Metabolic flux analysis of cultured hepatocytes exposed to plasma

Hepatic metabolism can be investigated using metabolic flux analysis (MFA), which provides a comprehensive overview of the intracellular metabolic flux distribution. The characterization of intermediary metabolism in hepatocytes is important for all biotechnological applications involving liver cells, including the development of bioartificial liver (BAL) devices. During BAL operation, hepatocytes are exposed to plasma or blood from the patient, at which time they are prone to accumulate intracellular lipids and exhibit poor liver-specific functions. In a prior study, we found that preconditioning the primary rat hepatocytes in culture medium containing physiological levels of insulin, as opposed to the typical supraphysiological levels found in standard hepatocyte culture media, reduced lipid accumulation during subsequent plasma exposure. Furthermore, supplementing the plasma with amino acids restored hepatospecific functions. In the current study, we used MFA to quantify the changes in intracellular pathway fluxes of primary rat hepatocytes in response to low-insulin preconditioning and amino acid supplementation. We found that culturing hepatocytes in medium containing lower physiological levels of insulin decreased the clearance of glucose and glycerol with a concomitant decrease in glycolysis. These findings are consistent with the general notion that low insulin, especially in the presence of high glucagon levels, downregulates glycolysis in favor of gluconeogenesis in hepatocytes. The MFA model shows that, during subsequent plasma exposure, low-insulin preconditioning upregulated gluconeogenesis, with lactate as the primary precursor in unsupplemented plasma, with a greater contribution from deaminated amino acids in amino acid-supplemented plasma. Concomitantly, low-insulin preconditioning increased fatty acid oxidation, an effect that was further enhanced by amino acid supplementation to the plasma. The increase in fatty acid oxidation reduced intracellular triglyceride accumulation. Overall, these findings are consistent with the notion that the insulin level in medium culture presets the metabolic machinery of hepatocytes such that it directly impacts on their metabolic behavior during subsequent plasma culture.     

Analysis of amino acid supplementation effects on hepatocyte cultures using flux balance analysis

When cultured hepatocytes are exposed to challenging environments such as plasma, they frequently suffer a decline in liver-specific functions. Media supplements are sought to reduce or eliminate this effect. A rational design approach for amino acid supplementation in hepatocyte culture has been developed in our prior work, and designed amino acid supplementation (DAA) was found to increase urea and albumin production. To fully characterize the metabolic state of hepatocytes under different amino acid supplementations, a number of metabolite measurements are performed in this work and used in a metabolic network flexibility analysis framework including thermodynamic constraints to determine the range of values for the intracellular fluxes. A metabolic objective prediction model is used to infer the metabolic objectives of the hepatocytes and to quantify the intracellular flux distribution for three different amino acid supplementations. The results illustrate that DAA leads to greater fluxes in the tricarboxylic acid cycle (TCA) cycle, urea cycle, and fatty acid oxidation concomitant with lower fluxes in intracellular lipid metabolism compared with empirical amino acid and no amino acid supplementation for hepatocytes during plasma exposure. It is also found that hepatocytes exhibit flexibility in their metabolic objectives depending on the composition of the amino acid supplementations. By incorporating both experimental data and thermodynamic constraints into the mathematical model, the proposed approach leads to identification of metabolic objectives and characterization of fluxes' variability and pathway changes due to different cultured conditions.     

Application of multivariate analysis to optimize function of cultured hepatocytes

Understanding the metabolic and regulatory pathways of hepatocytes is important for biotechnological applications involving liver cells, including the development of bioartificial liver (BAL) devices. To characterize intermediary metabolism in the hepatocytes, metabolic flux analysis (MFA) was applied to elucidate the changes in intracellular pathway fluxes of primary rat hepatocytes exposed to human plasma and to provide a comprehensive snapshot of the hepatic metabolic profile. In the current study, the combination of preconditioning and plasma supplementation produced distinct metabolic states. Combining the metabolic flux distribution obtained by MFA with methodologies such as Fisher discriminant analysis (FDA) and partial least squares or projection to latent structures (PLS) provided insights into the underlying structure and causal relationship within the data. With the aid of these analyses, patterns in the cellular response of the hepatocytes that contributed to the separation of the different hepatic states were identified. Of particular interest was the recognition of distal pathways that strongly correlated with a particular hepatic function. The hepatic functions investigated were intracellular triglyceride accumulation and urea production. This study illustrates a framework for optimizing hepatic function and a possibility of identifying potential targets for improving hepatic functions.     

