Un nuovo studio, presente su PUBMED e pubblicato a Marzo su Alternative Therapies in health and medicine, correla un risultato positivo del test ALCAT® agli zuccheri fruttosio, canna da zucchero barbabietola da zucchero, alla mutazione del gene TCF7L2, legata all’insulino-resistenza e alla predisposizione al diabete di tipo II.

La resistenza all’insulina (RI) è definita come l’incapacità di una quantità nota di insulina esogena o endogena di aumentare l’assorbimento e l’utilizzo del glucosio. Diversi meccanismi sono stati proposti come possibili cause alla base dello sviluppo della RI e della sindrome RI. La resistenza insulinica fa parte di un gruppo di anomalie metabolico-cardiovascolari comunemente note come “Sindrome metabolica”. Essa può portare allo sviluppo di diabete di tipo 2, aterosclerosi, ipertensione, dismenorrea, irsutismo e sindrome dell’ovaio policistico, a seconda del background genetico del singolo individuo. Lo scopo di questo studio è stato quello di valutare, in 123 donne e 35 maschi (età media, 42 y ± 10.3, range 19-75 y) volontari se la resistenza insulinica potesse essere in parte correlata ad un’intolleranza allo zucchero nella dieta e se ci potesse essere una correlazione tra il test di intolleranza ALCAT e una mutazione del gene TCF7TL2 . Tale gene promuove infatti la trascrizione del proglucagone e svolge un ruolo chiave nello sviluppo delle isole di Langherans. I risultati hanno evidenziato, in maniera statisticamente significativa, che i soggetti con intolleranza allo zucchero presentano anche un’alterazione (completa o parziale)  del gene TCF7TL2. Sulla base di questi risultati, il nostro studio ha dimostrato che esiste una correlazione clinica tra il test di intolleranza alimentare ALCAT e la resistenza insulinca. La positività al test ALCAT di uno degli zuccheri testati (fruttosio, canna da zucchero e barbabietola da zucchero) indica, nella maggior parte dei soggetti, la presenza di una mutazione del gene TCF7L2 e potrebbe contribuire alla prevenzione e al trattamento della RI.

Leggi l’articolo originale su Pub Med

Altern Ther Health Med. 2019 Mar;25(2):22-38.

Pompei PGrappasonni IScuri SPetrelli FTraini ESorrentino SDi Fede G.

10 October 2018 STUDI

Non-Celiac Gluten Sensitivity in patients with severe abdominal pain and bloating: The accuracy of ALCAT 5

Background and aims: Non-Celiac Gluten Sensitivity (NCGS) is a recently proposed clinical condition causing both intestinal and extra-intestinal symptoms, without gastrointestinal lesions, which improve on avoiding gluten intake, in the absence of celiac disease and wheat allergy. The prevalence of this condition is still a matter of debate, in part due to the very recent introduction of an accepted diagnostic test, a double-blind, placebo controlled gluten challenge. However, this is a lengthy and cumbersome procedure, theoretically burdened by a significant reduction of patient compliance. ALCAT 5 is an automated in vitro test evaluating the toxic effect of gluten on neutrophils by the exposure of these cells to a gluten-containing extract of gluten-containing cereals. The test is very simple to perform, the results are rapidly obtained, and might represent, if sufficiently accurate, a promising alternative to diagnose gluten intolerance. The aim of this study was the comparison of ALCAT 5 results with those of a double- blind, placebo-controlled, gluten challenge, in a group of patients with clinically-suspected NCGS. Methods: Twenty-five patients (M/F 3/22, mean age 32 ± 4 yrs) with severe functional abdominal pain and bloating, who had previously undergone the ALCAT 5 test, were enrolled. All the subjects reported their symptoms on a gluten-containing diet and considered gluten the causal agent. Following the Salerno Experts’ Criteria, they underwent a double-blind, placebo controlled trial with gluten vs placebo. A mean value during gluten ingestion >30% of the value during placebo was considered as indicative of gluten sensitivity.

