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PubMed2000

Measurements of macromolecule-bound and ultra-filtrable polyamines in rat liver homogenized without buffer.

Ultra-filtrable and macromolecule-bound polyamines in rat liver homogenates, made without buffer, were determined, using Potter-Elvehjem homogenizer and commercially available, pressure-aided ultrafiltration device with a membrane pore size that allows passage of particles of molecular weight no larger than 5000. About 90% of polyamines in the liver were shown to be equilibrated with externally added 15N-labeled polyamines, based on the difference in the ratio of the natural to 15N-labeled polyamine in the liver homogenate and the ultrafiltrate. The entire amount of ultrafiltrate in the homogenized liver, required for calculation of the amounts of ultra-filtrable and macromolecule-bound polyamines, was estimated to be about 0.25 g in one gram of the homogenate, using a limited dilution curve of spermine in the ultrafiltrate with phosphate buffered saline and distilled water. With this value, ultra-filtrable polyamines in normal rat liver homogenate were calculated as about 25%, 8%, and 2% of the total amount of putrescine, spermidine, and spermine, respectively. The method was then used to measure ultra-filtrable and macromolecule-bound polyamines in regenerating rat liver homogenates, to examine possible changes of polyamines during cell growth. The method was also applied to measure other ultra-filtrable compounds such as amino acids and inorganic ions in rat liver homogenate.

Amino Acids↗
PubMed2001

Effect of physical and chemical methods of homogenization on inflammatory mediators in sputum of asthma patients.

BACKGROUND: Dithiothreitol (DTT), which is used for sputum homogenization, may split S-S bonds of the bronchial mucins as well as other proteins and, thus, may have a detrimental effect on inflammatory mediators that are present in sputum. OBJECTIVE: To evaluate the effects of physical sputum homogenization, using ultrasonic and chemical (ie, DTT) means, on the concentrations of eosinophil cationic protein (ECP), eosinophil protein X (EPX), eosinophil peroxidase (EPO), and myeloperoxidase (MPO) in the sputum of patients with asthma. METHODS: The collection of sputum samples from nine patients with asthma was induced by their inhaling a sterile 3% saline solution for 10 min from an ultrasonic nebulizer. One half of the sputum sample was homogenized by ultrasound, and the other half was liquefied by DTT. The supernatant of the ultrasonically homogenized specimen was divided into the following three portions: (1) immediately frozen; (2) stored for 15 min at 37 degrees C; and (3) additionally treated with DTT. The supernatant of the sputum sample that was liquefied by DTT was divided into the following two portions: (1) immediately frozen; and (2) additionally subjected to ultrasound. The concentrations of ECP, EPO, EPX, and MPO in the sputum samples were measured using immunoassays. RESULTS: Statistically significant differences were found between the ultrasonically homogenized specimens that had been either processed immediately or stored at 37 degrees C and those treated by DTT, but only for concentrations of EPO and MPO (p < 0.005). No effect of temperature on the mediators in the ultrasonically homogenized specimens could be detected. Ultrasonic homogenization had no influence on the mediators in the samples liquefied by DTT. However, the addition of DTT to the cell-free supernatant of the ultrasonically homogenized sputum samples caused a significant fall in measured EPO and MPO concentrations. CONCLUSIONS: The sputum processing by DTT caused a statistically significant fall in EPO and MPO concentrations but did not significantly influence the measured concentrations of ECP and EPX.

Adult↗
PubMed2003

Comparison of homogenization methods for recovering Salmonella Enteritidis from eggs.

