Pathophysiology of
2.5 Anaemias
Anaemia (a, not; haima, blood) is a misnomer because without
blood, life is not possible. What we actually mean by anaemia
is a reduction in the concentration of haemo-globin in blood.
In practice, when the blood haemoglobin is low, the red cell
count and packed cell volume (PCV) are also generally low.
Reduction in haemoglobin level reduces the oxygen carrying
capacity of blood. In a normal person at rest, only about 25%
of the oxygen carried by arterial blood is extracted by the tis-
sues, leaving an enormous reserve for exercise. Therefore, the
metabolic needs at rest can be met satisfactorily even in an
anaemic individual. But when the anaemic person takes exer-
cise, the poor oxygen content of blood becomes a handicap.
The person’s cardiorespiratory reexes try to compensate for
the oxygen lack by increasing the respiratory rate and depth,
and the rate and force of contraction of the heart. Therefore,
the anaemic person feels breathless and gets palpitation during
exercise. His exercise tolerance is also reduced; he gets tired
quickly, and cannot undertake very heavy exercise.
Anaemia may be due to various causes. Some clue to the
cause of anaemia may be obtained from simple investigations
such as peripheral smear, packed cell volume, RBC count, and
the haemoglobin level. The most valuable investigation of these
is the peripheral smear. These simple investigations are enough
to make sure whether the person is anaemic; to calculate the
MCV so that we know whether the red cells are normal in size
(normocytic), small (microcytic) or large (macrocytic); and
to calculate the MCHC so that we know whether the haemo-
globin concentration in red cells is normal (normochromic) or
below normal (hypochromic). These variables are affected in
a characteristic manner in different types of anaemia.
AETIOLOGICAL CLASSIFICATION OF ANAEMIA
The aetiology (aitia, cause; logos, discourse) of a disease is its
underlying cause. Hence aetiological classication of anaemia
groups different types of anaemia based on their cause. It is
quite logical that red cell population may be reduced if their
rate of production is reduced, or if their rate of destruction is
increased, or if there are abnormal losses of blood.
I. Decreased Production of RBC
This basic defect may be due to one out of several causes.
1. Decient Supply of Essential Nutrients
There are nutrients which serve as raw materials for synthe-
sis of haemoglobin, or those which function as cofactors in
haemoglobin synthesis or maturation of precursors of erythro-
cytes. Deciency of any of these nutrients gives rise to anae-
mia. Important examples of this category are the anaemias due
to deciency of iron, folic acid, or vitamin B12.
2. Inactivity of the Bone Marrow
Production of RBC may be reduced if there is a ‘strike’ in
the factory where they are produced. This type of anaemia is
generally called aplastic (a, not; plassein, to form) anaemia,
but it would be more accurate to call it hypoplastic anaemia.
Hypoplasia of the marrow may be due to irradiation or antican-
cer drugs, or due to some unknown factor.
3. Chronic Renal Disease
Chronic renal disease is often associated with anaemia possibly
because kidneys are the major source of erythropoietin.
4. Chronic Inammatory Disease
Any chronic inammatory disease may also be asso-ciated
with anaemia due to ill-understood factors which depress
erythropoiesis.
5. Hypothyroidism
Hypothyroidism is associated with anaemia possibly because
of the reduced metabolic activity in the bone marrow.
6. Inltration of Bone Marrow
Production of red cells may be impaired because there is insuf-
cient space in the bone marrow for erythropoiesis. The space
may be crowded out by expansion of precursors of the granu-
locytic series (as in leukemia), or by a large number of tumor
cells which might have travelled from any site in the body.
The process of systemic spread of tumor cells in this fashion is
called metastasis.
II. Increased Destruction of RBC
It may be quite logically deduced that increased rate of destruc-
tion of RBC may be due to one out of two basic defects. Either
there may be some defect within the RBC which makes them

liable to premature destruction, or there may be some defect
outside the RBC which is responsible for their excessive
destruction. The defect outside the RBC may be in the imme-
diate environment of the RBC (i.e. the plasma), or it may be
hyperactivity of the normal destructive mechanisms (e.g. an
overactive spleen).
1. Corpuscular Defect
Here a defect inherent in the structure or function of RBC may
make them more vulnerable to normal destructive mechanisms.
