Holo-transcobalamin is an indicator of vitamin B-12 absorption in
healthy adults with adequate vitamin B-12 status1–3
Kristina M von Castel-Roberts, Anne Louise Morkbak, Ebba Nexo, Claire A Edgemon, David R Maneval,
Jonathan J Shuster, John F Valentine, Gail PA Kauwell, and Lynn B Bailey
ABSTRACT
Background: It has been hypothesized that the response of holo-
transcobalamin (holo-TC) to oral vitamin B-12 may be used to assess
absorption. To develop a reliable clinical absorption test that uses
holo-TC, it is necessary to determine the optimal timeline for vitamin
B-12 administration and postdose assessment.
Objective: The objective of this study was to assess the magnitude
and patterns of change in the postabsorption response of holo-TC to
oral vitamin B-12.
Design: Adult (18 – 49 y) male and female participants (n ҃ 21) with
normal vitamin B-12 status were given three 9-␮g doses of vitamin
B-12 at 6-h intervals beginning early morning (baseline) on day 1.
Blood was drawn at 17 timed intervals over the course of 3 d for the
analysis of holo-TC and other indicators of vitamin B-12 status.
Results: Mean holo-TC increased significantly (P 쏝 0.001) from
baseline at 6 h (11%) and 24 h (50%). TC saturation increased
significantly (P 쏝 0.001) from baseline at 12.5 h (33%) and 24 h
(50%). The mean cobalamin concentration changed significantly
(P 쏝 0.001) from baseline at 24 h (15%) and 48 h (14%). The ratio
of holo-TC to cobalamin increased significantly (P 쏝 0.001) at
24 h (32%).
Conclusions: The greatest increase in holo-TC was observed 24 h
after ingestion of three 9-␮g doses of vitamin B-12. Our results
indicate that a vitamin B-12 absorption test based on measurement of
holo-TC after administration of three 9-␮g doses of vitamin B-12
should run for 24 h. Am J Clin Nutr 2007;85:1057– 61.
KEY WORDS Transcobalamin, holo-transcobalamin, holo-
TC, vitamin B-12, vitamin B-12 absorption, healthy adults
INTRODUCTION
Vitamin B-12 is an essential nutrient functioning as a coen-
zyme for methionine synthase and methylmalonyl CoA mutase.
Circulating vitamin B-12 is bound to 1 of 2 carrier proteins,
haptocorrin (HC) or transcobalamin (TC). Although the majority
of vitamin B-12 (앒80%) is bound to HC (holo-HC), only TC-
bound vitamin B-12 (holo-TC) can be taken up by body cells (1).
TC has a half-life of 앒18 h and is sensitive to changes in vitamin
B-12 intake (2). Newly ingested vitamin B-12, as holo-TC, can
first be detected in the blood 3 h after intake with a maximum
plasma concentration occurring at 8 –12 h. Once in circulation,
holo-TC is taken up into cells within minutes (2, 3).
Am J Clin Nutr 2007;85:1057– 61. Printed in USA. © 2007 American Society for Nutrition
Depletion of total body cobalamin occurs slowly and is often
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a result of malabsorption, which is difficult to diagnose clinically
(4 –7). It is important, however, to detect and treat vitamin B-12
deficiency in the early stages before significant damage occurs.
