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1 Introduction: Muscle Wasting as the Main Feature of Cancer Cachexia

1 Introduction: Muscle Wasting as the Main Feature of Cancer Cachexia

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272



19. SULFUR AMINO ACIDS METABOLISM FROM PROTEIN SYNTHESIS TO GLUTATHIONE



4. The reactions of the cycle itself fulfill three additional requirements: (1) TM reaction; (2) the recycling of

methyltetrahydrofolate; and (3) the catabolism of choline (betaine) via RM.

5. Cysteine can spare methionine in only one of these functions; the synthesis of cysteine and its derivatives by

means of TS.

6. The residual methionine requirement after cysteine supplementation represents the need for protein synthesis,

the obligatory synthesis of cystathionine (if relevant) and methionine used in the process of RM secondary to

inefficient conservation (since these two enzymes are utilized in Hcy conservation).

The methionine-sparing effect of cysteine is based on the redistribution of Hcy between competing reactions,

notably an increase in RM relative to TS. While the absolute rates of RM remain unchanged, there is a marked

decrease in TS as the rates of flow of metabolites through Cystathionase β-synthase reaction decrease. The determinant of this metabolic pattern is represented by a reduction in the liver enzymes together with a decrease in

SAM which is an effector of Cystathionase β-synthase (Finkelstein et al., 1988).



19.5. SAA REQUIREMENT

19.5.1 Definitions of Dietary Requirements With Respect to the SAA

Methionine is clearly accepted as a dietary indispensable amino acid and cysteine as a dietary dispensable

AA, which can be entirely replaced with dietary methionine. This has been confirmed in all animal species examined to date.



19.5.2 Total SAA Requirement

Because methionine can be converted to cysteine as required and can meet 100% of the metabolic requirement

for cysteine (Rose et al., 1955; Di Buono et al., 2001b), the Total SAA (TSAA) requirement is defined as:

the methionine intake, in the absence of cysteine, that satisfies all of the physiological requirement for both methionine and cysteine

(eg, growth, nitrogen balance, TS, methyl donation, glutathione synthesis, taurine synthesis etc) (Ball et al., 2006).



19.5.3 Minimum Obligatory Requirement for Methionine

The sparing effect of cysteine on the methionine requirement means that cysteine is capable of replacing some

proportion of the TSAA requirement. The indispensable nature of methionine means that there is a Minimum

Obligatory Requirement for methionine, representing the quantity of the methionine requirement that cannot be

replaced by cysteine. This can be defined as

the intake of methionine that cannot be replaced by cysteine and that will not be reduced by addition of any methyl donor, cofactor

or any other metabolite (Ball et al., 2006).



19.5.4 Cysteine Sparing of Methionine

The definition of cysteine sparing of methionine can be derived from the definition of TSAA as follows:

Cysteine sparing is:

The proportion of the TSAA (as described above) that can be met with dietary cysteine.



Determination of the above three sets of requirements requires the conduction of a minimum of three experiments: (1) the TSAA requirement (measured by feeding graded intakes of methionine and zero dietary cysteine);

(2) The minimum obligatory methionine requirement, (measured by feeding an excess of dietary cysteine and

graded intakes of methionine); and (3) the cysteine sparing effect (measured by feeding the minimum obligatory

methionine requirement and graded intakes of cysteine; Ball et al., 2006).



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19.5. SAA REQUIREMENT



273



19.5.5 SAA Requirement Using Nitrogen Balance

Using nitrogen balance technique, Rose et al. (1950) were the first to determine the TSAA requirement in

young adult males. The minimal and safe requirements were estimated at 1.1 and 2.2 g/day respectively, representing a requirement of 13.25 mg/kg per day to keep all subjects in positive nitrogen balance. The minimum

methionine requirement was also estimated in young men (Rose and Wixom, 1955), and found to be 0.1À0.2 g/

day. This demonstrated a sparing effect of cysteine on the methionine requirement of 89% and 80%.

The TSAA requirement of the human infant was estimated using the nitrogen balance technique with nitrogen balance and growth as the criterion of adequacy (Snyderman et al., 1964; Fomon et al., 1986; Albanese

et al., 1948). Estimates of 80À88 and 44À49 mg/kg per day were derived for TSAA and minimum methionine

requirement respectively. This represents a 55À60% sparing effect of cysteine on the methionine requirement

of infants.

The nitrogen balance method has many practical limitations and flaws (Fuller and Garlick, 1994; Young and

Bier, 1987). Nitrogen balance is a relatively small value, obtained by subtracting a relatively large value of nitrogen losses from a similarly large value of nitrogen intake. This result in overestimation in the prediction of nitrogen balances and a tendency toward a falsely positive nitrogen balance because of overestimation of intake, and

underestimation of losses. This leads to underestimation of the requirement. Due to the slow turnover of the

body urea pool, nitrogen balance also require a prolonged adaptation time (minimum of 7 days) (Fuller and

Garlick, 1994; and Young and Bier, 1987), to the test diets for the dietary change to be reflected in the urinary

nitrogen excretion. This makes the method unfit for use in vulnerable populations like children and pregnant

women because it is unethical to maintain them on deficient intakes for such prolonged periods. The result is

that nitrogen balance studies do not allow for the evaluation of sufficient levels of intake of the AA in order to

determine the requirement. This is problematic because since the requirement is defined for the individual, each

individual needs to be studied at several levels of intake (at least three) both above and below the predicted

requirement in order to be able to estimate the individual requirement (Rand et al., 1976; Zello et al., 1990). Nitrogen

balance calculations and estimation of nitrogen can be significantly affected by miscellaneous and dermal losses,

therefore, they must be included in the calculation. However, they are very difficult to measure and vary with

environmental conditions (eg, ambient temperature) (Calloway et al., 1971; Rand and Young, 1999).

