Advances In Therapy 9 No 5: 280-289, Sep.Oct.1992

Comparative Study of Subjects’ Net Nitrogen Utilization while Receiving Son, a Nutritional Amino Acid-Formula, or High Biologic-Value Egg Protein, or Egg Protein  Amino Acid-Formula

Dr. Maurizio Lucà-Moretti

Director of Research, Program Against Malnutrition, of the Inter American Medical and Health Association (USA). Director of Research of the International Nutrition Research Center (INRC) (USA).

address: International Nutrition Research Center. INRC Building. 401 W. Linton Blvd, Suite 201. Delray Beach,  

Florida 33444, USA

Abstract

This comparative, double blind, triple crossover Net Nitrogen Utilization study has determined the percentage of Anabolized Amino Acids (UAA), also known as Net Nitrogen Utilization (NNU), and of Catabolized Amino Acids (UCA) in 66 subjects during a twelve-week period. The subjects were alternatively given the amino acid formula of the Master Amino Acid Pattern (Diet A), amino acid formula of the hen whole-egg protein (Diet B), and hen whole-egg protein (Diet C). The comparative results showed that Diet A (MAP) induced a UAA (NNU) equivalent to 99%. That means that 99% of its constituent amino acids acted as precursors of body protein synthesis and that Diet A induced a UCA equivalent to 1%. That means that only 1% of its constituent amino acids originated nitrogen catabolites. The results also showed that the UAA in the 66 subjects, while receiving Diets B and C, was 28% and 32% lower respectively than while receiving diet A (MAP). These results confirm the discovery of MAP, meaning the ideal amino acid formula for human nutrition, capable of inducing a UAA (NNU) higher than the hen whole-egg protein, officially considered the protein with the highest biologic-value according to the Chemical Protein Score Tables.

Key words: Essential amino acids, nutritional rehabilitation, high biologic-value proteins, utilization of anabolized amino acids, and utilization of catabolized amino acids.

Introduction

The scientific discoveries related to proteins and their constituent amino acids began in 1820 when Braconnot discovered the essential amino acid Leucine. In 1838, Mulder identified and classified protein for the first time and remarked its importance for human life. In 1881, Schultze and Barbieri discovered Phenylalanine, and in 1889 Drechsel discovered Lysine. Between 1892 and 1897, Atwater calculated the energetic value of the proteins (extracorporeal) through the use of the calorimeter and also estimated its energetic metabolic value (corporeal). In 1901, Fischer discovered Valine, and Hopkins and Cole discovered Tryptophane. In 1904, Ehrlich discovered Isoleucine and in 1922, Muller discovered Methionine. Finally in 1935, Rose concludes the identification of the eight essential amino acids with the discovery of Threonine. In 1946, Rose estimates for the first time the daily requirements of essential amino acids for humans (Table I). Also in 1946, Block and Mitchell suggested that the biological value of the dietary proteins depends on its constituent amino acids and show that if the eight essential amino acids are not available simultaneously at the time of the protein synthesis, the intracellular deficit would limit the protein synthesis of the body (1). Since 1947, there have been numerous unsuccessful attempts to discover a protein that could induce a Utilization of Anabolized Amino acids (UAA, also known as Net Nitrogen Utilization) higher than the one induced by the hen whole-egg protein, officially considered the protein with the highest biologic value with a hypothetical value of 100 according to the “Chemical Protein Score Tables” (2).

This study has determined the UAA and the Utilization of Catabolized Amino Acids (UCA) in 66 subjects while receiving diet A (amino acid formula of MAP); diet B (amino acid formula of whole-hen egg protein); and diet C (hen whole-egg protein).

Table I. Daily Requirement of Amino Acids, W. Rose (1946)

The study population was comprised of 66 healthy subjects, 33 men and 33 women, with a mean age of 27 years (SD = 3.4) (range 22-34). The subjects were selected if they satisfied all inclusion and no exclusion criteria.

• Inclusion criteria were: (a) good health (b) age between 21 and 40 years (c) either male or female subjects.
• Exclusion criteria were:   (a) underweight (b) pregnancy or lactation (c) current disease, which could alter the N balance (d) phenylketonuria.

