**Advance in Gluten-Free Diet**

Editor

**Paolo Usai-Satta**

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*Editor* Paolo Usai-Satta Brotzu Hospital, Piazzale Alessandro Ricchi Cagliari, Italy

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This is a reprint of articles from the Special Issue published online in the open access journal *Nutrients* (ISSN 2072-6643) (available at: https://www.mdpi.com/journal/nutrients/special issues/gluten).

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## **Contents**


**Massimo Bellini, Sara Tonarelli, Maria Gloria Mumolo, Francesco Bronzini, Andrea Pancetti, Lorenzo Bertani, Francesco Costa, Angelo Ricchiuti, Nicola de Bortoli, Santino Marchi and Alessandra Rossi**

Low Fermentable Oligo- Di- and Mono-Saccharides and Polyols (FODMAPs) or Gluten Free Diet: What Is Best for Irritable Bowel Syndrome?

Reprinted from: *Nutrients* **2020**, *12*, 3368, doi:10.3390/nu12113368 .................. **87**

## **About the Editor**

**Paolo Usai-Satta** (MD) is a gastroenterologist with expertise in celiac disease, alimentary intolerance, and digestive pathophysiology. He is a clinical physician at the Gastroenterology Unit of Brotzu hospital in Cagliari (Italy). His research is focused on Irritable bowel syndrome, alimentary intolerances, celiac disease, and digestive motility disorders. He is a member of the European Society of NeuroGastroenterology and Motility and the Italian Group of Digestive Motility. He is also advisor board member and general secretary of the Italian Association of Hospital Gastroenterology (AIGO).

### *Editorial* **New Perspectives on Gluten-Free Diet**

#### **Paolo Usai-Satta 1,\* and Mariantonia Lai <sup>2</sup>**


Received: 9 November 2020; Accepted: 17 November 2020; Published: 18 November 2020

Celiac disease (CD) is a permanent, chronic, gluten-sensitive disorder characterized by small intestinal inflammation and malabsorption in genetically predisposed individuals [1]. In addition, a self-reported gluten/wheat sensitivity without the diagnostic features of CD has recently been named non-celiac gluten/wheat sensitivity (NCGWS) [2].

The only effective and safe treatment for CD and gluten-related disorders (GRD) is a lifelong, strict exclusion of gluten, the so-called gluten-free diet (GFD). In this respect, there are new concepts and perspectives regarding GFD and its impact on clinical practice.

This Special Issue, entitled "Advance in Gluten-Free Diet", comprises eight peer-reviewed papers reporting on different points of view regarding GFD in different clinical conditions.

In detail, the interplay between irritable bowel syndrome (IBS) and GRD, the role of GFD compared to low fermentable oligo/di/monosaccharides and polyols (FODMAP) diet (LFD) in IBS and functional dyspepsia (FD), the role of a low nickel diet in CD on GFD with persistent IBS-like symptoms, the efficacy of high-iron diet in CD with iron deficiency without anemia, the current reformulation of gluten-free food composition in Spain, the nutritional value of GFD in Polish CD prisoners and the symptoms worsening after wheat ingestion in familial Mediterranean fever are discussed in this Special Issue.

IBS is frequently associated with CD, and IBS symptoms may also overlap and be similar to those associated with NCGWS. In addition, many patients with CD have persistent digestive symptoms despite a strict GFD. This can be due to a higher frequency of IBS in CD patients compared to the general population. On the other hand, many different dietary approaches have been recently suggested for IBS and a GFD is considered a therapeutic option in a subset of IBS patients [3].

In their review, Bellini et al. [4] discuss the evidence regarding two of the most advised diets for IBS, the GFD and the LFD. A GFD is less restrictive and easer to follow than LFD. On the other hand, according to recent evidence, LFD is the most effective dietary intervention suggested for treating IBS, and it is included in the most updated guidelines. Unfortunately, the clinical trials regarding the dietary intervention for IBS are of low quality. The problem is the difficulty in setting up randomized double-blind controlled trials which objectively evaluate clinical results without the risk of a nocebo/placebo effect.

Similarly to IBS, both GFD and LFD could improve symptoms in patients with FD. In a double-blind, randomized, placebo controlled pilot trial, Potter et al. [5] have evaluated the role of this diet (specifically gluten and fructan) in patients with FD. A combined GFD–LFD led to an overall improvement in dyspeptic symptoms but this result was not significant. Otherwise, a specific food trigger was not identified. The authors consequently suggest further larger studies to confirm these data.

As hypothesized by Borghini et al. [6], a nickel-rich diet could exacerbate or relapse IBS-like symptoms in CD patients on strict GFD. In fact, many gluten-free foods are high in nickel content. In their study, 20 celiac patients on GFD, with persistent digestive symptoms and with positive patch test for nickel-mucositis, consumed a low-nickel diet. The result was an overall improvement in digestive symptoms in CD patients, with significant effects for 10 out of 24 symptoms (according to

Gastrointestinal Symptom Rating Scale modified questionnaire). The impact of a nickel-rich diet on CD could be a clinical and scientific challenge for further studies to address.

Iron deficiency without anemia is a common clinical scenario in CD despite a strict GFD. A recommended approach to this condition is not yet defined. Scricciolo et al. [7] have compared a 12-week iron-rich diet to iron supplementation with ferrous sulfate in 22 celiac adult women. At the end of the treatments, both well tolerated, ferritin levels were statistically higher in the ferrous sulfate group. An iron-rich diet can be, however, recommended in patients who do not tolerate pharmacological supplementation.

The objective of the paper by Fajardo et al. [8] was to develop a nutritional food composition database including cereal-based gluten-free products available in Spain. A comprehensive database of 629 products was achieved. Gluten-free products were primarily composed of rice and/or corn flour. The most common added fat was sunflower oil, followed by palm fat, olive oil and cocoa. Xanthan gum was the most frequently employed fiber. Nutritional deficiencies have been described for CD patients on GFD and an updated quality assessment of available products is needed for further improvement in gluten-free product development.

A special clinical setting for CD patients can be represented by the prison population. The risk of nutritional deficiencies may be a real problem for CD prisoners due to the limited possibilities of external quality control. In the study by Kosendiak et al. [9], the nutritional value of GFD and regular diet meals served in 10 Polish prisons were assessed. GFD was characterized by lower average energy content in 11 out 14 essential nutrients. Greater quality control of GFD meals served in catering facilities should be recommended.

Finally, Carroccio et al. [10] have identified a clinical association between self-reported NCGWS and familial Mediterranean fever (FMF). In their pilot randomized trial, the authors have evaluated clinical and innate immune responses to wheat (compared to rice) challenge. In six NCGWS/FMF female patients, wheat ingestion exacerbated clinical and immunological features of FMF. These findings may suggest new clinical scenarios in the management of FMF.

In conclusion, the different perspectives presented in this Special Issue confirm that the gluten-free diet is currently a clinically and scientifically challenging topic. We would like to thank all the authors and the editorial team of *Nutrients* for their precious contributions.

**Funding:** This review received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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## *Article* **Gluten-Free Diet in Prisons in Poland: Nutrient Contents and Implementation of Dietary Reference Intake Standards**

#### **Aureliusz Kosendiak 1, Piotr Stanikowski 2,\*, Dorota Domagała <sup>3</sup> and Waldemar Gustaw <sup>2</sup>**


Received: 15 August 2020; Accepted: 12 September 2020; Published: 16 September 2020

**Abstract:** The gluten-free diet (GFD) requires special attention from nutritionists due to the potential risk of nutrient deficiencies in its users. This risk may be greater when this type of nutrition is implemented in prisons due to the limited possibilities of external control, a low catering budget for meals, and insufficiently defined recommendations regulating nutrition for prisoners. The aim of the present study was to assess the nutritional value of GFD and regular diet meals served in some Polish prisons and to compare the values to the dietary reference intake (DRI) standards. Using a specialized computer program, 7-day menus of both types of diet provided in 10 prisons were analyzed. The percentage coverage of the DRI was calculated based on the recommendations of the Polish National Food and Nutrition Institute. GFD was characterized by lower average contents of energy and 11 out of 14 essential nutrients, i.e., protein, carbohydrates, dietary fiber, starch, ash, sodium, calcium, iron, zinc, folate, and vitamin B12. The average content of phosphorus, niacin, and riboflavin in the gluten-free diet was higher than that in the regular diet. It was shown that the meals in GFD and the regular diet did not provide the recommended amounts of calcium (38 and 44% DRI, respectively), vitamin D (29 and 30% DRI), vitamin C (86 and 76% DRI), and folate (51 and 56% DRI). In turn, the supply of sodium, phosphorus, copper, and vitamins A and B6 substantially exceeded the recommended levels. The results indicate a need for greater quality control of GFD meals served in catering facilities. It is also necessary to develop legal provisions that will regulate more specifically the nutrition for prisoners in terms of an adequate supply of minerals and vitamins.

**Keywords:** gluten-free diet; celiac disease; dietary reference intake; prison diets

#### **1. Introduction**

Gluten is a general term given to the following fractions of protein: gliadins, glutenins, hordein, and secalin. These protein fractions are found in four grains, i.e., wheat, rye, barley, and triticale. Oats are inherently gluten-free but may be contaminated with wheat during growing or processing [1]. The ingestion of gluten can trigger an array of conditions; they are designated by a broader term "gluten-related disorders". They are divided into disorders with autoimmune pathogenesis, including celiac disease (CD), disorders characterized by allergic mechanisms, which include wheat allergy, and the controversial non-celiac gluten sensitivity, whose causes are neither autoimmune nor allergic in nature [2].

CD is a common chronic immune-mediated small bowel enteropathy resulting from gluten exposure in genetically susceptible individuals [3]. It is generally acknowledged that about 1% of the general population have CD [4]. In the US population, a higher proportion of persons living at latitudes of 35◦ North or greater have CD or avoid gluten than persons living south of this latitude, independent of the race or ethnicity, socioeconomic status, or body mass index [5]. Unfortunately, there are no data about the prevalence rate of CD and other gluten-related disorders in the health statistics reports on prisoners [6–8].

Currently, the only effective treatment available for CD is a strict life-long gluten-free diet(GFD), since it leads to resolution of intestinal and extraintestinal symptoms, negativity of autoantibodies, and regrowth of intestinal villi. In addition, the diet exerts a partial protective effect on several complications. However, these crucial advantages are accompanied by some disadvantages, including a negative impact on the quality of life, psychological problems, fear of involuntary/inadvertent gluten contamination, increased cardiovascular risk, and frequent severe constipation [9]. Gluten-free food products are substantially more expensive than regular equivalents. Replacement of commonly consumed cereal staple foods in GFDs with gluten-free equivalents may be associated with an increased supply of fat, saturated fatty acids, salt, and sugar [10]. GFD may lead to possible nutrient deficiencies of fiber [11,12], folate [11–14], vitamin D [11,14,15], calcium [11,12,14,16], magnesium [11,12,16], iron [12,14], zinc [16], selenium [16], and iodine [14]. To increase the supply of nutrients, it is recommended to include legume and pseudo-cereal products (especially amaranth, quinoa, and soybeans) in GFD. They are a better source of fat, fiber, high-quality protein, and minerals than the frequently served corn and rice [17].

