Clinical Metabolomics: the next stage of clinical biochemistry

Introduction

Centuries of scientific advances have paved the way for the relatively recent great strides in clinical biochemistry, a field which mainly relies upon biochemical analyses of various body fluids, prime amongst which are urine¹, blood and cerebrospinal fluid². Technological innovation, through the introduction of cutting edge instrumentation has enabled decades of substantial improvements in the field of standard analytical chemistry in the clinical setting.

At its dawn, clinical biochemistry relied on rudimental approaches, such as in the case of Richard Bright’s (1789–1858) test for proteinuria in cases of suspected renal disease, in which a candle flame was used to heat urine in a tablespoon³.

Only minor technological improvements could date back to the early twentieth century. As reported by Olukoga et al.⁴, the equipment of a clinical pathology laboratory within a 200-bedded American hospital in 1920 listed “a centrifuge, a urinometer, two monocular microscopes, two small substage microscope lights, a Bunsen burner, a Dubosq colorimeter, a basal metabolic rate machine, an electro-cardiograph, a microtome, a knife, a paraffin bath, a few antisera and an assortment of test tubes, beakers and pipettes”.

The dawn of blood collection

Other than testing, collection of blood samples was challenging as well, since only small blood volumes could be obtained by finger prick or either collected by “cut-down” to expose the vein, with subsequent venesection and cupping⁶. Indeed, the first hypodermic needle was created in 1840s by Francis Rynd for local injection of opiate in the treatment of neuralgia⁷: it was made of steel and accompanied by a hard rubber hub. Subsequent strides in the field of blood collection are to be attributed to the introduction of new syringe materials for the collection tube, since the rubber was replaced with glass to allow syringes to be reused. Finally, the Luer-Lok syringe provided a convenient method of attaching and removing the hypodermic needle from the glass syringe⁴.

Collection tubes containing small volumes of additives (e.g. anticoagulants) have represented a standard in blood collection procedures since their first appearance in the 1950s. Along the last fifty years, manufacturers have introduced only minor modifications to collection tubes, including the use of plastic as the primary tube component, and the addition of polymer gel or clot activator⁸.

Early analytical methods

In the history of clinical chemistry, separation technologies held a key role, with the centrifuge being invented in 1883 by the Swedish engineer Carl Gustav Patrik de Laval (1845–1913)^4,9.

Further advancements in analytical chemistry were then due to the theoretical and practical foundation of emission spectroscopy (a technique could be used to identify elements by means of the characteristic spectra of their free atoms), which dates back to the 1820s when John Frederick William Herschel (1792–1871) and Talbot^10,11. However, it was only in 1860 that this phenomenon could be fully explained by Robert Bunsen and Gustav Robert Kirchoff (1824–1887). Therefore, early strides in analytical methods in clinical biochemistry mostly stem from the brilliant work of Robert Wilhelm Bunsen (1811–1899), who introduced spectroanalysis and the concept of coefficient of extinction and, thereby, spectroscopy, which allowed detecting the various spectrogenic pigments in blood^4,9.

Analytical application of emission spectroscopy can be attributed to the work of Henrik Gunnar Lundgardh (1888–1969), who introduced the flame photometer for a direct estimation of the concentrations of specific elements. The rudimental version of the photometer was based on a mixture of air and acetylene (energy source), while the emitted light was dispersed by a quartz prism and captured on a photographic plate⁴. This instrument was used to perform electrolyte determinations in body fluids, overcoming the cumbersome titrimetric or colorimetric assays which had hitherto represented the traditional approach. Nevertheless, it was only in 1955 that the technique was applied in a clinical setting for quantitative elemental analysis¹².

Other than spectroscopy, electrophoresis is one of the most widely diffused methods to investigate proteins in the clinical setting. The first electrophoresis apparatus was devised in 1937 by Arne Wilhelm Tiselius (1902–1971)¹³.

Finally, chromatography was introduced by the Russian botanist Mikhail Tsvett (1872–1919) who described the absorption chromatography in 1906 in the frame of a research based upon the separation of plant pigments into their constituent parts¹⁴.

