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© 2007 American Society for Nutrition J. Nutr. 137:1874-1879, August 2007

Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Application of the Indicator Amino Acid Oxidation Technique for the Determination of Metabolic Availability of Sulfur Amino Acids from Casein versus Soy Protein Isolate in Adult Men1,2

Mohammad A. Humayun3,4, Rajavel Elango3,4, Soenke Moehn5, Ronald O. Ball4,5 and Paul B. Pencharz3–5,*

3 Research Institute, Hospital for Sick Children, Toronto, ON, Canada M5G 1X8; 4 Department of Nutritional Sciences, University of Toronto, Toronto, ON Canada M5S 2Z9; and 5 Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5

* To whom correspondence should be addressed: E-mail: paul.pencharz{at}sickkids.on.ca.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Our objective was to determine the metabolic availability (MA) of sulfur amino acids in dietary proteins using the indicator amino acid oxidation (IAAO) technique. Five to seven men received graded levels (20, 40, 60, and 70%) of the mean total sulfur amino acid (TSAA) requirement of 13 mg · kg–1 · d–1 as a crystalline AA mixture, casein, and soy protein isolate (SPI) (40, 50, 60, and 70%), respectively. Five of these subjects received 40% of TSAA requirement from SPI supplemented with methionine to the level of 40% of requirement. These 5 subjects also repeated the level of 60% TSAA requirements from both casein and SPI to assess repeatability. The mean MA of TSAA from SPI (71.8 ± 3.6%) was lower than from casein (87.4 ± 3.8%, P < 0.05). Supplementation of SPI with methionine decreased the IAAO (11.5 ± 0.3% administered dose) compared with unsupplemented SPI (12.8 ± 0.5% administered dose, P < 0.05). IAAO was similar for repeated measurements of casein and SPI, respectively, at the 60% TSAA intake level (10.8 ± 1.0 vs. 10.7 ± 1.2% for casein; 12.7 ± 1.3 vs. 12.9 ± 2.6% for SPI). In conclusion, the IAAO technique can be used to determine the MA of AA for protein synthesis in test proteins for humans.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
The nutritional value of dietary proteins is related to their content of metabolically available amino acids (AA)6 for growth and maintenance (1,2). This availability of dietary proteins is dependent not only on their digestibility, but also on the subsequent utilization of absorbed AA inside the body, i.e., the fraction of absorbed amino acids that are incorporated into protein synthesis. The nutritional quality of dietary proteins is classically determined by long-term balance studies in animals and human subjects, who are given various levels of proteins in their diets (3,4). Balance studies require long periods to conduct and thus are limited by ethics as well as cost.

The method adopted by the WHO and the FDA to determine protein quality is the protein digestibility corrected amino acid score (PDCAAS). This method considers both the AA composition and the digestibility of the protein, with respect to AA requirements based on 2- to 5-y-old children. The PDCAAS compares the ratio of each indispensable AA in dietary protein to the same AA in a reference AA pattern (expressed in mg · g protein–1), and corrects the value for estimated protein digestibility (in %). Although the PDCAAS is currently the preferred procedure for assessing protein quality of food products for human nutrition, there are concerns about this method (5). The limitations of PDCAAS include 1) an inability to apply correction values for digestibility of individual AA in proteins, thus ignoring the large differences between digestibility values for the entire proteins and individual AA (6); 2) an underestimation of the lower availability of specific AA (e.g., lysine, methionine) in heat-treated proteins (7); and 3) the inability to predict true AA availability by considering the subsequent metabolism of absorbed AA inside the body.

