Clinical chemistry became a field of science at the end of the 18th century (Berzelius, 1812; Rosenfeld, 2002). Many fundamental discoveries and developments have been made during the last two centuries, which enabled the use of analytical chemistry to detect diseases, and as a result, clinical laboratories have become an essential part of practice of medicine. It has been estimated that medical laboratory testing plays a significant role in 60–70% of all decisions related to establishing patients' diagnoses, and in selection and monitoring of treatments (http://www.mayo.edu/mshs/lab-career.html, 2008, accessed February 5, 2008). In part, this is related to the fact that many diseases have a similar clinical presentation, and testing often helps to differentiate between diseases, and leads to more efficient treatments and better outcomes.
The effectiveness of a diagnostic testing depends on the appropriateness of the utilized markers of diseases. Successful markers are features that could be objectively measured and evaluated as indicators of biological and pathological processes (Biomarkers Definitions Working Group, 2001; Sahab, Semaan, & Sang, 2007). Biomarkers can be anatomic, physiologic, or biochemical in nature and must be associated with a disease. To be medically useful, a biomarker must be detectable and measurable with objective techniques such as physical examination, imaging, or an analytical measurement. Biochemical markers are endogenous compounds that are either not present in a normal physiological state (e.g., certain tumor markers) or present within certain range of concentrations (e.g., intermediates and products of metabolic pathways). Biomarkers are important because accurate diagnoses and treatment monitoring make the foundation for successful outcomes. Biomarkers might serve for early diagnostic needs, as indicators of severity of a diseases, response to a treatment, recurrence of the diseases, or to determine patient's prognosis (Fig. 1).
A variety of biomarkers utilized in contemporary human diagnostics, range from DNA and RNA, to proteins, lipids, polysaccharides, and small molecules. One of the main tasks of genetics is to predict a difference in biological systems based on genetic variations. A single gene is translated into messenger RNA, which encodes multiple proteins. The proteome is more complex than the genome, because protein expression (with over 200 known post-translational modifications) might differ between different cells, at different times and physiological conditions. One example is a biomarker, alkaline phosphatase. There are at least four different alkaline phosphatase genes in humans: tissue-non-specific, intestinal, placental, and germ cell. The tissue-non-specific alkaline phosphatase occurs in at least three forms (liver, bone, kidney), differs in carbohydrate content, and is produced by post-translational processing of the same gene products. The relative distribution of the bone and liver alkaline phosphatases is age-dependent, and concentrations of the different forms of alkaline phosphatase correlate with a number of normal and pathologic conditions, including pregnancy, altered liver function, bone disorders, and certain tumors (Roberts et al., 2005).
Knowledge of the gene and protein expression is very useful, but alone it cannot detect and explain all phenotypic variations and changes. As a result of protein expression, many biochemical interactions and changes occur in living organisms and produce physiological functions that lead to the formation of active and inactive metabolites. These metabolites, along with proteins, often serve as markers of diseases and physiological conditions. Genetic and protein analyses provide information about systemic regulation on higher levels, whereas metabolites and intermediates of biochemical pathways represent at a physiological response to the regulation.
The Role of Mass Spectrometry in Clinical Diagnostic Laboratories
Modern clinical laboratories use diverse techniques and instrumentation that vary in reliability and specificity. Since the introduction of tandem mass spectrometry in clinical laboratories, it proved to be one of the most specific analytical techniques available for clinical diagnostics.
Clinical applications of tandem mass spectrometry could be sub-divided into two groups: screening and target analysis. Typically, screening methods are intended to detect multiple markers of diseases, drugs, or toxins (e.g., newborn screening for metabolic diseases, toxicology screening). In these applications, the goal is to achieve high throughput of testing and a low false-negative rate. In target analysis, the main focus is on accurate and precise quantitation and the assurance of the analyte identity.
In all analytical applications measurement accuracy is important. Errors encountered in the clinical diagnostics, however, are especially costly compared to other fields, because they might lead to a misdiagnosis, mistreatment, patient injury, and even to the loss of life (Plebani and Carraro, 1997; Witte et al., 1997; Plebani, 2006). Therefore, highly specific methods are required for clinical diagnostic testing, and strict guidelines must be followed with respect to the quality control (QC) and management of pre- and post-analytic variables.
The challenges of clinical diagnostic testing are related to the complexity of the biological samples, the large diversity of classes of molecules present in the samples, variability of the sample matrices among individuals, and the wide range of concentrations of the constituents in the samples. Figure 2 (based on Roberts et al., 2005) shows some of the clinically useful diagnostic biomarkers along with their biologically relevant concentrations, which span over 10 orders of magnitude. Such a diversity of concentrations suggests a need for highly sensitive and specific instruments to enable an accurate measurement of minor sample constituents in the presence of excessive amounts of other endogenous substances.
The main emphasis of this review is on mass spectrometry-based diagnostic methods, which were developed in our laboratories for the measurement of biochemical markers of endocrine and metabolic diseases.