A rapid method for the preparation of ganglioside Glac2 (GD3)

A method is described for the preparation of ganglioside G_lac2 [(II³(NeuAc)₂-LacCer, GD3] from cream of bovine milk using liquid-phase extraction with methanol or ethanol followed by anion exchange chromatography. The method is rapid and inexpensive; 1 kg cream, centrifuged from 14–15 L of bovine milk, yields approximately 70 mg of pure ganglioside G_lac2. The sialic acid constituent of ganglioside G_lac2 isolated from bovine milk cream consists solely of theN-acetylneuraminic acid derivative. The major components of its ceramide consist of octadecasphing-4-enine and the 22∶0 (behenic acid) and 23∶0 fatty acids. Short hand notations for gangliosides are according to Wiegandt (Ref. 1). G_lac2, (II³(NeuAc)₂-LacCer) is NeuAcα,8NeuAcα,-3Galβ,4Glcβ,1Cer; G_tet1, (II³NeuAc-Gg₄Cer) is Gal\,3GalNAcβ,-4(NeuAcα,3)Galβ,4Glcβ,1Cer.     

Lactational changes in the fatty acid composition of human milk gangliosides

The objectives of this work were to study the FA composition of milk gangliosides, as well as to gain further insight into the characterization of human milk gangliosides. The potential capacity of human milk gangliosides to adhere to human enterotoxigenic Escherichia coli (ETEC-strains) was also studied. Human milk gangliosides were isolated and identified by high-performance TLC or immunoassay. The latter also was used to assay bacterial adhesion. The FA composition of gangliosides was studied by GC. The presence of O-acetyl GD3 (Neu5,9Ac₂α2–8 NeuAcα2–3Galβ1–4GlcCer) and trace amounts of GM1 [Galβ1–3GalNAcβ1,−3(NeuAcα2–3)Galβ1–4GlcCer] in human milk was confirmed. Medium-chain FA were almost absent in colostrum, whereas in the subsequent stages they rose to 20%. The levels of long-chain FA decreased after colostrum. With respect to the degree of saturation, gangliosides from colostrum were richer in monounsaturated FA than gangliosides synthesized during the rest of the lactation period, opposite to the pattern for PUFA. A human-ETEC colonization factor antigen II-expressing strain showed binding capacity to human milk GM3 (NeuAcα2–3Galβ1–4GlcCer). New data on human milk gangliosides have been gathered. A thorough knowledge of their composition is needed since they may have important biological implications in regard to newborns' defense against infection. The ganglioside nomenclature of Svennerholm (34) is followed.     

Sialylation of lacto-N-neotetraosyl ceramide by a solubilized sialyltransferase(s) from chicken skeletal muscle: Effect of phosphatidylcholine and sphingomyelin

The sialytransferase(s) that transfers sialic acid to lacto-N-neotetraosylceramide and other glycosphingolipids with a galactose nonreducing terminus has been successfully solubilized from embryonic chicken skeletal muscle. The enzyme can be stored in 50 mM HEPES (pH 6.8), 1% Triton CF-54, and 20% glycerol at −70°C for as long as six months. Addition of phosphatidylcholine or sphingomyelin (0.167%) readily reactivates the stored inactive enzymes and such activity persists for about two weeks at 0°–4°C with the peak activity occurring at 1 to 2 days. Sphingomyelin from chicken muscle, which contains mainly C16∶0 and C18∶0, is 2.1-fold more effective than bovine brain sphingomyelin at the same concentration (0.4%). Abbreviations: GM₃, GM₁, GD_1a, GD_1b, GT_1b were designated according to Svennerholm, L. (1963)J. Neurochem. 10, 613–623. GgOse₄Cer and nLcOse₄Cer are short designations recommended by the IUPAC-IUB Commission on Lipid Nomenclature (1977)Eur. J. Biochem. 79, 11–21. Other abbreviations used are IV³ NeuAcnLcOse₄Cer; nLcOse₆Cer, V⁴Gal, IV³ GlcNAc-nLcOse₄Cer; NeuAc-nLcOse₆Cer, VI³ NeuAc, V⁴ Gal, IV³ GlcNAc-nLcOse₄Cer; PC, phosphatidylcholine; SM, sphingomyelin, SAT, sialyltransferase; Hepes, 4-2(hydroxyethyl)1-piperazineethane sulfanic acid. Structures of glycolipids: nLcOse₄Cer, Gal(β1→4)GlcNAc(β1→3)Gal(β1→4)-GlcCer; GgOse₄Cer, Gal(β1→3)GalNAc(β1→4)Galβ(1→4)GlcCer.     

