Liquid Chromatography Postcolumn Research Articles

Redox state of human serum albumin predicts 1-year prognosis in patients with heart failure: a single center prospective cohort study

Abstract Background As chronic inflammation would be a fundamental underlying mechanism in the pathogenesis in patients with heart failure (HF), oxidative stress has a possibility to closely associate with the prognosis. However, little is known about the association of oxidative stress with the prognosis in HF patients. Purpose In this study, we assessed the association of the fraction of human mercapto-albumin [f(HMA)], an index of the redox state of human serum albumin, with prognosis in patients hospitalized for HF with the aim to find the possible biomarker available on the prediction for adverse events in HF patients. Methods A single-center prospective cohort study was conducted. In the cohort, 290 ambulate patients admitted for acute HF or exacerbation of chronic HF were registered. Of these, 182 who were successfully followed-up patients were analyzed (median age: 79 years, men: 68.3%. median follow-up: 365 days). The primary outcome was a composite of HF rehospitalization and all-cause mortality. The high-performance liquid chromatography postcolumn bromocresol green method was used to part human serum albumin into human nonmercaptalbumin (oxidized form) and human mercaptalbumin (HMA, reduced form). We divided patients into two groups by median f(HMA) level. Kaplan-Meier Curve and the Cox proportional hazards model was used to examine the significance of f(HMA) as a prognostic indicator of HF patients, adjusting for potential confounding factors. Results Median level of f(HMA) was 57.2%. During 50,965 person-days of follow-up, 52 patients (28.6%) were re-hospitalized due to HF exacerbation, and 25 patients (13.7%) died due to cardiac or noncardiac reasons. Log-rank analysis showed that a high f(HMA) group reduced adverse events (Fig. 1). In the Cox proportional hazards model, a high level of f(HMA) was associated with a reduced risk of composite outcomes (hazard ratio [HR] 0.64; 95% confidence interval [CI] 0.39-1.07). In the subgroup analysis, high f(HMA) showed an event-reducing effect in HF rehospitalizations (HR 0.53; 95% CI 0.30-0.94), but not in all-cause mortality (HR 0.61; 95%CI 0.27-1.38). Conclusion The findings of this study support that f(HMA) could be a novel prognostic biomarker for HF patients.Fig. 1

Association Between the Redox State of Human Serum Albumin and Exercise Capacity in Patients With Cardiac Disease

The redox state of human serum albumin (HSA) is reported to be an oxidative stress biomarker; however, its clinical use in cardiac disease has not yet been examined. This study aimed to investigate the relation between the redox state of HSA and exercise capacity, which is a robust prognostic factor, in patients with cardiovascular disease. This cross-sectional study included outpatients with cardiac disease. Exercise capacity was assessed by peak oxygen consumption (peakVO2) measured using symptom-limited cardiopulmonary exercise testing. The high-performance liquid chromatography postcolumn bromocresol green method was used to part HSA into human nonmercaptalbumin (oxidized form) and human mercaptalbumin (HMA, reduced form). The fraction of human mercaptalbumin found in HSA (f[HMA]) was calculated as an indicator of the redox state of HSA. The association between peakVO2 and f(HMA) was examined using the Spearman correlation coefficient and multivariate linear regression analysis. A total of 70 patients were included (median age 76years; 44 men; median peakVO2 15.5ml/kg/min). The f(HMA) was positively correlated with peakVO2 (r=0.38, p <0.01). Even after controlling for potential confounders, this association remained in the multivariate linear regression analysis (standardized beta=0.24, p <0.05). We found a positive association between f(HMA) and peakVO2, independent of potential confounders in patients with cardiac disease, suggesting that f(HMA) may be a novel biomarker related to exercise capacity in cardiac disease. Longitudinal studies are required to further examine the prognostic capability of f(HMA), the responsiveness to clinical intervention, and the association between f(HMA) and cardiac disease.

