Investigation and validation of the affinity chromatography method for measuring glycated albumin in serum and urine.

Affinity chromatography on m-aminophenyl boronate columns together with albumin measurement by radioimmunoassay has been validated as a method for determining glycated albumin in serum and urine. Optimisation of sample volume and of elution buffer composition and volume ensured reproducibility of results. Fructosamine assay confirmed the absence of glycated albumin species from the non-glycated fraction. It was possible to elute the glycated fraction from the affinity columns with Tris or glycine which do not contain 1,2 diols but have similar functional groups. Column affinity was, therefore, not specific for glycated protein moieties. Inhibition of binding by glucose, and other small molecules in urine, necessitated ultrafiltration or dialysis of samples before analysis. Reference ranges for glycated albumin in non-diabetic subjects were 0.6-1.8% in serum and 0.9-2.6% in urine. In patients with diabetes mellitus, glycated albumin ranged from 1.4-10.9% in serum and from 1.5-12.5% in urine.

Intrinsic susceptibility of the kidney to acetaminophen toxicity in middle-aged rats.

Acetaminophen (APAP)-induced nephrotoxicity is age-dependent in male Sprague-Dawley (SD) rats: middle-aged (9-12 months old) rats exhibit nephrotoxicity at lower dosages of APAP than do young adults (2-3 months old). The present study was designed to test the hypothesis that the intrinsic susceptibility of renal tissue to APAP toxicity is increased in middle-aged rats. APAP toxicity was evaluated in renal slices from naive 3- and 12-month-old male SD rats incubated with 0-50 mM APAP for 2-8 h. Renal slice glutathione (GSH) and APAP concentrations were determined; renal function was assessed by organic anion (para-aminohippurate, PAH) and cation (tetraethylammonium, TEA) accumulation; and cell viability was assessed by lactate dehydrogenase (LDH) leakage. At each concentration of APAP tested, accumulation of APAP by renal slices was similar in 3- and 12-month-olds. APAP toxicity in renal slices from both 3- and 12-month-old rats was characterized by concentration-dependent increases in LDH leakage. In contrast to APAP nephrotoxicity in vivo, APAP toxicity in renal slices was accompanied by decreased accumulation of PAH and TEA. Additionally, APAP produced marked reductions in renal slice GSH content in a concentration-dependent manner: however, in contrast to APAP nephrotoxicity in vivo, APAP-induced GSH depletion in vitro did not precede cytotoxicity. No consistent age-dependent differences in the time- and concentration-response curves for APAP nephrotoxicity were observed. These data suggest that APAP cytotoxicity in vitro is not increased in 12-month-old rats. However, since the pattern (and mechanisms) of APAP cytotoxicity in vitro appears to be different from that observed in vivo, extrapolation of in vitro cytotoxicity to in vivo nephrotoxicity is limited. Therefore, age differences in intrinsic susceptibility of the intact kidney cannot be excluded as a mechanism contributing to enhanced APAP nephrotoxicity in middle-aged rats.

Binding and degradation of NH2-terminal parathyroid hormone by opossum kidney cells.

The binding and cellular processing of NH2-terminal parathyroid (PTH) hormone by confluent monolayers of opossum kidney (OK) cells was characterized using radiolabeled PTH peptide analogues. Time- and temperature-dependent specific binding of 125I-labeled (Nle-8,18, Tyr-34)-NH2-bovine(b)PTH-(1-34) was accompanied by the appearance of degraded radiolabel in the cell medium. Degrading activity was observed to be a specific consequence of binding by PTH receptors. Degrading activity was inhibited by monensin, chloroquine, and NH4+ but not by chymotrypsin inhibitors. Acid washing demonstrated that greater than 80% of total cell-associated specific binding at equilibrium was located in a rapidly internalized (acid-resistant) pool. Monensin pretreatment led to increased acid-resistant binding, presumably through inhibition of turnover of internalized receptor ligand and indicated that the degradation of radiolabel was probably associated with processing of the receptor-ligand complex. Release of intact radiolabel from the acid-resistant pool indicated that some of the internalized peptide was recycled out of the cell in an undegraded form (retroendocytosis). Acid-resistant binding and degradation of 125I-(Nle-8,18, Tyr-34)-NH2-bPTH-(3-34) was minimal, indicating that this ligand was not internalized. It is concluded that the binding and internalization of PTH-(1-34) fragment by confluent OK cells is a specific receptor-mediated process. Cellular processing of PTH-(1-34) conforms to established models of internalization by receptor-mediated endocytosis.

Measurement of circulating parathyroid hormone.

