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Carbamylated proteins formed in renal insufficiency from the spontaneous decomposition of urea exert a variety of metabolic effects. Here we examined the effects of carbamylated proteins on glomerular mesangial cells to determine whether urea retention in early renal insufficiency may itself promote glomerular sclerosis and hasten the progression to kidney failure. To this effect we carbamylated fetal bovine serum proteins in vitro and tested their effect on mesangial cell proliferation (by tritiated thymidine uptake), de novo protein synthesis (by tritiated leucine uptake), collagen I and collagen IV accumulation (by avidin-biotin enzyme immunoassay), and gelatinase levels in the medium (by zymography and quantitative fluorescence assay). Carbamylated fetal bovine serum at concentrations present in uremia increased tritiated thymidine incorporation by 50% without altering tritiated leucine incorporation, and it increased collagens I and IV in the monolayer by 150% to 300%. Gelatinase activity was unchanged. We conclude that carbamylated proteins can activate mesangial cells to a profibrogenic phenotype. From a clinical perspective, the carbamylation of proteins by elevated urea levels may accelerate the progression to kidney failure and thus set up a vicious cycle in which the nitrogen retention itself would cause further progression of fibrosis and deterioration of kidney function.

We have previously found that carbamylated hemoglobin (carHb) levels are increased in chronic renal failure and correlate positively with blood urea nitrogen (BUN) levels and with the duration of exposure to urea. In a fashion analogous to glycosylated hemoglobin in diabetic patients, it is possible that carHb may better reflect BUN levels before hemodialysis (preBUN) and also between hemodialysis sessions. We therefore tested the hypothesis that carHb could be a better index of adequacy of hemodialysis than the urea reduction ratio (URR). Fifty hemodialysis patients had carHb measured every 2 months for 14 months; the carHb level was compared with URR and preBUN levels, as assessed by changes in absolute numbers and trends of the BUN levels between hemodialyses. Mean URR was above 61% throughout the 14 months. Mean carHb levels did not change significantly during the study and were only weakly correlated with URR. However, there was a much better correlation between predialysis BUN and carHb, suggesting that carHb levels reflect more accurately the changes in BUN between hemodialysis sessions. To further test this hypothesis, we subdivided the patients arbitrarily, depending on the change in preBUN between two consecutive carHb measurements. We found significantly lower carHb levels when BUN decreased or remained stable than when it increased or was persistently high. In patients with decreasing or stable BUN, carHb was significantly lower than in patients with persistently high or increasing BUN (carHb 81.5 +/- 3.6 microg valine hydantoin [VH]/g Hb v 123.7 +/- 11.7 microg VH/g Hb, respectively; P0.001). URR was not different between groups. In addition to changes in BUN levels, carHb was correlated by multiple regression analysis with the presence of diabetes, weight, and plasma HCO3. The relationship between diabetic patients and carHb levels was complex because such patients tend to have higher preBUN levels, higher protein catabolic rate, and lower HCO3 levels. These results demonstrate that carHb reflects the changes between dialysis BUN and may serve as a more accurate index of uremia control. Clinically, it appears that well-dialyzed patients have carHb levels lower than 100 microg VH/g Hb.

We have identified the primary endogenous fluorescent substance, which has characteristic excitation/emission maxima at 380/440 nm and 400/460 nm, found in the sera of patients with chronic renal failure (Clin Chem 32: 1276, 1988). Preliminary studies, using thin layer chromatography (with cellulose) in conjunction with pteridine standards, indicated that the compound is an unconjugated pteridine. Characterization by gas chromatography-mass spectrometry (electron impact), direct probe-mass spectrometry (electron impact/chemical ionization), and Fourier Transform Infrared analysis showed this compound to be xanthopterin (2-amino 4,6 pteridinedione), an unconjugated pteridine known to be present in man in trace quantities. An authentic sample of this compound had a retention time with high-performance liquid chromatography (HPLC) identical to that of the purified fluorophore. The physiological role of xanthopterin in the pathogenesis of uremia has yet to be elucidated.

