J. Lewis*

Jaroslav Heger*

A. J. Lewis*

Department of Animal Science, University of Nebraska, Lincoln, Nebraska, USA

Introduction

Among the 20 amino acids that constitute the primary structure of proteins, two, methionine and cysteine, contain a sulphur atom. Because of their interconversions, these two amino acids are usually classified together as sulphur- containing amino acids or sulphur amino acids.

Methionine, whose name is derived from its chemical name, 2-amino-4-(methyl- thiol)butyric acid, is nutritionally essential for all animal species. Cysteine, like methionine, is incorporated into proteins based on the genetic code, however, from a nutritional point of view cysteine is classified as conditionally dispensable in most animal species, including pigs (Table 1.1). Cysteine is unstable in solution and is read- ily oxidized to the dimer form, cystine. Thus when proteins are hydrolysed cystine is pro- duced, with the number of moles of cystine being equal to half the number of moles of cys- teine within the protein structure. For this rea- son, it is cystine that is normally considered in a nutritional context, and the term sulphur amino acids usually means methionine + cystine.

Cystine was first isolated from urinary bladder calculi and named from the Greek word (kystis), meaning bladder. The structures of methionine and cysteine are illustrated in Fig. 8.1, and the conversion of cysteine into cystine is shown in Fig. 8.2. The term ‘cyst(e)ine’ is used to refer to cysteine and/or cystine.

Analytical Difficulties

One of the challenges in reviewing the nutri- tion of methionine and cystine for pigs is the difficulty in analysis for these two amino acids.

As discussed by Williams (1994), methionine and cyst(e)ine undergo oxidation during the

‘standard method’ of hydrolysis. Substantial amounts of methionine and cyst(e)ine are lost.

To circumvent this, the sulphur amino acids must be protected before hydrolysis. The usual method is a controlled oxidation of methionine to methionine sulphone and cyst(e)ine to cys- teic acid (Schram et al., 1954). Performic acid is used to oxidize the amino acids and hydro- gen bromide is added as the reducing agent to destroy the excess performic acid when the oxidation is complete (Moore, 1963).

In addition, the complete removal of HCl from the sample is critical when lithium- based, ion-exchange chromatography is used. The resolution of the early eluted peaks, which include cysteic acid, aspartic acid, methionine sulphone, and threonine, is very sensitive to the pH of the injected sam- ple (Mondino et al., 1972). A small change in sample pH can change the shape and size of the aspartic acid peak and can also change the retention times of both the methionine sulphone and threonine peaks affecting their resolution (Pickering, 1989;

Grunau and Swiader, 1992).

© CAB International2003. Amino Acids in Animal Nutrition,

2nd edition (ed. J.P.F. D’Mello) 143

*E-mail address: alewis@unlnotes.unl.edu

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Analysis of methionine and cysteine in plasma and serum is also sensitive to the meth- ods used before the chromatographic analysis.

Plasma and serum should be deproteinized as soon as possible after collection because deproteinizing before freezing helps to prevent loss of methionine and cysteine (Stein and Moore, 1954; DeWolfe et al., 1967).

Difficulties in analysis for the sulphur amino acids make it crucial that analytical methods are described in research papers. Unfortunately, much of the older research, and some more current research, failed to use adequate analysis methods and therefore the results of experi- ments can be difficult to interpret.

Metabolic Conversions

The metabolic relationships between methio- nine and cysteine are well established (Fig.

8.3). Methionine can be activated by ATP to S-adenosylmethionine. This compound read- ily donates its methyl group to a wide variety of acceptors. The resulting compound, S- adenosylhomocysteine, is then hydrolysed to homocysteine and adenosine. Homocysteine is a key intermediate because it can be remethylated to methionine or can condense with serine to form cystathionine and then cysteine. An important feature of these con- versions is that the conversion of homocys- teine into cysteine is not reversible. The net effect of these metabolic pathways is that methionine can be converted into cysteine, but cysteine cannot be converted into methio- nine. Several of the steps in the activated methyl cycle (in which methionine is demethylated to homocysteine and homocys- teine is then remethylated to methionine) require B-vitamin coenzymes. Thus, there are important relationships between the sulphur amino acids and other nutrients.

Nutritional Essentiality

The first study in which one of the sulphur amino acids was added to the diet of pigs was described by Bell et al.(1950). These authors reported that the addition of methionine at 2.0 g kg¹to a semipurified diet in which soy- bean meal provided the sole source of protein improved biological value. The basal soybean meal diet contained 100 g kg¹ protein and 0.7 g kg¹ methionine, but the cystine con- tent of this diet was not given. The biological value of the methionine-supplemented diet and the weight gain of the pigs fed this diet were equal to those of pigs fed a whole egg protein diet with 100 g kg¹ protein and 2.7 g kg¹ methionine. This study was the first to show that methionine was an essential amino acid for pigs. Previous research had shown that methionine was essential for rats, mice, chicks and humans.

