nutrients

Article
14 C-Cobalamin Absorption from Endogenously
Labeled Chicken Eggs Assessed in Humans Using
Accelerator Mass Spectrometry
Marjorie G. Garrod 1 , Heidi A. Rossow 2 , Christopher C. Calvert 2 , Joshua W. Miller 3 ,
Ralph Green 4 , Bruce A. Buchholz 5 and Lindsay H. Allen 1, *
1 USDA, ARS Western Human Nutrition Research Center, Davis, CA 95616, USA
2 Population Health and Reproduction, University of California, Davis, CA 95616, USA
3 Department of Nutritional Sciences, Rutgers University, New Brunswick, NJ 08901, USA
4 Department. of Medical Pathology and Laboratory Medicine, University of California, Davis, Sacramento,
CA 95817, USA
5 Center for Accelerator Mass Spectrometry, Lawrence Livermore National Laboratory, Livermore,
CA 94550, USA
* Correspondence: Lindsay.allen@ars.usda.gov; Tel.: +1-530-752-5268




Received: 4 August 2019; Accepted: 4 September 2019; Published: 8 September 2019


Abstract: Traditionally, the bioavailability of vitamin B-12 (B12) from in vivo labeled foods was
determined by labeling the vitamin with radiocobalt (57 Co, 58 Co or 60 Co). This required use of
penetrating radioactivity and sometimes used higher doses of B12 than the physiological limit of B12
absorption. The aim of this study was to determine the bioavailability and absorbed B12 from chicken
eggs endogenously labeled with 14 C-B12 using accelerator mass spectrometry (AMS). 14 C-B12 was
injected intramuscularly into hens to produce eggs enriched in vivo with the 14 C labeled vitamin.
The eggs, which provided 1.4 to 2.6 µg of B12 (~1.1 kBq) per serving, were scrambled, cooked and fed
to 10 human volunteers. Baseline and post-ingestion blood, urine and stool samples were collected
over a one-week period and assessed for 14 C-B12 content using AMS. Bioavailability ranged from 13.2
to 57.7% (mean 30.2 ± 16.4%). Difference among subjects was explained by dose of B12, with percent
bioavailability from 2.6 µg only half that from 1.4 µg. The total amount of B12 absorbed was limited
to 0.5–0.8 µg (mean 0.55 ± 0.19 µg B12) and was relatively unaffected by the amount consumed.
The use of 14 C-B12 offers the only currently available method for quantifying B12 absorption in
humans, including food cobalamin absorption. An egg is confirmed as a good source of B12, supplying
approximately 20% of the average adult daily requirement (RDA for adults = 2.4 µg/day).

Keywords: cobalamin; vitamin B12; bioavailability; eggs; endogenous label; human; accelerator
mass spectrometry

1. Introduction
Access to methods for quantifying the absorption of vitamin B12 (B12) from foods is important in
setting dietary requirements for the vitamin, and for assessing food cobalamin malabsorption which
can cause B12 depletion and deficiency, especially in the elderly. No methods are currently available
for quantifying B12 absorption from foods in humans [1]. The only method for detecting clinical B12
malabsorption, the CobaSorb test, is qualitative and does not provide data on the % of B12 absorbed [2].
The only natural dietary sources of B12 are animal products. However, B12 in animal source foods
is bound to protein and its bioavailability from these foods depends on factors such as the complexity
of different food protein structures and the body’s ability to release B12 from food proteins in the
stomach [3,4]. In the Framingham Study [5], strong associations were observed between plasma B12

Nutrients 2019, 11, 2148; doi:10.3390/nu11092148 www.mdpi.com/journal/nutrients

Nutrients 2019, 11, 2148 2 of 15

concentrations and intake of the vitamin from dairy products, eggs, meat, and seafood, but the data
suggested differences in the relative bioavailability of B12 from these sources.
In the past, to determine the bioavailability of B12 from foods animals were injected with or fed
radiocobalt (57 Co, 58 Co or 60 Co) labeled B12, which became incorporated into the liver, meat, or eggs
of the animal. These endogenously labeled tissues were then fed to humans and the amount of labeled
B12 appearing in plasma, urine, and stool was measured to determine bioavailability [6–15]. Gamma
ray counters used to measure radiocobalt isotopes measure the decay of the radiosubstrates rather
than the actual amount present in a sample. Consequently, limitations in sensitivity and precision
required the use of radiosubstrates with relatively high specific activity and often a dose of labeled
B12 an order of magnitude larger than the limit of B12 absorption through the physiologic, intrinsic
factor-mediated pathway (i.e., ≈2–3 µg from a single meal) [8,9,16,17].
Accelerator mass spectrometry (AMS), a highly sensitive technology for the detection and
quantification of 14 C-labeled substrates at attomole (10−18 ) concentrations [18], directly counts 14 C
atoms rather than radioactive decay events [18] and is approximately one million-fold more sensitive
than scintillation counting. The synthesis of 14 C-labeled B12 (14 C-B12) [19] provides a means to utilize
AMS technology in B12 bioavailability studies in humans. AMS typically requires samples containing
only 0.5 mg of carbon, enabling analysis in microliter-sized biological samples even when the dose
consists of a small amount of substrate with low specific activity [20]. Absorption studies can be carried
out with minimal radiation exposure for the participant. Finally, the long half-life of 14 C (5730 y)
enables sample collection over a longer period of time, which increases the accuracy of the study [21].
The aim of the study was to determine in humans the bioavailability and absorbed B12 from
chicken eggs endogenously labeled with 14 C-B12 using AMS. The yolk of a single large raw egg
contains up to 20% of the Recommended Dietary Allowance (RDA) of 2.4 µg [22]. Hens readily deposit
large amounts of labeled B12 in egg yolk when injected with the vitamin [23,24], potentially producing
eggs with sufficient 14 C-B12 label for use in bioavailability and absorption studies.

2. Materials and Methods

2.1. 14 C-B12 Synthesis

14 C-B12
was synthesized by S. enterica (serovar Typhimurium) strain TT24733, genotype
cbiD24::MudJ. The bacteria were grown aerobically on ethanolamine and incubated with
dicyanocobinamide and 14 C-dimethylbenzimidazole (DMB), which under these growth conditions
are dedicated precursors for the biosynthesis of B12 [19]. After a 24 h to 48 h incubation, the newly
synthesized 14 C-B12 was extracted and purified by high performance liquid chromatography
(HPLC). The product was confirmed as B12 by ultraviolet-visible spectrophotometry and liquid
chromatography-mass spectrometry [19]. The synthesis incorporates a cyanation step to convert all
forms of B12 to cyanocobalamin with the 14 C label located as shown in Figure 1.

