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PubMed1992

Comparative analysis of normal and growth-retarded placentas with phosphorus nuclear magnetic resonance spectroscopy.

OBJECTIVE: Phosphorus 31 magnetic resonance spectroscopy studies were carried out on placentas from normal vaginal and elective cesarean deliveries without antenatal complications and from pregnancies complicated by intrauterine growth retardation of unknown cause to determine differences. STUDY DESIGN: Perchloric acid extraction was performed on frozen tissue, and quantitative analysis was carried out for well-resolved resonances representing adenosine triphosphate, sugar phosphate, inorganic phosphorus, diphosphoglycerate, glycerophosphorylethanolamine, and glycerophosphorylcholine. RESULTS: Adenosine triphosphate levels were highest in the growth-retarded group. There were significantly higher levels of sugar phosphate, diphosphoglycerate, and glycerophosphorylcholine in the placentas of the growth-retarded pregnancies compared with those from normal placentas. CONCLUSION: These differences may represent a response to hypoxia and an increase in the amount of blood in the placenta. The results demonstrate the utility of nuclear magnetic resonance spectroscopy for studying the pathology of abnormal placentas to gain a better understanding of the pathology and represent early steps toward in vivo spectroscopic studies of the placenta.

Cesarean Section↗
PubMed2003

Nucleotide profile of mouse liver: response to 2'-O-dibutyryl cytidene 3',5'-cyclic monophosphate.

The effect of 2'-O-dibutyryl cytidene 3',5'-cyclic monophosphate (dibutyryl cCMP) on the nucleotide profile of mouse liver was examined. Dibutyryl cCMP caused an increase in the amount of CTP in mouse liver. Perchloric acid extracts of liver tissue were neutralized with tri-N-octylamine in trichlorotriflouroethene and, after removal of CLO(4-), subjected to preliminary purification on a Cu2+-loaded column of Chelex 100. A high-pressure liquid chromatographic anion-exchange procedure was used and gave good resolution of the free nucleotides on a single column.

Animals↗
PubMed1986

31P NMR spectra of rodent and avian fibroblasts transformed by Rous or Kirsten sarcoma viruses.

31P NMR spectra of normal rodent and avian fibroblasts were compared to those of the same cells transformed either by the Rous sarcoma virus (RSV) or by the Kirsten sarcoma virus (Ki-MSV). Under physiological conditions, the spectra of living or perchloric acid extracted chicken embryo fibroblasts, rat cell line FR3T3 and mouse cell line C127 did not differ from those of their counterparts transformed by RSV or Ki-MSV. However, in the case of FR3T3 cells, on shifting from 37 degrees C to 20 degrees C, and particularly if PBS replaced serum growth medium, a different, though transitory, response of the transformed cells was detected. They then showed, within few minutes, a more rapid ATP depletion with accumulation of fructose 1,6-diphosphate (FDP), as compared to normal control cells.

Animals↗
PubMed1987

Carcinoembryonic antigen (CEA) and two crossreacting antigens of 165 kD and 105 kD isolated from meconium exhibit identical amino terminal sequences.

Two antigens of 165 kD and 105 kD crossreacting with the carcinoembryonic antigen (CEA) were isolated from human meconium by perchloric acid extraction, differential immunoaffinity chromatography, and size exclusion HPLC. The sequences of the 30 amino terminal amino acids of both antigens are identical with the corresponding sequence of CEA.

Amino Acid Sequence↗
PubMed1999

Murine pharmacokinetics of 6-aminonicotinamide (NSC 21206), a novel biochemical modulating agent.

The pyridine nucleotide 6-aminonicotinamide (6AN) was shown recently to sensitize a number of human tumor cell lines to cisplatin in vitro. The present studies were undertaken to compare the drug concentrations and length of exposure required for this sensitization in vitro with the drug exposure that could be achieved in mice in vivo. Human K562 leukemia cells and A549 lung cancer cells were incubated with 6AN for various lengths of time, exposed to cisplatin for 1-2 hr, and assayed for Pt-DNA adducts as well as the ability to form colonies. K562 cells displayed progressive increases in Pt-DNA adducts and cisplatin sensitivity during the first 10 hr of 6AN exposure. An 18-hr 6AN exposure was likewise more effective than a 6-hr 6AN exposure in sensitizing A549 cells to cisplatin. HPLC analysis of 6AN and its metabolite, 6-amino-NAD+, permitted assessment of exposures achieved in vivo after i.v. administration of 10 mg/kg of 6AN to CD2F1 mice. 6AN reached peak serum concentrations of 80-90 microM and was cleared rapidly, with T1/2alpha and T1/2beta values of 7.4 and 31.3 min, respectively. Bioavailability was 80-100% with identical plasma pharmacokinetics after i.p. administration. At least 25% of the 6AN was excreted unchanged in the urine. The metabolite 6-amino-NAD+ was detected in perchloric acid extracts of brain, liver, kidney, and spleen, but not in serum. Efforts to prolong systemic 6AN exposure by administering multiple i.p. doses or using osmotic pumps resulted in lethal toxicity. These results demonstrated that 6AN exposures required to sensitize tumor cells to cisplatin in vitro are difficult to achieve in vivo.

