The Vitamins Fifth Edition Fundamental Aspects in Nutrition and Health Gerald F Combs JR All Chapter

The Vitamins, Fifth Edition:
Fundamental Aspects in Nutrition and

Health Gerald F. Combs Jr.
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The Vitamins
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The Vitamins
Fundamental Aspects in Nutrition and Health
Fifth Edition
Gerald F. Combs, Jr., Ph.D.

Professor Emeritus
Cornell University
Ithaca, NY
James P. McClung, Ph.D.

Westborough, MA
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Dedication
To the students and professionals who have used this book

and whose comments and suggestions have helped us
produce this fifth edition
–The Authors, Fifth Edition
and
to Barbara, ma fleur
–Jerry
and
to Holly, Elliott, and Adeline

–James

Contents
Preface to the Fifth Edition xi 8. Study Questions and Exercises 58

How to Use This Book xiii Recommended Reading 58
4. Vitamin Deficiency
1. The Concept of Vitamin Deficiency 60
Part I 2. Clinical Manifestations of Vitamin
Perspectives on the Vitamins in Deficiencies61
Nutrition 3. Causes of Vitamin Deficiencies 65
4. Study Questions and Exercises 78
1. What Is a Vitamin? Recommended Reading 78
1. Thinking About Vitamins 3 5. Vitamin Needs and Safety

2. Vitamin: A Revolutionary Concept 3
3. An Operating Definition of a Vitamin 4 1. Dietary Standards for Vitamins 80
4. The Recognized Vitamins 5 2. Vitamin Allowances for Humans 87
5. Study Questions and Exercises 5 3. Vitamin Allowances for Animals 89
4. Uses of Vitamins Above Required
Levels89
2. Discovery of the Vitamins
5. Hypervitaminoses 96
1. The Emergence of Nutrition as a Science 8 6. Safe Intakes of Vitamins 102
2. The Process of Discovery in Nutritional 7. Study Questions and Exercises 105
Science8 Recommended Reading 105
3. The Empirical Phase of Vitamin Discovery 8
4. The Experimental Phase of Vitamin
Discovery12 Part II
5. The Vitamine Theory 14 Considering the Individual Vitamins
6. Elucidation of the Vitamins 18
7. Vitamin Terminology 28 6. Vitamin A
8. Other Factors Sometimes Called Vitamins 28
1. Significance of Vitamin A 110
9. Modern History of the Vitamins 29
2. Properties of Vitamin A 111
3. Sources of Vitamin A 112
Recommended Reading 30
4. Absorption of Vitamin A 115
5. Transport of Vitamin A 118
3. General Properties of Vitamins 6. Metabolism of Vitamin A 125
1. Vitamin Nomenclature 34 7. Metabolic Functions of Vitamin A 129
2. Chemical and Physical Properties of the 8. Biomarkers of Vitamin A Status 137
Vitamins36 9. Vitamin A Deficiency 139
3. Physiological Utilization of the Vitamins 43 10. Vitamin A in Health and Disease 147
4. Metabolism of the Vitamins 50 11. Vitamin A Toxicity 153
5. Metabolic Functions of the Vitamins 51 12. Case Studies 156
6. Vitamin Bioavailability 52 13. Study Questions and Exercises 158
7. Vitamin Analysis 52 Recommended Reading 159
vii
viii Contents
7. Vitamin D 4. Absorption of Vitamin C 272

5. Transport of Vitamin C 272
1. Significance of Vitamin D 162 6. Metabolism of Vitamin C 274
2. Properties of Vitamin D 163 7. Metabolic Functions of Vitamin C 275
3. Sources of Vitamin D 164 8. Biomarkers of Vitamin C Status 283
4. Enteric Absorption of Vitamin D 170 9. Vitamin C Deficiency 284
5. Transport of Vitamin D 171 10. Vitamin C in Health and Disease 286
6. Metabolism of Vitamin D 173 11. Vitamin C Toxicity 292
7. Metabolic Functions of Vitamin D 176 12. Case Studies 293
8. Biomarkers of Vitamin D Status 190 13. Study Questions and Exercises 295
9. Vitamin D Deficiency 192 Recommended Reading 295
10. Vitamin D in Health and Disease 198
11. Vitamin D Toxicity 202
11. Thiamin
12. Case Studies 204
13. Study Questions and Exercises 205 1. The Significance of Thiamin 298
Recommended Reading 205 2. Properties of Thiamin 298
3. Sources of Thiamin 299
8. Vitamin E 4. Absorption of Thiamin 301
5. Transport of Thiamin 302
1. Significance of Vitamin E 208
6. Metabolism of Thiamin 303
2. Properties of Vitamin E 208
7. Metabolic Functions of Thiamin 304
3. Sources of Vitamin E 210
8. Biomarkers of Thiamin Status 308
4. Absorption of Vitamin E 212
9. Thiamin Deficiency 309
5. Transport of Vitamin E 214
10. Role of Thiamin in Health and Disease 312
6. Metabolism of Vitamin E 219
11. Thiamin Toxicity 313
7. Metabolic Functions of Vitamin E 221
8. Biomarkers of Vitamin E Status 227
9. Vitamin E Deficiency 229
10. Vitamin E in Health and Disease 231
11. Vitamin E Toxicity 239
12. Riboflavin
13. Study Questions and Exercises 241 1. The Significance of Riboflavin 316
Recommended Reading 241 2. Properties of Riboflavin 316
3. Sources of Riboflavin 317
9. Vitamin K 4. Absorption of Riboflavin 318
1. The Significance of Vitamin K 244 5. Transport of Riboflavin 319
2. Properties of Vitamin K 244 6. Metabolism of Riboflavin 320
3. Sources of Vitamin K 245 7. Metabolic Functions of Riboflavin 322
4. Absorption of Vitamin K 249 8. Biomarkers of Riboflavin Status 323
5. Transport of Vitamin K 249 9. Riboflavin Deficiency 323
6. Metabolism of Vitamin K 250 10. Riboflavin in Health and Disease 327
7. Metabolic Functions of Vitamin K 253 11. Riboflavin Toxicity 328
8. Biomarkers of Vitamin K Status 258 12. Case Study 328
9. Vitamin K Deficiency 259 13. Study Questions and Exercises 329
10. Vitamin K Health and Disease 262 Recommended Reading 329
11. Vitamin K Toxicity 262
12. Case Studies 263 13. Niacin
1. The Significance of Niacin 332
2. Properties of Niacin 332
3. Sources of Niacin 333
10. Vitamin C 4. Absorption of Niacin 334
1. The Significance of Vitamin C 268 5. Transport of Niacin 335
2. Properties of Vitamin C 268 6. Metabolism of Niacin 336
3. Sources of Vitamin C 269 7. Metabolic Functions of Niacin 340
Contents ix
8. Biomarkers of Niacin Status 342 10. Pantothenic Acid in Health and Disease 396
9. Niacin Deficiency 343 11. Pantothenic Acid Toxicity 397
10. Niacin in Health and Disease 344 12. Case Study 397
11. Niacin Toxicity 348 13. Study Questions and Exercises 398
12. Case Study 348 Recommended Reading 398
Recommended Reading 349 17. Folate
14. Vitamin B6 1. The Significance of Folate 400
2. Properties of Folate 400
1. The Significance of Vitamin B6352 3. Sources of Folate 402
2. Properties of Vitamin B6352 4. Absorption of Folate 404
3. Sources of Vitamin B6352 5. Transport of Folate 406
4. Absorption of Vitamin B6353 6. Metabolism of Folate 408
5. Transport of Vitamin B6355 7. Metabolic Functions of Folate 413
6. Metabolism of Vitamin B6356 8. Biomarkers of Folate Status 419
7. Metabolic Functions of Vitamin B6358 9. Folate Deficiency 420
8. Biomarkers of Vitamin B6 Status 365 10. Folate in Health and Disease 425
9. Vitamin B6 Deficiency 366 11. Folate Toxicity 427
10. Vitamin B6 in Health and Disease 367 12. Case Study 427
11. Vitamin B6 Toxicity 369 13. Study Questions and Exercises 428
12. Case Studies 369 Recommended Reading 428
18. Vitamin B12
15. Biotin 1. Significance of Vitamin B12432
2. Properties of Vitamin B12432
1. The Significance of Biotin 372
3. Sources of Vitamin B12433
2. Properties of Biotin 372
4. Absorption of Vitamin B12435
3. Sources of Biotin 372
5. Transport of Vitamin B12436
4. Absorption of Biotin 374
6. Metabolism of Vitamin B12439
5. Transport of Biotin 374
7. Metabolic Functions of Vitamin B12440
6. Metabolism of Biotin 376
8. Biomarkers of Vitamin B12 Status 443
7. Metabolic Functions of Biotin 376
9. Vitamin B12 Deficiency 444
8. Biomarkers of Biotin Status 380
10. Vitamin B12 in Health and Disease 450
9. Biotin Deficiency 380
11. Vitamin B12 Toxicity 450
10. Biotin in Health and Disease 382
12. Case Study 450
11. Biotin Toxicity 383
12. Case Study 383
Recommended Reading 384 19. Vitamin-Like Factors
1. Is the List of Vitamins Complete? 454
16. Pantothenic Acid 2. Choline 455
1. The Significance of Pantothenic Acid 388 3. Carnitine 462
2. Properties of Pantothenic Acid 388 4. Myo-Inositol469
3. Sources of Pantothenic Acid 388 5. Ubiquinones 474
4. Absorption of Pantothenic Acid 389 6. Lipoic Acid 477
5. Transport of Pantothenic Acid 390 7. Nonprovitamin A Carotenoids 480
6. Metabolism of Pantothenic Acid 391 8. Flavonoids 487
7. Metabolic Functions of Pantothenic 9. Orotic Acid 494
Acid393 10. Unidentified Factors 495
8. Biomarkers of Pantothenic Acid 11. Case Study 496
Status395 12. Study Questions and Exercises 496
9. Pantothenic Acid Deficiency 395 Recommended Reading 497
x Contents
Part III 3. Vitamin Status of Human Populations 534

4. Global Undernutrition 541
Using Current Knowledge of the 5. Study Questions and Exercises 543
Vitamins Recommended Reading 543
20. Sources of the Vitamins

1. Vitamins in Foods and Feedstuffs 501
Appendix A: Current and Obsolete Designations
2. Vitamin Bioavailability 508
of Vitamins (Bolded) and Other
3. Vitamin Losses in Foods 509
Vitamin-Like Factors 545
4. Vitamin Fortification 511
Appendix B: O riginal Reports for Case Studies 549
5. Biofortification 513
Appendix C: A Core of Current Vitamin Literature 551
6. Vitamin Labeling of Foods 516
Appendix D: Vitamin Contents of Foods (units per
7. Vitamins in Human Diets 517
100 g Edible Portion) 559
8. Vitamin Supplementation 521
Appendix E: Vitamin Contents of Feedstuffs
9. Vitamins in Livestock Feeding 523
(units per kg) 589
10. Case Study 528
Index593
21. Assessing Vitamin Status

1. Nutritional Assessment 531
2. Biomarkers of Vitamin Status 533
Preface to the Fifth Edition
Understanding the vitamins is key to understanding nutri- which prompted several changes that we believe enhanced
tion. The history of their discovery and the continuing eluci- the book. We reorganized several chapters, which reduced
dation of their roles in health is the history of the emergence their number. We emphasized roles of the gut microbiome
of nutrition as a science from the areas of physiology, bio- in several places of importance. We added sections on
chemistry, medicine, and agriculture. biomarkers of vitamin status and modestly expanded the
Capturing the understanding that grew out of that his- section on biofortification. We added, redrew, and updated
tory is both a challenge and a privilege. For us, it involved several tables and figures. We used extensive footnoting as
months of reviewing thousands of publications and look- a means of including explanatory notes as well as for citing
ing for clear ways to present complex information without primary sources.
overstating present understanding. We are grateful for the professional assistance from edi-
Producing this fifth edition of The Vitamins benefitted tors, Ms. Jaclyn Truesdell, Ms. Megan Ball, and Ms. Caroline
from the inclusion of a coauthor, which we believe brought Johnson of Elsevier.
a new prospective to the text. James studied the first edition We enjoyed writing this fifth edition of The Vitamins
of the The Vitamins as a masters student at the University together. We hope you will find it useful.
of New Hampshire in 1997. He encountered the second
edition of the text as Jerry’s student at Cornell University in Gerald F. Combs, Jr.
2001. We are hopeful that the dynamic relationship we have Topsham, Maine
enjoyed, as student/mentor, colleagues, friends, and now James P. McClung
coauthors, has resulted in the most effective edition of this Westborough, Massachusetts
text, as both a reference and a teaching aid. June 2016
In writing this fifth edition of The Vitamins, we were
mindful of comments from users of previous editions,
xi

How to Use This Book
TO THE HEALTH PROFESSIONAL Next, read through the Vocabulary list and mark any terms
that are unfamiliar or about which you feel unsure. Then,
The Vitamins is designed as a one-stop source of compre- make a list of your own questions about the topic of the
hensive, current information on the vitamins. In it you will chapter.
find information on the history of vitamin discovery, the As you read through the text, look for items related to
chemical properties of the vitamins and their isomers and your questions and for unfamiliar terms. You will be able to
metabolites, the utilization and metabolism of vitamins, find key terms in bold-faced type, and you should be able to
the consequences of their deficient and excessive intakes, get a good feel for their meanings from the contexts of their
biomarkers of vitamin status, and the health roles if par- uses. If this is not sufficient for any particular term, then
ticular vitamins in beyond the traditional deficiencies. You look it up in a medical dictionary. Do not wait to do this.
will find examples of classical and current research findings Cultivate the habit of being bothered by not understand-
as well as citations to recent key publications in the foot- ing something—this will help you enormously in years to
notes. You may find Appendix particularly useful, as it lists come.
the vitamin contents of a most common foods. Please let us As you proceed through the text, note what information
know of any ways you see we might enhance The Vitamins. the layout is designed to convey. First, note that the major
sections of each chapter are indicated with a bold heading.
TO STUDENTS AND INSTRUCTORS This is done to help you scan for particular information.
The Vitamins is also intended as a teaching text for an upper- Also note that the footnoted information is largely supple-
level college course within a nutrition or health-related cur- mentary and not essential to the understanding of the key
riculum; however, it will also be useful as a workbook for concepts presented. Therefore, the text may be read at two
self-paced study of the vitamins. It has several features that levels: at the basic level, one should be able to ignore the
are designed to enhance its usefulness to students as well as footnotes and still get the key concepts; at the more detailed
instructors. Here is how we suggest using it. level, one should be able to pick up more background, par-
To the student When you use this text, make sure to ticularly key citations to the primary literature, from the
have by your side a notebook, pencil (not pen—you may footnotes. Refer back frequently to your own list of ques-
want to make changes in the notes you take). Then, before tions and “target” vocabulary words; when you find an
reading each chapter, take a few moments to go over the answer or can make a deduction, make a note. Do not be
“Anchoring Concepts and Learning Objectives” on the reluctant to write in the book, particularly to put a concept
chapter title page. Anchoring Concepts are the ideas funda- into your own words, or to note something you find impor-
mental to the subject matter of the chapter, the concepts to tant or do not fully understand. Studies show that to be an
which the new ones presented in the chapter will be related. effective learning technique.
Those in the first several chapters should already be very When you have completed a chapter, take sometime to
familiar to you; if not, then it will be necessary for you to list what you see as the key points—those that you would
do some background reading or discussion until you feel cover in a formal presentation. Then, skim back over the
comfortable in your understanding of these basic ideas. You chapter.
will find that most chapters are designed to build upon the You will find that Chapters 6–19 each have one or
understanding gained through previous chapters; in most more Case Studies comprised of more clinical case reports
cases, the Anchoring Concepts of a chapter relate to the abstracted from the medical literature. For each, use the
Learning Objectives of previous chapters. Pay attention to associated questions to focus your thinking on the fea-
the Learning Objectives; they are the key elements of under- tures that relate to vitamin functions. As you do so, try to
standing what the chapter is intended to support. Keeping ignore the obvious connection with the subject of the chap-
the Learning Objectives in mind as you go through each ter; put yourself in the position of the attending physician
chapter will help you maintain focus on those elements. who was called upon to diagnose the problem without prior
xiii
xiv How to Use This Book
knowledge that it involved any particular nutrient, much will build to an array of descriptors that, collectively, are
less a certain vitamin. The Case Study in Chapter 21 is dif- more relevant to vitamin A than any is individually. Most of
ferent; it is a fictional but highly plausible scenario that calls the answers, by far, will relate to the clinical symptoms of
for a nonobvious decision. Additional case studies are listed vitamin A deficiency and the sources of vitamin A in diets.
in Appendix B. Catch each answer by dashing it on to a large sticky note and
Take sometime and go through the Study Questions then stick the note haphazardly to a blackboard or wall. If
and Exercises at the end of each chapter. These, too, are you hear something complex or a cluster of concepts, make
designed to direct your thinking back to the key concepts of sure to question the contributor until you hear one or more
the respective chapter and to facilitate integration of those individual concepts, which you can record on individual
concepts with those you already have. sticky notes. This approach never fails to stimulate further
We have made a point in Chapter 1 of using the tech- answers, and it is common that a group of 15–20 students
nique of concept mapping do demonstrate the integration will generate a list of twice that number of concepts before
of complex subject matter. We have found the concept map the momentum fades. Having used sticky notes, it is easy
to be a powerful teaching/learning tool. If you have had no to move them into clusters and, thus, to use the activity to
previous experience with this device, then it will be worth construct a concept map of “Vitamin A” based solely on the
your while to consult Learning How to Learn.1 knowledge that the students, collectively, brought into the
When you have done all of this for a chapter, then deal room. This exercise can demonstrate an empowering idea
with your questions. Discuss them with fellow students or that, having at least some background on the subject and
look them up. To assist you in the latter, a short reading list being motivated (by any of a number of reasons) to learn
is included at the end of each chapter. With the exception of more, every learner brings to the study of the vitamins a
Chapter 2, which lists papers of landmark significance to unique perspective which may not be readily apparent.
the discovery of the vitamins, the reading lists consist of key We are convinced that meaningful learning is served
reviews in prominent scientific journals. These reviews and when both instructor and students come to understand each
the papers cited in the footnotes will help you find primary others’ various perspectives. This has two benefits in teach-
research papers on topics of specific interest. ing the vitamins. First, it is in the instructor’s interest to
After you have followed all of these steps, reread the know the students’ ideas and levels of understanding con-
chapter. You will find this last step to be extraordinarily cerning issues of vitamin need, vitamin function, etc., such
useful in gaining a command of the material. that these can be built upon and modified as may be appro-
Last, but certainly not least, have fun with this fascinat- priate. Second, many upper-level students have interesting
ing aspect of the field of nutrition! experiences (through personal or family histories, their own
To the instructor The format of this text reflects the research, information from other courses, etc.) that can
way GFC taught a course called “The Vitamins” for some be valuable contributions to classroom discussions. These
29 years at Cornell University. To that end, some experi- experiences are assets that can reduce the temptation to
ences in using The Vitamins as a text for my course may be fall back on the “instructor knows all” notion, which we all
of interest to you. know to be false. To identify student perspectives, it is use-
I have found that every student comes to the study of ful to assign on the first class period, for submission at the
the vitamins with some background knowledge of the sub- second class, a written autobiographical sketch. Distribute
ject, although those backgrounds are generally incomplete your own as a model, and ask each student to write “as much
and frequently include areas of misinformation. This is true or as little” as he or she cares to, recognizing that you will
for upper-level nutrition majors and for students from other distribute to the class copies of whatever is submitted. The
fields, the difference being largely one of magnitude. This biographical sketches will range from a few sentences that
is also true for instructors, most of whom come to the field reveal little of a personal nature to longer ones that provide
with specific expertise that relates to only a subset of the many good insights about their authors; everyone will help
subject matter. you to get to know your students personally and to get a bet-
You can demonstrate this in the following exercise, best ter idea of their understandings of the vitamins and of their
done of the first day of class. Raise your index finger (best expectations of the course. The exercise serves the students
done with a bit of dramatic flair) and say “vitamin A.” Hold in a similar manner, thus promoting a group dynamic that
that pose for 10 s and then ask “What came to mind when I facilitates classroom discussions.
said ‘vitamin A’?” Without fail, someone will say “vision” The Vitamins can be used as a typical text from which
or “carrots,” and then an older graduate student may add you can make regular reading assignments as preparation
“toxic.” When it looks safe to chime in, others will add what for each class. This will free you of the need for lecturing
in favor of an open discussion format. In fact, this approach
1. Novak, J.D., Gowin, D.B., 1984. Learning How to Learn. Cambridge, allows more information to be covered, as even a brilliant
University Press, New York, NY, pp. 199 lecturer simply cannot cover the vitamins in any real depth
How to Use This Book xv
within the limits of traditional class periods. This was the the Case Studies are abstracted from actual clinical reports;
original motivation for putting that information into this students enjoy and do well on these assignments.
text, which has allowed shifting responsibility for learning The model we used in teaching The Vitamins at Cornell
to the student to glean from assigned reading. This allows was to evaluate student’s performance on the basis of class
class time to be used to facilitate learning through discus- participation, weekly written assignments, a review of a
sions of issues of student interest or concern. Often, this recent research paper, and either one or two examinations.
means that certain points were not clear upon reading or To allow each student to pursue a topic of specific individ-
that the reading itself stimulated questions not specifically ual interest, students were asked to review a research paper
addressed in the text. Usually, these questions are nicely published within the last year, using the style of Nutrition
handled by eliciting the views and understandings of other Reviews. Students were asked to make a short (10 min) pre-
students and by your giving supplementary information. sentation of each in class. Their reviews were evaluated on
With this approach, the instructor’s class preparation the basis of critical analysis and on the importance of the
involves the collation of research data that will supplement paper to the field. This assignment was also well received.
the discussion in the text, and the identification of questions Because many students are inexperienced in research and
that can initiate discussions. In developing questions, it may will, thus, feel uncomfortable in criticizing it, it is helpful
be useful to prepare your own concept maps of the subject to conduct in advance a discussion of the general principles
matter and to ask rather simple questions about the link- of experimental design and statistical inference. Exams
ages between concepts, e.g., “How does the mode of enteric were also concept-oriented: students were given brief case
absorption of the tocopherols relate to what we know about descriptions and actual experimental data, and were asked
its physiochemical properties?” If you are unfamiliar with to lay out diagnostic strategies, develop hypotheses, design
concept mapping, then consult “Learning How to Learn” means of hypothesis testing and interpretation of results,
and experiment with the technique to determine whether it etc. Many students may prefer the more familiar short-
can assist you in your teaching. answer test; such inertia can be overcome by using exam-
The Study Questions and Exercises or Case Studies can ples in class discussions and or homework assignments.
be used to give weekly written assignments to keep stu- The Vitamins was been of great value in enhancing the
dents focused on the topic and prevent them from letting teaching of the course by that name at Cornell. Thus, it is
the course slide until exam time. More importantly, there is our sincere wish that it will assist you similarly in your
learning associated with the thought that necessarily goes teaching. Please let us know how it meets your needs and
into such written assignments. To support that learning, how we might enhance it for that purpose.
make a point of going over each assignment briefly at the
beginning of the class at which it is due and return it by the Gerald F. Combs, Jr.
next class with your written comments. You will find that James P. McClung

