Biochem for boards Super Set

Mitochondria

• Site of oxidation phosphorylation: via the electron transport chain embedded in the inner mito membrane

  • Produce ATP

• The other biochemical processes occur in the Mito:

  • Pyruvate oxidation

  • Krebs Cycle

  • Fatty Acid Beta-Oxidation

Oxidative Phosphorylation

The electron transport chain

• Inner membrane has 5 distinct protein complexes embedded: some encoded by nuclear DNA, some encoded by Mito DNA

• Use NADH + FADH coming form Krebs cycle break down of Actyl-CoA

  • Fatty Acids (generated via F.A. B-Oxidation)

  • Pyruvate (generated via glycolysis)

• Electron transport down the chain of complexes: creates gradient by pumping IN H+ ions

• Complex V uses gradient to Generate ATP as H+ ions move OUT

Mitochondrial Disorders

Oxidative Phosphorylation/Electron Transport chain dysfunction

Two varieties:

• Secondary Mitochondrial dysfunction: Non-genetic conditions

  • Hypoxemia (inadequate Oxygen for Oxidative Phosphorylation)

  • Medication: valproic acid, HIV meds

  • Toxins: cyanide, rotenone

• Primary Mitochondrial Disease

  • mitochondrial DNA itself or nuclear DNA mutations

Mitochondrial Genome

The Mitochondrial Chromosome: encodes 37 genes

• only 3% of Mito. proteins are encoded by mito DNA

• 97% are encoded by nuclear DNA and imported into mitochondria

Complex 1

46 total proteins

MtDNA encoded: 7

nuDNA: 39

• Leigh Syndrome

• Leukodystrophy

Complex 2

4 proteins: ALL nuDNA ENCDOED

• Leigh Syndrome

• Paraganglioma

• Pheochromocytoma

Complex 3

11 proteins

MtDNA: 1

nuDNA: 10

• Leigh syndrome

• GRACILE syndrome

Complex 4

mtDNa: 3

nuDNA: 10

• Leigh Syndrome

• Hepatopathy

• Cardioencephalomyopathy

• Leukodystrophy/tubulopathy

Complex V

mtDNA: 2

nuDNA: 14

Maternal Inheritance

mtDNA mutations can only be inherited through the mother

• all mito provided by the ovum

• no mito contriubted by the sperm

Heteroplasmy

Mito genomes can differ between mitochondria in a given cell and % of mutant mtDNA can vary in an individual from cell-to-cell and tissue-to-tissue

• Each cell has up to 1000 mitochondria, each with their own copy of the mito genome

• mtDNA mutation rate is 10-20x nuclear DNA mutation rate

Threshold Effect.

• energy requirements vary between tissues

• mtDNA mutation burden varies tissue to tissue (heteroplasmy)

• Tissue specific % mutant mtDNA threshold for disease

• Phenotypic variability results

Example: Brain and Muscle have a lower threshold than Skin and Kidney

Mitochondrial Disorders: Presentation

Can present in almost any way and vary from person to person, but 3 general categories

• “Classic” Mitochondrial diseases: reproducible, multi-organ pattern

• Unexplained multi-organ dysfunction:

  • Hearing loss short stature

  • Diabetes + hypertrophic cardio myopathy

  • ophthalmoplegia +ptosis

• Unexplained single organ syndrome: just hearing loss, epilepsy, GI

Often elevated Lactic acid in Blood or CNA and Mitochondrial proliferation in muscle

Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke like episodes (MELAS)

• Age of Onset: before 40yo (average 5-15)

• Clinical

  • Stroke like episodes + Epilepsy, Dementia

  • Muscle weakness (myopathy), Cardiomyopathy, Lactic Acidosis

  • Hearing-Loss, Retinopathy, Diabetes

• CT/MRI: Infarcts→ but not seen in vasuclar regions: infarct occurs due to region engery insufficney from Mitocondrial

• Etiology: heterogeneous mtDNA mutations (Often mt-t RNA) → VERY dependent on Heteroplasmy with individual

Myoclonic Epilepsy with Ragged Red Fibers (MERRF)

• Adolescent onset

• Clinical manifestations

  • Epilepsy (myoclonic)

  • Muscle weakness (myopathy), Lactic acidosis, Ataxia

  • Encephalopathy, Hearing Loss

• EMG

• EEG:

• Muscle Biopsy: (if done on affected muscle) will show ‘ragged red fibers’ caused by mitochondria proliferation

• Etiology: Single mtDNA-tRNA mutation 80 to 90%

Leber’s Hereditary Optic Neuropathy (LHON)

• Age of onset 20-24 yo

• Clinical:

  • Acute or sub-acute bilateral central vision loss→ Rapid progression to blindness (usually confined to optic nerve)

  • Rarely: heart block, dystonia, MS-like symptoms

• Fundoscopy: early tortuous retinal arteries, followed by optic atrophy

• Etiology: 95% mtDNA “ND” (electron transport subunit) gene mutations MATERNAL INHERITANCE

  • ****4:1 M:F ration → X-linked modifier genes that make females less affected****

Chronic Progressive Ophthalmoplegia (CPEO)

Chronic Progressive Ophthalmoplegia (CPEO)

• External ophthalmoplegia (eye weakness) → can’t look in certain directions

• bilateral ptosis (eyelid drooping)

• mild myopathy (limb weakness)

• Onset ***AFTER*** 20yo (slowly progressive)

Etiology:

• Mainly mtDNA deletions→ can be smaller or larger chunks of mtDNA (smaller =CPEO, larger=KSS)

