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Most of the proposed electron accelerator projects for future FELs, ERLs or 4th generation light sources require electron beams with an unprecedented combination of high brightness, low emittance, and high average current. In all projects photoguns will be applied: DC-photoguns, normal conducting RF-photoguns (NC-guns), and superconducting RF photoguns (SRF-guns). While the concepts of DC- and NC-guns are well proofed, the SRF-gun development still possesses a high risk. Challenges are the design of the superconducting cavity, the choice of the photocathode type, its life time, a possible cavity contamination, the difficulty of coupling high average power into the gun, and, finally, the risk of beam excitation of higher-order cavity modes. In combination with SRF linacs, the SRF-guns seem to be the best solution for high average currents. Several R&D projects of SRF-gun have been launched. In this paper, we will give an overview of the progress of the SRF photoinjector development. In detail, the technical concept, the performance and the status of the Dresden Rossendorf SRF-gun project, a collaboration of BESSY, DESY, MBI and FZD, will be presented. The main design parameters of this SRF-gun are the final electron energy of 9.5 MeV, 1 mA average current, and transverse normalized emittances (rms) of 1 mm mrad at 77 pC and 2.5 mm mrad at 1 nC bunch charge. The 1.3 GHz cavity consists of three TESLA-shaped cells, a specially designed half-cell where the photocathode is placed and a choke filter in order to prevent RF losses at the cathode side. The normal-conducting photocathode with a Cs2Te photoemission layer is cooled by liquid nitrogen. The SRF-gun cryostat consists of a stainless steel vacuum vessel, a warm magnetic shield, a liquid nitrogen-cooled thermal shield and a titanium He tank with a two-phase supply tube. The 10 kW fundamental power coupler is adopted from the ELBE cryomodule. In a first commissioning and test period the gun will be operated in parallel to the accelerator. A diagnostic beamline will allow beam parameter measurement and further optimization of the SRF-gun. In a final step, the gun will be connected to the ELBE superconducting linear accelerator to deliver an improved electron beam to the user labs.

This paper presents results of the photocathode cooling system test of the 3 1 2 cell SRF gun at the Forschungszentrum Rossendorf. The SRF gun will produce short electron pulses with high bunch charges and low transverse emittance. The requirement for the superconducting electron linear accelerator in Rossendorf (ELBE) is to provide a low emittance electron beam up to 1 mA current and 9.5 MeV energy. Additionally, it will easily operate in continuous wave (cw) mode because of the low RF power losses in the superconducting material. Therefore, the normal conducting copper cathode must be cooled by liquid nitrogen in order to preserve the temperature of the cavity at 2.2 K. The estimated power input from the RF field into the cathode could be more than 10 W [P. vom Stein, Thesis, TU-Dresden, 1998]. First results of temperature measurements of the photocathode, respectively, from the cooling system at a heat load up to 30 W are presented.

Heterogenous nucleation on small molecule crystals causes a monoclinic crystal form of bacteriorhodopsin (BR) in which trimers of this membrane protein pack differently than in native purple membranes. Analysis of single crystals by nano-electrospray ionization-mass spectrometry demonstrated a preservation of the purple membrane lipid composition in these BR crystals. The 2.9-Å x-ray structure shows a lipid-mediated stabilization of BR trimers where the glycolipid S-TGA-1 binds into the central compartment of BR trimers. The BR trimer/lipid complex provides an example of local membrane thinning as the lipid head-group boundary of the central lipid patch is shifted by 5 Å toward the membrane center. Nonbiased electron density maps reveal structural differences to previously reported BR structures, especially for the cytosolic EF loop and the proton exit pathway. The terminal proton release complex now comprises an E194-E204 dyad as a diffuse proton buffer.

The human multidrug resistance protein (MRP1) confers resistance of cells to a number of different cytostatic drugs and functions as an export pump for glutathione S-conjugates, glucuronides and other amphiphilic anions. The present study details for the first time MRP1-mediated ATP-dependent transport of various glutathione S-conjugates of the bifunctional alkylating agents chlorambucil and melphalan. In membrane vesicles prepared from cells expressing recombinant MRP1, the conjugates were transported at rates in the following order: monoglutathionyl chlorambucil > bisglutathionyl chlorambucil > monohydroxy monoglutathionyl chlorambucil and monoglutathionyl melphalan > monohydroxy monoglutathionyl melphalan. In addition, we show that membranes from chlorambucil-resistant GST-alpha-overexpressing CHO cells as well as from their parental cells express the hamster homologue of MRP1. With both CHO cell membrane preparations, we observed ATP-dependent transport of monoglutathionyl chlorambucil and of leukotriene C4, a glutathione S-conjugate and high-affinity substrate of MRP1. The transport rates measured in the resistant cells were only two- to three-fold higher than those measured in the control cells. These results together with cytotoxicity assays comparing MRP1-overexpressing cell pairs with the CHO cell pair indicate that, although MRP1-mediated transport is active, it may not be the rate-limiting step in chlorambucil resistance in these cell lines. Images Figure 3

