Journal of the American College of Nutrition, Vol. 17, No. 1, 48-53 (1998)
Published by the American College of Nutrition
Biotin Status and Its Correlation with Other Biochemical Parameters in the Elderly People of Japan
Toshiaki Watanabe, PhD, DSc,
Seiji Yasumura, MD, PhD,
Hiroshi Shibata, MD, PhD and
Toru Fukui, BPhar
Department of Hygiene and Preventive Medicine (T.W.), Yamagata University School of Medicine, Yamagata
Department Public Health (S.Y.), Yamagata University School of Medicine, Yamagata
Department of Community Health, (H.S.), Tokyo Metropolitan Institute of Gerontology, Tokyo, JAPAN
Clinical Laboratory, Byotai Seiri Laboratory (T.F.), Tokyo, JAPAN
Address reprint requests to: Toshiaki Watanabe, PhD, DSc, Department of Hygiene and Preventive Medicine, Yamagata University School of Medicine, Iida Nishi 2-2-2, Yamagata, 99023, JAPAN
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ABSTRACT
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Objective: Biotin plays important roles in carbohydrate and lipid metabolism as well as in the decarboxylation of amino acids. In this study, to determine the biotin status in elderly people, we determined the levels of biotin and other biochemical variables in their serum.
Methods: Blood was collected from 685 elderly people aged 65 years and over (284 men and 401 women) and from 2,004 reference people. Biotin levels in the serum were microbiologically quantified by the agar plate method and other biochemical variables were recorded using the autoanalyzer.
Results: The serum biotin level in elderly people was 10.2 ± 7.20 pmol/ml (2.5 ± 1.76 ng/ml), the distribution of which was skewed to the right compared to the reference group (9.4 ± 1.43 pmol/ml) (2.3 ± 0.35 ng/ml). However, serum biotin levels did not change with age in the elderly people and no sex-related differences were detected. On the basis of the correlation coefficients among the biochemical variables in the serum, biotin levels were correlated positively with the total cholesterol level. A negative correlation was found between the biotin level and the serum albumin, triiodothyronine, phosphate, and calcium levels. On the other hand, 5.8% of the elderly people had biotinyl IgG in their serum, which did not differ from the percentage in the reference group (4.1%). However, in the elderly people the level of biotinyl IgG positivity was significantly lower in women than in men (4.1% vs. 8.1%). In the biotinyl IgG positive elderly, the levels of parathyroid hormone and total cholesterol were lower, whereas the uric acid level was higher compared to the biotinyl IgG negative elderly.
Conclusions: Although serum biotin levels are not affected by aging, the serum biotin levels of elderly people vary greatly.
Key words: biotin, biotinyl IgG, serum, elderly people, Japan
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INTRODUCTION
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Biotin is a water-soluble vitamin generally included in the vitamin B group. This vitamin is widely distributed in animals and plants, and plays important roles in carbohydrate and lipid metabolism as well as in the decarboxylation of amino acids [1]. Although a biotin inadequacy does not spontaneously occur in humans, biotin deficiencies cause growth retardation, alopecia, dermatitis, and neurological impairments in experimental animals and humans [2]. In addition, biotin is important in the normal reproductive performance and embryonic growth and development of mammals [3–5]. Recently biotin has been found to be correlated with certain diseases such as diabetes mellitus, liver and skin disorders, immunological and neurological abnormalities, and epilepsy [6–10]. On the other hand, Fukui and Oizumi [11] identified a G-class immunoglobulin antibody that binds to biotin in an immune specific fashion in human serum, which is referred to as "biotinyl IgG." This protein was found in a high incidence in patients with allergic and autoimmune disorders. The existence of biotinyl IgG in the peripheral blood has been suggested to be associated with the etiology of these disorders [12].
In a series of epidemiological studies to clarify the nutritional factors modulating events in life, we have examined elderly people living in various regions. Intakes of such micronutrients as calcium, iron, retinol, thiamin, riboflavin, ascorbic acid, and the proportion of energy from proteins and fats were significantly higher in Okinawa Prefecture (where life expectancies at birth and 65 years are the longest in Japan) than in Akita Prefecture (where the life expectancies were much shorter) [13,14]. In a study in Koganei, an urban community in Tokyo, there were no changes in the relationship between aging and biochemical variables in the serum. However, elderly males had significantly higher bone mineral densities on average than elderly females [14,15].
