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BACKGROUND Wheat bran (WB) has been associated with improved gastrointestinal health and a reduced risk of metabolic disorders. Reducing the particle size of WB might increase its fermentability and facilitate cross-feeding between the gut bacteria and in this way produce health effects. OBJECTIVES We investigated the impact of WB with reduced particle size (WB RPS) on colonic fermentation and host health in normal-weight (NW) and obese (OB) participants compared with placebo (PL). METHODS During 1 mo, 36 NW and 14 OB participants daily consumed 20 g WB RPS or PL (maltodextrin). Before and after the intervention, fasting serum and fecal SCFAs, fecal metabolite profiles, and microbiota composition were measured as fermentation parameters. Fecal output, fecal dry weight (%), fat excretion, transit, stool consistency, intestinal permeability, and serum total cholesterol, triglyceride, and C-reactive protein concentrations were measured as health parameters. The impact of WB RPS on the fermentation of other carbohydrates was assessed by quantifying postprandial cumulative serum 13C-SCFA after a challenge with 13C-inulin. RESULTS WB RPS increased fasting serum acetate (P

Wheat bran (WB) is a constituent of whole grain products with beneficial effects for human health. Within the human colon, such insoluble particles may be colonized by specific microbial teams which can stimulate cross-feeding, leading to a more efficient carbohydrate fermentation and an increased butyrate production. We investigated the extent to which WB fractions with different properties affect the fermentation of other carbohydrates in the colon. Ten healthy subjects performed four test days, during which they consumed a standard breakfast supplemented with 10 g (13)C-inulin. A total of 20 g of a WB fraction (unmodified WB, wheat bran with a reduced particle size (WB RPS), or de-starched pericarp-enriched wheat bran (PE WB)) was also added to the breakfast, except for one test day, which served as a control. Blood samples were collected at regular time points for 14 h, in order to measure (13)C-labeled short-chain fatty acid (SCFA; acetate, propionate and butyrate) concentrations. Fermentation of (13)C-inulin resulted in increased plasma SCFA for about 8 h, suggesting that a sustained increase in plasma SCFA can be achieved by administering a moderate dose of carbohydrates, three times per day. However, the addition of a single dose of a WB fraction did not further increase the (13)C-SCFA concentrations in plasma, nor did it stimulate cross-feeding (Wilcoxon signed ranks test). ispartof: Nutrients vol:9 issue:1 ispartof: location:Switzerland status: published

Lactose-[15N, 15N]-ureide is used to study the fate of the colonic urea-nitrogen metabolism. During the passage through the gastrointestinal tract, lactose ureide is hydrolysed to glucose ureide, which is absorbed to a limited extent from the small intestine and is excreted urinarily. In the present study, a procedure has been developed to quantify the urinary excretion of glucose-[15N, 15N]-ureide. In addition, urine and faecal samples obtained during a dietary intervention study with the prebiotic lactulose were retrospectively analysed. The glucose ureide and lactose ureide content was measured by GC–MS in 19 healthy volunteers. After consumption of a standard test meal containing 75 mg lactose-[15N, 15N]-ureide, six healthy volunteers performed a fractionated 24 h urine collection to investigate the urinary excretion of glucose-[15N, 15N]-ureide. In 13 volunteers, the effect of lactulose administration on the urinary excretion of glucose-[15N, 15N]-ureide was analysed. The urinary excretion of glucose-[15N, 15N]-ureide reached its maximum level in the 3–6 h urine collection and decreased in the 6–9 h urine. The label was still detectable in the 9–24 h urine collection. The cumulative excretion of 15N-labelled glucose ureide after 24 h amounted 12.91%. No significant differences in glucose-[15N, 15N]-ureide excretion were found in either of the urine fractions after administration of lactulose, compared with baseline. In none of the urine samples lactose-[15N, 15N]-ureide was detected. In conclusion, the results obtained in the present study indicated that the percentage dose glucose-[15N, 15N]-ureide recovered in urine is rather constant and not influenced by the presence of lactulose.

