Nail antioxidant trace elements are inversely associated with inflammatory markers in healthy young adults.

Abstract

Antioxidant intake may be linked to a reduction of the chronic low-grade inflammatory state related to obesity and several accompanying disorders such as insulin resistance, cardiovascular diseases, and metabolic syndrome. So, the aim of this study was to evaluate the potential associations between nail trace elements and several indicators in healthy young adults, emphasizing on the putative effect of antioxidant trace element intake on inflammation-related marker concentrations. This study enrolled 149 healthy young adults, whose anthropometrical and blood pressure values as well as lifestyle features were analyzed. Fasting blood samples were collected for the biochemical and inflammation-related measurements (C-reactive protein, tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6, IL-18, and homocysteine). Nail samples were collected for the analysis of selenium, zinc, and copper concentrations. Our results showed that nail selenium was negatively associated with IL-18; nail zinc concentrations were inversely related to circulating IL-6, IL-18, and TNF-alpha, whereas nail copper (Cu) and Cu/selenium values were negatively correlated with homocysteine levels and the Cu/zinc ratio was unaffected. In conclusion, nail content on some trace elements related to antioxidant defense mechanisms seems to be associated with several inflammation-related markers linked to chronic diseases in apparently healthy young adults, which is of interest to understand the role of antioxidant intake.

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Nail Antioxidant Trace Elements Are Inversely
Associated with Inflammatory Markers
in Healthy Young Adults
Blanca Puchau & María Ángeles Zulet &
Helen Hermana Miranda Hermsdorff &
Íñigo Navarro-Blasco & J. Alfredo Martínez
Received: 24 March 2009 /Accepted: 23 June 2009 /
Published online: 7 July 2009
# Humana Press Inc. 2009
Abstract Antioxidant intake may be linked to a reduction of the chronic low-grade
inflammatory state related to obesity and several accompanying disorders such as insulin
resistance, cardiovascular diseases, and metabolic syndrome. So, the aim of this study was
to evaluate the potential associations between nail trace elements and several indicators in
healthy young adults, emphasizing on the putative effect of antioxidant trace element intake
on inflammation-related marker concentrations. This study enrolled 149 healthy young
adults, whose anthropometrical and blood pressure values as well as lifestyle features were
analyzed. Fasting blood samples were collected for the biochemical and inflammation-
related measurements (C-reactive protein, tumor necrosis factor-alpha (TNF-α), interleukin
(IL)-6, IL-18, and homocysteine). Nail samples were collected for the analysis of selenium,
zinc, and copper concentrations. Our results showed that nail selenium was negatively
associated with IL-18; nail zinc concentrations were inversely related to circulating IL-6,
IL-18, and TNF-α, whereas nail copper (Cu) and Cu/selenium values were negatively
correlated with homocysteine levels and the Cu/zinc ratio was unaffected. In conclusion,
nail content on some trace elements related to antioxidant defense mechanisms seems to be
associated with several inflammation-related markers linked to chronic diseases in
apparently healthy young adults, which is of interest to understand the role of antioxidant
intake.
Keywords Inflammation.Oxidativestress.Antioxidant.Traceelement.Nail.Selenium.
Zinc.Copper
Biol Trace Elem Res (2010) 133:304–312
DOI 10.1007/s12011-009-8443-5
B. Puchau:M. Á. Zulet:H. H. M. Hermsdorff:J. A. Martínez (*)
Department of Nutrition and Food Science, Physiology and Toxicology, University of Navarra,
Calle Irunlarrea 1, 31008 Pamplona, Spain
e-mail: jalfmtz@unav.es
Í. Navarro-Blasco
Department of Chemistry and Soil Science, University of Navarra,
Calle Irunlarrea 1, 31008 Pamplona, Spain

