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.

Sepsis-associated changes of the arachidonic acid metabolism and their diagnostic potential in septic patients.

OBJECTIVES: Sepsis-associated changes of the arachidonic acid metabolism and the utility of arachidonic acid metabolites for the diagnosis of sepsis have been poorly investigated so far. Therefore, the primary objective of our study was to screen for differentially regulated arachidonic acid metabolites in septic patients using a lipopolysaccharide whole-blood model and to investigate their diagnostic potential. DESIGN: Prospective, observational, single-center, clinical study. SETTING: Intensive care unit at University Hospital Leipzig. PATIENTS: Thirty-five patients (first cohort 25 patients, second cohort 10 patients) meeting the criteria for severe sepsis or septic shock were enrolled. Eighteen healthy volunteers (first cohort 15 subjects, second cohort 3 subjects) were enrolled as controls. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Arachidonic acid and its metabolites were investigated in supernatants of nonactivated (baseline) and lipopolysaccharide-activated heparinized whole blood of healthy subjects (n=15) and septic patients (n=25) by solid phase extraction and subsequent liquid chromatography-tandem mass spectrometry. Arachidonic acid, arachidonic acid analogues, and the cyclooxygenase-associated metabolites prostaglandin E2, 11-hydroxyeicosatetraenoic acid, and thromboxane B2 were identified as differentiating metabolites between septic patients and healthy subjects. Some of these compounds, including arachidonic acid, its analogues, and the cyclooxygenase metabolites prostaglandin E2 and thromboxane B2 differed at baseline. The inducibility of arachidonic acid and the cyclooxygenase metabolites 11-hydroxyeicosatetraenoic and prostaglandin E2 were reduced by 80% to 90% in septic patients. The degree of the inducibility was associated with severity of sepsis and clinical outcome. A reduced inducibility of COX-2 but preserved inducibility of mPGES-1 on gene expression level were confirmed in an independent cohort of septic patients (n=10) by quantitative reverse-transcription polymerase chain reaction compared to healthy controls (n=3). CONCLUSIONS: Arachidonic acid metabolism is markedly affected in patients with sepsis. Our data suggest that the analysis of arachidonic acid metabolites in an in vitro whole blood activation model may be a promising approach for risk estimation in septic patients that has to be further evaluated in subsequent large-scale clinical studies.

Fast liquid chromatography combined with mass spectrometry for the analysis of metabolites and proteins in human body fluids.

In the last decade various analytical strategies have been established to enhance separation speed and efficiency in high performance liquid chromatography applications. Chromatographic supports based on monolithic material, small porous particles, and porous layer beads have been developed and commercialized to improve throughput and separation efficiency. This paper provides an overview of current developments in fast chromatography combined with mass spectrometry for the analysis of metabolites and proteins in clinical applications. Advances and limitations of fast chromatography for the combination with mass spectrometry are discussed. Practical aspects of, recent developments in, and the present status of high-throughput analysis of human body fluids for therapeutic drug monitoring, toxicology, clinical metabolomics, and proteomics are 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.

Comparison of detection performance of UV and electrochemical detection in NACE for a range of different capillary inner diameters.

A systematic comparison of UV and electrochemical detection (ED) in conjunction with NACE has been performed for capillaries with different ids ranging from 75 down to 2 microm. Ferrocene, (ferrocenylmethyl)trimethylammonium acetate, atropine and scopolamine served as model analytes. All studies were done using an ACN-based nonaqueous electrophoresis buffer. For the comparison of detection performance, particular attention was paid to the absolute and relative LODs for decreasing capillary dimensions. In case of UV detection, a significant increase in relative LODs was typically observed for capillaries smaller than 25 microm in diameter, while the absolute LODs remained nearly constant over a wide range of capillary dimensions. In contrast, ED showed some increase in relative LODs for capillaries smaller than 10 microm in diameter, but the absolute LODs decreased over the complete range of capillary diameters studied. It could be demonstrated that the straightforward end-column ED with microdisk electrodes can be applied in conjunction with small id (2 microm) capillaries. Studies with (ferrocenylmethyl)trimethylammonium acetate using such small capillaries resulted in relative and absolute LODs of 7.5 x 10(-7) and 8 x 10(-18) mol/L, respectively.

