I last left you, my amazing readers, in Part I of my blog on protein. There, I introduced you to the concept and discussed the pitfalls of  calculating “the required amounts of protein.”

protein

Now, it’s time to dig a little deeper into these building blocks of life. Here goes…

All science is based on theory and disproving or proving a hypothesis.1 Ah, herein lies the rub – protein requirement estimates are peppered with theory. In the last blog on protein, we looked at the controversy surrounding the amount of protein needed in the diet. In order to properly evaluate this, I needed to effectively understand how the “requirements” were established in the first place.

What I discovered was an inexact science. This truly displayed the importance of going beyond a generalized recommendation and taking into account the patient’s individual needs. As I continued to dive deeper into this topic, I began to glean a better explanation of why studies on specific macronutrients, especially protein, are so conflicting. This is due to the fact that even if researchers correctly based estimates on a “verified” formula, they still may not be properly adjusting for satiation and physiological requirements due to the flaws inherent in guidelines.

I’ve included the various methods of estimates of adequate protein intake as indicated by the IOM (Institute of Medicine) below.2 So, get your geeky glasses on, grab your favorite herbal tea, and get ready to get your scientific geek on.

  1. The Factorial Method which is based on estimating nitrogen losses by incorporating a diet that meets energy needs but is essentially protein free. The protein requirement is determined to be protein needs (as nitrogen) equal the protein lost as nitrogen (nitrogen equilibrium), the zero balance point. This has several limitations. One is the actual difficulty in determining nitrogen losses.  Furthermore, this method doesn’t take into account adaption of various protein intakes in the organism. This means the efficacy of nitrogen (N) retention becomes less when approaching zero balance (dietary N intake=N losses). 1-3
  2. The Nitrogen Balance Model is calculated as the nitrogen intake subtracted from the excreted amount in urine, feces, skin, and various miscellaneous losses. This method also has several limitations including the fact that urea turnover in adults is slow and several days of adaption are needed for attaining a steady state of nitrogen excretion. Also, the execution of obtaining accurate nitrogen balance is complex and it is easy to overestimate intake and underestimate excretion. A third issue is that since the requirement is defined for the individual and there is limited evidence to account for protein needed to produce zero balance, individuals have to be studied at several levels of protein intake. Finally, the various forces of losses (dermal, miscellaneous) are difficult to measure.
  3. The Plasma Amino Acid Response Method is another means to measure protein requirements. It focuses on the physiology of individual amino acids. It is believed that when the intake of an amino acid is below the requirement, the circulating concentration will be low and will not increase with changes of intake until the target amino acid approaches the requirement level. This “constant” portion is the intersection between intake and plasma levels, and is considered an estimate of requirement. Another variation of this calculation is examining the changes of plasma concentration of the test amino acid from postabsorptive to absoptive (fed) state, whereas, the plasma concentration will only rise when the supply of the amino acid is greater than the requirement. The main disadvantage of this approach is the complexity of the factors that influence amino acid metabolism besides intake, including gastric emptying time. Furthermore, the relationship between intake and concentration is hard to calculate, as with the DAAO method below.
  4. The Direct Amino Acid Oxidation (DAAO) method uses the measurements of the carbon oxidation of a single amino acid as the indicator of adequacy. (Ahh, remember those lovely days of biochemistry and counting electrons for calculating oxidation states?) This is based on the theory that the nutritional role of an amino acid is a function of its inability to synthesize its carbon skeleton. This “13 C protein breath test4 uses a test amino acid labeled with 13C, and the production of 13CO2 in the breath is considered a good measure of the oxidative loss of the amino acid. There are several limitations to this method. The first is the reliance on low intake to test the amino acid in order to determine the breakpoint in the relationship between carbon catabolism of the amino acid and its intake. This can lead to errors of estimation. Another issue is that it has limited use, largely to only branched chain amino acids in which we have the ability to measure their carboxyl group in the body. A criticism of this method was that the measurements were only during a short period of time over hour intervals.
  5. The Twenty-Four Hour Amino Acid Balance method is based on the DAAO method which estimates the carbon balance of an amino acid over a 24-hour period. This would assist with the limitation of DAAO estimation being based on a short period of time; however, drawbacks to this method, as stated by the IOM, “arises from the unresolved questions related to the method’s theoretical basis.”  Due to the methodology of assessing oxidation rate for some amino acids, which have different metabolism pathways, underestimation could occur. Another drawback is that the method itself is labor intensive.
  6. The Indicator Amino Acid Oxidation (IAAO) method is based on work with neonatal pigs. Interestingly, it was also verified in piglets by comparing the estimates to growth and body composition, demonstrating some reliability in mammals. This method is also based on amino acid oxidation but uses a nonlimiting amino acid (“indicator amino acid”) as a carbon equivalent of nitrogen balance. It is based on reasoning that if this single amino acid is provided below its requirement, it limits retention of other nonlimiting amino acids and causes all amino acids, including itself, to be oxidized. Therefore, when the intake of the amino acid is zero, protein synthesis is minimized and oxidation of this indicator is highest.

