G.l.c. of O-methyloxime and alditol acetate derivatives of neutral sugars, hexosamines, and sialic acids: "one-pot" quantitative determination of the carbohydrate constituents of glycoproteins and a study of the selectivity of alkaline borohydride reductions

A new procedure for the quantification by g.l.c. of the carbohydrate constituents of glycoproteins is proposed which involves (a) simultaneous action of neuraminidase and neuraminic acid aldolase, (b) hydrolysis with 4M trifluoroacetic acid at 125 degrees for 1 h, and (c) conversion of the products into O-methyloxime acetates and g.l.c. The procedure has been successfully tested on fetuin, transferrin, alpha 1-acid glycoprotein, and mucin. The g.l.c. conditions used also enabled the complete separation of O-methyloxime and alditol acetate derivatives in one run, so that the release of carbohydrate chains from glycoproteins by treatment with alkaline borohydride can be investigated conveniently. There was complete release of O-linked oligosaccharides from fetuin on treatment with 0.1M NaOH/0.8M NaBH4 (68 h, 37 degrees) or 0.05M KOH/M KBH4 (24 h, 45 degrees) and also release of approximately 75% and 35-40%, respectively, of N-asparagine-linked chains. Reduced oligosaccharides were formed only from O-linked chains; the mechanism by which N-linked chains were released is still not clear.     

The effect of mild alkali and alkaline borohydride on the carbohydrate and peptide moieties of fetuin

In the light of recent reports, based on radioactive labelling studies, that substantial amounts of N-linked oligosaccharides are released from protein under the mild-alkaline borohydride degradation conditions that are usually used to release O-linked oligosaccharides, we have investigated by chemical methods the effects of alkali alone and alkaline borohydride on the carbohydrate and peptide moieties of fetuin. The chromatographic profiles on Sephadex G50 columns, of the hexose- and ninhydrin-positive components of the native and Pronase-treated glycoprotein have been compared with those obtained after treatment with mild alkali alone (0.05 M-NaOH, 50 degrees C, 16 h) or mild-alkaline borohydride (0.05 M-NaOH containing 1 M-NaBH4, 50 degrees C, 16 h). Composition and methylation analyses have been performed on carbohydrate-containing peaks and the following conclusions were drawn: mild alkali treatment alone liberated a minor hexose- and ninhydrin-positive component and mild-alkaline borohydride treatment gave a major hexose-containing peak: both of these co-chromatographed on a Sephadex G50 column with Pronase glycopeptides. The polypeptide backbone was totally broken down by the alkaline borohydride treatment. The presence of released N-linked chains after alkaline borohydride treatment was confirmed. However, from the carbohydrate composition it was calculated that no more than 10-20% of the N-linked chains were released from protein. The results of methylation analysis have raised the possibility that this release is in part due to cleavage of the chitobiosyl core.     

Studying O-linked protein glycosylations in human plasma

Recent investigations have implicated aberrant glycosylations in various malignancies, including epithelial ovarian cancer (EOC). The protocol here identifies O-linked carbohydrate patterns in EOC plasma glycoproteins through chemical cleavage and purification of these glycans. Dialyzed plasma is subjected to reductive beta-elimination with alkaline borohydride to release O-linked oligosaccharides from glycoproteins. Enrichment of released glycans, as well as removal of peptide and other contaminants, is followed by carbohydrate pattern analysis with MALDI-FT-ICR-MS.     

Sequential analysis of N- and O-linked glycosylation of 2D-PAGE separated glycoproteins

A robust method has been developed that allows analysis of both N- and O-linked oligosaccharides released from glycoproteins separated using 2D-PAGE and then electroblotted to PVDF membrane. This analysis provides efficient oligosaccharide profiling applicable to glycoproteomic analysis. The method involves the enzymatic release of N-linked oligosaccharides using PNGase F followed by the chemical release of O-linked oligosaccharides using reductive beta-elimination and analysis using LC-ESI-MS. Oligosaccharides from the major plasma glycoproteins with a pI between 4 and 7 were characterized from the glycoforms of haptoglobin, alpha2-HS-glycoprotein, serotransferrin, alpha1-antitrypsin, and alpha1-antichymotrypsin. It was shown that the separation of protein glycoforms evident in 2D-PAGE is partially due to the combined sialylation of the O-linked and N-linked oligosaccharides. Bi-, tri- and tetra-antennary N-linked structures, which had differing levels of sialylation and fucosylation, were found to be present on the glycoproteins analyzed, together with O-linked oligosaccharides such as mono-, and disialylated T-antigen and a disialylated core type 2 hexasaccharide. In addition, N-linked site-specific information was obtained by MALDI-MS analysis using tryptic digestion after PNGase F release of the oligosaccharides.     

