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G Cueille

Publications and source records attributed to G Cueille.

15 recordsLinked to original sources
PubMed1987

Large scale use of Spherosil ion exchangers in plasma fractionation.

Spherosil microbeads are spherical and made of porous silica. Their surface is coated with hydrophilic and/or hydrophobic polymers. They are specially designed for the separation of proteins on an industrial scale either by ion exchange or by bioaffinity chromatography. Since 1980, large columns have been used in Institut Mérieux for the purification of placental albumin and several vaccines. Here we describe a chromatographic process for the purification of human albumin and immunoglobulins (IgG) from 25 1 of plasma per cycle. First the plasma was freed of the coagulation factors and then clarified at pH 5.25. The corresponding supernatant was filtered and processed in sterile conditions. Albumin was then purified by ion exchange on 3 successive columns, respectively containing: 6.25 kg of DEAE SPHEROSIL W-1000; 3.5 kg of QMA SPHEROSIL PH-1000; 8 kg of COOH-SPHEROSIL W-1000. The concentration and pH of the buffers were selected to reduce, as much as possible, the total quantity of ion exchangers required per cycle. IgG were then purified from the filtrate of the first of the previous columns. 1 column of 6.25 kg of DEAE SPHEROSIL W-1000 was used at pH 6.8. The selected chromatographic parameters allowed us to demonstrate a total elimination of HBs Ag and HB Virus when voluntarily added to an initial sample (RIA determination and HBV-DNA analysis by molecular hybridization). A second column of a large pore anion exchanger: DEAE SPHEROSIL LP-3000 was added at the end of the IgG purification, as a final security to avoid any risk of transmitting the Hepatitis B virus. The yield and quality of the final products will be presented.

Chromatography, Gel↗
PubMed1984

Drug interaction in middle molecule analysis, with special reference to acetylsalicylic acid.

Concerning the middle molecules in uremia and other diseases, the potential artifact that can impede an accurate quantitation of middle molecules and that is related to the absorption of a very commonly used drug, namely aspirin, is discussed. Peak 7c and peak b 4-2 are two different middle molecules separated by gel permeation chromatography followed by anion-exchange chromatography. Oral ingestion of acetylsalicylic acid modifies the chromatographic pattern of the middle molecule fraction in normal subjects and uremic patients. Peak 7c is increased in the urine of healthy subjects, whereas this is not the case for peak b 4-2: With the b 4-2 technique, ingestion of acetylsalicylic acid produces a higher peak b 5. This is consistent with the previous demonstration that peak 7c was eluted as peak b 5. Structural analogies between salicylate metabolites and orthohydroxyhippuric acid beta-glucuronate (i.e., the main component of peak 7c) could explain this drug-related artifact.

Aspirin↗
PubMed1983

Improved separation and quantification of the "middle molecule" b4-2 in uremia.

The plasma of uremic patients usually contains high concentrations of the so-called middle molecules (molecular mass, 300 to 1500 Da), which exert various toxic effects. Among these numerous substances, only one, named peak b4-2, has been correlated with uremic neuropathy. We describe our improvement of a two-stage chromatographic method, gel permeation followed by anion-exchange chromatography (J. Chromatogr. 146: 55-65, 1978), for separation and quantification of b4-2 in body fluids. In analyzing more than 300 samples from 43 uremic patients and 12 healthy subjects, we found a linear correlation between peak area at 254 nm and b4-2 concentration in the range 0.8 to 32 mg/L. The coefficient of variation, including data acquired during seven changes of columns, was 9%. Analysis time (80 min) was shorter than required with other methods. Our results confirm previous data for urinary b4-2 excretion by healthy subjects and for b4-2 removal rate in uremic patients undergoing hemodialysis or hemofiltration. Patients treated with continuous ambulatory peritoneal dialysis have a higher b4-2 excretion than do healthy subjects, suggesting a higher production of this solute in uremic patients.

Ambulatory Care↗
PubMed1981

In vitro frog sural nerve test: a monitor for detecting neurotoxin solutes.

