high-yield-biochemistry.pdf

! High-Yield Clinical Vignettes ! Molecular ! Cellular ! Metabolism ! Laboratory Techniques ! Genetics ! Nutrition

H I G H - Y I E L D P R I N C I P L E S I N

Biochemistry

“Biochemistry is the study of carbon compounds that crawl.”

––Mike Adams

This high-yield material includes molecular biology, genetics, cell bi-ology, and principles of metabolism (especially vitamins, cofactors, minerals, and single-enzyme-deficiency diseases). When studying metabolic pathways, emphasize important regulatory steps and en-zyme deficiencies that result in disease. For example, understanding the defect in Lesch-Nyhan syndrome and its clinical consequences is higher yield than memorizing every intermediate in the purine sal-vage pathway. Do not spend time on hard-core organic chemistry, mechanisms, and physical chemistry. Detailed chemical structures are infrequently tested. Familiarity with the latest biochemical tech-niques that have medical relevance––such as enzyme-linked im-munosorbent assay (ELISA), immunoelectrophoresis, Southern blot-ting, and PCR––is useful. Beware if you placed out of your medical school’s biochemistry class, for the emphasis of the test differs from that of many undergraduate courses. Review the related biochemistry when studying pharmacology or genetic diseases as a way to reinforce and integrate the material.

BIOC H E M ISTRY—H IG H-YI E LD C LI N ICAL VIG N ETTES

! Full-term neonate of uneventful What is the diagnosis? PKU. delivery becomes mentally

retarded and hyperactive and has a musty odor.

! Stressed executive comes What is the mechanism? NADH increase prevents

home from work, consumes 7 gluconeogenesis by shunting

or 8 martinis in rapid succession pyruvate and oxaloacetate to

before dinner, and becomes lactate and malate.

hypoglycemic.

! 2-year-old girl has an ↑ in What is the diagnosis? Kwashiorkor. abdominal girth, failure to thrive,

and skin and hair depigmentation.

! Alcoholic develops a rash, What is the vitamin Vitamin B₃(pellagra). diarrhea, and altered mental deficiency?

status.

! 51-year-old man has black spots What is the diagnosis? Alkaptonuria. in his sclera and has noted that

his urine turns black upon standing.

! 25-year-old male complains What is the disease, Familial hypercholesterolemia; of severe chest pain and has and where is the defect? LDL receptor.

xanthomas of his Achilles tendons.

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! BIOC H E M ISTRY—MOLEC U LAR

Chromatin Condensed by (−) charged DNA looped twice Think of beads on a string.

structure around (+) charged H2A, H2B, H3, and H4

histone octamers (nucleosome bead). H1 ties nucleosomes together in a string (30-nm fiber). In mitosis, DNA condenses to form mitotic chromosomes.

Heterochromatin Condensed, transcriptionally inactive.

Euchromatin Less condensed, transcriptionally active. Eu= true, “truly transcribed.”

Nucleotides Purines (A, G) have 2 rings. Pyrimidines (C, T, U) PURe As Gold: PURines.

have 1 ring. Guanine has a ketone. Thymine has CUT the PY (pie):

a methyl. Deamination of cytosine makes uracil. PYrimidines.

Uracil found in RNA; thymine in DNA. THYmine has a meTHYl.

G-C bond (3 H-bonds) stronger than A-T bond (2 H-bonds). ↑ G-C content → ↑ melting temperature.

Nucleotides are linked by 3′-5′ phosphodiesterase bond.

Transition vs. Transition––substituting purine for purine or TransItion= Identical type.

transversion pyrimidine for pyrimidine.

Transversion––substituting purine for pyrimidine TransVersion= conVersion

or vice versa. between types.

Genetic code Unambiguous––each codon specifies only 1 amino acid.

features Degenerate––more than 1 codon may code for same amino acid.

Commaless, nonoverlapping (except some viruses).

Universal (exceptions include mitochondria, archaeobacteria, Mycoplasma, and some yeasts).

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Aspartate

Purine (A, G) Pyrimidine (C, T, U) CO2 Glycine N C N C C C N N C Glutamine N10–Formyl-tetrahydrofolate N10–Formyl-tetrahydrofolate N C N C C C Carbamoyl phosphate Aspartate Nucleosome core histones H2A, H2B, H3, H4 DNA Histone H1

! BIOC H E M ISTRY—MOLEC U LAR (continued)

Mutations in DNA Silent––same aa, often base change in 3rd position Severity of damage: nonsense

of codon (tRNA wobble). > missense > silent. Missense––changed aa (conservative––new aa is

similar in chemical structure).

Nonsense––change resulting in early stop codon. Stop the nonsense!

Frame shift––change resulting in misreading of all nucleotides downstream, usually resulting in a truncated protein.

Prokaryotic DNA Single origin of replication––continuous DNA DNA polymerase III has

replication and synthesis on leading strand and discontinuous 5′ → 3′ synthesis and

DNA polymerases (Okazaki fragments) on lagging strand. proofreads with 3′ → 5′

DNA topoisomerases create a nick in the helix to exonuclease.

relieve supercoils. DNA polymerase I excises

Primase makes an RNA primer on which DNA RNA primer with 5′ → 3′

polymerase III can initiate replication. exonuclease.

DNA polymerase III elongates the chain by adding

deoxynucleotides to the 3′ end until it reaches primer of preceding fragment. 3′ → 5′ exonuclease activity “proofreads” each added nucleotide.

DNA polymerase I degrades RNA primer. DNA ligase seals.

Eukaryotic DNA Eukaryotic genome has multiple origins of replication. Replication begins at a

polymerases consensus sequence of AT base pairs.

Eukaryotes have separate polymerases (α, β, γ, δ, ε) for synthesizing RNA primers, leading-strand DNA, lagging-strand DNA, mitochondrial DNA, and DNA repair.

DNA repair: single Single-strand, excision repair–specific glycosylase recognizes and removes damaged

strand base. Endonuclease makes a break several bases to the 5′ side. Exonuclease removes

short stretch of nucleotides. DNA polymerase fills gap. DNA ligase seals.

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3' 5' 3' 5' 5' 3' Leading strand Primase Okazaki fragment DNA ligase Lagging strand RNA primer DNA polymerase III Single-strand binding protein DNA polymerase III

DNA repair defects Xeroderma pigmentosum (skin sensitivity to UV light), ataxia-telangiectasia (x-rays), Bloom’s syndrome (radiation), and Fanconi’s anemia (cross-linking agents).

Xeroderma Defective excision repair such as uvr ABC Autosomal recessive.

pigmentosum endonuclease. Results in inability to repair

thymidine dimers, which form in DNA when exposed to UV light.

Associated with dry skin and with melanoma and other cancers.

DNA/RNA/protein DNA and RNA are both synthesized 5′ → 3′. Imagine the incoming

synthesis direction Remember that the 5′ of the incoming nucleotide nucleotide bringing a gift

bears the triphosphate (energy source for bond). (triphosphate) to the 3′ host. The 3′ hydroxyl of the nascent chain is the target. “BYOP (phosphate) from 5 Protein synthesis also proceeds in the 5′ to 3′ to 3.”

direction. Amino acids are linked N

to C.

Types of RNA mRNA is the largest type of RNA. Massive, Rampant, Tiny.

rRNA is the most abundant type of RNA. tRNA is the smallest type of RNA.

RNA polymerases

Eukaryotes RNA polymerase I makes rRNA. I, II, and III are numbered as RNA polymerase II makes mRNA. their products are used in RNA polymerase III makes tRNA. protein synthesis. No proofreading function, but can initiate chains. α-amanitin is found in death

RNA polymerase II opens DNA at promoter site cap mushrooms. (A-T-rich upstream sequence––TATA and CAAT).

α-amanitin inhibits RNA polymerase II. Prokaryotes RNA polymerase makes all 3 kinds of RNA.

