Celiac and Enzymes
last updated 5.7.06
Celiac is a very serious condition, which often goes undiagnosed. So if someone sees regression with gluten + enzymes, particularly strong proteases such as Peptizyde, they are advised to consider celiac as a possibility. One mother knew her children reacted to gluten among many, many other foods. She started giving Peptizyde and Zyme Prime and carefully re-introducing foods one at a time and noted the reactions. For a few weeks, her children were doing well with 1 slice of gluten bread once each day. About the third week, her children started to regress and developed a noticeable rash. She withdrew the gluten and compared her children’s rash to those of photos of the type of rash that often accompanies celiac, and concluded that her children were celiac. She has re-introduced many other foods, but remains gluten-free with the enzymes. She considers the enzymes to be very successful overall for her family. Two other mothers saw increased diarrhea with gluten + Peptizyde, but success with all other foods. There is a total of 12 reported similar cases
involving those with known celiac or suspected celiac where gluten + strong proteases made the reaction worse than if the proteases were not used.
It was found, in general, people with celiac reacted worse when taking gluten + Peptizyde than when consuming small amounts of gluten with no enzymes. Celiac is an autoimmune disease/condition with a genetic basis. There are certain sequences of peptides which cause a reaction in the small intestine of individuals with celiac disease. The peptides can be as small as 12 amino acids long (that's pretty small). These peptides are different than the
peptides that get absorbed into the bloodstream and cause the opiate problem which are called gliadiomorphs. When the peptides get to the small intestine, the celiac's body registers these peptides as The Enemy. A non-celiac's body would just see the peptide as from gluten and let it pass. Once the celiac’s body detects The Enemy, certain antigens are produced which increase T-cell production in the small intestine villi. This causes the villi to breakdown. There are three common theories being discussed as the cause for celiac and
the specific amino acid sequences have not been identified. It is an autoimmune reaction with a genetic basis.
There is a certain structure in the gliadin that the small intestine sees as toxic in celiac individuals. The protease enzymes are not breaking this down in a way so that it does not cause a reaction in celiacs. So, in fact, taking just a protease such as Peptizyde may be just making more of these little peptides (or whatever) available to the small intestine, and perhaps increasing the number of chances to provoke a reaction. There was some research on the www.celiac.com site which proposed this same thing with the use of barley enzymes although it also said this was just a working theory and there was no evidence to back it up. No two celiacs are alike in their dietary tolerances for gluten - some are very sensitive, some can tolerate a little at a time, some can't take oats or spelt or kamut, some can. So if a person
suspects celiac, takes Peptizyde and gluten and doesn't do well, that person should avoid gluten under all circumstances.
This is a different situation from the peptide/opiate problem which is caused by large, insufficiently broken down molecules and leaky gut. Those peptides have a certain structure that attaches to specific receptors in the brain. Celiac reactions are triggered by different tiny peptides which attach to receptors in the small intestine. Only celiacs will have intestines that react in this way.
Amylase/Glucoamylase May Help Celiacs with Gluten
After the above was written, the following article below was found and describes the situation with references. Apparently the part of the gliadin in gluten that causes problems to a person with celiac is not the protein or peptides derived from the protein, it is portions of gliadin carbohydrate. Although the protein can antagonize the situation (and cause the peptide probelm), the enzymes needed to break down the part of gliadin reactive to celiacs are the amylases and some subgroups of amylases (other enzymes that work on starch bonds such as glucoamylases).
This correlates exactly to what we have seen with many families using enzymes. The proteases create more, smaller pieces but would not necessarily break down the carbohydrate bonds (if at all), thus making the situation for a celiac eating gluten with protease enzymes worse than if they ate gluten without protease enzymes. It also follows that some people with suspected celiac have done better with the the amylase containing enzymes with gluten. So a person with suspected celiac should be taking enzymes for carbs with gluten contamination or infractions as well, not just the proteases. If anyone with celiac happens to have the <ahem> "opportunity" to try amylases with gluten, please post if this helps. If this is true and holds up, the guidelines for a person with celiac wanting to take enzymes for contamination should be recommended the amylase/glucoamylase products in addition to the proteases which work on the gluten protein peptides.