For example, haemoglobin in the RBC may differ in struc-
ture from normal haemoglobin. In sickle cell anaemia, the
haemoglobin in RBC is HbS, which differs from normal hae-
moglobin only in having valine instead of glutamic acid in the
sixth position of the beta chain. This small change alters the
properties of the molecule such that it tends to form insolu-
ble polymers. Polymerization of the haemoglobin molecule
leads to a sickle-shaped deformity of the red cells, specially
when deoxygenated. Repeated sickling possibly also damages
the red cell membrane. The abnormal shape and membrane
damage make the red cells prone to intravascular haemolysis
and also more susceptible to phagocytosis by macrophages.
In thalassemias, RBC contain an abnormal variant of haemo-
globin which, in some cases, is fetal haemoglobin (HbF). In
some cases, there is impaired or decient synthesis of specic
globin chains. In the absence of complementary globin chains,
the normal chains precipitate in the red cells, damage the cell
membrane, and lead to premature cell death.
In spherocytosis, the RBC membrane is excessively per-
meable to sodium, possibly due to a defect in membrane pro-
teins. As a result, RBC assume a biconvex shape. Hence they
are more prone to haemolysis when exposed to a hypotonic
solution, and also more likely to be destroyed when passing
through narrow spaces.
In G6PD deciency anaemia, RBC are decient in the
enzyme glucose-6-phosphate dehydrogenase (G6PD). This
enzyme is required in the pentose phosphate pathway during
which NADPH is generated. NADPH is required, in turn, to
maintain glutathione in the reduced state. In the absence of an
adequate concentration of reduced glutathione, the RBC mem-
brane is more prone to peroxidation damage. That is why RBC
are more prone to haemolysis. In the milder varieties of G6PD
deciency, the disease becomes apparent only when the patient
is given a drug which stresses the antioxidant mechanisms of
the body. Important drugs in this category are sulphonamide
and antimalarials.
2. Extracorpuscular Defect
If normal RBC are exposed to an abnormal environment, they
may die a premature death. The environment may be abnor-
mal due to the presence of some anti-RBC antibodies, drugs or
snake venoms in the plasma.
A category of anti-RBC antibodies, called incomplete anti-
bodies, may be detected by Coombs’ test. These antibodies
are incomplete in that they do not by themselves bring about
agglutination (clumping) of RBC. But if antibodies to these
antibodies are available, agglutination takes place. In order to
procure antibodies to human antibodies, human globulins are
CHAPTER 2.5: Pathophysiology of Anaemias
Fig. 2.5.1: Direct and indirect Coombs’ test.
injected into a rabbit. After a few weeks, the rabbit serum con-
tains anti-human globulins. When this rabbit serum is brought
in contact with patient’s washed RBC, agglutination takes
place if the patient’s RBC are coated with incomplete anti-RBC
antibodies which cannot be washed away (Fig. 2.5.1). This is
called the direct Coombs’ test. However, if the antibodies are
of the type that get washed away, some normal human RBC
may be exposed to the patient’s serum. As a result, the normal
RBC get coated with the patient’s incomplete antibodies. Now
if these RBC are exposed to rabbit serum containing antihuman
globulin, the RBC are agglutinated. This is called the indirect
Coombs’ test.
Alternatively, haemolytic anaemia may be due to an over-
active spleen which destroys RBC at a faster rate than normal.
The condition is known as hypersplenism and may be treated
by surgical removal of spleen (splenectomy).
III. Abnormal Loss of Blood
An acute injury associated with massive bleeding leads to a
short-lasting reversible anaemia which, by itself, does not
cause much concern. The situations in which anaemia forms a
dominant part of the clinical picture are those associated with
chronic blood loss. A few common examples are heavy loss of
blood during menstrual cycles, and intestinal blood loss due to
either piles or hookworm infestation.
The etiological classication of anaemia has been summa-
rized in Table 2.5.1. A few anaemias, selected on the basis of
their high prevalence or academic importance have been dis-
cussed on the next few pages.