One year later, Shelton et al.(1951) fed a semipurified diet containing 210 g kg¹ protein, 1.0 g kg¹ methionine, and 0.1 g kg¹ cysteine to growing pigs. They supple- mented the diet with either methionine, cys- tine, or a mixture of the two amino acids.

Supplementation with 6.0 g kg¹ cystine

144 A.J. Lewis

C H

S H CH₂ N

H₂ C OHO C

H

S CH₂ N

H₂ C OHO

CH₂

CH₃

Methionine Cysteine

Fig. 8.1. Structures of methionine and cysteine.

C H

S H CH₂ N

H₂ C OHO

C

H S H

C H₂ N

H₂ C OHO

C H

S CH₂ N

H₂ C OHO

C

H S

C H₂ N

H₂ C OHO

Cystine Cysteine

Fig. 8.2. Conversion of two molecules of cysteine into one molecule of cystine.

Amino Acids - Chap 08 12/3/03 12:25 pm Page 144

increased weight gain from 43 to 163 g day¹, whereas supplementation with 5.0 g kg¹methionine increased weigh gain to 572 g day¹. Furthermore, supplementation with 2.0 g kg¹methionine and 3.0 g kg¹cystine was as effective as supplementation with 5.0 g kg¹ methionine or 5.0 g kg¹ methionine plus 6.0 g kg¹ cysteine. These results con- firmed the essentiality of methionine and placed cystine in the ‘conditionally dispens- able’ category. These early results indicated that the total sulphur amino acid requirement of pigs weighing approximately 20 kg was 6.0 g kg¹of the diet and that all of this could be provided by methionine or half could be provided by methionine and half by cystine.

Although these conclusions were based on very limited numbers of animals, and there was no evidence of statistical analysis, current estimates of sulphur amino acid requirements are remarkably similar to these early values.

Bioavailability of D-Methionine and

D-Cysteine

Unlike most other amino acids, which are produced commercially by fermentation, crys- talline methionine is produced by chemical synthesis. This has an important biological implication because whereas fermentation

yields only the natural L-isomer, chemical syn- thesis yields a racemic (50:50) mixture of D- and L-isomers (DL-methionine). D-Amino acids are not used by animals for protein synthesis or for other metabolic purposes. Therefore, any ingested D-methionine must be converted into L-methionine before it can be utilized.

The conversion (or inversion) consists of two steps: (1) oxidative deamination to the -keto acid (2-keto-4-(methylthiol)butyric acid) and (2) transamination of an amino group from gluta- mate. Most animals except primates readily convert D-methionine into L-methionine, and this is true for pigs. Although an early study indicated that the D-form was used less effec- tively than the L-form by very young pigs (Kim and Bayley, 1983), later studies indicated that

DL-methionine and L-methionine were nutri- tionally equivalent (Reifsnyder et al., 1984;

Chung and Baker, 1992a). The results of Chung and Baker (1992a) show equal uti- lization of D-, L- and DL-methionine by 10-kg pigs (Fig. 8.4). Commercial feed-grade methionine is in the DL-form.

In contrast to methionine, the D-isomer of cysteine does not have bioactivity.

Apparently, there is no metabolic pathway from D-cysteine to the -keto analogue of cys- teine and therefore neither D-cysteine nor D- cystine has biological activity (Baker, 1994;

Lewis and Baker, 1995).

Methionine-Cystine Relationships in Pig Nutrition 145

Methionine

Cysteine S-Adenosylmethionine

S-Adenosylhomocysteine Homocysteine

Cystathionine

Fig. 8.3. Metabolic pathways of sulphur amino acids.

Amino Acids - Chap 08 12/3/03 12:25 pm Page 145

Bioavailability of Methionine Analogues

In addition to DL-methionine, which is mar- keted in a 99% pure feed-grade form (Degussa, 2001), other sources of supplemen- tal methionine activity are available to the ani- mal feed industry. In particular, 2-hydroxy-4-(methylthiol)butyric acid (HMB), commonly known as methionine hydroxy ana- logue, has been manufactured in both solid and liquid forms. The solid consists of two moles of HMB bound to calcium by the two carboxyl carbons and generally contains 86%

HMB. The liquid contains 88% HMB, which exists in monomer (77%), dimer (17.2%), trimer (4%), and oligomer (1.8%) forms (Novus International, 2001). All HMB prod- ucts are 50% D-HMB and 50% L-HMB.