Part I
Perspectives on the Vitamins

in Nutrition
1. What Is a Vitamin? 3 4. Vitamin Deficiency 59

2. Discovery of the Vitamins 7 5. Vitamin Needs and Safety 79
3. General Properties of Vitamins 33

Chapter 1
What Is a Vitamin?
Chapter Outline
1. Thinking About Vitamins 3 4. The Recognized Vitamins 5
2. Vitamin: A Revolutionary Concept 3 5. Study Questions and Exercises 5
3. An Operating Definition of a Vitamin 4
Anchoring Concepts
1. Certain factors, called nutrients, are necessary for normal 1. THINKING ABOUT VITAMINS
physiological function of animals, including humans. Some Among the nutrients required for the many physiologic
nutrients cannot be synthesized adequately by the host and functions essential to life are the vitamins. Unlike other
must therefore be obtained from the external chemical envi-
nutrients, the vitamins do not serve structural functions, nor
ronment; these are referred to as dietary essential nutrients.
2. Diseases involving physiological dysfunction, often accom-
does their catabolism provide significant energy. Instead,
panied by morphological changes, can result from insuffi- the physiologic functions of vitamins are highly specific,
cient intakes of dietary essential nutrients. and, for that reason, they are required in only small amounts
in the diet. The common food forms of most vitamins
require some metabolic activation to their functional forms.
Imagination is more important than knowledge. Although the vitamins share these general characteris-
tics, they show few close chemical or functional similari-
A. Einstein
ties; their categorization as vitamins is strictly empirical.
Consider also that, whereas several vitamins function as
enzyme cofactors (vitamins A, K, and C; thiamin; niacin;
LEARNING OBJECTIVES riboflavin; vitamin B6; biotin; pantothenic acid; folate;
and vitamin B12), not all enzyme cofactors are vitamins.1
1. To understand the classic meaning of the term vitamin as Some vitamins function as biological antioxidants (vita-
it is used in the field of nutrition. mins E and C), and several function as cofactors in meta-
2. To understand that the term vitamin describes both a bolic oxidation–reduction reactions (vitamins E, K, and C;
concept of fundamental importance in nutrition as well niacin; riboflavin; and pantothenic acid). Two vitamins
as any member of a rather heterogeneous array of nutri- (vitamins A and D) function as hormones; one of them (vita-
ents, any one of which may not fully satisfy the classic min A) also serves as a photoreceptive cofactor in vision.
definition.
3. To understand that some compounds are vitamins for
one species and not another, and that some are vitamins 2. VITAMIN: A REVOLUTIONARY
only under specific dietary or environmental conditions. CONCEPT
4. To understand the concepts vitamer and provitamin.
Everyday Word or Revolutionary Idea?
The term vitamin, today a common word in everyday
VOCABULARY language, was born of a revolution in thinking about the
interrelationships of diet and health that occurred at the
Vitamer
Vitamin 1. Other enzyme cofactors are biosynthesized, e.g., heme, coenzyme Q,
Provitamin and lipoic acid.
The Vitamins. http://dx.doi.org/10.1016/B978-0-12-802965-7.00001-0

Copyright © 2017 Elsevier Inc. All rights reserved. 3
4 PART | I Perspectives on the Vitamins in Nutrition
beginning of the 20th century. That revolution involved the in understanding human physiology and nutrition, the actual
growing realization of two phenomena that are now taken definition of a vitamin has evolved in consequence of that
for granted, even by the nonscientist: understanding.
1. Diets are sources of many important nutrients.
2. Insufficient intakes of specific nutrients can cause cer- 3. AN OPERATING DEFINITION
tain diseases. OF A VITAMIN
In today’s world each of these concepts may seem self- A vitamin is defined as follows (Fig. 1.1). A vitamin
evident, but in a world still responding to and greatly influ-
l is an organic compound distinct from fats, carbohy-
enced by the important discoveries in microbiology made in
drates, and proteins
the 19th century, each represented a major departure from
l is a natural component of foods in which it is usually
contemporaneous thinking in the area of health. Nineteenth-
present in minute amounts
century physiologists perceived foods and diets as sources
l is essential, also usually in minute amounts, for normal
of only four types of nutrients: protein, fat, carbohydrate,
physiological function (i.e., maintenance, growth, devel-
ash,2 and water. After all, these accounted for very nearly
opment, and/or production)
100% of the mass of most foods. With this view, it is under-
l prevents a specific deficiency syndrome, which occurs
standable that, at the turn of the century, experimental find-
when it is absent or underutilized
ings that now can be seen as indicating the presence of
l is not synthesized by the host in amounts adequate to
hitherto unrecognized nutrients were interpreted instead as
meet normal physiological needs.
substantiating the presence of natural antidotes to unidenti-
fied disease-causing microbes. This definition will be useful in the study of vitamins,
Important discoveries in science have ways of direct- as it effectively distinguishes this class of nutrients from
ing, even entrapping, one’s view of the world; resisting this others (e.g., proteins and amino acids, essential fatty acids,
tendency depends on critical and constantly questioning and minerals) and indicates the needs in various normal
minds. That such minds were involved in early nutrition physiological functions. It also denotes the specificity of
research is evidenced by the spirited debates and frequent deficiency syndromes by which the vitamins were discov-
polemics that ensued over discoveries of apparently benefi- ered. Further, it places the vitamins in that portion of the
cial new dietary factors. Still, the systematic development external chemical environment on which animals (includ-
of what emerged as nutritional science depended on a new ing humans) must depend for survival, thus distinguishing
intellectual construct for interpreting such experimental vitamins from hormones.
observations.
Some Caveats
Vitamin or Vitamine?
It will quickly become clear, however, that, despite its util-
The elucidation of the nature of what was later to be called ity, this operating definition has limitations, notably with
thiamin occasioned the proposition of just such a new con- respect to the last clause. Many species can, indeed, syn-
struct in physiology.3 Aware of the impact of what was a thesize at least some of the vitamins, although not always
departure from prevailing thought, its author, the Polish bio- at the levels required to prevent deficiency disorders. Four
chemist Casimir Funk, chose to generalize from his findings examples illustrate this point:
on the chemical nature of that “vital amine” to suggest the Vitamin C: Most animal species have the ability to syn-
term vitamine as a generic descriptor for many such acces- thesize ascorbic acid. Only those few that lack the enzyme
sory factors associated with diets. That the factors soon to l-gulonolactone oxidase (e.g., the guinea pig, humans) can-
be elucidated comprised a somewhat chemically heteroge- not. For those species, ascorbic acid is properly be called
neous group, not all of which were nitrogenous, does not vitamin C.
diminish the importance of the introduction of what was Vitamin D: Individuals exposed to modest amounts of
first presented as the vitamine theory, later to become a key sunlight can produce cholecalciferol, which functions as a
concept in nutrition: the vitamin. hormone. Only individuals without sufficient exposure to
The term vitamin has been defined in various ways. ultraviolet light (e.g., livestock raised in indoor confine-
While the very concept of a vitamin was crucial to progress ment, people spending most of their days indoors) require
dietary sources of vitamin D.
2. The residue from combustion, i.e., minerals. Choline: Most animal species have the metabolic capac-
3. This is a clear example of what T.H. Kuhn called a “scientific revolution”
(Kuhn, T.H., 1968. The Structure of Scientific Revolutions. University of
ity to synthesize choline; however, some (e.g., the chick,
Chicago Press, Chicago, IL.), i.e., the discarding of an old paradigm with the rat) may not be able to employ that capacity if they are
the invention of a new one. fed insufficient amounts of methyl donor compounds. In
What Is a Vitamin? Chapter | 1 5
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FIGURE 1.1 Concept map of a Vitamin.4
addition, some (e.g., the chick) do not develop that capacity 4. THE RECOGNIZED VITAMINS
completely until they are several weeks of age. Thus, for the
Thirteen substances or groups of substances are now gen-
young chick and for individuals of other species fed diets
erally recognized as vitamins (Table 1.1); others have been
providing limited methyl groups, choline is a vitamin.
proposed.6 In some cases, the familiar name is actually
Niacin: All animal species can synthesize nicotinic acid
the generic descriptor for a family of chemically related
mononucleotide from the amino acid tryptophan. Only
compounds having qualitatively comparable metabolic
those for which this metabolic conversion is particularly
activities. For example, the term vitamin E refers to those
inefficient (e.g., the cat, fishes) and others fed low dietary
analogs of tocol or tocotrienol7 that are active in preventing
levels of tryptophan require a dietary source of niacin.
such syndromes as fetal resorption in the rat and myopa-
With these counterexamples in mind, the definition of a
thies in the chick. In these cases, the members of the same
vitamin has specific connotations for animal species, stage
vitamin family are called vitamers. Some carotenoids can
of development, diet or nutritional status, and physical envi-
be metabolized to yield the metabolically active form of
ronmental conditions.5
vitamin A; such a precursor of an actual vitamin is called
a provitamin.
The “Vitamin Caveat” 5. STUDY QUESTIONS AND EXERCISES

l Some compounds are vitamins for one species and not
1. What are the key features that define a vitamin?
another.
l
2. What are the fundamental differences between vitamins
Some compounds are vitamins only under specific
dietary or environmental conditions. and other classes of nutrients… between vitamins and
hormones?
3. Detail, citing a specific example, a situation in which a
vitamin may be nutritionally essential for one species
4. The concept map can be a useful device for organizing thought, as but not another.
its discipline can serve to assist in identifying the nature and extent of 4. Using key words and phrases, list briefly what you know
concepts related to the one in question. A concept map should be laid out
about each of the recognized vitamins.
as a hierarchy of related concepts with the superordinate concept at the
top and all relationships between concepts identified with a verb phrase.
Thus, it can be “read” from top to bottom. One of the authors (GFC) has
used concept mapping in graduate-level teaching, both as a group exercise 6. These include such factors as inositol, carnitine, bioflavonoids, pangamic
and testing device. For a useful discussion of the educational value of the acid, and laetrile, for some of which there is evidence of vitamin-like
concept map, the reader is referred to Learning How to Learn, 1984, J.D. activity (Chapter 19).
Novak and D.B. Gowin, Cornell University Press, Ithaca, NY, pp. 199. 7. Tocol is 3,4-dihydro-2-methyl-2-(4,8,12-trimethyltridecyl)-6-chromanol;
5. For this reason, it is correct to refer to vitamin C for the nutrition of tocotrienol is the analog with double bonds at the 3, 7, and 11′ positions on
humans but ascorbic acid for the nutrition of livestock. the phytol side chain (Chapter 7).
TABLE 1.1 The Vitamins: Their Vitamers, Provitamins, and Functions
Group Vitamers Provitamins Physiological functions

Vitamin A Retinol β-Carotene Visual pigments; epithelial cell differentiation
Retinal Cryptoxanthin
Retinoic acid
Vitamin D Cholecalciferol (D3) Calcium homeostasis; bone metabolism; transcription
Ergocalciferol (D2) factor
Vitamin E α-Tocopherol Membrane antioxidant
γ-Tocopherol
Vitamin K Phylloquinones (K1) Blood clotting;
Menaquinones (K2) calcium metabolism
Menadione (K3)
Vitamin C Ascorbic acid Reductant in hydroxylations in the formation of collagen
Dehydroascorbic acid and carnitine, and in the metabolism of drugs and steroids
Vitamin B1 Thiamin Coenzyme for decarboxylations of 2-keto acids
(e.g., pyruvate) and transketolations
Vitamin B2 Riboflavin Coenzyme in redox reactions of fatty acids and the
tricarboxylic acid (TCA) cycle
Niacin Nicotinic acid Coenzyme for several dehydrogenases
Nicotinamide
Vitamin B6 Pyridoxol Coenzyme in amino acid metabolism
Pyridoxal
Pyridoxamine
Folic acid Folic acid Coenzyme in single-carbon metabolism
Polyglutamyl folacins
Biotin Biotin Coenzyme for carboxylations
Pantothenic acid Pantothenic acid Coenzyme in fatty acid metabolism
Vitamin B12 Cobalamin Coenzyme in the metabolism of propionate, amino acids,
and single-carbon units
Chapter 2
Discovery of the Vitamins
Chapter Outline
1. The Emergence of Nutrition as a Science 8 7. Vitamin Terminology 28
2. The Process of Discovery in Nutritional Science 8 8. Other Factors Sometimes Called Vitamins 28
3. The Empirical Phase of Vitamin Discovery 8 9. Modern History of the Vitamins 29
4. The Experimental Phase of Vitamin Discovery 12 10. Study Questions and Exercises 30
5. The Vitamine Theory 14 Recommended Reading 30
6. Elucidation of the Vitamins 18
Anchoring Concepts LEARNING OBJECTIVES

1. A scientific theory is a plausible explanation for a set of
1. To understand the nature of the process of discovery in
observed phenomena; because theories cannot be tested
the field of nutrition.
directly, their acceptance relies on a preponderance of sup-
porting evidence.
2. To recognize the major forces in the emergence of nutri-
2. A scientific hypothesis is a tentative supposition that is tion science.
assumed for the purposes of argument or testing and is thus 3. To understand the impact of the vitamine theory, as an
used in the generation of evidence by which theories can be intellectual construct, on that process of discovery.
evaluated. 4. To understand that the discoveries of the vitamins pro-
3. An empirical approach to understanding the world involves ceeded along indirect lines, most often through the
the generation of theories strictly by observation, whereas an seemingly unrelated efforts of many people.
experimental approach involves the undertaking of opera- 5. To recognize the key events in the discovery of each of
tions (experiments) to test the truthfulness of hypotheses. the vitamins.
4. Physiology is that branch of biology seeks to elucidate
6. To become familiar with the basic terminology of the
the processes, activities, and phenomena of life and liv-
vitamins and their associated deficiency disorders.
ing organisms, while biochemistry seeks to elucidate the
molecular bases for such phenomena.
5. The field of nutrition is derived from both of these disci-
plines; it seeks to elucidate the processes by which animals VOCABULARY
or plants take in and utilize food substances. Accessory factor
Anemia
When science is recognized as a framework of evolving con- Animal model
cepts and contingent methods for gaining new knowledge, Animal protein factor
we see the very human character of science, for it is cre- Ascorbic acid
ative individuals operating from the totality of their experi- β-Carotene
ences who enlarge and modify the conceptual framework Beriberi
of science. Biotin
J.D. Novak.1 Black tongue disease
Cholecalciferol
Choline
Dermatitis
1. Joseph D. Novak (b. 1932) is a prominent American educator known Ergocalciferol
for his research on human learning, knowledge creation, and knowledge
representation. Prof. Novak, spent most of his career at Cornell University
Fat-soluble A
where he and his colleagues developed the technique of Concept Mapping Filtrate factor
as a means of representing science knowledge. Flavin
The Vitamins. http://dx.doi.org/10.1016/B978-0-12-802965-7.00002-2
Copyright © 2017 Elsevier Inc. All rights reserved. 7
Folic acid participant may be that of a thicket of tangled hypotheses