Both amylase and glucoamylase need to be in a product. If you think of starches as a tree, the amylase will work on from the end of branch inward, til it comes to a fork, where it stops because the glycosidic linkage is different. Glucoamylase, however, cleaves the fork, which then allows amylase to continue. That is why amylase should always be accompanied by glucoamylase, otherwise, the starch cleavage is not very effective. Same with the other carbohydrases. They work on one type of glycosidic linkage, and a carb may have many kinds of linkages present. The carbohydrate on gliadin is very complex, consisting of xylose, glucose, galactose, and arabinose. It is typical of plant cell walls which contain hemicellulase, pectin, lignin, and other sugars. One needs a complex of carbohydases to extensively degrade them.
I checked several of the references at Pubmed and the articles are listed there.
On pages 598-599 of the Pharmacology of Natural Medicines, there is this information:
"It has been known since the 1950s that the gluten found in wheat, rye and other grains is the cause of intestinal damage in celiac disease, with the gliadin fraction of gluten being the source of its toxicity. [references 1,2] By the 1970s, fractionation studies had succeeded at identifying the components of gliadin involved in teh toxic mechanism. The carbohydrate moiety, consisting mainly of glucose, galactose, xylose and arabinose, is the source of gluten's gastrointestinal toxicity in the celiac patient, rather than the protein fraction as had been previously suspected. [3,4,5]
"Amylytic enzymes from Aspergillus species are effective in vitro in the treatment of celiac disease, as they enzymatically cleave the toxic carbohydrate portion of gliadin. Fungal carbohydrase preparations render grains like wheat and rye virtually harmless to
individuals with gluten enterophathy.
A 1977 study attempting to identify the source of gliadin toxicity used a preparation of amylytic enzymes from Aspergillus niger to remove the carbohydrate portion of gliadin in vitro.
To be certain of the variables being tested, native gliadin was chromatographed showing that carbohydrate was associated with four main protein bands. When the carbohydrase-treated gliadin was chromatographed, no alteration was detected in the protein pattern, but carbohydrate was completely absent.
To further establish that the protein make-up remained unchanged as compared with native gliadin, peptide mapping of the treated gliadin was carried out using electrophoresis followed by chromatography. Peptide maps showed no difference between the treated and untreated gliadin, confirming that no alteration had occurred in the primary structure of the protein.
Gliadin treated in this manner was baked into loaves of bread made with gluten-free flour. The study compared the effect of bread with treated versus untreated gliadin on four patients with previously diagnosed celiac disease. All four patients had been on gluten-free diets for at least 3 months prior to the study and were virtually symptom-free. Previously, their clinical and physical signs and symptoms had included the diarrhea, malabsorption, decreased body weight and height, anemia, tetany, impaired D-xylose absorption, decreased intestinal mucosal enzyme secretion flattened mucosal brush-border and subepithelial tissue lymphocytosis typical of celiac disease.
During the test period, patients 1,2, and 3 received a total of 50g of treated gliadin baked into loaves of bread (10g gliadin/450 g loaf). Xylose absorption tests and intestinal biopsies from jejunal villi were performed before and after each test period.
The celiac patient receiving untreated gliadin (patient no 4) experienced a return of signs and symptoms of celiac disease – diarrhea, abdominal pain, low values on xylose absorption studies, decreased mucosal enzyme secretion (alkaline phosphates, lactase, surcease) and characteristic histological damage (mucosal lumphocytosis and loss of enterocyte height). The patients who received the treated glidin remained symptom-free during the test period and showed no abnormalitites in histological parameters (i.e. general morphology, epithelial cell height, tissue lymphocytes were normal in these patients).
This study demonstrated that carbohydrate-digesting enzymes from Aspergillus sp. can be used in vitro to remove the toxicity of gluten to celiac patients and supports the hyypothsis that carbohydrate components of gliadin are responsible for its toxicity, rather than protein components as had been widely suspected. It appears that no controlled studies have been done to evaluate the effectiveness of amylytic fungal enzymes at reducing gluten toxicity
to celiac patients in vivo by administering these enzymes with gluten containing foods at mealtime.
