Ma1 J Nutr 12(1): 67-78, 2006 Urinary Amino Acids Profile of Vegetarians and Non-vegetarians

Chia Yoke Yin & Ton So Ha

Monash University Malaysia, No. 2 Jalan Kolej, Bandar Sunway, 46150 Petaling Jaya, Selangor Darul Ehsan, Malaysia

ABSTRACT

The objective of the study was to quantify and to profile the amino acids content in urine samples. The amino acids content in urine was determined in 162 individuals (62 young non-vegetarians aged 15-45 years, 24 elderly non-vegetarians aged 46-70 years, 40 young vegetarians and 36 elderly vegetarians) by high performance liquid chromatography (HPLC). The most common amino acids detected in the young and elderly individuals on vegetarian and non-vegetarian diets were phenylalanine, threonine, arginine and asparagine, while leucine, aspartic acid and alanine were not found in any urine samples in both groups. Isoleucine was not detected in the urine of vegetarians. The concentrations of the majority of essential amino acids were between 0.10 - 2.00 mgl24hrs except for histidine which had a range of 4.1 - 5.0 mgl24hrs. The concentrations of non-essential amino acids varied. Proline, glycine and tyrosine concentrations were between 0.10 - 1.00 mg/24hrs, while cysteine, glutamine, glutamic acid and cystine concentrations were between 11.0 - 21.0 mg124hrs. Asparagine and hydroxy-proline had a range of 0.10 - 5.00 mg/24hrs, while serine and arginine ranged between 31.0 - 50.0 mg124hrs. Isoleucine and serine were not detected in elderly vegetarians while histidine, glycine, glutamic acid and hydroxy-proline were not detected in elderly non-vegetarians. Isoleucine, glycine and hydroxy-proline were detected in young non-vegetarians but not in young vegetarians. The levels of amino acids showed no significant statistical differences between young vegetarians and non-vegetarians as well as between elderly vegetarians and non-vegetarians. Phenylalanine, threonine and trypthophan were commonly detected in the lacto-ovo and lacto vegetarians, while valine, cysteine, arginine and asparagine were commonly detected in vegans. In conclusion, except for isoleucine, general differences were seen in urinary amino acid excretions between vegetarians and non-vegetarians even though the differences were statistically not significant. Therefore lacto-ovo diets could be nutritionally adequate as the nutrients were substituted by dairy or plant products.

Correspondence author: Dr Ton So Ha, email address: [email protected] 68 Chia YY & Ton SH

INTRODUCTION variations may exist to the extent in which animal products are avoided (Griffith and Quantification of total and individ- Omar, 2003). ual amino acids in biological fluids such as As not much information could be plasma, urine and cerebrospinal fluid is an found in the scientific literature on the important diagnostic tool in laboratory urinary amino acids excretion in vegetari- medicine (Babu et al., 2002). The total ans and non-vegetarians, this study was amino acid contents and amino acid undertaken to determine the urinary profiles in plasma and urine also reflect amino acid profiles in individuals on a the nutritional/metabolic status of an indi- vegetarian or normal diet. Vegetarians in vidual (Babu et al., 2002). this study included lacto-ovo, lacto- Amino acids are the primary compo- vegetarians and vegans. nents of proteins and they are essential to life (Elliot and Elliot, 2001). Humans ingest far more protein (amino acids) than they MATERIALS AND METHODS need for replacement of endogenous proteins. These excess amino acids cannot Sampling be stored and thus are catabolised. The ultimate fate of the carbon skeletons The total number of subjects studied derived from these amino acids is convert- was 162 with 102 young volunteers and 60 ed to carbon dioxide (C02), water (H20) elderly volunteers. In the present study, and energy (ATP) via the citric acid cycle young individuals were between 15 - 45 and the respiratory chain (Nelson and years old while elderly individuals were Cox, 2000). between 46 - 70 years old. There were 62 The kidney plays a major role in young individuals and 24 elderly individ- amino acid metabolism and nutrition. uals who were on a normal diet. In the Amino acid reabsorption at the renal vegetarian group, there were 40 young proximal tubules is mediated by the individuals and 36 elderly individuals specialised amino acid transport systems. (Figure 1).In the present study, there were Amino acids are filtered and reabsorbed in 33 lacto-ovo volunteers, 19 lacto volun- the proximal tubule. Active transport is teers and 24 vegan volunteers. There were the key to the reabsorptive processes in the 46 female volunteers and 40 male volun- proximal tubule. Glucose and amino acids teers on a normal diet, while there were 34 are reabsorbed across the luminal face of female volunteers and 42 male volunteers the proximal tubule by sodium-coupled on a vegetarian diet. secondary active transport (Pesce et al., 24-hour urine samples were collected 1980). in clean plastic bottles with toluene as a The eating patterns of vegetarians preservative (Varley, Gowenlock and Bell, may vary considerably. The lacto-ovo 1998). All participants were in a good vegetarian diet is based on grains, vegeta- nutritional state, consuming their usual bles, fruits, legumes, seeds, nuts, dairy food; no special precautions were taken products and eggs but excludes meat, fish, regarding drug therapy during the time of and fowl. The lacto-vegetarian diet sample collections. Collected urine sam- excludes eggs as well as meat, fish, and ples were frozen at -20 OC until assayed, fowl. The vegan, or total vegetarian, eating usually within 2 weeks. pattern is similar to the lacto-vegetarian The volunteers were requested to pattern, as it is the complete exclusion of complete a self-administered food animal products (Griffith and Omar, 2003). frequency questionnaire. Frequencies of Even within these patterns, considerable consumption of food items (number of Urinary Amino Acids Profile of Vegetarians and Non-vegetarians 69

