Clinica Chimica Acta 326 (2002) 47 – 59

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Review
Vegetarian lifestyle and monitoring of vitamin B-12 status
Wolfgang Herrmann *, Jürgen Geisel
Department of Clinical Chemistry-Central Laboratory, University Hospital of the Saarland, Bld. 40, D-66421 Homburg/Saar, Germany
Received 17 May 2002; received in revised form 19 August 2002; accepted 5 September 2002

Abstract

Vegetarians are at risk to develop deficiencies of some essential nutrients, especially vitamin B-12 (cobalamin). Cobalamin
occurs in substantial amounts only in foods derived from animals and is essential for one-carbon metabolism and cell division.
Low nutritional intake of vitamin B-12 may lead to negative balance and, finally, to functional deficiency when tissue stores of
vitamin B-12 are depleted. Early diagnosis of vitamin B-12 deficiency seems to be useful because irreversible neurological
damages may be prevented by cobalamin substitution.
The search for a specific and sensitive test to diagnose vitamin B-12 deficiency is ongoing. Serum vitamin B-12
measurement is a widely applied standard method. However, the test has poor predictive value. Optimal monitoring of
cobalamin status in vegetarians should include the measurement of homocysteine (HCY), methylmalonic acid (MMA), and
holotranscobalamin II. Vitamin B-12 deficiency can be divided into four stages. In stages I and II, indicated by a low plasma
level of holotranscobalamin II, the plasma and cell stores become depleted. Stage III is characterized by increased levels of
HCY and MMA in addition to lowered holotranscobalamin II. In stage IV, clinical signs become recognizable like
macroovalocytosis, elevated MCV of erythrocytes or lowered haemoglobin. In our investigations, we have found stage III of
vitamin B-12 deficiency in over 60% of vegetarians, thus underlining the importance of cobalamin monitoring in this dietary
group.
D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Vegetarian; Cobalamin; Vitamin B-12; Holotranscobalamin II; Homocysteine; Methylmalonic acid

1. Introduction risk of diseases. Vegetarian diet has a beneficial effect

Diseases that are related to a modern lifestyle such
as hyperlipidemia, hypertension, obesity or cardiovas-
cular diseases (CVD) are common in industrialized
countries. There is a considerable body of evidence
that a vegetarian lifestyle is associated with a lower
* Corresponding author. Tel.: +49-6841-162-3070; fax: +49-
6841-162-3109.
E-mail address: kchwher@uniklinik-saarland.de
(W. Herrmann).
on some cardiovascular risk factors [1– 4] and reduces
coronary heart disease [5,6]. The beneficial effects
could be due to the diet as well as the healthy lifestyle,
which includes desirable weight, regular physical
activity, and abstinence from smoking, alcohol, and
drug abuse. On the other hand, a recent review article
concluded that the beneficial effects of fruits and
vegetables against coronary heart disease are unim-
pressive, with roughly equal number of articles report-
ing negative or no associations [7]. Therefore, the
estimation of effects of vegetarian lifestyle on coro-

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PII: S 0 0 0 9 - 8 9 8 1 ( 0 2 ) 0 0 3 0 7 - 8
48 W. Herrmann, J. Geisel / Clinica Chimica Acta 326 (2002) 47–59
nary heart disease is a much more difficult task than it
seems from the positive influence on blood choles-
terol. In a meta-analysis of five prospective studies
with 24,000 vegetarians, it was shown that vegans who
did not eat any animal products had a higher mortality
from coronary heart disease than lacto-ovo-vegetarians
[8]. This trend to a more increased coronary risk could
possibly reflect that more pronounced diet deficiencies
in vegans are associated with less cardiovascular
protection. In the present review, we give a short
overview about critical nutrients in a vegetarian diet.
The main focus of this report is vitamin B-12 supple-
mentation, metabolism and monitoring.
2. Nutritional adequacy of a vegetarian diet
Several styles of eating fall into the category of
vegetarianism. Each style differs in the types of foods
included, what foods are avoided, and what nutrient
deficiencies are of greatest concern. Four main var-
iants of vegetarianism can be distinguished. Vegan
diet includes only plant foods – grains, vegetables,
fruits, legumes, nuts, seeds, and vegetable fats. The
lacto-vegetarian diet consists of plant foods plus some
or all dairy products. The lacto-ovo vegetarian diet
consists of plant foods, milk, dairy products, and eggs.
