High resolution 1H NMR of a lipid cubic phase using a solution NMR probe.

The cubic mesophase formed by monoacylglycerols and water is an important medium for the in meso crystallogenesis of membrane proteins. To investigate molecular level lipid and additive interactions within the cubic phase, a method was developed for improving the resolution of (1)H NMR spectra when using a conventional solution state NMR probe. Using this approach we obtained well-resolved J-coupling multiplets in the one-dimensional NMR spectrum of the cubic-Ia3d phase prepared with hydrated monoolein. A high resolution t-ROESY two-dimensional (1)H NMR spectrum of the cubic-Ia3d phase is also reported. Using this new methodology, we have investigated the interaction of two additive molecules, L-tryptophan and ruthenium-tris(2,2-bipyridyl) dichloride (rubipy), with the cubic mesophase. Based on the measured chemical shift differences when changing from an aqueous solution to the cubic phase, we conclude that L-tryptophan experiences specific interactions with the bilayer interface, whereas rubipy remains in the aqueous channels and does not associate with the lipid bilayer.

Microbial acceleration of aerobic pyrite oxidation at circumneutral pH.

Pyrite (FeS2 ) is the most abundant sulfide mineral on Earth and represents a significant reservoir of reduced iron and sulfur both today and in the geologic past. In modern environments, oxidative transformations of pyrite and other metal sulfides play a key role in terrestrial element partitioning with broad impacts to contaminant mobility and the formation of acid mine drainage systems. Although the role of aerobic micro-organisms in pyrite oxidation under acidic-pH conditions is well known, to date there is very little known about the capacity for aerobic micro-organisms to oxidize pyrite at circumneutral pH. Here, we describe two enrichment cultures, obtained from pyrite-bearing subsurface sediments, that were capable of sustained cell growth linked to pyrite oxidation and sulfate generation at neutral pH. The cultures were dominated by two Rhizobiales species (Bradyrhizobium sp. and Mesorhizobium sp.) and a Ralstonia species. Shotgun metagenomic sequencing and genome reconstruction indicated the presence of Fe and S oxidation pathways in these organisms, and the presence of a complete Calvin-Benson-Bassham CO2 fixation system in the Bradyrhizobium sp. Oxidation of pyrite resulted in thin (30-50 nm) coatings of amorphous Fe(III) oxide on the pyrite surface, with no other secondary Fe or S phases detected by electron microscopy or X-ray absorption spectroscopy. Rates of microbial pyrite oxidation were approximately one order of magnitude higher than abiotic rates. These results demonstrate the ability of aerobic microbial activity to accelerate pyrite oxidation and expand the potential contribution of micro-organisms to continental sulfide mineral weathering around the time of the Great Oxidation Event to include neutral-pH environments. In addition, our findings have direct implications for the geochemistry of modern sedimentary environments, including stimulation of the early stages of acid mine drainage formation and mobilization of pyrite-associated metals.

Microbial Fe(III) oxide reduction potential in Chocolate Pots hot spring, Yellowstone National Park.

Chocolate Pots hot springs (CP) is a unique, circumneutral pH, iron-rich, geothermal feature in Yellowstone National Park. Prior research at CP has focused on photosynthetically driven Fe(II) oxidation as a model for mineralization of microbial mats and deposition of Archean banded iron formations. However, geochemical and stable Fe isotopic data have suggested that dissimilatory microbial iron reduction (DIR) may be active within CP deposits. In this study, the potential for microbial reduction of native CP Fe(III) oxides was investigated, using a combination of cultivation dependent and independent approaches, to assess the potential involvement of DIR in Fe redox cycling and associated stable Fe isotope fractionation in the CP hot springs. Endogenous microbial communities were able to reduce native CP Fe(III) oxides, as documented by most probable number enumerations and enrichment culture studies. Enrichment cultures demonstrated sustained DIR driven by oxidation of acetate, lactate, and H2 . Inhibitor studies and molecular analyses indicate that sulfate reduction did not contribute to observed rates of DIR in the enrichment cultures through abiotic reaction pathways. Enrichment cultures produced isotopically light Fe(II) during DIR relative to the bulk solid-phase Fe(III) oxides. Pyrosequencing of 16S rRNA genes from enrichment cultures showed dominant sequences closely affiliated with Geobacter metallireducens, a mesophilic Fe(III) oxide reducer. Shotgun metagenomic analysis of enrichment cultures confirmed the presence of a dominant G. metallireducens-like population and other less dominant populations from the phylum Ignavibacteriae, which appear to be capable of DIR. Gene (protein) searches revealed the presence of heat-shock proteins that may be involved in increased thermotolerance in the organisms present in the enrichments as well as porin-cytochrome complexes previously shown to be involved in extracellular electron transport. This analysis offers the first detailed insight into how DIR may impact the Fe geochemistry and isotope composition of a Fe-rich, circumneutral pH geothermal environment.

