Mammary metabolism of amino acids

Precursors for milk protein synthesis

The arteriovenous net balance technique has been used to monitor mammary uptake of AA. The ratio of AA removal to milk protein secretion has been used to indicate limiting AA. Based on this comparison, it would appear that valine, leucine, isoleucine, arginine, lysine and threonine are not limiting since their extractions generally exceed milk protein outputs. In vivo data have demonstrated that leucine and lysine, which are extracted in excess, can be oxidized by the udder (Fig. 19.1; Oddy et ai, 1988; Bequette et ai, 1996a,b; Mabjeesh et ai, 2000). Thus, a portion of the carbon arising from these AA is unavailable for synthesis of nonessential AA, which for the most part are not extracted in adequate quantities. The amino-group is largely available and appears to be adequate or nearly adequate to cover non essential AA synthesis (Hanigan and Baldwin, 1994; Hanigan et ai, 2001b). By contrast, phenylalanine, methionine, threonine and his-tidine have been consistently observed to be extracted in amounts less than milk protein outputs (Mepham, 1982; Guinard and Rulquin, 1994; Metcalf et ai, 1996; Bequette et ai, 1999). Early observations by Mepham and Linzell (1966) and Bickerstaffe et ai (1974) suggested that circulating blood free AA were the principal sources of AA for casein synthesis. More recently, however, studies in vivo and in vitro seem to support the view that the deficiency of free AA uptake is made-up from extraction of non-free AA sources (i.e. peptides or proteins: Backwell et ai, 1994; Bequette et ai, 1994, 1999).

Despite the simplicity of the net uptake to milk output measurement, there are compounding errors associated with measurement of mammary blood flow, arteriovenous concentration differences and milk protein output (for reviews see Mepham, 1982; Bequette et ai, 1998) that add variability to the mammary balance data. In order to reduce these errors and acquire more reliable estimates of free AA removal by the mammary gland, the arteriovenous difference methodology has been improved (Bequette et ai, 1999; Mabjeesh et ai, 2000). These authors surgically ligated venous and arterial vessels that may contribute non-mammary derived AA, and blood was withdrawn as integrated samples over 1hour periods to coincide with the integrated milk protein yield and blood flow measurements. Even when these measures were applied, net uptake of methionine and phenylalanine were less then required.

Direct quantification of peptides in the circulation has proved difficult to assess. Crude methodologies have been employed based on deproteinization of plasma, molecular-weight exclusion followed by acid hydrolysis of the resulting peptide fractions, and comparison of the plasma free and liberated AA contents. These techniques are limited in accuracy and reproducibly, and require very sensitive quantification of AA by ion-exchange or other AA analysis instrumentation (i.e. 1-3 jjlM arteriovenous difference). By using this technique, the arteriovenous difference across the mammary gland of AA in the plasma pep-

tide fraction (<1500 Da) was found to be positive and small for histidine, alanine, leucine, proline and phenylalanine (Backwell et al., 1996). More compelling evidence derives from studies in vitro where replacement of free lysine or methionine with peptides containing these AA either maintained or increased milk protein synthesis by cultured mouse mammary tissue explants (Wang et ai, 1994, 1996; Pan et ai, 1996).

Stable isotope-labelling techniques have also been used to indirectly demonstrate that the mammary gland in vivo can use synthetic dipeptides for casein synthesis (Backwell et al., 1994) and that the contribution of peptides to casein synthesis is probably significant (Bequette et al., 1994, 1999; Backwell et al., 1996; Mabjeesh et al, 2000). By infusing 1 Relabelled glycylphenylalanine and glycyl-leucine close-arterial to the mammary gland of goats, Backwell et al. (1994) found greater (10-20%) incorporation of phenylalanine and leucine derived from the dipeptides than from the peripherally infused free form of the AA. By employing the precursor-product labelling methodology in lactating goats (days 45-253), peptides were found to contribute 0-20% of phenylalanine, 13-25% of tyrosine, and 0-18% of methionine in casein (Fig. 19.1, Bequette et al., 1994, 1999; Backwell et al., 1996). Peptides were also found to contribute to the uptakes of lysine (4-16%) and variable (0-15%) amounts to leucine (Mabjeesh et al., 2000), which seems surprising since these AA are almost always extracted in excess. Chen et al. (1999) measured the mRNA abundance of the peptide transporter PepTl in tissues from lactating cows and, although the sheep probe hybridized to mRNA in the gastrointestinal tissues, no hybridization occurred in the mam-man; gland. However, close inspection of the data from Backwell et al. (1994) suggests that the peptides may have also been hydrolysed at the cell surface to free AA prior to uptake. This mechanism appears to be compatible with amino-peptidase N expression in mammary tissue of lactating goats (Mabjeesh et al., 2001). Future research will need to examine peptide use by the udder in more detail to determine whether peptide use is a significant physiological phenomenon, and one that can be manipulated to increase milk protein synthesis.