Germ theory and facts. The seemingly straightforward appearance of the
Hemorrhage emergent limb of discovery is but an illusion achieved by
Lactoflavin discarding the dead branches of false starts and unsupported
Niacin hypotheses, each of which can be instructive about the pro-
Night blindness cess of scientific discovery.
Ovoflavin With the discovery of the vitamins, therefore, nutrition
Pantothenic acid moved from a largely observational activity to one that
Pellagra relied increasingly on hypothesis testing through experi-
Polyneuritis mentation; it moved from empiricism to science. Both the
Prothrombin process of scientific discovery and the course of the devel-
Provitamin opment of nutrition as a scientific discipline are perhaps best
Purified diet illustrated by the history of the discovery of the vitamins.
Pyridoxine
Retinen 2. THE PROCESS OF DISCOVERY
Riboflavin
Rickets
IN NUTRITIONAL SCIENCE
Scurvy Empiricism and Experiment
Thiamin
Vitamin A History demonstrates that the process of scientific discov-
Vitamin B ery begins with the synthesis of general ideas about the
Vitamin B complex natural world from observations of particulars within it—
Vitamin B12 i.e., an empirical phase. In the discovery of the vitamins,
Vitamin B2 this initial phase was characterized by the recognition of
Vitamin B6 associations between diet and human diseases, namely
Vitamin C night blindness, scurvy, beriberi, rickets, and pellagra,
Vitamin D each of which was long prevalent in various societies.
Vitamin E The next phase in the process of discovery involved the
Vitamin K use of these generalizations to form hypotheses that could
Vitamine be tested experimentally—i.e., the experimental phase.
Vitamine theory In the discovery of the vitamins, this phase necessitated
Water-soluble B the development of two key tools of modern experimen-
Xerophthalmia tal nutrition: the animal model and the purified diet. The
availability of both of these tools proved to be necessary
for the discovery of each vitamin; in cases where an ani-
mal model was late to be developed (e.g., for pellagra), the
1. THE EMERGENCE OF NUTRITION elucidation of the identity of the vitamin was substantially
AS A SCIENCE delayed.
In the span of only five decades commencing at the very
end of the 19th century, the vitamins were discovered. Their 3. THE EMPIRICAL PHASE OF VITAMIN
discoveries were the result of the activities of hundreds of
DISCOVERY
people that can be viewed retrospectively as having fol-
lowed discrete branches of intellectual progress. Those The major barrier to entering the empirical phase of nutri-
branches radiated from ideas originally derived inductively tional inquiry proved to be the security provided by presci-
from observations in the natural world, each starting from entific attitudes about foods that persisted through the 19th
the recognition of a relationship between diet and health. century. Many societies had observed that human popula-
Subsequently, branches were pruned through repeated tions in markedly contrasting parts of the world tended to
analysis and deduction—a process that both produced and experience similar health standards despite the fact that
proceeded from the fundamental approaches used in experi- they subsisted on very different diets. These observations
mental nutrition today. Once pruned, the limb of discovery were taken by 19th-century physiologists to indicate that
may appear straight to the naive observer. Scientific discov- health was not particularly affected by the kinds of foods
ery, however, does not occur that way; rather, it tends to consumed. Foods were thought important as sources of
follow a zigzag course, with many participants contributing the only nutrients known at the time: protein, available
many branches. In fact, the contemporaneous view of each energy, and ash. While the “chemical revolution,” led by
Discovery of the Vitamins Chapter | 2 9
the French scientist Antoine Lavoisier,2 started probing the between diet and the diseases scurvy, rickets, pellagra, and
elemental components and metabolic fates of these nutri- night blindness.
ents, the widely read ideas of the German chemist Justus Scurvy has been known that scurvy, the disease involv-
von Liebig3 resulted in protein being recognized as the ing apathy, weakness, sore gums, painful joints, and mul-
only essential nutrient, supporting both tissue growth and tiple hemorrhages, could be prevented by including in the
repair as well as energy production. In the middle part of diet green vegetables or fruits. Descriptions of cases in such
the century, attention was drawn further from potential sources as the Eber papyrus (c.1150 BCE) and writings of
relationships of diet and health by the major discoveries of Hippocrates (c.420 BCE) are often cited to indicate that
Pasteur,4 Liebig,5 Koch,6 and others in microbiology. For scurvy was prevalent in those ancient populations. Indeed,
the first time, several diseases, first anthrax and then oth- signs of the disease are said to have been found in the skeletal
ers, could be understood in terms of a microbial etiology. remains of primitive humans. Scurvy was common in north-
By the end of the century, germ theory, which proved to be ern Europe during the Middle Ages, a time when local agri-
of immense value in medicine, directed hypotheses for the culture provided few sources of vitamin C that lasted through
etiologies of most diseases. The impact of this understand- the winter. In northern Europe, it was treated by eating
ing as a barrier to entering the inductive phase of nutritional cresses and spruce leaves. Scurvy was very highly prevalent
discovery is illustrated by the case of the Dutch physician among seamen, particularly those on ocean voyages to Asia
Christiaan Eijkman,7 who found a water-soluble factor during which they subsisted for months at a time on dried
from rice bran to prevent a beriberi-like disease in chickens and salted foods. The Portuguese explorer Vasco da Gama
(now known to be the vitamin thiamin) and concluded that reported losing more than 60% of his crew of 160 sailors in
he had discovered a “pharmacological antidote” against the his voyage around the Cape of Good Hope in 1498. In 1535–
beriberi “microbe” presumed to be present in rice. 1536, the French explorer Jacques Cartier reported that signs
of scurvy were present in all but three of his crew of 103 men
(25 of whom died) during his second Newfoundland expe-
Diseases Linked to Diet
dition. In 1595–1597, the first Dutch East Indies fleet lost
Nevertheless, while they appeared to have little effect on the two-thirds of its seamen due to scurvy. In 1593, the British
prevailing views concerning the etiology of human disease, admiral Richard Hawkins wrote that, during his career, he
by the late 1800s empirical associations had been made had seen some 10,000 seamen die of the disease.
The link between scurvy and preserved foods was long
2. Antoine-Laurent de Lavoisier (1743–1794) is often considered the evident to seafarers. The first report of a cure for the disease
“father of modern chemistry”, as his work changed that science from a appears to have been Cartier’s description of the rapidly
qualitative to a quantitative one. He is best known for his discovery of successful treatment of his crew with an infusion of the bark
oxygen and its role in combustion.
3. In his widely read book, Animal Chemistry, or Organic Chemistry in its
of Arborvitae (Thuja occidentalis) prepared by the indig-
Application to Physiology and Pathology, Liebig argued that the energy enous Hurons of Newfoundland. By 1601, the consumption
needed for the contraction of muscles, in which he was able to find no of berries, vegetables, scurvy-grass (Cochlearia officinalis,
carbohydrate or fat, must come only from the breakdown of protein. which contains as much ascorbic acid as orange juice), and
Protein, therefore, was the only true nutrient. citrus fruits or juices was recognized as effective in pre-
4. Louis Pasteur (1822–1895) was a French pioneering microbiologist.
He disproved the doctrine of “spontaneous generation” of microbial life
venting the disease. In that year, the English privateer Sir
and advanced “germ theory.” He discovered the principles of vaccination, James Lancaster introduced regular issues of lemon juice
fermentation and developed the process of heat-killing of microbes in (three spoonfuls each morning) on one of his found ships,
liquids is now called “pasteurization”. finding significantly less scurvy among treated sailors.
5. Justus von Liebig (1803–1873) was a German chemist who made Nevertheless, the prestigious London College of Physicians
major contributions to agricultural and biological chemistry, elucidated
the importance of nitrogen in plant nutrition, and introduced laboratory
viewed scurvy as a “putrid” disease in which affected tis-
experience in teaching chemistry. sues became alkaline and stated that other acids could be as
6. Robert Koch (1843–1910) was a German physician who identified the effective as lemon juice in treating the disease. Accordingly,
causative agents of tuberculosis, cholera and anthrax, and formulated in the mid-1600s British ship’s surgeons were supplied with
the general principles (“Koch’s Postulates”) for linking specific vitriol (dilute sulfuric acid).
microorganisms to specific diseases. In 1905, he received the Nobel Prize
for Physiology or Medicine.
Against this background, in 1747, James Lind, a Scottish
7. Christiaan Eijkman (1858–1930) was trained in the Netherlands and served physician serving in the British Royal Navy, conducted
as a medical officer in the Dutch Indies. After contracting malaria in 1885, he what has been cited as the first controlled clinical trial to
returned to Amsterdam where he worked in the laboratories of Forster and, compare various therapies recommended for scurvy in
then, Kock (Berlin). In Koch’s laboratory he met another Dutch physician British sailors at sea. Lind’s report, published 6 years later,
C.A. Pekelharing whom he assisted in a second period of service in the Indies
investigating beriberi. They proposed establishing a medical laboratory of
described 12 sailors with scurvy whom he assigned in pairs
which Eijkman was named director and Director of the Javanese Medical to 2-week regimens including either lemons and oranges,
School, which ultimately became the University of Indonesia. vitriol, vinegar, or other putative remedies. His results were
clear: the pair treated with lemons and oranges recovered knock knees, and curvatures of the upper and/or lower
almost completely within 6 days; whereas, no other treat- arms), swollen joints, and/or enlarged heads. It is gener-
ment resulted in any improvement. In 1753, he published his ally associated with the urbanization and industrialization
now-classic work “A Treatise on Scurvy,” which had great of human societies. Its appearance on a wide scale was
impact on the medical thought of the time, as it detailed past more recent and more restricted geographically than that of
work on the subject (most of which was anecdotal) and also either scurvy or beriberi. The first written account of the
presented the results of his experiments. Lind believed that disease is believed to be that of Daniel Whistler,9 who wrote
citrus contained “a saponaceous, attenuating and resolving on the subject in his medical thesis in 1645. A complete
virtue” that helped free skin perspiration that had become description of the disease was published shortly thereafter
clogged by sea air; however, his results were taken as estab- (in 1650) by the Cambridge professor Francis Glisson,10 so
lishing the value of fresh fruits in treating the disease. Still, it is clear that by the middle of the 17th-century rickets had
it was not until the 1790s that the British Navy had made it become a public health problem in England. However, rick-
a regular practice to issue daily rations of lemon juice to all ets appears not to have affected earlier societies, at least not
seamen—a measure that gave rise to the term “limey”8 as a on such a scale. Studies in the late 1800s by the Scottish
slang expression for a British seaman. In the early part of physician T.A. Palm11 showed that the mummified remains
the 19th century, there remained no doubt of a dietary cause of Egyptian dead bore no signs of the disease. By the latter
and cure of scurvy; even so, it would be more than a century part of the century, the incidence of rickets among children
before its etiology and metabolic basis would be elucidated. in London exceeded one-third; by the turn of the century,
Outbreaks of scurvy continued in cases of food shortages: estimates of prevalence were as high as 80% and rickets
in British prisons, during the California gold rush, among had become known as the “English disease.” Noting the
troops in the Crimean War, among prisoners in the American absence of rickets in southern Europe, Palm in 1888 was the
Civil War, among citizens during the Siege of Paris in 1871, first to point out that rickets was prevalent only where there
and among polar explorers in the early 20th century. is relatively little sunlight (e.g., in the northern latitudes).
It is said that signs consistent with beriberi (e.g., ini- He suggested that sunlight exposure prevented rickets, but
tial weakness and loss of feeling in the legs leading to others held that the disease had other causes—e.g., hered-
heart failure, breathlessness, and, in some cases, edema) ity or syphilis. Through the turn of the century, much of
are described in ancient Chinese herbals (∼2600 BCE). the Western medical community remained either unaware
Certainly, beriberi was an historic disease prevalent in many or skeptical of a food remedy that had long been popular
Asian populations subsisting on diets in which polished among the peoples of the Baltic and North Sea coasts, and
(i.e., “white” or dehulled) rice is the major food. For exam- that had been used to treat adult rickets in the Manchester
ple, in the 1860s, the Japanese navy experienced the disease Infirmary by 1848: cod liver oil. Not until the 1920s would
affecting 30–40% of its seamen. Interesting clinical experi- the confusion over the etiology of rickets become clear.
ments conducted in the 1870s with sailors by Dr Kanehiro Pellagra, the disease characterized by lesions of the
Takaki, a British trained surgeon who later became Director skin and mouth, and by gastrointestinal and mental dis-
General of the Japanese Naval Medical Service, first noted turbances, also became prevalent in human societies fairly
an association between beriberi and diet: Japanese sailors recently. There appears to have been no record of the dis-
were issued lower protein diets than their counterparts in ease, even in folk traditions, before the 18th century. Its first
European navies, which had not experienced the disease. documented description, in 1735, was that of the Spanish
Takaki conducted an uncontrolled study at sea in which he physician Gaspar Casal. His observations were dissemi-
modified sailors’ rations to increase protein intake by includ- nated by the French physician François Thiery, whom he
ing more meat, condensed milk, bread, and vegetables at the met some years later after having been appointed as physi-
expense of rice. This cut both the incidence and severity of cian to the court of King Philip V. In 1755, Thiery published
beriberi dramatically, which he interpreted as confirmation a brief account of Casal’s observations in the Journal de
of the disease being caused by insufficient dietary protein. Vandermonde; this became the first published report on the
The adoption of Takaki’s dietary recommendations by the
Japanese navy was effective—eliminating the disease as a 9. Whistler (1619–1684) was an English physician. His thesis at the Royal
College of Physicians was the first printed book on rickets.
shipboard problem by 1880—despite the fact that his con- 10. Francis Glisson (1599–1677) was a British physician and anatomist
clusion, reasonable in the light of contemporaneous knowl- who wrote a text on pediatric rickets.
edge, later proved to be incorrect. 11. Theobold A. Palm (1849–?) was a Scottish physician born to missionary
Rickets, the disease of growing bones, presents in chil- parents in Ceylon. After studying medicine at Edinburgh University, he
dren as deformations of the long bones (e.g., bowed legs, served as a medical missionary in Japan, where he noted the absence of
rickets, in marked contrast to the prevalence of that condition he found in
Britain on his return in 1884. In 1888, he commented on Britain’s “want
8. That lemons were often called limes has been a source of confusion to of light” in a letter to the British Medical Journal in which he went on to
many writers on this topic. recommend “the systematic use of sunbaths” as a rickets therapy.
disease. Casal’s own description was included in his book during or after the American Civil War (1861–1865), in
on the epidemic and endemic diseases of northern Spain, association with food shortages in the southern states. It is
Historia Natural y Medico de el Principado de Asturias, clear from George Searcy’s 1907 report to the American
which was published in 1762, i.e., 3 years after his death. Medical Association that the disease was endemic at least
Casal regarded the disease, popularly called mal de la rosa, in Alabama.14 By 1909, it had been identified in more
as a peculiar form of leprosy. He associated it with poverty than 20 states, several of which had impaneled Pellagra
and with the consumption of spoiled corn (maize). Commissions, and a national conference on the disease was
In 1771, a similar dermatological disorder was described held in South Carolina.
by the Italian physician Francesco Frapolli. In his work Since it first appeared, pellagra was associated with pov-
Animadversiones in Morbum Volgo Pelagrum, he reported erty and with the dependence on corn as the major staple
the disease to be prevalent in northern Italy. In that region food. Ideas were proffered that it was caused by a toxin asso-
corn, recently introduced from America, had become a pop- ciated with spoiled corn, yet by the turn of the century other
ular crop, displacing rye as the major grain. The local name hypotheses were also popular. These included the sugges-
for the disease was “pelagra,” meaning rough skin. There tion of an infectious agent with, perhaps, an insect vector.
is some evidence that it had been seen as early as 1740. Night blindness, the inability to see under low levels
By 1784 the prevalence of pelagra (now spelled pellagra) of light, was one of the first recorded medical conditions.
in that area was so great that a hospital was established in Writings of Ancient Greek, Roman, and Arab physicians
Legano for its treatment. Success in the treatment of pel- show that animal liver was known to be effective in both
lagra appears to have been attributed to factors other than the prevention and cure of the disease. The Eber papyrus
diet—e.g., rest, fresh air, water, and sunshine. Nevertheless, (c.1550 BCE)15 described its treatment by the squeezing of
the disease continued to be associated with poverty and the liquid from a lamb’s liver (now known to be a good source
consumption of corn-based diets. of vitamin A in well-nourished animals) directly into the
Following the finding of pellagra in Italy, the disease was eyes of the affected patient. The use of liver for the preven-
reported in France in 1829 by the French physician Jean- tion of night blindness became a part of the folk cultures
Marie Hameau. It was not until 1845 that another French of most seafaring communities. In the 1860s, the French
physician Théophile Roussel associated pellagra with physicians, Hubbenet and, later, Bitot, each noted the pres-
Casal’s mal de la rosa and proposed that these diseases, ence of small, foamy white spots on the outer aspects of the
including a similar disease called flemma salada,12 were conjunctiva of patients with night blindness—those lesions
related or identical. To substantiate his hypothesis, Roussel have become known as “Bitot’s spots.” Corneal ulceration,
spent 7 months of 1847 in the area where Casal had worked now known to be a related condition resulting in permanent
in northern Spain13 investigating mal de la rosa cases; on blindness, was recognized in the 18th and 19th centuries in
his return, he presented to the French Academy of Medicine association with protein energy malnutrition as well as such
evidence in support of his conclusion. Subsequently, pel- diseases as meningitis, tuberculosis, and typhoid fever. In
lagra, as it had come to be called, was reported in Romania Russia, it occurred during long Lenten fasts. In the 1880s,
by Theodari in 1858, and in Egypt by the British physician cod liver oil was found to be effective in curing both night
Pruner-Bey in 1874. It was a curiosity, not to be explained blindness and early corneal lesions; by the end of the cen-
for years, that pellagra was never endemic in the Yucatán tury, cod liver oil, meat, and milk were used routinely in
Peninsula, where the cultivation of corn originated. The dis- Europe to treat both conditions. It was not until the early
ease was not reported there until 1896. 1900s, however, that the dietary nature of night blindness,
It is not known how long pellagra had been endemic in and the corneal lesions that typically ensued, was under-
the United States; however, it became common early in the stood—not until the “active lipid” was investigated, i.e., the
20th century. In 1912, American physician J.W. Babcock factor in cod liver oil that supported growth and prevented
examined the records of the state hospital of South Carolina night blindness and xerophthalmia in the rat.
and concluded that the disease had occurred there as early
as 1828. It is generally believed that pellagra also appeared Ideas Prevalent by 1900
Thus, by the beginning of the 20th century, four different
12. Literally meaning “salty phlegm,” this condition involved
gastrointestinal signs, delirium, and a form of dementia. It did not, diseases had been linked with certain types of diet. Further,
however, occur in areas where maize was the major staple food; this, and
disagreement over the similarities of symptoms, caused Roussel’s proposal 14. Sercy, a physician at the Mount Vernon Insane Hospital in Mobile,
of a relationship between these diseases to be challenged by his colleague Alabama, reported 88 cases of pellagra at that institution in 1906.
Arnault Costallat. From Costallat’s letters describing flemma salada in 15. The Eber Papyrus, named for the German egyptologist who discovered
Spain in 1861, it is apparent that he considered it to be a form of acrodynia, it, is among the oldest extant medical papyri of ancient Egypt. Written in
then thought to be due to ergot poisoning. c.1550 BCE, the 20 m long scroll is thought to be copied from earlier texts.
13. Casal practiced in the town of Oviedo in the Asturias of northern Spain. It is housed at the University of Liepzig.
to be true (i.e., through deduction). Both the inductive and

TABLE 2.1 Diet–Disease Relationships Recognized by deductive approaches may be linked; that is, probable con-
1900 clusions derived from observation may be used as hypoth-
Recognized eses for testing deductively in the process of scientific
Disease Associated Diet Prevention experimentation.
Scurvy Salted (preserved) Fresh fruits,
foods vegetables Requirements of Nutrition Science
Beriberi Polished rice-based Meats, vegetables For scientific experimentation to yield informative results, it
Rickets Few “good” fats Eggs, cod liver oil must be both repeatable and relevant. The value of the first
point, repeatability, should be self-evident. Inasmuch as
Pellagra Corn-based None
natural truths are held to be constant, nonrepeatable results
Night blindness None Cod liver oil cannot be construed to reveal them. The value of the second
point, relevance, becomes increasingly important when it
is infeasible to test a hypothesis in its real-world context. In
by 1900, it was apparent that at least two, and possibly such circumstances, it becomes necessary to employ a rep-
three, could be cured by changes in diet (Table 2.1). resentation of the context of ultimate interest—a construct
Other diseases, in addition to those listed in Table 2.1, known in science as a model. Models are born of practical
had been known since ancient times to respond to what is necessity, but they must be developed carefully to serve as
now called diet therapy. Unfortunately, much of this knowl- analogs of situations that cannot be studied directly.
edge was overlooked, and its significance was not fully
appreciated by a medical community galvanized by the new Defined Diets Provided Repeatability
germ theory of disease. Alternative theories for the etiolo-
gies of these diseases were popular. Thus, as the 20th century Repeatability in nutrition experimentation became pos-
began, it was widely held that scurvy, beriberi, and rickets sible with the use of diets of defined composition. The
were each caused by a bacterium or bacterial toxin rather most useful type of defined diet that emerged in nutrition
than by the simple absence of something required for normal research was the purified diet. Diets of this type were for-
health. Some held that rickets might also be due to hypothy- mulated using highly refined ingredients (e.g., isolated pro-
roidism, while others thought it to be brought on by lack of teins, refined sugars and starches, refined fats) for which the
exercise or excessive production of lactic acid. These theo- chemical composition could be tested and quantified. It was
ries died hard and had lingering deaths. In explanation of the use of defined diets that facilitated experimental nutri-
the lack of interest in the clues presented by the diet–disease tion; such diets could be prepared over and over by the same
associations outlined above, Harris (1955) mused: “Perhaps or other investigators to yield comparable results. Results
the reason is that it seems easier for the human mind to obtained through the use of defined diets were repeatable
believe that ill is caused by some positive evil agency, rather and, therefore, predictable.
than by any mere absence of any beneficial property.”
Appropriate Animal Models Provided
Limitations of Empiricism Relevance
In actuality, the process of discovery of the vitamins had Relevance in nutrition research became possible with the
moved about as far as it could in its empirical phase. identification of animal models16 appropriate to diseases of
Further advances in understanding the etiologies of these interest in human medicine or to physiological processes of
diseases would require the rigorous testing of the various
hypotheses—i.e., entrance into the deductive phase of 16. In nutrition and other biomedical research, an animal model consists of
nutritional discovery. That movement, however, required the experimental production in a conveniently managed animal species of
tools for productive scientific experimentation—tools that biochemical and/or clinical changes that are comparable to those occurring
in another species of primary interest but that may be infeasible, unethical,
had not been available previously.
or uneconomical to study directly. Animal models are, frequently, easily
managed and rapidly growing species with small body weights (e.g.,
4. THE EXPERIMENTAL PHASE OF VITAMIN rodents, chicks, rabbits); however, they may also be larger species (e.g.,
monkeys, sheep), depending on the target problem and species they are
DISCOVERY selected to represent. In any case, background information on the biology
and husbandry should be available. The selection and/or development of an
In a world where one cannot examine all possible cases animal model should be based primarily on representation of the biological
(i.e., use strictly inductive reasoning), natural truths can problem of interest without undue consideration of the practicalities of cost
be learned only by inference from premises already known and availability.
interest in human medicine or animal production. The first Dutch military to allow Eijkman to continue working on the
of these was discovered quite by chance by keen observers beriberi problem.
studying human disease. Ultimately, the use of animal mod- The facilities used by the Commission at the Military
els would lead to the discovery of each of the vitamins, as Hospital Batavia became a new Laboratory for Bacteriology
well as to the elucidation of the nutritional roles and meta- and Pathology of the colonial government, and Eijkman was
bolic functions of each of the approximately 40 nutrients. named as director, with one assistant. His efforts in 1888 to
The careful use of appropriate animal models made pos- infect rabbits and monkeys with Pekelharing’s micrococ-
sible studies that would otherwise be infeasible or unthink- cus were altogether unsuccessful, causing him to posit that
able in human subjects or in other animal species of interest. beriberi must require a long time before the appearance of
signs. The following year, he started using chickens as his
animal model. Later in the year, he noted that many, regard-
Major Forces in the Emergence of Nutritional Science less of whether they had been inoculated, lost weight, and
l Recognition that certain diseases were related to diet started walking with a staggering gait. Some developed
l Development of appropriate animal models difficulty standing and died. Eijkman noted on autopsy no
l Use of defined diets abnormalities of the heart, brain, or spinal cord, but micro-
scopic degeneration of the peripheral nerves, particularly in
the legs. The latter were signs he had observed in people
dying of beriberi. He was unable, though, to culture any
An Animal Model for Beriberi
consistent type of bacteria from the blood of affected ani-
The analytical phase of vitamin discovery, indeed modern mals. It would have been easy for Eijkman to dismiss the
nutrition research itself, was entered with the finding of thought that this avian disease, which he called “polyneuri-
an animal model for beriberi in the 1890s. In 1886, Dutch tis,” might be related to beriberi.
authorities sent a commission led by Cornelius Pekelharing
to their East Indian colony (now Indonesia) to find the
cause of beriberi, which had become such a problem among
Serendipity or a Keen Eye?
Dutch soldiers and sailors as to interrupt military operations After persisting in his flock for some 5 months, the disease
in Atjeh, Sumatra. Pekelharing took an army surgeon sta- suddenly disappeared. Eijkman reviewed his records and
tioned in Batavia (now Jakarta), Christiaan Eijkman, whom found that in June, shortly before the chickens had started to
he had met when each was on study leave (Pekelharing from show paralysis, a change in their diet had been occasioned
his faculty post at the University of Utrecht, and Eijkman as by failure of a shipment of feed grade brown (unpolished)
a medical graduate from the University of Amsterdam) in rice to arrive. His assistant had used, instead, white (pol-
the laboratory of the great bacteriologist, Robert Koch. The ished) rice from the hospital kitchen. It turned out that this
team, unaware of Takaki’s work, expected to find a bac- extravagance had been discovered a few months earlier by
terium as the cause, and was therefore disappointed, after a new hospital superintendent, who had ordered it stopped.
8 months of searching, to uncover no such evidence. They When Eijkman again fed the chickens brown rice, he found
concluded, “Beriberi has been attributed to an insufficient affected animals recovered completely within days.
nourishment and to misery: but the destruction of the periph- With this clue, Eijkman immediately turned to the
eral nervous system on such a large scale is not caused by chicken as the animal model for his studies. He found
hunger or grief. The true cause must be something coming chicks showed signs of polyneuritis within days of being
from the outside, but is it a poison or an infection?” fed polished rice, and that their signs disappeared even
However, looking for a poison, they observed, would be more quickly if they were then fed unpolished rice. It was
very difficult, whereas they had techniques for looking for a clear that there was something associated with rice polish-
microorganism that had been successful for other diseases. ings that protected chickens from the disease. After discuss-
Thus, they tried to culture organisms from blood smears ing these results, Eijkman’s colleague Adolphe Verdeman,
from patients and to create the disease in monkeys, rabbits, the physician inspector of prisons in the colony, surveyed
and dogs by inoculations of blood, saliva, and tissues from the use of polished and unpolished rice and the incidence of
patients and cadavers. When single injections produced no beriberi among inmates. His results (Table 2.2), later con-
effects, they used multiple injection regimens. Despite the firmed by others in similar epidemiological investigations,
development of abscesses at the point of some injections, demonstrated the advantage enjoyed by prisoners eating
it appeared that multiple inoculations could produce some unpolished rice: they were much less likely to contract beri-
nerve degeneration in rabbits and dogs. Pekelharing con- beri. This information, in conjunction with his experimental
cluded that beriberi was indeed an infectious disease, but findings with chickens, allowed Eijkman to investigate, by
an unusual one requiring repeated reinfection of the host. means of bioassay, the beriberi-protective factor apparently
Before returning to Holland, Pekelharing persuaded the associated with rice husks.
Such a finding was first reported by the Russian surgeon