It should be noted that although celiac patients show intolerance to the carbohydrate portion of gliadin, this is likely not he only source of gluten-induced pathology. A number of studies suggest that protein components of gluten produce systemic allergic manifestations in some patients. It appears that both gastrointestinal intolerance and immunological hypersensititity are capable, either individually or in concert, of producing disease symptoms in susceptible individuals. Future studies may also show that both pathological mechanisms are amenable to treatment by hydrolysis of the offending portions of gluten with the appropriate orally administered fungal enzymes. This, however, remains to be proven."
1.Dicke WK. An investigation into the injurious constituents of wheat in connection with their action on patients with Coeliac disease. Acta Pediatr 1953; 42:223-231
2.Van De Kamer JH, Weijers HA, Coeliac disease: some experiments on the cause of the harmful effect of the gliadin. Acta Pediatr 1955; 44: 465-469
3.Phelan JJ, Stevens FM, McNicholl B et a. Coelic disease: the abolition of gliadin toxicity by enzymes from Asergillus niger. Clin Sci Molec Med 1977; 53: 35-43
4.McCarthy CF. Nutritional defects in patients with malabsorption. Proc Nutr Soc 1976; 35:37-40
5.Phelan JJ. The nature of gliadin toxicity in celiac disease. Biochem Soc Trans 1974; 2:1368-1370
6.Hekkens WTMJ et al. Antibodies to gliadin in serum of normals, celiac patients and schizophrenics. Nature 1963; 199; 259-261
Research on Enzymes for Celiac
Stanford researchers find cause, possible cure for gluten intolerance
Public Release: 26-Sep-2002
Stanford researchers find cause, possible cure for gluten intolerance A team of investigators led by Stanford University researchers have discovered the cause and a potential treatment for celiac sprue, an autoimmune disease that leads to an inability to digest gluten, a major protein in wheat, rye and barley products. - National Science Foundation
(Here's the article. Note the last line - "We think that this mode of therapy - peptidase supplementation - may offer hope in treating celiac sprue eventually, and we're going to test this hypothesis.")
Stanford researchers find cause, possible cure for gluten intolerance
Public release date: 26-Sep-2002
STANFORD, Calif. - A team of investigators led by Stanford University researchers have discovered the cause and a potential treatment for celiac sprue, an autoimmune disease that leads to an inability to digest gluten, a major protein in wheat, rye and barley products. The disease is estimated to afflict as many as 1 in 200 Americans. In the Sept. 27 issue of Science, researchers identify a fragment of gluten called gliadin as the celiac culprit. They showed that this fragment is resistant to digestion and is responsible for the intestine-damaging inflammatory response experienced by celiac patients. They also report the use of a dietary enzyme made by a bacterium that can break down the fragment into harmless bits, suggesting future treatment through dietary supplements.
"These findings are the first step to giving people with celiac disease real hope for a normal life," said Chaitan Khosla, PhD, professor of chemistry, chemical engineering and, by courtesy, of biochemistry. Lu Shan, a graduate student in Khosla's lab, was lead author on the paper. The team included other Stanford researchers as well as a group from the University of Oslo in Norway.
The lining of the small intestine is normally carpetlike, covered with small protrusions called villi. Celiac disease, however, results in a smooth, pipelike intestine. The reduced surface area keeps the body from absorbing nutrients. Often diagnosed in childhood, the disease can lead to the distended stomach and stunted growth typical of starvation.
"The only effective therapy for most people is a lifelong gluten-free diet, and that's fairly restrictive," explained co-author Gary M. Gray, professor of medicine, emeritus. The diet is essential over the long term both to restore normal intestinal function and to reduce the risk of developing osteoporosis, lymphoma or cancer of the small intestine, he added.
In the laboratory, Shan simulated the digestive process, exposing gliadin to digestive enzymes in test tubes. She identified a protein fragment made up of 33 amino acids that was resistant to further digestion and whose structure was known to be toxic. Most proteins are broken down into small peptides of between two and six amino acids or into single amino acids. She then repeated her study in rats and again in test tubes using tissue taken by biopsy from patients undergoing unrelated medical procedures. "Even with prolonged treatment (exposure to intestinal enzymes), the peptide doesn't lose the ability to induce the inflammatory response," Shan said.
When they looked more closely at the fragment, Shan and her colleagues found that it was made up of even smaller fragments already known to induce human T-cells to attack the intestine. The team in Norway then measured the ability of the gliadin fragment to induce autoimmune activity. "The response by T-cells was about 10 to 20 times higher than the smaller peptides themselves," Shan said.
