- Email: [email protected]
Uraemia: is urea more important than we think?
1438
ME, Smith GS,
state
REFERENCES 1. HM Chief Inspector of Prisons for
Brewer TF. Suicide mortality in the Maryland 1979 through 1987. JAMA 1989; 262: 365-69. 6. Burtch BE. Suicide in prison: a commentary. Br J Psychiatry 1979; 135: 90. 7. Hurley W. Suicides by prisoners. Med J Aust 1989; 151: 188-90. 8. Bland RC, Newman SC, Dyck RJ, Orn H. Prevalence of psychiatric disorders and suicide attempts in a prison population. Can J Psychiatry 5. Salive
We thank the New Zealand Departments of Justice, Statistics, and Health (Statistical Services) for data not included in published reports.
England and Wales. Suicide and self harm in prison service establishments m England and Wales. London: HM Stationery Office, 1990. 2. Report of the Committee of Inquiry into procedures used in certain psychiatric hospitals in relation to admission, discharge or release on leave of certain classes of patients. Wellington: Government Printer, 1988. 3. Dooley E. Prison suicide in England and Wales, 1972-87. Br J Psychiatry 1990; 156: 40-45. 4. Backett SA. Suicide in Scottish prisons. Br J Psychiatry 1987; 151: 218-21.
prison system,
1990; 35: 407-13. 9. Niemi T. The time-space distances of suicides committed in the lock-up in Finland in 1963-1967. Psychiatria Fennica 1975: 267-70. 10. Cox JF, McCarty DW, Landsberg G, Paravati MP. A model for crisis intervention services within local jails. Int J Law Psychiatry 1988; 11: 391-407. 11. Skegg K, Cox B. Suicide in New Zealand 1957-1986: the influence of age, period and birth-cohort. Aust NZ J Psychiatry 1991; 25: 181-90.
HYPOTHESIS Uraemia: is
urea more
Urea is accumulated as an osmolyte by some groups of animals even though it impairs protein function. These organisms can withstand high internal urea concentrations because they also accumulate other low-molecular-weight osmolytes, the methylamines, which can offset the effects of urea on proteins. Methylamines have also been found in the medulla of the mammalian kidney (where urea concentrations are high) and in the plasma of human subjects with chronic renal failure. These findings suggest that previous investigations of the potential contribution of urea to the syndrome of uraemia may have been confounded because of the presence of variable concentrations of protective substances. That naturally occurring methylamines or related substances may prove to have a useful therapeutic role in uraemia is also possible. Introduction Loss of functional nephrons in chronic renal failure leads uraemia, which is widely thought to be due to the accumulation of waste products of protein metabolism in the body fluids. These "uraemic toxins" are thought to produce their effects through their diverse actions on protein function, particularly by inhibition of various enzyme activities. Many uraemic toxins have been proposed, including urea, creatinine, guanidines, aliphatic and aromatic amines, phenols, indole, aromatic hydroxyacids, oxalic acid, uric acid, and possibly "middle molecules" ,2.3 all of which accumulate in uraemia and have various toxic effects. However, since no single compound, or family of compounds, correlates well with uraemic symptoms, the development of uraemia is generally attributed to the accumulation of many toxins, acting with electrolyte and other metabolic and hormonal disturbances. Of the potential uraemic toxins, the concentration of urea alone is much higher than that of all the others combined (up to
important than we think?
to tens
of mmol/1 vs a few mmol/1), but urea is conventionally
as one of the least toxic protein metabolites that accumulate in uraemia. This is partly because of the high levels of urea that can be tolerated. Also, plasma urea concentrations are variable (rising with high protein intake and in catabolic states) and hence are difficult to correlate with uraemic symptoms. Clinical studies have generally shown a poor correlation between plasma urea concentration and the clinical state of the patient. In one study, for example, haemodialysis against high concentrations of urea in the dialysate led to a good clinical response by uraemic patients despite negligible change in the blood urea concentration.4 By contrast, in another study when high plasma urea concentrations were produced by the addition of urea to the dialysate, malaise, apathy, drowsiness, and glucose intolerance followed.s The main difficulty with these studies, and similar studies that attempt to assess the role of urea in uraemia, is that they have been unable to control adequately for the simultaneous effects of treatment on electrolytes, hormones, and other variables. In dogs with bilateral nephrectomy, maintained on peritoneal dialysis, an accelerated uraemic syndrome develops if urea is added to the dialysate.6 Such data show that urea can have important toxic effects in uraemia. In fact, it would be surprising if urea were not implicated in the cause of uraemic syndrome, since in addition to its presence in high concentrations, it is also a well-known destabiliser of protein structure. That urea is also able to diffuse across cell membranes and enter cells suggests that it is likely to produce widespread effects on protein function.