IRON DEFICIENCY ANAEMIA
Iron deciency anaemia may be due to inadequate dietary
intake of iron, poor intestinal absorption of iron, abnormal
loss of iron from the body (which is generally due to abnormal
blood loss), or heavy requirements of iron (as during childhood
or pregnancy). 61
SECTION 2: Blood
Table 2.5.1: Aetiological classication of anaemia
I. DECREASED PRODUCTION OF RBC
1. Decient supply of essential nutrients
(a) Iron deciency anaemia (b) Folic acid deciency anaemia
(c) Vitamin B12 deciency anaemia (Pernicious anaemia)
2. Inactivity of the bone marrow: Hypoplastic anaemia
(a) Irradiation (b) Anticancer drugs (c) Cause unknown
3. Chronic renal disease
4. Chronic inammatory disease
5. Hypothyroidism
6. Inltration of bone marrow
II. INCREASED DESTRUCTION OF RBC
1. Corpuscular defect
(a) Sickle cell anaemia (b) Thalassemias
(c) Spherocytosis (d) G6PD deciency anaemia
2. Extracorpuscular defect
(a) Antibodies (b) Drugs
(c) Haemolysins in (d) Hypersplenism
snake venoms
III. ABNORMAL LOSS OF BLOOD
1. Acute loss
2. Chronic loss
(a) Heavy menstrual loss (b) Piles
(c) Hookworm infestation
Fig. 2.5.2: Some metabolic interactions between folate and vitamin
B12. (1), thymidylate is required for DNA synthesis; (2), 5, methyl THFA
Iron is a component of haemoglobin. Therefore haemo- accumulates in vitamin B12 deciency (methylfolate trap); (3), dietary
globin synthesis is impaired in iron deciency. Hence the RBC folate can circumvent methylfolate trap by forming THFA, which can
are small in size, and even for their small size contain less hae- be converted into 5, 10-methylene THFA.
moglobin. That is, iron deciency anaemia is microcytic and Note that conversion of THFA into 5,10-methylene THFA requires
hypochromic in character. vitamin B6, thereby bringing yet another B vitamin into the picture.
Treatment of iron deciency anaemia should aim at treat-
ing the underlying cause. For example, if the patient has hook- as megaloblastic anaemias. Out of the two, vitamin B12 de-
worm infestation or heavy menstrual blood loss these should ciency anaemia is more dangerous, and is therefore known as
be appropriately treated. In addition, iron may be given in the pernicious anaemia.
form of tablets. Ferrous sulphate is nearly as good as and much
less expensive than some other forms. Since absorption of any Metabolic Role of Folate and Vitamin B12
form of iron is poor, and is partly determined by iron require- Some of the metabolic functions of folate and vitamin B12 are
ments, very high doses of therapeutic iron should be avoided. interrelated (Fig. 2.5.2). They are both required for conversion
The unabsorbed iron is lost in the faeces, and if the quantity of deoxyuridylate into thymidylate. This reaction is vital for
lost is very high, it is likely to produce gastrointestinal distur- DNA synthesis, which in turn is specially important for rapidly
bances. There is rarely a situation where more than 50 mg iron dividing cells. The vitamin directly required for this reaction
per day would do much good. is folate in the form of 5, 10-methylene THFA (tetrahydrofolic
For a discussion on iron absorption, see Chapter 6.7, and acid). However, in order to keep up the supply of 5, 10-meth-
for the nutritional aspects of iron, see Chapter 7.7. ylene THFA, an adequate supply of vitamin B12 is also impor-
tant because vitamin B12 can regenerate THFA from 5, methyl
THFA. In vitamin B12 deciency, the body responds through
FOLIC ACID AND VITAMIN B12 DEFICIENCY
a chemical feedback mechanism by diverting metabolic reac-
ANAEMIAS tions towards formation of more 5, methyl THFA. This helps
preserve essential methylation reactions at the expense of DNA
Folic acid and vitamin B12 are both members of the group synthesis. As a part of the diversion process, methylenetet-
known as vitamin B complex, and their metabolic roles are rahydrofolate reductase activity increases, thereby reducing
interrelated. Therefore a deciency of either vitamin gives the availability of 5, 10-methylene THFA. Simultaneously,
rise to a similar anaemia. In both anaemias, there is matura- because of vitamin B12 deciency, 5, methyl THFA is not suf-
tion arrest during erythropoiesis. Therefore the precursors of ciently used for methylation of vitamin B12. Thus 5, methyl
erythrocytes keep growing in size but neither divide nor dif- THFA accumulates, trapping available folate in this form. The
ferentiate properly. The resulting cells are large, immature and situation has been called methylfolate trap. As can be seen, die-
poorly differentiated, and are known as megaloblasts. That is tary folate can temporarily overcome this trap by leading to the
62 why folate and vitamin B12 deciency anaemias are known synthesis of 5, 10-methylene THFA. However, folate will not

Castle’s classical experiments
When William B. Castle, a young resident physician, age
30, was working with Dr. Francis Peabody, Director of the
Thorndike Memorial Laboratory of the Boston City Hospital
in 1927, the following facts were known:
1. Patients having pernicious anaemia show gastric atrophy
and achlorhydra (absence of hydrochloric acid in the
stomach).