TABLE 2.2 Beriberi in Javanese Prisons c.1890 Nikolai Lunin, in 1888, who found that the addition of milk
Prevalence to a synthetic diet supported the survival of mice. Lunin con-
(Cases/10,000 cluded, “A natural food such as milk must, therefore, contain
Diet Population Cases People) besides these known principal ingredients small quantities
of other and unknown substances essential to life.”
Polished rice 150,266 4200 279.5
Lunnin’s finding was soon confirmed by several other
Partially 35,082 85 24.2 investigators. By 1912, Rhömann in Germany, Socin in
polished rice Switzerland, Pekelharing in The Netherlands, and Hopkins
Unpolished 96,530 86 8.9 in England had each demonstrated that the addition of milk
rice to purified diets corrected the impairments in growth and
survival that were otherwise produced in laboratory rodents.
The German physiologist Wilhelm Stepp took another
Antiberiberi Factor Is Announced experimental approach. He found it possible to extract, from
bread and milk, factors required for animal growth. Although
Eijkman used this animal model in a series of investiga-
Pekelharing’s 1905 observations, published in Dutch, lay
tions in 1890–1897 and found that the antipolyneuritis fac-
unnoticed by many investigators, his conclusions about what
tor could be extracted from rice hulls with water or alcohol,
Hopkins had called the accessory factor in milk alluded to
that it was dialyzable, but that it was rather easily destroyed
the modern concept of a vitamin: “If this substance is absent,
with moist heat. He concluded that the water-soluble factor
the organism loses the power properly to assimilate the well
was a “pharmacological antidote” to the “beriberi microbe,”
known principal parts of food, the appetite is lost and with
which, although still not identified, he thought to be pres-
apparent abundance the animals die of want. Undoubtedly
ent in the rice kernel proper. Apparently, Gerrit Grijns,17 who
this substance not only occurs in milk but in all sorts of food-
continued that work after Eijkman returned to Holland, came
stuffs, both of vegetable and animal origin.”
to interpret these findings somewhat differently. Grijns went
Perhaps the most important of the early studies with
on to show that polyneuritis could be prevented by including
defined diets were those of the Cambridge biochemist
mung bean (Vigna radiata) in the diet; this led to mung beans
Frederick Gowland Hopkins.18 His studies demonstrated
being found effective in treating beriberi. In 1901, Grijns sug-
that the growth-promoting activities of accessory factors
gested, for the first time, that beriberi-producing diets “lacked
were independent of appetite, and that such factors prepared
a certain substance of importance in the metabolism of the
from milk or yeast were biologically active in very small
central nervous system.” Subsequently, Eijkman came to
amounts.
share Grijn’s view; in 1906, the two investigators published
a now-classic paper in which they wrote, “There is present in
rice polishings a substance different from protein, and salts, Two Lines of Inquiry
which is indispensable to health and the lack of which causes Therefore, by 1912, two independently developed lines
nutritional polyneuritis.” of inquiry had revealed that foods contained beneficial
factor(s) in addition to the nutrients known at the time. That
5. THE VITAMINE THEORY these factor(s) were present and active in minute amounts
was apparent from the fact that almost all of the mass of
Defined Diets Revealed Needs for Accessory food was composed of the known nutrients.
Factors
The announcement of the antiberiberi factor constituted the
first recognition of the concept of the vitamin, although the Two Lines of Inquiry Leading to the Discovery of the
Vitamins
term itself was yet to be coined. At the time of Eijkman’s
studies, but a world removed and wholly separate, others l The study of substances that prevent deficiency diseases
were finding that animals would not survive when fed “syn- l The study of accessory factors required by animals fed
purified diets.
thetic” or “artificial” diets formulated with purified fats,
proteins, carbohydrates, and salts—i.e., containing all of
the nutrients then known to be constituents of natural foods.
18. Sir Frederick Gowland Hopkins (1861–1947), is known for his work at
Cambridge University, which involved not only classic work on accessory
17. Grijns (1865–1944) was a Dutch physician trained at the University growth factors (for which he shared, with Christiaan Eijkman, the 1929
of Utrecht. He assisted Eijkman in Batavia and continued that work when Nobel Prize in Medicine or Physiology), but also the discoveries of
Eijkman, having contracted malaria, returned to Holland in 1896. glutathione and tryptophan.
Comments by Hopkins in 1906 indicate that he saw con- are far less important than the focus the newly coined term
nections between the accessory factors and the deficiency gave to the diet–health relationship. Funk was not unaware
diseases. On the subject of the accessory growth factors in of the importance of the term itself; he wrote, “I must admit
foods, he wrote, “No animal can live on a mixture of pure that when I chose the name “vitamine” I was well aware that
protein, fat and carbohydrate, and even when the necessary these substances might later prove not all to be of an amine
inorganic material is carefully supplied the animal still can- nature. However, it was necessary for me to use a name that
not flourish. The animal is adjusted to live either on plant would sound well and serve as a ‘catch-word.’”22
tissues or the tissues of other animals, and these contain
countless substances other than protein, carbohydrates and
fats. In diseases such as rickets, and particularly scurvy, we Funk’s Vitamines
have had for years knowledge of a dietetic factor; but though l Antiberiberi vitamine
we know how to benefit these conditions empirically, the l Antirickets vitamine
real errors in the diet are to this day quite obscure … They l Antiscurvy vitamine
are, however, certainly of the kind which comprises these l Antipellagra vitamine
minimal qualitative factors that I am considering.”

Hopkins demonstrated the presence of a factor(s) in
milk that stimulated the growth of animals fed diets con- Impact of the New Concept
taining all of the then-known nutrients (Fig. 2.1).
The vitamine theory opened new possibilities in nutri-
tion research by providing a new intellectual construct
The Lines Converge for interpreting observations of the natural world. No
The discovery by Eijkman and Grijns had stimulated efforts longer was the elucidation of the etiologies of diseases to
by investigators in several countries to isolate the antiberiberi be constrained by the germ theory. Thus, Funk’s greatest
factor in rice husks. Umetaro Suzuki, of Imperial University contribution involves not the data generated in his labo-
Agricultural College in Tokyo, succeeded in preparing a con- ratory, but rather the theory produced from his thoughtful
centrated extract from rice bran for the treatment of polyneu- review of information already in the medical literature
ritis and beriberi. He called the active fraction “oryzanin” but of the time. This fact caused Harris (1955) to observe,
could not achieve its purification in crystalline form. Casimir “The interpreter may be as useful to science as the dis-
Funk,19 a chemist at the Lister Institute in London, concluded coverer. I refer here to any man23 who is able to take a
from the various conditions in which it could be extracted and broad view of what has already been done by others, to
then precipitated that the antipolyneuritis factor in rice husks collect evidence and discern through it all some common
was an organic base and, therefore, nitrogenous in nature. connecting link.”
When he appeared to have isolated the factor, Funk coined a The real impact of Funk’s theory was to provide a new
new word for it, with the specific intent of promoting the new concept for interpreting diet-related phenomena. As the
concept in nutrition to which Hopkins had alluded. Having educational psychologist Novak24 observed more recently,
evidence that the factor was an organic base, and therefore “As our conceptual and emotional frameworks change, we
an amine, Funk chose the term vitamine20 because it was see different things in the same material.”
clearly vital, i.e., pertaining to life. Still, it was not clear by 1912 whether the accessory
factors were the same as the vitamines. In fact, until 1915,
there was a considerable debate concerning whether the
Funk’s Theory growth factor for the rat was a single or multiple entity (it
In 1912, Funk published his landmark paper presenting was already clear that there was more than one vitamine).
the vitamine theory; in it he proposed, in what some have Some investigators were able to demonstrate it in yeast and
referred to as a leap of faith, four different vitamines. That the not butter; others found it in butter and not yeast. Some
concept was not a new one, and that not all of these factors showed it to be identical with the antipolyneuritis factor;
later proved to be amines (hence, the change to vitamin21) others showed that it was clearly different.
19. Funk (1884–1957) was born in Poland and studied in Switzerland, 22. Funk, C. (1912). The etiology of the deficiency diseases. J. State Med.
Paris and Berlin. 20, 341–368.
20. Harris (1955) reported that the word vitamine was suggested to Funk by 23. Harris’s word choice reveals him as a product of his times. Because it is
his friend, Dr Max Nierenstein, Reader in Biochemistry at the University clear that the process of intellectual discovery to which Harris refers does
of Bristol. not recognize gender, it is more appropriate to read this word as person.
21. The dropping of the e from vitamine is said to have been the suggestion 24. Novak, J.D. (1977) “A Theory of Education,” Cornell University Press,
of J.C. Drummond. Ithaca, NY.
1890
chick polyneuritis
1900 anti-polyneuritis factor
guinea pig scurvy
“vitamine” anti-scorbutic
1910 factors
failures of refined diets
fat-soluble A water-soluble B
anti-xerophthalmia
1920 rat: fetal vitamin B
human pernicious
resorption
anemia cured with
heat-labile anti- heat-stable anti- heat-labile anti-beri heat-stable, anti- liver
xerophthalmic factor rachitic factor beri vitamin F pellagra factor
Vitamin A Vitamin D
chick anti- Vitamin C
1930 Vitamin B1 dermatitis factor
S. cerevisiae
Vitamin E hemorrhagic Rhizobium
dog anti- growth factor
disease growth factor rat adermin riboflavin pellagra factors
egg white disease Vitamin B2
anti-hemorrhagic yeast “bios” factors pyridoxine Pantothenic chick anemia
nicotinamide
factor
single factor Vitamin B6 Acid
1940 Niacin factors M,U,Bc zoopterin, animal
Vitamin K protein factor, manure
L. casei growth factor factor, LLD factor
Biotin rhizopterin
Folic Acid
1950 Vitamin B12
FIGURE 2.1 The cascade of vitamin discovery.
the antixerophthalmic factor. Shortly, it was found that the

