Excretion of tryptophan-n iaci n metabolites young men: effects of tryptophan, leucine, vitamin B6 intakes12

J. I. Patterson, A. E. Harper,3 Ph.D., Ph.D. it it Brown, Ph.D., H. Linkswiler, Ph.D., and

by and

ABSTRACT investigate the college 245, 548, day the

Three relationship

human

metabolic between

studies, tryptophan

each intake provided proteins

35 and

days the

in

length, of

were dietary of

performed tryptophan

to

proportion

converted
Nine either

to niacin
men or

and
consumed 845

the
mg of

effect

of supplements
diet that from for in 24-hr on to small the

of L-leuclne
8 mg per day.

and
of

vitamin
niacin, each diet of no

B6 on this
1 mg plus vitamin significant metabolites over 35-day

conversion.
B, for day consistent and one study,

a basal tryptophan

vitamin 10 g of LB6 per or

During

15-day
leucine added and

period
per to quinolinic proportion

basal
was diets acid of

diet
of both

alone

was
and

consumed,

for another
last urine the niacin and 2157-2167, 5-day samples. excretion tended was

15-day
period, There of any to probably

period basal
20 mg were of the not

Downloaded from www.ajcn.org by guest on June 20, 2011

consumed, groups. measured were tryptophan this change

was

N-methylnicotinamide,

N-methyl-2-pyridone-5-carboxamide, measured. the range of

effects
The increased;

of L-leucme
however,

or vitamin B6 supplements
converted was

increase tryptophan as significant

consumption

tryptophan
72:1 in these

intakes

studied.
Am.

The

average

conversion

ratio

of tryptophan
1980.

to was approximately niacin

subjects.

I. Clin. Nutr. 33:

trials indicated that vitamin B6 adPellagra has long been recognized as clinical a could effectively cure pellagra multiple-factor nutritional disease rather than ministration (1 and associates have concluded simply a niacin deficiency syndrome (1, 2). 1). Gopalan the results summarized above along More recently, a high leucine intake has been from other information (3, 4, 1 1) that converimplicated in the pathogenesis of endemic with of tryptophan to niacin is decreased pellagra by Gopalan and associates (3, 4) andsion when a diet with a high leucine content is low vitamin B6 status has been implicated as a factor that aggravates the effects of high consumed and that this effect may be ampliby marginal or inadequate vitamin B6 leucine intake (5-8). In support of these con-fled intake. Thty also concluded that additional clusions are observations from investigations vitamin B6 increases the rate of clearance of of effects of leucine ingestion on excretion of niacin metabolites by human subjects. In oneleucine, thus counteracting most ofthe effects ofleucine on the conversion of tryptophan to study, normal subjects who consumed 5 to 10 macin (6). g supplements of L-leuclne daily excreted inConflicting results concerning these effects creased amounts of N-methylnicotinamide been obtained, however, in experiments (9); in another, a supplement of 10 g L- of have on both human subjects (12-14) and (15, rats leucine decreased urinary excretion of N16). Truswell and associates (12, 13) noted methyl-2-pyridone-5-carboxamide and increased that of quinolinic acid but did not From the Departments of Nutritional Sciences, Huaffect N-methylnicotinamide excretion (10). man Oncology and Biochemistry, University of WisconIn subsequent studies (5), when a 5 g supplesin-Madison, Madison, Wisconsin 53706. 2Supported by a grant from the National Dairy Counment of leucine was provided, urinary excretion of quinolinic acid increased but excretion cil, Chicago, Illinois and by United States Public Health National Institutes of Health Grants AM 10747, of nicotinic acid decreased; these effects were Service 20432 from the National Instireversed either by withholding leucine sup-CA 20908, and P01-CA tutes of Health. plements or by providing a 50-mg supplement 3Author to whom requests for reprints should be
I

of
The

vitamin
American

B6.
Journal

Lastly,

results

of

preliminary
OCTOBER

addressed. 1980, pp. 2157-2167. Printed in U.S.A. 2157

of Clinical

Nutrition 33:

2158

PATFERSON

ET

AL.

