Phenotyping
On Reaching Base Camp (1950-1975)

Donald S. Fredrickson, MD

The search for plasma lipoproteins began at the turn of the century. It was not until 1949 that a meeting
of the Faraday Society celebrated the separation of the alpha and beta lipoproteins. At that moment,
ultracentrifugists in Berkeley were already busily converting “a” to high density lipoprotein and “8” to
low density lipoprotein; the modern era of lipoproteins had begun. Over the succeeding 10 years, a quarrel
over whether the level of S, 0-20 or cholesterol was the more powerful risk factor ended with an eclipse
of the analytical ultracentrifuge and a surge of interest in the biological side of lipoproteins. The
postheparin clearing factor became lipoprotein lipase, and free fatty acids were discovered. In 1960,
abetalipoproteinemia and Tangier disease suggested that the apolipoproteins must be specific and
spurred a hunt for their number and nature. The first amino acid sequences aroused speculation of
“amphipathic helices.” By 1970, conversion of hyperlipidemia to five types of hyperlipoproteinemia led to
worldwide fascination with electrophoretic patterns, “floating beta,” and “the Friedewald formula” as
codes for genetic abnormalities leading to early coronary artery disease. A few years later, the appearance
of “familial combined hyperlipidemia” confounded the phenotyping, and the discovery of the low density
lipoprotein receptor heralded the coming of true genotypes. This is a Bethesda-based story of the “climb
to base camp” preceding the joining of molecular biology with the research on lipoproteins, dyslipopro-
teinemia, and atherosclerosis. (Circulation 1993;87[(suppl III]:II1-1-I11-15)

Kry Worps e lipoproteins « dyslipidemia ¢ hyperlipoproteinemia ¢ Tangier disease ¢
hyperlipidemia « phenotyping ¢ apolipoproteins ¢ free fatty acids e xanthomas e lipoprotein

IlI-1

lipase

hen the Faraday Society met in Birmingham,
W England, in 1949, its discussions constituted
the first international symposium on lipo-
proteins. The host was Alastair Frazier, father of one of
the prevailing theories on fat absorption and chylomi-
cron formation. Paradoxically, the American scientists
Oncley and Gurd! were not among the approximately
140 attendees. Their paper describing the reproducible
isolation of two distinct classes of lipid-protein com-
plexes from human serum by fractionation techniques
using alcohol at low temperatures and low salt concen-
trations? was delivered by Edwin J. Cohn. On the basis
of their migration on electrophoresis, these complexes
were named the “alpha and beta lipoproteins,” the
latter being much larger and considered to have a
molecular weight of possibly 1 million.3 Essentially all of
the cholesterol in plasma was accounted for by these
two lipoproteins.

There had been many earlier explorations of how
nonpolar lipids were able to remain soluble in the
extracellular waterways. Those studies were thoroughly
reviewed by Theorell* in 1930 and included references
from as far back as 1902 but omitted the 1901 report of

 

From the National Library of Medicine, National Institutes of
Health, Bethesda, Md.

Address for correspondence: Donald S. Fredrickson, MD, Na-
tional Library of Medicine, 1W22, National Institutes of Health,
Bethesda, MD, 20892.

The first Donald S. Fredrickson Lecture on Lipoprotein Re-
search was presented at the Ninth International Symposium on
Atherosclerosis, October 9, 1991, Chicago.

Nerking,> who determined that the removal of all the
lipid with ether required proteolysis. Macheboeuf,*’
while working at the Pasteur Institute in the 1920s, first
reported the reproducible precipitation of a ““cenapse,”’
or lipid-protein complex (most likely an alpha lipopro-
tein), by treating horse serum with ammonium sulfate.
Bennhold,’ who in 1932 echoed the conviction of earlier
colleagues that “die Globuline binden Cholesterin,”
was generously credited by Tiselius (the inventor of
electrophoresis) as being the first to attempt to isolate
lipid-protein complexes using a prototype of this im-
portant technique.°

Thus, it was through several of the by-products of the
blood fractionation scheme developed by Cohn during
World War II that the 50-year-long primordial “solvent
and salt era’’ drew to a climactic close. As the curtain
then promptly rose on the modern era of lipoproteins,
Barr and associates!©1! at the New York Hospital were
adapting Cohn fractionation to a clinical scale. By 1951,
they had reported an apparent association between
lower levels of alpha lipoproteins and a_ proclivity
toward coronary artery disease. At the time, zonal
electrophoresis was also being used for preparative
capture of lipoproteins by other workers!?-'* including
Esko Nikkila in Helsinki, who was beginning a distin-
guished career studying the metabolism of triglyceride-
rich lipoproteins that was curtailed by his death in 1990.

The limited interest in plasma lipoproteins in 1950
has grown enormously and has come to exert a great
influence on health practices related to cardiovascular
disease. This article is a review of some of the events in
III-2 Supplement III Circulation Vol 87, No 4 April 1993

VLDL

LDL ABL
HDL  "pBsier, At DF

Isease

 

7900 1949

1960

Cay

C-Iil A-IV

All ~ G

   
   

1970 1980

FIGURE 1. With artistic license, depiction of the major episodes in the 90-year history of the search for understanding of the
transport of fats and lipids in plasma. VLDL, very low density lipoproteins; LDL, low density lipoproteins; HDL, high density
lipoproteins; LPL, lipoprotein lipase; FFA, free fatty acids; ABL, abetalipoproteinemia; APOs, apolipoproteins; LDLR, low density

lipoprotein receptors. (See text for explanation of episodes.)

the first 25 years of the modern era and pays particular
attention to the first large-scale exercise in phenotyping
individuals who had familial abnormalities in plasma
concentrations of cholesterol and triglycerides that be-
gan in the intramural research program of the National
Heart Institute (NHI). This institute, the second
spawned in the early pluralization of the National
Institutes of Health (NIH, 1948), was given a federal
mandate to wage a nationwide war on chronic heart
diseases, of which atherosclerosis was a principal
component.

Within its first year, the NHI took over responsibility
for the Framingham Heart Project, which had started in
1946. Eventually, this long-running observation of citi-
zens in a typical New England community would pro-
vide the data that would form the basis of more than a
score of randomized interventional trials in the preven-
tion of coronary artery disease. Each of these studies
helped to highlight the importance of blood lipids and
lipoproteins in the search for ways to reduce the prev-
alence of premature atherosclerosis.

An episodic view of plasma lipoprotein research is
shown in Figure 1, representing some of the major
phases of scientific activity from the midcentury to the
introduction of molecular genetics around 1975. In this
lattermost period, the harvest in understanding of both
normal and aberrant mechanisms of fat transport and
metabolism has been dramatic, and there is as yet no
end in sight.

In all fields of science, technological changes have
dictated forward movement in research. The start and
decline of new paradigms, however, also depend on the
people who use the technology to find the answers to
particular questions. This is illustrated by the group of
scientists in California who succeeded in overthrowing
the alpha—beta paradigm of lipoproteins immediately
after its belated arrival.

The Analytical Ultracentrifuge or Schlieren Period

The word “Schlieren” refers to the optical patterns of
substances sedimenting (or floating) in the analytical
ultracentrifuge (AnUC) first described by Svedberg and
Rinde!s in Uppsala in 1924. In the mid 1930s, one of his
students, A.S. McFarlane,16-!8 improved the optics and
carried out lengthy investigations of the ultracentrifugal
behavior of serum proteins from many sources, includ-
ing patients with a variety of diseases. Many of his
AnUC plots, especially those from the clinical material,
contained a density- and time-sensitive component in
the region of the albumin boundary, which McFarlane
called the “X protein” to distinguish it from albumin
and globulin. Ten years later, K.O. Pederson!9 extended
these studies and concluded that this labile component,
which appeared soon after acceleration of the cell, was
a lipoprotein. He succeeded in partially isolating it by
altering the salt concentration of the serum in a prepar-
ative ultracentrifuge.

