Amino acids as energy substrates: amino-nitrogen sparing

One of the most significant differences between what our ancestors ate (i.e. the diet for which our digestive system and metabolic energy partitioning are geared and optimised) and the present-day diet is, in addition to the overwhelming abundance of lipid, the constant presence of protein, with a relatively high proportion of high-quality protein. The tandem lipid–animal protein is substituting progressively complex carbohydrate–low-quality plant protein as the main dietary staple. The proportion of protein energy v. total energy intake is not too much different today from the ancestral diet⁽Reference Frassetto, Schloetter and Mietus-Synder¹⁴⁾, but the total amount of energy (after correcting for exercise) is higher, as is the proportion of essential amino acids⁽Reference Eaton and Eaton¹⁵⁾, whilst the relationship between dietary amino acids v. glucose derived from dietary sources tends to change as a direct consequence of the substitution of starches by fats⁽Reference Brooks and Lampi¹⁶⁾.

Our starvation resistance-prone mechanisms of adaptation preserve the use of amino acids as energy substrate when there is sufficient energy in the gut⁽Reference Oke and Loerch¹⁷⁾. In addition, both amino acids and glucose are spared when (if) the availability of lipid is high⁽Reference Hart, Wolf and Zhang^18, Reference Kawaguchi, Osatomi and Yamashita¹⁹⁾. In consequence, a diet rich in energy and lipids, with a sizeable proportion of easily digestible protein, rich in essential amino acids, will necessarily result in difficulties to process and oxidise the amino acid surplus, since we are metabolically conditioned to actively preserve them. However, under excess-energy diets (including protein), it is no longer necessary to retain so much amino-N and essential amino acids. Amino acids are used for energy when in relative excess⁽Reference Stoll, Henry and Reeds^20, Reference Wolfram and Scharrer²¹⁾, but the even higher availability of energy from other sources strongly limits our metabolic machinery to do so⁽Reference Esteve, Rafecas and Fernández-López²²⁾.

Amino acid catabolism tends to retain 2-amino-N, largely because most amino acid hydrocarbon skeletons are oxidised after transamination (typically to 2-oxoglutarate/glutamate). However, a few amino acids yield directly ammonia (Table 1). A quantitative analysis of several common food proteins shows that, as expected, the theoretical direct yield of 2-amino-N is much higher than that of direct ammonium production (Table 2). Thus, the parity needed to synthesise our main N excretion product, urea, requires (in a dietary equilibrium) the additional conversion of a varying proportion of dietary 2-amino-N to ammonium to reach the required (1:1) balance. The ratio amino-N:ammonium-N in most dietary proteins is close to 2 (Table 2), which leaves a wide margin for preservation of N under conditions of starvation, but in the end requires the mineralisation of about half of all amino-N to ammonia under normal feeding conditions.

Table 1 Main amino acid (AA) catabolism pathways in man (adapted from Ferrer-Lorente et al. ⁽Reference Ferrer-Lorente, Fernández-López and Alemany¹³⁰⁾)*

THF, tetrahydrofolate; [trans to], transamination to; UC, urea cycle; AcCoA, acetyl-coenzyme A; [KC], Krebs or tricarboxylic acid cycle; pyr, pyruvate; [1-C], one-carbon pathways (essentially via THF); succ, succinate; OAA, oxaloacetate; [dehydrogen to], dehydrogenation to; [oxid to], oxidation to; PNC, purine nucleotide cycle; [BO], β-oxidation pathway; AcAc, acetoacetate; AcAcCoA, acetoacetyl-coenzyme A.

* All values are estimations based on the scant data available from human subjects and other mammals; flow of amino acid catabolism via a given path depends largely on the size of amino-N pool, energy availability, metabolic needs and the relative abundance of the amino acid (essential amino acids).

