Analytical challenges in the detection of peptide hormones for anti-doping purposes

Growth hormone (GH) and insulin-like growth factor I (IGF-I) measurements are widely used in the diagnosis of disorders of GH secretion, evaluation of children with short stature from multiple causes, management of disorders that lead to nutritional insufficiency or catabolism, and monitoring both GH and IGF-I replacement therapy. Therefore, there is an ongoing need for accurate and precise measurements of these 2 peptide hormones. Representatives of the Growth Hormone Research Society, the IGF Society, and the IFCC convened an international workshop to review assay standardization, requirements for improving assay comparability, variables that affect assay interpretation, technical factors affecting assay performance, assay validation criteria, and the development and use of normative data. Special attention was given to preanalytical conditions, the use of international commutable reference standards, antibody specificity, matrix requirements, QC analysis, and interference by binding proteins. Recommendations for each of these variables were made for measurements of each peptide. Additionally, specific criteria for IGF-I were recommended for age ranges of normative data, consideration of Tanner staging, and consideration of the effect of body mass index. The consensus statement concludes that major improvements are necessary in the areas of assay performance and comparability. This group recommends that a commutable standard for each assay be implemented for worldwide use and that its recommendations be applied to accomplish the task of providing reliable and clinically useful results.

Abstract The surface of all mammalian cells is covered with a dense and complex array of sugar chains, which are frequently terminated by members of a family of molecules called sialic acids. One particular sialic acid called N‐glycolylneuraminic acid (Neu5Gc) is widely expressed on most mammalian tissues, but is not easily detectable on human cells. In fact, it provokes an immune response in adult humans. The human deficiency of Neu5Gc is explained by an inactivating mutation in the gene encoding CMP‐N‐acetylneuraminic acid hydroxylase, the rate‐limiting enzyme in generating Neu5Gc in cells of other mammals. This deficiency also results in an excess of the precursor sialic acid N‐acetylneuraminic acid (Neu5Ac) in humans. This mutation appears universal to modern humans, occurred sometime after our last common ancestor with the great apes, and happens to be one of the first known human‐great ape genetic differences with an obvious biochemical readout. While the original selection mechanisms and major biological consequences of this human‐specific mutation remain uncertain, several interesting clues are currently being pursued. First, there is evidence that the human condition can explain differences in susceptibility or resistance to certain microbial pathogens. Second, the functions of some endogenous receptors for sialic acids in the immune system may be altered by this difference. Third, despite the lack of any obvious alternate pathway for synthesis, Neu5Gc has been reported in human tumors and possibly in human fetal tissues, and traces have even been detected in normal human tissues. One possible explanation is that this represents accumulation of Neu5Gc from dietary sources of animal origin. Finally, a markedly reduced expression of hydroxylase in the brains of other mammals raises the possibility that the human‐specific mutation of this enzyme could have played a role in human brain evolution. Yrbk Phys Anthropol 44:54–69, 2001. © 2001 Wiley‐Liss, Inc.

The development of mass spectrometry (MS)-based methodologies for high-throughput protein identification has generated a concomitant need for protein quantification. Numerous MS-based relative quantification methodologies have been dedicated to the extensive comparison of multiple proteomes. On the other hand, absolute quantification methodologies, which allow the determination of protein concentrations in biological samples, are generally restricted to defined sets of proteins. Depending on the selected analytical procedure, absolute quantification approaches can provide accurate and precise estimations. These analytical performances are crucial for specific applications such as the evaluation of clinical biomarker candidates. According to bioanalytical guidelines, accurate analytical processes require internal standards and quality controls. Regarding MS-based analysis of small molecules, isotope dilution has been recognized as the reference method for internal standardization. However, protein quantification methodologies which rely on the isotope dilution principle have been implemented in the proteomic field only recently. In these approaches, the sample is spiked with defined amounts of isotope-labeled analogue(s) of specific proteolytic peptide(s) (AQUA and QconCAT strategies) or protein(s) (PSAQ strategy). In this review, we present a critical overview of these isotope dilution methodologies.