The Severe Gout of Holy Roman Emperor Charles V
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《新英格兰医药杂志》
Holy Roman Emperor Charles V (who was also King Charles I of Spain) was one of the most powerful rulers of all time (Figure 1A). His 40-year reign (1516–1556) occurred during the transition from the Middle Ages to modern times. He was a global leader, governing territories in Europe, Africa, and Asia as well as the conquered territories of the Aztec Empire of Mexico and the Inca Kingdom of Peru. Given this reach, he was described as ruling an empire "in which the sun does not set." Despite this power, Charles V had to live within the limitations of very intense arthritic pain for most of his adult life and needed, for example, a special chair for transportation (Figure 1B). His physical suffering influenced decisions that affected the future of many countries. His physicians diagnosed his arthritic disease clinically as gout.1 However, to our knowledge, there has never been objective biologic confirmation of this diagnosis.
Figure 1. Charles V Seated, by Titian (Panel A), and a Special Chair Designed to Carry the Emperor (Panel B).
The portrait of Charles V in his last years shows him incapacitated by severe arthritic pain, with the emperor's swollen left hand in what seems to be a protective position on his lap. (Reprinted with permission of the Alte Pinakothek, Munich.) The chair belonging to Philip II was copied from the original chair designed for his father, Charles V. (Inventory number 10014120, Palacio de los Austrias, Real Monasterio de San Lorenzo de El Escorial, Spanish National Heritage.)
Charles V was buried, in accordance with his wishes, at the monastery of Yuste, but in 1574 his body was transferred to the monastery of El Escorial. Historical reports of this transfer state that the body had undergone spontaneous mummification and that the emperor was completely recognizable. Recently, one of us contacted the Delegation of the Patrimonio Nacional in San Lorenzo de El Escorial in order to study the terminal phalanx of the finger of Charles V, which has been kept outside the coffin in a small box (inventory number, 10044506, Spanish National Heritage) and preserved in the sacristy of the Royal Monastery of San Lorenzo de El Escorial. We report here the finding of massive gouty tophi with demonstrable urate deposits that confirm the clinical suspicion of severe gout in this unique sample from the mummified remains of Charles V.
Methods
A full protocol was developed with the objective of investigating the cause of death and other associated pathologic conditions. The protocol was approved by the Patrimonio Nacional (the Spanish National Heritage) and the institutional review board of the Hospital Clinic-Institut and the University of Barcelona. The remains of the phalanx of the emperor were transported to Barcelona in the original red velvet box. The office of the Spanish National Heritage established, on the basis of historical documents, that the fingertip was considered part of the remains of Charles V. It was not possible to obtain DNA evidence. The specimen consisted of part of the final phalanx of the fifth finger of one of the hands (laterality unknown). Externally, the finger fragment had a dark-brown color and leathery texture. The proximal part of the nail was attached to the ungual bed, whereas the outer half was completely detached (Figure 2A). A radiographic study was performed.
Figure 2. Macroscopic View of the Mummified Finger of Charles V.
Panel A shows the specimen consisting of part of the final phalanx of the fifth finger of one of the hands of Charles V. The finger fragment has a dark-brown color, which corresponded to the soft parts. The proximal part of the nail was attached to the ungual bed, whereas the outer half was completely detached. Panel B is a radiograph showing extensive erosion of the proximal epiphysis of the phalanx, which had irregular borders and soft-tissue calcifications. Panel C shows the proximal edge of the specimen, with a massive yellowish deposit that has a chalky appearance.
The specimen was rehydrated by total immersion in Sandison's solution2,3 for one hour under visual inspection. After rehydration, the most proximal portion, consisting of the epiphysis of the joint, was immediately processed for histopathological characteristics. Tissue samples were embedded in paraffin according to standard protocols used for fresh tissue. After removal of the paraffin, sections that were 4 μm thick were processed with graded alcohols and water, and routinely stained with hematoxylin and eosin, Masson trichrome, and Giemsa.
