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Endogenous nitrosation of L‐proline by dietary‐derived nitrate T.M. Knight
a c
a
b
, D. Forman , H. Ohshima & H. Bartsch
b
a
Imperial Cancer Research Fund, Cancer Epidemiology Unit , Radcliffe Infirmary , Oxford, OX2 6HE, United Kingdom b
Unit of Environmental Carcinogens and Host Factors , International Agency for Research in Cancer , Lyon, France c
Dept. of Surgery, School of Postgraduate Medicine and Biological Sciences , University of Keele , Thornburrow Dr., Hartshill, Stoke on Trent, ST4 7QB, United Kingdom Published online: 04 Aug 2009.
To cite this article: T.M. Knight , D. Forman , H. Ohshima & H. Bartsch (1991) Endogenous nitrosation of L‐proline by dietary‐derived nitrate, Nutrition and Cancer, 15:3-4, 195-203, DOI: 10.1080/01635589109514127 To link to this article: http://dx.doi.org/10.1080/01635589109514127
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Endogenous Nitrosation of L-Proline by Dietary-Derived Nitrate Downloaded by [Temple University Libraries] at 03:44 08 January 2015
T. M. Knight, D. Forman, H. Ohshima, and H. Bartsch
Abstract Two studies were conducted to assess the availability of normal dietary sources of nitrate for endogenous nitrosation of an amino acid substrate, L-proline, and to investigate the potential for dietary ascorbic acid to inhibit such nitrosation. In the first study, 16 subjects consumed a salad meal (containing about 172 mg nitrate) with and without a loading dose of proline. A significant increase (10.8 compared with 2.7 µg/24 hrs, p < 0.0001) in mean urinary N-nitrosoproline (NPRO) excretion following meal plus substrate ingestion was indicative of intragastric proline nitrosation by meal-derived nitrate. In the second study (19 different subjects), the mean urinary NPRO level was significantly decreased (15.8 µg compared with 28.4, p = 0.022) by inclusion of ascorbic acid sources in the meal. This demonstrated inhibition of proline nitrosation by dietary sources of the vitamin. Large interindividual variation in nitrosating ability was apparent that was associated with variation in meal nitrate to salivary nitrite conversion. Although meals containing fresh vegetables, as tested here, could provide sufficient nitrate to result in endogenous formation of N-nitroso compounds, there will be considerable inter- and intraindividual variation in the extent of this process. (Nutr Cancer 15, 195-203, 1991)
Introduction
JV-Nitroso compounds are an extensive group of chemicals, and the majority of those tested have been shown to be carcinogenic in a wide range of animal species (1). These compounds can be synthesized from the reaction of nitrite- and nitrogen-containing compounds such as amines, amides, amino acids, and ureas (2). Although iV-nitroso compounds can be ingested directly in certain foods (3), their formation can also take place in the human stomach (4). Intragastric synthesis of mutagenic A'-nitroso compounds from nitrogen compounds (derived from food, drugs, or bile acids) and nitrite (derived from the T. M. Knight and D. Forman are affiliated with the Imperial Cancer Research Fund, Cancer Epidemiology Unit, Radcliffe Infirmary, Oxford, OX2 6HE, United Kingdom. H. Ohshima and H. Bartsch are affiliated with the International Agency for Research in Cancer, Unit of Environmental Carcinogens and Host Factors, Lyon, France.
Copyright © 1991, Lawrence Erlbaum Associates, Inc.
