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Animal Science Department, University of Illinois, Urbana IL, and * Molecular and Human Genetics and Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX
2 To whom correspondence should be addressed. E-mail: jcmarini{at}uiuc.edu.
ABSTRACT
Stable isotope studies in conscious mice have been limited by the invasive catheterization procedures and relatively large sample size required. We developed minimally invasive catheterization protocols that together with the ability to analyze small samples have allowed for the study of urea kinetics in conscious mice. A single dose of 15N15N-urea followed by multiple sampling in mice (n = 6) showed that a primary pool of urea exchanged rapidly [70.65 ± 14.96 mmol/(kg·h)] with a secondary pool. The urea entry rate determined with this protocol was 3.36 ± 0.30 mmol/(kg·h). Continuous infusion of 15N15N-urea (n = 6) achieved plateau enrichment values at 3.3 ± 0.2.h from which the urea entry rate was determined by isotope dilution [3.24 ± 0.23 mmol/(kg·h)]. The urea entry rate measured by the single dose or continuous infusion protocol did not difffer (P = 0.76). The minimally invasive methods described allow us to study not only ureagenesis and urea cycle disorders in vivo, but also urea transport and transporter function and nitrogen metabolism in general in mouse models. This is especially relevant because mouse targeting technologies will likely facilitate the generation of organ and tissue specific nulls of the various urea cycle enzymes.
KEY WORDS: • metabolic phenotyping • stable isotope • urea kinetics
Urea is the main end product of nitrogen metabolism in mammals, and its production and excretion play a central role in the nitrogen economy of these animals. For this reason, urea entry rate (UER)3 has been used as indicator of amino acid usage and oxidation in humans (1,2).
Both primary and secondary disorders of ureagenesis in humans have been identified. Some are consequence of the general loss of liver function [e.g., cirrhosis; (3)], whereas others are caused by the deficiency of enzymes and transporters of the urea cycle [urea cycle disorders; (4–6)]. The inability to detoxify ammonia into urea results in elevated plasma ammonia concentration with sporadic hyperammonemic crises, which often result in coma and death. Chronic hyperammonemia also has many metabolic implications, ranging from hyperglutaminemia and branched-chain amino acid depletion to alterations in energy metabolism and neurotransmitter function (7). Finally, the study of the natural histories of genetic disorders of ureagenesis has raised new questions about the compartmentalization of nitric oxide production as it relates to the urea cycle.
Despite the fact that mouse models of urea cycle disorders have been available for the last 30 y (8), no studies on urea kinetics and urea production have been conducted in mice. Estimation of urea production has relied on concentrations (9) or urea excretion (10) rather than on tracer techniques commonly employed in other animals and humans.
For this reason, the establishment of stable isotopic techniques for the determination of urea kinetics in mice would not only allow for the metabolic phenotyping of urea cycle disorder models but also for the study of amino acid and nitric oxide metabolism in general.
MATERIAL AND METHODS
Animals and diet
The experiments were performed on B6EiC3Sn a/A-Otc+ (wild-type) male mice obtained from The Jackson Laboratory. Mice were housed in a specific pathogen–free facility, caged in pairs, and had access to a 19.9% crude protein autoclaved pelleted feed (Harlan Teklad LM-485 Autoclavable Rat/Mouse Diet) and autoclaved reverse osmosis water at all times. Mice were kept in a temperature- (22 ± 2°C) and humidity- (55 ± 5%) controlled environment with a 12-h light cycle. All animal procedures were authorized by the University of Illinois Institutional Animal Care and Use Committee.
The lateral tail vein catheterization procedure was described in detail elsewhere (11). In brief, intravascular catheters were made of polytetrafluoroethylene (0.15 mm i.d., 0.30 mm o.d., SUBL-120 Braintree Scientific) and silastic tubing (0.30 mm i.d., 0.64 mm o.d., Dow Corning). After the tail was warmed with warm water, a 27-gauge needle was inserted into the lateral tail vein. The needle was removed and the catheter introduced through the puncture and secured to the tail with cyanoacrylate glue. The tail was then rested on a heat pad to increase blood flow and facilitate sampling. Mice were restrained by the tail during the experiments. Mice were in the fed state when the catheterization procedure was performed, and feed and water were available at all times during the infusions.
Single dose and multiple sampling protocol
After a unique infusion/sampling catheter was put in place, mice (n = 6, age = 51.5 ± 0.5 d, weight = 21.3 ± 1.5 g) were heparinized with 50 µL of a 0.5 IU/L heparin-saline solution, and 3 background blood samples were collected (
3 µL each sample). The infusate was prepared by accurately weighing 15N15N-urea, heparin, and sterile water on a microbalance (Mettler Toledo) with a readability of 0.001 mg. An aliquot of the infusate was loaded into a 4-mm long polyethylene 10 tubing (dose tubing; Becton Dickinson) and weighed in the microbalance. A saline-filled syringe was then connected to the dose tubing. The bolus dose of 15N15N-urea (2.66 µmol in 9 µL pyrogen-free double-distilled H2O) was then injected, followed by 150 µL of a saline-heparin (0.15 IU/L) solution. This method ensured that the whole dose entered the mouse in a short time (catheter dead volume < 2.3 µL) and that the catheter was flushed and ready to use for sample collection. Sampling started immediately, usually by collecting 10 samples during the first 3 min. Timed samples, at 5, 10, 15, 20, 25, 30, 40, 50, and 60 min were then obtained. Blood samples (3 µL) were collected directly into a 1.5-mL Eppendorf tube, hemolyzed immediately with 100 µL of H2O, and frozen at –20°C.