Untreated deficiency may lead to neurologic damage and an
increased risk of birth defect–affected pregnancies even when
the deficiency is only moderate (8 –11). Pernicious anemia, the
specific vitamin B-12 deficiency condition caused by a lack of
intrinsic factor (IF), may result from an autoimmune response to
IF or gastric parietal cells, atrophy of the gastric mucosa, chronic
gastritis, and, in rare cases, a congenital defect. Currently, the
only available diagnostic tests for pernicious anemia are not
clinically practical. The Schilling test, which involves ingestion
of radioactively labeled vitamin B-12, a flushing dose of nonla-
beled vitamin B-12, and collection of urine over a period of 24 h,
requires meticulous adherence to protocol, making it error prone
and costly (12–14). Presence of parietal cell and IF antibodies can
be measured to diagnose pernicious anemia; however, parietal
cell antibodies can occur in other autoimmune diseases, and both
tests are only clinically meaningful in a subgroup of patients with
autoimmune conditions (15, 16). It has been hypothesized that
changes in holo-TC in response to a supplemental dose of vita-
min B-12 may be used to assess vitamin B-12 absorption (17–
19). Bor et al (18) reported a significant increase in holo-TC and
TC saturation 24 and 48 h after receiving three 9-␮g doses of oral
vitamin B-12. Because no blood was collected before 24 h (after
baseline), the magnitude and pattern of change of holo-TC during
the first 24 h could not be determined (18). In developing a
clinical diagnostic test, it is important to know the optimal time
after the dose at which to draw blood. The objective of this study
was to evaluate the postabsorption response of holo-TC to oral
vitamin B-12 relative to other indicators of vitamin B-12 status.
1
From the Departments of Food Science and Human Nutrition (KMvCR,
CAE, DRM, GPAK, and LBB), Health Policy and Epidemiology (JJS), and
Medicine (JFV), the General Clinical Research Center (JJS), University of
Florida, Gainesville, and the Department of Clinical Biochemistry, Nørre-
brogade, Aarhus Sygehus, Aarhus, Denmark (ALM and EN).
2
Supported by a grant from the National Center for Research Resources,
National Institutes of Health (M01 RR00082) and by a grant from the Shands
General Clinical Research Center (7591).
3
Address reprint requests to LB Bailey, University of Florida, Food Sci-
ence and Human Nutrition Department, Building 475, Gainesville, FL
32611. E-mail: lbbailey@ifas.ufl.edu.
Received June 28, 2006.
Accepted for publication November 17, 2006.
1057
1058 CASTEL-ROBERTS ET AL
FIGURE 1. Timeline of the intervention protocol.
SUBJECTS AND METHODS
Subjects
Twenty-one healthy adult men (n ҃ 13) and women (n ҃ 8)
aged 18 – 49 y from the Gainesville, FL, community were se-
lected on the basis of the following inclusion criteria: 1) serum
vitamin B-12 concentration 쏜350 pmol/L at the time of screen-
ing, 2) no use of vitamin B-12– containing supplements or vita-
min B-12 injections during the previous year, 3) no use of to-
bacco products, 4) no history of chronic disease, 5) no pregnancy
or lactation, 6) no anemia [hemoglobin 욷11 g/dL (7.4 mmol/L),
women; 욷12 g/dL (8.1 mmol/L), men], 7) normal blood chem-
istry profile, 8) body mass index (in kg/m2) between 18 and 29,
and 9) no blood donations within 30 d of the study.
Study design
All participants signed an informed consent form approved by
the University of Florida Institutional Review Board before the
initiation of the study. Participants had a fasting blood sample
drawn at the University of Florida Shands General Clinical Re-
search Center (GCRC). Subjects’ heights and weights were mea-
sured, and a medical history questionnaire was completed. Blood
analyses included serum vitamin B-12, blood chemistry profile,
hematologic indexes, and a pregnancy test for women. The pri-
mary objective of measuring vitamin B-12 in the screening pro-
cess was to ensure that no enrolled subjects had a severe vitamin
B-12 deficiency.
Eligible subjects were admitted to the GCRC the evening
before (day 0) the intervention. The following morning (day 1)
after an overnight fast, an indwelling catheter was inserted for all
blood collections during day 1. A total of 17 timed blood draws
were taken from day 1 to day 3, and three 9-␮g doses of vitamin
B-12 were administered at 6-h intervals on day 1, beginning after
the baseline blood draw (Figure 1). Immediately after taking
each vitamin B-12 dose, subjects consumed a piece of bread and
236 mL (8 oz) juice to improve absorption efficiency (20). In
addition to the bread and juice consumed with each vitamin B-12
dose, subjects were given a midmorning snack 2 h after and lunch
3.5 h after dose 1. Dinner was fed 4 h after dose 2, and an evening
snack was 3 h after dose 3. The Recommended Dietary Allow-
ance for vitamin B-12 was provided in the diet on day 1 and on
day 2. Take-home meals were provided on day 2 of the study.