Downloaded by [Temple University Libraries] at 03:44 08 January 2015
reduction of ingested or endogenous nitrate) may play a central role in human gastric carcinogenesis (5). A reliable technique for demonstrating endogenous nitrosation in the human stomach, developed by Ohshima and Bartsch (6), involves ingestion of a source of nitrate (originally beetroot juice) followed by a dose of the amino acid, L-proline, as substrate. The resulting noncarcinogenic compound, AT-nitrosoproline (NPRO), is excreted in urine. Work with laboratory mammals demonstrated that endogenously synthesized NPRO was relatively unchanged in vivo, over 80% of an administered dose being excreted in a 24-hour urine sample (4). Thus, measurement of urinary NPRO can provide a direct indicator of the amount of in vivo NPRO synthesis. The kinetics of endogenous NPRO formation follow those of in vitro acid-catalyzed amine nitrosation (6) and are therefore indicative of intragastric (aqueous and acidic) nitrosation. The NPRO technique has now been used in numerous studies to demonstrate endogenous nitrosation in human subjects (4). Ascorbic acid and vitamin E, known to be nitrosation inhibitors (7), partially restrict endogenous NPRO formation in humans when given as large, concentrated doses (often 1,000 and 500 mg tablets, respectively) together with the precursors (4). Although such studies have demonstrated the ability of individual dietary components to modify endogenous nitrosation, they do not simulate the more normal dietary situation, in which both nitrate and vitamins may be ingested in smaller amounts from less concentrated, more complex sources, in combination with a variety of other components. In a number of recent studies (4), investigators gave proline to individuals eating their usual unrestricted diets; the measurement of urinary NPRO excretion thereby indicates the nitrosating potential of the normal diet. In most of these studies, because diets were uncontrolled, it was not possible to estimate the contribution to endogenous nitrosation of quantified amounts of specific dietary components. In this paper, we report an experimental dietary study in which we also investigated the availability of nitrate in normal food sources for the in vivo nitrosation of proline in human volunteers. Standard amounts of nitrate were administered as a component of set test meals under controlled conditions. The effect of dietary ascorbic acid on nitrosation was also assessed. Materials and Methods
We report two studies, each a self-contained experiment, with subjects acting as their own controls. Sixteen subjects of both sexes aged 23-56 years, all nonsmokers, participated in the first study, and 19 (different) females aged 20-28 years, 5 of whom were current smokers, participated in the second study. All subjects were volunteers, either scientific colleagues or nutrition degree students. Two test meals were designed. Test Meal 1 was designed to supply nitrate accompanied by relatively low levels of ascorbic acid, vitamin E, preformed NPRO, and nitrite. Test Meal 2 consisted of the same items as Test Meal 1, with the addition of foods rich in ascorbic acid. All test meals were prepared from fresh ingredients purchased on the day of consumption and stored in a refrigerator until required. Test meals were prepared within two hours of consumption, and subjects consumed all test meal ingredients within a maximum of 45 minutes under supervision in afternoon sessions. All subjects avoided intake of cured meats and beer, potential sources of preformed NPRO (8,9), and vitamin supplements for the 72 hours before test meal consumption and throughout urine collection. Subjects also fasted for two hours before and two hours after test meal consumption. The compositions of the test meals are given in Table 1. The amounts of ascorbic acid, vitamin E, and free proline present were estimated by combining nutrient compositions from the British Food Tables (10) with portion weights. Each time test meals were prepared, extra meals were made, sealed immediately in foil containers, frozen, and stored at — 40°C until analysis for nitrate, nitrite, and NPRO.
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Table 1. Test Meal Compositions
Downloaded by [Temple University Libraries] at 03:44 08 January 2015
Weight, g
Ingredient Beetroot (boiled) Celery (raw) Radish (raw) Rice salad Boiled white rice Boiled (frozen) peas Canned sweetcorn Mushroom (raw) Green pepper (raw) Cottage cheese Whole-meal bread Butter Salad dressing Salad cream Natural yogurt Fresh raw apple Fresh raw strawberries Bottled mineral water Ribena Component Nitrate", mg Nitrite", mg NPRO", ng Ascorbic acid*, mg Vitamin Bb, mg Proline*, g
Test Meal 1 (high nitrate/ low ascorbic acid)
Test Meal 2 (high nitrate/ high ascorbic acid)
50 50 50
50 50 50
75 30 30 30
75 30 30 30 30 100 50 9
100 50 9 20 40 100
20 40
250
100 250 85
Mean amount (n = S) 172 (165-179) 0.04 (ND -0.1) ND (all samples)
Mean amount (n =2) 197 (Both samples) 0.1 (Both samples) ND (Both samples)
24 0.5 2.4
340 1.0 2.4
a: Mean values (range) are from analysis of extra samples of test meals from both parts of study. b: Values were estimated with nutrient compositions given in Britisl l Food Tables (10).
