Use of stable isotope-tagged thymidine and multi-isotope imaging mass spectrometry (MIMS) for quantification of human cardiomyocyte division.

Quantification of cellular proliferation in humans is important for understanding biology and responses to injury and disease. However, existing methods require administration of tracers that cannot be ethically administered in humans. We present a protocol for the direct quantification of cellular proliferation in human hearts. The protocol involves administration of non-radioactive, non-toxic stable isotope 15Nitrogen-enriched thymidine (15N-thymidine), which is incorporated into DNA during S-phase, in infants with tetralogy of Fallot, a common form of congenital heart disease. Infants with tetralogy of Fallot undergo surgical repair, which requires the removal of pieces of myocardium that would otherwise be discarded. This protocol allows for the quantification of cardiomyocyte proliferation in this discarded tissue. We quantitatively analyzed the incorporation of 15N-thymidine with multi-isotope imaging spectrometry (MIMS) at a sub-nuclear resolution, which we combined with correlative confocal microscopy to quantify formation of binucleated cardiomyocytes and cardiomyocytes with polyploid nuclei. The entire protocol spans 3-8 months, which is dependent on the timing of surgical repair, and 3-4.5 researcher days. This protocol could be adapted to study cellular proliferation in a variety of human tissues.

Urinary Metabolomics Profiles Associated to Bovine Meat Ingestion in Humans.

SCOPE: The impact of meat consumption on human health is widely examined in nutritional epidemiological studies, especially due to the connection between the consumption of red and processed meat and the risk of colon cancer. Food questionnaires do not assess the exposure to different methods of meat cooking. This study aimed to identify biomarkers of the acute ingestion of bovine meat cooked with two different processes. METHODS AND RESULTS: Non-targeted UPLC-MS metabolite profiling was done on urine samples obtained from 24 healthy volunteers before and 8 h after the ingestion of a single meal composed of intrinsically 15 N labelled bovine meat, either cooked at 55 °C for 5 min or at 90 °C for 30 min. A discriminant analysis extension of independent components analysis was applied to the mass spectral data. After meat ingestion, the urinary excretion of 1-methylhistidine, phenylacetylglutamine, and short- and medium-chained acylcarnitines was observed. 15 N labelling was detected in these metabolites, thus confirming their origin from ingested meat. However, no difference was observed in urinary metabolomic profiles according to the meat cooking process used. CONCLUSION: Meat ingestion led to the excretion of several nitrogen-containing compounds, but although a metabolic signature was detected for meat ingestion, the impact of the cooking process was not detectable at the level of urinary metabolic signature in our experimental conditions.

Pilot Study Examining the Influence of Potassium Bicarbonate Supplementation on Nitrogen Balance and Whole-Body Ammonia and Urea Turnover Following Short-Term Energy Restriction in Older Men.

With aging there is a chronic low-grade metabolic-acidosis that may exacerbate negative protein balance during weight loss. The objective of this randomized pilot study was to assess the impact of 90 mmol∙day-1 potassium bicarbonate (KHCO3) versus a placebo (PLA) on 24-h urinary net acid excretion (NAE), nitrogen balance (NBAL), and whole-body ammonia and urea turnover following short-term diet-induced weight loss. Sixteen (KHCO3; n = 8, PLA; n = 8) older (64 ± 4 years) overweight (BMI: 28.5 ± 2.1 kg∙day-1) men completed a 35-day controlled feeding study, with a 7-day weight-maintenance phase followed by a 28-day 30% energy-restriction phase. KHCO3 or PLA supplementation began during energy restriction. NAE, NBAL, and whole-body ammonia and urea turnover (15N-glycine) were measured at the end of the weight-maintenance and energy-restriction phases. Following energy restriction, NAE was -9.8 ± 27.8 mmol∙day-1 in KHCO3 and 43.9 ± 27.8 mmol∙day-1 in PLA (p < 0.05). No significant group or time differences were observed in NBAL or ammonia and urea turnover. Ammonia synthesis and breakdown tended (p = 0.09) to be higher in KHCO3 vs. PLA following energy restriction, and NAE was inversely associated (r = -0.522; p < 0.05) with urea synthesis in all subjects. This pilot study suggests some benefit may exist with KHCO3 supplementation following energy restriction as lower NAE indicated higher urea synthesis.

