in the name of godgdrc-cong.tums.ac.ir/images/slide/1-sarkheil.pdf · transamination scatabolism of...

In The Name of GOD

S

Homocystinuria &

Remthylation defects

Peymaneh Sarkhail M.D.

Pediatric Endocrinologist &

Metabolic Consultant

Amino Acid Metabolism Defects

Agenda

SAmino acid structure

SAmino acid metabolism

SAmino acid disorders

SHomocystinuria

SRemethylation defects

Amino acid structure

Amino acids

Essential Phenylalanine

Threonine

Methionine

Lysine

Tryptophan

Leucine

Isoleucine

Valine

Histidine

Non-essential Tyrosine

Aspartate

Asparagine

Alanine

Serine

Glycine

Cysteine

Glutamine

Glutamate

Proline

Arginine

Amino acid metabolism

S The small intestines, liver, kidneys, and muscle are organs that play an essential

role in amino acid metabolism. The main role of each is given as follows:

S Intestine:

S Amino acids from protein digestion are absorbed in the small intestine.

S Intestine preferably uses glutamine and asparagine as energy suppliers.

S Releases CO2, ammonium, alanine, citrulline as end products.

S The products formed, together with the remaining amino acids in the diet, are

sent to the liver via the portal vein.

S Liver:

S The liver is the major site of nitrogen metabolism in the body. The catabolism of

amino acids, except those with branched chains, starts in the liver.

S The amino group must be removed, as there are no nitrogenous compounds in

energy transduction pathways.

S In times of dietary surplus, the potentially toxic nitrogen of amino acids is

eliminated via transaminations, deamination, and urea formation.

S The α-ketoacids that result from the deamination of amino acids are metabolized so

that the carbon skeletons goes to the gluconeogenic or ketogenic pathways.


Many amino acids are purely glucogenic:

Glutamate, aspartate, alanine, glutamine,

asparagine,…

Some amino acids are both gluco- and ketogenic:

Threonine, isoleucine, phenylalanine,

tyrosine, tryptophan

Amino Acids are categorized as ‘Glucogenic’

or ‘ketogenic’ or both.

The only PURELY ketogenic Amino Acids:

leucine, lysine


S Glucogenic amino acids are those that give rise to a net production of pyruvate orTCA cycle intermediates, such as 2-α-ketoglutarate or oxaloacetate, all of which areprecursors to glucose via gluconeogenesis.

S A small group of amino acids comprised of Isoleucine, Phenylalanine, Threonine,Tryptophan, and Tyrosine give rise to both glucose and fatty acid precursors and arethus, characterized as being glucogenic and ketogenic

S Lysine and leucine are the only amino acids that are solely ketogenic, giving rise onlyto acetyl-CoA or acetoacetyl-CoA, neither of which can bring about net glucoseproduction

S Finally, it should be recognized that amino acids have a third possible fate.

S During times of starvation the reduced carbon skeleton is used for energy production,with the result that it is oxidized to CO2 and H2O.


Transamination

S Catabolism of Amino acids occurs in 4 steps; transamination, deamination,ammonia transport , and urea formation

S Transamination interconverts pairs of α-amino acids and α -keto acids.

S It take place in cytoplasm and then followed by oxidative deamination inmitochondria.

S The general process of transamination is reversible and is catalyzed byTransaminases, also called amino transferases (ALT, AST) that require B6-Phosphate as coenzyme.

S Most of the amino acids act as substrate for the transaminases but the amino acidslike Lysine, Threonine, Proline, and Hydroxy proline do not participate intransamination reactions.

O CCH C

H

NH3

2 2 2

aspartate

(-)CO

t ransaminat ionO CCH C

O

2 2 2

(-)CO

oxaloacetate

(-) (-)

(+)

O C CH C

H

NH3

2 2 2

glutamate

(-)

t ransaminat ionCH C

O2 2

(-)

-ketoglutarate

CH2

O C2CH

2CO CO(-) (-)

(+)

CH C

H

NH3

3 2

alanine

(-)CO

t ransaminat ionCH C

O

3 2(-)

CO

pyruvate(+)

Transamination

B6

B6

B6

Deamination

S Deamination of free amino acids leads to the production

of ammonia for Urea synthesis and a-keto acids

S A) Oxidative deamination

S Amino acid oxidases of liver and kidney convert amino

acids to an α -imino acid that decomposes to an α -keto

acid with release of ammonium ion.

S Mostly Glutamate which is a collection center of Amino

group goes for oxidative deamination by Glutamate

Dehydrogenase (GDH) which need NAD+ or NADP+

S This conversion liberate Ammonia and produce α –imino

glutarate then α-keto glutarate.

