Polyethylene glycol as an indigestible marker to estimate fecal output in dairy cows.

The objective of this study was to evaluate the accuracy of fecal output measurements using polyethylene glycol (PEG) as an external marker determined by near-infrared reflectance spectroscopy. In addition, the accuracy of dry matter intake predictions based on fecal output and digestibility estimated using an internal marker [indigestible neutral detergent fiber (iNDF)] was assessed. The experiment was conducted using 6 lactating dairy cows fed 2 different diets. Polyethylene glycol was administered twice daily into the rumen and the diurnal pattern of fecal concentrations and recovery in feces were determined. To evaluate the effects of alternative marker administration and sampling schemes on fecal output estimates, the passage kinetics of PEG in the digestive tract of dairy cows was determined and used for simulation models. The results indicate that PEG was completely recovered in feces and, thus, fecal output was accurately estimated using PEG. Good agreement between measured and predicted dry matter intake (standard error of prediction = 0.86 kg/d, R2 = 0.81) indicates good potential to determine feed intake using PEG in combination with iNDF. The precision of cow-specific digestibility estimates based on iNDF was unsatisfactory, but for a group of cows iNDF provided an accurate estimate of dry matter digestibility. The current study indicated that, to overcome inherent day-to-day variation in feed intake and fecal output, the minimum of 4 fecal spot samples should be collected over 4 d. Preferably, these samples should be distributed evenly over the 12-h marker administration interval to compensate for the circadian variation in fecal PEG concentrations.

Cow-specific diet digestibility predictions based on near-infrared reflectance spectroscopy scans of faecal samples.

This study was designed to obtain information on prediction of diet digestibility from near-infrared reflectance spectroscopy (NIRS) scans of faecal spot samples from dairy cows at different stages of lactation and to develop a faecal sampling protocol. NIRS was used to predict diet organic matter digestibility (OMD) and indigestible neutral detergent fibre content (iNDF) from faecal samples, and dry matter digestibility (DMD) using iNDF in feed and faecal samples as an internal marker. Acid-insoluble ash (AIA) as an internal digestibility marker was used as a reference method to evaluate the reliability of NIRS predictions. Feed and composite faecal samples were collected from 44 cows at approximately 50, 150 and 250 days in milk (DIM). The estimated standard deviation for cow-specific organic matter digestibility analysed by AIA was 12.3 g/kg, which is small considering that the average was 724 g/kg. The phenotypic correlation between direct faecal OMD prediction by NIRS and OMD by AIA over the lactation was 0.51. The low repeatability and small variability estimates for direct OMD predictions by NIRS were not accurate enough to quantify small differences in OMD between cows. In contrast to OMD, the repeatability estimates for DMD by iNDF and especially for direct faecal iNDF predictions were 0.32 and 0.46, respectively, indicating that developing of NIRS predictions for cow-specific digestibility is possible. A data subset of 20 cows with daily individual faecal samples was used to develop an on-farm sampling protocol. Based on the assessment of correlations between individual sample combinations and composite samples as well as repeatability estimates for individual sample combinations, we found that collecting up to three individual samples yields a representative composite sample. Collection of samples from all the cows of a herd every third month might be a good choice, because it would yield a better accuracy.

Determination of selenium in freshwaters by cathodic stripping voltammetry after UV irradiation.

An analytical method was developed for the determination of total dissolved selenium in fresh waters, using linear sweep cathodic stripping voltammetry (CSV) in combination with UV photolytic digestion. Both the CSV method, based on the electrodeposition and stripping of Cu(2)Se, and the UV irradiation procedure were investigated in detail. In the presence of dissolved organic substances, as in freshwaters, Se(VI) is reduced to Se(IV) by UV irradiation in 0.1M hydrochloric acid. Glucose can be used as the carbon source in samples low in natural dissolved organic carbon (DOC). The photolytic yields of Se(IV) were about 90% in both cases. Five freshwater samples were analysed for total selenium by CSV after UV photolysis, and by hydride generation atomic absorption spectrometry (HG-AAS) after oxidative digestion followed by reduction with hydrochloric acid. The results agreed well and the concentrations were in the range 70-190 ng/l., well above the detection limit of the CSV method at 2 ng/l.

Comparison of in vitro and in situ methods in evaluation of forage digestibility in ruminants.

