Effect of Cosmos caudatus (Ulam raja) supplementation in patients with type 2 diabetes: Study protocol for a randomized controlled trial

Shi-Hui Cheng
Amin Ismail
Joseph Anthony
Ooi Chuan Ng
Azizah Abdul Hamid
Barakatun-Nisak Mohd
Yusof
Effect of Cosmos caudatus (Ulam raja) supplementation in patients with type 2 diabetes: Study protocol for a randomized controlled trial
10.1186/s12906-016-1047-7S T U D Y P R O T O C O L Open Access
Background: Type 2 diabetes mellitus is a major health threat worldwide. Cosmos caudatus is one of the medicinal plants used to treat type 2 diabetes. Therefore, this study aims to determine the effectiveness and safety of C. caudatus in patients with type 2 diabetes. Metabolomic approach will be carried out to compare the metabolite profiles between C. Caudatus treated diabetic patients and diabetic controls. Methods and design: This is a single-center, randomized, controlled, two-arm parallel design clinical trial that will be carried out in a tertiary hospital in Malaysia. In this study, 100 patients diagnosed with type 2 diabetes will be enrolled. Diabetic patients who meet the eligibility criteria will be randomly allocated to two groups, which are diabetic C. caudatus treated(U) group and diabetic control (C) group. Primary and secondary outcomes will be measured at baseline, 4, 8, and 12 weeks. The serum and urine metabolome of both groups will be examined using proton NMR spectroscopy. Discussion: The study will be the first randomized controlled trial to assess whether C. caudatus can confer beneficial effect in patients with type 2 diabetes. The results of this trial will provide clinical evidence on the effectiveness and safety of C. caudatus in patients with type 2 diabetes. Trial registration: ClinicalTrials.gov identifier: NCT02322268

Background

Type 2 diabetes mellitus is one of the biggest health concerns in the world. In 2014, about 387 millions of people suffer from type 2 diabetes worldwide. The number is projected to rise to 592 million people by 2035 [1]. The prevalence of type 2 diabetes in Malaysia showed the same worrying trend. One in every five Malaysians age over 30 has type 2 diabetes [2]. Despite the drug prescription, a majority (78 %) of patients with type 2 diabetes in Malaysia still have poor glycemic control with mean HbA1C of 8.7 % [3]. Poor glycemic control is associated with an increased risk of diabetic microvascular complications including neuropathy, nephropathy and retinopathy, and macrovascular complications such as coronary heart disease [4].

Currently, the most common treatment for type 2 diabetes includes the prescription of oral anti-diabetic drugs such as metformin and sulphonylureas [5]. While the efficacy of sulphonylureas has been confirmed, their use is associated with side effects such as increased weight gain and elevated risk of hypoglycemia [6]. In addition, researchers have shown that long-term treatment with oral anti-diabetic drugs is ineffective in protecting the declining function of the pancreatic beta-cell [7,8]. The deterioration of pancreatic beta-cell function is associated with the elevated oxidative stress in type 2 diabetic patients [9,10]. Research indicated that oxidative damage was higher in patients with type 2 diabetes than healthy individuals [11]. Patients with type 2 diabetes had lower antioxidant capacity than healthy individuals [12]. Antioxidant potentially reduces the harmful effect of oxidative stress and consequently decreases the insulin resistance [13].

Medicinal plants have been used as traditional medicine for treating diseases, and their uses are considered safer than the oral anti-diabetic drugs [14]. The discovery of metformin, from the Galega officinalis has augmented the interest to quest for more natural products to treat diabetes [15]. Cosmos caudatus, or known locally as Ulam Raja, is a medicinal herb found in tropical countries [16]. It has been identified as one of the ten commonly used medicinal plants in Malaysia for treatment of type 2 diabetes [17]. C. caudatus contained a variety of bioactive compounds, such as ascorbic acid, quercetin, proanthocyanidin, chlorogenic acid and catechin [18][19][20][21]. C. caudatus has been reported to have an extremely high antioxidant capacity of about 2500 mg ascorbic acid equivalent antioxidant capacity (AEAC) per 100 g of fresh samples as compared to orange and guava which had 142 mg and 270 mg AEAC, respectively [22]. Its high antioxidant content may suggest its potential in reducing the oxidative stress [20].

In addition, C. caudatus have been shown to possess various medicinal benefits [16] including anti-diabetic [23], antihypertensive [24] and anti-inflammatory effect [25] in animal studies. Recent pre-clinical study in rats indicated a significantly reduction in fasting blood glucose, and a significant improvement in lipid profile after 4 weeks of supplementation [23]. Preliminary study in healthy volunteers has shown that there was a steady reduction in the postprandial blood glucose after consuming rice and fresh C. caudatus (unpublished data). However, the effect of C. caudatus in patients with type 2 diabetes remains unclear.

Metabolomics is an unbiased approach to measure metabolites disturbance within a biological system at a given time [26]. The profiling of these metabolite can provide detailed information on the metabolic pathways [27]. Additionally, metabolites levels are influenced by environmental factors such as diet and lifestyle factors [28]. Therefore, by using metabolomics approach, it provides a clearer understanding of the effect of C. caudatus in patients with type 2 diabetes.