verted to niacin when tryptophan is provided that N-methylnicotinamide excretion by from food proteins. normal subjects or by men accustomed to eating large quantities of maize, was unafprocedures fected by supplements of5 to 10 g of L-leucine Experimental per day. Nakagawa et at. (14) reported that Subjects no consistent changes in excretion of NNine college men volunteered for each of the three methylnicotinamide and N-methyt-2-pyri35-day studies. Their ages ranged from 19 to 28 years. done-5-carboxamide occurred when subjects Their heights and weights ranged from 167 to 187 cm, (Table I). There were no fed diets composed of highly purified com- and 66 to 91 kg, respectively differences in the mean age, height or weight ponents consumed an additional 8.8 g significant of of subjects in the three studies. Hematocrits, urinary pH, leucine. They also noted that urinary quinoglucose, ketones, and protein as determined by Ames linc acid excretion tended to decrease only in Labstix were within normal values for all subjects. No subjects who were switched from a vitamin subject had any known metabolic disorder. men participated in the three studies which B6-free basal diet to one that contained a Nineteen during the fall, winter, and spring. Two men supplement of 8.8 g of L-teucrne. In experi- took place participated in all three studies and three men were in ments on rats results have been inconsistent two consecutive studies. One subject dropped out of and, also, have not conformed with thosestudy 1 on day 20. Data from subject number 23 were from human experiments. In one series omitted of because of poor compliance with the protocol. experiments, rats fed high leucine diets cxDiet and experimental design creted more quinolinic acid (7, 17) and more The experimental design is shown in Table 2. During N-methylnicotinamide (17) than ad libitum each 35-day study the men consumed basal diets which or pair-fed controls. However, in other expercontained 8 mg of niacin, 1 mg ofpyridoxine, and either iments, rats fed similar diets excreted less N-245, 548, or 845 mg of tryptophan per day. Each 35-day methylnicotinamide than pair-fed controls study, during which dietary tryptophan content was kept constant, was divided into three periods. For one 15-day and similar levels of quinolinic acid (15, 16). period, basal diet alone was consumed and for the second In more recent studies (18, 19) leucine was 15-day period basal diet plus 10 g of L-leuclne per day not found to precipitate niacin deficiency in was consumed. In order to equalize the effect of order of rats, chicks or dogs, although earlier the dog feeding, four or five subjects were given L-leucine during were given (20) and the monkey (21) had been reported the first 15 days and the rest of the subjects L-leucine during the second 15 days. For the last 5-day to develop niacin deficiency after addition of period, both groups were given a daily supplement of 20 leucine to an otherwise adequate basal diet. mg of vitamin B6. Because of the inconsistencies in these re- The diets were composed of ordinary food products suits, we conducted a series ofmetabolic stud- (Table 3). The diet containing 245 mg oftryptophan had protein foods with gelatin as the major protein ies to investigate the effects of L-teuclne andlow source. In the second study the quantity of gelatin was vitamin B6 supplements on the excretion of reduced and cheese was added to bring the level of tryptophan-niacin metabolites by young men tryptophan up to 548 mg. L-Methionme was added to fed diets marginal but adequate in niacin andthe diets containing 245 and 548 mg of tryptophan to raise the level of sulfur-containing amino acids to 2 g/ vitamin B6. person per day, the amount provided by the diet conWe have also reexamined the question of taming 845 mg of tryptophan. In the third study, cheese the effectiveness of tryptophan as a precursor and reconstituted powdered milk supplied enough trypof niacin. Horwitt et at. (22) and Goldsmith tophan to increase the level to 845 mg. The basal diets of et at. (23) have reported that for each 60 mg of tryptophan consumed by human subjects, 1 enough is oxidized on the average to provide TABLE Subject description about 1 mg of niacin. Nakagawa et at. (24) Mean height Mean body reported that the proportion of tryptophan Study Mean age weight converted to niacin increased as tryptophan yr kg cm consumption increased in subjects fed highly 181 5 76.4 8.2 1 21.9 2.7 (9, 8)b purified diets. However, Goldsmiths results 74.16.0 1777 2 21.33.4(9) did not indicate that the ratio changed with 3 179 7 81.0 8.4 23.3 3.5 (8) changes in tryptophan intake. Therefore, anEach value represents the mean SD. 1) Values other objective of our studies was to investi- in parentheses indicate number of subjects. One subject gate effects of changes in tryptophan intake dropped out of study I on day 20. Data for him through day 15 were included in the calculations. on the extent to which tryptophan is con-

Downloaded from www.ajcn.org by guest on June 20, 2011

EXCRETION TABLE Experimental 2 design

Tryptophan sumod

OF

TRYPTOPHAN-NIACIN

METABOLITES

BY

YOUNG

MEN

2159

Days
COfF

Study

Subject 1-15 16-30 Diet sequence 31-35

mg/day

1
2 3

245 548 845

1-4 5-9 10-13 14-18 19-22 24-27 diet as B6 as described pyridoxine in

L-leu
D

D +
D

L-leu L-leu D

D +

D L-leu D D + L-leu D D + L-leu

D +

D + B6 L-leu + D + B6 D + L-leu + D + B6 D + L-leu + D + L-leuclne supplemented

B6 B6 B6 to the diet. B6

D signifies

signifies 20 mg

the basal of vitamin

Table 3. supplemented

L-leu

signifies to the diet.

10 g of

studies 1, 2, and 3 provided, respectively, 10.0, 9.25, and macro-Kjeldahl method (27). Food composites were an1 1.4 g of nitrogen per person per day. All diets provided alyzed for niacin (28), pyridoxine (29), and tryptophan about 2800 kcal/person per day. Calorie intake was (30) by the WARF Institute, Inc., Madison, Wis. (now adjusted for each individual by the addition of sugar Raltech, Inc.). Pyridoxine was also determined in our products in order to maintain body weight. Minerals and laboratory (3 1) and the two sets of values agreed closely. all Vitamins, except for niacin and pyridoxine, were Niacin and tryptophan values agreed with values calculated from food composition tables (32, 33). The urinary supplemented to meet 100% of the Recommended Dietary Allowances (25). Supplementary vitamins, minerals, niacin metabolites measured were N-methylnicotinamL-methiOlune, and L-leucine were divided among allide (34) by fluorescence (Fluoro-microphotometer, three meals. All meals were prepared and consumed in American Instrument Co., Inc., Silver Spring, Md. The a metabolic kitchen in the Department of Nutritional primary filter was Corning no. 7-39 and secondary the Sciences. filters were Corning no. 3-73, Wrathen A and no. 4-70) The human metabolic studies were designed to mimic and N-methyl-2-pyridone-5-carboxamide (35) by specas closely as possible the dietary conditions described by trophotometry (DuQuarts Spectrophotometer, Beckman Krishnaswamy et al. (5). Krishnaswamys standard Instruments, Inc., South Pasadena, Calif.). Urinary quindiet was calculated to provide daily, 1.5 to 1.8 mg olinic of acid was determined by high-pressure liquid chrovitamin B, 45 g of vegetable protein; and 2500 kcal. matography In (model 601 liquid chromatograph, Perkincomparison, our diet in study I provided somewhat moreElmer Corp., Norwalk, Conn.) with a spectrophotometric protein and energy because our subjects were probably detector (LC-55 spectrophotometer and model 250 ultraof larger body size. Protein quality cannot be compared violet absorption detector, Perkin-Elmer Corp., Norsince Krishnaswamy et al. (5) did not report this inforwalk, Conn.; Patterson, J. I. and R. R. Brown, unpubmation. The diet in study 1 was calculated to provide lished data). only 2.5 g of leucine daily, therefore, our subjects were Statistics given a supplement of 10 g of L-leuclne compared to the 5 g given the subjects in Krishnaswamys study. Gelatin Results from the three studies were tested for signifiis low in sulfur-containing amino acids, consequently the cance of differences using Duncans new multiple-range diets containing gelatin were supplemented with L-me-test for groups with unequal replications (36). Results thionine. Vitamin B6 levels were lower in our diets than were tested for significance of differences using a paired in those of Krishnaswamy et al. In Krishnaswamys t test when comparisons were made between dietary study subjects received 5 g of leucine in a single dose, treatments within a study (36). Results were also examwhereas in ours the 10 g of L-leucine was divided among ined by regression analysis (36) and lack of fit (37). all three meals. Methods

