Development and application of a simple radioimmunoassay for urinary aldosterone

A simple, economical and direct assay was developed to measure aldosterone in urine, using aldosterone antibody of high specificity and gamma labelled ligand. The assay allows the direct measurement of aldosterone in 100 μl aliquots of urine after acid hydrolysis. It does not require preliminary solvent extraction and purification steps and hence a large number of samples in a single batch can be assayed simultaneously. An excellent correlation was obtained between the results of the direct assay and the levels measured after extraction and paper chromatography (Y = 0.97X + 0.89, r = 0.99, p< 0.001) or after extraction alone (Y = 0.98X + 1.75, r = 0.99, p< 0.001). The coefficients of variation for inter-assay and intra-assay determinations of samples from normal and high urine pools were 4.2–6.5% and 5.6–9.8%, respectively. Total urinary aldosterone excretion in 21 normal subjects on unrestricted sodium diet ranged from 3.8–20.2 μg/24 h (10.5–55.0 nmol/24h) with a mean of 12.5 ± 4.6 (SD) μg/24 h (34.7 ± 12.8 (SD) nmol/24 h).     

Hyponatremia and hyperkalemia in relation to hyperglycemia in insulin-treated diabetic out-patients

Interrelations between glucose and electrolyte homeostasis were evaluated in 193 insulin-treated diabetic out-patiens. All had normal serum creatinine and were studied during their everyday metabolic control. Although the patients were selected to be without proteinuria and ketonuria, they exhibited wide ranges of blood glucose values (2.5–29.5 mmol/1) and urine glucose excretions (0–301 mmol/mmol creatinine). Patients with blood glucose values within 2.5–10 mmol/1 (n = 80) had entirely normal levels of serum sodium (140.6 ± 2.7 (SD) versus 141.0 ± 2.6 mmol/1) and potassium (4.35 ± 0.38 versus 4.40 ± 0.38 mmol/1) as compared with normals (n = 371). In contrast, diabetics with higher blood glucose concentrations (n = 113) showed hyponatremia (137.7 ± 2.6 mmol/1, p < 0.001) and a moderate increase of serum potassium (4.60 ± 0.39 mmol/1, p < 0.001). On stratification into classes of blood glucose, serum sodium declined from 142 to 135 mmol/1 (r = ?0.61, p < 0.001), whereas serum potassium rose from 4.33 to 4.87 mmol/1 (r = 0.37, p < 0.001). Despite these reciprocal changes the urinary excretion rates relative to creatinine of sodium, potassium and water rose with rising degrees of glycosuria (r = 0.24, p < 0.001; r = 0.28, p < 0.001; and r = 0.63,p < 0.001, respectively).The decline in serum sodium represents a well-known osmoregulatory response to hyperglycemia. However, the rising level of serum potassium in virtual absence of renal failure and ketonuria suggests an abnormality in potassium homeostasis. Diabetic dysregulation, or rather relative insulin deficiency may be its cause.     

Measurement of urine albumin using bromocresol green

A method for the estimation of urine albumin using bromocresol green (BCG), is described. The albumin value measured by the method correlates well with a turbidimetric method for urine protein estimation¹ (r = 0.99). Recoveries of added human albumin to urine samples varied between 97.4% and 100%. The method is simple, reproducible, sufficiently accurate and suitable for routine use. The method is also adaptable for automation on the Technicon AutoAnalyzer.     

