Use of Flame Atomic Absorption Spectroscopy and Multivariate Analysis for the Determination of Trace Elements in Human Scalp

Sayo O. Fakayode, Sri Lanka Owen, David A. Pollard, Mamudu Yakubu

Department of Chemistry, Winston-Salem State University, Winston-Salem, USA.

DOI: 10.4236/ajac.2013.47044                

Abstract

The analysis of trace elements in human hair for use as biomarkers continues to generate considerable interest in environmental and bioanalytical studies, medical diagnostics, and forensic science. This study investigated the concentrations of essential and toxic elements (Fe, Mg, Ca, Cu, Zn, Cr, Cd, and Pb) using flame atomic absorption spectroscopy (FAAS) in human scalp hair obtained from subjects living in Forsyth County, North Carolina, USA. The influence of age, sex, race, and smoking habits on the levels of trace elements in the hair samples were also investigated. Additionally, analyses were subjected to a statistical, regression, and principal component analysis to evaluate inter-elemental association and possible pattern recognition in hair samples. Furthermore, Ca/Mg and Zn/Cu ratios, which are often used to evaluate the degree of Ca and Cu utilization in humans and as markers for various health related issues including, atherosclerosis, hypertension, insulin sensitivity, and pancreatic cancer, were calculated. The overall mean concentrations of Fe (25 μg/g), Ca (710 μg/g), Mg (120 μg/g), Zn (190 μg/g), Cu (12 μg/g), and Cr (0.20 μg/g) were found in hair samples. The trace element concentrations varied widely in hair samples as demonstrated by large range of concentrations obtained for each element. However, levels of Cd and Pb elements of <0.030 μg/g were detected in hair sample. In general, the levels of the trace elements in hair samples were poorly correlated. However, significant correlations were found between Ca and Mg (r = 0.840, p = 0.05). The levels of Fe, Ca, Mg, Zn, Cu, and Cr in hair samples and the calculated Ca/Mg and Zn/Cu ratios were found to be largely correlated with age, race, sex, and smoking habits.

Keywords

Human Scalp Hair; Biomonitoring; Trace Element Concentration; Flame Atomic Absorption Spectroscopy; Regression and Principal Component Analysis; Inter-Element Association

1. Introduction

Trace elements, including Fe, Ca, Mg, Zn, and Cu, play critical roles in proper human body development and metabolic activity [1-3]. For example, Fe is required in the diet for proper functioning of the liver and hemoglobin, a protein in humans primarily responsible for the transportation and distribution of oxygen from the lungs to various human organs [3]. Iron is also a critical component of myoglobin, an oxygen storing heme protein residing in the cell and is responsible for the color of meat [4-7]. Iron deficiency may result in insomnia, stunted growth, decreased immune function and inhibition of hemoglobin synthesis, particularly in women and children, resulting in anemia [8,9]. Calcium and magnesium are necessary for effective teeth and bone structure development, facilitating transmission of nerves impulses, carbohydrate and protein metabolism, activation of various enzymatic reactions, and enhancement of the absorption of phosphorus and vitamins [3]. Zinc and copper promote normal body metabolism, are critical in normal genetic expression, and are essential co-enzymes, catalyzing various enzymatic reactions [1,10]. These trace elements are required at certain concentrations which can be primarily obtained from sufficiently balanced diet and food supplements.  

In contrast, heavy metals such as Pb and Cd are non essential elements and their presence in the human body can be harmful, with serious health consequences. For instance, health hazards, including reduction of child intelligence quotients, hypertension, depression, mental disorders, cancer diseases, and deoxyribonucleic acid damage have been associated with heavy metals at high concentrations [11-15]. Humans are often exposed to heavy metals through various routes, including the consumption of heavy metal contaminated foods or water, occupational exposure in working environments, lead paints, contaminated toys, industrial activity, vehicular emissions, and environmental exposure [16-30]. The critical role of essential elements in normal metabolic activity and the potential negative health consequences of toxic heavy metals necessitate the need for routine biomonitoring of the levels of trace metals elements.

Toward these efforts, various studies have utilized a wide range of human specimens including, human serum, blood, tissues, urine, and saliva as biomarkers for effective biomonitoring of trace element concentrations [31- 35]. However, these biomarkers have some major drawbacks including, sample susceptibility to contamination, sample decomposition, and instability. Additionally, some human specimens may involve invasive sample collection. Human scalp hair has lately become more preferable to the analysis of traditional human specimens because many trace elements are more concentrated with longer residence times in hair compared to other human specimens. Besides, hair samples are easy to collect, noninvasive, easy to store, and relatively inexpensive. Thus, human hair samples have been effectively used as biomarkers for the determination of trace element concentrations in environmental and bioanalytical studies, clinical and medical diagnosis, occupational health, recreational and therapeutic use, and forensic science [36-56].

We hypothesized that the combined use of analytical spectroscopy, multivariate analysis, statistics, and human scalp hair can be utilized to map the level of essential and potentially toxic trace elements in human subjects. We also hypothesized that race would possibly influence the level of trace elements in the scalp hair of human subjects. Consequently, this study reports for the first time, a comprehensive and combined use of analytical spectroscopy, statistics, multivariate analysis, and human scalp hair as a biomarker for assessing trace element (Fe, Mg, Ca, Cd, Pb, Cu, Zn, and Cr) concentrations in human subjects. The study also reports for the first time, the influence of race on the levels of trace element concentrations and the use of multivariate analysis for trace element pattern recognition in scalp hair of human subjects. Furthermore, the study reports for the first time the influence of race, age, race, sex, on the Ca/Mg and Zn/Cu ratios (which are often used to evaluate the degree of Ca and Cu utilization in human and markers for various health related issues including, atherosclerosis, hypertension, insulin sensitivity, and pancreatic cancer) in the scalp hair of human subjects. 