The objective of this trial is to investigate the effectiveness and safety of C. caudatus in patients with type 2 diabetes by addressing the following hypothesis (1) C. caudatus supplementation will improve the glycemic status in patients with type 2 diabetes (2) C. caudatus supplementation will improve inflammation marker, oxidative stress marker and lipid profile in patients with type 2 diabetes (3) C. caudatus supplementation is safe and will not result in adverse effects (4) the therapeutic effect of C. caudatus will result in changes in urine and serum metabolome.

Methods and design

Trial design This is a single center, randomized, parallel-controlled clinical trial. A flow chart of the study protocol is shown in Fig. 1. This study will be conducted at a tertiary government hospital, Malaysia.

Ethical approval

The study protocol has been approved by the Ethics

Randomization

Patients with type 2 diabetes will be randomly allocated to either C. caudatus (U) group or control (C) group. The permuted block randomization (block of 4 and 6) will be used in this study. However, researchers will be blinded to the size of each block to reduce the selection bias. All laboratory personnel will also blinded to the group allocation.

Sample size

A total of 50 patients are required in each group to detect a significant mean difference of 1 % HbA1C [29], 1.75 % standard deviation [29], 95 % confidence level and 80 % power, with additional drop-out rate of 30 %.

Study population

Patients with type 2 diabetes will be recruited from the outpatient medical clinic at tertiary hospital. The diagnosis of diabetes will be based on the medical record of the tertiary hospital. Eligible diabetic patients will be enrolled in the study and written informed consent will be obtained prior to recruitment at the medical clinic of the tertiary hospital.

Inclusion criteria

Men and women between the age of 30-65 years old who have confirmed diagnosis of type 2 diabetes for more than 6 months, last HbA1C value greater than 7 %, Body Mass Index (BMI) between 18.5 and 40 kg/m 2 , treated with stabilized dose of oral anti-diabetic drugs and expected to

Exclusion criteria

Patients with any of the following criteria will be excluded: 1) pregnant and lactating; 2) have any gastrointestinal disorder that interferes the bowel function, severe hepatic or renal disease (dialysis), an infection that requires antibiotics within three weeks; 3) on insulin regimen; 4) Individuals on the anticoagulant drugs such as warfarin and aspirin.

Intervention

Diabetic C. caudatus treated (U) group

The C. caudatus seeds were obtained from the Institute of Bioscience, Universiti Putra Malaysia (UPM) and planted at the Universiti Putra Malaysia Agricultural Park. A voucher specimen (No. SK 2400/14) has been deposited in the herbarium, Biodiversity Unit, Institute of Bioscience, Universiti Putra Malaysia. Fresh C. caudatus will be provided to the C. caudatus treated (U) group twice a week for 8 weeks. The C. caudatus will be vacuum packed in a zip-lock bag before distribution. Patients in C. caudatus treated (U) group will be asked to consume raw C. caudatus leaves (15 g) daily together with one of the main meal (either lunch or dinner) for 8 weeks.

C. caudatus supplementation will be given simultaneously with the usual diabetes care that includes maintenance of the oral anti-diabetic drugs dosage and compliance with the dietary and physical activity recommendations. In addition, patients in C. caudatus treated (U) group will be advised to perform self-monitoring blood glucose. Glucometer and strips will be given to the patients to record three values daily (after fasting, before a meal and two hours after a meal). All the patients will be educated on how to use the glucometer.

Diabetic control (C) group

The diabetic control (C) group will be asked to abstain from consuming C. caudatus for 8 weeks . Both groups will be given standard lifestyle interventions which include medical nutrition therapy and physical activity enforcement). In addition, both groups will be educated for the concepts of carbohydrate exchange and serving sizes. Patients in diabetic control (C) group will be encouraged to eat the same serving of fruits and vegetable as the C. caudatus treated (U) group. Likewise, patients in diabetic control (C) group will be advised to perform selfmonitoring blood glucose. Glucometer and strips will be given to both groups to record three values daily (after fasting, before a meal and two hours after a meal). All the patients will be educated on how to use the glucometer.

Adherence

To check patients' compliance during the 8 weeks, the researcher will interview them weekly by telephone. In C. caudatus treated (U) group, patients will be interviewed to discern whether they were consuming C. caudatus and to address all their concerns. In diabetic control (C) group, phone calls are made weekly to emphasize the importance of lifestyle modification.

Patient safety

Patients will be monitored weekly during the study period and all the occurrence of the adverse events will be assessed by patient interviews. All anticipated adverse events such as loose stools, abdominal discomfort, bloating, flatulence, sign of hypoglycemia, sign of hyperglycemia will be recorded. Additionally, liver profile and renal profile will be measured at every visit.

Study outcomes Primary outcome

The primary outcome of this trial is the changes in HbA1C.

