Carbamylated Proteins and Peptides in Health and in Uremia (1998)

There has been a considerable amount of work done in the field of carbamylation of proteins and peptides in the last 15 years, some of which has been brought to the awareness of the clinical nephrologist, but a large amount of important work has been sequestered in journals to which the English-speaking nephrologist will not have ready access, life being short and the mass of literature escalating exponentially.The purpose of this review is to give a reasonably balanced view of the field from the point of view of a clinician undertaking research in this field.Carbamylation of proteins, lipids, peptides and amino acids is widespread in health in mammals [1, 2, 3, 4, 5, 6, 7, 8] and is a natural physiological phenomenon.Carbamylation is dependent on the production of isocyanic acid in the body fluids and tissues. The source of the isocyanic acid is from the breakdown of urea, and the amount of isocyanate produced is very small, and, like NO, at low concentrations its toxicity is not important, unless physiological carbamylation is taken to be a toxic phenomenon. The level of isocyanate produced from urea in body fluids is directly proportional to the concentration of urea in both body fluids and tissues. Isocyanate is converted to isocyanic acid, and this combines with various amino acids to yield the carbamylated amino acid. In hemoglobin the amino acid involved is valine, resulting in the production of carbamylated hemoglobin [2, 4, 9]. In the lens gamma crystallin is carbamylated at the lysine site [10]. In the red cell lipid envelope the carbamyated lipids are phosphatidylethanolamine and serine [5]. The site of carbamylation of insulin, T3 and other plasma proteins is uncertain at the time of writing. Carbamylation is a nonenzymatic, irreversible reaction. Once a protein, lipid, polypeptide or amino acid is carbamylated the concentration of the carbamylated material can only fall through two possible mechanisms – firstly, catabolism with destruction of the modified protein, lipid, peptide or amino acid or, secondly, entry of newly synthesized noncarbamylated proteins, lipids, peptides or amino acids into the compartment under study, usually but not always, the blood or plasma. However, the concentration of carbamylated substrate can increase if there is greater exposure to urea, either by longer exposure of the proteins to the same concentration or exposure to higher urea concentrations [2, 6, 11, 12]. This is an important point in the similarity of interpretation of the concentration of carbamylated hemoglobin in uremia to the concentration of glycosylated hemoglobin in diabetes mellitus. Neither will show the troughs in concentration of urea or glucose, but will reflect the peaks and sustained high levels of plasma urea and glucose, respectively.Carbamylated hemoglobin initially will be considered for two reasons: the first is that hemoglobin is the protein in highest concentration in the blood, is easily obtained in a pure form, and the methodology for measurement of carbamylation in hemoglobin is relatively simple; the second reason was the vast amount of work done on carbamylation of hemoglobin in sickle cell anemia and its treatment using isocyanate and its precursors [9, 13, 14]. A brief consideration of the methodology of measuring carbamylated hemoglobin is necessary. The majority of modern methodologies involve acid hydrolysis of the hemoglobin which liberates carbamylated valine, this being converted to valine hydantoin. Valine hydantoin is separated by ethyl acetate partition and subsequently measured by high-precision liquid chromatography (HPLC) or by gas chromatography. In the HPLC method the temperature and the duration of incubation are critical in the yield of valine hydantoin which is a moderately heat-labile substance. Carbamylated hemoglobin is expressed as micrograms of valine hydantoin per gram of hemoglobin, so that the degree of anemia in uremic or sickle cell patients does not influence the results of carbamylated hemoglobin as expressed above.Carbamylated hemoglobin gives up its oxygen with difficulty when compared to noncarbamylated hemoglobin, and this has been the subject of some study with regard to uremic patients [15]. In sickle cell disease, in addition carbamylated hemoglobin reduces the chance of sickle cell hemoglobin precipitating in the sickle cell patient, particularly in anoxic conditions, so that its presence is a benefit to the patient threatened by frequent sickle cell crises [9, 13, 14, 16]. In non-sickle-cell uremic patients with hemoglobin A, carbamylated hemoglobin alters red cell rigidity and enables the red cell to pass through narrow capillaries and between the endothelial cells more readily than non-carbamylated red cells [17].Although carbamylated hemoglobin gives up its oxygen with greater difficulty than the