Helicobacter ISSN 1523-5378

Micronutrients (Other than iron) and Helicobacter pylori

Infection: A Systematic Review
Edith Lahner,* Severino Persechino and Bruno Annibale*
*Digestive and Liver Disease Unit, University Sapienza, SantAndrea Hospital, Via Grottarossa 1035, 00189 Rome, Italy, Department of Dermatol-
ogy, University Sapienza, SantAndrea Hospital, Via Grottarossa 1035, 00189 Rome, Italy

Keywords
Micronutrient deficiency, Helicobacter pylori,
ascorbic acid, iron, cobalamin, meta-analysis.
Reprint requests to: Bruno Annibale, MD,
Dipartimento medico-chirurgico di scienze
cliniche, tecnobiomediche e medicina
traslazionale, University Sapienza Ospedale
SantAndrea, Via di Grottarossa 1035, 00189
Roma, Italy.
E-mail: bruno.annibale@uniroma1.it
Abstract
Background: Many micronutrients depend on a healthy stomach for
absorption. Helicobacter pylori chronic gastritis may alter gastric physiology
affecting homeostasis of vitamins and minerals.
Objectives: Systematic review to assess whether H. pylori infection is associ-
ated with reduced micronutrient levels (other than iron) in the plasma or
gastric juice and whether low micronutrient levels are modified by eradica-
tion treatment.
Method: Medline was searched for relevant publications from inception to
June 2010. Studies describing micronutrient levels in H. pylori-infected and
not-infected adults and or the effect of eradication treatment on micronutrient
levels were included.
Findings: Fifty-two publications were selected: 46 investigated the associa-
tion between H. pylori infection and reduced micronutrient levels and 14 the
effect of eradication treatment on micronutrient levels. Sixty-four studies
investigated vitamins (23 ascorbic acid, four -carotene, 21 cobalamin,
11 folate, and five a-tocopherol) and 10 addressed minerals (one calcium,
one copper, one magnesium, one phosphorus, three selenium, and three
zinc). Pooled standardized mean differences in micronutrient levels showed
positive associations with H. pylori infection for ascorbic acid (gastric juice,
)1.087) and cobalamin ()0.744), and a positive effect of eradication treat-
ment, which increased ascorbic acid in the gastric juice ()1.408) and serum
cobalamin ()1.910). No significant association between infection and low
folate levels was observed. Meta-analyses for other micronutrients were not
performed owing to insufficient data.
Conclusions: Meta-analyses indicate that H. pylori infection is associated
with reduced levels of ascorbic acid and cobalamin, supported by the
positive effect of eradication treatment. For other micronutrients, further
studies are needed.

Helicobacter pylori (H. pylori) is a Gram-negative, spiral-
shaped, microaerophilic bacterium that infects the
stomach of more than half of the global population.
The incidence of H. pylori infection has been decreasing
in industrialized countries because of improved sanita-
tion, smaller family sizes and decreased overcrowding,
but developing countries still have a large proportion of
adults infected [14].
H. pylori infection is considered a major pathoge-
netic factor for chronic gastritis, peptic ulcer disease,
gastric cancer, and mucosa-associated lymphoid tissue
associated lymphoma [1,2] and has been linked to some
nongastric disorders, such as thrombocytopenic
purpura, iron deficiency anemia, and atherosclerotic
disease [5]. Studies have suggested that, beyond iron,
H. pylori infection may affect the homeostasis of other
micronutrients such as vitamins and trace elements
including vitamin B12, folic acid, vitamin C, a-toco-
pherol, and -carotene [68].
Micronutrients are essential for life acting as cofactors
of enzymes and as an organizer of the molecular struc-
tures of the cell. Micronutrient deficiencies influence
immune homeostasis, thus affecting infection-related
morbidity and mortality [9]. Deficiency of hemopoietic

