The survival of patients with high grade glioma from different ethnic groups in South East England.

Studies in the United States (US) have reported varying treatment and survival for patients with high grade glioma from different ethnic groups. This study investigates for the first time whether differences also exist in the United Kingdom (UK). This population-based cohort study used cancer registration data for 4,845 patients diagnosed in South East England between 2000 and 2009. Linked self-assigned ethnicity data within Hospital Episode Statistics were used to define White, Indian, Pakistani, Bangladeshi, Black Caribbean, Black African, Other and Not known groups. Logistic regression was used to generate odds ratios for a record of receipt of treatment (surgery, radiotherapy and chemotherapy), adjusting for sex, age, morphology, socioeconomic deprivation and comorbidity in each ethnic group. Hazard ratios were generated using Cox regression, adjusting for sex, age, morphology, socioeconomic deprivation, comorbidity and treatment. The overall one-year survival was 28.4 %. Ethnicity data was available for 3,793 (78 %) patients. Receipt of treatment was generally similar between different ethnic groups after adjustment for sex, age, morphology, socioeconomic deprivation and comorbidity. After adjustment for potential confounders, the Indian (HR 0.72, p = 0.037) and Other groups (HR 0.76, p = 0.003) had better survival, while the Not known group (HR 1.34, p < 0.0001) had worse survival than the White group. Patients from UK Indian groups have better survival than White patients while those from Black ethnic groups appear to have similar survival to White patients. These findings suggest the need to investigate possible contributing factors including the completeness of follow-up, clinical performance status and tumour biology.

Under-filled blood collection tubes containing K2EDTA as anticoagulant are acceptable for automated complete blood counts, white blood cell differential, and reticulocyte count.

Current laboratory standards from Clinical Laboratory Standards Institute (CLSI) and manufacturer's (Becton Dickinson) data indicate that under-filling K(2)EDTA blood collection tubes can result in erroneous hematology values. To accommodate under-filled tubes and reduce collection volumes while optimizing our automation, we explored the acceptable limit of under-filled tubes for hematology values. We collected 8.0 ml of blood from 30 normal adult volunteers. Each donation was aliquoted in the following volumes: 4.0, 2.0, 1.0, 0.5 ml x 2. These samples were analyzed within 1 h of blood collection on Sysmex XE-2100 (Sysmex America Inc., Mundelein, IL, USA) for complete blood count, reticulocyte, and white blood cell differentials. Results of the under-filled tubes were compared to those of the standard volume. The Deming regression analysis show excellent correlation for all parameters between each under-filled blood collection volume compared to a standard 4 ml volume. The Bland and Altman analysis shows good agreement between both 1.0 and 2.0 ml compared to a 4.0 ml volume. The 0.5 ml compared to a 4.0 ml volume, however, shows increased variation on many parameters. In addition all three collection volumes show negative bias compared to the standard volume for platelet count, but the difference is considered insignificant with a percent difference of 5.5%, 3.2%, and 1.5% for 0.5, 1.0, and 2.0 ml collection volume respectively. Finally for 0.5 ml collection volume we noticed a low level of false positive flagging rate for white blood cell. Acceptable complete blood count values of under-filled powdered K(2)EDTA tubes can be obtained with as little as 1.0 ml of blood.

ETHE1 mutations are specific to ethylmalonic encephalopathy.

Mutations in ETHE1, a gene located at chromosome 19q13, have recently been identified in patients affected by ethylmalonic encephalopathy (EE). EE is a devastating infantile metabolic disorder, characterised by widespread lesions in the brain, hyperlactic acidaemia, petechiae, orthostatic acrocyanosis, and high levels of ethylmalonic acid in body fluids. To investigate to what extent ETHE1 is responsible for EE, we analysed this gene in 29 patients with typical EE and in 11 patients presenting with early onset progressive encephalopathy with ethylmalonic aciduria (non-EE EMA). Frameshift, stop, splice site, and missense mutations of ETHE1 were detected in all the typical EE patients analysed. Western blot analysis of the ETHE1 protein indicated that some of the missense mutations are associated with the presence of the protein, suggesting that the corresponding wild type amino acid residues have a catalytic function. No ETHE1 mutations were identified in non-EE EMA patients. Experiments based on two dimensional blue native electrophoresis indicated that ETHE1 protein works as a supramolecular, presumably homodimeric, complex, and a three dimensional model of the protein suggests that it is likely to be a mitochondrial matrix thioesterase acting on a still unknown substrate. Finally, the 625G-->A single nucleotide polymorphism in the gene encoding the short chain acyl-coenzyme A dehydrogenase (SCAD) was previously proposed as a co-factor in the aetiology of EE and other EMA syndromes. SNP analysis in our patients ruled out a pathogenic role of SCAD variants in EE, but did show a highly significant prevalence of the 625A alleles in non-EE EMA patients.

