate the body’s acid-base balance and maintain the blood pH 1. Blood buffers 2. Respiratory mechanism 3. Renal mechanism. I. Blood buffers A buffer may be defined as a solution of a weak acid (HA) and its salt (BA) with a strong base. The buffer resists the change in pH by the addition of acid or alkali and the buffering capacity is dependent on the absolute concentration of salt and acid. The buffer cannot remove H+ ions from the body. It temporarily acts as a shock absorbent to reduce the free H+ ions. The H+ ions have to be ultimately eliminated by the renal mechanism. The blood contains 3 buffer systems. 1. Bicarbonate buffer 2. Phosphate buffer 3. Protein buffer. . Bicarbonate buffer system : The most important buffer system in the plasma is the bicarbonate-carbonic acid system (NaHCO3 /H2 CO3 ). It accounts for 65% of buffering capacity in plasma and 40% of buffering action in the whole body. Sodium bicarbonate and carbonic acid (NaHCO3 – H2CO3) is the most predominant buffer system of the extracellular fluid, particularly the plasma.  Phosphate buffer system : Sodium dihydrogen phosphate and disodium hydrogen phosphate (NaH2PO4 – Na2HPO4) constitute the phosphate buffer. It is mostly an intracellular buffer and is of less importance in plasma due to its low concentration.  Protein buffer system : The plasma proteins and haemoglobin together constitute the protein buffer system of the blood.  II. Respiratory mechanism for pH regulation Respiratory system provides a rapid mechanism for the maintenance of acid-base balance.  The CO2 diffuses from the cells into the extracellular fluid and reaches the lungs through the blood.  The rate of respiration (rate of elimination of CO2 ) is controlled by the chemoreceptors in the respiratory centre which are sensitive to changes in the pH of blood.  When there is a fall in pH of plasma (acidosis), the respiratory rate is stimulated resulting in hyperventilation. This would eliminate more CO2 , thus lowering the H2 CO3 level. The large volumes of CO2 produced by the cellular metabolic activity endanger the acidbase equilibrium of the body. But in normal circumstances, all of this CO2 is eliminated from the body in the expired air via the lungs.  The rate of respiration (or the rate of removal of CO2) is controlled by a respiratory centre, located in the medulla of the brain.  This centre is highly sensitive to changes in the pH of blood. Any decrease in blood pH causes hyperventilation to blow off CO2, thereby reducing the H2CO3 concentration.  Simultaneously, the H+ ions are eliminated as H2O. Respiratory control of blood pH is rapid but only a short term regulatory process. When breathing is increased, the blood carbon dioxide level decreases and the blood become more basic. When breathing is decreased, the blood carbon dioxide level increases and the blood becomes more Acidic. III. RENAL REGULATION OF pH An important function of the kidney is to regulate the pH of the extracellular fluid. Normal urine has a pH around 6.The pH is lower than that of extracellular fluid (pH = 7.4). This is called acidification of urine. The pH of the urine may vary from as low as 4.5 to as high as 9.8, depending on the amount of acid excreted. The major renal mechanisms for regulation of pH are: A. Excretion of H+ B. Reabsorption of bicarbonate (recovery of bicarbonate) C. Excretion of titratable acid (net acid excretion) D. Excretion of NH4 + (ammonium ions). 1. Excretion of H+; Generation of Bicarbonate i. This process occurs in the proximal convoluted tubules ii. ii. The CO2 combines with water to form carbonic acid, with the help of carbonic anhydrase. The H2 CO3 then ionizes to H+ and bicarbonate. iii. iii. The hydrogen ions are secreted into the tubular lumen; in exchange for Na+ reabsorbed. These Na+ ions along with HCO3 – will be reabsorbed into the blood. iv. iv. There is net excretion of hydrogen ions, and net generation of bicarbonate. So this mechanism serves to increase the alkali reserve. 2. Reabsorption of Bicarbonate  This is mainly a mechanism to conserve base. There is no net excretion of H+  The cells of the PCT have a sodium hydrogen exchanger. When Na+ enters the cell, hydrogen ions from the cell are secreted into the luminal fluid. The hydrogen ions are generated within the cell by the action of carbonic anhydrase  The hydrogen ions secreted into the luminal fluid is required for the reabsorption of filtered bicarbonate.  