The role of high rates of glycolysis and glutamine utilization in rapidly dividing cells

The rates of utilization of both glucose and glutamine are high in rapidly dividing ceils such as enterocytes, lymphocytes, thymocytes, tumour cells; the oxidation of both glucose and glutamine is only partial, glucose to lactate and glutamine to glutamate , alanine or aspartate ; and these partial processes are termed glycolysis and glutaminolysis respectively . Both processes generate energy and also provide precursors for important biosynthetic processes in such cells. However, the rates of utilization of precursors for macromolecular biosynthesis are very low in comparison to the rates oi partial oxidation, and energy generation per se may not be the correct explanation for high rates of glycolysis and glutaminolysis in these cells since oxidation is only partial and other fuels can be used to generate energy. Both the high fluxes and the metabolic characteristics of these two processes can be explained by application of quantitative principles of control as applied to branched metabolic pathway s (Crabtree & Newsholme, 1985). If the flux through one branch is greatly in excess of the other, then the sensitivity of the flux of the low-flux pathway to regulators is very high. Hence, it is suggested that, in rapidly dividing ceils, high rates of glycolysis and gtutaminolysis are required not for energy or precursor provision per se but for high sensitivity of the pathways involved in the use of precursors for macromolecular synthesis to specific regulators to permithigh rates of proliferation when required for example, in lymphocytes in response to a massive infection.     

Glutamine metabolism by lymphocytes, macrophages, and neutrophils: its importance in health and disease

Many aspects of the cell biology of lymphocytes, macrophages, and neutrophils have been studied extensively. Our recent work on these cells has investigated how fuel metabolism, especially glutamine metabolism, is related to the specific function of these cells in the inflammatory response. The high rate of glutamine utilization and its metabolism in such immune cells has raised the question of why glutamine is responsible for these functions. The macrophage has access to a variety of metabolic fuels both in vivo and in vitro. The quantitatively important role of glutamine in the processes of free radical and cytokine production has been established in our laboratories. Our current understanding of the rate of utilization and the pathway of metabolism of glutamine by cells of the immune system raises some intriguing questions concerning therapeutic manipulation of utilization of this amino acid, specifically the phagocytic and secretory capacities of cells of the defense system can be beneficially altered.     

Glutamine metabolism in endothelial cells: ornithine synthesis from glutamine via pyrroline-5-carboxylate synthase

L-Glutamine (the most abundant free amino acid in plasma and the body) is a potent inhibitor of endothelial NO synthesis. However, little is known about glutamine metabolism in endothelial cells (EC). As an initial step toward understanding the role of glutamine in endothelial physiology, the present study was conducted to quantify glutamine catabolism in microvascular, aortic and venous EC. For metabolic studies, EC were incubated for 1 h in Krebs bicarbonate buffer containing 5 mM glucose and 0.5-4 mM L-[U-(14)C]-glutamine. For enzymological studies, cell extracts and mitochondrial fractions were prepared to determine the activities of glutamine-degrading enzymes. Our results reveal extensive hydrolysis of glutamine to glutamate and ammonia in a concentration-dependent manner via phosphate-dependent glutaminase in all EC studied. In addition, both metabolic and enzymological evidence indicate a novel pathway for endothelial synthesis of ornithine from glutamine via pyrroline-5-carboxylate synthase. This new knowledge of glutamine metabolism may pave a new path for understanding the physiological role of glutamine in vascular function.     

Rates of utilization of glucose, glutamine and oleate and formation of end-products by mouse peritoneal macrophages in culture.

1. The metabolism of mouse thioglycollate-elicited peritoneal macrophages was studied in culture for up to 96 h. 2. The rates of glycolysis, lactate formation and glutamine utilization were approximately linear with time for at least 80 h of culture. 3. The rates of glucose and glutamine utilization by cultured macrophages were approx. 500 and 90 nmol/h per mg of protein respectively. This rate of glucose utilization is at least 50% greater than that previously reported for macrophages during 60 min incubation in a shaking flask; and it is now increased by addition of glutamine to the culture medium. The rate of glutamine utilization in culture is similar to that previously reported for macrophages during 60 min incubation. The major end-product of glucose metabolism is lactate, and those of glutamine metabolism are CO2, glutamate, ammonia and alanine. 4. Oleate was utilized by these cells: 14C from [14C]oleate was incorporated into CO2 and cellular lipid. The highest rate of oleate utilization was observed when both glucose and glutamine were present in the culture medium. The presence of oleate in the culture medium did not affect the rates of utilization of either glucose or glutamine. Of the [14C]oleate incorporated into lipid, approx. 80% was incorporated into triacylglycerol and only 18% into phospholipid. 5. The turnover rate for the total ATP content of the macrophage in culture is about 10 times per minute: the value for the perfused isolated maximally working rat heart is 22. This indicates a high metabolic rate for macrophages, and consequently emphasizes the importance of the provision of fuels for their function in an immune response.     