6-Aminonicotinamide↗
PubMed1998

Tolbutamide attenuates diazoxide-induced aggravation of hypoxic cell injury.

ATP-dependent potassium (KATP) channels of neurons are closed in the presence of physiological levels of intracellular ATP and open when ATP is depleted during hypoxia or metabolic damage. The present study investigates hypoxic alterations of purine and pyrimidine nucleotide levels supposed to intracellularly modulate KATP channels. In addition, the effects of the KATP channel activator diazoxide and its antagonist tolbutamide were investigated on ATP, GTP, CTP and UTP levels in slices of the parietal cortex. Hypoxia was evoked by saturation of the medium with 95% N2-5% CO2 instead of 95% O2-5% CO2 for 5 min. Nucleotide contents were measured by anion-exchange HPLC in neutralized perchloric acid extracts obtained from slices frozen immediately at the end of incubation. Hypoxia per se decreased purine and pyrimidine nucleoside triphosphate contents. Thus, ATP and GTP contents were reduced to 69.9 and 77.6% of the respective normoxic levels. UTP and CTP contents were even more decreased (to 60.9 and 41.6%),, probably because the salvage pathway of these pyrimidine nucleotides is less effective than that of the purine nucleotides ATP and GTP. While tolbutamide (30 microM) had no effect on the hypoxia-induced decrease of nucleotides, diazoxide at 300, but not 30 microM aggravated the decline of ATP, UTP and CTP to 51.8, 37.5 and 28.5% of the contents observed at normoxia; GTP levels also showed a tendency to decrease after diazoxide application. Tolbutamide (300 microM) antagonized the effects of diazoxide (300 but not 30 microM aggravated the decline of ATP, UTP and CTP to 51.8, 37.5 and 28.5% of the contents observed at normoxia; GTP levels also showed a tendency to decrease after diazoxide application. Tolbutamide (300 microM) antagonized the effects of diazoxide (300 MicroM). Nucleoside diphosphate (ADP, GDP and UDP) levels were uniformly increased by hypoxia. There was no hypoxia-induced increase of ADP contents in the presence of tolbutamide (300 microM). The ATP/ADP, GTP/GDP and UTP/UDP ratios uniformly declined at a low pO2. However, only the ATP/ADP ratio was decreased further by diazoxide (300 microM). The observed alterations in nucleotide contents may be of importance for long- and short-term processes related to acute cerebral hypoxia. Thus, hypoxia-induced alterations of purine and pyrimidine nucleotide levels may influence the open state of KATP-channels during the period of reversible hypoxic cerebral injury. Furthermore, alterations during the irreversible period of cerebral injury may also arise, as a consequence of decreased pyrimidine nucleotide contents affecting cell survival viaprotein and DNA synthesis.

Animals↗
PubMed1996

Cyclolaminarinose. A new biologically active beta-(1-->3) cyclic glucan.

A unique glucan has been isolated from a recombinant strain of a Rhizobium meliloti TY7, a cyclic beta-(1-->2) glucan mutant carrying a locus specifying beta-(1-->3; 1-->6) glucan synthesis from Bradyrhizobium japonicum USDA110. This compound, which appears to have considerable hydrophobic affinity, was separated from a perchloric acid cell extract by adsorption to a C-18 silica column. Unlike those cyclic glucans previously isolated from Rhizobium meliloti or Bradyrhizobium japonicum, this molecule contains neither phosphoglycerol nor phosphocholine substituents, respectively. 2D NMR, FAB mass spectrometric analysis and high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) confirmed that this glucan is a single, cyclic decasaccharide (cyclolaminarinose) in which one of the residues is substituted in its 6-position with beta-laminarabiose. This structural assignment was confirmed by mass spectral and NMR analyses of the product obtained from two consecutive Smith degradations. Unlike the complex 13C spectrum of the unoxidized material, the spectrum of this product consisted of only six resonances due to rapid time averaging of its symmetrical structure on the relatively slow NMR timescale. Synthesis of this newly described cyclic beta-glucan in the R. meliloti ndvB mutant restored the symbiotic and hypoosmotic adaptation characteristics of the R. meliloti wild type strain.

