Extraction and radioimmunoassay of urinary aldosterone in the rat

Results from studies of the rat renin-angiotensin-aldosterone axis have been inconsistent and oftentimes conflicting among various laboratories and have led many investigators to regard the rat as a poor model with which to study this sytem. Many of these discrepancies have been shown to be due to the use of anesthetic agents in experimental protocols. Studies of unanesthesized animals indicate that the rat bears a remarkable resemblance to other mammalian species, but unfortunately the unanesthesized rat lends itself well only to acute studies, usually requiring killing the animal. Herein is described a method which permits the estimation of aldosterone excretory rates in intact, nonstressed, unanesthesized rats. The method is based upon the excretion and radioimmunoassay of urinary "acid-labile" or 3-oxo-conjugate of aldosterone. It is very sensitive, permitting the detection of less than 10 pg of aldosterone conjugate in extracted samples, and when compared to the double-isotope-dilution method, it is relatively inexpensive and much less tedious. Radioimmunoassays of rat urinary aldosterone excretion during 14.75 days of sodium depletion reflected a brisk increase in urinary excretion rate after day 2 and a concomitant reduction in sodium excretion that bears a remarkable resemblance to the excretory patterns described for man.     

Simultaneous determination of urinary catecholamines and 5-hydroxyindoleamines by high-performance liquid chromatography with fluorescence detection

OBJECTIVE: The relationship between bronchopulmonary dysplasia (BPD) and neurodevelopmental outcome after extracorporeal membrane oxygenation (ECMO) has not been extensively reported. We compared the outcomes in a large series of infants with and without BPD after ECMO. STUDY DESIGN: Hospital charts and follow-up records of 145 infants treated with ECMO (1985 through 1990) were reviewed. Complete long-term respiratory and follow-up outcome data were available in 64 infants. BPD occurred in 17 survivors; the remaining 47 did not have BPD. RESULTS: Babies with BPD were more likely to have had respiratory distress syndrome. Mean (+/- SD) age at ECMO initiation was later for the BPD group (127+/-66 vs 53+/-39 hours, p < 0.001), and the duration of ECMO treatment was longer (192+/-68 vs 119+/-53 hours, p < 0.001). Bayley Scales of Infant Development scores at <30 months were lower in infants with BPD (p < 0.001), as were three of four Mullen Scales of Early Learning scores (> or = 30 months, p < 0.001 or p = 0.01). At 57+/-16 months 11 (64%) patients with BPD had mild neurologic disabilities, and 3 (18%) had severe disabilities. At a similar age (53+/-16 months, p = NS) 16 (34%) patients without BPD had mild disabilities, whereas 2 (4%) had severe disabilities (p < 0.01). CONCLUSIONS: The occurrence of BPD after ECMO is associated with adverse neurodevelopmental outcome. Patients with BPD after ECMO merit close long-term follow-up.     

Quality of life of women with stress urinary incontinence with or without pollakiuria]

BACKGROUND: The optimal management of low grade Papanicolaou (Pap) smear abnormalities remains controversial. This center's experience with recommending cytologic follow-up for women with atypical cells of undetermined significance (ASCUS) or low grade squamous intraepithelial lesions (LSIL) was reviewed to determine outcome and patient/physician compliance. METHODS: The records were reviewed on women with Pap smears reported as either ASCUS (320) or LSIL (112) who did not have a history of dysplasia. The cytologic and colposcopic follow-up for a 2-year period was obtained from the laboratory data base that includes the colposcopy and cancer referrals for this region. Repeat Pap smear in 6 months was recommended. If patients subsequently demonstrated high grade SIL (HSIL) or persistent ASCUS or LSIL over three time intervals, colposcopic evaluation was recommended. RESULTS: The outcome was determined by the most significant diagnosis among the follow-up Pap smears or colposcopic biopsies. 29% of patients were lost to follow-up. Of the remaining patients, 70.5% reverted to normal or benign cellular changes, 25.3% persisted as ASCUS or LSIL, and 5.2% progressed to HSIL. The majority of patients (68%) were referred for colposcopy for persistent mildly abnormal Pap smears. The timing of referral ranged from 3-30 months. CONCLUSIONS: These results suggest that cytologic follow-up of women with low grade Pap smear abnormalities will identify a large number whose smears will regress to normal. A small but significant proportion of women showed subsequent HSIL. Most HSIL was detected within 1 year of the initial abnormal Pap smear and the majority of intervening Pap smears also were abnormal. Approximately one third of patients did not have follow-up within the study system and their outcome was uncertain. Although the recommendations are standard, patterns of follow-up and referral to colposcopy varied widely, suggesting that the guidelines need to be reinforced to both patients and physicians. [See editorial on pages 1-4, this issue.]     