Characterization of gangliosides of porcine erythrocyte membranes: Occurrence of ganglioside GD3 as major ganglioside

Four major ganglioside species were isolated from porcine erythrocyte membranes by DEAE-Sephadex and Iatrobeads column chromatography. Treatment of the lipids with graded neuraminidase and β-galactosidase, gas chromatographic analysis of their carboyhydrates, sphingosine bases and molecular species of sialic acid revealed that the structure of these gangliosides were G_M3(NeuAc), G_M3(NeuGc), G_D3(NeuAc) and G_D3(NeuGc), each of which was 16±2 μg, 304±42 μg, 30±3 μg and 240±26 μg, respectively, per gram of the dry erythrocyte stroma. The amount of G_M3 and G_D3 accounted for more than 95% of total gangliosides of the erythrocytes. Porcine erythrocytes may provide a good source for large scale preparation of ganglioside G_D3 which recently was identified as a human melanoma-associated antigen. Gangliosides are named according to Svennerholm (1) and the recommendation of the IUPAC-IUB Commission on Biochemical Nomenclature (2).     

Behavior of gangliosides on dialysis

Mixed brain gangliosides or individual ganglioside GM₁ were dissolved in one of the following solvents: (a) water, (b) 0.1 M aqueous KC1, or (c) methanol-aqueous 0.1 M KC1 at concentrations ranging from 10⁻⁸ to 10⁻³ M, and were submitted to dialysis against distilled water for up to 4 days. No significant loss of gangliosides on dialysis was observed when gangliosides were dissolved in water or 0.1 M aqueous KC1, but a loss occurred when the ganglioside solution contained methanol; the loss was greater at the lowest ganglioside concentration. However, losses ceased to occur after 1 day when methanol was removed from the dialysis sac.     

Photooxidation of N,N-bis(3,5-di-tert.-butylsalicylidene)-1,2-diamino hexane-manganese(III) chloride (Jacobsen catalyst) in chloroform

Mn^III(J-salen)Cl (Jacobsen catalyst) with J-salen=N,N^′-bis(3,5-di-tert.-butylsalicylidene)-1,2-diaminocyclohexane dianion in CHCl₃ is photooxidized by the solvent to a Mn^IV(J-salen) complex, presumably Mn^IV(J-salen)Cl₂, with φ=0.002 at λ_irr=333 nm.     

New members in the [Mn10] supertetrahedron family

Two manganese complexes, [Mn^II₄Mn^III₆Cl₄(CH₃OCH₂CH₂O)₁₂ O₄][Mn^II₃Ti^IVCl₆(CH₃OCH₂CH₂O)₆] (1) and [Mn^II₄Mn^III₆Cl₄(CH₃OCH₂CH₂O)₁₂O₄] [Mn₄^II Cl₁₀(CH₃OCH₂CH₂OH)₄]∙0.5CH₃OCH₂CH₂OH, (2) have been obtained and characterized by single-crystal X-ray diffraction. Both structures consist of the decametallic dicationic [Mn^II₄Mn^III₆Cl₄(CH₃OCH₂CH₂O)₁₂O₄]^2 + core constructed by four vertex-sharing [Mn^III₃Mn^IIO]^9 + tetrahedra. Also, these compounds contain the different tetrametallic dianions: [Mn^II₃Ti^IVCl₆(CH₃OCH₂CH₂O)₆]^2 − (in complex 1) and [Mn₄^IICl₁₀(CH₃OCH₂CH₂OH)₄]^2 − (in complex 2). Magnetic dc and ac susceptibility measurements for compound (1) show that the dicationic decanuclear magnetic cluster possesses an S = 12 ± 1 spin ground-state.     