Thus the nitrogen balance method is cumbersome, and lacks precision. In addition it is burdensome to subjects

and totally unsuitable for use in vulnerable populations. These disadvantages pointed to a need for more

sensitive, less cumbersome, and minimally invasive methods to be developed.



19.5.6 SAA Requirement Using Stable Isotope Tracer Kinetics

19.5.6.1 Indicator Amino Acid Oxidation Technique

The most important contribution to our current knowledge of the SAA requirement using stable isotope tracer

kinetics have been by the combined Toronto/Alberta group headed by P. Pencharz and R. Ball and the MIT

group headed by the late V.R. Young and his collaborators in India, namely Kurpad et al. These two groups have

employed the techniques of indicator amino acid oxidation (IAAO) and IAAO-balance technique to determine

the TSAA and minimum methionine requirement. In contrast to nitrogen balance, only minimal adaptation time

to each test level of AA intake (6À8 h) is needed (Elango et al., 2009), which is likely due to the time needed for

equilibration in the acyl-tRNA pools. This means that several levels of AA intakes can be studied without putting

the subjects at risk because they are only exposed to a deficient or excess level of intake for hours rather

than days.

The IAAO technique is based on the principle that the partitioning of any indispensable AA between

oxidation and protein synthesis is sensitive to the level of the most limiting AA in the diet. When an indispensable AA is limiting in the diet for protein synthesis, all other (AAs) are in excess and therefore must be

oxidized (Zello et al., 1995). It follows that as the dietary level of the limiting AA is increased in graded

amounts, the uptake of all other (AAs) for protein synthesis increases, leading to a decrease in their oxidation, including the oxidation of the indicator AA. This decrease in oxidation occurs until the requirement is

met, after which further increase in the limiting AA (test AA) will have no effect on the uptake of other indispensable (AAs) (the indicator amino acid) for protein synthesis or oxidation (Fig. 19.5) (Ball and Bayley, 1984;

Kim et al., 1983a,b).



III. CELLULAR AND MOLECULAR ACTIONS OF AMINO ACIDS IN NON PROTEIN METABOLISM



274



19. SULFUR AMINO ACIDS METABOLISM FROM PROTEIN SYNTHESIS TO GLUTATHIONE



Decreasing IAAO represents

increasing utilization of limiting

amino acid for protein synthesis



Response



EAR



RDA



No change in IAAO as requirement

for limiting amino acid has been

met



+2SD



Amino acid intake

FIGURE 19.5 Schematic representation of the pattern of oxidation of amino acids in studies of amino acid requirement using the indicator

amino acid oxidation (IAAO) technique. Oxidation of the indicator amino acid decreases as intake of the test amino acid increases until the

requirement (breakpoint) is reached. After the breakpoint further increase in the test amino acid has no effect on the oxidation of the indicator

amino acid and oxidation remains constant. IAAO; Indicator amino acid oxidation, EAR; Estimated average requirement, RDA; Recommended

dietary allowance.



IAAO provides a functional approach to measuring AA requirements. It is safe, noninvasive, and can be used

in vulnerable groups like children and pregnant women. It applies the stochastic modeling technique (Waterlow

et al., 1978) for the measurement of amino acid kinetics. This model makes the following assumptions:

1. the size of the metabolic pool with respect to both labeled and unlabeled indicator AA is constant during the

course of the study (steady state)

2. there is no significant reentry of isotope into the metabolic pool—at steady state 13C is treated in the same way

as 12C

3. AAs derived from the diet and from the catabolism of protein are handled in the same way (Picou and TaylorRoberts, 1969).

The IAAO method was used to determine the TSAA requirement, and the sparing effect of cysteine in young

adult males (Di Buono et al., 2001a,b). The mean TSSA requirement estimated by the IAAO method is 12.6 mg/

kg per day. This mean requirement estimate is the breakpoint of the estimate in the oxidation curve of the indicator AA (Fig. 19.5) (Di Buono et al., 2001b). The population safe requirement calculated from the variance of the

individual requirement at two standard deviations above the mean was calculated at 21 mg/kg per day

(Di Buono et al., 2001b). The sparing effect of cysteine on the methionine requirement was determined by providing methionine at the safe intake of 21 mg/kg per day and varying the intakes of cysteine. This results show that

cysteine is capable of sparing 64% of the methionine requirement of adult humans (Di Buono et al., 2001a).

Various aspects of SAA metabolism were studies by varying the ratio of methionine and cysteine to represent

the ratios present in common foods (Di Buono et al., 2003). Using the model of Storch et al. (1988), healthy men

were fed three different diets in random order; diet A: 24 mg/kg methionine without cysteine, diet B: 13 mg

methionine plus 11 mg/kg cysteine, and diet C: 5 mg/kg methionine plus 19 mg/kg cysteine. Methionine kinetics

was measured in the fed state using an orally administered L-[1-13C, methyl-2H3]methionine.

Based on the results it is apparent that the ratio of cysteine to methionine regulates whole body SAA metabolism in adult humans. When TSAA intake is adequate and held constant at 24 mg/kg, replacement of methionine

with cysteine results in increased RM at the expense of TS, whereas at high methionine intakes, the methionine

pool is regulated by high rates of TS.

The development of the piglet model as a surrogate for the human neonate (Wykes et al., 1993) to study AA

requirement and metabolism led to increased knowledge on SAA requirements. With the piglet model, Shoveller

et al. (2003b) determined the TSAA as methionine only (methionine in the absence of cysteine) of the enterally

and parenterally fed neonatal piglet. Using IAAO technique the mean methionine requirement of the enterally

and parenterally fed neonatal piglet was estimated at 0.42 and 0.26 g/kg respectively. The methionine requirement of the total parenterally (TPN) fed piglet was 30% lower than the enteral requirement. The splanchnic tissue

(gut and liver) is very important in AA metabolism (Stoll et al., 1998; Bertolo et al., 1999, 2000) with



III. CELLULAR AND MOLECULAR ACTIONS OF AMINO ACIDS IN NON PROTEIN METABOLISM



19.5. SAA REQUIREMENT



275



approximately one-third of dietary essential AA being consumed by the splanchnic tissue on first-pass

metabolism (Stoll et al., 1998). When the gastrointestinal track is bypassed with intravenous feeding, whole

body nitrogen metabolism is decreased in intravenously fed animals compared with those fed enterally despite

similar weight gain (Bertolo et al., 1999). In addition, intestinal atrophy, characterized by decreased villous

height and crypt depth occurs in intravenously fed animals. These alterations in AA metabolism and decreased

nitrogen retention have been used as possible explanation for the decrease AA requirement observed in TPN

feeding.