Bicarbonate is filtered by the glomerulus. This is completely reabsorbed by the proximal convoluted tubule, so that the urine is normally bicarbonate free.  The bicarbonate combines with H+ in tubular fluid to form carbonic acid. It dissociates into water and CO2. The CO2 diffuses into the cell, which again combines with water to form carbonic acid.  In the cell, it again ionizes to H+ that is secreted into lumen in exchange for Na+. The HCO3 – is reabsorbed into plasma along with Na+.  Here, there is no net excretion of H+ or generation of new bicarbonate. The net effect of these processes is the reabsorption of filtered bicarbonate which is mediated by the Sodium-Hydrogen exchanger. But this mechanism prevents the loss of bicarbonate through urine. 3. Excretion of H+ as Titratable Acid In the distal convoluted tubules net acid excretion occurs. Hydrogen ions are secreted by the distal tubules and collecting ducts by hydrogen ion-ATPase located in the apical cell membrane. The hydrogen ions are generated in the tubular cell by a reaction catalyzed by carbonic anhydrase. The bicarbonate generated within the cell passes into plasma. Titratable acidity reflects the H+ ions excreted into urine which resulted in a fall of pH from 7.4 (that of blood). The excreted H+ ions are actually buffered in the urine by phosphate buffer.  H+ ion is secreted into the tubular lumen in exchange for Na+ ion. This Na+ is obtained from the base, disodium hydrogen phosphate (Na2HPO4). The latter in turn combines with H+ to produce the acid, sodium dihydrogen phosphate (NaH2PO4), in which form the major quantity of titratable acid in urine is present. As the tubular fluid moves down the renal tubules, more and more H+ ions are added, resulting in the acidification of urine. This causes a fall in the pH of urine to as low as 4.5. 4. Excretion of ammonium ions: This is another mechanism to buffer H+ ions secreted into the tubular fluid. The H+ ion combines with NH3 to form ammonium ion (NH4+). The renal tubular cells deamidate glutamine to glutamate and NH3. This reaction is catalysed by the enzyme glutaminase. The NH3, liberated in this reaction, diffuses into the tubular lumen where it combines with H+ to form NH4+. Ammonium ions cannot diffuse back into tubular cells and, therefore, are excreted into urine. NH4+ is a major urine acid.  DISTURBANCES IN ACID-BASE BALANCE Classification of Acid-Base Disturbances :  Acidosis (fall in pH) a. Respiratory acidosis: Primary excess of carbonic acid. b. Metabolic acidosis: Primary deficit of bicarbonate.  Alkalosis (rise in pH) a. Respiratory alkalosis: Primary deficit of carbonic acid. b. Metabolic alkalosis: Primary excess of bicarbonate 1. Metabolic acidosis: Metabolic Acidosis It is characterized by a decrease in pH and bicarbonate. May result from either an excess of acidor reduced buffering capacity due to a low concentration of bicarbonate. Excess acid may occur due increased production of organic acids, or more rarely, ingestion of acidic compounds. a) Excess H+ Production: This is the commonest cause of metabolic acidosis and results from the excessive production of organic acids (usually lactic or pyruvic acid) as a result of anaerobic metabolism. This may result from local or global tissue hypoxia. Tissue hypoxia may occur in the following situations:  Reduced arterial oxygen content: for example anaemia or reduced PaO2  Hypoperfusion: this may be local or global, any cause of reduced cardiac output may hypoperfusion in conditions such as ischaemic bowel or an ischemic limb may cause acdiosis.  Reduced ability to use oxygen as a substrate. In conditions such as severe sepsis and cyanide poisoning anaerobic metabolism occurs as a result of mitochondrial dysfunction. Another form of metabolic acidosis is diabetic ketoacidosis. Cells are unable to use glucose to produce energy due to the lack of insulin. Fats form the major source of energy and result in the production of ketone bodies (aceto – acetate and 3- hydroxybutyrate) from acetyl coenzyme. A Hydrogen ions are released during the production of ketones resulting in the metabolic acidosis often observed. B) Excessive Loss of Bicarbonate: Gastro-intestinal secretions are high in sodium bicarbonate. The loss of small bowel contents or excessive diarrhoea results in the loss of large amounts of bicarbonate resulting in metabolic acidosis. This may be seen such conditions as Cholera or Crohn’s disease. 