The first study was designed to determine the extent of endogenous nitrosation of proline by the nitrate supplied in Test Meal 1. Subjects first consumed Test Meal 1 without proline to determine background levels of NPRO excretion and then, one week later, an identical meal with 500 mg proline (Forum Chemicals, Redhill, UK). The proline, dissolved in 20 ml of water, was consumed one hour after the end of the test meal, at which time salivary nitrate reduction would be reaching a peak (11). Urine collections (24 hr) commenced immediately after proline ingestion. To establish the extent to which nitrite was being generated from the ingested test meal nitrate at the time of proline ingestion, salivary nitrate and nitrite concentrations were determined in samples collected in the second week immediately before consumption of the test meal and one hour later, immediately before proline ingestion. The second study was designed to determine the effect of dietary sources of ascorbic acid on the nitrosation of the proline by the test meal nitrate. Subjects therefore consumed the basic high-nitrate Test Meal 1 with 500 mg of proline (identical to second week of first study) and then, one week later, consumed Test Meal 2 (high nitrate, high ascorbic acid), also with 500 mg of proline. As in the first study, urine collections commenced one hour after test meal consumption, immediately after proline ingestion. Saliva samples were again taken imme-
Vol. 15, Nos. 3 & 4
197
Downloaded by [Temple University Libraries] at 03:44 08 January 2015
diately before each test meal and immediately before proline ingestion in both weeks of the study. All 24-hour urine samples were collected according to a standard protocol. Immediately before the collection period, subjects urinated and discarded the urine. From this time, all urine was collected into a 2.5-1 polypropylene container. At the end of the 24 hours, subjects urinated into the container. Sodium hydroxide (10 g) was placed in each urine container as a preservative before collection (6). During collection, the containers were kept at room temperature and away from direct heat and sunlight. Within 24 hours of completion, total urine volumes were recorded, and two 30-ml samples were aliquoted into polypropylene bottles. One aliquot was immediately frozen and stored at — 40°C for a maximum of three months before being analyzed for NPRO. The second aliquot was stored at 4°C in the dark for a maximum of one week before being analyzed for nitrate. Nitrate analyses were performed on urine aliquots from the second study. Saliva samples (5-10 ml) were collected in glass bottles containing 0.2 ml of 1 M NaOH as a preservative (12). These were kept at 4°C in the dark until analysis for nitrate and nitrite within one week of collection. Urinary NPRO was determined by an established method (6) after conversion to their methyl esters by diazomethane. Samples were analyzed with a gas chromatograph interfaced to a Thermal Energy Analyser, a nitrosamine-specific detector. Urinary and salivary nitrite were determined by using a modified Griess procedure (12). Nitrate concentrations in these samples were determined by bacterial reduction to nitrite followed by analysis for nitrite (12). Multiplication of NPRO and nitrate concentrations by urine volumes gave the total amount of each excreted over the 24-hour period. NPRO, nitrate, and nitrite in samples of the test meals were determined with established chemiluminescence methodology (13), within three months of preparation. Data were analyzed with the SPSSX Statistical Package (SPSS, Chicago, IL). Data on urinary levels of NPRO, nitrate, and salivary nitrate and nitrite concentrations were normalized by logarithmic transformation. Means are therefore geometric. All P values are two tailed. Results NPRO Excretion After Ingestion of High-Nitrate Test Meal For the 16 subjects in the first study, the mean urinary excretion of NPRO was 2.7 /*g, after ingestion of the high-nitrate Test Meal 1 without proline (background NPRO excretion). After ingestion of the same meal with proline, the mean NPRO excretion for the same subjects was 10.8 /xg. The difference between these two means was highly significant (p < 0.0001) (Table 2). If we assume that the difference (8.1 /tg) was NPRO synthesized from test meal nitrate and administered proline, this represents a conversion rate of nitrate to NPRO of 0.002% and of proline to NPRO of 0.001%. For the 19 subjects in the second study, the mean level of urinary NPRO after consumption of Test Meal 1 and proline was 28.4 jig. This was significantly higher (p = 0.0024) than the comparable mean from the first study (16 subjects consuming identical test meal plus proline) (10.8 fig). The five smokers in the second study had a higher mean NPRO excretion level (39.4 ng) than did the 14 nonsmokers (25.2 fig), but this difference was not significant (p = 0.42). Inhibition of Proline Nitrosation by Dietary Ascorbic Acid In the second study, mean urinary NPRO excretion after ingestion of Test Meal 2 with proline was significantly lower than that excreted by the same subjects after ingestion of Test Meal 1 with proline (15.8 ng compared with 28.4, p = 0.022) (Table 2). This occurred despite a higher nitrate intake from Test Meal 2 compared with Test Meal 1 (Table 1). The difference
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Nutrition and Cancer 1991
Table 2. Urinary Excretion of NPRO and Nitrate Geometric Mean (95% CI) of NPRO, pg/24 hrs
Geometric Mean (95% CI) of Nitrate, mg/24 hrs
2.7 (2.2,3.3) 10.8 (7.9, 14.9)
Nutrition and Cancer Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/hnuc20
Endogenous nitrosation of L‐proline by dietary‐derived nitrate T.M. Knight
a c
a
b
, D. Forman , H. Ohshima & H. Bartsch
b
a
Imperial Cancer Research Fund, Cancer Epidemiology Unit , Radcliffe Infirmary , Oxford, OX2 6HE, United Kingdom b
Unit of Environmental Carcinogens and Host Factors , International Agency for Research in Cancer , Lyon, France c
Dept. of Surgery, School of Postgraduate Medicine and Biological Sciences , University of Keele , Thornburrow Dr., Hartshill, Stoke on Trent, ST4 7QB, United Kingdom Published online: 04 Aug 2009.
