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 languageWeb lessons, resource pages, animations

• Resource sessionsComplement self-directed learningApplied to patient caseStart simple, discuss difficultBig picture, relevant detailsOverlap and redundancyBuild upon previous knowledge

• Clinical probes for content and conceptsSelf-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), O2 binding, HbS (defect), fibers (sickling and inflammation)

Red Blood Cell BiochemistryA 4-year-old African boy presents with a 2-day history

of painful extremities.

Red Blood Cell Biochemistry

Eric Niederhoffer

SIU-SOM

RBC Structure - size, spectrin, channels

Metabolism - glycolysis (2,3-BPG), pentosephosphate pathway (G6PDH, NADPH),

glutathione

Hemoglobin - Genes, heme, Mb/Hb (normal), O2 binding,HbS (defect), fibers (sickling and inflammation)

Devlin, T. M. (ed.). 2006. 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. 2000. Haematology at a glance, Blackwell Science, Malden, Mass.

Salway, J. G. 2006. 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)

G6PDHlactonase6PGDH

CO2

NADP+ + H+

GSH

GSSGGR

GP

H2O2 H2O

Glc

Pyr

G6P

1,3-BPG

3PG

HK

PGI

PK

F6P

G3P

PFK

aldolaseF16BP

DHAP

2PG

PEP

PGK

PGM

enolase

G3PDH

Glycolysis

LactateNo O2

LDH

RBC Metabolic Pathways

2,3-BPG

BPG mutase

2,3-BPG phosphatase

PPP

NADPH

6PG

3-7 C metabolites(R5P, F6P, G3P)

G6PDHlactonase6PGDH

CO2

NADP+ + H+

GSH

GSSGGR

GP

H2O2 H2O

Glc

Pyr

G6P

1,3-BPG

3PG

HK

PGI

PK

F6P

G3P

PFK

aldolaseF16BP

DHAP

2PG

PEP

PGK

PGM

enolase

G3PDH

Glycolysis

LactateNo O2

LDH

Glc: glucose HK: hexokinase G6P: glucose-6-phosphate G6PDH: glucose-6-phosphate dehydrogenase

PGI: phosphoglucose isomerase PFK: phosphofructokinase DHAP: dihydroxyacetonephosphate

BPG: bisphophoglycerate PEP: phosphoenolpyruvate Pyr: pyruvate PK: pyruvate kinase (2 genes, 4 isozymes)

NADP+/NADPH: nicotinamide adenine dinucleotide R5P: ribulose-5-phosphate F6P: fructose-6-phosphate

G3P: glyceraldehyde-3-phosphate GSH: reduced glutathione (GSH = Glu-Cys-Gly) GSSH: oxidized glutathione

LDH: lactate dehydrogenase PPP: pentose phosphate pathway 6PGDH: 6-phosphogluconate dehydrogenase

GR: glutathione reductase GP: glutathione peroxidase 3PG: 3-phosphoglycerate 6PG: 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-linked PGI - autosomal recessive PK - autosomal recessive

Sodium fluoride inhibits enolase, used to preserve blood samples for glucose determinations.

Students’ Notes

Hemoglobin Structure Changes

http://www.mfi.ku.dk/PPaulev/chapter8/images/8-3.jpg

Factors Affecting Binding of O2

Depends on pH ([H+]), CO2, 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 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 MuscleA 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-CoALactateNo O2

Production of ATP

G6P

GlucoseGlycolysis

Pyruvate

BCAAIle, Leu, Val

Krebscycle

ElectronTransport

Chain

GlycogenGlycogenolysis

Ca2+

PKa Ca2+

PDH

Ca2+

ISDH, KGDH

Fatty acids

-Oxidation

Ketone bodies

Regulation in Skeletal MuscleGlc Glycolysis

GlycogenGlycogenolysis

PDH

PK

PFK-1

cAMP

Acetyl-CoAPyr

F6P

F16BP

PEP

G6PPKA

ACATP

EpAR

PiIMPAMP

Ca2+

PKa PP

Ca2+

PDHPPDHK

PDHP

PDH

NH4+

AMPPi

PFK-2

F26BP

ATPCitrate

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

BCAAIle, Leu, Val

Krebscycle

ElectronTransport

Chain

Glycogen

Glycogenolysis

LactateNo O2

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 FindingsClinical findings AA OA UCD CD GSD FAD LSD PD MD

Episodic decompensation X + ++ + X + - - X

Poor feeding, vomiting, failure to thrive

X + ++ + X X + + +

Dysmorphic features and/or skeletal or organ malformations

X X - - X X + X X

Abnormal hair and/or dermatitis - X X - - - - - -

Cardiomegaly and/or arrhythmias - X - - X X + - X

Hepatosplenomegaly and/or splenomegaly

X + + + + + + X X

Developmental delay +/- neuroregression

+ + + X X X ++ + +

Lethargy or coma X ++ ++ + X ++ - - X

Seizures X X + X X X + + X

Hypotonia or hypertonia + + + + X + X + X

Ataxia - X + X - X X - -

Abnormal odor X + X - - - - - -

Laboratory Findings*

Primary metabolic acidosis X ++ + + X + - - X

Primary respiratory alkalosis - - + - - - - - -

Hyperammonemia X + ++ X - + - - X

Hypoglycemia X X - + X + - - X

Liver dysfunction X X X + X + X X X

Reducing substances X - - + - - - - -

Ketones A H A A L/A L A A H/A

Glycogen Storage DiseasePyruvate Dehydrogenase Complex Deficiency

Krebscycle

G6P

Glucose

GlycolysisGlycogenGlycogenolysisGlycogenesis F6P

F16BPPFK

Tarui diseaseGlycogen Storage Disease Type VII

Acetyl-CoAPyruvatePDH

PDH complex deficiency

R5P nucleotides

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, 22 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 (GM2 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 Ca2+ 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/glucagonPFK-2 (PKA, AMP-dependent PK)PK (PKA)PDHGS (PKA, PPK, GSK-3, PP-1)GP (PKA, PPK, PP)PEPCK (glucagon)G6Pase (glucagon)

• Regulatory differences among tissuesLiverMuscleCardiac 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 learningApplied to patient caseStart simple, discuss difficultBig picture, relevant detailsOverlap and redundancyBuild upon previous knowledge

• Clinical probes for content and concepts