Organic Synthesis Laboratory

3-Hydroxy-3-methylglutaryl-CoA Lyase Deficiency shows a characteristic pattern of elevated urinary concentrations of 3-hydroxy-3-methylglutaric acid, 3-hydroxyisovaleric acid, 3-methylglutaconic acid and 3-methylglutaric acid. Confirmation of the enzyme defect can be obtained by direct assay of the enzyme in leukocytes or fibroblasts. Quantification of 3-hydroxy-3-methylglutaric acid in amniotic fluid contributes to prenatal diagnosis of the disease.

Standards

3-hydroxy-3-methylglutaric acid
3-hydroxy-3-[2H3]methylglutaric acid

In Glutaric Aciduria Type I (glutaryl-CoA dehydrogenase deficiency) elevated urinary levels of 3-hydroxyglutaric acid are found. Although glutaric acid excretion may be increased as well, this is not always the case. Furthermore, whereas increased urinary glutaric acid is also found in other diseases, elevated excretion of 3-hydroxyglutaric acid is specific for Glutaric Aciduria Type I.

Standard

3-hydroxyglutaric acid

In addition to 3-hydroxy-3-methylglutaryl-CoA lyase deficiency, 3-hydroxyisovaleric acid is one of the major abnormal urinary metabolites in isovaleryl-CoA dehydrogenase deficiency and in biotin-responsive multiple-decarboxylase deficiencies. 

Standards

3-hydroxyisovaleric acid, sodium salt
[2H6]3-hydroxyisovaleric acid, sodium salt

Interconversion of acyl-CoA esters and acylcarnitines plays a key role in the transport process of fatty acids across the mitochondrial membrane. Acylcarnitine profiling in plasma has become indispensable for the diagnosis of a series of fatty acid ß-oxidation deficiencies, and of defects specifically related to the membrane transport process. Increasingly, acylcarnitine profiling in blood spots by MS/MS is included in national neonatal screening programs

Standard:  acetyl-L-carnitine.HCl
Labeled standard: [2H3]acetyl-L-carnitine.HCl

Standard: propionyl-L-carnitine.HCl
Labeled standard: [3,3,3-2H3]propionyl-L-carnitine.HCl

Standard:butyryl-L-carnitine.HCl
Labeled standard: [4,4,4-2H3]butyryl-L-carnitine.HCl

Standard: isobutyryl-L-carnitine.HCl
Labeled standard: [2H7]isobutyryl-L-carnitine.HCl

Standard: pivaloyl-L-carnitine.HCl

Standard: valeryl-L-carnitine.HCl
Labeled standard: [5,5,5-2H3]valeryl-L-carnitine.HCl

Standard: isovaleryl-L-carnitine.HCl
Labeled standard: [2H9]isovaleryl-L-carnitine.HCl

Standard: 2-methylbutyryl-L-carnitine.HCl
Labeled standard: 2-[2H3]methylbutyryl-L-carnitine.HCl

Standard: hexanoyl-L-carnitine.HCL
Labeled standard: [6,6,6-2H3]hexanoyl-L-carnitine.HCl

Standard: octanoyl-L-carnitine.HCl
Labeled standard: [8,8,8-2H3]octanoyl-L-carnitine.HCl

Standard: decanoyl-L-carnitine.HCl
Labeled standard: [10,10,10-2H3]decanoyl-L-carnitine.HCl

Standard: dodecanoyl-L-carnitine.HCl
Labeled standard: [12,12,12-2H3]dodecanoyl-L-carnitine.HCl

Standard: tetradecanoyl-L-carnitine.HCl
Labeled standard: [14,14,14-2H3]tetradecanoyl-L-carnitine.HCl

Standard: hexadecanoyl-L-carnitine.HCl
Labeled standard: [16,16,16-2H3]hexadecanoyl-L-carnitine.HCl

Standard: octadecanoyl-L-carnitine.HCl
Labeled standard: [18,18,18-2H3]octadecanoyl-L-carnitine.HCL

Synthesis and application:

CG Costa, EA Struys, A Bootsma, HJ ten Brink, L Dorland, I Tavares de Almeida, M Duran, C Jakobs. Quantitative analysis of plasma acylcarnitines using gas chromatography chemical ionization mass fragmentography. J Lipid Res 38 (1997) 173-182.

Carnitine-labelled standards:

[methyl-2H3]-L-carnitine.HCl

[trimethyl-2H9]-D,L-carnitine.HCL

[4,4,4-2H3]butyryl -[methyl-2H3]-L-carnitine.HCl

[8,8,8-2H3]octanoyl -[methyl-2H3]-L-carnitine.HCl

[16,16,16-2H3]hexadecanoyl -[methyl-2H3]-L-carnitine.HCl

Acylglycines result from the transesterification of acyl-CoA esters with glycine through the action of the enzyme glycine-N-acylase. In defects of the mitochondrial amino acid and fatty acid metabolism where accumulation of acyl-CoA esters occurs, increased urinary acylglycine excretion is directly related to the intramitochondrial accumulation of the corresponding acyl-CoA esters. 