Water and noncaffeinated, noncaloric beverages were allowed
ad libitum. Subjects remained in the GCRC overnight and were
allowed to leave on pass after a fasting blood sample the morning
of day 2. Subjects returned on the morning of day 3 at which time
they had the final fasting blood sample drawn.
Biochemical analysis
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At each blood collection, holo-TC, total TC, cobalamin, and
plasma albumin were measured. The ratios of holo-TC concen-
tration to total-TC concentration (TC saturation) and holo-TC
concentration to cobalamin concentration (holo-TC:cobalamin)
were determined to assess changes in these indicators in relation
to one another. Baseline concentrations of methylmalonic acid,
creatinine, serum folate, and homocysteine were also measured.
The vitamin B-12 supplement (9 ␮g cyanocobalamin) was pre-
pared by Westlab Pharmacy (Gainesville, FL). The vitamin B-12
content of the supplement was validated by an independent lab-
oratory (Analytic Research Laboratories, Oklahoma City, OK).
Blood samples were collected and stored in a freezer at Ҁ80 °C
before analysis. Serum vitamin B-12 and folate were assayed on
the Advia Centaur automated immunoassay system (Bayer, Ter-
rytown, NY) with a total imprecision 쏝10%.
Total-TC concentration was determined by a sandwich
enzyme-linked immunoabsorbent assay with a total imprecision
of 4 – 6% (intraassay imprecision: 앒 3%) (21). After removal of
the apolipoprotein TC with vitamin B-12– coated beads, holo-TC
was measured by the TC enzyme-linked immunoabsorbent as-
say. The total imprecision for measurement of holo-TC was
앒8% (22), and the intraassay imprecision was 앒4% (23). Albu-
min and creatinine were measured with a Cobas Integra 800
(Roche Diagnostics, Indianapolis, IN). Total imprecision was
앒2% for albumin and 쏝3% for creatinine.
Total homocysteine was measured by the immunologic
method on the IMMULITE 2000 (Diagnostic Products Corpo-
ration, Los Angeles, CA) (total imprecision: 쏝6%) (24), and
methylmalonic acid was measured by slightly modified stable-
isotope dilution capillary gas chromatography–mass spectrom-
etry (total imprecision: 쏝8%) (25).
Statistical methods
For each dependent variable of interest (eg, cobalamin), a
linear model was fitted with independent categorical variables,
subject (random effects) and time (fixed effects). The overall P
value for time was obtained by the F test that tests the null
hypothesis that the distribution of the dependent variable was the
same at all time points. Tukey’s method (26) of multiple com-
parisons was used for assessment of differences between time
periods. A least significant difference, as defined by Tukey’s
procedure, ensures that simultaneously every target population
paired difference in means will be within 앐 least significant
difference of the corresponding difference in sample means with
95% confidence.
