Structural differences between glycosylated, disulfide-linked heterodimeric Knob-into-Hole Fc fragment and its homodimeric Knob-Knob and Hole-Hole side products.

An increasing number of bispecific therapeutic antibodies are progressing through clinical development. The Knob-into-Hole (KiH) technology uses complementary mutations in the CH3 region of the antibody Fc fragment to achieve heavy chain heterodimerization. Here we describe the X-ray crystal structures of glycosylated and disulfide-engineered heterodimeric KiH Fc fragment and its homodimeric Knob-Knob and Hole-Hole side products. The heterodimer structure confirms the KiH design principle and supports the hypothesis that glycosylation stabilizes a closed Fc conformation. Both homodimer structures show parallel Fc fragment architectures, in contrast to recently reported crystal structures of the corresponding aglycosylated Fc fragments which in the absence of disulfide mutations show an unexpected antiparallel arrangement. The glycosylated Knob-Knob Fc fragment is destabilized as indicated by variability in the relative orientation of its CH3 domains. The glycosylated Hole-Hole Fc fragment shows an unexpected intermolecular disulfide bond via the introduced Y349C Hole mutation which results in a large CH3 domain shift and a new CH3-CH3 interface. The crystal structures of glycosylated, disulfide-linked KiH Fc fragment and its Knob-Knob and Hole-Hole side products reported here will facilitate further design of highly efficient antibody heterodimerization strategies.

Lung ventilation/perfusion SPECT for diagnosing pulmonary embolism. Is the required minimal P/V count rate ratio fulfilled in routine situation? Results from a multicentre survey.

UNLABELLED: V/P-SPECT is a sensitive and specific procedure for the detection or exclusion of pulmonary embolism. It is important to conform to the guidelines in order to obtain reliable results. The sequence usually starts with the ventilation, followed by the perfusion scan. According to the current guidelines the count rate of the second study should be at least 3 times that of the first study. It is not always easy to meet this requirement, particularly since the deposed quantity of ventilation activity is not well known. The aim was therefore to analyse data from a multicentre survey to determine whether this essential precondition was strictly met. METHODS: In a multicentre survey 48 tertiary hospitals were asked to submit projections of all V/P-SPECT studies performed in January 2014. In total, 286 complete data sets from 16 institutions could be evaluated. First, the count rates of the first projections from the V-studies were subtracted from those of the P-studies. The resulting count rates in the first SPECT projections were then divided to calculate the activity ratios between the pure perfusion and ventilation scans (P/V-ratio at least 3 according to the guidelines). RESULTS: The range of the P/V ratio was 0.57-78.71, the mean P/V ratio was 6.94 ± 9.56. For 71 of the 286 external V/P studies (about 25%) the P/V ratio was < 3, in 23 studies (about 8%) the ratio failed to even reach the factor 2. CONCLUSIONS: An activity ratio of 3 between the perfusion and ventilation scan was not reached in about 25% of the 286 V/P studies (in around 8% the P/V ratio was <2), so that V/P studies were performed inadequately in a considerable number of procedures. Controlling the count rate increase during the perfusion tracer application (e. g. by handheld monitor) is therefore essential to avoid insufficient data.

Preoperative assessment of relative pulmonary lobar perfusion fraction in lung cancer patients. A rather simple three-dimensional CT-based vs. planar image-derived quantification.

