Methanol-chloroform Extraction Research Articles

Potential role of N-acetyl glucosamine in Aspergillus fumigatus-assisted Chlorella pyrenoidosa harvesting

BackgroundAlgal harvesting is a major cost which increases biofuel production cost. Algal biofuels are widely studied as third-generation biofuel. However, they are yet not viable because of its high production cost which is majorly contributed by energy-intensive biomass harvesting techniques. Biological harvesting method like fungal-assisted harvesting of microalgae is highly efficient but poses a challenge due to its slow kinetics and poorly understood mechanism.ResultsIn this study, we investigate Aspergillus fumigatus–Chlorella pyrenoidosa attachment resulting in a harvesting efficiency of 90% within 4 h. To pinpoint the role of extracellular metabolite, several experiments were performed by eliminating the C. pyrenoidosa or A. fumigatus spent medium from the C. pyrenoidosa–A. fumigatus mixture. In the absence of A. fumigatus spent medium, the harvesting efficiency dropped to 20% compared to > 90% in the control, which was regained after addition of A. fumigatus spent medium. Different treatments of A. fumigatus spent medium showed drop in harvesting efficiency after periodate treatment (≤ 20%) and methanol–chloroform extraction (≤ 20%), indicating the role of sugar-like moiety. HR-LC–MS (high-resolution liquid chromatography–mass spectrometry) results confirmed the presence of N-acetyl-d-glucosamine (GlcNAc) and glucose in the spent medium. When GlcNAc was used as a replacement of A. fumigatus spent medium for harvesting studies, the harvesting process was significantly faster (p < 0.05) till 4 h compared to that with glucose. Further experiments indicated that metabolically active A. fumigatus produced GlcNAc from glucose. Concanavalin A staining and FTIR (Fourier transform infrared spectroscopy) analysis of A. fumigatus spent medium- as well as GlcNAc-incubated C. pyrenoidosa cells suggested the presence of GlcNAc on its cell surface indicated by dark red dots and GlcNAc-specific peaks, while no such characteristic dots or peaks were observed in normal C. pyrenoidosa cells. HR-TEM (High-resolution Transmission electron microscopy) showed the formation of serrated edges on the C. pyrenoidosa cell surface after treatment with A. fumigatus spent medium or GlcNAc, while Atomic force microscopy (AFM) showed an increase in roughness of the C. pyrenoidosa cells surface upon incubation with A. fumigatus spent medium.ConclusionsResults strongly suggest that GlcNAc present in A. fumigatus spent medium induces surface changes in C. pyrenoidosa cells that mediate the attachment to A. fumigatus hyphae. Thus, this study provides a better understanding of the A. fumigatus-assisted C. pyrenoidosa harvesting process.

Evaluation of sample preparation protocols for quantitative NMR-based metabolomics.

Quantification of metabolites in biological fluids and tissues by NMR spectroscopy is challenged by the presence of abundant macromolecules and lipoproteins in samples, which give broad signals in the NMR spectra. To improve the quality of NMR spectra the different protocols for protein and lipid removal from the sample are used. This work is aimed at the evaluation of the effectiveness of various methods of purification of blood serum from proteins and lipids for 1H NMR metabolomic profiling. The advantages and limitations of different methods of the sample preparation for NMR-based quantitative metabolomics have been compared, including ultrafiltration, methanol and ethanol extractions with and without additional lipid removal, and methanol-chloroform extraction. The concentrations of 30 abundant metabolites extracted from human blood serum have been measured. It is found that ultrafiltration provides the best lipid removal, but causes significant and inhomogeneous metabolite losses. Ethanol and methanol extractions demonstrate similar performance with the minimal metabolite losses, and are ideal for fluids and tissues with low lipid content. The additional purification of alcohol extracts from lipids allows for the significant improving of NMR spectra, but causes additional metabolite losses. The methanol-chloroform extraction seems to be an optimal method for tissues with the high lipid content, providing a satisfactory lipid removal and low metabolite losses. The ultrafiltration leads to large losses of metabolites (up to 60%) and for this reason is not suitable for quantitative analysis.