The chemical constitution of the deposits was analyzed by scanning electron microscopy (Energy Dispersive Analysis by x-ray , Cambridge StereoScan S360, Leica) and an x-ray microanalysis system (INCA 200, Oxford Instruments). After removal of the paraffin, a section that was 4 μm thick was carbon-coated for electrical conductivity and examined by electron microscopy. Another section of the phalanx that was 20 μm thick, after removal of the paraffin with xylene and absolute alcohol, was immersed in 3 ml of uric-acid reagent for automated analysis (Bayer Advia, Bayer Diagnostics) at room temperature. The reaction is based on the conversion of uric acid to allantoin and peroxide by uricase. The presence of urate is detected by the identification of a colored complex formed from hydrogen peroxide. The method has a limit of sensitivity of 0.1 mg per deciliter.
Results
The radiographic studies showed extensive erosion of the proximal epiphysis of the phalanx, which had irregular borders and soft-tissue calcifications (Figure 2B). Macroscopic examination disclosed a yellowish deposit with a chalky appearance that completely occupied the proximal edge of the specimen (Figure 2C).
The histologic analysis revealed well-preserved dermal collagen and bone, but no preserved cells of any type were detected, except isolated red cells. At low magnification, no bone could be identified in the proximal margin of the specimen. At this location, a massive deposit of weakly basophilic, cloudy, fine fibrillar masses that completely occupied the center of the specimen, eroding the bone and extending into adjacent soft tissue, was observed (Figure 3A and 3B). Large amounts of fuzzy and crystalline material were identified in the center of these masses. Under polarized light, strongly birefringent sheaves and stacks of needle-shaped crystals consistent with urate were observed (Figure 3C and 3D). Scanning electron microscopy showed a large deposit of crystalline structures that had eroded and destroyed the bone (Figure 3E). At higher magnification, these deposits had a needle-like shape (Figure 3F, inset). The elemental constitution of the crystalloid deposits as determined on scanning electron microscopy is shown in Figure 3F. Large amounts of sodium, as expected for monosodium urate, were detected. The chemical analysis with uricase gave a strong positive reaction (not shown).
Figure 3. Microscopical Study of the Finger.
Specimens shown in Panels A, B, C, and D were stained with hematoxylin and eosin. In Panel A, at low magnification, there is a large deposit of weakly basophilic masses completely occupying the center of the specimen and destroying the bone. Large amounts of crystalline material are identified in the center of these masses. In Panel B, at higher magnification, the weakly basophilic, cloudy, fine fibrillar masses seen are characteristic of gout. In Panels C and D, under polarized light, strongly birefringent sheaves and stacks of needle-shaped urate crystals can be identified. Panels E and F show the scanning electron microscopical analysis. A massive deposit of crystalline structures is identifiable (Panel E), which erode and destroy the bone. The elemental constitution of the crystalloid deposits is shown as determined on electron microscopy (Energy Dispersive Analysis by x-ray , Cambridge StereoScan S360, Leica) (Panel F). Large amounts of sodium, characteristic of monosodium urate, are detected; the inset shows the needle-like shape of the deposits at higher magnification.
Discussion
Gout has been recognized since ancient times. The Roman surgeon Galen described it as a discharge of drops (gutta in Latin) of the four humors of the body in unbalanced amounts in the joints. Gout was traditionally a wealthy person's disease, because the foods that contribute to its development were available in quantity only to the rich. It is one of the most commonly reported diseases in history. However, as the definitive diagnosis of gout relies on laboratory testing, confirmation of this diagnosis has been possible only during the past few decades.
Our findings are remarkable in two respects. First, the detection of urate crystals in the mummified tissue represents an exceptional finding in paleopathology. The diagnosis of gout in tissue of this type is usually achieved through the indirect but characteristic evidence provided by the erosions produced by urate deposits in bone. Thus, macroscopic and radiologic evidence are usually the only basis for the diagnosis of gout. With the use of this method, evidence of gout has been observed in different human populations4 and even in dinosaurs.5 In our case, the urate composition of the deposits is clearly demonstrated. The histologic features of the material; the characteristic needle-like shape of the birefringent crystals, still identifiable after a short formalin passage6,7; the specific biochemical reaction; and the scanning electron microscopical analysis firmly demonstrate the chemical nature of the crystal deposits as sodium urate.
