Klaus D. Wutzke

α-Lactalbumin-enriched low-protein infant formulas: a comparison to breast milk feeding

Tryptophan (TRP) is the limiting amino acid in low‐protein infant formulas. This is mainly due to lower α‐lactalbumin (αLA) content in cows milk whey as compared with human milk protein. To study the effect of αLA‐enrichment on the TRP supply, cross‐over studies were carried out in 20 healthy infants up to 3 months of age. In this study, two protein‐reduced (1.3%) infant formulas (moderate TRP content of 1.88% and higher TRP content of 2.10%) were alternately fed over a 2 week period in two groups of infants. Serum TRP levels of the formula‐fed infants with the higher TRP content did not differ significantly from an exclusively breastfed control group of 11 infants (10.5 ±4.8 versus 10.9±4.7mgl‐1, p= 0.841), whereas levels of the formula‐fed infants with the moderate TRP content were significantly lower (7.4 ± 3.9, p= 0.038). The supplementation of αLA resulting in a higher TRP supply to low‐protein diets is a further step towards the production of infant formulas more closely adapted to human breast milk.

Evaluation of oro-coecal transit time : a comparison of the lactose-[13C, 15N]ureide 13CO2- and the lactulose H2-breath test in humans

OBJECTIVE: The lactulose H2-breath test is the most widely used non-invasive approach for evaluation of oro-coecal transit time (OCTT). In the present study, doubly-labelled lactose-[13C, 15N]ureide (DLLU) was synthesized to investigate the OCTT in comparison to the conventional lactulose H2-breath test. Additionally, the bacterial breakdown rate (BBR) and rate of elimination and the metabolic pathways of the cleavage products of DLLU (13CO2, [15N]urea, and 15NH3) were investigated.DESIGN AND SUBJECTS: In a first study, DLLU was administered as a single oral-pulse-labelling (dosage: one gram) either without and after pretreatment of five grams of unlabelled lactoseureide (LU) on the day prior to the study to twelve healthy adult volunteers after breakfast. Breath and urine were collected in one and two hour-intervals, respectively, over a one-day period. 13C-enrichment in breath as well as 15N-enrichment in urine fractions were measured by continuous flow-isotope ratio mass spectrometry (CF-IRMS). In a second study, lactulose was administered to the same subjects (dosage: ten grams). Breath was collected in quarter, half and one hour-intervals over a ten hour-period. Hydrogen concentration in breath was analysed using an electrochemical detector.RESULTS: The comparison of the lactose-[13C]ureide 13CO2-breath test and the lactulose H2-breath test showed that the mean increase of the 13C-enrichment in CO2 occurred 1.18 h later than the mean increase of H2 in breath. The resulting OCTTs derived from the two methods were 3.02±1.4 and 1.84±0.5 h (P<0.05) and the corresponding BBRs were 9.63±3.4 and 6.07±1.7 h (P<0.01), respectively. The 15N-enrichment of urinary urea and ammonia without and after pretreatment with LU started between two and three hours after DLLU-administration. The cumulative percentage urinary excretion of the 15N- and 13C-tracer was 29.9% and 13.6% respectively, and was slightly increased after LU-pretreatment to 32.1% and 14.6% of the dose administered. A total of 35.2% of the 13C was found to be exhaled and remained approximately constant after LU-pretreatment (36.2%).CONCLUSIONS: The use of the lactulose H2-breath test for evaluation of the OCTT showed a statistically significant shortening of 1.18 h in comparison to the lactose-[13C]ureide 13CO2-breath test in healthy adults. The most important limitations of the lactulose H2-breath test are its low specificity and sensitivity due to dose-dependent accelerations of OCTT, interfering H2-rise from malabsorbed dietary fibre and H2-non-producers. In contrast, our lactose-[13C]ureide 13CO2-breath test was confirmed to avoid these disadvantages and to yield reliable results. This test is recommended especially if higher sensitivity and specificity is required, if IRMS-technique is available and if lactulose H2-tests lead to insufficient results.SPONSORSHIP: Deutsche Forschungsgemeinschaft (DFG).

