A Pilot Study on Zinc Levels in Patients
with Rheumatoid Arthritis
Artur Mierzecki & Dorota Strecker &
Received: 23 November 2010 /Accepted: 28 December 2010 /
Published online: 29 January 2011
# The Author(s) 2011. This article is published with open access at Springerlink.com
Abstract The aim of the study was to evaluate zinc levels in three biological compartments
(serum, erythrocytes and hair) in patients with rheumatoid arthritis (RA) as compared to
healthy individuals. Zinc levels in serum, erythrocytes and hair (in 74 patients with RA and
30 healthy individuals) were assessed by atomic absorption spectroscopy. The mean hair
zinc content was significantly lower in RA patients as compared to healthy individuals (p<
0.001). Moreover, a positive correlation was observed in the RA patient group between the
erythrocyte zinc levels and the prednisone dose (rs=0.48, p<0.05), and a negative
correlation was found in this population between the serum zinc levels and disease duration
(rs=−0.42, p<0.0006). In conclusion, it seems that hair may be a useful complementary
study material for evaluating “zinc status” in rheumatoid arthritis patients.
Biol Trace Elem Res (2011) 143:854–862
A. Mierzecki (*)
Independent Laboratory of Family Physician Education, Pomeranian Medical University, ul. Rybacka 1,
70-204 Szczecin, Poland
Rheumatology Outpatient Clinic in Szczecin, Szczecin, Poland
Department of Paediatric Nursing, Pomeranian Medical University, ul. Zolnierska 48,
71-210 Szczecin, Poland
Institute of Quantum Electronics, Military Technical Academy, ul. Kaliskiego 2,
00-908 Warsaw 49, Poland
Rheumatoid arthritis (RA) is a well-studied progressive autoimmune disease. However, no
factor responsible for the immune system activation in the course of the disease has been
identified to date . Among the many contributing agents that have been proposed to take
part in the pathogenesis of this condition, trace elements, including zinc, have also been
investigated [2–5]. The evidence for their involvement stems from the observation that
postmenopausal women consuming higher amounts of zinc are at a lower risk of RA (RR=
0.39, 95% CI=0.17–0.88, p<0.03) .
It has been shown that reactive oxygen species play a role in the aetiology of RA [1, 6–
10] and that one of them is the superoxide radical, which is eliminated by superoxide
dismutase—an enzyme containing zinc in its molecule [3, 5, 11]. It has also been found that
over 90% of this trace element present in erythrocytes is bound with carbonic anhydrase
and superoxide dismutase . Furthermore, zinc mobilises vitamin A, a potent
antioxidant, from the liver, thus maintaining its proper levels in the blood .
Zinc is crucial for the proper functioning of approximately 300 hormones and enzymes in
the human body[11, 12], the latter representing all the classes taking part in the synthesis and
degradation of proteins, carbohydrates, fats and nucleic acids. Zinc constitutes a structural
element of alkaline phosphatase (ALP), with four of its atoms being present in the enzyme.
Zinc also stimulates ALP synthesis in osteoblasts and plays an important role in bone
mineralisation . In addition, zinc is responsible for the latency of metalloproteinases (such
as collagenases, gelatinases, stromelysins, matrilysin, metalloelastases and membrane
metalloproteinases) by means of a coordination bond between a zinc atom in the centre of
the enzyme and a free cysteine–SH group present in the pro-peptide. Notably, the breaking of
this bond and the release of the pro-peptide result in the activation of metalloproteinases
responsible for articular cartilage destruction observed in RA [11, 12, 14].
Zinc deficiency has been previously studied in an experimental human model involving
males on a low-zinc diet (mild zinc deficiency) . The reported effects included lowered
plasma testosterone levels, oligozoospermia, decreased natural killer cell function, reduced
interleukin 2 production, low thymulin levels, hyperammonaemia, diminished sensitivity to
different tastes, impaired adaptation to the dark and decreased lean body mass.
