This paper shows that the linkage between basic science and clinical research has characterized the field of sulfur amino acid metabolism since 1810, when Wollaston isolated cystine from a human bladder stone. The nature and consequences of this relationship are discussed.
If Medline citations reveal the pace and intensity of interest in specific areas of scientific inquiry, then Figure 1 demonstrates the explosion of interest in homocysteine during the last 3 decades. The growth curve has not been uniform. In the period from 1966 to 1981, there was a slow but continuous increase from 36 to 87 citations each year. Between 1981 and 1982, there was an increase of 33%, following which there was a plateau at approximately 120 citations per year until 1991. From then to the present time (1998), an exponential increase of almost 300%--a veritable explosion of interest-has occurred.
This extraordinary growth merits an examination and an explanation. The Second International Conference on Homocysteine Metabolism provided an appropriate forum to explore both the substance and significance of this chronology. During the conference, the status of research in our field was assessed and directions for future investigations were projected. It is necessary to also address the history of the evolution of knowledge of homocysteine metabolism and how patterns, established in earlier years, continue to inform present efforts.
What combination of circumstances might account for the growth and development of our field? What has been the chronology of scientific discovery, the evolving stages of understanding, the impact of technology, and the relationship between scientific inquiry and clinical observations? This paper addresses the historic foundations of research in our field from 1810, when Wollaston discovered cystine, to 1980, by which time the intellectual foundations for the "homocysteine explosion" had been set down. Further exploration ofthe more recent history is a subject of current investigation.
The history, as we shall see, is complex and multifaceted. Perhaps its most notable features have centered on a remarkable synergy between the basic and clinical sciences, the importance of technology, and the evolving nature of collaborations that have enabled progress. The first will be the major theme. In fact, the history is replete with intellectual transfers in both directions between basic science laboratories and clinical research units. There are examples of clinical observations that have catalyzed major advancements in fundamental understanding. The converse occurs with equal frequency, and there are examples of the testing and validation of preclinical findings by studies of patients with rare inborn errors of metabolism. It is these types of relationships that will be explored.
The examples given unfortunately will not include all of the significant contributions of many individuals and laboratories. In truth, our history documents the change of pattern from work by a small number of prominent investigators, located primarily in North America, to our current situation-a truly collaborative and international effort.
Collecting the Pieces (1810-1930)
In 1810, when he isolated "cystic oxide" from two human bladder stones, Wollaston initiated the study of mammalian sulfur metabolism.1 In retrospect, the patient had cystinuria and this was the first example of a common and recurrent theme-the interaction of clinical observation and basic science. Wollaston's misnomer lasted until the work of Berzelius (1833) and Thaulow (1838) established the correct elemental formula and the name "cystine." Morner first isolated cystine from animal protein in 1899, and Neuberg and Friedmann, working independently, defined the chemical structure in 1902-1903.1
Previous histories trace the beginnings of the study of transmethylation to this same period.2 The essential first steps were the isolation and characterization of the relevant compounds. In 1835, Chevreul reported his studies of muscle creatine, and 20 years later Dessaignes demonstrated its methyl group. Strecker isolated choline from lecithin in 1849.3 In 1884, Wilhelm His Jr. fed pyridine to dogs and isolated N-methylpyridine from the urine. Although the analytical chemistry was excellent, we honor His for his insight when he emphasized the need to demonstrate both the origin of the methyl group as well as the means for its translocation to the pyridine.4 One decade later, Hofmeister defined what we now term "transmethylation," in the context of his studies of the metabolism of metallic substances.4
Several compounds are notable for their absence from this history; they arrived substantially later. Mueller isolated a new sulfur-containing amino acid from protein hydrolysates in 1922. In 1928, Banger and Coyne used a total synthesis to prove the structure of this compound, which they named "methionine."5 The isolation, identification, and synthesis of homocystine, homocysteine, and cystathionine are products of even more recent research and will be dealt with in a subsequent section. It is important, however, to note that ignorance of methionine and these other derivatives limited progress. The generally accepted position was that cystine was essential for mammalian nutrition and could be the sole source of dietary organic sulfur.6
The discovery of insulin by Banting and Best, in Toronto in 1921, was the catalyst for the remarkable discoveries of the next 40 years. Not only did it establish biochemistry (or physiologic chemistry as it was then termed) as a respectable and central discipline, but also it provided the specific focus for the development of studies in sulfur metabolism. In this regard, two questions were foremost. The first was the chemical nature of the sulfur in the insulin molecule. The second was the explanation for the observation that insulin treatment reduced the hyperglycemia, but only prolonged the lives of pancreatectomized dogs, which died within weeks with fatty livers.
