CBFM Learning Objectives (Final) PDF

WEEK 6 Macronutrients and Healthy Dietary Intake (1.5hr) 1. Define factors (biological, social, physiological, pathophysiological) that influence health. a. Biological factors → Genotype (sex), age, phase of life cycle, metabolism b. Physiological factors → Desire to eat, appe...

WEEK 6 Macronutrients and Healthy Dietary Intake (1.5hr) 1. Define factors (biological, social, physiological, pathophysiological) that influence health. a. Biological factors → Genotype (sex), age, phase of life cycle, metabolism b. Physiological factors → Desire to eat, appetite, palatability of food c. Social factors → Cultural eating habits, availability of food d. Pathophysiological factors → Presence of disease 2. Compare and contrast the basic chemical structures of carbohydrates, fats, and proteins, and how the structures of each result in different caloric outputs per gram of each. a. Carbohydrates, proteins, and fats supply 90% of dry weight and 100% energy i. Calories in each 1. 4 Calories per 1 g of carbohydrates and protein 2. 9 Calories per 1 g of fat b. Digested in the intestines, where they break into their basic chemical units i. Uses basic units for growth, maintenance, and activity 1. Carbohydrates → Simple sugars 2. Proteins → AA 3. Fats → FA and glycerol 3. List common food sources of the three (3) major macronutrients and the recommended calorie intake range (%) of each. a. Dietary carbohydrate (45%-65%) i. Simple sugars 1. Monosaccharide (glucose, fructose) – Corn syrup, honey 2. Disaccharides (sucrose, lactose, maltose) – Maple syrup, molasses, table sugar, mil, beer, and malt liquors ii. Complex carbohydrates 1. Polysaccharides – Starch implants, wheat, whole grains, potatoes, dried peas, beans, vegetables iii. Non-digestible carbohydrates 1. Dietary fibers b. Dietary Protein (10-35%) i. Oxidation of AA will produce CO2, H2O, and ammonium (NH4+) ii. Some used for fuel, most used for anabolic purposes c. Dietary Fats (20%-35%) i. Body’s main form of long-term energy  ii. Influence incidence of CHD iii. Evidence link with risk of cancer or obesity much weaker 4. Recognize the nomenclature and compare and contrast saturated, monounsaturated, polyunsaturated, and trans fatty acids. a. Nomenclature i. Numbering #1 C ii. Greek method – Alpha (1st C), beta (2nd C) iii. Greek omega – Last C b. Example of nomenclature → 18:3Δ9,12,15 = 18 C atoms long, 3 double bonds located at C atoms 9,12,15. c. Specific FA i. Saturated – No double bonds, solid @ room temperature 1. Animal fats, tropical oils ii. Monounsaturated – One double bond, liquid @ room temperature 1. Olive, canola, nuts, avocado iii. Polyunsaturated – Multiple double bonds, liquid @ room temperature 1. Omega 3 → Fish, flaxseed, walnuts 2. Omega 6 → Corn, safflower, sunflower, soybean iv. Trans fatty acids – Some natural (milk, meat), most artificial due to addition of H to liquid vegetable oils (unsaturated) to make them liquid 1. Partially hydrogenated d. **NOTE** – # of double bonds ↑ membrane fluidity i. Also controlled by cholesterol ii. Fxn of lipid dependent on FA chain length and degree of saturation 5. Explain calorie, DEE, BMR, and DIT and describe the factors that contribute to BMR. a. Calorie: Unit of heat (energy) i. 1 Calorie (food) = 1 kilocalorie or 1000 calories = amount of energy needed to raise the temp of 1 L of H20 by 1ºC b. DEE (daily energy expenditure): Daily amount of energy needed i. DEE = BMR + PA + AT 1. BMR = Basal metabolic rate 2. PA = Physical activity a. Sedentary = BMR x 1.2 b. Heavy active = BMR x 1.9 3. AT = Adaptive thermogenesis a. Diet or temp induced b. Regulated by brain c. Uncoupling proteins (UPCs): Endogenous protein found in inner mitochondria that mediates AT i. Dissipate H+ gradients by transporting H+ back into matrix, bypassing ATP synthase c. BMR: Measure of the energy required for biological functions; minimum amount of energy that a body requires when lying in physiological and mental rest. i. Proportionate to amount of metabolically active tissue and lean body mass ii. Expressed as kcal/day (Calories/day)  iii. What affects → Gender, body temp (↑ with fever), environmental temp (↑ in cold), thyroid status (hyperthyroid ↑, hypothyroid↓), age (↓ with age), body comp (↑ with muscle mass) iv. Calculation → 1. Male: (13.75 x weight in kg) + (5 x height in cm) – (6.76 x age) + 66 = BMR 2. Female: (9.56 x weight in kg) + (1.85 x height in cm) – (4.68 x age) + 655 = BMR d. DIT (diet-induced thermogenesis): Thermic effect of food or production of heat by the body i. ↑ 30% above resting level during digestion and food absorption ii. Slows down weight loss in individuals 6. Describe BMI, relate a calculated BMI to the patient weight category (underweight, ideal, obese, etc.), and describe and evaluate the weaknesses of BMI as a measure of body fat. a. BMI (body mass index): Determines an individual’s healthy body weight b. BMI= Wtkg / (Htmeters)2 OR BMI= (WtLbs * 704)/(Htinches)2 c. Weaknesses of BMI measuring body fat i. Assume excess weight is fat ii. Various factors (age, ethnicity, etc) can influence relationship between BMI and body fat iii. Cannot distinguish between excess fat, muscle, or bone mass 7. Identify the four categories of dietary reference intake (EAR, RDA, UL, and AI) and list the classes of compounds that have an RDA. a. Dietary Reference Intake (DRIs): Set of reference values for a healthy population based on the relationships between nutrient intake and health or the prevention of disease i. Estimated Average Requirement (EAR): Average daily dietary intake level that is sufficient to meet 50% of the nutrient requirement for healthy individuals ii. Recommended Daily Allowance (RDA): Average daily level of intake sufficient to meet the nutrient requirements of nearly all (97%-98%) healthy people 1. Carbs don't have RDA 2. Fats a. Essential fatty acids – α-linoleic acid, α-linolenic acid, EPA, DHA 3. Protein ≈ 0.8g x weight (kg) or 0.36 g x weight (pounds) a. High quality: Has all essential AA in sufficient amounts i. The 9 essential AA → Lys, Iso, Leu, Thr, Val, Trp, Phe, Met, His 4. Based on food we ingest  iii. Tolerable Upper Intake Level (UL): Highest daily nutrient intake level with no adverse effects to most individuals iv. Adequate Intake (AI): Based on observation or experimentally determined approximation of nutrient intake by a group(s) of healthy people 1. Used when RDA cannot be determined 8. Describe the different types of fibers and the role of dietary fiber in diet and health. a. 3 types of fibers i. Soluble fiber: Forms a viscous gel when mixed with a liquid 1. Role of dietary fiber in diet and health → a. ↓ absorption of dietary fat and cholesterol b. ↑ fecal loss of cholesterol c. Delays gastric emptying d. Generate sensation of fullness e. Reduces postprandial [blood glucose] f. OPTIMIZE DIGESTIVE HEALTH 2. Examples → Pectin, hemicellulose (prebiotic) 3. Sources → Legumes, oats, chia seeds, various fruits, and vegetables ii. Insoluble fiber (roughage): Passes through the digestive tract largely intact 1. Role of dietary fiber in diet and health → a. Don’t dissolve in water, but retain it b. Promote regular bowel movements by retaining water; prevent constipation and hemorrhoid formation c. Essential for systematic cleansing and detoxification d. Modulate blood sugar 2. Examples → Lignin, cellulose (prebiotic) 3. Sources → Whole grain, wheat, bran, nuts/seeds, vegetables, fruits iii. Functional fiber: Extracted or synthetic fiber and proven health benefits 9. Distinguish between prebiotics and probiotics and how they relate to a healthy microbiome. a. Prebiotics: Soluble fibers that are key to optimizing digestive tract health i. Lead to production of SCFA ii. Can be fermented in the large colon; allows for slow digestion → maximum mineral absorption iii. Associated with ↓ risk of colon cancer, aids in ↓ cholesterol iv. Foods → Bananas, apples, beans, artichoke, asparagus, dark leafy greens, allium family vegetables b. Probiotics: Naturally created by process of fermentation and control growth of harmful bacterial, keeping digestive system healthy i. Live beneficial bacteria ii. Foods → Yogurt, sauerkraut, miso soup, kimchi 10. Describe the current NIH dietary guidelines, a healthy eating plate, and the Mediterranean diet. a. Current NIH dietary guidelines i. Healthy eating plan → Provides nutrients body needs every day while staying in Calorie goal ii. ↓ risk of heart disease and other health conditions iii. To ↓ 1. Calories of added sugar  2. Daily sodium intake to 35 inches (F)) 5. High blood pressure (≥ 135/85 mm Hg) ii. Can occur in people with a healthy weight/BMI. 6. Explain how glycemic index relates to obesity. a. Glycemic index: Number (0 to 100) assigned to a food, with pure glucose arbitrarily given the value of 100 i. Relative rise in blood glucose levels 2 hours after consuming that food ii. Ranking 1. Low GI < 55 2. Medium GI 56-69 3. High GI >70 b. Relation to obesity i. Foods with high GI → High blood glucose → Fat storage → Obesity Vitamins** (2hr) 1. Classify each vitamin as water- or fat-soluble. a. Vitamins: Organic nutrients that are required in small amounts (from diet), but play essential roles in life i. Water-soluble vitamins: Directly absorbed from GI tract and distributed/used by cells and tissue 1. Easily secreted 2. Less seen in toxicity 3. Have shorter half-life 4. Types → B Vitamins ( B1, B2, B3, Biotin, Pantothenic acid, B6, Folate, B12), Vitamin C ii. Fat-soluble vitamins: Absorbed and transported with fat (lipoproteins) 1. Accumulation in the body 2. Easy to cause toxicity if in excess 3. Types → Vit A, Vit D, Vit E, Vit K 2. Identify the major dietary sources for each vitamin. a. *see chart* 3. Explain how vitamins are taken up (particularly B12), transported, and stored (if applicable). a. B12 is absorbed in the ileum (initially bound to Transcobalamin I in the gastric mucosa of the stomach and travel to the small intestine) i. Absorption into ileum requires IF created in parietal cells of stomach to bind to B12 ii. IF replaces Transcobalamin I b. *see chart for rest * 4. Identify the cofactor each vitamin becomes (e.g. Flavin → riboflavin) and describe the major metabolic functions of these cofactors or the enzymes utilizing them. a. Types of cofactors i. Organic cofactors = Derived from vitamins ii. Inorganic cofactors = Derived from metal or salt b. THF i. Source of one-carbon unit (serine, glycine, histidine, formaldehyde, fomrate) ii. One carbon pool (formyl, methylene, methyl) iii. Products of one-carbon (receiver) 1. dUMP to dTMP 2. Guanine to adenine 3. Homocysteine to methionine iv. B6 and B12 helps v. Pathways involved in 1. 5,10-methylene THF (FH4) a. Nucleotide and DNA 2. B12 is essential to rescue THF from 5-methyl-THF a. 5-methyl-THF donates C to homocysteine to form methionine (needs vit B12) 3. B12 deficiency → leads to methyl-THF trap 5. List the major types of oxidants and the major mechanisms the body uses to reduce them. a. Folate i. Food THF is hydrolyzed to folic acid in intestine before it’s absorbed (active transport) ii. Reduced to dihydrofolate then THF in intestinal mucosa using NADPH iii. Main folate in plasma is 5-methyl-THF iv. Conjugated into functional polyglutamate form in tissues 6. Explain the function of vitamins E and C as antioxidants. a. Vitamin E is stationary in cell membrane, but is a scavenger for free radicals i. Prevent free radical-caused membrane damages 1. Cancer risk ↓ 2. Coronary heart disease ↓ 3. Aging-related signs ↓ ii. Reducing agent iii. α-tocopherol = most potent member b. Vitamin C is mobile, also a scavenger for free radicals; soluble i. Prevent free radical-caused damages ii. Can regenerate reduced vitamin E iii. Anti-cancer effect 1. Prevent formation of nitrosamine, a carcinogenic reagent iv. Also protects cardiovascular system v. Preventing LDL oxidation c. Beyond vitamins C & E, body has enzymes to protect against ROS/RNS i. SOD ( peroxisomes, the cytoplasm and mitochondria), catalase (peroxisomes), glutathione reductase (mitochondria and cytosol) 7. List the name of the disease due to a deficiency or an excess for each vitamin (where applicable).  a. *see chart* 8. Describe the evidence supporting whether patients should be taking vitamins as supplements. a. Expenditures in US for total supplements estimated to run in the range of $21–25 billion per year b. Estimated ~97% of population needs met by healthy diet c. Depending on who is doing the research and where money is coming from i. Vitamins can cause adverse rxns d. Vit D supplementation supported for some populations e. Aging population will lead to ↑ in vitamin deficiencies f. Ultimately → Vitamins may be beneficial in a few instances, but healthy diet should provide what’s needed (in certain subpops) i. Subpopulations include – older adults, pregnant women, people who can’t eat a proper diet, chronic alcoholics, strict vegetarians & vegans, and people with specific health conditions. WEEK 7 Minerals** (1.5hr) 1. Identify the major dietary sources for each mineral and list storage sites in the body (where applicable) a. See chart 2. Describe the mechanism(s) of uptake and transport for each mineral discussed, and where applicable, what inhibits and/or stimulates uptake a. See chart 3. List the name of the disease due to a deficiency or an excess for each mineral (where applicable) a. See chart 4. Identify the cofactor(s) each mineral becomes (where applicable) and describe the major metabolic functions of these cofactors or the enzymes utilizing them a. See chart 5. Explain the link between iron and oxidative stress a. Iron – key role in MANY enzymes – catalysis and electron transfer, oxygen transport b. Highly toxic (and insoluble) in presence of oxygen – catalyzes the Haber-Weiss-Fenton Reactions: i. Fe+2 + O2 → Fe+3 + O2- ii. 2 O2- + 2H+ → H2O2 + O2 iii. Fe+2 + H2O2 → OH + OH- + Fe+3 c. The HIGHLY reactive hydroxyl radical (OH ) attacks proteins, lipids, EVERYTHING, and damages these molecules d. Fe proteins create these toxins (by accident) and also defend against them (e.g. superoxide dismutase) e. This can also happen with Cu and Mn (but not Zn) i. Copper assists with iron absorption**** Heme Synthesis and Degradation (1hr) 1. Describe the basic chemical structure of heme. a. Heme: Planar porphyrin cofactor found in different biological pathways  i. Porphyrin: A cyclic compound that contains 4 pyrrole rings linked by methenyl bridges. ii. Dependent on what's proteins surrounds them 1. Found in hemoglobin and in other proteins (cytochromes, catalases, peroxidase) b. When not associated with protein, heme → lipophilic pro-oxidant c. Conjugation double bonds responsible for color i. Oxygenated = red; Deoxygenated = blue (cyanosis) d. Protoporphyrin IX + ferrous iron (Fe2+) i. Fe2+ doesn't fit perfectly in plane puckers out to one side e. Iron binds i. 4 N in the 4 pyrrole rings of protoporphyrin IX ii. Histidine AA in globin protein iii. +/- O2 2. Describe the heme synthesis pathway, including cellular location and starting substrates, and explain how it is regulated, including regulation of ALA synthase isoenzymes and mechanisms to coordinate heme and globin synthesis. a. Most heme made in the liver (cytochromes) or bone marrow (erythrocytes); small amount in cells b. 1st rxn (succinyl Coa + glycine to ALA) and the last 3 (coproporphyrinogen III to heme) are in the mitochondria; Rest is in the cytosol. c. Heme synthesis pathway i. Starting substance → Succinyl Co-A (product of TCA cycle) ii. Subsequent rxns result in condensation of pyrrole rings (each with methyl, propionyl, or vinyl side chains) which combine to form heme d. δ-ALA Synthase & Dehydratase i. 1st rxn → Catalyzed by δ-ALA Synthase 1. Location – Mitochondria 2. Substrates – Succinyl-CoA and glycine 3. Cofactor is vitamin B6 (pyridoxal phosphate) 4. Feedback inhibition by heme & hemin a. Regulatory i. Other regulation of ALAS 1. Glucose inhibits, steroids ↑ synthesis, >100 drugs ↑ ALA synthase activity (especially those metabolized by cytochrome P450) 5. Product → δ-ALA 6. Rate-limiting step 7. 2 isozymes – ALAS1 & ALAS2 a. ALAS 1 → i. Liver, other tissue ii. Short half-life iii. Negative feedback by heme (↑ mRNA degradation, blacks translation to mitochondria) iv. Exquisitely sensitive to regulation by levels of heme. b. ALAS2 →  i. Erythroid, bone marrow (makes 85% of daily heme) ii. Regulated in response to levels of Fe (TL level, IRE in 5’UTR) iii. Responses to heme at a more leisurely rate than ALAS1 1. Indirect via regulation of Fe acquisition from transferrin iv. TS regulated by erythroid-specific promoter 1. Same TSFs that regulate globin synthesis c. Regulation of protein synthesis in response to iron levels through IREs in mRNA i. IREs in 5’ UTR 1. Ferritin, ALAS2 2. ↓ translation when intracellular levels of iron are low ii. IREs in 3’ UTR 1. Transferrin receptor, DMT1 (divalent metal transporter) 2. mRNA stabilization and ↑ translation when intracellular levels of iron are low ii. 2nd reaction → Catalyzed by δ-ALA dehydratase 1. Location – Cytosol 2. Inhibited by heavy metals (i.e. Lead) 3. Product – Porphobilinogen (PBG) iii. 6th reaction → Ferrochelatase 1. Location – Mitochondria 2. Catalyzes insertion Fe2+ into protoporphyrin IX 3. Inhibited by heavy metals (i.e. Lead) 4. Product → Heme (Fe2+ protoporphyrin IX) e. Coordinations of mechanisms for heme and globin synthesis i. Coordinated with iron