Recently, we showed that the indicator amino acid oxidation (IAAO) method can be used in pigs to determine the metabolic availability (MA) for protein synthesis of lysine in dietary proteins (8). The IAAO method is based on the observation that, when one AA is limiting for protein synthesis, all other AA are in excess (including the indicator AA, such as L-[1-13C]phenylalanine) and thus must be oxidized. Hence, changes in the oxidation of indicator AA following the intake of test and reference proteins will reflect the whole body MA of the limiting AA at the site of protein synthesis and, thus, account for all losses of dietary AA during digestion, absorption, and cellular metabolism. In other words, the higher the oxidation of the indicator AA, the lower the metabolic availability of the test AA for protein synthesis, and vice versa. The advantages of the IAAO method over balance studies are a shorter time period and the avoidance of the many difficulties associated with long-term balance studies in humans. Unlike the PDCAAS, the IAAO method accounts for differences in digestibility and availability of individual amino acids in the protein.

In this study, we adapted the IAAO method (8) to humans. The objective was to develop a new method, based on the IAAO technique, for determining the MA of individual AA in foodstuffs for humans. In the pig study, we tested only 1 level of protein. In this experiment, we chose to also test a range of protein intakes. For reasons yet to be proven, we did not see a response of the indicator to the range of protein intakes. In the Discussion, we reason that this was because of an inadequate length of adaptation. Hence, we were restricted to applying the model previously tested in pigs (8), namely, using a single protein intake test level. To support our use of the current model, with adaptation limited to 4–6 h, we conducted 2 additional experiments. The first showed that the model did respond to the addition of methionine, which we predicted was limiting in the soy-based diets. The second experiment was conducted to restudy the subjects at the chosen fixed level of intake, namely 60% of the mean total sulfur amino acid requirement (TSAA). We used, as an example, determination of MA of TSAA from casein and soy protein isolate (SPI) in adult men.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Subjects. Seven to five young, healthy, male adults completed each of the 3 experiments (on an outpatient basis) in the Clinical Investigation Unit at the Hospital for Sick Children, Toronto, Canada. Subject characteristics, body composition, and energy intakes are described in Table 1. None of the subjects had a history of recent weight loss or illness, and none was using any medication at the time of entry into the study. The Research Ethics Board of the Hospital for Sick Children approved all procedures. Informed written consent was obtained from the participating subjects. The subjects received financial compensation for inconvenience.


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  		TABLE 1 Subject characteristics of healthy young men1

 
    Experimental and diet design and tracer protocol. Three experiments were conducted. In Expt. 1, IAAO response was measured following the feeding of graded levels of TSAA (as methionine) from a reference protein [crystalline AA mixture (MIX)], or from the test proteins casein (Casec, Mead Johnson) and SPI (Supro XT12C, Enerex Botanical) (n = 5–7). Our objective was to determine the MA of TSAA by comparing the slopes of IAAO response following the intake of graded levels test proteins vs. the reference AA mixture, using the slope ratio method. As mentioned early in our animal study (8), a single test level of protein was used. In Expt. 2, the effect of methionine supplementation to SPI (which is deficient in methionine) on IAAO response was determined in 5 subjects (n = 5). In Expt. 3, oxidation was measured a second time at 60% TSAA intake from casein and SPI in 5 of these subjects to determine repeatability of the method.