If Toronto was the origin, a unique series of events focused much of the excitement at the University of Illinois, which had recruited two graduates of the pioneer program in physiologic chemistry that Chittenden had implemented at Yale. Both Howard Lewis and William Rose had been students in that department that included Lafayette Mendel and Thomas Osborne as faculty members. Working together, Mendel and Osborne had begun investigations of the dietary requirements for proteins and amino acids. Indeed, they had authored the paper stating the essentiality of cystine.6 Despite the relevance of this research to his own future efforts, Rose noted that he had little awareness of their work during his time in New Haven. Because all of the studies were carried out at an off campus location, the graduate students, who were not involved, knew little until the publication of the findings.7 Nevertheless, it seems apparent that the Yale experience informed the interests of both Lewis and Rose.
In 1921 at Illinois, Lewis taught the introductory course in biochemistry. More than 30 years later, Vincent du Vigneaud, a student in that class, noted in the preface to his autobiography, "Can it be an accident when one develops an interest in chemistry and metabolism of sulfur compounds if one has had such an enthusiastic and inspirational teacher as Lewis in one's first course?"2 Later in 1921, Lewis accepted the chairmanship at the University of Michigan, a position he held for almost 25 years. During that time he continued his studies of sulfur amino acid metabolism and became very interested in cystinuria, the first inborn error in humans known to involve this pathway.8
Du Vigneaud also recalls in the same preface a 1923 lecture that Rose gave shortly after his visit with Banting and Best in Toronto. He recounts "... the thrill of listening to him and the curiosity that was aroused in me as to the chemical nature of this compound that could bring about the miracles that he described."2 Both Lewis and Rose reappear in our history, but du Vigneaud dominates the narrative.
On the Trail with Du Vigneaud (1930-1960)
After leaving Illinois in 1924, du Vigneaud moved through several positions and fellowships. The chemistry of insulin was his focus. In 1927, he earned his Ph.D. from Rochester under the supervision of Professor J.R. Murlin, who had prepared active insulin extracts prior to the first announcement from Bunting and Best. Subsequently, du Vigneaud had fellowships with Abel (Johns Hopkins) and Barger (Edinburgh). He returned to Illinois for a short period before becoming chairman of the Department of Biochemistry at George Washington University in 1932. In 1938, he assumed the same position at the Cornell University Medical School in New York City.2,9
It is interesting that du Vigneaud's time in Edinburgh appears to coincide with Barger's work on the structure of methionine because one of his first problems was that cystine did not seem to account for the entire sulfur of insulin. This led him to test the chemical reactivity of other sulfur compounds-including the newly discovered methionine. Treatment of methionine with concentrated acid yielded homocystine, which Butz and du Vigneaud described in 1932.10 Having synthesized homocysteine and homocystine, du Vigneaud began a series of classical nutritional studies designed to test the nutritional roles of methionine, cyst(e)ine, and homocyst(e)ine. With the availability of methionine, Jackson and Block already had corrected the error of their mentor, Mendel, and had shown that methionine, not cystine, was the essential amino acid.11 Du Vigneaud et al. now demonstrated that homocystine could replace cystine.12 Other investigators joined the chase. Lewis, now at Michigan, along with Virtue, showed that homocystine might appear in the urine of a patient fed methionine and offered the possibility of a metabolic sequence that led by oxidative demethylation from methionine to homocysteine.13 Brand and his coworkers found that the administration of either methionine or homocysteine to patients with cystinuria resulted in the increased urinary excretion of cystine.14
Two significant findings occurred in 1936-1937. Rose created diets based on purified amino acids and not on extracted proteins. With these, investigators could avoid the limitation of the Osborne-Mendel formulation that employed purified casein as the protein source and thus contained significant methionine, even after exclusion of other sources of the amino acid. Using this tool, Rose and his coworkers proved that methionine was both essential and sufficient.15 Furthermore, cystine could replace part of the methionine requirement the "methionine-sparing effect of cystine."16 Concurrently, Brand and his colleagues suggested that cystathionine was an intermediate in the conversion of methionine to cysteine.17 The sum total of these studies was the conceptualization of a pathway from methionine through homocysteine and cystathionine to cysteine. In 1939, Tarver and Schmidt, using S^sup 35^, demonstrated the conversion in rats of the sulfur atom of methionine to that of cysteine.18
The existence of "transsulfuration" was unequivocal. It remained for du Vigneaud to tidy up some of the loose ends. The synthesis of cystathionine in 1941 provided both a nutrient for feeding experiments19 and a substrate for the in vitro demonstration of cystathionase, the enzyme that cleaves cystathionine to yield cysteine and alpha-ketobutyrate.20,21 Simultaneously, his laboratory showed that rat liver slices could catalyze the synthesis of cystathionine from homocysteine and serine.22 During the period from 1950 to 1965, much of the further work on both enzymes took place in the laboratory of David Greenberg at the University of California at San Francisco.23,24
An additional facet was the extension of the observations to species other than the rat. In 1947, Tarver and Schmidt repeated their S^sup 35^ studies in dogs with the same finding.25 Finally, du Vigneaud, in collaboration with clinicians at the New York Hospital, showed that when fed to a patient with cystinuria, both S^sup 35^-methionine and S^sup 35^-cystathionine were converted to S^sup 35^-cystine.2,26
Having established methionine as a metabolic precursor of homocysteine, in the late 1930s, du Vigneaud and others turned to the reverse relationship. Could homocysteine replace methionine in the diet? If so, was the mechanism the methylation of homocysteine to form methionine? Clearly, Rose's amino acid-based diet, which allowed the total elimination of methionine, was the essential starting point. In separate experiments, Brand27 as well as Rose (and Rice)28 found that homocystine could replace methionine, whereas du Vigneaud et al.29 found that it could not. The diets used in the three labs, however, differed in their vitamin formulations. Brand used dried yeast plus a milk concentrate, Rose and Rice employed a rice bran preparation (tikitiki) together with the milk concentrate, and du Vigneaud et al. used the known crystalline vitamins and ryzamin B. In a remarkable demonstration of cooperation, Rose and du Vigneaud exchanged diets in order to prove that the results derived from the nutrition and not the specific laboratory environment.
