Cerebrosides and sulfatides are the two most distinctive molecules of the myelin sheath. Normally, there are perhaps four cerebroside molecules for each sulfatide molecule. The sheath breaks down if it contains too many cerebrosides or too many sulfatide molecules. It crumbles as would a brick wall with too much sand and too little cement in the mortar. This degenerative process is termed a leukodystrophy.
Now the unknown lipids could be extracted out of MLD urine and concentrated. But what were they? On a trip back to ColumbiaPresbyterian Medical Center in New York, I looked up an old medical school classmate, Dr. Jerry Phillips. Bright and hard-working, Jerry had become a serious investigator in the lipid field. I mentioned to him that the unknown lipid was highly metachromatic, and that for this reason part of the molecule should carry a strong negative charge. Going by the analogy with other known molecules in biology, I favored a negatively charged sulfate group over a phosphate group. What did he know about sulfated lipids? This wasn't Jerry's metier, but he did recall a report in the Federation Proceedings some months back. I finally located the reference, a brief paper by Marjorie Lees' in Folch-pi's group at Harvard Medical School.
At this point, the good will of four other people played a crucial role in the story. First, there was another medical school classmate, Lowell Lapham. Lowell dug into his old pathology files and sent me a small portion of MLD kidney material to work with. This fragment, fixed in formaldehyde solution, had remained in Cleveland, left over from the autopsy of the first child in the Clausen family. The second person was Frank Witmer, who, as we shall soon see, added his invaluable expertise in infrared spectrophotometry. A third was Sigfried Thannhauser, who donated an authentic sample of a sulfated lipid in early 1957. Thereafter, his prototype compound served as our reference standard for the sulfatide molecule. The fourth person was Mrs. Thompson. It was she who firmly persuaded the rest of the family to consent to the postmortem examination when her son finally died of MLD.
I made thin frozen sections of the MLD kidney material sent by Lowell Lapham and stained them with toluidine blue. This was to be my first real glimpse of the disease I had worked on for so long. I awaited the result with mounting excitement. Looking into the microscope, I finally saw the MLD kidney tubules-packed with vast amounts of the abnormal lipids. The deposits stained a sensational mixture of red, redpurple and golden-brown colors, and I immediately recalled the stained glass windows of the Saint-Chapelle in Paris, which until that moment had been my ultimate visual experience. I have never forgotten the way this slide looked; I can still see it vividly in my mind's eye-an aesthetic delight!
If some symbolism is involved in this earlier work in MLD, surely it lies close to a profound appreciation of these colors. They are among my favorites, and working with them has always been deeply satisfying.
I've had favorite colors before, and the first I can clearly remember as a small child is the rich olive green color of a crayon. There is nothing subtle about how it became my favorite; it was the color of Palmolive shaving cream with which I watched my father lather up his beard each morning. Later on, reds and blues would echo throughout a series of other experiences. When I was about nine years old I received a chemistry set from my parents. The experiment I had the most fun with was the one in which a water clear solution of a base (like sodium hydroxide) is added to a clear alcoholic solution of phenolpthalein. The mixture-magically-turns a deep rich red (like "wine," the directions hinted). Then, in later years, I remember how impressed I was by the stained-glass windows in the churches in which I sang in the choir for many years.
Now, I was looking at the microscopic sections from MLD kidney, and they provided another satisfaction. I had long been puzzled by the fact that the material in the urine stained a golden-brown color. This was a new metachromatic color that hadn't been reported before. Why didn't the granular material in the urine stain the conventional red, or purple metachromatic colors? Now I could guess part of the answer from the kidney sections. They showed that the original material upstream in the tubules was indeed more reddish colored. But later on, and lower down in the tubules, when the material was poised to enter the urine, it turned golden-brown. Clearly, the lipids upstream had changed their staining characteristics toward golden-brown before they entered the urine. Extracted and spotted on filter paper, they turned red again.
