Abstract
Vitamin B12 deficit is one of the most common vitamin deficiencies. However, there is no consensus on the cut-off points for vitamin B12 and its co-markers, such as folate, holotranscobalamin, methylmalonic acid and homocysteine. In order to establish the state of the art about cut-off points used to determine vitamin B12 deficiency in the last decades, the database MEDLINE was used for searching studies published in adults between December 1992 and May 2014 (69 articles), using search terms like ‘vitamin B12’, ‘cobalamin’, ‘cut-off’, ‘deficiency’ alone or in combinations. Broad ranges of cut-off points for vitamin B12 and its biomarkers were identified: vitamin B12 ranged between 100 pmol/L and 350 pmol/L, holotranscobalamin 20–50 pmol/L, methylmalonic acid 0.210–0.470 μmol/L, homocysteine 10–21.6 μmol/L, serum folate 3.7–15.9 nmol/L and red blood cell 124–397 nmol/L. For the majority of studies, the potential influence of age, analytical methods, gender and fortified food consumption was not taken in account when choosing cut-off values. This could explain the discrepancies between studies on vitamin B12 and folate deficiency prevalences. We conclude that there is inconsistency in the literature regarding vitamin B12 cut-offs. It would be necessary to establish different reference cut-offs according to age, considering the analytical methods used.
Introduction
Vitamin B12 deficiency is still an important nutritional problem worldwide as subclinical deficiency affects well defined risk groups. Vitamin B12 levels decreases with age [1], which means that deficiency risk increases in parallel with age [2–4]. Therefore, subclinical deficiency is quite more prevalent in the elderly. Epidemiologic data shows that prevalence ranges from 6% to 40% [5]. However, at younger ages, a higher risk of developing vitamin B12 deficiency has been described among vegetarians [5, 6], patients with gastrointestinal disease [7], people with depression [8], people with high alcohol consumption [9], and people suffering from renal insufficiency [7]. The main manifestations of vitamin B12 deficiency are hematologic, neurologic and psychiatric disorders. Notably, vertigo, tiredness, malaise and cognitive impairment have been traditionally ascribed as ‘normal aging’ signs [10]. Most of these disorders and symptoms are usually seen as ‘normal aging’ signs. However, severe vitamin B12 deficiency causes an irreversible degeneration of the nervous system [11]. Thus, an early diagnosis and treatment of subclinical vitamin B12 deficiency is essential. In order to reach a more efficient diagnosis, a combination of several markers associated with vitamin B12 metabolism could be used in place of a single vitamin B12 measurement [5]. The most reliable markers are serum folate (sFolate), red blood cell folate (RBC folate), holotranscobalamin (HoloTC), methylmalonic acid (MMA) and homocysteine (Hcy).
One of the major problems when attempting to diagnose vitamin B12 deficiency is the choice of the cut-off values for each of the markers. For instance, due to the high variability of cut-off values across laboratories, vitamin B12 deficiency prevalence ranges from 3% for people aged ≥3 years to 67.6% for elderly (71–74 years) [12, 13]. This prevalence can increase up to 15.8% for institutionalized elderly, which presents a greater risk for vitamin deficiencies than the free-living elderly [1]. Epidemiologic data by Andres et al. confirmed that 20% of the elderly present megaloblastic anemia in developed countries [14]. Finally, the different analytical methods used to measure vitamin B12 and its co-markers introduce an additional dispersion factor for cut-off setting.
The objective of this study is to provide a review of the published biomarkers and cut-off values for the diagnosis of vitamin B12 deficiency with respect to age, gender and methods of detection. The lack of consensus worldwide calls for a re-examination of the reference values currently employed to detect mild, moderate and severe forms of vitamin B12 deficiency.
Materials and methods
The electronic database MEDLINE (http://www.ncbi.nlm.nih.gov) was searched for studies published between December 1992 and May 2014. Terms ‘vitamin B12’, ‘cobalamin’, ‘cut-off’, ‘deficiency’ as well as combinations out of these terms were entered in the database. In addition, references in relevant articles were also used to get further information.
