METHOD FOR USE IN THE DIAGNOSIS OF FOOD ALLERGIES AND/OR FOOD INTOLERANCES

Abstract: The measurement of the nitric oxide concentration in the rectum or in a gas sample taken from the rectum of a patient can be used as a rapid screening method and as an aid in the diagnosis of allergies and intolerance reactions. A measurement performed after a rectal provocation with a suspected allergen or intolerance causing agent will give an indication as to the sensitivity to that specific allergen or agent. In nitric oxide measurements, without prior provocation, elevated levels compared to normal levels, obtained form healthy controls, are taken as an indication of allergy or a predisposition for allergy. One or several repeated measurements after elimination of the suspected allergen from the patient's diet can be used to confirm the diagnose and to check patient compliance.

Description: METHOD FOR USE IN THE DIAGNOSIS OF FOOD ALLERGIES AND/OR FOOD INTOLERANCES The present invention relates to the field of allergy tests and in particular to a method for use in the diagnosis of food allergy and food intolerance. Background of the invention Food allergies: The occurrence of allergies has increased dramatically during the last decades, fortunately accompanied by the development of pharmaceuticals alleviating the symptoms. Although our knowledge about the causative mechanisms behind the various types of allergy has increased tremendously, all aspects are still not fully elucidated. One particularly difficult area is the area of gastrointestinal allergies, often called food allergies. Food allergies can be defined as the clinical symptoms resulting from an inappropriate immune response to specific food proteins or food additives. The broader term, food intolerance, on the other hand describes any adverse reaction to a food that is non-immune in nature (Burks and Stanley; Food Nllergy, Current Opinion in Pediatrics, 10(6):588-93, 1998). The diagnosis of food allergies is one field where many deficiencies still exist, regardless of the development of various tests. In many cases, the patients themselves notice a connection between their symptoms and certain allergens, for example by observing an immediate adverse reaction upon ingestion of the suspected allergen or by eliminating and re-introducing the source of allergens. N detailed anamnesis is therefore often very helpful in establishing a diagnosis. Different tests exist, such as provocation test using isolated allergens. In the so called skin puncture test (SPT), small droplets of different allergens in solution are placed on the skin of the patient. One saline droplet and one histamine droplet are used as negative and positive control, respectively. The skin is then lightly punctured under each droplet. Where an allergic reaction occurs, the histamine released in the skin produces a blister, the size of which is measured. In the widely used radio-allergosorbent test (the so called RNST, e.g. UniCNP™ Specific IgE or the Pharmacia CAP System™ RNST from Pharmacia & Upjohn Diagnostics AB, Uppsala, Sweden) the presence of specific substances is determined in a blood sample. The correspondence between skin puncture tests and RAST is very good. Further, many allergens have been identified and in some cases isolated and characterised down to molecular levels. In practise, however, it is often enough to identify a particular food stuff as source of allergens, e.g. cow milk containing several known allergenic proteins. Further, it is known that certain allergens often present in food, such as said cow milk proteins, hen egg albumin, fish and shell fish, nuts etc in some patients can cause an anaphylactic chock, which can be life-threatening for the patient. In other patients, the same allergens give only very weak or diffuse symptoms. In the field of allergy tests, however, problems arise in specific situations, e.g. where the allergic reaction is either delayed, very diffuse or part of a complex of clinical symptoms. In the first case, when the reaction is delayed, it becomes difficult to associate the problems with the particular food causing them. In the second case, where the reaction is diffuse, the patient voices general complaints, such as stomach ache. Finally, in the third case where the reaction is part of a complex of clinical symptoms, the patient is exhibiting multiple symptoms, such as skin rashes, asthma etc. In this latter case, it is difficult to distinguish the symptoms caused by the food allergen from similar or even identical symptoms caused or aggravated by another allergen. Often, the symptoms resemble the symptoms of common problems such as headache, fatigue and joint pains. It is also possible that the same substances produce different reactions or at least fail to produce an identical reaction every time. It is understandable that the establishing of a diagnosis is both difficult, time-consuming and often frustrating for both the physician and the patient. Food intolerance: Food intolerance is an adverse reaction to a food or food component that does not involve the IgE -mediated immune response seen in classic food allergies, e.g. cow milk allergy. Food