C18 Resin Research Articles

Direct determination of vitamin B1in seawater by solid-phase extraction and high-performance liquid chromatography quantification

Vitamin B1 (thiamine) is one of the organic micronutrients essential for growth of algae. However, there is no direct method for the quantification of this vitamin in natural waters. Bioassay is the only protocol currently available to measure B1 in the marine environment. We have developed a new method for the direct determination of B1, along with vitamin B12 (cobalamin) in seawater. Solid-phase extraction with a column of C18 resin followed by elution with methanol was used to extract dissolved B1 from seawater. The eluted vitamin was then analyzed by reversed phase high-performance liquid chromatography (HPLC) using ammonium acetate (0.05 M) and methanol as mobile phases in pressure gradient mode. Ultraviolet-visible (UV-Vis) detection at 270 nm was used to quantify B1 levels. The calibration curve was linear from 0 to 0.60 µM and the detection limit of the protocol was on the order of 0.083 pM. This new method was successfully applied to the determination of vitamin B1 in filtered coastal seawater, porewater, and river water samples. Concentrations of vitamin B1 in those aquatic environments ranged from 0.15 nM in river to 0.75 nM in porewaters, with spike recoveries ranging from 93% to 98%.

High Throughput Quantitative Analysis of Serum Proteins Using Glycopeptide Capture and Liquid Chromatography Mass Spectrometry

It is expected that the composition of the serum proteome can provide valuable information about the state of the human body in health and disease and that this information can be extracted via quantitative proteomic measurements. Suitable proteomic techniques need to be sensitive, reproducible, and robust to detect potential biomarkers below the level of highly expressed proteins, generate data sets that are comparable between experiments and laboratories, and have high throughput to support statistical studies. Here we report a method for high throughput quantitative analysis of serum proteins. It consists of the selective isolation of peptides that are N-linked glycosylated in the intact protein, the analysis of these now deglycosylated peptides by liquid chromatography electrospray ionization mass spectrometry, and the comparative analysis of the resulting patterns. By focusing selectively on a few formerly N-linked glycopeptides per serum protein, the complexity of the analyte sample is significantly reduced and the sensitivity and throughput of serum proteome analysis are increased compared with the analysis of total tryptic peptides from unfractionated samples. We provide data that document the performance of the method and show that sera from untreated normal mice and genetically identical mice with carcinogen-induced skin cancer can be unambiguously discriminated using unsupervised clustering of the resulting peptide patterns. We further identify, by tandem mass spectrometry, some of the peptides that were consistently elevated in cancer mice compared with their control littermates.

Robotic preparation of Sodium Acetate C 11 Injection for use in clinical PET

Sodium Acetate C 11 Injection is a radiopharmaceutical commonly used for clinical studies with positron emission tomography (PET). We have designed a fully-automated robotic system for the compounding of this 20-minute half-lived tracer in the clinical setting. The system is comprised of five modular workstations that are configured in a flexible manner to accommodate all of the steps in the production of the labeled drug. The Trapping Station isolates cyclotron-produced [ 11C]CO 2 gas from the target and directs carbonation of methylmagnesium Grignard in diethyl ether. The Heating Station hydrolyzes the intermediate, and removes ether and unreacted [ 11C]CO 2 from the mixture. The Extraction Station removes ionic and organic contaminants from the drug using solid-phase extraction (AG 11A8 and C18 resin). The Filtration Station sterilizes the radiopharmaceutical, and tests membrane patency post filtration. The Assay Station measures the weight and radioactivity content of the Final Product Container, facilitating calculation of activity concentration in a remote manner. Rapid quality control methodology has also been developed that is suitable for pre-release analysis of the drug product. For a 7.5 min irradiation with a 40 μA proton beam, 223–300 mCi of Acetate C 11 Injection with purity meeting USP standards is produced within 23 min. This robotic system is a reliable method for producing Sodium Acetate C 11 Injection, USP in the clinical setting with minimal personnel exposure. Moreover, its design flexibility permits synthesis of additional 11C- or 18F-labeled PET tracers within the same shielded hot cell.