Preparation of defined molecular species of lactosylceramide by chemical deacylation and reacylation withN-succinimidyl fatty acid esters

A procedure for the preparation of specific molecular species ofd-erythro-lactosylceramide involving deacylation and reacylation of lactosylceramide prepared from bovine brain gangliosides is described. Lactosylceramide wasN-deacylated by alkaline hydrolysis and the resulting four lysolactosylceramides, which contained d18∶1, d20∶1, d18∶0 and d20∶0 long-chain bases, were simultaneously re-N-acylated with theN-succinimidyl ester of either 16∶0, 18∶0, 20∶0, 24∶0, 20∶1, 22∶1 or 24∶1 fatty acid. The resulting lactosylceramide contained four molecular species of lactosylceramides, i.e., d18∶1, d20∶1, d18∶0 and d20∶0 long-chain bases coupled with the fatty acid that was introduced. Lactosylceramides prepared in this manner were separated into four individual molecular species by high-performance liquid chromatography (HPLC). Each of the purified molecular species of lactosylceramide was quantitated by HPLC after derivatization with benzoylchloride and was characterized by mass spectrometry. The yields of reacylated lactosylceramide were 38–58% relative to the starting lactosylceramide; the purity of each of the molecular species of lactosylceramide was greater than 95%. The glycosphingolipid nomenclature is as recommended by the IUPAC-IUB Commission on Biochemical Nomenclature (1). GalCer, galactosylceramide, Gal(β1-1)Cer; GlcCer, glucosylceramide, Glc(β1-1)Cer; LacCer, lactosylceramide, Gal(β1-4)GlcCer; GbOse₃Cer, globotriaosylceramide, Gal(α1-4)Gal(β1-4)GlcCer; GbOse₄Cer, globotetraosylceramide, GalNAc(β1-3)Gal(α1-4)-Gal(β1-4)GlcCer; GgOse₃Cer, gangliotriaosylceramide, GalNAc-(β1-4)Gal(β1-4)GlcCer; GgOse₄Cer, gangliotetraosylceramide, Gal(β1-3)GalNAc(β1-4)Gal(β1-4)GlcCer; GM3, (NeuAcα2-3)-Galβ1-4GlcCer; GM1, Galβ1-3GalNAcβ1-4(NeuAcα2-3)Galβ1-4-GlcCer. The molecular species abbreviations suggested by Breimeret al. (2) are used. For example, in the notation d18∶1−18∶0, the d18∶1 represents the long-chain base sphingosine (d-erythro-1,3-dihydroxy-2-amino-trans-4-octadecene) and 18∶0 represents the fatty acid (octadecanoic acid).     

Generation and Electron-Transfer Reactivity of the Long-Lived Photoexcited State of a Manganese(IV)-Oxo-Scandium Nitrate Complex

Photoexcitation of a manganese(IV)-oxo-scandium nitrate complex ([(Bn-TPEN)Mn^IV(O)]²⁺−Sc(NO₃)₃) in a solvent mixture of trifluoroethanol and acetonitrile (v/v=1 : 1) resulted in generation of the long-lived photoexcited state, which is detected by nanosecond laser transient absorption measurements. The transient absorption maximum (λ_max) of the ²E excited state of [(Bn-TPEN)Mn^IV(O)]²⁺−Sc(NO₃)₃ is observed at 620 nm with lifetimes of 7.1 μs. The λ_max value is blue-shifted and the lifetime becomes longer as compared with the previously reported values of λ_max (640 nm) and lifetime (6.4 μs) of the ²E excited state of the 1 : 2 complex between [(Bn-TPEN)Mn^IV(O)]²⁺ and Sc(OTf)₃ ([(Bn-TPEN)Mn^IV(O)]²⁺−(Sc(OTf)₃)₂). The electron-transfer reactivity of the ²E excited states of [(Bn-TPEN)Mn^IV(O)]²⁺−Sc(NO₃)₃ was similar to that of [(Bn-TPEN)Mn^IV(O)]²⁺−(Sc(OTf)₃)₂. The long lifetime and the high reactivity of the ²E excited state of [(Bn-TPEN)Mn^IV(O)]²⁺−Sc(NO₃)₃ provide an excellent photooxidant for oxidation of compounds, which would otherwise be impossible to be oxidized.     

