metabolic fate of an oral dose of 15a/-labelednitrate in

{CANCER RESEARCH 43, 1921-1925, April 1983]0008-5472/83/0043-OOOOS02.00

Metabolic Fate of an Oral Dose of 15A/-labeledNitrate in Humans:

Effect of Diet Supplementation with Ascorbic Acid1

David A. Wagner, Dale S. Schultz, William M. Deen, Vernon R. Young, and Steven R. Tannenbaum2

Departments of Nutrition and Food Science [D. A. W., V. R. Y., S. ft. T.] and Chemical Engineering [D. S. S., W. M. D.Â¡,Massachusetts Institute of Technology,Cambridge, Massachusetts 02139

ABSTRACT

The metabolic fate of a p.o. dose of 3.5 mmol 15A/-labeled

nitrate has been investigated in 12 healthy young adults. Samplesof urine, saliva, plasma, and feces were collected over a periodof 48 hr following administration of the dose. Subjects receivedeither 60 mg of ascorbic acid, 2 g of ascorbic acid, or 2 g ofsodium ascorbate per day. An average of 60% of the 15N03~

dose appeared in the urine as nitrate within 48 hr. Less than0.1% appeared in the feces. The 15A/label of nitrate was also

found in the urine (3%) and feces (0.2%) in the form of ammoniaor urea. The fate of the remaining 35% of the 15N03~ dose

administered is unknown. No effect of ascorbic acid or sodiumascorbate on the nitrate and nitrite levels of plasma, saliva, urine,or feces was observed. A one-compartment pharmacokinetic

model was used to describe the relationships between intake,plasma concentration, and urinary excretion of nitrate. The half-

life of nitrate in the body was found to be approximately 5 hr,and its volume of distribution was about 30% of body weight.Daily endogenous biosynthesis of nitrate was estimated to beabout 1 mmol/day.

INTRODUCTION

There is widespread concern over exposure to nitrate fromdietary and environmental origins and its potential risk to humanhealth. Several epidemiological studies have suggested an association between exposure to high levels of nitrate and increased incidence of stomach cancer (2,11, 35). While nitrate isa stable chemical species, it can be converted to nitrite bybacterial reduction in the oral cavity (29) or other areas of thebody containing high concentrations of bacteria (23, 27). Salivarynitrite concentration has been reported to be directly proportionalto the amount of nitrate ingested (25). The swallowing of salivaexposes the stomach to the nitrite formed in the oral cavity.Considerable evidence suggests that nitrite in the stomach hasthe potential to nitrosate nitrogenous constituents of food products, drugs, and agricultural chemicals to form W-nitroso compounds (4, 6, 16). Endogenous production of W-nitroso com

pounds has been demonstrated in humans by a number ofinvestigators (14, 21, 30). The specific involvement of N-nitroso

compounds in human disease has not been shown, but it is wellestablished that most W-nitroso compounds are potent carcino

gens in laboratory animals (17).Recent studies (7,8,15, 26,34) have confirmed earlier reports

'This investigation was supported by USPHS Grant 1-P01-CA26731-03,

awarded by the National Cancer Institute, Department of Health and HumanServices.

2To whom requests for reprints should be addressed, at, Massachusetts

Institute of Technology, Room 56-309, Department of Nutrition and Food Science.77 Massachusetts Avenue, Cambridge, Mass. 02139.

Received May 24, 1982; accepted January 7, 1983.

(19) that the diet is not the only source of nitrate but that nitrateis synthesized endogenously from reduced nitrogen compounds.Hence, not all of the nitrate excreted in the urine is of dietaryorigin, although during periods of large nitrate intake, the contribution of endogenous nitrate biosynthesis to urinary nitrate issmall. Urinary excretion of nitrate in humans after high nitrateintakes relative to typical dietary levels [1.2 mmol/day for theUnited States population (33)] range from 50 to 90% (5, 12, 22).The recovery of large p.o. doses of nitrate in laboratory animalshas been found to be between 35 and 92% (10, 13, 32). Sinceurinary recovery is incomplete and fecal excretion of nitrate hasbeen found to be negligible (23), it is clear that nitrate is metabolized to a significant extent in the body.