The fourth group are semi- or partial vegetarians. The
semi-vegetarians do not eat red meat, but may eat
chicken or fish with plant foods, dairy products, and
eggs. There are many variations.
Vegetarian diet has less energy and less percentage
of energy from fat and cholesterol than omnivorous
diet. Usually, vegetarians have, compared with omni-
vores, lower body weight [9]. A low energy diet can
modulate blood lipids and in this way reduce athero-
sclerosis and coronary artery disease [10]. Despite
nutrient intake in a vegetarian diet is, in general,
sufficient for energy and proteins, deficiencies could
be found in the areas of vitamins B-1, B-12 and D,
iron, calcium and zinc [11]. Considering the four
types of vegetarian diet, the likelihood of nutritional
deficiencies increases with more restricted forms.
2.1. Vitamin B-1 (thiamin)
Vitamin B-1 is required for the metabolism of
carbohydrates and the neurological function. Vitamin
B-1 is widely distributed in foods, but most contain
only low concentrations of vitamin B-1. Yeasts are
particularly good sources, but cereals represent the
most important dietary sources [12]. However, in a
vegetarian diet, there are a lot of heat-stable vitamin
B-1 antagonists, such as polyphenols (caffeic acid,
chlorogenic acid, tannic acid) and flavonoids (querci-
tin and rutin) that can inhibit vitamin B-1 absorption
[13]. Also, some plant foods, such as raw fish, tea,
betel nuts, blueberries, and red cabbage, contain
thiaminases, which are enzymes that can inactivate
vitamin B-1 by altering its structure [14]. However, in
general, vegetarians consume adequate vitamin B-1
quantities indicating a good vitamin status. In a report
of Vudhivai et al. [15], vitamin B-1 deficiency was
determined in 7.6% of vegetarians. The most widely
used method to detect thiamine deficiency is the
indirect measurement of thiamine diphosphate in
erythrocytes with either the transketolase activitation
test or the transketolase activity assay [16,17]. A
recently published HPLC method yields results sim-
ilar to the erythrocyte activation assay [18]. A higher
precision and standardisation are possible advantages
of the HPLC method.
2.2. Vitamin B-12
Vitamin B-12 occurs in substantial amounts only in
foods derived from animals. Therefore, in a vegetarian
diet, the vitamin B-12 supplementation by nutrient
intake is possibly the most critical point [19]. Because
vitamin B-12 is important in different cellular pro-
cesses and a deficiency could result in irreversible
neurological damages, different aspects of vitamin B-
12 metabolism are discussed in more detail later on.
Recent investigations have shown that the vitamin B-
12 level in plasma could predict the cellular supply
with vitamin B-12 only insufficiently, especially when
the plasma vitamin B-12 levels are in the lower third
of the reference value [20 –22]. The prognostic value
of plasma vitamin B-12 in comparison to newer
parameters of monitoring a cellular vitamin B-12
deficiency is discussed later on.
2.3. Vitamin D
There are only few natural food sources of vitamin
D (cheese, butter, margarine, cream, fish) and none
 W. Herrmann, J. Geisel / Clinica Chimica Acta 326 (2002) 47–59
present in any plant foods. However, vitamin D
deficiency is rarely found in vegetarians. Obviously,
sufficient exposure to sunlight provides an adequate
vitamin D supplementation. However, it has to be
considered that sun exposure could be inadequate in
some cases, for example, in dark-skinned individuals
living in cloudy or northern climates [23]. On the
other hand, infants on a macrobiotic diet revealed
symptoms of rickets in 28%, whereas the vitamin D
level was significantly decreased in comparison to
individuals without symptoms [24]. Therefore, vita-
min D deficiency has to be considered as possible rare
complication especially for children in a vegetarian
diet. The problem of vitamin D supplementation could
easily be overcome by using of vitamin-D-fortified
product, e.g. rice, milk, and breakfast cereals. To
monitor the vitamin D status, immunological methods
are available [25]. In plasma, the 25-hydroxy- and the
1,25-hydroxy-form of vitamin D could be determined.
25-Hydroxy-vitamin D is hydroxylated in the liver
and sufficient to quantify the vitamin D status. The
second hydroxylation takes place in the nephron and
the determination of the 1,25-dihydroxy-form mainly
indicated in cases of renal insufficiency [26].