availability 1. ALAS2 has iron response elements (IRE) in 5’UTR 2. Heme/hemin regulate Fe availability ii. Coordinated by globin synthesis 1. TS of ALAS2 under control of same TF that regulate globin synthesis 2. EPO ↑ TS of ALAS2 and α and β globin 3. Heme ↑ TS of globins and stabilize their mRNAs 4. Low levels of heme activate a kinase that causes inhibition of TL iii. Happens mostly before enucleation 3. Explain the molecular bases of acquired (lead poisoning) and genetic (porphyria cutanea tarda and acute intermittent porphyria) porphyrias. a. Lead poisoning i. Pb inhibits ALA dehydratase and ferrochelatase ii. ↑ ALA in blood; Coproporphyrin III and ALA accumulate in urine iii. Anemia due to ↓ Hb iv. Competes with Ca2+, particularly in heavy storage sites  1. Neurotoxic effects primarily due to inhibition of NT due to disruption of Ca2+ homeostasis v. Symptoms are age-, dose-, and duration-dependent b. Genetic porphyrias i. Porphyrias: Abnormal build-up of heme synthesis intermediates 1. Rare, with intermittent and non-specific symptoms 2. Dominantly inherited with low penetrance ii. Classified as erythropoietic or hepatic iii. Symptoms precipitated by – 1. Certain drugs (metabolized by cytochrome P450), EtOH, fasting/severe dieting, hormones, stress iv. Some specific types 1. Porphyria cutanea tarda: Chronic disease caused by deficiency in uroporphyrinogen decarboxylase a. Uroporphyrin accumulate in urine b. Most common type with clinical onset in 30s-40s c. Expressed mainly in pts with alcoholism or liver damage d. No neurologic or abdominal symptoms e. Pts are photosensitive i. Caused by porphyrins or porphyrinogens (later intermediates) reacting with sunlight to form reactive oxygen species f. Symptoms precipitated by – i. Sunlight, iron, alcohol, hepatitis, HIV g. Tx → Beta-carotene (anti-oxidant), abstain from EtOH, phlebotomy to reduce iron, avoid sunlight 2. Acute intermittent porphyria: Acute disease caused by deficiency in hydroxymethylbilane synthase a. Porphobilinogen and δ-aminolevulinic acid accumulate in urine b. Intermittent attacks i. Acute abdominal pain, constipation, muscle weakness, cardiovascular abnormalities ii. Neurological dysfunction → agitation, seizures, psychosis iii. Colored urine and stool on exposure to light and air iv. Episodes last few days to several months c. Pts NOT photosensitive i. Blockage d. Many have no symptoms except with precipitating factors e. Tx → Hemin + glucose – slow down the heme synthesis pathway c. Overall treatment → Inhibition of ALAS i. Hematin (stable derived of heme) ii. Carbohydrate-rich diet iii. Withdrawal of any precipitating drugs 4. Explain the heme degradation pathway and its regulation. a. Most of the Hb to be degraded comes from senescent RBC.  i. Smaller amount comes from turnover of immature RBC and from other heme-containing proteins in non-erythroid tissues. b. Goal = Preserve Fe and convert protoporphyrin to products that can be safely excreted c. Heme degradation pathway i. Heme → Bilirubin (Br) in macrophages 1. Heme oxygenase cleaves pyrrole ring to produce biliverdin a. Requires O2, NADPH b. Releases CO and Fe3+ 2. Biliverdin reductase a. Requires NADPH b. Produces bilirubin ii. Conjugation of Br 1. Unconjugated Br = insoluble and is carried on albumin to liver 2. In hepatocytes, solubility ↑ by conjunction to 2 molecules of UDP-glucuronate a. Occurs via bilirubin glucuronyl transferase 3. Conjugated Br (Br diglucuronide) actively transported by Multidrug Resistance Protein 2 (MRP2) into bile iii. In small intestine, bacterial hydrolases de-conjugate Br iv. Bilirubin is reduced to form urobilinogen v. Fate 1. Some urobilinogen is oxidized to urobilin (stercobilin) and excreted in feces a. Give stool its brown color 2. Some is reabsorbed to the blood via the portal vein to the liver, then enters the circulation and is excreted in urine d. Regulation → Occurs to prevent toxic build-up of heme and its breakdown products  i. First reaction is rate-limiting and subject to regulation 5. Define direct and indirect bilirubin and describe common causes of direct and indirect hyperbilirubinemia. a. Jaundice/icterus: Caused by deposits of Br in skin and sclerae when levels in blood ↑ i. Due to ↑ production or ↓ excretion of Br 1. Prehepatic (Hemolysis) 2. Hepatic (Neonatal, Hepatitis, Genetic) 3. Post-hepatic (Bile duct obstruction) b. Measurement of Br i. Direct = conjugated (usually about 4% of total) 1. Can easily be coupled to diazonium salts (azo dyes) in a direct van den Bergh rxn ii. Indirect = unconjugated 1. Br in non-covalent complex with albumin won’t react with the dyes until albumin is released by an organic solvent iii. Total – Direct = Indirect 1. First measure direct, then add methanol and measure total. Subtract to get direct. c. Hyperbilirubinemia i. Prehepatic (hemolytic) Jaundice – Indirect 1. Hemolysis only causes jaundice if amount of bilirubin being produced is so high that it exceeds the capacity of the liver to conjugate it 2. Causes a. ↑ production of Br, exceeding capacity of liver to conjugate b. ↑ urobilinogen in blood and urine due to ↑ conjugated Br reaching intestine c. No unconjugated Br in urine because it’s insoluble, carried by albumin ii. Intrahepatic (hepatocellular) Jaundice – Direct/indirect 1. Impaired hepatic uptake, conjugation, and/or secretion of Br 2. Conjugated bilirubin in urine a. Less conjugated Br going to the intestine, so less urobilinogen in stool makes them paler than normal. b. Urobilinogen ↑ in urine (and makes urine darker) because ↓ enterohepatic circulation means more stays in the blood and goes to kidneys. 3. Liver enzymes (ALT and AST) get released to the blood when hepatocytes die (direct)  iii. Posthepatic (obstructive) jaundice – Direct 1. Bile acids may also accumulate in plasma 2. Conjugated bilirubin in urine 3. No urobilinogens in stool 4. Prolonged obstruction can lead to liver damage and ↑ unconjugated (indirect) Br 6. Explain the molecular basis of physiological newborn jaundice and the treatment of phototherapy. a. Neonatal jaundice i. Quite common, usually self-limited ii. More common in premature infants iii. Bilirubin glucuronyl transferase is developmentally expressed, adult levels reached ~4 weeks after birth iv. If Br levels exceed binding capacity of albumin, Br can accumulate in the basal ganglia → Causes toxic encephalopathy called kernicterus v. Jaundice is abnormal if it occurs before 24 hr (possible hemolysis) or if it persists or occurs after 10 days (inborn error, structural defect in bile duct) vi. Resolves within 10 days b. Phototherapy i. Converts Br to more soluble isoforms that can be excreted into bile without conjugation 7. Explain the molecular basis and genetic causes of intrahepatic jaundice (Crigler-Najjar, Gilbert, and Dubin-Johnson syndromes). a. Crigler-Najjar Syndrome i. Rare deficiency bilirubin glucuronyl transferase (very severe) ii. Type 1 = total deficiency, Type 2 = less severe than type 1 iii. Severe unconjugated hyperbilirubinemia present at birth iv. Autosomal recessive b. Gilbert Syndrome i. Quite common (~5%), benign mild elevation indirect bilirubin ii. Mutation in promoter of bilirubin glucuronyl transferase gene, decreased expression (~1/3 of normal) iii. Autosomal recessive c. Dubin-Johnson syndrome i. Defective transport of conjugated bilirubin out of hepatocytes ii. Rare mutation in MRP2 iii. Autosomal recessive 8. Describe the roles of haptoglobin and hemopexin in scavenging hemoglobin fragments in the blood. Haptoglobin Hemopexins Binds αβ dimers in blood, produces a complex too Free Hb not bound by haptoglobin is oxidized to large to be filtered by the renal glomerulus metHb → dissociates to globin and metheme Delivers dimers to macrophages via specific receptor Hemopexin binds metheme, delivers it to the liver  [Serum haptoglobin] reflects degree of intravascular Delivery of heme to hepatocytes induces heme hemolysis oxygenase Levels ↓ with sustained hemolysis Hemopexin and its receptor are recycled, but may be depleted with prolonged hemolysis Haptoglobin → acute phase reactant, so levels may ↑ during inflammation or infection Hemoglobin and Oxygen Delivery (1.5hr) 1. Describe the structure of hemoglobin. a. 4 proteins – globins OR globin chains i. 8 helices interrupted by turns ii. 2 α type and 2 β type; arranged as 2 αβ dimers 1. α and β chains within a dimer are held together tightly by very stable hydrophobic bonds 2. 