Catecholamines in patients with 22q11.2 deletion syndrome and the low-activity COMT polymorphism.

OBJECTIVE: To investigate catecholamine phenotypes and the effects of a tyrosine hydroxylase inhibitor in individuals with the 22q11.2 deletion syndrome and low-activity catechol-O-methyltransferase (COMT). BACKGROUND: Many persons with the 22q11.2 deletion syndrome suffer severe disability from a characteristic ultrarapid-cycling bipolar disorder and associated "affective storms." One etiologic hypothesis for this condition is that deletion of the COMT gene from one chromosome 22 results in increased catecholamine neurotransmission, particularly if the undeleted chromosome 22 encodes a variant of COMT with low activity. METHODS: In a preliminary study, plasma, urine, and CSF catecholamines and catecholamine metabolites were measured in four teenage patients with a neuropsychiatric condition associated with 22q11.2 deletion and the low-activity COMT polymorphism on the nondeleted chromosome. In these four patients, and an additional institutionalized adult with the condition, an uncontrolled, open-label trial of metyrosine was administered in an attempt to lower catecholamine production and to alleviate symptoms. RESULTS: Mild elevations of baseline CSF homovanillic acid (HVA) were found in three of four patients and a moderate reduction in CSF HVA after metyrosine treatment in the patient with the highest pretreatment concentration. The course of the five patients during the clinical trial is described. CONCLUSIONS: In patients with the 22q11.2 deletion syndrome and low-activity COMT, controlled studies of pharmacologic agents that decrease catecholamine production, block presynaptic catecholamine storage, or enhance S-adenosylmethionine, the cosubstrate of COMT, are warranted.

Beta2-glycoprotein I-dependent anticardiolipin antibodies preferentially bind the amino terminal domain of beta2-glycoprotein I.

Many of the autoantibodies in antiphospholipid syndrome (APS) are directed against beta2-glycoprotein I (beta2-GPI). Recent studies from our laboratories have indicated that the immunodominant binding epitope(s) for high titer, affinity purified antibodies from 11 APS patients are localized to the amino terminal domain (domain 1) of beta2-GPI. The present study employed surface plasmon resonance to localize the immunodominant domain in serum samples from a large cohort of patients with GPL values ranging from 21 to 230 units (n = 106 patients). Eighty-eight percent of patients showed > or = threefold selectivity for beta2-GPI containing domain 1 relative to the domain deletion mutant that lacked domain 1. The domain 1 binding activity in patient serum was abolished by removing the IgG fraction from the serum and the binding activity could be fully reconstituted with the IgG fraction. Thus, analysis of serum samples from a large cohort of APS patients indicates that the immunodominant binding epitope(s) for anti-beta2 antibodies are localized to the amino terminal domain of beta2-GPI.

Biostability and pharmacokinetics of LJP 920, an octameric Gal (alpha1-3) Gal conjugate for the inhibition of xenotransplantation rejection.

Antibodies to an alpha-galactosyl saccharide structure present in human serum are associated with hyperacute rejection and delayed xenograft rejection after pig-to-primate xenotransplantation. To overcome this major barrier to the xenotransplantation, LJP 920, a galactosyl alpha1-3 galactose (Gal (alpha1-3) Gal) coupled to a non-immunogenic platform at a valency of eight Gal (alpha1-3) Gal molecules/platform, was synthesized to clear circulating antibodies and to inhibit their production by B cells that produce these antibodies. Herein we report on the stability of UP 920 in biological media and its pharmacokinetic profile. Incubation of LJP 920 with mouse serum or liver microsomes at 37 degrees C for 2 days showed no indication of degradation of the conjugate as detected by a reversed-phase HPLC method, indicating that the conjugate is not subject to enzymatic metabolism. After intravenous administration of LJP 920 to mice at the doses of 20 and 100 mg kg(-1), UP 920 serum concentration decreased rapidly, showing a biphasic pattern, with a distribution half-life of 3 min and an elimination half-life of more than 30 min, respectively. The serum-to-erythrocyte concentration ratio of UP 920 was 33- and 36-fold excess at 0.5 and 5 min, respectively, after intravenous administration (100 mg kg(-1)). Both Cmax and AUC values increased in a dose-proportional manner. UP 920 displayed a great distribution to well-perfused tissues. It was eliminated mainly through renal excretion in the unchanged form, which accounted for 23% of the total amount within 8 h of dosing.