For Salmonella Enteritidis (SE) detection, shell eggs have been homogenized with stomachers, with electric blenders, and by hand massaging. However, to date, there have been no published reports addressing whether the method of homogenization affects the recovery of SE from raw eggs. Three inoculum levels (10, 126, and 256 SE cells per pool of 10 eggs) were used to conduct three experiments. The 10-egg pools were homogenized by one of four homogenization methods--mechanical stomaching, electric blending, hand massaging, and hand stirring-for 30 s. The homogenized eggs were then incubated at 37 degrees C, and SE colonies were enumerated after 24 and 48 h of incubation. After 24 h of incubation, no SE was recovered from egg samples from stomached or electrically blended pools inoculated with <10 cells, while levels of 106 CFU/ml were found for samples from whipped or hand-massaged pools inoculated with <10 cells. Similarly, after 24 h of incubation, the numbers of SE cells recovered from hand-massaged or hand-stirred egg pools inoculated with 126 cells were significantly larger than the numbers recovered from stomached or electrically blended egg pools inoculated with 126 cells. The number of SE cells recovered from samples homogenized with a blender was still significantly smaller than the numbers recovered from samples homogenized by the other three methods when the inoculum level was increased to 256 CFU per pool. However, the SE count for all samples approached 9 log10 CFU/ml after 48 h of incubation. It is concluded that the detection of small SE populations in shell egg samples could be improved with the use hand massaging and hand stirring for homogenization.

Animals↗
PubMed2001

Evolution of methods for measurement of HDL-cholesterol: from ultracentrifugation to homogeneous assays.

BACKGROUND: Adoption of automated homogeneous assays for HDL-cholesterol (HDL-C) is increasing, driven by the need of clinical laboratories to cope with increasing workloads while containing costs. However, performance characteristics of homogeneous assays often differ in important aspects from those of the earlier precipitation methods. This review provides an overview of the new generation of homogeneous assays for HDL-C within the historical context of the evolution of methods and the efforts to standardize measurements of the lipoproteins. APPROACH: This is a narrative review based on method evaluations conducted in the laboratories of the authors as well as on relevant publications, especially comparative evaluation studies, from the literature. Publications considered here have been collected by the authors over the past 30 years of involvement as methods for HDL-C made the transition from their early use in lipid research laboratories to clinical laboratories and the recent emergence of homogeneous assays. CONTENT: The presentation includes descriptions of methodologies, including homogeneous, precipitation, electrophoresis, and ultracentrifugation assays. Reference methods and recommended approaches for assessing accuracy are described. Accuracy and imprecision are summarized in the context of the National Cholesterol Education Program (NCEP) standards for analytical performance. The effects of interfering substances and preanalytical sources of variation are presented. SUMMARY: Homogeneous assays have been shown to be reasonably well suited for use in routine clinical laboratories, generally meeting the NCEP criteria for precision, accuracy, and total error. However, discrepant results compared with the reference methods have been observed with some of the assays, and the sources of discrepancies are not well characterized. Some homogeneous reagents have not been thoroughly evaluated. At least three of the reagents have experienced successive adjustments in formulation; hence, the reagents may not yet be fully optimized. For these reasons, the homogeneous assays cannot be confidently recommended for use in long-term clinical trials and other research applications without thorough validation.

Chemical Precipitation↗
PubMed2002

[Clinical evaluation of two kinds homogenous assays for determination of high-density lipoprotein cholesterol].

OBJECTIVE: To evaluate the clinical efficacy of two kinds homogenous assays for direct determination of high-density lipoprotein cholesterol (HDL-C) based on the principle of polyanion polymer/detergent (PPD method) and polyethylene glycol-modified enzyme (PEGME) method. METHODS: The two homogenous methods were compared with the precipitation method (PTA-Mg2+ method), their precision, accuracy, specificity and interference were also analyzed. RESULTS: Both homogenous HDL-C assays were precise, having a within-run CV < 3%, day-to-day CV < 3% and total CV < 4%. The HDL-C values measured by the two homogenous methods correlated well with those by PTA-Mg2+ method (X): Y = 0.9316 X + 0.1063, r = 0.9762 for PPD method (Y); and Y = 0.9106 X + 0.1368, r = 0.9894 for PEGME method (Y). The linearity studies showed the two homogenous methods to be linear up to 4.14 mmol/L. The lowest detectable concentration of the two methods was apparently 0.08 mmol/L. Recoveries of the two methods were 94.1%-106.2%. Hemoglobin did not interfere with the HDL-C results in the two homogenous methods, whereas icteric samples with total bilirubin > 200 mg/L showed discrepancies. Lipemia up to triglyceride concentration of 17.0 mmol/L did not interfere with the two homogenous HDL-C assays. CONCLUSIONS: The two new homogenous HDL-C assays meet the requirements for accuracy, precision, ease of handling with massive sample, allow full automation, and are clinically useful.

