Organic Acids The basics - · PDF fileCarboxylic organic acids:- ... Confirmatory Tests Assay of 14C-propionate incorporation into cultured fibroblasts or of propionyl CoA carboxylase

Organic Acids Organic Acids

The basics The basics

Donna Fullerton, Nottingham City Hospital

What are organic acids?What are organic acids?•• Organic compounds with acidic propertiesOrganic compounds with acidic properties

•• Most common organic acids are carboxylic acids Most common organic acids are carboxylic acids whose acidity is attributable to the carboxyl group whose acidity is attributable to the carboxyl group (COOH)(COOH)

Carboxylic organic acids:Carboxylic organic acids:--

•• Low molecular weight water soluble acidsLow molecular weight water soluble acids•• Ninhydrin negativeNinhydrin negative•• Compounds detectable by urinary organic acid analysisCompounds detectable by urinary organic acid analysis

Where do they come from?Where do they come from?

ORGANIC ACIDS

Neurotransmitters

Purines and pyrimidines

CarbohydratesAmino acids

Drugs and special diets

Cholesterol

Microorganisms

Fatty Acids

Organic acids are the end products and intermediates of a wide range of metabolic pathways

How do we measure organic acids?How do we measure organic acids?

Gas-Chromatography Mass Spectrometry (GC-MS)

Organic Acid Analysis (1)Organic Acid Analysis (1)•• ExtractionExtraction--A volume of urine (adjusted for creatinine concentration) is aciA volume of urine (adjusted for creatinine concentration) is acidified and then saturated dified and then saturated

with sodium chloride. Acidification converts polar organic acidwith sodium chloride. Acidification converts polar organic acids to nons to non--polar organic polar organic acids which have greater affinity for the ethyl acetate phase, hacids which have greater affinity for the ethyl acetate phase, hence maximising ence maximising organic extraction. The sodium chloride reduces the solubility organic extraction. The sodium chloride reduces the solubility of the organic acid of the organic acid aqueous phase (salting out), further aiding extraction.aqueous phase (salting out), further aiding extraction.

--The organic acids are solvent extracted using ethyl acetate and The organic acids are solvent extracted using ethyl acetate and diethyl ether.diethyl ether.

--After evaporation of the ethyl extracts the organic acids are deAfter evaporation of the ethyl extracts the organic acids are derivatised.rivatised.

--The organic acids are derivatised to form trimethylsilyl (TMS) eThe organic acids are derivatised to form trimethylsilyl (TMS) esters using BSTFA (sters using BSTFA (N,ON,O--Bis(trimethylsilyl)trifluoroacetamide)Bis(trimethylsilyl)trifluoroacetamide) and pyridine. Derivatisation converts the and pyridine. Derivatisation converts the unstable, low volatile organic acids into stable, volatile TMS dunstable, low volatile organic acids into stable, volatile TMS derivatives which are erivatives which are suitable for separation by gas chromatography. suitable for separation by gas chromatography.

OrganicOrganic Acid Analysis (2)Acid Analysis (2)

•• Gas ChromatographyGas Chromatography--Mass Spectrometry (GCMass Spectrometry (GC--MS)MS)--The sample is pumped through a capillary across a high voltage aThe sample is pumped through a capillary across a high voltage and at a high nd at a high

temperature. This causes the liquid to evaporate into smaller dtemperature. This causes the liquid to evaporate into smaller droplets, until the roplets, until the charge becomes so great the electrons repel one another and undecharge becomes so great the electrons repel one another and undergo columbic rgo columbic explosion forming gaseous ions.explosion forming gaseous ions.

--The gaseous ions interact with a column and are separated accordThe gaseous ions interact with a column and are separated according to size. The ing to size. The smaller, volatile ions interact less with the column and are elusmaller, volatile ions interact less with the column and are eluted first.ted first.

--The gaseous ions enter the quadrupole mass filter of the mass spThe gaseous ions enter the quadrupole mass filter of the mass spectrometer. The ectrometer. The quadrupole is formed of 4 parallel rods in a strong vacuum to whquadrupole is formed of 4 parallel rods in a strong vacuum to which fixed DC and ich fixed DC and alternating RF voltages are applied. alternating RF voltages are applied.