2.4. Iron
Iron is an important trace mineral that is found in
every cell of the body. Iron is essential to the for-
mation of haemoglobin and myoglobin, which carry
the oxygen in the blood and the muscle. It also makes
up part of many protein and enzymes in the body. The
major part of body iron (70%) is bound to haemoglo-
bin. Only approximately 20% of the iron pool is
stored in form of ferritin to replace lost iron. Clinical
symptoms of iron deficiency are anaemia, general
fatigue, shortness of breath, and headache. The rec-
ommended daily iron dose is 10 mg for men and
postmenopausal women, and 15 mg for premeno-
pausal women [27]. Dried beans, dark green leafy
vegetables, blackstrap molasses, bulgur, and prune
juice are good vegetarian sources of iron. However,
it has to consider that only about 2 – 20% of the iron
available in vegetarian sources is absorbed by the
body [28]. Research shows that vegetarians adults
typically have lower iron stores than nonvegetarians,
although most studies indicate that the ferritin levels
are within a normal range [29].
49
2.5. Calcium
Calcium is needed for bones, nerve and muscle
function, and blood clotting. For adults, a daily dose
of 800 mg is recommended. Limited data suggest that
calcium intakes of vegetarians are below current
recommendations [30]. Although oxalates, phytates,
and fibre in plant food decrease calcium availability, it
is shown that absorption of calcium from many plant
foods is excellent. Calcium absorption from low-
oxalate vegetables, such as broccoli, kale, and collard
greens, ranges from 52% to nearly 59% [31]. Calcium
absorption from beans, nuts, and seeds is considerably
lower. Infants on a macrobiotic diet revealed, in 28%,
physical symptoms of rickets [24]. Besides vitamin D,
the low availability of calcium in the macrobiotic diet
was an important factor in causing the high prevalence
of rickets. Therefore, in a vegetarian diet, especially
for infants, recommendation about the calcium con-
tent of plant food and the availability should be
considered.
2.6. Zinc
Zinc is important for optimal cell growth, rapid
wound healing, and proper functioning of the immune
system. Animal products, especially meat, provide
70% of the zinc in the diet [32]. Plant sources of zinc
include legume, whole grain pasta, wheat germ, for-
tified cereals, nuts, and tofu. However, the bioavail-
ability of zinc from plant foods is reduced by phytate
in whole grains and legumes. The absorption in-
creased when whole grain is leavened and soyfood
is fermented as miso and tempeh [33]. There is some
evidence that zinc absorption is lower on vegetarian
diet than on omnivore diets; however, it has been
shown that zinc excretion decreases when dietary zinc
is low [34]. Mean serum zinc was significantly
decreased in several studies, despite a comparable
intake of zinc [35].
3. Approaches to vitamin B-12 deficiency in
vegetarians
The discovery of vitamin B-12, also known as
cobalamin, was deduced from studies of a previously
incurable disease, pernicious anaemia. In the early
50 W. Herrmann, J. Geisel / Clinica Chimica Acta 326 (2002) 47–59
1920s, Minot and Murphy demonstrated that they
were able to cure pernicious anaemia with a diet that
included whole liver. Vegetarians have long been
known to be at risk of cobalamin deficiency. Reduc-
tion of serum vitamin B-12 in vegetarians has been
reported in various studies [20,36,37]. A high preva-
lence of vitamin B-12 deficiency was also found in
infants aged 10 –20 months on a macrobiotic diet
almost devoid of animal products [38]. In subjects
with lowered vitamin B-12 [20], plasma homocys-
teine (HCY) will increase, which has been recognized
as an independent atherothrombotic risk factor [39].
Furthermore, infants born from vegetarian mothers
and fed on with macrobiotic diets have increased
plasma levels of HCY compared with controls [37].
3.1. Sources and chemistry
Vitamin B-12 is essential for one-carbon metabo-
lism and cell division [40 –42]. Vitamin B-12 synthesis
is very complex and restricted to microorganisms. The
main sources of vitamin B-12 for humans in a non-
vegetarian diet are meat (2 –5 Ag/100 g), fish (2 –8 Ag/
100 g), milk (1.5 Ag/100 ml), cheese (1 –2 Ag/100 g),
and eggs (2 Ag/100 g) [43]. In a vegan diet, main
resources for vitamin B-12 are omitted. The degree of
vitamin B-12 supply decreases with more hygienic
efforts in preparing the meal. An example of vegan life
style without developing a vitamin B-12 deficiency is
shown by the gorilla. However, in cross-sectional
studies, it was shown that about 50% of vegans have
vitamin B-12 levels below normal [44,45]. The vitamin
B-12 status is directly correlated with dietary intake and
length of time following a vegan diet [46,47].
