Clinical Chemistry 47:12 2166 2178 (2001)

History

Early Blood Chemistry in Britain and France

Noel G. Coley
I review here key research in the early years of the field of blood chemistry. The review includes successes and limitations of animal chemistry in the critical period of the eighteenth and nineteenth centuries. Eighteenth century medical theories emphasized the primacy of body solids. Body fluids were governed by the tenets of humoral pathology. After Boerhaave sparked interest in the chemistry of the body fluids, a new humoralism developed. With the rise of animal chemistry in the eighteenth century, two complementary ideas came into play. The concept of vital force was introduced in 1774, and the chemical composition of animal matters, including the blood, began to be investigated. In the early nineteenth century, the development of new methods of analysis encouraged such chemical studies. Prominent chemists led the field, and physicians also became involved. Physiologists were often opposed to the chemical tradition, but Francois Magendie recognized the importance of chemistry in physiology. Liebig linked the formation and functions of the blood to general metabolism and so extended the scope of animal chemistry from 1842. About the same time, microscopic studies led to discoveries of the globular structure of the blood, and Magendies famous pupil, Claude Bernard, began the animal chemistry studies that led him to new discoveries in hematology. This review addresses discoveries, controversies, and errors that relate to the foundations of clinical chemistry and hematology and describes contributions of instrumental investigators.
2001 American Association for Clinical Chemistry

harboring diseases, their possible involvement in pathology was largely ignored, and any changes in them that might be observed were regarded as symptoms, rather than causes of disease. According to ancient humoral doctrine, body fluids, or humours, were maintained in an internal balance. Their relative quantities were considered important, and medical treatments aimed to restore the balance of these humours when it became disturbed by sickness. Thus, purging, vomiting, cupping, venesection, and leeches figured prominently in the armory of the eighteenth century physician. Bloodpreserver of life and vitalitywas the most important of all body fluids (1 ). Mystical properties were often attributed to blood, and some ascribed its lifepreserving powers to its fluidity. Those who held that body fluids, unlike organs, did not possess vitality opposed this notion but accepted that blood was different from other body fluids. The functions of organs such as the liver, lungs, digestive organs, and brain were thought to depend on vital spirits conveyed to them by the blood.

a vital force
From the 1770s, some physicians began to focus on animal chemistry (2 ), analyzing animal substances and studying vital functions such as respiration and digestion. As it became clear that these vital functions involved complex chemical transformations that could not be replicated in the laboratory, it seemed that forces unknown to inanimate matter must control the chemistry of life. To describe these forces, Friedrich Casimir Medicus (1736 1808) introduced the term Lebenskraft, or vital force, to animal chemistry in 1774. The notion of vital force received support in 1796 when Johannes Reil (1759 1813) included it as one of five types of force in nature (3 ). By the end of the century, however, Reil had become convinced that vital functions would ultimately be explained in chemical terms and that the idea of vital force could be discarded. Nevertheless, the concept of vitalism had gained a following among physiologists and chemists that ensured its use well into the nineteenth century (4 ). It was invoked to account for the functions of living organs and tissues, the organized parts of the body. Although doubts remained about whether blood should be considered an organized fluid,

The British Account: Blood and Other Body Fluids

Throughout the eighteenth century, most physicians thought that diseases arose solely in organs and tissues, the solid parts of the body. By the last quarter of the century, solidism was firmly entrenched in medical theory. Body fluids were not thought capable of causing or

Honorary Visiting Research Fellow, The Open University, Milton Keynes, United Kingdom. Address for correspondence: 24 Kayemoor Road, Sutton, Surrey SM2 5HT, England. E-mail n.g.coley@surrey28.freeserve.co.uk. Received March 28, 2001; accepted September 10, 2001.

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there could be no question that blood was essential for the maintenance of life, and it was generally thought to possess special vital properties (5 ).1

The New Humoralism boerhaaves groundwork

In eighteenth century Britain, many physicians were educated at Edinburgh, or at Leiden in Holland where Hermann Boerhaave (1668 1738) (6 ) was the leading teacher of chemistry and medicine. Boerhaave based his chemistry on Newtonian physical principles. Like Albrecht von Haller (1708 1777), the great Swiss physiologist, Boerhaave eschewed the generalities of vitalism and preferred physico-mechanical explanations of the vital functions (7 ).2 For Boerhaave, fluids and solids were merely different states of matter; the body fluids were just as important as the solids. Moreover, recognizing the value of chemistry in clinical and pathologic observations, he used the chemical analysis of body fluids to aid diagnosis. Thus, Boerhaave prepared the ground for the rise of a new form of humoralism, one in which the chemical composition of body fluids in health and disease would figure prominently (9 ). Many of his students achieved important medical positions (10 ), and those who favored his new ideas were not slow to promote them.

cullens nerves
Not all were convinced by Boerhaaves arguments, however. One such was William Cullen (1710 1790) (11 ), who has been regarded as second only to Boerhaave himself as a teacher of chemistry and medicine. At Edinburgh, Cullen adopted a sophisticated chemical philosophy and disagreed with Boerhaaves emphasis on mechanisms in explaining the vital functions (12 ). For vital chemistry, he thought that Boerhaave had paid too little attention to the influence of the nerves. He argued that morbidity in organs and tissues caused the nerves to increase the flow of blood to the affected parts. For Cullen, the blood became merely an agent, acting under the influence of the nerves. In keeping with the solidists, he argued that signs of morbidity in the fluids were symptoms rather than causes of disease. Cullens influence on eighteenth century medicine was strong, but his ideas tended to inhibit the study of body fluids, and many of his pupils continued to adopt the traditional solidism.

