Effect of ionic additives on the elution of sodium aryl sulfonates in supercritical fluid chromatography.

Addition of a small amount of polar solvent (i.e., modifier) to CO2 in packed column supercritical fluid chromatography (SFC) has shown major improvements in both polar analyte solubility and interaction of the polar analyte with the stationary phase. Recently, the addition of an ionic component (i.e., additive) to the primary modifier by one of us has been shown to extend even further the application of SFC to polar analytes. In this work, the effect of various ionic additives on the elution of ionic compounds, such as sodium 4-dodecylbenzene sulfonate and sodium 4-octylbenene sulfonate, has been studied. The additives were lithium acetate, ammonium acetate, tetramethylammonium acetate, tetrabutylammonium acetate, and ammonium chloride dissolved in methanol. Three stationary phases with different degrees of deactivation were considered: conventional cyanopropyl, deltabond cyanopropyl, and bare silica. The effect of additive concentration and additive functionality on analyte retention was investigated. Sodium 4-dodecylbenzene sulfonate was successfully eluted using all the additives with good peak shape under isocratic/isobaric/isothermal conditions. Different additives, however, yielded different retention times and in some cases different peak shapes.

Study of the elution mechanism of sodium aryl sulfonates on bare silica and a cyano bonded phase with methanol-modified carbon dioxide containing an ionic additive.

The elution mechanism of sodium sulfonates on both Deltabond cyanopropyl and bare silica stationary phases with an isocratic mobile phase composed of methanol-modified CO2 wherein an ammonium salt additive was dissolved in the methanol has been studied. The presence of the additive was crucial concerning elution of the sulfonate salts. Solid state 29silicon nuclear magnetic resonance spectroscopy provided some insight concerning the interaction of the mobile phase additive with the silica-based stationary phase. Computational calculations concerning the charge distribution on various ammonium salts were performed in an effort to explain the elution behavior. Ammonium ions are believed to deactivate available silanol sites on both phases. In addition, ammonium ion is speculated to interact with the cyano groups on the bonded phase. For concentrations of additive greater than 2 mM, stationary phase coverage of ammonium ion is anticipated to exceed one monolayer for both bare and bonded silica. The acetate counter-ion is thought to facilitate elution of the anionic sulfonates from the positively charged stationary phase in a pseudo ion exchange mechanism.

Advantages and drawbacks of popular supercritical fluid chromatography/mass interfacing approaches--a user's perspective.

Mobile phases in supercritical fluid chromatography (SFC) have low viscosities and high diffusion coefficients with respect to those of traditional high performance liquid chromatography (HPLC). These properties allow higher mobile phase flow rates and/or longer columns in SFC, resulting in rapid analyses and high efficiency separations. In addition, chiral SFC is becoming especially popular. Mass spectrometry (MS) is arguably the most popular "informative" detector for chromatographic separations. Most SFC/MS is performed with atmospheric pressure ionization (API) sources. Unlike LC/MS, the interface between the SFC column and the API source must allow control of the downstream (post-column) pressure while also providing good chromatographic fidelity. Here we compare and contrast the popular interfacing approaches. Some are simple, such as direct effluent introduction with no active back-pressure-regulator (BPR) in high speed bioanalytical applications. The pressure-regulating-fluid interface is more versatile and provides excellent chromatographic fidelity, but is less user friendly. The pre-BPR- split interface and an interface which provides total-flow-introduction with a mechanical BPR are good compromises between user friendliness and performance, and have become the most popular among practitioners. Applications of SFC/MS using these various interfaces are also discussed.

Characterization of protective immunity induced against Schistosoma mansoni via DNA priming with the large subunit of calpain (Sm-p80) in the presence of genetic adjuvants.