Quantitative analysis of surface-located triacylglycerol in intact emulsion particles.

The amount of triacylglycerol (TG) in the surface monolayer of intact phospholipid-stabilized emulsions was determined using (13)C nuclear magnetic resonance ((13)C NMR). (13)C NMR spectra of emulsions composed of bulk long-chain or medium-chain TG were prepared with [(13)C(3)]-carbonyl-enriched triolein, tripalmitin, or trioctanoin, and were analyzed and compared with NMR spectra of phosphatidylcholine vesicles with and without added TG. Identification of carbonyl peaks intermediate between those of phosphatidylcholine carbonyls and bulk TG confirmed the presence of surface TG in each emulsion. The surface of emulsions contained 2.2 mol % tripalmitin and 1.4 mol % triolein, but significantly more medium-chain TG, 9.1 mol % trioctanoin, as predicted by measurements of TG in phospholipid vesicles. Thus, medium-chain TGs are more accessible than long-chain TGs to enzymes or pro-oxidants in the continuous phase of phospholipid-stabilized emulsion systems. The quantitative determination of surface-located TG in intact particles will advance the understanding of emulsion colloidal properties, physicochemical stability, and metabolic behavior.

Modified atmosphere packaged cheddar cheese shreds: influence of fluorescent light exposure and gas type on color and production of volatile compounds.

The influences of fluorescent light exposure and packaging atmosphere on the headspace volatiles and color of Cheddar cheese shreds were evaluated using gas chromatography-mass spectrometry and spectrocolorimetry, respectively. Cheddar cheeses were packaged under atmospheres of 100% carbon dioxide or 100% nitrogen and stored at 4 degrees C under fluorescent light for 6 weeks. Cheeses stored under carbon dioxide contained higher concentrations of aldehydes and fatty acids and lower concentrations of alcohols and esters than cheeses stored under nitrogen. Carbon dioxide atmospheres potentiated light-induced oxidation in shredded Cheddar cheeses, as evidenced by aldehyde and fatty acid headspace volatiles measured following storage. Color bleaching occurred only in cheeses packaged under carbon dioxide and exposed to light. The shift in color is proposed to be due to an interaction between carbon dioxide and high-intensity light, leading to the oxidation of the pigment molecule, bixin. The results have significant implications for procedures used to handle and store pigmented cheeses to ensure desirable flavor and consumer acceptability.

Kinetics of 2-monoacylglycerol acyl migration in model chylomicra.

In the metabolism of triacylglycerol (TG)-rich lipoproteins, 2-monoacylglycerols (2-MG) are produced by lipoprotein lipase (LPL) hydrolysis of TG. The metabolic fate of 2-MG is not known with certainty. 2-MG that accumulate on the chylomicra surface have been proposed to isomerize spontaneously to 1(3)-MG, which are then hydrolyzed by LPL to free fatty acids and glycerol. In this study the rate and the effect of acyl chain saturation on the spontaneous acyl migration of 2-MG in in vitro model chylomicra emulsions were determined. After 1 h of incubation at 37 degrees C, less than 20% of 2-monoolein (2-MO) or 2-monopalmitin (2-MP) spontaneously isomerized to 1(3)-MO or 1(3)-MP, respectively. Accordingly, it was concluded that spontaneous isomerization of 2-MG is not the major mechanism for 2-MG metabolism post-TG hydrolysis in chylomicra. Isomerization rates, expressed as decrease in percentage of 2-MG remaining per hour, were -5.12 and -5.86 in water, and -0.43 and -0.41 in hexane for 2-MO and 2-MP, respectively. There was no significant difference between the isomerization rates of 2-MO and 2-MP. Thus, in the present study, saturation of the MG acyl chain did not influence spontaneous acyl migration in either water or hexane, but isomerization of 2-MG was faster in water than in hexane.

Modeling the sorption kinetics of divalent metal ions to hematite.

