American Journal of Clinical Nutrition, Vol. 70, No. 3, 576S-578S,
September 1999
© 1999 American Society for Clinical Nutrition
Cyanocobalamin (vitamin B-12) status in Seventh-day Adventist ministers in Australia1,2
Bevan D Hokin and
Terry Butler
1 From the Pathology Department, Sydney Adventist Hospital and Adventist Health Department, Australia.
2 Reprints not available. Address correspondence to BD Hokin, Pathology Department, Sydney Adventist Hospital, 185 Fox Valley Road, Wahroonga 2076, Australia. E-mail: bevan{at}sah.org.au.
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ABSTRACT
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As part of the Adventist Ministers' Health Study, a series of cross-sectional surveys conducted in 1992, 1994, and 1997, the serum vitamin B-12 status of 340 Australian Seventh-day Adventist ministers was assessed in 1997. The ministers in the study participated voluntarily. Of this group, 245 were either lactoovovegetarians or vegans who were not taking vitamin B-12 supplements. Their mean vitamin B-12 concentration was 199 pmol/L (range: 58–538 pmol/L), 53% of whom had values below the reference range for the method used (171–850 pmol/L) and 73% of whom had values <221 pmol/L, the lower limit recommended by Herbert. Dual-isotope Schillings test results in 36 lactoovovegetarians with abnormally low vitamin B-12 concentrations indicated that dietary deficiency was the cause in 70% of cases. Data from the dietary questionnaires supported dietary deficiency as the cause of low serum vitamin B-12 in this population of lactoovovegetarians and vegans, 56 (23%) of whom consumed sufficient servings of vitamin B-12–containing foods to obtain the minimum daily maintenance allowance of the vitamin (1 µg).
Key Words: Vitamin B-12 • cyanocobalamin • lactoovovegetarian • vegan • Schillings test • recommended daily allowance • RDA • Seventh-Day Adventists • ministers
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INTRODUCTION
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Seventh-day Adventists as a group attract much research attention because their recommended lifestyle proscribes alcohol and tobacco use, discourages the consumption of tea and coffee, and strongly promotes a lactoovovegetarian diet. Seventh-day Adventist ministers were selected as a subset of Adventists who were likely to follow this lifestyle closely.
The Adventist Ministers' Health Study refers to a series of cross-sectional studies conducted in 1992, 1994, and 1997. Participation was voluntary and all subjects provided informed consent. This study was jointly undertaken by the Adventist Church Health Department in the South Pacific and Sydney Adventist Hospital Pathology Department.
The vitamin B-12 status of vegetarian Seventh-day Adventist ministers in Australia is relevant because Australians have a lifestyle and standard of living similar to that of North Americans. Hence, it is likely that observations from this group will relate to other Western countries. One significant exception is the limitation in food fortification with vitamin B-12. Unlike in the United States, in Australia, Canada, and much of Europe there is no or very limited food fortification permitted by their governments. Consequently, the popularly held view that "clinical cobalamin deficiency due to dietary insufficiency is rare" (1) is questioned in these vegetarian populations. It is hypothesized that serum vitamin B-12 is lower in vegetarians than in nonvegetarians, with many vegetarians having vitamin B-12 concentrations below the reference range, and that dietary inadequacy is the major cause.
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METHODS
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Three hundred and forty Australian Seventh-day Adventist ministers participated in the 1997 study. All participation was voluntary. All subjects provided informed consent, and the study complied with the ethical standards of the Australian National Health and Medical Research Council. Each subject completed a detailed dietary (daily recall and average trend recall over 3 mo) and lifestyle questionnaire; had height, weight and blood pressure measured; and had a fasting blood sample drawn for a 20-test biochemical profile which included determining lipid concentrations studies, full blood counts, and vitamin B-12 concentrations.
Biochemical analyses and full blood counts were completed on the day of collection. All samples were centrifuged (at 2550 x g for 15 min at 4 °C) and the serum was separated from cells and refrigerated within 3 h of collection. Vitamin B-12 was measured with the Abbott IMX analyzer, reagents, and calibrators (Abbott Laboratories, Abbott Park, IL).