2. Pernicious anaemia may follow gastrectomy (surgical
removal of the stomach).
3. George Minot and William Murphy had just reported
in 1926 that pernicious anemia could be cured if the
patient’s diet included half a pound (about 240 g) of beef
liver everyday.
On the basis of these facts, Castle asked the following
questions:
1. How can a normal person keep pernicious anemia away
without taking half a pound of beef liver every day?
2. Can some normal digestive process of the healthy stom-
ach substitute for half a pound of liver?
The excellent formulation of questions was the rst step
towards the success of Castle’s experiment. In an attempt
to answer these questions, Castle designed and performed a
series of dietary experiments on patients of pernicious anae-
mia. The experiments involved feeding the patient a speci-
ed item (besides his normal diet) every day for 10 days
continuously. What he did and what he found is summarized
in the table below:
Dietary Supplement Observation
1. Beef muscle (200 g) No improvement
2. Normal gastric juice No improvement
3. Beef muscle which had stayed in the Improvement
stomach of a normal person for 1 h
4. Beef muscle which had been incubated Improvement
in normal gastric juice
5. Beef muscle and normal gastric juice Improvement
together (without incubation)
6. Beef muscle after complete in vitro No improvement
digestion with pepsin
Try to put yourself in Castle’s position. Assuming that
you know only what Castle knew in 1927, try to think what
conclusions may be drawn from these experiments. Put
your conclusions down on a piece of paper before reading
any further.
The conclusions that may be drawn from Castle’s classi-
cal experiments are:
CHAPTER 2.5: Pathophysiology of Anaemias
1. Some interaction between beef muscle and normal
gastric juice is essential for improvement.
2. The patient’s gastric juice is not normal.
3. Digestion of beef muscle protein by pepsin is not suf-
cient for improvement.
On the basis of these conclusions, Castle proposed the fol-
lowing hypotheses:
1. Pernicious anaemia is due to the dietary deciency of
some substance present in beef muscle. Since this sub-
stance has to be supplied to the body from outside,
Castle called it the extrinsic factor.
2. The extrinsic factor was effective only after interaction
with some component of normal gastric juice. Since the
component is available within the body, Castle called it
the intrinsic factor.
Besides proper formulation of questions and Castle’s
hardwork and analytic attitude, other factors which contrib-
uted to his success were:
1. The fact that reticulocyte count is an early and sensi-
tive indicator of improvement in an anaemic patient was
already known. Knowing this indicator reduced the dura-
tion of experiments since the reticulocyte count goes up
sharply on the 6th day of appropriate treatment. Further,
the sharpness of the rise in reticulocyte count made the
assessment of improvement of the patient more reliable.
Availability of a suitable technique is a marvellous aid to
discovery. In many cases, one can even say that a discov-
ery has to wait till a suitable technique is available.
2. Castle used beef muscle instead of beef liver in his experi-
ments. He used it because Minot and Murphy had attrib-
uted success of liver therapy to liver proteins. Castle
thought muscle proteins are not very different from liver
proteins (which is quite right), and therefore chose to
use muscle instead of liver in his experiments. However,
the vitamin B12 content of muscle is much poorer than
that of liver. Therefore, if Castle had used 200 g liver,
the patients would have improved on liver alone (as in
Minot and Murphy’s experiments) as well as on liver plus
normal gastric juice. That would have made the experi-
ments more difcult to interpret.1 Thus Castle’s decision
to use muscle instead of liver turned out to be an unfore-
seen happy departure.
3. The concept of dietary deciency of nutrients required in
minute quantities had already been established for about
a quarter century. The right atmosphere can be quite
crucial for right interpretation of observations.2

1
The experiments could have become more informative if Castle would have chosen to reduce the quantity of liver. 5 g liver would be ineffective alone
but would cure pernicious anaemia if fed with normal gastric juice.
