The plasma lipidome in acute myeloid leukemia at diagnosis in relation to clinical disease features.

BACKGROUND: Early studies established that certain lipids were lower in acute myeloid leukemia (AML) cells than normal leukocytes. Because lipids are now known to play an important role in cell signaling and regulation of homeostasis, and are often perturbed in malignancies, we undertook a comprehensive lipidomic survey of plasma from AML patients at time of diagnosis and also healthy blood donors. METHODS: Plasma lipid profiles were measured using three mass spectrometry platforms in 20 AML patients and 20 healthy blood donors. Data were collected on total cholesterol and fatty acids, fatty acid amides, glycerolipids, phospholipids, sphingolipids, cholesterol esters, coenzyme Q10 and eicosanoids. RESULTS: We observed a depletion of plasma total fatty acids and cholesterol, but an increase in certain free fatty acids with the observed decline in sphingolipids, phosphocholines, triglycerides and cholesterol esters probably driven by enhanced fatty acid oxidation in AML cells. Arachidonic acid and precursors were elevated in AML, particularly in patients with high bone marrow (BM) or peripheral blasts and unfavorable prognostic risk. PGF2α was also elevated, in patients with low BM or peripheral blasts and with a favorable prognostic risk. A broad panoply of lipid classes is altered in AML plasma, pointing to disturbances of several lipid metabolic interconversions, in particular in relation to blast cell counts and prognostic risk. CONCLUSIONS: These data indicate potential roles played by lipids in AML heterogeneity and disease outcome. GENERAL SIGNIFICANCE: Enhanced catabolism of several lipid classes increases prognostic risk while plasma PGF2α may be a marker for reduced prognostic risk in AML.

Liquid chromatography-tandem mass spectrometry for the analysis of eicosanoids and related lipids in human biological matrices: a review.

Today, there is an increasing number of liquid chromatography tandem-mass spectrometric (LC-MS/MS) methods for the analysis of eicosanoids and related lipids in biological matrices. An overview of currently applied LC-MS/MS methods is given with attention to sample preparation strategies, chromatographic separation including ultra high performance liquid chromatography (UHPLC) and chiral separation, as well as to mass spectrometric detection using multiple reacting monitoring (MRM). Further, the application in recent clinical research is reviewed with focus on preanalytical aspects prior to LC-MS/MS analysis as well as applications in major diseases of Western civilization including respiratory diseases, diabetes, cancer, liver diseases, atherosclerosis, and neurovascular diseases.

Effect of biobanking conditions on short-term stability of biomarkers in human serum and plasma.

BACKGROUND: Liquid biobanking is an important tool for laboratory diagnostics in routine settings and clinical studies. However, the current knowledge about adequate storage conditions for different classes of biomarkers is incomplete and, in part, contradictory. Here, we performed a comprehensive study on the effects of different storage conditions on the stability of various biomarkers in human serum and plasma. METHODS: Serum and citrated plasma were aliquoted and stored at 4 °C, -20 °C, -80 °C, and <-130 °C for 0, 7, 30, and 90 days, respectively (5-10 pools/condition). Additionally, frozen aliquots were temporarily exposed to higher temperatures during storage to simulate removing individual samples. Stability was tested for 32 biomarkers from 10 different parameter classes (electrolytes, enzymes, metabolites, inert proteins, complement factors, ketone bodies, hormones, cytokines, coagulation factors, and sterols). RESULTS: Biobanking at -80 °C and <-130 °C for up to 90 days did not lead to substantial changes (defined as >3 interassay coefficients of variation and p<0.01) of any biomarker concentration. In contrast, storage at 4 °C and -20 °C induced substantial changes in single biomarker concentrations in most classes. Such substantial changes were increases (<20%) in electrolytes, metabolites, and proteins, and decreases (<96%) in enzymes, ketone bodies, cytokines, and coagulation factors. Biomarker stability was minimally affected by occasional short-term thermal exposure. CONCLUSIONS: Based on these results, we provide recommendations for storage conditions of up to 90 days for several biomarkers. Generally, storage at ≤-80 °C for at least 90 days including occasional short-term thermal exposure is an excellent storage condition for most biomarkers.

Quantification of seven apolipoproteins in human plasma by proteotypic peptides using fast LC-MS/MS.

PURPOSE: We investigated different sample pretreatment strategies and developed a standardized sample pretreatment protocol for absolute quantification of seven apolipoproteins (Apos) in human serum by LC-MS/MS using proteotypic peptides and corresponding stable isotope-labeled peptides as internal standards. EXPERIMENTAL DESIGN: Micro-LC was coupled with quadrupole-linear ion trap MS for quantification and peptide confirmation. Denaturation, reduction, alkylation, and tryptic digestion including ultrasound and microwave assistance were investigated. Method comparison of 50 plasma samples with an immunoassay was performed for Apo A-I and Apo B. RESULTS: Tryptic digestion times ranged between 5 min (Apo A-I, Apo E, Apo A-IV) and 16 h (Apo A-II). Ultrasound and microwave assistance did not improve the digestion yield. Linearity was found between 0.1 nmol/L and 100 mmol/L. The lower limits of quantification were ≤ 0.4 µmol/L for Apo A-I, Apo A-IV, Apo B-100, Apo C-I, Apo C-III, Apo E, and <1.4 µmol/L for Apo A-II. CV <13% were determined. Comparison with immunoassays showed a good agreement for Apo A-I and Apo B. CONCLUSION AND CLINICAL RELEVANCE: The validated preanalytical protocol enables a reliable simultaneous analysis of seven Apos in human serum without depletion. The method can now be applied in clinical studies to investigate the Apo distributions in cardiovascular diseases.

LC-MS-based metabolomics in the clinical laboratory.

The analysis of metabolites in human body fluids remains a challenge because of their chemical diversity and dynamic concentration range. Liquid chromatography (LC) in combination with tandem mass spectrometry (MS/MS) offers a robust, reliable, and economical methodology for quantitative single metabolite analysis and profiling of complete metabolite classes of a biological specimen over a broad dynamic concentration range. The application of LC-MS/MS based metabolomic approaches in clinical applications aims at both, the improvement of diagnostic sensitivity and specificity by profiling a metabolite class instead of a single metabolite analysis, and the identification of new disease specific biomarkers. In the present paper we discuss recent advances in method development for LC-MS/MS analysis of lipids, carbohydrates, amino acids and biogenic amines, vitamins and organic acids with focus on human body fluids. In this context an overview on recent LC-MS/MS based metabolome studies for cancer, diabetes and coronary heart disease is presented.

Challenges and developments in tandem mass spectrometry based clinical metabolomics.

'Clinical metabolomics' aims at evaluating and predicting health and disease risk in an individual by investigating metabolic signatures in body fluids or tissues, which are influenced by genetics, epigenetics, environmental exposures, diet, and behaviour. Powerful analytical techniques like liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) offers a rapid, effective and economical way to analyze metabolic alterations of pre-defined target metabolites in biological samples. Novel hyphenated technical approaches like the combination of tandem mass spectrometry combined with linear ion trap (QTrap mass spectrometry) combines both identification and quantification of known and unknown metabolic targets. We describe new concepts and developments of mass spectrometry based multi-target metabolome profiling in the field of clinical diagnostics and research. Particularly, the experiences from newborn screening provided important insights about the diagnostic potential of metabolite profiling arrays and directs to the clinical aim of predictive, preventive and personalized medicine by metabolomics.