The measurement of parathyroid hormone (PTH) is important in the investigation of disorders of mineral metabolism. Though the first immunoassay for PTH was described more than 25 years ago, it is only recently that methods have been developed which provide the required sensitivity and specificity to detect small changes in hormone secretion in normal subjects. These new methods take advantage of modern labelled antibody technology and have already been shown capable of yielding improvements in the diagnosis and monitoring of disorders which involve changes in PTH secretion. In addition, they are now providing fresh insight into the basic physiology of PTH secretion.

Elevated serum intact parathyroid hormone levels in elderly patients with hip fracture.

It has been postulated that secondary hyperparathyroidism contributes to bone loss and the high incidence of hip fractures in the elderly population, but there are no data on serum intact parathyroid hormone concentrations in these patients. In this study, serum intact parathyroid hormone (PTH) levels have been measured in 39 elderly patients with hip fracture; in addition, serum 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 concentrations have been measured. Twenty patients (51.3%) had elevated serum intact PTH concentrations whilst five (12.8%) had abnormally low serum 25-hydroxyvitamin D3 levels and serum 1,25-dihydroxyvitamin D3 was reduced in only two. These results provide the first direct evidence for secondary hyperparathyroidism in elderly patients with hip fracture. Vitamin D deficiency is unlikely to be the sole cause of secondary hyperparathyroidism in these subjects and calcium deficiency by itself may also contribute.

Hypotension induced by vasopressin antagonists in rats: role of mast cell degranulation.

SK&F 101926, a synthetic peptide, is a potent antagonist of vasopressin at both the V2 and the V1 receptors. Following intravenous administration of SK&F 101926 (5 mg/kg), mean arterial pressure (MAP) immediately fell 75 mm Hg. Heart rate increased approximately 50 beats/min. Cutaneous flushing and cyanosis appeared approximately 2 to 5 min after the SK&F 101926 administration. Three of the five rats died within 40 min with no improvement in either color or MAP. The two surviving animals slowly recovered from these symptoms. The hypotension and flushing recorded in these studies resembled the effects during hypotensive shock. SK&F 101926 degranulated rat peritoneal mast cells in vitro as measured by the liberation of histamine. Analogs of SK&F 101926 were identified having reduced activity to release histamine from mast cells in vitro. The activity of these analogs to release histamine in vivo was also tested, as reflected by rat paw edema. A positive correlation was found between the potency to produce edema in vivo and the potency to release mast cell histamine in vitro (r = 0.94, p less than 0.05). In addition, compounds that released mast cell histamine and induced rat paw edema also produced hypotension and death when administered intravenously, while analogs which produced minimal histamine release in vitro produced minimal or no cardiovascular changes or lethality in vivo at the same dosages (5 mg/kg). Finally, cyproheptadine (10 mg/kg), an antagonist at both the serotonin and the histamine receptors, blunted the effects of SK&F 101926 on MAP and blocked the lethality. Pretreatment with a combination of histamine (H1 and H2) antagonists provided little protection against the SK&F 101926-induced toxicity. These data indicate that the cardiovascular toxicity of SK&F 101926 (and related peptides) is mediated via the release of autocoids from mast cells. Serotonin appears to play a major role in mediating the cardiovascular toxicity of SK&F 101926.

Renal blood flow changes induced with endothelin-1 and fenoldopam mesylate at quantitative Doppler US: initial results in a canine study.

PURPOSE: To evaluate quantitative Doppler ultrasonography (US) for assessing renal blood flow changes induced with endothelin-1 (ET-1) and fenoldopam mesylate in conscious dogs. MATERIALS AND METHODS: A blood flow probe was surgically implanted around the renal artery in eight adult dogs. Color and power Doppler US images were acquired in conscious restrained dogs during intravenous infusion of ET-1 and fenoldopam mesylate. Simultaneous with imaging, blood flow through the renal artery was measured with the implanted probe. The color level of the images within the region representing the kidney was analyzed to derive flow indices. These indices were compared with direct-flow measurements. RESULTS: The flow indices, color-weighted flow area (CWFA), and percentage of area of color, derived from color and power Doppler US images, correlated linearly with direct flow. The mean color level of color and power Doppler US images correlated weakly with direct flow. Pre- versus postinfusion CWFA decreased with all ET-1 infusions (P < or =.032). Infusion of fenoldopam mesylate increased CWFA in all cases (P < or =.032). CONCLUSION: Quantitative Doppler US enabled successful measurement of the flow changes induced with ET-1 and fenoldopam mesylate. Quantitative Doppler US is potentially useful as a noninvasive surrogate endpoint in evaluating the action of various therapeutic agents.