Downloaded from www.ajcn.org by guest on June 20, 2011

Results

and

discussion

Twenty-four hour collections of urine were made by Effects ofL-leucine with or without vitamin B6 having subjects awaken each morning at the same time, of tryptophan-niacin void their bladders, and include this urine in the previous on the excretion days collection. Urines were preserved with a few millimetabolites liters of toluene and aliquots of 24-hr collections were Nitrogen analyses were performed on all frozen until analyzed. All feces were collected throughurine and fecat samples; however, for clarity out the studies. Brilliant blue dye was given orally to aid as a fecal marker and stool collections were made into results 5for only four time periods are reported day composites for analysis. in Table 4. Half the subjects who consumed Daily urine collections were analyzed for creatinine the diet containing 245 mg of tryptophan to determine completeness of collection (26). Nitrogen in positive nitrogen balance (i.e., nitrowas determined in urine, fecal composites, and repre-were sentative daily food composites by a modification of the gen balance greater than or equal to +0.2

2160 TABLE Daily grams 3 food per intake person in

PATTERSON

ET

AL.

Foods common to all studies: Bread: Flour (white, unenriched) Wheat starch Crisco vegetable shortening Yeast (dry) Sugar Salt (iodized) Rice casserole: Rice (white, unenriched) Onions Carrots Celery Tomato juice Potatoes (dry flake) Green beans Beets or pickle Pears Applesauce Orange juice 7-Up Study 1 Margarine Gelatin Sugar Kool-Aid Study 2b Margarine Gelatin Sugar Cheese Kool-Aid Study 3C Margarine Milk (dry) Cheese Sugar Kool-Aid

90 50 20 2 15 3 100 15 15 15 100 30 100 100/50 200 200 200 288

75 33 100 6.5 40

8
75

88
3.2 40 25 139.2 25 2.2

a Supplements were put in capsules and contained (per person per day): 1.77 g of magnesium acetate, g of calcium phosphate, 1. 12 mg of thiamin HC1, mg of riboflavin, 0.35 mg of folacin, 3 sg ofvitamin B,2, The effect of level of dietary 10.9 mg of calcium pantothenate, and I g of L-methionine. The 10 g of L-leucine were dissolved in boiling the conversion of tryptophan water, gelatin, sugar, Kool-Aid, and 1 teaspoon of fresh Pyridine nucteotides are pineapple juice and was consumed as a dessert. Supplements were put in capsules and conmans from three dietary

nitrogen per 24 hr) and almost all subjects who consumed the higher tryptophan diets were in positive nitrogen balance. In studies 2 and 3, supplementary L-leucine brought most subjects into more positive nitrogen balance. Vitamin B6 supplementation had no effect on nitrogen balance. Values for urinary excretion of tryptophanniacin metabolites on days 1, 15, 30, and 35 are shown in Table 5. Urines were analyzed for N-methylnicotinamide and N-methyl-2pyridone-5-carboxamide on days 7, 10, 22, and 25 also but for brevity the values are not included in Table 5. Excess teucine in the diet had no effect on the urinary excretion of Nmethylnicotinamide, N-methyl-2-pyridone5-carboxamide, or quinolinic acid. In addition, supplements of 20 mg ofvitamin B6 with or without the extra L-teucrne had no effect on the amounts of these metabolites excreted. Thus, excess dietary teucine did not affect the niacin status of these normal subjects who were consuming a diet that provided 54 to 69 g of protein, 8 mg of niacin, and 1 mg of vitamin B6. These results are in general agreement with those of others who have not observed niacin deficiency to be precipitated in either human subjects (12-14) or in animals (15, 18, 19) by dietary additions of teucine. It is not possible to establish whether some unique, but not obvious, factor in the experiments done on several species at the National Institute of Nutrition in Hyderabad (3) is responsible for the striking effects ofleucine additions on the development of pellagra observed there. It is clear, in any event, that it is not a readily 23.0 reproducible phenomenon. 1.50 tryptophan to niacin on