Secondary outcomes

Secondary outcomes in this study include changes in other glycemic parameters such as fasting glucose, insulin, fructosamine, changes in lipid profile (triglyceride, total cholesterol, HDL-cholesterol, LDL-cholesterol), changes in inflammation (high-sensitivity C-reactive protein) and oxidative stress marker (plasma malondialdehyde), changes in anthropometry parameters (BMI, waist circumference), changes in metabolite profile (urine, serum), changes in safety parameters (liver profile and renal profile), changes in blood pressure, dietary pattern and physical activity.

Procedure

After obtaining the informed consent, both groups will be required to attend clinical research center on 4 occasions-baseline, week 4, week 8 (end of study) and week 12 (post study follow-up). Subjects will be interviewed regarding their socio-demographic background at baseline. At each follow-up, all subjects will undergo a physical examination which include anthropometric, blood pressure, blood test, urine test and completed a dietary history questionnaire and international physical activity questionnaire (IPAQ).

Anthropometry measurements including weight, height and waist circumference will be measured. Body height will be measured using SECA stadiometer model 217 (SECA, Hamburg, Germany) to the nearest 0.1 cm. Body weight will be measured using a SECA electronic scale model 703 to the nearest 0.1 kg (SECA, Hamburg, Germany). Waist circumference in cm will be assessed midway between the lowest rib and the iliac crest using a non-stretchable tape measure. BMI will be calculated using World Health Organization classification [30]. Blood pressure will be measured using an automatic blood pressure monitor (Omron, Kyoto, Japan).

Blood sample will be drawn after an overnight fasting from the antecubital vein with patients in seated position. The volume of blood taken will be 10 ml, collected in four different pre-chilled tubes. A plain evacuated blood collecting tube will be used to collect 4 ml blood for metabolomic analysis. One milliliter will be taken in potassium oxalate/sodium fluoride tubes for fasting glucose. Another 1 ml blood sample will be taken in tubes containing ethylenediaminetetraacetic acid (EDTA) for HbA1C. The remainder 4 ml blood will be collected in the heparinized tube for other biochemistry parameters. Blood will be drawn by trained nurses and immediately placed on ice in the dim light environment. Blood sample that are collected will be centrifuged at 3000 rpm for 10 min at 4°C to obtain the serum and plasma. 15 ml urine samples will be collected in the polyethylene containers. Blood and urine sample that are collected will be stored at −80°C until further analysis.

Biochemical assessments

All assays will be performed using Architect ci8200 analyzer (Abbott Laboratories, USA) except glycated hemoglobin (HbA1C) and malondialdehyde. Fasting blood glucose will be performed with a hexokinasebased method (Abbott Laboratories, USA). HbA1C will be measured with turbidimetric inhibition immunoassay method (Roche Diagnostics, Germany) using Cobas Integra 800 (Roche Diagnostics, Germany). Serum total cholesterol, triglycerides, high-density lipoprotein (HDL) cholesterol, and low-density lipoprotein (LDL) cholesterol levels will be quantified by commercially available enzymatic assay kits (Abbott Diagnostics, USA). High sensitivity C-reactive protein will be determined using the quantitative immunoturbidimetric method. For renal function test, sodium, potassium and chloride will be determined by potentiometric using solid state ionselective electrode, urea will be determined by enzymatic method and creatinine will be determined using the kinetic method. For liver function test, total protein will be determined using biuret endpoint, albumin will be determined by direct colorimetric determination, total bilirubin will be determined by diazonium salt method whereas alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase will be determined by the kinetic method. Malondialdehyde will be measured by thiobarbituric acid reactive substances assay using UV spectrophotometer (UV-1601, Shimadzu, Japan) [31]. All biochemical assays will be performed by B. P. Clinical Lab.

Dietary intake assessment

Dietary intake will be assessed with a dietary history questionnaire. Patients will be requested to provide complete food descriptions including food and drinks (brand names), food preparation (ingredients) and cooking method (such as deep-frying, stir-frying, boiling, grilling, barbecue, roasting, and steaming) as detail as possible in a week. Pictures of food commonly consumed in Malaysia, together with a set of common household measurement tools (glass, cup, Chinese rice bowls, soup bowls, plates, teaspoon and tablespoon) will be provided to assist subjects in estimating the portion sizes of the food.

The nutrient analysis will be carried out using Nutritionist Pro ™ diet analysis software (version 2.5, Axxya Systems, USA). This software contains several databases including Nutrient composition of Malaysian foods and the United States Department of Agriculture (USDA). For food items unavailable in Nutritionist Pro diet analysis software, database will be obtained from Singapore Food Composition Guide (Health Promotion Board, 2003) and all the nutrients for food items will be manually added to Nutritionist Pro diet analysis software.

Physical activity levels assessment

International Physical Activity Questionnaire (IPAQ) will be administered to assess the physical activity level of the subjects. The items in the IPAQ form consist of scores on walking, moderate-intensity and vigorousintensity activity. The IPAQ's total score for physical activity level will be expressed as metabolic equivalents per minute (MET-min) per week, sums the duration (in minutes) and frequency of walking, moderate-intensity and vigorous-intensity activities. Levels of physical activity will be category into low, moderate and high as per IPAQ criteria [32].