2011 Blackwell Publishing Ltd, Helicobacter 17: 115 1

Micronutrients and H. pylori
micronutrients such as iron, cobalamin, or folate may
lead to deleterious effects because of anemia and its
clinical consequences including reduced work capacity,
cognitive impairment, reduced resistance to infec-
tions, and negative effects on pregnancy and growth
[10]. Micronutrients like vitamin C, a-tocopherol,
-carotene, selenium, and others are powerful antioxi-
dants and anticarcinogens [1113]. Many micronutri-
ents depend on a healthy stomach for their absorption.
Although the absorption process does not take place in
the stomach, this organ contributes to the process by
means of the secretion of hydrochloric acid and
enzymes, which during the digestive process are neces-
sary to release the micronutrients from the food matrix
and, in the case of essential minerals, to render them
soluble [14]. The chronic inflammatory response of the
gastric mucosa to H. pylori infection may alter gastric
physiology. Impaired gastric acid secretion is a common
consequence of H. pylori-induced gastritis: the chronic
inflammatory injury of the corporal mucosa may lead
to a functional inhibition of parietal cells by inflamma-
tory products, which may finally provoke atrophy of
gastric glands and hypochlorhydria [1517].
Previous narrative reviews have focused on the
question of the influence of H. pylori infection on
micronutrient deficiencies reporting limited data on
vitamin B12, vitamin A, and ascorbic acid [6,7,1821].
More recently, meta-analyses have been performed
reporting a positive association between H. pylori infec-
tion and iron deficiency anemia and a positive effect of
eradication treatment on iron deficiency and anemia
[2226]. To our knowledge, to date, no efforts have
been made to review systematically in adults the link
between H. pylori infection and deficiency of micro-
nutrients other than iron. Thus, to examine whether in
adults H. pylori infection is associated with reduced
levels of micronutrients other than iron, we reviewed
studies (cohort studies, cross-sectional studies, case
series, randomized controlled trials, and casecontrol
studies) that assessed: 1, the micronutrient levels in the
serum, plasma, gastric juice, and or gastric mucosa in
H. pylori positives and negatives and or 2, the effect of
eradication treatment by measuring micronutrient
levels before and after treatment in adults.
Subjects and Methods
Study Selection
The search was conducted by using PRISMA guidelines
[27]. The electronic database PubMed MEDLINE (U.S.
National Library of Medicine, Bethesda, MD, USA) was
systematically searched for relevant studies that
2 2011 Blackwell Publishing Ltd, Helicobacter 17: 115
Lahner et al.
included the search term H. pylori combined with the
following search terms by using AND OR constructions:
micronutrients; vitamin*; ascorb*; carotene; cobalamin;
folate; tocopherol; mineral; calcium; copper; iron;
magnesium; phosphate; selenium; zinc. The search
strategy excluded reviews, meta-analyses, case reports,
and animal studies. No publication date restrictions
were imposed. Only reports published in English
language were considered.
Clinical studies (cohort studies, cross-sectional stud-
ies, casecontrol studies, case series, and randomized
controlled trials) published up to June 2010 were
considered for inclusion if they described in adults
(18 years): 1, the levels of micronutrients assessed in
the serum, plasma, gastric juice, and or gastric mucosa
in H. pylori positives compared with H. pylori negatives
and or 2, the effect of eradication treatment on micro-
nutrient levels by measuring the levels of micro-
nutrients before and after eradication treatment, and if
they reported information regarding the diagnosis of
H. pylori infection by invasive (histology, culture, or
rapid urease test) or noninvasive (serology, urea breath
test, stool antigen test) methods and regarding micro-
nutrient status determined by assessment of plasma,
serum, or tissue levels. Pediatric patients were excluded
from this review to reduce confounding as these indi-
viduals have higher micronutrient requirements com-
pared with adults because of different metabolic status
according to age and growth [28].
Potentially relevant articles were screened for eligibil-
ity independently in an unblinded standardized manner
by the two reviewers, initially by abstract and then by
full text when necessary to determine whether they
met the inclusion criteria. Disagreement between
reviewers was resolved by discussion. The reference lists
of the identified articles as well as of identified relevant
reviews were manually searched for additional studies
that may have been missed using the computer-assisted
search strategy.
Data Extraction
We developed a data extraction sheet, pilot-tested it on
five randomly selected included studies, and refined it
accordingly. One review author extracted the data from
included studies, and the second author checked the
extracted data. Disagreements were resolved by discus-
sion between the two review authors. The agreement
between reviewers for the selection of relevant articles
achieved a weighted j of 0.894 (95% CI 0.8030.984).
The following information was extracted from each
included paper: 1, author and year of publication; 2,
characteristics of study participants (including H. pylori
Lahner et al.
status, age, and gender); 3, study type; 4, assessment of
micronutrient status, dietary intake and intragastric pH;
5, diagnosis of H. pylori infection; 6, data on H. pylori
gastritis; 7, eradication treatment schedules, follow-up,
assessment of efficacy of treatment; and 8, outcome
data (micronutrient levels assessed in H. pylori positives
and H. pylori negatives and or before and after eradica-
tion treatment).
Statistical Analysis
The outcome measure taken into consideration in this
systematic review was continuous, i.e., the micronutri-
ent levels in H. pylori positives and H. pylori negatives
and or before and after eradication treatment. To
estimate the overall effect of interest, we planned to
use as summary outcome measure the difference in
means. The Hedges g statistic as a formulation for the
standardized mean difference (SMD) under the fixed-
effects model was used. The extent of heterogeneity
was investigated by chi-square test (Cochrans Q). Next,
the heterogeneity statistic was incorporated to calculate
the summary SMD under the random-effects model
(DerSimonian and Laird), which is more appropriate
than the fixed-effects model when the heterogeneity
test yields a low p-value (p < .05) [29]. The total SMD
with 95% CI was calculated both for the fixed-effects
model and for the random-effects model.
Separate analyses were conducted for studies that
assessed the association of H. pylori infection with
reduced micronutrient levels and for studies that
assessed the effect of eradication treatment, and when
possible, for micronutrient levels measured in different
biological samples, i.e., plasma serum, gastric juice, or
gastric mucosa. Statistical significance was considered at
the 5% level (p < .05).
In some cases, we decided not to combine studies,
but to show single outcome data for each study in a
table form. This occurred when the study design and
reported outcome measures varied markedly, so we
focused on describing the study characteristics and their
outcomes on qualitative synthesis rather than meta-
analysis. The statistical analysis was carried out using a
dedicated software package (version 11.3; MedCalc
Software, Mariakerke, Belgium).
Results
The electronic search strategy identified a total of 1481
potentially relevant publications, from which 95 were
selected as fulfilling the inclusion criteria. Forty were
then discarded because after reviewing the full-text
publications, it appeared that these articles clearly did