All 66 subjects provided informed consent to participate in the study. The 33 men and 33 women selected were randomly integrated according to sex and number into 3 matched groups of 11 men and 11 women, identified as group 1, 2 and 3. The ages, heights and ideal weights are shown in Table II.  Table III shows the characteristics of all 66 subjects.

Table II. Subject’s Characteristics by Group

SD = Standard Deviation

Table III. Characteristics of the 66 Subjects of the Study

SD = Standard Deviation

Study Design and Diet Definition

The study was carried out during a 114-day-period in double blind conditions, using a triple crossover technique (3). This technique allowed each subject to receive as an N source, diets A, B and C in different sequences. The study was divided into the following two phases:

a) The preliminary phase was conducted during a 30-day period to equalize and stabilize subject’s protein and energy metabolism, thus avoiding metabolism differences that could affect their N balance. To achieve this, the 66 subjects received the Metabolism Equalizing & Stabilizing Diet (MESD) before giving diets A, B and C (Table IV).

b) The main phase was conducted during three consecutive four-week periods (84 days) at which time the subject’s N balances were assessed to determine their UAA and the UCA during diets A, B and C.

Group 1, 2 and 3 received diets A, B and C following the obligatory sequence (Table IV).

Table IV. Sequence of Diets by Group and Period

MESD = Metabolism Equalizing & Stabilizing Diet.

Diets A, B and C consisted of an identical composition of equal amounts of protein or amino acids, carbohydrates, fats, vitamins, minerals and trace elements, and had the following characteristics:

a) Diet A provided each subject an amino acid intake of 400 mg/kg per day (equivalent to 64 mg/kg per day of N) through the amino acid formula of MAP (Table V), plus an energy intake of 50 kcal/kg per day through essentially protein-free carbohydrates and fats (Table VI).

Table V. Amino Acid Formula of the Master Amino Acid Pattern (g)

Table VI. Essentially Protein-Free Carbohydrate and Fat Foods

b) Diet B provided each subject an amino acid intake of 400 mg/kg per day (equivalent to 64 mg/kg per day of N) through the amino acid formula of the hen whole-egg protein (4), plus an energy intake of 50 kcal/kg per day through essentially protein-free carbohydrates and fats. (Table VI).

c) Diet C provided each subject an amino acid intake of 400 mg/kg per day (equivalent to 64 mg/kg per day of N) through hen whole-egg protein (5), plus an energy intake of 50 kcal/kg per day through essentially protein-free carbohydrates and fats (Table VI).

The MESD, A, B and C diets were supplemented with vitamins and minerals in accordance with the U.S. recommended daily allowance (RDA).

Methods:

Determining daily protein requirements

The daily protein requirement of each subject was determined by subtracting a factor of 100 from the subject’s height in centimeters, then multiplying the result by 0.9 (men) or 0.8 (women) in accordance with the subject’s sex. The following formulas were applied:

Men’s daily protein requirement =  [(H -100) x 0.9] g

Women’s daily protein requirement  =  [(H -100) x 0.8] g

The result was rounded off to the nearest 0.5 g.

Determining Weight

The subject’s weight in kg was determined daily in the early morning before breakfast, after the subject’s evacuation and urination. The result was rounded off to the nearest 100 g.

Determining N Balance

To determine the UAA and the UCA of each one of the 66 subjects while given the diets A, B and C, the N balance methodology was utilized. This has been the fundamental analytical technique for nutrition studies during the last 150 years (6,7).  The following formulas were applied:

B = I - O

O = (U + F + S)

B = I - (U + F + S)

B = N balance; I = N intake; O = N output; U = N loss in urine; F = N loss in feces; S = N dermal losses.

N balance represents the difference between the intake (I) and output (O) of N. The N balance could be positive (I > O) when the N intake is higher than its output (N retention), as in active growth and during pregnancy; it could be negative (I < O) when N intake is less than its output (N loss), as it could occur with an inadequate protein intake or an excessive catabolism; or it could be in equilibrium (I = O) when the difference between the N intake and output is equal to zero (neither retention nor loss of N) as it happens with an adequate protein intake.