Two Novel Bimetallic Cyano-Bridged Coordination Polymers Containing the 2,2′-(Ethylenedioxy)bis (Ethylamine): Syntheses,Structural, Thermal and Magnetic Properties

Two new cyano bridged bimetallic polymeric complexes, [Ni(edbea)Ni(CN)₄]·1/2H₂O (1) and [Cu(μ-edbea)(μ-CN)₂ Ni(CN)₂]·H₂O (2) [edbea = 2,2′-(ethylenedioxy)bis(ethylamine)] have been synthesized by adding metal chloride (M = Ni^II and Cu^II), and edbea into [Ni(CN)₄]²⁻ in water–ethanol solution and then characterized by elemental analysis, infrared (IR) and electron paramagnetic resonance (EPR) (for only complex 2) spectra, variable temperature magnetic measurement, and thermal gravimetric analysis. The X-ray diffraction crystal structure of complex 2 shows a 2D polymeric chain –edbea(N5,O1)–Cu1–N1C1–Ni(CN)2–C4N6–Cu1–(N6,O2) edbea– in which the Cu^II centers are linked by two cyano and one edbea. The powder EPR spectrum of the complex 2 has shown that Cu^II ions are located in rhombically distorted octahedral sites. The magnetic properties of the coordination polymers have been studied in temperature range of 15–300 K. The magnetic behaviors investigation of complexes 1 and 2 indicated the presence of a very weak antiferromagnetic interaction.     

Photochemical reductive elimination of [PtIV(NH3)4(NH2)Cl]2 +: Photogeneration of hydrazine

When [Pt^IV(NH₃)₅Cl]^3 + is deprotonated the complex [Pt^IV(NH₃)₄(NH₂)Cl]^2 + is formed. Upon NH₂⁻ → Pt^IV LMCT excitation (λ_irr > 250 nm) a reductive elimination takes place: [Pt^IV(NH₃)₄(NH₂)Cl]^2 + → [Pt^II(NH₃)₃Cl]⁺ + N₂H₄ + H⁺. Since Pt(II) ammine complexes can be reoxidized to Pt(IV) by H₂O₂ it is suggested that in a cyclic process the overall reaction could proceed according to the equation: 2 NH₃ + H₂O₂ → N₂H₄ + 2 H₂O.     

Characterization of Glycosphingolipids in the Human Parathyroid and Thyroid Glands

As part of a systematic investigation of the glycosphingolipids in human tissues, acid and non-acid glycosphingolipids from human thyroid and parathyroid glands were isolated and characterized with mass spectrometry and binding of carbohydrate-recognizing ligands, with a focus on complex compounds. The glycosphingolipid patterns of the human parathyroid and thyroid glands were very similar. The major acid glycosphingolipids were sulfatide and the gangliosides GM3, GD3, GD1a, GD1b, GT1b and Neu5Ac-neolactotetraosylceramide, and the major non-acid glycosphingolipids were globotriaosylceramide and globoside. We also found neolactotetra- and neolactohexaosylceramide, the x₂ glycosphingolipid, and complex glycosphingolipids with terminal blood group O and A determinants in both tissues. A glycosphingolipid with blood group Le^b determinant was identified in the thyroid gland, and the parathyroid sample had a glycosphingolipid with terminal blood group B determinant. Immunohistochemistry demonstrated the expression of blood group A antigens in both the thyroid and parathyroid glands. A weak cytoplasmatic expression of the GD1a ganglioside was present in the thyroid, while the parathyroid gland had a strong GD1a expression on the cell surface. Thus, the glycosylation of human thyroid and parathyroid glands is more complex than previously appreciated. Our findings provide a platform for further studies of alterations of cell surface glycosphingolipids in thyroid and parathyroid cancers.     

Solvent-induced assembly of two supramolecular isomers of Mn thiophenedicarboxylate coordination polymers

Two supramolecular isomers of Mn^II thiophenedicarboxylate coordination polymers, namely, [Mn₂(tdc)₂(dmf)₂]_n (1) and [Mn₃(tdc)₃(dmf)₃]_n (2) (H₂tdc = 2,5-thiophenedicarboxylic acid, dmf = N,N-dimethylformamide), were synthesized with solvent-induced assembly method and the antiferromagnetic interactions between the Mn^II ions in their solid phase were measured.     

