Repletion Phase Research Articles

Extracellular acidosis minimizes actin cytoskeletal alterations during ATP depletion

Extracellular acidosis has been shown to be protective during ischemia in renal tubule cells. However, the mechanism of protection remains unknown. Since ischemia leads to disruption and polymerization of the cortical actin cytoskeleton, we hypothesized acidosis may better preserve the actin cytoskeleton during ischemia. Therefore, the purpose of our studies was to examine the effect of pH on the integrity of the actin cytoskeleton during ATP depletion and ATP repletion. To do this, we used an in vitro model of reversible ATP depletion in LLC-PK1 cells at extracellular pH values (pHo) of 6.9, 7.4, and 7.9. Immunofluorescent studies with rhodamine-phalloidin demonstrated more marked redistribution and clumping of cortical actin at pHo 7.9 and 7.4 vs. 6.9 after 90 min of chemical anoxia. After 15 min of ATP depletion, G-actin, quantified by the deoxyribonuclease assay, decreased from 53.7 +/- 0.8 to 43.2 +/- 1.5 microgram/mg protein at pHo 6.9 vs. 37.6 +/- 1.8 microgram/mg protein at pHo 7.4 (P < 0.001). After 60 min, there still was significantly less conversion of G-actin to F-actin at pHo 6.9 vs. 7.4, with a decrease from 55.9 +/- 2.0 to 39.6 +/- 2.0 micrograms/mg protein at 6.9 vs. 35.8 +/- 2.4 at 7.4 micrograms/mg protein (P < 0.05). Furthermore, extracellular acidosis during the phase of ATP repletion resulted in more rapid normalization of cellular G-actin levels (95 +/- 3% of control vs. 82 +/- 2% for pH 6.9 vs. 7.4, respectively, P < 0.01). Together, these findings indicate the actin cytoskeleton is better preserved in an acidic environment during ATP depletion.(ABSTRACT TRUNCATED AT 250 WORDS)

The development of a vitamin C-deficient diet using a selective menu design

The amount of vitamin C found in human plasma as a direct function of dietary ingestion is unknown. This relationship, which must be understood to predict the ideal vitamin C requirement, can be identified by assaying serum from hospitalized normal volunteers who are placed on a controlled, vitamin C-deficient diet. With the development of a unique assay for serum ascorbic acid analysis, an 18 week study was designed, requiring the creation of a vitamin C-deficient diet. A 14-day cycle, selective menu design was used to develop a diet restricted to ≤5 milligrams (mg) vitamin C daily. This approach depleted both plasma and dietary vitamin C, while controlling for other nutrients. The three volunteers who have completed this study to date depleted plasma vitamin C from >50 micromolar (umol/L) to 7.0 ± 1.0 umol/L (scorbutic range) within 1–3 weeks. The diet was maintained and daily intake was monitored throughout the repletion phase of the study, during which time controlled oral and intravenous vitamin C doses were administered. Plasma ascorbic acid increased from 7.0 ± 1.0 umol/L to 76 ± 18 umol/L with subsequently increased doses of vitamin C. Nutritional parameters remained within normal ranges. Mean dietary compliance, calculated by number of days with <5.0 mg vitamin C/total days on study, was 84.5 ± 8.7% over the 18 weeks. This rate is indicative of the high degree of diet acceptance achieved with a selective menu design. This successfully developed diet was essential to the study of ascorbic acid pharmacokinetics over a 4–5 month period.

A new definition of maternal depletion syndrome.

Although the term "maternal depletion syndrome" has been commonly used to explain poor maternal and infant health, whether such a syndrome actually exists remains unclear. This uncertainty may be due to the lack of a clear definition of the syndrome and the absence of theoretical frameworks that account for the many factors related to reproductive nutrition. We propose a new definition of maternal depletion syndrome within a framework that accounts for potential confounding factors. Our conceptual framework distinguishes between childbearing pattern and inadequate diet as causes of poor maternal health; hence, our definition of maternal depletion syndrome has both biological and practical meaning. The new definition is based on overall change in maternal nutritional status over one reproductive cycle in relation to possible depletion and repletion phases and in relation to initial nutritional status. The empirical application of this approach should permit the testing of the existence of maternal depletion syndrome in the developing world, and the distinction between populations where family planning will alleviate maternal depletion and those in which an improved diet is also necessary.