Downloaded from www.ajcn.org by guest on June 20, 2011

synthesized by husources: nicotinic tained (per person per day): 1.50 g of magnesium acetate, acid, nicotinamide, and tryptophan. There1.12 mg of thiamin HCI, 1.10 mg of riboflavin, 0.35 mg fore, for diet formulation and recommendaof folacin, 3 ig of vitamin B,,, 10.9 mg of calcium tions, a quantitative expression of the extent pantothenate, and 0.6 g of L-methiomne. The 10 g of Lwhich dietary tryptophan is used as a leucine were dissolved in boiling water, gelatin, sugar, to precursor of niacin is essential. The term and Kool-Aid and consumed as a dessert. Supplements were dissolved in water and contained (per person per conversion ratio, defmed as the milligram day): 0.89 g of magnesium acetate, 1.12 mg of thiamin of tryptophan which must be consumed for HC1, 1.40 mg of riboflavin, 0.35 mg of folacin, 3 ig of 1 mg of niacin to be formed in the body (25), vitamin B12, and 10.9 mg of calcium pantothenate. The for this purpose. It is important to 10 g of L-leucine were dissolved in boiling water, sugar, is used and Kool-Aid, cooled and consumed as a beverage. recognize, when such expressions are used,
C

EXCRETION TABLE Nitrogen 4 balances in

OF

TRYPTOPHAN-NIACIN

METABOLITES

BY

YOUNG

MEN

2161

of young tryptophan

men

consuming

diets differing content

Day Study Subject 1-5 11-15 g nh:rogen/24 26-30 hr 31-35

1(245 per

mg y)

of tryptophan

1-4
5_9b

-2.09 -2.12 of subjects balance/nC in +N-

0.59 1.2 0/9

+0.15 +0.37

0.43 1.3 3/9

+0.27 +0.44 4/8 +0.48 +0.60 8/9

0.40 +0.16 0.58 +0.14

3/8

0.75 0.45

No.

2 (548 per

mg day)

of tryptophan

10-13 14-18 No. of subjects balance/n 19-22 23-27 No. of subjects balance/n

-0.97 -0.75 in +N-

2/9

1.5 1.1

+0.65 +0.22

0.21 0.84 5/9

0.31 +0.25 0.29 +0.91

7/9

0.35 0.14

3 (845 per

mg day)

of

tryptophan

+0.36 -1.07 in +N-

4/8

1.1 1.7

+ 1.68 +0.04

7/8

0.69 1.1

+0.49 + 1.94 7/8

0.41 +0.54 1.0 + 1.83

0.79 0.61

Downloaded from www.ajcn.org by guest on June 20, 2011

7/8 subjects of study

Values that are underlined are for subjects who were consuming were given a supplement of 20 mg of vitamin B6 on days 31 to nitrogen + fecal nitrogen), expressed in grams. Values represent 1 on day 20, therefore his data are included only in days 1 through + 0.2 g nitrogen/24 hr. n indicates total number of subjects.

10 g of L-leucine during that period. All 35. Nitrogen balance = nitrogen - (urinary intake mean SD. One subject dropped out C N-balance 15. is defined as nitrogen balance

that dietary tryptophan is needed for protein after the subjects had consumed a supplement or 10 g of lactatsynthesis and that theoretically only one mol- of 100 mg of L-tryptophan ecule of tryptophan is needed for the synthe- bumin (about 200 mg of tryptophan). Goldsis of one molecule of NAD(H). smith, in 2 to 3 week feeding trials with volunteers, compared the incremental Presently, the best way of assessing macin female nutriture in humans is to measure urinary changes in combined urinary excretion of NN-methylnicotinamide and N-methyl-2methytnicotinamide and N1-methyt-2-pyripyridone-5-carboxamide, the two major niadone-5-carboxamide after her subjects had cin excretory products (Fig. 1). In estimating consumed 10 to 150 mg of nicotinamide with

the

amounts

of

these

metabolites

derived the

incremental

changes

in these

metabolites

the subjects had consumed 1 to 6 g of assume that reg-after excretion of pyri- DL-tryptophan. D-Tryptophan was assumed not to be oxidized to nicotinamide. Hankes is not affected by i.e., whether it et at. (39) is reported that very littte D-[7]-4C] or tryptophan. tryptophan was oxidized to CO2 as cornthe pyridine nupared with the L-isomer despite a loading cleotide pool is constant once subjects have dose of D-tryptophan, thereby supporting adjusted to the experimental dietary condi- Goldsmiths assumption that humans do not tions (38). oxidize D-tryptOphan appreciably to nicotinThe conversion ratio of tryptophan to ma- amide. Goldsmith found that the average cin in humans has been accepted as 60:1 (25)conversion ratio was similar with all tevels of based on the studies of Horwitt et at. (22) tryptophan in intake, but variation among in1956 (22) and of Goldsmith et a!. (23) in dividuals was large, ranging from 34: 1 to 86: 1961. Horwitt, in a 22-week study of male 1. In the studies of Horwitt and Goldsmith, mental patients, compared the incremental the basal diets consisted ofnaturat food prodchange in urinary N-methylnicotinamide exucts and provided 265 and 200 rng of trypcretion after the subjects had consumed a tophan per day, respectively, similar to those supplement of 10 mg of nicotinamide with in our experiment; but lower levels of niacin, the incremental change in this metabolite 4.7 to 5.7 mg/day, respectively. from tryptophan, one must ulation of degradation and dine nucteotide metabolites the nature of the precursor, nicotinamide, nicotinic acid One must also assume that

2162 TABLE Urinary by young differing content 5 excretion of tryptophan-niacin men consuming diets in tryptophan

PATTERSON

ET

AL.

metabolites

Day Study Subject I 15 tmoIes/24 hr 30 35

N-methylnicotinamide: I (245 mg of tryptophan

per

day)

1-4
59b

44.0 37.8 38.2 35.9 36.9 33.4

12.5 5.0 3.4 4.9 8.3 3.0

8.1 7.4 10.4 1 1.9 1 1.4 28.3

1.1 1.2 2. 1 0.7

4.7 4.2 9.8 13.8

1.6 1.1

4.9 4.3

1.4 0.9 1.1 2.3

2 (548

mg

of

tryptophan

per

day

10-13 14-18 19-22 24-27

1. 1 8.0 2.6 1 1.8 17.1 18.7

3 (845

mg

oftryptophan

per

day)