NHE4 is critical for the renal handling of ammonia in rodents

Ammonia absorption by the medullary thick ascending limb of Henle’s loop (MTALH) is thought to be a critical step in renal ammonia handling and excretion in urine, in which it is the main acid component. Basolateral Na⁺/H⁺ exchangers have been proposed to play a role in ammonia efflux out of MTALH cells, which express 2 exchanger isoforms: Na⁺/H⁺ exchanger 1 (NHE1) and NHE4. Here, we investigated the role of NHE4 in urinary acid excretion and found that NHE4^–/– mice exhibited compensated hyperchloremic metabolic acidosis, together with inappropriate urinary net acid excretion. When challenged with a 7-day HCl load, NHE4^–/– mice were unable to increase their urinary ammonium and net acid excretion and displayed reduced ammonium medulla content compared with wild-type littermates. Both pharmacologic inhibition and genetic disruption of NHE4 caused a marked decrease in ammonia absorption by the MTALH. Finally, dietary induction of metabolic acidosis increased NHE4 mRNA expression in mouse MTALH cells and enhanced renal NHE4 activity in rats, as measured by in vitro microperfusion of MTALH. We therefore conclude that ammonia absorption by the MTALH requires the presence of NHE4 and that lack of NHE4 reduces the ability of MTALH epithelial cells to create the cortico-papillary gradient of NH₃/NH₄⁺ needed to excrete an acid load, contributing to systemic metabolic acidosis.     

The effect of uncomplicated potassium depletion on urine acidification

Studies were performed on normal human subjects to determine the effects of potassium depletion on urine acidification. Depletion was induced by ingestion of a low potassium diet either alone or in combination with a potassium-binding resin, and the response of each subject to an acute ammonium chloride load in the potassium-depleted state was compared to his normal response. Urine pH was significantly higher during potassium deficiency if sufficient potassium depletion had been induced. No differences in blood acid-base parameters, urinary flow rate, or urinary fixed buffer excretion rate were found to account for this change; however, the increase in urine pH was accompanied by a concomitant increase in net acid and ammonium excretion. It is proposed that these changes during potassium depletion reflect an increase in ammonia diffusion into the urine, presumably as a result of increased renal ammonia production. In addition, it is speculated that changes in ammonia metabolism may be a physiologic control mechanism for potassium conservation.     

A novel method for total chlorine detection using machine learning with electrode arrays

Chlorine is a common natural water disinfectant, but it reacts with ammonia''s nitrogen to form chloramines, which affects the accuracy of free chlorine measurement. In this case, total chlorine can be used as an indicator to evaluate the content of the effective disinfectant. In this article, a novel method to detect total chlorine using an electrode array in water has been proposed. We made the total chlorine sensor and captured the cyclic voltammetry curve of the electrode at different concentrations of chlorine ammonia. Principal component analysis and a peak sampling method were used to extract cyclic voltammetry curves, and the total chlorine prediction model was established by support the vector machine and extreme learning machine. The results show that the best predicting power was achieved by support vector regression with principal component analysis (R² = 0.9689). This study provides a simple method for determining total chlorine under certain conditions and likely can be adapted to monitor disinfection and water treatment processes as well.

Establish soft measurement model of total chlorine: cyclic voltammetry curves, principal component analysis and support vector regression.     

Mass spectrometric measurement of urinary kynurenine-to-tryptophan ratio in children with and without urinary tract infection

BackgroundIndoleamine-2,3-dioxygenase (IDO) catalyzes the first step of tryptophan (Trp) catabolism, yielding kynurenine (Kyn) metabolites. The kynurenine-to-tryptophan (K/T) ratio is used as a surrogate for biological IDO enzyme activity. IDO expression is increased during Escherichia coli urinary tract infection (UTI). Thus, our objective was to develop a method for measurement of Kyn/Trp ratio in human blood and urine and evaluate its use as a biomarker of UTI.MethodsA mass spectrometric method was developed to measure Trp and Kyn in serum and urine specimens. The method was applied to clinical urine specimens from symptomatic pediatric patients with laboratory-confirmed UTI or other acute conditions and from healthy controls.ResultsThe liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was linear to 500 μmol/L for both Trp and Kyn. Imprecision ranged from 5 to 15% for Trp and 6–20% for Kyn. Analytical recoveries of Trp and Kyn ranged from 96 to 119% in serum and 90–97% in urine. No correlation was found between the K/T ratio and circulating IDO mass (r = 0.110) in serum. Urinary Kyn and Trp in the pediatric test cohort demonstrated elevations in the K/T ratio in symptomatic patients with UTI (median 13.08) and without UTI (median 14.38) compared to healthy controls (median 4.93; p < 0.001 for both comparisons). No significant difference in K/T ratio was noted between symptomatic patients with and without UTI (p = 0.84).ConclusionsMeasurement of Trp and Kyn by LC-MS/MS is accurate and precise in serum and urine specimens. While urinary K/T ratio is not a specific biomarker for UTI, it may represent a general indicator of a systemic inflammatory process.     

