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Effects of various washing procedures on the chromium content of human scalp hair
Analytica ChinzicaActa. 138 ( 1982) 361-364
Elsevier Scientific
Publishing
Company,
Amsterdam
-
Printed in The Netherlands
Short Communication EFFECTS OF VARIOUS WASHING PROCEDURES CONTENT OF HUMAN SCALP HAIR
JORMPL KUMPULAINEN*, Deportment HetsinJ:i
(Received
of Food
SEPPO SALMELA,
Chemistry
and Technology,
ERKKI
ON THE CHROMIUM
VUORI
Uniuersify
and JAR1 LEHTC!
of HetsinJ:i, SF-00710
71 (Finland)
30th October
19Sl)
Summary. The effects of four wash procedures on the concentration of chromium in a pooled sample of human scalp hair are compared. The wash solutions examined are Triton X-100, sodium Iauryi sulphate, acetone, and a mixture (I c 1) of herane and ethanol. The plateaux reached for hair chromium concentration are functions of the number of washings, and are characteristic recommended involves two 20-min washes tion after a hexane rinse.
for each wash solution. The wash procedure with aqueous 1% sodium Iauryl suIphate solu-
Doubts have recently been raised concerning the significance of the trace element content of hair as an indicator of trace element nutritional status [l-3]. This is primarily due to controversial results in the literature which indicate that the fundamental problem of how to wash hair samples in order to remove exogenous trace elements is still unsettled. Chromium, being ubiquitous, is particularly difficult; numerous procedures and solvents have been used to wash hair samples in analysis for chromium [ 4-ll] _ Rosson et al. [7] washed the hair sequentially with acetone, aqueous 1% sodium lauryl sulphate solution and distilled water. Hubert [8] reported that a hexaneTriton X-lOWwater treatment was more effective than hexane-water or Triton X-100 alone: water alone was the least effective in removing exogenous chromium from hair. Hambidge et al, [6] found that washing with a non-ionic detergent or hesane-ethanol gave similar results, removing on average 38% of the mean chromium value for unwashed samples. The hexane-ethanol washing procedure has been e_mployed in several metabolic and nutritional studies on chromium [9--111. However, neither of these comparative studies systematically investigated the effect on the chromium content of hair samples of repeated washing with different types of solutions. Salmela et al. [ 121 showed recently that after a certain number of successive washes, a concentration plateau was reached for each of the trace elements studied, the plateaux being characteristic for each of the wash solutions. The number of washes required to reach these plateaux varied considerably_ In the work reported here, the same protocol was used to establish how different wash procedures affect the concentration of chromium in human scalp hair_ 0003-2670/62/0000-Oo00fS02.75
o 1982 Elsevier Scientific
Publishing
Company
362
Experimental Samples.
Female scalp hair which had not been chemically treated :Ixcept for ordinary washing was homogenized by cutting it with stainless steel scissors into 2-3-mm pieces and mixing with a glass rod on a piece of filter paper. The weight of the mixed hair pool was 20 g. Wash prccedztres. Four wash solutions were selected: an ionic detergent (1% sodium lauryl sulphate; BDH), a non-ionic detergent (1% Triton X-100; BDH), a strongly polar solvent (acetone; Merck) and a less polar solvent mixture (1 + 1 hesane-ethanol). The hair portions were washed in 50-ml test tubes fitted with ground-glass stoppers and placed in a three-dimensional mechanical shaker (Desaga, Heidelberg) at the minimum speed at room temperature. Each wash period was 20 min and 30 ml of wash solution was used. The first step in each wash procedure, except for the acetone and hexaneethanol washes, was to rinse with 30 ml of hexane (9570, J. T. Balier) for 20 min to remove surface grease. Six portions, ear--hweighing ca. 300 mg, were weighed, rinsed with hesane and dried at ‘15°C. Five of these samples were then washed 1, 2, 4, 8, and 12 times (20 min