Human hair elemental analysis of patients with viral hepatitis and liver cirrhosis by X-ray fluorescence method

Scalp human hair is well known to accumulate metal concentrations over time, thus containing a higher concentration than found in blood, serum or urine samples [1-3]. In recent years considerable effort has been made to measure trace metal levels in various human tissues to determine inter element correlations with different diseases [3-5] by several analytical techniques [3-8]. TXRF (total reflection X-ray fluorescence) is a technique that has many advantageous features such as multielemental capability, high sensitivity and precision, short time of analysis as well as offering the possibility of direct sample analysis with the use of very small sample manipulation. Usually, an analysis by TXRF involves the dissolution of sample; an aliquot of the solution is placed on a quartz plate, and, after drying, the plate with the sample is used as an emitter. To analyze biological materials, a thin micro section of the material to be analyzed (a microtome) is sometimes prepared. Being placed on a quartz support, it is used as an emitter. This feature makes the technique very attractive for monitoring environmental pollution and in analyzing biological materials [9-10]. Viral hepatitis and liver cirrhosis are very common diseases among Mongolian people and number of morbidity due to liver disorders have being increased for the last years in through statistic survey in last 5 years in Mongolia [11].

In the present paper the TXRF method was applied to chemical analysis of the human hair of the acute viral hepatitis patients in Ulaanbaatar, capital city of Mongolia.

Experimental

Apparatus. TXRF spectrometer installed at Nuclear Research Center, National University of Mongolia. The spectrometer consists of a total reflection unit (Atominstitut Oesterreichischen Univ., Vienna, Austria), an X-ray spectrometry unit (a semiconductor Si(Li) detector, model S1 18080) (CAMBERRA, USA) 160 eV resolution at 5.9 keV Mn K α line, an X-ray generator, model ISO-3000 SEIFERT, and data was collected with a measurement time of 200 s in a PC based NUCLEUS computer (USA). The AXIL software provides the calculation of components using the internal standard method (Ga is used as a reference material). In the experiments, an X-ray tube with molybdenum anode operated under the following conditions: voltage V=50 kV, current i=10 mA.

Reagents. Analytical grade acetone and distilled water were used for washing human hair samples; 14N nitric acid (HNO 3 ) pure for analysis and 30% hydrogen peroxide (H 2 O 2 ) were used for acid digestion of human hair samples. The gallium (Ga) element standard, 1000 µ g/ml was served for internal standardization by TXRF.

Samples. The scalp hair samples were collected from the same place on the backside of head, namely from the occipital region using a pair of stainless steel scissors. The age range of individuals was 19 to 70 years (mean: 43.9). Samples were stored in sealed plastic bags, which were mar-ked with personal information regarding age, sex, hair color and doctor’s diagnosis of diseases. The main problem of using hair is external contamination. The samples were thoroughly washed to remove external contamination by 1% soap solution rinsed with distilled water, then placed for one hour in acetone and finally washed again by distilled water and air-dried. The weight of the samples was about 25-30 mg. Hair samples were dissolved in a mixture of HNO 3 and H 2 O 2 . The weighed hair sample (25-30 mg) was placed into the chemical beaker and added 0.3 mL ultra pure nitric acid and then heated in the microwave oven within 3 min at 120^oC. Then to the residue was added 0.1 mL of 30% hydrogen peroxide and heated less than 100^oC for 2 min. After this, the solution was evaporated almost to dryness and 0.6 mL of 0.1 M nitric HNO 3 added to the mixture. Finally the solution was again evaporated almost to dryness at 130-140^oC. The residue was dissolved in 1.0 mL of 0.1 M HNO 3 to produce a final sample solution.