Because the fragment is rich in the amino acid proline, investigators reasoned that a peptidase (an enzyme that breaks down proteins) with the ability to digest proline-rich chains might be able to break down the gliadin fragment, rendering it harmless to celiac patients. They have now shown that this is the case in test tubes and in rats. Because there are no animal models of celiac disease, testing this approach in humans is a long way off and will require further preclinical work, Khosla said. "We think that this mode of therapy - peptidase supplementation - may offer hope in treating celiac sprue eventually, and we're going to test this hypothesis."
Contact: Michelle Brandt
Stanford University Medical Center
Okay...so where do we get this carboxypeptidase stuff? These two enzymes together would explain why some research shows carbohydrate enzymes help and protease enzymes help.
Here is some other comments on the carboxy-stuff-ase:
"The body produces three large pro-compounds: proenkephalin, prodynorphin, and pro-opiomelanocortin. Endorphins can further decompose to small fragments, oligomers, which are still active. Oligomers pass the blood-brain barrier more readily. Enzymatic degradation of small-chain endorphins is accomplished by dipeptidyl carboxypeptidase, enkephalinases, angiotensinases, and other enzymes. This limits their lifetime in the unbound state.
Opioid receptors presynaptically inhibit transmission of excitatory pathways. These pathways include acetylcholine, the catecholamines, serotonin, and substance P. Substance P is a neuropeptide active in neurons that mediate our sense of pain; antagonists of substance P are currently under investigation as clinical antidepressants. Endorphins are also involved in glucose regulation."
If I am following this correctly, it says that if you are deficient in carboxypeptidase then you don't break down the opioids quickly are a 'typical' person would. So any opiates from any source hang around longer than usual. When this happens the opiates reduce the activity of several neurotransmitters, including serotonin. If substance P is inhibited, my guess is that you would have all these sensory problems because of all the pain irregularities. I bet I have inhibited substance P with all the over-sensitivity to pain I have! But anyway, here are more connections.
Since Peptizyde has tons 'o DPP IV and lots of people do great on it except those with celiac, maybe there is something in celiac condition that is essentially a carboxypeptidase deficiency. Theoretically, a celiac-zyme would need both of these components in it.
Intestinal digestive resistance of immunodominant gliadin peptides.
Hausch F, Shan L, Santiago NA, Gray GM, Khosla C.Am J Physiol Gastrointest Liver Physiol 2002 Oct;283(4):G996-G1003 PMID: 12223360 [PubMed - indexed for MEDLINE]
Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA.
Two recently identified immunodominant epitopes from alpha-gliadin account for most of the stimulatory activity of dietary gluten on intestinal and peripheral T lymphocytes in patients with celiac sprue. The proteolytic kinetics of peptides containing these epitopes were analyzed in vitro using soluble proteases from bovine and porcine pancreas and brush-border membrane vesicles from adult rat intestine. We showed that these proline-glutamine-rich epitopes are exceptionally resistant to enzymatic processing. Moreover, as estimated from the residual peptide structure and confirmed by exogenous peptidase supplementation, dipeptidyl peptidase IV and dipeptidyl carboxypeptidase I were identified as the rate-limiting enzymes in the digestive breakdown of these peptides. A similar conclusion also emerged from analogous studies with brush-border membrane from a human intestinal biopsy. Supplementation of rat brush-border membrane with trace quantities of a bacterial prolyl endopeptidase led to the rapid destruction of the immunodominant epitopes in these peptides. These results suggest a possible enzyme therapy strategy for celiac sprue, for which the only current therapeutic option is strict exclusion of gluten-containing food.
Prolyl endopeptidase-mediated destruction of T cell epitopes in whole gluten: chemical and immunological characterization.
J Pharmacol Exp Ther. 2005 Jan;312(1):19-26. Epub 2004 Sep 09. PMID: 15358813
Marti T, Molberg O, Li Q, Gray GM, Khosla C, Sollid LM. Celiac Sprue Research Foundation, 3181 Porter Dr., Palo Alto, CA 94304.