2. Metabolic Alkalosis Primary excess of bicarbonate is the characteristic feature. Alkalosis occurs when a) excess base is added, b) base excretion is defective or c) acid is lost.  All these will lead to an excess of bicarbonate, This results either from the loss of acid or from the gain in base.  Loss of acid may result from severe vomiting or gastric aspiration leading to loss of chloride and acid. Therefore, hypochloremic alkalosis results.  Hyperaldosteronism causes retention of sodium and loss of potassium.  Hypokalemia is closely related to metabolic alkalosis. In alkalosis, there is an attempt to conserve hydrogen ions by kidney in exchange for K+. This potassium loss can lead to hypokalemia.   Potassium from ECF will enter the cells in exchange for H+. So, in alkalosis, pH of urine remains a) Excess of H+ loss: Gastric secretions contain large quantities of hydrogen ions. Loss of gastric secretions, therefore, results in a metabolic alkalosis. This occurs in prolonged vomiting for example, pyloric stenosis or anorexia nervosa. b) Excessive Reabsorption of Bicarbonate: As discussed earlier bicarbonate and chloride concentrations are linked. If chloride concentration falls or chloride losses are excessive then bicarbonate will be reabsorbed to maintain electrical neutrality. Chloride may be lost from the gastro-intestinal tract, therefore, in prolonged vomiting it is not only the loss of hydrogen ions that results in the alkalosis but also chloride losses resulting bicarbonate reabsorption. Anion gap: is defined as the difference between the total concentration of measured cations (Na+ and K+) and that of measured anion (Cl– and HCO3–). The anion gap (A–) in fact represents the unmeasured anions in the plasma which may be calculated as follows, by substituting the normal concentration of electrolytes (mEq/l). 3. Respiratory Acidosis A primary excess of carbonic acid is the cardinal feature. It is due to CO2 retention as a result of hypoventilation. 4. Respiratory Alkalosis: Respiratory Alkalosis is associated with an increase in pH and a decrease in pCO2. Results from the excessive excretion of CO2. The primary cause is hyperventilation. This is commonly seen in conditions that stimulate respiratory center like Oxygen deficiency at high altitudeso Pulmonary disease and congestive heart failure – caused by hypoxia. Digestive Disorders in Non- Ruminants 1. Vomition  It is a complex reflex act, which results in the rapid, forceful ejection of gastric contents through the mouth.  A number of conditions can stimulate vomiting are presence of foreign objects, intussuception, neoplasia, pyloric stenosis, chronic gastritis, presence of parasites, acute nephritis, hepatic disease, presence of poisons.  Dog and cat vomit easily. In horse it is rare. It is mainly control by centres in brain. Biochemical changes during vomition  During vomition loss of water and HCl. These loses result in dehydration and metabolic alkalosis with increased level of bicarbonate ion and decreased level of chloride ion concentration.  Gastric vomition may also cause hypokalemia, which may be due to increased urinary excretion during alkalosis.  Gastric contents also contains potassium and loss due to vomiting may also contribute to potassium deficiency.  potassium deficiency and hypovolemic due to dehydration may cause renal tubular damage and kidney failure. 2. Diarrhoea  It is rapid elimination of watery fecal material with increased frequency and volume or both.  It is due to parasite, infection by bacteria or virus in the intestinal tract, feeding poor quality diet, sudden dietry change, food poison, heavy metal and presence of organophosphorus compound. Biochemical changes  Diarrhoea results in dehydration associated with H+ and electrolyte disturbances.  Dehydration cause haemoconcentration, which leads to hypovolemic shock, this is characterized by decreased excretion of hydrogen,over production of lactic acid, Hyperkalemia.  Hypoglycemia  Disturbance in absorption of all nutrients. 3. Gastric dilatation volvulus (GDV)  It is an acute GI tract disorder, which is due to the accumulation of gas and fluid in the stomach causing mechanical and functional disturbances to pyloric out flow.  The stomach distends and rotates causing obstruction due to which there is necrosis and perforation of the stomach wall.  There is hyperkalemia, hyperphosphotemia due to reduced renal flow. There is release of intracellular potassium from the damaged tissues.  