2011 Blackwell Publishing Ltd, Helicobacter 17: 115
Micronutrients and H. pylori
not meet the inclusion criteria. Three additional studies
were discarded because full text of the study was not
available (Fig. 1). Manual searching of reference lists of
potentially relevant papers and reviews did not add any
article. Thus, a total of 52 studies were identified, of
which 46 (88.5%) investigated the association between
H. pylori infection and reduced micronutrient levels and
14 (26.9%) the effect of eradication treatment on
micronutrient levels. Of the 52 included studies, 15
(28.8%) investigated multiple micronutrients, whereas
37 (71.2%) addressed one specific micronutrient only.
Among the included studies, 23 addressed ascorbic
acid [3052], four -carotene [40,49,53,54], 21 cobal-
amin [5575], 11 folate [55,58,61,63,65,66,6972,76],
and five a-tocopherol [40,49,53,54,77]; among the
included studies regarding minerals and trace elements,
one study investigated calcium [62]; one, copper [49];
one, magnesium [78]; one, phosphorus [62]; three, sele-
nium [49,79,80]; and three, zinc [49,81,82]. Table S1
shows the detailed characteristics of the included
studies.
Vitamins
Ascorbic acid
Study characteristics: Of the 23 included studies investi-
gating H. pylori infection and ascorbic acid [3052], 21
addressed the association between H. pylori infection
and ascorbic acid levels (four population-based cross-
sectional studies [46,4951], 16 cross-sectional studies
based on clinical setting [3044,52], and one case series
[48]) and eight, the effect of eradication treatment on
ascorbic acid levels (all case series [30,31,
3335,42,45,47]).
1481 records identified through
database searching
653 duplicates removed
828 of records screened
733 of records excluded:
not relevant (n = 558)
pediatric population (n = 50)
reviews, editorials, letters (n = 125)
95 of full-text articles assessed for
eligibility
43 of full-text articles excluded:
not relevant study population (n = 7)
not relevant intervention/exposure (n = 24)
not relevant study type (n = 3)
outcome data not extractable (n = 6)
publication could not be retrieved (n = 3)
52 of studies included
Figure 1 Flow diagram showing the flow for selection of studies to
be included.
3
Micronutrients and H. pylori
Overall, the 21 studies addressing the association
between H. pylori infection and ascorbic acid levels
involved a total number of 10,957 subjects (mean age
47 years, 53.5% women), of whom 42.5% had H. pylori
infection assessed by histology in 17 [3044,48,52], cul-
ture in five [33,38,43,52], biopsy urease testing in nine
[31,3338,41,42,53], urea breath test in two [31,34],
and finally serology in five studies [46,4951,53]. In 11
studies, only one of these diagnostic methods for
H. pylori infection was used [30,32,35,39,40,44,46,
4851], which was mainly histology, except for four
studies that used serology only [46,4951]. The study
population included dyspeptic [31,3344,48,52] and or
anemic [30,32] patients or subjects derived from cross-
sectional population-based studies [46,4951]. Infor-
mation about the gastritis associated with H. pylori
infection was given for the antral and corporal mucosa
in eight studies [30,32,35,36,39,40,43,44,48,52] and for
the antral mucosa only in two studies [41,42]. Intragastric
pH was assessed in 10 studies [30,32,35,37,4144,48,52]
and information about dietary vitamin C intake was
collected in six studies [35,37,39,46,49,51]. As outcome
measure, the level of ascorbic acid was measured in the
plasma or in the serum in 16 [3032,34,37,38,
4044,46,4851], in the gastric juice in 13 [3032,
3437,4144,48,52], and in the gastric mucosa in
four studies [33,34,39,52]. Most studies used high-
performance liquid chromatography for assessing ascorbic
acid levels, but in some studies, other methods such as
spectrophotometry [37,41,42], fluorometric assay [50,51],
or a single-step colorimetric method [38] were used.
Taken together, the eight studies addressing the effect
of eradication treatment on ascorbic acid levels involved
an overall number of 299 H. pylori-positive patients
(mean age 47 years, 52.2% women), in whom the
diagnosis of infection was assessed by histology in all
eight [30,31,3335,42,45,47], culture in two [33,45],
biopsy urease testing in five [31,33,34,42,45], and urea
breath test in two studies [31,34]. In three studies, only
one of these diagnostic methods for H. pylori infection
(histology) was used. The clinical setting was dyspepsia
in all but one study, which involved dyspeptic and or
anemic subjects [30]. In three studies, information was
given about antral and corporal gastritis [30,35,45], and
in one study about antral gastritis only [42]. The eradi-
cation treatment schedules were different in each single
study including metronidazole in all eight studies, bis-
muth preparations in six studies [30,31,33,34,42,45,47],
antisecretory agents in two studies [34,35], and amoxi-
cillin in five studies [30,31,35,42,45]. The efficacy of
treatment was assessed by histology in all but one [35],
culture in two [33,45], biopsy urease testing in three
[33,42,45], urea breath test in two studies [31,34], and
4 2011 Blackwell Publishing Ltd, Helicobacter 17: 115
Lahner et al.
by one method only in two studies, which was histol-
ogy in both cases [30,47]. The successful cure of
H. pylori infection was obtained in a total of 209
(69.9%) patients, and the median follow-up period was
3 months (range 112 months). As outcome measure,
the ascorbic acid level before and after eradication
treatment was measured in the plasma in six
[30,31,34,41,45,47], in the gastric juice in seven
[30,31,34,35,42,45,47], and in the gastric antral mucosa
in two studies [33,34] by using high-performance liquid
chromatography, except for one study that used spec-
trophotometry [42].
Meta-analyses: As shown in Fig. 2 by the pooled SMD
of ascorbic acid levels in the plasma from 15 studies
[3032,34,37,38,4043,46,4851] and in the gastric
juice from 13 studies [3032,3437,4143,45,48,52],
H. pylori infection was significantly associated with
lower levels of ascorbic acid in both the plasma (total
random effects of SMD )0.193, 95% CI )0.372 to
)0.015, Fig. 2A) and the gastric juice (total random
effects of SMD )1.087, 95% CI )1.794 to )0.379,
Fig. 2B). In contrast, all but one [52] of the four studies
[33,34,39,52] investigating the levels of ascorbic acid in
the gastric antral mucosa showed higher levels in the
H. pylori-positive patients compared with the H. pylori
negatives, but the total random effects of pooled SMD
did not reach statistical significance (Fig. 2C).
As far as regards the effect of eradication treatment
on ascorbic acid levels, the summary estimates pooled
from four studies [30,31,34,42,45] showed that the
pre-treatment levels of gastric juice ascorbic acid were
significantly lower compared with post-treatment levels
(total random effects of SMD )1.408, 95% CI )2.471 to
)0.346), indicating that eradication treatment yielded a
significant increase in the micronutrient level (Fig. 2D).
Eradication treatment on ascorbic acid assessed in the
plasma [30,31,34,45] and in the gastric antral mucosa
[33,34] had no clear effect (total random effects of
SMD )1.202, 95% CI )0.811 to 0.606, and SMD 0.133,
95% CI )0.207 to 0.471.
Two studies were excluded from meta-analysis
because outcome data were not appropriate for quanti-
tative synthesis [35,47]: in one report, no significant
change in gastric juice ascorbic acid levels after eradica-
tion was observed [35], and the other described a sig-
nificant increase in gastric juice ascorbic acid after
successful treatment, whereas plasma levels did not
change significantly with treatment [47].
-Carotene
Study characteristics and synthesis of study outcomes: All four
selected studies addressed the association between
Lahner et al. Micronutrients and H. pylori