Using these requirement estimates derived from the piglet model the TSAA requirement of the TPN fed

human neonate was predicted. Since piglets grow at five times the rate of the human infant, the TSAA requirement estimates of the TPN fed human neonate was predicted to be 52 mg/kg per day. When the IAAO technique

was applied to the determination of the TSAA of the TPN fed human neonate the mean and safe estimates were

49.0 and 58.0 mg/kg, respectively (Courtney-Martin et al., 2008a). This study provides evidence that the piglet

model is valid for the study of AA requirement of the human neonate.

The minimum methionine requirement (methionine in the presence of excess cysteine) of the enteral and TPN

fed neonatal piglet was also estimated using the IAAO method. A reduction of the requirement to 0.25 and

0.18 g/kg per day for enterally and parenterally fed piglets respectively (Shoveller et al., 2003a) demonstrated a

40% sparing effect of cysteine on the methionine requirement when cysteine was added to the diet. The sparing

effect was not affected by route of feeding (Shoveller et al., 2003a). This shows that dietary cysteine is equally

effective in producing a sparing effect on the TSAA requirement whether fed enterally or parenterally.

In healthy school-age children, the TSAA requirement and minimal methionine requirement estimated using

the IAAO method was 12.9 and 5.8 mg/kg, respectively. This represents a 55% cysteine sparing effect on the

methionine requirement. These requirements are similar to that of healthy adults (Di Buono et al., 2001a,b) which

shows that the maintenance requirements of children and adults are similar and represents the predominant part

of SAA requirement in school-aged children.

19.5.6.2 Twenty-Four Hour IAAO and Balance Technique

The 24 h IAAO and balance technique is based on the same fundamental principles of the IAAO method. In

addition, the indicator AA balance is calculated as an absolute balance or as a percentage of doses of the indicator

AA oxidized in response to the intake of the test AA. This method combines the feature of 24 h direct balance

with indicator oxidation approach. It measures indicator oxidation and balance in the fed and fasted states over a

24 h period. The point at which the indicator AA balance is closest to zero in response to the intake of the test

AA is deemed the mean AA requirement. The 24 h IAAO and balance method is favored over the IAAO method

because measurements are conducted over the full 24 h in the fed and fasted states whereas the IAAO protocol is

done only in the fed state. This method is therefore the chosen method of determining AA requirement when

data are available. This method however, has only been used in healthy adults because of the prolonged adaptation time of 5À7 days to the test level of AA intake.

The TSAA requirement was estimated using the 24-h IAAO and balance technique in healthy (Kurpad et al.,

2003), and chronically undernourished (Kurpad et al., 2004b) Indian men. The requirement estimates were

15 and 16 mg/kg, respectively. These estimates are in agreement with the estimates of 12.6 mg/kg per day

derived using the IAAO method (Di Buono et al., 2001b). These requirement estimates suggest that chronic

undernutrition in the absence of infection does not increase the requirement for SAAs.

The sparing effect of cysteine on the methionine requirement was assessed at two different cysteine intakes

(5 and 12 mg/kg) with seven different intakes of methionine ranging from 3 to 21 mg/kg per day (Kurpad et al.,

2004a). With a cysteine intake of 5 mg/kg per day, methionine breakpoint was 20 mg/kg per day. At a cysteine

intake 12 mg/kg per day, methionine breakpoint was 10 mg/kg per day. The authors concluded based on the

overall results that cysteine may spare methionine requirement in healthy men but that the amount of sparing is

difficult to quantify.

Based on the definitions under the heading “Definitions of dietary requirements with respect to the SA,” it is apparent that the intake of 5 mg/kg per day of cysteine was less than that required to arrest the flow of methionine

through the TS pathway and therefore the sparing effect was too small to measure relative to the total potential

effect of cysteine to replace methionine in providing the total SAA requirement (Ball et al., 2006). The decrease in

the methionine breakpoint to 10 mg/kg per day when cysteine was provided at 12 mg/kg per day suggest a

sparing effect of cysteine on the methionine requirement. However, the estimate of total SAA requirement

(corrected for molecular weight) was 25 mg/kg per day when 12 mg/kg per day of cysteine was fed. Since the



III. CELLULAR AND MOLECULAR ACTIONS OF AMINO ACIDS IN NON PROTEIN METABOLISM



276

TABLE 19.1



19. SULFUR AMINO ACIDS METABOLISM FROM PROTEIN SYNTHESIS TO GLUTATHIONE



Summary of studies on estimates of methionine requirement and cysteine sparing

Sulfur AA intake



References



Method of

estimation



Mg/kg

Met

Rose et al.

(1955)



10À33



Rose and

Wixom (1955)



a



Number of

diets used



Estimated requirements mean

TSAA



Minimum

Meth



Cys

Sparing (%)



Comments



Cys

0



N Balance



6



12.5 mg/kg



ND



ND



1.2À5a



9.5À13.5



N Balance



4À9



12.7 mg/kg



1.9 mg/kg



87À89



Di Buono et al.

(2001b)



0,6.5,13.0,19.5,

26.0,32.0



0



IAAO



6



12.6



ND



ND



Di Buono et al.