All of the above studies were based on the minimally invasive IAAO model (9), recently used in healthy adults (10–13) and children (14,15). Two days before the study day, subjects consumed a maintenance diet to equalize protein and AA (including phenylalanine) intake among subjects prior to measurement of phenylalanine oxidation. On the study day, following a 12 h fast, subjects randomly received one of the test diets. The AA compositions of reference AA MIX and test proteins are given in Table 2. In Expt. 1, 13 dietary levels of TSAA were provided, either as crystalline methionine from AA MIX (20, 40, 50, 60, and 70% of the mean TSAA requirement of 13 mg · kg–1 · d–1) or as TSAA (40, 50, 60, and 70% of the mean TSAA requirement) from casein and SPI, respectively. The TSAA levels were achieved by including casein at 0.202, 0.2525, 0.303, and 0.3535 g · kg–1 and SPI at 0.2468, 0.3085, 0.3702, and 0.4319 g · kg–1, respectively. Free amino acids were added to casein and SPI diets to provide equal intake of all amino acids, except TSAA. Amino acid intake was equalized to that received from the AA MIX (Table 2). The addition of other amino acids to the diet did not affect the digestibility and metabolic availability of the test AA. In Expt. 2, the test diets provided 40% of the TSAA requirement from SPI (0.247 g · kg–1 · d–1 SPI) without or with supplemental crystalline methionine (2.6 mg · kg–1). In Expt. 3, the diets providing 60% of the TSAA requirement from casein or SPI were tested again to determine repeatability of the estimate of MA. All the above diets were formulated to provide a protein intake of 1.0 g · kg–1 · d–1 and identical AA intake except for TSAA. Diets were consumed along with a protein-free formula to achieve an energy intake of 1.5 x resting metabolic rate (RMR) with 52, 36, and 12% of energy from carbohydrates, fat, and protein, respectively. The study day diet was consumed as 8 isonitrogenous and isocaloric hourly meals, with each meal representing one-twelfth of the subject's total daily protein and energy requirement. Subjects were not allowed to eat or drink anything else except for water. The study days were separated by ≥1 wk.


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  		TABLE 2 Amino acid composition of reference and test proteins1

 
Details of the tracer protocol were described in our previous studies (10–13). Briefly, the tracer protocol was started with the fifth meal to measure phenylalanine kinetics, using L-[1-13C]phenylalanine (99 atom % excess, Cambridge Isotope Laboratories), and it was continued for the remaining 3 h of the study. The amount of L-[1-13C]phenylalanine given during the study day was subtracted from the dietary provision of phenylalanine, such that the total intake of phenylalanine was 30.5 mg · kg–1 · d–1 with a tyrosine intake of 40 mg · kg–1 · d–1 (to ensure an excess of tyrosine).

    Preparation of study diets. Details of the maintenance diet are provided in our previous studies (10–13). Briefly the maintenance diet (energy: RMR x 1.7 and protein: 1.0 g · kg–1 · d–1) was provided in the form of milk shakes for the 2 d prior to study days in all experiments. RMR was measured by open-circuit indirect calorimetry (2900 Computerized Energy measurement system, Sensormedics). For the duration of all experiments, subjects also consumed a daily multivitamin supplement (Centrum, Wyeth) (10) to ensure more than adequate folate and B-vitamin intake as cofactors for the metabolism of methionine and homocysteine.

The study-day diet consisted of a protein-free liquid formula (protein-free powder, Product 80056, Mead Johnson) flavored with Tang and KoolAid crystals (Don Mills), corn oil, the crystalline AA MIX (for reference protein) or crystalline AA MIX plus casein/SPI (for test proteins), and protein-free cookies.

    Breath sample collection and analysis. Breath samples were collected as described previously (10–13). Briefly, for each study, 3 baseline breath samples were collected at 15, 30, and 45 min after intake of the fourth hourly meal (before tracer infusion) and 4 breath samples were collected again at timed intervals between 150 and 240 min of tracer infusion. During each study day, open-circuit indirect calorimetry (2900 Computerized Energy measurement system, Sensormedics) was performed for 20 min to measure the rate of carbon dioxide production (VCO2). Enrichment of 13C in breath was analyzed by continuous flow isotope ratio mass spectrometer (20/20 isotope analyzer, PDZ Europa).

    Calculation of results. Indicator oxidation was expressed as the percentage of label dose administered, [F13CO2 excreted in breath at steady state (µmol · kg–1 · h–1)/L[1-13C]phenylalanine administered (µmol · kg–1 · h–1) x 100]. MA was estimated by applying the model previously tested in pigs (8), namely, using a single protein intake test level to compare the IAAO response of test proteins with the reference protein (crystalline AA MIX). To quantify the label oxidation, we chose to present the data as the percentage of administered dose (% dose oxidized), rather than rate of label oxidation (µmol · kg–1 · h–1). Determination of MA availability using % dose oxidized or rate of label oxidation provides the same estimate of MA, as both use F13CO2 data, and the remaining variables are the same in the calculation.