Only articles containing vitamin B12 cut-offs were chosen. The rest of cut-offs (MMA, Hcy, HoloTC, sFolate and RBC folate) were collected when they were available. Additional data including analytical methods, geographic area, sample size and age were included if available, whereas health status of the population under study was not considered. Data on creatinine was also taken into account when authors gave this information to assess renal function. Articles were excluded if the cut-off for vitamin B12 was not included, or in case of a review, a systematic review or meta-analysis.
Results
A total of 69 articles were included in this review. Table 1 summarizes the main data in each study, ordered from lowest to highest cut-off points of plasma vitamin B12 concentration.
No. | References | City (country)a | n (all; m, f) | Age, year | B12b, pmol/L | HoloTCb, pmol/L | MMAb, μmol/L | Hcyb, μmol/L | sFolateb, nmol/L | RBC folateb, nmol/L | Creatinineb, μmol/L |
---|---|---|---|---|---|---|---|---|---|---|---|
1 | [16] | Wageningen (NL) | 120 | ≥70 | 100 | 0.260 | 120 | ||||
CLIA | GC-MS | ||||||||||
2 | [17] | Germany (DE) | 202; 52, 150 | 81±6g | 100 | 0.320 | 120 | ||||
ND | ND | ||||||||||
3 | [15] | (BE, DE, NL) | 1=99; 53, 46 | 1=young subpopulation 19–55 | 103 | 0.247 | 13.9 | 5.4 | 124 | ||
2=64; 20, 44 | 2=healthy elderly people 65–88 | RIA | GC-MS | GC-MS | RIA | ||||||
3=286; 115, 171 | 3=hospitalized elderly 61–97 | ||||||||||
4 | [37] | 13 centers (BE) | 285; 105, 180 | 65–96c | 103 | 0.247 | 13.9 | 5.4 | |||
RIA | GC-MS | GC-MS | RIA | ||||||||
5 | [38] | Skutskär (SE) | 224; 94, 130 | 78 (77.2–78.9)d | 115 | 0.370 | 15 | 3.7 | 275 | ||
RIA | ND | ND | RIA | ILA | |||||||
6 | [39] | Toronto (CA) | 711; 403, 308 | 58e | 120 | 15 | 215 | 120 | |||
CPB | HPLC | CPB | |||||||||
7 | [22] | Dublin (IE) | 700; 210, 490 | 63–97 | 123 | 20 | 0.36 | 15 | 6.8 | 340 | |
MBA | FPIA | GC-MS | FPIA | MBA | MBA | ||||||
8 | [40] | Cardiff (GB) | 49 | <75 | 125 | 38 | 0.470 | 15 | |||
EIA | RIA | GC-MS | FPIA | ||||||||
9 | [41] | Sydney (AU) | 2963 | ≥50 | 125 ND | 15 ND | 6.8 ND | ||||
10 | [24] | Copenhagen (DK) | 98; 0, 98 | 41–75c | 130 | 50 | 0.280 | 11.9 | 115 (f) | ||
CLIA | ELISA | GC-MS | FPIA | ||||||||
11 | [42] | Saarland (DE) | 232; 79, 153 | 69 (19–102)f | 132 | 0.271 | 15 | 6.4 | 106 | ||
ND | ND | ND | ND | ||||||||
12 | [25] | Oxford (GB) | 195; 195, 0 | 44 (18–78)f | 135 | 50 | 0.280 | 12 | 6.8 | ||
London (GB) | EIA | ELISA | GC-MS | GC-MS | EIA | ||||||