Recovery of intact 2-aminobenzamide-labeled O-glycans released from glycoproteins by hydrazinolysis

The initial step in quantitative analysis of O-linked glycans of glycoproteins is to release them in high yield, nonselectively, unmodified, and with a free reducing terminus. In contrast to other techniques, hydrazinolysis can meet these criteria. However, when analyzing pools of O-linked glycans as described in the accompanying article by L. Royle et al. (2002, Anal. Biochem. 304), some peeling of the glycans was observed. Critical steps in the sample preparation and glycan recovery were therefore evaluated by analyzing and identifying both intact O-glycans and degraded products. Synthetic O-glycopeptides were characterized by mass spectrometry. Released glycans were identical to those on the glycopeptide. O-Linked glycans from a range of glycoproteins of increasing complexity, namely, bovine serum fetuin, glycophorin A, and previously uncharacterized glycopeptides isolated from human salivary mucin Muc5B, were also analyzed. Quantitative analysis of the glycan profile confirmed that there was <2% peeling of O-glycans released by hydrazinolysis conditions of 60 degrees C for 6 h, and recovered using the optimised procedure now described. This demonstrated that O-glycans can be prepared by hydrazinolysis without degradation and, as part of an analytical strategy, makes the analysis of O-glycans attached to low-microgram levels of naturally occurring glycoproteins feasible.     

Precolumn labeling of reducing carbohydrates with 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone: analysis of neutral and sialic acid-containing oligosaccharides found in glycoproteins.

A convenient precolumn labeling method was developed for the analysis of neutral and sialic acid-containing oligosaccharides in glycoproteins using 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone (PMPMP). PMPMP reacts with a reducing oligosaccharide under slightly alkaline conditions (pH 8.3) to form a 2:1 adduct (bis-PMPMP derivative). Sialic acid residues in the oligosaccharides remain intact during the reaction. Tryptic glycopeptides digested with glycopeptidase A for oligosaccharide liberation can be directly derivatized with PMPMP without prior treatment. Separation of the labeled oligosaccharides was performed by reverse-phase high-performance liquid chromatography on a C-18 column with aqueous acetonitrile, and positional isomers such as isomeric triantennary tetradecasaccharides from bovine fetuin were completely resolved. The bis-PMPMP derivatives were labile in alkaline media to form mono-PMPMP derivatives; however, the mono-PMPMP derivatives could be easily reconverted to the original bis-PMPMP derivatives. The proposed method is simpler than the reductive pyridylamination method, and detection sensitivity could reach subnanomole range with a uv detector. Oligosaccharides from ribonuclease B (bovine pancreas), ovalbumin, thyroglobulin (porcine thyroid), fetuin (bovine), and transferrin (human) have been successfully analyzed to demonstrate the usefulness of this method as an alternative to the existing methods.     

Evidence for posttranslational O-glycosylation of fetuin

Fetuin, a major glycoprotein in the serum of fetal calves that contains three N-linked and three O-linked carbohydrate side chains, was found to be synthesized in the liver with an 18 amino acid signal peptide, Met-X-X-X-X-Leu-Leu-X-Cys-Leu-Ala-X-Leu-X-X-Cys-X-X, and to undergo cotranslational N-glycosylation. In order to examine O-glycosylation, fetuin peptidyl-tRNA was purified from liver and analyzed for O-linked carbohydrate by quantitating the released [3H]GalNAcitol produced after beta-elimination in the presence of NaB3H4. Within the limits of the assay, less than 1.3% of the O-linked chains had been initiated. Additionally, rough microsomes were used to program a cell-free protein synthesis system. A radiolabeled fetuin intermediate was isolated by immunoprecipitation and shown to contain N-linked carbohydrate by binding to concanavalin A and by susceptibility to cleavage by endoglycosidase H. However, this fetuin intermediate was not detectably bound (less than 1%) by GalNAc-specific lectins, which were shown to bind asialoagalactofetuin. These results suggest that O-glycosylation of fetuin is a posttranslational event.     