By now, I was enthusiastic about my research, but it was difficult at first to persuade others that it was worth supporting. At the time, there was no clear precedent for looking at urine under the microscope to diagnose a disease of the nervous system. When my application for funds went in, it faced considerable resistance, and the validity of the urine sediment findings themselves was open to question. To document the request, I was asked to send a slide off to New York City showing the metachromatic granules. This I could not do at the time, because the metachromatic color was fugitive-it usually faded within a few hours after it was first developed. The application was accordingly turned down.
The portion of MLD kidney sent by Lowell Lapham was useful for another reason. It finally afforded me enough tissue for some vital chemical experiments. I had to find out whether the sulfatide isolation procedure (designed for brain) could also work when applied to kidney.
How could a neurologist be more interested in kidney than brain? Strategy dictated the priority I gave to kidney. I chose kidney because it showed the most striking visual contrast. A normal patient had no metachromatic material in kidney; an MLD kidney was loaded with deposits. I reasoned that it would be simpler to isolate and identify the MLD lipid from kidney. Once the lipid was known, we should be closer to the cause of the disease.
I then isolated sulfatide fractions from kidneys from the MLD patient and controls. It was exciting to find that only the MLD sample yielded a large amount of metachromatic lipid. It was soluble in various solvents like sulfatide; it stained metachromatically like sulfatide. But was it really sulfatide? Couldn't it also be some other lipid, which only resembled sulfatide in its solubilities and staining reactions? I tried for weeks to resolve this question with a technique called paper chromatography, for which A. J. P. Martin and R. L. Synge received the Nobel Prize in chemistry in 1952. This is a method of identifying an unknown compound, based on the fact that each molecule in a solution of appropriate liquids moves at a characteristic rate on a strip of filter paper. In my hands the method didn't work with sulfatide and the literature on chromatography didn't help either. I failed in every attempt.
By now, only a few precious milligrams remained of the unidentified lipid. Under these circumstances there remained only one good technique to identify the whole molecule. This method was infrared spectrophotometry.
Enter Frank Witmer. I still recall the moment he came through the door and ambled to the left into the laboratory. Some years before, he had been enrolled as a medical student, then switched to go into chemistry and business. His wife then developed multiple sclerosis and became a patient of Roy Swank. Roy told him of my work. Now, Frank wanted a sense of personal involvement in neurological research. In particular he wanted to help out in diseases which, like multiple sclerosis, also destroy the myelin sheath. Furthermore, he wished to donate his services. This was helpful, because, lacking funds, I was operating on a research budget of a few hundred dollars. I explained that I was studying such a disease, that I had isolated some material which seemed likely to be a sulfated lipid, but which had not yet been identified. He readily accepted the challenge.
When chemical substances are exposed to a beam of infrared light they let certain wave lengths pass and block others. Each chemical compound has a characteristic identity in its pattern of transmission and absorption-something like a chemical "fingerprint." This method, infrared spectrophotometry, was Frank's area of expertise.
To start to measure small amounts of the unknown lipid, I now had to learn a sensitive new technique of weighing less than a milligram of material. I could do so only because Monte Greer, in a neighborin
g laboratory, had (as an act of faith) let me use his delicate microbalance.
Within weeks our infrared studies were completed. They told us that the metachromatic lipid isolated in MLD was identical with the sulfatide of Thannhauser. It was basically a lipid which contained sugar (figure 3). To the sugar there was attached a sulfate group. Luckily, this sulfate group caused a major distortion of infrared light at a certain wave length. The more deflection there was at this wave length, the more lipid was present. We could now identify and measure how much sulfatide we had.
Our analyses showed a major increase of sulfatide in MLD (figure 4). MLD kidney had up to nine times more sulfatide than did normal kidneys, and even the devastated white matter from MLD brain had twice as much sulfatide as normal. We studied other diseases which destroy the myelin sheaths of white matter (multiple sclerosis, for example). Here, in contrast to MLD, sulfatide values were decreased much below normal. The fact that sulfatides were increased in MLD white matter was unique.