Proteomic analysis of shrimp white spot syndrome viral proteins and characterization of a novel envelope protein VP466.

White spot syndrome virus (WSSV) is at present one of the major pathogens in shrimp culture worldwide. The complete genome of this virus has been sequenced recently. To identify the structural and functional proteins of WSSV, the purified virions were separated by SDS-PAGE. Twenty-four protein bands were excised, in-gel digested with trypsin, and subjected to matrix-assisted laser desorption ionization-time of flight mass spectrometry and electrospray ionization tandem mass spectrometry, respectively. Eighteen proteins matching the open reading frames of WSSV genome were identified. Except for three known structural proteins and collagen, the functions of the remaining 14 proteins were unknown. Temporal analysis revealed that all the genes were transcribed in the late stage of WSSV infection except for vp121. Of the newly identified proteins, VP466 (derived from band 16) was further characterized. The cDNA encoding VP466 was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Specific antibody was generated with the purified GST-VP466 fusion protein. Western blot showed that the mouse anti-GST-VP466 antibody bound specifically to a 51-kDa protein of WSSV. Immunogold labeling revealed that VP466 protein is a component of the viral envelope. Results in this investigation thus proved the effectiveness of proteomic approaches for discovering new proteins of WSSV.

Definitive evidence for the actual contribution of yeast in the transformation of neutral precursors of grape aromas.

Experiments were designed to demonstrate the actual contribution of yeast in the formation of the primary aroma during the vinification of neutral grapes. Ruché was chosen as the model wine to study because of its unique fragrance. A yeast strain specific for Ruché was selected using a new and rapid isolation method for red wines. The results of this study can be summarized as follows: Skins from nonaromatic white or red grapes apparently contain most of the primary aroma compounds that are revealed in the must only after contact with yeast cells under defined conditions. Similar results were obtained with the pulp and seeds fractions; however, the olfactory notes, although well characterized, differed from those obtained with skins alone. Clarification, filtration, and centrifugation of the pulp and seed fractions or sonification of the skins produce different and well-characterized olfaction notes during the contact with yeast. The primary aroma of nonaromatic white and red grapes contained in the skins can be revealed within 24-48 h of yeast contact in a synthetic nutrient medium (SNM). The primary aroma precursors extracted from the skins with methanol, water-saturated butanol, or aqueous buffer at pH 3.2, concentrated and eluted from a C18 resin column, can be transformed to the free form wine aroma markers within 6 h of contact with yeast cells in SNM. By contrast, prolonged maceration of the skins in aqueous alcoholic buffer at pH 3.2 or 1.1, at 50 or 70 degrees C did not release primary odors typical of wine. The individual primary aroma compounds, identified by GC-MS analysis in Ruché wine samples or in Ruché skin-yeast-SNM samples, could not explain the complexity of the typical Ruché wine odor. Only odors common to many wine varieties were identified by GC-olfactometry analysis.

Monitoring the expression and purification of recombinant proteins by MALDI-TOF mass spectrometry

1Samples coming from biologic sources usually contain several contaminants that interfere seriously with Mass Spectrometry (MS) measurements. In this paper we report the application of MALDI-TOF MS to monitor recombinant protein expression and purification. The technique is based on the use of a C18 resin to clean and concentrate proteins in batch. The utility of this method is demonstrated for samples coming from different bacterial cultures expressing secreted and intracellular proteins ranging from 4 to 53 kDa. MALDI-TOF MS of peptide and proteins can be accomplished directly from complex bacterial cultures or from any purification step in a few minutes using the conventional stainless steel sample targets, allowing for a nearly instantaneous monitoring of the nature and integrity of recombinant expression products.