(2001a)



0,2.5,5.0,7.5,

10.0,13.0



21



IAAO



6



ND



4.5



64



Kurpad et al.

(2003)



3,6,9,13,18,21,24 0



IAAO and

Balance

Technique



7



15



ND



ND



Kurpad et al.

(2004a)



3,6,9,13,18,21,24 5 or 12



IAAO and

Balance

Technique



7



ND



20 and 10



ND



Mean calorie intake

of 56 kcal/kg. 123À

150 mg/kg of N



a

DL-methionine used in study.

ND, not detected; IAAO, indicator amino acid oxidation; N balance, nitrogen balance.



addition of cysteine to the diet should not result in a higher estimate of total SAA requirement compared with

methionine alone, a possible explanation for this unusual result is that glycine and serine were used to balance

the nitrogen and AA in this experiment. Glycine and serine are involved in the RM of methionine and serine

donates the carbon skeleton for the synthesis of cysteine, therefore difference in intake of these AAs could affect

the rates of TS and or RM.

Table 19.1 presents a summary of some of the studies which have evaluated the sparing effect of cysteine on

the methionine requirement in humans.



19.5.7 SAA Metabolism: Effect of Route of Feeding

Enteral nutrition results in higher plasma cysteine and Hcy concentrations than TPN (Shoveller et al., 2004;

Stegink and Den Besten, 1972; Miller et al., 1995a) in piglets, human neonates, and adult humans. These data

show that the gut is a significant site of methionine TS and TM producing significant amounts of Hcy and cysteine for net release into the circulation in neonates as well as in adults.

Data on splanchnic metabolism of SAAs are generated mostly from studies conducted in the pig and piglet

models because they have similar physiology to man, especially gut physiology (Bauchart-Thevret et al., 2009).

The importance of the gut in SAA metabolism was demonstrated in piglets when the SAA requirement of enterally fed piglets was 30% higher than piglets fed parenterally (Shoveller et al., 2003b). In addition 20% of the dietary methionine intake and 25% of whole body methionine is metabolized by the gut via TM and TS (Riedijk

et al., 2007a).

With regards to cysteine, studies in pigs indicate the gastrointestinal tract is an important site for its utilization

(Stoll et al., 1998; Bos et al., 2003). Approximately 40% of cysteine intake is metabolized in the splanchnic tissue

during first-pass metabolism (Bauchart-Thevret et al., 2011). Of this, the gastrointestinal tract utilizes about 25%

of the dietary cysteine intake, which represents 53% of the splanchnic first-pass uptake (Bauchart-Thevret et al.,

2011), with approximately 25% consumed in nonoxidative pathways.

In the neonatal piglet, plasma Hcy is highest when methionine is provided enterally in the absence of cysteine,

compared to when methionine is provided enterally with cysteine, or parenterally (Shoveller et al., 2004).

Increases in Hcy also occurs in the TPN-fed human neonate when methionine was fed in the absence of cysteine



III. CELLULAR AND MOLECULAR ACTIONS OF AMINO ACIDS IN NON PROTEIN METABOLISM



19.5. SAA REQUIREMENT



277



(Courtney-Martin et al., 2008a). These data show that both routes of feeding and dietary supply of methionine

and cysteine affect plasma Hcy concentration. Since high plasma Hcy is a predictive marker for hemorrhagic and

ischemic stroke in infants and children (Hogeveen et al., 2002; van Beynum et al., 1999), the provision of the SAA

as a balance between methionine and cysteine with the minimum amount of methionine for its obligatory

requirements would be beneficial in TPN and enteral feeding of infants and children.



19.5.8 Is Cysteine a Conditionally Essential AA in Human Neonates?

Sturman and Gaull were among the first to report on the absence of cystathionase activity in the livers of premature and newborn infants (Sturman et al., 1970), and to make the suggestion that cysteine is a conditionally essential

AA in the newborn until sometime after birth. Cystathionase is the second enzyme in the TS pathway (Fig. 19.2).

When that enzyme is absent or underdeveloped, cystathionine concentrations are elevated. This idea was in question when it was observed that premature infants on cysteine-free TPN had adequate growth and nitrogen retention which was not improved by adding cysteine to cysteine-free TPN formulation (Zlotkin et al., 1981).

In later work, cystathionase activity was measured in samples of human liver tissue during postmortem examination of infants who died prior to 1 year of age (Zlotkin and Anderson, 1982b). Based on those results,

cystathionase activity in the liver is dependent of both gestational age and postnatal age. In addition, kidney and

adrenals have considerable activity which is not affected by postnatal age. In the full-term infant there is a gradual increase in liver cystathionase activity during the first 3 months of life whereas in the premature infant there

is a more marked increase during the first 2 weeks of life. This provided further evidence that cysteine is not

conditionally essential in the human neonate.

Nevertheless, the existence of several reports of low plasma cysteine concentration in neonates on TPN served

as existing evidence in the minds of clinicians and researchers alike that cysteine may be conditionally essential

in the human neonate (Winters et al., 1977; Kanaya et al., 1984; Pohlandt, 1974; Wu et al., 1986; Stegink and

Baker, 1971).

Using growth and nitrogen balance, the essentiality of cysteine was tested in term and preterm infants on

cysteine-free and cysteine-supplemented TPN (Zlotkin et al., 1981). There was no difference in the nitrogen retention between the unsupplemented and cysteine-supplemented group. Both groups showed similar positive nitrogen retention of 282 mg/kg per day which was 56% of nitrogen infused. These retentions paralleled the expected

nitrogen in utero retention. There was no difference in the weight change between groups. As expected, plasma

cysteine concentrations were higher in the cysteine supplemented group.

Still others have shown that cysteine supplementation to cysteine-free TPN (Malloy et al., 1984) did not

improve nitrogen retention or weight gain in the cysteine supplemented group when compared with the cysteine

unsupplemented group. These studies provided further evidence that cysteine is not indispensable in the human

neonate.