regarded
ADDRESSES: University Department of Pathology, Royal Victoria Infirmary, Newcastle upon Tyne NE1 4LP (J A. Lee, PhD); Department of Renal Medicine, St Mary’s Hospital, Milton Road, Portsmouth (Prof H A Lee, FRCP); Department of Chemistry, Birkbeck College, University of London, Christopher Ingold Laboratories and Gordon House, Gordon Square, London (P J Sadler, PhD), UK. Correspondence to Dr J.A Lee
1439
then has it been so difficult to correlate urea concentrations with uraemic symptoms? And, how can we explain an early related observation that if the ureters are transplanted into the jejunum or ileum, there is a pronounced increase in blood urea but symptoms of uraemia are not produced?7 New evidence from various sources provides a possible explanation of these findings and also suggests why tb--potential role of urea as a uraemic toxin may have been previously underestimated. But first we shall fill in some background.
Why
Background
by urea,13.1S yet they apparently function adequately in medullary cells. As in species that accumulate urea throughout their body fluids, it is the accumulation of methylamines that offsets the effects of urea in these cells11.1618 (and also, incidentally, helps balance the intracellular and extracellular osmolarities in the medulla). In the dog, the main medullary methylamines seem to be betaine and glycerylphosphorylcholine.ll.16 Concentrations of methylamines increase from the cortex to the inner medulla, in concert with the rising urea concentration19-a finding that suggests that synthesis of methylamines may be sensitive
to
the urea concentration.
A lesson from sharks
Many organisms that live
in osmotically challenging environments such as sea water accumulate low-molecularweight substances within their body fluids to maintain osmotic equilibrium.8-1° In cartilaginous fishes (eg, sharks, rays) the principal low-molecular-weight osmolyte is urea (up to several hundred mmol/1). If urea has such strongly perturbing effects on macromolecular structure, how do cartilaginous fishes cope with high concentrations of urea throughout their bodies? In principle there are two possible solutions.ll Firstly, the proteins in these animals could have evolved to be relatively insensitive to the effects of urea, or secondly, there might be some chemical means of offsetting the effects of urea on the proteins. A few proteins of cartilaginous fishes are indeed less sensitive to the effects of urea than are normal proteins. 12 However, the finding that most proteins of urea-rich fishes are as sensitive to the effects of urea as are the homologous proteins in species that do not accumulate urea focused attention on the other major class of organic osmolyte found in urea-accumulating speciesnamely, the methylamines--eg, trimethylamine-N-oxide [TMAO], betaine, glycerylphosphorylcholine, and
sarcosine.S-ll These compounds are potent in counteracting the effects urea on protein function,13 and these "antidote" properties are most pronounced when the ratio of summed methylamines to urea is about 1/2, which is that found in most species of urea-retaining fish. An important observation is that the urea-counteracting effects of methylamines are independent of the species source of the protein: cartilaginous fish, bony fish, amphibian, and mammalian proteins all respond similarly, irrespective of whether these solutes are usually present in vivo.13 Is this interesting story merely a curiosity or does it have relevance to mammalian (particularly human) physiology? The answer is that it does, because there is one place in mammals where the internal environment has striking similarities to that of a cartilaginous fish-namely, the renal medulla.
of
The renal medulla
Owing to the countercurrent flow mechanism for concentrating urine, there is a large osmotic gradient in the renal medulla, increasing in the direction of the inner renal medulla and renal papillae. The increased extracellular osmolarity is produced by high concentrations of sodium chloride and urea, which are concentrated in this region by the unique arrangement of the renal tubules and medullary blood supply. However, whereas the increased salt concentration occurs in the extracellular compartment, 14 the high urea concentration also occurs intracellularly because of the high lipid solubility of urea. In vitro, enzymes that serve important functions in the renal medulla are inhibited
Hypothesis potential importance of these findings for understanding the pathophysiology of uraemia can easily be appreciated. The presence of protective methylamines in man and their accumulation in uraemia might account not only for the low toxicity when blood urea reaches high concentrations, but also for the poor correlation between The
concentration and uraemic symptoms, since there is another important (protective) variable that has not previously been measured. Additionally, accumulation of methylamines as the urea concentration rises might explain the lack of uraemic symptoms seen in the ureteric transplantation experiment mentioned earlier. Nuclear magnetic resonance spectroscopy (NMR) has provided evidence that methylamines accumulate in chronic renal failure. Using 1H, 13C, and 14N NMR, we did not find methylamines in the plasma of healthy human subjects, but in plasma from patients with chronic renal failure we noted additional peaks-ie, a small peak identified as dimethylamine and a larger peak assigned to TMAO .20 We also found that the concentration of TMAO seemed to correlate with that of urea and creatinine: the extracellular ratio of TMAO to urea was about 1/100, but substantially higher concentrations of this charged compound would be expected in the intracellular compartment. The NMR data also showed that lactate interacts differently with plasma proteins in uraemic plasma than in normal plasma-ie, much less lactate was bound. More research is needed on this ability of urea to cause regiospecific changes in protein structure, especially in the case of plasma proteins. Although urea is best known to chemists as urea
a
global protein-unfolding agent (denaturant)
at
high
concentrations (1 1 mol/1), it is clear from the NMR data that important effects also occur at the lower concentrations seen in uraemia and are thus likely to be important in the pathophysiology of this condition. That urea can induce local structural changes (distinct from the complete removal of ordered structure from proteins) is evidenced by circular dichroism spectroscopy,21 which has shown that urea can induce a stereospecific stabilisation of a left-handed extended helix in a polypeptide epitope of a virus. The secondary and tertiary structures of proteins are vital in many protein recognition processes, such as receptor binding or antigen-antibody interactions, and even slight perturbation of these structures may have substantial pathophysiological effects. An important area for future research is to refine our understanding of the roles of both free and bound urea and methylamines in the production of these effects. Thus, we may uncover specific binding, transport, and delivery systems for these and other important small molecules and provide new targets for
specific drug design.
