Primed, constant infusion with [H-2(3)]serine allows in vivo kinetic measurement of serine turnover, homocysteine remethylation, and transsulfuration processes in human one-carbon metabolism

Background: One-carbon metabolism involves both mitochondrial and cytosolic forms of folate-dependent enzymes in mammalian cells, but few in vivo data exist to characterize the biochemical processes involved.

Objective: We conducted a stable-isotopic investigation to determine the fates of exogenous serine and serine-derived one carbon units in homocysteine remethylation in hepatic and whole-body metabolism.

Design: A healthy man aged 23 y was administered [2,3,3 H-2(3)]serine and [5,5,5-H-2(3)]leucine by intravenous primed, constant infusion. Serial plasma samples were analyzed to determine the isotopic enrichment of free glycine, serine, leucine, methionine, and cystathionine. VLDL apolipoprotein B-100 served as an index of liver free amino acid labeling.

Results: [H-2(1)]Methionine and [H-2(2)]methionine were labeled through homocysteine remethylation. We propose that [H-2(2)]methionine occurs by remethylation with [H-2(2)]methyl groups (as 5-methyltetrahydrofolate) formed only from cytosolic processing of [H-2(3)]serine, whereas [H-2(1)]methionine is formed with labeled one-carbon units from mitochondrial oxidation of C-3 serine to [H-2(1)]formate to yield cytosolic [H-2(1)]methyl groups. The labeling pattern of cystathionine formed from homocysteine and labeled serine suggests that cystathionine is derived mainly from a serine pool different from that used in apolipoprotein B-100 synthesis.

Conclusions: The appearance of both [H-2(1)]- and [H-2(2)]methionine forms indicates that both cytosolic and mitochondrial metabolism of exogenous serine generates carbon units in vivo for methyl group production and homocysteine remethylation. This study also showed the utility of serine infusion and indicated functional roles of cytosolic and mitochondrial compartments in one-carbon metabolism.

Utilisation of a novel ProteaseTag™ activity immunoassay for the specific measurement of neutrophil elastase in patients with Cystic Fibrosis

Neutrophil elastase (NE), a biomarker of infection and inflammation, correlates with the severity of several respiratory diseases including cystic fibrosis (CF) however, its detection and quantification in biological samples is confounded by a lack of robust methodologies. Standard assays using chromogenic or fluorogenic substrates are not specific when added to complex samples containing multiple proteolytic and hydrolytic enzymes, resulting in an over-estimation of the target protease. ELISA systems measure total protein levels which can be a mixture of latent, active and protease-inhibitor complexes. We have therefore developed a novel immunoassay (NE-Tag ELISA), incorporating an activity dependent ProteaseTag™ and a specific antibody step, which is selective and specific for the capture of active NE. The objective of this study was to clinically validate NE-Tag ELISA for the detection of active NE in sputum from CF patients. Sputum (n=45) was recovered from CF patients hospitalised for acute exacerbation. Sol was recovered and analysed for NE activity using the NE-Tag ELISA and two fluorogenic substrate-based assays [1. Suc-AAPV-AMC (Sigma) and 2. InnozymeTM Immunocapture assay (Calbiochem)]. NE activity between assays and with a range of clinical parameters was correlated.A highly significant correlation was shown between assays. NE activity (NE-Tag) further correlated appropriately with clinical parameters: inversely with FEV1 (p = 0.036) and positively with CRP (p = 0.035), neutrophils and total white cell counts (p < 0.001). The InnozymeTM assay showed similar correlations with the clinical parameters (with the exception of CRP). No correlations with any of the clinical parameters were observed when NE was measured using the standard fluorogenic substrate.

Exhaled breath temperature measurement and asthma control in children prescribed inhaled corticosteroids: A cross sectional study

BACKGROUND: Exhaled breath temperature (EBT) reflects airways (both eosinophilic and neutrophilic) inflammation in asthma and thus may aid the management of children with asthma that are treated with anti-inflammatory drugs. A new EBT monitor has become available that is cheap and easy to use and may be a suitable monitoring device for airways inflammation. Little is known about how EBT relates to asthma treatment decisions, disease control, lung function, or other non-invasive measures of airways inflammation, such as exhaled nitric oxide (ENO).

OBJECTIVE: To determine the relationships between EBT and asthma treatment decision, current control, pulmonary function, and ENO.