2 αβ dimers held together by ionic bonds, can be easily broken and reformed. iii. 1 heme prosthetic group bound to EACH globin 2. Explain how the structure of Hb supports its function with regard to O2 and CO2 transport. a. Dimers held together by ionic bonds i. Flexibility between 2 dimers ii. Important feature allowing HB binding affinity for O2 b. Each heme can reversibly bind one molecule of oxygen, for a total capacity of 4. i. When all 4 binding sites are bound by oxygen, Hb molecule =saturated. c. Binding of Heme to Globin i. Fe of heme binds to globin chain via proximal His on the F helix ii. Distal His on E helix helps stabilize interaction of O2 with Fe 2+ 1. Helps prevent oxidation of Fe2+ to Fe3+ and reduces Hb affinity go CO 3. Describe developmental regulation of globin chain synthesis, and explain how it results in different hemoglobin molecules. Compare and contrast HbA and HbF in terms of their structure and function; explain the difference in their p50s. a. Developmental regulation i. There are – 1. 2 α-type globins, ζ (zeta) and α, which are encoded by three genes (there are two α globin genes) on chromosome 16. 2. 3 β-type globins → ε (epsilon), γ (gamma) and δ (delta) encoded on chromosome 11. ii. Expression of the globin genes is regulated developmentally, both in terms of cell differentiation during formation of red blood cells and in development of the person. iii. Genes are TS only during a specific period (5-7 days) of RBC development from proerythroblast to enucleations iv. Genes can’t be transcribed after enucleation, but mRNAs are very stable. v. Globin protein synthesis continues and can still be regulated at the TL level even after enucleation. b. Subunits → The most highly expressed  i. Epsilon (ε) and zeta (ζ) embryonic globins 1. Form earliest form on hb, Hb Gower (ζ2ε2) ii. Predominant form in the fetus, HbF, is composed of α and γ chains (α2γ2). iii. α and β are the “adult” hemoglobins 1. β → Fetal liver, then bone marrow, and combines with α to make HbA (α2β2). a. As synthesis of β chain ↑, levels of γ ↓ i. ≈ 6 months after birth HbA accounts for 90%, HbF 2% and HbA2 is a minor form 1. Beta Thalassemia shows HbA2 fraction ii. After ≈3 months gestation, α is the only α-type globin c. HbA vs HbF i. Fetal = higher affinity for O2 → Must be able to pull oxygen away from mother hemoglobin 1. Has gamma chain that has fewer + charges than beta chain 2. HbF does not bind 2,3-BPG (missing one allosteric inhibitor) a. Curve to the left of HbA and less steep in the pO2 found in tissues ii. Birth = worse affinity = shift over to adult form so that hemoglobin more easily gives up oxygen to tissue d. P50 i. 20 mmHg in fetal ii. 26 mmHg in Adult e. 4. Compare and contrast Hb and Mb, including structure, cellular functions, and oxygen dissociation curves. a. Hemoglobin: Tetramer of polypeptides (globins), each containing a heme group 1. AA constituents of each globin molecule are very similar, containing hydrophilic AA on the outside surface, and hydrophobic AA on the inside with the exception of 2 hydrophilic Histidine residues 2. His residues are responsible for holding heme group in place b. Myoglobin: Oxygen storing molecule found in skeletal and cardiac muscle 1. Primary structure (AA sequence) between Mb and β – globin of Hb shares high homology 2. Secondary and tertiary structures between Mb and Hb subunits are nearly identical 3. Unlike Hb, Mb exists as a monomer w/ gives it completely different oxygen binding characteristics c. Summary i. Myoglobin 1. Shift to the left (higher affinity) w/ hyperbolic curve (one bind site) 2. Mb (monomer) more suited for oxygen storage 3. p50 =1 mmHg ii. Hemoglobin 1. Shift to the right (lower affinity) w/ sigmoid curve (cooperativity) 2. Hb (tetramer) more suited for oxygen delivery 3. p50 =26 mmHg 5. List the factors involved in cooperative binding in Hb, and describe the mechanism by which each affects cooperative binding. a. The quaternary nature of Hb provides it with cooperative binding  b. Mechanism i. In deoxyHb, the Fe is slightly out of plane with the heme group ii. Binding of oxygen forces the Fe back into the plane of the heme iii. In Hb (since it is a tetramer), this local shift causes a more global shift in how the globin molecules are arranged, which is why Hb has a cooperative O2 binding effect iv. 300x ↑ in affinity once transitioned from T to R state 6. Explain the process of moving from a fully deoxygenated T state to a fully oxygenated R state in Hb. a. Conformation → Each component of the Hb tetramer can exist in either a tense (T) or a relaxed (R) state i. Due to salt bridges which form between the deoxyHb tetramers to stabilize it ii. Takes more energy to bind first O2 to a deoxyHb because some of these salt bridges must be broken b. Moving between states i. Salt bridges formed = T = less oxygen binds (↓ affinity) 1. Fe puckered out from center, heme plane has slight dome shape, F helix not perpendicular ii. Transition – As O2 approaches Fe2+, it causes unpaired e- to “flip their spins” and pair up; Fe becomes smaller, gets pulled into the heme plane. 1. Pulls Fe2+ down into center of plane, creating strain iii. Salt bridges weaken = R = more oxygen binds (↑ affinity) 1. Movement of globin chain relieves strain by breaking ionic bonds between alpha-beta dimers (rotated 15º) 2. Next O2 has easier time binding = less strain to overcome 7. Compare and contrast the action of 2,3 BPG, proton binding/pH, and CO2 with regard to modulation of oxygen binding in Hb. Predict the effect of each on the oxygen dissociation curve. a. O2 dissociation curve i. Sigmoidal curve = allosteric regulation 1. O2 = allosteric activator 2. 2,3 bisphosphoglycerate (2,3-DPG or BPG), H+, CO2, Cl-, temp= allosteric inhibitor a. Help Hb unload O2 by ↓ Hb affinity for O2 by stabilizing T form ii. Don't have much effect in the lungs because [O2] high b. 2,3-BPG → Lowers hemes affinity for O2 i. Associates inside the ‘pocket’ formed by Hb tetramer 1. Stabilizes the T-structure of Hb via salt bridges with various AA residues within the globin sequence 2. Low pO2 = 2,3-BPG to be formed from the Rapoport-Luberin shunt ii. In tissues → more 2,3-BPG, so more oxygen will be released from Hb into the tissues iii. In lungs → less 2,3-BPG, so more oxygen will bind to Hb iv. In altitude and hypoxic environments, 2,3-BPG levels ↑  c.H+ → Lowers its affinity for O2 i. As T converts to R, H+ is released ii. Proton binding (Bohr Effect): 1. Blood pH ↓ (ie: proton concentration rises) because CO2 produced by metabolism gets converted into carbonic acid 2. Lung pH ↑ (ie: proton concentration decreases) because carbonic anhydrase exists and cleaves carbonic acid into H2O and CO2 which can then be exhaled iii. p50 at high pH (arterial blood) is lower than the p50 at lower pH (venous) 1. This means Hb is more prone to give up its oxygen when in venous blood, and more prone to keep its oxygen in arterial blood iv. Ex. 1. As pH ↓=O2 affinity ↓ = H stabilized T form = right shift a. Little effect in the lungs, where pO2 is high d. CO2 → As CO2 levels rise, hb binds H+ rather than oxygen (O2 go to tissue) i. CO2 is about 20X more soluble in blood than O2 ii. When it diffuses into an RBC, it immediately becomes hydrated by carbonic anhydrase to form H2CO3 1. H2CO3 then dissociates into bicarbonate (HCO3-) and a proton (H+) iii. Haldane Effect: Describes how an ↑ in Hb-O2 binding ↓ the amount of proton binding 1. Why when the blood gets to the lungs (high pO2) the bicarb and proton recombine to form CO2 + H20 and are exhaled iv. Some binds directly to Hb (on N terminal AA of globin chain) 1. Produces H+, promotes further release of O2 form Hb in tissues 8. Describe chloride shift – what it is, and what it does to/for the cell. a. Chloride Shift: For every HCO3- moved out, a Cl- moves into the cell i. As a lot Cl- moves into the cell in venous blood, it causes water to follow 1. Results in swelling of the cell 2. Why venous hematocrit is ~3% higher than arterial hematocrit ii. In lungs, as CO2 is reformed and leaves, chloride leaves the cell, and RBCs shrink b. Helps equalize charge in RBC and plasma, regulate pH 9. Define methemoglobin. Explain how the cell reduces the amount of methemoglobin, and which enzymes are involved. a. Methemoglobin: Formation of Fe3+ that occurs when oxidation exceeds capacity of reduction  b. Methemoglobinopathies: When too much methemoglobin (Fe3+) is in the blood, either through genetic defects or acquired i. The auto-oxidation of Fe2+ in hemoglobin (Hb) to Fe3+ produces metHb ii. Normally ~1% of all circulating Hb is metHb iii. Methemoglobin is not capable of transporting oxygen iv. RBCs have a NADH-cytochrome b5 MetHb reductase system to reduce metHb back to normal (Fe2+) Hb 1. Off-shoot from the glycolysis pathway c. Methemoglobinemia i. Causes “chocolate” blood, cyanosis ii. Certain drugs, local anesthetics ↑ the amount of metHb iii. Infants are especially vulnerable iv. Death if metHb levels reach > 70% v. Tx → Methylene blue 1. Reduce