Stable isotopes in gastroenterology and nutrition

Stable Isotopesin Gastroenterology and NutritionKlaus D. Wutzke, Ph.D.,., D.Sc.Children‘s s Hospital of the University of RostockResearch Laboratorywww.kinderklinik-rostock.derostock.de ‘Forschung‘

In 1990 actually a dream became reality:Stable isotope research at Cornell University

HeadlinesI would like to entertain you with the following items, namely:• Stable isotopes• Measuring methods•13 CO 2 -Breath tests•13 C-Urea breath test for Helicobacter pylori diagnosis•13 C-Methacetin and 13 C 2 -aminopyrine for liver function tests• Lactose-[ 13 C]ureide breath test for orocoecal transit timemeasurements• Impact of L-carnitineLon fat oxidation• Utilisation of doubly labelled Lactobacillus johnsonii yoghurt.

Stable isotopes• They are rare.• „Isostopos“ (Greek) „on the same place“ at the periodicsystem of the elements.• Features, peculiarities:• Non radioactive.• Equal number of protons and electrons• identical chemical properties.• Different number of neutrons• different physical properties (mass).

Stable carbon isotope-612 C-6+-++ + +++(98,9%) 13 C (1,1%)6 -+--neutronsprotonselectronsresemble-7 6 6 ---++ + +++----Isotope (Greek : : same; : : place)

Isotope-man/womandeliberately German suffix: … stoff1216.0 kg (23 %)13192.0 gA 70 kg-man contains314 gheavy stable isotopes.Stakeholders:2H, 13 C, 15 N, 17 O, 18 O1696.0 g 16181.82 kg(2.6 %)42.4 kg(61 %)1717.2 g7.00 kg(10 %)7.14 g12.10 gmodified from Wada

Basic requirements of the tracer technique• The application of a stable isotope labelled tracer substancenecessarily give rise to an increase of the isotopic enrichment of theorganism.• The metabolic fate of the tracer can be followed by measuring itsenrichment.• It is possible to draw conclusions for the respective unlabelledsubstance (biologically indistinguishable from the tracee).

Measuring methods• Analytical method• mass spectrometry( 2 H, 13 C, 15 N, 18 O)• CF-IRMS• CF-C-IRMS• nondispersive• infrared spectroscopy• nuclear magnetic resonancespectroscopy (NMR)• Accuracy [atom-%]• 0.01• 0.0001• 0.001

Equipment: Tracer Mass 20-20,20, SerCon, , Crewe, UK

Mass spectrometry on the interstateRostock 50 kmResearch Laboratorycross wind100 km/ hour1.8 t12C 18 O 16 O M=46Make an educated guess …1.2 t0.6 t13C 16 O 2 M=4512C 16 O 2 M=44speed: 100 km/ hourcrash barrier

FANci2, Fischer GmbH, Leipzig

13 CO 2 -Breath tests• Benefits, incentives:• Non invasive• Non radioactive• On site, ad hoc• Sensitive, specific• Quick and simple

from G. Wagner

Helicobacter pylori diagnosis

Degradation pathwayIngestion of 75 mg[ 13 C]urea dissolvedin 100 ml orangejuice after anovernight fast.from H. Fischer

Interpretation

13 C-Methacetin and 13 C 2 -aminopyrine for liver function testsBasis of the [ 13 C]methacetin breath testH 3 CCONH13O CH 313CH3The microsomial dealcylation of[ 13 C]methacetin (paracetamol derivative)to p-hydroxyacetanilide is catalysed by thecytochrome P450 oxygenase system.The cleaved methyl group is oxidised toformic acid and is finally exhaled as 13 CO 2 .

Degradation pathway[ 13 C 2 ]Aminopyrine is degraded bycytochrome P450 oxygenasemediated demethylation and oxidation toformaldehyde which is converted tobicarbonate and finally exhaled as 13 CO 2 .

CO 2 -exhalation without and after red wine drinking in healthy adultsin striking comparison to subjects suffering from liver cirrhosis13 CO[%]50403020100[13C]methacetin[13C]aminopyrinewithout with red wine (10 days: 0.4 g ethanol/ kg) cirrhosis

The impact of L-carnitineLsupplementation onfat oxidation and protein turnoverL-carnitine• Point out: The primary function of L-carnitineLis to „shuttle“fatty acids into the mitochondria where they can be brokendown by -oxidation.

