Hugh J. Cornell

A simple and rapid colorimetric method for the determination of amylose in starch products

A method for the determination of the amylose content in starch has been developed which is based on the colorimetric measurement of the iodine complexes formed with amylose and amylopectin. The method requires measurement at only one wavelength and avoids the use of harsh dispersants for the starch. Dimethyl sulphoxide is used as the dispersant and a wavelength of 600 nm can be used for measurement of the amylose content of starches from different botanical sources. A linear relationship was obtained between absorbance and amylose concentration for mixtures of amylose and amylopectin standards, and this forms the basis of the determination. The method is rapid, simple, accurate and does not require the use of multi-component analysis of spectra, since a wavelength is chosen that suits the particular starch being analysed. It can be adapted to a micro-scale method if necessary.

A Model for the Surface of Keratin Fibers

A model of the epicuticle membrane of keratin fibers shows that it is a heavily crosslinked protein containing approximately 25% by weight of fatty acid, predomi nantly 18-methyleicosanoic acid, acylated to the protein as a thioester. The conclusion that acylated fatty acids reside on the surface of and completely surround individual cuticle cells is supported by an analysis of the amount of fatty acid removed by alcoholic alkali against treatment time and the observed decreasing amount of bound fatty acid found as fiber diameters increase. Allworden sacs form only under acidic conditions, which also cleave bound fatty acids. Prior removal of bound fatty acids facilitates the rapid formation of Allworden sacs.

Diversity in oat potential immunogenicity: basis for the selection of oat varieties with no toxicity in coeliac disease

Background and aims Coeliac disease (CD) is triggered by an abnormal reaction to gluten. Peptides resulting from partially digested gluten of wheat, barley or rye cause inflammation of the small intestinal mucosa. Previous contradictory studies suggest that oats may trigger the abnormal immunological response in patients with CD. Monoclonal antibodies (moAbs) against the main immunotoxic 33-mer peptide (A1 and G12) react strongly against wheat, barley and rye but have less reactivity against oats. The stated aim of this study is to test whether this observed reactivity could be related to the potential toxicity of oats for patients with CD. Methods In the present study, different oat varieties, controlled for their purity and by their distinct protein pattern, were used to examine differences in moAb G12 recognition by ELISA and western blot. Immunogenicity of oat varieties was determined by 33-mer concentration, T cell proliferation and interferon γ production. Results Three groups of oat cultivars reacting differently against moAb G12 could be distinguished: a group with considerable affinity, a group showing slight reactivity and a third with no detectable reactivity. The immunogenicity of the three types of oats as well as that of a positive and negative control was determined with isolated peripheral blood mononuclear T cells from patients with CD by measurement of cell proliferation and interferon γ release. A direct correlation of the reactivity with G12 and the immunogenicity of the different prolamins was observed. Conclusions The results showed that the reactivity of the moAb G12 is proportional to the potential immunotoxicity of the cereal cultivar. These differences may explain the different clinical responses observed in patients suffering from CD and open up a means to identify immunologically safe oat cultivars, which could be used to enrich a gluten-free diet.

Avenins from different cultivars of oats elicit response by coeliac peripheral lymphocytes

Objective. The avoidance of oats in coeliac patients is still controversial. If oats is confirmed to be safe, it would be a valuable component and offer more variation in a gluten-free diet. The aim of this work was to evaluate whether avenins from different varieties of oats show different abilities in the activation of coeliac peripheral lymphocytes. Material and methods. In order to assess whether the immunogenic effect of oats varies according to the cultivar, peripheral lymphocytes from 10 coeliac children were exposed to avenins from four different oats varieties: Lampton, Astra, Ava and Nave. Lymphocyte proliferation and interferon-gamma (IFN-γ) release in the culture medium were measured as indexes of immune activation. Results. All the varieties of oats tested were immunogenic, with Lampton and Ava avenins inducing lymphocyte activation similar to that activated by wheat gliadin, while Astra and Nave avenins showed less immunogenicity, but still with a measurable effect. Conclusions. There are still concerns about the suitability of including oats in a gluten-free diet. Coeliac patients consuming oats-containing food should be carefully monitored, until there is more evidence to show the safety of oats and varieties of low-toxicity oats.

Enzyme therapy for management of coeliac disease

Objective. Enzyme therapy based on animal digestive extracts was investigated as a means of completely digesting toxic residues from gluten in the small intestine, thus providing a means of protection of the mucosa. Material and methods. A randomized, placebo-controlled, clinical trial of an encapsulated enzyme extract was conducted in 21 coeliac patients in remission who were challenged with a modest amount of gluten daily over 2 weeks. Enzyme extract (900 mg) in three divided doses was administered during this challenge to half the group and a placebo to the other half in a double-blind, crossover design. Symptoms were recorded in daily diaries; blood was taken for tissue transglutaminase antibodies (anti-tTG) at the start and at intervals up to 12 weeks. Duodenal biopsies were performed for histological assessment at the start and end of each challenge period for 6 patients chosen at random from volunteers. After a further 10 weeks, the groups were changed over, and the same assessments carried out. Results. Only 8 of the 21 patients (38%) had more than 5 episodes of moderate to severe symptoms during either of the gluten challenge periods, and in these, symptoms scores were ameliorated during enzyme therapy compared with the placebo period (p<0.02). Rises of 5 U/ml or more in anti-tTG occurred in only 5 patients at about 6–8 weeks after challenge, but were not correlated with symptoms. Conclusions. Only 1 of the 6 patients had normal histology at entry, thus focusing attention on the need for better management of the disease. By histological criteria, enzyme therapy offered better protection than placebo during the gluten challenges. The study supports the use of enzyme supplementation as a safeguard for patients with coeliac disease because of the difficulty of ensuring a strictly gluten-free diet.