metHb vi. Very rare congenital forms = 1. Hemoglobin M – Autosomal dominant mutation in heme-binding pocket 2. NADH cytochrome b5 reductase deficiency – Autosomal recessive inheritance a. Type I – only erythrocytes b. Type II – all cells 10. Compare and contrast the various causes of met hemoglobinopathy. a. Hemoglobinopathy: Disorders affecting structure, function or production of globin chains i. Usually codominant inheritance b. Types → i. α Thalassemias: When ratio of α and β globin subunits is not 1:1 1. Must have an equal molar ratio for Hb to form correctly 2. Bart's syndrome → All 4 α genes deleted (usually fatal) a. Hydrops fetalis– abnormal accumulation of interstitial fluid b. Hb Barts= γ4 (↑ affinity for O2, poor transporter) 3. H disease = 3 α genes deleted a. HbH== β4 ii. β Thalassemia: Excess α chains precipitate, cause destruction of RBC (hemolysis), anemia 1. β Thalassemia Minor a. Heterozygous b. Usually asymptomatic except for mild anemia 2. β Thalassemia Major (aka Cooley Anemia) a. Homozygous b. Transfusions for severe anemia c. Often suffer from iron overload iii. Sickle cell: A specific globin allele (HbS) which results from a single nucleotide substitution (glu [charges] to val [nonpolar]), and leads to the formation of fibrous polymers of Hb; only in deoxy state 1. Autosomal recessive  2. ↓ solubility, but only the deoxy form is so insoluble that it precipitates in RBC and causes sickling 3. Usually only homozygotes have symptoms – Hemolytic anemia, vaso-occlusion leading to tissue infarction, painful crises, ↑ risk of stroke 4. Precipitating factors include – Acidosis, Hypoxia, Infection, Stress iv. CO poisoning: An acquired hemoglobinopathy wherein CO displaces O2 in the hemoglobin, leading to hypoxia 11. Describe the effect of carbon monoxide on hemoglobin function. a. CO competes with O2 for binding to Hb b. ↑ O2 affinity of remaining sites (220x more in CO than O2) WEEK 8 Basics of Enzymes and Enzyme Regulations (2hrs) 1. Explain the process of substrate molecules being enzymatically transformed into products (including transition state complex and activation energy) and illustrate the process on an energy diagram. a. Important definitions i. Activation Energy: Energy needed for the reactants (substrates) to reach the transition state 1. New bonds are forming and old bonds are breaking (transition state complex) 2. Transition State Complex: Point where maximal strain is experienced on the bonds right before snapping a. I.e. the point in between substrate and product that is denoted by the peak on the graph b. Unstable 3. Catalyst can lower EA → Speeding up rxn (stabilize transition state) a. Doesn’t change overall ΔG values ii. Transition State (point): State corresponding to the highest energy along the reaction coordinate. It has more free energy in comparison to the substrate or product; thus, it is the least stable state. 1. There can be multiple transitions states for couples (successive) rxns iii. Active Site: Site (usually an opening or cleft) where the chemical reaction takes place; contains fxnal groups that actively involves with rxn (may help lower EA by making bonds and stabilizing transition-state complex) 1. If there is a cofactor, will bind near active site as well iv. Substrate Binding Site: Point where the substrate is bound to the enzyme  b. What’s occurring i. Bind: Substrate binds to the substrate binding site 1. Non covalent bonds form 2. Amino acids from enzyme or cofactors ii. Stabilize Transition State: Additional bonds form with the enzyme to stabilize the substrate in its transition state 1. Stabilization is how enzymes lower the activation energy iii. Product: Formed product is released and the enzyme is available for another substrate to bind 2. Describe lock and key and induced fit. a. Specificity: Enzymes have an extremely high degree of specificity, due to the chemical shapes/interactions within the substrate binding site i. Lock and Key: Substrate binding site creates a 3-D shape that is complementary to the substrate allowing the substrate to lock into place (non-covalent interactions) ii. Induced Fit: Substrate binding to the enzyme induces a conformational change inducing a closer and more appropriate shape to the substrate 1. Fxnal groups reposition to promote rxn 3. Differentiate between cofactors, coenzymes, and isozymes a. Cofactors: A component other than the protein portion of an enzyme (metal ions/minerals, vitamins, GSH, ATP, CoQ, etc) i. Prosthetic group: Cofactor that is tightly bound to the apoenzymes (i.e. many minerals) 1. Protein portion usually has to be denatured for prosthetic group to be removed from protein portion ii. Coenzymes: Organic cofactors that is loosely bound to the apoenzyme and can be easily separated from it (i.e. most vitamins) 1. A specific cofactor b. Isozymes: Enzymes that differ in primary AA sequence, but catalyze the same chemical rxn i. Many have different kinetic parameters (i.e. Km, Vmax), where they are expressed, and/or how they are regulated ii. Ex- ALAS 1/2, HO-1, COX 1/2, LDH 4. Identify the different functional groups (amino acid side chains, coenzymes,and metal ions) and explain how each contributes to an enzymatic reaction. a. Amino Acid side chains i. Ser, Cys, Lys, His → covalent catalysis ii. Polar AAs → nucleophilic catalysis b. Coenzymes: Non-protein organic molecules (vitamins) i. Activation-transfer: Form covalent bond with substrate, then activate it for transfer forming more complex molecules 1. Ex. a. Biotin → Uses its N to attach to -COO groups of carboxylases (water-soluble B7, covalently linked to Lys) b. Coenzyme A → Uses its sulfhydryl group to do nucleophilic attacks on carbonyl groups (requires vit B5) c. Thiamine pyrophosphate (TPP) → Contains carbon with dissociable proton (requires B1 – thiamine ) ii. Oxidation-reduction: Functional groups accept or donate electrons;  1. Oxidized = loses electrons (loss of H or gain of O); Reduction = gains electrons (Gain H or lose O) 2. Similar to activation-transfer but NO covalent bonds formed c. Metal ions i. Electrophiles: Substrate binding, stabilizing anions through ionic charges (+ / -), donate/accept electrons in redox reactions 5. Describe factors (pH, temperature, concentrations of enzyme and substrate) affecting the rate of a reaction. a. pH → Most active at a certain pH, usually correspond to cellular location i. ↑ in activity of an enzyme as the pH ↑ = usually due to the ionization of residues in the active site (protons will dissociate at higher pH). ii. ↓ of activity as the pH gets too high is usually due to the inappropriate ionization of AA, or the loss of ionization of amine groups. iii. Can serve as protection (i.e. stomach enzymes fxn at pH 2-3) b. Temperature → Most enzymes function optimally at 37°C i. Denaturation occurs as temperature increases ii. Rates ↑ with temperature 1. Double for every 10 degree increase in temperature. c. Substrate/Enzyme Concentration → i. If there is very little [substrate], average rate of all the enzymes is low 1. Most of the enzymes don’t have substrate to bind. ii. If [enzyme] stays the same, but [substrate] increases, average rate ↑ until all enzymes are saturated and working at a max speed. iii. If there is more enzyme, products accumulate faster. 6. Compare and contrast between Michaelis-Menten and Lineweaver Burkplots for enzyme inhibition, describe where Km, Vmax, Km, app, Vi are present on each plot and predict the effect of reaction perturbations (i.e. increases in substrate, increases in inhibitor, etc) on each plot. a. Michaelis Menten: Plotting the Velocity vs [Substrate] gives us hyperbolic curve i. Assumes enzymes is reversible (catalyzes the forward and reverse rxns) b. Lineweaver Burke: Taking the inverse (1/V vs 1/[S]) transforms it to a line i. Easier to make comparisons, determine type of inhibition occurring, etc. c. Variables i. Km: An expression of the affinity an enzyme has for a substrate 1. Numerically denoted by ½ Vmax 2. Smaller Km = Higher Affinity / Larger Km = Lower Affinity 3. Tends to use μM or nM ii. Vmax: Known saturation point, aka the fastest speed that an enzyme can catalyze a reaction with unlimited substrate iii. Km Apparent: Measurement of affinity in the presence of an inhibitory molecule iv. Initial Velocity: Eliminates the reverse reaction by immediately removing the product v. Kcat: Numerically shows how well the enzyme is functioning d. General Inhibition  i. Reversible 1. Competitive → Inhibitor binds to substrate-binding site (active site) a. Ex– Statin drugs (antiHLP) 2. Noncompetitive → Inhibitor binds enzyme other than the substrate-binding site (allosteric site) a. Can be before or after substrate binds b. Pure: Inhibitor binds in a different place than a given substrate (can have 1 or more substrates) c. Mixed: Inhibitor binds outside of substrate-binding sites d. Ex– Allopurinol at high concentrations, alanine, lead poisoning 3. Uncompetitive → Inhibitor only binds the ES complex (not enzyme alone) a. Only happens in multi-substrate reactions ii. Irreversible inhibitors: Binds to the active site of the enzyme and irreversibly reacts with the enzyme to permanently inactivate the enzyme 1. Continue to try to make products as normal, however, will create highly reactive compounds that is irreversibly bound with enzyme 2. Vmax ↓ (like noncompetitive), Km unchanged 3. Ex – aspirin, cyanide sulfides, organophosphates; also useful in pharmaceutical agents e. Perturbations i. ↑ substrate – Used to overcome a competitive inhibitor ii. ↑ inhibitor – Shows up graphically as a proportional shift as denoted f. MM vs LB plots i. Competitive inhibition → Vmax unaffected, Km,app ↑ with inhibitor 1. With unlimited substrate, results in [substrate] >> [inhibitor]. Enzyme will bind substrate every time 2. Why Vmax is the same a. Lines cross at some point on LB due to Vmax being calculated from y-intercept 3. Since it takes a much higher [substrate] to reach the Vmax value, Km value has ↑ (lower affinity). 