The aim of the present study was to assess the nutritional value of gluten-free and regular diet meals served in some Polish prisons and to compare the values to the dietary reference intake standards.

#### **2. Materials and Methods**

#### *2.1. General Information*

The study was approved by the Director-General of the Prison Service in Poland on 3 December 2018. Next, a request for access to the menus was sent in an electronic or paper form to all the institutions. In total, 88 prisons responded to the request. Ten independent prisons, all serving gluten-free diet, were selected for the investigations, i.e., detention centers in Gda ´nsk, Pozna ´n, Suwałki, and Wrocław and prisons in D ˛ebica, Gr ˛ady Woniecko, Iława, Nysa, Strzelce Opolskie (No. 2), and Włodawa. Most of the institutions were male prisons, whereas three facilities were female and male prisons. All prisons were designed for adults.

#### *2.2. Analysis of Energy and Nutrient Content*

The analysis involved 7-day GFD menus (from different seasons) and 7-day regular diet menus (from different seasons) provided by each prison in 2018. The regular diet was served to all healthy adult prisoners, while the GFD was prescribed by medical staff [18]. In the study, 140 all-day menus consisting of breakfast, lunch, and supper were analyzed. A typical GFD breakfast usually consisted of puffed rice cakes, margarine, sandwich meats, jam, and an apple/vegetable. Various types of soup and a dish composed of meat, potatoes/white rice, and side salad were served for lunch. Supper mostly included white rice/puffed rice cakes, margarine, and sandwich meats/cottage cheese. With each meal, prisoners made tea themselves. The calculations did not include food that prisoners were able to buy at least three times a month in the prison canteen or food parcels that prisoners received once a month from their relatives [19].

The quantitative analysis was carried out with the use of specialized software DietetykPro (DietetykPro, Wrocław, Poland), which mainly incorporates Polish Food Composition databases developed at the National Food and Nutrition Institute in Warsaw [20] and the database of the United States Department of Agriculture [21]. All food products specified in the menus and inventory reports were analyzed. The inventory reports included names of the food products and their quantity in kilograms/liters used in the kitchen to prepare all meals. Ready meals included in the software database were not taken into account in the analysis. The study involved assessment of 31 parameters

of daily food rations: energy value, total protein, total fat, total carbohydrate, dietary fiber, sucrose, starch, cholesterol, fatty acids (saturated, monounsaturated, polyunsaturated), ash, minerals (sodium, potassium, calcium, phosphorus, magnesium, iron, zinc, copper), and vitamins A (as retinol activity equivalents), retinol, B1, B2, niacin, B6, B12, C, D, E, and folate (as dietary folate equivalents). The results took into account averaged technological losses caused by heat treatment: folate and vitamin C, 50%; vitamin B1, 30%; vitamin B6, 25%; vitamins A, E, retinol, and niacin, 20%; and other parameters, 10%. Next, the percentage of the dietary reference intake (DRI) was calculated based on nutrition standards for the Polish population [22]. Since approximately 57% of male prisoners in Poland in 2019 were in the age range of 31–50 [8], the calculations were based on recommendations for this group. For calculation of energy requirement, the physical activity level (PAL) of 1.4 was adopted.

Next, the DietetykPro and Microsoft Excel software was used to analyze the consumption of food groups. The classification of the food groups and subgroups was based on the Polish Food Composition databases [20]. Certain modifications in the classification have been introduced for better presentation of the differences between the analyzed diets. In the "cereal products" group, a sub-group "puffed rice cakes" has been added. The "vegetables and vegetable products" group has been supplemented with a "starchy roots" sub-group, and the "legumes" sub-group has been removed and analyzed as a separate "legumes" group.

#### *2.3. Statistical Analysis*

The statistical analysis was carried out using the Microsoft Excel 2020 and Statistica 13.1 program (StatSoft, Cracow, Poland). Welch's test was used to check whether the type of diet had an effect on the average content of the analyzed nutrients, energy, and daily consumption of the food groups. In the next step, 95% confidence intervals for differences between the means of 15 components significantly differing between the gluten-free and regular diets were determined. Similarly, such intervals were calculated for the daily consumption of seven food groups which were differed significantly in both types of diet.

#### **3. Results**

#### *3.1. Energy and Macronutrients*

The daily energy supply met the recommendations of the Polish National Food and Nutrition Institute in the case of GFD, but exceeded the recommended values by 108 kcal in the regular diet (Table 1). The supply of saturated fatty acids (SFA) was 24.5 g in the case of GFD and 26.3 g in the case of the regular diet, which exceeded the recommended values. SFAs covered approx. 9.0% of total energy intake.

**Table 1.** Energy and macronutrients provided in prisons (*n* = 10) menus per person per day and the age-specific dietary reference intake (DRI).


<sup>1</sup> 30% of energy from fats; EER estimated energy requirement; RDA recommended dietary allowance; SFA saturated fatty acids; MUFA monounsaturated fatty acids; PUFA polyunsaturated fatty acids; AI adequate intake; N.A. not available.

#### *3.2. Micronutrients*

The average supply of micronutrients in the daily food ration is presented in Table 2. In comparison with the recommendations, excess consumption of three minerals, i.e., sodium, phosphorus, and copper, was recorded in both diets. A particularly high supply was recorded in the case of sodium. The consumption of potassium slightly exceeded the recommended values. The supply of magnesium, iron, and zinc was close to the reference values. The lowest supply of all minerals was recorded for calcium. Its intake in GFD was 378.7 mg, which covered 38% of DRI. The intake of this element in the regular diet was 440.3 mg, which represented 44% of DRI.

**Table 2.** Micronutrients provided in prisons (*n* = 10) menus per person per day and the age-specific dietary reference intake (DRI).


AI adequate intake; RDA recommended dietary allowance; N.A. not available.

The content of vitamins A and B6 in the analyzed menus substantially exceeded the recommended values. In turn, the supply of vitamin D in both diets was very low, i.e., 4.3 and 4.5 μg, respectively. This only covered 29% of the recommended values in GFD and 30% in the regular diet. Both diets were characterized by a low intake of folate, covering approximately half of the DRI value.

#### *3.3. Analysis of the Menus by Types of Diet*

The gluten-free and regular diets differed statistically significantly in the content of energy and 14 nutrients: protein, carbohydrates, dietary fiber, starch, ash, sodium, calcium, phosphorus, iron, zinc, riboflavin, niacin, folate, and vitamin B12. For the differences between the mean levels of the essential ingredients in the regular diet and GFD, 95% confidence intervals were determined (Table 3). GFD was characterized by lower average contents of energy and 11 of the 14 essential nutrients, i.e., protein, carbohydrates, dietary fiber, starch, ash, sodium, calcium, iron, zinc, folate, and vitamin B12. The average content of phosphorus, niacin, and riboflavin in GFD was higher than in the regular diet.


**Table 3.** Lower and upper endpoints of a 95% confidence interval for the difference between mean regular and gluten-free diet.

*xr*—mean value for regular diet, *xg f*—mean value for gluten-free diet.

#### *3.4. Analysis of the Food Group Consumption*

In both types of diet, no consumption of products from the groups and subgroups "frozen fruits", "fruit, dried", "nuts", "seeds", and "beverages" was recorded (Table 4). Products from the subgroup "mushrooms" were served in only three prisons. In GFD, no products from the subgroups "pasta" (including gluten-free pasta), "breads and rolls" (including gluten-free breads and rolls) were served, and "legumes" were noted in only one object. The regular diet was characterized by no consumption of products from the subgroup "puffed rice cakes".

**Table 4.** Distribution of food group and sub-group consumption (g/day).



**Table 4.** *Cont.*

Food groups are bolded in the table.

The regular diet and GFD differed significantly in terms of the consumption of products from the following food groups and subgroups: "cereal products", "groats", "vegetable products", "fish, fish products, and seafood", "fruits and fruit products", "fruit, raw", and "other products" (Table 5). In GFD, the consumption of products from the groups "groats", "fruits and fruit products", and "fruit, raw" was significantly higher than in the regular diet and significantly lower in the case of the other groups ("cereal products", "vegetable products", "fish, fish products and seafood", and "other products") than in the regular diet (Table 5).

**Table 5.** Lower and upper endpoints of a 95% confidence interval for the difference between mean regular and gluten-free diet.


*xR*—mean value for regular diet, *xGF*—mean value for gluten-free diet.

#### **4. Discussion**

The investigations conducted by our team revealed inadequate quality of meals served in the Polish prisons. Compared to the regular diet, GFD was characterized by a significantly lower average level of energy and 11 nutrients: protein, carbohydrates, dietary fiber, starch, ash, sodium, calcium, iron, zinc, folate, and vitamin B12. The mean content of phosphorus, niacin, and riboflavin was higher in GFD than in the regular diet.

The implementation of GFD involves exclusion of many food products. Wheat or mixed bread was found to be the basic food served for breakfast and supper in almost all diets available in the Polish prisons. It was mainly replaced with puffed rice cakes in the GFD meals. Therefore, the cost of GFD breakfast and supper was high, since puffed rice cakes were up to 10 times more expensive than bread, as shown by the inventory reports. Hence, prison meal planners tended to limit the amount of these products served for breakfast and supper even twice in comparison with the ration of bread served in these meals in the other diets. The difference in the consumption of cereal products between the regular diet and GFD, i.e., 243.66 g, probably had an impact on the supply of energy, protein, and carbohydrates, which was significantly higher in the regular diet. As suggested by Soto et al. [23], the difference in the energy value between GFD and regular diet meals may also be associated with the exclusion of breaded fried foods. Bread, rolls, and bread products contribute substantially to the

supply of many nutrients. In the average Polish diet, these products provide 48.6% of manganese, 36.3% of carbohydrates, 35.4% of fiber, 24.9% of iron, 22.1% of copper, 21.1% of zinc, 21% of magnesium, and 20.7% of folate [24]. Therefore, the exclusion of bread from GFD may have resulted in the considerably lower consumption of such nutrients as fiber, iron, zinc, and folate, in comparison with the regular diet.

The average energy value was 2405.5 kcal/day in the GFD menus and 2708.0 kcal/day in the regular diet. The energy value in the latter diet was higher than the nutritional standards recommended for the Polish population (2100–2600 kcal) [22]. As specified by the regulations on nutrition for prisoners in Poland, meals should provide at least 2600 kcal [18]. In our opinion, these recommendations require personalization, which is supported by the varied physical activity [25] and excessive body weight in Polish prisoners [26].