A rational design approach for amino acid supplementation in hepatocyte culture

Improvement of culture media for mammalian cells is conducted via empirical adjustments, sometimes aided by statistical design methodologies. Here, we demonstrate a proof of principle for the use of constraints‐based modeling to achieve enhanced performance of liver‐specific functions of cultured hepatocytes during plasma exposure by adjusting amino acid supplementation and hormone levels in the medium. Flux balance analysis (FBA) is used to determine an amino acid flux profile consistent with a desired output; this is used to design an amino acid supplementation. Under conditions of no supplementation, empirical supplementation, and designed supplementation, hepatocytes were exposed to plasma and their morphology, specific cell functions (urea, albumin production) and lipid metabolism were measured. Urea production under the designed amino acid supplementation was found to be increased compared with previously reported (empirical) amino acid supplementation. Not surprisingly, the urea production attained was less than the theoretical value, indicating the existence of pathways or constraints not present in the current model. Although not an explicit design objective, albumin production was also increased by designed amino acid supplementation, suggesting a functional linkage between these outputs. In conjunction with traditional approaches to improving culture conditions, the rational design approach described herein provides a novel means to tune the metabolic outputs of cultured hepatocytes. Biotechnol. Bioeng. 2009;103: 1176–1191. © 2009 Wiley Periodicals, Inc.     

Metabolic pre-conditioning of cultured cells in physiological levels of insulin: generating resistance to the lipid-accumulating effects of plasma in hepatocytes

Understanding the regulation of hepatocyte lipid metabolism is important for several biotechnological applications involving liver cells. During exposure of hepatocytes to plasma, as is the case in extracorporeal bioartificial liver assist devices, it has been reported that hepatic-specific functions, e.g., albumin and urea synthesis and diazepam removal, are dramatically compromised and hepatocytes progressively accumulate cytoplasmic lipid droplets. We hypothesized that the composition of hepatocyte culture medium significantly affects lipid metabolism during subsequent plasma exposure. Rat hepatocytes were cultured in medium containing either physiological (50 microU/mL) or supra-physiological (500 mU/mL) insulin levels for 1 week and then exposed to human plasma supplemented with or without amino acids. We found that insulin's anabolic effects, such as stimulation of triglyceride storage, were carried over from the pre-conditioning to the plasma exposure period. While hepatocytes cultured in high insulin medium accumulated large quantities of triglycerides during subsequent plasma exposure, culture in low insulin medium largely prevented lipid accumulation. Urea and albumin secretion, as well as the ammonia removal rate, were largely unaffected by insulin but increased with amino acid supplementation. Thus, hepatocyte metabolism during plasma exposure can be modulated by medium pre-conditioning and supplements added to plasma.     

In vitro effect of caffeine on some aspects of nitrogen metabolism in isolated rat hepatocytes

Long-term exposure to high doses of caffeine affects several aspects of nitrogen metabolism, such as purine, pyrimidine and urea synthesis. However, little is known about the mechanisms of these changes and if they occur at shorter term. We have studied in isolated hepatocytes: 1) the in vitro effect of high doses of caffeine on amino acid levels, 2) the main destination of ammonia and carbon chains from amino acid catabolism, and 3) the cytosolic and mitochondrial redox states. We have found that, whereas it has a small effect on urea synthesis and on the levels of the cofactors and intermediates, it decreases the levels of several amino acids, the gluconeogenesis and the redox state. Our results suggest that a longer exposure to caffeine is necessary to affect the normal functions of some metabolic pathways.     

Hepatocyte heterogeneity in the metabolism of amino acids and ammonia.