There has been considerable controversy about the biological value of HMB relative to

DL-methionine. The controversy has been fuelled by the commercial importance of methionine supplements, especially in poultry.

Although there has been a wide range of esti- mates, poultry seem to utilize HMB with lower molar efficiency than DL-methionine (see review by Lewis and Baker, 1995). In pigs, however, most, but not all, research sup- ports the conclusion that HMB and DL- methionine are equal sources of methionine

activity on a molar basis (Becker et al., 1955;

Urba´nczyk et al., 1981; Reifsnyder et al., 1984; Steinhart and Kirchgessner, 1985;

Roth and Kirchgessner, 1986; Chung and Baker, 1992a; Stockland et al., 1992; Knight et al., 1998; Römer and Abel, 1999).

Although, this conclusion continues to be challenged (Pack and Höhler, 2000), data of Chung and Baker (1992a; Fig. 8.4) and Knight et al. (1998; Fig. 8.5) provide clear illustrations of equal efficacy of DL-methionine and the liquid form of HMB.

Because HMB does not contain nitrogen, supplementation with HMB results in less excretion of urinary nitrogen than supplemen- tation with an equivalent amount of DL- methionine (Römer and Abel, 1999). This would obviously be advantageous when mini- mization of nitrogen excretion is important.

Sulphur Amino Acid Requirements

In pig diets based on cereal grains and oilseed meals, sulphur amino acids are usually the sec- ond, third or fourth limiting amino acids (Lewis, 2001). The other limiting amino acids are lysine, threonine and tryptophan. Because of this importance, numerous experiments have investigated the sulphur amino acid requirements of pigs. A comprehensive review

146 A.J. Lewis

0 100 200 300 400 500 600

Average daily gain (g)

a

b

c c c

c

Basal 0.25 g kg^–1

0.50 g kg^–1

0.50 g kg^–1

0.50 g kg^–1

0.57 g kg^–1

L-Met L-Met D-Met DL-Met DL-HMB

Fig. 8.4. Average daily gain of 10-kg pigs fed a basal diet (containing 1.9 g kg¹methionine and 10 g kg¹cystine) supplemented with various sources of methionine (Chung and Baker, 1992a). The amount of 0.57 g kg¹HMB (2-hydroxy-4-(methylthiol)butyric acid, also known as methionine hydroxy analogue) is equimolar to 0.5 g kg¹methionine. Bars without a common letter (a, b or c) differ (P<0.05).

Amino Acids - Chap 08 12/3/03 12:25 pm Page 146

of sulphur amino acid requirements was pub- lished by NRC (1998). A computer model is included that enables requirements to be calcu- lated for growing pigs with specific weights and lean growth rates or for sows with speci- fied levels of reproductive performance.

To adjust for differences in bioavailability in amino acids among different feedstuffs, the NRC computer model uses true (or standard- ized) ileal digestible amino acids. However, out- put is also provided in terms of apparent ileal digestible amino acids and also as total amino acids. The NRC requirements (on a true ileal digestible basis) for methionine + cystine decrease from 7.6 to 3.1 g kg¹of the diet, as pigs increase in weight from the 3- to 5-kg range up to the 80- to 120-kg range. This is an increased requirement from 1.9 to 9.5 g day¹over the same weight ranges. For sows, methionine + cystine requirements range from 3.1 to 3.3 g kg¹ (5.7–6.4 g day¹) during gestation and from 3.5 to 4.3 g kg¹ (13.9–26.0 g day¹) during lactation, depend- ing on the level of production.

Some additional research on sulphur amino acid requirements of pigs is available that was not included in the NRC review.

Kirchgessner et al. (1994a) reported that in 7- to 30-kg pigs the optimal ratio of methion-

ine + cystine:lysine depended on the dietary lysine concentration and thus the growth rates of the pigs. At the highest lysine concentra- tions fed (11 g kg¹) the optimal methionine + cystine:lysine ratio was 56–57%. This is identical to the NRC ratio for this weight range. In another experiment, Kirchgessner et al. (1994b) found that weight gain and feed efficiency of pigs weighing 20–60 kg were maximized when the total methionine content was 3.1 g kg¹(3.4 g kg¹of dry matter). For pigs weighing 60–95 kg, weight gains were maximized with 2.4 g kg¹ total methionine (2.6 g kg¹ of dry matter). These values are higher than the corresponding NRC values of 2.5 and 2.0 g kg¹, respectively. However the estimates of methionine + cystine require- ments (4.9 and 4.1 g kg¹) were somewhat lower than the NRC requirements (5.4 and 4.4 g kg¹). Obviously, this implies a discrep- ancy in the proportion of methionine + cys- tine requirement that can be provided by cystine. This issue will be discussed more in a later section.