Results: After blinded administration of gluten, 13 out of 25 (52%) patients showed an increase in the severity of abdominal pain, and 11 out of 25 (44%) showed an increase in the severity of abdominal bloating. Considering these two symptoms together, in 16 patients out of 25 (64%), blinded gluten administration induced an increase of abdominal pain and/or bloating. The ALCAT 5 test proved to be positive in 20 and negative in 5 patients. In sixteen patients out of 25 the result of ALCAT 5 agreed with the double-blind trial (64%). In particular, both tests were positive in 14 patients and negative in 2. Conclusions: In this subgroup of patients, ALCAT 5 could be used to support the clinical suspicion of the presence of NCGS and to address these patients to a blinded gluten challenge.


Non-Celiac Gluten Sensitivity (NCGS) is a recently proposed clinical condition causing both intestinal and extra-intestinal symptoms which improve on avoiding gluten intake, in the absence of gastrointestinal lesions, celiac disease and wheat allergy. The prevalence of this condition is still a matter of debate. The first descriptions of NCGS were characterized by very high fre- quency reporting [2], but recently more realistic figures were described [3,4]. This discrepancy may be explained by the recent introduction of an accepted diagnostic test, a double-blind, placebo controlled gluten challenge [5], whereas the patient merely self- reporting the causative relationship between gluten ingestion and symptom occurrence was previously considered sufficient to diagnose the condition [6]. Apart from some criticisms on the interpretation of the results of the double-blind, placebo controlled food challenge, the proposed gluten challenge seems very prom- ising to describe the real prevalence of this condition. However, it is a lengthy and cumbersome procedure, theoretically burdened by a significant reduction in patient compliance during the three weeks of substrate intake.

ALCAT 5 is an in vitro test evaluating the toxic effect of gluten on neutrophils by the exposure of these cells to a gluten-containing extract of gluten-containing cereals, i.e., wheat, rye, barley, and oats. Results of the test are obtained through an automated mea- surement of neutrophil size and volume modifications following their incubation with gluten-containing cereal extracts [7]. The test is considered positive when the automated analysis reveals, in com- parison with the basal value, a change in volume and shape of neu- trophils after exposure to a test food substance. The test data are expressed as a volume distribution curve and the mean ± SD modi- fication of the area under the curve, in comparison with basal volume, is considered positive. An area change between the mean volume and 1 SD is considered positive. The test is very simple to perform, the results are rapidly obtained, and might represent, if sufficiently ac- curate, a promising alternative to diagnose gluten intolerance.

The aim of this study was the comparison of ALCAT 5 results to those of double-blind, placebo-controlled, gluten challenge, in a group of patients with suspected NCGS, in order to evaluate the performance of this test.

2. Patients and methods

Twenty-five patients (22 females, mean age 32 ± 4 yrs) with a long history of abdominal symptoms were enrolled. All the patients reported their symptoms on a gluten-containing diet, considered gluten to be the causal agent, and judged bloating and abdominal pain as the most severe. These characteristics of symptoms were confirmed by the completion of a questionnaire, based on a visual analogue scale, before entry the study. The mean duration of these two main symptoms were 10 ± 4 yrs. In all the patients, the pres- ence of organic conditions was excluded by endoscopic or radio- logic procedures, routine blood tests including thyroid function tests, coeliac disease associated serology, and abdominal ultra- sound. None of the patients suffered from wheat allergy. In this group of patients the following diagnoses were previously made: functional dyspepsia (n 1⁄4 14), irritable bowel syndrome (n 1⁄4 18), functional bloating (n 1⁄4 12), functional diarrhoea (n 1⁄4 8), functional constipation (n 1⁄4 6). In individual patients, more than one diag- nosis was frequently made. Rome IV criteria for functional bloating proved to be positive for all the patients, but for all the other conditions a positivity was present in only six patients (5 females, one male; 1 irritable bowel syndrome with constipation, 2 func- tional constipation, 1 irritable bowel syndrome with diarrhoea, 2 functional diarrhoea) [8,9].

All the patients underwent the ALCAT 5 test in the 3 months before the study on gluten-containing diet. However, the results of this test were blinded to the investigators during the next phases of the study. During the period from the ALCAT 5 test to the study entry, patients followed a gluten-containing diet.

None of the patients was following therapy known to interfere with intestinal function during the month prior to the study, such as antibiotics, prokinetics or laxatives. Constipated patients were advised to use a gentle water enema when needed and, in patients with diarrhoea, loperamide was allowed, if strictly necessary.