METHODS: Cross-sectional prospective study on 159 children aged 5-16 years attending a pediatric respiratory clinic. EBT was compared with the clinician's decision regarding treatment (decrease, no change, increase), asthma control assessment (controlled, partial, uncontrolled), level of current treatment (according to British Thoracic Society guideline, BTS step), ENO, and spirometry.

RESULTS: EBT measurement was feasible in the majority of children (25 of 159 could not perform the test) and correlated weakly with age (R = 0.33, P = <0.01). EBT did not differ significantly between the three clinician decision groups (P = 0.42), the three asthma control assessment groups (P = 0.9), or the current asthma treatment BTS step (P = 0.57).

CONCLUSIONS & CLINICAL IMPLICATIONS: EBT measurement was not related to measures of asthma control determined at the clinic. The routine intermittent monitoring of EBT in children prescribed inhaled corticosteroids who attend asthma clinics cannot be recommended for adjusting anti-inflammatory asthma therapy.

A proposed simple method for measurement in the anterior chamber angle: biometric gonioscopy

OBJECTIVE:

To design a system of gonioscopy that will allow greater interobserver reliability and more clearly defined screening cutoffs for angle closure than current systems while being simple to teach and technologically appropriate for use in rural Asia, where the prevalence of angle-closure glaucoma is highest.

DESIGN:

Clinic-based validation and interobserver reliability trial.

PARTICIPANTS:

Study 1: 21 patients 18 years of age and older recruited from a university-based specialty glaucoma clinic; study 2: 32 patients 18 years of age and older recruited from the same clinic.

INTERVENTION:

In study 1, all participants underwent conventional gonioscopy by an experienced observer (GLS) using the Spaeth system and in the same eye also underwent Scheimpflug photography, ultrasonographic measurement of anterior chamber depth and axial length, automatic refraction, and biometric gonioscopy with measurement of the distance from iris insertion to Schwalbe's line using a reticule based in the slit-lamp ocular. In study 2, all participants underwent both conventional gonioscopy and biometric gonioscopy by an experienced gonioscopist (NGC) and a medical student with no previous training in gonioscopy (JK).

MAIN OUTCOME MEASURES:

Study 1: The association between biometric gonioscopy and conventional gonioscopy, Scheimpflug photography, and other factors known to correlate with the configuration of the angle. Study 2: Interobserver agreement using biometric gonioscopy compared to that obtained with conventional gonioscopy.

RESULTS:

In study 1, there was an independent, monotonic, statistically significant relationship between biometric gonioscopy and both Spaeth angle (P = 0.001, t test) and Spaeth insertion (P = 0.008, t test) grades. Biometric gonioscopy correctly identified six of six patients with occludable angles according to Spaeth criteria. Biometric gonioscopic grade was also significantly associated with the anterior chamber angle as measured by Scheimpflug photography (P = 0.005, t test). In study 2, the intraclass correlation coefficient between graders for biometric gonioscopy (0.97) was higher than for Spaeth angle grade (0.72) or Spaeth insertion grade (0.84).

CONCLUSION:

Biometric gonioscopy correlates well with other measures of the anterior chamber angle, shows a higher degree of interobserver reliability than conventional gonioscopy, and can readily be learned by an inexperienced observer.

Strategies to improve the accuracy of vision measurement by teachers in rural Chinese secondary schoolchildren: Xichang Pediatric Refractive Error Study (X-PRES) report no. 6.

OBJECTIVE: To assess and improve the accuracy of lay screeners compared with vision professionals in detecting visual impairment in secondary schoolchildren in rural China. METHODS: After brief training, 32 teachers and a team of vision professionals independently measured vision in 1892 children in Xichang. The children also underwent vision measurement by health technicians in a concurrent government screening program. RESULTS: Of 32 teachers, 28 (87.5%) believed that teacher screening was worthwhile. Sensitivity (93.5%) and specificity (91.2%) of teachers detecting uncorrected presenting visual acuity of 20/40 or less were better than for presenting visual acuity (sensitivity, 85.2%; specificity, 84.8%). Failure of teachers to identify children owning but not wearing glasses and teacher bias toward better vision in children wearing glasses explain the worse results for initial vision. Wearing glasses was the student factor most strongly predictive of inaccurate teacher screening (P < .001). The sensitivity and specificity of the government screening program detecting low presenting visual acuity were 86.7% and 28.7%, respectively. CONCLUSIONS: Teacher vision screening after brief training can achieve accurate results in this setting, and there is support among teachers for screening. Screening of uncorrected rather than presenting visual acuity is recommended in settings with a high prevalence of corrected and uncorrected refractive error. Low specificity in the government program renders it ineffective.