In 1995, Miller et al. published a stable isotope tracer technique to assess human neonatal AA synthesis using

13

D-[U- C]glucose (Miller et al., 1995b). With this technique the conversion of glucose carbon into seven nonessential (AAs) was assessed by measuring their isotopic enrichments in plasma using gas chromatography/mass

spectrometry (GC/MS). Using this technique they were unable to detect significant 13C enrichment in plasma

cysteine (Miller et al., 1995a,b). This led them to suggest that cysteine is an essential AA in parenterally fed premature neonates.

However, using the same method but a more sensitive end-point, apo B-100, 13C labeled cysteine was detected

in hepatically derived apo B-100 (Shew et al., 2005). The tracer/tracee ratios of the M 1 1 isotopomer of cysteine

derived from apo B-100 were significantly greater after the [13C6]glucose than at baseline. There was a direct

correlation between the increase in cysteine synthesis and birth weight. They concluded that a functional pathway exists for cysteine synthesis in premature neonates and that the minimum synthetic capacity of this pathway

is directly related to neonatal maturity.

Previous studies using growth and nitrogen balance in neonates fed cysteine-free, methionine-adequate TPN

provided evidence that the neonate is able to synthesize adequate cysteine from methionine, at least for protein

synthesis (Zlotkin et al., 1981; Malloy et al., 1984; Zlotkin and Anderson, 1982a). The results of a study using

IAAO method in enterally fed preterm neonate (Riedijk et al., 2007b) also revealed that cysteine is not a conditionally essential AA in the preterm neonate. Neonates were fed methionine-adequate formula, with graded

intakes of cysteine. There was no change in oxidation of the indicator AA in response to changes in the intake of

cysteine.



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278



19. SULFUR AMINO ACIDS METABOLISM FROM PROTEIN SYNTHESIS TO GLUTATHIONE



A more recent report using stable isotope tracers provided evidence that the neonate is able to synthesize adequate cysteine from methionine not only for protein synthesis but also for GSH synthesis (Courtney-Martin et al.,

2010). GSH synthesis was measured in erythrocytes of neonates fed TPN in which the TSAA was provided as

methionine only, or as methionine plus supplemental cysteine. Cysteine supplementation had no effect on erythrocyte GSH synthesis (Courtney-Martin et al., 2010). Notwithstanding, the increase in plasma Hcy when SAAs is

provided as methionine only (Courtney-Martin et al., 2008a; Shoveller et al., 2004), especially in enteral feeding,

is evidence that provision of the SAAs as a balance of methionine and cysteine is advantageous over the provision as methionine only.



19.6. GLUTATHIONE

19.6.1 Introduction to GSH Metabolism

The tripeptide GSH (gamma-glutamyl-cysteinyl-glycine:GSH) is synthesized de novo within all cells from glycine, cysteine, and glutamate (Reid and Jahoor, 2000). Although synthesized within all cells, the liver is the major

producer and exporter of GSH. It is synthesized primarily if not exclusively in the cytoplasm (Smith et al., 1996).

Therefore, most of the cellular GSH (85À90%) is present in the cytosol. Unlike the synthesis of larger peptides, no

RNA template is involved in GSH synthesis (Beutler, 1989).

There are two steps in the synthesis of GSH: first the enzyme γ-glutamyl cysteine synthetase catalyzes the formation of a peptide bond between the γ-carboxyl group of glutamic acid and the amino group of cysteine

(Fig. 19.6). This is the rate-limiting step in GSH synthesis (Meister and Anderson, 1983), and because the peptide

bond formed between glutamic acid and cysteine is at the γ rather than the α carboxyl group of glutamic acid,

the tripeptide is resistant to peptidase (Beutler, 1989). In the next step, glycine is joined to γ-glutamyl cysteine

Glutamate

ATP



1



Cysteine



ADP + PI

ι-Glutamyl-cysteine

2



ATP



Glycine



ADT + PI

Glutathione (GSH)

O2



H2 O2



NADP+



3

Peroxidase



Reductase



H2 O2

4

NADPH + H +



H2 O



5



GSSG



FIGURE 19.6 Glutathione metabolism. Reaction 1. γ-Glutamylcysteine synthetase; Reaction 2. Glutathione synthetase; Reaction 3.

Oxidation of GSH by O2; conversion of GSH to GSSG is also modified by free radicals; Reaction 4. GSH peroxidase; Reaction 5. GSSG

reductase.



III. CELLULAR AND MOLECULAR ACTIONS OF AMINO ACIDS IN NON PROTEIN METABOLISM



19.6. GLUTATHIONE



279



(Fig. 19.6) to form GSH. This reaction is catalyzed by glutathione synthetase (Beutler, 1989). GSH has an inhibitory influence on the first enzyme γ-glutamyl cysteine synthetase, which acts as feedback control for the regulation of GSH synthesis. In rare cases of hereditary deficiency of glutathione synthetase, the second reaction in

GSH synthesis is halted and large amounts of γ-glutamyl cysteine accumulates, which is then catabolized to

5-oxyproline and excreted in the urine (Beutler, 1989).

Most of the functions of GSH require its reduced form, (Fig. 19.6) in which state it has a free sulfhydryl group

and is designated GSH. However, the cysteine residue is easily oxidized nonenzymatically to glutathione disulfide (GSSG). Because most of the functions of GSH require its reduced form, an active enzyme mechanism exists

in the form of glutathione reductase for the reduction of GSSG to GSH (Fig. 19.6). This enzyme uses NADPH or

NADH as the hydrogen donor. Hence the activity of GSH is very dependent on the intake of riboflavin (Beutler,

1989). In addition, the concentration of GSH, as well as the enzymes involved in its metabolism, are markedly

influenced by diet (Beutler, 1989).