Effect of taurine on intracellular calcium dynamics of cultured myocardial cells during the calcium paradox.

The “calcium paradox” phenomenon, first described by Zimmerman and Hulsman1, occurs when hearts are reperfused with calcium after a short period of calcium-free perfusion. The Ca2+ repletion phase causes irreversible myocardial damage, characterized by reduced electrical activity, extensive ultrastructural damage, depletion of tissue high-energy phosphate content, massive release of intracellular constituents and an increase in cytosolic Na+ and Ca2+2.

Choline may be an essential nutrient in malnourished patients with cirrhosis

Elemental diets designed for nutritional support in protein-calorie malnutrition are often deficient in choline, a nonessential nutrient. Previously, malnourished patients on these diets were found to be at risk of developing plasma choline deficiency. We have now estimated the prevalence of this deficiency by determining fasting plasma levels of choline among cirrhotic and noncirrhotic malnourished male subjects maintained on regular hospital mixed food or elemental parenteral and enteral formulas. Plasma choline concentrations (μM, average ± SD) were as follows: (i) mixed foods, 11.3 ± 4.3 for cirrhotic (n = 22) and 9.3 ± 2.4 for noncirrhotic (n = 12) patients; (ii) parenteral formula, 5.3 ± 1.6 for cirrhotic (n = 5) and 8.6 ± 5.2 for noncirrhotic (n = 16) subjects; and (iii) enteral formula, 6.1 ± 1.2 for cirrhotic (n = 5) and 11.7 ± 1.9 for noncirrhotic (n = 4) subjects. The level for healthy normal subjects eating mixed foods was 12.0 ± 2.2. The prevalence of plasma choline deficiency, i.e., plasma levels ≥2 SD below the normal average, was as follows: parenteral formula, all cirrhotic and 10 of 16 noncirrhotic subjects; enteral formula, all cirrhotic and none of the noncirrhotic subjects. The reversibility of choline deficiency was examined in a longitudinal study of three phases involving 10 patients—5 with alcoholic cirrhosis (all on enteral formula); 5 noncirrhotic (1 on enteral and 4 on parenteral formula). During phase 1 (3-day equilibration period; ad libitum regular hospital diet), plasma choline levels were within the normal range for all subjects. During phase 2 (2 wk, choline depletion phase, elemental formulas), choline levels were subnormal in all cirrhotic subjects (5.1 ± 2.0 μM) on enteral formula and all noncirrhotic patients on parenteral formula (5.9 ± 1.3 μM). During phase 3 (2 wk, choline repletion phase, elemental formula + 6 g choline/day), the levels normalized in all patients (cirrhotic 11.4 ± 3.1 μM and noncirrhotic 11.9 ± 3.2 μM). Analyses of abdominal computed tomographic scans and plasma liver chemistries in the cirrhotic subjects during the three phases suggested a correlation between plasma choline deficiency and hepatic steatosis and abnormal liver enzyme levels in some patients. Therefore, choline may be an essential nutrient in malnourished cirrhotic patients and its deficiency may be associated with adverse hepatic effects.

Magnesium reabsorption in the juxtamedullary loop of Henle: effect of magnesium deprivation.

To determine the contribution of the juxtamedullary loop of Henle to magnesium reabsorption during magnesium deficiency, we performed two-phase micropuncture studies of end-descending limbs in a group of magnesium-deficient rats (n = 7) and in a pair-fed control group (n = 8) given MgCl2 in their drinking water. In the magnesium-deficient rats, daily excretion of magnesium fell to very low values (1.2 +/- 0.2 vs. 52 +/- 12 microM.day-1.100 g body weight-1, p less than 0.05). Plasma magnesium concentration and fractional magnesium excretion during the control phase were nearly 52 and 27%, respectively, of the values observed in pair-fed controls. Fractional magnesium delivery to the end-descending limb did not differ significantly between the two groups. During the acute magnesium repletion phase, fractional magnesium excretion and fractional magnesium delivery to the end-descending limb increased by a similar value in the two groups of rats, despite a lower filtered load of magnesium in the magnesium-deficient group. Absolute magnesium reabsorption upstream to the end-descending limb was lower in the magnesium-deficient rats but was otherwise tightly coupled to the filtered load of magnesium (Y = 0.91 + 0.37 x, r = 0.82, p less than 0.05). Similar observations were made with regards to whole kidney magnesium reabsorption. Our results suggest that, in young magnesium-deficient rats, magnesium reabsorption is tightly coupled to the filtered load of magnesium both in segments upstream to the juxtamedullary end-descending limb and in the whole kidney, and that a reabsorptive defect for magnesium is not evident in this setting.