13 | [43] | Finland (FI) | 2806; 1328, 1478 | 45–74c | 138 | ||||||
CLIA | |||||||||||
14 | [27] | Los Angeles (US) | 591; 345, 246 | >60 | 140 | 0.376 | 17.1 (m) 16.8 (f) | 5.7 | 133 (m) | ||
RA | HPLC | HPLC | RIA | 115 (f) | |||||||
15 | [44] | Perth (AU) | 299 | ≥75 | 140 | 15 | |||||
ND | ND | ||||||||||
16 | [45] | Tel-Hashomer (IL) | 1167 | ≥69 | 147 | 0.240 | 15 | 11 | 141 | ||
ELISA | HPLC | RIA | ELISA | ||||||||
17 | [14] | Strasbourg (FR) | 201; 57, 144 | 67±6g | 148 | 13 | 6.8 | 120 | |||
RIA | GC-MS | ND | |||||||||
18 | [46] | Florida (US) | 359; 0, 359 | 20–30c | 148 | 35 | 14 | ||||
RIA | RIA | HPLC | |||||||||
19 | [47] | Gothenburg (SE) | 209 | 76 (70–93)h | 148 | 0.340 | 16 | 8.6 | |||
RIA | GC-MS | HPLC | RIA | ||||||||
20 | [48] | Sacramento (US) | 1789; 751, 1038 | ≥60 | 148 | 35 | 0.350 | 13 | 363 | 124 (m) | |
RIA | RIA | LC-MS | HPLC | CLIA | 97 (f) | ||||||
21 | [49] | United States (US) | 1145 | ≥65 | 148 | 0.370 | 133 (m) | ||||
RIA | GC-MS | 115 (f) | |||||||||
22 | [1] | Granada (ES) | 218; 82, 136 | 79.2 (60–105)f | 148 | 45 | 0.300 | 12 | 6 | 397 | |
MEIA | RIA | GC-MS | FPIA | MEIA | MEIA | ||||||
23 | [5] | Granada (ES) | 218; 82, 136 | 65–90c | 148 | 35 | 0.300 | 13 | 15.9 | ≤396.4 | |
MEIA | RIA | GC-MS | FPIA | MEIA | MEIA | ||||||
24 | [50] | Boston (US) | 1458 | 70±0.32g | 148 | 0.210 | 131 (m) | ||||
RIA | GC-MS | 115 (f) | |||||||||
25 | [30] | Santiago de Chile (CL) | 491 | 65–67.9c | 148 | 7 | |||||
RIA | RIA | ||||||||||
26 | [51] | ND (US) | 255 | 28–82c | 148 | 0.376 | 12.2 | ||||
CLIA | GC-MS | FPIA | |||||||||
27 | [52] | Ontario (CA) | 75; 28, 47 | 80.7±1.5g | 148 | 13.3 | 370 | ||||
CLIA | FPIA | CLIA | |||||||||
28 | [19] | New York (US) | 548; 200, 348 | 77.5 (67–96)h | 148 | 0.376 | 21.3 | 5.9 | 124 (m) | ||
RIA | GC-MS | GC-MS | RIA | 106 (f) | |||||||
29 | [12] | Unites States (US) | 7233; 3689, 3544 | ≥3 | 148 | 0.370 | 9 | 6.8 | |||
RIA | GC-MS | FPIA | RIA | ||||||||
30 | [53] | Adelaide (AU) | 64; 64, 0 | 50–70c | 150 | 10 | 6.8 | 317 | |||
RIA | HPLC | RIA | RIA | ||||||||
31 | [54] | Lieto (FI) | 224; 92,132 | ≥65 | 150 | 37 | 0.450 | 19.0 | |||
CPB | RIA | GC-MS | FPIA | ||||||||
32 | [4] | Lausanne (CH) | 50; 0, 50 | 18–91c | 150 | 0.260 | 7 | 97 | |||
RIA | GC-MS | RIA | |||||||||
33 | [21] | Lund (SE) | 209; 91, 118 | 73±11g | 150 | 40 | 0.410 | 19.9 | 7 | 120 | |
TR-FIA | RIA | GC-MS | HPLC | RIA | |||||||
34 | [55] | Puna (IN) | 204; 169, 35 | 27–55c | 150 | 35 | 0.260 | 15 | 5 | 110 | |
RIA | RIA | GC-MS | HPLC | RIA | |||||||