The effect of glutamine concentration on the activity of carbamoyl-phosphate synthase II and on the incorporation of [3H]thymidine into DNA in rat mesenteric lymphocytes stimulated by phytohaemagglutinin.

The maximum catalytic activities of carbamoyl-phosphate synthase II, a limiting enzyme for pyrimidine nucleotide synthesis, are very much less than those of glutaminase, a limiting enzyme for glutamine utilization, in lymphocytes and macrophages; and the flux through the pathway for pyrimidine formation de novo is only about 0.4% of the rate of glutamine utilization by lymphocytes. The Km of synthase II for glutamine is about 16 microM and the concentration of glutamine necessary to stimulate lymphocyte proliferation half-maximally is about 21 microM. This agreement suggests that the importance of glutamine for these cells is provision of nitrogen for biosynthesis of pyrimidine nucleotides (and probably purine nucleotides). However, the glutamine concentration necessary for half-maximal stimulation of glutamine utilization (glutaminolysis) by the lymphocytes is 2.5 mM. The fact that the rate of glutamine utilization by lymphocytes is markedly in excess of the rate of the pathway for pyrimidine nucleotide synthesis de novo and that the Km and 'half-maximal concentration' values are so different, suggests that the glutaminolytic pathway is independent of the use of glutamine nitrogen for pyrimidine synthesis.     

Metabolic adaptation of MDCK cells to different growth conditions: effects on catalytic activities of central metabolic enzymes

Lactate and ammonia are the most important waste products of central carbon metabolism in mammalian cell cultures. In particular during batch and fed-batch cultivations these toxic by-products are excreted into the medium in large amounts, and not only affect cell viability and productivity but often also prevent growth to high cell densities. The most promising approach to overcome such a metabolic imbalance is the replacement of one or several components in the culture medium. It has been previously shown that pyruvate can be substituted for glutamine in cultures of adherent Madin-Darby canine kidney (MDCK) cells. As a consequence, the cells not only released no ammonia but glucose consumption and lactate production were also reduced significantly. In this work, the impact of media changes on glucose and glutamine metabolism was further elucidated by using a high-throughput platform for enzyme activity measurements of mammalian cells. Adherent MDCK cells were grown to stationary and exponential phase in six-well plates in serum-containing GMEM supplemented with glutamine or pyruvate. A total number of 28 key metabolic enzyme activities of cell extracts were analyzed. The overall activity of the pentose phosphate pathway was up-regulated during exponential cell growth in pyruvate-containing medium suggesting that more glucose-6-phosphate was channeled into the oxidative branch. Furthermore, the anaplerotic enzymes pyruvate carboxylase and pyruvate dehydrogenase showed higher cell specific activities with pyruvate. An increase in cell specific activity was also found for NAD(+)-dependent isocitrate dehydrogenase, glutamate dehydrogenase, and glutamine synthetase in MDCK cells grown with pyruvate. It can be assumed that the increase in enzyme activities was required to compensate for the energy demand and to replenish the glutamine pool. On the other hand, the activities of glutaminolytic enzymes (e.g., alanine and aspartate transaminase) were decreased in cells grown with pyruvate, which seems to be related to a decreased glutamine metabolism.     

Metabolism of glucose, glutamine, long-chain fatty acids and ketone bodies by murine macrophages.