Vitamin B-12 is actually a family of derivatives,
only some forms are active for humans and other
forms are not. Vitamin B-12 is used to describe
compounds of the cobalt corrinoid family, in partic-
ular, those of the cobalamin group. The final sub-
stances that occur in nature from vitamin B-12
biosynthesis are 5V-deoxyadenosylcobalamin and
methylcobalamin (MeCbl), while vitamin B-12 is
by definition cyanocobalamin (CNCbl), which is
the form manufactured by industry [48]. Interest-
ingly, animals including humans and protists need
cobalamin but do not synthesize it. Plants and fungi
are thought to neither synthesize nor to use it. Two
forms of vitamin B-12 have biological activity as
cofactors in enzyme reactions: adenosylcobalamin
and MeCbl [49]. Three groups of enzymatic reac-
tions are involved: intramolecular rearrangements
involving the transfer of a hydrogen atom from
one carbon atom to an adjacent carbon and its
replacement by another adjacent group, catalysed
by adenosylcobalamin; reduction of ribonucleotide
triphosphate to 2V-deoxyribonucleotide triphosphate,
catalysed by adenosylcobalamin; intermolecular
methyl transfer, catalysed by MeCbl. In mammalian
metabolism, vitamin B-12 is required for only two
key enzymatic reactions: synthesis of methionine
from homocysteine catalysed by methionine synthase
with MeCbl as cofactor, and isomerisation of meth-
ylmalonyl-CoA to succinyl-CoA catalysed by meth-
ylmalonyl-CoA mutase with adenosylcobalamin [50].
The latter reaction is part of the catabolism of odd-
chained fatty acids and several amino acids. A
reduced flux through the methylmalonyl-CoA mutase
reaction is discussed to contribute to subsequent
neurological tissue damage.
The active form of vitamin B-12 is of importance
to strict vegetarians because this vitamin is essential
for normal maturation and development of blood
cells but is not normally provided by eating pure
plant food. The ultimate source of vitamin B-12 is
derived at the microbiological level. Not all forms of
vitamin B-12 formed by microbes are metabolically
active for mammalian cells. Some forms are termed
‘‘analogs’’ or ‘‘corrinoids’’ because they are sufficient
for enzymatic reactions and the growth of micro-
organisms, but they are insufficient for the specific
roles in human metabolism [49]. According to Her-
bert [51,52], algae and other organisms produce
noncobalamin vitamin B-12 analogous that are
unavailable to the human organism and may even
block vitamin B-12 metabolism, as was shown for
spirulina. In vegetarian children consuming vitamin
B-12 from algae, no improvement of haematological
status was detectable while plasma vitamin B-12
concentrations increased [53]. This confirms that
the bioavailability of vitamin B-12 from algae and
other plants is questionable.
3.2. Metabolism of vitamin B-12
Absorption of vitamin B-12 from food is a com-
plex process [54]. Low intake of vitamin B-12 from
 W. Herrmann, J. Geisel / Clinica Chimica Acta 326 (2002) 47–59
food or intestinal malabsorption could lead to negative
balance and, finally, to functional deficiency when
tissue stores of vitamin B-12 are depleted. Vitamin B-
12 in food is released by the action of gastric acid and
proteolytic enzymes in the stomach. In the stomach,
vitamin B-12 is preferentially captured by haptocor-
rin, an r-binder protein made in the saliva and stom-
ach. In the upper small intestine, pancreatic enzymes
and an alkaline pH degrade the haptocorrin –cobalam-
min complex. The released vitamin B-12 is recaptured
by yet another protein the intrinsic factor. The intrinsic
vitamin B-12 complex is transported to the terminal
ileum where the complex is recognised and internal-
ised by specific membrane receptors of the enter-
ocytes. The absorption of vitamin B-12 by this
process is limited ( < 3 Ag per meal). In contrast to
this receptor-mediated absorption, about 1% of the
vitamin B-12 is absorbed by passive diffusion even in
the absence of intrinsic factor. After absorption of the
vitamin B-12 – intrinsic factor complex into the enter-
ocytes, the complex is degraded and vitamin B-12 is
transferred to a third binding protein, transcobalamin
II. The new formed complex is released into the portal
circulation and is subsequently recognised by specific
receptors present on all cell types. The part of vitamin
B-12 which is bound to transcobalamin is named
holotranscobalamin (holoTC) [55]. Only 6– 20% of
total plasma vitamin B-12 is present in the active form
bound to transcobalamin II, holoTC II [56]. The
remaining major part of vitamin B-12 is bound to
transcobalamins I and III [57]. It is supposed that this
fraction is involved in transporting redundant vitamin
B-12 from cells back to the liver. The liver contains
most of the body’s cobalamin. It is calculated that 2– 3
mg vitamin B-12 is stored in the liver [54]. Consid-
ering a daily recommended dietary dose of vitamin B-
12 of 2.4 Ag [58], several years of dietary deficiency
are usually necessary before conditions of vitamin B-
12 deficiency is apparent.