Despite advances in control via snail eradication and large-scale chemotherapy using praziquental, schistosomiasis continues to spread to new geographic areas particularly in sub-Saharan Africa. Presently, there is no vaccine for controlling this disease. We have concentrated on a functionally important schistosome antigen Sm-p80 as a possible vaccine candidate for schistosomiasis. Here we report the proliferation of spleen cells in response to the recombinant Sm-p80 protein and cytokine (IFN-gamma and IL-4) production by the splenocytes. These spleen cells were obtained from groups of mice that were vaccinated with a DNA vaccine formulation containing Sm-p80 and one of the Th-1 (IL-2 or IL-12) or Th-2 (GM-CSF, IL-4) enhancer cytokines. The splenocytes from the groups of mice vaccinated with Sm-p80 DNA in the presence of Th-2 enhancer cytokines showed moderate but detectable proliferation. The splenocytes obtained from mice vaccinated with Sm-p80 DNA with Th-1 enhancer cytokines IL-2 and IL-12 provided the highest proliferation. The IFN-gamma production by splenocytes was found to follow the similar pattern [(Sm-p80) < (Sm-p80 + IL-4) < (Sm-p80 + GMCSF) < (Sm-p80 + IL-12) < (Sm-p80 + IL-2)], as has been observed for the proliferation and protection data. However, the elevated IL-4 production was inversely correlated to Sm-p80-induced splenocyte proliferation or the protection. These results show again that protective immune response induced by Sm-p80 is of Th-1 type.

Comparison of slow oscillating versus fast balloon inflation strategies for coronary angioplasty.

Previous studies suggest that slow and/or oscillating balloon inflation during coronary angioplasty may decrease the incidence of coronary dissection and improve clinical outcomes. To compare the effect of slow oscillating versus conventional fast inflation techniques on the incidence of severe coronary dissection during angioplasty, 622 patients were randomized to slow oscillating inflation versus fast inflation. Angiographic outcomes of the procedures and in-hospital clinical events were recorded. The primary end point of severe (type C, D, E, F) dissection occurred in 7.7% of patients undergoing slow oscillation and 6.6% of patients undergoing fast inflation (p = 0.87). Major complications (death, urgent coronary artery bypass graft surgery, stroke, abrupt closure, or Q-wave myocardial infarction) occurred in 4.7% of patients undergoing slow oscillation and 3.5% of patients undergoing fast inflation (p = 0.45). The 2 inflation strategies did not differ in the pressure at which the balloon achieved full expansion, angiographic success rate, residual stenosis, and incidence of all minor and/or major complications. We conclude that there is no benefit of slow oscillating inflation over routine fast inflation in angioplasty. Slow oscillating inflation did not dilate lesions at lower pressures, decrease the incidence of dissection or severe dissection, or reduce the incidence of adverse clinical outcomes.

Characterization of low molecular weight alkoxylated polymers using long column SFC/MS and an image analysis based quantitation approach.

The utility of low viscosity mobile phases and long chromatographic columns for complex polymer analysis is demonstrated. We use long column supercritical fluid chromatography/mass spectrometry (SFC/MS) with electrospray ionization (ESI) to characterize a variety of complex, low molecular weight polymers. When quantitative analysis is desired, the resulting three-dimensional (time, intensity, and mass-to-charge ratio [m/z]) data are converted to images. Custom image analysis software is used to detect and integrate peaks in arbitrarily defined regions of the time-m/z map. These integrated peak volumes can be used to quantitate distinct component classes of the polymer mixtures.

Radiation therapy among atomic bomb survivors, Hiroshima and Nagasaki.