A superconducting radio frequency (RF) photoelectron injector (SRF gun) is under development at the Research Center Dresden–Rossendorf. This project aims mainly at replacing the present thermionic gun of the superconducting electron linac ELBE. Thereby the beam quality is greatly improved. Especially, the normalized transverse emittance can be reduced by up to one order of magnitude depending on the operating conditions. The length of the electron bunches will be shortened by about two orders of magnitude making the present bunchers in the injection beam line dispensable. The maximum obtainable bunch charge of the present thermionic gun amounts to 80 pC. The SRF gun is designed to deliver also higher bunch charge values up to 2.5 nC. Therefore, this gun can be used also for advanced facilities such as energy recovery linacs (ERLs) and soft X-ray FELs. The SRF gun is designed as a 3 1 2 cell cavity structure with three cells basically TESLA cells supplemented by a newly developed gun cell and a choke filter. The exit energy is projected to be 9.5 MeV. In this paper, we present a description of the design of the SRF gun with special emphasis on the physical and technical problems arising from the necessity of integrating a photocathode into the superconducting cavity structure. Preparation, transfer, cooling and alignment of the photocathode are discussed. In designing the SRF gun cryostat for most components wherever possible the technical solutions were adapted from the ELBE cryostat in some cases with major modifications. As concerns the status of the project the design is finished, most parts are manufactured and the gun is being assembled. Some of the key components are tested in special test arrangements such as cavity warm tuning, cathode cooling, the mechanical behavior of the tuners and the effectiveness of the magnetic screening of the cavity.

In the paper, we report on the status and progress of the superconducting RF gun project in Rossendorf. The gun is designed for cw operation mode with 1 mA current and 9.5 MeV electron energy, and it will be installed at the ELBE superconducting electron linear accelerator. The gun will have a 3½ cell niobium cavity operating at 1.3 GHz. The cavity consists of three cells with TESLA geometry and a specially designed half-cell in which the photocathode will be placed. The production of two Nb cavities, with RRR 300 and 40 respectively, has be finished at the beginning of 2005. After delivery, the RF tests will be performed and the preparation of the cavities will be started. At the same time, the design of the cryostat and the fabrication of its components are already finished. Further activities are the design of the diagnostic beam line, the testing of the new photocathode preparation system, and the upgrade of the 262 nm driver laser system.

A defective mucus composition represents a key pathogenetic factor for intestinal injury. Phosphatidylcholine (PC) is an essential component contributing to formation of a hydrophobic mucus layer. For evaluation of PC in the pathogenesis of inflammatory bowel disease, the concentration and composition of PC in the rectal mucus of patients with ulcerative colitis was determined. Electrospray ionization (ESI) tandem mass spectrometry (MS/MS) allows quantification of PC species and enables analysis of crude extracts.Lipid extracts of material obtained by light scrapings of the intestinal lumen were analysed quantitatively by nanoESI MS/MS with synthetic internal PC and lysophosphatidylcholine (LPC) standards. PC and LPC species from rectoscopically acquired mucus aliquots of patients with ulcerative colitis were compared to Crohn disease and control subjects.Patients with inactive ulcerative colitis showed significantly less PC and LPC (median 346 [IQR: 230-405] pmol total PC/mg dry weight) in rectal mucus compared to Crohn disease (median 126 [IQR: 465-1941] pmol total PC/mg dry weight) and control subjects (median 1285 [IQR: 850-1639] pmol total PC/mg dry weight) (P0.05). The molecular species of PC and LPC were not significantly different between the groups. The most abundant species were PC 16:0/18:1; PC 16:0/18:2; PC 18:0/18:1; PC 18:0/18:2; LPC 16:0; and LPC 18:0.NanoESI MS/MS is a suitable tool for analysing and quantifying small amounts of PC in human mucus. Patients with ulcerative colitis have significant less PC in their intestinal mucus despite a comparable PC molecular species composition pattern. This suggests that a low amount of protective mucus PC is a characteristic feature in ulcerative colitis and explains an increased susceptibility to luminal contents.