Determination of Blood Folate Using Acid Extraction and Internally Standardized Gas Chromatography–Mass Spectrometry Detection

Whole blood folate level is a superior indicator of folate nutritional status than serum/plasma level. Problems with and lack of confidence in results of current whole blood folate assays have limited its popularity for assessing folate nutritional status. Here, an acid extraction GCMS detection method that measures total folate whole blood is presented. Folates are released from the matrix of whole blood and cleaved to para-aminobenzoic acid (pABA) by acid hydrolysis in the presence of [13C6]pABA as internal standard (IS). The hydrolysate is passed over a C18 resin to remove heme. The pABA isotopomers are ethyl esterified, isolated on C18 resin, and trifluoroacetylated. Following normal-phase HPLC separation, the isotopomers are silylated to their tBDMS derivatives. The abundance of these derivatives are measured at m/z 324 for [13C6]pABA as IS and m/z 318 for pABA from whole blood folate. Our method uses readily available chemicals and our results agree well with those using Lactobacillus casei, the current gold standard reference assay. The presence of folate analogs (methotrexate) or antibacterials (sulfonamines) does not affect our method. This feature makes it useful in monitoring folate status of patients undergoing chemotherapy. Before using our method, pABA supplements must be discontinued for a few days.

Viral inactivation of fresh frozen plasma.

Viral inactivation of fresh frozen plasma.

BEHAVIORAL (FEEDING) RESPONSES OF THE CRAYFISH, Procambarus clarkii, TO NATURAL DIETARY ITEMS AND COMMON COMPONENTS OF FORMULATED CRUSTACEAN FEEDS

We have examined the behavioral (feeding) response of Procambarus clarkii to natural dietary items (zooplankton, live fishes, dead fishes, and fish eggs) and common components of formulated feeds used in the aquaculture industry (soybean meal, fish meal, corn meal, alfalfa meal, and vitamin C). The feeding response by P. clarkii was determined using an ordinally ranked, whole-animal bioassay that included the following behaviors: (1) movement of the maxillipeds for longer than three seconds, (2) increased movement of the walking legs with dactyl “probing,” (3) movement of walking legs to the mouth, and (4) orientation of the entire body towards the odor source. Feeding behavior was determined in response to intact items, bathwater containing aqueous leachates from intact items, water and methanol fractions of bathwater eluted through a C18 resin flash chromatography column, and size fractions of bathwater containing either molecules ≤10,000 Da or molecules >10,000 Da. All fractions tested were significantly stimulatory. Zooplankton was the most stimulatory of the natural dietary items tested. However, the C18 water fraction of the soybean meal bathwater before size fractionation (containing molecules both 10,000 Da) was the most stimulatory of the common feed components and more stimulatory than the zooplankton. Proximate analysis indicated that the compounds present in this fraction were ca. 51% soluble carbohydrate, 4% soluble protein, and 45% unknown (assumed to be insoluble carbohydrates, insoluble proteins, and ash). We hypothesize that the primary compounds in soybean meal responsible for eliciting a feeding response in P. clarkii are soluble carbohydrates and/or glycoproteins.

Debittering Casein Hydrolysates with Octadecyl‐Siloxane (C18) Columns

ABSTRACTThree casein hydrolysates (degree of hydrolysis (DH) of 23, 47, and 65.5 (%)) were debittered using three different hydrophobic adsorption columns, C18, C8, and phenolic resin (PR). Higher DH hydrolysates resulted in higher total nitrogen yields through the debittering process. The C18 and PR columns had more effective debittering than the C8 column, which had the highest processing yield. The PR column had much lower processing yield than the C18 column. Q‐value correlated with bitterness in the hydrolysates. Antigenicity of treated hydrolysates was reduced by the debittering process.

Protecting fibrinogen with rutin during UVC irradiation for viral inactivation.