The analysis of the menus did not show any disturbing observations regarding the supply of macronutrients in the diet. Only the consumption of SFAs exceeded the recommended values in both analyzed diets. However, no significantly higher consumption of this ingredient was detected in GFD, which is usually observed in patients with celiac disease [27]. This may be related to the higher SFA content in GF products than in their counterparts [28]. The lower supply of fiber in GFD compared to the regular diet indicated by the present results is in agreement with values reported by other authors [16,28,29]. The consumption of dietary fiber in GFD covered DRI, although it was significantly lower than in the regular diet. This may be related to the significantly higher consumption of groats, fruit, and fruit products in this diet compared to GFD, which we showed in the analysis of the consumption of the food groups. It is recommended that GFD meals should be enriched with fiber and minerals through consumption of legumes and pseudocereals [17]. The analysis conducted in the study showed very low consumption of legumes, which in the case of GFD were included in the menu in only one prison, whereas no pseudocereals were served. The main ingredient of lunch was white rice, while brown rice, which provides more fiber and many other health-beneficial food components [30], was not served at all. The analyzed GFD diets did not include oat, which is nutritious and a good source of fiber and can be safely consumed by patients with CD [31]. However, according to the recommendations of the Polish Association of People with Celiac Disease and the Gluten-Free Diet, oats and oat products in Poland are highly contaminated with gluten and therefore should not be used in the GFD [32].

To our knowledge, the large difference in the supply of sodium between the two analyzed diets was associated with the fact that the meal planners followed invalid provisions regulating the issue of nutrition in prisons in Poland [33]. These regulations recommended that the daily salt intake in therapeutic diets should be limited to 6 g per day. The analysis of the menus and inventory reports demonstrated that the regular diet and GFD were planned to contain 3 to 11 g and 0 to 3 g of table salt per day, respectively. We reported high levels of salt additions to prison meals in our previous investigations as well [34]. Besides the lower addition of salt to the dishes, the sodium content in GFD may also have been influenced by the exclusion of bread from the diet. Bread is a source of 17.5% of sodium in the average diet of the Polish population [24] and 23% of sodium in the diet of Polish hypertension patients [35]. As far as other minerals are concerned, a significantly low calcium intake was noted in both diets. The low calcium supply is reflected in the level of consumption of the respective food groups. The average consumption of milk and dairy products, which are the most important source of this mineral, was only 61.96 g in GFD and 56.14 g in the regular diet. An adequate supply of this mineral is particularly important in CD patients, due to the malabsorption of the nutrient, which may lead to development of bone diseases [36]. Individuals with undiagnosed and untreated CD are at the highest risk of malabsorption [37]. GFD was characterized by a significantly lower supply of calcium than the regular diet. There are inconsistent data showing differences in the supply of this component between GFD and regular diets. The results reported by Dall'Asta et al. [27] confirm our observations, whereas Wild et al. [16] suggest that patients with GFD may consume higher amounts of calcium than those with a non-GFD diet. There are also considerable differences in the supply of this

nutrient to CD patients depending on their sex and age [38]. Due to the high supply of phosphorus, the menus analyzed in the present study had a very unfavorable Ca:P ratio, i.e., 0.26:1 in GFD and 0.32:1 in the regular diet. The correct ratio of these two minerals is 1.5:1 [39].

The supply of vitamins in both diets was especially high in the case of vitamins A and B6. The consumption of vitamins C, vitamin D, and folate did not meet the recommended intake. An inadequate supply of these nutrients was also observed in other studies on the nutrition of Polish prisoners [34,40,41]. The high supply of vitamin A in the analyzed menus was probably related to the frequent use of margarine (30–90 g per day), which is obligatorily fortified with this vitamin in Poland. The large standard deviations in the case of the vitamin A supply were probably associated with the presence of fried pork liver in many menus for the convicts. The supply of vitamin B6 in both diets covered over 200% of DRI. It is generally lower in the Polish population, as 16% of males and 36% of females do not consume or supplement its recommended amounts [42]. Vitamin D, the consumption of which in the daily food ration did not cover even half of the recommended amount, is especially important for celiac disease patients. It plays a key role in the regulation of immune response and may have an impact on CD [43]. As shown by research conducted in the USA, only 31% of prisoners have normal levels of this component in blood [44]. The cutaneous synthesis of this vitamin in prisoners may be lower than in the general population due to the limited time spent outdoors. However, deficiencies of this component have even been detected in 90% of individuals imprisoned in places with high sun exposure [45]. In our opinion, the low supply of vitamin C was probably caused by the limitation of the assortment of vegetables to a few cheapest ones, which are not a good source of this vitamin (beetroot, carrots, onions, and potatoes). Similarly, the range of fruit served in the diets sporadically was limited to apples. The infrequent presence of vegetables in the diets probably contributed to the low supply of folate. A significantly lower supply of this nutrient was noted in GFD, which may have been associated with the exclusion of many cereal products from this diet.

Analyses of the present results should take into account that the quality of GFD meals served in prisons may be influenced by the low catering budget allowance. This was evidenced by the results of our previous investigations of the nutrition for Polish prisoners, which demonstrated that the limited financial means resulted in a large reduction in the range of fruit and vegetables served in prison meals [34]. The mean all-day purchase-only cost of prison-provided meals and beverages in the GFD was 6.65 ± 0.61 PLN (1.49 ± 0.14 EUR). In Polish supermarkets, this is a purchase price of only 250 g of gluten-free bread or 300 g of gluten-free pasta. Gluten-free (GF) products are more expensive than their standard equivalents [46]. As shown by some data, they may be on average 159% more expensive than regular products in the UK [10] and from 22 to even 334% more expensive in Greece [47]. Probably because of these costs, the menus in all prisons did not include certified GF products; instead, the meals were based on naturally GF products. Both groups of GF products, however, may contain various levels of gluten. Verma et al. [48] showed that its allowable value (20 ppm) was exceeded in 9% of GF products available in stores in Italy.

GFD requires special attention from nutritionists and kitchen staff not only in terms of the nutritional value of the meals served. The difficulties in providing this type of diet in food service establishments are associated with an appropriate supply of GF products, storage of these products, production processes, tools, and processing methods [49]. It has been shown that contamination of kitchen utensils or food-contact surfaces with gluten in school kitchens is promoted by the use of non-protease detergents, lack of rinsing with water immediately before use, storage in open containers, and washing in dishwashers (compared to manual washing) [50]. Therefore, there is a need to investigate GFD served in prisons, with focus on the aspect of proper meal processing in this diet.

#### *Limitations*

It should be emphasized that the present results are based solely on analysis of the menus, but they do not include food that prisoners can buy and receive from families. Another limitation in the results

is the lack of knowledge of whether all prisoners consume the same portions of meals. This is related to the complicated prison hierarchy and confinement in cells.

#### **5. Conclusions**

The results of our research indicate that GFD meals in Polish prisons provide significantly lower amounts of many micronutrients than regular diet meals. This is caused by the exclusion of gluten-containing food from this diet, which is an important source of, e.g., fiber, iron, zinc, and folates. Both analyzed types of diets exhibited excess levels of SFA, sodium, calcium, phosphorus, copper, and vitamins (A, B6, C, D and folates). The results regarding the supply of nutrients necessitate action from the Central Board of the Prison Service aimed at introduction of external controls and improvement of the quality of meals. There is also a need for a comprehensive discussion on the possibility of supplementation of prisoners, especially with vitamin D. Undoubtedly, the quality of meals is related to the limited financial resources in Polish prisons. This makes it difficult to plan balanced meals by prison nutritionists, especially in the case of GFD meals, which are more expensive.

**Author Contributions:** Conceptualization, A.K. and P.S.; methodology, A.K., P.S., D.D. and W.G.; software, A.K., P.S. and D.D.; formal analysis, A.K., P.S. and D.D.; data curation, P.S.; writing—original draft preparation, A.K., P.S., D.D. and W.G.; writing—review and editing, A.K. and P.S.; supervision, P.S. and W.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Updated Food Composition Database for Cereal-Based Gluten Free Products in Spain: Is Reformulation Moving on?**

#### **Violeta Fajardo, María Purificación González \*, María Martínez, María de Lourdes Samaniego-Vaesken, María Achón, Natalia Úbeda** † **and Elena Alonso-Aperte** †

Departamento de Ciencias Farmacéuticas y de la Salud, Facultad de Farmacia, Universidad San Pablo-CEU, CEU Universities, Urbanización Montepríncipe, Alcorcón, 28925 Madrid, Spain; violeta.fajardomartin@ceu.es (V.F.); mar.martinez1.ce@ceindo.ceu.es (M.M.); l.samaniego@ceu.es (M.d.L.S.-V.); achontu@ceu.es (M.A.); nubeda@ceu.es (N.Ú.); eaperte@ceu.es (E.A.-A.)

**\*** Correspondence: mpgonzal@ceu.es; Tel.: +34-913-724-719

† Natalia Úbeda and Elena Alonso-Aperte share senior authorship.

Received: 21 July 2020; Accepted: 5 August 2020; Published: 7 August 2020

**Abstract:** We developed a comprehensive composition database of 629 cereal-based gluten free (GF) products available in Spain. Information on ingredients and nutritional composition was retrieved from food package labels. GF products were primarily composed of rice and/or corn flour, and 90% of them included added rice starch. The most common added fat was sunflower oil (present in one third of the products), followed by palm fat, olive oil, and cocoa. Only 24.5% of the products had the nutrition claim "no added sugar". Fifty-six percent of the GF products had sucrose in their formulation. Xanthan gum was the most frequently employed fiber, appearing in 34.2% of the GF products, followed by other commonly used such as hydroxypropyl methylcellulose (23.1%), guar gum (19.7%), and vegetable gums (19.6%). Macronutrient analysis revealed that 25.4% of the products could be labeled as a source of fiber. Many of the considered GF food products showed very high contents of energy (33.5%), fats (28.5%), saturated fatty acids (30.0%), sugars (21.6%), and salt (28.3%). There is a timid reformulation in fat composition and salt reduction, but a lesser usage of alternative flours and pseudocereals.

**Keywords:** gluten-free products; celiac disease; gluten-free diet; gluten containing products; food composition database

#### **1. Introduction**

One percent of the general population in the Western world is affected by celiac disease (CD), one of the most common food intolerances in Europe [1]. CD is an autoimmune disorder with an aberrant response to gluten proteins with subsequent atrophy of intestinal villi, impaired intestinal absorption, and malnutrition. Extra-intestinal symptoms such as fatigue, iron deficiency, and neurological/psychological disorders (e.g., depression) may also be present. Long-term risks associated with CD, such as lymphoma, osteoporosis, and anemia, have also been reported [2].