In the late 1940s, John Gofman, a physician and
physical chemist, gained a space in the Atomic Energy
Commission facilities at the Donner Laboratory, Uni-
versity of California at Berkeley, where he mobilized
resources to build an AnUC, and enlisted several grad-
uate students, including Frank Lindgren,2° in a cam-
paign to master the X protein, which had eluded the
Swedish workers (see “Note 1”). Over the next 5-year
period, Gofman and coworkers?!-2° drove their prepar-
ative and analytical ultracentrifuges day and night to
develop a totally new system of plasma lipoprotein
analyses and, at the same time, to explore the relations
between lipoprotein concentrations and certain aspects
of health and disease. Within a short time, they had
replaced the alpha and beta nomenclature with new
designators based on the densities and flotation charac-
teristics of lipoproteins in the ultracentrifuge. The Gof-
man group identified multiple subclasses of lighter
lipoproteins designated as “S,” (Svedberg units of flo-
tation) that soon came to be known as low density
lipoproteins (LDL) and very low density lipoproteins
(VLDL); they also discovered that the larger of these
lipoproteins were converted into those that were
smaller and denser. High density lipoproteins (HDL)
were also isolated,24 and the concentrations of HDL,
and HDL, subclasses were measured, which resulted in
the observation that the average concentration of HDL,
was much higher in women.”

Enlisting clinical colleagues to provide samples from
a large number of patients, Gofman and coworkers
undertook an early exploration into the relation be-
tween specific classes of lipoproteins and a propensity
for premature coronary artery disease. They examined
patients with “hyperlipoproteinemia,” thereby intro-
ducing this term into the literature.2” A compendium of
the findings of the Gofman group was published in the
obscure journal Plasma in 1954.29 Copies of this archive
became valuable collector’s items that novitiates such as
myself zealously guarded as if they were breviaries.

Within a year of his first publication—and unfortu-
nately, as events would prove, before their methodology
was perfected—Gofman and colleagues”* suggested
that certain lipoproteins in themselves and not their
lipid components might be the key cause of atheroscle-
rosis. It was intended as a challenge to an establishment
already comfortable with the cholesterol hypothesis that
had been promulgated around 40 years earlier by An-
itschkow?! from studies in cholesterol-fed rabbits (see
“Note 2”). The National Advisory Heart Council moved
quickly to set up a prospective cooperative study of
lipoproteins and atherosclerosis to compare cholesterol
and lipoprotein measurements as predictors of new
(coronary) events.

Five to six bumpy years later, the final judgment was
bitter and divided, with the majority choosing the
cholesterol concept (see “Note 3”).32 One unfortunate
outcome was Gofman’s premature departure from the
field he had opened so zestfully. It was not unlike the
sudden disappearance of a brilliant but brief civiliza-
tion, and there followed a period when elements of the
Donner achievements had to be rediscovered and re-
learned. Today, there is no quarrel over the measure-
ment of LDL rather than total serum cholesterol as the
more powerful indicator of coronary risk. Similarly, the
riddle of the relations between the various subgroups of
HDL and the metabolism of both cholesterol and
triglycerides continues to hold keys to as yet poorly
understood but no doubt important mechanisms (see
“Note 4”).

Physiology and Metabolism
Bethesda

The brief Schlieren period, with its intense focus on
the AnUC, served as a prism, splitting a single beam to
play upon a number of more basic aspects of lipoprotein
biology and lipid transport. One of the places housing
more diffuse interests was the Bethesda Laboratory of
Cellular Physiology and Metabolism, where the NHI
intramural research on atherosclerosis was concen-
trated, beginning around 1950. In characteristic fashion,
the then NHI scientific director, James A. Shannon,
chose as the leader of this effort a protein chemist,
Christian B. Anfinsen, who had a genius for allowing

Fredrickson Dyslipoproteinemias: Phenotyping I-3

young scientists to discover themselves (sce “Note 5”).
I arrived at this laboratory in 1953 as one of eight
clinical associates picked by Shannon for the NHI. I had
come from the laboratories of Ivan D. Frantz at the
Massachusetts General Hospital, where I had learned
how to measure cholesterol as the digitonide and to
handle the newly available isotopes of carbon and
tritium.*?

We clinical associates all realized that we were the
fortunate beneficiaries of converging opportunities. The
clinical center had unparalleled resources for combining
fundamental laboratory work with clinical investigation.
The “in” topics of the 1950s included the structure and
synthesis of proteins, and Anfinsen’s laboratories were
equipped with the latest instrumentation and research-
ers familiar with their use. The new Moore-Stein amino
acid analyzers shared space with the blocks of starch,
hoary with urea crystals as protection against denatur-
ation during the isolation of proteins. Enzymatic diges-
tion was followed by high-voltage electrophoresis for
identification and further isolation of peptide frag-
ments. The ninhydrin stains on all hands were consid-
ered to be the fingerprints of the future.

In such a milieu, those who had matriculated in
scientific experiments dominated by lipid solvents also
became keenly aware of the potential for studying
proteins, including those that mysteriously assisted the
solubilization and metabolism of cholesterol and triglyc-
erides. Among the other technologies that had recently
arrived, the use of radioactive isotopes offered unlim-
ited opportunities to explore previously hidden territo-
ries and to participate in charting metabolic routes
accessible for the first time, with the promise of unusual
opportunities.

One trend was predictable. The AnUC, with its
superior yet very demanding precision, was again re-
garded as more useful for the study of proteins than
lipoproteins. Excursions into the physiological and bio-
chemical realms of lipoproteins required the capture of
larger amounts for characterization and analysis. Clini-
cal investigations also benefited from the use of the
preparative ultracentrifuge to quantify lipoproteins in
different density fractions. After the overnight runs, the
plastic tubes were sliced and their contents drawn off for
chemical measurements. Adaptation of Lindgren’s early
methods*4 for preparative ultracentrifugation was one
of the first tasks undertaken in Bethesda by Havel,
Eder, and Bragdon,>4 and their method remains a staple
of lipoprotein laboratories today.

As relatively virgin territory, lipoprotein research was
marked by numerous leaps forward. Partly because they
involved Bethesda, but also because they were so fun-
damental in nature, two achievements of this period are
briefly described here.

Lipoprotein Lipase

One of the earlier landmarks was the unraveling of
the mystery surrounding the “‘anti-chylomicron effect”
of heparin, which was first observed in the early 1940s.°¢
Heparinized blood given intravenously to dogs caused a
rapid disappearance of the turbidity in postprandial
plasma. Gofman’s group”? had previously shown that
this clearing was accompanied by a swift cascade of
lighter triglyceride-rich lipoproteins into VLDL of
Iil-4 Supplement III Circulation Vol 87, No 4 April 1993

smaller size and higher density and that, later, there
were often lesser rises in the LDL region.

Anfinsen, Boyle, and Brown?’ showed that what they
called “the heparin-clearing factor” was a tissue com-
ponent that had the properties of a protein and was
therefore possibly an enzyme; a plasma cofactor also
appeared to be required for the clearing action. The
problem was then assigned to Edward Korn,38-* a
postdoctoral student newly arrived in Bethesda. Within
2 years, Korn had isolated the factor released by hepa-
rin from tissues and had proved that it was an enzyme;
this he termed lipoprotein lipase (LPL). The plasma
cofactor remained undiscovered at this time.

