Simultaneous determination of ethylene glycol, propylene glycol, 1,3-butylene glycol and 2,3-butylene glycol in human serum and urine by wide-bore column gas chromatography

A method has been developed for the separation and measurement of ethylene glycol and three other glycols (propylene glycol, 1,3-butylene glycol and 2,3-butylene glycol) in biological samples by wide-bore column gas chromatography with a flame ionization detector. The method used 1,3-propylene glycol (1,3-propanediol) as an internal standard. The method was linear at least from 2 to 1000 μg/ml, with a detection limit of 1 μg/ml. Analytical recoveries were 89–98% for the different concentrations. Precision studies showed coefficients of variation of 1.5–7.7% for the different concentrations. The assay was applied to the analysis of biological samples from two patients who had ingested ethylene glycol and/or other glycols in a suicide attempt.     

Membrane permeability of the human granulocyte to water,dimethyl sulfoxide,glycerol, propylene glycol and ethylene glycol

Granulocytes are currently transfused as soon as possible after collection because they rapidly deteriorate after being removed from the body. This short shelf life complicates the logistics of granulocyte collection, banking, and safety testing. Cryopreservation has the potential to significantly increase shelf life; however, cryopreservation of granulocytes has proven to be difficult. In this study, we investigate the membrane permeability properties of human granulocytes, with the ultimate goal of using membrane transport modeling to facilitate development of improved cryopreservation methods. We first measured the equilibrium volume of human granulocytes in a range of hypo- and hypertonic solutions and fit the resulting data using a Boyle-van’t Hoff model. This yielded an isotonic cell volume of 378 μm³ and an osmotically inactive volume of 165 μm³. To determine the permeability of the granulocyte membrane to water and cryoprotectant (CPA), cells were injected into well-mixed CPA solution while collecting volume measurements using a Coulter Counter. These experiments were performed at temperatures ranging from 4 to 37 °C for exposure to dimethyl sulfoxide, glycerol, ethylene glycol, and propylene glycol. The best-fit water permeability was similar in the presence of all of the CPAs, with an average value at 21 °C of 0.18 μm atm⁻¹ min⁻¹. The activation energy for water transport ranged from 41 to 61 kJ/mol. The CPA permeability at 21 °C was 6.4, 1.0, 8.4, and 4.0 μm/min for dimethyl sulfoxide, glycerol, ethylene glycol, and propylene glycol, respectively, and the activation energy for CPA transport ranged between 59 and 68 kJ/mol.     

Preparation of radioactive diacetyl,acetoin, and 2,3-butylene glycol

Toluene-treated cells of Streptococcus diacetilactis produced large amounts of diacetyl and acetoin without 2,3-butylene glycol. With Na-[3-¹⁴C]pyruvate added to reaction mixtures in place of unlabeled pyruvate, diacetyl with specific activity of 6.1 × 10⁴ cpm/μmol and acetoin with specific activity of 6.8 × 10⁴ cpm/μmol were harvested. Growing cells of Enterobacter aerogens incubated 48 h at 30°C in a complex medium produced large amounts of 2,3-butylene glycol without acetoin or diacetyl. With uniformly labeled [¹⁴C]glucose added to the medium in place of unlabeled glucose, 2,3-butylene glycol with specific activity of 10.8 × 10⁴ cpm/μmol was harvested. The radioactive chemicals were tested and found to be chromatographically homogeneous. Storage frozen in capped containers was especially important for diacetyl, which was found to evaporate rapidly from capped containers at room temperature.     

Separation of diacetyl, acetoin, and 2,3-butylene glycol by ion-exchange chromatography

Mixtures of diacetyl, acetoin, and 2,3-butylene glycol were quantitatively separated by ion-exchange chromatography on Dowex 1-X8 resin in the bisulfite form. Initial elution with water removed 2,3-butylene glycol from the column. Further elution with 0.1 m NaCl separated acetoin from diacetyl. Sulfite in the eluates was deactivated with $\text{I}{}_2\text{KI}$ reagent. After oxidation by bromine, 2,3-butylene glycol was measured as acetoin. Excess bromine was neutralized by addition of 40% NaOH and saturated Na₂S₂O₅. After separation and conversion of the glycol to acetoin, the Westerfeld colorimetric method was used to determine the three components quantitatively.     

