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At least 1,423 records · Page 79Linked to original sources
PubMed2006

Monitoring of in vitro fat digestion by electron paramagnetic resonance spectroscopy.

PURPOSE: The distribution of drugs between water, oil and mixed micelles after the oral application of lipid-based drug delivery systems affects their absorption rate. Since it has not been previously possible to monitor this process online during in vitro lipolysis, it was our aim to develop a suitable real-time method. MATERIALS AND METHODS: To follow the fate of a co-administered drug during fat digestion, the spin probe tempol benzoate was incorporated as a lipophilic model drug into a long-chain triglyceride (olive oil) and an in vitro digestion test was combined with electron paramagnetic resonance (EPR) spectroscopy (X-Band). Additionally the progression of digestion was determined by means of high performance thin layer chromatography (HPTLC). RESULTS: The spectral shape of the EPR spectrum changed significantly during the digestion process. EPR spectra at all times could be simulated with three species indicating a redistribution of the lipophilic model drug between olive oil, phosphate buffer and mixed micelles formed by bile salts and phospholipids. CONCLUSION: This in vitro real-time analysis could be a very helpful tool to monitor the digestibility of novel lipid-based drug nanocarriers which is an important step to optimize and to predict drug delivery processes. In future the EPR monitoring of fat digestion will be transferred to in vivo experiments.

Buffers↗
PubMed1988

Amino acid composition of peptides remaining after in vitro digestion of a gliadin sub-fraction with duodenal mucosa from patients with coeliac disease.

Sub-fraction 9-2, derived from fraction 9 of a peptic-tryptic-pancreatinic digest of wheat gliadin and previously shown to be toxic to individuals with coeliac disease, was digested in vitro with duodenal mucosa from patients with coeliac disease in remission and with mucosa from normals. Digestion products from coeliac mucosa caused damage to rat liver lysosomes in contrast to digestion products from normal mucosa which had practically no effects on the lysosomes. Ion exchange chromatography of the digestion products, followed by gel permeation chromatography to remove tissue proteins and amino acids allowed the separation of small peptides. Purification of the peptide residues by reversed-phase HPLC on a C18 column resulted in four subfractions, two of which were cytotoxic to rat liver lysosomes. Amino acid analysis of these latter peptide fractions showed that they were both rich in glutamine/glutamic acid, proline, serine and tyrosine. The results support the hypothesis of defective mucosal digestion as being the aetiology of coeliac disease and suggest that the causative agents are small peptides of apparent Mr congruent to 700 Da, with amino acid analysis corresponding to (Glx)3, (Pro)2, Ser and (Glx)3, (Pro)2, Tyr. These hexapeptides are likely to be H-Pro-Ser-Glx-Glx-Glx-Pro-OH and H-Glx-Glx-Pro-Tyr-Pro-Glx-OH.

Amino Acids↗
PubMed1983

Differences in chromatin from normal and leukemic human cells as shown by digestion with restriction nucleases.

Restriction endonuclease MspI digested significantly more than HpaII the DNA and chromatin from normal and leukemic human cells, although both enzymes digested DNA more than chromatin. Moreover, DNA and chromatin from normal cells showed higher digestion by HpaII compared to DNA and chromatin from leukemic cells indicating higher frequency of Cm5CCG in latter DNA. EcoRII and BstNI, which have the recognition sequence CCATGG but cut at different points, digested all DNAs significantly, as did BstNI for chromatin from all sources. However, chromatin from normal cells showed only limited digestion by EcoRII which significantly digested chromatin from leukemic cells. This could result from subtle differences in the conformation of normal and leukemic cell chromatin involving recognition sequences for EcoRII.

Chromatin↗
PubMed1993

Separation and tryptic digest mapping of normal and variant haemoglobins by capillary electrophoresis.

Characterization of haemoglobin (Hb) through whole protein separations and tryptic digest mapping allows the identification of structural Hb variants which result in haemoglobinopathies. Tryptic digest mapping by conventional two-dimensional paper chromatograph-electrophoresis provides high resolution but requires 48 h, while gradient elution reversed-phase high-performance liquid chromatography (HPLC) is faster (1.5 h), there is decreased resolution. CE analysis provides a fast separation with high resolution. We have used CE to optimise the tryptic digestion of globin purified from normal human haemoglobin A and to analyze tryptic digests from normal Hb. The separations were optimised and peak identification performed using UV scanning detection. In the optimised tryptic digest separation up to 28 peaks could be resolved in < 20 min. These peaks were identified as far as possible and a high-resolution map of the digest was constructed. The optimised analytical conditions were used to observe the separation pattern obtained from normal adult haemoglobin (HbA), common variant haemoglobins and some rarer haemoglobin variants.