Cholesterol, HDL↗
PubMed1981

Comparative studies on detergents - chlorhexidinum gluconicum, ditalan wo hc, sodium-laurylsulphate, laurosept, nekal bx - used for homogenization of diagnostic specimens in the microbiological diagnostic of tuberculosis.

Between 1974 and 1978 there were carried out comparative studies concerning the value of several detergents used to homogenization of diagnostic specimens in six countries (Bulgaria, Czechoslovakia, German Democratic Republic, Poland, The Soviet Union and Yugoslavia). In the first study it was confirmed that the homogenization of diagnostic materials by detergents gives good results and is more economical than conventional Petroff's method. In the majority of centers the highest detection of tubercle bacilli was found after homogenization by Sodium-Laurylsulphated-technique. Remarkable differences in the time of growth of tubercle bacilli after homogenization of the same samples by Laurylsulphate, Laurosept and Petroff's lye method were not observed. While, after homogenization by Nekal BX the time of growth was a little bit retarded. In the lowest percentage of contamination was observed after homogenization by Nekal BX. In the second study the value of Polish detergents (Chlorhexidinum gluconicum and Laurosept) was compared with Ditalan OW hc made in GDR and with other routinely in the participating laboratories used techniques. The best results were obtained after homogenization of specimens by Chlorhexidinum gluconicum. But the comparison to Laurosept and Ditalan WO hc showed no significant differences. Homogenization of sputa was better and number of contaminations lower after using laurosept or Ditalan WO hc than by means of Chlorhexidinum gluconicum.

Chlorhexidine↗
PubMed1993

[In vitro studies of the metabolism of paraquat and diquat using rat liver homogenates--isolation and identification of the metabolites of paraquat and diquat].

The metabolism of paraquat and diquat was studied in vitro using rat liver homogenates, and the resulting metabolites were identified. Rat liver was homogenized with three volumes of isotonic buffer, and aliquots of the homogenate were preheated in a boiling water bath for 5 min prior to use. One milliliter of a mixture including both paraquat and diquat in an isotonic buffer solution (10 micrograms ion/ml) was incubated with an equal volume of fresh or preheated homogenate for 1 to 60 min at 37 degrees C. Quantification of paraquat and diquat was carried out by high-performance liquid chromatography (HPLC). In the fresh homogenate, a gradual decrease of paraquat concentration (about a 30% decrease over 60 min of incubation) and a rapid decrease of diquat concentration (not detectable after 10 min of incubation) were observed, but the same phenomenon was not evident with the preheated homogenate. Analysis of the incubated mixture of fresh liver homogenate with paraquat and diquat revealed three unknown peaks on the HPLC chromatograms; these seemed to be breakdown products of paraquant and diquat. The products were isolated and purified from the mixture by Sep-Pak C18 cartridge extraction, HPLC, silica gel column chromatography and Sephadex LH-20 column chromatography. Analysis of the chemical structure of the purified compounds was performed by infrared spectroscopy, mass spectrometry and nuclear magnetic resonance spectroscopy. These analyses determined that paraquat-monopyridone (1',2'-dihydro-1,1'-dimethyl-2-oxo-4,4'-bipyridylium ion) was derived from paraquat, and that diquat-monopyridone (6,7-dihydro-4-oxodipyrido [1,2-a':2',1'-c] pyrazinium ion) and diquat-dipyridone (6,7-dihydrodipyrido [1,2-a:2',1'-c] pyrazine-4,9-dione) were derived from diquat. These results indicate that paraquat and diquat are metabolized by rat liver homogenate, diquat more readily so than paraquat. As the toxicity of these metabolites has been reported to be much lower than those of the parental compounds, it would seem that there is a system capable of detoxifying paraquat and diquat in rat liver.