Knowles et al. (1998) reported that for pigs from approximately 75 to 110 kg the ratio of total sulphur amino acids:lysine required was no greater than 47% to maxi- mize growth performance and carcass Methionine-Cystine Relationships in Pig Nutrition 147

0 100 200 300 400 500

Average daily gain (g)

Basal 0.50 g kg^–1

0.57 g kg^–1

1.00 g kg^–1

1.14 g kg^–1

DL-Met DL-HMB DL-Met DL-HMB

Fig. 8.5. Average daily gain of 4-kg pigs fed a basal diet (containing 2.3 g kg¹methionine and 4.8 g kg¹cystine) supplemented with DL-methionine or 2-hydroxy-4-(methylthiol)butyric acid (HMB) (Knight et al., 1998). The amounts of HMB (0.57 and 1.14 g kg¹) are equimolar to 0.5 and 1.0 g kg¹DL-

methionine. The contrasts basal vs. (0.50 g kg¹methionine + 0.57 g kg¹HMB) and (0.50 g kg¹ methionine + 0.57 g kg¹HMB) vs. (1.0 g kg¹methionine + 1.14 g kg¹HMB) were significant (P<

0.05); there was no difference between DL-methionine and HMB.

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muscling, although the ratio to minimize fat deposition was 65%. In comparison, the NRC value for 80–120 kg is 58%. Loughmiller et al. (1998) found that the apparent ileal digestible methionine requirement of gilts in the late finishing stages was 1.25 g kg¹ (≈3.0 g of apparent ileal digestible methionine day¹) or 25% of the ileal digestible lysine requirement. These values are all lower than the NRC requirements of 1.3 g kg¹, 4.1 g day¹, and 28% of lysine for apparent ileal digestible methionine.

Recently, Matthews et al. (2001c) deter- mined that the sulphur amino acid require- ment of pigs from 5 to 10 kg was 6.4 g kg¹ on an apparent ileal digestible basis. This is almost identical to the NRC requirement of 6.3 g kg¹.

Thus, some of these additional results suggest requirements higher than those of the NRC and some suggest lower requirements.

Overall, it seems that the NRC requirements are satisfactory, although there is some indica- tion that the requirements during late finishing stages may be lower than indicated by NRC.

Ileal Digestibility vs. Bioavailability

Batterham et al. (1993) found that ileal digestibility may not always provide a good estimate of methionine bioavailability, espe- cially for protein supplements that have been heat processed. The proportion of apparent ileal digestible methionine retained by pigs dif- fered among different protein sources. The proportions were 39% for cottonseed meal, 45% for meat and bone meal, and 47% for soybean meal. The authors concluded that in

‘heat-processed meals a considerable propor- tion of the methionine is absorbed in a form(s) that is (are) inefficiently utilized’.

Proportion of Sulphur Amino Acids That Can Be Provided by Cystine

An important question in the sulphur amino acid nutrition of animals is the extent to which cystine can meet the methionine + cystine requirements. Because methionine can be converted into cystine but there is no conver-

sion of cystine into methionine, it has been assumed that methionine can fulfil the need for both methionine and cystine but that cys- tine can only fulfil the cystine need and, there- fore, only a portion of the methionine + cystine requirement. This issue has important practical implications because most feedstuffs included in swine diets are higher in cystine than methionine (NRC, 1998). Thus, diets can be relatively high in total sulphur amino acids but low in methionine. In addition, it has been suggested that a high cystine content may increase the methionine requirement (Kirchgessner et al., 1994b). Although the question about the maximum proportion of total sulphur amino acids that can be provided by cystine seems simple at first sight, many issues have confounded estimates during the 50 years that this has been investigated.

Some of the issues are methodological such as inappropriate response criteria, underesti- mation of sulphur amino acid content of diets because of improper analysis, and differences in bioavailability of sulphur amino acids in dif- ferent feedstuffs (Chung and Baker, 1992b).