Adult↗
PubMed1976

Caffeine enhancement of digestion of DNA by nuclease S1.

The activity of Aspergillus orzae nuclease S1 on DNA has been investigated under varying pH and metal ion conditions. Nuclease S1 was found to preferentially digest denatured DNA. With native DNA as substrate the enzyme could only digest the DNA when caffeine was added to the reaction mixture. The enzyme was more active in sodium acetate buffer (pH 4.5), than in either standard saline citrate (PH 7.0) or sodium phosphate buffer (pH 6.8). Caffeine was also found to affect the thermal stability of DNA, resulting in a melting profile characterized by two transitions. The first transition (poorly defined) was below the normal melting temperature of the DNA, while the next transition was at the normal melting temperature of the DNA, while the next transition was at the normal melting temperature of the DNA. The susceptibility of caffeine-treated DNA to nuclease digestion seems to be a result of the local unwinding that caffeine causes in the regions of DNA that melt in the first transition. This selective destabilization presumably sensitizes the unwound regions to nuclease hydrolysis. The hydrolysates of the DNA digested by nuclease S1 were subjected first to ion exchange chromatography followed by paper chromatography. The results from this partial characterization of the digestion products showed that they contain mononucleotides as well as oligonucleotides of varying lengths. The base composition of the mononucleotide digests suggests that caffeine has greater preference for interacting with A-T base-pairs in DNA.

Animals↗
PubMed1983

Nuclease digestibility of chromatin is affected by nuclei isolation procedures.

Experiments using nucleases as probes of chromatin structure take place in two stages: (1) nuclei isolation, and (2) nuclease digestion. The parameters of the nuclease digestion stage are usually strictly controlled because of nuclease sensitivity to them. However, there have been no reports on whether parameters in the nuclei isolation stage affect the subsequent nuclease digestions. We have evaluated a typical nuclei isolation technique with respect to how changes in the isolation parameters affect nuclease digestion kinetics. Our observations point out that various parameters encountered in the nuclei isolation stage have a significant effect on the subsequent nuclease digestion kinetics of DNAase I. These parameters include the concentration of cells, divalent cations and phosphatase inhibitors. The pH, concentration of NaCl and concentration of detergent had little effect. Micrococcal nuclease was relatively unaffected by changes in the nuclei isolation parameters. The importance of this report lies in the demonstration that lack of control of seemingly insignificant parameters, such as cell concentration during the nuclei isolation stage, leads to subsequent irreproducible results in the DNAase I digestion. These findings indicate that great care must be exercised in the nuclei isolation stage if reproducible work is to be performed with DNAase I.

Animals↗
PubMed2004

A quantitative in silico analysis calculates the angiotensin I converting enzyme (ACE) inhibitory activity in pea and whey protein digests.

Angiotensin I converting enzyme (ACE) inhibitory peptides can induce antihypertensive effects after oral administration. By means of an ACE inhibitory peptide database, containing about 500 reported sequences and their IC(50) values, the different proteins in pea and whey were quantitatively evaluated as precursors for ACE inhibitory peptides. This analysis was combined with experimental data from the evolution in ACE inhibitory activity and protein degradation during in vitro gastrointestinal digestion. Pea proteins produced similar in silico scores and were degraded early in the in vitro digestion. High ACE inhibitory activity was observed after the simulated stomach phase and augmented slightly in the simulated small intestine phase. The major whey protein beta-lactoglobulin obtained the highest in silico scores, which corresponded with the fact that degradation of this protein in vitro only occurred from the simulated small intestine phase on and resulted in a 10-fold increase in the ACE inhibitory activity. Whey protein obtained total in silico scores of about 124 ml/mg, compared to 46 ml/mg for pea protein, indicating that whey protein would be a richer source of ACE inhibitory peptides than pea protein. Although beta-lactoglobulin is only partially digested, a higher ACE inhibitory activity was indeed found in the whey (IC(50) = 0.048 mg/ml) compared to the pea digest (IC(50) = 0.076 mg/ml). In silico gastrointestinal digestion of the highest scoring proteins in pea and whey, vicilin and albumin PA2, and beta-lactoglobulin, respectively, directly released a number of potent ACE inhibitory peptides. Several other ACE inhibitory sequences resisted in silico digestion by gastrointestinal proteases. Briefly, the quantitative in silico analysis will facilitate the study of precursor proteins on a large scale and the specific release of bioactive peptides.