Celiac Sprue is a widely prevalent immune disease of the small intestine induced by dietary gluten intake in genetically susceptible individuals. It has been suggested that prolyl endopeptidases (PEPs) may be useful catalysts for gluten detoxification. We have investigated this hypothesis using food-grade gluten as the target antigen, and a combination of mass spectrometry and patient-derived T cells as quantitative assay systems. Spectrometric characterization of physiologically proteolyzed gluten revealed a number of 10 to 50 residue peptides containing known T cell epitopes involved in Celiac Sprue pathogenesis. Several of these peptides were multivalent, suggesting they may be potent triggers of the inflammatory response to gluten in celiac patients. Treatment of proteolyzed gluten with recombinant bacterial PEP decreased the number of potentially immunostimulatory peptides. Substantially reduced immunogenicity was also quantified in 12 of 14 intestinal polyclonal T cell lines from celiac patients. Kinetic investigations using eight T cell clones showed rapid destruction of alpha-gliadin epitopes, but less complete processing of gamma-gliadin epitopes. Given the difficulty associated with a strict lifelong gluten-exclusion diet, the ability of a single enzyme to greatly reduce the antigenic burden of grocery store gluten reinforces the case for developing oral peptidase therapy against Celiac Sprue.
Oats in the treatment of childhood coeliac disease: a 2-year controlled trial and a long-term clinical follow-up study. Aliment Pharmacol Ther. 2006 May 15;23(10):1463-72. PMID: 16669961
Holm K, Maki M, Vuolteenaho N, Mustalahti K, Ashorn M, Ruuska T, Kaukinen K.
Summary Background The exclusion of oats from the diet in coeliac disease is controversial. Aim To study the long-term safety of oats in the treatment of children with coeliac disease. Methods Altogether 32 children with coeliac disease were enrolled in a 2-year controlled trial. Twenty-three children in remission were randomized either to oats or gluten challenge; when small bowel histological relapse was evident after gluten challenge, a gluten-free diet including oats was started. Furthermore, nine newly detected coeliac patients adopted an oat-containing gluten-free diet. Small bowel mucosal morphology, CD3+, alphabeta+ and gammadelta+ intraepithelial lymphocytes, human leucocyte antigen (HLA) DR expression and coeliac serology were determined. After the trial, the children were allowed to eat oats freely; follow-up was extended up to 7 years. Results In coeliac children in remission, oats had no detrimental effect on intestinal histology or serology during the 2-year trial. In contrast, the gluten-challenge group relapsed after 3-12 months. Complete recovery from the disease was accomplished in all relapsed and newly detected patients on an oat-containing gluten-free diet. After the trial, 86% of the children preferred to consume oats and they all remained in remission. Conclusion In most children with coeliac disease, long-term consumption of oats is well tolerated, and it does not result in small bowel mucosal deterioration or immune activation.
- Immunohistochemical analysis of coeliac mucosa following ingestion of oats. Clin Exp Immunol. 2006 May;144(2):197-203. PMID: 16634791
Srinivasan U, Jones E, Carolan J, Feighery C.
There is now considerable clinical evidence that oats do not activate coeliac disease. Nonetheless, a reluctance to include oats in the gluten-free diet remains. Because gluten-induced damage is accompanied by activation of the gastrointestinal immune system, the purpose of this study was to investigate if similar changes were induced by oats ingestion. Small intestinal histological sections from 10 patients who ingested 50 g of oats daily for 3 months were investigated for possible evidence of immune activation. Tissue obtained before and after oats challenge was stained with a series of antibodies directed against the following molecules: human leucocyte antigen D-related (HLA-DR), Ki-67, CD25, CD54 [intercellular adhesion molecule 1 (ICAM-1)] and mast cell tryptase. None of the patients developed clinical or laboratory evidence of adverse effects. The distribution of intestinal HLA-DR expression was not affected by oats ingestion and the crypt epithelium remained unstained. In the pre-oats biopsies, the percentage of Ki-67 positive enterocytes, 29.5 +/- 6.9 [95% confidence interval (CI) 13.9-45.0] did not differ significantly from that found in postoats biopsies, 41.2 +/- 3.7 (95% CI, 32.8-49.6), P = 0.19, not significant. Furthermore, oats ingestion did not alter the number of CD25 positive and tryptase positive cells. Finally, the distribution and intensity of ICAM-1 staining was unchanged by dietary oats. In summary, detailed immunohistological studies of biopsies from patients ingesting oats for 3 months did not reveal evidence of immune activation. Together with other reported findings, this study strengthens the view that oats can be included safely in the diet of gluten sensitive patients.
|Am J Physiol Gastrointest Liver Physiol. 2006 May 11;
Highly efficient gluten degradation with a newly identified prolyl endoprotease: implications for celiac disease.
