Recovery Of Sterols Research Articles

LC-MS/MS profiling of colon oxysterols and cholesterol precursors in mouse model of ulcerative colitis

Oxysterols and cholesterol precursors are being increasingly investigated in humans and laboratory animals as markers for various diseases in addition to their important functions. However, the quantitative analysis of these bioactive molecules is obstructed by high structural similarity, poor ionization efficiency and low abundance. The current assay methods are still cumbersome to be of practical use, and their applicability in different bio-samples needs to be evaluated and optimized as necessary. In the present work, chromatographic separation conditions were carefully studied to achieve baseline separation of difficult-to-isolate compound pairs. On the other hand, an efficient sample purification method was established for colon tissue samples with good recoveries of sterols, demonstrating negligible autoxidation of cholesterol into oxysterols. The developed UPLC-APCI-MS/MS method was thoroughly validated and applied to measure oxysterols and cholesterol precursors in colon tissue of dextran sulfate sodium (DSS)-induced mouse colitis models, and it is expected to be successfully applied to the quantitative determination of such components in other tissue samples.

Development and Validation of a Method to Simultaneously Determine Multiple Sterols in Diversiform Food Substrates with UPLC-MS/MS

Sterols play an essential part in physiological activities of almost all creatures. However, there is a lack of a widely applicable routine method to investigate various sterols with both saturated and unsaturated forms in diversiform food materials. In this study, a new UPLC-MS/MS method with wider extensive applicability has been developed to fill this blank, which is considered time and cost saving, and can improve labor efficiency. The Agilent Eclipse Plus C18 column and methanol-water gradient elution system were employed to improve the separation at 40 °C and the flow rate was 0.5 mL/min. The method validation parameters include precision, recovery, linearity, and limit of detection and quantification (LOD and LOQ). The recoveries of sterols in all food matrices and the inter-day and intra-day precisions represented by RSDs were all within the recommended range of chemical analysis. The LODs and LOQs in different types of samples were in range of 0.0036–0.68 μg/mL and 0.01–2.0 μg/mL, respectively. All the results demonstrated that this method was sensitive and accurate enough. Additionally, the acid hydrolysis was found closely related to the degradation of unsaturated sterols in the pretreatment.

Recovery of sterols from vegetable oil distillate by enzymatic and non-enzymatic processes

Sterols are a group of molecules found in plants and animals, which have a number of valuable applications. The deodorization residue, referred to as “deodistillate”, was previously considered as a waste but its economical value nowadays increased due to the presence of high concentrations of sterols, tocopherols and other secondary metabolites attractive for the cosmetic, pharmaceutical or food industry. Sterols can be extracted from vegetable oil deodistillate through a variety of physical and chemical separation processes or their combination. Recently, the use of lipase enzymes has been demonstrated to separate sterols more selectively in higher yields and in milder conditions. This article reviews these lipase-assisted sterol extractions and their main advantages and drawbacks in economic and environmental terms. • Sterol extraction from vegetable oil deodistillate. • Reviewing on enzymatic process to involve extraction. • Different strategies are discussed in an industrial operation approach.

Partitioning of predominant lipophilic bioactives (squalene, α‐tocopherol and β‐sitosterol) during olive oil processing

AbstractThe partitioning of predominant lipophilic olive bioactives (squalene, β‐sitosterol and α‐tocopherol) in olive oil processing was studied for the first time using common integrated olive milling plant. 7% of the oil and 5% of the squalene present in the olives was lost in the last pomace. β‐sitosterol and α‐tocopherol, which are mainly concentrated in the seed of the olive fruit, was recovered in virgin olive oil to a lesser extent (66%, 67%) while 10% were lost in last pomace. Thus, the loss of β‐sitosterol and α‐tocopherol was higher than that of squalene and oil, due to nonrecovered bound forms. 19% of the α‐tocopherol and 21% of the β‐sitosterol loss was unaccounted for, which can be attributed to degradation of α‐tocopherol and incomplete recovery of sterols from the olive and pomace matrices. This study provides a basis for process development studies.