Elderly on non- vegetarian diet Young on ln=24) vegetarian diet

Elderly on vegetarian diet (n=36)

vegetarian diet (n=62)

Figure 1. Distribution of different group of volunteers in present study servings) were reported on an increasing, filtration was done for the standard. 400 p1 four-level scale, including never, once a of Milli-Q water and 700 pl of internal day, 1-3 times a day and 4 or more times a standard were added to the sample prepa- day. The respondents indicated their aver- ration. The solution was mixed by inver- age portions of food sizes (i.e., small, sion. 25 pl of the solution was vacuum- medium and large) (Lindmark et al., 2005) dried using the ISSllO SpeedVacB System. based on the described standard food por- 10 pl of the re-drying solution tion in Table 1. However no quantification (methanol: 1M sodium acetate: triethy- of food intake was recorded. lamine; 2:2:1) was added, vortexed and vacuum-dried. 20 p1 of derivatising solu- High performance liquid chromatogra- tion (methanol: milli-Q water: triethy- phy procedure lamine: phenylisothiocyanate; 7:1:1:1) was added and it was allowed to stand at room In this study, a reversed-phase HPLC temperature for 10 minutes before being (RP-HPLC; Perkin Elmer) was used as it vacuum-dried. 50p1 of Pico-Tag diluent offered high sensitivity, versatility and (acetonitrile: sodium acetate trihydrate: speed of analysis (Griffith and Omar, milli-Q water; 2:1:97) was added before the 2003). Standard amino acids (2.5 standard / sample was analysed. The pmoleslml) were dissolved in 0.1N HC1. solution was mixed to ensure that all sub- 0.4mM methionine sulfone was prepared stances were dissolved completely. as an internal standard. 1.4 ml of urine The HPLC system was set up accord- sample was filtered using an Acrodisc CR ing to the manual (Pico-Tag method; PTFE syringe filter (0.45 pm; 13mm) and a Waters). The method was chosen accord- 1ml latex free syringe into a test tube. No ing to the conditions tested. A standard 70 Chia YY 6 Ton SH

Table 1. Standard food portion list used in this study

Food Groupl Food Item Food portion size (S, M and L) l Number of Servings Standard portion descripfion in a Day