Due to the leakage of fluid from the blood vessels into tissues, there is haemoconcentration, which results in increased blood urea nitrogen and creatinine values.  Due to degeneration of stomach cells and alteration of liver , the transaminases activities are increased.  There is increase lactic acid production, which cause metabolic acidosis. 4. Lactose Intolerance Definition Lactose intolerance-the inability to break down the lactose in milk due to defeciency of enzyme lactase secreted by the intestinal cells.  The ubiquitousness of this condition causes some to feel that it is not really a disease among adults.  Lactose malabsorption and milk products intolerance symptoms are the most common alimentary tract disorders.  Especially seen in young ones. Cause  Lactase is an intestinal enzyme that helps digest lactose, a sugar that is found in many foods, especially dairy products.  Diarrhea, gas, and abdominal pain can occur when there is not enough lactase to digest milk products.  Lactose intolerance was identified as the cause of bovine neonatal diarrhea  Although lactase deficiency is the most common carbohydrate malabsorption syndrome, other enzymes needed to absorb various sugars (disaccharides).  The clinical symptoms of lactose intolerance belongs: nausea, vomiting, abdominal distension, cramps, flatulence, flatus, diarrhea and abdominal pain. Laboratory Diagnosis  A lactose tolerance test-the administration of a lactose drink followed by monitoring for gastrointestinal symptoms-confirms the diagnosis.  During this test, the blood may also be tested for glucose (sugar), which rises in the lactosetolerant.  Other confirming tests include stool analysis for a high acid content, which signifies intolerance.  DIGESTIVE DISORDERS OF RUMINANTS.  True ruminants such as cattle have one stomach with four compartments: the rumen, reticulum, omasum, and abomasum.  The ruminant stomach occupies almost 75 percent of the abdominal cavity, filling nearly all of the left side and extending significantly into the right side.  The reticulo-rumen is home to a population of microorganisms (microbes or “rumen bugs”) that include bacteria, protozoa, and fungi. The purpose of these microbes is to ferment and break down plant cell walls into their carbohydrate fractions and produce volatile fatty acids (VFAs) from these carbohydrates. These VFAs are later used by the animal for energy. The rumen acts as a fermentation vat by hosting microbial fermentation.  About 50 to 65 percent of starch and soluble sugar consumed is digested in the rumen. Rumen microorganisms (primarily bacteria) digest cellulose from plant cell walls, digest complex starch, synthesize protein from non-protein nitrogen, and synthesize B vitamins and vitamin K. Rumen pH typically ranges from 6.5 to 6.8. The rumen environment is anaerobic (without oxygen). Gases produced in the rumen include carbon dioxide, methane, and hydrogen sulfide. The gas fraction rises to the top of the rumen above the liquid fraction.  The omasum is spherical in shape consistsof piles folds increase the surface area, which increases the area that absorbs nutrients from the feed and water.  The abomasum-true stomach produces hydrochloric acid and digestive enzymes such as pepsin (breaks down proteins) and receives digestive enzymes secreted from the pancreas such as pancreatic lipase (breaks down fats). DIGESTIVE DISORDERS OF RUMINANTS. (1) Acute rumen Indigestion (Rumen overload/lactic acidosis): It occurs mostly in sheep or cattle due to consumption of large amounts of grains and apples as they contain readily fermentable carbohydrates.  Simple indigestion is a minor disturbance in ruminant GI function that occurs most commonly in cattle and rarely in sheep and goats.  Simple indigestion is a diagnosis of exclusion and is typically related to an abrupt change in the quality or quantity of the diet. Etiology:  The disease is common in hand-fed dairy and beef cattle because of variability in the quality and quantity of their feed.  During drought, cattle and sheep may be forced to eat large quantities of poor-quality straw, bedding, or grain.  Simple indigestion can result from suddenly changing the feed, using spoiled or frozen feeds, introducing urea to a ration, turning cattle onto a lush cereal grain pasture, or introducing Feedlot Cattle To A High-Level Grain Ration. Biochemical changes:  Streptococcus bovis is the main rumen microflora responsible for rapid fermentation and production of large quintiles of lactic acid (by anaerobic glycolysis).  