In addition to biologic confirmation of the clinical suspicion of gout, our study also demonstrates the extreme severity of the emperor's arthritic disease through the identification of massive gouty tophi that completely destroyed the distal interphalanx joint and extended to the neighboring soft tissue. The first references to Charles V's arthritic pain were made when he was 28 years of age.8 In 1532, in a letter to his sister Mary of Hungary, he described how he suffered from "attacks of gout." His physicians recommended that he follow a strict diet, but the emperor had a voracious appetite, especially for meat. He also liked to drink large quantities of beer and wine, and he even ordered a specially designed four-handled drinking mug.9 Thus, his dietary habits were not at all beneficial to reducing the gout attacks. Charles V regularly had such episodes, and in his last years, the articular pain hindered both his mobility and even his ability to write. Letters to his son Philip in 1553 stated that "this is by the hand of Eraso as mine is not able to write long because of the gout"10; and this condition reached the state of ulceration, as revealed to his daughter Joanne of Austria: "this is not by my hand because the small holes of my little finger are open again."11 The destiny of many European countries was closely linked to the vicissitudes of the emperor's life, and his disease probably influenced decisions that affected the future of many countries.
Over time, the gout attacks increased in frequency and severity. In 1552, a terrible attack forced Charles V to postpone the attempt to recapture Metz. As a result, the arrival of the winter allowed the French city to resist the emperor's army. According to some scholars,12 a sense of guilt that his disease had led to this defeat made Charles V decide to abdicate. In 1556, he retired to the monastery of Yuste in western Spain, handing over his vast territories to his son, Philip II, and the Holy Empire to his brother, Ferdinand I. Charles V was only 58 years of age at his death, probably from malaria, in 1558, but he seemed an old and crippled man who could barely walk or use his hands (Figure 1A).
The evidence reported here confirms that Charles V had severe gout, which probably was a determining factor in his decision to abdicate, and illustrates how paleopathological studies can provide important information leading to an increased understanding of history.
This study was made possible by the kind collaboration of the Patrimonio Nacional (the Spanish National Heritage).
No potential conflict of interest relevant to this article was reported.
We are indebted to Montserrat Tortosa and Rafael Molina for the technical assistance; to Julia Esteban and the Serveis Científico-Tècnics de la Universitat de Barcelona for help with bibliographic research and scanning electron microscopy, respectively; to Antonio Martinez and Olga Balague for help with the artwork; to Adam Brooks for the English revision of the manuscript; and to Pablo Larrea for acting as a liaison with the Spanish National Heritage.
Source Information
From the Departments of Pathology (J.O., J.E., E.C., P.L.F.), Radiology (M.V.), and Biochemistry (E.M.), and the Center of International Health (P.L.A., J.Z.), Hospital Clínic d'Investigacions Biomèdiques August Pi i Sunyer, University of Barcelona, Barcelona.
References
Boonen A, van der Linden SJ. Case number 33: about being a famous European and suffering from gout. . . . Ann Rheum Dis 2005;64:528-528.
Sandison AT. Reconstitution of dried-up tissue specimens for histological examination. J Clin Pathol 1966;19:522-523.
Mekota AM, Vermehren M. Determination of optimal rehydration, fixation and staining methods for histological and immunohistochemical analysis of mummified soft tissues. Biotech Histochem 2005;80:7-13.
Rothschild BM, Heathcote GM. Characterization of gout in a skeletal population sample: presumptive diagnosis in a Micronesian population. Am J Phys Anthropol 1995;98:519-525.
Rothschild BM, Tanke D, Carpenter K. Tyrannosaurs suffered from gout. Nature 1997;387:357-357.
Darby AJ, Harness NF, Pritchard MS. Demonstration of urate crystals after formalin fixation. Histopathology 1998;32:382-383.
Eulderink F, Postma T. Demonstration of urate in formalin fixative as support for the histological diagnosis of gout. Histopathology 1997;30:195-195.
Fernandez-Alvarez M. Carlos V. Memorias. In: Fernandez-Alvarez M, ed. Corpus documental de Carlos V. Vol. 4. Madrid: Fundación Academia Europea de Yuste-Espasa, 2003:483-567.
Guerrero-Cabanillas V. Un paciente indisciplinado. In: Guerrero-Cabanillas V, ed. La salud de Carlos V. Yuste, Spain: Fundación Academia Europea de Yuste, 2005:203-10.
Fernandez-Alvarez M. Corpus documental de Carlos V. Vol. 3. Madrid: Fundación Academia Europea de Yuste-Espasa, 2003: 635-41.