Our project aimed to evaluate the relationship between the aging process and nutritional factors. In an effort to elucidate the status and the role of biotin in elderly people, we determined the levels of biotin and other biochemical variables in the serum in this study.
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MATERIALS AND METHODS
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Subjects
We undertook a baseline survey in elderly subjects living in a village in northeastern Japan. Nangai lies about 25 km west of Omagari City in Akita Prefecture and has been developed as a rice growing area for over 100 years (Fig. 1). This village had a population of 5,248 in an area of 10 km2 in 1991 and is an area which has rather short life expectancies at birth and at 65 years, compared with other areas in both Akita Prefecture and Japan. The proportion of the population aged 65 years and over has gradually increased and reached 14.5%. The outline of these surveys and studies has been described elsewhere [14].
The subjects were 881 eligible citizens living in Nangai in June, 1991. A baseline health examination was conducted on a total of 685 elderly people aged 65 years old and over. Forty-two percent were less than 70 years old, 44% were in their 70s and, 14% were over 80. Forty-one percent were men and 59% women. The baseline examination included a self-administered questionnaire, physical examination, and medical examination.
The reference subjects were volunteers selected from residents in some rural and urban communities and employees in some workplaces. Of these people, 2,004 had no abnormalities in their biochemical blood findings and agreed to participate in our studies on biotin. These participants were used as the reference population, whose age ranged from 17 to 77 years.
Analyses of Biotin and Biochemical Variables in the Serum
Biotin levels in the serum were microbiologically quantified by the agar plate method developed by Fukui et al [16]. Serum was treated with 4.5 N sulfuric acid at 120°C for 1 hour. After neutralization with 4.5 N sodium hydroxide, 10 µl of the serum sample was dropped in a small hole in an agar plate and inoculated with Lactobacillus plantarum ATCC 8014. After incubation at 37°C for 16 hours, biotin was determined by measuring the diameter of the bacterial growth area and was regarded as total biotin. This bioassay can determine biotin specifically without being affected by lipids or cholesterol in the serum. On the other hand, biotinyl IgG in the serum was determined by the immunofixation method [11,12]. Briefly, a cellulose acetate membrane was coated with antibodies specific for human immunoglobulin G, A, and M, and incubated with diluted sample sera in a moist box. The membrane was washed with saline and conjugated with avidin-alkaline phosphatase. After washing with saline, the sample was stained for alkaline phosphatase. Positive staining for alkaline phosphatase was evaluated with biotin binding immunoglobulin G, A, or M.
Clinical findings, such as urine findings, blood pressure, electrocardiographic findings, X-ray findings, and biochemical blood findings were examined. An autoanalyzer (7170, Hitachi, Ltd., Tokyo) was used to measure albumin, alkaline phosphatase, ß2-microglobulin, total cholesterol, HDL cholesterol, N-acetyl-ß-d-glucosaminidase, transaminase, triiodothyronine, parathyroid hormone, white blood cells, red blood cells, hemoglobin, hemoglobin A1c, hematocrit, calcium, phosphate, and uric acid levels in the blood.
Statistical Analyses
The statistical analysis of the relationships among biochemical variables was performed using simple and partial correlation coefficients [17]. To analyze the incidence of biotin binding immunoglobulin in elderly and reference people, the chi-square test was used. The Student’s t-test was used to determine significant differences in the mean values of serum biotin and other biochemical variables between biotinyl IgG positive and negative elderly. Multiple stepwise regression analysis was attempted with the serum biotin status as a dependent variable and the possible biochemical variables as independent variables. Statistical analyses of these data were conducted using SPSS software (ver. 6.1) on a Macintosh computer. Differences were considered significant at a probability level of p < 0.05 in all the analyses.