The revelation of LPL in 1955 became the center-
piece of what is still one of the most popular areas of
lipoprotein metabolism. At once, numerous reports
appeared of measurements of LPL activity in plasma,
most of which were nonvalid because none had reck-
oned on there being more than one enzyme released by
heparin.*-*3 The most important of these other lipases
was eventually traced to liver cell membranes*~*6 and is
now known as hepatic lipase. Measurement of LPL
release in plasma soon became critical to the phenotyp-
ing activities described below. The method developed in
our laboratory by Ronald Krauss in particular‘’ to
differentiate the various postheparin lipases became the
standard until immunoaffinity techniques began to
arrive.48.49

Another important boost to understanding LPL ac-
tivity came with the discovery of apoprotein C-II in
Bethesda in 1970.5°-52 Within a few months, three
independent laboratories recognized it as the missing
plasma cofactor considered by Anfinsen and coworkers
to be necessary for lipolysis of triglyceride-rich particles
by LPL.°3-55 Today, 20 years later, the LPL and C-II
genes have been cloned, and extensive study of chylo-
micronemic patients has revealed approximately a
dozen mutational changes in one protein or another
that are known to cause defective lipolysis.5¢

Free Fatty Acids

Another key discovery in the mid 1950s was that not
all of the key mechanisms of fat transport in the blood
involved lipoproteins. This knowledge was provided
independently by Vincent Dole*’ at the Rockefeller
Institute and Robert Gordon’* of the NHI Bethesda
contingent. A tiny fraction of the total mass of fats and
lipids in plasma proved to be the most sensitive mech-
anism for maintaining the body’s caloric economy in
balance.

The tidal movements of calories, tied mainly to the
digestive rhythms, are often far in excess of immediate
needs. The storage of excess calories as triglycerides
requires less water than glycogen, and the release of
ester bonds by hydrolysis demands less energy. When
summoned, the fatty acids race through plasma bound
to albumin and are available for immediate consump-
tion or are readily returnable to fat depots. Dole called
these moieties nonesterified fatty acids, and Gordon
preferred unesterified fatty acids. Steinberg of the
Journal of Lipid Research resolved the dilemma by
deciding upon free fatty acids (FFA), all sides agreeing
that harmony in nomenclature was worth the chemical
inaccuracy of the term.

It was deduced from arterial-venous differences that
these small amounts of FFA—for which very sensitive
methods of measurement were developed — had a tran-
sit time through plasma of seconds rather than minutes.
This was confirmed by study of the disappearance of
*C-labeled fatty acids, and the rapid reappearance of
the label in expired carbon dioxide indicated the ready
availability of FFA for metabolism.5? Dietary fatty
acids, touring the plasma as triglycerides in chylomi-
crons, also rapidly reemerged in FFA.

In the late 1950s, the accumulation of new knowledge
was so rapid that, accepting an invitation that Anfinsen
could not fulfil, Gordon and I wrote for Physiological
Reviews the first definitive analysis of the emerging
subject of the transport of fatty acids.©' It was yet
another sign that this was a renaissance, a time when
apprentices carried out the surplus work of their
masters.

Apolipoproteins and Mutations

Around 1960, I formed a small group called the
Section on Molecular Disease. (It was, as far as I am
aware, the first appearance of Pauling’s felicitous® term
among the tables of organization of the NIH.) The
purpose was to launch a long-range study of genetically
determined diseases involving plasma lipoproteins. The
theme had become important for several reasons. The
departure of Havel for the Cardiovascular Research
Institute at the University of California at San Francisco
had left me a legacy of patients with abnormal lipopro-
tein concentrations and a variety of xanthomas, some of
whom had been among the first patients studied at the
clinical center. Also around this time, I had agreed to
join John Stanbury and James Wyngaarden in writing a
book with the bold intention to take up from where
Garrod® had left his descriptions of inherited metabolic
disorders in 1908. The book was to be called The
Metabolic Basis of Inherited Diseases (MBID), and its
chapters were to cover the clinical and genetic manifes-
tations and all of the relevant biochemistry available on
such disorders as far as 1960. (In 1988, the sixth edition
of this ever-expanding volume appeared under a new
team of editors.)

On being immersed in both the care of patients with,
and gaining familiarity with the world literature on
xanthomatoses and lipidoses, I became acutely aware of
the inchoate state of diagnosis and minimal understand-
ing of these disorders. What we had seen in the preced-
ing 7 years in Bethesda convinced us that we were
entering a time of sweeping enlightenment in the area
of both lipid metabolism and genetics and, thus, oppor-
tunity for improvement in both taxonomy and
understanding.

Abetalipoproteinemia and Tangier Disease

In 1960, as we were embarking on studies of hyperli-
poproteinemia, two rare mutations were described that
caused hypolipoproteinemia and reinforced our grow-
ing faith in the uniqueness of the apolipoproteins. On
occasions, the elimination of an effective activity of a
particular protein by mutation is the only proof of its
function. These discoveries were a stimulus to divert
part of our activities to participate further in the study
of apolipoproteins.
The first disorder was described by Salt and cowork-
ers in England, who found a young girl whose ability to
absorb dietary fats was nearly abolished. On centrifu-
gation, LDL, VLDL, and chylomicrons were missing,
and plasma triglycerides were at correspondingly van-
ishing levels. Fortunately, by this time in the mid 1950s,
immunochemical methods were applied that could dis-
tinguish the two major antigens of the plasma lipopro-
teins.5-67 This patient lacked what came to be called
the “B” protein of beta-lipoprotein. This was the first
recognized instance of abetalipoproteinemia (ABL).

A scant few months after this report of ABL, we came
upon what appeared to be its mirror image.®° A
5-year-old boy diagnosed as having Niemann-Pick dis-
ease had been referred to the clinical center. Although
I had never previously seen a patient with this disease,
having scanned the world’s literature in preparation for
my definitive chapter on Niemann-Pick disease in the
MBID allowed me to assert that the diagnosis was
incorrect. The patient’s plasma cholesterol level was
low, whereas the triglycerides appeared to be somewhat
higher than normal. There were very low levels of HDL
or alpha-lipoproteins in the plasma. The most astonish-
ing abnormality —which had led to the erroneous diag-
nosis on histology—was the storage of mammoth
amounts of cholesteryl esters in reticuloendothelial
tissues throughout the body. The patient’s tonsils had
been removed, but we had the dramatic and rare
experience of observing the bright orange tonsils of the
boy’s only sibling. Among other things, we had discov-
ered the only lipidosis that can be diagnosed by looking
into the mouth! We called this condition Tangier dis-
ease, after the Chesapeake Bay island home of the
patient’s family. A carrier status characterized by half-
normal levels of HDL in obligate heterozygotes con-
firmed that this was an autosomal recessive disease.”

This finding stimulated a 10-year study of the minute
quantities of “A” apoprotein in the plasma of these
patients. We now know, however, that rather than being
due to a low level of HDL synthesis, these patients can
produce normal A-apolipoproteins (apo A), but the
turnover rate in plasma is so rapid that the levels remain
very low.”! The reasons for this as well as the exact
mechanism(s) of the attendant tissue storage of cho-
lesteryl esters as yet remain obscure, but, someday,
someone will reveal the important mechanisms for
removal of cholesterol from tissues. The discovery of
ABL and Tangier disease served for us believers as a
witness that lipids were not cadging rides on any old
globulins in plasma but used specially designed carriers
for this purpose (see “Note 6’). Exciting questions
were raised concerning these putative lipid—avid pro-
teins: How many were there, how were they able to
perform their tasks, and what had been their evolution?

The Hunt for Apolipoproteins

By the time the two new genetic disorders of apolipo-
proteins had been found, some effort to characterize
these proteins was already under way. Only the hardiest
experimentalist attempted to begin with the stubbornly
insoluble B-apoproteins (apo B), which appeared to
constitute all of the LDL protein. Most of the earliest
work concentrated on HDL or on the triglyceride-rich
particles. Initially, the main centers of this activity were
Berkeley, Bethesda, Chicago, and Oklahoma City. De-

Fredrickson Dyslipoproteinemias: Phenotyping III-5

termination of terminal amino acids in apoproteins
confirmed that the A and B proteins were different7>-*5
and suggested that VLDL might have other apoproteins
as well.?5 The meager amounts of protein in chylomi-
crons included the “fingerprints” of A and B and a third
unknown print, which Martin Rodbell and I’* called
simply “C.”