    Statistical analysis. We examined the effect of adding methionine by protein source (e.g., crystalline AA or casein or SPI) on phenylalanine oxidation using the procedure "MIXED" (16), with "subject" as a random variable. Covariates (age, weight, and VCO2) and their interactions with main effects were tested. A paired t test was applied to test the effect of methionine supplementation on IAAO. Results were expressed as means ± SEM, except for the repeatability study, where results were expressed as means ± SD. The repeatability of oxidation measurements was assessed using mean CV of measurements within subjects. Statistical significance was assumed at P < 0.05.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    Experiment 1: Linearity of response to increasing TSAA intake. Covariates (age, weight, and VCO2) and their interactions with main effects were tested, but they were not significant. As the TSAA (as methionine) intake from reference protein (AA MIX) increased from 20 to 70% TSAA requirement (100% TSAA requirement = 13 mg · kg–1 · d–1), the oxidation of L[1-13C]phenylalanine (% of dose) decreased linearly (Fig. 1A). Application of linear regression determined a negative slope of the best-fit line of –0.055 ± 0.01 (r2 = 0.91, P < 0.05). As the TSAA (methionine + cysteine) intake from casein increased from 40 to 70% TSAA requirement, the oxidation of L[1-13C]phenylalanine (% dose) was unchanged, and the slope did not differ from zero (Fig. 1B). As the TSAA (methionine + cysteine) intake from SPI increased from 40 to 70% TSAA requirement, the oxidation of L[1-13C]phenylalanine (% dose) was also unchanged (Fig. 1C).


Figure 1
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  		FIGURE 1  Oxidation responses of orally administered L-[1-13C]phenylalanine (% administered dose) following graded intake of methionine in AA MIX (A), TSAA in casein (B) and TSAA in SPI (C) (as % mean TSAA requirement of 13 mg · kg–1 · d–1) in healthy young men. Values are means ± SEM, n = 7. Equations represent linear regression on the data.

 
    MA of TSAA in casein and SPI. Because the IAAO response showed no significant slope for casein or SPI (Fig. 1), we conducted a second experiment, in which methionine was verified to be limiting by supplementing Met to SPI. Before applying this approach, we determined that in Experiment 1, the 60% TSAA intake level from casein and SPI had lower variability in IAAO responses than the other levels of intake (Table 3). The higher oxidation rate following intake of casein or SPI, compared with the reference protein, indicates a lower MA of TSAA for protein synthesis than in the reference protein. The IAAO was higher for SPI than casein (P < 0.05), indicating there was a lower MA of TSAA from SPI than from casein (P < 0.05) (Table 3).


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  		TABLE 3 Oxidation of orally administered L-[1-13C]phenylalanine as breath 13CO2 and metabolic availability of TSAA following the intake of AA MIX and test proteins in healthy young men1

 
    Experiment 2: Supplementation of SPI with methionine. SPI diet containing 2.6 mg · kg–1 · d–1 methionine and 2.6 mg · kg–1 · d–1 cysteine was supplemented with 2.6 mg · kg–1 · d–1 methionine. Oxidation of L[1-13C]phenylalanine (% dose) decreased compared with unsupplemented SPI (P < 0.05) (Fig. 2).


Figure 2
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  		FIGURE 2  Oxidation responses of orally administered L-[1-13C]phenylalanine (% administered dose) following the intake of SPI containing 2.6 mg · kg–1 · d–1 methionine and 2.6 mg · kg–1 · d–1 cysteine (representing 40% of mean TSAA requirement of 13 mg · kg–1 · d–1) and the same SPI supplemented with 2.6 mg · kg–1 · d–1 methionine in healthy young men. Values are means ± SEM, n = 7. *Different from SPI supplemented with methionine (P < 0.05).

