**Vitamin and Mineral Deficiencies Are Highly Prevalent in Newly Diagnosed Celiac Disease Patients**

**Nicolette J. Wierdsma 1,\*, Marian A. E. van Bokhorst-de van der Schueren <sup>1</sup> , Marijke Berkenpas <sup>1</sup> , Chris J. J. Mulder <sup>2</sup> and Ad A. van Bodegraven <sup>2</sup>**


*Received: 19 July 2013; in revised form: 13 September 2013 / Accepted: 13 September 2013 / Published: 30 September 2013* 

**Abstract:** Malabsorption, weight loss and vitamin/mineral-deficiencies characterize classical celiac disease (CD). This study aimed to assess the nutritional and vitamin/mineral status of current "early diagnosed" untreated adult CD-patients in the Netherlands. Newly diagnosed adult CD-patients were included (*n* = 80, 42.8 ± 15.1 years) and a comparable sample of 24 healthy Dutch subjects was added to compare vitamin concentrations. Nutritional status and serum concentrations of folic acid, vitamin A, B6, B12, and (25-hydroxy) D, zinc, haemoglobin (Hb) and ferritin were determined (before prescribing gluten free diet). Almost all CD-patients (87%) had at least one value below the lower limit of reference. Specifically, for vitamin A, 7.5% of patients showed deficient levels, for vitamin B6 14.5%, folic acid 20%, and vitamin B12 19%. Likewise, zinc deficiency was observed in 67% of the CD-patients, 46% had decreased iron storage, and 32% had anaemia. Overall, 17% were malnourished (>10% undesired weight loss), 22% of the women were underweight (Body Mass Index (BMI) < 18.5), and 29% of the patients were overweight (BMI > 25). Vitamin deficiencies were barely seen in healthy controls, with the exception of vitamin B12. Vitamin/mineral deficiencies were counter-intuitively not associated with a (higher) grade of histological intestinal damage or (impaired) nutritional status. In conclusion, vitamin/mineral deficiencies are still common in newly "early diagnosed" CD-patients, even though the prevalence of obesity at initial diagnosis is rising. Extensive nutritional assessments seem warranted to guide nutritional advices and follow-up in CD treatment.

**Keywords:** vitamins; minerals; celiac disease; deficiency; adult; Body Mass Index

#### **1. Introduction**

Celiac disease (CD) is the most common food intolerance in the Western population, and currently represents a major health care issue. The prevalence of CD has been estimate to be 0.5%–1% in different parts of the world [1]. CD is an inflammatory, immune-mediated chronic disease of the mucosa of the proximal small intestine due to irreversible gluten intolerance in genetically susceptible individuals. Gluten refers to a set of amino acid sequences found in the prolamine fraction of wheat, barley and rye. The characteristic histopathological finding is a varying degree of villous atrophy and crypt hyperplasia, primarily in the duodenum and jejunum, with inflammatory changes leading to malabsorption. The first-line, and up-till-now only, treatment is a lifelong strict adherence to a gluten free diet (GFD). All other treatment modalities suppress the intestinal inflammatory response and do not treat the intolerance [2,3].

CD is a multi-system disorder which leads to striking differences in its clinical presentation. When present, gastrointestinal symptoms, including clinically evident malabsorption, may facilitate the diagnosis of CD. Over the last few decades, there appeared to be a changing clinical presentation of CD from the classical malabsorptive picture (diarrhoea, malabsorption and weight loss) towards one of a non-classical presentation with milder, non-specific symptoms such as tiredness, hematologic abnormalities, constipation and/or abdominal distension [4–6]. Nowadays, many patients present with no or only minor extra-intestinal symptoms. Indeed, microcytic or macrocytic anaemia, or folate deficiency may occasionally be the only clinical symptom to suggest CD. This leads to a great extent of underdiagnoses in several countries [7]. Currently, 20%–40% of newly diagnosed CD-patients are even classified as overweight (Body Mass Index (BMI) > 25 kg/m2 ) instead of the anticipated underweight [8–12], although the prevalence of obesity seems lower than in the general population [13]. This makes the diagnosis of CD challenging. Greater clinical awareness—especially improved serological testing since the late 1990s, including that for anti-tissue transglutaminase (tTG) antibodies [14,15]—and the appearance of specialized centres has led to earlier recognition of CD-patients.

In the classically presenting CD-patients, malabsorption is frequently encountered [16], and micronutrient deficiencies may arise. Indeed, several studies demonstrate these deficiencies with varying results [17–21].

Our group recently demonstrated the specific functional insufficiency of the proximal small bowel in CD-patients by means of the citrulline generation test, in which the citrulline peak after glutamine administration and its conversion into citrulline in the enterocyte was delayed due to a decreased functional intestinal mass as a consequence of inflammatory changes [22].

Deficiencies of water-soluble vitamins, like B-vitamins, would be expected since they are absorbed in the proximal small bowel, which is the most prominent site affected in CD-patients. However, available data, in particular regarding vitamin B2 and B6 deficiencies, do not support this in untreated CD-patients [17,23]. Table 1 shows an overview of older and more recent literature on vitamin and mineral deficiencies in adult CD-patients.