19.6.2 Functions of GSH

The intracellular concentration of GSH in mammalian cells is in the millimolar range (0.5À10 mM) with

85À90% being present in the cytosol. The extracellular concentration (with the exception of bile acids which

contain up to 10 mM) is typically in the micromolar range, for example, 2À20 μM (Meister and Anderson, 1983).

GSH is therefore regarded as the most prevalent intracellular thiol (Meister and Anderson, 1983) and the most

important endogenous antioxidant and scavenger (Wernerman and Hammarqvist, 1999). The [GSH]:[GSSG] ratio

is often used as an indicator of the cellular redox state and is .10 under normal physiological conditions.

1. GSH is consumed in the detoxification of electrophylic metabolites and xenobiotics, converting the first step in

the conversion of formaldehyde (a toxic product of methanol oxidation) to formic acid (Beutler, 1989). It is an

effective free radical scavenger, protecting cells from the toxic effects of reactive oxygen compounds (Reid and

Jahoor, 2000).

2. Through the enzyme glutathione peroxidase (Fig. 19.6), GSH removes peroxides that could oxidize sulfhydrils

and participates in several reactions that serve to prevent oxidation of SH groups or to reduce them once they

have become oxidized (Beutler, 1989). This function is important to promote and protect the normal

functioning of proteins.

3. GSH is needed for the synthesis of leukotrienes, making GSH an important mediator of inflammation (Beutler,

1989).

4. GSH plays an important role in AA transport and is a source of cysteine reserve during food deprivation, and

a major source of cysteine for lymphocytes (Cho et al., 1981; Fukagawa et al., 1996; Malmezat et al., 2000b).

Irreversible cell damage occurs when the cell is no longer able to maintain its content of GSH (Reid and

Jahoor, 2000). Indeed poor prognosis is associated with decreasing GSH concentration in certain disease states.

Consequently an understanding of GSH metabolism and kinetics with particular emphasis/knowledge of

substrate needs for its synthesis is of importance in health as well as in disease states.



19.6.3 Physiological Aspects of GSH

19.6.3.1 Concentration Measurement

In the biological compartments, changes in GSH concentration are affected if there is a difference between the

rates of synthesis and the rates of disposal of GSH (Reid and Jahoor, 2000). A single concentration measure,

therefore, while it gives a static measure of previous kinetics and of amounts of GSH available or lacking in that

compartment and possible surrounding tissues, tells nothing about the rates of synthesis and loss of GSH.

Consequently, kinetic measures using radio and stable isotope tracers provide an opportunity to make meaningful interpretation of concentration measurements.

19.6.3.2 Kinetic Measurement

The first report in which GSH kinetics was measured (Dimant et al., 1955) used the rate of incorporation of

orally administered 15N glycine to estimate GSH synthesis in erythrocytes. This provided the first evidence that

erythrocytes synthesize GSH de novo. Recent reports suggest that erythrocyte contribute up to 10% of whole

body GSH synthesis in humans (Wu et al., 2004). Subsequent kinetic studies used intravenous (IV) injections of



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280



19. SULFUR AMINO ACIDS METABOLISM FROM PROTEIN SYNTHESIS TO GLUTATHIONE



supraphysiologic doses of GSH with measurement of loss from the plasma compartment; still others measured

incorporation of radio labeled precursors of GSH into in vitro systems. These methods were flawed because they

suggested that plasma GSH reflected interorgan, particularly hepatic GSH efflux. Venous plasma GSH concentration is higher than arterial glutathione concentration suggesting a limited role for plasma GSH in interorgan GSH

homeostasis (Reid and Jahoor, 2000). In addition, plasma GSH in vivo is much less than intracellular concentration (μmol vs mmol) (Reid and Jahoor, 2000), and plasma GSH is highly unstable, readily undergoing autooxidation to GSSG or protein GSH disulfides (Reid and Jahoor, 2000).

In 1995, with the development of a stable isotope precursor product model for measuring GSH synthesis

in vivo, (Jahoor et al., 1995), there began an opportunity for the more effective characterization of various aspects

of GSH metabolism and improvement in this body of knowledge.

19.6.3.3 The Precursor Product Model

The precursor product model for measuring GSH kinetics was developed by Jahoor et al. (1995). The minimum requirement for the calculation of the rate of synthesis of a protein or peptide with this model is the

measurement of the isotopic enrichment at two time points during the quasilinear portion of the exponential

increase in peptide-bound AA labeling (Reid and Jahoor, 2000). In addition, an estimate of the enrichment of AA

tracer in the precursor pool (the free pool of the tissue being studied) is necessary since the AA tracer should be

at isotopic steady state before a measurement of its incorporation into the protein/peptide is made.

19.6.3.4 Infusion Protocol

A primed continuous infusion (CI) of either 13C2 or 2H2 glycine is administered intravenously or intragastrically for 6 h in neonates and 7À8 h in adults (Reid and Jahoor, 2000). 13C2 glycine is used for glycine flux

measurements because of the loss of one or two of the deuterium when 2H2 glycine is used as the tracer

(Reid and Jahoor, 2000). Blood samples are collected at baseline and hourly during the infusion, with sampling

restricted to the last 3 h of infusion in neonates and small children (Reid and Jahoor, 2000). The rate of synthesis

of erythrocyte GSH is obtained from the rate of incorporation of 13C2 or 2H2 glycine into the GSH. Erythrocytefree glycine isotopic enrichment is used to represent the enrichment of the glycine precursor pool from which

erythrocytes make GSH (Reid and Jahoor, 2000).

19.6.3.5 Calculations

• The fractional synthesis rate (FSR) of erythrocyte GSH is calculated as follows:

• FSRGSH (%/h) 5 PEt2 2 PEt1 =IEP1 3 ðt2 2 t1 Þ 3 100

• Where PEt2 2 PEt1 5 the increase in the enrichment of GSH bound glycine over the period t2Àt1 of the

infusion.