35 | [56] | Boston (US) | 70; 70, 0 | 54–81c | 150 | 18.5 | 6.8 | ||||
RIA | HPLC | RIA | |||||||||
36 | [57] | Amsterdam (NL) | 1; 1, 0 | 51 | 150 | 18 | 6.5 | ||||
MEIA | FPIA | MEIA | |||||||||
37 | [58] | Busan (KR) | 184; 94, 90 | 61.6 (22–83)h | 150 | 35 | 12 | 6.8 | |||
MEIA | ND | FPIA | ND | ||||||||
38 | [59] | Leiden (NL) | 423 | ≥85 | 150 | 13.5 | 7 | ||||
DCSP | FPIA | DCSP | |||||||||
39 | [10] | Oxford (GB) | 1562; 618, 884 | ≥65 | 150 | 0.350 | 15 | 5 | 100 | ||
CPB | GC-MS | FPIA | MBA | ||||||||
40 | [29] | Great Britain (GB) | 1549 | ≥65 | 150 | 20 | 7 | ||||
CPB | FPIA | MBA | |||||||||
41 | [3] | Lieto (FI) | 1048 | 65–100c | 150 | 37 | ≥15 | ||||
CPB | RIA | FPIA | |||||||||
42 | [60] | Nijmegen (NL) | 105; 46, 59 | 76 (74–80)h | 150 | 0.320 | 19.9 | 90 (m) | |||
RIA | GC-MS | HPLC | 110 (f) | ||||||||
43 | [28] | Araihazar (BD) | 1650; 677, 973 | 20–65c | 151 | 11.4 (m) 10.4 (f) | 9 | ||||
RIA | HPLC | RIA | |||||||||
44 | [61] | Ibadan (NG) | 139; 61, 78 | 60–98c | 151 | ||||||
HPBC | |||||||||||
45 | [62] | India (IN) | 36 | 50.6 (16–80)h | 155.7 | ||||||
CLIA | |||||||||||
46 | [6] | Saarland (DE) | 545; 251, 294 | 57 (18–92)f | 156 | 35 | 0.271 | 12 | 7 | ||
CLIA | RIA | GC-MS | GC-MS | CLIA | |||||||
47 | [63] | Santiago (CL) | 108; 41, 67 | 74.4±3.7g | 165 | 14 | 6.8 | ||||
IAC | FPIA | IAC | |||||||||
48 | [64] | Oslo (NO) | 224 | 18–90c | 170 | 0.376 | 15.0 | 5 | 125 (m) | ||
RIA | CE | HPLC | RIA | 115 (f) | |||||||
49 | [65] | Uganda (UG) | 280 | >18 | 177 | ||||||
ND | |||||||||||
50 | [66] | Leiden (NL) | 185; 88, 97 | ≥65 | 184 | 124 | |||||
Glasgow (GB) | CLIA | CLIA | |||||||||
51 | [67] | Homburg (DE) | 228; 72, 156 | >65 | 196 | 29 | 0.280 | 14.1 | 11.1 | 106.1 (m) | |
CLIA | RIA | GC-MS | GC-MS | CLIA | 79.6 (f) | ||||||
52 | [68] | Aarhus (DK) | 937; 349, 588 | 72 (19–102)h | 200 | 40 | 0.280 | 11.9 | 350 | 133 (m) | |
CPB | RIA | GC-MS | FPIA | CPB | 120 (f) | ||||||
53 | [23] | Aarhus (DK) | 143; 51, 92 | 72 (24–90)h | 200 | 50 | 0.290 | 5 | 50 | 133 (m) | |
CPB | CPB | GC-MS | FPIA | CPB | 115 (f) | ||||||
54 | [69] | Gothenburg (SE) | 101; 35, 66 | 52 (18–80)h | 200 | 35 | 0.400 | 13 | |||
CPB | ND | GC-MS | HPLC | ||||||||
55 | [70] | Haukeland (NO) | 90; 69, 21 | 38–80c | 200 | 40 | 0.280 | ||||
EIA | ELISA | ND | |||||||||
56 | [71] | Oxford (GB) | 116; 43, 73 | 73e | 200 | 40 | 0.300 | 14 | 15 | 115 | |
ND | RIA | GC-MS | ND | ND | |||||||
57 | [72] | Oxford (GB) | 2403;986, 1417 | ≥65 | 200 | 0.45 | 124 (m) | ||||