2.2. Production of Labeled Eggs

All experimental procedures involving the use of animals were conducted in accordance with the
Universities Federation for Animal Welfare Handbook on the Care and Management of Laboratory
Animals [25] and approved by the Animal Use and Care Committee at the University of California,
Davis (#13036). Four White Leghorn laying hens aged 1.5 y were housed in individual laying cages
in the University of California, Davis, Avian Science Unit. Temperature was maintained at 24–25 ◦ C.
The 24-h light cycle was 20 h light and 4 h dark with light fixtures present on both ceiling and walls.
Air exchange was continuous. Hens received a commercial diet for laying hens (Layena, 5.5 µg B12 per
pound; Purina Mills, St. Louis, MO, USA) and water ad libitum.
Over a period of 4 d, three individual hens received a total injected dose in the thigh of 13.0,
14.8, or 17.8 kBq (350, 400, or 480 nCi) 14 C-B12 corresponding to 8.3 µg, 9.4 µg, and 11.3 µg total
B12, respectively. The dosing regimen was determined with the aid of a mathematical model for
respectively. The dosing regimen was determined with the aid of a mathematical model for
distribution of an intramuscular dose of B12 to other tissues and accumulation in eggs, which is
detailed in Appendix B. The fourth hen was used as the control and received no injections. Injections
were given at 09:00. Eggs were collected daily from each hen for a month, producing a total of 35
eggs. Eggs produced during the first 16 days were used for the feeding study. Total enrichment of
Nutrients 2019, 11, 2148 3 of 15
eggs with 14C-B12 was determined by counting samples to 1–2% precision in a Liquid Scintillation
Counter (Tri-Carb 2500 TR, Model 2700, Packard Instrument Company, Downers Grove, IL, USA).
Total B12 inofeggs
distribution was determined
an intramuscular dosebyof Covance
B12 to other Laboratories,
tissues andInc. (Madison, in
accumulation WI, USA)
eggs, using
which is
turbidometry to compare
detailed in Appendix B. Thethe growth
fourth response
hen was used asof the
a sample
control utilizing the no
and received bacterium
injections.Lactobacillus
Injections
delbrueckii
were givenagainst
at 09:00. the growth
Eggs wereresponse
collectedto a B12
daily fromstandard
each hen (coefficient of variation:
for a month, producing 9.91%)
a total[26].
of 35 eggs.
In the Metabolic Kitchen of the USDA, ARS Western Human Nutrition
Eggs produced during the first 16 days were used for the feeding study. Total enrichment Research Center, eggs
of eggs
were 14
with pooled
C-B12 from each hen, mixed
was determined with a samples
by counting hand blender,
to 1–2% portioned
precisioninto
in aservings, cooked in covered
Liquid Scintillation Counter
plastic containers by immersion in boiling water until the egg reached at
(Tri-Carb 2500 TR, Model 2700, Packard Instrument Company, Downers Grove, IL, USA). Total B12 least 71 °C, and stored at
−20 °C until used in the human bioavailability studies. Total B12 ranged
in eggs was determined by Covance Laboratories, Inc. (Madison, WI, USA) using turbidometry tofrom 1.82 to 2.65 μg/100 g
cooked
compareegg. the Each
growth serving contained
response approximately
of a sample utilizing the80 gbacterium
of egg, 1.1 kBq C-B12,
14
Lactobacillus and 1.44against
delbrueckii to 2.65 the
μg
total B12. The specific activity of the servings of cooked eggs
growth response to a B12 standard (coefficient of variation: 9.91%) [26]. varied from 429 to 781 Bq/μg B12 since
the 14C-B12 content varied amongst the eggs.

Figure 1.
Figure Chemical structure
1. Chemical structure of
of cyanocobalamin
cyanocobalamin with the 14
with the 14CC location
location indicated
indicated by
by the
the ** in
in the
the DMB
DMB
moiety. Image
moiety. Image modified
modified from
from Green
Green and
and Miller
Miller [27].
[27].

In the Metabolic
2.3. Human Kitchen
Subjects, Dosing andof the USDA,
Sample ARS Western Human Nutrition Research Center, eggs
Collection
were pooled from each hen, mixed with a hand blender, portioned into servings, cooked in covered
A correlation coefficient power calculation was completed to estimate the population sample
plastic containers by immersion in boiling water until the egg reached at least 71 ◦ C, and stored at
size ◦sufficient to detect a significant correlation between individual B12 bioavailability as
−20 C until used in the human bioavailability studies. Total B12 ranged from 1.82 to 2.65 µg/100 g
determined by the fecal excretion method. For this calculation, we made14the following assumptions
cooked egg. Each serving contained approximately 80 g of egg, 1.1 kBq C-B12, and 1.44 to 2.65 µg
based on previous studies in the literature: A null hypothesis correlation coefficient = 0; an
total B12. The specific activity of the servings of cooked eggs varied from 429 to 781 Bq/µg B12 since
alternative hypothesis correlation coefficient = 0.8; a significance level of p = 0.05; a power = 0.8; and a
the 14 C-B12 content varied amongst the eggs.

2.3. Human Subjects, Dosing and Sample Collection

A correlation coefficient power calculation was completed to estimate the population sample
size sufficient to detect a significant correlation between individual B12 bioavailability as determined
by the fecal excretion method. For this calculation, we made the following assumptions based on
previous studies in the literature: A null hypothesis correlation coefficient = 0; an alternative hypothesis
correlation coefficient = 0.8; a significance level of p = 0.05; a power = 0.8; and a one-sided analysis.
Nutrients 2019, 11, 2148 4 of 15

Based on these assumptions, the required sample size was calculated to be 8 subjects. A total of 12
subjects started the study and 10 subjects completed it.
Subject recruitment and study procedures were approved by the Human Subjects Review
Committees at the University of California, Davis (#260218-1) and Lawrence Livermore National
Laboratory (LLNL) (#08-103), and written informed consent was obtained from all participants.
The study is registered on ClinicalTrials.gov (#NCT01366937). The recruiting, enrollment and study
completion flow is described in Appendix A, Figure A1 Inclusion criteria included good overall
health based on pre-study survey self-report, adequate B12 status and assumed aborptive capacity as
determined by serum B12 assay above the cutoff for deficiency (>148 pmol/L), and the availability to
complete the protocol. Exclusion criteria included any chronic health disorder, anemia of any kind,
renal insufficiency, and pregnancy or lactation. The radiation dose from ingestion of 14 C-B12 was
calculated to be about 0.030 mSv, equivalent to the exposure during a 6 h commercial airline flight.
Human volunteers were each fed a single serving of the scrambled 14 C-B12 labeled egg (Table 1)
with dry toast and water. Individual doses ranged from 777 to 1158 Bq with total B12 per dose ranging
from 1.4 to 2.6 µg depending on the eggs used (Table 1). The adequacy of this dose was determined in
a pilot study in which a 2180 Bq dose of crystalline 14 C-B12 (1.5 µg B12) in water was given to one
subject and produced a very strong signal to background ratio in plasma, urine, and stool samples [19].
Upon analysis of the plasma samples of the first subject to complete the study (S2), subsequent doses
were adjusted to contain 1.1 kBq of 14 C. The total dose of B12 varied due the specific activity varying
between eggs and consequently servings.

