A multicenter trial evaluation of the fibrin/fibrinogen degradation products test for detection and monitoring of bladder cancer.

PURPOSE: Presently there is a lack of effective, noninvasive tests for the detection and monitoring of bladder cancer. Measurement of fibrin/fibrinogen degradation products in urine has been shown to be a useful indicator of bladder carcinoma. The objective of this study was to evaluate the AuraTek FDP rapid immunoassay device for the detection of urinary fibrin/ fibrinogen degradation products associated with bladder cancer. MATERIALS AND METHODS: A prospective multicenter study was conducted to compare AuraTek FDP with urinary cytology and hemoglobin dipstick for the detection of bladder cancer in 192 patients with a history of bladder cancer. RESULTS: AuraTek FDP was significantly more sensitive (68%) than conventional urinary cytology (34%, p < 0.001) or hemoglobin dipstick (41%, p < 0.001) in the detection of bladder tumors, particularly for low stage low grade disease. In subjects with invasive disease (T2-T4) the AuraTek FDP test had a sensitivity of 100%. Specificity of AuraTek FDP was 96% for healthy subjects, 86% in patients with urological disease other than bladder cancer and 80% for patients under surveillance for bladder cancer but with a negative cystoscopic finding at the time of assay. CONCLUSIONS: This simple, rapid (less than 7 minutes) point of care test is superior to conventional urine cytology and hemoglobin dipstick as an aid in the detection of bladder cancer.

Familial lipoprotein disorders in patients with premature coronary artery disease.

BACKGROUND: Genetic lipoprotein disorders have been associated with premature coronary artery disease (CAD). METHODS AND RESULTS: The prevalence of such disorders was determined in 102 kindreds (n = 603 subjects) in whom the proband had significant CAD documented by angiography before the age of 60 years. Fasting plasma cholesterol, triglyceride, low density lipoprotein (LDL) cholesterol, apolipoprotein (apo) B, and lipoprotein (a) [Lp(a)] values above the 90th percentile and high density lipoprotein (HDL) cholesterol and apo A-I below the 10th percentile of age- and sex-specific norms were defined as abnormal. An abnormality was noted in 73.5% of probands compared with 38.2% in age-matched controls (p less than 0.001), with a low HDL cholesterol level (hypoalphalipoproteinemia) being the most common abnormality (39.2% of cases). In these kindreds, 54% had a defined phenotypic familial lipoprotein or apolipoprotein disorder. The following frequencies were observed: Lp(a) excess, 18.6% (includes 12.7% with no other dyslipidemias); hypertriglyceridemia with hypoalphalipoproteinemia, 14.7%; combined hyperlipidemia, 13.7% (11.7% with and 2.0% without hypoalphalipoproteinemia); hyperapobetalipoproteinemia (elevated apo B only), 5%; hypoalphalipoproteinemia, 4%; hypercholesterolemia (elevated LDL only), 3%; hypertriglyceridemia, 1%; decreased apo A-I only, 1%. Overall, 54% of the probands had a familial dyslipidemia; unclassifiable lipid disorders (spouse also affected) were found in 3%. No identifiable familial dyslipidemia was noted in 43% of kindreds of those; nearly half (45%) had a sporadic lipid disorder. Parent-offspring and proband-spouse correlations for these biochemical variables revealed that lipoprotein and apolipoprotein levels are in part genetically determined, with Lp(a) showing the highest degree of parent-offspring correlation. CONCLUSIONS: Our data indicate that more than half of patients with premature CAD have a familial lipoprotein disorder, with Lp(a) excess, hypertriglyceridemia with hypoalphalipoproteinemia, and combined hyperlipidemia with hypoalphalipoproteinemia being the most common abnormalities.

Lipoprotein cholesterol, apolipoprotein A-I and B and lipoprotein (a) abnormalities in men with premature coronary artery disease.

