Mark C. Parkin

Spreading and Synchronous Depressions of Cortical Activity in Acutely Injured Human Brain

Background and Purpose— Cortical spreading depression (CSD) has been much studied experimentally but never demonstrated unequivocally in human neocortex by direct electrophysiological recording. A similar phenomenon, peri-infarct depolarization, occurs in experimental models of stroke and causes the infarct to enlarge. Our current understanding of the mechanisms of deterioration in the days after major traumatic or ischemic brain injury in humans has not yielded any effective, novel drug treatment. This study sought clear evidence for the occurrence and propagation of CSD in the injured human brain. Methods— In 14 patients undergoing neurosurgery after head injury or intracranial hemorrhage, we placed electrocorticographic (ECoG) electrodes near foci of damaged cortical tissue. Results— Transient episodes of depressed ECoG activity that propagated across the cortex at rates in the range of 0.6 to 5.0 mm/min were observed in 5 patients; this rate of propagation is characteristic of CSD. We also observed, in 8 of the 14 patients, transient depressions of ECoG amplitude that appeared essentially simultaneous in all recording channels, without clear evidence of spread. Conclusions— These results indicate that CSD or similar events occur in the injured human brain and are more frequent than previously suggested. On the basis of these observations, we suggest that the related phenomenon, peri-infarct depolarization, is indeed likely to occur in boundary zones in the ischemic human cerebral cortex.

Dynamic changes in brain glucose and lactate in pericontusional areas of the human cerebral cortex, monitored with rapid sampling on-line microdialysis: relationship with depolarisation-like events

The pathophysiology of peri-lesion boundary zones in acute brain injury is highly dynamic, and it is now clear that spreading-depression-like events occur frequently in areas of cerebral cortex adjacent to contusions in the injured human brain. An automated method to assay microdialysate from peri-lesion cerebral cortex in 11 patients with intracranial haematomas requiring surgery was used. Perfusate (2 μL/min) flowed directly into a flow-injection system for assay of glucose and lactate at intervals typically of 30 secs each. Four channels of electrocorticogram (ECoG) were recorded from a subdural strip adjacent to the catheter. Several patterns of change in metabolites were identified in different time domains. Overall, the number of transient lactate events was significantly correlated with the number of glucose events (r2=0.48, P=0.027, n=10). Progressive reduction in dialysate glucose was very closely correlated with the aggregate number of ECoG events (r2=0.76, P=0.0004, n=11). It is proposed that the recently documented adverse impact of low dialysate glucose on clinical outcome may be because of recurrent, spontaneous spreading-depression-like events in the perilesion cortex.

Iron(III) citrate speciation in aqueous solution

Citrate is an iron chelator and it has been shown to be the major iron ligand in the xylem sap of plants. Furthermore, citrate has been demonstrated to be an important ligand for the non-transferrin bound iron (NTBI) pool occurring in the plasma of individuals suffering from iron-overload. However, ferric citrate chemistry is complicated and a definitive description of its aqueous speciation at neutral pH remains elusive. X-Ray crystallography data indicates that the alcohol function of citrate (Cit4-) is involved in Fe(III) coordination and that deprotonation of this functional group occurs upon complex formation. The inability to include this deprotonation in the affinity constant calculations has been a major source of divergence between various reports of iron(III)-citrate affinity constants. However the recent determination of the alcoholic pKa of citric acid (H4Cit) renders the reassessment of the ferric citrate system possible. The aqueous speciation of ferric citrate has been investigated by mass spectrometry and EPR spectroscopy. It was observed that the most relevant species are a monoiron dicitrate species and dinuclear and trinuclear oligomeric complexes, the relative concentration of which depends on the solution pH value and the iron : citric acid molar ratio. Spectrophotometric titration was utilized for affinity constant determination and the formation constant for the biologically relevant [Fe(Cit)2]5- is reported for the first time.