--Under specific conditions ions of a specific massUnder specific conditions ions of a specific mass--toto--charge (charge (m/zm/z) ratio will oscillate ) ratio will oscillate through the quadrupole to the detector. The resultant chromatogthrough the quadrupole to the detector. The resultant chromatogram is compared to ram is compared to a library of known compoundsa library of known compounds

GCGC--MSMSOrganic acid extracts

Quadrupole of a Mass-Spectrometer

Organic Acid DisordersA block in the breakdown of an organic acid can lead to its accA block in the breakdown of an organic acid can lead to its accumulation umulation in the cell and its elevation in plasma and urine. The first trin the cell and its elevation in plasma and urine. The first transamination ansamination step and second dehydrogenation step of amino acid catabolism step and second dehydrogenation step of amino acid catabolism generate organic acids.generate organic acids.Often this leads to a metabolic acidosis but this is Often this leads to a metabolic acidosis but this is notnot always the case. It always the case. It may also be associated with and combination of hypoglycaemia, may also be associated with and combination of hypoglycaemia, hypocalcaemia, lactic acidaemia, hyperglycinaemia and hypocalcaemia, lactic acidaemia, hyperglycinaemia and hyperammonaemia. In some disorders one or more amino acids may hyperammonaemia. In some disorders one or more amino acids may also be elevated (eg Maple Syrup Urine Disease)also be elevated (eg Maple Syrup Urine Disease)Severe forms will present acutely in the neonatal period whereasSevere forms will present acutely in the neonatal period whereas other other forms may present chronically over a long period of time sometimforms may present chronically over a long period of time sometimes with es with acute episodes.acute episodes.There may be multisystem involvement or the brain may be the onlThere may be multisystem involvement or the brain may be the only y organ affected.organ affected.Some of the more common organic acid disorders are discussed in Some of the more common organic acid disorders are discussed in the subsequent slides.the subsequent slides.

Propionic and Methylmalonic AciduriaPropionic and Methylmalonic Aciduria

Propionyl CoA

D-Methylmalonyl CoA

L-Methylmalonyl CoA

Succinyl CoA

Propionyl CoA Carboxylase

Methylmalonyl CoA Racemase

Methylmalonyl CoA Mutase

Propionic and Methylmalonic acids are formed from the catabolism of branched chain amino acids, odd chain fatty acids and cholesterol. They are ultimately converted to succinyl CoA which feeds into the Kreb’s Cycle. Defects in any of the steps below result in their accumulation

Clinical Presentation Clinical Presentation •The more severe acute forms of Propionic aciduria (PA) and Methylmalonic aciduria (MMA) have similar clinical presentations:

•Neonatal presentation

-Ketoacidosis -Hypoglycaemia

-Vomiting -Lethargy

-Hypotonia

•Infancy

-Failure to thrive -Episodic illness associated with illness

-Reyes Syndrome like episode -Convulsions associated with hypoglycaemia

•Other presentations

-Chronic progressive neurological disease

-Ataxia, metabolic stroke, extrapyramidal signs (abnormal movements)

Common Laboratory Findings in PA/MMACommon Laboratory Findings in PA/MMA

•• HypoglycaemiaHypoglycaemia--caused by inhibition of pyruvate carboxylase and the transmitochcaused by inhibition of pyruvate carboxylase and the transmitochondrial malate ondrial malate shuffle by methylmalonyl CoAshuffle by methylmalonyl CoA

•• HyperammonaemiaHyperammonaemia--caused by inhibition of carbamoyl phosphate synthase by Propionycaused by inhibition of carbamoyl phosphate synthase by Propionyl CoAl CoA

•• HypocalcaemiaHypocalcaemia•• Elevated plasma lactate (and secondarily plasma alanine) Elevated plasma lactate (and secondarily plasma alanine)

with a high anion gap and a metabolic acidosiswith a high anion gap and a metabolic acidosis•• NeutropaeniaNeutropaenia•• Increased plasma/urine glycineIncreased plasma/urine glycine

--caused by inhibition of the glycine cleavage enzyme.caused by inhibition of the glycine cleavage enzyme.

Laboratory Tests for the Diagnosis of Propionic AciduriaLaboratory Tests for the Diagnosis of Propionic Aciduria

Propionic aciduria is caused by a defect in propionyl CoA carboxylase (PCC). Propionate metabolism is blocked in the absence of PCC activity and intermediary products accumulate. These are detected by:

Urine Organic Acid AnalysisUrine Organic Acid Analysis

--3OH3OH--propionatepropionate --MethylcitrateMethylcitrate

--PropionylglycinePropionylglycine --TiglyglycineTiglyglycine

Blood Acylcarnitine AnalysisBlood Acylcarnitine Analysis

--Increased PropionylIncreased Propionyl--carnitine (C3)carnitine (C3)

--Low free carnitine levelsLow free carnitine levels secondary to carnitine sequestrationsecondary to carnitine sequestration.