2
It is interesting that when Eijkman discovered in 1897 that a polished rice diet was the cause of beriberi, and that the condition could be cured by feed-
ing unpolished rice, he interpreted it as follows: rice husk contains an antidote to a nerve poison produced in the intestine by a starchy diet. Those were
the days dominated by many rapid discoveries of bacteria and toxins as the cause of disease, and no vitamin had yet been discovered. Hence it was
natural for Eijkman to arrive at an interpretation which today looks much more complex than the dietary deciency explanation. Thus the thinking of 63
scientists, like that of other human beings, is also coloured by the era in which they live.
SECTION 2: Blood
After the identication of vitamin B12 in 1948, Castle’s
associates repeated the experiments with the vitamin. Once
again, the results were the same. Small doses of vitamin B12
alone, or normal gastric juice alone, were ineffective. But
vitamin B12 and gastric juice together resulted in marked
improvement in patients of pernicious anaemia.
The question still remained as to what the nature of the
interaction between vitamin B12 and intrinsic factor was.
After struggling with several possibilities, intensive work
by several scientists has revealed that intrinsic factor is
required for absorption of vitamin B12 in the distal ileum.
The next question is why the stomach undergoes atrophy in
patients of pernicious anemia. The disease is now believed
to be autoimmune in nature. Work is still going on to nd
out whether the different type of antibodies seen in per-
nicious anaemia are the cause or the effect of the disease.
There is a possibility that a viral infection might trigger the
autoimmune process. All these are questions which future
work might answer.
be able to substitute for those functions of vitamin B 12 which
do not involve an interaction with folate. One such function is
conversion of methylmalonyl CoA to succinyl CoA. The others
are conversion of homocystein to methionine, and of methio-
nine to adenosylmethionine. Impairment of some of these con-
versions may be responsible for the neurological lesions of per-
nicious anaemia.
Clinical and Laboratory Features
Folic acid deciency is usually dietary in origin and occurs
most commonly in pregnant women due to enhanced
demands. It results in macrocytic, megaloblastic, normochro-
mic anaemia.
Vitamin B12 deciency is rarely dietary in origin. It is usu-
ally the result of poor intestinal absorption of vitamin B12.
Absorption of vitamin B12 requires a glycoprotein manufac-
tured by the parietal cells of the stomach. The protein is known
as intrinsic factor and forms a complex with vitamin B 12.
Specialized receptors for this complex occur in the mucosa
of the terminal ileum where vitamin B12 gets absorbed. If the
stomach has been surgically removed, or if there is gastric atro-
phy, intrinsic factor is not available and hence absorption of
vitamin B12 is impaired. Since parietal cells manufacture not
only intrinsic factor but also hydrochloric acid, intrinsic factor
deciency is associated with achlorhydria. The type of anaemia
seen in vitamin B12 deciency is also macrocytic, megaloblas-
tic and normochromic.
Anemia of any type stimulates erythropoietin production.
Therefore the relative area of bone marrow dedicated to the
erythroid series of cells expands, the absolute extent of bone
marrow involved in haemopoiesis expands and even extramed-
ullary sites such as liver and spleen may start manufactur-
ing blood cells. But in folate and vitamin B12 deciency, the
maturation of blood cells is arrested. Hence there is prepon-
derance of immature cells, which seem to be somewhat reluc-
64 tantly released into the peripheral circulation. Thus the marrow
shows much activity without achievement. The marrow is
overactive but underproductive. The peripheral blood has a low
RBC count. A large fractions of these RBC are large and imma-
ture (megaloblasts), and the remaining are neither uniform in
size (anisocytosis) nor in appearance (poikilocytosis).
In vitamin B12 deciency, besides anaemia there is also
neural involvement. The severity of neural lesions is not related
to the severity of anaemia. Neural lesions predominantly
involve nerve bers in dorsal and lateral columns of the spinal
cord. In addition, nerve bers in cerebral cortex and peripheral
nerves may also be affected. Since multiple nerve pathways are
involved, the complex is called subacute combined degenera-
tion or combined system disease.
Since folate can correct only some of the metabolic abnor-
malities of vitamin B12 deciency, it is important to distinguish
between folate deciency and vitamin B12 deciency. Besides
the patient’s history and clinical features, the following labora-
tory tests help distinguish between the two deciencies:
1. Serum Levels
Serum folate level is rather labile, and therefore unreliable as
an indicator of folate nutriture. Folate concentration of RBC is
a somewhat better indicator.