A strict and lifelong adherence to a gluten free diet (GFD) is the first-line treatment and, currently, the only effective therapy for celiac patients and all other gluten related disorders, such as non-celiac gluten sensitivity or wheat allergy [3]. Gluten originates from a family of proteins found in wheat (gliadins and glutenins), rye (secalins), barley (hordeins), and oats (avenins), or in their hybridized strains (e.g., spelt or kamut) [4]. A GFD comprises only naturally gluten free (GF) food products (e.g., legumes, fruit and vegetables, unprocessed meat, fish, eggs, dairy products, and GF cereals, such as rice or corn) and/or substitutes of wheat-based foods, specially manufactured without gluten

or having a gluten content lower than 20 ppm, as per European legislation [5]. For the traditional gluten-containing foods, such as bakery products, there is currently a wide variety of GF options available that use GF cereals (rice, corn, millet, and sorghum) and pseudocereals (quinoa, buckwheat, amaranth, and teff) as their base ingredients [6]. However, a GFD is difficult to follow because gluten is an ingredient widely used in the food industry, appearing in products that originally do not contain gluten such as meat, fish, and many other foodstuffs [7]. Hence, product labels and ingredient lists need to be carefully reviewed.

Consumer's interest and demand has led to a significant increase in the production and sales of GF products. Global market data indicate that GF product sales are forecasted to increase by a compound annual growth rate of 7.6% between 2020 and 2027 [8]. In the last decade, Spain has been the leader country increasing its production of GF goods (18.8%), compared to Western Europe and the rest of the world (13.6% and 15.4%, respectively), and has become the third world producer of this type of products, after U.S.A. and Brazil. In 2019, the European region held the maximum market share in the GF products market [7]. Reasons for this growth are not only due to purchases by those with CD or those with a gluten sensitivity but are also propelled by changes in consumer attitudes towards health. Mainstream consumers are experimenting with their diets for health-related reasons, and "free-from" foods (such as GF foods) are part of that trend [9].

However, comprehensive nutritional composition data of GF products, mainly vitamin and mineral content, are still scarce or limited [10,11]. More importantly, access to such data is even more restricted, since there is a broad lack of micronutrient data in food composition tables, databases, and food labels. This statement warrants the need of providing new data on mineral and vitamins in GF food products, to complete food composition tables or databases, to cover regulatory purposes, and/or to assess population dietary intakes [12]. Composition data are useful to evaluate the adequacy of nutrient intake of celiac patients, on which the debate is still open, and are, therefore, strongly needed [13].

The GFD has demonstrated benefits in managing some gluten-related disorders, although nutritional imbalances have been reported. Although a GFD is associated with being healthier by some authors [9,14], epidemiological studies indicate nutritional imbalances in different celiac populations following a GFD, both in children [13,15] and in adults [16,17]. They refer to both macronutrients and micronutrients, including minerals. Overall, nutritional imbalances include high lipid, high protein, and low fiber intakes, and lack of adequacy to reference intakes of vitamin D, calcium, and magnesium [13,15]. Celiac patients may also be at risk of iron and folate deficiencies [17]. Some authors state that nutritional deficiencies in CD patients may be due to GF products, which are made with highly refined flours and high amounts of fat and sugar to achieve a texture resembling the typical and unique viscoelastic properties of wheat [18,19].

To provide better consumer information, the objective of this paper is to develop a nutritional food composition database including cereal-based GF products available in Spain. For this purpose, we estimated the nutritional composition of the products considering both the nutritional information and the ingredients reported on the product label, focusing on the critical components that define the nutritional quality of a GFD (added flours, starches, fats, sugars, and fiber).

#### **2. Materials and Methods**

#### *2.1. Design and Data Collection*

The present study involved the compilation of cereal-based gluten-free (GF) products available in the Spanish market. Products and brands were gathered systematically from manufacturer websites and/or specialized retail stores and supermarkets with the highest market shares in Spain between September 2016 and March 2019. Retailers such as Carrefour, Hipercor, Mercadona, Alcampo, and Lidl, as well as smaller specialized stores, were visited. Information on ingredients and nutritional composition was retrieved from the food package labels. Major commercial and distribution brands

were selected. All products included in the study showed one of the following claims on the package: the European Crossed Grain Trademark, the Spanish Federation of Coeliac Associations (FACE) crossed grain symbol, or the "gluten free" claim.

#### *2.2. Food Database Development*

The cereal-based GF food database was developed according to LanguaL™ Thesaurus EuroFIR [20]. In total, 629 cereal-based GF food items were categorized into four groups, nine subgroups, and thirteen subgroup categories in the developed database, in consonance with the LanguaL™ classification. The four groups were beverages, milk, milk product, or milk substitutes, grain or grain products, and miscellaneous food products.

The grain or grain products group comprised six subgroups: bread and similar, breakfast cereals, cereal or cereal-like milling products and derivatives, fine bakery ware, pasta and similar products, and savory cereal dishes. Beverages (non-milk) included alcoholic beverages. The milk, milk product, or milk substitutes group included data from frozen dairy desserts. Finally, miscellaneous food products involved prepared food products.

Each cereal-based GF food item was assigned to one of the following subgroups and categories: beer or beer-like beverages, frozen dairy desserts, bread products, leavened breads, unleavened breads, crisp breads, and rusks, breakfast cereals and cereal bars, cereal or cereal-like milling products and derivatives, biscuits, sweets, and semi-sweets, pancakes or waffles, pastries and cakes, pasta and similar products, pasta dishes, pies, unsweetened, or pizzas, savory cereal dishes, and savory snacks (see Table 1). Bread products included breadcrumbs. Leavened breads included rolls, buns, breads baked in pans and French type. Cereal or cereal-like milling products and derivatives included flour and flour preparations for baking products.

#### *2.3. Food Composition in Terms of Ingredients*

The ingredient list used in the formulation of the GF products was analyzed. Four groups of critical ingredients were considered: starchy ingredients (i.e., flour or starch), fats (oils and fats), sugars (i.e., dextrose), and added fibers (i.e., xanthan gum). Ingredients were chosen according to their impact on the nutritional profile of GF products. In each case, the top ten most frequently used ingredients were considered.

#### *2.4. Nutritional Information Study*

The nutritional composition of each product item included in the database is given in terms of quantity of energy and nutrients per 100 g of product as sold. Energy expressed in kcal, macronutrients (fats (g), saturated fatty acids (g), carbohydrates (g), sugars (g), protein (g)), fiber (g), and salt (g) were the data on nutrient composition reported on the label of each product. Micronutrients, vitamin, and mineral contents were not declared on the label in hardly any GF products.

#### *2.5. Statistical Analysis*

Descriptive data on ingredients are expressed as frequency (number of products including a specific ingredient and percentage based on the total products within the category or the subgroup). Data on nutrient composition are expressed as average and standard deviation.

#### **3. Results**

GF products available in Spanish markets were systematically compiled between September 2016 and March 2019. In total, 629 cereal-based GF products were studied, and each food item was assigned to one of nine subgroups, based on LanguaL™ Thesaurus 2017 (Figure 1).

The main group was fine bakery ware (*n* = 229; 36.4%), followed by bread and similar products (*n* = 152; 24.2%) and pasta and similar products (*n* = 88; 13.9%). Minor categories were alcoholic beverages (*n* = 14; 2.2%) and frozen dairy desserts (*n* = 6; 0.9%). The targeted GF products belonged to more than 70 different commercial brands. Among the top five manufacturers of GF products (Schär, Santiveri, Airos, Adpan, and Proceli), four of them are Spanish companies. However, it should be noted that the leading producers were mostly different across different GF product categories. We developed an initial GF product database in 2016, including 271 cereal-based GF food products. Up to 10% of the foodstuffs found in the present update are no longer available, and 24.5% have been reformulated.

**Figure 1.** Cereal-based gluten free (GF) products included in the database (*n* = 629).

#### *3.1. Food Composition in Terms of Ingredients*

Cereal-based GF products were primarily composed of rice and/or corn flour, and almost 90% of them included added rice starch (Tables 1 and 2). Less than 10% of the GF products were formulated with other kinds of flours, such as buckwheat, soy and other legumes, brown rice, millet, or quinoa. Oatmeal, sorghum, amaranth, teff, guar, chia, chestnut, flax, or potato flours were very rarely present in the ingredient list. Corn starch was present in the formulation of 60% of the products, and other commonly added were rice and potato starches. Tapioca starch, modified starch, and potato maltodextrin were also found in some products. Barley malt was the main cereal used for beer-like beverages.

Considering bread products, wheat is the main flour used in Spain, but it must be substituted by rice flour, followed by corn flour, and frequently added with rice or corn starches when baking GF breads. Corn flour is more frequently used than rice flour when preparing GF breakfast cereals, pasta, and savory snacks. Soya flour was most frequently used in fine bakery ware, with 26% of biscuits, sweets, and semi-sweets containing this type of flour. Added starches were found in all products except for breakfast cereals and beer-like beverages.


*Nutrients* **2020** , *12*, 2369

flax, or potato.


*Nutrients* **2020** , *12*, 2369

One hundred thirty different combinations of added fats were found in the recorded GF food products. The most added fat was sunflower oil, which was present in almost one third of the products, followed by palm fat, olive oil, and cocoa, all used similarly in around 13% of the products. Other animal fats (butter, cream, or lard), margarines, rapeseed oil, and coconut oil were more seldom used (Table 3). Therefore, unsaturated fats were predominant in most of the GF foodstuffs considered in the database. However, palm fat was the main fat added to biscuits, sweets, and semi-sweets, classified in the fine bakery ware subgroup, in addition to other saturated fats such as cocoa and animal fats. When focusing on frozen dairy desserts, it was observed that only saturated fats were present (animal fats, coconut oil, palm oil, and cocoa). Both results are in accordance with the high amounts of fat and saturated fats found, respectively, in the subgroups in the macronutrient analysis (see Section 3.2). The three most used margarines were made by: palm, coconut, and sunflower; palm, coconut, and rapeseed; and coconut with sunflower. Bread and similar products and fine bakery ware were the subgroups in which margarines were more frequently used. Taken together, palm fat and margarines made up with palm oil were present in 22.8% of the GF products. Finally, no added fats or oils were used as ingredients in pasta and similar products, according to the labeling.

Only 154 GF foodstuffs (24.5%) had the nutrition claim "no added sugar" on the label [21], being pasta and similar products the most representative subgroup of this fact (Table 4). Among the added sugars, we found that 55.8 % of the GF products had sucrose in their formulation. Other sugars and sweeteners less employed were, in order of frequency, glucose, fructose, dextrose, and lactose. Other rich sugar ingredients such us non-refined or cane sugar, rice syrup, beetroot sugar syrup, and honey were also present in the GF products. Leavened breads, biscuits, sweets, semi-sweets, pastries, and cakes were the subgroups were GF products more frequently contained added sugars, with almost 100% of the products containing sucrose, dextrose or glucose, and fructose. Very few breakfast cereals and fine bakery ware (<3%) included no calorie sweeteners.