Table 1. Study subjects and dosing.

Serum B12 14 C-B12 Specific

Subject Age (y) 1 BMI 2 (kg/m2 ) B12 Dose Dose Activity
Sex
(pmol/L) (µg) (kBq) (Bq/µg)
S1 29 F 24.4 331 2.38 1.114 468
S2 43 M 28.2 441 1.66 0.777 468
S3 29 M 24.5 358 2.65 1.159 437
S4 24 F 21.2 323 2.65 1.158 437
S5 53 F 20.8 365 1.43 1.089 761
S6 25 M 23.9 371 1.40 1.083 774
S7 24 M 24.5 306 1.40 1.093 781
S8 26 M 23.8 325 1.44 1.089 756
S9 22 F 35.1 227 2.64 1.131 428
S10 24 M 23.1 386 2.64 1.131 428
1 F = female, M = male. 2 BMI = Body mass index.

Blood samples were taken at baseline, hourly through 12 h, at 15 h and 24 h, then daily at the
time of day the dose was given for an additional 7 d. Twenty-four hour urine and stool samples were
collected before dosing followed by collection of all post-dosing voids for 8 d. Meals were controlled
to provide no foods of animal origin or containing B12 during the first 24 h. Subjects returned to their
normal diet for the remainder of the study.
Stool collections were homogenized in order to obtain representative samples [28]. A stool
sample ≤ 350 g was placed in a 3.8 L paint can and an equivalent weight of water added. Fifty grams
of 8 mm 316 stainless steel balls was added to the can and the lid sealed with duct tape. The can was
shaken on a commercial paint shaker for 30 min, aliquots of 1.5 mL and 50 mL were retained and
the remaining stool discarded. The 1.5 mL tube was sent to LLNL for AMS analysis of 14 C content.
The 50 mL sample was retained for possible future analysis. Each sample was processed in a new
can with new stainless steel balls to prevent cross-contamination between samples. Aliquots of stool
homogenate, urine and plasma were promptly frozen and then shipped overnight to LLNL for graphite
preparation and AMS analyses.
Nutrients 2019, 11, 2148 5 of 15

2.4. Analysis of 14 C
Samples were prepared as graphite for measurement using standard procedures [29,30].
Since the carbon concentrations can vary widely, carbon concentrations of all urine samples and
stool homogenates were measured at LLNL using an Exeter Analytical CE440 carbon analyzer
(North Chelmsford, MA, USA) as described [28]. All 14 C AMS measurements were conducted on the
1-MV National Electrostatics Corporation (Middleton, WI) AMS system at LLNL and normalized to
four identically prepared IAEA C-6 isotopic standards [28,29,31]. An isotopic fractionation correction
of δ13 C = −25 per mil was used for all samples and results were reported as described previously [28].
All AMS data are reported as mean ±1 standard deviation.

2.5. Calculation of % Bioavailability

Normally, % bioavailability would be determined based on the difference between intake and
fecal excretion. However, we found that the percent of the dose of radioactivity excreted in urine is
1–2 orders of magnitude higher than that previously reported when the vitamin was labeled with
radioactive cobalt. This phenomenon was first observed by Carkeet et al. [19] who used a similar
dose of 14 C-B12 given in aqueous form. In that study, it was determined that the majority (>99%) of
radioactivity in the urine from the aqueous dose was not intact B12, indicating that the majority of 14 C
in the urine may be a degradation product of B12 that was absorbed, and not a bioavailable fraction of
the oral dose. Based on this observation % bioavailability was calculated using the sum of both urinary
and fecal excretion of 14 C:
14 C intake − [14 C excretion in feces + urine]
% Bioavailability = 14 C intake
× 100%.

2.6. Statistics
Data were examined for normality using the Shapiro–Wilk test. The alpha level was set at 0.05,
and a p-value > 0.05 is consistent with a normally distributed population. Mean % bioavailability
values in subjects receiving lower (1.43–1.66 µg) and higher (2.38–2.65 µg) doses of B12 were compared
using Student’s t-test. Both the Shapiro–Wilk test and the Student’s t-test were conducted using
Microsoft Excel.

