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PubMed1994

A method for the determination of the cellular phosphorylation potential and glycolytic intermediates in yeast.

A method is described for rapidly quenching metabolism in yeast and extracting metabolites for analysis. The saponin digitonin is used to permeabilize the yeast cell membrane, in conjunction with perchloric acid (PCA) to quench metabolism and extract metabolites. Using this digitonin-PCA quench and extraction procedure, we have determined ATP concentrations in Saccharomyces cerevisiae in the range 2.89-3.39 mM, [ATP]/[ADP] ratios of 4.5-6.6, and phosphorylation potentials of 48.7-49.9 kJ.mol-1. A direct comparison with the currently accepted freeze/grind PCA/thaw procedure for extraction of yeast cell metabolites shows that essentially the same values are obtained by both techniques. The digitonin-PCA quench extraction method, used in conjunction with automated enzymatic analyses of metabolites, allows rapid extraction and analysis of large numbers of samples and metabolites and hence permits detailed investigations of intermediary metabolism in yeast.

Acetates↗
PubMed1997

Sampling tube device for monitoring intracellular metabolite dynamics.

Continuous sampling of microorganisms from a controlled bioreactor with rapid inactivation of metabolism and extraction of metabolites using precooled -40 degrees C perchloric acid solution (35%) was achieved with a sampling tube, thus fixing fast dynamic reactions at a certain position in the tube. After sampling was stopped (200 s) the tube was frozen at -80 degrees C and divided into identical parts and the extracted metabolites were analyzed enzymatically. A high resolution in time was achieved due to the axial dispersion of the metabolites in the sampling tube: The events of 1 s in the cells of the reactor were represented by 15 parts of the sampling tube. Axial dispersion was determined quantitatively with tracer measurements. The performance of the sampling tube device was evaluated with dynamic investigations on glucose-metabolism of Zymomonas mobilis. The dynamics of intracellular glucose 6-phosphate, glyceraldehyde 3-phosphate, and 3-phosphoglycerate concentrations were monitored after adding a glucose pulse to a glucose-limited steady-state culture.

Bioreactors↗
PubMed1998

Enzyme-based microassay for accurate determination of soman in blood samples.

Successful medical therapy for nerve agent intoxication requires early diagnosis and treatment. Current clinical diagnostic methods do not permit early or definitive confirmation of intoxication. To improve the chances of successful medical therapy against nerve agent intoxication, a sensitive enzyme-based microassay for rapid and accurate quantification of residual soman levels in blood was developed. The new analytical technique is based on the linear correlation between residual eel acetylcholinesterase activities and the inhibitor concentration. Blood samples were deproteinized with perchloric acid, followed by immediate neutralization after deproteinization. The mixtures were centrifuged at 3000g and the supernatant was directly assayed for soman. The sensitivity of the technique (18-1820 pg/ml blood) is comparable to that attained by GC-FID analysis (250 pg/ml blood). To facilitate routine analysis, the linear range of the assay was optimized to span over a factor of 100 (0.1-10 nM), with a typical correlation factor of at least 0.999 (six standards). The assay accuracy, checked with four different concentrations of soman, was within +/- 10%. The assay capability in monitoring the pharmacokinetic of soman was validated using both in vitro and in vivo rat models.

Acetylcholinesterase↗
PubMed1998

Proteinase K digestion of proteins improves detection of bacterial endotoxins by the Limulus amebocyte lysate assay: application for endotoxin removal from cationic proteins.

Cationic proteins, such as lysozyme, ribonuclease A, and human IgG, impaired the detection of endotoxins with the Limulus amebocyte lysate assay (LAL assay) through formation of endotoxin-protein complexes, demonstrating pronounced masking of endotoxins. Methods, such as phenol extraction, dilution heating, and perchloric acid treatment failed to demask the endotoxins. Also, digestion with trypsin, chymotrypsin, or pronase recovered only 10 to 20% of the applied endotoxins. However, endotoxin recoveries up to 100% were obtained with proteinase K digestion of the samples prior to the LAL assay. This method was then applied to examine the impact of endotoxin masking on endotoxin removal from protein solutions by selective adsorption on membrane adsorbers. It was found that poly-L-lysine and poly(ethyleneimine) as endotoxin-selective ligands were able to pull endotoxins off the proteins studied, thereby guaranteeing successful decontamination.

