45,63-68(1978)

TOXICOLOGYANDAPPLIEDPHARMACOLOGY

Concentrations

of Nitrate in Normal Human Effect of Nitrate Ingestion

Urine and the

JACK L. RADOMSKI, CHRISTINE PALMIRI, AND WILLIAM Department

of Pharmacology, Received

School

of Medicine,

July 18,1977;

University

accepted

November

of Miami,

LEE HEARN

Miami,

Florida

33152

14,1977

Concentrations of Nitrate in Normal Human Urine and the Effect of Nitrate Ingestion. RADOMSKI,

J. L.,

PALMIRI,

C., AND

HEARN,

W.

L. (1978). Toxicol.

Appl. Pharmacol.

45,63-

68. A method suitable for the analysis of nitrate in human urine was developed. Normal urinary concentrations of nitrate in urine of human volunteers in Dade County, Florida, where the drinking water contains negligible amounts of nitrate, averaged 47.6 ppm of NO,- (SD = 17.3). On a vegetable and preserved-meat-free diet, the nitrate concentration was reduced (10 to 30 ppm of NO,-), but, on nitrate-supplemented drinking water, the urinary concentration rose to a range of 34-87 ppm of NO,-. A high vegetable diet resulted in peak urinary nitrate concentrations of 270-425 ppm. These results indicated that nitrate in drinking water is a factor in determining urinary nitrate concentration, but that vegetable ingestion is of greater significance.

Recent evidence suggests that patients with urinary tract infections may be suffering vesical exposure to nitrosamines because of the reduction of urinary nitrate to nitrite by the infecting organisms (Hawksworth and Hill, 1974; Radomski et al., 1978; Brooks et al., 1972). Since the concentration of nitrite and hence the amount of nitrosamine produced is correlated with the concentration of nitrate present, it becomes important to know something about the magnitude of urinary nitrate concentrations and the factors which influence it. Standard texts and compendia yielded only an unbelievably high value of 0.4% (Sollmann, 1957). One summary of the concentrations of 524 organic and inorganic substances in urine merely indicated in a footnote that nitrate was also present (Diem and Lentner, 1970). A review of the literature produced only one early study of human urinary nitrate concentrations reporting concentrations of 100-400 mg/liter. However, in this study a method was used in which nitrate was reduced to nitrogen dioxide, which was collected and measured by a Van Slyke procedure (Mitchell et al., 1916). This method is both cumbersome and likely to produce spurious results because of the reduction of other nitrogenous urinary components to nitrogen dioxide. A thorough investigation of methods for the analysis of nitrate concentrations in urine was therefore undertaken. The ion-selective electrode and various calorimetric procedures were tried. Eventually, a method was developed which gave satisfactory recoveries. In addition to a determination of the normal range of urinary nitrate concentrations, the question of the origin of urinary nitrate was addressed in this study. Drinking water, vegetables, and meats have been suggested as dietary sources. In addition, the possibility of inorganic nitrate being an end product of nitrogen metabolism has been 63

004 1-008X/78/045 l-0063 $02.0010 Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved. Printed in Great Britain

64 proposed. (Moreno, presented water and

RADOMSKI,

PALMIRI,

AND

HEARN

The negligible nitrate content of Miami drinking water (less than 1 ppm) H. R., Dade County Water Department, personal communication, 1976) us with the opportunity to investigate the influence of nitrate in drinking vegetable ingestion on human urinary nitrate concentrations. METHODS

Analysis of urinary nitrate. Use of the Orion selectively permeable nitrate electrode gave grossly high results, presumably the result of Cl- and NH, interference. Pretreatment of urine with Ag+ resulted in insufficient improvement. Calorimetric methods involving the reduction of nitrate to nitrite (Whelan, 1930; Straighton et al., 1960; Wegner, 1971) also gave high blanks and high recoveries due to interferences. A copper-cadmium column was also used to reduce nitrate to nitrite (Strickland et al., 1968), but results were too variable and recoveries were low. Apparently, materials present in the urine inhibited the catalyst. The method finally utilized was adapted from a procedure utilizing a 2,4-dimethylphenol and sulfuric acid (Vasak, 1965). In the presence of nitrate, the xylenol is nitrated. The nitrated phenol is steam distilled into sodium hydroxide producing a yellow color which could be measured calorimetrically. The specific procedure developed for these experiments is as follows: The urine specimen (1 ml) was diluted 1: 10 with saturated Ag,SO,, mixed, and centrifuged at 300 rpm for 15 min. Four milliliters of the supernatant was transferred to a test tube and chilled in ice while 7 ml of concentrated H,S04 was slowly added, followed by 3 drops of 2,4-dimethylphenol (Eastman Organics) solution in glacial acetic acid. The samples were allowed to stand at room temperature for 25 min, and then 25 ml of deionized water was added. The samples were distilled until 15 ml had been collected; 2.5 ml of NaOH (1 M) was added to the distillate, and the volume was adjusted with water to a final volume of 25 ml. The concentration was determined calorimetrically at 445 nm. Experimental diets. The low-nitrate diet used in this experiment involved the elimination of vegetables and preserved meats (e.g., ham, bacon, sausages, corned beef, etc.). The high-nitrate experimental diet consisted solely of vegetables reportedly high in nitrate (Kilgore et al., 1963), primarily broccoli, spinach, lettuce, tomatoes, and collard greens. Urine collection periods and meal times are indicated in the figures and in the descriptions of the individual experiments. Specific gravity measurements were made on all urines but attempts to correct the urinary nitrate measurements for differences in specific gravity introduced more, not less, variability. Experimental subjects. The subjects used in these experiments were all adult Caucasian volunteers, both males and females, ranging in age from 22 to 56 years and in good health. Nitrate Recovery Studies