From the first, there had been the suspicion that apo A
might be plural. In 1968, preliminary evidence of this by
Cohen and Djordjevich”? was amply confirmed by Ber-
nard and Virgie Shore,”® who separated and identified
the proteins according to their C-terminal residues as
C-Gln and C-Threo. The latter was corrected to C-GIn-II
in 1971 by Koster and Alaupovic,” who also noted that
the N-terminus of the second apo HDL was blocked,
providing one basis for the long period of confusion over
the number of major proteins present in HDL.

In 1965, Gustafson, Alaupovic, and Furman® used
nonpolar solvents to extract from human VLDL a third
protein that they called “C.” Between 1966 and 1969,
Virgil Brown in our laboratory set out to purify C in
VLDL and isolated three distinct apoproteins, namely,
apo VLDL-Val, apo VLDL-Glu, and apo VLDL-Ala
(now better known as apo C-I, apo C-II, and apo C-III,
respectively5°-52). As already noted, apo VLDL-Glu was
quickly identified as the LPL cofactor (see “Note 7’”).81.82

Apolipoprotein Nomenclature

Those who explore the older literature will be aware
that, by 1970, the naming of apolipoproteins was a
confused and contentious business. The Shores and our
group held to the C-terminal nomenclature. On the
other hand, Alaupovic and coworkers® introduced the
hypothesis that there were several apolipoprotein fam-
ilies, each characterized by the presence of a “single
apolipoprotein or its constituent polypeptides.”®*4 By
this reasoning, the several apoproteins isolated by
Brown from VLDL should all be termed “C” followed
by a different Roman numeral, as they are all related in
an as yet unelucidated manner. As neither side would
yield to the philosophical approach of the others, even-
tually, journal editors — and common sense — demanded
one system. As some of the initial C-terminal determi-
nations had to be corrected,”?85 and more than one
protein in a major lipoprotein class proved to have the
same carboxyl-terminal acid, theoretical objections gave
way to the practical. We conceded and urged adoption
of the ABC system in 1972 (see “Note 8”).8687

New Phenotypes of Hyperlipoproteinemia

Confusing Taxonomy

In the early 1960s, apolipoproteins did not offer a
rational approach to the first task assumed by the
Section on Molecular Disease: improvement of the
phenotyping of hyperlipoproteinemia. Patients with el-
evated cholesterol or triglyceride levels that baffled
their physicians came in increasing numbers to the
clinic. From the first, even good generic descriptors
were lacking. “Hyperlipemia,” for example, had become
synonymous with creamy plasma. I therefore chose to
use the term “hyperlipidemia” and inserted “familial”
as the key modifier in the title of the first MBID chapter,
but I lacked the courage to jettison the hallowed but
useless “essential” or “idiopathic.” I elected to relegate
III-6 Supplement III Circulation Vol 87, No 4 April 1993

 

ASH OENSTN
QEQPROTENS

\
\ ADE
WEA DENSITY
QPCR ROTENRS

\

DRUWA-BOVUND
VAESTTRRED FRUU KODS

CLINICAL No
STATE

 

—TENDON
XANTHOMAS

  
  
  

FIGURE 2. Graphic plot of lipo-
proteins and xanthomas con-
structed from the data of Gof-
man et aP° as it appeared in the
first edition of The Metabolic Ba-
sis of Inherited Disease,” 1960.
Reproduced with permission.

ERUPTIVE——

 

—TUBEROUS ———--~---~—

the xanthomatous lesions to secondary place, especially
after noting that, in the 19th century, biliary cirrhosis
had mocked nearly all the xanthomas found in hyper-
lipidemic patients.

In 1958, the doyen of the lipidoses was Siegfried
Thannhauser, at that time at Tufts University Medical
School (see “Note 9”). Thannhauser had left Germany
in the late 1930s, bringing with him a major work by
himself and Magendantz on the different groups of
xanthomatous diseases published in 1938.88 The work
became the core of Thannhauser’s authoritative book
published in 1940, its last revised edition appearing in
1958.89 In this edition, two syndromes of hyperlipidemia
were recognized: essential or idiopathic hyperlipemia
and essential hypercholesterolemia. Thannhauser was
aware from the reports of varying lipid patterns and the
different kinds of associated xanthomas in affected
patients that greater heterogeneity existed, but a new
approach did not appeal to him. In his last edition, he
wrote: “Gofman’s method is, from the heuristic point of
view of the investigation of the multiplicity of the serum
protein aggregates, very interesting, but it seems not
justified, or at least premature, to attribute the various
Svedberg units diagnostic significance in clinical
medicine.”8?

Toward the end of the 1950s, I visited Thannhauser to
convince him of his error. I had, by then, compiled
lipoprotein data from Gofman’s Plasma compendium2°
into a three-dimensional plot (Figure 2) to use as an
illustration in the first edition of MBID.® This proved to
have no power to tempt the ailing professor to recant,
however, and I returned to work on my own generation.

From the outset, we had restricted our studies to
patients with evidence of familial disorder. I will leave
to a footnote the details of methodology that relied
upon preparative ultracentrifugation with quantification
of the major lipoprotein classes in terms of their cho-
lesterol value (see “Note 10”).!-°2 It was soon evident
that other simpler methods for classification, particu-

larly in screening family members, would be helpful. In
1961, Ahrens and coworkers? at the Rockefeller Insti-
tute introduced the terms “fat-induced” and “carbohy-
drate-induced” hyperglyceridemia and added virtue to
the possibilities of rapidly distinguishing between the
triglyceride-rich lipoproteins WLDL (carbohydrate-in-
duced or endogenous triglycerides) and chylomicrons
(fat-induced or exogenous triglycerides).

Paper Electrophoresis

Around this time, a possible new approach for mak-
ing this distinction in a rapid and simple manner
literally came strolling through the door of the labora-
tory in the form of a new assistant, Robert S. Lees. At
the Massachusetts General Hospital, he and Frederick
Hatch had altered the conventional system for electro-
phoresis of plasma proteins and lipoproteins™ by adding
albumin to the usual barbiturate buffer, thereby ob-
taining as many as four lipoprotein bands from plasma
samples after the strips were dried and stained with a
fat-soluble dye. The other protein bands remained
invisible. When Lees joined us, I insisted that we were
interested only in quantitative methods, but I raised no
objection to demonstrations of his method.

The sideshow quickly captured our interest, however,
for the fluttering strips, stained with the scarlet dye oil
red O, made a fetching display. We performed a few
experiments to determine the nature of the bands that
often joined the usual alpha (HDL) and beta (LDL)
bands. It was shown that the prebeta bands, moving
between the beta and the faster alpha bands, were
VLDL. Material left at the origin represented chylomi-
crons.%° It appeared that here was a ready method for
distinguishing two different forms of triglyceride trans-
port, an obvious need in the population of abnormal
subjects we were investigating.

Soon, we were meeting each morning to “read the
wash” on the line where the stained strips were hung to
dry. A form of shorthand emerged from the laboratory
banter, numbered types evolving from the order of the
most prominent bands on the strips. A great splash of
red chylomicrons at the origin and often, little else in
the way of other lipoproteins, was called type I. A
strongly colored beta band (an excess of LDL) became
type I. Type IV was a prominent prebeta band (the
classification allowing a peek at the total concentration
of plasma triglycerides). Initially, type III was the
presence of both prominent beta and prebeta bands,
and type V became a useful handle for plasma samples
in which fairly high triglyceride concentrations were
accompanied by both a prebeta band and a chylomicron
smudge at the origin.

Evolution of Types

These original five types of hyperlipoproteinemia
were first publicly displayed to a group of cardiologists
in the mid 1960s.%7 In the chapter on hyperlipoprotein-
emia in the 1966 (second) edition of MBID, an impor-
tant adjustment to this scheme appeared as a last-
minute addendum. The genesis of the change was the
arrival of Robert I. Levy, who in the course of further
examining the nature of prebeta lipoproteins” had also
checked the completeness of the separations of VLDL
from LDL in the “‘beta-quants,” as we called the routine
analyses. He found that beta-migrating lipoproteins
were occasionally present in the supernatant layer after
the overnight centrifugation.