Procedures. The analysis of human hair was performed by using methodology previously developed by us [12]. To prepare emitters, an aliquot of the solution of up to 1,0 ml was taken and weighed in a special vessel using an AG204 electronic balance (Mettler-Toledo, Switzerland). Next, 10 µL of a standard solution of Ga with a concentration of 1000 ppm was added, and the solution was weighed again. After stirring, an aliquot of 5 µL was taken, applied to quartz reflector, dried using an infrared lamp, and used as an emitter. The acquisition time is 200 s. The performance parameters of the developed procedure were estimated. Limits of detection c min were calculated using the 3 µ -criterion [13], and the determination of standard deviation characterizing the scatter of background values was made using several blank emitters prepared by applying diluted HNO 3 (1:99) to quartz reflectors. The values of c min are 2, 8, 2, 10, 7 and 3 ng for Zn, Ca, Cu, K, Fe and Pb respectively. The reproducibility (s r,b ) of the procedure was estimated using the obtained results for hair analysis. The values of s r,b are 6,8,13,21 and 20% for Zn, Cu, Ca, K and Fe respectively. The accuracy was verified using an IAEA-086 certified reference sample of hair.

Results and discussion

The accuracy was checked by the analysis of a standard reference material, IAEA-086 unspiked human hair, including statistical and repeatable errors, whose elemental concentrations are presented in table 1. Errors mainly come from the preparation of targets for irradiation, spectrum fitting, and partially from the detection efficiency. The results obtained are in a good agreement with the certified values.

Table 1

Comparison of elemental concentrations ( µ g g^-1) in IAEA -086 standard sample and certified values1

Estimated concentrations of elements are presented in table 2, 3. From the present results, it can be seen the levels of elements Ca, Ti, Fe, and Cu, are higher and K and Zn are lower in the patients of chronic viral hepatitis and liver cirrhosis than those observed in the normal people.

Table 2 Results of human hair samples (B Virus)

The elemental concentrations in biological samples vary considerably due to geographical differences, nutritional status and environmental factors. The present results were compared with other results reported by several researchers and the ranges of word wide mean values. Table 4 shows the comparative results. The mean concentrations of Ca, Fe, Ti and Sr in this work were found higher than those of normal people in Japan, Mongolia, Iran and Indonesia. The level of elements Cu and Zn observed in human hair of liver cirrhosis and chronic viral hepatitis patients are in agreement with those determined in normal human hair of Japan, Mongolia, Iran and Indonesia. It is interesting to note that concentrations of Ca, Ti, Ni, As, Br and Sr in the human hair of the cirrhosis and chronic viral hepatitis patients were higher than Mongolian normal human hair which are have been analyzed by other researchers.

Some researchers have been observed that cancer of the respiratory tract can be attributed to the inhalation of nickel compounds. To determine the interaction between elements in hair correlation coefficients were calculated. Positive strong correlations were observed among the elements [Ti=f(Pb), f(Cu)], [Cu=f(K), f(Ti)] and [Sr=f(K), f(Fe)]. The correlation matrix gives us information about relationships between two metals but does not take into consideration of the presence of other elements.

Table 4.

Inter comparison of trace element concentrations ( µ g g^-1) in human hair

Element Our study,         Prev. study,          Iran1       Pakistan5     Indonesia16     Japan14

patients (TXRF) patients15 (PIXE) (INAA) (AAS) (AAS) (PIXE)

NA-not available

Conclusion

The trace elemental analysis is carried out in the human hair of the liver cirrhosis and chronic viral hepatitis patients by employing TXRF technique. It is confirmed that the present method of analyzing human hair can serve as a powerful one for evaluation of human environment. From the present results, it has illustrated that the levels of elements Ca, Fe, Ti and Sr higher in the human hair of the cirrhosis and chronic viral hepatitis patients than those observed by different researchers for normal people from Iran, Japan and Indonesia. The elements Cu and Zn observed in the in the hair of the liver cirrhosis and chronic viral hepatitis patients similar to those determined in normal human hair. Positive strong correlations were observed among the elements [Ti=f(Pb), f(Cu)], [Cu=f(K), f(Ti)] and [Sr=f(K), f(Fe)]. However, no visible correlation was determined. To provide more credible data more statistics (more populated cohort of patients) is needed.