intolerance reactions can be triggered by a physical reaction to a food or food additive. For example, people with lactose intolerance do not have enough of the enzyme lactase, to digest the lactose naturally present in milk. This is one of the most common food intolerances and can be managed through diet and the use of enzyme preparations, such as Lactaid ® caplets or drops. Another ailment is coeliac disease, where the patient reacts to the gluten in cereal foods. Further, certain food additives such as flavour enhancers (mono sodium glutamate) or preservatives (sulphates) used on foods may cause a food intolerance reaction in some people. The symptoms of food intolerance vary and can be mistaken for those of a food allergy or other ailments. Coeliac disease or malabsorption syndrome is a syndrome mainly affecting children, but also adults, caused by a reaction to gluten which prevents the small intestine from digesting fat. In adult coeliac disease, the adverse reaction to gluten is known to cause atrophy of the intestinal villi, so that the surface available for absorption becomes smaller. The symptoms of coeliac disease include a swollen abdomen, diarrhoea, abdominal pains and anemia. It is known that intolerance to wheat gluten is a major factor in the aetiology of the enteropathy observed in almost all children with coeliac disease and most adults with idiopathic steatorrhoea. Diagnosis has frequently been based on dietary changes, namely putting the patients on a gluten-free diet and observing the possible recovery / normalisation. The gold standard for the diagnosis of coeliac disease remains a jejunal biopsy showing typical mucosal lesions of the small intestine including villous atrophy. Biopsies may be taken before and after exclusion of gluten in the patient's diet. Following the introduction of a gluten-free diet, the intestinal mucosa will regenerate and the symptoms normally disappear. Controlled reintroduction of gluten or a rectal gluten challenge combined with colonoscopy or the examination of a biopsy sample has been experimentally used to confirm the diagnose. It has been suggested that coeliac disease can be diagnosed using jejunal biopsy, taken with a Watson/Crosby capsule before and after an oral FF3 challenge. FF3 (Frazer's fraction III) is a peptic-tryptic digest of gluten that is known to damage the mucosa of the small intestine in gluten-sensitive subjects. The biopsy samples are prepared and the number of intraepithelial lymphocytes is estimated. A result can be available within 24 - 48 h which prompts the authors to call this a "rapid confirmatory test of gluten sensitivity in patients already taking a gluten-free diet" (Loft, D. E. βt al, Lancet, 335 (1990) 1293-1295). Prior art When IgE -mediated food induced allergic reactions are suspected, both the SPT and the RNST tests provide useful methods for establishing whether the patient possesses IgE antibodies to specific foods. SPTs can be used to screen patients with suspected IgE-mediated food allergies. Glycerinated food extracts and appropriate controls are applied by the prick or skin puncture technique. Positive SPT responses indicate the possible association between the food tested and the patient's reactivity to that specific food. The positive predictive accuracy of SPTs are however less that 50 % compared with double blind placebo controlled food challenge tests (DBPCFC) according to Samson, H.N. in Food Nllergy. Part 2: Diagnosis and management (J Nllergy Clin Immunol, Vol. 103, no. 6, 1999, 981-9). The Phadiatop test (Pharmacia & Upjohn, Inc., USA) is a multi-RNST test which gives positive result if the sera of a patient contains IgE-antibodies against one or several of the most common airborne allergens. Another test is the Phadiatop Kombi (Pharmacia & Upjohn, Inc., USA) where the above Phadiatop is combined with the RNST-FX5 test, which detects IgE-antibodies against cow milk proteins, hen egg albumin, fish, peanut, wheat and soybean. The ECP test (eosinophile cationic protein) measures the degree of activation of the eosinophile granulocyte, which reflects the activity of the allergic reaction in e.g. asthma and eczema. Examples of commercial ECP tests are UniCNP™ ECP, the Pharmacia ECP RIA and the Pharmacia CAP System™ ECP FEIA (Pharmacia & Upjohn, Inc., USA). It is also possible to produce monoclonal antibodies against individual, isolated allergens. This has been done e.g. for the major mite allergen (Der pi), cat allergen (Fel dl) and dog allergen (Can fl). Using monoclonal antibodies, the presence of individual allergens can be detected, for example in the home environment of the patient. The presently used tests, based on the examination of a blood sample taken from a patient, are costly and time consuming, in particular if several allergens are suspected. Both when an allergy or intolerance is suspected, the anamnesis remains the most important diagnostic aid, preferably supplemented with a diet diary and physical examination. A tentative diagnosis can be confirmed by a diet eliminating the suspected allergen and/or by oral food challenges, in sensitive patients performed under medical observation. It is known through