All the mental steps in this process unfolded in perhaps two seconds. Proving the hypothesis took three years.
4
Microscopic Studies; New York City, 1953
A favorable condition for productivity in research is variety of experience, both one's own experience and that which may be derived through observation of others who are at work on different problems. Especially is this important during the early years of education and discipline. Thus insight into diverse methods is acquired, as well as acquaintance with ways in which they are applied. As an investigator continues in his career, accident will present him with unpredicted opportunities for research, perhaps in quite new directions. The early knowledge of various ways of solving problems provides him promptly with readiness and versatility of attack.
Walter Cannon
Every specialist, whatever his profession, skill or business may be, can improve his performance by broadening his base.
Wilder Penfield
If I never had examined urine before, the hypothesis and the subsequent experiments would never have occurred. But I could proceed straightaway for two reasons. Back in the summer of 1946, while I was a secondyear medical student, I had routinely examined urine sediments. Always drawn to the out-of-doors, I had taken a job as a "camp doctor" in New Hampshire during summer vacation. The job proved more than I bargained for. The camp was soon overcome by a major epidemic of virus peneumonia, and many were very sick for one or two weeks. Some had a second recurrence of fever, and I placed them on sulfadiazine, following a fashion of the time, in order to avoid secondary bacterial infections. The problem was that excessive concentrations of the sulfa drug could form harmful crystals in the kidney. Therefore, I had to search the urine sediment for sulfa crystals with the microscope.
I worried over my patients as only a second-year medical student can-like a mother hen over chicks. Would they get a bacterial pneumonia? I already knew what that meant in personal terms. Would the sulfa drug I was giving them plug up their kidneys? Kindled by my worries that summer, the memory traces that would lead to my using a microscope were glowing and ready for use whenever the appropriate occasion arose.
The second reason it was easy to look at urine sediment had to do with the nature of my internship. I was fortunate to intern at the Boston City Hospital at a time when interns themselves performed all basic urine and blood studies. I was already doubly primed, as it were, to regard body fluids as a source of medical information, and I turned to this approach quite naturally. If I had been limited to the microscopic experience that students and interns receive today, I probably would never had started, let alone continued, this line of research.
In March of 1953 two brothers whom I will call Clausen were admitted as patients to our child neurology ward at Columbia. Dr. Charles Poser, then a neurology resident, presented them at a conference. They were normal at birth but at the age of a year and a half had each developed a progressive neurological disorder. Their parents first noticed a clumsiness in walking. Gradually they became blind, and their arms and legs became paralyzed. Eventually, they lost the ability to talk, think, and feed themselves. The disease seemed to be genetically determined because their older brother had died earlier in Cleveland of exactly the same illness. Autopsy reports from the Cleveland hospital showed that this first brother had died of metachromatic leukodystrophy (MLD).'
The stage was now set to test the hypothesis developed at the library table ten months before. I centrifuged a urine sample from each brother, poured off all the lighter liquid at the top, and transferred the heavier sediment to a glass slide. With mounting excitement, I next added a few drops of dilute toluidine blue dye, stirred the mixture with a wooden stick, and then covered it with a glass cover slip. Under the microscope, the slide from each brother did show clumps of metachromatic material scattered throughout the urine sediment. It was red to reddishpurple in color. Large strands of this metachromatic material were quite obvious, even when I looked at the slide with the unaided eye.
Sometime during my intellectual development I acquired a passion to do carefully controlled studies. So, at the same time that I prepared sediments from the two brothers, I also made similar slides from several other (control) persons. I included my own urine sediment and also sediments from patients with various neurological illness. A search soon revealed that all of these subjects had variable amounts of reddishpurple metachromatic material in the urine sediment. The material from the control patients looked essentially the same as did that from the two Clausen children. Clearly, this metachromatic material was of no diagnostic use. The hypothesis appeared to be of no value.
Still, I looked again at fresh urine sediments the next day. I searched the slides and drew pictures of what I encountered. Gradually, my attention focused on something first casually interesting, soon arresting.
Material of another color was present. This special material occurred only in sediments from the two brothers. I had been expecting something which was reddish or reddish-purple. What I saw, instead, were shades of golden-brown. Furthermore, there was a distinctive structure to this material: it was globular or granular (see figure 3). Sometimes several globules were stuck together, resembling a cluster of grapes. In some instances, golden-brown granular material could be seen inside the cytoplasm of a cell. The appearance of the cell indicated that it had become detached from the kidney and had then found its way into the urine. In other instances, the material formed a large cylindrical cast conforming to the rounded shape of the inside of the kidney tubule.
Trial and error soon showed that the urine had to be fresh for best results. Even then, the amount of material fluctuated from day to day. But the older brother, whose disease was more advanced, almost always excreted much more material. As the size of the control series grew from ten to fifty and included more patients with other disorders, it became evident that abundant material like this, especially that in kidney cells, was not to be found in patients with other diseases. The urine findings seemed specific for metachromatic leukodystrophy' This gave us the first medical laboratory technique for diagnosing MLD during life.