As a follow-up to the two previous surveys of radiation therapy among the atomic bomb survivors, a large-scale survey was performed to document (1) the number of radiation therapy treatments received by the atomic bomb survivors and (2) the types of radiation treatments conducted in Hiroshima and Nagasaki. The previous two surveys covered the radiation treatments among the Radiation Effects Research Foundation Adult Health Study (AHS) population, which is composed of 20,000 persons. In the present survey, the population was expanded to include the Life Span Study (LSS), including 93,611 atomic bomb survivors and 26,517 Hiroshima and Nagasaki citizens who were not in the cities at the times of the bombings. The LSS population includes the AHS population. The survey was conducted from 1981 to 1984. The survey teams reviewed all the medical records for radiation treatments of 24,266 patients at 11 large hospitals in Hiroshima and Nagasaki. Among them, the medical records for radiation treatments of 1556 LSS members were reviewed in detail. By analyzing the data obtained in the present and previous surveys, the number of patients receiving radiation therapy was estimated to be 4501 (3.7%) in the LSS population and 1026 (5.1%) in the AHS population between 1945-1980. During 1945-1965, 98% of radiation treatments used medium-voltage X rays, and 66% of the treatments were for benign diseases. During 1966-1980, 94% of the radiation treatments were for malignant neoplasms. During this period, 60Co gamma-ray exposure apparatus and high-energy electron accelerators were the prevalent mode of treatment in Hiroshima and in Nagasaki, respectively. The mean frequency of radiation therapy among the LSS population was estimated to have been 158 courses/year during 1945-1965 and 110 courses/year during 1966-1980. The present survey revealed that 377 AHS members received radiation therapy. The number was approximately twice the total number of cases found in the previous two surveys. Thus the data on radiation therapy in the AHS members have been updated well by this survey, and will provide information for more precise dose-response analyses of radiation effects in the AHS population. The information extracted from the medical records of the hospitals consisted of hospital names, treatment periods, body sites treated, diagnoses, treatment doses, radiation sources, the field sizes of the exposures and the data for identification of the patients. These data will be essential in estimating the organ doses from scattered radiation and in evaluating the effects of therapeutic radiation.

Protein turnover in advanced lung cancer patients.

Understanding the extent to which changes in whole-body protein kinetics contribute to the commonly observed weight loss and decrease in lean body mass (LBM) in patients with cancer is currently obscured by conflicting reports in the literature. While several studies have reported significant increases in whole-body protein turnover (WBPT), synthesis (WBPS), and catabolism (WBPC) in patients with cancer, others have failed to confirm these observations. We have measured whole-body protein kinetics using a primed constant infusion of 15N-glycine in a homogenous group of 32 newly diagnosed advanced lung cancer patients with comparable staging and before any antineoplastic treatment, and in 19 normal healthy volunteer controls. Urinary urea and ammonia 15N enrichment was determined in individually collected urine samples obtained during the 24-hour study period and averaged for the determination of protein kinetics. During the last 6 hours of urine collection, samples were obtained hourly for determination of 15N plateau enrichment. Twenty-four-hour urinary nitrogen and creatinine excretion was determined from 24-hour pooled urine samples. Resting metabolic expenditure (RME) was determined by indirect calorimetry and LBM was estimated from deuterium oxide dilution. Age body weight, LBM, RME, and 24-hour urinary nitrogen excretion did not differ between cancer and control subjects. WBPT, WBPC, and WBPS (g/kg/d) were significantly increased in lung cancer patients. However, when the same results were expressed either per kilogram LBM or per gram 24-hour urinary creatinine excretion, WBPT, WBPC, and WBPS rates were not statistically different from those of the controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Corpus callosum surface area across the human adult life span: effect of age and gender.

This magnetic resonance (MR) imaging study analyzed and provides normative data on the midsagittal surface area of the corpus callosum (CC) across the human adult life span in 200 normal volunteers. Using MR imaging scans, CC midsagittal surface area was quantified, along with midsagittal intracranial surface area and total intracranial volume (measures of cranial size). Results demonstrate that when head size is controlled, no major changes associated with normal aging are evident in the CC during the more active years of adult life span (up through 65). Overall, females had significantly larger CC relative to total intracranial volume. Results are discussed in terms of potential gender differences in CC size and the clinical application of this normative data.

Increasing UV detection sensitivity in the supercritical fluid chromatographic analysis of alcohol polyethers.

Alcohol ethoxylates (AEOs) that contain a wide distribution of oligomers pose a challenge for ultraviolet (UV) absorbance detection due to the fact that the AEOs absorb strongly only in the range of commercial UV detectors between 190 and 200 nm. Most mobile phase components, with the exceptions of water and carbon dioxide, also absorb in this region. Ethoxylated hexadecanol and octadecanol were derivatized with disilazane-chlorosilane mixtures for the formation of phenyl containing silylethers. Derivatized samples were analyzed by supercritical fluid chromatography (SFC) coupled with both electrospray ionization mass spectrometry and UV absorbance detection. An increase in the number of phenyl groups incorporated into the derivatives increased the number of oligomers observed by UV detection. An increase in the number of oligomers detected increased the calculated average molar ethoxylate values. The average molar oligomer values calculated by SFC-UV for these alcohols were consistent with the nominal reported values.