3.3. Manifestation of vitamin B-12 deficiency
The clinical signs of vitamin B-12 deficiency
represent late stages of this disease including mega-
loblastic anaemia and neuropsychiatric disorders. The
condition is nearly identical to folate deficiency but
leads to irreversible degeneration of the nervous
system if left untreated. The staging of vitamin B-12
51
deficiency introduced by Herbert [40] suggests that
vitamin B-12 deficiency can be divided into four
stages. In stages I and II of vitamin B-12 deficiency,
the plasma and cell stores become depleted. These
stages are indicated by a low plasma level of holo-TC
II, in stage II, additionally by a low level of holohap-
tocorrin (HC) [59,60]. Stage III of vitamin B-12
deficiency is characterized by functional dysbalances.
At this stage, beside lowered holo-TC II, increased
plasma levels of HCY and MMA are observed. In
stage IV of vitamin B-12 deficiency, clinical signs
become recognizable like macroovalocytosis, elevated
MCV of erythrocytes, or lowered haemoglobin. An
early diagnosis of vitamin B-12 deficiency seems to
be useful because neurological damages are irrever-
sible and could possibly be prevented by early diag-
nosis followed by vitamin B-12 substitution [61]. An
elevated HCY level which precedes clinical signs of
vitamin B-12 deficiency is an earlier marker and
displays the metabolic consequence of vitamin B-12
deficiency [62]. Hyperhomocysteinemia is, for exam-
ple, associated with increased risk for cardiovascular
disease, venous thrombosis, neural tube defects or
preeclampsia, dementia, and Alzheimer’s disease
[63 – 70]. Because of substantial tissue stores, vitamin
B-12 deficiency takes years to become evident.
4. Monitoring of the vitamin B-12 status
4.1. Vitamin B-12, methylmalonic acid, homocysteine,
and holotranscobalamin II
The search for the optimal test to diagnose vitamin
B-12 deficiency is ongoing [20,42,71,72]. Metabolic
studies confirmed that most of low vitamin B-12
concentrations in asymptomatic patients or apparently
healthy subjects are representing functional relevant
cobalamin insufficiency. Additionally, the diagnostic
reliability of total vitamin B-12 in serum or plasma
was challenged by metabolic demonstrations of
falsely normal cobalamin concentrations [20,22].
Transcobalamin is the physiologically active form of
vitamin B-12 in plasma. Only in the complex form
cobalamin is taken up into the cells. Furthermore,
metabolic tests also have disadvantages. HCY, for
example, diagnoses vitamin B-12 deficiency, too,
unspecific because it is additionally increased in folate
52 W. Herrmann, J. Geisel / Clinica Chimica Acta 326 (2002) 47–59

and vitamin B-6 deficiency or renal insufficiency [21],
(Fig. 1). Methylmalonic acid (MMA) is more but not
fully specific for vitamin B-12 deficiency, is expen-
sive, and also increases in renal insufficiency [20,21].
Due to their life style, vegetarians normally have no
folate levels below the lower reference limit. There-
fore, vitamin B-12 deficiency caused by vegetarian
diet becomes a main factor influencing the HCY level.
Vitamin B-12 known to function as cofactor for
methionine synthase causes, in case of deficiency,
an inhibition in the remethylation of HCY to methio-
nine. Folate in form of 5-methyltetrahydrofolate
becomes trapped because the transfer of the methyl
group is inhibited (Fig. 2). Under this condition,
higher levels of folate are required in order to transfer
a sufficient amount of methyl groups from 5-methyl-
tetrahydrofolate to HCY, preventing hyperhomocys-
teinemia. However, due to folate trap causing relative
folate shortage, a substantial portion of vegetarians
develops hyperhomocysteinemia [20,73].