This “floating beta” and the broad beta band it
produced on paper electrophoresis proved to be not a
random error but the signature of what became type III.
Analysis of collections of such patients, who had a
unique tendency to collect lipid in the creases of the
palms as well as both peripheral and coronary artery
disease, led us to reexamine the earlier literature. As we
had acknowledged on introducing type III, this pheno-
type appeared to have a possible correspondence to the
abnormal Schlieren patterns described in patients with
xanthoma tuberosum in the AnUC studies of Gofman
and coworkers.”527 In those patients, S, 0-12 lipoprotein
(LDL) concentrations were sometimes depressed, and
variable increases in S, 12-20, 20-100, and 20-400
appeared. They noted that a few of their patterns could
not be distinguished from those with essential hyperli-
pemia. I had included these data in the graph (Figure 2)
prepared for my visit to Thannhauser (see “Note
117’).90.100-104 Jn 1969, we collaborated with Frank
Lindgren! to compare all the AnUC patterns in the
various types: The type III and xanthoma tuberosum
patterns corresponded.

Additional Types

By the time we thought we were ready to produce a
definitive description of the typing system, observations
of more patients required that several other adjust-
ments be made. The patients with type II, defined by
the excess of beta lipoprotein or LDL, frequently had
modest hypertriglyceridemia and distinct prebeta
bands. We subdivided type II into IIA and IIB, the
latter having a modest increase in triglyceride levels.
The bulk of these patients had the familial disorder first
described in the latter half of the 19th century! and
were diagnosed by Wilkinson and coworkers!© as hav-
ing essential familial hypercholesterolemia in 1949. The
presence or absence of mild hyperglyceridemia did not

Fredrickson Dyslipoproteinemias: Phenotyping Il-7

discriminate between the heterozygotes and homozy-
gotes in the data that we had collected from numerous
families.1°8

A fifth phenotypic pattern (type V) was added after
the observation of numerous patients with severe hy-
pertriglyceridemia who had both prebeta bands and
chylomicrons at the origin of the strips. Although a
number of the family members with these probands had
lesser degrees of hypertriglyceridemia, long observation
periods on the wards led us to consider that at least
some of the subjects with type IV and others with type
V were distinct phenotypes. These subjects responded
differently to diet and weight loss, and the type V
phenotypes had a higher prevalence of diabetes melli-
tus, coronary artery disease, attacks of abdominal pain,
and pancreatitis.

Moreover, as better tests for measuring postheparin
LPL activity were developed, it became readily appar-
ent that the familial hyperchylomicronemia expressed in
the type I pattern was biochemically different from the
others.*? In 1960, Havel and Gordon’ found posthep-
arin lipolytic activity (PHLA) to be very low in the
affected members of the first family with essential
hyperlipemia seen at the clinical center.!1° By 1965, we
had measured PHLA in 15 subjects with typical type I
and had confirmed that it was definitely low in 14 of
them. Virtually all of the type V subjects had normal
PHLA despite their gross chylomicronemia. The man-
agement of type I and type V was distinctly different,
but both were recognized to be potentially lethal dis-
eases because the bouts of pancreatitis could be life
threatening.

From the beginning, it was obvious that the pheno-
types being classified by this typing system (Figure 3)
were neither homogeneous entities nor true genotypes
but were frequently secondary to other conditions.
Although these phenotyping studies appeared to distin-
guish several new familial hyperglyceridemic syn-
dromes, the true usefulness of the approach lay in two
other directions. Large numbers of physicians, most of
whom had been unaware of lipoproteins, realized that a
better way to interpret the disturbing elevations of
cholesterol or triglycerides was needed, especially as
implications of risk for coronary artery disease began to
circulate. The translation of hyperlipidemia to hyperli-
poproteinemia attracted adherents who then gained
entry to the large body of information being reported
from the many laboratories working on lipoproteins.
Second, these designations into at least five groups were
not abstractions but the first step toward better diagno-
sis and management of hyperlipidemia, as there were
unique clinical features and different responses to both
diet and drug regimens in each phenotype and impor-
tant differences in familial expression and in the com-
plications of the hyperlipidemia.

In 1967, we!!! published a five-part review of all that
was then known of lipoproteins and dyslipoproteinemia
to assist clinicians in using this rapidly growing body of
information (see “Note 12”).112 Many of the basic
clinical aspects of the review, as deep as they went at the
time, have endured; the information on the lipoproteins
per se, however, was out of date by the time of
publication.
IlI-8 Supplement III Circulation Vol 87, No 4 April 1993

PHENOTYPING HVPERLIPOPROTEINEMIA

~ Hermal 1 T TU

  

Vv

 

 

Emergence of a New Speciality

The eagerness with which clinicians took to these
early exercises in lipidology and their generous referrals
of both questions and patients made the development of
a new nonspeciality of medicine a stimulating and
gratifying experience. Because of the pleomorphism of
the abnormalities accompanying hyperlipoproteinemia,
many aspects of medicine were involved that appeared
at first to be remote from the measurement of lipopro-
teins. We also occasionally violated the territories of
venerable medical specialities that had dealt with the
various clinical manifestations of hyperlipidemia. The
paramount group was the dermatologists under whose
dominion all patients with hyperlipidemia and xantho-
mas had been observed and managed for nearly a
century.

In the 1950s, some rare skin disease specialists began
to use the AnUC,!3 although the descriptive aspects
remained. In 1968, I gave back-to-back lectures to both
the American Academy of Dermatology and the Amer-
ican Dermatological Association without censure of the
cavalier treatment of the canons pertaining to xantho-
mas. Invitations to demonstrate phenotyping before
such diverse groups as geneticists, internists, ophthal-
mologists, orthopedists, otolaryngologists, pathologists,
and pediatricians led me to grateful appreciation of the
early operational arrangements of the NIH Clinical
Center that had allowed us to study patients vertically
by age, from infancy to senility, and horizontally across
the spectrum of organ manifestations, allowing the
broadest perspectives. Ultimately, of course, it was the
cardiovascular community that had the greatest need
for lipoprotein analyses and interpretations and re-
tained the closest interest in the core of our work. I
became a member of the American Society for the
Study of Arteriosclerosis at a time of anxiety over its
“marriage” to the American Heart Association (AHA).
The union has survived, and the major lipoprotein
forum in the world today is still the AHA meetings.

Phenotyping in Crescendo

The popularization of phenotyping as a means toward
better understanding and management of the patient
abnormalities grew even further when the AHA, the
NHI, and other groups became activists in enlisting
physicians into organized campaigns to reduce the risk of
premature coronary artery disease. After 1960, numer-
ous dietary trials—some on a large scale —were begun;
however, after a decision by the NIH in 1969 that a vast

FIGURE 3. The five major phe-

notypes of hyperlipoproteinemia
. as defined by electrophoresis,
_ circa 1967.

controlled clinical trial of dietary changes was impracti-
cal, drug trials became inevitable (see “Note 13”).114-118

Lipid Research Clinics

In 1970, a panel on hyperlipidemia and premature
atherosclerosis was convened by the then director of the
National Heart and Lung Institute (NHLI), Dr. Theo-
dore Cooper (see “Note 14”). The panel arrived at
several conclusions,'° including unanimous agreement
that translation of plasma lipid concentrations into
lipoproteins was desirable and that it was possible to
agree upon a standard classification of lipoprotein pat-
terns for diagnosis and management of hyperlipidemia.
Another decision was that a national network of lipid
research clinics should be established for the develop-
ment of population standards and final methodologies
over the next 5 years. A further conclusion was that one
of the purposes of the clinics would be to carry out a
randomized prospective trial to determine the effect of
the treatment of hyperlipidemia on the incidence of
coronary artery disease. Dr. Robert Levy became the
head of the new lipid research clinic program.