A Comparison Study of Phenolic Contents and &amp;lt;i&amp;gt;in Vitro&amp;lt;/i&amp;gt; Antioxidant Activities of Australian Grown Faba Beans (&amp;lt;i&amp;gt;Vicia faba&amp;lt;/i&amp;gt; L.) Varying in Seed Coat Colours as Affected by Extraction Solvents

Twelve Australian grown faba bean genotypes with seed colour ranging from white, different shades of buff/beige, green, red and purple, were extracted using 80% methanol (v/v) or 70% acetone (v/v). The findings showed that commonly consumed buff-coloured genotypes have comparable phenolic contents and antioxidant activities to those with exotic seed coat colour (red, green and purple). In general, the extraction yield for methanol and acetone extracts of faba bean genotypes were similar. However, the acetone extracts of faba bean varieties with coloured seed coats exhibited higher antioxidant activities than their methanol extracts counterparts. Phenolic compounds and antioxidant activities of the genotypes were compared in an array of chemical-based assays and profiled using an on-line high-performance liquid chromatography-post column derivatization (HPLC-PCD) system. The total phenolic content (TPC) and total flavonoid content (TFC) of acetone extracts were approximately twice those of methanol extracts. The acetone extracts contained six times higher levels of di(phenyl)-(2,4,6-trinitrophenyl) imi- noazanium radical scavenging activity (DPPH), six times higher Total Equivalent Antioxidant Capacity (TEAC) and two times higher Ferric Reducing Antioxidant Power (FRAP) than those of methanol extracts. In general, the methanol extracts of white-coloured faba bean genotype exhibited comparable phenolic contents and antioxidant activities to varieties with coloured seed coats. However, the TPC, TFC, DPPH, TEAC and FRAP of acetone extracts from white-coloured genotype were 2 - 4, 1 - 2, 5 - 9, 2 - 3 and 1 - 2 times lower than those of faba bean varieties with coloured seed coats. HPLC-PCD analyses showed substantial antioxidant responses, represented as a dense “hump” of peaks in the HPLC chromatograms of acetone extracts from coloured-genotypes. This “hump” was not detected in the chromatograms of white-genotype acetone extracts, or in chromatograms of methanol extracts regardless of genotype or seed coat colour. All coloured-beans had higher phenolic contents and antioxidant activities than the white-genotype. Hydroxybenzoic acids/flavanols, hydroxycinnamic acids and flavonols were dominant in coloured beans. The findings suggest potentials in selecting commonly consumed buff-coloured faba bean genotypes within the breeding programs for enhanced levels of phenolic compounds and antioxidant activities, potentially increasing their health-promoting properties. And, there might also be potentials in developing faba bean extracts for pharmaceutical or natural medicines.

Oxidized human serum albumin as a possible correlation factor for atherosclerosis in a rural Japanese population: the results of the Yakumo Study

BackgroundThe effect of the redox state of human serum albumin (HSA) on the antioxidant properties of the entire body has been a focus of recent research. The usefulness of HSA redox state as a biomarker for reducing oxidative stress has been investigated in clinical settings; however, evidence for its significance as a health index in non-clinical settings is yet to be established. This study aimed to examine the associations between HSA redox state and the atherosclerotic indices of carotid intima-media thickness (IMT) and plaque formation in a rural Japanese population.MethodsWe conducted a cross-sectional study as part of a health check-up program in the rural area of Hokkaido, Japan, at the end of August 2013. A total of 281 residents (124 men and 157 women) were included in the final analysis. Lifestyle-related data were obtained through a self-reported questionnaire, and ultrasound examinations were performed to measure IMT and determine plaque formation. The high-performance liquid chromatography postcolumn bromocresol green method was used to separate HSA into human nonmercaptalbumin and human mercaptalbumin (HMA).ResultsWe found a significant negative relationship between the fraction of HMA [f(HMA)] and IMT (standardized β = − 0.132, p = 0.03). Moreover, f(HMA) was significantly associated with plaque formation (p < 0.01) with an odds ratio of 0.89 (95% confidence interval, 0.81–0.97) for every 10% increment in f(HMA).ConclusionsWe found that the HSA redox state, as determined by f(HMA), was associated with atherosclerotic indices in Japanese subjects. These results suggest that the HSA redox state indicates the risk of developing atherosclerosis.