This method decreases the aggravation of assessing different amino acids, as carbon dioxide release applies only to the indicator amino acid. Limitations still exist; however, such as its only been studied in the fed state and not in the fasting state, dependence on total protein amount during isotope infusion hasn’t been established, and finally the choice of the best indicator was not established at the time, this method being based on the assumption on the general applicability of the amino acid that is used as the indicator.15

In older human studies, the IOM reports that it has been shown that it takes 7-10 days for urinary nitrogen to balance after a protein-free diet; whereas leucine reaches equilibrium is complete within 24 hours (90%). Therefore, it’s fair to assume that amino acid metabolism is quicker than nitrogen excretion.

The Food and Agriculture Organization of the United Nations states that the current use of nitrogen balance studies does constitute the most acceptable direct evidence short term, but that long-term nitrogen balance data is lacking:

None of the current evidence is entirely satisfactory because there is no method available for the independent validation of an optimal state of protein nutrition. The functional significance of larger and smaller total-body N pools and faster or slower protein turnover rates is unknown. Most biochemical markers (plasma retinolbinding protein, albumin, etc.) are either unchanged even after relatively long periods (30 days or more) of negative N balance or are not readily interpretable (e.g., enzyme changes). There are no functional indicators that can usefully be applied in experimental situations to detect protein inadequacy before clinically detectable changes occur. This area urgently requires further research….

….In the final analysis, one would wish to set protein allowances in accordance with such characteristics as health, growth, development, and longevity. 5

According to FAO:

  1. When energy intake rises, nutrients associated with an increase in protein in foods, such as the B-group vitamins and trace elements, increase as well.
  2. Populations with higher protein levels tend to be in healthier environments.
  3. “Thirdly, there are many different measures of health and wellbeing; the criteria are therefore complex and cannot easily be used to set physiological requirements for protein.”5

There’s a little bit of a caveat in the statement above, as there are measurements of plasma and urine amino acids that can indicate dietary deficiency current functional status. This can be considered in relationship to current symptomology as an aspect of physiological requirements. 6

Quality with Quantity

Another issue with determining a proper protein amount is the quality of protein one can assimilate and absorb. In 1989 the Joint FAO/WHO Expert Consultation on Protein Quality Evaluation suggested the use of the Protein Digestibility Corrected Amino Acid Score (PDCAAS) method for evaluating protein quality. According to the British Journal of Nutrition, “In calculating PDCAAS, the limiting amino acid score (i.e., ratio of first limiting amino acid in a gram of target food to that in a reference protein or requirement) is multiplied by protein digestibility. The PDCAAS method has now been in use for 20 years.”7

