PASS Proteins PDF

Chapter Four: Protein Structure and Function Chapter Sections: I. II. III. IV. V. VI. VII. VIII. Sequencing Proteins Secondary Proteins Pharmacokinetics Hemoglobin Organizing Metabolic Pathways Pharmacokinetics: CNS Depressants and Analgesic Collagen Other Quaternary Proteins Sequencing Proteins...

Chapter Four: Protein Structure and Function Chapter Sections: I. II. III. IV. V. VI. VII. VIII. Sequencing Proteins Secondary Proteins Pharmacokinetics Hemoglobin Organizing Metabolic Pathways Pharmacokinetics: CNS Depressants and Analgesic Collagen Other Quaternary Proteins Sequencing Proteins Now that we have learned about the amino acids, let us put them together and make proteins! 4 types of Protein Structures: 1. Primary – most basic level of organization—is the order, or sequence, of the amino acids in a protein; involves peptide bonds 2. Secondary – takes the shape of either an alpha helix or a beat-pleated sheet, held together by hydrogen bonds 3. Tertiary - three-dimensional (3-D) – contains a single polypeptide chain “backbone” in addition to one or more secondary protein structures 4. Quaternary – complexes of multiple polypeptide chains, also called subunits A peptide bond is formed when the carboxyl group of the amino acid on the left attacks the amino group of the amino acid on the right (see below) 67 Protein Structure and Function Figure 4.1 Primary Amino Acid Note: The amino terminal is always to your left and the carboxyl terminal is always to your right! The PEPTIDE BOND has 3 features to it: 1. Planar (flat) 2. Restricted mobility 3. R-groups are in trans configuration (face away from each other) to allow maximum mobility without instability. **Tighter than a single bond, but not as tight as a double bond Restriction enzymes: are currently used to sequence proteins The history of sequencing tells us the pros and the cons of each method (what they can tell us or not tell us). The amino terminal is always to your left, and the carboxyl terminal is always to your right! Methods of Protein Sequence: There are several laboratory methods used to determine the characteristics of a protein. These are tests that determine the type, amount, size, and charge of the amino acids in a protein. 68 Protein Structure and Function 1. 2. 3. 4. 5. Acid hydrolysis Gel electrophoresis Ninhydrin reaction Edman’s degradation Restriction peptidases Acid Hydrolysis • • • • • Determine types of amino acid in a protein, but not the sequence Performed by dipping the protein into acid, which denatures the protein Acids can denature proteins (melts a big protein into a little protein) Does NOT actually sequence the protein Turns asparagine into aspartate (acidic form) and glutamine into glutamate (acidic form) Con: can never tell how many asparagines, aspartates, glutamines, or glutamates are really in the protein Clinical Correlation If you have a protein in your body with lots of asparagines or glutamines (both are neutral), it is supposed to go to the liver. If they become charged after being dipped in acid, then the protein will never make it to the liver, eventually causing liver failure and renal failure. Therefore alcohol, which is acidic, can cause the hepatorenal syndrome. Gel electrophoresis • Uses agarose gel to separate proteins primarily by size • • It can also separate them by charge if you add electrodes Smaller proteins migrate further • Larger proteins stay closer to the start site 69 Protein Structure and Function • Does NOT sequence the proteins Pro: There is an electrophoretic pattern for any polypeptide, therefore gel electrophoresis can be used to detect it REMEMBER PROLINE is the oddball, responsible for twists, turns, kinks and bends! Figure 4.2 Gel Electrophoresis Ninhydrin Reaction • Is a chemical reaction which reacts with any amino acid on the amino terminal (left) • Reacts with all amino acids creating a purple color • Proline reaction creates a yellow color Con: Good ONLY for counting prolines Note: In all of biochemistry, there is only one enzyme that cuts to the left: CARBOXYPEPTIDASE 70 Protein Structure and Function Edman’s Degradation • Uses a reagent called phenyl isothiocyanate (PITC) • Reacts with ANY amino acid starting on the amino terminal • Amino acids are identified using thin-layer chromatography or highperformance liquid chromatography Con: Procedure is accurate ONLY up to 100 amino acids Restriction Peptidases • Determines a sequence by first cutting apart a protein into sets of amino acids • Restricted by what amino acids they can recognize • They are restricted in what they can recognize and cut • The proteins are chopped up and then we figure out how they must have been connected • Used to sequence proteins • You must derive the sequence • You must first know what amino acids the enzymes recognize ALL CUT TO THE RIGHT: • Trypsin: cuts to the right of LYS and ARG • Chymotrypsin: cuts to the right of the aromatic amino acids, PHE, TRP, TYR • Elastase: cuts to the right of GLY, ALA, SER, the 3 smallest amino acids • CNBr: cuts to the right of MET • Aminopeptidase: cuts to the right of the amino terminal amino acid • Mercaptoethanol: breaks up disulfide bond Clinical Correlation MERCAPTOETHANOL contains alcohol. Therefore, if you dip any of the 4 hormones with lots of disulfide bonds in alcohol, they will not work. This way, alcoholics can stop lactating or become diabetic after a while or why teenage alcoholics will stop growing. EXCEPTION: Carboxypeptidase: cuts to the left of any amino acid on the carboxyl terminal 71 Protein Structure and Function Restriction Enzymes ON THE TEST, they may ask you to determine sequence of a protein by giving you an enzyme and asking you to figure out the order in which the amino acids were cut off the protein. Each answer will give a list of sets, with each set containing several amino acids. The sets are not given in the order in which they came off the protein — e.g., Set 1 may or may not have come off before Set 2 etc. Additionally, the amino acids included within each set — the part inside the parentheses — also are not given in the order they were cut off —e.g., Phe and Arg are both in Set 1, however, Phe may or may not have come off before Arg. To determine the protein sequence, we suggest you first put the amino acids within each set in the correct order, and then put the completed sets in the correct order. Example: A researcher applied trypsin to a protein, causing three sets of amino acids to come off the protein: Set 1: (Phe, Arg, Val) Set 2: (Cys, Lys) Set 3: (Ser, Trp) In our example, you must determine the correct order of 7 fill-in-the-blank amino acids to get the overall protein sequence: __________________________________ Step 1: Pick a set-in parenthesis that contains an amino acid recognized by the enzyme mentioned in the question stem. Determine if that enzyme would have cut to the left or right of that amino acid. Use that information to put the amino acid in the correct position within that set. We know that trypsin cuts to the right of lysine and arginine, so we are looking for Lys or Arg. The first set of amino acids in parenthesis — Phe, Arg, Val — has an Arg. Since trypsin cuts to the right of that, then arginine must be the amino acid farthest to the right in Set 1. Step 2: Pick another set that