Substrate selectivity and other enzymic characteristics of two epidermis-derived lipoxygenases (LOXs), the epidermis-type (e) (12S)-LOX and (12R)-LOX, were compared with those of the platelet-type (p) (12S)-LOX. In contrast with p(12S)-LOX, e(12S)-LOX and (12R)-LOX exhibited no or very low reactivity towards the customary substrates linoleic acid and arachidonic acid but metabolized the corresponding fatty acid methyl esters, which, in contrast, were not accepted as substrates by p(12S)-LOX. Other esters of arachidonic acid and linoleic acid, including propan-2-yl and cholesterol esters, 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-linoleyl-sn-glycero-3-phosphoethanolamine, and ceramide 1 carrying an omega-linoleic acid ester, were not metabolized by these three LOX isoenzymes. Among various polyunsaturated fatty acids the isomeric eicosatrienoic acids were found to be oxygenated by e(12S)-LOX but not by (12R)-LOX. 4,7,10,13,16,19-Docosahexaenoic acid as a substrate was restricted to p(12S)-LOX. Variations in the pH and the Ca(2+) content of the incubation medium affected the catalytic potential only slightly. Whereas (12R)-LOX activity increased in the presence of Ca(2+) and with an acidic pH, Ca(2+) had no effect on p(12S)-LOX and e(12S)-LOX; an acidic pH decreased the catalytic activity of the latter two. However, the catalytic activity of the epidermis-type isoenzymes, but not of p(12S)-LOX, was found to be markedly increased in the presence of DMSO. Under these conditions, e(12S)-LOX and (12R)-LOX oxygenated 4,7,10,13,16,19-docosahexaenoic acid to 14-hydroxy-4,7,10,12,16,19-docosahexaenoic acid and 13-hydroxy-4,7,10,14,16,19-docosahexaenoic acid respectively. In addition, (9R)-hydroxyoctadeca-10,12-dienoic acid methyl ester was generated from linoleic acid methyl ester by (12R)-LOX. Independently of the substrate, the catalytic activity of e(12S)-LOX and (12R)-LOX was always at most 2% of that of p(12S)-LOX with arachidonic acid as substrate.

Analytically useful information from accurate mass data for peptides with an error of/=20 ppm is discussed. The deltamass (= mass value following the decimal point) distribution of natural peptides is extracted from a protein database. Compared with the random peptide data, the natural data show a higher average deltamass value and a smaller width of the mass distribution. This deviation can be ascribed to the non-random abundances of the standard amino acids. In particular, accurate mass data for peptides located near the edges of the natural mass distribution contain analytical information. Mass data near the edges generate very few hits in a protein database search and are therefore highly specific for protein identification. Mass signals near the low-mass edge indicate either a high probability that the peptide contains one or several cysteine sites, or that the peptide is highly acidic due to the presence of several D and/or E residues or that it is a glycopeptide. Mass data near the high-mass edge indicate a non-polar peptide with a high abundance of the non-polar amino acids leucine, isoleucine and valine. An Internet page is introduced that analyzes the deviation of a peptide mass from the average deltamass value and that supports the characterization of glycopeptides found near the low-mass edge of the mass distribution.

Leukotrienes are biologically highly active compounds derived from the 5-lipoxygenase pathway of arachidonic acid metabolism (Mayatepek and Hoffmann 1995; Samuelsson et al 1987). They comprise the cysteinyl leukotrienes (LTC 4 , LTD 4 , LTE 4 ) and LTB 4 . The first two committed steps in leukotriene synthesis are catalysed by the 5-lipoxygenase, which requires the presence of 5-lipoxygenase-activating protein (Figure 1). The resulting unstable epoxide intermediate LTA 4 can be further metabolized either to LTB 4 or to LTC 4 . The latter step is specifically catalysed by the enzyme LTC 4 synthase and requires the presence of glutathione. Subsequent formation of LTD 4 is catalysed by γ-glutamyl transpeptidase (y-GT). The rate-limiting step in the synthesis of cysteinyl leukotrienes is the conversion of LTA 4 to LTC 4 catalysed by LTC 4 synthase. Biosynthesis of leukotrienes is limited to a small number of human cells, including brain tissue (Simmet et al 1998). Besides their well-known function in the mediation of inflammation and host defence, cysteinyl leukotrienes have neuromodulatory and neuroendocrine functions in the brain (Lindgren et al 1984; Lammers et al 1996). We have investigated synthesis and metabolism of leukotrienes in two infants with severe neurological symptoms, later identified as LTC 4 synthesis deficiency, and two patients with γ-GT deficiency.