1440
One
of from
source
methylamines is diet; they
are
also
metabolism choline and of produced phosphatidylcholines by gut bacteria.22 In the gut, choline is converted to trimethylamine (and also, to a lesser extent, dimethylamine) before being internally oxidised to TMAO and then excreted in the bile and urine.22 After absorption, dietary TMAO is reduced and reoxidised again, and impaired N-oxidation can be inherited as a recessive trait.22,23 Also, it now seems likely that methylamines are also synthesised endogenously. In man, betaine is excreted in urine from the neonatal period until about the age of 2 years but is not excreted by adults, in whom dimethylamine is produced instead.24-26 Indeed, it has been suggested that dimethylamine excretion could be used to assess renal function since dimethylamine, like creatinine, is excreted at a constant rate during fasting.27 However, little else is yet known of methylamine physiology in human health and
disease. There
are numerous straightforward questions which require study. Is the accumulation of TMAO or other intracellular methylamines in uraemia strongly correlated
with urea concentration? Are the intracellular TMAO concentrations high enough to offset the toxic effects of urea? Does the plasma concentration of methylamines accurately reflect intracellular values? Does taking into account the plasma concentrations of TMAO and other methylamines improve the correlation between urea and symptoms of uraemia? Do other methods of increasing the plasma urea (such as ureteric transplantation) stimulate an increase in methylamines? Can symptoms be ameliorated by administration of TMAO or other methylamines? How does a reduction in dietary protein content, widely believed to decelerate deterioration of renal function in chronic renal failure ’28 29 affect the relation between urea and methylamines in chronic renal failure? What are the developmental influences on the relation between urea and methylamines? These and other questions await answers, and we hope that this article will stimulate interest in this little-explored area of human pathophysiology. REFERENCES 1. Knochel
Seldin DW. The pathophysiology of uraemia. In: Brenner MM, Rector FC, eds. The kidney. Philadelphia: Saunders, 1976: 1448-85. 2. Wills MR. Metabolic consequences of chronic renal failure. Aylesbury: HM & M Publishers, 1978. 3. Barsotti G. Uraemic toxins. In: Giovanetti S, ed. Nutritional treatment of chronic renal failure. Boston: Kluver, 1989: 33-40. 4. Merrill JP, Legrain M, Hoigne R. Observations on the role of urea in uraemia. Am J Med 1953; 14: 519-20. 5. Hutchings RM, Hegstrom RM, Scribner BH. Glucose intolerance in patients on long-term haemodialysis. Ann Intern Med 1966; 65: 275-85. 6. Gilboe DD, Javid MJ. Breakdown products of urea and uraemic syndrome. Proc Soc Exp Biol Med 1964; 115: 633-37. 7. Bollman JL, Mann FC. Nitrogenous constituents of the blood following transplantation of the ureters into different levels of the intestine. Proc Soc Exp Biol Med 1927; 24: 923-24. 8. Yancey PH, Clark ME, Hand SC, Bowlus RD, Somero GN. Living with water stress: evolution of osmolyte systems. Science 1982: 217: 1214-22. 9. Somero GN. Protons, osmolytes and fitness of internal milieu for protein function. Am J Physiol 1986; 251: R197-R213. 10. Chamberlin ME, Strange K. Anisosmotic cell volume regulation: a comparative review. Am J Physiol 1989; 257: C159-73 11. Somero GN. From dogfish to dogs: trimethylamines protect proteins from urea. News in Physiol Sci 1986; 1: 9-12. 12. Bonaventura J, Bonaventura C, Sullivan B. Urea tolerance as a molecular adaptation of elasmobranch hemoglobins. Science 1974; 186: 57-59. 13. Yancey PH, Somero GN. Counteraction of urea destabilisation of protein structure by methylamine osmoregulatory compounds of elasmobranch fishes Biochem J 1979; 183: 317-23.