UNLABELLED: Preoperative quantification of (relative) pulmonary lobar perfusion fraction using scintigraphy is established in predicting lung function after pulmonary surgery. Aim was to develop an easy and truly anatomical method for relative pulmonary lobar perfusion fraction quantification using SPECT/CT and to compare results with those from planar analyses in lung cancer patients. PATIENTS, METHODS: 36 patients with operable lung cancer, borderline lung function referred to pre-operative quantification. Perfusion SPECT-data were acquired p.i. of 163±9 MBq 99mTc-MAA, subsequent low-dose-CT (SymbiaT, Siemens). Iterative Flash3D-reconstruction, manual 3D segmentation of all lobes using PMOD. VOI transfer to coregistered perfusion SPECT-data, calculation of lobar fractions. Model-based calculation of relative lobar fractions based on planar data, analysis of planar vs. 3D results using t-test. RESULTS: Significant differences (p<0.05) between the results from 3D method and planar imaging were found for right upper and middle lobe and both lower lobes. Maximum differences ranged from 10.9% (left upper lobe) to 22.9% (right upper lobe). CONCLUSIONS: Relative pulmonary lobar perfusion fraction can easily be obtained by an anatomically driven 3D quantification. Results yielded by this method and the traditional planar approach differed greatly, possibly affecting eligibility for lung surgery in individual patients. Considering these results a 3D approach should be used whenever possible.

[Myocardial perfusion scintigraphy 2012 in Germany. Results of the 6th Query]. / Myokard-Perfusions-Szintigraphie 2012 in Deutschland. Ergebnisse der sechsten Erhebung.

AIM: The working group Cardiovascular Nuclear Medicine of the German Society of Nuclear Medicine presents the results of the 6th survey on myocardial perfusion scintigraphy (MPS) of the reporting year 2012. METHOD: 278 questionnaires (177 private practices (PP), 78 hospitals (HO), 23 university hospitals (UH)) were evaluated. RESULTS: MPS of 105,941 patients were reported. 95% [2005 = 80%] of MPS studies were conducted with (99m)Tc perfusion radiopharmaceuticals and only 5% with 201Tl. 79% [2009 = 76%] of the MPS were performed in PP, 15% [2009 = 17%] in HO, and 6% [2009 = 7%] in UH. Data from 108 centres which participated in all surveys from 2005 to 2012 showed an increase in MPS numbers of 4.0% (PP +6.1%, HO +18.2%, UH -18.3%). 29% of all participants (27% of PP, 31% of HO, and 26% of UH) noticed no change and 26% of all participants (28% of PP, 17% of HO and 35% of UH) an increase in their MPS requests since the 2009 query. The type of stress was pharmacological in 39% [2009 = 31%]. Of these 61% with adenosine (39% with exercise), 22% with regadenoson (51% with exercise), 14% with dipyridamole (60% with exercise), and 3% with dobutamine. Gated SPECT was performed in 73% [2009 = 56%] of all rest, in 70% [2009 = 56%] of all stress and in 67% [47%] of all stress and rest MPS. Only 36% [2009 = 33%] of the centres performed a quantification of all their studies with scores, whereas 41% [2009 = 52%] did not apply any quantification. 60% [2009 = 49%] of the MPS were requested by ambulatory care cardiologists. CONCLUSION: The survey on MPS in Germany reveals a good conformity of imaging procedures with the current guideline. A positive development in MPS practice and referral can be stated. However, there is still some potential of MPS processing considering the quantitative perfusion analysis.

[Myocardial perfusion scintigraphy - short form of the German guideline]. / Myokard-Perfusions-Szintigraphie. Kurzfassung der S1-Leitlinie.

This guideline is a short summary of the guideline for myocardial perfusion scintigraphy published by the Association of the Scientific Medical Societies in Ger-many (AWMF). The purpose of this guideline is to provide practical assistance for indication and examination procedures as well as image analysis and to present the state-of-the-art of myocardial-perfusion-scintigraphy. After a short introduction on the fundamentals of imaging, precise and detailed information is given on the indications, patient preparation, stress testing, radiopharmaceuticals, examination protocols and techniques, radiation exposure, data reconstruction as well as information on visual and quantitative image analysis and interpretation. In addition possible pitfalls, artefacts and key elements of reporting are described.

Potential hazards to embryo implantation: A human endometrial in vitro model to identify unwanted antigestagenic actions of chemicals.