Confirmatory TestsConfirmatory TestsAssay of 14CAssay of 14C--propionate incorporation into cultured fibroblasts orpropionate incorporation into cultured fibroblasts orof propionyl CoA carboxylase in cultured fibroblasts or leucocytof propionyl CoA carboxylase in cultured fibroblasts or leucocyteses

Organic Acid Trace of Propionic AcidaemiaOrganic Acid Trace of Propionic AcidaemiaIncreased excretion of 3OH propionate, Tiglyglycine and methylcitrate

Treatment of Propionic AcidaemiaTreatment of Propionic AcidaemiaTreatment of acute attacks involves using sodium benzoate to remTreatment of acute attacks involves using sodium benzoate to remove ove ammonia, correction of the metabolic acidosis, preventing propioammonia, correction of the metabolic acidosis, preventing propionate nate production by stopping protein intake and giving glucose and insproduction by stopping protein intake and giving glucose and insulin and ulin and removing propionate by giving carnitine.removing propionate by giving carnitine.Long Term treatment consists of a diet restricted in branched chLong Term treatment consists of a diet restricted in branched chain ain amino acids (the main source of propionate in the diet), preventamino acids (the main source of propionate in the diet), preventing ing carnitine depletion by giving carnitine, giving metronidazole tocarnitine depletion by giving carnitine, giving metronidazole to stop gut stop gut bacteria producing propionic acid and sodium benzoate to preventbacteria producing propionic acid and sodium benzoate to preventhyperammonaemia. In certain patients where management is difficuhyperammonaemia. In certain patients where management is difficult lt liver transplantation may be an option.liver transplantation may be an option.Despite treatment complications may occur such as mental retardaDespite treatment complications may occur such as mental retardation, tion, movement disorders, osteoporosis, cardiomyopathy and pancreatitimovement disorders, osteoporosis, cardiomyopathy and pancreatitiss..

Laboratory Tests for the Diagnosis of Methylmalonic Laboratory Tests for the Diagnosis of Methylmalonic AciduriaAciduria

Methylmalonic aciduria is caused by a defect in the activity of methylmalonyl CoA mutase . However in the majority of cases it is not the methylmalonyl CoA mutase enzyme itself that is deficient but its cofactor adenosylcobalamin synthesised from Vitamin B12. Propionyl CoA carboxylation is reversible and so propionate metabolites can also accumulate in methylmalonic aciduria. These are detected by:

•Urine Organic Acid Analysis

-Methylmalonate

-3OH propionate

-Methylcitrate

•Blood Acylcarnitine Analysis

-Raised propionylcarnitine but low free carnitine levels

Organic Acid Trace Organic Acid Trace -- Methylmalonic AcidaemiaMethylmalonic AcidaemiaIncreased excretion of MMA, 3OH-propionate and methylcitrate

Met

hyl c

itrat

e

3OH

pro

pion

ate

MM

A

MM

A

Hep

tade

cano

ate

IS

Methylmalonic Aciduria and Methylmalonic Aciduria and CobalaminCobalamin

Propionyl CoA

D-Methylmalonyl CoA

L-Methylmalonyl CoA

Succinyl CoA

Propionyl CoA Carboxylase

Methylmalonyl CoA Racemase

Methylmalonyl CoA MutaseAdenosylcobalamin (AdoCbl)

Cobalamin

Homocysteine Methionine

Methylcobalamin (MeCbl)

The common causes of increased urine and plasma methylmalonic acThe common causes of increased urine and plasma methylmalonic acid are as follows:id are as follows:--

AcquiredAcquiredVitamin B12 DeficiencyVitamin B12 Deficiency•• Secondary to dietary deficiency. Sometimes seen in breastfed chiSecondary to dietary deficiency. Sometimes seen in breastfed children of Vegan ldren of Vegan

parents. Mild deficiencies may not show anaemia or neurological parents. Mild deficiencies may not show anaemia or neurological symptoms.symptoms.

InheritedInherited

NB Serum vitamin B12 concentrations are normal.NB Serum vitamin B12 concentrations are normal.