Eggs (M) / 2 eggs (USDA, 2002) (2 servings) Meats / Poultry Chicken (M) / -3 ounces (USDA, 2002) (2 servings) Pork [3 ounces is estimated to be about the Beef size and thickness of a deck of playing cards (Allina Hospitals & Clinics, 2005)] Seafood Prawn (M) / -3 ounces (USDA, 2002) (2 servings) Crab [3 ounces is estimated to be about the Fish size and thickness of a deck of playing Squid cards (Allina Hospitals & Clinics, 2005)] Shell seafood (clam, etc) Vegetables Leaf vegetable (spinach, etc) (M) / 4 cups (USDA, 2002) (4 servings) Fungi/ Seaweed (M) / - 2 cups (USDA, 2002)[1 cup is Root vegetable (carrot, etc) estimated to be about the size of a fist Onion-family vegetables (Allina Hospitals & Clinics, 2005)l Tubers (potato, etc) Juices (M) / - 3 cups (USDA, 2002) Beans (2 servings) Soy beans (tofu, etc) (M) / - 1 cup (USDA, 2002) Sprouts Lentils Nuts (M) / - 2 / 3 cup (USDA, 2002) Others (peanut butter) (M) / 2 tablespoons (USDA, 2002) [I tablespoon is estimated to be about the size of the tip of a thumb (Allina Hospitals & Clinics, 2005)] Fruits(3 servings) (M) / 1 medium size (USDA, 2002) [I medium size is estimated to be about the size of a tennis ball (Allina Hospitals & Clinics, 2005)l Juices (M) / - 3 cups (USDA, 2002) Rice/ Noodles (M) / - 3 cups (USDA, 2002) (6 - 9 servings) Grains (M) / - 6 cups (USDA, 2002) (6 - 9 servings) Dairy Products Milk/ Cheese/ Yogurt (M) / - 2 cups of milk/yogurt or 1 M (2 - 3 servings) ounces of cheese (USDA, 2002) [I ounce is estimated to be about 4 stacked of dice (Allina Hospitals & Clinics, 2005)l Pastry / Bread (M) / - 6 slices (USDA, 2002) (6 - 9 servings) Snacks Chocolate/ Chips Soft Drinks / Alcoholic Drinks Supplements

(S) = small - half the standard portion, (M) = medium - standard portion, (L) = large - double the standard portion (Shimizu et al., 1999) Urinary Amino Acids Profile of Vegetarians and Non-vegetarians 71 gradient was created (Table 2) and was run was started. The phenylthiocarbamyl programmed into the software system amino acid derivatives were separated on (Total Chromatogram Navigator the C18 Pico-Tag physiological free amino Software-Perkin Elmer). An absorbance acid column [RP-18 endcapped- 5pm] detector was set at 254 nm. After the using a stated binary gradient at a flow method was set up, the pump was turned rate of 1.0 mllmin. on. The detector had to be auto-zeroed Amino acid derivatives were identi- before every run was carried out. 35 p1 of fied by their relative retention times based standardlsample were injected and the on the reference peaks for retention time of

Table 2. Standard gradient for amino acids in urine

Time Flow %A %B Curve Number

Table 3. Essential and non-essential amino acids in individuals on vegetarian and non- vegetarian diets

------Mean k S.D. (mg124hrs)

Vegetarian (n = 76) Non-vegetarian (n = 86) histidine isoleucine leucine lysine methionine phenylalanine threonine trypthophan valine cysteine proline glycine arginine asparagine serine glutamine cystine glutamic acid tyrosine hydroxy-proline alanine aspartic acid

(-) = not detectable 72 Chia YY G.' Ton SH

10.0 s vegetarian m non-vegetarian I A

4% A I

A A

I I I A. A 0' I Ab $ I 4 Z r L ,a ?U, I k I $'I I I A I a II i1 His Ile Met Phe Thr Val TT

Figure 2. Distribution of essential amino acids in urine of vegetarian and non-vegetarian individuals. His = Histidine; Ile = Isoleucine; Lys = Lysine; Met = Methionine; Phe = Phenylalanine; Thr = Threonine; Trp = Trypthophan; Val = Valine the internal standard (methionine sulfone, vegetarian and non-vegetarian, were which was added as an internal standard determined. Distribution patterns of solution). The ratio of the peak area of each essential and non-essential amino acids in amino acid to the internal standard the urine of vegetarian and non-vegetarian (methionine sulfone) was used to calculate individuals are shown in Figures 2 and 3. the amino acid concentrations and the As can be seen, leucine, aspartic acid and final results were expressed as alanine were not detected in both groups mg/24hours of amino acids in urine. while isoleucine was not detected in the vegetarian group. Statistical Analysis As shown in Table 3, individuals on vegetarian diets had a higher percentage The statistical analysis between the of frequency in the excretion of phenylala- groups was determined by student's t-test nine (68% vs. 35%), threonine (61% vs. and chi-square. A p-value 0.05 was con- 44%), cysteine (69% vs. 50%), glutamic sidered significant. acid (21% vs. 16%), tyrosine (13% vs. 9%) and hydroxy-proline (16% vs. 5%) while those on non-vegetarian diets had a higher RESULTS percentage of frequency in the excretion of lysine (31% vs. 26%), methionine (34% vs. In this study, urinary amino acids of 19%),proline (31% vs. 21%), arginine (68% two groups on different diets, namely Urinary Amino Acids Profile of Vegetarians and Non-vegetarians