The use of stable isotope labeled substrates and subsequent analysis of urinary nitrate, forms a noninvasive test for evaluation of the in vivo NO metabolism. The present paper describes a new method for simultaneous quantification of (15)N-nitrate and total nitrate with gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS). Nitrate, isolated from urine with a nitrate selective resin, was reduced to nitrite using copperized cadmium. Subsequently, Sudan I was formed by diazotation. Sudan II was added as internal standard, and both molecules were analyzed with GC-C-IRMS as tert-butyldimethylsilyl derivatives. The accuracy was determined during a recovery study of two different known nitrate concentrations and two (15)N-enrichments. A recovery of 101.6% and 103.9% for total nitrate and 107.6% and 91.2% for (15)N-nitrate was obtained, respectively. The validated method was applied on complete 72 h urine collections after intravenous administration of (15)N-nitrate and (15)N-arginine in humans. On average, 51.8% (47.0-71.0%) of administered (15)N-nitrate was excreted, while 0.68% (0.44-1.17%) of (15)N-arginine was metabolized to nitrate. In conclusion, this method can be used for accurate simultaneous determination of (15)N-nitrate and total nitrate concentrations in urine and can be applied in clinical studies for noninvasive evaluation of NO metabolism in vivo.

Background: Healthy colonic mucosa uses butyrate as the major energy source. In ulcerative colitis (UC) butyrate oxidation has been shown to be disturbed, but it remains unclear whether this is a primary defect. The aim of this study was to measure mucosal butyrate oxidation in UC (involved and noninvolved colon) and in pouchitis and to study the relationship with endoscopic as well as histological disease activity. Methods: Butyrate oxidation was measured in 73 UC patients, 22 pouchitis patients, and 112 controls (95 colon, 17 ileum) by incubating biopsies with 1 mM 14C-labeled Na-butyrate and measuring the released 14CO2. Results: Compared with that in normal colon, butyrate oxidation was significantly impaired in endoscopically active but not in quiescent disease or uninvolved colon segments. The severity of the metabolic defect was related to histological disease activity and decreased epithelial cell height. In active pouchitis, butyrate oxidation was significantly decreased compared with that in normal ileum and excluded pouches without inflammation. The histological pouchitis score correlated significantly with butyrate oxidation. Conclusions: Active UC and pouchitis show the same inflammation-related metabolic defect. Our data suggest that the defect is a consequence of inflammation and that pouchitis is metabolically similar to active UC. (Inflamm Bowel Dis 2008)

Burned greasy deposits were found inside shells of the large Nile bivalve Chambardia rubens, excavated in an eight- to tenth- century AD church of the Coptic monastery of Bawit, Egypt, and supposedly used as oil lamps. The residues were subjected to a combination of chromatographic residue analysis techniques. The rather high concentrations of unsaturated fatty acids, as analysed by gas chromatography (GC) in the methylated extract, suggest the presence of a vegetal oil. Analysis of the stable carbon isotopes (delta C-13 values) of the methyl esters also favoured plants over animals as the lipid source. In the search for biomarkers by GC coupled to mass spectrometry on a silylated extract, a range of diacids together with high concentrations of 13,14-dihydroxydocosanoate and 11,12-dihydroxyeicosanoate were found. These compounds are oxidation products of erucic acid and gondoic acid, which are abundantly present in seeds of Brassicaceae plants. Liquid chromatography coupled to mass spectrometry analysis showed low concentrations of unaltered triglycerides, but revealed sizeable amounts of triglycerides with at least one dihydroxylated acyl chain. The unusual preservation of dihydroxylated triglycerides and alpha,omega-dicarboxylic acids can be related to the dry preservation conditions. Analysis of the stereoisomers of the dihydroxylated fatty acids allows one to determine whether oxidation took place during burning of the fuel or afterwards. The results prove that the oil of rapeseed (Brassica napus L.) or radish (Raphanus sativus L.) was used as illuminant in early Islamic Egypt, and that not only ceramic lamps but also mollusk shells were used as fuel containers. ispartof: Analytical and Bioanalytical Chemistry vol:390 issue:2 pages:783-793 ispartof: location:Germany status: published

Three lipid analysis methods were applied to extracts of archaeological potsherds from the ancient city of Sagalassos, south-west Turkey. In the fatty acid methyl ester fraction, obtained by selective transesterification of acylglycerols, the ratio of methyl palmitate to methyl stearate was determined. Secondly, by means of gas chromatography‐ combustion‐isotope ratio mass spectrometry (GC‐C‐IRMS), stable carbon isotope ratios were measured for the same methyl esters. Finally, triglyceride composition was determined via high-performance liquid chromatography‐mass spectrometry (HPLC‐MS). All three methods can be used to trace the origin of animal fat. Agreement between them is reasonable, especially in recognizing ruminant adipose fat. One sample was found to be of dairy origin using both GC‐C‐IRMS and HPLC‐MS. The advantages of the various methods are discussed. Variations in lipid composition were observed between samples from different periods and excavated in diverse areas within the city of Sagalassos.