Fibrinogen solutions were irradiated with UVC (254 nm) to inactivate contaminating viruses. In order to protect fibrinogen during UVC irradiation, 0.5 mM rutin was added prior to UVC exposure and subsequently removed during processing. Viral kill by 0.1 J/cm2 UVC resulted in the following inactivation values (log 10): non-lipid-enveloped viruses: Parvo > or = 5.5; encephalomyocarditis virus > or = 6.5; hepatitis A virus > or = 6.5: lipid-enveloped viruses: human immunodeficiency virus > or = 5.7; vesicular stomatitis virus > or = 5.7. Fibrinogen irradiated with 0.5 mM rutin did not significantly differ from unirradiated material in terms of clot time and breaking strength. In the absence of rutin, UVC irradiation of fibrinogen at similar fluence led to loss of solubility, increased clot time and the cleavage of fibrino-peptides that reacted with dinitrophenyl hydrazine as a test for ketonic carbonyl groups. High-performance liquid chromatography and mass spectrometry data showed that rutin exposed to UVC formed numerous breakdown, oxidation and combinational products. Experiments with 3H-rutin showed that after UVC irradiation, subsequent processing by a C18 resin and alcohol precipitation removed > 99% rutin, representing < 10 ppm rutin in the final fibrinogen preparations. Residual 3H-rutin was not covalently bonded to the fibrinogen. Immunochemical studies with rabbit antisera to UVC irradiated (with rutin) fibrinogen showed the absence of neoimmungens. By all measures, rutin prevents fibrinogen degradation during virucidal UVC irradiation.

Urinary excretion of mutagens in coke oven workers.

The influence of occupational exposure to polycyclic aromatic hydrocarbons (PAHs) on urinary mutagenic activity was assessed in 75 coke oven workers, using a highly sensitive bacterial mutagen technique (extraction with C18 resin and liquid micro-preincubation test on strain TA98 of Salmonella typhimurium in the presence of metabolizing and deconjugating enzymes). Exposure to PAHs was assessed according to the urinary excretion of 1-pyrenol; the main confounding factors were checked by the number of cigarettes smoked per day and the levels of nicotine and its metabolites in urine, or by ascertaining whether recommended dietary restrictions had been followed. Of the 20 urine samples which turned out to be positive (producing at least double the number of spontaneous revertants), 19 (95%) belonged to smokers. Only one non-smoker had obvious urinary mutagenic activity, and was highly exposed occupationally to PAHs (urinary 1-pyrenol of 3.930 mumol/mol of creatinine). Of the five urine samples from subjects who had not followed the recommended diet, two (40%) were clearly mutagenic. Multiple regression analysis (n = 67) showed that the presence of samples positive for urinary mutagenic activity depended only on smoking habits, if this confounding factor was assessed according to the number of cigarettes smoked per day, while the significant influence of exposure to PAH could be shown when the confounding factor was objectively estimated according to the urinary levels of nicotine and its metabolites. Assessment of the mutagenic potency of urinary extracts (net revertants/mmol creatinine) confirmed the strong influence of smoking habits on urinary mutagenic activity (all smokers 2156 +/- 2691 versus non-smokers 939 +/- 947 net revertants/mmol creatinine; Mann-Whitney test: P < 0.01). In smokers highly exposed to PAHs, greater excretion of mutagens with respect to low-exposure smokers was revealed (3548 +/- 4009 versus 1552 +/- 1227 net revertants/mmol creatinine; Mann-Whitney test: P < 0.01). Multiple regression analysis showed that the mutagenic potency of urinary extracts of coke oven workers depended on exposure to PAHs, tobacco smoking habits, and consumption of fried, grilled or barbecued meat. Increased urinary mutagenic activity strengthens epidemiological evidence of the increased risk of renal and urinary tract tumours in these workers. The presence of mutagenic metabolites in urine as a result of occupational exposure to PAH may be demonstrated only by using highly sensitive techniques for assessing urinary mutagenic activity in studies which include careful checking of the main confounding factors.