S B) Non oxidative deamination

S Direct deamination without oxidation is carried

out by AA dehydratases.

S Serine, threonine, cysteine/cystine and Histidine

undergo non oxidative deamination to form

corresponding Alpha keto acids

S Deamination of Serine and Threonine is carried

out by PLP-dependent Dehydratase.

Deamination

S Muscle:

S The degradation of branched chain amino acids

mainly starts in skeletal muscle.

S The amine groups are transferred to pyruvate and

ketoglutarate to form alanine and Glutamine

S Alanine is used in the liver for gluconeogenesis.

S Glutamine is used in kidney for ammuniogenesis

and buffering system.


S Kidney:

S This organ captures glutamine released from

muscles.

S Reactions catalyzed by glutaminase and

glutamate dehydrogenase produce ammonia,

which is converted to ammonium ion and

excreted in urine, neutralizing anions.

S The ammoniagenesis is one of the mechanisms

used by the kidneys to maintain the body’s acid–

base balance


Amino acid metabolism disorders

S Inherited metabolic disorders (IMD) prevalence is 1 in 500-1500.

S More than 1000 IEMs have been reported , however, fortunately almost 100 such disordersare potentially treatable, if diagnosed at an earlier stage of life.

S Out of these conditions, 13 disorders are caused by the amino acid disturbance whichcombined incidence is 1:6000

S The inborn errors of amino acid metabolism are a family of genetic conditions in which anenzyme deficiency typically results in the accumulation of a ninhydrin-positive amino acid.

S They are conceptually identical to disorders caused by enzyme defects that result in theaccumulation of the organic acid intermediates.

S Recently some amino acid disorders due to synthesis defect has been recognizedBiochem Genet 2017

DOI 10.1007/s10528-017-9825-6

Amino acid synthesis deficiencies

S Serine deficiency

S Asparagine deficiency

S Glutamine deficiency

S Disorders of proline synthesis :

S Pyroline-5-carboxylate synthase (PSCS) deficiency

S Pyroline-5-carboxylate reductase 1 (PYCR1) deficiency

S Pyroline-5-carboxylate reductase 2 (PYCR2) deficiency

J Inherit Metab Dis (2017) 40:609–620

Primary amino acid disorders

S PKU

S Tyrosinaemia (I/II/III)

S Maple Syrup Urine Disease

S Homocystinuria

S Non-Ketotic Hyperglycinaemia

S Hyperprolinaemia (I/II)

S Sulphite oxidase deficiency

S Cystinuria

S Hartnup disease

S Lysinuric protein intolerance

S Iminoglycinuria

S Urea Cycle Disorders

OTC deficiency

CPS deficiency

Citrullinaemia

Argininosuccinic aciduria

Argininaemia

NAGS deficiency

S HHH

S OAT deficiency

http://upload.wikimedia.org/wikipedia/commons

http://farm6.static.flickr.com/5006/5253309531_02f0bea83f.jpg

Vincent du Vigneaud

1932 synthesis of Hcy

1955 Nobel prize-oxytocin

Homocystinuria

http://upload.wikimedia.org/wikipedia/commons/c/c5/L-methionine-B-3D-balls.png

http://upload.wikimedia.org/wikipedia/commons/c/c5/L-methionine-B-3D-balls.png

S Cystathionine beta-synthase (CBS) deficiency is a rare inherited disorder, alsoknown as classical homocystinuria (OMIM 236200).

S Homocysteine (Hcy) is a non-essential AA that is formed in the catabolic pathwayfor the essential AA, methionine (Met).

S Other causes of hyperhomocysteinemia include:S Inborn errors of Hcy remethylation (Non-classic Homocystinuria)S Vitamin deficiencies (especially B12), Folate deficiencyS Renal insufficiencyS Medication ( OCP, methotrexate)S HypothyroidismS MalignanciesS Smoking


Homocystinuria

Met

S-adenosyl

methionine

S-adenosyl

homocysteine

Glycine

Sarcosine

Homocysteine

FA

5-Methyl-THF

5,10-MethyleneTHF

Met

Synth

Adenosine

Cystathionine

Cys

Sulphate

Serine

CBS

CTH

Betaine

DiMeGly

MTHFR

SAHH

GNMT

MAT I/III

BMT MeCbl

Vitamin B12

CBS deficiency

B6

Prevalence

S The true frequency is unknown with estimates ranging from 1:1800 to 1:900,000

based on birth incidence of patients detected by newborn screening and/or

estimates from clinically ascertained patients

S The highest incidence has been reported in Qatar (1:1800), where there is a high

rate of consanguinity and a founder effect, with a carrier frequency of

approximately 2 %

S The prevalence may have been underestimated, however, the data also suggest

that some homozygotes for mutation c.833 T > C may be asymptomatic.