In the present study, the production and fate of bacterial metabolites in the colon were investigated in a direct way using two substrates labelled with stable isotopes: lactose [15N,15N]ureide as a source of labelled ammonia and egg proteins intrinsically labelled with [2H4]tyrosine as a precursor of [2H4]p-cresol. Both ammonia and phenolic compounds are believed to be carcinogenic. Stimulation of carbohydrate fermentation in order to prevent accumulation of these toxic metabolites was induced by inclusion of inulin in a test meal or by addition of inulin to the daily diet, allowing us to distinguish between changes induced by the actual presence of a fermentable carbohydrate and effects caused by a long-term dietary intervention. When a single dose of inulin was administered together with the labelled substrates, a significant increase in faecal15N excretion, accompanied by a proportional decrease in urinary15N excretion was observed, probably reflecting an enhanced uptake of ammonia for bacterial biosynthesis, since an increased concentration of labelled N in bacterial pellets was found. A statistically significant reduction of urinary [2H4]p-cresol excretion was also noted. Upon supplementation of inulin to the daily diet during 4 weeks, however, only a tendency towards decreased urinary excretion of both labelled and unlabelledp-cresol was noted. Further studies are warranted to confirm these results in a larger cohort.

Summary Background : A better substrate is needed for a hydrogen breath test to measure the orocaecal transit time. The currently used substrate, lactulose, accelerates the orocaecal transit time by increasing the osmolality of the gut contents. The recently developed lactose 13C-ureide breath test is reliable, but a hydrogen breath test is preferred, as it allows the simultaneous investigation of the digestion and absorption of nutrients by means of 13C-labelled compounds. Methods : The usefulness of different types of inulin as a substrate for a hydrogen breath test was studied. Raftilin HP (> 99% inulin with a degree of polymerization of between 5 and 60 and

Smoking reduces the non-steroidal anti-inflammatory drug (NSAID)-induced small intestinal permeability increase in healthy people. It also affects inflammatory bowel disease that is associated with a disturbed gut barrier function. To assess the role of nicotine on barrier function, its influence on basal and NSAID-induced intestinal permeability was studied in healthy volunteers.Thirty-one healthy non-smoker subjects performed permeability tests with 51Cr-EDTA and sugar markers (sucrose, lactulose, mannitol, sucralose) before and during 2 weeks of nicotine patch application, and with and without indomethacin intake, respectively. Since smoking has been described as affecting motility, transit measurements were also done with the sodium[13C]-octanoate and lactose-[13C]-ureide breath tests before and during nicotine exposure. Correlations between permeability markers were checked and the influence of gastrointestinal transit was assessed.Nicotine did not affect barrier function in vivo, nor gastric emptying, small-bowel transit time or orocaecal transit. 51Cr-EDTA and lactulose correlated in basal 0-6 h permeability testing (r = 0.529, P0.0001), as did 6-24 h excretion of 51Cr-EDTA and sucralose (r = 0.474, P0.001); 97% and 90% of the subjects had a permeability increase after indomethacin intake for 0-6 h and 6-24 h excretion of Cr-EDTA, respectively. This population proportion is 63% for lactulose/mannitol and 83% for sucralose.Short-term exposure to nicotine does not alter normal basal or NSAID-induced gut barrier function or transit. 51Cr-EDTA and the respective sugar markers correlate well in in vivo permeability testing in healthy humans. The radioactive test detects more NSAID-induced permeability increase than does the lactulose/mannitol ratio permeability test.