contains an amino acid recognized by the enzyme mentioned in the question stem. Determine if that enzyme would have cut to the left or right of that amino acid. Use that information to put the amino acid in the correct position within that set. We are still looking for Lys or Arg since they tell us where trypsin would have cut the protein. The second set of amino acids — Cys, Lys — has Lys. Since trypsin cuts to the right, we would put Lys in the position farthest to the right in Set 2, 72 Protein Structure and Function which is where it already is located. Meaning the order of Set 2 is: (Cys, Lys) Step 3: If the question gives multiple enzymes, reapply Steps 1 and 2 to each enzyme listed. Keep going until you have determined the amino acid order of as many sets as possible. Step 4: Now that you have the amino acids within each set-in correct order, it is time to put the sets in order. Start by trying to determine which set is the actual first set of overall protein sequence. Begin by taking Set 1. If Set 1 was the actual first set, that would mean that Arg would be in the third position: _ _ Arg _ _ _ _ Find all the answer choices with Arg in the third position. Next take Set 2. If Set 2 was the actual second set, then Cys and Lys would be in the fourth and fifth positions: _ _ Arg Cys Lys _ _ Now take Set 1. If Set 1 is the actual second set, that would mean that Arg would be in the fifth position: Cys Lys _ _ Arg _ _ Select the answer choice that fits this pattern. If there is not one, then start with Set 3, and so on. This is how you do it quickly, by elimination. Secondary Proteins Secondary Amino Acid Structure 2 types: 1. Alpha helix o Amino acids are in coiled or spiral conformation o Allows hydrogen bonds to form between N—H group of one amino acid with the C=O group of another amino acid four residues earlier o Some amino acids are more prone to form alpha helixes Examples: Met, Glu, Cys, His, Lys 73 Protein Structure and Function Question What tissue would have a whole lot of twists and turns? Hair, GI, and Blood Vessels 2. Beta pleated sheet o Amino acids are in a series of adjacent rows o Allows lateral hydrogen bonds to form between N—H group of one amino acid with the C=O group of another amino acid o When there is a kink in the pattern, that means there is a proline located there o Some amino acids are more prone to form beta pleated sheets Iso, Tyr, Trp, Val REMEMBER The amino acid that is recognized by the enzyme must be in the right-most position! Figure 4.3 Alpha Helix & Beta Pleated Sheet 74 Protein Structure and Function Figure 4.4 The 4 Levels of Protein Structure Secondary Structure - Proteins 2 Types: 1. Functional Made by: a. Bone Marrow – Immunoglobulins b. Liver – albumin, fibrinogen, alpha-1 antitrypsin etc. i. The LIVER produces 90% of your proteins. ii. They serve to create an osmotic gradient. Osmotic gradient is needed to pull fluid into and out of capillaries. Example: transferrin transports iron; ceruloplasmin transports copper, etc. 2. Acute phase reactants. a. They are made any time there is inflammation b. Nonspecific c. So, whenever we find serum proteins, they could be there to either serve a purpose or they could be there as a sign of inflammation in the body. Too many proteins in your plasma (hyperproteinemia): • • • • Reflected by an elevated ESR or CRP ESR is a blood test that determines the rate that RBCs sediment in one hour CRP is an opsonin that binds to surface of dead cells to activate complement system Indicates nonspecific inflammation 75 Protein Structure and Function • • • Does not tell which proteins are increased Falsely high ESR: anemia Falsely low ESR: sickle cell anemia; polycythemia Clinical Correlation PROCALCITONIN is a new test to reflect excess protein in the serum during infections. What good is the ESR? What information does a sedimentation rate tell us? Patient is ill and you want to know whether it is an inflammatory or noninflammatory process. Example: fever of unknown origin (FUO) Therefore, we get a SED rate in fever of unknown origin. If it is high, think of inflammatory illnesses. If it is normal, then it is a non-inflammatory illness The patient has a severe inflammatory disease, and you are about to treat them? Example: Takayasu, Temporal arteritis, SLE, Wegener’s. We can use SED rate to follow whether the inflammation is decreasing over time. IF the SED rate is not trending down, then your medicine is not working. Acute Phase Reactants • Caused mostly by IL-6 • Made by macrophages and T-helper cells 76 Protein Structure and Function • Too many acute phase proteins can be deposited anywhere in the body • Non-specific deposition is called AMYLOIDOSIS Two Types of Amyloidosis Primary: Autosomal Dominant: • • • • • • 50% chance of getting the disease if only one parent has it (one good gene, one bad gene) 75% chance of getting the disease if both parents have it You cannot go beyond 75% chance in AD If the patient has the gene but does not have the disease: decreased PENETRANCE The gene is having a hard time penetrating through the other genes, but it is still there. How to explain when several family members have the gene and the disease, but all look different: variable expression Clinical Correlation AUTOSOMAL DOMINANT diseases usually involve structural proteins. In Primary disease, the patient will not have a good life • • • Compliance of blood vessel walls will decrease, leading to less distensibility This makes the blood vessel more likely to rupture if the pressure goes up Massive intracerebral hemorrhage in a young person with no prior h/o HTN or trauma Diagnosis: • • Congo red stain Apple green birefringence 77 Protein Structure and Function Secondary Disease: • • Due to any chronic inflammatory disease Most common causes: REMEMBER The first A stands for amyloid The second letter stands for the type of protein 1. AA (90%): • A=acute phase reactants/acute inflammatory disease • chronic inflammatory disease • In developing countries AA is due to = chronic infection • In developed countries AA is due to = RA, SLE, IBD etc. Example: patient with SLE now develops enlarged organs. This is a sign that amyloid is now depositing in those organs. 2. AB: • • • • • • • B=Beta or Tau protein (older terminology) Found in both age-related and Downs syndrome-related Alzheimer disease Betalipoprotein E4 is more specific for Alzheimer’s disease currently In the general population, this protein gets oxidized, causing Alzheimer’s Neurofibrillary tangles are oxidized proteins building up (Tau Proteins) In Down’s syndrome, they are not able to cleave the betalipoprotein from the Amyloid Precursor Protein (APP) Build-up of APP causes early onset of Alzheimer’s in Down’s patients Question What vitamin can you give Alzheimer patients to prevent oxidization of the proteins? Vitamin E 78 Protein Structure and Function 3. AB-2: Chronic renal failure: • Short for Beta-2 macroglobulin, which is supposed to be excreted by the kidneys • Builds up over time because it is unable to cross the dialysis filter 4. AE and AF: MEN-II • E= endocrine • F= familial Clinical Correlation What are the tumors of MEN-2? Medullary carcinoma of the thyroid Pheochromocytoma +/- parathyroid tumors 5. AL: Multiple Myeloma • “L” is for light chains (Bence Jones proteins) • Plasma cells in bone marrow produce abnormal antibody light chains • Light chains become amyloid • In urine they are called Bence Jones Protein • Kappa (90%) or lambda (10%) 6. • • • Transthyretin: amyloid associated with aging alone Senile plaques MCC of hereditary familial amyloidosis Autosomal Dominant REMEMBER LIPOFUSCIN is an oxidized lipid (not protein)! 