• Majority are SPONTEOUS

Subacute Necrotizing Encephalopathy (Leigh Syndrome)

• Large spectrum of 75 genes (mito and nuc. but mostly nuc.) that cause an energy failure in the brain

• 6-12 months onset - death by 3-5 years (25% have later onset or slower forms)

• Clinical: (often abrupt decompensations/regression with infection/fever)

  • Developmental ***REGRESSION***

  • Seizures, Ataxia, Hypotonia, spasticity

  • Ophthalmoplegia, Nystagmus, Optic atrophy

• Diagnostic Testing

  • MRI: ***SYMETRIC LESIONS OF BASAL GANGLIA***

  • Elevated Lactic Acid in blood or Cerebral spinal fluid

  • 10% mtDNA mutation

  • 90% nDNA mutation

• Lower % of mitochondria with the mutant mtDNA→ have NARP instead of Leigh (HETEROPLASMY)

Leigh Etiology

Genetic Heterogeneity

• 10-30% mitochondrial DNA mutations → maternal inheritance

• 90-70% nuclear DNA mutations → Classic Mendelian

  • HETEROPLASMY AFFECT: If a lower # of mito. in a cell have these mutations = Later onset Neuropathy, Ataxia, Retinitis Pigmentosa (NARP)

Pyruvate Dehydrogenase Complex (PDHC) Deficiency: Clinical + Testing

Failure to convert Pyruvate to Actyl-CoA (via PDH)

Lactic Acid levels elevated (***PDHC most common cause of Lactic Acidosis***)

Point mutation in NUCLEAR DNA

Clinical Features: Progressive intermittent neurologic deterioration

• hypotonia, seizures, ataxia, ophthalmoplegia, dystonia

• Presents similar to mitochondrial dysfunction

Suggestive Abnormal Tests

• Plasma: increased Lactic Acid + Pyruvate, but normal ratio of Lactic Acid: Pyruvate

  • Distinguished from other Mitochondrial Disease: Pyruvate levels are NOT elevated

• Cerebral Spinal Fluid: increased Lactic Acid

Pyruvate Dehydrogenase Complex (PDHC) Deficiency: Metabolism +Etiology

• Failure to convert Pyruvate to Actyl-CoA (via PDH)

• Lactic Acid levels elevated (PDHC most common cause of Lactic Acidosis)

Etiology: PDHC is a multisubunit complex

• Catalytic components: E1, E2, E3

• Regulatory component: PDH Phosphatase

Confirmation:

• PDHC enzyme activity assay

• Sequencing of

  • E1 → PDHA1 : MOST COMMON , X-Linked (males only)

  • E2 → DLAT, Recessive

Mitodoncrial Diease: Work Up

• Serum levels: increased anion gap + metabolic acidosis

  • Lactic Acid: Pyruvate ratios (>30 Mito. Dis ; <10 PDHC Def.)

• Imaging: brain MRI, Spectroscopy ( LA peaks over brain regions( BasalGang)

  • Basal Ganglia hypodensities: generalized atrphy

  • Hypoplastic corpus callosum if fetal lactic acidosis

• Muscle Biopsy

• Genetic Testing

Mitochondrial Disease: Muscle Biopsy

Allows for:

• Detecting ragged red fibers (mito. proliferation)

• Abnormal mitochondria proliferation

• Detecting enzyme activity of the chain-genes

• Mutational analysis of mitoDNA

Pitfalls:

• need 1 gram of flesh (large amount)

• biopsy of moderately affected muscle

• may not distinguish exact genetic mechanisms

Genetic Testing

mtDNA:

• Leigh Syndrome

• LHON

• MERRF (blood/muscle)

• MELAS (blood/muscle)

• NARP (blood/muscle)

• KSS/CPEO (muscle)

nDNA (all in blood)

• Leigh syndrome

• MNGIE

• Mohr-Tranebjaerg

• Friedreich’s Ataxia

• AR spastic paraparesis

• AD PEO

Mitochondrial Disorders: Treatments

Less evidence for specific treatments that actually improve outcomes

• Trials with Vitamins that optimize Electron Transport chain function:

  • Carnitine

  • Biotin

  • thiamine

  • Riboflavin

• High Fat/ Low Carb diet: low carb→ less glycolysis→ less LA

• Avoid Mito toxic meds

• Reduce LA, control acidosis (dialysis/vent)

Organic Acidemias Background

• Primarily disorders of Amino Acid Catabolism: Mainly

  • Branch Chain Amino Acids (BCAA)

  • Lysine

• Toxicity comes from accumulation of ORGNIC ACIDS not from an A.A. acid accumulating

  • Causes metabolic acidosis with increased “Anion Gap”: Decrease in main anion Bicarbonate (HCO3-)

  • Secondary toxic effects of acidosis

    • Mitochondria→ Lactic acidemia

    • Urea Cycle → Hyperammonemia

    • Bone marrow→ Bone marrow suppression

    • CNS function→ Encephalopathy/Mental retardation

• Major Presentations: Neonatal encephalopathic acidosis, late chronic/intermediate

• All autosomal recessive

Metabolic Acidosis

• Blood pH low due to excess acid (H+) vs Base (HCO3-)

  • normal range pH 7.3-7.45 (measure via Atrial Blood Gas)