Comparison of total urinary oestrogens and total serum oestriol in pregnancy

The total oestrogens detected by fluorescence in 24-h urine and the concentration of total oestriol detected in serum by radioimmunoassay were estimated in 51 women during the second half of pregnancy. The statistical analysis showed a linear regression between urinary excretion and serum concentration according to the equation y = ?2.89 + 2.82x (y = serum oestriol). The correlation coefficient over all pairs of estimations (N = 567) was r = 0.69, which is very highly significant (P < 0.0001). The analysis of the individual pregnancies showed that the linear correlation coefficient was significant (P < 0.05) in 35 cases, poorly significant (0.05 < P < 0.1) in 3 cases and not significant (P > 0.1) in 5 cases. The correlation could not be calculated in 8 cases because the number of estimations was too small. These values were compared empirically (normal/pathological). There was agreement in 6 cases and no agreement in 2 cases.The results show on the one hand that the radioimmunological estimation of total oestriol in serum can be used in the same way as urinary excretion of oestrogens for monitoring the feto-placental unit. On the other hand, the study confirms that a “simple” fluorimetric Kober-Ittrich method can give at least the same clinical information as a radioimmunoassay of relatively high cost. It may be concluded indirectly from the results that a considerable diurnal rhythm or substantial day-to-day fluctuation in the production of oestriol are unlikely.     

Automated analyzer for the determination of potassium and sodium in whole blood

An automated analyzer for the measurement of potassium and sodium in whole blood has been developed and evaluated. The sensors used in this instrument are the sodium glass and potassium (valinomycin in polyvinyl chloride) membrane electrodes. The features of this system include automatic two-point calibration at fixed intervals or on command, automatic rinsing between measurements, digital display and printout of the emf data, and a sample volume requirement of 0.5 ml.In addition to the evaluation of the response of the system to aqueous and whole blood samples, a study was made of the correlation of data obtained on plasma samples with a flame photometer and the electrometric potassium/sodium analyzer. For both electrodes the correlation was very good (r = 0.998 for potassium and r = 0.991 for sodium). No anomalous effects were observed with blood samples from normal volunteers, however, no studies were performed with samples taken from patients which might show disease-induced changes in ion-binding properties or interferences from medications.From this study, it appears that the sodium and potassium electrodes are able to meet sensor requirements for a clinical blood-electrolyte instrument. Since an analyzer based on ion-selective electrode technology can determine sodium and potassium in whole blood, such an automatic instrument could be used outside of the central clinical laboratory for stat analyses. While the feasibility of this type of instrument has been demonstrated, much more work is required to establish its performance characteristics for clinical analysis.     

点尿法及24 h尿收集法估算高血压病患者24 h尿钠钾排泄量的应用比较

目的探讨利用点尿法及24 h尿收集法估算高血压病患者24 h尿钠钾排泄量的应用价值。 方法研究对象为2017年2月至2018年1月就诊于新疆医科大学第一附属医院高血压科的高血压病患者,共264例。收集患者24 h尿及相应的清晨空腹点尿,分别测定所有尿样的钠、钾、肌酐水平。采用Tanaka、Kawasaki和INTERSALT公式分别估算点尿法24 h尿钠钾排泄量,采用配对秩和检验比较公式估算值与实际测量值的差异;利用Spearman相关分析评价各公式估算的24 h尿钠钾排泄量与实测24 h尿钠钾排泄量的相关关系。 结果Tanaka法公式估算的24 h尿钠值(167.99 mmol/d)高于实际24 h尿钠值(157.73 mmol/d),差异无统计学意义(Z＝－0.23,P>0.05);Kawasaki法公式估算的24 h尿钠值(217.66 mmol/d)亦高于实际24 h尿钠值,差异有统计学意义(Z＝－8.81,P<0.01);INTERSALT法公式估算的24 h尿钠值(154.71 mmol/d)低于实际24 h尿钠值,差异有统计学意义(Z＝－3.53,P<0.01)。Tanaka、Kawasaki和INTERSALT公式估算的24 h尿钠值与实际24 h尿钠值的相关系数分别为0.68,0.55,0.58(均P<0.01);Tanaka公式估算的24 h尿钾值(39.51 mmol/d)低于实际测量值(42.90 mmol/d),差异有统计学意义(Z＝－3.47,P<0.05),相关系数为0.50(P<0.01)。 结论在高血压病患者中,公式法估算的24 h尿钠钾排泄量与实际测量值,存在不同程度的低估与高估,且相关性差。利用点尿法公式估算高血压病患者24 h尿钠钾排泄量存在一定程度的不准确性和局限性。     