S The rough estimate is 1/50,000 to 1/200,000.


Clinical presentations

S There is a wide spectrum of severity, from individuals who are currently asymptomatic to

those with severe multi-system disease, with a wide range of ages at presentation.

S The phenotype broadly relates to pyridoxine-responsiveness. Pyridoxine-responsive patients

generally have a milder phenotype and a later onset than the pyridoxine unresponsive

S Four main organs/systems can be involved:

S 1)Eye: Ectopia lentis and/or severe myopia

S 2)Skeleton: Excessive height and length of the limbs ( ‘marfanoid’ habitus), osteoporosis and

bone deformities, such as pectus excavatum or carinatum, genu valgum and scoliosis

S 3) CNS: Developmental delay/intellectual disability, seizures, psychiatric and behavioural

problems and extrapyramidal signs

S 4)Vascular system: ThromboembolismJ Inherit Metab Dis (2017) 40:49–74

Diagnosis

S Biochemical diagnosis

S Plasma total homocysteine (tHcy) should be the test for diagnosis of CBS deficiency.

S In untreated patients with CBS deficiency, tHcy concentrations are usually above 100μmol/L but they may be lower (mostly > 100 μmol/L).

S The diagnosis is very likely if elevated tHcy is accompanied by high plasma Met and furthersupported if plasma cystathionine concentrations is low with an increased Met to-cystathionine ratio

S Plasma free homocystine (fHcy) only becomes detectable at tHcy concentrations aboveapproximately 50-60 μmol/L

S Its measurement is not recommended because of its low sensitivity and reproducibility, andthe demanding pre-analytical requirements.


Diagnosis

S Pre-analytical requirements for biochemical testing

S The diagnosis can be masked in patients with mild disease who are taking pyridoxine or

pyridoxine-fortified multivitamins and foods prior to testing.

S In pyridoxine-responsive patients with some specific mutations (e.g. p.P49L), physiological

doses of pyridoxine as low as 2 mg per day may decrease the tHcy concentrations into the

reference range.

S It is important to avoid intake of any pyridoxine supplements for at least 2 weeks before

sampling plasma for tHcy measurement, although occasionally a wash-out period of up to 1-

2 months may be needed

S Differences in plasma tHcy concentrations due to diurnal variation, fed state, pregnancy or

posture are relatively minorJ Inherit Metab Dis (2017) 40:49–74

Diagnosis

S Pre-analytical requirements for biochemical testing

S Plasma should ideally be separated from whole blood within one hour and then can bestored at 4 °C for up to 1-2 weeks prior to analysis.

S The sample should be centrifuged within 1 hour if stored at room temperature since redblood cells generate Hcy at a rate of about 1-2 μmol/L/hr in unseparated whole blood orwithin 8 hours if blood with anticoagulants is stored at 4 °C; alternatively, serum may beused.

S After centrifugation the tHcy in plasma or serum is stable for at least 4 days at roomtemperature, for several weeks at 4 °C and several years at -20 °C

S In DBS, which are 30-40 % lower compared to plasma.


Confirmatory testing

S CBS deficiency should be confirmed by measurement of cystathionine synthase activity infibroblasts or plasma and/or by mutation analysis of the CBS gene.

S The confirming CBS deficiency is generally considered to be the determination ofcystathionine production from Hcy and serine in cultured fibroblasts using radioactive ordeuterium labelled substrates.

S The fibroblast CBS activity may, however, be normal in mild forms of the disease, despitebiochemical and clinical abnormalities and mutations in the CBS gene.

S Sequencing of the CBS gene is considered the gold standard in molecular diagnostics; Over160 disease causing genetic variants in the CBS gene are known

S however, pathogenic variants may not be detected in one of the parental alleles in up to 7-10 %of CBS deficient patients.

S In summary, if one of these techniques (enzyme or DNA analysis) does not confirm adiagnosis of CBS deficiency the other test should be done in a patient with metaboliteabnormalities suggestive of this disease.


Treatment

S Treatment aims to lower the plasma tHcy concentration to a safe level whilstmaintaining normal nutrition, including normal concentrations of methionineand other essential AA.

S In pyridoxine-responsive patients the target for plasma tHcy should be <50μmol/L and in un-responsive patients tHcy levels below 100 μmol/L isappropriate.