Embryo implantation is a crucial step in human reproduction and depends on the timely development of a receptive endometrium. The human endometrium is unique among adult tissues due to its dynamic alterations during each menstrual cycle. It hosts the implantation process which is governed by progesterone, whereas 17ß-estradiol regulates the preceding proliferation of the endometrium. The receptors for both steroids are targets for drugs and endocrine disrupting chemicals. Chemicals with unwanted antigestagenic actions are potentially hazardous to embryo implantation since many pharmaceutical antiprogestins adversely affect endometrial receptivity. This risk can be addressed by human tissue-specific in vitro assays. As working basis we compiled data on chemicals interacting with the PR. In our experimental work, we developed a flexible in vitro model based on human endometrial Ishikawa cells. Effects of antiprogestin compounds on pre-selected target genes were characterized by sigmoidal concentration-response curves obtained by RT-qPCR. The estrogen sulfotransferase (SULT1E1) was identified as the most responsive target gene by microarray analysis. The agonistic effect of progesterone on SULT1E1 mRNA was concentration-dependently antagonized by RU486 (mifepristone) and ZK137316 and, with lower potency, by 4-nonylphenol, bisphenol A and apigenin. The negative control methyl acetoacetate showed no effect. The effects of progesterone and RU486 were confirmed on the protein level by Western blotting. We demonstrated proof of principle that our Ishikawa model is suitable to study quantitatively effects of antiprogestin-like chemicals on endometrial target genes in comparison to pharmaceutical reference compounds. This test is useful for hazard identification and may contribute to reduce animal studies.

In vitro-Ishikawa cell test for assessing tissue-specific chemical effects on human endometrium.

The human endometrium is a fertility-determining factor. Its receptivity during the implantation window may be altered by chemicals. Since human embryo implantation is unique chemical risk assessment cannot be based solely on animal studies. We established a tissue-specific in vitro test based on human endometrial adenocarcinoma (Ishikawa) cells. Progesterone receptor (PR) was selected as primary target gene for estrogenic effects. Changes of mRNA levels were investigated by reverse transcription quantitative real-time PCR. Sigmoidal dose-response curves for up-regulation of PR mRNA and EC(50) values were established for 17beta-estradiol, diethylstilbestrol and the weak xenoestrogen bisphenol A. Nonylphenol also had a clear PR mRNA up-regulating effect. Several other chemicals were characterized as negative compounds. Among them was methoxyacetic acid which may produce false positive results in reporter gene assays. Up-regulation of PR protein by 17beta-estradiol, diethylstilbestrol, bisphenol A and nonylphenol was confirmed by Western Blotting.

[Stunning in radioiodine therapy of benign thyroid disease. Quantification and therapeutic relevance]. / Stunning in der Radioiodtherapie benigner Schilddrüsenerkrankungen: Effektquantifizierung und therapeutische Relevanz.

UNLABELLED: In radioiodine therapy of benign thyroid disease, a reduction of radioiodine uptake is known for consecutive administrations of 131I, which needs to be considered in therapy planning. AIM: Analysis of uptake reduction with regard on the time interval between radioiodine administration and the delivered dose to the thyroid tissue. PATIENTS, METHODS: 200 patients were enrolled in the study and distributed into two groups (matched for diagnoses), each containing 32 patients with Graves' disease (target dose 250 Gy), 24 with focal (400 Gy), 44 with disseminated thyroid autonomy (150 Gy). In one group, a second fraction of radioiodine was given after 48 h (2d) due to an unexpected low radioiodine uptake or effective half-life, whereas in the other group the second fraction was given after 96 h (4d). RESULTS: There was no significant difference between delivered doses due to the first fraction after four days: 2d: 86+/-48 Gy (extrapolated) vs. 4d: 87+/-41 Gy, p>0.05. In 2d, delivered dose at time of second administration was significantly lower (51+/-29 Gy) than in 4d (p<0.01). The radioiodine uptake of the second fraction relative to the initial uptake was significantly lower in the 4d (4d: 63+/-25% vs. 2d: 82+/-24%, p<0.01). In addition, a correlation between uptake reduction and delivered dose and an influence of the time interval between radioiodine administrations could be shown. CONCLUSIONS: Relative uptake of subsequent radioiodine fractions decreases with time after first administration and with increasing delivered dose to the thyroid. If a second fraction of 131I is given at an earlier time, the same therapeutic effect can be reached using lower amounts of activity, minimising radiation exposure and increasing efficiency of radioiodine therapy.