WO 96/17244 (Kjell Alving, Edward Weitzberg, Jan Lundberg and Jon Lundberg) that inflammatory states in the intestines can be detected by measuring the concentration of nitric oxide (NO) in a gas sample, taken from the intestinal lumen. This method has hitherto been implemented in the diagnosis of Crohn's disease and ulcerative colitis. Further, a method and device for the diagnosis of allergies has been disclosed in U.S. 5,983,899, which method comprises analysing a sample that has been taken rectally from the large intestine of a patient who is suspected of being allergic, after having provoked the mucous membrane of the patient's large intestine rectally with an allergen against which the suspected allergy of the patient is directed. The method is illustrated by examples, where nine patients suffering from coeliac disease were examined using a rectal perfusion technique using a six-channel PVC hose having an inner diameter of 10 mm, an outer diameter of 16 mm and a total length of 38 cm. Three inflatable balloons were fastened to the hose, to obtain a rectal perfusion segment of 8 cm in length. Two rectal perfusions were performed on each individual on one and the same day: a basal perfusion and a perfusion three hours after introducing gluten. The participants were studied after having fasted for 17 hours and 4 liters of an oral laxative solution was used as an enema. The disclosure of U.S. 5,983,899 also comprises an instrument for rectal insertion in the rectum, having an expandable part which surrounds a central channel which opens out on each side of the expandable part, and a separate channel through which expansion of the expandable part can be controlled such that when the instrument is applied in the rectum; the outer wall of the expandable part will be in direct contact with the mucous membrane of the rectum; and wherein a diffusible allergen and a receptor for an inflammation marker are present on an outer defining surface of the expandable part. In summary, it remains a problem to make available a simple, rapid, safe and reliable test for determining the occurrence of food allergy and food intolerance, and in particular a rapid method of screening patients exhibiting diffuse symptoms possible related to food allergy. Summary of the invention The above problems, among others, are solved by a method for use in the diagnosis of allergy or intolerance, according to which a patient is provoked rectally with a suspected allergen in an amount less than the amount which elicits a systemic reaction, after which provocation the concentration of NO is measured in a sample taken in or from the rectum. It was surprisingly shown by the present inventors, that the inventive method makes it possible to obtain an indication of allergy, and in particular food allergy, rapidly, reliably and with no or only minimal discomfort to the patient. Further embodiments and advantages of the present invention will be evident from the description and examples. Short description of the figures The invention will be illustrated in closer detail below, in the description, examples, claims, and in the attached figures, in which Fig. 1 shows the rectal NO concentration in seven patients following oral provocation with different allergens, and Fig. 2 shows the increase in rectal NO concentration in 18 patients, after rectal gluten provocation. Description The present inventors have surprisingly shown that the measurement of NO in the rectum or in a gas sample taken from the rectum, and in particular following provocation with the suspected allergen, is a useful tool in the diagnosis of allergy such as food allergy. It is of great importance to be able to rapidly and objectively screen patients in order to identify the individuals exhibiting a gastrointestinal allergic reaction. Regardless of the prevalence of food allergies and the notorious difficulties associated with establishing a correct diagnosis, very little advance had hitherto been made in this field. In summary, the method according to the present invention comprises the steps of obtaining a base line NO value for a patient, provoking the patient with the suspected allergen and obtaining a post-provocation NO value, which reflects how the patient reacts to the suspected allergen. The NO concentration can be measured directly in the rectum, using suitable sensors, adapted for rectal introduction, or measured in a sample, taken from the rectum. Rectal sampling results in NO values which measure both rectal and colonic reactions in case gas is allowed to freely spread in the intestinal canal. The sample can be taken through a catheter placed in the rectum and equipped with syringes for collecting the gas, or directly, for instance by aspirating rectal gas into a syringe. A sample is preferably taken by inserting an inflatable balloon, which is impermable to liquids but permeable to gases, and preferably selectively permeable to NO. When the balloon is filled with NO-free gas or with gas of a known composition, NO equilibrates over the membrane and the NO concentration in the space delimited by the membrane can be determined. The NO content may be determined/calculated as a