  • Normal HCo3- level: 22-26 mEq/L

• Mutiple etiologies for Metabolic Acidosis

  • Lowered HCo3- : loss through GI (diahrria), Renal tubule acidosis, Medications

  • Elevated H+: creation of abnormal acids in blood due to starvation, diabetes; Lactic acidosis due to mitochondrial dysfunction, Organic Acidosis

• Clinical Consequences

  • Neonatal: non-specfic, similar to UCDs presenations,

    • Lethargy, vomting, Tachypena, Hypotonia, Seizures, Coma, Death

  • Adult: Devleopmental Delay, Ataxia, Neurological Deficits, (then the neonatal presenations)

Organic Acidemias

Newborn Screening detects many Organic Acidemias

Organic Acidemias Treatment

• Restrict Dietary Protein disease specific amino acid free formulas

• Prevent Catabolism provide sufficient protein free calories

• Reverse Acidosis ± Hyperammonemia

  • Hemodialysis

  • Ammonia and lactic acid scavengers

    • Sodium bicarbonate, sodium benzoate, phenylbutyrate

• Cofactor therapy for specific Disorders

Propionic Acidemia Metabolism

• Failure of Propionyl-CoA carboxylase

• Step 11 of Isoleucine and Valine metabolism:

• Propionyl-CoA → Methylmalonyl-CoA via Propionyl-CoA carboxylase activity

  • Propinoyl-CoA: the activated mitochondrial form

  • Propionic Acid: free organic acid (interferes with NH3 removal, other stuff)

  • Propinolycarnatine (C3 - what NBS measures): the ‘detox’ / transport form that excess P-CoA gets converted to so it DOESN'T become Propionic Acid

Propionic Acidemia

• Also known as Ketonic hyperglycemia: high level of glycine and ketone bodies

• Autosomal Recessive

• Incidence 1:100,00 (higher in Saudia Arabia and Inuit)

Genetic Defect

• Propinyl-CoA Carboxylase (PCC) alpha or beta subunit genes

  • some genotype/phenotype correlation (null alleles/deletions more severe)

  • Biotin cofactor for PCC

• PA accumulation due to PA production from

  • MET/THR/VAL/ISO catabolism,

  • gut bacteria,

  • odd chain FAs

Untreated Propionic Acidemia

Classical Neonatal Encephalopathic Form

• Normal at birth

• Within a few days

  • Poor feeding, lethargy, vomiting hypotonia →encephalopathy, seizures, coma, death

Late-Onset Form

• Developmental delays/regression

• cyclic vomiting

• protein intolerance

• growth impairment

• hypotonia

• metabolic basal ganglia stroke

• cardiomyopathy

• Acute episode of toxic encephalopathy

Rare Cardiac Subtype isolated cardiomyopathy

Diagnosing Propionic Acidosis (PA)

Newborn Screening

• Elevated Propionyl Acylcarnitine and ratio to other carnitine species (Propionyl-CoA gets combined with Carnatine to try buffer high Prop-CoA levels)

  • other etiologies: Methylmalonic Acidemia, Cobalamin Defects, Maternal B12 Deficiency, False +

Confirmatory Testing

• Atrial Blood Gas: Elevated ammonia, low glucose, high acidosis, increased anion gap

• Complete blood count: suppression of bone marrow→ less blood cells

• Urine Organic Acid Analysis: High 3-OH-proprionate, mthylcitrate, tigly/proprionylglycine but NOT MMA

• Plasma Amino Acid profile:

  • elevated glycine + glutamine, not homocysteine (seen with Cobalamin defects)

• Acyl-Carnitine Profile: Elevated C3 acylcarnitine, not C4-DC unless SUCLA2 deficiency

• PCC enzyme activity: can measure PC enzyme in leukocytes or fibroblasts

PCC Genotyping

• Gene sequencing w del/dup analysis (99% detection rate)

Treating Propionic Acidosis

Acute Acidotic Encephalopathy

• Remove acids and ammonia hemodialysis

  • severe hyperammonia: ammonia scavengers

• Reduce PA production Protein restriction 24-25hr

• Prevent catabolism: glucose and lipids IV

• Enhance PA excretion: IV Carnitine

• Decreased PA production in Gut: Antibiotics (Metronidzole

• Biotin:

Chronic Treatment

• protein restriction and MTVI-free metabolic formula

• Oral Carantine, Biotin, and Antibiotics

• Avoid decompensation

• unresponsive to Tx → liver transplantation

Propionic Acidemia Deficiency Outcome

Treamtnet improves surivial, but invariable there is an affect to some degree

• Neurodevleopmatl disabilty

• metabolic basal ganglia stroke

• seiures

• pancreatisis

• cardiomyopathy

• gorwth impairment

• nuetorpnia, AA defience

• renal failure

• premature ovarian fialure

• hearing and vidual defecits (optic nerve atrphy)

Propionic Acidemia Deficiency Screening

• Carrier Screening:

• PRenatal Diaongis:

  • amontic fluid orgnaic acid measurment possible (some false negatives)

Methylmalonic Acidemia Pathway

• Isoleucine and Valine

• Methlymalonyl-CoA → Succinyl CoA via Methylmalonic-CoA mutase activity

• Methlymalonyl-CoA accumulates

Methylmalonic Acidemia

• Increased Methylmalonic Acid but not homocysteine (other forms of MMA have elevated homocysteine→ not primary MMA, but related to Adenosyl Cobalamin - A )

Genetic Defect: mutation of multiple genes cause similar phenotype

• 60% Methlymalonyl-Co mutase gene mutation (MUT)