Positron emission tomography study of regional cerebral blood flow and flow-metabolism coupling during general anaesthesia with xenon in humans.

BACKGROUND: The effects of xenon on regional cerebral blood flow (rCBF) are controversial. Moreover, the precise sites of action at which xenon exerts its effects in the human brain remain to be established. METHODS: rCBF was sequentially assessed by H(2)(15)O positron emission tomography in six volunteers. rCBF was determined at baseline and during general anaesthesia induced with propofol and maintained with one minimum alveolar concentration xenon. rCBF measurements were started after the calculated plasma concentration of propofol had decreased to subanaesthetic levels (<1.0 microg ml(-1)). Changes in rCBF were calculated for 13 cerebral volumes of interest by measurement of a semi-quantitative perfusion index (PI). In addition, voxel-wise changes in rCBF were analysed using statistical parametric mapping. RESULTS: Xenon had only minor effects on PI in grey matter volumes of interest. In contrast, PI was increased in white matter [from 1.01 (0.11) to 1.24 (0.15) kcnt ml(-1) MBq(-1), P=0.05, mean (SD)]. Voxel-based analysis showed an increase of rCBF in white matter and a relative decrease of rCBF during xenon anaesthesia in distinct grey matter regions, particularly the orbito- and mesiofrontal cortex, cingulate gyrus, thalamus, hippocampus and bilateral cerebellum (P<0.05 corrected). When correlating PI with cerebral metabolic rate of glucose (previously obtained in another group of six volunteers using (18)F-fluorodeoxyglucose as tracer), the flow-metabolism coupling was preserved during xenon anaesthesia. CONCLUSIONS: Xenon exerted distinct regional effects on CBF: relative decreases in several cortical, subcortical, and cerebellar areas were accompanied by an increase in white matter. Flow-metabolism coupling was not impaired during xenon anaesthesia.

EANM/ESC guidelines for radionuclide imaging of cardiac function.

Radionuclide imaging of cardiac function represents a number of well-validated techniques for accurate determination of right (RV) and left ventricular (LV) ejection fraction (EF) and LV volumes. These first European guidelines give recommendations for how and when to use first-pass and equilibrium radionuclide ventriculography, gated myocardial perfusion scintigraphy, gated PET, and studies with non-imaging devices for the evaluation of cardiac function. The items covered are presented in 11 sections: clinical indications, radiopharmaceuticals and dosimetry, study acquisition, RV EF, LV EF, LV volumes, LV regional function, LV diastolic function, reports and image display and reference values from the literature of RVEF, LVEF and LV volumes. If specific recommendations given cannot be based on evidence from original, scientific studies, referral is given to "prevailing or general consensus". The guidelines are designed to assist in the practice of referral to, performance, interpretation and reporting of nuclear cardiology studies for the evaluation of cardiac performance.

[3D volume and SUV analysis of oncological PET studies: a voxel-based image processing tool with NSCLC as example]. / Dreidimensionale Uptake- und Volumenanalyse von onkologischen PET-Studien: Voxel-basiertes Bildverarbeitungstool am Beispiel von NSCLC.