Evaluation of metabolite extraction protocols and determination ofphysiological response to drought stress via reporter metabolites in model plant Brachypodium distachyon

Metabolomics aims to systematically gather (quantitative) information on metabolites in the cell and is commonly viewed as the missing link between genomics, transcriptomics, and physiology. Typical metabolomics platforms consist of two main steps, the quenching and extraction of metabolites from plant material and the (un)targeted quantification of the extracted metabolites. Brachypodium distachyon, a native grass species of the Mediterranean region, is an attractive model plant to study temperate crops. In recent years, despite an increasing interest in genomic and transcriptomic studies, metabolomics studies for B. distachyon are still in their infancy. Drought, an abiotic stress factor, causes severe loss in plant productivity and it is therefore crucial to understand its effect on plant metabolism, in particular its metabolome. The objective of this study is to set up a quantitative plant metabolomics platform for B. distachyon to evaluate and optimize alternative metabolite extraction protocols (methanol and methanol-chloroform extraction). Focusing on three reporter metabolites (ATP, glucose, and starch) for metabolite quantification and two extraction protocols, we compared the drought stress response of 2 different genotypes from different geographical regions of Turkey. Results revealed that there is no one-protocol-fits-all, pointing to significant differences in metabolite recoveries upon different extraction protocols, though methanol extraction generally yielded higher recoveries. Drought stress resulted in higher glucose levels while ATP and starch levels did not show statistically significant or consistent changes. In conclusion, the impact of drought stress on B. distachyon metabolism was significant and this study could be the basis for further metabolomics studies on plant stress response.

Xanthan Gum Removal for 1H-NMR Analysis of the Intracellular Metabolome of the Bacteria Xanthomonas axonopodis pv. citri 306

Xanthomonas is a genus of phytopathogenic bacteria, which produces a slimy, polysaccharide matrix known as xanthan gum, which involves, protects and helps the bacteria during host colonization. Although broadly used as a stabilizer and thickener in the cosmetic and food industries, xanthan gum can be a troubling artifact in molecular investigations due to its rheological properties. In particular, a cross-reaction between reference compounds and the xanthan gum could compromise metabolic quantification by NMR spectroscopy. Aiming at an efficient gum extraction protocol, for a 1H-NMR-based metabolic profiling study of Xanthomonas, we tested four different interventions on the broadly used methanol-chloroform extraction protocol for the intracellular metabolic contents observation. Lower limits for bacterial pellet volumes for extraction were also probed, and a strategy is illustrated with an initial analysis of X. citri’s metabolism by 1H-NMR spectroscopy.

A quick isolation method for mutants with high lipid yield in oleaginous yeast

A novel method has been developed to easily isolate the mutants with high lipid yield after irradiating oleaginous yeast cells with carbon ions of energy of 80 MeV/u. Pre-selection of the mutants after ion irradiation was performed with culture medium in which the concentration of cerulenin, a potent inhibitor of fatty acid synthetase, was at 8.96 mu mol/l. Afterwards, lipid concentration in the fermentation broth of the pre-selected colonies was estimated by the sulfo-phospho-vanillin reaction instead of the conventional methanol-chloroform extraction. Two mutants with high lipid yield have been successfully selected out by the combined method. This easy and simple method is much less time-consuming but very efficient in the mutant isolation, and it has demonstrated great potential on mutation breeding in oleaginous microorganism.

Platelet-activating factor changes in phospholipid extracts from the plasma, peripheral blood mononuclear cells and bone marrow mononuclear cells of patients with acute leukemia--a 31P MRS in vitro study.

The aim of this investigation was to evaluate the changes in PAF concentrations in the plasma, PBMC and BMMC of patients with acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML). The plasma was from 23 healthy volunteers (HV) and 44 patients with AL (16 ALL, 28 AML). The PBMC were from 15 HV and 55 patients with AL (18 ALL, 37 AML), and the BMMC from 40 patients with AL (11 ALL, 29 AML). Methanol-chloroform phospholipid extraction from 60 × 106 cells (PBMC or BMMC) was performed according to a modified version of Folch’s method. 31P MRS data was obtained on an AMX 300 Bruker spectrometer (7.05 T). The PAF concentration in the plasma of the patients with ALL or AML was lower than that for the healthy volunteers. The PAF concentration in the plasma of the patients with ALL did not differ significantly from that of the patients with AML. In the case of both the PBMC and BMMC, the PAF concentration was significantly diminished in patients with ALL relative to the concentration for those with AML and for the healthy volunteers. No differences were observed in the PAF concentrations for the AML patients and the healthy volunteers.