In addition, however, there are two fundamen- tal issues that can confound interpretation of results. First it is important to clarify whether the replacement of methionine with cystine is on a weight or molar basis. Because the mole- cular weight of methionine (149) is greater than that of cysteine (121), equal weights of these two amino acids provide only 81% as many moles of methionine as cysteine (121/149 = 0.81). Thus, on a weight basis, increasing the methionine:cysteine ratio pro- vides a decreasing number of moles of sulphur amino acids. Almost all estimates of the maxi- mum portion of the methionine + cystine requirement that can be provided by cystine have been expressed on a weight basis and have ignored this issue. The second issue is that the proportion of methionine + cystine that can be provided by cystine is much greater for maintenance than for new tissue accretion. Consequently, the cystine replace- ment value increases as a pig matures and maintenance becomes a larger proportion of total amino acid need.

An initial estimate of the proportion of methionine + cystine that could be provided by cystine was proposed by Shelton et al.

148 A.J. Lewis

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(1951). Based on their experiment, they con- cluded that the methionine requirement of 20- kg growing pigs was 6.0 g kg¹ in the absence of cystine and 3.0 g kg¹in the pres- ence of adequate or excess cystine (6.0 g kg¹ added cystine in their experiment). On the basis of these results, they concluded that

‘approximately 50% of the methionine can be replaced with cystine in the diet of the wean- ling pig’. Although this study made an impor- tant initial contribution, both the number of animals and the number of treatments was very limited. There was no titration of either the methionine + cystine requirement or vari- ous methionine:cystine ratios.

In later work, Curtin et al. (1952a,b) found that the methionine + cystine require- ment of 15-kg pigs was approximately 7 g kg¹ of the diet and that 3.8 g kg¹ cystine could replace a corresponding amount of methionine, suggesting that cystine could sup- ply ≥ 50% of the methionine + cystine requirement. Becker et al. (1955) reported that the methionine requirement of 10-kg pigs was 2.5 g kg¹in the presence of 1.7 g kg¹ cystine. From this, they concluded that cystine could provide 40% of the methionine + cys- tine requirement. Again, however, in both of these experiments there was no titration of various methionine:cystine ratios and there- fore no direct determination of the maximum proportion of cystine that could be utilized.

During the 1960s, two additional papers were published by the research group at the University of Illinois. Mitchell et al. (1968) measured the nitrogen balance of 10-kg pigs.

They studied methionine:cystine ratios from 96:4 to 30:70. Although there were no signifi- cant differences, there was a tendency for nitrogen balance to increase as cystine replaced methionine. This would be expected because of the increase in the moles of sul- phur amino acids added. Based on these results, the authors concluded that ‘cystine can replace at least 70% of the methionine need without decreasing nitrogen retention’.

Despite this conclusion, there was a 6.5%

reduction in nitrogen balance when the pro- portion of cystine was increased from 57 to 70%. In the following year, the same research group (Baker et al., 1969) reported similar results for the nitrogen balance of 11-kg pigs.

Nitrogen balance tended to increase as the proportion of sulphur amino acids provided by cystine increased from 26 to 66%, although none of the differences were signifi- cant. In two growth assays, however, weight gain was reduced when the proportion of cys- tine was increased from 56 to 66%, leading the authors to conclude that ‘regardless of assay procedure, cystine can provide at least 56% of the requirement for total dietary sul- phur amino acids’. Differences between the two types of assays were attributed to the fact that feed intake was equalized in the nitrogen balance study, whereas ad libitum access to feed was allowed in the growth experiments.

In a comprehensive series of experi- ments, German researchers have also investi- gated methionine–cystine relationships. Based on growth experiments with pigs in weight ranges 30–60 kg and 60–90 kg, Roth and Kirchgessner (1987) concluded that the maxi- mum proportion of sulphur amino acids that could be provided by cystine was 55%. The authors cautioned, however, that at higher performance levels the maximum permissible proportion of cystine may be lower than this.

In a subsequent paper, Roth and Kirchgessner (1989) tested methionine:cystine ratios from 36:64 to 64:36. Pig performance improved as the methionine:cystine ratio increased from 36:64 to 40:60 to 45:55. There were no fur- ther significant increases in performance as ratios were increased to 50:50, 55:45, 60:40, and 64:36. These data support their earlier research, indicating that cystine can provide up to 55% of the methionine + cys- tine requirement. However, using quadratic regression analysis, the authors found that peak performance was obtained when the methionine:cystine ratio was 55:45, implying that not more than 45% of the methionine + cystine requirement should be furnished by cystine.

Chung and Baker (1992b) used a purified diet (to avoid issues of different bioavailability of amino acids among different feedstuffs) to study methionine:cystine ratios in 10-kg pigs.

In two experiments, methionine:cystine ratios from 100:0 to 40:60 were examined. The authors concluded that no more than 50% of the methionine + cystine requirement could be supplied by cystine.

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