HM B Lecture notes

Lecture 1: Diet-related problems in Australia Rosemary Stanton

Introduction • Integrated model (all are interrelated): o Health o What we eat o Social equity o Protection of land and water (climate change/sustainability)

Australians • Eat more: o Snack foods, fast foods, restaurants, soft drinks/energy drinks, instant noodles (fat/salt), cheese, wine Expense is an important factor • Eat less: o Fruit, vegetables, milk, breakfast cereal, bread (wholegrain) • Problems: o Overweight (kJ), underweight o Dental caries (refined carbs) o Coronary heart disease, high blood pressure, diabetes (saturated fat) o Cancers – bowel, breast, prostate (saturated fat) o Nutrient deficiencies – especially iodine and vitamin D o Gall stones (fat), constipation, osteoporosis

Dietary problems • Fat, especially saturated fats • Trans fats o Eg: oleic acid o Vegetable oils that have been partially hydrogenated for better shelf life o Increase LDL cholesterol and decrease HDL cholesterol • Poor quality carbohydrates • Salt • Alcohol

Deficiencies • Iodine – dairies have stopped using iodine for milk processing o Especially important in pregnant women o Bread, begun using iodised salt • Vitamin D – produced by sun exposure o Fat soluble, a hormone o People are scared of skin cancers, and thus avoid the sun completely Should come out before 11 and after 2-3pm o Deficiency can result in misshapen bones o Risk: elderly, cultural, religious • Omega 3 fats, calcium (especially teenage girls) • Dietary fibre – from fruit and veges o Results in constipation • Potential problems o Vegans: Vitamin B12: only found in animal products, need supplements or fortified soy Iron, calcium

Obesity • Reasons for: o Poor choices of fats, carbohydrates, and drinks • All ages and both genders are increasingly becoming fatter o Increase is rapid and continuing o Partially due to SES • People take no responsibility and are waiting for a miracle cure • Epidemiology: o Ages 2-18, 25-37% are overweight o Adults: male – 67%, female – 52% o Women worry, men do not (men: “it’s all muscle”)

Measurement • BMI – doesn’t account for different body shapes • Waist – a better measure for visceral (surface) fat that better correlates with disease risk • Risk of weight problems: o Skinny – osteoporosis o Fat – heart disease, diabetes, increased blood pressure, cancers, gall stones

Weight loss • Greatest problem is impatience – people have too high expectations o To lose 1kg, need a deficit of 32 000 kJ • Diets cause weight loss but not fat loss o Glycogen from muscles is lost taking water and the breakdown of proteins causes a diuresis and loss of water weight o No diet has been show to work in the long term Diets set off long term changes that are important in keeping the weight off o “quick fixes” that do not work • Advice: o Change habits (exercise) o Eat less, eat breakfast, don’t snack o Reduce sugar, alcohol o Avoid diets

Prevention and risk of obesity • Breast feeding vs • High fibre foods • TV, sedentary • Physical activity • Energy dense food, fast food • Soft drinks, cordials, juice (liquid kJs)

Strategies • Goals – to improve health rather than weight loss • Empowerment – look after own health/weight and understand it and how it happened • Hungry vs not hungry eating

Physical activity • Controls appetite • Advantages o Cardiovascular, decreased cancer (colon, breast), osteoporosis • Doesn’t have to be obvious exercise, general everyday activity counts o A pedometer is a good tool

Fats • Saturated fats – fatty meats and full fat dairy o Vegetable oils are hydrogenated for us in snacks, biscuits, spreads, etc Allows for better storage and reheating • Trans fats – not found in nature o Partially hydrogenated • Should decrease saturated fats, and eliminate trans fats • Good fats: o Fats in fish, omega 3 o Monounsaturated – olive oil, nuts, avocado o Polyunsaturated – soy bean, sunflower, walnut oil + margarines from these Omega 6, ok in small quantities, decreases LDL cholesterol

Carbohydrates • Glycaemic Index (GI) gives a measure of how fast carbohydrates are converted to blood glucose vs glucose (100) o Lower GI is better because energy is released slower over many hours This is especially important in DMII • Problems: o Low GI can be confused with low carbohydrate, ie chocolate has lower GI than carrots These are often fatty, sugary, low GI but not healthy o Doesn’t help with increased weight • Want: o Low GI, high nutrient food – fruit, wholegrains, peas, corn, sweet potato, legumes, milk and yogurt Wholegrain bread – slow fermentation, sour dough bread • Some carbs have useful nutrients and for this reason are important although they have high GI o Others do not like: soft drinks, sugar, confectionary

Drinks • Stick to: Tap water, tea (4-6 cups), coffee (2) o Avoid: juice (kJ, acidity – eats enamel), soft drinks (kJ, dental), alcohol (max 1-2/day)

Salt • >80% of salt is already in food – especially snack food and eg. soy sauce o Solutions: Eat more fresh foods, put less salt in cooking, buy no salt foods Use other things for flavour (eg. spices, pepper, garlic, herbs)

Deficiencies and solutions • Fruit/vegetables – eat them • Fibre – fruit and vegetables, good carbohydrates • Calcium – dairy, soy (fat reduced) • Iodine – fish, seafood, dairy, iodised salt • Omega 3 – fish (2x/week) • Vitamin D – sun/supplement

Social equity • Low income correlates with a bad diet and health o Dependent on: access to food, knowledge skills o Solutions: subsidies for healthy products, tax on junk food, lobbying

Sustainability issues • Agriculture produces a lot of greenhouse gases • Water • Soil fertility/fertiliser – superphosphate is running out • Processing, packaging, storage • Food crops for ethanol – food for cars or people? • Industrialised foods – wastage and overconsumption, preservatives, additives, lose origins of food Lecture 2: Design of metabolism Mike Edwards

Metabolism • Metabolism – all chemical reactions that take place in a cell/organism o Catabolism – metabolic pathways that release chemical energy breaking down complex molecules into simple ones Release energy (ADP ATP) o Anabolism – metabolic pathways for the synthesis of complex molecules from simple ones Use energy for biosynthesis (ATP ADP) o Metabolism in cells and organisms have ordered sequences of events metabolic pathways • Energy is stored within cells as a molecule: ATP ATP o ATP ADP + Pi ADP + Pi • Energy in and out o If energy in from food catabolism is greater than energy expenditure, excess is stored and mass increases o If energy out from exercise and cellular processes is greater than energy taken in, stores are used and mass decreases

• Respiration and fermentation o Amino acids and fats are broken down by respiration (O2-dependent) and are converted to CO 2 and H 2O o Carbohydrates have 2 pathways for breakdown Respiration – conversion to CO 2 and H 2O Fermentation – conversion of glucose to lactate (lactic acid, not ethanol!) • Only in skeletal muscle (intense exercise) and RBCs o Cell/tissues die in with a lack of oxygen Exceptions: RBCs, skeletal muscle Without O 2, cells can’t breakdown macronutrients to make ATP, thus they can’t function and die • Compartmentation o 2 compartments in the cell Cytosol (the factory) • Anabolism (synthesis) • ATP utilisation Mitochondria (the powerhouse) • Catabolism (breakdown) • ATP production

• Stages of catabolism o Stage 1 (digestion in GIT) – macromolecules are broken down into smaller molecules No ATP made o Stage 2 – fatty acids, glucose, amino acids are processed into Acetyl Coenzyme A via common pathways, Small amount of ATP made o Stage 3 – Acetyl CoA via the citric acid cycle (TCA) and redox reactions, 8e -s are produced Small amount of ATP made o Stage 3 – electrons activate the respiratory chain and by oxidative phosphorylation ATP produce ATP Large amount of ATP made ATP Inside the cell

Tissue utilisation • All cells/tissues use carbohydrates o Brain and related tissues (eg. retina) and RBCs can’t use fat or amino acids for energy Glucose dependent o All other tissues can break down fats or carbohydrates for energy (eg. liver, kidneys, skin, heart, lungs) Skeletal muscle generates ATP faster when breaking down carbohydrates vs fats of amino acids • Allows a higher intensity of exercise via carbohydrate catabolism • Muscle – heart and skeletal o Variable rates of catabolism to allow rapid and variable rates of ATP utilisation In skeletal muscle, there can be a change of 20x in rest vs exercise o Catabolism is aerobic in cardiac muscle Skeletal muscle is functionally anaerobic during intense, short duration exercise – producing lactate o Use glucose, fatty acids and ketones for fuel Cardiac muscle can also use lactate produced by skeletal muscle o Glucose can be stored by skeletal muscle as glycogen (the liver can also + some cardiac muscle) • Liver o High rates of catabolism with a rapid and constant rate of ATP utilisation o Supports other tissues by releasing metabolic fuels (glucose and ketone bodies) Released between meals into the blood stream o Uses glucose, fatty acids and lactate as metabolic fuels, but not ketone bodies o Stores glucose as glycogen and converts excess glucose and amino acids into fatty acids for storage • Brain o High rates of catabolism with a rapid and constant rate of ATP utilisation o Use glucose and ketone bodies (in prolonged starvation/fasting), not fatty acids • Adipose cells o Low rates of catabolism o Use glucose fatty acids and ketone bodies o Main site of triacylglycerol storage, release fatty acids into the blood during fasting and exercise • Red blood cells o Lack mitochondria o Low rates of catabolism o Can only use glucose – anaerobic break down (to lactate)

Dietary intake • NHMRC dietary recommendations for adults (in percentage of total energy, calorie intake): o ~15% protein, >55% carbohydrates, <30% fat o Most people have more fat than this • Process of macronutrients utilisation o Digestion (polymers monomers) [stage 1 of catabolism] o Absorption: uptake by intestinal epithelial cells, export from intestinal epithelial cells o Transport around the body via blood and lymph o Uptake by cells of different tissues/organs o Catabolism and/or storage inside cells [stages 2 and 3 of catabolism] ATP produced

Carbohydrates • Starch from plants and glycogen from animals o Broken down into a disaccharide (maltose, isomaltose, maltotriose) by α-amylase in the mouth and SI o Broken down into glucose by “maltase” in the small intestine “maltase” represents 4 enzyme complexes, because no single enzyme breaks down the disaccharides, combined activity is required • Other disaccharides: o Sucrose (cane sugar) “sucrase” glucose + fructose o Lactose (milk sugar) “lactase” glucose + galactose “sucrase” and “lactase” represent similar enzyme complexes as “maltase” These 4 complexes are responsible for breakdown of most dietary disaccharides and small oligosaccharides • Example: the sucrase-isomaltase complex o A glycoprotein attached the surface of epithelial cells Has 2 subunits with different specificities o The most active of the 4 enzyme complexes Makes up 100% of “sucrase” activity and ~80% of “maltase” activity

• Lactose intolerance o Also known as late-onset lactase deficiency Adults with lactase deficiency cannot digest dietary lactose • Symptoms: nausea, cramps, diarrhoea, bloating (bacteria breakdown lactose and produce gas) o Normally lactase activity is maximum at birth and declines after 1 month At the age of 6-7, adults levels are reached, 10% of infant activity Same pattern in all mammals In lactase deficiency, the level of activity continues to decline past the age of 6-7 resulting in intolerance o Not a genetic disease, lactose tolerance is the genetic defect (evolution theory) o Damage to absorptive cells of intestinal villi can also result in lactase deficiency

Transport of monosaccharides • Into the cell from GIT: 2 types of transporters o Na + glucose symport – uses sodium gradient to transport glucose and galactose into cell o Fructose and glucose facilitated transporters – use concentration gradient of monosaccharide • Into cells from the blood (and small intestine): o Facilitated uniports – GLUT family (1-5) GLUT 3 – glucose uptake into neurons, placenta and testes GLUT 4 – glucose uptake into adipose tissue, heart and skeletal muscle (insulin dependent) GLUT 5 – fructose uptake

Lecture 3: Abdominal wall and topography of the abdomen Dzung Vu

See prac notes and hand out from class *add in pictures from lecture slide + check info** Muscles of the abdomen • Posterior abdominal wall o Psoas – deeper, more medial o Quadratus lumborum – superficial, lateral • Anterolateral group o External oblique, internal oblique, transversus abdominis o External oblique aponeurosis: superficial inguinal ring, inguinal ligament o Conjoint tendon o Formation of rectus sheath, arcuate line, linea alba • Anterior group o Rectus abdominis • Actions o Compress the abdominal viscera to compress the diaphragm and aid in air expiration o Flex vertebral column o Laterally flex trunk o Rotate trunk (facilitated by muscles running in different directions) External oblique (coat pocket), internal oblique (perpendicular), transversus (transverse)

Topography of the abdomen • Most posterior structure of the abdomen is the kidneys, lie on the sides of the vertebral column o The right kidney is lower due to the liver, L: LV1, R: LV3 • Passing through the diaphragm: o Aorta: TV12, IVC: TV8, oesophagus: TV10 • Structures posterior to anterior: o Spine aorta/ kidneys IVC duodenum/pancreas spleen colon stomach o Pancreas is infront of the vertebral column and thus prone to injury

Peritoneum • Definitions o Peritoneum – membrane-lined cavity o Reflection – where the peritoneum leaves the organ o Meso – from abdominal wall to viscera (eg. mesentery, mesocolon) o Ligament – connects viscera to viscera (eg. lienorenal ligament) Or sometimes wall to viscera (eg. falciform ligament) o Omentum – comes from the curvatures of the stomach (greater and lesser) • Omental/epiploic are interchangeable o However, some conventions: omental bursa, epiploic foramen • Greater sac is the peritoneal cavity o Lesser sac lies behind the stomach/lesser omentum • Organs: free or retroperitoneal o Free in abdomen: Stomach, ileum, jejunum Transverse colon, sigmoid colon o Retroperitoneal Duodenum Ascending/descending colon

Spread of infection • Governed by: o Gravity o Position of body o Intraperitoneal pressure gradients o Mesenteric partitions and peritoneal recesses Lecture 4: Statistical tests and significance Rachel Thompson

See lectures 16 and 24 of HMA

Lecture 5: Utilisation of macronutrients 1 Mike Edwards

Glucose catabolism • Summary: o Digestion (from previous lecture) glycolysis (glucose to pyruvate) pyruvate utilisation (glycogen storage) o No cell has all of these pathways o Glycolysis is important in the catabolism of all dietary mono, di and polysaccharides

• Glucose catabolism – a universal metabolic pathway (in plants, animals, archaea, bacteria) o Glycolysis 1 – activation of glucose Hexokinase – normal enzyme • Kinases typically add phosphates from ATP • Relatively non-specific • Low Km (100uM) – thus works best at low concentrations of substrate Liver: Hexokinase has low activity instead has: glucokinase (an isozyme) • Specific for glucose • High Km (10mM) – allows the liver to take up glucose faster from the blood in high concentrations of glucose (ie. after eating) o Normal glucose concentration in the blood is 5mM Both enzymes need ATP to work • Process is making ATP, but need to invest ATP to make ATP: “need money to make money” Notice: phosphofructokinase – most important control point in glycolysis

This step causes formation of phosphate esters from the original sugars • 2 functions: o Traps sugars inside cells o Activates sugars for breakdown o Glycolysis 2 – splitting of 5 carbon fragments into 2x 3 carbon units 2 pathways: • Conversion into dihydroxyacetone phosphate (DHAP) • Conversion into Glyceraldehyde 3-phosphate (GAP) • DHAP and GAP can be interconverted by triose phosphate isomerase enzyme o GAP is used up, thus DHAP is converted to GAP Thus, overall reaction is F-1,6bisP 2 GAP • From here, everything occurs in 2x o Glycolysis 3 Redox reaction with a large standard free energy thus creating a high free energy bond: • Phosphate anhydride bond in 1,3-bisphosphoglycerate (has high free energy of hydrolysis) • This bond is broken to make ATP Pyruvate kinase converts phosphoenolpyruvate (PEP) into pyruvate and creates ATP • PEP is a phosphate ester with a very high free energy of hydrolysis • The irreversible direction results in ATP synthesis • This kinase is unique in that is the only kinase that catalyses an irreversible ATP synthesis reaction

o Pyruvate kinase explanations: 0 Overall: PEP pyruvate kinase pyruvate ΔG ’ = -62 000kj/mol Enzyme catalyses change of PEP into enolpyruvate with ΔG 0’ = -16000kj/mol • This undergoes non-enzyme spontaneous rearrangement to form pyruvate o ΔG 0’ = -46000kj/mol • Together, total of -62000kj/mol, combined energy is enough to synthesise ATP

Enzyme catalysed Glycolysis in red blood cells • Have no mitochondria and thus purely undergo anaerobic glycolysis o Also have extra pathways At the conversion of 1,3-bisPG into 3-PG, the Mutase enzyme has high activity and Phosphatase low activity • Thus there is the formation of 2,3-bisphosphglycerate (2,3-bisPG) same as 2,3 DPG o Decreases O 2 affinity of haemoglobin o Living at altitude increases 2,3-bisPG o Loss of 2,3-bisPG is a problem in stored blood

• Defect or malfunction of glycolysis in RBCs results in a problem in 2,3-bisPG and O 2 transport

Stoichiometry • Losses Gains 4- 3- + 2- 3- 4- + o Glucose + 2ATP + 4ADP + 2NAD + 2Pi 2pyr- + 2ADP + 4ATP + 2NADH + 2H + 2H 2O o Simplify: 3- + 2- 4- + Glucose + 2ADP + 2NAD + 2Pi 2pyr- + 2ATP + 2NADH + 2H + 2H 2O o Overall gain of 2ATP Achieved by redox reactions of NAD + + Pi NADH + H + • Forms new high energy phosphate anhydride bonds for ATP synthesis ATPs invested at start are recovered by pyruvate kinase via PEP

Glycogen • Glycogen metabolism o Glycogen is formed / broken down by addition and removal of glucose to glycogen chains Addition glycogen synthase, removal glycogen phosphorylase o Liver glycogen is broken down during fasting to increase blood glucose for other tissues o Skeletal muscle breaks down glycogen stores during exercise for itself Uses this preferentially to blood glucose • Glycogen is only important in the liver (1/3) and skeletal muscle (2/3) – make up 95% of glycogen metabolism

Utilisation of glycolysis products • ATP (-->ADP) is used as energy for cellular processes o Eg: muscle contraction, active transport, biosynthesis • Pyruvate and NADH reoxidation o Pyruvate has several pathways: Precursor for synthetic pathways (provides amino acids such as alanine) Aerobic metabolism (+O 2) forms acetyl-CoA [major pathway]

• Acetyl-CoA is converted to CO 2 and electrons to fuel the respiratory chain via the TCA cycle o Respiratory chain involves using oxygen to convert NADH back into NAD + Anaerobic metabolism (-O2) via reduction gives a waste product (eg. lactate, ethanol in yeast) • Uses NADH in reduction and returns to NAD + • Occurs in RBCs, exercising muscle o More detail on anaerobic disposal In yeast, conversion of acetaldehyde to ethanol has a reduction using NADH/NAD + In exercising muscle/RBCs, conversion of pyruvate to lactate by lactate dehydrogenase uses NADH/NAD +

Glycolysis control • 3 reactions in glycolysis are irreversible: phosphofructokinase, hexokinase and pyruvate kinase o These are important in controlling the pathway o Phosphofructokinase (PFK) is the rate-limiting step in glycolysis, thus is the major control point • Control of glycolysis o Inhibition when [ATP] is high o Activation when [ATP] is low o Signallers involved: ATP, ADP, AMP • PFK enzyme o Allosteric properties 2 different binding sites for ATP: substrate binding site and allosteric site AMP binding causes activation, ATP binding causes inhibition o Also many other factors controlling glycolysis and PFK, some are tissue dependent

Lecture 6: Utilisation of macronutrients 2 Mike Edwards

Fructose and galactose catabolism • Fructose and galactose and other ‘sugars’ don’t have their own catabolic pathways but are converted into intermediates along the glycolysis pathway o The energy yield (ATP) from glucose, galactose, fructose are identical • Fructose o Catabolism is different depending on the tissue Adipose tissue/ skeletal muscle: Hexokinase Liver: Fructokinase – major pathway • Forms DHAP and GAP o Defects: Essential fructosuria • Lack of fructokinase • Relatively benign Hereditary fructose intolerance • Lack of fructose-1-P adolase that splits fructose-1-P • Problem is in accumulation rather than lack o F-1-P interrupts metabolism, osmolarity and traps phosphates • Galactose o Catabolism involves formation of glucose-1-P and addition into glycolysis pathway as glucose-6-P o Overall reaction: Galactose + ATP glucose-1-P + ADP o Defects: Galactosemia • Lack of transferase resulting in accumulation of galactose-1-P • This leads to an alternative pathway forming galactitol which causes osmotic imbalances o In particular, accumulation in the lens of the eye can result in cataracts

Fatty acid catabolism • Fatty acids are activated to form acyl-CoA in the cytoplasm o This traps them in the cell o Then transported into the mitochondria where β-oxidation occurs (attached to mitochondrial matrix) This forms acetyl-CoA units that feed into the citric acid TCA cycle common with glucose - • A typical fatty acid is Palmitate (palmitic acid, C16): CH 3(CH 2)14 COO o Through catabolism, it is broken down into 8 acetyl-CoA units

Carnitine transport system • The carnitine system involves two pools of transferase enzymes that convert Acyl-CoA to CoA or CoA to Acyl-CoA o This changes carnitine into acyl-carnitine and visa versa Acyl-carnitine acts as a transport enzyme to bring the Acyl group into the mitochondria • The net effect is the transfer of Acyl-CoA from the cytoplasm into the mitochondria • Carnitine is widely found in meat o Often advertised as a dietary supplement for body builders and athletes and separately for weight loss No convincing evidence that it has any effect

β-oxidation • The process whereby a 2C unit is repetitively split from a long chain fatty acetyl-CoA o Occurs in the mitochondrial matrix • Process: o Oxidation with a cofactor to create a double bond o Addition of a water molecule o Oxidation with a cofactor to create a carbonyl group o Addition of a CoA group splitting the molecule into the an acetyl-CoA and the remaining fatty acyl-CoA • Eg. Palmitoyl-CoA (C16) undergoes 7 cycles of this oxidation resulting in 8x acetyl-CoA • ATP yield of palmitate (β-oxidation) o Activation to palmitoyl-CoA -2ATP 2 high energy bonds o Carnitine transport system 0 o β-oxidation 8 acetyl-CoA 7FADH 2 (=1.5xATP) +10.5ATP 7NADH (=2.5ATP) + 17.5ATP • Used by respiratory chain, reoxidation produces ATP o Further breakdown of 8 acetyl-CoA in TCA cycle +8ATP 24NADH +60ATP 8FADH 2 +12ATP o Total +106 ATP Vs. 2ATP from glucose and 30ATP aerobically later via carbohydrates Fatty acids are a good fuel, very energy rich (good for storage) • However, they are broken down slower than carbs • Rates of β-oxidation controlled by: o Cellular requirements for ATP Requires re-oxidation of NADH and FADH 2 which are reoxidised by ATP synthesis in the respiratory chain • Lack of ADP, this won’t happen o Fatty acid availability Release from adipose tissue is under hormonal control o Inhibition of carnitine transport by malonyl-CoA (a fatty acid synthesis precursor) Thus, in the liver as it synthesises fatty acids, malonyl-CoA prevents fatty acid breakdown

Glycerol catabolism • Triglycerides are made up of 3 fatty acids and a glycerol backbone • Glycerol is converted into dihydroxyacetone phosphate (DHAP) and thus can be further broken down as part of glycolysis o 2 step process involving addition of a phosphate to trap and activate the glycerol and then oxidation by NAD + or FAD o 2 possible enzymes – example of convergent evolution Different tissues have one more or less important • Eg: cytoplasmic – NAD +, mitochondrial: FAD

Triacylglycerol storage • Glucose/fats are stored by synthesis of triacylglycerol by the liver and adipose tissue o Glucose/glycerol is converted into glycerol 3-P o This has additions by fattyacyl-CoA units to form triacylglycerol Along the pathway, phosphatidic acid is formed which is important for forming phospholipids for cell membranes In the formation of triglycerides, phosphatidic acid loses a phosphate group and the final fattyacyl- CoA is added • Adipose tissue cells lack glycerol kinase which converts glycerol to glycerol 3-P o Thus, glycerol 3-P comes solely from the glucose DHAP glycerol 3-P pathway • Diets abuse the idea that some glycolysis is required for effective fat storage to promote the idea that if you eat carbs and fats separately, you can’t make fat o UNTRUE: Excess dietary carbs can still be converted into fatty acids Blood glucose concentration is tightly regulated and glucose is always available for adipose cells

Lecture 7: Physiology of diet Steve Boutcher

Obesity • Australian epidemiology o 20-25% males are borderline obese, 20-30% females o 56% adults overweight Defined as males: >25% of body weight is fat, females: >30% o In children, obesity has recently doubled • Measuring obesity o Body mass index (BMI) Underweight : <18.5, normal: 18.5-24.9, overweight: 25-29.9 Asians: <23 normal o Waist circumference (WC) High risk when BMI>25 and men >102cm, women >88cm o Waist-hip ratio (WHR) Ideally women: 0.80 or less, men: 0.95 or less • Children o Becoming more and more overweight and obese In the 80s, correlates with the introduction of computers and decreased PE in schools Current levels: >30%, top 3 in the world • 85% will be obese as parents • Obesity affects fertility, causes orthopaedic issues etc • Location of fat is important o Females hips, back of arms o Males visceral (worse for you) Increased blood flow to this adipose, metabolically active and metabolically dangerous Releases chemical messengers that induce insulin resistance o Subcutaneous fat (females) is not as dadngerous

Diets • If we eat less, the body stores energy as fat • Weight is an energy balance between: o Kcal – protein, carbohydrates, fats o Exercise – resting metabolic rate (RMR), thermic effect of meal (TEM), thermic effect of activity (TEA) • Amount of energy used depends on RMR, TEM and TEA o Proportions: TEA: 15-30%, TEM: 10%, RMR: 75% o Dieting causes RMR to decrease, body goes into starvation, storing energy mode • Common diets o Egs: Low fat diets Low carb diets – atkins, zone, protein power GI diets – south beach Meal replacement – slim-fast (shakes, meal bars) Meal providers – jenny craig, nutrisystem Group approaches – weight watchers Mediterranean eating plan o All work in the short term Use the principal of decreasing kJ and thus losing weight, want to lose fat weight, not water/muscle In long term, diets are unsustainable • Crash dieting, process: o Initially, glycogen stores are used up, also lose water Then starts to use amino acids – muscle mass Fat is stored o If diet fails and person returns to original weight, fat is put on, not muscle (unless exercising) Weight cycling (yoyo dieting)

Effects of starvation diets • Physical effects o Decreased RMR (brain conserves energy) o Drop in sex hormones o Bone loss o Hypothermia o Loss of lean body mass • Health effects o Loss of lean tissue (heart, liver, kidneys, muscle) o Bone loss o Loss of hair • Cognitive/emotional effects o Decreased concentration o Poor judgement o Apathy, depression and anxiety o Food cravings o Psychotic changes, social withdrawal o Sleep disturbance

Fat loss – the ideal diet • Fat loss is dependent on balance between fat intake and fat oxidation • Factors that impeded fat loss: • Optimal eating plan: o 55% carbohydrate (complex) – plants and fruits o 30% fat (unsaturated, medium chain free fatty acids) – olive oil, fish oils o 15% protein (plant proteins) o ~26Kcal (107kJ) / kg Eg: 60kg x 26 = 1560 kcal = 6040kJ + more depending on activity, most people ~7000kJ/day

Keeping the weight off • Diet and exercise • Eat breakfast (cereal, fruit) • Fast food no more than 1/week • Reduce energy intake (low calorie, low fat diet) • Consistent eating • Reduce TV watching

Nutrients • Fruits and plants are important for reducing heart disease risk • Soft drinks – high in fructose o Effects: Increases appetite Decreases leptin, decreasing satiety o Associated with: Greater energy intake and body weight Lower milk, calcium and other nutrients Increased risk of medical problems like diabetes and hypertension • Proteins from meat o Meat is treated with steroid hormones o Cooked in bad oil Acryl amide – carcinogen o Correlation between meat eating and rates of breast cancer, intestinal cancers, male lung cancer, lymphoma, ovarian cancer, prostate cancer

Nutrients continued • Saturated fat o Fats used to make fried foods etc o With higher levels of fat, skeletal muscle has lower blood flow • Good fats o Increased oxidation (breakdown) of fats o Bad fats – decreased oxidation of fats

Dietary guidelines and eating well • USDA guidelines o Eat a variety foods not sure whether it is better to eat good things or not eat bad things Shown that omega 3 reduces triglycerides o Balance food with physical exercise o Eat grains, vegetables and fruit o Eat low saturated fat, cholesterol diet o Eat moderate sugars (avoid fructose) o Eat moderate salt and sodium o Drink alcohol in moderation • Seven steps: to weight control o Set reasonable goals o Have realistic expectations o Balanced, low fat diets o Reshape bad eating habits o Regular exercise o Track progress – hard by yourself, may need someone to work with o Celebrate success

Example of high caloric food • McDonalds meal: o Burger: 710Kcal, fries: 540Kcal, drink: 310Kcal Total: 1560Kcal (6427kJ) o Ie. most of a day’s energy requirements in one meal!

Good and bad foods • Good o Spicy food (increased fat oxidation) - capsaicin o Alcohol in moderation (decreased atherosclerosis) o Exercise o Vegetables, fresh food o Dark chocolate o Caffeine, green tea – burn fat • Bad (6 worst) o Doughnuts – fried, sugar, trans fats (carcinogenic in rats/mice), few nutrients o Soft drinks – fructose, caffeine, food colouring, suphites, aspartame o French fries – high GI, trans fats, acrylamide o Chips (trans fats, high salt) o Non-fish seafood, fried – trans fats, acrylamide, mercury, parasites o Vegetables oils – oxidised, rancidity: fresh oil is important • Amount of fat can be easily reduced by choosing how to cook food and different similar food options o Eg: take the skin off chicken, boil vs fry egg, crumb, grill, batter fish

Summary • Adults should eat a wide range of healthy, unprocessed foods o Need a diet of low unhealthy saturated and unsaturated fat – healthy fats instead: olive, coconut and avocado oils o Small amount of sugar (low GI foods) o Low salt foods o Plenty of water, and physical activity Lecture 8: Utilisation of macronutrients 3 Noel Whitaker *** read text books*** Proteins • Broken down into amino acids o Amino acids then have their amino groups removed which enter the urea cycle o Remaining backbone is processed through the TCA cycle

• Digestion: o Polymer oligomer monomer o Protein oligopeptide amino acid o Nucleic acid oligonucleotide nucleotides nucleosides bases

Nitrogen • Synthesis: o Fixation of nitrogen bonds takes a lot of energy (16ATP) o Only occurs in eubacteria and cyanobacteria • We get nitrogen from the plants/animal cells that we eat o Thus, we need to salvage it, rather than make it • Balance: o For the non-growing adult, we need to maintain the level of nitrogen in our body Dietary nitrogen needs to be equal/day to the amount of nitrogen excreted (~5g N/day) Thus, the nitrogen balance/equilibrium is maintained o Nitrogen is important for maintaining the amino acid pool Exists in the lysosome and proteosome within the cell Used to make proteins for cellular function

Amino acids • There are essential (can’t make ourselves) and non-essential amino acids (can make ourselves) o Essential: Isoleucine, leucine, valine Methionine Lysine, threonine Phenylalanine, tryptophan Arginine, histidine (can be made slowly, essential in juveniles) o Non-essential: Alanine, asparagine, aspartate, cysteine Glutamate, glutamine, glycine Proline, serine Tyrosine (made from an essential amino acid) • Synthesis: o Formed from various precursors such as pyruvate and α-ketogluterate by transamination This explains why these are necessary o Example: synthesis of serine Transamination allows transfer of an amine group to the carbon backbone creating serine o Example: formation of alanine (transamination ) Pyruvate + glutamate alanine aminotransferase alanine + α-ketogluterate • Transamination o Creates amino acids o Transports NH 3 (ammonia groups) to the liver to form urea Amino acid metabolism • Proteins o Humans (and most animals) don’t store amino acids Thus, proteins (made up of AA) are continually degraded and synthesised • Nitrogen balance is important in the body’s ability to synthesise new protein • Essential and non-essential amino acids are also important in protein synthesis • Amino acids o Nitrogen is shuffled around by transamination to create amino acids o Amino acids are used to make proteins Excess amino acids results in deamination by transamination + • This leads to NH 4 biosynthesis in the liver feeding into the urea cycle and excretion • The remaining carbon backbone is used as metabolic fuel (α-keto acids) by feeding into the TCA cycle • Other nitrogenous molecules o Nucleic acids: pyrmidines, purines and uric acid o Creatine and creatinine Creatine is a high energy molecule that is phosphorylated in muscles for energy storage Creatinine is the breakdown product of creatine and regularly it is turned into a waste product • This enters the blood and is filtered by the kidneys • In dysfunction of the kidneys, can measure creatinine levels

Amino acids and energy • Excess amino acids are not stored or excreted but used as a metabolic fuel

o The amino group is removed (NH 4) and converted into urea (via the urea cycle) and excreted o Remaining carbon skeleton is used as fuel: CO 2 + H 2O glucose, acetyl-CoA, ketone bodies • Fuel storage o Triglycerides, muscle/liver glycogen are useful for storing energy o Total body protein can also be used as a fuel However, need to remove ammonia because otherwise, if the liver is overloaded, ammonia is toxic and can seep through the skin

Removal of nitrogen/ammonia groups • Aminotransferases o Catalyse transfer of an amino group from one amino acid to a ketoglutarate forming glutamate + o Glutamate is then oxidatively deaminated yielding NH 4 via glutamate dehydrogenase • Eg: Amino groups of amino acids are often transferred to α-ketoglutarate forming glutamate via transamination o Glutamate then is deaminated (by the liver’s glutamate dehydrogenase) reforming α-ketoglutarate and + giving up the NH 4 group to the urea cycle + • NH 4 is then converted to urea for excretion (in most terrestrial vertebrates)

Glutamine synthesis + • Acts as a storage/transfer for NH 4 allowing transfer to the liver + o Prevents build up of toxic NH 4 • Process: + o NH 4 + glutamate + ATP glutamine + ADP + Pi • At the liver, a glutaminase hydrolyses the glutamine removing the ammonia group for urea synthesis + o Glutamine + H 2O glutamate + NH 4

Glucose-alanine cycle • Glutamate in muscle is transaminated to alanine o Alanine is carried by the bloodstream to the liver where it is converted into pyruvate Pyruvate then feeds into the TCA cycle for gluconeogenesis

Urea cycle • Occurs in hepatocytes (cytoplasm and mitochondria) o Different products feed into the cycle at different points Alanine pyruvate aspartate Argininosuccinate is a short-lived intermediate Fumarate feeds into the TCA cycle Urea is formed and secreted as a waste product

Carbon skeletons • Carbon skeletons are left after amino acids undergo transamination/deamination o These skeletons often feed back into various points on the TCA cycle

Amino acid derivatives - uses

• Salvaged amino acids (and their NH 3) groups are used to make different bioactive products o Eg: Phosphocreatine – phosphorylated for energy storage in muscle Purine bases Pyrimidine bases

• Disease: Kwashiorkor o Due to a deficiency of protein in the diet while there is, however, adequate calories This results in decreased plasma proteins (especially albumin) • Osmotic effects result in increased interstitial fluid causing edema and a characteristic distended abdomen May also cause muscle wasting o Problems are furthered by decreased ability to produce digestive enzymes and new intestinal epithelial cells Thus can’t get protein from diet and problem gets worse o Deficiency in protein means amino acid groups and amino acids themselves are not present for: Providing groups (such as NH 3) important for activating molecules Production of new proteins

Liver functions • Heme formation • Carbohydrate formation • Pyrimidine and purine synthesis • Amino acid synthesis • Ketone body formation • Secretion of excess nitrogen

Nucleic acid metabolism • Often reused/salvaged o Most are used to form new molecules o Secreted as uric acid • Nucleic acids include DNA and RNA o DNA is quite stable, RNA is unstable with nucleases present targeting breakdown When we eat protein, we are essentially eating cells, we want RNA broken down to prevent translation of dietary RNA • Most nucleic acids are associated with protein o Dietary nucleoproteins are broken down by pancreatic enzyme and tissue nucleoproteins by lysosomal enzymes More specifically: nucleic acids are hydrolysed by: • Endonucleases (restriction enzymes) to form polynucleotides) • Exonucleases (cut ends of molecules) yielding mononucleotides • Overall, these are absorbed as nucleosides • Pyrimidines and purines (nucleotides) o These are not salvaged from tissue turnover, instead are catabolised and excreted (don’t yield much energy however) Pyrimidine – catabolised to beta-alanine (precursor of carnosine) Purines – little dietary purines are used, that which is absorbed is catabolised • Catabolised into uric acid (an antioxidant) • RNA and DNA can be reused o RNA to retranslate the same protein o DNA as part of the repair process

Purine bases to uric acid • Adenine and guanine nucleotides converge to form the intermediate xanthine o Adenine is oxidised to xanthine by xanthine oxidase (in liver, intestine) o Guanine is deaminated to xanthine (amino group released as ammonia) • Xanthine is oxidised again by xanthine oxidase to produce hydrogen peroxide and urate

o Urate is excreted and H 2O2 (a ROS) is degraded by catalase o Concentration of uric acid is higher in the cytosol than the serum

Uric acid • Product of nucleic acid metabolism (not protein) o Chief nitrogenous waste of insects, reptiles and birds • Produced in the peroxisomes o Size of lysosomes (0.5-1.5um) and resemble lysosomes: filled with enzymes, bound by a single membrane o Bud off the endoplasmic reticulum rather than the golgi body (lysosomes o Functions in the human liver: Breakdown excess fatty acids (oxidation) Breakdown H 2O2, a product of fatty acid oxidation via catalase Aids the synthesis of cholesterol via enzyme HMG-CoA reductase Aids bile acids synthesis Aids synthesis of lipids to make myeline Aids breakdown of excess purines (AMP, GMP) to uric acid • Slightly soluble in water, easily precipitates out to form needle-like crystals of sodium urate o Form kidney stones o Can also form in synovial fluid in joints – excruciating pain: gout • Kidneys reclaim uric acid filtered at the glomeruli and use it as an antioxidant to protect from ROS • Mammals have an enzyme: uricase (inactive in humans and apes) o Breaks down uric acid into the soluble allantoin

Pyrimidine catabolism

• Pyrimidines undergo ring cleavage, the usual end products being beta-amino acids + ammonia and CO 2 o Beta-amino isobutyrate from thymine or 5-methyl cytosine excreted o Beta-alanine from cytosine or uracil can be excreted or incorporated into brain and muscle dipeptides Ie: carnosine (his-beta-ala) or anserine (methyl his-beta-ala)

**look up pyrimidine and purine anabolism**

Lecture 9: Obesity and disease – a clinical perspective Nick Zwar

Definitions • Obesity – a chronic condition that requires long term management to correct an energy imbalance, manage disease and to prevent weight regain o Complex, its causes and consequences incompletely understood o Largely preventable • Risk of disease is a spectrum, based on fat mass o A small amount of fat still has risk

Measurement parameters • BMI o Problems: Doesn’t take into account muscle mass Varies based on race/genetics • Asians have a higher fat mass for a lower BMI, Tongans opposite o Good measure of total fat • Waist circumference o Less interpretation problems than BMI o Tricky to measure o A good measure of visceral fat (vs. subcutaneous fat) Visceral fat is a better indication of risk of disease o Male: >94cm risk, >102cm high risk, female: 80,88 • Waist height ratio o >0.5 increased CVD, T2DM risk Consistent for children and adults • Waist-hip ratio o >0.8 is indicates increased risk • Direct measures o Used more for research than for the clinic setting Costly, unnecessary exposure to radiation Can be used to validate other measures o CT scans, total body water (gold standard for body fat measurement), MRI, DEXA • Children o Still growing, need different ranges and definitions BMI decreases to the age of 4-5 before increasing again into adulthood o Clinically, for children use BMI for age charts Allows calculation of percentiles and tracking of changes

Epidemiology • Obesity is increasingly becoming a problem worldwide – not just in developing countries • Australia o Peak in the 45-64 age group (people at the peak of their careers, in their most productive) Obesity is also increasing in the whole population over time o More men than women: 60% vs 45% overweight, 17.4% vs 16% obese o Obesity is negatively associate with SES Indigenous people living in urban environments are more likely to be obese than non-indigenous o Children Increasing in boys and girls (more in boys) Next survey to be conducted next year • Puppy fat myths: o BMI is increasing, waist circumference is increasing with puppy fat Very few people loss puppy fat entirely and become lean o Behaviours that allow puppy fat to develop can lead to obesity, weight problems in later life 80% of obese children are obese as adults o The longer children keep excess fat, the more likely they are to have co-morbidities

Co-morbidities • Types: o Respiratory – asthma, sleep apnoea o Gastrointestinal – fatty liver disease, gall stones, reflux (pressure on stomach) o Musculoskeletal – complications with feet, knees, hips, ankles like osteoarthritis o Cardiovascular – hypertension, dyslipidaemia (high lipids), fatty streaks, raised insulin, IMT (intima-media thickness) o Endocrine – insulin resistance, T2DM, early puberty, polycystic ovary (fertility problems, increased androgen production) o Psychosocial – Bullying depression, low self-esteem • These lead to increased relative risk of: o Cancers, heart disease, T2DM, hypertension, gall bladder disease o This is not an unsubstantial risk increase

Fat distributions • Types: o Abdominal/visceral fat More metabolically active, more deleterious Results in: increased BP, insulin resistance Measured well by waist circumference o Subcutaneous Fat on the thighs/hips o Retroperitoneal • Exercise can result in gain in muscle mass, and thus not loss in weight o Person may be discouraged, but they are metabolically better off and healthier

Metabolic syndrome • Clustering of risk factors o Abdominal obesity o Increased triglycerides, decreased HDL cholesterol o Hypertension o Insulin resistance o Proinflammatory state Excess fat cells produce inflammatory mediators • Thought to be related to coronary artery disease and arterial vessel diseases • 47 million adults in the US have metabolic syndrome

Psychosocial co-morbidities • Examples: o Low self-esteem o Bullying, social isolation o Depression o Decreased marriage rates o Poorly paid jobs o Bias and prejudice from society (including medical professionals) • Often parents do not think their children are obese and that they will eventually get leaner. Majority not true.

