The n e w e ng l a n d j o u r na l of m e dic i n e

clinical implications of basic research

Dan L. Longo, M.D., Editor

The Clinical Implications of Basic Research series has focused on highlighting laboratory research that could lead
to advances in clinical therapeutics. However, the path between the laboratory and the bedside runs both ways:
clinical observations often pose new questions for laboratory investigations that then lead back to the clinic.
One of a series of occasional articles drawing attention to the bedside-to-bench flow of information is presented here,
under the Basic Implications of Clinical Observations rubric. We hope our readers will enjoy these stories of discovery,
and we invite them to submit their own examples of clinical findings that have led to insights in basic science.

basic implications of clinical observations

Vitamin B12 and Pernicious Anemia — The Dawn

of Molecular Medicine
H. Franklin Bunn, M.D.

“From bedside to bench and back again” is a
time-honored trajectory to which many medical
students, residents, and fellows aspire. Arguably
the earliest and most celebrated completion of
this circuit was the dietary cure of pernicious
anemia that drove the discovery of vitamin B12
(cobalamin) and its physiologic role.
The story begins in 1855 with Thomas
Addison’s initial report of a patient with the in-
sidious development of pale countenance, languor,
and “increasing indisposition to exertion.”1 Over
the next 30 years, it became apparent that many
patients with this clinical presentation had a
sore, beefy red, smooth tongue, and some had
digital numbness and tingling that occasionally
progressed to spasticity and ataxia. By the end
of the 19th century, patients with these clinical
manifestations were shown to have atrophy of
the gastric mucosa and absence of acid in the
gastric juice as well as an anemia characterized
by large, oval erythrocytes. The term “pernicious
anemia” crept into common medical parlance
for designating patients with this striking con-
stellation of clinical and laboratory findings.
At the beginning of the 20th century, perni-
cious anemia attracted the attention of a number
of inquisitive Boston physicians. In 1908, Richard
Cabot reported that the duration of survival
among 1200 patients was usually 1 to 3 years
after the onset of symptoms.2 Francis Weld
Peabody and George Richards Minot shared a
deep appreciation of the importance of cell
morphology in the diagnosis and monitoring of
blood disorders.3 Peabody’s observations on bone
marrow biopsy specimens from patients with
pernicious anemia led him to conclude that
their red-cell production was disordered and
ineffective. George Whipple at the University of
Rochester had recently explored the effect of
diet on the regeneration of red cells in dogs
after periodic phlebotomy.4 These experiments
prompted Peabody and Minot to independently
initiate dietary trials involving patients with
pernicious anemia.
Minot joined forces with William Murphy at
the Peter Bent Brigham Hospital. The two were
extraordinarily thorough, both at the bedside and
in the laboratory. Whipple’s results in dogs sug-
gested that liver should be the key component
of their dietary regimen. In 1926, Minot and
Murphy reported the results of studies involving
45 patients with pernicious anemia who had
been treated with “a special diet” consisting of
seared (nearly raw) liver, along with beef or
mutton and fresh fruit.5 By the end of the first
week of treatment, a striking increase in the
number of new red cells (reticulocytes) was
­accompanied by a clear improvement in well-
being. Within 2 to 4 months, the red-cell count
rose to normal levels in virtually all the patients

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 The n e w e ng l a n d j o u r na l of m e dic i n e

who were able to adhere to the prescribed diet.6
In many patients, there was a dramatic improve-
ment in the neurologic manifestations. In 1934,
the Nobel Prize in Physiology or Medicine was
bestowed on Minot, Murphy, and Whipple “in
recognition of their discoveries respecting liver
therapy in anaemias.”
The remarkable efficacy of this treatment of
a hitherto incurable and generally fatal disease
prompted a vigorous search for the active prin-
ciple in liver that was responsible for the cure.
In collaboration with Edwin Cohn, a chemist at
Harvard Medical School, Minot and Murphy were
able to test the efficacy of purified liver extracts
in patients with pernicious anemia. Biochemical
fractionation of these extracts coupled with the
development of a microbiologic assay eventually
led to the isolation and partial characterization
of vitamin B12 in 1948 by Karl Folkers at Merck.
In 1955, Alexander Todd and Dorothy Hodgkin
at Cambridge University in England collaborated
to solve the chemical and three-dimensional
structure of vitamin B12. In 1973, Robert Wood-
ward and his team at Harvard University
achieved a tour-de-force chemical synthesis of
this very complex molecule. The latter three scien-
tists were also Nobel laureates.
One year after the discovery of liver therapy