Start and stop AUG (or rarely GUG) is the mRNA initiation codon. AUG inAUGurates

codons Eukaryotes––AUG codes for methionine, which protein synthesis.

may be removed before translation is completed. Prokaryotes––the initial AUG codes for a formyl-methionine (f-met).

Stop codons––UGA, UAA, UAG. UGA= U Go Away.

UAA= U Are Away.

UAG = U Are Gone.

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Thymidine dimer 3' 5' 5' 3' UV

! BIOC H E M ISTRY—MOLEC U LAR (continued) Regulation of gene expression

Promoter Site where RNA polymerase and multiple other Promoter mutation commonly transcription factors bind to DNA upstream from results in dramatic ↓ in

gene locus. amount of gene transcribed.

Enhancer Stretch of DNA that alters gene expression by binding transcription factors. May be located close to, far from, or even within (in an intron) the gene whose expression it regulates.

Operator Site where negative regulators (repressors) bind.

Introns vs. Exons contain the actual genetic information INtrons stay IN the nucleus,

exons coding for protein. whereas EXons EXit and are

Introns are intervening noncoding segments of DNA. EXpressed.

Splicing of mRNA Introns are precisely spliced out of 1° mRNA transcripts. A lariat-shaped intermediate

is formed. Small nuclear ribonucleoprotein particles (snRNP) facilitate splicing by binding to 1° mRNA transcripts and forming spliceosomes.

RNA processing Occurs in nucleus. After transcription: Only processed RNA is

(eukaryotes) 1. Capping on 5′ end (7-methyl-G) transported out of the

2. Polyadenylation on 3′ end (≈ 200 A’s) nucleus. 3. Splicing out of introns

Initial transcript is called heterogeneous nuclear RNA (hnRNA).

Capped and tailed transcript is called mRNA.

tRNA structure 75–90 nucleotides, cloverleaf form, anticodon end is opposite 3′ aminoacyl end. All

tRNAs, both eukaryotic and prokaryotic, have CCA at 3′ end along with a high percentage of chemically modified bases. The amino acid is covalently bound to the 3′ end of the tRNA.

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DNA mRNA Exons Introns Transcription and splicing HO-AAAA 3' 5' Cap G_pppp Tail Coding Methionine Anticodon (CAU) UAC AUG ACC 3' 3' 5'

tRNA charging Aminoacyl-tRNA synthetase (1 per aa, uses ATP) Aminoacyl-tRNA synthetase scrutinizes aa before and after it binds to tRNA. If and binding of charged incorrect, bond is hydrolyzed by synthetase. The tRNA to the codon are aa-tRNA bond has energy for formation of peptide responsible for accuracy of bond. A mischarged tRNA reads usual codon but amino acid selection. inserts wrong amino acid.

tRNA wobble Accurate base pairing is required only in the first 2 nucleotide positions of an mRNA

codon, so codons differing in the 3rd “wobble” position may code for the same tRNA/amino acid.

Protein synthesis Met sits in the P site––peptidyl. The incoming ATP––tRNA Activation

amino acid binds to the A site––aminoacyl, (charging).

hydrolyzing Met’s bond to its tRNA while GTP––tRNA Gripping and

simultaneously forming a peptidyl bond between Going places (translocation).

the 2 amino acids. The ribosome shifts 1 codon toward the 3′ end of the mRNA, shifting the uncharged tRNA into the E position and the dipeptidyl tRNA into the P site.

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AA ATP AMP + PP_i OH 3' 5' Aminoacyl tRNA synthetase 3' 5' Ribosome 40S E P A 60S

! BIOC H E M ISTRY—C E LLU LAR Enzyme kinetics

The lower the K_m, the higher the affinity.

HINT: Competitive inhibitors cross each other

competitively, while noncompetitive inhibitors do not.

Enzyme regulation Enzyme concentration alteration (synthesis and/or destruction), covalent modification

methods (e.g., phosphorylation), proteolytic modification (zymogen), allosteric regulation (e.g.,

feedback inhibition), and transcriptional regulation (e.g., steroid hormones).

Cell cycle phases M (mitosis: prophase–metaphase– G stands for Gap or Growth; S

anaphase–telophase) for Synthesis.

G₁(growth)

S (synthesis of DNA) G₂(growth)

G₀(quiescent G₁phase)

G₁and G₀are of variable duration. Mitosis is usually shortest phase. Most cells are in G₀. Rapidly dividing cells have a shorter G₁.

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Vmax Vmax Km [S] Noncompetitive inhibitor Uninhibited Competitive inhibitor slope = 1 −Km Km Vmax 1 Vmax 1 [S] 1 [S] 1 V 1 V V elocity (V) 1 2 Km = [S] at Vmax1 2 Competitive Noncompetitive inhibitors inhibitors

Resemble substrate Yes No

Overcome by ↑ [S] Yes No

Bind active site Yes No

Effect on V_max Unchanged ↓

Effect on K_m ↑ Unchanged G2 Mitosis G1 S phase Interphase (G₁, S, G₂)

Rough RER is the site of synthesis of secretory (exported) Mucus-secreting goblet cells of

endoplasmic proteins and of N-linked oligosaccharide addition the small intestine and

reticulum (RER) to many proteins. antibody-secreting plasma

cells are rich in RER.

Nissl bodies Nissl bodies (in neurons)––rough ER; not found in axon or axon hillock.

Synthesize enzymes (e.g., ChAT) and peptide neurotransmitters.

Smooth SER is the site of steroid synthesis and detoxification Liver hepatocytes and

endoplasmic of drugs and poisons. steroid hormone–producing

reticulum (SER) cells of the adrenal cortex

are rich in SER.

Functions of Golgi 1. Distribution center of proteins and lipids from I-cell disease is caused by the

apparatus ER to the plasma membrane, lysosomes, and failure of addition of

secretory vesicles mannose-6-phosphate to

2. Modifies N-oligosaccharides on asparagine lysosome proteins, causing 3. Adds O-oligosaccharides to serine and threonine these enzymes to be secreted

residues outside the cell instead of

4. Proteoglycan assembly from proteoglycan core being targeted to the

proteins lysosome. Characterized by

5. Sulfation of sugars in proteoglycans and of coarse facial features and selected tyrosine on proteins restricted joint movement. 6. Addition of mannose-6-phosphate to specific

lysosomal proteins, which targets the protein to the lysosome

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RER SER Cell membrane Cell membrane trans face cis face

(Reproduced, with permission, from Junqueira L, Carneiro J. Basic Histology, 10th ed. New York:McGraw-Hill, 2003.)

! BIOC H E M ISTRY—C E LLU LAR (continued)

Microtubule Cylindrical structure 24 nm in diameter and of variable Drugs that act on microtubules:

length. A helical array of polymerized dimers of α- 1. Mebendazole/thiabendazole and β-tubulin (13 per circumference). Each dimer (antihelminthic)

has 2 GTP bound. Incorporated into flagella, cilia, 2. Taxol (anti–breast cancer) mitotic spindles. Grows slowly, collapses quickly. 3. Griseofulvin (antifungal) Microtubules are also involved in slow axoplasmic 4. Vincristine/vinblastine

transport in neurons. (anti-cancer)

5. Colchicine (anti-gout) Chédiak-Higashi syndrome is due to a microtubule polymerization defect resulting in ↓ phagocytosis.

Cilia structure 9 + 2 arrangement of microtubules. Kartagener’s syndrome is due

Dynein is an ATPase that links peripheral to a dynein arm defect, 9 doublets and causes bending of cilium by resulting in immotile cilia. differential sliding of doublets. Dynein = retrograde.

Kinesin = anterograde.

Plasma membrane Plasma membranes contain cholesterol (≈ 50%, promotes membrane stability),

composition phospholipids (≈ 50%), sphingolipids, glycolipids, and proteins. High cholesterol or

long saturated fatty acid content → ↑ melting temperature. Only noncytoplasmic side of membrane contains glycosylated lipids or proteins (i.e., the plasma membrane is an asymmetric, fluid bilayer).