Maturation and secretion of lipoprotein lipase in cultured adipose cells. II. Effects of tunicamycin on activation and secretion of the enzyme

The effects of N-linked glycosylation on the activation and secretion of lipoprotein lipase were studied in Ob17 cells. The cells were first depleted of any activity and enzyme content by cycloheximide treatment and of precursors of oligosaccharide chains by tunicamycin. The repletion of lipoprotein lipase content was studied in these cells maintained in the presence of tunicamycin after cycloheximide removal. During the repletion phase, the EC 50 values of inhibition by tunicamycin (approx. 0.2 μg/ml) of the incorporation of labeled glucose, mannose or galactose into trichloroacetic acid-insoluble material were found to be identical. Under these conditions, the rate of protein synthesis was maximally decreased by 30%. The results showed clearly that the recovery in lipoprotein lipase activity was parallel to the recovery in hexose incorporation, no activity being recovered in the absence of glycosylation. An inactive form of lipoprotein lipase from tumicamycin-treated cells was detected by competition experiments with mature active lipoprotein lipase for the binding to immobilized antilipoprotein lipase antibodies, as well as by immunofluorescence staining. SDS-polyacrylamide gel electrophoresis and Western blots of cellular extracts and of extracellular media, obtained after tunicamycin-treated cells were exposed to heparin, revealed a single immunodetectable M r 52000 protein, whereas a single M r 57000 protein was detected in control cells. Therefore, the results indicate that the acquisition by lipoprotein lipase of a catalytically active conformation is linked directly or indirectly to glycosylation. Despite this lack of activation, the lipoprotein lipase molecule was able to migrate intracellularily and to undergo secretion after heparin stimulation of the tunicamycin-treated cells.

Maturation and secretion of lipoprotein lipase in cultured adipose cells. I. Intracellular activation of the enzyme.

The intracellular pathway and the activation of lipoprotein lipase have been examined in differentiated Ob17 cells. These adipose cells were previously shown to secrete lipoprotein lipase during exposure to heparin. Treatment of the cells with cycloheximide and heparin leads to enzyme depletion, as shown by activity measurement and immunofluorescence microscopy. The repletion phase has been studied in the presence of monensin or carbonyl cyanide m-chlorophenylhydrazone, ionophores known to affect the intracellular transport of membrane and secretory proteins. Monensin-treated cells synthesize fully active lipoprotein lipase. Under these conditions the antigen accumulates in the Golgi apparatus and the heparin-stimulated enzyme release is extensively reduced. Carbonyl cyanide m-chlorophenylhydrazone-treated cells do not contain any enzyme activity but show detectable antigen which accumulates in the endoplasmic reticulum. Competition for binding to immobilized anti-lipoprotein lipase antibodies of mature and endoplasmic reticulum-sequestered antigens is observed. Carbonyl cyanide m-chlorophenylhydrazone removal is rapidly followed by a transient burst of enzyme activity and a redistribution of the antigen in the different subcellular compartments. Therefore, the results show that the activation of lipoprotein lipase is an intracellular event taking place after the enzyme exits from the endoplasmic reticulum and before it reaches the trans-Golgi cisternae.

Effect of protein deficiency on growth and plasma zinc concentration in genetically lean and obese swine.

