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The purpose of this study was to determine the potential toxicity of docosahexaenoic acid (DHA)-rich microalgae (DRM) from Schizochytrium sp., administered in the diet of growing swine. DRM was administered in the diet to groups of castrated male growing pigs (mixed commercial breeds, Landrace & Large White) reared from early weaned (weighing approximately 20 lbs) to approximately 250–270 lbs. Over the course of the 120 day study, animals were fed ad libitum four DRM treatment diets, each designed to optimize weight gain over the growing cycle, and a control diet. DRM was incorporated into the diet of the first treatment group at a level delivering 2.680 kg DRM per pig over the course of 120 days (a constant, whole-life exposure) equating to 598 g DHA per pig. DRM was incorporated into finisher diets only (administered over the last 42 days of the growing cycle) to treatment groups 2, 3, and 4 delivering 1.169, 3.391, and 5.746 kg DRM per pig (261, 756, and 1281 g DHA per pig). These levels represent approximately 1, 3, and 5 times the anticipated commercial dose and were delivered in a feeding strategy designed to mimic commercial use. Vitamin E was added to all diet groups to provide supplementary dietary antioxidant given the high content of polyunsaturated fat in DRM. There were no statistically significant treatment-related effects in clinical observations, body weights, food consumption, mortality, hematologic values, gross necropsy findings, organ weights or histopathology. The only DRM treatment-related changes were higher weight gain and feed conversion efficiency, anticipated results based on the increased fat content in the experimental DRM treatments. This study demonstrates that administration of DRM (at up to five times the anticipated commercial dose) did not produce any treatment-related adverse effects in commercial strains of swine.

Reductive cleavage of fully methylated, partially O -carboxymethylated cellulose had previously been shown to produce 4- O -acetyl-1,5-anhydro-2,3,6-tri- O -methyl-, -2- O -(methoxycarbonylmethyl)-3,6-di- O -methyl-, -3- O -(methoxycarbonylmethyl)-2,6-di- O -methyl-, -6- O -(methoxycarbonylmethyl)-2,3-di- O -methyl-, -2,3-di- O -(methoxycarbonylmethyl)-6- O -methyl-, -2,6-di- O -(methoxycarbonylmethyl)-3- O -methyl-, -3,6-di- O -(methoxycarbonylmethyl)-2- O -methyl-, and -2,3,6-tri- O -(methoxycarbonylmethyl)- d -glucitol. Described herein is the independent synthesis of these derivatives, except for the first, which had been reported. In addition, their 1 H-n.m.r. spectra, chemical-ionization (NH 3 ) mass spectra, and electronionization mass spectra are tabulated.

A two-step procedure is described in this paper to identify the position of O-acetyl groups in S-657 polysaccharide. Reductive-cleavage experiments performed on the fully methylated (base-catalyzed) polysaccharide, followed by acetylation of anhydroalditols, identified individual sugar residues and their position of linkage. In a second experiment, the polysaccharide was methylated under neutral conditions leaving native acetate groups intact. Reductive cleavage of the neutral methylated polysaccharide using CF3SO3SiMe3 as a catalyst, followed by acetylation in situ, identified sugar residues containing native acetate groups and established their position of substitution. Using this two-step procedure of analysis, S-657 polysaccharide is shown to contain O-acetyl groups on the 2-position and the 2,6-positions of 3-linked glucopyranosyl residues.