non-carbamylated hemoglobin, and tissue PO2 falls in inverse proportion to the concentration of carbamylated hemoglobin, this is offset in uremic patients by the effect of carbamylated hemoglobin on 2,3 diphosphoglycerate [15].There are several papers investigating carbamylated hemoglobin in acute renal failure, nondialyzed chronic renal failure, in patients on regular hemodialysis and on CAPD, in prerenal azotemia and after transplantation [1, 2, 3, 4, 6, 10, 11, 12, 18, 19, 20].In acute renal failure the data are shown in table 1. Davenport et al. [18] investigated the value of carbamylated hemoglobin in differentiating acute from chronic renal failure. They found that those patients with acute renal failure of 10 days or less duration had a lower median carbamylated hemoglobin than those with a longer duration (29 vs. 72 µg of valine hydantoin per gram hemoglobin). They also observed that the carbamylated hemoglobin levels in pure acute renal failure were lower than in acute or chronic renal failure. Stim et al. [20] also found that the carbamylated hemoglobin levels were lower in acute than in chronic renal failure and in patients on dialysis. Han et al. [6] also found similar results. All investigators found that the values of carbamylated hemoglobin were higher in acute renal failure patients than in controls. Stim et al. [20] also concluded from in vitro studies that exposure to increased urea concentrations produced a progressive rise in carbamylated hemoglobin in the first 9 days, explaining perhaps the findings of Davenport et al. [18] with regard to the length of acute renal failure being related to the elevation in carbamylated hemoglobin.Several groups have examined the concentration of carbamylated hemoglobin in nondialyzed chronic renal failure. Fluckiger et al. [2] found a significant correlation between valine hydantoin/Hb tetramer, expressed in molar terms, and time-averaged BUN values in 7 non-dialyzed chronic renal failure patients, using a gas-chromatographic method. They pointed out, presciently, that the red cell life is shortened in uremia, and this would tend to lower the carbamylated hemoglobin levels, as would the frequent transfusions given then in chronic renal failure patients before the advent of recombinant erythropoietin. Kwan et al. [3] found similar results using an HPLC method they introduced, in 55 stable, nondialyzed chronic renal failure patients. Davenport et al. [18] and Stim et al. [20] reported similar results and found the concentrations of carbamylated hemoglobin to be higher in chronic renal failure than in any of the other renal disease groups. These are shown in table 2 (see next paragraph).Smith et al. [4] reported in 1988 that carbamylated hemoglobin was elevated in 31 hemodialysis patients (100 ± 28 µg of valine hydantoin per gram hemoglobin), and in 34 CAPD patients a slightly higher figure (127 ± 39). These figures were lower than those found in non-dialyzed chronic renal failure (137 ± 63). Stim et al. [20], using HPLC – in contradistinction to Smith et al. [4] who used gas chromatography – found a mean of 106 (SD 7) in hemodialysis patients. Kwan et al. [21] reported the results in 20 patients; they divided their patients according to the adequacy of their dialyses and found in 11 with adequate dialysis that carbamylated hemoglobin was elevated (142 ± 29 µg of valine hydantoin per gram of hemoglobin), but in 6 of the underdialyzed group on hemodialysis the concentration was significantly higher (197 ± 30; p = 0.002). We have found in a group of adequately dialyzed hemodialysis patients, in whom the incubation was done at a carefully controlled 90°C, an elevated figure (89.6 ± 22) but lower than the values of Kwan et al. [7], although our controls at this temperature were similar in value [Berlyne and Caruso, unpubl. data].Davenport et al. [12] studied 55 regular dialysis patients and like Kwan et al. [21] found elevated carbamylated hemoglobin levels in both well-dialyzed and poorly dialyzed patients (120 ± 8 vs. 99 ± 7; p < 0.01).Both Kwan et al. [21] and Davenport et al. [12] showed that in hemodialyzed patients adequacy of dialysis was inversely related to the carbamylated hemoglobin concentration. Davenport et al. [12] pointed out that carbamylated hemoglobin was a useful marker of uremia, and it appears that carbamylated hemoglobin is a rather expensive and less accurate index of the time-averaged urea concentration. It is certainly not a practical alternative to measurement of urea reduction rate or Kt/V by any of the numerous methods of measuring the latter. Carbamylated hemoglobin is an interesting, indirect, less accurate, more expensive and less than justifiable method of determining adequacy of dialysis when compared to standard methods using BUN measurements.In a group of patients with carefully documented prerenal azotemia, Frazao et al. [19] found elevated carbamylated