 
    Experiment 3: Repeatability of MA estimate. To determine the repeatability of the measurement of MA, we repeated the 60% TSAA intake from casein and SPI in 5 of the 7 subjects. The repeated measurements of each protein (casein or SPI) did not differ significantly in terms of 1) oxidation of L[1-13C]phenylalanine (% dose), 2) CV for L[1-13C]phenylalanine oxidation (% dose), or 3) MA of TSAA (Table 4). These results suggest that the repeatability is high for measurements conducted to determine MA on 2 separate occasions under similar conditions.


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  		TABLE 4 Repeatability of oxidation of orally administered L-[1-13C]phenylalanine as breath 13CO2 and MA of TSAA from test proteins in healthy young men1

 

   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
The PDCAAS is currently the recommended method of assessing the quality of dietary protein in humans (17). However, the PDCAAS does not account for variation among AA in digestibility within a protein, nor does it consider the MA of absorbed AA. Given our knowledge that the PDCAAS estimation of protein quality, using only a single digestibility factor and composition of AA, is incorrect, it should no longer be considered adequate. Protein synthesis is affected by the availability of AA at the site of synthesis; therefore, the IAAO method is preferable to the PDCAAS, because it assesses the quality of proteins in terms of MA of absorbed AA for protein synthesis. This new method determines the quality of proteins by considering all losses during AA digestion, absorption, and subsequent metabolism, prior to protein synthesis. Other technically advanced methods are also available to determine protein quality in humans (18,19). However, these methods use foods intrinsically labeled with isotopes (18,19) and, hence, are not suitable for routine analysis of the protein quality of regular foods consumed by humans.

Our goal in this study was to adapt our recently developed method of determining MA of AA in pigs (8) for humans. We determined the MA of TSAA in test proteins by comparing the IAAO response of test proteins with a reference protein at a fixed test AA level (60% TSAA requirement level). The test diets were supplemented with all amino acids, other than the test AA, to ensure that they did not become limiting. By ensuring that only the test AA was limiting in the test proteins, it became the only factor driving the IAAO response. In the first instance, we had hoped to observe a linear decrease in IAAO, with graded increases in TSAA, from the 2 protein sources. However, this was not the case, for reasons that are discussed below. Because the linearity of IAAO response could not be demonstrated for the test proteins, we applied the approach that was successful in pigs (8) to determine the MA of TSAA. In the pig study (8), the chosen protein level provided 80% of the NRC level for the limiting AA, lysine. This level was selected because it is ~2 SD less than the NRC mean dietary requirement for pigs. In our earlier work (10), we found that the mean TSAA requirement is 12.6 mg · kg–1 . d–1, with a 95% CI of 21 mg · kg–1 · d–1, for young men. In this study, we chose a 60% TSAA intake level to determine MA. Our reasons for choosing this level include the following: 1) the 60% TSAA intake level represents ~1 SD less than the mean TSAA requirement (10), and all the subjects were still below their TSAA requirement; 2) the 60% TSAA intake level had the least variability in IAAO responses; and 3) the reliability of the change in oxidation measurements at this intake level was confirmed in the subsequent experiment (Expt. 3).

The results of applying the above approach indicated that the IAAO of casein, SPI, and the reference AA MIX were different at the 60% TSAA intake level (P < 0.05). The MA of TSAA in casein and SPI were calculated to be 87 and 72%, respectively. These estimates of MA for casein (87%) and SPI (72%) are similar to the reported net protein utilization (NPU) of 80–85% for milk proteins (18,20,21), and of 71–78% for soy proteins (19,22). Therefore, our current estimate is supported by other methods and appears to be an acceptable approach to determining the MA of amino acids in proteins for humans. The overall magnitude of CV of ~10% was similar to that found in pigs (8). The practical implication of the repeatability of the IAAO measurements at a single test intake level is that reliable and rapid experiments can be conducted to determine MA of AA in human food. However, we feel that further refinement in the method is required before this approach can be widely recommended for application.