Isolation, purification, and amino acid sequence of lactobin A, one of the two bacteriocins produced by Lactobacillus amylovorus LMG P-13139.

Lactobacillus amylovorus LMG P-13139, isolated from corn steep liquor, produces two bactericidal peptides with respective estimated molecular masses of 4.5 and 6.0 kDa upon denaturing sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The antimicrobial activity detected in the fermentation supernatant fraction of L. amylovorus LMG P-13139 was heat stable (20 min, 121 degrees C), displayed a narrow inhibitory spectrum, and was sensitive to proteinase K, trypsin, and alpha-chymotrypsin but insensitive to alpha-amylase, lysozyme, catalase, and lipase. The 4.5-kDa bacteriocin was purified and characterized and designated lactobin A. Lactobin A was isolated as a floating pellicle from culture supernatant brought to 35% saturation with ammonium sulfate. Upon this ammonium sulfate treatment, crude lactobin A was incorporated, together with Tween 80 as a major contaminant, in high-molecular-mass complexes sized at approximately 670 kDa by gel filtration chromatography. Contaminating fatty acids were removed from these micelles by a simple one-step methanol-chloroform extraction without loss of activity. Both inhibitory peptides were separated in an isocratic isopropanol gradient on a PepRPC 5/5 reversed-phase column, and both peptides retained activity towards Lactobacillus helveticus ATCC 15009 upon separation. Lactobin A has a molecular mass determined by electrospray mass spectrometry of 4,879 +/- 0.69 Da. Its peptide chain contains 50 unmodified amino acids, of which 26% are glycine residues and 40% are hydrophobic residues (A, V, L, I, and P). It displays the highest structural homology (42% identity and 28% similarity) with the lafX gene product, encoded by the second open reading frame of the lactacin F operon. These data strongly indicate that lactobin A belongs to the class IIb bacteriocins according to the classification of Klaenhammer.

Iron acquisition by oral hemolytic spirochetes: isolation of a hemin-binding protein and identification of iron reductase activity.

Oral anaerobic spirochetes (OAS) have been implicated in the etiology of periodontal disease. To adapt to the environment of the subgingiva, OAS must be able to acquire iron from limited sources. OAS have previously been shown not to produce siderophores but are beta-hemolytic and can bind hemin via a proteinaceous 47-kDa outer membrane sheath (OMS) receptor. Present studies show that [3H]hemin is not transported into the cytoplasm, that hemin and ferric ammonium citrate, as the sole iron sources, can support the growth of OAS and that protoporphyrin IX and Congo red are inhibitory, thereby implying an important in vivo role for hemin as an iron source. Treponema denticola ATCC 35405 produces an iron reductase. The iron reductase can reduce the central ferric iron moiety of hemin. The 47-kDa OMS hemin-binding protein has been purified to apparent homogeneity by methanol-chloroform extraction of cellular lipoproteins and the use of a hemin-agarose bead affinity column. A model of iron acquisition by OAS is presented.

Automated sample preparation for cholesterol determination in foods

An automated sample preparation system has been developed for the determination of cholesterol in a wide range of matrices. Isolation of cholesterol is performed with a robotic arm coupled with a series of modular stations. Samples are introduced into the system which adds the appropriate reagents, carries out the saponification, pH adjustment, solid-phase extraction and drying steps. This system was evaluated using 15 different food matrices. The average recovery for NIST standards exceeded 97%. A solution of n-hexane-2-propanol was substituted for the traditional methanol-chloroform extraction. Manual pH adjustment was replaced with a buffer. Manual and automated methods were compared and no difference was observed at the 95% confidence level.

A simplified method for determining the gross chemical composition of pinniped milk samples