Due to the fact that the methodology of the nitrogen balance is valid only when it is carried out in an accurate manner (8,9), the following precautions were taken during the study:

a) To avoid, or reduce to a minimum, the possible differences in the retention of N caused by a determined dietetic sequence, the triple crossover technique was utilized (3), considering the fact that the N retention increases after a negative nitrogen balance (10). Therefore, each subject received the same three diets in different sequences.

b) To avoid error common to the energetic intake, which could influence the nitrogen balance (11-13), and taking into consideration that carbohydrates have a protein saving effect (5), the diets MESD, A, B and C gave each subject a constant energy intake equivalent to 50 kcal/kg per day during the whole period of the study.

c) To achieve an energy intake equivalent to the fat and carbohydrates contained in the hen egg (14) (diet C), 900 mg of fat and 70 mg of carbohydrates were added per gram of MAP (diet A) and for each gram of the amino acid formula of the hen egg protein (diet B).

d) To avoid errors in the N intake which could influence the N balance, the carbohydrates and fat of the diets MESD, A, B and C were chosen from a list of essentially protein-free foods. (Table VI).

e) To avoid an excessive N intake in mg/kg per day, which could influence the N balance (15), the protein requirements were calculated according to the ideal weight of each subject.

f) To avoid an overvaluation of the N intake, due to an incomplete ingestion of the diet MESD, A, B and C, the total consumption of each designated portion was reached.

g) In order to maintain the double blind condition of the study, the diets A, B and C were given with the same fruit shake from foods selected from Table VI.

h) To avoid a possible error of interpretation, the N daily output of each subject, which is not usually lineal, was averaged during a four-week period corresponding to each diet.

Determining UAA and UCA

The UAA and the UCA of a specific protein can be determined through the N balance in a subject while that particular protein is given. To better understand UAA and UCA, in relation to a specific protein, it is necessary to analyze the following:

When a protein is digested, it is enzymatically hydrolyzed in the digestive tract. The protein releases its constituent amino acids. After their absorption in the small intestine, they can follow one of the following two metabolic pathways:

a) Anabolic pathway, in which the amino acids act as precursors of body protein synthesis (primary function of amino acids) without providing either energy or nitrogen catabolites.

b) Catabolic pathway, in which the amino acids are deaminated, meaning that their amino groups (NH2) are separated from their carboxylic groups (COOH) providing energy and nitrogen catabolites (secondary function of amino acids).

Determining N Balance

To determine the UAA and the UCA of each one of the 66 subjects while given the diets A, B and C, the N balance methodology was utilized. This has been the fundamental analytical technique for nutrition studies during the last 150 years (6,7).  The following formulas were applied:

B = I - O

O = (U + F + S)

B = I - (U + F + S)

B = N balance; I = N intake; O = N output; U = N loss in urine; F = N loss in feces; S = N dermal losses.

N balance represents the difference between the intake (I) and output (O) of N. The N balance could be positive (I > O) when the N intake is higher than its output (N retention), as in active growth and during pregnancy; it could be negative (I < O) when N intake is less than its output (N loss), as it could occur with an inadequate protein intake or an excessive catabolism; or it could be in equilibrium (I = O) when the difference between the N intake and output is equal to zero (neither retention nor loss of N) as it happens with an adequate protein intake.

Due to the fact that the methodology of the nitrogen balance is valid only when it is carried out in an accurate manner (8,9), the following precautions were taken during the study:

a) To avoid, or reduce to a minimum, the possible differences in the retention of N caused by a determined dietetic sequence, the triple crossover technique was utilized (3), considering the fact that the N retention increases after a negative nitrogen balance (10). Therefore, each subject received the same three diets in different sequences.

b) To avoid error common to the energetic intake, which could influence the nitrogen balance (11-13), and taking into consideration that carbohydrates have a protein saving effect (5), the diets MESD, A, B and C gave each subject a constant energy intake equivalent to 50 kcal/kg per day during the whole period of the study.

c) To achieve an energy intake equivalent to the fat and carbohydrates contained in the hen egg (14) (diet C), 900 mg of fat and 70 mg of carbohydrates were added per gram of MAP (diet A) and for each gram of the amino acid formula of the hen egg protein (diet B).