A few weeks after the Bethesda meeting, the World
Health Organization issued a report recommending
the phenotyping system as a worldwide standard (see
“Note 15’).120

Continued Furbishes

As we entered the 1970s, we continued in our at-
tempts to make the phenotyping system more practical
for physicians.!2!\22 No practitioner had access to a
preparative ultracentrifuge, and few were able to obtain
satisfactory electrophoretic patterns in type III, al-
though local or regional laboratories all over the world
began to enjoy a “land-office” business in lipoprotein
patterns. The diagnoses on printouts sent to hospitals
and physicians from these laboratories assumed an
undeserved authority.

For this reason, various algorithms and other devices
were sought to encourage thinking about lipoproteins
while using readily available information and a mini-
mum of extra laboratory analyses. An example was the
popularization of the practice of examining the patient's
plasma after allowing it to sit overnight at 2°C. The
finding of a creamy layer floating over the clear infra-
natant layer was an infallible hallmark of full-blown
type I. In type V, the cream was always overlying a
milky layer of variable turbidity.

Diagnosis of Type III
Type III, the rarest form of hyperlipoproteinemia,
received much attention whenever it was recognized. It
 

was quickly found to carry a high risk of both coronary
artery and peripheral vascular disease. Once recognized
and treated, its extraordinary xanthomatous skin depos-
its melted away within a few weeks. A precise diagnosis
of type III was available only to specialized laboratories
such as ours, and our search for other rules of thumb to
assist the clinicians sometimes approached the baroque
and were focused on using plasma lipid analyses alone
for screening purposes. A ratio of cholesterol to triglyc-
eride approaching 1 was, and still remains, a valid clue
to the presence of type II. In 1975, after a review of
about 100 type III patients out of a total of nearly 1,000
patients in our registry,!23 we concluded that the ratio of
VLDL cholesterol to plasma triglycerides clearly distin-
guished the abnormal VLDL of type III.!24+ This was
consistent with a previous report by Hazzard and col-
leagues,!25 which used the slightly more cumbersome
measurement of VLDL lipids only.

Fortunately, at about this time, the mystery of this
rare and always intriguing phenotype began to clear due
to interventions involving apoproteins. During 1971-
1972, again with particular thanks to the sleuthing of the
Shores,’26 a new “arginine-rich peptide” was detected in
VLDL apolipoproteins.!2”7 Two years later, Havel and
Kane!28 reported a predominance of arginine-rich pro-
tein in type III plasma (see “Note 16”). Gerd Uter-
mann?!?9 then isolated what he called apoprotein E from
the abnormal VLDL. Utermann’s discovery included
the separation of E by isoelectric focusing into three
major components: E,, E;, and E,. The discovery of the
inheritance of these phenotypes, the appearance of the
E,/E, homozygote in type III, and the single point
mutations in the E proteins that alter the catabolism of
lipoproteins bearing the mutant E make a fascinating
story but unfortunately are beyond the scope of this
essay.

The Friedewald Formula

Of all of these physicians’ do-it-yourself lipoprotein
guides, the most durable was published in 1972. Judging

Fredrickson Dyslipoproteinemias: Phenotyping IlI-9

FIGURE 4. Three examples of the
diets developed for each phenotype
of hyperlipoproteinemia and the ac-
companying handbook published by
the National Heart Institute!3! and
sent to physicians on request. An
estimated 7 million copies were
distributed.

from the composition of average VLDL particles pro-
vided by numerous laboratories and from observations
made in hundreds of patients with hypertriglyceridemia,
we sought a way to estimate LDL concentrations with-
out an ultracentrifuge. Levy and I popularized a crude
rule (obvious to the cognoscenti) stating that, from the
concentrations of total plasma cholesterol (C) and
triglycerides (TG) and the concentration of HDL cho-
lesterol (HDL-C), the amount of LDL cholesterol
(LDL-C) can be estimated as LDL-C=C—[HDL-C
+TG/5], provided that the patient does not have type
III. This was hardly higher mathematics but, to establish
the validity of this rule, we asked William Friedewald, a
biometrics expert in the NHLI, to check this formula
independently. Even after the designation of type III
will have faded from memory, the Friedewald formu-
la}30 may be remembered forever as the most durable
relic of the phenotyping period.

Our Best Sellers

One of the most useful features of the phenotyping
system was that it allowed guidance to more specific
treatments for each type. The fundamental first step in
therapy was to adjust the diet, including a focus on the
patient’s body weight. Physicians, including ourselves,
are inept at changing diets, but we were fortunate at the
clinical center to have the ample and invaluable help of
nutritionists, including, in particular, Ernestine Bou,
Mamie Bonnell, Nancy Ernst, and Edith Jones.'3! Their
standard diets were often sought by physicians who
made their requests in large quantities. Through the
courtesy of the U.S. Government Printing Office, we
had five different diet booklets (Figure 4) in five differ-
ently colored covers designed to respond to the calls
from the clinics. When the distribution was finally
suspended in the late seventies, more than 7 million
(royalty-free) copies are believed to have been re-
quested and distributed.
I-10 Supplement II Circulation Vol 87, No 4 April 1993

Evolution

At a meeting on lipids in Greece in the late 1960s,
Hugh Sinclair, a don of Magdelene College, was presid-
ing in his inimitable way at an informal session of
lipidologists. While gazing at the Temple of Poseidon at
Sunion, he intoned, “Regard these magnificent ruins
still standing after 2,000 years, and you, Fredrickson,
your classification will be lucky to survive another five!”
He was partly right; the end was predictable although it
endured for more than 20 years. Its limitations included
having ignored HDL, having no claim to genotype, and
relying on a nonstandardized test for the diagnosis of
type III; we stood naked in the path of the march of
molecular genetics toward a modern basis for
genotyping.

While an old-timer in the laboratory can make use of
the shorthand for an abnormal pattern, the departure of
the typing system from the curricula of medical schools
here and abroad has spelt relief for many students.

The long-desired specificity of mechanisms as a re-
placement for this durable expedient arrived gloriously
with the revelation of the LDL receptor (LDLR, see
Figure 1) in 1974.!32133 Several years earlier, the ap-
pearance of familial combined hyperlipidemia and its
mockery of phenotypic variation within families'34-136
further weakened the foundations of a structure that
even I had ceased to support, although my disillusion-
ment had been of another kind.

By 1970, I had lectured on phenotyping literally
around the world. On such a journey visiting tiny
hamlets in New Zealand, great cities in Australia, clinics
in India, and many other places between those and
America, I had found some things to cheer. Certainly,
one was the nearly ubiquitous photographs on the
laboratory walls of the six tubes of plasma, one for each
type. Decidedly unpleasant, however, was the booming
commercial market everywhere for electrophoretic
strips stained with oil red O, and they were badly
interpreted. Two years later, even institutes in the
Soviet Union had fallen under the spell, and yet the
conventional laboratory was unable to confirm a diag-
nosis of type III. In 1975, I dispatched an editorial to
Circulation entitled “It’s time to be practical”:

“Ten years ago, we recommended in this journal the
use of lipoprotein patterns for identification of different
groups of familial hyperlipidemia .. . Technical short-
comings (still) exist ... Commercial laboratories cannot
provide reliable or quantitative lipoprotein patterns,
and preparative ultracentrifugation is still necessary to
make a certain diagnosis of type III hyperlipoprotein-
emia ... Upon finding a subject with hyperlipidemia,
the physician is likely to wonder if he should now obtain
a lipoprotein electrophoretic analysis. At present, my
answer, directed to the generalist, is negative .. .”1°7

By the time the editorial appeared, I was occupying
the chair of the director of the NIH. Those exhilarating
days among the lipoproteins were over. As I reflect
upon the grand and competitive struggle that it has
been, I realize that it had been a salient advance,
working upward over newly opened routes to establish a
base camp. From there would begin the scaling of the
high peaks that slowly became visible as we climbed.
Our successors in these steeper ascents needed the
magic of molecular biology, a technology beyond our

imagining in the early 1950s. The result has been an
astounding trail of achievements already left by our
successors — the LDL receptor, the determination of the
structure of apoprotein B and the regulation of the
synthesis of its Broo and B, components, the emerging
view of the relation between HDL and triglyceride-rich
remnants as subtended by the cholesterol transfer pro-
tein, and the chromosomal localization of the apopro-
tein genes and abnormalities under genetic control —
these are only some of the enormously gratifying
scientific advances since 1975.