The administration of 15A/-labelednitrate allows one to study

the metabolic fate of ingested nitrate more directly. Wang eÃal.(31) administered 15A/-labelednitrate to rats and recovered approximately 60 to 70% of the 15/Vlabel in the urine, about one-

half of which was in the form of nitrate. We reported previouslythat about one-half of the 15A/-labeled nitrate administered to

humans appeared in the urine as nitrate (7); however, the urineor feces was not analyzed for the presence of 15/Vlabel in other

nitrogen-containing compounds.

In addition to collections of urine, feces, and saliva, the studiesreported in this paper included blood sampling up to 48 hr afteringestion of Na'5NO3. This permits a comparison of urinary nitrate

clearance with total nitrate clearance from the body with the useof a one-compartment pharmacokinetic model and also is helpful

in interpreting salivary nitrate and nitrite data. Since supplementing the diet with ascorbic acid has been suggested as a possiblemeans of reducing endogenous /V-nitrosation (18), the effect of

ascorbic acid on the clearance of nitrate from the body and onthe nitrate and nitrite levels of saliva was investigated.

MATERIALS AND METHODS

The protocols for work described were screened by the Committeeon the Use of Humans as Experimental Subjects of M. I. T. Subjectswere selected for experiments on the basis of a medical history, thoroughphysical examination, and normal routine blood and urine clinical chemistry analysis. Volunteers on these studies were selected from the M. I.T. student population and participated on an outpatient basis in the M.I. T. Clinical Research Center but consumed their meals in the Department Diet Kitchen.

The first study was conducted on 6 young adult men, ages 20 to 28years, with a mean body weight of 79 kg. Subjects received a low-nitrate

diet for 7 days with complete mineral and vitamin supplements to meetall National Academy of Sciences/National Research Council recommendations, including trace elements. The diet, which has been describedpreviously (7), consisted of a soy protein diet, supplying 0.8 g of protein(W x 6.25) per kg per day, to which egg and milk protein was added toincrease protein intake to 1.5 g of protein per kg per day. The remainingcalories were derived from protein-free cookies, comstarch dessert, and

carbonated and sucrose beverages. Energy intake to provide energy

APRIL 1983 1921

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D. A. Wagner et al.

equilibrium was calculated from a dietary history interview and maintainedwithout change during the study. During the final 48 hr of this period, a15NO3~pharmacokinetic study was conducted as described below. A

break period of 1 week followed in which subjects could choose a free-choice diet, but each subject took a 500-mg ascorbic acid supplementwith each meal and evening snack (4 times per day = a 2-g total dailydose). After the break period, a final 1-week period followed, consistingof the low-nitrate diet plus a 2-g supplement of ascorbic acid. The 15NO3~

pharmacokinetic study was repeated at the end of this period. Theascorbic acid supplements were taken at each meal during the pharmacokinetic studies.

A second study investigated nitrate pharmacokinetics in young adultsusing 3 male subjects and 3 female subjects (the group of 6 werebetween the ages of 19 and 24 years, with a mean weight of 64 kg).The protocol was similar to that of the first study, except that 500 mg ofL-proline were administered to subjects 1 hr after the nitrate dose. Theformation of nitrosoproline in these subjects will be reported elsewhere.3

An additional third diet period of 1 week was investigated in thesesubjects, consisting of a 2-g (500 mg 4 times a day) supplementation ofsodium ascorbate (Hoffmann-LaRoche Inc., Nutley, N. J.). A 15NO3"

kinetic study was done at the end of this period.A study of 15NO3~kinetics involved giving a p.o. dose of 3.5 mmol of

Na15NO3 (99% atom excess 15N; KOR Isotopes, Cambridge, Mass.) in

10 ml of distilled water, followed by 200 ml of distilled water. Completeurine collections were made for the periods 0 to 1, 1 to 3, 3 to 6, 6 to12,12 to 24, and 24 to 48 hr after dosing. Samples of whole saliva werecollected by expectoration without stimulation just before the administration of nitrate and 0.5, 1, 2, 3, 6, 12, 24, and 48 hr afterwards. Bloodsamples were taken just prior to the nitrate dose and at 1, 3, 6,12, 24,and 48 hr after dosing. Fecal samples were collected following the dosefor 3 days and pooled.

