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BACKGROUND: The clinical significance of a trace protein reading on urinalysis is unclear, and such a result is often ignored by the clinician. METHODS: We examined 185 samples of urine with trace proteinuria by both Chemstrips and sulfosalicylic acid testing, and compared the results with those of urinary albumin and total protein concentrations. RESULTS: Taking for the purposes of this study an arbitrary upper limit of normal of 20 mg/l for albumin and 100 mg/l for total protein concentration, we found abnormal albumin excretion in 87% and abnormal total protein excretion in 88% of trace samples. In this study, a negative urinalysis for protein excluded microalbuminuria in 87% and proteinuria in 78% of cases. CONCLUSION: Qualitative testing for protein by urinalysis has a high sensitivity and specificity for diagnosing or ruling out microalbuminuria. Trace proteinuria usually means microalbuminuria; negative proteinuria tends to rule it out.

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Urinary total protein (UTP) determinations are notoriously inaccurate, poorly reproducible, and difficult to interpret in early renal disease, causing many investigators to measure urinary albumin instead. In this study, we compare a new nonimmunologic fluorescent dye (AB-dye) for measuring albumin with the more expensive and cumbersome radioimmunoassay. We tested 207 urine specimens from patients with variable protein concentrations and divided the results into five arbitrary ranges (0 to 20, 21 to 50, 51 to 100, 101 to 200, and 201 to 400) for chi-square analysis. There was a high degree of correlation between the two methods (chi-square = 260. 8 with 16 degrees of freedom; P < 0.001). The correlation was also high when analyzed by linear regression (R = 0.86; F < 0.01). Based on our comparison of total protein and albumin concentration in the same urine samples, we hypothesized that patients with mild proteinuria may not necessarily have microalbuminuria. Urine samples with UTP between 150 and 400 microg/mL were tested for albumin by the AB-dye. Of 41 samples in this range, 18 (44%) had normal albumin levels. We conclude that measuring urinary albumin with the AB-dye is comparable in performance to radioimmunoassay and could replace UTP determinations, especially for patients with borderline elevations of UTP, many of whom do not have microalbuminuria.

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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.

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We re-addressed the question of whether routine total urinary protein determinations can be used to predict the presence of microalbuminuria by studying 61 patients who attended a diabetic clinic and tested negative or had one positive protein by dipstick. Total urinary protein was measured by the Biorad dye-binding method in undialyzed urine (UND), in dialyzed urine (DIAL), and in dialyzed urine in which albumin and globulins were separated, measured separately with albumin and globulin standards and the results added together to obtain total urinary protein (A + G). The results were compared with albumin measurements obtained by radioimmunoassay (RIA). Compared to DIAL, urinary protein measurements were 43% higher with A + G and 22% higher with UND. Microalbuminuria correlated moderately with UND (r =0.81) and better with the other methods (r=0.87 for DIAL, r=0.91 for A + G). None of the methods predicted microalbuminuria reliably. Taking a protein-to-creatinine ratio of 0.15 and an albumin-to-creatinine ratio of 0.03 as upper limits of normal, we found that UND had a 72% positive predictive value (28% false positives) and 85 % negative predictive value (15% false negatives). DIAL had 90% positive predictive value (10% false positives) and 78% negative predictive value (22% false negatives). A + G had 65% positive predictive value (35% false positives) but 91% negative predictive value (9% false negatives). A + G, which uses the correct standards, would be the most suitable method for screening, having the least number of false negatives, but has more false positives because it is more sensitive. In practice, most routine chemical laboratories find it expedient to use only UND, but physicians interpreting the results of this method should be aware of its limitations.

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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; P < 0.001). 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.

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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 micrograms 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.