A Clinical Features
Anemia Sore Tongue Autoimmune Gastritis Numbness and Ataxia
Hypersegmented Atrophic glossitis Atrophy of parietal cells Demyelination of posterior
neutrophils Loss of papillae Achlorhydria and lateral columns
Megaloblastic Intrinsic factor reduced
maturation

B Hematologic Response C William B. Castle

Figure 1. Pernicious Anemia.

Panel A shows clinical features of patients with pernicious anemia. Panel B shows hematologic responses in two
patients with pernicious anemia, as assessed by William Castle (Panel C).8 One patient (solid diamonds) received
oral liver treatment, and the other (open circles) received an intragastric infusion of the contents of a normal stom-
ach of a human after the ingestion of 300 g of beef. Dashed lines indicate red cells (RBC) per cubic millimeter, and
solid lines the percent of reticulocytes (retics).

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 Basic Implications of Clinical observations

for pernicious anemia, William B. Castle joined
Minot’s clinic and laboratory at Boston City
Hospital. Keenly aware of the nearly universal
presence of atrophic gastritis in pernicious ane-
mia, Castle wondered whether the extrinsic fac-
tor in liver that cured these patients interacted
with an intrinsic factor normally present within
the stomach.7 He tested this hypothesis by aspi-
rating his own gastric juice after the ingestion
of a hamburger and transferring it into the
stomach of patients with pernicious anemia. As
shown in Figure 1, a brisk rise in reticulocytes

Gastric
Gastric lumen
parietal
cell Intrinsic
factor

H+/K+ Intrinsic
factor B12
ATPase
ATP H+
ADP

Dendritic
cell

Immune
attack

Distal ileum

H+/K+–
ATPase reactive
CD4 T cell

Paragastric
Lymphocytes lymph node

Figure 2. Pathogenesis of Autoimmune Atrophic Gastritis in Pernicious Anemia. COLOR FIGURE

Draft 3 cells produced

The autoimmune attack is directed by dendritic cells in the stomach that clear apoptotic parietal 2/3/14
Bunnwhere they acti-
Authornodes,
during the normal turnover of gastric mucosa. The dendritic cells include paragastric lymph
Fig # 2
vate CD4 T cells that recognize H+/K+ ATPase expressed in gastric parietal cells.9
Title

ME
DE Longo
Artist Knoper
AUTHOR PLEASE NOTE:
Figure has been redrawn and type has been reset