CLIA | GC-MS | 97 (f) | |||||||||
58 | [13] | Norway (NO) | 6946; 3075, 3871 | (47–49 adults)c | 150;200;400 | 0.280 adults | 106 (m) | ||||
(71–74 elderly)c | MBA | 0.360 elderly | 87 (f) | ||||||||
GC-MS | |||||||||||
59 | [73] | Albuquerque (US) | 100; 50, 50 | 68–96c | 221 | 0.271 | 16.2 | 5 | |||
ND | ND | ND | ND | ||||||||
60 | [26] | Denver (US) | 152 | 65–99c | 221 | 0.376 | 21.6 | ||||
RA | GC-MS | GC-MS | |||||||||
61 | [20] | Seattle (US) | 315; 203, 112 | 65–100c | 221 | 0.271 | 13.9 | 7.3 | 109 | ||
ND | ND | ND | ND | ||||||||
62 | [74] | Mashhad (IR) | 244; 126, 154 | ≥65 | 243.5 | 15 | 14.7 | ||||
RIA | ELISA | RIA | |||||||||
63 | [75] | Georgia (US) | 103; 21, 82 | 76±8g (60–95)c | 258 | 0.271 | 13.9 | 6.8 | 295 | 127 | |
RIA | GC-MS | GC-MS | RIA | RIA | |||||||
64 | [76] | (US) and (ES) | 1350; 656, 694 | 65–90 | 258 | 15 | 14 | ||||
ND | CLIA | ND | |||||||||
65 | [77] | London (GB) | 421; 215, 206 | 66 (37–90)f | 258 | 0.271 | 14 | ||||
EIA | GC-MS | HPLc | |||||||||
66 | [78] | Baltimore (US) | 762; 0, 762 | ≥65 | 258 | 0.271 | 13.9 | 11.4 | 100 | ||
CPB | GC-MS | GC-MS | CPB | ||||||||
67 | [2] | Hanover (DE) | 178; 0, 178 | 60–70c | 258 | 0.271 | 12 | 7 | 320 | ||
CLIA | GC-MS | FPIA | CLIA | CLIA | |||||||
68 | [79] | Chicago (US) | 121 | ≥65 | 258 | 0.271 | |||||
CDI | GC-MS | ||||||||||
69 | [18] | Molise (IT) | 240; 161, 79 | 18–66 | 350 | 10 | 15 | 305 | |||
ILA | HPLC | ILA | ILA |
aISO 3166-1 country code elements; bSI conversion factors: To convert vitamin B12 (pg/mL) to pmol/L, multiply with 0.738; vitamin B12 and HoloTC to ng/L, multiply with 1.3554; folate to ng/mL, multiply with 2.266; MMA to mg/L, divide by 8.475; Hcy to mg/L, divide by 7.397; creatinine to g/dL, divide by 88.4; crange; dmean (95% CI); emean; fmean (range); gmean ± SD; hmedian (range). CDI, competitive displacement immunoassay; CE, capillary electrophoresis; CLIA, chemiluminescent immunometric assay; CPB, competitive protein binding; DCSP, dual count solid phase no boil-assay; EIA, enzyme immunoassays; ELISA, enzyme-linked immunosorbent assay; f, female; FPIA, fluorescence polarization immunoassay; GC-MS, gas chromatography-mass spectroscopy; HPLC, high pressure liquid chromatography; IAC, ion capture assay; ILA, immunoligand assay; LC-MS, liquid chromatography-mass spectroscopy; m, male; MBA, microbiological assay; MEIA; microparticle enzyme immunoassay; ND, there was no date or no specific method; RA, radiodilution assay; RIA, radioimmunoassay; TRFIA, time-resolved fluoroimmunoassay.