Lectin affinity chromatography of proteins bearing O-linked oligosaccharides: application of jacalin-agarose.

The lectin jacalin immobilized on agarose was found to bind a variety of glycoproteins known to contain typical O-linked oligosaccharides, including human IgA, C1 inhibitor, chorionic gonadotropin, plasminogen, bovine protein Z, bovine coagulation factor X, and fetuin. These proteins were eluted from columns of jacalin-agarose specifically by alpha-galactopyranosides such as melibiose and alpha-methylgalactopyranoside but not by lactose or other sugars. Treatment of asialofetuin with endo--alpha--N--acetylgalactosaminidase eliminated its affinity for the lectin column, and other proteins known to contain only N-linked oligosaccharides such as ovalbumin, transferrin, and alpha 1-acid glycoprotein were not retained by the lectin. Binding of proteins with O-linked oligosaccharides to the lectin column did not require divalent cations and was affected little by changes in pH and ionic strength over a wide range. Virtually all of the glycosidically linked oligosaccharides of fetuin, chorionic gonadotropin, and plasminogen are known to be sialated. Thus, binding of these glycoproteins to jacalin, which is known to have affinity for the core disaccharide, 1-beta-galactopyranosyl-3-(alpha-2-acetamido-2-deoxygalactopyranoside ), in O-linked oligosaccharides of these proteins, was not prevented by the presence of sialic acids. Affinity of oligosaccharides for jacalin did appear to be reduced by occurrence of sialic acids as it was found that higher concentrations of melibiose were required to elute asialofetuin than fetuin from jacalin-agarose. Results of the present study indicate that affinity chromatography using this lectin is a widely applicable technique for identifying and purifying proteins bearing O-linked oligosaccharides.     

Application of aqueous hydrazine solution for beta-elimination of O-glycans from gastric mucin

O-Glycans from pig gastric mucin were released by beta-elimination in 0.2 M triethylamine and 50% aqueous hydrazine solution. The released glycan hydrazides were isolated using Centricon 10 separators, brought to their reducing form and reductive by labelled with p-aminobenzoic acid ethyl ester (ABEE). Labelled products were fractionated into neutral and acid fractions on a Bio-Gel P4 column, calibrated with a mixture of dextran oligosaccharides, labelled according to the same procedure.     

IgA1 is the premier serum glycoprotein recognized by human galectin-1 since T antigen (Galbeta1-->3GalNAc-) is far superior to non-repeating N-acetyl lactosamine as ligand

Human heart galectin-1 (HHL) was separated by high pressure liquid chromatography from endogenous glycoproteins co-purified with it during affinity chromatography. These glycoproteins offered excellent ligands for HHL binding and were rich in T antigen (Galβ1 → 3 GalNAc-) of O-linked oligosaccharides. In enzyme linked lectin assay and hemagglutination inhibition assay, human IgA1, bovine fetuin and other O-glycosylated T antigen-bearing glycoproteins bound to the lectin efficiently in contrast to single N-acetyl lactosamine (LacNAc)-bearing N-linked oligosaccharides released from them and to IgG which is not O-glycosylated. HHL binding to IgA1 and fetuin was unaffected by removal of their N-linked oligosaccharides by -mannosidase. When immobilized, O-glycosylated serum proteins but not IgG could capture HHL from its solutions. Desialylated or polymeric IgA1 was better inhibitor than monomeric IgA1. The findings suggest a possible role for galectin-1 in anchoring of microbial and cancer cells known to be rich in T antigen, in high serum IgA1 turn over and in tissue sequestering of IgA1 immune complexes especially after their microbial desialylation in IgA nephropathy and other immune complex-mediated disorders.     

