Biomedical subjects
M D Shepard
Publications and source records attributed to M D Shepard.
The kinetics of sulfobromophthalein uptake by rat liver sinusoidal vesicles.
The kinetics of bromo[35S]sulfophthalein (35S-BSP) binding by and uptake across the hepatocyte sinusoidal membrane were investigated using isolated rat liver sinusoidal membrane vesicles containing K+ as the principal internal inorganic cation. Uptake of 35S-BSP into vesicles was found to be temperature dependent, with maximum uptake between 35 and 40 degrees C; only binding occurred at or below 15 degrees C. Uptake at 37 degrees C was saturable and resolvable by Eadee-Hofstee analysis into two components: one with high affinity (Km = 53.1 microM) but low capacity, and the second of low affinity (Km = 1150 microM) but high capacity. By pre- or post-incubation, respectively, with unlabelled BSP, trans-stimulation and counter transport of 35S-BSP could also be demonstrated in these vesicles. Uptake was inhibited competitively using 5 microM Rose bengal and 10 microM indocyanine green, and non-competitively using 10 microM DIDS. Taurocholate did not inhibit uptake, and actually enhanced transport at concentrations greater than or equal to 250 microM. Imposition of inwardly directed inorganic ion gradients resulted in the enhancement of 35S-BSP transport when chloride ions were part of this gradient, irrespective of the cation employed whereas there was no apparent cation effect. However, substitution of 10 mM Na+ for 10 mM K+ as the internal cation resulted in a significant increase in uptake in the presence of external K+ as compared to Na+ gradients. This effect was not observed when 10 mM Tris+ was employed as the internal cation. The kinetics of 35S-BSP uptake by isolated sinusoidal membrane vesicles are indicative of facilitated transport. While the observed inorganic ion effects suggest a possible electrogenic component, the driving forces for hepatic BSP uptake remain uncertain. Isolated sinusoidal membrane vesicles provide a useful technique for studying hepatic uptake processes independent of circulatory or subsequent cellular phenomena.
Performance of quantitative urine analysis in Australasia critically assessed.
The standard of quantitative urine analysis in Australasia has been assessed over a three-year period (1984-1986) through a national interlaboratory quality-assurance program, conducted jointly by the Royal College of Pathologists of Australasia and the Australian Association of Clinical Biochemists. We investigated the precision and accuracy of individual methods and measurement systems routinely used in 14 urine assays. Assays of sodium, potassium, creatinine, glucose, and chloride were performed satisfactorily. Further improvement is required in assays of urinary osmolality, phosphate, and 5-hydroxyindoleacetic acid. Determinations of oxalate, urea, calcium, and 4-hydroxy-3-methoxymandelic acid are improving, but assays of protein and urate in urine present major problems for Australasian laboratories. In this program the sulfosalicylic acid turbidimetric method for urinary protein has consistently displayed poor precision plus a significant positive bias and should be abandoned. In contrast, the trichloroacetic acid-Ponceau S manual method has continued to perform well.
A new recycling technique for human placental cotyledon perfusion: application to studies of the fetomaternal transfer of glucose, inulin, and antipyrine.
A previously described technique has been modified to permit the continuously recirculating perfusion of the separate maternal and fetal circulations of an isolated cotyledon of human placenta. Viability of the perfused cotyledons was established by measurements of oxygen consumption (average, 0.18 ml/gm/hr), glucose utilization (average, 1.0 mg/gm/hr), and lactate production (less than 0.01 mumol/gm/hr), and integrity of the placental barrier by the failure of India ink, 125I-albumin, or 35S-sulfobromophthalein to cross from fetal to maternal circulation. Clearance of 3H-inulin from the fetal circuit, 0.0059 +/- 0.0005 (SE) ml/min/gm, corresponded to 2.5% of its clearance by the adult human kidney. Clearance of 14C-antipyrine was 0.013 +/- 0.003 ml/min/gm. After introduction into the fetal circuit, the observed appearance of both inulin and antipyrine in the maternal circuit closely paralleled curves predicted by a simple mathematical model. The use of a continuously recirculating perfusion system is technically feasible, and has advantages over the single-pass technique for studying transplacental transfer of metabolites with a low efficiency of extraction.
Changes in renal cortical sialic acids and colloidal iron staining associated with exercise.
The purpose of this study was to determine if exercise was associated with alterations in renal cortical sialic acid content or glomerular capillary anionic character. These factors have been shown to be important, insofar as they contribute to an electrostatic barrier which prevents the filtration of negatively charged macromolecules. In unilaterally nephrectomized dogs (n=7), exercised kidneys tended to have increased amounts of sialic acids and a decrease in glomerular anionic character, as evaluated by the intensity of colloidal iron staining, however, nephrectomy alone also caused similar changes. Additional experiments were conducted using rat litter mates assigned to control or treadmill-exercise groups. Exercised animals were run for 60-80 min. Renal cortical sialic acids were 2.74 +/- 0.07 mumol/g for controls (n=12) and 3.03 + 0.09 mumol/g for the exercised animals (n=10) (P less than 0.05). Colloidal iron staining, rated on a 0-3 scale (0=no uptake, 3=maximum staining) was 2.5 +/- 0.1 and 1.3 +/- 0.3 for the controls and exercised animals, respectively (P less than 0.05). Colloidal iron staining remained below control levels for 24 h post-exercise. These data suggest that exercise increases glomerular sialic acid content and decreases colloidal iron staining. This latter effect may decrease the glomerular capillary electrostatic barrier and thereby may be an important factor in causing exercise proteinuria.
Analytical goals for quantitative urine analysis: a clinical view.
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