3.1 5.1

16.6 2.7 15.0 1.5

2.3 3.8

N-methyl-2-pyridone-5-carboxamide: I (245 mg oftryptophan

Downloaded from www.ajcn.org by guest on June 20, 2011

per

day)

1-4
5_9b

147 153

38 34 15

16.7 14.4

2.3 2.2 3.0

10.3 11.0 17.7 22.73.7 38.9 34.5

1.8 2.4 2.2

1 1.1 11.5

2.3 2.2 1.3

2 (548 mg

of

tryptophan

per

day)

10-13 14-18 19-22 24-27

99.5 11628 134 81.4

24.6 31.25.5 29.8 55.4

19.2 20.22.6 38.7 39.2

3 (845

mg

oftryptophan

per

day)

41 3.9

6.7 14.0

5.7 4.3

3.2 6.8

Quinolinic I (245 mg

acid: oftryptophan

per

day)

1-4

5-8 2 (548
mg oftryptophan per day) 10-13 14-18 19-22 24-27

24.0 26.9 21.5 32.0 31.0 39.5

1.0 1.9 1.7 1.7 2.2 6.7

21.3 23.6 24.8 34.3 33.7 33.8

3.0 2.9 1.5 2.6

18.5 23.0 27.1 32.5 32.0 35.0

2.2 3.7

18.6 26.7 26.3 32.0 35.6 30.5

3.9 3.0

2.7 3.9 6.7 4.9

1.9 2.9 4.3 3.2 All subjects dropped were

3 (845

mg

oftryptophan

per

day)

3.9 2.0

a Values supplemented out of study

that

are underlined are for subjects consuming 10 g of L-leucine with 20 mg of vitamin B6 on days 3 1 to 35. Values represent mean I on day 20, therefore his data are included only in days 1 through

on that day. b SEM. subject One 15.

Nakagawa et at. (24) catcutated tryptophan Our experiments were designed to investithe change, if any in the extent of conto niacin conversion ratios from the amounts gate of tryptophan to niacin when tryptoof supplementary tryptophan and niacin thatversion phan was supplied from protein in amounts produced the same total urinary excretion of niacin metabolites. They reported that that as might ordinarily be consumed by humans. There were no significant effects of tryptophan intake increased, the proportion of L-teuclne or vitamin B6 on of tryptophan converted to niacin increased; supplements hence, the conversion ratio decreased. They the urinar excretion of N-rnethytnicotinused highly purified diets containing crystal- amide, N -methyt-2-pyridone-5-carboxamline amino acids in their experiments. The ide, or quinolinic acid; therefore, all values included in the statistical analysis of basal diet to which supplementary trypto- were without distinction as to whether or phan was added contained no niacin, whereas results had consumed supplementary Lthe basal diet to which niacin was added not subjects or vitamin B6. Subjects had adjusted provided 250 mg oftryptophan per day. Four teucine to the different tryptophan intakes by about levels of both tryptophan and niacin were day 10 or 15, using as criteria the leveling off tested, however, each period in which a difof N-methylnicotinamide and ferent supplement was given was only 5 daysof excretion to in duration with two or three subjects partic- N-methyt-2-pyridone-5-carboxamide constant values. For each subject, values for ipating.

EXCRETION

OF

TRYPTOPHAN-NIACIN

METABOLITES

BY

YOUNG

MEN

2163

each of these metabolites for days 22, 25, 30, 30, and 35 for each subject were averaged and 35 were averaged (Table 6). The sum of and these values were used to calculate the the average values for N-methylnicotinarnmean for each study. Urinary quinolinic acid ide and N-methyl-2-pyrtdone-5-carboxamexcretion also increased with increasing trypide are also given for each study. Total excre- tophan intake. tion of these two metabolites increased with Since dietary niacin content was kept conincreasing tryptophan intake. stant throughout the three studies, the Table 6 also provides a summary of urinary amount of urinary N-methylnicotinamide
quinolinic acid results. Values for days 15, and arising termined.

Tryptophan

Nicotinic

Acid

Quinolinic

Acid

not be deHowever, Goldsmith et at. (23) reported that an average of 57% of 10 to iSO mg of mcotinamide included in a diet contaming either 200 or 1000 rng of tryptophan could be recovered as these two urinary metabolites. Her studies involved 14 subjects in
19 trials and the value of 57% agreed closely

N-methyi-2-pyridone-5-carboxamide from dietary niacin could

Nicotinic Acid Mononucleotide

Downloaded from www.ajcn.org by guest on June 20, 2011

C
N

Nicotinic Acid Dinucleotide

Adenine

with her previous estimate recovery of the mcotinamide urinary niacin metabolites
by the size of the mcotinamide

(40). The percent suppternents as was not affected

supplement;

Nicotinamide Adenine Dinucleotide Nicotinamide Mononuci eotide

however, ranging

Nicotinamide

individual variation was large, from 23 to 103% (23). Using the average value for recovery of niacin metabolites of 57%, tryptophan to macm conversion ratios were calcutated by two methods. Using the method as described in Table 7, the conversion ratiowas calculated from the difference between the amounts of urinary macrn metabolites excreted in successive experiments. The results indicated that

N1-methylnicotinamide

-methyl-2-pyridone-5-carboxamide 1. Biosynthesis in rat liver (34). of nicotinamide adenine dinu-

FIG. cleotide

the tryptophan to niacin conversion ratio decreased with increasing tryptophan intake. Thus, 96 mg of dietary tryptophan was calculated to be needed for formation of 1 mg
of niacin when the tryptophan content of the

