Oral Mucosa Patch Test: A New Tool to Recognize and Study the Adverse Effects of Dietary Nickel Exposure

Abstract

On contact with the skin, nickel may cause allergic contact dermatitis, which can be diagnosed by an epicutaneous patch test. Nickel exposure via the intestinal mucosa can induce diarrhea, abdominal pain, and swelling. The aim of the present study was to investigate the relationship between these symptoms and nickel intake by means of a novel oral mucosa patch test. Eighty-six patients with intestinal symptoms related to ingestion of nickel-containing foods were submitted to epicutaneous and oral mucosa patch tests for nickel. All patients with positive oral mucosa patch test results were subject to a low-nickel diet and monitored over time. Skin lesions were observed in 33 out of 86 (38.4%) patients evaluated by the epicutaneous patch test. Mucosal lesions were seen in 53 out of 86 (61.6%) patients given the oral mucosa patch test. After 2 months of a low-nickel diet, 52 out of 53 (98.1%) patients showed an improvement of their symptoms. There is a significant correlation between response time of the oral mucosa patch test and the latency of symptoms after ingestion of nickel-containing foods. Consequently, the oral mucosa patch test can be used to recognize and study the adverse effects of dietary nickel exposure that could be defined as allergic contact mucositis. A low-nickel diet is also shown to be an effective treatment for this condition.

Full text

Oral Mucosa Patch Test: A New Tool to Recognize
and Study the Adverse Effects of Dietary Nickel Exposure
Antonio Picarelli &Marco Di Tola &Anna Vallecoccia &
Valerio Libanori &Mirella Magrelli &Marta Carlesimo &
Alfredo Rossi
Received: 2 November 2009 /Accepted: 10 February 2010
#Springer Science+Business Media, LLC 2010
Abstract On contact with the skin, nickel may cause allergic contact dermatitis, which can be
diagnosed by an epicutaneous patch test. Nickel exposure via the intestinal mucosa can induce
diarrhea, abdominal pain, and swelling. The aim of the present study was to investigate the
relationship between these symptoms and nickel intake by means of a novel oral mucosa patch
test. Eighty-six patients with intestinal symptoms related to ingestion of nickel-containing
foods were submitted to epicutaneous and oral mucosa patch tests for nickel. All patients with
positive oral mucosa patch test results were subject to a low-nickel diet and monitored over
time. Skin lesions were observed in 33 out of 86 (38.4%) patients evaluated by the epicutaneous
patch test. Mucosal lesions were seen in 53 out of 86 (61.6%) patients given the oral mucosa
patch test. After 2 months of a low-nickel diet, 52 out of 53 (98.1%) patients showed an
improvement of their symptoms. There is a significant correlation between response time of the
oral mucosa patch test and the latency of symptoms after ingestion of nickel-containing foods.
Consequently, the oral mucosa patch test can be used to recognize and study the adverse effects
of dietary nickel exposure that could be defined as allergic contact mucositis. A low-nickel diet
is also shown to be an effective treatment for this condition.
Keywords Allergic contact dermatitis .Allergic contact mucositis .Celiac disease .
Epicutaneous patch test .Intestinal symptoms .Nickel .Low-nickel diet .
Oral mucosa patch test
Introduction
Nickel (Ni) is a transition metal found in the environment, some drugs, cosmetics,
detergents, jewelry, watches, keys and other commonly used metallic utensils. Contact with
Biol Trace Elem Res
DOI 10.1007/s12011-010-8652-y
A. Picarelli (*):M. Di Tola :A. Vallecoccia :V. Libanori :M. Magrelli
Center for Research and Study of Celiac Disease—Department of Clinical Sciences, Policlinico Umberto
I—Sapienza University, Viale del Policlinico, 155, 00161 Rome, Italy
e-mail: antonio.picarelli@uniroma1.it
M. Carlesimo :A. Rossi
Department of Skin-Venereal Diseases and Plastic Reconstructive Surgery, Policlinico Umberto I—
Sapienza University, Viale del Policlinico, 155, 00161 Rome, Italy