Zinc protoporphyrin/heme ratio for diagnosis of preanemic iron deficiency.

OBJECTIVE: Iron deficiency anemia is known to impair cognitive and psychomotor development. The zinc protoporphyrin/heme (ZPP/H) ratio is a simple, accurate, and sensitive laboratory screening test that detects early iron depletion before the onset of anemia. The objective of this work was to evaluate this test in a primary pediatric practice setting. METHODS: The iron status of a cohort of 361 children was screened during routine examinations at a community pediatric practice. Whole blood hemoglobin concentration, hematocrit ratio, serum transferrin saturation, ferritin concentration, and the ZPP/H ratio were measured. The ZPP/H ratio then was evaluated as a single indicator of iron status by comparing it with other tests for detecting the onset of iron deficiency and for monitoring recovery after iron supplementation. RESULTS: Significant age- and sex-related differences in the ZPP/H ratio were found. In this cohort, serum ferritin concentration and the ZPP/H ratio independently identified the same fraction of iron-deficient patients (3%-4%), and both tests were more specific than was either hemoglobin or hematocrit. A concordance of three iron status parameters changed the prediction of iron deficiency to

Ahomocysteinemia in molybdenum cofactor deficiency.

We report an infant with molybdenum cofactor deficiency (MCD) and a unique clinical presentation of hemiplegia, hypotonia, dystonia, and bilateral basal ganglia changes. Biochemistry revealed absent serum homocysteine, low concentrations of plasma cystine, high levels of urinary S-sulfocysteine and sulfite, and high levels of oxypurines in serum and urine. The depletion of cysteine and cystine through reaction with sulfite suggests that other thiols and thiol-dependent proteins may be similarly depleted. Ahomocysteinemia may be a clue to the mechanism of cytotoxicity in MCD.

Complement receptor type 1 (CR1, CD35) is a receptor for C1q.

Molecular definition of the cellular receptor for the collagen domain of C1q has been elusive. We now report that C1q binds specifically to human CR1 (CD35), the leukocyte C3b/C4b receptor, and the receptor on erythrocytes for opsonized immune complexes. Biotinylated or radioiodinated C1q (*C1q) bound specifically to transfected K562 cells expressing cell surface CR1 and to immobilized recombinant soluble CR1 (rsCR1). *C1q binding to rsCR1 was completely inhibited by unlabeled C1q and the collagen domain of C1q and was partially inhibited by C3b dimers. Kinetic analysis in physiologic saline of the interaction of unlabeled C1q with immobilized rsCR1 using surface plasmon resonance yielded an apparent equilibrium dissociation constant (K[eq2]) of 3.9 nM. Thus, CR1 is a cellular C1q receptor that recognizes all three complement opsonins, namely, C1q, C3b, and C4b.

Lipid transfer from human epithelial cells to Pneumocystis carinii in vitro.

Pneumocystis carinii lipids are similar to host lipids, but it is not known if some of these lipids are acquired from host cells. The ability of P. carinii to incorporate a fluorescent fatty acid analogue (Bodipy-C12) was analyzed, the metabolism of the incorporated lipid by P. carinii was characterized, and lipid transfer from human alveolar epithelial cells (A549) to P. carinii was investigated. Both P. carinii and A549 cells incorporated exogenous Bodipy-C12 in a concentration-dependent manner. Biochemical analysis of labeled P. carinii revealed incorporation of Bodipy-C12 into complex lipid classes. Incubation of unlabeled P. carinii with Bodipy-C12-labeled A549 cells demonstrated lipid transfer to P. carinii, a process facilitated by attachment. These data suggest that P. carinii can incorporate and modify an exogenous fluorescent lipid. The observed transfer of lipid from A549 cells to P. carinii provides important insight into the interaction of this organism with alveolar epithelial cells.

Fetal origin of amniotic fluid polymorphonuclear leukocytes.

