Rikard Landberg

A rapid gas chromatography-mass spectrometry method for quantification of alkylresorcinols in human plasma.

Alkylresorcinols (ARs) are phenolic lipids that among foods are found almost exclusively in whole grain and bran products based on wheat and rye. They have been suggested to be used as selective biomarkers of whole grain wheat and rye intake and, thus, may serve as an alternative/complement to commonly used dietary assessment methods in epidemiological studies. For such investigations where analysis of large numbers of valuable samples is required, rapid, sensitive, and repeatable methods are essential. In this article, we describe a rapid and sensitive gas chromatography-mass spectrometry (GC-MS) method for quantification of AR homologues C17:0, C19:0, C21:0, C23:0, and C25:0 in human plasma. The method uses Oasis MAX solid phase extraction cartridges for sample cleanup. A plasma sample of 0.2 ml could be used without preincubation with water. Samples in the range of 7 to 8750 nmol total AR/L were successfully analyzed with the method described, and the average total AR recovery within the reported range was 92+/-12%. The within- and between-day precision values of total AR concentration in a quality control sample, determined as the coefficients of variation, were on average 7 and 10%, respectively. Approximately 30 to 50 samples could be analyzed within 1 day. The improved GC-MS method presented can be used for rapid analysis of ARs in relatively small sample volumes.

Rye Whole Grain and Bran Intake Compared with Refined Wheat Decreases Urinary C-Peptide, Plasma Insulin, and Prostate Specific Antigen in Men with Prostate Cancer

Rye whole grain and bran intake has shown beneficial effects on prostate cancer progression in animal models, including lower tumor take rates, smaller tumor volumes, and reduced prostate specific antigen (PSA) concentrations. A human pilot study showed increased apoptosis after consumption of rye bran bread. In this study, we investigated the effect of high intake of rye whole grain and bran on prostate cancer progression as assessed by PSA concentration in men diagnosed with prostate cancer. Seventeen participants were provided with 485 g rye whole grain and bran products (RP) or refined wheat products with added cellulose (WP), corresponding to ~50% of daily energy intake, in a randomized controlled, crossover design. Blood samples were taken from fasting men before and after 2, 4, and 6 wk of treatment and 24-h urine samples were collected before the first intervention period and after treatment. Plasma total PSA concentrations were lower after treatment with RP compared with WP, with a mean treatment effect of -14% (P = 0.04). Additionally, fasting plasma insulin and 24-h urinary C-peptide excretion were lower after treatment with RP compared with WP (P < 0.01 and P = 0.01, respectively). Daily excretion of 5 lignans was higher after the RP treatment than after the WP treatment (P < 0.001). We conclude that whole grain and bran from rye resulted in significantly lower plasma PSA compared with a cellulose-supplemented refined wheat diet in patients with prostate cancer. The effect may be related to inhibition of prostate cancer progression caused by decreased exposure to insulin, as indicated by plasma insulin and urinary C-peptide excretion.

Plasma Alkylresorcinol Concentrations Correlate with Whole Grain Wheat and Rye Intake and Show Moderate Reproducibility over a 2- to 3-Month Period in Free-Living Swedish Adults

Plasma alkylresorcinols (AR) are useful as dietary biomarkers of wheat and rye whole grain (WG) during interventions but need to be validated in free-living populations. This study estimated the medium-term reproducibility and relative validity of plasma AR as biomarkers of WG and cereal fiber intake. Seventy-two Swedish adults kept 3-d weighed food records on 2 occasions 2-3 mo apart. Of these men and women, 51 provided a fasting blood sample at the end of each occasion. In addition, 18 participants provided 3 fasting and 3 nonfasting samples for 3 consecutive days on the first and second occasions, respectively. Dietary and blood variables did not differ between the 2 occasions. Nonfasting plasma total AR concentration [210 nmol/L (95% CI: 140, 314)] was higher than fasting [99 nmol/L (95% CI: 72, 137)] (P < 0.0001). Mean WG intake was 70 ± 61 g/d (41% from rye) and the intra-class correlation coefficient was 0.44 (95% CI: 0.26, 0.63) for total WG intake and 0.47 (95% CI: 0.27, 0.67) for the fasting plasma total AR concentration, suggesting moderate reproducibility. Fasting plasma total AR moderately correlated with WG rye + wheat (r(s) = 0.53; P < 0.001) and cereal fiber intake (r(s) = 0.32; P < 0.05) when using mean values from both occasions. This suggests that plasma AR concentration in fasting samples can be used as a biomarker of rye + wheat WG intake in free-living populations with a high and consistent WG intake.