TABLE 6 Urinary N-methylnicotinamide 5-carboxamide (MPCA), excretion by young men different amounts of tryptophan
Study

(NMe), and quinolinic consuming

N-methyl-2-pyridoneacid

Tryptophan

NMe

MPCA tmoIes/24 hr

NMe

MPCA

Quinolinic

acid

1 2 3 Correl
a

245 548 845 ation coefficient

5.4 11.6 17.1

0.9 1.3 l.6C 0.80

11.7 20.2 38.6

0.86

1.3 1.8 3.lC

17.1 31.8 55.6

0.86

2.0 2.76 45C

22.0 29.9 334

2.2 1.86 2.56 0.61

NMe and MPCA, values values for each subject for days supplements of L-leucine and/or metabolite were tested for significance values with different superscripts 0.05; and for NMe + MPCA, P <0.01. study 1 significantly different from

For

for each subject for days 22, 25, 30, and 35 were averaged. For quinolinic acid, 15, 30, and 35 were averaged. The values for all subjects regardless of whether vitamin B6 were consumed, were averaged for each study. The means for each of difference using Duncans new multiple-range test. Within column, each are significantly different from each other. ForP < NMe, 0.01; for MPCA, P < For quinolinic acid: study 1 significantly different from study <0.05 2, and P study P < 0.01. 3,

2164 TABLE 7 Calculation of amount required to provide 1 mg of niacin

PATFERSON

ET

AL.

of

tryptophan

were and

anlyzed Wannemacher

separately (41)

(Table showed

6). that

Powanda for mice

and
ship
From study 1 to study 2 From study 2 to study 3

rats,
between

there NAD

was
level study

a positive
of dietary suggest

linear
tryptophan

relationand

hepatic
Powandas

concentration.

Our
that

study
over

and
these

Increase in tryptophan sumed Increase in niacin lites excreted in Increase in niacin lites excreted in Dietary niacin to tabolite excretionr mg tryptophan:mg

con- 303 metabopinoles metabomg equal meniacin 14.7 1.80

mg sm mg 3.16

297 23.8 2.92 mg 5.12 58:1

mg m mg mg

ranges of tryptophan intake the proportion dietary tryptophan converted to niacin not change significantly. By using the
of the line in that the quantity Figure of 2 and metabolites the

of does slope
in

assumption recovered

96:1

the the

urine was equivalent average conversion

to 57% of the niacin, ratio over this range

of intake was calculated to be 72: a Tryptophan intake per person per day was 245 mg tryptophan for study 1, 548 mg for study 2, and 845 mg for study 3. b Mg of niacin excreted = (jtmoles of N-methylIn our calculation of tryptophan to niacin nicotinamide + zmoles of N-methyl-2-pyridone-5-carconversion ratios, the contribution from enboxamide) x 0.1226 mg/imole. The molecular weight dogenous tryptophan is a factor to be considof niacin was assumed to be 122.6 g/mole. C asAs Vivian (42) has discussed, loss of 1 sumption was made that the amount of niacin recovered ered. in the urine as N-methylnicotinamide plus N-methylg of nitrogen per day would represent about 2-pyridone-5-carboxamide was approximately 57% of 6.25 g of protein or about 62.5 mg of tryptothe total amount of niacin metabolites that could come phan. If a 60: 1 ratio is assumed, this would from dietary niacin (23).

Downloaded from www.ajcn.org by guest on June 20, 2011

be equivalent 57% recovery,

to about 1 mg of niacin and at would be equivalent to about

diet was increased from 245 to 548 conversion ratio decreased to 58: 1 tryptophan content of the diet was from 548 to 845 mg. The sequential for the two subjects who participated three studies support this conclusion. ratio for one subject was 105: 1
tryptophan 548 mg and intake was 34: 1 when increased tryptophan

mg. The 4 tmotes of urinary niacin metabolites. Dcwhen thespite the fact that five of eight subjects in increased study 1 were in negative nitrogen balance, the values amount of tryptophan arising from protein in all degradation would not be large enough to The contribute significantly to the urinary niacin when his metabolites.
in inter-

acid increased from 548 to 845. along with other subject were 178: 1 and 105: 1, respec- the changes in tryptophan content which tivety. The values for these two subjects rewere accomplished by including cheese and flect the large individual variation in this dry powdered milk in the diet and reducing ratio, as was also found by Goldsmith and by the amount of gelatin. The objective of the
Horwitt. study was to simulate

from 245 to Lastly, a point to be considered intake was preting these results is that the Ratios for the composition of the diets changed

amino

Since
investigate

our
the

experiments
possibility

were
of

designed
in

a change

to which in tryptophan the would be supplied

a true-to-life and other

amino

situation acids

from

proteins

and

not

as

conversion the results

crystalline amino acids. Although the sulfurcontaining amino acid intake was kept conanalysis (Table 6 and Fig. 2). There was stant a at 2 g/day in all studies, levels of other significant linear correlation (r = 0.86, P < amino acids were not adjusted. Therefore, the 0.01) and no evidence of lack of fit between possibility that other amino acids could have

of tryptophan to were also examined

nicotinamide, by regression

the amount of tryptophan consumed per dayhad some effect on tryptophan metabolism and the amount of total niacin metabolites cannot be excluded. Despite these differences measured in the urine per day. This would in amino acid composition and protein conindicate that differences between the ratios tent, our results agreed quite well with those reported in Table 7 are not statistically sig- Horwitt of et at. (22) who used both crystalnificant, possibly owing to the large individ- line amino acids and lactalbumin as tryptoual variations. A similar relationship was phan sources and those of Goldsmith et at. crystalline DL-tryptophan. found when values for each niacin metabolite (23) who used

EXCRETION

OF

TRYPTOPHAN-NIACIN

METABOLITES

BY

YOUNG

MEN

2165

>S

70
.