Virus Inactivation of Fresh Frozen Plasma by a Solvent Detergent Procedure: Biological Results

In order to increase the safety of blood products, we have developed a procedure for the virus inactivation of fresh frozen plasma. Several batches have been prepared and with the first 10 batches, each of them composed of 60 litres of plasma, we have determined a set of biological parameters. Virus inactivation was realised using TnBP (1%) and Octoxynol 9 (1%). After their elimination with castor oil using chromatography on insolubilized C18 resin, glycine was added and the pH of the plasma was adjusted to 7.4. Plastic bags were aseptically filled with a mean volume of 200 ml of plasma. The mean levels of coagulation factors were all over 0.7 U/ml and their recovery from initial plasma was nearly the same as total protein except for factor VIII:C. The net loss in factor VIII:C was 16%, when including the dilution of plasma. In vivo and in vitro tests demonstrated that in the final product there were no activated factors. As in fresh frozen plasma, the protein concentration was over 50 g/l and the potassium level lower than 5 mmol/l. According to these results, virus-inactivated plasma has the same qualities of fresh frozen plasma and could now replace it.

Sensitivity of different bacterial assays in detecting mutagens in urine of humans exposed to polycyclic aromatic hydrocarbons

The urine mutagenicity and excretion of 1-hydroxypyrene (1-OH PYR) in non-smoking psoriatic patients treated topically with coal-tar-based ointments were analysed in order to find the most appropriate procedure for monitoring occupational PAH exposure. The bacterial mutagenicity assays used were the plate incorporation, macro-scale fluctuation and microsuspension tests, all on Salmonella typhimurium strain TA98 in the presence of S9 mix and β-glucuronidase. The sensitivities of the three assays in detecting mutagenic urinary PAH metabolites were compared. The efficiencies of XAD-2 and C18 resins for concentrating PAH urinary mutagens were evaluated in the microsuspension assay. The plate and fluctuation tests on XAD-2 urine extracts were shown to be insufficiently sensitive to detect low urinary levels of mutagens, being positive on urine samples with very high PAH metabolite content, estimated as more than 30 μg/g of creatinine of 1-OH PYR. The microsuspension assay on XAD-2 or, even better, on C18 urine extracts was very sensitive in detecting up to 5 μg/g of creatinine of 1-OH PYR. It therefore seems to be applicable to the biological monitoring of most occupational low exposures to coal tar.

Solvent/detergent-treated plasma: a virus-inactivated substitute for fresh frozen plasma

Fresh frozen plasma (FFP) is prepared in blood banks world-wide as a by- product of red blood cell concentrate preparation. Appropriate clinical use is for coagulation factor disorders where appropriate concentrates are unavailable and when multiple coagulation factor deficits occur such as in surgery. Viral safety depends on donor selection and screening; thus, there continues to be a small but defined risk of viral transmission comparable with that exhibited by whole blood. We have prepared a virus sterilized FFP (S/D-FFP) by treatment of FFP with 1% tri(n-butyl)phosphate (TNBP) and 1% Triton X-100 at 30 degrees C for 4 hours. Added reagents are removed by extraction with soybean oil and chromatography on insolubilized C18 resin. Treatment results in the rapid and complete inactivation of greater than or equal to 10(7.5) infectious doses (ID50) of vesicular stomatitis virus (VSV) and greater than or equal to 10(6.9) ID50 of sindbis virus (used as marker viruses), greater than or equal to 10(6.2) ID50 of human immunodeficiency virus (HIV), greater than or equal to 10(6) chimp infectious doses (CID50) of hepatitis B virus (HBV), and greater than or equal to 10(5) CID50 of hepatitis C virus (HCV). Immunization of rabbits with S/D-FFP and subsequent adsorption of elicited antibodies with untreated FFP confirmed the absence of neoimmungen formation. Coagulation factor content was comparable with that found in FFP. Based on these laboratory and animal studies, together with the extensive history of the successful use of S/D-treated coagulation factor concentrates, we conclude that replacement of FFP with S/D-FFP, prepared in a manufacturing facility, will result in improved virus safety and product uniformity with no loss of efficacy.