Idem. Corpus documental de Carlos V. Vol. 4. Madrid: Fundación Academia Europea de Yuste-Espasa, 2003:332-3.
Guerrero-Cabanillas V. La abdicación imperial. In: Guerrero-Cabanillas V, ed. La salud de Carlos V. Yuste, Spain: Fundación Academia Europea de Yuste, 2005:157-68.(Jaume Ordi, M.D., Pedro L)
Figure 1. Charles V Seated, by Titian (Panel A), and a Special Chair Designed to Carry the Emperor (Panel B).
The portrait of Charles V in his last years shows him incapacitated by severe arthritic pain, with the emperor's swollen left hand in what seems to be a protective position on his lap. (Reprinted with permission of the Alte Pinakothek, Munich.) The chair belonging to Philip II was copied from the original chair designed for his father, Charles V. (Inventory number 10014120, Palacio de los Austrias, Real Monasterio de San Lorenzo de El Escorial, Spanish National Heritage.)
Charles V was buried, in accordance with his wishes, at the monastery of Yuste, but in 1574 his body was transferred to the monastery of El Escorial. Historical reports of this transfer state that the body had undergone spontaneous mummification and that the emperor was completely recognizable. Recently, one of us contacted the Delegation of the Patrimonio Nacional in San Lorenzo de El Escorial in order to study the terminal phalanx of the finger of Charles V, which has been kept outside the coffin in a small box (inventory number, 10044506, Spanish National Heritage) and preserved in the sacristy of the Royal Monastery of San Lorenzo de El Escorial. We report here the finding of massive gouty tophi with demonstrable urate deposits that confirm the clinical suspicion of severe gout in this unique sample from the mummified remains of Charles V.
Methods
A full protocol was developed with the objective of investigating the cause of death and other associated pathologic conditions. The protocol was approved by the Patrimonio Nacional (the Spanish National Heritage) and the institutional review board of the Hospital Clinic-Institut and the University of Barcelona. The remains of the phalanx of the emperor were transported to Barcelona in the original red velvet box. The office of the Spanish National Heritage established, on the basis of historical documents, that the fingertip was considered part of the remains of Charles V. It was not possible to obtain DNA evidence. The specimen consisted of part of the final phalanx of the fifth finger of one of the hands (laterality unknown). Externally, the finger fragment had a dark-brown color and leathery texture. The proximal part of the nail was attached to the ungual bed, whereas the outer half was completely detached (Figure 2A). A radiographic study was performed.
Figure 2. Macroscopic View of the Mummified Finger of Charles V.
Panel A shows the specimen consisting of part of the final phalanx of the fifth finger of one of the hands of Charles V. The finger fragment has a dark-brown color, which corresponded to the soft parts. The proximal part of the nail was attached to the ungual bed, whereas the outer half was completely detached. Panel B is a radiograph showing extensive erosion of the proximal epiphysis of the phalanx, which had irregular borders and soft-tissue calcifications. Panel C shows the proximal edge of the specimen, with a massive yellowish deposit that has a chalky appearance.
The specimen was rehydrated by total immersion in Sandison's solution2,3 for one hour under visual inspection. After rehydration, the most proximal portion, consisting of the epiphysis of the joint, was immediately processed for histopathological characteristics. Tissue samples were embedded in paraffin according to standard protocols used for fresh tissue. After removal of the paraffin, sections that were 4 μm thick were processed with graded alcohols and water, and routinely stained with hematoxylin and eosin, Masson trichrome, and Giemsa.
The chemical constitution of the deposits was analyzed by scanning electron microscopy (Energy Dispersive Analysis by x-ray , Cambridge StereoScan S360, Leica) and an x-ray microanalysis system (INCA 200, Oxford Instruments). After removal of the paraffin, a section that was 4 μm thick was carbon-coated for electrical conductivity and examined by electron microscopy. Another section of the phalanx that was 20 μm thick, after removal of the paraffin with xylene and absolute alcohol, was immersed in 3 ml of uric-acid reagent for automated analysis (Bayer Advia, Bayer Diagnostics) at room temperature. The reaction is based on the conversion of uric acid to allantoin and peroxide by uricase. The presence of urate is detected by the identification of a colored complex formed from hydrogen peroxide. The method has a limit of sensitivity of 0.1 mg per deciliter.