79 Protein Structure and Function Tertiary Structure • • • • • • Can consist mostly of: o Alpha helixes – like hemoglobin or intermediate filaments o Beta-pleated sheets – fatty acid transporters or antibodies o Alpha helixes & beta-pleated sheets – some enzymes 3-D structure (square or circle, etc.) Most important factor that determines structure is hydrophobic and hydrophilic interactions Fat-soluble will fold inside, away from contact with water Water-soluble will fold outside, in contact with water Where covalent bonds are made Quaternary Structure • • • • • • Two or more proteins are interacting with each other = COOPERATIVITY The term cooperativity, when applied to enzymes, is called ALLOSTERISM. Allosteric enzyme is called the rate-limiting enzyme because it controls everything. It means that this is the enzyme that the others must cooperate with all the time. If it goes slower, all the others must go slower; if it goes faster; then all the others must go faster. If it stops, then all the others must stop. It is the slowest enzyme (not the fastest). REMEMBER Most genetic diseases are due to deficiency of the rate limiting enzyme. REMEMBER Apply this to the KIDNEY: If this is the kidney and we are filtering through a bunch of holes, there is no way to saturate holes. Whatever comes, a certain percentage of it will just fall through those holes. This is what gives the GFR a straight line. 80 Protein Structure and Function Pharmacokinetics First-Order vs. Zero-Order Elimination Rates Every rate-limiting enzyme in the body will cause the kinetic curve to be Sigmoidal shaped shows COOPERATIVITY Pharmacodynamics – has to do with the concentration of the drug at the site of action and what pharmacologic effect it has Pharmacokinetics – about the drug’s movement in, around and out of the body First-Order Elimination The drug action sites, which can be referred to as enzymes, but could be receptors or target organs. They are not saturated with the drug. As substrate concentration goes up, Vmax goes up. (Substrate = drug concentration) That means that, for as many active sites that you have, you have not saturated them all yet. There is room for more. Which is why, as you add more substrate, you get more Vmax. For that reason, you are metabolizing a constant fraction or proportion, which gives the slope of the line (20%, 30%, etc.). Test-Taking Strategy: You know that something is firstorder elimination because they will use the word “proportion” or give a percent eliminated per hour Zero-Order Elimination Says that substrate concentration no longer affects Vmax All the active sites are now filled up. 81 Protein Structure and Function You are now metabolizing a constant number rather than fraction. Whenever you have passive movement in the kidney, it follows a straight line— you cannot saturate filtration. Even if you send more liters of fluid to the kidney, it will still filter 20%. Therefore, GFR is 20%. There is no way to saturate the glomerular filtration. But for things that need transport proteins, like reabsorption and secretion, there is a finite number of transport proteins. Whenever transport proteins are involved, you will reach a saturation point called zero-order. It cannot go any faster than that. We can use this to figure out the transport maximum of things that are normally reabsorbed in the kidney. REMEMBER Transport maximum in the kidney to stay under zeroorder, you must reabsorb as much as you filter. Test-Taking Strategy: You know that something is zero-order elimination because they will give an amount—such as mg/ml—eliminated per hour 82 Protein Structure and Function First-Order Kinetics Zero-Order Kinetics Substrate Concentration still affects Vmax linearly All active sites are saturated (Vmax stays the same) All active sites are not yet saturated Substrate concentration no longer affects Vmax (flat) The substrate is metabolized at a constant fraction. Therefore, look at slope because it is constant. Metabolize a constant amount or number Figure 4.5 Zero-Order vs. First-Order Clinical Correlation Transport Max of GLUCOSE in the kidney is 126. If you keep the patient’s glucose is under 126, no glucose will be found in the urine. At 127, glucose will spill into the urine. Above 126, excess glucose will be floating around destroying the kidney, the retina, and the limbs. Because of this, current management of diabetes is to keep serum glucose levels under 126. Therefore, prediabetes is referred to as serum glucose between 100 and 126. 83 Protein Structure and Function Can there be zero-order without first-order? No. The first-order for these chemicals lasts for such a short period of time that you will not see them as the physician. Any drug can be pushed to zero-order. How do we bypass the first-order? Loading dose. Especially useful for drugs that treat deadly diseases. Once zero-order is reached, the drug is at a steady state. At zero-order is when the drug will start being most effective. Why is it better to keep a drug at first-order? Drugs can still be broken down if they are still in first-order. Zero-order drugs have great potential to do significant harm. Why would it be okay in medicine to use a drug in zero-order, knowing that it will harm your patient? When the disease is far worse than the harm you might do to your patient; when the disease is deadly; and when the benefit outweighs the risk. Clinical Correlation DRUGS THAT ARE ZERO-ORDER Some drugs reach zero-order way more quickly than others, and therefore must be carefully administered. This includes: • Alcohol • Aspirin • Phenytoin ***Zero-order elimination quickly in patients with liver or kidney disease since they physically have less ability to eliminate the drug. 84 Protein Structure and Function REACTION RATES But what does it matter how fast a reaction goes or if all the enzymes are saturated? It is a question of toxicity. We want to give a patient the lowest amount of a drug. How much drug we give depends on how well the drug binds to its target enzyme. COMPETITIVE vs. NON-COMPETITIVE Km is measured when half the enzyme's active sites are occupied by drug. Km = ½ Vmax Km tells you affinity—which is about the enzyme—and concentration—which is about the drug Vmax tells you the speed—which is about the drug—and saturation—which is about the enzyme. REMEMBER Vmax: how fast the reaction goes (Efficacy) [S]: substrate concentration Figure 4.6 Michaelis-Menten Graph 85 Protein Structure and Function Figure 4.7 Lineweaver- Burk Graph Test-Taking Strategy: They sometimes will show you an enzyme-kinetic graph called Michaelis-Menten Graph or Lineweaver-Burk graph. The visual difference between the two types of graphs has to do with the plot line—it is curved on a Michaelis-Menten graph and straight on a Lineweaver-Burk Graph. Competitive Inhibition Non-Competitive Inhibition Substrate and Inhibitor have similar properties and therefore bind to the same site Substrate and Inhibitor DO