4.2. Distributions in vegetarians
In an investigation on vegetarians, we studied the
course of HCY, MMA, and total vitamin B-12 from
omnivorous persons to lacto- (LV)/and lacto-ovo
vegetarians (LOV) up to vegans who refuse all kinds
of animal products [20]. There was a consistent
significant increase of HCY and MMA from omniv-
orous subjects via LV/LOV up to vegans, while
vitamin B-12 decreased only slightly (Fig. 3). This
study and the findings of other investigations [22,54,
74,75] confirmed that MMA is the most sensitive
studied marker of a functional intracellular vitamin B-
12 shortage. The scatter plot shown in Fig. 4 demon-
strates that, within the normal range up to 360 pmol/l
vitamin B-12 in serum, a greater number of subjects
have increased MMA >271 nmol/l. Thus, serum
vitamin B-12 concentrations within the normal do
not exclude a functional vitamin B-12 deficiency,
and conversely, low serum vitamin B-12 does not
confirm functional vitamin B-12 deficiency. Subjects
with serum vitamin B-12 concentrations up to 360
pmol/l and increased MMA may have functional
vitamin B-12 deficiency, which could be attributable
to a lowered fraction of holo-TC II. Using the cut-off
values for vitamin B-12 and MMA, we found in
vegetarians increased MMA in 25%, whereas lowered
serum vitamin B-12 occurred in only 8%. It is con-
cluded by use of total vitamin B-12 as marker for

Fig. 1. Metabolism of homocysteine. SAM, S-adenosyl-methionine; SAH, S-adenosyl-homocysteine; HCY, homocysteine; CYS,
cystathionine; a-KBT, a-ketobutyrate; PRP-CoA, propionyl-CoA; MMA, methylmalonic acid; D-MM-CoA, D-methylmalonyl-CoA; L-
MM-CoA, L-methylmalonyl-CoA; SUC-CoA, succinyl-CoA; THF, tetrahydrofolate; 5-MTHF, 5-methyltetrahydrofolate; 5,10-MTHF, 5,10-
methylenetetrahydrofolate; 1, methionine synthase; 2, serine-hydroxy-methyltransferase; 3, N5,N10-methylene-tetrahydrofolatreductase; 4,
cystathionine-h-synthase; 5, cysta-thionase; 6, L-methylmalonyl-CoA-mutase; AA, amino acids; FA, fatty acids.
 W. Herrmann, J. Geisel / Clinica Chimica Acta 326 (2002) 47–59 53

Fig. 2. Homocysteine metabolism in vitamin B-12 deficiency: Low vitamin B-12 causes an inhibition of the homocysteine (HCY) remethylation
and thus induces trapping of 5-methyltetrahydrofolate (5-MTHF). The increased folate concentration inhibits the transmethylation reaction
leading to elevated S-adenosyl-methionine (SAM) levels. High SAM concentrations activate the transsulfuration pathway (SAH, S-adenosyl-
homocysteine; CYS, cystathionine; THF, tetrahydrofolate; 5,10-MTHF, 5,10-methylenetetrahydrofolate; 1, methionine synthase; 2, N5,N10-
methylene-tetrahydrofolatereductase; 3, cystathionine-h-synthase; 4, glycin-N-methyltransferase).

vitamin B-12 status that a greater number of subjects
with functional vitamin B-12 deficiency would be
missed. Additionally, the scatter plot (Fig. 4) also
shows that increased MMA concentrations in vegeta-
rians may occur within the normal range of HCY
starting as low as 8 Amol/l. Thus, normal HCY also
does not exclude functional vitamin B-12 deficiency.