Xanthan gum is the extracellular polysaccharide produced by the bacterium Xanthomonas campestris. Its structure has been shown’,’ to consist of a (1 -+ 4) linked /3-D-glucan backbone with P-D-Manp-(I + 4)-P-D-GlcpA-(1 -+ 2)(U-DManp-(1 + side chains substituted at O-3 of alternating glucose residues. In addition, the side chains may contain 0-acetyl and pyruvic acid acetal groups, which are located at the 6-position of the internal mannose residue and at the 4,6-position of the terminal mannose group, respectively. The amount of noncarbohydrate substituents vary depending on the strain of bacteria, the fermentation conditions and the recovery procedure3-5. Samples of xanthan gum with high acetyl content have been observed6. The proportion of acetyl groups was shown to exceed the theoretical value of one residue per pentasaccharide repeat unit. Although the location of additional acetyl groups is uncertain, it has been speculated that multiple acetylation of the internal mannosyl residue is possible in some xanthan gum samples, as is acetylation of the terminal mannosyl group’. In X. campestris, three genes have been identified8,9 that encode enzymes which catalyze modifications of mannose residues. It was later shown” that one of these genes encodes an acetylase activity, termed acetylase II, directing acetylation of the terminal mannose group; however, the position of substitution was not determined. We now report the location of a second 0-acetyl group in xanthan gum using the reductive-cleavage technique”. A sample of xanthan gum was hydrolyzed using trifluoracetic acid and analyzed for the presence of organic acids by high-performance liquid chromatography (HPLC). The results of this analysis indicated the presence of acetic acid (6.8%) and pyruvic acid (2.7%). Assuming a sodium form of xanthan gum containing an average of 1.0 acetyl groups and 0.5 pyruvic acid acetal substituents per repeat unit, the theoretical content for acetic acid and pyruvic acid is 6.3 and 4.4%

Permethylated alginic acids comprised of 4-linked D-mannopyranosyluronic acid and 4-linked L-gulopyranosyluronic acid residues undergo reductive cleavage to yield, after acetylation, methyl 3-O-acetyl-2,6-anhydro-4,5-di-O-methyl-D-mannonate (2b) and methyl 3-O-acetyl-2,6-anhydro-4,5-di-O-methyl-D-gluconate (3b) as major products. Small amounts (ca. 13%) of ring-contracted products, namely methyl 2-O-acetyl-3,6-anhydro-4,5-di-O-methyl-D-mannonate (9) and methyl 2-O-acetyl-3,6-anhydro-4,5-di-O-methyl-D-gluconate (10), were also observed in these experiments. These results are in marked contrast to previous results on the reductive cleavage of 4-linked D-glucopyranosyluronic acid residues, wherein the ring-contracted product was formed exclusively. Formation of the ring-contracted products could be completely eliminated by reduction (LiAlH4) of ester groups in the permethylated alginic acid prior to reductive cleavage. In the latter experiments, 4,6-di-O-acetyl-1,5-anhydro-2,3-di-O-methyl-D-mannitol (5b) and 4,6-di-O-acetyl-1,5-anhydro-2,3-di-O-methyl-L-gulitol (6b) were the sole products of reductive cleavage of the 4-linked ManA and 4-linked GulA residues, respectively. However, in the previous experiments it was noted that low yields of permethylated alginic acids were obtained and that extensive depolymerization occurred under methylation conditions. Depolymerization could be avoided and higher yields of permethylated polysaccharides could be obtained, by reduction of the carboxyl groups of the alginic acids prior to methylation. Reductive cleavage of the latter polysaccharides yielded the products expected from 4-linked D-mannopyranosyl and 4-linked L-gulopyranosyl residues, namely 4-O-acetyl-1,5-anhydro-2,3,6-tri-O-methyl-D-mannitol (13b) and 4-O-acetyl-1,5-anhydro-2,3,6-tri-O-methyl-L-gulitol (14b), respectively. Using the latter analytical strategy, it was established that the Macrocystis pyrifera alginate was comprised of 60% 4-linked ManA and 40% 4-linked GulA residues, whereas the Pseudomonas aeruginosa alginate was comprised of 80% 4-linked ManA and 20% 4-linked GulA residues.

The applicability of the reductive-cleavage method to the analysis of polysaccharides bearing pyruvic acid acetals has been demonstrated. Direct reductive cleavage of fully methylated gum xanthan yielded the expected products, including 1,5-anhydro-4,6- O -[( S )-1-methoxycarbonylethylidene]-2,3-di- O -methyl- d -mannitol. The latter product was not observed when reductive cleavage was performed subsequent to reduction of ester groups in the fully methylated polysaccharide and mild hydrolysis to remove pyruvic acid acetal substituents. Instead, the latter experiment yielded 1,5-anhydro-2,3-di- O -methyl- d -mannitol, establishing the presence in the polysaccharide of terminal (nonreducing) d -mannopyranosyl groups bearing 4,6- O -(1-carboxyethylidene) substituents. The products of reductive cleavage were characterized, where appropriate, by comparison of the gas chromatographic retention times and chemical ionization- and electron ionization-mass spectra of their acetates to those of authentic standards. Alternatively, the products of reductive cleavage could be characterized without resort to comparison with authentic standards by analysis of the 1 H-n.m.r. spectra of their benzoates, which were obtained in pure form by high-performance liquid chromatography. By either method of product characterization, this two-step procedure of analysis reveals the presence of pyruvic-acetal residues in polysaccharides and establishes both the identity of the sugar residue to which they are attached and their positions of attachment.