Growth failure was associated with fatty livers. Remembering the work of Best et al.30 that had defined "lipotropic" substances, du Vigneaud et al. tested the effect of choline. Addition of this compound, or of betaine, made the homocystine diets equivalent to the methionine diets in both promotion of weight gain and prevention of hepatic steatosis.31 In turn, this observation provoked the hypothesis that the transfer of a methyl group from choline or betaine allowed the conversion of homocysteine to methionine. The converse observation, that adequate dietary methionine made choline unnecessary, suggested that one fate of the methyl group of methionine was the incorporation into choline.
Taken together, these nutritional experiments provided the basis for the concept of transmethylation-the process of the direct transfer of methyl groups between compounds. In 1949, in a series of elegant studies that once again proved his extraordinary talents as an analytic and synthetic organic chemist, du Vigneaud, along with Rachele, demonstrated this bidirectional movement of the intact methyl group between methionine and choline.32 The need for the oxidation of choline to betaine prior to the donation of its methyl group emerged from the combined work of Dubnoff,33 Borsook and Dubnoff,34 and Muntz 35 The final step occurred in the mid-1950s with the isolation of the enzyme betaine-homocysteine methyltransferase by Ericson and colleagues.36
The emphasis on lipotropes had obscured another possibility: that some other component of the vitamin supplement had provided an additional source of methyl groups. Indeed, this had been implicit in du Vigneaud's observation that a small minority of his rats fed his homocystine diet resumed growth after a variable period of stunting. In the period from 1940 to 1945, Bennett, Toennies, and Medes pursued this lead. They found that sulfa antibiotics, when added to the diet, prevented the spontaneous resumption of growth. Conversely, a crude liver extract that contained little methionine or choline promoted growth in homocystine-fed animals. The investigators concluded that "there may be vitamin factors, of either dietary or intestinal origin, the presence of which may enable the animal to compensate for the absence of dietary methyl donors ..."37 Following isolation of both folic acid38 in 1943 and vitamin B^sub 12^ by several groups39-41 in 1948, Bennett proved that these two vitamins, when added to homocystine, could replace methionine in the diets of rats.42 In turn, this led directly to the concept of the de novo synthesis of methyl groups and ultimately, in the early 1960s, to the identification of the central enzyme: methyltetrahydrofolate-homocysteine methyltransferase (methionine synthase).43,44
Knowledge of this reaction provided the basis for understanding the relationship between folic acid and cobalamin, a question that had been stimulated by the similarity of the clinical states that result from deficiency of either vitamin. In 1962, two groups, Noronha and Silverman45 and Herbert and Zalusky,46 conceptualized the "folate trap," a hypothesis that could explain how cobalamin deficiency resulted in functional folate insufficiency.
Although du Vigneaud had proven the transfer of the methyl group of methionine to creatine (or creatinine), the mechanism for this process remained obscure. Borsook and Dubnoff47 provided significant insight with their finding that rat liver preparations could synthesize creatine from methionine and guanidoacetate (now termed guanidinoacetate), either under aerobic conditions or in an anaerobic system if ATP was present. In 1951, Cantoni resolved the problem when he showed that the necessary first step was the synthesis of S-adenosylmethionine (AdoMet),48,49 which was the essential methyl donor in numerous reactions, each catalyzed by a specific enzyme. Subsequently, Cantoni and colleagues studied methionine adenosyltransferase, the enzyme that forms AdoMet;50 S-adenosylhomocysteine (AdoHcy), the product following methyl transfer; and adenosylhomocysteinase, the enzyme that cleaves AdoHcy.51
The ready availability of AdoMet, derived from its synthesis by yeast grown in a medium rich in methionine, facilitated world: in many labs. Important studies identified the numerous AdoMet-dependent methyltransferases. Of particular importance were Axelrod's studies of the methylation of neurotransmitters52 and the investigations by Bremer and Greenberg of the conversion of phosphatidylethanolamine to phosphatidylcholine.53 More recent research focuses on the role of methylation in the regulation of DNA expression.
