Eric Niederhoffer SIU-SOM Making basic science clinically relevant for learners: the biochemistry example

Considerations Wants and needs Curriculum design, objectives, goals; USMLE Biochemistry as a foreign language Web lessonsWeb lessons, resource pages, animationsresource pagesanimations Resource sessions Complement self-directed learning Applied to patient case Start simple, discuss difficult Big picture, relevant details Overlap and redundancy Build upon previous knowledge Clinical probes for content and concepts Self-assessment questionsSelf-assessment questions, examinations Glucose metabolism as an example

RBC Structure - size, spectrin, channels Metabolism - glycolysis (2,3-BPG), pentose phosphate pathway (G6PDH, NADPH), glutathione Hemoglobin - Genes, heme, Mb/Hb (normal), O 2 binding, HbS (defect), fibers (sickling and inflammation) Red Blood Cell Biochemistry A 4-year-old African boy presents with a 2-day history of painful extremities.

Devlin, T. M. (ed.) Textbook of biochemistry with clinical correlations, 6th ed. John Wiley & Sons, Inc., New York. This is very good for most of what you need. Mehta, A. B., and A. V. Hoffbrand Haematology at a glance, Blackwell Science, Malden, Mass. Salway, J. G Medical biochemistry at a glance, 2nd ed. Blackwell Science, Malden, Mass. This is very good for general principles and topics, and metabolic pathways and regulation. Good focused clinical correlations. Students’ Notes

RBC Metabolic Pathways 2,3-BPG BPG mutase 2,3-BPG phosphatase PPP NADPH 6PG 3-7 C metabolites (R5P, F6P, G3P) G6PDH lactonase 6PGDH CO 2 NADP + + H + GSH GSSG GR GP H2O2H2O2 H2OH2O Glc Pyr G6P 1,3-BPG 3PG HK PGI PK F6P G3P PFK aldolase F16BP DHAP 2PG PEP PGK PGM enolase G3PDH Glycolysis Lactate No O 2 LDH

Glc: glucoseHK: hexokinaseG6P: glucose-6-phosphateG6PDH: glucose-6-phosphate dehydrogenase PGI: phosphoglucose isomerasePFK: phosphofructokinaseDHAP: dihydroxyacetonephosphate BPG: bisphophoglyceratePEP: phosphoenolpyruvatePyr: pyruvatePK: pyruvate kinase (2 genes, 4 isozymes) NADP + /NADPH: nicotinamide adenine dinucleotideR5P: ribulose-5-phosphateF6P: fructose-6-phosphate G3P: glyceraldehyde-3-phosphateGSH: reduced glutathione (GSH = Glu-Cys-Gly)GSSH: oxidized glutathione LDH: lactate dehydrogenasePPP: pentose phosphate pathway6PGDH: 6-phosphogluconate dehydrogenase GR: glutathione reductaseGP: glutathione peroxidase3PG: 3-phosphoglycerate6PG: 6-phosphogluconate Defect in HK, PGI, aldolase, or BPG mutase/2,3-BPG phosphatase  decreased [2,3-BPG]; defect in PK  increased [2,3-BPG] BPG mutase(or synthase)/2,3-BPG phosphatase is a bifunctional enzyme (one protein, two activities), regulated by hypoxia and T3 MIultiple inositol polyphosphate phosphatase acts on 2,3-BPG to give 2-PG Fetal Hb - lower affinity for 2,3-BPG compared with adult Hb; 2,3-BPG binds to and stabilizes deoxyHb; it is easily displaced from oxyHb Common deficiencies:G6PDH - X-linkedPGI - autosomal recessivePK - autosomal recessive Sodium fluoride inhibits enolase, used to preserve blood samples for glucose determinations. Students’ Notes

Hemoglobin Structure Changes

Factors Affecting Binding of O 2 Depends on pH ([H + ]), CO 2, BPG (DPG), Temp pH _ BPG or T  ; right shift pH  BPG or T _; left shift

Review Questions What metabolic pathways are used in erythrocytes? What clinical observations would you make concerning patients with SCD?