concentration value, absolute amount or relative some internal or external standard. The content may be expressed as values normalised against components that natively are present in intestinal gas together with NO, preferably in fairly constant levels (internal standards). The NO content may also be calculated as amount secreted per time unit when taking air flow into account. The sample or samples can be taken in a clinic, hospital or home environment, either by medically trained personnel or by the patient him/herself. Samples can be extracted, treated, stored and/or transported for later measurements at a different location, provided that the integrity of the sample is guaranteed or that the chemical changes taking place in the sample are known. Methods for the determination of NO in gas samples are well known in the field. When low NO-levels are concerned, NO gas analysers based on chemiluminescent detection are preferred at the priority date. See for instance Archer, FASEB J., 7 (1993) 349-60 and Alving et al, WO 9502181. One commercially available NO analyser is the MBA" β or NIOX"' apparatus (Aerocrine AB, Solna, Sweden). The NO concentration in a sample can also be determined using laser or IR based analysers or electrochemical sensors. According to one embodiment, the method of the present invention comprises the following steps: a) a device for measuring or monitoring NO is inserted into the rectum of a patient; b) the NO-concentration in the rectum is determined; c) the patient is provoked with the suspected allergen by introducing the suspected allergen in the rectum of the patient; d) after a predetermined incubation time, the NO concentration is determined and compared to the base line value determined in a) - b), and e) an elevated value is taken as an indication of food allergy, triggered by the suspected allergen of step c). According to another embodiment, the method of the present invention comprises the following steps: a) a catheter having an inflatable, NO-permeable portion is inserted into the rectum of a patient; b) the inflatable portion of the catheter is inflated with NO-free gas, e.g. NO-free air, preferably using a syringe; c) the inflated catheter is kept in place for an amount of time allowing an equilibrium between the NO-concentration in the rectum and in the inflated portion of the catheter to be reached; d) a gas sample is extracted from the catheter, e.g. using a syringe; e) the NO-concentration in the gas sample is determined; f) the patient is provoked with the suspected allergen by introducing the suspected allergen in the rectum of the patient; g) after an incubation time the steps a) through e) are repeated and the later measured NO- concentration is compared to the base line value determined in a) - e), and h) an elevated value is taken as an indication of food allergy, triggered by the suspected allergen of step f). Alternatively, the method starts by the introduction of the allergen and the value obtained after provocation is compared to compiled values representative for healthy controls; and an elevated value, compared to healthy controls, is taken as an indication of food allergy. Likewise, an elevated value, compared to previously measured values for the same patient, is taken as an indication of a worsened condition and a decreased value as an indication of an improved condition, all other circumstances being the same. According to one embodiment of the invention, the patient is given a suppository containing the suspected allergen or intolerance causing agent, and instructed to insert the suppository a predetermined time before the scheduled time for measurement. For example, the patient is scheduled for a NO measurement in the morning, and is instructed to insert the suppository the previous evening. Alternatively, the patient is given a device for measuring and recording NO, and instructed to insert both the device and the allergen containing suppository at predetermined times, respectively. Based on the result of the above determination and the anamnesis, established in co-operation with the patient or, in the case of small children, a parent or other person, responsible for the patient, a diagnosis can be made. The diagnosis can be further confirmed by ordering the patient to follow a diet, eliminating the suspected allergen. If the suspected allergen has been effectively eliminated from the patient's diet, the NO-levels obtained at a later date should be lover than the levels initially measured and also lower than the levels measured after provocation. A repeated NO-measurement can thus help to confirm or contradict the diagnosis. As it is not uncommon for patients to outgrow their allergies, there exists a need for later controls as to weather a previously diagnosed allergy still exists. The present invention offers an easy and repeatable method for checking or confirming, whether an allergy still exists. Further, the inventive method is useful for testing patient compliance. A patient following a diet, eliminating the source of allergen, should exhibit low or normal NO concentrations in intestinal gas samples. High NO concentrations during ongoing elimination diet