AIM: The standardized uptake value (SUV) of 18FDG-PET is an important parameter for therapy monitoring and prognosis of malignant lesions. SUV determination requires delineating the respective volume of interest against surrounding tissue. The present study proposes an automatic image segmentation algorithm for lesion volume and FDG uptake quantitation. METHODS: A region growing-based algorithm was developed, which goes through the following steps: 1. Definition of a starting point by the user. 2. Automatic determination of maximum uptake within the lesion. 3. Calculating a threshold value as percentage of maximum. 4. Automatic 3D lesion segmentation. 5. Quantitation of lesion volume and SUV. The procedure was developed using CTI CAPP and ECAT 7.2 software. Validation was done by phatom studies (Jaszczak phantom, various "lesion" sizes and contrasts) and on studies of NSCLC patients, who underwent clinical CT and FDG-PET scanning. RESULTS: Phantom studies demonstrated a mean error of 3.5% for volume quantification using a threshold of 41% for contrast ratios >or=5 : 1 and sphere volumes >5 ml. Comparison between CT- and PET-based volumetry showed a high correlation of both methods (r = 0.98) for lesions with homogeneous FDG uptake. Radioactivity concentrations were underestimated by on average -41%. Employing an empirical threshold of 50% for SUV determination, the underestimation decreased to on average -34%. CONCLUSIONS: The algorithm facilitates an easy and reproducible SUV quantification and volume assessment of PET lesions in clinical practice. It was validated using NSCLC patient data and should also be applicable to other tumour entities.

Quantification of left ventricular volumes and ejection fraction from 16- and rebinned 8-frame gated 99mTc-tetrofosmin SPECT. Comparison of 4D-MSPECT and QGS.

AIM: Using 8-frames/cardiac cycle with gated SPECT underestimates end-diastolic volumes (EDV) and ejection fractions (LVEF), and overestimates end-systolic volumes (ESV). However, using 16-frames/cardiac cycle significantly decreases the signal-to-noise-ratio. We analyzed 16-frames and rebinned 8-frame gated SPECT data using common 4D-MSPECT and QGS algorithms. PATIENTS, METHODS: 120 patients were examined using gated SPECT on a Siemens Multispect 3 (triple-head gamma camera) 60 minutes after intravenous administration at rest of about 450 MBq (two-day protocol) or about 750 MBq (one-day protocol) (99m)Tc-tetrofosmin. Reoriented short axis slices (16-frames) were summed framewise (1+2,3+4, etc.) yielding 8-frame data sets. EDV, ESV and LVEF were calculated for both data sets using 4D-MSPECT and QGS. RESULTS: QGS succeeded with 119, 4D-MSPECT with 117 patients. For the remaining 116 patients, higher EDV (+0.8ml/+3.8 ml) and LVEF (+1.5%/+2.6%; absolute) and lower ESV (-1.7ml/-0.9 ml) (4D-MSPECT/QGS) were found for 16-frame runs. Bland-Altman limits were smaller for QGS than 4D-MSPECT [EDV 32/12 ml, ESV 21/10 ml, LVEF 17/7% (4D-MSPECT/QGS)]. CONCLUSION: Both algorithms showed the expected effects. Contour finding using QGS failed with only one data set, whereas contour finding using 4D-MSPECT failed with three data sets. Since the effects observed between the 8- and the 16-frame studies are relatively small and quite predictable, 8-frame studies can be employed in clinical routine with hardly any loss at all, plus contour finding appears less susceptible to error.

Dipyridamole/aspirin combination in secondary stroke prevention: effects on absolute myocardial blood flow and coronary vascular resistance.