 HOLO-TRANSCOBALAMIN AND VITAMIN B-12 ABSORPTION 1059
TABLE 1 TABLE 2
Values of vitamin B-12–related indicators at baseline1 Concentrations of measured indicators unadjusted for albumin at
scheduled intervals1
Variable Value Reference interval
Variable
Holo-TC (pmol/L) 85 앐 38 (41–208)2 40–1503
Cobalamin (pmol/L) 407 앐 118 (241–710) 200–600 Time from baseline (h) Holo-TC Cobalamin Total TC
TC saturation (%) 0.12 (0.05–0.27) 0.05–0.203
Holo-TC:cobalamin (%) 0.22 (0.08–0.44) 0.15–0.513 pmol/L
Hcy (␮mol/L) 6.6 앐 1.4 (3.9–9.3) 4.5–11.94 0.0 85 앐 38 407 앐 118 712 앐 135
MMA (␮mol/L) 0.134 앐 0.060 (0.08–0.32) 0.08–0.284 0.5 84 앐 38 395 앐 113 688 앐 134
Folate (nmol/L) 32.7 앐 7.3 (22.2–54.4) 쏜6.0 1.5 85 앐 39 397 앐 109 706 앐 135
Creatinine (␮mol/L) 69 앐 11.7 (48–87) 50–100 2.5 89 앐 43 409 앐 114 723 앐 136
1
Holo-TC, holo-transcobalamin; Hcy, homocysteine; MMA, methyl- 3.5 91 앐 43 421 앐 113 743 앐 138
malonic acid. 4.5 96 앐 43 431 앐 126 746 앐 147
2
x៮ 앐 SD; range in parentheses (all such values). 5.5 97 앐 41 414 앐 109 763 앐 1442
3
From Nexo et al (22). 6.0 99 앐 452 423 앐 117 752 앐 141

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4
From Rasmussen et al (27). 7.0 97 앐 42 426 앐 117 755 앐 128
8.0 95 앐 41 424 앐 102 773 앐 1392
9.0 96 앐 38 428 앐 102 772 앐 1442
RESULTS 10.0 96 앐 38 432 앐 116 757 앐 143
11.0 97 앐 39 423 앐 114 738 앐 154
Mean baseline values for all analytes were within normal
11.5 99 앐 41 429 앐 112 739 앐 136
ranges (Table 1) (22, 27). Some subjects had values that were 12.5 100 앐 392 411 앐 107 739 앐 139
somewhat outside the normal range; however, none of the sub- 24 124 앐 462,3 456 앐 1102 715 앐 129
jects were severely vitamin B-12 deficient. Measurement of vi- 48 102 앐 372 456 앐 1152 758 앐 145
tamin B-12 at screening was done with the use of a different assay
All values are x៮ 앐 SD. Values are not reported as a ratio to albumin.
1
from the one used at baseline of the study and likely explains the
Holo-TC, holo-transcobalamin; TC, transcobalamin.
variation rather than a true change in vitamin B-12 status. Plasma 2
Significantly different from baseline, P 쏝 0.001 (ANOVA, Tukey’s test).
albumin fluctuated throughout the intervention period, suggest- 3
Significantly different from all other values, P 쏝 0.001 (ANOVA,
ing a change in hydration status throughout day 1 and among the Tukey’s test).
mornings of days 1, 2, and 3 (data not shown). Holo-TC, cobal-
amin, and total-TC values are reported as a ratio to albumin to
adjust for diurnal changes in overall body protein concentration
resulting from changes in hydration status. Unadjusted (for al- 24 h (Figure 2). As observed with holo-TC concentration, the
bumin) means and statistical differences for holo-TC, cobal- mean TC saturation and percentage change at 24 h were signif-
amin, and TC saturation are reported in Table 2. All time points icantly greater than at all other time points with 48% and 15%
are reported relative to baseline. Of all of the analytes, only increases from baseline and 12.5 h, respectively (Figure 4).
holo-TC and TC saturation changed significantly on day 1. Among all subjects, the percentage change from baseline ranged
Mean holo-TC concentration increased steadily after baseline from 7% to 104% with 19 of 21 subjects having a value of 욷22%.
and fluctuated throughout day 1. Statistically significant in- The ratio of holo-TC to cobalamin did not increase significantly
creases were observed in mean holo-TC during the first 24 h of until 24 h with absolute and percentage increases of 0.15% and
the intervention, although these small increases were not main- 32%, respectively. The range for percentage change in this ratio
tained. Mean holo-TC concentration reached a maximum value among all subjects was Ҁ7% to 109%; 15 of 21 subjects had an
at 24, h which was a significant increase relative to baseline and increase of 욷23% at 24 h.