Effect of subcritical water and steam explosion pretreatments on the recovery of sterols, phenols and oil from olive pomace

Hydrothermal pretreatments including steam explosion and subcritical water (SCW) facilitate hydrolyses of plant cell wall materials and supply environmentally friendly extraction solvents to recover value added compounds. In this study, effect of steam and SCW and temperature (160, 180 and 200 °C, 5 min) on yield and β-sitosterol content of pomace oil and phenolics were compared for value added utilization of olive pomace. Compared to acid hydrolysis, hydrothermal pretreatments yielded similar oil recovery. 54–76% of the bound oil and 18–32% of the bound β-sitosterol of the pomace were recovered by hydrothermal pretreatments. Steam pretreatment was more effective than SCW pretreatment at lower temperatures, however SCW showed increasing trend on oil yield related to steam pretreatment. As further research on process development, use of sequential temperature might be investigated, starting with steam explosion (<180 °C), followed by SCW pretreatment (>200 °C) to obtain multiple, aqueous and meal, fractions for total valorization of olive pomace.

Evaluation of different protocols for the analysis of lipophilic plant metabolites by gas chromatography–mass spectrometry using potato as a model

In the analysis of lipophilic plant metabolites by gas chromatography–mass spectrometry a step is required to release fatty acids and other analytes from complex molecules. Seven alternative methods were compared to the standard method of 1% H2SO4/50°C/16 h using Desiree and Phureja potato tubers as models. With two sodium methoxide alkali-catalysed methods (0.5 M NaOCH3/50°C/1 and 16 h) recoveries of ferulic acids increased, long chain fatty acids and sterols decreased, 2-hydroxy acids were negligible, solanidine was absent and Δ5-avenasterol isomerisation was minimal. Using a harsh alkali hydrolysis (1.0 M KOH/120°C/24 h) followed by a mild methylation (1% H2SO4/50°C/1.5 h), recoveries of polyunsaturated fatty acids were poor, sterols decreased but Δ5-avenasterol isomerisation was minimal. With a mild alkali hydrolysis (0.5 M NaOH/100°C/5 min) followed by methylation with boron trifluoride (14%BF3/100°C/30 min) recoveries of sterols and 2-hydroxy fatty acids were similar to the standard method and Δ5-avenasterol isomerisation was high. Lower ferulic acid recoveries, absence of solanidine and overestimation of fatty alcohols were evident in both methods involving alkali hydrolysis. Three different methods using hydrochloric acid (1.00 M HCl/70°C/5 h, 0.63 M HCl/110°C/2 h and 2.00 M HCl/50°C/24 h) all gave increased recoveries of 2-hydroxy acids, ferulic acids, solanidine and sterols, although Δ5-avenasterol isomerisation increased. Hydrochloric acid methods are recommended for studies requiring quantitative determinations (i.e. concentration of metabolite in sample). Either the hydrochloric acid methods or the standard sulphuric acid method are suggested for determining relative concentrations between samples, although there is a requirement for further studies.

Rapid screening of phytosterols in orange juice by solid-phase microextraction on polyacrylate fibre derivatisation and gas chromatographic–mass spectrometric

The potential of solid-phase microextraction on polyacrylate coated fibre, with sequential or simultaneous trimethylsilyl derivatisation followed by gas chromatographic–mass spectrometric analysis, was evaluated for a rapid determination of the distribution of the phytosterols in aqueous food matrixes. Influences of different parameters (bis(trimethylsilyl)trifluoro-acetamide and sterol exposure time, sterol concentration and experimental protocol) on the recovery of sterols were investigated to determine optimum conditions which were tested for sterol extraction and analysis from orange juice. Best selectivity, sterol recovery and derivatisation yields were obtained by extraction and simultaneous derivatisation through immersion of the SPME-PA fibre in the orange juice (10min, 65°C) after headspace absorption of BSTFA (30min, 65°C) on the fibre. Nevertheless the method developed cannot be used for quantitative analysis. But the possibility to effect rapid screen of phytosterol containing in complex media have been shown.

Influence of salinity and natural organic matter on the solid phase extraction of sterols and stanols: Application to the determination of the human sterol fingerprint in aqueous matrices