Statistical analysis

All clinical data will be performed using SPSS version 21 for windows (SPSS Inc, Chicago, USA). Data will be expressed as mean ± SD for continuous parameters, and percentage for categorical parameters. Normality tests assessed through Shapiro-Wilk tests will be carried out on each parameter before analysis. For baseline characteristic, independent t-test will be used to compare the difference in the study outcomes between the diabetic treatment and control group. All outcome measurements will be evaluated based on intention-to-treat analysis using all randomized patients who have attended the blood test at baseline. Missing data will be imputed using "last observation carried forward" method. The comparison within each group will be carried out using paired t-test, while the changes between the two groups will be conducted using independent t-test. A p-value of less than 0.05 will be considered as significant. Data will be reported with adherence to the CONSORT 2010 guidelines [33].

NMR Measurements

Thawed 400 μL urine samples will be mixed with 200 μL of a 0.75 M phosphate buffer solution (containing 0.5 % sodium trimethylsilylpropionate-d4 (TSP)) prepared in D 2 O and transferred to a 5 mm NMR tube. Thawed 200 μL blood serum samples will be mixed with 400 μL D 2 O and transferred to a 5 mm NMR tube. All 1 H NMR spectra will be collected on a Varian 500 MHz NMR spectrometer (Agilent technologies, Santa Clara, United States) equipped with a 5 mm PFG One NMR probe. One dimensional(1D) NOESY-Presaturation pulse sequence for urine and 1D Carr-Purcell Meiboom-Gill (cpmg) experiment for blood serum will be used. Each free induction decay (FID) will be collected using a total of 128 scans with a relaxation delay of 2 s.

Multivariate data analysis for metabolomics study

All 1 H NMR spectra will be manually phased, baselinecorrected and reference to TSP (0.0 ppm) using ACD software (ACD/Labs, Ontario, Canada). Region containing water (4.5-5.2 ppm) and TSP (0.0-0.2 ppm) will be excluded. Each 1 H NMR spectra will be segmented in bins of 0.04 ppm. Urine spectra will be normalized based on creatinine peak and serum spectra will be normalized based on the glucose peak area. All binned data will be imported into SIMCA (Version 14.0, Umetrics, Umea, Sweden). The binned data will be pareto-scaled prior to multivariate statistical analysis. Multivariate data analysis including unsupervised principal component analysis (PCA), and supervised partial least square discriminant analysis (PLS-DA) will be performed. PCA will be used to obtain a general overview of the metabolites and to detect outliers. PLS-DA will be performed to separate different groups. The quality of the model will be described by the parameter of R 2 and Q 2 which represent model fitness and predictive ability. All PLS-DA models will be validated using the permutation test. An overall p-value of 0.05 based on the Bonferroni correction will be used.

Discussion

Type 2 diabetes is a common chronic disease worldwide. Medicinal plants are gaining tremendous resurgence of interest among researchers recently because of their health benefits [14,34]. C. caudatus is widely consumed among the local Malays in South East Asia and its potential medicinal benefits have been reported [16]. The strength of this trial is that, it is the first randomized controlled trial to determine the effectiveness of C. caudatus on the outcomes measurement such as glycemic status, lipid profile, oxidative stress marker, and inflammation marker which are important parameters in patients with type 2 diabetes.

To the best of knowledge, there is no established data on herb-drug interaction of C. caudatus. Therefore, an interim analysis will be carried out to monitor the safety of consuming C. caudatus throughout the study. The results of this trial will provide clinical evidence on the effectiveness and safety of C. caudatus supplementation in patients with type 2 diabetes. Moreover, metabolomics is a powerful tool to study the altered metabolism, identify short-term changes in biological fluids and serve as biomarker detection [26]. Metabolomics approach used in this study will fill in the gap and provide a better understanding of metabolite profile following the supplementation of C. caudatus. Therefore, results from this study will contribute to the knowledge on the therapeutic effect mechanism of C. caudatus.

To date, the most commonly used metabolomics technologies for metabolomics study are nuclear magnetic resonance (NMR) spectroscopy, and mass spectrometry (MS) such as liquid chromatography-MS (LC-MS) and gas chromatography-MS (GC-MS) [26]. GC-MS and LC-MS are robust technologies that offer high sensitivity in metabolites detection [26]. However, both of the techniques have some drawbacks including relatively slower acquisition time, requires sample derivatization (GC-MS), consumption of sample (both LC-MS and GC-MS), chromatography can drift during a sample run, which makes data processing difficult (LC-MS), and peak assignment is a major challenge (LCMS) [35,36]. On the other hand, NMR offers some advantages such as its ability to provide high-resolution spectra, requires relatively simpler sample preparation hence more rapid analysis and more importantly non-destructive of the sample. However, NMRbased metabolomics has a lower sensitivity compared to MS [35][36][37].