Do we excrete what we eat? Analysis of stable nitrogen isotope ratios of human urinary urea.

RATIONALE: Natural stable nitrogen isotope ratios (δ15 N) are frequently used for the determination of provenance and dietary assessment of recent and ancient humans. Although individual δ15 N values typically correspond to the dietary δ15 N composition, they are also affected by metabolic conditions. Preferred matrices for the measurement of human δ15 N values have been hair, nail or blood. The goal of this study was to validate a novel approach for the assessment of the δ15 N values from urinary urea, the principal end-product of human N metabolism. METHODS: The method, which involves the precipitation of urea from urine using xanthydrol, was validated using fortified urea solutions. Intra- and inter-individual variance of the δ15 N values of urinary urea was determined from samples obtained from multiple human subjects. RESULTS: Precipitation with xanthydrol did not alter the δ15 N values of urea. The mean δ15 N value in urinary urea from human subjects from Germany was +4.4 ± 0.6 ‰, which corresponds to the estimated dietary composition. It falls below previously reported δ15 N values for human tissue and blood samples. Longitudinal analyses over 7 days illustrate short-time changes linked to varying protein intake. CONCLUSIONS: Our results indicate that δ15 N values can be measured reliably from human urine and that the method is suitable to monitor rapid dietary and metabolic changes of an individual. Our findings further confirm that urinary urea is depleted in 15 N compared with human tissue but within the range of the δ15 N composition of the diet. Copyright © 2017 John Wiley & Sons, Ltd.

Measures of Total Energy Expenditure and Its Components Using the Doubly Labeled Water Method in Rehabilitating Burn Children.

BACKGROUND: A persistent hypermetabolic state delays anabolism and growth in burned children. However, our own clinical experience has been that resting energy expenditure (REE) is not increased during the rehabilitative phase, suggesting other contributing factors. We measured total energy expenditure (TEE) and its components in rehabilitating pediatric burn patients to identify the basis for accelerated energy metabolism in this population. MATERIALS AND METHODS: Children admitted with initial burns of 20% of their total body surface area (TBSA) or greater were enrolled into this prospective, descriptive study. TEE was measured using the doubly labeled water method over a 7-day period. During that period, REE was measured on 2 days by indirect calorimetry. Activity energy expenditure (AEE) was assessed using a physical activity monitoring device for a 24-hour period. TEE and REE were compared with sex-specific, age-matched, and weight-matched norms using the Dietary Reference Intakes (DRI) standards. RESULTS: Ten children with an average burn size of 53.7% ± 20% (range, 27%-82%) of TBSA completed this study. Their mean age and weight were 10.4 ± 5.5 years and 35.8 ± 16.4 kg, respectively. Daily TEE averaged 66 kcal/kg and was 1.08% of reference DRI. REE was 92% ± 25% of predicted basal metabolic rate, not exceeding 120% as a maximum value in any child. CONCLUSIONS: TEE and REE in rehabilitating burn children are comparable to reference standards. Increased REE was not typical in our population, but measures of AEE were commonly high.

The effect of dietary water soluble carbohydrate to nitrogen ratio on nitrogen partitioning and isotopic fractionation of lactating goats offered a high-nitrogen diet.

The objective of this study was to investigate the relationship between nitrogen (N) partitioning and isotopic fractionation in lactating goats consuming diets with a constant high concentration of N and increasing levels of water soluble carbohydrate (WSC). Eight lactating goats were offered four different ratios of WSC : N in the diet. A two-period incomplete cross-over design was used, with two goats assigned to each treatment in each period. N balance measurements were conducted, with measurement of feed N intake and total output of N in milk, faeces and urine. Treatment, period and infusion effects were tested using general ANOVA; the relationships between variables were analysed by linear regression. Dietary treatment and period had significant effects on dry matter (DM) intake (g/day). DM digestibility (g/kg DM) and N digestibility (g/kg N) increased as the ratio of WSC : N increased in the diet. No treatment effect was observed on milk urea N concentration (g/l) or urinary excretion of purine derivatives (mM/day). Although dietary treatment and period had significant effects on N intake, the change of N intake was small; no effect was observed for N partitioning among faeces, milk and urine. Milk, plasma and faeces were enriched in 15N compared with feed, whilst urine was depleted in 15N relative to feed. No significant relationship was established between N partitioning and isotopic fractionation. This study failed to confirm the potential to use N isotopic fractionation as an indicator of N partitioning in dairy goats when diets provided N in excess to requirements, most likely because the range of milk N output/N intake and urinary N output/N intake were narrow.