all other time points (Figure 2). The mean percentage increase
from baseline was also greater at 24 h than at all other time points
with a 49% increase relative to baseline and a 29% increase DISCUSSION
relative to 12 h (Figure 3). This peak at 24 h was observed for In this intervention study, the changes in markers of vitamin
almost all subjects, with an increase of 욷22% (22– 85%) for all B-12 status were measured hourly during and after administra-
but 1 subject. By 48 h, mean holo-TC concentration decreased tion of three 9-␮g doses of oral vitamin B-12. In previous studies,
significantly relative to 24 h (33%); however, it was still signif- the changes in response to similar vitamin B-12 doses were
icantly greater than baseline (Figure 2). measured after 24 h; however, no data were collected before that
Mean serum cobalamin concentration did not increase signif- time point (17–19). The data from the present study indicate that
icantly relative to baseline on day 1, although there were fluctu- a series of three 9-␮g doses of oral vitamin B-12, given over 12 h,
ations in concentrations throughout the day. At 24 h mean serum led to small fluctuations in holo-TC concentration during the first
cobalamin concentration was significantly greater than baseline study day followed by the previously observed maximum in-
(Figure 2). Overall, the percentage change in cobalamin concentra- crease in holo-TC concentration 24 h after the first vitamin B-12
tion was smaller than for holo-TC throughout the intervention pe- dose was given. A similarity was observed in the overall pattern
riod with ranges of Ҁ2% to 15% and Ҁ1% to 50%, respectively. of change in holo-TC, cobalamin, and TC saturation, with a
Mean total-TC concentration did not change significantly dur- gradual increase over the first day with the most pronounced
ing the study, varying 쏝6% from baseline at all time points (data increase 24 h after the initial vitamin B-12 dose and 13 h after the
not shown). Mean TC saturation began to increase significantly final vitamin B-12 dose. Because no measurements were taken
relative to baseline at 12.5 h, with the most significant increase at between 12.5 and 24 h, we cannot unequivocally conclude that
1060 CASTEL-ROBERTS ET AL
FIGURE 2. Mean holo-transcobalamin (TC) concentrations (n ҃ 21),
cobalamin concentrations (n ҃ 21) relative to albumin, and TC saturation
(n ҃ 21) at scheduled intervals after oral vitamin B-12 (B12) intake. Holo-TC
increased significantly from baseline at 6 –7 and 11– 48 h (P 쏝 0.001) and
significantly from all other time points at 24 h (P 쏝 0.001). Cobalamin
increased significantly from baseline at 24 h (P 쏝 0.001). TC saturation
increased significantly from baseline at 12.5– 48 h and from all other time
points at 24 h (P 쏝 0.001; ANOVA, Tukey’s test). Error bars represent least
significant differences.
the true maximum increase in concentration for all markers oc-
curred at 24 h. The timing of vitamin B-12 absorption and me-
tabolism may explain the pattern of change observed in holo-TC
concentration during the first 12 h of the intervention. After
ingesting vitamin B-12, an increase in holo-TC is first measur-
able in the blood at 3– 4 h, and holo-TC can be taken up by cells
within minutes (2). It is hypothesized that until cells are saturated
with holo-TC, most of it will be taken up so quickly that no large
changes would be observed initially in the blood. When intake is
sufficient to saturate the cells with vitamin B-12, significant
changes in holo-TC can then be measured. One potential limita-
tion of the current protocol is that a person with a low vitamin
B-12 concentration because of dietary deficiency alone may re-
quire more vitamin B-12 to saturate tissues before significant
changes in holo-TC can be measured. Future investigations
should compare the response to this same intervention protocol
in persons with vitamin B-12 deficiency but no problems with
vitamin B-12 absorption with persons with adequate vitamin
B-12 status.
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FIGURE 3. Mean percentage changes in holo-transcobalamin (holo-TC;
■) and cobalamin (F) relative to albumin at scheduled intervals after oral
vitamin B-12 (B12) intake (n ҃ 21). The increases in holo-TC and cobalamin
from baseline to 24 h were significantly larger than the changes at all other
time points (P 쏝 0.001; ANOVA, Tukey’s test). Error bars represent least
significant differences.