Summary Background : An inverse relationship has been established between serum magnesium and serum lipid levels. By means of breath tests, we tested the hypothesis that magnesium inhibits intraluminal lipid digestion and subsequently causes changes in lipid metabolism. We also investigated the influence of the administration of magnesium chloride on protein digestion and gastric emptying. Methods : Five healthy volunteers performed simultaneous breath tests for gastric emptying and intraluminal lipid digestion, and six others for gastric emptying and protein digestion. Each test was performed in basal conditions and after the intake of 800 mg of magnesium chloride dissolved in water. Breath samples were taken at regular time intervals and analysed for 13CO2 and 14CO2 enrichment in order to calculate gastric emptying and lipid and protein digestion rates. Results : The oral administration of a single dose of magnesium chloride resulted in a diminished rate of intraluminal lipid and protein digestion. The most pronounced effect of magnesium chloride, however, was a decreased gastric emptying rate of both test meals. After correction for gastric emptying, no differences were noted in intraluminal lipid or protein digestion. Therefore, the lower lipid levels noted after magnesium supplementation are unlikely to be the result of altered lipid assimilation. Conclusion : Magnesium chloride slows gastric emptying but does not influence lipid digestion.

Lactose-[(15)N, (15)N]-ureide is used to study the fate of the colonic urea-nitrogen metabolism. During the passage through the gastrointestinal tract, lactose ureide is hydrolysed to glucose ureide, which is absorbed to a limited extent from the small intestine and is excreted urinarily. In the present study, a procedure has been developed to quantify the urinary excretion of glucose-[(15)N, (15)N]-ureide. In addition, urine and faecal samples obtained during a dietary intervention study with the prebiotic lactulose were retrospectively analysed.The glucose ureide and lactose ureide content was measured by GC-MS in 19 healthy volunteers. After consumption of a standard test meal containing 75 mg lactose-[(15)N, (15)N]-ureide, six healthy volunteers performed a fractionated 24 h urine collection to investigate the urinary excretion of glucose-[(15)N, (15)N]-ureide. In 13 volunteers, the effect of lactulose administration on the urinary excretion of glucose-[(15)N, (15)N]-ureide was analysed.The urinary excretion of glucose-[(15)N, (15)N]-ureide reached its maximum level in the 3-6 h urine collection and decreased in the 6-9 h urine. The label was still detectable in the 9-24 h urine collection. The cumulative excretion of (15)N-labelled glucose ureide after 24 h amounted 12.91%. No significant differences in glucose-[(15)N, (15)N]-ureide excretion were found in either of the urine fractions after administration of lactulose, compared with baseline. In none of the urine samples lactose-[(15)N, (15)N]-ureide was detected.In conclusion, the results obtained in the present study indicated that the percentage dose glucose-[(15)N, (15)N]-ureide recovered in urine is rather constant and not influenced by the presence of lactulose.

International audience; Background/Objectives: Lactose-[N, N]-ureide is used to study the fate of the colonic urea-nitrogen metabolism. During the passage through the gastrointestinal tract, lactose ureide is hydrolysed to glucose ureide, which is absorbed to a limited extent from the small intestine and is excreted urinarily. In the present study, a procedure has been developed to quantify the urinary excretion of glucose-[N, N]-ureide. In addition, urine and faecal samples obtained during a dietary intervention study with the prebiotic lactulose were retrospectively analysed. Subjects/Methods: The glucose ureide and lactose ureide content was measured by GC-MS in nineteen healthy volunteers. After consumption of a standard test meal containing 75 mg lactose-[N, N]-ureide, 6 healthy volunteers performed a fractionated 24h urine collection to investigate the urinary excretion of glucose-[N, N]-ureide. In 13 volunteers, the effect of lactulose administration on the urinary excretion of glucose-[N, N]-ureide was analysed. Results: The urinary excretion of glucose-[N, N]-ureide reached its maximum level in the 3-6h urine collection and decreased in the 6-9h urine. The label was still detectable in the 9-24h urine collection. The cumulative excretion of N-labelled glucose ureide after 24h amounted 12.91 %. No significant differences in glucose-[N, N]-ureide excretion were found in either of the urine fractions after administration of lactulose compared to baseline. In none of the urine samples lactose-[N, N]-ureide was detected. Conclusions: In conclusion, the results obtained in the present study indicated that the percentage dose glucose-[N, N]-ureide recovered in urine is rather constant and not influenced by the presence of lactulose.