Because sulfatides were increased not only in MLD brain and kidney, but also in other tissues, we could conclude that MLD was a generalized sulfatide deposition disease. Another term for this is sulfatide lipidosis (figure 5). Somehow, the excess of this lipid caused the nervous system to break down (figure 4).
In this respect, MLD resembled other disorders which had been described for years-the so-called lipid storage diseases. One useful feature of these deposition diseases is that the stored molecules clump together; under the microscope the chemical quarry is in full sight when the tissue section is properly stained. I had heard about these illnesses in medical school, but, like all medical students, I found it hard to remember which molecule was increased in which disease. It would have been completely impossible for me to believe, while still a medical student, that ten years later I would help identify the lipid in another storage disease, and my teachers would have been equally incredulous.
Up to this point, the search itself had provided a steadily accelerating excitement. But, now that the sulfatide proof was in, I reached a stage of intellectual exhilaration. I remember vividly the hours when the sulfatide discovery made its full impact on me. I lay awake that night. Images raced and tumbled through my mind-colors, people, places, contingencies. Foremost among my thoughts were two I can recall even now. To a biologist, one of these thoughts was deeply gratifying. It seemed as though I would probably be worth my grain of salt on some eternal time scale. Our work had added one fact to the mass of information in the universe. The other thought served to counterbalance any possible sense of smugness. It was the nagging awareness that the hard central question still lay ahead: what kind of metabolic error caused the increase in sulfated lipids in MLD?
Figure 3
The granular body and its chemical background. Under the microscope, the granular body of MLD looks something like a raspberry or cluster of grapes. It is composed of many molecules of sulfatide (cerebroside-sulfate). Normally, an enzyme (sulfatase A: cerebroside sulfatase) splits the sulfate group from the sulfatide molecule. If the enzyme is deficient, as in MLD, deposits of cerebroside-sulfate build up. The raised negative sign ( ) in , OS-O- indicates that the sulfate group is a locus of negative charge. The sulfate group is hooked to galactose; this sugar is linked to sphingosine, which in turn is attached to a fatty acid.
Figure 4
A normal nerve fiber and the way it is affected in metachromatic leukodystrophy. The normal nerve fiber shown at the top has a layer of fatty insulation, myelin (M), surrounding its axon. This layer is formed by a sheath cell (S.C.) which envelops it.
In metachromatic leukodystrophy, sheath cells and myelin itself develop abnormally high levels of sulfatide. The myelin sheaths and some of their axons then break down. The nerve fiber no longer conducts impulses. The sulfated lipid (sulfatide) accumulates in granular deposits, termed granular bodies, inside large scavenger cells (phagocytes). Granular deposits of the lipid also build up in sheath cells.
Figure 5
Sequences in the metachromatic leukodystrophy story
6
Molecules and Meanderings, 1957
Anybody who looks back over an experimental development which.. .. has continued for many years, can hardly fail to notice that it has pursued an exceedingly wobbly course. If the surveyer is himself an experimenter, he will know that the recorded wanderings are fewer and less extensive than those which actually occurred.
Frederick Bartlett
My report that sulfatides were increased in MLD created little stir when it was first submitted in 1957. At the time, there was no good precedent for thinking that a primary disease of the myelin sheath was caused by an increase in one of its molecules, and the program committee of the American Neurological Association did not accept the paper for presentation. But even in 1957, it was reasonable to postulate that an enzyme deficiency caused the increase in sulfatides. By then, separate enzyme deficiencies were increasingly being pinpointed as the cause of genetically determined diseases.'
