Abstract

Following mini project is based on determination of zinc level in human hair. Total number of 10 samples collected and their zinc level analyzed and compared with respect to two parameters: 1. Gender 2. Dyed and non-Dyed hairs Each sample was analyzed using Flame Atomic Absorption Spectrometry to determine its zinc concentration. Calculation of zinc level for each sample was carried out based on the calibration line corresponding to the standard zinc solutions, which were prepared according to the procedure.

Introduction
Zinc is an essential trace mineral required by living organisms as wide ranging as microorganisms, plants and animals. It is a component in many enzymes, numbering from 100 to 300 specific enzymes and is the only metal, which appears in all enzyme classes. Furthermore it is the second most abundant transition metal in organisms after iron. Among some notable functions of zinc are RNA and DNA metabolism, signal transduction and gene expression; affects synaptic plasticity in brains; regulates apoptosis; and the functioning of the male prostate gland. The interconversion of blood carbon dioxide and bicarbonate in respiration is carried out by the zinc-containing enzymes carbonic anhydrase and carboxypeptidase. Salivary glands, the prostate, the immune system and intestine cells use zinc signaling as cellular communication. The distribution of zinc is found to be highest in the choroid of the eye and optic nerve, followed by the prostate, bone, liver and kidneys, muscles, heart, spleen, testes, brain, and adrenal glands. The skin also has a high concentration of zinc and can often be a sensitive indicator of zinc levels. The role of zinc in physiology are numerous, hence biomedical studies attempt to establish a link between zinc levels and health, or major disorders such as depression, immune deficiency or genetic diseases. A relation between low concentrations of zinc and mental health problems, especially in at risk populations has been demonstrated in a study by Whittle, N. (2009). Another study by Pfeiffer (1975) showed how severe zinc deficiency is seen in acrodermatitis enteropathica, a genetic disorder occuring in children and how zinc supplements improved their conditions. Hence, there is a wide range of research in zinc levels that could potentially give important insights in human physiology and disease treatment.

A survey of studies conducted on zinc levels will indicate that the samples in studies used are typically plasma, serum, blood, and hair, nails and saliva. Hair is the most non-invasive biological marker and carries the least risk of contamination. According to Hashem and Abed (2007), hair is a complex matrix of inorganic and organic materials formed over a period of time. It can provide a more permanent record of trace element associated with normal and abnormal metabolism as well as those assimilated from the environment. Plasma and serum zinc levels regarded as being less reliable information since they are regulated by the bodies homeostasis system and will be reduced during acute illnesses. Hair zinc levels poses some difficulty in interpretations since the underlying mechanisms of zinc transfer and deposition to and from hair is not clearly known. For example, high hair zinc can indicate low body levels, but low hair zinc also correlates well with low red blood cell and total body zinc. Significantly higher hair zinc is usually interpreted as a sign of zinc wasting and low body zinc levels. In this study, the hair zinc levels in males and females in the age group 20-30 as well as in females in the same age group with dyed or non-dyed hair were determined by atomic absorption spectrometry (AAS)

Procedure Apparatus
Beaker Pipettes Volumetric Flask Spatula Oven Flame AAS

Chemicals
Detergent Water Methanol Concentrated Nitric Acid Perchloric Acid Standard Zinc Solutions

Hair samples were collected from male and female volunteers in the age range of 20-30 years old and sorted into dyed and undyed categories. Each hair sample was rinsed a few times with distilled water, followed by three rinses of methanol.

Fig 1. Washing and soaking the hair samples in methanol.

The hair samples were oven dried at 100 C for 45 minutes to remove solvent traces. After cooling, 0.3 g of each sample was weighed into a conical flask. 10 ml of concentrated nitric acid was added to each sample and the mixture was boiled gently in the fumehood until its volume halves. The samples were cooled to room temperature before 2 ml of perchloric acid was added to each. The samples were boiled further until around 1-2 ml remained. (Fig.2).

Fig. 2. Boiling samples to 1-2 ml.

The samples were cooled and made up to 100 ml in volumetric flasks. (Fig.3). A calibration curve was constructed through measuring the absorption of zinc standards of concentrations 0.25, 0.5, 0.75, 1.00, 1.50 and 2.00 ppm by AAS. The measurement from each sample was plotted on the curve and the concentration of zinc in each was determined, (Fig.4).

Fig 3. Hair Samples.

Fig 4. FAAS machine.