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RESULTS
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Serum Biotin Levels in the Elderly People
Fig. 2 presents a histogram of the serum biotin levels in elderly and reference people. In 685 elderly people (284 men and 401 women), the serum biotin levels ranged from 2.5 to 96.2 pmol/ml. The mean and standard deviation (SD) of serum biotin in the elderly was 10.2 ± 7.20 pmol/ml (2.5 ± 1.76 ng/ml, coefficient of variance, CV: 70.4%), the distribution of which was skewed to the right compared with 9.4 ± 1.43 pmol/ml (2.3 ± 0.35 ng/ml, CV: 15.2%) seen in the 2,004 reference people. The numbers of elderly people with extreme amounts of serum biotin, less than 5.1 pmol/ml and more than 13.6 pmol/ml (mean ± 3SD in reference people) were 84 (12.2%) and 145 (21.1%), respectively, and they had no clinical characteristics or complaints. In addition, the mean and SD of biotin levels did not differ between men and women, and did not change with age in the elderly (Table 1).
Correlation between Biotin and Other Biochemical Variables
Table 2 shows the Pearson’s and partial correlation coefficients between the serum biotin level and other biochemical variables in the elderly. The biotin levels correlated positively with the total cholesterol levels (r = 0.151, p < 0.01) in men. A negative correlation was found between the biotin level and serum albumin (r = -0.169, p < 0.01) or calcium level (r = -0.195, p < 0.01) in both sexes, and between the biotin level and the triiodothyronine (r = -0.191, p < 0.01) or phosphate level (r = -0.132, p < 0.01) in women.
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Table 2. Correlation Coefficients between Biotin Levels and Biochemical Variables in Serum in Elderly Men and Women
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Multiple Stepwise Regression Analyses
Multiple stepwise regression analysis revealed significant relationships between the biochemical variables related to bone metabolism (parathyroid hormone, calcium and phosphate), thyroid function (albumin, total cholesterol and triiodothyronine), and the serum biotin status of both sexes as shown in Table 3. Although a significant relationship between biochemical variables affecting lipid metabolism (total and HDL cholesterol) and the serum biotin status was obtained in elderly men, there was no such significant relationship in elderly women.
Biotinyl IgG among Elderly People
The levels of biotinyl IgG positivity in the serum of elderly and reference people are shown in Table 4. Of the elderly people, 5.8% were positive for biotinyl IgG, which did not differ from the 4.1% observed in the reference people. However, the level of biotinyl IgG positivity was significantly lower in elderly women than in elderly men (4.1% vs. 8.1%). In the biotinyl IgG positive elderly, the levels of parathyroid hormone and total cholesterol were lower, whereas the uric acid level was higher compared to the biotinyl IgG negative elderly. However, there was no difference in the serum biotin level or other biochemical variables between the biotinyl IgG-groups in either sex, except for the significantly lower levels of parathyroid hormone in biotinyl IgG positive elderly men (19.5 pg/ml vs. 24.0 pg/ml).
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Table 4. Difference in Biotin Levels and Biochemical Variables in Serum among Elderly People with and without Biotinyl IgG
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DISCUSSION
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We observed large variations in serum biotin levels in elderly people living in a rural area compared with that in reference people in this study. However, there was no difference in the mean biotin level in the serum between the two groups. This finding is not in agreement with a previous study by Bonjour [1], in which the elderly had a significantly lower plasma biotin level than the controls, although sample size was very small. The normal range of serum biotin concentrations in reference people was determined to be between 6.5 and 12.1 pmol/ml (mean ± 2SD) in this study. The lowest and highest concentrations of serum biotin were 4.5 and 28.3 pmol/ml respectively. This is higher than the normal value of 3.3 to 6.1 pmol/ml reported in the literature [18,19]. The difference is partly due to the fact that the bioassay using L. plantarum can be affected by other compounds, as can all bioassays. The incidence of elderly people with extreme amounts of biotin were 12.2% below the lower limit, and 21.1% above the upper limit, which was much higher than in the reference people (0.4% and 5.7%, respectively). However, even elderly people with extremely low amounts of serum biotin showed no clinical findings or complaints.