The protocol was approved by the local Ethical Committee and all the subjects gave their written informed consent.

2.1. NCGS diagnosis

Before the beginning of the study, and after ALCAT 5 test, all the patients had followed a period of gluten-free diet (GFD) which was even longer than the 6-week period suggested by the Salerno protocol [5], and all declared a clear improvement of symptom severity. Accordingly, we decided to avoid the repetition of this phase. However, to avoid an effect of gluten ingestion during the days immediately before the beginning of the protocol in sensitized patients, we prescribed a 2-week period of GFD, as a run in period, followed by the 3-week period of the blinded procedure suggested by the Salerno protocol [5], composed of two weeks of capsule ingestion, separated by one week of wash-out period (Fig. 1). After the run in period, the patients continued a strict GFD and were asked to fill in a daily questionnaire to rate the severity of both intestinal and extraintestinal symptoms, considered as gluten- dependent in NCGS (Table 1). Then, in a random order, following a randomization list generated by a computer, the patients were selected to follow a one week period during which gluten (10 capsule/day, 500 mg/capsule) or placebo capsules (rice starch, 10 capsule/day, 500 mg/capsule) were ingested. Rice starch was cho- sen due to its rapid absorption in comparison with other complex carbohydrates. At the end of the first week of capsule ingestion, the patients followed a 1-week wash-out period, without taking cap- sules, but continuing the completion of the daily questionnaires. Then, at the end of the wash-out period, the second period of capsule intake began (Fig. 1).

The daily questionnaire was administered to evaluate the presence and severity of abdominal and extra-intestinal symptoms, indicated in Table 1. The questionnaires used a 100 mm-long visual analogue scale (VAS) and patients were instructed to indicate the severity of each symptom on the line, considering the left end of the line as “absence of symptom” and the right end as “severe symp- tom” [10]. The score of the symptom was calculated by the sum of the score of the single days of each week. In accordance with the Salerno criteria, a mean value of the severity of abdominal pain and bloating during gluten ingestion >30% the value during placebo was considered as indicative of NCGS.

2.2. Statistics

To evaluate agreement between the gold standard test for NCGS and ALCAT 5 we used Cohen’s k. We judged the extent of the agreement according to Landis and Koch [11]. Agreement was considered: poor if k < 0.00; slight if 0.00 k 0.20; fair if 0.21
k 0.40; moderate if 0.41 k 0.60; substantial if 0.61 k 0.80; almost perfect if k > 0.80. Additionally, we calculated the sensi- tivity, specificity, and positive and negative predictive values of ALCAT 5: for these calculations we considered a subject to be positive if she/he displayed a worsening in pain or bloating at the blinded administration of gluten.

3. Results

After blinded administration of gluten, 13 out of 25 patients showed an increase in the severity of abdominal pain >30% than after placebo capsules (52%), and 11 out of 25 showed an increase in the severity of abdominal bloating >30% than after placebo cap- sules (44%).

Alcat Results

Alcat 5Considering symptom severity after gluten challenge, in the whole group of patients we found a significant correlation between the severity of abdominal pain and abdominal bloating (r 1⁄4 0.64, p < 0.0005) (Fig. 2), and between the modification of abdominal pain and the modification of bloating (r 1⁄4 0.54, p < 0.005) (Fig. 3).

Among the other symptoms we included in the questionnaire, none of the extraintestinal symptoms worsened after gluten chal- lenge (data not shown). Gluten challenge worsened the severity of

flatulence in 11 patients, the severity of nausea in 4 patients, the severity of diarrhoea in 1 patient, and the severity of borborygmus in 1 patient. The number of bowel movements and the Bristol score during gluten challenge were not significantly different than pla- cebo challenge.

ALCAT 5 test proved to be positive in 20 and negative in 5 pa- tients. The comparison between ALCAT 5 and blinded gluten challenge results showed the two tests were concordant in 12 out of 25 patients (48%) when we considered abdominal pain as the main symptom and in 13 out of 25 patients (52%) when we considered bloating as the main symptom. However, the concordance of the two tests improved if the modification of both symptoms was considered: in 16 out of 25 patients (64%) the severity of at least one of the two symptoms during gluten capsule intake was >30% than its severity during placebo capsule intake. In this case, ALCAT 5 and blinded gluten challenge results were concordant in 16 out of 25 patients (64%).