Faecal sterols have been proposed as direct chemical markers for the determination of faecal contamination in inland and coastal waters. In this study, we assess the impact of (a) the concentration of dissolved organic carbon (DOC), (b) the nature of DOC, (c) the salinity and (d) the concentration of sterols and stanols on their solid phase extraction. When natural organic matter (NOM) is modelled by humic acid, increasing DOC concentration from 2.7 to 15.4 mg/L has no significant impact on the recovery of sterols and stanols. The modelling of NOM by a mixture of humic acid and succinoglycan induces a significant (24%) decrease in the recovery of sterols and stanols. For all concentrations of target compounds, no significant increase in recovery is associated with increasing the salinity. Moreover, an increase in the recovery of target compounds is induced by an increase in their concentration. The nine target compounds and the recovery standard (RS) exhibit the same behaviour during the extraction step. Thus, we propose that (a) the concentration of target compounds can be corrected by the RS to calculate more realistic concentrations without modifying their profile and (b) the sterol fingerprint can be investigated in the colloidal fraction of aqueous samples without altering the information it could provide about the source. The application of this analytical method to waste water treatment plant influent and effluents yields results in agreement with previous studies concerning the use of those compounds to differentiate between sources of faecal contamination. We conclude that this analytical method is fully applicable to the determination of sterol fingerprints in the dissolved phase (<0.7 μm) of natural aqueous samples.

Quantitative determination of cholesterol, sitosterol, and sitostanol in cultured Caco-2 cells by liquid chromatography–atmospheric pressure chemical ionization mass spectrometry

In this study, we describe a simple liquid extraction (methanol/choloroform, 1:1, v/v) method for endogenous free cholesterol and administered sterols extracted from cultured Caco-2 cells. To quantify sterol contents in Caco-2 cells, a new HPLC–APCI-MS method was developed. All the sterols were baseline separated using reversed-phase column (C 8, 2.1 mm × 150 mm, 3.5 μm) and isocratic conditions (90%, v/v, methanol–water mixture containing 0.2 mM ammonium acetate). The full scan mass spectra of sterols were measured by an ion trap mass spectrometer equipped with an APCI ion source. The intense fragment ions resulting from the loss of water [M + H–H 2O] + ( m/ z 369, 395, 397 and 399 for cholesterol, stigmasterol, sitosterol, and sitostanol, respectively) were used for determinations. The absolute extraction recovery of sterols from the spiked cell samples were 109.7 ± 26.2, 105.7 ± 5.1, 109.8 ± 5.0 and 99.0 ± 7.0% for cholesterol, stigmasterol, sitosterol, and sitostanol, respectively. Furthermore, no significant matrix effect was observed for the sterols in the cell samples. The sample assay was based on the internal standard method using stigmasterol as an internal standard. The method was linear over the concentration ranges of 0.45–9.0 μM (cholesterol) and 0.225–7.2 μM (sitosterol and sitostanol). The within- and between-day precision was less than 7% and accuracy ranged from 93.51 to 101.77%. The lowest limit of quantitation (LLOQ) was 0.225 μM for sitosterol and sitostanol, and 0.45 μM for cholesterol. The accuracy range was 95–106% and precision was lower than 9% for all LLOQ values.

Recovery of sterols as fatty acid steryl esters from waste material after purification of tocopherols

Tocopherols are purified industrially from soybean oil deodorizer distillate by a process comprising distillation and ethanol fractionation. The waste material after ethanol fractionation (TC waste) contains 75% sterols, but a purification process has not yet been developed. We thus attempted to purify sterols by a process including a lipase-catalyzed reaction. Candida rugosa lipase efficiently esterified sterols in TC waste with oleic acid (OA). After studying several factors affecting esterification, the reaction conditions were determined as follows: ratio of TC waste/OA, 1:2 (wt/wt); water content, 30%; amount of lipase, 120 U/g-reaction mixture; temperature, 40 degrees C. Under these conditions, the degree of esterification reached 82.7% after 24 h. FA steryl esters (steryl esters) in the oil layer were purified successfully by short-path distillation (purity, 94.9%; recovery, 73.1%). When sterols in TC waste were esterified with FFA originating from olive, soybean, rapeseed, safflower, sunflower, and linseed oils, the FA compositions of the steryl esters differed somewhat from those of the original oils: The content of saturated FA was lower and that of unsaturated FA was higher. The m.p. of the steryl esters synthesized (21.7-36.5 degrees C) were remarkably low compared with those of the steryl esters purified from high-b.p. soybean oil deodorizer distillate substances (56.5 degrees C; JAOCS 80, 341-346, 2003). The low-m.p. steryl esters were soluble in rapeseed oil even at a final concentration of 10%.