The sorption kinetics of the divalent metals Zn, Co, Ni, and Cd to hematite were studied in single sorbate systems with high sorbate/sorbent ratios (from 1.67 to 3.33mol sorbate/mol sorption sites) in 10mM Na-piperazine N,N'-bis 2-ethane sulfonic acid (Na-PIPES) solution at pH 6.8. The experimental data showed a rapid initial sorption (half-time about 1min) followed by slower sorption that continued for 1-5 days. The sequence of fast to slow sorption kinetics was modeled by slow inner-sphere (IS) complexation in equilibrium with outer-sphere (OS) complexes. Although the OS reaction was fast and considered to be in equilibrium, the extent of OS complexation changed over time due to increased surface potential from the IS complexes. For example, the model showed that the dimensionless OS complexation function, K(os), decreased from 0.014 initially to 0.0016 at steady state due to sorption of 4x10(-5)M Zn(II) to 2gL(-1) hematite. Sorption rate constants, k(ads), for the various divalent metals ranged from 6.1 to 82.5M(-1)s(-1). Desorption rate constants, k(des), ranged from 5.2x10(-7) to 6.7x10(-5)s(-1). This study suggests that the conversion from OS to IS complex was the rate-determining step for the sorption of divalent metals on crystalline adsorbents.

Iron isotope fractionation during microbial dissimilatory iron oxide reduction in simulated Archaean seawater.

The largest Fe isotope excursion yet measured in marine sedimentary rocks occurs in shales, carbonates, and banded iron formations of Neoarchaean and Paleoproterozoic age. The results of field and laboratory studies suggest a potential role for microbial dissimilatory iron reduction (DIR) in producing this excursion. However, most experimental studies of Fe isotope fractionation during DIR have been conducted in simple geochemical systems, using pure Fe(III) oxide substrates that are not direct analogues to phases likely to have been present in Precambrian marine environments. In this study, Fe isotope fractionation was investigated during microbial reduction of an amorphous Fe(III) oxide-silica coprecipitate in anoxic, high-silica, low-sulphate artificial Archaean seawater at 30 °C to determine if such conditions alter the extent of reduction or isotopic fractionations relative to those observed in simple systems. The Fe(III)-Si coprecipitate was highly reducible (c. 80% reduction) in the presence of excess acetate. The coprecipitate did not undergo phase conversion (e.g. to green rust, magnetite or siderite) during reduction. Iron isotope fractionations suggest that rapid and near-complete isotope exchange took place among all Fe(II) and Fe(III) components, in contrast to previous work on goethite and hematite, where exchange was limited to the outer few atom layers of the substrate. Large quantities of low-δ(56)Fe Fe(II) (aqueous and solid phase) were produced during reduction of the Fe(III)-Si coprecipitate. These findings shed new light on DIR as a mechanism for producing Fe isotope variations observed in Neoarchaean and Paleoproterozoic marine sedimentary rocks.

A thermodynamically-based model for predicting microbial growth and community composition coupled to system geochemistry: Application to uranium bioreduction.

'Bioimmobilization' of redox-sensitive heavy metals and radionuclides is being investigated as a way to remediate contaminated groundwater and sediments. In one approach, growth-limiting substrates are added to the subsurface to stimulate the activity of targeted groups of indigenous microorganisms and create conditions favorable for the microbially-mediated reductive precipitation ('bioreduction') of targeted contaminants. We present a theoretical framework for modeling this process that modifies conventional geochemical reaction path modeling to include thermodynamic descriptions for microbial growth and may be called biogeochemical reaction path modeling. In this approach, the actual microbial community is represented by a synthetic microbial community consisting of a collection of microbial groups; each with a unique growth equation that couples a specific pair of energy yielding redox reactions. The growth equations and their computed standard-state free energy yields are appended to the thermodynamic database used in conventional geochemical reaction path modeling, providing a direct coupling between chemical species participating in both microbial growth and geochemical reactions. To compute the biogeochemical reaction paths, growth substrates are reacted incrementally with the defined geochemical environment and the coupled equations are solved simultaneously to predict reaction paths that display changing microbial biomass, community composition (i.e. the fraction of total biomass in each microbial group), and the aqueous and mineral composition of the system, including aqueous speciation and oxidation state of the targeted contaminants. The approach, with growth equations derived from the literature using well-known bioenergetics principles, was used to predict the results of a laboratory microcosm experiment and an in situ field experiment that investigated the bioreduction of uranium. Predicted effects of ethanol or acetate addition on uranium concentration and speciation, major ion geochemistry, mineralogy, microbial biomass and community composition were in qualitative agreement with experimental observations although the available data precluded rigorous model testing. While originally developed for use in better understanding of bioimmobilization of heavy metals and radionuclides, the modeling approach is potentially useful for exploring the coupling of microbial growth and geochemical reactions in a variety of other basic and applied biotechnology research settings.