Measurement of serum vitamin B-12 concentration
has been widely applied as standard method for test-
ing cobalamin deficiency. However, the test has poor
positive and negative predictive values because of
problems of sensitivity and specificity [76], which is
confirmed by our investigations on vegetarians
[20,73]. One important point for the low specificity
of vitamin B-12 in the prediction of vitamin B-12
status is the lower cut-off value of the reference
interval. The reference interval is founded by the
twofold standard deviation of an obviously healthy
population. However, as shown by measuring of
MMA as a functional marker of cellular vitamin B-
12 deficiency, the obvious healthy group includes
individuals with clinically not obvious vitamin B-12
deficiency. To create an improved reference interval
for vitamin B-12 in serum, only individuals with
normal MMA levels should be included. Based on
our results, we recommend a lower reference limit of
at least 360 pmol/l. Increased levels of MMA were
very rarely observed above this cut-off level. Another
critical point is the distribution of circulating cobala-
min among its several binding proteins. To get more
specificity and sensitivity in diagnosing vitamin B-12
deficiency, the concept of measuring the subfraction
of vitamin B-12 (cobalamin), which is attached to
transcobalamin (TC) and is called holo-TC II, has
reached great interest [42,60,77]. Holo-TC II repre-
sents the biologically active vitamin B-12 fraction
because only TC II promotes specific uptake of its
vitamin B-12 by all cells. The quantitatively much
larger fraction haptocorrin (HC) seems to be metabol-
ically inactive and no cellular receptors are known for
holo-HC [71,78]. The blood holo-TC II concentration
might be modulated by several influences. These
include the amount of absorbed cobalamin, the rates
of hepatic and renal uptake of holo-TC II, the pro-
54 W. Herrmann, J. Geisel / Clinica Chimica Acta 326 (2002) 47–59
Fig. 3. Course of vitamin B-12 (a), HCY (b), and MMA (c) in
vegetarian subgroups. , Median with 95% and 5% percentile;
HCY, homocysteine; MMA, methylmalonic acid; HME, high meat-
eaters; LME, low meat-eaters; LOV/LV, lacto-ovo/lacto vegetarians.
Statistical analysis was performed with the Mann – Whitney test
(*p < 0.05, **p < 0.01).
duction and release of ileal and possibly renal holo-
TC II, tissue requirements for cobalamin, and perhaps
other unknown factors [71]. Some clinical evidence
supports the concept that the measurement of holo-TC
II provides a better index of cobalamin status [49]. A
low holo-TC II concentration is hypothesized to be
seen as an early sign of general cobalamin insuffi-
ciency or specific evidence of decreased absorption of
cobalamin. There is no convincing evidence that low
holo-TC II favours cobalamin deficiency or impaired
absorption [71]. In vegetarians, however, cobalamin
deficiency is the most prominent problem.
In a second study on vegetarians, we only found a
few subjects with a normal vitamin B-12 status, while
a majority was categorized as stage III of vitamin B-
12 deficiency according to Herbert, with elevated
HCY and/or MMA as well as lowered holo-TC II
[73]. Stage III of vitamin B-12 deficiency indicates
that the vitamin B-12 stores are depleted (holo-TC II
is very low, compared with omnivores, it decreased by
>80%) and the two vitamin B-12-dependant enzyme
reaction (methionine synthase and methylmalonyl-
CoA mutase) are impaired. However, not all vegeta-
rians with stage III of vitamin B-12 deficiency have
simultaneously elevated metabolites HCY and MMA.
A higher folate level may compensate the folate trap
induced by moderate vitamin B-12 deficiency, hyper-
homocysteinemia is still prevented but MMA is
elevated. An isolated lowering of holo-TC II without
elevation of the functional markers HCY and/or MMA
was found in f 20% of the vegetarians. At this stage,
the vitamin B-12 stores are still sufficient to catalyse
the vitamin B-12-dependant reactions properly, no
metabolic dysbalances are detectable. Stage IV of
vitamin B-12 deficiency is the most severe stage
and is characterized by clinical signs of B-12 defi-
ciency. In our studies on vegetarians, we did not see
stage IV of vitamin B-12 deficiency. However, in
vegans, the MCV as morphological marker increased
slightly within the normal range. The combined use of
storage as well as of metabolic markers for monitoring
vitamin B-12 status as suggested by Herbert [40]
allows, on the one hand, a deeper insight in the
severity of this deficiency and, on the other hand,
helps to control the plausibility of the results drawn by
the different markers. The latter is also of importance
because no single marker has the power to diagnose
vitamin B-12 deficiency with sufficient reliability. We
found few vegetarians having an isolated slightly
elevated MMA but normal HCY and holo-TC II. This
constellation of the different B-12 markers indicates
 W. Herrmann, J. Geisel / Clinica Chimica Acta 326 (2002) 47–59 55

Fig. 4. Scatter plots illustrating the relationship between MMA and vitamin B-12 (a), and between MMA and HCY (b). 5, All vegetarians; +,
high meat-eaters; z, increased; #, decreased; ! , normal; HCY, homocysteine; MMA, methylmalonic acid.