We used NMR in this study as NMR-based metabolomics can detect many different metabolites simultaneously such as carbohydrate, amino acids, organic and fatty acids, amines and lipids. In addition, NMR-based metabolomic have shown high reproducibility and identification of the metabolites is relatively easy due to a good library of spectra [36]. The most prominent metabolites in the 1 H NMR blood serum spectra are fat (methylene-and methyl-moieties from the lipoproteins), amino acids, lactate and glucose [38]. Meanwhile, creatinine, creatine, trimethylamide N-oxide, dimethylamide, citrate and lactate are the most significant metabolites found in the 1 H NMR urinary spectra [39]. Recently, studies have revealed that there is a change in the amino acid metabolism in addition to the changes in glucose and fatty acid metabolism in patients with type 2 diabetes . Raised branched-chain amino acids including leucine, valine, isoleucine and glycerol were associated with the insulin assistance [40]. In the current study, NMR-based metabolomics is used to investigate the metabolic perturbation due to C. caudatus supplementation in patients with type 2 diabetes. To complement the current study, future studies involve MSbased metabolomics would provide further insight into the fatty-acid profiling in patients with type 2 diabetes.

However, there are limitations in this trial. Placebo is not used in this study. The reason is because there is no identical vegetable that can mimic the taste of C. caudatus. In addition,, there is also no blinding among the patients because they would know once they allocated in the treatment arm as they will receive the supplementation of C. caudatus. Therefore, the possibility of bias cannot be ruled out. In conclusion, findings from this study will provide significant evidence of using C. caudatus as an adjuvant therapy in patients with type 2 diabetes.

Committee for Research involving Human Subjects Universiti Putra Malaysia (JKEUPM) (FPSK_Ogos (13)05), Herbal Medicine Research Centre, Institute for Medical Research Malaysia (version 1, 8/2014) and Medical Research and Ethics Committee Ministry of Health Malaysia (NMRR-13-1344-18177). This protocol will be conducted in compliance with Helsinki declaration.

Fig. 1
1Trial flow chart remain on this oral anti-diabetic drugs throughout the duration of the study.
AcknowledgementsThis trial is supported by grants from Agro-Biotechnology Institute under Ministry of Science, Technology, and Innovation Malaysia (No. 6370700).Competing interestsThe authors declare that there is no conflict of interest.Authors' contributions SHC contributed to the study design, acquisition of data, prepared and revised the draft of this manuscript. BNMY participated in the study design and reviewed the manuscript. JA and OCN participated in study design, acquisition of data and reviewed the manuscript. AI and AAH participated in study design and reviewed the manuscript. All authors read, approved the final manuscript and agreed for all aspects of the work.• We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal Submit your next manuscript to BioMed Central and we will help you at every step:
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Exhaled nitric oxide decreases after positive food- allergen challenge

Avigael H Benhamou
Alice Koehli
Isabelle Rochat
Alexander Moeller
Philip Taramarcaz
Roger P Lauener
Philippe A Eigenmann
Exhaled nitric oxide decreases after positive food- allergen challenge
R E S E A R C H Open Access
Background: Exhaled nitric oxide (FeNO) is a well described marker of airway inflammation in asthma and is also known to increase after chronic exposure to inhaled allergens. It is not known whether monitoring FeNO could be useful during food challenges to detect early or subclinical reactions.Methods: Forty children aged 3 to 16 years undergoing an allergen-food challenge at two centres were prospectively recruited for this study. FeNO was assessed before and repeatedly after the food-challenge. Results: Data were obtained from a total of 53 challenges (16 positive, 37 negative) and were compared between the two groups. Half of the patients with a positive food challenge exhibited clinical upper respiratory symptoms. The FeNO significantly decreased in 7 of 16 patients with a positive challenge test within 60 to 90 minutes after the first symptoms of an allergic reaction. Conclusion: Our results show a significant decrease in FeNO after a positive food challenge suggesting involvement of the lower airways despite absence of clinical and functional changes of lower airways. Prospective blinded studies are needed to confirm these results. Figure 4 Individual changes in FeNO values measured throughout the test in patients with a negative food-antigen challenge.

Introduction

The current prevalence of food allergy in childhood varies between 6-8% during the first 3 years of life [1]. Food challenges are the gold standard for the diagnosis, in particular when the patient's history and specific IgE test results do not correlate, as well as for follow-up assessment. Among other potential symptoms, lower respiratory symptoms can be elicited by a positive challenge; they constitute a major risk factor for severe lifethreatening anaphylaxis. It has also been observed that up to 40% of children and adolescents with food allergy but without asthma have concomitant asymptomatic bronchial hyperreactivity (BHR) to methacholine, in general without lung function changes [2,3].

There is in agreement with the observation that fractional exhaled nitric oxide (FeNO) reflects bronchial allergic inflammation, and the measurement of FeNO has been proposed as a diagnostic tool for asthma [4], both in adults and children [5]. In addition, FeNO is related to the degree of bronchial inflammation in asthma and provides a non-invasive measure to monitor the anti-inflammatory treatment of asthmatic patients [6]. However it is unknown, whether FeNO changes occur early during food challenges in allergic children and if monitoring FeNO could serve to detect positive responses during such an allergen challenge.