TABLE 2.3 McCollum’s Rat Growth Factors so-called water-soluble B material was not only required for
Factor Found in Not Found in normal growth of the rat but also prevented polyneuritis in
the chick. Therefore, it was clear that water-soluble B was
Fat-soluble A Milk fat, egg yolk Lard, olive oil
identical to or at least contained Funk’s antiberiberi vita-
Water-soluble B Wheat, milk, egg yolk Polished rice mine; hence, it became known as vitamine B.
There Is More Than One Accessory Factor Accessory Factors Are the Same as Vitamines
The debate was resolved by the landmark studies of the With these discoveries, it became apparent that the bio-
American biochemist Elmer McCollum25 and his volunteer logical activities of the accessory factors and the vitamines
assistant Marguerite Davis26 at the University of Wisconsin were likely to be due to the same compounds. The concept
in 1913–1915. Using diets based on casein and lactose, they of a vitamine was thus generalized to include nonnitroge-
demonstrated that at least two different additional growth nous compounds, and the antipolyneuritis vitamine became
factors were required to support normal growth of the rat. vitamin B.
One factor could be extracted with ether from egg or but-
terfat (but not olive or cottonseed oils) but was nonsaponi-
fiable; it appeared to be the same factor shown earlier by
Elucidation of the Vitamines
Wilhelm Stepp,27 and by Thomas Osborne28 and Lafayette So it was, through the agencies of several factors, a use-
Mendel29 in the same year, to be required to sustain growth ful new intellectual construct, the use of defined diets, and
of the rat. The second factor was extractable with water and the availability of appropriate animal models, that nutrition
prevented polyneuritis in chickens and pigeons. McCollum emerged as a scientific discipline. By 1915, thinking about
called these factors fat-soluble A and water-soluble B, diet and health had been forever changed, and it was clear
respectively (Table 2.3). that the earlier notions about the required nutrients had
been incomplete. Therefore, it should not be surprising to
find, by the 1920s, mounting interest in the many questions
Accessory Factors Prevent Disease
generated by what had become sound nutritional research.
Subsequent studies conducted by McCollum’s group That interest and the further research activity it engendered
showed that the ocular disorders (i.e., xerophthalmia30) resulted, over the brief span of only five decades, in the
that developed in rats, dogs, and chicks fed fat-free diets development of a fundamental understanding of the identi-
could be prevented by feeding them cod liver oil, butter, or ties and functions of about 40 nutrients, one-third of which
preparations of fat-soluble A, which then became known as are considered vitamins.
25. Elmer Verner McCollum (1879–1967) received his doctorate at Yale and
worked on dietary protein quality with Osborne and Mendel there. In 1909, he Crooked Paths to Discovery
joined the faculty of the University of Wisconsin, where his work on growth-
promoting factors in deproteinized milk led to the recognition of vitamin A.
The paths leading to the discovery of the vitamins wan-
In 1917, he moved to Johns Hopkins University. McCollum opposed Funk’s dered from Java with the findings of Eijkman in the 1890s,
term “vitamine” on the basis that all essential nutrients were vital. to England with Funk’s theory in 1912, to the United States
26. Marguerite Davis (1887–1967) was a graduate student with McCollum. with the recognition of fat-soluble A and water-soluble B in
When she and McCollum had shown that water-soluble B was not a single 1915. By that time the paths had already branched, and for
compound, she gave the components letter names, thus, starting that
tradition of naming vitamins.
the next four decades they would branch again and again as
27. Wilhelm Stepp (1882–1964) was a professor of medicine at several scientists from many laboratories and many nations would
German Universities (Strassburg, Jena, Breslau and Munich). pursue many unexplained responses to diet among many
28. Thomas Burr Osborne (1859–1929) was a professor of chemistry types of animal model. Some of these pursuits appeared
who spent his career at the Connecticut Agricultural Experiment Station to fail; however, in aggregate, all laid the groundwork of
studying protein quality and nutritional requirements. His collaboration
with Mendel led to the recognition of the essentiality of amino acids.
understanding on which the discoveries of those factors
29. Lafayette Benedict Mendel (1872–1935) was a professor of now recognized to be vitamins were based. When viewed
physiological chemistry at Yale University who worked with Osborne in retrospect, the path to that recognition may seem decep-
to determine why rats could not survive on diets of only purified tively straight—but it most definitely was not. The way was
carbohydrates, fats and proteins. branched and crooked; in many cases, progress was made
30. Xerophthalmia, from the Greek xeros (“dry”) and ophthalmos (“eye”),
involves dryness of the eyeball owing to atrophy of the periocular glands,
by several different investigators traveling in apparently dif-
hyperkeratosis of the conjunctiva, and, ultimately, inflammation and ferent directions. The following recounts the highlights of
edema of the cornea, which leads to infection, ulceration, and blindness. the exciting search for the elucidation of the vitamins.
6. ELUCIDATION OF THE VITAMINS hexuronic acid,33 now called ascorbic acid. Szent-Györgi
had isolated it in crystalline form from adrenal cortex, while
New Animal Model Reveals New King had isolated it from cabbage and citrus juice.34 After
Vitamin: “C” Szent-Györgi returned to Hungary to take a professorship,
Eijkman’s report of polyneuritis in the chicken and an ani- he was joined by an American-born Hungarian, J. Svirbely,
mal model for beriberi stimulated researchers Axel Holst who had been working in King’s laboratory. Szent-Györgi
and Theodor Frölich at the University of Christiana in had isolated c.500 grams of crystalline hexuronic acid from
Oslo, who were interested in shipboard beriberi, a com- peppers, and then 25 g of the vitamin from adrenal glands,
mon problem among Norwegian seamen. Working with making samples available to other laboratories. On April 1,
pigeons, they found a beriberi diet to produce the polyneu- 1932, King and Waugh reported that their crystals protected
ritis described by Eijkman; however, they considered that guinea pigs from scurvy; 2 weeks later, Svirbely and Szent-
condition very different from the disease of sailors. In 1907, Györgi reported virtually the same results. The following
they attempted to produce the disease in another experimen- year, the chemical structure of ascorbic acid was elucidated
tal animal species: the common Victorian household pet, by the groups of Haworth in Birmingham and Karrer in
the guinea pig. Contrary to their expectations, they failed Zurich, both of which also achieved its synthesis.
to produce, by feeding that species a cereal-based diet, any-
thing resembling beriberi; instead, they observed the famil- Fat-Soluble A: Actually Two Factors
iar signs of scurvy. Eijkman’s work suggested to them that,
like beriberi, scurvy too might be due to a dietary deficiency. Pursuing the characterization of fat-soluble A, by 1919
Having discovered, quite by chance, one of the few pos- McCollum’s group35 and others had found that, in addition
sible animal species in which scurvy could be produced,31 to supporting growth for the rat, the factor also prevented
Holst and Frölich had produced something of tremendous xerophthalmia and night blindness in that species. In 1920,
value—an animal model of scurvy32—showing that lesions Drummond called the active lipid vitamin A.36 This factor
could be prevented by feeding apples, (unboiled) cabbage, was present in cod liver oil, which at the turn of the century
potatoes, and lemon juice. had been shown to prevent both xerophthalmia and night
This finding led Henriette Chick and Ruth Skelton of the blindness—which Bitot, some 40 years earlier, had con-
Lister Institute, in the second decade of the 20th century, cluded had the same underlying cause.
to use the guinea pig to develop a bioassay for the deter-
mination of the antiscorbutic activity in foods, and S.S. Vitamin A Prevents Rickets?
Zilva and colleagues (also at the Lister Institute) to isolate
from lemons the crude factor that had come to be known as Undoubtedly influenced by the recent recognition of vita-
vitamin C. It was soon found that vitamin C could reduce min A, Edward Mellanby, who had worked with Hopkins,
the dye 2,6-dichloroindophenol, but the reducing activ- undertook to produce a dietary model of rickets. For this
ity determined with that reagent did not always correlate he used puppies, which the Scottish physician Findley
with the antiscorbutic activity determined by bioassay. found developed rickets if kept indoors.37 Mellanby fed a
Subsequently, it was found that the vitamin was reversibly low-fat diet based on oatmeal with limited milk intake to
oxidized, but that both the reduced and oxidized forms had puppies that he kept indoors; the puppies developed the
antiscorbutic activity.
In 1932, Albert Szent-Györgi, a Hungarian scientist 33. It is said that when Szent-Györgi first isolated the compound, he was
working in Hopkins’ laboratory at Cambridge University, at a loss for a name for it. Knowing it to be a sugar, but otherwise ignorant
and Glen King at the University of Pittsburgh established of its identity, he proposed the name ignose, which was disqualified by
that the antiscorbutic factor was identical with the reductant an editor who did not appreciate the humor of the Hungarian chemist.
Ultimately, the names ascorbic acid and vitamin C, by which several
groups had come to refer to the antiscorbutic factor, were adopted.
31. Their finding was, indeed, fortuitous, as vitamin C is now known to 34. The reports of both groups (King, C.G., Waugh, W.S., 1932. Science
be an essential dietary nutrient only for the guinea pig, primates, fishes, 75, 357–358; Svirbely, J.L., Szent-Györgi, A., 1932. Biochem. J. 26,
some fruit-eating bats, and some passeriform birds. Had they used the 865–870) appeared within 2 weeks of one another in 1932. In fact, Svirbely
rat, the mouse or the chick in their study, vitamin C might have remained had recently joined Szent-Györgi’s group, having come from King’s
unrecognized for perhaps quite a while. laboratory. In 1937, King and Szent-Györgi shared the Nobel Prize for
32. In fact, scorbutic signs had been observed in the guinea pig more than their work in the isolation and identification of vitamin C.
a decade earlier, when a U.S. Department of Agriculture pathologist noted 35. In 1917, McCollum moved to the newly established School of Public
in an annual report: “When guinea pigs are fed with cereals (bran and oats Health at Johns Hopkins University.
mixed), without any grass, clover or succulent vegetables, such as cabbage, 36. In 1920, J.C. Drummond proposed the use of the names vitamin A and
a peculiar disease, chiefly recognizable by subcutaneous extravasation vitamin B for McCollum’s factors, and the use of the letters C, D, etc., for
of blood, carries them off in four to eight weeks.” That this observation any vitamins subsequently to be discovered.
was not published for a wider scientific audience meant that it failed to 37. Exposing infants to sunlight is a traditional practice in many cultures
influence the elucidation of the etiology of scurvy. and had been a folk treatment for rickets in northern Europe.
marked skeletal deformities characteristic of rickets. When Robeson crystallized retinol and several of its esters; in
he found that these deformities could be prevented by feed- 1947, they crystallized the 13-cis-isomer. Isler’s group in
ing cod liver oil or butterfat without allowing the puppies Basel achieved the synthesis of retinol in the same year and
outdoors, he concluded that rickets, too, was caused by a that of β-carotene 3 years later.
deficiency of vitamin A, which McCollum had discovered
in those materials.
The Nature of Vitamin D
McCollum’s discovery of the antirachitic factor he called
New Vitamin: “D”
vitamin D in cod liver oil, which was made possible through
McCollum, however, suspected that the antirachitic factor the use of animal models, was actually a rediscovery, as that
present in cod liver oil was different from vitamin A. Having material had been long recognized as an effective medicine
moved to the Johns Hopkins University in Baltimore, he for rickets in children. Still, the nature of the disease was the
conducted an experiment in which he subjected cod liver subject of considerable debate, particularly after 1919, when
oil to aeration and heating (100°C for 14 h), after which he Huldschinsky, a physician in Vienna, demonstrated the effi-
tested its antixerophthalmic and antirachitic activities with cacy of ultraviolet light in healing rickets. This confusion was
rat and chick bioassays, respectively. He found that heat- clarified by the findings in 1923 of Goldblatt and Soames,
ing had destroyed the antixerophthalmic (vitamin A) activ- who demonstrated that when livers from rachitic rats were
ity, but that cod liver oil had retained antirachitic activity. irradiated with ultraviolet light, they could cure rickets when
McCollum called the heat-stable factor vitamin D. fed to rachitic, nonirradiated rats. The next year, Steenbock’s
group demonstrated the prevention of rickets in rats by ultra-
β-Carotene, a Provitamin violet irradiation of either the animals themselves or their
food. Further, the light-produced antirachitic factor was
At about the same time (1919), Steenbock in Wisconsin associated with the fat-soluble portion of the diet.39
pointed out that the vitamin A activities of plant materials
seemed to correlate with their contents of yellow pigments.
He suggested that the plant pigment carotene was respon-
Vitamers D
sible for the vitamin A activity of such materials. Yet the The ability to produce vitamin D (which could be bioas-
vitamin A activity in organic extracts of liver was colorless. sayed using both rat and chick animal models) by irradi-
Therefore, Steenbock suggested that carotene could not be ating lipids led to the finding that large quantities of the
vitamin A, but that it may be converted metabolically to vitamin could be produced by irradiating plant sterols.
the actual vitamin. This hypothesis was not substantiated This led Askew’s and Windaus’s groups, in the early
until 1929, when von Euler and Karrer in Stockholm dem- 1930s, to the isolation and identification of the vita-
onstrated growth responses to carotene in rats fed vitamin min produced by irradiation of ergosterol. Steenbock’s
A-deficient diets. Further, Moore in England demonstrated, group, however, found that while the rachitic chick
in the rat, a dose–response relationship between dietary responded appropriately to irradiated products of cod
β-carotene and hepatic vitamin A concentration. This liver oil or the animal sterol cholesterol, that animal did
proved that β-carotene is, indeed, a provitamin. not respond to the vitamin D so produced from ergos-
terol. On the basis of this apparent lack of equivalence,
Vitamin A Linked to Vision Wadell suggested in 1934 that the irradiated products of
ergosterol and cholesterol were different. Subsequently,
In the early 1930s, the first indications of the molecular Windaus’s group synthesized 7-dehydrocholesterol
mechanism of the visual process were produced by George and isolated a vitamin D-active product of its irradiation.
Wald, of Harvard University but working in Germany at the In 1936, they reported its structure, showing it to be a side
time, who isolated the chromophore retinen from bleached chain isomer of the form of the vitamin produced from
retinas.38 A decade later, Morton in Liverpool found plant sterols. Thus, two forms of vitamin D were found:
that the chromophore was the aldehyde form of vitamin ergocalciferol (from ergosterol), which was called vita-
A—retinaldehyde. Just after Wald’s discovery, Karrer’s min D2,40 and cholecalciferol (from cholesterol), which
group in Zurich elucidated the structures of both β-carotene
and vitamin A. In 1937, Holmes and Corbett succeeded in
crystallizing vitamin A from fish liver. In 1942, Baxter and 39. This discovery, i.e., that by ultraviolet irradiation it was possible to
induce vitamin D activity in such foods as milk, bread, meats, and butter,
led to the widespread use of this practice, which has resulted in the virtual
38. For this and other discoveries of the basic chemical and physiological eradication of rickets as a public health problem.
processes in vision, George Wald was awarded, with Haldan K. Hartline 40. Windaus’s group had earlier isolated a form of the vitamin he had
(of the United States) and R. Grant (of Sweden), the Nobel Prize in called vitamin D1, which had turned out to be an irradiation-breakdown
Chemistry in 1967. product, lumisterol.
was called vitamin D3. While it was clear that the vitamers had been isolated earlier by Auhagen. That many research
D had important metabolic roles in calcification, insights groups were actively engaged in the research on the anti-
concerning the molecular mechanisms of the vitamin polyneuritis/beriberi factor is evidence of intense interna-
would not come until the 1960s. Then, it became appar- tional interest due to the widespread prevalence of beriberi.
ent that neither vitamer was metabolically active per se; The characterization of thiamin clarified the distinc-
each is converted in vivo to metabolites that participate tion of the antiberiberi factor from the antipellagra activity.
in a system of calcium homeostasis that continues to be The latter was not found in maize (corn), which contained
of great interest to the biomedical community. With this appreciable amounts of thiamin. Goldberger called the two
understanding, it became apparent that vitamin D3 was substances the “A-N factor” (antineuritic) and the “P-P
actually a steroid hormone.41 factor” (pellagra-preventive). Others called these factors
vitamins F (for Funk) and G (for Goldberger), respectively,
but these terms did not last.43 By the mid-1920s the terms
Multiple Identities of Water-Soluble B
vitamin B1 and vitamin B2 had been rather widely adapted
By the 1920s, it was apparent that the antipolyneuritis fac- for these factors, respectively; this practice was codified
tor, called water-soluble B and present in such materials as in 1927 by the Accessory Food Factors Committee of the
yeasts, was not a single substance. This was demonstrated British Medical Research Council.
by the finding that fresh yeast could prevent both beriberi
and pellagra. However, the antipolyneuritis factor in yeast
Vitamin B2: A Complex of Several Factors
was unstable to heat, while such treatment did not alter
the efficacy of yeast to prevent dermatitis in rodents. This That the thermostable second nutritional factor in yeast,
caused Goldberger to suggest that the then-called vitamin which by that time was called vitamin B2, was not a single
B was actually at least two vitamins: the antipolyneuritis substance, and was not immediately recognized, giving rise
vitamin and a new antipellagra vitamin. to considerable confusion and delay in the elucidation of its
In 1926, the heat-labile antipolyneuritis/beriberi factor chemical identity (identities). It should be noted that efforts
was first crystallized by Jansen and Donath, working in the to fractionate the heat-stable factor were guided almost
Eijkman Institute (which replaced Eijkman’s simple facili- exclusively by bioassays with experimental animal mod-
ties) in Batavia. They called the factor aneurin. Their work els. Yet, different species yielded discrepant responses to
was facilitated by the use of the small rice bird (Munia maja) preparations of the factor. When such variation in responses
as an animal model in which they developed a rapid bioas- among species was finally appreciated, it became clear that
say for antipolyneuritic activity.42 Six years later, Windaus’s vitamin B2 actually included several heat-stable factors.
group isolated the factor from yeast, perhaps the richest Vitamin B2, as then defined, was indeed a complex.
source of it. In the same year (1932), the chemical structure
was determined by R.R. Williams, who named it thiamin—
i.e., the vitamin containing sulfur (thios, in Greek). Noting Components of the Vitamin B2 Complex
that deficient subjects showed high blood levels of pyruvate l The P-P factor (preventing pellagra in humans and
and lactate after exercise, in 1936 Rudolph Peters of Oxford pellagra-like diseases in dogs, monkeys, and pigs)
l A growth factor for the rat
University used, for the first time, the term “biochemical
l A pellagra-preventing factor for the rat
lesion” to describe the effects of the dietary deficiency.
l An antidermatitis factor for the chick
Shortly thereafter, methods of synthesis were achieved by
several groups, including those of Williams, Andersag and
Westphal, and Todd. In 1937, thiamin diphosphate (thiamin
Vitamin B2 Complex Yields Riboflavin
pyrophosphate) was isolated by Lohmann and Schuster,
who showed it to be identical to the cocarboxylase that The first substance in the vitamin B2 complex to be eluci-
dated was the heat-stable, water-soluble rat growth factor,
which was isolated by Kuhn, György, and Wagner-Jauregg
41. 1,25-dihydroxycholecalciferol meets the standard definition of at the Kaiser Wilhelm Institute in 1933. Those investigators
a hormone in as much as it is produced and transported through the
found that thiamin-free extracts of autoclaved yeast, liver, or
circulation to exert biological activity in distal organs.
42. The animals, which consumed only 2 grams of feed daily, showed rice bran prevented the growth failure of rats fed a thiamin-
a high (98+%) incidence of polyneuritis within 9–13 days if fed white supplemented diet. Further, they noted that a yellow-green
polished rice. The delay of onset of signs gave them a useful bioassay fluorescence in each extract promoted rat growth, and that
of antipolyneuritic activity suitable for use with small amounts of
test materials. This point is not trivial, inasmuch as there is only about
a teaspoon of thiamin in a ton of rice bran. The bioassay of Jansen and 43. In fact, the name vitamin F was later used, with some debate as to the
Donath was sufficiently responsive for 10 μg of active material to be appropriateness of the term, to describe essential fatty acids. The name
curative. vitamin G has been dropped completely.
the intensity of fluorescence was proportional to the effect including the nurses and physicians who cared for patients;
on growth. This observation enabled them to develop a rapid this suggested to him that pellagra was not an infectious
chemical assay that, in conjunction with their bioassay, they disease. Noting that the food available to the professional
exploited to isolate the factor from egg white in 1933. They staff was much different from that served to the inmates
called it ovoflavin. The same group then isolated, by the (the former included meat and milk not available to the
same procedure, a yellow-green fluorescent growth-promot- inmates), Goldberger suspected that an unbalanced diet
ing compound from whey (which they called lactoflavin). was responsible for the disease. He secured funds to sup-
This procedure involved the adsorption of the active factor ply meat and milk to inmates for a 2-year period of study.
on fuller’s earth,44 from which it could be eluted with base.45 The results were dramatic: pellagra soon disappeared, and
At the same time, Ellinger and Koschara, at the University no new cases were reported for the duration of the study.
of Düsseldorf, isolated similar substances from liver, kid- However, when funds expired at the end of the study and
ney, muscle, and yeast, and Booher in the United States the institution was forced to return to its former meal pro-
isolated the factor from whey. These water-soluble growth gram, pellagra reappeared. While the evidence from this
factors became designated as flavins.46 By 1934, Kuhn’s uncontrolled experiment galvanized Goldberger’s convic-
group had determined the structure of the so-called flavins. tion that pellagra was a dietary disease, it was not suf-
These substances were thus found to be identical; because ficient to affect a medical community that thought the
each contained a ribose-like (ribotyl) moiety attached to disease likely to be an infection.
an isoalloxazine nucleus, the term riboflavin was adopted. Over the course of two decades, Goldberger worked to
Riboflavin was synthesized by Kuhn’s group (then at the elucidate the dietary basis of pellagra. Among his efforts
University of Heidelberg) and by Karrer’s group at Zurich to demonstrate that the disease was not infectious was the
in 1935. As the first component of the vitamin B2 complex, it exposure, by ingestion and injection, of himself, his wife,
is also referred to as vitamin B2; however, that should not be and 14 volunteers to urine, feces, and biological fluids from
confused with the earlier designation of the P-P factor. pellagrins.48 He also experimented with 12 male prison-
ers who volunteered to consume a diet (based on corn and
other cereals, but containing no meat or dairy products) that
Vitamin B2 Complex Yields Niacin
he thought might produce pellagra: within 5 months half of
Progress in the identification of the P-P factor was retarded the subjects had developed dermatitis on the scrotum, and
by two factors: the pervasive influence of the germ theory some also showed lesions on their hands.49 The negative
of disease and the lack of an animal model. The former results of these radical experiments, plus the finding that
made acceptance of evidence suggesting a nutritional origin therapy with oral supplements of the amino acids cysteine
of the disease a long and difficult undertaking. The latter and tryptophan was effective in controlling the disease, led,
precluded the rigorous testing of hypotheses for the etiol- by the early 1920s, to the establishment of a dietary origin
ogy of the disease in a timely and highly controlled manner. of pellagra. Further progress was hindered by the lack of an
These challenges were met by Joseph Goldberger, a U.S. appropriate animal model. Although pellagra-like diseases
Public Health Service bacteriologist who, in 1914, was put had been identified in several species, most proved not to
in charge of the Service’s pellagra program. be useful as biological assays (indeed, most of these later
proved to be manifestations of deficiencies of other vita-
Pellagra: An Infectious Disease? mins of the B2 complex and to be wholly unrelated to pel-
lagra in humans).
Goldberger’s first study47 is now a classic. He studied a The identification of a useful animal model for pellagra
Jackson, Mississippi, orphanage in which pellagra was came from Goldberger’s discovery in 1922 that maintain-
endemic. He noted that whereas the disease was preva- ing dogs on diets essentially the same as those associated
lent among the inmates, it was absent among the staff, with human pellagra resulted in the animals developing a
necrotic degeneration of the tongue called black tongue
disease. This animal model for the disease led to the final
44. Floridin, a nonplastic variety of kaolin containing an aluminum solution of the problem.
magnesium silicate. The material is useful as a decolorizing medium. Its
name comes from an ancient process of cleaning or fulling wool, in which
a slurry of earth or clay was used to remove oil and particulate dirt.
45. By this procedure, the albumen from 10,000 eggs yielded c.30 mg of 48. People with pellagra.
riboflavin. 49. Goldberger conducted this study with the approval of prison authorities.
46. Initially, the term flavin was used with a prefix that indicated the source As compensation for participation, volunteers were offered release at the
material; for example, ovoflavin, hepatoflavin, and lactoflavin designated end of the 6 mo. experimental period, which each exercised upon the
the substances isolated from egg white, liver, and milk, respectively. conclusion of the study without evaluation. For that reason, Goldberger
47. See the listing of papers of key historical significance, in Recommended was unable to demonstrate to a doubting medical community that the
Reading at the end of this chapter. unbalanced diet had, indeed, produced pellagra.
Impact of an Animal Model for Pellagra formerly been called acrodynia or rat pellagra, which was
a symmetrical florid dermatitis spreading over the limbs
This finding made possible experimentation that would lead
and trunk, with redness and swelling of the paws and ears.
rather quickly to an understanding of the etiology to the
His definition effectively distinguished these signs from
disease. Goldberger’s group soon found that yeast, wheat
those produced by riboflavin deficiency, which involves
germ, and liver would prevent canine black tongue and pro-
lesions on the head and chest, and inflammation of the eye-
duce dramatic recoveries in pellagra patients. By the early
lids and nostrils. The focus provided by György’s definition
1930s, it was established that the human pellagra and canine
strengthened the use of the rat in the bioassay of vitamin B6
black tongue curative factor was heat-stable and could be
activity by clarifying its end point. Within 2 years, partial
separated from the other B2 complex components by filtra-
purification of vitamin B6 had been achieved by his group;
tion through fuller’s earth, which adsorbed only the latter.
and in 1938 (only 4 years after the recognition of the vita-
Thus, the P-P factor became known as the filtrate factor.
min), the isolation of vitamin B6 in crystalline form was
In 1937, Elvehjem isolated nicotinamide from liver extracts
achieved by five research groups. The chemical structure of
that had high antiblack tongue activity and showed that
the substance was quickly elucidated as 3-hydroxy-4,5-bis-
nicotinamide and nicotinic acid each cured canine black
(hydroxymethyl)-2-methylpyridine. In 1939, Folkers
tongue. Both compounds are now called niacin. In the same
achieved the synthesis of this compound, which György
year, several groups went on to show the curative effect of
called pyridoxine.
nicotinic acid against human pellagra.
It is ironic that the antipellagra factor was already well
known to chemists of the time. Some 70 years earlier, the B2 Complex Yields Pantothenic Acid
German chemist Huber had prepared nicotinic acid by the In the course of studying the growth factor called vitamin
oxidation of nicotine with nitric acid. Funk had isolated the B2, Norris and Ringrose at Cornell described, in 1930, a
compound from yeast and rice bran in his search for the antipellagra-like syndrome of the chick. The lesions could be
beriberi factor; however, because it had no effect on beriberi, prevented with aqueous extracts of yeast or liver, then rec-
nicotinic acid remained, for two decades, an entity with unap- ognized to contain the B2 complex. In studies of B2 com-
preciated biological importance. This view changed in the plex-related growth factors for chicks and rats, Jukes and
mid-1930s, when Warburg and Christiaan isolated nicotin- colleagues at Berkeley found positive responses to a ther-
amide from the hydrogen-transporting coenzymes I and II,50 mostable factor that, unlike pyridoxine, was not adsorbed
giving the first clue to its importance in metabolism. Within by fuller’s earth from an acid solution. They referred to it as
a year, Elvehjem had discovered its nutritional significance. their filtrate factor.
At the same time, and quite independently, the
B2 Complex Yields Pyridoxine University of Texas microbiologist R.J. Williams was pur-
suing studies of the essential nutrients for Saccharomyces
During the course of their work leading to the successful
cerevisiae and other yeasts. His group found a potent
isolation of riboflavin, Kuhn and colleagues noticed an
growth factor that they could isolate from a wide variety
anomalous relationship between the growth-promoting and
of plant and animal tissues.52 They called it pantothenic
fluorescence activities of their extracts: the correlation of
acid, meaning “found everywhere,” and also referred to
the two activities diminished at high levels of the former.
the substance as vitamin B3. Later in the decade, Snell’s
Further, the addition of nonfluorescent extracts was neces-
group found that several lactic and propionic acid bacteria
sary for the growth-promoting activity of riboflavin. They
require a growth factor that had the same properties. Jukes
interpreted these findings as evidence for a second com-
recognized that his filtrate factor, Norris’s chick antider-
ponent of the heat-stable complex—one that was removed
matitis factor, and the unknown factors required by yeasts
during the purification of riboflavin. These factors were
and bacteria were identical. He demonstrated that both his
also known to prevent dermatoses in the rat, an activity
filtrate factor and pantothenic acid obtained from Williams
called adermin; however, the lack of a critical assay that
could prevent dermatitis in the chick. Pantothenic acid
could differentiate between the various components of the
was isolated and its chemical structure was determined by
B2 complex led to a considerable confusion.
Williams’s group in 1939. The chemical synthesis of the
In 1934, György proffered a definition of what he called
vitamin was achieved by Folkers the following year. The
vitamin B6 activity51 as the factor that prevented what had
52. The first isolation of pantothenic acid employed 250 kilograms of
50. Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine sheep liver. The autolysate was treated with fuller’s earth; the factor was
dinucleotide phosphate (NADP), respectively. adsorbed to Norite and eluted with ammonia. Brucine salts were formed
51. György defined vitamin B6 activity as “that part of the vitamin and were extracted with chloroform–water, after which the brucine salt of
B-complex responsible for the cure of a specific dermatitis developed by pantothenic acid was converted to the calcium salt. The yield was 3 grams
rats on a vitamin-free diet supplemented with vitamin B1, and lactoflavin.” of material with c. 40% purity.
that the fat-soluble factor was a new vitamin, which he

TABLE 2.4 Factors Leading to the Discovery of called vitamin E.
Pantothenic Acid
Factor Bioassay A Classic Touch in Coining Tocopherol

