Nutrient Requirements
JUDY D. RIBAYA-MERCADO,
of Elderly
ROBERT M. RUSSELL,3 NADINE SAHYOÃœN,
FRANK D. MORROW AND STANLEY N. GERSHOFF U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts university, Boston, MA 02111 and the Tufts university School of Nutrition, Medford, MA 02155 Increased excretion of xanthurenic acid (XA) in 24-h urine after a tryptophan load also has been re ported (3, 9). Although the vitamin B-6 requirements of elderly men and women have not been determined, the recommended daily intakes of vitamin B-6 for persons 51 y and older and for younger adults (from age 15 y for males and from age 19 y for females) are the same, i.e., 2.0 and 1.6 mg of vitamin B-6 for males and females, respectively (15). The present studies were conducted to determine the vitamin B-6 require ments of elderly men and women and the effects of two levels of dietary protein intake on these require ments.
ABSTRACT The vitamin B-6 requirements of 12 men and women over 60 y old were studied. The protocol consisted of a 5-d baseline period and four experimental periods during which the subjects successively received 0.003, 0.015, 0.0225 and 0.03375 mg of vitamin B-6/ (kg body wt-d). Dietary protein was 1.2 or 0.8 g/(kg body wt-d). At 5- or 6-d intervals, xanthurenic acid (XA) after a 5-g L-tryptophan load and 4-pyridoxic acid (4PA) in 24-h urine, erythrocyte aspartate aminotransferase activity coefficient (EAST-AC) and plasma pyridoxal-5'-phosphate (PLP) were measured. These measurements were abnormal during vitamin B-6 depletion but returned to normal during repletion. Men who ingested -120 g protein/d required 1.96 ±0.11 mg of vitamin B-6 to normalize XA; women who ingested 78 g protein/d required 1.90 ±0.18 mg of vitamin B-6 to normalize XA. To attain normal levels of EAST-AC and 4-PA in men, 2.88 ±0.17 mg of vitamin B-6 were needed; to normalize PLP, 1.96 ±0.11 mg of vitamin .B-6 were required. Women required 1.90 ±0.18 mg or more of vitamin B-6 to normalize these measurements. Vitamin
B-6
requirements
were
not decreased
MATERIALS AND METHODS Subjects. The 12 healthy subjects (six men and six women) who completed this study were at least 60 y of age. They were recruited by advertisements, mail and presentations to local senior citizen groups. The volunteers underwent two screening procedures. Ini-
in two of
three subjects who ingested 54 g of protein daily. Thus, vitamin B-6 requirements of elderly men and women are about 1.96 and 1.90 mg/d, respectively. J. Nutr. 121: 1062-1074, 1991. INDEXING KEY WORDS:
•vitamin B-6 •xanthurenic acid •aspartate aminotransferase •pyiidoxal-5'-phosphate •4-pyrtdoxlc acid •humans
'An abstract describing preliminary data was presented at the meeting of the Federation of American Societies for Experimental Biology, May 1-5, 1988, Las Vegas, NV [Ribaya-Mercado, J. D., Russell, R. M., Sahyoun, N., Morrow, F. D. &.Gershoff, S. N. (1988) Vitamin B6 requirements of the elderly. FASEB J. 2: A847 (abs. 3203)]. 1This study was supported by the U. S. Department of Agricul ture, Contract Number 53-3K06-5-10. The contents of this paper do not necessarily reflect the views or policies of the U. S. Department of Agriculture nor does mention of trade names or commercial products imply endorsement by the United States government *To whom correspondence should be addressed. ^Abbreviations used: EAST-AC, erythrocyte aspartate amino transferase activity coefficient; EEC, electroencephalogram; ERP, event-related potential; 4-PA, 4-pyridoxic acid; HNRC, Human Nutrition Research Center; PLP, plasma pyridoxal-5'-phosphate; XA, xanthurenic acid.
Many studies of the elderly have indicated that the various biochemical indices used to assess vitamin B-6 nutriture frequently are abnormal. Thus, lower levels of plasma pyridoxal-S'-phosphate (PLP)*(1-7), serum pyridoxal (8), plasma total vitamin B-6 (6), aspartate aminotransferase (2-5, 9-12) and alanine aminotransferase (10, 13, 14) have been reported among elderly people compared with young adults. 0022-3166/91
$3.00 ©1991 American Institute of Nutrition. Received 19 March 1990. Accepted 19 November 1990.
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Vitamin B-6 Requirements Men and Women1'2
VITAMIN B-6 REQUIREMENTS
1063
dietary component of the study was designed to provide an isoenergetic diet, with the deficiency phase followed by the gradual addition of vitamin B-6 during the repletion phases. The amounts of vitamin B-6 ingested daily during the three repletion phases were calculated per kilogram body weight as follows: vitamin B-6-repletion I, 0.015 mg vitamin B-6/kg; vitamin B-6-repletion U, 0.0225 mg vitamin B-6/kg; vitamin B-6-repletion DI, 0.03375 mg vitamin B-6/kg. The diet during the repletion stages contained -0.5 mg of vitamin B-6 (pyridoxine) and the rest of the vitamin was provided as a supplement in the form of pyridoxine hydrochloride (Seltzer Chemicals, Carlsbad, CA) mixed in water. A 4-d final phase fol lowed the third repletion phase. During this phase the subjects ingested 50-mg supplements of pyridoxine hydrochloride (Lederle Laboratories, Pearl River, NY) daily before they were discharged. This amount of vitamin B-6 supplement is equivalent to 41.13 mg of pyridoxine. hi this report, the total amounts of vi tamin B-6 intake from the diet plus supplements are given as total pyridoxine intake. The protein content of the diet was constant throughout the study and was either 0.8 g/(kg body wt-d) or an amount 50% higher, 1.2 g/(kg body wt-d). Throughout the study, the subjects ingested adequate energy to maintain their body weight and adequate amounts of all nutrients other than vitamin B-6. Diets. During the deficiency stage, the diet con sisted primarily of a liquid formula (Ensureâ„¢)5pre pared courtesy of Ross Laboratories (Columbus, OH). It was devoid of vitamin B-6 but complete in all other nutrients. The rest of the menu was semi-synthetic and consisted of a limited number of food items. A microcrystalline cellulose product, Avicelâ„¢ (FMC, Philadelphia, PA), was used to provide fiber. Multiminerals6 (Sundown Vitamins, Hollywood, FL) and a specially prepared multivitamin7 supplement devoid of vitamin B-6 (Arther, Mountain Lakes, NJ) were provided. Sodium cascinale (Western Foods, Louis ville, KY), Pro-Modâ„¢(Ross Laboratories), whey, egg white powder and gelatin were used as the sources of
5The liquid formula diet devoid of vitamin B-6, Ensureâ„¢, con tained the following nutrients (per 1000 mL): protein, 37.2 g, fat, 37.2 g; carbohydrates, 145 & retinyl palmitate, 909 ug; cholecalciferol, 5.28 ug; all-rac-a-tocopheryl acetate, 31.7 mg; phylloquinone, 148 ug; ascorbic acid, 161 mg; folie acid, 211 ug; thiamin, 1.6 mg; riboflavin, 1.8 mg; cyanocobalamin, 6.3 mg; niacin, 21.1 mg; choline, 550 mg; biotin, 160 u& pantothenic acid, 5.3 mg; Na, 846 m& K, 1564 mg; Cl, 1436 mg; Ca, 550 mg. P, 550 mg, Mg, 211 mg; I, 80 u& Mn, 2.1 mg, Cu, 1.1 m& Zn, 15.9 mg, Fe, 9.5 mg. *The multimineral supplements provided iodine (from ocean kelp), 112.5 ug; ferrous sulfate, 9 m& calcium (elemental), 250 mg; magnesium oxide, 200 mg; copper gluconate, 500 ug; zinc sulfate, 7 mg; potassium chloride, 49.5 mg; manganese carbonate, 7 mg; selenium (primary yeast, organically bound), 5 ug; chromium chlo ride, 50 ug.