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.

Preanalytical Investigation of Polyunsaturated Fatty Acids and Eicosanoids in Human Plasma by Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: Preanalytical variables have a great impact on sample matrices and are a source of laboratory errors. The effect of cryobanking, which is gaining great importance recently, requires systematic investigation. The arachidonic acid metabolism is useful as a quality marker since eicosanoids are easily subjected to in vitro oxidation processes. MATERIALS AND METHODS: Polyunsaturated fatty acids (PUFAs) and related metabolites were analyzed by online solid-phase extraction coupled to liquid chromatography-tandem mass spectrometry. The influence of different plasma anticoagulants, as well as serum, freeze-thaw cycles (n = 5), short-term storage at 4°C, room temperature up to 120 minutes, and long-term storage at -20°C, -80°C, and -150°C up to 180 days, were investigated. We further investigated the influence of protein depletion, antioxidants, and shock-freezing on plasma. RESULTS: PUFA metabolites were stable at 4°C in ethylenediaminetetraacetic acid (EDTA)-stabilized whole blood for 120 minutes and in EDTA-plasma for 30 minutes. Plasma stability at 4°C could be further increased up to 7 days after protein depletion, while addition of antioxidants such as butylated hydroxytoluene or coverage with nitrogen had no protective effects. Repeated freeze-thaw cycles (n > 1) resulted in eicosanoid formation up to 63%. Long-term storage at -20°C led to substantial eicosanoid increases after 30 days, which could be prevented by depleting proteins before storage. Cryobanking at -80°C and -150°C revealed decreased concentrations of eight eicosanoids after 180 days. An advantage of shock-freezing with liquid nitrogen could not be confirmed compared to conventional freezing. CONCLUSION: Defined preanalytical conditions for eicosanoid analysis in human matrices are required to minimize in vitro data variability.

Comparison of eight routine unpublished LC-MS/MS methods for the simultaneous measurement of testosterone and androstenedione in serum.

BACKGROUND: Liquid-chromatography tandem mass spectrometry (LC-MS/MS) has become the method of choice in steroid hormone measurement. However, little information on the mutual agreement of LC-MS/MS methods is available. We compared eight routine unpublished LC-MS/MS methods for the simultaneous measurement of testosterone and androstenedione. METHODS: Sixty random serum samples from male and female volunteers were analysed in duplicate by eight routine LC-MS/MS methods. We performed Passing-Bablok regression analyses and calculated Pearson's correlation coefficients to assess the agreement of the methods investigated with one published method known to be accurate. Intra-assay CV of each method was calculated from duplicate results, recoveries for each method were calculated from six spiked samples. Furthermore, a CV between the investigated methods was calculated. RESULTS: The concentrations ranged from 0.05-1.26 nmol/L, 6.15-24.44 nmol/L and 0.15-4.78 nmol/L for testosterone in females, testosterone in males and androstenedione, respectively. The intra-assay CVs were between 3.7-16.0%, 0.9-5.2% and 1.2-9.5% for testosterone in females, testosterone in males and androstenedione, respectively. The slopes of the regression lines ranged between 0.90-1.25, 0.87-1.24 and 0.94-1.31 for testosterone concentrations in females, all testosterone values and androstenedione, respectively. Inter-method CVs were 24%, 14% and 29% for testosterone for concentrations in females and males and androstenedione, respectively. These compare unfavourably to the variation found earlier in published methods. CONCLUSION: Although most routine LC-MS/MS methods investigated here showed a reasonable agreement, some of the assays showed a high variation. The observed differences in standardization should be taken into account when applying reference values, or should, preferably, be solved.

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.

Fast liquid chromatography-quadrupole linear ion trap-mass spectrometry analysis of polyunsaturated fatty acids and eicosanoids in human plasma.

Profiling of polyunsaturated fatty acids (PUFAs) and their oxidized metabolites, mainly eicosanoids, in human plasma by fast liquid chromatography-mass spectrometry is described. Sample preparation involved protein precipitation of 200µL plasma followed by on-line solid-phase extraction. 7 PUFAs and 94 oxidized metabolites were separated utilizing a C-18 column packed with 2.6µm core-shell particles in 7min. The analytes and deuterium-labeled standards were detected via scheduled multiple reaction monitoring transitions (123 sMRM). Simultaneously, linear ion trap fragment spectra were acquired for confirmation, if necessary. The lower limit of quantitation ranged between 200 and 1000ng/mL for the PUFAs and 10-1000pg/mL for the metabolites. The method was applied to a study on plasma samples from 50 healthy subjects.