Stepniak D, Spaenij-Dekking L, Mitea C, Moester M, de Ru A, Baak-Pablo R, van Veelen P, Edens L, Koning F.
Dept. of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, Netherlands.
Celiac disease is a T cell-driven intolerance to wheat gluten. The gluten derived T cell epitopes are proline-rich and thereby highly resistant to proteolytic degradation within the gastrointestinal tract. Oral supplementation with prolyl oligopeptidases has therefore been proposed as a potential therapeutic approach. The enzymes studied, however, have limitations as they are irreversibly inactivated by pepsin and acidic pH, both present in the stomach. As a consequence, these enzymes will fail to degrade gluten before it reaches the small intestine, the site where gluten induces inflammatory T cell responses that lead to celiac disease. We have now determined the usefulness of a newly identified prolyl endoprotease from Aspergillus niger for this purpose. Gluten and its peptic/tryptic digest were treated with prolyl endoprotease and the destruction of the T cell epitopes was tested using mass spectrometry, T cell proliferation assays, ELISA, rpHPLC, SDS-PAGE and Western blotting. We observed that the A. niger prolyl endoprotease works optimally at pH 4-5, remains stable at pH 2 and is completely resistant to digestion with pepsin. Moreover, the A. niger derived enzyme efficiently degraded all tested T cell stimulatory peptides as well as a peptic/tryptic digest of gluten and intact gluten. On average the endoprotease from A. niger degraded gluten peptides 60 times faster than a prolyl oligopeptidase. Together these results indicate that the enzyme from A. niger efficiently degrades gluten proteins. Future studies are required to determine if the prolyl endoprotease can be used as an oral supplement to reduce gluten intake in patients.
Public release date: 23-Jun-2006
Contact: Heidi Hardman
New therapy may mean less dietary restrictions for celiac sufferers
Scientists have discovered what may be a successful non-dietary therapy for celiac sprue, an inherited inflammatory disorder of the small intestine that impacts an estimated 1 in 200 people around the world. Two research studies, published in the June issue of Chemistry and Biology, pave the way for clinical testing with an oral enzyme therapy that may prevent the many symptoms and complications of this widespread disease.
People with celiac sprue, also called celiac disease, cannot tolerate the protein gluten in their diet. Gluten is present in grains like wheat, barley, and rye. When gluten is ingested by a celiac patient, it sets off an inflammatory reaction that damages the small intestine, leading to malabsorption, an autoimmune-like response, and many other complications. The only effective therapy for celiac disease is complete dietary exclusion of gluten. However, the ubiquitous nature of gluten poses a constant threat to celiacs, and a majority of celiac patients who adopt a restrictive diet still exhibit structural and functional gut abnormalities.
"Non-dietary therapies that allow celiac patients to safely incorporate low-to-moderate levels of gluten into their daily diet would be of considerable benefit," explains study leader Dr. Chaitan Khosla, from Stanford University and Celiac Sprue Research Foundation. "Having demonstrated earlier that certain types of enzymes can detoxify gluten, our laboratory set out to devise an optimal oral enzyme therapy for celiac sprue by borrowing from nature. In germinating barley seed, gluten serves as a nutritious storage protein that is efficiently digested by enzymes. One enzyme, EP-B2, plays a crucial role in this process by breaking gluten proteins after glutamine residues, which comprise one-third of all amino acid residues in gluten."
Dr. Khosla's group used recombinant bacteria to produce a form of EP-B2 that only activates under acidic conditions similar to the conditions found in the human stomach. The researchers demonstrated that EP-B2 efficiently digested gluten protein under gastric conditions and, importantly, EP-B2 was most specific for those parts of gluten that are known to trigger celiac pathogenesis. In a second study, the researchers went on to devise an even more potent double enzyme therapy for detoxifying gluten.