PDCAAS is corrected for fecal nitrogen digestibility and values are truncated to 100%. This truncation has caused many to question the validity of ignoring that values above 100% could provide additional benefit. This also creates issues in evaluation of the nutritional significance of proteins as part of mixed diets. Furthermore, this method’s fecal digestibility doesn’t account for loss from the colon of indispensable amino acids not absorbed in the ileum. Anti-nutritional factors, such as lectins and trypsin inhibitors, were also not accounted for.7-9

In 2011, the FAO recommended replacing the PDCAAs to the digestible indispensable amino acid score; DIAAS. DIAAS is defined as: “DIAAS % = 100 x [(mg of digestible dietary indispensable amino acid in 1 g of the dietary protein) / (mg of the same dietary indispensable amino acid in 1g of the reference protein)]. Their key findings included:

  1. Dietary amino acids should be treated as individual nutrients when evaluating for protein quality.
  2. The DIAAS method recommendation with digestibility based on true ileal digestibility, preferably determined in humans, verses fecal.
  3. Recommended amino acid scoring patterns for infants, young children and older children.

The authors further support the complexity of the issue of determining optimal intake, “While DIAAS, combining ileal amino acid digestibility with predicted bioavailability identified as the amino acid score, is a step forward it is still dependent on the score accurately predicting the biological value of the absorbed amino acid mixture and hence the overall protein quality. Because the actual metabolic demand and requirement for amino acids is complex and not fully understood, any approach to predicting protein quality will likely be imperfect to a greater or lesser extent.” 10

What the Heck Did All That Mean?

Here’s my takeaway, after pulling out several hairs, I’ve concluded that recommendations for protein intake are not an exact diet and “high protein” diets may in fact, be a relative term. If anything, my hope is that you’ve seen the flaws in calculating “recommendations” for protein, something to keep in mind when determining your optimal food choices. In my upcoming blog (blogs if I get into too much research), I want to discuss why they may assist with weight optimization and the factors to consider when selecting supplementation.

References

  1. Garland T. The Scientific Method as an Ongoing Process. University of CA, Riverside. March 20, 2015. 2.
  2. 10 Protein and Amino Acids.” Dietary Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Fatty Acids, Cholesterol, Protein, and Amino Acids (Macronutrients) . Washington, DC: The National Academies Press, 2005. http://books.nap.edu/openbook.php?record_id=10490&page=612
  3. Tome’ D, Bos C. Dietary Protein and Nitrogen Utilization. J. Nutr. July 1, 2000 vol. 130 no. 7 1868S-1873S
  4. Ghoos Y, Beaufrere B. 13C protein breath tests. Gut. 1998 Nov; 43(Suppl 3): S23–S24.
  5. FAO Corporate Document Repository. Energy and Protein Requirements: Chapter 5: Principles of Estimating Protein Requirements. Available at: http://www.fao.org/docrep/003/aa040e/AA040E05.htm
  6. Genova. Amino Acids Analysis Application Guide. Genova Diagnostics. 2009.
  7. Boye J, Wijesinha-Bettoni R, Burlingame B. Protein quality evaluation twenty years after the introduction of the protein digestibility corrected amino acid score method. Br J Nutr. 2012 Aug;108 Suppl 2:S183-211. doi: 10.1017/S0007114512002309.
  8. Schaafsma G. Advantages and limitations of the protein digestibility-corrected amino acid score (PDCAAS) as a method for evaluating protein quality in human diets. Br J Nutr. 2012 Aug;108 Suppl 2:S333-6. doi: 10.1017/S0007114512002541
  9. Schaafsma G. The Protein Digestibility-Correct Amino Acid Score. J. Nutr. July 1, 2000. 130(7):1865S-1867S. http://jn.nutrition.org/content/130/7/1865S.full
  10. Food and Agriculture Organization of the United Nations. Report of an FAO Expert Consultation. March 31-April 2, 2011. Auckland, New Zealand. 2013. http://www.fao.org/ag/humannutrition/35978-02317b979a686a57aa4593304ffc17f06.pdf

 

Note: Published originally On Designs For Health Blog and Republished with Permission.