NOT have similar properties The Substrate and Inhibitor both bind to the same ACTIVE site The Inhibitor binds to site other than active, called Allosteric site. Causes enzyme to change conformation; substrate therefore cannot bind 86 Protein Structure and Function ↑Km = substrate concentration, affinity decreases No change in Km, affinity stays the same ↑Km will overcome inhibitor ↑Km will NOT overcome inhibitor Vmax stays the same, efficacy stays the same Vmax decreases, efficacy of the substrate decreases Km = 1/Potency, as Km increases potency decreases. Vmax=efficacy, which stays the same Km stays the same, so there is no change in Potency. Vmax decreases, therefore, efficacy decreases Majority of drugs (90%) are competitive inhibitors b/c if patient overdoses, the effect can be reversed These types of drugs are used in diseases that are far worse in prognosis than the effects of the drug ½ Vmax = Km If Vmax is 100 molecules per second, you just find where the Vmax is at 50 molecules per second. Then find how much substrate it took to do that. Km equals one over affinity or one over potency. • • If the km is high, then affinity and potency are low If km is low, the potency and affinity are high Clinical Correlation Name an organ where you will find the lowest km for any substrate (highest affinity or potency) The brain KIDNEY: • The kidney is for water-soluble compounds. 87 Protein Structure and Function • • Drugs that go to the kidney are acids and are charged. The best way to evaluate the kidney is via the creatinine clearance, but it is an impractical method because it requires a 24-hour hospital-stay. Now we use the peak and trough drug levels at any one moment in time. • • • • • • Peak is the highest concentration. It corresponds with the dose given. If the peak is too high, then you need to lower the dose. If the peak is too low, then you need to increase the dose of the drug. Peaks is measured 4 hours after a dose is administered. Trough corresponds to frequency of dosing. If the trough is too high, then give the drug less often (increase time between dosing). Trough is usually measured 1 or 2 hours before the next dose. • LIVER: Fat-soluble drugs are headed for the liver. There are large and small fat-soluble molecules. Fat soluble drugs are large molecules. Certain enzymes in the liver will take fat-soluble molecules and make them more water-soluble. They are called mixed function oxidases (most use oxygen to make them more soluble): • • • Sulfur Nitrogen Oxygen The liver itself can now get rid of it, or it can be sent to the kidney for excretion. Example: bilirubin The p450 system is the most important of these. Function decreases with age, so older people have problem metabolizing fat-soluble drugs. Definitions CONJUGATE: Adding a large molecule like glucose to bilirubin 88 Protein Structure and Function For large fat-soluble molecules, conjugation of a large water-soluble molecule: • • • • • • Acetylation is used 90% of the time Most common because the body already has a lot of acetyl-coA in the mitochondria from the Krebs’s cycle Adenylation Sulfate Phosphorylate Alcohol clearance and tolerance: The zero-order number for alcohol is 100 ml/dl. You can clear your blood of 100 mg of alcohol per hour. If the blood alcohol level is 100 ml (0.1), then it will take one hour for it to be cleared, 200mg, 2 hours, 250 mg, 2.5 hours. What can you do to speed it up? Nothing. So, there is no way to sober-up faster. Therefore, we let alcoholics in the ER sleep off their drunken stupor. After that point, they can sign out AMA if they refuse treatment. Alcohol would need to spend more time in first-order for it to be cleared faster. How can I get that to happen? Induce more enzymes, so there will be more sites for it to bind. This is called building up tolerance. How do you induce more enzymes? Ingest small amounts over a long period of time. Therefore, a person can drink 3x as much as another person before they even get buzzed. It has nothing to do with the size of the person. Cross-Tolerance • • • Any other chemical that reminds your body of the first chemical, your body will be used to that one too. Alcohol is an acid—how will it do damage to you? It will induce GABA Any other drug that enhances GABA, your body will be used to it. Therefore, alcoholics require even higher doses of benzodiazepines to be sedated. Definitions Drug abuse: If patient experiences social impairment Drug addiction: If patient has any withdrawal symptoms when the drug is taken away from them Chemical dependence: If patient needs the chemical just to feel normal 89 Protein Structure and Function Clinical Correlation 4 effects of acidosis: • • • • GABA increase Denatures proteins Causes hyperkalemia leading to arrhythmias Kussmaul breathing (rapid, deep breathing) Hemoglobin The first quaternary protein discovered that is not an enzyme. Hb is composed of 4 subunits. Each hemoglobin subunit has two components: • • Heme Molecule – contains an iron atom held in the middle of a honeycomb of carbon rings A globin – which means ‘globe’ or ‘ball’--protein chain, which wraps around the heme and holds it in place Hb A (adult): 2 alpha – 2 beta chains 98% of what we have Hb A2: 2 alpha – 2 delta chains 2% of what we have Hb F: 2 alpha – 2 gamma chains Fetal Figure 4.8 Hemoglobin 90 Protein Structure and Function Hemoglobin F Main hemoglobin made by the fetus during pregnancy. Production decreases after birth, reaching adult level in 6 months because it has a low affinity for 2,3, DPG or 2,3BPG. Hb F has a high affinity for oxygen. Hb F holds onto oxygen very tightly, except in severe hypoxia of the fetus. 2,3-DPG is found inside RBCs and physically sits in the hemoglobin molecule. 2,3 DPG will weaken hemoglobin’s grasp on oxygen in hypoxic situations. • • • COPD Anemia High Altitude In hypoxic conditions, the body will increase the amount of 2,3-DPG to get more oxygen to tissues. In utero Hb F ignores 2,3-DPG and holds onto oxygen very tightly. Why? Because the Hb F in the baby’s blood cells must forcibly take oxygen off Hb A in mom’s blood cells, which are on the opposite side of the placental membrane. Clinical Correlation Management: Hydroxyurea Why does HB F not work for normal people? What if I could induce Hb F in a patient? The oxygen in the blood would rise because hemoglobin could not drop it off. So, the RBCs would see that all the time and be stopped from sickling. It fools the RBCs. This is how hydroxyurea works in the sickle-cell patient. Hydroxyurea will wipe out the entire bone marrow. It does not drop off oxygen in an adult. Clinical Correlation Normal Lab Values (ABG): • • • • PO2 is 60-100 PCO2 is 40 (35-45 range) HCO3- is 24 (22-26) pH 7.40 (7.35-7.45) What is the first gene expressed? Hb F. 91 Protein Structure and Function Oxygen Saturation Dissociation Curve Figure 4.9 Hemoglobin-Oxygen Dissociation Oxygen-Hb Dissociation Curve • • • • • • Graph shows what happens to oxygen after blood leaves the lungs and reaches the tissues. Called an oxygen-hemoglobin dissociation curve. Normal Hb has a sigmoidal curve. When the pO2 is at 60, the oxygen saturation of Hb will be 90%. These are the only 2 numbers that correlate with one another. When the saturation is above 90%, you are telling me that the pO2 is at 60 or more. If the pO2 is 60 or more, then oxygen delivery to the 3 most important organs is adequate. Therefore, doctors order the nurse to keep the oxygen saturation above 90%. Hb will remain fully saturated until the pO2 drops below 60. Once the pO2 drops below 60, hemoglobin will desaturate so that oxygen will fall off into the blood and raise the pO2 of the tissue back up, causing the curve to shift to the right. When they say shift the curve to the right, it means that oxygen is being delivered to tissues. 