Recent studies of lactation in wild mammals have emphasised the need for accurate information on milk composition, requiring the analysis of large numbers of samples. A simplified method for determining milk composition in pinnipeds was assessed using samples collected from Antarctic fur seals (Arctocephalus gazella). The stoichiometric relationships between elemental C, H, and N in the ash-free dry mass were used to determine the organic composition and energy content of samples. The results were compared with those obtained by conventional biochemical analyses. Stoichiometric estimates of lipid content were positively correlated with those obtained by methanol–chloroform extraction (r2 = 0.62, P &lt; 0.0001) but consistently lower (P &lt; 0.0005). The mean difference between the results of the two methods was 3.56 ± 0.89% of total milk volume (range −16.98 to +7.47%). Variation between replicates was significantly lower when the stoichiometric method was used (P &lt; 0.05). Gross energy content, calculated from stoichiometric estimates of milk composition, was closely correlated with, though consistently higher than (P &lt; 0.0005), that determined by bomb calorimetry (r2 = 0.96, P &lt; 0.0001). The difference between the two methods, however, was negligible (1.8 ± 0.08%, range −5.0 to +5.6%). The sum of the milk components measured by the stoichiometric method accounted, on average, for 96.5% of the wet mass and varied inversely with lipid content (r2 = 0.29, P &lt; 0.001). This small error is most likely due to retained water in the oven-dried milk. The stoichiometric method for determining organic composition allows the processing of samples at a much faster rate, requiring much smaller volumes, than conventional techniques and should prove useful in ecological studies of lactation.

IgE-challenged human lung mast cells excite vagal sensory neurons in vitro.

Substances released from immunoglobin (Ig) E-stimulated mast cells are likely to be among the chemical mediators responsible for changes in the vagal component of airway reactivity. We have attempted to identify a direct role for mast cell mediators in the control of visceral afferent excitability by examining intracellular electrophysiological changes in vagal neurons after application of extracts prepared from purified and IgE-stimulated human lung mast cells (HLMC). HLMC's, applied by superfusion or by focal pressure ejection from micropipettes, reversibly enhanced the excitability of a subpopulation of rabbit visceral sensory C-fiber neurons by 1) abolishing the slow Ca2+-dependent post-spike after hyperpolarization that uniquely resides in these neurons and controls their spike frequency, 2) depolarizing the cell membrane potential, and 3) increasing membrane input resistance. Control HLMC prepared by subjecting purified human lung mast cells to normal goat serum had no measurable affects on neuronal excitability. The immunologically released constituents responsible for these excitability changes are likely to be lipid mediators, because essentially all biological activity is extractable into an organic phase after methanol-chloroform solvent extraction of the HLMC preparations. These results provide the first unambiguous evidence that products released from immunologically challenged HLMC's directly affect visceral afferent nerve cell function.

Biophysical characterization of macrophage migration enhancement factor (MEF)

Macrophage migration enhancement factor (MEF), a lymphokine produced in the spleen by suppressor-like lymphoid cells, may be an important immunoregulatory molecule of macrophage function. MEF appears to be a potent positive chemokinetic factor and is unusual in that it lacks chemotactic activity. To aid in the development of a purification scheme for MEF we have employed biophysical characterization techniques to define its physical properties. Using the technique of velocity sedimentation in isokinetic sucrose gradients, the S 20w for MEF was determined to be 2.25. The Stoke's radius for MEF was determined by Sephadex G-100 gel filtration to be 28.9 Å. From these measurements the D 20w was calculated to be 7.55 × 10 −7cm 2/sec, the mol. wt was calculated to be 28,000, the frictional ratio (|/| 0) was calculated to be 1.45, the axial ratio was calculated to be 1:8, and the dimensions of the molecule were estimated to be 20̊ × 160 Å. Using the technique of isoelectric focusing in liquid density gradients, the isoelectric point for MEF was estimated to be 8.8. We have also determined by enzyme treatment that MEF is resistant to DNase and RNase and susceptible to proteinase K and L-fucosidase. In addition, MEF partitioned to the aqueous phase during methanol-chloroform extraction procedures. MEF was inactivated at pH 12; at 100°C MEF was stable for 10 min but was inactive after 1 hr. Collectively, these data will facilitate the development of a purification scheme for MEF which will ultimately permit the analysis of the molecule and its function.

Partial purification and characterization of an insulin-like material from spinach and Lemna gibba G3.