Recovery of phytosterols from sunflower oil deodorizer distillates

AbstractPhytosterols are usually recovered by crystallization from the deodorizer distillate (DD) of vegetable oils. In this work, the impact of the principal process variables (viz., solvents and cosolvents, cooling rate, crystallization temperature, and ripening time) on the quality and yield of the recovered phytosterols was studied by using a sunflower oil DD “enriched” (i.e., preconcentrated) via transesterification with ethanol (EDD) as a feedstock and commercial hexane as solvent (S), with S/EDD mass ratios of 3 to 5. Water (0 to 4.5 wt%) and ethanol (0 to 10 wt%) were used as cosovents, with crystallization temperatures between 0 and −20°C and crystallization times from 4 to 96 h. The cooling rate was either −20°C/h or “brisk chilling” from 40 to −5°C. The nature and composition of the EDD solvent and cosolvent composite arose as the most important process variable, strongly influencing both the percentage of sterol yield and the purity of the crystals, as well as their filterability and washability. Water‐saturated hexane sufficed to give good crystallization, yet the beneficial effect of adding water as the single cosolvent was enhanced by adding small and precise amounts of ethanol. A recovery of sterols as high as 84% (with 36% purity) was achieved by using a single‐stage batch crystallization of the S/EDD mixture (S/EDD=mass ratio 4).

Solid-phase extraction–thin-layer chromatography–gas chromatography method for the detection of hazelnut oil in olive oils by determination of esterified sterols

The sterol composition of extra virgin olive oil is very characteristic and, thus, has become a helpful tool to detect adulterations with other vegetable oils. Special attention has been addressed to the separate determination of the free and esterified sterol fractions, since both have different compositions and can thus provide more precise information about the actual origin of the olive oil. In the case of admixtures with small amounts of hazelnut oil, this approach can be extremely useful, because the similarity between the fatty acid compositions of both oils hampers the detection of the fraud. A hyphenated chromatographic method was developed for a sensitive and precise determination of esterified sterols in olive oils. The oil was subjected to silica solid-phase extraction (SPE) fractionation, cold saponification of the collected fraction and purification on silica TLC. The sterol band was then injected into an SPB-5 (30 m×0.25 mm I.D., 0.25 μm film thickness) and the ratio [% campesterol×(% 7-stigmastenol) 2]/(% 7-avenasterol) was calculated. The method was tested on extra virgin olive oil; good sterol recoveries and repeatability were obtained. The results were compared with another method, which has a different sample preparation sequence (silica column chromatography, hot saponification and silica TLC). Similar results were achieved with both methods; however, the SPE–cold saponification–TLC–capillary GC was faster, required less solvent and prevented sterol decomposition. The SPE-method was applied to an admixture with 10% of hazelnut oil and to a screening of 11 oils (husk oil, virgin and refined olive oils) from different Mediterranean countries.

Supercritical fluid extraction and gas chromatographic electron capture detection method for sterol analysis of environmental water samples

A standard additions technique was carried out by spiking standard sterol mixtures onto environmental water filtering glass fibre filters, which were then extracted by supercritical fluid extraction, derivatized into their pentafluorophenyldimethylsilyl ethers and analyzed by gas chromatography using electron capture detection. Calibration curves obtained from directly derivatized sterol standards, and also sterol standards spiked both onto blank filters and filters used to filter environmental water, were all r2 > 0.99 linearity. Spiked sterol recoveries ranged from 80 to 120% with good precision, allowing application of the method to the determination of sterol levels in environmental water samples. For a sample size of 0.5 l, the limit of detection of coprostanol was estimated to be 200 ng l–1. Water from the Prahran main drain outfall in Melbourne was analyzed and the coprostanol concentration was found to be about 400 ng l–1, which exceeds the equivalent primary contact limit (60 ng l–1) for this sterol in recreational waters in Australia.

Simultaneous determination of sterols in edible oils by use of a continuous separation module coupled to a gas chromatograph

A new analytical method that combines on-line isolation-preconcentration, extraction and gas-liquid chromatography was developed for the determination of sterols in oils and fats. A silica gel column permits the isolation of sterols together with triglycerides (25%) which were further transesterified with acetyl chloride-methanol to fatty acid methyl esters. The proposed method was applied to the determination of cholesterol, campesterol, β-sitosterol and stigmasterol in vegetable oils and lard; olive oil was also mixed with sunflower-seed oil and lard in order to check for potential adulteration of vegetable oils. The average recoveries of sterols added to olive oil at concentrations between 13.6 and 40.8 mg per 100 g of oil ranged from 91.4 to 103.9%.