Synthesis and Characterization of Eu3+‐Doped Transparent Glass–ceramics Containing Nanocrystalline SrIINbIVO3

The glass–ceramics containing a rarely achievable nanocrystalline Sr^IINb^IVO₃ phase in the 53.75SiO₂–18.25K₂O–9Bi₂O₃–9SrO–9Nb₂O₅–0.5CeO₂–0.5Eu₂O₃ (mol%) glass system were prepared by the melt‐quench technique followed by a two‐stage controlled heat treatment. The unusual oxidation state of Nb in Sr^IINb^IVO₃ crystal is 4+ and upon heat treatment of the samples at lower temperature of 500°C for several hours, the glass composition and chemical environment around Nb ions played a key role for the formation of Sr^IINb^IVO₃ in the glass–ceramics. The microstructure of the glass–ceramics was studied using TEM and FESEM. The TEM images advocate 10–40 nm crystallite size of Sr^IINb^IVO₃. FTIR study confirms that all the samples consist of SiO₄, BiO₃, BiO₆, and NbO₆ structural units. The refractive index at different wavelengths was found to vary in the range 1.7105–1.7905 and increase with increase in heat‐treatment time. The luminescence spectra of Eu³⁺‐doped glass and glass–ceramics were recorded at 465 nm excitation wavelength and the luminescence intensity is found to be increased with heat‐treatment time due to increase in crystallinity. The high intensity ratio of ⁵D₀→⁷F₂ to ⁵D₀→⁷F₁ indicates that the Eu³⁺‐doped nanocrystalline Sr^IINb^IVO₃ glass–ceramics are promising candidate materials as red‐light source.     

Alkene oxidation catalyzed by transition metal substituted Keggin-type heteropolyanions

Alkene oxidations with various oxidants (tert-butyl hydroperoxide, iodosylbenzene and molecular oxygen in the presence of isobutyraldehyde (IBA)) catalyzed by transition metal monosubstituted heteropolyanions, PW₁₁MO ₃₉ ^n– (PW₁₁M; M=Co^II, Mn^II, Cu^II, Ti^IV, Ru^IV, V^V and Nb^V), have been studied. Orders of catalytic activity of PW₁₁M are different for the oxidants studied. Radical chain mechanisms are proposed fort-BuOOH and O₂/IBA. Preliminary coordination of the oxidant to PW₁₁M is not a necessary step of its homolytic activation. Epoxidation with PhIO requires its coordination to the catalyst and most likely includes the formation of active metal-oxo species.     

GM3 Ganglioside Linked to Neurofibrillary Pathology in a Transgenic Rat Model for Tauopathy

Glycosphingolipids (GSLs) are amphipathic lipids composed of a sphingoid base and a fatty acyl attached to a saccharide moiety. GSLs play an important role in signal transduction, directing proteins within the membrane, cell recognition, and modulation of cell adhesion. Gangliosides and sulfatides belong to a group of acidic GSLs, and numerous studies report their involvement in neurodevelopment, aging, and neurodegeneration. In this study, we used an approach based on hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution tandem mass spectrometry (HRMS/MS) to characterize the glycosphingolipid profile in rat brain tissue. Then, we screened characterized lipids aiming to identify changes in glycosphingolipid profiles in the normal aging process and tau pathology. Thorough screening of acidic glycosphingolipids in rat brain tissue revealed 117 ganglioside and 36 sulfatide species. Moreover, we found two ganglioside subclasses that were not previously characterized—GT1b-Ac2 and GQ1b-Ac2. The semi-targeted screening revealed significant changes in the levels of sulfatides and GM1a gangliosides during the aging process. In the transgenic SHR24 rat model for tauopathies, we found elevated levels of GM3 gangliosides which may indicate a higher rate of apoptotic processes.     

Voltammetric study of electrocatalysis for dioxygen reduction by trinuclear rutheniumammine complex confined in a polymer membrane

Electrocatalytic O₂ reduction was studied using a modified electrode coated with a Nafion membrane (Nf) dispersing a trinuclear ruthenium ammine complex ([(NH₃)₅Ru^IIIORu^IV(NH₃)₄ORu^III(NH₃)₅]Cl₆, Ru-red). When measuring cyclic voltammogram under O₂ atmosphere (at 0.5 mV s⁻¹), catalytic currents due to O₂ reduction were found to develop below −0.2 V (vs. Ag/AgCl). Since Ru-red undergoes irreversible decomposition into the mononuclear complexes via the reduced state (Ru^III-Ru^III-Ru^III) (∼−0.1 V), it is suggested that the electrocatalysis originates from the decomposed species (initial active species: Ru^II(NH₃)₅(OH₂) and Ru^II(NH₃)₄(OH₂)₂) rather than from the Ru-red. Although the present electrocatalyst was also applied to H₂O₂ reduction system, the catalytic activity was found to be poor from the voltammetric behavior. It appeared that the kinetics of the electrocatalysis is much faster in the O₂ reduction than in the H₂O₂ one. A selective and direct catalysis for O₂ reduction into H₂O was suggested from a ring-disk voltammogram to take place by an aggregate of the mononuclear ruthenium complexes in the polymer matrix. In addition, it was found that electrocatalytic O₂ reduction involves a slow kinetic process, so that factors affecting the overall kinetics were discussed in terms of the catalysis mechanism.     