d) To avoid errors in the N intake which could influence the N balance, the carbohydrates and fat of the diets MESD, A, B and C were chosen from a list of essentially protein-free foods. (Table VI).

e) To avoid an excessive N intake in mg/kg per day, which could influence the N balance (15), the protein requirements were calculated according to the ideal weight of each subject.

f) To avoid an overvaluation of the N intake, due to an incomplete ingestion of the diet MESD, A, B and C, the total consumption of each designated portion was reached.

g) In order to maintain the double blind condition of the study, the diets A, B and C were given with the same fruit shake from foods selected from Table VI.

h) To avoid a possible error of interpretation, the N daily output of each subject, which is not usually lineal, was averaged during a four-week period corresponding to each diet.

Determining UAA and UCA

The UAA and the UCA of a specific protein can be determined through the N balance in a subject while that particular protein is given. To better understand UAA and UCA, in relation to a specific protein, it is necessary to analyze the following:

When a protein is digested, it is enzymatically hydrolyzed in the digestive tract. The protein releases its constituent amino acids. After their absorption in the small intestine, they can follow one of the following two metabolic pathways:

a) Anabolic pathway, in which the amino acids act as precursors of body protein synthesis (primary function of amino acids) without providing either energy or nitrogen catabolites.

b) Catabolic pathway, in which the amino acids are deaminated, meaning that their amino groups (NH2) are separated from their carboxylic groups (COOH) providing energy and nitrogen catabolites (secondary function of amino acids).

Therefore, the percentage of Amino Acid Utilization (AU) of a specific protein is equal to the sum of its UAA & UCA, according to the following equation:

AU = (UAA + UCA) %

Thus, the UAA corresponds to the percentage of the constituent amino acids of a specific protein that follow the anabolic pathway, while the UCA corresponds to the percentage of its constituent amino acids that follow the catabolic pathway.

Consequently, the higher the percentage of UAA of a specific protein, the lesser would be its energetic supply, and the lesser the percentage of nitrogen catabolites would originate.  On the contrary, the higher the percentage of UCA of a specific protein, the higher its energetic supply would be and the higher the percentage of nitrogen catabolites that would originate.

To determine the UAA in each of the 66 subjects during diets A, B and C, it was taken into consideration that:

a) When the nitrogen balance is negative (I < O), or it is in equilibrium (I = O), the UAA can be calculated.

b) When the nitrogen balance is positive (I > O), the UAA can not be calculated.

In order to determine the percentage of UAA and UCA, the following procedure was applied:

Knowing the negative nitrogen balance (mg/kg/day) corresponding to each subject, its percentage was determined utilizing the following equation:

Nitrogen Balance (%)  =     (I - O) mg/kg/day     x 100

                                                                                              I   mg/kg/day

Knowing the negative nitrogen balance percentage corresponding to each subject, the UAA was determined utilizing the following equation:

  UAA = 100 + (nitrogen balance %)

Knowing the percentage of UAA corresponding to each subject, the UCA percentage was determined utilizing the following equation:

UCA = 100 - UAA %

Determining N intake

To determine the N intake, the following formula was applied:

Quantity of dietary proteins = Quantity of dietary N x 6.25

In which the utilization of 6.25 implies that the mean N content of a dietary protein is equivalent to 16%.

Determining N output

The urine (U) and the feces (F) of each subject were collected daily during the four-week period corresponding to each diet to determine its N content through the analytic method of Kjeldahl (17).  To avoid error in the calculation of the N output, an enema was given to each subject before and after the MESD, A, B and C diets. Due to the fact that the measurement of the direct N output through the skin and other minor paths (S) is not a common procedure, such output was calculated in each subject with the help of a constant utilizing the following formula:

S = 5 mg x weight (actual) of the subject (kg) x day

Determining loss of lean tissue

The loss of lean tissue in each subject was determined multiplying the N loss of each subject by the 6.25 and then by the 5 factor. The following formula was applied:

Lean tissue = (N x 6.25 x 5) g

Considering that the N content of the different body proteins are equivalent to 16%, the loss of 1 g of N represents a loss of 6.25 g of body protein. The intracellular protein is found in the lean tissue in an aqueous solution at a concentration of 20 to 25%. Therefore, for each 5 g of hydrated lean tissue there is 1 g of protein. Consequently, the elimination of 1 g of N represents a loss of 31.25 g of lean tissue.