S In pyridoxine unresponsive patients, the good outcomes can be achieved if plasmafHcy levels are maintained below 11 μmol/L. This corresponds to a tHcyconcentration of about 120 μmol/L.

S In general, the aim is to keep the Hcy concentration as close to normal aspossible.


Pyridoxine-responsive homocystinuria

S To assess pyridoxine responsiveness after infancy, giving 10 mg/kg/day pyridoxine up to a maximumof 500 mg/day for 6 weeks; the plasma tHcy concentration should be measured at least twice beforetreatment and twice on treatment.

S The test should not be done if the patient is catabolic. The protein intake should be normal, folatesupplements should be given and vitamin B12 deficiency should be corrected prior to testing.

S Patients who achieve plasma tHcy levels below 50 μmol/l on pyridoxine are clearly responsive anddo not need any other treatment.

S If the tHcy falls >20% but remains above 50 μmol/L, additional treatment should be considered (i.e.diet and/or betaine).

S If tHcy falls by <20% on pyridoxine, the patient is likely to be unresponsive.

S Patients detected by newborn screening rarely respond to pyridoxine and, in this group,

S To avoid delaying effective treatment, we recommend giving neonates a relatively high pyridoxinedose (e.g. 100 mg/day) for at least 2 weeks, with measurement of the plasma tHcy at the end of thefirst and second weeks. J Inherit Metab Dis (2017) 40:49–74


Pyridoxine-responsive homocystinuria

Treatment

S All patients should receive adequate folate supplementation. Vitamin B12 should besupplemented if deficient.

S There are several reports of vitamin B12 and folate deficiencies in patients with CBSdeficiency

S This may be due to increased flux through the remethylation pathway and use of thecofactors, or inadequate intake of the vitamins in patients on restricted diets

S All patients need low-dose folate supplements (1 mg/d orally) and vitamin B12 (1mg 1–3 timesa month).

S Administration of vitamin C is reported to improve endothelial function in CBS-deficientpatients.

S The use of antithrombotic agents, for example, aspirin and/or dipyridamole, has no provenlong-term benefit and should be considered on an individual basis


Dietary management

S Approaches to dietary treatment

S Dietary treatment should be considered for all patients with CBS deficiency unlesstarget Hcy levels are achieved entirely by pyridoxine supplementation.

S Most pyridoxine-unresponsive patients require a diet that is very low in naturalprotein, with supplements of a Met-free L-AA mixture.

S The approach is analogous to the management of phenylketonuria (PKU).

S The majority of patients on dietary treatment require a low Met diet with a cystine-enriched, Met-free L-AA supplement

S UK guidelines recommend a starting allowance of 90-120 mg Met/day (or 30mg/kg/day if weight <3 kg). The Met allowance is then titrated against the patient’splasma tHcy levels.


Betaine treatment

S Betaine (trimethylglycine) is formed in the body from choline and small amounts are presentin the normal diet .

S Betaine should be considered as adjunctive treatment in patients who cannot achieve targetlevels of Hcy by other means.

S It lowers Hcy concentrations in CBS deficiency by donating a methyl group and convertingHcy to Met. Betaine can increase cysteine levels

S In children, the initial dose is 100 mg/kg/day, divided into twice daily doses, and thenadjusted according to response (typically increased weekly by 50 mg/kg increments up to250 mg/kg).

S Betaine has a half-life of 14 hours so twice daily dosing is adequate

S The maximum licensed dose is 3 grams twice daily and this is the usual dose in adults buthigher doses have sometimes been used with anecdotal evidence of biochemical benefit.


S Side effects of betaine

S Higher doses have been associated with a fishy odor. This is probably due to

inadequate activity of flavin containing monooxygenase 3 and may respond to

riboflavin.

S Betaine is generally safe but some people dislike the taste and compliance may be

poor .

S Cerebral edema has also been seen in a few patients with high levels of

Met(>1000 μmol/L).


Betaine treatment

Remethylation defects

cblJ

The combined remethylation defects

S The cblC, cblF, cblJ, and cblD (MMA-Hcy) and cblX defects

S Mutations in the LMBRD1 gene cause the cblF defect and ABCD4 mutations the cblJ defect.

S The defect in both disorders appears to be due to trapping of endocytosed Cbl in thelysosomes following degradation of TC. Cbl accumulates in the lysosomes and is notavailable for conversion to either AdoCbl or MeCbl

S The cblC defect, caused by mutations in the MMACHC gene is the most common of thesedefects.

S The product of the MMACHC gene appears to act as a molecular chaperone and to play arole in processing of Cbl after it has left the lysosome.