Serum B12 is low in vitamin B12 deciency, but may be low
also in folate deciency. The test may be repeated after admin-
istration of folate. If folate does not correct the low serum B12
level, it indicates a genuine B12 deciency. If folate brings the
serum B12 level to normal, the deciency was possibly that of
folate.
2. Urinary Metabolites
If a vitamin is deficient, the substrates of the reactions in
which the vitamin acts as a cofactor accumulate. Accumulation
of some of the substrates may be reected in their increased
urinary excretion. Based on this principle, B 12 deficiency
may be assessed from methylmalonate excretion, and
folate deficiency from formiminoglutamic acid (FIGLU)
excretion.
a. Methylmalonate excretion
Vitamin B12 acts as a cofactor in the reaction
Methylmalonyl CoA Succinyl CoA
Hence urinary methylmalonate excretion is increased in
vitamin B12 deciency.
b. FIGLU excretion
Folate acts as a cofactor in the reaction.
Histidine FIGLU Glutamic acid
Hence urinary FIGLU excretion is increased in folate
deciency.
3. Deoxiuridine Suppression Test
If tritiated thymidine is added to cultured bone marrow cells, it
gets incorporated into DNA (control). However, if we rst add
deoxiuridine (dU) and then tritiated thymidine, the dividing
cells convert dU into thymidine and use it for DNA synthesis.
Therefore they do not have much use for tritiated thymidine.
Hence tritiated thymidine incorporation in this case is less than
10% of that in control. This is the degree of dU suppression if
the bone marrow cells are from a normal person.

Since conversion of dU into the thymidine requires
5, 10-methylene THFA, the conversion is impaired in folate
as well as B 12 deciency. Therefore in case of either de-
ciency, incorporation of tritiated thymidine into DNA is not as
much suppressed as in normal individuals. Addition of folate
to the culture medium restores the normal degree of suppres-
sion in either deciency because it can overcome the methyl-
folate trap even in B12 de-ciency. But addition of B12 to the
culture medium restores normal dU suppression only in B12
deciency.
4. Vitamin B12 Absorption Test (Schilling’s Test)
This test has been discussed in Chapter 6.11.
Treatment of Folate Deciency Anaemia
It would be ideal to prevent folate deciency by incorporating
enough of green leafy vegetables in the diet. But if deciency
does occur, it can be speedily corrected by using folic acid tab-
lets. As the metabolic role of folic acid and vitamin B12 shows,
folic acid deciency will not respond to administration of vita-
min B12.
Treatment of Vitamin B12 Deciency Anaemia
If vitamin B12 deficiency is due to intrinsic factor defi-
ciency, which is usually the case, it cannot be treated by
oral administration of vitamin B 12. It is, however, very
conveniently treated by intramuscular injections of vitamin
B12. Since vitamin B12 can be stored in the liver, one injec-
tion of a large dose can supply several months’ supply of the
vitamin.
CHAPTER 2.5: Pathophysiology of Anaemias
However, it is important not to treat vitamin B12 deciency
anaemia with folate. Dietary folate can temporarily overcome
the effects of methylfolate trap, and thereby improve the anae-
mia. But folate cannot perform the role of vitamin B12 in the
nervous system. Therefore if vitamin B12 deciency is treated
with folate, there is a deceptive improvement in anaemia while
the neural lesions continue to progress.
Fortunately pernicious anaemia is not very common, and is
rarer in India than in some western countries. But folate de-
ciency is quite common in India, specially among pregnant
women. But still we have discussed pernicious anaemia also
in considerable detail because of its academic importance: we
know quite a lot about it, and the history of the discovery of
intrinsic factor is very interesting and instructive.
QUESTIONS AND PROBLEMS
1. How can half a pound of liver per day cure pernicious anae-
mia even without intrinsic factor.
ANSWERS AND SOLUTIONS
1. Vitamin B12 is absorbed in the intestine by two mecha-
nisms. One is an efcient and specic receptor-mediated
mechanism which needs intrinsic factor. The other is the
nonspecic mechanism of diffusion which is very inef-
cient but does not need intrinsic factor. Half a pound of beef
liver contains about 250 micrograms of vitamin B12. From
this intake, the daily requirement of 1 microgram can be
absorbed even by the passive process of diffusion.
65