Thirty-two genetically lean and 32 genetically obese weanling (4 to 5 wk old) pigs from inter se matings of crossbreds produced by within-line matings of Duroc and Yorkshire breeds, selected for low or high backfat, were fed the following four diets for 8 wk (eight lean and eight obese pigs/diet): 10% protein -- 100 ppm Zn, 10% protein -- 200 ppm Zn, 18% protein -- 100 ppm Zn, 18% protein -- 200 ppm Zn. After 8 wk, all pigs were fed a standard 16% protein corn-soybean meal-type growing diet to a slaughter weight of about 87 kg. Lean pigs were more severely affected than obese pigs by low protein diets as measured by weight gain, plasma total protein and plasma albumin. Plasma Zn concentration was reduced in lean and obese pigs fed low protein diets. Supplemental Zn failed to increase plasma Zn in protein-deficient pigs. During repletion, weight gain of lean and obese pigs previously fed the 10% protein -- 100 ppm Zn diet was less than that of pigs previously fed other diets. The lower level (100 ppm) of Zn fed during protein depletion appeared to be associated with a negative effect on repletion weight gain of lean and obese pigs. Lean and obese pigs responded similarly during the repletion phase to earlier protein restriction. We conclude that supplementary dietary Zn does not maintain plasma Zn concentration in growing pigs fed protein-deficient diets and that repletion from dietary protein restriction is not associated with compensatory growth in genetically lean pigs.

The temperature-sensitivity of slow channel calcium blockers in relation to their effect upon the calcium paradox

The isolated perfused rat heart preparation was used to investigate the interrelationship between temperature and the ability of calcium antagonists to reduce protein leakage in the calcium paradox. Exploiting the fact that although the calcium depletion phase of calcium paradox-injury is highly temperature sensitive, the calcium repletion phase, during which time the injury is manifested, is temperature independent. Verapamil (4 mumol l-1) included in a 10 min period of calcium depletion (37 degrees C) and a 20 min period of calcium repletion (37 degrees C) was known to reduce cumulative protein leakage by 22 +/- 3%. With calcium depletion again at 37 degrees C but repletion at 21 degrees C verapamil failed to achieve any reduction in protein leakage despite the fact that leakage in the 21 degrees C control group did not differ significantly from that in the 37 degrees C control group. Temperature-response studies (repletion temperature of 4, 10, 21, 23, 25, 28, 31, 34 and 37 degrees C) revealed an abrupt loss of drug effect below 25 degrees C. Studies with D600 (10 mumol l-1), nifedipine (4 mumol l-1), terodiline (4 mumol l-1) fendiline (20 mumol l-1) and diltiazem (2 mumol l-1) revealed that while all of these drugs reduced protein leakage at 37 degrees C (24 +/- 3%, 31 +/- 2%, 31 +/- 3%, 23 +/- 3% and 29 +/- 3%, respectively), like verapamil they had no effect at 21 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Slow calcium channel blockers and the calcium paradox: comparative studies in the rat with seven drugs.

In studies of the calcium paradox, the isolated perfused rat heart was used to characterize the relationship between myocardial protein leakage and the concentration of calcium antagonist (verapamil and D600) or calcium in the perfusion fluid during a cycle of calcium depletion and repletion. The results indicated a dose-dependency such that protein leakage could be progressively reduced by decreasing the concentration of calcium during calcium repletion and/or by increasing the concentrations of drug. Detailed dose-response studies with seven calcium antagonists (verapamil, D600, nifedipine, terodiline, diltiazem, fendiline and prenylamine) and a calcium concentration of 1.0 mmol/l during a 20 min period of calcium repletion following a 10 min period of calcium depletion revealed complex dose-response characteristics for each drug. In the dose range studied (0 to 40 mumol/l) all drugs with the exception of prenylamine were able to reduce protein leakage by up to 25 to 30% Optimal concentrations for verapamil, nifedipine, D600, diltiazem and terodiline were all between 2.0 and 4.0 mumol/l. With the exception of D600, which provided a constant reduction of protein leakage at all concentrations above this optimum, all drugs exhibited bell-shaped dose-response curves with a loss of efficacy at higher concentrations. Fendiline also had a bell-shaped dose-response curve with 23% as a maximal reduction of leakage; however, the optimal concentration for this drug was 21.0 mumol/l. Additional studies with verapamil revealed that the drug acted during the calcium repletion phase and did not influence events during calcium depletion. Simultaneous use of two drugs, verapamil and nifedipine, at their optimal concentrations failed to improve the reduction in protein leakage over and above that observed with one drug alone.

Plasma active and inactive renin in the rabbit: effect of dietary sodium depletion and repletion.