PHIPA 3-10 [13-(4'-iodophenyl)-3-(p-phenylene)tridecanoic acid] is a p-phenylene-bridged, radioiodinated omega-phenyl fatty acid that has recently been developed to study coronary artery disease or cardiomyopathies. Here, we demonstrate that PHIPA 3-10 exhibits the characteristics of a long-chain fatty acid, including its ability to be efficiently taken up by myocytes and to function as a substrate for beta-oxidation before it is trapped.Myocardial metabolism of carrier-added and carrier-free 131I-PHIPA 3-10 preparations were investigated in rats in vivo and in isolated Langendorff rat hearts. Heart extracts were analyzed by high-performance liquid chromatography, negative-ion electrospray mass spectrometry and investigation of intracellular distribution using density-gradient centrifugation.A single, rapidly formed metabolite was found in the heart extract and also, surprisingly, in the hydrolyzed lipids. The total amount of metabolite increased from 43% to 51% between 15 and 60 min postinjection. By high-performance liquid chromatography comparison with synthetic potential catabolites, the metabolite was assigned the name PHIPA 1-10 [11-(4'-iodophenyl)-1-(p-phenylene)undecanoic acid] and was the product of one beta-oxidation cycle. Additional proof was obtained from the mass spectrometric analysis of the metabolite formed in vivo. The formation of this metabolite could be suppressed by Etomoxir, a carnitine palmitoyl transferase I inhibitor, indicating beta-oxidation of 131I-PHIPA 3-10 in mitochondria. Final evidence for the involvement of mitochondria in the degradation of 131I-PHIPA 3-10 was obtained by density-gradient centrifugation of homogenized rat heart tissue. The position of the labeled free PHIPA 3-10 and free metabolite peaked within the fraction containing mainly mitochondria.In spite of its bulky structure, 131I-PHIPA 3-10 is extracted by the myocardium in a manner similar to the extraction of the unmodified fatty acid analog, IPPA. The retention of PHIPA 3-10 in heart muscle results from the presence of the p-phenylene group, which prevents more than one beta-oxidation cycle. Intracellular free PHIPA 3-10 and free PHIPA 1-10 are present in the mitochondria, whereas most of the esterified metabolite was found in the cytosolic lipid pool. Hence, the rapid appearance of PHIPA 1-10 in the lipid pool must be accounted for by mitochondrial leakage or by an unknown in-out transport system.

In summary, these models provide a unifying stereochemical summary of the dioxygenation reactions catalyzed by (S)-type lipoxygenases or for the first dioxygenation step of prostaglandin H synthases.

Conjugation chemistry and kit formulated binding of the NHS ester of 6-(4'-(4"-carboxyphenoxy)butyl)-2, 10-dimercapto-2,10-dimethyl-4,8-diazaundecane (NHS-BAT ester) to monoclonal antibodies (MAbs) was investigated. The functionalities of the resulting BAT conjugated and 99mTc-labeled MAbs BW 431/26, MAb 425 and bispecific MDX210 (fragment construct) were tested by immunoreactivity and immunoscintigraphy.The kinetics and chemistry of the conjugation reaction were monitored by high-performance liquid chromatography, size-exclusion chromatography and positive fast-atom-bombardment mass spectra (FAB-MS). The 99mTc BAT-MAbs were tested with various immunoreactivity assays. The biodistribution of 99mTc-BAT-BW 431/26 in rats was compared with directly labeled BW 431/26.At pH 8.5 and 25 degrees C, the reactivity of the NHS-BAT ester was high with 90% completion after 30 min. The conjugation yield of 19 microM MAb and 228 microM NHS-BAT ester amounted to 30%. Higher NHS-BAT ester concentrations afforded higher BAT-to-MAb ratios. According to FAB-MS, the conjugation competing hydrolysis surprisingly occurred at the NHS ring. Almost quantitative 99mTc labeling was achieved after 5 min at 25 degrees C. Immunoreactivity of the 99mTc-BAT antibodies showed90% recovery and proved to be insensitive to BAT-to-MAb ratios of up to 10. The 99mTc-BAT-BW 431/26 showed similar organ distribution but revealed less urinary excretion compared with the directly labeled BW 431/26. Immunoscintigraphy with 99mTc-labeled and BAT-BW 431/26 and BAT-MAb 425 showed the respective biological function in vivo.According to straightforward conjugation chemistry, the ease of 99mTc labeling and the application of a simple ultrafiltration technique, the NHS-BAT ester represents a nondestructive, universally applicable biofunctional ligand to introduce stable 99mTc protein binding sites. Kit formulated conjugation/labeling can be performed with little time requirements and laboratory experience.