Separation of derivatized alcohol ethoxylates and propoxylates by low temperature packed column supercritical fluid chromatography using ultraviolet absorbance detection.

Supercritical fluid chromatography (SFC) is capable of separating oligomers of alcohol ethoxylates (AEOs) and propoxylates (APOs) samples with pure carbon dioxide. The instrumental conditions, however, needed for separation necessitate both high temperature and high pressure. Derivatization of alcohol polyether samples with an UV absorbing agent has been achieved with a phenylated disilazane in hopes of employing a solvent-modified CO2 mobile phase in conjunction with both lower CO2 pressure and lower temperature for oligomer separation. A silylether containing a single phenyl group was formed via the derivatization of the hydroxyl termini of AEO and APO samples. The derivatized polyethers were detected at 215 nm with little or no interference from the mobile phase. Octadecylsilica (ODS) and a polar embedded alkyl bonded silica stationary phase were studied with the organic solvent-modified CO2 mobile phase. The combination of an ODS phase and the polar embedded phase, tandemly stacked, produced the best chromatographic separation of oligomeric species. Data from SFC-UV separations combined with peak assignments from SFC with electrospray ionization-mass spectrometric (ESI-MS) detection produced average molar oligomer values for each surfactant sample.

Determination of alcohol polyether average molar oligomer value/distribution via supercritical fluid chromatography coupled with UV and MS detection.

Supercritical fluid chromatography (SFC) was used for the analysis of ethoxylated and propoxylated surfactants. Samples were derivatized to phenylated silyl ethers with a disilazane-chlorosilane mixture. Addition of a phenyl group to the surfactant allowed UV-absorbance detection of each oligomer. Acetonitrile and methanol were evaluated as mobile phase modifiers. Better peak shape was realized with methanol-modified CO2 on an octadecyl silica bonded phase than with acetonitrile-modified CO2. Peak assignments were made via SFC coupled with electrospray ionization-mass spectrometry (ESI-MS) in the positive ion mode. A sulfonamide-embedded alkyl stationary phase was also evaluated for separation of the derivatized samples. SFC-UV and SFC-ESI-MS data were jointly used for calculation of average molar oligomer values which were then compared to values calculated from 1H NMR data of non-derivatized samples. The derivatization or separation method using the sulfonamide embedded phase required no preliminary cleanup and yielded reproducible oligomer values that were consistent with those of the manufacturer's nominal values.

Glucose metabolism in advanced lung cancer patients.

Although it is generally accepted that altered nutrient intake and metabolism are responsible for the progressive loss of body weight observed in most advanced cancer patients, there is still considerable controversy regarding the contributory role of changes in both resting energy expenditure (REE) and glucose metabolism. Several studies suggest increases in both REE and glucose appearance in advanced cancer patients compared with healthy control subjects, whereas others revealed no changes in either metabolic parameter. We measured REE with indirect calorimetry and glucose kinetics with a primed constant infusion of D-[U-14C]glucose and D-[6-3H]glucose over the last 4 h of a 24-h fast in 32 advanced lung cancer patients immediately after diagnosis and before any chemotherapy or radiotherapy and in 19 healthy volunteer subjects. REE for the lung cancer group was not significantly different from that in the control group (1535.8 +/- 78.0 vs. 1670.2 +/- 53.9 kcal/day, respectively, p = 0.151). When REE was expressed as a function of body weight, or lean body mass, no differences between the two groups were observed. The rate of glucose appearance was 9.88 +/- 0.36 mumol.kg-1.min-1 in the cancer patients and 10.15 +/- 0.53 mumol.kg-1.min-1 in control subjects (p = 0.667), of which 50.4 versus 58.2%, respectively, was oxidized. The amount of glucose recycled was 13.54 +/- 1.22% in cancer patients and 15.08 +/- 0.99% in control subjects (p = 0.394). The amount of VCO2 from direct oxidation of glucose was 23.39 +/- 0.74% in cancer patients and 27.45 +/- 1.36% in control subjects (p = 0.006).(ABSTRACT TRUNCATED AT 250 WORDS)

Stability of postconcussion symptomatology differs between high and low responders and by gender but not by mild head injury status.