• IEpl 5 isotopic enrichment at plateau of erythrocyte free glycine

• Absolute synthesis rate (ASR) of GSH is calculated as follows:

• ASR 5 GSHmass 3 FSRGSH

• Where GSHmass 5 the product of the cell volume (or cell number or cell protein) and the concentration of

GSH in the cell.



19.6.4 GSH Metabolism and Synthesis Rates

19.6.4.1 In Healthy States

Cysteine is the rate-limiting factor for GSH synthesis. When healthy adults were fed a diet containing protein

of 1.0 g/kg per day or the same protein intake but devoid of methionine and cysteine (Lyons et al., 2000), their

erythrocyte FSR and ASR of GSH fell significantly when the diet was devoid of methionine and cysteine.

However, GSH concentration did not differ between groups. Therefore, GSH concentration alone cannot be used

as an isolated marker of GSH metabolism, since it does not reflect changes in GSH synthesis within the cell.

A decrease in GSH synthesis has also been observed in response to a 30% decrease in protein intake in healthy

adults (Jackson et al., 2004). Erythrocyte GSH synthesis was measured in healthy adult males while consuming

their habitual protein intake (1.13 g/kg per day), and while consuming a diet providing the safe WHOrecommended amount of dietary protein of 0.75 g/kg per day (FAO/WHO/UNU, 1985). Both FSR and ASR

decreased significantly from baseline despite maintenance of nitrogen balance.



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19.6. GLUTATHIONE



281



Animals are capable of retaining sulfur beyond that required for protein synthesis when fed a diet low in

protein, and infused with methionine. Pigs fed an adequate protein (AP) diet and low protein (LP) diet have similar weight gain (Hou et al., 2003). After receiving a methionine infusion, sulfate excretion was significantly lower

in the LP pigs than the pigs who received AP resulting in a more positive sulfur balance after methionine infusion but lower nitrogen to sulfur ratio compared to the AP group. In the AP piglets the entire methionine load

was catabolized and excreted in the urine whereas only 69% of the infused methionine was excreted in the LP

piglets. This sulfur retention was not due to increased methionine uptake for protein synthesis because there was

no increase in nitrogen balance in the face of the increased sulfur balance (Hou et al., 2003).

On the other hand, when the TSAA requirement is provided as methionine only in the presence of an

adequate protein intake, additional graded intakes of supplemental cysteine do not increase erythrocyte GSH

synthesis in healthy adults (Courtney-Martin et al., 2008b). Rather, the increased cysteine intake results in a linear

increase in sulfate excretion (Courtney-Martin et al., 2008b). The protein intake in that study was provided as

crystalline (AAs) and the methionine was provided at 14 mg/kg per day.

In healthy states therefore, consumption of protein at intakes of 1.0À13 g/kg per day provided in the form of

native protein in subjects habitual diet, is adequate for the maintenance of nitrogen balance as well as for synthesis of the most abundant intracellular antioxidant, GSH. The same level of protein intake from a crystalline AA

mixture designed to provide the TSAA at its requirement of 14 mg/kg per day, is also adequate for maintenance

of GSH synthesis. A protein intake of 0.75 mg/kg per day, although sufficient to maintain nitrogen balance, is

inadequate for maintenance of GSH synthesis.

19.6.4.2 In Stress/Disease/Aging

GSH concentration is reduced in several disease states, including HIV infection (Jahoor et al., 1999), liver

cirrhosis (Bianchi et al., 1997, 2000), diabetes (Ghosh et al., 2004), Sickle cell disease (Reid et al., 2006), and

Alzheimer’s disease (Liu et al., 2005). GSH concentration is also found to be reduced in surgical trauma (Luo

et al., 1998), septic patients (Lyons et al., 2001), premature infants (Vina et al., 1995), as well as in children with

severe childhood undernutrition (SCU) (Badaloo et al., 2002; Reid et al., 2000). The mechanism surrounding this

decreased concentration was believed to be increased utilization. However, protein-deficient animals subject to

the stress of inflammation are unable to maintain GSH homeostasis and ASR, while piglets fed adequate protein

maintained GSH homeostasis even when subjected to the stress of inflammation (Jahoor et al., 1995). In addition,

cysteine and methionine supplementation was shown to modulate the effect of TNF-α on protein and GSH

synthesis in animals fed a low protein diet (Hunter and Grimble, 1994). Survival of guinea pig pups subjected to

oxidative stress was improved by feeding nutritional substrate for GSH synthesis (Chessex et al., 1999).

In HIV-infected individuals, GSH deficiency is due in part, to reduced synthesis, secondary to cysteine deficiency (Jahoor et al., 1999). GSH fractional and ASR were measured in symptom-free HIV-infected subjects before

and after a 1-week supplementation with N-acetylcysteine (NAC) (Jahoor et al., 1999). After NAC supplementation, both fractional and ASR of GSH experienced a significant increase in the HIV-infected individuals which

was similar to that observed in healthy control subjects.

Earlier publications had shown that children with SCU have lower total free plasma concentrations of (AAs)

including glutamine and cysteine (Edozien et al., 1960) and a 60% lower plasma methionine concentration than

healthy children (Roediger, 1995). This knowledge led to a series of investigations in children with edematous and

nonedematous SCU in which the precursor product model was used to measure erythrocyte GSH synthesis at three

time points during hospitalization: shortly after admission when the children were infected and malnourished

(phase 1); 8 days postadmission when they were no longer infected (phase 2); and when they had recovered (phase

3). Children with edematous (SCU) have significantly lower erythrocyte GSH concentrations and lower ASR of

erythrocyte GSH than those with nonedematous SCU (Reid et al., 2000) during phases 1 and 2 of study. During

those two phases, the group with edematous SCU had lower erythrocyte GSH concentrations and lower ASR than

at recovery (phase 3). Plasma and erythrocyte free cysteine concentration were lower in the children with edematous SCU during phases 1 and 2 than at recovery (phase 3). On the other hand, erythrocyte GSH concentration,

rates of GSH synthesis, and plasma and erythrocyte free cysteine concentrations of the nonedematous group were

similar at all three points and were greater at phase 1 and 2 than in the edematous group. Those results led to the

conclusion that GSH deficiency in children with edematous SCU is due to decreased synthesis as a result of cysteine deficiency both as preformed cysteine and from methionine (Reid et al., 2000; Jahoor, 2012).

