Dietary Effects of Sulfur Amino Acids
Condition: Healthy
Study type: Observational

Summary: 
      The purpose of this study is to determine the level of sulfur amino acids in the diet that is
      needed to maintaining glutathione antioxidant status. This information could prove very
      useful in medicine because it could allow identification of an optimal diet for control of
      antioxidant levels. This diet could be used in conjunction with other therapies in order to
      prevent or delay oxidative stress and any diseases that may result from this. Total study
      participation will include a screening visit and a 14-day study period where dietary intake
      of sulfur amino acids will be controlled. We aim to enroll 12 normal subjects at Emory
      University Hospital.The primary hypothesis is that steady-state GSH/GSSG and CYS/CYSS redox
      in human plasma will become oxidized due to SAA intake at 0% of the RDA and reduced due to
      SAA intake at 3X the RDA.
    

Detailed description: 
      The long-term objectives of this research are to improve the definition of optimal human SAA
      intake. This will include defining Dietary Reference Intakes (DRI), estimated average
      requirement (EAR), and the upper limit (UL) of intake by improving the understanding of the
      physiologic and pathologic responses to variatiation in SAA intake. Using high-resolution
      1H-NMR, we plan on developing comprehensive metabolic profiles that can indicate when SAA
      intake is inadequate.

      The specific aims of the current protocol are:

        1. To determine whether steady-state GSH/GSSG or Cys/CySS redox in human plasma is oxidized
           due to sulfur amino acid intake at 0% of the RDA.

        2. To determine whether steady-state GSH/GSSG or Cys/CySS becomes more reducing during
           dietary sulfur amino acid repletion with SAA at 3X the RDA.

        3. To determine whether low or high sulfur amino acid intakes induce primary and secondary
           metabolic effects detectable by high-resolution 1H-NMR.

      B. BACKGROUND AND SIGNIFICANCE:

      Sulfur amino acids are involved in central metabolic processes. The biologically useful
      sulfur is derived almost exclusively from the amino acids methionine and cysteine. Methionine
      (Met) is an essential amino acid and is metabolized in individuals by the transulfuration
      pathway to form cysteine (Cys) (1). In addition to use in the primary sequence of most
      proteins, both Met and Cys are required for other metabolic functions. Met is converted to
      S-adenosylmethionine, which is used for methylation reactions (2) for structural and
      functional modifications of proteins, RNA and DNA, as well as synthesis of phospholipids and
      signaling molecules. The carbon skeleton of Met is also used for biosynthesis of polyamines,
      which are required for cell division and cell growth (3). Cysteine is used for biosynthesis
      of glutathione (GSH), coenzyme A, taurine and sulfate (4). GSH functions in redox regulation
      (5) and detoxification of oxidants and reactive electrophiles (6). Coenzyme A is central to
      fatty acid metabolism and the citric acid cycle; taurine is utilized for bile acid synthesis
      and osmotic regulation (7); sulfate is used as a structural component of oligosaccharides
      (8), transport of steroid hormones (9) and detoxification of foreign compounds (10). Thus,
      there is a requirement for adequate sulfur amino acid intake that extends beyond the need for
      adequate amounts to maintain normal protein synthesis and turnover.

      Sulfur amino acid intake in humans is variable and optimal SAA intake has not been adequately
      defined. The average American diet contains about 100 g of protein daily (4); the mean SAA
      intake is about 2.4 g (11). Daily intakes range from < 0.3 g to > 5 g (11). Most of the SAA
      is derived from animal protein, which constitutes about 2/3 of the total protein intake. The
      content of SAA in legumes and some types of nuts is about half of that in animal protein
      while most other plant-derived foods contain only 10-20% of the SAA found in an equivalent
      amount of animal protein. Because plant-derived foods are low in total protein, individuals
      who do not consume animal protein are at risk of SAA deficiency. Conversely, if excess SAA
      has associated health risks due to H2S or other toxic metabolites generated by colonic flora,
      individuals consuming diets high in animal protein would be at risk (12). Hence, Dietary
      Reference Intake values for SAA are needed as a foundation for improved dietary guidelines.

      Differences in quantity and timing of SAA intake can result in substantial effects on Met,
      Cys and GSH homeostasis. Animal studies show that there is a diurnal variation in hepatic GSH
      in which the level increases following consumption of foods high in Cys and decreases in the
      post-absorptive period (16-17). The total amount of Cys present in GSH in the human body is
      about 3 g, roughly equal to the SAA requirement for 3 d (13) so that the Cys
      supply/utilization pattern can be considered in 4 phases. In the first phase, systemic Cys
      supply is from dietary Cys and metabolism of dietary Met. During this phase, excess Cys
      signals enhanced catabolism (18). Under post-absorptive conditions, probably for a period of
      about a day, systemic supply of Cys occurs principally from hepatic release of GSH (14).
      Under these conditions, Cys metabolism continues to support biosynthetic needs for taurine
      and sulfate. Although not explicitly established for humans, diurnal variation in hepatic GSH
      in mice ceases after 24 h of fasting so that systemic Cys supply probably enters a third
      phase after about 24 h. In this phase, an increased fraction of total systemic supply is
      likely to be derived from non-hepatic tissues (e.g., skeletal muscle; 15). Because sulfate
      and taurine production normally occurs in the intestines and liver (19), a decline in
      utilization of Cys for these functions is expected during this phase. A fourth phase appears
      likely to occur after about 3 days in which conservation of Met and Cys would be maximal.