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tially a detailed medical history was obtained. Those with a family history of epilepsy, chronic sleep prob lems, nephrolithiasis, diabetes and recent alcohol abuse, and those who had taken vitamin pills con taining vitamin B-6 or medications that may affect vitamin B-6 or tryptophan metabolism during the month before the interview were excluded from par ticipating. The prospective volunteers tasted the food to be served during the study to ensure compliance with the diet. A physical examination, psychosocial assessment, electrocardiogram, chest X-ray and a va riety of blood and urine tests were performed. The tests included an eight-parameter hematological pro file, white cell differential, 16-parameter clinical chemistry profile and a complete urinalysis. Subjects whose first screening tests were normal underwent a second screening to further exclude subjects who might be prone to epileptic seizures, i.e., a clinical electroencephalogram (EEG) with measurements of event-related potentials (ERP). An oral glucose tol erance test also was given. Those with abnormal EEG, ERP or oral glucose tolerance test results were not eligible to participate in the study. Participation was not limited to any ethnic group. However, all subjects were Caucasians. Their characteristics were as fol lows: the six men had a mean age ±SEMof 64.8 ±1.2 y (range, 61-70 y); the six women had a mean age ± SEMof 65.5 ±1.4 y (range, 61-71 y). The men had a mean body weight ±SEMof 94.8 ±6.4 kg; the women had a mean body weight ±SEMof 65.7 ±4.1 kg. The mean height ±SEMwas 186.7 ±2.1 cm for the men and 156.6 ±4.6 cm for the women. The mean ±SEM body mass index (weight in kilograms divided by height in meters squared) was 27.2 ±1.7for men and 26.9 ±1.6for women. All of the subjects were either occasional or noningesters of alcoholic drinks and either former smokers or nonsmokers. Experimental design. The screening and experi mental procedures were reviewed and approved by the Human Investigation Review Committee of Tufts University. Informed consent was obtained from par ticipants. Three days before admission to the Meta bolic Research Unit of the Human Nutrition Research Center (HNRC) on Aging at Tufts University, the subjects were asked to keep a 3-d food diary. At the HNRC where the subjects resided for about 3 mo, the protocol was as follows: a 5-d baseline period during which the subjects ate self-selected diets was followed by a vitamin B-6-depletion period of up to 20 d. During this period, the subjects ingested diets con taining no more than 0.003 mg of vitamin B-6/(kg body wt-d). A person was considered vitamin B-6 deficient when the 24-h urinary XA excretion after a 5-g L-tryptophan load was 300 mg or more. To com plete neuropsychologjcal tests, the subjects were con tinued on the vitamin B-6-deficient diet for an addi tional 5 d. Three stages of vitamin B-6-repletion, each lasting 21 d, followed the B-6-depletion period. The
OF ELDERLY HUMANS
1064
RJBAYA-MERCADO
carbohydrates. For men and women consuming the lower protein level, to maintain body weight the energy intakes were 11,527 ±1895 kj (2755 ±453 kcal) and 8025 ±126 kj (1918 ±30 kcal), respectively. Ten percent of total energy was provided by protein, 38% by fat and 52% by carbohydrates. Biochemical, physiological and statistical anal yses. Biochemical analyses on fasting blood samples and 24-h urinary specimens were undertaken throughout the study. Blood specimens were collected into evacuated blood tubes (Becton-Dickinson, Rutherford, NJ) containing either no additive, EDTA or heparin anticoagulants as required for a given assay. Specimens were processed within 60 min of venipuncture by centrifugation (3000 x g for 20 min) and stored in the dark at 4*C. Serum, plasma or washed erythrocytes were stored at -70°C in cryotubes (Vangard International, Neptune, NJ) until analysis. The 24-h urine specimens were collected into opaque plastic jugs containing thymol and indomethacin as preservatives. The urine specimens were stored for up to 26 h at 5°Cprior to aliquot storage in the dark at -20*C. A colorimetrie method (17) was used to measure 24-h urinary XA excretion after a 5-g L-tryptophan (Tryptacin™, Arther, Mountain Lakes, NJ) load. The tryptophan was ad ministered 2 h after breakfast, and tests were repeated at 5- or 6-d intervals. Also measured at 5- or 6-d intervals were erythrocyte aspartate aminotransferase (EC 2.6.1.1, L-aspartate: 2-oxoglutarate-aminotransferase) activity coefficients (EAST-AC) by the method of Williams (18), plasma pyridoxal-5'-phosphate (PLP) by the radioenzymatic method of Rey nolds (19), and 24-h urinary 4-pyridoxic acid (4-PA) excretions by fluorometric reverse-phase liquid chromatography (20). Urinary creat in me was determined using a commercially available kit (Roche Diagnostic Systems, Montclair, NJ) based on a kinetic alkaline picrate procedure (21). To study the effects of vitamin B-6 status on hematopoiesis and iron metabolism, the following blood analyses were performed at the end of each study phase: serum iron and iron-binding capac ities by a commercial kit using a colorimetrie ferrozine-based method (Diagnostic Chemicals, Monroe, CT), ferritin by a 2-site immunoradiometric assay kit (Ciba Coming Magnetic Immunochemistries, Medfield, MA), and hematological measurements on an automated hematology analyzer (Baker 9000, SeronoBaker, Allentown, PA). Plasma ascorbate was mea sured using a colorimetrie procedure (22). Procedures
7The multivitamin
supplements
without
vitamin B-6 provided
ascorbic acid, 30 mg; thiamin mononitrate, 0.6 mg; riboflavin, 0.7 mg; niacin, 8 mg; folie acid, 0.2 mg; cyanocobalamin, 1.5 |i& biotm, 0.1 mg; pantothenic acid, 3.5 mg; rctinyl acetate, 86 u& cholccalciferol, 0.0625 \ig¡a-tocopheryl succinate, 4.79 mg; phylloquinone, 70 ng.