To test this hypothesis, two groups of children with edematous SCU were supplemented with either NAC or

alanine for 7 days (Badaloo et al., 2002). Supplementation with NAC resulted in higher erythrocyte cysteine and

GSH concentration as well as ASR of GSH. Importantly, in the cysteine supplemented group edema resolved



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19. SULFUR AMINO ACIDS METABOLISM FROM PROTEIN SYNTHESIS TO GLUTATHIONE



approximately 5 days sooner than in the control group. However, both groups had similar rates of weight gain,

yet the clinically important loss of edema occurred quicker in the cysteine supplemented group. This observation

is supportive of prior reports in healthy subjects that SAA is partitioned more toward protein synthesis when

intakes are low (Jackson et al., 2004; Storch et al., 1988). Similar results are obtained in protein or SAA-deficient

animals subjected to the stress of inflammation (Jahoor et al., 1995; Hunter and Grimble, 1994) that SAA is partitioned more into proteins than into GSH when SAA intake is low.

Recent evidence shows that GSH concentration and synthesis are lower in elderly subjects than younger adults

and that cysteine and glycine supplementation lowers both oxidative stress and oxidative damage in the elderly

(Sekhar et al., 2011). The suggestion is that cysteine deficiency is a characteristic of aging, and can be alleviated

by supplementation. The mechanism underlying the deficiency is unknown but decreased intake as a result of

inadequate dietary protein intake is a possible explanation.

The results from all the above studies suggest an increased need for SAA particularly cysteine during illness,

infection, disease, and even aging. This increased need for cysteine can be partly explained by the increased need

for GSH synthesis; a lack of which can lead to slower recovery time and prolonged illness (Badaloo et al., 2002).

Animal studies demonstrate an increased requirement for cysteine in infection (Malmezat et al., 2000a,b).

Cysteine flux, as well as the activity of the enzymes of GSH synthesis (γ-glutamyl cysteine synthetase, and glutathione reductase) are increased in infected rats compared to controls. GSH synthesis accounts for at least 40% of

the enhanced cysteine flux during inflammation. In addition methionine TS and methionine flux are increased

during sepsis but to a less extent than cysteine flux (Malmezat et al., 2000b). The increased cysteine flux observed

in inflammation is higher than predicted from estimates of protein turnover suggesting that the increase cysteine

flux is due to increased GSH synthesis. This is believed to be the case because previous work in humans have

shown that up 50% of cysteine flux in the fasted state is due to GSH breakdown (Fukagawa et al., 1996).

In addition, human (Sekhar et al., 2011; Mercier et al., 2006) and animal (Vidal et al., 2014) studies support an

increased requirement for cysteine with aging. Elderly humans have lower cysteine and GSH concentrations as

well as lower fractional and ASR of GSH (Sekhar et al., 2011) when compared to younger controls. After cysteine

and glycine supplementation, all markers of GSH homeostasis improved with no observed differences between

elderly and young subjects.



19.7. CONCLUSIONS

The SAAs are important nutrient substrates for protein synthesis. In addition, they have important roles

outside of protein synthesis; methionine via its metabolite SAM is the most important methyl donor in vivo and

cysteine is the rate-limiting substrate for synthesis of the most abundant intracellular antioxidant GSH.

Deficiency of SAA therefore, results in impaired growth and protein synthesis but also compromises methylation

reactions and host antioxidant status. Deficient SAA and low protein intake results in decrease in GSH synthesis

in healthy adults. In addition, GSH deficiency is a characteristic feature of multiple disease states including HIV,

diabetes, sepsis, and severe childhood undernutrition. The mechanism underlying GSH deficiency is cysteine

deficiency. Existing evidence points to hierarchy regarding cysteine utilization by the body; cysteine is preferentially used for protein synthesis when intakes are low but intakes in excess of that required for protein synthesis

are partitioned into GSH synthesis, then taurine, and finally oxidized to sulfate when intakes are high.



References

Albanese, A.A., Holt Jr., L.E., et al., 1948. The sulfur amino acid requirement of the infant. Fed. Proc. 7 (1 Pt 1), 141.

Badaloo, A., Reid, M., Forrester, T., Heird, W.C., Jahoor, F., 2002. Cysteine supplementation improves the erythrocyte glutathione synthesis

rate in children with severe edematous malnutrition. Am. J. Clin. Nutr. 76 (3), 646À652.

Baker, D.H., 2006. Comparative species utilization and toxicity of sulfur amino acids. J. Nutr. 136 (6 Suppl), 1670SÀ1675S.

Ball, R.O., Bayley, H.S., 1984. Tryptophan requirement of the 2.5-kg piglet determined by the oxidation of an indicator amino acid. J. Nutr. 114

(10), 1741À1746.

Ball, R.O., Courtney-Martin, G., Pencharz, P.B., 2006. The in vivo sparing of methionine by cysteine in sulfur amino acid requirements in

animal models and adult humans. J. Nutr. 136 (6 Suppl), 1682SÀ1693S.

Bauchart-Thevret, C., Stoll, B., Burrin, D.G., 2009. Intestinal metabolism of sulfur amino acids. Nutr. Res. Rev. 22 (2), 175À187.

Bauchart-Thevret, C., Cottrell, J., Stoll, B., Burrin, D.G., 2011. First-pass splanchnic metabolism of dietary cysteine in weanling pigs. J. Anim.

Sci. 89 (12), 4093À4099.



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