Targeted Metabolomics

In assays, from pharmacokinetics to proteomics, tandem mass spectrometry (known as MS/MS) coupled with high-pressure liquid chromatography (HPLC) is the analytical technique of choice. It is commonly used during drug discovery alongside protein, peptide and oligonucleotide analysis. One often overlooked application though, is its use in clinical biochemistry and toxicology for laboratory screening of inborn errors of metabolism (IEM).

The technique, which can be used with or without chromatographic separation, offers increased analytical sensitivity and selectivity by reducing the interference from other sample components. It also facilitates the development of better analytical methods for complex mixtures – ones that are rapid, require less painstaking sample preparation and separation (so they consume less solvent), and enable higher overall sample throughput.

MS/MS can be used to gain structural information about a compound by simple collision-induced dissociation of molecules, followed by scanning of the resultant fragment ions. When pieced together, this information allows researchers to generate a putative structure of the parent, or intact molecule. This feature is particularly important in toxicology and screening for drugs of abuse. However, MS/MS using stable isotope dilution, is a powerful quantitative tool in the majority of clinical laboratory diagnoses.

At the forefront of bioanalysis

IEM comprise a rare group of genetic disorders that can have serious clinical consequences for neonates, children, and adults. Left undiagnosed and untreated, they can cause physical disability, irreversible mental retardation, and neurological damage and, in certain cases, can be fatal. Although, as rare disorders their incidence is infrequent, their potentially devastating medical effect has made them of considerable significance to public health.

An expert in the field of MS/MS for IEM screening is Neil Dalton, professor of pediatric biochemistry, King’s College London, director of the WellChild Laboratory, Evelina London Children’s Hospital and a founding director of SpOtOn Clinical Diagnostics. Dalton is one of a number of experts who were instrumental in introducing the routine use of MS in newborn screening in the UK. “The role of an inherited metabolic disease laboratory is to provide accurate, rapid, and cost-effective screening and confirmatory diagnostic tests for an expanding range of rare disorders with common clinical presentations. The advent of generic diagnostics has allowed us to maximize disease coverage while minimizing costs, laboratory logistics, and the requesting dilemmas of clinicians,” he says.

The intrinsic analytical specificity of multiple reaction monitoring acquisition with MS/MS, combined with the quantitative precision of stable isotope dilution, has placed the technology at the forefront of bioanalysis. Nonetheless, the obvious impact of it on newborn dried blood spot (DBS) screening for inherited metabolic diseases is yet to be fully recognized – plasma/urine amino acid and urine organic acid analyses (and an increasing plethora of disease-specific tests) remain the diagnostic methods of choice.

Screening: the government stance

In the UK, governmental policy has supported research into extending the use of MS/MS as an IEM diagnostic, but progress has been slow. Back in 1997, the NHS R&D Health Technology Assessment (HTA) program commissioned two reviews of neonatal screening for inherited metabolic disorders (1,2). Both reviews called for further studies on the application of MS/MS for neonatal screening of IEM, with certain caveats. At the time, it was recommended that widespread introduction of the technology should be withheld pending further evaluation; a position supported by the Child Health Subgroup of the National Screening Committee (NSC). Since then, a 2004 systematic review, which incorporated economic modeling and built on the two HTA reports, has brought the evidence base more up-to-date (3). The review supported the introduction of MS/MS into the UK neonatal screening program for phenylketonuria (PKU) and medium-chain acyl-coA dehydrogenase deficiency (MCADD) combined, although the “wholesale inclusion of all disorders detectable by MS/MS” was not recommended. Currently all babies in England are screened for sickle cell disease, cystic fibrosis, congenital hypothyroidism as well as PKU and MCADD via expanded newborn DBS screening.

Searching for the evidence

Between 2004 and 2006 Carol Dezateux, her team at University College Hospital in London and numerous UK laboratories and metabolic services, participated in the UK Collaborative Study of Newborn Screening (UKCSNS), funded by the Department of Health and the NSC. In their MCADD study, they trialed a prospective multicenter pilot screening service with testing at age five to eight days, standardized screening, and management protocols using MS/MS (4). Around 60 babies each year (or one in every 10,000 born in England) is currently diagnosed with MCADD by newborn screening. The majority of MCADD variants detected during the trial were predicted to be of definite clinical importance, but this was found to vary according to ethnic group (with certain clinically important variants most commonly seen in Asian babies). The initial UKCSNS findings therefore supported MCADD screening, but highlighted the need to take into account the ethnic diversity of the population tested at implementation.

Another arm of the UKCSNS study has addressed how MCADD screening programs can estimate their sensitivity directly, because at present only individuals with a positive result undergo a definitive diagnostic test. The study group proposed a framework incorporating a Bayesian model, which simultaneously combined available prevalence data on the most common mutation of MCADD (c.985A>G) in screened and non-screened populations by using the relationship between true and apparent prevalence of disease to overcome this limitation (4). True prevalence of c.985A>G homozygotes in England was found to be 6.2 per 100,000 individuals and the sensitivity of the screening program was 94 percent (95 percent confidence interval [CI]: 74, 100 percent) compared with a detection rate in non-screened areas of 48 percent (95 percent CI: 30, 68 percent) by the age of 5 years. Hence, the screening program detected (95 percent CI: 30, 60 percent) an additional 47 percent of cases compared with no screening. The researchers concluded that, owing to the high sensitivity of England’s screening program, their estimation approach could be adapted to inform other rare diseases and national screening initiatives.