We also evaluated the agreement between the two tests. The agreement between the ALCAT 5 and positive pain symptoms after blinded gluten administration was poor (Cohen’s k 1⁄4 0.06). The agreement between the ALCAT 5 and positive bloating symptoms after blinded gluten administration was slight (Cohen’s k 1⁄4 0.03). Similarly, if we considered positive pain or bloating symptoms after blinded gluten administration, agreement between the two tests was slight (Cohen’s k 1⁄4 0.04).

The ALCAT 5 has a sensitivity of 81% but a poor specificity of 22.2%. The positive predicted value (PPV) of the ALCAT 5 was 65%, while the negative predicted value (NPV) was 40%.

Alcat 5 test

Alcat 5

4. Discussion

Whether symptoms attributed to NCGS are indeed due to gluten intake is still a matter of debate [12]. On pathophysiological grounds, this is a very important topic, as the exact definition of the main actor inducing symptom onset in this condition will allow for a specific treatment. Accordingly, it is important to clarify whether gluten or wheat [13] should be the target of our attention, but also FODMAPs [14] or amylase-trypsin inhibitors [15]. Consequently, the same uncertainty is also present on diagnostic grounds. As far as the role of gluten is concerned, a double-blind, placebo-controlled, gluten challenge was recently proposed [5]: resembling that already adopted in the diagnostic algorithm of food allergy, a blinded administration of gluten or placebo together with a strict symptom occurrence monitoring is today considered an accurate test to diagnose NCGS. We and others [3,4] have recently applied this protocol in patients self-reporting the gluten dependence of their symptoms and some drawbacks should be reported. First of all, the protocol suggests a preliminary 6-week period of GFD to evaluate if symptom improvement may be achieved, to select the subgroup of patients who need a blinded gluten challenge. Since this preliminary phase is unblinded, it is possible that a placebo or nocebo effect may have a role in the improvement of symptoms. The length of this period might be responsible for an excessive extension of the whole protocol and, therefore, it could reduce the patient’s compliance. Second, the organoleptic characteristics of gluten make true blindness very difficult: it must be not visible to the patients and, therefore, it must inevitably be hidden in capsules. Moreover, to avoid excessive capsule size, due to the physical characteristics of gluten, a maximum amount of 500 mg can be contained in one capsule and this causes the need for a high number of capsules to be ingested in a day in order to administer a dose of gluten sufficient to induce symptoms. The ingestion of 10 capsules a day for two weeks, one for the gluten test and the other for the placebo test, i.e. 140 capsules in total, might cause a reduction in patient compliance. Third, daily questionnaires for symptom occurrence monitoring administered over many days might cause a reduction of the patient’s attention, in particular during the last period of the test. Consequently, it is evident that, even if the blinded food challenge is considered the gold standard

in the diagnosis of many adverse reactions to food [16], this test is largely absent in a clinical setting [17].

Accordingly, the availability of alternative tests is advisable. ALCAT 5 is a simple, automated test that could represent a valid option in the diagnosis of NCGS. This is the first evaluation of the accuracy of ALCAT 5 in human disorders: the principle of the test is derived from the ALCAT test, adopted for food intolerance [7], and it was applied to discriminate the role of gluten in self-reported NCGS patients.

Our results show that in a group of patients suffering in particular from functional severe abdominal pain and bloating and without organic diseases, wheat allergy or celiac disease, the prevalence of NCGS is higher than previously reported [3,4], sug- gesting that, in a gastroenterological outpatient clinic, abdominal symptoms are more indicative than extra-intestinal symptoms to guide physicians towards a correct diagnostic algorithm for this condition. All the enrolled patients have previously undergone the ALCAT 5 test, and the comparison between ALCAT 5 and blinded gluten challenge showed that in 64% of cases a concordance was evident. The concordance between the two tests was less accurate when abdominal pain or bloating were considered separately. However, the strict correlation between postechallenge severity of these two symptoms permits the adoption of a combination of them, thus optimizing the performance.