Table 5 shows the type of fibers used in the formulation of GF products. Xanthan gum was the most frequently employed fiber, appearing in 34.2% of the GF products, followed by other commonly used such as hydroxypropyl methyl cellulose (23.1%), guar gum (19.7%), vegetable gums (psyllium, bamboo, chicory, potato, rice, pea, corn, etc.) (19.6%), and sodium carboxymethyl cellulose (6.4%). Fibers less frequently found in GF products were citrus fiber, carrageenan, pectin, cellulose, locust bean gum, and apple fiber (appearing in 1.6 to 2.7% of the products). The least fiber enriched products were breakfast cereals and pasta and similar products, whereas the most frequently supplemented were bread and similar products, fine bakery ware, and savory cereal dishes. Macronutrient analysis (Table 6) revealed that 25.4% of the products could be labeled as a source of fiber (>3 g/100 g), mostly breads, breakfast cereals, milling products, and fine bakery ware.

#### *3.2. Nutritional Information*

The highest amount of energy, total fats and sugars was found in the fine bakery ware subgroup (426.1 ± 77.7 kcal/100 g, 20.5 ± 6.8 g/100 g and 22.2 ± 9.2 g/100 g, respectively). The highest content of saturated fats was found in frozen dairy desserts (8.8 ± 4.2 g/100 g); proteins in savory cereal dishes (7.7 ± 2.0 g/100 g); carbohydrates in pasta and similar products (76.5 ± 5.9 g/100 g); and fiber and salt in bread and similar products (5.2 ± 2.2 g/100 g, 1.5 ± 0.5 g/100 g, respectively). Average salt content in all products was 0.6 ± 0.4 g/100 g. Highest contents were found in bread and similar products, savory cereal dishes, and prepared food products (Table 6).


*Nutrients* **2020** , *12*, 2369

> **Table 3.** Fat ingredients

 used in the formulation

 of gluten free (GF) products.


**Table 4.** Sugar addition and types of sugars and sweeteners used in the formulation of gluten free (GF) products. Results are expressed as frequency (*n*, number of products including a specific ingredient) and (percentage based on the total products within the category or the subgroup). sugarsincludeisomaltose,fructose,glucose,agavesyrup,goldensyrup,maltodextrin,orhighmaltosecornsyrup.

#### *Nutrients* **2020** , *12*, 2369



gums include psyllium, bamboo, chicory, potato, rice, pea, or corn. \*\* Extract from apple or other fruits.


**6.**Energyandnutrientcomposition100ofglutenfree(GF)products,basedthenutritionalinformationthe

Data are expressed as average ± standard deviation. SFA, saturated fatty acids.

#### *Nutrients* **2020** , *12*, 2369

Many of the considered GF food products showed very high contents of: energy (33.5%), defined as >400 kcal/100 g; fats (28.4%), defined as >17.5 g/100 g; saturated fatty acids (30.0%), defined as >5 g/100 g; sugars (21.6%), defined as >22.5 g/100 g; and salt (28.3%), defined as >500 mg of sodium or the equivalent value for salt /100 g. On the other hand, 25.4% could be labeled as a source of fiber (>3 g/100 g) [21,22].

#### **4. Discussion**

This cross-sectional study of 629 cereal-based GF products represents the largest comparative nutrient analysis of packaged Spanish GF food products and their ingredients, up to date. Most of the considered GF food products (~ 30%) showed very high contents of energy, fats, saturated fatty acids, sugars, and salt. In contrast, 25.4% could be labeled as a source of fiber [21,22]. Compared to other recent studies, our food composition database showed similar or slightly lower nutrient values than others [23–27].

It is important to mention that there is not an unequivocal nutritional profile for GF food products worldwide. Differences from country to country, from brand to brand, and among food categories have been asserted. Furthermore, differences could be attributable to different methodology (product selection) between studies. Nutritional values of each food item included in the present database were calculated as average of all the single similar foods from each brand included in each category. Therefore, nutritional composition variability for each food item due to its ingredient formulation has been considered.

Regarding ingredients, we found that the main fat component of GF products was sunflower oil, followed by palm fat, olive oil, and cocoa fat. This result differs to that shown by Calvo-Lerma et al. [25] in a similar study conducted in Spain, in which they found that GF products were largely composed of palm oil. Our database was developed up to March 2019 and the data collection in the study by Calvo-Lerma et al. [25] was conducted between March and October 2017. In this sense, this could indicate recent food reformulation to improve the nutritional quality of fat, thus providing a healthier food choice for celiac patients. In fact, recent research on palm fat and oil has brought up intriguing health issues, due to the presence of toxic contaminants generated in the processing of palm oil and other vegetable oils. This has promoted an update on the tolerable daily intakes (TDI) for toxic contaminants (2- and 3-monochloropropanediols and glycidyl esters [28,29]), but has also posed some misunderstandings in mass communication [28]. Consumers have been aware of these issues through the media, demanding and purchasing only palm oil free biscuits, especially for children. Processed foods are constantly changing as manufacturers try to protect or increase market share and profits and respond to policy changes dictated by a combination of government policies and consumer pressure, e.g., reduction of sugar, salt, saturated, and trans fatty acids [30]. General awareness of the role of diet on health is boosting the rate of changes in composition and foods consumed in many countries.

In the case of breads, we found that fat was commonly added to leavened breads, being it sunflower oil in more than 50% of the cases, followed by olive oil and a margarine made with palm, coconut, and sunflower oils. Consequent nutritional composition renders a considerably high amount of fat (5.7 %), most of it unsaturated (61%). Our data are slightly lower than those given by Calvo Lerma et al. [25] for total fat in a study of 619 GF products conducted in 2017. Miranda et al. [10] also studied Spanish GF commercialized breads in a study of 206 GF products, undertaken between 2012 and 2013, and stated that these contained less protein and double the fat content, (being this fat mainly SFA), in contrast to their gluten containing counterparts. Again, producers may be reducing fat in leavened breads, although data show that there is a large variability in fat composition when comparing different brands.

Fat content in pastries, cakes, pancakes, or waffles was 30% saturated and the most commonly added fat was palm fat. Results are in agreement with other studies [10,25].

GF products are made with high amounts of fat and sugar to achieve a texture resembling the typical and unique wheat viscoelastic properties [18,19]. Fat ingredients are indeed useful in bakery products for the stabilization of gas bubbles and the reduction of kneading resistance and swelling of starch granules [31]. Moreover, emulsifiers can be used to increase dough stiffness, improve bread structure, and decrease the speed of staling. In pasta products, emulsifiers act as lubricants in the extrusion process and provide firmer consistency and a less sticky surface, as they control starch swelling and leaching phenomena during cooking [32]. Other components such as sugars (sucrose, glucose, and fructose syrup), starch (corn starch, rice flour, and corn flour), and fibers (xanthan gum, hydroxypropyl methyl cellulose, and guar gum) were also present in GF products in our database.

It is interesting to point out that almost all breads contained added sugars (98% of leavened breads and 79% of unleavened breads, crisps, and rusks). Sugar is not a common ingredient in bread. In Spain, normal bread is solely composed of flour (usually wheat), salt, baker's yeast, and water [33], although sugar may be added to special breads. Sugar addition in bread is normally a matter of concern since bread is not usually associated with sugar consumption in the population. Due to the sugar addition, simple carbohydrate composition raised to values around 5.2 g per 100 g. This amount is similar to that described by Miranda et al. [10] in 2012–2013, and somewhat smaller than that described by Calvo Lerma et al. [25] in 2017. Nonetheless, both authors state that there is no significant difference in sugar content when comparing GF breads with their gluten containing counterparts. Therefore, gluten-containing flour may be contributing to sugar composition on a higher amount as compared to GF flour. To prove this idea, sugar content in GF pasta, without added sugars, was quite low (below 1%), and other authors have demonstrated that GF pasta contains a significantly lower amount of sugars as compared to gluten containing pasta [10,25].

Cereal-based GF products were primarily composed of rice and/or corn flour. Less than 10% of the GF products were formulated with other kind of flours, such as buckwheat, soy and other legumes, brown rice, millet, or quinoa. Therefore, the type of flour used results in a high glycemic index, because of the high content (70–80%) of amylopectin and related glucose polymers. The use of pseudocereals is still small, although several authors present them as good gluten free alternatives. According to Jastrebova and Jägerstad [34], the best way to develop nutritious healthy GF products with high content of proteins, fibers, micronutrients, and antioxidants, is natural fortification by using nutritious ingredients such as whole grain flours of GF cereals/pseudocereals, protein-rich flours of soy, lupin, chick-pea, chestnut, and different seeds, as well as bioprocessing, such as germination or fermentation with yeast and/or sourdough. Other authors suggest the use of pseudocereals such as amaranth, quinoa, or buckwheat because of their content in thiamine, vitamin E, or carotenoids [35] or the nutritional quality of their protein, fat, fiber, and minerals [36].

In our study, soy, legume, and quinoa flours were present in 8.4%, 7.3% and 4.1%, respectively, of the analyzed products. Breakfast cereals were the group with the most frequent inclusion of alternative cereals such as teff, oatmeal, sorghum, and other flours coming from chia, amaranth, or flaxseeds. Again, manufacturers seem to be timidly introducing the use of nutritious pseudocereal and legume flours in the formulation of GF products.

Xanthan gum and hydroxypropyl methylcellulose are the most popular hydrocolloids that are used in GF products. They display thickening properties through the binding of water and, as a result, the viscosity of the gluten-free dough is enhanced and gas is better retained, improving loaf volume and structure [14].

Differences between GF products and their equivalents with gluten have also been described in other studies. The most recent surveys on the nutritional quality of GF food products currently available on the market, and recently reviewed by Melini and Melini [18], show key inadequacies—a low protein content and a high fat and salt content—compared to their equivalent gluten-containing products. However, an interesting trend towards some improvements has emerged. More adequate levels of fiber and sugar than in the past have been reported in the surveys of the last two years, although the composition in terms of fiber and sugars is highly variable between the different product categories. Further studies are nevertheless required to investigate the micronutrient content of GF food products, since very few reported data exist. Kulai and Rashid [37] and Jamieson et al. [26]

informed of a significant lower iron and folate content in GF products compared to gluten containing food. Potassium content was also significantly lower in GF food products [27]. Furthermore, only 5% of GF breads were fortified with all four mandatory fortification nutrients (calcium, iron, nicotinic acid or nicotinamide, and thiamin), and 28% of GF breads were fortified with calcium and iron only in UK [24]. Fortified GF products represent only 10% of GF staple foods in Europe, because the use of starches (with low levels of many essential micronutrients) as main ingredient in many GF foods makes it difficult to implement common fortification with single micronutrients [34]. This lack of fortification may increase the risk of micronutrient deficiency in coeliac sufferers according to these authors. GF choices could account in unanticipated health disorders for CD patients based on the limited labeling description and narrow range of nutritionally balanced products and brands currently available [25].

