A common presentation of IEMs is an apparently healthy child presenting acutely with a short history of illness, which is worsening progressively. The symptoms could be as mild as a fever, or as severe as encephalopathy or status epilepticus. Few common clinical presentations are (i) acute (and recurrent) episodes of symptoms such as vomiting, respiratory distress, ataxia, lethargy or coma with or without seizures, hypotonia, hypoglycemia or hyperammonemia; (ii) chronic and progressive symptoms such as developmental delay, epilepsy or other neurological illness, or (iii) organ specific presentation (single or multiple) such as hepatopathy, cardiomyopathy, neuro-muscular, renal, gastroenterology or hematological illness. These children should be evaluated for a metabolic cause particularly with a past history or family history of similar illness in a sibling. Presence of consanguinity makes the suspicion of an IEM stronger. A complete discussion on the various clinical approaches to diagnosis of IEMs is out of scope of this review; the readers are suggested to refer to other resources [6-8].

IEMs can be detected on routine tests such as blood gas, blood glucose, blood ammonia and ketones in urine, which can be followed by more advanced tests. The tests utilized for diagnosing an IEM are detailed below.

Some tests on blood and urine can be performed at the bedside or quickly in a routine laboratory. These tests are helpful in screening for many IEMs, but are not diagnostic by themselves.

Urine Analysis

The urine is an excellent source of crucial metabolites as the excess pathological metabolites in the body are excreted out in urine. Urine should be examined for the following observations before subjecting it to advanced biochemical testing [8-12].

Color: Yellow (light to dark) may indicate bilirubin/drug metabolites, while coffee or cola colored may indicate rhabdomyolysis, and darkening on standing (exposure to light) may be observed in porphyria and alkaptonuria (Table I).

TABLE I Interpreting Abnormal Color and Odour of Urine 

Odor: Sweet odor in ketosis, maple syrup/ burnt sugar in maple syrup urine disease (MSUD) and mousy in phenylketonuria (PKU) have been described. Table I outlines the odors described in the various IEMs.

Urinalysis: Urinary pH, specific gravity, creatinine concentration, glucose/reducing substances, protein/albumin/micro-albumin, ketonuria are important preliminary investigations in IEMs.

Blood Tests

Blood Glucose, Electrolytes, Lactate, Acid base balance/arterial blood gas, Ammonia, and Ketosis (Acronym- GELAK): Stringent conditions need to be met with regards to the transport and analysis of both lactate and ammonia. Ammonia estimation should be done from a free flowing blood sample obtained from either arterial or venous puncture. The sample should be collected in a preferably pre-chilled tube containing EDTA (for ammonia) as an anticoagulant. Patient should be fasting (or at least 4-6 hours after feeding) and non-stressed such that it is not collected after a difficult venipuncture. This, however, would not hold true for an acutely sick child when samples should be collected immediately upon arrival or in acutely decompensated state, irrespective of feeding status. Samples should be transported on ice, separated within 15 minutes of collection, and analyzed immediately to prevent artefactual increase in generation of ammonia from RBC degradation or deamination of amino acids by enzymes such as gamma glutamyl transferase. Normal levels of ammonia are <110 µmol/L (neonates), <80 µmol/L (infants), <50 µmol/L (older children) and <35 µmol/L (adults). Lactate analysis can be done from whole blood or plasma collected in a sodium fluoride or heparinized vial. Sample should be transported on ice slurry or an ice pack within 30 minutes of collection. Once aliquoted, the plasma is stable on room temperature for 8 hours. Blood collected from artery is ideal but a free flowing venous blood sample is also acceptable. The lactate value on blood gas is reliable if the machine is calibrated. There are two units used frequently for lactate values – mg/dL and mmol/L. Normal range of lactate (both in blood in CSF) are 2-20 mg/dL and 0.2–2.0 mmol/L at all ages.