• 37% Cobalamin A,B,D2 (MMAA, MMAB, MMADHC)→ the upstream vitamins that will be converted into Adenosyl Cobalamin→ leads to dysfunctional MM-Co mutase

Untreated Methylmolaynic Acdiemia

Infantile Subtype: Most common mut0, cblB mutations

• Normal at Birth

• Within days to weeks: poor feeding, lethargy, vomiting, hypotonia, encephalopathy→ progress to seizures, coma, death

Intermediate phenotype: mut-, cblA, cblD2

• Normal for month to years: fialure to thrive, devleopmental delay, hypotonia, poriten aversion→ risk of carastrophic decompensations

Benign Adult form: typically asymptomatic, can decomapnste

Diagnosing Methylmolaynic Acdiemia

Newborn screening: Elevated Propinoyl Acylcarnitine (and ratios) → but non specific

Confirmatory testing

• Atrial Blood Gas, Ammonia Levels, Completel blood ocunt:

  • Hi AG metabolic acidsosi

  • Elevated ammonia

  • Low gluclose

  • pancytopenia

• Urine Organic Acid: High MMA

• Plasma Amino Acid profile: high glycine + glutamine, no Homocystine (Hcy)

  • CblC/D/F - Hcf + MMA high ;

  • cblD2/E/G - Just hcf High

• Enzyme activity: fibroblasts

Genotyping on genes = 95%

Treating Methylmolonic Acidemia

Treat acute acidotic encephalopathy

• Remove acids+amonia: hemodialysis

• Reduce MMA production: protein restriction

• Prevent catabolism: IV glucose and lipids

• Severe hyperammonemia: Amonia scavengers

• Decrease gut bacteria: Antibotics

• HYDOXYCOBALAMIN (B12) injects: cofactor

Chronic Treatment

• protien restriciton and MTVI-free meatolibc fomumal

• L-Carnitine + OH-B12

• Avoid decompensation

Methylmalonic Acidemia Treatment outcome

• Most patient will have some degree of mental impairment, long term affects

Methylmalonic Acidemia Diagnosis

• Prenatal/Preimplantation:

  • Ammonitic organic acid fluid analysis possible

  • Enzyme activity of CVS and amniocentesis

Iso-valeric Acidemia

• issues with the Isovaleryl-CoA dehygroenase enzyme

• LEUCINE PATHWAY ONLY

• Build up of Isovalryl-CoA (Isovaleric Acid)

• Disorder of Leucine metabolism only (unlike PA or MMA)

Genetic Defect

• IsoValeryl-CoA Dehydrogenase (IVD) Gene Mutation

  • results in increased Isovaleric Acid

  • Sweaty feet odoer is prominent

Untreated Isovaleric Acidemia

Severe Neonatal Onset form

• Normal at birth

• Within day: poor feeding, lethargy, hypotonia, Sweaty feet order → encephalopathy, seizure, coma, death

Mid/Late Onset Form

• unexplained failure to thrive and developmental delay

Benign Adult Form: Typically, asymptomatic but can mildly decompensate

Diagnosing Isovaleric Acidemia

Newborn screening: elevated Isovalerylcarnitine (C5 acylcarnitine) - used to ‘buffer’ Isovaleric Acid that builds up when Acylvaleryl-CoA builds up

Confirmatory testing

• Blood tests:

  • High ammonia

  • Low glucose

  • High metabolic acidosis

• Urine organic acid

  • High IVA

  • High isovaleryl glycine

• Plasma AA levels:

  • High glycine

  • High glutamine

• Enzyme activity: Fibroblast

Genotyping: exact genes unknown

Treating Isovaleric Acidemia

Treat Acute Acidotic encephalopathy

• Remove acids and ammonia: hemodialysis

• Reduce IVA production: protein restriction 24-26hrs

• Prevent Catabolism: IV glucose and lipids

• Enhance IVA excretion: IV carnitine

• If hyper ammonia: ammonia scavengers

• GLYCINE SUPPLMENTAITON-BINDS IVA

Chronic Treatment

• Protein rection and LEUCINE-free metabolic formula

• Oral L-Carnitine and L-Glycine

• Avoid decompensation

Isovaleric Acdiemia Otucome

• Outlook with Treatment is one of the best if treatment done early and effecetively enough

  • can be comepltely asymptomatic as long condition is monitored

  • Leucine tolerance gets better with age

• Even if diaognsis is after neonatal period, and evne with major encaplapthic event in neonatal period—> longer term out look is vairable : CAN BE OK

Isovaleric Acidemia Prenatal Diagnosis

• Ammniotic fluid can be checked for organic acids

Biotinidase Deficiency Pathway

• Biotin is a vital cofactor for of number of different enzymes:

  • ALL ARE CARBOXYLASES

• 3-Methylcrontoyl-CoA carboxylase (Leucine)

• Propinoyl-CoA Carboxylase (Isoleucine and Valine)

• Malonyl-CoA decarboxylase

When there are mutations in the BIOTINADASE gene: Biotin is not properly recycled → these blocks develop

Biotinidase Deficiency (BTD)

• Late multiple Carboxylase Deficiency

• Slightly increased incidence in Hispanic and Middle Easter

Gene Defect: Biotinidase (BTD) gene

• failure to recycle biotin = biotin deficiney

• Biotin co-factor for the carboxylases: cannot combine and make function enzyme

Untreated Biotinidase Deficiency

Affect depends on the residual enzymatic activity when biotin absent

Profound Deficiency (<10% enzyme)