Methylmalonic AciduriaMethylmalonic Aciduria•• Methylmalonic Mutase Deficiency:Methylmalonic Mutase Deficiency:

--Complete deficiency (Complete deficiency (mutmutoo)/Partial )/Partial deficiency(mutdeficiency(mut--))•• Adenosylcobalamin Synthesis defectsAdenosylcobalamin Synthesis defects

--Mitochondrial Cobalamin reductase deficiency (Mitochondrial Cobalamin reductase deficiency (cblAcblA))--Mitochondrial Mitochondrial Cob(I)alaminCob(I)alamin adenosyltransferaseadenosyltransferase deficiency (deficiency (cblBcblB))

Combined Methylmalonic Aciduria and HomocystinuriaCombined Methylmalonic Aciduria and HomocystinuriaDefects in Defects in AdenosylAdenosyl andand Methylcobalamin synthesisMethylcobalamin synthesis--CblCCblC, , CblDCblD and and CblFCblF

Methylmalonic Aciduria and CobalaminMethylmalonic Aciduria and Cobalamin

Additional Laboratory TestsAdditional Laboratory Tests

•• Confirmation of the diagnosis can be made by measuring the incorConfirmation of the diagnosis can be made by measuring the incorporation of poration of radiolabelled propionate into fibroblast cell protein and Vitamiradiolabelled propionate into fibroblast cell protein and Vitamin B12 responsiveness n B12 responsiveness can be tested by adding this to the cells.can be tested by adding this to the cells.

•• Further delineation of the primary defect is more difficult. MetFurther delineation of the primary defect is more difficult. Methylmalonyl CoA mutase hylmalonyl CoA mutase can be assayed directly although these account for only a small can be assayed directly although these account for only a small proportion of cases. proportion of cases. Since direct enzyme assays for the enzymes of adenosylcobalamin Since direct enzyme assays for the enzymes of adenosylcobalamin and and methylcobalamin biosynthesis are difficult, the usual approach imethylcobalamin biosynthesis are difficult, the usual approach is to determine the s to determine the affected gene by fusing the fibroblasts with known patient cell affected gene by fusing the fibroblasts with known patient cell lines and seeing if they lines and seeing if they normalise activity (complementation analysis). Once identified tnormalise activity (complementation analysis). Once identified then mutation hen mutation screening can be performed.screening can be performed.

Treatment of Methylmalonic AciduriaTreatment of Methylmalonic Aciduria

••Acute illness Acute illness

The treatment is identical to that for propionic aciduriaThe treatment is identical to that for propionic aciduria

••Long term treatmentLong term treatmentIn cases of isolated methylmalonyl malonic aciduria the patientsIn cases of isolated methylmalonyl malonic aciduria the patients are put on a diet restricted are put on a diet restricted in branch chain amino acids. Patients with combined methylmaloniin branch chain amino acids. Patients with combined methylmalonic aciduria and c aciduria and homocystinuria also have to have a methionine and threonine reshomocystinuria also have to have a methionine and threonine restricted diet. As with tricted diet. As with propionic acidaemia carnitine and metronidazole are used. Betainpropionic acidaemia carnitine and metronidazole are used. Betaine may also be given in the e may also be given in the case of homocystinuria to help reduce the homocysteine concentracase of homocystinuria to help reduce the homocysteine concentration by the action of a tion by the action of a liver specific enzyme that can convert homocysteine to methioninliver specific enzyme that can convert homocysteine to methionine using betaine as its e using betaine as its methyl donor.methyl donor.

Patients with partial defects in adenosylcobalamin synthesis somPatients with partial defects in adenosylcobalamin synthesis sometimes respond to etimes respond to supraphysiological doses of Vitamin B12. Responding patients aresupraphysiological doses of Vitamin B12. Responding patients are continued on this often continued on this often with additional folate supplements. As one would expect patientswith additional folate supplements. As one would expect patients who respond have a better who respond have a better prognosis. However, despite this they can develop developmental prognosis. However, despite this they can develop developmental delay, metabolic strokes delay, metabolic strokes and learning difficulties.and learning difficulties.

NonNon--responding patients, despite treatment, can develop renal probleresponding patients, despite treatment, can develop renal problems secondary to the ms secondary to the toxic effects of methylmalonic acid on the kidney.toxic effects of methylmalonic acid on the kidney.

Glutaric Acidaemia (Type I)Glutaric Acidaemia (Type I)

•• Autosomal recessive condition caused by a deficiency in the enzyAutosomal recessive condition caused by a deficiency in the enzyme me glutarylglutaryl--CoA dehydrogenase which is involved in lysine/tryptophan CoA dehydrogenase which is involved in lysine/tryptophan metabolismmetabolism

Tryptophan

2-Amino, 3-Carboxymuconic Semialdehyde (Picolinic acid)

Aminomuconic Acid Quinolinic Acid

Glutaryl-CoA

Crotonyl-CoA

Nicotinic Acid

Nicotinamide

Glutaryl-CoA Dehydrogenase

Clinical Presentation of Glutaric Aciduria Type 1Clinical Presentation of Glutaric Aciduria Type 1