Liver function tests (LFT): The list of IEMs with liver involvement is exhaustive–ranging from small molecule disorders such as urea cycle, amino acid or organic acid disorders to mitochondriopathies and large molecule disorders such as peroxisomal and lysosomal storage disorders. For more in-depth information on IEMs related to liver disease readers are directed towards other reviews [13,14].

Renal function tests: Serum creatinine indicates renal function derangement, if high; and creatine synthetic or transporter defect, if low.

Uric acid: High levels in presence of features such as intellectual disability and self-mutilation is highly suggestive of Lesch-Nyhan Syndrome (HPRT deficiency – purine recycling disorder). High levels may also suggest disorders of carbohydrate disorders such as glycogenolysis or gluconeogenesis. A low level may point towards xanthine/hypoxanthine disorder or a molybdenum cofactor deficiency (MoCD).

Creatine kinase: It is elevated in metabolic disorders affecting the muscle causing glycogenolysis, or rhabdomyolysis, and in disorders of energy production such as fatty acid oxidation defects, gluconeogenesis defects or a mitochondrial disorder.

Lipid profile: This is deranged in glycogen storage disorders, lipoprotein disorders, transient infantile hypertriglyceridemia. Dyslipidemia is also noted in Wolman disease, fructose-1-6-bisphosphatase deficiency and lipid storage disorders such as Niemann-Pick disease.

Prolactin levels: This may be elevated in neurotransmitter disorders (dopamine synthesis). Normal level in serum is 5 to 20 ng/mL (5 to 20 µg/L).

Copper levels (in plasma): This may be decreased in Wilson disease, Menkes, aceruloplasminemia, and MEDNIK syndrome; and increased in peroxisomal disorders.

Iron levels: Increased serum iron/ferritin is observed in hemochromatosis and peroxisomal disorders

These are biochemical reactions utilized for making a specific diagnosis of a single or group of disorders. These tests measure specific metabolite, enzyme or a cofactor. Specific indications for these tests with classification of IEMs are provided in Table II. The classification is proposed by the Society for study of inborn errors of metabolism (SSIEM) according to biochemical basis [7].

TABLE II Metabolic Tests for Diagnosis of IEMs (According to SSIEM Classification ) [7]

Enzyme Assay

Testing for activity of an enzyme requires recreating/simulating the actual enzymatic reaction outside of body using appropriate body fluids/tissue (leukocytes, plasma/serum, skin fibroblasts or liver biopsy specimens), and using artificial or natural substrates [15].

Lysosomal storage disorders: More than 24 enzymes are known, and can be tested in blood (leucocytes, sometimes plasma) and cultured skin fibroblasts. Examples are enzymes for Gaucher disease, Niemann Pick disease [13]. Usual requirement for testing is 4-5 mL blood in heparin vial (or sometimes EDTA vial). Lysosomal enzymes can also be measured on a dried blood spot [14]

Galactosemia: Galactose-6-phosphate uridyl transferase (GALT), Galactokinase (GALK), and Galactose-1-phosphate uridyl epimerase (GALE) can be performed on red blood cells (RBCs) only; therefore, blood in heparin vacutainers is required. Semi-quantitative testing can also be performed on dried blood spots (Beutler spot test), and is useful in newborn screening.

Biotinidase deficiency: This enzyme assay can be performed in serum (quantitative) or on dried blood spots (semi quantitative), and is useful in newborn screening.

Other small molecule disorders: Enzyme assays for fatty acid oxidation defects (FAOD), gluconeogenesis (hereditary fructose intolerance, fructose 1-6-biphosphatase ), glycogen storage disorders (GSD), urea cycle defects or organic acidurias, tyrosinemia, cystinosis, and porphyrias used to be the standard diagnostic modalities until molecular diagnosis became widely available. There are limited accredited laboratories for enzyme assays [16]. Majority of these disorders would require specialized tissues such as cultured skin fibroblasts, frozen liver biopsy or muscle biopsy specimen [6]. The diagnosis of these disorders relies heavily upon specific metabolite analysis using methods described below and molecular genetic testing involving sequencing of particular gene or group of genes.

