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 insidious 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 constellation of clinical and laboratory findings. At the beginning of the 20th century, pernicious 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 suggested 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 wellbeing. Within 2 to 4 months, the red-cell count rose to normal levels in virtually all the patients
n engl j med 370;8 nejm.org february 20, 2014
The New England Journal of Medicine Downloaded from nejm.org at CALIFORNIA STATE UNIV FRESNO on May 29, 2014. For personal use only. No other uses without permission. Copyright © 2014 Massachusetts Medical Society. All rights reserved.
773
The
n e w e ng l a n d j o u r na l
who were able to adhere to the prescribed diet.6 In many patients, there was a dramatic improvement 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 principle 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
of
m e dic i n e
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 Woodward and his team at Harvard University achieved a tour-de-force chemical synthesis of this very complex molecule. The latter three scientists were also Nobel laureates. One year after the discovery of liver therapy
A Clinical Features Anemia
Sore Tongue
Autoimmune Gastritis
Numbness and Ataxia
Hypersegmented neutrophils Megaloblastic maturation
Atrophic glossitis Loss of papillae
Atrophy of parietal cells Achlorhydria Intrinsic factor reduced
Demyelination of posterior and lateral columns
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 stomach 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).
774
n engl j med 370;8
nejm.org
february 20, 2014
The New England Journal of Medicine Downloaded from nejm.org at CALIFORNIA STATE UNIV FRESNO on May 29, 2014. For personal use only. No other uses without permission. Copyright © 2014 Massachusetts Medical Society. All rights reserved.
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 anemia, Castle wondered whether the extrinsic factor in liver that cured these patients interacted
with an intrinsic factor normally present within the stomach.7 He tested this hypothesis by aspirating 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 lumen
Gastric parietal cell
Intrinsic factor Intrinsic factor
H+/K+ ATPase ATP
B12
H+
ADP
Dendritic cell Immune attack Distal ileum
H+/K+– ATPase reactive CD4 T cell
Lymphocytes
Paragastric lymph node
COLOR FIGURE Figure 2. Pathogenesis of Autoimmune Atrophic Gastritis in Pernicious Anemia. Draft 3 cells produced 2/3/14 The autoimmune attack is directed by dendritic cells in the stomach that clear apoptotic parietal Authornodes, Bunnwhere they actiduring the normal turnover of gastric mucosa. The dendritic cells include paragastric lymph 2 Fig # vate CD4 T cells that recognize H+/K+ ATPase expressed in gastric parietal cells.9
Title ME
DE Artist
Longo
Knoper AUTHOR PLEASE NOTE:
n engl j med 370;8
nejm.org
february 20, 2014
Figure has been redrawn and type has been reset Please check carefully
Issue date 2/20/14
The New England Journal of Medicine Downloaded from nejm.org at CALIFORNIA STATE UNIV FRESNO on May 29, 2014. For personal use only. No other uses without permission. Copyright © 2014 Massachusetts Medical Society. All rights reserved.
775
Basic Implications of Clinical observations
was observed within approximately 5 days, followed by a rise in the red-cell count.8 The timing and extent of this hematologic response was similar to that achieved with oral liver treatment. He then showed that the intragastric administration 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 factor within stomach juice is a protein that is both necessary and sufficient for the absorption of small (dietary) amounts of vitamin B12 in the distal small intestine (Fig. 2). Castle’s discovery of this intrinsic factor not only paved the way for our current understanding 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 serum antibodies against gastric parietal cells, and approximately 50% have antibodies against intrinsic factor. This autoimmune attack is directed by dendritic cells in the stomach that clear apoptotic parietal cells produced during the normal turnover of gastric mucosa (Fig. 2). These dendritic 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 diseases 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 autoimmune thyroiditis, a combination designated as the autoimmune polyglandular syndrome type 3B. In addition, atrophic gastritis is often encountered 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 pernicious anemia. A sizable minority of patients
776
with documented vitamin B12 deficiency are infected 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 insufficiency. George Minot was a lean man who had brittle diabetes, and he was one of the first patients in Boston successfully treated with insulin. If he had lived another decade or so, he would have been at substantially higher risk for autoimmune 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 reticulocytes 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 pernicious 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;42:26978. 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.
n engl j med 370;8 nejm.org february 20, 2014
The New England Journal of Medicine Downloaded from nejm.org at CALIFORNIA STATE UNIV FRESNO on May 29, 2014. For personal use only. No other uses without permission. Copyright © 2014 Massachusetts Medical Society. All rights reserved.
