Multiple urea transporter proteins in the kidney of holocephalan elephant fish (Callorhinchus milii)

Reabsorption of filtered urea by the kidney is essential for retaining high levels of urea in marine cartilaginous fish. Our previous studies on the shark facilitative urea transporter (UT) suggest that additional UT(s) comprising the urea reabsorption system could exist in the cartilaginous fish kidney. Here, we isolated three cDNAs encoding UTs from the kidney of elephant fish, Callorhinchus milii, and termed them efUT-1, efUT-2 and efUT-3. efUT-1 is orthologous to known elasmobranch UTs, while efUT-2 and efUT-3 are novel UTs in cartilaginous fish. Two variants were found for efUT-1 and efUT-2, in which the NH₂-terminal intracellular domain was distinct between the variants. Differences in potential phosphorylation sites were found in the variant-specific NH2-terminal domains. When expressed in Xenopus oocytes, all five UT transcripts including the efUT-1 and efUT-2 variants induced more than a 10-fold increase in [¹⁴C] urea uptake. Phloretin inhibited dose-dependently the increase of urea uptake, suggesting that the identified UTs are facilitative UTs. Molecular phylogenetic analysis revealed that efUT-1 and efUT-2 had diverged in the cartilaginous fish lineage, while efUT-3 is distinct from efUT-1 and efUT-2. The present finding of multiple UTs in elephant fish provides a key to understanding the molecular mechanisms of urea reabsorption system in the cartilaginous fish kidney.

Morphological and molecular investigations of the holocephalan elephant fish nephron: the existence of a countercurrent-like configuration and two separate diluting segments in the distal tubule

In marine cartilaginous fish, reabsorption of filtered urea by the kidney is essential for retaining a large amount of urea in their body. However, the mechanism for urea reabsorption is poorly understood due to the complexity of the kidney. To address this problem, we focused on elephant fish (Callorhinchus milii) for which a genome database is available, and conducted molecular mapping of membrane transporters along the different segments of the nephron. Basically, the nephron architecture of elephant fish was similar to that described for elasmobranch nephrons, but some unique features were observed. The late distal tubule (LDT), which corresponded to the fourth loop of the nephron, ran straight near the renal corpuscle, while it was convoluted around the tip of the loop. The ascending and descending limbs of the straight portion were closely apposed to each other and were arranged in a countercurrent fashion. The convoluted portion of LDT was tightly packed and enveloped by the larger convolution of the second loop that originated from the same renal corpuscle. In situ hybridization analysis demonstrated that co-localization of Na(+),K(+),2Cl(-) cotransporter 2 and Na(+)/K(+)-ATPase α1 subunit was observed in the early distal tubule and the posterior part of LDT, indicating the existence of two separate diluting segments. The diluting segments most likely facilitate NaCl absorption and thereby water reabsorption to elevate urea concentration in the filtrate, and subsequently contribute to efficient urea reabsorption in the final segment of the nephron, the collecting tubule, where urea transporter-1 was intensely localized.

Monitoring urea levels during haemodialysis with a pulsed-flow chemiluminescence analyser

We have developed a rapid and robust method for the determination of urea in spent haemodialysis fluid as a measure of the efficiency of haemodialysis treatments. A novel flow analysis instrument (which generates a pulsed solution flow) was coupled with a chemiluminescence detection system, based on the oxidation of urea with hypobromite. The ‘pulsed-flow chemiluminescence analyser’ exhibited high precision (1.6% relative standard deviation (R.S.D.) for a 1×10−5 M urea standard, n=10) and good limit of detection (9×10−7 M, S/N=3) as a result of the rapid and reproducible mixing of small volumes of reagent and sample at the point of detection. The proposed chemiluminescence technique and an established urease-based laboratory procedure were compared, and showed a very similar trend for the change in urea concentration during a typical haemodialysis treatment. The relative chemiluminescence response from the oxidation of species with similar structure has revealed the inherent selectivity of the light producing pathway, but a positive interference was obtained from protein when this technique was applied to the determination of urea in serum samples. Arginine was identified as the predominant source of this interference.

Analytical methodology for the determination of urea: current practice and future trends

The determination of urea is important in a wide range of fields, including clinical diagnostics, environmental monitoring and food science. Numerous analytical techniques have been developed for the determination of urea, with no single technique dominant in all areas because of the diversity of applications. An overview of the existing analytical methodologies for urea is presented, and some new approaches are discussed, particularly those based on chemiluminescence detection to improve the sensitivity and the selectivity for the determination of this important analyte.

