Background

The clinical significance of water soluble vitamin B12 (cobalamin) and of folate, and the importance of their routine analysis, is supported by recent findings in physiology regarding their function [1–8]. The simple yet biologically important role of support by these vitamins in catalysis of methyl group transfer is essential for life maintenance [9]. The function of folate overlaps with vitamin B12 both of them being essential in methylation reactions. With telomere shortening emerging as a research topic of senescence, it has been shown that the one-carbon metabolism pathway may affect telomere length through DNA-methylation [10].

Vitamin B12 deficiency later in life is estimated to affect 10 % of people over the age of 60 [11, 12]. Recent metaanalytic surveys [13, 14] confirmed the finding of vitamin B12-, and folate- deficiencies in elderly patients [15]. Optional but frequently practiced fortification of grain-based food supply is done with folic acid and vitamin B12 (www.blv.admin.ch).

Next to vitamin B12, folates are essential in eukaryotic cells for single carbon transfer reactions. Folate receptors transport their ligand via endocytosis [16] and jejunal folate resorption is of no clinical concern unless viral infection interferes [44, 45, 53–55]. With the recent discoveries regarding the indispensable functions of vitamin B12 and folate in living organisms [1, 2, 7], it comes as little surprise that vitamin B12 and, to a lesser extent, folate, maintain their levels throughout our life cycle or else the participating subjects would not have been able to enter the study in advanced age under the classification ‘healthy’.

This was a community-dwelling cohort and haematinic sufficiency [56] can be assumed among participants whose BMI was normal and who were omnivorous, well fed and unlikely to be vegetarians. Marginal vitamin B12 haematinic provision remains a topic in daily health care. Switzerland counts only around 20.000 lactovegetarians distributed over all age groups: inadequate dietary intake of these two vitamins cannot be regarded as a relevant issue in this study. The effect of food fortifications on the levels of vitamin B12 and folate could not be addressed. With the Canadian Health Measures Survey on healthy folate replete subjects, the prevalence of folate deficiency was close to zero while vitamin B12 deficiency (cutoff < 148 pmol/L) came up to 5 % [43] but without effect of advanced age in older adults (60–79 years). In Switzerland, intentional fortification of food with vitamin B12 and folic acid is not enforced by law unless vitamin supplementation is a recommended component of pregnancy care. We thus might assume that dietary intake by the healthy elderly studied here is adequate.

The drop in folate but not RBC folate levels observed here may be linked to an intracellular maintenance of folic acid entrapped in RBCs but no full explanation can be forthcoming until half-life studies in both body compartments, plasma and RBC, have been conducted. Some authors hold that free and RBC folate are equivalent [57] and guidelines from the UK judge serum folate measurement as sufficient for clinical purpose [58]. Some are of the opinion that estimation of RBC folate is required to appreciate general tissue folate supply; should the latter view prevail for interpretation of our observations, then increasing age at least is not connected to folate deficiency.

In line with acknowledged understanding, our study links the surrogate markers of vitamin B12 status, i.e., MMA & Hcy, more closely to waning kidney function than to age. It is widely confirmed that with advancing age and particularly over age 50, functional kidney performance declines by > 1 ml/min/year/1.73 m2 eGFR [59–61]. Our cohort confirms receding kidney function with progressing age ≥60 years using state-of-the art evaluation with creatinine and corrected cystatin C levels [20]. Thus logic would dictate that there are limits to applicability of MMA and Hcy as surrogate markers in geriatrics. Unhelpfully, quite a few studies have used claims of high levels of MMA and Hcy to underscore their findings of vitamin B12/folate reductions without testing for impaired kidney function, which is now well acknowledged, to increase MMA and Hcy levels [62].

It is noteworthy that significantly lower, but still normal, vitamin B12 median levels emerge in elderly males by comparison with females in the 60–79 years age range. Although no such sex difference showed up in a Canadian-HMS cycle 1 [43], it was observed that elderly males clearly were at higher risk for Vitamin B12/folate deficiency in a NHANES survey on 1770 elderly persons [47] and in our study women maintain sufficient vitamin B12/folate levels from their reproductive life cycle onwards.

In our study we have seen an increment in MCV with increasing age. Although the index values steadily increase by merely 1 fl from the youngest to the oldest age group, the difference is statistically significant because of the narrow distribution. This observation confirms previous reports indicating a link between macrocytosis and poorer age-related cognitive performance [18]; the selection criteria prior to study entry used here preclude such an association in the cohort studied here [52]. Out of curiosity, we also estimated the Mentzer index, now undergoing a revival as a test for safety of stem cell apheresis from healthy donors [35]. The values here obtained confirm those observed with MCV and in our case served as a validity criterion for our observations.

In an earlier report on a small subsample of the present cohort, we reported a borderline difference of eGFR in participants with low and normal holoTC [38]. In line with other investigations, we now could demonstrate that holoTC is not influenced by kidney function [63]. The fact that we found holoTC to possess the highest accuracy in recognizing an insufficient vitamin B12 status together with the independence of the parameter from reduced kidney function (a problem often occurring in the elderly) and from age seems to make holoTC an attractive first line choice for assessing vitamin B12 status in the elderly.

There are several possibilities to interpret markers of vitamin B12 and folate status. The most often used approach is the comparison with reference intervals. We were able to report reference intervals for all relevant parameters relating to the investigation of vitamin B12 and folate status in the elderly. Each of the reported stratum provides results from far more than 120 subjects, a lower limit generally accepted for evaluation of valid reference intervals [39]. Reference intervals have been reported from substantially smaller collectives [22]. The present investigation to the best of our knowledge is the largest collective of elderly persons, where reference intervals for vitamin B12 and folate status have been derived in an integral approach.

Another way of interpreting laboratory results is the comparison with pathophysiological conditions. We also have taken this approach with ROC curve analysis, which allows to describe areas of relative certainty (i.e., above or below two cut-offs) together with a greyzone. Interestingly, the lower limits of the reference intervals are similar to the specific but insensitive cut-offs. Applying classically evaluated reference intervals for assessment of vitamin B12 deficiency can thus be regarded as very specific and relatively insensitive. Within the difficult context of diagnosing vitamin B12 and folate deficiency, we think that using an approach with decision limits provided in Table 2 is preferable to the use of reference intervals. Unfortunately, a functional score for detecting folate deficiency in analogy to the Fedosov wellness quotient in vitamin B12 deficiency is lacking so far.

Our study contains limitations:

  1. (i)

    The medication and supplementation intake of the participants was self-reported. It might be that some patients had supplements and medications leading to erroneous inclusion of a participant in this analysis. We can therefore not rule out the possibility that a portion of our participants have normal vitamin B12 levels because supplemented. As impaired cognition was an exclusion criterion, it can be assumed that the number of affected individuals is low.

  2. (ii)

    It is unfortunate that the questionnaire for study subjects did not include more details on eating habits

Conclusions

Whereas the vitamin B12 and holoTC levels remain steady after 60 years of age, we observed a significant increment in MMA levels accompanied by increments in Hcy; the latter is better explained by age-related reduced kidney function than by vitamin B12 insufficiency. Total serum folate levels but not RBC folate levels decreased with progressing age. The present work evaluated decision limits for the use of these parameters in the elderly.