Carbohydrate metabolism in erythrocytes of copper deficient rats.

Dietary copper deficiency is known to adversely affect the circulatory system of fructose-fed rats. Part of the problem may lie in the effect of copper deficiency on intermediary metabolism. To test this, weanling male Long-Evans rats were fed for 4 or 8 weeks on sucrose-based diets containing low or adequate copper content. Copper deficient rats had significantly lower plasma and tissue copper as well as lower plasma copper, zinc-superoxide dismutase activity. Copper deficient rats also had a significantly higher heart:body weight ratio when compared to pair-fed controls. Direct measurement of glycolysis and pentose phosphate pathway flux in erythrocytes using (13)C NMR showed no differences in carbon flux from glucose or fructose to pyruvate but a significantly higher flux through the lactate dehydrogenase locus in copper deficient rats (approximately 1.3 times, average of glucose and glucose + fructose measurements). Copper-deficient animals had significantly higher erythrocyte concentrations of glucose, fructose, glyceraldehyde 3-phosphate and NAD(+). Liver metabolite levels were also affected by copper deficiency being elevated in glycogen and fructose 1-phosphate content. The results show small changes in carbohydrate metabolism of copper deficient rats.

Vibrational and NMR spectroscopic study of aged flurazepam mono- and dihydrochloride salts for content identity.

Archival samples of flurazepam monohydrochloride and "hydrochloride" (i.e., the dihydrochloride) were examined by Fourier transform infrared and Raman spectroscopy to determine evidence of degradation during storage for 13-15 years. No degradation of the three different batches of monohydrochloride salts was detected, but various degrees of degradation of the eight specimens of flurazepam hydrochloride diprotonated salts were indicated by enhanced intensities (IR 1635, 1509, 1226; Raman 1636, 1408, 1149 cm-1) and new features (IR 1742, 943, 755; Raman 1554, 837, 742 cm-1). All of these features, except the 1742 cm-1 IR band, were attributed to the presence of the hydrolysis product 5-chloro-2-[[2-(diethylamino)ethyl]amino]-2'-fluorobenzophenone hydrochloride whereas the 1742 cm-1 band was attributed to glycine hydrochloride, the other hydrolytic moiety. The flurazepam hydrochloride samples were also examined in deuterated dimethyl sulfoxide solution by proton nuclear magnetic resonance (1H-NMR) spectroscopy to verify the presence of the degradation products and to estimate the levels of degradation (approximately 3-36%) of the drug. IR and Raman spectra of the "benzophenone" hydrochloride in the "fingerprint" region are compared with two samples of flurazepam dihydrochloride (slightly and highly degraded) and their features discussed. Vibrational assignments are made and discussed for the observed IR and Raman wavenumbers for the "benzophenone" hydrochloride.

Toxicity of structurally related anthraquinones and anthrones to mammalian cells in vitro.

Anthraquinones and structurally related compounds were cytotoxic to mammalian cell lines using cloning efficiency and MTT reduction as endpoints. In V79 cells, the concentration of chemical causing 50% inhibition ranged from 0.21 to 21.6 mug/ml for cloning efficiency and from 0.86 to 14.6 mug/ml for MTT reduction. The anthrones anthralin and chrysarobin were 4.1 and 3.2 times more toxic, respectively, in the cloning efficiency assay than in the MTT assay. In contrast, the anthraquinones danthron and emodin were 2.8 and 2.1 times more toxic, respectively, in the MTT assay than in the cloning efficiency assay. Among the four mammalian cell lines tested using the MTT assay, the human leukaemia cell line (K562) was the most sensitive to the test chemicals. In contast, anthraquinone toxicity was reduced in rat hepatoma (H4IIE) cultures. In general, structures with carbonyl groups in positions 9 and 10 on the anthracene skeleton (anthraquinones) were less toxic than structures with carbonyl groups in position 9 only (anthrones). Toxicity was also influenced by the position of hydroxy substituents on the tricyclic skeleton. The results suggested that in vitro cytotoxicity assays are useful in elucidating the relationships between structure and biological activity for anthraquinones and related compounds.

Utilizing nuclear magnetic resonance (NMR) spectroscopy for assessing nadolol racemate composition.

