Development of a second-tier method for C4, C5 and C2 acylcarnitine analysis in plasma.

INTRODUCTION: Acylcarnitines are typically analyzed using either a flow injection analysis (FIA) method or liquid chromatography-mass spectrometry (LC-MS/MS) methods. The FIA method is a fast, efficient method, however it does not have the capability to separate compounds with the same molecular weight. These isobaric interferences can be removed by chromatographic separation with LC-MS/MS. In this study, we aimed to develop and optimize a qualitative LC-MS/MS method to separate the isobaric interferences for two-, four- and five-carbon acylcarnitines. METHODS: The samples were first prepared by acylcarnitine derivatization with butanolic HCl. The developed LC-MS/MS method is a combination of isocratic and gradient elution used to separate acylcarnitines. Multiple reaction monitoring was used for determination of precursor and product ions for each acylcarnitine species as well as known interferences used in our study. We used this method to analyze quality assurance and patient samples with elevated two-, four- and five-carbon acylcarnitines. RESULTS: Butyryl- and isobutyrylcarnitines as well as valeryl- and isovalerylcarnitines were successfully separated using the developed method. This method was able also to separate and distinguish acetylcarnitine from glutamate interference that has been causing overestimation of acetylcarnitine. In patients, the dominant five-carbon acylcarnitine was found to be isovalerylcarnitine. We confirmed that the majority of analyzed patient samples had additional carnitine adducts present but not valerylcarnitine. Butyryl- and isobutyrylcarnitines, in variable ratios, were present in every patient sample. CONCLUSION: We developed a qualitative LC-MS/MS method for butyl-ester derivatized acylcarnitines, which can be used as a second-tier method for diagnosis and monitoring of various inborn errors of metabolism in our hospital network.

Alerting to acute kidney injury - Challenges, benefits, and strategies.

Acute Kidney Injury (AKI) is a complex heterogeneous syndrome that often can go unrecognized and is encountered in multiple clinical settings. One strategy for proactive identification of AKI has been through electronic alerts (e-alerts) to improve clinical outcomes. The two traditional criteria for AKI diagnosis and staging have been urinary output and serum creatinine. The latter has dominated in aiding identification and prediction of AKI by alert models. While creatinine can provide information to estimate glomerular filtration rate, the utility to depict real-time change in rapidly declining kidney function is paradoxical. Alerts for AKI have recently been popularized by several studies in the UK showcasing the various use cases for detection and management by simply relying on creatinine changes. Predictive models for real-time alerting to AKI have also gone beyond simple delta checks of creatinine as reviewed here, and hold promise to leverage data contained beyond the laboratory domain. However, laboratory data still remains vital to e-alerts in AKI. Here, we highlight a select number of approaches for real-time alerting to AKI built on traditional consensus definitions, evaluate impact on clinical outcomes from e-alerts, and offer critiques on new and expanded definitions of AKI.

Analysis of serum tranexamic acid in patients undergoing open heart surgery.

BACKGROUND: Tranexamic acid is a drug used during open cardiac surgery to prevent blood loss. The blood levels of 10-100 µg/mL are reported to be in the therapeutic range and higher levels are linked to increased incidence of adverse effects. The aim of this study was to optimize and validate an LC-MS/MS method for serum tranexamic acid and measure its levels in patients from the DEPOSITION Pilot trial in order to prove the concept that topical administration will yield lower serum concentration. METHODS: The method development was carried out in several steps including sample preparation, and optimization of chromatography and tandem mass spectrometry parameters. Method validation including day-to-day precision with 4 QC levels, limit of detection, sample stability, carryover, and concentration-signal linearity was carried out. Ninety patient samples were analyzed using the validated method. RESULTS: Fast and efficient LC-MS/MS method for analysis of tranexamic acid in serum was developed. The run time was 7 min with the total time of one hour including the sample preparation. The method precision was acceptable (%CV = 10.5-12.6%) with no sample carryover observed. The matrix effect on the analytical sensitivity was negligible and the lower limit of detection was 0.5 µg/mL. The difference in the mean adjusted concentrations between topical (45 patients) and intravenous (45 patients) groups was statistically significant (0.1154 µg/mL/kg vs. 0.2542 µg/mL/kg, p < 0.0001) CONCLUSIONS: Rapid and simple LC-MS/MS method for analysis of tranexamic acid was optimized and validated. The laboratory has played a crucial role in proving the concept that topical administration yields significantly lower systemic levels of tranexamic acid, and thus decreases the risk of adverse outcomes in patients undergoing open cardiac surgery.

The potential of reducing AST testing in hospital settings.