Mechanisms of Involvement of Eicosanoids and their Precursors in the Pathophysiology and Treatment of Schizophrenia.

The pathophysiology of schizophrenia has not been fully elucidated but there are converging leads to understanding this complex psychiatric disorder. One family of molecules that may play a crucial role in the development of schizophrenia is the eicosanoids. Review of the literature on eicosanoids in patients with schizophrenia points to findings in three areas: precursor molecules such as polyunsaturated fatty acids (PUFAs) and specifically arachidonic acid (AA), the actions of specific eicosanoids such as thromboxane A2 (TxA2), thromboxane B2 (TxB2) and prostaglandin E2 (PGE2), and enzymes with important functions in eicosanoid metabolism such as cyclooxygenase 2 (COX-2). It has also been found that classical as well as second generation antipsychotics, drugs used to treat schizophrenia, influence eicosanoid metabolism. For example, clozapine and its metabolite N-desmethylclozapine (NDMC) decreased TxB2 production in vitro. Eicosanoids and the enzymes involved in their metabolism may provide novel future drug targets. Therapeutic response to COX-2 inhibitors has already been demonstrated in patients at an early stage of schizophrenia. COX-2 inhibitors may exert this therapeutic action through their effects in reducing PGE2, type-2 cytokine and kynurenic acid production and strengthening glutamatergic neurotransmission.

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.

Metabolomics of dietary fatty acid restriction in patients with phenylketonuria.

BACKGROUND: Patients with phenylketonuria (PKU) have to follow a lifelong phenylalanine restricted diet. This type of diet markedly reduces the intake of saturated and unsaturated fatty acids especially long chain polyunsaturated fatty acids (LC-PUFA). Long-chain saturated fatty acids are substrates of mitochondrial fatty acid oxidation for acetyl-CoA production. LC-PUFA are discussed to affect inflammatory and haemostaseological processes in health and disease. The influence of the long term PKU diet on fatty acid metabolism with a special focus on platelet eicosanoid metabolism has been investigated in the study presented here. METHODOLOGY/PRINCIPAL FINDINGS: 12 children with PKU under good metabolic control and 8 healthy controls were included. Activated fatty acids (acylcarnitines C6-C18) in dried blood and the cholesterol metabolism in serum were analyzed by liquid chromatographic tandem mass spectrometry (LC-MS/MS). Fatty acid composition of plasma glycerophospholipids was determined by gas chromatography. LC-PUFA metabolites were analyzed in supernatants by LC-MS/MS before and after platelet activation and aggregation using a standardized protocol. Patients with PKU had significantly lower free carnitine and lower activated fatty acids in dried blood compared to controls. Phytosterols as marker of cholesterol (re-) absorption were not influenced by the dietary fatty acid restriction. Fatty acid composition in glycerophospholipids was comparable to that of healthy controls. However, patients with PKU showed significantly increased concentrations of y-linolenic acid (C18:3n-6) a precursor of arachidonic acid. In the PKU patients significantly higher platelet counts were observed. After activation with collagen platelet aggregation and thromboxane B(2) and thromboxane B(3) release did not differ from that of healthy controls. CONCLUSION/SIGNIFICANCE: Long-term dietary fatty acid restriction influenced the intermediates of mitochondrial beta-oxidation. No functional influence on unsaturated fatty acid metabolism and platelet aggregation in patients with PKU was detected.

Impact of clozapine, N-desmethylclozapine and chlorpromazine on thromboxane production in vitro.