Adsorption↗
PubMed1999

13C-Isotopic enrichment of glutathione in cell extracts determined by nuclear magnetic resonance spectroscopy.

An NMR method was developed for measuring the isotopic enrichment of glutathione in extracts of cells fed a medium containing [3, 3'-13C2]cystine. Two sublines of human mammary adenocarcinoma MCF-7 cells were exposed to growth medium containing the labeled cystine for varying periods, treated with monobromobimane, harvested, and extracted with perchloric acid. The glutathione-bimane adduct was partially purified by solid-phase extraction before analysis by 1H NMR spectroscopy. The isotopic enrichment of the beta-carbon of the cysteinyl residue of glutathione was determined directly in the cell extracts without further purification. These isotopic enrichment data can be used to determine the rate of synthesis of glutathione in cell and tissue extracts.

Adenocarcinoma↗
PubMed1999

Automated sampling device for monitoring intracellular metabolite dynamics.

An automated sampling device coupled to a stirred tank reactor was developed for monitoring intracellular metabolite dynamics. Sample flasks fixed in transport magazines were moved by a step engine in a way that each sample flask was filled within 220 ms, resulting in a sampling rate of 4.5 s-1. Rapid inactivation of the metabolism was achieved by spraying the samples into 60% methanol at -50 degrees C. After centrifugation of the quenched cells at -20 degrees C the metabolites were extracted with perchloric acid and analyzed biochemically or with HPLC. The automated sampling device was applied for investigation of the intracellular metabolite dynamics of glycolysis in Escherichia coli after rapid glucose addition to a glucose-limited steady-state culture. For the first time oscillations of intracellular metabolite concentrations like glucose-6-phosphate, phosphoenolpyruvate, glyceraldehyde 3-phosphate, dihydroxyacetonphosphate, 3-phosphoglycerate, and pyruvate were quantified on a subseconds to seconds scale in E. coli. As an example, the kinetics of the decomposition of fructose 1, 6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetonphosphate were investigated by use of a well-known mechanistic kinetic model and the measured in vivo metabolite dynamics.

Biosensing Techniques↗
PubMed1999

Identification and quantitation of phosphorus metabolites in yeast neutral pH extracts by nuclear magnetic resonance spectroscopy.

(31)P NMR spectroscopy offers a possibility to obtain a survey of all low-molecular-weight phosphorylated compounds in yeast. The yeast cells have been extracted using chloroform into a neutral aqueous phase. The use of high fields and the neutral pH extracts, which are suitable for NMR analysis, results in well-resolved (31)P NMR spectra. Two-dimensional NMR experiments, such as proton-detected heteronuclear single quantum ((1)H-(31)P HSQC) and (31)P correlation spectroscopy ((31)P COSY), have been used to assign the resonances. In the phosphomonoester region many of the signals could be assigned to known metabolites in the glycolytic and pentose phosphate pathways, although some signals remain unidentified. Accumulation of ribulose 5-phosphate, xylulose 5-phosphate, and ribose 5-phosphate was observed in a strain lacking transketolase activity when grown in synthetic complete medium. No such accumulation occurred when the cells were grown in yeast-peptone-dextrose medium. Trimetaphosphate (intracellular concentration about 0.2 mM) was detected in both cold methanol-chloroform and perchloric acid extracts.

Culture Media↗
PubMed1999

Assay of dihydrofolate reductase activity by monitoring tetrahydrofolate using high-performance liquid chromatography with electrochemical detection.