RESULTS

Since nitrate-free urine was not available, recovery studies were performed by measuring the concentration of nitrate in urine specimens before and after addition of a known amount of nitrate. The results of these recovery studies are presented in Table 1. Since these data involve errors in both the original measurements and the final measurements after added nitrate and 50% of the time the errors could be expected to be

NITRATE

CONCENTRATIONS

IN HUMAN

URINE

65

TABLE 1

Concentration of NO,- in (ppm) Urine (A)

Added (W

60 60 60 60 60 60 25 46 46 49 38.5 112

Expected (A + B)

76 76 53 53 32 32 60 37 62 48 75 124

136 136 113 113 92 92 85 83 108 97 113.5 236

Obtained 105

Recovery (%I 77 74 81 94 96 87 79 82 82 97 85 84

100 92 106 88 80 67 68 89 94 97 199

Overall average 85 additive, these recoveries are necessarily more variable and inaccurate than the actual determinations. Nevertheless, the accuracy is sufficient for our purposes and clearly superior to the other procedures we tried. Normal Concentrations of Nitrate in the Urine of Dade County Residents The normal concentration of nitrates in urine was determined in seven residents of Dade County, Florida. Multiple samples were taken on different days and at different times during the day. No attempt was made to control the diet or the time of the urine collection, except that in no case was an overnight urine used. In all, 24 measurements were made (Table 2). The means of the nitrate determinations in all seven individuals ranged from 41.0 to 55.6 ppm (pug/ml), a surprisingly narrow range. The individual TABLE 2

Urine concentration (ppm) Subject

Sex

Age

1

M

2 3 4 5

F

30 28 25 56 30 22 38

6 7

M M M F F

Number of samples

Mean

Range

3 6 3 4

1

55.3 46.5 41.0 46.0 53.0

31-65 32-56 30-58 19-98

2 5

36.0 55.6

20-52 46-64

-

66

RADOMSKI,

PALMIRI,

AND

HEARN

measurements have a wider range but ail values were below 100 ppm. The overali mean of the measurements was 47.6 ppm with a SD of 17.32. Effects of Nitrate Ingestion on Concentrations of Urinary Nitrate In order to evaluate the effects of nitrate ingestion on urinary nitrate concentrations, an experiment was conducted on four individuals. These individuals were first placed on a low-nitrate diet. They were then provided with nitrate-containing drinking water (40 ppm of NO,- as their sole source of liquid intake, the volume of which was recorded, and the amount of nitrate ingested was calculated. Measurements of the concentration of the nitrate in the urine were made when the subject was on the low-nitrate diet and for the 2 days the subject drank nitrate-supplemented water. A single measurement was TABLE CONCENTRATIONS

3

OF NITRATE IN URINE IN SUBJECTS ON A LOW-NITRATE EFFECT OF NITRATE IN DRINKING WATER

DIET

AND

THE

Nitrate concentration (ppm) Low In subjects on

Subject 1 2 3 4 5

In subjects on nitrate-containing drinking water

low-nitrate diet 17.0 + 2.1 (4)b

10.0 + 2.0 (3) 24.0 & 1.7 (4) 17.0 & 3.5 (3) 29.0 + 0.5 (3)

Day 2

Day 1 60.0 34.3 60.0 87.3 54.3

f * f & +

4.9 5.0 17.0 15.9 14.3

(3) (4) (5) (3) (3)

48.0 78.3 86.0 66.0 57.0

+ + + f +

10.8 5.4 12.0 13.4 18.1

Total

nitrate intake” Day 3 (mg of NO,-) (3) (4) (3) (3) (3)

31 74 51 44

(1) (1) (1) (1) 60 (1)

305 178 131 146 182

’ Above that present in the basal, low-nitrate diet. b Mean k SE. Number in parentheses indicates number of urine samples measured.

made the morning of the third day of the experiment. On the low-nitrate diet individuals, a relatively consistent depression of the nitrate concentration in the urine was evident (Table 3). The overall mean of all the individuals on the low-nitrate diet was 19.6 ppm. Each individual responded with an increased urinary nitrate concentration as a result of being placed on nitrate supplemented drinking water. There was no consistent cumulative effect on the second day. In two of the subjects the mean concentration of urinary nitrate increased and in the other two it decreased. These concentrations are what one might expect for individuals drinking water containing the usual governmentapproved concentration of 40 ppm of nitrate. The overall mean of all of the measurements taken on these four individuals for 2 days was 64.0 ppm, a value somewhat higher than observed in the first experiment in which no control of the dietary nitrate intake was attempted. The experiment was not designed to be a study in nitrate balance, so all of the urine excreted was not collected and measured. Approximate calculations, however, indicated that, during the course of the experiment, less than half of the ingested nitrate was recovered.