Reproducibility of Plasma Alkylresorcinols during a 6-Week Rye Intervention Study in Men with Prostate Cancer

Alkylresorcinols (AR), phenolic lipids exclusively present in the outer parts of wheat and rye grains, have been proposed as concentration biomarkers of whole-grain wheat and rye intake. A key feature of a good biomarker is high reproducibility, which indicates how accurately a single sample reflects the true mean biomarker concentration caused by a certain intake. In this study, the short- to medium-term reproducibility of plasma AR was determined using samples from a crossover intervention study, where men with prostate cancer (n = 17) were fed rye whole-grain/bran or refined wheat products for 6-wk periods. AR homologs C17:0 and C21:0 differed between the treatments (P < 0.001). The reproducibility determined by the intraclass correlation coefficient (ICC) was high (intervention period 1: ICC = 0.90 [95% CI = 0.82-0.98], intervention period 2: ICC = 0.88 [95% CI = 0.78-0.98]). The results show that a single fasting plasma sample could be used to estimate the mean plasma AR concentration during a 6-wk intervention period with constant intake at a precision of +/- 20% (80% CI). This suggests that the plasma AR concentration can be used as a reliable short- to medium-term biomarker for whole-grain wheat and rye under intervention conditions where intake is kept constant.

Long-term reproducibility of plasma alkylresorcinols as biomarkers of whole-grain wheat and rye intake within Northern Sweden Health and Disease Study Cohort

Background/Objectives:Alkylresorcinols (AR) have been suggested as specific biomarkers of whole-grain (WG) and bran intake from wheat and rye. Before using plasma AR as biomarkers in prospective cohort studies, the long-term reproducibility needs to be determined in order to judge how well a single plasma sample reflects the long-term concentration. The objective was therefore to estimate the reproducibility of plasma AR concentrations over 0.1–3.9 years.Subjects/Methods:The concentrations of AR homologues were analysed in plasma samples, drawn>8 h since last meal, 0.1–3.9 years apart (mean ∼2 years) in 74 participants in the Swedish prospective Västerbotten Intervention Project cohort. Reproducibility was estimated by calculating the intra class correlation coefficient (ICC).Results:Fasting plasma AR concentrations were similar between the first and second measurements. The ICC for total AR was 0.54 (95% confidence interval (CI)=0.38–0.69] overall, 0.34 (95% CI=0.13–0.64) for men and 0.73 (95% CI=0.56–0.85) for women, respectively. Somewhat higher ICCs were obtained for shorter AR homologues.Conclusion:In summary, the reproducibility of plasma AR over 0.1–3.9 years was high for women and moderate for men within this population. Together with previous data showing high validity of plasma AR as biomarkers of wheat and rye in different populations, the current finding suggest that this biomarker is stable over a long-time period and is therefore probably useful for assessment of long-term WG intake in populations with a wide intake range and a frequent intake.