The existence in invertebrates, unicellular eukaryotes, and prokaryotes of materials that resemble several vertebrate peptide hormones led to the suggestion that these peptide messengers may have arisen earlier in evolution than had previously been thought. Consistent with this hypothesis, we describe here material in two plants, spinach and Lemma gibba G3, that is very similar to mammalian insulin, yet distinctive. In each of the early purification steps, which consisted of acidic methanol chloroform extraction and sequential chromatography on C-18 hydrophobic resin, Sephadex G-50, CM-Sepharose, and a short C-3 high performance liquid chromatography column, the immunoactive material from plants resembled the common vertebrate insulins. The protein nature of the material was suggested by its destruction by Pronase but not by the inactivated enzyme. In addition, on TSK chromatography it eluted in a position similar to that of insulin, i.e. equivalent to a protein of 6000 daltons. Using an isocratic high performance liquid chromatography system, the plant immunoactivity eluted earlier, and thus was more hydrophilic, than most of the common mammalian insulins, including pork insulin. The interaction of the plant material with anti-insulin antibodies in a radioimmunoassay was confirmed by using an affinity column of anti-insulin antibodies which adsorbed the plant immunoactivity at neutral pH, and released the material with acid elution. Using a quantitative double radioimmunoassay, the plant insulin-like material was distinguished immunologically from chicken insulin. Although the plant insulin-like material is clearly distinct from pork insulin chromatographically, and from chicken insulin immunologically, it resembles vertebrate insulins in its overall configuration. The plant insulin-like material bound to insulin receptors on IM-9 lymphocytes and stimulated glucose oxidation and lipogenesis in isolated adipocytes from young rats. The bioactivity was neutralized in the presence of anti-insulin antibodies, but not in the presence of normal guinea pig IgG. The role of this insulin-like material in plants is unknown but its existence is consistent with an early evolutionary origin of the insulin messenger peptide family. Alternatively we cannot exclude a later convergent development of this family or introduction of vertebrate DNA into plants.

Metabolism of 25-hydroxycholecalciferol in human promyelocytic leukemia cells (HL-60). Isolation and identification of (5Z)- and (5E)-19-nor-10-oxo-25-hydroxycholecalciferol.

Human promyelocytic leukemia cells incubated with 25-hydroxy[26,27-methyl-3H] cholecalciferol (1 microCi) or non-radioactive 25-hydroxycholecalciferol (550 micrograms) produced significant quantities of two vitamin D3 metabolites. The two metabolites were isolated and purified by methanol chloroform extraction and a series of chromatographic procedures. The metabolite purification and elution positions on these columns were followed by radioactivity and their ultraviolet absorption at 310 nm. The two metabolites have been unequivocally identified as (5Z)- and (5E)-19-nor-10-oxo-25-hydroxycholecalciferol by ultraviolet absorption spectrophotometry, mass spectrometry, Fourier-transform infrared spectrophotometry and co-chromatography with synthetic compounds on a high-performance liquid chromatograph. (5E)- but not (5Z)-19-nor-10-oxo-25-hydroxycholecalciferol was able to induce HL-60 cell phenotypic and functional differentiation. However, these two metabolites of 25-hydroxycholecalciferol did not bind specifically to the chick intestinal 3.7 S. receptor protein for 1 alpha,25-dihydroxycholecalciferol. The precise biological role of these metabolites is as yet unclear.

Isolation and identification of 1 alpha,25-dihydroxy-24-oxovitamin D3, 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, and 1 alpha,24(S),25-trihydroxyvitamin D3: in vivo metabolites of 1 alpha,25-dihydroxyvitamin D3.

Three new in vivo metabolites of 1 alpha,25-dihydroxyvitamin D3 were isolated from the serum of dogs given large doses (two doses of 1.5 mg/dog) of 1 alpha,25-dihydroxyvitamin D3. The metabolites were isolated and purified by methanol-chloroform extraction and a series of chromatographic procedures. By cochromatography on a high-performance liquid chromatograph, ultraviolet absorption spectrophotometry, mass spectrometry, Fourier-transform infrared spectrophotometry, and specific chemical reactions, the metabolites were identified as 1 alpha,25-dihydroxy-24- oxovitamin D3, 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, and 1 alpha,24(S),25-trihydroxyvitamin D3. According to these procedures, the total amounts of the isolated metabolites were as follows: 1 alpha,25-dihydroxyvitamin D3, 23.6 micrograms; 1 alpha,25-dihydroxy-24- oxovitamin D3, 1.8 micrograms; 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, 9.2 micrograms; 1 alpha,24(R),25-trihydroxyvitamin D3, 15.4 micrograms; 1 alpha,24(S),25-trihydroxyvitamin D3, 1.0 microgram. With recovery corrections, the serum levels of each metabolite were approximately 49 ng/mL for 1 alpha,25-dihydroxyvitamin D3, 3.7 ng/mL for 1 alpha,25-dihydroxy-24- oxovitamin D3, 19 ng/mL for 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, 32 ng/mL for 1 alpha,24(R),25-trihydroxyvitamin D3, and 2.1 ng/mL for 1 alpha,24(S),25-trihydroxyvitamin D3.