Phosphatidylcholine Phosphatidylcholine (lecithin) is a major component of RBC membranes, of myelin, of

function bile, and of surfactant (DPPC––dipalmitoyl phosphatidylcholine). Also used in

esterification of cholesterol (LCAT is lecithin-cholesterol acyltransferase).

Sodium pump Na+_-K+_{ATPase is located in the plasma membrane Ouabain inhibits by binding to}

with ATP site on cytoplasmic side. For each ATP K+_{site. Cardiac glycosides}

consumed, 3 Na+_{go out and 2 K}+_{come in. (digoxin, digitoxin) also}

During cycle, pump is phosphorylated. inhibit the Na+_-K+_ATPase,

causing ↑ cardiac contractility.

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24 nm Microtubule doublets Dynein ATPase Cytosolic

side 3Na+ ATP ADP

3Na+ 2K+ 2K+ Extracellular side P P

G-protein-linked 2nd messengers

Receptor G-protein class

HAVe 1 M&M.

MAD 2s.

Collagen types Collagen is the most abundant protein in the Be Cool, Read Books.

human body. Functions to organize and strengthen extracellular matrix.

Type I (90%)––Bone, tendon, skin, dentin, fascia, Type I: BONE. cornea, late wound repair.

Type II––Cartilage (including hyaline), vitreous Type II: carTWOlage. body, nucleus pulposus.

Type III (Reticulin)––skin, blood vessels, uterus, fetal tissue, granulation tissue.

Type IV––Basement membrane or basal lamina. Type IV: Under the floor

Type X––epiphyseal plate. (basement membrane).

Collagen synthesis Inside fibroblasts:

and structure 1. Collagen α chains (preprocollagen)

translated on RER––usually Gly-X-Y polypeptide (X and Y are proline, hydroxyproline, or hydroxylysine) 2. ER → hydroxylation of specific proline

and lysine residues (requires vitamin C) 3. Golgi → glycosylation of pro-α-chain

lysine residues and formation of

procollagen (triple helix of 3 collagen

α chains)

4. Procollagen molecules are exocytosed into extracellular space

Outside fibroblasts:

5. Procollagen peptidases cleave terminal regions of procollagen, transforming procollagen into insoluble tropocollagen 6. Many staggered tropocollagen molecules

are reinforced by covalent lysine-hydroxylysine cross-linkage (by lysyl oxidase) to make collagen

fibrils

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Receptor Lipids PIP2 Gq H1, α1, V1, M1, M3 IP3 [Ca2+]_in DAG Protein kinase C Phospholipase C G s β1, β2, D1, H2, V2 Protein kinase A ATP cAMP Receptor Adenylyl cyclase M2, α2, D2 Gi Receptor + – mRNA Nucleus Glycosylation (pro α chain)

Triple helix (procollagen) Osteogenesis imperfecta Collagen fibrils with crosslinks OH OH OH OH ER DNA Golgi Ehlers-Danlos Scurvy Hydroxylation Cell membrane Peptide cleavage c(1-)

! BIOC H E M ISTRY—C E LLU LAR (continued)

Ehlers-Danlos Faulty collagen synthesis causing:

syndrome 1. Hyperextensible skin

2. Tendency to bleed (easy bruising) 3. Hypermobile joints

10 types. Inheritance varies. Associated with berry aneurysms.

Osteogenesis Primarily an autosomal-dominant disorder caused May be confused with child

imperfecta by a variety of gene defects, resulting in abnormal abuse.

collagen synthesis. Clinically characterized by: Type II is fatal in utero and in 1. Multiple fractures occurring with minimal the neonatal period.

trauma (brittle bone disease), which may Incidence is 1:10,000. occur during the birth process

2. Blue sclerae due to the translucency of the connective tissue over the choroid

3. Hearing loss (abnormal middle ear bones) 4. Dental imperfections due to lack of dentition

Immunohistochemical stains

Stain Cell type

Vimentin Connective tissue

Desmin Muscle

Cytokeratin Epithelial cells

Glial fibrillary acid proteins (GFAP) Neuroglia

Neurofilaments Neurons

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! BIOC H E M ISTRY—M ETABOLISM Metabolism sites

Mitochondria Fatty acid oxidation (β-oxidation), acetyl-CoA production, Krebs cycle.

Cytoplasm Glycolysis, fatty acid synthesis, HMP shunt, protein synthesis (RER), steroid synthesis (SER).

Both Gluconeogenesis, urea cycle, heme synthesis.

Summary of pathways

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Glycogen UDP-glucose Glucose-1-phosphate Glucose Glucose-6-phosphate 6-phosphogluconolactone Fructose-6-phosphate Fructose-1,6-bisphosphate Glyceraldehyde-3-P DHAP 1,3-bis-phosphoglycerate 3-phosphoglycerate 2-phosphoglycerate Phosphoenolpyruvate (PEP) Pyruvate Acetyl-CoA Glyceraldehyde Ribulose-5-phosphate F1P Fructose NH4 + CO2 Carbamoyl phosphate Citrulline Aspartate Argininosuccinate Urea cycle Ornithine Urea H2O Arginine Fumarate Oxaloacetate Malate _TCA cycle Succinate Citrate Isocitrate α-ketoglutarate Succinyl-CoA Methylmalonyl-CoA Propionyl-CoA Odd-chain fatty acids Acetoacetate β-hydroxybutyrate Mevalonate Galactose Galactose-1-phosphate HMP shunt Glycolysis Lactate Acetoacetyl-CoA HMG-CoA Malonyl-CoA Fatty acids

Cholesterol 1 2 3 4 ₅ 6 7 8 9 11 12 Gluconeogenesis 15 14 16 17 18 10 1 Galactokinase (mild galactosemia)

2 Galactose-1-phosphate uridyltransferase (severe galactosemia)

3 Hexokinase/glucokinase

4 Glucose-6-phosphatase (von Gierke’s) 5 Glucose-6-phosphate dehydrogenase (G6PD) 6 Transketolase

7 Phosphofructokinase 8 Fructose-1,6-bisphosphatase 9 Fructokinase (essential fructosuria) 10 Aldolase B (fructose intolerance) 11 Pyruvate kinase 12 Pyruvate dehydrogenase 13 HMG-CoA reductase 14 Pyruvate carboxylase 15 PEP carboxykinase 16 Citrate synthase 17 α-ketoglutarate dehydrogenase 18 Ornithine transcarbamylase 13

! BIOC H E M ISTRY—M ETABOLISM (continued)

ATP Base (adenine), ribose, 3 phosphoryls. 2 phosphoanhydride bonds, 7 kcal/mol each.

Aerobic metabolism of glucose produces 38 ATP via malate shuttle, 36 ATP via G3P shuttle. Anaerobic glycolysis produces only 2 net ATP per glucose molecule.

ATP hydrolysis can be coupled to energetically unfavorable reactions.

Activated carriers Phosphoryl (ATP).

Electrons (NADH, NADPH, FADH₂). Acyl (coenzyme A, lipoamide). CO₂(biotin).

1-carbon units (tetrahydrofolates). CH₃groups (SAM).

Aldehydes (TPP). Glucose (UDP-glucose). Choline (CDP-choline).

S-adenosyl- ATP + methionine → SAM. SAM transfers methyl SAM the methyl donor man.

methionine units to a wide variety of acceptors (e.g., in

synthesis of phosphocreatine, a high-energy phosphate active in muscle ATP production). Regeneration of methionine (and thus SAM) is dependent on vitamin B₁₂.

Signal molecule ATP → cAMP via adenylate cyclase.

precursors GTP → cGMP via guanylate cyclase.

Glutamate → GABA via glutamate decarboxylase (requires vitamin B₆). Choline → ACh via choline acetyltransferase (ChAT).

Arachidonate → prostaglandins, thromboxanes, leukotrienes via cyclooxygenase/ lipoxygenase.

Fructose-6-P → fructose-1,6-bis-P via phosphofructokinase (PFK), the rate-limiting enzyme of glycolysis.