Microbial production of isotopically light iron(II) in a modern chemically precipitated sediment and implications for isotopic variations in ancient rocks.

The inventories and Fe isotope composition of aqueous Fe(II) and solid-phase Fe compounds were quantified in neutral-pH, chemically precipitated sediments downstream of the Iron Mountain acid mine drainage site in northern California, USA. The sediments contain high concentrations of amorphous Fe(III) oxyhydroxides [Fe(III)(am)] that allow dissimilatory iron reduction (DIR) to predominate over Fe-S interactions in Fe redox transformation, as indicated by the very low abundance of Cr(II)-extractable reduced inorganic sulfur compared with dilute HCl-extractable Fe. delta(56)Fe values for bulk HCl- and HF-extractable Fe were approximately 0. These near-zero bulk delta(56)Fe values, together with the very low abundance of dissolved Fe in the overlying water column, suggest that the pyrite Fe source had near-zero delta(56)Fe values, and that complete oxidation of Fe(II) took place prior to deposition of the Fe(III) oxide-rich sediment. Sediment core analyses and incubation experiments demonstrated the production of millimolar quantities of isotopically light (delta(56)Fe approximately -1.5 to -0.5 per thousand) aqueous Fe(II) coupled to partial reduction of Fe(III)(am) by DIR. Trends in the Fe isotope composition of solid-associated Fe(II) and residual Fe(III)(am) are consistent with experiments with synthetic Fe(III) oxides, and collectively suggest an equilibrium Fe isotope fractionation between aqueous Fe(II) and Fe(III)(am) of approximately -2 per thousand. These Fe(III) oxide-rich sediments provide a model for early diagenetic processes that are likely to have taken place in Archean and Paleoproterozoic marine sediments that served as precursors for banded iron formations. Our results suggest pathways whereby DIR could have led to the formation of large quantities of low-delta(56)Fe minerals during BIF genesis.

Mercury methylation by dissimilatory iron-reducing bacteria.

The Hg-methylating ability of dissimilatory iron-reducing bacteria in the genera Geobacter, Desulfuromonas, and Shewanella was examined. All of the Geobacter and Desulfuromonas strains tested methylated mercury while reducing Fe(III), nitrate, or fumarate. In contrast, none of the Shewanella strains produced methylmercury at higher levels than abiotic controls under similar culture conditions. Geobacter and Desulfuromonas are closely related to known Hg-methylating sulfate-reducing bacteria within the Deltaproteobacteria.

Dissimilatory Fe(III) Reduction by the Marine Microorganism Desulfuromonas acetoxidans.

The ability of the marine microorganism Desulfuromonas acetoxidans to reduce Fe(III) was investigated because of its close phylogenetic relationship with the freshwater dissimilatory Fe(III) reducer Geobacter metallireducens. Washed cell suspensions of the type strain of D. acetoxidans reduced soluble Fe(III)-citrate and Fe(III) complexed with nitriloacetic acid. The c-type cytochrome(s) of D. acetoxidans was oxidized by Fe(III)-citrate and Mn(IV)-oxalate, as well as by two electron acceptors known to support growth, colloidal sulfur and malate. D. acetoxidans grew in defined anoxic, bicarbonate-buffered medium with acetate as the sole electron donor and poorly crystalline Fe(III) or Mn(IV) as the sole electron acceptor. Magnetite (Fe(3)O(4)) and siderite (FeCO(3)) were the major end products of Fe(III) reduction, whereas rhodochrosite (MnCO(3)) was the end product of Mn(IV) reduction. Ethanol, propanol, pyruvate, and butanol also served as electron donors for Fe(III) reduction. In contrast to D. acetoxidans, G. metallireducens could only grow in freshwater medium and it did not conserve energy to support growth from colloidal S reduction. D. acetoxidans is the first marine microorganism shown to conserve energy to support growth by coupling the complete oxidation of organic compounds to the reduction of Fe(III) or Mn(IV). Thus, D. acetoxidans provides a model enzymatic mechanism for Fe(III) or Mn(IV) oxidation of organic compounds in marine and estuarine sediments. These findings demonstrate that 16S rRNA phylogenetic analyses can suggest previously unrecognized metabolic capabilities of microorganisms.