It has been shown earlier that FeNO increases after specific bronchial allergen provocation in adults [7], whereas no changes in FeNO have been reported after a nasal allergen challenge in children [8]. More recently, no change in FeNO was found during a milk food challenge in infants [9].

We hypothesised, that a food-induced, IgE-mediated allergic reaction might provoke a modification in FeNO during or early after food challenges and hence, FeNO may serve as an early objective marker of a positive reaction involving the respiratory tract.

Materials and methods

Patients were recruited from two separate paediatric allergy centres in a prospective study with the primary aim of measuring FeNO changes in children undergoing food challenges. All data were treated confidentially and available only to the investigating physicians. The study was reviewed and approved by the Ethics Committee of the Department of Paediatrics of the participating institutions and was subjected to patient and/or parental written consent.

Patients

Children were recruited at the Paediatric Allergy Clinics at the Geneva University Hospital and at the University Children's Hospital Zurich, Switzerland between May 2007 and March 2009. Inclusion criteria were children between 3 to 16 years of age, with or without a history of asthma, undergoing a diagnostic or follow-up oral food-allergen challenge. Exclusion criteria were any asthma treatment current or within the previous four weeks (oral or inhaled steroids, inhaled bronchodilators and/or leukotriene receptor antagonists), or a history of an acute respiratory tract infection in the previous four weeks. Sensitisation to common aeroallergens (grass and tree pollen, animal dander, dust mites and moulds) was determined by skin prick tests with commercially available extracts (Allergopharma, Reinbek, Germany), and/ or by specific IgE measurement by UniCAP™ System (Thermo Fisher Scientific, Uppsala, Sweden).

Food challenges

Oral food challenges were performed according to previously published guidelines [10], for diagnosis in patients with a suspicion of IgE-mediated food allergy, or for food allergy follow-up in children previously diagnosed. Two different challenge protocols were used according to the clinical history. Patients with immediate-type reactions were tested by an open food challenge, and those with atopic dermatitis or potentially presenting with subjective, but immediate-type reactions were tested by double-blind, placebo-controlled food challenges (DBPCFC), with the two challenges (placebo and tested food) done the same day, with a free interval of minimum 2 hours and only if the morning challenge was negative. Oral anti-histamines were stopped at least 5 days before the challenge test. The challenged food was administered in five increasing doses every 15 to 30 minutes. Test foods were milk, hen's egg, sesame, hazelnuts and tree nuts, peanuts, soy, fish, wheat, and mushroom. The challenge test was stopped, when a) there was a convincing clinical reaction (positive test), such as an urticarial rash, laryngeal symptoms, vomiting, a marked decreased activity, an acute rhinitis or cough, wheezing or chest tightness, as described in the guidelines [10] or b) when the final dose of the test was achieved without a clinically subsumable reaction (negative test). In children with a positive test, the reaction was graded from 1 [very mild] to 5 [anaphylactic shock] [11], and a treatment was administered according to the severity of the symptoms [12]. All children were observed for at least 2 hours after the end of the challenge test.

Measurement of FeNO

Exhaled nitric oxide (FeNO) was measured in all patients before the food challenge, every 30 minutes during the 2 hours observation period, and after the completion of the food challenge. According to American Thoracic Society/European Respiratory Society recommendations [13], FeNO was collected in a single breath offline collector system with controlled expiratory flow and NO filter and analyzed in a chemiluminescence analyser (CLD77TM; Ecomedics, Duernten, Switzerland). A change of FeNO of more than 6 ppb from baseline was considered as significant , as previous studies have shown that FeNO measurement where consistent within 0.5 ppb of the results using chemiluminescence technique [14] and that FeNO results for a same volunteer are reproducible within 2 ppb, with no diurnal variation or learning effect [15].

Statistical Analysis

Comparison of FeNO levels in children with positive or negative challenge tests were analysed by a non-parametric test (Wilcoxon rank-sum test). The in-patient difference from the sequentially measured FeNO values (at 30, 60, 90 or 120 min) and the baseline FeNO [FeNOt-FeNOb] were analysed by Wilcoxon signedrank test. A Fisher's exact t-test was used for in-between patient group analysis. Pulmonary function test results (pre-test compared to post-test in the same patient) were analyzed by the t test for paired samples. Data are presented as median and ranges, or as mean with standard deviations when normally distributed. Differences were considered to be statistically significant when the p-value was <0.05.

