Volume 31, Issue 3

Surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF-MS) is one of the most important proteomic technologies currently available for the high-throughput analysis of complex protein samples. In the last few years, several studies have demonstrated that comparative protein profiling using SELDI-TOF-MS breaks new ground in diagnostic protein analysis, particularly with regard to the identification of novel biomarkers. Importantly, researchers have acquired a better understanding of the limitations of this technology and the pitfalls in biomarker discovery. Bearing these limitations in mind, great emphasis must be placed on the development of rigorous standards and quality control procedures for the preanalytical and analytical phases and subsequent bioinformatics applied to analysis of the data. To avoid the risk of false significant results, studies have to be carefully designed and control groups have to be accurately selected. In addition, data should be analyzed with appropriate tools already established for analysis of highly complex microarray data. Finally, validation of the significance of any biomarker candidate derived from pilot studies is mandatory in appropriately designed prospective multi-centre studies; the reproducibility of the clinical results must be shown over time and in different diagnostic settings. SELDI-TOF-MS-based studies that are in compliance with these requirements are now required and few have been published so far. In the meantime, further evaluation and optimization of both technique and marker validation strategies are called for before MS-based proteomic algorithms can be translated into routine laboratory testing.

Decades after its first description by Berg in 1963, lipoprotein(a) [Lp(a)] is an established independent risk marker of cardiovascular diseases. The main difference between Lp(a) and LDL is the apo(a) residue which is covalently bound to apoB. Apo(a) is a glycoprotein displaying a large genetic polymorphism. The latter is caused by variations of the kringle 4-type-2 repeats of the protein, characterized by structural homology to plasminogen. Lp(a) plasma concentrations in the population are highly skewed and are predominantly determined (>90%) by genetic factors. In healthy subjects, Lp(a) concentrations are associated with its synthesis rather than its catabolism. Plasma concentrations of Lp(a) are affected by disease states (e.g., diseases of liver and kidney), hormonal factors (e.g., sex steroids, glucocorticoids, thyroid hormones), and individual and environmental factors (e.g., age, smoking), as well as drugs (e.g., nicotinic acid) and therapeutic intervention (lipid apheresis). Even though a large number of studies on Lp(a) have been performed, many details of its physiological function and regulation of plasma concentrations are not yet understood. In addition, studies demonstrated contradictory results owing to large differences in patients included, the use of insufficiently validated tests, and analysis of frozen samples. The aim of this review is to describe the function and physiological regulation of Lp(a), as well as therapeutic factors influencing its plasma concentration.

Asymmetric dimethyl arginine (ADMA) is an endogenous amino acid that inhibits all three isoforms of nitric oxide synthase. High concentrations of ADMA in endothelial cells interfere with release of the vasodilator signalling molecule nitric oxide and may therefore facilitate the development of atherosclerotic lesions. It has been shown that ADMA accumulates in plasma in parallel with the progression of chronic renal failure, diabetes mellitus, hypertension and coronary artery disease. The concentration of ADMA in plasma is low, while a number of structurally similar compounds occur at much higher concentrations. Determination of ADMA hence remains an analytical challenge. Pathological concentrations should always be considered by comparison with a suitable control group in which ADMA has been determined using identical methodology. This article summarizes the current state of knowledge on the biological effects, production, metabolism and elimination of ADMA. Exemplified by original results, the importance of ADMA as a prognostic factor for cardiovascular and total mortality is discussed.

Free fatty acids (FFAs) serve as physiologically important energy substrates and their release from adipose tissue by lipolysis is regulated according to the energy demands of the body. Blood FFA concentrations are increased in obese patients and contribute to type 2 diabetes, hepatic steatosis and several cardiovascular disorders. In patients with heart failure and acute coronary syndrome, elevated FFA concentrations are considered a consequence of increased lipolysis due to the action of catecholamines and natriuretic peptides. FFAs contribute to myocardial dysfunction and are proarrhythmic, and their oxidation requires more oxygen compared to the metabolic use of glucose. Therapeutic approaches involving so-called metabolic therapy have emerged for coronary artery disease and heart failure that aim to reduce the metabolism and/or oxidation of fatty acids in the myocardium. The routine measurement of FFAs as a diagnostic tool is limited by their pronounced variability, which is strongly influenced by nutrition and the impact of hormones. In addition, it remains to be clarified whether fasting or postprandial levels or dynamic measurements, including changes in FFA concentrations induced by stress, are the preferred parameter for evaluating cardiovascular risk. In this review, we present an overview of the metabolism and role of FFAs as a cardiovascular risk factor and discuss the potential of FFAs in the diagnosis and treatment of cardiovascular disorders.

In Europe the multi-system disorder Lyme borreliosis is caused by at least four different Borrelia burgdorferi sensu lato species. Therefore, genetic and antigenic heterogeneity is a major challenge for development of diagnostic methods. Serological diagnosis should follow the principle of a two-step procedure: for screening a sensitive IgM and IgG differentiating ELISA is recommended that should be confirmed by immunoblot in the case of reactivity. Detection of specific antibodies can only be judged if the immunoblot is positive. Sensitivity of antibody detection is 20%–50% in stage I, 70%–90% in stage II, and nearly 100% in stage III. The use of recombinant antigens – e.g., primarily in vivo expressed proteins such as VlsE or homologues of immunodominant antigens of different strains such as DbpA – leads to an improvement in serological diagnosis. Detection of the aetiological agent from skin biopsies, cerebrospinal fluid, and synovial fluid or synovial tissue by culture or PCR should be confined to specific indications. Sensitivity is 50%–70% for skin biopsies and synovial fluid (the latter only by PCR), and 10%–30% for cerebrospinal fluid. Methods that are not recommended for diagnostic purposes include antigen tests in body fluids, PCR for urine or whole blood/serum, lymphocyte transformation test, or testing of ticks removed from humans to deduce an indication for antibiotic prophylaxis.

Borrelia -specific antibodies are not detectable until several weeks after infection and their presence alone is not proof of an active infection. The sensitivity of culture methods and PCR for the confirmation or exclusion of Lyme borreliosis is too low. Therefore, a method is required that detects an active Borrelia infection as early as possible. For this purpose, a lymphocyte proliferation test (LPT) using three endogenous Borrelia antigens ( Borrelia burgdorferi sensu stricto , Borrelia afzelii , and Borrelia garinii ) and recombinant OspC was developed and validated by investigating seronegative (n=100) and seropositive (n=36) healthy individuals, as well as seropositive (n=44) patients with clinically overt borreliosis. The sensitivity of the Borrelia LPT in clinical borreliosis before the administration of antibiotic treatment was 91%, while the specificity was 94%. In 820 patients with clinical suspicion of borreliosis, positive and negative results by serology and LPT were in agreement in 77.3% of cases. Some 165 patients (20.1%) were serologically positive and negative by LPT. These were mainly patients with borreliosis after antibiotic therapy. Furthermore, 21 patients (2.6%) had negative serology and a positive LPT result, seven of whom had erythema migrans. Following antibiotic treatment, the LPT becomes negative or borderline in patients with early manifestations of borreliosis, whereas in patients with late symptoms it demonstrates a regression while remaining positive. Follow-up investigations over a period of 1 year yielded one reactivation among six patients with early manifestations, in contrast to eight out of ten patients with late symptomatology that exhibited frequent episodes of reactivation and/or persistently positive LPT reactions. Therefore, we propose follow-up monitoring of disseminated Borrelia infections as the main indication for the Borrelia LPT.