3. Results

3.1. Appearance of 14 C Label in Eggs

In the Edwards study [23], peak enrichment was noted in eggs produced 5–7 d after dosing when
% dose incorporated ranged from 9.6–10.3%. In the present study, the nCi concentration and range
of enrichment in peak eggs was similar to that observed by Edwards [23]. Peak enrichment in each
injection protocol was achieved in eggs produced on d 6–8 in all hens and ranged from 7.1 to 12.2% of
the total dose given (Figure A4). The initial delay in enrichment is due to the time (approximately 24 h)
that the egg remains in the ovary, where the B12 is incorporated into the yolk. Each hen incorporated
a different percentage of the total dose into the eggs produced over the initial 16-d time period; 48%,
56%, and 39% for doses of 13.0, 14.8, and 17.8 kBq, respectively. The efficiency of B12 incorporation
into the eggs enables endogenous 14 C-B12 labeling in normal sized servings (Figure 2).
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Figure 2. Time course of 14C-B12 enrichment of study eggs. The series indicate the 14C-B12 doses
Figure 2. Time course of 14 C-B12 enrichment of study eggs. The series indicate the 14 C-B12 doses
received by different hens.14
received
Figure 2.byTime
different hens.
course of C-B12 enrichment of study eggs. The series indicate the 14C-B12 doses
received by different
14
hens.
3.2. Appearance
3.2. Appearance of
of C
C Label
Label in
14
in Plasma, Uurine, and
Plasma, Uurine, and Feces
Feces
1414C first appeared
C first appeared
3.2. Appearance in in
of 14C Label in plasma
plasma approximately
approximately
Plasma, Uurine, and 4Feces 4 hingestion
h after after ingestion of theegg
of the labeled labeled egg in all
in all volunteers
volunteers
although although
peak appeared peak plasma
plasma concentrations 14
concentrations of
of C varied among
14C varied among participants (Figure 3). Most
participants (Figure 3). Most excess
14C first in plasma approximately 4 h after ingestion of the labeled eggplasma
in all
14excess
C was plasma
cleared
14C was cleared by 72 h although a low-level elevation above baseline remained
by 72 h although a low-level elevation above baseline remained throughout the
volunteers although peak plasma concentrations14of C varied among participants (Figure 3). Most
14
throughout
collection the14collection
period. Plasma peak 14 C Plasma
period. peak (Cmax)
concentration C concentration
ranged from(Cmax)
17.4 ranged
to 60.3 fromplasma
Bq/L 17.4 towith
60.3
excess plasma C was cleared by 72 h although a low-level elevation above baseline remained
Bq/L
meanplasma
athroughout± SDthe withvalue
peak a mean ± SD
of period.
34.9 peakmBq/mL.
± 12.0 value of 1434.9 ± area
12.0under
mBq/mL. Plasma areafor under the curve
collection Plasma peakPlasma C concentration the curve
(Cmax) (AUC)
ranged from0–24
17.4h ranged
to 60.3
(AUC) for
from to 0–24 h ranged fromwith
250 atomean
649 Bq·h/Lofplasma with a mean AUCarea of 404 ± 106theBq·h/L.
Bq/L 250plasma 649 Bq·h/L
with a meanplasma± SD peak valueAUC
of 34.9 404 ± 106
± 12.0 Bq·h/L.
mBq/mL. Plasma
Plasma under
area under the curve
curve
Plasma
(AUC) area
for under
0–168 h the
rangedcurve (AUC)
from 657 for
to 0–168
1785 h ranged
Bq·h/L plasmafrom 657
with ato 1785
mean Bq·h/L
AUC plasma
of 1294 ± with
327 a mean
Bq·h/L.
(AUC) for 0–24 h ranged from 250 to 649 Bq·h/L plasma with a mean AUC of 404 ± 106 Bq·h/L.
AUC
The of
plasma1294 ± 327 Bq·h/L. The plasma AUC for all study subjects is given in Table 2.
Plasma areaAUCunderfortheallcurve
study(AUC)
subjectsforis0–168
givenhinranged
Table 2.from 657 to 1785 Bq·h/L plasma with a mean
AUC of 1294 ± 327 Bq·h/L. The plasma AUC for all study subjects is given in Table 2.

Figure3.3. Time
Figure Time course of 1414C-B12 in plasma after ingestion of in vivo
course of vivo labeled
labeled egg
egg (A)
(A) for
for the
the entire
entire
study,
study,and
and(B)(B)for
forthe
theinitial
initial 24
24h.
h. The
The error
error bars
bars for
for individual
individual data
data points
points are
are 11 SD
SD and
and generally
generally
Figure 3. Time course of 14C-B12 in plasma after ingestion of in vivo labeled egg (A) for the entire
smaller
smallerthan
thanthe
thesymbol.
symbol.
study, and (B) for the initial 24 h. The error bars for individual data points are 1 SD and generally
smaller than the symbol.
Nutrients 2019, 11, 2148 7 of 15

Nutrients 2019, 10, x FOR PEER REVIEW 7 of 15

Table 2. Plasma absorption and clearance parameters for study subjects S1–S10.

Table B12 14 C-B12 and clearance

2. Plasma absorption Cmax parameters
Tmax for studyPlasma
subjects S1–S10. Plasma
Dose B12 Dose PlasmaTmax Plasma 1 AUC 1 0–168 h
Subject 14C-B12 Cmax PlasmaAUC 0–24 h
Plasma
(µg) Dose
Subject Dose
(kBq) Plasma
(Bq/L)Plasma (h)AUC 1 0–24 h(Bq·h/L)
AUC 1 0–168 h(Bq·h/L)
(μg) (kBq) (Bq/L) (h) (Bq•h/L) (Bq•h/L))
S1 2.38 1.114 21.9 12 323 940
S2 S1 1.66 2.38 1.114
0.777 21.917.4 12 15 323 250 940 657
S3 S2 2.65 1.66 0.777
1.159 17.460.3 15 9 250 649 657 1745
S4 S3 2.65 2.65 1.159
1.158 60.334.9 9 7 649 386 1745 1241
S5 S4 1.43 2.65 1.089
1.158 34.932.5 7 11 386 410 1241 1649
S6 S5 1.40 1.43 1.083
1.089 32.537.2 11 8 410 363 1649 1185
S7 S6 1.40 1.40 1.093
1.083 37.236.2 8 8 363 357 1185 1291
S8 S7 1.44 1.40 1.089
1.093 36.240.2 8 8 357 417 1291 1336
S9 S8 2.64 1.44 1.131
1.089 40.232.6 8 8 417 399 1336 1308
S10 S9 2.64 2.64 1.131
1.131 32.637.6 8 8 399 483 1308 1585
S10 2.64 1.131 1 AUC = Area under
37.6 8 the curve. 483 1585
1 AUC = Area under the curve.
In all volunteers, the majority of 14 C label appeared in the first two 24 h urine collections
In all volunteers, the majority of 14C label appeared in the first two 24 h urine collections (Figure
(Figure 4). However, the amount of label recovered in the first 48 h varied widely among the subjects
4). However, the amount of label recovered in the first 48 h varied widely among the subjects with
with the cumulative % dose recovered in urine ranging from 5.1 to 62.0% (mean 26.2 ± 18.9%).
the cumulative % dose recovered in urine ranging from 5.1 to 62.0% (mean 26.2 ± 18.9%). 14C
14 C concentrations in urine returned to near background values by the fifth 24 h urine collection (120 h).
concentrations in urine returned to near background values by the fifth 24 h urine collection (120 h).
The majority of fecal 14 C label appeared in the stool by Day 4. However, as with the urine, the amount
The majority of fecal 14C label appeared in the stool by Day144. However, as with the urine, the
of label recovered varied widely among the subjects (Figure 5). C concentration in stool returned to
amount of label recovered varied widely among the subjects (Figure 5). 14C concentration in stool
background concentrations by the fifth 24 h stool collection (120 h). The cumulative % dose recovered
returned to background concentrations by the fifth 24 h stool collection (120 h). The cumulative %
in feces ranged from 24.7 to 74.1% with a median 34.2%. The total (urine + stool) excreted 14 C ranged
dose recovered in feces ranged from 24.7 to 74.1% with a median 34.2%. The total (urine + stool)
from 43.2 to 86.7% with a mean of 69.9 ± 16.4% (Figure 6).
excreted 14C ranged from 43.2 to 86.7% with a mean of 69.9 ± 16.4% (Figure 6).