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Introduction
Oxidative stress has been suggested as a potential promoter of inflammatory processes and
to be involved in the susceptibility to develop obesity and related diseases [1]. Thus, the
role of inflammation and oxidative stress processes on several chronic diseases is receiving
increasing attention due to their association with atherosclerosis, obesity, or type 2 diabetes
[2, 3]. The cause–effect relationships between these mechanisms and clinical manifestations
are not fully clear, but a number of studies have associated such pathological conditions
with higher plasmaconcentrations of inflammatory biomarkers [4, 5].
In this context, the intake of dietary antioxidants has been found to be related with
lower C-reactive protein (CRP) levels in women, suggesting a possible anti-inflammatory
role for antioxidant nutrients [6]. Furthermore, a high selenium (Se) and zinc (Zn) intake
has been hypothesized to reduce the risk of diseases induced by oxidative stress and
inflammation [7].
The aim of this study was to evaluate the possible associations of nail Se, Zn, and copper
(Cu) with several anthropometrical, biochemical, lifestyle, and circulating inflammatory
indicators in healthy young adults, emphasizing on the putative effect of antioxidant trace
element intake or related ratios on inflammation-related marker concentrations.
Materials and Methods
Subjects
One hundred and forty-nine healthy Caucasian subjects were recruited to participate in the
study (101 women and 48 men; age 20.9±2.7 years). Initial enrolment screening
evaluations included medical history, physical examination, and fasting blood profile to
exclude subjects with evidence of any disease at baseline related to chronic inflammation,
oxidativestress,hydricimbalance,nutrientabsorption,ornutrientmetabolism.Otherexclusion
criteria werechronicdrug ordietetic treatmentupto 6 monthsbeforeparticipation inthis study.
At the time of the initial interview, data about lifestyle features (smoking, vitamin
supplementation, physical activity) were retrieved. In accordance with the Declaration of
Helsinki, all subjects gave written informed consent to participate, which was previously
approved by the Ethics Committee of the University of Navarra (ref:79/2005).
Anthropometry and Body Composition
All anthropometric measurements were carried out with the subjects who were barefoot,
wearing only their underwear and after an overnight fast following standardized protocols
[8]. All these measurements were done three times, but not consecutively. Body weight was
measured to the nearest 0.1 kg and body fat to the nearest 0.1% by using a Tanita TBF 300
(NY, USA). Body mass index (BMI) was calculated according to body weight divided by
squared height (kilogram per square meter).
Analyses of Biological Samples
All serum blood samples were drawn after an overnight (12-h) fast, immediately
centrifuged for 15 min at 3,500 rpm and 4°C, and stored at −80°C. Serum glucose,
triacylglycerols, total cholesterol, and high-density lipoprotein cholesterol (HDL-c) were
Nail Trace Elements Associated with Inflammatory Markers305