Table 2 Amino acid (AA) content of a number of common food proteins, showing the -NH₂:NH₃ ratio that would be theoretically generated from the complete oxidation in the body of its constituent amino acids*

* The data have been calculated from standard protein amino acid composition tables⁽¹³¹⁾ and the yield in free ammonium and transaminable 2-amino groups resulting from the complete catabolism of these amino acids (Table 1). Since urea excretion requires equal proportions of amino and ammonium, any -NH₂:NH₃ ratio above 1·00 represents a relative excess of amino groups, which may be further converted to ammonium via the purine nucleotide cycle or (in certain tissues and physiological conditions) by glutamate dehydrogenase. The ammonium yield of the proteins listed may be underestimated, since most of the data gathered give a combined Glu + Gln (Glx) value in the overall analyses. From the analysis of whole rat protein⁽Reference Rafecas, Esteve and Fernández-López¹³²⁾ in which amide-N was analysed separately, we assumed, conservatively, and for the sake of these calculations only, that half the Glx values corresponded to Gln and half to Glu; this correction has been included in the calculations and is reflected in the data shown in the Table.

When active (exercise), muscle uses most of the body energy available: its standard feed is glucose, but blood lipids (fatty acids) limit glucose uptake and favour fatty acid oxidation (insulin resistance)⁽Reference Cahová, Vavrínková and Kazdová²³⁾. Some amino acids are oxidised in muscle (especially branched-chain⁽Reference Shinnick and Harper²⁴⁾) in the postprandial state to save glucose, but excess energy hampers the process and its timing, since in fact there is no real scarcity of glucose. Amino-N conversion into ammonia is largely done in the liver and muscle through the purine nucleotide cycle⁽Reference Goodman and Lowenstein²⁵⁾, and its operation is both linked to active glycolysis⁽Reference Tornheim and Lowenstein²⁶⁾ and increased AMP levels (i.e. low ATP availability, partly compensated by the action of adenylate kinase)⁽Reference Atkinson and Walton²⁷⁾; thus, under excess energy availability, the cycle is largely idle. Glutamate dehydrogenases play a key role in ammonium metabolism in micro-organisms⁽Reference Núñez de Castro, Arias-Saavedra and Machado²⁸⁾, and in the muscle of invertebrates⁽Reference Batrel and Regnault²⁹⁾. However, Their role in ammoniagenesis in mammalian muscle is limited, because of the predominance of the purine nucleotide cycle⁽Reference Lowenstein³⁰⁾ in this role, and the low presence of the enzyme compared with the liver⁽Reference Arola, Palou and Remesar³¹⁾, unaltered under starvation⁽Reference Remesar, Arola and Palou³²⁾. In the liver (and kidney), the activity of glutamate dehydrogenase is considerable⁽Reference Arola, Palou and Remesar³¹⁾, but its function is clearly that of resynthesising glutamate from 2-ketoglutaratre and excess ammonia, as determined by direct studies and the analysis of its kinetic constrictions⁽Reference McGivan and Chappell^33–Reference Bailey, Bell and Bell³⁵⁾.

Equilibrium between amino-nitrogen and ammonia for urea synthesis

Thus, in muscle there is no other major way to produce ammonia than the purine nucleotide cycle⁽Reference Lowenstein³⁰⁾. In consequence the muscle cannot use amino acids as an energy source in significant amounts⁽Reference Flanagan, Holmes and Sabina³⁶⁾ and to use their N to produce and release glutamine. This is an important question, since glutamine is the main form of blood transport of ammonia, towards the splanchnic bed⁽Reference Marliss, Aoki and Pozefsky³⁷⁾, i.e. intestine and kidney; there, glutaminases free the ammonia again for its ultimate disposal as urea⁽Reference Wu³⁸⁾, or as urinary ammonium ion⁽Reference Good and Burg³⁹⁾. Lower muscle synthesis of glutamine results, then, in lower splanchnic synthesis of carbamoyl-P, insufficient to maintain an adequate flow of urea synthesis (Fig. 1). Alternative sources of ammonium, such as the microbiota⁽Reference Karasawa and Nakata^40, Reference Wrong and Vince⁴¹⁾, which is in part derived from glutamine⁽Reference Anderson, Bennett and Alleyne^42, Reference Weber, Veach and Friedman⁴³⁾, and the direct ammoniagenic amino acids cited above (serine, threonine, glycine), help maintain a steady albeit diminished rate of urea synthesis in the intestine–liver system, a rate insufficient to cope with the excess 2-amino-N pool generated by the diet and limited amino acid disposal.