Complete daily 24-hr urine samples were collected in prewashed 2-

liter polypropylene bottles containing the following preservatives: 100 mlof pure ethyl alcohol, 10 g of NaH2PO4, and 1.5 g of Na2HSO3. Loweramounts but similar ratios of the preservatives were added to bottlescollecting urine for shorter collection periods. Saliva was collected insterile polypropylene centrifuge tubes containing 100 n\ of 1.0 N NaOHas a preservative. Saliva was immediately centrifugea at 10,000 x g for15 min after collection in order to move any microbiological sediment orlarge debris. The supernatant was removed and stored frozen (-20Â°)

until analyzed. Whole blood was collected in heparinized tubes andcentrifuged for 30 min at 1000 x g. Plasma was removed and stored at-20Â° until analyzed.

Nitrate in urine and plasma and nitrate and nitrite in saliva wereanalyzed by an automated Griess procedure (9). Diet samples wereanalyzed for nitrate according to the method of Sen and Lee (24). Eachdiet component (10 g) was extracted for 30 min into an alkaline solutionat 50Â°. After protein precipitation by 10 ml of 1.4 M ZnSO4 and then

centrifugation for 15 min (2500 x g), the resulting supernatant wasplaced on a short (10 cm) anion-exchange column (Dowex 1, 50 to 100

mesh). Nitrate was eluted with 20 ml of 4 M NaCI. Fecal samples wereanalyzed for nitrate similarly to food samples but with the addition of afinal reverse-phase Sep-Pak (Waters Associates, Inc., Milford, Mass.)

cleanup step (23).15A/-Labeled nitrate in urine was measured by nitration of benzene to

form [15A/]nitrobenzene, followed by gas chromatography-mass spec-

trometry (HP5992 system from Hewlett-Packard) with selected ion mon

itoring at a mass:charge ratio of 123 (M) and 124 (M + 1) (9). The amountof [15N]nitrate in feces was analyzed in an identical manner. The total 15N

enrichment of both ammonia and urea in urine was assayed by treating5 ml of urine with urease in the outer well of a Conway dish (1). After asaturated K2CO3 solution is added to liberate gaseous ammonia, theammonia is trapped in 1% H2SO4 in the center well of the Conway dish.The resulting ammonium sulfate solution was analyzed by isotope ratio

mass spectrometry [Nuclide 3-60 RMS (8)]. The 15N enrichment of all

nitrogen-containing compounds in feces was determined by Kjeldahl

digestion of 5 g of feces (20) followed by steam distillation of ammoniainto 1% H2SO4. Isotope ratio mass spectrometry was used to quantltate

RESULTS

The daily urinary nitrate excretion for 12 subjects on a 5-daylow-ascorbic acid diet (60 mg/day) was 0.78 Â±0.22 (S.D.) mmol/

day. No significant effect of ascorbic acid or sodium ascorbate(both given at 2 g/day) on daily urinary nitrate excretion wasfound; the average daily nitrate excretion for each 5-day diet

period was 0.70 Â±0.26 and 0.68 Â±0.36 mmol/day, respectively.However, on all 3 diets, more nitrate was excreted in the urinethan was ingested. Since the average daily intake of nitrate fromthe diet for all subjects was 0.15 mmol/day, this representsexcretion of about 0.6 mmol nitrate/day in excess of dietaryintake.