The prevalence of abnormalities of lipoprotein cholesterol and apolipoproteins A-I and B and lipoprotein (a) [Lp(a)] was determined in 321 men (mean age 50 +/- 7 years) with angiographically documented coronary artery disease and compared with that in 901 control subjects from the Framingham Offspring Study (mean age 49 +/- 6 years) who were clinically free of coronary artery disease. After correction for sampling in hospital, beta-adrenergic medication use and effects of diet, patients had significantly higher cholesterol levels (224 +/- 53 vs. 214 +/- 36 mg/dl), triglycerides (189 +/- 95 vs. 141 +/- 104 mg/dl), low density lipoprotein (LDL) cholesterol (156 +/- 51 vs. 138 +/- 33 mg/dl), apolipoprotein B (131 +/- 37 vs. 108 +/- 33 mg/dl) and Lp(a) levels (19.9 +/- 19 vs. 14.9 +/- 17.5 mg/dl). They also had significantly lower high density lipoprotein (HDL) cholesterol (36 +/- 11 vs. 45 +/- 12 mg/dl) and apolipoprotein A-I levels (114 +/- 26 vs. 136 +/- 32 mg/dl) (all p less than 0.005). On the basis of Lipid Research Clinic 90th percentile values for triglycerides and LDL cholesterol and 10th percentile values for HDL cholesterol, the most frequent dyslipidemias were low HDL cholesterol alone (19.3% vs. 4.4%), elevated LDL cholesterol (12.1% vs. 9%), hypertriglyceridemia with low HDL cholesterol (9.7% vs. 4.2%), hypertriglyceridemia and elevated LDL cholesterol with low HDL cholesterol (3.4% vs. 0.2%) and Lp(a) excess (15.8% vs. 10%) in patients versus control subjects, respectively (p less than 0.05). Stepwise discriminant analysis indicates that smoking, hypertension, decreased apolipoprotein A-I, increased apolipoprotein B, increased Lp(a) and diabetes are all significant (p less than 0.05) factors in descending order of importance in distinguishing patients with coronary artery disease from normal control subjects. Not applying a correction for beta-adrenergic blocking agents, sampling bias and diet effects leads to a serious underestimation of the prevalence of LDL abnormalities and an overestimation of HDL abnormalities in patients with coronary artery disease. However, 35% of patients had a total cholesterol level less than 200 mg/dl after correction; of those patients, 73% had an HDL cholesterol level less than 35 mg/dl.

Prevalence of lipoprotein (a) [Lp(a)] excess in coronary artery disease.

Lipoprotein (a) [Lp(a)] is composed of 1 low-density lipoprotein (LDL) particle, to which 1 molecule of apolipoprotein (a) is covalently linked. Elevated levels of Lp(a) have been associated with coronary artery disease (CAD) and Lp(a) has been shown to be highly heritable. Our purpose was to determine the prevalence of familial Lp(a) excess in patients with CAD. We determined plasma levels of Lp(a) in 180 patients (150 men and 30 women) with angiographically documented CAD before age 60 years, and in 459 control subjects (276 men and 183 women) clinically free of cardiovascular disease. In addition, Lp(a) levels were determined in families of 102 of the CAD probands (87 men and 15 women). No gender differences in Lp(a) levels were observed between men and women (patients or control subjects). Patients with CAD had higher Lp(a) levels than did control subjects (19 +/- 21 vs 13 +/- 15 mg/dl, p less than 0.001). The prevalence of Lp(a) excess (defined as greater than 90th percentile of controls) was 17% in patients with CAD (p less than 0.05). Lp(a) levels were not correlated with cholesterol, LDL cholesterol, high-density lipoprotein (HDL) cholesterol or apolipoproteins A-I or B. There was a weak correlation between Lp(a) and triglycerides (r = 0.166, p less than 0.05) in patients and control subjects. Stepwise discriminant analysis revealed that Lp(a) was a risk factor for the presence of CAD in men, independent of smoking, hypertension, diabetes, LDL and HDL cholesterol, or apolipoprotein A-I and B levels. Family studies revealed that Lp(a) levels are strongly genetically determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoprotein B-48 is the product of a messenger RNA with an organ-specific in-frame stop codon.

The primary structure of human apolipoprotein (apo) B-48 has been deduced and shown by a combination of DNA excess hybridization, sequencing of tryptic peptides, cloned complementary DNAs, and intestinal messenger RNAs (mRNAs) to be the product of an intestinal mRNA with an in-frame UAA stop codon resulting from a C to U change in the codon CAA encoding Gln2153 in apoB-100 mRNA. The carboxyl-terminal Ile2152 of apoB-48 purified from chylous ascites fluid has apparently been cleaved from the initial translation product, leaving Met2151 as the new carboxyl-terminus. These data indicate that approximately 85% of the intestinal mRNAs terminate within approximately 0.1 to 1.0 kilobase downstream from the stop codon. The other approximately 15% have lengths similar to hepatic apoB-100 mRNA even though they have the same in-frame stop codon. The organ-specific introduction of a stop codon to a mRNA appears unprecedented and might have implications for cryptic polyadenylation signal recognition and RNA processing.

Monoclonal antibodies as probes of high-density lipoprotein structure: identification and localization of a lipid-dependent epitope.