**Table 1.** Literature overview on vitamin and mineral status in newly diagnosed adult celiac disease (CD)-patients.


**Table 1.** *Cont.*

It has been suggested that the current "early diagnosis" of CD might be associated with less vitamin and mineral deficiencies at the moment of diagnosis than the classical CD. Mineral and trace-element status of untreated CD-patients has not been widely studied. There is even a lack of recent reports in the literature (see Table 1) indicating which deficiencies should be checked in newly diagnosed celiacs in Western Europe. Therefore, we aimed to measure essential serum nutritional variables in order to assess the prevalence of vitamin and mineral deficiencies in untreated adult CD-patients from a tertiary referral Celiac Disease Centre, consuming a (gluten containing) standard Dutch (Western) diet before diagnosis. Secondly, we studied the nutritional status and differences in prevalence of vitamin and mineral deficiencies between patients with different grades of intestinal histological damage, nutritional status, and sex.

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

#### *2.1. Patients*

Eighty consecutively diagnosed adult patients (aged 18–75 years) with newly diagnosed CD were recruited from the Outpatient Clinic of the VU University Medical Centre, Amsterdam, the Netherlands, during the period 2005–2012. All patients consumed a normal (gluten-containing) Dutch-Western diet until inclusion. The mean daily gluten intake in the Netherlands is around 13 g [32]. Duodenal biopsy specimens were harvested to determine the grade of histological damage due to gluten sensitive enteropathy as classified by Marsh [33] and modified by Rostami [34,35]. Gastric (corpus) biopsies were routinely harvested to determine whether atrophic gastritis was present. CD associated antibodies, *i.e.*, anti-endomysial antibodies (EMA) and anti-tTG antibodies, were determined [36,37]. In addition, HLA-genotyping was performed, to analyse the presence of DQ2 and DQ8 (heterozygote or homozygote) as a prerequisite for a definitive diagnosis [38]. The diagnosis of CD was based on these histopathological, serological and genetic criteria.

Histopathological classification according to Marsh was used: intraepithelial lymphocytosis, crypt hyperplasia and villous atrophy Marsh IIIA, B and C (respectively, partial, subtotal and total villous atrophy) with or without elevated antibodies. Also, a group was added with low grade histopathological abnormalities Marsh I or Marsh II (lymphocytic enteritis with crypt hyperplasia) with gluten-dependent disorders, in case of elevated antibodies and the HLA-DQ2 or HLA-DQ8 genotype.

Blood samples were collected as part of routine clinical care. Patients were excluded if they had an established or suspected gastrointestinal abnormality other than CD, such as inflammatory bowel disease (IBD). A representative sample of 24 healthy Dutch subjects (comparable for sex, age and BMI) was added to compare concentrations of vitamin A, B6, folic acid and B12 (see control subjects characteristics [39]). Both groups were studied in the same period, were living in a similar environment, and measurements were performed at the same clinical chemistry laboratory.

The study protocol was approved by the Medical Ethics Committee (2005, project code 05.153) of the VU University Medical Centre Amsterdam, The Netherlands.

#### *2.2. Nutritional Status*

Patient characteristics and demographic data (including age (year), body height (m), body weight (kg) and self-reported involuntary weight loss in the past 1 and 6 months) were collected, BMI was calculated and biochemical analyses were performed following diagnosis and before any dietary advice to initiate a GFD. Patients were subsequently classified as "malnourished" when they unintentionally lost more than 10% of their bodyweight in the past 6 months or more than 5% in the past month prior to diagnosis or as having "risk of malnutrition" when 5%–10% of the bodyweight was unintentionally lost in the 6 months before diagnosis. Moreover, patients were classified into 3 groups on the basis of baseline BMI; less than 18.5 kg/m<sup>2</sup> (underweight), 18.5–25.0 kg/m2 (normal weight) and more than 25 kg/m2 (overweight, or even "obese" in case of BMI above 30 kg/m<sup>2</sup> ) (according to the definition of the World Health Organisation (2000)).

#### *2.3. Biochemical Analysis*

Fasting venous blood samples were drawn and subsequently analysed at the endocrine and clinical chemistry laboratories of the VU University Medical Centre, Amsterdam. Samples for serum folic acid and serum vitamin B12 were analysed by competitive immunoassay (Luminescence, Abbott, IL, USA). Serum vitamin B6 and vitamin A status were determined by high-performance liquid chromatography (HPLC). Vitamin (25-hydroxy) D was assessed with a competitive binding protein assay (Diasorin, Stillwater, MN, USA). Zinc status was assessed using Flame Atomic Absorption Spectroscopy (FAAS), serum haemoglobin by colorimetric methods (Cell Dyn Sapphire, Abbott, IL, USA) and ferritin values by electro-chemiluminescence immunoassay "ECLIA" (Roche, Mannheim, Germany) or "ACS CENTAUR" (Bayer, Mijdrecht, The Netherlands).