The extension of scientific knowledge and epidemio-
logical data concerning lipoproteins into health prac-
tices is, of course, an even more significant contribution
to humanity. The paths yet to be traversed remain an
impressive challenge. I owe a great debt to those who
climbed with me (“Note 17”); I don’t think any of us
would have missed it for the world.

Notes
Note 1

To John Gofman, Pederson’s lengthy report of his
struggles with the X protein was an irresistible chal-
lenge. His successful conquest of this problem was also
attributable to the talents of E.N. Pickel for building his
first instrument and to the support of J.H. Lawrence,
head of the Donner Laboratory, who found a way to pay
for another centrifuge and donated space in his labora-
tory. Lindgren, a physical chemist and Gofman’s very
able “right hand,” has described? how the way to
resolve Pederson’s difficulties with the pile-up distor-
tions at the albumin boundary came to him one night
when he had fallen asleep on the AnUC while working
in the Donner Laboratory. In 1990, the laboratory met
once again, and Gofman left a valuable transcript
describing old memories from the early 1950s. I am
grateful to Ron Krauss for an opportunity to peruse
these memoirs.

Note 2

I had the opportunity to meet academician N.N.
Anitschkow in 1963 as a member of one of the first
cardiovascular groups to visit the USSR in the post-
Stalin period. We walked up the street of life from the
obstetrics pavilion to the morgue at the medical school
in Leningrad. There, dressed in a white surgical gown
and cap, Anitschkow handed out rabbit aortas stained
with oil red O. When asked about his constant cigarette
smoking, he said “Cholesterin ist Alles.”

Note 3

Some called it the “‘uncooperative cooperative
study.” Four laboratories —the Donner, the Cleveland
Clinic, the University of Pittsburgh, and Harvard School
of Public Health—examined more than 15,000 men
aged 40-59 years and studied 4,914 of them in a 4-year
prospective study. The cast of characters involved was
stellar. The new events were judged by no less a panel
than Paul D. White, Samuel Levine, and Howard
Sprague.

The challenge had been given and accepted prema-
turely. The Gofman team was forced to change the rules
as they gained more knowledge. Starting with the S,
10-20 class, they then insisted that 12-20 was the
proper one; later, they insisted on inclusion of the
12-100 band. Corrections for the effect of concentra-
tions on flotation rates were also eventually demanded,
and, finally, only the Donner group was measuring S;
100-400 lipoproteins.

The divided conclusion pitted the opinion of labora-
tories in the East against those in the West, although
there appeared to be agreement that cholesterol ap-
peared to be the most effective in separating out the
new events, and S; 12-20 was the least so. With Gofman
in a minority position, his introduction of an “athero-
genic index” triggered further polemics.

Note 4

After 1955, the Donner Laboratory continued to
produce important studies of lipoproteins and is still
doing so today. Lindgren has recently retired, but
Alexander Nichols and Trudi Forte, who made some of
the first electronmicroscopic studies of lipoproteins, are
still there, and Ronald Krauss, one of our valued
coworkers in Bethesda, is now in charge. During the
1960s and 1970s, Bernard and Virgie Shore, at the
Livermore Laboratory, collaborated with the Donner
group in many key advances in our knowledge of
apolipoproteins.

Note 5

Dr. Shannon became the director of the NIH in 1955
and served until 1968, the period of the most rapid
growth of the extramural programs of the NIH. Anfin-
sen was awarded a Nobel Prize for Chemistry in 1972
for studies showing how the primary structure of a
protein also dictated its conformation. Before we clini-
cal associates arrived, Anfinsen’s recruits had included
Raymond Brown, Edwin Boyle, Joseph Bragdon, Daniel
Steinberg, Martha Vaughan, James Baxter, Howard
Goodman, and Majorie Horning. Soon, Howard Eder
joined us from New York and Edward Korn arrived with
a fresh degree in biochemistry, later followed by Martin
Rodbell, who shared the lab with Korn.

Note 6

In 1965, I was invited to Mosbach by Professor Ernst
Klenk to address the annual gathering of the Gesell-
schaft ftir Physiologische Chemie on the subject of lipo-
proteins.7”2 I took along the figures showing lipoproteins
that today appear absurdly simple and used ABL and
Tangier disease again to emphasize the special functional
nature of these “no doubt” specific proteins, called
simply A and B. After I had finished, Professor Klenk’s
docent, Willi Stoffel—now Klenk’s successor—said to
me, “You did all right, but they don’t believe a thing you
said!” At a later meeting in Cologne in 1970, I was
gratified when Klenk introduced me with the admission
that I had “made him believe in lipoproteins.”

Note 7

Eventually, we were to reap the reward for having
devoted so much laboratory space to equipment and to
other preparations for sequencing an apoprotein that
might finally be caught; this was considered one of the
higher prizes of the competition in those days. The first
two sequences (apo A-II and C-IJ) became lab trophies
in 1972-—1974,81.82

Fredrickson Dyslipoproteinemias: Phenotyping WI-11

Note 8

In preparing this history, I noted from a Harvey
Lecture® and another presentation®’ in 1972 that I was
using both terms for each apoprotein while not sparing
the reasons for our reluctance to adopt a system that
was somewhat metaphysical. When I telephoned Petar
Alaupovic recently to jog my memory of the precise
moment of surrender, he immediately and graciously
gave me the answer. Apparently, I had sent a coworker
to deliver a paper at a meeting on serum lipoproteins in
Austria in October 1973. Alaupovic has even supplied
me with a copy of the abstract containing the conces-
sion. Noting that (Peter Herbert’s) “presentation was
very well delivered and accepted by all participants, but
especially my colleagues and myself,” Alaupovic gives
more details of the great feast afterward and of Her-
bert’s toast, in which the latter purportedly said, “You
know, Fredrickson sent me into this ‘enemy’ camp to
deliver his message that I had not even had time to
digest properly on the flight ... I was really scared at
first, but now I find you to be quite all right, at least after
a few glasses of wine and beer.” (P. Alaupovic, 1991;
personal communication). I suppose that we may de-
note this brief footnote in the history of lipoproteins as
the Concordance of Graz.

Note 9

My first meeting with Thannhauser was in 1949 at the
instigation of my prospective mother-in-law, who was
visiting from the Hague. I shall never forget when, over
the dinner table at his home, the professor was asked by
Mevrouw Eekhof-Bos what it meant that I intended to
pursue a career in biomedical research. “It means,
Madame, that your daughter must learn a gainful
occupation!” replied Thannhauser.

Note 10

All our diagnostic samples were obtained after an
overnight fast, with the patients retaining a more or less
stable weight. Samples from NIH staff volunteers and
several younger cohorts were used to set 90% cutoff
points above and below the means in 10-year age
increments as normal standards. All values for VLDL,
LDL, and HDL were expressed in terms of the content
of cholesterol.

We also arbitrarily eliminated all patients with fasting
triglyceride levels >200 mg/dL, as there was little
normal data available at the time. When, 15 years later,
the Lipid Research Clinics compiled their normal stan-
dards for thousands of patients, only our triglyceride
levels deviated significantly from the means of the new
standards.

Occasional technical improvements have allowed a
reduction in the complexity and tedium of the original
analytical systems. One of the most useful was the
discovery by Bernfield®! and Burstein, the latter at
the Centre Transfusion in Paris, and with whom I
visited for firsthand observation of his work. It ap-
peared that polyelectrolytes could be used to precipi-
tate LDL and most of the triglyceride-rich lipopro-
teins, reducing the routine analyses to one overnight
ultracentrifugation of plasma at its own density. The
tubes were sliced, and the cholesterol in the superna-
tant was VLDL. LDL was precipitated from the
IH-12 Supplement II Circulation Vol 87, No 4 April 1993

infranatant fraction, leaving the HDL cholesterol to
be determined. LDL was calculated as the difference
between plasma C—(VLDL-C+HDL-C).