92 Protein Structure and Function • • Anything that means, “hypoxia,” will shift the curve to the right. As exercise continues, hemoglobin will continue to desaturate until it has only a little bit of oxygen remaining. Clinical Correlation OXYGEN CONTENT = bound oxygen (Hb) + dissolved oxygen (pO2) So, when you measure a person’s blood oxygen, you are only measuring the dissolved portion. When you measure the Hemoglobin, you are measuring the bound oxygen. If the patient is anemic, the oxygen content will go down because the Hb goes down; but notice that the PO2 will stay the same. So, anemia does not equal hypoxia In late/extreme exercise • • • Hemoglobin has desaturated almost fully The body is now hypoxic, creating lots of CO2, 2,3 DPG, H+ (lactic acid) These 3 chemicals force hemoglobin to give up any remaining oxygen that is bound to it, therefore shifting the curve to the right Now you are in a period of 40-60, there is not enough oxygen. If you keep running, at pO2 40, Hb F or myoglobin will desaturate and give you a whole bunch of oxygen. This is called the second wind of exercise. But you must go through a period of pain (as muscles denature) to reach that point. That is why we say, “No pain, no gain!” REMEMBER ENDURANCE TRAINING: Over time, it will increase the pain threshold before getting to the second wind. Develop tolerance to pain. 93 Protein Structure and Function MYOGLOBIN: As muscle degenerates what is inside of the cell will leak out When CPK leaks out, it is called myositis (one muscle) or polymyositis (more than one muscle). Causes: • • • • Most common cause is hypothyroidism. Cushing's is another endocrine cause of myositis #2 cause is drugs (statins, INH, rifampin, steroids) Infection (Trichinella Spiralis – from raw bear meat or taenia solium from raw pork) When myoglobin leaks out, it is called rhabdomyolysis (severe form of myositis) • • Most common cause is trauma – crush injury from vehicle accidents #2 is drugs (statins) Definitions Myoglobin: A reserve source of energy for muscle. Only found in muscle. A weak acid. It will damage kidneys first. High Altitude Hypoxia will cause the Hb to desaturate so that it drops off more oxygen to the tissues, shifting the curve to the right. • • Increased temperature Raises the basal metabolic rate (BMR) In Summary: • • To shift to the right, there must be an increase in everything, except the pH. To shift the curve to the left, there will be a decrease in everything except the pH. Shifts to right: Increase in CO2, Acid (low pH), 2,3-DPG, Exercise and Temperature 94 Protein Structure and Function Clinical Correlation HIGH-ALTITUDE TRAINING Less oxygen in the atmosphere will force the body into a hypoxic state, leading to 3 adaptations: 1. Polycythemia: Erythropoietin is released, which expands first-order by creating more RBCs. 2. Increased mitochondrial density: Makes more ATP, spending more time in first-order. They never get into anaerobic metabolism 3. Increased angiogenin, so they grow more vessels going to the muscles, so that they get more oxygen going to them Once they return to sea level, they have more erythropoietin and hemoglobin than they need for the race, so they do not become hypoxic during the race! Clinical Correlation Blood Doping In sports look for high hematocrit level with a normal or low erythropoietin. Clinical Correlation Clubbing Chronic hypoxia will be felt most severely in the parts furthest from the heart—the fingers and toes. As angiogenesis occurs in response to the hypoxia, the new blood vessels are very leaky. As the vessels leak, this will cause swelling of the fingertips, which is called clubbing. 95 Protein Structure and Function Dermatomyositis • • • • • • • • • • • • Symmetrical proximal muscle weakness Skin presentation “Gottron’s papules” and “heliotropic rash” Heliotrope eruption – purple skin of upper eyelid with swelling Gottron’s papules – purple scaly papules on the back of the fingers and hand Shawl sign – erythema of shoulder/neck Fascia around groups of muscle fibers—called the perimysium—show inflammation with CD4-positive T cells Elevated levels of muscle enzymes = ↑CPK ↑aldolase Abnormal EMG Anti-Jo-1 antibodies Positive ANA Follow-up for occult visceral malignancy o Order CT Abd and CXR Polymyositis • • • • • • Symmetrical proximal muscle weakness, often involving shoulders Muscle fiber, necrosis, degeneration, regeneration, and inflammatory cell infiltrate Fascia around individual muscle fibers—called the endomysium—show inflammation with CD8-positive T cells Elevated levels of muscle enzymes = ↑CPK, ↑ lactate dehydrogenase, ↑ aldolase, ↑AST and ALT Anti-Jo-1 antibodies Positive ANA Erythropoiesis The process of making erythrocytes (RBCs). In adults it occurs in the bone marrow. Average adult has about 5 Liters of blood. Lifespan of RBCs: 120 days In the fetus, the location changes according to gestation age: • • • • Begins in the yolk sac at 4 months gestation 6 mo. gestation: moves into the liver, spleen, and flat bones 8 mo. gestation: moves into the long bones 1 year of age: liver, spleen, and flat bones close 96 Protein Structure and Function Clinical Correlation Polycythemia in the Newborn In the birth canal, the baby becomes hypoxic, causing him/her to undergo erythropoiesis. The newborn will typically have polycythemia. If you lose the long bones after 1 year, the spleen can reopen, causing massive splenomegaly. Massive splenomegaly will occur because the spleen will have to do the work of all the bone marrows. Only diseases that wipe out the entire bone marrow can cause this. Organizing Metabolic Pathways Metabolic Pathways Now we start discussing pathways. We will go through the steps of the pathways you need to know. We will not go through the steps of the pathways you do not need to know to save time. For those pathways, we will note bypassed steps with multiple arrows. There are 3 things that you must know, however, for every pathway: 1. 2. 