14. Beck F, Dorge A, Rick R, Thurau K. Intra- and extracellular elemental concentrations of rat renal papilla in antidiuresis. Kidney Int 1984; 25: 397-403. 15. Gutman Y, Katzper-Shamir Y. The effects of urea, sodium and calcium on microsomal ATPase activity in different parts of the kidney. Biochim Biophys Acta 1971; 233: 133-36. 16. Balaban RS, Knepper MA. Nitrogen 14 nuclear magnetic resonance spectroscopy of mammalian tissues. Am J Physiol 1983; 245: C438-
C444. 17.
Bagnasco S, Balaban RS, Fales H, Yong Y-M, Burg M. Identification of intracellular organic osmolytes in renal inner medulla. J Biol Chem
1986; 201: 5872-77. 18. Balaban RS, Burg MB. Osmotically active organic solutes in the renal inner medulla. Kidney Int 1987; 31: 562-64. 19. Yancey PH, Burg MB. Distribution of major organic osmolytes in rabbit kidneys in diuresis and antidiuresis. Am J Physiol 1989; 257: F602-07. 20. Bell JD, Lee JA, Lee HA, Sadler PJ, Wilkie DR, Woodham RH. Nuclear magnetic resonance studies of blood plasma and urine from subjects with chronic renal failure: identification of trimethylamine-N-oxide. Biochim Biophys Acta 1991; 1096: 101-07. 21. Siligardi G, Drake AF, Mascagni P, Rowlands P, Brown F, Gibbons WA. A CD strategy for the study of polypeptide folding/unfolding: a synthetic foot-and-mouth disease virus immunogenic peptide. Int J Peptide Protein Res (in press). 22. Al-Waiz M, Ayesh R, Mitchell SC, Idle JR, Smith RL. A genetic polymorphism of the N-oxidation of trimethylamine in humans. Clin Pharmacol Ther 1987; 42: 588-94. 23. Al-Waiz M, Ayesh R, Mitchell SC, Idle JR, Smith RL. Disclosure of the metabolic retroversion of trimethylamine-N-oxide in humans: a pharmacogenetic approach. Clin Pharmacol Ther 1987; 42: 608-12. 24. Davies SEC, Chalmers RA, Randall EW, Iles RA. Betaine metabolism in human neonates and developing rats. Clin Chem Acta 1988; 178: 241-50. 25. Bell JD, Sadler PJ, Morris VC, Levander DA. Effect of aging and diet on proton NMR spectra of rat urine. Magnetic Resonance Med 1991; 17: 414-22. 26. Brown JCC, Miels GA, Sadler PJ, Walker V. 1H NMR studies of urine from premature and sick babies. Magnetic Resonance Med 1989; 11: 193-201. 27. Bates JR, Bell JD, Nicholson JK, Sadler PJ. 1H NMR studies of urine during fasting: excretion of ketone bodies and acetylcarnitine. Magnetic Resonance Med 1986; 3: 849-56. 28. Diamond JR. Effects of dietary interventions on glomerular pathophysiology. Am J Physiol 1990; 27: F1-8. 29. Brenner BM, Meyer TW, Hostetter TH. Dietary protein intake and the progressive nature of kidney disease. N Engl J Med 1982; 307: 652-59.
From The Lancet Clinical
teaching
JP,
In relation to the profession, hospitals are intended to supply to the student a knowledge of disease; they are the laboratories in which the various manipulations and processes necessary for the cure of physical suffering are conducted. Within their walls human misery displays a thousand varied shapes; the student is appalled at the immensity of the labour that seems spread before him, and n.’ friendly hand is stretched forth to aid him through his difficulties. Clinical teaching has become a mere word, without signification; and the few hasty remarks made at the bedside are directed to those who are already informed upon the subject, and tend only to perplex still more the junior student. The too frequent effect, on the mind of the student, of the difficulties met with in comprehending the nature of disease, from its protean manifestations, and of the absence of all assistance or stimulus, excepting that distant and little regarded one-a sound knowledge of his profession-is a distaste for the duties of the hospital, and, as a consequence, gradual neglect of their performance-a neglect in which the well-disposed are as apt to take part as the idle. In those hospitals in which a casebook is kept, the industrious student finds a great resource in the perusal of its pages... We trust that all who have any influence in the hospitals will consider it to be a duty... to devote some portion of their time to the consideration of the manner in which so important a benefit to those institutions could be obtained, as would be conferred by the universal record of their proceedings in hospital case-books.