• Normal at birth

• Symptoms develop after few months

  • Developmental delay, seizures, hypotonia, ataxia, hearing loss, visual problems, ***alopecia***, ***eczema*** (unique to BTD)

Partial Deficiency (10-30% enzyme)

• intermittent symptoms with stress

Symptoms can be irreversible once present

Diagnosing Biotinadase

Newborn Screening: Elevated C5-OH Acylcarnitine, but not specfic to BTD

Confirmatory Testing

• Blood:

  • High ammonia

  • High acidosis

  • Low gluclose

• Urine Organic Acids: multiple organic acids b/c Bitonaisde affects multiple enzymes —> refered to as ‘Multiple Carboxylase Defeicieny’ (MCD) on uOA

  • ( )

• Elevated Hydroxy-Isovalyrl-carnatine (C5-OH)

• Enzyme activity: IMPORTANT STEP: blood sample

  • If Biotinadase activity is normal→ then issue is probably Holocarboxylase Deficiency (presents the same way but is earlier)

Genotyping: sequencing 99% detection

Treating Biotinadase Deficeincy

Rarely severyl acidotic or hyperammonemic

• may occasionally need sodium bi-cabonate (adress acidty)

• may occasionally need amonia scavnerge (adress amonia levels)

• Insitute Biotin therapy immediately

Chronic treatment

• Biotin

• No protien restction

• Avoid raw egg whites (has protein that binds Biotin)

Biotinadase Deficiency outcome

• Extremely great outlook for patients (one of the best for Organic Acidemias)

• As long as treatment is implemented BEFORE the development of severe symptoms

• If detected after symptoms, some are irreversible: optic atrophy, hearing loss, developmental delay can presist

Biotinadase Deficiency Prenatal diagonsis

• Biotinadase enzyme activity can also be measuredin the amniocytes and the amniotic fluid

Glutaric Acidemia Type 1 (GA1) Metabolism

• NOT a Branch Chain Amino Acid metabolism disorder

• Breakdown of LYSINE and TRYPTOPHANE

• Lysine + Tryptophane → Alpha ketoadipic→ Glutyrl-Coa

• Glutyrl-Coa→ Glutaconyl-CoA (shunt to Glutaontic Acid) via Glutaryl-CoA Dehydrogenase activity

Glutaric Acidemia Type 1 (GA1)

• “Cerebral” Organic Acidemia: Often normal

Genetic Defect: Glutaryl-CoA Dehydrogenase (GCDH) gene mutation causing defective Lysine + Tyrptohan metabolism

Glutaric Acidemia Type 1 (GA1) Symptoms

• Often normal at birth or only macrocephalic

• symptoms often begin prior to 2 years of age

  • May start with a Sudden neurologic decompensation: 75% by 14months—> fever, illness, metabolic stress

Primary symptoms

• Stress-induced encephalopathy

• Ataxia

• Epilepsy

• Myoclonus

• Storke-like episodes

Glutaric Acidemia Type 1 (GA1) Diagnosis

Newboarn screening: Elevated C5-DC (glutaryl) Acylcarnitine

• many False negatives

Confirmatory Testing

• Blood: elvated ammonia, low gluclose, Aciditiy

• Plasma + Urine: C5-DC glutaryl acylcarnitine + glutaric acid

• Enzyme activity: fibroblast

• CT/MRI: Cerberallar atrophy, basal ganlia infact and hemorrhage

Genotyping

Glutaric Acidemia Type 1 (GA1) Treatment

Reverse/Prevent Catabolism When sick: protien free calroeis during metaoblic stress

Dietary Mdofication

• Low LYSINE and TRYOPTHAN

MEdicaitons

• B2 (Riboflavin) is a COFATOR

• Carntine: Binds Glutaric acid and remvoes it

Avoid Valproate (Bind Carnitine)

The Urea Cycle

• 6 Major Enzymatic reactions that occur in the liver within the Hepatocytes: the mitochondria + cytoplasm

• 2 Major functions

  • removal of nitrogenous waste (produced mainly from protein catabolism) → Ammonia incorporated into Urea for Excretion

  • Synthesis of amino acids: Arginine, Ornithine and Citrulline (become ESSENTIAL A.A. in deficiencies of the UREA CYCLE ENZYMES)

• Major presentations of Urea Cycle Disorders:

  • Severe Neonatal Hyperammonemic encephalopathy (exception: Argine deficiency + late/mild onset variants)

• All Autosomal Recessive except for Ornithine transcarboxylase deficiency (OTC):

  • OTC is X-Linked Recessive → only affects Males

The 6 Urea Cycle Disrders

Correspond to the 6 steps of the Urea Metabolic cycle: taken individual relatively uncommon, but all together 1:8-35,000

• N-Acetyl Glutamate Synthetase Deficiency

• Carbamoyl Phosphate Synthetase (CPS1) Deficiency

• ****Ornithine Transcarbamylase (OTC) Deficiency****—> *****MOST COMMON*****

• Arginosuccinic Acid Synthetase I (ASS1) Deficiency → also called Citrullinemia I

• Arginosuccinic Acid Lyase (ASL) Deficiency

• Arginase (ARG) Deficiency

Ammonia (NH3)

• Ammonia is the product of the metabolism/catabolism of protiens/amino acids

  • Normal serum ammonia levels

    • Adults <35 mmol/L

    • Neonates <100 mmol/L (immature liver cells + increased tissue catabolism surrounding delivery)

• Hyperammonemia: happen with great degree with IEMs of Urea Cycle But also seen with other metabolic disorders like Organic Acidemias (excess acid decreased Urea Cycle activity, lesser extent that UCDs)

