CHROMATOGRAPHY–MASS SPECTROMETRY:
SIGNIFICANCE AND PERSPECTIVE
Hormonal analysis is indispensable to monitoring endocrine diseases. In the diag- nosis of enzyme defects of steroid biosynthesis, hormonal testing and molecular
336 Wudy et al.
biology have been shown to constitute important complementary techniques.
Therefore, the development and improvement of reliable quantitative analytical methods for hormone determination presents a decisive field of endocrinology.
In the field of steroid analysis, analytical techniques based on MS offer highest specificity. Therefore, the major advantage of GC–MS steroid profiling is the high degree of proof for every steroid analyzed.
Urinary steroid profiling using GC–MS is a nonselective multicomponent analysis of very high diagnostic potential. It allows definitive delineation of prac- tically all disorders of steroid metabolism. It is best suited as a confirmatory technique after positive screening values (elevated 17α-hydroxyprogesterone) in screening neonates for 21-hydroxylase deficiency. Furthermore, the technique is noninvasive and rapid. The constellation of urinary steroid metabolites allows the diagnosis of most steroid-related disorders from spot urine samples, whereas determination of excretion rates of steroid metabolites requires 24-hr urine sam- ples. In newborns and patients with steroid-secreting tumors, unusual steroids are produced for which specific serum assays are not available, but their metabo- lites can be monitored by urinary profiling.
The potential of ID-GC–MS in clinical quantitative plasma steroid analysis has not yet been exploited to its full extent. Drawbacks concerning the use of ID-GC–MS in a clinical setting have primarily been the huge cost of the sophisti- cated instrumentation, complex sample workup procedures, and the unavailability of most stable isotope–labeled internal standards. However, during the last de- cade, vast technical improvements have rendered the development of reliable labor- and cost-effective GC–MS instruments possible. With respect to appro- priate internal standards, several suitable pathways leading to nonradioactive in- ternal standards have been published. Our method for profiling seven steroids by ID-GC–MS has been found to be clinically applicable. Regarding the possible number of samples to be analyzed, GC–MS cannot compete with direct immuno- assays. The latter techniques require only minimal sample preparation and allow analysis of numerous samples in batch assays in contrast to only serial assays possible with GC–MS. Plasma steroid analysis by GC–MS will not replace im- munoassays in general. Besides its role as a reference methodology, we suggest application of ID-GC–MS in a clinical setting, whenever problems from matrix effects or cross-reactivity are likely to arise, and/or when suspicious results need to be rechecked. The GC–MS technique should provide the chance of a comple- mentary analytical technology with highest specificity.
To keep costs within a reasonable limit, we suggest establishing priorities for urinary and plasma steroid analysis by GC–MS. Thus, steroid analyses are at best carried out in a small number of specialized supra-regional laboratories (reference centers) equipped with the analytical instrumentation and where sup- port of specialist biochemists or clinicians is available.
Over the last decade, steroid determination by immunoassays has faced
Clinical Steroid Analysis 337
considerable loss of reliability due to preference of direct immunoassays. Most assays currently available cannot be used in analytically critical periods such as the neonatal period. For steroids rarely requested, hardly any assays are offered any more. This development has been favored by uncritical efforts of saving money in the biomedical disciplines. In this context, it is important to point out that clinical steroid analysis by GC–MS fulfills all criteria of a good clinical assay. It still has to be realized that quality is not equivalent to luxury.
ACKNOWLEDGMENTS
Stefan A. Wudy and Janos Homoki acknowledge the research grants that have been awarded to them from the Deutsche Forschungsgemeinschaft (DFG). The authors gratefully acknowledge the help of their Ph.D. students Michaela Hart- mann, Claudia Solleder, and Ulrich Wachter. The skilled technical assistance of Heide Pinzer and Edith Ambach, the graphical support of Frank Wo¨rsinger and the expert secretarial work of Heidrun Richter are gratefully acknowledged.
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13
Gas Chromatography–Mass Spectrometry for Selective Screening for Inborn Errors of Metabolism
Jo¨ rn Oliver Sass
Leopold Franzens University Innsbruck, Innsbruck, Austria Adrian C. Sewell
Johann Wolfgang Goethe University, Frankfurt, Germany
1. INTRODUCTION
Inborn errors of metabolism represent a group of diseases, which are individually rare, but collectively numerous [1]. In order to detect treatable inborn diseases as early as possible, during the last decades most European and North American countries have established neonatal laboratory screening tests, which search only for a small number of inborn errors of metabolism [2]. However, for the great majority of metabolic diseases, biochemical investigations are only performed in selected cases, after clinical symptoms or family history have indicated that a metabolic disease might be present (selective screening). Selective screening may involve the analysis of amino acids in plasma and urine, organic acids, purines, pyrimidines, oligosaccharides, and mucopolysaccharides in urine; as well as free carnitine, acylcarnitines, and very long-chain fatty acids in plasma. Among the positive results of selective screening, detection of organic acidurias accounts for a major portion [3]. More than 50 phenotypically different diseases are known to present with organic aciduria at least during metabolic decompensation [4].
A variety of clinical features are known. Patients may present with clinical symp- toms including vomiting, muscular hypotonia, failure to thrive, or a sepsis-like 341
342 Sass and Sewell
picture. If an organic aciduria is suspected on clinical grounds, organic acid analy- sis is a vital part of the investigations and represents the main application of gas chromatography–mass spectrometry (GC–MS) in selective screening.
Organic acids, as understood in this chapter, are intermediates in the degra- dative metabolic pathways of amino acids, fats, and carbohydrates. Organic acids contain one or more carboxylic or acidic phenolic groups, but do not contain any primary amino groups which might react with ninhydrin. Therefore, although their transamination products (oxoacids) are addressed, amino acids per se are not considered here. Very long-chain fatty acids and inorganic metabolites are likewise not considered. Organic acids may also result from exogenous sources, such as diet, drugs, or bacterial contamination. In addition to the naturally oc- curring urinary organic acids which may be increased in various disease states, there are many abnormal acids, the presence of which is indicative of a particular disease (e.g., propionic acidemia) (Fig. 1), together with glycine [5] and glucuro- nide conjugates [6].
Since most organic acids undergo efficient renal excretion and urine is often obtained more easily from children than other body fluids, urine represents the preferred sample for organic acid analysis. However, in special cases or if no other sample should be available, plasma, serum, cerebrospinal fluid, or vitreous
Figure 1 Urinary organic acid chromatogram of a patient with propionic acidemia. Peak A is 3-hydroxypropionic acid, B is 2-methyl-3-hydroxybutyric acid, C is 3-hydroxy-iso- valeric acid, D is 3-hydroxy-n-valeric acid, peaks E represent propionylglycine, peak F is tiglylglycine, peaks G are methylcitric acid. IS⫽internal standard (tricarballylic acid).
Inborn Errors of Metabolism 343
humor (the last obtained post mortem) can also be analyzed. Urinary concentra- tions of organic acids are usually adjusted for the creatinine content of a sample.