(July 10, 1841)
ME, Smith GS,
state
REFERENCES 1. HM Chief Inspector of Prisons for
Brewer TF. Suicide mortality in the Maryland 1979 through 1987. JAMA 1989; 262: 365-69. 6. Burtch BE. Suicide in prison: a commentary. Br J Psychiatry 1979; 135: 90. 7. Hurley W. Suicides by prisoners. Med J Aust 1989; 151: 188-90. 8. Bland RC, Newman SC, Dyck RJ, Orn H. Prevalence of psychiatric disorders and suicide attempts in a prison population. Can J Psychiatry 5. Salive
We thank the New Zealand Departments of Justice, Statistics, and Health (Statistical Services) for data not included in published reports.
England and Wales. Suicide and self harm in prison service establishments m England and Wales. London: HM Stationery Office, 1990. 2. Report of the Committee of Inquiry into procedures used in certain psychiatric hospitals in relation to admission, discharge or release on leave of certain classes of patients. Wellington: Government Printer, 1988. 3. Dooley E. Prison suicide in England and Wales, 1972-87. Br J Psychiatry 1990; 156: 40-45. 4. Backett SA. Suicide in Scottish prisons. Br J Psychiatry 1987; 151: 218-21.
prison system,
1990; 35: 407-13. 9. Niemi T. The time-space distances of suicides committed in the lock-up in Finland in 1963-1967. Psychiatria Fennica 1975: 267-70. 10. Cox JF, McCarty DW, Landsberg G, Paravati MP. A model for crisis intervention services within local jails. Int J Law Psychiatry 1988; 11: 391-407. 11. Skegg K, Cox B. Suicide in New Zealand 1957-1986: the influence of age, period and birth-cohort. Aust NZ J Psychiatry 1991; 25: 181-90.
HYPOTHESIS Uraemia: is
urea more
Urea is accumulated as an osmolyte by some groups of animals even though it impairs protein function. These organisms can withstand high internal urea concentrations because they also accumulate other low-molecular-weight osmolytes, the methylamines, which can offset the effects of urea on proteins. Methylamines have also been found in the medulla of the mammalian kidney (where urea concentrations are high) and in the plasma of human subjects with chronic renal failure. These findings suggest that previous investigations of the potential contribution of urea to the syndrome of uraemia may have been confounded because of the presence of variable concentrations of protective substances. That naturally occurring methylamines or related substances may prove to have a useful therapeutic role in uraemia is also possible. Introduction Loss of functional nephrons in chronic renal failure leads uraemia, which is widely thought to be due to the accumulation of waste products of protein metabolism in the body fluids. These "uraemic toxins" are thought to produce their effects through their diverse actions on protein function, particularly by inhibition of various enzyme activities. Many uraemic toxins have been proposed, including urea, creatinine, guanidines, aliphatic and aromatic amines, phenols, indole, aromatic hydroxyacids, oxalic acid, uric acid, and possibly "middle molecules" ,2.3 all of which accumulate in uraemia and have various toxic effects. However, since no single compound, or family of compounds, correlates well with uraemic symptoms, the development of uraemia is generally attributed to the accumulation of many toxins, acting with electrolyte and other metabolic and hormonal disturbances. Of the potential uraemic toxins, the concentration of urea alone is much higher than that of all the others combined (up to
important than we think?
to tens
of mmol/1 vs a few mmol/1), but urea is conventionally
as one of the least toxic protein metabolites that accumulate in uraemia. This is partly because of the high levels of urea that can be tolerated. Also, plasma urea concentrations are variable (rising with high protein intake and in catabolic states) and hence are difficult to correlate with uraemic symptoms. Clinical studies have generally shown a poor correlation between plasma urea concentration and the clinical state of the patient. In one study, for example, haemodialysis against high concentrations of urea in the dialysate led to a good clinical response by uraemic patients despite negligible change in the blood urea concentration.4 By contrast, in another study when high plasma urea concentrations were produced by the addition of urea to the dialysate, malaise, apathy, drowsiness, and glucose intolerance followed.s The main difficulty with these studies, and similar studies that attempt to assess the role of urea in uraemia, is that they have been unable to control adequately for the simultaneous effects of treatment on electrolytes, hormones, and other variables. In dogs with bilateral nephrectomy, maintained on peritoneal dialysis, an accelerated uraemic syndrome develops if urea is added to the dialysate.6 Such data show that urea can have important toxic effects in uraemia. In fact, it would be surprising if urea were not implicated in the cause of uraemic syndrome, since in addition to its presence in high concentrations, it is also a well-known destabiliser of protein structure. That urea is also able to diffuse across cell membranes and enter cells suggests that it is likely to produce widespread effects on protein function.