A large sample of undergraduates (N = 2,326) were screened for general health. The screen discretely included items assessing postconcussion symptomatology and neurologic history. A subsample of experiment-blind participants whose self-reported postconcussion symptom levels were >0.5 SD below the screening sample mean (low symptomatic; n = 45) or >0.5 SD above the screening sample mean (high symptomatic; n = 53) were re-evaluated for postconcussion symptomatology 3 to 90 days later. The sample included 50 participants who reported history of mild head injury (MHI) and 48 controls. MHI was defined as loss of consciousness of < or =20 minutes within the past 10 years, but no more recent than 3 months. Persons with history of >1 head injury or with other neurologic history were not included in the study. The stability of postconcussion symptoms differed by high symptomatic versus low symptomatic presentation (high symptomatic presentation was more variable) and by gender (females were more variable), but not by actual history of MHI. Sex differences in self-reported postconcussion symptomatology are consistent with gender-related differences in reporting of other physical and psychological symptoms. In addition to demonstrating differences in the stability of self-reported postconcussion symptomatology, the current study provides normative test-retest data for the Postconcussion Symptom Checklist for extreme responders by gender and MHI status.

Quantitative PET scan findings in carbon monoxide poisoning: deficits seen in a matched pair.

Quantitative positron emission tomography (PET) was utilized to establish the degree and localization of central nervous system dysfunction in 2 adult patients 3 years status postchronic carbon monoxide poisoning. The individual PET scans were compared against a composite scan made up of 32 normals using a z transformation statistical parametric map. Neuropsychological findings indicated marked anterior frontal lobe syndrome in the context of far-above-average intelligence, memory, and language. They also showed manifest frontal symptoms in activities of daily living that resulted in vocational disability in each case. PET analysis revealed substantially decreased metabolism in the orbitofrontal and dorsolateral prefrontal cortex as well as in areas of the temporal lobe for each individual. Individual scans were very similar and consistent with patient's presenting symptoms, and changed life circumstances. This report represents the first quantitative functional neuroimaging study relevant to carbon monoxide poisoning.

Incidence rates of salivary gland tumors: results from a population-based study.

Salivary gland tumors are uncommon, and their epidemiology has not been well described. We conducted a descriptive epidemiologic study of parotid, submaxillary, and sublingual gland tumors newly diagnosed in Jefferson County, Alabama, hospitals from 1968 to 1989. Incidence rates were estimated with a population-based subset of cases diagnosed during the years 1979 to 1980, 1983 to 1984, or 1987 to 1988. Among 248 incident cases, 84.3% were benign and 15.7% were malignant. Eighty-six percent of cases arose in the parotid gland, and 14% arose in the submaxillary gland. No sublingual gland tumor was identified. The benign mixed tumor was the most frequent tumor (65.6%), followed by Warthin's tumor (29.2%). Mucoepidermoid carcinoma was the most frequent malignant tumor (51.3%). The average annual age-adjusted incidence rate per 100,000 was 4.7 for benign tumors and 0.9 for malignant tumors. Incidence rates for both benign and malignant tumors increased with age until ages 65 to 74 years and then declined. Benign mixed tumors occurred more frequently in female patients, whereas Warthin's tumors and malignant tumors occurred more frequently in male patients (P < 0.05). Warthin's tumor was rare in black patients (P < 0.001). We conclude that salivary gland tumors are an uncommon but epidemiologically diverse group of tumors. Their causes are also likely to differ.

Psychopharmacology and mental retardation: a 10 year review (1990-1999).

We conducted a 10 year review of the literature pertaining to psychopharmacology and mental retardation. Studies were included or excluded from the review based on meeting one or more of the methodological criteria normally considered fundamental for sound scientific research. The vast majority of studies conducted in the last 10 years in this area had major methodological flaws. While a large number of medications were prescribed for various psychological disorders and behavior problems, most drug administrations were not based in science, were not evaluated appropriately, and generally did not follow best practices for treatment of persons with mental retardation. Very few medications prescribed were behavior or psychiatric symptom specific; that is, most medications were given to suppress a myriad of aberrant behaviors thus chemically restraining the individual in question. Practices such as these present serious problems for service providers due to the deleterious side effects of many psychotropic medications and the federal government's intervention into the care-provision practices of developmental centers, community homes, and other living arrangements for persons with mental retardation. Implications of our review are discussed.

Feasibility of supercritical fluid chromatography/mass spectrometry of polypeptides with up to 40-mers.

Supercritical fluid chromatography (SFC) provides a number of advantages over traditional HPLC such as speed, practical use of longer columns, a normal-phase retention mechanism, and reduced use of organic solvents. Yet, it has been a technique traditionally limited to relatively nonpolar compounds. The nature of SFC mobile and stationary phases did not allow the elution of ionic compounds or of peptides, except, in the latter case, for the most hydrophobic peptides. The characterization of peptides is critically important for drug discovery and development in the pharmaceutical industry, as well as for a variety of other important applications. Here, for the first time to our knowledge, we show that relatively large peptides (at least 40 mers), containing a variety of acidic and basic residues, can be eluted in SFC. We used trifluoroacetic acid as additive in a CO2/methanol mobile phase to suppress deprotonation of peptide carboxylic acid groups and to protonate peptide amino groups. A 2-ethylpyridine bonded silica column, which was specifically developed for SFC, was used for the majority of this work. The relatively simple mobile phase was compatible with mass spectrometric detection.

Comparison of LC/MS and SFC/MS for screening of a large and diverse library of pharmaceutically relevant compounds.

The search for greater speed of analysis has fueled many innovations in high-performance liquid chromatography (HPLC), such as the use of higher pressures and smaller stationary-phase particles, and the development of monolithic columns. Alternatively, one might alter the chromatographic mobile phase. The low viscosity and high diffusivity of the mobile phase in supercritical fluid chromatography (SFC) allows higher flow rates and lower pressure drops than is possible in traditional HPLC. In addition, SFC requires less organic, or aqueous-organic, solvent than LC (important in preparative-scale chromatography) and provides an alternative, normal-phase retention mechanism. But fluids that are commonly used as the main mobile-phase component in SFC, such as CO2, are relatively nonpolar. As a result, SFC is commonly believed to only be applicable to nonpolar and relatively low-polarity compounds. Here we build upon recent work with SFC of polar and ionic compounds and peptides, and we compare the LC/MS and SFC/MS of a diverse library of druglike compounds. A total of 75.0% of the library compounds were eluted and detected by SFC/MS, while 79.4% were eluted and detected by LC/MS. Some samples provided strong peaks that appeared to be related to the purported compound contained in the sample. When these were added to the "hits", the numbers rose to 86.7 and 89.9%, respectively. A total of 3.7% of the samples were observed by SFC/MS, but not by LC/MS, and 8.1% of the samples were observed by LC/MS, but not by SFC/MS. The only compound class that appeared to be consistently detected in LC/MS, but not in SFC/MS under our conditions, consisted of compounds containing a phosphate, a phosphonate, or a bisphosphonate. The SFC/MS method was at least as durable, reliable, and user-friendly as the LC/MS method. The APCI source required less cleaning during the SFC/MS separations than it did during LC/MS.

Human T and B lymphocyte migration under agarose: differences in the characteristics of migrating cells and migration patterns.

The morphologic and migration characteristics of separated human peripheral blood T and B lymphocytes were evaluated by means of an agarose plate technique. The motile T cells migrated in a tightly packed and regularly arranged monolayer, while the B cell monolayer appeared much more disorganized with the cells forming a loose spongy network. Fixed and stained preparations following 2-3 days of incubation showed that the T cells maintained mature lymphocyte features throughout the period of migration. Although many of the B cells were indistinguishable from the T cells, many were irregular in size and shape with monocytic characteristics. It is suggested that the differences in T and B cell in vitro migration patterns may reflect fundamental differences in the mechanisms of locomotion for these cells.