Arch oral Biol. Vol. 36, N’a. 12, pp. 881-884, Printedin GreatBritain.All rightsreserved
1991
0003-9969/91 $3.00 + 0.00
Copyright0 I!391PergamonPressplc
A STLJDY OF THE RELATIONSHIP BETWEEN TRACE ELSEMENTS IN SALIVA AND DENTAL CARIES IN CHILDREN M. S. DUGGAL,’ H. S. CIUWLA’ and M. E. J. CURZON’ ‘Department of Child Dental Health, University of Leeds, U.K. and *Department of Dentistry, Post Graduate Institute of Medical Education and Research, Chandigarh, India (Accepted
2 July 1991)
Summary-Absout 8 ml of saliva were collected from each of 272 children, living in rural areas in North India, equally distributed into age groups 47 and 12-16 yr and stratified into low, moderate and high caries groups. The salivas were analysed for Zn, Cu, Fe, Mn by atomic absorption spectrophotometry and for F with a fluoride-sensitive electrode. The elements Cu and F had a consistent, inverse relationship with caries experience. The concentration of Zn, Fe and Mn in saliva did not have any consistent
relationship with caries experience. Key words: saliva, trace elements (Zn, Cu, Fe, Mn, F), canes.
INTRODUCIlON
Elements present in only minute quantities in animal tissues are called trace elements regardless of their abundance in nature. Some of these, such as fluoride, iron, iodine, zinc, manganese, molybdenum and copper, are physiologically essential. There are many others that are probatbly present in biological systems only as contaminants and appear to have no specific function. Despite considerable interest in trace element concentrations in water (Losee and Bibby, 1969; Adkins and Losee, 1970; Curzon, Spector and Iker, 1978) and enamel (Curzon and Cracker, 1978) as determinants of dental caries, there have been comparatively few attempts to analyse the trace element composition of saliva (Dreizen and Levy, 1970; Cutress, 1972; Schamschula et al., 1978). Even fewer studies have attempted to correlate human caries susceptibility with the salivary concentration of trace elements (Schamschula et al., 1978; Dreizen and Levy, 1970). This is surprising because saliva constantly bathes the teeth and is important in the prevention and remineralization of early enamel caries. An understanding of the mineral components of saliva is therefore of great importance in the prevention of dental caries. Our aim now was to examine any correlation between the concentration of five trace elements in saliva, zinc (Zn), colpper (Cu), iron (Fe), manganese (Mn) and fluoride (E) and the dental caries status of children. MATERIALS AND METHODS
Selection of subjects
The population sample was from five rural areas of the north of India with known and similar concentrations of trace elements in their water. All the available children (a,bout 500) in two age groups of 4-7 and 12-16yr were initially examined for dental AOB36112-B
caries, using the World Health Organization index (1977), and stratified into low caries (O-2 DMFT/ deft), moderate caries (3-4 DMFT/deft) and high caries (5 and above DMFT/deft) groups. The 276 children who were equally distributed between the age and caries groups were then randomly selected for trace element analysis. The saliva of four children was lost during transportation, leaving 272 children in the study. The two age groups were selected in order to study the primary and the permanent dentition. Only those children who were in ‘continuous residence’ in the study areas, as determined by questioning the parents, were included. Children were considered to be in ‘continuous residence’ if they had been born and lived their lives in that area except for short intervals of no more than 3 weeks-for example, holidays. None of the children in the study had access to fluoride supplements or received any fluoride- or zinc-containing toothpastes; their dental restorations did not contain copper cement or copper amalgam. Collection of saliva
About 8 ml of unstimulated saliva were collected from each child, who drooled into acid-cleaned, dry, 20-ml Corning glass tubes with a screw cap of inert plastic. Care was taken to avoid contact between fingers and saliva to minimize the chance of contamination. Preparation of saliva samples