Filtrate factor Chick growth
Soon, Evans was able to prepare a potent concentrate of
Chick antidermatitis Prevention of skin lesions and poor vitamin E from the unsaponifiable lipids of wheat germ
factor feather development in chicks oil; others prepared similar vitamin E-active concentrates
Pantothenic acid Growth of Saccharomyces cerevisiae from lettuce lipids. By the early 1930s, Olcott and Mattill
and other yeasts at the University of Iowa had found that such prepara-
tions, which prevented the gestation resorption syndrome
in rats, also had chemical antioxidant properties that could
be assayed in vitro.55 In 1936, Evans isolated from unsa-
various factors leading to the discovery of pantothenic acid ponifiable wheat germ lipids allophanic acid esters of three
are presented in Table 2.4. alcohols, one of which had very high biological vitamin E
activity. Two years later, Fernholz showed that the latter
A Fat-Soluble, Antisterility Factor: Vitamin E alcohol had a phytyl side chain and a hydroquinone moi-
ety and proposed the chemical structure of the new vita-
Interest in the nutritional properties of lipids was stimu-
min. Evans coined the term tocopherol, which he derived
lated by the resolution of fat-soluble A into vitamins A and
from the Greek words tokos (“childbirth”) and pherein (“to
D by the early 1920s. Several groups found that supple-
bear”);56 he used the suffix -ol to indicate that the factor is
mentation with the newly discovered vitamins A, C, and
an alcohol. He also named the three alcohols α-, β-, and
D and thiamin markedly improved the performance of ani-
γ-tocopherol. In 1938, synthesis of the most active vitamer,
mals fed purified diets containing adequate amounts of pro-
α-tocopherol, was achieved by the groups of Karrer, Smith,
tein, carbohydrate, and known required minerals. However,
and Bergel. A decade later another vitamer, δ-tocopherol,
H.M. Evans and Katherine Bishop, at the University of
was isolated from soybean oil; not until 1959 were the toco-
California, observed that rats fed such supplemented diets
trienols described.57
seldom reproduced normally. They found that fertility was
abnormally low in both males (which showed testicular
degeneration) and females (which showed impaired pla- Antihemorrhagic Factor: Vitamin K
cental function and failed to carry their fetuses to term).53 In the 1920s, Henrik Dam, at the University of
Dystrophy of skeletal and smooth muscles of the uterus Copenhagen, undertook studies to determine whether
was also noted. In 1922, these investigators reported that cholesterol was an essential dietary lipid. In 1929, Dam
the addition of small amounts of yeast or fresh lettuce to reported that chicks fed diets consisting of food that had
the purified diet would restore fertility to females and pre- been extracted with nonpolar solvents to remove sterols
vent infertility in animals of both sexes. They designated developed subdural, subcutaneous, or intramuscular hem-
the unknown fertility factor as factor X. Using the preven- orrhages, anemia, and abnormally long blood-clotting
tion of gestation resorption as the bioassay, Evans and times. A similar syndrome in chicks fed ether-extracted
Bishop found factor X activity in such unrelated materials fish meal was reported by McFarlane’s group, which at
as dried alfalfa, wheat germ, oats, meats, and milk fat, from the time was attempting to determine the chick’s require-
which it was extractable with organic solvents. They dis- ments for vitamins A and D. They found that nonextracted
tinguished the new fat-soluble factor from the known fat- fish meal completely prevented the clotting defect. Holst
soluble vitamins by showing that single droplets of wheat and Holbrook found that cabbage prevented the syndrome,
germ oil administered daily completely prevented gestation which they took as evidence of an involvement of vitamin
resorption, whereas cod liver oil, known to be a rich source C. By the mid-1930s, Dam had shown that the clotting
of vitamins A and D, failed to do so.54 In 1924, Sure, at the
University of Arkansas, confirmed this work, concluding
55. Although the potencies of the vitamin preparations in the in vivo (rat
gestation resorption) and in vitro (antioxidant) assays were not always well
53. The vitamin E-deficient rat carries her fetuses quite well until a fairly correlated.
late stage of pregnancy, at which time they die and are resorbed. This 56. Evans wrote in 1962 that he was assisted in the coining of the name for
syndrome is distinctive; it termed gestation resorption. vitamin E by George M. Calhoun, Professor of Greek and a colleague at
54. In fact, Evans and Bishop found that cod liver oil actually increased the University of California. It was Calhoun who suggested the Greek roots
the severity of the gestation resorption syndrome, a phenomenon now of this now-familiar name.
understood on the basis of the antagonistic actions of high concentrations 57. The tocotrienols differ from the tocopherols only by the presence of
of the fat-soluble vitamins. three conjugated double bonds in their phytyl side chains.
defect was also prevented by a fat-soluble factor present and Feiser’s groups in the same year. Soon, Doisy’s group
in green leaves and certain vegetables, and distinct from isolated a second form of the vitamin from putrified fish
vitamins A, C, D, and E. He named the fat-soluble factor meal; this vitamer (called vitamin K2) was crystalline.
vitamin K.58 Subsequent studies demonstrated that this vitamer too dif-
At that time, Herman Almquist and Robert Stokstad, at fers from vitamin K1 by having an unsaturated isoprenoid
the University of California, found that the hemorrhagic side chain at the 3-position of the naphthoquinone ring; in
disease of chicks fed a diet based on ether-extracted fish addition, putrified fish meal was found to contain several
meal and brewers’ yeast, polished rice, cod liver oil, and vitamin K2-like substances with polyprenyl groups of dif-
essential minerals was prevented by a factor present in ether fering chain lengths. Syntheses of vitamins K2 were later
extracts of alfalfa, and that was also produced during micro- achieved by Isler’s and Folker’s groups. A strictly synthetic
bial spoilage of fish meal and wheat bran. Dam’s colleague, analog of vitamers K1 and K2, consisting of the methylated
Schønheyder, discovered the reason for prolonged blood- head group alone (i.e., 2-methyl-1,4-naphthoquinone), was
clotting times of vitamin K-deficient animals. He found shown by Ansbacher and Fernholz to have high antihemor-
that the clotting defect did not involve a deficiency of tissue rhagic activity in the chick bioassay. It is, therefore, referred
thrombokinase or plasma fibrinogen, or an accumulation to as vitamin K3.
of plasma anticoagulants; he also determined that affected
chicks showed relatively poor thrombin responses to exog- Bios Yields Biotin
enous thromboplastin. The latter observation suggested
inadequate amounts of the clotting factor prothrombin, a During the 1930s, independent studies of a yeast growth
factor already known to be important in the prevention of factor (called bios IIb60), a growth- and respiration-pro-
hemorrhages. moting factor for Rhizobium trifolii (called coenzyme R),
In 1936, Dam partially purified chick plasma prothrom- and a factor that protected the rat against hair loss and skin
bin and showed its concentration to be depressed in vita- lesions induced by raw egg white feeding (called vitamin
min K-deficient chicks. It would be several decades before H61) converged in an unexpected way. Kögl’s group isolated
this finding was fully understood.59 Nevertheless, the clot- the yeast growth factor from egg yolk and named it biotin.
ting defect in the chick model served as a useful bioassay In 1940, György, du Vigneaud, and colleagues showed that
tool. When chicks were fed foodstuffs containing the new vitamin H prepared from liver was remarkably similar to
vitamin, their prothrombin values were normalized; hence, Kögl’s egg yolk biotin.62 The chemical structure of biotin
clotting time was returned to normal and the hemorrhagic was elucidated in 1942 by du Vigneaud’s group at Cornell
disease was cured. The productive use of this bioassay led Medical College;63 its complete synthesis was achieved by
to the elucidation of the vitamin and its functions.
60. Bios IIb was one of three essential growth factors for yeasts that had
Vitamers K been identified by Wilders at the turn of the century in response to the
great controversy that raged between Pasteur and Liebig. In 1860, Pasteur
Vitamin K was first isolated from alfalfa by Dam in collabo- had declared that yeast could be grown in solutions containing only water,
ration with Paul Karrer at the University of Zurich in 1939. sugar, yeast ash (i.e., minerals), and ammonium tartrate; he noted, however,
the growth-promoting activities of albuminoid materials in such cultures.
They found that the active substance, which was a yellow
Liebig challenged the possibility of growing yeast in the absence of such
oil, was a quinone. The structure of this form of the vita- materials. Although Pasteur’s position was dominant through the close of
min (called vitamin K1) was elucidated by Doisy’s group the century, Wilders presented evidence that proved that cultivation of yeast
at the University of St Louis, and by Karrer’s, Almquist’s actually did require the presence of a little wort, yeast water, peptone, or
beef extract. (Wilders showed that an inoculum the size of a bacteriological
loopful, which lacked sufficient amounts of these factors, was unsuccessful,
58. Dam cited the fact that the next letter of the alphabet that had not whereas an inoculum the size of a pea grew successfully.) Wilders used the
previously been used to designate a known or proposed vitamin-like term bios to describe the new activity required for yeast growth. For three
activity was also the first letter in the German or Danish phrase koagulation decades, investigators undertook to characterize Wilders’s bios factors. By
facktor, and was thus a most appropriate designator for the antihemorrhagic the mid-1920s, three factors had been identified: bios I, which was later
vitamin. The phrase was soon shortened to K factor and, hence, vitamin K. identified as meso-inositol; bios IIa, which was replaced by pantothenic
59. It should be remembered that, at the time of this work, the biochemical acid in some strains and by β-alanine plus leucine in others; and bios IIb,
mechanisms involved in clotting were incompletely understood. Of the which was identified as biotin.
many proteins now known to be involved in the process, only prothrombin 61. György used the designation H after the German word haut (skin).
and fibrinogen had been definitely characterized. It would not be until the 62. For a time, the factors obtained from egg yolk and liver were called
early 1950s that the remainder of the now-classic clotting factors would be α-biotin and β-biotin, respectively. They were reported as having different
clearly demonstrated and that, of these, factors VII, IX, and X would be melting points and optical rotations. Subsequent studies, however, clearly
shown to be dependent on vitamin K. While these early studies effectively demonstrated that such differences do not exist, nor do preparations from
established that vitamin K deficiency results in impaired prothrombin these sources exhibit different activities in microbiological systems.
activity, that finding would be interpreted as indicative of a vitamin 63. du Vigneaud was to receive a Nobel Prize in Medicine for his work on
K-dependent activation of the protein to its functional form. the metabolism of methionine and methyl groups.
to develop anemia, leukopenia,66 ulceration of the gums, diar-

TABLE 2.5 Factors Leading to the Discovery of Biotin rhea, and increased susceptibility to bacillary dysentery. They
Factor Bioassay found that the syndrome did not respond to thiamin, ribofla-
vin, or nicotinic acid; however, it could be prevented by daily
Bios IIb Yeast growth
supplements of either 10 grams of brewers’ yeast or 2 grams
Coenzyme R Rhizobium trifolii growth of a dried hog liver–stomach preparation. Day named the pro-
Vitamin H Prevention of hair loss and skin lesions in tective factor in brewers’ yeast vitamin M (for monkey).
rats fed raw egg white
Factors U and R, and Vitamin Bc

In the late 1930s, three groups (Robert Stokstad’s at the
Folkers in the following year. A summary of the factors
University of California, Leo Norris’s at Cornell, and Albert
leading to the discovery of biotin is presented in Table 2.5.
Hogan’s at the University of Missouri) reported syndromes
characterized by anemia in chicks fed highly purified diets.
Antianemia Factors The anemias were found to respond to dietary supplements
of yeast, alfalfa, and wheat bran. Stokstad and Manning
The last discoveries that led to the elucidation of new vita-
called this unknown factor U; Baurenfeind and Norris at
mins involved findings of anemias of dietary origin. The
Cornell called it factor R. Shortly thereafter, Hogan and
first of these was reported in 1931 by Lucy Wills’s group
Parrott discovered an antianemic substance in liver extracts;
as a tropical macrocytic anemia64 observed in women in
they called it vitamin Bc.67 At the time (1939), it was not
Bombay, India, which was often a complication of preg-
clear to what extent these factors may have been related.
nancy. They found that the anemia could be treated effec-
tively by supplementing the women’s diet with an extract
of autolyzed yeast.65 Wills and associates found that a Yeast Growth Related to Anemia?
macrocytic anemia could be produced in monkeys by feed-
At the same time, the microbiologists Snell and Peterson,
ing them food similar to that consumed by the women in
who were studying the bios factors required by yeasts,
Bombay. Further, the monkey anemia could be cured by
reported the existence of an unidentified water-soluble fac-
oral administration of yeast or liver extract, or by paren-
tor that was necessary for the growth of Lactobacillus casei.
teral administration of extract of liver; these treatments
This factor was present in liver and yeast, from which it
also cured human patients. The antianemia activity in these
could be prepared by adsorption to and then elution from
materials thus became known as the Wills factor.
Norit;68 for a while they called it the yeast Norit factor, but it
quickly became known as the L. casei factor. Hutchings and
Vitamin M? colleagues at the University of Wisconsin further purified
Elucidation of the Wills factor involved the convergence the factor from liver and found it to stimulate chick growth;
of several lines of research, some of which appeared to be this suggested a possible identity of the bacterial and chick
unrelated. The first of these came in 1935 from the studies factors. The factor from liver was found to stimulate the
of Day and colleagues at the University of Arkansas Medical growth of both Lactobacillus helveticus and Streptococcus
School, who endeavored to produce riboflavin deficiency in fecalis R.,69 whereas the yeast-derived factor was twice
monkeys. They fed their animals a cooked diet consisting of as potent for L. helveticus as it was for S. fecalis. Thus, it
polished rice, wheat, washed casein, cod liver oil, a mixture became popular to refer to these as the “liver L. casei factor”
of salts, and an orange; quite unexpectedly, they found them and the “yeast (or fermentation) L. casei factor.”
Snell’s group found that many green leafy materials were
potent sources of something with the microbiological effects
64. A macrocytic anemia is one in which the number of circulating of the Norit eluate factor—extracts promoted the growth of
erythrocytes is below normal, but the mean size of those present is greater both S. fecalis and L. casei. They named the factor, by virtue
than normal (normal range, 82–92 μm3). Macrocytic anemias occur in such of its sources, folic acid. In 1943, a fermentation product
syndromes as pernicious anemia, sprue, celiac disease, and macrocytic
anemia of pregnancy. Wills’ studies of the macrocytic anemia in her monkey
model revealed megaloblastic arrest (i.e., failure of the large, nucleated,
embryonic erythrocyte precursor cell type to mature) in the erythropoietic 66. Leukopenia refers to any situation in which the total number of
tissues of the bone marrow, and a marked reticulocytosis (i.e., the presence leukocytes (i.e., white blood cells) in the circulating blood is less than
of young red blood cells in numbers greater than normal [usually <1%], normal, which is generally c.5000 per mm3.
occurring during active blood regeneration); both signs were eliminated 67. Hogan and Parrott used the subscript c to designate this factor as one
coincidentally on the administration of extracts of yeast or liver. required by the chick.
65. Wills’s yeast extract was not particularly potent, as they needed to 68. A carbon-based filtering agent.
administer 4 g two to four times daily to cure the anemia. 69. Streptococcus fecalis was then called Streptococcus lactis R.
was isolated that stimulated the growth of S. fecalis but not L.

casei; this was called the SLR factor and, later, rhizopterin. TABLE 2.6 Factors Leading to the Discovery of Folic
Acid
Who’s on First? Factor Bioassay

Wills’ factor Cure of anemia in humans
It was far from clear in the early 1940s whether any of these
factors were at all related, as folic acid appeared to be active Vitamin M Prevention of anemia in monkeys
for both microorganisms and animals, whereas concentrates Vitamin Bc Prevention of anemia in chicks
of vitamin M, factors R and U, and vitamin Bc appeared to be
Factor R Prevention of anemia in chicks
effective only for animals. Clues to solving the puzzle came
from the studies of Mims and associates at the University Factor U Prevention of anemia in chicks
of Arkansas Medical School, who showed that incuba- Yeast Norit factor Growth of Lactobacillus casei
tion of vitamin M concentrates in the presence of rat liver
L. casei factor Growth of L. casei
enzymes caused a marked increase in the folic acid activity
(i.e., assayed using S. casei and Streptococcus lactis R.) of SLR factor Growth of Rhizobium species
the preparation. Subsequent work showed such “activation” Rhizopterin Growth of Rhizobium species
enzymes to be present in both hog kidney and chick pancreas.
Folic acid Growth of Streptococcus fecalis and
Charkey, of the Cornell group, found that incubation of their L. casei
factor R preparations with rat or chick liver enzymes pro-
duced large increases in their folic acid potencies for micro-
organisms. These studies indicated for the first time that at
least some of these various substances may be related. real breakthrough toward understanding the etiology of
pernicious anemia did not come until 1926, when Minot
and Murphy found that lightly cooked liver, which the
Derivatives of Pteroylglutamic Acid prominent hematologist G.H. Whipple had found to
The real key to solving what was clearly the most compli- accelerate the regeneration of blood in dogs made ane-
cated puzzle in the discovery of the vitamins came in 1943 mic by exsanguination, was highly effective as therapy
with the isolation of pteroylglutamic acid from liver by for the disease.51,71,72 This indicated that liver contained
Stokstad’s group at the Lederle Laboratories of American a factor necessary for hemoglobin synthesis.
Cyanamid, Inc., and by Piffner’s group at Parke-Davis, Inc.
Stokstad’s group achieved the synthesis of the compound Intrinsic and Extrinsic Factors
in 1946. Soon it was found that pteroylmonoglutamic acid
was indeed the substance that had been variously identified Soon, studies of the antipernicious anemia factor in
in liver as factor U, vitamin M, vitamin Bc, and the liver liver revealed that its enteric absorption depended on yet
L. casei factor. The yeast L. casei factor was found to be another factor in the gastric juice, which W.B. Castle,
the diglutamyl derivative (pteroyldiglutamic acid) and the in 1928, called the intrinsic factor, to distinguish it
liver-derived vitamin Bc was the hexaglutamyl derivative from the extrinsic factor in liver. Biochemists then com-
(pteroylhexaglutamic acid). Others of these factors (the SLR menced a long endeavor to isolate the antipernicious
factor) were subsequently found to be single-carbon metab- anemia factor from liver. The isolation of the factor was
olites of pteroylglutamic acid. These various compounds necessarily slow and arduous for the reason that the only
thus became known generically as folic acid. A summary of bioassay available was the hematopoietic response of
the factors leading to the discovery of folic acid is presented human pernicious anemia patients, which was frequently
in Table 2.6. not available. No animal model had been found, and a
bioassay could not be replaced by a chemical reaction
or physical method because, as is now known, this most
potent vitamin is active at exceedingly low concentra-
Antipernicious Anemia Factor
tions. Therefore, it was most important to the elucida-
The second nutritional anemia that was found to involve tion of the antipernicious anemia factor when, in 1947,
a vitamin deficiency was the fatal condition of human Mary Shorb of the University of Maryland found that it
patients that was first described by J.S. Combe in 1822
and became known as pernicious anemia.70 The first 71. Minot and Murphy treated 45 pernicious anemia patients with
120–240 g of lightly cooked liver per day. The patients’ mean erythrocyte
70. This condition has also been called Addison’s anemia after T. count increased from 1.47 × 106/mL before treatment to 3.4 × 106/mL
Addison, who described it in great detail in 1949, and Biemer’s anemia, and 4.6 × 106/mL after 1 and 2 mos. of treatment, respectively.
after A. Biemier, who reported the disease in Zurich in 1872 and coined the 72. Whipple, Minot, and Murphy shared the 1934 Nobel Prize in Medicine
term pernicious anemia. for the discovery of whole liver therapy for pernicious anemia.
was also required for the growth of Lactobacillus lac-

tis Dorner.73 With Shorb’s microbiological assay, isola- TABLE 2.7 Factors Leading to the Discovery of
tion of the factor, by that time named vitamin B12 by the Vitamin B12
Merck group, proceeded rapidly. Factor Bioassay
Extrinsic factor Cure of anemia in humans
Animal Protein Factors LLD factor Growth of L. lactis Dorner
At about the same time, animal growth responses to factors Vitamin B12 Growth of L. lactis Dorner
associated with animal proteins or manure were reported
Animal protein factor Growth of chicks
as American animal nutritionists sought to eliminate
expensive and scarce animal by-products from the diets of Factor X Growth of rats
livestock. Norris’s group at Cornell attributed responses of Zoopherin Growth of rats
this time to an animal protein factor; the factor in liver
necessary for rat growth was called factor X by Cary and
zoopherin74 by Zucker and Zucker. It soon became evi-
dent that these factors were probably identical. Stokstad’s TABLE 2.8 Timelines for the Discoveries of the Vitamins
group found the factor in manure and isolated an organism
Structure Synthesis
from poultry manure that would synthesize a factor that
Vitamin Proposed Isolated Determined Achieved
was effective both in promoting chick growth and in treat-
ing pernicious anemia. That the antipernicious anemia fac- Thiamin 1906 1926 1932 1933
tor was produced microbiologically was important, in that Vitamin C 1907 1926 1932 1933
it led to an economical means of industrial production of
Vitamin A 1915 1937 1942 1947
vitamin B12.
Vitamin D 1919 1932 1932 (D2) 1932
Vitamin B12 Isolated 1936 (D3) 1936