each) with the selected wash solution, rinsed with deionized water until a clear solution was obtained, and dried at 105°C. Each sample was divided into three portions Xeasrtremen t procedztre. which were transferred to 20-ml Vitreosil quartz crucibles and weighed on an automatic electronic balance fSartorius type 1201~MP2). The samples were then dry-ashed overnight at 500°C. If the ash still contained carbon, it was treated with a Iittle concentrated sulphuric acid and 30% hydrogen peroxide (both analytical-reagent grade; Merck) as described elsewhere [ 13]_ The ash was dissolved in 1 ml of 1 M HCl (Suprapur; Merck). After 30 min, the chromium concentration was determined by graphite-furnace atomic absorption spectrometry against chromium standards. Working standards in the range of l-10 ng Cr ml -’ in 1 M HCl were prepared daily from a 1 mg Cr 1-l stock standard (potassium dichromate), The Perkin-Elmer 5000 atomic absorption spectrometer used was equipped with a background corrector (tungsten-iodide lamp), a graphite furnace (HGA-500), an autosampler (AS-40), a printer (PRS-10) and a chart recorder (Model 56). The analytical conditions used were as described elsewhere r141. The validity of the entire measurement procedure was checked by employing the National Bureau of Standards (NBS) S+mdard Reference Material Bovine Liver (SRM 15’77). The blanks fat least three for each run) contained 0.5 ng Cr ml-’ on average, which was
and discussion
The precision of the measurement procedure was tested by determining chromium in samples (ca 100 mg) of the pooled homogeneous hair before and after the hexane wash. The precision was good. &washed hair showed a
363
mean chromium content of 168 ng g-l (s-d. = I1 ng g-r; r.s.d. = 6.5%; n = 9) and hexane-washed hair gave a mean chromium content of 151 ng g-’ (s.d. = 8 ng g-’ ; r.s.d_ = 5.3%; n = 5). Table 1 summarizes the results of the wash experiments. As expected, the hexane rinse removed Iittle chromium from the hair sample. The least effective wash solution was Triton X-100, removing 48% of the chromium originally present, while hexane-ethanol and sodium Iauryl sulphate were the most effective solvents, both removing slightly above 70% of the original hair chromium content. Successive acetone washes resulted in a slowly declining concentration, where the first wash removed 49% and the total 12 washes removed 59% of the chromium in the unwashed hair. In the case of Triton X-100 only one wash was required to reach the approximate plateau value, whereas for hexane-ethanol and sodium lauryl sulphate two washes were necessary. On the basis of these results, it is not possible to judge which, if any, of the wash procedures is ideal. However, some useful conclusions can be drawn. First, it is obvious that not onIy the wash solution but also the wash time must be considered in analyses for chromium. Secondly, the acetone wash recommended by the International Atomic Energy Agency (IAEA) [IL51 as a standard method, is unsuitable for hair chromium because the chromium concentration continues to decline on lengthy washing and with other solvents. ThirdIy, valid hair chromium comparisons between populations and Iaboratories are possible only if very simiIar wash procedures have been followed, It seems logical
to consider
that sodium
Iauryl sulphate
and hesane-
ethanol are the most refiable washing agents for hair intended for chromium determinations. Both yield essentially the same results although one is an ionic detergent and the other is a mixture of organic solvents. Even if these TABLE
I
The concentration of chromiuzn different solutions Wash solution
in the hair pool before and after successive washes with
No. of successive washes 1
0
2
4
8
12
Cr content (ng g-l)” Hexane Triton X-100 Acetone Hexane-ethanol Sodium lauryl sulphate
168 t 11
151 r8
-
-
-b
90 f 4 86 + 1 94 2 8
94 * 4 86 f. 3 46 t I.
_b
79 +4
52+8
86 78 56 53
_b
-b
_+6 I8 f 7 c6
-
-
97 + 5 75 ” 2 37 _e2
8’7 69 46 48
38 t 2
r5 +7 t 5 +1
%fean and standard error (II = 3 except for hexane). “All mashes were preceded by a hexane rinse.