Detection of ketamine and its metabolites in urine by ultra high pressure liquid chromatography-tandem mass spectrometry

Current analytical methods used for screening drugs and their metabolites in biological samples from victims of drug-facilitated sexual assault (DFSA) or other vulnerable groups can lack sufficient sensitivity. The application of liquid chromatography, employing small particle sizes, with tandem mass spectrometry (MS/MS) is likely to offer the sensitivity required for detecting candidate drugs and/or their metabolites in urine, as demonstrated here for ketamine. Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) was performed following extraction of urine (4 mL) using mixed-mode (cation and C8) solid-phase cartridges. Only 20 microL of the 250 microL extract was injected, leaving sufficient volume for other assays important in DFSA cases. Three ion transitions were chosen for confirmatory purposes. As ketamine and norketamine (including their stable isotopes) are available as reference standards, the assay was additionally validated for quantification purposes to study elimination of the drug and primary metabolite following a small oral dose of ketamine (50 mg) in 6 volunteers. Dehydronorketamine, a secondary metabolite, was also analyzed qualitatively to determine whether monitoring could improve retrospective detection of administration. The detection limit for ketamine and norketamine was 0.03 ng/mL and 0.05 ng/mL, respectively, and these compounds could be confirmed in urine for up to 5 and 6 days, respectively. Dehydronorketamine was confirmed up to 10 days, providing a very broad window of detection.

Resolving dynamic changes in brain metabolism using biosensors and on-line microdialysis

A tight coupling exists in the brain between the activity of neurons, the energetic consequences of this activity and the local blood flow. This makes the monitoring of the levels of energy metabolites, such as glucose and lactate, in the fluid surrounding the neurons a particularly effective means of studying brain function. This review outlines why rapid monitoring of brain metabolism in vivo is effective, and demonstrates how the in vivo techniques of implanted biosensors and on-line microdialysis can be used to study the brain in the laboratory and in brain-injury patients.

Interlaboratory Agreement of Insulin-like Growth Factor 1 Concentrations Measured by Mass Spectrometry

BACKGROUND Insulin-like growth factor 1 (IGF-1)(7) is a key mediator of growth hormone (GH) action and a well-characterized biomarker of GH abuse. Current immunoassays for IGF-1 suffer from poor concordance between platforms, which makes comparison of results between laboratories difficult. Although previous work has demonstrated good interlaboratory imprecision of LC-MS/MS methods when plasma is supplemented with purified proteins, the interlaboratory imprecision of an endogenous protein in the nanogram-per-milliliter concentration range has not been reported. METHODS We deployed an LC-MS/MS method to quantify serum IGF-1 in 5 laboratories using 5 different instruments and analyzed 130 healthy human samples and 22 samples from patients with acromegaly. We determined measurement imprecision (CV) for differences due to instrumentation, calibration curve construction, method of calibration, and reference material. RESULTS Instrument-dependent variation, exclusive of digestion, across 5 different instrument platforms was determined to be 5.6%. Interlaboratory variation was strongly dependent on calibration. Calibration materials from a single laboratory resulted in less variation than materials made in individual laboratories (CV 5.2% vs 12.8%, respectively). The mean imprecision for 152 samples between the 5 laboratories was 16.0% when a calibration curve was made in each laboratory and 11.1% when a single-point calibration approach was used. CONCLUSIONS The interlaboratory imprecision of serum IGF-1 concentrations is acceptable for use of the assay in antidoping laboratories and in standardizing results across clinical laboratories. The primary source of variability is not derived from the sample preparation but from the method of calibration.

On-line monitoring in neurointensive care Enzyme-based electrochemical assay for simultaneous, continuous monitoring of glucose and lactate from critical care patients

This paper describes the development, verification and use of the first dual on-line electrochemical assay system for use with clinical microdialysis. The assay is a flow injection assay using mediated enzyme beds with electrochemical detection. The on-line assay has been specifically designed for use with head trauma patients in neurointensive care, allowing simultaneous measurement of the levels of glucose and lactate present in the dialysate with high time resolution (30 s sampling intervals). The assay is compared with the Yellow Springs 2300 glucose/lactate analyser in simple analyses and during zero net flux experiments. The on-line assay has greater accuracy and precision particularly at low concentrations of glucose and lactate. The rapid nature of this dual on-line system makes it valuable in studying the roles of energy metabolites in the brain extracellular fluid (ECF), and as an important new method for routine clinical monitoring of patients in the neurointensive care unit. Data is shown for monitoring during surgery and in intensive care.

Use of human microsomes and deuterated substrates: an alternative approach for the identification of novel metabolites of ketamine by mass spectrometry.