Dietary fibre-rich resistant starches promote ammonia detoxification in the human colon as measured by lactose-[¹5N2]ureide.

Three resistant starches (RSs), namely fibre of potatoes (FP), wrinkle pea starch (WPS), and high amylose maize starch (HAMS) with different dietary fibre contents, were supplemented in adults to evaluate their effects on urinary nitrogen and ammonia excretion as well as on faecal nitrogen excretion by means of lactose-[(15)N2]ureide ((15)N-LU) degradation. Twenty subjects received a regular diet either without or with the supplementation of FP, WPS, and HAMS in a randomized order. After administration of (15)N-LU, urine and faeces were collected over 48 and 72 h, respectively, whereas blood was collected after 6 h. The (15)N-abundances were measured by isotope ratio mass spectrometry. In comparison to the dry run, supplementation with RS significantly lowered renal (15)N-excretion (dry run: 43.2%, FP: 34.6%, WPS: 37.9%, HAMS: 36.4%) as well as the corresponding (15)NH3-excretion (dry run: 0.08%, FP: 0.06%, HAMS: 0.05%), clearly indicating a reduced colonic nitrogen generation at high dietary fibre intake.

Isotope-labelled urea to test colon drug delivery devices in vivo: principles, calculations and interpretations.

This paper describes various methodological aspects that were encountered during the development of a system to monitor the in vivo behaviour of a newly developed colon delivery device that enables oral drug treatment of inflammatory bowel diseases. [(13)C]urea was chosen as the marker substance. Release of [(13)C]urea in the ileocolonic region is proven by the exhalation of (13)CO2 in breath due to bacterial fermentation of [(13)C]urea. The (13)CO2 exhalation kinetics allows the calculation of a lag time as marker for delay of release, a pulse time as marker for the speed of drug release and the fraction of the dose that is fermented. To determine the total bioavailability, also the fraction of the dose absorbed from the intestine must be quantified. Initially, this was done by calculating the time-dependent [(13)C]urea appearance in the body urea pool via measurement of (13)C abundance and concentration of plasma urea. Thereafter, a new methodology was successfully developed to obtain the bioavailability data by measurement of the urinary excretion rate of [(13)C]urea. These techniques required two experimental days, one to test the coated device, another to test the uncoated device to obtain reference values for the situation that 100 % of [(13)C]urea is absorbed. This is hampered by large day-to-day variations in urea metabolism. Finally, a completely non-invasive, one-day test was worked out based on a dual isotope approach applying a simultaneous administration of [(13)C]urea in a coated device and [(15)N2]urea in an uncoated device. All aspects of isotope-related analytical methodologies and required calculation and correction systems are described.

The metabolic effect of resistant starch and yoghurt on the renal and faecal nitrogen and ammonia excretion in humans as measured by lactose-[(15)N2]ureide.

Resistant starch (RS) and Lactobacillus acidophilus yoghurt (LC1) were supplemented simultaneously in healthy adults to evaluate the effect on the urinary and faecal nitrogen and ammonia excretion by means of lactose-[(15)N2]ureide ((15)N-LU) degradation. Nineteen subjects received a regular daily diet either without or with supplementation of an RS-LC1-mixture composed of fibre of potatoes (RS type 1), wrinkle pea starch (RS type 2), and LC1 over a 20-day period in randomised order. Thereafter, (15)N-LU was administered together with breakfast. Urine and faeces were collected over a period of 48 and 72 h, respectively. The (15)N abundances were measured by isotope ratio mass spectrometry. The intake of the pre- and probiotic mixture composed of RS of type 1, type 2 and of LC1 significantly lowered the colonic generation and the renal excretion of toxic (15)NH3 and functioned as an ammonia shift from urinary to faecal (15)N excretion when using (15)N-LU as a xenobiotic marker.