this ubiquitous element may easily occur by inhalation, ingestion, or through the skin. Its
absorption depends on the solubility of the chemical form of Ni. In the blood, Ni is mainly
bound to albumin. Its circulating levels reflect the degree of exposure to soluble compounds
rather than insoluble salts or unabsorbed deposits of Ni metal in the lungs. The distribution
of Ni in tissues depends on the type and length of exposure, with the kidneys as the primary
route of elimination [1].
The main clinical manifestation caused by skin contact with Ni is allergic contact dermatitis
(ACD), for which the epicutaneous patch test (ePT) is considered the gold standard for its
diagnosis [2]. Similarly, Ni exposure through the intestinal mucosa following the ingestion of
Ni-containing foods can result in other clinical conditions, including contact stomatitis [2,3].
Several studies have also reported that even small amounts of Ni ingested with a normal diet
are enough to flare up a preexistent ACD [4–6]. However, these observations are occasional
and fragmentary, so the sensitivity to dietary Ni is a condition still poorly understood.
In our own experience, several intestinal symptoms not attributable to any known
pathological condition, such as diarrhea, abdominal pain, and swelling, are often reported
by patients after the ingestion of Ni-containing foods. The aim of the present study was to
investigate the relationship between intestinal symptoms and Ni intake by a novel oral
mucosa patch test (omPT), based on recent studies that have shown a close association
between oral and intestinal mucosal compartments in patients with celiac disease, a
complex autoimmune enteropathy induced by dietary gluten [7–9]. The omPT is described
for the first time in the present study.
Materials and Methods
Patients
Eighty-six subjects (six male and 80 female), with a mean age of 40.8 years (range 14–
66 years), were chosen from outpatients from our gastrointestinal unit (GU) during the
period from December 2006 to May 2008.
Patients who presented intestinal and extra-intestinal symptoms related to the ingestion
of Ni-containing foods were included in the study. Informed consent was obtained from
each patient and all procedures in this study were in accordance with the ethical standards
of the institutional committee for human experimentation.
The patients carried a detailed food diary and were given a full medical evaluation to
assess the adverse effects of dietary Ni exposure, as well as the presence of other clinical
conditions (e.g., celiac disease and lactase deficiency). The main clinical data of
participants are given in Table 1. Both ePT and omPT for Ni were given to each patient.
Those who tested positive for omPT were put on a low-Ni diet and were monitored over
time to follow the course of their symptoms.
Patch Tests for Nickel
Epicutaneous Patch Test
A patch containing 5% solution of Ni sulfate (NiSO
4
·6H
2
O) in Vaseline was applied on the
upper back of patients. After 48 h the patch was removed to look for any lesion or reaction
in the test site, repeating the inspection at 96 h. The presence of erythema, edema, and/or
vesicles on the test site was considered a positive result.
Picarelli et al.

Oral Mucosa Patch Test
The omPTwas performed inside the upper lip after removal of excess saliva with sterile gauze.
Briefly, a 5-mm filter paper disk saturated with a 5% solution of Ni sulfate in Vaseline was
applied on the test site and held in place by an adhesive transparent film (Tegaderm, 3M) that in
turn, was covered with a small absorbent pad. After 2 h, the patch was removed and the site of
application was closely observed to determine the presence of any lesion or reaction, repeating
the inspection at 24 and 48 h. Even the occurrence of any general reaction was carefully
evaluated. If the patients showed adverse effects before 2 h, the patch was immediately
removed, the reaction time was annotated, and the presence of any local and/orgeneral reaction
was assessed. Specifically, the appearance of erythema, edema, and/or vesicles on the test site,
as well as itching and dermographia, were considered as positive result.
Low-Nickel Diet and Follow-Up
The ubiquity of Ni makes it virtually impossible to eliminate it from the diet. To prevent the
adverse effects caused by the ingestion of this element, a low-Ni diet is the only resource at hand.
For this reason, patients showing positive omPT results received a list of foods containing high
amounts of Ni (Table 2), with the recommendation to introduce these in very limited quantities
and to not consume more than one Ni-containing food in the same meal [10]. All patients
assessed the adherence to the low-Ni diet by means of a food diary compiled daily, while the
clinical effects of the dietary Ni restriction were carefully evaluated after 2 months of treatment.
Statistical Analysis
The differences between qualitative data were assessed by means of the chi-square test,
while differences between quantitative data were evaluated by the Student two-tailed ttest
Table 1 Clinical Data of the Participants
Intestinal
symptoms
Pts/total (%) Extra-intestinal
symptoms
Pts/total (%) Other
diseases
Pts/total (%)
Abdominal swelling 56/86 (65.1%) Itching 15/86 (17.4%) CD 18/86 (20.9%)
Abdominal pain 45/86 (52.3%) Hives 11/86 (12.8%) LD 13/86 (15.1%)
Diarrhea 23/86 (26.7%) Headache 6/86 (7.0%) T 5/86 (5.8%)
Constipation 9/86 (10.5%) Erythema 5/86 (5.8%) IgAD 3/86 (3.5%)
Stomatitis 5/86 (5.8%) Dizziness 4/86 (4.7%) OAs 3/86 (3.5%)
Dyspepsia 4/86 (4.7%) Asthenia 2/86 (2.3%) ACD to X 2/86 (2.3%)
Nausea 4/86 (4.7%) Conjunctivitis 2/86 (2.3%) P 2/86 (2.3%)
Meteorism 3/86 (3.5%) Dermographia 2/86 (2.3%) V 2/86 (2.3%)
Vomiting 3/86 (3.5%)
Belching 2/86 (2.3%)
Each patient presented at least one intestinal symptom referable to the ingestion of Ni-containing foods, but
not everyone had an extra-intestinal Ni-related symptom or was suffering from another disease. The
symptoms as well as other diseases with a frequency less than 2% were not included in this table
CD celiac disease, LD lactase deficiency, Tthyreopathy, IgAD IgA deficiency, OAs other allergies (to eggs,
wheat, and atopic dermatitis), ACD to X allergic contact dermatitis to metals other than Ni (cobalt and
palladium), Ppsoriasis, Vvitiligo
Oral Mucosa Patch Test for Ni-Sensitivity

