Gas chromatography coupled-to Fourier transform orbitrap mass spectrometer for enantioselective amino acid analyses: Application to pre-cometary organic analog.

Gas chromatography (GC) is a separation technique commonly developed for targeted in situ analyses in planetary space missions. It is coupled with low-resolution mass spectrometry to obtain additional structural information and allow compound identification. However, ground-based analyses of extraterrestrial samples have shown the presence of large molecular diversities. For future targeted in situ analyses, it is therefore essential to develop new technologies. High resolution mass spectrometry (HRMS) is currently being spatialized using FT-orbitrap-MS technology. In this contribution, the coupling of gas chromatography with FT-orbitrap-MS is studied for targeted amino acid analyses. The method for enantioselective separation of amino acids was optimized on a standard mixture comprising 47 amino acid enantiomers. Different ionization modes were optimized, chemical ionization with three different reactive gasses (NH3, CH4 and NH3/CH4) and electron impact ionization at different electron energies. Single ion and full scan monitoring modes were compared, and detection and quantification limits were estimated by internal calibration under the optimized conditions. The GC-FT-orbitrap-MS demonstrated its ability to separate 47 amino acid enantiomers with minimal co-elution. Furthermore, due to the high mass resolution and accuracy of FT-orbitrap-MS, with mass extraction, the S/N is close to zero, allowing average LOD values of 10⁻7 M, orders of magnitude lower than conventional GC-MS techniques. Finally, these conditions were tested for enantioselective analysis of amino acids on an analog of a pre-cometary organic material showing similarities to that of extraterrestrial materials.

Insurance status and mortality among patients with AIDS.

OBJECTIVES: The aim of the study was to evaluate risk factors for mortality, including health care insurance status, among patients with AIDS in the era of modern combination antiretroviral therapy (cART). METHODS: This study was part of the prospective, multicentre, observational Longitudinal Study of the Ocular Complications of AIDS (LSOCA). Patients were classified as having private health care insurance, Medicare, Medicaid, or no insurance. Hazard ratios (HRs) for death were calculated using proportional hazards regression models and staggered entries, anchored to the AIDS diagnosis date. RESULTS: Among 2363 participants with AIDS, 97% were treated with cART. At enrolment, 31% of participants had private insurance, 29% had Medicare, 24% had Medicaid, and 16% were uninsured. Noninfectious, age-related diseases, such as hypertension, diabetes, and renal disease, were more frequent among persons with Medicare than among those with private insurance. Compared with those who were privately insured, mortality was greater among participants with Medicare [adjusted HR (HRadj ) 1.35; 95% confidence interval (CI) 1.08-1.67; P = 0.008]. Among participants with a suppressed HIV viral load, compared with those who were privately insured, HRadj values for mortality were 1.93 (95% CI 1.08-3.44; P = 0.02) for those with Medicare and 2.09 (95% CI 1.02-4.27; P = 0.04) for those with Medicaid. Mortality among initially uninsured participants was not significantly different from that for privately insured participants, but these participants typically obtained ART and insurance during follow-up. Compared with privately insured participants, time-updated HRadj values for mortality were 1.34 (95% CI 1.05-1.70; P = 0.02) for those with Medicare, 1.34 (95% CI 1.01-1.80; P = 0.05) for those with Medicaid, and 1.35 (95% CI 0.97-1.88; P = 0.05) for those who were uninsured. CONCLUSIONS: In persons with AIDS, compared with those with private insurance, those with public insurance had increased mortality, possibly as a result of a greater burden of noninfectious, age-related diseases.

Attenuated kallikrein-related peptidase activity disrupts desquamation and leads to stratum corneum thickening in human skin equivalent models.