Figure 4. Time course of % dose recovered in urine after ingestion of in vivo labeled egg.
Figure 4. Time course of % dose recovered in urine after ingestion of in vivo labeled egg.
Nutrients 2019, 10, x FOR PEER REVIEW 8 of 15
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Figure 5. Time
Figure course
5. Time of of
course %% dose
doserecovered
recovered in stoolafter
in stool afteringestion
ingestionof of
in in vivo
vivo labeled
labeled egg.egg.
Figure 5. Time course of % dose recovered in stool after ingestion of in vivo labeled egg.

Figure 6. Time course of total % dose excreted after ingestion of in vivo labeled egg.
Figure 6. Time course of total % dose excreted after ingestion of in vivo labeled egg.
Figure 6. Bioavailability
3.3. Calculation of Time course of total % dose excreted after ingestion of in vivo labeled egg.
3.3. Calculation of Bioavailability
The % of
3.3. Calculation bioavailability in the 10 volunteers, calculated using the sum of the fecal and urinary 14 C
Bioavailability
excretion, ranged from 13.2
The % bioavailability in theto 10
57.7% with a mean
volunteers, of 30.2 using
calculated ± 16.4% the(psum
= 0.19, Shapiro–Wilk
of the test for 14C
fecal and urinary
The %ranged
excretion, bioavailability
normality). Mean in to
the57.7%
10 volunteers,
% bioavailability
from 13.2 values
with in calculated
subjects
a mean 30.2using
ofreceiving thedoses
low
± 16.4% sum of theShapiro–Wilk
(p = (1.43–1.66
0.19, fecal
µg)and urinary
of total B12 14C
test for
and in ranged
excretion,
normality). subjects
Mean % receiving
from 13.2high
to doses
57.7%
bioavailability (2.38–2.65
values µg) of total
withina subjects
mean B12 ±were
ofreceiving
30.2 significantly
16.4%
low (p = 0.19,
doses different (40.3
Shapiro–Wilk
(1.43–1.66 ± 17.8%
μg) of testB12
total for
and vs. 20.1 ±
normality).
in subjects 5.4%,
Mean %respectively;
bioavailability
receiving = 0.04,
highpdoses Student’s
values t-test)
in subjects
(2.38–2.65 μg) of(Figure
total7A,C).
receivingB12lowThe
were total
doses amount
(1.43–1.66
significantly of B12
μg)absorbed
of total
different B12
(40.3 ±
and retained was consequently very similar, ≈0.5–0.8 µg for most subjects (mean = 0.55 ± 0.19 µg;
17.8% vs. 20.1 ± 5.4%, respectively; p = 0.04, Student’s t-test) (Figure 7A,C). The total amount of B12±
and in subjects receiving high doses (2.38–2.65 μg) of total B12 were significantly different (40.3
17.8% 0.78,
p =vs. 20.1Shapiro–Wilk test for normality),
± 5.4%, respectively; p = 0.04, across the doses administered
Student’s (Figure 7B,D).
absorbed and retained was consequently very similar,t-test) (Figure
≈0.5–0.8 μg for7A,C).
mostThe total amount
subjects (mean = of B12
0.55 ±
absorbed and retained was consequently very similar, ≈0.5–0.8 μg for most
0.19 μg; p = 0.78, Shapiro–Wilk test for normality), across the doses administered (Figure 7B,D). subjects (mean = 0.55 ±
0.19 μg; p = 0.78, Shapiro–Wilk test for normality), across the doses administered (Figure 7B,D).
 excretion, ranged from 13.2 to 57.7% with a mean of 30.2 ± 16.4% (p = 0.19, Shapiro–Wilk test for
normality). Mean % bioavailability values in subjects receiving low doses (1.43–1.66 μg) of total B12
and in subjects receiving high doses (2.38–2.65 μg) of total B12 were significantly different (40.3 ±
17.8% vs. 20.1 ± 5.4%, respectively; p = 0.04, Student’s t-test) (Figure 7A,C). The total amount of B12
Nutrients
absorbed2019,
Nutrients and
2019, 2148
11,11,
retained
2148 9 of9 15
was consequently very similar, ≈0.5–0.8 μg for most subjects (mean = 0.55 ±of 15
0.19 μg; p = 0.78, Shapiro–Wilk test for normality), across the doses administered (Figure 7B,D).