hemoglobin levels as soon as 4 days after the documented beginning of BUN elevation (table 3). The measurement of carbamylated hemoglobin was unable to distinguish between prerenal azotemia and acute renal failure, and simple clinical and biochemical estimations were found to be more efficient at this differentiation.Oimomi et al. [1] have shown that plasma proteins are carbamylated in renal failure in a similar way to hemoglobin and point out that there is good reason to extrapolate from carbamylation of plasma proteins to tissue proteins which are most likely carbamylated because of the ubiquitous presence of urea in cells. How this effects their function is at the moment unknown, but the same group has pursued this matter in the peptides insulin, triiodothyronine and ACTH [8, 22, 23].Insulin: Insulin was carbamylated in vivo and its immunological activity and its bioactivity were determined. They authors found that the biological activity of insulin was reduced to one fifth of that of uncarbamylated insulin. In addition, they found that immunologically carbamylated insulin was only immunoprecipitable with insulin antibodies to one third of the amount precipitable by non-carbamylated insulin. The latter is of dubious pathophysiological significance, for it depends on the part of the carbamylated insulin molecule that the antibodies recognize, and this may have no relevance to in vivo activity.Triiodothyronine has similarly been carbamylated in vitro, and its biological activity determined in the maturation of the tadpole tail. There is a statistically significant decrease in the activity of the carbamylated triiodothyronine in vivo which could be remotely related to the situation in uremic man, but the majority of patients in advanced uremia do not display hypothyroidism, so the significance of carbamylation experiments in vitro or in vivo is not clear. A possible problem arises from the fact that the hormones are in vivo present in very tiny concentrations, so that we cannot demonstrate readily with present methods whether they are significantly carbamylated in uremia in vivo.In addition ACTH has been carbamylated, but there is no published literature that I have been able to find apart from the paper of Ishikawa et al. [8], in which the effect of carbamylation of ACTH is discussed. It should be borne in mind that in vivo these are relatively short half life substances (insulin, T3 and ACTH) and that they may not be in circulation a sufficient time to get significantly carbamylated before they affix to the appropriate receptor.Major plasma proteins and hemoglobin, however, have a considerable half life measurable in days, are demonstrably carbamylated, and the physiological consequences of carbamylation have been studied. Let us look at some of the effects of carbamylation of hemoglobin.Oxygen carriage is altered. This is illustrated by the fact that the binding of oxygen in carbamylated hemoglobin is such that O2 is not given up by the red cell as readily, so that tissue pO2 falls. However, this is not significant in vivo, because the effect of uremia on DPG cancels out the effect of carbamylated hemoglobin on oxygen retention in the red cell [15].In addition, the effect on erythrocyte viscosity of carbamylation has been reported by Colin et al. [17] in 1987. They found a demonstrable increase in the deformability of red cells with carbamylation, and there was an associated decrease in hemoglobin viscosity, in vitro. However, in uremia there is a decrease in erythrocyte deformability in vivo, so the uremic state must have factors other than carbamylation responsible for this. Trepanier and Thibert [5] studied erythrocyte membrane aminophospholipids in vitro and in vivo in uremics. They found the presence of carbamylated phosphatidylethanolamine and suggested that this could be involved in the hematological consequences of uremia on the erythrocyte.In sickle cell anemia, there has been considerable experience in the use of isocyanate to prevent or treat sickle cell disease and its crises. Carbamylation of sickle cell blood decreases sickling and decreases the crises by up to 80% [13]. There is considerable discussion of the possible mechanisms [14, 26].Lens proteins are maybe carbamylated. Liu and Li [10] found carbamylation of human lens gamma-crystallin and this may be related to cataract formation in uremia. However, in contrast, Smith et al. [24] demonstrated that alpha crystallins (obtained from 4 uremic patients’ lenses) when digested into peptides, glutathione adducts were found at Cys 131 and Cys 132. These were not found in two normal donor lenses. Unlike carbamylation of hemoglobin, lysine was not carbamylated nor was cysteine in alpha crystallins. Hence the authors concluded that no carbamylation was detected in uremic alpha crystallins, unless it was present in amounts less than their analytical sesitivity was capable of