OBJECTIVE: Although polymorphonuclear leukocytes are the inflammatory cells most frequently recovered from the amniotic cavity in cases of suspected intrauterine infection, the source of these cells has not been definitively determined. We took advantage of the gender difference between the mother and her male fetus, and we report four cases in which amniotic fluid polymorphonuclear leukocytes were identified as fetal by fluorescence in situ hybridization with probes specific for X and Y chromosomes. Fetal membranes were intact at the time amniotic fluid was obtained in all cases. STUDY DESIGN: Amniotic fluid was obtained from women with male fetuses in premature labor with clinical or laboratory evidence of infection. Cytospin preparations of amniotic fluid samples with polymorphonuclear leukocytes were prepared and sequentially stained with fluorescent reagents. To determine which cells were polymorphonuclear leukocytes, all replicate samples were stained with the fluorescent nuclear stain 4'-6-diamidino-2-phenyl-indole. This allowed definition of the characteristic multilobed polymorphonuclear leukocytes nuclear morphologic features. The sample was then probed with a rhodamine-labeled probe specific for the X chromosome and a fluorescein-labeled probe specific for the Y chromosome to assess whether the polymorphonuclear leukocytes were male or female. RESULTS: Ninety percent to 99% of polymorphonuclear leukocytes identified by normal multiple lobed nuclear morphologic study on 4'-6-diamidino-2-phenyl-indole staining had an X and Y chromosome and were therefore fetal cells. CONCLUSIONS: These data demonstrate a fetal response during intraamniotic infection. Further investigation of the roles for maternal and fetal polymorphonuclear leukocytes in chorioamnionitis may provide valuable information about the critical interaction of the two immune responses in this setting.

D-2-hydroxyglutaric aciduria: hypotonia, cortical blindness, seizures, cardiomyopathy, and cylindrical spirals in skeletal muscle.

An infant girl was demonstrated to have D-2-hydroxyglutaric aciduria, the fifth case described and the first with muscle biopsy of this rare organic aciduria that differs clinically and genetically from the more common L-2-hydroxyglutaric aciduria. Her clinical features included mildly dysmorphic facies, developmental delay, generalized hypotonia, myoclonic seizures, cortical blindness, and dilated cardiomyopathy requiring treatment. Muscle biopsy demonstrated only excessive glycogen histochemically, but ultrastructural examination revealed subsarcolemmal cylindrical spirals and normal mitochondria. Because of the metabolism of D-2-hydroxyglutaric aciduria, we regard valproic acid as contraindicated in the treatment of epilepsy in this disease.

Plasma homocysteine and methionine concentrations in children with neural tube defects.

Mild to moderate homocysteinemia in women has been associated with an increased frequency of pregnancies with neural tube defects (NTD). Homocysteinemia is also an independent risk factor for premature vascular disease. In addition to folic acid, supplemental Vitamin B12, Vitamin B6 and betaine may normalize homocysteine metabolism, decrease the risk for NTD formation, and correct related metabolic imbalances in children with NTD. By means of automated amino acid analysis, we assessed total non-fasting homocysteine and methionine in plasma from 24 children with myelomeningocele. This study group (mean age 10.5 +/- 4.9 years) included 12 girls and 12 boys randomly selected from our Birth Defects Clinic. Homocysteine concentrations in our patients (4.7 +/- 1.8 mumol/L) did not differ from those of 20 randomly selected child controls (5.1 +/- 2.6 mumol/L). The mean homocysteine concentration for 36 adult controls (9.3 +/- 3.0 mumol/L) was significantly higher than the mean for either group of children (p < 0.0001). Linear regression analysis revealed negative correlation of total plasma homocysteine with serum folate (r = -0.53; p = 0.01), but not of homocysteine with either methionine or B12. Plasma methionine concentrations from our patients did not differ from adult reference values. Elevated homocysteine in some mothers of children with NTD has been attributed to defective methylation of homocysteine. These preliminary results do not indicate such a defect in the children themselves. A more comprehensive study of homocysteine, methionine and related metabolites in children with NTD and age-matched controls will be required to determine the clinical significance of these findings.

Purification of human lung leukotriene C4 synthase and preparation of a polyclonal antibody.

Leukotriene (LT) C4 synthase is an integral membrane protein that catalyzes the conjugation of LTA4 to reduced glutathione to form LTC4. LTC4 synthase has been cloned and characterized from transformed cell lines, but the protein has not been defined from a tissue source. LTC4 synthase was purified to homogeneity from human lung tissue, utilizing S-hexyl glutathione chromatography followed by LTC4 affinity chromatography. A greater than 100,000-fold purification with a yield of 8 to 25% (n = 4) was achieved. The purified LTC4 synthase migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as an 18-kD protein, and its 19 N-terminal amino acid sequence is identical to that of purified LTC4 synthase from KG-1 myeloid cells or from expression cloning of a KG-1 library in COS cells. Using a rabbit polyclonal IgG raised against purified LTC4 synthase, SDS-PAGE immunoblotting of LTC4 synthase from human lung tissue, eosinophils, KG-1 cells, and platelets showed an 18-kD protein. Immunofluorescence staining of alveolar macrophages in human lung sections with the anti-LTC4 synthase IgG revealed LTC4 synthase to be largely perinuclear in distribution. Thus, LTC4 synthase, the biosynthetic enzyme responsible for the formation of cysteinyl LTs, is present in lung tissue in a form apparently identical to that of hematopoietic cells.