indicate either non-compliance from the side of the patient or that one or more further, unidentified allergen or allergens are present. The provocation of step f) is preferably done with an isolated allergen or any known allergen or substance, suspected for causing an allergenic reaction in the patient to be examined. Importantly, said substance is administered to the patient in an amount, sufficient to trigger a response in the intestinal mucosa but not so large, that a systemic response is achieved. This amount can be empirically determined for each allergen, groups of allergens and with respect to different patient groups, e.g. different age groups. The allergen is preferably administered as liquid, semi-liquid or solid composition comprising physiologically acceptable carriers and adjuvants chosen among carriers and adjuvants, normally used in pharmaceutical preparations. The allergen is most preferably administered in the form of a composition, suitable for rectal administration. Examples of suitable preparations include solutions, gels, pastes, suppositories and foams. Preferably the allergen is administered in the form of a suppository or a foam, inserted in the rectum. The allergen is preferably complex-bound, or physically and/or chemically encapsulated in order to limit or entirely prevent its uptake by the intestinal mucous membrane. Further, the sensitivity of the determination can be increased by adding L-arginine to the preparation or by administering the substrate L-arginine perorally, intravenously, or locally e.g. via a colonoscope or a catheter to enable the detection of low grade reactions. According to a preferred embodiment, the suspected intolerance causing substance is comprised in a suppository, further comprising L-arginine and suitable carriers and adjuvants, e.g. microcrystalline cellulose. According to another preferred embodiment, the suspected intolerance causing substance is comprised in a foam or foam forming composition, further comprising L-arginine. The present invention further encompasses the use of rectal NO measurements, with or without provocation, as a method for use in screening patients exhibiting allergy related symptoms. This is based on the preliminary finding, that elevated NO values in rectally sampled air - without prior provocation - reflects systemic inflammation and - with prior provocation - local and systemic reaction to the specific agent used in the provocation. For example in very young patients with suspected atopic symptoms during the first year, a rectal NO measurement could give an indication as to the degree, character and severity of the allergy. Preliminary results also indicate, that rectal NO can be used as a predictory instrument in the examination of young patients with symptoms of allergy. High NO levels as compared to healthy individuals would indicate a disposition for the development of allergies. One embodiment is thus a method for use in the screening of allergic patients, exhibiting gastrointestinal problems, and where non-allergy related diseases are ruled out or held to be unlikely, according to which method the NO-concentration is measured in a gas sample taken from the rectum of a patient and an elevated value, compared to values obtained for healthy controls, is taken as an indication of allergy as an underlying cause of the gastrointestinal problems of said patient. Another embodiment of the invention is a method for use in the screening of patients, exhibiting allergy-like symptoms in the airways, according to which method the NO- concentration is measured in a gas sample taken from the rectum of a patient and an elevated value, compared to values obtained for healthy controls, is taken as an indication of allergy as an underlying cause of the airway symptoms. In both the above methods, part of the diagnosis is based on excluding other sources of NO in the intestines, e.g. inflammatory intestinal diseases. Depending on the overall clinical picture, the age and condition of the patient, the absence of other signs, indicating other diseases, supports the conclusion that an increased NO level is caused by allergy. A related application, also encompassed by the present invention, concerns the differentiation between cold-related asthma and true asthma. A patient and in particular a young patient exhibiting breathing difficulties similar to those encountered in asthmatic patients would, according to the invention, be subjected to a rectal NO measurement. An elevated value, compared to healthy individuals, would be an indication of an inflammatory reaction and thus an indication of true asthma or the predisposition for the development of asthma. Further, the rectal NO measurement can be used to monitor the effect of treatment, e.g. steroid treatment and/or patient compliance. The invention will now be illustrated by means of examples, which are intended to show embodiments of the invention, but not to limit the scope of the inventive concept as set forth in the description and claims. Examples Example 1. Rectal NO levels in patients with gastrointestinal symptoms The inventive method was been tested on a group of young patients of both sexes (age 0.5 to 9 years, n - 5) with manifest gastrointestinal problems. A gas sample was obtained by inserting the tip of a conventional urological catheter into the rectum, inflating the cuff with NO-free air and extracting said air after 10 minutes. The NO concentration in the gas sample was measured with a Medical Breath Analyser (Aerocrine AB, Stockholm, Sweden). In all but two patients, the NO concentration was significantly elevated. The two patients exhibiting low values in parity with the NO concentrations obtained for healthy controls, were patients on an elimination diet. Thus the inventive method is also useful for checking patient compliance. The results are summarised in table 1. Table 1. NO levels in rectal gas samples from patients with gastrointestinal symptoms Patient no. Age Sex Clinical picture NO- (y) concentration (ppb) 1 2 M Asthma, chronic gastrointestinal problems, 920 cow milk allergy, hen egg allergy 2 0.5 M Eczema, asthma, colitis 5000 3 9 F Atypic, diffuse gastrointestinal problems 1700 4 8 F Coeliac disease, on elimination diet 10 5 4 M Coeliac disease, on elimination diet 30 As a comparison it can be mentioned, that normal NO values in healthy controls (n = 1 1, age 5 - 14 years) were in the range of 72 ± 12 ppb (mean ± sem). Thus the results obtained indicate that NO in rectal gas samples correlates with clinical symptoms indicating food allergy. The low values observed in patients no. 4 and 5 indicate that the NO values return to normal when no allergen is present and gluten is avoided. Example 2. Rectal NO levels following oral provocation with allergen Seven allergic patients of both sexes, age 1 to 19 years, were admitted to the clinic in the morning and received the suspected allergen orally. Allergy against egg, milk or oats was tested by administering these allergens orally in increasing doses. NO was measured rectally, using a NO-permeable balloon, before provocation, two hours after provocation, and the following morning. The results are presented in Table 2 and Fig. 1. Table 2. Rectal No concentration (ppb) after oral allergen provocation Patient (allergen) Pre-provocation 2 hours post- Following day provocation (ppb) (PPb) (PPb) PI (egg) 75 105 90 P2 (milk) 303 303 212 P3 (oats) 445 399 P4 (egg) 450 450 445 P5 (egg) 113 58 825 P6 (milk) 360 255 690 P7 (milk) 580 1325 540 Among these patients, P7 reacted very strongly and was given steroid treatment. Another patient, P3 did not return the following day for the final measurement. The basal values range from 75 to 580 ppb, which probably is due to a more or less pronounced inflammatory state already before the provocation. The results show that allergic patients exhibit increased rectal NO values after oral provocation (patients P5, P6, and P7) and that the NO level is reduced by steroid treatment. During the tests, patients PI, P4, P5, P6 and P7 showed adverse reactions to the allergen (swollen tounge, rashes, nausea, vomiting etc.), which probably is a direct result of the provocation being oral and not rectal. Example 3. Rectal NO measurements after rectal gluten provocation The concentration of nitric oxide was determined in a gas sample taken from the rectum of patients subjected to local provocation with gluten. A group of patients with diagnosed coeliac disease (n = 18, age 24 - 75 years) and a group of healthy controls (n = 5, age 24 - 44 years) were subjected to a provocation and NO-measurement according to the inventive method. A soluble gluten extract (2 g Frazer's fraction dissolved in 10 ml saline) was introduced 15 cm into the rectum via a catheter. The gluten extract was kept in place 1 hour before starting the measurements. The concentration of NO in the rectal gas was measured by introducing NO-free air in the inflatable cuff of the catheter and waiting 10 minutes, allowing NO to penetrate into the cuff. The air was withdrawn from the cuff and the concentration of NO measured using a conventional NO analyser (MBA, Aerocrine AB, Danderyd, Sweden). The NO-concentration was determined at 0, 2, 4, 8 and 24 hours. The results of this study are presented in table 3. Notably none of the controls (N = 5) exhibited increased NO concentrations. Among the 18 patients with diagnosed coeliac disease, 1 1 exhibited more than a two-fold increase after 24 hours. The average increase, exhibited by the patients was 126 times the starting value. Surprisingly as many as 9 patients (50 %) exhibited an increase above 10 times the starting value. Table 3: Rectal gluten provocation study Subject Base line 24 h Change NO factor Patient 1 51 4783 94 Patient 2 310 137 neg Patient 3 601 14000 23 Patient 4 82 152 1.8 Patient 5 870 803 neg Patient 6 22 17 neg Patient 7 36 1600 44 Patient 8 76 14000 184 Patient 9 27 750 30 Patient 10 95 273 2.9 Patient 11 119 1022 8.6 Patient 12 43 23000 535 Patient 13 32 36 1.1 Patient 14 28 8050 288 Patient 15 20 17 neg Patient 16 129 21000 163 Patient 17 60 35 neg Patient 18 8 98 12.3 Average: 145 4987 34 Responders (Factor >2) n = = 11 ( > 60%) Responders (Factor >10) n = = 9 ( 50%) Control 1 230 160 neg Control 2 103 51 neg Control 3 200 44 neg Control 4 130 118 neg Control 5 76 70 neg As evident from