SUMMARY. Anderson and co-workers' recently described a simple high-performance liquid chromatographic/fluorimetric assay for serotonin and tryptophan. in which deproteinised samples were directly injected. Using this method routinely. we frequently observed interference which could not sufficiently be resolved from serotonin to allow accurate quantitation. However, when the amine was isolated by ion-exchange chromatography prior to HPLC separation as advocated by Koch and Kissinger.i chromatograms were obtained with practically no extraneous peaks. In this paper the application of this method for the determination of serotonin in CSF, plasma, serum and urine is described.
SUMMARY The development and validation of two different assays for serum angiotensin-converting enzyme are reported. The first step in both analytical systems is based on enzymatic cleavage of the synthetic substrate, hippuryl-histidyl-Ieucine, under conditions advocated by Cushman and Cheung.1 The hippuric acid released is then assayed either by colorimetry following an azlactone condensation reaction with an aromatic aldehyde, or by reversed-phase ion-pair high-performance liquid chromatography (HPLC). Both procedures reveal good linearity in the range 0-80 nmol/ml per min, with an inter-assay coefficient of variation of 6· 2 %for the colorimetric assay and 4· 5 %for the HPLC-assisted assay. Recovery values measured for the colorimetric method ranged from 97 % to 102%and for the HPLC-assisted method from 98 %to 101 %. The colorimetric method is suitable for performance in small hospital laboratories since it can be carried out with simple analytical instrumentation. It is obvious nevertheless; that the HPLC-assisted assay reveals better validation criteria. The method is also simple and rapid and it has successfully been used in our laboratory for the last two years.Angiotensin-converting enzyme (ACE) (kinase II. tripeptides such as hippuryl-histidyl-Ieucine (Hip-EC 3.4.15.1), recently reviewed by Erdos,2 is an His-Leu) and hippuryl-glycyl-glycine (Hip--Glyexopeptidase which catalyses cleavage of carboxy-Gly) or on cleavage of fluorogenic substrates terminal dipeptidyl residues.P'" It catalyses cleavage consisting of a highly fluorescent group coupled to of histidyl-leucine (His-Leu) from angiotensin I to a quenching nitrophenyl moiety.l920 Enzymatic yield its more potent vasopressive octopeptide hydrolysis of these fluorogenic substrates results in angiotensin II and also inactivates bradykinin by liberation from the quenching interaction and cleavage of the Pro 7-Phe8 bond. Hence the enzyme release of fluorescence. Hydrolysis of Hip--His-Leu not only inactivates hypotensive kinins but also or Hip-Gly-Gly is followed by a wide range of activates hypertensive angiotensin. It is a mem-analytical methods. A fluorescent complex can be braneous glycoprotein constituent of the vascular formed by reaction of e-phthalaldehyde'" 22 or endothelial cells located in juxtaposition to the fluorescarnine'" with the dipeptide residues; hippuric circulating bloodstream.vP Although the lung and acid released during the assay is extracted with ethyl kidney are physiologically important sites of con-acetate and determined by spectrophotometry at version, the enzyme is also found in many other 228 nm,112 by a differential spectrophotometric organs.'! In some diseases involving pulmonary method after reaction with 2,4,6-trichloro-Schanges, such as sarcoidosis, silicosis, and triazine.i" by condensation of hippuryl azlactone asbestosis.Pv'" and also in some non-pulmonary with an aromatic aldehyde after thin-layer chromatogranulomatous diseases such as Gaucher's disease graphic separation of hippuric acid 25 or by HPLC. 26 and lepro...
An automated determination for xanthurenic acid in urine was devised based upon its reaction with 2,6-dichloroquinonechlorimide. The separation of xanthurenic acid from interfering substances, such as urine chromogens and phenolic compounds other than xanthurenic acid, was performed by using Sephadex G-10. The effluents containing the xanthurenic acid were determined automatically. As a result of the high sensitivity of this procedure, concentration technics are unnecessary.
A study of the alterations in lactate dehydrogenase and creatine kinase isoenzyme patterns in isolated grey and white matter edema fluid and in peripheral blood is described with a view to possible clinical use in severe brain-injury. The breakdown of the blood-brain barrier, caused by the infliction of a cold injury to the cerebral cortex of cats, resulted in a shift in the LD isoenzyme distribution in favour of the faster moving fractions and the appearance of brain-type CK in the peripheral blood. Total lactate dehydrogenase activity demonstrated no statistical significant changes during the experiment and total creatine kinase showed only slight increased values in the samples collected simultaneously with the infliction of a cold lesion. This strongly suggests that measurements of these parameters in peripheral blood are of little value as indicators of brain damage. Alterations in isoenzyme patterns observed in edema fluids are reflected in peripheral blood. The presence, therefore, of brain-type creatine kinase accompanied by a relative high amount of lactate dehydrogenase H-sub unit in peripheral blood, can be considered as adequate indices of severe brain damage.
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