Maximum activities of some key enzymes of metabolism were studied in elicited (inflammatory) macrophages of the mouse and lymph-node lymphocytes of the rat. The activity of hexokinase in the macrophage is very high, as high as that in any other major tissue of the body, and higher than that of phosphorylase or 6-phosphofructokinase, suggesting that glucose is a more important fuel than glycogen and that the pentose phosphate pathway is also important in these cells. The latter suggestion is supported by the high activities of both glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. However, the rate of glucose utilization by 'resting' macrophages incubated in vitro is less than the 10% of the activity of 6-phosphofructokinase: this suggests that the rate of glycolysis is increased dramatically during phagocytosis or increased secretory activity. The macrophages possess higher activities of citrate synthase and oxoglutarate dehydrogenase than do lymphocytes, suggesting that the tricarboxylic acid cycle may be important in energy generation in these cells. The activity of 3-oxoacid CoA-transferase is higher in the macrophage, but that of 3-hydroxybutyrate dehydrogenase is very much lower than those in the lymphocytes. The activity of carnitine palmitoyltransferase is higher in macrophages, suggesting that fatty acids as well as acetoacetate could provide acetyl-CoA as substrate for the tricarboxylic acid cycle. No detectable rate of acetoacetate or 3-hydroxybutyrate utilization was observed during incubation of resting macrophages, but that of oleate was 1.0 nmol/h per mg of protein or about 2.2% of the activity of palmitoyltransferase. The activity of glutaminase is about 4-fold higher in macrophages than in lymphocytes, which suggests that the rate of glutamine utilization could be very high. The rate of utilization of glutamine by resting incubated macrophages was similar to that reported for rat lymphocytes, but was considerably lower than the activity of glutaminase.     

Cancer cell metabolism: the essential role of the nonessential amino acid,glutamine

Biochemistry textbooks and cell culture experiments seem to be telling us two different things about the significance of external glutamine supply for mammalian cell growth and proliferation. Despite the fact that glutamine is a nonessential amino acid that can be synthesized by cells from glucose‐derived carbons and amino acid‐derived ammonia, most mammalian cells in tissue culture cannot proliferate or even survive in an environment that does not contain millimolar levels of glutamine. Not only are the levels of glutamine in standard tissue culture media at least ten‐fold higher than other amino acids, but glutamine is also the most abundant amino acid in the human bloodstream, where it is assiduously maintained at approximately 0.5 mM through a combination of dietary uptake, de novo synthesis, and muscle protein catabolism. The complex metabolic logic of the proliferating cancer cells' appetite for glutamine—which goes far beyond satisfying their protein synthesis requirements—has only recently come into focus. In this review, we examine the diversity of biosynthetic and regulatory uses of glutamine and their role in proliferation, stress resistance, and cellular identity, as well as discuss the mechanisms that cells utilize in order to adapt to glutamine limitation.     

Transport of glutamine and glutamate in kidney mitochondria in relation to glutamine deamidation

1. In the absence of added ADP glutamine is transformed by pig kidney mitochondria to ammonium glutamate, which appears in the external medium. This reaction is stimulated only slightly by the addition of ADP, but under these conditions about 20% of the glutamate is oxidized to aspartate. 2. Externally added glutamate is oxidized to aspartate, and at about the same rate as glutamine. 3. The net rates of glutamine and glutamate influx into the intramitochondrial compartment are very slow. 4. The phosphate-dependent glutaminase activity of intact mitochondria is stimulated by the provision of energy. 5. The provision of energy also decreases the concentration of glutamate and increases the concentration of glutamine in the intramitochondrial compartment. These energy-linked changes in the glutamine and glutamate concentrations are of equal magnitude. 6. It is suggested that transport of glutamine and glutamate across the inner membrane of kidney mitochondria occurs by an obligatory exchange between the two metabolites, and is electrogenic. The existence of an electrogenic glutamine-glutamate anti-porter is proposed.     

miR-137 inhibits glutamine catabolism and growth of malignant melanoma by targeting glutaminase

Glutamine catabolism is considered to be an important metabolic pathway for cancer cells. Glutaminase (GLS) is the important rate-limiting enzyme of glutamine catabolism. miR-137 functions as a tumor suppressor in many human malignant tumors. However, the role and molecular mechanism of miR-137 and GLS in malignant melanoma has not been reported. In this study, we showed that miR-137 was decreased in melanoma tissue, and the low miR-137 level and high GLS expression are independent risk factor in melanoma. miR-137 suppressed the proliferation and glutamine catabolism of melanoma cells. GLS is crucial for glutamine catabolism and growth of malignant melanoma. We also demonstrated that miR-137 acts as a tumor suppressor in melanoma by targeting GLS. This result elucidates a new mechanism for miR-137 in melanoma development and provides a survival indicator and potential therapeutic target for melanoma patients.     