  • Causes Neuronal excitotoxin increased extracellular glutamate +overexcitation of NMDA receptors→ Cell death and Cerebral Edema

• Clinical Consequence

  • Acute severe elevation: seizures, coma, death

  • Mild chronic elevations: Brain atrophy, cognitive impairment

Classic UCD Presentation: Early

Neonatal Hyperammonemia Encephalopathy

• In utero: protected my maternal urea Cyle activity of liver cells

• At Birth in first 48hours: Ammonia levels rise quickly

  • Decreased feeding w/ vomiting

  • Lethargy

  • Tachypnea (rapid breathing)

  • Seizure activity

• Followed by Rapid

  • Encephalopathy/Coma

  • Respiratory Failure

  • Cerebral Edema and Death

Late-Onset UCD Presentations

• Variable age of onset and severity of Chronic and/or recurrence/Fluctuating symptoms:

  • Headache, Vomiting Ataxia and incoordination

  • Psychiatric/Behavioral disturbance: Delirium, ASD, ADD/ADHD, Manic episodes

  • Cognitive impairment: DD/MR, executive processing defects, early dementia

• Often exacerbated/precipitated by:

  • Fever, Illness, fasting, post-partum, protein load (self restrict protein)

• STILL AT RISK FORM HYPERAMMONEMC ENCEPHALOPATHY:

  • even if previously asymptomatic, can still be fatal

Mutiple Etiologies for Hyperammonemia

Things besides UCDs (MOST COMMON CAUSE) that can also cause the accumulation of excess ammonia:

• Generalized Liver Deiasie (Acute or Chronic)

  • Non-genetic Causes: infections, Toxins, Trauma, Ischemia etc.

  • Genetic Causes

    • Non-IEM: Alpha-1 Antitrypsin (accumulation in liver=chrossis), Alagille syndrome etc

    • Non-UCD IEM

      • Aminoacidopathies: Tyrosinemia Type I

      • Organic Acidemia: elevated Lactic acid which inhibit NAGS

      • Primary Mitochondrial disorders

      • Fatty Acid oxidation Defects: Reye-Like syndrome

      • Carbohydrate Metabolic defects: Galactosemia, Fructosemia

      • Metal Processing defects WILSONS DIEASE, hemochromatosis

  • Primary UCDs

OTC Deficeiny Metabolism

X-Linked Recessive Form

Within the Mitochondria of Liver Cell:

• Carbamyl Phosphate + Ornithine → Citrulline via Ornithine transcarbamylase (OTC) activity: a Block at OTC = decreased Citrulline, increased Arginine and Aringnosuccinate (along the cycle)

  • Ammonia + Orotic Acid also increased (run-off product of carbamyl phosphate NOT becoming citrulline, not usually accumlated)

OTC Deficiency

• Orthine Trancarbamylase (OTC) Deficiency:

  • Most Common UCD

  • X-Linked Recessive Inheritance

  • Incidence 1:56,000 (not increased in any specific group)

Genetic Defect: Orthine Transcarbamylase (OTC) Gene mutation

• High Orotic Acid + Ammonia : Low Cirtrulline+ASS+ARGE

• Female carriers most common, with symptomatic male probands

• Non-carrier females: 3-4% germline mosaicism rate (apprecaible)

Untreated OTC Deficiency

Classic (usually males, only very rarely females)

• At Birth to 24/48 Hours: Asymptomatic

• 2-3 Days old: Progressive hyperammonemia Encephalopathy

  • Poor feeding

  • Vomiting

  • Lethargy

  • Hyperventilation

  • Seizures

• By 1 week Old: Lethal hyperammonemia Encephalopathy

  • Cerebral edema

  • Hypothermia

  • Coma

  • Respiratory failure

  • Death

MILD (Mild mutation males/symptomatic Females - 15%)

• Episodic hyperammonemia Symptoms: ranging form mild to life-threatening

• Chronic symptoms

  • Protein avoidance

  • recurrent headache

  • neuropsychiatric difficulty

Diagnosing OTC Deficiency

Newborn Screening: not done in all states (like NY) but ME, MAN,RI,VT

• LOW CITRULLINE

Diagnostic Evaluation

• Ammonia Level: symptomatic >100, Encephalitic >200

• Blood gas/Metabolic profile/lactic acid levels: exclude other IEMs

• Plasma Amino Acid Profile + Urine Organic Acid Profile

  • High glutamine/alanine + citrulline/ASA/ARG low in blood

  • High Orotic acid in urine

  • ****Allopurinol given to female carriers can expose OTC by preserving Orotic Acid and show Orotic Acidemia****

Confirmatory Testing

• Enzyme activity analysis: needs liver biopsy and not 100% in females due to x-inactivation in the liver

• OTC genotyping via sequencing +del/dup analysis: 60-90% detection

Treating OTC Deficiency

Acute Encephalopathy treatment

• Radpidly remove ammonia: hemodialysis/hemofiltration

• Reduce production: protein cessation 12-24hours, decrease nitrogenous waste

• Prevent Catabolism: high calorie, protein free - IV glucose + lipids and IVE arginine (become essential AA b/c it cannot be synthesized ‘like normal’)

• Provide other ammonia removal agents: IV ammonia scavengers Sodium-Benzoate + Sodium-Phenylbutyrate