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assessed by an automated colorimetric assay (Cobas Mira, Roche, Switzerland) with
specific commercial kits (ABX Pentra, Roche, Switzerland). The reported plasma low-
density lipoprotein cholesterol (LDL-c) data were calculated by the Friedewald equation as
described elsewhere [9]. Plasma circulating concentrations of CRP (Immundiagnostik AG,
Bensheim, Germany), interleukin (IL)-6 (R&D Systems, Minneapolis, MN, USA), tumor
necrosis factor-alpha (TNF-α; R&D Systems), and IL-18 (Medical & Biological
Laboratories Co., Naka-ku Nagoya, Japan) were evaluated by specific enzyme-linked
immunosorbent assay procedures. Plasma homocysteine concentrations were measured by a
colorimetric assay (Cobas Mira, Roche) by using a commercially available kit (Demeditec
Diagnostics GmbH, Kiel-Wellsee, Germany). Nail samples were collected at the time of
interview and stored at room temperature in clean polypropylene bags labeled with subject
identification numbers. Fingernail and toenail samples were treated with subboiling
nitric acid in a high-pressure Teflon digestion vessel using a microwave digestion system
(Ethos Plus, Millestone, Sorisole, Italy). A Perkin Elmer AAnalyst 800 atomic absorption
spectrometer (Norwalk, CT, USA), equipped with transverse-heated graphite atomizer,
Zeeman background corrector, and AS-800 autosampler, was used for the measurement
of Se at 196.0 nm with a spectral band width of 2.0 nm as used elsewhere [10]. An
electrodeless discharge lamp (Perkin Elmer) was used as a light source operated at 280 mA.
Pyrolytically coated graphite tubes with end caps supplied by Perkin Elmer were used. Zn
and Cu concentrations in digested acid solutions were analyzed by flame atomic absorption
spectrophotometry (Perkin Elmer). Zn and Cu hollow cathode lamps (Perkin Elmer),
providing resonance lines of 213.9 and 324.8, operated both at 15 mAwith a slit width seat
at 0.7 nm were used. The measured concentration values were adjusted for the sample
weight and expressed as microgram per gram (µg/g) of nail.
Statistical Analysis
The Shapiro–Wilk test was used to determine variable distribution. Results are presented as
mean ± standard deviation and p<0.05 was considered statistically significant. Cut-off
values were defined as 0.42, 113.3, and 5.9 μg/g of sample for selenium, zinc, and copper
and 0.05, 13.5, and 262.1 for Cu/Zn ratio, Cu/Se ratio, and Zn/Se ratio, respectively.
Statistical analyses (Mann–Whitney U test, Spearman and partial correlations) were
performed by using SPSS version 15.0 (SPSS Inc., Chicago, IL, USA) for Windows XP
(Microsoft, Redmond, WA, USA).
Results
Anthropometrical characteristics and lifestyle features of subjects categorized according to
nail Se, Zn, and Cu median concentrations and Cu/Se ratio median value (cut-off values
categorization) are described in Table 1. Men and women distribution had only statistical
differences between Cu/Se groups. However, we did not find any statistically significant
change for any studied variable according to Cu/Zn and Zn/Se ratios (data not shown).
Moreover, sex distribution showed a similar trend for Zn concentrations. Significantly
lower values for subjects with high concentrations of nail Zn and Cu were found for body
weight. Similar trends (p<0.10) were followed by BMI values. The only statistically
significant differences found in lifestyle markers were achieved for physical activity when
split by nail Zn and Cu concentrations. Also, an inverse statistical trend (p<0.10) was found
for smoking concerning Se and Cu, Cu/Zn, and Cu/Se nail concentrations.
306 Puchau et al.