      In addition, detoxification of foreign compounds by conjugation with GSH, sulfate or taurine
      requires utilization of Cys. Such use can impose a substantial SAA demand; for instance,
      metabolism of 4 doses of maximum strength acetaminophen, i.e., 4 g daily, will consume the
      equivalent of up to 250 mg Cys for GSH conjugation and 640 mg Cys for sulfate conjugation
      (13). This amount is very similar to the RDA for SAA. Thus, the combination of dietary and
      environmental factors affecting SAA homeostasis and the complex metabolic effects that can
      result from variation in SAA homeostasis emphasize the need for better means to evaluate
      adequacy of SAA nutrition. This need is present for otherwise healthy individuals and is
      accentuated in acute or chronic illness and during convalescence and metabolic/nutritional
      stabilization during recovery from illness. The lower intake level was selected because it is
      similar to the lower intake levels estimated from food frequency analysis (11). This level
      could occur with a vegetarian diet with a poor selection of foods, but would not be as
      extreme as found with prolonged fasting or starvation. The upper SAA intake level was
      selected because 3X RDA is about 20% above the average intake of SAA in this country, thus
      representing a somewhat high but otherwise normal SAA intake.

      High resolution 1H-NMR and mass spectrometry provides an approach to investigate complex
      metabolic effects of diet. Traditional methods to investigate dietary and nutritional
      components have relied upon isolation and purification of substances with specific biologic
      activities. However, the very large number of compounds in the diet, the multiple effects of
      individual compounds, the metabolic changes in the compounds that occur after consumption and
      the complex interactions of the compounds limit the ability to unravel complex effects such
      as those due to variation in SAA intake during health, illness and recovery from illness.

      Available high resolution metabolic analysis (21) such as 1H-NMR and mass spectrometry (MS)
      combined with bioinformatic tools provides a way to identify and establish a pattern of
      metabolic changes attributed to SAA deficiency and excess. We believe that if the environment
      is controlled and a dietary change (e.g. different level of SAA intake) is induced in an
      individual, observed metabolic changes can be ascribed to the dietary change. High resolution
      1H-NMR and MS are well suited for investigating changes in body fluid composition because a
      large number of metabolites are present in body fluids, many metabolites can be detected
      simultaneously and these metabolites can be analyzed and quantified with minimal or no sample
      preparation. Examples are available from the studies of Nicholson and coworkers.

      The use of multivariate statistical analysis of NMR data to evaluate metabolic responses of
      living systems is under development for toxicologic screening of pharmacologic compounds
      (24). The principles for detection of diet-related effects on health are the same as for the
      toxicologic use; in effect, nutritional deficiencies, excesses or intolerances can be viewed
      as toxicologic responses that are expressed in the metabolic profile. Thus, we believe that
      the information-rich character of the NMR spectra with MS data can provide a powerful
      approach to investigate both direct and indirect effects of nutritional variation in SAA
      intake. Given the fundamental roles of SAA metabolites in cell growth regulation, digestion,
      detoxification, antioxidant defense and hormonal signaling, and the potential risks of excess
      SAA intake, metabolic profiling by NMR could provide a useful approach to define optimum SAA
      intake and, in the future, to diagnose and treat SAA malnutrition.

      Considerable information is available concerning the assignment of NMR spectra of urine and
      plasma (e.g., Ref 22-24, 38-39); this information will provide a useful guide to possible
      metabolites which could vary as a function of SAA intake. 2-D NMR and other techniques are
      available for peak identification. However, the goals of this project do not require
      identification of which chemical species change, only whether changes at specific frequencies
      can be attributed to variation in SAA intake.

      Significance. The sulfur amino acids, Met and Cys, are highly variable in human foods and
      there are risks associated with both deficiency and excess. Both are required for physiologic
      processes in addition to maintenance of protein synthesis and nitrogen balance, and, in
      principle, excess of either could lead to generation of toxic sulfur metabolites in the
      colon. The present study will test 2 novel approaches to define dietary sulfur amino acid
      needs (i.e., plasma redox and metabolic analyses by NMR and MS). The results are expected to
      establish the feasibility of using these approaches for improved definition of Dietary
      Reference Intake values for these important nutrients.
    

Observational model: Cohort
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        Healthy volunteers
      
Keywords: oxidative stress; amino acids; dietary requirements