Metabolism and roles of amino acids

It has long-been recognized that the mammary gland is a site of extensive synthesis and degradation of AA. Tracer studies with the perfused mammary gland (Verbeke et ah, 1968, 1972; Roets et ai, 1974), tissue explants and cell culture systems (Jorgensen and Larson, 1968) have been instrumental in identifying many of the metabolic transformations of AA. These pathways are the same, or very similar, to pathways that occur in other tissues. However, these may be more prominent for the mammary gland with net uptake of non-essential AA by the udder far less than required for milk protein synthesis, compared to muscle tissue where a stoichiometric relationship mostly exists. In recent years, a wider range of radio- and stable-isotope labelled AA has become available at an affordable price, which has led to a number of metabolic studies on the lactating cow and goat mammary gland in vivo (see Fig. 19.1). Traditionally, AA and their metabolism have been categorized according to the balance between net arteriovenous uptake and milk casein-AA output. Excess uptake is assumed to represent catabolism, and, for this reason, those AA taken up in excess are usually not considered to be limiting for milk protein synthesis. This point has also been argued on the basis that the Km values for activation of acyl-tRNA synthetases are 100-fold lower than those for catabolic enzymes (1 x 10~6 vs. 1 x 10-4; Rogers, 1976) and, therefore, catabolism should only proceed once the acyl-tRNA have become fully charged (DePeters and Cant, 1992). This argument assumes that none of the products of AA catabolism serve as rate-limiting substrates or regulators for protein synthesis. It also assumes homogeneity of intracellular concentrations, particularly at the sites of tRNA loading and AA degradation, which may not be the case.

Leucine, valine, and isoleucine are catab-olized by mammary cells along pathways found in other tissues to yield organic acids (keto and iso acids, propionate, acetate and citrate), carbon skeletons for non-essential AA synthesis, and C02 (Chapter 4). The first reaction of the branched-chain AA catabolic pathway involves transamination (Fig. 4.13). Substantial transamination of branched-chain AA occurs in the mammary gland of the goat where reamination of the keto acid represents 20-50% of leucine (Bequette et al, 2002) and 10% of valine (Roets et al, 1974) flux. In theory, the supply of these AA to the udder can be supplemented by removal of the branched-chain keto acids from blood; however, net fluxes of the keto acid of leucine represent <3% of leucine net flux (Bequette et al., 1996b). The second, rate-limiting step is decarboxylation of the respective keto acid, catalysed by the branched-chain keto acid dehydrogenase (EC This dehydrogenase is shared by all branched-chain AA and methionine (Harper et al., 1984). Regulation of branched-chain keto acid dehydrogenase is dependent on phosphorylation status. When insulin levels or tissue sensitivity are high or when branched-chain AA concentrations are low (Randle et al, 1984), the enzyme is inactive (phosphorylated) and catabolism is inhibited. In lactating goats, insulin depresses mammary leucine oxidation and transamination, but this appears to be overridden by arterial leucine supply (Bequette et al., 2002). Net catabolism of the branched-chain AA results in a contribution of amino-groups to non-essential AA synthesis.

Oxidation of leucine by the udder is lower (0.08 vs. 0.34 of leucine uptake) in early lactation goats yielding 4.3 kg milk day-1 than in late lactation goats yielding 1.5 kg milk day-1 (Oddy et al, 1988). In the dairy cow, both the fractional (0.047 vs. 0.136) and absolute (5 vs. 18 g leucine day-1) rates of leucine oxidation are increased by dietary protein supplementation (Bequette et al, 1996b). These studies suggest an inverse relationship between milk protein output and leucine catabolism. This association was dismissed by Bequette et al (1996a) when they showed that leucine oxidation could be reduced (20% vs. 3%) substantially without affecting milk protein synthesis. A more tenable relationship may be one where AA oxidation is a function of the differential between supply and demand.

Although lysine is often thought to be first- or second-limiting on most maize-based dairy rations, lysine presents an anomaly because it is almost always taken up in excess by the udder. From 16 to 34% of lysine is oxidized by the mammary gland of lactating goats (Mabjeeshet et al, 2000), and furthermore, levels of oxidation are higher in late than in early lactating animals, similar to observations for leucine (Oddy et al, 1988). Lysine is ketogenic (Fig. 4.14), but it is not known whether its oxidation by the udder serves to provide ketogenic substrate.