Combining sterol and fatty acid profiles for the characterization of fungi

Forty two strains of fungi belonging to 16 species and 11 genera within Phycomycetes, Ascomycetes and Basidiomycetes were cultivated on agar plates and analysed for their fatty acids by a slightly modified method commonly used in the investigation of bacteria. Several fungal sterols were also detected in the fatty acid extracts following an extended gas chromatographic separation. Differences between fatty acid profiles and combined fatty acid+sterol profiles of the fungi were evaluated by multivariate discriminant analysis. Not all genera could be separated on the basis of their fatty acid profiles alone. Considerable improvement was achieved when the sterol profiles were included in the statistical analysis. This enabled even the differentiation of two sibling species of Heterobasidion annosum (type S and P). The ergosterol and ergosta-7,22-dien-3-ol content proved to be especially valuable for grouping of these fungi. Compared to untreated mycelia, better recovery of sterols was obtained when fungal mycelial samples were frozen in liquid nitrogen and homogenized in a ball mill prior to extraction. Small temperature deviations (±3 °C) during cultivation of fungi may affect fungal identification using this method. There are no earlier reports on the fatty acid composition of four genera (Ascocoryne, Gremmeniella, Neobulgaria and Amylostereum) investigated.

Enzymatic pretreatment of deodorizer distillate for concentration of sterols and tocopherols

Separation of sterols and tocopherols from fatty acids in deodorizer distillate was facilitated through lipase‐catalyzed modification of fatty acids in canola, mixed and soya deodorizer distillates. The fatty acid esterification with methanol catalyzed by SP‐382 (an immobilized nonspecific lipase) proceeded rapidly, with conversion of fatty acid to methyl ester in 5 h being 96.5, 83.5 and 89.4%, respectively. A model mixture of pure oleic acid and dl‐α‐tocopherol was used to study any potential side reactions that may lower the tocopherol content during the esterification reaction. Under the conditions employed, the loss of tocopherol was less than 5%. Simple vacuum distillation (1–2 mm Hg) was employed to remove the volatile fraction (methyl esters of fatty acids, some fatty acids and other volatiles) of the esterified deodorizer distillate, leaving behind sterols, sterol esters and tocopherols. Sterols and tocopherols were almost completely retained in the residue fraction with recoveries in the range of 95%. Overall recoveries of sterols and tocopherols after esterification and distillation were over 90% for all the deodorizer distillate samples.

Application of radiometric and isotope dilution-mass spectrometric techniques to the certification of edible oil and fat reference materials

A method for the isotope dilution-mass spectrometric (ID-MS) determination of butyric acid C4 in butter fat (RM164) was developed in order to support the data gathered from nine experienced European laboratories within the final certification exercise. The ID-MS results (3,46±0,06 g C4/100 g fat) were in very good agreement with those obtained by “classical” GLC and HPLC techniques. (RM164 was finally certified at 3,49±0,06 g C4/100 g fat). This paper reports briefly on a previous preliminary study undertaken to validate a procedure (agreed by the BCR-sterol group) for the isolation of the sterol from fats and oils. By use of labelled sterols and radiometric measurements it was shown that sterol recoveries were superior to 96%.

Validation of a quantitative procedure for the extraction of sterols from edible oils using radiolabelled compounds.

The work described in this paper is integrated in an analytical programme organised by the Community Bureau of Reference with the aim of developing reference materials certified for sterol content. Preliminary inter-comparison of methods showed that the level of agreement of the results was insufficient for certification purposes. Errors could occur in the different steps before the final determination by gas-liquid chromatography. It was, therefore, decided to validate a quantitative procedure for the isolation of sterols. A well defined saponification-extraction method was tested using labelled sterols ([3H]cholesterol and [3H]cholesteryl oleate) and radiochemical measurements. The study has shown that total cholesterol recovery reached 100.5 +/- 1.4%, that cholesteryl ester was saponified quantitatively and that there were no appreciable amounts of degradation products. The procedure has been used as the basis for the certification of three reference materials and it has been shown that the saponification and extraction procedure leads to the quantitative recovery of sterols regardless of the nature of the fatty material tested.