Cost • Australia, direct costs: $876 million, indirect (loss of income/productivity): $2891 million o A lot of money is also spent on weight loss programs, USA: $3.3 billion/year • Examples of cost: o Obese people may need special hospital equipment, eg. larger operating tables o Airline seats, should they be bigger? Discrimination?

Clinical assessment • Calculate BMI, measure waist circumference, • Assess and treat co-morbidities – eg. back pain, osteoarthritis, T2DM • Assess readiness to change – theoretical model of change: preconteplators contemplators ready (decision) process relapse • Assess why/how energy imbalance occurred o Dietary/physical activity Hx o Hx of weight, same or changed, pregnancy? • Set realistic goals • Referral to allied health professionals? Medications? o Eg. dieticians, exercise physiologists (medical available)

Fat metabolism • Dietary fat is broken down (digested by pancreatic lipase etc) absorbed chylomicrons muscles/liver/adipose liver packages and returns to blood stream for body cells to use • Fat cell is an endocrine gland (especially visceral fat): o Release: Angiotensinogen, leptin, IL-6, TNF-α, cortisol, adiponectin, resistin, stored triglyceride

Interventions • Need to primarily correct the energy imbalance o Some are predisposed have increased/decreased metabolic rates • Clinical management: o Pyramid: Bottom – population education (everyone) how to eat well Up a level – individual education/allied health referral (overweight, obese no risk factors) Level up – behaviour modification (bad eating habits) group/individual programs Top – bariatric surgery/medication (risk factors)

Intervention methods • Traditional methods: o Reduce energy intake Need long term changes – ie, a pattern of eating that is sustainable Diets can be successful in the short term o Increase energy expenditure Increased physical activity – can be ordinary tasks: walking vs car, stairs vs lift o Realistic goals with good management of relapse/regain of weight • Behavioural methods: o Self-monitoring – intake of food/calorie counting o Stimulus control emotional eating, comfort food o Problem solving – source of stimulus, self-image, depression o Contingency management – relapse management o Cognitive restructuring – link to problem solving o Social support – eg. weight watchers, group support • Pharmacological methods: - none are perfect o Anorexic agents Sibutramine (reductil) – 5-HT and NA reuptake inhibitor • Increases satiety • Shown to reduce weight over 6 months • Should be combined with a dietary plan o Inhibition of fat reabsorption Orlistat (xenical) • Reduces fat absorption by 30% • Shown to cause significant weight loss and improve risk factors o Rimonabant – cannabinoid receptor antagonist (used to prevent smoking/hunger cravings) • Bariatric surgery o Adjustable lap band – can control stomach size and thus lessen the ability to eat o Normally last resort – used for morbidly obese people with high risk factors Increases weight loss and decreases risk factors o High cost, most patients are in private hospitals

Health benefits of weight loss • Decreased BP (1% weight loss, 1% SBP decrease, 2% DBP decrease) • Decreased LDL cholesterol (1%/1kg) • 15-20% weight loss can reverse the morbidity of T2DM o Especially if weight is lost pre-onset • Decreased CVD risk o Especially if reductions are in visceral fat • Loss of 5-10% weight loss is sufficient to achieve clinically significant benefits

Lecture 10: Cellular respiration 1 Rebecca LeBard

Introduction • TCA (tricarboxylic acid) cycle is like a round about – many different entry and exit points o Also known as the citric acid cycle or the Kreb’s cycle • Revision of where TCA cycle fits in catabolism: o Catabolism – breakdown of complex molecules to simple ones releasing energy Stage 1 – digestion: large molecules into small molecules Stage 2 – small molecules broken down into acetyl CoA Stage 3 – TCA cycle and oxidative phosphorylation oxidising the acetyl group of acetyl CoA to produce ATP • Central pathway (roundabout) that facilitates recovery of energy from metabolic fuels (via electrons)

• Carbohydrate catabolism o Aerobic catabolism (respiration) yields more ATP than anaerobic catabolism (fermentation) o Glucose is processed by glycolysis to pyruvate Anaerobic: pyruvate is converted to lactate or ethanol Aerobic: pyruvate is transported from site of glycolysis in cytosol to mitochondria • Facilitated by a transport protein

Pyruvate dehydrogenase (PDH) • Links glycolysis and the TCA cycle o Found in the mitochondrial matrix o Catalyses the oxidative carboxylation of pyruvate to acetyl CoA + + Pyruvate + CoA + NAD Acetyl CoA + CO 2 + NADH + H

• Produces CO 2 and NADH captures energy in oxidation via reduction • Large enzyme complex – 4-10 million Daltons o From a family of homologous complexes including α-ketoglutarate dehydrogenase (TCA cycle) o Multi-enzyme complex advantages: Minimises the distance substrate has to diffuse between reactions Minimises side reactions Reactions are co-ordinately controlled Ie. like a manufacturing plant, instead of separate factories • Structure: o Made up of: 3 enzymes (E1, E2, E3) 5 cofactors (thiamine pyrophosphate: TPP, lipolic acid, FAD, CoA, NAD +)

• Mechanism of action o Pyruvate is decarboxylated forming hydroxyethyl-TPP o Lipoamide arm of E2 moves to active site of E1 o E1 catalyses transfer of 2 carbon group to lipoamide group forming acetyl-lipoamide complex o E2 catalyses transfer of acetyl moiety to CoA forming acetyl CoA, arm swings to E3 o Lipoamide oxidised by FAD o NADH produced as FADH 2 is reoxidised to FAD

TCA cycle • Overall reaction: + + o Acetyl CoA + 3NAD + FAD + GDP + Pi + 2H 2O 2CO 2 + 3NADH + GTP + 2H + CoA + CO 2 is breathed out, NAD is reduced to NADH

• Process: o Oxaloacetate and acetyl CoA condense to form citrate o Citrate is isomerised to isocitrate o Oxidative carboxylation of isocitrate to α-ketoglutarate Produces NADH + CO 2 o Oxidative carboxylation of α-ketoglutarate to succinyl CoA Produces NADH + CO 2 Reaction catalysed by α-ketoglutarate dehydrogenase (homologous to PDH) α-ketoglutarate also found as a by-product of protein catabolism o Thioester bond of succinyl CoA is cleaved allowing phosphorylation of GDP Forms succinate • Succinate is symmetric, thus can’t identify where carbons originated o Ie: CO 2 lost may or may not come from the 2 carbon acetyl CoA Reaction catalysed by Succinyl CoA synthetase (succinate thiokinase) o Oxaloacetate is regenerated Succinate oxidised to fumarate by succinate dehydrogenase Fumarate hydrated to form malate by fumarase Malate oxidised to oxaloacetate by malate dehydrogenase

Fate of electrons • TCA cycle: + o Electrons are transferred from acetyl CoA to NAD and FAD forming NADH and FADH 2 These act as electron carriers to the respiratory chain (electron transport chain) • Respiratory chain: + o NADH and FADH 2 are reoxidised to NAD and FAD which are then free to oxidise more reactions

Control of TCA cycle • Primarily governed by ATP and NADH concentrations o If we have lots of ATP, don’t need to use pyruvate to make more Acetyl CoA to make NADH to make ATP • First level of regulation: o E1 of PDH is inactivated by phosphorylation by a specific kinase Can be reactivated by a specific phosphatase Kinases and phosphatases generally turn enzymes on and off by (de)phosphorylation o Kinase and phosphatase in this case are controlled by ATP/ADP ratio, acetyl CoA and NADH levels • Second level of regulation o Isocitrate dehydrogenase is allosterically stimulated by ADP, inhibited by ATP, NADH • Third level of regulation o α-ketoglutarate dehyodrgenase is inhibited by products succinyl CoA and NADH, and ATP

Amount of products generated

• NADH and FADH 2 o 1 glucose molecule produces 10NADH and 2FADH 2 molecules Glycolysis 2NADH PDH 2x 1NADH TCA cycle 2x 3NADH, 2x 1FADH 2 • ATP o 1 glucose molecule produces 2ATP, 2GTP Glycolysis 2ATP TCA 2x 1GTP

Transport into the mitochondria • Outer membrane is quite permeable, inner membrane is impermeable to most molecules o Cytoplasmic NADH formed from glycolysis must be transported for use NADH is not itself transported, but electrons are transferred by the glycerol 3-phosphate shuttle • In the heart and liver, cytoplasmic NADH transfers electrons into the mitochondria via the malate-aspartate shuttle

Summary questions • Primary function of TCA cycle o Oxidation of metabolic fuels to produce NADH, FADH 2 These act as electron carriers to transfer electrons to O 2 driving ATP production • Metabolic fuels for TCA cycle o Fatty acids and glycerol, amino acids, CHO • TCA cycle needs aerobic conditions o Needs NAD +, FAD o In anaerobic conditions, NADH can’t pass on electrons to O 2 • Murder or mystery? o Darwin, Napoleon, Pharlap: arsenic found on them at death Arsenite binds to the lipoamide bond (arm) • Thus the enzyme can’t function and PDH enzyme is disabled – Acetyl CoA not produced o TCA cycle disabled Used to be used as a treatment for syphilis because it binds better to microorganisms than humans • Given in tonics as an antimicrobial – murder? Or poisoning?

• Cytoplasmic NADH yields less ATP than mitochondrial NADH because: o The glycerol 3-phosphate shuttle transfers the electrons as FADH 2 which has a lower yield of ATP (1.5) than NADH (2.5)

Lecture 11: Hepatobiliary anatomy Dzung Vu

The liver • Anterior border o Sharp o Fundus of gall bladder o Notch for ligamentum teres (that connects to the falciform ligament) • Porta hepatis – entrance to the liver o Portal triad: hepatic artery, bile duct, portal vein Bile duct to the right of hepatic artery Surrounded by the lesser omentum • Divisions o 4 lobes R, L, caudate and quadrate o 8 segments Divided functionally for surgery Minimises risk of biliary peritonitis and excessive blood loss • Caudate lobe (posterior) o Encloses IVC o Lies at the root of the epiploic foramen o Independent – not fused to other liver lobes • Liver suspended to diaphragm at the back, not from the top • Identify: o Caudate lobe o Quadrate lobe o Porta hepatis o Gall bladder o Ligamentum venosum (remnant of ducts venosus) o IVC • Peritoneum: o Falciform ligament o Coronary ligament o R and L triangular ligaments o Bare area of liver o Less omentum • Relations o Esophagus, stomach o IVC o Right kidney and suprarenal gland o Duodenum o Right colic flexure

• Blood supply o To the liver: Portal vein • Splenic vein + superior mesenteric vein join to form portal vein • Drains blood from intestine to liver • Branches after entering liver (L and R) Coeliac trunk common hepatic hepatic proper L and R hepatic ( R cystic artery) o From the liver: L, R and Middle hepatic veins ( IVC)

The biliary tree • R and L hepatic ducts join to form the common hepatic duct o Gall bladder drains via the cystic duct o Common hepatic and cystic ducts merge to form bile duct o Travels in front of the IVC, behind the pancreas and joins with the pancreatic duct entering the duodenum • Gall bladder o Related to the duodenum and colon o GB stones inflammation as stones try to push through the bile duct (can block, colic pain) Can enter the duodenum or colon and block the intestine • Identify: o R and L hepatic ducts o Common hepatic ducts o Gall bladder cystic duct o Common bile duct

The pancreas • Comes over the vertebral hump • Identify: o Head o Neck o Body o Tail o Uncinate process • Relations o Head duodenum, bile duct o Uncinate process has superior mesenteric artery and vein crossing it o Anterior surface transverse mesocolon o Splenic artery has a tortuous route along the superior border of the pancreas neck to tail o Splenic vein (posterior) • Pancreatic ducts o Major and accessory o Openings of the bile duct and pancreatic ducts at the major and minor duodenal papillae Guarded by the hepatopancreatic ampulla and sphincter • Blood supply o Arteries: Gastroduodenal (head of pancreas) Splenic Superior mesenteric • Porto-systemic anastomosis

Lecture 12: Pharmacology of obesity Margaret Morris

Obesity: facts/definitions • Obesity is the result of a chronic imbalance between energy uptake and expenditure o Most common disease of affluence Will soon overtake smoking as the most common cause of preventable death o Bodyweight is multifactorial: genetic, metabolic, behavioural, environmental, cultural, socioeconomic • BMI definition: o >30 obese, 25-29.9 overweight

Diseases linked to obesity • Sleep apnoea • Coronary heart disease • Pulmonary disease • Diabetes • Fatty liver disease • Hypertension • Gall bladder disease • Dysplipidemia • Osteoarthritis • Kidney failure • Gout • Cancer – breast, cervix, colon, endometrial, ovarian, • Stroke prostate

Causes of obesity • Interaction between nature and nurture o Largely due to genetics Twin studies: similarities between pairs, differences across pairs o Genetic causes – monogenic syndromes (rare) and susceptibility genes o Environmental causes – maternal obesity, stress, food availability, energy expenditure habits Environmental factors have changed in the last decade – explains obesity epidemic • Factors: o Parents – genetics, maternal/postnatal feeding (breast feeding is protective, leptin?) o Society – enriched food choice, advertising, social stress (emotional eating) o You – diet and exercise, drug therapy (iatrogenic)

Monogenic syndrome: leptin deficiency • Leptin is made in fat, circulates in the blood o Involved in satiety o 16 000MW Similar to a cytokine o Shows that fat is an endocrine organ • Mice: leptin deficient ob/ob mice homozygous ob deletion o Increased food intake and body weight o Low energy expenditure o Increased glucose asnd insulin o Reduced reproductive function Decreased Leptin thought to explain infertility in anorexia • Leptin deficiency, however, is rare and cannot explain the obesity epidemic

Energy balance equation • Intake vs Expenditure o Intake – hunger, satiety, nutrient absorption o Expenditure – metabolic rate, thermogenesis, activity

Control of eating • Multifactorial o Combination of: Cultural factors Hormones – leptin, insulin Neural input, gut peptides (detected by neural receptors) Fuel (glucose) Psychological factors • These multi-factors alter central control o Mediated by various NTs: Increase appetite • NPY, orexin (hypothalamus), cannabinoids Decrease appetite • MSH (melanocyte stimulating hormone) o Lack means obesity • CRF (corticotropin-releasing factor, same as CRH) o Linked to stress, causes us to not want to eat • Serotonin • Neural circulates of appetite regulation (pic 2) o Hypothalamus is central Ventral part of the brain where BBB is thinner so hypothalamus can sample blood • Detects leptin and insulin levels based on adiposity signals • Stimulus from gut and liver via sympathetics and vagus afferents Arcuate nucleus increases release eof neurotransmitters that signal other nuclei such as the paraventricular nucleus to release NTs that give feelings of satiety or hunger (POMC, NPY, CRF) o Redundancy Many different NTs do the same thing in a complex pathway web Circuit is complex and multiple such that if one doesn’t work, we can still function • Leptin and insulin (pic 1) o Released by adipose cells into the blood– amount of signalling is based on amount of fat cells Lets us known how fat we are o If fat cells increase in number, increased leptin in the blood stimulates the arcuate nucleus to decrease NPY hormone release decreasing appetite Humans are very good at not listening to their appetites, lack of homeostasis and obesity

Summary of peptides and hormones • Peptides that increase feeding (orexigenic) o Central – Neuropeptide Y, Melanin concentrating hormone, Agouti-related peptide, Orexin A and B, Endocannabinoids o Peripheral – Ghrelin (stomach) • Peptides that decrease feeding (anorexigenic) o Central – α-melanocyte stimulating hormone, CART, Urocortin, Corticotrophin releasing hormone, Neurotensin, Noradrenaline/serotonin o Peripheral – leptin, insulin, cholecystokinin (stim by bolus of food in duodenum), gastrin-releasing peptide Treatments • Basic principles: o Decrease energy intake (food and drink) and increase energy expenditure (basal metabolic rate, activity) o Exercise alone, however, is only moderately effective for weight loss • Pharmacological strategies: o Food intake Central – amines, NPY, PMC, AGRP, CB Peripheral – leptin, ghrelin, PYY o Fat Absorption – lipase inhibitors Metabolism o Thermogenesis Thyroid hormones (abused) β3 agonists (induces lipolysis) Uncoupling proteins • Current therapeutic options: o Very low calorie diet Mechanism: decreases energy intake Useful for committed individuals o Phentermine Mechanism: decreases NA reuptake o Sibutramine (Reductil) Mechanism: decreases NA, 5HT reuptake o Orlistat (Xenical) Decreases fat absorption o Rimonabant Antagonist at CB1 Not approved for use in Australia due to SE (suicidal thoughts, depression) o Need to be combined with lifestyle intervention (diet and exercise) Large problem is relapse/regain after weaning off drug

Phentermine • Derivative of amphetamine o Mechanism: reduces NA re-uptake in CNS Also causes a small increase in NA release o Acts as a sympathomimetic, lengthening the effect of NA Suppresses appetite • Uses o BMI>30, useful in the short term only (12 weeks max) • Adverse effects o Increased BP, HR, insomnia, nervousness, headache, constipation, etc o Can’t be combined with other weight loss drugs, antidepressants, pregnancy o Not recommended in the long term

Sibutramine • Mechanism: reduces NA and 5HT reuptake in CNS o Suppresses appetite and increases thermogenesis • Uses o BMI>30 or >27 with other risk (such as hyperlipidemia, high cholesterol) • Causes loss of ~5-9% of body weight at 12 months • AE o Increased BP, HR, headache, insomnia, nervousness o Avoid in hypertension, angina Monitor BP (average increase SBP 1.7mm, HR 4.5bpm) • Body weight often increases again on discontinuation

Orlistat (Xenical) • Mechanism: inhibits gastrointestinal lipase o Binds serine residue in active site of gastric and pancreatic lipase o Decreases dietary fat absorption by ~30% • Uses o BMI>30, combined with diet • AE o Bloating, explosive diarrhoea, faecal leakage, increase in BP, oily stools (steatorrhea) o Must combine with low fat diet Side effects may encourage dietary compliance, but discourage drug compliance o May need to take vitamins to prevent deficiency – fat soluble vitamin malabsorption

New therapeutic options • NPY antagonists o Mechanism: inhibit appetite – hypothalamic Shown to work in rats and mice o Trials disappointing (MERC lost billions)

• PYY 3-36 (peptide YY) o Hypothalamic, however, hard to get it into the brain o Phase 1 trial underway • Growth hormone fragment o Increases muscle deposition

Bariatric surgery • Most effective weight loss therapy in severely obese o Less weight regain • However: complications and expensive

Children • 25% of Australian children are overweight or obese • Children’s BMI is associated with maternal BMI o 30% women had pre-pregnancy BMI obese/overweight o Thus, mums are fatter and consequently kids are going to be fatter

Overview • Obesity is a growing health problem that needs safe and effective drug therapies • Understanding chemical regulation allows us to find better obesity drugs • A polypharmacy approach may be needed (redundancy, may need >1 approach) • Any treatment of obesity should include a healthy eating and exercise plan

Recent findings • FTO gene – associated with obesity o Variation in gene group weighed 3kg more than control and had 1.7x risk of obesity Risk is present in children at age 7 • However, diet easily can induce obesity Lecture 13: Cellular respiration 2 Mike Edwards

Respiratory chain

• The process of reoxidising cofactors and transferring electrons to molecular O 2 • Redox reactions: o Reducing power is transferred downhill from NADH to 3 cofactors down to oxygen in a series of reactions Reactions flow in a thermodynamically favourable direction allowing energy to be harnessed o Overall free-energy change is ~52kcal/mol This is spread over smaller steps Overall, requires 7kcal/mol to make ATP • Thus, can make 7ATP from each NADH this is not the case however because energy can’t be directly transferred to ATP production • Overall reaction is very thermodynamically favourable, however • Components – all located in the mitochondrial inner membrane o Multi-subunit protein complexes Complex I – NADH-Q oxidoreductase Complex III – Q-cytochrome c oxidoreductase Complex IV – Cytochrome c oxidase o Coenzyme Q and Cytochrome c Mobile electron carriers – sit in the inner membrane and can move between the multi-protein complexes o Fe-S iron-sulfur protein o FMN flavoprotein (similar to FAD) o Cyt cytochrome groups (containing heme) • Process: o Multi-protein complex I oxidises NADH and reduces CoQ CoQ transfers electrons to multi-protein complex III o Multi-protein complex III oxidises CoQ and reduces Cyt c Cyt c transfers electrons to multi-protein complex IV o Multi-protein complex IV oxidises Cyt c and reduces O 2 to H 2O

Multiprotein complex II and equivalents

• FADH 2 reoxidation by respiratory chain o Succinate-Q oxidoreductase (complex II, same as succinate DH in TCA cycle) o Acyl-CoA-Q oxidoreductase (part of beta-oxidation) • Other o Mitochondrial FAD dependent enzyme for glycerol -3-P  DHAP

• These are FAD/FADH 2-dependent dehydrogenase enzymes o Also donate electrons to CoQ

FADH 2 Structure of complex I • Determined by electron microscopy o Made up of an inner domain and trans-membranous domain – spans the mitochondrial inner membrane • Process: o NADH + H + NAD + at the inner domain in the mitochondrial matrix Causes a conformational change in complex causing CoQ to be reduced to CoQH 2 CoQH 2 can then travel in the inner membrane to the next complex

ATP synthase • Multiprotein complex made up of subunits o Domain spanning the inner membrane – has channel and rotor structures o Domain in the mitochondrial matrix made up of a ball-on-stalk structure o Has alpha and beta subunits for the binding of ATP and ADP On isolation, can hydrolyse ATP ADP + Pi

Chemiosmosis • Discovered by Peter Mitchell (rich guy) who won the nobel prize o Links the respiratory chain with ATP synthase • Process: o Respiratory chain oxidises NADH to reduce oxygen to water Energy from this process is used to pump protons out of the mitochondrial matrix producing a proton gradient o Proton gradient is used to drive ATP synthesis (oxidative phosphorylation) • Evidence of this process: o If mitochondria are isolated and incubated at pH 8, then shifted to pH 4, a burst of ATP synthesis is measurable

This occurs without the presence of respiratory chain activity (O 2 consumption) Shows that: an H + gradient is sufficient to give ATP synthesis o If inner mitochondrial membrane is made leaky to protons, no ATP is synthesised with respiratory chain function (O 2 is used) Shows that: formation of H + gradient is essential for ATP synthesis linked to the respiratory chain o Photorhodopsin proteins and ATP synthase are incorporated into an artificial phospholipid vesicle Photorhodopsin is extracted from the bacterium H. halobium ATP synthase from beef heart mitochondria If light is shone on the photorhodopsin, it absorbs the light and pumps proteins across the membrane • this resulted in ATP synthesis • Chemiosmosis has since been shown to extend to other areas of biochemistry: chloroplasts, etc. o Shown to be used as an effective link between chemical energy and ATP synthesis

Relation to the respiratory chain • Complexes I, III and IV (but not II) pump H + across the inner membrane o Numbers are controversial but present understanding is: Complex I 4H + Complex III 2H+ Complex IV 4H+ These protons are not those used for redox reactions (ie. NADH + H NAD +) o Complex II and other equivalent complexes that use FADH 2 for reoxidation do not pump proteins

ATP synthase mechanism • Like hydroelectricity • Process: o H+ gradient turns a rotor (chemical energy into rotational energy) o Rotor turns shaft that causes conformational changes in the ball on stick part This allows binding of ADP and Pi before conversion to ATP • Can be demonstrated by attaching an actin filament coated with fluorescent material to the rotor o Shown to rotate

Amount of ATP made • NADH o Synthesis of ATP requires: 3H + to return to matrix for rotation 1H + via associated transport of ADP and Pi in and ATP out via specific transporter • Thus, net movement of 4H + is required for ATP synthesis o Reoxidation of NADH causes pumping of 10H + out of the mitochondrial matrix (I: 4, III:2, IV:4) Thus, 1NADH is equivalent to the synthesis of 2.5ATP

• FADH 2 o Inward movement of 4H + required for ATP synthesis + o Reoxidation of FADH 2 causes pumping of 6H out of mitochondrial matrix (III: 2, IV:4) Thus, 1FADH 2 is equivalent to synthesis of 1.5ATP (regardless of where FADH 2 comes from)

Uncoupling proteins • Proteins that are deliberately leaky to protons o Means that concentration gradient can’t be set up, and thus ATP can’t be made o Instead, fuel is burned and heat generated • Especially found in metabolically active tissues • ULP-1 (thermogenin) o Found in the mitochondria of brown adipose tissue in some newborn animals (incl. humans), animals that hibernate, animals adapted to the cold o Converts mitochondria into heat generating organelles • 2 other types of ULPs found in other tissues o May be involved in homeostasis • May be a good target for obesity medications – make cells burn more energy

The fluid membrane • Membrane is fluid in that it allows protein components to move and interact o Allows functioning of the respiratory chain via transport of H + and electrons • Phospholipid bilayer is also impermeable to ions allowing creation of the proton gradient • Membrane allows sidedness – asymmetry of membrane proteins o Permits pumping of protons in the respiratory chain in the correct direction to set up the proton gradient

Lecture 14: Physiology of gastric digestion Paul Bertrand

GIT motility revision • Effective digestion and absorption needs optimal time exposure to the different parts of the GIT o Motility involves mixing and propulsion o Function is highly regulated by neural, hormonal, local chemical and physical factors • GIT made up of smooth muscle that contracts with Ca2+ o Contains intrinsic pacemakers but is also controlled by nervous system and hormones Dual neural innervation: • Intrinsic – enteric nervous system • Extrinsic – sympathetic (inhibitory), parasympathetic (excitatory) • Major motor patterns: o Peristalsis, segmentation, pyloric pump, migrating motility complex

GIT digestion and absorption revision • Dietary nutrients are complex molecules (carbohydrates, lipids, proteins) o Need enzymatic breakdown o Ions, minerals, vitamins do not require digestion • All molecules need transport across GI wall and then into blood stream/lymphatics • Small intestine has a structure that encourages maximum surface absorption o Physical and chemical nature of nutrients determines how they are absorbed o Transport and carrier mechanisms are necessary for movement of some nutrients

Stomach overview • 4 regions: cardia, fundus, body, (pyloric) antrum • Functions: o Temporary food storage o Control of rate that food enters duodenum o Acid secretion and antibacterial action o Fluidisation of stomach contents o Preliminary digestion with pepsin/lipases etc o Other motor functions – see BGDB

Gastric secretions • Acid (H +, protons) parietal cells o Converts pepsinogen to pepsin o Bacteriostatic o Initiates digestion of protein (with pepsin) • Pepsin (as pepsinogen) chief, zymogen cells - • Mucus (including HCO 3 ) – mucus neck cells, surface mucus cells o Lubricates and protects against physical damage o Maintains a near neutral pH at the lining of stomach protecting stomach wall • Intrinsic factor – parietal cells o Protects vitamin B12 and facilitates its absorption in the ileum o Lack leads to pernicious anaemia • Hormones (gastrin – G cells, somatostatin – D cells, histamine – ECL cells) endocrine cells

Glands of the stomach • Oxyntic gland area and IF o Body and fundus (upper 80%) o Acid secreting parietal cells • Pyloric gland area o Antral region (lower 20%) Somatostatin (D cells), histamine (ECL cell) o Gastrin secreting G cells

Acid secretion • Biochemistry: - + o H2O OH + H H+ is then pumped actively into the lumen - - o OH + CO 2 carbonic anhydrase HCO 3 - HCO 3 diffuses to blood • Parietal cells: o 2 active transports: H+/K + ATPase on luminal side Na +/K + ATPase on basal side • K+ diffuses back out to lumen and blood via potassium channels - - o Carbonic anhydrase produces HCO 3 which diffuses out into the blood in exchange for Cl Cl - diffuses via a transporter into the lumen • Isotonic composition of gastric chyme o Relationship between Na + and H + with respect to flow rate With increased rate of gastric secretion, Na + decreases and H + increases in the chyme

Control of acidic secretion • Parietal cells are controlled by 3 methods (3 receptor types) o Acetylcholine (ACh) Muscarinic receptors Work by increasing intracellular Ca 2+ o Gastrin Gastrin/CCK-B receptors Work by increasing Ca 2+ o Histamine H2 receptors Work by increasing cAMP • Vagal tone stimulates: o Parietal cells via M3 Increases intracellular Ca 2+ o Enterochromaffin-like (ECL) cells via M1 Increases histamine released • Food stimulates: o Endocrine cells Gastrin is released o Chief cells Pepsinogen is released (and converted to pepsin by H + in stomach lumen) • Gastrin stimulates: o ECL cells via CCK-B receptor Increases histamine release o Parietal cells via CCK-B Increases intracellular calcium • Histamine stimulates: o Parietal cell via H2 receptor Increases cAMP which up-regulates production of the proton pump protein

• Stimulants in combination are greater than individually there is much complex interplay between them

Phases of gastric secretion • Summary: o Cephalic phase – parasympathetics excite pepsin and acid production o Gastric phase – local nervous secretory reflexes, vagal reflexes, gastrin-histamine stimulation o Intestinal phase – nervous mechanisms, hormonal mechanisms inhibiting secretion o Interdigestive phase – low pH in stomach

• Interdigestive phase o Stomach contains low volume, thus pH >2.0 This inhibits gastrin release from G cells and limits excessive acid production • Secretion rate is 10% maximal capacity o Gastro-esophageal reflux occurs in this phase • Cephalic phase – 20-30% secretory response o Preparatory phase o Initiated by taste/smell via mechano and chemoreceptors Afferent and efferent information via vagus nerve causing reflex: • Stimulates parietal cells (directly: muscarinic) • Stimulates G-cells (indirectly: gastrin releasing peptide (GRP)) via enteric neurons o Studied hy sham feeding (via esophageal and gastric fistulas • Gastric phase – >50% of secretory response o Food enters stomach causing pH to rise (pH 6) inhibition on gastrin stopped o Distension of stomach walls via mechano-receptors initiates short (local) and long (vaso-vagal) reflexes Cholinergic transmission o G cells stimulated by amino acids, calcium, caffeine (independent of vagus) Increases gastrin release o Generally: gastrin is stimulated by GRP (enteric neurons) and inhibited by local somatostatin (SOM, SST) from D cells

• Intestinal phase – 5% of secretory response o Amino acids are thought to increase gastrin release, also other hormones o Inhibits acid secretion/controls action

Control of other secretions • Pepsinogen – pro-enzyme released from chief cells o Stimulus is vagally-mediated via ACh occurring in cephalic and gastric phases H+ locally stimulates an enteric cholinergic reflex also causing release • H+ is required to activate (pH 5 is inhibitory) • Intrinsic factor (IF) o Secreted by parietal cells o Required for the protection/transport of vitamin B12 for uptake in the terminal ileum Lack of B12 leads to pernicious anaemia – important cofactor in producing DNA, without it, RBCs can’t be produced o Lack of IF also associated with achlorhydria (low production of gastric acid) and a lack of parietal cells • Mucus secretion o Soluble mucus Produced by mucus neck cells • Stimulated by ACh via vagus Lubricates by mixing with gastric contents o Insoluble(surface) mucus Produced in response to chemical/mechanical stimuli - Forms a protective gel layer trapping HCO 3 and cellular debris to prevent acidic breakdown of stomach wall

Summary • Gastric juice is made up of acid, pepsinogen, mucus, intrinsic factor and some electrolytes o Acid activates pepsinogen, initiates breakdown of food and is bacteriostatic • Secretion of acid involves H +/K + ATPase and Na +/K + ATPase active transport o Stimulating factors include: hormone gastrin, NT ACh, locally released histamine These factors work together in complex ways to stimulate acid secretion • Cephalic phase – vagal stimulation of parietal cells and gastrin release from antral G cells • Gastric phase – distension stimulates short and long reflexes causing acid secretion and gastrin release o Protein digestion also stimulates G-cells • Somatostatin (released when pH <2-3) inhibits acid secretion o Duodenal mechanisms also inhibit • Intrinsic factor secretion by parietal cells is required for absorption of vitamin B12 in terminal ileum

Notes • Gastrin is a peptide hormone produced by G cells in the stomach, duodenum and pancreas

Lecture 15: Creation of health Adrienne Torda

Public health ethics • Public health is the instrument for promotion and enhancement of health and well-being of the individual as well as the community o Public health ethics gives priority to health of the collective group over the individual EG: Swine flu – stay home o Vs. Autonomy – the right for an individual to be free and decide what to do with themselves o Human rights of patients Not to be discriminated against by shape, age, culture etc To make their own decisions about their own health

Smoking • Consequences: o Cancer, heart disease, cerebrovascular disease, peripheral vascular disease • Prevention measures: o Banned in public, in cars with children, warnings on cigarette packets o Social stigma (vs. 50 years ago, where it represented social freedom and culture) • Problems for healthcare: o Recalcitrant (stubborn) smokers – ie. patients who don’t want to stop Does society have the responsibility to pay for t heir healthcarea? In recent times, GPs now have an item number for preventative medicine to encourage them to help these smokers etc

Obesity • High prevalence (also in young people: children and adolescents) o 53% women, 67% men overweight or obese o Prevalence is increasing rapidly, especially in children • Linked to diseases: o IHD, DM, endocrine, GIT, dermatological o Some surgeons don’t perform surgery until weight loss is achieved Risk is higher with obesity, and surgery can often fail • Weight loss is a powerful healthcare measure o 2/3 no longer need DM treatment after weight loss o Decreases – hypertension, sleep apnoea, depression, cholesterol Increases fertility • Management o History, examination and investigation o Weight loss (3-12 months) Lifestyle modification, diet o Weight management o Bariatric surgery • Public health ethics o Need to treat anyway, treat in most cost effective way – best for community Vs: responsible for own health and education o Health shouldn’t just be for the wealthy o Money could be better spent? – prevention etc • Ethical issues o Orthodox medicine doesn’t seem to work – preventative measures are not working o Health messages can be discriminatory – schools, work, jobs o Health messages can be harmful o Whose responsibility – virtue ethics o Public health (community good) vs individual rights • Other issues o Psychological health o Social worth, self image • Community, doctor and the individual Beauty • Not as easily defined as smoking/obesity • A range of medical options are available o Should these be allowed to advertise o Cosmetic tourism – safety, infection, follow up, insurance • Issues: o Social pressures to stay beautiful and stay young o Informed consent – not just procedure, but also meaning of body change At what age can we make informed consent? o Medicare subsidy – item numbers exist for correcting disfigurement, but not cosmetics? Should cosmetics be subsidised – benefits: self esteem? o Hospital beds o Prices – should they be capped, or can people charge what they want o Beauty changes with time

Healthy • What is healthy? • What is the doctor’s role? • Alternative therapies

Happy • Depression is common o Youth suicide rates are high • Antidepressants are common in the US o Prozac – used to top up mood in cases where antidepressants probably aren’t necessary

Creation of health • Interplay between medicine, the society and the media • PH vs. human rights and autonomy

Lecture 16: Fuel synthesis H. Robert Yang

Energy use • The liver takes in various products including glucose, alanine/lactate, fatty acids o These are processed before different products are excreted: glucose, fatty acids, ketone bodies • The brain and heart use a lot of energy, but do not produce any • Glucose o Some tissues are glucose dependent: Brain (120g/day) Red blood cells Kidney medulla Lens/cornea of eye Testis o The liver stores 190g of glucose, these glucose dependent tissues use ~160g glucose/day 190g is enough for just over 24 hours o Supply of glucose is maintained by gluconeogenesis

Gluconeogenesis • Definition – the synthesis of glucose by non-carbohydrate precursors (amino acids, lactate, pyruvate, glycerol etc.) o Important in supporting the metabolism of tissues that use glucose as their primary energy source Ie: brain, RBCs, kidney medulla, lens, cornea and testis) o Particular important during starvation when no new glucose is introduced into the body • Basically allows creation of glucose (necessary for brain etc function) from alternate (ie. not dietary glucose - carbohydrate) forms of energy • Lactate, pyruvate, glycerol and amino acids feed into the gluconeogenesis pathway at different points o Lactate is converted directly to pyruvate o Amino acids can be converted to pyruvate or oxaloacetate o Fatty acids are broken down to glycerol which feeds into as a triose phosphate o From here, pyruvate is converted to oxaloacetate then triose phosphates then glucose • Amino acids are used via their amino group-stripped carbon skeletons o Glucogenic – give rise to increased levels of pyruvate (etc) feeding into gluconeogenesis o Ketogenic – give rise to Acetyl CoA, not pyruvate

Gluconeogenesis continued • Location: o Liver o Kidney cortex o Cytosol – but first step is in mitochondria • Time o After intense skeletal muscle activity – lactate surge (thus removes lactate) o Fasted state (starvation) Parallels with beta-oxidation – both occur when there is little glucose

Gluconeogenesis and glycolysis • Overall, gluconeogenesis is the reverse process of glycolysis o Glycolysis: 1 glucose 2 pyruvate o Gluconeogenesis: 2 pyruvate 1 glucose • However, glycolysis is an irreversible process (with a large negative free energy change) o Thus is facilitated by 3 irreversible steps Gluconeogenesis is different to glycolysis in that it has to bypass these 3 steps

Bypass reaction 1

• Forward reaction: PEP pyruvate kinase pyruvate o Bypass reaction: 1: Pyruvate Oxaloacetate (OAA) 2: Transport of oxaloacetate out of mitochondria via Malate 3: Oxaloacetate Phosphoenolpyruvate (PEP) + • 1: Pyruvate + CO 2 + ATP + H 2O pyruvate carboxylase Oxaloacetate + ADP + Pi + 2H o Anaplerotic reaction (forms intermediates of the TCA cycle) o Enzyme pyruvate carboxylase contains biotin (vitamin H, B7) Biotin is a prosthetic group that carries activated CO 2 Found in the mitochondrial matrix Allosteric activated by acetyl-CoA • Ie. increased levels of acetyl-CoA signal synthesis of more OAA • Acetyl-CoA can commonly come from beta-oxidation • 2: Oxaloacetate is reduced within the mitochondria to form malate o Malate is then transported out of the mitochondria by a transport system o Here it is reoxidised to OAA

• 3: Oxaloacetate + GTP Phosphoenolpyruvate carboxykinase (PEPCK) Phosphoenolpyruvate + GDP + CO 2 • OVERALL: Pyruvate + ATP + GTP PEP + ADP + GDP + Pi o Uses energy (GTP, ATP)

Bypass reaction 2

• Forward reaction: Fructose 6-phosphate Phosophofructokinase Fructose 1,6-bisphophate o Bypass reaction: Fructose 1,6-bisphophate + H 2O Fructose 1,6-bisphophatase Fructose 6-phosphate + Pi o This continues to Glucose 6-phosphate

Bypass reaction 3

• Forward reaction: Glucose Hexakinase Glucose 6-phosphate o Bypass reaction: Glucose 6-phosphate + H 2O Glucose 6-phosphatase Glucose + Pi o This continues enters the blood for use by other tissues via the hepatic vein

Summary

Energy costs of GNG • Overall reaction: + + + o 2 Pyruvate + 4ATP + 2GTP + 2NADH + 2H + 6H 2O Glucose + 4ADP + 2GDP + 6Pi + 2NAD + 4H ΔG o’ = ~-38 Kj/mole o Glycolysis: ΔG o’ = ~-85Kj/mole • Cost of GNG is 6ATP, allows us to turn an unfavourable reaction into a favourable one

Control of gluconeogenesis • Using reaction 2 as an example: o Gluconeogenesis/glycolysis are activated/inhibited by reciprocal things • Glycolysis: o Phosphofructokinase Stimulated: AMP, Fructose 2,6 bisphophate Inhibited: Citrate, ATP • Gluconeogenesis o Fructose 1,6-bisphosphatase Stimulated: citrate Inhibited: Fructose 2,6-bisphosphate, AMP