One-pot nonreductive O-glycan release and labeling with 1-phenyl-3-methyl-5-pyrazolone followed by ESI-MS analysis

A novel one-pot procedure for the nonreductive release of O-linked glycans from glycoproteins and the simultaneous derivatization of released glycans with 1-phenyl-3-methyl-5-pyrazolone (PMP) is described. Unlike the traditional reductive β-elimination, which produces alditols, this new method employs PMP/ammonia aqueous solution as the reaction medium. The O-glycans are released from glycoproteins and derivatized with PMP nonreductively, specifically, and quantitatively. Samples can be easily purified from ammonia, excess PMP, and peptide residues by evaporation, chloroform extraction, and solid-phase extraction (SPE) column fractionation for HPLC, CE, or MS analysis. The procedure has been elaborated with two purified glycoproteins, porcine stomach mucin and bovine fetuin, and successfully applied to O-glycan profiling of a challenging biological specimen, healthy human plasma. This new procedure has shown methodological significance in O-glycan analysis.     

Development of an apparatus for rapid release of oligosaccharides at the glycosaminoglycan-protein linkage region in chondroitin sulfate-type proteoglycans

An apparatus, AutoGlycoCutter (AGC), was developed as a tool for rapid release of O-linked-type glycans under alkaline conditions. This system allowed rapid release of oligosaccharides at the glycosaminoglycan-protein linkage region in proteoglycans (PGs). After digestion of PGs with chondroitinase ABC, the oligosaccharides at the linkage region were successfully released from the protein core by AGC within 3 min. The reducing ends of the released oligosaccharides were labeled with 2-aminobenzoic acid and analyzed by a combination of capillary electrophoresis (CE) and matrix-assisted laser desorption time-of-flight mass spectrometry. In addition, the unsaturated disaccharides produced by chondroitinase ABC derived from the outer parts of the glycans were labeled with 2-aminoacridone and analyzed by CE to determine the disaccharide compositions. We evaluated AGC as a method for structural analysis of glycosaminoglycans in some chondroitin-sulfate-type PGs (urinary trypsin inhibitor, bovine nasal cartilage PG, bovine aggrecan, bovine decorin, and bovine biglycan). Recoveries of the released oligosaccharides were 57-73% for all PGs tested in the present study. In particular, we emphasize that the use of AGC achieved ca. 1000-fold rapid release of O-glycans compared with the conventional method.     

The degradation of glycoproteins with lithium borohydride. Isolation and analysis of O-glycopeptides with reduced C-terminal amino acid residue

By the example of fetuin and a blood-group-specific mucin from porcine stomach, we showed that, under conditions of reductive degradation of glycoproteins with LiBH4-LiOH in 70% aqueous tert-butyl alcohol, the reduction and cleavage of amide bonds occur much faster than the simultaneous beta-elimination of carbohydrate chains O-linked with Ser and Thr residues of the peptide chain. The major degradation products containing the O-linked glycans are the O-glycosylated derivatives of 2-aminopropane-1,3-diol and 2-aminobutane-1,3-diol (the products of reduction of glycosylated Ser and Thr) and the glycopeptides containing 2-4 amino acid residues with reduced C-terminal amino acid. Seventeen homogeneous O-glycopeptides were isolated from the fetuin degradation products by ion-exchange and reversed-phase HPLC. Their structures were determined by MALDI-TOF mass spectrometry and by analyses for amino acids, amino alcohols, and carbohydrates. The application of the reaction for characterization of O-glycans and localization of O-glycosylation sites in O- and N,O-glycoproteins is discussed.     

Characterization of Glycoprotein Oligosaccharides Using Surface Plasmon Resonance

Surface plasmon resonance was used to characterize the carbohydrate moieties of bovine fetuin. This technique, requiring no sample derivitization or labeling, identified the presence and composition of both N- and O-linked oligosaccharides, using a combination of lectin probes and in situ glycosidase digestion. A complete analysis was achieved using 1.4 μg of pure bovine fetuin, and was fully automated using Pharmacia′s BIAcore. The presence or absence of specific oligosaccharide structures was determined by the binding of a panel of unlabeled lectins. Monosaccharide order and linkages were determined by sequential digestion using a range of specific exoglycosidases. This novel method implementing in situ digestion was achieved using a modification to the BIAcore software, allowing the flow of reagent over the sensor chip to be stopped for variable lengths of time, thereby permitting enzymatic digestion to occur. This technique can be applied to other commercial SPR biosensors currently available. Glycoanalysis by SPR uses approximately 100- to 1000-fold less protein than comparable analyses using alternative techniques such as gel permeation chromatography of released oligosaccharides, labeled lectin binding, or mass spectrometry.     