Results
The radiographic studies showed extensive erosion of the proximal epiphysis of the phalanx, which had irregular borders and soft-tissue calcifications (Figure 2B). Macroscopic examination disclosed a yellowish deposit with a chalky appearance that completely occupied the proximal edge of the specimen (Figure 2C).
The histologic analysis revealed well-preserved dermal collagen and bone, but no preserved cells of any type were detected, except isolated red cells. At low magnification, no bone could be identified in the proximal margin of the specimen. At this location, a massive deposit of weakly basophilic, cloudy, fine fibrillar masses that completely occupied the center of the specimen, eroding the bone and extending into adjacent soft tissue, was observed (Figure 3A and 3B). Large amounts of fuzzy and crystalline material were identified in the center of these masses. Under polarized light, strongly birefringent sheaves and stacks of needle-shaped crystals consistent with urate were observed (Figure 3C and 3D). Scanning electron microscopy showed a large deposit of crystalline structures that had eroded and destroyed the bone (Figure 3E). At higher magnification, these deposits had a needle-like shape (Figure 3F, inset). The elemental constitution of the crystalloid deposits as determined on scanning electron microscopy is shown in Figure 3F. Large amounts of sodium, as expected for monosodium urate, were detected. The chemical analysis with uricase gave a strong positive reaction (not shown).
Figure 3. Microscopical Study of the Finger.
Specimens shown in Panels A, B, C, and D were stained with hematoxylin and eosin. In Panel A, at low magnification, there is a large deposit of weakly basophilic masses completely occupying the center of the specimen and destroying the bone. Large amounts of crystalline material are identified in the center of these masses. In Panel B, at higher magnification, the weakly basophilic, cloudy, fine fibrillar masses seen are characteristic of gout. In Panels C and D, under polarized light, strongly birefringent sheaves and stacks of needle-shaped urate crystals can be identified. Panels E and F show the scanning electron microscopical analysis. A massive deposit of crystalline structures is identifiable (Panel E), which erode and destroy the bone. The elemental constitution of the crystalloid deposits is shown as determined on electron microscopy (Energy Dispersive Analysis by x-ray , Cambridge StereoScan S360, Leica) (Panel F). Large amounts of sodium, characteristic of monosodium urate, are detected; the inset shows the needle-like shape of the deposits at higher magnification.
Discussion
Gout has been recognized since ancient times. The Roman surgeon Galen described it as a discharge of drops (gutta in Latin) of the four humors of the body in unbalanced amounts in the joints. Gout was traditionally a wealthy person's disease, because the foods that contribute to its development were available in quantity only to the rich. It is one of the most commonly reported diseases in history. However, as the definitive diagnosis of gout relies on laboratory testing, confirmation of this diagnosis has been possible only during the past few decades.
Our findings are remarkable in two respects. First, the detection of urate crystals in the mummified tissue represents an exceptional finding in paleopathology. The diagnosis of gout in tissue of this type is usually achieved through the indirect but characteristic evidence provided by the erosions produced by urate deposits in bone. Thus, macroscopic and radiologic evidence are usually the only basis for the diagnosis of gout. With the use of this method, evidence of gout has been observed in different human populations4 and even in dinosaurs.5 In our case, the urate composition of the deposits is clearly demonstrated. The histologic features of the material; the characteristic needle-like shape of the birefringent crystals, still identifiable after a short formalin passage6,7; the specific biochemical reaction; and the scanning electron microscopical analysis firmly demonstrate the chemical nature of the crystal deposits as sodium urate.
In addition to biologic confirmation of the clinical suspicion of gout, our study also demonstrates the extreme severity of the emperor's arthritic disease through the identification of massive gouty tophi that completely destroyed the distal interphalanx joint and extended to the neighboring soft tissue. The first references to Charles V's arthritic pain were made when he was 28 years of age.8 In 1532, in a letter to his sister Mary of Hungary, he described how he suffered from "attacks of gout." His physicians recommended that he follow a strict diet, but the emperor had a voracious appetite, especially for meat. He also liked to drink large quantities of beer and wine, and he even ordered a specially designed four-handled drinking mug.9 Thus, his dietary habits were not at all beneficial to reducing the gout attacks. Charles V regularly had such episodes, and in his last years, the articular pain hindered both his mobility and even his ability to write. Letters to his son Philip in 1553 stated that "this is by the hand of Eraso as mine is not able to write long because of the gout"10; and this condition reached the state of ulceration, as revealed to his daughter Joanne of Austria: "this is not by my hand because the small holes of my little finger are open again."11 The destiny of many European countries was closely linked to the vicissitudes of the emperor's life, and his disease probably influenced decisions that affected the future of many countries.