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 Basic Implications of Clinical observations
was observed within approximately 5 days, fol-
lowed by a rise in the red-cell count.8 The tim-
ing and extent of this hematologic response was
similar to that achieved with oral liver treat-
ment. He then showed that the intragastric ad-
ministration of normal stomach juice and beef
muscle was effective only if given together or
within a 12-hour period. Subsequent studies by
Castle and others showed that the intrinsic fac-
tor within stomach juice is a protein that is both
necessary and sufficient for the absorption of
small (dietary) amounts of vitamin B12 in the dis-
tal small intestine (Fig. 2).
Castle’s discovery of this intrinsic factor not
only paved the way for our current understand-
ing of the subtle and elegant mode of absorption
of vitamin B12 in the gastrointestinal tract but
also provided a crucial clue to the autoimmune
pathogenesis of pernicious anemia. More than
90% of patients with pernicious anemia have se-
rum antibodies against gastric parietal cells, and
approximately 50% have antibodies against in-
trinsic factor. This autoimmune attack is directed
by dendritic cells in the stomach that clear apop-
totic parietal cells produced during the normal
turnover of gastric mucosa (Fig. 2). These den-
dritic cells travel to paragastric lymph nodes,
where they activate CD4 T cells that recognize
hydrogen–potassium ATPase expressed in gastric
parietal cells.9
Pernicious anemia is but one of a host of dis-
eases that arises because of the interplay between
nature and nurture. The distribution of certain
HLA polymorphisms is substantially skewed in
persons with chronic atrophic gastritis as well
as those with autoimmune thyroiditis. Indeed,
atrophic gastritis is strongly associated with auto-
immune thyroiditis, a combination designated as
the autoimmune polyglandular syndrome type 3B.
In addition, atrophic gastritis is often encoun-
tered with other autoimmune disorders such
as type 1 diabetes, vitiligo, Addison’s disease
(chronic primary adrenal insufficiency), and
Graves’ disease (hyperthyroidism).
The environment appears to play a crucial,
independent role in the pathogenesis of perni-
cious anemia. A sizable minority of patients
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with documented vitamin B12 deficiency are in-
fected with Helicobacter pylori. One study reported
that approximately half the infected patients had
a complete and generally durable hematologic
remission with antibacterial therapy alone.10
The illustrious history of pernicious anemia,
from bedside to bench and back again, invites
speculation. If Thomas Addison’s patient with
severe anemia had been blessed with vitamin
B12 treatment, in later life he would have been
at increased risk for Addisonian adrenal insuf-
ficiency. George Minot was a lean man who had
brittle diabetes, and he was one of the first pa-
tients in Boston successfully treated with insulin.
If he had lived another decade or so, he would
have been at substantially higher risk for auto-
immune atrophic gastritis and pernicious anemia.
Disclosure forms provided by the author are available with the
full text of this article at NEJM.org.
I thank Dr. Paul Anderson, Dana–Farber Cancer Institute,
and Dr. Scott Lovitch, Brigham and Women’s Hospital, Harvard
Medical School, for helpful suggestions.
From the Division of Hematology, Department of Medicine,
Brigham and Women’s Hospital, Boston.
1. Addison T. On the constitutional and local effects of disease
of the suprarenal capsules. London: Samuel Highley, 1855:1-43.
2. Cabot RC. Pernicious and secondary anemia, chlorosis and
leukemia. In: Osler W, Mac CT, eds. Modern medicine. Vol. 4.
Philadelphia: Lea and Febiger, 1908:612-8.
3. Rackemann FM. The inquisitive physician: the life and times
of George Richards Minot. Cambridge, MA: Harvard University
Press, 1956.
4. Corner GW. George Hoyt Whipple and his friends.
Philadelphia: J.B. Lippincott, 1963.
5. Minot GR, Murphy WP. Treatment of pernicious anemia by a
special diet. JAMA 1926;87:470-6.
6. Minot GR, Murphy WP, Stetson RP. The response of the re-
ticulocytes to liver therapy: particularly in pernicious anemia.
Am J Med Sci 1928;175:581-99.
7. Castle WB. The conquest of pernicious anemia. In: Wintrobe
MM, ed. Blood, pure and eloquent. New York: McGraw-Hill, 1980:
284-317.
8. Idem. The effect of the administration to patients with perni-
cious anaemia of the contents of the normal human stomach
after ingestion of beef muscle. Am J Med Sci 1929;178:748-63.
9. Toh BH, Chan J, Kyaw T, Alderuccio F. Cutting edge issues in
autoimmune gastritis. Clin Rev Allergy Immunol 2012;42:269-
78.
10. Kaptan K, Beyan C, Ural AU, et al. Helicobacter pylori — is
it a novel causative agent in vitamin B12 deficiency? Arch Intern
Med 2000;160:1349-53.
DOI: 10.1056/NEJMcibr1315544
Copyright © 2014 Massachusetts Medical Society.