To cite this article: T.M. Knight , D. Forman , H. Ohshima & H. Bartsch (1991) Endogenous nitrosation of L‐proline by dietary‐derived nitrate, Nutrition and Cancer, 15:3-4, 195-203, DOI: 10.1080/01635589109514127 To link to this article: http://dx.doi.org/10.1080/01635589109514127
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Endogenous Nitrosation of L-Proline by Dietary-Derived Nitrate Downloaded by [Temple University Libraries] at 03:44 08 January 2015
T. M. Knight, D. Forman, H. Ohshima, and H. Bartsch
Abstract Two studies were conducted to assess the availability of normal dietary sources of nitrate for endogenous nitrosation of an amino acid substrate, L-proline, and to investigate the potential for dietary ascorbic acid to inhibit such nitrosation. In the first study, 16 subjects consumed a salad meal (containing about 172 mg nitrate) with and without a loading dose of proline. A significant increase (10.8 compared with 2.7 µg/24 hrs, p < 0.0001) in mean urinary N-nitrosoproline (NPRO) excretion following meal plus substrate ingestion was indicative of intragastric proline nitrosation by meal-derived nitrate. In the second study (19 different subjects), the mean urinary NPRO level was significantly decreased (15.8 µg compared with 28.4, p = 0.022) by inclusion of ascorbic acid sources in the meal. This demonstrated inhibition of proline nitrosation by dietary sources of the vitamin. Large interindividual variation in nitrosating ability was apparent that was associated with variation in meal nitrate to salivary nitrite conversion. Although meals containing fresh vegetables, as tested here, could provide sufficient nitrate to result in endogenous formation of N-nitroso compounds, there will be considerable inter- and intraindividual variation in the extent of this process. (Nutr Cancer 15, 195-203, 1991)
Introduction
JV-Nitroso compounds are an extensive group of chemicals, and the majority of those tested have been shown to be carcinogenic in a wide range of animal species (1). These compounds can be synthesized from the reaction of nitrite- and nitrogen-containing compounds such as amines, amides, amino acids, and ureas (2). Although iV-nitroso compounds can be ingested directly in certain foods (3), their formation can also take place in the human stomach (4). Intragastric synthesis of mutagenic A'-nitroso compounds from nitrogen compounds (derived from food, drugs, or bile acids) and nitrite (derived from the T. M. Knight and D. Forman are affiliated with the Imperial Cancer Research Fund, Cancer Epidemiology Unit, Radcliffe Infirmary, Oxford, OX2 6HE, United Kingdom. H. Ohshima and H. Bartsch are affiliated with the International Agency for Research in Cancer, Unit of Environmental Carcinogens and Host Factors, Lyon, France.
Copyright © 1991, Lawrence Erlbaum Associates, Inc.