Hemolytically inactive C5b67 complex: an agonist of polymorphonuclear leukocytes.

The activity of hemolytically inactive C5b67, designated iC5b67, was evaluated as an agonist for functional responses of human polymorphonuclear leukocytes (PMN). C5b67 was formed from purified human complement components and decayed in phosphate-buffered saline (PBS) until it had no lytic activity for sheep erythrocytes in a standard assay. iC5b67, at nanomolar concentrations, stimulated PMN chemotaxis and Ca2+ fluxes, but inhibited superoxide production and failed to upregulate CR1 and CR3. There was no significant contamination of the iC5b67 with C5a to explain these results. Neither isolated C5b6 nor C7 alone exhibited the activities of iC5b67, while insolubilized anti-C7 could remove the PMN agonist activity from the iC5b67 preparation. Binding studies to define a specific receptor for iC5b67 on PMN were hampered by the very hydrophobic nature of the ligand. 125I-iC5b67, by contrast to hemolytically active 125I-C5b67, was unable to insert in erythrocytes, suggesting that iC5b67 need not insert in the PMN membrane to induce signaling. Two lines of evidence suggest that iC5b67 and C5a and FMLP share common steps in intracellular signaling (1) pretreatment of PMN with iC5b67 deactivates PMN for C5a- and FMLP-induced chemotaxis; and (2) pretreatment of PMN with pertussis toxin inhibits iC5b67-induced chemotaxis. Thus, iC5b67 has important effects on the activity of PMN and G-proteins and Ca2+ are involved in the signaling.

Diamniotic placentation associated with omphalopagus conjoined twins: implications for a contemporary model of conjoined twinning.

We have studied omphalopagus conjoined twins with a diamniotic monochorionic placenta. Although conjoined twins usually present in a single amniotic sac, one other example of diamniotic placenta has been reported in omphalopagus twins [Weston et al., 1990: Am J Med Genet 37:558-561]. Most theories concerning the pathogenesis of conjoined twinning exclude the possibility of diamniotic placentation. However, Spencer [1992: Teratology 45:591-602] recently elaborated a model for conjoined twinning based on duplication of organizing centers (primitive streaks) during gastrulation. We have considered the fate of embryonic membranes according to this model of omphalopagus twinning and show that diamniotic placentation is a predictable outcome.

Regulation of C1q receptor expression on human polymorphonuclear leukocytes.

The C1q receptor (C1qR) is expressed on a variety of cells, including polymorphonuclear leukocytes (PMN), in which stimulation by the C1qR leads to activation as measured by superoxide production. To investigate the expression and modulation of the C1qR on PMN, the binding of biotinylated C1q to PMN in suspension was measured by flow cytometry. Biotinylated C1q bound in a saturable and specific manner to PMN and the use of low ionic strength buffers enhanced binding. Covalent coupling of C1q to Sepharose beads allowed the affinity precipitation of a single 125-kDa band from surface iodinated PMN. The apparent molecular mass of the C1qR increased to 135 kDa upon reduction. Freshly isolated PMN had a uniform expression of C1qRs and phorbol myristate acetate induced a unimodal up-regulation of receptors. The inflammatory peptide FMLP rapidly up-regulated receptors by up to fivefold, and the high level of expression remained constant over 45 min. Taxol inhibited the FMLP induction of C1qR up-regulation, indicating that the ability to move the intracellular stores of C1qR depends on normal microtubule functioning. Thus, the C1qR is a constitutively expressed protein of the human PMN plasma membrane and it is rapidly up-regulated from a discrete intracellular pool of preformed molecules with the same kinetics as CR1 and CR3. It is likely that the C1qR is a component of the PMN complement receptor exocytic vesicle (CREV), in which CR1 and CR3 are also stored.

The role of complement component C3b and its receptors in sperm-oocyte interaction.