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Regarding biochemical data, only statistical differences in HDL-c were found with nail
Zn concentrations categorized by the median (Fig. 1). However, a statistically significant
decrease was found for IL-18 when split according to Se concentrations, for TNF-α
according to nail Zn values, and for homocysteine regarding Cu/Se values (Fig. 2).
Moreover, similar trends were followed by CRP according to nail Se (p<0.10), by IL-6
when split by Zn and Cu concentrations, and by homocysteine regarding Cu values. To
further explore the relationships between nail antioxidant trace elements and inflammatory
markers, correlations analyses were performed. Nail Se was negatively associated with
IL-18 (rs=−0.187; p=0.026); Zn concentrations were inversely related to circulating IL-6
(rs=−0.169; p=0.042), IL-18 (rs=−0.177; p=0.036), and TNF-α (rs=−0.227; p=0.006);
homocysteine levels were negatively correlated with nail Cu (rs=−0.165; p=0.049) and
values (rs=−0.166; p=0.047), whereas Cu/Se ratio was positively associated with CRP
concentrations (rs=0.163; p=0.050).
Discussion
The food content in trace elements changes geographically and depends on the soil and
water characteristics, as well as on the use of fertilizers containing trace elements [11–13].
Thus, the dietary intake evaluation of some trace elements is not a reliable assessment of
their exposure [11] and is traditionally assessed through specific biomarkers [12]. Thus,
urine and blood Se concentrations reflect recent intake: several days for urine samples [14]
and several weeks for blood measurements [15]. In contrast, nail samples report the last
6- to 12-month exposures, being considered a long-term marker [11]. Since the aim of our
study was to evaluate habitual intake, nail samples were considered as a reliable option in
Table 1 Anthropometrical and Lifestyle Data (Mean ± SD) for Young Adults Categorized by the Median of
Nail Trace Elements and Their Cu/Se Ratio
SeleniumZincCopperCu/Se ratio
<0.42
(n=74)
>0.42
(n=74)
<113.3
(n=74)
>113.3
(n=73)
<5.9
(n=76)
>5.9
(n=72)
<13.5
(n=73)
>13.5
(n=74)
Sex (n males (%)) 23 (31.1)
Body weight (kg)
Body mass
index (kg/m2)
Body fat (%)
Vitamin
supplementation
users (n (%))
Smokers (n (%))
Smoking habits
(cigarettes/day)
Physical activity
(h/week)
24 (32.4)29 (39.2)18 (24.7)29 (38.2)19 (26.4) 32 (43.8)15 (20.3)*
63.3±12.6 62.2±10.5 65.2±11.2 60.8±11.6* 64.9±12.4 60.7±10.4* 64.5±12.0 61.1±11.1
22.2±2.722.1±2.5 22.5±2.3 21.9±2.822.5±2.721.8±2.5 22.3±2.622.0±2.6
20.4±6.2
25 (33.8)
20.0±7.1
25 (33.8)
20.4±6.3
28 (37.8)
20.1±6.9
21 (28.8)
20.2±7.2
28 (36.8)
20.2±6.0
22 (30.6)
18.9±7.0
28 (38.4)
21.6±6.0*
22 (29.7)
22 (29.7)
3.5±5.6
12 (16.2)
1.7±4.4
15 (20.3)
2.3±4.8
18 (24.7)
2.9±5.4
13 (17.1)
2.3±4.9
21 (29.2)
3.0±5.3
12 (16.4)
2.0±5.0
22 (29.7)
3.2±5.2
29.9±19.0 28.7±17.2 25.2±17.0 33.2±18.0* 26.4±17.7 32.7±18.1* 30.1±21.0 28.4±14.4
Cut-off values were 0.42, 113.3, and 5.9 μg/g of sample for selenium (n=148), zinc (n=147), and copper
(n=148) and 13.5 for Cu/Se ratio (n=147), respectively
*p value<0.05 for χ2test and for Mann–Whitney U test between low and high nail concentrations of trace
elements
Nail Trace Elements Associated with Inflammatory Markers 307

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order to analyze trace element concentrations in relation with dietary intake. Moreover, nail
collection is a non-invasive method that allows an easy long-term storage [16].
Trace elements are known to interact with each other because of their tendency to form
similar coordination complexes with other ions. This means that an excess of one trace
element may saturate carriers that two elements have in common and thus inhibit the
absorption of the other element [17]. Moreover, the ratio of Cu/Zn suggests an inverse
correlation between these micronutrients as reported elsewhere [18]. In this sense,
bioavailability of Cu is affected by other trace element supplementation [19]. Since those
interactions, represented by ratios such as serum Cu/Se, Zn/Se, but mainly Cu/Zn occur in
humans and have been shown to be different in health and disease [20–23], we studied Se,
Zn, and Cu ratios in relation with inflammatory-related markers without relevant findings.
However, it has been described that blood Cu/Zn serves as a better supportive evidence
Fig. 1 Circulating total
cholesterol (a), HDL-c (b), and
LDL-c (c) concentrations
depending on nail Zn values.
Concentrations of total
cholesterol, HDL-c, and LDL-c
are means and standard
deviations of the mean (n=147).
Nail Zn was categorized as low
and high values, according
to the median value (cut-off
113.3 μg/g). *p values (p<0.05)
are from the Mann–Whitney U
test
308 Puchau et al.