Solvent/Detergent-Treated Plasma: A Virus-Inactivated Substitute for Fresh Frozen Plasma

Fresh frozen plasma (FFP) is prepared in blood banks world-wide as a by-product of red blood cell concentrate preparation. Appropriate clinical use is for coagulation factor disorders where appropriate concentrates are unavailable and when multiple coagulation factor deficits occur such as in surgery. Viral safety depends on donor selection and screening; thus, there continues to be a small but defined risk of viral transmission comparable with that exhibited by whole blood. We have prepared a virus sterilized FFP (S/D-FFP) by treatment of FFP with 1% tri(n-butyl)phosphate (TNBP) and 1% Triton X-100 at 30 degrees C for 4 hours. Added reagents are removed by extraction with soybean oil and chromatography on insolubilized C18 resin. Treatment results in the rapid and complete inactivation of greater than or equal to 10(7.5) infectious doses (ID50) of vesicular stomatitis virus (VSV) and greater than or equal to 10(6.9) ID50 of sindbis virus (used as marker viruses), greater than or equal to 10(6.2) ID50 of human immunodeficiency virus (HIV), greater than or equal to 10(6) chimp infectious doses (CID50) of hepatitis B virus (HBV), and greater than or equal to 10(5) CID50 of hepatitis C virus (HCV). Immunization of rabbits with S/D-FFP and subsequent adsorption of elicited antibodies with untreated FFP confirmed the absence of neoimmungen formation. Coagulation factor content was comparable with that found in FFP. Based on these laboratory and animal studies, together with the extensive history of the successful use of S/D-treated coagulation factor concentrates, we conclude that replacement of FFP with S/D-FFP, prepared in a manufacturing facility, will result in improved virus safety and product uniformity with no loss of efficacy.

Virus Inactivation of Fresh Frozen Plasma by a Solvent Detergent Procedure: Biological Results

In order to increase the safety of blood products, we have developed a procedure for the virus inactivation of fresh frozen plasma. Several batches have been prepared and with the first 10 batches, each of them composed of 60 litres of plasma, we have determined a set of biological parameters. Virus inactivation was realised using TnBP (1%) and Octoxynol 9 (1%). After their elimination with castor oil using chromatography on insolubilized C18 resin, glycine was added and the pH of the plasma was adjusted to 7.4. Plastic bags were aseptically filled with a mean volume of 200 ml of plasma. The mean levels of coagulation factors were all over 0.7 U/ml and their recovery from initial plasma was nearly the same as total protein except for factor VIII:C. The net loss in factor VIII:C was 16%, when including the dilution of plasma. In vivo and in vitro tests demonstrated that in the final product there were no activated factors. As in fresh frozen plasma, the protein concentration was over 50 g/l and the potassium level lower than 5 mmol/l. According to these results, virus- inactivated plasma has the same qualities of fresh frozen plasma and could now replace it.

Small particle aerosols of enviroxime-containing liposomes

Enviroxime inhibits the replication of all rhinoviruses tested in vitro at very low concentrations (10–100 ng/ml), but evaluations in humans have not consistently shown efficacy. Lack of an appropriate method for administering this water-insoluble drug may have contributed to the latter result. The present report describes the characteristics and utilization of small particle aerosols to continuously deliver enviroxime-containing liposomes (LE) throughout the respiratory tract. The enviroxime content of liposomes and biological fluids of exposed individuals was quantified by high performance liquid chromatography using C18 resin, a mobile phase of 60:40 acetonitrile:water, and monitoring at 215 nm. Small particle aerosols of LE generated by Puritan-Bennett nebulizers had mass median diameters ranging from 2.4 to 3.1 μm. The concentration of enviroxime in aerosol particles was proportional to the reservoir concentration; during the first hour of operation, the mean concentration was 20 μg of enviroxime/l of aerosol. Liposome particles in the reservoir, although initially heterogeneous in size (<0.1 to >1 μm), were processed by passage through the nebulizer to smaller, more homogeneous particles; the majority were less than 0.2 μm. In a preliminary study to evaluate short term tolerance and toxicity, five volunteers were exposed to small particle aerosol of LE for 1 h. At 1 h post-treatment, large amounts of enviroxime were still present in the nasal wash as determined both by HPLC and biological assay. Enviroxime was not detected in any urine sample and was detected in only 1 of 5 serum samples. No side effects were noted. This data suggest that liposome aerosols offer a method for the delivery of hydrophobic compounds for the treatment of respiratory diseases.