Fig. 1 Effect of excess dietary lipid on the main paths of N catabolism, driving to a decrease in the operation of the urea cycle because of lack of conversion of 2-amino-N to ammonium.

High amino acids in conjunction with high energy availability can generate a paradoxical scarcity of ammonia, retaining a large and unshrinkable pool of amino-N because the mechanisms that protect its conversion to ammonia remain unaffected, and are both efficient and effective (and potentially crippling). In the metabolic syndrome (and in general, in energy-rich feeding), urea synthesis is decreased⁽Reference Barber, Viña and Viña⁴⁴⁾, but there is not – either – a massive accumulation of body-N⁽Reference Esteve, Rafecas and Remesar⁴⁵⁾. Amino acids tend to be preserved in spite of excess energy availability⁽Reference Serra, Gianotti and Palou⁴⁶⁾, but in any case, the excess N is eventually lost, albeit not in the canonical way of urea formation⁽Reference Esteve, Rafecas and Remesar⁴⁷⁾. A small but significant proportion of N is excreted as N₂ gas⁽Reference Costa, Ullrich and Kantor^48, Reference Cissik, Johson and Rokosch⁴⁹⁾ by means of, so far, unknown pathways. In addition there is an increased (but relatively small) loss of dietary amino-N in the form of urinary nitrate and nitrite⁽Reference Green, Ruiz de Luzuriaga and Wagner⁵⁰⁾. Obligatory N losses also include urinary losses of uric acid (from purine catabolism⁽Reference Sutton, Toews and Ward⁵¹⁾), creatinine and small proportions of peptides and amino acids, as well as the ammonium ion, excreted by the kidney (especially in acidosis)⁽Reference Desir, Bratusch-Marrain and DeFronzo⁵²⁾. Small amounts of ammonium may be also excreted by the lungs⁽Reference Robin, Travis and Bromberg⁵³⁾.

Muscle also accumulates fat, near mitochondrial clusters (C Cabot, K Pouillot, S Roy, MM Romero, R Vilà, MM Grasa, M Esteve, JA Fernández-López, M Alemany and X Remesar, unpublished results) and adapts itself to the utilisation of this main substrate (as well as to glucose, but to a lower extent)⁽Reference Turner, Bruce and Beale^54, Reference Lam, Hatznikolas and Weir⁵⁵⁾. Exercise facilitates the massive utilisation of energy and streamlines the oxidation of fats⁽Reference Bruce, Thrush and Mertz⁵⁶⁾, but also restores in part the production of ammonium via the purine nucleotide cycle⁽Reference Graham, Bangsbo and Saltin⁵⁷⁾, thus increasing the flow of glutamine to the splanchnic bed. However, a large proportion of glucose, lipids and amino acids can not be taken up by any of the above cited systems, leaving them unused and in high serum concentrations, waiting for their storage as fat in the last-recourse energy pool: white adipose tissue.

The nitric oxide pathway

NO^∙ is synthesised from arginine by NO^∙ synthases, yielding citrulline⁽Reference Moncada, Palmer and Higgs⁵⁸⁾. Excess N availability increases the synthesis of ornithine⁽Reference Mouillé, Robert and Blachier⁵⁹⁾, including the intermediate step of acetyl-glutamate synthesis⁽Reference Tujioka, Lyou and Hirano⁶⁰⁾, which is also a key regulator of carbamoyl-P synthase 2, and thus also participates in the regulation of ammonium disposal⁽Reference Saheki, Ohkubo and Katsunuma⁶¹⁾. In consequence, higher amino-N levels may increase those of arginine, irrespective of low carbamoyl-P (i.e. low ammonium) availability, shunting the NO^∙ cycle from arginine to citrulline and leaving out ornithine (and the synthesis of urea) (Fig. 2 (a) and (b)) ⁽Reference Moncada and Higgs⁶²⁾. In cells that do not have a fully operative urea cycle, the eventual regulation is even easier since it is largely dependent on arginine availability⁽Reference McCall, Boughton-Smith and Palmer⁶³⁾.