Nutrients 2019, 10, x FOR PEER REVIEW 9 of 15

Figure
Figure
Figure Relationship
7. 7.
7. Relationship
Relationship between
between
between bioavailability
bioavailability
bioavailability and
andandtotal dosedose
dose
total of B12.
of B12. (A)Percent
(A)
of B12. Percent bioavailability
bioavailability
(A) Percent values
values
bioavailability
for subjects
forvalues
subjects S1–S10.(B)
forS1–S10.
subjects (B)The
S1–S10.Therelationship
relationship
(B) betweenbetween
between
The relationship absorbed
absorbed dose
doseand
absorbed and total
total
dose dose
anddose ofofB12.
total B12.The
dose The
of symbols
B12.symbols
The
for the
forsymbols subjects
the subjects follow
follow
for the subjects the convention
thefollow
convention of earlier
of earlierof
the convention figures.
figures. (C)
earlier (C) Mean
Mean
figures. % bioavailability
(C)%Mean
bioavailability values in subjects
valuesvalues
% bioavailability in subjects
in
receiving
receiving
subjects lowerdoses
lower
receiving doses
lowerof total
of dosesB12
total of in
B12 inthe
totaltheeggs
B12 in(1.43–1.66
eggs (1.43–1.66
the eggsµg, n = 5)
n and
(1.43–1.66
µg, 5)inn
=μg, those
and receiving
= 5)inand
those higher
receiving
in those doses
higher
receiving
of total
doses
higher B12 in
of total
doses ofthe
B12 ineggs
totalthe (2.38–2.65
B12eggs (2.38–2.65
in the eggs 5) were
n =µg;
µg; (2.38–2.65 n =μg;compared
5) were
n withcompared
= 5) compared
were Student’s
with witht-testStudent’s
(p = 0.04).
Student’s (D)=(p
t-testt-test
(p The
0.04).
=
absorbed
(D)0.04). dose
The absorbed of B12 was
dose of B12
(D) The absorbed similar
dosewas between
similar
of B12 low
wasbetween and high administered
low and low
similar between highand doses
administered when compared
doses when
high administered doses using
compared
when
Student’s
using Student’s
compared t-test (pStudent’s
t-test
using 0.69).
= (p = 0.69).
t-test (p = 0.69).
4. Discussion
4. 4.
Discussion
Discussion
The use of 14 C-labeled B12 in this study allowed for a much lower dose of radioactive label for
The use 14 C-labeled
ofof 14C-labeledB12
The use B12in in this
this study
study allowed
allowed for foraamuch
muchlower lowerdose doseofofradioactive
radioactive label
label forfor
determining bioavailability, compared to past studies using radiolabeled cobalt [8]. For example,
determining
determiningbioavailability,
bioavailability,comparedcompared to to past
past studies using radiolabeled
studies using radiolabeledcobalt cobalt[8].[8].ForFor example,
example,
Doscherholmen et al. used doses as high as 18.5 kBq of 57 Co labeled B12 (half-life ~270 d) to determine
Doscherholmen
Doscherholmen et al.
et used
al. useddosesdosesas high as
as high 18.5as kBq
18.5ofkBq57 Cooflabeled B12 (half-life ~270
57Co labeled B12 (half-life ~270 d) to
d) to determine
B12 bioavailability from scrambled eggs in human subjects [6]. In the present study, a 1.11 kBq dose of
B12determine
14bioavailability from scrambled
B12 bioavailability
C labeled B12 (half-life ~5700 y)eggs was in
from scrambled human eggssubjects
sufficient
in human
to raise the[6].concentration
In the present
subjects
ofstudy,
[6]. In the
C fara enough
14present 1.11 kBqabove
study, adose
1.11 of
14 C
kBq dose of
labeled B12for
14C labeled B12 (half-life ~5700 y) was sufficient to raise the concentration
(half-life 14 of 14C far
background accurate~5700 detectiony) was sufficient
by AMS in all to raise thefluids
biological concentration
analyzed. of C far enough
In addition, 57 Co emitsabove
enough above
background for background
accurate for accurate
detection by AMS detection
in all by AMSfluids
biological in allanalyzed.
biologicalInfluids
addition, 57 Co emits
analyzed. In
penetrating high energy gamma radiation which is far more damaging than the low energy beta
addition,
penetrating
57Co emits penetrating high energy gamma radiation which is far more damaging than the
high energy
radiation emitted by C.gamma radiation which is far more damaging than the low energy beta
14
low energy
radiation beta byradiation
14 C. emitted by 14C.
Theemitted
bioavailability of B12 from eggs in this study (mean = 30.2%) is similar to the 27.5% reported
The bioavailability of B12 from eggs in this study (mean = 30.2%) is similar to the 27.5%
The bioavailability
by Doscherholmen et al. when of B12 from eggs in thiswas
bioavailability study (mean based
calculated = 30.2%) is similar
on intake minusto the
fecal27.5% reported
excretion of
reported
57 Co-B12 by Doscherholmen et al. when bioavailability was calculated based on intake minus fecal
by Doscherholmenlabeled et
in al.
the when
corrin bioavailability
ring [6]. Active was calculated
absorption of B12 based
from on
the intake minus
gastrointestinal fecal
tractexcretion
involves of
excretion of 57Co-B12 labeled in the corrin ring [6]. Active absorption of B12 from the gastrointestinal
57 Co-B12
a complex mechanism
labeled that requires
in the corrin ring [6].the gastricabsorption
Active productionofofB12 intrinsic
from the factor (IF). While thetract
gastrointestinal capacity of
involves
tract involves a complex mechanism that requires the gastric production of intrinsic factor (IF).
the IF-dependent
a complex mechanism pathway is limited
that requires the [16],
gastrictheproduction
binding specificity
of intrinsic of IF is very
factor high
(IF). Whileandthe requires
capacity an of
While the capacity of the IF-dependent pathway is limited [16], the binding specificity of IF is very
intact
thehigh B12 molecule,
IF-dependent pathwaylimiting the absorption of analogues.
and requires an intactisB12 limited
molecule,[16],limiting
the binding specificityofof
the absorption IF is very high and requires an
analogues.
intact B12 Previous reports
molecule, indicated
limiting the that urinaryofexcretion
absorption analogues. of an oral dose of intact B12 with radiocobalt
Previous reports indicated that urinary excretion of an oral dose of intact B12 with radiocobalt
label is extremely
Previous reports low—far
indicatedless than
thatthan1% of theexcretion
urinary dose givenofunless
an it isdose
oral followed by aB12 largewith
intramuscular
label is extremely low—far less 1% of the dose given unlessofitintact
is followed byradiocobalt
a large
flushing
label is dose
extremely as in the
low—far Schilling
less than test
1% [16].
of In
the this
dose study,
given no flushing
unless it is dose
followed was administered,
by a large yet 5 to
intramuscular
intramuscular flushing14dose as in the Schilling test [16]. In this study, no flushing 14 dose was
62% (mean 25%) of the C dose was found in 14 urine. In parallel work using an oral dose of C-B12 in
flushing dose asyet
administered, in the5 to Schilling
62% (mean test [16].
25%) of In
thethis C study,
dose was no found
flushing dose was
in urine. administered,
In parallel work using yetan5 to
water, the amount of isotope
14 C dose appearing in urine without a flushing dose administered was similar to
62%oral(mean
dose25%) of the in
of 14C-B12 water, was found inofurine.
the amount isotopeIn parallel
appearing work usingwithout
in urine an oral dose of 14 C-B12
a flushing dose in
water, the amount
administered wasofsimilar
isotopetoappearing
that observed in urine without
in this study,a and
flushing
HPLC dose administered
fractionation wasthat
verified similarthe to
that observed in
radioactivity in this
urine study,
was notandintact
HPLCC-B12
14 fractionation verified that
[19]. It is assumed the radioactivity
that this is also the casein urine
with thewas samenot
label found in the urine of the current egg study volunteers. Therefore, in calculating bioavailability
in this study, it was assumed that the substantial amount of urinary 14C detected was not originally
absorbed as intact 14C-B12.
The mean bioavailability from doses of 1.4 to 2.6 μg of B12 was 30%. A large egg contains
approximately 0.6 μg of the vitamin [32], all in the yolk—a lower amount than in our study because
Nutrients 2019, 11, 2148 10 of 15