Furthermore, dual-isotope Schillings tests (2) using the Dicopac kit (Amersham Health Care, Buckinghamshire, United Kingdom) were performed on 36 consecutive Adventist lactoovovegetarians who were referred by their physician for further testing because their vitamin B-12 concentrations were below the reference range. The Dicopac Schillings test uses 2 cobalt isotopes to achieve a differential diagnosis of the causes of low vitamin B-12 concentrations. Free cyanocobalamin was labeled with 58Co. Absorption of this, and hence the appearance of 58Co in urine, is impaired if the patient has intrinsic factor (human gastric juice) deficiency. Cyanocobalamin, which is bound to intrinsic factor, was labeled with 57Co. Absorption of this, and the subsequent concentration of 57Co in urine, is independent of the patient's ability to secrete intrinsic factor but is dependent on the patient's ability to absorb the vitamin in the ileum. The patients were given an oral tracer dose of both the free (58Co) and the bound (57Co) vitamin. Absorbed isotope-labeled vitamin was flushed from the blood stream by simultaneous intramuscular injection of a relatively large amount (1000 µg) of nonradioactive cyanocobalamin. Urine was collected over the next 24 h and 57Co and 58Co were measured in aliquots. The 2 isotopes were easily measured in the presence of each other (with a good-quality 
counter) and the percentage of each tracer dose excreted in 24 h was readily calculated.
Presence of pernicious anemia is usually evident if the ratio of 57Co (%) to 58Co (%) is >1.3, and malabsorption is assumed if the amount of tracer excreted in 24 h is <14% of the initial dose. A normal Schillings test result suggests that the cause of a low vitamin B-12 concentration is inadequate dietary intake.
Statistical analysis of data was performed by using Wilcoxon's rank sum test, a nonparametric analysis for skewed data.
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RESULTS
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Of the 340 subjects in the sample, 331 were men and 9 were women. Their average age was 46 y (range 22–80 y). Forty-two of the subjects were taking vitamin B-12 supplements (tablets or injections) and were excluded. Another 53 subjects who consumed one or more servings of flesh products per week (fish, poultry, or red meat) were used as control subjects. Of the remaining subjects, 234 were lactoovovegetarians and 11 were vegans, none of whom were taking vitamin B-12 supplements. This group had been vegetarians from 3 to 64 y (mean: 32 y). Two-hundred and fourteen of the 245 had been lactoovovegetarians or vegans for 
20 y.
The reference range for serum vitamin B-12 used by the laboratory was 171–850 pmol/L. Herbert (3) recommended a lower limit of the normal range of 221 pmol/L. Sixty-two percent of all subjects (vegetarian and nonvegetarian combined) had serum vitamin B-12 concentrations below the Herbert reference range (Table 1
). Seventy-three percent of the vegetarians but only 40% of the nonvegetarians had serum vitamin B-12 concentrations below Herbert's recommended lower limit.
Analysis of these data showed that the vitamin B-12 concentrations of the vegetarians (
± SE: 199 ± 126 pmol/L; range: 58–538 pmol/L) were significantly lower than those of the nonvegetarian control subjects ( ± SE: 292 ± 145 pmol/L; range: 134–721 pmol/L; P < 0.0005). Similarly, the vegans (n = 11), as a subset of the vegetarians, had vitamin B-12 concentrations significantly lower ( ± SE: 166 ± 74 pmol/L; range: 73–311 pmol/L) than those of the nonvegetarian control subjects (P < 0.003). A comparative analysis of vitamin B-12 concentrations of the vegans and lactoovovegetarians in the group showed no significant differences. The distributions of vitamin B-12 concentrations for vegetarians and nonvegetarians are shown in Figure 1.
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FIGURE 1. Frequency distribution of vitamin B-12 concentrations among vegetarian (n = 245) and nonvegetarian (n = 53) subjects in the Adventist Ministers' Health Study.