3. The product-Tells us why we even run this pathway The rate-limiting enzyme—Which are the source of most genetic diseases Substrate that starts the pathway Rule: • • To synthesize something, do it in the cytoplasm (anabolic) To break something down (catabolism), do it in the mitochondria Exceptions: • • Glycolysis Purely catabolic but occurs in the cytoplasm because it must feed into other pathways. It must be centrally located 97 Protein Structure and Function Clinical Correlation 5 pathways occur in both compartments: • • • • • Heme synthesis Gluconeogenesis Urea cycle Fatty acid synthesis Pyrimidine synthesis Heme Synthesis Occurs in both the mitochondria and the cytoplasm Because we start out with Succinyl-CoA, which comes from the Krebs cycle: • • • • • • • Succinyl-Coa is used to make ringed structures (ex: cholesterol, ringed amino acids) D-ALA Synthase is the rate-limiting enzyme At the end is ferrochetalase, which adds the iron to the ring Glycine is the main amino acid we need. Vitamin we need is B6 Each ring is called a porphyrin ring Ferrochetalase will add iron 2+ in the middle; now it is called heme (4 porphyrin rings with an iron in the middle) Methemoglobinemia (Fe3+) • • • We need the iron in the 2+ configuration, so Vitamin C will keep it from getting oxidized in the GI system, and vitamin E protects it in the blood. If protons attack it, histidine will act to buffer it. Cannot pick up oxygen, so it will decrease oxygen saturation, but pO2 will remain normal (there is oxygen there in the bloodstream, but you cannot pick it up). Cyanide Poisoning • • • Oxygen saturation low, pO2 is normal Cyanide causes a configurational change to hemoglobin, so that it cannot pickup oxygen, causing noncompetitive inhibition, so the active site does not work Treatment of methemoglobinemia: • Reduce the iron back to 2+, since it is oxidized, with methylene blue The most common cause of oxidized iron: 98 Protein Structure and Function • • Free radicals caused by infections; made by neutrophils (NADPH-Oxidase) Drugs: Sulfa drugs are most common (ex: trimethoprim sulfa), Metronidazole INH, anti-malarias Management: MCC of Cyanide Poisoning in America-SODIUM NITROPRUSSIDE Treatment: 1. Amyl Nitrite to oxidize the iron. Turns it into MetHb on purpose because it will bind up the CN so that it cannot enter the cells to impair the ETS 2. Vitamin B12 to bind the cyanide and excretes through the kidney 3. B12 cyanocobalamin also binds to cyanide (most current management) 4. Methylene blue to reduce the iron back to 2+ 5. Transfuse Iron deficiency anemia • • • • • • • • • • • • • • Most common cause of microcytic hypochromic anemia The number one type of anemia There is less hemoglobin than normal Most common cause is iron deficiency and chronic disease Without iron, you cannot make heme, so therefore cannot make hemoglobin Best source of iron is red meat In children: Mcc is inadequate intake In young adults: Mcc is still inadequate intake In adults aged 20 to 40: IBD > age 40: mucosal bleeding For men, GI bleeding (rule out colon cancer) For women, endometrial bleeding (rule out cancer) Tx: ferrous sulphate Administer with vitamin c to protect it in the GI and Vitamin E to protect it in the bloodstream from free radicals 99 Protein Structure and Function Clinical Correlation Mucosal cells store 90% of the ferritin (stored iron). So, when you slough of the mucosa (i.e., GI bleed), then you are losing ferritin. Therefore, all chronic mucosal bleeds lead to an iron-deficiency anemia. Lab Values for Iron Deficiency Anemia: • • • • Serum Fe: low (because there is not any) Saturation (saturation of iron on hemoglobin): low (because it is not there to attach to the hemoglobin) Ferritin (stored iron): low (if you do not have any, how can you store it?) If you cannot store it, now what do you want transferrin to do? Bring more or absorb less? Bring more, so TIBC (transferrin) is high Question What is the most common IBD in middle aged men? Crohns In middle aged women? Crohns Chronic Disease 1. 2. 3. The number one problem is that RBCs die faster. Normal RBC life span is 120 days. In chronic disease, they die in 60-90 days. Something in the plasma destroys the RBCs Hepcidin is an acute phase protein that interferes with transferrin’s ability to absorb iron. This is why anemia of chronic disease and iron-deficiency have similar labs. There is decreased production. Bone marrow is turned off to save energy for the main illness. This occurs at about 3 weeks. 100 Protein Structure and Function REMEMBER Do not just look at the serum iron because it will not help to separate iron deficiency from chronic disease. Instead, look at the ferritin or the TIBC. In chronic disease, the iron is in the bone marrow. But you are not using it to make anything! The body tries to conserve energy. Serum iron could be low if hepcidin is already out there or it could be normal if early. We look at the ferritin (stored iron). It will be high because you are not using it. The bone marrow is shut down as the body is fighting the chronic disease, such as SLE or rheumatoid arthritis or an infection. So, the iron will pile up. It could even be normal early on, then become high later. In iron deficiency, it is never normal or high. In that case, the TIBC will be low. Iron deficiency never has low TIBC. Ferritin will differentiate between iron deficiency and chronic anemia. Lead Poisoning o Can cause delayed language development and irreversible neurobehavioral dysfunction in children. In the U.S., lead was used in paint until the 1970s. Most children will present without symptoms. o Si/Sx: Lead lines at the junction of the teeth and gums are rarely seen, encephalopathy, anemia, wrist/foot drop o Lead inhibits delta ALA dehydratase (the second enzyme of the pathway) as well as ferrochetolase (last enzyme of the pathway) o Cannot add iron to the porphyrin ring, so the porphyrin rings will float around inside the RBC Note: Lead used to be the most common cause of mental retardation. (See more below) Lab: Free erythrocyte protoporphyrins (FEP) will be extremely high: Best test MCC: Eating peeling paint from old buildings Classic clue: Basophilic stippling (porphyrin rings stuck onto RBCs) Elevated FEP (free erythrocyte protoporphyrin’s) Heme Synthesis is completely blocked 101 Protein Structure and Function Lead levels to know: There is no safe or nontoxic blood lead level (BLL) The CDC (Center for Disease Control) currently designates a blood level of 10 Normal: <5 µg/dl (still at risk for neurological development) (MILD) If between 5-14 µg/dl:     Recheck level in 6 months Educational intervention for parents; try to find source by good history If between 15-44 µg/dl: • Notify Public Health Department (usually done by the lab) • NO CHELATION THERAPY! 1-3mth follow-up (MODERATE) If 45- 69 µg/dl:    Remove child from environment Educational intervention for parents Notify Public Health Department (usually done by the lab) Treatment: Meso-2,3-Dimercapto Succinic Acid (DMSA) also called Succimer Succimer is a water soluble, non-toxic substance. It does not remove Pb in CNS and cannot cross Blood Brain Barrier (BBB). Clinical Correlation Penicillamine used to chelate: o Copper o Lead BAL used to chelate: o Lead o Arsenic 102 Protein Structure and Function (SEVERE) If above 70 µg/dl:      Neurological signs may already be present. Need to determine if lead can be mobilized or is already incorporated into the tissues. No longer a need for a Ca-EDTA challenge. Hospitalize and treat with one drug if no neurological symptoms. Treat with two drugs if there are neurological symptoms. Drugs used are EDTA, Dimercaprol (BAL), and Penicillamine. Clinical Correlation Ca-EDTA will bind anything with a 2+charge: o o o o o Calcium Iron Lead Copper Magnesium **EDTA treatment leads to these 5 deficiencies MCC Mental retardation: 1. 2. 