Downloaded by [Temple University Libraries] at 03:44 08 January 2015
reduction of ingested or endogenous nitrate) may play a central role in human gastric carcinogenesis (5). A reliable technique for demonstrating endogenous nitrosation in the human stomach, developed by Ohshima and Bartsch (6), involves ingestion of a source of nitrate (originally beetroot juice) followed by a dose of the amino acid, L-proline, as substrate. The resulting noncarcinogenic compound, AT-nitrosoproline (NPRO), is excreted in urine. Work with laboratory mammals demonstrated that endogenously synthesized NPRO was relatively unchanged in vivo, over 80% of an administered dose being excreted in a 24-hour urine sample (4). Thus, measurement of urinary NPRO can provide a direct indicator of the amount of in vivo NPRO synthesis. The kinetics of endogenous NPRO formation follow those of in vitro acid-catalyzed amine nitrosation (6) and are therefore indicative of intragastric (aqueous and acidic) nitrosation. The NPRO technique has now been used in numerous studies to demonstrate endogenous nitrosation in human subjects (4). Ascorbic acid and vitamin E, known to be nitrosation inhibitors (7), partially restrict endogenous NPRO formation in humans when given as large, concentrated doses (often 1,000 and 500 mg tablets, respectively) together with the precursors (4). Although such studies have demonstrated the ability of individual dietary components to modify endogenous nitrosation, they do not simulate the more normal dietary situation, in which both nitrate and vitamins may be ingested in smaller amounts from less concentrated, more complex sources, in combination with a variety of other components. In a number of recent studies (4), investigators gave proline to individuals eating their usual unrestricted diets; the measurement of urinary NPRO excretion thereby indicates the nitrosating potential of the normal diet. In most of these studies, because diets were uncontrolled, it was not possible to estimate the contribution to endogenous nitrosation of quantified amounts of specific dietary components. In this paper, we report an experimental dietary study in which we also investigated the availability of nitrate in normal food sources for the in vivo nitrosation of proline in human volunteers. Standard amounts of nitrate were administered as a component of set test meals under controlled conditions. The effect of dietary ascorbic acid on nitrosation was also assessed. Materials and Methods
We report two studies, each a self-contained experiment, with subjects acting as their own controls. Sixteen subjects of both sexes aged 23-56 years, all nonsmokers, participated in the first study, and 19 (different) females aged 20-28 years, 5 of whom were current smokers, participated in the second study. All subjects were volunteers, either scientific colleagues or nutrition degree students. Two test meals were designed. Test Meal 1 was designed to supply nitrate accompanied by relatively low levels of ascorbic acid, vitamin E, preformed NPRO, and nitrite. Test Meal 2 consisted of the same items as Test Meal 1, with the addition of foods rich in ascorbic acid. All test meals were prepared from fresh ingredients purchased on the day of consumption and stored in a refrigerator until required. Test meals were prepared within two hours of consumption, and subjects consumed all test meal ingredients within a maximum of 45 minutes under supervision in afternoon sessions. All subjects avoided intake of cured meats and beer, potential sources of preformed NPRO (8,9), and vitamin supplements for the 72 hours before test meal consumption and throughout urine collection. Subjects also fasted for two hours before and two hours after test meal consumption. The compositions of the test meals are given in Table 1. The amounts of ascorbic acid, vitamin E, and free proline present were estimated by combining nutrient compositions from the British Food Tables (10) with portion weights. Each time test meals were prepared, extra meals were made, sealed immediately in foil containers, frozen, and stored at — 40°C until analysis for nitrate, nitrite, and NPRO.
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Table 1. Test Meal Compositions
Downloaded by [Temple University Libraries] at 03:44 08 January 2015
Weight, g
Ingredient Beetroot (boiled) Celery (raw) Radish (raw) Rice salad Boiled white rice Boiled (frozen) peas Canned sweetcorn Mushroom (raw) Green pepper (raw) Cottage cheese Whole-meal bread Butter Salad dressing Salad cream Natural yogurt Fresh raw apple Fresh raw strawberries Bottled mineral water Ribena Component Nitrate", mg Nitrite", mg NPRO", ng Ascorbic acid*, mg Vitamin Bb, mg Proline*, g
Test Meal 1 (high nitrate/ low ascorbic acid)
Test Meal 2 (high nitrate/ high ascorbic acid)
50 50 50
50 50 50
75 30 30 30
75 30 30 30 30 100 50 9
100 50 9 20 40 100
20 40
250
100 250 85
Mean amount (n = S) 172 (165-179) 0.04 (ND -0.1) ND (all samples)
Mean amount (n =2) 197 (Both samples) 0.1 (Both samples) ND (Both samples)
24 0.5 2.4
340 1.0 2.4
a: Mean values (range) are from analysis of extra samples of test meals from both parts of study. b: Values were estimated with nutrient compositions given in Britisl l Food Tables (10).