Metabolism in Skeletal Muscle and Nervous Tissue Metabolism in skeletal muscle Pathways overview Regulation in skeletal muscle Metabolism in nervous tissue Pathways overview Clinical aspects Clinical/laboratory findings GSD, PDHCD Glycogen storage disease type VII Pyruvate dehydrogenase complex deficiency Inborn errors of metabolism

Glycolysis Glycogenolysis  -oxidation (ketone bodies) Krebs (tricarboxylic acid) cycle Branched-chain amino acids Electron transport chain Calcium regulation Key enzyme regulation Metabolism in Skeletal Muscle A 21-year-old woman comes to the physician with pain in her right mid-arm. A 5-year-old boy is brought to the physician to have sutures removed.

Pathways Overview Acetyl-CoA Lactate No O 2 Production of ATP G6P Glucose Glycolysis Pyruvate BCAA Ile, Leu, Val Krebs cycle Electron Transport Chain Glycogen Glycogenolysis Ca 2+ PKa Ca 2+ PDH Ca 2+ ISDH,  KGDH Fatty acids  - Oxidation Ketone bodies

Regulation in Skeletal Muscle Glc Glycolysis Glycogen Glycogenolysis PDH PK PFK-1 cAMP Acetyl-CoAPyr F6P F16BP PEP G6P PKA AC ATP Ep  AR Pi IMP AMP Ca 2+ PKa PP Ca 2+ PDHP PDHK PDH P PDH NH 4 + AMP Pi PFK-2 F26BP ATP Citrate

Metabolism in Nervous Tissue Glycolysis Glycogenolysis (stress)  -oxidation (ketone bodies) Krebs (tricarboxylic acid) cycle Branched-chain amino acids Electron transport chain A 21-year-old woman comes to the physician with pain in her right mid-arm. A 19-year-old man is brought to the emergency department after a diving accident. A 63-year-old woman is brought to the physician for her “parkinsonism.”

Pathways Overview Acetyl-CoA Lactate (glial) Production of ATP Glycolysis G6P Glucose Pyruvate BCAA Ile, Leu, Val Krebs cycle Electron Transport Chain Glycogen Glycogenolysis Lactate No O 2 Fatty acids  -oxidation Ketone bodies

Clinical Aspects for Inborn Errors of Metabolism in Muscles Toxic accumulation disorders Protein metabolism disorders (amino acidopathies, organic acidopathies, urea cycle defects) Carbohydrate/intolerance disorders Lysosomal storage disorders Energy production/utilization disorders Fatty acid oxidation defects Carbohydrate utilization, production disorders (glycogen storage, gluconeogenesis, and glycogenolysis disorders) Mitochondrial disorders Peroxisomal disorders Metabolic acidosis (elevated anion gap) Hypoglycemia Hyperammonemia

Clinical Aspects for Inborn Errors of Metabolism in Nervous Tissue Evidence of familial coincidence Progressive decline in nervous functioning Appearance and progression of unmistakable neurologic signs General symptoms State of consciousness, awareness, reaction to stimuli Tone of limbs, trunk (postural mechanisms) Certain motor automatisms Myotatic and cutaneous reflexes Spontaneous ocular movements, fixation, pursuit; visual function Respiration and circulation Appetite Seizures

Clinical/Laboratory Findings Clinical findingsAAOAUCDCDGSDFADLSDPDMD Episodic decompensationX++++X+--X Poor feeding, vomiting, failure to thrive X++++XX+++ Dysmorphic features and/or skeletal or organ malformations XX--XX+XX Abnormal hair and/or dermatitis-XX Cardiomegaly and/or arrhythmias-X--XX+-X Hepatosplenomegaly and/or splenomegaly X++++++XX Developmental delay +/- neuroregression +++XXX++++ Lethargy or comaX++ +X --X SeizuresXX+XXX++X Hypotonia or hypertonia++++X+X+X Ataxia-X+X-XX-- Abnormal odorX+X Laboratory Findings* Primary metabolic acidosisX++++X+--X Primary respiratory alkalosis HyperammonemiaX+++X-+--X HypoglycemiaXX-+X+--X Liver dysfunctionXXX+X+XXX Reducing substancesX KetonesAHAAL/ALAAH/A

Glycogen Storage Disease Pyruvate Dehydrogenase Complex Deficiency Krebs cycle G6P Glucose Glycolysis Glycogen Glycogenolysis Glycogenesis F6P F16BP PFK Tarui disease Glycogen Storage Disease Type VII Acetyl-CoA Pyruvate PDH PDH complex deficiency R5Pnucleotides Pentose Phosphate Pathway