Detection of Metabolites

These tests detect specific metabolites through simple chemical reactions with precision, sometimes alleviating the need for molecular genetic testing. Urine metabolic tests can be done easily in most laboratories. Table II depicts the preliminary simple bedside tests that can be used for the diagnosis of inborn metabolic errors. Among the specialized biochemical tests, three methods are described in more detail; whereas, other tests are mentioned in Table III.

Tandem mass spectrometry (TMS), also known as mass spectrometry-mass spectrometry (MS/MS) as two mass spectrometers are situated in tandem. This is a revolutionary technology devised in early 90s [17], which can detect multiple independent metabolites/analytes in a single test. The basic principle of TMS relies on the ionization and fragmentation of each molecule or metabolite into specific ions coupled with a robust detection system which is computerized to provide results [18]. Fig. I provides a simplified diagrammatic representation of MS/MS equipment [19]. A sample is first ionized and made to pass through a series of chambers, which serve specific purpose of further ionization, collision-induced dissociation and further ionization of daughter ions before reaching a detection chamber that recognizes individual ions based on specific mass-to-charge ratio. The computer then interprets the data and provides highly accurate results of each analyte. The utility of a TMS is multi-fold and its applications are expanding. It measures amino-acids, organic acids and fatty acids in form of their acyl-carnitine esters. This particular technology forms the core of expanded newborn screening throughout the developed world [20]. Table IV provides interpretations of few metabolites [21]. TMS does not screen for many IEMs such as mitochondriopathies, purine and pyrimidine disorders, neurotransmitters, congenital disorders of glycosylation (CDG) and very long chain fatty acids. TMS using dried blood spot is highly sensitive but not specific, and thus remains a screening test for majority of disorders. Its use for prenatal diagnosis is not recommended. Higher efficacy equipments such as liquid chromatography-MS/MS (LC-MS/MS) are now employed for better accuracy in biological samples, including CSF and plasma.

Fig. 1 Basic steps of tandem mass spectrometry.

GC-MS is gas chromatography-mass spectrometry technology to detect metabolites specific for small molecule disorders (typically organic acids and related) in the urine [22]. The organic acids are volatile and tend to evaporate without preservation, which requires samples to be frozen. Detection of organic acids in urine should ideally be done in frozen sample or reliably if transported under ambient temperatures not exceeding 25°C. Transportation is stable when urine sample is put on a special grade filter paper that is able to soak in all metabolites and fix or stabilize it preventing it from evaporation [22]. They can be eluted back from filter paper into liquid solution using simple techniques. Interpretation of results of urine organic acid analysis by GC-MS requires expertise and biochemical background training, and thus should be taken up only by specialized genetic laboratories or institutes. The modality can also measure metabolites in bile, plasma and other body fluids including postmortem samples when there is a failure to collect urine.

The measurement of specific amino acids in body fluids such as plasma/serum, urine or CSF is being utilized widely for diagnosing many amino-acid disorders such as phenylketonuria, tyrosinemia type 1/2/3, maple syrup urine disease, and homocystinuria (methionine as well as homocysteine which requires a different algorithm for identification) [23]. There are multiple methods for quantifying amino acids – chromatographic as well as electrophoresis. Most laboratories currently employ either HPLC or UPLC technique because of the relative ease and high specificity. All samples (plasma not whole blood, urine or CSF) for chromatography or electrophoresis should be transported in frozen conditions using dry ice. Samples exposed to high temperatures and harsh conditions would destroy the amino acids and give erroneous or inaccurate results. Samples can also be collected in a sulfosalicylic acid (SSA) pretreated vial to ensure stability. CSF samples should be paired with plasma samples for accurate interpretation. Diagnosis of non-ketotic hyperglycinemia will be made when both CSF and plasma levels of glycine are elevated. Isolated elevation of glycine may be due to other non-genetic causes such as perinatal asphyxia or bloody tap (due to contamination of CSF with blood). Interpretation for some common and important metabolite/amino acid derangement is provided in Table III. Detection of amino acids qualitatively in the urine (and sometimes both urine as well as plasma) has also been used traditionally for the diagnosis amino acid disorders. The test is done by a thin-layer chromatography technique that is a simple test using filter paper. The pattern of excretion of amino acids is interpreted to get to a diagnosis.