The absolute and percentage increases in cobalamin concen-
tration were smaller, occurred later, and were maintained longer
than those for holo-TC. This finding is not surprising because
total serum cobalamin primarily consists of holo-HC, and the
slower rate of HC metabolism relative to TC metabolism leads to
a slower overall turnover of serum cobalamin and a slower re-
sponse to changes in intake (1, 28, 29). When comparing these 2
measures among the individual subjects, holo-TC had the most
consistent pattern with only 1 subject not having a change of
욷20% at 24 h. In addition the mean percentage change at 24 h
was 3 times that of cobalamin. Holo-TC is clearly a more sensi-
tive indicator of change in vitamin B-12 intake and absorption
than is serum cobalamin because it increased earlier after sup-
plementation, increased relatively more, and decreased earlier
after supplementation ceased.
FIGURE 4. Mean percentage changes in transcobalamin (TC) saturation
(
) and in the ratio of holo-transcobalamin to cobalamin (Œ) at scheduled
intervals after oral vitamin B-12 (B12) intake (n ҃ 21). A significantly larger
percentage increase was observed in TC saturation and in the ratio of holo-
transcobalamin to cobalamin at 24 h than at all other time points compared
with baseline (P 쏝 0.001; ANOVA, Tukey’s test). Error bars represent least
significant differences.
 HOLO-TRANSCOBALAMIN AND VITAMIN B-12 ABSORPTION
Total TC did not change significantly during the intervention
period. TC saturation increased in a similar manner to holo-TC
(Figure 4). Both holo-TC concentration and TC saturation had
comparable results even when considering individual subjects.
Of all subjects, 95% and 90% had increases of 쏜22% at 24 h for
holo-TC and TC saturation, respectively. In a previous study, a
larger change in TC saturation (at 24 h) than for holo-TC was
observed which was due to a drop in total TC at this time point
(18). No such conclusion can be made from these data because no
significant difference was observed in total TC at any time point.
Because TC saturation is a calculated rather than a direct mea-
sure, the potential error in this value is greater than that for
holo-TC. Therefore, holo-TC may be the better indicator to use.
This is the first study to monitor hourly changes in holo-TC in
response to oral intake of vitamin B-12. The most significant
change in holo-TC occurred at 24 h, indicating that this is the
optimal time after dose at which to measure holo-TC. The three
9-␮g vitamin dose sequence used in this study has previously
been chosen to minimize passive absorption and to maximize the
amount of actively absorbed vitamin B-12 (17, 18). This aspect
of the protocol would be important in a clinical vitamin B-12
absorption test, because it is the capacity to actively absorb vi-
tamin B-12 that is being assessed. Further studies evaluating the
necessity of 3 doses and the exact timing of the doses are war-
ranted. In addition, it is possible that the peak in holo-TC con-
centration at 24 h could be due to a decrease in cellular uptake at
this time point. The TC receptor could undergo a similar refrac-
tory period as is observed in IF, so that fewer receptors are
available for a certain time after being saturated with holo-TC,
resulting in an increase of holo-TC remaining in circulation.
Future studies focusing on the capacity of TC receptor to take up
holo-TC might help identify whether this is indeed occurring;
however, note that even if the peak at 24 h was due to a reduction
in cellular uptake, if significant changes in holo-TC do not occur
in the malabsorbers as was reported, the current intervention
protocol would still be useful in diagnosing a vitamin B-12 mal-
absorption condition (17, 18).
In conclusion, holo-TC increases measurably in response to ad-
ministration of oral vitamin B-12 within 6 h with a maximum peak
at 24 h after an overnight period. Our results indicate that a vitamin
B-12 absorption test based on measurement of holo-TC after 3 doses
of 9 ␮g oral vitamin B-12 should run for 24 h.
KMvCR designed the experiment, collected and prepared the samples,
analyzed the data, and wrote the manuscript. ALM and EN designed the
experiment, analyzed the sample, interpreted the data, and wrote the manu-
script. CAE supervised the clinical protocol, designed the clinical diet, and
edited the manuscript. DRM managed the overall laboratory operations,
sample collection, and preparation. JJS performed the statistical analyses and
wrote the manuscript. JFV performed the physical examination and super-
vised the subjects. GPAK designed the clinical diet and edited the manu-
script. LBB designed the experiment, interpreted the data, and wrote and
edited the manuscript. None of the authors had a conflict of interest.
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