About 3 ml of saliva from each individual were set aside for fluoride estimation. The remainder was prepared by the Willis’ technique modified for analy sis of trace elements in saliva (Gow, 1965). Concentrated, analytical grade, nitric acid (0.5 ml) was added to each sample to bring the pH below 2 so as to prevent any microbial growth and enzymatic changes. The samples were coded by one of us 881
882
M. S. DUGGAL et al. Table 1. Mean concentration of trace elements in saliva Means + SD (mg/l)
Caries status (deft/DMFT f SD)
n
Zn
CU
Fe
Mn
F
47yr Age Group 0.53 + 0.15 0.35 f 0.10 0.27 kO.17
5.84 & 3.21 8.61 * 1.10 6.39 + 3.90
0.22 + 0.12 0.30 &-0.24 0.28 + 0.06
0.03 + 0.02 0.01 &-0.01 0.00 * 0.01
12-16yr Age Group 4.73 * 2.64 0.38 k 0.09 5.32 + 0.88 0.31 + 0.17 4.05 f 2.17 0.23 f 0.16
0.23 + 0.02 0.31 & 0.16 0.23 f 0.16
0.02 * 0.01 0.01 f 0.01 0.01 f 0.01
Low (0.72 + 0.32) Mod. (3.70 f 0.61) High (6.91 f 2.54)
45 44 45
0.85 + 0.30 0.89 f 0.09 0.73 * 0.59
Low (1.14 f 0.83) Mod. (3.46 If: 0.41) High (6.72 + 3.32)
46 46 46
0.87 f 0.51 0.76 f 0.42 0.83 + 0.61
(H.S.C.) so that at the time of analysis the principal investigator (M.S.D.) was not aware of the caries status of the children. The acidified samples were transferred to a laboratory about 20 miles from the collection site, packed in ice. On receipt the samples were centrifuged to remove particulate material and mucin clot that might choke the capillary tube of the atomic absorption spectrophotometer, and the supernatant was used for the estimation of trace elements. Estimation of trace elements other than fluoride
About 5 ml of the saliva prepared as described above were used to determine the concentration of Zn, Cu, Fe and Mn. Standards covering the appropriate concentration range and containing the same volume and concentration of reagents as those used for sample solutions were made before the start of analysis. Estimation of the four trace elements was done in an atomic absorption spectrophotometer (Vat-ion AA 175 ABQ) with a IO-cm, air-acetylene flame. Estimation of fluoride
Fluoride estimation was by the direct method first described by Gron, McCann and Brudevold (1968). About 3 ml of saliva were treated with equal amount of low-level, total ionic strength adjuster. This was prepared by adding 57 ml of glacial acetic acid and 58 g of sodium chloride to 500 ml of distilled water in a beaker. After cooling this solution in a water bath the pH was adjusted to 5.0-5.5 with 5 M sodium hydroxide and the solution made up to 1 1. The estimation was then made with an Orion fluoride electrode coupled to an Orion ion analyser. This allowed the determination of low levels of fluoride
and has been used by many other investigators for salivary fluoride (Yao and Gron, 1970). The validity of this method was, however, checked by adding known amounts of fluoride to known volumes of saliva before the test samples were analysed to make sure that the observed increases in concentration were equal to the added concentration of fluoride. Statistical analysis
After an analysis of variance, the StudentNewman-Keul’s procedure was used to analyse differences between the mean concentrations of trace elements in the different caries groups. RESULTS
The mean deft and DMFT in the various groups are shown in Table 1. The concentration ranges of the five trace elements in saliva (mg/l) were: Zn: ~0.10-3.82; Cu: 0.20-7.05; Fe: 1.50-29.00; Mn: ~0.05-1.76 and F: 0.05.
Zn
cu
k?yr Age Group ns
1991
0003-9969/91 $3.00 + 0.00
Copyright0 I!391PergamonPressplc
A STLJDY OF THE RELATIONSHIP BETWEEN TRACE ELSEMENTS IN SALIVA AND DENTAL CARIES IN CHILDREN M. S. DUGGAL,’ H. S. CIUWLA’ and M. E. J. CURZON’ ‘Department of Child Dental Health, University of Leeds, U.K. and *Department of Dentistry, Post Graduate Institute of Medical Education and Research, Chandigarh, India (Accepted
2 July 1991)
Summary-Absout 8 ml of saliva were collected from each of 272 children, living in rural areas in North India, equally distributed into age groups 47 and 12-16 yr and stratified into low, moderate and high caries groups. The salivas were analysed for Zn, Cu, Fe, Mn by atomic absorption spectrophotometry and for F with a fluoride-sensitive electrode. The elements Cu and F had a consistent, inverse relationship with caries experience. The concentration of Zn, Fe and Mn in saliva did not have any consistent
relationship with caries experience. Key words: saliva, trace elements (Zn, Cu, Fe, Mn, F), canes.