Animals↗
PubMed2000

Gelation of chicken breast and thigh muscle homogenates: effect of pH and time of aging.

Gelation properties of chicken breast and thigh muscle homogenates were studied at various pH values. Breast muscle homogenates showed a greater protein extractability at pH 5.8-6.6 and gel strength at pH 6.3-6.6 than thigh muscle homogenates. At pH 5.8-6.0 breast muscle homogenate gels were weaker than thigh muscle homogenate. The pH for optimum gelation, indicated by the highest stress at failure was 6.3 for breast and 6.0 for thigh muscle homogenates. The pH dependence of chicken breast and thigh muscle homogenate gel strengths, influenced by the muscle fibre type, probably was due to altered protein-protein and other meat component interactions and changes in protein extractability.

Animals↗
PubMed1994

Dithiothreitol homogenization of prefixed sputum for lung cancer detection.

The technique of chemical homogenization of sputum for cancer detection was revisited. The mucolytic agent dithiothreitol (DTT) had been used by the authors on fresh specimens. In this study, its effectiveness in homogenizing prefixed sputum was investigated. Fifty-seven positive samples were examined: 28 were prefixed with 2% carbowax in 60% ethanol and 29 in 3% carbowax in 60% ethanol. Each specimen was divided equally into three parts and homogenized immediately, 3 and 7 days later, respectively, with 0.2% (0.013 mol/L) DTT in the respective prefixative. Five samples were prefixed for 4 wk before homogenization. The homogenization time varied from 30 min to 48 hr. The cellular morphology was compared to directly smeared controls from the same samples. It was found that the cellular morphology was well preserved and best with the 3% carbowax in 60% ethanol. The specimen could be prefixed for up to 4 wk before homogenization and the homogenization process could last from 30 min to 48 hr without any damaging effect on morphology. Furthermore, screening was made easy with the mucus lysed and its obscuring effect removed and the cells concentrated and evenly distributed. The method was versatile and could be of value in enhancing the detection rate of cancer in sputum.

Adenocarcinoma↗
PubMed1989

Relaxometry of lens homogenates. II. Temperature dependence and comparison with other proteins.

We have extended our earlier work (C.F. Beaulieu, J.I. Clark, R.D. Brown III, M. Spiller, and S.H. Koenig, Magn. Reson. Med. 8, 45 (1988] on the magnetic field dependence of 1/T1 (NMRD profiles) of calf lens nuclear homogenates, at 25 degrees C, to 5 degrees C, and to other protein systems as well. These include concentrated solutions of myoglobin and bovine serum albumin, both globular proteins, the first compact and roughly spherical, the other extended, flexible, and with weak internal bonding; chicken lens homogenate, for which the dominant crystallins (lens proteins) are approximately 70% alpha-helical compared with calf crystallins, which are essentially all beta-sheet; and hen egg white, both native and heat-denatured. Our earlier conjectures regarding a reversible change in protein organization of the calf lens crystallins as a function of solute protein concentration is given added support. Our findings suggest that cytoplasmic homogenate can be characterized as a heterogeneous and polymorphic solution of crystallins. At high concentrations the NH moieties of the protein backbone become accessible to solvent with water (not NH proton) exchange rates greater than 10(4) s-1. This conclusion is based on two aspects of the observed NMRD profiles. At low crystallin concentration, the profiles of calf and chicken lens homogenates are similar in form to those of myoglobin and native hen egg white, a form that has been studied previously for a range of diamagnetic globular proteins and has been demonstrated to arise from the rotational thermal motion of the solute molecules. At high crystallin concentrations, the NMRD profiles of the lens homogenates develop a monotonic background (high rates at low fields), much like that of the heat-denatured egg-white sample and those of most tissues. In addition, there is a set of peaks in the central part of the profiles of the concentrated crystallins, seen also in the denatured egg white and some tissues but not in the myoglobin sample, which is known to arise from cross-relaxation interactions between the water protons and (through the intermediary of the NH proton) the 14N quadrupolar levels. The magnitude of these peaks, which is larger by an order of magnitude for native calf lens homogenates than for any tissue, requires that the majority of the NH moieties be accessible to water. Finally, going to 5 degrees C for the native calf lens homogenate takes the sample below the temperature of reversible phase separation, and it becomes opaque.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1999