3. Fetal alcohol syndrome o Alcohol poisons the process of nuclear division o Affects the philtrum Fragile X syndrome o Large testicles after puberty. Pill shaped head with large ears before puberty Down’s syndrome Porphyrias If an enzyme in the heme synthesis pathway is defective, the resulting disease is called a “porphyria.” Most porphyrias are inherited autosomal dominant. • Acute—porphyrias have nonspecific “neurovisceral” symptoms but not of the skin 103 Protein Structure and Function • • • • • • Cutaneous—porphyrias have skin symptoms, but not neurovisceral. Skin symptoms always related to sun exposure, called photosensitivity. Due to porphyrin rings in the skin becoming activated by UVA A group of enzyme deficiencies Synthesize too many porphyrin rings or inadequate metabolism Porphyrin rings in the urine make it red Dipstick negative for blood Three types are most important: 1. Porphyria Cutanea Tarda o Porphyria Cutanea Tarda if onset is more than one year of age o Patient presents with skin blisters in reaction to sunlight o Enzyme deficiency— uroporphyrinogen decarboxylase Treatment:         2. Protect them from light Can only go outside at night Use red, blue light Hemolytic reactions, so need blood transfusions Risk of blindness (light must hit retina by 3 mo. of age) Risk of vitamin D deficiency, so supplement with Vitamin D Phlebotomy, if necessary Anti-malarials (low dose) --removes excess porphyrins from liver Acute Intermittent Porphyria (AIP): o Autosomal dominant o Enzyme deficiency—Uroporphyrinogen I Synthase (also called porphobilinogen deaminase or uroporphyrinogen decarboxylase) o A buildup of porphyrin rings o Porphyrin rings are deposited into visceral organs and around nerves o Recurrent severe abdominal pain and neuropathy o Mnemonic 5 Ps: o Abdominal Pain o Polyneuropathy o Psychological abnormalities o Pink urine o Precipitated by drugs: Alcohol, Barbiturates and OCPs 104 Protein Structure and Function Treatment:      3. Help them deal with stress Opiates for acute pain Isotonic fluids (IV normal saline) to flush rings out of kidney Glucose (conjugate glucose to bilirubin will help excrete it faster) Hematin (block delta-ala-synthase, the rate limiting enzyme) to decrease synthesis of porphyrins rings Erythrocytic Protoporphyria (early onset PCT) o Erythrocytic Protoporphyria if onset is less than one year of age o Enzyme deficiency--Ferrochetalase o Porphyrin rings are deposited underneath the skin o Light reacts with the rings causing a release of heat which leads to burns o Mcc of death: skin infections Treatment:       Protect them from light Can only go outside at night Use red, blue light Hemolytic reactions, so need blood transfusions Risk of blindness (light must hit retina by 3 mo. of age) Risk of vitamin D deficiency, so supplement with Vitamin D Genetics—Hardy Weinberg Equation If family history, then both parents must be carriers (carry the little “a”): Aa x Aa = • • • A a A AA Aa a As aa 1/4 chance of being diseased (aa) 3/4 remaining (AA, Aa, Aa) Of these, 2 carry the gene (Aa, Aa), therefore 2/3 chance of being a carrier (67%) 105 Protein Structure and Function If no family history, then use the Hardy-Weinberg equation: AA = P2 Aa = 2pq aa = q2 P + Q = 1; because only 2 genes involved Incidence of disease = q2 Frequency of the gene = q Carrier = 2q Management: Chronic Pain Any chronic pain or neuropathy syndrome, treat with: Amitriptyline - A tricyclic antidepressant MOA: 1. Block reuptake of catecholamines, increasing sympathetic tone 2. Strongly anti-cholinergic, increasing sympathetic (with hot, dry skin & hyperthermia) 3. Block alpha-1-receptors, decreasing blood pressure 4. Blocks AV conduction, increasing ventricular arrhythmias risk, prolong QT 5. Blocks Na channels 6. Antihistaminic Note: If patient has a history of heart disease, use Gabapentin (avoids sympathetic tone). If the pain becomes shooting and stabbing, Carbamazepine is superior Pharmacokinetics: CNS Depressants and Analgesic Opiates Main actions 1. 2. 3. CNS Depressants Muscle Relaxants Analgesics S Side effects o Stimulate the Edinger-Westphal nucleus (CN 3—pupillary constriction)  Causes pinpoint pupils 106 Protein Structure and Function o Greatest sedating effect is on smooth muscle  During withdrawal, causes cramps, and diarrhea, and excessive urination Receptors for opiates: Mu receptors: brain Kappa receptors: spinal cord Opiates • • • Heroin Loperamide Methadone Diphenoxylate Morphine Fentanyl Meperidine Pentazocine Codone Tincture of Opium Oxycodone Opiate Receptor blockers: Codeine Naloxone Dextromethorphan Suboxone Buprenorphine Naltrexone Methylnaltrexone Benzos, Barbs, Alcohol and Opiates all have the same pattern: CNS depressants = will slow you down. o MCC of death while toxic is respiratory failure. Withdrawal of these drugs will cause things to speed up. o MCC of death during withdrawal is arrhythmias Alcohol withdrawal leads to formication (sensation of insects crawling underneath the skin) o Also caused by cocaine intoxication Hemoglobinopathies • • Hemoglobin S disease—also known as Sickle Cell Disease Hemoglobin C disease 107 Protein Structure and Function Hemoglobin S disease (Sickle Cell Disease) • • • • • • Autosomal recessive High prevalence in Africa (natural selection) Substitution of valine (fat-soluble) for glutamic acid (water-soluble) at position 6 of beta chain o Glutamate is usually out on the surface of the protein. Instead, valine is there. o Oxygen keeps the valine pushed out on top of the protein interacting with water; once oxygen disappears, the valine will sink back into the inside of the protein, causing the cell to sickle. o Hydrophobic interaction Hypoxia causes cells to sickle leading to vasocclusion Vaso-occlusive crises Presents after 6 mo. of age with dactylitis REMEMBER Incidence of any uncommon disease in the general population: 1-3% Add 1 risk factor: 10% Add 2 risk factors: >20% 108 Protein Structure and Function Vaso-occlusive Crises Can lead to: 1. 2. 3. 4. CVA (cerebrovascular accident) Pulmonary infarction Splenic sequestration Priapism Clinical Correlation 4 functions of the spleen: 1. 2. 3. 4. Removes old RBCs (spherocytes) Removes oddly- shaped RBCs Removes nuclear remnants from old RBCs (Howell-Jolly bodies) Removes encapsulated organisms • Strep pneumonia • HiB • Meningococcus Treatment: 1. 2. 3. Oxygen Exchange transfusion for CVA; no need to give TPa or aspirin Opiates for pain Aplastic Crisis o o o o Complete bone marrow suppression Always check the reticulocyte count Tells you if bone marrow is working Mcc: parvovirus B-19 A Few Points to Remember… • • • Functional asplenia by age 6 due to infarcts Susceptible to encapsulated organisms after functional asplenia Give pneumovax any time after age 2 Infections are mcc for crises 109 Protein Structure and Function • • • Hydroxyurea increases HbF, decreasing chance for hypoxia Use opiates for pain Transfuse when anemic and symptomatic Hemoglobin AS disease (sickle cell trait) • • • • • • Generally asymptomatic, except in severe hypoxia Autosomal recessive Hemoglobin C Disease Substitution of lysine for glutamic acid at position 6 of beta chain No sickling occurs since both amino acids are hydrophilic Missense mutation of the beta-globin subunit Thalassemia • • • • • • Represent gene deletions o Alpha thalassemia has 4 genes Beta thalassemia has 2 genes Autosomal recessive Common in Mediterranean people Minor: at least one gene remaining Major: no genes remaining Figure 4.10 Hemoglobins (HbA, HbA2, HbF) Alpha Thalassemia 4 genes, so each one accounts for 25% of the hemoglobin chain 110 Protein