Chemiluminescence from the oxidation of urea and ammonia with hypobromite and N-bromosuccinimide

The spectral distribution for the chemiluminescent oxidation of ammonia with hypobromite is significantly different to that for the oxidation of ammonia with N-bromosuccinimide. Therefore, in contrast to the assumptions of several authors, the action of N-bromosuccinimide is not solely derived from the in situ formation of hypobromite. Neither the oxidation of urea with hypobromite nor the oxidation of urea with N-bromosuccinimide involves an initial hydrolysis of urea to ammonia in the alkaline solution. However, these two reactions lead to a common emitter. The addition of xanthene dyes, such as dichlorofluorescein, enhance the chemiluminescence intensity by energy transfer to the efficient fluorophore, but reaction between the sensitiser and hypobromite can result in a significant increase in the background signal. A list of potential interferences has been compiled; particular attention was paid to guanidino compounds, as the chemiluminescence accompanying the oxidation of this functional group has not been previously discussed.

The determination of urea in soil extracts and related samples - a review

Although the dominant methods for the determination of urea in clinical applications incorporate selective enzymatic hydrolysis of urea, the determination of urea in soil extracts is complicated by the presence of urease inhibitors. The spectrophotometric determination of urea with an acidic solution diacetyl monoxime and semicarbazide is a viable option but traditional manual procedures are time-consuming. New variations on these procedures, based on microplates or flow-injection analysis methodologies, allow a far greater number of samples to be analysed with high precision and sensitivity.

The determination of urea with hypobromite: a theme for the discussion of numerous fundamental concepts in chemistry

Over the past century, numerous aspects of the reaction between urea and hypobromite have been exploited to quantify urea in clinical and industrial process samples. A review of these analytical approaches provides an interesting illustration of changes in a chemical system that indicate a reaction has occurred-the production of a gas, a color change, the release of heat, and the emission of light-and a variety of instruments that were developed to measure these changes and quantify a reacting species. In this paper we describe how we have used this material in a tutorial class for first-year undergraduate (freshman) students and a follow-up assignment, which we have included in the supporting material. In addition to the concepts exemplified by the above phenomena, we discuss the reaction pathway, which includes examples of ion and atom transfer. These are
often overlooked in favor of electron transfer in the teaching of redox chemistry.

The determination of urea in wine – a review

The concentration of urea in wine is not routinely measured in Australian laboratories, but has been examined in studies of yeast metabolism and the formation of ethyl carbamate, a known carcinogen. For alcoholic beverages that may contain high levels of urea, steps have been taken to reduce the concentration of urea and therefore prevent ethyl carbamate production. Methods for the determination of urea in wine can be grouped into three categories that indicate how selectivity for urea is achieved; those based on colour-forming reactions, enzymatic hydrolysis and chromatographic separation. The two dominant methods used by research groups over the past fifteen years for the determination of urea in wine are based on the urea/ammonia test kit available from Boeringer Mannheim/R-Biopharm and the reaction of urea with 1-phenyl-1,2-propanedione-2-oxime; both are time-consuming and labour-intensive, but involve relatively straightforward and well-established procedures. However, other options are available that may be better suited to the desired application and the instrumentation available in any particular laboratory.

Cross-adaptation and bitterness inhibition of L-Tryptophan, L-Phenylalanine and urea : further support for shared peripheral physiology

A previous study investigating individuals' bitterness sensitivities found a close association among three compounds: L-tryptophan (L-trp), L-phenylalanine (L-phe) and urea (Delwiche et al., 2001, Percept. Psychophys. 63, 761-776). In the present experiment, psychophysical cross-adaptation and bitterness inhibition experiments were performed on these three compounds to determine whether the bitterness could be differentially affected by either technique. If the two experimental approaches failed to differentiate L-trp, L-phe and urea's bitterness, then we may infer they share peripheral physiological mechanisms involved in bitter taste. All compounds were intensity matched in each of 13 subjects, so the judgments of adaptation or bitterness inhibition would be based on equal initial magnitudes and, therefore, directly comparable. In the first experiment, cross-adaptation of bitterness between the amino acids was high (>80%) and reciprocal. Urea and quinine-HCl (control) did not cross-adapt with the amino acids symmetrically. In a second experiment, the sodium salts, NaCl and Na gluconate, did not differentially inhibit the bitterness of L-trp, L-phe and urea, but the control compound, MgSO4, was differentially affected. The bitter inhibition experiment supports the hypothesis that L-trp, L-phe and urea share peripheral bitter taste mechanisms, while the adaptation experiment revealed subtle differences between urea and the amino acids indicating that urea and the amino acids activate only partially overlapping bitter taste mechanisms.

