Biosynthesis of D-glucaric acid in mammals: a free-radical mechanism?

In the presence of iron salts and hydrogen peroxide, D-glucuronic acid was converted into D-glucaric acid. The reaction was strongly inhibited by free-radical scavengers and is ascribed to the action of the hydroxyl radical. The formation of D-glucarate was dependent upon pH and occurred in the presence of some iron-complexing agents. The first product of oxidation was a lactone that was a strong inhibitor of beta-D-glucuronidase and assumed to be D-glucaro-1,5-lactone. Microsomal preparations in the presence of NADPH also produced D-glucarate from D-glucuronic acid, presumably due to formation of hydrogen peroxide, and the product was an inhibitor of beta-D-glucuronidase. Superoxide did not produce D-glucarate from D-glucuronate. The cytochrome P450 system is more likely than "glucuronolactone dehydrogenase" to be responsible for the production of D-glucaric acid in vivo.

An enzymatic determination of D-glucaric acid by conversion to pyruvate.

A method was developed for determination of D-glucaric acid by treatment with a bacterial extract containing glucarate dehydratase and ketodeoxyglucarate aldolase. This led to the quantitative formation of pyruvate, which was then assayed by use of lactate dehydrogenase. Measurements of D-glucarate in individual samples of human urine by this technique were compared with those by the commonly used method of beta-glucuronidase inhibition, and gave values for D-glucarate content which were about 25% higher, but with otherwise good correlation.

Fibronectin: its relationship to basement membranes. II. Ultrastructural studies in rat kidney.

Fibronectin, basement membrane and type I collagen antigens have been localized in normal rat kidney by electron immunohistochemical methods. Immunoreactive fibronectin was found in the interstitial connective tissue matrix and on collagen fibers, while tubular, endothelial and smooth muscle basement membranes throughout the kidney were consistently negative. In the glomerulus immunoreactive fibronectin was abundant in the mesangial matrix. The peripheral glomerular basement membrane was occasionally reactive in a spotty, irregular manner. These findings suggest that fibronectin antigens are probably not a constituent of basement membranes. It is proposed that some fibronectin antigen may be trapped in the glomerular filter, and that normal glomerular cleansing mechanisms would transport this trapped fibronectin toward the mesangial areas where it would be eventually processed.

Glomerular basement membrane: lamina rara, lamina densa.

Electron immunohistochemical and histochemical studies of glomerular and tubular basement membranes were undertaken to elucidate the relationship between lamina rara and lamina densa. Our results indicate that the lamina rara is not a fixation artifact and it has common antigenic determinants with lamina densa (type IV collagen antigens). Therefore, lamina rara has to be considered an integral component of basement membranes, and biochemical preparations containing only lamina densa should not be considered representative of the whole basement membrane.

An electron-microscope study of beta-glucuronidase crystals.

beta-Glucuronidase from rat preputial glands was crystallized as thin sheets having p6 symmetry in projection with a equal 20.2nm. A filtered image was produced by Fourier methods to a resolution of 2.2 nm by averaging information from six areas. This suggests an approximately triangular molecular outline in projection, and this is taken to indicate a probable tetrahedral arrangement of the four subunits of the beta-glucuronidase molecule.

A rapid manual method for routine assay of ascorbic acid in serum and plasma.

A method is described for the assay of ascorbic acid in either serum or heparinized plasma. 1. The assay is based on the reduction of ferric chloride by ascorbic acid with the resulting ferrous ion quantitated by the addition of 2,4,6-tripyridyl-s-triazine to form a purple colour with a maximum absorbance at 595 nm. 2. Uric acid interference has been eliminated by the use of a high molarity acetate buffer and by optimising the amount of TPTZ and ferric chloride used. 3. Protein was found to cause rapid fading of the final colour; it was therefore necessary to remove the protein, by addition of 10% trichloroacetic acid, from the specimen prior to the final assay. This had the added advantage of assisting to stabilize the ascorbic acid prior to final assay. 4. All reagents used are easily obtained and no special equipment is required.

Variant angina pectoris. Pain and arrhythmias controlled after postoperative myocardial infarction.

A 30-year-old man with variant angina pectoris and ventricular arrhythmias had an angiographically demonstrable 60% obstructive lesion of the proximal left anterior descending coronary artery that was observed to progress to 100% during spasm. Control of pain and arrhythmia by pharmacologic means was unsuccessful. Aortocoronary saphenous vein-internal mammary coronary bypass was associated with an anteroseptal wall myocardial infarction and relief from both angina pectoris and arrhythmias. It is suggested that infarction of the ischemic myocardium played a role in the successful management of this case.

Golgi beta-glucuronidase of androgen-stimulated mouse kidney.

Subcellular fractions were prepared from mouse kidney homogenates by differential and sucrose-gradient centrifugation. A fraction enriched in Golgi apparatus was obtained, which had considerably enriched galactosyltransferase and thiamin pyrophosphatase activities, and was morphologically typical of Golgi material. This preparation also had high beta-glucuronidase activity, which increased concomitantly with microsomal beta-glucuronidase activity during the specific stimulation of the enzyme in male mouse kidney after androgen administration. The degree of stimulation was much greater in the Golgi fraction. Gel-electrophoretic patterns of Golgi beta-glucuronidase resembled more closely those of the enzyme located within lysosomes, but contained minor bands similar to those described previously (Swank & Paigen, 1973) as characteristic of the microsomal enzyme. It was concluded that the Golgi complex is involved in the distribution of the enzyme after its synthesis to both lysosomal and microsomal fractions.

Metabolism of D-glucuronolactone in mammalian systems. Inhibitory properties of the products of D-glucuronolactone-dehydrogenase action.

1. d-Glucuronolactone was converted into d-glucaro-(1-->4)-lactone, a beta-glucuronidase inhibitor, probably via the intermediate d-glucaro-(1-->4)-(6-->3)-dilactone, by a dehydrogenase in human-liver extracts. 2. Similar experiments with mouse-liver extracts appeared to yield only d-glucaric acid or a non-inhibitory lactone. 3. A strong beta-glucuronidase inhibitor was present in mouse liver after the intraperitoneal administration of d-glucuronolactone. 4. d-Glucarodilactone decomposed spontaneously in aqueous solution to give 25% of d-glucaro-(1-->4)-lactone; the percentage conversion was unaffected by human-liver homogenates. but considerably increased by mouse-liver or pig-liver fractions.