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protein. The vitamin B-6-repletion diet was a 2-d cycle menu and consisted of rice casseroles with vegetables, selected fruits and fruit juice, cheese, mar garine and other foods such as cereal, omelettes, muffins and biscuits (Table 1). All food items used for each subject were from the same lot number to avoid fluctuations in vitamin B-6 content. The recipes were developed in the research kitchen of the Metabolic Research Unit. During the repletion phases, the sub jects also received Ensure™and the multimineral and multi vitamin supplements devoid of vitamin B-6. All ingredients in the menu were analyzed for nitrogen by the Nutrition Evaluation Laboratory of the HNRC using a standard Kjeldahl procedure and for vitamin B-6 by Dr. James E. Leklem's laboratory at Oregon State University using a microbiological assay that employed Sacchaiomyces uvarum (16). Vitamin B-6-depletion and -repletion diets for a woman who weighed 68 kg, ingested 8908 kj (2129 kcal) daily and had a protein intake of 1.2 g/(kg body wt-d) are given in Table 1. Table 2 shows the actual amounts of vitamin B-6 ingested (as pyridoxine) from the diet plus supple ments during the various stages of the study. The study was designed so the total vitamin B-6 ingested was dependent on the subject's body weight. The mean ±SEMbody weight of the four men and four women who ingested 1.2 g protein/(kg body wt-d) were 99.2 ±5.5 (range, 87-112 kg) and 65.0 ±6.5 kg (range, 55-82 kg), respectively. The two men who ingested 0.8 g protein/(kg body wt-d) had a mean body weight of 86.2 kg (individual values, 68 and 104 kg); the two women had a mean body weight of 67.1 kg (individual values, 65 and 69 kg). The actual amounts of pyridoxine ingested (mean ±SEM)during the three vitamin B-6-repletion stages by men whose protein intake was maintained at 1.2 g/(kg body wt-d) were 1.34 ±0.08, 1.96 ±0.11 and 2.88 ±0.17mg/d, respec tively; for the women they were 0.89 ±0.08, 1.29 ± 0.12, and 1.90 ±0.18 mg/d, respectively. The corres ponding values for men whose protein intake was maintained at 0.8 g/(kg body wt-d) were 1.16 ±0.22, 1.70 ±0.34 and 2.50 ±0.50 mg vitamin B-6/d, respec tively,- for the women they were 0.91 ±0.02, 1.33 ± 0.03 and 1.95 ±0.05 mg vitamin B-6/d, respectively. The protein intake was dependent on the subject's body weight. The actual protein intake (mean ±SEM) of men who were assigned to the higher protein level of 1.2 g/(kg body wt-d) was 119.6 ±7.2 g (range, 104.0-135.5 g); for women, this was 77.8 ±7.7 g (range, 65.1-98.3 g). For the two men assigned to the protein intake level of 0.8 g/(kg body wt-d), the mean value was 69.1 g (individual values, 54.4 and 83.8 g),for the two women, the mean value was 54.0 g (in dividual values, 52.8 and 55.1 g). Body weight was maintained during the study with energy intakes (means ±SEM)of 12,765 ±971 kj (3051 ±232 kcal) and 8284 ±502 kj (1980 ±120 kcal) for men and women, respectively, who ingested the higher protein level. Fifteen percent of total energy was provided by protein, 35% by fat and 50% by
ET AL.
VITAMIN B-6 REQUIREMENTS
the exception of the PLP assay (6.7%). In addition, a variety of other blood and urinary analyses were per formed to determine the effects of vitamin B-6 status on the immune system and on glucose and insulin metabolism. Preliminary data have been reported (23, 24), and final results of these substudies will be re ported separately. Blood pressure was measured daily. In addition, studies were conducted in the Neuropsychology Labo ratory, Department of Neurology at the Tufts Uni versity School of Medicine to evaluate the effects of
TABLE 1 Sample vitamin B-6 depletion and vitamin B-6 repletion menus Sample vitamin B-6-depletion menu itemsBreakfastEnsureâ„¢1Pro-Modâ„¢2Avicelâ„¢ Food
Sample vitamin B-6-repletion menu
B-6mg0.00880.00380tracetracetrace0.0174trace0.00320.00370.00880.00380.00370.0174trace0.00 B-6rag0.00270.0361tra itemsBreakfastEnsureâ„¢1Orange
juiceMargarineOmeletteSwiss powder3Instant coffeeNondairy regular creamerSugarBiscuitMargarineHigh
cheeseBiscuitsInstant coffeeNondairy regular creamerSugarAvicelâ„¢
powder3LunchEnsureâ„¢1CaseinPro-Modâ„¢2High-protein puddingCaseinLunchEnsureâ„¢1Pro-Modâ„¢2CaseinBiscuitMargarineJelloPeach protein
puddingAvicelâ„¢ powder*DinnerEnsureâ„¢1CaseinPro-Modâ„¢2Vegetable
slicesDinnerEnsureâ„¢1CaseinPro-Modâ„¢2Avicelâ„¢ casseroleRiceMargarineCarrotsOnionsSwiss powder3Instant coffeeNondairy regular creamerSugarHigh-protein
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were modified as necessary to allow for precise auto mated analysis on a Cobas FarÃn centrifugal analyzer (Roche Diagnostic Systems). Quality control was pro vided by pooled specimens processed and stored using the same procedure used for the study specimens. Assays were rejected when a quality control result exceeded ±2so from an established mean value. Whenever feasible, specimens from a given subject were batched and analyzed concurrently at the end of a given study period. The within-assay coefficient of variation for all biochemical analyses was < 5%, with
OF ELDERLY HUMANS
cheeseChickenCaseinBiscuitsPear
puddingSnackEnsureâ„¢1Pro-Modâ„¢2CookiesWeight8250571.63083051315250553051741002504571.6308131250560Protein88.103.79000.2001.900.014.90 halvesHot chocolateAvicelâ„¢ powder3SnackHot
chocolateWeightg90.092.310.0113.010.030.01.630.08.010.0158.02.54.1131.0 is a vitamin B-6-deficient liquid formula (Ross Laboratories, Columbus, OH). 2Pro-Modâ„¢is a protein source (Ross Laboratories, Columbus, OH). 3Avicelâ„¢powder is a cellulose product (FMC, Philadelphia, PA) used to provide fiber.
