A gas chromatography-mass spectrometry method for the quantitation of N-nitrosoproline and N-acetyl-S-allylcysteine in human urine: application to a study of the effects of garlic consumption on nitrosation

Abstract

Biomarkers in urine can provide useful information about the bioactivation of chemical carcinogens and can be used to investigate the chemoprotective properties of dietary nutrients. N-Nitrosoproline (NPRO) excretion has been used as an index for endogenous nitrosation. In vitro and animal studies have reported that compounds in garlic may suppress nitrosation and inhibit carcinogenesis. We present a new method for extraction and sensitive detection of both NPRO and N-acetyl-S-allylcysteine from urine. The latter is a metabolite of S-allylcysteine, which is found in garlic. Urine was acidified and the organic acids were extracted by reversed-phase extraction (RP-SPE) and use of a polymeric weak anion exchange (WAX-SPE) resin. NPRO was quantified by isotope dilution gas chromatography-mass spectrometry (GC-MS) using [13C5]NPRO and N-nitrosopipecolinic acid (NPIC) as internal standards. This method was used to analyze urine samples from a study that was designed to test whether garlic supplementation inhibits NPRO synthesis. Using this method, 2.4 to 46.0 ng NPRO/ml urine was detected. The method is straightforward and reliable, and it can be performed with readily available GC-MS instruments. N-Acetyl-S-allylcysteine was quantified in the same fraction and detectable at levels of 4.1 to 176.4 ng/ml urine. The results suggest that 3 to 5 g of garlic supplements inhibited NPRO synthesis to an extent similar to a 0.5-g dose of ascorbic acid or a commercial supplement of aged garlic extract. Urinary NPRO concentration was inversely associated with the N-acetyl-S-allylcysteine concentration. It is possible that allyl sulfur compounds found in garlic may inhibit nitrosation in humans.

Figure 1

Feeding study design - nitrate dosing began on day 7 following a run-in diet and continued for seven days. Subjects in the treatment arms received supplementation on day 9 and continued for five days. Urine was collected on day 10, day 12, and day 14.

Figure 2

Total ion chromatogram (TIC) for a 5ml urine sample prepared as described in the methods. Panel (A) shows a 5 minute window of the ion current intensity with 20pg/ul NPRO spiked into the sample. The peak for NPRO is identified with an arrow. Panel (B) shows the mass spectra of a 2pg/ul quantity of NPRO standard. Quantitation was carried out on the molecular ion (m/z 143) at a retention time of 12.466 minutes.

Figure 3

Representative linear standard curve of the NPRO-PFB response for a concentration range of 0.1-80 pg/ul. The linear regression equation for this standard curve was: y=-54.8 + 403.6(x). The R² for the fit was 0.9996.

Figure 4

The graph shows the concentration of ¹³C₅ NPRO that was added at 5, 10 and 15 ng/ml of urine (x-axis) versus the ¹³C₅ NPRO concentration (ng/ml) found following extraction and derivatization (y-axis). The percent return from these levels was 77, 84 and 89%, respectively. Data points and error bars are the mean (± SD) of 3 replicate measurements. The regression coefficient for linear fit was 0.944 and the goodness-of-fit for the regression model was R²= 0.9997.

Figure 5

The graph shows the correlations between the NPRO concentrations quantified by the internal standard method versus the isotope dilution method for each sample. The regression coefficient was 0.9 and the adjusted R² equaled 1.

Figure 6

The graph shows the mean (±SEM) of the NPRO output in each of the groups. The concentrations were averaged for each subject. The groups, identified on the x-axis, are as follows: control (no supplement); 1 gram garlic supplement; 3 gram garlic supplement; 5 gram garlic supplement; 3 gram Kyolic supplement; 0.5 gram ascorbic acid supplement. The NPRO concentration (ng/ml) is on the y-axis.

Figure 7

The abundance of the extracted ion current for N-acetyl-S-allylcysteine (y-axis), and the mass spectra (inset) for the molecular ion m/z=202 is shown. Panel (A) shows the EIC of approximately 90ng/ul of synthesized standard. Panel (B) shows a similar concentration quantified in 5 ml of extracted urine which was collected from a subject that consumed a 5 gram quantity of a fresh garlic supplement for one day.

Figure 8

The graph plots the average total NPRO concentrations versus the average total concentration of N-acetyl-S-allylcysteine concentrations that were excreted in the urine samples of each subject. The regression line is for all the data points.