•• Incidence 1:30,000 Incidence 1:30,000 –– 1:100,0001:100,000•• Macrocephaly at birthMacrocephaly at birth•• Acute encephalopathic episodes Acute encephalopathic episodes -- typically during period of illnesstypically during period of illness•• Sudden onset of hypotonia and severe movement disorders followinSudden onset of hypotonia and severe movement disorders following an g an

acute episodeacute episode

Neurological findingsNeurological findings•• Often an initial period of normal development with Often an initial period of normal development with ““softsoft”” neurological neurological

signs eg. Irritability/jitterinesssigns eg. Irritability/jitteriness•• Acute episodes associated with striatum destructionAcute episodes associated with striatum destruction•• Frontal/temporal atrophy Frontal/temporal atrophy •• White matter changes may be observedWhite matter changes may be observed•• Basal ganglia changes may be evident after an encephalopathic epBasal ganglia changes may be evident after an encephalopathic episodeisode

Laboratory Findings in Glutaric Aciduria Type 1Laboratory Findings in Glutaric Aciduria Type 1

NB Metabolic abnormalities may be fluctuating and inconsistentNB Metabolic abnormalities may be fluctuating and inconsistentIn acute episodes there may be hypoglycaemia, metabolic acidosisIn acute episodes there may be hypoglycaemia, metabolic acidosis and hyperammonaemiaand hyperammonaemia..

•• Urine Organic AcidsUrine Organic Acids--Increased glutaric acidIncreased glutaric acid--Increased 3Increased 3--hydroxyglutaratehydroxyglutarate(NB. A small group of patients may not show increased levels of (NB. A small group of patients may not show increased levels of glutaric acid and only glutaric acid and only trace levels of 3OH glutarate. Glutarate and 3OH glutarate can trace levels of 3OH glutarate. Glutarate and 3OH glutarate can be nonbe non--specifically specifically elevated due to bacterial contamination)elevated due to bacterial contamination)

•• Blood AcylcarnitinesBlood Acylcarnitines--Increased Glutaryl carnitineIncreased Glutaryl carnitine--Free carnitine levels may be lowFree carnitine levels may be low

•• Enzyme studies (Definitive test)Enzyme studies (Definitive test)--Glutaryl CoA dehydrogenase activity in cultured fibroblastsGlutaryl CoA dehydrogenase activity in cultured fibroblasts(Typically <5(Typically <5--10% normal activity level in affected individuals10% normal activity level in affected individuals))

•• Mutation AnalysisMutation Analysis--No common mutations except in certain ethnic isolates.No common mutations except in certain ethnic isolates.

Organic Acid Trace of Glutaric Aciduria Type IOrganic Acid Trace of Glutaric Aciduria Type I3OH GlutarateGlutarate Heptadecanoate IS

Treatment of GA1Treatment of GA1

Acute EpisodesAcute EpisodesThe hypoglycaemia, metabolic acidosis and hyperammonaemia need tThe hypoglycaemia, metabolic acidosis and hyperammonaemia need to be o be managed and an emergency protocol is required to deal with thesemanaged and an emergency protocol is required to deal with these situations.situations.

Long Term ManagementLong Term ManagementThe aim of dietary treatment is to reduce the accumulation of thThe aim of dietary treatment is to reduce the accumulation of the presumed toxic e presumed toxic agent glutaric acid by reducing its rate of formation. To this eagent glutaric acid by reducing its rate of formation. To this effect patients can be put ffect patients can be put on a low protein diet which specifically restricts lysine and tron a low protein diet which specifically restricts lysine and tryptophan. Alternatively yptophan. Alternatively they can be put on a lysine free diet however they will need trythey can be put on a lysine free diet however they will need tryptophan supplements ptophan supplements for protein biosynthesis.for protein biosynthesis.Patients are also given carnitine to avoid carnitine depletion sPatients are also given carnitine to avoid carnitine depletion secondary to the econdary to the excretion of glutarylcarnitine.excretion of glutarylcarnitine.

Questions

1. Elevated urine 3-hydroxypropionate2. Elevated urine methylcitrate3. Elevated urine 3-Methylmalonic acid4. Elevated 3-hydroxyglutarate5. Hyperammonaemia6. Hyperlactic acidaemia7. Hypoglycaemia8. Hyperglycinaemia9. Low plasma carnitine

Which of these biochemical abnormalities occur with the following disorders:-

Glutaric Aciduria

Propionic Aciduria

Methylmalonic Aciduria

Answers

Glutaric Aciduria4, 5 , 6 , 7 & 9

Propionic Acidaemia1, 2, 5, 6, 7, 8, & 9

Methylmalonic Aciduria1, 2, 3, 5, 6, 7. 8 & 9