Liquid Chromatography Post-Column Oxidation (PCOX) Method for the Determination of Paralytic Shellfish Toxins in Mussels, Clams, Oysters, and Scallops: Collaborative Study

Sixteen laboratories participated in a collaborative study to evaluate method performance parameters of a liquid chromatographic method of analysis for paralytic shellfish toxins (PST) in blue mussels (Mytilus edulis), soft shell clams (Mya arenaria), sea scallops (Placopectin magellanicus), and American oysters (Crassostrea virginicus). The specific analogs tested included saxitoxin, neosaxitoxin, gonyautoxins-1 to -5, decarbamoyl-gonyautoxins-2 and -3, decarbamoyl-saxitoxin, and N-sulfocarbamoyl-gonyautoxin-2 and -3. This instrumental technique has been developed as a replacement for the current AOAC biological method (AOAC Official Method 959.08) and an alternative to the pre-column oxidation LC method (AOAC Official Method 2005.06). The method is based on reversed-phase liquid chromatography with post-column oxidation and fluorescence detection (excitation 330 nm and emission 390 nm). The shellfish samples used in the study were prepared from the edible tissues of clams, mussels, oysters, and scallops to contain concentrations of PST representative of low, medium, and high toxicities and with varying profiles of individual toxins. These concentrations are approximately equivalent to 1/2 maximum level (ML), ML, or 2xML established by regulatory authorities (0.40, 0.80, and 1.60 mg STX diHCl eq/kg, respectively). Recovery for the individual toxins ranged from 104 to 127%, and recovery of total toxin averaged 116%. Horwitz Ratio (HorRat) values for individual toxins in the materials included in the study were generally within the desired range of 0.3 to 2.0. For the estimation of total toxicity in the test materials, the reproducibility relative standard deviation ranged from 4.6 to 20%. A bridging study comparing the results from the study participants using the post-column oxidation (PCOX) method with the results obtained in the study director's laboratory on the same test materials using the accepted reference method, the mouse bioassay (MBA; AOAC Official Method 959.08), showed that the average ratio of results obtained from the two methods was 1.0. A good match of values was also achieved with a new certified reference material. The results from this study demonstrated that the PCOX method is a suitable method of analysis for PST in shellfish tissue and provides both an estimate of total toxicity, equivalent to that determined using the MBAAOAC Official Method 959.08, and a detailed profile of the individual toxin present in the sample.

Age-related impairment of HDL-mediated cholesterol efflux

Our aim in this study was to investigate the effect of aging on the capacity of HDLs to promote reverse cholesterol transport. HDLs were isolated from plasma of young (Y-HDL) and elderly (E-HDL) subjects. HDL-mediated cholesterol efflux was studied using THP-1 and J774 macrophages. Our results show that E-HDLs present a lower capacity to promote cholesterol efflux than Y-HDLs (41.7 +/- 1.4% vs. 49.0 +/- 2.2%, respectively; P = 0.013). Reduction in the HDL-mediated cholesterol efflux capacity with aging was more significant with HDL(3) than HDL(2) (Y-HDL(3), 57.3 +/- 1% vs. E-HDL(3), 50.9 +/- 2%; P = 0.012). Moreover, our results show that ABCA1-mediated cholesterol efflux is the more affected pathway in terms of cholesterol-removing capacity. Interestingly, the composition and structure of HDL revealed a reduction in the phosphatidylcholine-sphingomyelin ratio (E-HDL, 32.7 +/- 2.7 vs. Y-HDL, 40.0 +/- 1.9; P = 0.029) and in the phospholipidic layer membrane fluidity in E-HDL compared with Y-HDL as well as an alteration in the apolipoprotein A-I structure and charge. In conclusion, our results shown that E-HDLs present a reduced capacity to promote cholesterol efflux, principally through the ABCA1 pathway, and this may explain the increase of the incidence of cardiovascular diseases observed during aging.

Hepatic SR-BI-mediated cholesteryl ester selective uptake occurs with unaltered efficiency in the absence of cellular energy

Scavenger receptor class B type I (SR-BI) plays a critical role in the delivery of HDL cholesterol and cholesteryl esters (CEs) to liver and steroidogenic tissues by a selective process that does not result in significant degradation of HDL protein. Recently, SR-BI-mediated endocytosis and recycling of HDL have been demonstrated. However, it remains unclear whether efficient SR-BI-mediated selective uptake occurs strictly at the plasma membrane or at additional sites along its endocytic itinerary. To examine the requirement for SR-BI endocytosis in HDL selective uptake, we determined the effects of energy depletion on the levels of cell-associated HDL protein and CE in primary mouse hepatocytes. Compared with CHO cells, we observed a much larger energy-dependent effect on CE uptake in primary mouse hepatocytes. Although varying the levels of caveolin-1 and carboxyl ester lipase altered the efficiency of selective uptake, neither was able to account for the energy-dependent component of HDL-CE uptake. Finally, we demonstrate that the hepatocyte-specific, energy-dependent effects on HDL-apolipoprotein A-I and -CE uptake are independent of SR-BI and are not required to achieve efficient SR-BI-mediated selective uptake of CE. Together, these data support the conclusion that neither the intracellular trafficking of HDL nor any energy-dependent cellular process affects the ability of the cell to maximally acquire CE through SR-BI-mediated selective uptake from HDL.