regarded
ADDRESSES: University Department of Pathology, Royal Victoria Infirmary, Newcastle upon Tyne NE1 4LP (J A. Lee, PhD); Department of Renal Medicine, St Mary’s Hospital, Milton Road, Portsmouth (Prof H A Lee, FRCP); Department of Chemistry, Birkbeck College, University of London, Christopher Ingold Laboratories and Gordon House, Gordon Square, London (P J Sadler, PhD), UK. Correspondence to Dr J.A Lee
1439
then has it been so difficult to correlate urea concentrations with uraemic symptoms? And, how can we explain an early related observation that if the ureters are transplanted into the jejunum or ileum, there is a pronounced increase in blood urea but symptoms of uraemia are not produced?7 New evidence from various sources provides a possible explanation of these findings and also suggests why tb--potential role of urea as a uraemic toxin may have been previously underestimated. But first we shall fill in some background.
Why
Background
by urea,13.1S yet they apparently function adequately in medullary cells. As in species that accumulate urea throughout their body fluids, it is the accumulation of methylamines that offsets the effects of urea in these cells11.1618 (and also, incidentally, helps balance the intracellular and extracellular osmolarities in the medulla). In the dog, the main medullary methylamines seem to be betaine and glycerylphosphorylcholine.ll.16 Concentrations of methylamines increase from the cortex to the inner medulla, in concert with the rising urea concentration19-a finding that suggests that synthesis of methylamines may be sensitive
to
the urea concentration.
A lesson from sharks
Many organisms that live
in osmotically challenging environments such as sea water accumulate low-molecularweight substances within their body fluids to maintain osmotic equilibrium.8-1° In cartilaginous fishes (eg, sharks, rays) the principal low-molecular-weight osmolyte is urea (up to several hundred mmol/1). If urea has such strongly perturbing effects on macromolecular structure, how do cartilaginous fishes cope with high concentrations of urea throughout their bodies? In principle there are two possible solutions.ll Firstly, the proteins in these animals could have evolved to be relatively insensitive to the effects of urea, or secondly, there might be some chemical means of offsetting the effects of urea on the proteins. A few proteins of cartilaginous fishes are indeed less sensitive to the effects of urea than are normal proteins. 12 However, the finding that most proteins of urea-rich fishes are as sensitive to the effects of urea as are the homologous proteins in species that do not accumulate urea focused attention on the other major class of organic osmolyte found in urea-accumulating speciesnamely, the methylamines--eg, trimethylamine-N-oxide [TMAO], betaine, glycerylphosphorylcholine, and
sarcosine.S-ll These compounds are potent in counteracting the effects urea on protein function,13 and these "antidote" properties are most pronounced when the ratio of summed methylamines to urea is about 1/2, which is that found in most species of urea-retaining fish. An important observation is that the urea-counteracting effects of methylamines are independent of the species source of the protein: cartilaginous fish, bony fish, amphibian, and mammalian proteins all respond similarly, irrespective of whether these solutes are usually present in vivo.13 Is this interesting story merely a curiosity or does it have relevance to mammalian (particularly human) physiology? The answer is that it does, because there is one place in mammals where the internal environment has striking similarities to that of a cartilaginous fish-namely, the renal medulla.
of
The renal medulla
Owing to the countercurrent flow mechanism for concentrating urine, there is a large osmotic gradient in the renal medulla, increasing in the direction of the inner renal medulla and renal papillae. The increased extracellular osmolarity is produced by high concentrations of sodium chloride and urea, which are concentrated in this region by the unique arrangement of the renal tubules and medullary blood supply. However, whereas the increased salt concentration occurs in the extracellular compartment, 14 the high urea concentration also occurs intracellularly because of the high lipid solubility of urea. In vitro, enzymes that serve important functions in the renal medulla are inhibited
Hypothesis potential importance of these findings for understanding the pathophysiology of uraemia can easily be appreciated. The presence of protective methylamines in man and their accumulation in uraemia might account not only for the low toxicity when blood urea reaches high concentrations, but also for the poor correlation between The
concentration and uraemic symptoms, since there is another important (protective) variable that has not previously been measured. Additionally, accumulation of methylamines as the urea concentration rises might explain the lack of uraemic symptoms seen in the ureteric transplantation experiment mentioned earlier. Nuclear magnetic resonance spectroscopy (NMR) has provided evidence that methylamines accumulate in chronic renal failure. Using 1H, 13C, and 14N NMR, we did not find methylamines in the plasma of healthy human subjects, but in plasma from patients with chronic renal failure we noted additional peaks-ie, a small peak identified as dimethylamine and a larger peak assigned to TMAO .20 We also found that the concentration of TMAO seemed to correlate with that of urea and creatinine: the extracellular ratio of TMAO to urea was about 1/100, but substantially higher concentrations of this charged compound would be expected in the intracellular compartment. The NMR data also showed that lactate interacts differently with plasma proteins in uraemic plasma than in normal plasma-ie, much less lactate was bound. More research is needed on this ability of urea to cause regiospecific changes in protein structure, especially in the case of plasma proteins. Although urea is best known to chemists as urea
a
global protein-unfolding agent (denaturant)
at
high
concentrations (1 1 mol/1), it is clear from the NMR data that important effects also occur at the lower concentrations seen in uraemia and are thus likely to be important in the pathophysiology of this condition. That urea can induce local structural changes (distinct from the complete removal of ordered structure from proteins) is evidenced by circular dichroism spectroscopy,21 which has shown that urea can induce a stereospecific stabilisation of a left-handed extended helix in a polypeptide epitope of a virus. The secondary and tertiary structures of proteins are vital in many protein recognition processes, such as receptor binding or antigen-antibody interactions, and even slight perturbation of these structures may have substantial pathophysiological effects. An important area for future research is to refine our understanding of the roles of both free and bound urea and methylamines in the production of these effects. Thus, we may uncover specific binding, transport, and delivery systems for these and other important small molecules and provide new targets for
specific drug design.