Chronic Treatment: after acute encephalopathy has resolved

• Protein restriction + essential AA formula

• Oral cituralline or arginine and ammonia scavenrs (Sodium-Benzoate + Sodium-Phenylbutyrate provide alternative pathways for ammonia excretion)

• Avoid Valproic Acid, fasting, Fever, steroid, protein load

• Liver transplant: only if neurologically intact after EPISODE + poor response to chronic treatment

OTC Deficiency Outcome

Outcome with Treatment: Variable

• Neurocongtive delvepmn depens on intiala hyperammoneicm encephalyopth udration and control of subsquent amonia/glutamate leves

• Ofente ID, ADHA, executive defcits, brain attrophy (even in midl males and symptomatic females)

OTC Carrier Screening + Prenatal Diag

Carrier Screening:

• Molecuelar if mutation known in proband

• Allopurinol Loading test show Orotic Acid elevations

Prenatal implantation: No enzyme/metabolite testing is useful, activity of OTC contained to liver cells

Fatty Acids

• Under fasting conditions, fatty-acids are the stores used once 8-hour of carb derived glycogen is used up

  • Lipolysis: fatty acids get released into blood stream to be used by the body and converted into energy as Actyl-CoA and gluconeogenesis

  • Fatty acids can also produce ketone bodies via Bet-Oxidation to serve as energy for the BRAIN

• Fatty acids:

  • 3 acyl groups with attached glycerol group + carbon chain

  • Many fatty acids based upon the length of their carbon chain + the saturated double bonds

  • Most dietary fat stored as Palmaric acid (C16) or Stearic acid (C18)

Fatty-Acid Metabolism Pathways: C14-C18 transport step

Fatty acids needs to be converted to Acetyl-CoA

• C14-C18 Fatty acids → transport across plasma membrane via Long chain F.A transporter

• Converted to Actyl-CoA (But cannot enter inner mitochondrial membrane)

• Acyl-CoA + Carnitine → Acylcarnitine (of varying lengths) via CPT1 activity (TRANSPORT PREP STEP)

• Acyl Carnatine moved into inner membrane in exchange free cranatine out via CACT activity (TRANSPORT STEP)

• Acylycratine → Actyl CoA + Carnitine via CPTII activity (LIBERATION STEP)

Acyl-CoA can now enter into BETA OXIDATION

Fatty-Acid Metabolism Pathways: C14-C20 B-Ox

• Special Cleavage Steps: Only done for fatty acidsC14 or longer

• Acyl 2 carbon is cleaved from it via the Mitochondrial Trifunctional Protein

• the Acyl-2C’s liberated can then undergo Beta-Oxiadtion

  • Each turn liberated a 2C Acytl CoA group

Fatty-Acid Metabolism Pathways: C4-C12 B-Ox

• Smaller fatty acids can diffuse freely across all membrane of the cell + mitochondria: no need for the transporter or Beta-oxidation steps

  • Each turn liberated a 2C Acytl CoA group

Fatty Acid Oxidation Defects: Pathophysiology

• Defective utilization of Fatty Acids (Acyl groups) for energy

  • Rapid glycogen depletion→ hypoglycemia in fasting sate (can’t switch over to FA B-Ox to maintain blood glucose levels)

  • Deficiency of energy substrate for muscles and brain

    • Muscles: F.A → Acetyl-CoA for energy

    • Brain: F.A → Acetyl-CoA → Ketone bodies (ketogenesis step)

  • Accumulation of unmetabolized F.A in liver and muscle

    • Liver disease

    • Myopathy

    • Cardiomyopathy

Fatty Acid Oxidation Defects: Major Clinical phenotypes

Hypoketotic + hypoglycemic

• Glucose levels drop quickly, Ketone bodies made at low levels

  • Cognitive and developmental insults to the brain

• Body TRIES to breakdown F.A., but can’t convert smaller chunks to the 2-Acetyl-CoA via B Ox

  • F.A.s liberated into blood stream = buildup within muscle and liver → dysfunction

  Myopathy + Cardiomyopathy

  Hepatic failure/Liver dysfunction (Reye Syndrome)

• Maternal liver disease → affected fetus can create issue in the mother w F.A build up

Fatty Acid Oxidation Defects: Etiologies

• Disease of the Carnitine pathway:

  • primary: defects in the protein pathways

  • secondary: nutrition deficiencies

• Disease of Fatty Acid B-Oxidization

L-Carnitine Amino Acid

• Binds to Acyl (organic +inorganic) residues (F.A) in the blood and enables transport into cells + their elimination → (why we supplement with Carnitine in Organic Acidemias: for their elimination w carnitine)

  • Long chain (>C12) needs it to get their long “acyl” chains into the inner mitochondria membranes where “Digestion” step + B-Ox can occur

Sources: we can make a-little, but not enough on its own

• Diet: Milk + meat

• Reabsorption: Kidneys via Caratine trnasporter protien

Carnitine Testing

• Plasma Carnatine levles: Free, unbound and Acyl-Cartines (boudn to Fas and organic acids)

• Plasma Acyl Carnatine prolei: quantity the diffrent types of bound-caratine resdiues

Fatty Acid Oxidation Defects: Key Concepts

• All Autosomal Recessive

• Spectrum of Severity

• Phenotypes vary but always have

  • Hypoketotic + Hypoglycemia

  • Myopathy and/or Cardiomyopathy

  • Liver failure (Reye Syndrome)

    • SCIDs can ocure fruently: overnight fast casues sudden hypoglycemi even that kills them

Fatty Acid Oxidation Defects: Therapy

Acute Illness/Fastine

• IV Dextrose (immediate)

• Carnitine (some FOADs): overdrive membrane import of FA and remove excess FAs

• Monitor for

  • hypoglycemia

  • liver failure

  • Muscle breakdown

Chronic

• Avoid prolonged fasting

  • frequent feedings: CORNSTARCH (McArdles?)