The first study was designed to determine the extent of endogenous nitrosation of proline by the nitrate supplied in Test Meal 1. Subjects first consumed Test Meal 1 without proline to determine background levels of NPRO excretion and then, one week later, an identical meal with 500 mg proline (Forum Chemicals, Redhill, UK). The proline, dissolved in 20 ml of water, was consumed one hour after the end of the test meal, at which time salivary nitrate reduction would be reaching a peak (11). Urine collections (24 hr) commenced immediately after proline ingestion. To establish the extent to which nitrite was being generated from the ingested test meal nitrate at the time of proline ingestion, salivary nitrate and nitrite concentrations were determined in samples collected in the second week immediately before consumption of the test meal and one hour later, immediately before proline ingestion. The second study was designed to determine the effect of dietary sources of ascorbic acid on the nitrosation of the proline by the test meal nitrate. Subjects therefore consumed the basic high-nitrate Test Meal 1 with 500 mg of proline (identical to second week of first study) and then, one week later, consumed Test Meal 2 (high nitrate, high ascorbic acid), also with 500 mg of proline. As in the first study, urine collections commenced one hour after test meal consumption, immediately after proline ingestion. Saliva samples were again taken imme-
Vol. 15, Nos. 3 & 4
197
Downloaded by [Temple University Libraries] at 03:44 08 January 2015
diately before each test meal and immediately before proline ingestion in both weeks of the study. All 24-hour urine samples were collected according to a standard protocol. Immediately before the collection period, subjects urinated and discarded the urine. From this time, all urine was collected into a 2.5-1 polypropylene container. At the end of the 24 hours, subjects urinated into the container. Sodium hydroxide (10 g) was placed in each urine container as a preservative before collection (6). During collection, the containers were kept at room temperature and away from direct heat and sunlight. Within 24 hours of completion, total urine volumes were recorded, and two 30-ml samples were aliquoted into polypropylene bottles. One aliquot was immediately frozen and stored at — 40°C for a maximum of three months before being analyzed for NPRO. The second aliquot was stored at 4°C in the dark for a maximum of one week before being analyzed for nitrate. Nitrate analyses were performed on urine aliquots from the second study. Saliva samples (5-10 ml) were collected in glass bottles containing 0.2 ml of 1 M NaOH as a preservative (12). These were kept at 4°C in the dark until analysis for nitrate and nitrite within one week of collection. Urinary NPRO was determined by an established method (6) after conversion to their methyl esters by diazomethane. Samples were analyzed with a gas chromatograph interfaced to a Thermal Energy Analyser, a nitrosamine-specific detector. Urinary and salivary nitrite were determined by using a modified Griess procedure (12). Nitrate concentrations in these samples were determined by bacterial reduction to nitrite followed by analysis for nitrite (12). Multiplication of NPRO and nitrate concentrations by urine volumes gave the total amount of each excreted over the 24-hour period. NPRO, nitrate, and nitrite in samples of the test meals were determined with established chemiluminescence methodology (13), within three months of preparation. Data were analyzed with the SPSSX Statistical Package (SPSS, Chicago, IL). Data on urinary levels of NPRO, nitrate, and salivary nitrate and nitrite concentrations were normalized by logarithmic transformation. Means are therefore geometric. All P values are two tailed. Results NPRO Excretion After Ingestion of High-Nitrate Test Meal For the 16 subjects in the first study, the mean urinary excretion of NPRO was 2.7 /*g, after ingestion of the high-nitrate Test Meal 1 without proline (background NPRO excretion). After ingestion of the same meal with proline, the mean NPRO excretion for the same subjects was 10.8 /xg. The difference between these two means was highly significant (p < 0.0001) (Table 2). If we assume that the difference (8.1 /tg) was NPRO synthesized from test meal nitrate and administered proline, this represents a conversion rate of nitrate to NPRO of 0.002% and of proline to NPRO of 0.001%. For the 19 subjects in the second study, the mean level of urinary NPRO after consumption of Test Meal 1 and proline was 28.4 jig. This was significantly higher (p = 0.0024) than the comparable mean from the first study (16 subjects consuming identical test meal plus proline) (10.8 fig). The five smokers in the second study had a higher mean NPRO excretion level (39.4 ng) than did the 14 nonsmokers (25.2 fig), but this difference was not significant (p = 0.42). Inhibition of Proline Nitrosation by Dietary Ascorbic Acid In the second study, mean urinary NPRO excretion after ingestion of Test Meal 2 with proline was significantly lower than that excreted by the same subjects after ingestion of Test Meal 1 with proline (15.8 ng compared with 28.4, p = 0.022) (Table 2). This occurred despite a higher nitrate intake from Test Meal 2 compared with Test Meal 1 (Table 1). The difference
198
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Table 2. Urinary Excretion of NPRO and Nitrate Geometric Mean (95% CI) of NPRO, pg/24 hrs
Geometric Mean (95% CI) of Nitrate, mg/24 hrs
2.7 (2.2,3.3) 10.8 (7.9, 14.9)