The objective of this study was to compare the application of different in vitro and in situ methods in empirical and mechanistic predictions of in vivo OM digestibility (OMD) and their associations to near-infrared reflectance spectroscopy spectra for a variety of forages. Apparent in vivo OMD of silages made from alfalfa (n = 2), corn (n = 9), corn stover (n = 2), grass (n = 11), whole crops of wheat and barley (n = 8) and red clover (n = 7), and fresh alfalfa (n = 1), grass hays (n = 5), and wheat straws (n = 5) had previously been determined in sheep. Concentrations of indigestible NDF (iNDF) in all forage samples were determined by a 288-h ruminal in situ incubation. Gas production of isolated forage NDF was measured by in vitro incubations for 72 h. In vitro pepsin-cellulase OM solubility (OMS) of the forages was determined by a 2-step gravimetric digestion method. Samples were also subjected to a 2-step determination of in vitro OMD based on buffered rumen fluid and pepsin. Further, rumen fluid digestible OM was determined from a single 96-h incubation at 38°C. Digestibility of OM from the in situ and the in vitro incubations was calculated according to published empirical equations, which were either forage specific or general (1 equation for all forages) within method. Indigestible NDF was also used in a mechanistic model to predict OMD. Predictions of OMD were evaluated by residual analysis using the GLM procedure in SAS. In vitro OMS in a general prediction equation of OMD did not display a significant forage-type effect on the residuals (observed - predicted OMD; P = 0.10). Predictions of OMD within forage types were consistent between iNDF and the 2-step in vitro method based on rumen fluid. Root mean square error of OMD was least (0.032) when the prediction was based on a general forage equation of OMS. However, regenerating a simple regression for iNDF by omitting alfalfa and wheat straw reduced the root mean square error of OMD to 0.025. Indigestible NDF in a general forage equation predicted OMD without any bias (P ≥ 0.16), and root mean square error of prediction was smallest among all methods when alfalfa and wheat straw samples were excluded. Our study suggests that compared with the in vitro laboratory methods, iNDF used in forage-specific equations will improve overall predictions of forage in vivo OMD. The in vitro and in situ methods performed equally well in calibrations of iNDF or OMD by near-infrared reflectance spectroscopy.

Benchmarking: a case report.

In mid 1993, administrators and physicians at Bristol Medical Center teams up with HCIA to perform clinical pathway analysis on five diagnosis related groups. The major goal of this project was to establish a partnership between the hospital administration and the medical staff to meet or beat existing benchmarks.

Elimination of high-voltage field effects in end column electrochemical detection in capillary electrophoresis by use of on-chip microband electrodes.

The influence of the separation voltage on end column electrochemical detection (EC) in capillary electrophoresis (CE) has been investigated using an electrochemical detector chip based on an array of microband electrodes. It is shown, both theoretically and experimentally, that the effect of the CE electric field on the detection can be practically eliminated, without using a decoupler, by positioning the reference electrode sufficiently close to the working electrode. In the present study, this was demonstrated by using an experimental setup in which neighboring microband electrodes on a chip, positioned 30 microns from the end of the CE capillary, were used as working and reference electrodes, respectively. The short distance (i.e., 10 microns) between the working and reference electrode ensured that both of the electrodes were very similarly affected by the presence of the CE electric field. With this experimental setup, no significant influence of the CE voltage on the peak potentials for gold oxide reduction could be seen for CE voltages up to +30 kV. The detector noise level was also found to be reduced.

Immunochemical partial identity between two independently identified and isolated major allergens from Alternaria alternata (Alt-I and Ag 1).

Partially purified preparations of Alt-I, the main allergenic fraction of Alternaria alternata isolated by Yunginger, and of Ag 1, shown in crossed radioimmunoelectrophoresis (CRIE) to be the dominating major allergen of A. alternata (Løwenstein, Nyholm), were compared by tandem crossed immunoelectrophoresis (CIE), RAST inhibition, and the CRIE-related technique, single radial radioimmunodiffusion (SRRID). The two allergen preparations showed reaction of identity in tandem-CIE and indistinguishable specific IgE binding in CRIE and SRRID, regardless of antibodies and serum pools used. In RAST inhibition, the relative potencies of the allergen preparations and of the crude extracts correlated well with their Alt-I/Ag 1 content as estimated by rocket immunoelectrophoresis. Moreover, all inhibition curves were parallel, confirming identical IgE binding by Alt-I and Ag 1 with the serum pools used. A second preparation of Alt-I, isolated from another strain of Alternaria, showed reaction of partial identity with Ag 1 in tandem-CIE, indicating that different variants of Alt-I (Ag 1) may exist in different strains of A. alternata.