In order to evaluate the in vitro neurotoxicity of MM fractions obtained by chromatographic analytical methods from plasma of uremic polyneuropathic patients and urine of healthy subjects, we performed an in vitro test on isolated frog sural nerve. The nerve is incubated in various media and stimulated by a rectangular shock (0.05ms, 1Hz, intensity supramaximal). Action potential are recorded. Results were expressed by the inhibition index Ii = 1 divided by t 1/2 x 100 where t 1/2 is the time (in min) necessary to reduce the 1/2 the spike amplitude. When the nerve is immerged in Ringer solution or plasma of healthy subjects Ii congruent to 0. Among MM fractions obtained by gel chromatography Sephadex G-15 from uremic polyneuropathic subjects' plasma and healthy subjects' urine, only fraction b gives a positive response (Ii congruent to 0.86 +/- 0.02, n = 4). From the 6 sub-fractions obtained by anion exchange chromatography DEAE Sephadex A-25 of fraction b, only sub-fraction b4-2 exhibits a reduction of the spike amplitude, Ii = 0.54 when concentration b4-2 is 6 mg/L, Ii = 1.30 when b4-2 plasma concentration is 14 mg/L. These values increase with the severity of neurologic symptoms. A positive correlation was found between Ii and various b4-2 concentrations of standard solutions and plasma ultrafiltrate (y = 0.089x + 0.046, r = 0.890, n = 13). In vitro sural nerve test demonstrated its sensitivity as a tool for monitoring analytical and preparative procedures developed for isolation of neurotoxic solutes.

Animals↗
PubMed1981

Uremic middle molecules: analytical study of middle molecular weight fractions subpeak b4-2.

Fractions containing substances weighing less than 1800 daltons have been obtained from hemofiltrate and peritoneal dialysis fluid. Oligosaccharides and glycopeptides were evidenced in these fractions by paper chromatography. This analytical procedure has been applied to a fraction containing peak b4-2 obtained by molecular exclusion chromatography on Sephadex G-15 followed by ion exchange chromatography on Sephadex A-25 (Cueille et al.). Preparative paper chromatography of this fraction evidenced 3 carbohydrate and 3 peptidic fractions. Study of the carbohydrate fraction (01, 03, 04) by gas-chromatography and mass spectrometry shows that they are composed of glucuronoconjugates whose aglycons have not been identified. Fraction 04 which contains subpeak b4-2 has been found to have an inhibitory effect on the action potentials of the sural nerve of the frog.

Chromatography, Gas↗
PubMed1981

Characterization of sub-peak b4.2, middle molecule.

The Middle Molecules (MM) within the molecular weight (MW) range of vitamin B12 (1355 daltons) are assumed to be partly responsible for uremic toxicity. We have isolated a solute, b4.2, the purity of which is controlled by thin layer chromatography on silica gel. It correlates with active clinical polyneuropathy. The Stockholm group is dealing with a MM they call peak 7c. After exchange of purified solutes between the Stockholm group and us, comparative analyses demonstrate that 7c and b4.2 are different. The b4.2 solute is a glucuronide but it is impossible to obtain the aglycon moiety after enzymatic or acidic hydrolysis. Desorption chemical ionization and electron-impact ionization mass spectrometry results of b4.2 after transformation in methyl ester trimethylsilyl derivative are compatible with a b4.2 MW of 568 daltons (or 526 in native form) corresponding to a glucuronoconjugate of an aglycon with a MW 392 daltons (or 350 in native form). Moreover mass spectrometry confirms that b4.2 isolated from normal human urine and from uremic RP6 hemofiltrate fluid are identical.

Gas Chromatography-Mass Spectrometry↗
PubMed1981

Technical aspects on middle molecules: separation, isolation, and identification.

For the last few years we have attempted to separate and isolate from biological fluids the uremic Middle Molecules (MM). The first step in the treatment of plasma is an ultrafiltration through AN69 membrane. The samples are fractionated by Sephadex G-15 chromatography into 9 peaks "a" to "i' under precise conditions that have been selected in order to exhibit different patterns in the MM range (peak b and c) between uremic and uremic polyneuropathic patients. Further separation of peak b is performed by ion exchange chromatography with Sephadex DEAE A-25 into 7 sub-peaks b; sub-peak b 4-2 is the only one to correlate with neuropathy. Prior to b 4-2 identification studies, specific large scale isolation procedures are necessary. The purity of the product is monitored at each step by thin-layer chromatography on silica gel.

Chromatography, Gel↗
PubMed1980

Experimental and clinical activity of a new anthracycline derivative: detorubicin (14-diethoxyacetoxydaunorubicin).