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 insidious 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 constellation of clinical and laboratory findings. At the beginning of the 20th century, pernicious 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 suggested 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 wellbeing. Within 2 to 4 months, the red-cell count rose to normal levels in virtually all the patients
n engl j med 370;8 nejm.org february 20, 2014
The New England Journal of Medicine Downloaded from nejm.org at CALIFORNIA STATE UNIV FRESNO on May 29, 2014. For personal use only. No other uses without permission. Copyright © 2014 Massachusetts Medical Society. All rights reserved.
773
The
n e w e ng l a n d j o u r na l
who were able to adhere to the prescribed diet.6 In many patients, there was a dramatic improvement 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 principle 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
of
m e dic i n e
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 Woodward and his team at Harvard University achieved a tour-de-force chemical synthesis of this very complex molecule. The latter three scientists were also Nobel laureates. One year after the discovery of liver therapy
A Clinical Features Anemia
Sore Tongue
Autoimmune Gastritis
Numbness and Ataxia
Hypersegmented neutrophils Megaloblastic maturation
Atrophic glossitis Loss of papillae
Atrophy of parietal cells Achlorhydria Intrinsic factor reduced
Demyelination of posterior and lateral columns
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 stomach 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).
774
n engl j med 370;8
nejm.org
february 20, 2014
The New England Journal of Medicine Downloaded from nejm.org at CALIFORNIA STATE UNIV FRESNO on May 29, 2014. For personal use only. No other uses without permission. Copyright © 2014 Massachusetts Medical Society. All rights reserved.
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 anemia, Castle wondered whether the extrinsic factor in liver that cured these patients interacted
with an intrinsic factor normally present within the stomach.7 He tested this hypothesis by aspirating 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 lumen
Gastric parietal cell
Intrinsic factor Intrinsic factor
H+/K+ ATPase ATP
B12
H+
ADP
Dendritic cell Immune attack Distal ileum
H+/K+– ATPase reactive CD4 T cell
Lymphocytes
Paragastric lymph node
COLOR FIGURE Figure 2. Pathogenesis of Autoimmune Atrophic Gastritis in Pernicious Anemia. Draft 3 cells produced 2/3/14 The autoimmune attack is directed by dendritic cells in the stomach that clear apoptotic parietal Authornodes, Bunnwhere they actiduring the normal turnover of gastric mucosa. The dendritic cells include paragastric lymph 2 Fig # vate CD4 T cells that recognize H+/K+ ATPase expressed in gastric parietal cells.9
Title ME
DE Artist
Longo
Knoper AUTHOR PLEASE NOTE:
n engl j med 370;8
nejm.org
february 20, 2014
Figure has been redrawn and type has been reset Please check carefully
Issue date 2/20/14
The New England Journal of Medicine Downloaded from nejm.org at CALIFORNIA STATE UNIV FRESNO on May 29, 2014. For personal use only. No other uses without permission. Copyright © 2014 Massachusetts Medical Society. All rights reserved.
775
Basic Implications of Clinical observations
was observed within approximately 5 days, followed by a rise in the red-cell count.8 The timing and extent of this hematologic response was similar to that achieved with oral liver treatment. He then showed that the intragastric administration 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 factor within stomach juice is a protein that is both necessary and sufficient for the absorption of small (dietary) amounts of vitamin B12 in the distal small intestine (Fig. 2). Castle’s discovery of this intrinsic factor not only paved the way for our current understanding 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 serum antibodies against gastric parietal cells, and approximately 50% have antibodies against intrinsic factor. This autoimmune attack is directed by dendritic cells in the stomach that clear apoptotic parietal cells produced during the normal turnover of gastric mucosa (Fig. 2). These dendritic 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 diseases 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 autoimmune thyroiditis, a combination designated as the autoimmune polyglandular syndrome type 3B. In addition, atrophic gastritis is often encountered 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 pernicious anemia. A sizable minority of patients
776
with documented vitamin B12 deficiency are infected 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 insufficiency. George Minot was a lean man who had brittle diabetes, and he was one of the first patients in Boston successfully treated with insulin. If he had lived another decade or so, he would have been at substantially higher risk for autoimmune 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 reticulocytes 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 pernicious 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;42:26978. 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.
n engl j med 370;8 nejm.org february 20, 2014
The New England Journal of Medicine Downloaded from nejm.org at CALIFORNIA STATE UNIV FRESNO on May 29, 2014. For personal use only. No other uses without permission. Copyright © 2014 Massachusetts Medical Society. All rights reserved.