Vitamin B6 and folic acid (both proximally absorbed) and vitamin B12 (distally absorbed) were considered to represent the water-soluble vitamin status. Vitamin A and vitamin (25-hydroxy) D were considered to represent the fat-soluble vitamin status, although sun exposure, even in The Netherlands, may have a strong influence on serum levels of vitamin D. Haemoglobin and ferritin levels below the reference ranges listed were used to establish a respective diagnosis of anaemia, iron-deficiency or iron-deficiency anaemia when both haemoglobin and ferritin were below reference ranges. Patients with a serum value below the lower limit of the reference value were considered "deficient". Reference values for the different parameters are displayed in Table 3.

#### *2.4. Statistical Analysis*

Data were tested for normal distribution and presented as means ± SD. The percentage of patients with values below the reference value and absolute number of deficient patients were additionally calculated for all assessed serum vitamin and mineral concentrations.

Data were analysed for the total group, and in stratified subgroups by sex, histological damage (Marsh classification) and BMI. To determine differences with regard to gender, a Student's *t*-test was applied in case of continuous data and Pearson's Chi-~
 `#
 proportions (% of deficient patients). Analysis of variance (ANOVA), with a Bonferroni correction when a statistical significant difference was achieved, was used to compare more than two groups. A Mann-Whitney U test (Wilcoxon) or in case of more than 2 variables, a Kruskal-Wallis test was applied for variables not found in a normal distribution. The level of statistical significance was determined a priori at *p* < 0.05. Statistical analyses were performed using SPSS (Statistical Package for Social Sciences Inc., Chicago, IL, USA—Windows version 20.0).

#### **3. Results**

#### *3.1. Patient Characteristics and Nutritional Status*

Patient characteristics are shown in Table 2; two-thirds of the population was female. Male patients were significantly older than female patients (*p* = 0.006). Approximately 46% (37/80) of the patients had partial villous atrophy (Marsh IIIA) and 42.5% showed subtotal (20/80) or total villous atrophy (14/80) (Marsh IIIB or IIIC, respectively). Histologically and serologically atrophic (corpus) gastritis was ruled out by a pathologist experienced in intestinal histology. Some of the CD patients reported to have used vitamin and mineral supplements before diagnosis of CD was made: 18 (22.5%) a multivitamin, folic acid or vitamin B-complex, 7 (8.8%) iron supplements and 14 (17.5%) a calcium supplement. The anthropometric data of 24 healthy controls were comparable to those of the patients included.

Patients had, on average, lost 2.4% (±6.3%) of their bodyweight during the 6 months prior to diagnosis. Approximately 17% was classified as malnourished (>10% weight loss) and 5% as being at risk of malnutrition (5%–10% previous weight loss). Six out of 80 patients (7.5%), and only females, were classified as underweight, whereas 29% (23/80) of patients were classified as overweight (female:male ratio =1:1). Of the overweight patients, 26% were even obese (6/80 of the total group, with female:male ratio = 1:1).


**Table 2.** Patient characteristics of untreated adult CD-patients by gender and compared to healthy controls.

BMI: Body Mass Index, EMA: anti-endomysial antibodies, tTG: anti-tissue transglutaminase, ^ Significantly different from men (*p* < 0.05) by Student's *t*-test, n.d. (not determined), # low grade histopathological abnormalities with HLA-DQ2 and/or DQ8 and elevated antibodies (EMA and/or tTG), \* variables NS (not statistically significant from CD-patients (*p* < 0.05) by Mann-Whitney U test), \*a statistical trend *p* = 0.05.


#### *3.2. Biochemical Analyses*

Serum concentrations of vitamins and minerals of the untreated CD-patients are shown in Table 3. CD-patients were most frequently deficient for folic acid (20%, 16/80), followed by vitamin B12 (19%, 15/79), vitamin B6 (14.5%, 9/62), vitamin A (7.5%, 4/53) and vitamin (25-hydroxy) D (4.5%, 1/21), respectively. Approximately 67% (26/39) of the patients had zinc deficiency, 32.4% (23/71) had anaemia, 46.2% (18/39) had insufficient iron storage evidenced by low ferritin and 25% (8/40) had iron-deficiency anaemia. CD-patients had lower values of vitamin A and folic acid than healthy controls. Overall, vitamin deficiencies were barely seen in healthy controls, with the exception of vitamin B12. None of the healthy controls showed deficient levels (below the reference values) for vitamin A and vitamin B6 and only one for folic acid.

Ten patients (12.5%) were not deficient for any of the assessed vitamins and minerals. These patients had similar base-line characteristics to the rest of the CD group. The remaining 70 patients (87.5%) were deficient in at least one of the nutritional parameters and 43 (53.8%) for two or more parameters.

Table 3 depicts the proportion of deficient patients, stratified by sex. As anticipated, serum haemoglobin concentrations were lower in women than in men (*p* < 0.001). While, on the other hand, serum folic acid concentrations were lower (*p* = 0.040) in men when compared to women. No statistically significant difference was found for any of the other nutritional parameters when comparing males and females. Notwithstanding, multivitamin use (including folic acid or vitamin B supplement)

at their own volition or prescribed by the GP was more prevalent in women than men (30% *vs.* 13%). A statistical trend was seen for vitamin B6, suggesting that men might be more often deficient than women (*p* = 0.097).