Continuous ultrasound-assisted extraction coupled to on line filtration–solid-phase extraction–column liquid chromatography–post column derivatisation–fluorescence detection for the determination of N-methylcarbamates in soil and food

A dynamic ultrasound-assisted method for the extraction of N-methylcarbamates (oxamyl, dioxacarb, metolcarb, carbofuran, carbaryl and isoprocarb) from soils and foods is proposed. The main factors affecting the extraction efficiency have been optimised by means of a central composite design. Pure water can be used as leaching agent. A flow injection manifold coupled to the extractor allows automation of the several steps involved in the analytical process. The method allows extraction of the carbamates from soil and food at 1 μg/g spiked level, with recoveries similar to those provided by the EPA 8318 method, without degradation of the target compounds during the extraction and with drastic shortening of the time required for this step (2 min vs. 4 h). Recoveries of the target analytes were 77–95% for spiked soil and 85–101% for spiked food. The detection and quantification limits were 12 and 40 ng/g, respectively, for all analytes, except carbaryl (detection and quantification limits 3 and 10 ng/ng, respectively). The relative standard deviations for repeatability and within-laboratory reproducibility were 3.1 and 7.5%, respectively.

Heterogeneous post-column immunoreaction detection using magnetized beads and a laboratory-constructed electromagnetic separator.

The nature of immune reactors allows development of quantitative analytical methods that are highly selective and can often be used directly with complex biological matrixes such as blood, plasma or urine. A major limitation of immunoassay is that antibodies are sometimes unable to discriminate structurally similar species such as drug metabolites and synthetic analogs. The problem associated with the lack of discrimination can be circumvented by coupling immunoassay with liquid chromatography post-column. The most commonly used separation method in post-column immunoreaction detection is the affinity column. Affinity columns may create undesired effects such as a compromise of the chromatographic separation efficiency, the requirement for an antibody with fast reaction kinetics and the need for flushing the column. This paper reports a post-column immunoreaction detection system coupled with a laboratory-constructed on-line magnetic separation flow chamber that is designed to overcome these problems. The system uses disposable magnetic beads as a solid-phase support for separation that can be easily removed from the system. The model analytes chosen for this study were digoxin and its metabolites due to the commercial availability of monoclonal antibodies for these compounds. Digoxin was separated using a chromatographic method prior to being interfaced through a liquid handler system to the immunoreactor. Compatibility of the HPLC mobile phase was determined to be acceptable with a mixing ratio of 1:3 between the LC fraction and immunoreagent solution. The dynamic range of the calibration curve in digoxin-spiked phosphate buffer was found to be 0.25-12 ng/ml and a quadratic fit was found to provide the best fit to the data with a correlation coefficient of 0.9974. The residual error for all standards was less than 15%. The percentage RSDs for the two controls, 2 and 10 ng/ml, were 6.88 and 4.82% (n = 6) and the percentage errors were 7.07 and -6.89% (n = 6), respectively.

Fluorimetric determination of isatin in human urine and serum by liquid chromatography postcolumn photoirradiation.

For the fluorimetric determination of isatin in human urine and serum, HPLC-postcolumn photoirradiation using a mobile phase has been developed. Isatin in the urine or serum sample was separated on a Capcell Pak C1 column (250 x 4.6 mm id). The mobile phase consisted of 70 mmol l-1 phosphate buffer (pH 7.2)-tetrahydrofuran (85 + 15% v/v) containing 5 mmol l-1 hydrogen peroxide, which was irradiated with germicidal light to induce fluorescence (lambda ex 302 nm, lambda em 418 nm). The addition of tetrahydrofuran to the mobile phase led to the peaks showing good separation as well as increased sensitivity. The calibration graph for isatin was linear over the range of 0.16-10.7 ng. The pretreatment of the acidified urine or serum samples consisted of diluting steps or deproteinizing steps using perchloric acid, respectively. The mean recovery of isatin from urine and serum was greater than 94%.