Eight stable murine monoclonal antibodies (mabs) were raised against human high-density lipoproteins (HDL). Three different antibody reactivities were demonstrated by immunoblotting. A group of five antibodies were specific for apolipoprotein A-I (apoA-I) and bound to similar or overlapping epitopes. The second type of reactivity, shown by mab-32, was specific for apoA-II. In the third group, two antibodies showed high reactivity with apoA-II and slight cross-reactivity with apoA-I. The properties of two antibodies, mab M-30 specific for apoA-I and mab M-32 specific for apoAII, were characterized in detail as probes of HDL structure. The association of 125I-labeled HDL or synthetic complexes of apoA-I and phosphatidylcholine with mab M-30 was lipid dependent. Mab M-32 binding to apoA-II was independent of lipid. The lipid-dependent epitope bound by mab M-30 has been localized to an 18 amino acid synthetic apoA-I peptide. Moreover, studies with HDL2, HDL3, and immunoadsorbed HDL subfractions indicate that binding of mab M-30 to HDL is influenced by some component within the microenvironment individual HDL particles. These lines of evidence suggest that the molar ratio of apoA-I to apoA-II is the critical determinant. Binding of mab M-32 to HDL increased the reactivity of HDL to mab M-30 in a dose-dependent manner, indicating an unusual form of cooperativity between two mabs that recognize different proteins in HDL. These monoclonal antibodies will be valuable in studies of the metabolic significance of protein-protein and lipid-protein interactions in HDL.

Isolation and characterization of a high molecular weight protein phosphatase from rabbit skeletal muscle.

A high molecular weight protein phosphatase (phosphatase H-II) was isolated from rabbit skeletal muscle. The enzyme had a Mr = 260,000 as determined by gel filtration and possessed two types of subunit, of Mr = 70,000 and 35,000, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On ethanol treatment, the enzyme was dissociated to an active species of Mr = 35,000. The purified phosphatase dephosphorylated lysine-rich histone, phosphorylase a, glycogen synthase, and phosphorylase kinase. It dephosphorylated both the alpha- and beta-subunit phosphates of phosphorylase kinase, with a preference for the dephosphorylation of the alpha-subunit phosphate over the beta-subunit phosphate of phosphorylase kinase. The enzyme also dephosphorylated p-nitrophenyl phosphate at alkaline pH. Phosphatase H-II is distinct from the major phosphorylase phosphatase activities in the muscle extracts. Its enzymatic properties closely resemble that of a Mr = 33,500 protein phosphatase (protein phosphatase C-II) isolated from the same tissue. However, despite their similarity of enzymatic properties, the Mr = 35,000 subunit of phosphatase H-II is physically different from phosphatase C-II as revealed by their different sizes on sodium dodecyl sulfate-gel electrophoresis. On trypsin treatment of the enzyme, this subunit is converted to a form which is a similar size to phosphatase C-II.

The association of phosphorylase kinase with rabbit muscle T-tubules.

Evidence is presented for the association of a phosphorylase kinase activity with transverse tubules as well as terminal cisternae in triads isolated from rabbit skeletal muscle. This activity remained associated with T-tubules throughout the purification of triad junctions by one cycle of dissociation and reassociation. The possibility that the presence of phosphorylase kinase in these highly purified membrane vesicle preparations was due to its association with glycogen was eliminated by digestion of the latter with alpha-amylase. The phosphorylase kinase activity associated with the T-tubule membranes was similar to that reported for other membrane-bound phosphorylase kinases. The enzyme had a high pH 6.8/pH 8.2 activity ratio (0.4-0.7) and a high level of Ca2+ independent activity (EGTA/Ca2+ = 0.3-0.5). The kinase activated and phosphorylated exogenous phosphorylase b with identical time courses. When mechanically disrupted triads were centrifuged on continuous sucrose gradients, the distribution of phosphorylase kinase activity was correlated with the distribution of a Mr 128,000 polypeptide in the gradients. This polypeptide and a Mr 143,000 polypeptide were labeled with 32P by endogenous and exogenous protein kinases. These findings suggest that the membrane-associated phosphorylase kinase may be similar to the cytosolic enzyme. Markers employed for the isolated organelles included a Mr 102,000 membrane polypeptide which followed the distribution of Ca2+-stimulated 3-O-methylfluorescein phosphatase activity, which is specific for the sarcoplasmic reticulum. A Mr 72,000 polypeptide was confirmed to be a T-tubule-specific protein. Several proteins of the triad component organelle were phosphorylated by the endogenous kinase in a Ca2+/calmodulin-stimulated manner, including a Mr ca. 72,000 polypeptide found only in the transverse tubule.

Isolation and characterization of rabbit skeletal muscle protein phosphatases C-I and C-II.