The major gangliosides of the bovine pineal body

Acetone powders of fresh-frozen pineals were extracted with chloroform/methanol mixtures. By column chromatography on silicic acid, mild alkaline methanolysis, ion-exchange high performance liquid chromatography and a final thin layer chromatography on silicic acid, the major glycosphingolipids were purified from the extracts of a total of 300 bovine pineal bodies. Chromatographically purified fractions were characterized by gas chromatographic analysis. The most prominent glycosphingolipid appeared to be cerebroside. In addition, five different gangliosides were found in detectable levels. The two major gangliosides have the chromatographic and component characteristics of GD₃ and GM₃, with disialoganglioside predominating. Gangliosides indistinguishable from purchased standards of GM₁ and GD_1a were third and fourth, respectively, in amount. The fatty acid profiles of the two lactosyl gangliosides are similar and significantly different from those of the two gangliotetraose gangliosides. The fifth most prominent ganglioside, present at a level of 1.09% of total recovered ganglioside sialic acid, appears to be a novel trisialoganglioside, called GT_x. This new molecule has a component ratio of gal:glc:sialic acid:amino sugar of approximately 1∶2∶3∶1. Similarities between bovine pineal and rod outer segments are discussed.     

Enterotoxigenic <Emphasis Type="Italic">Escherichia coli</Emphasis> strains bind bovine milk gangliosides in a ceramide-dependent process

Diarrhea caused by enterotoxigenic Escherichia coli (ETEC) is the main infectious disease of newborn calves. The first step of infection involves bacterial attachment to the intestinal mucosa. This adhesion is mediated by fimbriae that recognize some glycoconjugates on the host cell surface, in particular, several gangliosides. Because milk also contains gangliosides, these have been suggested to serve as ligands for bacterial fimbriae and thus prevent the bacterial attachment to mucosa. The most relevant ETEC strains in calves, including those with K99 and F41 fimbriae, were assayed to determine whether they are able to bind gangliosides isolated from several stages of bovine lactation. Both GM3 and GD3, the main gangliosides of milk, were recognized by ETEC strains, although the different fimbriae showed diverse levels of affinity. Unexpectedly, the adhesion to colostral gangliosides was considerably weaker than that to gangliosides from the other stages of lactation. Because the carbohydrate moiety did not change and because differences in the percentages of unsaturated FA and sphingosine between colostrum and other stages were observed, we conclude that the differences in adhesion could be due to a different composition of the ganglioside caramide.     

Liquid Chromatography–High-Resolution Mass Spectrometry for Quantitative Analysis of Gangliosides

Gangliosides are a large family of glycosphingolipids that are abundant in the brain, and have been shown to affect neuronal plasticity during development, adulthood, and aging. We developed a fast, efficient, and sensitive liquid chromatography–mass spectrometry method to quantify eight different classes of gangliosides (GM₁, GM₂, GM₃, GD₃, GD_1a, GD_1b, GT_1b, GQ_1b) in the brains of 2-day-old and 80-day-old Wistar rats. The gangliosides were extracted from rat brain using a modified Svennerholm and Fredman method. After ganglioside class separation using a hydrophilic high performance liquid chromatography (HPLC) column, the resolving power of the LTQ-Orbitrap™ mass spectrometer was used to extract and sum the major species of each ganglioside class, generating fully resolved extracted ion current peaks for both standards and samples. The flexibility and the specificity of this method are such that it can be applied to the analysis of other ganglioside species/classes not discussed in this paper, provided appropriate standards are available. The method had good repeatability (coefficient of variation 4.8–12.3%) and mean recoveries in the range 92–107%.