Urea Space Research Articles

Ontogeny of blood-brain barrier permeability to, and cerebrospinal fluid sink action on, [14C]urea

To ascertain the ontogeny of CSF sink action on a small hydrophilic solute, we investigated the rate and extent of uptake of [14C]urea by lateral ventricle choroid plexus (CP), cisternal cerebrospinal fluid (CSF), cerebral cortex, and cerebellum in 0.5- to 4-wk-old etherized nephrectomized rats. Five hours after ip injection, radiourea penetrated the entire H2O of tissue and CSF in 1-wk-old brain; moreover, for animals 1-4 wk old there was a marked inverse relationship between age and magnitude of steady-state [14C]urea space in all regions. However, there was lack of progression (with advancing age) in distribution times to steady state, undoubtedly a reflection of the complexity of maturational factors (CSF secretion, barrier permeability, biphasic changes in cerebral blood flow, etc.) that affect solute penetration into central nervous system. Analysis of [14C]urea uptake curves for various regions at different stages of development (1 vs. 2 wk) revealed half times (slow component) that were significantly greater at 1 wk (1.2-1.4 h) than at 2 wk (0.5-0.8 h). Steady-state concentration gradients for tracer urea, plasma to CP to CSF, indicated negligible molecular sieving of urea by CP 1 wk after birth; however, after the 2nd postnatal wk CSF sink action on urea became manifest due to development of CSF secretion and molecular sieving at the blood-CSF and blood-brain barriers.

Measurement of intracellular volume in monolayers of cultured cells

Methods have been developed for measuring the intracellular water space (ICS) in cultured diploid fibroblast monolayers. The values obtained have been compared to similar measurements of ICS in suspended fibroblasts using an oil spin method. Markers commonly used for measurement of total water space (TWS) ([3H]H2O, [14C]urea) and extracellular space (ECS) ([3H]inulin, [14C]L-glucose, [3H]sucrose, [3H]D-mannitol) were investigated for use in cell monolayers. Monolayer incubations were terminated by rapidly rinsing the culture dishes three times with ice cold buffer. The distribution volume of the TWS marker [14C]urea plateaued at 10 to 15 min and was independent of urea concentration. [3H]H2O was not a suitable marker for measuring total water space in cell monolayers because of rapid loss of intracellular label during rinsing. Intracellular space was calculated by subtracting [3H]sucrose space (5 min) from [14C]urea space (20 min) after incubation of fibroblasts with both markers. Values obtained for ICS (mean +/- SE: microliter/10(6) cells) of fibroblasts from two cell lines measured in monolayer (1.74 +/- 0.11 and 1.60 +/- 0.10) and in suspension (1.88 +/- 0.07 and 1.78 +/- 0.11) was approximately 10% smaller than the values for cell size (2.01 and 2.22) obtained from Coulter Counter sizing. Thus, the methods developed for measurement of ICS in monolayer fibroblasts yield data comparable to those obtained with the more standard oil spin method. Furthermore, the methods can be applied to measurement of ICS in other types of adherent cells.

THE USE OF UREA DILUTION AND ULTRASONIC BACKFAT THICKNESS TO PREDICT THE CARCASS COMPOSITION OF LIVE LAMBS AND CATTLE

Thirty-eight lambs (22 rams, 16 ewes), 25 Holstein cows and 30 steers were evaluated ultrasonically for subcutaneous fat thickness. Urea space was also estimated, using the dilution principle, by a single injection of a known amount of urea and taking a single blood sample 12 min later. All lambs and cattle were slaughtered within 2 days and the half-carcasses were separated into fat, lean and bone. Urea space (R2 = 0.10) and fat thickness (R2 = 0.18) in lambs were poorly related to the weight of half-carcass lean tissue. Neither urea space nor fat thickness improved the level of explained variation in half-carcass lean tissue weight over that explained by liveweight alone (R2 = 0.73). Urea space showed a larger association with half carcass lean weight in cows (R2 = 0.55) than in steers (R2 = 0.14), but again did not improve the relationship provided by liveweight alone (R2 = 0.60). Fat thickness provided nonsignificant regressions (P < 0.05) with half-carcass lean weight both in cows and in steers. Liveweight was the dominant independent variable (R2 = 0.33) for predicting total fat in lamb half-carcasses; urea space (R2 = 0.08) and fat thickness (R2 = 0.13) did not improve the prediction given by liveweight alone. Fat thickness was poorly related to total fatness both in steers (R2 = 0.12) and in cows (nonsignificant regression). A multiple regression equation combining fat thickness and liveweight provided the best prediction of half-carcass fat in cows, whereas a similar equation with the addition of urea space gave the best prediction of half-carcass fat in steers. The measurement of urea space and fat thickness to predict the weight of carcass tissues (lean, fat) in live lambs and cattle over the weight (41.9 + 9.7 kg(SD) lambs, 624 ± 62.8 kg cows and 466 ± 63.2 kg steers) and fatness (19.9 ± 3.27% (SD) lambs, 21.9 ± 2.18% cows and 20.9 ± 3.98% steers) ranges studied was of limited value. Key words: Urea dilution, ultrasound, live animal evaluation, carcass composition