NMR methods were developed for the determination of the racemate composition in nadolol raw materials. With high-field instruments (400 MHz or greater) the racemate ratio may be determined by the relative heights of the t-butyl peaks, which are well enough resolved for this determination. For lower field spectrometers, the t-butyl peaks are not resolved. An NMR method has been developed which involves preparation of the tribenzoate derivative of the drug. Seven lots of nadolol raw material, as well as several standards, were analysed for their racemate content. Three lots of raw material did not meet the USP limits of 40-60% for racemate A. Of these, two were granular in appearance and were found to vary markedly in racemate composition in successive analyses. The results for all the materials of uniform content agree very well with those from the HPLC method, as well as for the USP IR method using the absorbance at the corrected wavelength.

Chromatographic and spectroscopic characterization of sulphur-bound dimetridazole and ranidazole derivatives.

The 5-nitroimidazoles, dimetridazole and ronidazole, two important veterinary drugs, were reacted under reductive conditions with the sulfhydryl-containing substrates cysteine and glutathione to yield 5-amino-4-S-substituted imidazoles. After purification by reversed-phase liquid chromatography (RP-LC), the four adducts were characterized by RP-LC with photodiode array detection using conditions where their parent drugs were not eluted from the column. Structural identification was conducted by spectroscopic techniques, mainly 1-dimensional and 2-dimensional NMR. While the dimetridazole adducts were found to be monosubstituted at the C-4 position, the two ronidazole products contained two units of the sulfhydryl substrate, located at the C-4 and C-6 positions.

Multinuclear NMR (1H, 13C and 19F) spectroscopic re-examination of the solvolytic behaviour of flurazepam dihydrochloride.

The dissolution of reference and archival samples of flurazepam dihydrochloride (2) was studied in DMSO-d6 and in D2O by 1H-, 13C- and 19F-NMR spectroscopy to identify and distinguish solvated species of the parent drug (2), the "benzophenone" (4) and glycine (5) hydrochloride degradation products. In DMSO-d6, for most samples, only the ring intact form (2) could be detected by 13C-NMR whereas the inherently greater sensitivity of 19F-NMR allowed detection of initial trace amounts (< 1%) of the open-ring form (3). 19F-NMR spectroscopy also afforded the best means of quantifying the various entities in solution, including the increase towards equilibrium levels of the open-ring entity and detection/quantitation of a new equilibrium species, possibly the cis/trans rotamer of the open-chain entity (3). Various chemical shifts for flurazepam dihydrochloride and USP flurazepam related reference standards C and F are reported for DMSO-d6 solutions. The bases for 1H- and 19F-NMR assay of DMSO-d6 solutions of (2) for (4) are discussed with comparative data. The solvation characteristics of (2) in D2O at 0 and 27 degrees C were found to be too complex to follow by 13C-NMR; however, 19F-NMR studies at these temperatures permitted one to clearly discern that no additional formation of entity (4) occurred beyond whatever initial levels were present in degraded samples while the open-ring entity (3) was observed to increase to an equilibrium level of 56% over 24 h at 27 degrees C. Dissolution in D2O at either 0 or 27 degrees C does not contribute to solvolytic degradation of (2) to (4) over 24 h.

Preparative liquid chromatographic separation of the oligomers of nonoxynol-9 and their characterization by 1H-NMR and mass spectroscopy.

Conditions are described for the preparative LC separation of a bulk nonoxynol-9 material into 16 components. 1H-NMR analyses of these fractions provided evidence for all but the first of the components to be oligomers of nonylphenoxypolyethoxyethanol (nonoxynol) with n-values for (OCH2CH2)n ranging consecutively from 3 to 17, corresponding to the LC fractions 2-16. Mass spectral analysis of the separated LC fractions confirmed the oligomeric sizes deduced from 1H-NMR spectral data, and provided EI fragmentation information for these oligomeric substances.

Spectral characterization of fluconazole.