Fatty acid metabolism • Fatty acid metabolism is interconnected with glucose metabolism o Acetyl-CoA which is formed from pyruvate and leads into the TCA cycle has an alternate pathway to form fatty acids and triacylglycerides This is stimulated by: insulin, a fed state • Net stoichiometry (using palmitate, C16, as an example): o 8 Acetyl-CoA + 7ATP + 14 NADPH Palmitate + 7ADP + 7Pi + 14NAD + + 8CoA • Acetyl-CoA needs to be transported out of the mitochondria into the cytosol o Process: Acetyl-CoA is transformed into citrate which passes through the membrane Citrate is processed in the cytosol to remove the Acetyl-CoA group reforming OAA In this same process, NADH is transformed to NADPH (Also necessary for palmitate synthesis)

Fatty acid synthesis step 1 • Acetyl CoA carboxylase processes Acetyl-CoA into Malonyl-CoA - o Acetyl-CoA + ATP + HCO 3 Acetyl CoA carboxylase Malonyl-CoA + ADP + Pi • This is the rate limiting step

Fatty acid synthesis steps 2-6 • 2: CoA groups are swapped for ACP groups o Acetyl-CoA + ACP Acetyl-ACP + CoA o Malonyl-CoA + ACP Malonyl-ACP + CoA • 3: Condensation • 4: Reduction • 5: Dehydration • 6: Reduction • Steps 3-6 are repeated as necessary to form the right length chain o Ie. in this case, repeated 7x to get C16 o These steps require the enzyme complex fatty acid synthase

• From here, palmitate can be converted to other long chain carbons via fatty acid elongases and desaturases o Fatty acids are important for synthesising: Phospholipids – cell membranes Steryl esters – collect in BV walls Triacylglycerols – fat cells

Overall reactions of lipogenesis • Has a distinct sequence of reactions that is not a direct reversal of beta-oxidation • Occurs in the cytosol o In the liver, mammary gland and ?adipose tissue? • Summary of process: o Acetyl CoA carboxylase (with biotin) transfers carboxyl to acetyl-CoA forming malonyl-CoA o Fatty acyl intermediates are linked to an acyl carrier protein (ACP) o Fatty acid synthase (polypeptide chain multi-enzyme complex) cycles through several reactions that add 2 carbon units to chain This reaction is reduced by NADPH Elongation in this manner stops at C16 (palmitate) • Control: o Acetyl CoA Carboxylase (ACC) is activated by glucose, citrate o Malonyl CoA inhibits beta-oxidation o Long chain FA inhibit ACC

Fatty acid synthesis vs beta-oxidation

Lecture 17: Diabetes Mellitus Rakesh Kumar

Definition • Diabetes mellitus – a syndrome resulting from a functional deficiency of insulin o Results in impaired glucose utilisation and hyperglylcaemia o Methods of deficiency: Increased insulin demand Decreased insulin supply Insulin doesn’t work Physiological opposition to insulin action o More than one mechanism may be important • Insulin is a critical metabolic hormone o Important for carbohydrate, protein and lipid metabolism • In a population, diabetes mellitus (DM) is defined by a glucose tolerance test o Subjects are given a 75g oral load of glucose and levels are measured before administration and at 2 hours o Diabetes exists as a continuum, risk increases as you progress down the scale Normal Impaired fasting glucose – fasting glucose is high, processing of glucose is normal Impaired glucose tolerance – fasting glucose is moderate and glucose processing is high Diabetes – fasting glucose is high, glucose processing is high

Epidemiology • 7.5% of the Australian population has been diagnosed with DM o ~700 000 people, thought to be 1 undiagnosed for every diagnosed • Every year 1% of people age 55 develop DM • Every day, 275 adults develop diabetes o Most had a previous prediabetic state – impaired glucose tolerance, impaired fasting glucose • Risk increased if overweight (2x) and obese (4x) • Risk associated with ethnic groups – ATSI have 3x non-indigenous

Complications and diseases • DM causes 8% of burden of disease o Causes 3% of deaths and contributes to 6% more • Complications: o Common cause for needing renal dialysis (for chronic renal failure) o Common cause of blindness <60 o Common cause of non-traumatic lower-limb amputation o Major cause of cardiovascular disease (3x MI, 3x strokes) Exaggerates atherosclerosis causing CVD and CeVD • Common chronic disease in children (along with asthma)

Classification • Type 1 o Not the most common – 10% (quite rare) Most spectacular, abrupt onset, dramatic reduction in insulin output o Disease of children (esp. <20) o Triad of symptoms: Polyuria – excessive urination Polydipsia – excessive thirst Polyphagia - excessive hunger o Significant risk of ketoacidosis • Type 2 o Major form – 85-90% Gradual onset, reduction in insulin output as well as response to insulin Over time, just as severe as type 1 o Disease of adults >30 (especially obese) o Ketoacidosis not common • Other types (uncommon): o Single gene defects [note genes can commonly contribute to susceptibility] Problems in: • Insulin synthesis • Receptor regulation o Damaged pancreas Pancreas removal Hemochromatosis, pancreatitis, infection o Endocrine disorders Hypersecretion of growth hormone, glucocorticoids o Gestational diabetes Transient state during pregnancy Estimated to occur in 5% of pregnant women

Pathogenesis • Type 1 o Results from autoimmune destruction of >90% of beta-cells Normally involves genetic predisposition (susceptibility or resistance, to do with HMC class II genes) Also environmental factors – eg. viral infection o Twin studies show ~50% concordance, thus not all genetics, also environmental factors important o Picture : insulitis Autoimmune inflammation of pancreatic islets leading to destruction of beta-cells

Pathogenesis (continued) • Type 2 o Large genetic input High twin concordance rate and increased risk in first degree relatives Explains high prevalence in certain populations o Also associated with environmental factors such as obesity and metabolic syndrome Obesity causes insulin resistance which causes hyperglycaemia Hyperglycaemia leads to increased ineffective insulin production by beta-cells which become exhausted These cells die and insulin levels again fall o Picture: amyloid deposition Twisted beta-pleated sheets May be seen in non-diabetics • Unknown whether this deposition is cause or effect o End up with low insulin output and failure of cells to respond to insulin Intracellular mechanisms contributing to insulin resistance are largely not understood • Theories: lipid metabolites, endoplasmic reticulum function

Clinical effects • Can be an interaction between the underlying condition and a transient state (eg. diabetes and pregnancy/infection) o Type 1: Severe insulin depletion, metabolic complications o Type 2: Long term complications DM remains undetected until it is quite far along Tight control is very good at reducing risk • Metabolic abnormalities: o Catabolic state (type 1) o Ketoacidosis (type 1) High rates of lipolysis in fat cells produces FFA which are converted to ketones in the liver • This can lead to a metabolic acidosis • Acidity in blood causes CNS depression leading to coma and death o Hyperlipidaemia (both) o Hyperglycaemia (both) Causes diuresis of extra and intracellular compartments • Cells become dehydrated and blood sticky o Especially a problem in the elderly who may not be able to get water easily

Clinical effects (continued) • Accelerated atherosclerosis (CVD 2x-4x more common) o Ischaemic heart disease Picture 1: notice scar tissue at top, and compensatory hypertrophy at bottom Women are normally protected from heart disease in their fertile years by estrogen • With diabetes, there is a substantial increase in heart disease o Cerebrovascular accidents Picture 2: notice liquefactive necrosis on left o Peripheral vascular disease Poor peripheral circulation can result in lactic acidosis Severe ischaemia often results in atrophy and then gangrene o Renal ischaemia Stenosis of renal vessels

• Other chronic complications o Nephropathy (kidney disease overall 4x more common) Sustain hyperglycaemia allows glucose to bind non-enzymatically to proteins – glycosylation (glycation) Glucose can thus come together and form ring molecules advanced glycosylation end products (AGE) Proteins become cross-linked • Eg: lipoproteins, external matrix collagens Cross-linked proteins accumulated in the basement membrane and make it leaky - glomerulosclerosis • Thus they disrupt the balance in the BM and hyperfiltration occurs chronic renal disease Picture 3: hyaline change – diffuse thickening due to protein accumulation Picture 4: diffuse glomerulosclerosis in diabetic nephropathy Picture 5: Advanced glomerulosclerosis with nodular and diffuse lesions • Pink nodules were once glomeruli

o Eye disease (retinopathy – most common cause of blindness adults 20-74, cataracts, glaucoma) Diabetic microvascular disease where haemorrhages into the retina cause areas of microinfarct • From here, neovascularisation and scarring of macula may lead to retinal detachment o Poor healing Impaired leukocyte function Poor blood supply and thus poor granular response o Neuropathy Somatosensory and autonomic • Can injure feet and not know • Sweating impaired – dry skin • Poor proprioception – abnormal weight bearing and pressure Not being able to feel, skin being dry and abnormal weight bearing can lead to ulcers • These don’t heal, can get infected, and become gangrenous Clinical effects (continued) • Infections o Suppurative (eg. periodontal disease – tissue around the tooth) Eg. Paronychia Eg. acute on chronic pyelonephritis with papillary necrosis o Non-suppurative (eg. mycobacterial, fungal) Eg. Fibrocaseous tuberculosis (mycobacterium) Eg. Oral candidiasis (fungi) Eg. Mucor fungus (bread mould) – can cause a retro-orbital infection causing proptosis • Infection known as mucormycosis o Other: pneumonias, gingivitis o Mechanism: Cross-linking of proteins in the membranes of leukocytes causes neutrophils and macrophages to be ineffective Thus diabetics are more susceptible to infection and commonly get disease from pathogens that don’t normally cause disease (opportunistic) • Other long term problems: o Complicated pregnancy o Fetal abnormalities

Mortality • People with diabetes have 2x increased mortality than people with normal glucose o This mortality is similar to smokers or people who have had previous cardiovascular disease o Pre-diabetes (impaired fasting glucose, impaired glucose tolerance) had a 1.5x increased mortality risk • 2/3 of CVD mortality occurred in people with diabetes or pre-diabetes • Causes of death: o Myocardial infarction > chronic renal failure > cerebrovascular accidents > infections > metabolic coma

Lecture 18: Metabolism – integration and regulation I: organs Robert Yang

Key concepts – revision • Anaerobic respiration o Glycolysis without the presence of oxygen resulting in lactate/ethanol production o Relatively ATP inefficient IE. cancer cells use this pathway and consume a lot of energy • Aerobic respiration o Glycolysis in the presence of oxygen converging on the TCA cycle and then oxidative phosphorylation o Most efficient way to produce ATP • Cellular compartments: o Cytosol events: glycolysis, pentose phosphate pathway, fatty acid synthesis o Mitochondria: citric acid cycle, oxidative phosphorylation, β-oxidation of fatty acids, ketone body formation o Both compartments – gluconeogenesis, urea synthesis • 7 major metabolic pathways o Glycolysis o Gluconeogenesis o Glycogen degradation and synthesis o Fatty acid synthesis and degradation o Citric acid cycle o Oxidative phosphorylation o Amino acid synthesis and degradation • Crossroads compounds: o Acetyl-CoA o Pyruvate • Other notes: o Polymers  monomers o ATP is the energy currency o NAD + and FAD are important in oxidative metabolism o NADPH is important in reductive biosynthesis (eg. FA) o Biosynthesis and degradation pathways are not simple reversals o Not every tissue has every pathway

Organ specialisation • Different organs have different roles in energy storage and usage and are linked by the bloodstream • Summary: o Brain (the spoilt child: “I want glucose”) Glucose-dependent o Skeletal muscles (Mr strong: glycogen for own use but can be persuaded to share protein”) Resting/aerobic exercise: FA Strenuous exercise: phosphocreatine, glucose (anaerobic) o Heart muscle – fatty acids o Adipose tissues (expandable warehouse: saving for a rainy day) Storage of fatty acids o Liver (Mr nice guy: is everyone provided for?) Maintains appropriate levels of circulating fuels for other tissues/organs o Pancreas (Sensor: plenty or empty?)

Brain • Glucose-dependent o Can use ketone bodies during prolonged starvation • Needs a steady supply of glucose o Has little storage capacity – relies on blood glucose • Very important: o Only 2% of body mass but uses 20% of body’s O 2 consumption o Lack of glucose can result in brain dysfunction, coma, death Skeletal muscle • Can use glucose, fatty acids, ketone bodies • Stores a large amount of glycogen o Preferred fuel for bursts of energy o Store is for own use only – can’t export glucose Lacks glucose-6-phosphatase and thus glucose 6-phosphate can’t get out of the cell o Can provide glucose via proteolysis of alanine Ie. breakdown of protein stores in prolonged starvation (after glucose/fatty acid stores used) o In resting muscle and aerobic metabolism (long distance exercise), fatty acids are the main fuel • Muscle contraction o Driven by ATP hydrolysis ATP is provided initially by phosphocreatine (4s) Next is usage of glycogen store by glycolysis • High exertion – anaerobic glycolysis producing lactate o Ie. with high exertion (sustained peak contraction), blood vessels are suppressed and oxygen supply decreased thus resulting in anaerobic metabolism o Lactate produced can be recycled using the Cori cycle • Cori cycle: o Lactate is produced from anaerobic glycolysis Not pyruvate because lactate dehydrogenase converts pyruvate to regenerate NAD + o Lactate is transported to the liver where it is used via gluconeogenesis to form glucose again • Muscle fatigue o Definition – the inability for muscles to maintain a given power output o Maximum exertion can be maintained for only 20seconds This is the time it takes for the muscles to deplete ATP levels to its limit • Limit is defined by the point where there is enough ATP left to start glycolysis (to produce more ATP) and to maintain cell function Mechanism that causes this fatigue and prevents cell death is unknown • Thought to be due to glycolysis increasing intracellular protons and decreasing pH and thus decreasing the activity of PFK

Adipose tissue • Function – storage and release of fatty acids to store/release energy o Storage is as triglycerides (triacylglycerols) o Also produces adipokines/hormones • Synthesis of triglycerides o Requires glycerol backbone (byproduct of glycolysis) Glucose …~~~… fructose 1,6-bisphosphonate DHAP Glycerol-3-phosphate TG • Note: glucose uptake is insulin stimulated o Fatty acids come from diet and from the liver as VLDL or chylomicrons • Degradation of triglycerides o Can be broken down into glycerol and fatty acids in adipose tissue to provide energy for other organs (especially the liver)

Heart muscle • Continuous activity relying entirely on aerobic respiration o Has a large number of mitochondria • Preferred fuel is fatty acids o Has a store of glycogen – limited o Can also use ketone bodies, glucose

Liver • Acts as the body’s central clearing house o Regulates the level of fuel available to the brain muscle and other organs via the blood o Processes all nutrients absorbed by the intestines via the portal vein Except FA in chylomicrons o Exports: FA-VLDL, glucose, ketone bodies (in starvation) • For own energy – main fuel is fatty acids • Glucose metabolism o Uptake of glucose by hepatocytes is unique in that it is independent of insulin All other cells: Hexokinase – high affinity (Km~0.1mM) for glucose, inhibited by G-6-P • High activity in low concentrations of glucose (3.6-5.8mM blood glucose) Liver cells: Glucokinase - low affinity (Km~6mM)for glucose, not inhibited by G-6-P • High activity in high concentrations of glucose o Thus, when excess glucose, takes and stores in various ways • Allosterically regulated, synthesis induced by insulin • Use of glucose in the liver (glucose-6-phosphate) o Isomerisation to glucose 1-phosphate and processing to glycogen o Glycolysis to pyruvate and then to Acetyl CoA which can lead to the TCA cycle or fatty acid synthesis o Pentose phosphate pathway resulting in synthesis of NADPH and ribose-5-phosphate o Processing to glucose for export to other organs

• Fatty acid metabolism in the liver o Processing to liver lipids (phospholipids) – for cell membranes o Beta oxidation to acetyl coA and then to the TCA cycle o Beta oxidation to acetyl coA and then processing to ketone bodies for export to other organs o Beta oxidation to acetyl coA and then processing to cholesterol for bile salts and steroid hormones o Packaging into plasma lipoproteins to deliver TG to body tissues o Free fatty acids can be bound to serum albumin in the blood • Fatty acid metabolism depends on the situation: o Time of plenty: FA TG VLDL adipose tissue o Starvation: FA acetyl CoA ketone bodies

Ketone bodies • Produced by the liver in times of starvation o Exported to provide fuel for other tissues (esp. muscle + brain) o Liver cannot use ketone bodies because it lacks the enzyme 3-ketoacyl CoA transferase • Normal conditions – carbohydrates (glucose) provides >50% of energy o Low glucose, increased beta-oxidation of fatty acids increased acetyl CoA produced This cannot enter TCA cycle because oxaloacetate is deficient in starvation, gluconeogenesis is activated and uses up oxaloacetate Excess acetyl CoA is processed into ketone bodies: acetoacetate, D-3-hydroxybutyrate, acetone o Conditions that produce this are: fasting, starvation, diabetes mellitus, low carb diet • Problems with this: o Ketosis – if levels of ketone bodies get too high (esp. acetoacetate nad hydroxybutyrate which are acidic), pH of the body can change causing an acidosis If untreated, this can lead to: • Coma and death • Dehydration – excessive urination to remove acids • Electrolyte imbalance • Bad breath • Protein degradation

Amino acid metabolism in liver • Excess amino acids are transported to the liver for use o Synthesis of liver proteins/plasma proteins o Amino acids in blood for tissue synthesis of proteins o Synthesis of nucleotides, hormones o Excretion via urea and backbone used for energy production • Muscles can donate protein (amino acids) to the liver for breakdown to produce energy o Glucose-alanine cycle

Metabolic control • Two levels of control o Local Based on the immediate requirements of the individual cell Commonly involves regulation by allosteric enzymes o Global Integration of individual cell metabolism into the context of the whole body Commonly involves regulation by hormones, receptors, signalling pathways and changes to enzymes via covalent modification • Hormonal control in this way often makes use of G proteins, second messengers (eg. cAMP), protein kinase A (modifies enzymes by de/phosphorylation) • EG: local – glycolysis o PFK is regulated allosterically by: (+) AMP, fructose 2-6-P; (-)ATP, citrate, H + PFK makes more energy in the presence of AMP, fructose 2-6-P • EG: whole body – regulation of glycolylsis o PFK is activated by fructose-2,6-bisphosphate Fructose-2,6-bisphophate is made by enzyme PFK2 from fructose 6-phosphate • Activity of this enzyme is regulated by insulin (hormone) o Insulin is stimulated by high blood glucose levels o THUS: PFK is regulated by blood glucose levels, high blood-glucose activation and processing of glucose

Metabolic homeostasis • Definition – the balance between fuel availability and the needs of different tissues for different fuels o Regulation/control is by: hormones (cell signalling) and the CNS • Glucose is especially important because many tissues rely largely on glucose: o Ie. Brain, RBC, lens of eye, kidney medulla, exercising skeletal muscle need glycolysis for part of energy req

Pancreas • Senses blood glucose levels and secretes regulatory hormones as appropriate: o β-cells produce insulin – stimulated in high blood glucose o α-cells produce glucagon – stimulated in low blood glucose • EG: fed state – high insulin and low glucagon (high insulin/glucagon ratio) • EG: starved state – low insulin and high glucagon (low insulin/glucagon ratio)

Lecture 19: Metabolism – integration and regulation II: insulin and glucagon Robert Yang

Signal transduction • Hormones such as insulin regulate cell function by signal transduction • Process: o Hormone binds to an extracellular receptor o Receptor causes a relay of signals (often via second messengers like cAMP) o This signal is amplified and modified o Signal diverges to multiple targets and regulates various functions such as: Metabolic pathways Gene expression Cytoskeleton changes

Insulin • A polypeptide anabolic hormone synthesised as a preprohormone by pancreatic β-cells • Functions: o Promotes fuel storage after a meal – glycogen synthesis, FA synthesis o Promotes growth – amino acid uptake, protein synthesis • Stimulated by increased blood glucose levels (80mg/dL)

Insulin signalling • Insulin binding to tyrosine kinase receptor and activates the beta- subunit o Tyrosine residues of beta-subunits are autophosphorylated o Also, tyrosine kinase receptor phosphorylates other proteins such as insulin-receptor substrate-1 These phosphorylated proteins (kinases/phosphatases) lead to biological activity • Effects: o Induction/repression of specific enzyme synthesis o General stimulation of protein synthesis o Stimulates glucose and amino acid uptake • Overall effects: o Increased glucose uptake o Increased glycogen synthesis o Increased lipogenesis o Decreased lipolysis

Glucose uptake • Insulin activates GLUT4 which transports glucose into the cell o GLUT 4 is present on adipocytes and skeletal muscle cells, but not in the liver Thus, insulin regulates glucose uptake here. The liver’s uptake of glucose is independent of insulin • Glucose transporters (GLUTs) – many different isoforms o GLUT 2 – liver and pancreatic beta-cells (Km = 15-20mM, low affinity) Pancreas – regulates insulin secretion Liver – removes excess glucose from the blood o GLUT 4 – skeletal muscle and adipocytes (Km = 5mM, high affinity) Uptake of glucose is affected by insulin o GLUT 3 – brain and nerve tissues (Km = 1mM) Allows for basal glucose uptake

Glucose storage • Insulin promotes glycogen synthesis – synthase is active, phosphorylase is inactive o Insulin causes dephosphorylation of glycogen synthase and phosphorylase

Lipogenesis and lipolysis • Insulin regulates (liver): o Inhibits hormone sensitive lipase – breaks down triglycerides (adipocyte) o Inhibits carnitine acyl-transferase – important for beta-oxidation o Stimulates Acetyl CoA carboxylase – important for fatty acid biosynthesis (liver) o Stimulates lipoprotein lipase – used to breakdown triglycerides in chylomicrons for storage (adipose) • Lipoprotein lipase (LPL) – found in capillaries of adipose tissues and skeletal muscles o Digests TAGs found in chylomicrons to release glycerol and free fatty acids • Hormone sensitive lipase (HSL) o Activated by cAMP dependent phosphorylation o Breaksdown TAGs into free fatty acids • Insulin stimulates the expression and secretion of adipose LPL and inhibits HSL thereby promoting lipogenesis and inhibiting lipolysis

• FA biosynthesis involves the step: o Acetyl-CoA acetyl CoA carboxylase Malonyl CoA Allosteric regulation: • (-) palmitoyl CoA (end product, feedback inhibition) • (+) citrate Hormonal regulation • (+) insulin dephosphorylation activating enzyme • (-) glucagon phosphorylation inactivating the enzyme • A high insulin/glucagon ratio induces enzyme synthesis Summary, insulin: • Signals in times of plenty (fed state) • Activates glucose uptake in adipose and muscle • Activates glycogen synthesis in liver and muscle • Activates lipogenesis in the liver and adipose Glucagon • Polypeptide synthesised as an inactive precursor by the pancreatic α-cells • Functions: o Mobilise fuels – glycogenolysis (liver), FA release (adipose) o Maintains blood glucose levels during fasting - gluconeogenesis • Secretion is inhibited by glucose and insulin and stimulated by amino acids • Overall effects: o Glycogenolysis in the liver o Release of fatty acids from the adipocytes o Gluconeogenesis in the liver Glycogenolysis in the muscles is controlled by adrenaline, glucagon has no effect

Glycogenolysis • Glucagon causes phosphorylation of glycogen synthase and phosphorylase o This causes glycogen to be broken down by glycogen phosphorylase • This process is regulated by phosphorylation o Glucagon binds to G-protein receptors which activates G-proteins o G-proteins activate adenylate cycle which produces cAMP o cAMP activates protein kinase A o Protein kinase A phosphorylates phosphorylase kinase This phosphorylates glycogen phosphorylase which activates glycogen breakdown o Protein kinase A phosphorylates glycogen synthase inhibiting glycogen synthesis

2nd messengers • A common second messenger is cyclic AMP, synthesised via adenylate cyclase o ATP + H 2O adenylate cyclase cAMP + PPi • Other common second messengers: o Note: Viagra inhibits the breakdown of cyclic GMP

Glucagon on fatty acid metabolism • Overall effects: o Inhibition of FA synthesis and TAG synthesis o Increased breakdown of TAG • Effects on hepatocyte o Inhibits acetyl-CoA carboxylase and thus prevent fatty acid biosynthesis • Effects on adipose cells o Deactivates LPL and activates HSL o Thus decreases TAG in VLDL and increases breakdown of TAG into FA and glycerol

Cortisol • Provides for long term changing requirements o Stimulates: Gluconeogenesis Mobilisation of amino acids from muscle protein FA release from adipose tissue

Adrenaline • Mobilises fuel during acute stress o Glucose production from glycogen (muscle, liver) o FA release from adipose tissue • Process of glycogen breakdown is similar to that of glycogenolysis in the liver but in the muscle

Summary of adrenaline and glucagon • Both increase amount of cAMP: glucagon via glucagon receptor, adrenaline via beta-adrenergic receptor o This causes increased glycogenolysis and decreased glycogen synthesis, decreased lipogenesis • Fasted state of stress: o Glucagon causes lipolysis in the adipose tissue, glycogenolysis and gluconeogenesis in the liver o Adrenaline causes lipolysis in the liver, glycogenolysis and gluconeogenesis in the liver and glycogenolysis in the muscle

Meals • High carbohydrate meal o Glucagon drops o Insulin rises then drops o Blood glucose rises then drops • Low carbohydrate/high protein meal o Nitrogen rises and remains high o Glucose remains low o Insulin increases then drops off Enables amino acid uptake and protein synthesis in muscles – levels not high enough to inhibit gluconeogenesis o Glucagon rises and remains high Stimulates gluconeogenesis in the liver to maintain blood glucose

Summary: insulin, glucagon, adrenaline

Fat is good • Congenital generalised lipodystrophy – no fat on body o If eat fat, fat overflows and lipotoxicity leads to the muscles having insulin resistance and death of beta-cells • Other extreme: obesity: fat cells have reached capacity for storage o Fat overflows and lipotoxicity causes diabetes, as above

Pentose phosphate pathway • An oxidative pathway like glycolysis o Generates NADPH and the ribose component of nucleotides (5 carbon sugars) • Can use up to 30% of the glucose in the liver • NADPH is important for other pathways: o Fatty acid, cholesterol, neurotransmitter, and nucleotide biosynthesis o Reduction of oxidised glutathione and cytochrome p450 monooxygenases detoxification

Lecture 20: Metabolism – integration and regulation III: Fed, fasting and exercise states Robert Yang

Early fasting • Liver glycogen is the main fuel source to maintain blood glucose o Glucagon stimulates the liver glycogen breakdown, gluconeogenesis Gluconeogenesis involves lactate and alanine from ala and cori cycles o Ala cycle Amino group is removed and disposed of by urea cycle Carbon backbone is converted to glucose via gluconeogenesis o Cori cycle Lactate from glycolysis is converted to glucose via gluconeogenesis • Note: adipose tissue is intact and no ketone bodies are produced

Prolonged fasting • No fuel from diet and liver glycogen store is used up o Glucagon is high o Liver: undergoes gluconeogenesis and continues breakdown of glycogen. Ketone bodies are also produced o Skeletal muscles: undergo proteolysis releasing amino acids feeding into the ala cycle o Adipose tissue: lipolysis occurs releasing fatty acids and glycerol Hormone sensitive lipase is active o Brain: after 3 days, induces synthesis of enzymes that allow it to use ketone bodies (so it doesn’t die due to lack of glucose) • Adipose is broken down into fatty acids that are processed via beta-oxidation and ketone bodies are produced

Types of fuel during starvation • As phases of starvation continue: o Blood glucose goes from exogenous to glycogen breakdown to gluconeogenesis o Tissue using glucose goes from all to all except liver to brain, RBCs, renal medulla and some muscle to just brain, RBCs and renal medulla o Major fuel goes from: glucose to glucose and ketone bodies to ketone bodies • As starvation continues, plasma levels change: o Glucose decreases, free fatty acids increase, ketone bodies greatly increase

Fuel reserves • Notice: o 70kg man has 15kg fat, 6kg protein, 150g muscle glycogen, 75g liver glycogen, 20g circulating glucose o In a marathon, liver glycogen would last 18 minutes, muscle glycogen 71 minutes, adipose 4012 minutes, proteins 1786 minutes and free glucose 4 minutes

Oxygen consumption • Trends: o Skeletal muscle increases with work o Abdominal organs, kidneys, brain stay the same o Heart and skin increase moderately o Brain uses more oxygen than the heart at rest

Fuel use in late starvation (days 3-40) • Trends: o Glucose use by brain decreases o Ketone bodies use increases o Lipolysis doesn’t change, proteolysis decreases o Glucose output decreases and ketone body output stays the same

The starve-fed cycle • Humans eat regular meals during the day o After the evening meal, there is an overnight fast (enter a state of early fasting) o At breakfast, re-enter the re-fed state

Feeding • Early re-fed state – while insulin is increasing and glucagon is decreasing after a meal o Liver carries out glycogen synthesis Doesn’t use blood glucose Remains in gluconeogenic mode using glucose-6-phosphate produced from this process to make glycogen • Lactate and glucogenic amino acids are still used for gluconeogenesis As time progresses(insulin/glucagon ratio rises), rate of gluconeogenesis declines and liver uses blood glucose for glycogen synthesis

Feeding (continued) • Well fed state o Insulin is main hormonal stimulus o Liver Increased protein synthesis, increased glycogen synthesis and increased FA synthesis o Adipose tissues Increased glucose uptake, increased TAG synthesis and synthesis and secretion of LPL to breakdown VLDL TAG o Muscles Increased glucose uptake and increased protein synthesis o Cori cycle is interrupted and lactate instead is converted back into pyruvate before conversion to acetyl CoA for fatty acid synthesis

Exercise • Muscle contraction *** repeated from lecture 18 *** o Driven by ATP hydrolysis ATP is provided initially by phosphocreatine (4s) Next is usage of glycogen store by glycolysis • High exertion – anaerobic glycolysis producing lactate o Ie. with high exertion (sustained peak contraction), blood vessels are suppressed and oxygen supply decreased thus resulting in anaerobic metabolism o Lactate produced can be recycled using the Cori cycle • Choice of fuel is determined by intensity and duration of activity o ATP – used as a fuel for 1-4 seconds in vigorous exercise Eg: high jump, power lift, shot put, tennis serve ATP + H 2O 00> ADP + Pi o Creatine phosphate – used as a fuel for up to 10 seconds of vigorous exercise Eg: 100m sprints Creatine-phosphate + ADP  Creatine + ATP Either from diet or synthesised from amino acids (arginine, glycine, methionine) • Broken down into creatinine – a waste product o Glycolysis – used for up to 1.5 minutes Eg. 200-400m race, 100m swim Involves glucose lactate o Oxidative phosphorylation – hours Eg. race beyond 500m Use of TCA cycle o ATP, Phosphocreatine, glycolysis are anaerobic, oxidative phosphorylation is aerobic

Anaerobic metabolism • Useful for 1.5 minutes o Glycogen break down stimulated by adrenaline • Pathway: o Glycogen glucose pyruvate + ATP lactate • Examples: Sprinting or weight lifting • Found in type 2 muscle fibres o Low blood supply, few mitochondria o Low TCA cycle activity, mostly glycolysis and creatine kinases • Little organ cooperation – at peak contraction, blood vessels are compressed and muscle is isolated from body • Fuel o ATP o Phosphocreatine – high energy source of ATP used until glycogenolysis and glycolysis set in o Muscle glycogen – produces glucose for anaerobic glycolysis Anaerobic glycolysis – main source of ATP produces lactate • Before and after exercise o Muscle ATP decreases, muscle creatine decreases, blood lactate increases, blood pH decreases o Lactate, if gets too high can lead to lactate acidosis

Aerobic • Useful for hours o Makes use of the TCA cycle • Pathway: o Glucose pyruvate actyl-CoA oxidative phosphorylation o Fat acetyl-CoA oxidative phosphorylation • Fuel: o Production of pyruvate/acetyl CoA: Muscle glycogen (main) Liver glycogen Fatty acid breakdown from adipose tissue o Glucose is then broken down by TCA cycle and oxidative phosphorylation • Type 1 muscle fibres used o Good blood supply, active mitochondria o High TCA activity and beta-oxidation followed by oxidative phosphorylation • Note: o Low blood glucose causes a lower insulin/glucagon ratio leading to: Increase in glycogenolysis Mobilisation of TG from adipose tissues • TG is broken down by beta-oxidation and acetyl-CoA used to feed the TCA cycle • Any ketone bodies produced are used by the muscle The use of FA spares glucose: acetyl coA produced inhibits PDH allowing there to be glucose remaining after exercise has finished to ensure cell survival

Aerobic vs anaerobic summary:

Migratory birds • Fly non-stop over water for 2400km o Have a large store of fat (obese at time of migration) under skin, in abdominal cavity, in muscles and liver Use up 2/3 of this store in flight Fat store provides energy and water (to replenish that lost through respiration o Use fat instead of glycogen because: TAG catabolism provides more energy (glycogen storage of equivalent energy would be 6x heavier) TAG is anhydrous, glycogen is not Lecture 21: Blood glucose homeostasis and diabetes Tony O’Sullivan

Glucose homeostasis • Overnight, your blood glucose is maintained by gluconeogenesis in the liver o In diabetics with badly controlled blood glucose, blood glucose can increase overnight • Insulin o Hormone that binds to the transmembrane insulin receptor This activates intracellular action that activates glucose transporters (GLUT4 – active transport) o Varies throughout the day: Glucose levels rise after eating, insulin rises in response and causes glucose to drop Height of peak after eating is based on contents of the meal • High GI food causes a higher peak, low GI causes a lower, longer peak • Glucose o Can be utilised to produce energy (ATP) o Can be stored as glycogen/fat o Ratio of storage/energy utilisation depends on physical activity • EG: meal o 50% fat, 50% carbohydrates generally the fat is stored and the carbohydrates burnt as energy

Diabetes mellitus (DM) • Comparison: type 1 vs type 2 Parameter Type 1 – juvenile onset Type 2 – adult onset Prevalence <0.3% Up to 8% Onset <40 >30 Cause Insulin deficiency Insulin resistance Twins 50% concordance 90% concordance Insulin therapy Always 25% Obesity related No Yes • Note: o ½ people with type 2 diabetes don’t know they have it • Diagnosis: o Random glucose >11.1 mmol/L o Fasting glucose >7.0, 5.6-7 = impaired fasting glucose o 2 hour value >11.1, 5.6-7 = impaired glucose tolerance

Pancreas • Exocrine and endocrine function o Exocrine – digestion: enzymes etc o Endocrine- hormones (islets of Langerhans – sit within exocrine cells) • Removal: o Loss of endocrine and exocrine function

Type 1 diabetes: pathogenesis • Autoantibodies can be produced to: o Islet cells (Islet cell antibodies ICA) o Glutamic acid decarboxylase (GAD) o Insulin (Insulin autoantibodies IAA) o Only 60-80% of patients at diagnosis have these antibodies • Antibodies result in insulinitis which causes scarring of cells and cell death – thus decreased insulin production o Thus, there is a high fasting glucose level If patient eats, get hyperglycaemia (can’t uptake glucose into cell via the active transports, no insulin activation) Glucose is an osmotic diuretic resulting in polyuria

Aetiology • Genetic predisposition environmental trigger (virus?) loss of insulin secretion (6-12 month progression) o This leads to T1DM with marked hyperglycaemia • Insulin deficiency results in reduced anabolism (and increased catabolism), and reduced glucose transport o Thus also decreased glycogen synthesis in the muscles • Symptoms: o Hyperglycaemia, polyuria, polydipsia o Ketoacidosis coma, death

Ketone bodies

• Glucose insulin uptake, energy production • Fat can be used for storage or energy o If used for energy, ketone bodies are produced (in starvation, most are produced by the liver) • In insulin deficiency, there is no glucose uptake, so fat is used instead for energy o This causes production of ketones Ketones are metabolised by the brain, heart and skeletal muscle If there’s too much ketones than the body can metabolise, blood becomes acidotic – ketoacidosis • Ketoacidosis: o Symptoms: Polyuria, polydipsia, dehydration Sweet smelling breath (nail polish) Nausea, vomiting, abdominal pain o Causes 1st presentation of DMT1 Infections – GE, pneumonia Surgical procedures when nil by mouth (2-4 days) Non-compliance with insulin therapy

Insulin replacement • Mimic physiological secretion of insulin o Given in a basal-bolus regimen that mimics the pattern of insulin throughout the day o Involves short and long acting insulin doses

Type 2 DM • Associated with obesity o Obesity results in insulin resistance – thus there is a decreased biological response to insulin • Measurement of insulin response: o Give a set dose of insulin and ensure it remains constant Give glucose, and see how much is necessary to maintain homeostatic glucose levels – reduced in insulin resistance • Insulin resistance is a syndrome due to: o Impaired glucose tolerance (DMT2) o Central obesity o Dyslipidaemia (decreased HDL, increased LDL, increased TG) o Hypertension o Increased uric acid o Increased plagminogen activator inhibitor-1 • Many of these conditions are also risk factors for atherosclerosis o Many are interrelated and dependent on each other • Following insulin resistance, beta cells can become “exhausted” and this can result in insulin deficiency o Decline of insulin sensitivity is gradual, however, and T2DM is unlikely to experience ketoacidosis Management of DM • Diet o Hard in the context of western society o Need to ensure a good balanced diet to not exacerbate problem • Exercise o Non-strenuous (increased compliance) 5x30-40minutes/week o Advantages: Reduces body fat (esp. visceral fat) Increases insulin sensitivity (independent of body fat loss) Improves physical fitness • Education/monitoring of glucose levels • Oral medications (T2DM) • Insulin therapy (T1DM, 25% T2DM: ie, if become insulin deficient)

Prevention • Lifestyle change (diet and exercise) is the best way of preventing DM o Vs metformin (increases insulin sensitivity) and placebo o Only small amounts of weight loss are necessary for noticeable results

1st hand experience: interview • T1DM o Hypoglycaemia – body releases catecholamines to stimulate glycogen breakdown Tingling Sweating, loss of consciousness Nauseous o Ketoacidosis – due to GI virus Dehydration Breath/taste

Note: hepatic glucose output • Post-absorptive (fasting) – eg. while sleeping o Liver is major source of endogenous glucose production via glycogenolysis and gluconeogenesis • Post-prandial (following a meal) – eg. after eating o Glucose disposal is mostly as muscle glycogen

Lecture 22: Management of Diabetes Mellitus and its complications Tony O’Sullivan

Approach to management • Education – importance of monitoring and documenting of blood glucose • Dosage adjustment of medications and insulin • Regular review of complications and risk factors o Especially monitor CV risk factors • Multidisciplinary approach o GP, endocrinologist, psychiatrist, dietician, exercise physiologist, educators, podiatrist

Diet and exercise: see lecture before (21)

Monitoring • Methods: o Finger prick home glucose monitoring – blood from finger put on electrode o Glycosylated haemoglobin Glucose binds to HbA1c • Want levels <7% to prevent complications Changes to Hb occur slowly, thus reflects control over the last 2-3 months. Good measure for doctors to see how well patient DM was controlled (not just immediately before consultation)

Pharmacological therapies • Oral hypoglycaemic pharmacotherapies o Sulphonylureas – stimulate pancreas insulin secretion o Metformin – increases insulin sensitivity o Biguanides, eg. metformi (thiazolidinediones) – decrease glucose production in the liver, increase glucose uptake peripherally – increase insulin sensitivity o α-glucosidase inhibitors – slow the rate of carbohydrate digestion in the gut Metformin o Drug of choice especially for overweight patients Decreases appetite o Increases insulin sensitivity of the liver and muscles Lower HbA1c by 1-2% o Contraindications – renal failure (secreted in urine unchnaged) • Sulphonylureas o Increases insulin secretion by increasing the β-cell response to glucose Shorter action – glicazide (diamicron), glipizide (minidiab) Longer action – glibenclamide (daoninl), glimepiride (amaryl) • Higher incidence of hypoglycaemia, some active metabolites excreted by kidney o Renal excretion o Useless for type 1 DM • Combination therapy can be useful • Thiazolidinediones (rosiglitazone, pioglitazone) o Improve insulin sensitivity Reduce HbA1c by 0.5-1.4% o Side effects (rosiglitazone): Leads to fluid retention and adipose tissue generation • Thus increases hypertension and cholesterol increased CV events • Contraindicated in CV disease o Mechanism of action Unknown: thought to be a conscription factor activating GLUT genes and LPL genes • Thus increases glucose uptake and TG clearance

• Acarbose – α-glucosidase inhibitor o Glucosidase is an enzyme for breakding down carbohydrates Drug delays carbohydrate absorption thus reducing blood glucose post-meal Reduces HbA1c by 0.5-1% o AE: flatulence, diarrhoea (bacteria in colon use excess carbs and produce gas) Thus not popular drug

Insulin • Important factors: type, timing, delivery • Types: o Rapid acting: 1-2 hours, given before meal Nonrapid, humalog,apidra o Short acting: 2-4 hours Actrapid, Humulin R o Intermediate acting: 4-8 hours Humulin NPH, protophane o Longer acting: 18-36 hours Levemir, Lantus o Premixed Combination of injections to reduce injections Useful for children • Regimens: o Basal bolus Eg: short acting after meals, long/intermediate action overnight/+ one in the morning if need Can work out a flexible regime based on meal size and exercise etc o Twice daily – premixed Mostly used in T2DM One given at breakfast, one at night