2. Materials and Methods

2.1. Sample Collection and Sample Preparation

Human scalp hair samples were collected in three cities (Winston-Salem, Clemmons, and Kernersville) in Forsyth County, North Carolina, USA. Forsyth County is 409.60 square miles in area, with a population of 350,670 [57]. Females and males account for 52% and 48%, respectively of the population. In addition, people under 18 years of age account for 24.6% of the sample population while those older than 18 years are make-up of 73.4% of the population in Forsyth County. The demographic racial composition of Forsyth County consists of Caucasians (majority 62.3%) and non-Caucasians (minority, 37.7%). Non-Caucasians were made up of African Americans, 26%; Hispanics, 11.9%; and Asians, 1.9%. Scalp hair samples were collected from 125 participants using stainless steel scissors during regular haircut periods at various barber shops and beauty salons over a period of three weeks in June 2011. The sample population consists of 60 males and 65 females aged between 5 and 70 years old. In addition, the hair samples were collected from different races and demographics consisting of Caucasian (n = 87) and non-Caucasians (n = 38). Non-Caucasians were made up of African Americans, Hispanics, and Asians. Only eighteen of the people from whom hair samples were collected declared themselves as tobacco product smokers while 107 identified themselves as nonsmokers.    

The collected hair samples were immediately placed in cleaned polyethylene plastic bags, properly labeled, and transported to a dust free laboratory, where the samples were sequentially washed with acetone and ethanol, and thoroughly rinsed with deionized water to remove pharmaceutical products, particulates, and other exogenous materials. The washed hair samples were subsequently dried in an oven at 90˚C for approximately 20 minutes and stored in pre-nitric acid washed Teflon sample containers. Approximately 1.0 g of dried hair sample from each participant was accurately weighed and digested with 15 mL HNO₃ (trace metal grade, purity, 99.999%) in a digestion flask. Hair sample digestions were initiated at relatively low temperatures to prevent a violent reaction until all the hair samples were fully dissolved in the nitric acid. The temperature was then subsequently increased until the solution turned pale yellow to ensure complete sample digestion. The nitric acid digested hair samples were filtered into a 25 mL volumetric flask using a Whatman filter paper (ashless) and diluted to the mark with deionized water.  

2.2. Calibration Curve, Trace Element Analysis, Method Validation, and Quality Assurance

Working range standard solutions used to construct calibration curves for each element were prepared by serial dilution of 1000 ppm standard stock solutions of each element purchased from Fisher Scientific. The calibration curve for each element was constructed by plotting the absorbance obtained from FAAS analysis of the standard solution versus the element concentration. The nitric acid digested hair samples were subjected to trace element analysis using a flame atomic absorption spectrophotometer (Shimadzu, AA-6300) using a pre-mixed burner air-acetylene flame. The flow rates of the fuel and oxidant gases were always carefully optimized for each metal analysis. Other routine instrumental checks and calibrations were always performed on the spectrometer before use to ensure the accuracy, reliability and consistent performance of the spectrometer. Each sample was analyzed in triplicate and the averages of the trace element concentrations in the hair samples were calculated using the constructed calibration curves and listed in Table 1. All necessary precautions were also observed to ensure the accuracy of the results of this study. First, the acetone and ethanol used for the hair wash were of spectroscopic grade. Second, the highest purity HNO₃ acid (purity, 99.999%) was used for the hair digestion and standard solution preparations. Also, all glassware including the sample containers, digestion flasks, and volumetric flasks were pre-soaked in 6M nitric acid for three days and thoroughly rinsed with deionized water before use to remove impurities and contaminants. Additionally, all trace element sample analyses were blank subtracted.

A recovery study was also performed for each metal to further evaluate the accuracy and reliability of the results of the heavy metal concentrations obtained in the analysis. The recovery study was performed by randomly spiking ten previously analyzed hair samples with a known concentration of metal standard solution. The spiked samples were then subjected to HNO₃ digestion and metal analysis procedures under the same experimental conditions as previously used for hair sample analysis. The recovery of the metals in the spiked samples was evaluated by comparing the known concentration of the spiked metal with the concentration detected using FAAS spectrometer.

2.3. Multivariate Analysis: Regression and Principal Component Analysis

Linear regression and multivariate principal component analysis for pattern recognition of the levels of the metals in human scalp hair was performed using chemometric software (The Unscrambler, CAMO Inc., 9.4).

3. Results and Discussion

3.1. Calibration Curves

Table 1 presents the results of the calibration curve parameters constructed for the investigated trace elements in the hair samples. The table indicates the wavelength used for the AAS elemental analysis, the limits of detection (LOD), and limits of quantitation (LOQ) of the trace elements analyzed. The LOD, which is defined as the minimum detectable amount of metal, was calculated using the equation: LOD = 3 s/m; where s is the signal of the blank and m is the slope of the calibration curve. The limit of quantitation, defined as the lowest measurable concentration of analytes (trace elements in this study), was evaluated using the formula: LOQ = 10 s/m. The parameters used to calculate LOQ were as previously defined for LOD. The high values of the correlation coefficients (r) obtained in Table 1 demonstrate good linear correlation of the absorbance with trace element concentrations.

Conflicts of Interest

The authors declare no conflicts of interest.

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