Carbohydrate Conformation↗
PubMed2002

In vivo assessment of nodularin-induced hepatotoxicity in the rat using magnetic resonance techniques (MRI, MRS and EPR oximetry).

Acute nodularin-induced hepatotoxicity was assessed in vivo, in rats using magnetic resonance (MR) techniques, including MR imaging (MRI), MR spectroscopy (MRS), and electron paramagnetic resonance (EPR) oximetry. Nodularin is a cyclic hepatotoxin isolated from the cyanobacterium Nodularia spumigena. Three hours following the intraperitoneal (i.p.) administration of nodularin (LD50), a region of 'damage', characterized by an increase in signal intensity, was observed proximal to the porta hepatis (PH) region in T2-weighted MR images of rat liver. Image analysis of these regions of apparent 'damage' indicated a statistically significant increase in signal intensity around the PH region following nodularin administration, in comparison with controls and regions peripheral to the PH region. An increase in signal intensity was also observed proximal to the PH region in water chemical shift selective images (CSSI) of nodularin-treated rat livers, indicating that the increased signal observed by MRI is an oedematous response to the toxin. Microscopic assessment (histology and electron microscopy) and serum liver enzyme function tests (aminotransferase (ALT) and aspartate ALT (AST)) confirmed the nodularin-induced tissue injury observed by MRI. In vivo and in vitro MRS was used to detect alterations in metabolites, such as lipids, Glu+Gln, and choline, during the hepatotoxic response (2-3 h post-exposure). Biochemical assessment of perchloric acid extracts of nodularin-treated rat livers were used to confirm the MRS results. In vivo EPR oximetry was used to monitor decreasing hepatic pO2 (approximately 2-fold from controls) 2-3 h following nodularin exposure. In vivo MR techniques (MRI, MRS and EPR oximetry) are able to highlight effects that may not have been evident in single end point studies, and are ideal methods to follow tissue injury progression in longitudinally, increasing the power of a study through repeated measures, and decreasing the number of animals to perform a similar study using histological or biochemical techniques.

Alanine Transaminase↗
PubMed1978

Evaluation of a direct radioimmunoassay for carcinoembryonic antigen.

1. A radioimmunoassay for CEA using commercially available reagents (CEA-Roche), which avoids the perchloric acid and dialysis steps used in the CEA-Roche assay, has been devised. 2. Comparison of results on 746 plasma samples using our assay with those obtained using the CEA-Roche assay revealed a highly significant correlation coefficient (0.72; p less than 0.00001). 3. A further comparison of results on 373 plasma samples using our assay, but including a preincubation step with goat serum, with those obtained using the CEA-Roche assay also revealed a highly significant correlation coefficient (0.89; 0.00001). The goat serum preincubation step eliminated most of the discordant results in which our assay value was higher than CEA-Roche assay, presumably because of anti-goat antibodies in the patient plasma sample.

Animals↗
PubMed1982

Comparison of RIA and IRMA methods for measurement of carcinoembryonic antigen (CEA).

We present data comparing the Roche CEA radioimmunoassay (RIA) utilizing ultrafiltration and the Abbott CEA immunoradiometric assay (IRMA) methods for standard curve sensitivity, analytical reproducibility, recovery (pre and post extraction), dilution linearity, and patient correlation. The Roche intra- and inter-assay precision figures for this assay were 8% and 15%, respectively, for a CEA concentration of 4.0 ng/mL. The Abbott assay gave comparable precision values of 9% and 16%, respectively, for a CEA concentration of 2.0 ng/mL. Recovery for plasma spiked with CEA stock preparations was commercial-source-dependent when assayed by the Roche assay, but independent when measured by the Abbott assay. The Roche assay did not recover both preparations quantitatively, while the Abbott assay did. Plasma dilution studies over a wide range of CEA concentrations gave linear results for both assays provided the Roche assay utilizes the extraction method with dialyzate dilution for CEA values greater than 20 ng/mL. A difference (60%) was observed in linear regression slopes of the measured values between the Roche "indirect" and "direct" assays. Exposure of CEA to perchloric acid seems to play a critical role in recovery of CEA material when assayed by the Roche method. A large number of the patient samples assayed gave widely discrepant results when comparing the RIA to the IRMA methods. The potential significance of these discrepancies is discussed.