Evidence for Colonic Absorption of Protein Nitrogen in Infants

The absorption of protein nitrogen by the colon was assessed in 6 infants with colostomy by giving 15N yeast protein in a dosage of 5–20 mg 15N/kg (92.4 atom‐%15N). The absorption of 15N ranged between 87.1 and 98.1% of the administered dose, and the retention in the protein pool ranged between 79.0 and 94.2%. The incorporation of 15N in the plasma proteins was demonstrated by 15N excess values between 0.02 and 0.10 atom‐%. The results suggest that the colon can assimilate proteins when insufficient absorption of protein nitrogen in the small intestine occurs. The breakdown of protein is thought to result from the action of colonic flora.

Whole-body protein parameters in premature infants: a comparison of different 15N tracer substances and different methods.

[15N]glycine, [15N]leucine, and [15N]yeast protein thermitase hydrolysate (YPTH) as tracers for investigating the protein turnover rates in premature infants were studied in nine human milk-fed neonates (born after 32 to 34 wk of gestation) by paired comparison of the tracers. The 15N enrichment of total urinary nitrogen and ammonia after administration of a single oral dose of 15N was measured by emission spectrometry. Flux rates were calculated using a three-compartment model and the ammonia end product method. The mean whole-body protein synthesis rates, as determined by the three-compartment model derived from the three 15N tracers, differed significantly (p < 0.01) among [15N]glycine (15.9 g/kg/d), [15N] leucine (9.1 g/kg/d), and 15N-YPTH (5.9 g/kg/d). When the corresponding rates were determined from the excretion of label in ammonia, the results showed the opposite tendency; the lowest apparent synthesis rates were found after [15N]glycine (7.5 g/kg/d), followed by [15N]leucine (14.4 g/kg/d), and the highest figure resulted after [15N] YPTH (16.7 g/kg/d). The results of this comparison substantiate the assumption that there are methodologic errors in connection with the use of different tracers and models for the calculation of whole-body protein parameters in preterm infants, with respect to the main requirement for tracer kinetic studies; the tracer nitrogen must be representative of total amino acid nitrogen. Seen in this light, mixtures of completely labeled amino acids such as YPTH may represent the most reliable tracer substance. On the basis of the data obtained with 15N-labeled YPTH and with total 15N as the end product, there is no major difference in the order of magnitude of the protein flux between preterm and full-term infants.

CLOSTRIDIUM INNOCUUM : A GLUCOSEUREIDE-SPLITTING INHABITANT OF THE HUMAN INTESTINAL TRACT

Glycosylureides were recently described as non-invasive markers of intestinal transit time. The underlying principle is an enzymatic splitting of (13)C-labelled ureides by intestinal bacteria. The (13)CO(2) released from the urea moiety of the glycosylureides can be measured in breath samples when the ingested tracer substrate reaches the caecum that is colonised with microbes. To date, the microbes that degrade glycosylureides are unknown. In order to identify the glucoseureide (GU)-splitting bacteria, 174 different strains of intestinal microbes obtained from five healthy adults were checked for their ability to degrade GU. The results of the microbial cultures and thin layer chromatography revealed that GU was exclusively degraded by Clostridium innocuum, belonging to the normal human intestinal microflora. C. innocuum probably synthesises a yet unknown enzyme that splits the glucose-urea bond. We suggest that the term glucoseureidehydrolase is the appropriate designation for this enzyme.

The effect of pre- and probiotics on the colonic ammonia metabolism in humans as measured by lactose-[15N2]ureide