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The Schillings test study in 36 lactoovovegetarians to assess relative frequency of dietary deficiency, intrinsic factor abnormalities, and ileal malabsorption showed that 25 subjects (70%) had normal results, suggesting that inadequate dietary intake was the likely cause of their deficiencies; 10% had an intrinsic factor–related abnormality and 20% showed some degree of malabsorption.
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DISCUSSION
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Clearly, the causes of vitamin B-12 deficiency are multiple. The 6 common causes of gradual-onset vitamin B-12 deficiency are shown in Table 2 (1). Schillings test data revealed that of these causes, dietary inadequacy was the most common (70%) in the population studied, followed by inadequate absorption (30%) due to either malabsorption in the terminal ileum or to intrinsic factor–related problems.
The mean vitamin B-12 concentration of vegetarian Adventist ministers was significantly lower than that of the nonvegetarian Australian population (P < 0.0001). The cumulative mean vitamin B-12 concentration of the last 2000 patients (mostly nonvegetarians) referred to this laboratory was 310 ± 80 pmol/L, and it should be remembered that physicians who refer patients for vitamin B-12 assays usually expect the result to be low. Consequently, the mean concentration in the general public is likely to be even higher.
Persons with normal Schillings test results and adequate dietary intake should have serum vitamin B-12 concentrations >220 pmol/L. Vegetarians should determine their average daily vitamin B-12 intake to ensure it exceeds the minimum maintenance amount of 1 µg/d, and preferably the recommended dietary allowance of 2 µg/d (Table 3; 4, 5)
All vitamin B-12 is manufactured by bacteria and appears "second hand" in meats and animal products. There is no vitamin B-12 in plant sources, unless by contamination from bacterial sources (6). Reports of vitamin B-12 in organically grown spinach (7) should be viewed with caution until confirmed because the original research is subject to serious question because of flawed experimental design and mathematical errors (8).
Lactoovovegetarians obtain their vitamin B-12 from milk, yogurt, cheese, and eggs (Table 4). Vegans can only obtain reliable sources of vitamin B-12 from supplements or fortified foods (meat analogues, soymilk, cereals, and fortified yeast products). Vitamin B-12–supplemented foods are common in the United States, where extensive food fortification is permitted. Vegans face a greater problem in other parts of the world where more restrictive food fortification regulations are in place. Caution should be exercised in selecting some food supplements promoted as containing vitamin B-12 (eg, tempeh and spirulina). The form of vitamin B-12 present may be an analogue, capable of blocking the absorption of biologically useful forms of the vitamin (6).
Conclusion
As many as 73% of Australian vegetarians have serum vitamin B-12 concentrations below recommended amounts. Schillings test results suggest that 70% of such deficiencies are due to inadequate dietary intake. Those who follow a vegetarian diet should consume an adequate amount of vitamin B-12, either from foods containing vitamin B-12, from vitamin B-12–fortified foods, or from supplements.
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Machlin L, Huni J, eds. Vitamins basics. 1st ed. Basel, Switzerland: F Hoffman-LaRoche Ltd, 1994.
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Katz JH, Mase J, Donaldson RM. Simultaneous administration of gastric juice-bound and free radioactive cyanocobalamin: rapid procedure for differentiating between intrinsic factor deficiency and other causes of vitamin B-12 malabsorption. J Lab Clin Med 1963;61:266–71.
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Herbert V. Vitamin B12. In: Ziegler EE, Filer LJ Jr, eds. Present knowledge in nutrition. 7th ed. Washington, DC: International Life Sciences Institute Press, 1996:191–2051.
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National Research Council. Recommended dietary allowances. 10th ed. Washington, DC: National Academy Press, 1989.
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Joint FAO/WHO Expert Group. Requirements of vitamin A, iron, folate and vitamin B12. Rome: Food and Agriculture Organization, 1988. (FAO Food Nutr Ser no. 23.)
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Herbert V. Vitamin B-12: plant sources, requirement, and assay. Am J Clin Nutr 1988;48(suppl):852–8.[Abstract/Free Full Text]
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Mozafar A. Enrichment of some B-vitamins in plants with application of organic fertilisers. Plant Soil 1994;167:305–11.
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ABSTRACT
INTRODUCTION