Fig. 2 Possible mechanism of activation of the NO^∙ shunt under high energy availability–low ammonium production. (a) Urea cycle function under full operation, i.e. enough ammonium to produce carbamoyl-P and an adequate supply of 2-amino-N through aspartate. (b) Enhancement of the operation of the NO^∙ shunt of the urea cycle under limited supply of ammonium (in the form of carbamoyl-P), but maintained supply of 2-amino-N through aspartate. Pi, inorganic phosphate; PPi, inorganic pyrophosphate.

It may be expected, then, that under high energy and amino-N availability, the low ammonia concentrations⁽Reference Herrero, Angles and Remesar⁶⁴⁾ can not sustain an effective excretion of N through the urea cycle⁽Reference Roig, Esteve and Remesar⁶⁵⁾, indirectly favouring an increased activity of the NO^∙ synthesis shunt. The excess NO^∙ in blood vessels (derived from the activity of erythrocyte and endothelial NO^∙ synthases)⁽Reference Kleinbongard, Schultz and Rassaf⁶⁶⁾ may initially raise the blood flow, at least locally, increasing the availability of oxygen and substrates to the surrounding cells⁽Reference Sureda, Tauler and Aguiló⁶⁷⁾.

NO^∙ is highly reactive and interacts with specific proteins, such as guanylate cyclase⁽Reference Arnold, Mittal and Katsuki⁶⁸⁾, increasing the production of cyclic GMP which activates protein kinase G (PKG)⁽Reference Francis, Busch and Corbin⁶⁹⁾ which, in turn, relaxes the smooth muscle of small vessels and thus increases blood flow and lowers arterial tension⁽Reference Lincoln, Komalavilas and Cornwell⁷⁰⁾. This is the main recognised function of NO^∙(Reference Sessa⁷¹⁾, but NO^∙ is also able to bind cysteine residues of other proteins, such as protein kinase A (PKA)⁽Reference Ferro, Coash and Yamamoto⁷²⁾, which may induce a phantom adrenergic stimulation (i.e. without the intervention of catecholamines or cAMP)⁽Reference Burgoyne and Eaton⁷³⁾.

Cytochrome c also efficiently oxidises NO^∙ to nitrite⁽Reference Torres, Sharpe and Rosquist⁷⁴⁾. Most of the NO^∙, however, rapidly reacts with oxyhaemoglobin, eventually oxidising NO^∙ to nitrate⁽Reference Gow, Luchsinger and Pawloski⁷⁵⁾. Other highly reactive NO_x compounds, such as peroxynitrite⁽Reference Huie and Padmaja⁷⁶⁾, are formed by further oxidation with reactive oxygen species. Part of these nitrogen oxides react with proteins, fatty acids and other compounds yielding nitro-derivatives⁽Reference Trostchansky and Rubbo^77, Reference Rubbo and Radi⁷⁸⁾, often short-lived, but which can cause permanent structural changes⁽Reference Jain, Siddam and Marathi⁷⁹⁾.