Amino Acid Sequence↗
PubMed2004

Evaluating heavy metal contents in nine composts using four digestion methods.

The production and application of compost potentially contaminate the environment with heavy metals. The heavy metal contents of composts must be exactly determined. Therefore, this study aims to understand the heavy metal contents in composts, using different digestion methods and to recommend the most appropriate digestion method making this measurement. Nine composts from different sources, including swine manure, poultry litter, food waste, municipal sewage sludge, soybean meal, wood residues, flesh-bone dust, fish-bone meal and guano, were selected to be digested by four methods to determine the contents of Cd, Cr, Cu, Mn, Ni, Pb and Zn by AAS. The four digestion methods were nitric acid, dry ashing, nitric-perchloric acid and sulfuric acid methods. Analytical results indicated that the nitric acid procedure was the most efficient for recovering Cd, Mn and Ni from most compost samples. The sulfuric acid procedure yielded the lowest recovery of Pb from the certified reference material (BCR 146) and the compost samples, especially from the composts with high pH and EC values potentially high in Ca. The nitric acid procedure was recommended as the method for digesting the composts herein, based on recovery analysis, cost and time taken. Dry ashing was recommended as a flexible method. Nitric-perchloric acid procedure was not recommended because perchloric acid is potentially hazardous during digestion and it recovers relatively little heavy metal.

Chemistry Techniques, Analytical↗
PubMed2005

Anaerobic digestion of animal waste: effect of mixing.

Six laboratory scale biogas mixed anaerobic digesters were operated to study the effect of biogas recycling rates and draft tube height on their performance. The digesters produced methane at 0.40-0.45 L per liter of digester volume per day. A higher methane production rate was observed in unmixed digesters, while increased biogas circulation rate reduced methane production. However, different draft tube heights caused no difference in the methane production rate. Air infiltration (up to 15% oxygen in the biogas) was observed in the digesters mixed by biogas recirculation. Slight air permeability of tubing or leakage on the vacuum side of the air pump may have caused the observed air infiltration. The similar performance of the mixed and unmixed digesters might be the result of the low solids concentration (50 g dry solids per liter of slurry) in the fed animal slurry, which could be sufficiently mixed by the naturally produced biogas.

Analysis of Variance↗
PubMed2006

Characterization of food waste as feedstock for anaerobic digestion.

Food waste collected in the City of San Francisco, California, was characterized for its potential for use as a feedstock for anaerobic digestion processes. The daily and weekly variations of food waste composition over a two-month period were measured. The anaerobic digestibility and biogas and methane yields of the food waste were evaluated using batch anaerobic digestion tests performed at 50 degrees C. The daily average moisture content (MC) and the ratio of volatile solids to total solids (VS/TS) determined from a week-long sampling were 70% and 83%, respectively, while the weekly average MC and VS/TS were 74% and 87%, respectively. The nutrient content analysis showed that the food waste contained well balanced nutrients for anaerobic microorganisms. The methane yield was determined to be 348 and 435 mL/gVS, respectively, after 10 and 28 days of digestion. The average methane content of biogas was 73%. The average VS destruction was 81% at the end of the 28-day digestion test. The results of this study indicate that the food waste is a highly desirable substrate for anaerobic digesters with regards to its high biodegradability and methane yield.

Anaerobiosis↗
PubMed2006

Pronase digestion of brush border membrane-bound Cry1Aa shows that almost the whole activated Cry1Aa molecule penetrates into the membrane.