0 0 0

60

a, a,
U

$
.

50

a, 4
0 0.

40

+ as
30

z
(I)

I
20
.

a, 0

Downloaded from www.ajcn.org by guest on June 20, 2011

--

245

548

845

mg
FIG. excreted subject 0.01. 2. Total per day as described imo1es of as a function in Table

tryptophon

consumed

/doy
(MPCA) value for one of 0.86, < P

N-methylnicotinamide (NMe) plus N-methyl-2-pyridone-5-carboxamide of milligram tryptophan consumed. dot Each represents the 6. By linear regression 0.0642x y = - 0.131 with a correlation

average coefficient

Our

resutts

show

that

the

proportion

tryptophan converted crease as tryptophan

however, this change as determined was small

to niacin consumption
and

one would predict that the conversion ratio of of tryptophan to niacin required to cover tended to inwould be 89: 1 and increased, 97.5% of the population

our results, 1 1 1:1. by linear regression from there was a tendency for the propornot significant over Since tion of tryptophan converted to niacin to the range of tryptophan intakes studied. increase as tryptophan intake increased, the 60:1 ratio is probably appropriate for the What is an appropriate tryptophan to niacin American adult consuming a diet containing conversion ratioforpublic health 800 to 1000 mg of tryptophan per day. Howrecommendations? ever, for adults consuming low protein diets A question arises as to whether the average or for growing infants and children, the ap-

ratio of 60: 1 as recommended by the Foodpropriateness and Nutrition Board of the National Re- calculating In search Council (25) is the appropriate ratio uted by the
to use Goldsmith mg of

ofthis

ratio

may

be questioned.

mation give a The variation

lites in

the amount of niacin contribtryptophan in a diet that is marfor public health recommendations. ginal in protein for a potentially pellagrous reported that between 34 and 86population, a ratio of between 90 and 110 rng tryptophan was required for the forof dietary tryptophan to provide 1 rng of of 1 mg of niacin (23), which would macin should be high enough to include those coefficient of variation of about 30%. at the upper end of the conversion ratio range.

in excretion
and

of niacin

metabo-

our

studies

in

Horwitts

studies References

El
F0LLIS, R. H. Deficiency Disease. Springfield, Ill.: Charles C Thomas Publisher, 1958. 2. SARE1-r, H. P. Effect of dietary tryptophan imbalance upon the metabolism of tryptophan and nicotinic acid in man. J. Biol. Chem. 182: 659, 1950. 1.

(personal communication) was also approximately 30%. Therefore, some individuals would be expected to convert proportionately
more cutating smith tryptophan to niacin than from the values obtained and assuming a normal

others. Catby Golddistribution,

2166 3.
GOPALAN,

PATTERSON C.,
AND

ET 22. 505,

AL.

4.

5.

6.

7.

8.

9. 10.

1 1.

12. 13.

14.

15.

16.

17.

18.

19.

20.

21.