RIBAYA-MERCADO
1066
RESULTS The 3-d food diaries kept by the subjects prior to their admission to the HNRC were analyzed by the HNRC Scientific Computing Department using the Grand Forks database (U.S. Department of Agri culture-Agriculture Research Service, Grand Forks Human Nutrition Research Center, Grand Forks, ND). The reported energy intakes per day were 9184 ± 2293 kl (2195 ±548 kcal) and 6075 ±397 kj (1452 ±95 kcal) for men and women, respectively. However, the actual energy provided during the study to maintain body weights were 8 to 44% greater than these re ported amounts. Thus, the subjects underreported their energy intakes. The protein intakes reported were 18% of energy intakes, i.e., 101 ±20 g and 65 ± 7g for men and women, respectively. Men reported ingesting 38% and 44% of their total energy as fat and carbohydrates, respectively; women reported 33% and 49% of their intake as fat and carbohydrates, respec tively. Reported vitamin B-6 intakes were 1.61 ±0.38 mg for men and 1.36 ±0.03 mg for women. However, the nutrient database does not have vitamin B-6 values for all foods so the actual vitamin B-6 intakes may be underestimated. The dietary regimen did not interfere with the usual physical activities of the subjects, and there was no evidence that the regimen had any deleterious effect on their health. Except for one man who deve loped a mild reaction (nausea) to tryptophan, all the subjects tolerated the tryptophan load well. Figure 1 shows 24-h urinary XA excretions after a 5-g L-tryptophan load during the various stages of the study for men and women whose diets contained 1.2 g protein/(kg body wt-d). At baseline, the men had an XA excretion of 7.0 ±1.2 mg/24 h. As vitamin B-6-deficiency progressed, urinary XA excretion in creased up to a maximum value of 386.4 ±55.0 mg/24 h during d 15 of the deficiency period. On vitamin B-6-repletion, XA excretion promptly decreased. Baseline value was not attained during the first vi tamin B-6-repletion stage when 1.34 ±0.08 mg of vitamin B-6 was ingested; it was attained by d 20 of the second vitamin B-6-repletion stage. Thus, for these men, who ingested -120 g of protein daily, the amount of vitamin B-6 required to normalize the XA
level was the amount given during the second repletion stage. The vitamin B-6 ingested during this period was 1.96 ±0.11 mg/d (range, 1.71-2.22 mg/d; Table 2). For women, baseline urinary XA excretion after a 5-g L-tryptophan load was 21.7 ±13.6 mg/24 h. On vitamin B-6 depletion, XA excretion rose promptly to a maximum value of 538.4 ±81.5 mg/24 h after 17 d of vitamin B-6 deficiency. On vitamin B-6 repletion, urinary XA decreased promptly; the baseline level was not attained during the second vitamin B-6-repletion stage when 1.29 ±0.12 mg vitamin B-6 was ingested. For these women who ingested -78 g of protein daily, the vitamin B-6 intake required to nor malize the XA level was 1.90 ±0.18 mg/d (range, 1.61-2.37 mg/d), the amount given during the third vitamin B-6-repletion stage (Table 2). For both men and women, the XA values were not further changed by the ingestion of 42 mg of pyridoxine during the final phase of the study. Tryptophan load test results were obtained from three of the four subjects assigned to the lower protein intake level of 0.8 g/(kg body wt-d). One of two men fed this diet was inadvertently given placebo instead of tryptophan pills due to the manufacturer's error. The other male subject ingested 54 g of protein during the study. His baseline XA value after a 5-g L-tryptophan load was 5.2 mg/24 h; on vitamin B-6 depletion, values peaked at 351.5 mg/24 h. On vitamin B-6 repletion, urinary XA excretion decreased promptly; however, the baseline level was not quite attained at the end of the third vitamin B-6-repletion phase when 1.99 mg vitamin B-6/d was ingested, nor even after 3 d of ingesting 42 mg of pyridoxine at the final phase of the study. For this person, the XA levels at these stages were 16.3 and 10.7 mg/24 h, respec tively. One woman who ingested 53 g of protein had a baseline XA value of 8.7 mg/24 h; at the end of vitamin B-6 depletion, it was 373.4 mg/24 h. Values returned to baseline during the second vitamin B-6-repletion stage when she ingested 1.30 mg of vi tamin B-6. The second woman who ingested 55 g of protein had a baseline XA value of 7.2 mg/24 h; at the end of vitamin B-6 depletion, it was 388.2 mg/24 h. Repletion with 2.00 mg of vitamin B-6 brought xanthurenic acid excretion to baseline levels.
Tecce, J. J., Raber, S. R., O'Brien, M. E., Russell, R. M., RibayaMercado, J. D., Sahyoun, N., Morrow, F. D., Sadowski, J. A &. Gershoff, S. N. (1987) Vitamin B-6 depletion and L-tryptophan effects on brain functioning. Presented at the meeting of the American College of Neuropsychopharmacology, San Juan, Puerto Rico. ^ecce, J. J., Kosta, S. M., Mazaheri, S. T., Russell, R. M., RibayaMercado, J. D. & Gershoff, S. N. (1988) Tryptophan and dividedattention effects on event-related brain potentials (CNV). Presented at the meeting of the American College of Neuropsychopharma cology, San Juan, Puerto Rico.
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vitamin B-6 depletion and repletion on brain func tioning, psychophysiological activity and alertness. Weekly mood ratings of the subjects were obtained. Preliminary results of the neuropsychological tests have been reported at scientific meetings.8-9 ANOVA and the Neumann-Keuls test were used for statistical analyses. The t test was used to compare data for men vs. women; differences were considered significant when P > ffiC
0.5n
IT*B6
oÕ(A)-
r\(B)J.ÕIA IITiI2.01.51.00.5r\ « i »B6• B6^2.88l0.17mgB6-1.96i0.11mgB6L
fBLL4 F 1.69 î 0.02
II-1.29\A.iui|
mg
»se» B61-1.90
|F L 4 1.62 10003
mg
i0.18mgB6Ã-O. B6mgB60.1710.01
1.341 0.08 mg B6m-M-
12mg
B6mg 0.89 10.08 mg 0.1010.01f•B(125.4 B6
FIGURE 4 Urinary 4-pyridoxic acid (mg/24 h) at baseline, BL; vitamin B-6-depletion, -B6; three stages of vitamin B-6-repletion, +B6 I, +B6 ÕÕ, +B6 IH; at the final phase, F. Values are means ±SEM for four men (A) and four women (B) who consumed 1.2 g protein/(kg body wt-d); means ±SEM, 119.6 ±7.2 g protein for men and 77.8 ±7.7 g for women. Each bar represents measurement obtained every 5 or 6 d.
turnover time for RBC of -120 d. The provision of 1.34 mg of pyridoxine (the first repletion dose) was insufficient to normalize XA measurements after a tryptophan load in elderly men who ingested -120 g of protein,- 1.96 mg of the vi tamin (the third repletion dose) was required. Simi larly, 0.89 and 1.29 mg of vitamin B-6 (the first and second repletion doses) were insufficient to normalize post-tryptophan XA measurements in elderly women who ingested 78 g of protein,- 1.90 mg of the vitamin (the third repletion dose) was required. These vitamin B-6 requirements expressed per gram of protein are 0.0163 and 0.0244 mg vitamin B-6/g protein for men and women, respectively. Studies have shown that the requirement for vi tamin B-6 is directly related to protein intake (27); the protein effect is mainly the result of its methionine content (28). La this study, the effects of lower protein intakes (54 g) on XA measurements and vitamin B-6 requirements were not consistent; only one of three subjects showed a reduced requirement compared with the subjects fed the higher protein level. Thus, based on XA excretion after a tryptophan load, our results indicate that the estimated vitamin B-6 re quirements of elderly men vs. elderly women with a wide range of protein intake are not greatly different. The women excreted larger amounts of XA and deve loped abnormalities in their metabolism of tryp tophan faster than the men. Similar observations have been reported in younger adults (29, 30). Although we did not study the vitamin B-6 require ments of younger adults, a comparison of our data
with reports in the literature indicates that the vi tamin B-6 requirements of elderly men and women are higher than the requirements reported for younger men and women. Baker et al. (31) found that for young men with a low protein intake (30 g), the minimal daily vitamin B-6 requirement was slightly more than 1.0 mg; for young men with a high protein intake (100 g), the minimal daily requirement was 1.5 mg. Miller and Linkswiler (32) showed that, during vitamin B-6 depletion after a 2-g L-tryptophan load, young men consuming diets containing 150 g of protein excreted larger abnormal amounts of tryp tophan metabolites than those consuming diets con taining 54 g of protein. In both instances, 1.5 mg of pyridoxine normalized tryptophan metabolite excre tion. Sauberlich (33) concluded that the normal young adult consuming 100 g of protein requires a minimum of 1.5 to 1.75 mg vitamin B-6/d,-these requirements are increased (or decreased) somewhat when more (or less) than 100 g of protein is consumed. Horwitt (34) concluded that, for young male adults and adolescents consuming 60 g of protein, 1.4 mg of vitamin B-6 should be adequate,- for those whose diets contained 100 g of protein, 1.8 mg of vitamin B-6 is more than adequate, and a 2-mg allowance should be generous. The vitamin B-6 requirements of young adult women also have been studied. Donald et al. (35) proposed that the vitamin B-6 requirement for adult women consuming a moderate protein diet (57 g) was 1.5 mg/d. Shin and Linkswiler (29) showed that, in young adult women, abnormal urinary tryptophan
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1.5
JUDY D. RIBAYA-MERCADO,
of Elderly
ROBERT M. RUSSELL,3 NADINE SAHYOÃœN,
FRANK D. MORROW AND STANLEY N. GERSHOFF U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts university, Boston, MA 02111 and the Tufts university School of Nutrition, Medford, MA 02155 Increased excretion of xanthurenic acid (XA) in 24-h urine after a tryptophan load also has been re ported (3, 9). Although the vitamin B-6 requirements of elderly men and women have not been determined, the recommended daily intakes of vitamin B-6 for persons 51 y and older and for younger adults (from age 15 y for males and from age 19 y for females) are the same, i.e., 2.0 and 1.6 mg of vitamin B-6 for males and females, respectively (15). The present studies were conducted to determine the vitamin B-6 require ments of elderly men and women and the effects of two levels of dietary protein intake on these require ments.