Continuous infusion and multiple sampling protocol
A double catheterization procedure was employed, infusing through the proximal and sampling from the distal catheter, placed on the right and left lateral tail vein, respectively. Mice (n = 6, age = 49 ± 3.8 d, weight = 24 ± 1.2 g) were heparinized as described previously and 3 background blood samples were collected. A 6-h infusion of 15N15N-urea [113 µmol/(kg·h)] at a rate of 50 µL/h was performed utilizing syringe pumps (Harvard Apparatus). Samples were collected from the distal catheter at 1.5, 3, 4, 5, and 6 h, after the catheter was flushed with saline-heparin. Samples were immediately hemolyzed with 100 µL double-distilled H2O and frozen until analysis.
Sample analysis
Samples were deproteinized by adding 100 µL acetone, mixing on a vortex, and placing them overnight at 4°C. Samples were then centrifuged at 1500 x g for 15 min, and the supernatant transferred to a new Eppendorf tube. After the addition of 50 µL of isooctane, samples were emulsified by mixing on a vortex, followed by a second centrifugation at 1500 x g for another 15 min. The bottom layer containing the urea was transferred to a 1-mL V-vial (Wheaton) and the acetone was evaporated in a dry heat bath at 80°C under a continuous flow of nitrogen gas. The remaining residue was derivatized with 30 µL of a 1:1 solution of MTBSTFA (Sigma Aldrich) and acetonitrile at 80°C for 20 min in the tightly capped V-vials. The analysis was performed in a 5973 Agilent GC mass selective detector in selected ion monitoring mode, monitoring m/z ions 231 and 233.
Calculations
Urea kinetics from single tracer dose experiments
A multiexponential model was fitted to the urea tracer disappearance data
 | (1) |
where Et is the observed blood urea enrichment [mole percent excess (mpe)] at time t, n is the number of terms and i is the term number; the constants Ei and gi are the fitted parameters. The best fit was determined by visual inspection of the residuals rather than by "improvement" of the coefficient of determination.
A compartmental analysis of urea plasma enrichment disappearance was performed as described by Shipley and Clark (12) and Matthews and Downey (13). The compartmental model allows for the calculation of pool sizes and the transfer of urea among the pools.
Stochastic measurement of urea entry rate from single tracer dose experiments.
Urea entry rate can also be calculated from fitted data to Eq. 1 by using stochastic methods. In this way, no assumption about the underlying model is required. Urea production is calculated by dividing the tracer dose by the area under the urea enrichment-time curve (AUC), which is calculated by integrating Eq. 1 from zero to infinity as
 | (2) |
where Ei, gi, i and n are as defined for Eq. 1, UER is the urea entry rate [mmol/(kg·h)], and D is the single dose of the tracer (mmol 15N15N-urea).
Calculation of urea entry rate from continuous infusion of tracer.
Urea production was calculated from the isotopic dilution of the tracer at plateau enrichment, as
 | (3) |
where UER is the urea entry rate [mmol/(kg·h)], R is the infusion rate [(15N15N-urea mmol/(kg·h)] and E is the 15N15N-urea enrichment at plateau (mpe).
Statistical Analyses
Nonlinear regression [NLREG; (14)] was used to fit the multiexponential model (Eq. 1) to the single tracer dose data.
For the continuous infusion data, the following broken-line model was fitted to determine the plateau enrichment and the time to reach the plateau
 |
where Et is the observed blood urea enrichment (mpe) at time t; a is the intercept, b is the slope of the function, and tplateau is the breakpoint of the function (time to reach plateau).
Mean comparison of UER estimated by single dose and continuous infusion was done by Student's t test (15). A paired t test was conducted on blood and plasma enrichment from the continuous infusion protocol. Differences were considered significant at P < 0.05.
RESULTS
Best-fitted multiexponential curves for the single bolus dose of 15N15N-urea resulted in 2- (n = 4 mice) and 3-exponential (n = 2 mice) decay curves. Best-fitted 2- and 3-exponential curves, as well as their residuals for the enrichment data from mouse #266-B showing a 3-exponential decay curve are shown in Figure 1. Although the improvement in the coefficient of determination was very small, from 0.9862 to 0.9984, for the 2- and 3-exponential, respectively, there was a marked improvement in the distribution of the residuals. Residuals for the 3-exponential curve were distributed randomly and were smaller than 0.2 mpe, which is considered the analytical limit for GC MS analysis.