A C

B D

Figure 2 Forest plot of standardized mean differences of ascorbic acid levels assessed in the plasma (A), gastric juice (B), and gastric mucosa (C)
in Helicobacter pylori positives and H. pylori negatives, and of ascorbic acid levels assessed in the gastric juice (D) before and after eradication
treatment. (A) Test for heterogeneity: Q = 278.05; df = 14; p < .0001. (B) Test for heterogeneity: Q = 752,46; df = 12; p < .0001. (C) Test for hetero-
geneity: Q = 35.21; df = 3; p < .0001. (D) Test for heterogeneity: Q = 80.39; df = 4; p < .0001.

H. pylori infection and -carotene [49,53,54] or retinol
[40]. Three were case series [40,53,54] and one a popu-
lation-based cross-sectional study [49]. Overall, these
four studies involved a total number of 246 subjects
(mean age 49 years, 49.1% women), of whom 54.9%
had H. pylori infection. None of these studies reported
data on intragastric pH and in one study only detailed
data on antral and corporal gastritis are given [54].
Only one study [49] collected information about
vitamin A dietary intake. We did not identify any
study addressing the effect of eradication treatment on
-carotene levels.
Table 1 gives the summary results of these studies
separately for plasma, gastric juice, and gastric antral
mucosa -carotene levels. As far as regards plasma,
serum, or gastric antral mucosa levels of -carotene and
retinol, none of the studies showed a significant differ-
ence between H. pylori-positive and H. pylori-negative
subjects. In contrast, the only study that investigated the
-carotene levels in the gastric juice [54] showed a
significant difference between infected and not-infected
dyspeptic subjects (2.9 vs 4.6 nmol L, p = .01), suggest-
ing a possible influence of H. pylori infection on this
micronutrient.
Cobalamin
Study characteristics: Of the totally 21 selected studies
addressing H. pylori infection and cobalamin [5575],
17 investigated the association between H. pylori infec-
tion and cobalamin levels (nine cross-sectional studies
mainly based on clinical setting [55,58,61,62,66,
7072,74] and eight case series [56,57,59,60,
67,69,73,75]), and five case series addressed the effect
of eradication treatment on cobalamin levels [59,
6365,68].

2011 Blackwell Publishing Ltd, Helicobacter 17: 115 5

Micronutrients and H. pylori
Table 1 Association between Helicobacter pylori infection and -carotene levels
-Carotene levels First author year Method
Plasma serum Phull 1998 (40) HPLC
Sanderson 1997 (53) HPLC
Toyonaga 2000 (49) HPLC
Zhang 2000 (54) HPLC
Gastric antral mucosal Sanderson 1997 (53) HPLC
Zhang 2000 (54) HPLC
Gastric juice Zhang 2000 (54) HPLC
a
Retinol levels.
b
Approximate data abstracted from a plot.
c
Serum levels.
NS, not significant; HPLC, high-performance liquid chromatography.
Overall, the 17 studies addressing the association
between H. pylori infection and cobalamin levels in-
volved a total number of 2454 subjects (mean age
58 years, 57.1% women), of whom 47.8% had H. pylori
infection assessed by histology in six [5962,67,71],
culture in one [59], biopsy urease testing in one [67],
urea breath test in six [56,58,59,62,70,72], stool anti-
gen test in two [70,72], and finally serology in eight
studies [5557,66,69,7375]. In 11 studies, only one of
these diagnostic methods for H. pylori infection was
used, which was histology in three [60,61,71], urea
breath test in one [58], and serology in seven studies
[55,57,66,69,7375]. The clinical setting of the study
populations was heterogeneous including not only dys-
peptic patients [6062,67], cobalamin-deficient patients
[56,57,59], and asymptomatic subjects [66,69,70,73]
but also patients with peripheral arterial disease [55]
and type II diabetes [58], subjects with diagnosed or
suspected coronary heart disease [71,74], and alcohol-
dependent subjects [75].
Information about the type of H. pylori gastritis was
given for the antral and corporal mucosa in six studies
[5962,71,73] and for the antral mucosa only in one
study [67]. Intragastric pH was assessed only in one study
[59], and also information about dietary cobalamin
intake was collected in only one study [73]. Cobalamin
levels were measured in the plasma in three [55,58,73]
and in the serum in 11 studies [6062,66,67,
6972,74,75] by radioimmunoassay [55,7173], immu-
noenzymatic assays [61,67,69,74,75] as well as electron
chemiluminescence immunoassay [70]. Three studies
determined the cobalamin levels by the egg-yolk absorp-
tion test (urinary cobalamin excretion of test dose in
24 hours) [56,57,59]. In four studies, the method of
cobalamin assessment was not reported [58,60,62,66].
The five studies addressing the effect of eradication
treatment on cobalamin levels involved an overall
6 2011 Blackwell Publishing Ltd, Helicobacter 17: 115
Lahner et al.
Median mean; unit Hp+ Hp- p
Median; lg dla 74.1 81.3 NS
Mean; nmol Lb 310 300 NS
Mean; lg dLc 28.0 27.8 NS
Median; lmol Lc 0.3 0.2 NS
Mean; nmol kgb 190 180 NS
Median; nmol g 0.4 0.5 NS
Median; nmol L 2.9 4.6 0.01
number of 283 H. pylori-positive patients (mean age
55 years, 61.7% women), in whom the diagnosis of
infection was assessed by histology in all five [59,
6365,68], culture in two [59,64], biopsy urease testing
in three [6365], and by urea breath test in two studies
[59,64]. In one study, only histology was used for the
diagnosis of H. . pylori infection [68]. The clinical setting
was cobalamin deficiency in three studies [59,63,64]
and dyspepsia with minimal or without gastric atrophy
in the remaining two studies [65,68]. In four studies,
information was given about antral and corporal gastri-
tis [59,63,64,68], and in the remaining study, gastric
biopsies were taken but data on gastritis were not
reported [65]. The prescribed eradication treatment
schedules were different in each study including proton
pump inhibitors in four [6365,68], amoxicillin in three
[63,65,68], clarithromycin in four [6365,68], and
metronidazole in two studies [59,63]. The efficacy of
treatment was assessed by histology and urea breath
test in two studies [59,64], and only by histology in the
remaining three studies [63,65,68]. The successful cure
of H. pylori infection was obtained in a total of 173
(61.1%) patients, and the median follow-up period was
1 month (range 0.2512 months).
As outcome measure, in four studies the serum
cobalamin levels before and after eradication treatment
were assessed [6365,68], and in one study, the egg-
yolk absorption test was performed, which measures
the urinary cobalamin excretion of test dose in
24 hours [58].
Meta-analyses: As shown in Fig. 3A, the forest plot of
the pooled SMD of cobalamin levels assessed in the
plasma, serum, or urine from 14 studies [5559,61,
62,66,67,6972,75], H. pylori-positive subjects showed
significantly lower cobalamin levels than H. pylori
negatives (total random effects of SMD )0.744, 95% CI
)1.147 to )0.340). Despite significant heterogeneity
Lahner et al.
A increase cobalamin levels, though within the limits of a
B between H. pylori infection and folate levels involved a
Figure 3 Forest plot of standardized mean differences of cobalamin
levels assessed in Helicobacter pylori positives and H. pylori negatives
(A) and before and after eradication treatment (B). (A) Test for hetero-
geneity: Q = 341.91; df = 8; p < .0001. (B) Test for heterogeneity:
Q = 177.98; df = 4; p < .0001.
between studies (p < .0001), these results indicate a
role of H. pylori infection in cobalamin deficiency. Three
studies [60,73,74] were excluded from meta-analyses,
because continuous outcome data of cobalamin levels
were not shown. Two studies [60,73], instead, reported
the proportions of patients with and without cobalamin
deficiency among H. pylori positives and H. pylori nega-
tives, which, however, in both reports were not signifi-
cantly different, and also the remaining study [74]
reported no significant association between H. pylori
infection and cobalamin serum levels (p = .28).
In Fig. 3B, pooled data on the effect of eradication
treatment on cobalamin levels from five studies [59,
6365,68] are reported: cobalamin levels were signifi-
cantly lower before eradication treatment compared
with post-treatment levels (total random effects of SMD
)1.910, 95% CI )3.358 to )0.463). This finding sug-
gests that eradication treatment is able to significantly
2011 Blackwell Publishing Ltd, Helicobacter 17: 115
Micronutrients and H. pylori
significant between-study heterogeneity (p < .0001)
and with the low number of studies included.
Folate
Study characteristics: Overall, 11 studies addressing H. pylori
infection and folate were selected [55,58,61,63,65,66,69
72,76]: nine investigated the association between
H. pylori infection and folate levels (six cross-sectional
based on clinical setting [55,58,61,66,71,72] and three
case series [69,70,76]), and two case series, the effect of
eradication treatment on folate levels (two case series
[63,65]).
Overall, the nine studies addressing the association
total number of 1566 subjects (mean age 55.3 years,
59.8% women), of whom 48.9% had H. pylori infection
assessed by histology in two [61,71], urea breath test in
four [58,70,72,76], stool antigen test in two [70,72],
and serology in four studies [55,66,69,76]. In six
studies, only one of these diagnostic methods for
H. pylori infection was used, which was histology in
two [61,71], urea breath test in one [58], and serology
in three studies [55,66,69]. The clinical setting of
the study populations was heterogeneous including
dyspeptic [61,76] and asymptomatic subjects [66,69,
70], as well as patients with diabetes [58], heart [71] or
renal disease [72].
Information about the type of H. pylori gastritis was
given for the antral and corporal mucosa in two studies
[61,71]. None of these studies reported data about
intragastric pH or information about dietary folate
intake. As outcome measure, the folate levels were
measured in the plasma in two [55,58], in the serum in
seven [61,66,6972,76], and in the erythrocytes in one
study [72] by using radioimmunoassay [55,71,72],
chemiluminescent enzyme assay [61], or electron
chemiluminescence immunoassay [70]. In four studies,
the method of folate assessment was not reported
[58,66,69,76].
We identified only two studies investigating the effect
of eradication treatment on folate levels [63,65], which
involved 150 patients (mean age 50 years, 70.1%
women), 77 cobalamin-deficient [63] and 73 dyspeptic
patients [65], respectively. In both studies, the diagnosis
of H. pylori infection was assessed by histology and
biopsy urease testing, the prescribed eradication treat-
ment was based on proton pump inhibitors together
with amoxicillin and clarithromycin, and the efficacy of
treatment was assessed by histology. The successful
cure of H. pylori infection was obtained in 31 77
(40.2%) and 41 73 (56%) patients, and the follow-up
7
Micronutrients and H. pylori Lahner et al.