Reference 1H, 19F and 13C NMR, mass, IR and Raman spectra are provided to the open literature for the first time for the potent antifungal agent fluconazole, alpha-(2,4-difluorophenyl)-alpha-(1H-1,2,4-triazol-1-ylmethyl++ +)-1H-1,2, 4-triazole-1-ethanol. The 1H, 19F and 13C NMR spectra were analyzed in detail to attribute shifts, including 19F chemical shifts and C-F and F-F coupling constants. The EI mass spectrum, although rich in fragment ions, lacked a molecular ion. FAB and MS/MS experiments were undertaken in support of the structure in order to validate the EI spectrum as a reference mass spectrum. IR and Raman spectra are compared to show the complementary nature of their features and discussed in terms of principal group vibrations. NMR and vibrational data together with assignments are summarized in tabulated form for convenience of use. All these data are consistent with the structure of fluconazole.

Fenofibrate raw materials: HPLC methods for assay and purity and an NMR method for purity.

HPLC methods for drug content and HPLC and NMR methods for related compounds in fenofibrate raw materials were developed. The HPLC methods resolved 11 known and six unknown impurities from the drug. The HPLC system was comprised of a Waters Symmetry ODS column (100 x 4.6 mm, 3.5 microm), a mobile phase consisting of acetonitrile water trifluoroacetic acid 700/300/l (v/v/v) at a flow rate of 1 ml min(-1). and a UV detector set at 280 nm. Minimum quantifiable amounts were about 0.1% for three of the compounds and less than 0.05% for the other eight. Individual impurities in 14 raw materials ranged from trace levels to 0.25%, and total impurities from 0.04 to 0.53% (w/w). Six unknown impurities were detected by HPLC, all at levels below 0.10%, assuming the same relative response as fenofibrate. An NMR method for related compounds was also developed and it was suitable for 12 known and several unknown impurities. It requires an NMR of 400 MHz, or greater, field strength. Individual impurities in the raw materials analyzed ranged from trace levels to 0.24%, and total impurities from trace levels to 0.59%. Several lots contained small amounts of unknown impurities at trace levels. Three lots, all from the same manufacturer, contained an unknown impurity, not detectable by HPLC, which was not present in the other raw materials. It was estimated to be present at a level greater than 0.2%. The results for related compounds by the two techniques were consistent. The main differences stem from the low sensitivity of the HPLC method for some of the related compounds at 280 nm, or from the higher limits of quantitation by the NMR method for several other impurities using the conditions specified. A fifteenth raw material was not homogeneous in its content of impurity VI, a synthetic intermediate and possible degradation product. The HPLC/MS results provided information on the peak purity (number of components) for minor HPLC peaks, as well as structural data such as the molecular ions and diagnostic fragment ions. The HPLC/MS results showed that there were five unknown drug related impurities, for which there were no standards available. Results for the assay of 15 raw materials by HPLC were within the range 98.5-101.5%.

Parthenolide content and bioactivity of feverfew (Tanacetum parthenium (L.) Schultz-Bip.). Estimation of commercial and authenticated feverfew products.

Three physicochemical methods (HPLC, NMR spectroscopy, and HPLC of a derivative) have been used to measure parthenolide in authenticated Tanacetum parthenium (feverfew) and in several commercial purported feverfew products. A bioassay based on inhibition of the secretory activity of blood platelets by extracts of feverfew in comparison with parthenolide was also used. Similar results were obtained for all three physicochemical assays and also for the bioassay. Thus different methodologies yield consistent values for parthenolide content of feverfew preparations. Parthenolide appears to be mainly responsible for the antisecretory effects of extracts of feverfew. Authenticated Tanacetum parthenium grown in the UK contained a high level of parthenolide in leaves, flowering tops and seeds but a low level in stalks and roots. The level of parthenolide in powdered leaf material fell during storage. The purported feverfew products varied widely in their parthenolide content and in some products parthenolide was not detected. Possible reasons for the variation in parthenolide content are discussed. Since therapeutic efficacy has only been demonstrated for preparations of feverfew that contain parthenolide, it is suggested that manufacturers of feverfew products should use measurements of parthenolide as a means of standardization and quality control.

Chiral identification and determination of ephedrine, pseudoephedrine, methamphetamine and methcathinone by gas chromatography and nuclear magnetic resonance.