Quantitative determination of leukocyte esterase with a paper-based device

The commercially-available colorimetric urine dipstic for the early detection of urinary tract infection (UTI) has several limitations. The quantitative determination of urinary leukocyte esterase (LE) for predicting UTI remains uncertain. This study presents a paper-based analytical device to detect LE (LE-PAD) as a point-of-care quantitative test for UTI. The LE-PAD is composed of a coating of mixed 3-(N-tosyl-L-alaninyloxy)-5-phenylpyrrole (PE) and 1-diazo-2-naphthol-4-sulfonic acid (DAS) deposited onto a silver conducting film (Ag film). The LE/urine reacts with the PE and DAS, and the resulting products in turn react with the silver coating, causing a change in resistivity. The quantitative calibration curve was established in this study and has been used to analyse urine samples from inpatients with urinary catheters (n = 21). The results revealed that the level of LE determined by LE-PADs was predictive of UTI diagnosis with an area under the receiver operating characteristic curve of 0.875 (95% confidence interval, 0.704–1.000). Using an appropriate cut-off value, the sensitivity and specificity of UTI diagnosis by LE-PAD were 87.5% and 92.3%, while the LE-positivities of urine dipstics were 62.5% and 76.9%, respectively. For UTI diagnosis, the LE-PAD demonstrated positive and negative likelihood ratios of 11.38 and 0.14, suggesting that the novel LE-PAD is a reliable test.

A novel chemiresistive method for quantitative determination of leukocyte esterase.     

Association between FSH,E1, and E2 levels in urine and serum in premenopausal and postmenopausal women

ObjectiveWe aimed to establish correlations for the levels of follicle-stimulating hormone (FSH), estrone (E1) and estradiol (E2) between urine and serum in premenopausal and postmenopausal women using immunoassays.MethodsIn this study of 92 women (61 postmenopausal, 31 premenopausal), both urine and blood specimens were collected on the same day and stored at 4 °C for analysis by chemiluminescent immunoassay, radioimmunoassay and/or electrochemiluminescent immunoassay.ResultsThere were correlations in the levels of FSH, E1 and E2 between urine and serum in both postmenopausal (r = 0.96 for FSH, r = 0.91 for E1, r = 0.80 for E2) and premenopausal (r = 0.98 for FSH, r = 0.92 for E1, r = 0.90 for E2) women. It is indicated that the correlations were stronger in the premenopausal group compared with the postmenopausal group, especially for FSH.ConclusionThe levels of FSH, E1 and E2 in urine correlated with those in the serum in premenopausal and postmenopausal women. Urine samples could be used instead of serum samples to measure hormone levels, which would reduce the difficulty of conducting large survey studies.     

A solid-phase fluoroimmunoassay of tobramycin

A solid-phase fluoroimmunoassay has been developed for the determination of tobramycin in serum. The principle of this method is based on direct competition of a fluorescein-labeled and unlabeled hapten for a fixed amount of spec ific antibody immobilized on a dipstick sampler. The bound and free labeled hapten are separated simply by removing the sampler from the reaction mixture. The fluorescence signal from the bound fluorescein-labeled hapten is measured with a FIAX^® fluorometer.The fluorscein-labeled tobramycin, which is easily prepared, has excellent shelf life. The assay is fast and simple, involves only one pipetting step, and avoids the use of radiochemicals. Within-assay coefficients of variation are less than 10%. The assay correlates well with radioimmunoassay (r = 0.97) and radioenzymatic assay (r = 0.96).     

