The determination of prolamins in gluten-free food. Introductory remarks.

This paper gives the considerations on the Codex Alimentarius Standard on gluten free food and the levels that are proposed as a new standard. Background for the level of tolerance for gliadin in coeliac patients is given as well as the arguments for the choice of an enzyme immunochemical method for the determination of gliadin in gluten free food.

Elevated level of IgA gliadin antibodies in patients with rheumatoid arthritis.

The possibility that dietary antigens contribute to the pathogenesis of rheumatoid arthritis (RA) has been proposed. Moreover, occasional patients have been described in whom coeliac disease and RA coincide. Furthermore, most RA patients are treated with non-steroidal anti-inflammatory drugs (NSAIDs), which are known to increase gut permeability. For these reasons antibodies against gliadin were measured in a group of 43 patients with rheumatoid arthritis (RA) and a group of 43 age- and sex-matched controls. The median IgA antigliadin ELISA index was 7.1 (range 2.1-22.4) for the RA group and 3.1 (range 0.3-34.9) for the controls (p = 0.0001). The median IgG and IgM antigliadin indexes for the RA group didn't differ significantly from those of the controls. In the RA group, the level of antigliadin antibodies did not correlate with the daily dose of NSAIDs. The elevated IgA antigliadin titre in the RA group might be ascribed to the use of NSAIDs, which are harmful to the gut, but the immunological trigger effect of gluten cannot be ruled out.

Immunocrossreactivity of antisera against grain prolamines of wheat, rye, barley and oats.

Prolamines extracted from wheat (gliadin), rye (secalin), barley (hordein) and oats (avenin) were used to raise antibodies in rabbits. The four prolamines were separated by SDS-PAGE and blotted onto nitrocellulose. The immunocrossreactivity of the separated prolamines with the four antisera was studied. The close botanical relationship between wheat and rye, and to a lesser extent barley, is clearly established. The crossreactivity of gliadin, secalin and hordein with anti-avenin serum was found to be weak. In contrast, avenin shows a strong crossreactivity with anti-gliadin serum.

Isolation and analysis of peptidic fragments of alpha-gliadin using reversed-phase high-performance liquid chromatography.

Peptidic fragments of alpha-gliadin were obtained by peptic-tryptic-pancreatic (PTP) digestion of the alpha-gliadin fraction isolated by ion-exchange chromatography on a sulphopropyl-Sephadex C-50 column. The proteolytic digest was fractionated by ultrafiltration into three subfractions, PTPa1-PTPa3. The subfraction PTPa2 was then analysed and individual peaks were separated using reversed-phase high-performance liquid chromatography (RP-HPLC) using a gradient of acetonitrile in 0.1% trifluoroacetic acid and a Separon SGX-C18 sorbent. A 100-mg amount of the PTPa2 subfraction was separated in a single analysis by preparative RP-HPLC and twenty peaks were obtained for further characterization. The molecular mass in range 300-3000 was established for individual peptidic fragments by gel-permeation chromatography on a TSK-G2000 SW column.

Monoclonal antibodies reacting with gliadin as tools for assessing antigenic structure responsible for exacerbation of celiac disease.

Monoclonal antibodies reactive with gliadin were prepared by fusion of spleen cells isolated from gliadin-immunized BALB/c mice with myeloma cells. The reactivity of mAbs with different preparations of gliadin and their enzymatic digest were measured using ELISA method. The mAb produced by GL 1 clone was shown to react preferentially with alpha-gliadin and its enzymatic digest.

Influence of gliadin on fetal chick intestine in tissue culture.

The purpose of this study was to develop a model system for detecting biological effects of gliadin which may be related to coeliac disease. The technique applied was tissue culture of chicken duodenum at several stages of fetal development. A normally occurring increase in disaccharidase activities in cultured tissue explants was diminished by the presence of peptic-tryptic digested gliadin or of a tryptic fragment of alpha-gliadin (alpha-GT 18,000). The effect of peptic-tryptic digested gliadin was only demonstrable in the early phase of fetal development (days 10-14), and disappeared at day 16. The release of enzymes into the culture medium was decreased by gliadin at the 12th day of fetal development. The results suggest that gliadin inhibits the differentiation processes of the fetal intestine.

Influence of a dorsomedial hypothalamus lesion on the circadian changes in the enzyme development and activity of intestinal brushborder membranes in the rat.

Circadian changes in activities and electrophoretic pattern of the intestinal brushborder enzymes alkaline phosphatase (EC 3.1.3.1), isomaltase (EC 3.2.1.10) and sucrase (EC 3.2.1.26) of normal rats and of rats with a lesion of the dorsomedial hypothalamus (DMH-rats) were studied. In contrast to the rhythm found in normal rats, there are no significant differences between the average enzyme activities in the light or dark period for the DMH-rats. The peak ratio in the electrophoretic pattern for sucrase-isomaltase (SI) over pro-sucrase-isomaltase (pro-SI) changes over 24 hr with higher values during the active (dark) period of the normal rat. In DMH-rats this ratio is much higher, but no significant difference could be demonstrated between the values near the acrophase in these rats.

The specificity of wheat protein reactivity in coeliac disease.

The purpose of this study was to compare the effects of seven wheat protein fractions on the cell-mediated immune response of coeliac patients and normal individuals, by means of the leukocyte migration inhibition factor (LMIF) assay. Two preparations of milk protein were used as control antigens. Whereas milk protein had no effect on the release of LMIF by cells from either normal or coeliac patients, wheat protein preparations stimulated two types of response. A non-specific reaction was elicited from both coeliac and normal cells by crude preparations such as gliadin and Frazer's fraction III (an enzyme digest of gluten), whereas purified fractions (alpha gliadin and alpha-pel) stimulated a specific response from the cells of coeliac patients only. These results suggest that only a pure wheat protein preparation such as alpha gliadin or alpha-pel is of value in studying immunological parameters in coeliac disease.