ABSTRACT The vitamin B-6 requirements of 12 men and women over 60 y old were studied. The protocol consisted of a 5-d baseline period and four experimental periods during which the subjects successively received 0.003, 0.015, 0.0225 and 0.03375 mg of vitamin B-6/ (kg body wt-d). Dietary protein was 1.2 or 0.8 g/(kg body wt-d). At 5- or 6-d intervals, xanthurenic acid (XA) after a 5-g L-tryptophan load and 4-pyridoxic acid (4PA) in 24-h urine, erythrocyte aspartate aminotransferase activity coefficient (EAST-AC) and plasma pyridoxal-5'-phosphate (PLP) were measured. These measurements were abnormal during vitamin B-6 depletion but returned to normal during repletion. Men who ingested -120 g protein/d required 1.96 ±0.11 mg of vitamin B-6 to normalize XA; women who ingested 78 g protein/d required 1.90 ±0.18 mg of vitamin B-6 to normalize XA. To attain normal levels of EAST-AC and 4-PA in men, 2.88 ±0.17 mg of vitamin B-6 were needed; to normalize PLP, 1.96 ±0.11 mg of vitamin .B-6 were required. Women required 1.90 ±0.18 mg or more of vitamin B-6 to normalize these measurements. Vitamin
B-6
requirements
were
not decreased
MATERIALS AND METHODS Subjects. The 12 healthy subjects (six men and six women) who completed this study were at least 60 y of age. They were recruited by advertisements, mail and presentations to local senior citizen groups. The volunteers underwent two screening procedures. Ini-
in two of
three subjects who ingested 54 g of protein daily. Thus, vitamin B-6 requirements of elderly men and women are about 1.96 and 1.90 mg/d, respectively. J. Nutr. 121: 1062-1074, 1991. INDEXING KEY WORDS:
•vitamin B-6 •xanthurenic acid •aspartate aminotransferase •pyiidoxal-5'-phosphate •4-pyrtdoxlc acid •humans
'An abstract describing preliminary data was presented at the meeting of the Federation of American Societies for Experimental Biology, May 1-5, 1988, Las Vegas, NV [Ribaya-Mercado, J. D., Russell, R. M., Sahyoun, N., Morrow, F. D. &.Gershoff, S. N. (1988) Vitamin B6 requirements of the elderly. FASEB J. 2: A847 (abs. 3203)]. 1This study was supported by the U. S. Department of Agricul ture, Contract Number 53-3K06-5-10. The contents of this paper do not necessarily reflect the views or policies of the U. S. Department of Agriculture nor does mention of trade names or commercial products imply endorsement by the United States government *To whom correspondence should be addressed. ^Abbreviations used: EAST-AC, erythrocyte aspartate amino transferase activity coefficient; EEC, electroencephalogram; ERP, event-related potential; 4-PA, 4-pyridoxic acid; HNRC, Human Nutrition Research Center; PLP, plasma pyridoxal-5'-phosphate; XA, xanthurenic acid.
Many studies of the elderly have indicated that the various biochemical indices used to assess vitamin B-6 nutriture frequently are abnormal. Thus, lower levels of plasma pyridoxal-S'-phosphate (PLP)*(1-7), serum pyridoxal (8), plasma total vitamin B-6 (6), aspartate aminotransferase (2-5, 9-12) and alanine aminotransferase (10, 13, 14) have been reported among elderly people compared with young adults. 0022-3166/91
$3.00 ©1991 American Institute of Nutrition. Received 19 March 1990. Accepted 19 November 1990.
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Vitamin B-6 Requirements Men and Women1'2
VITAMIN B-6 REQUIREMENTS
1063
dietary component of the study was designed to provide an isoenergetic diet, with the deficiency phase followed by the gradual addition of vitamin B-6 during the repletion phases. The amounts of vitamin B-6 ingested daily during the three repletion phases were calculated per kilogram body weight as follows: vitamin B-6-repletion I, 0.015 mg vitamin B-6/kg; vitamin B-6-repletion U, 0.0225 mg vitamin B-6/kg; vitamin B-6-repletion DI, 0.03375 mg vitamin B-6/kg. The diet during the repletion stages contained -0.5 mg of vitamin B-6 (pyridoxine) and the rest of the vitamin was provided as a supplement in the form of pyridoxine hydrochloride (Seltzer Chemicals, Carlsbad, CA) mixed in water. A 4-d final phase fol lowed the third repletion phase. During this phase the subjects ingested 50-mg supplements of pyridoxine hydrochloride (Lederle Laboratories, Pearl River, NY) daily before they were discharged. This amount of vitamin B-6 supplement is equivalent to 41.13 mg of pyridoxine. hi this report, the total amounts of vi tamin B-6 intake from the diet plus supplements are given as total pyridoxine intake. The protein content of the diet was constant throughout the study and was either 0.8 g/(kg body wt-d) or an amount 50% higher, 1.2 g/(kg body wt-d). Throughout the study, the subjects ingested adequate energy to maintain their body weight and adequate amounts of all nutrients other than vitamin B-6. Diets. During the deficiency stage, the diet con sisted primarily of a liquid formula (Ensureâ„¢)5pre pared courtesy of Ross Laboratories (Columbus, OH). It was devoid of vitamin B-6 but complete in all other nutrients. The rest of the menu was semi-synthetic and consisted of a limited number of food items. A microcrystalline cellulose product, Avicelâ„¢ (FMC, Philadelphia, PA), was used to provide fiber. Multiminerals6 (Sundown Vitamins, Hollywood, FL) and a specially prepared multivitamin7 supplement devoid of vitamin B-6 (Arther, Mountain Lakes, NJ) were provided. Sodium cascinale (Western Foods, Louis ville, KY), Pro-Modâ„¢(Ross Laboratories), whey, egg white powder and gelatin were used as the sources of
5The liquid formula diet devoid of vitamin B-6, Ensureâ„¢, con tained the following nutrients (per 1000 mL): protein, 37.2 g, fat, 37.2 g; carbohydrates, 145 & retinyl palmitate, 909 ug; cholecalciferol, 5.28 ug; all-rac-a-tocopheryl acetate, 31.7 mg; phylloquinone, 148 ug; ascorbic acid, 161 mg; folie acid, 211 ug; thiamin, 1.6 mg; riboflavin, 1.8 mg; cyanocobalamin, 6.3 mg; niacin, 21.1 mg; choline, 550 mg; biotin, 160 u& pantothenic acid, 5.3 mg; Na, 846 m& K, 1564 mg; Cl, 1436 mg; Ca, 550 mg. P, 550 mg, Mg, 211 mg; I, 80 u& Mn, 2.1 mg, Cu, 1.1 m& Zn, 15.9 mg, Fe, 9.5 mg. *The multimineral supplements provided iodine (from ocean kelp), 112.5 ug; ferrous sulfate, 9 m& calcium (elemental), 250 mg; magnesium oxide, 200 mg; copper gluconate, 500 ug; zinc sulfate, 7 mg; potassium chloride, 49.5 mg; manganese carbonate, 7 mg; selenium (primary yeast, organically bound), 5 ug; chromium chlo ride, 50 ug.