Thromboxane A2 (TxA2) and the activation of its receptor have been shown to modulate vasoconstriction and platelet aggregation as well as dopaminergic and serotonergic signalling. Dopaminergic and serotonergic systems play a crucial role in the pathophysiology of schizophrenia and these systems are the main targets of antipsychotics (APs). As the first antipsychotic (AP) chlorpromazine (CPZ) has already been shown to reduce TxA2, we hypothesized that the AP clozapine and its metabolite N-desmethylclozapine (NDMC) might also influence TxA2 production. We measured levels of thromboxane B2 (TxB2), the metabolite of the very unstable molecule TxA2, in unstimulated and stimulated blood samples of 10 healthy female subjects in a whole blood assay using toxic shock syndrome toxin-1 (TSST-1) and monoclonal antibody against surface antigen CD3 combined with protein CD40 (OKT3/CD40) as stimulants. Blood was supplemented with the APs CPZ, clozapine or NDMC in one of four different concentrations. Additionally, thromboxane levels were measured in blood without the addition of APs under different stimulation conditions. Under TSST-1 as well as OKT3/CD40 stimulation, mean TxB2 concentrations were significantly (p < 0.05) decreased by clozapine over all applied concentrations. NDMC led to a decrease in TxB2 levels under unstimulated conditions as well as under TSST-1 stimulation. CPZ reduced TxB2 production at low concentrations under unstimulated and TSST-1- stimulated conditions. Clozapine, NDMC and CPZ possibly act on neurotransmitter systems via modulation of TxA2 or TxB2 production. Additionally, known side effects of APs such as orthostatic hypotension may be a result of changes in the concentrations of TxA2 or TxB2.

Preclinical challenges in steroid analysis of human samples.

Preclinical challenges in the analysis of steroid hormones are primarily determined by biological factors involved in the physiology and pathophysiology of hormone secretion. Major biologically influencing factors like age, sex, pubertal stage, pregnancy, phase of the menstruation, and diurnal rhythm have to be considered in the definition of reference ranges for steroids and their clinical interpretation. Hitherto, in clinical routine laboratories steroids were mainly determined by direct immunoassays applied on automated platforms, which are simple, rapid and cheap if a high number of samples are measured. However, technical factors like cross-reactivity of related steroid metabolites or limited analytical ranges have to be taken in account and may impair accuracy and precision of these direct methods. The actual development of mass spectrometry based analytical platforms for the determination of single steroid or steroid patterns seems to be an alternative analytical approach combining multi-parametric analysis, high sensitivity and specificity as well simple sample pre-treatment, robustness and low running costs for steroid analysis. This short review will give an overview about biological influencing factors and technical disturbing factors of routinely used immunoassay for the analysis of steroids. The application of LC-MS/MS as an alternative routine high-throughput platform for steroid analysis and its perspective role in the standardization and harmonisation of steroid measurements in clinical routine application will be discussed.

Rapid quantification of steroid patterns in human serum by on-line solid phase extraction combined with liquid chromatography-triple quadrupole linear ion trap mass spectrometry.

BACKGROUND: The determination of steroids is important for the diagnosis and monitoring of endocrine diseases and infertility workup. We developed a rapid and reliable mass spectrometric method for the simultaneous quantification of steroid patterns in human serum. METHODS: An on-line solid phase extraction (SPE)-liquid chromatography-triple quadrupole linear ion trap (LC-QTrap) method utilizing atmospheric pressure chemical ionization was developed. Following protein precipitation of 100 microL serum, on-line SPE and chromatographic separation was performed for 13 steroids in 1.8 min. Analytes were confirmed by the characteristic fragment patterns. RESULTS: The total run time of the method was 4 min. Detection limits ranged between 0.02 microg/L (testosterone) and 9 microg/L (dehydroepiandrosterone sulfate). The method was linear up to 7000 microg/L for dehydroepiandrosterone sulfate, 500 microg/L for cortisol, 125 microg/L for 11-deoxycortisol, and 25 microg/L for aldosterone, 17-hydroxyprogesterone, progesterone, testosterone, androstenedione and beta-estradiol, respectively. Accuracy ranged between 80 and 114%. Between-day variance at three different concentration levels was <15%. Excellent correlations with immunoassays were observed for testosterone, cortisol and beta-estradiol with Pearson's correlation coefficient r=0.967, 0.963, and 0.998, respectively. CONCLUSION: The novel on-line SPE-LC-MS/QTrap platform offers a very fast, reliable, and sensitive quantification of steroid patterns and fulfils the quality criteria for routine laboratory application.