Carcinoembryonic Antigen↗
PubMed1986

Choline in plasma measured by liquid-chromatography with electrochemical detection.

We have developed a simple, specific, and sensitive method for plasma choline measurement based on the HPLC procedure of Potter et al. (J Neurochem 1983; 41: 188) for the measurement of acetylcholine and choline in neuronal tissue. The effluent from a reverse-phase column is mixed with choline oxidase in a post-column reaction coil to produce hydrogen peroxide which is monitored electrochemically. Plasma samples are prepared by deproteinization with perchloric acid. Choline is recovered quantitatively from the plasma, but an internal standard (homocholine) is added to compensate for any variation in electrode response. Choline can be measured in plasma samples containing less than 1 mumole per litre of plasma; the method response is linear in the 1-20 mumol/L range. Catecholamines and ascorbic acid do not interfere. The chromatography, enzymatic reactions, and electrochemistry all contribute to the specificity of the method.

Alcohol Oxidoreductases↗
PubMed1989

Partial purification and characterization of CA19-9 antigen from the ascitic fluid of a patient with pancreatic cancer.

CA19-9 immunoreactive protein was partially purified from the ascitic fluid of a patient with pancreatic cancer by perchloric acid fractionation, gel chromatography and Affi-gel Blue column chromatography, resulting in a purified sample of 5.0 x 10(6) CA19-9 units per milligram of protein (3700-fold purification). Western blotting analysis of this purified sample revealed a single band of molecular weight 210 kDa. Although the original ascitic fluid showed a high CA125 immunoreactivity, this purified sample had no CA125 immunoreactivity. The elution pattern for CA19-9 activity on Affi-gel Blue column is quite distinct from that for CA125. These results suggest that CA19-9 antigen in carcinoma patients may be identical or very similar to that recently purified from the culture media of the colorectal cell line SW1116 and is distinct from CA125 antigen.

Antigens, Tumor-Associated, Carbohydrate↗
PubMed1989

Phosphatic metabolites in keratoconus.

Sixteen corneal buttons were obtained from 16 patients with keratoconus at the time of penetrating keratoplasty (mean age, 34 +/- 3.2 yr) and prepared for phosphorus-31 nuclear magnetic resonance spectroscopy following perchloric acid extraction. The corneas were compared with age-matched corneas from fresh eye-bank eyes with normal slit-lamp biomicroscopic appearance. Higher levels (P less than 0.05) of nucleoside monophosphates and choline phosphate and lower levels (P less than 0.05) of adenosine diphosphate were detected in keratoconus than in eye-bank corneas. The level of an unidentified metabolite at 3.96 delta, present in eye-bank corneas, was ninefold higher in keratoconus corneas. Another unidentified metabolite at 3.31 delta, accounting for 1.5% of the total detected phosphatic metabolites, appeared in keratoconus but not in eye-bank cornea spectra. These findings of altered phosphatic metabolites are consistent with altered corneal metabolism. The unidentified, unknown metabolites in the spectral profile uniquely distinguish keratoconus from eye-bank corneas.

Adenine Nucleotides↗
PubMed2003

Early changes in glucose and phospholipid metabolism following apoptosis induction by IFN-gamma/TNF-alpha in HT-29 cells.

The effects of apoptosis induction on glucose and phospholipid metabolite levels in cancer were studied using human colon adenocarcinoma cells (HT-29). Apoptosis was induced by co-incubation with 200 U/ml tumor necrosis factor (TNF)-alpha for 4, 8 or 15 h, after sensitization with 500 U/ml interferon (IFN)-gamma for 7 h. Perchloric acid extracts were analyzed by (1)H and (31)P nuclear magnetic resonance (NMR) spectroscopy. Significantly increased lactate and NTP (all nucleoside 5'-triphosphates) signals were detected 4 h after apoptosis-inducing IFN-gamma/TNF-alpha treatment, but not in cells which were TNF-alpha-treated without IFN-gamma preincubation. Simultaneous lactate and NTP changes, if confirmed in vivo, may serve as early, non-invasive markers of treatment response in some tumors.

Apoptosis↗
PubMed1997

C-terminal truncation of thymosin beta10 by an intracellular protease and its influence on the interaction with G-actin studied by ultrafiltration.