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3 min, and this reflects the dilution of the tracer by newly synthesized urea. The mean urea production based on stochastic analysis was 3.36 ± 0.30 mmol/(kg·h) (Table 1).
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3.3 ± 0.2 h. The mean urea production rate was 3.24 ± 0.23 mmol/(kg·h) (Table 3). Urea enrichment did not differ (P = 0.34) between plasma (3.47 mpe) and blood (3.40 mpe) samples.
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DISCUSSION
The injected labeled urea equilibrated rapidly with the urea pool as indicated by the g1 parameter (Fig 1; Table 1). Although this process took 1 h in humans (13), it was completed in 3 min in mice. In mice, it was shown that 14C-urea was distributed in all tissues, with the exception of the central nervous system and pregnant uterus contents, within 2 min of injection (17). The steep decline in enrichment, with a rate constant g1 > 100 h–1, after the tracer injection implies that a small error in the timing of the sample has a great effect on the estimation of parameter E1. Because we obtained multiple consecutive samples, we were able to determine the transit time of the blood sample inside the catheter and adjust accordingly. Other difficulties associated with the determination of the size of the first pool were outlined previously (12). Multiple samples have to be collected immediately after tracer injection to describe the steeper component of the decay curve; however, diffusion of the tracer into the blind compartment usually occurs before mechanical mixing in the blood is completed, resulting in a larger primary pool. Another implicit assumption was that tail blood enrichment represented the enrichment in the compartment. Tails were placed on a heat pad to increase blood flow, but that might not have been sufficient in 2 of the mice, and resulted in an apparently larger primary pool (25 and 40% of the total urea pool). In the remaining 4 mice, the primary pool represented 9% of the total pool, consistent with the intravascular compartment (18). In humans, a large primary pool (50% of the total) has been reported, which suggests that the primary pool in this case corresponded to extracellular water (13).
The exchange between the primary and secondary compartments (Fab and Fba) was several times greater than the urea produced (Foa; Table 2). Transport systems that facilitate the exchange/transport of urea in and out of the cells were identified in most tissues (19). These urea transporters (UT) are involved in the salvage of urea from excretion and the transfer of urea into the gut; they are also responsible for the ability of the kidney to concentrate urine (20). Upregulation of UT-A in UT-B knockout mice seems to indicate that maintaining a rapid exchange of urea between tissues might be of importance for the homeostasis of the mouse (21).
The determination of urea production by stochastic analysis requires no assumptions on the internal configuration of the system (12). Furthermore, because the area associated with the fast exponential decay contributes on average 4.3% of the total area under the curve, errors associated with its determination affect the urea production estimates only slightly. Thus, the precision of the urea production measurement relies on defining the parameters of the slow portion of the curve (E2 and g2). Regardless of the approach used, compartmental or stochastic, the same rate of urea production is obtained.
Blood urea enrichment reached an apparent plateau between 3 and 4 h after the initiation of a continuous infusion of the tracer solution. The calculated urea production rate was on average 3.24 mmol/(kg·h), not different (P = 0.76) from that obtained by the single-dose method [3.36 mmol/(kg·h)]. The advantage of the continuous infusion method is that a large terminal blood sample can be obtained at the end of the infusion (or a spot urine sample), and that a single sample at plateau is required to calculate urea production; however, a longer period of restraint has to be imposed on the experimental mice. A primed-continuous infusion is a valid alternative to reduce the time the mice are restrained; based on the data presented in Table 2, we estimated that a prime equivalent to a 1-h infusion would achieve plateau values within 15 min. Plasma and blood samples showed the same enrichment; thus, either one can be used to determine urea production. The single tracer dose method allows for additional information on the urea metabolism of the mice, but more samples, and of very small volume, have to be analyzed. If the stochastic approach is preferred, early samples (first 5 min after injection) can be omitted and the sampling protocol reduced to 3 or 4 samples within 1 h after the injection (13,22).
To the best of our knowledge no data on urea kinetics in mice have been published. The urea entry rate in mice, on a body weight basis, was an order of magnitude greater than that reported in humans [0.22 mmol/(kg·h); (5,13)] or growing piglets [0.31 mmol/(kg·h); (22)]. This is consistent with the high metabolic activity of mice and their higher protein intake on a body weight basis.
The methods described in this paper for the determination of urea entry rate would allow not only for the study of urea cycle disorders in vivo, but also urea transport and transporter function, nitrogen metabolism in general, and the relation between the urea and nitric oxide cycles in mouse models. This is especially relevant because mouse targeting technologies will likely facilitate the generation of organ and tissue specific nulls of the various urea cycle enzymes.
FOOTNOTES
1 Supported in part by the Mental Retardation and Developmental Disabilities Research Center (HD024064), and the National Institutes of Health (DK54450, DK54991). 
3 Abbreviation used: D, 15N15N urea dose; Ei, fitted parameter (intercept); Et, observed enrichment at time t; gi, fitted parameter (exponent); mpe, mole percent excess; R, 15N15N urea infusion rate; UER, urea entry rate.
Manuscript received 28 September 2005. Initial review completed 11 October 2005. Revision accepted 24 October 2005.
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