Subcutaneous insulin pump • Mobile phone sized cartridge attached to a cannula that sits under the skin o Supplies a constant basal rate of insulin o Programmable to supply boluses with meals • Disadvantages: o Need to measure blood glucose levels accurately o Expensive o No as of yet integration between measuring blood glucose levels and adjusting dose automatically

Delivery devices • Disposable flexipens • Innolet – clockface to give ~units o Useful for elderly, those lacking dexterity, poor eye sight • Lantus solostar

Incretins • GLP-1 and GIP produced by the gut o Stimulate insulin production from the pancreas • Drugs work on these: o Exenatide (Byetta) – glucagon-like peptide analogue Given by subcutaneous injection • Long acting 1/week injection in development Presently not on the PBS AE: nausea, pancreatitis o Sitagliptin (Januvia)– prevents breakdown of incretins by enzyme Dipetidyl peptidase-4 Body weight neutral • Vs sulphonylureas that increase weight Incretins help regulate insulin secretion and glucagon secretion • By breaking down the enzyme, sitagliptin increases the action of GLP-1 and GIP thereby increasing the production of insulin

Going out with diabetes • Take insulin with you • Plan meals/snacks • Care with alcohol o Hypoglycaemia – missed meals and alcohol Similar effects to intoxication, can lead to seizures/coma o Hyperglycaemia – soft drinks o Hangovers – not eating, miss insulin • Driving – should measure BGL before • Recreational drugs o Stimulants increase appetite and increase metabolism – altering blood glucose o Increased physical activity, altered sleep patterns – interrupt regular blood-glucose patterns • Smoking

Complications • CV • Nephropathy o Glomerulosclerosis Increased creatinine/urea o May need dialysis • Neuropathy o Hyperglycaemia results in nerve damage • Retinopathy o Haemorrhages New vessel growth can easily haemorrhage o Can result in blindness • Foot ulcers o Loss of sensation, proprioception, dry skin o Sore can be unnoticed and become infected Complications: osteomyelitis, infection gangrene amputation • Peripheral vascular disease o Vascular supply is bad, ischaemia (combined with infection) amputation

Lecture 23: Diabetes: an indigenous perspective Laurie Clay *** check notes when put on web CT** Aboriginal medical services • A community health service set up and community controlled • Medicare: bulk-billed

Closing the gap • Mortality and morbidity, 20 year lower life expectancy than non-indigenous population

Aboriginal culture • Totems o Remnant from hunter-gatherer society o Each tribe has its own totem: an animal that it can’t hunt Prevents the wiping out of species

Bush medicine • Still currently used and practised o Eg. treat a cut with spiderwebs

Issues • Education – levels of reading o Reading/words are not as important in Aboriginal culture o Perceptions of words and meanings Eg: 1 tablet 3x/day • Diseases brought into the country • Men’s and women’s health • Beliefs o Borrowing tablets o Taking more tablets o AB vs metformin • Unemployment • Compliance and adherence o Not because don’t want to, but because they can’t • Doctor-shy/embarrassed • Chemist can be not face-to-face • Drug and alcohol – higher priority than food, medicine • Housing – overcrowding • Refrigeration • Confidentiality – DM, false visits • Obesity lifestyle problem: eating is a reward (from hunter gatherer culture) o Hard to change these habits • Mental health o Eg. schizophrenia is seen as a curse, not a disease • Need to read between the lines • Culture and shame o Eg. walking is seen as shameful, like you don’t have enough money to pay for the bus/car • Time and distance to hospitals and other healthcare services • Education in schools – a global service o Nutrition – breakfast clubs o Truancy

The way forward • Acknowledge past, embrace future • Take responsibility for own health • Money doesn’t help • Need community cooperation and collaboration o (NACHC, NHMRC)

Interventions into the communities • Elders • Support groups • Workshops

Lecture 24: Epidemiology of alcohol Robyn Richmond

The standard drink • One standard drink is 10g of ethanol o 375ml can or bottle of light beer (2.8% alcohol) o 285ml middy of regular beer (5% alcohol) o 100ml white or red wine (12% alcohol) o 60ml fortified wine (port, sherry: 40% alcohol) o 30 ml nip of spirits (40% alcohol)

Guidelines to reduce health risks of drinking • 4 Australian guidelines to reduce risks from alcohol consumption o Population health approach used focussing on health of entire community o Based on concept: risk of harm increase with amount of alcohol consumed Both amount drunk on one occasion and drinking on a regular basis Ie: the more you drink, the higher the risk o Don’t work to prescribe, but define risk and allow people to make informed choices Based on life-time risk of alcohol-related injury or disease due to: • Many drinking occasions (guideline 1) • Immediate risk in a single occasion (guideline 2) o Guidelines are for both men and women Also outline guidelines for pregnant/breastfeeding women More conservative than before • Guideline 1 o Aims to reduce risk of alcohol-related harm over a lifetime o Healthy men and women: no more than 2 standard drinks on any day reduces lifetime risk or alcohol- related disease or injury As the volume of alcohol consumption and frequency of drinking increases, lifetime risk of alcohol- related death increases • Risk triples when increase from 2 to 3 standard drinks/day • Guideline 2 o Aims to reduce risk of injury on a single occasion of drinking o Healthy men and women: no more than 4 drinks on a single occasion (a binge: ≥4 drinks in one occasion) As volume consumed increases, risk of injury increases • Risk doubles over 4 drinks in a single occasion • Guideline 3 o Children and young people below 18 should not drink o Parents and carers should not let children under 15 drink Ages 15-17, parents and carers should delay initiation of drinking as long as possible • Guideline 4 o Women who are pregnant or planning a pregnancy should not drink Fetal alcohol spectrum disorder (FASD) – caused by women drinking alcohol during pregnancy • These mothers often also use other harmful agents such as: nicotine, cocaine, heroin, solvents, methadone, marijuana • Many of these pregnancies are unplanned • Features: facial abnormalities, growth retardation, CNS damage, cardiac malformations o Can be difficult to diagnose at birth o Most clearly expressed at age 2-10 years • Occurs more frequently in disadvantaged communities and remote indigenous communities o Often affects siblings o Women who are breastfeeding should not drink Should avoid alcohol for first month until breastfeeding is well established After: avoid drinking directly beforehand, may need to consider expressing milk in advance Australia • Majority of Australians have tried alcohol o 90% in lifetime, 83% in past year, 41% weekly, 8% daily • Alcohol is more readily available now than 20 years ago: supermarkets + extending trading hours • Amount consumed in Australia has remained stable over time o However: pattern of consumption has changed – preference to pre-mixed drinks and spirits (especially young people)

Reasons for drinking • Social • Cultural • Religious • Pleasure, relaxation, mood alteration, enhanced creativity • Intoxication, addiction • Boredom, habit • To overcome inhibitions • To forget, to “drown sorrows”

Alcohol metabolism • Affects the brain in 5 minutes o Blood alcohol concentration (BAC) reaches peak in 30-45 minutes after 1 STD o Takes 1 hour for body to clear 1 STD • Rate of metabolism depends on: o Liver size, body mass, alcohol tolerance, genes (controlling expression of alcohol-metabolising enzymes), age, health conditions • Eating slows increase in BAC, food reduces alcohol absorption o Drinking coffee, cold showers, vomiting and exercise do not reduce BAC • With heavy drinking, it can take a long time for BAC to return to 0

Subpopulations at risk • Adolescents – binge drinkers o Alcopops – aim to start young people drinking at a young age Made so they mask the taste of alcohol and are easy to drink • Occupations o Risk industries: storage, fishing and hunting, air transport, labouring etc Corporate culture – alcohol expected to show clients a good time Celebrities – set bad examples Doctors too are drinkers The army/police force has a culture of drinking o Alcohol is the cause of a lot of absenteeism from work • Indigenous drinkers o Many do not drink, but those that do drink a lot more than the average population o Many more deaths due to alcohol in indigenous people than in non-indigenous poeple • Non-metropolitan areas o More deaths caused by alcohol in non-metropolitan areas than metropolitan areas • NT o Higher amount of alcohol drunk each year on average (15L vs 9L pure alcohol) o Consequences of drinking: Homicide, rape, domestic violence, aggravated assault, alcohol-related violence, crime

Effect of alcohol on health • Protective effects: o J curve – low levels of consumption, alcohol has health benefits Reduces risk of CVD and cerebrovascular disorders by ~30-50% Relevant from middle-age and older

• Other effects: o Depend on BAC: Trends: decreased inhibitions, loss of judgement, loss of coordination, nausea and vomiting, memory loss, vision problems, potential for aggression • Diseases: o Liver diseases: eg. cirrhosis o Chronic pancreatitis o Gastritis o Anorexia o Hypertension heart disease o Short term memory loss Particular important in teenagers – due to binges o Brain damage 1/8 Australians at risk of brain permanent brain damage (problem solving, visual working memory, cognitive flexibility, spatial planning) • Some already suffer from alcohol-related brain damage but don’t know yet o Cancers of: oropharynx, larynx, oesophagus, stomach, liver, bowel, breast, prostate, colon o Vasodilation and flushing o Loss of libido, impotence Alcohol increases production of estrogen in males • Male vs female: o Alcohol affects women faster than men because they have a higher fat/water ratio and they are generally smaller in size

Costs of drinking • Most of total cost to society is through: o Road accidents o Lost productivity • Makes up a significant proportion of healthcare costs: o Hospitalisations o Much injury is in young people • Other costs to society: o Psychiatric presentations, injuries, assaults, fire injuries, falls and drownings, car accidents, child abuse, suicides, industrial accidents

Lecture 25: Ethanol metabolism Mike Edwards

Ethanol catabolism • Ethanol is converted via 3 steps to acetyl-CoA which is then metabolised via the TCA cycle o 2 redox reactions: Ethanol +NAD + acetaldehyde + NADH + H + Acetaldehyde + NAD + acetate + NADH + H + o Reaction like first step in FA catabolism Acetate (a 2 carbon fatty acid) + CoA-SH + ATP acetyl-coA + AMP + PPi • Our bodies are not really designed to use ethanol as a major metabolic fuel o Enzymes that catalyse the reactions in ethanol catabolism are part of a family of isoenzymes that have much variation Variation accounts for different rates of alcohol clearance, degree of inebriation and susceptibility to alcohol-induced liver disease in the population • Catabolism occurs in different compartments: o Ethanol to acetaldehyde is almost exclusively liver o 90% of acetaldehyde formed in liver is transported into the mitochondria in the liver and converted to acetate o Liver is not very good at metabolising acetate and thus most is released into the blood Acetate is then broken down to acetyl-CoA in the mitochondria of body cells: especially sk muscle • Note similarity to ketone bodies: ethanol is an equivalent metabolic fuel – however acetate cannot be used by brain

Alcohol dehydrogenases – stage • 2 possible pathways to convert alcohol to acetaldehyde o Major: A family of cytosolic NAD +/NADH-dependent isoenzymes Different isoenzyme families have different specificities for specific chain lengths of alcohols

• Class I ADHs – highest specificity for ethanol (lowest K ms 0.05-4.0) o Most active in liver, adrenal, absent in brain and heart o 3 genes for class I ADH subunits (ADH 1, ADH2, ADH3), each with allelic variants Genetic differences account for observed differences in ethanol elimination rates in populations Genetic variation examples:

• ADH2*2 allele encodes for ADH with a relatively high V max (a high capacity to metabolise alcohol) o Associated with a decreased susceptibility to alcoholism – ie, breakdown faster and thus drink less o Also increases susceptibility to alcohol liver disease due to increased accumulation of toxic products o This allele is common in east asian populations, low in white Europeans • ADH2*1/2*1 genotype is a risk factor for developing Wernicke-Korsoakoff syndrome o Associated with alcoholism o Minor: MEOS (microsomal ethanol oxidising system) The more alcohol consumed, the more ethanol metabolised via this route • Normally accounts for 10-20% of ethanol oxidation in a moderate drinker Involves an enzyme: CYP2E1 – a cytochrome-P450 mixed function oxidase enzyme • Also part of the system to breakdown xenobiotics

Aldehyde dehydrogenase – stage 2 • Conversion of acetaldehyde to acetate • Accumulation of acetaldehyde causes nausea and vomiting o ALDH2*2 – a common variant has an increased K m for acetaldehyde (x260) and thus a decreased V max (x10) This means that this enzyme is very underactive until very high concentrations of acetaldehyde Homozygous people have absolute protection from alcoholism, ie: get sick very quickly when drink alcohol, and thus can’t drink much – can’t even enjoy a modest amount o Principle is the target for disulfiram

Acetate activation – stage 3 • Occurs away from the liver (mostly in skeletal muscle) o In the liver, this enzyme is cytosolic and is used for producing acetyl-CoA for cholesterol and fatty acid biosynthesis Thus, acetate entering these pathways is highly regulated and not much is used – thus enters blood o Heart and skeletal muscle have a mitochondrial isoenzyme allowing acetyl-coA to directly enter TCA cycle Enzymes are often GTP (rather than ATP) dependent

Acetaldehyde toxicity • Many toxic effects from chronic alcohol consumption are from accumulation of acetaldehyde o Highly reactive, spontaneously binds covalently and forms adducts with amino and thiol groups, nucleotides, phospholipids Ie: proteins, DNA, membranes – basically everything • 2 mechanisms of toxicity: o Formation of adducts with tubulin thus decreasing secretion of serum proteins (VLDL, lipoproteins, albumin) that are synthesised in the liver These products accumulate and cause an increase in osmolarity causing water influx and cell swelling • Thus results in loss of structure, portal hypertension and possibly ethanol-induced hepatitis o Adduct formation enhances free radical damage Reacts with glutathione compromising protection (a free radical scavenger, especially H 2O2) Damages mitochondria increasing free radical production

NADH/NAD + ratio • Increased NADH/NAD + ratio inhibits GNG o Ethanol catabolism increases the NADH/NAD + ratio o NAD + is needed to: Convert lactate to pyruvate Convert malate to oxaloacetate o Thus, with an increased ratio: Alanine that is converted to pyruvate is converted to lactate Lactate and glycerol GNG sources are interrupted Oxaloacetate is converted to malate o Thus, acute effects: Hypoglycaemia (no GNG), lactic acidosis (lots of lactate), ketoacidosis (ketone body production)

NADH/NAD + ratio • Increased NADH/NAD + ratio causes fatty liver o Fatty acid oxidation in liver is decreased Beta-oxidation requires NAD +, this is used in the mitochondria by acetaldehyde dehydrogenase and indirectly in the cytosol by alcohol dehydrogenase o Triacylglycerol synthesis is increased NADH is required for conversion of DHAP into glycerol 3-P and fatty acyl-CoA o Ethanol also causes a general increase in activity of all endoplasmic reticulum enzymes (proliferation of ER) These also favour TAG synthesis

Free radicals • Cytochrome P450 enzymes (eg. CYP2E1 from MEOS-pathway) produce free radicals as by-products . o Ethanol metabolism, hydroxyethyl free radical is produced (CH 3CH 2O ) • CYP2E1 activity varies: o Between individuals – 20x variation o Chronic alcohol consumption – 5-10x increase (+ 2-4x increase in other cytochrome P450s) This increase in enzymes causes ER proliferation • explains increased TAG synthesis • explains why with more drinking, more is catabolised via MEOS

Patterns of ethanol metabolism • Small amounts of ethanol are efficiently metabolised without significant MEOS involvement and without significantly elevating the NADH/NAD + ratio o In chronic alcohol consumption, MEOS activity is increased and a higher proportion of ethanol is metabolised by this route thus clearing alcohol from the blood faster Rate of acetaldehyde metabolism, however is not increased – thus leading to accumulation • MEOS activity causes: o Increased acetaldehyde accumulation adduct damage, free radicals o Free radicals from CYP2E1 activity

Hepatic fibrosis – a proposed model • Chronic alcohol consumption leads to an increase in acetaldehyde o Acetaldehyde activates Kupffer cells (tissue macrophages in sinusoids of liver) Kupffer cells release TGF-beta and produce ROS, NO via respiratory burst • Stellate cells are stimulated by TGF-beta and ROS o Stimulated stellate cells release extracellular matrix, collagen, and metalloproteases (model ecm) Thus leading to fibrosis (accumulation of CT) and loss of tissue function

Cirrhosis • At this point, liver injury is irreversible • Process: o Initially liver can be enlarged, full of fat and cross-linked with collagen fibres (fibrosis) o Later as function is lost, liver becomes shrunken • Loss of function: o Synthesis of blood proteins (+ tubulin protein release from liver prevented) o Urea cycle – leads to toxic accumulation of ammonia o Bilirubin metabolism and excretion – leading to jaundice

Nutritional factors • Alcohol-induced liver disease can occur in an otherwise well-nourished individual and other nutritional deficiencies can enhance disease progression o Alcohol beverages are empty calories – high on calories, low on essential nutrients o Ethanol ingestion can decrease GIT absorption of nutrients (eg. vitamins, essential fatty acids and essential amino acids) Thiamine – megaloblastic anaemia Pyridoxine – sideroblastic anaemia • Secondary malabsorption can occur through GIT complications and pancreatic insufficiency

Lecture 26 & 27: Risky drinking, alcohol dependence and management

Part 1: Prevention of risky alcohol consumption Robyn Richmond

Definitions of prevention • Measures that: o Prevent or delay the onset of alcohol use and abuse o Protect healthy development of children o Reduce harms associated with alcohol • Prevention aims to reduce probability of harm in individuals, families and communities • Paradox: o More harm is prevented by focussing on majority of people who are less seriously involved, but as a group have more problems vs individual high risk drinkers who have very large problems

Levels of prevention • Primary prevention o Aims to reduce to incidence/prevalence of alcohol misuse and problems related to drinking behaviours o Eg: Education about not driving with BAC >0.05 Designated driver Education about safe drinking levels and dangers of binging Media campaigns about unsafe sex related to excessive drinking Breathalysers tests on the road • Secondary prevention o Aims to diagnose early the symptoms of the disease (risky alcohol consumption) such that it can be treated early to slow progress and prevent complications Focuses on screening and early intervention o Egs: Brief interventions by GPs who identify problem drinking in patients Brief intervention in the workplace (occupational physicians) eg. people with regular Mondayitis • Tertiary prevention o Aims to rehabilitate and help patients adapt to their disability Particularly useful for chronic diseases: cirrhosis, chronic pancreatitis, cancer, FASD, CVD o Egs: Re-training in self=support skills Medications – pharmacotherapies prescribed by GP/specialist to aid in moderating heavy drinking Harm minimalisation – rather than quitting, minimising damage • Summary:

Prevention activities • Advocacy and lobbying • Legal control and policy formation • Expert advice and consultancy • Health promotion campaigns • Public education raising awareness • Community focussed strategies • Technologies that let drivers know they are over the limit • Treatment with counselling and pharmacotherapies • prevention is about regulation and law enforcement, not just education and persuasion

Primary prevention of risky drinking • Laws: o Alcohol tax Price regulation on alcohol o Regulation of licensed premises o Increased legal age of drinking • Drinks o Promotion of low alcohol beers as an alternative o Specify number of drinks on drinks • Advertising o Counteract alcohol advertising and promotion Change sponsorship of events by alcohol industry • Education o Education campaigns to raise awareness of risky drinking consequences o Programs for health education and health promotion (tv ads?) • Drink driving o Random breath testing o Designated drivers o Enforcement of zero BAC for probationary drivers, punishments for drink driving • Communities o Community and workplace interventions o Dry zones

Most effective: tax on alcohol • Shown to reduce social, health and economic costs of excess drinking in Australia o Reduces amount drunk, hospital admissions • Rates of taxation should be the same between and within beverage types o Based on alcohol content, rather than type of alcohol Ie: low alcohol beers should have lower tax because there is a lower alcohol content o Stop duty free alcohol • Money saved (from hospitals), and from taxes can be reinvested – hypothecation o Money can be used to: Fund prevention and treatment campaigns Fund research

Regulations - strategies • Regulate licensed premises o Reconsider distribution of liquor outlet density o Reduce availability of alcohol by reducing number of alcohol outlets o Restricting hours of opening Late trading hours (12-3am are associated with alcohol related violence) o Ensure a safe environment o Restrictions on access to alcohol to increase responsible serving of alcohol • Responsible service of alcohol o Training staff to recognise and act when a person is intoxicated o Avoid supporting irresponsible alcohol promotions o Staggering closing times to reduce numbers of intoxicated people gathering at one time o Replacement of drinking glasses with plastic o these reduce incidence of intoxication and subsequent alcohol-related harm • Policing o Currently police only attend a venue if there is a problem or disturbance Consider regular police checks

Low alcohol beers • Make up a small amount of the total beer market at present due to cost o Introduction of low alcohol beers has, however, decreased the amount of beer drunk, but increased the amount of wine drunk o However, has reduced morbidity and mortality of drinking

Advertising, laws and education • Alcohol advertising problems: o Minimal regulation on alcohol advertising and promotion o Aimed at young people o Contributes to drinking behaviour as well as attitudes to drinking o Includes: commercials, points of sale, internet marketing, marketing at young people’s venues • Prevention strategies targeting alcohol advertising: o Changing mentality such that it is not normal to drink alcohol in a risky way o Code regulating advertising o Strengthening of the regulatory system on tv advertising o Setting up of a system to allow complaints about tv advertising (like cigarettes) o Ensure equal say for community and alcohol industry • RBTs o Australia is the world leader o Used to target crashes at risky times of day Use highly visible vehicles Used in a variety of situations and incorporation with other traffic regulations o Success: Tests all drivers, high visibility of policing Public advertising and negative attitudes to drink driving Rapid and assured punishment On the spot licence loss

Preventing drink driving • Evidence for success: o Community awareness o Improved road conditions o Improved car safety • Other strategies: o Zero BAC for inexperienced drivers 0.05 BAC limit for full licence holders o Ignition interlocks that require breath sample before starting car Prevent starting of the car with a reading above 0.02 Used to prevent repeat drink-driving offenders from starting their car Use c an be ordered by magistrates in some Australian states o Education of learners o Gear stick know has a sensitive dour sensor that detects alcohol in the perspiration causing immobilisation of the vehicle and warning against drink driving o Odour sensors in the driver and passenger seats that give a warning if alcohol is detected in the air o Camera that detects drowsiness and issues alerts and tugs the seatbelt • Punishments for drink driving o Licence disqualification o Fines o Severe and repeat offence – prison sentence (no proven deterrent effect) o Increase drink-driving penalties o Treatment programs – combination of treatment and re-education programs with licence suspension Treatment reduces relapse (recidivism), more effective interventions with less severe negative consequences needed (eg. ignition locks, improved public transport) Early intervention programs in Australia • EGS: o Gatehouse project – promotes mental health through a multi-level school approach o Booze bus blitz – RBTs in Melbourne + satellite police patrol cars catching those avoiding the bus o Non-govt agency ARBIAS ran a campaign of awareness and provided treatment for alcohol related brain injury o Control of supply: ban of alcohol in NT Aboriginal communities, creation of dry zones o Drug education week – raise awareness of drinking during pregnancy o NSW Youth Alcohol Campaign – drink drunk the difference is U

Secondary prevention activities • Brief and early interventions o 5 As: Ask – identify the risky drinker Assess – assess alcohol dependence and readiness to change Advise – advise to cut down Assist – assist via motivational interviewing Arrange – arrange follow up

Part 2: Alcohol and public health Anthony Shakeshaft

Harms and benefits • Morbidity (DALYs) o Tobacco > alcohol > illicits o Indigenous > non-indigenous (especially in terms of alcohol, but not so much the others) • Diseases increase with consumption o J curve – applicable from middle age onwards Small amount of consumption reduces relative risk of CVD No health benefit for young people, only middle age onwards o Health risks CHD, DM, hypertension, congestive heart failure, stroke, dementia o Suspected reasons for J-curve: Insulin sensitivity increased, HDL cholesterol increased Glucose metabolism altered Antioxidants, platelet function o Related to alcohol – no specific drink

Individual vs population intervention • Number who drink too much is related to harm distribution in the whole population o If this is true, target whole population and reduce harm, thereby reducing number of individuals above the harm line o If this is not true, target individuals and specifically reduce harm o True for parameters such as: Body weight, BP, salt intake Alcohol if reduce by 2STD/week, 10% reduction in harm – therefore there is a relationship • Ideally, want a stepped intervention approach o Treat existing problem in population and target high risk o If solely target high risk, low effect because it is hard to shift a habit Much government money is spent on this o Prevention is better prevent people reaching this stage with whole population interventions • Types of drinkers o Only 5% at the top of pyramid are dependent, need to treat all, and reduce average harm

Things that work in the whole population • Taxation (hard to sell politically) o Different types of alcohol are taxed differently • Advertising bands • Minimum legal drinking age • RBTs • Licensing controls

Doctor prevention – what we can do • Primordial – decreasing underlying risk factors • Primary – advocacy for tax system, advocacy for decreased availability and alcohol restrictions • Secondary – brief and early intervention o 5 mins frequently and regularly, results in a 30% reduction o Problem is getting people to do it • Tertiary – treating dependent drinkers • Accident and emergency – hard to give intervention while drunk screen and mail intervention?

Self-reporting prevalence • Long term drinkers 10% • Binge drinkers – drinking to intoxication o Last month 30% o Last 12 months 40%

Harm • Crime o Assault, malicious damage • Hospitalisations o Alcohol disorders (long term problems) o Acute intoxication • Traffic crashes

Treatment for the individual • 20% of people are found on screening (by GPs) o 64% receive intervention • Problems: o Doctors respond to late complications rather than early signs o Doctors don’t quantify ‘social drinking’ etc o Stereotype resistant alcohols who don’t respond to intervention • Ways to improve: o Quantify every patient Standardised question tests – Audit o Don’t worry about blood tests o Assistance if intervention not working

Part 3: Dependency and management of patients with alcohol problems Robert Graham

Thorley’s balls • Alcohol dependence, alcohol problems and excessive drinking are interlinked

Alcohol problems • Physical health o Alcohol affects all body systems When younger – acute, older – chronic • Some chronic problems can present earlier, others only with end stage disease Dose-response relationship Cirrhosis is a major problem • Mental health o Cause or effect? Alcohol causes more depression than the other way around Alcohol often exacerbates depression and other MH problems • MH problems improve without treatment if patient abstains o Depression is an important mental health issue o Need to treat both MH and alcohol problems • Relationships o Family, marital, social isolation • Employment – can lose job • Finance o Not just alcohol, also other associated costs • Legal o Drink driving – often an early warning sign o Assaults o Indecency in public places • Homelessness – affects few, but is very visible

Alcohol dependency – features • Narrowing of drinking repertoire – lose ability to vary beverages o Like a particular brand of beer etc o 1 is too many, a thousand too few – once get started, can’t drink enough Need abstinence to treat, controlled drinking will be ineffective • Salience drink-seeking behaviour o Alcohol is more important than anything else and other priorities/responsibilities • Subjective awareness of compulsion to drink o Drinker has very strong cravings • Withdrawal o Symptoms: Anorexia, nausea, vomiting, dry retching Excessive sweating, tremor Profound dysphoria anxiety + depression If need a straw to get first drink – indicator of severe dependence • Relief of withdrawal symptoms with alcohol o Time of first drink is important – drink becomes earlier and earlier based on dependence • Tolerance o Drink more and more to get same effect, or get a smaller effect from the same amount • Reinstatement after abstinence (relapse) o If relapse after many years, symptoms return quickly and thus quickly return to previous drinking levels Only takes months instead of years o Due to a change in the hardwiring of the brain? o In old age, people solve the problem by themselves by their own resolution reduces harm significantly • Persistent desire/ efforts to reduce/stop o Recognition of problem • Using despite harm – physical, psychological, social Importance of alcohol dependence • Important to be less judgemental – it is a medically defined problem • Spectrum of severity – mild to moderate • Biological and psychological elements – both are interrelated

Alcohol abuse • A maladaptive pattern of alcohol use causing clinically significant distress or impairment of social or occupational functioning • Defined by one or more of: o Failure to fulfil major role obligations o Exposure to physical hazards o Legal problems o Social or interpersonal problems • Controlled drinking is a valid tool in this situation

High risk drinking patterns • Relentless excessive – rarely intoxicated o Chronic disease main risk o Behaviour disturbances are rare • Sporadic excessive – often intoxicated o Acute trauma main risk o Behaviour disturbances are common • Combinations of the two • Pathological drinking: secretive, alone, hiding alcohol, denial of drinking

Harm reduction • Aim to reduce health, social, economic costs without necessarily reducing consumption o Some are unwilling to stop, or reduce consumption – need to reduce harm • Better to aim for suboptimal goals than utopian unrealistic ones – ie. get 90% of something rather than 100% of nothing • EG: road trauma o Well lit roads with big signs o Safer cars – belts etc

Alcohol industry vs public health • 80:20 rule – 20% of drinkers drink 80% of the product • Industry often opposes important public health measures o Politically powerful • Similar to gambling, smoking, food packing industries

Clinical • History, examination • Biological markers (important in combination) o Increased LFTs, AST>ALT, γGT increased o Low urea, K +, Mg 2+ o MCV – mean cell volume o Platelets o INR – international normalised ratio

Management • Brief intervention o Facilitative, advice, goals, follow up • Get patient to look at pros and cons of drinking/not drinking • Get patient to make own goals (short term + reassess) o Options: same, cut down, abstain • Set start and finish dates • Avoid being directive Controlled drinking • Establish drinking days each week o What and how much • Not recommended if life threatening disease – need abstinence • Drinking diary o Prospective and retrospective (how much planned to drink, and how much did drink) o Document behaviour/triggers for drinking • Strategies to controlling drinking: o Sip don’t gulp o Take hand off glass between sips o Plan amount, and when? o Don’t quench thirst with alcohol – start with non-alcoholic drinks o Alternate alcohol/non-alcoholic • Other: o Identify non-drinking ways to achieve the same thing o Take up hobbies o Reward and punish if meet, don’t meet goals o Review and monitor

Abstinence • Preferably used in older, moderate-severe dependence • Refer alcoholics anonymous o Self-help o Reference to higher powers • SMART recovery – CBT • Strategies: o Keep a drinking diary o Offer detoxification Process of withdrawal syndrome • Not treatment – predictable response • Prelude to treatment Can be done at home, in hospital, with medications etc (valium, diazepam) Like a pit stop before attempting to abstain

Medications • Acamprosate (campral) o Blocks subtype of glutamate receptors (NMDA receptors) 2x chance of abstinence by 12 months o Side effects – head ache, abdominal bloating, diarrhoea, normally well tolerated If gradually introduced, SE reduced o PBS listed • Naltrexone (revia) o Blocks release of endogenous opioids Prevents gratification and thus reduces cravings o SE – headache, diarrhoea, abdominal discomfort o PBS listed • Disulfiram (antabuse) o Causes acute sensitivity to alcohol Inhibits ALDH and causes accumulation of acetaldehyde o Last resort – use for resistant patients, avoid in frail patients o Needs supervision – spouse, clinic etc o SE – flushing, hypotension, syncope, impending doom o Not on PBS • Drugs are on the whole underused o Dose for 6-12 months o Need combination with a comprehensive alcohol management plan Good counselling and follow up is very useful Lecture 28: Introduction to drug chemistry and absorption Ross Grant

Revision • Pharmacokinetics o What the body does to the drug Absorption Distribution Metabolism Excretion • Pharmacodynamics o What the drug does to the body The effect on target receptor

Cell membranes • Absorption, distribution, metabolism and excretion of drug involve passage across cell membranes • Cell membranes are made up of the phospholipid bilayer o Phospholipids are made up of a hydrophilic head and two hydrophobic tails • Drugs and cell membranes o For transport (ie in the blood) drugs need to dissolve in water, to go between different compartments, need to dissolve in cell membranes (fatty) o Drugs traverse cell membranes in 4 different ways: Pinocytosis • Part of cell membrane encloses drug and is retained as it is taken intracellularly o From here, there is intracellular fusion with lysosomes to release contents • Not a major drug delivery method Passive diffusion • Drugs can have the ability to dissolve in fat and water • Major drug delivery method Filtration • Used for molecules <100 da o Move through pores • Not major delivery method for drugs Active transport • Can pump things in or out of the cell o Eg: p-glycoprotein can pump drugs back out of the cell

• Passive diffusion: o Most drugs pass through membrane by passive diffusion of unionised molecule o Rate is dependent on the lipid-water partition coefficient Amount a drug dissolves in lipid vs water Drug accumulates in membrane until this ratio is found at both the extracellular and intracellular borders • Eg: 20:80:20 • Want a higher L-W partition coefficient to get more drug inside a cell

pH effect on drug absorption and distribution • Most drugs are either weak acids or weak bases o Thus they easily lose/gain protons to become charged ions o Charged ions easily dissolve in water (due to water’s dipole) but not easily in lipid Ionised molecules do not cross membranes easily o EGS: Acids, with carboxyl groups that can lose H + – aspirin, penicillin Bases, with amine groups that can gain H + – Morphine, Nicotine, amphetamine: MDMA (ecstasy) • Changing the pH affects the ionisation of drugs o Mantra: pH < pKa drug will be protonated acid: unionised, base: ionised • pH of the GIT varies, thus drugs are absorbed in different areas o stomach: pH = 1.3, duodenum: pH = 6, small intestine: pH = 7.8 o CSF = 7.35, serum = 7.4, saliva = ~6, urine = ~5 • Theory of mantra: o Henderson-Hesselbach equation BH +(acid) B (base) + H + [] pH = pK a + Log ( ) []

• pK a is the pH at which half the drug is ionised • EG: diazepam (weak base) o Below pH 3, ions accepted onto ammonia group Thus most of drug is ionised in the stomach and absorption occurs further down the GIT • EG: aspirin (weak acid) o Above pH 3, protons donated, thus in the stomach exists in a non-ionised form and is absorbed

• Summary: o Changing the pH affects ionisation of drugs o Ionised molecules don’t cross membranes easily o pH < pKa then drug protonated, acid – unionised, base – ionised

Drug absorption and distribution • EG: MDMA o 3,4-methylenedioxymethamphetamine pKa of ~9.9, basic • Proportionally more ionised at pH < 9.9 • Thus, not much absorbed in stomach, most in the small intestine • pH may not be the major determinant in drug absorption in the SI (large SA) o May only need 0.1% of drug in non-ionised form to sufficiently absorb drug o Also, passage of drug in the SI may be slow enough for complete absorption

Drug distribution • Depends on lipophilic-hydrophilic partitioning o Pharmacodynamic effects may not occur until drug reaches target tissue • EG: MDMA o Target – 5-HT, NE, DA membrane transporters in CNS o Clinical effect takes ~15-30 minutes 1.5 hours peak plasma concentration • Increasing the speed of distribution: increase the pH of stomach o Increasing speed of excretion: decrease the pH of the kidneys Reabsorption if unionised, ionised is excreted (not re-uptaken into blood from kidney tubules)

Absorption and distribution of EtOH • pKa = 15.9 o thus, doesn’t donate protons at physiological pH • Peak blood EtOH occurs in about 60 minutes o Depends on: Speed of consumption, Presence of food Rate of gastric emptying Body habitus – physique • Women have a smaller volume of distribution: higher fat:water ratio, thus attain higher blood EtOH faster o This is because EtOH is water soluble Most drugs are lipophilic, thus in these cases, the reverse is true o Older, fatter people have a higher volume of adipose tissue, and thus drugs have a higher half life

Other factors affecting drug access to cells • Molecular size and shape • Degree of ionisation • Relative lipid solubility of ionised and non-ionised forms • Binding affinity to receptor proteins (membrane transport) • Extracellular concentration o Le Chatelier’s and Fick’s: things move down concentration gradient, the higher the gradient the faster the diffusion

Methods of administration • Oral o Advantages Most common, convenient, safe and economical o Disadvantages Limited absorption (some drugs) Emesis due to GIT irritation Digestive enzymes, low pH may destroy some drugs Irregular absorption due to variables such as food/other drugs Metabolism by intestinal flora • Can decrease drug concentration, can convert to toxic metabolites/carcinogens Methods of administration (continued) • Intravenous o Advantages Absorption is circumvented Complete/rapid bioavailability Controlled and accurate drug delivery Suitable for large MW and irritant drugs (eg. proteins that would be digested in GIT) Useful in emergencies o Disadvantages Increased risk of adverse effects – miscalculation, poor response, anaphylaxis Not good for oily/insoluble substances – get formation of oil droplets that can block small BVs Septicaemia – due to infection • Intramuscular o Advantages Rapid absorption (allows oil to diffuse) Reduced risk of septicaemia – has first line defence measures o Disadvantages Increased absorption can occur if muscle injection site is exercised and blood flow increased o Injection into commonly the vastus lateralis (quadriceps), deltoids, or gluteus maximus • Subcutaneous o Rate of absorption is constant and slow – gives a sustained effect o Only suitable for non-irritating drugs otherwise can cause an erythema that will affect rate of absorption • Transdermal (percutaneous) o Easy to apply o Only useful for a limited number of applications – epidermis is relatively impermeable (stratum corneum) Works better if moist o Factors: Drug concentration, surface area, drug attraction to skin vs to vehicle, vehicle, skin hydration, rubbing or inunction, thickness of stratum corneum, time of application, multiple applications • Per rectal (vascular) o Advantages: Useful when oral ingestion is hard Approx 50% drugs avoid 1 st pass metab (liver) o Disadvantages: Absorption can be irregular/incomplete Can cause irritation of rectal mucosa Bacteria can metabolise drugs • Sublingual o Adv: good absorption of high lipid soluble drugs (eg. nitroglycerin for angina), avoid 1 st pass metab o Disadv: small area for absorption, some drugs have unpleasant taste • Pulmonary o Gaseous and volatile drugs inhaled and absorbed, large SA rapid access to circulation Avoids 1 st pass metabolism o Dosage variability – overcome by metered pumps o May cause irritation • Ophthalmic o Mostly used for local effects, drug can go systemic with drainage via nasolacrimal canal Avoids 1 st pass metabolism o Local effects need absorption via the cornea, corneal infection/trauma can increase absorption Suspension and ointments may prolong duration of action and minimise drainage to systemic • Route of administration changes the serum concentration of drug o Oral rises due to absorption then decreases due to distribution and metabolism/excretion o Intramuscular is similar but absorption is much slower, and metabolism/distribution slower o IV starts high but decreases rapidly due to distribution and metabolism

Lecture 29: Drug metabolism Ross Grant

Bioavailability • The proportion of administered dose that reaches the systemic circulation intact, and thus to target tissue o Barriers to pass in most routes are: GIT absorption, 1 st pass metabolism (portal vein drainage to liver)

Phase 1 and 2 metabolism • Drug metabolism involves either of two important changes to drug: o Biotransformation and excretion – make drug inactive o Polarisation of drug and excretion – make drug polar EG: lipophilic drugs circulation liver for processing, made polar kidney (excretion) • 1st pass metabolism o Metabolism by the liver after drainage from the GIT via the portal vein before entering the systemic circulation o Eg: Statins, as much as 95% is lost to liver metabolism • Most drug metabolism occurs in the liver o Most drug elimination is in the urine • Drug metabolism occurs through phase I and Phase II reactions o May have multiple of either, and may not necessarily be in order phase 1 phase 2 Both are fairly generic reactions and are capable of dealing with many different compounds • Phase 1 metabolism o Occurs in the endoplasmic reticulum o Functionalisation reactions – changes function by changing functional groups o Involves Cytochrome P450 enzyme system ~1000 known CYP450 enzymes ~50 are functionally active in humans Important ones: CYP3A (metab~50% drugs), CYPE1 (alcohol), CYP2D6 (~25%) • Phase 2 metabolism o Occurs in the cytosol o Conjugation reactions – add a functional group

Examples • MDMA (ecstasy) o Phase 1: demethylation Phase 2: methylation • Phase 2: conjugation (glucouronide/sulphate) o Urine excretion • Caffeine o Combination of phase 1 and 2 with various pathways

• Alcohol (EtOH) o Eliminated at a rate of 7-10g/hour Depends on: blood [EtOH], [metabolising enzymes], efficiency of excretion Rate limiting factor is NAD + (cofactor) in the reaction alcohol acetaldehyde o Zero-order kinetics Rate of drug elimination is constant and independent of plasma drug concentration A small increase in dosage results in a disproportionate increase in plasma drug concentration • Moderate alcohol levels are 1000x greater than in non-alcohol consuming persons o Note: hangovers are due to accumulation of acetaldehyde, NAD + is also important in DNA repair and longevity genes • Overall, absorption/distribution metabolism excretion is controlled by phase I and phase II enzymes o The order and pathways can vary