1,3-BPG → 2,3-BPG via bisphosphoglycerate mutase.

Universal electron Nicotinamides (NAD+_{, NADP}+_{) and flavin NADPH is a product of the}

acceptors nucleotides (FAD+_{). HMP shunt.}

NAD+_{is generally used in catabolic processes to}

carry reducing equivalents away as NADH.

NADPH is used in anabolic processes (steroid NADPH is used in:

and fatty acid synthesis) as a supply of reducing 1. Anabolic processes

equivalents. 2. Respiratory burst

3. P-450

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NH2 O _P O _P O _{P O} -HO HO N N N N O O O O O- _O- _O -~ ~

Oxygen-dependent respiratory burst

Hexokinase vs. Hexokinase is found throughout body. Only hexokinase is feedback

glucokinase GLucokinase is primarily found in the Liver inhibited by G6P.

(lower affinity [↑ K_m] but higher capacity Glucokinase phosphorylates

[↑ V_max]). excess glucose (e.g., after a

meal) to sequester it in the liver as G6P.

Glycolysis D-glucose Glucose-6-phosphate Glucose-6-P ".

regulation, Hexokinase/glucokinase*

irreversible Fructose-6-P Fructose-1,6-BP ATP ", AMP ⊕, citrate ",

enzymes Phosphofructokinase-1_{(rate-limiting step)} fructose-2,6-BP ⊕.

Phosphoenolpyruvate Pyruvate ATP ", alanine ",

Pyruvate kinase _{fructose-1,6-BP ⊕.} Pyruvate Acetyl-CoA ATP ", NADH ",

Pyruvate _acetyl-CoA ".

dehydrogenase

* Glucokinase in liver; hexokinase in all other tissues.

Glycolytic enzyme Hexokinase, glucose phosphate isomerase, aldolase, RBCs metabolize glucose

deficiency triosephosphate isomerase, phosphate glycerate anaerobically (no

kinase, enolase, and pyruvate kinase deficiencies mitochondria) and thus are associated with hemolytic anemia. depend solely on glycolysis.

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Phagolysosome Neutrophil cell membrane O₂ ↓ O2• ↓ H₂O₂ ↓ HOCl• ↓ NADPH NADP+ H2O2 H2O GSH GSSG NADP+ _NADPH G6P 6PG Cl- GSH/GSSG = glutathione (reduced/oxidized) HOCl• = bleach Bacteria

1 NADPH oxidase (deficiency = chronic granulomatous disease) 2 Superoxide dismutase 3 Myeloperoxidase 4 Catalase 5 Glutathione reductase 6 Glucose-6-phosphate dehydrogenase (G6PD) 1 2 3 4 5 6

! BIOC H E M ISTRY—M ETABOLISM (continued)

Pyruvate The complex contains 3 enzymes that require 5 The complex is similar to the

dehydrogenase cofactors (the first 4 B vitamins plus lipoic acid): α-ketoglutarate

complex 1. Pyrophosphate (B₁, thiamine; TPP) dehydrogenase complex

2. FAD (B₂, riboflavin) (same cofactors, similar

3. NAD (B₃, niacin) substrate and action).

4. CoA (B₅, pantothenate) 5. Lipoic acid

Reaction: pyruvate + NAD+_{+ CoA → acetyl-CoA +}

CO₂+ NADH. Activated by exercise:

↑ NAD+_{/NADH ratio}

↑ ADP ↑ Ca2+

Pyruvate Causes backup of substrate (pyruvate and alanine), Lysine and Leucine––the only

dehydrogenase resulting in lactic acidosis. Can be seen in purely ketogenic amino

deficiency alcoholics due to B₁deficiency. acids.

Findings: neurologic defects.

Treatment: ↑ intake of ketogenic nutrients (e.g., high fat content or ↑ lysine and leucine).

Pyruvate metabolism 6 ATP equivalents are needed

to generate glucose from pyruvate.

Alanine serves as carrier of amino groups from muscle to liver.

Oxaloacetate can be used to replenish TCA cycle or in gluconeogenesis.

Cori cycle Transfers excess reducing

equivalents from RBCs and muscle to liver, allowing muscle to function anaerobically (net 2 ATP).

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MUSCLE LIVER B L O O D Glucose 2ATP Pyruvate Lactate dehydrogenase Lactate Pyruvate Lactate Lactate dehydrogenase 6ATP Glucose Lactate Glucose Cytosol Mitochondria Alanine NADH + H+ _NAD+ NADH + H+ Acetyl-CoA Oxaloacetate NAD+ CO2 + ATP CO2 Pyruvate ALT LDH PC PDH

TCA cycle Produces 3 NADH, 1 FADH₂, 2 CO₂, 1 GTP per acetyl-CoA = 12 ATP/acetyl-acetyl-CoA (2× everything per glucose) α-ketoglutarate dehydrogenase

complex requires same cofactors as the pyruvate dehydrogenase complex.

Can I Keep Selling Sex For Money, Officer?

Electron transport chain and oxidative phosphorylation

Electron transport 1 NADH → 3 ATP; 1 FADH₂→ 2 ATP. chain

Oxidative 1. Electron transport inhibitors (rotenone, antimycin A, CN−_{, CO) directly inhibit}

phosphorylation electron transport, causing a ↓ of proton gradient and block of ATP synthesis. poisons 2. ATPase inhibitor (oligomycin) directly inhibits mitochondrial ATPase, causing an

↑ of proton gradient, but no ATP is produced because electron transport stops. 3. Uncoupling agents (2,4-DNP) ↑ permeability of membrane, causing a ↓ of

proton gradient and ↑ O2consumption. ATP synthesis stops. Electron transport

continues.

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Acetyl-CoA NADH Malate Fumarate Succinate Succinyl-CoA CO2 + NADH α-ketoglutarate Isocitrate cis-aconitate Citrate Citrate synthase Isocitrate dehydrogenase α-KG dehydrogenase GTP + CoA FADH2 Oxalo-acetate Pyruvate Pyruvate dehydrogenase Succinyl-CoA NADH ATP -ATP NADH ADP -+ -ATP Acetyl-CoA NADH -ATP -CO2 + NADH Oligomycin

Complex I Complex III Complex IV Complex V ADP + P_i NADH ATP + H₂O 2,4-Dinitrophenol NAD+ H+ H+ H+ H+ CoQ CoQ 1_/ 2O2 H2O

! BIOC H E M ISTRY—M ETABOLISM (continued) Gluconeogenesis, irreversible enzymes

Pyruvate carboxylase In mitochondria. Pyruvate → oxaloacetate. Requires biotin, ATP.

Activated by acetyl-CoA.

PEP carboxykinase In cytosol. Oxaloacetate → phosphoenolpyruvate. Requires GTP.

Fructose-1,6- In cytosol. Fructose-1,6-bisphosphate →

bisphosphatase fructose-6-P. _{Pathway Produces}

Glucose-6- In cytosol. Glucose-6-P → glucose. Fresh Glucose.

phosphatase

Above enzymes found only in liver, kidney, intestinal epithelium. Muscle cannot participate in gluconeogenesis.

Hypoglycemia is caused by a deficiency of the key gluconeogenic enzymes listed above (e.g., von Gierke’s disease, which is caused by a lack of glucose-6-phosphatase in the liver).

Pentose phosphate Produces ribose-5-P from G6P for nucleotide synthesis.

pathway (HMP Produces NADPH from NADP+_{for fatty acid and steroid biosynthesis and for}

shunt) maintaining reduced glutathione inside RBCs.

All reactions of this pathway occur in the cytoplasm. No ATP is used or produced. Sites: lactating mammary glands, liver, adrenal cortex––all sites of fatty acid or steroid

synthesis.

Glucose-6- G6PD is a rate-limiting enzyme in HMP shunt G6PD deficiency is more

phosphate (which yields NADPH). NADPH is necessary prevalent among blacks.

dehydrogenase to keep glutathione reduced, which in turn Heinz bodies––altered

deficiency detoxifies free radicals and peroxides. ↓ NADPH Hemoglobin precipitates

in RBCs leads to hemolytic anemia due to poor within RBCs.