The differences in the MA of TSAA in casein and SPI are probably related to 2 important factors: 1) lower digestibility of methionine and cysteine in SPI than casein, and 2) reduced metabolic availability of methionine and cysteine following digestion, due to compounds formed by heat processing. Although the true ileal digestibility of milk protein is higher than SPI (95 vs. 91%) (19,21), this difference is less than the difference observed in feeding or NPU studies. SPI contains TSAA in quantities (21 mg · g protein–1) comparable to that provided by casein (26 mg · g protein–1); however, the methionine content in SPI (10.5 mg · g–1) is less than half of that in casein (23 mg · g–1) (Table 2). Because SPI is heat processed, some of the cysteine in SPI is not available for protein synthesis, because of heat-related losses of cysteine and the formation of cysteine-crosslinks (23).

In Expt. 1 of this study, we provided graded levels of crystalline methionine in an AA MIX (20–70% of TSAA requirement) and TSAA as casein and SPI, respectively (40–70% of TSAA requirement). Our results indicated that the L[1-13C]phenylalanine oxidation decreased linearly after graded levels of TSAA intake (as methionine) from the reference AA MIX but not in the test protein. This was very surprising, because we had observed consistent linear differences in oxidation with varying intakes of protein in pigs (24). Phenylalanine (and tyrosine) intake was identical for all treatments. Ileal digestibility of phenylalanine in casein and SPI is very high—between 98 and 100% (25); therefore, differences in the proportion of phenylalanine intake from protein-derived or free phenylalanine did not affect the phenylalanine oxidation response. This had been demonstrated previously (8,24). The lack of a significant decrease in indicator oxidation following graded levels of casein or SPI in humans would occur if either the TSAA were not first-limiting, or the MA of TSAA in these foodstuffs was close to zero. However, both of these possibilities are unlikely. Supplementation of methionine to the SPI diet decreased indicator oxidation (Fig. 2), which indicates that methionine was the limiting AA. The published NPU of casein and soy proteins is >70% (18–22); zero availability of TSAA would result in an NPU close to zero as well. One possible reason for the lack of the expected linear response in this experiment could be the large variability in IAAO responses in humans consuming test proteins, compared with the responses in subjects consuming the reference AA MIX. Further, the adaptation period of the subjects to the diets may have affected the results. In this experiment, as in most of our previous studies on AA requirements, subjects consumed the same liquid diet for 2 d and then consumed the test diet for 8 h, with oxidation being measured during the final 2 h. Because a mixture of free and protein-bound amino acids was used, inadequate adaptation might have affected the tracer/tracee ratio for the indicator amino acid, phenylalanine. We have shown that AA oxidation in pigs adapts quickly to changes in intake (26). However, a longer period of feeding test diets may be required in humans when studying oxidation response to intact protein sources.

In conclusion, we demonstrated that the IAAO technique could be used to estimate the MA of TSAA for protein synthesis in casein and SPI. Before this method can be widely applied to determine the MA of amino acids within dietary protein, it is necessary to define the optimal period of dietary adaptation to the intact test protein and study the influence of free vs. protein-bound amino acids.


   FOOTNOTES
 
1 Supported by the Canadian Institutes for Health Research (grant MT 10321). Mead Johnson Nutritionals (Canada) donated the protein-free powder for the experimental diets. Back

2 Author disclosures: M. A. Humayun, R. Elango, S. Moehn, R. O. Ball, and P. B. Pencharz, no conflicts of interest. Back

6 Abbreviations used: AA, amino acid; IAAO, indicator amino acid oxidation; MA, metabolic availability; MIX, mixture; NPU, net protein utilization; PDCAAS, protein digestibility corrected amino acid score; RMR, resting metabolic rate; SPI, soy protein isolate; TSAA, total sulfur amino acid. Back

Manuscript received 23 January 2007. Initial review completed 14 February 2007. Revision accepted 14 June 2007.


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 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
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R. Elango, R. O. Ball, and P. B. Pencharz
Indicator Amino Acid Oxidation: Concept and Application
J. Nutr., February 1, 2008; 138(2): 243 - 246.
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