Factors affecting drug metabolism • Age o Children have a decreased fat/water ratio o Newborns when converting Hb to adult Hb, porphyrin needs glucuronidation if doesn’t work get excess bilirubin and thus jaundice o Increased age: Reduced CO reduced blood flow the liver decreased drugs entering the liver decreased drug metabolism and increased action of drug Decreased gastric emptying time decreased liver metabolism higher fat/water ratio better ability to store fat soluble drugs • Gender o Water ratio o Pregnancy – increase in drug metabolising enzymes • Genetic variation (pharmacogenetics) o Individual genetic makeup partially determines the response to a drug • Induction/inhibition of drug metabolising enzymes • Nutrition o Undernourished can result in decreased drug metabolism (especially in protein defieicny) • Drug/drug interactions o >4 drugs at once can reduce metabolising ability of liver • Disease o Decreased CO, decreased kidney function, hypo/hyperthyroidism changing basal metabolic rate o Viral infection – can increase and decrease basal metabolic rate

Induction/inhibition of CYP450 enzymes (phase 1) • Inhibition o Inhibition of enzymes o Decreases metabolism of drugs and thus increases the drug concentration for the same dose Thus there is potential that this could exceed the toxic dose o EGS, inhibitors of CYP3A: Antibiotics: clarythromycin, troleandomycin, erythromycin Antihistamines: cimetidine Antifungals: fluconazole, ketaconazole, itraconazole Herbal medicines etc: grape fruit juice • Induction o Increased gene transcription o Increases amount of metabolising enzymes thus increasing metabolism of drugs and thus decreasing the drug concentration May result in lack of therapeutic efficacy o EGS: Induction of CYP3A: • Anticonvulsant/mood stabiliser: carbamazepine • Antibiotic for TB: rifampicin, rifabutin • Antiretroviral: ritonavir • Herbal medicine: St John’s wort(for treating depression) Induction of CYP1A2: tobacco smoke Induction of CYP2E1: alcohol

Pharmacogenetics EG: • Poor metabolism of nortriptyline debrisoquin o Due to deficiency in expression of CYP2D6 (gene as ~80 allelic variants) Autosomal recessive 6-10% of caucasians, <1% Asians, 2-5% Africa-americans • Most common mutation is the CYP2D6*3A allele with a single adenine deletion in exon 5 o Treatment – reduce dose • Type of metabolisers: o Extensive metabolisers – normal people o Poor metabolisers – decreased activity o Ultra-rapid meatbolisers – 5-6 number of copies of gene, increased metabolism and reduced clinical benefit from drugs

Renal excretion • Process: o Glomerular filtration – glomerulus Molecules <60 000da o Active tubular secretion – proximal tubule Anions: non-specific transport systems Can be saturated, drugs can compete Passive tubular reabsorption – proximal and distal tubulesAffected by concentration in filtrate and lipid solubility (affected by pH) • Effect of pH o Urinary pH fluctuates between ~4.5-8 Acidic drug: [AH] [A -] + [H +] • Decreasing the pH increases concentration of non-ionised molecule, and thus less excreted Basic drug: [B] + [H +] [BH +] • Increasing pH increases concentration of non-ionised molecule, thus less excreted • EG drugs o Bases cleared rapidly with acidic urine: amphetamine, chloroquine, imipramine, levorphanol, mecamylamine, quinine o Acids cleared more rapidly with alkaline urine: acetazolamide, nitrofurantoin, Phenobarbital, probenecid, salicylates, sulfathiazole Lecture 30: Confidence and clinical significance Dr Rachel Thompson

See lectures 16 and 24 of HMA

Lecture 31: Viral hepatitis Peter White

Hepatitis presentation • All hepatitis viruses replicate in the liver and thus have similar clinical presentations • Incubation period – 1-6 weeks after exposure o Acute hepatitis with marked onset of fever, headache, weakness, myalgia, anorexia Followed by: dark urine, jaundice, clay-coloured faeces o Physical examination Enlarged, tender liver

Definitions • Hepatitis – elevated serum amino transferases o Acute – short term/severe; typically – malaise, anorexia, vomiting, right upper quadrant pain o Chronic – lingering or lasting >6 months – defined by liver function test abnormal without cirrhosis o Fulminant – quickly developing, high mortality o Cirrhosis – scarring o Jaundice – yellowing of skin/eyes due to raised levels of bilirubin

Causes of hepatitis • Drugs • Manifestation of systematic illness • Connective tissue disease • Viruses o Hepatitis A, B, C, D, E o EBV, HCMV, HSV, HHV6, yellow fever virus

Viruses – overview • All viruses are from different families and are not related except in that they cause hepatitis o A and E are related – faecal-oral transmission, only cause acute infection o B and D are related – parenteral transmission, D is dependent on B for replication o C – parenteral, no vaccine available • Other trends: o All have long incubation periods o All are RNA viruses except for HBV • Serum diagnosis: o HBV – IgM core, others – IgM (+) o Liver function tests: Peak ALT is important, often ~1000, normal <30. • Epidemiology overview: o HCV more prevalent than HBV in USA and AUS, reversed in developing countries

Hepatitis C virus (HCV) • Initially known as hepatitis non-A non-B • Virology o Flaviviridae family (also includes yellow fever virus, Dengue fever virus) o 6 genotypes that have geographical distribution Virus mutates readily Some genotypes are easier to treat than others • Genotype 1a/1b makes up 70%, type 3 20% • Australia – 1a, 1b, 3 are most prevalent Can have co-infection with 2 viruses, often one will become dominant • Epidemiology o 200 million chronic carriers in the world Australia, India and US, 1-2% of population infected o Most common reason for a liver transplant in Australia and USA • Transmission o Parenteral NOT via arthropod vectors (like other flaviviruses) IVDU, haemophiliacs, recipients of unscreened blood transfusions • Screening of blood products from 1990 decreased transmission Sexual and mother-to-baby not common (requires blood, so MSM) • Average rate at delivery is 6% (higher with HIV co-infection: 17%) o Not associated with delivery method, breastfeeding • Infected infants do well – severe hepatitis is rare • Morphology o Spherical, 50nm diameter o Lipid envelope • Genome o ssRNA that codes for: protease (to break up polyprotein), RNA polymerase, envelop proteins (E1 and E2), core capsule o Highly variable nucleotide sequence – can escape immunity/drugs easily Some share only 60% of nucleotide sequence o High mutation rate can easily result in several populations of the virus in the same person – quasi-species Diversity allows easy adaptation of change • Primary infection o Viraemia is at its highest at primary onset Antibodies appear 6-12 weeks after hepatitis onset • Seroconversion can take up to a year o Liver damage is immunologically mediated – thought to be due to activation of virus specific CD8 cells • Disease course (untreated) o 70% infected progress on to chronic hepatitis Many of these are asymptomatic (75%) Other 30% resolve disease o After 10-30 years of chronic hepatitis (which is often clinically silent) 30-50% will develop cirrhosis This has a risk of 2-6%/year for hepatocellular carcinoma o Spontaneous resolution of chronic infection is rare (<2%) o 6% of HCV +ve people die from infection

Hepatitis C virus (continued) • Serological patterns o Measure ALT (alanine transferase) levels – indicate lysis of liver cells o Acute HCV with recovery (30%) Rise in ALT after incubation period lasting as long as symptoms, then drop to normal levels as Anti- HCV appear o Progression to chronic infection (70%) Rise in ALT after incubation period which drops off but never full returns to normal • Periodic flare-ups cause spikes in ALT levels

• Diagnosis o Serology (not useful in acute phase – no antibodies) EIA for proteins – measures infection, not immunity HCV load – a predictor for therapy response (low titre often gets a better response) • Also used to monitor how treatment is progressing • Management o Assess biochemistry for chronic liver disease o Assess severity of disease and decide appropriateness of treatment Vaccinate against HBV, HAV o Counsel to maximise liver health Limit/abstain from alcohol • Treatment o Pegylated-interferon + ribavirin Sustained response in 50-80% of people, can’t cure 20% of people Reduces chance of progression to HCC o Viral factors: Old, male, fat – worse chance to clear Race, genotype (type 2-3 respond better), quasispecies, viral load o Viral load can be analysed periodically to assess efficacy of treatment (want a 2 log loss) o No vaccine

Hepatitis A • Epidemiology o Most common cause of hepatitis in developing countries (endemic) 90% of 5 years olds have caught disease o Faecal oral spread, very contagious o Prevalence correlates with sanitary standards • Classification o Similar genome to rhinovirus • Clinical importance: o Clinical presentation is variable: Many asymptomatic: 90% childhood infections, 25-50% of adult o Incubation period 10-50 days Self-limited infection with no viraemia – no liver damage Can get liver damage in some cases – occurs after viral replication has stopped o 99% cases recovery, 0.1% cases – fulminant disease • Morphology/genome o ssRNA, forms a large polyprotein that is cleaved sections: protease, capsid, RNA polymerase o naked capsid virus, spherical – 28nm • Diagnosis o Not distinguishable from other viral hepatitis o Liver function tests (ALT/AST) o Enzyme immunoassays (EIA) Detection of HAV-IgM antibody – indicates present infection • Vaccine o Inactivated HAV vaccine (Havrix, Vaqta) o Safe, well tolerated and effective 95-100% have protective levels of antibody within 1 month of first dose, nearly 100% protective in healthy people o Previous therapy: administration of normal human immunoglobluin

Hepatitis E • Epidemiology o If found in developed countries, most are from history of travel of HEV-endemic areas o Prevalence follows levels of sanitation Ie: endemic in Africa, India, Asia, Middle East, South America o Has relatives that infect monkeys and pigs • Transmission o Faecal-oral transmission Waterborne spread is particular important o Less contagious than HAV • Normal course results in acute infection similar to HAV o Exception: in pregnancy (3 rd trimester) causes 15-30% mortality for mother and baby • Diagnosis o IgG and IgM HEVAb by EIA • Morphology o Similar to norovirus, naked capsid virus

Hepatitis B • Epidemiology o Kills 1 million/year, 2 billion infected world wide o Often related to HIV infection due to sexual transmission • Transmission o Serum – blood transfusions, transplants, IVDU o Sexually o Mother to child • Incubation period of 45-120 days • Morphology o Spherical enveloped particles 42-47nm o DNA virus with 1.5 strands • Genome o Genes: Core protein (c), polymerase (p), surface antigens (s), transactivator of viral transcription Note: also: “e” antigen which is produced as part of “c” protein, but is released into circulation during active replication o If someone is HBeAg +ve, want to induce seroconversion to clear infection • Pathogenesis o 3 outcomes: Acute course recovery and immunity (90% clearance in the adult) Chronic infection carrier state with viral persistence (10%) Fulminant hepatitis (~1%) with liver failure and death o Acute infection has 3 outcomes: Subclinical, icteric hepatitis, fulminant hepatitis o Chronic infection has 3 outcomes: Asymptomatic, chronic persistent, chronic active cirrhosis HCC

• Treatment o IFN alpha – 16 week course o Lamivudine – 12 months (nucleoside analogue) o Adefovir dipivoxil – rescue therapy with LMV resistance • Prevention o Recombinant HBsAg vaccine made in yeast

Hepatitis D virus • HDV is a defective transmissible pathogen that is dependent on HBV for replication o 10 million infections worldwide o Increases the chance of fulminant hepatitis (10x) • Features o ssRNA, circular, lacks RNA polymerase o Infectiosn results from co-infection (both HBV and HDV)or superinfection (HBV already present) • Treatment is by treating HBV infection Lecture 32: Hepatic physiology Karen Gibson

Functional anatomy • The liver is made up of lobules o Hexagonal in shape and 0.8-2mm in diameter, several mm thick o Centrally: central vein o At each corner between lobules: portal spaces which contain – portal veins, hepatic arteries and bile ducts • Liver cells – hepatocytes o Arranged in cellular plates that radiate from the central vein Cellular plates are 2 cells thick Surrounded by sinusoids At centre have intralobar bile ducts • Bile canaliculi – formed from a specialised part of the hepatocyte cell membrane o Run between adjacent cells and drain into the intralobar bile ducts interlobar bile ducts in portal space o Bile flows in a centrifugal direction through the lobule • Sinusoidal blood comes from: o Portal venules (venous outflow of GIT) o Hepatic arterioles Also supplies arterial blood to septal tissue and the biliary duct system Empty into sinusoids 1/3 distance away from fibrous septa (within hexagon) o Blood flows in a centripetal direction towards the central vein of the lobule hepatic veins IVC Time in sinusoids is ~8.4s • Relationships between sinusoids and hepatocytes o Blood easily contacts hepatocytes Large gaps in endothelial cells of sinusoids (pores up to 1um diameter) • Allows passage to large plasma proteins into space of Disse and near hepatocytes Hepatocytes are directly adjacent to the sinusoid • Kupffer cells line sinusoids • Space of Disse o Narrow tissue space beneath the endothelial lining o Connects with the lymphatic vessels in the interlobular septa • Bile o Made in hepatocytes, enters canaliculi intra/interlobular bile ducts Leaves the liver via the L and R hepatic ducts common hepatic duct • Joins the cystic duct from the gall bladder forming the common bile duct o Enters the duodenum at the duodenal papilla (often unites with main pancreatic duct here) Entrance is protected by the sphincter of Oddi

Liver blood flow • Liver has a dual blood supply o 1000ml/minute from portal vein Portal venous pressure ~10mmHg o 500ml/minute from the hepatic artery Mean hepatic arterial pressure ~90mmHg • There is a pressure drop in the hepatic arterioles before entering the sinusoids such that blood flows into the sinusoids and not back out through the portal vein o The pressure in the portal vein is higher than in the sinusoids Resistance in the sinusoids is low, central vein pressure being ~0mmHg o This pressure drop adjusts such that an inverse relationship is established between hepatic arterial and portal venous pressure Mechanism: adenosine accumulation • Produced by hepatocyte metabolism at a constant rate • Increase in portal blood flow washes away adenosine and decreases hepatic blood flow • Decrease in portal blood flow causes adenosine accumulation and increased hepatic blood flow due to a vasodilator effect on the hepatic arterioles (via vasoconstrictor innervation) • Oxygenation zones o Blood flow within lobule is not uniform Hepatocytes closer to the portal space get the best blood supply (zone 1) • Important for oxidative metabolism (that requires oxygen) Hepatocytes closest to the central vein are prone to anoxic injury (zone 3) • Important for biotransformations, drug detoxifications etc Intermediate zone is between these • Effect of systemic venous pressure and shock o If systemic venous pressure rises Portal venous system dilates and increases the amount of blood entering the liver o If arterial blood pressure drops (eg. haemorrhage) Blood in liver enters the systemic circulation (via portal system) Arterioles constrict diverting blood away from the liver o Severe shock may cause patchy necrosis in the liver due to diversion of too much hepatic artery blood • Lymph o Much formed in the liver (sinusoid endothelium is very permeable) ~50% of lymph in the body at rest o Lymph formed in the liver has a high protein count (6g/100ml) because endothelial pores are large o If hepatic venous pressure (hepatic veins) rises (3-7mmHg) above normal, can get excessive lymph formation – ie, reduced outflow from liver Excess lymph can ‘sweat’ from the free surface of the liver and cause ascites If venal caval pressure is 10-15mmHg above normal, lymph flow is increased 20x

Functions of the liver • Formation and secretion of bile o Important for fat digestion and excretion of waste products like bilirubin and cholesterol • Metabolism o Carbohydrate Storage of glycogen, GNG, glycogenolysis Conversion of galactose and fructose to glucose Maintains normal glucose levels (glucose buffer) o Fat Oxidation of fatty acids, production of ketone bodies Formation of lipoproteins, synthesis of cholesterol and phospholipids Conversion of carbohydrates and proteins to fat o Protein Deamination of amino acids, formation of urea (to detoxify NH 3), Interconversions of amino acids and other compounds, formation of nonessential amino acids, Formation of plasma proteins Death can result if protein is not metabolised by liver (NH 3 poisoning) Functions of the liver (continued) • Synthesis of plasma proteins o Forms 90% of plasma proteins, essentially all except for gamma globulins (formed by plasma cells) o Including – albumin, acute phase proteins, binding proteins, clotting factors Also produces bile salts that facilitate the absorption of vitamin K (important for clotting factors) • Inactivation of substances o Drugs and hormones With decreased liver function, may not inactivate estrogen hormone in males gynecomastia • Excretion via the bile of endogenous and foreign organic molecules and trace metals • Storage o Vitamins: A (10 month supply ), D (3-4 month) , B 12 (12) o Iron (as ferritin) • Immunity o Kupffer cells – phagocytose bacteria and other foreign material in sinusoids Reduce bacterial load that reaches the systemic circulation to 1% o Immunoglobulin A released into the intestine • Vascular functions – storage and filtration of blood o Liver can expand (from 450ml blood volume in normal) 0.5-1L in right heart failure Acts as a reservoir for blood in excess blood volume and can supply extra blood in low blood volume o Destroys old erythrocytes • Endocrine functions o Secretion of insulin=like growth factor I (IGF-I) o Contributions to the activation of vitamin D o Formation of T 3 from T 4 o Secretion of angiotensinogen o Metabolism of hormones

Formation and secretion of bile • Bile is made by the liver and concentrated in the gall bladder o Bile secreted by the liver is hepatic bile, after concentration and modification in the GB – gall bladder bile • Composition of bile: o Bile salts o Bile pigments o Other substances (cholesterol, fatty acids, lecithin, fat, alkaline phosphatase) o All in an alkaline electrolyte solution • Secretion o 500-1200ml of hepatic bile secreted each day o 2 stages: Initial portion secreted by hepatocytes – containing bile acids, cholesterol and organic constituents As bile flows along duct system, watery solution of sodium and bicarbonate added by epithelial cells • Doubles the quantity of bile o 97% water at liver

Bile salts • The sodium and potassium salts of conjugated bile acids • Bile acids o Synthesised from cholesterol o Primary bile acids secondary bile acids Cholic acid deoxycholic acid Chenodeoxycholic acid lithocholic acid Converted to secondary bile acids by bacteria in the colon: o All bile acids are conjugated to glycine (most) or taurine (derivative of cysteine, less) in the liver After being conjugated, bile acids form potassium and sodium salts in alkaline hepatic bile • Eg: glycocholic acid, taurocholic acid Actions of bile salts • 2 functions: o Emulsification Reduces surface tension of fat particles • Aided by phospholipids and monoglycerides Separates fat particles from food into small minute particles that can easily be attacked by lipase enzymes o Transport (ferrying) Amphipathic – have hydrophobic and hydrophilic domains Form micelles (once past a critical concentration) • Transport digested fat end products through the intestinal mucosal membrane Without bile: 50% of ingested fat appears in faeces and severe malabsorption of fat soluble vitamins

Enterohepatic circulation • Bile is 90-95% reabsorbed in the terminal ileum o Mechanism: secondary active Na + bile salt cotransporter ASBT (apical sodium-bile transporter) Uses Na + gradient that is replenished by the Na +/K +/ATPase on the basolateral membrane o There is evidence of at least one more types of transporter • 5-10% remaining enters colon and salts are converted to salts of deoxycholic acid and lithocholic acid (2 o acids) o Deoxycholate absorbed, lithocholate relatively insoluble, and only 1% absorbed • Absorbed bile salts returned to liver via the portal vein o Mostly absorbed on first passage into hepatocytes and are re-excreted as bile • Bile lost in the stool is replaced by synthesis o Normal synthesis rate: 0.2-0.6g/day o Total pool of 3.5g of bile that recycles repeatedly through the enterohepatic circulation 2x/meal, 6-8x/day • If enterohepatic circulation is interrupted (eg. by disease of terminal ileum, Crohn’s) amount of fat in stools increases because liver can’t increase rate of bile salt production enough to compensate

Choleretics • Substances that increase bile secretion o Bile salts Absorption from intestine inhibits synthesis of new bile but they are quickly secreted increasing bile flow Equivalent to 7-10uL/umol of bile salts o Secretin (increases water and bicarbonate secretion by ducts) o Vagal activity (weak) • Note: humans have littler or no bile acid independent flow

Bilirubin metabolism • Bile is normally golden yellow due glucuronides to bile pigments, biliverdin and bilirubin o Biliverdin – formed in reticuloendothelial tissues when old RBCs destroyed (eg spleen) Globin molecule is split off heme converted to biliverdin (iron atom lost, cyclic tetrapyrrole group opened) Biliverdin is rapidly reduced to bilirubin • Bilirubin in the circulation is bound to albumin o Can dissociate into the liver and enter liver cells to be bound by cytoplasmic proteins Then, reacts with 2 uridine diphosphoglucuronic acid molecules in smooth ER via enzyme glucuronyl transferase to form bilirubin diglucuronide • This is more water soluble than free bilirubin and actively transported into the bile canaliculi o Some escapes into the blood and is excreted bound to albumin in the urine o rest remains in bile (as conjugated bilirubin) o 80% bilirubin is converted to bilirubin glucuronide, 10% bilirubin sulfate, 10% other

Bilirubin excretion • Conjugated bilirubin cannot permeate intestinal mucosa, however, unconjugated bilirubin and urobilinogen can o Urobilinogen is formed by bacterial action in intestine from conjugated bilirubin • Urobilinogen can thus be reabsorbed or further metabolised o Reabsorbed into portal circulation and again excreted by the liver (95%) or via the kidneys (5%) o In urine: Exposure to air causes oxidation to urobilin • Gives urine the yellow colour o In faeces Alteration to stercobilinogen and oxidation to stercobilin • Gives stools their brown colour

Cholesterol • 1-2g/day of cholesterol is secreted into bile o Formed as a byproduct of bile salt formation and secretion (no function known) • Combines with bile salts and lecithin forming micelles in bile • Excess cholesterol in bile can result in gall stones

The gallbladder • Bile is continuously secreted by the liver, thus it is stored in the gall bladder until needed o Bile is concentrated by removal of water (liver bile: 97% water, gall bladder bile: 89%) ~5x, but as much as 20x concentrated o Bile is acidified (liver bile: pH~7.5, GB bile: ~6-6.5) • Max volume is 30-60ml, can store 12h of bile secretion o Epithelium of bladder absorbs water, sodium, chloride, small electrolytes and thus concentrates bile Via sodium transport with secondary absorption of chloride/water • Bile remains isotonic despite sodium ions increasing in concentration up to 300mM overnight o Mechanism: micelle formation allows high concentrations of bile acids and cations to remain Thus, less osmotically active particles than indicated by concentration • If block bile duct and cystic duct o Intrabiliary pressure reaches 320mmbile in 30 minutes and bile secretions stops from liver • If block just bile duct o Water is reabsorbed in gall bladder and intrabiliary pressure rises ~100mmbile in a few hours • Overnight fasting: o Half hepatic bile salts enter gall bladder Rest enters small intestine and is reabsorbed in ileum and resecreted o The longer the fast, the greater the fraction of the body’s bile acids stored in GB

Cholecystagogues/cholagogues • Substances that cause gallbladder contraction o Cholecystokinin (CCK, main) Released when fatty acids and AA enter duodenum Has other effects: stimulation of pancreatic secretion o Vagus and enteric nerve supply • Also need relaxation of sphincter of Oddi o 3 relaxation factors: CCK Peristaltic waves in common bile duct Peristaltic waves in the duodenum (most important) • Causes bile to enter duodenum in squirts in synch with relaxation phase of peristalsis • Emptying of gallbladder o Not much fat – empties poorly o Adequate fat – empties completely in an hour and remains contracted so bile is not stored in GB during digestion • Gallbladder is not essential: o If not removed, bile duct dilates and has storage potential

Gallstones • Very common • Made of cholesterol or calcium bilirubinate • Formation promoters: o Bile stasis o Supersaturation with cholesterol o Nucleation factors – inflammation or bacterial precipitate that stones form around • Treatment: o Ursodeoxycholic acid (minor secondary bile acid in humans) 1-2g taken/day for a few ears Causes dissolution and reabsorption of gallstone Mechanisms: • Increases volume of bile decreasing concentration of cholesterol • Increases concentration of bile acid which makes cholesterol more soluble • Reduces secretion of cholesterol into bile (less bile produced) Lecture 33: Body fluids and composition Karen Gibson

Properties of solutions • Definitions o Mole – number of particles equal to avogadro’s number (6.022x10 23 ) One mole of substance weighs the molecular weight of the substance in grams o Molarity – number of moles of substance per litre of solution (mol/L, M) o Molality – number of moles of substance per kg of solvent (mol/kg) Molality and molarity are often used interchangeably • Biological solutions: o Water is the solvent Molality is moles/kg H20, thus molality ~molarity o Concentrations are small, so often expressed as mM • Molecules in solution o Some molecules liberate one or more particle in solution EGs:

• glucose (s) = glucose (aq) + - • NaCl (s) = Na (aq) + Cl (aq) 2 osmotically active particles + 2- • Na 2SO 4 = 2Na , SO 4 3 OAP o The number of osmoles = moles x number of free moving particles liberated by each molecule Eg: 1mole NaCl = 2osmoles o Osmolarity – number of osmoles per litre of solution o Osmolality – number of osmoles per kg of solvent Osmolality more often used in body fluids because mass stays the same at different temperatures, but volume doesn’t Osmolality is measure most often used in body fluids because they often have a mix of substances, but we don’t care what the substances are • 290-300 mOsm/kg osmolality in body fluids o Osmosis – flow of water across a semi-permeable membrane from lower solute concentration to higher Ie: flows from side with higher concentration of solvent to side with lower solvent • Osmolality gradient causes water to move by osmosis Water flows from 100 mOsm/kg 200 mOsm/kg compartments by osmosis

Colligative properties • Osmolarity depends on colligative properties o Ie: depends on number of particles in solution, not their chemical nature (mass, charge, size) • Other things with colligative properties: o Freezing point depression Pure water freezes at 0 oC, with solutes freezes lower (1osmol/kg solution freezes at -1.86oC • Thus, machine measuring osmolality measures freezing point of solution and relates it to osmolality of solution Body fluids - -0.558 oC, ~300 mOsm/kg o Boiling point elevation, vapour pressure lowering, osmotic pressure

Osmosis and osmotic pressure • Osmotic pressure o The pressure that needs to be applied to stop the movement of water to the more concentrated solution o Calculated with Van’t Hoff’s law • Van’t Hoff’s law: o π = nCRT π = osmotic pressure (atmospheres, 1atm = 760mmHg), n = no. of dissociable particles/molecules, C = total solute concentration (mol/L), R = Gas constant, T = Absolute temperature ( oK) o At body temperature (310K) π (mmHg) = 19.3 x osmolality (mOsm/kg) • eg. osmolality = 300 mOsm/kg, thus π = 5790mmHg Comparing solutions • Osmolality o Isosmotic (iso-osmotic) – same osmolality Eg. 0.9% NaCl (300mOsm/kg) = ~1.8% urea o Hypo-osmotic – lower osmolality Eg. 0.45 NaCl is hypo-osmotic to 0.9% NaCl o Hyperosmotic – higher osmolality Eg. 1.8% NaCl vs 0.9% NaCl • Tonicity o If a normal body cell is placed in solution: Isotonic – cells don’t change size Hypotonic – cells swell as water goes in Hypertonic – cells shrink as water goes out o Based on osmolar concentration of solution (vs cells) and whether solute can permeate the cell Impermeant solutes (although concentration may be lower/higher in cell) eg. NaCl • Isosmotic – isotonic • Hypoosmotic – hypotonic • Hyperosmotic – hypertonic Permeant solutes – not always match o EG: 0.9% NaCl and 1.8% urea Iso-osmotic NaCl: • RBCs isotonic (impermeable although concentration lower in cell) • RBCs hypotonic o urea goes into cell because intracellular concentration is lower (0.03% urea) and cell membrane is permeable to urea o thus, more osmotically active particles inside cell, water flows in swelling

Colloid osmotic pressure = oncotic pressure • Osmotic pressure exerted by proteins and accompanying cations o Proteins stay in the plasma (capillary walls only let small molecules cross) • Plasma oncotic pressure is ~25-28mmHg of the total ~5000mmHg osmotic pressure o Makes up a small proportion, but is important because it acts at the capillary wall (where proteins don’t cross)

Volumes of body fluid compartments • 70kg young man: o Water – 60% (50-70%) o Proteins and related substances – 18% o Minerals – 7% o Fat – 15% • Total body water varies with: o Age Newborn babies are 75% water o Obesity With more fat, water is a lower percentage of body weight Fat has very little water (10-20%), organs are normally 70-80%, bone is 25% o Gender Women have a higher percentage of fat Women have a lower body weight

Volumes of body fluid compartments (continued) • Compartments: o Total body water (60% of body weight, 42L) Division plasma membranes • Intracellular water (40% body weight, 2/3 TBW, 28L) • Extracellular water (20% BW, 1/3 TBW, 14L) o Division walls of BVs Plasma (5% BW, ¼ ECV, 3.5L) Interstitial fluid (15% BW, 3/4ECV, 10.5L) – outside cells and vessels, milieu of cells • Slowly exchanging – bone, dense CT (cartilage, tendons) • Rapidly exchanging – rest • Special cases: o Blood – mix of intracellular (RBCs, WBCs) and extracellular (plasma) o Lymph – special part of interstitial fluid, 2-3% BW o Transcellular fluid – fluid separated from plasma by epithelial cells (rather than endothelial cells of BVs) Egs: CSF, intraocular fluid, endolymph of inner ear, fluid in pericardial, pleural space, sinovial fluid in joints Composition can be similar to interstitial fluid, others can be unique

Measurement of volumes • Indicator dilution technique o Method: Dye or marker of known amount (Q) is added to body compartment Time given for mixing Concentration of marker measured (c) Volume of distribution calculated using V = Q/c o Marker can be metabolised/excreted during mixing/equilibrium time Then: Q = amount injected – amount lost o Good markers: Non-toxic Mix evenly in compartment, not change size in compartment Easily and accurately measured Able to account for loss Excreted, able to be destroyed o EGs: 3 2 Total body water – Tritium water ( H2O), deuterium oxide (heavy water: H2O), antipyrine • Often easier to just weigh patient to get a good indication Extracellular fluid – 22 Na (overestimates because tends to get inside cells), inulin (polysaccharide, doesn’t penetrate well), 125 I-iothalamate, thiosulfate Intracellular fluid – calculated from: total body water – extracellular fluid volume Plasma volume – 125 I-albumin (plasma protein), globulin (more stays in plasma), Evan’s blue dye (binds to albumin) Blood volume – 51 Cr-labelled RBCs, Tc-99 (shorter half life, less exposure), calculated, BV = Plasma volume/1-haematocrit) Interstitial fluid – calculated as extracellular fluid – plasma

Composition • Notes: o Measured in milliequivalents to take charge into account, however within the compartment, charge is irrelevant – electro-neutrality Osmolality is the same in all compartments – 290-300mOsmkg 1 mol Na + = 1mEq, 1 mol Ca 2+ = 2mEq o Extracellular Plasma + - - • Cations – Na , anions: HCO 3 , Cl , protein o Protein has many charges, thus plasma has more mEq than interstitial, has more protein (6-7g/100ml), vs interstitium (1.5-2g/100ml) o Protein is also negative at body pH, thus comes brings more cations with it Interstitial • Similar to plasma with less protein Intracellular (sk muscle) • Cations – K+, Mg 2+ • Anions – organic phosphates (ATP creatinine phosphate, GMP etc), protein o Note: no chlorine or bicarbonate in sk muscle In RBCs up to 90mEq/L, epithelium 20 mEq, lymph 40mEq o Protein is polyvalent, thus increased mEq intracellular, but osmolality same as others

Control of composition • Plasma electrolyte composition is controlled in the kidney o Indirectly, this controls electrolyte composition in other compartments • Plasma vs interstitial composition (ie between inside/outside BVs) maintained by: o Capillaries hold protein (plasma protein) o Gibbs-Donnan effect proteins are negatively charged, hold cations • Extracellular vs intracellular composition (ie between inside and outside cell) maintained by: o Cell membrane (selective permeability) o Selective ion pumps (eg. Na +/K +/ATPase pumps out 3Na +, in 2K +) o Gibbs-Donnan effect – cells have protein, -ve charge thus more +ves)

Control of volume • Total body water controlled by thirst, and urine excretion (kidney, ADH) • Plasma vs interstitial compartments o Starling forces Ie: balance between hydrostatic and colloid osmotic pressures in capillary and interstitial space + lymphatics • Extracellular vs intracellular compartments o Amount of solute in each Water is freely permeable and moves via osmosis to maintain osmolality of both compartments

Functions of kidney + + - 2+ 2- 3- • Regulates water and electrolyte (eg. Na , K , Cl , Mg , SO 4 , PO 4 ) balance o Not including Zn, Fe, and some Ca 2+ • Regulation of acid-base balance • Excretion of metabolic waste products (eg. urea, uric acid, creatinine) • Excretion of foreign chemicals and other bioactive substances (eg. pesticides, food additives) • Regulation of arterial blood pressure o Salt and water balance o Vasoactive hormones (renin angiontensinogen 2: vasoconstrictor; PG: vasodilator) • Regulation of RBC production o Proximal tubular cells produce erythropoietin that causes bone marrow to produce RBCs Renal failure leads to anaemia • Regulation of Vitamin D production o Makes the final active 125-Vitamin D 3 o • GNG

Lecture 34: Fungal infections and treatment Hazel Mitchell

Introduction • More than 70 000 species of fungi, ~300 cause disease o Often don’t appear as we expect (most often not mushrooms)

Classification • Filamentous fungi o Grow as multinucleate branching hyphae A mass of hyphae is a mycelium o Reproduce by spores that often sit atop hyphae o Cause superficial mycoses and cutaneous mycoses Mycoses – fungal infections • Yeasts o Grow as ovoid or spherical single cells o Reproduce by budding o Cause systemic mycoses • Fungal diseases: o Superficial Infects: hair shaft, dead layer of skin Diseases: pityriasis versicolour, tinea nigra, piedra Causative agents: Malassezia, Trichosporon, Exophiala o Cutaneous Infects: epidermis, hair, skin Disease: tinea (ringworm) Causative agents: Microsporum, Epidermophyton, Trichophyton o Sub-cutaneous Infects: dermis, subcutis Diseases: Sporotrichosis, Mycetoma Causative agents: Sporothrix, several other genera o Systemic Infects: internal organs Diseases: coccidioidomycosis, histoplasmosis, blastomycosis Causative agents: Coccidioides, Histoplasma, Blastomyces o Opportunistic Infects: internal organs Diseases: Cryptomycosis, candidiasis, aspergillosis, Pneumocystis pneumonia Causative agents: Cryptococcus, candida, aspergillus, pneumocystis

Sites of infection • Outermost layers of the skin (stratum corneum – dead layer), hair shafts o Superficial mycoses • Keratinised layers of epidermis, nails and hair o Cutaneous mycoses • Dermal layers of skin o Subcutaneous mycoses

Superficial mycoses • Pityriasis versicolour – common fungal infection of skin o Flaky discoloured patches Confined to the trunk/proximal parts of limbs • Uncommon in other areas Hypo or hyperpigmented lesions • Coalesce forming scaling plaques Not itchy Often spontaneously resolve o Causative agent: yeast Malassezia furfur Common skin inhabitant • Changes from yeast to hyphal form of fungus as it changes from commensal to pathogen o Spaghetti and meatball appearance o Phase change stimulus unknown

Cutaneous mycoses • Tinea (ringworm) – cutaneous infection o Causative agents: Dermatophyta – Epidermophyton, Trichophyton, Microsporum Source of infection: • Humans, soil, animals • Infection is spread by contact with arthrospores (asexual spores) Infection: • Arthrospores attach to keratinocytes and invade • Dermatophytes use keratin as a nutrient source (via keratinase) • Doesn’t invade living tissue, simply colonises keratinised stratum corneum o Presence here of fungus and metabolic products causes allergic and inflammatory eczematous response Diagnosis • Clinical assessment • Specimen collection – scraping of leading edge of lesion o Microscopy – examine structure of fungus o Culture – Sabouraud’s agar (pH 5, high sugar) Look for pigment and mycelial formation • Tinea corporis o Presentation: Annular lesions Scaling and raised margin • Sample from the edge of the lesions because fungi is only active at the margin o Symptoms: itching • Tinea pedis (athlete’s foot) o Presentation: Erythema of first and second toes Toe-web maceration (wasting) o Transmitted from feet through desquamated skin scales shed in carpet, mats, showers • Tinea barbae – beard area • Tinea capitis – affects hair and skin of scalp removing hair • Onychomycosis o Infection of keratinised layer of nails o 3% of population, 5% in elderly

Diagnosis of fungal infections • Clinical assessment • Sampling and processing o Potassium hydroxide breaks down the skin cells, leaving the fungi • Microscopy o Look for the presence/absence of asexual spores (conidia), size, shape and location o Can identify fungi from pattern of these • Culture o Examine colour (bottom of plate) and mycelia (top of plate) Eg 1: Trichophyton rubrum on SAB appears red Eg 2: Epidermiphyton floccosum on SAB appears yellow with crusty mycelia Eg 3: Microsporum canis on SAB is orange with cloud-like mycelia Eg 4: Trichohyton mentagrophytes on SAB appears black with orange radiances and dried out mycelia

Subcutaneous • Sporotrichosis o Caused by Sporothrix schenkii, Found in soil and plants • Particularly important in gardeners, farmers o Clinical presentation In 1 month, small papules (small subcutaneous nodules) at the site of trauma • This spreads by the lymphatics Dissemination can occur (especially in the immunosuppressed) • Mycetoma o Caused by Madurella species o Chronic infection of subcutaneous tissue tumour-like formation Can invade contiguous tissue, especially bone o Endemic infection in Africa

Systemic • Coccidioidomycosis o Causative agent: Coccidioides immitis Dimorphic fungi – environment: filamentous, body: yeast-like o Pulmonary disease due to inhalation of conidia (spores) 60% benign chest infection 40% typical symptoms of pulmonary fungal disease – anorexia, weight loss, cough, hemoptysis • Often confused with TB o Progression to systemic disease occurs in 5% Affects: meninges, bones, joints, subcutaneous and cutaneous tissues o Endemic in soil in south-western USA, N Mexico and various areas of South America • Blastomycosis o Causative agent: Blastomyces dermatitidis Dimorphic o Symptomatic in 50% Primary pulmonary involvement progressing slowly to chronic granulomatous disease • Haematogenous spread (70%) causes cutaneous lesions in skin or bone o Found in N America, Africa, Asia, Europe • Histoplasmosis o Causative agent: Histoplasma capsulatum Dimorphic Intracellular infection of reticuloendothelial system o Acquisition Inhalation of conidia from soil enriched with chicken, starling and bat excreta o 95% of cases are inapparent (no symptoms), subclinical (no clinical signs) or benign (harmless) 5% of cases lead to chronic progressive lung disease, chronic cutaneous or systemic disease or acute fulminating fatal systemic disease (esp. immunosuppressed) o Found worldwide, endemic in Mississippi-Ohio River Valley, USA Opportunistic fungal infections • Candidiasis o Caused by members of g enus Candida C. albicans: 47%, parapsilosis: 19.3%, glabrate: 17.8% o Clinical manifestations Vary: acute, subacute, chronic, episodic o Locations: Mouth, throat, skin, scalp, vagina, fingers, nails, bronchi, lungs, GIT Systemic septicaemia, endocarditis, meningitis o Healthy individuals: Infection is often due to impairment of epithelial barrier functions Occurs in all age groups • More common in newborn and elderly (decreased immune function) Remains superficial Responds well to treatment o Systemic candidiasis Often seen in: • Cell-mediated immune deficiency • Aggressive cancer treatment, immunosuppression, transplantation therapy o Special cases: Newborn babies • Characteristic patches of creamy white to grey pseudomembrane (membranous exudate) • Factors: o Low pH of newborn mouth may encourage C. albicans proliferation o Infection may be acquired at birth – vaginal thrush in birth canal o Nursing mothers may contract C. albicans infection of nipples – pain while feeding Ping-pong effect, transmits mother baby mother Immunosuppressed adults • Characteristic oral and oesophageal candidiasis • Rarely seen in healthy adults o Occurs in 5% newborn infants, 10% elderly o Broad spectrum antibiotics • Predisposing factors: o Immunological impairment due to: DM, leukemia, lymphoma, malignancy, neutropenia, HIV, corticosteroids, cytotoxic drugs, radiation therapy Vaginal thrush • Causative agent: Candida albicans • Clinical presentation – curd-like discharge • Usually an opportunistic infection in this case o Common: 75% women have ≥ one occurrence in lifetime, relates to ph of vagina • Can be sexually transmitted o Penile colonisation in 20% of men with partner with infection Men normally asymptomatic, if have symptoms: balanitis Cutaneous candidiasis • Intertrigo, diaper candidiasis, onychomycosis • Locations: Axillae, groin, inter and sub-mammary folds, intergluteal folds, interdigital spaces, umbilicus • Predisposing factors: moisture, heat, friction, maceration of skin • Flaking of skin, rash o Diagnosis Microscopy wet mount and gram stain looking for yeast cells and pseudohyphae, • Appears similar to gram +ve Culture Sabouraud’s medium • Colonies smell like yeast Germ tube test • C. albicans is positive, glabrata, parapsilosis is negative • Yeast is placed in serum broth, production of germ tubes is a positive test Opportunistic fungal infections (continued) • Cryptococcosis o Main causative agent: Cryptococcus neoformans Encapsulated yeast Source: bird faces – especially pigeons o Transmission – inhalation of airborne cells Results in primary infection in lung pneumonia o Commonest cause of fungal meningitis In immunsuppressed patients, invades bloodstream from primary site resulting in meningitis • Increased chance if CD4 lymphocytes <200/mm 3 • 7-10% of AIDS patients affected Symptoms develop slowly over months • Initially: headache, then: drowsiness, dizziness, irritability, confusion, nausea, vomiting, neck stiffness, focal neurological defect • Later stage: diminishing visual acuity, coma o Lab diagnosis Microscopy – India inc preparation • Ovoid yeast cell + capsule o Allows survival in blood and protection from phagocytes Culture – Sabouraud’s dextrose agar Colonies – cream-coloured, smooth, mucoid Cryptococcal antigen test • Rapid latex agglutination test to detect capsular polysaccharide antigens • Test serum and CSF • Aspergillosis o Common causative agents: Aspergillus fumigatus, Aspergillus flavus o Can be non-invasive and invasive Non-invasive pulmonary disease • Predisposed: patients with pre-existing lung cavities or chronic pulmonary disorders • Aspergillus colonises cavity fungal ball (aspergilloma) which blocks alveoli Invasive pulmonary disease • Predisposed: immune-compromised • Lung infection, can lead to haematogenous dissemination o Predisposed: severe immunosuppression, IVD users o Can result in thrombosis, infarction • Pneumocystis pneumonia (PCP) o Causative agent: pneumocystis jiroveci (carinii) – yeast-like fungus o Results in interstitial pneumonia o Important disease in immunocompromised patients: AIDS • AIDS defining illness, reduced illness following HAART therapy introduction Immunosuppressant medication • Solid organ transplantation • Bone marrow transplantation o Symptoms High fever, non-productive cough, shortness of breath, weight loss, night sweats Limited sputum production o Rare in people with normal immune systems May be found but doesn’t cause disease