Two beta-thymosins are expressed in most mammalian tissues. We detected small amounts of a third peptide in extracts of rabbit spleen. The portion of this peptide increased when the tissue was first frozen and then thawed at 4 degrees C. Small amounts of the peptide are also present in cells from suspension cultures homogenized immediately in diluted perchloric acid. By means of amino acid analysis and MALDI-mass spectroscopy this peptide was identified to be a C-terminally truncated form of thymosin beta10. Having studied the formation in more detail we found that after a 4-h thaw at 4 degrees C all thymosin beta10 was truncated to thymosin beta10(1-41), which was further degraded during the next 20 h. On the other hand, thymosin beta4Ala, the second beta-thymosin being present in rabbit spleen, was not truncated or degraded even after 22 h. It might be possible that in vivo a truncated form of thymosin beta10 is formed by a carboxydipeptidase while thymosin beta4Ala is rather stable against proteolytic modification. By using a newly designed ultrafiltration assay, we determined the dissociation constants of the complexes of G-actin and these three beta-thymosins to be 0.28, 0.72, and 0.94 microM for thymosin beta4Ala, beta10, and thymosin beta10(1-41), respectively. The complex with beta4Ala is unambiguously more stable than the complex with beta10 or beta4 (0.81 microM). The change in the dissociation constant generated by the truncation of the two C-terminal amino acid residues of beta10 is small but statistically significant. This demonstrates that even the very last amino acid residues at the C-terminus of beta-thymosins are involved in the interaction with G-actin.

Actins↗
PubMed1998

Heterologous expression of human H1 histones in yeast.

The complete set of seven human H1 histone subtype genes was heterologously expressed in yeast. Since Saccharomyces cerevisiae lacks standard histone H1 we could isolate each recombinantly expressed human H1 subtype in pure form without contamination by endogenous H I histones. For isolation of the H1 histones in this expression system no tagging was needed and the isoforms could be extracted with the authentic primary structure by a single extraction step with 5%(0.74 M) perchloric acid. The isolated H1 histone proteins were used to assign the subtype genes to the corresponding protein spots or peaks after two-dimensional gel electrophoresis and capillary zone electrophoresis, respectively. This allowed us to correlate transcriptional data with protein data, which was barely possible until now.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1980

Rapid high-performance liquid chromatographic method for the measurement of amiodarone in blood plasma or serum at the concentrations attained during therapy.

A simple high-performance liquid chromatographic method has been developed for the measurement of the antiarrhythmic drug amiodarone in small (200 microliter) volumes of plasma or serum. After addition of 2 mole/l phosphate solution, pH 4.5 (20 microliter), containing the internal standard, the sample is vortex-mixed with diisopropyl ether (200 microliter) for 30 sec. A portion (100 microliter) of the resulting extract is analysed on a microparticulate (5 micron) silica column using methanol-diethyl ether (85:15) containing perchloric acid (0.02% v/v) as the mobile phase, and the absorption of the column effluent is monitored at 240 nm. No endogenous sources of interference have been observed, and interference from other drugs is minimal. The procedure is rapid, an analysis in duplicate taking less than 15 min to complete. The limit of sensitivity of the assay is 0.05 mg/l, and the concentrations of amiodarone measured in plasma samples from patients under treatment with this compound ranged from 0.15 to 4.5 mg/l.

Amiodarone↗
PubMed1981

Rapid high-performance liquid chromatographic method for the measurement of verapamil and norverapamil in blood plasma or serum.

A simple high-performance liquid chromatographic method for the simultaneous measurement of plasma verapamil and norverapamil concentrations has been developed. The sample (100 microliters) is vortex-mixed for 30 sec with 4 M sodium hydroxide solution, pH 13 (50 microliters), internal standard solution (aqueous 5,6-benzoquinoline, 0.20 mg/l) (50 microliters) and methyl tert.-butyl ether (200 microliters). After centrifugation at 9950 x g for 2 min, a portion (100 microliters) of the resulting extract is analysed on a microparticulate (5 microns) silica column using a methanolic solution of potassium bromide (3.0 mM) and perchloric acid (0.37 mM) as the mobile phase, and the column effluent is monitored by fluorescence detection using an excitation wavelength of 203 nm. A specimen, together with a quality control sample, can be analysed, in duplicate, within 30 min. The limit of accurate measurement of the assay is 2 micrograms/l, and no potential sources of interference have been identified. The method has advantages of speed, small sample requirement and complete resolution of the three major metabolites of verapamil.

Chromatography, High Pressure Liquid↗