Endogenous cysteinyl leukotriene synthesis was assessed in 10 patients with Kawasaki disease and 10 healthy controls by measuring excretion of leukotriene E4 (LTE4) in urine. LTE4 was increased more than fivefold in patients with Kawasaki disease compared with controls (median (range) 55.3 (31.8-120.6) v 10.2 (7.1-14.9) nmol/mol creatinine); this suggests that cysteinyl leukotrienes are involved in the pathophysiology of Kawasaki disease. Leukotriene synthetase inhibition or receptor antagonism may therefore offer a new potential therapeutic approach in children with this disease.

Upon induction of metamorphosis, larvae of the marine hydroid Hydractinia echinata release [14C]-arachidonic acid from previously labeled endogenous sources. The lipoxygenase inhibitors nordihydroguaiaretic acid and 5,8,11,14-eicosatetraynoic acid inhibited metamorphosis induced by Cs+ and 1,2-sn-dioctanoylglycerol, whereas the inhibitors of cyclooxygenase, indomethacin, and acetylsalicylic acid were ineffective, suggesting a role for lipoxygenase metabolites of arachidonic acid in induction of metamorphosis. Lipoxygenase products in Hydractinia echinata were isolated and identified by gas chromatography/mass spectrometry. 8- and 12-HETE were the most abundant metabolites. In cytosolic fractions from larvae activity of an arachidonic acid metabolizing enzyme, presumably a lipoxygenase, was found. The metabolic product was identified by 1H-NMR and chiral phase HPLC as 8(R)-HETE. Its production was strongly inhibited by NDGA, but not by indomethacin. © 1994 Wiley-Liss, Inc.

Im Misch-, Parotis- und Submandibularisspeichel von 16 Patienten mit Plattenepithelkarzinomen des oberen Aerodigestivtrakts wurden die Spiegel von 5-, 8-, 9-, 11-, 12- und 15-Hydroxyeicosatetraensaure (HETE) und 9- und 13-hydroxyoktadekadiensaure (HODE) als Produkte der Lipoxygenasen sowie 12-Heptadekatriensaure (12-HHT) als Marker fur den Zyklooxygenasemetabolismus mit gaschromatographisch-massenspektrometrischen Methoden bestimmt. Als Kontrollgruppe dienten 8 gesunde Probanden. Der Mischspeichel von gesunden Personen zeigte eine 12(S)-Lipoxygenaseaktivitat, die vermutlich auf der Anwesenheit von polymorphkernigen Leukozyten in der Mundhohle beruht. Speichel aus der Glandula parotis und aus der Glandula submandibularis zeigte bei der Kontrollgruppe keine Lipoxygenaseaktivitat, von den monohydroxylierten Fettsauren waren nur Spuren nachweisbar.

The degradation of leukotrienes by beta-oxidation from the omega-end proceeds in peroxisomes (Jedlitschky et al. J. Biol. Chem. 1991. 266:24763-24772). Peroxisomal degradation of leukotrienes was studied in humans by analyses of endogenous leukotrienes in urines from eight patients with biochemically established peroxisome deficiency disorder and eight age- and sex-matched healthy infant controls. Leukotriene metabolites were separated by high-performance liquid chromatography, quantified by radioimmunoassays, and identified as well as quantified by gas chromatography-mass spectrometry. Urinary leukotriene E4 (LTE4) and N-acetyl-LTE4 excretions, relative to creatinine, were increased > 10-fold in the patients in comparison to healthy infants. The beta-oxidation product omega-carboxy-tetranor-LTE3 averaged 0.05 mumol/mol creatinine in the controls but was not detectable in the patients. However, omega-carboxy-LTE4 (median 13.6 mumol/mol creatinine) was significantly increased in the patients' urine, whereas LTB4 (median 0.07 mumol/mol creatinine) and omega-carboxy-LTB4 were detected exclusively in the urines of the patients. These data indicate an impairment of the inactivation and degradation of both LTE4 and LTB4 in patients with peroxisomal deficiency. The increased levels of the biologically active, proinflammatory mediators LTE4 and LTB4 might be of pathophysiological significance in peroxisome deficiency disorders. This is the first and so far only condition with a pronounced urinary excretion of omega-carboxy-LTE4, omega-carboxy-LTB4, and LTB4. This impaired catabolism of leukotrienes and the altered pattern of metabolites may be of diagnostic value. These findings underline the essential role of peroxisomes in the catabolism of leukotrienes in humans.