Table 2 Association between Helicobacter pylori infection and

a-Tocopherol levels

a-Tocopherol First author Median mean;

levels year unit Hp+ Hp) p

Plasma serum Phull 1998 (40) Median; mg dL 11.1 10.0 NS

Phull 1996 (77) Median; mg L 11.2 10.4 NS
Sanderson lmol L 30 26 NS
1997 (53)
Toyonaga Mean; mg dla,b 1.19 1.06 NS
2000 (49)
Zhang 2000 (54) Median; lmol Lb 21.4 23.1 NS
Gastric juice Zhang 2000 (54) Median; nmol L 22.3 29.9 NS
Gastric mucosa Phull 1996 (77) Median; mg Lc 4.7 3.0 NS
Median; mg Ld 8.6 16.4 <.05
Sanderson Mean; lmol L 20 18 NS
Figure 4 Forest plot of standardized mean differences of folate levels 1997 (53)
assessed in Helicobacter pylori positives and H. pylori negatives. Test Zhang 2000 (54) Median; nmol g 12.6 20.4 .01
for heterogeneity: Q = 341.91; df = 8; p < .0001.
a
Serum levels.
b
Vitamin E.
period was 1 month. As outcome measure, both studies c
Antral mucosa.
measured the serum folate levels before and after eradi- d
Corporal mucosa.
cation treatment, and one study [65] assessed also the NS, not significant.
erythrocyte folate levels.
Meta-analyses: As shown in Fig. 4, the pooled SMD of in only one study [54]. We did not identify any study
folate levels derived from nine studies [55,58,61,66, addressing the effect of eradication treatment on
6972,76] showed that H. pylori infection was associated a-tocopherol levels.
with lower folate levels, but the total random effects of Table 2 gives the summary results of these studies
SMD, which we considered owing to the presence of separately for plasma and serum, gastric juice, and
heterogeneity (p < .0001), missed to show statistical gastric mucosa a-tocopherol levels. As far as regards
significance (total random effects of SMD )0.433, 95% a-tocopherol vitamin E levels measured in the plasma
CI )0.943 to 0.078). or serum, none of the five studies [40,49,53,54,77]
Cure of H. pylori infection had no effect on serum showed a significant difference between H. pylori-
folate levels, which were not different before and after positive and H. pylori -negative subjects. The a-tocoph-
eradication treatment (25 vs 20 nmol L by radioimmu- erol levels assessed in the gastric juice were not
noassay [63] and 5.6 vs 6.0 ng mL by electron chemi- different in infected and not-infected subjects [54]. The
luminescence immunoassay [65]), but a significant results of a-tocopherol levels in the gastric mucosa were
increase in erythrocyte folate levels after cure of infec- conflicting: one study assessed only the antral mucosal
tion was observed (539 vs 442 ng mL, p = .024), which, levels and showed a significant difference between
however, was performed by only one study [65]. infected and not-infected subjects (20.4 vs 12.6 nmol g,
p = .01) [54]; another study, which measured the
a-tocopherol levels in both the antral and corporal
a-Tocopherol
mucosa, showed that in the corporal mucosa, the levels
Study characteristics and synthesis of study outcomes: All five were significantly higher in the H. pylori-negative sub-
selected studies addressed the association between jects compared with H. pylori-positive subjects
H. pylori infection and a-tocopherol [40,53,54,77] and (16.4 mg L vs 8.6 mg L, p < .05), whereas there were
vitamin E [49]. All but one study were case series no difference in the antral mucosa [77].
[40,53,54,77], and one was a cross-sectional study [49].
These five studies involved a total number of 276
Minerals and Trace Elements
subjects (mean age 49.6 years, 47.3% women), of
whom 55.8% had H. pylori infection. None of these Calcium, copper, magnesium, phosphorus, selenium,
studies reported data on intragastric pH, and two stud- and zinc.
ies collected data on vitamin E dietary intake [49,77]. Study characteristics: Few studies investigating the influ-
Detailed data on antral and corporal gastritis were given ence of H. pylori infection on minerals and trace