OBJECTIVE: In the European Stroke Prevention Study (ESPS 2), oral administration of a fixed combination of 200 mg extended-release dipyridamole and 25 mg aspirin (twice daily) after ischemic stroke or transient ischemic attack, significantly reduced the risk of stroke compared to placebo as well as compared to aspirin or dipyridamole alone. However, the i.v. application of dipyridamole over 4 - 6 min is known to increase myocardial blood flow up to 6-fold, and thereby potentially provoke ischemic wall motion abnormalities in patients with coronary artery disease. We therefore assessed the cardiac side effects of the dipyridamole/aspirin combination on absolute myocardial blood flow (MBF) and coronary vascular resistance (CVR). METHODS: MBF and CVR were measured using 150-water positron emission tomography in 24 patients after stroke or transient ischemic attack, before and 6.7 +/- 1.9 days after starting the dipyridamole/aspirin combination (Aggrenox) therapy. RESULTS: Resting MBF increased by 39% (max. 112%), from 0.92 +/- 0.13 (ml x g(-1) x min(-1)) at baseline to 1.28 +/- 0.27 (ml x g(-1) x min(-1)) under ongoing dipyridamole/aspirin combination therapy (p < 0.0005). CVR consecutively decreased from 105.3 +/- 16.9 to 74.1 +/- 16.5 (mmHg x ml(-1) x g x min) (p < 0.0005). The relative increase in MBF correlated negatively with the body surface area. No correlation was found between relative MBF increase and duration of dipyridamole/aspirin combination therapy (range 4 - 10 days). CONCLUSIONS: Orally administered dipyridamole/aspirin combination therapy in secondary stroke prevention increases MBF and decreases CVR significantly. These cardiac side effects of the dipyridamole/aspirin combination should be taken into account in stroke patients with proven or suspected coronary artery disease, particularly in combination with a small body surface area.

Radionuclide imaging of the painful hip arthroplasty: positron-emission tomography versus triple-phase bone scanning.

Two major complications of hip replacement are loosening and infection. Reliable differentiation between these pathological processes is difficult since both may be accompanied by similar symptoms. Our aim was to assess the diagnostic ability of triple-phase bone scanning (TPBS) and positron-emission tomography (PET) to detect and differentiate these complications in patients with a hip arthroplasty. Both TPBS and PET were performed in 63 patients (92 prostheses). The radiotracer for PET imaging was (18)F-fluorodeoxyglucose (FDG). Image interpretation was performed according to qualitative and quantitative criteria although the final diagnosis was based upon either surgical findings or clinical follow-up. The sensitivity, specificity and accuracy of PET was 0.94, 0.95 and 0.95 respectively, compared with 0.68, 0.76 and 0.74 for TPBS. We found that an image interpretation based exclusively upon quantitative criteria was inappropriate because of its low selectivity. The histological examination indicated that increased periprosthetic uptake of FDG in patients with aseptic loosening was caused by wear-induced polyethylene particles and the subsequent growth of aggressive granulomatous tissue.

Complications associated with traction on the hip during arthroscopy.

We studied 16 hips (eight cadaver specimens) using arthrography, arthroscopy and anatomical dissection, under incremental traction of up to a maximum of 64 kg, to determine the relationship of the portals to nearby neurovascular structures. The distance of each arthroscopic portal (anterior, anterolateral, and posterolateral) to the associated neurovascular structures was measured after the application of 23 kg of traction. Traction of up to 64 kg on the lower limb failed to produce evidence of labral or capsular injury. Furthermore, traction of 23 kg resulted in little change in the position of adjacent neurovascular structures relative to the standard arthroscopic portals.

mRNA expression profiles for corticotrophin-releasing hormone, urocortin, CRH-binding protein and CRH receptors in human term gestational tissues determined by real-time quantitative RT-PCR.