Evaluation of radioimmunological methods for assay of plasma and urinary aldosterone

Two radioimmunological methods for assay of plasma and urinary aldosterone were carefully evaluated. In the plasma method a radioimmunoassay is preceded by chromatography on a Sephadex LH-20 column. The method for urine includes a preextraction, hydrolysis of the acid-labile conjugates of aldosterone, and a radioimmunoassay. Both methods fulfill the criteria of reliability and are suitable for both routine and demanding research assays. The plasma method, using columns of double length, is also applicable to analysis of aldosterone in plasma of newborn children, and pregnant females and in cord plasma. The concentration of plasma aldosterone in healthy subjects on an ad lib salt diet was 162 +/- 93 (S.D.) pmol/1 in the supine position and 312 +/- 217 (S.D.) pmol/1 upright. The urinary excretion of aldosterone in healthy subjects was 28.3 +/- 16.7 (S.D.) nmol/24 h.     

Electrokinetic chromatography without electroosmotic flow

Otx1 and Otx2 genes are mouse cognates of a Drosophila head gap gene orthodenticle. The homozygous mutants have previously indicated that Otx2 is essential to development of structures anterior to rhombomere 3, probably reflecting its expression around the early primitive streak stage. Otx2 mutation also exhibits craniofacial defects by haplo-insufficiency. Affected structures correspond to the most anterior and most posterior parts of the Otx2 expression where Otx1 is not, or is only weakly, expressed at the time of brain regionalization. No apparent defects are found in early brain development by the Otx1 mutation, suggesting that the Otx1 and Otx2 functions overlap in the regions where both are expressed. To demonstrate this, the Otx1/Otx2 double heterozygous phenotype was examined in this study. Analyses with molecular markers at 9.5 days post coitus suggested the failure in development of mesencephalon and caudal diencephalon with the expansion of anterior metencephalon. Genes expressed in isthmus exhibited a characteristic lateral stripe normally, although rostrally shifted, except that Fgf8 expression was expanded dorsally. The defects were apparent at the 6-somite stage, but not at the 3-somite stage. Broad Fgf8 expression at the 3-somite stage took place normally, but it did not concentrate into a spot corresponding to future isthmus. The double heterozygous phenotype implicates a previously unsuspected mechanism for development of the mes/metencephalic territory; at the 3- to 6-somite stage Otx1 cooperates with Otx2 to establish the mes/diencephalic domain, allowing for the correct development of isthmus/ rhombomere 1.     

Removal of contaminating substances before gas-liquid-chromatographic determination of aldosterone in urine

This paper deals with removal of contaminants before gas-chromatographic determination of aldosterone in urine. Urine is incubated with beta-glucuronidase, which hydrolyzes all beta-glucuronides except aldosterone-18-glucuronide. The contaminants (the aglycones released and other methylene chloride-soluble substances) are extracted with methylene chloride. Solvolysis of the aqueous phase liberates aldosterone from aldosterone-18-glucuronide, which then is extracted with methylene chloride and oxidized by use of periodic acid. The resulting lactone can be easily separated by one-dimensional thin-layer chromatography and determined by gas-liquid chromatography.     

The application of the modified method of determination of aldosterone in plasma

Concentrations of triglycerides, free fatty acids (FFA) and glycerol were measured in umbilical venous blood from 99 infants with a birth weight of between 1100-2700 g and a gestational age of 27-41 weeks. Thirty infants were small for gestational age (SGA), 58 were appropriate (AGA) and 11 were of uncertain gestational age. In AGA infants with a gestational age of less than or equal to 35 weeks. FFA values were lower than in those with a gestational age of less than 35 weeks; otherwise concentrations of triglycerides, FFA and glycerol were independent of birth weight and gestational age in AGA infants. In SGA infants, higher FFA values were found compared with both AGA and term infants of normal birth weight. Triglyceride values were higher in SGA than in AGA infants. In SGA infants, a significant positive correlation was found between gestational age and concentrations of both FFA and triglycerides. No differences in FFA, glycerol and triglyceride concentrations were seen between asphyxiated and non-asphyxiated AGA infants.     

Rapid separation of urinary acids by high-performance liquid chromatography

Over a hundred acidic urinary constituents were separated within 30 min by using 5-micron octadecyl-silica columns and gradient elution with increasing acetonitrile concentration in dilute aqueous phosphoric acid solution at 70 degrees. The column effluent was monitored with a UV detector at 280 nm or with a fluorescence detector at 260 nm excitation and 340 nm emission wavelengths. The high sensitivity and speed of analysis, the excellent reproducibility and adequate resolution obtained suggest that this technique may be useful to obtain metabolic profiles in routine clinical work.