Despite analytical methods were available decades before, it was only with the introduction of automation by Leonard Skeggs (1957) that clinical chemistry started taking actual advantage of these analytical approaches¹⁵. As reviewed by Rocks and Riley¹⁶, the AutoAnalyzer was characterized by a single-channel, continuous flow, batch analyser that provided one result per analyte for each specimen at a rate of 40–60 specimens per hour^15,16.

However, greater number of samples increase the complexity of the data handling process (collection, validation and interpretation) in the clinical setting, which represented a challenge until the introduction of computers and ad hoc software into laboratory work. In recent times (last two decades), the capillarity of internet connections has thus allowed further easing centralization and distribution of clinical data.

Despite the above mentioned implementations in the field of clinical chemistry, the introduction of novel technologies, such as metabolomics will likely enough add up to the analytical strategies currently at disposal of clinical experts.

Metabolomics

One of the main analytical advancements over the last decades has been represented by the introduction of Omics disciplines, that is to say those disciplines which investigate only certain classes of biomolecules in their entirety in biological matrices¹⁷. Omicoriented strategies have been designed as to delve into biological complexity as a whole (e.g. proteins in proteomics, mRNAs in transcriptomics), rather than dissecting biological samples through targeted analysis of single molecules¹⁷. While at the beginning of the third millennium genomics (investigating the whole genome compartment) represented perhaps the leading science, during the last ten years it has been possible to observe the dramatic expansion of the fields of proteomics (proteins), lipidomics (lipids) and metabolomics (metabolites)^18–27. Metabolomics investigates the metabolome within a specific biological matrix (biological fluid, tissue, cells), that is to say the molecular complement to the genome and proteome below the 1.5 kDa range¹⁹. Being closer to the phenotype than any other omics discipline, metabolomics and metabolic patterns have been in depth investigated in many fields of basic and applied research, including toxicology³¹, pharmaceutical research^32–35 and fertility research^36–39. More recently, a role has been proposed for metabolomics in clinical biochemistry^18–27 and personalized medicine^40–50, in that whether an experimental connection will emerge between metabolome profiles and specific diseases, metabolomics could soon become a reliable and robust analytical approach in predictive medicine. Indeed, the origins of metabolomics share consistent traits with clinical biochemistry, which has historically pursued determination of standard and anomalous parameters (i.e. absolute concentration, relative abundance, etc.) of small molecular compounds in blood and its components (plasma/serum and cellular fractions).

Metabolomics: historical perspectives and future directions

Citing Roux et al.²⁰, “biochemists have long been doing metabolomics, just like the Bourgeois Gentilhomme was speaking prose without knowing it” (Molière - Bourgeois Gentilhomme II. 4).

Despite early applications in 1960’s, it was only in 1971 that Pauling, Robinson et al. conceived the core concept of modern metabolomics, which posits that “information-rich data reflecting the functional status of a complex biological system resides in the quantitative and qualitative pattern of metabolites in body fluids”⁵¹.

The metabolome is also referred to as the set of small molecular mass organic compounds found in a given biological media, which includes endogenous compounds (all organic substances naturally occurring from the metabolism of the studied living organism), and xenobiotics (and their catabolic products). Metabolic analyses at first relied upon nuclear magnetic resonance (NMR), although recent improvement in the field of mass spectrometry (MS) made available two complementary methods which allow detecting from a few hundreds to thousands of signals related to both genetic and environmental contributions²¹. Each technique holds specific advantages over the other: while NMR was favoured by (i) machine accessibility; (ii) established data handling; and (iii) the nondestructive nature of the analysis; MS has gradually complemented NMR owing to its (i) higher sensitivity; (ii) improved metabolite discrimination; (iii) coverage of the metabolome space; and (iv) modularity to perform compound-class-specific analyses; other than to (v) a dramatically reduced demand for starting material necessary to perform an extensive analysis^52,53. MS also allows to perform (vi) targeted analyses, through monitoring of one (or a handful) of metabolites through isolation and fragmentation of precursor ion and subsequent isolation of the fragmented transitions, a workflow which is known as selected/multiple reaction monitoring (SRM or MRM)⁵⁴. Direct monitoring of specific metabolites allows quantitation throughout a wide spread range of linear concentrations (from mM to nM, down to picomole quantities, depending on the characteristics of the MS instruments) and results in less demanding requirements for analyte volumes (0.5 μl as in the case of blastocoele fluid³⁶).