The urinary excretion of a 3.5-mmol p.o. dose of 15NO3~isshown in Table 1. About 90% of all 15N03~ appearing in urine

was excreted within 24 hr. Even during elevated nitrate intake,excess urinary nitrate excretion above dietary intake still occurs,as shown by the excretion of 14N(V . A high supplementation of

ascorbic acid (2 g/day) or sodium ascorbate (2 g/day) had nosignificant effect on urinary excretion of the 15NO3~dose (data

not shown).The average fasting nitrate level in plasma was 0.03 mivi for

the low-ascorbic acid diet period (Table 2). After the administra

tion of nitrate, the plasma levels rose sharply during the first hr,a 6-fold increase above fasting levels. Nitrate levels in plasmadid not fully return to initial base-line levels until 24 to 48 hr later.

The average fasting level or rate of decay of nitrate in plasmawas not altered by supplementation of ascorbic acid or sodiumascorbate (data not shown).

As shown in Table 2, the fasting salivary nitrate and nitriteconcentrations for 12 individuals on the low ascorbic acid diet,measured before the nitrate dose, averaged 0.20 and 0.09 HIM,respectively. The mean nitrate and nitrite levels in mixed salivafollowing the dose are also given in Table 2. Wide variations inthe concentration of nitrate and nitrite in saliva were observedamong individuals. The peak concentration of salivary nitrateand nitrite occurred in the vicinity of 1 hr. The peak level forsalivary nitrate was roughly 10 times the fasting level, whereasthe nitrite peak was about 5 times that of the fasting salivarynitrite level. The S:P ratio4 varied between 12 and 20, the higher

values occurring shortly after the dose (Table 2). The S:P ratioaveraged 17 following the dose, but 2 individuals showed S:Pratios of up to 50. The average ratio of salivary nitrite to salivarynitrate varied from 0.19 to 0.42 (Table 2). This ratio was highestat fasting but dropped significantly following the nitrate dose.Ascorbic acid or sodium ascorbate did not alter nitrate and nitritelevels in saliva.

The recovery of 15N after the 15NO3~ dose revealed thatapproximately 60% (2.0 mmol) of the 15N03~appears as unme-

tabolized nitrate in urine within 48 hr after the dose. Less than0.1% (0.001 mmol) of the nitrate dose appears in the fÃ¨ces as15NO3". Three % (0.11 mmol) of the administered 15NO3~ap-

3 D. A. Wagner, D. E. G. Shuker, and S. R. Tannenbaum. Endogenous nitroso

proline formation in man, manuscript in preparation.

'The abbreviation used is: S:P ratio, the average ratio of the sum of salivary

nitrate and nitrite to plasma nitrate.

1922 CANCER RESEARCH VOL. 43



Fate of 15A/03~Â¡nHumans

Table 1Urinary excretion of nitrate following a 3.5-mmol p.o. dose of 15N03~

Urinary nitrate excretion for 12 subjects on the low-ascorbic acid diet (60 mg/day). The average amount of nitrate (14N(V) ingested from the diet was 0.15 mmol

during the testing period.

Rate of urinary excretion Cumulative urinary excretion

Time (hr) afterdose0-1

1-33-6

6-1212-2424-48Total

NO3~(mmol/hr)0.39

Â±0.06a

0.25 Â±0.050.19 Â±0.040.12 Â±0.020.06 Â±0.010.04 Â±0.0215NO3-

(mmol/hr)0.28

Â±0.050.19 Â±0.040.14 Â±0.020.08 + 0.020.03 Â±0.000.01 + 0.0014NO3-

(mmol/hr)0.1

2 Â±0.080.05 + 0.010.05 + 0.010.04 Â±0.010.03 Â±0.010.04 Â±0.02Total

NO3-(mmol)0.39

+ 0.060.88 Â±0.13

1.5 Â±0.172.2 Â±0.272.9 Â±0.284.0 Â±0.5715NO3'

(mmol)0.28

+ 0.050.66 Â±0.12

1.1 Â±0.151.6 + 0.251.9 + 0.272.1 Â±0.28'"NOr

(mmol)0.11

Â±0.020.22 Â±0.030.40 Â±0.050.60 Â±0.07

1.0 + 0.171.9 Â±0.63

Mean Â±S.D.