RBC defense against oxidizing agents (fava beans, X-linked recessive disorder. sulfonamides, primaquine) and antituberculosis

drugs.

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NADP+ 2 GSH (reduced) GS–SG (oxidized) NADPH Glutathione reductase Glucose-6-phosphate dehydrogenase G6P 6PG 2H2O H2O2

Disorders of fructose metabolism

Fructose intolerance Hereditary deficiency of aldolase B (recessive). Fructose-1-phosphate accumulates, causing a ↓ in available phosphate, which results in inhibition of glycogenolysis and gluconeogenesis.

Symptoms: hypoglycemia, jaundice, cirrhosis, vomiting.

Treatment: must ↓ intake of both fructose and sucrose (glucose + fructose). Essential fructosuria Involves a defect in fructokinase and is a benign, asymptomatic condition.

Symptoms: fructose appears in blood and urine.

Disorders of galactose metabolism

Galactosemia Absence of galactose-1-phosphate uridyltransferase. Autosomal recessive. Damage is caused by accumulation of toxic substances (including galactitol) rather than absence of an essential compound.

Symptoms: cataracts, hepatosplenomegaly, mental retardation.

Treatment: exclude galactose and lactose (galactose + glucose) from diet.

Galactokinase Causes galactosemia and galactosuria, galactitol accumulation if galactose is present in diet. deficiency

Lactase deficiency Age-dependent and/or hereditary lactose intolerance (blacks, Asians).

Symptoms: bloating, cramps, osmotic diarrhea. Treatment: avoid milk or add lactase pills to diet.

Essential amino Ketogenic: Leu, Lys. All essential amino acids:

acids Glucogenic/ketogenic: Ile, Phe, Trp. PriVaTe TIM HALL.

Glucogenic: Met, Thr, Val, Arg, His. Arg and His are required during periods of growth.

FRUCTOSE METABOLISM (LIVER)

Fructose Fructose-1-P

Fructokinase Aldolase B Dihydroxyacetone-P Glyceraldehyde

Glyceraldehyde-3-P Glycolysis

Glycerol Deficiency = fructose intolerance

• Deficiency = essential fructosuria

NADH Triose kinase ATP ADP _ATP ADP NAD

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GALACTOSE METABOLISM Galactose Galactose-1-P Galactokinase Aldose reductase Galactitol Uridyl transferase Glycolysis/ gluconeogenesis 4-epimerase Glucose-1-P ATP

! BIOC H E M ISTRY—M ETABOLISM (continued)

Acidic and basic At body pH (7.4), acidic amino acids Asp and Glu Asp = aspartic ACID, Glu =

amino acids are negatively charged; basic amino acids Arg glutamic ACID.

and Lys are positively charged. Basic amino Arg and Lys have an extra acid His at pH 7.4 has no net charge. NH₃group.

Arginine is the most basic amino acid. Arg and Lys are found in high amounts in histones, which bind to negatively charged DNA.

Transport of ammonium by alanine and glutamine

Urea cycle Degrades amino acids into amino groups. Accounts Ordinarily, Careless Crappers

for 90% of nitrogen in urine. Urea cycle occurs Are Also Frivolous About

in the liver; carbamoyl phosphate incorporation Urination.

occurs in the mitochondria; the remaining steps occur in the cytosol.

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CO2 + NH4+ Carbamoyl phosphate Mitochondria Cytoplasm (Liver) Citrulline Ornithine Arginine Fumarate Argininosuccinate Aspartate Urea H2O Amino acids (NH3) (NH3) α-ketoacids α-ketoglutarate Glutamate Alanine Pyruvate Glucose Alanine Pyruvate Glucose α-ketoglutarate Glutamate Urea Muscle Liver Glutamine NH4+ NH4+ Glutamate NAD(P)+ _NAD(P)H α-ketoglutarate (NH3) (NH3) (NH3) (NH3)

Amino acid derivatives

Phenylketonuria Normally, phenylalanine is converted into tyrosine Screened for at birth.

(nonessential aa). In PKU, there is ↓ phenylalanine Phenylketones––phenylacetate, hydroxylase or ↓ tetrahydrobiopterin cofactor. phenyllactate, and

Tyrosine becomes essential and phenylalanine phenylpyruvate.

builds up, leading to excess phenylketones in urine. Autosomal-recessive disease. Findings: mental retardation, growth retardation, Incidence ≈ 1:10,000.

fair skin, eczema, musty body odor. Disorder of aromatic amino Treatment: ↓ phenylalanine (contained in acid metabolism → musty

aspartame, e.g., NutraSweet) and ↑ tyrosine in diet. body odor.

Alkaptonuria Congenital deficiency of homogentisic acid oxidase in the degradative pathway of

tyrosine. Resulting alkapton bodies cause urine to turn black on standing. Also, the connective tissue is dark. Benign disease. May have debilitating arthralgias.

Albinism Congenital deficiency of either of the following: Lack of melanin results in an

1. Tyrosinase (inability to synthesize melanin ↑ risk of skin cancer. from tyrosine)

2. Defective tyrosine transporters (↓ amounts of tyrosine and thus melanin)

Can result from a lack of migration of neural crest cells.

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Tryptophan

Niacin NAD+_/NADP+ Melatonin

Serotonin

Phenylalanine NE

Thyroxine

Tyrosine Dopa Dopamine

Histidine Histamine

Glycine Porphyrin Heme

Epi Arginine Urea Nitric oxide Creatine Melanin Glutamate GABA Phenylalanine THB DHB NADP+ _NADPH Phenylalanine hydroxylase Dihydropterin reductase Tyrosine

! BIOC H E M ISTRY—M ETABOLISM (continued)

Homocystinuria 3 forms: Results in excess homocysteine

1. Cystathionine synthase deficiency (treatment: in the urine. Cysteine ↓ Met and ↑ Cys in diet) becomes essential.

2. ↓ affinity of cystathionine synthase for Can cause mental retardation, pyridoxal phosphate (treatment: ↑↑ vitamin osteoporosis, tall stature,

B₆in diet) kyphosis, lens subluxation

3. Methionine synthase deficiency (downward and inward), and atherosclerosis (stroke and MI).

Cystinuria Common (1:7000) inherited defect of renal tubular COLA.

amino acid transporter for Cystine, Ornithine, Treat with acetazolamide to

Lysine, and Arginine in kidneys. Excess cystine alkalinize the urine.

in urine can lead to the precipitation of cystine kidney stones.

Maple syrup urine Blocked degradation of branched amino acids Urine smells like maple syrup.

disease (Ile, Val, Leu) due to ↓ α-ketoacid dehydrogenase. I Love Vermont maple syrup.

Causes ↑ α-ketoacids in the blood, especially Leu. Causes severe CNS defects, mental retardation, and

death.

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Homocysteine Methionine THF CH₃ THF Methionine synthase Cystathionine synthase Cystathionine Cysteine B₁₂ B₆ via SAM CH3

Purine salvage deficiencies

Adenosine ADA deficiency can cause SCID. Excess ATP and SCID––severe combined deaminase dATP imbalances nucleotide pool via feedback (T and B) immunodeficiency deficiency inhibition of ribonucleotide reductase. This disease. SCID happens to

prevents DNA synthesis and thus ↓ lymphocyte kids (remember “bubble

count. 1st disease to be treated by experimental boy”). human gene therapy.

Lesch-Nyhan Purine salvage problem owing to absence of LNS––Lacks Nucleotide

syndrome HGPRTase, which converts hypoxanthine to Salvage (purine).

inosine monophosphate (IMP) and guanine to guanosine monophosphate (GMP). X-linked recessive. Results in excess uric acid production. Findings: retardation, self-mutilation, aggression,

hyperuricemia, gout, and choreoathetosis.