S Mutations in the HCFC1 gene on chromosome Xq28 causes cblX deficiency . The product ofthe HCFC1 gene is a transcriptional regulator that can interact with other proteins tostimulate expression of specific genes, including MMACHC J Inherit Metab Dis (2015) 38:1007–1019

S Depending on type and location of the mutation in the MMADHC gene, the cblD defect eitherpresents as isolated methylmalonic acid (MMA)-uria (cblD-MMA), combined MMA andhomocystinuria (cblD-MMA-Hcy) or isolated homocystinuria (cblD-Hcy)

S The function of the MMADHC protein remains unknown, although it is known to bind toMMACHC.

S The variability in the cblD phenotype suggests that MMADHC plays a role in directinginternalized Cbl to the mitochondria and methylmalonyl- CoA mutase, or to the cytoplasm andmethionine synthase.

S All defects affect the synthesis of methylcobalamin, the co-factor for the cytosolic enzymemethionine synthase, and of adenosylcobalamin, the coenzyme for the intra-mitochondrialenzyme methylmalonyl-CoA mutase, which metabolizes MMA



S Main clinical symptoms of the early-onset form (<12 months, 88 % of cases)

include:

S FTT, hypotonia, developmental delay, dysmorphic features, thinned corpus

callosum,, pulmonary hypertension, HUS, macrocytic anemia and decreased visual

acuity due to pigmantary retinopathy but no lens dislocation.

S Late-onset patients may present any time during childhood or adult life, typically

with myelopathy, dementia, psychiatric symptoms, HUS, thromboembolism, and

pulmonary hypertension; retinopathy occurs rarely

S In early-onset forms, overall survival, hematological symptoms, and failure to thrive

improve while eye disease and cognitive impairment often progress under treatment



Treatment

S Treatment of the cblC defect — which has also been applied in cblF, cblJ, and cblD-

Hcy-MMA defects—consists of B12 (OH-Cbl), oral betaine and folate, rarely

combined with Met supplementation.

S Many centres use 1 mg of parenteral OHCbl daily in neonates (assuming a body

weight of 3 kg, the dosage is 0.33 mg/kg/day).

S In conclusion, OHCbl treatment is usually started at a dosage of 1 mg IM daily and

then it should be titrated individually based on metabolic response

S Levocarnitine facilitates the excretion of MMA and prevents carnitine deficiency

S Since the de novo synthesis of carnitine depends on methionine and may be decreased

in cblC patients.


S lower MMA excretion upon protein restriction (methionine-free, threonine-free, valine-

free, isoleucine-low formula or XMTV formula).

S Met is essential in patients with remethylation defects and its plasma levels should be

maintained in the normal range.

S For these reasons, methionine-free formulas and protein restriction leading to low

methionine plasma levels is contraindicated in cblC patients


Treatment

Isolated remethylation defects

S The cblE, cblG, and cblD-Hcy defects

S The cblG defect affects the enzyme methionine synthase (MTR) whilemethionine synthase reductase (MTRR) is defective in the cblE defect

S Initial symptoms in the first year of life or in early childhood includemegaloblastic anemia, failure to thrive and feeding difficulties, cognitivedysfunction, nystagmus, impaired visual acuity but no lens dislocation, andseizures are frequent.

S Microangiopathy and hemolytic uremic syndrome have only very rarely beendescribed

S Treatment encompasses folic or folinic acid, betaine and a variety of cobalamin(OH-Cbl or methylcobalamin) preparations.


S MTHFR deficiencyS The enzyme MTHFR catalyzes the reduction of 5,10 methylene- tetrahydrofolate to 5-

methyltetrahydrofolate, which serves as a methyl donor for the methylation of Hcy to Met

S In severe MTHFR deficiency, developmental delay, seizures, and movement disorders are frequent inchildren.

S Adolescents and adults may present with psychiatric symptoms, neuropathy or thromboembolicevents. Retinopathy has only rarely been reported.

S Individuals with MTHFR deficiency do not usually present with haematological abnormalities

S Treatment with folate, folinic acid, Met, pyridoxine, has generally been considered unsuccessful .

S However, daily doses from 5 to 30 mg, divided in 2–3 administrations/week of folate or folinic acidhave been used. It has recently been shown that folic acid may exacerbate cerebral CH3-THFdeficiency and it has therefore been suggested to avoid folic acid and preferably use folinic acid or 5-CH3-THF

S Betain and Met supplementation improved the course in an early onset case

Isolated remethylation defects


References

S J Inherit Metab Dis (2017) 40:21–48



Thank you

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