Evaluation of Urea Space and Ultrasonic Measurement as Selection Criteria for Beef Animal Composition

Evaluation of Urea Space and Ultrasonic Measurement as Selection Criteria for Beef Animal Composition

Quantative evaluation of total body fluid by measuring Urea Space

Quantative evaluation of total body fluid by measuring Urea Space

Reversal of insulin effects in fat cells may require energy for deactivation of glucose transport, but not for deactivation of phosphodiesterase

The reversal of insulin effects on sugar transport and phosphodiesterase in fat cells was studied after arresting further actions of insulin with KCN, NaN 3, 2,4-dinitrophenol, or dicumarol. These agents rapidly lower the ATP concentration and concomitantly block the actions of insulin added later. Contrary to our expectation, the above inhibitors failed to initiate deactivation of the hormone-stimulated transport system. Instead, in the presence of the agents the transport system remained activated even after cells had been washed with an insulin-free buffer. This effect of the inhibitors was reversed when cells were washed with an inhibitor-free buffer containing glucose or pyruvate. The above inhibitors also blocked the deactivation of sugar transport stimulated by mechanical agitation. The effects of the inhibitors could not be explained by their possible effects on the basal transport activity, the intracellular urea space, or the cell count. The insulin-stimulated phosphodiesterase activity was rapidly lowered when cells were exposed to the above inhibitors. Apparently, these agents did not denature phosphodiesterase itself since the latter could be reactivated by insulin when inhibitor-treated cells were washed with a glucose-containing buffer. None of the above agents, except dicumarol, significantly inhibited phosphodiesterase activity in a cell-free system. It is suggested that the effects of insulin on sugar transport and phosphodiesterase are reversed by different mechanisms. ATP or metabolic energy may be involved in the deactivation of sugar transport, but not in that of phosphodiesterase.

Metabolic characteristics of pancreatic β-cells exposed to calcium-transporting ionophores

The effects of the ionophores A-23187 and X-537A on glucose metabolism, ATP content and sucrose permeability in pancreatic islets microdissected from obese-hyperglycemic mice were studied. The formation of 14CO 2 from 10 mM D-[U- 14C]glucose was inhibited by omission of Ca 2+ from the medium. A-23187 (10 μM) induced a further decrease of 14CO 2 formation whereas X-537A (10 μM) had no effect. At 20 mM glucose both A-23187 (48 μM) and X-537A (43 μM) decreased the 14CO 2 formation in the absence of CA 2+ whereas only X-537A inhibited in the presence of Ca 2+. X-537A (43 μM) also decreased the formation of 3H 2O from 20 mM D-[5- 3H]glucose. The islet content of ATP was not changed after incubation in media deficient in either Mg 2+ or Ca 2+. However, omission of both Mg 2+ and Ca 2+ resulted in about 50% decrease of the ATP content. A-23187 and X-537A induced dose-dependent decreases of the islet ATP content. X-537A was much more potent than A-23187. Both ionophores induced stronger depression of the ATP content when CA 2+ was omitted. X-537A (43 μM) but no A-23187 (48 μM) increased the β-cell membrane permeability as indicated by an increased sucrose space in relation to the urea space of islets. Such an effect was not obtained with X-537A at 1 μM or by ommision of Ca 2+. It is suggested that the marked metabolic effects of the ionophores reflect an impaired mitochondrial metabolism. These metabolic changes should be considered in interpretations of ionophore action on insulin secretion.

Ionic effects on the uptake of sulfonylurea (glibenclamide) by pancreatic islets.

The accumulation of glibenclamide was studied in the pancreatic islets of non-inbred ob/ob-mice. Microdissected islets were incubated in media of different ionic composition and the accumulation measured as the uptake of drug not accounted for by equilibration in the urea space. In 12 mM N-hydroxyethylpiperazine-N'-2-ethane sulphonic acid (HEPES) buffer the accumulation was only half of that in Krebs-Ringer bicarbonate buffer. Addition of 50 mM NaCl, Na2SO4, KCl, or LiCl to the HEPES buffer restored the accumulation to the level seen in Krebs-Ringer buffer. Studies with different concentrations of NaCl and Na2SO4 showed that the glibenclamide accumulation was sensitive to Na+ with little or no effect being attributable to the anions. CaCl2 or MgCl2 had much stronger effects than NaCl. It is concluded that cations participate in the binding of glibenclamide to beta-cells. The mechanism could be an increased ionic attraction between cell surfaces and hydrophilic regions of the drug, or a shielding of fixed surface anions allowing stronger hydrophobic interactions between drug and beta-cells.