Treatment of fungal infections • Fungi are eukaryotic and thus have much in common with human cells o Antibiotics have no effect Need to use specialised antifungal agents that can be toxic • Cell membrane and wall is made up of: o Phospholipid bilayer o Chitin o Sugars o Mannoproteins • Sites of action: o Polyenes – cell membrane o Flucytosine – in nucleus o Azoles – mitochondria and cell membrane

Azoles • Used for: o Superficial mycoses o Candidiasis • Mechanism of action o Blocks production of ergosterol (yeast cholesterol) causing accumulation of toxic sterol intermediates Ie. blocks lanosterol C14 demethylase This puts stress on the cell membrane • Preparations o Oral, eg: fluconazole Used for: candida oesophagitis, vaginal candidiasis (if unresponsive to other treatment), severe oral candidiasis, onychomycosis, recalcitrant superficial mycoses o Topical, eg: bifonazole, clotrimazole, econazole, miconazole, Ketaconazole – used for seborrhoeic dermatitis and dandruff • Tinea – use topical, if doesn’t work: use oral

Polyenes • Mechanism of action: inhibits cell membrane function o Preferentially binds to ergosterol, but cholesterol binding causes toxicity • EGS: o Amphotericin B IV Used for serious systemic infections, eg. pneumonias, cryptococcal meningitis Very toxic, to limit: saline administration prior to amphotericin B • Side effects: renal toxicity o Nystatin Topical, not well absorbed in the GIT, thus oral administration can be useful: non-toxic • Not absorbed through unbroken skin or mucous membranes Used for candida albicans (vaginal thrush) as a cream/pessary

5-Flucytosine • 1950s developed as an anticancer agent o Found to have antifungal activity • Mechanism of action: inhibits RNA and DNA synthesis • Only active against yeasts o Used for cryptococcal infection • Resistance develops quickly o Can be used in combination with amphotericin B

Lecture 35: The liver in health and disease Andrew Lloyd

Functional anatomy • Located on the R side of the body lying underneath the diaphragm o Broadly can be divided into 2 lobes o Large: 1400-1600g (2.5% body weight) • Dual circulation o 60%: portal vein – gut drainage containing nutrients, toxins, poor oxygenation o 40%: hepatic artery – oxygenated supply from the aorta o Due to dual circulation, hard to get ischaemic insult in the liver • Structural unit is the lobule o 1-2mm, hexagonal o Central vein, portal tracks at periphery o Made up of hepatocytes organised in sheets or plates Separated by sinusoids and bile canaliculi • Functional unit is the acinus o Tissue supplied by terminal branches of the hepatic artery and portal vein o Demarcated by blood supply • Zones of oxygenation o Zone 1: 1/3 closest to hepatic artery/portal vein o Zone 3: 1/3 closest to central vein o Zone 2: intermediate between these o Significance: Systemic venous hypertension (RHF) • Zone 3 receives even less new blood than before cardiac cirrhosis (nutmeg liver) Haemorrhage/shock • Blood is diverted away from liver, zone 1 undergoes ischaemic injury • Sinusoids are lined by a unique endothelium that has large pores o Allows proteins, peptides etc to move through into the liver • Space of Disse – space between sinusoids and hepatocyte plates o Contains Kupffer cells that differentiated from monocytes by factors in the liver – tissue macrophages o Contains stellate cells (Ito cells) – lay down fibrous tissue, store fat soluble vitamins

Physiology • Detoxification of endogenous metabolic waste, excretion of bilirubin and bile acids o Fat soluble wastes excreted via bile acids, non-fat soluble via blood drainage • Processing of dietary proteins, carbohydrates, lipids, vitamins and drugs • Storage of glycogen • Synthesis of serum proteins (incl. albumin, coagulation factors), synthesis of vitamins • Phagocytosis of particulate material from gut • Immune response

Hepatitis: pathophysiology • Liver has a vast functional reserve o Can function with 90% lost, <5% remaining have problems • Liver responds to injury in various ways: o Steatosis – intracellular fata accumulation o Necrosis and inflammation (chronic and acute) Acute • Neutrophil accumulation or mononuclear cell accumulation (lymphocytes) • Oedema, congestion Chronic • Scar tissue formation possibly leading to cirrhosis • Simultaneous tissue injury and repair o Regenerative nodules of hepatocytes are formed structure lost, however o Fibrosis – part of chronic inflammation • Cirrhosis – progressive fibrosis and regenerative nodule formation (as part of chronic inflammation) • Liver function failure causes: o Acute fulminant hepatitis Develops over days-weeks o Chronic hepatitis with cirrhosis Develops over months to decades, eg. HBV chronic o Manifests with disorders due to physiological function loss: eg. jaundice, coagulopathy Liver function tests • Biochemical assays that reflect liver performance o Transaminases – AST, ALT Released into the circulation when hepatocytes die There is a normal level due to apoptosis of hypatocytes, in hepatitis, greatly elevated levels o GGT (gamma glutamyl transferase), SAP (serum alkaline phosphatase) Reflect biliary obstruction or injury, eg. gallstones, cholestasis o Bilirubin – elevated in biliary obstruction o Albumin, prothrombin time (coagulation factors 2, 7, 9, 10) – reflect protein synthesis

Acute hepatitis • Major causes: o Hepatitis viruses (most common to least): A, B, E, C, D o Alcohol • Clinical features o Variable: asymptomatic to liver failure Often involves: malaise, anorexia, abdominal discomfort, distaste for smoking, jaundice, dark urine • Jaundice: sclerectasias, bilirubin 2-3x normal Live failure: bruising, bleeding, ascites, coma • Pathology o Diffuse liver cell damage Elevated transaminases 10-100x upper limit of normal (40-50) • Alcohol: AST>ALT, virus ALT>AST o Scattered necrotic eosinophilic hepatocytes (Councilman bodies) o Portal inflammation or lobular inflammation or both o Pattern of necrosis can be bridging – portal-portal, central-portal etc Ie. spread of necrosis beginning in centrizonal area towards portal areas

Chronic hepatitis • Major causes: o Hepatic viruses: B, C, D B – significant mother-baby transmission at birth • In developed countries: screening and immunisation has almost eliminated • In developing countries: endless cycle o Alcohol • Clinical features o Predominantly asymptomatic until complications of cirrhosis (liver failure, HCC) arise May have malaise, fatigue without other symptoms o Signs of chronic liver disease: Spider naevi (disturbance in estrogen metabolism), palmar erythema, leukonychia (low albumin), jaundice, splenomegaly (obstruction of portal drainage, portal hypertension), ascites (high portal HTN high back pressure, liver sweating), caput medusae • Pathology o Mild transaminase elevation (2-3x ULN) o Progressive reduction in albumin (late) o Progressive elevation of bilirubin (late) o Progressive increase in prothrombin time o Fibrosis including bridging and nodule formation on histopathology

Cirrhosis • Diffuse fibrosis causing conversion of normal hepatic micro-architecture into an abnormal formation with nodule formation o Macronodular (>3mm) – viral o Micronodular (<3mm) – alcohol • Functional consequences o Impaired metabolic function: Deficient albumin synthesis – important for oncotic pressure, thus results in swelling: ascites and peripheral oedema Deficient synthesis of coagulation factors Impaired metabolism of drugs and hormones Impaired bilirubin excretion Impaired metabolism of nitrogenous waste encephalopathy, confused, comatose o Clinical presentations: Pitting oedema Gynecomastia Spider naevi

o Portal hypertension, pressure in the portal-venous anastomoses Oesophageal, gastric, rectal varices Ascites Splenomegaly Caput medusae

Hepatocellular carcinoma • Primary malignant neoplasm of hepatocytes o Most often arises in cirrhotic liver • Cause: o Hepatitis B > Hepatitis C > Alcohol • Typically diagnosed late • High mortality

Lecture 36: Clinical aspects of liver disease Stephen Riordan

Anatomy and physiology • Liver is the largest internal body organ (1/50 total body weight adult) • Dual blood supply o Portal vein (75%) o Hepatic artery (25%) o Each provides 50% of oxygen for liver despite differences in volume • Composition of liver: o Hepatocytes, non-parenchymal cells (Kupffer cells, stellate cells, sinusoidal endothelial cells), bile ductules • Functions o Synthetic, biotransformatory and toxin-scavenging

Pathology: overview • 2 broad categories: o Hepatocellular: acute/chronic o Biliary: acute/chronic • Clinical syndromes can be very severe – high mortality o Depends on: Degree of liver damage Capacity for liver regeneration

Acute hepatocellular disorders • Hepatitis viruses o Types A-E AE • Faecal-oral transmission • Short incubation • Acute only BCD • Parenteral transmission • Long incubation • Acute and chronic states o Symptoms: Prodromal illness: • Malaise, anorexia, nausea, RUQ pain, rash (rare), arthritis (rare) Leading to jaundice o Signs: Tender hepatomegaly, jaundice • Other viruses o EBV, CMV, VZV, HSV, Adenovirus, Papilloma virus, Paramyxoviruses, Haemorraghic fever viruses o No progression to chronicity • Drug-induced o Drug history is important Any drug may cause Important to establish a temporal relationship – ie. timing of drug dosage and symptoms o EGs: paracetamol, anti-epileptics, anti-tuberculosis, antibiotics

Chronic hepatocellular disorders • Viral: hepatitis B, C • Alcohol-related • Non-alcoholic fatty liver/steatohepatitis (NASH, NAFL) • Immune-mediated: autoimmune hepatitis • Metabolic o Haemochromatosis (iron overload) o Wilson’s disease (copper overload) o Alpha-1-antitrypsin deficiency

Hepatitis B • 350 million chronically infected worldwide with HBV o >75% are Asian o 15-20% die prematurely from liver disease 9th leading cause of death worldwide

Hepatitis C • Epidemiology o 1.1% of Australian population has chronic infection Most unaware 3% population worldwide choric infection o New infection rate: 15000/year o >70% of IV drug users are infected most become infected within the first year of taking • Pathogenesis o Immune-mediated liver damage • Clinical features – due to immune reaction and liver failure o Most don’t present with jaundice, dark urine, pale stools, RUQ pain or other constitutional symptoms Instead: lethargy and non-specific symptoms (eg. nausea) o Often detected incidentally (eg. with cholesterol, PSA test, find elevated liver enzymes levels) Or after presenting with complications of cirrhosis o Extra-hepatic manifestations Cryoglobulinemia – vasculitic rash, arthralgia Glomerulonephritis – mesangiocapillary, membranous Autoimmune phenomena –haemolytic anaemia, thrombocytopenia Lymphoproliferative disorder – Non-Hodgkin’s lymphoma, Waldenstrom’s macroglobulinemia (IgM proliferation) • Risk factors o IVDU o Blood, plasma transfusion (before 1990), haemodialysis o Sharing razors, toothbrushes, tattooing o Immunisation in developing countries o Occupational – doctors/nurses o Sexual – less important o Vertical – pregnancy/childbirth (rate 3-5%)

Alcoholic liver disease • Fatty liver o TG within cells Decreased liver function o Reversible • Alcoholic hepatitis o 3 month mortality rate is 20% in most severe form • Cirrhosis o Irreversible but progress can be halted • Only 15% of heavy drinkers every develop liver disease and have symptoms o Genetic factors are important

Non-alcoholic fatty liver • Associations o Obesity o NIDDM o Hyperlipidaemia o Rapid weight loss o Drugs (eg. Amiodarone, Methotrexate, Corticosteroids) • Generally benign o A subgroup can have steato-hepatitis which can lead to cirrhosis

Autoimmune hepatitis • Epidemiology o Mostly occur in young to middle aged women o Can be associated with other autoimmune disorders, eg. coeliac disease • Disease can be aggressive and progress to cirrhosis early despite immunosuppression

Genetic diseases • Haemochromatosis o Autosomal recessive (chromosome 6) o Common – 1/500 o Due to decreased duodenal absorption of iron Results in iron overload – liver, heart, pancreas, skin, pituitary o Signs Increased serum transferrin saturation, serum ferritin, hepatic iron (on liver biopsy) o Can be diagnosed before iron overload occurs Gene testing (family screening) look for C282Y mutation on HfE gene o C282Y mutation Prevalence of heterozygotes: 1/7-1/12 in Australia Most homozygotes develop iron overload • Only 20% of these develop liver fibrosis/cirrhosis o Fibrosis, cirrhosis rare <40, serum ferritin <1000ug/L • Wilson’s disease o Autosomal recessive (chromosome 13) o Prevalence 1/30 000 o Due to impaired biliary excretion of copper Results in increased copper deposition – brain, liver, eye (cornea) o Onset of clinical syndromes occurs at age 5-40 (liver, neurological symptoms) o Signs Reduced serum ceruloplasmin Kayser-Fleischer rings (eyes) Increased hepatic copper (liver biopsy) Haemolysis – deposition in RBCs, possibly resulting in anaemia • Alpha-1-antitrypsin deficiency o Autosomal dominant (chromosome 14) o Affects lung and liver (often in isolation) 10-20% homozygotes develop liver disease Most with liver disease do not have lung emphysema o Can be due to >80 different mutations Thus genetic screening is hard Diagnosis – measurement of a1-antitrypsin in blood

Biliary (cholestatic) disorders • Intrahepatic (small bile ducts in liver) o Drugs o Primary biliary cirrhosis (mostly women) • Extrahepatic (large bile ducts that connect liver to gallbladder to pancreas) o Intrinsic Choledocholithiasis (gallstones) Sclerosing cholangitis (immune-mediated disease affecting bile ducts) Cholangiocarcinoma (malignancy of bile duct epithelium, risk increased by sclerosing cholangitis) Trauma (operative or other) o Extrinsic Pancreatitis, pancreatic malignancy or lymphadenopathy causing compression

Cirrhosis • Histopathology o Nodules of liver cells form loss of sinusoids, fibrosis surrounding nodules in different bridging patterns • Clinical signs (hepatobiliary cirrhosis): o Reduced hepatic span o Leukonychia – decreased albumin o Palmar erythema o Spider naevi o Gynecomastia o Testicular atrophy o Pruritus – itch: scratch marks • Complications o Portal hypertension Ascites • Treated with diuretics and salt restricted diet o Effective in 90% • In other 10%, renal function can cause treatment to be ineffective o Thus: local drainage of peritoneal cavity used to treat Variceal haemorrhage • Treatment: vasoactive drugs ASAP o Reduce portal hypertension and pressure in varices o Endoscopic banding/fibrosing o Bone disease Liver and kidney activate vitamin D by hydroxylation • Liver not functioning, reduced vitamin D Malabsorption of vitamin D due to loss of bile o Hepatocellular carcinoma Incidence 1-6%/year in cirrhosis Symptoms occur late in disease (pain, weight loss) • Pain only occurs when it disrupts/distends capsule • Poor prognosis if present with symptoms Screening is performed if at risk • 6 monthly ultrasound and alpha-fetoprotein level in blood measured to detect small lesions • Problem: may have cirrhosis without knowing o Loss of liver function: liver failure

Liver failure • Signs o Jaundice o Hypoalbuminemia – pitting oedema (swelling), leukonychia o Coagulopathy – bruising, bleeding o Hepatic encephalopathy – NH 3 toxicity o Other: Hemodynamic instability – tachycardia, low blood pressure Renal failure Metabolic derangement Susceptibility to infection – esp. bacterial (impaired complement system synthesis and Kupffer cells) Cause: o Clear precipitant (that indicates liver failure) Sepsis • Eg. spontaneous bacterial peritonitis (SBP) – a common infective complication o Presents in 30% of cirrhotics admitted to hospital o Commonly infection with bowel bacteria o Poor prognosis – implies that liver cell function is very low Gastrointestinal bleeding (eg. varices) HCC o Progressive liver damage (cirrhosis)

Liver transplantation • Indications: o Advanced, uncontrolled functional decompensation (ie. severe loss of liver function) o Episode of SBP (previous) o Uncontrolled complications of portal hypertension Ascites, variceal haemorrhage (recurrent despite therapy) o HCC if small tumour and confined to liver • Problems o Limited donors Even if giving 1 liver to 2 people o Long waiting lists (many die while on list) o Need for long term immuno-suppression o Disease can reoccur in new liver

Acute liver failure • Definition – onset of encephalopathy in absence of previous clinical liver disease o Mostly due to acute viral hepatitis or other toxin (eg. paracetamol) • Rapidly progressive and rapidly fatal without emergency transplantation o Hard to predict who will survive without a transplant and thus who needs the transplant

Lecture 37: Alcohol-related diseases Nick Hawkins

Alcoholic liver disease • Leading cause of liver disease in western countries • Manifests in several ways o There are 3 reaction patterns of liver response to injury, liver can interchange between these: Fatty liver (fatty change, hepatic steatosis) • Fatty change Alcohol hepatitis (acute alcoholic hepatitis, steatohepatitis) • Necrosis (apoptosis) • Inflammation Alcoholic cirrhosis • Fibrosis • Regeneration

Fatty liver • Cause: accumulation of TAG within hepatocytes o Appears pale yellow, greasy vs brick-red o Manifestations: asymptomatic enlargement of liver • Completely reversible • Several non-alcohol causes o Starvation, DM, malnutrition • Histopathology o Lobule structure interrupted Oxygenated blood flows from outside to inside of lobules, injury is compounded by relative hypoxia at centre vein • Thus, more fatty change at centre of lobules o Lipid is found as cytoplasmic vacuoles within hepatocytes • Pathogenesis – more fat coming into liver, less fat packaged and removed o Ethanol causes: Increased breakdown of fat peripherally Increased lipid biosynthesis in hepatocytes Decreased oxidation of fatty acids by mitochondria o Ethanol has a toxic effect on hepatocytes that decreases ability to transport lipoproteins out of cell

Acute alcoholic hepatitis • Cause o Effect of alcohol and its metabolites on the liver • Histology o Fatty change o Focal hepatocellular necrosis with neutrophil (alcohol) or lymphocyte infiltration (viral) o Mallory bodies (Mallory hyaline) – reflects damage o Fibrosis development, especially around central vein (perivenular fibrosis) which can with time lead to cirrhosis • Clinical effects o May have: fever, liver tenderness, jaundice (obstructed bile flow and hepatocyte necrosis) Severe forms have a mortality of 20% Presentation varies greatly o Can resolves with cessation of drinking • Note: necrotic debris near PMNs

Non-alcoholic steatohepatitis (NASH, NAFL) • Becoming more common in Australia o Present in 30% of men in US • Histologically identical to alcoholic steatohepatitis • Causes: o Obesity, insulin resistance, T2DM • Asymptomatic with abnormalities in LFTs o Can lead to cirrhosis in ?10-30% of cases

Cirrhosis • End stage reaction pattern witih: o Diffuse hepatic fibrosis Perisinusoidal and perivenular due to collagen deposition by stellate (Ito) cells in spaces of Disse o Nodular regeneration (vs. nodule which has neat organisation) o Disruption of normal hepatic architecture • No microscopic features that indicate ethanol as the cause • Causes: o 60-70% alcohol (only 10-15% of chronic heavy drinkers get cirrhosis) Commonly thought to present with micronodular cirrhosis (<3mm) o 10% Hepatitis B and C infection Affects portal track outside of lobule and bridges portal to portal, thus macronodular (>3mm) o 10-15% unknown: cryptogenic, possible NASH, NAFL o 5% Haemochromatosis o Other causes

Cirrhosis (continued) • Images o Pic 1: micronodular pattern of cirrhosis: finely granular nodular surface <3mm If stop drinking, can develop macronodular – thus distinction is not that important o Pic 2: close up of micronodular pattern o Pic 3: cirrhosis: irregular bands of fibrosis (blue) with regenerating nodules (pink) Fibrosis means: • Hard to get blood to percolate hypoxic injury • Portal hypertension o Pic 4: cirrhosis medium power o Pic 5: cirrhosis high power • Note: o Initially, liver can be large due to inflammation (acute steatohepatits), but in cirrhosis, liver has shrunken

Complications of cirrhosis • Portal venous hypertension o Cause – collagenation of the space of Disse causing changes to blood flow in liver Reverses porto-systemic anastomoses o Results in: Splenomegaly – congestion of spleen due to splenic vein hypertension Ascites – Starling forces interrupted causing fluid to flow into surrounding tissue • Impaired liver function o Decreased albumin – plasma oncotic pressure reduction exacerbates ascites o Decreased clotting proteins o Impaired drug and hormone metabolism o Acute liver failure and hepatoc encephalopathy • Hepatocellular carcinoma

GIT effects of alcohol abuse • Gastritis o Irritation and thus imflammation of stomach lining by alcohol Progressing possibly to erosion with interruption of stomach lining o Presents with haematemesis When acid mixes with blood black Thus called: coffee-grounds vomitus • Gastric ulceration o Erosion past the stomach mucosa o Particularly seen in people with cirrhosis changes to blood flow to stomach reduces ability to repair and some protective functions • Mallory-Weiss tear o During strenuous vomiting, a tear in mucosal layer at cardio-esophageal junction can occur • Oesophageal varices (ruptured) o When arteries bleed, bleeding can be reduced by arterial spasm Veins don’t have enough muscle in their walls and thus are hard to stop .

Note: councilman bodies are apoptotic hepatocytes in acute hepatitis

Alcohol and the pancreas • Acute pancreatitis o 20-60% is related to ethanol o Pancreatic tissue is destroyed by tryptases, elastases and lipases o Clinical presentation (morbidity and mortality high): Acute severe illness with abdominal pain and shock Elevated lipase • Chronic pancreatitis o 70-80% related to chronic ethanol use o Pathology Protein precipitation and/or calcification of ducts of the exocrine pancreas Atrophy and fibrosis of pancreatic tissue o Clinical Recurrent abdominal pain Exocrine and endocrine pancreatic insufficiency

Alcohol and the nervous system • Acute effects o CNS depression, violence, disinhibition o Chronic alcoholics can get acute withdrawal Opposite effect to drug – anxious agitated, upset/violent; delirium tremens (tremors, hallucinations) • Chronic effects o CNS: Wernicke-Korsakoff syndrome (Thiamine deficiency, vitamin B1 deficiency) Wernicke encephalopathy (acute) and Korsakoff syndrome (chronic) • Pathology: haemorrhage and necrosis of mamillary bodies • Clinical: Severe memory loss, confabulation o PNS: peripheral neuropathy

Alcohol and the CVS • Cardiomyopathy o Degenerative disease of heart muscle leading to cardiac failure Due to toxic effect of ethanol • Hypertension o More common in chronic alcoholics o Due to Vasopressor effects of ethanol • Cardioprotective effects o 1-2 drinks/day protective o Increased HDL, decreased platelet aggregation

Fetal alcohol syndrome • Syndrome: Growth retardation (LBW, FTT), microcephaly, motor and mental retardation, facial dysmorphology • Unknown how many drinks/day necessary o One of the most common preventable causes of mental retardation • Possibly due to acetaldehyde action in fetal tissues

Alcohol and cancer • Increased incidence of: o Oral cavity cancer, pharynx, oesophagus Causally associated: 10x increased risk vs no drinking o Liver (although, often associated with HBV and HCV) o Other – breast, colorectal Association, not causal: eg. 20% increase = 0.2x risk

Other diseases • Trauma secondary to alcoholic intoxication: violence, falls, motor vehicle accidents, drownings • Diseases associated with destitution (which is associated with alcohol) o Pneumonia and other infections, nutritional deficiency syndromes Lecture 38: Renal and urinary tract structure Dzung Vu

Etymology and definitions • Reno – kidney (latin) • Nephro – kidney subunit (greek) • Pyelo – pelvis • Vesico/cysto – bladder

Background • Muscles o Diaphragm o Psoas Relations: • Genitofemoral from the lumbosacral plexus passes through and onto the anterior surface • Anterior surface – renal arteries, testicular/ovarian arteries, ureter o Quadratus lumborum o Transversus abdominis, internal, external oblique etc • Nerves o Subcostal o Iliohypogastric o Ilioinguinal

Kidneys • R kidney is lower than L o R has a cortically shaped adrenal gland such that upper pole of both kidneys about the same • Retroperitoneal, the most posterior abdominal structure o Lie on the side of the lumbar vertebrae • Superior poles are closer together than lower poles o In not this relation: abnormality eg. horseshoe kidney • Tail of pancreas is infront of the L kidney o Head of pancreas/duodenum are infront of the right kidney

Vessels • T12 aortic hiatus, descending aorta branches descending order: o 2 branches to inferior surface diaphragm o Coeliac trunk Related to the median arcuate ligament of the diaphragm o Renal arteries Pelvis is posterior to renal arteries • Ureter o Found infront of the genitofemoral nerve o Found behind renal arteries • IVC (diaphragm at T8) o Anterior to aorta Thus: Renal veins are anterior to renal arteries o Anterior to R suprarenal gland • Superior mesenteric clamps down L renal vein o Posterior to anterior: L renal artery, superior mesenteric, splenic vein, splenic artery, pancreas

Kidney conceptual overview • Yellow sandpit o Put cones on the sand Fill sand up again • Attach suction cups to cones o Roll up • Original layer of sand is the cortex o Cones are pyramids , cords of the medulla (collecting tubules) In between cords is continuous with cortex renal columns • Suction cups are papillae and connect to minor and major calices (pl. calyx) o Centre of rolled is renal sinus and renal pelvis Contains BVs, nerves and lymphatics • Arteries and veins o Renal artery Segmental arteries (5) • (1 is posterior and larger) • Each into lobar arteries o Become interlobar when go between pyramids At junction of medulla/cortex become arcuate arteries • No anastomoses (arcuate veins do) o Predisposes to infarct

Renal fascia • CT band • Fused: o Together at the top o Laterally as it descends over the psoas (fusing with the psoas fascia) Thus: medially not fused, infection can track down from kidney to psoas/groin) o analogy. “I am the kidney, I put a garbage bag over my head” • Partition between adrenal gland and kidney – controversial: some think there, others do not • Fascia is continuous on both sides o Medial border not fused, fused to CT in the midline o Controversial research that says communication between perirenal spaces • 2 layers o Anterior and posterior layers Parietal peritoneum lies on top of the anterior layer • Pararenal spaces o Around perirenal space, between renal fascia and peritoneum/posterior abdominal wall

Ureter • Constrictions o Pelvic ureteric junction o Iliac artery o Bladder wall • Relations o Psoas major o Bifurcation of common iliac artery o Deep to uterine artery Significant during hysterectomy • 2 ureters are connected by fascia • Kidney stones o Pain from muscle pushing stone through ureter at constriction

Urethra • Male o Long o 1: prostatic o 2: membranous – urogenital diaphragm o 3: penile • BPH – prostate pushes on bladder

Lecture 39: Renal histology P De Permentier

Urinary system • Originates from the mesoderm • Made up of: o 2 bean-shaped kidneys o 2 ureters (muscular urine conduction tubes) o Urinary bladder (fibromuscular bag for temporary urine storage) o Urethra (passageway for urine to outside)

Kidneys – overview • Functions: o Excretory Elimination of impurities from blood – nitrogenous, excesses in electrolytes, water o Homeostatic Ultrafiltration of blood plasma Selective reabsorption o Endocrine Erythropoietin – accelerates erythropoiesis • General features o Thin capsule enclosing entire kidney – made of collagen o Cortex – wide outer rim, beneath capsule o Medulla – radially striated in appearance, beneath cortex Made up of pale, streaked pyramids (lobes) • 10-15 in humans o Arcuate vessels – found at the junction of the two zones o Minor calyces extend from the papillae of the pyramids and join together to form major calyces These join to form the renal pelvis – expanded upper ureter o Hilus – indentation that marks the entrance and exit for ureter, renal vein and artery, lymph vessels, nerves • Finer details o Medulla is striated due to straight tubular and vascular structures that loop down into the medulla and then back up toe the cortex Includes straight portions of proximal and distal tubules o Granular appearance of cortex is due to glomeruli (capillary bundles) Function as ultrafilters that separate water, ions, nitrogenous waste from blood plasma into urine

Blood supply • Renal artery feeds into kidney at the hilum o Divides into: interlobar, arcuate, interlobular arteries o Interlobar arteries then divide into intralobular arterioles ( afferent arterioles ) These then further divide before forming the glomerulus (tuft of capillaries) • Made up of 20-50 capillary loops Afferent arteriole cells on entering the glomerulus change: • Typical smooth muscle cell of tunica media enlarge, become glandular myo-epithelioid cells • Polyhedral in shape and with a larger diameter than smooth muscle cells – juxtaglomerular cells (JG cells) o Produce renin – vasoconstrictor increasing BP o Efferent arteriole drains glomerulus Small diameter than afferent arteriole Few JG cells Branches into capillary network that feeds the convoluted tubules in the region • Also gives off recurrent capillary tubules – vasa recta o These run parallel into the medulla along medullary rays o Venous return drains into interlobular veins before leading into the renal vein

Nephron • Functional unit of the kidney (1 million in each human kidney) o Made up of renal corpuscle and renal tubule • Renal corpuscle o Glomerular capsule (of Bowman) o Afferent arteriole – feeds into glomerulus o Glomerulus Glomerular capsule • Double-layered membrane made up of 2 simple squamous epithelial layers o Outer parietal, inner visceral • 2 poles o Vascular pole – afferent and efferent arterioles enter and leave o Urinary pole – renal tubule leaves capsule Squamous parietal cells thicken and become cuboidal cells of proximal convoluted tubule Capillary network interrupting path of artery • Connected to afferent and efferent arterioles o Afferent enters giving off several capillaries that each supply a lobule of the glomerulus Loops unite to form smaller efferent arteriole Filtration membrane • Plasma leaves the glomerular capillaries passing through fenestrated endothelial cells o Fenestrations (pores) 60-100nm Allows passage of non-cellular elements into blood such as free haemoglobin, but not albumin • Filtration is dependent on: size, charge, configuration o Efferent arteriole – drains the glomerular capillaries • Renal tubule o Involved in resorption and secretion o Begins in cortex at the urinary pole Proximal convoluted tubule – twisted course Descending straight tubule – straight course from cortex to medulla Thin loop of Henle – hairpin loop Ascending loop – returns to cortex Thick ascending limb of Henle’s loop – wall thickens Distal convoluted tubule – becomes tortuous again as it nears the glomerulus of origin

Proximal convoluted tubule and descending straight tubule • PCT o Arises at the urinary pole of the renal corpuscle as an extension of the parietal epithelium of Bowman’s capsule o Longest, most convoluted tubule, makes up most of the cortex Epithelium: simple cuboidal with an apical brush border of microvilli (1um long) • Increases SA for absorption (much absorption occurs in PCT) – not cilia • Descending straight tubule o Begins in a medullary ray of the cortex and ends in the upper medulla Here it becomes the thin limb of Henle’s loop when cells become simple squamous epithelium • Functions o Resorption of 75-80% of water and sodium ions from glomerular ultrafiltrate o Resorption of glucose, amino acids, vitamin C and chloride ions

Loop of Henle o Thin descending and ascending loops are continuations of straight portions of proximal tubules (descending straight tubule) o Arise in medulla and return to cortex after a hairpin bend o Upon returning to cortex, become continuous with thick segments of distal convoluted tubules o Functions: further concentration of ultrafiltrate by osmosis Distal convoluted tubules (DCT) • Shorter, and less convoluted than PCT • 3 parts: o Thick ascending portion Starts in the medulla at the termination of the ascending limb of Henle’s loop • Begins as the thick ascending segment of Henle’s loop • Squamous epithelium of thin loop becomes cuboidal • Outer diameter increases to 50um with a similar increase in luminal diameter o Macula densa Thick ascending portion ascends into the cortex to the vascular pole of the parent glomerulus – macula densa Made of closely packed, modified, narrow cells in a palisade configuration • Dark staining nuclei are close together (like a string of black beads) • Close to the JG cells allowing exchange of materials (eg. sodium ions) o Convoluted segment • Thick ascending limb and convoluted potion of distal tubule are similar to corresponding structures in PCT o Exceptions: Basal region • Extensive lateral infoldings of plasmalemma envelope and long mitochondria causing basal striations Lumen is wider in DCT (30-50um) and has a smooth surface with a lack of brush borders on epithelial apices • Functions o Resorption of sodium ions and 9% of water from tubular fluid o Secretion of K + and hydrogen ions into lumen Regulates acid-base and electrolyte balance of body (along with collecting tubule) o Aldosterone has influence

Collecting tubules • Continues from the DCT in the cortex o Begin as short, arched tubules that converge forming bundles of straight tubules o Travel towards medulla (making up medullary rays) In the medulla, tubules join together to form large ducts that open onto the apex of the renal papillae as the papillary ducts of Bellini • From here, enter into minor calyces etc • 40um diameter lined by cuboidal epithelium • Presence of ADH makes collecting ducts permeable to water creating a hypertonic urine in tubules

Juxtaglomerular apparatus (JGA) • 3 parts: JG cells, Macula densa, Plokissen (mesangial cells) • Juxtaglomerular cells (JG cells) o Modified smooth muscle cells in afferent arteriole walls before they enter Bowman’s capsule at vascular pole o Granules in cytoplasm contain renin Respond to changes in blood pressure in afferent arteriole releasing renin • Decrease in BP causes renin release o Renin-angiotensin system Renin release reacts with plasma globulin angiotensinogen producing inactive polypeptide angiotensin I • Angiotensin I is converted by angiotensin converting enzyme to angiotensin II o Angiotensin II is a vasoconstrictor of arterioles that increases systemic blood pressure o Angiotensin II also causes release of aldosterone from adrenal cortex Aldosterone acts on kidney tubules causing sodium retention and thus fluid increasing BP o Synthesises erythropoietin – glycoprotein stimulating RBC production in bone marrow

JGA continued • Modified epithelial lining cells of DCT (macula densa) o Lies adjacent to afferent arteriole containing JG cells Made up of small, darkly stained round nuclei arranged like a string of beads o Function: monitors sodium concentrations in distal tubule • Polkissen (mesangial) cells [german: pole cushions] o Small, clear cells lying between afferent, efferent arterioles and macula densa o Function: may be macrophages that cleanse basal lamina of foreign material that accumulates on the membrane during filtration

Urinary passageways • Minor and major calyces, renal pelves, ureters, bladder and urethrae conduct urine o the outside o 3 tunicas: Mucosa Muscularis Adventitia • Mucosa o Transitional epithelium (only found in urinary system) Stratification changes • 2-3 cell layers in calyces and pelvis • 4-5 in ureter • 6-8 in relaxed bladder (3-4 squamous in distension) Superficial surface is large and dome-shaped giving a scalloped appearance to mucosal surface Often binucleate with prominent nucleoli o Bladder: Cells slide past each other on bladder filling reducing number of cell layers • Due to interdigitating cell junctions (plaques) act as hinges • On bladder filling, hinges open and allow extension of cells without damage to cell surfaces Plasmalemma (cell membrane) is thick making it impervious to urine • Creates a blood-urine barrier Tight junctions exist between cells • Aid creation of blood-urine barrier • Lamina propria o Calyces and renal pelvis Elastic and reticular fibres (fibroblasts, collagen) with few lymphocytes (neutrophils) No glands o Ureters and bladder Diffuse lymphoid tissue with scattered collagenous and elastic fibres Capillaries Bladder: lymph nodules and mucous glands near internal sphincter Ureter: mucosa has longitudinal folds (star-shaped lumen • Bladder (relaxed): mucosal folds are thick and irregular • Muscularis o Calyces and pelvis Circular outer smooth muscle fibres, inner longitudinal band of smooth muscle (reverse of GIT) o Ureter Outer circular and inner longitudinal become discrete layers Lower third of ureter, 3 rd layer envelops circular muscle – outermost longitudinal layer o Layers increase in size descending into the bladder • Adventitia o Fibro-elastic covering blending with surrounding CT o Pelvis – continuous with capsule of kidney o Ureters and urinary bladder Retroperitoneal except superior surface of bladder (has peritoneum) • Here, outer coat is a serosa – not adventitia o Contains BVs, nerves, lympahtics

Male urethra • Mucous membrane tube 20cm long • Functions: o Transports urine and sperm to the outside • Prostatic part (4cm) o Next to the bladder Embedded in the prostate gland Related to the ejaculatory ducts (terminations of the ductus deferentes) o Prostate gland empties secretions here: Seminal fluid (fructose, sucrose, PGs, seminal plasmin (AB), semen motility factors) o Lined with transitional epithelium, distally becomes pseudostratified or stratified columnar LP contains elastic fibres intertwining with venules Mucous urethral glands of Littre empty into lumen • Membranous segment (1-2cm) o Where the urethra pierces the urogenital diaphragm (fibromuscular layer between pelvic bones) Acquires skeletal muscle here • Forms circular muscle that makes up the external (voluntary) sphincter o Pseudostratified or stratified columnar epithelium o Urethral glands increase in size and number • Cavernous (penile) urethra (~15cm) o Stratified or pseudostratified columnar epithelium At dilated opening: stratified squamous

Female urethra • Shorter tube (3-5cm) • Lined with transitional epithelium only near bladder o Distal portion is stratified squamous that is continuous with epidermis of skin o Glands open onto mucosal surface • Thin LP with elastic fibres • Outer circular and inner longitudinal layer of smooth muscle o Some circular fibres form internal sphincter o At lower end, other smooth muscle fibres are reinforced by skeletal muscle to form voluntary external sphincter urethrae • Women get more UTIs: o Exposed o Shorter o Moist environment o Uterus is on top of the bladder - pregnancy

Lecture 40: Renal blood flow, filtration and clearance Karen Gibson

Formation of urine • Glomerular filtration – fluid passes from glomerular capillary into the tubule at the bowman’s capsule o Tubular secretion – fluid passes from efferent arteriole/peritubular capillary into the tubule o Tubular reabsorption – fluid passes from tubule into arteriole Excretion – fluid continues in tubule and is excreted as urine • processes above combine in different ways for processing of different substances • Different patterns of urine formation: o Glomerular filtration involves a fraction of plasma Eg. 20% of plasma is filtered 80% remains in arteriole and leaves via renal veins o A: (eg. inulin) – no secretion/reabsorption o B: (eg. vitamin C) – some reabsorption o C: (eg. glucose, normal) – complete reabsorption o D: (eg. Para amino hippurate) – complete secretion

Blood to kidney • Renal artery 5 segmental arteries interlobar arcuate (parallel to cortex) interlobular afferent arteriole glomerulus efferent arteriole peritubular capillaries veins o Efferent arteriole diameter is smaller than afferent (more downstream)

Renal blood flow • ~1200ml/min in males, 980ml/min in females o 20-25% of CO • High blood flow compared to other organs o 4ml/min/g vs brain 0.5, heart 0.8 • Needs blood for filtration, rather than for oxygen requirements o A-V oxygen difference is low: 63umol/100ml vs brain 275, heart 500 • Distribution: 90% cortex, 10% medulla, 1-2% papillae o Papillae are deep in the medulla, however are not unperfused- get similar amount of blood as resting muscle • Renal plasma flow = renal blood flow * (100-Hct)/100 o Ie, the fraction of renal blood flow which is plasma (therefore available for filtration Renal blood flow and pressure • Renal artery starts at systemic arterial pressure o Decreases in afferent arteriole ~40-45% systemic in glomerular capillary (high for capillaries in general) o Decreases again in efferent arteriole ~8mmHg in peritubular capillaries, down to nothing in renal vein o THUS: afferent and efferent arterioles provide resistance to blood flow • Flow is dependent on resistance created by arteriolar tone o Any organ: Q = P/R Q: flow, P: A-V hydrostatic pressure difference, R: vascular resistance o Thus, if perfusion pressure is constant, Constriction of afferent/efferent arterioles = fall in RBF Dilation of afferent/efferent arterioles = rise in RBF • Autoregulation – relative constancy of RPF, and thus GFR despite variations in arteriole pressure o Pressure is often not constant, for a wide range of blood pressures (autoregulatory range) kidney can keep RPF, GFR constant o Based on afferent, efferent constriction/dilation Intrinsic to kidney, independent of systemic nerves/hormones • If you paralyse the smooth muscle, lose autoregulation Thought to be a myogenic reflex – a response to stretch by the smooth muscle