Nitrite and other forms of nitrogen excretion

In the obese, the overall production and levels of NO^∙ are increased⁽Reference Asl, Ghasemi and Azizi⁸⁰⁾, as is its loss in the air breathed⁽Reference Maniscalco, de Laurentiis and Zedda⁸¹⁾, but there is a marked decrease in the excretion of urea⁽Reference Barber, Viña and Viña⁴⁴⁾. A significant part of the difference in the N balance is made up of N₂ gas⁽Reference Esteve, Rafecas and Remesar^45, Reference Esteve, Rafecas and Remesar^47, Reference Cissik, Johnson and Hertig⁸²⁾. A possible source is the reaction of nitrite and free amino acids, which in an acidic medium yield N₂ gas and 2-hydroxyacids⁽Reference Schmidt⁸³⁾; this reaction has been described to occur in the stomach lumen⁽Reference Yoshida and Kasama⁸⁴⁾. However, this reaction can hardly explain the large discrepancies found in N balances. There must be another – larger – source of N₂ gas integrated in the amino acid metabolism, which so far has not been discovered. We can hypothesise the existence of an ‘emergence’ pathway, shunting the action of NO^∙ synthases towards the reaction of arginine with nitrite, yielding citrulline and N₂ gas under acidotic conditions. This way, nitrite, the main active product of NO^∙ synthesis, would be rapidly removed and, at the same time, the excess 2-amino-N would be decreased at the expense of aspartate-derived arginine guanido-N; unfortunately, no enzyme has been found (so far) able to carry out this reaction, which nevertheless is known to proceed spontaneously under low pH conditions⁽Reference Schmidt⁸³⁾.

Glucocorticoids may elicit counteractive actions⁽Reference Worrall, Misko and Sullivan⁸⁵⁾ to inhibit NO^∙ synthesis, but catecholamines increase its production⁽Reference Lin, Tsai and Huang⁸⁶⁾. It is unclear whether NO^∙ overproduction in the obese can be a consequence of leptin-related catecholamine vasoconstriction⁽Reference Hall⁸⁷⁾, a consequence of enhanced NO^∙ synthesis through activation of endothelial or inducible NO^∙ synthases⁽Reference Elizalde, Rydén and van Harmelen⁸⁸⁾, or a lower bioavailability of NO^∙ favouring its increased synthesis⁽Reference Blouet, Mariotti and Mathe^89, Reference Williams, Wheatcroft and Shah⁹⁰⁾.

Nitrite is considered a stabilised form of NO^∙(Reference Tsuchiya, Kanematsu and Yoshizumi⁹¹⁾, which can yield NO^∙ under hypoxic conditions by reacting with Hb⁽Reference Piknova, Keszler and Hogg^92–Reference Lundberg, Weitzberg and Gladwin⁹⁴⁾, thus helping increase blood flow to hypoxic areas⁽Reference Ingram, Pinder and Bailey⁹⁵⁾. Nitrite is also a source of NO^∙ in the alimentary canal⁽Reference Eckmann, Jaurent and Langford^96, Reference Lundberg, Weitzberg and Lundberg⁹⁷⁾; it is largely the product of reduction by the oral biota of nitrate secreted by salivary glands⁽Reference Chen, Ren and Lu⁹⁸⁾. Nitrite-derived NO^∙ also kills bacteria in the stomach⁽Reference Iijima, Fyfe and McColl⁹⁹⁾; in this acidic medium, nitrite reacts with free amino acids yielding N-nitroso-proline from arginine⁽Reference Ishibashi and Kawabata¹⁰⁰⁾, as well as N₂ as indicated above⁽Reference Yoshida and Kasama⁸⁴⁾.

The ‘obese’ microbiota⁽Reference Cani, Possemiers and van de Wiele^101, Reference Brignardello, Morales and Diaz¹⁰²⁾ is probably a consequence of this magnified effect of NO_x⁽Reference Dykhuizen, Frazer and Duncan¹⁰³⁾; changes in the gut microbial ecosystem and composition also influence the relationships with the host through modulation of the immune response⁽Reference Manco, Putignani and Bottazzo^104, Reference Vaarala, Atkinson and Neu¹⁰⁵⁾. The relative abundance of protein debris in the intestine, a consequence of diets rich in lipid and protein, together with relatively scarce fibre and polysaccharides, also affects the composition of the microbiota, increasing the share of amino acid-related catabolism in the process of formation of stool⁽Reference Harmon, Becker and Jensen^106, Reference Hughes, Magee and Bingham¹⁰⁷⁾. The resulting higher pH, and the production of amines through amino acid decarboxylation⁽Reference Hughes, Magee and Bingham¹⁰⁷⁾, higher proportions of amines, ammonium, as well as amine- and sulfide-related catabolites may also help induce the development of intestinal cancer⁽Reference Bingham, Pignatelli and Pollock¹⁰⁸⁾.