for independent data. Linear regression was used to evaluate the correlation index between
quantitative data. In all statistical tests, the significance level was set at p≤0.05.
Results
Appearance of erythema, edema, and/or vesicles on the epicutaneous test site was observed
in 33 out of 86 (38.4%) patients. The omPT resulted in erythema, edema, and/or vesicles on
the test site (Fig. 1a–c), as well as itching and dermographia (Fig. 1d) in 53 out of 86
(61.6%) patients. The percentage of patients presenting positive results was significantly
higher (p=0.004) for the omPT than for those undergoing the ePT (Fig. 2).
The number of Ni-related symptoms was significantly higher in patients who gave
positive results in either of the patch tests than that of patients with negative results (p=
0.010 for the ePT and p< 0.001 for the omPT). The number of Ni-related symptoms
reported by patients with negative ePT was significantly higher (p=0.002) than for patients
with negative omPT (Fig. 3).
Of the 86 patients included in the study, 18 (20.9%) and 13 (15.1%) had a previous diagnosis
of celiac disease and lactase deficiency, respectively (Table 1). The percentage of patients with
celiac disease or lactase deficiency giving positive results for both patch tests is not significantly
different from that of patients with the same illnesses and with negative results (Table 3).
Among the 53 patients with positive omPT results, 39 (73.6%) showed local lesions and/
or general reactions after 2 h. In eight cases (15.1%), the response to Ni was evident after
more than 2 h and in six (11.3%) within 30 min from the test start (Fig. 4). A significant
correlation (p<0.0001) was found between the omPT response time and the latency of
symptoms described by the patients after ingesting Ni-containing foods (Fig. 5).
Furthermore, after the omPT, all patients showing positive results reported a relapse of
the intestinal and extra-intestinal symptoms previously registered in their clinical history.
All participants completed the study with excellent compliance of the low-Ni diet. After
2 months of dietary Ni exposure reduction, 52 out of 53 (98.1%) patients who had positive
omPT results showed an improvement of their intestinal and extra-intestinal symptoms.
Discussion
In the present study, the omPT is introduced as a new tool to recognize and study the
adverse effects of dietary Ni exposure.
Table 2 Foods That Contain a High Amount of Nickel
Foodstuffs Ni-containing foods
Fishes Herring, mackerel, salmon, shellfish, tuna
Vegetables Fresh and dried legumes (chickpeas, lentils, peanuts, peas, red kidney beans, soya beans
and soy products), garlic, green leafy vegetables (spinach), onion, raw carrots, tomatoes
Fruits Fresh and dried fruits (almonds, hazelnuts, walnuts)
Cereals Buckwheat, maize, millet, oat, rye, whole grain, whole wheat
Beverages Beer, coffee, initial water flow from the tap (especially in the morning), red wine, tea
Others Baking powder, canned foods, cocoa and chocolate, foods cooked in stainless steel utensils
(especially if acidic foods as tomatoes), gelatin, linseeds, marzipan, Ni-containing vitamin
supplements, strong licorice, sunflower seeds
Picarelli et al.