NITRATE

CONCENTRATIONS

IN HUMAN

URINE

67

Eflect of the Ingestion of Vegetables on Nitrate Excretion After maintaining a group of five volunteers, three males and two females, on a lownitrate diet with no vegetables, preserved meats, or sausages, the subjects were fed meals consisting of both cooked and raw vegetables. A series of three such meals (one lunch, one evening meal, and a lunch the following day) were consumed. All urine was collected and analyzed for nitrate (Fig. 1). The results obtained showed a dramatic response to the nitrate content of the vegetable meals, with peak urinary concentrations ranging from 270 to 425 ppm. The magnitude of the response was appreciably greater than that observed in the previous experiment in which subjects were placed on nitratecontaining drinking water.

FIG. 1. The effects of vegetableingestion on urinary nitrate (NO,-) concentrations in five human volunteers. Arrows and vertical dashedlines indicatetimes when vegetableswere consumed.

DISCUSSION

An attempt has been made both to determine the levels of nitrate which can be expected in the urine of humans and to investigate someof the factors which control or influence this concentration. Existence of a local population consuming water essentially free of nitrate presented a good opportunity to control nitrate intake. Vegetablessuch as cauliflower, spinach, and collard greenshave been determined to contain a considerable amount of nitrate, especially if they are produced with the utilization of excessive quantities of nitrate fertilizers (Kilgore et al., 1963). Nitrates are also used in conjunction with nitrites in the preservation of meats and the preparation of sausages (Wolff and Wasserman, 1972). With virtually no nitrates in the water, elimination of high-nitrate foods reduced the nitrate concentrations of urine to approximately 20 ppm. This amount cannot, however, be considered to be endogenously produced becauseit may well representresidual nitrate in the diet. When our subjects were placed on drinking water containing 40 ppm of nitrate, the government-recommended maximum, a modest elevation in nitrate content occurred.

68

RADOMSKI,

PALMIRI,

AND

HEARN

However, when the subjects were fed meals containing large amounts of vegetables, particularly collard greens, cauliflower, and broccoli, sharp increases in the urinary nitrate content were produced, in some cases to 300 ppm and above. It appears from these results that nitrates in the vegetables are more apt to constitute an important source of urinary nitrate than is drinking water. Extrapolating from our results, one predicts that inhabitants of a city in which the drinking water contains the usual concentrations of 20 to 40 ppm would probably produce urine with nitrate concentrations of about 60 to 80 ppm. REFERENCES J. B., CHERRY, W. B., THACKER, L., AND ALLEY, C. C. (1972). Analysis by gas chromatography of amines and nitrosamines produced in vivo and in vitro by Proteus miribilis. J. Infec. Dis. 126, 143-153. DIEM, K., AND LENTNER, C. (1970). ScientiJic Tables. J. R. Geigy, Switzerland. HAWKSWORTH, G. M., AND HILL, M. J. (1974). The in vivo formation of N-nitrosamines in the rat bladderandtheir subsequent absorption.Brit. J. Cncer 29, 353-358. KILGORJZ, L., STASCH, A. R., AND BARRENTINE, B. F. (1963).Nitrate contentof beets,collards, turnip greens.J. Amer. Diet. Ass. 43, 39-42. MITCHELL, H. H., SHOULE, H. A., AND GRINDLEY, H. S. (1916).The origin of nitratesin urine. J. Biol. Chem. 24,46 l-490. RADOMSKI, J. S., GREENWALD, D., HEARN, W. L., BLOCK, N. L., AND WOODS, F. M. (1978). Nitrosamineformation in bladderinfectionsand its role in the etiology of bladdercancer.J. Toxicol. Appl. Pharmacol., in press. SOLLMANN, T. (1957).A Manual of Pharmacology, p. 1168.W. B. Saunders,Philadelphia,Pa. STRAIGHTON, W. R., TRAVIS, I. G., AND HIATT, E. (1960). Quantitative determinationof nitrate by enzymereductionto nitrite. J. Lab. Clin. Med. 41, 157. STRICKLAND, J. D. H., AND PARSONS, T. R. (1968). A Practical Handbook of Sea Water Analysis, pp. 71-80. QueensPrinter, Ottawa. VASAK, V. (1965). Determinationof nitrate in urine as a test of exposureto diethyleneglycol dinitrate.Prac. Lek. 17,47-50. WEGNER, T. N. (1971). Simpleand sensitiveprocedurefor determiningnitrate and nitrite in mixturesin biologicalfluids.J. Dairy Sci. 55,642-644. WHELAN, M. (1930). A calorimetric method for quantitative determinationof nitrates and nitritesin biologicalfluids.J. Biol. Chem. 86, 189-197. WOLFF, I. A., AND WASSERMAN, A. E. (1972). Nitrates, nitrites andnitrosamines.Science 177, 15-19. BROOKS,