Plasma betaine concentrations correlate with plasma cortisol but not with C-reactive protein in an elderly population.

BACKGROUND: Low plasma betaine concentrations are a feature of seriously ill patients. Increased dietary betaine intake has been associated with lowered systemic inflammation. We aimed to compare plasma cortisol (a stress marker) and C-reactive protein (an inflammation marker) as statistical predictors of plasma betaine concentrations. METHODS: Plasma carnitine, cortisol and C-reactive protein concentrations, other biochemical measures and urine betaine excretion, were compared with plasma betaine concentration by correlation and in multiple regression models, using morning blood and urine samples from 64 ambulant elderly subjects and from 55 patients admitted to hospital with hip fractures. RESULTS: In the ambulant elderly without acute trauma, plasma cortisol (with negative coefficients) and carnitine (with positive coefficients) statistically predicted plasma betaine concentrations. C-reactive protein was not a predictor. In the patients, the significant predictors were plasma carnitine (positive coefficient) and plasma homocysteine (negative coefficient) and C-reactive protein again was not a predictor. In regression models using combined patient and control data there were large ranges of both cortisol and especially C-reactive protein; cortisol and homocysteine (negative coefficients) and carnitine (positive coefficient) were significant predictors but C-reactive protein was not significant. CONCLUSIONS: Stress rather than inflammation may affect plasma betaine concentrations.

Monitoring chloramines and bromamines in a humid environment using selected ion flow tube mass spectrometry.

The selectivity and sensitivity of selected ion flow tube mass spectrometry (SIFT-MS) for individual breath analysis of haloamines has been improved by heating the flow tube in a commercial instrument to around 106 degrees C. Data is presented showing the marked reduction in the number density of water clusters of product ions of common breath metabolites that are isobaric with the product ions from monochloramine and monobromamine that are used to monitor the haloamine concentrations. These results have direct relevance to the real-time monitoring of chloramines in drinking water, swimming pools and food processing plants. However, once the isobaric overlaps from water cluster ions are reduced at the higher temperatures, there is no conclusive evidence showing the presence of haloamines on single breath exhalations in the mid parts per trillion range from examination of the breaths of volunteers.

Validation of (1)H NMR spectroscopy as an analytical tool for methylamine metabolites in urine.

BACKGROUND: Methylamines have many metabolic roles and there is an increasing demand for their measurement. Glycine betaine is an important osmolyte, and a reservoir for methyl groups. Proline betaine and trigonelline are important dietary betaines. Trimethylamine, derived from gut flora, is normally converted to trimethylamine oxide but in 'fish odour syndrome' is excreted as TMA. These compounds are all suitable for quantification by (1)H NMR spectroscopy as they all have methyl protons. METHOD: Urine samples are acidified and (1)H NMR spectra are obtained using presaturation for water suppression. Peak integrals or heights are compared to an internal standard of acetonitrile. RESULTS: Inter- and intra-assay CV's were <5% for TMAO and creatinine, and <10% for the other analytes. Responses were linear from 50 to 1000 microM for all metabolites, and recoveries were > or =97%. Limits of detection using NMR are slightly higher than alternative HPLC assays (15-25 microM). However, sensitivity is adequate for the detection of raised levels in urine, and sample analysis was complete in less than 5 min. CONCLUSION: (1)H NMR spectroscopy is a convenient, rapid and economical option for the determination of betaines and related compounds in urine in a single analysis.

Separation of cationic aracyl derivatives of betaines and related compounds.

Cationic aracyl esters of betaines can be formed by alkylation with aracyl halides or trifluoromethanesulfonates. HPLC on a non-endcapped strong cation exchange (SCX) column gave high retention of these derivatives. Cation exchange HPLC may be carried out on a normal-phase (silica or alumina) column using a polar organic solvent (acetonitrile, propan-2-ol) containing an aqueous buffer with an organic cation and a hydrophilic anion. Selectivity is affected by the choice of organic solvent and buffer, e.g. alcohols decrease the retention times of hydroxybetaines such as carnitine. Retention is reduced by increasing the water content and the buffer concentration. Capillary electrophoresis migration times are affected by the choice of buffer anion, with low pH citrate buffers favoured.