The investigators’opinionsonthezincstatusinthehuman bodyvary,particularlyinrespect
to this element’s serum, plasma and erythrocyte levels. Relatively few publications addressing
the zinc content in hair are available [16–19], and there seems to be no reports on its
assessment in RA patients. Thus, the authors of the present study undertook the task of
evaluating the content of this bioelement in serum, erythrocytes as well as hair of patients
with rheumatoid arthritis.
The aim of this work was to examine the differences between the levels of zinc present
in three fundamental biological compartments in patients with RA and in healthy
individuals, with a particular focus on hair.
Material and Methods
Seventy-four patients treated for RA were evaluated in two specialist Rheumatology
Outpatient Clinics in Szczecin, Poland. The studied group comprised 20 men and 54
women aged 29–50 (mean age, 39.8±6.1 years). The diagnosis of RA was based on
Zinc Levels in Patients with Rheumatoid Arthritis855
meeting at least four of the seven criteria given by the American College of Rheumatology
RA patients were divided into four subgroups (listed below) depending on the received
pharmacological treatment during the previous 6 months prior to the study:
MTX=patients treated with methotrexate (MTX) at a weekly dose range from 7.5 to
12.5 mg/week and non-steroidal anti-inflammatory drugs (NSAIDs);
MTX+GC=patients treated as above but also receiving glucocorticoids (GC),
prednisone, at a dose of 5.0–12.5 mg daily;
MTX+GC+Ca+D3=patients treated as above but also receiving calcium and vitamin
D3(as means of preventing osteoporosis); and
SAS=patients receiving sulphasalazine (SAS) at a dose of 1,500–2,000 mg/daily and
The control group consisted of 30 healthy individuals reporting successively to the clinic
of two general practitioners in Szczecin for reasons unrelated to RA. This group comprised
8 men and 22 women aged 27–50 (mean age, 38.2±8.3 years).
The inclusion criteria applied to both groups were as follows:
1.No diuretics, hypotensive agents, anticonvulsants, antibiotics, vitamin supplements
containing studied elements or oral contraceptives (females) taken for at least 6 months
prior to the study;
Nutrition—following a normal diet;
No evident gastrointestinal or urinary disturbances or endocrine conditions;
Age below 50 and regular menstruation (females); and
Informed consent to take part in the study.
All study participants had their medical history taken and underwent a physical examination
and had their zinc levels measured: in the extracellular fluid (serum) and in the intracellular
compartment (erythrocytes and hair).
The study has been accepted by the Bioethical Commission (Ethics Board) of the
Pomeranian Medical University in Szczecin for studies in human subjects.
Serum and Erythrocyte Zinc Levels
Zinc levels in serum and in erythrocytes were measured by atomic absorption spectroscopy
samples were prepared by mineralisation in a 5:2 mixture of ultrapure nitric acid and perchloric
quantitatively into a volumetric flask and filled up to the final volume with deionised water.
Hair Zinc Content
Hair sections of 3–4 cm in length, measured from the skin, were collected from six to eight
scalp regions with stainless steel scissors to avoid contamination with other heavy metals.
856Mierzecki et al.
The total hair sample weight was 0.2–0.3 g. For at least 6 months prior to the study, no hair
treatment was applied that might potentially alter the biological hair content of the studied
bioelements (the hair was not dyed, permanent waved, conditioned, etc.). The hair samples
were washed with a water solution of non-metal detergent, rinsed with deionised water and
dried at 60°C. Next, they were rewashed with a mixture of anhydrous alcohol and acetone
for 25 min, dried at 60°C and placed in a desiccator for 24 h. Subsequently, the hair
samples were weighed using analytical scales and subjected to mineralisation with a
mixture of ultrapure concentrated nitric acid and perchloric acid at a ratio of 5:2 (v/v). The
mineralisation process was conducted at the boiling point of the acid mixture, with water
and excess acids being distilled off to approximately one third of the original sample
volume. The remaining solution was transferred quantitatively to 25-cm3glass flasks and
filled to volume with water distilled three times with a quartz distillation apparatus .