Other factors influencing RBF • Factors can override autoregulation: o Angiotensin II o Catecholamines o Anti-diuretic hormone

Renin-angiotensin system • Angiotensinogen (protein made in the liver) is cleaved to angiotensin I by renin (made in JG cells of kidney) o Angiotensin I (10AA, no biological activity) • Angiotensin I is cleaved by Angiotensin II converting enzyme (ACE) o Target for blood pressure lowering drugs: ACEI o Inactivates bradykinin (vasodilator) o Angiotensin II (8AA, active peptide) • Angiotensin II acts at AT1 and AT2 receptors • Recent discoveries: o ACE II (converts ang I and ang II into various products) o Prorenin receptor that gives prorenin activity

Renin-angiotensin system - continued • Juxtaglomerular apparatus o Granular cells in the afferent arteriole • Distal tubule – when it comes back up communicates with its source glomerulus o Macula densa communicates with JG cells • Intraglomerular mesangial cells o Change the filtration SA, sit between glomeruli loops • Control of renin release o Renal baroreceptor Decreased perfusion pressure increases renin o Macula densa Measure NaCl delivered to distal tubule, decreased NaCl, increased renin o Renal sympathetic nerves Stimulated, increases renin; catecholamine stimulate renin via beta receptors o Feedback Intravascular volume, blood pressure and renal perfusion – inhibition AngII acts on J-G cells inhibiting release (short loop) o Others Increase – chronic hypokalaemia, PGE2 Decrease – acute hypernatraemia, ADH, ANF (atrial natriuretic factor) • Actions of angiotensin II o Vasoconstriction of arterioles (systemic and kidney) o Renal Vasoconstriction Proximal sodium reabsorption Contraction of mesangial cells – decrease area of absorption decreasing GFR o Adrenal - aldosterone o Neural Facilitates sympathetic activity, increases BP Inhibits vagus Increases ADH secretion Increases ACTH secretion Dipsogen – makes you thirsty

Sympathetic nerves • Many sympathetic (NA) neurones o No parasympathetic neurones • Effects: o Vasoconstriction via α-adrenoreceptors o Renin release via β-adrenoreceptors on JG cells o Increased sodium and water reabsorption by tubules • Small amount of tonic discharge at rest o However, not very important transplants, no nerve supply, work fine

Glomerular filtration • Blood flowing through the glomerulus is filtered into the urinary space o Forms an ultrafiltrate (no RBCs or proteins) Similar composition of small MW substances as plasma Formation rate is the glomerular filtration rate (GFR) • Normal GFR is 125ml/min for males o = 7.5L/hour, 180L/day • Filtration barrier o 3 layers: Endothelial cells of the capillary • Fenestrations (60-80nm) – no diaphragm Basement membrane (3 layers) Epithelial cells of the tubule • Foot processes of podocytes o Constrict to change the filtration SA o Unclog the filter – phagocytosis o Hormone release • Covered in –ve charges (glycosialoprotein coat) which prevents protein filtration o Filtration is based on 2 things: Size: doesn’t let larger than 70nm diameter through Charge:–ve charge on epithelial layer stops protein • Forces involved (similar to systemic capillaries): o Single nephron glomerular filtration rate (SNGFR) = Kf * [net filtration pressure]

= Kf * [(P GC – PT) – (π GC – πT)] P: hydrostatic pressure; π: colloid osmotic pressure; GC: glomerular capillary; T: tubule Kf: glomerular ultrafiltration coefficient = effective surface area (A) * capillary wall conductivity (k) o Hydrostatic pressure pushes blood out into tubule Colloid oncotic pressure due to plasma proteins aims to suck water back in (=~25mmHg) o EG values: Notes:

• πT is negligible because there is very little protein in the tubule • Filtration stops when colloid osmotic pressure rises to equal net hydrostatic pressure

Filtration fraction • Not all plasma that flows in capillaries filters into Bowman’s space o If it did, blood would have no fluid/volume • Filtration fraction = GFR/RPF o Eg: 125ml/min / 600ml/min o FF = 0.16-0.20

Measurement of GFR • Use a substance that is freely filtered at the glomerulus and not reabsorbed or secreted down the tubule o Thus, everything filtered ends up in the urine • Also needs: o Not protein bound o Not metabolised o Not stored in kidney o Non-toxic o No effect on filtration rate o Easy to measure in plasma and urine Measurement of GFR – continued • Calculations: o Amount filtered = GFR * P ay Pay is the arterial plasma concentration of y

o Amount excreted = U y *

UY: urinary concentration of y; : urine flow rate (volume of urine produced/unit time, ml/min) o Thus:

GFR * P ay = U y *

GFR = U y * /P ay • Substance used: o Gold standard – inulin Inert starch-like polymer of fructose found in artichokes, dahlias, chickory

o Thus: GFR = U INULIN * /P INULIN (inulin clearance) o However: inulin needs to be infused Thus, more convenient to use creatinine clearance as an estimate • Produced from muscle metabolism • Plasma creatinine is not sufficient, thus take 24 hour urine sample also • Creatinine o GFR can decrease by 50% before plasma creatinine shows a significant difference Can show useful measurements at end stage renal failure o Plasma creatinine gives a large range of creatinine clearance values Depends on muscle mass

Measurement of RPF • Uses the Fick principle o Amount of substance x entering kidney is equal to the amount of substance x leaving (assuming no metabolism or synthesis) • Calculations: o Entering = RPF a * P ax

o Leaving = RPF v * P vx + U x * RPF V ~ RPF a (ie. probably 600 vs 599)

• Thus: RPF = U x * /(P ax – Pvx ) o Use a substance that is completely cleared by the kidney, renal venous blood concentration can be assumed as zero Thus:

• RPF = U x * /P ax EG: para-amino hippuric acid (PAH), radio-opaque substances (eg. diodrast)

• Thus: RPF = U PAH * /P aPAH (PAH clearance) • Effective renal plasma flow (ERPF) o Some PAH appears in renal vein (ie, blood from the capsule, pelvis, perirenal fat and medulla) Thus Clearance PAH strictly gives ERPF which is ~90% of true RPF o For most purposes, ERPF is adequate

Renal clearance • One function of the kidney is remove (clear) substances from the plasma o Can measure clearance success by looking at how much plasma is cleared/unit time • Definition: renal clearance of a substance is the minimal volume of plasma which could have supplied the amount of substance excreted in urine per unit time o Measured in volume of plasma/unit time (eg. ml/min) o Formula:

Clearance A = C A = U A * /P A

• UA: urinary concentration of A; : urine flow rate (volume/time); P A: plasma conc of A • EG: A and B excrete urea at 0.28mmol/min o Plasma urea A: 4mmol/l; B: 40mmol/l A: 0.28 mmol of urea in one minute is the amount of urea excreted in 70ml plasma (0.28/4) B: 0.28 mmol of urea in one minute is the amount of urea excreted in 7ml of plasma (0.28/40) o Thus, both were excreting same amount per minute, A had better kidney function If B had normal kidney function, excretion should have been more • Note: this is a virtual minimum volume, not a real volume o A small amount of urea is removed from every ml rather than from a certain number of mls o CINULIN and C PAH are special examples of the clearance formula • Signifcance o Clearance tells us how kidney handles substances CX < GFR, x is reabsorbed (if filtered freely) Cx = 0, substance is completely reabsorbed (eg. glucose) • In a non-diabetic, no glucose is excreted (U =0) Cx = 0, substance is not filtered (eg. protein) + Cx >GFR, substance undergoes net secretion (eg: K )

• Upper limit of clearance is C PAH

Lecture 41: Introduction to kidney disease George Mangos

Kidney – ugly but interesting • Converts 1700L of blood into 1.5L of specialised fluid – urine o Excretes waste products o Maintains acid-base balance o Maintains salt-water balance • Produces hormones: EPO, renin, prostaglandins

Imaging • Ultrasound o Advantages: non-invasive Disadvantages: resolution o Can measure Thickness of the kidney parenchyma Length of the kidney (diagnose CKD) See obstructions • IV urogram (pyelogram) o Iodine contrast given IV o Advantages: Nephrogram – cortex/medulla Pyelogram – ureters, calyxes, bladder o Disadvantages: Contrast Preparation time Ionising radiation o Mostly replaced by CT scan • CT scan o Advantages: resolution, sensitive o Disadvantages: radiation, contrast (non-contrast scans are possible)

Nephron • Glomerulus o Ultrafiltration of plasma Depends on size and charge: • <3.5nm (albumin 3.6nm) • -ve charge not allowed through o 144L primitive urine/day o Podocyte has foot processes all over outside of capillaries with various functions, in particular has a negatively charged coat that prevents proteins diffusing through • Tubules and interstitium o Reabsorption 99% of glomerular ultrafiltrate (Na, K, PO4, AA, proteins) o Secretion + K, H , NH 3 o Second-rate blood supply Dehydration, potential necrosis of tubules acute tubular necrosis (acute renal failure) • Vessels become contracted when BP lost

GFR • Main measure of renal function • Normal GFR is 90-120ml/min (equates to 144L/day) • Kidney failure reduced GFR o Chronic kidney injury (chronic renal failure) – longstanding and irreversible o Acute kidney injury (acute renal failure) – sudden and potentially reversible • Hyperfiltration increased GFR o Eg. pregnancy, diabetes (early stage of diabetes nephropathy) Measuring GFR • Need a substance that is freely filtered by the glomerulus and then not handled by the tubules o Inulin clearance (laboratory) Not convenient, = UV/P o Serum creatinine (clinical) Formed by muscle metabolism • 10% tubular reabsorption • Varies with muscle mass, age, sex, weight Inversely proportional to GFR Needs a large change in GFR to show abnormality (insensitive marker) o Creatinine clearance 24 hours urine measurement o 99mTc-DTPA (Diethylene Triamine Penta-acetic acid) GFR measurement • Can estimate GFR from serum [Cr] eGFR o Equations that relate GFR to serum Cr Cockroft Gault equation, MDRD equation o More useful than 24 hour urine creatinine clearance • Best estimate of GFR is MDRD eGFR

Assessing tubular function • Urine o Acid urine (pH usually <6) o Protein – normal excretion (<300mg/day) Protein can leak through damaged glomerulus o Normally concentrated o No glucose • Serum o Normal Ca, PO4, K, Na • Use the dipstick • Tubular failure: o Pass a lot of urine o Eg. obstructive uropathy chronic urinary retention

Failure to concentrate • Diabetes insipidus o Failure of ADH release – central o Failure of kidney to respond – nephrogenic Eg. lithium treatment for bipolar • Presents with polyuria • Can be progressive • Causes are often unknown

Failure to acidify • Renal tubular acidosis o Alkaline urine o Systemic acidosis • Cause o Failure to reabsorb bicarbonate (proximal renal tubular acidosis) o Failure to excrete H + (distal renal tubular acidosis)

Determining normal kidney function (summary) • Normal structure US, IVP, CT • Normal function Serum Cr + eGFR • Normal urine negative dipstick

Major syndromes • Acute renal failure o Sudden reduction in GFR (hours-weeks), rise in serum Cr o Usually reversible o Eg causes: Shock (haemorrhage), crush injuries – myoglobin release from muscles) o 3 mechanisms Pre ARF – before kidney, eg. cut femoral artery, dehydration Intra ARF – glomerulus, nephron; eg. DM, nephritis, polycystic kidney Post (obstructive) ARF – renal pelvis, ureter, bladder, urethra; eg stones, prostate • Chronic renal failure and chronic kidney disease o Slow and irreversible reduction in GFR Definitions, based on eGFR (ml/min): >90: stage 1 (normal); 60-89: stage 2 (mild CRF); 30-59: stage 3 (moderate CRF); 15-29: stage 4 (severe CRF); <15: stage 5 (end stage renal failure) o Causes: DM, glomerulonephritis, APKD (adult polycystic kidney disease), vesico-ureteric reflux, hypertension, analgesics o Mortality Rate of death greatly increases with decreased GFR, especially death by CV disease o Affects kidney function: Decreased urine concentration Waste products – make you sick Acid builds up – acidosis Salt/water balance lost Hormone problems – EPO decreased anaemia • EPO, Renin, PG can be given synthetically o Risk factors: >50, smoke, obesity, DM, ATSI, family Hx, HBP • Other: o Nephrotic syndrome, Microscopic haematuria, Proteinuria, Hypertension, UTI, Nephrolithiasis, Renal tubular defects

Chronic renal failure - effects • Cardiovascular disease o Hypertension, Left ventricular hypertrophy, premature vascular disease o Increased mortality • Anaemia o Erythropoietin deficiency o Iron deficiency • Bone disease o Hypocalcaemia, hyperphophataemia Phosphate cause low calcium, parathyroid senses low calcium and causes osteoclasts to break down bone • High turnover bone disease • Calcium can deposit in wrong location Secondary hyperparathyroidism o Vitamin D deficiency Osteomalacia o Mixed bone disease o Adynamic bone disease

Abnormal results on dipstick • Haematuria o Can be due to lesion anywhere in urinary system o Microscopic haematuria is common 6% Australians Most benign Always needs evaluation cystoscopy/CT • Proteinuria o Often indicates glomerular disease o Quantitate by 24 hour urine collection Normal range <300mg/day Greater amounts worse o Cause can be tubular or glomerular • Blood and protein o Glomerular disease – glomerularnephritis Immune mediated disease causing inflammation of glomerulus and commonly renal failure • Heavy proteinuria o >3g/day o Always glomerular Diabetes Glomerulonephritis o Can be associated with hypoalbuminaemia and oedema Nephrotic syndrome: • Heavy proteinuria • Low albumin • Oedema • Leukocytes and nitrites (with/without blood and protein) o Urinary tract infection Cystitis, pyelonephritis Common in women, 90% due to e. coli Suspected, send MSU to lab for culture

Other abnormalities • Renal colic o Severe flank pain radiating to iliac fossa and groin Cause: Stone in kidney or ureter • Stone can spontaneously pass or may require surgery o Associated with Microscopic or macroscopic haematuria • Polyuria o Often a tubular problem Causes: drugs, poisons, diseases

Structural abnormalities • Cysts – benign • Vesico-ureteric reflux o Congenital failure of ureter entering bladder o Can cause ESKD • Adult polycystic kidney disease o Cystic fluid dark on CT • Pelviureteric junction (PUJ) obstruction • Renal cell carcinoma – Grawitz tumour • Hydronephrosis – obstruction of renal pelvis by obstruction leading to progressive atrophy of kidney

Summary • Think of disorder as aberration of normal function

Lecture 42: Tubular reabsorption and secretion Karen Gibson

Modification of filtrate • Blood is filtered to remove toxins, then important substances need to be reclaimed o EG: sodium, - a lot reabsorbed; glucose – all reabsorbed

Filtered Urine Reabsorption Volume/Water 180L/day 1-2L/day 178 -179L/day Sodium 25200mmol/day (GRF x plasma 150 mmol/day 25050 mmol/day sodium, eg. 140mmol) Glucose 720 mmol/day 0 720 mmol/day Osmolality 300 mosm/kg 50 -1200 mosm pH 7.4 4.4 -8.4

Reabsorption • Passive reabsorption o Eg. water, urea o Follow osmotic gradient/concentration gradients • Tubular maximum (transport maximum) reabsorption o Involves a carrier (can be saturated) Thus there is a fixed maximum transport rate o Eg: glucose, phosphate, vitamin C, amino acids • Active gradient-time limited reabsorption o Rate of transport is limited by: The gradient that is established across the tubular wall The time the fluid is in contact with the epithelium o Eg: sodium

Secretion • Passive secretion + o Eg. NH 3 (diffusion trapping: trapped in the lumen by addition of H ) o Active tubular maximum (transport maximum) limited Eg. secretion of organic acids (PAH, diodrast, penicillin, bile acids, uric acid), organic bases (histamine, creatinine, catecholamines) o Gradient –time limited Eg. H +

Sodium reabsorption • Gradient-time limited • Tubule wall is like yogurt tubs lined up with lids on luminal side • Methods of transport: o Transcellular – through yogurt Active transport, pushed into the cell by concentration gradient that is set up by Na/K/ATPase pumps • Na/K/ATPase pumps are on basolateral side o Paracellular – through gaps between tubs Passive diffusion through tight junctions between cells • Diffusion is based on the gradient created by the Na/K/ATPase pumps on basolateral side • Can run in both directions

Parts of the tubule • Different parts secrete/reabsorb differently o Glomerulus proximal convoluted tubule straight proximal tubule descending thin limb of Henle’s loop ascending thin limb of Henle’s loop thick ascending limb of Henle’s loop Distal convoluted tubule connecting tubule cortical collecting duct medullary collecting duct papillary collecting duct • Nephros come in 2 types: functionally different o Cortical – 75% Superficial nephrons • Short loops of Henle that end at the junction of the inner and outer medulla • No thin ascending limb Midcortical nephrons • Either short or long loops of Henle o Juxtamedullary – 25% Long loops of Henle Have thin ascending limb

Proximal tubule • Most reabsorption occurs here o All glucose, amino acids o >2/3 of Na, Cl and water in isosmotic amounts (proportions maintained so osmolality doesn’t change)

o Most HCO 3, K (80-90%), 2/3 Ca o 30% Mg, 80% phosphate o 50% urea o Lactate, citrate, Kreb’s cycle intermediates, vitamins • Other functions: o Secretion of organic acids and bases o Synthesis of ammonium • Segments o S1 – early proximal convoluted o S2 – remaining cortical proximal convoluted tubule o S3 – straight convoluted tubule (pars recta) o Early segments: Rapid transcellular transport, leaky paracellular More mitochondria, increased SA with microvilli and thus increased solute reabsorption o Later segments: Slower transcellular reabsorption, tighter paracellular pathways Decreased SA o Functions of all segments are quite similar, S2 predominates in organic acid and base secretion • Solute concentrations in proximal tubule o TF = tubular fluid, P = plasma o Inulin – concentration increases 3x because water is reabsorbed and inulin doesn’t reabsorb o Chloride – reabsorption starts ¼ way through tubule, thus initial rise, then isosmotic o Na + - reabsorbs in proportion with water - Often reabsorbs with amino acids/ glucose/HCO 3 o Osmolality – small drop, although said to be isosmotic (small drop means osmolality in plasma >TF) o AA, glucose – drop as these are completely reabsorbed o PAH – secretion in S2, thus concentration increases to 15x higher than plasma o Potential difference – negative initially because Na + taken out of lumen, then Cl - are taken out causing it to go positive This difference is small because the paracellular pathways don’t allow a large change Glomerulotubular balance • Peritubular capillary has a high colloid osmotic pressure and low hydrostatic o This has high colloid osmotic because it’s been to glomerular capillaries already and had fluid taken out • The fraction of sodium reabsorbed by the proximal tubule remains relatively constant despite changing GFR o Ie: more filtered, the more reabsorbed o Related to changes in peritubular Starling’s forces The higher the GFR, the more water taken out of peritubular capillary, thus increasing colloid osmotic pressure in capillary in proportion causing increase in forces that drive reabsorption

Glucose reabsorption • Filtered load of glucose depends on plasma glucose o This is reabsorbed to a certain amount until the glucose carriers become saturated (tubular maximum) Tubular maximum is approx 2mmol/min o From here, excess is excreted (glucose filtration has passed the renal threshold: ~14mmol/L ) • Definitions: o Tubular maximum – maximum rate of reabsorption that is possible when carrier is completely saturated Glucose, Tm ~2mmol/min in males o Renal threshold – plasma concentration where solute begins to appear in the urine Glucose, Rt ~14mmol/l Theoretical threshold = Tm/GFR (~17mmol/l) • Actual threshold is slightly lower due to splay o Ie. carrier doesn’t work at maximum until it absolutely has to o Not every nephron is identical o Normal plasma glucose: 3.5-6mmol/l Thus in a non-diabetic, glucose doesn’t normally appear in urine • Transporters: o Early proximal tubule cell SGLT2 (pushes glucose into cell using the Na + concentration gradient) • Glucose intracellularly is only 70x outside GLUT 2 puts glucose into interstitial fluid o Late proximal tubule cell SGLT1 (pushes glucose into cell using 2Na + and concentration gradient) • Glucose intracellularly is 4900x outside GLUT 1 puts glucose into interstitial fluid

Loop of Henle • Thin descending limb o Permeability (thin epithelium) Very permeable to water Impermeable/slightly permeable to Na, Cl, urea No active transport o Filtration Water leaves the lumen Osmolality increases o End product – 300mOsm/kg at end of proximal tubules Cortical nephrons • Osmolality: 600mOsm/kg (electrolytes 560, urea 40) • Volume of filtrate has been halved Juxtamedullary nephrons • Osmolality: 1200mOsm/kg (electrolytes 1120, urea 80) • Volume of filtrate has been quartered • Interstitium of medulla has a high osmolality and thus drains water easily • Thin ascending limb – only in juxtamedullary nephrons o Permeability Impermeable to water Highly permeable to Na and Cl Moderately permeable to urea No active solute transport o Filtration NaCl leaves passively via the concentration gradient Urea enters Fluid becomes more dilute • More NaCl leaves than urea enters and volume stays the same (no water permeability) o End product – 1200mOsm/kg at end of thin descending limb Osmolality: 500mOsm/kg (electrolytes 400, urea 100) • Thick ascending limb o Permeability Impermeable to water Low urea permeability Active sodium chloride transport High permeable paracellular pathway + Reabsorbs HCO 3 by H secretion o End product – 500mOsm/kg at end of thin ascending limb; 600mOsm/kg at end of thin descending limb Juxtamedullarly nephrons • Osmolality: 200mOsm/kg (electrolytes 100, urea 100) Cortical nephrons • Osmolality: 140mOsm/kg (electrolytes 100, urea 40) Average • Osmolality: 150mOsm/kg (electrolytes 100, urea 50) o Transporters Na/2Cl/K symporter – uses Na + concentration gradient • Na/K/ATPase pumps out Na + from cell creating gradient • Furosemide blocks this symporter and increases urine excretion of sodium thereby reducing BV to treat congestive heart failure and oedema On the whole, at this level we are taking more negative ions than positive ions from lumen and thus creating a positive potential on the luminal side • This encourages paracellular diffusion of positive ions: Na +, K +, Ca 2+ , Mg 2+

Distal convoluted tubule and connecting tubule • Functionally similar o Connecting tubule is sensitive to hormone such as parathyroid hormone: Ca 2+ , phosphate levels • Permeability: o Water impermeable – filtrate remains same volume o Low urea permeability o Active sodium chloride reabsorption o Tight junctional complexes • Filtration o 8-10% of filtered sodium is reabsorbed here (2/3 proximal tubules, 20% loop of Henle) • End product – 150mOsm/kg at end of loop of Henle o Osmolality: 100mOsm/kg (electrolytes 50, urea 50) • Transporters: o Distal convoluted tubule Na/Cl symporter • Blocked by thiazides (milder diuretic than furosemide because less Na reabsorbed here) • Uses Na + gradient

o Collecting duct Principal Intercalated 3 parts: cortical, medullary, papillary collecting duct Cortical cd 70 -80% 20 -30% 2 cell types Medullary cd >90% <10% • Principal Papillary cd 100% 0% o Light cells o Na reabsorption and K secretion o Respond to aldosterone (Na and K reabs) and ADH (water reabs) • Intercalated o Dark cells o α-intercalated cells reabsorb K, secrete H o β-intercalated cells secrete HCO 3 ratio α: β depends on acid-base balance o Principal cells: ENaC – epithelial sodium channel Aldosterone acts here o Alpha-intercalated: H/ATPase; H/K/ATPase pump • Acidic blood increases transporters Beta-intercalated is similar cell but reversed in direction

Overall trends of collecting ductubles • Water permeability o Absence of ADH, collecting tubule is impermeable to water (but sodium is taken out) o ADH present, permeable to water • Urea o Cortical and medullary segments impermeable to urea Papillary segment permeable (inner medullary connecting ductule) • Apical membrane: UTa1, basolateral: UTa4 • ADH stimulates UT1 • Final urine o Maximum ADH urinary osmolality is 1200mOsm/kg (non-urea: 600, urea: 600), flow: 15ml/min o No ADH osmolality 60-70 mosm/kg (non-urea: 20, urea: 50), flow: 15 ml/min

Diagram: note: % is percentage of filtrate remaining

Lecture 43: Water balance Karen Gibson

Balance • In water balance, want a zero balance: inputs = outputs (don’t want shrinking/swelling cells) o Outputs > inputs +ve balance o Inputs > outputs -ve balance • Inputs and outputs: o Water inputs: Food ~800-1000ml/day • Oxidation of food ~300-400ml/day Liquid intake ~1000-2000ml/day • Varies considerably from <1000ml to >20L Total: ~2100-3400ml/day o Water outputs: Insensible losses (breathing, transpiration through skin) ~800-1000ml/day Sweat ~200ml/day Faeces ~100ml/day Urine ~1000-2000ml • Varies considerably from 500ml to 20L/day • 500-600ml is an obligatory loss such that we can removal all solutes necessary Total: ~2100-3400ml/day

Regulation of water balance • Thirst and drinking • Kidneys o Excess water can be excreted and urine diluted o Scarce water can be retained and urine concentrated • Inhibition and stimulation of ADH o Controls renal water excretion

Thirst • Regulated by hypothalamus and cerebral cortex • Stimulated by: o Increased osmolality (1-2% increase), if osmolality increases, want to increase BV to equalise o Fall in extracellular volume (not as sensitive as osmolality: 10% increase) o Activation of renin-angiotensin system (AngII) o Dryness of mouth/throat o can satisfy thirst before time for plasma osmolality to equalise, thus thought that there are receptors in the oral cavity

Dilute urine • Solute is taken out of tubule and water left behind • Location: o Occurs in the thin ascending loop of henle and onwards Thin aLH, thick aLH and dct are water impermeable (thus can take out Na, but leave water) o No ADH, collecting duct is water impermeable • Medullary interstitium osmolality is ~600mOsm/kg in diuresis (high production of dilute urine) o Vs trying to concentrate urine, 1200mOsm/kg

Concentrated urine • Remove water from tubule when there is excess solute • Requirements: o High medullary osmolality Rises progressively from outer to inner medulla o ADH • Filtration o Urine passes through medulla in collecting ductules on the way to the renal pelvis If collecting ductules are permeable to water (ADH present), water flows out down osmotic gradient • Water flow continues until luminal osmolality = medullary interstitial osmolality o ADH makes collecting ductule permeable to water Thus, water flows out along osmotic gradient concentrating urine • Diagram notes: o Medullary interstitium osmolality is created by half Na +, half urea

Medullary interstitium gradient • Countercurrent mechanism o 3 components: Active countercurrent multiplication – outer medulla Passive countercurrent multiplication – inner medulla Countercurrent exchange – vasa rectae

Active countercurrent multiplication – loop of henle • Movement of fluid and dispersion of solutes from tubule into interstitial space o Proximal side of loop is permeable to water and thus osmolality equalises with interstitium o Distal side of loop has NaCl pumps and thus osmolality decreases by ~200 • As fluid moves through, proximal and distal sides do their actions and cause a gradient to occur between top and bottom o Thus medullary interstitium gradient is established

Passive countercurrent multiplication • Loop of henle has: 1200mOsm/kg (80 urea, 1120 NaCl) o Interstitium has 1200mOsm/kg (600 urea, 600 NaCl) o Thus, sodium freely diffuses into the interstitium from the loop of henle Adds to sodium gradient • Urea recycling o Thin ascending LH, some urea moves into the tubules but not more than NaCl moving out This facilitates NaCl movement out by diluting tubule fluid somewhat o In collecting ductule, urea is concentrated by water reabsorption o At the medullary connecting ductules, urea moves by passive diffusion back into the interstitium via urea transporters (activated by ADH) Increases osmolality and aids interstitial gradient

Countercurrent exchange • Prevents dissipation of medullar interstitial gradient • Capillaries also have loops o Equilibration occurs down loop relative to level of interstitium Fluid starts at 300 and loses water as it loses solutes down the loop solutes are taken away in fractions Fluid equilibrates at bottom as 1200mOsm In the ascending loop, water is gained such that osmolality decreases again to ~300 • May ~325, slight solute excess is due to lag time in equilibration o Want a slow flow in capillaries to maximise time for equilibration, otherwise get a higher excess in solute removed o Thus, blood is supplied and solutes in interstitium are not interrupted

Factors affecting renal concentrating • Length of the loop of henle o % of long loops • Availability of urea – higher protein diet, easier to concentrate urea • Rate of flow in loop of henle – increased flow, increased water reabsorption • Rate of flow in collecting duct – increased flow, increased water reabsorption • Blood flow in the vasa recta – high flow, high water reabsorption (less water resecreted) • Loop diuretics – eg. furosemide which inhibits transporter and causes more sodium excretion • Diseases that affect structures in the medulla

Antidiuretic hormone • Other names – ADH, arginine, vasopressin, AVP • 8AA peptide o Produced by nerve cells in supraoptic and paraventricular nuclei of hypothalamus Near the areas that control thirst o Axons extend down into the posterior pituitary • ADH is released into the blood by action potential from axons

Control of ADH • Osmotic control: o ADH nerves act as osmoreceptors Osmolality rises, cells shrink and ADH release stimulated • Small rise in plasma osmolality causes a large increase in ADH (sensitive) Osmolality falls, cells swell, ADH release inhibited • Moderate f all in plasma osmolality causes ADH to go undetectable • Haemodynamic control: o Fall in blood volume or blood pressure stimulates release o Detected by low pressure and high pressure baroreceptors Low pressure: right atrium, great veins, pulmonary vessels High pressure: carotid sinus, aortic arch o With a low BV, need to retain water to increase BP, need a 10% drop before significant rise • Osmotic control is more sensitive • Interaction: o When 2 oppose, volume overrides tonicity Eg: major haemorrhage and drink water

• Other stimulus of release o Cold, surgery, anaesthesia, haemorrhage, pain, emotional stress, nausea, vomiting o Angiotensin II o Drugs (eg. narcotics: morphine, tricyclic antidepressants, nicotine) • Other inhibition of release o ANP (atrial natriuretic hormone) o Drugs (eg. alcohol, narcotic antagonists)

Actions of ADH • Kidney o Controls permeability of apical cell membrane of principal cells of collecting duct to water Acts via V 2 receptor on basolateral membrane second messengers AQP2 Causes insertion of pre-formed water channels (Aquaporin 2: AQP2) into apical membrane • Without ADH, channels are withdrawn by endocytosis o Stimulates UT1 to transport urea into interstitium • Cardiovascular o Potent vasoconstrictor increasing arterial pressure Receptor: V 1A o Stimulates vagus, thus slowing heart rate (and not compensating for increasing BP) • Other o ACTH release stimulated (via V 1B [V 3]) receptor

Aquaporins • Allow water to pass from the lumen into the cell (free water) o Many types (AQP1, 2, 3, 4, 6, 7, 8) Only AQP2 responds to ADH stimulus • All parts of the nephron that are water permeable (proximal tubules, descending LH, collecting ductules)

Renal medulla and hypertonicity • Hypertonicity in the renal cell medulla can lead to cell shrinkage and death o This is prevented by: osmotic osmolytes (eg. sorbital, inositol, taurine, betaine) Sit in the cell and help maintain cell volume • Urea is also toxic and can cause cell death if it accumulates in the cell o Heat shock protein 70 protects cells from urea

Lecture 44: Urinary tract infection Hazel Mitchell

Epidemiology • One of the most common site of infection

Classification • Uncomplicated urinary tract infections – mostly adult non-pregnant women o Acute uncomplicated cystitis o Acute pyelonephritis • Complicated urinary tract infections – anatomical, functional abnormalities o Increased risk of serious complications, treatment failure, repeat infection • Source: o Community acquired – most common type o Hospital acquired – 30-40% of hospital acquired infections

Community acquired UTI • Colonises urethra then bladder produces symptoms o Urine is a good media for gram –ve bacteria (pH 5-6, has nutrients • Route of infection can be ascending or haematogenous o Most common is ascending pathway o Occasional haematogenous spread to kidney

Host defences • Mechanical processes – prevent bacterial colonisation o Flushing effect of urination o Epithelial cell shedding o Sphincter action at entrance • Biological o Immune system is less important than mechanical processes

Susceptibility • More common in females because: o Females have a shorter urethra o Urethra is close to source of e.coli (gut) • Predisposing factors o Disruption of normal urine flow o Impairment of urine drainage o Injury to mucosa of urinary tract o Facilitation of access of organisms to bladder • Most susceptible: o Women 20-40 (sexual activity enhances faecal bacteria entry to urinary tract) o Pregnant women (hormonal changes and growing fetus) o Children (anatomical abnormalities, uncircumcised) o Males >60 (prostate) • Other predisposing factors: o Calculi – in any part of the tract (kidney stones) Damaged, blocked o Loss of neurological control of bladder (eg. spina bifida, paraplegia, MS) Flushing lost o Incomplete bladder emptying o Urinary reflux – leading to pyelonephritis o Tumours – can cause retention, reflux o Contraceptive diaphragms – pressure on urethra o Diabetes – more sever UTIs o Instrumentation of lower urinary tract

Signs and symptoms • Cystitis – Inflammation of the bladder o Dysuria (burning pain on urination) o Frequency o Urgency o Suprapubic pain • Pyelonephritis – inflammation of kidneys o Begins like cystitis o Systemic symptoms: fever, chills, nausea, vomiting (organism in blood) o Possible flank pain o Rapid onset of symptoms • If organism enters bloodstream, can get rapid septicaemia • Young children – non-specific symptoms o Fever, irritability, incontinence, diarrhoea, anorexia • Urethral syndrome o Symptoms of cystitis: frequency, dysuria, suprapubic discomfort, difficulty starting, slow stream, incomplete bladder emptying Sterile urine culture – no bacteria present o Cause unknown Possible: hormone imbalances, urethral stenosis, environmental chemicals, traumatic sex • Asymptomatic bacteruria o Multiplication of bacteria in urine/bladder without infection o Detected by screening o Occurs in: Pregnant women, young children, patients before urological procedures (people who are screened) o Signs: Bacteria in urine, asymptomatic

Pregnancy • Pregnant women have an increased risk of UTI o Progesterone induces ureteral dilation o Expanding uterus puts pressure on ureters • Early can be asymptomatic, increased risk of pyelonephritis septicemia premature delivery o Thus we screen during pregnancy

Hospital acquired UTI • Catheterisation is the major predisposing factor o During insertion bacteria carried into bladder o In situ catheter facilitates bacterial access Lumen of catheter Tracking between catheter and urethral wall • Catheter can become colonised with bacteria o Biofilm (a polysaccharide) formed bacteria that allows it to track up catheter • Long term catheterisation (>5 days) has a 50% chance of infection o Treatment is removal of catheter to clear infection, may have just been a bacteruria rather than infection

Causative agent • Community acquired UTI: o E coli: 80%; Coag -ve staph: 10%; other gram +ves: 2%; proteus mirabilis: 5%, other gram –ves: 3% • Hospital acquired UTI o E coli: 40%; coag –ve staph: 3%; other gram +ves: 16% (catheter); candida: 5% (biofilm formed, found in vagina); proteus mirabilis: 11%; other gram negatives: 25%

Urinary pathogenic e. coli (UPEC) • Colonisation o Requires: Source of UPEC in colon (not all e. coli are the same, some strains have different virulence factors) Entry and colonisation of vagina/urethra • Adhesion o Use Adhesins – proteins that protect bacterium from urinary lavage and thus allow them to multiply and invade o E. coli: Type 1 pili • Allows attachment to bladder – associated with uroplakins and mannose receptors Pyelonephritis associated pili (P pili) • Common in pyelonephritis associated e. coli • Attach to receptors in kidney • Inflammatory response o LPS (lipopolysaccharide) stimulates cytokines inflammation o Exotoxins (alpha haemolysin) damages kidney cells inflammation

Clinical presentation • 23 yo female, 2 day history of frequency, dysuria, slight haematuria, suprapubic pain o No vaginal discharge • Action: o Take a MSU and send for analysis

Sampling • MSU – midstream urine o Reduces contamination of urine with bacteria from urethra • Procedure o Clean surroundings with saline swabs (not antiseptics) o Allow first 20-30ml to pass o Collect next 50ml • Number of organisms is important, thus don’t want bacteria from urethra • Babies/young children: o Bag urine + nappy possible contamination (longer urine in bag, more bacteria) o Suprapubic aspirate avoids contamination but invasivfe and traumatic o Catheterised patient urine drawn from catheter port

Storage and transport • Urine should be stored immediately at 4 oC o Prevents growth of organisms in urine • Number of bacteria in urine is important for correct diagnosis o Antibiotics need to be noted on request form

Urinalysis • Dipstick o Detects: blood, WBC, protein, nitrite, pH, glucose, etc

Diagnosis • Macroscopic appearance o Detects blood in urine and evidence of bacterial growth (cloudy) • Microscopic appearance – wet preparation o Bacterial count – number of bacteria o White cell count – pyuria, indicating infection PMNS, granular >100x10 6/L abnormal o Red blood cell count – haematuria, indicating bleeding Circular, nongranular Causes: • Infection of urinary tract, endocarditis, renal trauma, calculi, urinary carcinomas, clotting disorders, thrombocytopenia, menstruation, catheterisation, instrumentation, aspirin, anticoagulants o Epithelial cell count – high number indicates contamination >20 epithelial cells per high power field is contamination o Presence of casts – may indicate renal tubule involvement Eg: renal epithelial cell, red cell, white cell • May indicate renal tubule injury, pyelonephritis, glomerulonephritis Dehydration can cause hyaline? casts • Culture o Function: Quantitation Identification of bacteria present and causative agent o MacConkey agar – bile salts + lactose Streaking Quantitation using urostrips • Filter paper strips of a given size that take up a given volume • Dip and culture, then count colonies o Sensitivity testing – resistance testing • Contamination o Urine is often contaminated thus, number of bacteria is important 10 8/L is diagnostic

Findings • UTI: o >10 8 organisms/L o 1 bacterial species isolated o Polymorphs elevated >100x10 6/L Without this elevation, colonisation without infection o Do sensitivity testing • Likely UTI: o 2 bacterial species, one >10 8/L o Polymorphs >100x10 6/L o Do sensitivity testing • Probable contamination o 2 species, both > 10 8 o Polymorphs not elevated o Increased squamous epithelial cell present o Reinvestigate only if symptoms persist

Treatment • Uncomplicated cystitis o Non-pregnant women Empirical treatment: • Trimethoprim (toxic to fetus) OR: • Cephalexin (cephalosporin) OR • Amoxicillin (penicillin) + clavulanate (breaks down beta-lactamase) OR • Nitrofurantoin (urinary antiseptic, only works in bladder) o Pregnant woman MSU collected and tested • Cephalexin OR amoxicillin+clavulanate OR nitrofurantoin Urine culture repeated 48 hours after completion of treatment o Men Similar to women Investigate for obstruction/abnormality (eg. prostate) o Children Trimethoprim, cephalexin or amoxicillin+clavulanate Females <5, males any age investigate abnormality • Prophylaxis until imaging complete • Pyelonephritis o Exclude underlying abnormality o Culture and sensitivities o Mild infection (low grade fever, no nausea, vomiting) Empiric cephalexin, amoxicillin+clavulanate or trimethoprim o Severe infection (sepsis, vomiting) Immediate empiric therapy gentamicin IV + amoxicillin/ampicillin IV • Gentamicin: toxic but effective, rapidly bacteriocidal to gram –ve bacteria • Amoxicillin: kills enterococci Allergy to penicillin • Gentamicin alone Switch to oral therapy as soon as possible • Recurrent UTI o Common in young healthy women 27-44% women with initial UTI experience 1 recurrent in 6 months despite antibiotics o Causes: Reinfection Persistent infection – bacteria remain dormant in epithelial cells, may have reduced rather than cleared bacteria • Catheter-associated infection o Treat if systemic symptoms – often colonisation rather than infection o Catheterise for shortest time possible Remove catheter when possible intermittent rather than continuous use Change infected catheter o Increase fluid intake