The recent improvements in the field of metabolomics are not only to be attributed to technical advancements, but also to the creation of specific software and databases which now allow mapping and interpreting metabolic fluctuations with relative ease^55–57. These improvements have opened brand new scenarios in the field of red blood cell investigations (basic science systems biology^58–60, red blood cell cold liquid-^61,62 or cryo-storage⁶³ for transfusion purposes), being erythrocyte both one of the simplest biological cell models and a unique treasure trove of either direct or indirect biological signatures (biomarkers) for most various diseases and pathological conditions.

Integrated Omics and Clinical Chemistry - the four paths of the new couple or the guidelines for a happy marriage

One main goal in the ambitious agenda of both metabolomics (or “omics”, in general) researchers and clinician experts is to rapidly configure a rendez-vous point and propose future directions which deserves further joint explorations. Hereby we propose four main objectives (Figure 1) that are already technically feasible and could be at hand within the next few years.

Figure 1.

An overview of the likely applications of metabolomics and other omics disciplines in the future of clinical chemistry and transfusion medicine.

Nanotechnology

Theoretical and technical improvements have recently allowed separation of biological compounds at the nanoscale. Rapid resolution HPLC approaches of nano-fluxes and MS instruments with over 1×10⁶ resolving power⁶⁴ have boosted Omics disciplines, including metabolomics, which could soon be elicited as investigative approaches indicating biomarkers to implement nanosensors⁶⁵ to most various biological compounds (in like fashion to glucose nanosensors for diabetes⁶⁶), thus dramatically enhancing the sensitivity of clinical chemistry assays.

Predictive medicine

Enhanced sensitivity and improved metabolite coverage also translates into faster and more accurate predictive capacity. In a society where ageing is rapidly becoming the main challenge of 21^st century both in western and rapidly developing countries, it is becoming mandatory to tackle the life-long quality issue. In this frame, prevention and therefore predictive medicine have been erected as the watch-tower in the strive to guarantee a better quality life for a longer period to the greatest possible portion of the population worldwide. Early diagnoses indeed result in more effective therapies and lower costs for the whole healthcare systems, and could pave the way for a healthier (other than older) working population.

Nutraceuticals

When it comes to prevention, a balanced diet is pivotal. Since we are what we eat, technologies which guarantee food safety (such as proteomics and metabolomics⁶⁷) are not to be underestimated. It is also worthwhile to stress that food contains bioactive compounds (nutraceuticals) which have been shown to play a central role in each and every aspect of our life, including health pitfalls such as metabolic homeostasis and tumor prevention. From antioxidant vitamins to flavonoids and polyphenols, curcumin and resveratrol (for a detailed review the interested reader is referred to D’Alessandro et al.⁶⁸) everything we eat might hold potential benefits for our health. Metabolomics and omics-oriented investigations could help clinicians further our understanding of those molecules which have positive effects on our health and shed light on the molecular mechanisms behind, in order to design more balanced diets or envisage nutraceutical-enriched food.

Regenerative medicine

Where diagnoses and routine therapies will not succeed, regenerative medicine might represent an eligible solution. Application of trans-differentiated adult stem cells has been growingly playing a role in transplantation treatments (as in the case of peripheral blood hematopoietic stem cells⁶⁹) and will likely represent a valid alternative strategy in the transfusion setting as well⁷⁰. Metabolomics and other omics disciplines (proteomics, lipidomics) will represent a valid asset in the assessment of the correct transdifferentiation of stem cells in the biological path towards the acquisition of the desired phenotype.

Conclusions

In the next few years, we will testify a big stride in the field of clinical chemistry, since the valuable expertise accumulated in laboratory science (mostly in the field of “omics” disciplines, such as metabolomics, and their integration in “Systems biology”¹⁹) will endow clinicians with new and powerful analytical technologies. In this ambitious agenda, the four yet undisclosed paths hereby proposed (nanotechnology, predictive medicine, nutraceuticals and regenerative medicine) will likely become the pillars of a new era, the next stage of clinical chemistry.