Results

Food challenges

Fifty-three food challenges were performed in 40 children (18 girls, 22 boys). Eleven children underwent 2 or 3 challenges, because of a double-blind, placebo-controlled procedure or because 2 different foods were tested. No challenge was performed the same day after a positive food challenge. Sixteen of the challenges were positive, in fifteen patients (16/53, 30%) (Table 1), and 1 patient had two positive challenges, with raw egg and peanuts, done at a 5 months interval. All patients presented clinical reactions interpreted as mild to moderate [11]. Among these patients, eight had respiratory symptoms such as nasal congestion, sneezing, rhinorrhea, sensation of throat tightness. None of the patients exhibited a severe respiratory reaction with overt symptoms of the lower respiratory tract (no coughing, wheezing, dyspnoea and/or cyanosis). Non-respiratory symptoms in the remaining positive challenges included cutaneous or gastro-intestinal reactions, and/or a marked decreased activity.

Allergy tests

Sensitisation to common aeroallergens was assessed by skin pricks tests and/or specific IgE measurements. The tests were positive to one or more aeroallergens in 14/ 15 (93%) patients with positive food challenges, and in 21/28 (75%) of those with negative challenges (p = n.s). When pre-challenge FeNO measurements were compared to sequential measurements after the food challenges a significant decrease in FeNO was measured in positive patients at 60 and 90 minutes (Figure 2). When a change of FeNo superior of 6 ppb was considered, a significant change was observed in 3 out of 16 (19%) of children with a positive challenge at 30 minutes after the completion of the test (p = 1), in 7/15 (47%) children (p = 0.003) and 7/14 (50%) children (p = 0.005) after 60 and 90 minutes, respectively. After 120 minutes a decrease of > 6 ppb from baseline was still observed in 5 of 15 (33%) patients, however without statistical significance (p = 0.08) ( Table 2). Neither pre-challenge FeNO levels, nor FeNO changes did correlate to the clinical severity of the allergic reaction during the challenge test. The type of clinical reaction to the challenge test, respiratory or non-respiratory was not associated with changes in FeNO.

In patients with negative food challenges, there was no significant change in FeNO throughout the observation period, with variations of more than 6 ppb (increase or decrease) observed only in 5 of 36 patients (14%) at 30 minutes, 4 of 37 patients (11%) at 60 and 90 minutes, and 2 of 33 patients (6%) at 120 minutes post challenge. Individual changes in FeNO throughout the test are presented in Figure 3 (positive challenges), and Figure 4 (negative challenges).

Discussion

It is well recognized that respiratory reactions may potentially lead to life-threatening reactions of food allergy. In this study, we investigated whether measurement of FeNO was useful for early detection of reactions during food challenges. A significant number of positive food-allergen challenges were linked to a decreased FeNO, when compared to baseline, at 60 and 90 minutes after the first symptoms of allergy, in patients with and without objective lower respiratory tract symptoms. These changes were seen in patients regardless of objective lower respiratory tract symptoms. No specific patient characteristics (e.g. age, food challenged or type of symptoms) were linked to FeNO changes.

Convincing evidences indicate that allergic reactions to foods may, in addition to clinical reactions, also elicit local inflammation with a direct consequence on functional tests. An early decrease in lung function parameters has been previously described after respiratory allergen challenge [16]. In addition, increased bronchial hyperreactivity demonstrated by a methacholine challenge before and after a food challenge may occur despite a normal FEV 1 immediately after the food challenge [17]. These two studies clearly showed that allergen-induced allergic reactions can induce airway changes, without lower respiratory tract symptoms in some patients, and that airway inflammation during allergen challenges is probably largely unrecognized. FeNO in relation to food allergy has so far only been studied in infants with milk allergy [9]. No variation in FeNO could be observed, although most patients suffered from a late-type allergic reaction, probably not with an IgE-mediated mechanism.

Late increase of FeNO after nasal or bronchial allergen challenge or after natural allergen exposure [18], suggests that FeNO may also increase similarly after a positive food-allergen challenge. All our patients with a positive challenge experienced an early, IgE-type allergic reaction, and a significant part of these patients showed, within 60 to 90 minutes after the clinical reaction, a decrease in FeNO, which is probably in relation with the early phase of inflammation. There were no significant changes of FeNO in children with negative food challenges. Khatri et al. have reported temporal association of nitric oxide levels after whole lung allergen challenge and observed, similarly to us, that FeNO levels decreased significantly from baseline, between 2 to 5 hours after the challenge, and returned to normal levels at 24 hours. They hypothesized that the FeNO decrease first observed might be possibly due to consumption by reactive oxidant species into peroxynitrite [16]. Similarly, Pedroletti et al. observed a decrease in  FeNO 6 hours after an allergen nasal challenge in asthmatic children [8]. However, this change was similar in the non-allergic control group and no increase was observed in either group after 24 h. The authors suggested that the single dose nasal challenge might not have been potent enough to induce a prolonged inflammation. Previous studies in which FeNO was increasing mainly explored FeNO changes in late phase reactions in airway allergen challenges [4,19,20], while our patients were tested during the early phase of inflammation. A secondary increase in FeNO would have been possible in our patient population, but could not be measured here due to the food challenge schedule with a discharge 2 hours after completion of the procedure.