Glycoproteomics of milk: differences in sugar epitopes on human and bovine milk fat globule membranes

Oligosaccharides from human and bovine milk fat globule membranes were analyzed by LC-MS and LC-MS/MS. Global release of N-linked and O-linked oligosaccharides showed both to be highly sialylated, with bovine peak-lactating milk O-linked oligosaccharides presenting as mono- and disialylated core 1 oligosaccharides (Galbeta1-3GalNAcol), while human milk had core type 2 oligosaccharides (Galbeta1-3(GlcNAcbeta1-6)GalNAcol) with sialylation on the C-3 branch. The C-6 branch of these structures was extended with branched and unbranched N-acetyllactosamine units terminating in blood group H and Lewis type epitopes. These epitopes were also presented on the reducing terminus of the human, but not the bovine, N-linked oligosaccharides. The O-linked structures were found to be attached to the high molecular mass mucins isolated by agarose-polyacrylamide composite gel electrophoresis, where MUC1 and MUC4 were present. Analysis of bovine colostrum showed that O-linked core 2 oligosaccharides are present at the early stage (3 days after birth) but are down-regulated as lactation develops. This data indicates that human milk may provide different innate immune protection against pathogens compared to bovine milk, as evidenced by the presence of Lewis b epitope, a target for the Helicobacter pylori bacteria, on human, but not bovine, milk fat globule membrane mucins. In addition, non-mucin-type O-linked fucosylated oligosaccharides were found (NeuAc-Gal-GlcNAc1-3Fuc-ol in bovine milk and Gal-GlcNAc1-3Fuc-ol in human milk). The O-linked fucose structure in human milk is the first to our knowledge to be found on high molecular mass mucin-type molecules.     

Improved nonreductive O-glycan release by hydrazinolysis with ethylenediaminetetraacetic acid addition

The study of protein O-glycosylation is receiving increasing attention in biological, medical, and biopharmaceutical research. Improved techniques are required to allow reproducible and quantitative analysis of O-glycans. An established approach for O-glycan analysis relies on their chemical release in high yield by hydrazinolysis, followed by fluorescent labeling at the reducing terminus and high-performance liquid chromatography (HPLC) profiling. However, an unwanted degradation known as “peeling” often compromises hydrazinolysis for O-glycan analysis. Here we addressed this problem using low-molarity solutions of ethylenediaminetetraacetic acid (EDTA) in hydrazine for O-glycan release. O-linked glycans from a range of different glycoproteins were analyzed, including bovine fetuin, bovine submaxillary gland mucin, and serum immunoglobulin A (IgA). The data for the O-glycans released by hydrazine with anhydrous EDTA or disodium salt dihydrate EDTA show high yields of the native O-glycans compared with the peeled product, resulting in a markedly increased robustness of the O-glycan profiling method. The presented method for O-glycan release demonstrates significant reduction in peeling and reduces the number of sample handling steps prior to release.     

A general approach to desalting oligosaccharides released from glycoproteins

Desalting of sugar samples is essential for the success of many techniques of carbohydrate analysis such as mass spectrometry, capillary electrophoresis, anion exchange chromatography, enzyme degradation and chemical derivatization. All desalting methods which are currently used have limitations: for example, mixed-bed ion-exchange columns risk the loss of charged sugars, precipitation of salt by a non-aqueous solvent can result in co-precipitation of oligosaccharides, and gel chromatography uses highly crosslinked packings in which separation of small oligosaccharides is difficult to achieve. We demonstrate that graphitized carbon as a solid phase extraction cartridge can be used for the purification of oligosaccharides (or their derivatives) from solutions containing one or more of the following contaminants: salts (including salts of hydroxide, acetate, phosphate), monosaccharides, detergents (sodium dodecyl sulfate and Triton X-100), protein (including enzymes) and reagents for the release of oligosaccharides from glycoconjugates (such as hydrazine and sodium borohydride). There is complete recovery of the oligosaccharides from the adsorbent which can also be used to fractionate acidic and neutral glycans. Specific applications such as clean-up of N-linked oligosaccharides after removal by PNGase F and hydrazine, desalting of O-linked glycans after removal by alkali, on-line desalting of HPAEC-separated oligosaccharides and -eliminated alditols prior to electrospray mass spectrometry, and purification of oligosaccharides from urine are described.     