We developed a method to determine dihydrofolate reductase (DHFR) activity at pH 7.4 (37 degrees C) by monitoring its product, tetrahydrofolate (H(4)folate), using HPLC with electrochemical detection. After the assay mixture was deproteinized by 0.5 M perchloric acid, the H(4)folate concentration was measured. Using sodium ascorbate at 20 mM, H(4)folate was stable in our assay system. The enzyme activity was also stable. The detection limit of this method was less than 1 nM of H(4)folate in the enzyme assay system, which was 1/100 lower than those for the NADPH-spectrophotometric assay, which is commonly used for analysis of DHFR activity. This value of 1 nM allowed us to control the conversion from dihydrofolate (H(2)folate) to H(4)folate less than 10% of initial substrate concentrations during assay, when we used a concentration around K(m) values reported for DHFR from various sources. The rate of reduction showed a linearity at concentrations around the K(m). The reduction rate must be evaluated exactly around the K(m), in order to obtain an accurate profile of Michaelis-Menten kinetics. This assay method has a sensitivity high enough to determine the reduction rate at H(2)folate concentrations around K(m). In addition, the assay procedure is very simple. Therefore, our method may be useful for studying DHFR.

Animals↗
PubMed2001

A highly sensitive method for measurement of myosin ATPase activity by reversed-phase high-performance liquid chromatography.

A new method for measurement of myosin ATPase activity has been developed utilizing reversed-phase high-performance liquid chromatography (HPLC), which detects as low as 0.05 nmol of ADP hydrolyzed from ATP. After termination of the ATPase reaction by addition of perchloric acid, the hydrolysate ADP and substrate ATP were separated by reversed-phase HPLC. The absorbance of ADP was monitored at 259 nm, and the amount of ADP was quantified from its peak area on the chromatogram by use of the NIH Image computer software. Our method showed linearity over a wide range from 0.05 to 10 nmol of ADP per 20 microl with a coefficient of determination (r(2)) of 0.99. Myosin ATPase activities determined by the HPLC method were almost identical to those determined by the malachite green method, a widely used spectrophotometric method with range of detection from 1 to 8 nmol of phosphate. Because our method requires only a small volume of reaction solution, it will be a powerful tool for measuring ATPase activity of motor proteins, which are difficult to obtain in large amount.

Adenosine Diphosphate↗
PubMed2001

Quantification of intracellular metabolites in Escherichia coli K12 using liquid chromatographic-electrospray ionization tandem mass spectrometric techniques.

The quantitative comprehension of microbial metabolic networks is a prerequisite for an efficient rational strain improvement ("metabolic engineering"). It is therefore necessary to accurately determine the concentration of a large number of reactants (i.e., metabolites, nucleotides, cofactors) in order to understand "in vivo" reaction kinetics. Quantification of intracellular concentrations of glycolytic intermediates and nucleotides in Escherichia coli K12 using a perchloric acid extraction and an LC-ESI-MS method was achieved. Intracellular metabolites (e.g., glucose 6-phosphate, fructose 1,6-bisphosphate, 6-phospho gluconate, acetyl-CoA, adenine nucleotides) were quantified under defined (glucose-limited steady-state) growth conditions. The method was verified by comparing the intracellular metabolite concentrations measured via LC-ESI-MS with enzymatic determinations. It is thus possible to identify and quantify more than 15 intracellular metabolites in parallel with a minimal amount of sample volume.

Acetyl Coenzyme A↗
PubMed2001

Assay of the concentration and 13C-isotopic enrichment of malonyl-coenzyme A by gas chromatography-mass spectrometry.

We developed gas chromatography-mass spectrometry assays for the concentration and mass isotopomer distribution of malonyl-CoA in tissues. The assay involves perchloric acid extraction of the tissue, spiking the extract with [U-13C3]malonyl-CoA or dimethylmalonyl-CoA internal standard, isolation of short-chain acyl-CoA fraction on an oligonucleotide purification cartridge, alkaline hydrolysis to malonate, trimethylsilyl derivatization, and analysis of the mass isotopomer distribution of malonate. The assay was applied to labeling of malonyl-CoA from various [13C]substrates in perfused rat livers and hearts. In livers perfused with [1,2-13C2]acetate, malonyl-CoA is doubly labeled from [1,2-13C2]acetate and singly labeled from 13CO2. In livers perfused with either NaH13CO3 or [3-13C]lactate + [3-13C]pyruvate, the half-lives of singly labeled malonyl-CoA were less than 20 s and 6.95 min, respectively. In rat heart, the half-life of malonyl-CoA, traced with NaH13CO3, was about 1.25 min. Thus, our assay allows us to measure the turnover of tissue malonyl-CoA, the contribution of various [13C]substrates to its production in lipogenic and nonlipogenic organs, and the cycling between acetyl-CoA and malonyl-CoA in nonlipogenic organs.