Chain shortening via beta-oxidation from the omega-end has been recognized as the major pathway for the degradation of cysteinyl leukotrienes as well as leukotriene B4 (LTB4). The metabolic compartmentation of this pathway was studied using peroxisomes purified from normal and clofibrate-treated rat liver. beta-Oxidation products of omega-carboxy-LTB4, including omega-carboxy-dinor-LTB4 identified by gas chromatography-mass spectrometry, were formed by the isolated peroxisomes. The reaction was dependent on CoA, ATP, and NAD and was stimulated by FAD. NADPH was necessary for the further metabolism of omega-carboxy-dinor-LTB4. Together with microsomes a degradation of omega-carboxy-LTB4 also proceeded in isolated mitochondria in the presence of CoA, ATP, and carnitine. beta-Oxidation of the cysteinyl leukotriene omega-carboxy-N-acetyl-leukotriene E4 was observed only with isolated peroxisomes in combination with lipid-depleted microsomes. Direct photoaffinity labeling using omega-carboxy-[3H] LTB4 and omega-carboxy-N-[3H]acetyl-LTE4 served to identify peroxisomal leukotriene-binding proteins. The bifunctional protein (EC 4.2.1.17 and 1.1.1.35) and 3-ketoacyl-CoA thiolase (EC 2.3.1.16) of the peroxisomal beta-oxidation system were the predominantly labeled polypeptides as revealed by precipitation with monospecific antibodies. In vivo studies with N-acetyl-[3H2]LTE4, N-acetyl-[3H8]LTE4, and N-[14C]acetyl-LTE4 after treatment with the peroxisome proliferator clofibrate indicated formation and biliary excretion of large amounts of metabolites more polar than omega-carboxy-tetranor-N-acetyl-LTE3 including omega-carboxy-tetranor-delta 13-N-acetyl-LTE4 and omega-carboxy-hexanor-N-acetyl-LTE3. Increased formation of beta-oxidized catabolites of N-acetyl-LTE4 and LTB4 was also observed in hepatocytes isolated after clofibrate treatment. Our results indicate that peroxisomes play a major role in the beta-oxidation of leukotrienes from the omega-end. Whereas omega-carboxy-LTB4 was beta-oxidized both in isolated peroxisomes and mitochondria, the cysteinyl leukotriene omega-carboxy-N-acetyl-LTE4 was exclusively degraded in peroxisomes.

An enzymatic activity has been found in cytosolic preparations from mouse epidermis which catalyzes the formation of 8-hydroperoxyeicosatetraenoic acid/8-hydroxyeicosatetraenoic acid (8-HPETE/8-HETE) from arachidonate. In contrast to 12-lipoxygenase this enzyme activity was not detectable in normal (untreated) mouse skin but only after in vivo treatment with the phorbol ester tumor promoter TPA (12-O-tetradecanoylphorbol-13-acetate). The induction showed a maximum at 24 h after TPA treatment strictly depended on the age of the mice and the TPA dose and was prevented by cycloheximide. The primary product formed from arachidonic acid was 8-HPETE, and the enzyme seems not to possess a significant peroxidase activity. This result as well as studies with specific inhibitors and its cytosolic localization indicates this enzyme to be a member of the lipoxygenase family. Most of the 8-lipoxygenase activity is located in cells of the suprabasal compartment of the epidermis. In spite of being a cytosolic enzyme 8-lipoxygenase appeared to be lipophilic to some extent and was activated by lecithin. The enzyme did not require calcium ions or ATP and showed a pH optimum at 7.5-8.0. 8-HPETE/8-HETE levels in mouse epidermis in vivo were determined by gas chromatography-mass spectrometry and found to be strongly increased after phorbol ester treatment, in agreement with the induction of 8-lipoxygenase observed.

Preliminary clinical experience with early amniocentesis is reported. Fifty-two amniocenteses were performed before the end of the 14th week following the last menstrual period. Cytogenetic and biochemical analyses (AFP, AChE) were performed. Increasing experience with amniotic fluid samples containing small-cell populations and the use of combined culture media improved the poor initial results to a 100% level, thus enabling the use of this technique for diagnostic purposes. The sampling technique and the post-procedural evolution of twelve amniocenteses in pregnant women, whose pregnancy is to be continued, are presented. The possibility of performing amniocentesis in early pregnancy is discussed with reference to anatomical aspects (amniotic and chorionic cavity).

Cassmer hat 1959 als erster auf das endokrine Zusammenwirken im Ostrogenstoffwechsel von Mutter, Plazenta und Fetus hingewiesen.

Field desorption mass spectrometry is a new and versatile technique which expands the range of applicability of mass spectrometry to include highly polar and thermally labile compounds. The simplicity of the FD mass spectra of organic compounds, containing in each case an intense signal for the intact ionized or cationized molecule is the basis for the applicability of the technique to analytical problems in environmental, biochemical, and biomedical research.