Vitamin E 1922 1936 1938 1938
By the late 1940s, Combs75 and Norris, using chick growth
as their bioassay procedure, were fairly close to the isola- Niacin 1926 1937 1937 1867a
tion of vitamin B12. However, in 1948, Folkers at Merck, Vitamin B12 1926 1948 1955 1970
using the L. lactis Dorner assay, succeeded in first isolating
Biotin 1926 1939 1942 1943
the antipernicious anemia factor in crystalline form. This
achievement was accomplished in the same year by Lester Vitamin K 1929 1939 1939 1940
Smith’s group at the Glaxo Laboratories in England (who Pantothenic 1931 1939 1939 1940
found their pink crystals to contain cobalt), assaying their acid
material on pernicious anemia patients in relapse.76 The
Folate 1931 1939 1943 1946
elucidation of the complex chemical structure of vitamin
B12 was finally achieved in 1955 by Dorothy Hodgkin’s Riboflavin 1933 1933 1934 1935
group at Oxford with the use of X-ray crystallography. Vitamin B6 1934 1936 1938 1939
In the early 1960s, several groups accomplished the par-
aMuch of the chemistry of nicotinic acid was known before its nutritional
tial synthesis of the vitamin; it was not until 1970 that the roles were recognized.
de novo synthesis of vitamin B12 was finally achieved by
Woodward and Eschenmoser. A summary of the factors
leading to the discovery of vitamin B12 is presented in Vitamins Discovered in Only Five Decades
Table 2.7.
Beginning with the concept of a vitamin, which emerged
with Eijkman’s proposal of an antipolyneuritis factor in
1906, the elucidation of the vitamins continued through the
73. For a time, this was referred to as the LLD factor. isolation of vitamin B12 in potent form in 1948 (Table 2.8).
74. The term zoopherin carries the connotation: “to carry on an animal
species.”
Thus, the identification of the presently recognized vitamins
75. Characterization of the animal protein factor was the subject of the was achieved within a period of only 42 years! For some
senior author’s father’s doctoral thesis in Norris’s laboratory at Cornell in vitamins (e.g., pyridoxine) for which convenient animal
the late 1940s. models were available, discoveries came rapidly; for others
76. Friedrich (1988) has pointed out that it should be no surprise that the (e.g., niacin, vitamin B12) for which animal models were
first isolations of vitamin B12 were accomplished in industrial laboratories,
as the task required industrial-scale facilities to handle the enormous
late to be found, the pace of scientific progress was much
amounts of starting material that were needed. For example, the Merck slower (Fig. 2.1). These paths of discovery were marked by
group used a ton of liver to obtain 20 mg of crystalline material. nearly a dozen Nobel Prizes (Table 2.9).
TABLE 2.9 Nobel Prizes Awarded for Research on Vitamins
Year of
Award Recipients Discovery
Prizes in Medicine and Physiology
1929 Christiaan Eijkman and Frederick G. Discovery of the antineuritic vitamin; discovery of the growth-stimulating
Hopkins vitamins
1934 George H. Whipple, George R. Discoveries concerning liver therapy against pernicious anemia
Minot, and William P. Murphy
1937 Albert von Szent-Györgi and Charles Discoveries in connection with the biological combustion, with especial refer-
G. King ence to vitamin C, and the catalysis of fumaric acid
1943 Henrik Dam and Edward ADoisy Discovery of vitamin K; discovery of the chemical nature of vitamin K
1953 Fritz A. Lipmann Discovery of coenzyme A and its importance in intermediary metabolism
1955 Hugo Theorell Discoveries relating to the nature and mode of action of oxidizing enzymes
1964 Feodor Lynen and Konrad Bloch Discoveries concerning the mechanism and regulation of cholesterol and fatty
acid metabolism
Prizes in Chemistry
1928 Adolf Windaus Studies on the constitution of the sterols and their connection with the vitamins
1937 Paul Karrer and Walter N. Haworth Research on the constitution of carotenoids, flavins, and vitamins A and B;
researches into the constitution of carbohydrates and vitamin C
1938 Richard Kuhn Work on carotenoids and vitamins
1957 Alexander Todd Work on the structure of nucleotides (including vitamin B12)
1964 Dorothy C. Hodgkin Elucidation of the structure of vitamin B12
1965 Robert B. Woodward Chemical synthesis of vitamin B12
1967 George Wald, H.K. Hartline, and R. Discoveries of the basic chemical and physiological processes in vision
Grant
7. VITAMIN TERMINOLOGY all called B vitamins. In the case of folic acid, certainly
the name survived its competitors by virtue of its rela-
The terminology of the vitamins can be as daunting as that
tively attractive sound (e.g., vs rhizopterin). Therefore,
of any other scientific field. Many vitamins carry alpha-
the accepted designations for the vitamins, in most cases,
betic or alphanumeric designations, yet the sequence of
have relevance only to the history and chronology of their
such designations has an arbitrary appearance by virtue
discovery and not to their chemical or metabolic similari-
of its many gaps and inconsistent application to all of the
ties. The discovery of the vitamins left a path littered with
vitamins. This situation notwithstanding, the logic under-
designations of “vitamins,” “factors,” and other terms,
lying the terminology of the vitamins becomes apparent
most of which have been discarded (see Appendix A for a
when it is viewed in terms of the history of vitamin dis-
complete listing).
covery. The familiar designations in use today are, in most
cases, the surviving terms coined by earlier researchers on
the paths to vitamin discovery. Thus, because McCollum 8. OTHER FACTORS SOMETIMES CALLED
and Davis used the letters A and B to distinguish the lipid-
soluble antixerophthalmic factor from the water-soluble
VITAMINS
antineuritic and growth activity that was subsequently Several other factors have, at various times or under cer-
found to consist of several vitamins, such chemically tain conditions, been called vitamins. Many remain today
and physiologically unrelated substances as thiamin, only as historic markers of once incompletely explained
riboflavin, pyridoxine, and cobalamins (in fact, all water- phenomena, now better understood. Today, some factors
soluble vitamins except ascorbic acid, which was desig- would appear to satisfy, for at least some species, the oper-
nated before the vitamin B complex was partitioned) are ating definition of a vitamin; although in practice that term
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refrigerator. He has the appetite and stomach capacity of an ostrich.
What do you say?"
"Possible," McGinity concurred. "Let's go."
As I crept stealthily downstairs, with the reporter at my side, I fully
expected, at any moment, to be confronted with a long hairy arm,
stretching out from some dark corner, to clutch at my throat. My
feeling of nervousness increased when, in the midst of our search on
the ground floor, my flashlight suddenly failed. We had just stepped
into the dining room. I was reluctant to switch on the wall, or ceiling
lights, for fear of alarming the servants, or attracting the attention of
the night watchman who patrolled the grounds. Under no
consideration could we afford to arouse the household, especially
Henry.
So we elected to sit down in the darkness and wait for something to
happen, possibly the discovery of the prowling Mr. Zzyx. I marveled
at the instinct which enabled him to move about so freely in the dark.
It was so quiet in the dining room that we could hear the ticking of
the grandfather's clock in the library. There we sat, waiting, in the
utter silence of the night. One o'clock struck—then half-past.
All the time we were seated there, I fancied I heard a sound quite
distinct above the ticking of the clock; a faint, crackling sound, like a
dog makes when it is crunching bones between its teeth. I made no
mention of it to McGinity, but my heart was going in great sickening
thumps. Another ten minutes of strained silence in the darkness, and
my nerves were stretched to the limit.
As it turned out, McGinity had heard the same mysterious sound.
Also, his eyes becoming more quickly adjusted to the darkness,
being so much younger than mine, he saw something that had so far
escaped my notice. Flecks of white on the floor, just to the right of
us, in front of the heavily curtained French window.
Suddenly, he put his hand on my arm warningly. I heard him draw his
breath sharply as he slowly rose, and tiptoed a few steps beyond
where we were seated. As I craned forward to try to see what he
was up to, he lit his cigarette lighter.
"My God! Look!" I heard him breathe; and, rising, I saw, in the
flickering glimmer, a lot of white feathers on the floor. As his lighter
quickly burned itself out, the room once more was in darkness. But I
had seen enough in that momentary flash to realize that at last we
had found something we were waiting for.
"What do you make out of those white feathers?" McGinity
whispered, gripping my arm.
"It wouldn't surprise me a bit," I replied, "to find that Mr. Zzyx has
killed Pat's white cockatoo."
"Yes; and I'll bet you anything he's somewhere close by, in one of
these rooms, enjoying a cold bird, bones and all," his quick whisper
came back. "That will account for the funny sound I've been hearing
all the time we've been sitting here. Let's turn on the light—take a
chance. What do you say?"
My memory of what immediately followed is rather blurred and
confused. I have some memory of feeling my way through the library
doorway, although how I accomplished it in the dark is more than I
can figure out. What I most remember clearly is the strange, eerie
sight that met our startled gaze after I had turned on the lights.
Crouching on the floor of the library, confronting us, was Mr. Zzyx, in
his pajamas, and surrounded by feathers and bits of bone of the
cockatoo, which obviously he had so cruelly slaughtered and
devoured. And as we stood there gazing at him, he snarled at us like
a wild beast defending its spoils.
Quick as a flash, McGinity's hand went to his hip pocket, but I
restrained him. "Leave him alone," I advised. "Let him finish his
feast."
"Pat's heart will be broken," McGinity sighed. "How could he do such
a horrible, cruel thing?" He lit a cigarette.
"Because he's more animal than man," I answered; "a very
dangerous and vicious animal."
We continued to watch him in silence until he had finished crunching
the last bone. Then he got to his feet, and started to walk towards
McGinity, round whose head spirals of tobacco smoke were curling.
There was a dark stain all around the creature's mouth, which made
him more repellent and disgusting.
"He wants a cigarette," I suggested.
McGinity gave him one, and lit it. And then, to our amazement, he
followed us meekly as we led the way upstairs, and opened the door
of his bedroom. He climbed into bed, and pulled the bedclothes over
his head, like a child who has been caught in the act and is ashamed
of his wrong-doing. Presently he was fast asleep.
XXIII
The after-midnight experience, especially the cruel killing of Pat's pet
cockatoo, distressed me terribly. I was still feeling nervous and
depressed the next morning when, after a hasty breakfast, I caught
an early train for the city. I took a taxi-cab at the railroad terminal,
and drove straight to the office of the Royal Parchment Paper
Company, in Beekman Street, which is in the downtown district.
I did not reveal my identity—there was no reason why I should—
simply explaining that I was interested in seeking out the maker of a
very interesting scroll that had come into my possession, the
parchment paper of which bore the firm's water-mark. In less than
ten minutes, a point of high importance was settled. The parchment
paper used in the scroll was identified as a heavy grade formerly
manufactured by this concern but discontinued about three years
before.
As it was a wholesale house, selling its products only in bulk to
retailers, I was beginning to lose hope that I should ever be able to
track this particular brand of parchment paper when, by great good
luck, one of the assistant officials recalled having sold a small
quantity of it, from the remaining store-room stock, to an aged, silver-
haired man, four months back.
He remembered the transaction very well indeed because the
customer explained that he had been looking for some time for this
special, heavy grade of parchment paper, as he made it a business
of transforming newly manufactured parchment into ancient-looking
family and historical documents, for which he found a ready market
among dealers in antiques.
He walked out of the office carrying his purchase, leaving no name
and no address. As it was raining heavily at the time, the assistant
official, because of the customer's age and apparent infirmities,
followed him to the door with an umbrella, and politely volunteered to
call a taxi. After he had done this, he put the old man into the cab.
And now, after an interval of four months, he recalled the address
the customer had given the taxi-driver: "Stuyvesant Place and
Twelfth Street."
This remarkable display of memory sent me off at once to another
field of inquiry. In a small, musty, corner curio and book shop, at
Stuyvesant Place and Twelfth Street, I found a courteous, white-
haired old man, looking rather shaggy and unkempt, who recognized
the scroll at once as a sample of his own handicraft. He did not
know, of course, he said, for what purpose it was to be used, nor did
he seem to care; and he appeared equally unconcerned over the
strange inscriptions it contained. He seemed both surprised and
grieved when I showed him the water-mark. Apparently, he believed
that I had been taken in by some antique dealer, in the purchase of
the scroll as an ancient document.
"I do not often make a slip like that," he said, "and I am very sorry
indeed if I've caused my customer any embarrassment. He did not
specify that it should look old, but just different from the usual run of
scrolls. For instance, he requested me to perfume the gum that holds
the parchment securely to the ebony roller."
"That's all very interesting," I said, as calmly as I could. "Now, there's
just one more question—did your customer reveal his name?"
The old bookseller shook his head. "I have no idea who he is," he
replied; "no idea at all of his actual identity. He paid me a pretty stiff
price in advance for my work. That's all that interested me."
"Can you describe him?" I asked.
He took off his spectacles, and wiped them carefully on a frayed,
white silk handkerchief. "No," he said, finally and slowly. "I'm afraid I
can't describe him. My memory and eye-sight are both failing fast. If
you were to leave here now, and an hour later, some one was to ask
me to describe your appearance, I would be utterly at a loss. I do
recall, however, that he was middle-aged, well-dressed, and well
bred—a gentleman, I should say."
"And that is all you know of him?" I persisted.
"That's all I know of him," the old man assented. "Well, yes, I do just
remember one other thing. The day he called for the scroll, he
apologized for his hurried departure, saying that he had only a few
minutes in which to keep an important engagement in Radio Center,
and make his train."
"Ah!" I breathed. "Did he mention taking a train on any particular
railroad?"
"No," was the reply. "But after he'd gone, I found a Long Island
Railroad time-table on my desk. Evidently he had left it behind—
forgotten it in his haste."
After some further questioning, I went away. At noon, I met McGinity,
having promised the night before to join him at lunch, in town, and
submit all the evidence I had collected about the water-mark in the
scroll. The restaurant was one of his own choosing, a cheerful but
obscure eating-place in the Times Square section, noted for its
home-cooking and excellent beer, and largely patronized by
newspaper reporters working in that district.
The reporter listened to my story with signs of ever increasing
interest, as we sat together in a dining nook, and when it came to an
end, he exclaimed: "'Middle-aged—well-dressed—well bred!' You
see? Olinski! without a doubt."
"No, I don't see it at all," I answered. "The old bookseller's vague
description of the man who gave him the order for the scroll, in my
opinion, doesn't fit Olinski. True, there's evidence that the man left
hurriedly for an important engagement at Radio Center, and later, to
catch a train on the Long Island Railroad, but that proves nothing
against Olinski. Why should he rush off to keep an appointment in
Radio Center, when he spends most of his time in his laboratory
there? Besides, he's perfectly familiar with the time schedules of the
railroad, so far as Sands Cliff is concerned."
"Then, if it wasn't Olinski, it must have been his accomplice,"
McGinity persisted. "There's more than one person mixed up in this."
"Undoubtedly you're right," I concurred. "But it would be just as easy
and logical to suspect Prince Matani. Personally, I would suspect the
Prince of doing anything, short of murder, for money. He's been
trying to force Henry's hand for some time, in regard to Pat, and
failing, this may have been his revenge. But acting only as a paid
agent for a superior intellect, who put the thing over in a much bigger
way, perhaps, than the Prince had anticipated."
"If this is true, then it will account for the Prince falling in a fit the first
time he set eyes on Mr. Zzyx," the reporter suggested. "He expected
to find a small baboon, and he finds a monster. Then, he vanishes.
Very odd that he should go off to California—disappear like that."
"You've taken the words right out of my mouth," I rejoined, with a
benign smile. "However, as matters are now shaping themselves, it's
my opinion that any suspicions we may have regarding either Olinski
or the Prince are coming to a quick end. We might as well attach
suspicion to Mamie Sparks, our colored laundress."
"Well, at any rate, Mr. Royce," said McGinity, "one thing is pretty well
established in my mind, and that is—if the perpetrator of this gigantic
fraud isn't a lunatic, he's certainly been carried away by some
strange fanatical motive."
"The facts of the case are all very strange, and very puzzling," I
observed. "I have been reflecting on the matter for the last hour or
so, since leaving the old bookseller, and I'm beginning to feel that
we're up against a pretty difficult task—perhaps an impossible one."
"Oh, please, don't say that, Mr. Royce!" the reporter said, earnestly.
"We may be all tangled up in this web of mystery, but we've got a
start—just a thread of a clue—haven't we? Not such a big one but
still a start. If we keep on the alert, we may run into something else
that will put us in possession of another thread of a clue. That'll be
two threads, won't it?"
"Yes," I replied, lugubriously; "but as far as I can see at present,
things look pretty hopeless, and we might as well stop now with our
investigations, and let matters take their natural course."
"That I'll never do," said McGinity, bringing his fist down on the table,
as though to accentuate his determination to see things through.
"You must remember, I've been taken in, as well as your brother
Henry, and on my shoulders rests the responsibility of all those
printed articles of mine."
"And not inventions, either," I said, "but stories founded on facts. You
can excuse—facts."
"Not if they're fictitious facts," said McGinity; "and it's my duty now to
expose their falsity to the public. No, Mr. Royce, we can't turn back
now!"
As he spoke the last word, a boy selling a special extra of an
afternoon paper, entered the restaurant, and came straight to our
table. "Want a paper, Mister?" the boy asked me. I shook my head,
but as soon as McGinity had glimpsed the big, front page headline
he snatched a paper out of the boy's hand.
Within a second he was directing my attention to the glaring
headline, which read: "Martian Rocket Disappears—Stolen!" and
then to a space in the center of the page, headed: "Very Latest
News," under which appeared a few lines, printed in red ink.
Together we read them:
"New York police notified today by officials of New York Museum of
Science that the Martian rocket, found on Long Island, near the
estate of Henry Royce, millionaire scientist, and placed on exhibition
in the museum, was stolen from its glass-case during the night.
Watchman found bound and drugged. While nothing uncovered so
far to establish clue to identity of daring thieves, police have obtained
information showing theft was committed by two men, who were
seen to leave the museum, carrying the rocket, and drive off in a
small van, about three o'clock this morning."
Before I could speak, McGinity jumped to his feet, and made for a
telephone booth. Five tense minutes passed, then he burst out of the
booth, and came to me.
"Ah!" he said, excitedly; "now, we're getting somewhere."
"What do you mean?" I asked.
"Just this," the reporter replied. "The theft of this rocket proves
conclusively that the superior intellect, the master mind, is back on
the job. Something has forced him out of hiding—out of the unknown
into which he passed about three months ago. He's getting scared.
He realizes that the finger of suspicion, sooner or later, will be
pointed at him, and he's trying to destroy all evidence of his guilt."
"That is, of course, a possibility," I agreed. "But this theft of the
rocket, now. Why, to me, it makes the whole thing seem more and
more of a puzzle."
"It's the best thing that could have happened," McGinity observed. "It
will prepare the public for the exposé, which is bound to come now,
and put your brother in right. Public sentiment is always with the man
who has been duped."
"Does this mean that we will not go on with our investigations?" I
inquired.
McGinity shook his head. "My instructions from the Desk," he
answered, "which I just received on the phone, are to continue with
our private investigations. And my first job is to make contact as
soon as possible with your brother Henry. And let me say, right here,
that I think it highly important that we keep nothing back from him.
We must give him a clear, succinct account of the whole matter as
we know it up to this moment."
"Whew!" I exclaimed. "You don't know Henry. He would consider any
move like that, on our part, as highly meddlesome, even offensive."
"But in enlisting his services in tracking down the stolen rocket—ten
to one, it's been dumped in the East River, which is only a few blocks
from the museum—we must acquaint him with all the particulars that
have come to light. Tell what we know and suspect in the matter.
He's got to know sometime—why not now?"
"Very well, then," I assented; adding, with an amused chuckle:
"Looks like we've got a very busy afternoon ahead of us."
"Busy isn't the word," McGinity rejoined, as he began making some
hasty notes on a bunch of copy-paper, which he always kept handy
in his coat pocket. "However, this is only the beginning."
"What are you making notes for?" I asked, curiously. "Are they for
your paper, or the police?"
"Neither," replied McGinity. "They are intended for broadcasting.
After I'd talked to my City Editor, I got Mr. Scoville of the NRC on the
phone, and he's promised to have a good description of the rocket
put on the air at three o'clock this afternoon, again at six, and at nine
this evening."
"Excellent idea," I said, enthusiastically. "I only wish there was
something I could do. What can I do?"
"Well, Mr. Royce," replied McGinity, as he finished making his notes,
and gave me a smile and roguish wink that meant much, "a reward
is always useful in these matters. Money can do things that mere
words can't do."
"I see what you mean," I responded slowly. I thought a moment, and
then said: "If my belief's correct, the sooner we lay hands on the two
men who stole the rocket the better! Yes? Well, Mr. McGinity, I'm
quite willing to help out on this, in a small way, of course. I'll offer a
reward of $5,000—"
"Five thousand dollars!" McGinity interrupted, gleefully. "That's a
whole lot of money, Mr. Royce, and I'm sure it's going to help solve
the mystery. And say—here's an idea that occurs to me. Why not
phone Olinski now, at once, and get a detailed description of the
rocket from him. And then ask him—also for me—if he ever visited a
certain curio and bookseller's shop at the corner of Stuyvesant Place
and Twelfth Street. If he doesn't answer you directly, and begins to
question you—well, just hang up. Better hurry now!"
I am easily excited, and I certainly felt my heart thump as I hurried
into one of the compartments of the telephone booth, to carry out the
reporter's suggestions, while McGinity stepped quickly into the
adjoining section, to conclude the necessary arrangements by
telephone for broadcasting the $5,000 reward.
I smiled to myself as I impatiently awaited a response to my call.
There I was, a staid member of society, a pillar of the church, holding
dignified offices in at least a dozen of the most exclusive and
conservative clubs of New York—tracking down an ingeniously
concocted scheme to ruin my brother's reputation as a scientist, with
the self-possession of a Hercule Poirot, or any other equally
distinguished detective of fiction; lunching at a reporters' hangout,
and, now, about to perform a rather dirty trick on my good friend,
Olinski—altogether putty in the hands of a very audacious but
ingratiating reporter.
Luckily for me, Olinski was reported "out" at his laboratory. In fact, he
hadn't been in for two days; obviously his staff was worried.
"Of course, Olinski's out," muttered McGinity, when I told him; "he's
got other business to attend to—pressing business." And then he
proceeded to begin preparations to leave. "Now, we'd better get
along to Sands Cliff—quick! Our next job's there."
The reporter's car was waiting for us, and in less than an hour we
were outside our lodge-gate. The big iron gate is usually kept open
during the day-time, but now it was closed. As there was no sign of
the lodge-keeper, McGinity got out and opened the gate. When we
rolled through, the radiator was spouting hot water and steam like a
miniature Yellowstone Park geyser. The reporter had whirled me
along country roads and through villages, in the drab light of a cloudy
November afternoon, at a speed not at all to my liking.
Parking the car just inside the gate, we drew near the gray-walled
castle. Something ominous was in the air. A deadly chill, floating in
across the terrace from the dark waters of the Sound, seemed to
penetrate to our very bones. Everything was weirdly silent. No sign
of life. I grew very anxious and uncomfortable, although the
incredible truth did not dawn upon me. Why was everything so
horribly silent? Where were the usual sounds and stir of a big
country estate? Why this tomb-like castle?
I was surprised to find the front door open. Within a few seconds we
had entered, and were standing in the great, vaulted entrance hall,
now dark and gloomy. Not a sound, nor a movement!
And then, suddenly, in the gloom and silence, we saw something
that struck terror in our hearts. Jane—dear, lovable old Jane—lying,
still as death, face downward, on the floor, at the base of the great
staircase. Showing vividly on the stone steps, from top to bottom,
were blotches of dark red. They looked like bloody footprints.
XXIV
I have often wondered, since all this occurred, how it happened that
McGinity and I arrived at the castle at this very critical moment,
which, afterwards, proved to be the crucial stage of our adventures
in trying to detect and trace the utterly unscrupulous scoundrel who
perpetrated the Martian hoax. Seconds—or minutes—later, and I
might now be recording a much more terrible series of events. It was
all horrible enough, God knows!
To our great relief, we found that Jane had fainted from shock. She
showed signs of returning to consciousness as the reporter and I
sprang to her side. She was, of course, the first person to give us the
news. After we had assisted her to her feet, we partly carried her to a
big easy chair, propping her up with sofa pillows. Luckily, her
smelling salts were in her handbag, which I had picked up from the
floor, and as I waved the vial of sal volatile to and fro under her nose,
I urged her gently to tell us what had happened.
"Where's Henry?" was my first question.
"He went away—er—after lunch," Jane replied, slowly and painfully.
She was still breathing with difficulty, and her words came in little
gasps.
"Did he say where he was going?"
"No place—in particular. He was completely fagged out. I think he
went for a drive."
"And Pat? Where is she?"
"She went out about an hour ago. I begged her not to go. She's been
crying all day—about her white cockatoo."
"Did she say where she was going, Miss Royce?" McGinity asked.
"She said something about the dock. What she did say was that she
felt that some fresh air, and a little exercise, would do her good."
"Did she mention any particular kind of exercise?" McGinity
questioned again.
"No—she merely said—oh, yes—she spoke of rowing—that was it."
"Pat's very fond of rowing," I explained to McGinity, "and frequently
goes over to the island, and potters about the old lighthouse ruins."
Then I turned again to Jane: "Now, Jane, tell us—what about Mr.
Zzyx?"
At the mention of the creature's name, she turned more pale and
sank back in the chair, gasping. I thought she was going to faint
again. Between us, McGinity and I rubbed her hands and forearms
briskly. Quickly rallying, she murmured, with quivering lips: "He went
mad—or something—stark crazy!"
I glanced at McGinity, and whispered: "He must have gone on a
rampage—just as I feared."
"Where are all the servants, Miss Royce?" McGinity asked, as Jane
recovered some semblance of her natural poise.
She smiled a little grimly. "I guess they've all been frightened away,"
she answered. "You see, I don't know about everything that
happened, but it's my belief that all the servants have locked
themselves up in the service wing. Oh, neither of you can
comprehend the utter reign of terror we've just passed through. Here
I was, by myself—Henry and Pat both out—the servants fleeing in
alarm. Naturally, at first, I was in a state of absolute despair as to
what to do."
"Let's begin at the beginning, Jane," I counselled, softly. "When did
you first hear of Mr. Zzyx acting up?"
"About half an hour ago," she replied. "I was in my room, reading,
when Schweizer knocked at my door. His face was as white as a
sheet. He said a great commotion was going on in the State
Apartment, and hadn't he better call the police. But I advised him to
summon all the men-servants in the place, as I felt they could handle
the situation, whatever it might be."
"Then what?"
"The butler had not been gone two minutes when I heard that
dreadful thing, screeching—oh, terrible to hear!—and running up and
down the hall, outside my room, and smashing the furniture. Then
everything became quiet. He must have gone downstairs, for, a few
minutes later, I heard the woman servants screaming—such
screaming as I never heard before and never want to hear again."
"What did you do, then?"
"As soon as the screaming had subsided, I decided that something
must be done, for I suddenly realized that all the men on the place
had gone off on a half holiday. Besides, the telephone extension on
the second floor went out of order this morning. My intention, as I
slipped out of my room, was to go downstairs to the library, lock
myself in, and phone the police. As none of the servants, not even
my personal maid, had shown themselves, and viewing the awful
wreckage that creature had made of the tables, chairs and tapestries
in the hall, I was convinced that something terrible was going on."
"But how did you happen to be lying at the foot of the staircase?"
"I will tell you." She drew a long, painful breath, and then continued.
"All went well until I reached the bottom of the stairs, when I heard
heavy footsteps above me. I turned and looked. Mr. Zzyx was
coming down towards me, chattering, and showing his teeth, rolling
his head, and waving his arms convulsively, like he had a fit. I was
frozen with terror to the spot. I couldn't move. I remember seeing
blood on his hands and clothes, as he came nearer to me. I recall
receiving a heavy blow on my arm. After that, I remember nothing."
"Thank God! you escaped without a scratch," I breathed. "But where
do you suppose he's gone? The front door was open when we
arrived. He may have gone out that way."
"I have no idea," Jane said. Then she wailed: "Oh, what are we to
do?"
"We'll do something," I replied, and immediately went into action. I
had a police whistle in my pocket, and, leaving Jane in charge of
McGinity, I went quickly into the library. Opening the window that
gave on to the terrace, I blew the whistle. Just then, I saw Schweizer
coming from the servants' wing. I waved a hand to him, and he came
hurrying on to the terrace and up to the window.
"What's become of everyone?" I inquired.
"The women have barricaded themselves in their quarters, sir," the
butler replied. "That hairy fellow nearly scared the life out of 'em.
Mamie Sparks went into a faint, and isn't out of it yet."
"Isn't there a strong-armed man left on the place?"
"None, sir," Schweizer replied. "The two chauffeurs went with your
brother on a drive. I was afraid to tackle that hairy thing unarmed and
single-handed, and ever since leaving Miss Jane locked up in her
room, I've been searching everywhere for a gun."
"Did you see Mr. Zzyx leaving by the front door?"
"I saw nothing, sir, after I went back to the servants' wing to look for
a weapon. If I'd found one, I meant to shoot that fellow dead. He
surely made a mess of things with his tearing and smashing."
"Yes, I know," I rejoined, glancing back over my shoulder. The dining
room looked as though a small whirlwind had struck it. "Better come
inside, Schweizer, and help us get things straightened out."
"Just a word, sir," the butler said, coming up closer to the window,
and speaking in little more than a whisper. "I think murder's been
committed."
"Oh, I don't believe that," I replied, "but we'll soon see."
While alarmed and mystified at first over the red blotches on the
marble staircase, it was my belief now that Mr. Zzyx must have cut
himself severely during his rampage, which would account for the
blood stains. But after the butler had joined us, and had told of
hearing Niki screaming, during the commotion in the State
Apartment, that put a different complexion on the matter. Leaving
Schweizer to guard Jane, McGinity and I hastened upstairs.
It was my earnest hope that Niki was in hiding somewhere. I could
not picture a person of his athletic prowess being outmatched, even
by a strong-limbed creature like Mr. Zzyx. First, I tried Mr. Zzyx's
door. It was locked on the inside. Then I knocked on the door which
opened into the room occupied by Niki, a double room, one half of
which was fitted as a bedroom. There was no response. Dead
silence followed each knock—an eerie silence that caused my blood
to run chill.
In a moment I had opened the door, and we were standing in his
room. There were unmistakable traces of some sort of struggle.
Several chairs and a reading-table were overturned, rugs
disarranged, and books and magazines scattered over the floor. But
no sign of Niki. I called him by name. "Niki! Niki!" my voice echoing
weirdly from the high ceiling.
Then, at McGinity's suggestion, I opened the door connecting Niki's
apartment with Mr. Zzyx's luxurious sleeping quarters. I gave one
glance into the room, then recoiled with an exclamation of horror.
The reporter leapt forward to look. The sight that met our gaze
stayed with me for many days afterwards.
Niki was lying on the bed, on his back, his clothes almost torn to
tatters, and the upper part of his body and head hidden under pillows
and bedclothes, which bore crimson stains. I made no comment at
the moment. My thoughts were going back to the performance of
Verdi's "Otello" at the Metropolitan-Civic Opera House; the night I
had studied Mr. Zzyx attentively as he watched, as if spellbound, the
smothering to death of Desdemona by the jealous and enraged
Moor. Had my surmises at that time been right? Had this violent
climax of the opera taken hold of his primitive mind and obsessed
him until it had quickened him to this deed of incredible violence?
Beyond any reasonable doubt, Niki had been overcome and
smothered to death after a terrific fight with this hairy monster. The
wreckage of the furnishings of the room bore evidence of such a
struggle.
McGinity spoke first. "Awful!" he said in a faint voice.
"Poor Niki!" I said, in a tone which I scarcely recognized as my own.
"If that fiend smothered Niki to death, how do you account for all that
—" McGinity checked himself as his voice choked.
"As Niki's face bears only scratches," I replied, "it's possible that Mr.
Zzyx cut himself seriously while smashing window-panes and
picture-glass. That will account for the bloodstains on the pillows and
bedclothing."
"Then he must have killed Niki after going on a rampage through the
castle," McGinity suggested.
"No, I don't think so," I replied. "I figure that he killed Niki first. He
must have returned to the second floor by the back stairs, and by
some strange instinct, re-enacted the killing with his cut and bleeding
hands, to make sure his victim was dead."
"A cruel, murderous affair any way you look at it," said McGinity.
"Better call the police at once."
"No," I demurred. "I mean to keep things quiet until Henry returns."
"In that case, then," the reporter suggested, "we'd better split up. You
go and find Pat, and I'll start looking for Mr. Zzyx. It's my belief that
he's escaped into the thick woods, back of the castle."
"Be careful, young man," I advised, in assenting to his proposed plan
of action. "That fellow is mad—desperate, and likely to show fight."
"He'll not escape me, don't you worry," the reporter rejoined, his
hand moving instinctively to his hip pocket. "I'll take no chances in
tackling that bird. So now," he concluded, "whatever it is we're in for
—"
He had no time to finish that sentence. The butler's voice broke in,
coming from the hall. "Come, quickly, Mr. Royce! Come, at once, sir!"
the butler shouted.
We left the chamber of death, taking good care to lock the door, and
hurried down the hall to join Schweizer, who had only come to the
head of the stairs, so as not to let Jane out of his sight. He had
surprising news to tell. The gate-keeper, who had deserted his post
at the first alarm, had come running up from his hiding-place, behind
the terrace wall, at the brink of the cliff, to report that he had seen Mr.
Zzyx go down to the dock, and, a few minutes later, cast off in a
runabout, heading for the island.
The effect of this news was terrifying. The same thought must have
struck McGinity and myself at the same instant. Pat was on the
island. To be caught there—alone—by—It was too terrible to
contemplate.
If the effect of the news was terrifying to us both, it was also
electrical, so far as McGinity was concerned. Without uttering a
word, he dashed out of the castle, ran across the terrace, and
disappeared down the steps to the dock.
Apprehensive of the effect of this news, as well as the killing of Niki,
on Jane, who was now comfortably ensconced on a divan in the hall,
with her personal maid in attendance, I gave Schweizer a quick,
whispered account of what we had found, and enjoined him to
secrecy.
"Then there was murder, sir?" he said, in a low voice. "Niki
murdered! Murder, says you, murder!" His mind couldn't seem to
grasp it. "Lord help us!" he added. "I hope that reporter person gets
that hairy, murdering thing, and gets it good!"
Increasingly disturbed and anxious about Pat's fate, I left the butler,
signaling to him as I went outside, to stay back and look after Jane.
Emerging on the terrace, another surprising sight met my gaze,
giving a startling and dramatic turn to the tragic proceedings of the
afternoon.
The shanty, which stood on the island, near the lighthouse ruins, was
on fire. The bitterly cold, north-east wind was already whipping the
flames and sending them upward in long, red tongues, which
seemed to lick the lowering November sky. Cold and biting as the
wind was, I was not sure that the quiver which shook me from head
to foot was more from cold than from the dread anticipation of what
was at hand.
Shaking and shivering, I somehow managed to get to the dock.
McGinity had already cast off, and, as I breathed a prayer for the
safety of Pat, I watched him struggling against the wind and
incoming tide in a big, unwieldy dory, the only boat available at the
dock. A flat-bottomed boat with high flaring sides, largely used on the
New England coast, and by American fishing vessels, and
christened "The Tub" by our servants, who used it for fishing
excursions.
Mr. Zzyx must have reached the island with incredible speed. The
runabout was tied up at the tiny dock, on the far side of which Pat's
row-boat rocked with the tide. The flames from the burning shanty
were mounting still higher, their reflection turning the expanse of
surrounding water into turbulent pools of fiery red. Still, no movement
was noticed on the island that would indicate the presence there,
either of Pat, or of the maddened creature, Mr. Zzyx. I was beginning
to be more alarmed than ever, when suddenly things began to take
shape.
First, I saw McGinity beach his boat at the far end of the island,
where there was a small, pebbly beach. Then came a flutter of
something white—Pat's scarf, or handkerchief—at the pinnacle of
the ruins.
At that moment the flames died down, and myriads of sparks flew
upward as the walls of the shanty collapsed. Visibility became
obscure on account of the smoke. Presently I saw McGinity running
up the steps, cut in the rocks, to the door of the lighthouse, the lower
part of which was practically intact. I saw him enter the doorway.
Then everything became indistinct in a cloud of smoke, and out of
that obscurity, I saw a black figure come stealthily around the ruins,
moving from the ledge of rock on the side next to the Sound, as
though it had been in hiding. As it crept into the doorway, and
disappeared into the dark interior of the ruins, I cried, "Oh, God!" It
was Mr. Zzyx. Pat and McGinity were trapped in the lighthouse.
Standing there alone on the dock, in the biting cold and gathering
gloom, and helpless to assist Pat and McGinity in their perilous
position, I passed into a state of anxiety bordering on frenzy. It was
only my abounding faith in the courage and resourcefulness of the
reporter in meeting the situation that kept me sane. Also, I felt sure
then, as I do still, that Mr. Zzyx did not go to the island in pursuit of
Pat. By no possible means could he have known that she was there.
Mad with fury, and out to wreck and kill, he was winding up his
abnormal excitation with all the mischief he could do on the island.
It is natural to assume that when he rushed out of the castle and
reached the dock, he saw in the runabout a means to further satisfy
his madness for excitement; or the boat may have suggested a
means of escape. As I learned afterwards, he had gone with Niki for
a spin in the runabout, directly after lunch. The engine may still have
been warm, for he seemed to have had no difficulty in starting it
himself, and he had long ago become proficient in casting off and
tying up. The fact remains that he got to the island.
Of course, from the dock, I could not see what was transpiring inside
the lighthouse. But I know now what happened. As Pat told her story
afterwards, she had spent about an hour on the island when she
decided to row back to the mainland. The exercise of rowing, the
cold, bracing wind, and quiet moments spent in wandering about the
ruins, had refreshed her wonderfully. She was walking down the
rocky slope to the island dock, when she saw the runabout
approaching. Naturally, she suspected nothing out of the way.
"At first, I thought," she said, after it was all over, "that it must be
either one of our servants, or—improbable as it seemed—Mr.
McGinity. The runabout was halfway across before I recognized Mr.
Zzyx.
"My first horrified thought was that he was coming after me," she
went on, her voice still strained by excitement. "And to me that
meant only one thing: that he was going to make an attempt on my
life, using the same tactics as he had employed when he so cruelly
killed my white cockatoo. He'd always seemed mild to me, and while
I was afraid of him, I never considered him really dangerous. I had
developed a sort of fondness for him, as I would for a big dog. But
after killing my poor bird—well, that settled everything. I had decided
not to spend another night in the castle while he was in it, and I was
prepared to give Uncle Henry my ultimatum, and stay with friends in
town, if he didn't rid the premises at once of that—killer.
"I was scared into a fit, too scared for a minute or so to think of
anything to do. Then I thought of setting fire to the shanty. That's a
thought that might occur to anybody in the same fix. I counted on the
fire bringing someone, quickly, from the castle to the island, for I had
told Aunt Jane I was going for a row, and I believed the fire would
indicate that I was at the lighthouse, and in danger. I had been inside
the shanty, and had noticed a barrel filled with waste paper and
pasteboard boxes—probably gathered up from one of the picnic
parties trespassing on the island during the summer. So I ran back,
into the shack, and threw a burning match into the barrel. The flames
leapt up so quickly, it was a close call getting outside without getting
singed.
"I was pretty shaky by this time, so I decided to hide in the ruins. Mr.
Zzyx was tying up at the dock. I could hear him chattering; he was
acting very queerly. I got down, and crawled on my hands and
knees, behind the rocks, until I reached the lighthouse doorway. I
don't believe he saw me.
"When I got inside the ruins it was so dark I had to light a match to
find my way. As I did so, something rushed at me from above, and
struck me on the head. It was a big bat. I screamed, and ran up the
winding, stone stairway as far as I could go. I crawled behind one of
the larger stones that had fallen inside, on the third landing, and
stayed there until I got my breath. The clouds were hanging so low
over my head, I felt I could almost reach up and touch them. This
feeling suggested something, so I climbed up on one of the
dislodged stones, leaned over the broken ledge of the circular wall,
and waved the white silk scarf I had been wearing under my wool