Combined biochemical and ultrastructural analysis of aflatoxin B1action on the endomembrane system of plant cells

SummaryAflatoxin B1 inhibits bean germination, elongation of both attached and excised, cultured roots and the latter's ability to intraccllularize 14C-leucine. Thus, B1 may alter plamalemma structure and, or, function. To determine whether 18h incubation of excised roots in B1—containing medium could affect membrane chemical content, endomembranes were isolated at 1 000 (1), 40 000 (40) and 80 000 (80) xg and characterized chemically. Statistically significant differences (SD) in acid-insoluble protein between treated (T) and non-treated (NT) roots were observed for both 40 and 80 xg pellets (P). Protein and sterol recoveries were 81 (T) and 84 (NT)% for the former and 77 (T) and 79 (NT)% for the latter. Lipid phosphorus recoveries were 87, 3 (T) and 136 (NT) with and 96 (T) and 83 (NT) without membrane stabilization.Protein: sterol: lipid phosphorus were: - 35, 7: 4, 5: 1 (1 xg); 18, 9: 3, 6: 1 (40 xg); 26, 3: 4, 6: 1 (80 xg); 1, 010: 29: 1 (80 supernatant S) for NT.- 36, 9: 3, 3: 1 (1 xg); 23, 1: 3, 8: 1 (40 xg); 36, 2: 4, 8: 1 (80 xg); 1, 053: 21, 7: 1 (80 xg), for T roots.SD in RNA contents between T and NT roots were found for both 1 and 40 xg P but not for 80 xg pellet and its S. Whereas a SD increase in 1 xg pellet DNA content occured upon treatment, a decrease was noted for the 40 xg pellet but not for the 80 xg pellet and its S. Similar pH 6 (plasmalemma marker) and 9 (mitochondrial marker) k+—stimulated ATPase activities were demonstrated for 40 and 80 xg P. The 1 xg pellet contained 50% for the NADH-cytochrome C reductase activity (ER marker) recovered from fractions examined for activity (absent from 40 xg pellet). Both the 80 xg pellet and its S possessed activity.

Aflatoxin - induced alteration in the levels of membrane chemical of subcellular organelles isolated from excised, incubated glycine max, cv. "Essex" roots. I. Non-enriched organelles.

Isolates of aflatoxin-producing strains of Aspergillus grow on autoclaved and field-grown (lesser extent) Glycine max beans. Both mixed and aflatoxin B1 inhibit G. max, cv. ‘Essex’ bean germination and elongation of either attached or excised cultured roots. Because B1 impairs the latter roots' ability to intracellularize [14C]-leucine, it may alter plasmalemma structure and/or function. To determine whether incubation of excised roots for 18 hours in toxin-containing medium could affect cellular membrane chemical content, organelles were isolated by differential centrifugation (1 000, 40 000, and 80 000 xg) of homogenates and characterized chemically. Statistically significant differences between treated and untreated roots in acid insoluble protein but not either sterol or lipid phosphorus levels were observed for both 40,000 and 80,000 xg pellets. Protein and sterol recoveries were 81 (treated) and 84 (untreated) % for the former and 77 (treated) and 79 (untreated) % for the latter. Lipid phosphorus recoveries were 87.3 (treated) and 136 (untreated) % with and 96 (treated) and 83 (untreated) without membrane stabilization. Protein:sterol:lipid phosphorus were 35.7∶4.5∶1 (1 000 xg), 18.9∶3.6∶1 (40000 xg), 26.3∶4.6∶1 (80 000 xg) and 1,010∶29∶1 (80 000 xg supernatant) for untreated and 36.9∶3.3∶1 (1,000 xg), 23.1∶3.8∶1 (40 000 xg), 36.2∶4.8∶1 (80 000 xg) and 1,053∶21.7∶1 (80 000 xg supernatant) for treated roots. Significant differences in RNA content between treated and untreated roots were found for both 1 000 and 40 000 xg pellets but not for the 80 000 xg pellet and its supernatant. Whereas a significant increase in the 1 000 xg pellet occurred upon treatment, a decrease was noted for the 40 000 xg pellet but not for the 80 000 xg pellet and its supernatant. Similar pH 6 (plasmalemma marker enzyme) and 9 (mitochondrial marker enzyme) K+-stimulated ATPase activities were demonstrated for 40 000 and 80 000 xg pellets. The 1 000 xg pellet contained greater than 50% of the NADH-cytochrome c-reductase activity (endoplasmic reticulum marker enzyme) recovered from fractions examined for this activity which was absent from the 40 000 xg pellet. Both the 80 000 xg pellet and its supernatant possessed equivalent reductase activities. Inosine diphosphatase activity (dictyosome marker enzyme) was not present in 1 000 xg pellets obtained from either treated or untreated roots but was in both 40 000 and 80 000 xg pellets. Based on these results, a tentative assignment of organelles to each fraction (xg force) is reported.