Oxidation of leukotrienes at the omega end: demonstration of a receptor for the 20-hydroxy derivative of leukotriene B4 on human neutrophils and implications for the analysis of leukotriene receptors.

Leukotriene B4 [LTB4; (5S,12R)-5,12-dihydroxy-6,14-cis-8,10-trans-icosatetraenoic acid] and its 20-hydroxy derivative [20-OH-LTB4; (5S,12R)-5,12,20-trihydroxy-6,14-cis-8,10-trans-icosatetraenoic acid] are principal metabolites produced when human neutrophils (hPMNs) are stimulated by the calcium ionophore A23187. These compounds were purified to homogeneity by Nucleosil C18 and silicic acid HPLC and identified by UV absorption and gas chromatographic/mass spectral analyses. 20-OH-LTB4 is considerably more polar than LTB4 and interacts weakly with the hydrophobic Nucleosil C18 resin, whereas LTB4 interacts strongly, reflecting the hydrophobic C13-C20 domain in LTB4. Specific binding of highly purified [3H]LTB4 and [3H]20-OH-LTB4 to hPMNs was assessed. Binding of [3H]20-OH-LTB4 could be largely displaced by an excess of nonlabeled LTB4 or 20-OH-LTB4 but not by 15-hydroxyicosatetraenoic acid (15-HETE), (5S,12S)-5,12-dihydroxy-6,10-trans-8,14-cis-icosatetraenoic acid [(5S,12S)-diHETE], or the 6-trans stereoisomer of LTB4 at 1 microM. In contrast, [3H]LTB4 displays a high level of nonspecific binding to human PMNs, which makes assessment of the Kd for LTB4 binding unobtainable. Binding measurements for [3H]LTB4 were performed in a buffer containing bovine serum albumin, and under these conditions significantly less nonspecific binding was observed. The apparent Kd for high-affinity binding sites on human PMNs at 0 degrees C was 31.3 X 10(-9) M for LTB4 and 14.3 X 10(-9) M for 20-OH-LTB4. In addition, we observed a saturable low-affinity receptor for 20-OH-LTB4 with a Kd of approximately 100 X 10(-9) M and 2 X 10(5) receptors per cell. The data from this study suggest that omega oxidation represents a major pathway for metabolism of LTB4 as well as other arachidonate metabolites. LTB4 and 20-OH-LTB4 express similar functional activities and share common binding properties to hPMNs but differ significantly in their physical properties. It is the unique physical characteristics of 20-OH-LTB4 that suggest that arachidonate metabolites oxidized at the omega position may be more important agents in inflammation than LTB4.

A Novel Method for the Quantitation of Warfarin and its Metabolites in Plasma

Abstract A procedure for the rapid, quantitative recovery of warfarin and its metabolites (diastereoisomeric alcohol, 4′-, 6-, 7-, 8-benzylic-hydroxywarfarin and dehydrowarfarin) from plasma with Sep-Pak C18 cartridges has been developed. A solution of warfarin and its metabolites in plasma was acidified with NH4OAc buffer (pH 4.85), adsorbed on the Sep-Pak C18 resin, washed free of polar constituents and eluted with methanol. Dilution of the eluate with buffer followed by gradient high performance liquid chromatography permitted accurate quantitation of the desired compounds when detected at 313 nm. The recovery of warfarin and each metabolite was greater than 95% over an investigated range of 0.5–10.0 μg/ml of plasma and the limit of quantitation by the assay was 0.1 μg/ml of plasma. For more rapid quantitation of warfarin, without simultaneous analysis of metabolites, the chromatographic parameters were modified so that elution of warfarin occurred within 13 minutes, and the sensitivity of the assay increased to 0.03 μg of warfarin/ml of plasma. The quantitative recovery of warfarin and its metabolites coupled with the chromatographic versatility of the method make it ideally suited for either detailed pharmacokinetic studies or routine plasma analysis of warfarin.