In vitro experiments have shown that fermentation of carbohydrates prevents accumulation of nitrogen in the colon. Variable results have been obtained on modulation of dietary intakes in vivo. Lactose[15N,15N]-labeled ureide has been proposed as a tool to study colonic nitrogen metabolism. However, on oral administration of the marker, different urinary excretion patterns of the 15N label have been found. In this study, 50 mg lactose[15N,15N]ureide was directly instilled in the colon through an orocecal tube to investigate the colonic handling of this molecule in a direct way. In basal conditions, 42% (range, 37–48%) of labeled nitrogen administered as lactose[15N,15N]ureide was retrieved in urine after 72 h. A substantial variability in total urinary excretion of the label was found, but the urinary excretion pattern of the label was similar in all volunteers. When inulin, a fermentable carbohydrate, was administered together with the labeled marker, a significant decrease in urinary excretion of 15N after 72 h was found, to 29% (range, 23–34%). The effect of a smaller dose of inulin (250 mg) on colonic handling of lactose[15N,15N]ureide (50 mg), was investigated in another group of volunteers, and this time, fecal excretion of the marker was also evaluated. The results seem to indicate that fermentation of inulin causes an increased fecal excretion of the marker, thereby reducing urinary excretion but not retention in the human nitrogen pool. This instillation study shows that lactose[15N,15N]ureide is a tool with good properties to investigate the effect of different types of carbohydrates on nitrogen metabolism in the proximal colon in vivo.

A better substrate is needed for a hydrogen breath test to measure the orocaecal transit time. The currently used substrate, lactulose, accelerates the orocaecal transit time by increasing the osmolality of the gut contents. The recently developed lactose 13C-ureide breath test is reliable, but a hydrogen breath test is preferred, as it allows the simultaneous investigation of the digestion and absorption of nutrients by means of 13C-labelled compounds.The usefulness of different types of inulin as a substrate for a hydrogen breath test was studied. Raftilin HP (99% inulin with a degree of polymerization of between 5 and 60 and0.5% glucose, fructose and sucrose) was further evaluated and compared with lactulose with regard to its effects on gastric emptying and the digestion of protein and lipids.A good correlation was found between the orocaecal transit times using Raftilin HP (338 min; interquartile range, 300-383 min) and lactose 13C-ureide (353 min; interquartile range, 285-375 min) (r=0.85; P0.001). The administration of 5 or 10 g Raftilin HP had no influence on the orocaecal transit time, whereas lactulose significantly shortened the orocaecal transit time. Neither inulin nor lactulose had a significant influence on gastric emptying or protein or lipid assimilation.Raftilin HP is an ideal substrate for a hydrogen breath test to measure the orocaecal transit time.

As an octanoic acid 13CO2 breath test is frequently used to test gastric emptying of solid food, the purpose of the present study was to study whether oxidative breakdown of octanoic acid is affected by severe liver disease. The design of our study was twofold. First, cirrhotic patients (n = 82) of varying severity were compared with healthy controls (n = 17). Values of half-time, time point of maximal expiration and cumulative recovery of octanoic acid breath tests (OBT) were not significantly different between them. Secondly, cirrhotic patients (n = 10) were studied before placement of transjugular intrahepatic portosystemic shunt, 4-7 days later and 1-2 months later. Values of half-time, time point of maximal expiration and cumulative recovery of consecutive OBTs did not change significantly. The OBT may therefore be a suitable test in the future to detect delayed gastric emptying of solids in cirrhotic patients with reduced liver function and portal hypertension.

An inverse relationship has been established between serum magnesium and serum lipid levels. By means of breath tests, we tested the hypothesis that magnesium inhibits intraluminal lipid digestion and subsequently causes changes in lipid metabolism. We also investigated the influence of the administration of magnesium chloride on protein digestion and gastric emptying.Five healthy volunteers performed simultaneous breath tests for gastric emptying and intraluminal lipid digestion, and six others for gastric emptying and protein digestion. Each test was performed in basal conditions and after the intake of 800 mg of magnesium chloride dissolved in water. Breath samples were taken at regular time intervals and analysed for 13CO2 and 14CO2 enrichment in order to calculate gastric emptying and lipid and protein digestion rates.The oral administration of a single dose of magnesium chloride resulted in a diminished rate of intraluminal lipid and protein digestion. The most pronounced effect of magnesium chloride, however, was a decreased gastric emptying rate of both test meals. After correction for gastric emptying, no differences were noted in intraluminal lipid or protein digestion. Therefore, the lower lipid levels noted after magnesium supplementation are unlikely to be the result of altered lipid assimilation.Magnesium chloride slows gastric emptying but does not influence lipid digestion.