Neurotransmitter assays: This is a specialized test performed only on CSF using sophisticated LC-MS/MS and GCMS technique [10] that only few laboratories have standardized and thus authorized to perform.

Serum transferrin isoforms for glycosylation pattern: This is a widely used screening test for CDGs, the diagnosis of which requires genetic testing. The test was initially established using iso-electric focusing (IEF) [24] followed by capillary zone electrophoresis (CZE) in the nineties [25]. There are two abnormal patterns of transferrins identified in CDGs leading to their classification into either CDG type 1 (showing a type 1 pattern of derangement of transferrin isoforms) or type 2. However, not all CDGs would show up with these patterns [26]. The test can also be done using HPLC, MALDI-TOF-MS/MS or LC-MS/MS [27].

Metabolic biomarkers: Testing for chitotriosidase, CCL18/PARC, heparin thrombin cofactor ii, lyso GB3 are available in few specialized laboratories. Multiple biomarkers are in use for diagnosis and prognosis of many metabolic disorders. A holistic approach is recommended to detect and interpret certain (specific) pattern of metabolites in body fluids to make a metabolic diagnosis. This new system also known as the metabolomics approach is rapidly advancing and holds promise for the future [28]

Molecular Genetic Testing

The molecular genetic test entails testing of either single or multiple genes together depending upon the suspicion of the disease. For disorders where a specific diagnosis is already made using other techniques such as GALT deficiency, only one gene (GALT) may be tested. However, for other disorders like mitochondrial disorders, multiple genes are to be tested together for lack of evidence towards any specific genes (barring few exceptions). There are two methods employed for molecular genetic testing: a traditional, well established and more precise method of Sanger sequencing [29], and a more advanced method known as Next generation sequencing (NGS). Both methods allow for sequencing of genes, determining the sequence of base pairs in the DNA of the exons/exon-intron boundaries or coding regions of a gene. However, Sanger sequencing is time consuming, laborious and expensive as it is performed fragment wise, one by one for each fragment of a gene. The NGS utilizes the multiplexing of all the sequencing fragments and thus has the capacity to sequence any number of DNA fragments simultaneously. The NGS can sequence from one gene to whole exome or even genome which is about 3 billion base pairs. By multiplexing the sequencing there is considerable saving of time as well as cost. It is now becoming the standard diagnostic methodology in most of genetic laboratories, including for metabolic disorders.

An entire battery of test has been developed over the years to diagnose IEMs. The basic and advanced metabolic test would diagnose majority of IEMs, except few. TMS can detect only three groups of disorders (fatty acid oxidation, organic and few amino acidurias) and not mitochondrial disorders. A biochemical test may show normal metabolite levels as they are not always deranged. In such cases, the metabolic tests should be repeated during acute sickness.

Inborn errors of metabolism are disorders often encountered in pediatric emergencies presenting with common symptoms. As many of them are treatable, a high index of suspicion can lead to their early recognition. Early and accurate treatment is the key to a fruitful outcome. IEMs can be screened by basic biochemical tests. Appropriate samples should also be collected and stored at the time of acute illness for advanced genetic tests, which can be carried out later. All IEMs have a genetic basis: hence, an accurate genetic counseling should be provided to the families for prevention in future births. With knowledge of the basic and advanced metabolic tests, the pediatrician would be empowered to diagnose and treat most IEMs.

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