With respect to hepatocyte heterogeneity in ammonia and amino acid metabolism, two different patterns of sublobular gene expression are distinguished: 'gradient-type' and 'strict- or compartment-type' zonation. An example for strict-type zonation is the reciprocal distribution of carbamoylphosphate synthase and glutamine synthase in the liver lobule. The mechanisms underlying the different sublobular gene expressions are not yet settled but may involve the development of hepatic architecture, innervation, blood-borne hormonal and metabolic factors. The periportal zone is characterized by a high capacity for uptake and catabolism of amino acids (except glutamate and aspartate) as well as for urea synthesis and gluconeogenesis. On the other hand, glutamine synthesis, ornithine transamination and the uptake of vascular glutamate, aspartate, malate and alpha-ketoglutarate are restricted to a small perivenous hepatocyte population. Accordingly, in the intact liver lobule the major pathways for ammonia detoxication, urea and glutamine synthesis, are anatomically switched behind each other and represent in functional terms the sequence of the periportal low affinity system (urea synthesis) and a previous high affinity system (glutamine synthesis) for ammonia detoxication. Perivenous glutamine synthase-containing hepatocytes ('scavenger cells') act as a high affinity scavenger for the ammonia, which escapes the more upstream urea-synthesizing compartment. Periportal glutaminase acts as a pH- and hormone-modulated ammonia-amplifying system in the mitochondria of periportal hepatocytes. The activity of this amplifying system is one crucial determinant for flux through the urea cycle in view of the high Km (ammonia) of carbamoylphosphate synthase, the rate-controlling enzyme of the urea cycle. The structural and functional organization of glutamine and ammonia-metabolizing pathways in the liver lobule provides one basis for the understanding of a hepatic role in systemic acid base homeostasis. Urea synthesis is a major pathway for irreversible removal of metabolically generated bicarbonate. The lobular organization enables the adjustment of the urea cycle flux and accordingly the rate of irreversible hepatic bicarbonate elimination to the needs of the systemic acid base situation, without the threat of hyperammonemia.     

Natural Isotopic Signatures of Variations in Body Nitrogen Fluxes: A Compartmental Model Analysis

Body tissues are generally ¹⁵N-enriched over the diet, with a discrimination factor (Δ¹⁵N) that varies among tissues and individuals as a function of their nutritional and physiopathological condition. However, both ¹⁵N bioaccumulation and intra- and inter-individual Δ¹⁵N variations are still poorly understood, so that theoretical models are required to understand their underlying mechanisms. Using experimental Δ¹⁵N measurements in rats, we developed a multi-compartmental model that provides the first detailed representation of the complex functioning of the body''s Δ¹⁵N system, by explicitly linking the sizes and Δ¹⁵N values of 21 nitrogen pools to the rates and isotope effects of 49 nitrogen metabolic fluxes. We have shown that (i) besides urea production, several metabolic pathways (e.g., protein synthesis, amino acid intracellular metabolism, urea recycling and intestinal absorption or secretion) are most probably associated with isotope fractionation and together contribute to ¹⁵N accumulation in tissues, (ii) the Δ¹⁵N of a tissue at steady-state is not affected by variations of its P turnover rate, but can vary according to the relative orientation of tissue free amino acids towards oxidation vs. protein synthesis, (iii) at the whole-body level, Δ¹⁵N variations result from variations in the body partitioning of nitrogen fluxes (e.g., urea production, urea recycling and amino acid exchanges), with or without changes in nitrogen balance, (iv) any deviation from the optimal amino acid intake, in terms of both quality and quantity, causes a global rise in tissue Δ¹⁵N, and (v) Δ¹⁵N variations differ between tissues depending on the metabolic changes involved, which can therefore be identified using simultaneous multi-tissue Δ¹⁵N measurements. This work provides proof of concept that Δ¹⁵N measurements constitute a new promising tool to investigate how metabolic fluxes are nutritionally or physiopathologically reorganized or altered. The existence of such natural and interpretable isotopic biomarkers promises interesting applications in nutrition and health.     

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders.In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via β-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying β-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of β-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.     

Metabolic flux rearrangement in the amino acid metabolism reduces ammonia stress in the α1-antitrypsin producing human AGE1.HN cell line

This study focused on metabolic changes in the neuronal human cell line AGE1.HN upon increased ammonia stress. Batch cultivations of α(1)-antitrypsin (A1AT) producing AGE1.HN cells were carried out in media with initial ammonia concentrations ranging from 0mM to 5mM. Growth, A1AT production, metabolite dynamics and finally metabolic fluxes calculated by metabolite balancing were compared. Growth and A1AT production decreased with increasing ammonia concentration. The maximum A1AT concentration decreased from 0.63g/l to 0.51g/l. Central energy metabolism remained relatively unaffected exhibiting only slightly increased glycolytic flux at high initial ammonia concentration in the medium. However, the amino acid metabolism was significantly changed. Fluxes through transaminases involved in amino acid degradation were reduced concurrently with a reduced uptake of amino acids. On the other hand fluxes through transaminases working in the direction of amino acid synthesis, i.e., alanine and phosphoserine, were increased leading to increased storage of excess nitrogen in extracellular alanine and serine. Glutamate dehydrogenase flux was reversed increasingly fixing free ammonia with increasing ammonia concentration. Urea production additionally observed was associated with arginine uptake by the cells and did not increase at high ammonia stress. It was therefore not used as nitrogen sink to remove excess ammonia. The results indicate that the AGE1.HN cell line can adapt to ammonia concentrations usually present during the cultivation process to a large extent by changing metabolism but with slightly reduced A1AT production and growth.     