Research proliferates,'- and for a simple reason: as one question is answered, at least two new ones arise. The new questions soon arose. Unstained sediment showed nothing; the material itself was colorless and only took on the golden-brown shade when stained with the toluidine blue dye. Only then did it become metachromatic. But why did the material have this curious golden-brown color, particularly when the deposits described in the earlier article by Brain and Greenfield were shades of reddish-purple? These unanswered questions nagged me for almost two more years. The questions prompted other experiments, most of which yielded answers different from those sought.
A peripheral issue also remained to be settled. Both brothers had a yellow-orange color to their skin. Their blood plasma also had a yellowish-brown color. Why? It turned out that at home they had been fed large amounts of vegetables and other strained foods. Having ingested large quantities of yellow vegetable pigments (carotene) they had developed carotenemia. Concentrated carotene has a golden-brown color. Could the carotene in the blood somehow have been deposited in the kidney, then become decolorized and finally recolorized by toluidine blue? This all seemed unlikely. Still, I had to eliminate it as a possibility.
No human patients with carotenemia showed up to test this point. So I became a rat doctor for the first time. I fed rats huge amounts of carotene daily. They failed to show any golden-brown deposits in the kidney or in the urine resembling those found in MLD urine. Therefore, there was no reason to think the urine findings in human MLD were caused by the carotenemia.
The simpleminded rat studies seemed a diversion at the time. However, they were my first really personal demonstration of how animal experiments might help clarify the mechanisms involved in a human disease. The rat experiments also introduced me to the useful technique of making thin sections of frozen tissues that could be looked at under t
he microscope. To do this, I found I had to purchase a tank of carbon dioxide and scrounge old frozen section equipment from the anatomy department. I had no technician to do these things for me. As a result, I had the opportunity to learn the basic histological skills which were to become essential for work with lipids later on.
The six months' fellowship in neuropathology was soon over. My primary responsibility now was to care for patients on the wards and in the out-patient clinics. If I was going to continue my research, I had to add it on to an already hectic neurology residency. I had an even more practical problem. I badly needed a refrigerator, both to delay the fading of the golden-brown color on the slides and to keep the urine from spoiling. The refrigerator space in neuropathology was already spoken for. Besides, one could anticipate no great enthusiasm in neuropathology for a neurology resident who was working on his own. So I did most of my research after hours or during the evenings when I was on duty that night. I did the rest of the work during daylight hours in whatever inches of space I could beg away from the technicians in the crowded clinical laboratory. Naturally, these good women protested mildly when increasing volumes of urine started to flood the laboratory and clutter up their refrigerator. However, they seemed mollified by three things: I was enthusiastic about what I was doing and took the time to demonstrate the abnormal urine material to them; I also enclosed these urine bottles in brown paper bags to mask their prosaic contents; and finally, I demonstrated my own high regard for the aesthetic aspects of their refrigerator by choosing to keep my own luncheon sandwiches in it. Nevertheless, my status as a persona grata in the laboratory remained precarious for the next two years.
It is not easy to persuade harried nurses on busy wards to collect urine samples for research. Then, too, there was the matter of wishing to study children who appeared to have a hopeless disease. What practical good could come out of all the bother? Gradually, to my surprise, I found that it was really difficult to conduct clinical research in a busy hospital. Medically oriented people, even in university teaching centers, are not automatically receptive to new ideas in research. The investigator must endure subtle harassments, both major and minor, in order to prevail. But all of this became relatively easy because of my own growing enthusiasm for the project. Directly ahead lay the big intriguing question: What was the chemical nature of the metachromatic material?
A neurology resident acting in a vacuum could never have approached a problem of this dimension. What made the difference were the friends, collaborators, and colleagues who publish in the scientific literature. They helped generate ideas about how to pursue this research and provided all kinds of other assistance.
For example, I had one other important chance encounter in the library at the Neurological Institute of New York. While browsing there one day, I happened to meet Dr. Robert Katzman, a fellow resident. Bob had had some earlier training in neurochemistry before he started to specialize in neurology. I told him that my solubility studies showed the unknown material was some kind of a lipid, a fatty substance. In fact, I had just found that the material dissolved slightly in alcohol but not in ether. Bob made the helpful suggestion that I look up the writings of Dr. Jordi Folch-pi and his group of chemists at Harvard. These workers had recently been concentrating on lipids in the nervous system. Lipids, I recalled, were the distinctive constituents of the brain, making up half its total dry weight. There were more lipids in the nervous system than in any other tissue. In my reading, I found that the Harvard investigators could readily dissolve these lipids in a mixture of two parts of chloroform to one part of methanol (wood alcohol). Perhaps this solvent mixture would help free the metachromatic material from MLD tissues so that I could then identify it.