Direct application of an enzyme-linked immunosorbent assay method for carbaryl determination in fruits and vegetables. Comparison with a liquid chromatography–postcolumn reaction fluorescence detection method

The insecticide carbaryl was quantified in fruit and vegetable juices obtained from seven crops without any previous treatment. A previously developed enzyme-linked immunosorbent assay (ELISA) was applied by simply analysing the fruit and vegetable juices diluted in the buffer assay and adjusting the pH of the final solution to the optimum value (pH ∼7.5), in case of citric samples. The similarity and parallelism between the standard curves obtained from the diluted crop juices and the assay buffer indicate that the matrix interference is minimal for most samples. Limits of detection, calculated at the pesticide concentration giving 90% of the B 0 (working range), varied from 0.13 to 6.05 μg l −1 in the crop juices. Accuracy of carbaryl ELISA method from crops spiked at 10 and 100 μg l −1 was comparable to liquid chromatography–postcolumn fluorescence method. Recoveries averaged 95% (85–108%) for immunoassays and 103% (100–106%) for LC analysis.

Cholesterol hydroperoxides in erythrocyte membranes of alcoholic patients.

Evidence for the presence of 5alpha-hydroperoxycholest-6-en-3beta-ol (cholesterol 5alpha-hydroperoxide, Ch 5alpha-OOH) and 7alpha- and 7beta-hydroperoxycholest-5-en-3beta-ols (cholesterol 7-hydroperoxides: Ch 7alpha-OOH and Ch 7beta-OOH, respectively) in human erythrocyte membrane was found. Blood samples were collected from alcoholic patients and healthy volunteers (controls), and their cholesterol hydroperoxides were analyzed by high-performance liquid chromatography postcolumn chemiluminescence and roughly identified by liquid chromatography-mass spectrometry. Ch 7alpha-OOH and Ch 7beta-OOH were present in each sample, being significantly higher in alcoholic samples than in control samples. Ch 5alpha-OOH was present in some alcoholic samples, but not in the control ones. The accumulation of cholesterol hydroperoxides suggests enhanced lipid peroxidation by active oxygen species and/or a reduced elimination system for lipid peroxide in alcoholic patients.

Fluorometric determination of N1-methylnicotinamide in human urine and serum by liquid chromatography postcolumn photoirradiation with a mobile phase containing 18-crown-6

For the determination of N 1-methylnicotinamide in human urine and serum, liquid chromatography (LC)–postcolumn photoirradiation using a mobile phase containing 18-crown-6 has been developed. The mobile phase consisted of 35 mmol l −1 potassium dihydrogenphosphate containing 5 mmol l −1 hydrogen peroxide and 25 mmol l −1 18-crown-6. Addition of 18-crown-6 to the mobile phase led to peaks having a good shape, earlier elution and increased sensitivity. Moreover, the assay succeeded in separation of N 1-methylnicotinic acid. The calibration graph for N 1-methylnicotinamide was linear over the range of 5.0–20.4 pmol using an injection volume of 20–100 μl. The pretreatment of the urine or serum samples consisted of diluting–filtering steps or deproteinizing steps, respectively. The mean recovery of N 1-methylnicotinamide from urine and serum was over 95%.

29] Fluorophore-linked assays for high-performance liquid chromatography postcolumn reaction detection of biotin and biocytin

Publisher Summary This chapter describes high-performance liquid chromatography (HPLC) postcolumn reaction detection systems in which two types of protein-binding assay principles are used. In a competitive-binding approach, the fluorescent probe 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS) competes with the analyte (biotin or biocytin) for the same binding sites on avidin. Two noncompetitive binding approaches are also described, in which the interaction that takes place is between the analyte (biotin or biocytin) and avidin or streptavidin. In these cases (strept)avidin is labeled with fluorescein isothiocyanate (FITC). On binding with the analyte, the fluorescence intensity of (strept)avidin–FITC increases in a manner proportional to the concentration of the analyte. The coupling of the binding assays with HPLC results in a highly selective detection of biotin and its derivatives owing to their selective interaction with (strept)avidin. These types of approaches also lead to enhanced sensitivity because the HPLC detection is fluorescence based rather than the conventional ultraviolet (UV) absorbance-based detection. The chapter also describes an absorbance-based postcolumn reaction detection system. In this system, biotin competes with and displaces the dye 2-(4'-hydroxyphenylazo)benzoic acid (HABA), where the free and bound HABA have different absorbance characteristics.