8 2011 Blackwell Publishing Ltd, Helicobacter 17: 115

Lahner et al. Micronutrients and H. pylori

elements other than iron were identified: calcium [62],
copper [49], magnesium [78], and phosphorus levels
[62] were addressed by only one study, and selenium
[49,79,80] and zinc levels [49,81,82] by three studies.
All but one of these studies addressed the association
between H. pylori infection and mineral levels, and only
one, the effect of eradication treatment on selenium
levels [80]. Detailed study characteristics are given in
Table S1. None of these studies assessed intragastric pH,
and information about dietary intake of the study
mineral and or trace elements was collected in only
two studies [49,79]. Data about antral and corporal gas-
tritis associated with H. pylori infection were reported in
four studies [62,78,79,81], and on antral gastritis, only
in one study [80].
Study outcomes: The low number of identified studies
did not allow pooling of outcome data (Table 3). As far
as regards calcium [62], cuprum [49], and phosphorus
[62] in the serum, and zinc in the serum and in the
gastric antral and corporal mucosa [49,81,82], no differ-
ent levels between H. pylori-infected and not-infected
subjects were shown. As far as regards magnesium, the
serum levels did not differ in the H. pylori-positive and
H. pylori-negative groups, but the levels in the erythro-
cytes and the gastric antral mucosa were significantly
lower in the infected subjects (1.81 vs 2.14 mm L for
erythrocyte and 510.6 vs 729.2 lg g for antral mucosa
magnesium, p < .001) [78]. Also, the selenium levels
assessed in the serum [49,79] or plasma [80] did not
differ between H. pylori-positive and H. pylori-negative
subjects, but the selenium levels measured in the gas-
tric antral mucosa were significantly higher in the
H. pylori positives compared with the negatives (20.17
vs 2.83 lg g, p = .001) [80]. This result was confirmed
after eradication treatment, which led to a significant
decrease in selenium levels in the gastric antral mucosa
(20.17 vs 7.4 lg g, p < .05), whereas the plasma levels
remained unchanged (71 vs 71.6 lg L) [80]. Table 4
gives an overview of the results of meta-analyses.
Discussion
H. pylori infection is a thoroughly investigated patho-
genetic factor for several diseases as chronic gastritis,
peptic ulcer, and gastric cancer [1,2]. Less attention has
been paid to its potential role in micronutrient deficien-
cies. The findings of this systematic review and meta-
analyses support in adults the association between
H. pylori infection and reduced levels of some micro-
nutrients as ascorbic acid and cobalamin, which may be
restored after cure of infection (Table 4). To date, this is
the most comprehensive systematic review on the
possible link between H. pylori infection and reduced
micronutrients beyond iron. Our findings show a posi-
tive association between H. pylori positivity and low
ascorbic acid levels in the plasma (SMD )0.2) and in
the gastric juice (SMD )1.1) and a recovery after cure
of infection in the gastric juice (SMD )1.4). We also
report for the first time meta-analytic data showing a
positive association between H. pylori infection and

Table 3 Helicobacter pylori infection and minerals and trace elements other than iron: calcium, copper, magnesium, phosphorus, selenium, and
zinc

Mineral trace element First author year Mineral assessment Median mean; unit Hp+ Hp) p

Association with H. pylori infection

Calcium (ionic) Kakehasi 2009 (62) Serum Mean; mmol L 1.24 1.26 NS
Cuprum Toyonaga 2000 (49) Serum Mean; lg dL 86.6 84.4 NS
Magnesium Abbasciano 2003 (78) Serum Mean; mm L 0.81 0.81 NS
Erythrocytary Mean; mm L 1.81 2.14 <.001
Gastric antral mucosa Mean; lg g 510.6 729.2 <.001
Phosphorus Kakehasi 2009 (62) Serum Mean; mg dL 3.8 3.6 NS
Selenium Camargo 2008 (79) Serum lg dL NRa NRa NS
Toyonaga 2000 (49) Serum Mean; lg dL 12.7 12.8 NS
Ustundag 2001 (80) Plasma Mean; lg L 71 68 NS
Gastric antral mucosa Mean; lg g 20.17 2.83 .001
Zinc Sempertegui 2007 (81) Gastric antral + corporal mucosa Mean; ng mg 256.4 321.2 NS
Toyonaga 2000 (49) Serum Mean; lg dL 95.5 87.4 NS
Zullo 2000 (82) Serum Mean; lg dL 81.7 87.3 NS
Effect of eradication treatment Pre-tx Post-tx
Selenium Ustundag 2001 (80) Plasma Mean; lg L 71 71.6 NS
Gastric antral mucosa Mean; lg g 20.17 7.4 <.05

a
In the original paper the lack of difference is stated but data are not shown.
NS, not significant; NR, not reported.