Nine studies involved a sample size ≤100 persons, 39 studies between 100 and 500, eight articles between 500 and 1000 and 13 with more than 1000 participants. The overall number of participants was 48,868 subjects. Forty-two studies specified the gender of the participants, 18 studies did not provide gender information and nine were only performed in one gender. In terms of age, 41 studies recruited subjects ≥60 years and 25 studies subjects ≤60 years. Three studies divided the population in different age groups [12, 13, 15] and only one depending on racial-ethnic group [12]. Forty studies were carried out in European countries, 20 in American countries, four in Asiatic countries, three in oceanic countries and two in Africa.
Vitamin B12
The current standard clinical screening test for vitamin B12 deficiency is the determination of total vitamin B12 concentration in plasma or serum. Some authors considered deficiency when vitamin B12 concentration was below the low limit of indeterminate interval (grey zone), and others did not specify if they took the low limit of this indeterminate interval or the low limit of the reference range.
Cut-offs employed to define vitamin B12 ranged from 100 pmol/L [16, 17] to 350 pmol/L [18]. Thirteen authors chose 150 pmol/L to establish vitamin B12 deficiency and 12 reported a value of 148 pmol/L. Therefore, 37% of authors used nearly the same value.
Lindenbaum et al. chose a serum vitamin B12 of 258 pmol/L as a cut-off value based on high serum MMA concentrations in subjects with vitamin B12 values below this number [19]. Wolters et al. chose a cut-off value of 258 pmol/L for their study population of 178 senior females (60–70 years) [2]. They admitted that it might be too high, because the MMA concentrations in subjects in the second quartile (227–≤269 pmol/L) of serum vitamin B12 were not significantly different from those in subjects in the third (270–≤330 pmol/L) and fourth quartiles (<331 pmol/L). Rajan et al. suggested 221 pmol/L was a more appropriate cut-off [20]. Others recommend to restructure the normal range and to raise the vitamin B12 cut-off point for elderly to a much higher value than 220–260 pmol/L [21]. However, recently, Valente et al. used the lower limit of the 95% central reference interval (123 pmol/L) as established from their reference population [22].
Holotranscobalamin
Cut-offs for HoloTC ranged between 20 and 50 pmol/L. The lower one was established by Valente et al. [22] by using FPIA measurement and the higher one was set by Hvas and Nexo [23], Bor et al. [24] and Llody-Wright et al. [25] using CPB and ELISA analytical methods, respectively. The principal methodology run was RIA assay (65%) and the corresponding cut-off range was between 35 and 40 pmol/L. Only 20 authors considered HoloTC in their studies.
Methylmalonic acid
A total of 44 articles included MMA as biomarker to assess vitamin B12 deficiency. Reported values ranged from 0.210 to 0.470 μmol/L. The majority of GC-MS users (77.3%) reported very close cut-offs: from 0.26 to 0.28 μmol/L. Only one author disclosed different MMA cut-off for adults and elderly [13].
Homocysteine
Hcy was measured in 53 studies and cut-off values ranged from 10 μmol/L [18] with HPLC to 21.6 μmol/L [26] with GC-MS. Only seven studies did not specify the analytical method used. FPIA was the preferred method (39%) to determine Hcy levels, followed by HPLC (32.6%) and GC-MS (21.7%). Fifteen studies set Hcy cut-off at 15 μmol/L and this value was the most commonly used. Two studies divided samples by gender, reporting 17.1 μmol/L and 16.8 μmol/L [27], and 11.4 μmol/L and 10.4 μmol/L for males and females, respectively [28].
sFolate
This variable was measured in 40 studies and the method mostly used for analysis was RIA (53%). The most common cut-off used was 6.8 nmol/L (22.5% of studies), followed by 7 nmol/L (17.5%), although values ranged from 3.7 nmol/L (RIA) to 15.9 nmol/L (MEIA).
RBC folate
Only 14 articles (20.6%) included RBC folate measurement. All studies used different cut-off concentrations which ranged between 124 nmol/L (CLIA) and 397 nmol/L (MEIA). The most common methodology was CLIA.
Creatinine
Creatinine values were separated by gender in 13 articles, reporting a cut-off range from 90 μmol/L to 141 μmol/L for males and 79.6 μmol/L to 120 μmol/L for females. Fifteen articles did not separate data by gender, giving a cut-off interval from 97 μmol/L to 141 μmol/L. The majority of studies have considered 120 μmol/L or 124 μmol/L as high reference range limit for males and 115 μmol/L for females.