Table 3 and as also shown in Figure 2, 50 % of patients reacted with significantly increased NO concentrations. Based on this, it is held that this method has clinical importance. The fact that none of the controls responded with increased NO values is also a strong indication that the inventive method could avoid false positive diagnosis. On the other hand, the fact that some of the patients with diagnosed coeliac disease showed a negative change at the 24 hour measurement can have several reasons. It is possible that the previously made diagnosis was incorrect or that the disease has receded thanks to other treatment, medication or dietary changes. It is also possible that these patients exhibited a peak NO value earlier or later than the 24 hour measurement, that their NO increase was only very short and temporary and escaped detection in the present study. It is also possible that the FF3 challenge was not optimal for eliciting the right reaction in these patients. It is for example possible, that the exposure time was too short for the specific reaction pattern in these patients. Also the dose and composition of the gluten extract used in the experiments could have been less than optimal for these patients and thus be a source of error. It is further possible that sub-groups exist among patients suffering from coeliac disease and that these subgroups exhibit different reaction patterns in a test according to the invention. In that case, the present method could make possible the differentiation between different patient groups, suffering from similar symptoms or within a group, diagnosed to suffer from coeliac disease. The present method could also function as a screening to distinguish between patients having symptoms, unrelated to gluten, and patients meriting a further examination. In general, the inventive method has proven to be a rapid test, easy to perform and well accepted by the patients. The test causes no or very slight discomfort to the patient, comparable to a rectal temperature measurement. In particular with small children, exhibiting diffuse symptoms, the method has proven to be superior to previously used in vitro tests or food challenge tests. The results obtained by the present inventors indicate, that an increase in rectal NO occurs only after a delay of several hours after provocation. The measurement of NO according to the invention has therefor an additional advantage in that it can be performed repeatedly or continuously over a long time period, as the method is painless and easy, causing no or very little discomfort to the patient. Example 4. Rectal NO measurements in children with atopic and non-atopic food allergy Children aged below 12 years were examined for nitric oxide in the rectum. A silicone urinary catheter was inserted into the rectum and inflated with 10 ml of air. The catheter was kept inflated inside the rectum for 10 min. and after that air was aspirated into a 10 ml syringe and analysed for NO by direct aspiration into the sampling catheter of the NIOX"' system (Aerocrine AB, Solna, Sweden). A plateau level of NO was read directly from the screen of the chemiluminiscens part of the system. Three groups of children were studied. 1.: A group of 18 children with an atopic food allergy where at least one major food was of importance. These children were known from clinical records to be RNST-positive and/or skin test positive to common foods and were on more or less broad elimination diets. 2.: N miscellaneous group of 17 children with less well defined food sensitivities, where one or several foods had been taken away in clinical practice due to reported symptoms indicative of hypersensitivity. 3.: N control group of 9 healthy children. Median values and ranges for rectal NO the three groups are given in table 4. Table 4. Median values and ranges for rectal NO Atopic group Non-atopic group Controls Median 244 97 57 Range 48-3200 17-3280 19-288 Non-parametric tests were used. NO was statistically higher in the atopic group as compared to the non-atopic group (p<0.05) and the control group (p=0.002). Current asthma and eczema, respectively were not associated with elevated rectal NO and neither were reported bowel symptoms (abdominal pain and loose stools). Children with atopic food hypersensitivity had significantly higher rectal NO values than healthy controls. Among cases with a less well defined food sensitivity, where foods had been eliminated but atopic allergy had not been demonstrated there was only a tendency to elevated rectal NO and occasional cases did exhibit levels which appeared to be considerable. The elevated values were found despite ongoing elimination of known offending foods and may indicate ongoing eosinophilic inflammation in the rectum of patients with an atopic disease. Although the invention has been described with regard to its preferred embodiments, which constitute the best mode presently known to the inventors, it should be understood that various changes and modifications as would be obvious to one having the ordinary skill in this art may be made without departing from the scope of the invention which is set forth in the claims appended hereto.