Comparison of Liquid Chromatography–Atmospheric Pressure Chemical Ionization/Mass Spectrometry and Liquid Chromatography–Postcolumn Fluorometry for Determination of Carbamates in Food

Abstract Two detection methods, postcolumn derivatization with fluorescent detection and atmospheric pressure chemical ionization/mass spectrometry (MS) were compared for determination of N-methyl carbamates in food by liquid chromatography. Five commodities were spiked with 11 carbamates at levels from 10 to 100 ppb, extracted, and cleaned up by using a method based on the AOAC official method. With each detection method, sample blanks showed false-positive responses, indicating that neither method alone provides definitive identification. Except for aldicarb sulfoxide determined by MS, recoveries were linear between 10 and 100 ppb. Agreement between the 2 methods was good for determination of recoveries at 25 ppb. Coefficients of variation were 11–33% for fluorescence detection and 9–26% for MS.

Automated Enzyme Analysis by High-Performance Liquid Chromatography Postcolumn Substrate Reaction: Application to Trypsin and Chymotrypsin

An automated enzyme analysis was designed for trypsin and chymotrypsin. The technique consisted of a hydrophobic chromatography of the enzyme and coupled postcolumn substrate reaction. In a desalted aqueous medium, trypsin and chymotrypsin were adsorbed on a phenyl modified adsorbent gel by hydrophobic interaction, and eluted by the concentration gradient of an electrolyte. After the substrate for the enzyme was reacted in a postcolumn reaction coil, the product was spectrophotometrically detected at 410 nm. The separatory determination of trypsin and chymotrypsin was carried out by using the suitable substrate solution. Each calibration plot was a good straight line at 5–100 µg of enzyme. The relative standard deviation of 50 ug trypsin and the same quantity of chymotrypsin was 2% and 3.5% (n=5, within-run) respectively. The method was applied to the determination of trypsin and chymotrypsin activity of rat pancreatic juice.

Immunoaffinity Column Coupled with Solution Fluorometry or Liquid Chromatography Postcolumn Derivatization for Determination of Aflatoxins in Corn, Peanuts, and Peanut Butter: Collaborative Study

An AOAC/IUPAC (International Union of Pure and Applied Chemistry) collaborative study was conducted to evaluate the effectiveness of an immunoaffinity column for the determination of aflatoxin. The test portion is extracted with methanol-water (7 + 3), filtered, diluted to less than 30% methanol with water, and applied to the affinity column. The column is washed with water and the concentrated aflatoxins are eluted with methanol. Total aflatoxins are determined by solution fluorometry with bromine (SFB), and individual toxins are determined by reverse-phase liquid chromatography with postcolumn derivatization with iodine (PCD). Corn naturally contaminated with aflatoxins, and peanuts, peanut butter, and corn containing added aflatoxins (B1:B2:G1:G2 = 7:1:3:1) were sent to 24 collaborators in the United States, France, Canada, and the Republic of South Africa. Twelve collaborators used the SFB method, 9 used the PCD method, and 3 used both SFB and PCD methods. Twenty collaborators completed the study (10 used the SFB method, 7 used the PCD method, and 3 used both SFB and PCD methods). Test portions were spiked at 10, 20, and 30 ng/g. For SFB analyses, recoveries of total aflatoxins were 123, 105, and 107%, respectively; the relative standard deviation for repeatability (RSDr) ranged from 11.75 to 16.57%, and the relative standard deviation for reproducibility (RSDR) ranged from 10.97 to 33.09%. For PCD analyses, recoveries were 81, 81, and 83%, respectively; the RSDr ranged from 5.20 to 17.22%, and the RSDR ranged from 4.68 to 50.77%. The RSDr for aflatoxins B1 and G1 for spiked test portions ranged from 5.45 to 23.55%, and the RSDR ranged from 4.21 to 57.28%.(ABSTRACT TRUNCATED AT 250 WORDS)