Acetates↗
PubMed2002

Assay of the concentration and (13)C isotopic enrichment of propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA by gas chromatography-mass spectrometry.

We developed gas chromatography-mass spectrometry assays for the concentration and mass isotopomer distribution of propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA in tissues. The assays involves perchloric acid extraction of the tissue, spiking the extract with [(2)H(5)]propionyl-CoA and [(2)H(4)]succinyl-CoA internal standards, and isolation of short-chain acyl-CoA fraction on an oligonucleotide purification cartridge. Propionyl-CoA is reacted with sarcosine and the formed N-propionylsarcosine is assayed as its pentafluorobenzyl derivative. Methylmalonyl-CoA and succinyl-CoA are hydrolyzed and the corresponding acids assayed as tert-butyl dimethylsilyl derivatives. The assay was applied to a study of [U-(13)C(3)]propionate metabolism in perfused rat livers. While propionyl-CoA is only M3 labeled, succinyl-CoA is M3, M2, and M1 labeled because of isotopic exchanges in the citric acid cycle. Methylmalonyl-CoA is M3 and M2 labeled, reflecting reversal of S-methylmalonyl-CoA mutase. Thus, our assays allow measuring the turnover of the coenzyme A derivatives involved in anaplerosis of the citric acid cycle via precursors of propionyl-CoA, i.e., propionate, odd-chain fatty acids, isoleucine, threonine, and valine.

Acyl Coenzyme A↗
PubMed1994

A simple HPLC method for the determination of S-adenosylmethionine and S-adenosylhomocysteine in rat tissues: the effect of vitamin B6 deficiency on these concentrations in rat liver.

A simple method using isocratic HPLC with ultraviolet detection was established for the simultaneous measurement of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) in rat tissues. The method provides rapid resolution of both compounds in a single run by direct injection of the perchloric acid extract of tissue, so that sampling procedures and analytical errors can be reduced in determining the SAM/SAH ratio, a measure of transmethylation reactions. The assay has a detection limit of 25 pmol and is linear in the range 50-200 pmol. This procedure was applied to determine the change of SAM/SAH ratio in liver caused by vitamin B6 deficiency. The marked reduction of the SAM/SAH ratios in vitamin B6-deficient rats were due to a drastic rise in SAH concentration with a concurrent striking decrease in SAM concentration.

Animals↗
PubMed2001

(31)P NMR of apicomplexans and the effects of risedronate on Cryptosporidium parvum growth.

High-resolution 303.6 MHz (31)P NMR spectra have been obtained of perchloric acid extracts of Plasmodium berghei trophozoites, Toxoplasma gondii tachyzoites, and Cryptosporidium parvum oocysts. Essentially complete resonance assignments have been made based on chemical shifts and by coaddition of authentic reference compounds. Signals corresponding to inorganic pyrophosphate were detected in all three species. In T. gondii and C. parvum, additional resonances were observed corresponding to linear triphosphate as well as longer chain polyphosphates. Spectra of P. berghei and T. gondii also indicated the presence of phosphomonoesters and nucleotide phosphates. We also report that the pyrophosphate analog drug, risedronate (used in bone resorption therapy), inhibits the growth of C. parvum in a mouse xenograft model. When taken together, our results indicate that all the major disease-causing apicomplexan parasites contain extensive stores of condensed phosphates and that as with Plasmodium falciparum and T. gondii, the pyrophosphate analog drug risedronate is an inhibitor of C. parvum cell growth.

Animals↗
PubMed1996

Proton nuclear magnetic resonance spectroscopy of lactate production in isolated rat liver during cold preservation.