On the Mechanism of Polyuria in Potassium Depletion: THE ROLE OF POLYDIPSIA

The association of potassium (K) depletion with polyuria and a concentrating defect is established, but the extent to which these defects could be secondary to an effect of low K on water intake has not been systematically investigated. To determine whether hypokalemia has a primary effect to increase thirst and whether any resultant polyuria and polydipsia contribute to the concentrating defect, we studied three groups of rats kept in metabolic cages for 15 days. The groups were set up as follows: group 1, normal diets and ad lib. fluids (n = 12); group 2, K-deficient diet on ad lib. fluids (n = 12); and group 3, K-deficient diet and fluid intake matched to group 1 (n = 14). Daily urine flow and urinary osmolality of groups 1 and 3 were not significantly different throughout the study. In contrast, as of day 6, group 2 rats consistently had a higher fluid intake (P < 0.0025), higher urine flow (P < 0.001), and lower urinary osmolality (P < 0.001) than the other two groups. These alterations in fluid intake and urine flow preceded a defect in maximal concentrating ability. On day 7, maximal urinary osmolality was 2,599±138 msmol/kg in rats on K-deficient intake and 2,567±142 msmol/kg in controls. To determine whether this primary polydipsia is itself responsible for the development of the concentrating defect, the three groups of rats were dehydrated on day 15. Despite different levels of fluid intake, maximal urinary osmolality was impaired equally in groups 2 and 3 (1,703 and 1,511 msmol/kg, respectively), as compared to rats in group 1 (2,414 msmol/kg), P < 0.001. We therefore conclude that K depletion stimulates thirst, and the resultant increase in water intake is largely responsible for the observed polyuria. After 15 days of a K-deficient diet, the impaired maximal urinary concentration in hypokalemia, however, was not related to increased water intake, since fluid restriction did not abolish the renal concentrating defect.     

Fructosamine: A new approach to the estimation of serum glycosylprotein. An index of diabetic control

The development of a novel manual method designed to measure serum glycosylprotein as an index of diabetic control is described. The method relies on the ability of ketoamines (fructosamines) to act as reducing agents in alkaline solution. Conditions are described for a simple colorimetric procedure which permits assay of both a synthetic fructosamine and purified albumin while severely limiting the contribution of interfering substances. Applied to whole sera, the measurement is linear with volume of serum assayed. It allows clear discrimination of normal and diabetic populations (p < 0.001), and is significantly correlated with fasting blood glucose concentration (r = 0.72) and with a thiobarbituric acid procedure for measuring glycosylprotein-derived hydroxymethylfurfural (r = 0.58). The method is rapid (at least 12 samples per hour) and demands only simple equipment.     

Potassium permanganate used to eliminate interference with the Porter-Silber reaction in the butanol extraction method for determination of urinary 17-hydroxycorticosteroids

We describe a simple, rapid, and less interference-susceptible method for using the Porter-Silber reaction for determination of urinary 17-hydroxycorticosteroids. The procedure is based on butanol extraction of the steroids after the urine sample is treated with potassium permanganate and sodium bisulfite. These treatments, together with an additional acid- and alkali-washing of the extract, could eliminate most of the substances in urine that interfere with the Porter-Silber reactions. Values so obtained correlated well (r = 0.95) with those by Furuya's method in which beta-glucuronidase is used. Our method may be useful and suitable for a screening test of adrenocortical and pituitary functions.     