A simple binding assay for the direct determination of biotin in urine.

BACKGROUND: We have devised a simple assay to detect adequate biotin intake, which uses an alternative configuration from most existing assays. METHODS: The assay depends on the competition of streptavidin peroxidase for immobilized biotin or soluble biotin in standards or samples. Immobilized streptavidin peroxidase is detected using tetramethylbenzidine, and the plates are read at 450 nm. The assay was normalised by determining the biotin/creatinine ratio in the urine of healthy adults. Urinary biotin excretion was measured in unsupplemented pregnant women. The half-life of biotin excretion after a single oral supplement was determined for healthy volunteers. RESULTS: Urinary biotin excretion in unsupplemented pregnant women was 2.9+/-1.9 micromol/mol creatinine (mean+/-S.D.) and was significantly lower (p<0.001) than those of healthy males and females, which were 9.0+/-5.4 and 7.0+/-2.1 micromol/mol creatinine (mean+/-S.D.), respectively. The half-life of a single oral biotin supplement was 30-40 h, with excretion returning to basal levels at 70 h. CONCLUSION: We have devised a novel binding assay for the direct determination of total biotin excretion in urine, which is suitable for routine clinical laboratory. The assay is inexpensive, simple, rapid, and could be fully automated.

A high performance liquid chromatographic method for the measurement of total carnitine in human plasma and urine.

Total carnitine in plasma and urine can be measured by high performance liquid chromatography (HPLC) using the novel fluorescent derivatisation reagents 6'-methoxynaphthacyl trifluoromethanesulfonate and 2'-phenanthrenacyl trifluoromethanesulfonate. Sample preparation for total carnitine analysis involves: extraction of plasma and urine in methanol, the optional addition of serine betaine as an internal standard, saponification of acyl carnitines with calcium hydroxide, followed by derivatisation with 6'-methoxynaphthacyl trifluoromethanesulfonate or 2'-phenanthrenacyl trifluoromethanesulfonate. The derivatives were separated using an alumina column and measured by fluorescence detection. The coefficient of variation was below 5% using internal standard calibration, and recoveries of acyl carnitines after saponification were over 90%. The total carnitine method was shown to be linear at biological levels for plasma (over the range 30-130 micromol/l) and urine (over the range 80-180 micromol/l). Advantages of this method include good precision, accuracy and linearity, the use of fluorescence to gain sensitivity, the small sample volume required and a relatively low sample preparation time.

Detection of volatile compounds produced by microbial growth in urine by selected ion flow tube mass spectrometry (SIFT-MS).

Selected ion flow tube-mass spectrometry has been used to measure the volatile compounds occurring in the headspace of urine samples inoculated with common urinary tract infection (UTI)-causing microbes Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumoniae, Enterococcus faecalis, or Candida albicans. This technique has the potential to offer rapid and simple diagnosis of the causative agent of UTIs.

Increased concentrations of breath haloamines are not detectable in airways inflammation using SIFT-MS.

The haloamines, including the chloramines (H(2)NCl, HNCl(2)) and bromamine (H(2)NBr), are diffusible gases that are likely to be produced during inflammation and so may be present as markers of lung inflammation on breath. Although haloamines are quite reactive, it is possible to measure these compounds in humid samples using SIFT-MS. Until recently the quantification of haloamines in breath suffered from interference from other common breath compounds. This was overcome by heating the flow tube which removed major water cluster product ions. Despite the improvements to the method, previous attempts to measure the haloamines in breath samples from normal volunteers had found no evidence to support their presence. Since it is proposed that the haloamines may be present in higher concentrations during airways inflammation we have attempted to detect the compounds in the exhaled breath of patients with airways inflammatory conditions. On-line and off-line breath samples were analyzed; however, there was no discernable change to any of product ions when compared to ambient air or normal subjects. This suggests that despite sensitivity in the mid part per trillion range haloamines are not significantly raised in airways inflammation.