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tially a detailed medical history was obtained. Those with a family history of epilepsy, chronic sleep prob lems, nephrolithiasis, diabetes and recent alcohol abuse, and those who had taken vitamin pills con taining vitamin B-6 or medications that may affect vitamin B-6 or tryptophan metabolism during the month before the interview were excluded from par ticipating. The prospective volunteers tasted the food to be served during the study to ensure compliance with the diet. A physical examination, psychosocial assessment, electrocardiogram, chest X-ray and a va riety of blood and urine tests were performed. The tests included an eight-parameter hematological pro file, white cell differential, 16-parameter clinical chemistry profile and a complete urinalysis. Subjects whose first screening tests were normal underwent a second screening to further exclude subjects who might be prone to epileptic seizures, i.e., a clinical electroencephalogram (EEG) with measurements of event-related potentials (ERP). An oral glucose tol erance test also was given. Those with abnormal EEG, ERP or oral glucose tolerance test results were not eligible to participate in the study. Participation was not limited to any ethnic group. However, all subjects were Caucasians. Their characteristics were as fol lows: the six men had a mean age ±SEMof 64.8 ±1.2 y (range, 61-70 y); the six women had a mean age ± SEMof 65.5 ±1.4 y (range, 61-71 y). The men had a mean body weight ±SEMof 94.8 ±6.4 kg; the women had a mean body weight ±SEMof 65.7 ±4.1 kg. The mean height ±SEMwas 186.7 ±2.1 cm for the men and 156.6 ±4.6 cm for the women. The mean ±SEM body mass index (weight in kilograms divided by height in meters squared) was 27.2 ±1.7for men and 26.9 ±1.6for women. All of the subjects were either occasional or noningesters of alcoholic drinks and either former smokers or nonsmokers. Experimental design. The screening and experi mental procedures were reviewed and approved by the Human Investigation Review Committee of Tufts University. Informed consent was obtained from par ticipants. Three days before admission to the Meta bolic Research Unit of the Human Nutrition Research Center (HNRC) on Aging at Tufts University, the subjects were asked to keep a 3-d food diary. At the HNRC where the subjects resided for about 3 mo, the protocol was as follows: a 5-d baseline period during which the subjects ate self-selected diets was followed by a vitamin B-6-depletion period of up to 20 d. During this period, the subjects ingested diets con taining no more than 0.003 mg of vitamin B-6/(kg body wt-d). A person was considered vitamin B-6 deficient when the 24-h urinary XA excretion after a 5-g L-tryptophan load was 300 mg or more. To com plete neuropsychologjcal tests, the subjects were con tinued on the vitamin B-6-deficient diet for an addi tional 5 d. Three stages of vitamin B-6-repletion, each lasting 21 d, followed the B-6-depletion period. The
OF ELDERLY HUMANS
1064
RJBAYA-MERCADO
carbohydrates. For men and women consuming the lower protein level, to maintain body weight the energy intakes were 11,527 ±1895 kj (2755 ±453 kcal) and 8025 ±126 kj (1918 ±30 kcal), respectively. Ten percent of total energy was provided by protein, 38% by fat and 52% by carbohydrates. Biochemical, physiological and statistical anal yses. Biochemical analyses on fasting blood samples and 24-h urinary specimens were undertaken throughout the study. Blood specimens were collected into evacuated blood tubes (Becton-Dickinson, Rutherford, NJ) containing either no additive, EDTA or heparin anticoagulants as required for a given assay. Specimens were processed within 60 min of venipuncture by centrifugation (3000 x g for 20 min) and stored in the dark at 4*C. Serum, plasma or washed erythrocytes were stored at -70°C in cryotubes (Vangard International, Neptune, NJ) until analysis. The 24-h urine specimens were collected into opaque plastic jugs containing thymol and indomethacin as preservatives. The urine specimens were stored for up to 26 h at 5°Cprior to aliquot storage in the dark at -20*C. A colorimetrie method (17) was used to measure 24-h urinary XA excretion after a 5-g L-tryptophan (Tryptacin™, Arther, Mountain Lakes, NJ) load. The tryptophan was ad ministered 2 h after breakfast, and tests were repeated at 5- or 6-d intervals. Also measured at 5- or 6-d intervals were erythrocyte aspartate aminotransferase (EC 2.6.1.1, L-aspartate: 2-oxoglutarate-aminotransferase) activity coefficients (EAST-AC) by the method of Williams (18), plasma pyridoxal-5'-phosphate (PLP) by the radioenzymatic method of Rey nolds (19), and 24-h urinary 4-pyridoxic acid (4-PA) excretions by fluorometric reverse-phase liquid chromatography (20). Urinary creat in me was determined using a commercially available kit (Roche Diagnostic Systems, Montclair, NJ) based on a kinetic alkaline picrate procedure (21). To study the effects of vitamin B-6 status on hematopoiesis and iron metabolism, the following blood analyses were performed at the end of each study phase: serum iron and iron-binding capac ities by a commercial kit using a colorimetrie ferrozine-based method (Diagnostic Chemicals, Monroe, CT), ferritin by a 2-site immunoradiometric assay kit (Ciba Coming Magnetic Immunochemistries, Medfield, MA), and hematological measurements on an automated hematology analyzer (Baker 9000, SeronoBaker, Allentown, PA). Plasma ascorbate was mea sured using a colorimetrie procedure (22). Procedures
7The multivitamin
supplements
without
vitamin B-6 provided
ascorbic acid, 30 mg; thiamin mononitrate, 0.6 mg; riboflavin, 0.7 mg; niacin, 8 mg; folie acid, 0.2 mg; cyanocobalamin, 1.5 |i& biotm, 0.1 mg; pantothenic acid, 3.5 mg; rctinyl acetate, 86 u& cholccalciferol, 0.0625 \ig¡a-tocopheryl succinate, 4.79 mg; phylloquinone, 70 ng.