The effects of dietary nitrogen to water-soluble carbohydrate ratio on isotopic fractionation and partitioning of nitrogen in non-lactating sheep.

The main objective of this study was to investigate the relationship between partitioning and isotopic fractionation of nitrogen (N) in sheep consuming diets with varying ratios of N to water-soluble carbohydrate (WSC). Six non-lactating sheep were offered a constant dry matter (DM) allowance with one of three ratios of dietary N/WSC, achieved by adding sucrose and urea to lucerne pellets. A replicated 3 dietary treatments (Low, Medium and High N/WSC) × 3 (collection periods) and a Latin square design was used, with two sheep assigned to each treatment in each period. Feed, faeces, urine, plasma, wool, muscle and liver samples were collected and analysed for ¹5N concentration. Nitrogen intake and outputs in faeces and urine were measured for each sheep using 6-day total collections. Blood urea N (BUN) and urinary excretion of purine derivative were also measured. Treatment effects were tested using general ANOVA; the relationships between measured variables were analysed by linear regression. BUN and N intake increased by 46% and 35%, respectively, when N/WSC increased 2.5-fold. However, no indication of change in microbial protein synthesis was detected. Results indicated effects of dietary treatments on urinary N/faecal N, faecal N/N intake and retained N/N intake. In addition, the linear relationships between plasma δ¹5N and urinary N/N intake and muscle δ¹5N and retained N/N intake based on individual measurements showed the potential of using N isotopic fractionation as an easy-to-use indicator of N partitioning when N supply exceeds that required to match energy supply in the diet.

Carbon and nitrogen isotopic ratios of urine and faeces as novel nutritional biomarkers of meat and fish intake.

PURPOSE: Meat and fish consumption are associated with changes in the risk of chronic diseases. Intake is mainly assessed using self-reporting, as no true quantitative nutritional biomarker is available. The measurement of plasma fatty acids, often used as an alternative, is expensive and time-consuming. As meat and fish differ in their stable isotope ratios, δ(13)C and δ(15)N have been proposed as biomarkers. However, they have never been investigated in controlled human dietary intervention studies. OBJECTIVE: In a short-term feeding study, we investigated the suitability of δ(13)C and δ(15)N in blood, urine and faeces as biomarkers of meat and fish intake. METHODS: The dietary intervention study (n = 14) followed a randomised cross-over design with three eight-day dietary periods (meat, fish and half-meat-half-fish). In addition, 4 participants completed a vegetarian control period. At the end of each period, 24-h urine, fasting venous blood and faeces were collected and their δ(13)C and δ(15)N analysed. RESULTS: There was a significant difference between diets in isotope ratios in faeces and urine samples, but not in blood samples (Kruskal-Wallis test, p < 0.0001). In pairwise comparisons, δ(13)C and δ(15)N were significantly higher in urine and faecal samples following a fish diet when compared with all other diets, and significantly lower following a vegetarian diet. There was no significant difference in isotope ratio between meat and half-meat-half-fish diets for blood, urine or faecal samples. CONCLUSIONS: The results of this study show that urinary and faecal δ(13)C and δ(15)N are suitable candidate biomarkers for short-term meat and fish intake.

Studies of the protein and the energy metabolism in man during a wintering in Antarctica.