Note 11

There were 23 patients with tuberous xanthomas
included in the Donner data, and two were siblings,
proving the familial occurrence of the syndrome.?5 The
Gofman reports, as did our own, applied reproducible
chemical analyses to descriptions of skin lesions that
were subjective, historically old in origin, changing over
time, and of various etiology.

The vitilgoidea tuberosa of the mid 19th century!™ was
due to obstructive biliary disease. At the end of the
century, Pollitzer,!0.102 a renowned New York City der-
matologist, categorically distinguished xanthoma tubero-
sum from xanthoma multiplex, and, then, in 1912, pub-
lished several papers on xanthoma tuberosum multiplex.

We found that tuberous xanthoma lesions over the
skin of the elbows and other surfaces were not unique to
type III but occurred in patients with familial hypercho-
lesterolemia. Far more characteristic of type III are the
yellow-orange deposits of lipid in the creases of the palms
and other limbs, which we called planar xanthomas.

Later, following the advice of the Dutch dermatolo-
gist Polano, we used the term “xanthoma striatum
palmaris.”°3 Using a classification scheme that was
different from and not compatible with ours, they also
found these lesions in several phenotypes.'™ By what-
ever name, however, these lesions melted away quickly
when the patients were treated by diet or administration
of clofibrate.

Note 12

For several years, Joseph Garland of the New England
Journal of Medicine had been requesting that we write a
review of our work. The rapid development of the field
and the priority given to the MBID foreclosed a re-
sponse for many months, but something had to be done.
In 1966, I went as usual to the Atlantic City Federation
of American Societies for Experimental Biology meet-
ings although this time, I remained in a motel room,
filling sheets of foolscap paper and making frequent
calls to Levy and Lees for information to fill in the gaps.
Garland’s acceptance note of the product, with a line
stating “Your ms has passed from loving hand to loving
hand” was the nicest comment I had ever received from
an editor.

The New England Journal of Medicine had, at that
time, an agreement with Little, Brown & Co. that
offered authors an opportunity to reproduce the longer
reviews as a monograph, provided that no reprints were
issued. I requested that Robert Berliner allow us to
order 10,000 reprints instead. He agreed, and 95% were
gone within a year. In Garfield’s “citation classics” for
the years 1961-1975, I was flabbergasted to note that, no
doubt as a result of this publication, I had emerged as
the “most cited physiologist” during that period.!!2

Note 13

The Framingham Study added LDL and HDL mea-
surements to its annual analyses in the late 1960s. The
key question at this time was whether the diet of the
population should be swayed toward more unsaturated
fats. In 1969, the first such attempt to compare polyun-

saturated with ordinary fat in a diet was reported in a
Veterans Administration domiciliary population.14

Before this report, the council of the NHI had created
an executive committee on diet—heart disease, which
recommended that a trial of diet in free-living popula-
tions should be undertaken in America.!!5 I was partic-
ularly involved because during 1966-1968, I had as-
sumed the directorship of the NHI while still running
my laboratory, with the able help of Robert Levy. By
this time, Irvine Page was undertaking feasibility stud-
ies in 2,000 subjects as preparation for a massive trial of
perhaps 100,000 volunteers. Doubts as to the cost-
benefit of such an undertaking arose, and I convened a
diet-heart review panel under the chairmanship of
Edward H. Ahrens Jr. to reconsider the feasibility of the
full trial. The panel concluded that such a trial was
economically unwarranted.!!6 This, in turn, was consid-
ered by an NHLI Task Force on Arteriosclerosis, which
decided in 1971 that such field trials should also involve
other agents than diet alone to lower cholesterol.11” This
opened the way to large-scale drug trials of prevention
of coronary artery disease. During this time, the famed
NHLI biometrician, Jerome Cornfield, a man without
an advanced degree, was the most influential person on
the scene and, for the institute director, an invaluable
tutor of the art of clinical trials.1!8

Note 14

I left the directorship of the NHI to resume full-time
work at the laboratory in 1969, although I was then also
director of intramural research of the institute, succeed-
ing Robert Berliner. When Theodore Cooper asked me
to convene and chair the panel on hyperlipidemia to
explore the question of setting up the lipid clinics, I took
pains to invite critics as well as aficionados of the
phenotyping system. Among the former, I counted my
valued colleague Richard Havel. In addition to Havel,
the decision makers included Edwin Bierman, David
Blankenhorn, William Castelli, William Connor, Gerald
Cooper, Seymour Dayton, Howard Eder, Ivan Frantz,
Tony Gotto, DeWitt Goodman, William Friedewald,
Frederick Hatch, Peter Kuo, Robert S. Lees, Robert I.
Levy, Robert P. Noble, Isadore Rosenfeld, and Daniel
Steinberg. A questionnaire was issued to the members
in advance and written to afford opportunity for sharp
criticisms. We had no desire to include the NHLI
Molecular Disease Branch, but we were proud that it
was the basic model for the new network.

Note 15

Dr. Zdanek Fajfar, a Czechoslovakian cardiologist, as
the head of the cardiovascular desk, convened a meeting
consisting of J.L. Beaumont, a professor of medicine
from Paris, Lars Carlson, head of the King Gustave
Institute, Gerald Cooper of the Centers for Disease
Control (CDC), T.R. Strasser of the World Health
Organization, and myself. It was the humid season in
Geneva, which added to the heated discussions on the
validity and usefulness of the typing system. Beaumont,
then a dean of a medical school, was of the persuasion
that much of hyperlipoproteinemia had an immunolog-
ical basis. Carlson, the proper Swedish skeptic, was not
enthusiastic about lipoproteins. Cooper, who was the
head of the standards group at the CDC, was helpful in
smoothing tempers as we slowly achieved some accord.
A year or two after our affirmative report was issued, I
visited Carlson’s laboratories in Stockholm and was
pleased to be invited to the conference room where,
surrounded by piles of lipoprotein strips, everyone was
engaged in “reading the wash.”

Note 16

Havel and Kane used the term dysbetalipoprotein-
emia for this syndrome rather than type III. Today, the
general usage is to reserve the former term for the
apoprotein abnormality in the far more numerous mem-
bers of the families of the very few probands that
express the full type II] phenotype with hyperlipidemia
and its frequent accompaniments. The reason for the
expression of the abnormal phenotype in apparently
genotypically identical family members is a mystery not
yet penetrated.

Note 17

I am grateful for the collegiality and contributions of
the following who worked in molecular disease: Paul H.
Altrocchi, Gerd Assmann, Thomas P. Bersot, David W.
Bilheimer, Mariel Birnbaumer, Conrad Blum, Jan L.
Breslow, H. Bryan Brewer Jr. (now chief of the labora-
tory), Nancy Briggs, W. Virgil Brown, James Carter,
L.L. Davis, Steven J. Demosky, Schlomo Eisenberg,
Charles J. Glueck, Antonio M. Gotto, Heiner Greten,
Robert J. Heinen, Lloyd O. Henderson, Peter Herbert,
Anne Houser, Kathryn John, Antonio Jover, Ronald M.
Krauss, Peter O. Kwiterovich Jr., Terry Langer, John D.
LaRosa, Robert S. Lees, Robert I. Levy, Samuel E. Lux,
Robert Mahley, Betty H. Masket, Duncan L. McCol-
lester, Michael B. Mock, Joel Morganroth, Arne Nor-
man, Katsuto Ono, Nancy Priddy, Stephen H. Quar-
fordt, Allen Rider, Basil Rifkind, Rosemary Ronan, T.
Shiratori, Richard Shulman, Howard R. Sloan, Neil J.
Stone, Katherine Wehrly, Lee Witters, and Oscar
Young.

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