Membrane fusion due to dehydration by polyethylene glycol, dextran, or sucrose

To determine whether polyethylene glycol (PEG) causes growth of liposomes by affecting them directly or indirectly, vesicles composed of phosphatidylcholine were exposed to increasing concentrations of Mr 15 000-20 000 PEG or Mr 40 000 dextran either by direct mixing or across a dialysis membrane. After incubation at room temperature and dilution below at least 5% (w/w) polymer, the vesicles were monitored for fluorescence energy transfer and for absorbance at 400 nm. PEG induced the same levels of dequenching or lipid mixing and increased turbidity, regardless of whether the vesicles had been mixed directly with or dialyzed against PEG. These changes occurred within 5-15 min of polymer application. It is concluded that the increased lipid mixing and/or increased turbidity, indicating vesicle growth, resulted from an indirect effect of PEG on the vesicles--most likely dehydration. Dextran, in contrast to PEG, induced less dequenching and/or less turbidity increase when vesicles were directly mixed with, as opposed to dialyzed against, dextran. Although dextran not in contact with vesicles and with osmotic activity comparable to PEG was able to cause a degree of membrane fusion similar to that of PEG, therefore, the dehydrating effect of dextran could be mitigated if it were allowed to interact with vesicles. In further support of membrane dehydration as a precursor to membrane fusion, lipid mixing among sonicated and sonicated, frozen-thawed vesicles dialyzed against sucrose increased as a function of sucrose concentration. Vesicle morphology generally determined the maximal degree of membrane fusion inducible by the polymers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhancement of enzymatic production of cyclodextrins by adding polyethylene glycol or polypropylene glycol

Enzymatic production of cyclodextrins (CDs) from soluble starch was studied using either Bacillus macerans or Bacillus ohbensis cyclomaltodextrin glucanotransferase (CGTase). The production yield of CDs was found to be increased up to 1.5–2 times by the addition of low molecular weight polyethylene glycol (PEG 400) or polypropylene glycol (PPG 425) to the reaction medium. Such results were interpreted as being due to a conformational change of the substrate as well as reduction of hydrolytic activity of the enzyme in the presence of these additives.     

Effect of ethylene glycol and polyethylene glycol on the acid-unfolded state of trypsinogen

Effect of increasing concentrations of two of the polyols, ethylene glycol (EG) and polyethylene glycol (PEG), was studied by near and far circular dichroism (CD), fluorescence emission spectroscopy, and binding of hydrophobic dye, 1-anilino-8-naphthalene sulfonic acid (ANS). Far-UV CD spectra show the transition of acid-unfolded trypsinogen from an unordered state to an intermediate state having ordered secondary structure. Interestingly, near-UV CD spectra show some amounts of stabilizing effect on the tertiary structure of the protein also. Tryptophan fluorescence studies indicate the change in the environment of the tryptophan residues on addition of EG and PEG. Maximum ANS binding occurs in presence of 80% EG and 90% PEG (v/v), suggesting the presence of an intermediate or molten globule-like state at high concentrations of the two polyols.     

Effect of stress on sulfated glycol, metabolites of brain norepinephrine.