Modelling acute renal failure using blood and breath biomarkers in rats.

This paper compares three methods for estimating renal function, as tested in rats. Acute renal failure (ARF) was induced via a 60-min bilateral renal artery clamp in 8 Sprague-Dawley rats and renal function was monitored for 1 week post-surgery. A two-compartment model was developed for estimating glomerular filtration via a bolus injection of a radio-labelled inulin tracer, and was compared with an estimated creatinine clearance method, modified using the Cockcroft-Gault equation for rats. These two methods were compared with selected ion flow tube-mass spectrometry (SIFT-MS) monitoring of breath analytes. Determination of renal function via SIFT-MS is desirable since results are available non-invasively and in real time. Relative decreases in renal function show very good correlation between all 3 methods (R²=0.84, 0.91 and 0.72 for breath-inulin, inulin-creatinine, and breath-creatinine correlations, respectively), and indicate good promise for fast, non-invasive determination of renal function via breath testing.

Accurate, reproducible measurement of acetone concentration in breath using selected ion flow tube-mass spectrometry.

Using selected ion flow tube-mass spectrometry (SIFT-MS) for on-line analysis, we aimed to define the optimal single-exhalation breathing manoeuvre from which a measure of expired acetone concentration could be obtained. Using known acetone concentrations in vitro, we determined the instrument's accuracy, inter-measurement variability and dynamic response time. Further, we determined the effects of expiratory flow and volume on acetone concentration in the breath of 12 volunteers and calculated intra-individual coefficients of variation (CVs). At acetone concentrations of 600-3000 ppb on 30 days over 2 months there was an instrument measurement bias of 8% that did not change over time, inter-day and intra-day CVs were 5.6% and 0.0%, respectively, and the 10-90% response time was 500 ± 50 ms (mean ± SE). Acetone concentrations at exhalation flows of 193 ± 18 (mean ± SD) and 313 ± 32 ml s(-1) were 619 ± 1.83 (geometric mean ± logSD) and 618 ± 1.82 ppb in the fraction 70-85% by volume of exhaled vital capacity (V(70-85%)) and 636 ± 1.82 (geometric mean ± logSD) and 631 ± 1.83 ppb in V(85-100%). A difference was observed between acetone concentrations in the V(70-85%) and V(85-100%) fractions (p < 0.01), but flow had no effect. Median intra-individual CVs were 1.6-2.6%. On-line SIFT-MS measurement of acetone concentration in a single exhalation requires control of exhaled volume but not flow, and yields low intra-individual CVs and is potentially useful in approximating blood glucose and monitoring metabolic stress.

In vitro SIFT-MS validation of a breath fractionating device using a model VOC and ventilation system.

The measurement of volatile organic compounds in exhaled breath is becoming recognized as a method of disease diagnosis and therapeutic monitoring. The aim of this study was to validate the collection of breath from intubated patients in the intensive care setting. This was done by assembling a system of ventilators and humidification to emulate the human respiratory system. A known concentration of acetone was spiked into the system to simulate alveolar and dead-space 'breath'. Selected-ion flow tube mass spectrometry (SIFT-MS) was used to directly measure gas at two separate points (headspace, distal circuit end) and from Tedlar bags collected using a remote breath fractionator. The mean acetone concentration for headspace, distal circuit end and Tedlar bag concentrations were calculated. The fractionator was effective in separating the early (dead space) and late phases of exhaled breath. Results from the late Tedlar bag samples collected by the remote breath fractionator showed a clear correlation with headspace and distal circuit end acetone concentrations. The collection for remote analysis of breath samples from immobile patients is made possible using the breath fractionator in conjunction with SIFT-MS analysis.