During the 29th Soviet Antarctic Expedition in Novolazarevskaya from March 1984 to March 1985, the protein and energy metabolisms were studied in six expeditioners from the German Democratic Republic. The investigations were carried out at the beginning of the expedition (May), during the polar night (July) and during the polar day (December). The effect of a special stress situation (sledge trek in April 1984) was investigated in one subject. The stable nitrogen isotope (15)N was used to study the protein metabolism. The assessment of the energy metabolism was based on the oxygen consumption, which was determined by means of a spirograph. In addition, the vital capacity, the breath minute volume, the blood pressure, etc. were measured. The following results were obtained: During the polar night, the utilisation of the dietary proteins and the whole body protein synthesis calculated by means of the (15)N excretion of the total nitrogen in urine were greater (73.6±0.9 % and 3.48±0.17 g protein d(-1) kg(-1), n=3) than the respective values during the polar day (69.7±1.2, p<0.05, n=3 and 3.05±0.07, p<0.05, n=3) and at the beginning of the expedition (69.6±1.4, p<0.02, n=5 and 2.81±0.09, p<0.01, n=5). The lowest values (58.0 % and 2.43 g protein d(-1) kg(-1)) were obtained in the subject after the trek. The resting metabolic rate (in kJ d(-1) m(-2)) was decreased during the polar night (45.6±5.0, n=4) in comparison with the polar day (61.5±11.3, n=3) and the beginning of the expedition (52.3±9.6, n=4) with p<0.01 in both cases.

Tracer kinetics and metabolic models in medicine.

In the present paper, a survey of methods applied to the interpretation and evaluation of tracer kinetic data is given. For their mathematical description, both compartmental and non-compartmental models, such as the modified model of Sprinson and Rittenberg, the San Pietro-Rittenberg model, two models of the albumin metabolism and a 10-pool model of the N and protein metabolism were used. By means of single or multiple pulse, infusion and priming techniques, the N and protein metabolism in various metabolic states (e.g. healthy man, pathological and stress conditions, therapeutic treatments) were studied.

A non-invasive, low-cost study design to determine the release profile of colon drug delivery systems: a feasibility study.

PURPOSE: Conventional bioavailability testing of dosage forms based on plasma concentration-time graphs of two products in a two-period, crossover-design, is not applicable to topical treatment of intestinal segments. We introduce an isotope dual-label approach ((13)C- and (15)N(2)-urea) for colon drug delivery systems that can be performed in a one-day, non-invasive study-design. METHODS: Four healthy volunteers took an uncoated or a ColoPulse-capsule containing (13)C-urea and an uncoated capsule containing (15)N(2)-urea. In case of colon-release (13)C-urea is fermented and (13)C detected as breath (13)CO(2). Absorbed (13)C-urea and (15)N-urea are detected in urine. RESULTS: C and (15)N in urine released from uncoated capsules showed a ratio of 1.01 ± 0.06. The (13)C/(15)N-recovery ratio after intake of a ColoPulse-capsule was constant and lower >12 h post-dose (median 0.22, range 0.13-0.48). The (13)C/(15)N-ratio in a single urine sample at t ≥ 12 h predicted the 24 h non-fermented fraction (13)C of <26 %. Breath (13)CO(2) indicated delayed (>3 h) release and a fermented fraction (13)C >54 %. CONCLUSIONS: Breath and urine (13)C and (15)N data describe the release-profile and local bioavailability of a colon delivery device. This allows non-invasive bioavailability studies for evaluation of colon-specific drug delivery systems without radioactive exposure and with increased power and strongly reduced costs.

Development and application of 15N-tracer substances for measuring the whole-body protein turnover rates in the human, especially in neonates: a review.

Our research group of the Children's Hospital of the University of Rostock (Rostock group) has long-time experience in (15)N-labelling and in using yeast protein and its hydrolysates for tracer kinetic studies to evaluate parameters of the whole-body protein metabolism in premature infants. The particular advantage of applying an economically convenient, highly (15)N-enriched, and completely labelled yeast protein for evaluating protein turnover rates is the fact that the (15)N dose is spread among all proteinogenic amino acids. The absorption has been improved by hydrolysing [(15)N]yeast protein with thermitase into a mixture of amino acids, dipeptides and tripeptides so that faecal analysis becomes unnecessary when determining turnover rates. The review shows that, in contrast to the application of single (15)N-labelled amino acids with resulting overestimation of protein turnover rates, the (15)N-labelled yeast protein thermitase hydrolysate represents the amino acid metabolism more closely without causing amino acid imbalances. The (15)N-labelled yeast protein thermitase hydrolysate leads to the estimation of reliable protein turnover rates, particularly in premature infants.