The present study examined the effect of footshock stress on the formation of the two major metabolites of rat brain norepinephrine (NE) - the sulfate conjugates of 3-methoxy-4-hydroxyphenylglycol (MOPEG-SO₄) and 3,4-dihydroxyphenylglycol (DOPEG-SO₄). Rats receiving intraventricular injections of either ³HNE or Na₂³⁵SO₄ prior to 0.5 hour of footshock showed significant and comparable increases in both sulfated glycols labeled with ³H or ³⁵SO₄. Elevations were greatest in the hypothalumus using Na₂³⁵SO₄. In pheniprazine pretreated rats footshock did not increase the production of MOPEG-³⁵SO₄ from intraventricular labeled sulfate given alone or in combination with various doses of exogenous MOPEG. The results indicate that neuronally released brain NE is metabolized to form both MOPEG-SO₄ and DOPEG-SO₄. The increase in these metabolites results from an increased glycol production and not from a stress-induced activation of brain sulfation mechanisms.     

Determination of alpha-amylase activity in dextran,ficoll and polyethylene glycol solutions

Summary A new insoluble chromolytic substrate for the spectrophotometric determination of alpha-amylase activity (starch cross-linked with 1,4-butanediol diglycidyl ether in the presence of black drawing ink; “black starch”) has been shown to work well also in the presence of dextran, Ficoll and polyethylene glycol; on the other hand these phase-forming polymers interfere with some commonly used alpha-amylase assays.     

Biotinidase radioassay using an 125I-biotin derivative, avidin, and polyethylene glycol reagents.

A radioassay for determining biotinidase activity in human serum was developed, using N-[beta-(4-OH-3-125I-phenyl)ethyl]-biotinamide in combination with biocytin as the substrate, avidin as a binding protein, and polyethylene glycol as a separation reagent. The gamma-emitting 125I-biotinamide (= tracer) was synthesized by coupling (pH 8.5, 20-22 degrees C, 90 min) N-hydroxysuccinimidobiotin to 125I-tyramine. Using polyethylene glycol as a separation reagent, it was possible to eliminate several problems that were encountered when other separation reagents were used. Biotinidase activity was evaluated following the cleavage of the 125I-biotinamide and expressed in fmol of tracer cleaved.min-1.ml-1 in the presence of 9 nmol of biocytin. Under the conditions used, the time response of the assay was linear up to 3 h. The method is simple to perform, more sensitive than the previously described methods, and reproducible (intra- and interassay CVs of 4.9 and 10.2%, respectively) and allows the simultaneous handling of more than 100 samples in less than 3 h.     

Novel microbial screen for detection of 1,4-butanediol, ethylene glycol, and adipic acid

A novel microbial-screening procedure was developed for separate detection of 1,4-butanediol, ethylene glycol, and adipic acid, three commercially important oxychemicals potentially derivable from bacterial omega-oxidation of n-butanol, ethanol, and hexanoic acid, respectively. The screening method involved postproduction addition of one of several specific Pseudomonas strains which produce a soluble fluorescent pigment during growth on the product of interest. A mutation and selection procedure was developed for isolation of specific strains with phenotypes for growth and pigment production on the desired product (e.g., 1,4-butanediol), but not on its bioconversion substrate (e.g., n-butanol), common by-products (e.g., n-butyrate), or product isomers. Pigment production was growth associated and required cultivation of the screening strains under limiting Fe3+ concentrations. The pigments resembled well-characterized, iron-chelating siderophores produced by other fluorescent pseudomonads. The sensitivity of the assay for product accumulation was enhanced by (i) conducting the screening in microtiter dishes to permit examination of individual isolates of putative producers and to control product diffusion, (ii) using a wavelength cutoff filter to reduce background source light, and (iii) using adapted screening strains which grew at lower (0.3 mM) concentrations of test compounds. The potential utility of the method for detecting a variety of oxidative catabolic products is discussed.     