intact 14 C-B12 [19]. It is assumed that this is also the case with the same label found in the urine of the
current egg study volunteers. Therefore, in calculating bioavailability in this study, it was assumed
that the substantial amount of urinary 14 C detected was not originally absorbed as intact 14 C-B12.
The mean bioavailability from doses of 1.4 to 2.6 µg of B12 was 30%. A large egg contains
approximately 0.6 µg of the vitamin [32], all in the yolk—a lower amount than in our study because
injecting the chickens with B12 increased the total amount of B12 in the eggs that we used. Mean
bioavailability from 1.44 µg B12 in egg (the equivalent of 2 to 3 normal eggs) in this study was closer to
50% in the 4 subjects given this dose. Because bioavailability decreased with increasing B12 intake our
overall estimate of 30% bioavailability may substantially underestimate the actual bioavailability from
a single egg.
Most of the wide range in % bioavailability among the 10 human subjects (13.2 to 57.7%) was
explained by the fact that different amounts of total B12 were given to the volunteers. The dose of egg
was determined based on the experimental decision to supply approximately 1.1 kBq to each subject.
While this supplied the same dose of 14 C-B12 (~1.1 kBq), the amount of total B12 varied between
doses because the eggs were derived from different hens, different injection protocols, and contained
different amounts of the total vitamin—approximately 1.4, 2.4 and 2.6 µg per dose. This protocol,
using different doses of total B12, revealed an inverse relationship between the dose of total B12 and
bioavailability, which is consistent with the well-established phenomenon of saturation of the intrinsic
factor-cobalamin receptor at higher doses [16]. It was surprising, however, how strong this saturation
effect was across the range of relatively low amounts of B12 provided in the single servings of eggs,
causing the amount of absorbed vitamin to be very similar across the dose range used.
The Recommended Dietary Allowance for B12 established by the Institute of Medicine
is ≈0.5 to 2.4 µg/d across the age range from young children to adults. This recommendation assumes
that the usual bioavailability of the vitamin from foods such as meat and fish is 50%, i.e., that the
average adult requirement for absorbed B12 is 1.2 µg/d. The current study shows that B12 is absorbed
as well from egg as has been reported from fish and meat [1,3,8,13,17]. An egg containing 0.5 µg will
therefore provide ≈100% and 20% of the daily recommended intake of the vitamin for children and
adults, respectively.
The method for producing eggs endogenously labelled with 14 C-B12 worked well. The amount
14
of C-B12 in individual eggs dropped such that eggs laid after Day 16 did not have sufficient label
for human servings. Endogenous labels are the best way to trace natural nutrient absorption in
food sources. The approach we used works well for small animals and in a ‘tissue’ such as egg that
concentrates the nutrient under study. Although not endogenously labeled, it is possible that 14 C-B12,
added to eggs at the point of preparation, could be used to detect food cobalamin malabsorption,
following the approach used in the egg yolk cobalamin absorption modification of the Schilling test [1].
Tracing the 14 C-B12 in plasma, urine and stool was easily accomplished using AMS, but the level of
14 C is too low for conventional decay counting techniques. Recent developments using cavity ring

down spectroscopy [33,34] to measure 14 C in samples containing a few mg of carbon could be applied
to samples such as those generated in absorption studies. Based on the data obtained, it is likely the
sampling could have been truncated. Most of the activity was excreted in urine by 48 h and in the stool
by Day 4. A limitation of the study was the small sample size (n = 10), but it is noted that the sample
size was calculated to be sufficient to provide sufficient power for statistical analysis, and that the
calculated mean % bioavailability was very similar to that reported by Doscherholmen et al. [6] using
57 Co-B12, as discussed above. This provides some confidence that our method using endogenously

labeled 14 C-B12 containing eggs gives a reasonable estimate of % bioavailability of the vitamin from
eggs. The study sample also was fairly diverse in age, sex, and BMI, suggesting that the findings may
be fairly generalizable to other healthy adults with putatively intact absorptive capacity.
Nutrients 2019, 11, 2148 11 of 15

5. Conclusions
We confirm that eggs are a good source of B12, and that the experimental approach described
here provides a method for quantifying the absorption of the vitamin from in vivo labeled food and
for detecting food-bound cobalamin malabsorption, using extremely low doses of radioactivity and
low doses of B12. Since urinary and fecal excretion of the label were relatively constant after 48–72 h,
the period and frequency of sample collection can be reduced in any future studies. These results
also show that more attention needs to be paid to the effect of the amount of total B12 in foods on
bioavailability. The latter is especially important for interpreting relationships among intake data,
biomarkers of B12 status, and requirements for the vitamin.

Author Contributions: M.G.G., H.A.R., C.C.C., J.W.M., R.G., B.A.B., L.H.A. designed research; M.G.G., C.C.C.,
B.A.B., conducted research; M.G.G., J.W.M., R.G., B.A.B., L.H.A., analyzed data; M.G.G., L.H.A. wrote the original
draft; M.G.G., L.H.A., H.A.R., C.C.C., B.A.B., J.W.M., R.G., L.H.A. reviewed & edited the paper; L.H.A. had
primary responsibility for final content. All authors read and approved the final manuscript.
Funding: This research was supported by the American Egg Board, NIH SBIR 2R44DK077563-02, NIGMS
8P41GM103483 and USDA, ARS Project #2032-51000-003-00-D.
Acknowledgments: This work was performed in part under the auspices of the U.S. Department of Energy
by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344. Reviewed and released as
LLNL-JRNL-757858. USDA, ARS is an equal opportunity employer.
Conflicts of Interest: Miller, Green, and Buchholz hold the following patent: “Assay for Vitamin B12 Absorption
and Method of Making Labeled Vitamin B12”, Patent Number: 8,202,507. Garrod, Rossow, Calvert, and Allen, no
conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation
of data; inNutrients
the writing
2019, 10,of the PEER
x FOR manuscript,
REVIEW or in the decision to publish the results. 12 of 15

Appendix A. Recruitment,
Appendix A. Screening and Participation of Subjects
The Recruitment,
protocol included
Screeninga week of participation,
and Participation so finding a study population willing and able to
of Subjects
complete all the
The protocol included a week of participation, so finding aa study
blood, urine, and stool collections required relatively largewilling
population pool.and
Of able
the to
12 subjects
who started
complete all the blood, urine, and stool collections required a relatively large pool. Of the 12 subjects analyses.
the protocol, 10 completed it. The data for those 10 are used in the reported
A flowchart
who showing
started the the recruitment,
protocol, screening
10 completed and
it. The data forparticipation ofinthe
those 10 are used the population is presented
reported analyses. A in
flowchart
Figure A1. showing the recruitment, screening and participation of the population is presented in
Figure A1.