• Supplment with Medium-Chain-Triglycrides (“MCT”) oil <C10 for some (not MCAD/SCAD) → doesn’t need transporter or Digestion steps before B-Ox

• Supplment with L-Cartine for some (not LCHAD)

• Avoid liver toxic or carantine lowerin medication (valproic acid, salicylates, some anethetics)

Overview of FAOD Clinical Issues

OCTN2 Deficiencey: Metabolism

• OCTN1 and OCTN2 transport Carnitine across plasma membrane

  • OCTN1: Primarily in liver

  • OCTN2: Primarily in Muscle + Kidney

• No OCTN2 = No carnitine = Long F.A.s don’t undergo B-Oxidation in the Muscle

  • Also: Kidney→ not enough Carnitine is re-absorbed and therefore too much excreted = very low levels of carnitine

OCTN2 Deficiency

• Also called Primary Carnitine Uptake Defiencey

Etiology

• SLC22A5 gene mutation→ reduced Carn. uptake by Kidney +muscle→ low blood and muscle Carn+ Acyl-Carn

Can also just be caused by LOW-Diet CARTNATINE

OCTN2 Deficiency: presentation

Forms: Severe Infantile, Mild Child/late Onset

• Infancy:

  • Hypoketotic hypoglycemia

  • Liver failure

• Child:

  • Myopathy + Cardio myopathy

  • Liver affects are less: when Carn is severe enough, B-Ox affected through whole body → but Child type, Carn is enough to avoid defieciny inthe liver but not the rest of the body

• Late

  • mild myopathy or asymptomatic

OCTN2 Deficiency: Diagnosis

NBS: Low C0 and Acyl-Carnitine levels (Low level of ALL the species) → NEED TO TEST MATERNAL LEVELS AS WELL

Confirmation

• Carnitine and Acyl-Carnitine levels

• Enzyme activity: Fibroblasts

• Molecular: finds 70%

OCTN2 Deficiency: Treatment and outcome

Treatment

• high-dose Carn

• Avoid fasting

• IV Dextrose

Outcome

• Good if treated before severe decompensation

Very Long-Chain Acyl-CoA Dehydrogenase (VLCAD) Deficeny

• VLCAD Drives the initial steps of fatty acid beta oxidation for the long chain fatty acids (>C14)

VLCAD Deficiency: Pathophysiology

• ACADVL mutation → Deficient C14-20 B-oxidation

• Generally more mild, later onset, exercise

VLCAD Def: Presentation

Infant:

• Hypoketoitic hypoglycemia

• Liver failure

• myopathy

• cardiomyopathy

Child: Cardio myopathy

Late: *******EXCERCISE MYOPATHY: WAY MORE COMMON FORM *****

VLCAD Def: Diagnosis

NBS: Elevated C14:1 ratio and longer chain Acyl-carnitine

Confirmation:

• Carnatine and AC

• Enzyme activity: Skin, white blood cell, aminotic fluid

• Moelcualr: 85-93%

VLCAD Def: Treatment + Outcome

Treatment:

• Avoid fasting with frequent feeding and IV dextrose when ill

• Cornstarch

• MCT Oil

• +/- Carnitine

• High-carb/low-fat diet

Outcome

• good if treated before severe decompensation

Medium-Chain Acyl-CoA Dehydrogenase (MCAD) Deficiency

• Most common FAOD and the first to be added to NBS

• the meatbolism of C8-C10 length acyl-CoA Fatty Acid groups

  • These can move freely acros the inner mitocondrial memebrane WITH OUT then eed for Carnatine complex (so no Acylcarantine needed

• Once inside,C8-C10 Acyl-CoAs → *****BETA OXIDATION via MCAD****→ C4-C6 AcylCoAs

MCAD Deficiency = C8-C10 Buildup in blood and tissue + C8 Octanoyl Acycl Carainatase (Toxic)

MCAD Def: Pathophysiolo

ACADM gene mutation → lowered C6-C10 Fatty acid B-Oxidation = build up of C8-C10 in tissues (C8 more prevlant vs C6 or C10)

• Also alternative FA metablism pathway (Omega Oxidaiton) creates new species (dicarboxylic acid)

MCAD Def: Presentation:

Typical presentation at 3-24months old with Fasting/Illness

• Hypoketotic hypoglycemic

• Liver disease

• Often Sudden Infant Death syndrome→ usually the first personation you will see (sleeping longer through the night, greater chance for fasting affects to kick in)

MCAD Def: Diagnosis

NBS: Elevated C8, lesser C6 and C10 : (C8 >C6/C10)

• C8 (Octanoylcarnatine)

• C6 (Hexanoylglycine)

• Dicarboxylic Acid (Alternative FA Metabolsim Product)

Confirmation

• Carnitine

• AC

• uAG

• Enzyme activity: Skin, WBC, amino/CVS

• Molecualr: 90%

MCAD Def: Treatment + Outcome

Treatment

• Fasting with Frequent feeding and IV dextrose when ill

• Cornstarch

• +/- Carnitine

• High carb/low-fat diet

• ****NO MCT OIL!!!!****

Outcome

• Good if treated before severe decompensation

• Maternal liver disease reported (AFLP/HELLP)