Analysis of 8-hydroxy-2'-deoxyguanosine in urine using high-performance liquid chromatography-electrospray tandem mass spectrometry

8-hydroxy-2'-deoxyguanosine (8-OHdG) is a widely used biomarker of oxidative stress in research related to DNA, protein damage as well as lipid peroxidation. HPLC-MS/MS with electrospray ionization (ESI) and the use of isotopically labelled 8-OHdG as an internal standard allows a simple quantification of 8-OHdG in urine samples. HPLC separation utilized the peak cutting technique and a 1.5 mmx120 mm analytical anion exchange column. Novel method entails only minimal sample handling including the addition of a buffer and an internal standard followed by centrifugation before the samples are ready for analysis. The levels of 8-OHdG in human urine samples (n=246) varied from 0.16 to 16.48 microg/L and the corresponding creatinine-normalized values were ranged from 0.49 to 14.27 microg of 8-OHdG/g creatinine. The correlation between the developed HPLC-MS/MS method and the existing HPLC-EC method was good with an R2 value of 0.8707.     

4-Phenylbutanoyl-2(S)-acylpyrrolidines and 4-phenylbutanoyl-L-prolyl-2(S)-acylpyrrolidines as prolyl oligopeptidase inhibitors

New 4-phenylbutanoyl-2(S)-acylpyrrolidines and 4-phenylbutanoyl-L-prolyl-2(S)-acylpyrrolidines were synthesized. Their inhibitory activity against prolyl oligopeptidase from pig brain was tested in vitro. In the series of 4-phenylbutanoyl-2(S)-acylpyrrolidines, the cyclopentanecarbonyl and benzoyl derivatives were the best inhibitors having IC(50) values of 30 and 23 nM, respectively. This series of compounds shows that the P1 pyrrolidine ring, which is common in most POP inhibitors, can be replaced by either a cyclopentyl ring or a phenyl ring, causing only a slight decrease in the inhibitory activity. In the series of 4-phenylbutanoyl-L-prolyl-2(S)-acylpyrrolidines the cyclopentanecarbonyl and benzoyl derivatives were not as active as in the series of 4-phenylbutanoyl-2(S)-acylpyrrolidines. The hydroxyacetyl derivative did however show high inhibitory activity. This compound is structurally similar to JTP-4819, which is one of the most potent prolyl oligopeptidase inhibitors. The acyl group in the two series of new compounds seems to bind to different sites of the enzyme, since the second series of new compounds did not show the same cyclopentanecarbonyl or benzoyl specificity as the first series.     

Atrial natriuretic peptide, B-type natriuretic peptide, and serum collagen markers after acute myocardial infarction.

Experimental data suggest that atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) act locally as antifibrotic factors in heart. We investigated the interrelationships of natriuretic peptides and collagen markers in 93 patients receiving thrombolytic treatment for their first acute myocardial infarction (AMI). Collagen formation following AMI, evaluated as serum levels of amino terminal propeptide of type III procollagen, correlated with NH(2)-terminal proANP (r = 0.45, P < 0.001), BNP (r = 0.55, P < 0.001) and NH(2)-terminal proBNP (r = 0.50, P < 0.01) on day 4 after thrombolysis. Levels of intact amino terminal propeptide of type I procollagen decreased by 34% (P < 0.001), and levels of carboxy terminal cross-linked telopeptide of type I collagen (ICTP) increased by 65% (P < 0.001). ICTP levels correlated with NH(2)-terminal proBNP (r = 0.25, P < 0.05) and BNP (r = 0.28, P < 0.05) on day 4. Our results suggest that ANP and BNP may act as regulators of collagen scar formation and left ventricular remodeling after AMI in humans. Furthermore, degradation of type I collagen is increased after AMI and may be regulated by BNP.     

Synthesis and antileishmanial activity of novel buparvaquone oxime derivatives

Novel oxime derivatives (2, 3 and 5) of buparvaquone (1) and O-methyl-buparvaquone (4) were synthesized and their in vitro activities against Leishmania donovani, the causative agent of visceral leishmaniasis (VL), were determined. Buparvaquone-oxime (2) was also studied as a bioreversible prodrug structure of buparvaquone (1). Buparvaquone-oxime (2) released buparvaquone (1) in vitro when it was incubated with induced rat liver microsomes, which suggests that the oxime-structure is a useful prodrug template for developing novel prodrugs of buparvaquone and other hydroxynaphthoquinones. Moreover, the formation of NO(2)(-) , formed via oxidation of NO, was confirmed during the bioconversion. The release of NO from buparvaquone-oxime (2) may provide an additional therapeutic effect in the treatment of leishmaniasis. Buparvaquone-oxime (2) and buparvaquone-O-methyloxime (3) demonstrated moderate activity against amastigotes of the Leishmania species that causes VL. However, the studied oximes (2, 3) most probably did not release buparvaquone (1) and NO during the present in vitro experiment. Further in vivo studies are needed to verify the biological activity of buparvaquone-oximes in the treatment of leishmaniasis.     

Distribution of Immunoreactive Prolyl Oligopeptidase in Human and Rat Brain

Prolyl oligopeptidase (POP) is a serine endoprotease that hydrolyses peptides shorter than 30-mer. POP may have a role in inositol 1,4,5-triphosphate (IP₃) signaling and in the actions of antidepressants, and POP inhibitors have exhibited antiamnesic and neuroprotective properties. However, little is known about the distribution of POP protein in the brain. We used immunohistochemistry to localize POP enzyme in the human whole hemisphere and in the rat whole brain. In humans, the highest POP densities were observed in caudate nucleus and putamen, hippocampus and cortex. In the rat, the highest POP densities were found in substantia nigra, hippocampus, cerebellum and caudate putamen. In general, the distribution of POP in human and rat brains was very similar and resembled that of IP₃ receptors. Our findings are support for a role of POP in movement regulation, cognition and possibly in IP₃ signaling. The expression of POP in processing nuclei further supports its function beyond neuropeptide metabolism. Dr. Erkki Tupala M.D. sadly passed away during the research project.     

Deletion of the Laminin α4 Chain Leads to Impaired Microvessel Maturation

The laminin alpha4 chain, a component of laminin-8 and -9, is expressed in basement membranes, such as those beneath endothelia, the perineurium of peripheral nerves, and around developing muscle fibers. Laminin alpha4-null mice presented with hemorrhages during the embryonic and neonatal period and had extensive bleeding and deterioration of microvessel growth in experimental angiogenesis, as well as mild locomotion defects. Histological examination of newborn mice revealed delayed deposition of type IV collagen and nidogen into capillary basement membranes, and electron microscopy showed discontinuities in the lamina densa. The results demonstrate a central role for the laminin alpha4 chain in microvessel growth and, in the absence of other laminin alpha chains, in the composition of endothelial basement membranes.