Comparison of metabolic flux distributions for MDCK cell growth in glutamine- and pyruvate-containing media

In mammalian cell cultures, ammonia that is released into the medium as a result of glutamine metabolism and lactate that is excreted due to incomplete glucose oxidation are both known to essentially inhibit the growth of cells. For some cell lines, for example, hybridoma cells, excreted ammonia also has an effect on product formation. Although glutamine has been generally considered as the major energy source for mammalian cells, it was recently found that various adherent cell lines (MDCK, CHO-K1, and BHK21) can grow as well in glutamine-free medium, provided glutamine is substituted with pyruvate. In such a medium the level of both ammonia and lactate released was significantly reduced. In this study, metabolic flux analysis (MFA) was applied to Madin Darby Canine Kidney (MDCK) cells cultivated in glutamine-containing and glutamine-free medium. The results of the MFA allowed further investigation of the influence of glutamine substitution with pyruvate on the metabolism of MDCK cells during different growth stages of adherent cells, e.g., early exponential and late contact-inhibited phase. Pyruvate seemed to directly enter the TCA cycle, whereas most of the glucose consumed was excreted as lactate. Although the exact mechanisms are not clear so far, this resulted in a reduction of the glucose uptake necessary for cellular metabolism in glutamine-free medium. Furthermore, consumption of ATP by futile cycles seemed to be significantly reduced when substituting glutamine with pyruvate. These findings imply that glutamine-free medium favors a more efficient use of nutrients by cells. However, a number of metabolic fluxes were similar in the two cultivations considered, e.g., most of the amino acid uptake and degradation rates or fluxes through the branch of the TCA cycle converting alpha-ketoglutarate to malate, which is responsible for the mitochondrial ATP synthesis. Besides, the specific rate of cell growth was approximately the same in both cultivations. Thus, the switch from glutamine-containing to glutamine-free medium with pyruvate provided a series of benefits without dramatic changes of cellular metabolism.     

Metabolic profiling based quantitative evaluation of hepatocellular metabolism in presence of adipocyte derived extracellular matrix

The elucidation of the effect of extracellular matrices on hepatocellular metabolism is critical to understand the mechanism of functional upregulation. We have developed a system using natural extracellular matrices [Adipogel] for enhanced albumin synthesis of rat hepatocyte cultures for a period of 10 days as compared to collagen sandwich cultures. Primary rat hepatocytes isolated from livers of female Lewis rats recover within 4 days of culture from isolation induced injury while function is stabilized at 7 days post-isolation. Thus, the culture period can be classified into three distinct stages viz. recovery stage [day 0-4], pre-stable stage [day 5-7] and the stable stage [day 8-10]. A Metabolic Flux Analysis of primary rat hepatocytes cultured in Adipogel was performed to identify the key metabolic pathways modulated as compared to collagen sandwich cultures. In the recovery stage [day 4], the collagen-soluble Adipogel cultures shows an increase in TriCarboxylic Acid [TCA] cycle fluxes; in the pre-stable stage [day 7], there is an increase in PPP and TCA cycle fluxes while in the stable stage [day 10], there is a significant increase in TCA cycle, urea cycle fluxes and amino acid uptake rates concomitant with increased albumin synthesis rate as compared to collagen sandwich cultures throughout the culture period. Metabolic analysis of the collagen-soluble Adipogel condition reveals significantly higher transamination reaction fluxes, amino acid uptake and albumin synthesis rates for the stable vs. recovery stages of culture. The identification of metabolic pathways modulated for hepatocyte cultures in presence of Adipogel will be a useful step to develop an optimization algorithm to further improve hepatocyte function for Bioartificial Liver Devices. The development of this framework for upregulating hepatocyte function in Bioartificial Liver Devices will facilitate the utilization of an integrated experimental and computational approach for broader applications of Adipogel in tissue e engineering and regenerative medicine.     

Metabolic adjustments during semi-aestivation of the marble swamp eel (Synbranchus marmoratus, Bloch 1795)--a facultative air breathing fish.

