U. Redweik

Mass fragmentography of dopamine and 6-hydroxydopamine: Application to the determination of dopamine in human brain biopsies from the caudate nucleus

A method is described for the determination of dopamine and 6-hydroxydopamine in human brain biopsies (ca. 1 mg) using mass fragmentography of N.O-trifluoroacetyl derivatives. The sensitivity limit is 10 pg of dopamine and 20 pg of 6-hydroxydopamine. Untreated phenylketonuric patients show very low dopamine concentrations (? 1.9 ng per 100 μg of protein) in the caudate nucleus compared with control persons (ca.) 12 ng per 100 μg of protein). The deficiency of catecholamines in the brain, probably caused by a competitive inhibition of tyrosine 3-hydroxylase by phenylalanine, is believed to be an important factor in the pathogenesis of the neurological symptoms and of the mental retardation observed.

Mass fragmentography of 5-hydroxytryptophol and 5-methoxytryptophol in human cerebrospinal fluid

A specific and very sensitive method for the determination of 5-hydroxy-tryptophol (5-HTOL) and 5-methoxytryptophol (5-MTOL) in extracts from human cerebrospinal fluid (CSF) involving the use of mass fragmentography and pentafluoropriopionyl derivatives is described. 5-HTOL and 5-MTOL were determined in human CSF of three patients with leukaemia and from nine patients with neurological disorders. The concentration of free 5-HOT in CSF was in the range of 0.1-33 ng/ml and that of 5-MTOL was 0.3-13.9 ng/ml. For the first time the presence of these compounds in human material has been shown. The concentration of these two alcohols in CSF is markedly lower than the concentration of 5-hydroxyindoleacetic acid. These results suggest that human cerebral 5-hydroxytryptamine is preferentially metabolized to 5-HTOL-hydroxyindoleacetic acid rather than to 5-HTOL and 5-MTOL.

Quantitation of deuterated and non-deuterated phenylalanine and tyrosine in human plasma using the selective ion monitoring method with combined gas chromatography-mass spectrometry : Application to the in vivo measurement of phenylalanine-4-monooxygenase activity

A specific method is described for the quantitative analysis of deuterated and non-deuterated phenylalanine and tyrosine in human plasma by gas chromatography-mass spectrometry using selective ion monitoring. From the several derivatives investigated, the N- or N,O-trifluoroacetyl methyl esters were found to be the most suitable for our purposes. DL-Phenylalanine-4-d1 and L-tyrosine-d7 were used as internal standards. The sensitivity of this method permits the measurement of amounts as small as ca. 2.5 ng/ml in plasma for both phenylalanine and tyrosine. The coefficients of variation were found to be ca. 1.6% (n = 12) for phenylalanine and 3.0% (n = 12) for tyrosine. Using this method, an in vivo determination of phenylalanine-4-monooxygenase activity in humans is possible by loading the subjects with deuterated L-phenylalanine-d5 (accepted as substrate by phenylalanine-4-monooxygenase E.C. 1.14.16.1) and the subsequent measuring of deuterated L-tyrosine-d4 formed and residual L-phenylalanine-d5.

Purification of 6-pyruvoyl-tetrahydropterin synthase from human liver

The enzyme which catalyzes the first step in the conversion of dihydroneopterin triphosphate to tetrahydrobiopterin has been purified approx. 40,000-fold from human liver to apparent homogeneity. The enzyme has a native molecular weight of approximately 83,000 and consists of four identical subunits, each of which has a molecular weight of approximately 19,000. It contains carbohydrates and is remarkably stable to heat treatment. In the presence of purified sepiapterin reductase, Mg2+, and NADPH, this enzyme catalyzes efficiently the formation of tetrahydrobiopterin from dihydroneopterin triphosphate. This indicates that these two proteins are sufficient for the overall conversion.

Mass fragmentographic determination of endogenous glycine and glutamic acid released in vivo from the pigeon optic tectum. Effect of electric stimulation of a midbrain nucleus

Various investigations suggest glycine to be an inhibitory transmitter in the pigeon optic lobe in a pathway originating in the nucleus isthmi, pars parvocellularis (Ipc) terminating in the optic tectum. In order to obtain additional evidence for this hypothesis the in vivo release of endogenous glycine in the optic tectum upon electrical stimulation of Ipc was investigated. By perfusing the upper strata of the optic tectum with Ringer solution using a push-pull cannula endogenous amino acids released from the surrounding tissue were collected. Concentration of glycine and glutamic acid in the perfusates were determined by mass fragmentography of their N-pentafluoropropionyl hexafluoroisopropyl esters. Deuterium-labeled glycine and glutamic acid were used as internal standards for quantitative measurements. The resting release of glycine and glutamic acid was 2.9 pmol/min and 1.4 pmol/min, respectively. Electrical stimulation of Ipc was found to induce a 2--40-fold increase of the glycine efflux into the perfusate whereas the efflux of glutamic acid remained at a constant level. These findings strongly support the hypothesis that glycine is a transmitter in Ipc-tectal neurons.

Mass fragmentographic determination of cholecalciferol and 25-hydroxycholecalciferol in human serum

The serum extracts were purified by column-, thin layer- and high pressure liquid chromatography. Deuterated cholecalciferol and deuterated 25-hydroxycholecalciferol were used as internal standards. The quantitative analysis was performed using GC-mass fragmentography technique of TMS-ethers.

Co-ordinate Expression of GTP Cyclohydrolase I and Inducible Nitric Oxide Synthase in Rat Mesangial Cells

The synthesis of NO from L-arginine by NO synthase (NOS) is an important intracellular and intercellular signalling pathway originally discovered in macrophages and endothelial cells (1). Molecular cloning and sequence analyses revealed the existence of at least 3 main types of NOS isoforms. The brain and endothelial enzmes are constitutively expressed and are regulated by Ca2+ -mobilizing agonists that trigger an increase in intracellular free Ca2+, which binds to calmodulin, and thus activates NOS. A third type of NOS has been cloned from murine macrophages and is not constitutively expressed, but is induced by y-interferon and lipopolysaccharide, and is Ca2+ -independent. The macrophage NOS is regulated on a transcriptional level and once induced is active for hours and days. Not surprisingly, therefore, the inducible macrophage enzyme produces amounts of NO that are several hundred times higher than the ones produced by the constitutive endothelial cell and brain enzymes which are only active during the short periods of elevated intracellular Ca2+ observed after exposure of cells to Ca2+ -mobilizing agonists (1). We have shown that cytokines, such as interleukin 1 (IL-I) and tumour necrosis factor a (TNFa),

Cyanogen Bromide Cleavage of Proteins on Blots and Subsequent Separation of the Fragments by Polyacrylamide Gel Electrophoresis Directly from those Blots

Since nucleic acid sequencing is now quick and accurate, the usual goal of protein sequencing is no longer to determine an entire primary structure. A partial sequence is sufficient for the design of the oligonucleotide probes, needed to obtain starting material for sequence determinations on the nucleic acid level.

Bestimmung von Oxocarbonsäuren im Urin

A method for the quantitative determination of urinary keto acids is described. The keto acids are converted into their 2,4-dinitrophenylhydrazones at pH 2.5 to 3, reduced to their corresponding amino acids by means of Ha/PtC^ in ethanol and quantitatively determined according to STEIN and MOORE. The recovery of 11 keto acids was measured. The results obtained with this method are presented for a normal subject, a patient with phenylketonuria and a patient with leucinosis (branched-chain oxoaciduria).