Lipids labelled with deuterium or carbon-14 have been investigated by field desorption mass spectrometry for determination of their degree of labelling. This application is demonstrated for free fatty acids, cholesterol, cholesteryl esters, triglycerides, and l-α-phosphatidylcholines. Comparison of the molecular ion groups of the non-labelled and of the labelled compounds enables a fast and reliable determination of the degree of labelling. For multiply labelled compounds the label distribution is also obtained from the molecular ion group. In addition, for cholesteryl esters and for phosphatidylcholines structurally significant fragment ions provide information about the position of the label. Several hundred nanograms of the compound are typically required for a single analysis with a relative standard error of 0.5–2 % in the value calculated for atom % hydrogen-2 or for the specific carbon-14 activity.

In this work, the applicability of field desorption mass spectrometry for the determination of lithium in mineral and tap water, wine and high-purity solvents has been examined. Because of the outstanding sensitivity of field desorption mass spectrometry for alkali cations and the high specificity of mass spectrometry, it was possible to determine lithium from microlitre samples by using the method of stable isotope dilution. Lithium, at levels of 10−7–10−4 mg/ ml, was determined in the samples without any pretreatment. The time required for one analysis was about 20–30 min. The results from quantitative analyses of mineral water were compared with others obtained by atomic-absorption spectrophotometry and were in good agreement. The very small concentrations of lithium in wine and high-purity solvents, which are too low to be measured by conventional atomic-absorption techniques, can be determined accurately without difficulty by field desorption mass spectrometry.

Bei drei Patientinnen am Ende der Graviditat (zwei Erstgebarende und eine Zweitgebarende) wurde eine stark erniedrigte Ausscheidung der Gesamtostrogene im 24-Std-Harn festgestellt. Nach dem Dehydroepiandrosteronsulfatbelastungstest lies sich die Ostrogenausscheidung nicht verbessern. Der Plasmaspiegel des humanen placentaren Lactogens lag dagegen im Normbereich. Da die ubrigen perinatalen Uberwachungsparameter (USD, Cardiotokogramm) ebenfalls normale, der Gestationszeit entsprechende Befunde ergaben, wurde die Verdachtsdiagnose 3-Sulfatasemangel der Placenta gestellt. Die beiden Erstgebarenden musten wegen einer totalen cervikalen Dystokie durch Kaiserschnitt entbunden werden. Alle Neugeborenen waren gesund und mannlichen Geschlechts. Die in vitro-Inkubationsversuche des Placentagewebes (Homogenat oder Mikrosomenfraktion) mit radioaktiv markiertem Ostronsulfat oder Dehydroepiandrosteronsulfat bestatigten den vermuteten Enzymdefekt gegenuber den Ergebnissen aus Inkubationen mit normalem Placentagewebe. Die placentaren Enzyme 17β-Hydroxysteroiddehydrogenase, Δ4/5-Isomerase und aromatisierendes System, wiesen dagegen eine normale Aktivitat auf.

Fetal adrenal glands were obtained from legal abortions in the 14--22 gestational weeks. The adrenal cortex was separated from the medulla using a micromanipulator. The cortex was homogenized in a glass-teflon homogenizer and the microsomal fraction was isolated by centrifugation. Tissue slices were also prepared by the method of Deutsch. The microsomal fraction or the slices were incubated with (4-14C) pregnenolone or (4-14C) cholesterol in the presence of an NADPH regenerating system and oxygen. ACTH or HCG was added. After extraction and paperchromatography radioactivity was determined in a scanner. The conversion of pregnenolone to 17 alpha-hydroxypregnenolone and dehydroepiandrosterone was increased by ACTH. In the presence of HCG dehydroepiandrosterone production only was increased,--and this occurred in slices only. In the microsomal fraction HCG was without effect on steroid biogenesis from dehydroepiandrosterone.