Lactate-edited 1H NMR difference spectra have been acquired from intact rat liver tissue following flushing and preservation in ice. A peak, initially at 1.26 ppm, was seen to increase in the liver tissue with preservation time. This peak was assigned to lactate, despite the fact that its chemical shift was initially shifted by approximately -0.1 ppm relative to an externally added standard. The assignment was based on the following: (a) the peak increased over a 24-h ischemic storage period; (b) it was coupled to a signal 2.78 +/- 0.02 ppm upfield; and (c) a parallel increase in lactate was noted in perchloric acid extracts of tissue from the same liver. An additional peak, assigned to alanine, was also observed during storage and was also shifted by approximately -0.1 ppm. Inclusion of dimethyl sulfoxide, which readily permeates liver tissue, demonstrated that this chemical shift alteration was a tissue-specific effect. These results demonstrate that 1H NMR spectroscopy of intact liver tissue during hypothermic ischemia is possible, though chemical shift assignments should be made with caution.

Animals↗
PubMed1998

Metabolic effects of citrate in liver during cold hypoxia studied by 1H NMR spectroscopy.

We propose the use of 1H nuclear magnetic resonance (NMR) spectroscopy to investigate metabolite fluxes in the mammalian liver during cold hypoxia. Rat livers were flushed with one of four different preservation solutions and stored on ice in the same solution. The preservation solutions were: Marshall's hypertonic citrate (HC); carnosine modified HC (HC-C); modified University of Wisconsin (mod UW); and Bretschneider's histidine--typtophan--ketoglutarate (HTK). Liver biopsies were taken before and at 1, 2.5. 4, 24, and 48 h after storage, and freeze-clamped. The liver was extracted with perchloric acid and analyzed by 1H NMR spectroscopy. Components of the individual preservation solutions, such as citrate, histidine, mannitol, and raffinose, were detected in the extracts. Lactate was increased over the first 4 h in all stored livers, but only continued to increase in those stored in HC-C and HTK, reaching significantly high levels of 15 and 14 mumol/g, respectively, by 48 h storage (P < 0.05 and P < 0.01, respectively). Levels of succinate and fumarate in all livers were generally unchanged in the first 0-4 h of storage. However, after 4 h of storage, succinate levels rose in the HC and HC-C livers, while remaining unchanged in mod UW and HTK livers. The presence of citrate in the preservation solutions appeared to enhance the late hepatic synthesis of succinate. Fumarate levels were significantly decreased by 48 h of cold storage, indicating continued fumarate consumption at low temperatures. Despite cold hypoxic conditions, some carbon-substrate cycling appears to continue in mammalian liver via pathways other than glycolysis, and citrate from the preservation solution appears to influence this.

Adenosine↗
PubMed1998

Dexamethasone and dexamethasone phosphate detected by 1H and 19F NMR spectroscopy in the aqueous humour.

To apply nuclear magnetic resonance (NMR) spectroscopy to study the penetration of dexamethasone phosphate into the aqueous humour from rabbit following topical administration. After topical administration of 0.1%, 1.0% and 10% dexamethasone phosphate solutions, respectively, samples of aqueous humour were aspirated, freeze-dried, redissolved in deuterium oxide and analyzed by high resolution 1H and 19F NMR spectroscopy. In order to study the lipophilic and hydrophilic metabolites of the drug, samples obtained after application of 1% dexamethasone phosphate were extracted with methanol/chloroform, and then extracted with perchloric acid. In all samples obtained from eyes denuded of the corneal epithelium prior to administration of dexamethasone, signals corresponding to the chemical shifts of the drug were identified in 19F NMR spectra. In the experiments performed with 1% dexamethasone phosphate, both dexamethasone and dexamethasone phosphate were detected in the aqueous humour. Using 10% dexamethasone phosphate solutions, signals from the drug were detected in 1H NMR spectra simultaneously with signals from about twenty other substances present in the aqueous humour. NMR spectroscopy appears to be a valuable method for studying dexamethasone metabolism and penetration into ocular tissues. It provides simultaneous detection of both the drug metabolites and other substances in the sample and might offer a complementary approach to other analytical methods.

Administration, Topical↗