It should be noted that our study suffers from some limitations, inherent to the food challenges. Due to the various foods tested and to individual responses in positive food challenges, it is not possible to have a "standard" positive responder. However, the values were decreasing in all patients, in whom a modification in FeNO was observed, constantly indicating a change towards the same direction. In addition, no other markers of inflammation have been measured, thus not allowing further elaborating on potential mechanisms for FeNO changes.

In summary, we could show an early decrease in FeNO between 60 and 90 minutes after a positive food allergen challenge, regardless of objective respiratory symptoms. Our study clearly demonstrates that IgE-specific allergic reactions to foods are involving the lung, with FeNO changes observed also in absence of overt clinical respiratory symptoms. Our study does not provide an explanation for the decreased FeNO measures after positive food allergen challenges; further studies should explore our current findings.

Abbreviations

FeNO
Median pre-challenge FeNO values were higher in patients with positive food challenges (median 20.7 ppb [range 6.4-54.8]) compared to those with negative challenges (median 14.5 ppb [range 1.8-109.3]) (p = 0.04) (Figure 1). At different time points after the challenge (30, 60, 90 and 120 minutes, respectively) there were no significant differences in FeNO between children presenting with or without a positive challenge test.

Figure 1
1FeNO measurements in ppb before (0 min), and 30, 60, 90 and 120 minutes after the last dose of a food-antigen challenge. The results are grouped at the different time points by food-antigen challenge results, negative (Neg0, Neg30, Neg60, Neg90 or Neg120) or positive (Pos0, Pos30, Pos60, Pos90 or Pos120), and plotted as median value with lower and upper quartiles.

Figure 2
2FeNO change from baseline (ΔNO) at 30, 60, 90 and 120 minutes after the last dose of a food-antigen challenge. The results are grouped by food-antigen challenge results, negative (Neg30, Neg60, Neg90 or Neg120) or positive (Pos30, Pos60, Pos90 or Pos120), and plotted as median value with lower and upper quartiles.

Figure 3
3Individual changes in FeNO values measured throughout the test in patients with a positive food-antigen challenge.

Table 1
1Characteristics of patients with a positive food challengeAge
(y.m)

Gender Food
Food-
specific IgE
titres
(kU/L)

Reaction Score (1 [very mild]
to 5 [anaphylactic shock]*)

Respiratory reaction
(as defined in the
text)

Decreased FeNO at
60 minutes(>6 ppb)

Decreased FeNO at
90 minutes (>6 ppb)

9.9
F
Peanut
0.95
2
Y
Y
Y

9.8
F
Peanut
8.28
3
Y
Y
N

8.6
M
Peanut
1.07
1
N
N
N

10.1
F
Peanut
18.6
2
N
N
Y
6.8
M
Peanut
31.6
3
Y
Y
Y

6.9
M
Peanut
2.93
3
Y
Y
Y

6.5
M
Peanut
6.71
2
N
N
N

5.1
M
Milk
4.75
2
N
N
N

5.1
F
Hazelnut
7.04
2
N
N
Y

3.5
M
Hazelnut
<0.35
1
N
N
N

7.1
M
Tree nut
2.50
3
Y
N.A.
N.A.

5.1
F
Tree nut
1.91
3
Y
Y
Y
9.2
F
Raw
egg

1.58
2
Y
N
N

9.9
F
Raw
egg

2.07
2
N
Y
Y

5.7
F
Boiled
egg

2.92
2
N
Y
N.A.

7.5
F
Boiled
egg

8.6
2
Y
N
N

M: male, F: female, Y: yes, N: no
* according to (11).

Table 2
2Number of food challenges with or without a significant decrease in FeNO (> 6 ppb) Pos. Neg. Pos. Neg. Pos. Neg. Pos. Neg.Time after last
antigen dose

30 min
60 min
90 min
120 min

Outcome of food
challenge

ΔFeNO >6 ppb
3
5
7
4
7
4
5
2

No change in FeNO 13
30
8
33
7
33
10
31

P=
1 (n.s.)
0.003
0.005
0.08 (n.s.)

Pos.: positive food challenge; Neg.: negative food challengen.s.: non significant
AcknowledgementsThe authors wish to thank M Beghetti, JS Lacroix and M Hugentobler for facilitating NO analysis. The study was internally funded by both institutions.Authors' contributions AHB participated in the study coordination, contributed to the recruitment of patients, data acquisition and analysis and manuscript drafting. AK participated in the study design and data acquisition and analysis. IR participated in the study design and coordination and helped to draft the manuscript. DI participated in data acquisition and revising the manuscript. AM participated in the study design and coordination and helped to draft the manuscript. PT participated in the study design and coordination. RL participated in the study design and coordination and revising the manuscript. PAE participated in the study design and coordination, contributed to the recruitment of patients, data analysis and manuscript revising. All authors read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.
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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution. Benhamou, Clinical and Translational Allergy. 14Exhaled nitric oxide decreases after positive food-allergen challenge. Submit your manuscript at www.biomedcentral.com/submitCite this article as: Benhamou et al.: Exhaled nitric oxide decreases after positive food-allergen challenge. Clinical and Translational Allergy 2011 1:14. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit