Treatment with acarbose, an alpha-glucosidase inhibitor, reduces increased albumin excretion in streptozotocin-diabetic rats.

1. We examined the effect of the alpha-glucosidase inhibitor acarbose on urinary albumin excretion (UAE) in streptozotocin diabetic rats. 2. Treatment with acarbose for 8 weeks after induction of diabetes prevented the significant increase in UAE observed in untreated diabetic rats relative to nondiabetic controls. 3. Acarbose significantly reduced integrated glycemia, which correlated with albumin excretion rates, and exerts a salutary effect on diabetic renal dysfunction.

Glycated albumin: a marker of glycaemic status in rats with experimental diabetes.

Monoclonal antibodies that specifically recognize non-enzymatically glycated epitopes residing in albumin were used to measure levels of glycated albumin in the plasma of control and streptozotocin diabetic rats. Standard or sample was incubated with monoclonal antibody immobilized onto preactivated tubes, and binding was detected with enzyme-conjugated polyclonal antibody to rat albumin and substrate. Rat glycated albumin exhibited a linear dose response in the assay, and plasma samples contained between 30-100 micrograms/50 microliters. Levels were significantly increased in diabetic compared with control rats, and correlated positively and significantly with mean blood glucose concentrations measured in the fasting and fed states. The results indicate that glycated albumin levels measured by immunoassay reflect recent integrated glycaemia and provide and objective index of glycaemic status in the rat experimental model of diabetes.

Evaluation of new rapid office test for microalbuminuria and its comparison to fully quantitative radioimmunoassay.

A novel enzyme-linked immunosorbent assay (ELISA [Dialbumin]) for rapid office measurement of microalbuminuria was evaluated and its performance compared with that of a commercially available radioimmunoassay (double-antibody albumin). Urine samples containing between 0.75 and 1800 micrograms/ml of albumin were obtained from 31 diabetic patients and assayed by both methods. A comparison of the paired values obtained from the two methods gave a correlation coefficient of greater than 0.99. The Dialbumin assay, which used detachable eight-well strips (1 strip/sample), 10-min incubation, tap water wash, and a 2-min color development step, was read on both an ELISA reader and a hand-held analytical device (Acc-U-Dial) designed specifically for this test. The findings of this study indicate that the Dialbumin assay, used in conjunction with the Acc-U-Dial device, affords a rapid, convenient, and sensitive method for quantitative determination of a broad range (0.3-1280 micrograms/ml) of urinary albumin levels in the office setting.

Prostaglandins in inflammatory bone pathology: mechanism and therapeutic benefit of etodolac.

To investigate the role of PGE2 in the development of bone and joint pathology in rat adjuvant arthritis, hindlimb paws were evaluated by calcified tissue histologic techniques focusing on histochemical visualization of cartilage and bone lesions. Case studies of hindlimbs from normal, adjuvant arthritic, and etodolac-treated arthritic rats demonstrated the association of disease severity with inflammation, chondromalacia, replacement of adipose bone marrow with a fibroid marrow, osteoclastic bone resorption, synovial cysts, and pannus formation within the joints. Extensive periosteal intramembranous bone formation was temporally associated with joint destruction and medullary tissue pathology. In vivo data were correlated with in vitro effects of inflammatory mediators (IL-1, PGE2) on bone resorption. Etodolac blocked bone explant PGE2 accumulation at concentrations of 10(-7) M and higher, and inhibited bone resorption at concentrations of 10(-5) M and higher. The data indicate that in vitro and in vivo models of bone metabolism are well correlated regarding prostaglandin synthesis; that the inflammatory mediator PGE2 is largely responsible for the involvement of skeletal tissue in the adjuvant arthritis model; and that the effects of etodolac are specifically mediated by its ability to inhibit PGE2 accumulation in vivo.

Improved competitive enzyme-linked immunoassay (ELISA) for albuminuria.