Liver: fed state vs. fasting state

In the PHasting state,

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Nucleic acids Guanylic acid (GMP) Guanosine Guanine Inosinic acid (IMP) Hypoxanthine Inosine Adenosine Adenylic acid (AMP) Adenine Nucleic acids Xanthine Uric acid 2 4 1 3 1 HGPRT + PRPP 2 APRT + PRPP

3 Adenosine deaminase (ADA) 1 4 4 Xanthine oxidase VLDL HMP shunt TCA cycle

FED STATE FASTING STATE

Digestive system

Glucose Amino Chylomicrons

acids Fatty_acids

Amino acids glycerol lactate Glycogen G6P Pyruvate Acetyl-CoA Glu Ketone bodies Fats Glu Glu-6-P Glycogen Glycolysis / TCA cycle CM Fatty acids Protein Glucose AA

! BIOC H E M ISTRY—M ETABOLISM (continued)

Insulin Made in β cells of pancreas. Required for adipose Insulin moves glucose Into cells.

and skeletal muscle uptake of glucose. BRICK L (don’t need insulin

GLUT2 receptors are found in β cells and GLUT4 for glucose uptake): in muscle and fat. Insulin inhibits glucagon Brain

release by α cells of pancreas. RBCs

Serum C-peptide is not present with exogenous Intestine

insulin intake. Cornea

Anabolic effects of insulin: Kidney

1. ↑ glucose transport Liver

2. ↑ glycogen synthesis and storage 3. ↑ triglyceride synthesis and storage 4. ↑ Na retention (kidneys)

5. ↑ protein synthesis (muscles)

Insulin vs. Glucagon phosphorylates stuff → turns glycogen synthase OFF and phosphorylase ON.

glucagon Insulin dephosphorylates stuff → turns glycogen synthase ON and phosphorylase OFF.

Glycogen storage 12 types, all resulting in abnormal glycogen metabolism and an accumulation of glycogen

diseases within cells.

Type I Von Gierke’s disease––glucose-6-phosphatase The liver becomes a muscle.

deficiency. (Think about it.)

Findings: severe fasting hypoglycemia, ↑↑ glycogen in liver, hepatomegaly, ↑ blood lactate.

Type II Pompe’s disease––lysosomal α-1,4-glucosidase Pompe’s trashes the Pump

deficiency. (heart, liver, and muscle).

Findings: cardiomegaly and systemic findings, leading to early death.

Type III Cori’s disease––deficiency of debranching enzyme

α-1,6-glucosidase.

Findings: milder form of type I with normal blood lactate levels.

Type V McArdle’s disease––skeletal muscle glycogen McArdle’s: Muscle.

phosphorylase deficiency.

Findings: ↑ glycogen in muscle but cannot break it Very Poor Carbohydrate

down, leading to painful cramps, myoglobinuria Metabolism.

with strenuous exercise.

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C peptide S S -COOH Human proinsulin NH2- α chain β chain Cys Cys Cys _S S Cys Cys S S Cys

Lysosomal storage Each is caused by a deficiency in one of the many lysosomal enzymes.

diseases

Accumulated

Disease Findings Deficient enzyme substrate Inheritance

Fabry’s disease Peripheral neuropathy of α-galactosidase A Ceramide XR

hands/feet, angiokeratomas, trihexoside

cardiovascular/renal disease

Gaucher’s disease Hepatosplenomegaly, β-glucocerebrosidase Glucocerebroside AR aseptic necrosis of femur,

bone crises, Gaucher’s cells (macrophages)

Niemann-Pick Progressive neurodegeneration, Sphingomyelinase Sphingomyelin AR disease hepatosplenomegaly,

cherry-red spot (on macula)

Tay-Sachs disease Progressive neurodegeneration, Hexosaminidase A GM₂ganglioside AR developmental delay,

cherry-red spot, lysozymes with onion skin

Krabbe’s disease Peripheral neuropathy, β-galactosidase Galactocerebroside AR developmental delay,

optic atrophy

Metachromatic Central and peripheral Arylsulfatase A Cerebroside sulfate AR leukodystrophy demyelination with ataxia,

dementia

Hurler’s syndrome Developmental delay, α-L-iduronidase Heparan sulfate, AR

gargoylism, airway dermatan sulfate

obstruction, corneal clouding,

hepatosplenomegaly

Hunter’s syndrome Mild Hurler’s + aggressive Iduronate sulfatase Heparan sulfate, XR

behavior, no corneal dermatan sulfate

clouding

No man picks (Niemann-Pick)

his nose with his sphinger (sphingomyelinase). Tay-SaX (Tay-Sachs)

lacks heXosaminidase.

Hunters aim for the X

(X-linked recessive).

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GM1 GM2 GM3 Lactosyl cerebroside Glucocerebroside Cerebroside Tay-Sachs Gaucher's Sulfatides Galactocerebroside Metachromatic leukodystrophy Globoside Sphingomyelin Krabbe´s Niemann-Pick Fabry´s Ceramide trihexoside

! BIOC H E M ISTRY—M ETABOLISM (continued) Fatty acid

metabolism sites

Ketone bodies In liver: fatty acid and amino acids → acetoacetate + Breath smells like acetone

β-hydroxybutyrate (to be used in muscle and brain). (fruity odor). Urine test for Ketone bodies found in prolonged starvation and ketones does not detect diabetic ketoacidosis. Excreted in urine. Made from β-hydroxybutyrate (favored HMG-CoA. Ketone bodies are metabolized by the by high redox state). brain to 2 molecules of acetyl-CoA.

Cholesterol Rate-limiting step is catalyzed by HMG-CoA Lovastatin inhibits

HMG-synthesis reductase, which converts HMG-CoA to CoA reductase.

mevalonate. 2_⁄

3of plasma cholesterol is esterified

by lecithin-cholesterol acyltransferase (LCAT).

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Fatty acid synthesis

Malonyl-CoA

Acetyl-CoA

Acetyl-CoA

Inner mitochondrial Citrate Carnitine

membrane shuttle shuttle

Mitochondrial matrix

Fatty acid + CoA

Acyl-CoA Acyl-CoA β-oxidation (breakdown to acetyl-CoA groups) Malonyl-CoA

Fatty acid degradation occurs where its products will be consumed—in the mitochondrion. Cell cytoplasm CO₂ (biotin)

-Fatty acid CoA synthetase

Lipoproteins

Pancreatic lipase––degradation of dietary TG in small intestine.

Lipoprotein lipase––degradation of TG circulating in chylomicrons and VLDLs. Hepatic TG lipase––degradation of TG remaining in IDL.

Hormone-sensitive lipase––degradation of TG stored in adipocytes.

Major A-I––Activates LCAT.

apolipoproteins B-100––Binds to LDL receptor.

C-II––Cofactor for lipoprotein lipase. E––Mediates Extra (remnant) uptake.

Chylomicron TG CE VLDL Chylomicron remnant Lipoprotein lipase Lipoprotein lipase Modified LDL IDL E E Atherosclerotic plaque

TG = triglyceride, CE = cholesterol, FFA = free fatty acid Small intestine Hepatic triglyceride lipase LDL CE B-100 B-100 B-100 C-II CE B-100 E Receptor for B-100 Less TG CE TG B-48 C-II A E TG FFA B-48 Eat TG Pancreatic lipase FFA

Intestinal cells convert FFA back to TG and package in chylomicrons

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! BIOC H E M ISTRY—M ETABOLISM (continued)

Lipoprotein functions Lipoproteins are composed of varying proportions of cholesterol, triglycerides,

and phospholipids.

Function and route Apolipoproteins

Chylomicron Delivers dietary triglycerides to peripheral tissues and B-48 mediates secretion. dietary cholesterol to liver. Secreted by intestinal A’s are used for formation of epithelial cells. Excess causes pancreatitis, lipemia new HDL.

retinalis, and eruptive xanthomas. C-II activates lipoprotein lipase. E mediates remnant uptake by

liver.