Data Analysis

Data were analyzed using the analysis of variance (ANOVA), followed by the Student-Newman-Keuls test.

Results:

Nitrogen Balance

Table VII summarizes the N balance results of the subjects by group while receiving diets A, B and C. Table VIII shows the results of the N balance of the 66 subjects while receiving such diets.

Table VII. Nitrogen Balance (mg/kg per day) Results by Group and Diet

N = nitrogen; SD = Standard Deviation; N Balance = N intake – N Output.

The comparison of the mean and output differences within groups 1, 2 and 3 between diets A, B and C was statistically significant (P < 0.001). The comparison of the mean and output differences by each diet (A, B and C), between groups, had a high statistical significance (P < 0.001) for diets B and C in each case while having a lower or no statistical significance for diet A (MAP).

Effectiveness

Each subject belonging to groups 1, 2 and 3 while receiving MAP (diet A) showed the lowest N output and a UAA significantly higher (P < 0,001). Also while given diet A, each one of the subjects of this group kept a nitrogen balance in equilibrium (Table VII and VIII).

Table VIII. Nitrogen Balance (mg/kg per day) by Diet of all 66 Subjects

N = nitrogen; I = N intake; O = N Output; B = N Balance.

The variance related to the mean N output of the subjects while receiving diet A (MAP) is extremely low and constant (SD = 0,001). This indicates a high UAA during diet A (MAP) in comparison with diets B and C.

Safety and Tolerance

While receiving diet A (MAP), none of the 66 subjects reported any side effects and none showed adverse effects on blood parameters.

Discussion

Historically, recommendations for protein intake have been based on the concept that involuntary daily loss of N resulting from the catabolism of the body proteins should be replaced with an equivalent amount of N (proteic) through the ingestion of dietary proteins (18). The 66 subjects of the study maintained a N balance in equilibrium, while receiving 400 mg/kg per day of the amino acid formula of MAP (Diet A). This is equivalent to 64 mg/kg per day of N per subject. The attainment of a nitrogen balance in equilibrium does not itself permit the conclusion that 400 mg/kg per day of the amino acid formula of MAP (diet A) was nutritionally adequate. This is due to the fact that a nitrogen balance in equilibrium could also be obtained through a decrease of the body protein turnover (19, 20). The results of this study have demonstrated that even though diets A, B and C gave each subject the same N intake per kg/day, and the same energy intake, the 66 subjects obtained: 

a) A UAA equivalent to 99% while receiving diet A (MAP) (Table IX) in addition to a nitrogen balance in equilibrium.

b) A UAA 28% lower than diet A (MAP) while receiving diet B in addition to a negative nitrogen balance equivalent to a mean N loss of 18.0 mg/kg/day (SD = 0.4) (Table IX). This means a lean tissue loss of 562.5 mg/kg/day.

c) A UAA 32% lower than diet A (MAP) while receiving diet C in addition to a negative nitrogen balance with a mean N loss of 20.7 mg/kg/day (SD = 0.4) (Table IX). This means a lean tissue loss of 646.8 mg/kg/day.

Table IX. Utilization of Anabolized Amino Acids (UAA) by Diet

Conclusion

The comparative results of this double blind triple crossover study have confirmed the discovery of the Master Amino Acid Pattern (MAP), meaning the ideal amino acid formula for human nutrition, the only one capable of inducing a UAA, namely a Net Nitrogen Utilization (NNU), equivalent to 99%, which means that 99% of its constituent amino acids acted as precursors of the body’s protein synthesis. This UAA (NNU) is higher than that of any known dietary protein, even the hen whole-egg protein, until now officially considered the highest biologic-value protein. The comparative results have shown that MAP generated a UCA equivalent to 1%. This means that only 1% of its constituent amino acids generated energy and nitrogen catabolites. This UCA is the lowest compared to that of any known dietary protein. As a result, MAP provides only 0.4 Kcal per gram.