Competition between Fe(III)-reducing and methanogenic bacteria for acetate in iron-rich freshwater sediments.

The kinetics of acetate uptake and the depth distribution of [2-14C]acetate metabolism were examined in iron-rich sediments from a beaver impoundment in northcentral Alabama. The half-saturation constant (Km) determined for acetate uptake in slurries of Fe(III)-reducing sediment (0.8 mM) was more than 10-fold lower than that measured in methanogenic slurries (12 mM) which supported comparable rates of bulk organic carbon metabolism and Vmax values for acetate uptake. The endogenous acetate concentration (Sn) was also substantially lower (1.7 mM) in Fe(III)-reducing vs methanogenic (9.0 mM) slurries. The proportion of [2-14C]acetate converted to 14CH4 increased with depth from ca 0.1 in the upper 0.5 cm to ca 0.8 below 2 cm and was inversely correlated (r2 = 0.99) to a decline in amorphous Fe(III) oxide concentration. The results of the acetate uptake kinetics experiments suggest that differences in the affinity of Fe(III)-reducing bacteria vs methanogens for acetate can account for the preferential conversion of [2-14C]acetate to 14CO2 in Fe(III) oxide-rich surface sediments, and that the downcore increase in conversion of [2-14C]acetate to 14CH4 can be attributed to progressive liberation of methanogens from competition with Fe(III) reducers as Fe(III) oxides are depleted with depth.

Suboxic deposition of ferric iron by bacteria in opposing gradients of Fe(II) and oxygen at circumneutral pH.

The influence of lithotrophic Fe(II)-oxidizing bacteria on patterns of ferric oxide deposition in opposing gradients of Fe(II) and O(2) was examined at submillimeter resolution by use of an O(2) microelectrode and diffusion microprobes for iron. In cultures inoculated with lithotrophic Fe(II)-oxidizing bacteria, the majority of Fe(III) deposition occurred below the depth of O(2) penetration. In contrast, Fe(III) deposition in abiotic control cultures occurred entirely within the aerobic zone. The diffusion microprobes revealed the formation of soluble or colloidal Fe(III) compounds during biological Fe(II) oxidation. The presence of mobile Fe(III) in diffusion probes from live cultures was verified by washing the probes in anoxic water, which removed ca. 70% of the Fe(III) content of probes from live cultures but did not alter the Fe(III) content of probes from abiotic controls. Measurements of the amount of Fe(III) oxide deposited in the medium versus the probes indicated that ca. 90% of the Fe(III) deposited in live cultures was formed biologically. Our findings show that bacterial Fe(II) oxidation is likely to generate reactive Fe(III) compounds that can be immediately available for use as electron acceptors for anaerobic respiration and that biological Fe(II) oxidation may thereby promote rapid microscale Fe redox cycling at aerobic-anaerobic interfaces.

Composition of Non-Microbially Reducible Fe(III) in Aquatic Sediments.

The production of small quantities of Fe(II) during the initial phase of microbial Fe(III) reduction greatly increased the amount of Fe(III) that could be extracted from freshwater sediments with oxalate. This finding and other evidence suggest that the oxalate-extractable Fe(III) that is unavailable for microbial reduction in anoxic sediments is not in the form of mixed Fe(III)-Fe(II) forms, as was previously suggested, but rather is in the form of highly crystalline Fe(III) oxides.

Changes in bacterial species composition in enrichment cultures with various dilutions of inoculum as monitored by denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis revealed changes in the bacterial species obtained from enrichment cultures with different inoculum dilutions. This inoculum dilution enrichment approach may facilitate the detection and isolation of a greater number of bacterial species than traditional enrichment techniques.

Microbially catalyzed nitrate-dependent oxidation of biogenic solid-phase Fe(II) compounds.