UvrABC nuclease complex repairs thymine glycol, an oxidative DNA base damage

The UvrABC nuclease complex recognizes a wide spectrum of DNA lesions including pyrimidine dimers, bulky chemical adducts and O6-methylguanine. In this study we have demonstrated that the UvrABC complex is also able to incise PM2 DNA containing the oxidative DNA lesion, thymine glycol. However, DNA containing dihydrothymine, a lesion with a similar structure to thymine glycol, was not incised. The UvrABC complex was also able to incise DNA containing reduced apurinic sites or apurinic sites modified with O-alkyl hydroxylamines, but not DNA containing apurinic sites or urea residues. In vivo, in the absence of base-excision repair, nucleotide excision repair was operable on phi X-174 RF transfecting DNA containing thymine glycols. The level of the repair was found to be directly related to the level of the UvrABC complex. Thus, UvrABC-mediated nucleotide excision repair appears to play a role in the repair of thymine glycol, an oxidative DNA-base lesion that is produced by ionizing radiation or formed during oxidative respiration.     

Bacterial degradation of glycol ethers

Assimilation of ethyleneglycol (EG) ethers by polyethyleneglycol-utilizing bacteria was examined. Ethyleneglycol ether-utilizing bacteria were also isolated from soil and activated sludge samples by enrichment-culture techniques. Three strains (4-5-3, EC 1-2-1 and MC 2-2-1) were selected and characterized as Pseudomonas sp. 4-5-3, Xanthobacter autotrophicus, and an unidentified gram-negative, non-spore-forming rod respectively. Their growth characteristics were examined: Pseudomonas sp. 4-5-3 assimilated EG (diethyleneglycol, DEG) monomethyl, monoethyl and monobutyl ethers, DEG, propanol and butanol. X. autotrophicus EC 1-2-1 grew well on EG monoethyl and monobutyl ethers, EG and primary alcohols (C₁-C₄), and slightly on EG monomethyl ether. The strain MC 2-2-1 grew on EG monomethyl ether, EG, primary alcohols (C₁-C₄), and 1,2-propyleneglycol (PG). The mixed culture of Pseudomonas sp. 4-5-3 and X. autotrophicus EC 1-2-1 showed better growth and improved degradation than respective single cultures towards EG monomethyl, monoethyl or monobutyl ethers. Intact cells of Pseudomonas sp. 4-5-3 degraded various kinds of monoalkyl ethers, which cannot be assimilated by the strain. Metabolic products were characterized from reaction supernatants of intact cells of Pseudomonas sp. 4-5-3 with EG or DEG monoethyl ethers: they were analyzed by thin-layer chromatography and GC-MS and found to be ethoxyacetic acid and ethoxyglycoxyacetic acid. Also, PG monoalkyl ethers (C₁-C₄), dipropyleneglycol monoethyl and monomethyl ethers and tripropyleneglycol monomethyl ether were assimilated by polypropyleneglycol-utilizing Corynebacterium sp. 7.     

Inducible or constitutive polyethylene glycol dehydrogenase involved in the aerobic metabolism of polyethylene glycol

2, 6-Dichlorophenolindophenol (DCIP)-dependent polyethylene glycol (PEG) dehydrogenase activity was found in the particulate fractions of cell-free extracts prepared from PEG-utilizing bacteria (Pseudomonas and Flavobacterium species). This result suggested that PEG dehydrogenase is linked to the respiratory chain of each bacterium and that the enzyme plays a major role in the aerobic metabolism of PEG. Enzyme activities were strongly inhibited by 1, 4-benzoquinone. No metal ion was indispensable for the enzyme activities. Enzyme activities of PEG-utilizing bacteria were induced by PEG except for the activity of PEG 4000-utilizing Flavobacterium sp. no. 203 which had a constitutive enzyme. Although PEG-utilizing bacteria had different growth substrate specificities toward PEGs 200–20,000, their PEG dehydrogenases oxidized the same molecular wt. range of PEGs (dimer-20,000). Cell-free extracts of PEG 400-, 1000- or 4000-utilizing bacteria oxidized PEG 6000 and 20,000 though these bigger PEGs could not be utilized as the sole carbon and energy sources by the bacteria. Methanol, ethylene glycol and glycerol were not or only barely dehydrogenated by all the enzyme preparations.