The assessment of the degree of agreement between the double blinded gluten challenge and ALCAT 5 tests showed very low values of Cohen’s k. Considering the single symptoms separately or together, results indicate that the agreement between the tests was no better than would be expected by chance. However, these results are similar to those obtained from a faecal occult blood test and a colonoscopy for the screening of left-sided colon cancer [18,19]. Accordingly, the importance of our results relies on the possibility of using the ALCAT 5 test preliminary to the blinded gluten chal- lenge. Due to the complexity of clinical manifestations of NCGS, frequently characterized by many concomitant symptoms, both intestinal and extraintestinal [2], as often reported by the patients, it may be difficult in many of them to correctly focus on the de- scriptions and the importance of any single symptom during the interpretation of the whole clinical presentation. Therefore, ALCAT 5 might be used to preliminarily screen patients with severe abdominal pain and bloating in order to select, in this subgroup of References patients, which of them should undergo the blinded gluten challenge, at least on the basis of these two main symptoms.

A limit of the study was the absence of a healthy control group, to test for in vitro modification of neutrophils after oral gluten challenge. In a subsequent study, it should be important to test for this modification and to evaluate also the reproducibility of the procedure.

In conclusion, we have compared ALCAT 5 and double-blind, placebo-controlled, gluten challenge results in a group of self- reported NCGS with severe abdominal pain and bloating, to eval- uate if a simplification of diagnostic procedures is possible in these patients. In this subgroup of patients, ALCAT 5 could be used to support the clinical suspicion of the presence of NCGS and to direct these patients to a blinded gluten challenge.

Conflict of interest
The authors have no conflict of interest to declare.

Funding sources

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Guarantor of the article

Michele Di Stefano.

Specific author contributions

Michele Di Stefano planned and conducted the study, inter- preted data, drafted the manuscript.

Eugenia Vittoria Pesatori, Giulia Francesca Manfredi, Giacomo Grandi, Alessandro Gabriele, Davide Iozzi conducted the study, collected and interpreted data, drafted the manuscript.

Mara De Amici conducted the study, collected data and drafted the manuscript.

Giuseppe Di Fede planned the study and drafted the manuscript.

All the mentioned Authors approved the final version of the manuscript.



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15 February 2016 NOTIZIESTUDI