A series of six major urinary conjugates in the metabolism of antipyrine in man and rat has been investigated. A preparative isolation procedure has been developed using chromatography of methanolic extracts from urine on silica gel. In a two-step chromatographic procedure, methanolic extracts are first separated in 1) a "free fraction", containing unconjugated phase-I-metabolites and unchanged antipyrine, 2) a sulfate fraction and 3) a glucuronide fraction. Sulfate and glucuronide fraction, respectively, are each subjected to a second run for separation into their three components. Thus, the following conjugates have been prepared: 4-hydroxy-antipyrine sulfate, norantipyrine sulfate, 4,4'-dihydroxy-antipyrine sulfate, and 4-hydroxy-antipyrine glucuronide, norantipyrine glucuronide, 3-hydroxymethyl-antipyrine glucuronide. Methodology is also applicable to bile fluid and liver perfusate. Stability of isolated conjugates against acid hydrolysis has been studied to show that strongly marked differences exist in this series of conjugates. Field desorption mass spectrometry has been used for the direct identification of intact conjugates in an underivatized form. Using 13C-NMR, the structure of norantipyrine glucuronide has been established as an 5-enol glucuronide. By analogy, a structure of 5-enol sulfate is proposed for norantipyrine sulfate. From a semiquantitative examination by TLC of urine extracts from man and rat, it becomes apparent, that in the rat, at the dose level studied, sulfate formation is the predominant conjugation pathway. In man, glucuronides are the most prominent type of conjugates. Formation of sulfates is minimal up to a dose of 15 mg/kg antipyrine.

The utility of field desorption mass spectrometry for quantitative metal cation analysis in forensic sciences is demonstrated by the determination of a lethal thallium level in the brain tissue of an experimental animal. Stable isotope dilution and accumulation of the electrically recorded field desorption ion currents with a multi-channel analyzer allowed a direct estimation of thallium in homogenized tissue samples without further pretreatment. Experiments with standard solutions revealed the limit of detection for thallium to be about 10pg of the metal cation.

We report a patient who had a marked exacerbation of idiopathic thrombocytopenic purpura at 33 weeks of pregnancy. The platelet count rose in response to treatment with high doses of glucocorticoids and it was possible to allow the pregnancy to continue until 39 weeks. The patient was then delivered by elective Caesarean section and she had a splenectomy at the same time. A transfusion of platelets was administered as soon as the splenic vessels had been clamped. The resultant excess of platelets necessitated treatment with acetylsalicylic acid. At 18 months after Caesarean section and splenectomy the thrombocytopenia had not recurred. The infant had transient neonatal thrombocytopenia but required no active treatment for this.

The influence of bromocriptine, a prolactin antagonist, on maternal plasma and amniotic fluid prolactin (PRL) was investigated in two pregnancies at the 20th week with medical indication for abortion. Voluntary consensus of the patients was obtained. Blood sample determinations demonstrated that bromocriptine inhibits the secretion of PRL both in plasma and amniotic fluid. Since no changes were observed in peripheral maternal steroid concentrations of dehydroepiandrosterone, dehydroepiandrosterone-sulfate, delta 4-androstene-3, 17-dione, testosterone, estradiol, estriol, and cortisol, it is concluded that PRL does not seem to affect maternal and fetal adrenal cortex as supposed in amenorrhoic patients with hyperprolactinemia.

The glucuronide conjugates of the two isomeric antipyrine phase I metabolites of antipyrine in man, 4-hydroxyantipyrine and 3-hydroxymethylantipyrine have been analysed by field desorption and fast atom bombardment mass spectrometry. These isomers could be clearly distinguished on the basis of their fragmentation behaviour which was found to correlate with that observed under pyrolysis electron impact conditions.

Quantitative ultratrace analysis (10 nmol to 10 /cmol/1) of cesium in biological samples such as human body fluids and animal tissues is performed without any prior purification or concentration steps. The normal level of cesium ions in heart cells was determined. After poisoning these cells with high concentrations of the alkali cation much higher levels were found inside the cells then had been suggested previously. It is demonstrated that field desorption mass spectrometry is a unique tool for the qualitative and quantitative investigations of metal cations in biological material.

Carbon-14 and tritium labelled ionic organic compounds such as quaternary ammonium salts, steroid sulphates, bile acid conjugates, and oligopeptides have been analyzed for their label distribution and for their specific radioactivity by fast atom bombardment and field desorption mass spectrometry. No significant differences between the quantitative results with both techniques are found. The minimal specific radioactivities detectable by this approach are about 20 MBq mmol−1 or 10 GBq mmol−1 for compounds labelled with one atom of carbon-14 or one atom of tritium per molecule, respectively. Specific radioactivity determinations of highly labelled biochemicals are characterized by a precision and an accuracy in the region between 1% and 5%.

Field Desorption Mass Spectrometry, a-Amino Acid Methionine The fragmentation of methionine in field desorption mass spectrometry has been studied. Decomposition mechanisms are described which are based on stable isotope labelling, low and high resolution field desorption, and the application of field desorption kinetics. The combined use of these techniques for the study of some fundamental fragmentations in field desorption is demonstrated successfully. Further, a comparison with the fragmentation pattern of methionine under electron impact, chemical ionization and Curie point pyrolysis is given.