4. When there is excess [inhibitor] or even equal [inhibitor] as [substrate], some of the enzymes bind to the inhibitor instead of substrate. a. Average velocity of all enzymes is lower because some enzymes aren’t catalyzing anything. 5. Comparison – a. On the MM plot, this is shown by a less steep line leading up to the Vmax value. b. On the LB plot, the x-intercept gets closer to 0. ii. Noncompetitive inhibition  1. Pure → Vmax ↓, Km unaffected a. Inhibitor binding in a different place than a given substrate b. Because inhibitor can bind to enzyme alone, Vmax ↓ i. Some enzyme is bound to inhibitor and can't bind substrate, even if [substrate] ↑ c. On the LB plot, it crosses the y-axis higher up. i. Should be written as Vmax, but it often is not. d. Km does not change because the substrate still has access to its binding site (active site) i. Both lines cross the x-axis at the same point. ii. Only true for pure noncompetitive, not mixed 2. Mixed → Vmax ↓, Km changes (may ↑ or ↓ ) a. Binding of the inhibitor changes the affinity for the substrate, so Km will be affected now. i. As to whether Km,app is ↑ or ↓ depends on the inhibitor. iii. Uncompetitive → Vmax ↓, Km,app ↓ 1. Inhibitor only binds ES complex, can’t bind to enzyme alone 2. As the inhibitor binds to and stabilizes the ES complex, makes it more difficult for S to dissociate or be converted to product, ↑ the enzyme’s affinity for S, thus ↓ the Km value (higher affinity). SUMMARY → 7. Contrast KD and KM a. KM - Michaelis-Menton constant i. Kinetic constant ii. Measures impact of [substrate] on speed of rxn iii. Can be used as an indirect measure of affinity iv. Determined by substrates binding affinity and how quickly enzyme-substrate complex is turned over into product 1. Turnover numbers (catalytic constant): Measure of the max catalytic activity when the enzyme is saturated with substrate.  a. Catalytic activity: # of substrate molecules converted into product per enzyme molecule per unit time i. Represents the kinetic efficiency of the enzyme. ii. Formula 1. kcat = Vmax / [E]total (Units = sec-1) b. KD - Dissociation constant i. Thermodynamic constant ii. True measure of affinity of substrate for binding site on enzyme iii. Under usual conditions, gives the [ligand] at which half of the protein molecules have ligand bound. iv. Ex – At what [] will substrate want to dissociate from the enzyme binding pocket? 1. Doesn’t address speed of rxn c. For both, the smaller the number, the bigger the affinity 8. Explain the importance of a regulatory enzyme in a metabolic pathway using examples. a. Catalyze i. Irreversible metabolic reactions ii. First committed step in a metabolic pathway b. Enzyme activity can be regulated by i. Levels of substrate and products – immediate effects 1. Substrate availability (velocity changes) 2. Products inhibition (VMAX and/or Km will change, depends on if inhibitor is competitive or noncompetitive) ii. Allosteric effector molecules iii. Hormonal regulations c. Regulation via inhibition i. Inhibitors - ↓ the rate of an enzymatic reaction 1. Reversible – diffuse away at a significant rate (i.e. not covalent!) 2. Irreversible – ‘suicide inhibitors’ a. Covalent (or very, very tight non-covalent) bonds to enzyme b. ↓ amount of active enzyme available c. Ex. aspirin, penicillin ii. Transition-state analogs 1. Best inhibitors 2. Bind more tightly to enzyme than substrate or products 3. Many pharmacologic examples (penicillin, allopurinol, etc) d. Regulation of each step permits efficient regulation of flux of metabolites through that specific pathway i. Example → Phosphofructokinase 1. Allosteric enzyme 2. Sigmoidal response to its substrate 3. Activated by fructose-2,6-P 4. Inhibited by citrate 5. Rate limiting step of glycolysis e. Placement of regulated steps can vary; oftentimes near start of a pathway or at a branch point. 9. Compare and contrast the mechanisms for regulating enzymes (feedback inhibition, allosteric, hormonal regulation via covalent modification, and induction/repression), and relate to the time required for each. a. Feedback loops (positive or negative) i. Product acts on an upstream enzyme ii. Can be covalent modification or allosteric modification iii. Quick form of feedback (seconds) 1. Ex. Heme had negative feedback on ALA synthase-1 for heme synthesis pathway. b. Allosteric regulation (activators or inhibitors) i. Allosteric enzymes change their confirmation upon binding of allosteric effector 1. Multiple active sites with cooperative binding for the substrate a. Slow rate of rxn initially; as you occupy more active sites, enzyme undergoes conformational changes, rate of rxn changes b. Number of subunits equals # of active sites ii. Sigmoidal curve is dependent on [substrates], vary greatly in catalytic output in response to small changes in effector or ligand concentration iii. Allosteric effector (inhibitors and activators) bind to the allosteric site of the enzyme (not the active site) 1. Inhibitor → Binds to allosteric site, causes a conformational change in the active site, preventing the enzyme from binding to the substrate 2. Activator → Binds to allosteric site, causes conformational change in active site, making it more available to the substrate to bind a. “Lock and Key” iv. Can see effects within seconds c. Covalent modification i. Physical change to the enzyme 1. Example – Addition or removal of a phosphate group → alters protein function a. Kinase: Adds a phosphate group to the hydroxyl portion of the enzyme b. Phosphatase: Removes phosphate group from hydroxyl portion of enzyme c. Phosphorylation by covalent modification may lead to an ↑ in activity while others may be inactivated ii. Takes sec to minutes d. Hormone regulation via covalent modification i. Hormones bind receptors and cause activation of kinases or phosphatase → Regulate enzyme pathways ii. Cellular response (covalent: sec – min; hormone: min – months) iii. Ex – Insulin binding to its receptors and activating PKB, which phosphorylates glycogen synthase kinase, making it inactive. e. Induction/repression of genes i. Hormone changes the expression of the enzyme 1. Binds to DNA and influence expression of the enzyme 2. Changes the synthesis of the protein (min to hrs. to days)  a. Over-expression = induction i. Changes physiological events that are to occur b. Under-expression =repression ii. Many pathways lead to activation of TF (activate or inhibit transcription of DNA to RNA) or proteins that affect translation (i.e. IRE-BP binding to 5’ or 3’-UTR). iii. Slower process in the cell, on the scale of minutes to hours (even days if hormones are involved). 