Cantoni had predicted that AdoMet would be able to donate any of the three moieties attached to the sulfonium center.50 In 1958, Tabor's group54 showed that the propylamine chain, remaining after the decarboxylation of AdoMet, was transferred sequentially to putrescine in order to form spermidine and to spermidine to form spermine in bacterial systems. Williams-Ashman and Raina demonstrated similar enzymatic reactions in mammals.54 (It remains unproven that the transfer of adenosine from AdoMet occurs in mammalian systems.)
The achievements of du Vigneaud, Cantoni, and their contemporaries were enormous. By 1960, we knew virtually all of the metabolites and reactions involved in the metabolism of methionine and homocysteine. However, the texts of that time show a common pattern: the tendency to deal with transsulfuration and homocysteine methylation as two separate pathways.
Before concluding this portion, we should note that this remarkable productivity began with a misinterpretation of the initial findings. In fact, cystine does account for all of the sulfur of insulin. Du Vigneaud alludes to this in his autobiography, on page 21: "It is apparent that the entire homocystine work leading to the studies of transulfuration (sic) and of transmethylation would not have taken place if we had been able to account for the sulfur of insulin at the beginning and if the absence of methionine in insulin had been known at that time. The paths which research may take are indeed curious."2
Homocystinuria and "Rarer Forms of Disease" (1962- )
Cystathionine Synthase Deficiency
If insulin was the first catalyst, then perhaps homocystinuria was the second. In 1962, Carson and Neill55 in Belfast and Gerritsen et al.56 in Wisconsin reported the occurrence of this apparent inborn error of metabolism. Independently, Barber and Spaeth in Philadelphia had identified another patient, a seven-year-old girl with mental retardation and subluxation of the ocular lenses, whom they referred to Leonard Laster at the National Institutes of Health (NIH) for further study. His section included James Finkelstein, who had completed his training in gastroenterology at Columbia and shared his interest in inborn errors of metabolism. Laster has described how he assembled a team that included Fil Irreverre, who was an expert analytical chemist, and Harvey Mudd, a physician who had worked with Cantoni before becoming chief of his own laboratory.57
The initial intellectual challenge was to identify a single defect that could explain the increased concentrations of both methionine and homocystine in urine and plasma. Other investigators had suggested impaired membrane transport; however, the NIH team favored an enzyme defect. Taken alone, neither the transsulfuration pathway nor the homocysteine methylation provided a basis for the observed abnormalities, but considered together they did. A defect in cystathionine synthase would lead to an increase in homocysteine that would be diverted to methionine synthesis via one or both of the remethylation reactions. Despite the development of sensitive assays for two of the relevant enzymes, preliminary studies indicated that only the liver was an adequate source. A percutaneous liver biopsy or a laparotomy would be necessary. After detailed discussions, the parents agreed to the former procedure. In order to minimize the risk, there was a week of rehearsal with the patient. Each day they progressed one step further. During the actual biopsy, tears rolled down her cheeks but the patient did not move, and the biopsy was uncomplicated and successful. The assay results confirmed the hypothesis of a defect in cystathionine synthase, and the integrated pathway, combining transsulfuration and remethylation (Figure 2), may appear for the first time in that paper.58 Subsequently, similar studies involving liver biopsies from the parents and from other families allowed for the definition of a recessive pattern of inheritance.59 Clearly, the courage and trust of these first families were the essential and unique contribution.
In the preface to his text, Victor McKusick quotes from a letter written in 1657 by William Harvey.60 "Nature is nowhere accustomed more openly to display her secret mysteries than in cases where she shows traces of her workings apart from the beaten path; nor is there any better way to advance the proper practice of medicine than to give our minds to the discovery of the usual law of Nature by careful investigation of cases of rarer forms of disease."
The definition of the discrete metabolic defect in cystathionine synthase provided the opportunity to test specific facets of methionine metabolism in human subjects. Furthermore, it was a model for future studies of patients with other genetically defined abnormalities in the pathway. Although many scientists participated independently or as his coworkers, there is no doubt that Harvey Mudd became the master of this experimental format. In this first instance, studies of patients with cystathionine synthase demonstrated that:
Cystathionine synthesis is essential for transsulfuration from methionine to cysteine.
The formation of homocysteine may exceed the capacity for cystathionine synthesis. Excess homocysteine is cleared in part by remethylation that may result in increases in both methionine and adenosylmethionine.
The adenosylhomocysteinase reaction is reversible in vivo. Excess homocysteine is associated with an increase in S-adenosylhomocysteine.
Most significantly, these results served to validate the concept of the integrated pathway encompassing both transsulfuration and homocysteine methylation.61
Thus the pattern of synergy between preclinical laboratory science and clinical investigations, already an established and recurrent theme in the history of this field, was expressed once more. Rather than provide a comprehensive review, this paper will offer only a few additional examples based on the author's personal experiences.