A Ni patch is able to determine lesions of the oral mucosa including erythema, edema,
and vesicle formation. This is consistent with reports of gingival abnormalities caused by
Ni-based alloy prosthesis in orthodontic patients sensitive to Ni [11].
Recent studies have also shown a close association between oral and intestinal mucosa
in patients with celiac disease, a complex autoimmune enteropathy induced by dietary
gluten [7–9]. Consequently, the Ni-patch-induced oral mucosa lesions are probably related
to bowel damage that occurs after ingestion of Ni-containing foods that in turn, causes the
Fig. 1 Local and general reactions observable after the oral mucosa patch test for nickel. Appearance of
erythema (a), edema (b), and vesicles (c) on the test site, as well as development of itching and dermographia
(d) after performing the oral mucosa patch test in Ni-sensitive patients
0%
20%
40%
60%
80%
100%
ePT results omPT results
Posi tive Negative
χ
2
=8.395
p=0.004
Fig. 2 Percentage of patients
showing positive patch tests for
nickel. The percentage of patients
presenting positive results for the
epicutaneous and oral mucosa
patch tests was plotted in this
graph. The χ
2
and pvalues refer
to chi-square test applied between
these parameters. ePT epicutane-
ous patch test, omPT oral mucosa
patch test
Oral Mucosa Patch Test for Ni-Sensitivity

intestinal symptoms frequently reported by Ni-sensitive patients such as diarrhea,
abdominal pain, and swelling.
A Ni patch may also cause development of general reactions such as itching and
dermographia, suggesting that the omPT can effectively identify Ni-sensitive patients.
Furthermore, besides reinforcing the observations that Ni intake causes contact stomatitis
[2,3] and may trigger a preexistent ACD [4–6], our results show that the omPT is more
sensitive than the epicutaneous version in recognizing the adverse effects of ingesting Ni-
Pts ePT+ Pts ePT- Pts omPT+ Pts omPT-
0
1
2
3
4
5
(n=33) (n =53) (n=53) (n=33)
t=2.650, p=0.010 t=6.228, p<0.001
t=3.229, p=0.002
No. of Ni-related symptoms
Fig. 3 Number of nickel-related symptoms in relation to the results of patch tests. The number of Ni-related
symptoms reported by participants was plotted in this graph (mean±SD) in relation to the results of
epicutaneous and oral mucosa patch tests. The tand pvalues refer to the Student two-tailed ttest for
independent data applied between these parameters. Pts ePT+ patients presenting positive epicutaneous patch
test results, Pts ePT−patients with negative epicutaneous patch test results, Pts omPT+ patients showing
positive oral mucosa patch test results, Pts omPT−patients with negative oral mucosa patch test results
Table 3 Percentage of Patients with Celiac Disease or Lactase Deficiency in Relation to the Results of Patch
Tests for Nickel
Pts No pts χ
2
p
CD
Pts ePT þn¼33ðÞ 8 (24.2%) 25 (75.8%) 0.104 0.747 (ns)
Pts ePT n¼53ðÞ 10 (18.9%) 43 (81.1%)
Pts omPT þn¼53ðÞ 9 (17.0%) 44 (83.0%) 0.754 0.385 (ns)
Pts omPT n¼33ðÞ 9 (27.3%) 24 (72.7%)
LD
Pts ePT þn¼33
ðÞ 4 (12.1%) 29 (87.9%) 0.091 0.762 (ns)
Pts ePT n¼53ðÞ 9 (17.0%) 44 (83.0%)
Pts omPT þn¼53ðÞ 10 (18.9%) 43 (81.1%) 0.849 0.357 (ns)
Pts omPT n¼33ðÞ 3 (9.1%) 30 (90.9%)
The χ
2
and pvalues refer to chi-square test applied between these parameters
CD celiac disease, LD lactase deficiency, Pts ePT+ patients presenting positive epicutaneous patch test
results, Pts ePT−patients with negative epicutaneous patch test results, Pts omPT+ patients showing positive
oral mucosa patch test results, Pts omPT−patients with negative oral mucosa patch test results, ns not
significant
Picarelli et al.