Over time, the gout attacks increased in frequency and severity. In 1552, a terrible attack forced Charles V to postpone the attempt to recapture Metz. As a result, the arrival of the winter allowed the French city to resist the emperor's army. According to some scholars,12 a sense of guilt that his disease had led to this defeat made Charles V decide to abdicate. In 1556, he retired to the monastery of Yuste in western Spain, handing over his vast territories to his son, Philip II, and the Holy Empire to his brother, Ferdinand I. Charles V was only 58 years of age at his death, probably from malaria, in 1558, but he seemed an old and crippled man who could barely walk or use his hands (Figure 1A).
The evidence reported here confirms that Charles V had severe gout, which probably was a determining factor in his decision to abdicate, and illustrates how paleopathological studies can provide important information leading to an increased understanding of history.
This study was made possible by the kind collaboration of the Patrimonio Nacional (the Spanish National Heritage).
No potential conflict of interest relevant to this article was reported.
We are indebted to Montserrat Tortosa and Rafael Molina for the technical assistance; to Julia Esteban and the Serveis Científico-Tècnics de la Universitat de Barcelona for help with bibliographic research and scanning electron microscopy, respectively; to Antonio Martinez and Olga Balague for help with the artwork; to Adam Brooks for the English revision of the manuscript; and to Pablo Larrea for acting as a liaison with the Spanish National Heritage.
Source Information
From the Departments of Pathology (J.O., J.E., E.C., P.L.F.), Radiology (M.V.), and Biochemistry (E.M.), and the Center of International Health (P.L.A., J.Z.), Hospital Clínic d'Investigacions Biomèdiques August Pi i Sunyer, University of Barcelona, Barcelona.
References
Boonen A, van der Linden SJ. Case number 33: about being a famous European and suffering from gout. . . . Ann Rheum Dis 2005;64:528-528.
Sandison AT. Reconstitution of dried-up tissue specimens for histological examination. J Clin Pathol 1966;19:522-523.
Mekota AM, Vermehren M. Determination of optimal rehydration, fixation and staining methods for histological and immunohistochemical analysis of mummified soft tissues. Biotech Histochem 2005;80:7-13.
Rothschild BM, Heathcote GM. Characterization of gout in a skeletal population sample: presumptive diagnosis in a Micronesian population. Am J Phys Anthropol 1995;98:519-525.
Rothschild BM, Tanke D, Carpenter K. Tyrannosaurs suffered from gout. Nature 1997;387:357-357.
Darby AJ, Harness NF, Pritchard MS. Demonstration of urate crystals after formalin fixation. Histopathology 1998;32:382-383.
Eulderink F, Postma T. Demonstration of urate in formalin fixative as support for the histological diagnosis of gout. Histopathology 1997;30:195-195.
Fernandez-Alvarez M. Carlos V. Memorias. In: Fernandez-Alvarez M, ed. Corpus documental de Carlos V. Vol. 4. Madrid: Fundación Academia Europea de Yuste-Espasa, 2003:483-567.
Guerrero-Cabanillas V. Un paciente indisciplinado. In: Guerrero-Cabanillas V, ed. La salud de Carlos V. Yuste, Spain: Fundación Academia Europea de Yuste, 2005:203-10.
Fernandez-Alvarez M. Corpus documental de Carlos V. Vol. 3. Madrid: Fundación Academia Europea de Yuste-Espasa, 2003: 635-41.
Idem. Corpus documental de Carlos V. Vol. 4. Madrid: Fundación Academia Europea de Yuste-Espasa, 2003:332-3.
Guerrero-Cabanillas V. La abdicación imperial. In: Guerrero-Cabanillas V, ed. La salud de Carlos V. Yuste, Spain: Fundación Academia Europea de Yuste, 2005:157-68.(Jaume Ordi, M.D., Pedro L)