Glycogen Storage Disease Type VII (Tarui Disease) Classic, infantile onset, Late onset Exercise intolerance, fatigue, myoglobinuria Phosphofructokinase Tetramer of three subunits (M, L, P) Muscle/heart/brain - M4; liver/kidneys - L4; erythrocytes - M4, L4, ML3, M2L2, M3L General symptoms of classic form Muscle weakness, pronounced following exercise Fixed limb weakness Muscle contractures Jaundice Joint pain Laboratory studies Increased serum creatine kinase levels No increase in lactic acid levels after exercise Bilirubin levels may increase Increased reticulocyte count and reticulocyte distribution width Myoglobinuria after exercise Ischemic forearm test - no lactate increase with ammonia increase

Neonatal, infantile, childhood onset Abnormal lactate buildup (mitochondrial disease) Pyruvate dehydrogenase complex E1 -  (thiamine dependent) and  subunits,  2  2 tetramer E2 - monomer (lipoate dependent) E3 - dimer (riboflavin dependent) common to  KGDH and BCAKDH X protein - lipoate dependent Pyruvate dehydrogenase phosphatase Nonspecific symptoms (especially with stress, illness, high carbohydrate intake) Severe lethargy, poor feeding, tachypnea Key feature is gray matter degeneration with foci of necrosis and capillary proliferation in the brainstem (Leigh syndrome) Infants with less than 15% PDH activity generally die Developmental nonspecific signs Mental delays Psychomotor delays Growth retardation Laboratory studies High blood and cerebrospinal fluid lactate and pyruvate levels Elevated serum and urine alanine levels If E2 deficient, elevated serum AAs and hyperammonemia If E3 deficient, elevated BCAA in serum,  KG in serum and urine Pyruvate Dehydrogenase Complex Deficiency

Inborn Errors of Metabolism Carbohydrates (Glycogen storage diseases) Amino acids (Maple syrup urine disease) Organic acids (Alkaptonuria) Mitochondrial function (Pyruvate dehydrogenase deficiency) Purines and pyrimidines (Lesch-Nyhan disease) Lipids (Familial hypercholesterolemia) Porphyrins (Crigler-Najjar syndromes) Metals (Hereditary hemochromatosis) Peroxisomes (X-linked adrenoleukodystrophy) Lysosomes (G M2 gangliosidoses - Tay Sachs disease) Hormones (hyperthyroidism) Blood (Sickle cell disease) Connective tissue (Marfan syndrome) Kidney (Alport syndrome) Lung (  1 -antitrypsin deficiency) Skin (Albinism)

Review Questions How does muscle produce ATP (carbohydrates, fatty acids, ketone bodies, branched-chain amino acids)? How is skeletal muscle phosphofructokinase-1 regulated? What are the key Ca 2+ regulated steps? How does nervous tissue (neurons and glial cells) produce ATP (carbohydrates, fatty acids, ketone bodies, branched-chain amino acids)? How do glial cells (astrocytes) assist neurons? What are some key clinical features (history, physical, laboratory test results) associated with defects in metabolism that affect muscles and nervous tissue?

Carbohydrate Metabolism in Diabetes For the third example taken from the ERG Unit, what would you choose for the resource session? A 59-year-old man is brought to the emergency department for evaluation of his semiconsciousness and minimal responsiveness

Carbohydrate Metabolism in Diabetes Regulation of glycolysis, glycogenesis, glycogenolysis, gluconeogenesis by insulin/glucagon PFK-2 (PKA, AMP-dependent PK) PK (PKA) PDH GS (PKA, PPK, GSK-3, PP-1) GP (PKA, PPK, PP) PEPCK (glucagon) G6Pase (glucagon) Regulatory differences among tissues Liver Muscle Cardiac muscle Key clinical features (history, physical, laboratory test results) associated with carbohydrate metabolism that occur in diabetes

Summary Remember curriculum wants and needs Practice new language skills Use resource sessions effectively Complement self-directed learning Applied to patient case Start simple, discuss difficult Big picture, relevant details Overlap and redundancy Build upon previous knowledge Clinical probes for content and concepts