10. Compare the catalytic mechanism of an allosteric enzyme to an enzyme that follows Michaelis-Menten kinetics. a. Allosteric enzymes DON'T obey MM kinetics i. Velocity curve is SIGMOIDAL rather than hyperbolic when plotted with identical axes ii. Due to allosteric molecules have more than one subunit and them changing conformation when reacting with substrate 1. Multiple active sites exhibit cooperativity iii. Influenced by [substrate] 1. Ex. a. R state (high [], more enzymes found in this state) b. T state (favored when there is insufficient substrate to bind to enzyme) b. Allosteric effectors: Small molecules that bind to enzyme at sites other than active site and regulate enzymatic activity i. At branch points, or committed steps, in a pathway ii. Ex. 2,3-BPG, pH, CO2 for hemoglobin affinity c. Ex – Hemoglobin (protein NOT enzyme though) Basics of Hormonal Regulation (1hr) 1. Compare and contrast the properties and mechanisms whereby hormones exert effects on target tissues a. Communication i. Nervous system → Electrical impulses 1. Neuroendocrine hormones: Secreted by neurons into circulating blood  ii. Endocrine system → Chemical signals – hormones 1. Endocrine hormones: Released by glands or specialized cells into circulating blood b. Hormones i. Cholesterol derivatives ii. Tyrosine derivatives iii. Peptides/polypeptides c. Transport of hormones i. Water-soluble hormones: Travel free in blood 1. Receptors located on outer membrane of cell 2. Easier to target for degradation 3. Stored in secretory vesicles until signal indicated time to release a. Some produced as prohormones (i.e. insulin) ii. Lipid-soluble hormones: Bind to proteins in the blood 1. Produced by liver 2. Free hormone = Active and act on receptors a. Hormones will pass through the membrane of a target cell and act upon a receptor located in cytosol or nucleus 3. Stabilized as they are bound to carrier proteins 4. Synthesized as needed (except thyroid hormone) 2. List the hormones (insulin, glucagon, epinephrine, norepinephrine, cortisol, thyroid hormone) produced by endocrine glands and their broad functions Hormone Function Insulin Released into blood when blood [glucose] levels are high Promotes the uptake of glucose in cells ○ Can use eventually for energy or store for later Metabolically affects liver, muscle, adipocytes Mechanistically affects enzymatic activity (phosphorylation status) and amounts of enzymes (inducing expression) Glucagon Controls plasma [glucose] during fasting, exercise and hypoglycemia by ↑ hepatic glucose output to circulation Acts on liver to ↑ glucose production ○ 1st by glycogenolysis, then gluconeogenesis Stimulates breakdown of lipids in liver (lipolysis) and ↑ production of ketone bodies Epinephrine Released from adrenal medulla in response to stress ○ Exercise, exposure to cold, emergencies, hypoglycemia Causes a prompt ↑ in blood [glucose] in postabsorptive state ○ Has similar effects as glucagon in metabolism (hepatic glycogenolysis, gluconeogenesis, increased FAs oxidation) In skeletal muscles, activated breakdown of glycogen so lactate can be formed and transported to liver to convert into glucose *Opposing effects of epinephrine on heart and liver! Norepinephrine Released from adrenal medulla and nerve endings (majority) in response to stress (i.e. exercise, exposure to cold, emergies, hypoglycemia) ↑ hepatic gluconeogenesis ○ Due to combined ↑ in lactate and glycerol released by peripheral tissues net hepatic gluconeogenic efficiency Can stimulate thermogenesis in BAT Hepatic glycogenolysis is less responsive Major response is vasoconstriction (↑BP) Cortisol Released from adrenal cortex and promotes the mobilization of energy stores ○ Regulated by ACTH in a diurnal rhythm ○ ACTH regulated by CRH from hypothalamus (HPA axis) Cortisol binds corticosteroid-binding globulin (CBG, aka transcortin) and albumin in the blood CBG transports cortisol through blood Hormone (H) diffuse to the cytosol and binds to its receptor (R) H-R translocates to the nucleus interacts with hormone responsive element (HRE) Mediates gene expression Reduce inflammation (inhibits phospholipase A2) ↑ degradation of proteins in muscles to transport glucogenic AA to liver for increased gluconeogenesis Promotes lipolysis in adipose tissue, ↑ transport of FAs and glycerol to liver Enhance activity of glucagon Thyroid hormone ↑ metabolic rate and consumption of O2 ○ Clearance of cholesterol from plasma ○ Glucose absorption in small intestine Iodine is necessary for thyroid hormone synthesis 3. Describe the basic structure and function of the pancreas a. Has endocrine and exocrine portion b. Structure i. Islet of Langerhans: Set of specialized cells that produce and secrete hormones directly into bloodstream 1. Alpha cells produce glucagon a. Localized near periphery of islets 2. Beta cells produce insulin a. Localized near the middle 3. Delta cells produce somatostatin (gastrin) 4. F cells produce pancreatic polypeptide c. Function i. Cells sense levels of glucose in blood and secrete appropriate hormone into blood 4. Describe how a meal activates insulin release and how insulin activates tissues a. mRNA → Preproinsulin → proinsulin → insulin → Ca2+ and glucose → secretory granules i. C-peptide is secreted together with insulin b. Insulin release i. GLUT2 → Only active in high [glucose] 1. When high, glucose enters into pancreatic BETA cells and glycolysis occurs → TCA cycle and oxidative phosphorylation to ↑ intracellular [ATP] 2. High [ATP] inhibits ATP-gated K+ channel, which leads to depolarization causing Ca2+ to enter through voltage-gated channel 3. Intracellular ↑ in [Ca2+] leads to insulin secretion to the blood. c. Insulin activation i. Soluble insulin binds to RTK (an insulin receptor) 1. 2 receptors must dimerize and activate each other by autophosphorylation 2. This allows RTKs to phosphorylate other proteins ii. Activated PI3K (Activated phosphatase (PP1), mobilization of GLUT4 transporters) or MAPK pathway (promote gene expression) d. GLUT4 and Insulin i. Glucose uptake in skeletal and cardiac muscles, diaphragm and adipose tissues cells → Regulated by insulin-stimulated exocytosis of membranous vesicles containing GLUT4 (insulin dependent) 1. Process is reversed by endocytosis 5. Describe how fasting activates glucagon release and its functions a. Preproglucagon → preglucagon → glucagon i. Secretion is inhibited by 1. Insulin (inversly proportional) 2. Glucose ii. Secretion is enhanced by 1. AA 2. Catecholamine 3. Glucocorticoids 4. Nervous system b. Glucagon release  i. The levels of ATP inside pancreatic ALPHA cells indicate the levels of [glucose] in the blood ii. When blood [glucose] gets low, not much glucose is transported inside the alpha cell through the GLUT1 transporter 1. Means glycolysis is not occurring and the levels of ATP in the cell ↓. iii. Low level of ATP causes the ATP-sensitive K+ channels to close, resulting in depolarization of the membrane and opening of the voltage-dependent Ca2+ channels to open iv. Ca2+ enters the alpha cell and results in vesicles containing glucagon to be released from the alpha cells into the bloodstream. v. Regulators 1. Major → Glucose, insulin, AA 2. Minor → Cortisol, neural (stress), Epi c. Glucagon activation i. Glucagon receptors are GPCRs that activate either Gq or Gs pathways 1. Located in liver 2. Gq stimulates PLC, which produces IP3 and DAG (Ca2+). 3. The Gs pathway activates AC to produce cAMP and activates PKA. ii. REMEMBER → Glucagon activates PKA, and given it is a kinase, glucagon will phosphorylate regulatory enzymes in metabolic pathways. WEEK 9 Broad Overview of Metabolism (1 hr) 1. List the end product of food oxidation, and describe the process of respiration a. Fuel metabolism i. Body is programmed to store any excess dietary fuel so it can be used at a later time. ii. Various tissues are specific for storing different types of molecules. 1. Adipose cell store TAGs 2. Muscles store glycogen and protein 3. Liver stores glycogen. iii. All the eaten and stored fuel sources can be oxidized to produce energy, usually in the form of ATP. iv. Byproducts – Heat (energy lost/wasted), CO2 (elimination of carbons), and H2O (elimination of electrons) b. Respiration: Oxidation of food fuels to generate ATP i. Sources: Metabolites 1. Glucose (carbohydrates) 2. Fatty acids (fats) 3. Amino acids (protein) ii. Result: Energy 1. All are oxidized down to acetyl CoA which is a precursor of the TCA cycle 2. Ultimately results in electron transfer, ATP production 2. Compare and contrast between catabolism and anabolism, and list the key components/molecules that link metabolic pathways a. Catabolism: Series of chemical rxns that breakdown (oxidizes) complex molecules into smaller units, usually releasing energy in the process b. Anabolism: Sequence of chemical rxns that construct/synthesize molecules from smaller units, usually requiring the input of energy (ATP) in the process  c. Liver → only organ/tissue that can carry out the major metabolic pathways d. Key linker molecules between metabolic pathways – Metabolites that are used as a substrate for multiple pathways allows them to be key links between pathways. i. Glucose-6-Phosphate → can become glycogen, pyruvate, or ribose-5-phosphate ii. Pyruvate → Aerobic or anaerobic metabolism iii. Acetyl CoA 3. Describe delta G and how it impacts whether a reaction will move forward or not a. Work: A measure of energy transfer that occurs when an object is moved over a distance by an external force b. Efficiency: A measure of how much