containing foods in a population of symptomatic patients. In this regard, the significant
portion of Ni-sensitive patients identified by the omPT, as well as the substantial clinical
differences between positive and negative omPT patients further support this simple test as
an effective diagnostic tool.
It is interesting to note that several individuals with positive omPT results are celiac
disease or lactase deficiency patients, even if there is no evidence of a causal association.
The high prevalence of celiac disease and lactase deficiency among the participants in this
study could be simply due to a clinical bias because most of the patients that refer to our
GU are suffering from these conditions. However, given that gluten-free diet usually
include foods rich in Ni, the elective treatment for celiac disease could unmask a sub-
clinical form of Ni-sensitivity. The latter hypothesis should be carefully considered in the
monitoring of patients suffering from celiac disease.
The different response time to Ni among patients presenting positive omPT results is
also of interest. Most patients react to Ni 2–48 h from the test start and are classified as late
responders. There are also early responders, who have a response time of 30 min or less. It
010 20 30 40 50
0
10
20
30
40
50
r=0.705
p<0.0001
Latency of Ni-relate d symptoms
(hrs)
Response time to Ni
(hrs)
Fig. 5 Response time to nickel in relation to the latency of nickel-related symptoms. The response time to
Ni observed during oral mucosa patch test and the latency of symptoms described by the patients after
ingestion of Ni-containing foods were plotted in this graph. The rand pvalues refer to linear regression used
to evaluate the correlation index between these parameters
<30 min
2 hrs*˚
>2 hrs (6 hrs )
>2 hrs (12 hrs )
>2 hrs (24 hrs )
>2 hrs (48 hrs )
χ χ
2
=39.542, p<0.001 vs <30 min °
2
=34.403, p<0.001 vs >2 hrs
∗
Fig. 4 Percentage of patients with different response times to nickel. The percentage of patients showing
different response times to Ni during oral mucosa patch test (<30 min, 2 h, and >2 h) was plotted in this
graph. The response times >2 h were split in other four time points (6, 12, 24, and 48 h). The χ
2
and pvalues
refer to chi-square test applied between these parameters
Oral Mucosa Patch Test for Ni-Sensitivity

is possible that these results are due to differences in the response mechanism. Late
responders could react to Ni mainly by a delayed-type hypersensitivity or Th1 response,
while early responders could do so predominantly by an immediate-type hypersensitivity or
Th2 response.
For a long time, the scientific community has considered that the Ni-induced ACD
response is of the Th1-type. By analyzing the cytokine secretion profile in peripheral blood
of ACD patients, it has been shown that the majority of Ni-specific CD4+ T lymphocytes
produce Th1 cytokines such as interleukin (IL)-2 and interferon (IFN)-γ[12]. Other authors
have stressed the crucial role of Ni-specific CD8+ T cells in the pathogenesis of ACD [13–
15]. Beyond the hypothesis based on the Th1-type mechanism, it has also been shown that
Ni-specific CD4+ T lymphocytes from both peripheral blood and skin lesions of ACD
patients are able to produce Th2 cytokines such as IL-4 and IL-5 [16–18]. Concurrently,
other authors have shown that serological and urinary Ni levels correlate with the amounts
of CD3+CD25+ and CD4+CD45RO+ T cells, CD5+CD19+ B cells, and IL-13 that may be
found in peripheral blood of ACD patients in a pattern that reminds a Th2 response [19]. A
more recent study has finally shown that Ni exposure leads to a concomitant production of
Th1 (IFN-γ), Th2 (IL-4, IL-5, IL-13), and regulatory (IL-10) cytokines by peripheral blood
mononuclear cells isolated from ACD patients [20].
Although the aforementioned studies are somewhat contradictory, they suggest that an
imbalance among Th1, Th2, and regulatory pathways could be responsible for the
immunological response that takes place in ACD patients during Ni exposure. Likewise,
this mechanism could be taken as a model to explain the different response times to Ni
observed in our investigation. Other studies will follow to fully elucidate the molecular
bases of mucosal response to Ni.
Our data also highlighted that, in the course of omPT, patients showing positive results
respond to Ni with the same latency than that described following ingestion of Ni-
containing foods. Inversely, after a low-Ni diet, patients presenting positive omPT results
show symptom relief. In this manner, we show for the first time a close relationship
between Ni intake and intestinal symptoms commonly reported by Ni-sensitive patients.
Finally, although previous data on the usefulness of dietary Ni restriction in ACD patients
are slightly contradictory [21–23], we have shown that a low-Ni diet is effective in restoring
the patient’s general state of health.
In conclusion, the omPT can be used to recognize and study the adverse effects of dietary Ni
exposure that could be defined as allergic contact mucositis (ACM). We suggest that patients
with this condition be placed in a diet consisting of foods with the lowest possible Ni content
over a reasonable period of time, during which the patient’s response should be periodically
evaluated. The high prevalence of Ni-sensitivity in North America and Western Europe, recently
estimated to be 8.6% [24], stresses the relevance of the new acquisitions. However, the
molecular role of Ni in ACM as well as in other intestinal disorders is still to be elucidated.
Acknowledgment The authors are grateful to Dr. Oralia Lopez for revision of the manuscript and assistance
with the English style.
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