We describe the purification and initial characterization of a hitherto unrecognized fluorescence (excitation/emission maxima at 380/440 nm and 400/460 nm) reported from this laboratory in patients with chronic renal failure (Clin Chem 31: 1988, 1985). Purification was achieved using Sephadex G-10 gel chromatography combined with reverse phase and ion exchange high-performance liquid chromatography (HPLC). Purity of the "blue-green" fluorescent compound was determined to be greater than 99% by HPLC, and two-dimensional thin layer chromatography using an acidic and basic solvent system. The excitation/emission maxima were shown to be 390 nm/456 nm, and ultraviolet scans, at pH 1.0, 7.0, and 13.0, gave absorbance optima at 261 nm/356 nm, 278 nm/390 nm, and 255 nm/394 nm, respectively. The isoelectric point of 4.05 in conjunction with the fluorescent and ultraviolet spectra suggests that the fluorophore belongs to the class of compounds known as pteridines.

Factors determining hemoglobin carbamylation in renal failure. Carbamylated hemoglobin (carhb) is formed by the reaction of hemoglobin with cyanate, a product of in vivo urea dissociation. It is found in high levels in patients with renal failure and may be useful in their clinical evaluation. Accordingly, we measured carhb by HPLC after acid hydrolysis in 73 patients with renal failure and 11 controls. Mean carhb levels, (expressed as μg valine hydantoin/g Hb), were highest in chronic renal failure (CRF, 146 ± 13), intermediate in end-stage renal disease on hemodialysis (ESRD, 106 ± 7), and lowest in acute renal failure (ARF, 80 ± 12) when compared to normal subjects (27 ± 2). In all patients carhb was significantly correlated with BUN but not with creatinine, bicarbonate, or phosphate. For any level of BUN above 80 mg/dl, carhb was substantially higher in CRF than in ARF. Predialysis BUN and urea reduction ratio (URR) were significant predictors of carhb in ESRD. To investigate the effect of time of exposure and BUN level on the rate of carbamylation of hemoglobin, blood from normal subjects and dialysis patients was incubated in vitro with urea equivalent to BUN levels of 50, 100, 150, and 200 mg/dl and assayed for carhb at 0, 5, 9, and 14 days. Carhb increased linearly over the first nine days of urea exposure and leveled off thereafter. The rate of carbamylation increased as BUN increased and was significantly higher in hemoglobin from dialysis patients than from normal subjects. These results show that the higher the level of carhb at baseline, the higher the rate of carbamylation upon exposure to increasing urea concentrations. We conclude that carhb formation is dependent on urea concentration and length of exposure to urea. The rate of carhb formation for a given urea concentration is greater in hemoglobin already carbamylated, and this may explain why carhb is higher in CRF than in ARF at BUN levels greater than 80 mg/dl. Carhb may thus be a useful index of the duration and degree of exposure to high blood urea levels in patients with renal failure, and may potentially serve as an index of the adequacy of dialysis.

To further define the platelet abnormality responsible for uremic bleeding, we studied platelet aggregation with adenosine diphosphate, ristocetin, and collagen in serum fractions obtained by Sephadex G-15 chromatography. We found that uremic patients had considerable inhibition in several peaks of middle molecular range, but the findings were inconsistent and not clearly related to the degree of uremia.

Cutinase from pollen grains of Tropaeolum majus was purified by Sephadex G-100 gel filtration, QAE-Sephadex chromatography, and isoelectric focusing. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The molecular weight of the enzyme was estimated to be 40,000 by both Sephadex G-100 gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This cutinase was found to be a glycoprotein containing about 7% carbohydrate and the isoelectric point of this enzyme was 5.45. It catalyzed hydrolysis of p-nitrophenyl esters of C2 to C18 fatty acids with similar Km and V. The purified cutinase showed an optimum pH of 6.8 with cutin as the substrate, whereas with p-nitrophenyl esters of fatty acids the optimum pH was 8.0. This enzyme did not show any metal ion requirement. Unlike the previously studied fungal cutinases, the present pollen enzyme was strongly inhibited by thiol-directed reagents such as N-ethylmaleimide and p-hydroxymercuribenzoate whereas it was totally insensitive to the active serine-directed reagent, diisopropylfluorophosphate. The purified pollen cutinase showed preference for primary alcohol esters, but it did not catalyze hydrolysis of tripalmitoyl or trioleyl glycerol at significant rates. The properties of the pollen enzyme are, in general, in sharp contrast to those of the fungal cutinase, and the present results strongly suggest that the pollen enzyme belongs to a new class of cutinases. Another esterase which preferentially hydrolyzed p-nitrophenyl acetate was also found in the extracellular fluid. This enzyme, separated from cutinase, showed a pI of 5.6 and it was sensitive to diisopropylfluorophosphate, but not to SH-directed reagents.