Average salt content in all products was 0.6 g/100 g. As compared to other studies [10], we found much lower amounts of salt in pasta, cereal milling products, and fine bakery ware. Other studies [25] do not show data on salt content since salt content in nutritional information labels was only introduced as compulsory from December 2016 [38]. As with fat quality, salt reduction could be another of the reformulation targets that are being assessed in GF products.

Several population studies, in different countries, have investigated the nutritional status of CD patients adhering to a GFD. In published studies, CD patients consumed more fats (especially saturated), protein, and simple carbohydrates (sugars) but less fiber and micronutrients, such as iron, calcium, and vitamin D than recommended [11,17,39,40], and also compared to healthy subjects [16,41,42]. A research group from our laboratory recently showed no relevant differences in the general nutrient quality of the diet of children and adolescents following a GFD, as compared to matched controls, in contrast to previous studies, with the exception of polyunsaturated fatty acids, folate, and calcium intakes. These were significantly lower in coeliac as compared to non-coeliac children and adolescents, as well as low when compared to the recommended intakes for these nutrients [13]. Adequacy of vitamin D intake to recommendations was dramatically low, for both coeliac and non-coeliac children and adolescents; however, only coeliac girls presented a significantly lower level of plasmatic vitamin D (below reference values, <30 ng/mL), as compared to non-coeliac controls, although without clinical repercussion in bone mass density [13]. Therefore, we consider that vitamin D fortification in GF products could be a strategy of great importance to minimize adverse health effects associated to vitamin D deficiency.

#### **5. Conclusions**

In conclusion, the present study represents an attempt to build a systematic composition database of GF products based on the ingredients listed on the label and the nutritional information provided by the manufacturer. This type of study is a priority, since CD patients include this type of products in their diets, and studies assessing CD patient's diets need to use updated data on GF product composition. Moreover, since nutritional deficiencies have been described for CD patients and it has been shown that nutritional quality of GF products is lower, updated quality assessment of available products is needed for further improvement in GF product development. We describe 629 cereal-based GF products available in the Spanish market, in terms of ingredients and strategic nutrients. However, information on micronutrient composition is a still pending question.

#### *5.1. Strengths*

Studies that evaluate the formulations of commercially available GF products are scarce. In our study, we included an important number (629) of products and we describe them using a standardized classification (LanguaL™ Thesaurus EuroFIR), in order to make them comparable to other studies. Brands have also been recorded. Our data are likely to be just as accurate as most data reported for any kind of food product present on the market.

#### *5.2. Limitations*

Some limitations related to our data should be considered. First, since the nutritional composition data of GF products has been estimated, it cannot substitute a direct analysis. Direct chemical analysis is the gold standard to estimate the nutrient composition of food. Additionally, nutrient data shown on food labels provided by the food industry may be based on estimations from the ingredients rather than direct chemical analysis of the food products. Finally, another limitation is related to the lack of information on micronutrient content (minerals and vitamins) in GF products.

**Author Contributions:** E.A.-A. and N.Ú. designed, wrote the manuscript, supervised, and carried out the project administration. E.A.-A. and N.Ú., and V.F. were responsible for interpretation and discussion of the results. V.F., M.P.G., and M.d.L.S.-V. contributed to the review of the study protocol, design, and methodology. V.F., M.P.G., M.d.L.S.-V. and M.M. were responsible for the careful software, resources, formal analysis, and investigation. V.F., and M.P.G. designed, wrote—reviewed and edited the manuscript. E.A.-A., N.Ú., and M.A. revised the manuscript, and shared funding acquisition. E.A.-A., the Principal Investigator, was responsible for the design, protocol, methodology, and follow-up/checking of the study. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by a Grant FUSPBS-PPC08-2015 from Universidad CEU San Pablo, Madrid (Spain).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

## **Beneficial E**ff**ects of a Low-Nickel Diet on Relapsing IBS-Like and Extraintestinal Symptoms of Celiac Patients during a Proper Gluten-Free Diet: Nickel Allergic Contact Mucositis in Suspected Non-Responsive Celiac Disease**

#### **Ra**ff**aele Borghini 1, Natascia De Amicis 1, Antonino Bella 2, Nicoletta Greco 1, Giuseppe Donato <sup>1</sup> and Antonio Picarelli 1,\***


Received: 29 June 2020; Accepted: 24 July 2020; Published: 29 July 2020

**Abstract:** Background and Aim: Nickel (Ni)-rich foods can induce allergic contact mucositis (ACM) with irritable bowel syndrome (IBS)-like symptoms in predisposed subjects. Ni ACM has a high prevalence (>30%) in the general population and can be diagnosed by a Ni oral mucosa patch test (omPT). Many celiac disease (CD) patients on a gluten-free diet (GFD) often show a recrudescence of gastrointestinal and extraintestinal symptoms, although serological and histological remission has been achieved. Since a GFD often results in higher loads of ingested alimentary Ni (e.g., corn), we hypothesized that it would lead to a consequent intestinal sensitization to Ni in predisposed subjects. We wanted to (1) study Ni ACM prevalence in still symptomatic CD patients on a GFD and (2) study the effects of a low-Ni diet (LNiD) on their recurrent symptoms. Material and Methods: We recruited 102 consecutive CD patients (74 female, 28 male; age range 18–65 years, mean age 42.3 ± 7.4) on a GFD since at least 12 months, in current serological and histological remission (Marsh–Oberhuber type 0–I) who complained of relapsing gastrointestinal and/or extraintestinal symptoms. Inclusion criteria: presence of at least three gastrointestinal symptoms with a score ≥5 on the modified Gastrointestinal Symptom Rating Scale (GSRS) questionnaire. Exclusion criteria: IgE-mediated food allergy; history of past or current cancer; inflammatory bowel diseases; infectious diseases including *Helicobacter pylori*; lactose intolerance. All patients enrolled underwent Ni omPT and followed a LNiD for 3 months. A 24 symptoms questionnaire (GSRS modified according to the Salerno Experts' Criteria, with 15 gastrointestinal and 9 extraintestinal symptoms) was administered at T0 (free diet), T1 (GFD, CD remission), T2 (recurrence of symptoms despite GFD), and T3 (GFD + LNiD) for comparisons. Comparisons were performed using Wilcoxon signed-rank test. RESULTS: Twenty patients (all female, age range 23–65 years, mean age 39.1 ± 2.9) out of 102 (19.6%) were finally included. All 20 patients enrolled (100%) showed positive Ni omPT, confirming an Ni ACM diagnosis. A correct GFD (T0 vs. T1) induced the improvement of 19 out of the total 24 (79.2%) symptoms, and 14 out of 24 (58.3%) were statistically significant (*p*-value <0.0083 according to Bonferroni correction). Prolonged GFD (T1 vs. T2) revealed the worsening of 20 out of the total 24 (83.3%) symptoms, and 10 out of 24 (41.7%) were statistically significant. LNiD (T2 vs. T3) determined an improvement of 20 out of the total 24 (83.4%) symptoms, and in 10 out of 24 (41.7%) symptoms the improvement was statistically significant. Conclusions: Our data suggest that the recrudescence of gastrointestinal and extraintestinal symptoms observed in CD subjects during GFD may be due to the increase in alimentary Ni intake, once gluten contamination and persisting villous atrophy are excluded. Ni overload can induce Ni ACM, which can be diagnosed by a specific Ni omPT. Improvement of symptoms occurs after a proper LNiD. These encouraging data should be confirmed with larger studies.

**Keywords:** celiac disease; refractory celiac disease; remission; gluten-free diet; nickel allergy; allergic contact mucositis; irritable bowel syndrome (IBS); low-nickel diet

#### **1. Introduction**

Celiac disease (CD) is a chronic inflammatory bowel disease triggered by the ingestion of gluten in genetically susceptible individuals, who test positive for human leukocyte antigen (HLA) DQ2 and/or DQ8. Its prevalence is about 1%, and since the small intestine is its main target organ, CD can have gluten-related gastrointestinal manifestations, such as bloating, abdominal pain, diarrhea, and constipation [1–3]. What is more, CD is a multisystem disorder, and patients can also complain of extraintestinal signs and symptoms [4,5]. CD diagnosis in adults is usually based on positive results of specific serological tests for anti-endomysial antibodies (EMA) and anti-tissue transglutaminase (tTG) antibodies performed during a free diet and then confirmed by the finding of intestinal villous atrophy on histological examination of duodenal biopsies. The only treatment currently available is a lifelong and strict gluten-free diet (GFD) but, nevertheless, many CD patients complain about the persistence or relapse of symptoms even during GFD [6]: in this case, interviews with gastroenterologists and nutritionists are necessary in order to investigate a proper adherence to the GFD; moreover, repetition of serological tests and duodenal biopsies are mandatory to exclude ongoing intestinal damage and gluten exposure. When persistent damage in the duodenal mucosa is found despite a correct GFD, refractory CD and possible complications such as intestinal lymphoma must be investigated [7].

Moreover, in clinically non-responding CD, other possible overlapping diagnoses should be considered, such as inflammatory bowel diseases (IBD), and irritable bowel syndrome (IBS)-like disorders (e.g., lactose intolerance), but many cases seem to remain unsolved [8]. Recently, a diet low in fermentable oligo-, di-, and monosaccharides and polyols (FODMAPs) has been proposed as an ex adiuvantibus treatment to reduce IBS-like symptoms in CD patients following a GFD, although there is no specific indication or supporting diagnostic test [9,10].

More recently, nickel (Ni) allergic contact mucositis (ACM), which is linked to the ingestion of Ni-rich foods, has been added to IBS-like disorders. Together with Ni allergic contact dermatitis (ACD), Ni ACM is an expression of "systemic Ni allergy syndrome" (SNAS) and can have both gastrointestinal and extraintestinal manifestations. According to the European Surveillance System on Contact Allergy (ESSCA), the prevalence of an epicutaneous patch test positive to Ni may reach 30% in some European countries, but Ni ACM prevalence may even be higher [11]. Patients affected by Ni ACM show a low-grade intestinal inflammation with a local adaptive response to Ni-containing foods: this mucositis seems to be characterized by increased lymphocyte trafficking (type IV immune response) [12,13]. Ni ACM diagnosis is currently based on a Ni oral mucosa patch test (omPT), which has already proved good sensitivity and specificity [13], and a low-Ni diet (LNiD) can be thus suggested in this condition in order to significantly reduce both Ni-related gastrointestinal and extraintestinal symptoms [14–19].

