Margaret Acara

QUANTITATIVE TWO-DIMENSIONAL THIN-LAYER CHROMATOGRAPHY OF DEOXYRIBONUCLEIC ACID COMPONENTS.

Abstract In studying the various parameters of normal and malignant mammalian tissues, it became apparent that there was a need for a rapid quantitative procedure for the determination of deoxyribosides and deoxyribotides. A new technique, thin-layer chromatography (1), has shown great versatility in the separation of different types of chemical compounds. Application of this technique for the separation of various ribonucleosides and ribonucleotides has been recently reported by Randerath (2, 3), but his procedure does not give sufficient resolution for quantitative determination. We wish to report a two-dimensional thin-layer chromatographic method for the quantitative determination of deoxyribosides and their 5′-phosphates.

Brain osmoregulation during extreme and moderate dehydration in a rat model of severe DKA

To determine if osmoprotective molecules accumulate in the brain during severe DKA with extreme (DKA-E) and moderate (DKA-M) dehydration, Fischer 344 rats (250-350g) were given STZ 45 mg/kg (i.p.) and allowed food and water ad lib. DKA-M received NaCl 77 mmol/L 60 ml/kg (i.p.) q 4 hrs. on day 2. All rats were anesthetized and sacrificed at 48 hrs. Half of each brain was used to measure water content (BWC) and half to measure Na+, K+, and organic osmoles by HPLC. Just prior to expiration, values for mean concentration of serum glucose (mmol/L) percent weight loss and median blood pH for DKA-E were 33.4, 19%, 6.98; for DKA-M, 16.8, 7.5% and 6.84, respectively. Means +/- SEM were compared by Students t-test. Percent BWC was 76.3, 77.3 and 77.6 in DKA-E, DKA-M and normal controls, respectively (NS). Brain Na+ and K+ were increased in DKA-M compared to controls (p < .05) but not significantly different in DKA-E compared to controls. Of organic osmoles measured (umol/g wet weight) taurine was significantly increased (p < .01) in DKA-E and DKA-M (8.04 +/- .39 and 9.73 +/- .78, respectively) as compared to controls (5.92 +/- .35) as was myoinositol in DKA-E compared to controls (9.96 +/- .39 vs. 8.87 +/- .28) (p < .05) and urea in DKA-E as compared to controls (14.24 +/- 3.9 vs. 4.14 +/- .52) (p < .01). DKA-M were not significantly different for brain myoinositol or urea as compared to control animals. There was no significant difference in brain glutamine between either study group and controls. Preservation of brain water despite systemic dehydration can be partly explained by increased brain concentrations of osmoprotective molecules. Such adaption in the clinical setting of DKA warrants a cautious repair of dehydration and hyperosmolality.

Magnetic resonance imaging (MRI) and pathophysiology of the rat kidney in streptozotocin-induced diabetes.

Proton magnetic resonance imaging was performed on rats before induction of diabetes with streptozotocin (STZ) and at 2 and 12 days postinduction. Images revealed an increase in maximal longitudinal and axial dimensions of the kidneys at 2 days and a further increase at 12 days. Similarly, an increase in the size of the remaining kidney was seen in a rat which underwent uninephrectomy as a positive control. Two major differences were observed between the kidney undergoing compensatory hypertrophy and those developing diabetic nephropathy: (i) Expansion of the renal vasculature was seen only in images of the diabetic rat; (ii) A loss in conspicuity of the normal corticomedullary junction was seen in the T2-weighted images of the diabetic rat but not in the uninephrectomized rat. Histologic examination revealed that the medulla increased to a size greater than the cortex during diabetic nephropathy whereas the medullary volume was less than that of the cortex during compensatory hypertrophy. In vitro T1 relaxation times in cortex, outer medulla and inner medulla of kidneys from control rats were measured and compared with the same respective regions in diabetic rats. When these values were correlated with tissue water content, a linear increase in relaxation rate versus percent water content from cortex to inner medulla was found in the control kidneys, but this correlation was absent in diabetic nephropathy. These studies demonstrate that MRI is an effective noninvasive tool for studying the course of renal hypertrophy and hydration changes in the development of renal disease in STZ-induced diabetes in the rat.

