Low grade histopathological abnormalities with HLA-DQ2 and/or DQ8 and elevated antibodies (EMA and/or tTG), \* gender specific Females I/II 8.2 ± 0.29; IIIA 7.4 ± 0.95; IIIB 7.3 ± 0.84; IIIC 7.8 ± 0.97 and Male I/II 8.9 ± 0.78; IIIA 8.9 ± 0.96; IIIB 9.0 ± 0.67; IIIC 8.8 ± 0.72, ^ statistically significantly different (*p* = 0.041) by Kruskal-Wallis test.

### *3.4. Association between Vitamin and Mineral Concentrations and Nutritional Status*

Table 5 depicts the mean serum concentrations of vitamins and minerals per BMI stratum. A trend was observed for underweight patients having a slightly higher serum folic acid concentration than patients with normal weight or overweight patients (*p* = 0.058). Besides, patients with >10% unintentional weight loss in the past 6 months had higher vitamin A and (a trend for) higher vitamin B6 (*p* = 0.09) levels than patients without weight loss. Otherwise, no differences were observed between the different classes of nutritional status.



^ NS (not statistically significant (*p* < 0.05) by Kruskal-Wallis test, # statistically significantly different from well-nourished patients (Student *t*-test), *p* = 0.001.

#### **4. Discussion**

The present study showed that the majority of an "early diagnosis" adult untreated CD patient group (with so-called non-classical presentation) in the Netherlands, had at least one, and often several, serum vitamin or mineral deficiencies at diagnosis. Almost 90% of CD-patients were found to be deficient in at least one or more of the assessed nutritional parameters, and half of patients were deficient for two or more nutritional serum variables. This was observed to be unrelated to severity of clinical presentation, nutritional status or (semi-quantified) histopathological damage (score).

In this study, no statistically significant difference in serum vitamin and mineral concentration was found between men and women, although (multi)vitamin use prior to diagnosis was more prevalent in women than men (30% *vs.* 13%). Information is lacking whether they took the vitamins at their own volition, or if they were prescribed by the general practitioner. In addition, the assessed serum deficiencies were independent of Marsh stratum (Table 4), nutritional status (Table 5) and age (data not shown). Folic acid deficiency was observed in 20% of the untreated CD-patients in this study. The prevalence of folate deficiency varies from 18% to 90% in varying older and newer reports of CD-patients [17,18,24,26]. In studies from Scotland and Finland, folate deficiency was reported in 42% [18] and 37% [27] of the untreated CD-patients, respectively. The difference in prevalence might at least partially be explained by technical aspects of measurement of "folate" (which is the natural form of folic acid) and "folic acid" since bioavailability of folic acid is twice that of folate [40]. Macrocytic anaemia in untreated CD-patients is usually caused by folate deficiency. In two large European studies, anaemia, mostly attributed to malabsorption, was reported to be present in 20%–34% of untreated CD-patients [20,30]. This corresponds with our results in which 25% of the CD-patients suffered from iron-deficiency anaemia.

The presence of vitamin B6 deficiency has been reported in two studies, albeit in children with "acute celiac disease". A decreased pyridoxal phosphate was reported in serum samples and in duodenal mucosa, suggestive of vitamin B6 deficiency [41,42] and indicative for decreased levels of vitamin B6 in untreated CD children. We found water-soluble vitamin deficiencies (B6, folic acid and B12) in approximately one in seven (B6) to one in five (folic acid and B12) untreated CD-patients. This was despite the fact that more than 20% of the patients were using a prescribed or over-the-counter multivitamin/vitamin B-complex or a folic acid supplement before diagnosis.

Vitamin B12 deficiency was frequently observed in our CD-patient group (19%), in accordance with earlier studies, notably also in those without atrophic gastritis [17,18,26–28]. Intriguingly, this vitamin is typically absorbed in the terminal ileum. Apparently, the distal small bowel is functionally more affected than previously believed, based on patho-histological analysis of distal small bowel biopsy samples [43]. Vitamin B12 deficiency in untreated CD-patients has been confirmed in several previously conducted European studies [18,25–28], ranging from 12% up to 41%. It may be hypothesized that vitamin B12 deficiency is a result of a dysfunctional intrinsic factor [44]. Dickey reported that low vitamin B12 concentrations in CD were not due to auto-immune gastritis [28]. In this study, none of the patients had histologically or serologically demonstrated atrophic gastritis and, therefore, this was unlikely to be responsible for vitamin B12 deficiencies in CD.

In various small studies in Europeans, deficiencies of the fat soluble vitamins A [23,27], E [19,29] and D [45] in untreated CD-patients have been previously reported. The latter has been associated with osteomalacia. In clinical practice, many believe that the clinically relevant lower limit for vitamin D deficiency should be increased. As a consequence, the displayed (relatively low) deficiency percentages can therefore be an underestimation. The observed 7.5% of vitamin A deficient patients is less than the percentage reported in Finland (14%) for this vitamin [27]. From healthy subject studies, it is known that the vitamin A body storage is usually stable and sufficient for approximately one to two years [46]. This is confirmed by our healthy sample, in which none of the subjects showed a deficiency.

A majority of our untreated CD-patients had a zinc deficiency, which can probably be explained by increased endogenous losses of zinc, rather than abnormal zinc absorption [20]. Clinical relevance of zinc deficiency remains inconclusive and additional research is warranted. However, it is known that CD is associated with a wide array of skin lesions and manifestations, which may be partly ascribed to zinc deficiency [47–50], and cell mediated immunity and antioxidant buffer capacity may be compromised due to it as well [51].