Results and Discussion:

Table 1: Males hair samples: No Age Dyed 1 2 3 4 5 24 26 25 24 25 no no no no no

Mass of sample (g) 0.3608 0.3463 0.3433 0.3514 0.3526

Table 2: Females hair samples: No Age Dyed 6 7 8 9 10 23 22 22 22 23 yes no no yes yes

Mass of sample (g) 0.3617 0.3584 0.3363 0.3588 0.3552

Table 3: Atomic absorbances of standard solutions No Standard Entered Calculated solutions concentrati concentration on (m g/L) (m g/L) 1 Blank 0.00 0.000 2 Standard 1 0.25 0.323 3 Standard 2 0.50 0.646 4 Standard 3 0.75 0.904 5 Standard 4 1.00 1.126 6 Standard 5 1.50 1.495 7 Standard 6 2.00 1.780

Mean of absorbance 0.000 0.162 0.324 0.454 0.566 0.751 0.895

Standard deviation 0.0000 0.0009 0.0007 0.0013 0.0021 0.0023 0.0025

Relative standard deviation 0.00 0.54 0.21 0.29 0.37 0.31 0.28

Fig 5: Calibration curve with absorbance versus concentration of standard solutions

Table 4: Atomic absorbances of male sample solutions No Mean of Standard Relative Mean of concentration deviation standard absorbance (m g/L) (m g/L) deviation (m g/L) 1 1.257 0.004 0.284 0.782 2 0.772 0.002 0.208 0.480 3 1.088 0.004 0.339 0.676 4 1.054 0.008 0.794 0.655 5 1.174 0.004 0.366 0.730 Table 5: Atomic absorbances of female sample solutions No Mean of Standard Relative Mean of concentration deviation standard absorbance (m g/L) (m g/L) deviation (m g/L) 6 1.280 0.006 0.488 0.796 7 0.998 0.004 0.419 0.621 8 1.347 0.005 0.400 0.837 9 1.552 0.008 0.506 0.965 10 1.054 0.007 0.632 0.655

Standard deviation

Relative standard deviation 0.28 0.21 0.34 0.79 0.37

0.0022 0.0010 0.0023 0.0052 0.0027

Standard deviation

Relative standard deviation 0.49 0.42 0.40 0.51 0.63

0.0039 0.0026 0.0033 0.0049 0.0041

Calculation of actual hair samples zinc concentration: Since 100 ml of sample solution was prepared, therefore the mass of zinc in hair: Sample 1: Mass of zinc in hair Zinc concentration in hair