Figure 1 shows the main foods with the highest Ni content, and it is easy to observe that many of them (e.g., corn) are consumed in large quantities by CD patients on a proper and strict GFD [15,16]. It is therefore possible that a high load of alimentary Ni may induce or exacerbate a "Ni sensitivity" in predisposed subjects, especially in CD patients on a long-term GFD.

On these premises, our aims were (1) to study the prevalence of Ni ACM in CD patients in serological and histological remission with relapsing symptoms; (2) to evaluate the effects of an LNiD on gastrointestinal and extraintestinal symptoms in these patients.

**Figure 1.** Foods with high nickel (Ni) content and their possible overlap with foods rich in fermentable oligo-, di-, and monosaccharides and polyols (FODMAPs). Here we report some of the main foods belonging to specific categories. To be noted is the overlap between Ni-rich foods and foods with high FODMAP content, as well as the overlap between foods high in FODMAPs and lactose content [10,15–19].

#### **2. Materials and Methods**

#### *2.1. Patients*

Study design: pilot study. We recruited 102 consecutive CD patients (74 female, 28 male; age range 18–65 years, mean age 42.3 ± 7.4) on a GFD since at least 12 months, with current serum EMA and anti-tTG antibodies negative results and histological remission (Marsh–Oberhuber type 0–I) who complained of relapsing or persisting gastrointestinal and/or extraintestinal symptoms. They referred to our Gastroenterology Unit from January 2017 to December 2019. Their CD diagnosis had been previously made according gluten-related signs and symptoms, serum EMA and anti-tTG antibodies positive results, and the histological finding of duodenal villous atrophy (Marsh–Oberhuber type IIIA, B, or C) [20–22].

Inclusion criteria: presence of at least three gastrointestinal symptoms with a score ≥5 on the modified GSRS questionnaire completed at study recruitment, in order to exclude patients with less significant clinical pictures. Exclusion criteria: IgE-mediated food allergy (diagnosed by skin prick test or laboratory tests (ImmunoCAP) for serum allergen-specific IgE antibodies); history of past or current cancer; inflammatory bowel disease; infectious diseases including *Helicobacter pylori* (HP); lactose intolerance (by means of the lactose breath test and genetic evaluation of lactase-gene polymorphism [23,24]).

The study was performed in compliance with the Declaration of Helsinki. Approval of the local ethics committee was obtained (study approval: report 8.2.0 06/2020 of the Board of the Department of Translational and Precision Medicine—Sapienza University of Rome). Written informed consent was obtained from all patients.

#### *2.2. Symptom Questionnaire*

The Gastrointestinal Symptom Rating Scale (GSRS) questionnaire modified according to "Salerno's experts' criteria" is a standardized tool used in the diagnostic protocol for non-celiac gluten sensitivity, and it has also been employed to objectively evaluate the clinical status in other IBS-like disorders such as Ni sensitivity [15]. It consists of a list of gastrointestinal and extraintestinal symptoms

associated to a numeric scale (score ranging from 0 to 10), which represents the intensity perceived by the patients during a specific dietary regimen [25]. Gastrointestinal symptoms include abdominal pain, heartburn, acid regurgitation, bloating, nausea, borborygmus, swelling, belching, flatulence, decreased or increased evacuations, loose or hard stools, urgent need for defecation, oral/tongue ulcers. Extraintestinal symptoms include dermatitis, headache, foggy mind, fatigue, numbness of the limbs, joint/muscle pain, fainting [15].

According to our standard outpatient treatment protocol for the management of CD, the questionnaire had been previously administered at CD diagnosis (T0) and after at least 12 months of GFD, when serological and histological remission had been achieved (T1). After at least three further months on a GFD, a third questionnaire was administered to those CD patients who complained of a relapse of gastrointestinal and extraintestinal symptoms, despite confirmed negative serological and histological results to exclude refractory CD (T2, study recruitment). The last questionnaire was administered after 3 months of LNiD in addition to GFD (T3).

#### *2.3. Nickel Oral Mucosa Patch Test*

Once enrolled in the study (T2), all patients underwent an Ni omPT to detect the presence of Ni ACM. Ni omPT is a 5 mm filter paper disk saturated with a 5% solution of Ni sulfate in Vaseline (0.4 mg Ni-sulfate/8 mg Vaseline). It is applied on the upper lip mucosa and held in place by a transparent adhesive film (Tegaderm, 3M, St. Paul, MN - USA). For appropriate diagnostic purposes, a control test with only 8 mg Vaseline is also provided and applied. Local Ni-induced type IV hypersensitivity reactions (e.g., edema, hyperemia, aphthous/vesicular lesions) can be evident after just 2 h of exposure or even after 24–48 h as late reactions. Late general symptoms triggered by omPT (e.g., swelling, abdominal pain, diarrhea, headache, foggy mind, itching) should also be considered as positive test results [14,15].

#### *2.4. Low-Nickel Diet*

All enrolled patients followed a balanced GFD with the addition of an LNiD for 3 months after a visit with trained dieticians, who verified correct adherence to both diet regimens by means of a daily dietary diary and biweekly telephone interviews. Ni is an element abundantly present in many foods, with a certain concentration variability depending on the type of soil and plant species, irrigation water, fertilizers, and pesticides. Thus, since its total elimination from the diet is impossible, we recommended to avoid only foods with an estimated high content of Ni (Ni > 100 μg/kg) (Figure 1). The use of stainless-steel utensils and pots has also been discouraged, in order to reduce Ni contamination during cooking [15,19,26].

#### *2.5. Statistical Analysis*

Data obtained during the present study were both qualitative (omPT results) and quantitative (modified GSRS questionnaire). Qualitative data were expressed as frequencies (both absolute and relative). The symptoms' scores (GSRS scale: 0 = absent, 10 = maximum intensity) were summarized by median, and Wilcoxon's signed-rank test was used to compare each symptom at different times (T0, T1, T2, T3). Applying the Bonferroni correction, *p*-value <0.0083 (alpha = 0.05/6 comparisons) was considered statistically significant. Statistical analysis was performed using the Stata software, version 16.0 (Stata Cooperation, College Station, TX, USA).

Study arrangement and patient enrollment are summarized in Figure 2a,b.

Study management, Ni omPT, administration of the modified GSRS questionnaire, patient follow-up, and final data processing were performed at the Department of Translational and Precision Medicine.

**Figure 2.** Flow charts of the study: (**a**) study arrangement; (**b**) patient enrollment. Legend: CD, celiac disease; GFD, gluten-free diet; GSRS, Gastrointestinal Symptom Rating Scale; LNiD, low-nickel diet; Ni omPT, nickel oral mucosa patch test; pts, patients.

#### **3. Results**

#### *3.1. Patients*

Of the 102 patients recruited, 17 patients were excluded since they did not meet the criterion of at least three gastrointestinal symptoms with a score ≥5 in the GSRS questionnaire completed at T2. Sixty-two out of the remaining 85 patients were also excluded: 54 were lactose intolerant, 7 were affected by HP infection, 1 was affected by overlapping active ulcerative colitis. Three out of the remaining 23 patients dropped out of the study, reporting that they no longer wanted to follow further food restrictions. Therefore, a total of 20 patients (all female, age range 23–65 years, mean age 39.1 ± 2.9, median age 40) completed the study (Figure 2b).

#### *3.2. Nickel Oral Mucosa Patch Test*

All 20 patients studied (100%) showed Ni omPT positive results and received an Ni ACM diagnosis. They all showed evident local mucosal alterations induced by Ni (erythema, edema, and/or vesicles) within 2 h after patch application (Figure 3a,b). What is more, all 20 patients showed at least one additional gastrointestinal or extraintestinal systemic symptom within 48 h after Ni omPT.

**Figure 3.** Nickel oral mucosa patch test (Ni omPT) results: Ni-sensitive patients before Ni omPT application (**a**) and after Ni omPT removal (2 h) (**b**).

#### *3.3. Symptom Questionnaire*

A correct GFD (T0 vs. T1) induced an improving trend in 19 out of the total 24 (79.2%) symptoms, and 14 out of 24 (58.3%) were statistically significant (*p*-value < 0.0083).

The prolonged GFD (T1 vs. T2) revealed a worsening trend in 20 out of the total 24 (83.3%) symptoms, and 10 out of 24 (41.7%) were statistically significant: abdominal pain, bloating, nausea, swelling, loose stools, dermatitis, fatigue, numbness of the limbs, and muscle and joint pain.

Once an Ni ACM diagnosis was obtained, an LNiD (T2 vs. T3) determined an improving trend in 20 out of the total 24 (83.4%) symptoms, and in 10 out of 24 (41.7%) symptoms the improvement was statistically significant. In detail, 12 out of 15 (80%) gastrointestinal symptoms improved, and 7 out of 15 (46.7%) showed a statistically significant improvement. In the same interval, 8 out of 9 (88.9%) extraintestinal symptoms showed an improvement, and 3 out of 9 (33.3%) significantly improved.

More details about gastrointestinal and extraintestinal symptoms during the different intervals analyzed are reported in Figures 4 and 5 and Table 1.

**Figure 4.** Variation of gastrointestinal symptoms in celiac patients during different stages of the study. The *p*-value was calculated using the Wilcoxon signed-rank test (statistically significant *p*-value < 0.0083

according to Bonferroni correction). Legend: GSRS, Gastrointestinal Symptom Rating Scale; T0, baseline, during gluten-containing diet; T1, after ≥12 months of proper gluten-free diet; T2, after ≥3 months of prolonged gluten-free and Ni-rich diet; T3, after 3 months of low-nickel and gluten-free diet.

**Figure 5.** Variation of extraintestinal symptoms in celiac patients during different stages of the study. The *p*-value was calculated using the Wilcoxon signed-rank test (statistically significant *p*-value < 0.0083 according to Bonferroni correction). Legend: GSRS, Gastrointestinal Symptom Rating Scale; T0, baseline, during gluten-containing diet; T1, after ≥12 months of proper gluten-free diet; T2, after ≥3 months of prolonged gluten-free and nickel-rich diet; T3, after 3 months of low-nickel and gluten-free diet.


**Table 1.** Gastrointestinal and extra-intestinal symptoms during the different intervals analyzed. The table shows for each category of symptoms how many of themimproved, worsened or remained stable in the different ranges of time considered. Data are reported in both absolute and percentage values. Legend: CD, celiac

#### **4. Discussion**

The persistence or recurrence of gastrointestinal and/or extraintestinal symptoms in CD patients during GFD is a very common condition and is a topic of great relevance. The causes of this problem are to be initially searched in an incorrect adherence to GFD and this is what can happen in those patients who still show persistently positive antibody titers and significant duodenal histological alterations, even despite quite a long period of GFD (>12 months). Furthermore, the possibility of refractory CD is always to be taken into consideration [4,6–8].