EP-B2 was tested in combination with another well-characterized enzyme called PEP that breaks gluten protein after proline residues. Like glutamine, proline is also abundant in inflammatory gluten peptides. At very high gluten loads, where neither PEP nor EP-B2 alone could detoxify gluten quickly enough to prevent inflammation, a PEP and EP-B2 combination completely abolished gluten immunotoxicity within ten minutes under simulated gastric and duodenal conditions.
In this tag-team therapy, EP-B2 first cleaved gluten into small pieces under gastric conditions that were then easier for PEP to fully detoxify under duodenal conditions. "Our results suggest that recombinant EP-B2 should be effective as supportive therapy to help celiacs cope with the 'hidden' gluten in everyday life, and that a two-enzyme cocktail containing PEP and EP-B2 may even allow celiacs to resume a more normal diet in the future," offers Dr. Khosla.
Seigel et al.
The researchers include Matthew Siegel, Michael T. Bethune, Jiang Xia, Alexandre Johannsen, Tor B. Stuge, and Peter P. Lee of Stanford University in Stanford, CA; Jonathan Gass, Jennifer Ehren, Gary M. Gray, and Chaitan Khosla of Stanford University in Stanford, CA and Celiac Sprue Research Foundation in Palo Alto, CA. This research was supported by a grant from the National Institutes of Health (R01 DK63158 to C.K. and Mary Hewitt Loveless, MD Pilot-Project Grant to P.P.L.).
Siegel et al.: "Rational Design of Combination Enzyme Therapy for Celiac Sprue." Publishing in Chemistry & Biology 13, 649–658, June 2006 DOI 10.1016/j.chembiol.2006.04.009 www.chembiol.com
Bethune et al.
The researchers include Michael T. Bethune, Yinyan Tang, and Chaitan Khosla of Stanford University in Stanford, CA; Pavel Strop of Howard Hughes Medical Institute and Stanford University in Stanford, CA; Ludvig M. Sollid of University of Oslo and Rikshospitalet University Hospital in Oslo, Norway. This research was supported by R01 DK063158 to C.K. M.T.B. is a recipient of a National Institutes of Health Cellular and Molecular Biology Training Grant through Stanford University.
Bethune et al.: "Heterologous Expression, Purification, Refolding, and Structural-Functional Characterization of EP-B2, a Self-Activating Barley Cysteine Endoprotease." Publishing in Chemistry & Biology 13, 637–647, June 2006 DOI 10.1016/j.chembiol.2006.04.008 www.chembiol.com
Celiac Success: New Enzyme Efficiently Degrades Gluten In 'Human Stomach' Environment
A new enzyme originally developed for commercial food processing turns out to also quickly and nearly-completely break down whole gluten molecules as well as the T cell stimulatory peptides that cause celiac disease, a digestive disease with no current effective treatment other than avoiding wheat, barley or rye products.
In addition, the enzyme operates best in just the kind of physiological environment found in the human stomach and works 60 times faster than an earlier promising enzyme, which was not effective in acidic conditions and was inactivated by pepsin, both of which are found in the stomach.
"On the basis of our results, there now is a realistic chance that oral supplementation with an enzyme can ensure gluten degradation in the stomach before reaching the small intestine, where it causes problems for people with celiac disease," according to Frits Koning, researcher at the Leiden University Medical Center, The Netherlands, who headed the team that has published a new research paper on its work.
The paper, "Highly efficient gluten degradation with a newly identified prolyl endoprotease: implications for celiac disease," is in the online American Journal of Physiology- and Liver Physiology, published by The American Physiological Society. Research was by Dariusz Stepniak, Liesbeth Spaenij-Dekking, Cristina Mitea, Martine Moester, Arnoud de Ru, Renee Baak-Pablo, Peter van Veelen and Frits Koning of Leiden University Medical Center, the Netherlands, and Luppo Edens of DSM Food Specialties, Delft.
Clinical trials are likely next step
The new prolyl endoprotease (PEP) that was studied is derived from Aspergillus niger (AN), a common fungus. Strains of A. niger are used in industrial production of citric and gluconic acid as well as producing several food grade enzymes.
Because there are no animal models of celiac disease, "the in vivo efficacy of AN-PEP for gluten detoxification will ultimately have to be addressed in clinical studies involving celiac patients. AN-PEP appears to be a prime candidate for such clinical trials," the paper concluded. As for the timing of any such trials, Koning said: "This is an option the team hopes to explore in the future."