= 1.257 mg/L x 100/1000 L = 0.1257 mg = 0.1257 mg/0.3608 g = 0.3484 g/g

Sample2: Mass of zinc in hair Zinc concentration in hair

= 0.772 mg/L x 100/1000 L = 0.0772 mg = 0.0772 mg/0.3463 g = 0.2229 g/g

Sample 3: Mass of zinc in hair Zinc concentration in hair

= 1.088 mg/L x 100/1000 L = 0.1088 mg = 0.1088 mg/0.3433 g = 0.3169 g/g

Sample 4: Mass of zinc in hair Zinc concentration in hair

= 1.054mg/L x 100/1000 L = 0.1054mg = 0.1054 mg/0.3514 g = 0.2999 g/g

Sample 5: Mass of zinc in hair Zinc concentration in hair

= 1.174 mg/L x 100/1000 L = 0.1174 mg = 0.1174 mg/0.3526 g = 0.3330 g/g

Table 6: Male Age 24 26 25 24 25

Absorbance 0.782 0.480 0.676 0.655 0.730

conc/ppm 1.257 0.772 1.088 1.054 1.174

con/ g/g 0.3484 0.2229 0.3169 0.2999 0.3330

Sample 6: Mass of zinc in hair Zinc concentration in hair

= 1.280 mg/L x 100/1000 L = 0.1280 mg = 0.1280 mg/0.3617 g = 0.3539 g/g

Sample 7: Mass of zinc in hair Zinc concentration in hair

= 0.998mg/L x 100/1000 L = 0.0998 mg = 0.0998 mg/0.3584 g = 0.2785 g/g

Sample 8: Mass of zinc in hair Zinc concentration in hair

= 1.347 mg/L x 100/1000 L = 0.1347 mg = 0.1347 mg/0.3363 g = 0.4005 g/g

Sample 9: Mass of zinc in hair Zinc concentration in hair

= 1.552 mg/L x 100/1000 L = 0.1552 mg = 0.1552 mg/0.3588 g = 0.4326 g/g

Sample 10: Mass of zinc in hair Zinc concentration in hair

= 1.054 mg/L x 100/1000 L = 0.1054 mg = 0.1054 mg/0.3552 g = 0.2967 g/g

Table 7: Female Age 23 22 22 22 23

Absorbance 0.796 0.621 0.837 0.965 0.655

conc/ppm 1.280 0.998 1.347 1.552 1.054

con/ g/g 0.3539 0.2785 0.4005 0.4326 0.2967

Discussion
In this experiment, concentration of zinc in 10 hair samples comprising of male and female of age group 20-30 was analyzed, the results of which are given in Table 6 and Table 7. Two parameters were chosen for comparison; sex (Male vs Female) and dyed versus non-dyed females. Absorbance for these samples were measured using an AAS machine and the concentration was found using the absorbance graph for a series of standard solutions of zinc (Fig.5). Six standard solutions, excluding the blank were used to plot this graph, to give a standard deviation of 1.0, which shows the accuracy of calibration curve. The First comparison was done to find the difference (if any) in the zinc content between male and female of same age group. As shown in Fig. 6, and Tables-6 and 7, the concentration of zinc present in the female samples was found to be 7.7% greater than that in the male samples. These results could be explained by several reasons. This maybe caused due to variation in food habits due to diets that includes zinc. The major sources of zinc are red meat, poultry, fish and seafood, whole cereals and dairy products. Thus taking food rich in zinc would ensure higher concentration of zinc in the body. According to Reinhold, Kfoury, Ghalambor and Jean (1966) the dietary levels of some essential microelements have been reported to correspond to hair concentration of the elements. Moreover, Contiero and Folin (1994) have said the analysis of Zn in hair represents an addition to conventional materials in the assessment of the nutritional status of groups of individuals.

Another reason for higher zinc concentration in female than in male is the possibility of exposure to various chemical substances causing variation in zinc concentration level. This is in reference to care and maintenance of hair; the type of shampoo used, cosmetic treatments and how often hair is washed, all these factors will contribute in to this. For an example, it is proven that using anti-dandruff shampoo can increase the concentration of zinc in hair while chelating agent present in permanent waving lotion

can decrease the zinc level in hair as they easily remove the zinc during treatment process. There are also other hair treatments, which reduce the zinc concentration in hair.

In reviews of environmental contamination and toxicity (175) there are several factors influencing the levels of elements in body, which is hair in this case. These factors can be categorized in to four groups: biological, personal, environmental and analytical/methodological factors. Dying the hair can be considered as an environmental factor. In this experiment, dyed and non-dyed female hair was also compared to see the zinc content. It was found out that the dyed hair has 4% more zinc concentration than non-dyed. (Fig.7). This is because of the presence of high levels of zinc (together with other elements) in the dye products. For an example, the brand Garnier has zinc added in to its products.

Last, but not the least factor to be considered is the method by which the zinc concentration was analyzed. In this project Flame Atomic Absorption Spectroscopy was used to determine the zinc level in each sample solution. The technique of flame atomic absorption spectroscopy (FAAS) requires a liquid sample to be aspirated, aerosolized, and mixed with combustible gases, such as acetylene and air or acetylene and nitrous oxide. The mixture is ignited in a flame whose temperature ranges from 2100 to 2800 oC. During combustion, atoms of the element of interest in the sample are reduced to free, unexcited ground state atoms, which absorb light at characteristic wavelengths. One of the disadvantages of this method is that the flame should be set manually and may be altered during the experiment, which may cause error in results. Also the flame may blow away some of the atoms before they could be analyzed by the beam, therefore affecting the absorbance value given. This problem could be overcome by using a Graphite Furnace instead of the flame, which uses graphite-coated furnace to vaporize the sample. Thus minimizing the above-mentioned problems associated with FAAS.

Conclusion
A total 10 hair samples of male and females were analyzed to find out concentration of zinc. It was found out that the concentration of zinc present in the female samples were 7.7% greater than that in the male samples while the comparison between dyed and non-dyed female hair samples showed that the dyed hair has 4% more zinc concentration than non-dyed. To conclude, concentration of zinc in human hair sample is not a good method of measuring zinc in body as the zinc level in hair can be altered by several factors. The alternative, more accurate way of measuring zinc level in body could be achieved by using nail samples.

References:
1. http://ods.od.nih.gov/factsheets/Zinc-HealthProfessional/ 2. http://medfac.mans.edu.eg/english/forensic/january2009/S. %206_. pdf 3. Cutler, A.H Hair Test Interpretation: Finding Hidden Toxicities

4. http://www.traceelements.com/Docs/The%20Nutritional%20Relationships%20of%20Zinc.pdf 5. http://calosol.com/zinc.php 6. AR Hashem and KF Abed, Aluminum, Cadmium and Microorganisms in Female Hair and Nails from Riyadh, Saudi Arabia Journal of Medical Sciences Volume 7, Number 2, 263-266, 2007 7. Pfeiffer CC: Mental and Elemental Nutrients. Keats Pub. New Canaan, Conn. 1975.

8. Whittle, N.; Lubec, G. and Singewald, N. (2009) : "Zinc deficiency induces enhanced depression-like behaviour and altered limbic activation reversed by antidepressant treatment in mice". Amino Acids; 36 (1):147-158. 9. Reinhold, J. G.; Kfoury, G.A.; Ghlambor, M. A.; Jean, C.: Zinc and Copper concentrations in hair of Iranian Villagers. AM J Clin Nut 1966; 18. 10. Contiero E, Folin M., Trace elements nutritional status. Use of hair as a diagnostic tool, Biological Trace Element Res. 1994 Feb; 40(2): 151-60. 11. Sukumar A. Factors influencing levels of trace elements in human hair ReviewsEnvironment Contamination Toxicology, 175(2002): 47-78.