Background:The evaluation of ammonia detoxification by pre- and probiotics by means of colonic lactose-[15N2]ureide (15N-LU) degradation is of great interest both scientifically and in terms of nutrition physiology.Objective:Pre- and probiotics were supplemented in healthy adults to evaluate the effect of the ammonia metabolism in the human colon by means of 15N-LU.Methods:A total of 14 participants aged 20–28 years daily received a regular diet either without (no treatment) or with supplementation of 30 g fibre of potatoes (FPs), 30 g wrinkle pea starch (WPS, resistant starch content: 12 and 70%, respectively) and 375 g Lactobacillus acidophilus (LC1) yoghurt, over a 10-day period in a randomised order. After 1 week, 5.7 mg/kg body weight 15N-LU was administered together with breakfast. A venous blood sample was taken after 6 h. Urine and faeces were collected over a period of 48 and 72 h, respectively. The 15N abundances were measured by isotope ratio mass spectrometry.Results:The mean renal 15N-excretion differed significantly between the supplementation of FP and no treatment (32.5 versus 46.3%, P=0.034), FP and LC1 (32.5 versus 51.6%, P=0.001), and WPS and LC1 (38.5 versus 51.6%, P=0.048). The mean faecal 15N-excretion amounted to 42.7% (no treatment), 59.7% (FP), 41.8% (WPS) and 44.0% (LC1). In comparison with no treatment, the urinary 15NH3-enrichment was significantly decreased at 16 h after FP supplementation.Conclusion:The prebiotic intake of FP and WPS lowered the colonic generation and the renal excretion of toxic 15NH3, respectively, when using 15N-LU as a xenobiotic marker.

Triglyceride oxidation in cystic fibrosis: a comparison between different 13C-labeled tracer substances.

BACKGROUND For indirect evaluation of pancreatic lipase activity in cystic fibrosis, different 13C-labeled triglycerides may be used. METHODS Triglyceride oxidation in patients with cystic fibrosis was investigated after administration of different 13C-labeled triglycerides by comparing 13CO2 breath exhalation. In the comparative study, five patients with cystic fibrosis (age, 8-15 years; body weight, 22.5-39.8 kg) were treated with Pangrol (individual dosages: 1-3 capsules per morning meal; Berlin-Chemie, Berlin, Germany). [1,1,1-13C3]Glyceryl tripalmitate and [1,1,1-13C3]glyceryl trioleate were administered as a single oral pulse at 8:00 A.M. (dosage, 4 mg/kg each) with the standard diet Fresubin (dosage, 10 ml/kg; Fresenius, Bad Homburg, Germany). Alternately, the same subjects were given the synthetic mixed triglyceride 1,3-distearyl, 2[13C]octanoyl glycerol (dosage, 12.5 mg/kg) contained in the standard diet Nutri-Mix (dosage, 10 ml/kg; Nutricia, Zoetemeer, The Netherlands). Breath samples were taken in 15- and 30-minute intervals over 8 hours. The 13CO2 enrichment was measured by continuous-flow isotope ratio mass spectrometry. RESULTS After administration of the 13C-labeled tripalmitin-triolein mixture and the mixed triglyceride, mean maximum 13CO2 enrichments were 4.70 and 7.37 delta over baseline, occurring at 7.0 and 3.5 hours, respectively. The corresponding percentage cumulative 13CO2 exhalations were 12.25% and 29.19%, respectively, and differed significantly in the five paired subjects (p = 0.003). CONCLUSIONS After using different 13C-labeled triglycerides the resultant 13CO2 exhalation reflected the triglyceride hydrolysis and subsequent oxidation. It is concluded that the different cumulative 13CO2 exhalations were mainly caused by the rate-limiting step of triglyceride hydrolysis to free fatty acids and 2-monoglycerides and by fat deposition. Noninvasive 13C breath tests using different 13C-labeled triglycerides can be used for evaluation of pancreatic lipase activity before and during enzyme supplementation.

The use of 13C-labelled glycosyl ureides for evaluation of orocaecal transit time.

Objective: In the present study, cellobiose-[13C]ureide and glucose-[13C]ureide were synthesized and tested as alternative substrates for noninvasive evaluation of the orocaecal transit time (OCTT).Design: Experimental study.Interventions: In total, 1 g cellobiose-[13C]ureide was administered together with a continental breakfast either without or after predosing of 5 × 1 g unlabelled cellobiose ureide on the day prior to study commencement. After 2 weeks, the same subjects ingested glucose-[13C]ureide (dosage: 0.57 g) either without or after predosing of the respective unlabelled ureide under identical conditions. Expired air samples were taken over 10 h. 13CO2-enrichment was measured by isotope ratio mass spectrometry (PDZ Europa, Sandbach, UK). The OCTT was calculated from the interval between 13C-ureide administration and the detection of a significant and sustained 13C-rise of 2 delta over baseline in breath.Setting: University of Rostock, Childrens Hospital, Research Laboratory.Subjects: Eight healthy adults aged 22–55 y.Results: After application of cellobiose-[13C]ureide and glucose-[13C]ureide OCTTs of 401 and 415 min, respectively, were measured. The predosing resulted in higher and steeper 13C-enrichments and caused a significant shortening of OCTTs of 265 and 287 min, respectively (P=0.012 and 0.017).Conclusions: The onset of 13CO2-enrichment reflected the degradation of glycosyl-[13C]ureides by glucose ureide hydrolase. The predosing with unlabelled ureides prior to pulse labelling with cellobiose-[13C]ureide and glucose-[13C]ureide (the latter is the key substance of the enzymatic sugar-ureide degradation) led to an induction of enzyme activity and resulted in a more precise and similar estimation of the OCTT when using both 13C-labelled ureides.