Due to such characteristics, MAP could be highly beneficial in the dietary treatment of patients with:

a) Protein-Energy Malnutrition (PEM)
b) Anorexia
c) Bulimia
d) Renal insufficiency
e) Hepatic insufficiency
f) Anemia due to insufficient erythropoiesis
g) Lack of appetite
h) Vomiting
i)  Uncontrollable diarrhea
j)  Excessive catabolism
k) Obesity

It can also be concluded that the discovery of MAP allows, for the first time, the calculation of the specific UAA  (NNU) and UCA of a nutritional protein in an accurate and specific manner (with an error margin less than 1%).
 

References

1) Block RJ, Mitchell HH. The Correlation of the Amino-Acid Composition of Protein with their Nutritive Value. Nutr. Abstr. Rev. 1946; 16:249-278.
2) WHO/FAO/UNU Report. Energy and Protein Requirements. Genève, Switzerland; WHO Technical Report Series, 1965; 301; 48.
3) Cochran WG. Experimental Designs. New York, NY: John Wiley and sons: 1957; 130.
4) Orr ML. Watt BK. Amino Acid Content of Foods. US Department of Agriculture, 1957.
5 )INCAP-ICNND Report. Composition of Foods. Interdepartmental Committee on Nutrition for National Defense, National Institutes of Health. Bethesda, Maryland, USA, 1961.
6) Peters JP, Van Slyke DD. Quantitative Clinical Chemistry Interpretations. Vol. 1, 2a ed. Baltimore, Md: Williams & Wilkins; 1946.
7) Munro HN. Carbohydrate and Fat as Factors in Protein Utilization and Metabolism. Physiol. Rev. 1951; 31: 449-488.
8) Sukhatme PV, Margen S. Models for Protein Deficiency. Am. J. Clin. Nutr. 1978; 31:1237-1256.
9) Oddoye EA, Margen S. Nitrogen Balance Studies in Humans: Long Term Effect of High Nitrogen Intake on Nitrogen Accretion. J. Nutr. 1979; 109:363-377.
10 Lusk G. The Physiological Effect of Undernutrition. Physiol. Rev. 1921; 1:523-552.
11) Felig P. Inter-organ Amino Acid Exchange. En: Waterlow JC, Stephen JML. Eds Nitrogen Metabolism in Man. London, England: London Applied Science Publishers, 1981; 45-62.
12) Kinney JM. Energy Metabolism. En: Fisher JE. ed. Surgical Nutrition. Boston, Mass; Little, Brown and Co; 1983; 97-126.
13) Contaldo F, Presta E, Di Biase G, Mancini M. Nitrogen Loss and Urinary Creatinine Excretion During Fasting in Massive Obesity. Int. J. Obesity: 1982: 6:97-100.
14) Strang JM, McCluggage HB, Evans FA. The Nitrogen Balance During Dietary Correction of Obesity. Am. J: Med. Sci. 1931; 181: 336-349.
15) Wallace WM. Nitrogen Content of the Body and its Relation to Retention and Loss of Nitrogen. Fed Proc 1959; 18:1125-1130.
16) Nutritional Evaluation of Protein Foods. Washington D.C.; National Academy of Sciences, 1978.
17) Steyer Mark, A., Albert, H.K., Alvise, V.A., et al. Anal. Chem. 23:523-528. 1951.
18) WHO/FAO/UNU Report. Energy and Protein Requirements. Genève, Switzerland; WHO Technical Report Series, No. 522; 1973.
19) Golden MHN, Waterlow JC, Picou D. Protein Turnover, Synthesis and Breakdown Before and After Recovery from Protein Energy Malnutrition. Clin. Sci. Mol. Med. 1977; 53:473-477.
20)    Rennie MJ, Edwards RHT, Emery PW et al. Depressed Protein Synthesis is the Dominant Characteristic of Muscle Wasting and Cachexia. Clin. Physiol. 1983; 3: 387-398.

ALTRI LAVORI SCIENTIFICI SVOLTI SUL M.A.P.