Possible explanations for the high prevalence of nutritional deficiencies in untreated CD-patients might be an insufficient nutritional intake. This is supported by the presence of malnutrition in this cohort: 17% was malnourished based on the usual definition of >10% involuntary body weight loss prior to diagnosis and 7.5% had a BMI <18.5 kg/m<sup>2</sup> . However, this seemed unlikely since patients did not report any changes (intentional or involuntary) in their habitual diet before diagnosis. Moreover, the observed presence of high serum folate levels, particularly in those classified as underweight, were contradictory as well. On the other hand, increased faecal losses of nutrients as a result of malabsorption might (partially) explain the high prevalence of deficiencies. However, since most patients did not report any clinical sign of malabsorptive diarrhoea, losses via the stools were thought to be of limited importance as well. Nevertheless, the observed findings in this study, sharing deficiencies in water and fat soluble vitamins, zinc and iron, indicate that maldigestion, malabsorption or a structurally moderately inadequate intake might have been present long before the clinical diagnosis of CD was established. It is known that the delay in diagnosing CD can be more than a decade [52]. One may hypothesize that this might be due to functional changes of the intestinal tract or due to changes in the intestinal microbiome [53]. In this study, even patients with mild CD (low grade pathohistological abnormalities) showed nutritional deficiencies and weight loss. A finding that was recently corroborated in a large Italian cohort showed that mild (histopathological) enteropathy did not necessarily mean mild intestinal dysfunction since, also in this group, alterations in bone marrow density and laboratory parameters were reported [54]. Remarkably, only female patients presented as underweight (approximately one in four females), while the female-male ratio was similar in the overweight and obese patients. A statistical trend for higher serum vitamin A and B concentrations was observed in patients classified as malnourished (low BMI or >10% unintentional weight loss prior to diagnosis). This may be explained by their more so-called classical CD presentation, which may trigger use of supplements due to a greater physicians' or patients' awareness when observing clinical signs of malnutrition.

Some considerations arise when interpreting the presented results: not all data were available in all subjects and some subgroups were relatively small, precluding detection of small effects or the drawing of firm conclusions in subgroups. Vitamin or mineral supplement use, whether at the patient's own initiative or prescribed by the GP, was based on self-reported information and can therefore be underestimated if patients forget to mention these "medicinal supplements". However, real numbers and frequencies of deficiencies or anaemia might be underestimated, since some patients were already taking these supplements before the initial diagnosis of CD, potentially leading to misclassification as "non-deficient" in this study. Besides, serum values of almost all vitamins or minerals do not fully represent total body stock or physiological function.

Based on our experience and supported by a recently published guideline on CD [3], we suggest monitoring body weight at diagnosis and nutritional serum parameters; at least vitamin B6, folic acid, B12 and zinc and in any case (25-hydroxy) D of the fat soluble vitamins (due to its connection with presence of osteomalacia). Moreover, we suggest follow-up until serum values are at satisfying levels or upon indication (for instance, if bone density deviations, chronic diarrhoea, or skin lesions are present). Practically, a standard complete multivitamin supplement (100%–300% of RDA) should be considered for every newly diagnosed CD-patient. Continuation time has yet to be determined, since patients are at risk for vitamin deficiencies even after 10 years of a GFD [55]. Evidently, hypervitaminosis should be avoided, in particular regarding pyridoxine and iron [56,57]. It is demonstrated that bone-mineral density and nutritional status can improve after a GFD treatment [58] as well as that of general well-being, which can improve after vitamin B supplementation in CD-patients on a GFD [59].

#### **5. Conclusions**

In conclusion, deficiencies of vitamins or minerals are frequently observed in untreated adult Dutch CD-patients using a Western diet, although they are currently diagnosed earlier than in the previous century. This was observed even in obese patients. Almost 90% of the newly diagnosed CD-patients had one or more nutritional deficiencies. Malnutrition, expressed as an involuntary weight loss or being underweight, was found in 16% and 7.5% of patients, respectively, while overweight status (BMI > 25 kg/m2 ) was present in almost 30% of the patients. Therefore, these results indicate that extensive nutritional assessment of body weight and serum nutritional parameters should be an integral part of celiac disease treatment to guide nutritional advices and follow-up in CD-treatment by means of an adequately composed individual-based, gluten free diet.

#### **Acknowledgments**

The authors thank the Department of Clinical Chemistry of the VU University Medical Centre in Amsterdam for their help with the analyses. And student Priyantha Malhoe for her assistance at the beginning of this manuscript.

#### **Authorship Statement**

NJW, AAvB and CJJM designed the protocol, NJW and MB conducted the research, NJW analyzed the data, NJW and AAvB wrote the paper, and all authors critically reviewed and approved the final version of the paper. NJW is primarily responsible for the contents of the paper and is acting as the submission 

## **Financially Support**

This study was funded in part by a grant received from the Dutch Society for Gastroenterology (Gastrostart). Gastrostart had no involvement in any of the study tasks.

### **Conflicts of interest**

The authors declare no conflict of interest.

#### **References**




Reprinted from *Nutrients*. Cite as: Ciacci, C.; Siniscalchi, M.; Bucci, C.; Zingone, F.; Morra, I.; Iovino, P. Life Events and the Onset of Celiac Disease from a Patient's Perspective. *Nutrients* **2013**, *5*, 3388-3398.

*Concept Paper* 
