Biochemical Diagnostic Testing

Biochemical diagnostic testing measures analytes (metabolites, enzymes, transport proteins) to identify inborn errors of metabolism (IEM). Biochemical assays are typically the first-line workup for a suspected IEM because they are fast, inexpensive, and reflect real-time pathway function. They identify the disease class (for example, organic acidemia versus fatty acid oxidation defect), and molecular testing is then used to confirm the precise gene and variant for prognosis, family testing, and reproductive planning.

Quantitative analysis of plasma amino acids by ion-exchange chromatography or tandem mass spectrometry. Used for aminoacidopathies and urea cycle disorders.

• Phenylketonuria (PKU, PAH): elevated phenylalanine, low or normal tyrosine; elevated Phe/Tyr ratio.
• Maple syrup urine disease (BCKDHA/B, DBT): elevated branched-chain amino acids (leucine, isoleucine, valine) and alloisoleucine (pathognomonic).
• Tyrosinemia type 1 (FAH): elevated tyrosine and methionine; succinylacetone in plasma or urine is diagnostic.
• Citrullinemia type 1 (ASS1): markedly elevated citrulline; low arginine; hyperammonemia.
• Argininosuccinic aciduria (ASL): elevated argininosuccinic acid and citrulline.
• OTC deficiency: low citrulline, hyperammonemia, elevated urinary orotic acid (X-linked).
• Homocystinuria (CBS): elevated methionine and homocysteine.

GC-MS analysis of urine organic acids. Used for organic acidemias and several other IEMs.

• Methylmalonic acidemia (MUT, MMAA, MMAB, MMADHC): massively elevated methylmalonic acid.
• Propionic acidemia (PCCA, PCCB): elevated 3-hydroxypropionate, methylcitrate, propionylglycine, tiglylglycine.
• Isovaleric acidemia (IVD): elevated isovalerylglycine and 3-hydroxyisovaleric acid; "sweaty feet" odor.
• 3-Methylcrotonyl-CoA carboxylase deficiency (MCCC1, MCCC2): elevated 3-methylcrotonylglycine and 3-hydroxyisovalerate.
• Glutaric acidemia type 1 (GCDH): elevated glutaric and 3-hydroxyglutaric acid.

Tandem mass spectrometry (MS/MS) of acylcarnitine species in plasma or dried blood spot. The basis of expanded newborn screening and the first test in suspected fatty acid oxidation defects and organic acidemias.

• Carbon-chain nomenclature: C0 (free carnitine), C2 (acetylcarnitine), C3 (propionylcarnitine), C4, C5, C5-DC, C8, C14:1, C16, C18.
• MCAD deficiency (ACADM): elevated C8 (octanoylcarnitine), elevated C8/C10 ratio. Most common FAO disorder.
• VLCAD deficiency (ACADVL): elevated C14:1 (tetradecenoylcarnitine).
• LCHAD/TFP (HADHA, HADHB): elevated C16-OH and C18-OH hydroxyacylcarnitines.
• CPT1 deficiency: elevated C0 with low long-chain species.
• CPT2 / CACT deficiency: elevated C16 and C18:1.
• Propionic / methylmalonic acidemia: elevated C3.
• Isovaleric acidemia: elevated C5.
• Glutaric acidemia type 1: elevated C5-DC (glutarylcarnitine).
• Primary carnitine deficiency (SLC22A5): low C0 (free carnitine).

First-line for peroxisomal disorders. Measured as C26:0 absolute level and C26:0/C22:0 and C24:0/C22:0 ratios.

• X-linked adrenoleukodystrophy (ABCD1): elevated VLCFA in males; about 15-20% of female heterozygotes have normal VLCFA, so molecular testing is required to rule out carrier status.
• Zellweger spectrum disorders (PEX genes): elevated VLCFA plus elevated phytanic and pristanic acids, low plasmalogens.

Direct measurement of enzyme activity in leukocytes, fibroblasts, or dried blood spot. Used to confirm lysosomal storage disorders.

• Tay-Sachs disease: low hexosaminidase A (HEXA) activity. Pseudodeficiency alleles (p.R247W, p.R249W) reduce activity against synthetic substrate in vitro without causing disease, so molecular testing distinguishes true carriers.
• Gaucher disease: low glucocerebrosidase (GBA) activity in leukocytes; elevated chitotriosidase as a marker.
• Fabry disease: low alpha-galactosidase A (GLA) activity in males; female heterozygotes can have normal enzyme levels, so molecular testing is required.
• MPS I (Hurler): low alpha-L-iduronidase (IDUA).
• Pompe disease (GAA): low acid alpha-glucosidase; pseudodeficiency alleles common in East Asian populations.
• Krabbe disease (GALC): low galactocerebrosidase.

First-line screen for congenital disorders of glycosylation (CDG). Isoelectric focusing or mass spectrometry of serum transferrin reveals abnormal glycoform patterns.

• Type 1 pattern (PMM2-CDG and other N-linked assembly defects): increased disialo- and asialotransferrin.
• Type 2 pattern (processing defects): increased trisialo- and monosialotransferrin.
• Follow-up with specific enzyme assays and molecular testing.

Most US newborn screening programs use MS/MS-based acylcarnitine and amino acid panels plus targeted assays (immunoassays for endocrine disorders, isoelectric focusing for hemoglobinopathies, enzyme assays for select LSDs, fluorometric assays for galactosemia, biotinidase) on a dried blood spot. The Recommended Uniform Screening Panel (RUSP) lists about 35 core conditions and additional secondary conditions. Positive screens are presumptive only and require diagnostic confirmation with quantitative biochemical and molecular testing.

• Always confirm a biochemical diagnosis with molecular testing. Biochemical assays identify a metabolic class, but molecular testing yields the specific gene and variant needed for:
  • Genotype-phenotype prediction (for example, severe vs attenuated MPS I).
  • Family cascade testing and carrier identification.
  • Reproductive options (PGT-M, prenatal diagnosis).
  • Eligibility for variant-specific therapies (for example, sapropterin response in PKU, ivacaftor in CFTR-G551D, antisense oligonucleotides).