Standards 

acetylglycine - [13C2]acetylglycine
propionylglycine - [3,3,3-2H3]propionylglycine
butyrylglycine - [4,4,4-2H3]butyrylglycine
isobutyrylglycine - [2H7]isobutyrylglycine
pivaloylglycine 
valerylglycine - [5,5,5-2H3]valerylglycine
isovalerylglycine - [2H9]isovalerylglycine
2-methylbutyrylglycine - [2H9]2-methylbutyrylglycine
hexanoylglycine - [6,6,6-2H3]hexanoylglycine
heptanoylglycine 
phenylpropionylglycine - [2H5]phenylpropionylglycine
tiglylglycine 
suberylglycine 

Synthesis and application

CG Costa, WS Guérand, EA Struys, U Holwerda, HJ ten Brink, I Tavares de Almeida, M Duran, C Jakobs. Quantitative analysis of urinary acylglycines for the diagnosis of ß-oxidation defects using GC-NCI-MS. J Pharm Biomed Anal 21 (2000) 1215-1224.

The main metabolic pathway for the bile acid intermediates di- and trihydroxycoprostanic acid is through peroxisomal ß-oxidation. Patients with either a peroxisome biogenesis defect or a specifically bile acid ß-oxidation defect accumulate these DHCA and THCA in blood and bile.

Measurement of DHCA and THCA in body fluids contributes to the diagnosis of such defects, both postnatal and prenatal. 

Standards

dihydroxycoprostanic acid
[27,27,27-2H3]dihydroxycoprostanic acid 
trihydroxycoprostanic acid 
[27,27,27-2H3]trihydroxycoprostanic acid 

Synthesis and application

DW Johnson, HJ ten Brink, RC Schuit, C Jakobs. Rapid and quantitative analysis of unconjugated C27 bile acids in plasma and blood samples by tandem mass spectrometry. J Lipid Res 42 (2001) 6-16.

In the mitochondrial ß-oxidation cycle, the conversion of straight-chain acyl-CoAs into 2-trans-enoyl-CoAs is catalyzed by acyl-CoA dehydrogenases, differing in chain length specificities. Phenylpropionyl-CoA is specificly accepted by Medium-Chain Acyl-CoA Dehydrogenase yielding cinnamoyl-CoA as product. In a variety of cell types MCAD can be assayed spectrophotometrically by measuring at 308 nm the formation of cinnamoyl-CoA from phenylpropionyl-CoA in the presence of an artificial electron acceptor.

Standards

3-phenylpropionyl-CoA
cinnamoyl-CoA

Application

Yao K-W, Schulz H. Specific assay of Medium-Chain Acyl-CoA Dehydrogenase based on the spectrophotometric maesurement of product formation. Anal Biochem 214 (1993) 528-534.

Deficiency of guanidinoacetate methyltransferase results in a low creatine and creatinine formation. Diagnosis of this deficiency can reliably be made by quantification of the precursor of creatine, guanidinoacetic acid, in urine, plasma and cerebrospinal fluid. 

Standard

guanidino-[13C2]acetic acid 

Synthesis and application

EA Struys, EEW Jansen, HJ ten Brink, NM Verhoeven, MS van der Knaap, C Jakobs. An accurate stable isotope dilution gas chromatographic - mass spectrometric approach to the diagnosis of guanidinoacetate methyltransferase deficiency. J Pharm Biomed Anal 18 (1998) 659-665.

In Canavan disease a deficiency of aspartoacylase leads to accumulation of N-acetyl-aspartic acid in urine, blood and cerebrospinal fluid. Prenatal diagnosis of the disease is possible via quantification of the acid in amniotic fluid.

Standard

N-[2H3]acetylaspartic acid

Synthesis and application

C Jakobs, HJ ten Brink, SA Langelaar, T Zee, F Stellaard, M Macek, K Srsnová, S Srsen, WJ Kleijer. Stable isotope dilution analysis of N-acetylaspartic acid in CSF, blood, urine and amniotic fluid: accurate postnatal diagnosis and the potential for prenatal diagnosis of Canavan disease. J Inher Metab Dis 14.

In order to improve the performance of laboratories in organic acid measurements and harmonization of laboratory results, it is essential that control materials are available.

Following advice of the ERNDIM Advisory Board a mixture of organic acids was composed having - after reconstitution with 5 mL water – the following concentrations:

The 2 sets of mixtures are provided as lyophilized products. After reconstitution, products should be processed as patient samples according to existing procedures for organic acid analysis.