End-column amperometric detection in capillary electrophoresis: influence of separation-related parameters on the observed half-wave potential for dopamine and catechol.

Capillary electrophoresis (CE) was coupled to a micro-electrode-based end-column amperometric detector. The influences of separation voltage, CE buffer concentration, and capillary-to-electrode distance on the observed hydrodynamic voltammetry of dopamine and catechol were studied using a separation capillary with an i.d. of 25 microns. It was found that an increased CE voltage, increased buffer concentration, or decreased capillary-to-electrode distance resulted in a positive shift of the observed half-wave potentials for both dopamine and catechol. At a constant separation current of 1.6 microA, the observed half-wave potential was found to increase with applied separation voltage. Furthermore, when experiments were carried out with a platinum quasi-reference electrode instead of a Ag/AgCl reference electrode, similar shifts in half-wave potential were observed. These results indicate that the observed shifts are an effect of the separation voltage rather than the separation current or a change in the reference potential. The characteristics of end-column detection with and without a fracture decoupler were compared. It was found that the effects of separation voltage, CE buffer concentration, and capillary-to-electrode distance were minimized by the use of a decoupling device. The observed half-wave potentials for dopamine and catechol were more positive when a CE capillary without a decoupler was employed compared to when a decoupler was used. Additionally, using the fracture decoupler, the observed half-wave potentials for both dopamine and catechol were approximately the same as when no CE voltage was applied (i.e., when the hydrodynamic voltammograms were recorded under flow injection conditions).

Development of an amperometric detector for packed capillary column supercritical fluid chromatography.

Amperometric detection at a naked platinum microelectrode is shown to be compatible with pressure-programmed packed capillary column supercritical fluid chromatography (SFC) using carbon dioxide modified with as little as 1% acetonitrile. Amperometric detection in modified carbon dioxide is, hence, possible without addition of salts, which otherwise may limit the stability of the chromatographic system. The detection, which can been based on either oxidations or reductions, is also compatible with the use of methanol as a sole modifier. Picogram amounts of ferrocene could be detected after separations both at a constant pressure and under pressure-programmed conditions. The detection limit for ferrocene, at a constant pressure of 200 atm, was found to be approximately 20 pg. The response of the amperometric detector was observed to increase with decreased mobile phase density and increased amount of added modifier. The injection of increasing amounts of analytes resulted in increased peak tailing, most likely due to the limited solubility of the oxidation products in the mobile phase. The influence of this effect was, however, small for the amounts of analytes relevent in capillary column SFC.

Characterization of the atmospheric pressure ionization mass spectrometric process obtained using a fused-silica emitter with the high voltage applied upstream

The atmospheric pressure ionization process obtained when a mixture of methanol and water (90:10, v/v) also containing 50 microM sodium hydroxide is dispersed from a fused-silica emitter was studied. A combination of a high electric field and a nebulizer gas with the high voltage applied upstream in the liquid flow was utilized to facilitate the spray process. By comparing the dependences of the spray current and ion signals on the spray potential, it was found that electrical corona discharges were obtained for potentials higher than about 2.6 kV, which resulted in a mixed electrospray and chemical ionization process. By introducing vapour from a solvent, such as benzene or toluene, with a low ionization energy into the nebulizing gas, it was found that the appearance of the corresponding molecular ion was correlated with a change in the slope of the spray current-potential curve. This indicates that the breakpoints in the spray current-potential curves observed were correlated with the onsets of corona discharges. It was shown that the mixed ionization process gives rise to increased amounts of protonated solvent molecules and assists in the formation of sodiated adduct ions from an uncharged fatty acid methyl ester.

Influence of mixed diffusional, migrational, and convective mass transport on the response of a wall-tube microelectrode in a flow injection system.