1. Active and acid-activatable (inactive) renins were measured in rabbit plasma under control conditions and during sodium depletion with subsequent repletion. 2. Active renin increased by 97% during sodium depletion and returned to control levels on repletion. Both changes were complete within 1 day of changing the diet. 3. During dietary sodium depletion inactive renin levels initially fell to zero and then increased until, after 13 days, inactive renin was again 10% of total renin levels, a proportion comparable to the control values. 4. Sodium repletion caused plasma inactive renin to return to control levels over about 13 days, a quite different time course to active renin. Therefore, in the first phase of repletion the proportion of total renin in the inactive form rose to 19%. 5. These changes are discussed in relation to concurrent changes in sodium, potassium and water metabolism.

Breed differences in copper metabolism in sheep

SUMMARYEvidence was sought for genetic differences in the absorption or systemic use of copper by comparing responses in plasma Cu concentration to oral and intravenous Cu repletion. Lambs of the North Ronaldsay (Orkney), Scottish Blackface and Welsh Mountain breeds and crosses of these with the North Ronaldsay were used.In two oral repletion phases average Cu intakes/kg live weight were 0·87 and 0·63 mg/week and similar for all breed groups. Plasma Cu concentrations of Scottish Blackface lambs showed little or no increase while those of North Ronaldsay lambs increased rapidly, and those of Welsh Mountain lambs increased moderately. The plasma Cu concentrations of the cross-breds increased more rapidly in the first than in the second phase; their rate of change lay between the pure breeds but was closer to that of the North Ronaldsay in the first phase.The rates of increase in plasma Cu concentration during intravenous repletion were relatively uniform for the different breeds and crosses except the Blackface which showed a lower rate. It was concluded that the large differences between groups in response to oral Cu repletion were largely attributable to differences in the efficiency of Cu absorption.

The oxygen paradox and the calcium paradox: Two facets of the same problem?

An isolated rat heart preparation was used to study and compare the sudden tissue damage which may result from calcium repletion after a period of calcium-free perfusion with the effects of reoxygenation after a period of anoxic perfusion and with reperfusion after a period of ischaemia. Striking similarities in morphological and enzyme release characteristics exist for all three phenomena. Our results suggest that the primary lesion in the calcium paradox is the separation of the basement membrane from the sarcolemma and the energy-dependent loss of intracellular calcium to the extracellular space during the phase of calcium depletion. These changes, coupled with separation of opposing faces of the intercalated disc predispose the cell to the major damage which results from an energy independent, sudden, cellular influx of calcium during the phase of calcium repletion. In the case of the oxygen paradox the primary lesion would be the accumulation of cytoplasmic calcium and the loss of cellular and subcellular membrane control. While these anoxia or ischaemia-induced processes are related to the cellular availability of energy, the sudden damage induced by oxygen repletion would appear to be initiated by an energy-independent mitochondrial calcium uptake which is triggered by the reoxidation of the electron transport chain. Studies of such factors as the critical duration of calcium or oxygen depletion, the effects of stepwise calcium or oxygen repletion or the striking effects of hypothermia, reinforce the association between the calcium and oxygen paradoxes. While the basic principles underlying the cause of damage may be similar in both cases, fine differences exist in the progression, the nature and the extent of damage.

Influence of infestation rate of tick-paralysis in sheep induced by rhipicephalus evertsi evertsi neumann, 1897

The paralysis-inducing potential of Rhipicephalus evertsi evertsi was investigated by infesting sheep of different weights and ages with 80–100 ticks of both sexes. This paralysis was characterized by a constant incubation time, determined by the repletion phase and a temporal parallelism between infestation and persistence of the clinical signs. The intensity of the tick-paralysis was directly related to the infestation level and inversely proportional to age and weight of the host animals.

Effect of experimental malnutrition on corticosteroids and liver cytoplasmic receptors in rats

Adrenocortical function was studied in experimentally induced malnutrition in rats. Plasma corticosterone levels, liver cytosol binding capacity, and affinity binding constants were serially estimated while on a low-protein diet and later when replenished with a normal-protein diet. Plasma corticosterone levels and liver cytosol binding capacity remained within normal limits both during the hypoproteinemic phases as well as during repletion phases. Adrenal glands were hypertrophied, indicating a compensatory mechanism to maintain normal steroid levels.