Chemical structure of L-carnitineLH 3 CH 3 C–N + – CH 2 – CH – CH 2 – COO -H 3 COH

Aim of the studyEvaluation of the metabolic effect of L-carnitineLsupplementation on• whole-body protein turnover• [ 15 N]glycine, three-compartment model along with• fat oxidation• [U- 13 C]algae lipid mixture,• in slightly obese subjects.

Composition of 13 C-algae fatty acid mixture“Comparatively representative for fatty acid metabolism”[%]505040302720109130palmitic acidC16:0oleic acidC18:1palmitoleicacid C16:1linoleic acidC18:2

ResultsCO 2 -enrichmet13 CODOB141210864200 2 4 6 8 10 12 14time [hours]without with L-carnitine

Cumulative 13 CO 2 -exhalation25201519,3 %15,8 %[%]10500 2 4 6 8 10 12 14time [hours]without with 3 g/day L-carnitine

Protein turnover rates[g/kg/day]43,532,521,510,503,72 3,65synthesis3,41 3,41breakdownwithoutwith L-carnitine

Interpretation, conclusion• Anyway, it turned out that L-carnitineLseemingly increased thefat oxidation in slightly obese subjects, whereas proteinturnover remains unchanged.

Glycosyl-[ 13 C]ureides for measuring theorocoecal transit time[ 13C]-Sugarureides13 C]• Address: No enzymatic degradation of the sugar-urea urea bond inthe small intestine alleged.• Clostridium innocuum is accounted for the exclusivedegradation.• Ubiquitous present in the coecum (Mohr, BBA;1998).• Launched in 1993 by Heine, AJG.• Experienced with own laboratory synthesis: Lactose-[ 13 C]ureide: : marker for measuring orocoecal transit time(OCTT). Credit: Wutzke, EJCN;1997, 2004, 2010.

Lactose-[ 1313 C, 1515 N 2 ]ureide (DLLU)CH 2 OHOCH 2 OHOO13CHOHHH15NH15NH 2galactoseOglucoseHOHHHOHHHHOHHOH

Gradual degradation of lactose-[ 13lactose-[ 13 C, 15 N 2 ]ureide13 C, 1515 N 2 ]ureideß-galactosidasesmall bowelgalactose +glucose-[ 13 C, 15 N 2 ]ureideglucose ureide hydrolaseClostridium innocuumcoecumglucose + [ 13 C, 15 N 2 ]ureaurease: Klebsiella, Bacteroidescolon13CO 2 + 2 15 NH 3lungkidney13C, 15 N-measurementIRMS

Measurement of the orocoecal transit time with lactose-[ 13 C]ureide8765DOB432cut-off: > 2 DOB100 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1513C-LUOCTT time [h]13C-LU

Utilisation of doubly stable isotope labelled Lactobacillusjohnsonii yoghurt in humans• In the face of Lactobacillus johnsonii (La1), a probioticlactobacillus strain of human origin, is evidently able to adhereto the intestinal mucosa.• One of the important properties of probiotics is the ability tosurvive the dangerous journey through the intestine.• For evaluation of this issue (phenomenon), La1 was used fordoubly labelling with 15 N and 13 C to follow the metabolic fate oforally administrated doubly stable isotope 13 C-, 15 N-labelled La1in humans.

Aim of the study• Investigation of the metabolic fate of doubly 13 C-, 15 N-labelledLactobacillus johnsonii• the 13 CO 2 -exhalation,• the urinary and faecal 13 C- and 15 N-excretion, respectively,• and the corresponding isotopic enrichment of specific bloodplasma fractions.

Material and methods• Universal labelling of La1 with 13 C and 15 N• by fermentation (Biostad(Biostad, , Braun, Melsungen, , Germany) in amedium containing• [U- 15 N]yeast extract (prior labelled by [ 15 N]H 4 Cl) ) and• [U- 13 C 6 ]glucose (CamproScientific, Berlin)

Fermenter Biostad, , B. Braun, Melsungen

13 CO 2 -Enrichment and cumulative percentage exhalation8[DOB][%]1068644220OCTT0 2 4 6 8 10 12 14time [h]Keep an eye on it![DOB] [%]0

Urinary total and ammonia 15 N-enrichment0,35[at%exc]0,30,250,20,150,10,050OCTT0 6 12 18 24 30 36 42 48time [h]total-15N 15NH3

Isotope incorporation in different fractions of the blood0,20,18[at%exc]15N13C0,160,140,120,10,080,060,040,020supernatant fibrinogen plasma proteinprecipitate

Percentage total 15 N-excretion and 15 N-incorporation50.012.437.6renal faecal incorporation

Percentage total 13 C-excretion and 13 C-incorporation8.61.350.239.9expiratory renal faecal incorporation