This report concerns the experimental activity and the preliminary clinical results obtained with detorubicin, an anthracycline. Experimentally, in comparison with doxorubicin, detorubicin is less toxic, less immunodepressive, and less mutagenic, its experimental antitumor activity is equal or superior, and differences are found in pharmacokinetics. Its most interesting activities in clinical cancer therapy are found in non-Hodgkin lymphomas, in carcinomas, and in soft-tissue sarcomas.

Animals↗
PubMed1980

Uremic neurotoxin in the middle molecular weight range.

Among the middle molecule fractions obtained by high performance gel chromatography on Sephadex G-15 combined with gradient ion exchange chromatography on DEAE Sephadex A-25 from plasma ultrafiltrates of six polyneuropathic patients, only peak b4-2 was at a significantly higher concentration than that obtained from uremic patients with neuropathy. Purification of the b4-2 solute allows its quantitative determination in biological fluids. The b4-2 plasma concentrations are 1 mg/L in healthy subjects (n=30), 4.6 +/- 0.2 mg/L in uremic patients (n=67) and 13-19 mg/L in six polyneuropathic patients. The 24-hr urinary excretion is 13 +/- 1 mg. The weekly removal rates in hemofiltration or in hemodialysis, using high permeability membrane three times a week, are 38-40 mg. In an in vitro sural nerve test for the evalution of middle molecule neurotoxicity, the b4-2 solute exhibits a neurotoxic effect at concentrations similar to those found in plasma of neuropathic patients. Preliminary results of an attempt to identify the neurotoxin indicate that it is an acid-polyol derivative.

Action Potentials↗
PubMed1978

[Determination of "middle molecules" presenting vitamin B12 molecular size in normal and uremic body fluids (author's transl)].

Uremic solutes with the molecular size of vitamin B12 are assumed to be toxic. An analytical method is proposed to detect and separate these solutes in body fluids using two combined techniques: gel filtration on Sephadex G-15 and ion-exchange chromatography on DEAE-Sephadex A-25. The vitamin B12 molecular size has been localized by ultrafiltration through membranes with a defined cut-off. Normal and uremic body fluids (urine, plasma, hemodialysis fluid) have been separated into 9 ultraviolet-absorbing peaks (a to i) by high-speed gel filtration. Peaks b and c present the molecular size of vitamin B12, 10--15 A molecular diameter in pH7 aqueous solution. Peak b, which correlates with uremic neuropathy, is separated into 6 sub-peaks (b1 to b6) by ion-exchange chromatography, sub-peak b4.2 is the only one to correlate with uremic neuropathy. The coefficient of variation in the integrated area of a single peak is 16%. This method gives the chromatographic prolife of the vitamin B12 molecular size from 500 microliter of uremic plasma or 100 microliter of normal urine within one hour.

Chromatography, Gel↗
PubMed1978

Evaluation of plasma neurotoxin concentration in uraemic polyneuropathic patients.

Purification of b4-2 sub-peak obtained on DEAE Sephadex A25 chromatography gave us the possibility of quantifying the plasma concentration of the neurotoxin present in uraemic patients with active polyneuropathy. From the purified neurotoxin isolated by kieselguhr and cellulose chromatography we calibrated analytic columns for b4-2 analysis. Plasma concentration, measured in 6 uraemic neuropathic patients, is between 13 and 19 mg/litre. In 52 uraemic patients without neuropathy, the plasma concentration is between 3 and 9 mg/litre. In 20 healthy subjects the plasma concentration is less than 1 mg/litre. The weekly neurotoxin removal in uraemic patients without neuropathy, treated by a five hours RP6 session 3 times a week, is of the same order of magnitude as the weekly urinary excretion in healthy subjects. Preliminary results of a tentative identification of this purified product indicate that it is not a polypeptide but an acid-polyol with carbohydrate structure.

Chromatography, Ion Exchange↗
PubMed1976

Inhibition of bovine renal adenylate cyclase by urinary products.

The secondary hyperparathyroidism in uremic patients is due to the modification plasma electrolyte concentrations accompanied by a renal resistance to PTH action. We postulate that the retention of a uremic toxin could be at least partly responsible for this resistance. We have tested this hypothesis "in vitro" by measuring the action of Middle Molecules on the adenylate cyclase activity stimulated by NaF, PTH and isoproterenol.

Adenylyl Cyclase Inhibitors↗