Analysis of sarcoplasmic reticulum Ca2+ transport and Ca2+ ATPase enzymatic properties using mouse cardiac tissue homogenates.

Recent studies have focused on developing transgenic mouse models to explore the physiological roles of sarcoplasmic reticulum (SR) calcium handling proteins. The goal of this study was to develop methodology to measure SR Ca2+ transport function and enzymatic properties of SR Ca2+ ATPase (SERCA) in individual mouse hearts. We describe here the procedures to specifically measure SR Ca2+ uptake, the formation and decomposition of SERCA phosphoenzyme intermediate (E-P) in mouse cardiac homogenates. The specificity of SERCA enzymatic activity in cardiac homogenates was established by (a) the selective inhibition of SERCA enzyme by inhibitor-thapsigargin, and (b) comparison of the kinetic parameters of SERCA activity between homogenates and isolated microsomes. Here we show that the apparent affinity of SERCA for Ca2+ and ATP, the time to reach steady-state levels of E-P, and the rate of E-P decomposition (turnover rate of SERCA enzyme) are similar in homogenates and microsomes. These studies demonstrate that SERCA Ca2+ transport and enzymatic properties can be accurately measured in mouse cardiac tissue homogenates. Additionally, we show that frozen cardiac homogenates can be used without significant loss of enzymatic activity. In conclusion, we have developed and established the methods to employ tissue homogenates to study SR Ca2+ transport function in individual mouse hearts.

Animals↗
PubMed1997

Coalescence of Protein-Stabilized Emulsions in a High-Pressure Homogenizer

Coalescence rate constants in a high-pressure homogenizer were inferred for pure emulsions of a neutrally buoyant mixture of carbon tetrachloride and benzene dispersed in aqueous medium, as well as for emulsions stabilized by sodium caseinate or whey protein, from the temporal evolution of number of drops per unit volume in the exit stream of the homogenizer in a recirculating system consisting of the homogenizer and a stirred tank when subjected to a negative step change in applied homogenizer pressure (Ph). The homogenization pressure was varied in the range 6.9 to 41.4 MPa, pH in the range 5 to 7, dispersed phase fraction in the range 0.05 to 0.15, ionic strength in the range 0.01 to 0.05 M, and protein concentration in the range 0.01 to 0.05 wt%. Functional dependence of drop coalescence rate on the homogenizer pressure was derived for pure emulsions for mechanisms of drop collisions due both to turbulence and to shear. The inferred collision rate constant for pure emulsions was found to be proportional to Ph0.722, which was closer to the functional dependence for drop collisions due to turbulence. The coalescence rate constant was found to be higher for higher homogenizer pressures, larger drop sizes, and higher dispersed phase fractions. For emulsions stabilized by proteins, drop coalescence rate constant was found to be higher at lower protein concentrations, near the isoelectric point of the protein, and higher ionic strengths because of the smaller interdroplet colloidal repulsive forces, thus clearly demonstrating the effect of colloidal forces on drop coalescence. Sodium caseinate was found to result in a lower coalescence rate constant than whey protein.