Previous studies have shown that human sperm that have undergone the acrosome reaction express a unique tissue-specific variant of the complement component 3 (C3)-binding molecule membrane cofactor protein (MCP, CD46) and that damaged or dead sperm activate the alternative pathway of complement and bind C3 catabolites. In this study we provide evidence that MCP on sperm that have undergone the acrosome reaction specifically binds dimeric C3b and that human sperm acrosomal proteases released during the acrosome reaction directly cleave C3, facilitating its binding to MCP. Furthermore, human and hamster oocytes can activate the alternative pathway of complement and also bind human C3 fragments. Monoclonal antibodies specific for complement receptors type 1 (CD35) and type 3 (CD11b/CD18) bind to the human oocyte plasma membrane, indicating that specific complement-binding molecules may play a role in the attachment of C3 catabolites to oocytes. Subsaturating concentrations of dimeric C3b (0.01-1 microM) promoted penetration of hamster oocytes by human sperm, whereas saturating doses (> 10 microM) inhibited this process. In addition, antibodies to both MCP and C3 significantly inhibited penetration of hamster oocytes by human sperm. These data provide evidence that regulated gamete-induced generation of C3 fragments and the binding of these fragments by selectively expressed receptors on sperm and oocytes may be an initial step in gamete interaction, leading to membrane fusion and fertilization.

Lipid and membrane protein transfer from human neutrophils to schistosomes is mediated by ligand binding.

Attachment of human neutrophils to schistosomula of Schistosoma mansoni involves leukocyte receptors recognizing carbohydrate, complement and/or IgG ligands on the parasite surface. Here, we examined the transfer of a fluorescent fatty acid analog (BOFA) from human neutrophils to schistosomula coated with concanavalin A (Con A), immune serum or nonimmune serum under co-culture conditions by fluorescence confocal laser scanning microscopy (CLSM). Coating schistosomes with Con A or immune serum and co-culturing them for 24 hours with BOFA-labeled neutrophils resulted in a specific lipid transfer to the surface tegument of the schistosomes. Tegumental labeling was absent when nonimmune serum was used. No significant difference (P < 0.001) was found in the number of neutrophils bound to the worm surface between Con A-coated schistosomes (4.1 +/- 0.345 cells/worm) and worms incubated in immune serum (4.261 +/- 0.362). The number of neutrophils bound to the schistosomula (2.7 +/- 0.223) was significantly reduced in the presence of nonimmune serum (P < 0.0001). The viability of the schistosomula was 98% in nonimmune treated co-cultures, and 91% in cocultures treated with immune serum. HPLC analysis of labeled neutrophils demonstrated that BOFA was incorporated into both phospholipids and neutral lipids, which were almost exclusively triglycerides and, after 18 hours of culture, all of the fatty acid analog was incorporated into complex lipids. Double-label experiments in which schistosomula bearing Con A were first incubated with BOFA-labeled neutrophils and subsequently immunolabeled revealed that the neutrophil membrane proteins, MHC class I, CR1 and CR3 were co-transferred with neutrophil lipids to the parasite tegument.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of human leukotriene C4 synthase.

Leukotriene (LT) C4 synthase, the enzyme that catalyzes the conjugation of LTA4 with reduced glutathione to form LTC4, was purified to homogeneity from the KG-1 myeloid cell line after solubilization of the microsomes utilizing a combination of 0.4% sodium deoxycholate and 0.4% Triton X-102. The solubilized enzyme was then applied to an S-hexyl-glutathione-agarose column that was eluted by the use of 7.5 mM probenecid. After removal of the probenecid by sequential concentration and dilution in an Amicon concentrator, the enzyme was additionally purified and concentrated by binding to and elution from approximately 75 mg of S-hexyl-glutathione-agarose. The enzyme was further resolved by electrophoresis with a nondenaturing Tris-glycine gel, and the LTC4 synthase activity was localized to slices 3 and 4. When the remainder of the eluate from the nondenaturing gel was precipitated by acetone and analyzed by 14% SDS/PAGE with silver staining, a single protein band of 18 kDa was associated with LTC4 synthase activity and was not present in the eluates of slices lacking activity. The overall recovery was 12.5%. In a separate preliminary purification, in which the yield was only approximately 1%, the eluates of the nondenaturing gel had also revealed a single protein of 18 kDa by SDS/PAGE, which was present only in the eluates with LTC4 synthase activity. These data identify LTC4 synthase as a protein of 18 kDa, a size consistent with its membership in the microsomal glutathione S-transferase family.