Table 2Nitrate levels in plasma and nitrate and nitrite levels in saliva after a 3.5-mmol dose of nitrate

Nitrate and nitrite levels in plasma and saliva for 12 subjects on a low-ascorbic acid diet (60 mg/day). Dietary NO3~ was 0.15 mmol/day.

Time (hr) after dose Plasma NO3 (ITIM)Salivary NO3~ (ITIM) Salivary NO2 (ITIM)

S:P ratio Salivary NCV/NCV

00.512361224480.03Â±0.01a0.1

7Â±0.030.14

Â±0.020.10+0.010.06Â±0.010.04Â±0.010.03Â±0.020.20

0.092.32.52.31.90.810.380.430.760.770.870.780.180.200.190.28

0.250.09

Â±0.040.53Â±0.220.48+0.240.52Â±0.270.49Â±0.200.29+0.160.15+0.060.12+0.070.10Â±0.0813Â±920

Â±720131215767512

50.420.230.190.230.300.380.380.300.180.100.080.110.140.160.170.150.42Â±0.25a Mean Â±S.D.

peared in the urine as either ammonia or urea. The 15Ncontent

of Kjeldahl-digested feces was considerably less (only 0.2% ofthe administered dose). Therefore, approximately 35% of the 15N

was not recovered as either excreted nitrate, ammonia, or urea.

DISCUSSION

Nitrate balance studies as presented here and those conducted previously (7, 8, 15,19, 26, 34) show net urinary nitrateexcretion which cannot be attributed to dietary intake. Duringperiods of low dietary nitrate intake (0.15 mmol/day), urinarynitrate excretion in excess of dietary intake is approximately 0.6mmol/day. We have suggested that endogenous nitrate biosynthesis in man must account for this excess urinary nitrate excretion (7). When a high intake (3.5 mmol) of nitrate, as 15N03~,was

presented to subjects, this endogenous production of nitrate stilloccurred; excretion of 14N03~averaged 1 mmol/day.

When the diet was supplemented with 2 g of ascorbic acid orsodium ascorbate, there was no significant effect on endogenousproduction of nitrate. Furthermore, while ascorbic acid effectivelycompetes with nitrogen-containing compounds for nitrosating

species derived from nitrate metabolism (18), it appears to haveno influence on nitrate excretion. Following the administration of3.5 mmol of 15NO3~,the amount of nitrate appearing in the urine,

plasma, or saliva was not different in supplemented and nonsup-

plemented ascorbic acid diet periods.Fasting salivary nitrite levels averaged 0.09 mw, as has been

reported previously for individuals consuming a low-nitrate diet

(25,29). A wide range of salivary nitrate and nitrite concentrationsamong the individuals was found following the nitrate dose. Thevariability found in this study may be associated with individualdifferences in salivary flow rates, oral cavity flora (25), and thecapacity of the active transport mechanism for enriching salivawith nitrate (3). The ratio of nitrite to nitrate in saliva tended toaverage about 0.33, which is slightly higher than the value of0.25 reported by Spiegelhalder ef al. (25). This ratio appears

lower at earlier times following the dose (high salivary nitratelevels) and is high at low salivary nitrate concentrations. Apossible explanation is that the nitrate reductases of the oralbacteria are approaching saturation at the peak salivary nitrateconcentrations achieved following the dose (2.0 to 3.0 mw).

The mean recovery of 15NO3~in urine was 60% of the admin

istered dose. Previously reported recovery of nitrate in urine inhuman studies is between 50 and 90% (5, 7, 12, 22). Theexcretion of nitrate in the feces was negligible (less than 0.1%),as was similarly reported by Saul ef al. (23). Excretion of nitrateÂ¡nthe feces has also been reported to be low in the rat followingadministration of nitrate (8, 31). It was found that nitrate under-

goe , reduction to reduced nitrogen, with 3% of administerednitrate appearing as either ammonia or urea in urine and 0.2% infeces. This is much less than the value of 16% obtained fromstudies with rats (8). About 35% of the 15NO3"dose cannot be

recovered as excreted nitrogen-containing compounds in the

urine or feces. This nitrate may undergo metabolism to gaseousproducts which are exhaled in the breath or appear in flatus.

