Quantitative high performance liquid chromatography/tandem mass spectrometric analysis of the four classes of F2-isoprostanes in human urine

Isoprostanes (iPs) are free radical catalyzed prostaglandin isomers. Analysis of individual isomers of PGF2α—F2-iPs—in urine has reflected lipid peroxidation in humans. However, up to 64 F2-iPs may be formed, and it is unknown whether coordinate generation, disposition, and excretion of F2-iPs occurs in humans. To address this issue, we developed methods to measure individual members of the four structural classes of F2-iPs, using liquid chromatography/tandem mass spectrometry (LC/MS/MS), in which sample preparation is minimized. Authentic standards of F2-iPs of classes III, IV, V, and VI were used to identify class-specific ions for multiple reaction monitoring. Using iPF2α-VI as a model compound, we demonstrated the reproducibility of the assay in human urine. Urinary levels of all F2-iPs measured were elevated in patients with familial hypercholesterolemia. However, only three of eight F2-iPs were elevated in patients with congestive heart failure, compared with controls. Paired analyses by GC/MS and LC/MS/MS of iPF2α-VI in hypercholesterolemia and of 8,12-iso-iPF2α-VI in congestive heart failure were highly correlated. This approach will permit high throughput analysis of multiple iPs in human disease.

RNA analysis by ion-pair reversed-phase high performance liquid chromatography

Ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC) is presented as a new, superior method for the analysis of RNA. IP RP HPLC provides a fast and reliable alternative to classical methods of RNA analysis, including separation of different RNA species, quantification and purification. RNA is stable under the analysis conditions used; degradation of RNA during the analyses was not observed. The versatility of IP RP HPLC for RNA analysis is demonstrated. Components of an RNA ladder, ranging in size from 155 to 1770 nt, were resolved. RNA transcripts of up to 5219 nt were analyzed, their integrity determined and they were quantified and purified. Purification of mRNA from total RNA is described, separating mouse rRNA from poly(A)+ mRNA. IP RP HPLC is also suitable for the separation and purification of DIG-labeled from unlabeled RNA. RNA purified by IP RP HPLC exhibits improved stability.

Measurement of 8-hydroxy-2′-deoxyguanosine in DNA by high-performance liquid chromatography-mass spectrometry: comparison with measurement by gas chromatography-mass spectrometry

Measurement of 8-hydroxy-2′-deoxyguanosine (8-OH-dGuo) in DNA by high-performance liquid chromatography/mass spectrometry (LC/MS) was studied. A methodology was developed for separation by LC of 8-OH-dGuo from intact and modified nucleosides in DNA hydrolyzed by a combination of four enzymes: DNase I, phosphodiesterases I and II and alkaline phosphatase. The atmospheric pressure ionization-electrospray process was used for mass spectral measurements. A stable isotope-labeled analog of 8-OH-dGuo was used as an internal standard for quantification by isotope-dilution MS (IDMS). Results showed that LC/IDMS with selected ion-monitoring (SIM) is well suited for identification and quantification of 8-OH-dGuo in DNA at background levels and in damaged DNA. The sensitivity level of LC/IDMS-SIM was found to be comparable to that reported previously using LC-tandem MS (LC/MS/MS). It was found that approximately five lesions per 106 DNA bases can be detected using amounts of DNA as low as 2 µg. The results also suggest that this lesion may be quantified in DNA at levels of one lesion per 106 DNA bases, or even lower, when more DNA is used. Up to 50 µg of DNA per injection were used without adversely affecting the measurements. Gas chromatography/isotope-dilution MS with selected-ion monitoring (GC/IDMS-SIM) was also used to measure this compound in DNA following its removal from DNA by acidic hydrolysis or by hydrolysis with Escherichia coli Fpg protein. The background levels obtained by LC/IDMS-SIM and GC/IDMS-SIM were almost identical. Calf thymus DNA and DNA isolated from cultured HeLa cells were used for this purpose. This indicates that these two techniques can provide similar results in terms of the measurement of 8-OH-dGuo in DNA. In addition, DNA in buffered aqueous solution was damaged by ionizing radiation at different radiation doses and analyzed by LC/IDMS-SIM and GC/IDMS-SIM. Again, similar results were obtained by the two techniques. The sensitivity of GC/MS-SIM for 7,8-dihydro-8-oxoguanine was also examined and found to be much greater than that of LC/MS-SIM and the reported sensitivity of LC/MS/MS for 8-OH-dGuo. Taken together, the results unequivocally show that LC/IDMS-SIM is well suited for sensitive and accurate measurement of 8-OH-dGuo in DNA and that both LC/IDMS-SIM and GC/IDMS-SIM can provide similar results.

Optical imaging of functional domains in the cortex of the awake and behaving monkey

As demonstrated by anatomical and physiological studies, the cerebral cortex consists of groups of cortical modules, each comprising populations of neurons with similar functional properties. This functional modularity exists in both sensory and association neocortices. However, the role of such cortical modules in perceptual and cognitive behavior is unknown. To aid in the examination of this issue we have applied the high spatial resolution optical imaging methodology to the study of awake, behaving animals. In this paper, we report the optical imaging of orientation domains and blob structures, approximately 100–200 μm in size, in visual cortex of the awake and behaving monkey. By overcoming the spatial limitations of other existing imaging methods, optical imaging will permit the study of a wide variety of cortical functions at the columnar level, including motor and cognitive functions traditionally studied with positron-emission tomography or functional MRI techniques.

The hippocampal formation participates in novel picture encoding: evidence from functional magnetic resonance imaging.

Considerable evidence exists to support the hypothesis that the hippocampus and related medial temporal lobe structures are crucial for the encoding and storage of information in long-term memory. Few human imaging studies, however, have successfully shown signal intensity changes in these areas during encoding or retrieval. Using functional magnetic resonance imaging (fMRI), we studied normal human subjects while they performed a novel picture encoding task. High-speed echo-planar imaging techniques evaluated fMRI signal changes throughout the brain. During the encoding of novel pictures, statistically significant increases in fMRI signal were observed bilaterally in the posterior hippocampal formation and parahippocampal gyrus and in the lingual and fusiform gyri. To our knowledge, this experiment is the first fMRI study to show robust signal changes in the human hippocampal region. It also provides evidence that the encoding of novel, complex pictures depends upon an interaction between ventral cortical regions, specialized for object vision, and the hippocampal formation and parahippocampal gyrus, specialized for long-term memory.