detecting.However, the ferroxidase activity of ceruloplasmin, an important antioxidant defense mechanism in man, is impaired by carbamylation, and Roxborough et al. [25] have noted that this may be one of the causes of the reduced antioxidant capacity in uremia.Lipoproteins can be carbamylated. If they are mildly carbamylated, as would be expected in most chronic renal failure patients, there is a decreased clearance of LDL by as much as 41%. Human uremic LDL injected into rabbits was cleared more slowly than LDL from normal controls. [26].Elasmobranchs have normal levels of BUN in the range of 1,000 mg/dl, an important physiological fact explained by its use to maintain plasma osmolality in the presence of the large influx of ions from sea water. The hemoglobin in sharks and other elasmobranchs differs considerably from mammalian hemoglobins, and it is likely that the hemoglobin and other protein peptides and amino acids would be highly carbamylated. We attempted to measure carbamylated hemoglobin in blood from two sharks obtained for us by fish merchants in the Fulton Fish Market, in New York, but one was grossly hemolyzed and the second clotted, such that although the clot had high concentrations of valine hydantoin we were unable to express the results in terms of valine hydantoin per gram of hemoglobin. The dangers of attempting to obtain shark blood in reasonable quantity from live sharks are obvious, and we were unable to obtain the cooperation of a local aquarium to this end.The technical problems in measuring carbamylated hemoglobin should not be underestimated. The major paper in the literature [3] gives few technical details of the heat lability of valine hydantoin above 100°C, although the time factor has been considered. We found that the heating block purchased from the same manufacturers as described by Kwan et al. [3] was grossly inaccurate and was only calibrated up to 105°C. Using a stirred oil bath and with both thermocouple and mercury thermometer measurements of temperature we were able to show that above 95°C there was heat lability of valine hydantoin, and that at 110°C we were unable to recover any. Similarly, incubation at temperatures of 70°C and less was inefficient in producing valine hydantoin. It appears to us that the temperature of 90°C is optimum for yield of valine hydantoin. The choice of a relatively heat-stable standard, carbamyl-nor-valine, is unwise at a temperature of 110°C because it is destroyed at this temperature, and we were unable to recover carbamyl-nor-valine added as a standard after 2 h incubation at this temperature. If table 4 is studied, the wide range of values in controls using the original method of Kwan et al. [3] gives rise to some surprise. Thus when we changed the temperatures, we obtained quite surprisingly different values in the normals, clear evidence of a methodology problem hitherto unresolved apparently.Clearly the methodology needs further development, but, however it is done, it is still a fairly tedious and moderately expensive analysis and is best suited for research laboratories rather than routine chemical laboratories. It is of great interest physiologically, but there are no micromethods available at the moment which enable us to examine the effect of carbamylation in vivo on the majority of hormones due to lack of sensitivity of the currently available tests; these require at least a hundred milligrams of the native protein for the routine HPLC assay and thus femtogram quantities of the various hormones are at present out of reach of the majority of research workers carrying out in vivo or ex vivo studies.Carbamylation is a universal phenomenon affecting many proteins, peptides, amino acids and lipids in physiological control circumstances due to the generation of isocyanate from urea in aqueous solution in the body fluid and cells. In renal failure the increase in urea concentrations is responsible for the increase in carbamylation of hemoglobin, plasma proteins, various hormones and peptides. Various physiological effects of carbamylation of proteins and peptides are observed. In chronic non-dialyzed renal failure the levels of carbamylated hemoglobin are generally higher than in the regular dialysis patients or in those suffering from acute renal failure. Carbamylated hemoglobin affords a rough indication of the adequacy of dialysis, but the difficulties in using a research tool in the general clinical laboratory need to be overcome before it can be recommended as a routine test for determining dialysis adequacy in the majority of the dialysis population. Similarly, carbamylated hemoglobin is significantly related to the mean urea or time-averaged urea, but this is only necessary when a patient is being worked up whose records containing prior data are not available. Otherwise the urea figures are readily obtainable, are cheaper and give the requisite data directly.