Plasma analysis of phytanic acid and pristanic acid is an important tool in the biochemical diagnosis of peroxisomal disorders. In addition to measurement of absolute values, establishing pristanic acid / phytanic acid ratios is of value for the differential diagnosis of Refsum Disease, Rhizomelic Chondrodysplasia Punctata, Peroxisome Deficiency Disorders and isolated peroxisomal ß-Oxidation Deficiencies. Alternatively diagnosis of some of these diseases can be obtained by application of the method to stored blood spots.

Quantification of both acids is usefully combined with the analysis of very long chain fatty acids.

Standards

phytanic acid
pristanic acid
[3-methyl-2H3]phytanic acid
[2-methyl-2H3]pristanic acid

Synthesis

HJ ten Brink, C Jakobs, JL van der Baan, F Bickelhaupt. Synthesis of deuterium labelled analogues of pristanic acid and phytanic acid for use as internal standards in stable isotope dilution analysis. In: Synthesis and Applications of Isotopically Labelled Compounds 1988 (TA Baillie, JR Jones, eds.). Elsevier Science Publishers BV, Amsterdam (1989) 717-722.

Application

HJ ten Brink, F Stellaard, CMM van den Heuvel, RM Kok, DSM Schor, RJA Wanders, C Jakobs. Pristanic acid and phytanic acid in plasma from patients with peroxisomal disorders: stable isotope dilution analysis with electron capture negative ion mass fragmentography. J Lipid Res 33 (1992) 41-47.

HJ ten Brink, CMM van den Heuvel, E Christensen, C Largillière, C Jakobs. Diagnosis of peroxisomal disorders by analysis of phytanic and pristanic acids in stored blood spots collected at neonatal screening. Clin Chem 39 (1993) 1904-1906.

In hepatorenal tyrosinemia type 1 (HT1) tyrosine breakdown is impaired due to a deficiency in fumarylacetoacetate hydrolase. As a consequence, fumarylacetoacetate is converted into succinylacetone (4,6-dioxoheptanoic acid) which is elevated in HT1 patients. Since succinylacetone is unknown to occur in any other metabolic pathway, it is a specific diagnostic marker for the disease. It can be reliably measured in plasma, urine, amniotic fluid and dried blood spots.

Standard

[1,2,3,4-13C4]succinylacetone

Synthesis and application

Al-Dirbashi OY, Rashed MS, ten Brink HJ, Jakobs C, Filimban N, Al-Ahaidib LY, Jacob M, Al-Sayed MM, Al-Hassnan Z, Faquih E. Determination of succinylacetone in dried blood spots and liquid urine as a dansylhydrazone by liquid chromatography tandem mass spectrometry. J Chromatogr B 831 (2006) 274-280

Johnson DW, Gerace R, Ranieri E, Trinh M-U, Fingerhut R. Analysis of succinylacetone, as a Girard T derivative, in urine and dried bloodspots by flow injection electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spectrom 21 (2007) 59-63.

Plasma analysis of very long chain fatty acids C22:0, C24:0 and C26:0, together with phytanic and pristanic acids, is the first step in the investigation of patients suspected to suffer from a peroxisomal disorder.

In an adapted form, the method is applicable for diagnosis in stored blood spots collected at neonatal screening, and prenatally in amniotic fluid, chorion biopsy material and cultured amniocytes.

As long as for unsaturated VLCFA the specific labelled analogs are not available, the saturated standards can be used for quantification of the unsaturated metabolites.

Standards

[3,3,5,5-2H4]docosanoic acid
[3,3,5,5-2H4]tetracosanoic acid
[3,3,5,5-2H4]hexacosanoic acid

Synthesis

HJ ten Brink, C Jakobs, F Bickelhaupt. Synthesis of deuterium labelled analogues of very long chain fatty acids C22:0, C24:0 and C26:0 for use as internal standards in stable isotope dilution analysis. In: Synthesis and Applications of Isotopically Labelled Compounds 1988 (TA Baillie, JR Jones, eds.). Elsevier Science Publishers BV, Amsterdam (1989) 723-726.

Application

F Stellaard, HJ ten Brink, RM Kok, L van den Heuvel, C Jakobs. Stable isotope dilution analysis of very long chain fatty acids in plasma, urine and amniotic fluid by electron capture negative ion mass fragmentography. Clin Chim Acta 192 (1990) 133-144.

C Jakobs, CMM van den Heuvel, F Stellaard, C Largillière, F Skovby, E Christensen. Diagnosis of Zellweger syndrome by analysis of very long chain fatty acids in stored blood spots collected at neonatal screening. J Inher Metab Dis 16 (1993) 63-66.