15N tracer kinetic studies on the validity of various 15N tracer substances for determining whole-body protein parameters in very small preterm infants.

Reliable 15N tracer substances for tracer kinetic determination of whole-body protein parameters in very small preterm infants are still a matter of intensive research, especially after some doubts have been raised about the validity of [15N]glycine, a commonly used 15N tracer. Protein turnover, synthesis, breakdown, and further protein metabolism data were determined by a paired comparison in four preterm infants. Their post-conceptual age was 32.2 ± 0.8 weeks, and their body weight was 1670 ± 181 g. Tracer substances applied in this study were a [15N]amino acid mixture (Ia) and [15N]- glycine (Ib). In a second group of three infants with a post conceptual age of 15N-labeled 32.0 ± 1.0 weeks and a body weight of 1,907 ± 137 g, yeast protein hydrolysate (II) was used as a tracer substance. A three-pool model was employed for the analysis of the data. This model takes into account renal and fecal 15N losses after a single 15N pulse. Protein turnovers were as follows: 11.9 ± 3.1 g kg-1 d-1 (Ia), 16.2 ± 2.5 g kg-1 d-1 (Ib), and 10.8 ± 3.0 g kg-1 d-1 (II). We were able to demonstrate an over-estimation of the protein turnover when Ib was used. There was an expected correspondence in the results obtained from Ia and II. The 15N-labeled yeast protein hydrolysate is a relatively cheap tracer that allows reliable determination of whole-body protein parameters in very small preterm infants.

Gastrointestinal pH and Transit Time Profiling in Healthy Volunteers Using the IntelliCap System Confirms Ileo-Colonic Release of ColoPulse Tablets

Introduction ColoPulse tablets are an innovative development in the field of oral dosage forms characterized by a distal ileum and colon-specific release. Previous studies in humans showed release in the ileo-colonic region, but the relationship between gastrointestinal pH and release was not experimentally proven in vivo. This information will complete the in vivo release-profile of ColoPulse tablets. Materials and Methods Release from ColoPulse tablets was studied in 16 healthy volunteers using the dual label isotope strategy. To determine gastrointestinal pH profiles and transit times the IntelliCap system was used. A ColoPulse tablet containing 13C-urea and an uncoated, immediate release tablet containing 15N2-urea were taken simultaneously followed by a standardized breakfast after three hours. Five minutes after intake of the tablets the IntelliCap capsule was swallowed and pH was measured until excretion in the feces. Breath and urine samples were collected for isotope analysis. Results Full analysis could be performed in 12 subjects. Median bioavailability of 13C -urea was 82% (95% CI 74–94%, range 61–114%). The median lag time (5% release of 13C) was 5:42 h (95% CI 5:18–6:18 h, range 2:36–6:36 h,) There was no statistically significant difference between lag time based on isotope signal and colon arrival time (CAT) based on pH (median 5:42 vs 5:31 h p = 0.903). In all subjects an intestinal pH value of 7.0 was reached before release of 13C from the ColoPulse tablet occurred. Discussion and Conclusions From the combined data from the IntelliCap system and the 13C -isotope signal it can be concluded that release from a ColoPulse tablet in vivo is not related to transit times but occurs in the ileo-colonic region after pH 7.0 is reached. This supports our earlier findings and confirms that the ColoPulse system is a promising delivery system for targeting the distal ileum and colon. Trial Registration ISRCTN Registry 18301880