Identification of energy consumption and nutritional stress by isotopic and elemental analysis of urine in bonobos (Pan paniscus).

A mounting body of evidence suggests that changes in energetic conditions like prolonged starvation can be monitored using stable isotope ratios of tissues such as bone, muscle, hair, and blood. However, it is unclear if urinary stable isotope ratios reflect a variation in energetic condition, especially if these changes in energetic condition are accompanied by shifts in dietary composition. In a feeding experiment conducted on captive bonobos (Pan paniscus), we monitored urinary δ(13)C, δ(15)N, total C (carbon), total N (nitrogen), and C/N ratios and compared these results with glucocorticoid levels under gradually changing energy availability and dietary composition. Measurements of daily collected urine samples over a period of 31 days showed that while shifts in urinary isotope signatures of δ(13)C and δ(15)N as well as total C were best explained by changes in energy consumption, urinary total N excretion as well as the C/N ratios matched the variation in dietary composition. Furthermore, when correcting for fluctuations in dietary composition, the isotope signatures of δ(13)C and δ(15)N as well as total C correlated with urinary glucocorticoid levels; however, the urinary total N and the C/N ratio did not. These results indicate for the first time that it is possible to non-invasively explore specific longitudinal records on animal energetic conditions and dietary compositions with urinary stable isotope ratios and elemental compositions, and this research provides a strong foundation for investigating how ecological factors and social dynamics affect feeding habits in wild animal populations such as primates.

Whole body protein kinetics measured with a non-invasive method in severely burned children.

UNLABELLED: Persistent and extensive skeletal muscle catabolism is characteristic of severe burns. Whole body protein metabolism, an important component of this process, has not been measured in burned children during the long-term convalescent period. The aim of this study was to measure whole body protein turnover in burned children at discharge (95% healed) and in healthy controls by a non-invasive stable isotope method. Nine burned children (7 boys, 2 girls; 54±14 (S.D.)% total body area burned; 13±4 years; 45±20 kg; 154±14 cm) and 12 healthy children (8 boys, 4 girls; 12±3 years; 54±16 kg; 150±22 cm) were studied. A single oral dose of (15)N-alanine (16 mg/kg) was given, and thereafter urine was collected for 34 h. Whole body protein flux was calculated from labeling of urinary urea nitrogen. Then, protein synthesis was calculated as protein flux minus excretion, and protein breakdown as flux minus intake. At discharge, total protein turnover was 4.53±0.65 (S.E.)g kg body weight(-1) day(-1) in the burned children compared to 3.20±0.22 g kg(-1) day(-1) in controls (P=0.02). Expressed relative to lean body mass (LBM), the rates were 6.12±0.94 vs. 4.60±0.36 g kg LBM(-1) day(-1) in burn vs. healthy (P=0.06). Total protein synthesis was also elevated in burned vs. healthy children, and a tendency for elevated protein breakdown was observed. CONCLUSION: Total protein turnover is elevated in burned children at discharge compared to age-matched controls, possibly reflecting the continued stress response to severe burn. The oral (15)N-alanine bolus method is a convenient, non-invasive, and no-risk method for measurement of total body protein turnover.

Simultaneous measurement of [15N]nitrate and [15N]nitrite enrichment and concentration in urine by gas chromatography mass spectrometry as pentafluorobenzyl derivatives.

Quantification of (15)N-labeled nitrate and determination of the (15)N-enrichment in urine upon administration of (15)N-labeled precursors such as L-[guanidino-(15)N(2)]arginine is a suitable approach to study formation and metabolism of nitric oxide (NO) and its metabolites in vivo. Previously, we have reported on the simultaneous derivatization and accurate quantification of nitrite and nitrate in various biological fluids using pentafluorobenzyl bromide and GC/MS. We report here on a modification of this method that allows for the simultaneous determination of (15)N-enrichment of [(15)N]nitrate and [(15)N]nitrite and the simultaneous quantification of [(15)N]nitrate, [(14)N]nitrate, [(15)N]nitrite, and [(14)N]nitrite in human urine. In a pilot study, using the carbonic anhydrase inhibitor drug acetazolamide at therapeutical oral doses (5.4 and 5 mg per kg bodyweight) and by oral intake of [(15)N]nitrite (0.31 and 0.5 micromol per kg bodyweight) by two healthy volunteers, we demonstrate for the first time that renal carbonic anhydrase activity is mainly responsible for the reabsorption of nitrite from the primary urine and confirm previous findings on nitrate.