Journal Article↗
PubMedDate not supplied

Codon equilibrium I: Testing for homogeneous equilibrium.

We present theoretical considerations that suggest that synonymous-codon usage might be expected to be close to an equilibrium distribution given a very homogeneous process of silent substitution. By homogeneous we mean that substitution depends only on the two bases involved, so that 12 base-substitution rates completely describe the silent substitution process. We have developed a method of statistically testing for such homogeneous equilibrium and applied it to reported data on the codon usages of different classes of organisms. Weakly expressed bacterial sequences and both mammalian and nonmammalian eukaryotic sequences deviate significantly from a random pattern of codon usage, in the direction of homogeneous equilibrium. On the other hand, highly expressed bacterial sequences do not exhibit homogeneous equilibrium, which may be correlated with recent experimental results showing that they are optimized to accept the most abundant tRNAs. To examine the effect of amino acid replacements on the homogeneous model of silent substitution, we divided the amino acids with degenerate codes into two classes, those with high mutabilities and those with low, and performed the same analysis on bacterial and eukaryotic data sets. The codon sets of the highly mutable class of amino acids are not further from homogeneous equilibrium than are the codon sets of the class with low mutabilities. We also found for the eukaryotic data that these independent classes of codon sets show very similar equilibrium patterns. The various results suggest a high level of uniformity in the process of silent fixation in the different synonymous-codon sets, especially in eukaryotes.

Amino Acid Sequence↗
PubMed1994

GABAA receptors in mouse cortical homogenates are phosphorylated by endogenous protein kinase A.

Biochemical, molecular, and electrophysiological studies suggest that phosphorylation of beta subunits of the GABAA receptor (GaR) by exogenous protein kinase A inactivates the receptor channels. We have developed a method which for the first time allows the study of GaR phosphorylation in brain tissues by endogenous PKA. Desalted homogenates or crude synaptic membranes from mouse cerebral cortex were incubated with [gamma-32P]ATP and 8-Br-cAMP or chlorophenylthio-cAMP. Extracts from these incubations were immunoprecipitated by polyclonal antibodies against native GaR and analyzed by SDS-gel electrophoresis and autoradiography. In both homogenates and membranes, cAMP-dependent incorporation of 32P was observed for a 57-kDa peptide, and to a lesser extent 51- to 53-kDa peptides. Phosphorylation of affinity-purified GaR by the catalytic subunit of PKA also produced a major 57-kDa phosphopeptide and a minor 51-kDa phosphopeptide. Limited digestion by S. aureus V-8 protease of the 57-kDa phosphopeptide from the desalted homogenates or from purified receptors produced a major 32P-labeled fragment of 11 kDa, suggesting that the phosphorylation site is similar to that shown previously to reduce GaR function. The phosphorylation of GaRs in homogenates was time dependent and blocked by H-89 or protein kinase inhibitor 5-24, specific inhibitors of protein kinase A. Prolonged incubations resulted in dephosphorylation of the 57-kDa phosphoprotein by a microcystin-LR sensitive phosphatase. In cortical homogenates the level of cAMP-dependent phosphorylation of the 57-kDa GaR peptide was more than 5 times that obtained with washed synaptic membranes. However, assays of PKA using the heptamer kemptide as substrate showed that the specific activity in the particulate fraction was 57% that of the homogenate. This suggests that GaRs on synaptic membranes are preferentially phosphorylated by a cytoplasmic form of protein kinase A. By comparing the [3H]flunitrazepam-photolabeled 53-kDa GaR subunit with the 51-57 kDa [32P]peptides from cortical homogenates, the molar ratio of [32P]/[3H] was estimated at 0.43, suggesting that a substantial fraction of the GaR pool is phosphorylated under these conditions.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2001

Dose homogeneity as a function of source activity in optimized I-125 prostate implant treatment plans.