Cys Pro Gly Arg Asn Ser Gln Css Glu Tyr Hyp

Figure 3. Distribution of non-essential amino acids in urine of vegetarian and non-vegetarian individuals. Cys = Csyteine; Pro = Proline; Gly = Glycine; Arg = Arginine; Asn = Asparagine; Ser = Serine; Gln = Glutamine; Css = Cystine; Glu = Glutamic acid; Tyr = Tyrosine; Hyp = Hydroxy-proline vs. 51%), asparagines (54% vs. 43%), serine histidine which had a range of 4.1 - 5.0 (12% vs. 3%), and cystine (40% vs. 37%). mgl24hrs. The concentrations of non- However, no differences were observed in essential amino acids in both groups all amino acids between vegetarians and varied. Proline, glycine and tyrosine non-vegetarians (p>0.05). ranged between 0.10 and 1.00 mg/24hrs, Among the vegetarians, histidine, while cysteine, glutamine, glutamic acid glycine and tyrosine were only found in and cystine ranged between 11.0 and 21.0 lacto-ovo and vegans while serine was mg124hrs. Asparagine and hydroxy- only detected in lacto individuals and not proline had a range of 0.10 - 5.00 in the lacto-ovo and the vegans. The other mg/24hrs, while serine and arginine amino acids were commonly detected in ranged between 31.0 and 50.0 mg124hrs. lacto-ovo, lacto and vegan individuals. Cysteine, glutamine and hydroxy-proline The concentrations of the majority of were found to have differences between essential amino acids in both groups were the two groups, where cysteine and gluta- between 0.10 and 2.00 mg/24hrs, except mine had a higher concentration range in 74 Chia YY & Ton SH vegetarians while hydroxy-proline had a A comparison of mean and standard higher concentration range in non- deviation computed from individuals on vegetarians (p>0.05). vegetarian and non-vegetarian diets, and Young individuals on non- percentage frequency (presence of amino vegetarian diets excreted isoleucine, acid in a group/ total of samples in a glycine and hydroxy-proline in their urine, group) of both essential and non-essential which were not found in the young vege- amino acids in individuals on vegetarian tarians (Table 4). Isoleucine and serine and non-vegetarian diets are seen in were found in the urine of elderly non- Tables 3, 4 and 5 respectively. vegetarian individuals but not in elderly Concentrations of lysine, phenylalanine, vegetarian individuals. However threonine, trypthophan, valine, cysteine, histidine, glycine, glutamic acid and glycine, asparagine, serine, glutamine, hydroxy-proline were not found in the cystine, glutamic acid and tyrosine were urine of elderly'non-vegetarians but were seen to be higher among vegetarian indi- found in elderly vegetarians. viduals while histidine, methionine,

Table 4. Essential and non-essential amino acids in young and elderly individuals on vegetarian and non-vegetarian diets.

Mean + S.D.

Young Young Elderly Elderly non-vegetarian vegetarian non-vegetarian vegetarian (n = 62) (n = 40) (n = 24) (n = 36) histidine isoleucine leucine lysine methionine phenylalanine threonine trypthophan valine cysteine proline glycine arginine asparagine serine glutamine cystine glutamic acid tyrosine hydroxy-proline alanine aspartic acid

-- -- -

(-) = not detectable Urinary Amino Acids Profile of Vegetariaizs and Non-vegetarians 75

Table 5. Percentage detection frequency of both essential and non-essential amino acids in individuals on vegetarian and non-vegetarian diets. I Detection frequency (%)

Vegetarian (iz = 76) Non-vegetarian (n = 86) p-value histidine isoleucine leucine lysine methionine phenylalanine threonine trypthophan valine cysteine proline glycine arginine asparagine serine glutamine cystine glutamic acid tyrosine hydroxy-proline alanine aspartic acid