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The Vitamins, Fifth Edition:

Fundamental Aspects in Nutrition and

Gerald F. Combs, Jr., Ph.D.

James P. McClung, Ph.D.

AMSTERDAM • BOSTON • HEIDELBERG • LONDON • NEW YORK • OXFORD • PARIS

Academic Press is an imprint of Elsevier

Library of Congress Cataloging-in-Publication Data

British Library Cataloguing-in-Publication Data

For information on all Academic Press publications

Publisher: Nikki Levy

Typeset by TNQ Books and Journals

To the students and professionals who have used this book

to Holly, Elliott, and Adeline

Preface to the Fifth Edition xi 8. Study Questions and Exercises 58

1. Thinking About Vitamins 3 5. Vitamin Needs and Safety

7. Vitamin D 4. Absorption of Vitamin C 272

Part III 3. Vitamin Status of Human Populations 534

20. Sources of the Vitamins

21. Assessing Vitamin Status

Perspectives on the Vitamins

1. What Is a Vitamin? 3 4. Vitamin Deficiency 59

The Vitamins. http://dx.doi.org/10.1016/B978-0-12-802965-7.00001-0

The “Vitamin Caveat” 5. STUDY QUESTIONS AND EXERCISES

TABLE 1.1 The Vitamins: Their Vitamers, Provitamins, and Functions

Group Vitamers Provitamins Physiological functions

Discovery of the Vitamins

Anchoring Concepts LEARNING OBJECTIVES

Folic acid participant may be that of a thicket of tangled hypotheses

to be true (i.e., through deduction). Both the inductive and

Such a finding was first reported by the Russian surgeon

minimal qualitative factors that I am considering.”

1900 anti-polyneuritis factor

guinea pig scurvy

the antixerophthalmic factor. Shortly, it was found that the

that the fat-soluble factor was a new vitamin, which he

Factor Bioassay A Classic Touch in Coining Tocopherol

to develop anemia, leukopenia,66 ulceration of the gums, diar-

Factors U and R, and Vitamin Bc

was isolated that stimulated the growth of S. fecalis but not L.

Who’s on First? Factor Bioassay

was also required for the growth of Lactobacillus lac-

Vitamin B12 Isolated 1936 (D3) 1936

TABLE 2.9 Nobel Prizes Awarded for Research on Vitamins

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Preface to the Fifth Edition xi 8. Study Questions and Exercises 58

1. Thinking About Vitamins 3 5. Vitamin Needs and Safety

7. Vitamin D 4. Absorption of Vitamin C 272

Part III 3. Vitamin Status of Human Populations 534

20. Sources of the Vitamins

21. Assessing Vitamin Status

1. What Is a Vitamin? 3 4. Vitamin Deficiency 59

The “Vitamin Caveat” 5. STUDY QUESTIONS AND EXERCISES