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Xanthopterin, a metabolic end product of the nonconjugated pterins dihydrobiopterin and tetrahydrobiopterin, is present in many organs and is known to inhibit the proliferation and growth of conconavalin-stimulated lymphocytes. We have developed a simple fluorometric method to measure xanthopterin in the blood and have validated the method by high pressure liquid chromatography (HPLC). Serum levels were 14 +/- 7 nmol/l in normal subjects and 141 +/- 51 nmol/l in hemodialysis patients (p < 0.02). Intermediate levels from patients with renal insufficiency not on dialysis correlated with serum creatinine levels (p < 0.001). Xanthopterin (MW 179) was cleared by hemodialysis at a slightly lower rate than creatinine. It is bound to protein, but the binding, 90 +/- 5% in normal subjects, is decreased in uremia to 60 +/- 15%, p < 0.01. Red cell levels of xanthopterin were five times higher than those of plasma in normal subjects (69 +/- 15 vs. 14 +/- 7 nmol/l, p < 0.001), but uremic patients had lower levels in red cells than in plasma (101 +/- 24 vs. 141 +/- 51 nmol/l, p < 0.05). Slight or moderate hemolysis induced by mechanical stress increased plasma xanthopterin levels by 35%, the effect being more pronounced when hemolysis was severe. We conclude that xanthopterin is increased and its binding to protein is decreased in chronic renal failure. The altered ratio of red cell/plasma xanthopterin levels may reflect an abnormality of the red cell membrane in uremia. We are conducting further studies to amplify our preliminary findings that xanthopterin inhibits cellular growth in vitro.

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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.

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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.

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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 HC1 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.

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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 less than 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 (HC1) hydrolysis. In addition, treatment with 6N HC1 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.

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The results of conservative surgical treatment of tubal pregnancy (salpingotomy in 10 cases, partial salpingectomy and anastomosis in two cases) were compared with radical treatment in seven cases. The subsequent pregnancy rate was 55% and 71% for the conservative and radical groups, respectively. There were no recurrent tubal pregnancies in either group. Prior presence of pelvic adhesions did not have an effect on fertility in these patients. The rate of multiparity was significantly higher in patients who became pregnant after the surgery than those patients who did not become pregnant.

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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.

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Using the model of the isolated perfused rat ovary, we have found that highly purified ovine follicle-stimulating hormone (FSH) preparations cause ovulation and that this effect is not due to luteinizing hormone (LH) contamination. Ovine FSH-13 at a concentration of 1.5 mU/ml induced ovulations in all perfused ovaries (8.8 +/- 2.3 ovulations/ovary), as did a more purified preparation, ovine FSH-211B, at concentrations of 0.5 mU/ml (15.0 +/- 6.4 ovulations/ovary) and 5 mU/ml (11.3 +/- 2.6 ovulations/ovary). This ovulation-inducing effect of FSH is accompanied by a marked stimulation of estradiol levels in the perfusion medium without stimulation of progesterone levels. Furthermore, a purified rat FSH preparation (15 mU/ml) also induced ovulation in all ovaries (13.8 +/- 2.2 ovulations/ovary) as well as a stimulation of both estradiol and progesterone in the medium. These data clearly confirm the direct ovulatory effect of FSH on the ovary.

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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.

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Gel filtration and thin layer chromatography were conducted on sera from uremic patients and normal subjects for the isolation of nitrogenous substances unique to uremia. Many ninhydrin-positive substances were found in greater amounts in uremic patients compared to normal subjects. Some of these ninhydrin-positive substances were also detected by staining with chlorine-tolidine. Amino acid analysis of these substances showed considerable qualitative and quantitative differences, perhaps reflecting interference with enzymatic activity by the uremic environment.

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Sperm antibody titers were evaluated in the serum of 73 infertile couples by the F-D and Kibrick agglutination tests, to study the relationship of the quality of post-coital tests and semen analysis. Thirty-eight couples had poor post-coital tests, fifteen couples had normal post-coital tests, and twenty couples revealed abnormal semen pictures of the males. In the first two groups, semen analysis was normal. The incidence of sperm antibodies was 10.5%, 6.6% in men, and 18.4%, 20.0% in women, with poor and normal post-coital tests, respectively. This suggests that sperm antibody testing is equally indicated in infertile couples with poor post-coital tests. In addition, there was no difference in the incidence of sperm antibodies in men with poor semen pictures and those with normal semen analysis in this infertile population. This suggests the significance of sperm antibody testing in infertile men with abnormal and normal semen pictures after correcting other infertility factors, in order to detect those patients who could benefit from treatment of such immunologic problems.

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In order to characterize the spectrum of possibly toxic retention compounds in uremia we have developed a simple reproducible method of separating fractions of uremic serum by Sephadex G-15 column chromatography. This technique, which requires no prior deproteinisation and is carried out at ambient temperatures, allowed the separation of uremic serum into several well defined fractions. Subsequent thin layer chromatography (TLC) showed that each peak represented a mixture of peptides, and that there were qualitative and quantitative differences between the plasma of normal and uremic patients as well as between patients with acute renal failure and chronic renal failure.

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