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protein. The vitamin B-6-repletion diet was a 2-d cycle menu and consisted of rice casseroles with vegetables, selected fruits and fruit juice, cheese, mar garine and other foods such as cereal, omelettes, muffins and biscuits (Table 1). All food items used for each subject were from the same lot number to avoid fluctuations in vitamin B-6 content. The recipes were developed in the research kitchen of the Metabolic Research Unit. During the repletion phases, the sub jects also received Ensure™and the multimineral and multi vitamin supplements devoid of vitamin B-6. All ingredients in the menu were analyzed for nitrogen by the Nutrition Evaluation Laboratory of the HNRC using a standard Kjeldahl procedure and for vitamin B-6 by Dr. James E. Leklem's laboratory at Oregon State University using a microbiological assay that employed Sacchaiomyces uvarum (16). Vitamin B-6-depletion and -repletion diets for a woman who weighed 68 kg, ingested 8908 kj (2129 kcal) daily and had a protein intake of 1.2 g/(kg body wt-d) are given in Table 1. Table 2 shows the actual amounts of vitamin B-6 ingested (as pyridoxine) from the diet plus supple ments during the various stages of the study. The study was designed so the total vitamin B-6 ingested was dependent on the subject's body weight. The mean ±SEMbody weight of the four men and four women who ingested 1.2 g protein/(kg body wt-d) were 99.2 ±5.5 (range, 87-112 kg) and 65.0 ±6.5 kg (range, 55-82 kg), respectively. The two men who ingested 0.8 g protein/(kg body wt-d) had a mean body weight of 86.2 kg (individual values, 68 and 104 kg); the two women had a mean body weight of 67.1 kg (individual values, 65 and 69 kg). The actual amounts of pyridoxine ingested (mean ±SEM)during the three vitamin B-6-repletion stages by men whose protein intake was maintained at 1.2 g/(kg body wt-d) were 1.34 ±0.08, 1.96 ±0.11 and 2.88 ±0.17mg/d, respec tively; for the women they were 0.89 ±0.08, 1.29 ± 0.12, and 1.90 ±0.18 mg/d, respectively. The corres ponding values for men whose protein intake was maintained at 0.8 g/(kg body wt-d) were 1.16 ±0.22, 1.70 ±0.34 and 2.50 ±0.50 mg vitamin B-6/d, respec tively,- for the women they were 0.91 ±0.02, 1.33 ± 0.03 and 1.95 ±0.05 mg vitamin B-6/d, respectively. The protein intake was dependent on the subject's body weight. The actual protein intake (mean ±SEM) of men who were assigned to the higher protein level of 1.2 g/(kg body wt-d) was 119.6 ±7.2 g (range, 104.0-135.5 g); for women, this was 77.8 ±7.7 g (range, 65.1-98.3 g). For the two men assigned to the protein intake level of 0.8 g/(kg body wt-d), the mean value was 69.1 g (individual values, 54.4 and 83.8 g),for the two women, the mean value was 54.0 g (in dividual values, 52.8 and 55.1 g). Body weight was maintained during the study with energy intakes (means ±SEM)of 12,765 ±971 kj (3051 ±232 kcal) and 8284 ±502 kj (1980 ±120 kcal) for men and women, respectively, who ingested the higher protein level. Fifteen percent of total energy was provided by protein, 35% by fat and 50% by
ET AL.
VITAMIN B-6 REQUIREMENTS
the exception of the PLP assay (6.7%). In addition, a variety of other blood and urinary analyses were per formed to determine the effects of vitamin B-6 status on the immune system and on glucose and insulin metabolism. Preliminary data have been reported (23, 24), and final results of these substudies will be re ported separately. Blood pressure was measured daily. In addition, studies were conducted in the Neuropsychology Labo ratory, Department of Neurology at the Tufts Uni versity School of Medicine to evaluate the effects of
TABLE 1 Sample vitamin B-6 depletion and vitamin B-6 repletion menus Sample vitamin B-6-depletion menu itemsBreakfastEnsureâ„¢1Pro-Modâ„¢2Avicelâ„¢ Food
Sample vitamin B-6-repletion menu
B-6mg0.00880.00380tracetracetrace0.0174trace0.00320.00370.00880.00380.00370.0174trace0.00 B-6rag0.00270.0361tra itemsBreakfastEnsureâ„¢1Orange
juiceMargarineOmeletteSwiss powder3Instant coffeeNondairy regular creamerSugarBiscuitMargarineHigh
cheeseBiscuitsInstant coffeeNondairy regular creamerSugarAvicelâ„¢
powder3LunchEnsureâ„¢1CaseinPro-Modâ„¢2High-protein puddingCaseinLunchEnsureâ„¢1Pro-Modâ„¢2CaseinBiscuitMargarineJelloPeach protein
puddingAvicelâ„¢ powder*DinnerEnsureâ„¢1CaseinPro-Modâ„¢2Vegetable
slicesDinnerEnsureâ„¢1CaseinPro-Modâ„¢2Avicelâ„¢ casseroleRiceMargarineCarrotsOnionsSwiss powder3Instant coffeeNondairy regular creamerSugarHigh-protein
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were modified as necessary to allow for precise auto mated analysis on a Cobas FarÃn centrifugal analyzer (Roche Diagnostic Systems). Quality control was pro vided by pooled specimens processed and stored using the same procedure used for the study specimens. Assays were rejected when a quality control result exceeded ±2so from an established mean value. Whenever feasible, specimens from a given subject were batched and analyzed concurrently at the end of a given study period. The within-assay coefficient of variation for all biochemical analyses was < 5%, with
OF ELDERLY HUMANS
cheeseChickenCaseinBiscuitsPear
puddingSnackEnsureâ„¢1Pro-Modâ„¢2CookiesWeight8250571.63083051315250553051741002504571.6308131250560Protein88.103.79000.2001.900.014.90 halvesHot chocolateAvicelâ„¢ powder3SnackHot
chocolateWeightg90.092.310.0113.010.030.01.630.08.010.0158.02.54.1131.0 is a vitamin B-6-deficient liquid formula (Ross Laboratories, Columbus, OH). 2Pro-Modâ„¢is a protein source (Ross Laboratories, Columbus, OH). 3Avicelâ„¢powder is a cellulose product (FMC, Philadelphia, PA) used to provide fiber.