A method is described for the analysis of positions of substitution of O-carboxylated groups in commercial samples of O-carboxymethylcellulose. Sequential permethylation of the polymer and reductive cleavage gives eight products, which are analyzed as their O-acetyl derivatives by gas-liquid chromatography.

The 4,6- O -(1-methoxycarbonylethylidene), -(hydroxyisopropylidene), and -(methoxyisopropylidene) acetals of methyl 2,3-di- O -methyl-α- d -glucopyranoside were subjected to reductive cleavage in the presence of triethylsilane and trimethylsilyl methanesulfonate-boron trifluoride etherate (Me 3 SiOMs-BF 3 ·Et 2 O), BF 3 ·Et 2 O, or trimethylsilyl trifluoromethanesulfonate (Me 3 SiOSO 2 CF 3 ) and the mole fractions of products were determined as a function of reaction time. The 4,6-(1-methoxycarbonylethylidene) acetal was quite stable to reductive-cleavage conditions but isomerization of the initial R,S mixture of diastereomers to the more-stable S diastereoisomer was noted. In addition, a slow, regiospecific, reductive ring-opening of the acetal was observed to give 6- O -[1-(methoxycarbonyl)ethyl] derivatives. The 4,6-(hydroxyisopropylidene) acetal was very unstable under reductive-cleavage conditions. Both Me 3 SiOMs-BF 3 ·Et 2 O and Me 3 SiOSO 2 CF 3 catalyzed complete removal of the group, via the intermediate 6-[1-(hydroxymethyl)ethyl] ether, but BF 3 ·Et 2 O gave a mixture of products. The 4,6-(methoxyisopropylidene) acetal was also very labile under reductive-cleavage conditions; Me 3 SiOMs-BF 3 ·Et 2 O catalyzed complete removal of the acetal, via the intermediate 6-[1-(methoxymethyl)ethyl ]ether, but the intermediate ether was quite stable in the presence of either BF 3 ·Et 2 O or Me 3 SiOSO 2 CF 3 . It is concluded from these studies that polysaccharides bearing 4,6- O -(1-carboxyethylidene) substituents can be analyzed directly by sequential permethylation and reductive cleavage. It is proposed that the identity of the substituted monomer and the positions of substitution of the acetal can be determined by sequential permethylation, ester reduction, and reductive cleavage.

A method is described for the analysis of positions of substitution of O-methyl and O-ethyl groups in commercial samples of O-methylcellulose and O-ethylcellulose, respectively. The method requires perethylation of O-methyl-cellulose and permethylation of O-ethylcellulose. Subsequent reductive cleavage of both polymers gives the same eight products, which are analyzed as their O-acetyl derivatives by gas-liquid chromatography.

Reductive cleavage of fully methylated, partially O -ethylated cellulose or fully ethylated, partially O -methylated cellulose and subsequent acetylation had previously been shown to produce 4- O -acetyl-1,5-anhydro-2,3,6-tri- O -methyl-, -6- O -ethyl-2,3-di- O -methyl-, -3- O -ethyl-2,6-di- O -methyl-, -2- O -ethyl-3,6-di- O -methyl-, -2,3-di- O -ethyl-6- O -methyl-, -2,6-di- O -ethyl-3- O -methyl-, -3,6-di- O -ethyl-2- O -methyl-, and-2,3,6-tri- O -ethyl- d -glucitol. Described herein is the independent synthesis of these derivatives, except for the first (which had been reported); and their 1 H-n.m.r. spectra, chemical-ionization (NH 3 ) mass spectra, and electron-impact mass spectra are tabulated.