2011 Blackwell Publishing Ltd, Helicobacter 17: 115 9

Micronutrients and H. pylori Lahner et al.

Table 4 Summary of meta-analyses of standardized mean differences (SMD) of micronutrient levels between Helicobacter pylori positives and
H. pylori negatives and or before and after eradication treatment

Micronutrient Studies (References) SMD (total random effects) 95% CI Heterogeneity (Q; p)

Vitamins
Ascorbic acid, plasma Ass (3032,34,37,38,4043,46,47,4951) )0.193 )0.372 to )0.015 278.05; <.0001
EffTx (30,31,34,45) )0.102 )0.811 to 0.606 22.75; <.0001
Ascorbic acid, gastric juice Ass (3032,3437,4143,45,48,52) )1.087 )1.794 to )0.379 752.46; <.0001
EffTx (30,31,34,42,45) )1.408 )2.471 to )0.346 80.39; <.0001
Ascorbic acid, gastric mucosa Ass (33,34,39,52) 0.129 )0.357 to 0.615 35.21; <.0001
EffTx (33,34) 0.133 )0.207 to 0.473 1.47; .22
-Carotene Ass (40,49,53,54) ND ND ND
Cobalamin Ass (5659,61,62,66,67,6972,75) )0.744 )1.147 to )0.340 435.09; <.0001
EffTx (59,6365,68) )1.910 )3.358 to )0.463 177.98; <.0001
Folate Ass (55,58,61,66,6972,76) )0.433 )0.943 to 0.078 341.91; <.0001
a-Tocopherol Ass (40,49,53,54,77) ND ND ND

Minerals trace elements

Calcium Ass (62) ND ND ND
Copper Ass (49) ND ND ND
Magnesium Ass (78) ND ND ND
Phosphorus Ass (62) ND ND ND
Selenium Ass (49,79,80); EffTx (80) ND ND ND
Zinc Ass (49,81,82) ND ND ND

Ass, studies investigating the association between H. pylori infection and low micronutrient levels; EffTx, studies investigating the effect of eradica-
tion treatment on micronutrient levels. Data in bold are statistically significant.

cobalamin deficiency (SMD )0.7), which may be
restored after eradication treatment (SMD )1.9).
The mechanisms by which H. pylori infection may
compromise the homeostasis of some micronutrients
have not been well established. The observed positive
association between H. pylori infection and ascorbic acid
and cobalamin may plausibly be explained by a com-
mon mechanism that involves reduced gastric acid
secretion leading to hypochlorhydria in the presence of
a particular pattern of H. pylori gastritis involving the
corporal acid-secreting mucosa [7,8]. Impaired gastric
acid secretion is a common consequence of H. pylori-
induced gastritis: the chronic inflammatory injury of
the corporal mucosa may lead to a functional inhibition
of parietal cells by inflammatory products, which may
finally provoke gastric gland atrophy and hypochlor-
hydria [1517]. Gastric acidity seems to play a role in
the homeostasis of ascorbic acid, the reduced form of
vitamin C, because this compound is very unstable in
the presence of an increased pH and is converted to the
less active form of dehydroascorbic acid [68], as
reported in the presence of H. pylori gastritis associated
with corporal mucosa inflammation [30,31,52]. Thus,
hypochlorhydria may decrease the stability and the bio-
availability of this vitamin. Moreover, ascorbic acid is
an important promoter of iron absorption, and
decreased bioavailability of this micronutrient may, in
turn, have negative effects on iron absorption [7,8].
Also, the absorption of cobalamin from food requires
the action of gastric acid for at least two steps of this
complex process: to release in the stomach the strictly
protein-bound dietary cobalamin and to increase the
affinity of free, unbound cobalamin for salivary R pro-
teins instead of intrinsic factor [83]. Thus, it is plausible
that hypochlorhydria caused by H. pylori infection may
contribute to food-cobalamin malabsorption [7,84,85].
Cobalamin deficiency may result also as a consequence
of gastric atrophy of the corporal mucosa and conse-
quent intrinsic factor deficiency, i.e., pernicious anemia,
which in turn is a long-standing consequence of
H. pylori infection [86,87]. The biological plausibility of
the hypothesized link between impaired gastric acid
secretion and reduced micronutrient levels is further
supported by the observation that the long-term ther-
apy with proton pump inhibitors may have negative
effects on ascorbic acid and cobalamin levels [8890].
Unfortunately, the underlying mechanisms possibly
linking together H. pylori infection, low intragastric
acidity, and micronutrient deficiency were addressed
only by a part of the included studies: data on the dis-
tribution and type of the associated H. pylori gastritis
were reported in about one-third of studies (mean
28%) and data on intragastric pH were assessed in
about half of the ascorbic acid studies and 6% of the