Rabbit antibody to cutinase-I, isolated from Fusarium solani f. pisi, was conjugated to ferritin. With this ferritin-conjugated antibody it was shown that germinating spores of this fungus excreted cutinase during the penetration of the host pisum sativum. This result constitutes the most specific and strongest evidence for an enzymic penetration of a plant cuticle by a pathogen during infection.

We measured the fluorescence, at various excitation (Ex) and emission (Em) wavelengths, of serum ultrafiltrates and fractions of serum resolved by chromatography on Sephadex G15, studying both normal subjects and patients in chronic renal failure requiring hemodialysis. We found hitherto undescribed fluorescence at Ex 380 nm/Em 440 nm and Ex 400 nm/Em 460 nm, the intensity being greatly increased in patients with chronic renal failure in comparison with normal subjects (p less than 0.005). This fluorescence persisted unaltered when serum was filtered through membranes having cutoffs ranging from 10 000 to 500 Da. Each serum fraction resolved by gel chromatography demonstrated a characteristic fluorescence, which was generally much more intense in uremics. The most intense fluorescence (Ex 380 nm/Em 440 nm and Ex 400 nm/Em 460 nm) was emitted in the higher-Mr fractions.

We evaluated the biochemical characteristics of endogenous fluorescent substances. Ex 380 nm/Em 440 nm and Ex 400 nm/Em 460 nm, present in sera of patients with chronic renal failure (Clin. Chem. 31:1988, 1985). Sera from 23 patients with chronic renal failure (CRF) and from 10 normal subjects were filtered through ultrafiltration membranes (cutoff limit of 500 Da). Fluorescence intensity of the aforementioned substances was significantly elevated as compared to normals (p < 0.001). Fluorescence characteristics of these substances remained unaltered after ultrafiltration and treatment with beta-glucuronidase. Extraction of these fluorescent compounds with organic solvents (dichloromethane, ethyl acetate, chloroform:methanol) could not be achieved after ultrafiltrates were subjected to 6N hydrochloric acid (HCl) hydrolysis. In addition, treatment with 6N HCl enhanced fluorescence intensity without altering fluorescence excitation/emission maxima. Removal of fluorescence could be accomplished in toto by adsorption onto activated charcoal with subsequent recovery from charcoal by treatment with sodium hydroxide, pH 12 (Ex 380 nm: 51.1%, Ex 400 nm: 91.8%). Analysis of alkali-treated specimens by high performance liquid chromatography demonstrated that peptides associated with these fluorescent substances were denatured, although fluorescence at these previously described excitation/emission maxima persisted. Our studies indicate that the unique fluorescence observed in the sera of patients with CRF is not an intrinsic characteristic of a specific peptide or its amino acids, but rather an inherent property of fluorescent molecules which may bind to these peptides.

In order to characterize the spectrum of small peptides retained in chronic renal failure, we carried out high pressure liquid chromatography (HPLC) of serum ultrafiltrates from patients with chronic renal failure (CRF), acute renal failure (ARF), and normal subjects. HPLC patterns in CRF resolved into more than twenty peaks; those in ARF contained fewer peaks and resembled that of normals. We carried out amino acid analysis of HPLC fractions after hydrolysis with 6N HCl of four patients with CRF, one patient with ARF, and one normal subject. Following hydrolysis each HPLC fraction yielded several amino acids. Glycine, leucine, serine, phosphoserine, glutamic acid, and phenylalanine were found in greatest frequency in the four CRF patients.

The present study involves the intracellular distribution of acid phosphatase (AP) in the root meristem ofAlfalfa vulgaris. Root tips, 1-2 mm long, were excised from 24 h old seedlings and fixed in 3% gluteraldehyde in 0.1M PO4buffer at pH 7.2 for 1 h. The fixed roots were washed in the same buffer 3X (15 min each), then incubated for 30 min at 37°C in either Gomori or Barka and Anderson's lead medium with β-glycerophosphate as substrate at pH 5.0. These roots were subsequently washed in the respective buffers of the incubation mixtures and were post-fixed for 30 min in Millonig's OSO4. After brief washing in distilled water the roots were dehydrated in short steps through ethanol, and embedded in Epon for routine Ultramicrotomy. The controls consisted of roots incubated in the incubation mixture lacking substrate and of heat-inactivated (95°C for 15 min) and NaF-treated (0.1M for 2 h) roots incubated in the complete reaction mixture.