Bacillus thuringiensis insecticidal proteins, Cry toxins, following ingestion by insect larvae, induce insecticidal effect by penetrating the brush border membranes (BBM) of midgut epithelial cells. Purified, activated B. thuringiensis Cry1Aa bound to Bombyx mori BBMV or unbound Cry1Aa were vigorously digested with Pronase. Both digests were compared by Western blotting. Free Cry1Aa was digested to alpha-helix and/or to amino acids at 1 mg Pronase/mL within 2.4 h at 37 degrees C. Whereas, BBMV-bound Cry1Aa was very resistant to Pronase digestion and even at 2 mg for 24 h, 7.5 kDa and approximately 30 kDa peptide were detected by alpha-2,3 antiserum, and alpha-4,5 and alpha-6,7 antisera, respectively. Another approximately 30 kDa peptide was also detected by beta-6-11 and domain III antisera. These fragments are believed either to be embedded in or to strongly interact with the BBMV. The 7.5 and former approximately 30 kDa peptides are thought to be derived from alpha-2,3 helix and stretch of alpha-4 to alpha-7 helices. Furthermore the latter approximately 30 kDa was thought to include the stretch of beta-6 to domain III. Moreover, the embedded Cry1Aa molecule appears to be segregated in some areas of beta-1-5 sheets, resulting in the above two approximately 30 kDa peptides. From these digestion patterns, we proposed new membrane insertion model for single Cry1Aa molecule. On the other hand, in digestion of BBMV-bound Cry1Aa, 15 kDa peptide which was recognized only by alpha-4,5 antiserum was observed. This fragment must be dimeric alpha-4,5 helices and we discussed the origin of this peptide.

Animals↗
PubMed2005

Exploring human plasma proteome strategies: high efficiency in-solution digestion protocol for multi-dimensional protein identification technology.

Multi-dimensional protein identification technology (MudPIT) is becoming a prevalent proteomic approach due to its high-throughput separations and accurate mass detection. Prior to MudPIT analysis, complicated samples required in-solution digestion. Unlike in-gel digestion, in which enzymes work on just a few proteins, in-solution digestion involves simultaneous digestion of hundreds or thousands of proteins. In-solution digestion protocols must therefore be very efficient. Few investigations have evaluated the efficiency of in-solution digestion protocols. The present research compared three such protocols. Results suggest that a protocol utilizing trifluoroethanol (TFE) as denaturant is most efficient.

Blood Proteins↗
PubMed2005

Rapid protein digestion and identification using monolithic enzymatic microreactor coupled with nano-liquid chromatography-electrospray ionization mass spectrometry.

A novel monolithic enzymatic microreactor was prepared in the fused-silica capillary by in situ polymerization of acrylamide (AA), N-acryloxysuccinimide (NAS) and ethylene dimethacrylate (EDMA) in the presence of a binary porogenic mixture of dodecanol and cyclohexanol, which could offer very low back pressure, enabling the fast digestion of proteins. The performance of the monolithic microreactor was demonstrated by digesting cytochrome c at high flow rate, and the comparisons between the in-solution digestion and on-column reaction were made by a nano-high performance liquid chromatography-mass spectrometry (nano-HPLC-MS) system. The performance of the monolithic microreactor was demonstrated with the digestion of cytochrome c at the fast flow rate of 1 microL/min, which afforded a residence time of 7s, yielding a sequence coverage of 54.81% using strict multiple database searching thresholds. Future more, a mixture of four standard proteins was digested and analyzed using the on-line digestion and nano-HPLC-MS system. The results showed the promising of such a system in the analysis of protein mixture.

Bioreactors↗
PubMed2006

Correlation between digestion of the lipid phase of smedds and release of the anti-HIV drug UC 781 and the anti-mycotic drug enilconazole from smedds.

The present studies were conducted primarily to compare the drug release process of the anti-HIV drug UC781 from three different smedds to the smedds digestion profile. The influence of every formulation component on the digestion process, measured as the release of fatty acids, was determined. In addition, the release of the antimycotic drug enilconazole from a smedds was investigated as well in order to study the influence of the type of incorporated drug on oil digestion. Simulsol 1292, Tween 80, Cremophor RH40, ethanol and both drugs reduced the fatty acid release. C8, C10 and C12 fatty acids, originating from oil hydrolysis, were able to reverse the inhibitory effect of phospholipids present in the release medium. Similarly Cremophor RH40 lost its inhibitory capacities in combination with Captex 200P hydrolysis. In addition, UC781 did not decrease fatty acid release in combination with a Captex 200P-Tween 80-ethanol mixture. The release of UC781 from smedds significantly increased compared to the dissolution of the pure drug. The drug release profiles were characterized by rapid and complete release followed by precipitation. In order to detect possible correlations between drug release and oil digestion, release results were compared to those of vehicle digestion experiments. Contrary to what one would assume, a higher extent of fatty acid liberation did not enhance drug release. In other words, drug release does not seem to be driven by the extent of lipid digestion.