The zinc content was assessed by AAS using either an acetylene–air or acetylene–nitrous
oxide flame. The measurements were conducted with a Pay-Unicam SP-9 spectrophotometer
interfaced with a computer.
The levels of zinc in serum, erythrocytes and hair were analysed at the Institute of
Quantum Electronics, Military Technical Academy, Warsaw, Poland.
Statistical analysis was performed using the StatSoft Polska v. 9.0 package (StatSoft Inc.,
Tulsa, Oklahoma, USA).
The distribution type of the studied parameters within the analysed groups was
determined based on the Shapiro–Wilk test for normality (N), sample size (n), range of
values (range), median (Me), mean x ð Þ and standard deviation (SD). The results from two
independent groups were compared using the Student’s t test (when the distribution of a
given parameter was not significantly different statistically from the normal distribution in
any of the comparison groups) or the Mann–Whitney U test (when the distribution of a
given parameter was significantly different statistically from the normal distribution in at
least one of the groups being compared).
In order to determine a statistical relationship between two variables, either the Pearson’s
(linear) correlation coefficient (when both variables had normal distribution) or Spearman’s
rank correlation coefficient (with at least one variable not having the normal distribution)
In all comparisons, the adopted significance threshold was p value ≤0.05.
Table 1 shows the distribution patterns of the quantitative parameters in RA patients: mean
disease duration, RA clinical activity such as the Ritchie articular index for joint tenderness
 as well as the subjective joint pain score based on the visual analogue scale (VAS), and
haematology test results for erythrocyte sedimentation rate (ESR) and haemoglobin (Hb)
level. Additionally, the disease activity of RA was determined with the disease activity
score including 28 joints using ESR (DAS28-ESR) .
Table 2 presents the comparison of zinc levels in serum, erythrocytes and hair from
patients with RA and from healthy individuals. Statistically significant lower zinc
levels were found in RA patients versus control group, but only for the hair
Zinc Levels in Patients with Rheumatoid Arthritis857
Within the RA patient group, no statistically significant differences were found in the
zinc levels between the treatment subgroups irrespective of the evaluated biological
compartment (Table 3).
Table 4 presents the values of Spearman’s rank correlation coefficients (rs) for statistical
dependence between the zinc levels in studied biological compartments and the doses of
administered MTX and prednisone (GC). A positive correlation was observed between the
erythrocyte zinc levels and the glucocorticoid dose (rs=0.48, p<0.05).
Table5 presents the relationship between the evaluated parameters and the levels of zinc in
each of the biological compartments. The only statistically significant correlation (a negative
one) was observed between the serum zinc levels and the disease duration (rs=−0.42, p<
0.0006). No correlation was established between the zinc levels in the analysed biological
compartments and disease activity determined by DAS28.
Our evaluation of the zinc content in the hair of patients with RA is a pioneering study on
“zinc status” in RA patients. The available literature offers reports focussing solely on the
examination of zinc levels in plasma/serum, urine and, rarely, in erythrocytes of patients
with RA [2, 24–29]. The hair is a practical material for conducting bioelement analysis
since, similar to skin, myocardium and skeletal muscles, it constitutes a more stable depot
of elements than blood serum or erythrocytes [11, 16, 17, 19, 23]. Growing at a rate of 1.0–
2.0 cm per month, hair along its entire length constitutes a specific reflection of all the
biochemical events occurring in the body [17, 18]. Unlike tissues, hair is the end metabolic
product, with various elements incorporated into its composition as it grows. Upon reaching
the skin surface, the hair hardens and keratinises, and the elements accumulated during its
formation become embedded in its protein structure . The present study demonstrates
Table 1 Study group characteristics (patients with RA, n=74)
ParameterNormality of distributionRangeMedianMean ± SD
Mean disease duration (years)
Ritchie articular index (points)
Table 2 Comparison of mean zinc levels in serum, erythrocytes and hair of patients with RA versus the
Type of sample in which mean zinc
levels/content were assessed
RA patients (n=74)Control group (n=30)p value
Hair (μg/g d.h.m.)
d.h.m. dry hair mass
858 Mierzecki et al.