以肾脏损伤为首发症状甲基丙二酸血症患儿17例临床分析

目的分析以肾脏损伤为首发症状的甲基丙二酸血症（methylmalonic acidemia,MMA）患儿临床特点及实验室检查结果。方法以肾脏损伤为首发症状的MMA患儿17例,检测血、尿常规、尿有机酸分析、血气分析及生化相关指标。结果 17例MMA患儿14例合并同型半胱氨酸血症,3例为单纯MMA;尿液甲基丙二酸水平为（0.100-214.143）mmol/（mmol·Cr）;其中11例尿潜血阳性,9例尿蛋白阳性;尿β2-微球蛋白、微量白蛋白、微量IgG、视黄醇结合蛋白、N-乙酰-β-D-氨基葡萄糖苷酶异常例数分别为10、6、8、10、11例;血氧、血乳酸、尿素氮的异常例数分别为9、11、10例;尿中甲基丙二酸水平与患儿血pH呈负相关（r=-0.493,P=0.022）,与血清谷草转氨酶和谷丙转氨酸水平呈正相关（r=0.455,P=0.000;r=0.826,P=0.033）。结论以肾损伤为首发症状的甲基丙二酸患儿多合并同型半胱氨酸升高;以尿潜血和尿蛋白异常为主;肾小管早期损伤指标中N-乙酰-β-D-氨基葡萄糖苷酶最灵敏,β2-微球蛋白、视黄醇结合蛋白次之;血氨、血乳酸可作为诊断该病的辅助指标;当尿中甲基丙二酸水平明显增高时,应警惕酸中毒和肝损伤发生。     

Comparison of a simplified radioimmunoassay with a gas-liquid chromatographic method for urinary aldosterone

A simplified radioimmunoassay for the measurement of aldosterone in urine, requiring only an extraction without any further purification is described.The results obtained on 47 urine specimens were compared with those obtained by a gas-liquid Chromatographic method. An excellent correlation was found (r = 0.932) and values obtained by both methods were similar so that this RIA has been satisfactorily adopted in our laboratory for one year.Some discrepancies, however, led us to consider the problem of preservation and storage of biological fluids.     

Inverse relationship between diastolic blood pressure and urinary excretion of potassium in girls aged 8 to 9 years--a preliminary communication

In order to investigate the association of urinary excretion of sodium and potassium with blood pressure, 72 prepubertal healthy children between 8 and 9 years were investigated in a cross sectional study. We determined anthropometric data, diastolic (DBP), and systolic blood pressure (SBP) and urinary excretion of sodium and potassium in 24-hour urine samples. No relationship was found between urinary sodium or potassium excretion and systolic or diastolic blood pressure, neither with regard to simple, nor multiple regression analysis. However, on simple linear and multiple regression analysis a significant inverse association between the excretion of potassium and diastolic blood pressure (r = -0.53; p less than 0.01 and r = -0.60; p less than 0.001) was observed in girls. From our data we conclude that in normal weight, healthy children urinary excretions of sodium and potassium are not associated with systolic blood pressure. However, a high 24-hour urinary excretion of potassium might be associated with lower diastolic blood pressure in prepubertal girls.     

Hyperammonemia in idiopathic epileptic seizure

ObjectiveIt is known that patients with convulsion often present hyperammonemia. The elevation of ammonia levels in convulsion is considered to occur along with extensive muscle contractions, but the details remain unclear. In emergency pathologies, such as cardiopulmonary arrest or hemorrhagic shock without muscle contraction, red blood cells are known to produce ammonia through acidosis, leading to hyperammonemia. A similar effect would be considered to be involved in idiopathic epileptic seizure patients as well.MethodsWe retrospectively analyzed the cases of epileptic seizure that were transported to the emergency medical care center of Ohta Nishinouchi Hospital and diagnosed by neurologist as idiopathic epileptic seizure or epilepsy due to cerebrovascular disorder. Forty-four patients were idiopathic epilepsy, and 8 had epilepsy due to cerebrovascular disorder. Those with hepatic encephalopathy, metabolic disorder, alcohol consumption, tumor, and patients taking oral valproic acid were excluded.ResultsHigh ammonia levels (> 35 μmol/L) were observed in 22 cases. Maximum ammonia level was 506 μmol/L. Significant differences were observed in the pH (r = 0.838, P < .0001) and base excess (BE) (r = 0.863, P < .0001), the values suggesting a strong negative correlation between the ammonia level and pH/BE.ConclusionIdiopathic epileptic seizures can present with prominent hyperammonemia with acidosis. Because high ammonia level in epileptic seizure was strongly correlated with pH and BE, we speculate that hyperammonemia is not only because of extensive muscle contractions but is also related to ammonia production in the red blood cells through acidosis like other emergency conditions.