In vivo prediction of body composition in cattle from urea space measurements.

SummaryUrea space was measured in cattle by infusing (iv) a known quantity of urea (approximately 130 mg/kg body wt) and withdrawing blood samples at varying times after infusion for plasma urea analysis. The relation between urea space determined at 12 or 15 min after urea infusion in 12 steers was highly correlated with percentage empty body fat and percentage empty body water.Assistance rendered by Drs. V. R. Cahill and C. R. Weaver is gratefully acknowledged.

Amino acid metabolism of experimental granulation tissue in vitro.

1. The intracellular volume in granulation tissue was about 15% of the total urea space. 2. The experimental granuloma has a greater ability to retain amino acids during the proliferation phase than later during the synthesis of collagen. 3. The synthesis of collagen and other proteins by granulation tissue is related to the concentrations of proline and glutamic acid in the medium. 4. The rate of synthesis of proline from glutamic acid in granulation-tissue slices is greatest during collagen synthesis. It is enhanced by lactate. 5. Extracellular cations influence the synthesis of collagen and ouabain is inhibitory. Synthesis of other proteins is less sensitive in this respect. 6. It is suggested that the synthesis of collagen is related to the supply of certain amino acids, especially proline, and hence to the redox balance, and also to the function of the cell wall.

The effect of keto-analogues of essential amino acids in severe chronic uremia.

Alpha keto-analogues of valine, leucine, isoleucine, methionine, phenylalanine, and (in one instance) tryptophan and histidine, along with the remaining essential amino acids, were administered orally to 10 patients with severe chronic uremia fed a diet low in protein but adequate in calories. Ketoacid dosage varied from 6 to 14 g daily, as sodium or calcium salts. Net nitrogen intake, calculated as intake minus urinary protein nitrogen, averaged 1.8 g/day. The urea space was either estimated or measured with [(14)C]urea and daily changes in the body urea pool were calculated. Urea appearance was measured as the sum of urea excretion and the change in urea pool. If these ketoacids were converted to amino acids and utilized for protein synthesis, a fall in urea nitrogen appearance should occur. In five subjects, ketoacids were given for 15-18 days and then withdrawn. Urea nitrogen appearance increased 1.55 g/day on withdrawing ketoacids, and corrected nitrogen balance decreased by 1.73 g/day. In two other subjects ketoacid administration was followed, on two occasions each, by a period of administration of nine essential amino acids. In three of these four instances, urea appearance rose significantly with amino acids. In four patients studied at high blood urea levels, ketoacid treatment was relatively ineffective; two of these patients responded more favorably when studied again after peritoneal dialysis. One of these improved enough clinically to be managed as an out-patient for short intervals, despite virtual anuria. No accumulation of ketoacids in plasma or urine could be detected, and no toxicity was identified.

Metabolism of urea in late pregnancy and the possible contribution of amino acid carbon to glucose synthesis in sheep.

1. Metabolism of urea in non-pregnant and pregnant sheep (1–25 d from term) has been examined. Injections of [14C]urea were used to estimate urea entry rate, urea pool size and urea space in sheep given 1000 g of a diet of equal parts of crushed oats and chaffed lucerne hay (daya) and in the same sheep 4 d after the ration had been reduced to 250 g (dayb).2. On both experimental days (aandb), mean pool size was greater (14% on day α, 29% on dayb) and urea space was greater (54% on day α, 24% on dayb) in pregnant animals than in non-pregnant animals; mean plasma urea concentrations were lower (35%) in the pregnant animals on day a but were not significantly different on dayb.3. The entry rate of urea was similar in all the animals on daya, but was significantly higher (34%) in pregnant than in non-pregnant animals on dayb. There was a significant decrease in urea entry rate in both pregnant (33%) and non-pregnant (86%) animals on dayb.4. The rate of excretion of urea was lower (26% on daya, 35% on dayb) in pregnant animals, indicating a higher (31% on day α, 40% on dayb) rate of degradation of urea in the digestive tract of pregnant as compared with non-pregnant sheep.5. Measurements of urea entry rate have been used to calculate the upper limit of amino acid deamination in pregnant and non-pregnant sheep, and this has been used as an indication of the potential availability of amino acid carbon for glucose synthesis. It is suggested that, at a maximum, amino acids may have contributed the carbon required for 63 g/d and 52 g/d of glucose on daysaandbrespectively.