But, by then, my neurology residency in New York was almost over. Judy and I now had two young children to think about. I had to interrupt my studies. Where to go? What was the best way to combine teaching, research, and patient care responsibilities, yet still be able to feed, clothe, and house a family of four? I had seen California while in the Navy, and the vision of the West Coast was still a very attractive one. A new opportunity at Portland, Oregon, was particularly appealing. There, at the University of Oregon Medical School, Dr. Roy Swank had just opened up a new Division of Neurology. Roy was an energetic, friendly, open, and generous man who approached neurological diseases through biochemistry, and I was increasingly sympathetic to this point of view. After visits to, and correspondence with, various medical centers, I happily accepted a position at Oregon.
July, 1955, found us on the road, heading West, immensely excited and full of wonder and curiosity about what the future would bring. My starting salary as a full-time faculty member, seven years after graduating from medical school, would amount to the heady figure of $8,000.
5
Sulfated Lipids; Portland, Oregon, 1955
New medical observations are generally made by chance; if a patient with a hitherto unknown illness enters a hospital where a physician comes for consultation, it is surely by luck that the doctor encounters this patient.
Claude Bernard
The new job posed some stimulating challenges. When I arrived in Oregon, no MLD patients were known to live there. I had no freshfrozen or formalin-fixed autopsy material to study, and knew essentially nothing about the practical aspects of neurochemistry. I started with no laboratory space as such. My work bench was a cluttered corner of a borrowed laboratory space still used for demonstrations in physiology. Furthermore, to practice clinical medicine in Oregon, I first had to pass a certifying examination in the preclinical sciences. My knowledge of biochemistry was fragmentary, and l had to study a great deal of basic chemistry to pass the exam. This was painful but very important. I found that I could learn, and this gave me the minimal confidence I needed to approach a neurochemical problem that was really way beyond me.
On the positive side, the academic setting at the medical school was favorable. The biochemical approach to neurological disorders was exemplified both in Dr. Swank's work and in that of Dr. Jack Fellman, a helpful young neurochemist who had also joined us. Then, too, there was the intervention of chance.
Two months after we arrived in Portland, a boy was admitted to the medical school hospital. I will call him Warren Thompson. His symptoms were ominous. When he was five years old his parents noted a slight limp affecting his right leg. By age seven, walking was much impaired and climbing stairs was impossible. He became clumsy and developed thick speech. In brief, the pattern of his reflex changes (some were increased, some were decreased) was consistent with a disorder causing widespread breakdown of the myelin sheath (figures 1 and 2). The sheath is an insulating layer of lipid that normally surrounds each nerve fiber in the brain, the spinal cord, and the peripheral nerves. When it breaks down, the fiber stops transmitting its impulses. All neurological functions (motor, speech, vision, etc.) then gradually shut down. Because the Thompson child was older when his symptoms began, his history and examination differed somewhat from the Clausen brothers in New York. Still, it seemed possible that he might have the kind of MLD that starts later in childhood.
I stained his urine sediment. Again, I saw the distinctive goldenbrown material. Here was a new case of MLD and fresh lipid material with which to work. The trouble was that urine sediment is a potpourri. Knowing that this small amount of material existed was only the first step toward understanding the disease. If I ever wanted to identify this unknown lipid, my first job was to separate the minute amounts of it from the vast quantity of other material in the urine sediment.
After much trial and error, I discovered three things: (1) The mixture of chloroform and methanol I had read about in New York did indeed dissolve the metachromatic lipid. (2) When this mixture was exposed to water, it separated into three layers as had been described by Folch-pi. Lipids (fats) in the middle layer could be suctioned up with a pipette, placed on filter paper and then stained with toluidine blue. (3) In normal patients the lipids did not show a str
ong red metachromasia. However, lipids from the Thompson child with MLD always gave a strong red color.
Figure 2
The normal myelin sheath and three of its molecules, magnified 57 million times. The nerve fiber (axon) normally conducts nerve impulses down to innervate muscles or other nerve cells. Surrounding it is an insulating fatty coat, the myelin sheath.
The myelin sheath, like a jellyroll, is made up of many individual layers. Each layer is composed of molecules of lipids, protein and water. In this diagram, part of the molecular architecture of one layer is expanded and shown schematically.
The layer resembles a double-decker sandwich: the two lipid "fillings" are contained within three protein (P) layers. If you look carefully at each lipid filling, you will see that it contains two cholesterol molecules. (The gourd-like structures approximate the actual shape of a cholesterol molecule.) The two other molecules shown are either two sulfatide or two cerebroside molecules. Sulfatide molecules (also termed cerebroside sulfate) differ from cerebroside only in that they possess a sulfate group. This group is a locus of negative charge, indicated by a minus sign.
Chase, Chance, and Creativity Page 3