Microalbuminuria has been established as a marker strongly predictive of diabetic nephropathy. The appearance of microalbuminuria (30-150 micrograms/min) is considered to herald incipient nephropathy, and the progression to clinical proteinuria (greater than 200 mg/24 h) is believed to reflect a shift from reversible to irreversible renal damage in diabetic patients. Periodic monitoring of albumin excretion could thus detect diabetic renal disease at an early, potentially reversible stage. However, this is not routinely done, largely due to cumbersome collection and methodologic constraints. We therefore have developed a simplified competitive immunoassay (ELISA) that is sensitive to 10 ng and reproducibly quantifies urinary albumin levels between 0.1-10 micrograms/ml, a range appropriate to that demarcating normal from microalbuminuric patients. Examination of results obtained with aliquots of 24 h and fractional urine collections, without and with correction for creatinine (albumin: creatine ratio), in basal and post-exercise states indicates that (a) this assay can effectively measure urine albumin concentration in single void specimens, and albumin excretion rates in fractional collections, and (b) conversion to albumin:creatinine ratio is not necessary to discriminate normal from microalbuminuric states.

An ELISA for PGE2 utilizing monoclonal antibody.

An ELISA for PGE2 has been developed which is sensitive to concentrations of 0.5 to 20.0 ng PGE2/ml. Mouse monoclonal anti-PGE2 ascites is utilized in a binding competition between the test sample and an adsorbed conjugate of PGE2-BSA. The antibody which remains bound to the solid phase is quantitated colorimetrically by incubation with alkaline phosphatase-conjugated goat anti-mouse IgG followed by incubation with p-nitro-phenylphosphate. PGE1, PGA1, PGA2, PGB2, 6-keto-PGF1 alpha, PGF2 alpha, 13,14-dihydro-15-keto-PGE2, thromboxane B2 and arachidonic acid showed minimal cross-reactivity with the anti-PGE2. The PGE2 ELISA permits the quantitative analysis of large numbers of samples at a fraction of the cost and time required to process a commercial RIA kit. When linked to the appropriate computer software, data collection and analysis can be performed in less than 10 minutes per 96-well plate. Furthermore, the use of an ELISA system eliminates the radioactive and toxic chemical waste generated by RIA methods.

Inhibition of prostaglandin biosynthesis by etodolac. I. Selective activities in arthritis.

Etodolac is the first anti-inflammatory drug belonging to the tetrahydropyranoindole class. In contrast to several other common anti-inflammatory drugs, etodolac exhibited an unusually high potency as an inhibitor of established adjuvant arthritis relative to its activity against carrageenan paw edema in the rat. This phenomenon led us to investigate whether the ability of NSAIDs to inhibit prostaglandin biosynthesis differed between cultures of macrophages, which are present in inflammatory exudates, and cultures of synoviocytes and chondrocytes, which contribute to inflammation of the articulating joint. Although other anti-inflammatory drugs were found to be equally active in all three cell types, etodolac was found to be much more effective on the cells of the joint than on the macrophage. This differential activity may be responsible for the striking efficacy of etodolac as an anti-arthritic drug.

Patterns of proteoglycan degradation by a neutral protease from human growth-plate epiphyseal cartilage.

The hypothesis is widely held that proteolytic degradation of proteoglycans in the lower hypertrophic zone of the growth plate may be involved in the initiation of mineralization in the zone of provisional calcification. However, a neutral protease that is responsible for the degradation of proteoglycans in the growth plate has not been identified, isolated, and characterized. In the work reported here, neutral protease activity in the growth plate is demonstrated for the first time, and some of the properties of the enzyme are described. Proteoglycans subunits were prepared from bovine nasal cartilage and calf costal cartilage by equilibrium density-gradient centrifugation under dissociative conditions. The proteoglycan subunits were labeled with 14C-formaldehyde. Homogenates from human growth plates were examined for neutral protease activity using the proteoglycan subunits as substrates. Following incubation of the proteoglycan subunits with growth-plate homogenates at pH 5.3 and at pH 7.5 in the presence and absence of ten-millimolar magnesium chloride and calcium chloride, the digestion products were examined by gel chromatography on Sepharose-2B and 6B columns. Column eluants containing proteoglycan-subunit degradation products were monitored for uronic acid, hexose, and radio-activity. Maximum extensive degradation of proteoglycan subunits occurred at pH 7.5 in the presence of ten-millimolar magnesium chloride and calcium chloride.