364
wash solutions leach some of the endogenous chromium, valid comparisons can still be made between hair populations if all have been subjected to the same standardized wash procedure. If esogenous chromium is present because of inadequate wash solutions or procedures, such comparisons are impossible. -4s sodium lauryl sulphate (SLS) is a detergent and is easier to use than hesane-ethanol, a minimum of two 20-min washes with SLS after the hexane rinse is recommended as the standard wash procedure for chromium in hair. This procedure is currently being used in a study of chromium in hair as an indicator of dietary chromium intake. REFERENCES 1 3 3 1 5
Ii. B. Bradfield and K. hl. Hambidge, Lancet, 16 (1980) 363. G. S. Xssarian and D. Oberleas, Clin. Chem., 23 (1977) 1771. &I_ R. Spivey Fox and S. H. Tao, Fed. Proc., t0 (1981) 2130. D. C. Hildebrand and D. H. White. Clin. Chem., 20 (197-I) 148. R. F. Coleman, F. H. Cripps, _X Stinson and H. D. Scott, Atomic Weapons Research Establishment Rep. No. O-86/66. 1967. 6 K. M. Hambidge, RI. L. Frankiin and hI. A. Jacobs, Am. J. Clin. Nutr., 25 (1972) 383. 7 J. W’. Rosson, K. J. Foster, R. J. Walton, P. P. Alonro, T. G. Taylor and K. G. bl. 31. Xlberti. Clin. Chim. Acts, 93 (1979) 299. 8 J. Hubert, in D. Shapcott and J. Hubert (Eds.), Proc. Conf. Chromium in Nutr. and LIetab., July 1979. Eisevier, -4msterdam. pp. 15-30.
9 10 11 1‘2 13
K. i\I. Hambidge and J. D. Baum, Am. J. Clin. Nutr., 25 (19’72) 3’76. K. &I. Hambidge, 31. L. Franklin and RI. A. Jacobs, Am. J. Clin. Nutr., 25 (1972) 380. J. R. hlahaiko and hl. Bennion. Am. J. Clin. Xutr., 29 (1976) 1069. S. Salmein, E. Vuori and J. 0. Kilpi6, Anal. Chim. Acta, 125 (1981) 131. J. Kumpulainen, \\‘. Wolf, C. Veillon and W’. Hertz, J. Agric. Food Chem., 27 (19i9) -190.
l-1 J. Kumpulainen, E. Vuori, S. Xikinen and R. Kara, Br. J. Xutr., 44 (1980) 257. 15 Activation analysis of hair as an indicator of contamination of man by environmental trace element pollutants, Report IAE_4/RL/-llH, 1977, p. 10.
Elsevier Scientific
Publishing
Company,
Amsterdam
-
Printed in The Netherlands
Short Communication EFFECTS OF VARIOUS WASHING PROCEDURES CONTENT OF HUMAN SCALP HAIR
JORMPL KUMPULAINEN*, Deportment HetsinJ:i
(Received
of Food
SEPPO SALMELA,
Chemistry
and Technology,
ERKKI
ON THE CHROMIUM
VUORI
Uniuersify
and JAR1 LEHTC!
of HetsinJ:i, SF-00710
71 (Finland)
30th October
19Sl)
Summary. The effects of four wash procedures on the concentration of chromium in a pooled sample of human scalp hair are compared. The wash solutions examined are Triton X-100, sodium Iauryi sulphate, acetone, and a mixture (I c 1) of herane and ethanol. The plateaux reached for hair chromium concentration are functions of the number of washings, and are characteristic recommended involves two 20-min washes tion after a hexane rinse.
for each wash solution. The wash procedure with aqueous 1% sodium Iauryl suIphate solu-
Doubts have recently been raised concerning the significance of the trace element content of hair as an indicator of trace element nutritional status [l-3]. This is primarily due to controversial results in the literature which indicate that the fundamental problem of how to wash hair samples in order to remove exogenous trace elements is still unsettled. Chromium, being ubiquitous, is particularly difficult; numerous procedures and solvents have been used to wash hair samples in analysis for chromium [ 4-ll] _ Rosson et al. [7] washed the hair sequentially with acetone, aqueous 1% sodium lauryl sulphate solution and distilled water. Hubert [8] reported that a hexaneTriton X-lOWwater treatment was more effective than hexane-water or Triton X-100 alone: water alone was the least effective in removing exogenous chromium from hair. Hambidge et al, [6] found that washing with a non-ionic detergent or hesane-ethanol gave similar results, removing on average 38% of the mean chromium value for unwashed samples. The hexane-ethanol washing procedure has been e_mployed in several metabolic and nutritional studies on chromium [9--111. However, neither of these comparative studies systematically investigated the effect on the chromium content of hair samples of repeated washing with different types of solutions. Salmela et al. [ 121 showed recently that after a certain number of successive washes, a concentration plateau was reached for each of the trace elements studied, the plateaux being characteristic for each of the wash solutions. The number of washes required to reach these plateaux varied considerably_ In the work reported here, the same protocol was used to establish how different wash procedures affect the concentration of chromium in human scalp hair_ 0003-2670/62/0000-Oo00fS02.75
o 1982 Elsevier Scientific
Publishing
Company
362
Experimental Samples.