In vitro biosynthesis using pooled human liver microsomes was applied to help identify in vivo metabolites of ketamine by liquid chromatography (LC)-tandem mass spectrometry. Microsomal synthesis produced dehydronorketamine, seven structural isomers of hydroxynorketamine, and at least five structural isomers of hydroxyketamine. To aid identification, stable isotopes of the metabolites were also produced from tetra-deuterated isotopes of ketamine or norketamine as substrates. Five metabolites (three hydroxynorketamine and two hydroxyketamine isomers) gave chromatographically resolved components with product ion spectra indicating the presence of a phenolic group, with phenolic metabolites being further substantiated by selective liquid-liquid extraction after adjustments to the pH. Two glucuronide conjugates of hydroxynorketamine were also identified. Analysis by LC-coupled ion cyclotron resonance mass spectrometry gave unique masses in accordance with the predicted elemental composition. The metabolites, including the phenols, were subsequently confirmed to be present in urine of subjects after oral ketamine administration, as facilitated by the addition of deuterated metabolites generated from the in vitro biosynthesis. To our knowledge, phenolic metabolites of ketamine, including an intact glucuronide conjugate, are here reported for the first time. The use of biologically synthesized deuterated material as an internal chromatographic and mass spectrometric marker is a viable approach to aid in the identification of metabolites. Metabolites that have particular diagnostic value can be selected as candidates for chemical synthesis of standards.

Application Of Rapid-Sampling, Online Microdialysis To The Monitoring Of Brain Metabolism During Aneurysm Surgery

OBJECTIVE: To introduce rapid-sampling microdialysis for the early detection of adverse metabolic changes in tissue at risk during aneurysm surgery. METHODS: A microdialysis catheter was inserted under direct vision into at-risk cortex at the start of surgery. This monitoring was sustained throughout the course of the operation, during which intraoperative events, for example, temporary arterial occlusion or lobe retraction, were precisely documented. A continuous online flow of dialysate was fed into a mobile bedside glucose and lactate analyser. This comprises flow-injection dual-assay enzyme-based biosensors capable of determining values of metabolites every 30 seconds. RESULTS: Eight patients underwent clipping or wrapping of intracranial aneurysms and were monitored. Time between events and detection: 9 minutes. Mean change in metabolite value ± standard deviation: temporal lobe retraction lactate, +656 ± 562 &mgr;mol/L (n = 7, P < 0.05); glucose, -123 ± 138 &mgr;mol/L (n = 6, P = 0.08). Glucose intravenous bolus infusion glucose, +512 ± 244 &mgr;mol/L (n = 5, P < 0.01); peak at mean time after bolus, 16 minutes. Temporary proximal clip lactate, +731 ± 346 &mgr;mol/L (n = 6, P < 0.01); glucose, -139 ± 96 &mgr;mol/L (n = 5, P < 0.05); mean clip time, 8.6 minutes. CONCLUSION: The technique detects changes 9 minutes after intraoperative events occur (limited only by probe-to-sensor tubing length and dialysate flow rate). This provides reliable information to the surgeon and anesthetist promptly. It is a useful method for monitoring glucose and lactate in dialysate, particularly when rapid, transient changes in brain analyte levels need to be determined and the alternative offline methodology would be inadequate.

An introduction to mass spectrometry based proteomics-detection and characterization of gonadotropins and related molecules

This review introduces fundamental aspects of mass spectrometry (MS) based proteomics and illustrates how MS is an effective tool for the analysis of glycoprotein hormones. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and electrospray ionization (ESI) MS are complementary approaches that have been applied for the analysis of gonadotropins, e.g. to characterize differences in the oligosaccharide distribution of commercial human chorionic gonadotropin preparations, for isolated nicked beta-subunit, and identification of a metabolite of placental transforming growth factor in pharmaceutical hCG preparations. Immunoaffinity trapping and concentration of digested sample extract prior to MS analysis confers analytical sensitivity akin to immunoassay. A desirable objective would be to develop for clinical purposes a rapid procedure for MS detection and characterization of gonadotropins. Refinement of on-target immobilization and digestion for subsequent ionization by MALDI may eventually help to provide this capability. The advent of hybrid mass spectrometers will further advance the characterization of these complex molecules.