Discussion
A major problem when comparing vitamin B12 deficiency prevalence across studies consisted on the variability of cut-off values used. Several factors can be addressed in this context. The first one was the method used to establish cut-offs values since means, medians, two or three SD or percentiles and the analytical methods used to measure biomarkers were diverse. Another issue was the application of values obtained from younger subjects to the elderly. All these factors contributed to scattered cut-off values. This is the first review to group information on cut-off values for vitamin B12 deficiency from 1992 to 2014, taking into account age and analytical detection methods.
Regarding the literature, the prevalence of vitamin B12 deficiency in British elderly ranged from 5% (age 65–74 years) to 10% (age >75 years) with 150 pmol/L as a cut-off [29]. Sanchez et al. found 12% B12 deficiency using 148 pmol/L and 25.4% with cut-off value of <221 pmol/L [30]. Meanwhile, Palacios et al. [5] found 17.4% (≤148 pmol/L) and Loikas et al. [3] observed 6.1% considering 150 pmol/L as cut-off. Two extreme B12 cut-offs were recorded: the lowest one (123 pmol/L) was used by Valente et al. and observed 8% of B12 deficiency [22]. However, Zapacosta et al. using the highest value (350 pmol/L) found that only 16.3% of the Italian adults presented an adequate vitamin B12 concentration [18]. A detailed study on vitamin B12 status was carried out by Vogiatzoglou et al.; using 150 pmol/L as cut-off, they found a prevalence of 0.4% for adults and 1.7% for elderly [13].
Only four authors used vitamin B12 as a single marker, while the rest of them made a combination of markers, as already proposed by Valente et al. [22] and Palacios et al. [5]. The biomarker most included was Hcy (78%) followed by MMA (65%), sFolate (59%), HoloTC (30%) and RBC folate (21%). The small number of studies including RBC folate was unexpected, as erythrocyte folate is considered a more precise physiological marker of folate concentrations than sFolate. This may be due to the preparation of samples for RBC folate measurement which requires more laborious steps than for sFolate. Additionally, both folate markers should be measured to assess if there is a mixed deficiency between folate and vitamin B12 [1]. However, MMA measurement is important to distinguish between vitamin B12 and folate deficiency. Elevated MMA is a specific marker of vitamin B12 deficiency while Hcy rises in both vitamin B12 and folate deficiencies [2, 31].
The majority of studies did not take into account age or gender of participants when they applied the cut-offs values. For example, none of the studies modified the cut-offs for vitamin B12 depending on age and only two for MMA. Vogiatzoglu et al. [13] set different cut-off values for middle-ages (47–49 years; 0.280 μmol/L) and elderly (71–74 years; 0.360 μmol/L) due to different creatinine and vitamin B12 concentrations. In the same way, Clarke et al. took in consideration both age and renal function to adapt cut-offs because their study was carried out in elderly [10]. In the elderly, impaired renal function could be an important confounding factor for Hcy and MMA [32]. For this reason, we have included in the Table 1 the creatinine cut-off values when available. In the same way, none of the studies included different cut-offs for Hcy depending on age. Only Carmel et al. [27] and Gamble et al. [28] reported values for males (m) and females (f) separately: 17.1 μmol/L (m) and 16.8 μmol/L (f), and 11.4 μmol/L (m) and 0.4 μmol/L (f). Age and gender did not seem to influence the choice of cut-offs values for both sFolate and RBC folate.
MMA was predominantly measured using GC-MS methods. This method is expensive and not available in all the routine laboratories. Therefore, Palacios et al. proposed an algorithm to differentiate vitamin B12 from folate deficiency with no need for MMA measurement [5].