Metabolic changes, principally in intermediary metabolism and nitrogen excretion, were investigated in the marble swamp eel (Synbranchus marmoratus) after 15 and 45 days of artificially induced semi-aestivation. Glucose, glycogen, lactate, pyruvate, free amino acids, triglycerides, ammonia, urea, and urate contents were determined in liver, kidney, white muscle, heart, brain, and plasma. Lactate dehydrogenase, glutamate dehydrogenase, malate dehydrogenase, aspartate amino transferase, alanine amino transferase, glutamine synthase, ornithine carbamoyl transferase, and arginase enzymes were assayed. The teleost S. marmoratus maintained initial energetic demands by lipid oxidation. The course of normal oxidative processes was observed through tissue enzyme profiles. After the lipid stores were exhausted, the fish consumed body proteins. Constant values of hematocrit during induced semi-aestivation suggested that the water balance remained normal. Therefore, the surrounding water was probably did not trigger the semi-aestivation in this teleost. Decrease of ammonia and increase of renal urea synthesis after 45 days of semi-aestivation led to the assumption that an alternative form of eliminating ammonia exists. Metabolic changes entailed by starvation were proposed to explain the biosynthesis of small molecules involved in the semi-aestivation of S. marmoratus.     

A window into cellular metabolism: hepatic metabolism of (15)N-labelled substrates

It is now apparent that many of the subtleties of cellular metabolism are intrinsically associated with cell structure and that their physiological study requires techniques that respect the integrity of cells and organs. We have used 15N-substrates to examine urea synthesis in the intact perfused rat liver. This work permits us to determine the extent to which different amino acids donate nitrogen atoms to the two nitrogens of urea. It is apparent that alanine and the amino group of glutamine provide nitrogen for urea synthesis primarily via cytoplasmic aspartate, whereas mitochondrial ammonia is the preferred route of entry for nitrogen from pre-formed ammonia or from the amide nitrogen of glutamine. Most importantly, this methodology permits us to explore for the occurrence of metabolic channels in such a highly organised, physiological system. Our studies indicate that a metabolic channel does not exist between glutaminase and carbamoylphosphate synthetase 1.     

A Peptide-Based Method for 13C Metabolic Flux Analysis in Microbial Communities

The study of intracellular metabolic fluxes and inter-species metabolite exchange for microbial communities is of crucial importance to understand and predict their behaviour. The most authoritative method of measuring intracellular fluxes, ¹³C Metabolic Flux Analysis (¹³C MFA), uses the labeling pattern obtained from metabolites (typically amino acids) during ¹³C labeling experiments to derive intracellular fluxes. However, these metabolite labeling patterns cannot easily be obtained for each of the members of the community. Here we propose a new type of ¹³C MFA that infers fluxes based on peptide labeling, instead of amino acid labeling. The advantage of this method resides in the fact that the peptide sequence can be used to identify the microbial species it originates from and, simultaneously, the peptide labeling can be used to infer intracellular metabolic fluxes. Peptide identity and labeling patterns can be obtained in a high-throughput manner from modern proteomics techniques. We show that, using this method, it is theoretically possible to recover intracellular metabolic fluxes in the same way as through the standard amino acid based ¹³C MFA, and quantify the amount of information lost as a consequence of using peptides instead of amino acids. We show that by using a relatively small number of peptides we can counter this information loss. We computationally tested this method with a well-characterized simple microbial community consisting of two species.     

Novel quantitative tools for engineering analysis of hepatocyte cultures in bioartificial liver systems

Extracorporeal bioartificial liver devices (BAL) are perhaps among the most promising technologies for the treatment of liver failure, but significant technical challenges remain in order to develop systems with sufficient processing capacity and of manageable size. One key limitation is that during BAL operation, when the device is exposed to plasma from the patient, hepatocytes are prone to accumulate intracellular lipids and exhibit poor liver-specific functions. Based on hepatic intermediary metabolism, we have utilized mathematical programming techniques to optimize the biochemical environment of hepatocyte cultures towards the desired effect of increased albumin and urea synthesis. To investigate the feasible range of optimal hepatic function, we have obtained a Pareto optimal set of solutions corresponding to liver-specific functions of urea and albumin secretion in the metabolic framework using multiobjective optimization. The importance of amino acids in the supplementation and the criticality of the metabolic pathways have been investigated using logic-based programming techniques. Since the metabolite measurements are bound to be patient specific, and hence subject to variability, uncertainty has to be integrated with system analysis to improve the prediction of hepatic function. We have used the concept of two stage stochastic programming to obtain robust solutions by considering extracellular variability. The proposed analysis represents a new systematic approach to analyze behavior of hepatocyte cultures and optimize different operating parameters for an extracorporeal device based on real-time conditions.     