Along with lysine, methionine is often considered to be one of the limiting AA of maize-based rations, particularly when heated soybeans make up most of the protein source. In addition to incorporation into protein, methionine is involved in multiple pathways leading to synthesis of phospholipids, carnitine, creatine (Fig. 4.8) and polyamines (Fig. 4.3). At the same time, methionine provides methyl groups for a number of transmethylation reactions involved in regulation of DNA activity and oncogene status, and it provides sulphur for cysteine synthesis. In goats, 28% of the methionine methyl group contributes to the plasma choline pool, and 10% is irreversibly lost through oxidation (Emmanuel and Kelly, 1984). One consequence of this catabolism is the synthesis of cysteine. In the goat udder, 10% of methionine-sulphur contributes to cysteine synthesis (Lee et al, 1996).

Attempts to increase milk protein yield by increasing the supply of methionine by addition of rumen-protected methionine have given mixed results. One side effect of providing excess methionine is that it is one of the most toxic AA. As an alternative, 4-thiomethyl-2-hydroxybutanoic acid (HMB), the hydroxy analogue of methionine, has been considered. The HMB does not appear to be toxic and since it has no known mammalian transporter it readily diffuses into tissues. The analogue does not appear to be removed by the gut and liver tissues to the same extent as methionine, and several tissues in growing sheep are able to convert HMB into methionine via transamination (Wester et al, 2000). In dairy cows, 20% of methionine in casein was derived from HMB when infused (Lobley and Lapierre, 2001).

Arginine is extracted in the greatest quantities relative to milk protein outputs (150-200% in excess). Arginine has other metabolic functions in addition to being a precursor for protein synthesis. Recently, its role as a precursor of nitric oxide (Reaction [4.11]) has received attention because of the potential role of nitric oxide in regulating mammary tissue nutrient perfusion (Lacasse et al., 1996). Mammary vascular endothelial cells and the epithelium lining alveoli and ducts exhibit nitric oxide synthase III (EC activity. Thus, secretory cells may be capable of regulating their own local nutrient environment by altering arginine catabolism. The mammary gland possesses a partial urea cycle where an intermediary role for arginine and other intermediates (ornithine, citrulline) of the cycle may be important in mammary function. In rat mammary tissue, arginase (EC, which hydrolyses arginine to form ornithine and urea (Fig. 4.2), increases threefold in activity in lactation (Jenkinson and Grigor, 1996). The activity of this pathway may be important for the synthesis of proline. In the sheep and goat udder, citrulline, arginine and ornithine contribute -20% to casein-proline synthesis (Verbeke et al., 1968; Roets et al., 1974). This pathway may serve to provide an alternative and perhaps critical supply of proline, an AA that is typically not extracted in adequate quantities for casein synthesis. The synthesis of proline from arginine may be inherently limited, however. In bovine mammary tissue, the key enzyme in this pathway, ornithine-S-transferase (EC, has a high Km (8.4 mM), which would require high intracellular concentrations of ornithine to maintain maximal rates of conversion through this pathway (Basch et al., 1995). Alternatively, the requirement for de novo synthesis of proline may restrict the availability of arginine for other functions (e.g. polyamine synthesis). None the less, evidence in vitro (Harduf et al., 1985) and in vivo (dairy cows; Bruckental et al., 1991) seem to suggest that there may be constraints either in mammary intracellular arginine supply or in the conversion of arginine into proline. Unfortunately, since the report by Bruckental et a I. (1991), there have been no follow-up studies to confirm their observed responses in milk production to supplemental proline.

Observations that cultured bovine mammary tissues do not require tyrosine to synthe size casein suggested that sufficient tyrosine could be generated from phenylalanine via the phenylalanine hydroxylase (EC pathway (Chapter 4) (Jorgensen and Larson, 1968). Studies with perfused sheep udder estimated that 10% of casein-tyrosine could be derived via phenylalanine hydroxylation (Verbeke et a I., 1972). Recent tracer studies in lactating goats (Bequette et al., 1999) have estimated that 5-9% of phenylalanine is converted into tyrosine. On a whole body basis, however, 10-18% of phenylalanine is converted into tyrosine, most of this probably occurring in the liver. In the whole body and the mammary gland, this conversion was increased by phenylalanine supply.

Was this article helpful?

0 0
Diabetes Sustenance

Diabetes Sustenance

Get All The Support And Guidance You Need To Be A Success At Dealing With Diabetes The Healthy Way. This Book Is One Of The Most Valuable Resources In The World When It Comes To Learning How Nutritional Supplements Can Control Sugar Levels.

Get My Free Ebook

Post a comment