10 2011 Blackwell Publishing Ltd, Helicobacter 17: 115

Lahner et al.
cobalamin studies, whereas the other studies did not
perform efforts to link the micronutrient deficiency to
possible alterations of gastric acid secretion or other
mechanisms. Thus, the possible role of reduced gastric
acid secretion leading to reduced micronutrient levels
in the presence of H. pylori infection mechanisms is still
too poorly investigated to draw definite conclusions,
and other mechanisms different from hypochlorhydria
linking together H. pylori and low micronutrient levels
are possible.
Beyond ascorbic acid and cobalamin, our findings do
not support a clear association between H. pylori infec-
tion and deficiencies of other vitamins and minerals.
One reason for this may be the lack of appropriate,
well-designed studies. Ascorbic acid and cobalamin
were investigated by 44% and 40% of the overall
selected 52 studies, respectively, whereas other vitamins
and minerals were addressed by a lower number of
reports that did not permit to compute pooled outcome
estimates. As a consequence, a possible role of H. pylori
could emerge for those micronutrients, which had been
investigated by a sufficient number of studies by using
accurate study design and methods, whereas for poorly
investigated micronutrients, this relationship possibly
could have remained hidden, albeit single well-
conducted studies showed a positive link with the
infection. For some micronutrients, the study design
and the method of assessment were pivotal for showing
or not an association between H. pylori infection and
the micronutrient level, thus further complicating the
pooling of study outcomes.
The data of our meta-analyses did not support the
association between H. pylori positivity and low folate
levels, addressed in 21% of the selected studies. A
negative effect of H. pylori infection on folic acid absorp-
tion decrease may be explained as a consequence of a
pH increase and or a vitamin C concentration decrease
in gastric juice, a situation frequently observed in
H. pylori-infected patients [6,91]. A possible reason for
this may be the fact that a low number of studies
assessed erythrocyte folate levels, which have been
reported to better reflect the folate status than serum
levels [10].
The interpretation of our findings deserves some cau-
tion. The strength of evidence varied markedly between
the single micronutrients and even within the same
micronutrient assessed at different levels, i.e., plasma
and gastric juice. Moreover, a considerable heterogene-
ity was observed (Table 4), impairing the strength of
observed evidence. We tried to overcome this problem
expressing the pooled outcome estimates as random-
effects model, which gives more weight to the results of
smaller studies than does the fixed-effects analysis and
2011 Blackwell Publishing Ltd, Helicobacter 17: 115
Micronutrients and H. pylori
considers both the within- and between-study variabil-
ity. It is likely that one of reasons for the observed
heterogeneity may be the highly variable number of
participants, particularly among the studies on the asso-
ciation between H. pylori infection and micronutrient
levels, which included large population-based seroepi-
demiological studies as well as smaller case series.
As far as regards the quality of included studies, there
are some concerns that may influence the weight of
our observations. All studies investigating the effect of
eradication treatment on micronutrient levels were case
series; studies addressing the association between
H. pylori infection and micronutrient levels were mainly
cross-sectional based on clinical setting and case series.
Thus, the vast majority of selected studies were of
moderate quality with a limited level of evidence [92].
Moreover, the clinical setting of included patients was
heterogeneous, including patients with different clinical
conditions as anemia, dyspepsia, renal or cardiac
diseases as well as asymptomatic subjects. This may
possibly impair the strength of the observed evidence,
making data less homogeneous, but, at the same time,
the broad spectrum of included H. pylori-infected adult
subjects might better reflect real life.
Moreover, the dietary intake of the investigated
micronutrient was assessed in a low proportion of stud-
ies, thus missing adjustment for an important
confounder. It should further be considered that dietary
habits are highly variable among different regions of
the world and even within regions, a fact that may
importantly influence the measurable micronutrient
levels in subjects from different origins. However, the
geographic provenance of the study populations was
mainly from regions with rather similar dietary habits,
because the majority (68%) of selected studies was per-
formed in Europe, USA, and Australia.
Regarding H. pylori infection, the proportion of
H. pylori positivity was ranging from 42% in ascorbic
acid studies to 49% in folate studies fitting with the
mean age of included subjects ranging from 44 years in
the folate studies to 58 years in the cobalamin studies
according to the cohort effect of the prevalence of this
infection [3,4]. An accurate diagnosis of H. pylori
infection with at least two diagnostic methods, as rec-
ommended [93], was performed in a median of 35% of
observational studies, ranging from 33% in folate
studies to 48% in ascorbic acid studies. In the studies
on eradication treatment, the accuracy of H. pylori diag-
nosis was higher, reaching a median of 90% of studies
that used at least two diagnostic methods, but the effi-
cacy of eradication treatment was assessed by at least
two tests in a median of 57% of studies. Subjects were
followed up for very short periods, median 1 month,
11
Micronutrients and H. pylori
thus information about long-term outcome was not
available. We are not able to exclude that these pitfalls
regarding the quality of selected studies may interfere
with the validity of our findings.
Some limits of our approach needs to be considered:
- This systematic review addressed a broad topic
addressing the link between H. pylori infection and the
micronutrients all together, thus reporting a huge
amount of data. A criticism which may emerge is that
it would have been simpler to approach singly each
individual micronutrient, thus reducing data and find-
ings. In our opinion, our comprehensive approach
should have been able to give a more appropriate
answer to the question whether H. pylori infection
needs to be considered as a possible causal factor in the
case of micronutrient deficiencies, which in the clinical
practice may not necessarily present separately, but
often in various combined forms.
- Owing to logistic reasons, only one database for
electronic search was used, and it is thus possible that
not all the relevant studies were identified from com-
puterized searching. However, we do not think that this
should represent an important pitfall of our work,
because, firstly, the database used is a highly qualified
and reliable source for electronic searching, and, sec-
ondly, because our search strategy, beyond electronic
searching, was based on an extensive manual search-
ing, which has been considered a key part of the search
process in any systematic review [94]. We performed a
systematical handsearching of reference lists of reviews
and editorials judged as relevant according to our topic
as well as of all potentially relevant selected full papers,
thus considerably increasing the accuracy and efficiency
of our search strategy.
Considering the worldwide high prevalence of
H. pylori infection, its interference with micronutrient
malnutrition may have important clinical implications:
over 50% of people in the world are infected by
H. pylori and in Africa, South America, and Central
America, H. pylori infection reaches 7090% of the pop-
ulation [3,4]. In developing and poorly industrialized
regions, there is a concomitant high prevalence of both
H. pylori infection and micronutrient malnutrition [95].
To date, we are not able to know to which extent
micronutrient deficiencies may be facilitated by H. pylori
infection, but, according to our findings, in the regions
of the world in which micronutrient deficiencies are a
major concern and in which H. pylori infection is highly
prevalent, intervention programs might take into con-
sideration the need of diagnosis and treatment of
H. pylori infection.
In conclusion, the findings from our meta-analyses
indicate that H. pylori infection is associated with
12
Lahner et al.
reduced levels of ascorbic acid and cobalamin, sup-
ported by the positive effect of eradication treatment.
For other micronutrients, data are insufficient to draw
conclusions and further studies are needed.
Acknowledgements and Disclosures
We thank Ms Felicia Proietti and her collaborators, University
Library, 2nd Medical School, as well as Mr Michele
Putignano, University Library, Department of Internal
Medicine, University Sapienza Rome, Italy, for their support
in the search of hard copies of the reviewed papers. This
work was supported by grants from the Italian Ministry for
University and Research 2007 and University Sapienza Rome
2007 2009.
Authors Contributions
Lahner E designed and conducted research, analyzed data and
performed statistical analyses, and wrote the paper; Persichino
S contributed to conduct the research; Annibale B designed
and overviewed research and had primary responsibility for
final content. All authors read and approved the final
manuscript.
Conflict of Interests
The authors declare that there is no conflict of interest with
regard to this work that they should disclose.
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Table S1 Characteristics of the selected studies.
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