To measure the fractional and absolute synthesis rates of GSH, an antioxidant that protects cells from damage, in the erythrocytes of healthy school-aged children, young adults and old adults in response to graded intakes of protein in order to determine if increasing the protein intake above the current recommended DRI produces increases in the GSH synthesis rates in erythrocytes of these individuals. Participants will consume specially formulated diets with varying protein levels.

The current dietary reference intake (DRI) recommendations for protein in children (6-11 years old) and adults (>19 years old) are based on nitrogen balance data and set at a mean (EAR) of 0.76 and 0.66 g/kg/day respectively and population safe (RDA) recommendation of 0.95 and 0.80 g/kg/day respectively. Using the indicator amino oxidation (IAAO) method we estimated higher protein requirements in these populations. This suggests that current recommendations are underestimated. Studies in adults have shown that protein recommendation set on the basis of nitrogen balance data, while sufficient to maintain nitrogen balance, does not maintain GSH status.

Glutathione (GSH) is most important intracellular antioxidant and scavenger and its deficiency has been shown to compromise recovery in acute as well as chronic stressors. Deficiency of GSH results when protein and/or cysteine intake is inadequate. Inadequate protein intake in children not only compromises growth, but could increase susceptibility to, and compromise recovery from regular childhood illness. Similarly, in young and older adults' inadequate intakes may result in increased oxidative stress and compromised immune function. Measurement of GSH synthesis provides a functional method whereby the current protein recommendation can be tested against higher estimates derived using the IAAO method. The purpose of this study therefore is to measure GSH synthesis in healthy school aged children (6-11 years), and healthy young (19 - 40 years) and old (60 - 90 years) adults in response to current recommended protein intakes and in response to protein requirement estimates derived using the IAAO method.

In healthy school-aged children, each level of protein will be studied over three days. For two days (adaptation days), participants will eat some low protein foods plus a milkshake drink. On the study day, participants will consume 8 hourly protein drinks and special cookies at SickKids. In healthy young and old adults, each level of protein will be studied over three days. For two days (adaptation days), young adults will eat some low protein foods plus a milkshake drink. On the one study day, young adults will consume 10 hourly milkshake diets at SickKids. In old adults, each level of protein will be studied over four days. For three days (adaptation days), old adults will eat some low protein foods plus a milkshake drink. On the one study day, old adults will consume 10 hourly milkshake diets at SickKids.

Inclusion Criteria in:

1. Healthy school-aged children

   • Body weight ≥ 22 kg
   • No history of anemia
   • Willingness to participate

2. Healthy Young Adults

   • Consent provided
   • Aged 19 - 40 years old
   • In good general health as evidenced by medical history, physical health and blood draw
   • Fasting blood glucose, hemoglobin A1c (HbA1c), urea, creatinine, vitamin-B6, vitamin-B12 and folate levels within normal ranges for age.
   • Willingness to participate in the study.
   • BMI <25 kg/m2.

3. Healthy Old Adults

   • Consent provided
   • Aged 60- 90 years old
   • In good general health as evidenced by medical history, physical health and blood draw
   • Fasting blood glucose, hemoglobin A1c (HbA1c), urea, creatinine, vitamin-B6, vitamin-B12 and folate levels within normal ranges for age.
   • Willingness to participate in the study.
   • BMI <30 kg/m2.

Exclusion Criteria in:

1. Healthy school-aged children

   • HIV
   • Diabetes
   • Anaemia
   • Significant weight loss within 30 days of study
   • On weight loss diets
   • Body weight less that 22kg
   • Inability to tolerate diet
   • Unwilling to have blood drawn

2. Healthy Young and Old Adults:

   • Presence of chronic disease and/or acute illness known to affect GSH and/or protein/amino acid metabolism (e.g. HIV, diabetes, cancer, liver or kidney disease, acute cold or flu).
   • Anemia (15 - 20% outside lower limit of normal lab reference ranges)
   • Taking medications known to affect GSH and/or protein/AA metabolism (e.g. acetaminophen, steroids).
   • Significant weight loss during the past month or consumption of weight reducing diets.
   • Significant caffeine consumption (>2 cups per day).
   • Significant consumption of alcohol (>1 drink per day i.e. 1 beer or ½ glass of wine).
   • Inability to tolerate the diet
   • Unwilling to have blood drawn from a venous access during the isotope infusion day.