no vitamin B-12 deficiency but an MMA elevation by
other reasons. Achlorhydria and the intestinal blind
loops resulting from gastrectomy may provide sides
for bacterial overgrowth which may be another source
of elevated MMA and may also contribute to cobala-
min deficiency through microbial gluttony for vitamin
B-12. Additionally, renal insufficiency and hypovole-
mia are further reasons for moderate elevation of
MMA and HCY without vitamin B-12 deficiency
[21,79,80]. Other discordant results were obtained in
only some subjects showing increased levels of HCY
and MMA together with a high level of holo-TC II.
This constellation could be caused by an intracellular
functional vitamin B-12 deficiency, indicated by high
56 W. Herrmann, J. Geisel / Clinica Chimica Acta 326 (2002) 47–59
levels of HCY and MMA, combined with a disturbed
cellular uptake of vitamin B-12, indicated by high
levels of holo-TC II. This has been reported for
patients with chronic renal failure [81,82]. It has been
suggested that the kidney plays an important role in
regulating cobalamin flux [83,84]. However, the
mechanisms responsible for high cobalamin levels in
renal failure are still unknown. An abnormal distribu-
tion of holotranscobalamins in renal failure [81], a
disturbed receptor activity for renal TC II uptake, and
the possibility that TC II is functionally altered by
renal failure offer partial explanation for a failure of
renal uptake of holo-TC II in renal disease.
A common reason for a decreased serum level of
holo-TC II may be malabsorption or deficiency of
cobalamin [60,71]. Therefore, gastrointestinal condi-
tions compatible with cobalamin malabsorption have
to be differentiated from deficiencies by diet. A
vegetarian lifestyle, however, is most probably com-
patible with a vitamin B-12 deficiency. Stage I/II of
vitamin B-12 deficiency, which is characterized by a
low level of holo-TC II but normal metabolite con-
centrations (HCY and MMA), was found in our
studies in a substantial portion of vegetarians. How-
ever, it should be taken in consideration that a
transient lowering of holo-TC II without vitamin B-
12 shortage by diet can also be caused by a transient
malabsorption, for example, by temporary exposure to
drugs (colchicines or omeprazole) or by alcohol
abuse. A repeated measurement of holo-TC II con-
centration and a patient’s interview may help to clarify
the situation and to improve the diagnostic value of
the result.
5. Conclusions
The vegetarian lifestyle, especially the strict vegan
lifestyle, is at risk to develop deficiencies of some
essential nutrients, for example, trace elements and
vitamins [8]. Investigation in vegetarians revealed a
high prevalence of vitamin B-12 deficiency. Vitamin
B-12 deficiency contributes to the development of
hyperhomocysteinemia, which has been recognized as
atherothrombotic risk factor, but also as risk factor for
dementia [70], which may counteract the otherwise
health beneficial lifestyle of vegetarians. Furthermore,
S-adenosyl-methionine, which primarily serves as a
universal donor of methyl groups, is lowered by B-12
deficiency and is the reason for hypomethylation of
DNA, RNA, proteins, myelin, or neurotransmitters.
As clinical consequence, the development of psychi-
atric and neurological disorders, like cognitive dys-
function, dementia, or Alzheimer disease, are
discussed [61,70,85,86]. Therefore, an early diagnosis
of vitamin B-12 deficiency is urgent.
An optimal monitoring of vitamin B-12 status in
vegetarians should include the measurement of HCY,
MMA, and holo-TC II. Additionally, the levels of
folate and vitamin B-6 should be controlled. If holo-
TC II is not available, the serum level of total vitamin
B-12 should be carried out instead. A serum or plasma
HCY concentration < 12 Amol/l or better, < 10 Amol/
l, should be aimed. Holo-TC II should be >45 pmol/l
and MMA < 271 nmol/l. A serum level of vitamin B-
12 >156 pmol/l is normal but only a B-12 concen-
tration above 360 pmol/l does prevent functional
vitamin B-12 deficiency with more reliability. The
efficacy of vitamin substitution should be controlled
by sufficient laboratory tests. At least one annual
check of vitamin B-12 status should be recommended.
Acknowledgements
We would like to thank Jean-Pierre Knapp and
Heike Schorr for supporting the preparation of this
manuscript.
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