Glutamine synthetase is a genetic determinant of cell type-specific glutamine independence in breast epithelia

Although significant variations in the metabolic profiles exist among different cells, little is understood in terms of genetic regulations of such cell type-specific metabolic phenotypes and nutrient requirements. While many cancer cells depend on exogenous glutamine for survival to justify the therapeutic targeting of glutamine metabolism, the mechanisms of glutamine dependence and likely response and resistance of such glutamine-targeting strategies among cancers are largely unknown. In this study, we have found a systematic variation in the glutamine dependence among breast tumor subtypes associated with mammary differentiation: basal- but not luminal-type breast cells are more glutamine-dependent and may be susceptible to glutamine-targeting therapeutics. Glutamine independence of luminal-type cells is associated mechanistically with lineage-specific expression of glutamine synthetase (GS). Luminal cells can also rescue basal cells in co-culture without glutamine, indicating a potential for glutamine symbiosis within breast ducts. The luminal-specific expression of GS is directly induced by GATA3 and represses glutaminase expression. Such distinct glutamine dependency and metabolic symbiosis is coupled with the acquisition of the GS and glutamine independence during the mammary differentiation program. Understanding the genetic circuitry governing distinct metabolic patterns is relevant to many symbiotic relationships among different cells and organisms. In addition, the ability of GS to predict patterns of glutamine metabolism and dependency among tumors is also crucial in the rational design and application of glutamine and other metabolic pathway targeted therapies.     

Alteration of Mitochondrial Proteome Due to Activation of Notch1 Signaling Pathway

The Notch signaling pathway, a known regulator of cell fate decisions, proliferation, and apoptosis, has recently been implicated in the regulation of glycolysis, which affects tumor progression. However, the impact of Notch on other metabolic pathways remains to be elucidated. To gain more insights into the Notch signaling and its role in regulation of metabolism, we studied the mitochondrial proteome in Notch1-activated K562 cells using a comparative proteomics approach. The proteomic study led to the identification of 10 unique proteins that were altered due to Notch1 activation. Eight of these proteins belonged to mitochondria-localized metabolic pathways like oxidative phosphorylation, glutamine metabolism, Krebs cycle, and fatty acid oxidation. Validation of some of these findings showed that constitutive activation of Notch1 deregulated glutamine metabolism and Complex 1 of the respiratory chain. Furthermore, the deregulation of glutamine metabolism involved the canonical Notch signaling and its downstream effectors. The study also reports the effect of Notch signaling on mitochondrial function and status of high energy intermediates ATP, NADH, and NADPH. Thus our study shows the effect of Notch signaling on mitochondrial proteome, which in turn affects the functioning of key metabolic pathways, thereby connecting an important signaling pathway to the regulation of cellular metabolism.     

Present status and significance of the glutamine cycle in neural tissues

Evidence derived from various types of neurochemical experiments indicates that in the CNS of vertebrates there is a net flux of glutamate and GABA from neurons to astroglia and a metabolic conversion of these amino acids to glutamine. This glutamine is apparently released into the interstitial fluid and is in part taken up neurons and converted back into glutamate and GABA. This process, which is frequently referred to as “the glutamine cycle”, probably reflects the involvement of astrocytes in maintaining very low extracellular levels of glutamate and GABA, and the role of glutamine as a metabolic precursor of the transmitter pools of glutamate and GABA. The synthesis and release of glutamine by astrocytes may also reflect the role of these cells in ammonia detoxification. The quantitative importance of glutamine as a precursor of the neurotransmitter pools of glutamate and GABA has yet to be established. Other potential metabolic precursors such as α-ketoglutarate have not yet been evaluated adequately.     

The transport and metabolism of glutamine by kidney-cortex mitochondria from normal and acidotic rats.