Studies on intestinal fluid transport II. The location of fluid accumulated in the wall of rat jejunum during incubation in vitro

1. 1. The quantity of fluid accumulated in the wall of jejunal sacs incubated in vitro was directly proportional to the rate of fluid transport. 2. 2. The uptake of fluid was not reflected in an expansion of the serosal PEG 4000 space, but did expand the urea space. 3. 3. The uptake of fluid could not be accounted for as an expansion of an extracellular space which could be demonstrated structurally, either as the development of sub-epithelial lakes, or as the distension of the lateral channels of the epithelial cells. 4. 4. The dimensions of epithelial cells were shown to vary linearly with the gut fluid uptake. 5. 5. It is suggested that the swelling of the epithelial cells indicates a specialized mechanism of fluid transport at the luminal cell border.

The Urea Space in Adult Cattle

The Urea Space in Adult Cattle

Metabolism of urea in sheep

1. The entry rates of urea into the urea pool of the body fluids have been measured in sheep given rations varying in crude protein percentage from 3.5 to 27.3.2. Results obtained with a single injection and with continuous infusions of [14C]urea were essentially the same.3. The difference between the entry rate and the rate of excretion of urea in the urine was taken to indicate the quantity of urea degraded in the alimentary tract.4. Plasma concentrations and urea entry rates were significantly and linearly related.5. The relationship between excretion rate and plasma urea concentration was best described by a cubic equation.6. Degradation of urea in sheep was found to be extensive in all the animals studied; as the protein intake increased, the quantity of urea degraded also increased but the percentage of urea entering the body pool that was degraded was decreased. Animals given a ration containing 3.5% crude protein degraded 76–92% of the urea entering the body pool.7. A rectilinear relationship was found between pool size and plasma urea concentration. The urea space in animals given low-protein rations was significantly less than in animals on high-protein rations.8. The effects of starvation for 2, 4 and 6 days on urea metabolism in sheep were investi-gated. In a11 the sheep starved for 2 days there was a significant increase in urea pool size, but the entry rate was markedly depressed indicating a retention of urea in the body pool on starvation.9. A significant amount of nitrogen was found to go through the system: rumen ammonia → portal blood ammonia→blood urea→rumen ammonia.10.Urea excretion rate, urea clearance by the kidney, urine flow rate and the ratio of the concentration of urea in urine to that in plasma (urea U:P ratio) were also examined.11. There were significant correlations between urine flow rate and urea excretion and between plasma urea concentration and urine flow rate.

Distribution of body water in the camel (Camelus dromedarius).

The distribution of water in the different body compartments were determined in the adult male Indian camels with one hump. The average distributions were as follows: serum volume, 5.9% of body wt.; blood volume, 8.3% of body wt.; total body water (urea space), 68.7% of body wt.; extracellular water (thiocyanate space), 23.3% of body wt.; intracellular water, 45.4% of body wt.; and interstitial water, 17.4% of body wt. The serum and blood volumes of the camel were higher than similar values in other ruminants. When these values were compared with similar values in man, it was observed that the camel had significantly higher serum volumes, total body water, and intracellular water volumes. The capacity of the camel to hold more water might help the animal in withstanding the deprivation of water for a prolonged period in comparison to other animals.

Transport of D-xylose and sugar space in Baker's yeast.

The cell volume fraction ofSaccharomyces cerevisiae accessible tod-xylose and other nonmetabolized sugars was found to correspond to 100% of cell water in resting yeast over a range of osmolarities of the medium. This sugar space was decreased to 60% by nitrogen deficiency and to 82% by phosphorus deficiency. The space of N-deficient cells was significantly raised by pre-incubation with ammonium chloride, amino-acids and nucleotides from the yeast extract and by adenosine triphosphate while that of P-deficient cells was not significantly altered by analogous treatment. The xylose space was found to be markedly lower in growing than in resting cells. Urea (0.3–6%) had a depressing effect on the sugar space size while a variety of metabolic inhibitors were without influence. TheKm andV values ofd-xylose uptake bySaccharomyces cerevisiae R XII at 30° were found to be 180–192 mM and 680–730 mg. xylose/ml. cell volume/hr., respectively. These values were not affected by N-deficiency and by enrichment with adenosine triphosphate. The xylose space size was shown to be practically identical with that of urea space and its decrease was related to the increase of “bound” water within the cell under various conditions studied. The changes of space are tentatively attributed to changes of hydration and dehydration of cellular components.