Structure and Function Clinical Correlation Alpha Thalassemia Minor o One gene missing  Asymptomatic – (Hb 12) 75% o Two genes missing  IF sedentary: asymptomatic  IF active: symptomatic (Hb 7.5) 50% o Three genes missing  Symptomatic in ALL (Hb 4 to 5) 25% Alpha Thalassemia Major o No genes remaining o Unable to make any hemoglobin  All hemoglobins require alpha chains o Hydrops Fetalis  Heart failure in utero o Hemoglobin Bart (4 gamma chains)  Not allosteric o Hemoglobin H (4 beta chains)  Not allosteric Baseline labs: RBC Mass: 3.5 to 4.5 million Hemoglobin/Hct: 15/45% REMEMBER One problem HbAS will complain about: Bleed from ureters (hematuria) REMEMBER #1 reason for transfusion in USA: Symptomatic anemia Clinical Correlation Beta chains needed by Hb A. Body tries to compensate by making more Hb A-2 and Hb F; Hb F is of no use after 6 months of age Beta Minor (Hb 7.5) Beta Major One gene missing Both genes are missing If Sedentary: asymptomatic Able to make only Hb A2 an Hb F If active: symptomatic Asymptomatic until 6 months of age Increased in HbA2 and Hb F Transfusion dependent Ineffective erythropoiesis 111 Protein Structure and Function Management: Blood Products Blood products: 1. Whole blood • Rarely given to patients because, oftentimes, it can be harmful • Giving primarily for acute hemorrhage/trauma 2. Fresh frozen plasma (FFP) • Plasma, but no RBCs (plasma = the yellow liquid portion of blood) • Contains ALL clotting factors • Used for any acute bleeding disorder 3. Packed Red blood cells (PRBCs) • Only RBCs, no plasma • Used for the Anemic patient • For every 1 unit of packed cells, give 1 unit of fresh frozen plasma, and one 1 unit of platelets • One unit will raise Hb by 1-2 grams 4. Platelets • For bone marrow suppression mainly • DO NOT transfuse into destructive processes unless pre-op or if platelet count falls <10K 5. DDAVP • Synthetic ADH • Releases Factor 5, 8 and vwF by contracting endothelium • For Mild Hemophilia A or Von Willebrand’s Disease • For Severe enuresis 6. Factor 8 concentrate • Severe bleeding in hemophilia A • Made in the lab 7. Albumin • 2 types 5% = isotonic • Volume-expander • 25% = hypertonic; To pull fluids back into circulation • Only if deficient • IV albumin only has 6hr half life 8. Cryoprecipitate • Contains Factor 8, fibrinogen and vWF and only small amounts of Factor 7 • For moderate bleeding in Hemophilia A, Von Willebrand’s Disease and fibrinogen problems 9. Immunoglobulins • Passive Immunity • Blocking Antibodies • Hypo/Agammaglobulinemic Patients 112 Protein Structure and Function Blood Products Blood Transfusions: • • • • Done only when patient is symptomatic. Done in heart failure (keep Hb 1012) Done in Renal failure (keep Hb 10-12) o One unit of PRBCs: Raises Hg by 1 to 2 grams (3 to 6 HCT) Delivers 3.4 grams of iron REMEMBER If the reticulocyte count is high, let the bone marrow take care of it. REMEMBER Anytime you transfuse a foreign protein into a patient, you run the risk of an allergic immune reaction. Transfusion Related Reactions • Anaphylaxis o Selective IG-A Deficiency o Acute: stop the transfusion; epinephrine, use filter for future transfusions • Acute Febrile Reaction o White Blood Cell Antigens • ABO Incompatibility o Most common o Stop transfusion o Re-type and cross 113 Protein Structure and Function • Transfusion Related Acute Lung Injury (TRALI) o From release of cytokines, causes ARDS picture, treat with steroids • Transfusion Associated Circulatory Overload (TACO) o Vigorous diuresis Note: Multiple transfusions can lead to Iron overload Iron Overload Hemosiderosis: bone marrow is overwhelmed by iron Hemochromatosis: iron overload has involved other organs Skin: bronze pigmentation Liver: bronze cirrhosis Pancreas: bronze diabetes Heart: restrictive cardiomyopathy Joints: arthritis Treatment: o Deferoxamine o Any drug that chelates 2+ (penicillamine or EDTA-BAL) Hemochromatosis Primary Hemochromatosis Autosomal Recessive Secondary Hemochromatosis Too many transfusions Too much iron absorption from MCC of death in first 10 years: d/t duodenum transfusion related infections HLA A3 on Chromosome 6 MCC of death after 10 years: CHF Question What is the stain for iron? Prussian Blue 114 Protein Structure and Function Transfusion Related Infections • • • • • • • • • • • HIV Hepatitis B Hepatitis C Hepatitis D EBV CMV Hemorrhagic viruses Bacterial infections Malaria Babesiosis Syphilis Collagen Collagen • • • • • Collagen is the most abundant protein in the body, accounting for 30% of all proteins It is made by fibroblasts and epithelial cells More than a quaternary structure It is a TRIPLE HELIX COLLAGEN SYNTHESIS: • • • • • • • Glycine: every third amino acid Lysine Proline—makes up 17% of collagen OH-Proline OH-Lysine Vitamin C Copper 4 Types of collagens: • • • • Type 1: Skin Type 2: Connective tissue Type 3: Arteries Type 4: Basement membrane 115 Protein Structure and Function REMEMBER Liquids, such as aqueous humor, synovial fluid and blood are all connective tissue! COLLAGEN SYNTHESIS There are 2 groups of ribosomes: Free ribosomes make proteins that are used right there in the cytoplasm of the cell. Rough ER make proteins that need to be packaged, whether for another organelle or another cell. At the rough ER, we start with a pre and pro sequence attached to the protein. They are both recognition sequences. Pre-Sequence → guides the protein into the RER. The protein is made on the RER, but it needs permission to enter inside the RER. Collagen is the only protein completely modified in the RER. The only modification that can go on in the RER for anybody is called, N-acetylation. This means adding an acetyl group onto Nitrogen. Pro-Sequence → guides the protein to the Golgi apparatus. It comes in on the concave side and exits through the convex side. Therefore, you have a chaperone to show you the way (HSP-90, heat shock protein). This is where all proteins, except collagen, are modified. The pro-sequence is degraded here in the Golgi apparatus, except for one protein— INSULIN. The prosequence of insulin gets packaged with insulin and we measure it in the plasma as Cpeptide. Therefore, we use it as a measure of endogenous insulin production. If I see high insulin and high C-Peptide, this means that the patient made it. Either they are on a drug that made it, or they have a tumor that made it. High insulin and low CPeptide → exogenous insulin. If you add a mannose-6-phosphate label at the Golgi, it will redirect the protein to the lysosome. 116 Protein Structure and Function Figure 4.11 Collagen Synthesis PROTEINS tagged with Mannose-6-Phosphate → goes to LYSOSOME Proteins in the lysosome are generically called ACID HYDROLASES. If you are missing the mannose-6 phosphate label, then no enzymes will end up in the lysosomes, which will be empty. This disease is called I-CELL DISEASE. Acid hydrolases will end up in the plasma. If proteins do not get labeled correctly, they will by default end up secreted into the plasma. Short amino-terminal sequence: a sequence is added to the amino terminal of the protein while on the RER, to end up going to the mitochondria. The chaperone for the mitochondria is HSP-70. 117 Protein Structure and Function MITOCHONDRIA • • • Has all its own genetic material Mitochondrial genes are circular Only given ge