PURPOSE: In conventional treatment planning for permanent I-125 prostate implants, it has been suggested that lower seed activities result in more homogeneous dose distributions and also less overdose of the critical structures. We sought to determine if this hypothesis holds by analyzing treatment plans constructed using an automated optimized approach. METHODS AND MATERIALS: We studied treatment plans for 10 patients using mixed-integer programming and the branch-and-bound method. Two mixed-integer models (that yielded somewhat different treatment plans) were developed: a "basic" model and a "dose homogeneity" model. For each resulting treatment plan, we examined dose homogeneity (by evaluating the dose non-uniformity ratio [DNR] and the full-width half-maximum [FWHM] of the differential dose-volume histogram [DVH]) as a function of three different source activities (0.35 mCi, 0.44 mCi, and 0.66 mCi). In addition, target coverage and critical structure dose distributions were evaluated. Plans using multiple source activities were also evaluated for resulting dose inhomogeneities. RESULTS: The homogeneity model results in a more homogeneous dose distribution than the basic model. DNR is lowered by an average of 42% (standard deviation [SD] = 19%), 39% (SD = 21%), and 33% (SD = 21%) for the 0.35 mCi, 0.44 mCi, and 0.66 mCi seeds, respectively, when the homogeneity model is employed over the basic model. Corresponding average decreases in the FWHM of the DVH for 0.35 mCi, 0.44 mCi, and 0.66 mCi, respectively, are 29 Gy (SD = 28 Gy), 24 Gy (SD = 22 Gy), and 27 Gy (SD = 13 Gy). Seeds of 0.35 mCi and 0.44 mCi result in the lowest DNR and narrower FWHM of the DVH relative to 0.66 mCi seeds. In general, the 0.44 mCi seeds produce greater target coverage and require fewer seeds and needles than the 0.35 mCi seeds. Although 0.66 mCi seeds result in the greatest target coverage, they yield highest critical structure doses. They also yield solutions requiring the least number of seeds and needles. However, the dose distributions from 0.66 mCi seeds are highly inhomogeneous. Multiple source activities in the same treatment plan produce dose distributions that are comparable in homogeneity to 0.44 mCi seed implants. CONCLUSIONS: Even when an optimization model that seeks to minimize dose inhomogeneity is employed, all factors involved in seed implants make 0.44 mCi the best activity choice in comparison with 0.35 mCi and 0.66 mCi.

Algorithms↗
PubMed1997

Reduction of pantethine in rabbit ocular lens homogenate.

In several animal models, preliminary studies have indicated that pantethine may inhibit cataract formation. Therefore, preclinical trials need to be conducted to study the pharmacology of pantethine in the ocular lens and to establish its efficacy. Since pantethine, which is a disulfide, can undergo a variety of chemical modifications such as reduction and formation of mixed disulfides, a detailed study was first conducted to determine the stability of pantethine in rabbit lens homogenate. A knowledge of the stability of pantethine in lens homogenate was necessary to establish if pantethine could be metabolized in the time it takes to harvest and homogenize a lens. The results of this study will be used to establish a protocol for harvesting and homogenizing lens samples. Pantethine (100 microM) is completely reduced to pantetheine in rabbit lens homogenate in about 16 min. About 1.5% of the pantethine added to lens homogenate forms a mixed disulfide with lens proteins, and the remainder is found in the supernatant. The supernatant pantethine concentration decreases exponentially as a function of time, and the terminal half-life for this process is 3.3 min. The free supernatant pantetheine concentration increases in pseudo first order manner as a function of time with a rate constant of 4.3 min. Pantethinase activity is not significant, because the free supernatant pantetheine concentration did not decrease. The exact mechanism of pantethine reduction in rabbit lens homogenate remains to be determined.

Animals↗
PubMed1996

Slurry preparation by high-pressure homogenization for the determination of heavy metals in zoological and botanical certified reference materials and animal feeds by electrothermal atomic absorption spectrometry.