1. The oxidation of glutamine by kidney-cortex mitochondria from normal and acidotic rats was not inhibited by avenaciolide, which did inhibit glutamate uptake and oxidation. The oxidation of glutamine by these mitochondria was always greater than that of glutamate. Direct measurements of the metabolism of [1-14C]glutamine in the presence of glutamate, and of [1-14C]glutamate in the presence of glutamine, demonstrated that the uptake and metabolism of external glutamate is insufficient to account for the observed rate of glutamine uptake and metabolism. Thus the postulated glutamine/glutamate antiport does not play a quantitatively important role in the metabolism of glutamine by renal mitochondria. 2. Rapid swelling of these mitochondria was observed in iso-osmotic solutions of L-glutamine and L-glutamyl-gamma-monohydroxamate but not in D-glutamine or L-isoglutamine (1-amido-2-aminoglutaric acid). Thus a relatively specific glutamine uniport exists in these mitochondria. 3. The utilization of glutamine was increased about 3-fold in mitochondria from chronically acidotic rats. Thus mitochondrial adaptations play an important part in the renal response to metabolic acidosis.     

Cationic amino acid transport across the blood-brain barrier is mediated exclusively by system y+

Cationic amino acid (CAA) transport is brought about by two families of proteins that are found in various tissues: Cat (CAA transporter), referred to as system y+, and Bat [broad-scope amino acid (AA) transporter], which comprises systems b0,+, B0,+, and y+L. CAA traverse the blood-brain barrier (BBB), but experiments done in vivo have only been able to examine the BBB from the luminal (blood-facing) side. In the present study, plasma membranes isolated from bovine brain microvessels were used to identify and characterize the CAA transporter(s) on both sides of the BBB. From these studies, it was concluded that system y+ was the only transporter present, with a prevalence of activity on the abluminal membrane. System y+ was voltage dependent and had a Km of 470 +/- 106 microM (SE) for lysine, a Ki of 34 microM for arginine, and a Ki of 290 microM for ornithine. In the presence of Na+, system y+ was inhibited by several essential neutral AAs. The Ki values were 3-10 times the plasma concentrations, suggesting that system y+ was not as important a point of access for these AAs as system L1. Several small nonessential AAs (serine, glutamine, alanine,and glycine) inhibited system y+ with Ki values similar to their plasma concentrations, suggesting that system y+ may account for the permeability of the BBB to these AAs. System y+ may be important in the provision of arginine for NO synthesis. Real-time PCR and Western blotting techniques established the presence of the three known nitric oxide synthases in cerebral endothelial cells: NOS-1 (neuronal), NOS-2 (inducible), and NOS-3 (endothelial). These results confirm that system y+ is the only CAA transporter in the BBB and suggest that NO can be produced in brain endothelial cells.     

Inhibition of glutamine synthetase in the mouse kidney: a novel mechanism of adaptation to metabolic acidosis

As part of a study on the regulation of renal ammoniagenesis in the mouse kidney, we investigated the effect of chronic metabolic acidosis on glutamine synthesis by isolated mouse renal proximal tubules. The results obtained reveal that, in tubules from control mice, glutamine synthesis occurred at high rates from glutamate and proline and, to a lesser extent, from ornithine, alanine, and aspartate. A 48 h, metabolic acidosis caused a marked inhibition of glutamine synthesis from near-physiological concentrations of both alanine and proline that were avidly metabolized by the tubules; metabolic acidosis also greatly stimulated glutamine utilization and metabolism. These effects were accompanied by a large increase (i) in alanine, proline, and glutamine gluconeogenesis and (ii) in ammonia accumulation from proline and glutamine. In the renal cortex of acidotic mice, the activity of phosphoenolpyruvate carboxykinase increased 4-fold, but that of glutamate dehydrogenase did not change; in contrast with what is known in the rat renal cortex, metabolic acidosis markedly diminished the glutamine synthetase activity and protein level, but not the glutamine synthetase mRNA level in the mouse renal cortex. These results strongly suggest that, in the mouse kidney, glutamine synthetase is an important regulatory component of the availability of the ammonium ions to be excreted for defending systemic acid-base balance. Furthermore, they show that, in rodents, the regulation of renal glutamine synthetase is species-specific.     