Inhibition of hepatocyte proteolysis and lactate gluconeogenesis by chloroquine

Chloroquine (50 μm) is rapidly taken up by isolated hepatocytes in a temperature-dependent manner. It inhibits glucose synthesis from lactate, but not from pyruvate or dihydroxyacetone. The inhibition is reversed by lysine or ammonia but not by oleate or carnitine. Ammonia inhibits chloroquine uptake by the hepatocytes but lysine does not. Chloroquine also inhibits urea synthesis, the release of ninhydrin-reacting substances, the accumulation of amino acids, and the lactate-dependent accumulation of glutamate. Ethanol oxidation in the presence of lactate is also inhibited, and this too is reversed by lysine. Chloroquine increases the redox state of the cytosolic compartment, as evidenced by lactate-to-pyruvate ratios, of hepatocytes prepared from both 48-h fasted and meal-fed rats. The above findings are consistent with chloroquine entering the lysosomes of the hepatocytes and inhibiting proteolysis by raising the lysosomal pH. Isolated hepatocytes are deficient in amino acids and, chloroquine inhibition of proteolysis prevents replenishment of the amino acid pools. Thus, chloroquine prevents reconstitution of the malate-aspartate shuttle required for the movement of reducing equivalents into the mitochondrion during lactate gluconeogenesis, ethanol oxidation, and glycolysis. The metabolic competency of freshly isolated hepatocytes, therefore, depends on the replenishment of amino acid pools by lysosomal breakdown of endogenous protein. Furthermore, chloroquine uptake may be an index of lysosomal function with isolated hepatocytes.     

Control by pH of urea synthesis in isolated rat hepatocytes

Control by pH of urea synthesis has been studied in isolated rat hepatocytes incubated with a physiological mixture of amino acids. Inhibition of urea synthesis by decreasing the pH of the medium was caused by diminished production of ammonia and not, as suggested in the literature, by inhibition of entry of ammonia into the ornithine cycle. The decrease by low pH of the rate of degradation of the added amino acids, that of alanine being quantitatively the most important, was accompanied by a decrease in their intracellular concentration. It is concluded that inhibited transport of amino acids across the plasma membrane of the hepatocyte is responsible, at least in part, for the fall in urea synthesis with decreasing pH. It is proposed that inhibition by low pH of other steps in the ureogenic pathway, distal to the production of ammonia, does not affect flux through the ornithine cycle per se, but rather contributes to the buffering of the intrahepatic concentration of ammonia.     

Nitrogen metabolism and excretion in the aquatic chinese soft-shelled turtle, Pelodiscus sinensis, exposed to a progressive increase in ambient salinity

This study aimed to determine effects of 6-day progressive increase in salinity from 1 per thousand to 15 per thousand on nitrogen metabolism and excretion in the soft-shelled turtle, Pelodiscus sinensis. For turtles exposed to 15 per thousand water on day 6, the plasma osmolality and concentrations of Na+, Cl- and urea increased significantly, which presumably decreased the osmotic loss of water. Simultaneously, there were significant increases in contents of urea, certain free amino acids (FAAs) and water-soluble proteins that were involved in cell volume regulation in various tissues. There was an apparent increase in proteolysis, releasing FAAs as osmolytes. In addition, there might be an increase in catabolism of certain amino acids, producing more ammonia. The excess ammonia was retained as indicated by a significant decrease in the rate of ammonia excretion on day 4 in 15 per thousand water, and a major portion of it was converted to urea. The rate of urea synthesis increased 1.4-fold during the 6-day period, although the capacity of the hepatic ornithine urea cycle remained unchanged. Urea was retained for osmoregulation because there was a significant decrease in urea excretion on day 4. Increased protein degradation and urea synthesis implies greater metabolic demands, and indeed turtles exposed to 15 per thousand water had significantly higher O2 consumption rate than the freshwater (FW) control. When turtles were returned from 15 per thousand water to FW on day 7, there were significant increases in ammonia (probably released through increased amino acid catabolism) and urea excretion, confirming that FAAs and urea were retained for osmoregulatory purposes in brackish water.