The accumulation of lactic acid is more rapid than it is absorbed. This leads to fall in rumen pH and Ruminal atony. There is production of racemic mixture of both ‘D’ and ‘L’ forms of lactic acid.  Some ‘L ‘ form is absorbed and metabolized by the liver and other tissues, but the D lactate cannot be utilized and contributes significantly to the acid load of the body.  Ruminal acidity extends to the blood causing metabolic acidosis, resulting in reduced blood bicarbonate and finally a fall in urine pH. Fluid accumulates in the rumen because of the increase osmolality of the rumen fluid. This causes haemoconcentration, resulting in hypovolumic shock and death. It affected animal survive, there will be chemical ruminants induced by the excess lactic acid and hypoerosmolarity and further metastatic hepatic abscesses may occur. Diagnosis:  A diagnosis of simple indigestion is based on a history.  The diagnosis is confirmed by collection and examination of ruminal fluid, which may have an abnormal pH (7), decrease in the numbers and size of protozoa, or prolonged methylene bluereduction time (a measure of bacterial metabolic activity). (2)Bloat or acute rumen tympany: Ruminal tympany is abnormal distension of the rumen and reticulum caused by excessive retention of the gases of fermentation. Either in the form of a persistent foam mixed with the rumen contents (Primary ruminal tympany) or as free gas separated from the ingesta (secondary ruminal tympany); (Ingestion of bloating forages or interference with eructation mechanism). Normally, gas bubbles produced in the rumen coalesce, separate from the rumen contents to form pockets of free gas above the level of the contents, and finally are eliminated by eructation. (Regular fermentation by the rumen produces about 1.2 to 2.0 liters of gas per day (average is 1.5), which include CO, and CH4 and these gases are continuously removed by eructation. Any disruption in the eructation process leads to the accumulation of gas in the rumen. The reasons for interruption in the normal release of gas may either be (1) Physiological (i.e. mechanical obstruction of the esophagus or (2) Neurological (i.e. interruption of eructation reflex). This results in acute tympany of the rumen (bloat). There are two types of bloat.)  Primary Ruminal Tympany (Frothy Bloat): This is caused due to consumption of large amount of leguminous plants by the ruminant, which contains considerable levels of the polysaccharide pectin and its hydrolytic enzyme pectin methyl esterase. The enzyme pectin methyl esterase hydrolyses pectin into pectic acidand galactouronic acid (a mucopolysaccharide-sticky in nature). Due to sticky nature of Galacto-uronic acid it greatly increases the viscosity of rumen fluid and adheres the food particles in rumen. This results in increasing the surface tension. (i.e.decreases inintermolecular space) which will not allow free flow of gasses, leading to entrapping the gas produced in normal amounts. Further production of gas bubbles in rumen contents forming froth, which further exaggerates the condition forming highly sable foam. Some bacteria overproduce mucopolysaccharids causing bloat. Another enzyme present in the feed of the animal is ribulose diphosphate carboxylase which also produces mucopolysaccharides forming stable bloat.  Secondary ruminal tympany (free-gas bloat): Feedlot bloat may also be of the free-gas type based on the observations that gas may be easily released with a stomach tube. Feedlot cattle are susceptible to esophageal obstruction, esophagitis, ruminal acidosis, rumenitis, overfill, and ruminal atony, each of which can interfere with eructation and cause secondary ruminal tympany and free-gas bloat. (Frothiness of the ruminal contents interferes with function of the cardia and inhibits the eructation reflex. Rumen movements are initially stimulated by the distension, and the resulting hypermotility increase the frothiness of the ruminal contents. Terminally there is a loss of muscle tone and ruminal motility. The most distinctive aspect of bloated cattle is abdominal distension, particularly the left abdomen, caused by distension of the rumen. When intraruminal pressure increases; the pressure on the diaphragm is very high, which results in reduced lung capacity and death from hypoxia.) 