Regulation of Na+,K+-ATPase by chronic ethanol exposure of PC 12 cells

The effects of chronic ethanol exposure on Na+, K(+)-ATPase were investigated in PC 12 cells. Inclusion of ethanol in the Na+, K(+)-ATPase assay (i.e. in vitro addition of ethanol) inhibited enzyme activity. Conversely, intrinsic Na+, K(+)-ATPase activity was increased after chronic ethanol exposure of the cells. This increase in Na+, K+ pumps occurred without any alteration in the inhibitory effects of in vitro ethanol. A similar response was observed when the chronic treatments were carried out using serum-free defined medium. The effects of other agents, which like ethanol decrease membrane order, were investigated. The addition of ketamine and tert-butanol in vitro caused a concentration-dependent inhibition of Na+, K(+)-ATPase activity. However, chronic exposure of the PC 12 cells to tert-butanol or ketamine did not alter either intrinsic Na+, K(+)-ATPase activity or the inhibitory effects of ethanol in vitro. Maintenance of PC 12 cells in medium containing ethanol resulted in an increase in the intracellular content of Na+ without any change in the K+ levels. In contrast, maintenance of the cells in medium containing tert-butanol did not alter intracellular levels of Na+ or K+. The present study shows that the ethanol-induced increase in Na+, K+ pumps involved an increase in the intracellular content of Na+. This increase in Na+ content did not appear to be secondary to an inhibition of Na+, K(+)-ATPase activity.

Magnetic resonance imaging and histopathology of hydronephrosis in the rat

Magnetic resonance imaging revealed conspicuously hyperintense regions in the papillary area of kidneys of three untreated rats. When the kidneys were examined histologically, a hydronephrosis associated with the presence of bacteria was found. This study relates magnetic resonance images of an early stage of hydronephrosis to its histological picture.

Renal pressor effect of ethanol in the isolated perfused rat kidney

These experiments were performed to detect changes in renal function produced by acute infusions of small amounts of ethanol into the isolated kidney of the rat. Ethanol was infused for 10 min beginning at 40 min to reach a final concentration of approximately 80 mg/100 ml in the recirculating perfusate. Control kidneys were perfused for 90 min without the addition of ethanol. Control and ethanol infused kidneys were compared with respect to the following measurements: glomerular filtration rate, urine volume, urine protein concentration, pressure and fractional excretion of sodium, chloride, potassium, calcium and magnesium. Ethanol concentration in the perfusate was measured by gas chromatography. The only parameter affected by these concentrations of ethanol was pressure. During the ten min ethanol infusion, the pressure in the system rose significantly (P less than 0.01) from 110 +/- 0.3 to 120 +/- 2.8 mmHg. After the ethanol infusion, the pressure decreased towards pre-ethanol levels at a faster rate than the decrease in ethanol concentration in the perfusate.

Effect of Renal Transplantation on the Levels of Choline in the Plasma of Uremic Humans

Plasma choline levels were measured in patients who received a kidney transplant, in donors who underwent nephrectomy and in nonrenal surgical patients. Choline was measured using a choline kinase assay. Choline levels in patients receiving a kidney fell from 29.8 +/- 1.86 microM before transplantation to 15.7 +/- 2.32 1 day later; this normal level was maintained for at least 7 months and in a single case for 2 years. Kidney donors and nonrenal surgery patients showed a significant decrease in plasma choline on the day following surgery but choline levels returned to normal by 3 days after surgery. Thus a transplanted functional kidney reduced the high plasma choline levels, associated with uremia, to normal and maintained these normal levels throughout the period of observation.

800 – THE BIPHASIC EFFECT OF ORGANIC CATIONS ON THE EXCRETION OF OTHER ORGANIC CATIONS

The renal excretion of 14C-choline or 14C-acetylcholine was increased by the infusion of another organic cation at low rates but was decreased by infusion of the same added organic cation at higher rates with the Sperber technique in hens. The range of low rates of infusion was from 1 X 10(-15) to 1 X 10(-8) mol/min. At infusion rates greater than 1 X 10(-8) mol/min, inhibition of tubular excretion was found. At the low infusion rates, thiamine, lysine, quinine, atropine, acetylcholine and methylguanidine were found to increase 14C-choline excretion. The same compounds with the exception of lysine and acetylcholine inhibited 14C-choline excretion at the higher infusion rates. A biphasic effect on 14C-acetylcholine excretion was also observed with added atropine, thiamine and choline over the same infusion range. Increases in 14C-choline excretion occurred during a choline infusion rate that normally produced an excretory tubular maximum for choline whereas increases in 14C-acetylcholine excretion occurred during infusion of tracer amounts of 14C-acetylcholine. The effect of the addition of organic cations was selective for cations since the tubular excretion of organic anions was not affected by the addition of organic cations. The tubular excretion ratio of 14C-thiamine/p-aminohippuric acid increased from 0.25 to 0.95 when the infusion rate of added unlabeled thiamine was increased from 1 X 10(-11) to 1 X 10(-8) mol/min. Enhanced tubular excretion of 14C-thiamine may represent the effect of the increased load of unlabeled thiamine to protect the labeled thiamine from conversion to a nontransportable metabolite. Enhancement of excretion of 14C-choline and 14C-acetyocholine produced by very small amounts of other organic cations may represent either inhibition of tubular reabsorptive transport or induction of tubular excretory transport.