The current response of a 10-µm wall-tube microelectrode in a flow injection system under the conditions of low ionic strength has been examined experimentally for several redox systems such as ferrocene in methanol, undiluted methanol, and water in acetone. The examination involved the dependence of the current on the positioning of the microelectrode relative to a 760-µm-i.d. capillary outlet, flow rate, potential pulse time, and ratio between the concentrations of the supporting electrolyte and electroactive species (C(el)/C(redox)). For C(el)/C(redox) ratios smaller than ∼0.001 and a flow rate of 200 µL/min, the dependencies of the current on the flow rate and the positioning of the microelectrode versus the capillary tip were reversed compared to the presence of excess supporting electrolyte. The current was thus found to decrease with increasing flow rate while a local current maximum could be seen when the microelectrode was center-aligned with the capillary tip. The changes in the current are the results of local differences in convective transport. These differences alter the rate of migrational accumulation of counterions at the electrode surface. It is shown that results similar to those obtained for the excess supporting electrolyte case can be obtained for a low value of C(el)/C(redox) and a given flow rate if the electrode potential and time scale of the experiments are chosen appropriately.

Evaluations of the stability of sheathless electrospray ionization mass spectrometry emitters using electrochemical techniques.

The processes that cause the failure of sheathless electrospray ionization (ESI) emitters, based on different kinds of gold coatings on fused-silica capillaries, are described and explained. The methods chosen for this study include electrochemical methods, ICPMS analysis of the electrolytes used, SEM studies, and electrospray experiments. Generally, the failure occurs by loss of the conductive coating. It is shown that emitters with sputter-coated gold lose their coatings because of mechanical stress caused by the gas evolution accompanying water oxidation or reduction. Emitters with gold coatings on top of adhesion layers of chromium and nickel alloy withstand this mechanical stress and have excellent durability when operating as cathodes. When operating as anodes, the adhesion layer is electrochemically dissolved through the gold film, and the gold film then flakes off. It is shown that the conductive coating behaves as a cathode even in the positive electrospray mode when the magnitude of a superimposed reductive electrophoretic current exceeds that of the oxidative electrospray current. Fairy-dust coatings developed in our laboratory (see Barnidge, D. R.; etal.Anal. Chem. 1999, 71, 4115-4118,) bygluing gold dust onto the emitter, are unaffected by the mechanical stress due to gas evolution. When oxidized, the fairy-dust coatings show an increased surface roughness and decreased conductivities due to the formation of gold oxide. The resistance of this oxide layer is however negligible in comparison with that of the gas phase in ESI. Furthermore, since no flaking and only negligible electrochemical etching of gold was found, practically unlimited emitter lifetimes may be achieved with fairy-dust coatings.

A simple and robust conductive graphite coating for sheathless electrospray emitters used in capillary electrophoresis/mass spectrometry.

A graphite-polyimide mixture was used as a conductive coating for sheathless electrospray emitters. The coating procedure described is simple and inexpensive compared to previously described methods. An investigation of the stability of the conductive coating carried out by electrochemical methods revealed good performances during oxidative stress. In addition, no decrease in emitter performance was seen during continuous electrospray in the positive electrospray mode for two weeks. Fast capillary electrophoresis with attomole sensitivity demonstrated the excellent performance of the described sheathless interface when used in conjunction with an orthogonal time-of-flight mass spectrometer. The overall simplicity, stability and low cost of this type of sheathless emitter make the described approach highly suitable for any on-column coupling of low flow rate separation techniques to a mass spectrometer.

2-Amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) in human hair as biomarker for dietary exposure.

Heterocyclic aromatic amines (HAAs), formed during cooking of meat, are multipotent rodent carcinogens and are suspected to cause cancer in humans. In a search for suitable biomarkers for human dietary exposure to HAAs, we have investigated the concentration of a common fried food mutagen, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), in human hair. Fourteen volunteers participated in the study, each contributing hair from a regular haircut, and completing a questionnaire about consumption of fried/grilled meat and smoking habits. Hair samples were treated with 1 N NaOH at 100 C, and the HAAs extracted under alkaline and acidic conditions, derivatized and analysed by gas chromatography-mass spectrometry. PhIP was found in 12 out of 14 hair samples, in amounts from approx. 50 to 5000 pg g(-1) hair, while two samples were below the detection limit (<50 pg g(-1) hair). Grey/white hair straws from subjects with a mixture of coloured and grey/white hair had about a 50% reduction in PhIP concentration as compared with the natural hair mixture from the same person. This demonstrates that melanin, responsible for hair colour and spectrophotometrically characterized in the samples, participates in PhIP binding. Thus, when HAA binding components of the hair are taken into account, hair seems like an interesting object of further investigation as a biomarker for human exposure to dietary PhIP.