Effects of Processing on the Bound and Free Fatty Acid Levels in Wool

Bound and free fatty acids in degreased wool fibers were affected to varying degrees by processing treatments. Scouring and dyeing both removed significant amounts of bound and free fatty acids from wool. Free fatty acids were reduced by dissolution into the treatment liquor, whereas bound fatty acids were hydrolyzed under the hot aqueous conditions. Chlorination at pH levels below 3 released over 50% of the bound fatty acids. Chlorine treatments cleave only thioesters but not oxygen esters or amides under these conditions, indicating that a significant proportion of the bound fatty acid is linked to wool by a thio ester bond.

The activity of wheat gliadin peptides in in vitro assays for coeliac disease

A fraction of a peptic-tryptic-pancreatinic digest of wheat gliadin (fraction 9), known to be toxic to individuals with coeliac disease, together with synthetic peptides containing key gliadin sequences, were tested for their effects on foetal chick intestine and on rat liver lysosomes. Fraction 9 and a dodecapeptide corresponding to residues 75-86 of A-gliadin (RPQQPYPQPQPQ) were the only peptides to display appreciable activity in both assays. A synthetic hexapeptide corresponding to residues 77-82 was only weakly toxic to lysosomes and non-toxic to chick duodenum. A decapeptide corresponding to residues 76-85 was non-toxic in both assays. Two serine-containing peptides containing the key sequence PSQQ were also tested but were found to be non-toxic, as was the hexapeptide PSQQQP. The results suggest that the key sequences QQQP and PSQQ are not sufficient by themselves to cause activity. Further tests on synthetic peptides will be necessary to define the sequences of highest toxicity.

Characterization of the gliadin-derived peptides which are biologically active in coeliac disease

Reversed-phase HPLC on C18 silica gel at pH 3.5 was used to separate peptides in fraction 9, a mixture of peptides of unknown composition obtained from an enzymic digest of wheat gliadin. This fraction, which has been shown to be toxic to individuals with coeliac disease, yielded a principal peak as well as many minor peaks after HPLC. The significant peaks were subjected to amino acid analysis. The principal peak obtained was purified by rechromatography at pH 6.0 and shown to contain a dodecapeptide of sequence H-Arg-Pro-Gln-Gln-Pro-Tyr-Pro-Gln-Pro-Gln-Pro-Gln-OH. This peptide may have been derived from regions in the A-gliadin molecule corresponding to amino acids numbered 75-86 or from homologous regions in other gliadin molecules. Preliminary results indicate that it is active in two in vitro models of coeliac disease and that it could be the source of one of the undigested peptides (Hexapeptide II, (Glx)3, (Pro)2, Tyr) obtained from coeliac mucosal digestion of fraction 9. Some active serine-containing peptides were also obtained from chromatography at pH 3.5 and attempts are being made to correlate these with the other undigested peptide (Hexapeptide I) of composition (Glx)3, (Pro)2, Ser, obtained after coeliac mucosal digestion of fraction 9.

In vitro tests indicate that certain varieties of oats may be harmful to patients with coeliac disease

Background:  The presence of oats in gluten‐free diet is controversial. The aim of this work is to evaluate if different varieties of oats exert different toxicity in coeliac disease.

In vitro mucosal digestion of synthetic gliadin-derived peptides in celiac disease

Two celiac-active synthetic peptides derived from the A-gliadin structure corresponding to residues 8–19 (LQPQNPSQQQPQ) and to 11–19 were digestedin vitro with small intestinal mucosa from children with celiac disease in remission and from normal children. The products of digestion were separated into two fractions on the basis of Mr<400 and Mr>400 by gel permeation chromatography and subjected to amino acid analysis. After digestion of the dodecapeptide with celiac mucosa, 71±14% (molar) of the total digestion products remained in the Mr>400 fraction. Glutamine, proline, serine, and asparagine were the major amino acids present. Glutamine, proline, and leucine were the major amino acids in the Mr<400 fraction. The Mr>400 fraction from the celiac mucosal digestion of the nonapeptide was of similar composition to the corresponding fraction from the dodecapeptide and represented 78±15% of the total products. Digestion of the two peptides with normal mucosa gave lower amounts of products in the Mr>400 fraction, but they were of similar composition to the corresponding fractions from the celiac mucosal digestion. Peptides such as NPSQQQP and QNPSQQQ may be present in the Mr>400 fractions since glutamine and proline are present in the approximate ratio of 2∶1, respectively. The results indicate a defect in the mucosal digestion of peptides which are active in an animal model of celiac disease.