Figure Flowchart
A1. A1.
Figure Flowchartof
ofparticipants’ progress
participants’ progress through
through study.study.

Appendix B.

Egg Labeling Model

To estimate the 14C-B12 dose necessary and to optimize hen dosing schedules, B12 kinetics and
Nutrients 2019, 11, 2148 12 of 15

Appendix B. Egg Labeling Model

To estimate the 14 C-B12 dose necessary and to optimize hen dosing schedules, B12 kinetics and
transfer into egg were estimated by developing a mathematical model. The model used data from
six previously reported dose–response studies [23,24]. Edwards [23] used radioactive B12 while
Denton et al. [24] used a dose of the unlabeled vitamin. Differential equations estimated B12 intake
and absorption by gut, and transfer of 14 C-B12 from an intramuscular dose to other tissues, muscle,
and accumulation in eggs from blood (Figure A2). Exchanges between state variables were based on
mass action kinetics and estimated using Advanced Continuous Simulation Language with a 4th-order
Runge-Kutta algorithm for numerical integration of differential equations and an integration interval
of 0.01 d2019,
Nutrients (acslXtreme 2007,REVIEW
10, x FOR PEER Aegis Technologies, Huntsville, AL, USA). 13 of 15

14 hens. 14hens.
B1. Model
Figure A2.
Figure Modeldiagram
diagram representing
representingC-B12 kinetics
14C-B12 in laying
kinetics in laying C-B1214is transferred
C-B12 between
is transferred
blood, muscle, gut, all other tissues, and deposited in each egg from egg number 1 to N.
between blood, muscle, gut, all other tissues, and deposited in each egg from egg number 1 to N.

The mathematical model was able to replicate experimental results from Denton et. al. [24] and
The mathematical model was able to replicate experimental results from Denton et. al. [24] and
Edwards [23] (Table A1). Observed (literature) and predicted (model) percentage accumulation of B12
Edwards [23] (Table A1). Observed (literature) and predicted (model) percentage accumulation of
dose in all eggs (ODE and PME, respectively), and observed and predicted peak B12 peak eggs (OPE
B12 dose in all eggs (ODE and PME, respectively), and observed and predicted peak B12 peak eggs
and PPE, respectively) were very close for all experiments (Table A1). Several different 14 C-B12 doses
(OPE and PPE, respectively) were very close for all experiments (Table A1). Several different
14
14C-B12
were simulated to determine which design or dosing schedule would result in the most C-B12 label
doses were simulated to determine which design or dosing schedule would result in the most
accumulation in eggs (Figure A3). The dosing regimen of 100 nCi/d (3.7 kBq/d, 2.36 µg B12/d) for 4 d
14 C-B12 label accumulation in eggs (Figure B2). The dosing regimen of 100 nCi/d (3.7 kBq/d, 2.36 μg
predicted the maximum percent of dose transferred to eggs. Figure A4 depicts the 14 C activity per egg
B12/d) for 4 d predicted the maximum percent of dose transferred to eggs. Figure B3 depicts the 14C
for the eggs collected from the three hens dosed with 14 C-B12.
activity per egg for the eggs collected from the three hens dosed with 14C-B12.
Table A1. Summary of model simulation results for experimental protocols.
Table B1. Summary of model simulation results for experimental protocols.

1
ODE
ODE PMEPME OPEOPE PPE
PPE ODB
ODB PMB
PMB
Reference
Reference Protocol
Protocol Dose
Dose 1
(%)(%) (%)(%) (%)(%) (%)
(%) (%)
(%) (%)
(%)
Edwards
Edwards[23]
[23] 11 0.50.5 μCi
µCi
57Co-B12 (IM)
57 Co-B12 (IM) 52 52 52 52 8 8 1111 35
35 37
37
Edwards
Edwards[23]
[23] 22 0.50.5
µCi 57 Co-B12
μCi 57 Co-B12 (FED)
(FED) 33 33 32 32 5 5 44 18
18 19
19
Edwards[23]
Edwards [23] 3 0.072
0.072 μCi
µCi 60 Co-B12 (IM)
60Co-B12 (IM)
63 63 59 59 1111 1212 45
45 41
41
Edwards[23]
Edwards [23] 4 0.072
0.072 μCi
µCi 60 Co-B12 (FED)
60Co-B12 (FED)
36 36 37 37 7 7 55 30
30 27
27
Denton[24]
Denton [24] 55 3 µg/d
3 μg/dcrystal
crystalB12
B12(IM)
(IM) 33 33 28 28 4 4 44 NA
NA NA
NA
Denton[24]
Denton [24] 66 3 µg/d
3 μg/dcrystal
crystal B12
B12(FED)
(FED) 20 20 15 15 1.51.5 1.3
1.3 NA
NA NA
NA
11 Abbreviations:
Abbreviations: IM
IM= intramuscular, ODEODE
= intramuscular, = observed data eggs,
= observed dataPME
eggs, predicted
= PME (model) eggs,
= predicted OPE eggs,
(model) = observed
OPE
peak egg, PPE = predicted peak egg, ODB = observed data body, PMB = predicted model body, NA
= observed peak egg, PPE = predicted peak egg, ODB = observed data body, PMB = predicted model = not available.

body, NA = not available.

 Denton [24] 5 3 μg/d crystal B12 (IM) 33 28 4 4 NA NA
Denton [24] 6 3 μg/d crystal B12 (FED) 20 15 1.5 1.3 NA NA
1 Abbreviations: IM = intramuscular, ODE = observed data eggs, PME = predicted (model) eggs, OPE

= observed peak egg, PPE = predicted peak egg, ODB = observed data body, PMB = predicted model
body,
Nutrients NA
2019, 11,=2148
not available. 13 of 15

Nutrients 2019, 10, x FOR PEER REVIEW 14 of 15

Figure A3.
Figure Estimated (model)
B2. Estimated (model) accumulation of 1414CCB12
accumulation of B12in
ineggs
eggswith
withdifferent
differentdosing
dosingschedules.
schedules.

Figure A4. Time course of average 14 C activity per egg. The series indicate 14 C B12 doses delivered to
Figure B3. Time course of average 14C activity per egg. The series indicate 14C B12 doses delivered to
different hens.
different hens.
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