In 1965, Finkelstein, now at the Veterans Administration Hospital in Washington, DC, began studies of the nature of the regulatory control of the integrated pathway for methionine metabolism. Because regulation was implicit in the Womack and Rose finding of a methioninesparing effect of cystine,16 the experiments defined the metabolic consequence of this nutritional exchange in rats. Clearly, regulation could be achieved by changing the rate of cycling of methionine and/or the fraction of homocysteine diverted to cystathionine during each cycle.62 Both the tissue content of the relevant enzymes as well as their intrinsic kinetic properties provide the mechanism for regulation. Subsequently, the same conclusions were derived from both observations in isolated perfused rat livers63 and methyl balance studies in human subjects.64
The studies of patients with methionine adenosyltransferase (MAT) deficiency provide another example of this synergy. In 1973, studies of AdoMet formation by an extract from the liver of a patient with asymptomatic hypermethioninemia demonstrated enzyme kinetics that were nonlinear. The defect was far greater at higher concentrations of the substrate, methionine.65 The conclusion that the likely defect was impairment of allosteric adaptation to higher substrate concentrations proved to be wrong. However, Liau et al.66 cited those observations among the factors that prompted the studies that first demonstrated the isoenzymes of mammalian MAT. Now we know that an absence of the higher Km MAT I/III is the basis for this form of inherited hypermethioninemia. That some of these patients are asymptomatic tends to support the concept that the lower Km isoenzymes catalyze the synthesis of AdoMet sufficient for essential transmethylation reactions. These isoenzymes, however, are inadequate for the removal of excess methionine.
Subsequent studies with patients with MAT deficiency also confirmed two other observations that were based on animal experimentation. In 1971, Kutzbach and Stokstad made the important observation that AdoMet inhibited methylenetetrahydrofolate reductase and thus the availability of methyltetrahydrofolate for homocysteine methylation.67 Later it was shown that AdoMet activated cystathionine synthase68 but inhibited betaine-homocysteine methyltransferase.69 Linkage of these findings led to the proposal that AdoMet was a metabolic "switch," with high concentrations facilitating transsulfuration.70-72 This was supported when a metabolic balance study in a MAT deficient patient demonstrated a pattern of methionine "conservation" despite the obvious excess of this compound owing to impaired synthesis of AdoMet.73
In addition, Tangerman and his colleagues73 used these patients to demonstrate the likely occurrence of methionine transamination, but only at very high concentrations of that metabolite. This confirmed the previous animal experiments by Case and Benevenga.74
Impaired Homocysteine Methylation
The integrated pathway for methionine metabolism contained two reactions for the remethylation of homocysteine. Questions of the relative importance as well as the specific functions of these two methylases remained unresolved in the mid-1960s. Du Vigneaud emphasized the importance of preformed methyl groups and, therefore, of the betaine-homocysteine methyltransferase. His concept was that de novo synthesis of methyl groups took place primarily in animals fed methyl-deficient diets.2 Subsequent findings did not support this position. The betaine enzyme had a very limited tissue distribution in mammals, being restricted to liver (and to kidney is some species).72 Furthermore, the content of the enzyme increased with increasing dietary methionine.75 By contrast, the 5methyltetrahydrofolate-homocysteine methyltransferase (methionine synthase) was ubiquitous,72 and the hepatic content increased with dietary methionine restriction.75 Concurrent studies of a new form of homocystinuria resolved this issue.
An infant with homocystinuria but low levels of methionine died at the Massachusetts General Hospital. Af terward, Mudd, Levy, and their collaborators76 defined the biochemical lesion-impaired homocysteine methylation resulting from a defect in the synthesis of the methylcobalamin coenzyme essential for the methionine synthase reaction. Clearly the latter reaction was necessary to sustain the tissue concentration of methionine. Conversely, the betaine-homocysteine methyltransferase alone was inadequate to prevent homocysteine accumulation-at least in the absence of supplemental dietary betaine or choline. Subsequently, studies in patients with methylenetetrahydrofolate reductase deficiency, a third form of homocystinuria, confirmed the essential metabolic role of methionine synthase.77
The child with impaired cobalamin metabolism stimulated two additional significant fields of inquiry. Studies of additional patients with this impairment and others with a variety of related defects have enabled Rosenblatt and his colleagues to dissect and define the pathway for normal cobalamin metabolism,78 once again fulfilling the prophecy of William Harvey.