1440
One
of from
source
methylamines is diet; they
are
also
metabolism choline and of produced phosphatidylcholines by gut bacteria.22 In the gut, choline is converted to trimethylamine (and also, to a lesser extent, dimethylamine) before being internally oxidised to TMAO and then excreted in the bile and urine.22 After absorption, dietary TMAO is reduced and reoxidised again, and impaired N-oxidation can be inherited as a recessive trait.22,23 Also, it now seems likely that methylamines are also synthesised endogenously. In man, betaine is excreted in urine from the neonatal period until about the age of 2 years but is not excreted by adults, in whom dimethylamine is produced instead.24-26 Indeed, it has been suggested that dimethylamine excretion could be used to assess renal function since dimethylamine, like creatinine, is excreted at a constant rate during fasting.27 However, little else is yet known of methylamine physiology in human health and
disease. There
are numerous straightforward questions which require study. Is the accumulation of TMAO or other intracellular methylamines in uraemia strongly correlated
with urea concentration? Are the intracellular TMAO concentrations high enough to offset the toxic effects of urea? Does the plasma concentration of methylamines accurately reflect intracellular values? Does taking into account the plasma concentrations of TMAO and other methylamines improve the correlation between urea and symptoms of uraemia? Do other methods of increasing the plasma urea (such as ureteric transplantation) stimulate an increase in methylamines? Can symptoms be ameliorated by administration of TMAO or other methylamines? How does a reduction in dietary protein content, widely believed to decelerate deterioration of renal function in chronic renal failure ’28 29 affect the relation between urea and methylamines in chronic renal failure? What are the developmental influences on the relation between urea and methylamines? These and other questions await answers, and we hope that this article will stimulate interest in this little-explored area of human pathophysiology. REFERENCES 1. Knochel
Seldin DW. The pathophysiology of uraemia. In: Brenner MM, Rector FC, eds. The kidney. Philadelphia: Saunders, 1976: 1448-85. 2. Wills MR. Metabolic consequences of chronic renal failure. Aylesbury: HM & M Publishers, 1978. 3. Barsotti G. Uraemic toxins. In: Giovanetti S, ed. Nutritional treatment of chronic renal failure. Boston: Kluver, 1989: 33-40. 4. Merrill JP, Legrain M, Hoigne R. Observations on the role of urea in uraemia. Am J Med 1953; 14: 519-20. 5. Hutchings RM, Hegstrom RM, Scribner BH. Glucose intolerance in patients on long-term haemodialysis. Ann Intern Med 1966; 65: 275-85. 6. Gilboe DD, Javid MJ. Breakdown products of urea and uraemic syndrome. Proc Soc Exp Biol Med 1964; 115: 633-37. 7. Bollman JL, Mann FC. Nitrogenous constituents of the blood following transplantation of the ureters into different levels of the intestine. Proc Soc Exp Biol Med 1927; 24: 923-24. 8. Yancey PH, Clark ME, Hand SC, Bowlus RD, Somero GN. Living with water stress: evolution of osmolyte systems. Science 1982: 217: 1214-22. 9. Somero GN. Protons, osmolytes and fitness of internal milieu for protein function. Am J Physiol 1986; 251: R197-R213. 10. Chamberlin ME, Strange K. Anisosmotic cell volume regulation: a comparative review. Am J Physiol 1989; 257: C159-73 11. Somero GN. From dogfish to dogs: trimethylamines protect proteins from urea. News in Physiol Sci 1986; 1: 9-12. 12. Bonaventura J, Bonaventura C, Sullivan B. Urea tolerance as a molecular adaptation of elasmobranch hemoglobins. Science 1974; 186: 57-59. 13. Yancey PH, Somero GN. Counteraction of urea destabilisation of protein structure by methylamine osmoregulatory compounds of elasmobranch fishes Biochem J 1979; 183: 317-23.