High-pressure homogenization was evaluated for the preparation of slurries suitable for the determination by ETAAS of Cr, Cu, Fe, Mn, Ni and Se in soft organ tissues (liver and kidney), certified reference materials of biological and botanical origin and animal feeds. Frozen fresh organ tissue, (2 g) or certified reference material or dried, ground plant material (0.1 g) was blended, at high speed with 20 ml of ethanol-water (1 + 9 v/v) containing 0.25% m/m of tetramethylammonium hydroxide and the resulting mixture was subjected to homogenization at 38.9 MPa. After four passes through the homogenizer, the resulting solution was suitable for analysis by ETAAS. Capping the flat valve head of the homogenizer with a ruby disc appreciably reduced (but did not eliminate) metal contamination introduced by the processing. Homogenization of botanical reference materials or dried animal feeds resulted in preparations with variable amounts of residual fibres and particulate matter in the resulting suspensions. Nonetheless, all the Cu and Mn and virtually all of the Fe had been transferred to the supernatant fraction and remained with that fraction for at least 10 d. The addition of EDTA to the solvent modestly increased the mobilization of Fe from the matrix but also increased the contamination from the homogenizer. The slopes of the calibration curves generated by the method of standard additions were not significantly different from those of calibration curves generated with aqueous standards in a homogenized blank indicating that there was no significant matrix effect for any of the analytes in the nine reference materials, liver or kidney or the five animal feed samples and that aqueous standards could be used to calibrate the instrumental response.

Animal Feed↗
PubMed1968

The redox state of the nicotinamide-adenine dinucleotides in rat liver homogenates.

1. The redox state of the NAD couple of rat liver mitochondria, as measured by the [beta-hydroxybutyrate]/[acetoacetate] ratio, rapidly changed in the direction of oxidation during the preparation of homogenates in a saline medium. The value of the [beta-hydroxybutyrate]/[acetoacetate] ratio fell from 2.3 to 0.15 in 10min. EDTA diminished the fall and succinate prevented it. 2. The redox state of the rat liver cytoplasm, as measured by the [lactate]/[pyruvate] ratio, changed slightly in the direction of reduction during the preparation of homogenate. This was prevented by succinate. 3. In unsupplemented homogenates the differences in the redox states of mitochondria and cytoplasm decreased. Succinate and EDTA together maintained the differences within the physiological range. A measure of the ability of the mitochondria to maintain different redox states in mitochondria and cytoplasm is the value of the expression [lactate][acetoacetate]/[pyruvate][beta-hydroxybutyrate]. If there are no differences in the redox states of the NAD in the two cell compartments the value of the expression is 444 at 37 degrees . The value in the intact rat liver is between 4.7 and 21. 4. alpha-Oxoglutarate or glutamate were still more effective than succinate in maintaining high [beta-hydroxybutyrate]/[acetoacetate] ratios in the homogenates because these substrates supply a reducing agent of NAD(+) and, through succinate, an inhibitor of the oxidation of NADH. 5. When supplemented with alpha-oxoglutarate and EDTA, homogenates readily adjust the redox state of the beta-hydroxybutyrate dehydrogenase system after it has been upset by the addition of either acetoacetate or beta-hydroxybutyrate. 6. Amytal and rotenone raised the value of the [beta-hydroxybutyrate]/[acetoacetate] ratio. This is taken to indicate that the reduction of acetoacetate in the homogenates was not an energy-linked process. 7. 2,4-Dinitrophenol shifted the [beta-hydroxybutyrate]/[acetoacetate] ratio in the presence of succinate in favour of oxidation because it inhibited the oxidation of succinate and accelerated the oxidation of NADH. 8. Rotenone increased the rate of ketone-body formation of liver homogenates, though it decreased the rate of oxygen uptake.

Acetoacetates↗