The potential for microbially catalyzed NO3(-)-dependent oxidation of solid-phase Fe(II) compounds was examined using a previously described autotrophic, denitrifying, Fe(II)-oxidizing enrichment culture. The following solid-phase Fe(II)-bearing minerals were considered: microbially reduced synthetic goethite, two different end products of microbially hydrous ferric oxide (HFO) reduction (biogenic Fe3O4 and biogenic FeCO3), chemically precipitated FeCO3, and two microbially reduced iron(III) oxide-rich subsoils. The microbially reduced goethite, subsoils, and chemically precipitated FeCO3 were subject to rapid NO3(-)-dependent Fe(II) oxidation. Significant oxidation of biogenic Fe3O4 was observed. Very little biogenic FeCO3 was oxidized. No reduction of NO3- or oxidation of Fe(II) occurred in pasteurized cultures. The molar ratio of NO3- reduced to Fe(II) oxidized in cultures containing chemically precipitated FeCO3, and one of the microbially reduced subsoils approximated the theoretical stoichiometry of 0.2:1. However, molar ratios obtained for oxidation of microbially reduced goethite, the other subsoil, and the HFO reduction end products did not agree with this theoretical value. These discrepancies may be related to heterotrophic NO3- reduction coupled to oxidation of dead Fe(III)-reducing bacterial biomass. Our findings demonstrate that microbally catalyzed NO3(-)-dependent Fe(II) oxidation has the potential to significantly accelerate the oxidation of solid-phase Fe(II) compounds by oxidized N species. This process could have an important influence on the migration of contaminant metals and radionuclides in subsurface environments.

Bacterial reductive dissolution of crystalline Fe(III) oxide in continuous-flow column reactors.

Bacterial reductive dissolution of synthetic crystalline Fe(III) oxide-coated sand was studied in continuous-flow column reactors in comparison with parallel batch cultures. The cumulative amount of aqueous Fe(II) exported from the columns over a 6-month incubation period corresponded to (95.0 +/- 3.7)% (n = 3) of their original Fe(III) content. Wet-chemical analysis revealed that only (6.5 +/- 3.2)% of the initial Fe(III) content remained in the columns at the end of the experiment. The near-quantitative removal of Fe was visibly evidenced by extensive bleaching of color from the sand in the columns. In contrast to the column reactors, Fe(II) production quickly reached an asymptote in batch cultures, and only (13.0 +/- 2.2)% (n = 3) of the Fe(III) oxide content was reduced. Sustained bacterial-cell growth occurred in the column reactors, leading to the production and export of a quantity of cells 100-fold greater than that added during inoculation. Indirect estimates of cell growth, based on the quantity of Fe(III) reduced, suggest that only an approximate doubling of initial cell abundance was likely to have occurred in the batch cultures. Our results indicate that removal of biogenic Fe(II) via aqueous-phase transport in the column reactors decreased the passivating influence of surface-bound Fe(II) on oxide reduction activity, thereby allowing a dramatic increase in the extent of Fe(III) oxide reduction and associated bacterial growth. These findings have important implications for understanding the fate of organic and inorganic contaminants whose geochemical behavior is linked to Fe(III) oxide reduction.

A simple, efficient method for the separation of humic substances and DNA from environmental samples.

Three different gels (Sepharose 4B, Sephadex G-200, and Sephadex G-50) were evaluated as a means of removing humic contaminants from DNA extracts of environmental samples. Sepharose 4B gave superior separation of DNA from humics, and DNA purified in this way showed consistently greater amplification than DNA purified by the other materials.

Serum albumin binds beta- and alpha-monoolein in vitro.

We investigated the interaction of bovine serum albumin (BSA) and monoolein (MO) and estimated the number of BSA binding sites for the alpha- and beta-isomers of MO. The turbidity of increasing concentrations of aqueous dispersions of alpha-MO and beta-MO in the presence and absence of BSA was measured in triplicate by absorption spectrophotometry. Aqueous dispersions of [13C(1)]MO and [13C(1)]MO/BSA mixtures at molar ratios of 1:1, 3:1 and 5:1 were analyzed in duplicate by [13C]nuclear magnetic resonance (NMR) at pH 7.4 and 36 degrees C. BSA bound significantly more beta-MO than alpha-MO at 15 min: 5.4 +/- 0.42 and 3.3 +/- 0.60 mol MO/mol BSA, respectively (P: < 0.05). [13C]NMR spectra of the 1:1 molar ratio of [13C(1)]MO /BSA exhibited a single carbonyl peak at 175.19 ppm, whereas spectra of 3:1 and 5:1 molar ratios exhibited three peaks between 172 and 174 (ppm), each distinct from carbonyl resonances of either [13C(1)]MO dispersed in water, 176.72 (ppm) or BSA alone. The intensities of individual peaks, but not their chemical shift values, varied between 3:1 and 5:1 molar ratios, indicating that BSA has at least three MO binding sites and may bind up to five molecules of MO per molecule. This study confirms that serum albumin binds MO in vitro and supports the theory that albumin transports monoglycerides produced by lipoprotein lipase hydrolysis of triglyceride.