A disease of many paradoxes
Celiac disease affects about 2 million Americans and is also found in Europe, India and parts of the Middle East. It's caused by an uncontrolled immune response to wheat gluten and similar proteins of rye and barley that cause diarrhea, malnutrition and failure to thrive because it inhibits nutritional uptake.
"It's a Caucasian disease with a wide spectrum of symptoms; not all patients are equally affected, but we do not understand why this is the case," Koning said. "It is known to be associated with the HLA-DQ2 gene," he noted, "but while about 25% of the white population has this gene, only about one in 100 get the disease, so it's really a quite puzzling disease in many ways."
Currently the only way to elude the disease symptoms is by avoiding wheat, barley and rye products. "It sounds easy, but gluten especially is widespread in Western diets," Koning said. Gluten is often used as a food additive because it adds protein content inexpensively and also gives dough its elasticity and stickiness, which helps in manufacturing. For instance, Koning said: "Celiac patients can eat potato chips, but not if they have added paprika or other spices because they're 'glued' to the chip with gluten."
AN-PEP outstrips earlier enzyme by 60-fold
Earlier attempts at finding non-human proteases for gluten detoxification (first proposed in the late 1950s) focused on prolyl oligopeptidases (POP), most notably FM-POP, which was able to break down gluten sequences in vitro. However FM-POP's optimal operating pH is between 7 and 8, so it didn't work well in the more acidic stomach pH that goes down to 2 at one stage. A combination of pH 2 and pepsin "immediately inactivated FM-POP," the paper said. AN-PEP, on the other hand, is active from pH 2-8, with optimum effect around pH 4. The combination of pH 2 and pepsin didn't affect AN-PEP activity.
"An effective enzymatic treatment for celiac diseases requires means of destroying all or at least the vast majority of gluten derived T cell stimulatory sequences," the paper said. The key to this is to break the large gluten molecules (large peptides and intact proteins) into smaller pieces before they leave the stomach. Because food stays in the stomach one to four hours, speed of protein degradation is also important. Mass spectrometry comparisons showed that "degradation of gluten peptides by AN-PEP was on average [about 4 minutes, or] 60 times faster than degradation by FM-POP," the paper reported.
In addition to its ability to perform as a potential oral enzymatic therapy because it "is capable of degrading intact gluten molecules and T cell stimulatory epitopes from gluten into harmless fragments" AN-PEP has several additional commercial advantages, the paper said: "The enzyme is extremely stable and can be produced at acceptable cost at food grade quality in an industry setting."
Celiac disease is an HLA-linked disease related to Type 1 diabetes and rheumatoid arthritis in which autoimmune reactions cause the disease; similarly, immune reactions can lead to organ transplant rejection. Koning said it "isn't likely that AN-PEP would be of any therapeutic value in any of these HLA-associated diseases" because Type 1 diabetes and rheumatoid arthritis are real autoimmune diseases, where the immune system attacks parts of the body. In celiac disease, it is the gluten that is the target, not the body.
Reminder warning on early introduction of gluten products
Koning said feeding wheat (or barley or rye) products to infants before they're 6 months old isn't recommended because once an immune response develops "immuno-memory builds up and it doesn't go away." Indeed, Koning noted that in Sweden some years ago gluten was introduced into baby food, which led to a five-fold increase in celiac disease. The problem disappeared when gluten was removed.
Source and funding/support
The paper, "Highly efficient gluten degradation with a newly identified prolyl endoprotease: implications for celiac disease," is in the online American Journal of Physiology-Gastrointestinal and Liver Physiology, published by The American Physiological Society. Research was by Dariusz Stepniak, Liesbeth Spaenij-Dekking, Cristina Mitea, Martine Moester, Arnoud de Ru, Renee Baak-Pablo, Peter van Veelen and Frits Koning, Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, the Netherlands, and Luppo Edens of DSM Food Specialties, Delft, a subsidiary of Koninklijke DSM N.V., a Dutch chemical conglomerate.
Research supported by Netherlands Organization for Scientific Research, Celiac Disease Consortium, Centre for Medical Systems Biology (the latter two partly backed by the Netherlands Genomics Initiative). DSM supplied A. niger-derived PEP (AN-PEP) on which it holds a patent.