INTRODUCIlON
Elements present in only minute quantities in animal tissues are called trace elements regardless of their abundance in nature. Some of these, such as fluoride, iron, iodine, zinc, manganese, molybdenum and copper, are physiologically essential. There are many others that are probatbly present in biological systems only as contaminants and appear to have no specific function. Despite considerable interest in trace element concentrations in water (Losee and Bibby, 1969; Adkins and Losee, 1970; Curzon, Spector and Iker, 1978) and enamel (Curzon and Cracker, 1978) as determinants of dental caries, there have been comparatively few attempts to analyse the trace element composition of saliva (Dreizen and Levy, 1970; Cutress, 1972; Schamschula et al., 1978). Even fewer studies have attempted to correlate human caries susceptibility with the salivary concentration of trace elements (Schamschula et al., 1978; Dreizen and Levy, 1970). This is surprising because saliva constantly bathes the teeth and is important in the prevention and remineralization of early enamel caries. An understanding of the mineral components of saliva is therefore of great importance in the prevention of dental caries. Our aim now was to examine any correlation between the concentration of five trace elements in saliva, zinc (Zn), colpper (Cu), iron (Fe), manganese (Mn) and fluoride (E) and the dental caries status of children. MATERIALS AND METHODS
Selection of subjects
The population sample was from five rural areas of the north of India with known and similar concentrations of trace elements in their water. All the available children (a,bout 500) in two age groups of 4-7 and 12-16yr were initially examined for dental AOB36112-B
caries, using the World Health Organization index (1977), and stratified into low caries (O-2 DMFT/ deft), moderate caries (3-4 DMFT/deft) and high caries (5 and above DMFT/deft) groups. The 276 children who were equally distributed between the age and caries groups were then randomly selected for trace element analysis. The saliva of four children was lost during transportation, leaving 272 children in the study. The two age groups were selected in order to study the primary and the permanent dentition. Only those children who were in ‘continuous residence’ in the study areas, as determined by questioning the parents, were included. Children were considered to be in ‘continuous residence’ if they had been born and lived their lives in that area except for short intervals of no more than 3 weeks-for example, holidays. None of the children in the study had access to fluoride supplements or received any fluoride- or zinc-containing toothpastes; their dental restorations did not contain copper cement or copper amalgam. Collection of saliva
About 8 ml of unstimulated saliva were collected from each child, who drooled into acid-cleaned, dry, 20-ml Corning glass tubes with a screw cap of inert plastic. Care was taken to avoid contact between fingers and saliva to minimize the chance of contamination. Preparation of saliva samples
About 3 ml of saliva from each individual were set aside for fluoride estimation. The remainder was prepared by the Willis’ technique modified for analy sis of trace elements in saliva (Gow, 1965). Concentrated, analytical grade, nitric acid (0.5 ml) was added to each sample to bring the pH below 2 so as to prevent any microbial growth and enzymatic changes. The samples were coded by one of us 881
882
M. S. DUGGAL et al. Table 1. Mean concentration of trace elements in saliva Means + SD (mg/l)
Caries status (deft/DMFT f SD)
n
Zn
CU
Fe
Mn
F
47yr Age Group 0.53 + 0.15 0.35 f 0.10 0.27 kO.17
5.84 & 3.21 8.61 * 1.10 6.39 + 3.90
0.22 + 0.12 0.30 &-0.24 0.28 + 0.06
0.03 + 0.02 0.01 &-0.01 0.00 * 0.01
12-16yr Age Group 4.73 * 2.64 0.38 k 0.09 5.32 + 0.88 0.31 + 0.17 4.05 f 2.17 0.23 f 0.16
0.23 + 0.02 0.31 & 0.16 0.23 f 0.16
0.02 * 0.01 0.01 f 0.01 0.01 f 0.01
Low (0.72 + 0.32) Mod. (3.70 f 0.61) High (6.91 f 2.54)
45 44 45
0.85 + 0.30 0.89 f 0.09 0.73 * 0.59
Low (1.14 f 0.83) Mod. (3.46 If: 0.41) High (6.72 + 3.32)
46 46 46
0.87 f 0.51 0.76 f 0.42 0.83 + 0.61
(H.S.C.) so that at the time of analysis the principal investigator (M.S.D.) was not aware of the caries status of the children. The acidified samples were transferred to a laboratory about 20 miles from the collection site, packed in ice. On receipt the samples were centrifuged to remove particulate material and mucin clot that might choke the capillary tube of the atomic absorption spectrophotometer, and the supernatant was used for the estimation of trace elements. Estimation of trace elements other than fluoride
About 5 ml of the saliva prepared as described above were used to determine the concentration of Zn, Cu, Fe and Mn. Standards covering the appropriate concentration range and containing the same volume and concentration of reagents as those used for sample solutions were made before the start of analysis. Estimation of the four trace elements was done in an atomic absorption spectrophotometer (Vat-ion AA 175 ABQ) with a IO-cm, air-acetylene flame. Estimation of fluoride
Fluoride estimation was by the direct method first described by Gron, McCann and Brudevold (1968). About 3 ml of saliva were treated with equal amount of low-level, total ionic strength adjuster. This was prepared by adding 57 ml of glacial acetic acid and 58 g of sodium chloride to 500 ml of distilled water in a beaker. After cooling this solution in a water bath the pH was adjusted to 5.0-5.5 with 5 M sodium hydroxide and the solution made up to 1 1. The estimation was then made with an Orion fluoride electrode coupled to an Orion ion analyser. This allowed the determination of low levels of fluoride
and has been used by many other investigators for salivary fluoride (Yao and Gron, 1970). The validity of this method was, however, checked by adding known amounts of fluoride to known volumes of saliva before the test samples were analysed to make sure that the observed increases in concentration were equal to the added concentration of fluoride. Statistical analysis
After an analysis of variance, the StudentNewman-Keul’s procedure was used to analyse differences between the mean concentrations of trace elements in the different caries groups. RESULTS
The mean deft and DMFT in the various groups are shown in Table 1. The concentration ranges of the five trace elements in saliva (mg/l) were: Zn: ~0.10-3.82; Cu: 0.20-7.05; Fe: 1.50-29.00; Mn: ~0.05-1.76 and F: 0.05.
Zn
cu
k?yr Age Group ns