This issue becomes even more difficult to decipher and solve when CD patients reach serological and histological remission, but symptoms are still present or show a new peak despite a correct GFD. Furthermore, some of these patients may even report the appearance of new symptoms never complained about before. Other overlapping disorders, such as IBS or IBS-like disorders may be the causes of these symptoms and, in this regard, encouraging results have been obtained by ex adiuvantibus use of a low-FODMAP diet, although this approach revealed some limitations that will be discussed further [9,10].

On the other hand, many gluten-free foods consumed by CD patients are high in Ni content. Therefore, once a CD diagnosis has been obtained, the progressive Ni load induced by the GFD can trigger a relapse of symptoms in subjects predisposed to Ni allergy. Ni is present in many foods with different concentrations. It can be responsible for SNAS, which can have both gastrointestinal and extraintestinal manifestations. Specifically, Ni ACM is estimated to be one of the most common IBS-like disorders, and its diagnosis can rely on an Ni omPT, more sensitive and specific than the epicutaneous patch test [11]. What is more, excellent clinical results have already been obtained by Ni omPT and LNiD in the management of IBS-like and extraintestinal symptoms of women suffering from endometriosis who were still symptomatic despite different treatments [15,17].

Based on these considerations, we investigated the prevalence of Ni ACM in still symptomatic CD subjects after appropriate GFD and studied the effects of an LNiD on their gastrointestinal and extraintestinal symptoms.

Firstly, we selected symptomatic CD patients on a proper GFD who had no more serological and histological signs of disease activity. Then, we excluded those who did not meet the minimum clinical criterion by means of the GSRS questionnaire: in this way, we eliminated the less disabling and most confounding clinical pictures, even if this has led to a reduction in the number of patients studied. Other possible overlapping confounding pathologies have been excluded, such as lactose intolerance, HP infection and IBDs.

Our results showed an Ni ACM prevalence of 100% in the final 20 patients actually enrolled (Figure 3a,b): this percentage may appear extraordinarily high, but it is mandatory to consider not only the high prevalence of Ni ACM in the general population (estimated to be even greater than 30%) but also the strict exclusion criteria previously applied [11]. These 20 patients with Ni omPT positive results should be contextualized among the 85 CD patients who had a significant symptomatic picture: thus, Ni ACM should have a prevalence of at least 23.5% in our study. This percentage could have been higher considering not only the dropouts but also those patients affected by other pathologies who had been excluded in recruitment phase: in fact, Ni ACM can also easily overlap with other disorders, especially lactose intolerance, and in our study we excluded 54 lactose intolerant patients (about 63.5% of the 85 patients with a significant clinical picture) [11].

Afterward we focused on the effects of the different diet regimens on the symptoms.

First, we confirmed a general clinical improvement after CD diagnosis and a correct GFD (T0 vs. T1): once serological and histological remission were achieved, about 80% of the 24 total symptoms improved (almost 60% was also statistically significant).

On the other hand, a prolonged strict GFD (T1 vs. T2) resulted in a general clinical relapse involving more than 80% of all symptoms (the worsening was statistically significant in more than 40% of the symptoms). This negative change may be attributed to a GFD-related load of Ni in already sensitive or predisposed subjects.

This theory seemed to be confirmed after a balanced restriction of Ni-rich foods: in only three months, GFD plus LNiD (T2 vs. T3) induced an improvement of more than 80% of the symptoms and in the half of the cases the improvement was statistically significant, including the most complained about and disabling symptoms, such as abdominal pain, swelling, increased evacuations, and loose stools. Moreover, many of them got even better compared to the initial GFD alone (T1 vs. T3), although this difference was not statistically significant. As regards extraintestinal symptoms in the T2 vs. T3 range, dermatitis, headache, and fatigue statistically improved. Dermatitis deserves a special mention, as it showed a very peculiar trend: at the beginning (T0 and T1) it was almost totally absent, then it was significantly exacerbated reaching an acute peak at T2 and finally significantly reduced/resolved after GFD plus LNiD at T3. The curve of dermatitis' clinical course can further suggest the interference of an "alimentary trigger factor" during prolonged GFD, which is not related to gluten contamination, as demonstrated by the negative serological and histological results. The dietary profile of CD patients on a GFD and the impressive results of both Ni omPT and LNiD, would confirm that alimentary Ni overload is able to unmask/exacerbate not only gastrointestinal but also systemic symptoms, over a medium to long-term time period.

It should be emphasized that no symptoms significantly improved or worsened by comparing T0 (free diet) with T2 (Ni overload during GFD), suggesting a close clinical similarity between these two times: this is what often makes gastroenterologists think that the cause of the clinical relapse is a new gluten contamination.

The comparison between free diet and GFD + LNiD (T0 vs. T3) led to an improving trend of more than 70% of the symptoms: this is a very good percentage, although slightly lower than the almost 80% obtained from GFD alone, before Ni overload (T0 vs. T1). This may mean that, although LNiD is very effective in achieving a new clinical remission, Ni-sensitive patients cannot completely eliminate Ni, therefore, Ni-related symptoms, from their GFD.

The comparison between the well-being obtained by the initial GFD alone and the well-being obtained by the addition of the LNiD (T1 vs. T3) is also interesting: these two stages showed no statistically significant difference in symptoms' intensity perceived. This means that after the relapse peak in T2 (Ni load), a correct dietary intervention (LNiD) is able to completely restore well-being again.

The absence of a trial design was a limit of our study. Furthermore, it was carried out in a single center, the final sample size was quite small, and finally resulted in including only female patients. In addition, the very high prevalence of Ni omPT positive results (100%) may seem misleading.

Firstly, our results must be contextualized in the initial larger pool (102 total patients): the choice to exclude from the study those patients with less marked symptomatic pictures certainly led to the underdiagnosis of many other Ni-sensitive patients. In addition, it should be considered that Ni ACM can coexist with other disorders and can overlap with them from the symptomatic point of view [11]: in our study many HP-positive and lactose intolerant patients (almost 60% of the 102 patients initially recruited) were excluded for methodological correctness, and, thus, many other Ni-sensitive patients were probably lost among them.

The fact that the final 20 patients studied were only females is probably due to the greater prevalence of females in CD: in literature the female/male ratio is estimated to be about 3:1 and this proportion is approximately preserved in the 102 patients recruited at the beginning [27]. Furthermore, it has been described that Ni can act as a metalloestrogen and, thus, may have a greater influence in women with both extraintestinal and gastrointestinal clinical manifestations [15,28].

Given the strict differential diagnosis previously performed, this would explain such a high percentage (100%) of Ni omPT positive results. Moreover, the specific and successful treatment by LNiD seemed to confirm the appropriateness of our assumptions and supported the Ni omPT positive results: as above mentioned, more than 80% of symptoms improved after LNiD and about the half of them were statistically significant. In this regard, if we had not used the Bonferroni correction (*p* < 0.05 instead of *p* < 0.0083), the improvements of some other important symptoms (borborygmus, foggy mind, muscle pain, and joint pain) would have resulted statistically significant. We hope that future trials with larger populations will be able to confirm these preliminary observations.

Another weak point of our study, as well as possible obstacle for future trials, may be the impossibility to accurately measure Ni contained in foods and biological samples from patients studied. There is some variability of Ni content in foods and, to date, there are still no standard methods to measure it routinely: if these methods existed, we would have the possibility to prescribe highly personalized diets, more effectively monitor the intake of Ni-rich foods, and make even more appropriate comparisons. As recently demonstrated, we can successfully overcome this limit by prescribing patients a balanced LNiD on the base of an estimated average content of Ni in foods and under direct control of trained dieticians. A daily dietary diary and a detailed interview were also used to verify correct adherence to the GFD and LNiD.

It has already been described in literature that a low-FODMAP diet as an ex adiuvantibus treatment can benefit still symptomatic CD patients on a GFD. However, this dietary intervention has not so far been supported by specific diagnostic tests [9]. Moreover, it is known that many foods with estimated high content of FODMAPs may also cause other IBS-like disorders, such as lactose intolerance. It is therefore possible that during a low-FODMAP diet, other underlying and unrecognized diseases are treated. In this regard, we also observed a significant overlap between FODMAP-rich and Ni-rich foods (Figure 1), especially corn and other gluten-free foods consumed by CD subjects. It is therefore possible that the benefits of a low-FODMAP diet can depend on a concomitant involuntary LNiD in unrecognized Ni-sensitive patients. Given the high prevalence of Ni ACM and our results, this is more than only a hypothesis. On the other hand, our study can claim among its strengths an accurate preliminary differential diagnosis with other common IBS-like disorders and organic diseases. In addition, a targeted LNiD was prescribed after a reliable specific diagnostic test (Ni omPT), thus avoiding unnecessary dietary exclusions [15].

It may also be interesting to discuss the possible effects of close contact with patients during follow-up: frequent and extensive dietary interviews can have a placebo effect in clinical setting, capable even of inhibiting a symptom. On the other hand, they are essential methodological tools for an adequate quality assessment of the diet followed, as well as for the determination of the patient's clinical status. Close clinical contact also seemed to play a relevant role in supporting such "delicate" patients who had to follow two strict diet regimens: GFD and LNiD. This was confirmed not only by the encouraging clinical results obtained but also by the very low number of drop-outs. In addition, close clinical contact appeared even more necessary for such a study spread over a long period of time: the long time span of observation could have led to misestimation of the beneficial or harmful effects of dietary interventions. Finally, the inclusion of a significant number of symptoms in the GSRS standardized test for clinical evaluation has most probably helped to further reduce possible placebo/nocebo effects and misestimation of the results.

#### **5. Conclusions**

In conclusion, our findings show for the first time that Ni-rich foods and Ni ACM can frequently be the cause of relapsing gastrointestinal and extraintestinal symptoms in CD patients, even/especially during a correct GFD. Furthermore, our study not only confirms the usefulness of Ni omPT in making an Ni ACM diagnosis but also highlights that a balanced LNiD in addition to a correct GFD can offer a significant clinical improvement in this category of patients.

Further studies with larger populations should be carried out to confirm these important data, which may change the clinical management of CD patients.

**Author Contributions:** Conceptualization, R.B. and A.P.; data curation, R.B., N.D.A., and A.B.; formal analysis, R.B., N.D.A., A.B., N.G., and G.D.; investigation, R.B. and A.P.; methodology, R.B., A.B., and N.G.; project administration, R.B. and A.P.; software, A.B.; supervision, R.B., G.D., and A.P.; validation, R.B., G.D., and A.P.; visualization, R.B., N.G., and A.P.; writing—original draft, R.B.; writing—review and editing, R.B. and A.P. Guarantor of article: A.P. is the author who is acting as the submission's guarantor. All authors have read and agreed to the published version of the manuscript.

**Funding:** No institutional, private, or corporate financial support for the work was received. This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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