Khmer McCully catalyzed the second line of study. By 1965, it was well known that children with cystathionine synthase deficiency were at risk for serious, often fatal, thrombovascular events. The issue was whether the pathology related to the increase in homocystine or the increase in methionine. The answer would have therapeutic implications. Whereas some patients responded to pyridoxine, the nonresponders were treated with a lowmethionine, high-cystine diet, often with a cofactor to enhance homocysteine methylation. Trials employed choline, betaine, and folate. However, treatments that lowered homocysteine by synthesizing methionine might be harmful if methionine rather than homocysteine was the proximate toxin.61 McCully79 found that arteriosclerosis was common to both the one patient with impaired cobalamin metabolism as well as one case of cystathionine synthase deficiency in the files of the Massachusetts General Hospital, although thromboses were prominent only in the latter. Based on these two cases, both with increased homocysteine but different changes in methionine concentration, McCully argued that the former compound was the basis for the premature and extensive arteriosclerosis. Furthermore, he questioned whether arteriosclerosis in individuals free of known enzyme deficiencies might be related to the concentrations of homocysteine and homocysteine derivatives.79
Hyperhomocysteinemia, the Expanding Relevance (1975- )
In the years immediately following the description of homocystinuria owing to cystathionine synthase deficiency, investigators focused most attention on mental retardation. This was a common clinical finding and, given the frequency of the metabolic defect, it was suggested that homocystinuria owing to cystathionine synthase deficiency would join phenylketonuria as an inborn error commonly associated with mental retardation. The biochemistry was compelling. Cystathionine accumulated in the brains of normal mammals, which contain cystathionine synthase but lack cystathionase.72 Furthermore, the concentration of cystathionine increased as one ascended the phylogenetic tree.80 Thus it was reasonable to suggest that cystathionine was linked to cerebral function. The fact that impaired cystathionine synthesis resulted in mental retardation appeared confirmatory. Thomas Huxley pointed out "the tragedy of Science-the slaying of a beautiful hypothesis by an ugly fact." In this case, the ugly fact was that the original high frequency of mental retardation was due to an ascertainment bias-mentally retarded individuals were disproportionately represented in the populations screened initially. Indeed, the function of cystathionine in the nervous system remains unknown.
In any event, McCully's observation encouraged the evolving change in emphasis.79 More investigators addressed the question of the relationship between the chemical abnormalities and thrombovascular events. The inability to establish an animal model despite the employment of extraordinary doses of homocysteine forms unlikely to occur in the diet, however, impaired progress and required the development of both new in vitro systems and surrogate markers for vascular dysfunction in intact animals. These themes continue to the present time.
The need for the development of systems to separate and quantitate the relevant metabolites also impeded the clinical studies. Existing assays for tissue metabolites were both insensitive and labor intensive. Indeed the serum concentrations of homocysteine and its metabolites that we currently label "hyperhomocysteinemia" and consider to be abnormal were below the limits of detection by routine methodologies until the late 1980s. Nevertheless, in 1976, Wilcken and Wilcken reported the finding that has energized much of the work during the last 2 decades. They found evidence for abnormal methionine metabolism in patients with unexplained and premature coronary artery disease.81 Their method, the measurement of plasma cysteine-homocysteine-mixed disulfide after a methionine load, was used subsequently by several research units but was not adaptable to large surveys. An immediate question was whether this was the appropriate metabolite for study. In 1979, Kang and his coworkers showed that homocysteine bound to protein through disulfide bonds was the dominant species in the plasma of nonhomocystinuric subjects.82 The acid-soluble fraction, which contains homocystine, cysteine-homocysteine disulfide, and homocysteine, constituted a much smaller portion.83 A more difficult problem was the need for sensitive, specific, efficient, and affordable assay methods that measured the sum of all homocysteine forms (now termed "tHcy"). Ueland, Refsum, Stabler, Malinow, Andersson, and Allen, a list of authors that includes most of the major contributors, provide an excellent summary of the history of the development of several techniques that are now in use.84 With the availability of these methods, the studies of hyperhomocysteinemia accelerated in the middle and late 1980s, thus explaining the dramatic growth spurt illustrated in Figure 1.
Another question emerged from the Wilckens' observation:81 the need to identify and characterize the group of patients with abnormal methionine metabolism. An early thought was that they were the heterozygotes for cystathionine synthase deficiency.81,85 Studies of the natural history of the cardiovascular risk in obligatory heterozygotes and grandparents of affected children, however, did not support that possibility.86 In 1988, Kang and his coworkers87 described the occurrence of a thermolabile variant of methylenetetrahydrofolate reductase (MTHFR) and suggested that homozygotes for this allele might substantially contribute to the patients with hyperhomocysteinemia. The latter, owing to improved technology, became the marker for abnormal methionine metabolism.87 It became apparent, however, that neither the estimation of tissue concentrations of metabolites nor enzyme assays in readily available tissue and tissue culture could define the genetic groups within the population at risk. In turn, this failure encouraged the development of methods for the determination of the genotypes of individual subjects. The fact that the enzymes most studied are cystathionine synthase and MTHFR is a direct index of the impact of the unresolved clinical issue.