Furthermore, a 2014 review of mass spectrometry in clinical analysis (5) explained, that “there is consistent evidence of benefits from newborn screening for many disorders detected by MS/MS as well as for congenital hypothyroidism, cystic fibrosis, congenital adrenal hyperplasia by immune-enzymatic methods”. The author went on to explain that real-time PCR tests have been proposed for the detection of certain severe combined immunodeficiencies (SCID) along with MS/MS for adenosine deaminase deficiency (ADA) and purine nucleoside phosphorylase deficiency (PNP) SCID, although initial cost-benefit analyses are still ongoing. According to the author, fundamental to the success of a newborn screening program is using specific biomarkers to avoid false negatives and conducting second-tier tests to minimize the false positive rate.

A targeted metabolomic approach

Dalton explains that a targeted metabolomic approach using MS/MS would help rationalize the clinical diagnosis of IEM. According to him, at the Evelina London Children’s Hospital, the strategy has been to develop validated analytical methods for amino acids, acylcarnitines, organic acids, purines and pyrimidines, and disease-specific tests, which are then multiplexed into a single assay: essentially an IEM metabolite analyzer. The further diagnostic and research potential of this approach has been augmented by the recent introduction of an API6500 Q trap instrument, which has an increased linear range and true positive-negative ion switching, to include novel infection, specific organ function, and tumor biomarkers.

Dalton provides a genuine example of the rapid nature of MS/MS in practice: “The Evelina London received an acute IEM screen request over the phone for a baby with severe acidosis and hyperammonemia at another major London hospital at 15.10 one Friday. By 16.48, the sample had been transported to the laboratory, MS/MS analysis performed, and an Evelina London consultant had phoned the other hospital to report increased propionylcarnitine (m/z 218) with absence of methylmalonylcarnitine (m/z 262), normal methylmalonic acid and increased methylcitrate. A reported diagnosis of propionic acidemia was subsequently confirmed by enzymology results,” he explains. This illustrates the strength of MS/MS as a metabolomic approach to diagnose IEM – it facilitates rapid diagnosis, earlier treatment intervention, better patient outcome and immediate reassurance for the parents.

Expanding the screening program

In May 2014, the NSC announced its recommendation to screen every baby in the UK for an additional four IEMs as part of newborn blood spot screening (6). These diseases are maple syrup urine disease (MSUD), isovaleric acidemia (IVA), glutaric aciduria type 1 (GA1), homocystinuria (pyridoxine unresponsive) (HCU). Six of the 13 inherited metabolic disease laboratories in England took part in a pilot study and are already screening for these conditions; the other seven English laboratories began screening in January 2015 and Wales will also begin to screen early this year.

Dalton and his team support expanded IEM screening and believe that analytical platforms which can evaluate multiplexed metabolites, proteins and enzymes from a single DBS, would hold great value for this application.

Diagnostic sensitivity of these tests can be improved using new and established biomarkers and comparison of contentious biomarkers, for example in the case of type 1 tyrosinemia, porphobilinogen synthase activity versus tyrosine versus succinylacetone, or for homocystinuria, total homocysteine versus methionine.

Point proven?

Yvonne Daniel (lead scientist and operational lead – hematology, Viapath) and colleagues at the WellChild Laboratory at Guy’s & St Thomas’ NHS Foundation Trust (of which Dalton is a director) have developed an MS/MS method for newborn hemoglobinopathy screening. This screen is usually undertaken using HPLC or isoelectric focusing (IEF). The team devised a rapid and specific electrospray MS/MS method using multiple reaction monitoring (MRM) based peptide analysis, for simultaneous detection of the clinically significant hemoglobinopathies: hemoglobin (Hb)S, HbC, HbE, HbD-Punjab and HbO-Arab (7), and subsequently for the detection of Hb Lepore and HbA2 quantitation (8).

The method has been validated successfully by comparing its performance with the conventional IEF methods in 40,000 newborn DBS in collaboration with Leeds Teaching Hospitals NHS Trust. This study was commissioned by the NHS Sickle Cell and Thalassaemia Programme Centre to evaluate its potential in England’s newborn screening program. To facilitate uptake of the technique the number of processing steps required for conventional tryptic digestion of proteins was reduced and, together with a 30-minute digestion, sample preparation for MS/MS was modified so that it is no more onerous than for IEF. Furthermore, the consumable costs associated with MS/MS can be offset by high-throughput integrated with current IEM MS/MS screening. The specificity of MRM analyses implies that hemoglobinopathy detection can be limited to specified conditions – based on agreed screening policies – and can eliminate the need for costly and time-consuming second-line testing. In addition, subsequent product ion scanning on linear ion trap instruments provides unequivocal sequence data.

Dalton concludes, “There are obvious challenges that limit the introduction of routine clinical diagnostic multiplexed assays: calibration, quality control, technical competence, and interpretation skills. However, large scale metabolomic studies have not only served to emphasize these challenges but they’ve also demonstrated how analytically robust modern MS/MS platforms can be.”