3) UREA POISIONING:  Urea is normal component of animal fee. Ruminal microflora contains the enzyme urease which acts upon ureasplitting it into CO2 and NH3.  Ammonia is utilized by the microbes in the rumen for their protein synthesis.  If more than 3% of urea is consumed in the diet by the ruminant, then it leads to toxicity . Excess amount of urea produces more amount of ammonia resulting in its accumulation in the rumen and its subsequent entry into the bloodstream. From the blood, ammonia enters the brain and gets protonated to ammonium ion which cannot be released back into circulation.  Accumulation of ammonia in the brain disrupts the functioning of the brain leading to nervousness, depression, slurring of the voice,blurring of vision, tremors, finally to coma and if situation continues it leads to death. Biochemical Control:  Ammonia exists in two forms depending upon the pH i.e. NH3 and NH4+.  NH3 is hydrophobic and so is easily diffusible through the cell membrane.  NH4+ is more hydrophilic and so cannot easily diffuse across the cell membrane. Hence In order to inhibit the absorption of ammonia from the rumen cells it should be converted to NH4+ form for which the {H+} concentration in the rumen should be increased.  So oral administration of acetic acid is taken up without altering the Ruminal pH, which decreases the absorption of free NH3.  Addition of acetic acid in the feed itself, depending upon the urea content is recommended to avoid ammonia intoxication or urea poisoning.  Biochemistry of Oxidative stress and shock A free radical is a molecule or molecular fragment that contains one or more unpaired electrons in its outer orbital. Oxidation reactions ensure that molecular oxygen is completely reduced to water. The products of partial reduction of oxygen are highly reactive and create havoc in the living systems. Hence, they are also called Reactive oxygen species or ROS.. The following are members of this group: i. Superoxide anion radical (O2 – ) ii. Hydroperoxyl radical (HOO ) iii. Hydrogen peroxide (H2 O2 ) iv. Hydroxyl radical (OH ) v. Lipid peroxide radical (ROO ) vi. Singlet oxygen ( 1O2 ) vii. Nitric oxide (NO ) viii. Peroxy nitrite (ONOO– ) Important characteristics of the ROS are: a. Extreme reactivity b. Short lifespan c. Generation of new ROS by chain reaction d. Damage to various tissues. HARMFUL EFFECTS OF FREE RADICALs ;Free radicals are highly reactive, and are capable of damaging almost all types of biomolecules (proteins, lipids, carbohydrates, nucleic acids).Free radicals beget free radicals i.e. generate free radicals from normal compounds which continues as achain reaction. Proteins : Free radicals cause oxidation of sulfhydryl groups, and modification of certain aminoacids (e.g. methionine, cysteine, histidine, tryptophan, tyrosine). Lipids : Polyunsaturated fatty acids (PUFA) are highly susceptible to damage by free radicals. Carbohydrates : At physiological pH, oxidation of monosaccharides (e.g. glucose) can produce H2O2 and oxoaldehydes. Nucleic acids : Free radicals may cause DNA strand breaks, fragmentation of bases and deoxyribose. Such damages may be associated with cytotoxicity and mutations leading to carcinogenesis. ANTIOXIDANTS IN BIOLOGICAL SYSTEM: To mitigate the harmful/damaging effects of free radicals, the aerobic cells have developed antioxidant defense mechanisms. Antioxidants may be considered as the scavengers of free radicals. The production of free radicals and their neutralization by antioxidants is a normal bodily process. Various antioxidants in biological system:  Antioxidants that will block the initial production of free radicals e.g. catalase,glutathione peroxidase.  Antioxidants that inhibit lipid peroxidation e.g. superoxide dismutase, vitamin E, uric  acid.  Plasma antioxidants e.g. E-carotene, ascorbic acid, bilirubin, uric acid, ceruloplasmin,  transferrin.  Cell membrane antioxidants e.g. D-tocopherol.  Intracellular antioxidants e.g. superoxide dismutase, catalase, glutathione peroxidase. Oxidative stress (oxidative burst ) occurs when excess oxygen radicals are produced in cells that becomes beyond the normal antioxidant capacity of the cell.  