14. Beck F, Dorge A, Rick R, Thurau K. Intra- and extracellular elemental concentrations of rat renal papilla in antidiuresis. Kidney Int 1984; 25: 397-403. 15. Gutman Y, Katzper-Shamir Y. The effects of urea, sodium and calcium on microsomal ATPase activity in different parts of the kidney. Biochim Biophys Acta 1971; 233: 133-36. 16. Balaban RS, Knepper MA. Nitrogen 14 nuclear magnetic resonance spectroscopy of mammalian tissues. Am J Physiol 1983; 245: C438-
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Bagnasco S, Balaban RS, Fales H, Yong Y-M, Burg M. Identification of intracellular organic osmolytes in renal inner medulla. J Biol Chem
1986; 201: 5872-77. 18. Balaban RS, Burg MB. Osmotically active organic solutes in the renal inner medulla. Kidney Int 1987; 31: 562-64. 19. Yancey PH, Burg MB. Distribution of major organic osmolytes in rabbit kidneys in diuresis and antidiuresis. Am J Physiol 1989; 257: F602-07. 20. Bell JD, Lee JA, Lee HA, Sadler PJ, Wilkie DR, Woodham RH. Nuclear magnetic resonance studies of blood plasma and urine from subjects with chronic renal failure: identification of trimethylamine-N-oxide. Biochim Biophys Acta 1991; 1096: 101-07. 21. Siligardi G, Drake AF, Mascagni P, Rowlands P, Brown F, Gibbons WA. A CD strategy for the study of polypeptide folding/unfolding: a synthetic foot-and-mouth disease virus immunogenic peptide. Int J Peptide Protein Res (in press). 22. Al-Waiz M, Ayesh R, Mitchell SC, Idle JR, Smith RL. A genetic polymorphism of the N-oxidation of trimethylamine in humans. Clin Pharmacol Ther 1987; 42: 588-94. 23. Al-Waiz M, Ayesh R, Mitchell SC, Idle JR, Smith RL. Disclosure of the metabolic retroversion of trimethylamine-N-oxide in humans: a pharmacogenetic approach. Clin Pharmacol Ther 1987; 42: 608-12. 24. Davies SEC, Chalmers RA, Randall EW, Iles RA. Betaine metabolism in human neonates and developing rats. Clin Chem Acta 1988; 178: 241-50. 25. Bell JD, Sadler PJ, Morris VC, Levander DA. Effect of aging and diet on proton NMR spectra of rat urine. Magnetic Resonance Med 1991; 17: 414-22. 26. Brown JCC, Miels GA, Sadler PJ, Walker V. 1H NMR studies of urine from premature and sick babies. Magnetic Resonance Med 1989; 11: 193-201. 27. Bates JR, Bell JD, Nicholson JK, Sadler PJ. 1H NMR studies of urine during fasting: excretion of ketone bodies and acetylcarnitine. Magnetic Resonance Med 1986; 3: 849-56. 28. Diamond JR. Effects of dietary interventions on glomerular pathophysiology. Am J Physiol 1990; 27: F1-8. 29. Brenner BM, Meyer TW, Hostetter TH. Dietary protein intake and the progressive nature of kidney disease. N Engl J Med 1982; 307: 652-59.
From The Lancet Clinical
teaching
JP,
In relation to the profession, hospitals are intended to supply to the student a knowledge of disease; they are the laboratories in which the various manipulations and processes necessary for the cure of physical suffering are conducted. Within their walls human misery displays a thousand varied shapes; the student is appalled at the immensity of the labour that seems spread before him, and n.’ friendly hand is stretched forth to aid him through his difficulties. Clinical teaching has become a mere word, without signification; and the few hasty remarks made at the bedside are directed to those who are already informed upon the subject, and tend only to perplex still more the junior student. The too frequent effect, on the mind of the student, of the difficulties met with in comprehending the nature of disease, from its protean manifestations, and of the absence of all assistance or stimulus, excepting that distant and little regarded one-a sound knowledge of his profession-is a distaste for the duties of the hospital, and, as a consequence, gradual neglect of their performance-a neglect in which the well-disposed are as apt to take part as the idle. In those hospitals in which a casebook is kept, the industrious student finds a great resource in the perusal of its pages... We trust that all who have any influence in the hospitals will consider it to be a duty... to devote some portion of their time to the consideration of the manner in which so important a benefit to those institutions could be obtained, as would be conferred by the universal record of their proceedings in hospital case-books.
(July 10, 1841)