The enantiomers of the related substances methamphetamine, ephedrine, pseudoephedrine and methcathinone were determined by both gas chromatography after derivatization and by nuclear magnetic resonance using a chiral solvating agent. For GC the substances were derivatized with (R)-(+)-alpha-methoxy-alpha-(trifluoromethyl)phenylacetic acid (MTPA) to give diasteromeric derivatives. Resolution (baseline) of at least 1.6 was obtained between all derivatives. NMR determination of the enantiomers was conducted in a chiral environment by the addition of the chiral solvating agent, (R)-(+)-1,1'-bi-2-naphthol, to NMR solutions of the substances. Racemization of methcathinone was demonstrated to be facile by exposure to alkaline solutions for varying periods of time. Enantiomeric ratios of some products derived from the oxidation of ephedrine were determined.

Proposed phytic acid standard including a method for its analysis.

A method is described that accurately and rapidly quantifies the free and total phosphorous content of a commercially available, purified, phytic acid preparation. This allows its use as a standard for phytic acid determinations in foods. The method involves a wet ashing step followed by phosphorous measurement with a 1-amino-2-naphthol-4-sulfonic acid-molybdate reagent in a microplate reader at 660 nm. The procedure can be performed in 3 h with as little as 50 mg sample.

Immunocytochemical localization of transient DNA strand breaks in differentiating myotubes using in situ nick-translation.

We have localized DNA strand breaks during in vitro chicken myogenesis by repairing nicks in nuclei of fixed cell monolayers in situ with biotin-11-dUTP, followed by immunocytochemical detection of incorporated biotin with rabbit anti-biotin and FITC-labeled goat anti-rabbit antibodies. No accumulations of biotin sufficient for immunocytochemical detection were observed in 23-hr cultures of dividing cells. In 33- and 43-hr cultures, biotin was first detected in only 3% of the nuclei, all of which appeared to be in fusing myoblasts or small myotubes. In contrast, cultures of young, highly fused myotubes (56 hr) exhibited 18% biotinylated nuclei; virtually all of these nuclei, most of which were grouped as aggregates, were within myotubes. In older cultures (73 and 94 hr) incorporation of biotin into myotube nuclei markedly decreased, while increases were noted in nuclei of mononuclear cells. These results indicate that extensive single-stranded DNA nicking occurs in nuclei of young myotubes, followed by repair as terminal differentiation ensues.

Analysis of piperazine drug formulations for N-nitrosamines.

A quantitative method is described for the measurement of N-mononitrosopiperazine (NPIP) and N,N'-dinitrosopiperazine (DNPIP) in drug formulations containing piperazine, using a gas chromatograph interfaced to a thermal energy analyzer (GC/TEA). The method has detection limits of 20 ppb for NPIP and 12 ppb for DNPIP. In a survey of 6 products available on the Canadian market, all contained NPIP at levels of 0.38-15.3 micrograms NPIP/g piperazine and none contained any detectable amount of DNPIP.

Analysis of selected drug formulations for volatile nitrosamines.

A number of selected drug formulations on the Canadian market were analyzed for N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) content by using a gas chromatograph interfaced to a thermal energy analyzer (GC/TEA). Of the 34 formulations analyzed, NDMA was found in 21 at levels up to 12 ppb and NDEA was found in 11 at levels up to 0.6 ppb. In many cases, the drug entity in the formulation did not contain the precursors for the observed nitrosamines.

Affinity isolation of transcriptionally active murine erythroleukemia cell DNA using a cleavable biotinylated nucleotide analog.

We have developed an affinity technique to obtain active gene domains from murine erythroleukemia cell nuclei, based on the differential sensitivity of potentially active and inactive chromatin to DNase I. Nuclei isolated from potentially active noninduced cells and transcriptionally active induced MEL cells were treated with DNase I at concentrations which did not digest the beta-globin gene, followed by repair using a typical nick translation reaction during which a cleavable biotinylated nucleotide analog, 5-[N-biotinamido)hexanoamido-ethyl-1,3-dithiopropionyl -3-aminoallyl]-2'- deoxyuridine 5'-triphosphate (Bio-19-SS-dUTP), was inserted into DNA sequences. Following purification and digestion with EcoRI restriction endonuclease, biotinylated sequences were affinity isolated by sequential binding to streptavidin and biotincellulose. The streptavidin/biotin-cellulose complex bound up to 80% of the nick-translated DNA, which comprised a small percentage of the total nuclear DNA. Cleavage of the disulfide bond in the linker arm of the biotinylated nucleotide resulted in elution of virtually all of the affinity isolated sequences. Hybridization analysis of this fraction of DNA revealed up to a 16-fold enrichment for the active beta-globin gene, as compared with DNA which did not bind to the biotincellulose. Conversely, the inactive alpha-fetoprotein gene was barely detectable in affinity isolated DNA from noninduced cells and was 2-fold depleted in samples from induced cells.