the eighteenth century there were signs of a move toward a new humoral pathology. For example, in 1839 1840, Henry Ancell, surgeon to the Western Dispensary in London, discussed the rise of fluidism in a historical survey of the physiology and pathology of animal fluids given at the school of anatomy adjoining St. Georges Hospital (13 ). In addition, Richard Davies, a Bath physician writing in 1760 on the value of blood analysis, identified three of its constituents as serum, gluten, and red globules (14 ). George Fordyce (1736 1802), a physician at St. Thomass Hospital, although a pupil and friend of Cullen, attempted a chemical study of body fluids. He identified some components of saliva, gastric juice, pancreatic fluid, and bile, but he was unclear about the functions of these fluids; e.g., the role of ferments in saliva and gastric juice. His ideas about the formation of chyle were vague, although he found components in it reminiscent of blood, including white globules and a coagulable fluid (15 ).3 William Hewson (1739 1774), a pupil and associate of William and John Hunter, studied the blood itself. In the 1770s, Hewson, who had attended Fordyces chemical lectures, began to study some of the metabolic changes in which the blood is involved. He discovered a lymphatic system in birds and reptiles previously thought not to possess one, and he accepted William Hunters view that the absorption of nutrients is a function of the lymph glands. Because the lymphatic system has connections with blood vessels, it seemed to Hewson that blood must also be involved in nutrition, and he therefore began to study the blood itself (16 19 ). Hewson examined red globules, which he found in the serum to be flat rather than globular (20 ), and described leukocytes (white blood globules), which he observed by diluting blood with serum instead of water. However, his most important contribution revealed some essential features of blood coagulation, which he attributed to a coagulable lymph, now known to be the nucleoprotein fibrinogen. As a result of his discoveries, Hewson has sometimes been called the father of hematology.4 At the end of the century, James Corrie, an Edinburgh physician, still suggested that the humoral pathology so long discarded may yet [. . .] be revived (22 ).

The Role of the Animal Chemists in Clinical Chemistry the nature of blood
As is evident from the foregoing, attempts to understand the nature and functions of the blood were not confined to

of globules and ferments: a clinical chemistry hematology connection

Some British physicians did begin to study the chemical properties of the blood, however, and in the second half of

1 In a series of public lectures for a general audience at the Gresham Institution, John Spurgin (17971866), physician and medical writer, adopted the notion of blood as a vital fluid without question.

3 Based on Fordyces Gulstonian Lecture at the Royal College of Physicians in 1789. Fordyces life and work are reassessed in my paper, George Fordyce MD, FRS (1738 1802), physician-chemist and eccentric. Notes Rec R Soc Lond 2001;55:395 409.

For a fuller discussion of the mechanist-vitalist controversy, see Heim (8 ).

4 The word itself was first used in 1743 by the Dutch physician Thomas Schwencke (16931733). The epithet has also been applied to others (21 ).

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medicine alone. Progress also depended on developments in chemistry as a whole as well as the introduction of new analytical methods. During the eighteenth century, chemists relied on the theory of phlogiston, but the last decade of the century saw the introduction of Lavoisiers theory concerning oxygen. This led to experimental observations on respiration that revealed links among oxygen, the products of digestion, animal heat, and the components of the blood (23 ). In the early nineteenth century, chemists introduced improved analytical techniques to replace the traditional method of destructive distillation (24 ). Animal substances, including body fluids, became legitimate subjects for analysis by animal chemists, and the empirical foundations for physiologic and clinical chemistry were laid. The blood, available in quantity although far from easy to analyze, was prominent in these studies because it was thought to be created from the food and to contain the constituents of all the other components of the body. Antoine Fourcroy (17551809) suggested that chyle was converted into blood by a process beginning in the subclavian vein and continuing in the lungs. He argued that in digestion and assimilation, there must be a stage at which inanimate food was converted into living matter in a process that he designated animalisation (25 ). Fourcroy also examined human blood in subjects of different ages in an effort to relate its fibrous content to the state of the muscles. He found that in infants, in whom muscles were weak, the fibrous content of the blood was low; blood from old men, whose muscles contained more fibrous matter, had a higher fibrous content (26 ). Fourcroy also identified three main constituents of animal tissuesalbumin, gelatin, and fibrin by chemical tests (27 ).

were met with strong objections by vitalists who could see no place for chemistry in the study of the vital functions.5

the chemical composition of animal fluids, including blood

Animal chemistry attracted attention from other young physicians, notably the Liverpool physician John Bostock, the Younger (17731846). While investigating the chemical composition of animal fluids, Bostock showed that albumin could be distinguished from gelatin (33, 34 ). He found that heat and mercuric chloride coagulated albumin, whereas gelatin liquefied when heated and was coagulated by tannin (35 ). Gelatin was known to be an important constituent of animal tissues, and Fourcroy (36 ) had asserted that it occurred in blood serum. When Bostock added tannin to serum from which the albumin had been removed, however, he observed no further precipitation. He therefore concluded that blood did not contain gelatin (37 ).6 Bostock, studying other chemical characteristics, attributed the alkalinity of blood to the presence of free soda (38 ), which was in agreement with Berzelius and Alexander Marcet (1770 1828), physician at Guys Hospital. When George Pearson (17511828), a physician at St. Georges Hospital, stated that the alkali was potash rather than soda, a controversy followed (39 42 ). In 1817, Bostock moved to London, where he ceased to practice medicine and instead turned to chemistry and physiology. The first of Richard Brights collaborators, he later lectured at Guys Hospital medical school, following Marcet in 1820 (43 ). A need for better information on the chemical composition of animal substances was recognized by Berzelius in Sweden, and in 1806 1808 he published a two-volume textbook of animal chemistry in Swedish (44 ). Humphry Davy at once realized the importance of the work and resolved to have it translated into English. The project seemed eminently suitable for the newly formed Animal Chemistry Club, a special interest group within the Royal Society. Unfortunately, mainly for financial reasons, no English translation of the work was ever published (45 ). Marcet later persuaded Berzelius to publish some of his analyses of animal fluids in English, which became an important source of data for British animal chemists (46 ). In his analysis of the blood, Berzelius separated the solid and liquid parts by coagulation and examined each part separately. In the clot, he found compounds of carbon, phosphorus, calcium, magnesium, and iron (Table 1). The serum consisted of water containing albumin,

a place for chemistry in physiology?

Further experimental work on animal fluids was made possible by new analytical techniques introduced by J.J. Berzelius (1779 1848) in Sweden and Justus von Liebig (18031873) in Germany. These methods were improved by William Prout (17851850), the London physician and animal chemist best remembered for his atomic weight hypothesis (28 ). All of these analytical techniques involved the oxidation of organic matter to carbon dioxide and water. Prout determined the composition of many animal substances more accurately than any of his contemporaries and investigated the processes of animal nutrition from the digestion of food to the fabrication of blood (29 ). He initially thought that digestive juice contained lactic acid, but he was forced to revise his ideas in 1824 when he discovered free hydrochloric acid in the stomach (30 ). He reexamined the digestive processes and, after another 10 years of careful investigation, proposed a general theory of digestion and assimilation (31 ). Prout was a powerful advocate of the value of chemistry in physiology (32 ), but his repeated calls for physiologists to become chemists

5 Prouts first call for physiologists to become chemists came in 1816, but was repeated in his Gullstonian Lectures of 1831. A debate between Prout and Wilson Philip followed. Wilson Philip argued that chemistry and the science of the vital functions were so different in kind that study of the one would seriously affect that of the other.

Berzelius later confirmed the absence of gelatin in blood serum.

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Table 1. Berzeliuss analysis of blood clot (1812).a

Proportion, parts per 100

Oxide of iron Subphosphate of iron Phosphate of lime and a little magnesia Pure lime Carbon dioxide and loss Total
a

50.0 7.5 6.0 20.0 16.3 100.0

From Berzelius (46 ).

various salts, and some animal matter. His analysis of the serum confirmed results obtained by Marcet (Table 2). Berzelius also found that bile, saliva, mucous fluids, the humors of the eye, and serous fluids contained the same salts in roughly the same proportions as in blood serum. He thought that this result showed that all animal secretions were ultimately derived from the blood. Thus, by the second decade of the nineteenth century, the new humoralism had already produced useful observations on the properties and constituents of the blood.

The New Humoralism in Physiologic Chemistry chemical properties of blood

By the third decade of the nineteenth century, chemical changes in blood began to attract the attention of some physicians. In 1832, George Leith Roupell (17971854), a physician at St. Bartholomews Hospital, described chemical changes in the blood of cholera victims in his Croonian Lectures at the Royal College of Physicians in London. Conscious of the work that had been done on animal fluids since the late eighteenth century, he said, it is to a more exact acquaintance with the chemical changes of the
Table 2. Comparison of analyses of blood serum by A. Marcet and J.J. Berzelius.a
Marcet Proportion, parts per 1000

fluids in diseases that we are chiefly to look for the future advancement of physic as a science (47 ). In the same year, William Stevens (1786 1868) described changes he observed in the composition of the blood in fevers, cholera, and other diseases during his practice as a physician in the West Indies. Stevens acknowledged Prouts contributions to animal chemistry and recognized blood as the source of secretions, such as bile and gastric and pancreatic juices, and excretions, such as perspiration and urine. He also thought that the blood nourished the solid parts of the body and conveyed gases that it held in the free state. Despite his belief in the importance of the chemical properties of the blood, Stevens, like so many of his contemporaries, remained a vitalist (48 ). It had long been suspected that the vitality of the blood was related to its property of coagulation. John Hunter argued that coagulation occurred under the influence of the vital force (49 ), and Fourcroy even suggested that coagulation of the blood might be the mechanism by which animal fibers were formed in the living body (50 ). However, because coagulation occurred only after the blood had left the body, others such as Charles Thackrah (17951833),7 a physician in Leeds, argued that the process resulted from the loss of vitality (51 ). In that case, the vital force in living blood must maintain the fluidity of blood (52 ).

the scarlet color

The transformation from dark venous to bright arterial blood as a result of contact with the air was another puzzling characteristic of this fluid. Because the change took place mainly in the lungs, it obviously depended on respiration; it also seemed linked to the production of animal heat (53 ). [Adair Crawford (1748 1795) explained animal heat as the release of phlogiston (54 )]. Comparing the body to a heat engine, Lavoisier thought that animal heat was produced in the lungs because of the oxidation of nutrients in the blood, but Stevens observed that this could not account for the clinical phenomena of fevers. Stevens remarked that there was often a cold stage in fevers during which patients sometimes died. If patients recovered, heat began first in the extremities, and although the patient still felt cold, the skin might become burning hot. This convinced him that heat was generated throughout the body rather than in the lungs alone, and he suggested that in all warm-blooded animals carbon combined with oxygen in arterial blood and that animal heat was evolved in consequence of the chemical combination of these two agents (55 ). By the 1840s, it was widely accepted that the source of animal heat was in the blood itself, greatest in the lungs where arterial blood is formed, but proceeding wherever arterial blood meets oxidizable carbon in the tissues (56 ). Although this is

Water Albumin Muriate of soda and potash Muco-extractive matter Sodium carbonate Potassium sulfate Earthy phosphates Total
Berzelius

900.00 86.80 6.60 4.00 1.65 0.35 0.60 1000.00 905.00 80.00 6.00 4.00 4.10 0.90 1000.00

Water Albumin Muriate of soda and potash Lactate of soda and animal matter Soda and phosphate of soda Loss Total
a

From Lecanu LR. Ann Chim 1831;48:316.

7 Thackrah later became known for studies of the industrial diseases of his home town.

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broadly correct, the true origins of animal heat remained unrecognized throughout the nineteenth century (57 ). By a strange chance, the color of venous blood led to one expression of the mechanical equivalent of heat. In 1840, Julius R. Mayer (1814 1878), a German physician working on a Dutch ship bound for Java, noticed that venous blood drawn from European sailors was unusually bright red. He thought the heat of the tropics must cause a lower metabolic rate, requiring less oxygen than in a colder climate. Mayer decided that this confirmed the theory of animal heat from the oxidation of food. Moreover, he suggested that the heat evolved in muscular exertion must also be derived from the chemical energy latent in food; the intake of food and the output of force (i.e., energy) must be balanced. After returning home in 1841, Mayer wrote an account of his ideas that was published by Liebig in his Annalen in 1842, the same year in which Leibigs own theories of animal chemistry appeared (58, 59 ). Others favored different explanations of the color of blood. Stevens had ascribed the reddening of arterial blood to the absorption of oxygen. He began to doubt this when he observed that certain salts also produced the scarlet color and that oxygen did not cause reddening of a black clot free from salts. He decided that carbonic acid turned venous blood dark. When this compound was removed during respiration, it was not oxygen, but the saline ingredients in the blood that turned it scarlet (60 ). It had been known since the mid-eighteenth century, however, that the blood contained iron, and Fourcroy (61 ) ascribed the red color of blood to a subphosphate of iron, a view accepted by many chemists. William Charles Wells (17571817), a physician at St. Thomass Hospital, attributed the red color of the blood to a peculiar animal substance (62 ).8 In 1812, William Thomas Brande (1788 1866), professor of chemistry at the Royal Institution, showed that the proportion of iron in the coloring matter of the blood was no greater than that in other animal substances (64 ). [This was later confirmed by Vauquelin (65 ).] Thus, although iron was present in the coloring matter, there was also a large proportion of animal matter in the blood, a remarkably astute conclusion that the chemistry of the period was incapable of pursuing. Later, in 1824, Sir Charles Scudamore (1779 1849), a fashionable London physician, published a chemical study of the blood (66 ) in which he confirmed Brandes observation that the red coloring matter of the blood was an animal principle containing a small proportion of iron.

ing interest. Sir Everard Home (1756 1832), a surgeon at St. Georges Hospital and brother-in-law of John Hunter, had observed this some years earlier (67, 68 ), but John Davy (1790 1868), Humphry Davys younger brother, disputed this (69 ). Scudamore, in 1824, confirmed the evolution of carbon dioxide, stating that it was evidently an essential circumstance in the process of coagulation, as the same causes which retain the carbonic acid in the blood, delay coagulation (70 ). He also mentioned the possible evolution of heat and extended his investigations to include the effects of electricity, galvanism, and various salts and other chemical agents on the process. Thackrah confirmed the presence of small amounts of albumin in blood serum, together with mineral salts, including the carbonate, chloride, phosphate, and acetate salts of sodium; potassium chloride and potassium sulfate; calcium phosphate; and free alkali. In the clot, he found three components, red corpuscles, fibrin, and albumin, and observed that the last had often been overlooked.9 He could not confirm the release of heat during coagulation that had been reported earlier by Gordon (73 ) and John Davy ( 74 ) and concluded that those who claimed that heat was evolved in the process were mistaken (75 ). He agreed that carbon dioxide was usually given off, but he was not convinced that this was either a necessary condition for or a cause of coagulation. Having decided that none of the substances he had examined explained the process of coagulation satisfactorily, Thackrah then turned to the effects of the nerves whose influence on the blood had been suggested by Cullen, Berzelius, and others. By confining samples of blood in live leeches and in sections of veins taken from dead animals, Thackrah found that so long as irritability remained, blood contained in such vessels did not coagulate, although it did so quite rapidly after the loss of vital force. Stevens agreed with Thackrah that vitality and the motion of the blood in the living body prevented coagulation, but he still thought the fundamental cause of coagulation resided in the chemical changes that occurred when blood was drawn from the system and exposed to air (76 ). Thackrah disagreed with Stevens and asserted that the vital or nervous influence was the source of fluidity in the blood. The loss of that influence thus caused coagulation (77 ). Moreover, he argued that the rate of coagulation of extravasated blood was a diagnostic indicator of the vitality of a patient. These conflicting views demonstrate the difficulty of achieving certainty in blood studies in the early years of the nineteenth century.

the beginnings of medical chemistry clots and conflicts

Scudamore repeated the curious observation that carbonic acid was evolved during coagulation, a process of increasAmong British physicians of the period, George Owen Rees (18131889), a physician at Guys Hospital, was one

According to William Henry (63 ), LJ Thenard (17771857) also attributed the color of the blood to a special animal coloring matter.

9 Compare this with Prouts On Diabetes (71 ), where albumin was not mentioned, and Henrys The Elements of Experimental Chemistry (72 ), which also failed to mention albumin.

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of the foremost champions of the new humoralism (78 ). In 1833, while still a student, he showed that urea could be identified in diabetic blood serum (79 ). R.H. Brett and Golding Bird, his colleagues at Guys Hospital, disagreed with this finding (80 ), and a controversy ensued in which Rees showed a clear grasp of chemical analysis (81 ). Rees developed his skill as an analyst while assisting Richard Bright at Guys Hospital. Later, as he developed the chemical analysis of blood and urine, he advocated his methods as an aid to diagnosis and aimed to simplify them for the ordinary medical practitioner. He deplored the lack of interest in the chemistry of the blood shown by most physicians (82 ). He admitted that although animal chemistry had developed rapidly, it had not produced the predicted medical advances, and physicians used this deficiency to support their lack of interest in chemical methods. Cheered by renewed interest in blood chemistry, however, Rees said, The philosophical revival of a humoral pathology bids fair to render the analysis of diseased blood one of the most useful adjuncts to our medical knowledge (83 ). Rees based his own analyses on Berzeliuss work, but he was aware that others, especially Lecanu in France, had applied new chemical methods to blood analysis. Rees applauded such efforts, arguing that the chemical composition of healthy blood should be determined for use as a comparative guide in cases in which disease caused noticeable changes. For example, in cholera the blood contained a lower proportion of water, but in diabetes there was an excess of fatty matter and urea. In other cases, cholesterine, the coloring matter of the bile, was found, but because of inadequate analytic techniques, these compounds had often been missed altogether. Rees emphasized the need to determine each constituent of the serum by a separate procedure and criticized earlier analysts for attempting to determine all of the constituents by a single analysis (84 ). In 1838, he described a method for isolating sugar from diabetic blood serum, which has been hailed as a major contribution to medical chemistry (85 ).

microscopy for physicians (89 ). Some years later, George Gulliver, assistant surgeon to the Royal Regiment of Horse Guards, suggested that pus globules, detected in the blood in cases of extensive suppuration or great inflammatory swelling, were modified red corpuscles. In addition, recognizing the value of quantitative studies, Gulliver measured the diameters of the red corpuscles in various species (90 ). Gullivers quantitative measurements preceded the techniques of blood-cell counting and hemoglobin estimation (91 ).

controversy prompts research

In 1842, Liebig, having turned his attention from pure organic chemistry to its applications in agriculture and physiology, introduced a new approach to the chemistry of the blood and its role in animal nutrition (92 ). Assuming that the chemical composition of the blood was identical to that of flesh, Liebig assigned the empirical formula C48N6H39O15 to both. His chemical account of physiologic phenomena grossly oversimplified the intricacies of metabolism, and his equations, in which empirical formulas were balanced arithmetically, provided no more than an overall summary of the changes that they were intended to explain. Like Mayer, Liebig sought to balance the intake of food against the output of muscular energy and animal heat (93 ). Mayer accused Liebig of plagiarism, but it is more likely that the similarities between them were the result of common influences, although Liebig was a vitalist and Mayer was not. Liebig agreed with Mayer that the oxidation of carbohydrates yielded animal heat, but held that the oxidation of muscle tissue was the source of animal energy. He also thought that the excreted urea and other nitrogen compounds resulted from the degradation of muscle tissue and were a measure of the work done by the animal. He also discussed the formation of the blood and its function as a carrier of nutrients and excretory products. Liebigs ideas, influential among British animal chemists, provoked controversies between physiologists and chemists that stimulated valuable research into animal metabolism [see Holmes (94 ), which is an exhaustive summary of the experimental investigation of Liebigs theories up to 1870].

the microscopic story

In the 1840s, microscopic studies of the blood began to reveal some new facts. Rees, working with Samuel Lane, a colleague at Guys Hospital, investigated the microscopic structure and properties of red corpuscles. They found that each red corpuscle was surrounded by a membrane, and they carried out experiments on the osmotic effects of various solutions (86 ). At about the same time, William Addison (18021881), a physician in Malvern not to be confused with his famous namesake, Thomas Addison, observed rough or granulated colorless capsules (granulocytes) and loose or independent molecules (platelets) in the blood (87, 88 ). The value of microscopy in the study of disease was also recognized by the French physician Alfred Donne (18011878), who from the early 1840s in Paris, organized classes in clinical

the qualitative vs the quantitative

Some found that qualitative data came under fire. Rees investigated the relationships between chyle, lymph, and blood, and his analyses led him to agree with those, like Prout and Liebig, who regarded the formation of blood as a culminating stage in nutrition and assimilation (95 ). Rees analyzed chyle taken from the bodies of newly executed prisoners for Samuel Lane, who had been commissioned to supply an article including this topic for Todds Cyclopaedia of Anatomy and Physiology (96 ). He found that the fatty matter was similar to that of the blood, except that the latter contained phosphorus (97 ). As a result of these observations, he returned to the idea

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that chyle might be an intermediate product in the formation of blood, although by this time he had become suspicious of the suggestion that the incipient reddening of chyle as it changed into blood could be observed (97 ).10 Five years later, Rees proposed a new theory to explain the color changes of the blood. He suggested that the red globules of venous blood contained phosphorized fat in a serum deficient in alkaline phosphates, whereas in arterial blood the red globules contained no such fats but the serum was richer in alkaline phosphates. He suggested that the reddening of the blood occurred when tribasic sodium phosphate formed by the oxidation of phosphorized fats in venous blood reacted with hematosine. We may see here a hint of later developments regarding the role of phosphorus in cell respiration. However, Rees failed to provide quantitative results to support his new theory, and his assertion that qualitative observations were sufficient to establish its truth did not convince his critics. If correct, the theory seemed to imply that nearly all of the alkaline phosphates formed in arterial blood must be discharged before the blood reached the veins. This would necessitate a large constant supply of phosphorus to the venous blood and a more copious elimination of phosphates than had hitherto been suspected. Reess paper was read at a meeting of the Royal Society on June 3, 1847, and was printed in the Proceedings (98 ). He was asked to supply more convincing experimental evidence before the paper could be accepted for publication in the Philosophical Transactions. After testing his theory further, Rees produced more qualitative observations to support it, but he was unable to demonstrate its truth beyond doubt.11 The simple tests that had served him so well in other areas of clinical chemistry, including his work on the blood, were inadequate to explain the complex reactions involved in one of the bloods chief functions. At about the same period, a new book on animal (and human) chemistry by the young German animal chemist Johann Franz Simon (18071843) appeared posthumously.12 Simon adopted many of Liebigs ideas, including his use of empirical formulas, his notion of the metamorphosis of tissues, and his equations (102 ). He began with the idea that the blood nourishes cells and organs, supplying them with albumin, fibrin, and fat. The cells would select appropriate nutritive matter and form decomposi-

tion products to be excreted.13 He named urea, bilin, and carbonic acid among the substances formed by the vital energy of the blood corpuscles nourished by oxygen, albumin, and fat, but he thought that the most important product of the blood was animal heat, released during the combination of oxygen with the carbon of globulin (104 ). He thought that the blood conveyed the excretory products either to the lungs or via the kidneys to the urine and that the quantity of urea excreted was directly related to the evolved animal heat. Simons work, which showed great promise, was highly regarded by many British animal chemists; sadly, he died at the age of 36.

The French Version the beginnings of animal chemistry

In France, animal chemistry developed rapidly after the introduction of Lavoisiers antiphlogistic theory. Fourcroy, assisted by L.N. Vauquelin (17631829), investigated many aspects of animal chemistry, including the nature and properties of the blood. Vauquelin, an outstanding experimental chemist, later worked alone and produced several hundred papers on chemical topics, many of which were linked to animal and physiologic chemistry (105, 106 ). Animal fluids such as blood and urine were examined, but physicians largely ignored this chemical research. In France, as in Britain, medical theorists emphasized the importance of the organs and tissues, and by the last quarter of the eighteenth century, solidist theories of disease were firmly entrenched in French medicine. In Paris, Francois Joseph Victor Broussais (17721838) and his follower Jean Baptiste Bouillaud (1796 1881), proponents of a system called physiological medicine, regarded the gastrointestinal organs as the chief seat of disease (107, 108 ). Rene Theophile Hyacinthe Laennec (17811826), inventor of the stethoscope, focused attention on the heart, lungs, and thoracic organs in a system designated pathological anatomy. At Montpellier, however, diseases were regarded as the cause rather than the effect of pathologic changes in the organs. By the early nineteenth century, some French physicians, like their British counterparts, began to develop an interest in the chemical composition and properties of animal fluids. In the 1820s, Jean Baptiste Dumas (1800 1884) and Jean Louis Prevost (1790 1850) studied the blood of various animals in relation to vital phenomena such as respiration and digestion (109, 110 ). In a mainly physiologic study, they were concerned with the shape, size, and contents of the red globules and the effects of various diseases on them. Like Wells and Brande earlier, they concluded that the coloring matter of the blood consisted of an animal substance, possibly albumin, combined with iron peroxide. In 1833, the pharmacist Felix Henri Boudet (1806 1878)

10 Even the great German physiologist Johann Muller claimed to have observed this change in the chyle of a horse, but Rees failed to confirm it.

11 The revised paper was later published in Philosophical Magazine (99 ) and Erdmanns Journal fur Praktische Chemie (100 ).

12 Simon became a private tutor at the University of Berlin in 1843, but his death later that year cut short his brief career (101 ).

13

Rees had also compared the analysis of blood with that of urine (103 ).

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decided that a more precise chemical analysis of the blood was needed (111, 112 ). He discarded ill-defined terms such as osmazome and muco-extractive matter for constituents of the serum. Suspecting that the quantities of many constituents of the blood were very small, Boudet thought it necessary to examine large volumes of serum to reveal more detail about its composition (113, 114 ). By evaporating a large volume of blood to dryness, he was able to extract small translucent, noncrystalline plates from the residue. He considered this a new constituent of blood and named it seroline. Its quantity was very small, but from this residue he extracted a white, alcoholsoluble substance, which he suspected to be cholesterine. Although Prosper Sylvain Denis (1799 1863) (115, 116 ), a physician at Commercy, stated that he had found cholesterine in diseased blood (117 ), Boudet thought that Deniss crystalline product was probably a phosphorylated fat crystallized in a form resembling cholesterine (114 ).

the solid foundations of blood chemistry

Louis Rene Lecanu (1800 1871) (118, 119 ), professor of pharmacy at LEcole de Pharmacie in Paris, also studied the chemistry of the blood in the 1830s. Whereas Boudet had concentrated on serum, however, Lecanu began with the red coloring matter, for which he adopted Chevreuls term hematosine (120 ) (Table 3). The compound of hematosine and albumin found in blood globules he called globuline to distinguish it from hematosine itself. Uncertainty about the chemical nature of the coloring matter of the blood led Lecanu to devise fresh experiments on globuline in which he confirmed the presence of iron. From many comparative analyses, he concluded that the proportion of water in the blood varied with sex, age, and temperament (121 ). The proportion of water was higher in male than in female blood, but for individuals of the same sex this proportion remained roughly the same between the ages of 20 and 60. He also found that the proportion of albumin was about the same in male and female blood and in individuals of different ages and temperaments but that the number of globules varied with sex and age. He found more globules in male than female blood and more in younger than older individuals of the same sex. Lecanus experiments are the earliest comparative analyses of healthy blood (122 ). He submitted his observations for an MD degree in the Faculty of Medicine of the University of Paris in 1837 (123 ).

About the same time, Denis published an analysis of human blood in which he described seven groups of components (124 ) (Table 4). He found the properties of hematosine different from those of albumin and therefore separated the two compounds. This view was in contrast to that of Berzelius, who grouped hematosine with albumin and fibrin as albuminous substances. Denis made a thorough study of the fibrin of the blood (125 ) and found that it was dissolved in the blood and solidified only on coagulation, although others considered fibrin to be a solid constituent of blood globules even before coagulation. Lecanu stated that fibrin lost four-fifths of its weight on desiccation (Berzelius had said three-fourths), but Denis found that fibrin took up variable proportions of water in the presence of salts, such as barium chloride, potassium nitrate, sodium sulfate, or potassium sulfate, that held it in solution. He criticized Lecanu for adopting an error attributable to Chevreul, who thought that chlorides and some other salts formed weaker compounds with fibrin. Denis further showed that there was no gelatin in the blood, in spite of its abundance in all the body tissues and suggested that blood albumin changed into gelatin as it entered the tissues and organs (126 ). He found cholesterine absent from healthy blood and showed that fibrin and albumin had almost identical chemical properties, an observation later confirmed by Liebig. In 1856, Denis added globulin as a constituent of blood (127 ). He found the properties of globulin very different from those of fibrin, with which it was combined. He also described the presence of globules of chyle and lymph in blood, as well as the red and white globules.

the apogee
Boudet, Lecanu, and Denis had placed blood chemistry on a sound foundation, but important as their discoveries were, it was Gabriel Andral (17971876) who brought the subject to its apogee in the mid-nineteenth century. First appointed to the Chair of Hygiene in the Faculty of ` Medicine at Paris in 1828, Andral became professor of pathologie interne in 1830 and followed Broussais as professor of general pathology in 1839 (128 130 ). Two important early works in which he compared the composition of the blood directly with the solid parts of the body brought him rapid recognition (131 ). In his studies, he combined microscopic with chemical procedures. Although at first he was a follower of Broussaiss system of

Table 3. Summary of Lecanus analyses of blood.a

Whole blood Proportion, parts per 1000 Serum Proportion, parts per 1000 Globules Proportion, parts per 1000

Serum Globules Total

a

869.1547 130.8453 1000.00

Water 25 mineral and organic substances Albumin Total

790.3707 10.9800 67.8040 869.1547

Fibrin Hematosine Albumin Total

2.9480 2.2700 125.6273 130.8453

From Lecanu LR. Ann Chim 1838;67:64 5.

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Table 4. Components of human blood according to Denis (124 ).

Fluids and gases Globules Albuminous substances Coloring matter Fatty matter Odorous matter Saline matter Imponderables Water, oxygen, carbon dioxide, nitrogen, and so forth Red, white, chyle, and lymph Fibrin and albumin; globulin Hematosine, yellow biliary matter, blue matter Cerebrine, seroline, cholesterine, fatty acids Alliaceous matter Alkalis, soluble salts Electricity, heat

physiologic medicine, his clinical observations at the hospital of La Charite in Paris led him to oppose it and join the partisans of pathologic anatomy. In his Precis dAnatomie Pathologique (132 ), he began to set out a new, more scientific approach to the study of disease. Andral derived his belief from the system of pathologic anatomy and his notion of lesions of the blood from Jean Cruveilhier (17911874), who embraced the system in 1829 (133, 134 ). Andral recognized a direct comparison between the pathology of the blood and that of the organs and tissues and distinguished between medical theories based on the primacy of organic lesions and those depending on physical changes in the humors. He also urged the chemical analysis of the blood, especially in morbid conditions, and justified the revival of humoralism by asserting that the solids of the body and the blood were complementary parts of a single whole. He discussed the role of the blood in plethora, anemia, pyrexia, and so-called organic diseases such as cardiac hypertrophy. Unlike Cruveilhier, who held that phlebitis dominated all pathology (135 ), Andal did not exaggerate the role of the blood in pathology. Like Alfred Donne (1801 1878), who observed blood platelets in 1842, Andral relied heavily on microscopic observations at a time when many of their contemporaries viewed such observations with suspicion and regarded the microscope as an amusing toy rather than a scientific instrument (136 ). In 1835, Andral reviewed the work done on the blood in France in a joint article with C.P. Forget (1800 1861), a professor of clinical medicine at Strasbourg, that appeared in the Dictionnaire de Medecine et de Chirurgie Practique (137 ).

use organic chemistry, feeble though its light may be (139 ), to discover the composition of the blood and investigate the manner in which morbid changes are produced. He advocated the study of animal chemistry, and although he recognized that the subject had yielded very little new knowledge, he believed that it would come to exercise great influence on physiology (140 ). In his own work he combined chemical experiments with vivisection. Calling his approach experimental medicine, he insisted that most problems associated with the causes and mechanisms of disease were connected with the chemical, physical, and vital properties of the blood. Magendie remarked that his former pupil P.S. Denis has made some very curious researches on the chemical composition of the blood; among other important facts it would appear to result from his labours that the fibrin is nothing more than albumen combined with different salts (141 ). Although Magendie decided to reserve his judgment on Deniss findings, he admired the careful experimental approach and always avoided reliance on systems or hasty hypotheses. Thus, Magendies physiologic work supported Andrals medical approach to blood studies.

animal chemistry, clinical chemistry, and medicine

In 1840, Andral, with his colleague Louis Denis Jules Gavarret (1809 1890), published quantitative studies of the composition of the blood (142 ).14 Their work showed the value of animal chemistry as a means of confirming diagnoses. Although there were objections to their methods and doubts were raised about accuracy, they stoutly defended themselves (145 ). They showed that the composition of the blood varied in different pathologic conditions. For example, in fevers they found no increase in fibrin; in serious cases there was a noticeable decrease, but in inflammation they observed a definite increase (146 ). This discovery was an important one confirming the distinction between these conditions. Andral insisted that experimental observation was the only sound basis for advance and thought that progress in hematology would be made only when the blood of a great number of patients shall have been submitted both to a chemical investigation, and that by the microscope (147 ). These ideas were extended to animals when Henri-MamertOnesime Delafond (18051861), professor of physiology at the veterinary college at Alfort, joined Andral and Gavarret to perform experiments on animal blood (148 ). These studies showed that the composition of the blood varied in different animals and in humans under different conditions. Andrals discoveries were important, not least because he provided experimental evidence to confirm them

pioneering experimental medicine

While Andral and his medical colleagues were studying blood in relation to disease, Francois Magendie (1783 1855), the pioneer of experimental physiology, was similarly engaged. In 18371838, he gave a course of lectures at the College de France on blood and the changes it ` undergoes in disease; Magendies lectures were reported in full in The Lancet (138 ). He was critical of contemporary medical theories such as pathologic anatomy (supported by Andral and Cruveilhier) and solidism; he also expressed contempt for vitalism. Instead, he proposed to

14 Chauffard (143 ) claims that this work marked the birth of modern hematology. For information on Gavarret, see Fruton (144 ).

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(149 ).15 He brought an objective approach to the study of disease and placed the new humoralism on a scientific basis that recognized the importance of chemical analysis as a means of confirming clinical observations. His concept of lesions of the blood supported the notion that body fluids are as important in pathology as tissues and organs. By the 1840s, the medical value of blood chemistry was beginning to be recognized, and the foundations of modern hematology had been laid (151 ). Animal chemistry had shown both its potential and its limitations for the improvement of medicine, and with the rise of animal chemistry the importance of clinical chemistry was also realized.

7.

8. 9.

10. 11.

the milieu interieur

In the 1840s, Magendies famous pupil Claude Bernard (18131878) turned his attention to animal chemistry (152 ). As he studied the problems of nutritional processes, he discovered details about the composition and functions of the blood. Bernard believed that the vital processes occurred at the sites where the blood and tissues interacted. He wanted to determine these minute changes as they occurred, whereas chemical analysis was capable only of deducing the overall changes that had transpired during nutrition. He began to realize that chemical analysis was not yet precise enough to detect minute changes in the blood as it interacted with the tissues. In the 1840s, his aims were unattainable. Two decades of careful experimentation and deduction led him to the important concept of the milieu interieur (1865). According to this con cept, the blood, as it bathes all the cells and tissues of the body, creates an internal environment in which nutrients and oxygen are transferred from the blood to the cells and waste products are removed from them. Thus, the notion of blood as an agent and carrier was extended. Bernards grand concept was beyond the understanding of most of his contemporaries, however, and detailed exploration of its implications would have to await developments in twentieth century biochemistry.
12. 13.

14. 15. 16. 17.

18. 19. 20.

21.

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