Hair protein and amino acid 13C and 15N abundances take more than 4 weeks to clearly prove influences of animal protein intake in young women with a habitual daily protein consumption of more than 1 g per kg body weight.

A high protein or meat intake might be a risk factor for metabolic disorders. Stable isotopic abundances (SIA) of hair can be used as biomarkers for animal protein intake due to characteristic isotopic patterns of food proteins. We investigated if an additional meat intake (M, 200 g pork fillet/day) or an omission of meat and meat products (NOM) can influence the natural (15)N and (13)C SIA within 4 weeks in hair and plasma of young women. The daily protein intake (means +/- SD) was 1.40 +/- 0.29, 2.25 +/- 0.35, and 1.15 +/- 0.26 g/kg at baseline, during M, and during NOM, respectively. At baseline the animal protein intake correlated with bulk SIA of hair ((15)N: R(2) = 0.416; (13)C: R(2) = 0.664; n = 14). However, isotope ratio mass spectrometry (IRMS) analyses have not shown that hair and plasma SIA were changed significantly after M or NOM. Possible reasons were discussed. Urinary SIA were significantly lower after M than after NOM ((15)N: p = 0.039; (13)C: p = 0.006) and close to those of pork fillet. Characteristic patterns of SIA were measured in individual amino acids (AA) by gas chromatography/combustion isotope ratio mass spectrometry (GC/C/IRMS). The results confirmed considerable differences in SIA between AA (delta(15)N, up to 22 per thousand; delta(13)C, up to 31 per thousand). Plots of (15)N versus (13)C abundances in hair revealed characteristic differences between indispensable and dispensable AA. The intervention-dependent changes of AA-specific SIA were not as clear as expected. Although the AA-specific SIA may reveal more detailed characteristics of physiological conditions, further methodological research is required. We suggest that the SIA of leucine can be potential markers of protein intake. The reliability of SIA as biomarkers of protein intake still have to be tested in longer lasting intervention studies in humans. The results may have implications in the assessment for possible benefits and risks of protein consumption.

Does the biomarker 15N-lactose ureide allow to estimate the site of fermentation of resistant starch?

We evaluated the effect of resistant starch (RS) and resistant starch with wheat bran (RS+WB) on the colonic ammonia metabolism in healthy volunteers using the biomarker (15)N-lactose ureide ((15)N-LU). Particularly, it was investigated whether this biomarker allowed to estimate differences in the site of fermentation. Ten volunteers were included in a placebo-controlled crossover study. They consumed in random order 2 x 15 g RS/day for 2 weeks and placebo for 2 weeks separated by 2 weeks wash-out. At baseline, on the first day of each intake period and after each intake period, they consumed a (15)N-labelled test meal and collected all urine in different fractions for 48 h. In ten other volunteers, the effect of 2 x 15 g RS/day and of 2 x 15 g RS + 2 x 6 g WB was compared. These volunteers collected urine and feces for 72 h. (15)N-content of urine and feces was measured using combustion-isotope ratio mass spectrometry. RS exerted a significant decrease in cumulative urinary (15)N-excretion which was different from placebo. The effect was most pronounced in the 6-24 h urine fraction which suggest fermentation in the proximal colon. The effect of RS+WB on cumulative urinary (15)N-excretion was not significantly different from the effect of RS. A less pronounced decrease in the 6-24 h fraction was observed suggesting less fermentation in the proximal colon whereas no indications for more distal fermentation were observed. Since about 80% of the cumulative urinary (15)N was recovered within 24 h, it was concluded that the biomarker (15)N-LU was useful to monitor processes in the proximal colon rather than in the distal colon.