Let us briefly review what an enzyme is in order to appreciate what happens when an enzyme is deficient. We may think of an enzyme as a protein molecule which greatly speeds up a chemical reaction. Were it not for the hustling, catalytic action of many enzymes, each orchestrated beautifully with the others, our body's vital metabolic reactions would come to an abrupt halt. The reason is that some toxic compounds would reach damagingly high levels, while the supply of other essential molecules would dwindle to harmfully low levels.
What kind of an enzyme might be deficient in MLD? There were two clues. One was the presence of the sulfate group itself-the distinctive feature of the sulfatide molecule (see figure 3). The other was a report I had read indicating that a phosphatasc' enzyme deficiency existed in quite a different disease. I wondered, therefore, if MLD might be an analogous situation-a disorder caused, in this instance, by a sulfatase enzyme deficiency.
I knew nothing about sulfatases. However, I soon read everything available in the library. A sufatase, I found, was an enzyme that helped split off sulfate (SO4) groups (see figure 3). The function of the enzyme, therefore, was to keep the number of sulfated molecules down to an appropriate biological level. Different sulfatases were measured by using special chemical reagents. Each reagent had a sulfate group linked to it, and the point of attachment served in a sense as the "target" on which the enzyme acted. When the sulfatase split off the sulfate group, the reagent turned a new color. By measuring the amount of this new color, one knew how much sulfatase activity was present.
Formidable technical problems remained. What sulfatase method should I use, and which tissue should be studied? The answers are simple now, but getting them at that time took almost four years. In the interim, there was considerable meandering around. To illustrate, one day I was browsing in the library looking for information about metachromatic compounds and various storage diseases. I happened across a report by Lahut Uzman and took it back to my desk in the laboratory. Uzman had been studying Hurler's disease, a separate disorder in which the patients had both mental and bony abnormalities.' His report happened to mention that the white blood cells of these patients contained abnormal deposits of stored material. This was new to me. The article alerted me to the possibility that storage products might accumulate in readily accessible cells, such as circulating white blood cells. Accessibility is an important consideration for the researcher. A surgical operation to sample a piece of the human central or peripheral nervous system is troublesome for the patient and the physician. It is always preferable to diagnose a disease by studying cells that are easily obtained, as are white cells in a drop of blood.
While reading about white cells in Uzman's article, the idea struck me to study white blood cells in MLD. Perhaps they too contained metachromatic deposits. If they did, then it might be practical to test these cells for sulfatase activity. It took perhaps four seconds for all the visual steps in this hypothesis to unfold together with the methods for testing it out. I reme
mber being swept up in a moment of intense heightened awareness. The effect was something like having the volume turned up on your television set-yet with. a corresponding reduction in static-while at the same time perceiving the sounds of a deeper bass, a higher treble, and an image with enhanced colors in sharper focus. Even now, writing these lines, I retain a clear visual image of the scene at the desk where these ideas burst forth. This time, in a kind of mental double vision, I am both a participant in the process and a spectator hovering over a point about five feet up and ten feet directly to the rear.
There was a precedent for my looking at white blood cells (just as there had been earlier for examining urine sediment). Eleven years before, in medical school, I first became interested in blood cells and collected boxes full of slides showing the typical changes in white blood cells in different diseases. I had long viewed the making, staining, and examining of a blood smear as a natural extension of the clinical method of diagnosis.
By this time we had discovered two more Oregon patients with MLD-a brother and sister in the McLean family. Their symptoms began when they were about a year old. [ promptly made a blood smear on each and stained it myself with a standard technique. The results were startling; their white blood cells were stuffed with many abnormal granules!
I worry whenever a hypothesis is too quickly confirmed, and immediately suspect that something must be amiss. This lingering anxiety helped spark the usual long period that followed of examining control blood smears from many other sick children the same age. Only after looking at these controls could I conclude that the abnormal white cell granules were confined to just these two children with MLD. In fact, even the other MLD patients did not have the abnormalities. This ambiguity was puzzling, but at least now [ had some accessible cells that could be tested for a sulfatase deficiency. The question was, how to do so?
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