Specific gene expression of ATP-binding cassette transporters and nuclear hormone receptors in rat liver parenchymal,endothelial, and Kupffer cells

Hepatic cholesterol(ester) uptake from serum coupled to intracellular processing and biliary excretion are important features in the removal of excess cholesterol from the body. ATP-binding cassette (ABC) transporters play an important role in hepatic cholesterol transport. The liver consists of different cell types, and ABC transporters may exert different physiological functions dependent on the individual cell type. Therefore, in the current study, using real time PCR we compared the mRNA expression of ABC transporters and genes involved in the regulation of cholesterol metabolism in liver parenchymal, endothelial, and Kupffer cells. It appears that liver parenchymal cells contain high expression levels compared with endothelial and Kupffer cells of scavenger receptor class BI ( approximately 3-fold), peroxisome proliferator-activated receptor (PPAR)alpha and PPARgamma (8-20-fold), cholesterol 7alpha-hydroxylase A1 (>100-fold), and ABCG5/G8 ( approximately 5-fold). Liver endothelial cells show a high expression of cholesterol 27-hydroxylase, liver X receptor (LXR)beta, PPARdelta, and ABCG1, suggesting a novel specific role for these genes in endothelial cells. In Kupffer cells, the expression level of LXRalpha, ABCA1, and in particular ABCG1 is high, leading to an ABCG1 mRNA expression level that is 70-fold higher than in parenchymal cells. It can be calculated that 51% of the total liver ABCG1 expression resides in Kupffer cells and 24% in endothelial cells, suggesting an intrahepatic-specific role for ABCG1 in Kupffer and endothelial cells. Because of a specific stimulation of ABCG1 in parenchymal cells by a high cholesterol diet, the contribution of parenchymal cells to the total liver increased from 25 to 60%. Our data indicate that for studies of the role of ABC transporters and their regulation in liver, their cellular localization should be taken into account, allowing proper interpretation of metabolic changes, which are directly related to their (intra)cellular expression level.     

Expression of glutamine synthetase in macrophages.

We studied the expression of glutamine synthetase in liver macrophages (Kupffer cells, KCs) in situ and in culture. Glutamine synthetase was detectable at the mRNA and protein level in freshly isolated and short-term-cultured rat liver macrophages. Enzyme activity and protein content were about 9% of that in liver parenchymal cells. In contrast, glutamine synthetase mRNA levels in liver macrophages apparently exceeded those in parenchymal liver cells (PCs). By use of confocal laser scanning microscopy and specific macrophage markers, immunoreactive glutamine synthetase was localized to macrophages in normal rat liver and normal human liver in situ. All liver macrophages stained positive for glutamine synthetase. In addition, macrophages in rat pancreas contained immunoreactive glutamine synthetase, whereas glutamine synthetase was not detectable at the mRNA and protein level in blood monocytes and RAW 264.7 mouse macrophages. No significant amounts of glutamine synthetase were found in isolated rat liver sinusoidal endothelial cells (SECs). The data suggest a constitutive expression of glutamine synthetase not only, as previously believed, in perivenous liver parenchymal cells but also in resident liver macrophages.     

Amine metabolomics of hyperglycemic endothelial cells using capillary LC-MS with isobaric tagging

Intracellular amine metabolite changes were quantified from hyperglycemic human aortic endothelial cells (HAECs) as a model for macrovascular complications of diabetes. Amines were selectively tagged using the N-hydroxysuccinimide ester (NHS) based isobaric tag DiART (Deuterium isobaric Amine Reactive Tag), synthesized in house. DiART labeling improved chromatographic resolution of derivatized amines, resulted in 100-fold signal-to-noise enhancement in mass spectrometry (MS) analyses, and allowed multiplex quantification of four samples concurrently through tandem MS fragmentation. Targeted measurement of 31 DiART-tagged amines demonstrated the limits of detection below 10 nM/100 amol and averaged RSDs less than 5%. Examination of endothelial cells exposed to short-term hyperglycemia resulted in significant changes to alanine, proline, glycine, serine, and glutamine compared to osmotic controls. Discovery of proline elevation in hyperglycemic endothelial cells suggests a role of proline in hyperglycemia-mediated oxidative stress. Exposure of endothelial cells to high glucose for 7 days resulted in reduced cell number and significant changes to 21 amines relative to cell number. Prominent amine elevation from long-term hyperglycemia include aminoadipate as a sign of lysine breakdown through oxidative stress; cystathionine, hypotaurine, and proline indicating an antioxidant response; and glutamine/glutamate as substrate level activators of additional metabolic pathways. This report is the first investigation of amine changes to hyperglycemic endothelial cells and offers new insights into the pathophysiology of diabetic complications.