Increasing maternal plasma levels of corticotrophin-releasing hormone (CRH) during the last weeks of pregnancy suggest that this stress hormone plays an important role in the control of human parturition. Little is known about the quantitative contribution of gestational tissues (other than placenta) to intrauterine formation of CRH, urocortin and CRH-binding protein (CRH-BP), or about the distribution of CRH receptors within the uterus. We have investigated the mRNA expression of CRH, urocortin, CRH-BP and CRH receptors 1 and 2 (CRH-R1 and -R2) in gestational tissues by real-time RT-PCR. Placenta, myometrium and choriodecidua were collected after uncomplicated pregnancies at term, before the onset of labour. Distribution of CRH-R1 and CRH-R2 protein was also investigated by immunostaining with receptor subtype-specific antibodies. The placenta was identified as the main site of CRH and CRH-BP mRNA expression, displaying mRNA levels >1000 and >20 times higher than those found in the myometrium and choriodecidua respectively (P<0.05 in each case). mRNA expression of urocortin was low in all tissues investigated. Myometrium and choriodecidua expressed relevant amounts of both receptor subtypes, whereas the CRH receptor population in placenta consisted mainly of CRH-R2. The high expression of CRH in placenta and the substantial expression of CRH receptors in choriodecidua and myometrium suggested that CRH derived from placenta exerts direct or indirect actions on these tissues. Neither CRH produced by myometrium or choriodecidua nor urocortin from other intrauterine sources seem to play a major role in the control of labour.

Radioiodine therapy in patients with hyperthyroid disorder: standard versus dosimetric activity application.

Due to its high success rate and non-invasive character, an increasing demand for radioiodine therapy can be seen. This study was conducted to determine whether standardized 131I activities can be used to facilitate management of patients with hyperthyroid disorder or whether a pre-therapeutic radioiodine test is advisable to determine an adequate therapeutic activity. The therapeutic uptake of 218 patients with benign thyroid disorders were determined and compared with 24 h and 48 h test uptake measurements as well as with calculated standard uptake values. Since there is a linear relationship between iodine uptake and delivered radiation dose, the effect of the different therapeutic approaches on the latter parameter was analysed. Special care was taken to assess possible differences between the various thyroid disorders. A mean deviation between pre-therapeutic test uptake and actual therapeutic uptake of 14.7% was observed in contrast to one of 29.1% when using disease specific standard values per millilitre of thyroid tissue. Furthermore, the proportion of patients with large deviations of more than 40% increased drastically when using standard uptake values (with radioiodine test, 4.1%; with standard values, 18.8%). In conclusion, the dosimetric approach with a pre-therapeutic radioiodine test proved to be the most accurate therapeutic procedure. Both the 24 h and 48 h test uptake measurements gave analogous results and yielded a correlation coefficient of 0.91 when compared with the therapeutic uptake. While it may be tempting to use standard activities to facilitate patient management, the findings of this study confirm that, for precise therapy planning, a pre-therapeutic radioiodine test is advised. Since no significant difference could be found between the 24 h and 48 h test uptake values, an early measurement 24 h after administration of the test activity is recommended.

Characterization and identification of cytochrome P450 metabolites of arachidonic acid released by human peritoneal macrophages obtained from the pouch of Douglas.

Cytochrome P450 metabolism of arachidonic acid (AA) was investigated in human peritoneal macrophages which play a central role in chronic pelvic diseases in women (for example in endometriosis). The formation of eicosanoids other than prostaglandins (PGs) by these cells is still unknown. In non-activated macrophages obtained from women in the reproductive age, the main [(3)H]-AA metabolites coeluted with epoxyeicosatrienoic acids, dihydroxyeicosatrienoic acids (DHETs) and hydroxyeicosatetraenoic acids (HETEs) in reverse-phase HPLC. After zymosan activation a shift to PGs pathway was observed. Treatment with low doses of 2,3,7,8-tetrachlorodibenzo- p -dioxin increased the formation of a metabolite coeluting with 5,6-DHET. By gas chromatography/mass spectrometry 5,6-DHET (after beta-naphthoflavone induction), and 14,15-DHET as well as 11,12-DHET (after AA stimulation) were identified as major epoxygenase metabolites, respectively. The enantioselective formation of 12(S)-HETE was demonstrated by chiral-phase HPLC. Our findings demonstrate that non-activated peritoneal macrophages produce substantial amounts of bioactive cytochrome P450 metabolites of AA.