Previous studies have shown that phosphorylase phosphatase can be isolated from rabbit liver and bovine heart as a form of Mr approximately 35,000 after an ethanol treatment of tissue extracts. This enzyme form was designated as protein phosphatase C. In the present study, reproducible methods for the isolation of two forms of protein phosphatase C from rabbit skeletal muscle to apparent homogeneity are described. Protein phosphatase C-I was obtained in yields of up to 20%, with specific activities toward phosphorylase a of 8,000-16,000 units/mg of protein. This enzyme represents the major phosphorylase phosphatase activity present in the ethanol-treated muscle extracts. The second enzyme, protein phosphatase C-II, had a much lower specific activity toward phosphorylase a (250-900 units/mg). Phosphatase C-I and phosphatase C-II had Mr = 32,000 and 33,500, respectively, as determined by sodium dodecyl sulfate disc gel electrophoresis. The two enzymes displayed distinct enzymatic properties. Phosphatase C-II was associated with a more active alkaline phosphatase activity toward p-nitrophenyl phosphate than was phosphatase C-I. Phosphatase C-II activities were activated by Mn2+, whereas phosphatase C-I was inhibited. Phosphatase C-I was inhibited by rabbit skeletal muscle inhibitor 2 while phosphatase C-II was not inhibited. Both enzymes dephosphorylated glycogen synthase and phosphorylase kinase, but displayed different specificities toward the alpha- and beta-subunit phosphates of phosphorylase kinase (Ganapathi, M. K., Silberman, S. R., Paris, H., and Lee, E. Y. C. (1980) J. Biol. Chem. 246, 3213-3217). The amino acid compositions of the two proteins were similar. Peptide mapping of the two proteins showed that they are distinct proteins and do not have a precursor-proteolytic product relationship.

Purification and properties of pyruvate dehydrogenase phosphatase from bovine heart and kidney.

Pyruvate dehydrogenase phosphatase was purified to apparent homogeneity from bovine heart and kidney mitochondria. The phosphatase has a sedimentation coefficient (S20,w) of about 7.4 S and a molecular weight (Mr) of about 150 000 as determined by sedimentation equilibrium and by gel-permeation chromatography. The phosphatase consists of two subunits with molecular weights of about 97 000 and 50 000 as estimated by sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Phosphatase activity resides in the Mr 50 000 subunit, which is sensitive to proteolysis. The phosphatase contains approximately 1 mol of flavin adenine dinucleotide (FAD) per mol of protein of Mr 150 000. FAD is apparently associated with the Mr 97 000 subunit. The function of this subunit remains to be established. The phosphatase binds 1 mol of Ca2+ per mol of enzyme of Mr 150 000 at pH 7.0, with a dissociation constant (Kd) of about 35 microM as determined by flow dialysis. Use of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetate (EGTA) at pH 7.6 in conjunction with flow dialysis gave a Kd value for Ca2+ of about 8 microM. In the presence of both the phosphatase and the dihydrolipoyl transacetylase (E2) core of the pyruvate dehydrogenase complex, two equivalent and apparently non-interacting CA2+-binding sites were detected per unit of Mr 150 000, with a Kd value of about 24 microM in the absence and about 5 microM in the presence of EGTA. In the presence of 0.2 M KCl, which inhibits phosphatase activity about 95%, the phosphatase exhibited only one Ca2+-binding site, even in the presence of E2. The phosphatase apparently possesses an "intrinsic" Ca2+-binding site, and a second Ca2+-binding site is produced in the presence of E2. The second site is apparently altered by increasing the ionic strength. It is proposed that the second site may be at the interface between the phosphatase and E2, with Ca2+ acting as a bridging ligand for specific attachment of the phosphatase to E2.

Dephosphorylation of rabbit skeletal muscle phosphorylase kinase. Evidence against the operation of the "second-site phosphorylation" mechanism of regulation.

The dephosphorylation of rabbit skeletal muscle phosphorylase kinase was studied using two purified rabbit skeletal muscle protein phosphatases. The first enzyme (Mr = 32,000) corresponds to the form we have previously termed protein phosphatase C. Phosphorylase kinase was found to be rapidly dephosphorylated by this enzyme. The site of dephosphorylation was examined, and it was shown that this enzyme was relatively specific for the dephosphorylation of the beta-subunit phosphate, as compared to the alpha-subunit phosphate, of phosphorylase kinase. Phosphate release from the beta-subunit was approximately 100-fold faster than from the alpha-subunit. More importantly, dephosphorylation of the beta-subunit phosphate was not significantly affected by phosphorylation of the alpha-subunit. The dephosphorylation of phosphorylase kinase by a second low molecular weight protein phosphatase, Mr = 33,500, was also studied. The specific activity of this enzyme toward phosphorylase kinase was only a fraction of that exhibited by the Mr = 32,000 phosphatase. This enzyme removed phosphate from both the alpha- and beta-subunits but more rapidly (about 4-fold) from the alpha-subunit. With neither of these enzyme preparations was there any evidence for the regulation of beta-subunit dephosphorylation by phosphorylation of the alpha-subunit as proposed by Cohen and Antoniw ((1973) FEBS Lett. 34, 43-47).