The 13C-methacetin breath test (MBT) has been proposed for the noninvasive evaluation of the hepatic mixed function oxidase activity. Up to now, stable isotope analysis of carbon dioxide of the MBT has been carried out with isotope ratio mass spectrometry (IRMS). The aim of the present study was to test a recently developed isotope-selective nondispersive infrared spectrometer (NDIRS) in comparison to IRMS in healthy volunteers and patients with liver cirrhosis. Ten healthy volunteers (range 22 to 76 years) and ten patients with histologically proven liver cirrhosis (range 47 to 71 years; Child Pugh score A = 5, B = 3, C = 2) were studied. After an overnight fast each subject received 2 mg/kg BW of 13C-methacetin dissolved in 100 ml of tea. Breath samples were obtained before substrate administration and after 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, 120, 150, 180 min. The 13C/12C-ratio was analyzed in each breath sample both by NDIRS (IRIS, Wagner Analysen Technik, Worpswede, Germany) and CF-IRMS (ABCA, Europa Scientific, Crewe, UK). Results were expressed as delta over baseline (DOB [/1000]) and as cumulative percentage doses of 13C recovered (cPDR [%]) at each time interval. Correlations between IRMS and NDIRS were tested by linear regression correlation. For measuring agreement an Altman-Bland-plot was performed. Applying correlation analysis a linear correlation was found (DOB: y = 1.068 +/- 0.0012.x + 2.088 +/- 0.2126, r = 0.98, p0.0001; cPDR: y = 1.148 +/- 0.0109.x + 0.569 +/- 0.172; r = 0.99, p0.0001). For DOB the mean difference (d) was 2.9/1000 and the standard deviation (SD) of the difference was 2.7/1000. The limits of agreement (d +/- SD) were -2.5/1000 and 8.3/1000. The comparison of DOB- and cPDR-values by NDIRS and IRMS shows a high linear correlation. However, the distance of the limits of agreement is wide. Consequently, the validity of the MBT could be influenced which could make MBT by NDIRS unprecise for exact evaluation of hepatocellular dysfunction. Further studies are necessary to determine sensitivity and specifity of the MBT with NDIRS in larger study populations.

The 13C mixed-triglyceride breath test (MTB) has been proposed for the non-invasive assessment of duodenal pancreatic lipase activity. Until now, stable isotope analysis of CO2 of the MTB has been carried out with isotope ratio mass spectrometry (IRMS). The aim of the present study was to compare MTB results by using the new non-dispersive infrared spectrometry (NDIRS) and the IRMS.Ten healthy volunteers and 10 patients with chronic pancreatitis and exocrine insufficiency were studied. After an overnight fast each subject received a test meal containing 250 mg 1,3 distearyl, 2[13C] octanoyl glycerol. Breath samples were taken at base line and at 30-min intervals over a period of 6 h postprandially. The 13C/12C ratio was determined in each breath sample by NDIRS and CF-IRMS as delta values. Results were expressed as delta over base line (DOB (per 1000)) and as cumulative percentage dose of 13C recovered (cPDR (%)). Correlations between IRMS and NDIRS were tested by linear regression analysis. For measuring agreement an Altman-Bland plot was performed.A linear correlation was found (DOB: y = 0.645 +/- 0.040 x + 1.496 +/- 0.089, r = 0.70, P0.0001; cPDR: y = 1.269 +/- 0.031 x + 2.010 +/- 0.353, r = 0.93, P0.0001). For DOB the mean difference (d) was 1.0/1000, and the standard deviation (s) of the difference was 1.3/1000. The limits of agreement (d +/- 2 s) were -1.6/1000 and 3.6/1000.The comparison of DOB and cPDR values by NDIRS and IRMS shows a moderate to good linear correlation. However, the distance of the limits of agreement is rather wide. Consequently, the validity of the MTB is diminished, which makes MTB by NDIRS less suitable for exact evaluation of non-invasive assessment of duodenal pancreatic lipase activity. Further studies are necessary to determine sensitivity and specificity of the MTB with NDIRS in larger study populations.