Neural Tube Defects
The story of the relationship between folic acid, homocysteine, and neural tube defects contains many similarities and analogies to that of thrombovascular dysfunction. However, the major observation linking folate nutrition and embryopathy was in 1965, and most of the subsequent work emerged after 1985. Thus it belongs more to our present than to our history. Nevertheless, it is appropriate to note the many major contributions by the University of Nijmegen. Professor T K.A.B. Eskes, who birthed and nurtured this multidisciplinary effort, retired as Professor of Obstetrics on April 24, 1998. His valedictory address captures the history of the studies of embryopaty.88
Lessons from the Past
Despite its brevity, this historical overview suggests the need for continued inquiry into three interesting and relevant areas: the determinants of the rate of growth of an area of research, the movement of investigators to a research area, and the characteristics of the synergy between laboratory and clinical research.
The Growth of Knowledge
Any evaluation of the rate of progress in the study of sulfur amino acid metabolism needs to be situated in the context of the evolution of the chemical and biologic sciences. For the most part, progress was orderly and moved through a sequence of phases as new information kept pace with new methodologies and technology. A chronological listing of the essential technical achievements is illustrative. This category includes the: (1) isolation and characterization of cystine, methionine, homocysteine, and cystathionine; (2) development of diets based on purified amino acids; (3) availability of radioisotopes for in viv(, studies; (4) establishment of techniques of protein chemistry necessary for the isolation and characterization of individual enzymes; (5) development of the methodology necessary for efficient and accurate measurement of relevant metabolites in biologic samples; and (6) advancement of molecular genetic techniques for the identification of genotypes. Clearly most technical and intellectual limitations are general, and, in fact, only the first item is unique to the study of sulfur amino acid metabolism.
This list can be used in the search for an explanation of the growth curve illustrated in Figure 1. Unfortunately, our data begin in 1966 and the first four technical achievements antedate that. It is hoped that future studies will track back to an earlier time and allow us to judge the impact of these first events. Despite these limitations, it is likely that the early, gradual growth from 1966 through 1980 reflects a response to the description of the homocystinurias with the attraction and recruitment of additional participants. The increase from 1980 to 1982 may relate to the work of the Wilckens, whose data indicated a broader relevance for abnormal metabolism 8' However, we must also include the impact of Kang et al.'s identification of the importance of protein binding of homocysteine because this provided a metabolite that was more easily estimated.82 This explanation finds some support in the fact that citations for "plasma homocysteine" represent a disproportionate percentage of the increase in total citations from 1980 to 1982. Inadequate technology impaired this extension of interest from a few individuals with rare genetic diseases to a larger, more general population. Full expression of that interest awaited the general availability of the appropriate analytic techniques and instrumentation. That development took place during the middle and late 1980s, and deployment was in progress by the end of the decade.84 This is apparent in the dramatic growth in total citations that began in 1991 (Figure 1 ). Once again, it is relevant to note that citations for "plasma homocysteine" increased from 23 to 167 annually during the period 1991-1998.
These observations indicate that the limits on the growth of a research area are likely to result as much from the need for new methodologies as from a need for new concepts.
Recruitment of New Investigators
The change in the number and distribution of active investigators is another index of the pattern and rate of growth. Whereas the early analytic work was performed in Europe, most of the activity from 1920 to 1960 was conducted in North America by a relatively small number of investigators. Furthermore, although there were contributions by individuals, participants tended to be members of a limited number of units or groups. Charles Best remained in Toronto, and there was a historical relationship between the programs at Yale, Illinois, and Michigan. Du Vigneaud, a product of Illinois, moved from George Washington University to Cornell University Medical School. The University of California at San Francisco/Berkeley also had a productive department, led by C.L.A. Schmidt, whose interest in biliary taurine, fostered by Allen O. Whipple, evolved into a more general concern with sulfur metabolism. Schmidt recruited Harold Tarver and David Greenberg, who has described the growth of the department.89 The Lakenau Hospital in Philadelphia housed M.A. Bennett, Cz Medes, and G Toennies. During the 1950s, a group centered on Giulio Cantoni formed at NIH.
This initial pattern differs markedly from our present situation, which is characterized by multiple participants, often in multidisciplinary groups from many countries, including Spain, Norway, Ireland, and the Netherlands. The processes that led to this dramatic change must interest us because this apparently self directed adaptation to a new area of interest reaffirms the principles of investigator-initiated research. To study this question, we must find out who is here, where they came from, and why they came. That consideration is in progress. This shorter history, however, provides some insight.
Both clinical medicine and animal husbandry stimulated a growth in the area of folic acid nutrition and metabolism. Stokstad describes the evolution of his own interest.38 The work of Bennett and her colleagues related this field to that of sulfur amino acid metabolism. Under specific nutritional conditions, folate and cobalamin together allowed homocystine to replace methionine in the diet.42 Subsequently, it was found that in most mammalian tissues homocysteine methylation used methyltetrahydrofolate rather than betaine and that folate was essential for normal methionine metabolism.72 Kutzbach and Stokstad discovered that the converse was also true; folate metabolism was regulated by the concentration of AdoMet as well as by the availability of homocysteine.67 Both the more complete definition of the "AdoMet switch"68-70 and its reaffirmation by Krebs et al.90 formalized the merger of the two fields-folate and methionine metabolism. As a consequence, not only basic scientists but also clinician investigators interested in hematology, gastroenterology, oncology, and obstetrics moved into this newly relevant area and were introduced to transmethylation and transsulfuration. Multidisciplinary units-combining clinical and laboratory investigations-were the logical outcome. These relationships will be discussed in the last section.