Lecture 45: Sodium and potassium balance Karen Gibson

Sodium balance • Na and its salts make up 90% of the osmotically active particles in the ECF o Thus, the amount of sodium determines the ECF size (ECFV) • Sodium intake o Diet: ~100-400mmol/day Low Na diets: <10mmol/day; liberal with salts: >600mmol/day • Sodium output o Sweat – negligible at rest in cool environment (may be increased with exercise/sweating) o Faeces – negligible (may be significant in diarrhoea, eg. cholera) o Urine - ~100-400mmol/day, regulated according to need • If kidney couldn’t increase/decrease Na excretion: o If we lose sodium – drop in ECFV, thus drop in BV and circulatory collapse o If we have too much sodium – salt retention: oedema

Detection of changes in extracellular volume (ECFV) • Increased sodium intake increased plasma osmolality o Increased thirst – increased water intake o Increased ADH release – increased water retention increased volume of extracellular compartment • With increases and decreases in sodium intake, body adapts and can reach a new steady-state over 3-5 days o Results in similar changes to: Sodium excretion Weight (which reflects ECFV)

Effective circulating volume • ECFV is not a measurable and distinct compartment, instead the body measures effective circulating volume o Defined as portion of the ECFV that is in the vascular system and perfusing tissues Related to changes in volume/pressure in the CVS and cardiac output • Normally ECFV parallels changes in effective circulating volume o Distorted in disease: eg. congestive cardiac failure, ascites Expanded ECFV but ECV is contracted and appears low, thus body retains sodium and exacerbates disease • Detection of changes: o Low pressure volume receptors Pulmonary vasculature, atria, great veins o High pressure (arterial) baroreceptors Carotid sinus, aortic arch, juxtaglomerular apparatus o CNS receptors, liver receptors (less important) • Pathways: o Afferent mechanisms (receptors of ECFV and ECV) hypothalamus Efferent mechanisms: • Alteration of sympathetic activity o Increased salt intake increases BP, decreases sympathetic tone which leads to Na excretion o Increased sympathetic tone (due to low salt intake) causes a drop in GFR, increase in renin and increase in NaCl reabsorption • Alteration of hormonal activity o See later Physical factors • High sodium, increases BP: hydrostatic high (aids GFR) which increases excretion • Colloid osmotic pressure in BV lower due to diluted volume, filter more (less pp keeping fluid in vessel) aiding excretion and reabsorption reduced in proximal tubule (same reason)

Glomerular filtration and sodium excretion • The higher the GFR, the more sodium excreted assuming reabsorption remains constant (which it doesn’t) o This is not a main control mechanism because: GFR is autoregulated constant with different BPs Glomerulotubular balance we reabsorb a constant fraction of filtered load (rather than a constant amount) Tubulogloerular feedback macula densa (in DCT) detects Na and changes to GFR and thus with increased Na, it decreases GFR to give a longer time for absorption • Thus, to control sodium excretion, we must alter tubular reabsorption

Note: -proximal tubule is the course tuner (2/3 reabsorption) -loop of henle is the medium tuner (20- 25% reabsorption) -collecting tubule/DCT is fine tuner (2/10% reabsorption)

Hormonal control of sodium excretion • Excretion is decreased by: o Renin-angiotensin system o Aldosterone o Noradrenaline Affects blood flow, similar to angiotensin II Directly changes reabsorption in proximal tubules (decreases blood flow increased oncotic pressure and decreased hydrostatic pressure thus increasing passive reabsorption) • Excretion is increased by: o Atrial natriuretic peptide o Prostaglandins Produced locally in the kidney and increase excretion Counter-regulatory role • High during high loads of conservation hormones (eg. angiotensin II, ADH) Eg. if inhibit prostaglandins (eg. via NSAIDs), get intense kidney vasoconstriction because angII is unopposed

Angiotensin II – sodium conserver • Decreases Na excretion by: o Stimulating aldosterone production o Direct effect on proximal epithelium via AT1 receptors Even with low AngII (10 -10 M) reabsorption enhanced and thus excretion decreased o Renal vasoconstriction Medullary blood flow reduced, thus medullary osmotic gradient enhanced and passive Na reabsorption in thin ascending loop of henle enhanced • Lose water in the descending limb, thus greater concentration gradient higher in ascending

Aldosterone – sodium conserver • Steroid hormone produced from zona glomerulosa of adrenal cortex • Increases Na reabsorption and K and H secretion o Works in the colon, sweat glands, salivary glands and collecting ductules o Controls reabsorption of ~2% sodium filtered (seems like not much, but 2% ~500mmol/day of the 3L ECFV) • Acts on principal cells of collecting duct (and DCT) o Immediate effects (non-genomic) Increase permeability of apical (luminal) membrane to K +, increasing secretion • Not via mineralocorticoid receptor o Latent effects (genomic) Take 60-90 minutes to occur Act via mineralocorticoid receptor in cytoplasm and nucleus • Increase serum glucocoid inductible kinase (sgk) that increases the number of active epithelium sodium channels (ENaCs) o ENaCs increase sodium reabsorption from tubule lumen o Associated with H + pumping as well • Increases Na/K/ATPase pumps in basolateral membrane that pumps sodium into interstitium and into the blood and K + into cell for secretion Amiloride • Diuretic that blocks ENaCs and thus prevents sodium reabsorption and thus increases excretion of sodium and water • Stimuli for aldosterone release o AngII o Rise in plasma K (very sensitive to this) In response, increases K + excretion o Fall in plasma Na (relatively weak effect) Often don’t see large changes in Na anyway, water dissipates to moderate change o ACTH – increases aldosterone production However, more important for cortisol

Atrial natriuretic peptide (ANP) – sodium excretor • Peptide hormone (28AA) produced by atrial myocytes o Atrial distension causes fusion of secretory granules with plasma membrane and thus ANP release Distension can be due to eg: increased BV; venous pressure • Actions: o Natriuresis (sodium excretion) and diuresis (increased urine flow rate) Due to: increased GFR and inhibition of NaCl reabsorption in collecting duct Affects mesangial cells that change the filtration SA o Lower BP by vasodilation o Reduces smooth muscle responsiveness to vasoconstrictors o Decreases renin (conserve Na), aldosterone (conserve Na, via cGMP) and AVP (ADH: water conserver) levels

Potassium introduction • Most prevalent cation In all body fluids (ie. most in intracellular, intracellular largest) o Very important for: Electrical polarisation of excitable cells (nerves, muscles) • Low K + causes hyperpolarisation, can result in muscle paralysis, arrhythmias • High K + can cause more excitable cells, which can also lead to cardiac arrest Normal intracellular functioning (eg. cell growth, enzyme function) o Most is intracellular (3500mEq vs 56 mEq extracellular) 56 is from 14L x 4mmol/L in plasma o Plasma volumes: Hyperkalaemia is >5.5mmol/L; hypokalaemia is <3.5 mmol/L Potassium balance • Potassium intake o Average diet: ~50-100mmol/day Vegetarians: ~600mmol/day o Not physiologically regulated in man • Potassium output o Sweat: ~<5mmol/day (unless exercising etc) o Faeces: ~5-10mmol/day (unless diarrhoea etc) o Urine: ~45-90mmol/day regulated according to need

Short term potassium control • Involves exchange between ECF and ICF o Important to prevent sudden increase in plasma potassium after ingestion (eg. rockmelon: 50mmol K) Rockmelon would raise plasma K + by 3mmol/L, fatal • Physiological control – keeps plasma K + normal o Insulin – causes K + to enter cells Treatment for hyperkalaemia: insulin + glucose (don’t want hypoglycaemia) o Adrenaline – keeps K + constant in exercise o Aldosterone – upregulates Na/K/ATPase pump • Pathophysiologic – displaces plasma K from normal o Acid-base balance H+ concentration changes K + Metabolic acidosis, H + goes into cell, K + out in exchange increasing K + plasma Metabolic alkalosis, opposite o Plasma osmolality Cells shrink in high plasma osmolality, thus cell concentration of K + increases which enhances passive K + release from cell o Cell lysis K+ escapes o Exercise K+ pushed out as part of action potential/contraction

Renal handling of potassium • Freely filtered o Proximally, net reabsorption of >80% regardless of deficient/overload o Distally, net reabsorption or secretion according to need Thus, body K + levels are controlled by collecting tubule • Normally, excrete 5-15% of filtered K + load o Can vary from 1% to >200% • Collecting duct cells: o Reabsorbed by intercalated cells, constant despite dietary K o Secretion by principal cells according to need • Secretion – the 4 determinants o (1) [K] in cytoplasm of principal cells o (2) [K] in tubular fluid o (3) Transepithelial potential difference The more negative the lumen, the more K + into the lumen o (4) Permeability of the luminal membrane to K Enhanced by aldosterone Apical is already more permeable to K than basal

Renal handling of potassium - continued • Factors that influence secretion: o Increase in aldosterone, (1, 3, 4) 1: increase Na/K/ATPase pumps; 3: increase Na + reabsorption; 4: immediate effects o K intake, (1, 3, aldosterone) Aldosterone: more sodium transport, more Na/K/ATPase o Distal nephron flow rate (2) 2: increased flow, K + build up is limited o Distal nephron Na delivery (3, flow rate) 3: Na + necessary for K secretion via Na/K/ATPase o Impermeant anions in tubular fluid (3) 3: more –ve anions in tubule - - Distal nephron more permeable to Cl than HCO 3 , 2- SO 4 or nitrates, thus these stay in the urine and attract K + • Diagram: K + secretion increases with tubular flow rate because K + build up in tubule is limited, thus decreases K + in tubule

K+ and systemic acid-base status • Acute alkalosis, K secretion increases o More bicarbonate in urine which acts as an impermeant anion o K and H exchange pushes H + out of cells to neutralise blood, but increase intracellular K + in principal cells and thus K + secretion o Higher pH of tubular fluid increases permeability of luminal membrane to K + • Acute acidosis, K is retained (opposite to above) • Chronic acidosis K secretion increases o Proximal reabsorption is inhibited (as in acute acidosis) to such an extent that, more fluid gets to distal nephrons which increases secretion K+ can become depleted and secretion decreases

Lecture 46: Dialysis and end stage kidney failure George Mangos

Risk factors for chronic kidney disease (7) • Modifiable: o Smoking o Diabetes o HBP High blood pressure damages kidneys which exacerbates high blood pressure Kidney damage causes high blood pressure that exacerbates kidney damage o Obesity • Non-modifiable: o Over 50 years o Family history of kidney disease Not just PKD, other environmental factors etc o Aboriginal or Torres Strait Islander heritage • These help us identify people for screening o Screen with the dipstick for proteinuria Want <300mg/day protein in urine

End stage kidney disease (ESKD) • Defined as GFR <15ml/min o irreversible • Cause: o Usually due to progression of CKD o Can be due to rapid progressive acute kidney injury (AKI) • Usually symptomatic: o Nausea, insomnia, termor o Lethargy, dyspnoea, anaemia o Reduced urine output (can have normal urine output in first few years of ESKD) • Causes: o Diabetes > glomerulonephritis > hypertension > miscellaneous > analgesics > PKD > reflux • Results in uraemia – failure of homeostasis: o Salt, water, acid-base balances Water balance fluid retention and hyponatremia (Na excreted in attempt to lower volume) Sodium balance oedema, congestive heart failure, hypertension Potassium balance hyperkalemia Bicarbonate balance metabolic acidosis, osteodystrophy Magnesium balance hypermagnesemia Phosphate balance hyperphosphatemia, osteodystrophy o Excretion of nitrogenous end products – create symptoms Urea, creatinine, uric acid, amines, guanidine derivativfes ?anorexia, nausea, pruritus, pericarditis, polyneuropathy, encephalopathy, thrombocytopathy, lethargy o Endocrine-metabolic Conversion of vitamin D (1) to active metabolite (125) - osteomalacia, osteodystrophy Production of erythropoietin anaemia Renin hypertension (increased renin) • Complications: o Symptomatic CV disease, anaemia, renal osteodystrophy, malnutrition, growth retardation in children, fluid overload, pericarditis o Metabolic/risk factor Hypertension, lipid abnormalities, LV hypertrophy, hyperkalaemia, acidosis

Management • Renal replacement therapy o Peritoneal dialysis o Haemodialysis o Transplantation • No dialysis o “non-dialysis pathway” conservative measures Symptomatic treatment to increase QOL Dialysis may not be effective, especially with high other comorbidities thus don’t live very long

Dialysis • Treatment for ESKD, not a cure o Involves 2 processes: Ultrafiltration • Removal of fluid (water) by convection Dialysis • Removal of solute by diffusion (eg. K +, acid, phosphate) • Diffusion: o Membrane is used as filter (haemo: artificial kidney, peritoneal: tissue) On one side, blood, other dialysate o Remove bicarbonate, creatinine, urea, equilibrate Na, and K • When to start dialysis: o Precise GFR unknown Thought to be GFR 5-7 ml/min GFR 12-15ml/min o Varies with age: Older people, start dialysis later, can still be functional Younger people, have more muscle and more catabolic function (and therefore waste products), thus may need a higher GFR to function o Symptomatic indication Nausea, vomiting, encephalopathy, intractable fluid overload, K + balance When these symptoms become unmanageable by medications

Haemodialysis • Dialysis: o Countercurrent mechanism Membrane is permeable to small molecules and water o Blood travels down a thin fibre membrane Dialysate travels in other direction • Ultrafiltration: o Convection, dialysate fluid has a negative pressure allowing water movement following pressure gradient • Fistula o Communication between artery and vein created artificially by a surgical procedure Allows increased blood flow to 200-300ml/minute of blood removed/replaced from circulation 2 lines are inserted: input and output lines o Complications Infection, thrombosis • Management of patients o Fluid balance, dry weight dialyse down to calculated weight o BP control BP often corrects when weight decreases o Diet Protein, K + decreased, watch fluids o Depression, social factors o Ca, PO 4, PTH o Hb – anaemia risk o Adequacy of dialysis o Other vascular risk factors o Dialysis access (fistula condition) Peritoneal dialysis • Tube (permanent peritoneal catheter) is inserted into abdominal cavity o Uses peritoneum as a membrane: capillary network exists on the other side of the membrane • Dialysis: o Dialysate fluid (2-2.5L) is put into the cavity Equilibrates with Na, K, Ca; removes creatinine, bicarbonate o Left for several hours o Fluid drained, and replaced every few hours (continuous peritoneal dialysis) • Ultrafiltration: o Osmosis by making the dialysate solution hypertonic (increased glucose osmolality) • Normally dialysis of once/day for a few hours is sufficient

Comparison • Haemodialysis o Benefits 3x5 hours sessions/week Possible at home No catheter o Complications Fistula problems – infection, stenosis, blockage Diet and fluid restrictions Needles • Peritoneal dialysis o Benefits Noctural or awake (automated by machine at night) At home Easier technique o Complications Infection – especially staph infections causing peritonitis Glucose load – increased calorie intake due to glucose Osmolite Permanent catheter • Even with dialysis, mortality is still high

Renal transplantation • Process o Natural kidneys not removed, new kidney is placed in pelvis Connected to iliac artery, iliac vein and ureter tunnelled into bladder • Survival QOL, life expectancy better than for dialysis o not in older patients • Needs immunosuppression o Triple therapy: prednisolone, ?calcin urine inhibitor, antimetabolite: microfenelate?; + (monoclonal antibody therapy) o Side effects • Long term side effects o Cardiovascular disease o Cancer and infection • Problems: o Major surgery – cardiac, respiratory, infectious complications o Graft function: 90% after 1 year are still functioning, 80% after 5 years o Side effects of immunosuppression Cancer Infection especially in fluid collections in the localised area o Limited organs

Donating • Waiting list o 5 years in NSW, limited supply of organs ESRF 16000 people: 9000 on dialysis, 7000 transplants (650 new transplants/year, 230 NSW) o Ethical issues: who should receive limited number of organs (should the elderly?) • Donations from: o Living: relatives, friends (need same blood group) o Cadaveric: licence, donor registry o Illegal human organ sales internationally (poor people, prisoners etc) Often not screened well

New transplant possibilities • Xenografts o Pig (knock-out pigs, that lack pig antigens) Problems: • Pig antigens • Hyperacute rejection • Animal cross-species infection • New immunosuppressive agents • Monoclonal antibody therapies o Basiliximab – to reduce acute rejection • Lower dose combination therapy – less toxicity • Paired exchange o To match blood groups

Lecture 47: Drugs and the kidney Margaret Morris

Roles of the kidney • Regulate water, electrolyte balance • Eliminate endogenous waste • Eliminate exogenous waste (eg. metabolites of drugs etc.) • Endocrine organ (renin, EPO)

Drug elimination • Many drugs metabolised in liver (oxidation, reduction, hydrolysis etc.) • Some drugs are eliminated by kidneys o Rate of renal elimination (and thus adequacy of kidney function) determines duration of action • Renal processes: o Glomerular filtration – bulk flow o Tubular secretion – carrier-mediated, active, utilises energy, saturable, can be inhibited o Tubular reabsorption – carrier-mediated, active, main: passive back diffusion (based on concentration gradient)

Glomerular filtration • Depends on: o Molecular size of drug o Extent of plasma protein binding (protein bound isn’t filtered) o Drug concentration in plasma

Tubular reabsorption • Depends on: o Lipid solubility of drug (unionised vs ionised) – if soluble can pass through membrane back into blood o Renal clearance rate – slower the flow rate, more time to reabsorb, the lower clearance o pH of tubule fluid acid drugs are more rapidly excreted in alkaline urine basic drugs are more rapidly excreted in acidic urine Can take advantage of pH to increase/decrease clearance

Tubular secretion • Occurs in proximal tubule • Occurs mostly by active transport o Active transport moves drug against concentration gradients from blood into tubule o 2 major types of transporters: Weak acids (-ve charge) • Penicillins, probenecid, salicylic acid Weak bases (+ve charge) • Amiloride (diuretic), morphine, quinine o Plasma protein-bound drugs can still be cleared, based on equilibrium: • Free drug + free protein  drug-protein complex o Competition for transport mechanisms by drugs can be imnmportant Can cause drug-drug interactions (eg. penicillin and probenecid) o Transport can be saturated • EG: penicillin and probenecid o Probenecid competes with penicillin for active transport sites and is preferentially secreted Thus action of penicillin is prolonged

Other drug interactions

Overall: elimination = filtration + secretion – reabsorption

Assessment of renal elimination • Measure: o Blood, urine levels of drug o Clearance: volume of blood cleared of drug per unit time Clearance = C uVu/C p (urine conc x urine flow / plasma conc) o If clearance > GFR, drug is secreted

Renal dysfunction: a special case • Creatinine measured to assess renal function • In renal failure: o Kidney function is reduced and thus renal-drug excretion is reduced May need to reduce dose rate o Need to take into account drug-drug interactions that may compete for transporters and have multiplied prolonged effects

The kidney as a target of drugs: diuretics • Increase sodium and water excretion o Several classes with several mechanisms (target different transporters) o Widely used Eg. HTN, congestive heart failure, ascites o Adverse effects: Relate to ion/fluid balance problems • Na + loss, K + changes, metabolic effects o Overall effect is to reduce BV • Loop diuretics – most potent o Inhibit Na +, Cl - reabsorption in thick ascending limb of LH Block Na +/2Cl -/K + cotransporter o EG: frusemide (Lasix) • Thiazides – less potent, widely used for HBP o Inhibit Na +, Cl - reabsorption in early DCT Block Na +/Cl - cotransporter o EG: chlorothiazide • Potassium sparing diuretics – sparingly used, modest effect o Spironolactone Aldosterone antagonist (blocks K + loss and sodium reabsorption) thus causes K + retention and sodium loss Can be useful in patients with high aldosterone, and heart failure o Amiloride Na channel inhibitor o Can be used in combination therapy Monotherapy has risks: hyperkalemia, hyponatremia

Toxic effects of drugs • A toxic effect is produced when a substance reaches appropriate sites in the body for a sufficient length of time o Depends on: Properties of agent Dose of agent Route of absorption Susceptibility of subject (eg. existing renal function) o Kidney is particular important because it is a major method of clearing the blood of toxic agents and in failure can lead to toxic agent accumulation • Kidney factors: o Unique anatomy/physiology Receives 25% of CO, sees all blood o Chemicals can be concentrated in kidney cells o Site-selective injury o Patient factors (blood volume, hydration, co- morbidities) • Chemical induced renal toxicity o Certain drug classes have a certain clinical liability Antibiotics Angiotensin converting enzyme inhibitors Non-steroidal anti-inflammatory drugs

Toxic effects of drugs - continued o EGs: Aminoglycosides (eg. gentamycin) • Used for treating gram –ve infections • AE: Can cause renal dysfunction – decreased GFR, increased creatinine • Mech: highly polar cation that binds to the –ve phospholipids of PCT brush border o Endocytosis lysosomes, swelling of cell, rupture • Can lead to acute tubular necrosis Cyclosporin • Used to prevent graft rejection (immunosuppressive agent) • AE: nephrotoxicity, acute renal dysfunction, renal failure • Mechanism: vasoconstriction of renal arterioles, fibrosis, tubular atrophy o Can be difficult to distinguish between nephrotoxicity and transplant rejection

Summary • Kidney is important in elimination of some drugs o Dose adjustment may be required in kidney disease • Diuretics act in the kidney to reduce Na and water reabsorption to reduce blood volume • Some drugs are nephrotoxic

Lecture 48: Renal regulation of acid-base balance Karen Gibson pH revision • pH = -log[H +] o As [H +] increases, pH decreases • Normal arterial pH ~7.40 (7.35-7.43) o Venous pH is slightly lower: ~7.37 (due to increased CO 2 concentration) o Thus, body fluids are slightly alkaline of neutral However, alkalosis and acidosis are relative to 7.40 pH rather than “neutral” • pH tightly regulated o important for metabolic functions: eg: enzymes, blood clotting, muscle contraction

Acids and bases – definitions • Acid – releases protons when dissolved in solution: a proton donor o EG: hydrochloric acid (HCl); carbonic acid (H2CO 3); carboxyl groups (R-COOH) • Base – ion or molecule that combines with protons: a proton acceptor - - o EG: hydroxyl ions (OH ); ammonia (NH 3); bicarbonate (HCO 3 ); amino groups (R-NH 2) • Acid/base strength o Stronger the acid, the more it dissociates Acid HA: HA  H+ + A - [][] KA = [ ] The stronger the acid, the higher the K A o pK A = -log[K A] the stronger the acid, the lower the pK A o Important pK A’s Bicarbonate system: pK = 6.1 Phosphate system: pK = 6.8 Ammonium system: pK = 9.27 o Derived from following equations: Weak acid  weak base + H + - + H2CO 3  HCO 3 + H  CO 2 + H 2O - 2- + H2PO 4  HPO 4 + H + + NH 4  NH 3 + H

Buffers • Definition: any substance that reversibly consumes or releases H + o Help minimise the change in pH that occurs when an acid or base is added to a system • Most effective when its pK is within 2 (preferably 1) of the pH it is called to defend

Body acid-base balance • Normal metabolism of the body produces acids o Cellular metabolism of carbohydrates and fats Produces 15-20mol/day of CO 2 (volatile acid) • This assumes adequate insulin and tissue perfusion, otherwise production of non-volatile beta-hydroxybutyric and lactic acid can occur

CO 2 can be blown off by respiratory regulation of pH o Also produces small amounts of non-volatile (fixed) acids Examples: • Sulfuric acid – metabolism of methionine and cysteine • Hydrochloric acid – metabolism of lysine, arginine and histidine • Phosphoric acid – metabolism of phospholipids, nucleic acids etc Overall, production of 50-100 mEq/day (~ 70mEq/day = ~1mEq/kg/day of fixed acid ) • Can vary with diet: eg. vegetarians can produce less due to less due to less amino acid in diet • Normal pH is maintained despite production of these acids (need pH 6.8-7.8 for life) o pH maintained by: buffering (plasma proteins); respiratory regulation (S&H A); renal regulation

Renal regulation – overview • Volatile acids: o CO 2 is blown off to maintain pH of body fluids, this is effectively similar to losing bicarbonate Thus kidneys generate new bicarbonate • Fixed acids: o ~70mEq produced/day Kidney excretes acid equivalent to this amount (titratable acid and ammonium) • Other than generating new bicarbonate, kidneys recover filtered bicarbonate by reabsorption o Filtered bicarbonate is significant: 24mmol/L (plasma) * 180L/day = 4320 mEq/day • If body pH is too high, bicarbonate can be secreted to equalise balance

Net acid excretion • Amount of acid excreted to counter production of fixed acids each day o Net acid excretion = ammonium excretion + titratable acid excretion – bicarbonate excretion

o NAE = U NH4+- + U TITA- – UHCO3- Bicarbonate lost in the urine is equivalent to adding H + to the body • Very little acid is excreted as free protons o Maximum H + in urine is pH 4.0 (0.1mmol free H +/L) Thus, if you wanted to secrete 70mEq, need 700L Consequently, protons are excreted as NH 3 and titratable acid

Bicarbonate reabsorption • Overview: o 85% in proximal tubule Luminal hydrogen ATPase pump and Na/H antiport o 10% thick ascending loop of henle o 5% collecting tubules Independent of sodium • Proximal tubule o Na +/H + antiport uses Na gradient to push H + out of cell o H+ ATPase uses ATP to push H + out of cell + - In the lumen, H combines with HCO 3 to form H 2CO 3 o Carbonic anhydrase in apical membrane converts H 2CO 3 to CO 2 and water - CO 2 diffuses into the cell and is converted by intracellular carbonic anhydrase into HCO 3 which is then transported back into the body + - o Net result is: transport of sodium into cell, H into tubule and HCO 3 reabsorption + - o In acidosis, there is more H in the cells and thus reabsorption of HCO 3 is increased

Bicarbonate reabsorption - continued • Factors that affect reabsorption: o Expansion of vessels Overloaded body volume means Na + reabsorption will be decreased to lose fluid - • Since HCO 3 reabsorption is dependent on sodium reabsorption, it is decreased - o Ie: increased HCO 3 excretion and decreased reabsorption

o Partial pressure of CO 2 in blood Increased (ie. increased acidity of blood) causes increased - HCO 3 reabsorption o Chloride ions Both are negative anions, thus high plasma chloride - concentration prevents HCO 3 reabsorption

Excretion of protons with non-bicarbonate buffers • Overall o H+ is pumped out into the lumen and combines with a buffer Buffer can be: 2- • Phosphates (HPO 4 )

• Ammonia (NH 3) Thus, H + is bound to buffer which can’t be reabsorbed (ionised) and thus is excreted o As H + is secreted, more bicarbonate is made • Non-bicarbonate buffers can be sorted into titratable acids and ammonium

Titratable acid • Acid in the urine that can be neutralised back to glomerular filtrate pH (plasma pH) by titration with a strong base o Eg. urine pH 6; how much NaOH needed to increase pH to 7.4 • Excretion of these titratable acids is due to: [ie. form acids by combination with these weak bases] o Phosphates o Beta-hydroxybutyrate and acetoacetate in diabetic ketoacidosis o Creatinine • If pH of urine >7.4 no titratable acid

Phosphates – urinary buffer forming titratable acid • Derived from diet • Amount excreted (as titratable acid) depends on (filtered load - reabsorption) o ~75% reabsorbed, 36mmol/day available for titration Total ~70mmol/day, thus excretes 50% of fixed acid • Reabsorption: o Acidosis causes adaptive inhibition of phosphate reabsorption, amount is modest o Phosphate reabsorption is regulated to maintain phosphate balance Thus its use to manage acid-base change is limited Ammonia – most important urinary buffer • Produced by the kidneys o Synthesis varies in acidic-basic environments More made in acidic urine and in acidosis • Produced in proximal tubular cells from glutamine o Glutamine enters cells via Na + coupled co-transport from both luminal and peritubular fluid + Yield of 2NH 4 from each glutamine o Process: + Glutamine glutaminase glutamate + NH 4 + Glutamate glutamic dehydrogenase alpha-ketoglutarate + NH 4 o Acidosis, glutaminase and glutamic hydrogenase enzymes are upregulated Can take several days Opposite in alkalosis • Further process: + + o NH 4 is transported into the tubule lumen by Na facilitated transporter in the proximal tubule + o In the thick aLH, NH 4 is reabsorbed Na +/K +/2Cl - transporter Paracellular pathway, +ve ion reabsorbed + + o Equilibrates based on NH 4 NH 3 + H NH 3 can pass into the collecting ductules by passive secretion (diffusion) NH 3 can pass into the proximal tubule and thin dLH to be recycled NH 3 (a small amount) can be washed out + o NH 3 that passes into collecting ductules can have H added and thus be trapped in tubules as NH 4 Diffusion trapping H+ comes from further up the track or secretion by collecting ductule cells + + o NH 4 trapped in lumen is excreted and takes away H ions • Notes: o Renal venous NH 3 higher than renal arterial + - o NH 4 must be excreted or it negates HCO 3 generation + + + o The lower the urinary pH, the more NH 4 trapped, ie: the more H to attach to NH 3 trapping it as NH 4 + pH 7.2, ratio of NH 3/NH 4 is 1:100, at pH5: more NH 4 excreted o Ammonium does not contribute to titratable acid pK is higher than 7.4

Acidosis • Secretion of H + by nephron - o Causes entire load of HCO 3 is reabsorbed o Net acid excretion increased (phosphates, NH 4)

• Stimulation of production and excretion of NH 4 • New bicarbonate is generated and returns to plasma • Examples: o Health - ~70mEq of H + excreted/day o Diabetic acidosis increased excretion as titratable acids due to other weak bases used to carry hydrogen o Chronic renal disease Can’t excrete all acid + + • Less acid excreted and NH 4 is decreased (loss of NH 4 production)

Alkalosis • Secretion of H + by nephron is inhibited - - o HCO 3 reabsorption reduced (more HCO 3 in urine) o Net acid excretion reduced - • HCO 3 secretion by intercalated cells in cortical collecting duct stimulated (Type B cells)

Lecture 49: Transplant immunology Bruce Pussell

Transplants in Australia • Kidney: 400 cadaveric; 250 live o Other: heart, lung, liver, pancreas o Cornea: 1300 o Hemopoietic stem cell: 360 • Australia has the lowest donor rate in the western world o Only a small increase of last few years o Increases in live-related and unrelated donors as these become more well known and popular methods Now make up ~35% of total

Success of transplants • For the most part, very successful o 6000 people living with transplanted kidneys in Australia currently o Recipient survival is 85-90% at 5 years Vs dialysis which as a 15% annual mortality o Surgical skills have been refined and risk is low May have laparoscopic surgery • National transplant registry aids location of viable kidneys • Transplants have gained ethical acceptance in the population

Challenges in organ transplantation • Rejection: hyperacute, acute, chronic o Mechanisms: antibody, t-cell mediated, immune based • Complications of rejection o Immunosuppression infections, cancer • Shortage of donors

Definitions • Host – recipient • Graft – tissue from donor o Low complication, organ accepted: Grafts within individuals (autograft) • eg. skin, autologous transfusion before surgery, autologous hemopoietic stem cell transplantation before chemo Same genetics, twins (isograft) o Possible rejection, need immunosuppression Genetically different animals of same species (allograft) Between animals of different species (xenograft)

Immune system • Innate immune system o Broad recognition of pathogen Process, present antigen to adaptive immune system o Mechanisms: “toll-like” receptors, respond to cytokines Cells: neutrophils, mast cells, NK cells Complement system: important in causing inflammatory response in allografts • Adaptive immune system o Antigen driven o T and B cells respond via CD8 T cells and antibodies from plasma cells • Antigen presenting cells – dendritic cells: o Sample the environment and process antigens Shuttle antigens from periphery to lymph node and present to lymphocytes via MHC o Stimulate lymphocyte response

Immune system - continued • T-lymphocyte o CD8 – effector, cytotoxic o CD4 – regulator, helper TH1 cell mediated TH2 humoral immunity (antibody) Suppressor cells Have an auto-stimulation effect • B-lymphocyte o Activation by TH2 or directly (have antigen receptors: surface-bound antibodies) Activation results in differentiation into plasma cells/memory cells and the production of specific antibodies o Can act as APCs • Antibodies o Opsonisation – coating of antigen in antibody and allowing phagocytosis by reticuloendothelial tissue o Complement activation o Links acquired and innate immunity

MHC – major histocompatibility complex • Group of genes and their cell surface proteins o Coded on chromosome 6 short arm • Important factor in determining acceptance/rejection of grafts • Major human MHC is HLA o Normal function is to present antigens to T cells o Found on cell surfaces (essentially all except RBCs) MHC I on most cells, presents to CD8 cells MHC II on specialised antigen-presenting cells (eg. dendritic cells) – presents to CD4 cells • Activates B-cells

MHC and transplantation • MHC genes and proteins are highly polymorphic o ~100 alleles of HLA genes • Normal function, self-MHC presents microbial antigens to T-cell receptors o MHC also presents self-antigens which the body becomes used to over time o After transplant, foreign MHC presents these tissue antigens to T-cell receptors and causes an immune response (if there is MHC mismatching) A single foreign MHC can stimulate >1% of T cells (microbial antigens only stimulate <1 in 10 000 T cells • MHC and rejection o Prior blood transfusions and pregnancy may expose body to mismatched MHC proteins and cause formation of anti-HLA antibodies cause hyperacute reaction o After transplant, foreign MHC proteins stimulate T-cells resulting in acute rejection • Genome is made up of 2A, 2B and 2D sections from each parent o Thus there are 6 HLA antigens to match Perfect match is a significant transplant advantage

Interaction between MHC and CD4 cells • Signal 1: antigen presentation o Signal 2: co-stimulatory pathway that pushes the cell over the activation threshold Signal 3: recruitment of other T-cells • Actions: o Cytokine release modulated especially IL-2 which attracts other lymphocytes o Receptor regulation

Hyperacute reaction • Occurs within minutes of a transplant and destroys the target organ o Recipient antibodies attack graft endothelium MHC recognised as antigens, adaptive immune response mounted ABO blood group antigens, adaptive immune response mounted o Complement system is activated which causes endothelial damage thrombosis death of organ • To prevent: o Recipient serum is tested against donor cells and antibody status determined If antibodies are present, often transplant is not performed • ABO status o A, B, O blood group antigens are found on surface molecules of RBCs and endothelium Rules are similar for blood transfusion – see table Want compatibility or risk organ rejection o System is distinct from MHC status

Acute rejection • Occurs within a few days o Mediated by T-cells (CD4 and CD8) o Risk and severity are proportional to number of MHC mismatches • Can be controlled with immunosuppressive drugs o Thus zero mismatches is not necessary • Rejection is due to parenchymal cell damage and interstitial inflammation o Activation of T-cells causes inflammation o Accumulation of inflammatory cells and debris blocks vessels leading to ischaemic injury and cell death

Chronic rejection • Occurs in months to years after transplantation o Associated with development of recipient antibodies to graft MHC I antigens Transplant glomerulopathy • Characterised by vessel wall thickening (smooth muscle proliferation) and narrowing of lumen o This causes ischaemia and loss of graft function

Treatment • T-cell inhibition o Combination therapy is an option to lower dose and reduce side effects o Glucocorticoids – antiinflammatory inhibiting cytokine etc production; many SE o Cytotoxics – decrease DNA replication (eg. azathioprine); inhibits T-cell proliferation and all other rapidly dividing cells o Cyclosporin A – inhibits T cell activation and proliferation; SE: nephrotoxicity o Monoclonal antibodies • Complications o Immunsuppression Increased risk of infections and cancers • Opportunistic infections • Cancers (esp. skin cancers, lymphoma) that are virus-associated o Nephrotoxicity – small therapeutic window

Organ shortage • Policies, education to increase organ donation • Alternatives that don’t use human organs o Xenotransplantation Pigs, transgenic pigs High risk of hyperacute rejection Trans-species viruses risk o Mechanical organs o Stem cells

Allogeneic haemopoietic stem cell transplantation (bone marrow) • Used to treat: o leukaemia/lymphoma o congenital disorders of blood formation • Used to be called bone marrow transplantation, now use peripheral blood donation o Drug treatment to increase circulating haemopoeitic stem cells • Fewer ethical issues than organ transplantation.

Graft-versus-host disease • Major cause of death after alloHSCT • If host and donor differ genetically, donor may pass on T-cells o If host is immunosuppressed, these may attack body Particularly affects the skin, intestine, liver • More likely to occur if donor and recipient have a high number of mismatches o Siblings have a ¼ chance of perfect MHC match In developing countries, 1/3 potential recipients have MHC-matched sibling o Worldwide registry of MHC types has been established to aid location of donors ¼ alloHSCT are performed across national boundaries

Lecture 50: Organ donation and allocation Ian Kerridge

Hx of organ donation • 1954, 1 st kidney transplant o Then heart liver, lung pancreas cornea • Quite clinically successful these days o Increased survival rates and increased quality of life

Ethical issues • Economics • Procurement of organs • Stem cells and regenerative medicine • Decision-making in donation and procurement (especially from families) • Death and brain death • Survivorship

Problems • 2000 Australians waiting for solid organ transplants o 160-200 from diseased donors/year o Average wait is 3 years, 20-30% die waiting • Large gap between supply and demand in Australia

Sources of organs • Many sources o Living – related, unrelated (altruistic) o Cadaveric – non-heart beating o After cardiac death (2minutes of asystole) o Brain-dead donors o Make organs, steal/buy organs, animal organs

Altruism • Organ donation in Australia relies on goodwill of people to donate to society o Skin, bones, eyes are often exceptions that people want ot keep Disfigurement issues • Discussions with family o Family can object to donations, lose 20-30% of donations this way • Encouraging donation o Governmental campaigns have been largely unsuccessful o Perhaps an “opt out” system is better than our current “opt in” Everyone is a donor unless otherwise indicated System in use in parts of Europe

Families • Should we legislate away families? – enforcing the rights of the dead o Need to consider the rights of the individual vs rights of the family o Problems: Ignores medical culture of tending to the living Ignores concern for those left behind Overemphasises autonomy Other cases, family allowed to express religious and cultural objections

Stem cells • Have the capacity to differentiate and proliferate o Totipotent – everything o Pluripotent – cross lineage lines o Multipotent – limited, specific types of cells o Unipotent – one type of cell (Eg. cornea) • Biology o Embryonic vs somatic stem cells o Adult stem cells Found in essentially every tissue (especially bone marrow) Activation is important Multipotent stem cells from adults can be used for transplants • Transdifferentiation o Cells have potential to differentiate advantageously depending on factors of the environment Grow organs? o Induced pluripotent stem cells Successful in early animal models Creating life problematic? • Stem cells have been used for treatment of Parkinson’s disease, degenerative diseases, heart failure etc. • Debate has moved into the public domain • Issues to consider: o Business, hope, hype, church, cloning fears, embryos, public opinion and morals, politics

Living donation • Risk borne by donor, recipient has benefit o What is acceptable risk? • Free consent, is the donor (especially family donors), are they free to make choices o Acceptable risk may be higher o Coercion • Growing body parts: children for spare parts

Xenografting • Coagulation proteins and cytokines come from goats, pigs o Animals can be humanised with human genes o Heart valves continue to come from pigs • Issues: o Animal rights o Risks (infective/immune) o Interference with nature

Fetal tissue • Issues: o Morality of termination of pregnancy o May encourage termination o Symbolism of the fetus, utilising a person for another’s end o Often unsuccessful

Organ commerce • Transplant tourism – condemned by WHO o Established processes in India, Malaysia, China Moves dependent on source availability • Donors o Often have intergenerational debt (get 5% of money, owner gets 30%, vender gets 10%) o Often illiterate o Health problems, consequences • Reasons for: o Is it acceptable to let 20-30% on waiting list die? o Monetary benefit to donors, autonomy over own body • Against: o Intrinsic, integrity of body: doesn’t extend to autonomy o Slavery – cannot decide to sell organs in these situations, when there is no other options o Consequences Killing of vulnerable Risks to donors and recipients o Enhances inequity Rich get richer, poor get poorer

Brain death – a convenient fiction • “legal death” – heart stops or brain dead o Permits donation to occur Allows heart/lungs to be taken out without committing ‘murder’ • Problems: o Biological ‘Brain dead’ can be kept alive for 14-18 years • Females can be impregnated, gestate a child and give birth Possible persistent brain function • Anaesthetic given before organ removal o Community/families Have issues and concerns that person wasn’t dead • A convenient definition to increase numbers of organs available for transplant • Arguments over definition: o All brain function or just high cortical? • Law change: o 2 minutes after asystole is considered for donation after cardiac death (DCD) donors • Black and white vans • Ways of arguing it to patients so they are not fooled: o No one survives brain death Disconnect and die OR Donate organs and die • Choose.

Allocation of organs • Criteria are controversial, egs: o Biological factors (HLA, ABO, liver size) o Need, outcome, aetiology, time on waiting list o Compliance o Age, co-morbidities, social utility/ dependents o Race • Fairest way: uniform set of medical criteria and then a lottery

Survivorship • Many people who survive are not better off: o 30-60% fall apart existential crises: identity, body fears, relationships, isolation • Need to ensure provision of care caters for this phenomenon Lecture 51: Wrap up lecture Rachel Thompson

Scenario 1: • Obesity and nutrition • Biochemistry

Scenario 2: • Alcohol • Histopath • Pharmacology

Scenario 3: • Physiology • Histopath • Micro