Female scalp hair which had not been chemically treated :Ixcept for ordinary washing was homogenized by cutting it with stainless steel scissors into 2-3-mm pieces and mixing with a glass rod on a piece of filter paper. The weight of the mixed hair pool was 20 g. Wash prccedztres. Four wash solutions were selected: an ionic detergent (1% sodium lauryl sulphate; BDH), a non-ionic detergent (1% Triton X-100; BDH), a strongly polar solvent (acetone; Merck) and a less polar solvent mixture (1 + 1 hesane-ethanol). The hair portions were washed in 50-ml test tubes fitted with ground-glass stoppers and placed in a three-dimensional mechanical shaker (Desaga, Heidelberg) at the minimum speed at room temperature. Each wash period was 20 min and 30 ml of wash solution was used. The first step in each wash procedure, except for the acetone and hexaneethanol washes, was to rinse with 30 ml of hexane (9570, J. T. Balier) for 20 min to remove surface grease. Six portions, ear--hweighing ca. 300 mg, were weighed, rinsed with hesane and dried at ‘15°C. Five of these samples were then washed 1, 2, 4, 8, and 12 times (20 min each) with the selected wash solution, rinsed with deionized water until a clear solution was obtained, and dried at 105°C. Each sample was divided into three portions Xeasrtremen t procedztre. which were transferred to 20-ml Vitreosil quartz crucibles and weighed on an automatic electronic balance fSartorius type 1201~MP2). The samples were then dry-ashed overnight at 500°C. If the ash still contained carbon, it was treated with a Iittle concentrated sulphuric acid and 30% hydrogen peroxide (both analytical-reagent grade; Merck) as described elsewhere [ 13]_ The ash was dissolved in 1 ml of 1 M HCl (Suprapur; Merck). After 30 min, the chromium concentration was determined by graphite-furnace atomic absorption spectrometry against chromium standards. Working standards in the range of l-10 ng Cr ml -’ in 1 M HCl were prepared daily from a 1 mg Cr 1-l stock standard (potassium dichromate), The Perkin-Elmer 5000 atomic absorption spectrometer used was equipped with a background corrector (tungsten-iodide lamp), a graphite furnace (HGA-500), an autosampler (AS-40), a printer (PRS-10) and a chart recorder (Model 56). The analytical conditions used were as described elsewhere r141. The validity of the entire measurement procedure was checked by employing the National Bureau of Standards (NBS) S+mdard Reference Material Bovine Liver (SRM 15’77). The blanks fat least three for each run) contained 0.5 ng Cr ml-’ on average, which was
and discussion
The precision of the measurement procedure was tested by determining chromium in samples (ca 100 mg) of the pooled homogeneous hair before and after the hexane wash. The precision was good. &washed hair showed a
363
mean chromium content of 168 ng g-l (s-d. = I1 ng g-r; r.s.d. = 6.5%; n = 9) and hexane-washed hair gave a mean chromium content of 151 ng g-’ (s.d. = 8 ng g-’ ; r.s.d_ = 5.3%; n = 5). Table 1 summarizes the results of the wash experiments. As expected, the hexane rinse removed Iittle chromium from the hair sample. The least effective wash solution was Triton X-100, removing 48% of the chromium originally present, while hexane-ethanol and sodium Iauryl sulphate were the most effective solvents, both removing slightly above 70% of the original hair chromium content. Successive acetone washes resulted in a slowly declining concentration, where the first wash removed 49% and the total 12 washes removed 59% of the chromium in the unwashed hair. In the case of Triton X-100 only one wash was required to reach the approximate plateau value, whereas for hexane-ethanol and sodium lauryl sulphate two washes were necessary. On the basis of these results, it is not possible to judge which, if any, of the wash procedures is ideal. However, some useful conclusions can be drawn. First, it is obvious that not onIy the wash solution but also the wash time must be considered in analyses for chromium. Secondly, the acetone wash recommended by the International Atomic Energy Agency (IAEA) [IL51 as a standard method, is unsuitable for hair chromium because the chromium concentration continues to decline on lengthy washing and with other solvents. ThirdIy, valid hair chromium comparisons between populations and Iaboratories are possible only if very simiIar wash procedures have been followed, It seems logical
to consider
that sodium
Iauryl sulphate
and hesane-
ethanol are the most refiable washing agents for hair intended for chromium determinations. Both yield essentially the same results although one is an ionic detergent and the other is a mixture of organic solvents. Even if these TABLE
I
The concentration of chromiuzn different solutions Wash solution
in the hair pool before and after successive washes with
No. of successive washes 1
0
2
4
8
12
Cr content (ng g-l)” Hexane Triton X-100 Acetone Hexane-ethanol Sodium lauryl sulphate
168 t 11
151 r8
-
-
-b
90 f 4 86 + 1 94 2 8
94 * 4 86 f. 3 46 t I.