A micromethod for the estimation of oligosaccharides containing glycosidically linked sialic acid or hexoses, or both, in glycoproteins

The peeling reaction, the process by which oligosaccharides are degraded in alkali, was used as the basis for an assay to provide structural information about glycosidically linked oligosaccharides in glycoproteins. Glycoproteins were treated with 0.05 M NaOH at 50 degrees to induce release, and subsequent degradation ("peeling"), of glycosidically linked, but not of N-glycosydically linked, oligosaccharides. Among the degradation products generated from O-linked chains were three 3-deoxy sugar acids whose formation was correlated with certain structural features of the oligosaccharides. N-Acetylneuraminic acid was released from terminal positions in the oligosaccharides, and iso- and meta-saccharinic acids were derived from the degradation of 4-O- and 3-O-substituted hexoses, respectively. All of these sugar acids were detected colorimetrically by periodate oxidation and reaction of the product with 2-thiobarbituric acid. The ability of the method to generate 3-deoxy sugar acids was tested in 8 alkali-treated glycoproteins. 3-Deoxy sugar acids were detected only in those glycoproteins whose glycosidically linked carbohydrates contained N-acetylneuraminic acid, or 3-O- or 4-O-substituted hexoses, or both. As little as 0.12 microgram of 3-deoxy sugar acid produced from 5 micrograms of human chorionic gonadotropin was sufficient for detection. This method is novel in its ability to distinguish sialylation of glycosidically linked carbohydrates. Furthermore, it combines the specificity of beta-elimination with the sensitivity of the 2-thiobarbituric acid assay in targeting degradation products of the peeling reaction as candidates for an assay method.     

Oligosaccharide chains of herpes simplex virus type 2 glycoprotein gG.2

gG.2 glycoprotein was purified by H966 monoclonal antibodies linked to Sepharose from herpes simplex virus type 2-infected HEp-2 cells labeled with [3H] glucosamine. The glycoprotein was subjected to Pronase digestion and the glycopeptides were fractionated by Con A-Sepharose in a major fraction (88.5% of total radioactivity) unbound to the lectin gel and in a minor species which bound to the lectin as a N-linked diantennary oligosaccharide. Mild and strong acid hydrolysis of Con A-unbound and Con A-bound fractions revealed that (i) both species were highly sialylated; (ii) the Con A-unbound fraction contained mainly labeled N-acetylgalactosamine, as is the case for O-linked oligosaccharides; and (iii) the Con A-bound fraction carried the vast majority of the labeled N-acetylglucosamine present in gG.2. Three size classes of oligosaccharides were separated from mild alkaline borohydride-treated Con A-unbound glycopeptides, which accounted for about 80% of the radioactivity present in the fraction. Galactosaminitol was recovered as the major labeled product in the strong acid hydrolyzates of the oligosaccharides generated by reductive beta-elimination, indicating that they were O-glycosidically linked to the peptide backbone. Thin-layer and DEAE-Sephacel chromatography of the three O-linked oligosaccharide species indicated that disialylated tetrasaccharides and monosialylated trisaccharides were the major components, whereas neutral disaccharide was a minor component. Digestion with neuraminidase and beta-galactosidase of the O-linked oligosaccharides supported the idea that the common disaccharide core was mainly of the structure beta-galactosyl-N-acetylgalactosamine. The large occurrence of O-linked oligosaccharides differentiates this type 2-specific herpes simplex virus glycoprotein from the type-common herpesvirus glycoproteins gB, gC, and gD.     

Fluorometric assay of O-linked glycoproteins by reaction with 2-cyanoacetamide

A simple assay for O-glycosylated glycoproteins involving the liberation of oligosaccharides by beta-elimination with dilute alkali and the subsequent derivatization of the reducing end with 2-cyanoacetamide is reported. The method can be used to quantitate microgram amounts of mucin within 30 min. The assay is 30 times less sensitive to protein or N-linked glycoproteins and 100 times less sensitive to DNA than to the corresponding weight of canine tracheal mucin. Of the substances tested, only cesium chloride and potassium thiocyanate caused substantial interference, but in neither case was this sufficiently serious to prevent the method being used for monitoring mucin purification schemes utilizing these reagents. The coefficients of variation for replicate analyses of canine tracheal mucin (14.0, 5.0, and 2.0 micrograms) were 3.6, 6.5, and 12.3%, respectively.