Shock Inadequate Blood Circulation: - Will result fall in peripheral blood pressure leads to reflex sympathetic vaso-constriction, which in turn leads to deficient blood supply to brain. To conserve fluid ATP, Renin - Angiotensin Aldosterone system mechanism are stimulated but vaso- constriction causes renal ischemia and damage leading to finally death of the animal. Shock due to toxin of Bacterial or plant origin: - Are absorbed from the gut, come into the systemic circulation and result into the vasodilatation. These toxins cause trauma to tissues to liberate histamine like substance, dilatation of capillaries, and increased permeability of blood vessels leading to diapedesis and decreased blood volume. Hypovolumia: - Stimulates antidiuretic hormone vasopressin. There is contraction of spleen and venous blood vessels, which cause increased peripheral vascular resistance and increased heart rates to compensate and maintain cardiac output and blood perfusion through coronary and cerebral blood vessels. Water shifts: - During shock interstitial tissue fluid is depleted causing loss of elasticity of skin. Peripheral vasoconstriction leads to continued hypovolumia, falling of cardiac out put and blood pressure. In response to this there is opening of arterio venous shunt, decreased perfusion of tissues and organs having less oxygen content will produce tissue acidosis, hypoxia, anoxia, dysponea and peripheral vascular failure, unconsciousness some times comma and shock. Septic shock: - In normal healthy animals intestinal mucosa will act as a effective barrier to prevent absorption of endotoxins and if toxins are in small quantity will be detoxified by liver, do not reach systemic circulation and shock will be prevented. In early hyper dynamic stage of sepsis and endotoxaemia there is increased demand for oxygen leading to pulmonary hypertension. Increase transvascular fluid filtration in the lung, clinically manifested by pulmonary oedema, dysponea and shock. Due to trauma or entry of endotoxin into systemic circulation will produce. Acidosis in Shock. Most metabolic derangements that occur in shocked tissue can lead to blood acidosis all through the body. This results from poor delivery of oxygen to the tissues, which greatly diminishes oxidative metabolism of the foodstuffs. When this occurs, the cells obtain most of their energy by the anaerobic process of glycolysis, which leads to tremendous quantities of excess lactic acid in the blood. In addition, poor blood flow through tissues prevents normal removal of carbon dioxide. The carbon dioxide reacts locally in the cells with water to form high concentrations of intracellular carbonic acid; this, in turn, reacts with various tissue chemicals to form still other intracellular acidic substances. Thus, another deteriorative effect of shock is both generalized and local tissue acidosis, leading to further progression of the shock itself. Biotransformation of Xenobiotics Biotransformation is the process whereby a substance is changed from one chemical to another by a chemical reaction within the body. Biotransformation also serves as an important defense mechanism in that toxic xenobiotics and metabolites are converted into less harmful substances that can be excreted from the body. In general, biotransformation reactions generate more polar metabolites, that are readily excreted from the body. The liver plays the most important role in the biotransformation reactions. The biochemical processes whereby the noxious substances are rendered less harmful and more water soluble, are known as detoxification. Lipophilic toxicants are hard for the body to eliminate and can accumulate to hazardous levels. Xenobiotics are compounds which may be accidentally ingested or taken as drugs or compounds produced in the body by bacterial metabolism. (Greek, xenos = strange). Molecules to be eliminated/detoxified are called xenobiotics. Biotransformation is not exactly synonymous with detoxification, since in many cases, the metabolites are more toxic than the parent substance. This is known as bioactivation or toxication. The compounds that are detoxified include: a. Compounds accidentally ingested like preservatives, food additives and adulterants b. Drugs taken for therapeutic purposes. c. Compounds produced in the body which are to be eliminated, e.g. bilirubin and steroids. Bilirubin is toxic to the brain of newborns and may cause irreversible brain injury. d. Compounds produced by bacterial metabolism, e.g. amines produced by decarboxylation of amino acids:  Histidine → Histamine  Lysine → Cadaverine  Ornithine → Putrescine