VLDL Delivers hepatic triglycerides to peripheral tissues. B-100 mediates secretion. Secreted by liver. Excess causes pancreatitis. C-II activates lipoprotein lipase.

E mediates remnant uptake by liver.

IDL Formed in the degradation of VLDL. Delivers triglycerides and cholesterol to liver, where they are degraded to LDL.

LDL Delivers hepatic cholesterol to peripheral tissues. B-100 mediates binding to cell Formed by lipoprotein lipase modification of surface receptor for

VLDL in the peripheral tissue. Taken up by target endocytosis. cells via receptor-mediated endocytosis. Excess

causes atherosclerosis, xanthomas, and arcus corneae.

HDL Mediates centripetal transport of cholesterol (reverse A’s help form HDL structure. cholesterol transport, from periphery to liver). Acts A-I in particular activates as a repository for apoC and apoE (which are LCAT (which catalyzes needed for chylomicron and VLDL metabolism). esterification of cholesterol). Secreted from both liver and intestine. CETP mediates transfer of

cholesteryl esters to other lipoprotein particles. LDL and HDL carry most cholesterol. LDL HDL is Healthy.

transportscholesterol from liver to tissue; HDL LDL is Lousy.

transports it from periphery to liver.

Familial dyslipidemias

Elevated

Type Increased blood levels Pathophysiology

I––hyperchylomicronemia Chylomicrons TG, cholesterol Lipoprotein lipase deficiency or altered apolipoprotein C-II

IIa––hypercholesterolemia LDL Cholesterol ↓ LDL receptors

IIb––combined hyperlipidemia LDL, VLDL TG, cholesterol Hepatic overproduction of VLDL III––dysbetalipoproteinemia IDL, VLDL TG, cholesterol Altered apolipoprotein E

IV––hypertriglyceridemia VLDL TG Hepatic overproduction of VLDL

V––mixed hypertriglyceridemia VLDL, chylomicrons TG, cholesterol ↑ production/↓ clearance of VLDL and chylomicrons

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Heme synthesis Underproduction of heme causes microcytic hypochromic anemia. Accumulation of intermediates causes porphyrias.

Porphyrias

Lead poisoning Inhibits ferrochelatase and ALA dehydrase. Coproporphyrin and ALA accumulate in urine.

Acute intermittent Deficiency in uroporphyrinogen I synthetase. Symptoms = 5 P’s: Painful porphyria Porphobilinogen and δ-ALA accumulate in urine. abdomen, Pink urine, Porphyria cutanea Deficiency in uroporphyrinogen decarboxylase. Polyneuropathy,

tarda Uroporphyrin accumulates in urine (tea-colored). Psychological disturbances,

Photosensitivity. Precipitated by drugs.

Heme catabolism Heme is scavenged from RBCs and Fe2+_{is reused. Heme → biliverdin → bilirubin (sparingly}

water soluble, toxic to CNS, transported by albumin). Bilirubin is removed from blood by liver, conjugated with glucuronate, and excreted in bile. In the intestine it is processed into its excreted form. Some urobilinogen, an intestinal intermediate, is reabsorbed into blood and excreted as urobilin into urine.

Hemoglobin Hemoglobin is composed of 4 polypeptide subunits Carbon monoxide has 200×

(2 α and 2 β) and exists in 2 forms: greater affinity than O₂ 1. T (taut) form has low affinity for O₂. for hemoglobin. 2. R (relaxed) form has high affinity for O₂

(300×). Hemoglobin exhibits positive cooperativity and negative allostery (accounts for the sigmoid-shaped O₂dissociation curve for hemoglobin), unlike myoglobin.

Hemoglobin structure ↑ Cl−_{, H}+_{, CO}

2, 2,3-BPG, and temperature favor When you’re Relaxed, you do

regulation T form over R form (shifts dissociation curve to your job better (carry O₂).

right, leading to ↑ O₂unloading). Fetal hemoglobin (2α and 2γ subunits) has lower affinity for 2,3-BPG than adult hemoglobin (HbA) and thus has higher affinity for O₂.

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β2 β1 α1 α2 Heme

Succinyl CoA + Glycine

δ-Aminolevolinic acid (ALA) Porphobilinogen Pre-uroporphyrinogen Committed step ALA synthetase Lead poisoning Heme Protoporphyrin Coproporphyrinogen Uroporphyrinogen III Porphyria cutanea tarda Fe2 Acute intermittent porphyria (−)

! BIOC H E M ISTRY—M ETABOLISM (continued)

Methemo- Iron in hemoglobin is in a reduced state (ferrous, Administer nitrites in cyanide

globinemia Fe2+_{). Methemoglobin is an oxidized form of poisoning to oxidize}

hemoglobin (ferric, Fe3+_{) that does not bind O}

2 hemoglobin to

as readily but has ↑ affinity for CN–_{. methemoglobin form.}

Treat toxic levels of

METHemoglobin with METHylene blue.

CO₂transport in CO₂binds to amino acids in globin chain (at N CO₂must be transported from

blood terminus) but not to heme. CO₂binding favors T tissue to lungs, the reverse

(taut) form of hemoglobin (and thus promotes O₂ of O₂(occurs primarily in the

unloading). form of bicarbonate).

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Specific IgG in patient’s blood Peroxidase enzyme generates color Specific antigen in patient’s blood Test antibody 1. 2. Test antigen

! BIOC H E M ISTRY—LABORATORY TEC H N IQU ES

Polymerase chain Molecular biology laboratory procedure that is used to synthesize many copies of a desired

reaction (PCR) fragment of DNA.

Steps:

1. DNA is denatured by heating to generate 2 separate strands

2. During cooling, excess premade DNA primers anneal to a specific sequence on each strand to be amplified

3. Heat-stable DNA polymerase replicates the DNA sequence following each primer These steps are repeated multiple times for DNA sequence amplification.

Molecular biology techniques

Southern blot A DNA sample is electrophoresed on a gel and then SNoW DRoP:

transferred to a filter. The filter is then soaked in a Southern = DNA denaturant and subsequently exposed to a labeled Northern = RNA DNA probe that recognizes and anneals to its Western = Protein complementary strand. The resulting double-

stranded labeled piece of DNA is visualized when the filter is exposed to film.

Northern blot Similar technique, except that Northern blotting involves radioactive DNA probe binding to sample RNA.

Western blot Sample protein is separated via gel electrophoresis and transferred to a filter. Labeled antibody is used to bind to relevant protein.

Enzyme-linked A rapid immunologic technique testing for ELISA is used in many

immunosorbent antigen-antibody reactivity. laboratories to determine

assay (ELISA) Patient’s blood sample is probed with either whether a particular

1. Test antigen (coupled to color-generating antibody (e.g., anti-HIV) is enzyme)––to see if immune system present in a patient’s blood

recognizes it; or sample. Both the sensitivity

2. Test antibody (coupled to color-generating and the specificity of ELISA enzyme)––to see if a certain antigen is approach 100%, but both

present false positive and false

If the target substance is present in the sample, negative results do occur. the test solution will have an intense color

reaction, indicating a positive test result.

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! BIOC H E M ISTRY—G E N ETICS Genetic terms

Variable expression Nature and severity of the phenotype varies from 1 individual to another. Incomplete Not all individuals with a mutant genotype show the mutant phenotype.

penetrance

Pleiotropy 1 gene has > 1 effect on an individual’s phenotype.

Imprinting Differences in phenotype depend on whether the mutation is of maternal or paternal origin (e.g., AngelMan’s syndrome [Maternal], Prader-Willi syndrome [Paternal]). Anticipation Severity of disease worsens or age of onset of disease is earlier in succeeding generations

(e.g., Huntington’s disease).

Loss of heterozygosity If a patient inherits or develops a mutation in a tumor suppressor gene, the complementary allele must be deleted/mutated before cancer develops. This is not true of oncogenes. Dominant negative Exerts a dominant effect. A heterozygote produces a nonfunctional altered protein that

mutation also prevents the normal gene product from functioning.