In this study, the fact that a large number of elderly had a high level of serum biotin is of great interest, although they did not take any supplementary biotin. It is not obvious why biotin accumulates in the serum of elderly people. Biotin is widely distributed in foodstuffs of both plant and animal origin. However, the high level of biotin seen in the serum can not be explained by dietary biotin intake alone in elderly people, even if they consumed many foods rich in biotin. Bitsch et al [19] demonstrated that maximal concentrations of plasma biotin were reached only 1 hour after oral loading with 600 µg biotin. This dose of biotin is comparable to about 10 times the dietary intake. The elimination half life of the administered biotin dose was 2 hours [20,21]. Therefore, serum biotin levels may be influenced more by the turnover (recycling) and the retention capacity of biotin in the tissue rather than the daily dietary intake of biotin.
On the other hand, biotin deficiencies are rare in humans. However, a low level of biotin (less than 5.1 pmol/ml) was also frequently observed in elderly people (12.2%) in this study. Although the clinical significance of low biotin levels in humans is not clear, inadequate food intake may partly contribute to a reduced nutritional status and influences the health of elderly people.
Uncertainties regarding biotin requirements and intake in humans make assessment of the status difficult [22]. Although the recommended dietary allowance (RDA) has not been formulated, a safe and adequate dietary intake for humans has been proposed, which is based on the average food content and national consumption habits in general populations. The daily requirement of biotin has been recommended to be around 100 to 200 µg for adults in the USA [23,24]. However, the estimated daily intake of biotin is 30 to 100 µg for adults in the USA [25]. The dietary intake of biotin has also been estimated to be below 100 µg/day in Swiss, Canadian, and British populations [1,26–28]. In addition, a prevalence of undernutrition has been reported in the elderly population [29]. Therefore, inadequate food intake, including micronutrients in the elderly, may partly contribute to a lowered nutritional status, resulting in specific metabolic changes brought about by a dietary biotin insufficiency.
Calcium and phosphate are important components of the bone. Ninety-eight percent of calcium and 80% of phosphate exists in the form of hydroxyapatite. In addition, parathyroid hormone plays an important role in the regulation of calcium levels in the serum. Watkins and his colleagues [30,31] suggest that biotin and certain factors are of importance for maintaining skeletal growth and bone modeling in broiler chicks. In this study, serum biotin levels had a significantly negative relationship with albumin, triiodothyronine, parathyroid hormone, calcium, and phosphate in the elderly, but a positive relationship with total cholesterol. These findings indicate that biotin may influence the bone mineral homeostasis in humans as well.
Biotin also plays an important role in the regulation of immunological functions which are responsible for abnormal antibody formation, decreased T-cell cytotoxicity, and increased susceptibility to infection [32,33]. The incidence of biotinyl IgG positivity in elderly women was much lower than that in elderly men. The role and the induction mechanisms of biotinyl IgG are not known in detail. However, we demonstrated that a prevalence of biotinyl IgG was markedly higher in Grave’s disease (47%) compared with control subjects [12]. This disease is known to result from autoimmune dysfunction in the thyroid gland. In addition, this protein was frequently found in the serum of children with atopic dermatitis, and biotin treatment was very effective in biotinyl IgG positive patients [11]. Therefore, biotinyl IgG may be associated with autoimmune and allergic disorders. In this study, we observed a significant relationship between some parameters related with thyroid function and serum biotin levels. However, it is unclear how serum biotin affects the function of the thyroid gland in the elderly.
In conclusion, we have shown that serum biotin levels are not affected by aging, although the serum biotin levels in elderly people vary greatly. Also, biotin may affect some metabolic functions in elderly people. Further investigations to determine the role of biotin and biotinyl IgG in the elderly are necessary.
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ACKNOWLEDGMENTS
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The authors thank the public health nurses and local staffs of the Health Center for their cooperation. This study is one of the research projects, "Longitudinal multidisciplinary study on aging", conducted by the Tokyo Metropolitan Institute of Gerontology.
Received October 1, 1996.
Revised May 1, 1997.
Accepted May 1, 1997.
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ABSTRACT
INTRODUCTION