Energy intake and the appearance of 5,8,11-eicosatrienoic acid in serum lipids during parenteral nutrition without fat.

During parenteral nutrition without fat, biochemical changes in fatty acid composition of serum lipids may occur, such as a decrease in the concentration of linoleic and arachidonic acid and an increase of 5,8,11-eicosatrienoic acid. In various reports on essential fatty acid deficiency in parenteral nutrition, the rates and degrees of these changes in fatty acid composition of serum lipids are different. We have tested the hypothesis that a relationship exists between the energy intake and the increase in 5,8,11-eicosatrienoic acid in parenteral nutrition without fat. The hypothesis is based on the assumption that mobilization of lipids from body fat, and thus of stored essential fatty acids, only takes place when energy intake is below requirements. Seventeen patients with gastrointestinal diseases were fed parenterally without fat during 1 to 5 weeks (mean 3.5 week). The energy supply to the patients varied from 88 to 222 kilo Joules per kilogram per day. In all patients, the fatty acid pattern of the serum phospholipid fraction was determined weekly. It appeared that those patients with the highest energy intake per kilogram body weight showed the lowest increase in 5,8,11-eicosatrienoic acid. In ten patients, energy was given as glucose and fructose, in seven patients as sorbitol. The type of carbohydrate investigated did not influence the changes in the fatty acid pattern. Results show further that the individual values of the concentrations of 5,8,11-eicosatrienoic acid and of arachidonic acid provide more information concerning the essential fatty acid status of a patient than the commonly used ratio of these two fatty acids.

[Diagnosis in celiac dis]. / Diagnostiek bij coeliakie.

The work of Dicke, Weyers and Van de Kamer in the fifties caused a breakthrough in the treatment of Coeliac Disease. The pathogenesis of the disease however is still unknown in spite of great research efforts. Research nowadays is mainly directed to chemical analysis of wheat proteins and to immunological aspects of the disease. A close relationship to the HLA-system has been demonstrated. This article deals with the situation of the research and the consequences thereof for the diagnosis of the disease.

Studies on the determination of extracellular galactosyltransferase in human intestinal tissue.

The determination of extracellular galactosyl transferase (EC 2.4.1.38) activity in human intestinal tissue by assessment of the incorporation of label after incubation with UDP[3H]galactose was evaluated. Intestinal biopsy specimens were incubated with membrane-permeable L-[1-14C]fucose and non-permeable UDP-D-[6-3H]galactose (UDP[3H]Gal). Comparison of the amounts of 3H- and 14C-label incorporated into subcellular fractions showed uptake and incorporation of galactose formed by the hydrolysis of UDP[3H]Gal by brush-border enzymes. The results indicate that incorporation of galactose after incubation of the tissue with UDP[3H]Gal is not exclusively attributable to extracellular galactosyl transferase.

The determination of ammonium in Kjeldahl digests using the gas-sensing ammonia electrode. Comparison of the direct method with the known-addition method.

The efficacy of the ammonia electrode for analysis of the nitrogen content of a large series of Kjeldahl digests was investigated. By using this electrode, two methods for the measurement of ammonium concentrations were compared, the direct method and the known-addition method. When the direct method was used, a marked shift in the electrode potential occurred within a few hours, causing errors of 9-17% in the results. When the ammonium concentrations were calculated from the difference in electrode potential before and after addition of a known amount of an ammonium standard solution (known-addition method), it was possible to carry out reproducible measurements and the shift in the electrode potential did not influence the results. In two series of identical samples the coefficient of variation was respectively 1.45% and 0.80%.

Lysosomal damage by gliadin and gliadin peptides; an activity not related to coeliac disease.

Rat-liver lysosomes have been used to determine the toxicity of gliadin fractions in relation to coeliac disease. In this study we compared the activity in acid phosphatase release from rat-liver lysosomes by casein, gliadin and by their peptic-tryptic digests. The release of acid phosphatase is not specific for gliadin.

Wheat grains: a substrate for the determination of gluten antibodies in serum of gluten-sensitive patients.

A simple method of detecting gluten antibodies in serum is described. Cryostat sections of wheat grains proved to be an excellent substrate in the immunofluorescence technique. Rabbit antisera to gliadin and alpha-gliadin, and high percentage of sera from patients with gluten-induced enteropathy had antibodies that reacted with an internal structure of wheat grains. These antibodies could be absorbed with gliadin and with alpha-gliadin.

Transient paraproteinaemia in a patient with coeliac disease.

A case is reported of a 43 year old man who suffered from a grass pollen allergy and a malabsorption syndrome and in whom a paraproteinaemia was found. The grass pollen hypersensitivity was abolished by desensitization. The malabsorption syndrome was found to be due to coeliac disease--that is, a "flat" mucosa of the jejunum with an almost normal ileal mucosa--followed by clinical recovery and morphological improvement on a gluten-free diet. A short period of gluten reintroduction caused deterioration of the jejunum. The monoclonal immunoglobulin (IgG-gamma) diminished and disappeared in the course of three years. Although it has not been possible to demonstrate that this paraprotein had anti-gliadin activity, it is suggested that the constant stimulation of the gut reticuloendothelial system by gluten might bear some relation to the appearance of the paraproteinaemia.