Countries that currently mandate folic acid fortification as well as individuals under multivitamin supplementation may require specific reference values for the diagnosis of vitamin B12 deficiency [33]. Vitamin B12 supplements or food fortification should be taken into account in countries that use this kind of supplementation. A study by Sanchez et al. showed that a daily dose of 1.4 ug of vitamin B12 was not sufficient to improve vitamin B12 status [30]. Supplementation with higher doses of vitamin B12 (>500 ug/day) have proven effective in Latino populations residing in USA [34] and institutionalized Spanish elderly increased significantly from 308.4 pmol/L to 558.3 pmol/L (p<0.001) [35]. Also, Favrat et al. showed that oral vitamin B12 treatment normalized the metabolic markers [4]. However, this response did not persist for an additional 3 months following cessation of therapy. These discrepancies may be the result of employing the same cut-offs for diagnosing vitamin B12 deficiency in populations exposed to different basal levels of vitamin B12 and folate. Vitamin B12 is not the only vitamin affected since folate is also added to food in some countries. In these countries with food folate fortification or populations taking folic acid supplements, the upper reference limit for Hcy is usually 20%–25% lower than in non-fortified populations [36]. Clarke et al. suggested as ultimate gold standard for vitamin B12 deficiency the reduction in increased Hcy or MMA concentrations and improvement in clinical symptoms or signs in response to vitamin B12 treatment [10].
Limitations of this review comprise the lack of information on preanalytical procedures, analytical performance and diagnostic accuracy, even if these factors may have a deep impact on cut-off values choice. Unfortunately, the majority of articles did not specify these kinds of data. Therefore, we were not able to include them in our review.
Conclusions
There is no consensus in the literature about cut-off points for blood vitamin B12 reference ranges and its associated metabolites. In most of the studies, age and gender were not taken into account when measuring any of these biomarkers, nor the use or consumption of fortified food. Published data in the literature regarding deficiency percentage should be considered with caution. For future studies on vitamin B12 status, it would be necessary to establish different reference cut-offs according to age and gender, considering the analytical methods used.
About the authors
Raquel Aparicio Ugarriza, born in 1989 in Alicante (Spain), studied Physical Activity and Sport Sciences from 2007 to 2012 at the University of Elche. She has a Master degree in Education and a Master Degree in Health and Sport Performance, both at the same university. Currently, she is doing a PhD at the Technical University of Madrid and she is assistant researcher and member of the Imfine Research Group. She is interested to research the relationship between physical activity and biomarkers (i.e., vitamin B12 deficiency, vitamin D, folate, homocysteine) and stress oxidative parameters in relationship to ageing, fitness and health.
Gonzalo Palacios, born in Madrid (Spain) in 1960, completed a PhD in Biochemistry at the University of Geneva, Switzerland. He returned to Madrid to work for more than 15 years at Abbott Diagnostics, where he had responsibilities on analytical and clinical evaluations of in vitro diagnostics products, mainly for metabolism, hematology and cancer diseases. At present, he is a Senior Researcher at the Faculty of Physical Activity and Sport Sciences, Technical University of Madrid, working on pathophysiological biomarkers related to fitness, physical activity and aging.
Monika Alder Novotni, born in 1977 in Zürich (Switzerland) studied Nutrition Science from 1999 to 2007 in Bonn (Germany). Her practical diploma thesis was completed in Madrid (Spain). She is now working as a Product Manager for Laboratory Products in an International company in Chur (Switzerland).
Marcela González-Gross, PhD in Pharmacy and Master Degree in Nutrition. Full Professor for Sports Nutrition and Exercise Physiology at the Department of Health and Human Performance, Technical University of Madrid, Spain. She is the Head of the Nutrition, exercise and healthy lifestyle research group (ImFINE). For more than 20 years she has been analyzing the nutritional and fitness status and its impact on both physical and cognitive function of healthy subjects with different levels of physical activity, especially adolescents and the elderly. Her main research aspects include early diagnosis of subclinical vitamin deficiency, optimal hydration, improvement of life quality and health education. She has received several research awards and published over 180 articles in JCR journals.
Acknowledgments
We would like to thank Prof. Klaus Pietrzik, Institute of Nutrition and Food Science, University of Bonn for constructive discussion and assistance.
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Financial support: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
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