Claims: Claims 1. A method in the diagnosis of allergy or intolerance in a human patient, characterized in that the concentration of nitric oxide (NO) is determined in a gas sample, taken from the rectum of a patient, after provocation with a suspected allergen in an amount less than the amount required to cause a systemic reaction, and an increased concentration, compared to a normal concentration, obtained from healthy controls or from the same patient before provocation, is taken as an indication of allergy or intolerance. 2. A method in the diagnosis of allergy or intolerance in a human patient, characterized in that the concentration of nitric oxide (NO) is determined in the rectum of a patient, after provocation with a suspected allergen in an amount less than the amount required to cause a systemic reaction, and an increased concentration, compared to a normal concentration, obtained from healthy controls or from the same patient before provocation, is taken as an indication of allergy or intolerance. 3. A method in the diagnosis of allergy in a human patient comprising the following steps: a) the patient is provoked with a suspected allergen; b) the NO-concentration is measured in the rectum or in a gas sample taken from the rectum of the patient; c) the NO-concentration is compared to a base line value obtained earlier for the same patient, or to values, representative for healthy controls; and d) an elevated value is taken as an indication of allergy. 4. Method in the diagnosis of allergy in a human patient comprising the following steps: a) the NO-concentration is measured in the rectum or in a first gas sample taken from the rectum of the patient; b) the patient is provoked with a suspected allergen; c) whereafter the NO-concentration is measured in a second gas sample taken from the rectum of the patient; d) the later measured NO-concentration is compared to the base line value obtained in a) or to values, representative for healthy controls; and e) an elevated value is taken as an indication of allergy. 5. A method according to claim 3, characterized in that the provocation of step b) is performed by introducing the suspected allergen in the rectum of the patient. 6. A method for the confirmation of diagnosed allergy, characterized in that the NO- concentration is measured in a gas sample taken from the rectum of a patient after said patient has been on a diet, eliminating the suspected allergen, and a lower value than previously obtained values for the same patient is taken as a confirmation, that the eliminated allergen was the causative agent, and an unchanged or increased value is taken as an indication that the diagnosis was wrong or that further allergens are involved. 7. A method for use in the screening of allergic patients, exhibiting gastrointestinal problems, and where non-allergy related diseases are ruled out or held to be unlikely, characterized in that the NO-concentration is measured in a gas sample taken from the rectum of a patient and an elevated value, compared to values obtained for healthy controls, is taken as an indication of allergy as an underlying cause of the gastrointestinal problems of said patient. 8. A method for use in the screening of patients, exhibiting allergy-like symptoms in the airways, characterized in the NO-concentration is measured in a gas sample taken from the rectum of a patient and an elevated value, compared to values obtained for healthy controls, is taken as an indication of allergy as an underlying cause of the symptoms. 9. A method for the prediction of the development of atopic allergy in young patients, characterized in that the NO-concentration is measured in a gas sample taken from the rectum of a patient exhibiting allergic reactions, and an elevated value, compared to values obtained for healthy controls, is taken as an indication of a predisposition for the later development of atopic allergy. 10. A method for use in the diagnosis of food intolerance in a human patient, characterized in that the level of nitric oxide is measured in the rectum or in a sample taken from the intestinal lumen before, and after, subjecting said patient to a provocation, using a suspected intolerance causing substance, and an elevated value obtained after the provocation, is taken as an indication of an intolerance reaction towards the suspected intolerance causing substance. 11. A method according to claim 10, characterized in that the patient is provoked with the suspected intolerance causing substance by administering said substance rectally to said patient. 12. A method according to claim 10, characterized in that L-arginine is administered to the patient perorally, intravenously, or locally before or simultaneously with the administration of the suspected intolerance causing substance. 13. A method according to claim 1 1 , characterized in that the suspected intolerance causing substance is administered as a gel, paste, solution, foam, spray or suppository. 14. A method according to claim 1 1 , characterized in that the suspected intolerance causing substance is administered as a gel, paste, solution, foam, spray or suppository comprising L-arginine. 15. A method for use in the diagnosis of coeliac disease, characterized in that the level of NO in the rectum or in a gas sample taken from the rectum of a patient is determined after administering a gluten-containing composition rectally to a patient, and an increased NO level, in comparison to a value typical for healthy individuals or a value, obtained for said patient before the administration of said gluten fraction, is taken as an indication of coeliac disease. 16. A method according to claim 15, characterized in that the gluten-containing composition is Frazer's fraction III (FF3). 17. A method according to claim 15, characterized in that L-arginine is administered to the patient perorally, intravenously, or locally before or simultaneously with the administration of the gluten-containing composition. 18. Composition for use in the method according to claim 10, characterized in that said composition comprises a suspected intolerance causing substance and L-arginine. 19. Composition for use in the method according to claim 15, characterized in that said composition comprises gluten and L-arginine.

Inventors: ALVING KJELL, LUNDBERG JON, NORDVALL LENNART, WEITZBERG EDWARD, ALVING, KJELL, LUNDBERG, JON, NORDVALL, LENNART, WEITZBERG, EDWARD
Applicants: AEROCRINE AB, ALVING KJELL, LUNDBERG JON, NORDVALL LENNART, WEITZBERG EDWARD, ALVING, KJELL, LUNDBERG, JON, NORDVALL, LENNART, WEITZBERG, EDWARD
Application year: 2000
Main classification: G01N33
IPC codes: A61B5, G01N33