Determination of urea using high-performance liquid chromatography with fluorescence detection after automated derivatisation with Xanthydrol

A high-performance liquid chromatography (HPLC) method for the determination of urea that incorporates automated derivatisation with xanthydrol (9H-xanthen-9-ol) is described. Unlike the classic xanthydrol approach for the determination of urea, which involves the precipitation of dixanthylurea (N,N′-di-9H-xanthen-9-ylurea), the derivatisation procedure employed in this method produces N-9H-xanthen-9-ylurea, which remains in solution and can be quantified using fluorescence detection (λ_ex = 213 nm; λ_em = 308 nm) after chromatographic separation from interferences. The limit of detection for urea was 5 × 10⁻⁸ M (0.003 mg L⁻¹). This method was applied to the determination of urea in human and animal urine and in wine.

Conversion of urea to hydrazine: Hofmann reaction or Favorskii analogue? Exploring reaction mechanism through isotopic labeling studies

The power of isotopic substitution for the elucidation of a reaction mechanism is illustrated with reactions named after Hofmann and Favorskii. These reactions have important roles in synthetic chemistry; therefore, a wide range of experiments involving isotopic labeling or kinetic isotope effects were employed to establish their mechanistic pathways. The concepts introduced by these investigations are drawn together with an isotopic labeling study of the oxidation of urea with hypohalites. The two mechanisms proposed for this reaction have been described as a Hofmann rearrangement and a nitrogen analogue of the Favorskii rearrangement.

Feed intake, growth, plasma glucose and urea nitrogen concentration, and cacass traits of lambs fed isoenergetic amounts of canola meal, soybean meal, and fish meal with forage based diet.

This experiment was conducted to examine the effect of feeding small, isoenergetic amounts of supplements containing high protein and functional lipid components, rather than the greater amounts of cereal and/or legume grains usually fed during the dry season in Australia, on dry matter intake (DMI), growth performance, plasma metabolites, and fat deposition in lambs consuming low quality roughage. Thirty two crossbred wether lambs ([Merino × Border Leicester] × Poll Dorset) were divided into four groups by stratified randomization according to liveweight (26–33 kg). After a 7-day adaptation to a hay diet (lucerne hay:oaten hay; 30:70), lambs were allocated to four treatments consisting of (1) basal diet of lucerne hay:oat hay (20:80; metabolizable energy (ME) = 7.0 MJ/kg DM), Basal; (2) basal + canola meal (84 g per day), CM; (3) basal + soymeal (75 g per day), SM; or (4) basal + fishmeal (80 g per day), FM. Daily hay and supplement DMI, and weekly liveweight were recorded during a 53-day experimental study. Blood samples were taken on day 1 and pre- and post-feeding on days 30 and 53 to measure changes in plasma glucose and plasma urea nitrogen (PUN) concentration. At the end of the experiment, lambs were slaughtered and hot carcass weight (HCW) recorded; cold carcass fatness (total muscle and adipose tissue depth at 12th rib, 110 mm from midline; GR) was determined at 24 h postmortem. Total DMI was increased (P < 0.001) in CM, SM and FM treatments, but basal hay DMI intake was only increased (P < 0.01) in CM and FM treatments compared with Basal treatment. This resulted in significant (P < 0.01) increases in metabolizable energy (ME) and crude protein (CP) intakes in all supplemented treatments, with the highest intakes recorded in the FM treatment. Liveweight gain (LWG) was significantly increased in CM and SM (P < 0.05) and FM (P < 0.01) treatments but HCW was significantly (P < 0.01) heavier slaughter only in the FM treatment. Feed conversion efficiency (P < 0.001) and GR fat at depth (P < 0.05) was reduced in all supplement treatments compared with Basal. Plasma glucose concentration was significantly (P < 0.05) increased after feeding in all treatments but there was no treatment effect. PUN was significantly increased over time in the supplemented treatments compared with the Basal treatment; there was no significant difference between supplement treatments by day 53. Results show that feeding small amounts of high protein and lipid-containing supplements improves production responses and are beneficial in producing carcasses with more lean compared with carcasses from lambs fed a low quality hay diet.