Anilides↗
PubMed2004

Optimized sample-processing time and peptide recovery for the mass spectrometric analysis of protein digests.

Proteomics requires an optimized level of sample-processing, including a minimal sample-processing time and an optimal peptide recovery from protein digests, in order to maximize the percentage sequence coverage and to improve the accuracy of protein identification. The conventional methods of protein characterization from one-dimensional or two-dimensional gels include the destaining of an excised gel piece, followed by an overnight in-gel enzyme digestion. The aims of this study were to determine whether: (1) stained gels can be used without any destaining for trypsin digestion and mass spectrometry (MS); (2) tryptic peptides can be recovered from a matrix-assisted laser desorption/ionization (MALDI) target plate for a subsequent analysis with liquid chromatography (LC) coupled to an electrospray ionization (ESI) quadrupole ion trap MS; and (3) an overnight in-gel digestion is necessary for protein characterization with MS. These three strategies would significantly improve sample throughput. Cerebrospinal fluid (CSF) was the model biological fluid used to develop these methods. CSF was desalted by gel filtration, and CSF proteins were separated by two-dimensional gel electrophoresis (2DGE). Proteins were visualized with either silver, Coomassie, or Stains-All (counterstained with silver). None of the gels was destained. Protein spots were in-gel trypsin digested, the tryptic peptides were purified with ZipTip, and the peptides were analyzed with MALDI and ESI MS. Some of the samples that were spotted onto a wax-coated MALDI target plate were recovered and analyzed with ESI MS. All three types of stained gels were compatible with MALDI and ESI MS without any destaining. In-gel trypsin digestion can be performed in only 10-60 min for protein characterization with MS, the sample can be recovered from the MALDI target plate for use in ESI MS, and there was a 90% reduction in sample-processing time from overnight to ca. 3 h.

Mass Spectrometry↗
PubMed2006

Ultrasound-assisted digestion: a useful alternative in sample preparation.

Ultrasound-assisted digestion is a promising alternative in the analysis of solid samples when either simple dissolution or direct analysis is not applicable. However, the field of application of ultrasonic sample digestion is still small in comparison with classical digestion alternatives and, particularly, with microwave-assisted digestion. This fact can be justified by the scant knowledge analytical chemists have about the advantages ultrasonic energy provides to digestion. Among these, the strict control at low temperatures of ultrasound applications allows the implementation of ultrasonic-assisted steps in biochemical analyses. In this connection, two specific biological applications, ultrasonic enzymatic digestion and assistance of ultrasound for cell disruption, are also reviewed.

Cell Fractionation↗
PubMed2004

Impact of food industrial waste on anaerobic co-digestion of sewage sludge and pig manure.

The performance of an anaerobic digestion process is much dependent on the type and the composition of the material to be digested. The effects on the degradation process of co-digesting different types of waste were examined in two laboratory-scale studies. In the first investigation, sewage sludge was co-digested with industrial waste from potato processing. The co-digestion resulted in a low buffered system and when the fraction of starch-rich waste was increased, the result was a more sensitive process, with process overload occurring at a lower organic loading rate (OLR). In the second investigation, pig manure, slaughterhouse waste, vegetable waste and various kinds of industrial waste were digested. This resulted in a highly buffered system as the manure contributed to high amounts of ammonia. However, it is important to note that ammonia might be toxic to the micro-organisms. Although the conversion of volatile fatty acids was incomplete the processes worked well with high gas yields, 0.8-1.0 m3 kg(-1) VS.

Animals↗
PubMed2004

The effects of post-persulfate-digestion procedures on total phosphorus analysis in water.

There are differences between the EPA Method 365 and the APHA-AWWA-WEF's Standard Method 4500 with respect to the post-digestion treatment procedures of the persulfate-digested water. The effects on total phosphorus analysis of different post-digestion treatment procedures, such as neutralization and reacidification, and shaking/settling, were investigated in this study using the total phosphorus measurements of water samples from the Everglades Round Robin (ERR) study and comparing the results with the ERR study. The effects of the insoluble particles or phosphorus adsorption/precipitation on/with Al and Fe hydroxides in different post-digestion treatment procedures adequately accounted for the differences between the most probable value and the higher or lower total phosphorus measurements reported in the ERR study. Based on the results of this investigation we recommend that a clearly defined set of digestion and post-digestion treatment procedures be adopted as the standard for total phosphorus analysis using the ascorbic acid method.

Ecosystem↗