(-) = not detectable Detection frequency = Presences of amino acid in a group Total samples in a group

proline, arginine and hydroxy-proline Most foods contain at least some protein were higher among non-vegetarian (Nelson and Cox, 2000). Therefore, the individuals. No statistically significant amount of amino acids excreted in the differences were observed in any of the urine will be dependent on the food levels of amino acids between vegetarians consumed and its requirement by the indi- and non-vegetarians (p>0.05). vidual. The advantages of urine analysis are that they reveal more distinctive patterns DISCUSSION related to problems in enzymatic activity, nutrient cofactor adequacy and transport. Different foods contain different pro- In fact, urine is also not subjected to the teins, each with their own unique amino circadian rhythm variation in amino acids acid composition (Nelson and Cox, 2000). that are present in blood, and excesses or The proportions of essential amino acids deficiencies over a period of time can be in foods differ from the proportions need- more easily assessed (Carroll and Temte, ed by the body to synthesise proteins. 2000). 76 Chia YY & Ton SH

Amino acid analysis is an extremely of non-vegetarians, but not in the urine of difficult task as there are some 40 to 50 any vegetarians. This could be due to the amino acids or amino acid conjugates in consumption of seafoods by the non- biological fluids, with more than a 1000- vegetarians as seen from their diet, since it fold range of concentrations (Walker and contains a high concentration of valine Mills, 1995). However, there are only 20 and isoleucine (Dwyer, 1999). No leucine, amino acids which are necessary for alanine and aspartic acid were detected in proper human growth and functions both groups. These amino acids are found (Elliot and Elliot, 2001; Nelson and Cox, moderately in poultry and in low levels in 2000). In this study, 22 amino acids were soy products (USDA, 2005). Therefore, it analysed, as cystine and hydroxy-proline could be due to low consumption (small were added in the detection. serving size) of poultry among the non- A fresh random urine sample is vegetarians while among vegetarians it adequate for amino acid analysis. The could be due to the low content of these excretion rates of amino acids may vary amino acids in the food itself. independently over 24 hours. Therefore, a Neither cysteine nor cystine is essen- 24 hour collection is necessary if accurate tial in the diet of humans; cystine and quantitative data are required (Walker and cysteine are interconvertible and cysteine Mills, 1995). is synthesised in the body from serine and Very little work has been done on the methionine (Nelson and Cox, 2000). The amino acid profiles in the urine of vegetar- vegetarians had higher mean values ian and non-vegetarian individuals. The probably due to the consumption of more range of distribution in essential and non- cysteine-rich foods such as soy beans, essential amino acids concentrations in legumes, wheat germ, broccoli, brussel this present study varied among vegetari- sprouts and oats than the non-vegetarians ans and non-vegetarians. (Dwyer, 1999). The majority of the individuals on ,Proline and hydroxy-proline are vegetarian and non-vegetarian diets who amino acids found in collagen in tendons excreted phenylalanine were found to and ligaments (Nelson and Cox, 2000). have a dietary intake of eggs and dairy Therefore, non-vegetarian individuals products. The detection frequency of were found to have a higher mean value methionine was higher in individuals on compared to the vegetarians. This could be non-vegetarian diets. Methionine is due to the consumption of animal normally activated and converted to products like poultry and meats by the homocysteine (Gomber, Dewan and Dua, non-vegetarians (Dwyer, 1999). 2004). Homocysteine is then further con- Individuals who consumed dairy verted to either cysteine or methionine. products in the vegetarian group (lacto- Normally homocysteine is an intracellular ovo and lacto) excreted more phenylala- intermediate and is not detectable in plas- nine, cystine, cysteine and glycine than ma or urine (Gomber, Dewan and Dua, vegans. This is because phenylalanine can 2004). be found in milk, whereas cystine, cysteine Tryptophan was significantly higher and glycine are found in cheese, yogurt in vegetarians compared to non- and also in milk (Dwyer, 1999; Davis et al., vegetarians (p<0.05). This could be due to 1994). the consumption of different diets where As for lacto-ovo vegetarians who vegetarians in this study consumed more consumed eggs, they excreted higher soybeans than non-vegetarians (Dwyer, amounts of glutamine, cystine, histidine 1999). and phenylalanine. This could be due to Isoleucine was detected in the urine the consumption of eggs as it is the best Urinary Amino Acids Profile of Vegetarians and Non-vegetarians 77 sources of cystine and histidine (Dwyer, Babu SSV, Shareef MM, Pavan Kumar 1999; Davis et al., 1994).On the other hand, Shetty, A & Taranath Shetty K (2002). vegans were found to excrete a higher HPLC method for amino acids pro- amount of arginine and asparagine proba- file in biological fluids and inborn bly due to the fruits and vegetables they metabolic disorders of aminoaci- consumed, as these contain high concen- dopathies. lndian J Clin Biochem 17 trations of arginine and asparagine eg. (2): 7-26. broccoli, oranges and apples (Harris, 2001). Carroll MF & Temte JL (2000). Proteinuria In conclusion, concentrations and in adults: A diagnostic approach. Am patterns of amino acids excreted in the Fam Physician 62(6): 1333 - 1340. urine by non-vegetarians and vegetarians were similar except for isoleucine, which Davis TA, NguYen HV, Garcia-Bravo R, was not found in vegetarians but in non- Fiorotto ML, Jackson EM, Lewis DS, vegetarians. No significant correlations Lee DR & Reeds PJ (1994). Amino were observed between urinary amino acid composition of human milk is acids in both groups. However, trends of not unique. J Nutr 24(7):1126. differences in the patterns of amino acid excretion between vegetarians and non- Dwyer JT (1991). Nutrition Consequences vegetarians were seen. Concentrations of of Vegetarianism. Ann Rev Nutr 11: lysine, phenylalanine, threonine, tryptho- 61 - 91. phan, valine, cysteine, glycine, asparagine, serine, glutamine, cystine, glutamic acid Elliot WH & Elliot DC (2001). and tyrosine were seen to be higher among Biochemistry and Molecular Biology. vegetarian individuals while histidine, Oxford University Press, Australia methionine, proline, arginine and hydroxy-proline were higher among non- Gomber S, Dewan P & Dua T (2004). vegetarian individuals. Homocystinuria: a rare cause of Thus even when nutrient bioavail- megaloblastic anemia. Indian J Pediatr ability is compromised (meatless diets), 41: 941-943. lacto-ovo diets can be nutritionally ade- quate as they are substituting meat prod- Griffith J & Omar H (2003). Association ucts with meat alternatives, plant or dairy between vegetarian diet and men- products in their diets (Venti and strual problems in young women: A Johnston, 2002). case presentation and brief review. J Pediatr Adolesc Gynecol 16: 319 - 323. Acknowledgements Harris W (2001). USDA National Nutrient The authors are grateful to the Database for Standard Reference, volunteers who participated in this study Washington DC and the staff of Monasl~ University Malaysia. Lindmark U, Stegmayr B, Nilsson B, Lindahl B & Johansson I (2005). Food selection associated with sense of REFERENCES coherence in adults. Nutr J 4:9.