Linkage Tendency for certain alleles at 2 linked loci to occur together more often than disequilibrium expected by chance. Measured in a population, not in a family, and often varies in

different populations.

Mosaicism Occurs when cells in the body have different genetic makeup (e.g., lyonization–– random X inactivation in females).

Locus heterogeneity Mutations at different loci can produce the same phenotype (e.g., albinism).

Hardy-Weinberg If a population is in Hardy-Weinberg equilibrium, Hardy-Weinberg law assumes:

population then: 1. There is no mutation

genetics Disease prevalence: p2_{+ 2pq + q}2_{= 1 occurring at the locus}

Allele prevalence: p + q = 1 2. There is no selection for p and q are separate alleles; 2pq = heterozygote any of the genotypes at

prevalence. the locus

3. Mating is completely random

4. There is no migration into or out of the population being considered

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Modes of inheritance

Autosomal dominant Often due to defects in structural genes. Many Often pleiotropic and, in many generations, both male and female, affected. cases, present clinically after

puberty. Family history crucial to diagnosis.

Autosomal recessive 25% of offspring from 2 carrier parents are affected. Commonly more severe than Often due to enzyme deficiencies. Usually seen in dominant disorders; patients

only 1 generation. often present in childhood.

X-linked recessive Sons of heterozygous mothers have a 50% chance of Commonly more severe in being affected. No male-to-male transmission. males. Heterozygous females

may be affected.

X-linked dominant Transmitted through both parents. Either male or Hypophosphatemic rickets. female offspring of the affected mother may

be affected, while all female offspring of the affected father are diseased.

Mitochondrial Transmitted only through mother. All offspring of Leber’s hereditary optic inheritance affected females may show signs of disease. neuropathy; mitochondrial

myopathies.

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carrier

! BIOC H E M ISTRY—G E N ETICS (continued) Autosomal-dominant diseases

Adult polycystic kidney Always bilateral, massive enlargement of kidneys due to multiple large cysts. Patients

disease present with pain, hematuria, hypertension, progressive renal failure. 90% of cases are due to mutation in APKD1 (chromosome 16). Associated with polycystic liver disease, berry aneurysms, mitral valve prolapse. Juvenile form is recessive.

Familial Elevated LDL owing to defective or absent LDL receptor. Heterozygotes (1:500) have hypercholesterolemia cholesterol ≈ 300 mg/dL. Homozygotes (very rare) have cholesterol ≈ 700+ mg/dL, (hyperlipidemia severe atherosclerotic disease early in life, and tendon xanthomas (classically in the type IIA) Achilles tendon); MI may develop before age 20.

Marfan’s syndrome Fibrillin gene mutation → connective tissue disorders.

Skeletal abnormalities––tall with long extremities (arachnodactyly), hyperextensive joints, and long, tapering fingers and toes (see Image 109).

Cardiovascular––cystic medial necrosis of aorta → aortic incompetence and dissecting aortic aneurysms. Floppy mitral valve.

Ocular––subluxation of lenses.

Neurofibromatosis Findings: café-au-lait spots, neural tumors, Lisch nodules (pigmented iris type 1 (von hamartomas). Also marked by skeletal disorders (e.g., scoliosis),

Recklinghausen’s pheochromocytoma, and ↑ tumor susceptibility. On long arm of chromosome disease) 17; 17 letters in von Recklinghausen.

Neurofibromatosis Bilateral acoustic neuroma, optic pathway gliomas, juvenile cataracts. NF2 gene on type 2 chromosome 22; type 2 = 22.

Tuberous sclerosis Findings: facial lesions (adenoma sebaceum), hypopigmented “ash leaf spots” on skin, cortical and retinal hamartomas, seizures, mental retardation, renal cysts, cardiac rhabdomyomas. Incomplete penetrance, variable presentation.

Von Hippel–Lindau Findings: hemangioblastomas of retina/cerebellum/medulla; about half of affected disease individuals develop multiple bilateral renal cell carcinomas and other tumors.

Associated with deletion of VHL gene (tumor suppressor) on chromosome 3 (3p). Von Hippel–Lindau = 3 words for chromosome 3.

Huntington’s disease Findings: depression, progressive dementia, choreiform movements, caudate atrophy, and ↓ levels of GABA and ACh in the brain. Symptoms manifest in affected individuals between the ages of 20 and 50. Gene located on chromosome 4; triplet repeat disorder. “Hunting 4 food.”

Familial adenomatous Colon becomes covered with adenomatous polyps after puberty. Progresses to colon polyposis cancer unless resected. Deletion on chromosome 5; 5 letters in “polyp.”

Hereditary Spheroid erythrocytes; hemolytic anemia; increased MCHC. Splenectomy is curative. spherocytosis

Achondroplasia Autosomal-dominant cell-signaling defect of fibroblast growth factor (FGF) receptor 3. Results in dwarfism; short limbs, but head and trunk are normal size.

Autosomal- Cystic fibrosis, albinism, α₁-antitrypsin deficiency, phenylketonuria, thalassemias,

recessive sickle cell anemias, glycogen storage diseases, mucopolysaccharidoses (except Hunter’s),

diseases sphingolipidoses (except Fabry’s), infant polycystic kidney disease, hemochromatosis.

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Cystic fibrosis Autosomal-recessive defect in CFTR gene on Infertility in males due to absent chromosome 7. Defective Cl−_{channel → secretion vas deferens. Fat-soluble}

of abnormally thick mucus that plugs lungs, vitamin deficiencies (A, D, E, pancreas, and liver → recurrent pulmonary K). Can present as failure to infections (Pseudomonas species and S. aureus), thrive in infancy.

chronic bronchitis, bronchiectasis, pancreatic Most common lethal genetic insufficiency (malabsorption and steatorrhea), disease of Caucasians. meconium ileus in newborns. ↑ concentration Treatment: N-acetylcysteine of Cl−_{ions in sweat test is diagnostic. to loosen mucous plugs.}

X-linked recessive Fragile X, Duchenne’s muscular dystrophy, hemophilia A and B, Fabry’s, G6PD

disorders deficiency, Hunter’s syndrome, ocular albinism, Lesch-Nyhan syndrome, Bruton’s

agammaglobulinemia, Wiskott-Aldrich syndrome.

Female carriers of X-linked recessive disorders are rarely affected because of random inactivation of X chromosomes in each cell.

Muscular dystrophies

Duchenne’s Frame-shift mutation → deletion of dystrophin Duchenne’s = Deleted (X-linked) gene → accelerated muscle breakdown. Onset Dystrophin.

before 5 years of age. Weakness begins in pelvic Diagnose muscular dystrophies girdle muscles and progresses superiorly. by ↑ CPK and muscle Pseudohypertrophy of calf muscles due to biopsy.

fibrofatty replacement of muscle; cardiac myopathy. The use of Gowers’ maneuver, requiring assistance of the upper extremities to stand up, is characteristic (indicates proximal lower limb weakness).

Becker’s Mutated dystrophin gene is less severe than Duchenne’s.

Fragile X syndrome X-linked defect affecting the methylation and Triplet repeat disorder (CGG)_n

expression of the FMR1 gene. The 2nd most that may show genetic common cause of genetic mental retardation anticipation (germlike (the most common cause is Down syndrome). expansion in females). Associated with macro-orchidism (enlarged testes), Fragile X= eXtra-large long face with a large jaw, large everted ears, testes, jaw, ears. and autism.

Trinucleotide repeat Huntington’s disease, myotonic dystrophy, Try (trinucleotide) hunting

expansion diseases Friedreich’s ataxia, fragile X syndrome. May show for my fried eggs (X).

anticipation (disease severity ↑ and age of onset ↓ in successive generations).

Common congenital 1. Heart defects

malformations 2. Hypospadias

3. Cleft lip (with or without cleft palate) 4. Congenital hip dislocation

5. Spina bifida 6. Anencephaly

7. Pyloric stenosis Associated with projectile

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