RIBAYA-MERCADO
1066
RESULTS The 3-d food diaries kept by the subjects prior to their admission to the HNRC were analyzed by the HNRC Scientific Computing Department using the Grand Forks database (U.S. Department of Agri culture-Agriculture Research Service, Grand Forks Human Nutrition Research Center, Grand Forks, ND). The reported energy intakes per day were 9184 ± 2293 kl (2195 ±548 kcal) and 6075 ±397 kj (1452 ±95 kcal) for men and women, respectively. However, the actual energy provided during the study to maintain body weights were 8 to 44% greater than these re ported amounts. Thus, the subjects underreported their energy intakes. The protein intakes reported were 18% of energy intakes, i.e., 101 ±20 g and 65 ± 7g for men and women, respectively. Men reported ingesting 38% and 44% of their total energy as fat and carbohydrates, respectively; women reported 33% and 49% of their intake as fat and carbohydrates, respec tively. Reported vitamin B-6 intakes were 1.61 ±0.38 mg for men and 1.36 ±0.03 mg for women. However, the nutrient database does not have vitamin B-6 values for all foods so the actual vitamin B-6 intakes may be underestimated. The dietary regimen did not interfere with the usual physical activities of the subjects, and there was no evidence that the regimen had any deleterious effect on their health. Except for one man who deve loped a mild reaction (nausea) to tryptophan, all the subjects tolerated the tryptophan load well. Figure 1 shows 24-h urinary XA excretions after a 5-g L-tryptophan load during the various stages of the study for men and women whose diets contained 1.2 g protein/(kg body wt-d). At baseline, the men had an XA excretion of 7.0 ±1.2 mg/24 h. As vitamin B-6-deficiency progressed, urinary XA excretion in creased up to a maximum value of 386.4 ±55.0 mg/24 h during d 15 of the deficiency period. On vitamin B-6-repletion, XA excretion promptly decreased. Baseline value was not attained during the first vi tamin B-6-repletion stage when 1.34 ±0.08 mg of vitamin B-6 was ingested; it was attained by d 20 of the second vitamin B-6-repletion stage. Thus, for these men, who ingested -120 g of protein daily, the amount of vitamin B-6 required to normalize the XA
level was the amount given during the second repletion stage. The vitamin B-6 ingested during this period was 1.96 ±0.11 mg/d (range, 1.71-2.22 mg/d; Table 2). For women, baseline urinary XA excretion after a 5-g L-tryptophan load was 21.7 ±13.6 mg/24 h. On vitamin B-6 depletion, XA excretion rose promptly to a maximum value of 538.4 ±81.5 mg/24 h after 17 d of vitamin B-6 deficiency. On vitamin B-6 repletion, urinary XA decreased promptly; the baseline level was not attained during the second vitamin B-6-repletion stage when 1.29 ±0.12 mg vitamin B-6 was ingested. For these women who ingested -78 g of protein daily, the vitamin B-6 intake required to nor malize the XA level was 1.90 ±0.18 mg/d (range, 1.61-2.37 mg/d), the amount given during the third vitamin B-6-repletion stage (Table 2). For both men and women, the XA values were not further changed by the ingestion of 42 mg of pyridoxine during the final phase of the study. Tryptophan load test results were obtained from three of the four subjects assigned to the lower protein intake level of 0.8 g/(kg body wt-d). One of two men fed this diet was inadvertently given placebo instead of tryptophan pills due to the manufacturer's error. The other male subject ingested 54 g of protein during the study. His baseline XA value after a 5-g L-tryptophan load was 5.2 mg/24 h; on vitamin B-6 depletion, values peaked at 351.5 mg/24 h. On vitamin B-6 repletion, urinary XA excretion decreased promptly; however, the baseline level was not quite attained at the end of the third vitamin B-6-repletion phase when 1.99 mg vitamin B-6/d was ingested, nor even after 3 d of ingesting 42 mg of pyridoxine at the final phase of the study. For this person, the XA levels at these stages were 16.3 and 10.7 mg/24 h, respec tively. One woman who ingested 53 g of protein had a baseline XA value of 8.7 mg/24 h; at the end of vitamin B-6 depletion, it was 373.4 mg/24 h. Values returned to baseline during the second vitamin B-6-repletion stage when she ingested 1.30 mg of vi tamin B-6. The second woman who ingested 55 g of protein had a baseline XA value of 7.2 mg/24 h; at the end of vitamin B-6 depletion, it was 388.2 mg/24 h. Repletion with 2.00 mg of vitamin B-6 brought xanthurenic acid excretion to baseline levels.
Tecce, J. J., Raber, S. R., O'Brien, M. E., Russell, R. M., RibayaMercado, J. D., Sahyoun, N., Morrow, F. D., Sadowski, J. A &. Gershoff, S. N. (1987) Vitamin B-6 depletion and L-tryptophan effects on brain functioning. Presented at the meeting of the American College of Neuropsychopharmacology, San Juan, Puerto Rico. ^ecce, J. J., Kosta, S. M., Mazaheri, S. T., Russell, R. M., RibayaMercado, J. D. & Gershoff, S. N. (1988) Tryptophan and dividedattention effects on event-related brain potentials (CNV). Presented at the meeting of the American College of Neuropsychopharma cology, San Juan, Puerto Rico.
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vitamin B-6 depletion and repletion on brain func tioning, psychophysiological activity and alertness. Weekly mood ratings of the subjects were obtained. Preliminary results of the neuropsychological tests have been reported at scientific meetings.8-9 ANOVA and the Neumann-Keuls test were used for statistical analyses. The t test was used to compare data for men vs. women; differences were considered significant when P > ffiC
0.5n
IT*B6
oÕ(A)-
r\(B)J.ÕIA IITiI2.01.51.00.5r\ « i »B6• B6^2.88l0.17mgB6-1.96i0.11mgB6L
fBLL4 F 1.69 î 0.02
II-1.29\A.iui|
mg
»se» B61-1.90
|F L 4 1.62 10003
mg
i0.18mgB6Ã-O. B6mgB60.1710.01
1.341 0.08 mg B6m-M-
12mg
B6mg 0.89 10.08 mg 0.1010.01f•B(125.4 B6
FIGURE 4 Urinary 4-pyridoxic acid (mg/24 h) at baseline, BL; vitamin B-6-depletion, -B6; three stages of vitamin B-6-repletion, +B6 I, +B6 ÕÕ, +B6 IH; at the final phase, F. Values are means ±SEM for four men (A) and four women (B) who consumed 1.2 g protein/(kg body wt-d); means ±SEM, 119.6 ±7.2 g protein for men and 77.8 ±7.7 g for women. Each bar represents measurement obtained every 5 or 6 d.
turnover time for RBC of -120 d. The provision of 1.34 mg of pyridoxine (the first repletion dose) was insufficient to normalize XA measurements after a tryptophan load in elderly men who ingested -120 g of protein,- 1.96 mg of the vi tamin (the third repletion dose) was required. Simi larly, 0.89 and 1.29 mg of vitamin B-6 (the first and second repletion doses) were insufficient to normalize post-tryptophan XA measurements in elderly women who ingested 78 g of protein,- 1.90 mg of the vitamin (the third repletion dose) was required. These vitamin B-6 requirements expressed per gram of protein are 0.0163 and 0.0244 mg vitamin B-6/g protein for men and women, respectively. Studies have shown that the requirement for vi tamin B-6 is directly related to protein intake (27); the protein effect is mainly the result of its methionine content (28). La this study, the effects of lower protein intakes (54 g) on XA measurements and vitamin B-6 requirements were not consistent; only one of three subjects showed a reduced requirement compared with the subjects fed the higher protein level. Thus, based on XA excretion after a tryptophan load, our results indicate that the estimated vitamin B-6 re quirements of elderly men vs. elderly women with a wide range of protein intake are not greatly different. The women excreted larger amounts of XA and deve loped abnormalities in their metabolism of tryp tophan faster than the men. Similar observations have been reported in younger adults (29, 30). Although we did not study the vitamin B-6 require ments of younger adults, a comparison of our data
with reports in the literature indicates that the vi tamin B-6 requirements of elderly men and women are higher than the requirements reported for younger men and women. Baker et al. (31) found that for young men with a low protein intake (30 g), the minimal daily vitamin B-6 requirement was slightly more than 1.0 mg; for young men with a high protein intake (100 g), the minimal daily requirement was 1.5 mg. Miller and Linkswiler (32) showed that, during vitamin B-6 depletion after a 2-g L-tryptophan load, young men consuming diets containing 150 g of protein excreted larger abnormal amounts of tryp tophan metabolites than those consuming diets con taining 54 g of protein. In both instances, 1.5 mg of pyridoxine normalized tryptophan metabolite excre tion. Sauberlich (33) concluded that the normal young adult consuming 100 g of protein requires a minimum of 1.5 to 1.75 mg vitamin B-6/d,-these requirements are increased (or decreased) somewhat when more (or less) than 100 g of protein is consumed. Horwitt (34) concluded that, for young male adults and adolescents consuming 60 g of protein, 1.4 mg of vitamin B-6 should be adequate,- for those whose diets contained 100 g of protein, 1.8 mg of vitamin B-6 is more than adequate, and a 2-mg allowance should be generous. The vitamin B-6 requirements of young adult women also have been studied. Donald et al. (35) proposed that the vitamin B-6 requirement for adult women consuming a moderate protein diet (57 g) was 1.5 mg/d. Shin and Linkswiler (29) showed that, in young adult women, abnormal urinary tryptophan
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1.5