Clinical Relevance: Synergy or Stimulation
The relationships between research in basic science laboratories and investigations involving human subjects appear to be a dominant characteristic of this history. However, there are at least two important facets to such ties. In the first form, the clinical situation provides a research tool that generates new information or validates existing basic science findings. The second form becomes dominant when the importance of the relevant disease or dysfunction provides a catalyst for increasing interest and support, whereas the clinical studies may offer relatively little insight into fundamental normal or pathologic processes. Both represent a form of synergy, and both are present, but to varying degrees, in most of the examples in our history.
We began with cystinuria, which provides a relatively clear example of the intellectual synergy described by William Harvey. The chronology is clear. Wollaston isolated cystine from a patient with cystinuria. More than a century later, several investigators, most notably Brand, du Vigneaud, and Lewis, studied subjects with this transport defect as a means to elucidate the intermediary metabolism of sulfur amino acids-the urinary excretion of large amounts of cystine allowing relatively easy sampling of the tissue pool of that amino acid. Even more recent studies of this disease provided the basis for the definition of the membrane transport mechanism for cystine, lysine, arginine, and ornithine.
The effect of the discovery of insulin on studies of sulfur metabolism differed qualitatively from that outlined for cystinuria. The dramatic therapeutic success, the ability to treat a previously fatal disease, galvanized interest in the molecule. Insulin was a stimulus, whereas cystinuria was an experimental tool. The goal was to determine the nature and mechanism of action of the hormone. The intermediate steps toward these answers generated information that could be applied elsewhere. Du Vigneaud's quest for the chemical structure of insulin led to the synthesis of homocystine from the recently discovered methionine. Testing whether homocystine was a nutritional surrogate for cystine or methionine was irrelevant to the insulin study. Thus the concepts of transmethylation and transsulfuration evolved in parallel to the study of insulin chemistry, not as an integral part of that endeavor.
Studies of homocystinuria owing to cystathionine synthase deficiency, although containing both forms of the relationship between laboratory and clinical research, provided more of the experimental model. Some ofthe many exchanges of fundamental information in both directionsbetween laboratory and clinical research unit-have been noted. Although therapy for the individual patients was an important goal, the relative rarity of the disease would have limited both interest and support but for this unique opportunity to generate new information with potential relevance to mental retardation and thrombovascular disorders. This pattern was repeated with the studies of other genetically defined enzyme deficiencies: cystathioninuria owing to gamma-cystathionase deficiency, homocystinuria owing to methylenetetrahydrofolate reductase deficiency or impaired cobalamin metabolism, hypermethioninemia owing to impaired MAT 1/III, and sulfocysteinuria owing to sulfite oxidase deficiency.61
The transition to the current focus on hyperhomocysteinemia and its relationship to thrombovascular pathology moves the nature of the relationship back toward that of stimulus. The societal investment is more for an immediate remedy than a search for new knowledge. Indeed, we will be challenged to prove that additional science remains an essential prelude to the therapeutic goal. This task is complicated by the unfortunate abundance of public misinformation, if not overt propaganda, which heralds the completion of the story. Injudicious extrapolations from the studies of homocystinuria obscure the reality that we remain uncertain whether hyperhomocysteinemia causes, or merely marks, the presence of thrombovascular diseases. Consequently, we do not know whether reducing plasma concentrations of tHcy will affect the incidence of pathology in any or all of our patients. It is hoped that we will find the answers in the planned intervention trials or the development of new experimental models of the pathochemistry. However, our history should reassure us that even if we fail in the ultimate goal, we will generate significant information during our quest.
Acknowledgments. Mr. Robert Fendler, Medical Librarian at the Veterans Affairs Medical Center, facilitated our recovery of the relevant aged references. I am particularly grateful for his help in tracking down and obtaining the unpublished Greenberg manuscript. Ms. Diana Watson and Mr. John Martin added their expertise. Finally, the manuscript benefited substantially from Dr Harvey Mudd's review and critique.
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Dr. Finkelstein is Senior Clinician and Chief, Biochemistry Research Laboratory, Veterans Affairs Medical Center, and Professor of Medicine, George Washington University, Washington, DC 20422, USA. This essay is adapted from the Presidential Address to the Second International Conference on Homocysteine Metabolism, delivered on April 29, 1998, at Nijmegen, the Netherlands.
Copyright International Life Sciences Institute and Nutrition Foundation Jul 2000
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