Affinity isolation of active murine erythroleukemia cell chromatin: uniform distribution of ubiquitinated histone H2A between active and inactive fractions.

This laboratory recently reported the development of a biotin-cellulose/streptavidin affinity chromatography method based on the DNase I sensitivity of active chromatin to isolate a DNA fraction from murine erythroleukemia (MEL) cells that is more than 15-fold enriched in active genes (Dawson et al.: Journal of Biological Chemistry 264:12830-12837, 1989). We now report the extension of this technique to isolate and characterize chromatin that is enriched in active genes. In this approach, DNA in nuclei isolated from MEL cells was nicked with DNase I at a concentration that does not digest the active beta-globin gene, followed by repair of the nicks with a cleavable biotinylated nucleotide analog, 5-[(N-biotin-amido)hexanoamido-ethyl-1,3'-dithiopropionyl-3- aminoallyl]-2'- deoxyuridine 5'-triphosphate (Bio-19-SS-dUTP), during a nick-translation reaction. After shearing and sonication of the nuclei to solubilize chromatin, chromatin fragments containing biotin were separated from non-biotinylated fragments by sequential binding to streptavidin and biotin cellulose. The bound complex contained approximately 10% of the bulk DNA. Reduction of the disulfide bond in the biotinylated nucleotide eluted approximately one-half of the affinity isolated chromatin. Hybridization analysis of DNA revealed that whereas inactive albumin sequences were equally distributed among the chromatin fractions, virtually all of the active beta-globin sequences were associated with chromatin fragments which had bound to the affinity complex. Western blot assessment for ubiquitinate histones revealed that ubiquitinated histone H2A (uH2A) was uniformly distributed among active (bound) and inactive (unbound) chromatin fractions.

In vitro assessment of cytotoxicity and biotransformation of propranolol in Cunninghamella echinulata.

1. Biotransformation studies with five concentrations of racemic propranolol were conducted using the filamentous fungus Cunninghamella echinulata ATCC 9244. 2. The rate of formation and subsequent disappearance of a new major metabolite, 8-hydroxypropranolol, was dose-dependent. Desisopropylpropranolol and 4-hydroxypropranolol were also formed. 4-Hydroxypropranolol was the major fungal metabolite in earlier studies. 3. Propranolol exerted a dose-dependent response on biotransformation, fungal growth, dextrose utilization, ammonia formation and incubation broth pH. Determination of dextrose utilization and incubation broth pH would provide reliable, cost-effective and convenient alternative methods for cytotoxicological evaluation.

Temperature regulation of glucose metabolism in red blood cells of the freeze-tolerant wood frog.

The low-temperature metabolism of erythrocytes from the freeze-tolerant frog Rana sylvatica was investigated by (13)C and (31)P NMR spectroscopy. Erythrocytes readily took up high concentrations of the natural cryoprotectant, glucose, at both high (12 and 17 degrees C) and low (4 degrees C) temperatures but glucose was apparently not metabolized at 4 degrees C. Strong inhibition of glucose catabolism at low temperature would facilitate the maintenance of the very high concentrations of glucose (approximately 200 mM) that are accumulated to provide cryoprotection during freezing in wood frogs. Analysis of (13)C labeling of glycolytic intermediates at 4 degrees C showed mixing of label primarily in hexose (fructose) and hexose phosphate (glucose 6-phosphate, fructose 6-phosphate) pools but little label incorporation into triose phosphate intermediates. These data are consistent with a profound low-temperature-induced inhibition of phosphofructokinase (PFK). Investigations into potential PFK control mechanisms were undertaken. (31)P NMR analysis showed that the intracellular pH of erythrocytes increased from 7.0 to 7.3 as temperature decreased from 17 to 4 degrees C in a manner consistent with alphastat regulation. This change is exactly opposite to that expected if overall PFK activity was regulated by changes in cellular pH since PFK is less active at lower pH values in vitro. Other factors must, therefore, operate to regulate PFK at lower temperatures.