BACKGROUND: Aspartate aminotransferase (AST) is regularly ordered with alanine aminotransferase (ALT) to assess liver integrity. In many situations, AST testing provides little or no added clinical value, since ALT is more specific and the both enzyme activities highly correlate. The objective of this study is to determine the potential reduction in AST testing, if not performed when ALT results are within reference intervals (RI). METHODS: Results for patients >18 years of age for both AST and ALT from the same specimen were obtained for the period January 1, 2017 - December 31, 2017. We calculated frequency of AST and ALT results that had various combinations of results within and above the RI. We also investigated the clinical locations of origin for the samples. RESULTS: In total 87,704 paired samples with both AST and ALT test results were recovered. The total of 73.2% of AST tests for males and 66.9% for females would be eliminated if we performed AST testing only when ALT was increased. However, 7.4% of elevated AST tests would be missed for males and 3.8% for females due to ALT being within limits. Specifically in the outpatient clinics, 79% male and 73% females paired enzyme results were within RI. Only 4% of males and 3% of females had paired results where ALT was within RI while AST > RI. CONCLUSIONS: The rate of test results with increased AST while ALT is within the RI is low enough to recommend limiting AST testing only to cases where ALT is above the RI. Our recommendation for AST restriction is to begin with the hospitals outpatient clinics.

The potential role of a turbidimetric heart-type fatty acid-binding protein assay to aid in the interpretation of persistently elevated, non-changing, cardiac troponin I concentrations.

BACKGROUND: Elevated and non-changing high-sensitivity cardiac troponin (hs-cTn) concentrations may suggest a process other than acute injury, possibly due to chronic condition(s) causing the elevation, an analytical error/interference or the formation of macrocomplexes. Heart-type fatty acid binding protein (H-FABP) might be useful in this setting to identify the etiology of abnormally high and non-changing cTn concentrations which could aid clinical decision making in the hospital setting. METHODS: We analytically validated the H-FABP assay (Randox) on the Abbott ARICHTECTc8000 platform, testing imprecision, linearity, stability, and matrix comparison. Over the 2-month analytical validation; EDTA plasma samples from patients with a hospital visit with persistently elevated and stable cTnI concentrations (Abbott hs-cTnI≥52 ng/L or 2x99th percentile upper limit of normal (ULN = 26 ng/L) with change between results <20%) were collected and frozen (-20 °C). These samples were tested with the H-FABP assay, polyethylene glycol (PEG) precipitation, with the lowest estimated glomerular filtration rate (eGRF) during the hospital visit also obtained from these patients. RESULTS: The H-FABP assay was linear, with concentrations stable after 4 freeze/thaw cycles, up to 150 h at room temperature, and comparable between lithium heparin and EDTA plasma. During the validation there were 6 patients with eGFR ≥60 ml/min/1.73m2 identified (total population screened n = 917) with high and non-changing hs-cTnI concentrations. All 6 patients had H-FABP<2xULN; with 3 patients having a macrocomplex and a final diagnosis of not ACS. CONCLUSION: Testing of H-FABP in patients with an eGFR≥60 ml/min/1.73m2 with persistently high and stable cTn elevations may help to confirm prior cardiac injury or the presence of macrocomplexes as the source of these elevations.

Lipophilic fluorescent products of free radicals.

BACKGROUND: Fluorescent pigments are the end-products of reactions involving free radical attack on biological molecules and can be formed, for example, in reactions between lipid peroxidation products, mainly unsaturated aldehydes, with free amino groups. Their characteristic emission maximum was found to be at 420-470 nm after being excited at 340-390 nm. The mechanism of their formation and chemical identity has been revealed in many in vitro studies, in which reactive aldehydes were incubated with amino group-containing molecules. Owing to their intrinsic fluorescent properties and molecular stability these products are easily measured by means of spectrofluorimetry and are used as biomarkers of oxidative stress caused by various triggers. It has been found that the fluorescent products are formed in excess in conditions linked with increased free radical production, such as atherosclerosis, Alzheimer's disease and multiple sclerosis. METHODS: We searched the literature using "MEDLINE" and "Web of Science" in order to get an overview of the state of knowledge about fluorescent products of free radicals, that is, their analysis from in vitro studies, animal and human studies and their use as markers of oxidative damage. CONCLUSIONS: Although their chemical structure may not have been elucidated, the fluorophores formed in this way have found application as markers of oxidative stress in many animal and human studies. In vitro experiments using model reactions have given some clues as to how certain fluorescent pigments arise during oxidative reactions in vivo. Advances in analytical techniques should lead the chemical characterization of pigments of different origin to completeness.

Early postnatal development of rat brain is accompanied by generation of lipofuscin-like pigments.

The increased generation of free radicals results in the formation of fluorescent end-products of lipid peroxidation, lipofuscin-like pigments (LFPs). The authors observed that LFPs are generated in rat brain after a normal birth during 5 postnatal days. The experimental design of the study comprised 10 groups of animals. The authors measured prenatal values 1 day and 7 days before birth, and then the animals were sampled on postnatal day 1, 2, 5, 10, 15, 25, 35, and 90. Maximum LFP concentration is achieved on the postnatal day 2. Starting from postnatal day 10, LFP concentration returns to prenatal values. A new rise in LFP concentration is observed at 3 months of age. This is associated with the beginning of the aging process. LFPs were characterized by fluorescence spectroscopy using tridimensional excitation spectra, synchronous spectra and their derivatives, and HPLC with fluorescence detection. It was possible to discern several tens of fluorescent compounds of unknown structure that are generated and metabolized during early development. The authors suggest that LFPs are formed after respiratory burst of microglia phagocytosing apoptotic cells.