HoRwn-r, M. K., C. C. HARVEY, W. S. ROTHWELL, J. L. CUTLER AND D. HAFFRON. Tryptophan-niacin relationship in man. J. Nutr. 60: 1, 1956. GOPALAN, C., AND B. S. N. RAO. Experimental 23. GOLDSMITH, G. A., 0. N. MILLER AND W. G. UNniacin deficiency. Meth. Achievem. Exptl. Pathol. 6: GLAUB. Efficiency of tryptophan as a niacin precur49, 1972. sor in man. J. Nutr. 73: 172, 1961. KRISHNASWAMY, K., S. B. R&o, T. RAGHIJRAM C. 24. NAKAGAWA, I., T. AKAHASHI, T T. Suzutu AND Y. AND S. G. SRncirIA. Effect of vitamin B6 on leuMASANA. Effect in man ofthe addition of tryptophan cine-induced changes in human subjects. Am. J. or niacin to the diet on the excretion of their meCliii. Nutr. 29: 177, 1976. tabolites. J. Nutr. 99: 325, 1969. BAPURAO, S., AND K. KRISHNASWAMY. Vitamin B6 25. Recommended Dietary Allowances (8th ed). Washnutritional status of pellagrins and their leucine tolington, D.C.: National Academy of Sciences/Naerance. Am. J. Chin. Nutr. 31: 819, 1978. tional Research Council, 1974. BAPU RAO, S., T. C. RAGHURAM AND K. KRISHNAS26. CLARK, L. C., AND H. L. THOMPSON. Determination WAMY. Role of vitamin B6 on leucine-induced metof creatine and creatinine in urine. J. Biol. Chem. abolic changes. Nutr. Metabol. 18: 318, 1975. 21: 1218, 1949. KR!SHNASWAMY, K., AND S. BAPURAO. Effect of 27. SCALES, F. M., AND A. P. HARRISON. Boric acid leucine at different levels of pyridoxine on hepatic modification of Kjeldahl method for crop and soil quinolinate phosphonbosyl transferase (EC 2.4.2.19) analysis. J. Ind. Eng. Chem. 12: 350, 1920. 28. Official Methods of Analysis of the Association of and leucine aminotransferase (EC 2.6. 1.6) in rats. Brit. J. Nutr. 39: 61, 1978. Official Analytical Chemists (12th ed.) 43. 12 1, p. GOPALAN, C., AND S. G. SRIKANTIA. Leucine and 844, 1975. pellagra. Lancet I: 954, 1960. 29. ATKINS, L., A. S. SCHULTZ, W. L. WILLtai,iS AND C. BELAVADY, B., S. G. SRIKANTIA AND C. GOPALAN. N. FREY. Yeast microbiological methods for deterThe effect of the oral administration of leucine on mination of vitamins. Pyridoxine. Ind. Eng. Chem. the metabolism of tryptophan. Biochem. J. 87: 652, Anal. Ed. 15: 141, 1943. 1963. 30. WOOLEY, J. G., AND W. SEBRELL. H. Two microbiGOPALAN, C., AND K. KRISHNASWAMY. Effect of ological methods for the determination of l(-)-trypexcess leucine on tryptophan niacin pathway and tophan in proteins and other complex substances. J. pyridoxine. Nutr. Rev. 34: 3 18, 1976. Biol. Chem. 157: 141, 1945. TRUSWELL, A. S., G. A. GOLDSMITH AND W. N. 3 1. TOEPFER, E. W., AND J. LEHMANN. Procedure for PEARSON. Leucine and pellagra. Lancet 1: 778, 1963. chromatographic separation and microbiological asTRUSWELL, A. S. The role of maize in the pathogensay for pyridoxal, pyndoxine, and pyridoxamine in esis of pellagra. Effect of leucine on N-methylnicofood extracts. J. Assoc. Official Anal. Chem. 44: 426, tinamide excretion in human adults. South African 1961. 32. WATr, B. K., AND A. L. Maaniu. Composition of Med. J. 37: 253, 1963. NAKAGAWA, I., S. OHGURI, A.SASKI, M. KAJIMOTO Foods. Agricultural Handbook, no. 8. Washington, AND M. SASAKI. Effect of excess intake of leucine D.C.: United States Department of Agriculture, and valine deficiency on tryptophan and niacin me1963. tabolites in humans. J. Nutr. 105: 1241, 1975. 33. ORR, M. L., AND B. K. WAi-r. Amino Acid Content NAKAGAWA, I., AND A. SASAKI. Effect of an excess of Foods. Home Economics Research Report no. 4. D.C.: United States Department of Agintake of leucine, with and without additions of Washington, vitamin B6 and/or niacin, on tryptophan and niacin riculture, 1957. metabolism in rats. J. Nutr. Sci. Vitaminol. 23: 535, 34. VIVIAN, V. M., M. REYNOLDS S. AND J. M. PRICE. 1977. The use of ion-exchange resins for the determination LOJKIN, M. E. Tryptophan and nitrogen metabolism of N-methylnicotinamide in human urine. Anal. and pregnancy of the rat as affected by dietary Biochem. 10: 274, 1965. supplementations with leucine and with amino acid 35. PRICE, J. M. The determination of N-methyl-2-pyridone-5-carboxamide in human urine. J. Biol. Chem. mixtures. J. Nutr. 100: 51, 1970. RAGMURAMULU, N., B. S. N. Ro AND C. GOPALAN. 211: 117, 1954. Amino acid imbalance and tryptophan-niacin me36. STEEL, R. G. D., AND J. H. TORRIE. Principles and tabolism. I. Effect of excess leucine on the urinary Procedures of Statistics. New York: McGraw-Hill excretion of tryptophan-niacin metabolites in rats. J. Book Co., 1960. Nutr. 86: 100, 1965. 37. DRAPER, H. R., AND H. N. S.smi. Applied RegresMANSON, J. A., AND K. CARPENTER. J. The effect of sion Analysis. New York: John Wiley & Sons, 1966. high level of dietary leucine on the niacin status 38. of KELLER, J., M. LIERSCH AND H. GRIJMICKE. Studies chicks and rats. J. Nutr. 108: 1883, 1978. on the biosynthesis of NAD from nicotinamide and on the intracellular pyridine nucleotide cycle in isoMANSON, J. A., AND K. CARPENTER. J. The effect of lated perfused rat liver. European J. Biochem. 22: a high level of leucine on the niacin status of dogs. J. Nutr. 108: 1889, 1978. 263, 1971. GOPALAN, C., B. BELVADY AND KRISHNASMURK. 39. HANKES, L. V., R. R. BROWN, J. Lricws, M. THY. The role of leucine in the pathogenesis of canine SCmsAnutR AND J. JEsSEPH. Metabolism of C4 lablack-tongue and pellagra. Lancet 2: 956, 1969. beled enantiomers of tryptophan, kynurenine and hydroxykynurenine in humans with scleroderma. J. BELVADY, B., AND P. V. Rio. Production of nicotinic acid deficiency in monkeys fed leucine-suppleChin. Invest. Dermatol. 58: 85, 1972. mented diets. lnternat. J. Vitamin Res. 43:454, 1973 40. GOLDSMITH, G. A., H. L. ROSENTHAL, J. GIBBENS
JAYA RAO.

K.

S.

amino 1975.

acid

imbalance.

Vitamins

Pellagra Hormones

and 33:

Downloaded from www.ajcn.org by guest on June 20, 2011

EXCRETION
AND

OF Studies

TRYPTOPHAN-NIACIN

METABOLITES lion and for a

BY

YOUNG

MEN variation in

2167 tissue NAD

in in 41.

W. G. man. II. tryptophan.

UNGLAUB.

POWANDA,

Evidence of dietary

Requirement J. Nutr. M. C., AND for a linear tryptophan

of niacin requirement of wheat and corn low diets 56: 371, 1955. R. W. WANNEMACHER, JR. 42. correlation between the level and hepatic NAD concentra-

systematic

concentration in themouse and the rat. J. Nutr. 100: 1471, 1970. VIVIAN, V. M. Relationship between tryptophanniacin metabolism and changes in nitrogen balance. J. Nutr. 82: 395, 1964.

Downloaded from www.ajcn.org by guest on June 20, 2011