work is conserved in a process. i. Calculated by the energy output/energy input c. Delta G (Gibbs Free Energy): Amount of energy in a metabolic reaction that is required to perform work i. ΔG= ΔH {enthalpy – heat}+ (-TΔS) {entropy – disorder} 1. ΔG°’ – standard physiological conditions (25 °C, 1 M, pH 7) 2. ΔG – actual conditions for the molecules in the balanced equation ii. Conditions 1. Negative ΔG: Net loss of energy, rxn proceed spontaneously (exergonic). G substrate> G product, Keq >1 (more like catabolism) a. Less products 2. PositiveΔG: Net gain of energy, rxn does not proceed spontaneously (endergonic) G product> G substrate, Keq < 1 (more like anabolism) a. Less reactants 3. Δ G=0 means equilibrium is reached 4. Describe how cells use coupled reactions, the roles of ATP, NADH and NADPH, and contrast ATP energy with the “reducing power” of NAD(P)H a. ATP: Unit of energy generated by oxidations of fuels (glucose, FAs, aa), energy currency of the body i. Provides energy by cleaving off it’s gamma phosphate or sometimes both the beta and gamma phosphates. b. NAD(P)H: Coenzyme that provides a functional group through a redox reaction where it is the common redox agent/electron donor i. NADH → carries e- to the ETC (NAD+ for catabolism, NADH for anabolism) ii. NADPH → carries e- for biosynthesis (NADP+ for catabolism, NADPH anabolism) c. Endergonic and exergonic reactions are coupled → Ex. ATP hydrolysis i. Balance of reactions are required in a cell or organism ii. Synthetic operations coupled to energy production iii. Reflects free energy sum of individual coupled reactions 5. Predict how tissues such as liver, blood, kidney, skeletal muscle, brain, and adipose, would obtain energy under fed, fasting, and starving conditions a. Fed State: Glucose is the main fuel source, ingestion of carbs, lipids and proteins. Insulin and glucagon hormones regulate storage and retrieval i. Liver –Uses glucose (uptake via GLUT2), converts excess glucose to glycogen; produces Acetyl-CoA for triacylglycerol (TAG) storage via VLDL to be released into circulation, synthesizes protein from excess AA 1. GLUT2: Glucose transporter that has a high Km; only takes up glucose if there are high concentrations in the blood. ii. Blood – Use glucose (anaerobic glycolysis) iii. Kidney – n/a iv. Skeletal Muscle – Uses glucose (via GLUT4), stores excess as glycogen, synthesizes protein from excess AA 1. GLUT4: Glucose transporter that is insulin-dependent; insulin recruits the transporters to the membrane to uptake more glucose  v. Brain – Uses glucose vi. Adipose – Stores TAGs b. Fasting State: Blood glucose levels must be maintained, glucose levels ↓→ insulin ↓ i. Liver – Maintains glucose through glycogenolysis until stores depleted, and glycolysis. 1. Initiates gluconeogenesis with substrates (lactate-glycerol-AAs). 2. Converts nitrogen to urea (Excreted by kidneys). 3. Fatty acids are main fuel source. ii. Blood – Use glucose iii. Kidney – n/a iv. Skeletal Muscle – Uses glycogen, FAs, and KBs, and performs proteoglycans (AAs → liver) v. Brain – Use glucose vi. Adipose – Mobilized via lipolysis. 1. TGs converted to FAs and glycerol for liver to make ATP and KBs c. Starving Conditions i. Liver – Glycogen stores depleted quickly. GNG maintained to a lesser extent. 1. Converts FAs → KBs. 2. FAs are main fuel source. 3. Makes more KB for the brain. Glucose sparing. ii. Blood – Uses glucose for anaerobic glycolysis iii. Kidney – Excretes less urine. 1. Reduces acid load caused by ↑ production of KBs iv. Skeletal Muscle– ↓ use of glucose. 1. ↑ use of Fas. 2. Maintains a ↓ rate of proteoglycans. 3. Uses KBs for short period. 4. ↓ AAs for liver GNG → ↓ urea production/urine excretion. Protein sparing. v. Brain – ↓ use of glucose by 30%. 1. ↑ use of KBs, used exclusively after 2-3 weeks. vi. Adipose – Continues to mobilize TGs → FAs + glycerol Carbohydrates: digestion, absorption and glycolysis (1.5hrs) 1. Recognize the nomenclature (i.e. monosaccharides, polysaccharides, pentoses, etc.) and basic structure of carbohydrates, and the idea of enantiomers/isomers a. Carbohydrates: “Hydrate of carbons” i. Empirical formula = (CH2O)n b. Type of carbohydrate i. Monosaccharide → 1 monomeric unit 1. Most are in D form; AA are L form ii. Disaccharides → 2 monosaccharides 1. Formation of Disaccharides a. Combine through glycosidic bonds b. Reducing (anomeric C) vs. non-reducing end c. Maltose= 2 glucose i. More often written as Glc (α1→4) Glc d. Lactose= galactose and glucose e. Sucrose= glucose and fructose iii. Oligosaccharide → ~2-20 monosaccharides iv. Polysaccharide → > 20 or more repeating subunits 1. Ex. Amylose ( linear portion and is connected by α1→4 bonds only) and amylopectin (branches and has both α1→4 and β1→6 bond) a. One reducing end, many non-reducing ends  b. Starch, glycogen, cellulose i. Cellulose: Indigestible carbs for humans (component of dietary fiber). Strong fibrils formed by parallel chains of cellulose connected by hydrogen bonds 1. Has β1→4 bonds c. Triose: Smallest sugar i. Aldose vs. Ketose 1. Carbonyl group = aldehyde → aldose 2. Carbonyl group = ketone → ketose d. Ringed sugars i. Pentose: 5 carbon sugars ii. Hexose: 6 carbon sugars; more common e. Sugars attached to Proteins and Lipids i. Glycoprotein: Protein>sugar 1. Membrane bond, secreted ii. Proteoglycans: Sugar>protein 1. Mucins (mucus), lectins (cell-cell- interactions) iii. Glycolipid f. Enantiomers/isomers i. Chiral carbon: Carries 4 different atoms or groups ii. Enantiomers: Form non-superimposable mirror images iii. Isomers: Molecules have same molecular formula, but different arrangement of the atoms 1. Conformational isomers: Isomers are interconverted just rotating a group on a single bond (α vs β) 2. Describe the digestion and absorption of the major carbohydrates, including disaccharides and polysaccharides a. Cannot absorb long polymers of carbs, so we need to break them apart b. Digestion of Carbs i. Starch or glycogen begin digestion in the mouth ii. Saliva has α-amylase to start the breakdown 1. Starts to cleave α-1,4 glycosidic bonds to produce oligomers 6-10 in length iii. Go through esophagus to the stomach iv. High acidity in stomach to break down; inactivated α-amylase v. When gastric juices enters duodenum, pancreas secretes bicarbonate (HCO3-) down pancreatic duct 1. Neutralizes pH. 2. Pancreatic α-amylase also secreted in the duct and acts to further hydrolyze the α-1,4 glycosidic bonds. a. Forms maltose (Glc-α-1,4-Glc), isomaltose (Glc-β-1,4-Glc) and maltotriose (3 glucose with α-1,4 glycosidic bonds). vi. Other disaccharides (i.e. sucrose and lactose) and other maltoses → converted to monosaccharides by glycosidases attached to membrane in brush border of intestine. 1. Have different enzymes like maltase, isomaltase, sucrase, lactase, etc. to break down into monosaccharides (glucose, fructose, galactose) vii. Absorbed luminal side viii. Fiber take indigestible to the colon and excreted through feces 1. Bacteria metabolize saccharides, forming feces and SCFA (absorbed in colon mucosal cells and provide source of energy) c. Absorption – Take molecules from luminal side (apical) i. Fructose → GLUT5 ii. Glucose → GLUT1 iii. Galactose → Na+-glucose cotransporter (SGLT1) 3. Contrast the roles of the different glucose transporters, their location and identify which are insulin regulated a. Secondary Active Transporter SGLT-1 i. Glucose co-transported to the intestinal cells with sodium (free energy) ii. Coupled with Na/K-ATPase (consuming ATP) iii. SGLT2 in kidney cells for glucose reabsorption b. GLUT1 (constitutive) i. Glucose facilitated transport, Km= 1mM ii. In most tissues, brain c. GLUT2 i. Glucose facilitated transport, Km=~20mM (high) ii. Found in liver, kidney, pancreatic beta cells iii. Uptake promotes insulin secretion d. GLUT3 i. Glucose facilitated transport, Km= ~1 mM (low) ii. Found in brain, placenta, fetal muscle e. GLUT4 i. Glucose facilitated transport, insulin dependent, Km= 5mM 1. Exocytosis of membrane vesicles; reversed by endocytosis ii. Found in skeletal/heart muscle, adipocytes f. GLUT5 i. Fructose facilitated transports; Km=5mM ii. Found in small intestine, liver 4. List the 3 steps of glycolysis that are regulated, where they are located and explain how they are regulated a. Intro to Glycolysis i. Location – Cytoplasm ii. Anaerobic iii. 6 carbon molecule breaks into two 3 carbon molecules iv. 2 molecules of ATP are used, 1. 1 each by hexokinase/glucokinase (step 1) and PFK-1 (step 3). 2. 4 ATP are generated, 2 each by phosphoglucokinase (step 7) and pyruvate kinase (step 10). 3. Net yield → 2 ATP. b. Mechanism of glycolysis i. Preparatory phase – insulin facilitates uptake of glucose in skeletal muscles, cardiac m