Health consequences of hampered nitrogen excretion in the obese

The main problem posed by this question is the almost absolute lack of information about the human patterns of N excretion in overnutrition, obesity and the metabolic syndrome. It has been found that a relative increase in dietary protein at the expense of carbohydrates facilitates the loss of weight⁽Reference Lee, Lee and Bae^109, Reference Pasiakos, Mettel and West¹¹⁰⁾, but we only know the short-term macroscopic changes; the dynamics of 2-amino-N under these conditions has not been studied.

We have mechanisms to adjust amino acid catabolism to their relative abundance with respect to glucose⁽Reference Oke and Loerch¹⁷⁾, but the large presence of lipids in the diet alters everything. Ketogenic diets favour the excretion of ammonium in the urine to counter the acidosis produced by ketone bodies⁽Reference Schloeder and Stinebaugh^111, Reference Simon, Martin and Buerkert¹¹²⁾, and increase liver gluconeogenesis from amino acids⁽Reference Sherwin, Hendler and Felig¹¹³⁾, but the problem of conversion of amino-N to ammonium remains. The possible negative effects of a few truly hyperproteic (i.e. not ketogenic) diets⁽Reference Metges and Barth¹¹⁴⁾, and their limited effect on body fat point both to a generalised inefficiency of the so-called ‘high-protein’ diets⁽Reference Johnstone¹¹⁵⁾ and support the relative danger of their uncontrolled application.

One of the most important aspects of amino acid metabolism is the synergistic complementarity of the roles of a number of peripheral organs, the liver and the rest of splanchnic bed organs⁽Reference Aikawa, Matsutaka and Takezawa^116, Reference Felig¹¹⁷⁾. Hyperproteic diets may induce changes in their roles in the absence of energy overload, i.e. under conditions of active amino acid catabolism⁽Reference Morens, Gaudichon and Fromentin¹¹⁸⁾. However, it is highly improbable that the finely adjusted inter-organ relationships could be maintained under the pressure of high-energy diets, as the low urea output seems to indicate; as a consequence the whole body is affected by an excess of 2-amino-N.

There are few data on human subjects supporting an increase in the synthesis of NO^∙ in high-energy availability conditions, except for an increased breath release of NO^∙(Reference Maniscalco, de Laurentiis and Zedda⁸¹⁾ and the consequent formation of nitrite and nitrate⁽Reference Kim-Shapiro, Schechter and Gladwin^93, Reference Lundberg, Weitzberg and Gladwin⁹⁴⁾. Perhaps the high levels of nitrite and the easy interconversion of nitrite and NO^∙(Reference Iijima, Fyfe and McColl^99, Reference Gladwin, Crawford and Patel¹¹⁹⁾, a powerful vasodilator⁽Reference Rees, Palmer and Moncada¹²⁰⁾, may be related to the ‘obesity paradox’, i.e. a decreased severity of the consequences of heart failure in the obese⁽Reference Gruberg, Weissman and Waksman^121, Reference Badheka, Rathod and Kizilbash¹²²⁾. The large presence of NO_x in the alimentary canal and its profound influence on the microbiota has to produce, necessarily, changes in their properties and functions: at least a different way to cope with unused substrates and different relationships with the immune system-controlled intestinal barrier. The latter may be related to the higher levels of circulating lipopolysaccharide observed in the metabolic syndrome⁽Reference Brun, Castagliuolo and di Leo¹²³⁾, also linked to the maintenance of low-key inflammation⁽Reference Cancello and Clément^124, Reference Matsuo, Hashizume and Shioji¹²⁵⁾ caused by increased intestinal bacteria activity⁽Reference Sabate, Jouet and Harnois¹²⁶⁾. These findings hint to the postulated excess 2-amino-N, in agreement with the higher availability of amino acids and energy to increase protein turnover⁽Reference Robinson, Jaccard and Persaud^127, Reference Yuile, Lucas and Olson¹²⁸⁾ and to maintain a fully functional immune system⁽Reference Dunca and Schmidt¹²⁹⁾ observed in the metabolic syndrome.