stomaco ciboLo stomaco è un organo fondamentale del nostro corpo, la cui funzione principale consiste nel ridurre in minuscoli pezzetti il cibo: tutto quello che viene mangiato in un pasto viene accolto nella cavità dello stomaco e subisce un processo di frammentazione dovuto alla contrazione dei muscoli presenti nelle pareti che, circa 3 volte al minuto, lanciano i frammenti di cibo da una parte all’altra della cavità gastrica, favorendo lo spezzettamento meccanico degli alimenti e il lavoro degli enzimi digestivi. Alla fine del processo si ottengono frammenti di cibo di grandezza variabile tra 1 e 3 millimetri, dimensione sufficiente per il passaggio attraverso il piloro verso l’intestino tenue. Bisogna tuttavia ricordare che non tutto il nutrimento che arriva nello stomaco viene trattato in maniera uguale: per sua forma, infatti, lo stomaco assomiglia ad una piccola borsa sbilenca che presenta un lato più corto, una strada quindi più veloce per giungere all’intestino tenue. Questa scorciatoia viene solitamente presa dai liquidi: è questo il motivo per il quale si è soliti consumare brodini e minestrine “per stare leggeri” ed è difficile che l’acqua “rimanga sullo stomaco” come invece accade con gli alimenti solidi. Oltre al cibo liquido il nostro stomaco gradisce maggiormente il cibo di piccolo volume. Infatti, più i frammenti sono piccoli, meno fatica farà il nostro stomaco per ridurli in poltiglia. A tal proposito fondamentale importanza riveste la masticazione, considerata non a caso il primo passaggio della digestione. Anche riscaldare il cibo aiuta la nostra digestione, dal momento che il calore favorisce la rottura dei legami che tengono unite le molecole negli alimenti, aiutando quindi il lavoro degli enzimi digestivi. Il tempo in cui il cibo ingerito resta all’interno della cavità gastrica viene definito “digestione gastrica” e varia da alimento ad alimento: ad esempio i cereali in chicchi, se cotti, permangono nella cavità gastrica per poco meno di un’ora, la pasta per 3 ore, le proteine per un periodo di tempo ancora maggiore. Sfruttando la digestione gastrica è quindi possibile prolungare il senso di sazietà, se si consuma una piccola quota proteica ad ogni pasto. Un’eccessiva permanenza del cibo nella cavità gastrica, tuttavia, favorisce la nascita di fastidiosi disturbi come i rigurgiti di acido cloridrico dallo stomaco all’esofago, bruciori gastrici, eruttazione, nausea, dolori, crampi addominali, etc. Per evitare di incorrere in tali problematiche è doveroso adottare un corretto comportamento alimentare ed evitare i cibi che predispongono all’insorgenza di tali sintomatologie: tra i comportamenti alimentari corretti vi è ad esempio quello di mangiare lentamente, masticando il più a lungo possibile, evitare di guardare il computer o la televisione durante i pasti e concentrarsi su quanto si ha nel piatto. Per quanto riguarda le scelte alimentari, poi, bisognerebbe cercare di fare attenzione agli alimenti particolarmente acidi come le bevande alcoliche, i dolci, il latte e i suoi derivati, i cibi in scatola, i sottaceti, i sughi pronti, i dadi da brodo, le spezie piccanti, il cioccolato, la menta, i dolcificanti sintetici e le bevande gassate.
Anche un’alimentazione squilibrata o pro-infiammatoria contribuisce a peggiorare la sintomatologia gastrica. Per capire se il cibo che ingeriamo crea infiammazione, un aiuto ci viene dato dall’America attraverso il test per intolleranze ALCAT, approvato dalla Food and Drug Administration (FDA), che permette di segnalare le reazioni che alcuni alimenti, additivi chimici o additivi alimentari hanno sul nostro organismo
Il test è in grado di evidenziare anche una categoria di alimenti particolarmente importante nelle problematiche legate all’acidità di stomaco e al reflusso: i salicilati. Questi alimenti contengono acido salicilico, sostanza che può irritare la mucosa gastrica e alla quale molte persone risultano addirittura allergiche. Tra gli alimenti con più alta concentrazione di acido salicilico abbiamo ad esempio broccoli, cetrioli, cicoria, indivia verde, melanzane, patate dolci, peperoni, radicchio verde, ravanelli, spinaci, zucca e zucchine; tra i frutti, invece, troviamo albicocche, ananas, arachidi, arance, datteri, lamponi, mandorle, ribes neri, ribes rossi e uva passa. L’indicazione su tali alimenti, per chi soffre frequentemente di problemi di stomaco non legati a patologie, è quella di cercare di non consumarne più di un tipo di verdura o di frutta al giorno e mai nello stesso pasto.

Articolo di Davide Iozzi, Biologo nutrizionista, esperto in nutrizione umana, collaboratore dell’Istituto di Medicina Genetica Preventiva (I.M.Ge.P.) di Milano.

7 April 2015 STUDI

Titolo: Effetti clinici della correlazione tra Alcat Test e insulino-resistenza – Tesi Sperimentale in Farmacologia e Farmacoterapia II
Università degli Studi di Camerino, Scuola del Farmaco e dei Prodotti della Salute, Corso di Laurea in Farmacia Unità di Medicina Sperimentale e Sanità Pubblica
Relatore: Prof. Pierluigi Pompei / Relatori esterni: Dott. Sacha Sorrentino, Dott. Davide Iozzi, Prof. Giuseppe Di Fede A.A. 2013-2014
Tesi di: Flavia Colucci



7 April 2015 STUDI

Titolo: Analisi dell’influenza delle intolleranze alimentari sulla composizione corporea mediante l’utilizzo di ALCAT TEST e Bio-impedenziometria
Università degli Studi di Pavia – Dipartimento di Biologia e Biotecnologie “L. Spallanzani”
Master di II Livello in Nutrizione Umana
Relatori: Prof.ssa Paola Rossi Relatore esterno: Prof. Giuseppe Di Fede
Tesi di: Dott. Alessio Tosatto


7 April 2015 STUDI

Titolo: Analisi dell’andamento statistico e stagionale delle intolleranze alimentari, valutato per grandi gruppi alimentari, dai risultati dei Test ALCAT degli anni 2010, 2011 e 2012
Università degli Studi di Pavia – Dipartimento di Biologia e Biotecnologie “L.Spallanzani”
Master di II Livello in Nutrizione Umana
Tesi di: Dott.ssa Cecilia Pedroni