BACKGROUND: Epidermal homeostasis is maintained through the balance between keratinocyte proliferation, differentiation and desquamation; however, human skin equivalent (HSE) models are known to differentiate excessively. In native tissue, proteases such as kallikrein-related peptidase (KLK) 5 and KLK7 cleave the extracellular components of corneodesmosomes; proteins corneodesmosin, desmocollin 1 and desmoglein 1, loosening the cellular connections and enabling desquamation. The actions of KLK7 are tightly controlled by protease inhibitors, skin-derived antileucoproteinase (SKALP) and lymphoepithelial Kazal-type-related inhibitor (LEKTI), which also inhibits KLK5, localizing protease activity to the stratum corneum. OBJECTIVES: To investigate the mechanisms that inhibit the desquamation cascade in HSE models. METHODS: Human skin tissue and HSE models were investigated using gene microarray, real-time polymerase chain reaction (PCR), immunohistochemistry and Western blot analysis to examine key components of the desquamation pathway. To elucidate proteolytic activity in HSEs and native skin, in situ and gel zymography was performed. RESULTS: Histological analysis indicated that HSE models form a well-organized epidermis, yet develop an excessively thick and compact stratum corneum. Gene microarray analysis revealed that the desquamation cascade was dysregulated in HSE models and this was confirmed using real-time PCR and immunohistochemistry. Immunohistochemistry and Western blot indicated overexpression of LEKTI and SKALP in HSEs. Although KLK7 was also highly expressed in HSEs, zymography indicated that protease activation and activity was lower than in native skin. CONCLUSIONS: These findings demonstrate that stratum corneum thickening is due to inhibited KLK5 and KLK7 activation and a subsequent lack of corneodesmosome degradation in the HSE model epidermis.

Anisotropy spectra for enantiomeric differentiation of biomolecular building blocks.

All biopolymers are composed of homochiral building blocks, and both D-sugars and L-amino acids uniquely constitute life on Earth. These monomers were originally enantiomerically differentiated under prebiotic conditions. Particular progress has recently been made in support of the photochemical model for this differentiation: the interaction of circularly polarized light with racemic molecules is currently thought to have been the original source for life's biological homochirality. The differential asymmetric photoreactivity of particular small molecules can be characterized by both circular dichroism and anisotropy spectroscopy. Anisotropy spectroscopy, a novel derivative of circular dichroism spectroscopy, records the anisotropy factor g = Δε/ε as a function of the wavelength. Anisotropy spectroscopy promisingly affords the wavelength-dependent determination of the enantiomeric excess (ee) inducible into chiral organic molecules by photochemical irradiation with circularly polarized light. Anisotropy spectra of small molecules therefore provide unique means for characterizing the different photochemical behaviors between enantiomers upon exposure to various wavelengths of circularly polarized light. This chapter will: (1) present the theory and configuration of anisotropy spectroscopy; (2) explain experimentally recorded anisotropy spectra of selected chiral biomolecules such as amino acids; and (3) discuss the relevance of these spectra for the investigation of the origin of the molecular homochirality observed in living organisms. This review describes a new chiroptical technique that is of significance for advances in asymmetric photochemistry and that is also highly relevant for the European Space Agency Rosetta Mission, which will determine enantiomeric excesses (ees) in chiral organic molecules in cometary ices when it lands on Comet 67P/Churyumov-Gerasimenko in November 2014.

The use of MS classifiers and structure generation to assist in the identification of unknowns in effect-directed analysis.

Structure generation and mass spectral classifiers have been incorporated into a new method to gain further information from low-resolution GC-MS spectra and subsequently assist in the identification of toxic compounds isolated using effect-directed fractionation. The method has been developed for the case where little analytical information other than the mass spectrum is available, common, for example, in effect-directed analysis (EDA), where further interpretation of the mass spectra is necessary to gain additional information about unknown peaks in the chromatogram. Structure generation from a molecular formula alone rapidly leads to enormous numbers of structures; hence reduction of these numbers is necessary to focus identification or confirmation efforts. The mass spectral classifiers and structure generation procedure in the program MOLGEN-MS was enhanced by including additional classifier information available from the NIST05 database and incorporation of post-generation 'filtering criteria'. The presented method can reduce the number of possible structures matching a spectrum by several orders of magnitude, creating much more manageable data sets and increasing the chance of identification. Examples are presented to show how the method can be used to provide 'lines of evidence' for the identity of an unknown compound. This method is an alternative to library search of mass spectra and is especially valuable for unknowns where no clear library match is available.

Naproxen and celecoxib do not prevent AD in early results from a randomized controlled trial.

OBJECTIVE: To evaluate the efficacy and safety of naproxen and celecoxib for the primary prevention of Alzheimer disease (AD). METHODS: Randomized, placebo-controlled, double-masked clinical trial conducted at six US dementia research clinics. Volunteers aged 70+ years, with cognitive screening scores above designated cut-offs and a family history of AD, were randomly assigned to celecoxib 200 mg BID, naproxen sodium 220 mg BID, or placebo. Enrollment began in early 2001. The main outcome measure was diagnosis of AD after randomization. RESULTS: On December 17, 2004, treatments were suspended. Events while on treatment yielded hazard ratios vs placebo of 1.99 (95% CI 0.80 to 4.97; p = 0.14) for celecoxib and 2.35 (0.95 to 5.77; p = 0.06) for naproxen. Imperfect screening measures led to enrollment of 7 individuals with dementia and 46 others with milder cognitive syndromes. Their (prevalent) illness was detected at enrollment and diagnosed within 6 months following randomization. Secondary analyses that excluded the 7 cases of prevalent dementia showed increased hazard ratios for AD with both treatments. Neither treatment produced a notable effect on the incidence of milder cognitive syndromes. CONCLUSIONS: These results do not support the hypothesis that celecoxib or naproxen prevent Alzheimer dementia, at least within the early years after initiation of treatment. Masked long-term follow-up of these participants will be essential.

Factors affecting attrition in a longitudinal study of patients with AIDS.

Anecdotal data have suggested that retention of HIV-infected patients with immune recovery in longitudinal studies may be difficult as they resume normal activities. This study evaluated risk factors for attrition among patients with AIDS in a cohort study in the era of highly active antiretroviral therapy. Patients with AIDS enrolled in the Longitudinal Study of Ocular Complications of AIDS were evaluated every three months with demographic, clinical and laboratory data collected. Lost to follow-up was defined as any patient who missed all study visits and could not be contacted for 12 consecutive months, who had not died and who did not re-enter the study at a later date. Of the 1,052 patients studied, 77 (7.3%) were lost to follow-up (rate = 0.03/person year). In the multivariate analysis, factors associated with attrition were CD4+ T-cell count category (hazard ratio (HR) = 2.03; 95%CI: 1.01, 4.24; P = 0.05 for CD4+ count < or = 50 cells/microL and HR = 1.96; 95%CI: 1.12, 3.40; P = 0.02 for CD4+ count 51-200 cells/microL) and detectable HIV viral load (HR = 1.29; 95%CI: 1.07, 1.53; P < 0.001 for HIV viral load >400 copies/mL). These data suggest that patients with compromised immunologic status are at an increased risk for being lost to follow-up.

Double-masked randomized trial comparing alternate combinations of intraoperative anesthesia and postoperative analgesia in abdominal aortic surgery.

BACKGROUND: Improvement in patient outcome and reduced use of medical resources may result from using epidural anesthesia and analgesia as compared with general anesthesia and intravenous opioids, although the relative importance of intraoperative versus postoperative technique has not been studied. This prospective, double-masked, randomized clinical trial was designed to compare alternate combinations of intraoperative anesthesia and postoperative analgesia with respect to postoperative outcomes in patients undergoing surgery of the abdominal aorta. METHODS: One hundred sixty-eight patients undergoing surgery of the abdominal aorta were randomly assigned to receive either thoracic epidural anesthesia combined with a light general anesthesia or general anesthesia alone intraoperatively and either intravenous or epidural patient-controlled analgesia postoperatively (four treatment groups). Patient-controlled analgesia was continued for at least 72 h. Protocols were used to standardize perioperative medical management and to preserve masking intraoperatively and postoperatively. A uniform surveillance strategy was used for the identification of prospectively defined postoperative complications. Outcome evaluation included postoperative hospital length of stay, direct medical costs, selected postoperative morbidities, and postoperative recovery milestones. RESULTS: Length of stay and direct medical costs for patients surviving to discharge were similar among the four treatment groups. Postoperative outcomes were similar among the four treatment groups with respect to death, myocardial infarction, myocardial ischemia, reoperation, pneumonia, and renal failure. Epidural patient-controlled analgesia was associated with a significantly shorter time to extubation (P = 0.002). Times to intensive care unit discharge, ward admission, first bowel sounds, first flatus, tolerating clear liquids, tolerating regular diet, and independent ambulation were similar among the four treatment groups. Postoperative pain scores were also similar among the four treatment groups. CONCLUSIONS: In patients undergoing surgery of the abdominal aorta, thoracic epidural anesthesia combined with a light general anesthesia and followed by either intravenous or epidural patient-controlled analgesia, offers no major advantage or disadvantage when compared with general anesthesia alone followed by either intravenous or epidural patient-controlled analgesia.

Photographic measures of cytomegalovirus retinitis as surrogates for visual outcomes in treated patients.

OBJECTIVE: To evaluate photographic measures of cytomegalovirus (CMV) retinitis as surrogate outcomes for changes in vision in patients with CMV retinitis related to the acquired immunodeficiency syndrome. METHODS: Data from 3 clinical trials of CMV retinitis treatments were analyzed. Two photographic assessments of retinitis in eyes involved at baseline were evaluated: progression (lesion border movement > or = 750 microm or occurrence of a new lesion) and change in area of retina involved with retinitis. Vision measures were decline in best-corrected visual acuity and change in visual field. Photographic measures were evaluated as surrogate outcomes based on 4 criteria: (1) association with vision measure; (2) ability to account for treatment-related differences in vision measure; (3) data completeness; and (4) sample size requirements. RESULTS: Data from 1001 involved eyes (666 patients) were analyzed. Progression and change in area involved were predictive of declines in vision measures, accounted for 50% and 66% of the treatment effect on visual field, and were available from 93% and 64% of involved eyes, respectively. Sample size estimates for a clinical trial were smallest with progression as the design outcome. CONCLUSION: Progression and change in area involved met the first and second criteria for surrogate outcomes for visual field loss; a complete evaluation for visual acuity decline was not possible because treatment-related differences were not observed. Progression met the logistical and sample size criteria better than change in area of retina involved with retinitis.

Estimation of gender bias in clinical trials.

The perception is that the clinical trials enterprise has been biased in favour of males by devoting a disproportionate effort to males and to the diseases and conditions afflicting them - a perception reinforced by a few high profile male-only heart trials undertaken in the 1970s and 1980s. The perception was sufficient to cause the U.S.A. Congress to enact legislation to require that a clinical trial 'is designed and carried out in a manner sufficient to provide for a valid analysis of whether the variables being studied in the trial affect women ...differently than other subjects in the trial'. Observed effort differentials are based on counts of single-gender trials indexed in MEDLINE and published in U.S. journals. Differentials are compared to those expected using male-female differentials in mortality and years of potential life loss due to mortality before age 65 to estimate effort bias. The ratios of female-only to male-only published trials were 0.53, 0.89 and 0.95 for the decades of 1966-1975, 1976-1985 and 1986-1995, respectively. The expected ratios, if single-gender trials were done in proportion to female-male mortality differentials, would be 0.57, 0.56 and 0.57, respectively. The differences in observed versus expected female to male ratios correspond to a slight excess of male-only trials in the decade of 1966-1975 and to sizeable excesses in female-only trials in the decades of 1976-1985 and 1986-1995. The results do not support the perception that women have been understudied relative to males in clinical trials. Most differentials favour females, whether based on mortality or years of potential life loss due to mortality before age 65 years.

Patient notification and follow-up after suspension of treatment protocols. experience from four clinical trials of treatments for AIDS-related CMV retinitis.

We reviewed procedures for and data about patient notification of the suspension of the treatment protocol for four clinical trials. We also examined how data collected after the suspensions were used. All four trials were designed to evaluate treatments for cytomegalovirus retinitis in patients with AIDS and were conducted by the Studies of Ocular Complications of AIDS Research Group. Documentation that patients were notified of the results varied from 62--100% with three of the four studies documenting the process for > or =92% of patients. The median time between the recommendation for protocol suspension and patient notification varied from 7--49 days with three of the four trials at 12 days or fewer. Most of the analyses of data collected after the suspension of the treatment protocol did not focus on comparisons among treatment groups. Although they provided useful information about the long-term outcomes and the nature of the disease, they did not alter the conclusions about safety and efficacy from the randomized portion of the trial. Control Clin Trials 2001;22:62-68

Gender representation in trials.

The perception is that women have been understudied relative to men. It has been sufficient to cause Congress to enact legislation to require that a clinical trial must be "designed and carried out in a manner sufficient to provide for a valid analysis of whether the variables being studied in the trial affect women ellipsis differently than other subjects in the trial." We looked for evidence as to whether the perception has a basis in fact by looking at measures of gender-based research effort. Clinical trials, published between 1966 and 1998 in U.S. journals and indexed in MEDLINE, were classified by gender. Reports of trials appearing in five widely circulated medical journals (Annals of Internal Medicine, British Medical Journal, Journal of the American Medical Association, Lancet, and New England Journal of Medicine) in 1985, 1990, and 1995 were retrieved and read to obtain counts of the numbers of males and females represented in trials published in those journals. For reports of trials published in U.S. journals, the percent involving males and females, males only, females only, and those where gender was not specified were 55.2%, 12.2%, 11.2%, and 21.4%, respectively. Counts of males and females represented in the reports of trials appearing in the five aforementioned journals were 355,624 and 550,743, respectively. We did not find evidence of systematic effort bias against females.

Clinical review 115: effect of thyroxine therapy on serum lipoproteins in patients with mild thyroid failure: a quantitative review of the literature.

The objective of our study was to estimate the expected change in serum lipoprotein concentrations after treatment with T4 in patients with mild thyroid failure (i.e. subclinical hypothyroidism). Our data sources included MEDLINE, between January 1966 and May 1999, and review of references from relevant articles. There were 1,786 published studies identified, 461 abstracts reviewed, 74 articles retrieved, 24 articles evaluated against predetermined entry criteria, and 13 studies systematically reviewed and abstracted. All studies reported serum total cholesterol concentration changes during T4 treatment, 12 reported triglyceride changes, 10 reported high-density lipoprotein (HDL) cholesterol changes, and 9 reported low-density lipoprotein (LDL) cholesterol changes. There were 247 patients in 13 studies. The mean decrease in the serum total cholesterol concentration was -0.20 mmol/L (-7.9 mg/ dL), with a 95% confidence interval of -0.09 to -0.34. The decline in serum total cholesterol was directly proportional to its baseline concentration. Studies enrolling hypothyroid participants receiving suboptimal T4 doses reported significantly larger decreases in serum total cholesterol after thyroid-stimulating hormone normalization than studies enrolling previously untreated individuals with mild thyroid failure [-0.44 mmol/L (-17 mg/dL) vs. -0.14 mmol/L (-5.6 mg/dL), P = 0.05]. The change in serum LDL cholesterol concentration was -0.26 mmol/L (-10 mg/dL), with a 95% confidence interval of -0.12 to -0.41. Serum HDL and triglyceride concentrations showed no change. These results, although based on fewer than 250 patients, suggest that T4 therapy in individuals with mild thyroid failure lowers mean serum total and LDL cholesterol concentrations. The reduction in serum total cholesterol may be larger in individuals with higher pretreatment cholesterol levels and in hypothyroid individuals taking suboptimal T4 doses. There do not seem to be significant effects of T4 on serum HDL or triglyceride concentrations.

Risk factors for advancement of cytomegalovirus retinitis in patients with acquired immunodeficiency syndrome. Studies of Ocular Complications of AIDS Research Group.

OBJECTIVE: To identify ocular and systemic factors that predict advancement of cytomegalovirus (CMV) retinitis during treatment. METHODS: Patients with acquired immunodeficiency syndrome were enrolled in a multicenter clinical trial designed to evaluate foscarnet sodium and ganciclovir sodium as therapy for newly diagnosed CMV retinitis. Ocular characteristics at baseline and measurements of retinitis were assessed from fundus photographs by graders at a fundus photograph reading center. The following measures of advancement were assessed: (1) lesion border movement of at least 750 microm or development of a new lesion in involved eyes; (2) rate of increase in retinal area with CMV in involved eyes; and (3) development of retinitis in uninvolved eyes of patients with unilateral disease at baseline. RESULTS: In eyes with retinitis, risk factors at baseline for advancement while receiving treatment included smaller area involved, active margins of retinitis, and posterior location. Risk factors for development of retinitis in uninvolved fellow eyes included blood and urine cultures positive for CMV and lower CD8(+) T-lymphocyte count. CONCLUSIONS: Lesion characteristics can be used to predict advancement of preexisting disease, whereas only systemic factors are associated with development of bilateral disease. These analyses describe retinitis activity before the introduction of potent antiretroviral therapies but provide an important reference point for patients in whom CMV retinitis develops after failure or intolerance of antiretroviral agents. Arch Ophthalmol. 2000;118:1196-1204

The refractive status and vision profile: a questionnaire to measure vision-related quality of life in persons with refractive error.

OBJECTIVE/BACKGROUND: To describe the Refractive Status and Vision Profile (RSVP), a questionnaire that measures self-reported vision-related health status (symptoms, functioning, expectations, concern) in persons with refractive error. DESIGN: Cross-sectional study by survey. PARTICIPANTS: The RSVP was self-administered by 550 participants with refractive error (or history of refractive surgery) recruited from five refractive surgery practices and one optometric practice. Information on refraction, uncorrected and best-corrected visual acuity, and history of refractive surgery was obtained from physicians' records. METHODS: Internal consistency, test-retest reliability, agreement with global measures of vision (criterion validity), discriminant validity, content validity, and construct validity (associations of scale scores with patient status variables) were assessed using Cronbach's alpha, Spearman rank correlations, factor analysis, and multitrait analysis. OUTCOME MEASURES: Scores on the overall RSVP scale (S) and on eight RSVP subscales (functioning, driving, concern, expectations, symptoms, glare, optical problems, problems with corrective lenses) were calculated based on 42 items. RESULTS: Cronbach's alpha was 0.92 for S and ranged from 0.70 to 0.93 for RSVP subscales, indicating good internal consistency. Satisfaction with vision was more strongly associated with S than with refractive error or with visual acuity. Individuals with more refractive error had significantly lower (worse) scores for S and for subscales concern, functioning, driving, optical problems, and glare. Scores for S and for subscales concern, functioning, optical problems, and driving remained significantly associated with satisfaction with vision after adjustment for age, gender, corrective lens type, and refractive error. CONCLUSIONS: The RSVP measures a range of visual, functional, and psychologic impacts of refractive error that are likely to be important to patients. The RSVP would be a useful tool for evaluating interventions for correction of refractive error and may be useful for assessing refractive surgery candidates in clinical practice.

FDA censoring of manufacturers' postmarketing commitments in HIV/AIDS drug approval letters.

CONTEXT: Changing evidentiary standards and partial shift of the investigational phase of drug approval process to the postmarketing phase. OBJECTIVE: To determine the availability of information for independent researchers needed to examine accelerated drug approvals to determine how they differ from traditional drug approvals in the HIV/AIDS domain. DESIGN: Identification of all approved HIV/AIDS and AIDS-related conditions drugs between 1987 and 1999. Follow-up of postmarketing study requirements in the approval letters addressed to the manufacturers. SETTING: Accelerated approval has been expanded to other disease conditions in the past decade. INTERVENTION: Request of approval letters from the U.S. Food and Drug Administration for 76 regulatory actions including expanded access and accelerated and traditional approvals for 42 drugs under the Freedom of Information Act (FOIA) between September 1998 and October 1999. MAIN OUTCOME MEASURE(S): Obtainability of approval letters and uncensored postmarketing study requirements. RESULTS: Fifty-five approval letters were received. Postmarketing study commitments of manufacturers were censored in 25 letters received. We were unable to obtain uncensored copies of those approval letters as of May 2000. Censoring was associated with whether (1) the Prescription Drug User Fee Act of 1992 was applicable to the drug (odds ratio (OR) = 5.7, 95% confidence interval (CI) = 1.4- 23.7) and (2) the new drug application was for a new molecular entity or new drug formulation (OR = 4.2, 95% CI = 1.3 -13.6). CONCLUSIONS: Continued secrecy may stifle independent research and hinder health care providers and patients in making informed decisions.

Redesign of trials under different enrollment mixes.

A few large multi-centre male-only heart trials done in the 1970s and 1980s have been seen as ill-conceived because they did not include females. The purpose here is to revisit two of those trials and to consider consequences in terms of cost and power had they been designed to include females.