Allina Hospitals & Clinics (2005). Food Nelson DL & Cox MM (2000). Lehninger Portions Sizes. Allina Health System Principles of Biochemistry. Worth Press, Minneapolis Publishers, United States of America 78 Chia YY & Ton SH

Pesce AJ, Clyne DH, Pollak VE, Kant SK, National Agricultural Library: Food Foulkes EC & Selenke WM (1980). and Nutrition Information Center, Renal Tubular Interactions of Beltsville Protein. Clin Biochem 13(5):209 - 215. Varley H, Gowenlock AH & Bell M (1998). Shimizu H, Ohwaki A, Kurisu Y, Practical Clinical Biochemistry. Takatsuka N, Ido M, Kawakami N, William Heinimann. Interscience Nagata C & Inaba S (1999). Validity .Publishers, New York and reproducibility of a quantitative food frequency questionnaire for a Venti CA & Johnston CS (2002) Modified cohort study in Japan. Jpn J Clin food guide pyramid for lacto vege- Oncol29(1):38-44. tarians and vegans. ] Nutr 132: 1050 - 1054. USDA (2002). Dietary Guidelines for Americans, Center for Nutrition Walker V & Mills GA (1995). Quantitative Policy and Promotion, Washington methods for amino acid ana 1y si s DC in biological fluids. Ann Clin Biochem 32 (1):28 - 57. USDA (2005). USDA National Nutrient Database: Nutrient Data Laboratory.