_b
79 +4
52+8
86 78 56 53
_b
-b
_+6 I8 f 7 c6
-
-
97 + 5 75 ” 2 37 _e2
8’7 69 46 48
38 t 2
r5 +7 t 5 +1
%fean and standard error (II = 3 except for hexane). “All mashes were preceded by a hexane rinse.
364
wash solutions leach some of the endogenous chromium, valid comparisons can still be made between hair populations if all have been subjected to the same standardized wash procedure. If esogenous chromium is present because of inadequate wash solutions or procedures, such comparisons are impossible. -4s sodium lauryl sulphate (SLS) is a detergent and is easier to use than hesane-ethanol, a minimum of two 20-min washes with SLS after the hexane rinse is recommended as the standard wash procedure for chromium in hair. This procedure is currently being used in a study of chromium in hair as an indicator of dietary chromium intake. REFERENCES 1 3 3 1 5
Ii. B. Bradfield and K. hl. Hambidge, Lancet, 16 (1980) 363. G. S. Xssarian and D. Oberleas, Clin. Chem., 23 (1977) 1771. &I_ R. Spivey Fox and S. H. Tao, Fed. Proc., t0 (1981) 2130. D. C. Hildebrand and D. H. White. Clin. Chem., 20 (197-I) 148. R. F. Coleman, F. H. Cripps, _X Stinson and H. D. Scott, Atomic Weapons Research Establishment Rep. No. O-86/66. 1967. 6 K. M. Hambidge, RI. L. Frankiin and hI. A. Jacobs, Am. J. Clin. Nutr., 25 (1972) 383. 7 J. W’. Rosson, K. J. Foster, R. J. Walton, P. P. Alonro, T. G. Taylor and K. G. bl. 31. Xlberti. Clin. Chim. Acts, 93 (1979) 299. 8 J. Hubert, in D. Shapcott and J. Hubert (Eds.), Proc. Conf. Chromium in Nutr. and LIetab., July 1979. Eisevier, -4msterdam. pp. 15-30.
9 10 11 1‘2 13
K. i\I. Hambidge and J. D. Baum, Am. J. Clin. Nutr., 25 (19’72) 3’76. K. &I. Hambidge, 31. L. Franklin and RI. A. Jacobs, Am. J. Clin. Nutr., 25 (1972) 380. J. R. hlahaiko and hl. Bennion. Am. J. Clin. Xutr., 29 (1976) 1069. S. Salmein, E. Vuori and J. 0. Kilpi6, Anal. Chim. Acta, 125 (1981) 131. J. Kumpulainen, \\‘. Wolf, C. Veillon and W’. Hertz, J. Agric. Food Chem., 27 (19i9) -190.
l-1 J. Kumpulainen, E. Vuori, S. Xikinen and R. Kara, Br. J. Xutr., 44 (1980) 257. 15 Activation analysis of hair as an indicator of contamination of man by environmental trace element pollutants, Report IAE_4/RL/-llH, 1977, p. 10.