Isolation and chemical characterization of two new vitamin D metabolites produced by the intestine. 1,25-Dihydroxy-23-oxo-vitamin D3 and 1,25,26-trihydroxy-23-oxo-vitamin D3.

Two new vitamin D metabolites were isolated in pure form from incubations of 53 nM 1,25-dihydroxyvitamin D3 with homogenates of small intestinal mucosa of vitamin D-replete chicks. The birds were injected intravenously with 8 to 9 nmol of 1,25-dihydroxyvitamin D3/100 g body weight 5 to 8 h before death. The isolation involved methanol-chloroform extraction and four successive chromatographic procedures (Sephadex LH-20 and high performance liquid chromatography). Chemical structures of the metabolites are proposed on the basis of (a) their chromatographic behavior, (b) their mass spectra, and (c) ultraviolet absorption spectra. They are identified as 1 alpha,25-dihydroxy-23-oxo-vitamin D3 and 1 alpha,25,26-trihydroxy-23-oxo-vitamin D3. Neither of the two new metabolites is produced by the intestinal mucosa when 1,25S,26-trihydroxyvitamin D3 is used as a substrate.

23,24,25-Trihydroxyvitamin D3, 24,25,26-trihydroxyvitamin D3, 24-keto-25-hydroxyvitamin D3, and 23-dehydro-25-hydroxyvitamin D3: new in vivo metabolites of vitamin D3.

Four new in vivo metabolites of vitamin D3 were isolated from the blood plasma of chicks given large doses of vitamin D3. The metabolites were isolated by methanol-chloroform extraction and a series of chromatographic procedures. By use of mass spectrometry, ultraviolet absorption spectrophotometry, and specific chemical reactions, the metabolites were identified as 23,24,25-trihydroxyvitamin D3, 24,25,26-trihydroxyvitamin D3, 24-keto-25-hydroxyvitamin D3 and 23-dehydro-25-hydroxyvitamin D3.

Isolation of identification of 24(R)-hydroxyvitamin D3 from chicks give large doses of vitamin D3.

A new metabolite of vitamin D was isolated from the blood plasma of chicks given large doses of vitamin D3. The isolation involved methanol-chloroform extraction and four column chromatographic steps. The metabolite was identified by high- and low-resolution mass spectroscopy, chemical derivatization, an comigration with authentic standard as 3 beta, 24(R)-dihydroxy-9,10-seco-5,7,10(19)-cholestatriene [24(R)-hydroxyvitamin D3]. No detectable 24-(R)-hydroxyvitamin D3 was recovered from 16 L of plasma from chicks receiving physiologic levels of vitamin D3.

Isolation of a new metabolite of vitamin D produced in vivo, 1α,25-dihydroxyvitamin D 3-26,23-lactone

A new vitamin D metabolite was isolated in pure form from the blood of rats given oral doses of 50 μg/kg of 1α-hydroxyvitamin D 3. The isolation involved methanol-chloroform extraction and four successive column chromatographic procedures. A tentative structure of the metabolite is proposed on the basis of its column chromatographic behavior via mass spectrometry, ultraviolet absorption spectrophotometry, and as 1α,3β,25-trihydroxy-9,10 (19)-cholestatrieno-26,23-lactone. The trivial name 1α,25-dihydroxyvitamin D 3-26,23-lactone is suggested for this compound.

25-Hydroxyvitamin D3 26,23-lactone: a new in vivo metabolite of vitamin D

A major vitamin D metabolite was isolated in pure form from the blood plasma of chicks either maintenance levels or large doses of vitamin D3. The isolation involved methanol-chloroform extraction and five column chromatographic procedures. The metabolite purification and elution position on these columns were followed by a competitive protein binding assay. The metabolite was identified, using high- and low-resolution mass spectrometry, 270-MHz proton nuclear magnetic resonance spectrometry, ultraviolet absorption spectrophotometry, Fourier transform infrared spectrophotometry, and specific chemical reactions, as 3 beta,-25-dihydroxy-9,10-seco-5,7,10(19)-cholestatrieno-26,23-lactone. The trivial names 25-hydroxyvitamin D3 26,23-lactone or calcidiol 26,23-lactone are suggested for this compound.