Determination of alkylresorcinol metabolites in human urine by gas chromatography–mass spectrometry

Alkylresorcinols (ARs) are phenolic lipids present at high concentrations in the outer parts of rye and wheat kernels and have been proposed as biomarkers for intake of whole grain and bran products of these cereals. AR are absorbed in the small intestine and after hepatic metabolism two major metabolites, 3,5-dihydroxybenzoic acid (DHBA) and 3-(3,5-dihydroxyphenyl)-1-propanoic acid (DHPPA), are excreted in urine either as such or as conjugates. Urine samples from nine individuals were incubated with different enzymes to assess type and extent of conjugates. In comparison with DHBA, which was mostly found in the free form, the less polar DHPPA was conjugated to a greater extent and the major conjugates were glucuronides. In this method, urine samples were hydrolyzed using beta-glucuronidase from Helix pomatia and syringic acid was used as internal standard. Samples, silylated with BSTFA, were analyzed by GC-MS utilizing a BP-5 fused silica capillary column and single ion monitoring of molecular ions (m/z 370 [DHBA], m/z 398 [DHPPA]). Recoveries of DHBA and DHPPA were estimated to be 94% and 93%, respectively. The average intra-assay/inter-assay coefficients of variation were 4.9/5.7% for DHBA and 7.6/9.3% for DHPPA.

Dietary Flavonoid Intake and Esophageal Cancer Risk in the European Prospective Investigation into Cancer and Nutrition Cohort

We prospectively investigated dietary flavonoid intake and esophageal cancer risk in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. The study included 477,312 adult subjects from 10 European countries. At baseline, country-specific validated dietary questionnaires were used. During a mean follow-up of 11 years (1992-2010), there were 341 incident esophageal cancer cases, of which 142 were esophageal adenocarcinoma (EAC), 176 were esophageal squamous cell carcinoma (ESCC), and 23 were other types of esophageal cancer. In crude models, a doubling in total dietary flavonoid intake was inversely associated with esophageal cancer risk (hazard ratio (HR) (log₂) = 0.87, 95% confidence interval (CI): 0.78, 0.98) but not in multivariable models (HR (log₂) = 0.97, 95% CI: 0.86, 1.10). After covariate adjustment, no statistically significant association was found between any flavonoid subclass and esophageal cancer, EAC, or ESCC. However, among current smokers, flavonols were statistically significantly associated with a reduced esophageal cancer risk (HR (log₂) = 0.72, 95% CI: 0.56, 0.94), whereas total flavonoids, flavanols, and flavan-3-ol monomers tended to be inversely associated with esophageal cancer risk. No associations were found in either never or former smokers. These findings suggest that dietary flavonoid intake was not associated with overall esophageal cancer, EAC, or ESCC risk, although total flavonoids and some flavonoid subclasses, particularly flavonols, may reduce the esophageal cancer risk among current smokers.

Comparison of gas chromatography–mass spectrometry and high-performance liquid chromatography with coulometric electrode array detection for determination of alkylresorcinol metabolites in human urine

Alkylresorcinols (AR) are amphiphilic compounds present at high concentrations in the outer parts of wheat and rye kernels. Due to their specificity to whole grain and bran products of these cereals, AR and their metabolites have been proposed as biomarkers for intake of such foods. Two alkylresorcinol metabolites, 3,5-dihydroxybenzoic acid (DHBA) and 3-(3,5-dihydroxyphenyl)-1-propanoic acid (DHPPA), have previously been quantified in human urine using two different methodologies: high-performance liquid chromatography coupled to a coulometric electrode array detector (HPLC-CEAD) and gas chromatography in combination with mass spectrometry (GC-MS). In this study, these two methodologies were compared by analysing 114 urine samples from free-living Swedish subjects consuming their habitual diet. Data were evaluated by graphical investigation of difference-plots and statistical inference of agreement was assessed by weighted Deming regression analysis. The median DHBA concentrations were 11 μM (GC-MS) and 13 μM (HPLC-CEAD), respectively. Both difference-plot and regression analysis showed a small but statistically significant additive bias, with HPLC-CEAD resulting in a slightly higher DHBA concentration than GC-MS. The median concentration of DHPPA was 18 μM for both methods. Examination of the difference-plot of DHPPA did not indicate any systematic difference between the methods, but regression analysis showed small but statistically significant constant and proportional biases. The conclusion was that the two methodologies are equally suitable for analysis of alkylresorcinol metabolites in human urine and that any small systematic differences observed are most likely of limited practical importance.