A one-compartment pharmacokinetic model is a useful tool in

analyzing the plasma and urine data obtained in this study.Nitrate entry into the body can occur by 2 routes, namely, dietaryintake and endogenous synthesis. Nitrate is removed by urinaryexcretion and reaction to reduced forms of nitrogen. When thenitrate inputs to the body are taken to be constant and theremoval processes are assumed to be first order in nitrateconcentration, a one-compartment pharmacokinetic model leads

to the following equation to describe the plasma nitrate concentrations:

dt

where VD is the volume of distribution of the body, C is theplasma nitrate concentration, R is the net rate of input (primarilyendogenous synthesis), and kT is the total elimination constant(units of inverse time). The solution to this equation is:

APRIL 1983 1923



D. A. Wagner et al.

Cd) = â€” +

C0 is equal to the size of the dose divided by VD.â€”â€”is seen toKTVD

be the steady-state plasma nitrate concentration, CM. The aboveequation predicts that plotting [C(f) - Css] versus time semi-logarithmically should yield a straight line with slope -kT and

intercept C0.From the data in Table 2, it appears that the steady-state

concentration of nitrate in plasma has a mean value of about0.03 rriM. This value was subtracted from the mean plasmaconcentration following ingestion of the nitrate dose and plottedversus time on semilog coordinates. It was found that the removal of nitrate from the body was, indeed, primarily first orderin plasma nitrate concentration (data not shown). kT was foundto be 0.14 per hr, corresponding to a half-life for nitrate in the

body of 5 hr. C0 was determined by extrapolating the semilogplot to time 0 and was found to be 0.135 mw, indicating a volumeof distribution for nitrate of 21.1 liters (VD= dose/C0). Since the

mean weight of all 12 subjects was 71.4 kg, the nitrate space inhumans is thus about 30% of body weight. The data of Ellen efal. (5), who administered a dose of up to 130 mmol of nitrate,also shows an exponential decay in plasma nitrate concentrationfollowing ingestion and suggests a similar volume of distributionof about 30% of body weight.

The total clearance of nitrate from the body can be estimatedby multiplying kr and VD,which yields 2.9 liters/hr for the subjectsof this study. Urinary clearance was calculated by dividing theaverage rate of urinary excretion by the log mean plasma nitrateconcentration for each urine collection period, which yielded amean value of 1.6 liters/hr. This ratio of renal to total clearance(1.6/2.9), determined from the data for total nitrate (14+15N),

provides an independent prediction of the fraction of the nitratepresented to the body which will appear unmetabolized in urine.The urinary clearance was calculated to be 55% of total clearance, which is in good agreement with the recovery of 60% ofthe administered 15NO3~in urine as nitrate.

The daily nitrate excretion in the urine was found to be in therange of 0.50 to 0.90 mmol/day. However, this only represents60% of the total daily exposure to nitrate. Since the meanconsumption of dietary nitrate was 0.15 mmol/day in this study,the amount of endogenous nitrate biosynthesis in humans canbe estimated to be 0.60 to 1.4 mmol/day.

A one-compartment model is inadequate for addressing ques

tions concerning such matters as the generation of nitrite in theoral cavity and the fate of nitrite presented to the stomach.However, since the nitrate concentration of the blood is a majorfactor in determining the nitrate and nitrite levels of the rest ofthe body, the simple analysis presented here is an importantstep toward developing models which will yield insight into theseissues.

ACKNOWLEDGMENTS

The authors wish to thank Dr Nawfal Istfan for taking blood samples, JosephGlogowski for his excellent technical assistance, and Marta Wolfe, Shriner BurnsHospital, Boston, Mass., for analysis of 15NH3.

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Fate of 15N(V in Humans

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APRIL 1983 1925



1983;43:1921-1925. Cancer Res David A. Wagner, Dale S. Schultz, William M. Deen, et al. Humans: Effect of Diet Supplementation with Ascorbic Acid

-labeled Nitrate inN15Metabolic Fate of an Oral Dose of

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