Stable IsotopesStable Isotopesin Gastroenterology and Nutritionin Gastroenterology and Nutrition

Klaus D. Wutzke, Klaus D. Wutzke, Ph.DPh.D., ., D.ScD.Sc..

ChildrenChildren‘‘s Hospital of the University of Rostocks Hospital of the University of RostockResearch LaboratoryResearch Laboratory

www.kinderklinikwww.kinderklinik--rostock.derostock.de ‘‘ForschungForschung‘‘

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

HeadlinesHeadlines

I would like to entertain you with the following items, namely:I would like to entertain you with the following items, namely:Stable isotopesStable isotopesMeasuring methodsMeasuring methods1313COCO22--Breath testsBreath tests1313CC--Urea breath test for Urea breath test for Helicobacter pyloriHelicobacter pylori diagnosisdiagnosis1313CC--Methacetin and Methacetin and 1313CC22--aminopyrine for liver function testsaminopyrine for liver function testsLactoseLactose--[[1313C]ureide breath test for C]ureide breath test for orocoecalorocoecal transit time transit time measurementsmeasurementsImpact of LImpact of L--carnitinecarnitine on fat oxidationon fat oxidationUtilisationUtilisation of doubly of doubly labelledlabelled Lactobacillus Lactobacillus johnsoniijohnsonii yoghurt.yoghurt.

Stable isotopesStable isotopes

•• They are rare.They are rare.•• „„IsosIsos topostopos““ (Greek) (Greek) „„on the same placeon the same place““ at the periodic at the periodic

system of the elements.system of the elements.•• Features, peculiarities:Features, peculiarities:•• Non radioactive.Non radioactive.•• Equal number of protons and electronsEqual number of protons and electrons

•• identical chemical properties.identical chemical properties.•• Different number of neutronsDifferent number of neutrons

•• different physical properties (mass).different physical properties (mass).

Stable carbon isotopeStable carbon isotope

+

++ ++

+

-

-

--

-

-

1212CC+66

1313CC66

+

++ ++

+

-

-

--

-

-

-66 +77 66 -66

(98,9%)(98,9%) (1,1%)(1,1%)

Isotope Isotope (Greek (Greek : same; : same; : place): place)

neutronsneutrons

protonsprotons

electronselectrons

resemble

IsotopeIsotope--man/womanman/woman1313

1212

1

96.0 g96.0 g 161617171818

17.2 g17.2 g

42.4 kg42.4 kg(61 %)(61 %)

1.82 kg1.82 kg(2.6 %)2.6 %)

2.10 g2.10 g

16.0 kg16.0 kg (23 %)(23 %)

192.0 g192.0 g

7.00 kg7.00 kg(10 %)(10 %)

7.14 g

A 70 kg-man contains 314 gheavy stable isotopes.Stakeholders:2H, 13C, 15N, 17O, 18O

modified from Wada

deliberately German suffix: … stoff

Basic requirements of the tracer techniqueBasic requirements of the tracer technique

•• The application of a stable isotope The application of a stable isotope labelledlabelled tracertracer substance substance necessarily necessarily give risegive rise to an increase of the isotopic enrichment of the to an increase of the isotopic enrichment of the organism. organism.

•• The metabolic fate of the tracer can be followed by measuring itThe metabolic fate of the tracer can be followed by measuring its s enrichment.enrichment.

•• It is possible to draw conclusions for the respective It is possible to draw conclusions for the respective unlabelledunlabelledsubstance (biologically indistinguishable from the substance (biologically indistinguishable from the traceetracee).).

Measuring methodsMeasuring methods

•• Analytical methodAnalytical method•• mass spectrometry mass spectrometry

((22H, H, 1313C, C, 1515N, N, 1818O)O)•• CFCF--IRMSIRMS•• CFCF--CC--IRMSIRMS•• nondispersivenondispersive•• infrared spectroscopyinfrared spectroscopy•• nuclear magnetic resonance nuclear magnetic resonance

spectroscopy (NMR)spectroscopy (NMR)

•• Accuracy [atomAccuracy [atom--%]%]•• 0.010.01

•• 0.00010.0001

•• 0.0010.001

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

cross windcross wind100 km/ 100 km/ hourhour

spee

d: 1

00 k

m/ h

our

crash barrier

Mass spectrometry on the interstateMass spectrometry on the interstateMass spectrometry on the interstate

0.6 t

1.2 t

1.8 t

Rostock 50 kmRostock 50 kmResearch LaboratoryResearch Laboratory

Make an educated guess …

12C16O2 M=44

13C16O2 M=45

12C18O16O M=46

FANci2, Fischer GmbH, LeipzigFANci2, Fischer GmbH, Leipzig

1313COCO22--Breath testsBreath tests

•• Benefits, incentives:Benefits, incentives:•• Non invasiveNon invasive•• Non radioactiveNon radioactive•• On site, ad hocOn site, ad hoc•• Sensitive, specificSensitive, specific•• Quick and simpleQuick and simple

from G. Wagner

Helicobacter pyloriHelicobacter pylori diagnosisdiagnosis

Ingestion of 75 mg [13C]urea dissolved in 100 ml orange juice after an overnight fast.

Degradation pathwayDegradation pathway

from H. Fischer

Interpretation

O CH3

13NHCOH3C

13CH3

Basis of the [13C]methacetin breath test

The microsomial dealcylation of [13C]methacetin (paracetamol derivative) to p-hydroxyacetanilide is catalysed by the cytochrome P450 oxygenase system.The cleaved methyl group is oxidised to formic acid and is finally exhaled as 13CO2.

1313CC--Methacetin and Methacetin and 1313CC22--aminopyrine for liver function testsaminopyrine for liver function tests

Degradation pathwayDegradation pathway

[13C2]Aminopyrine is degraded by cytochrome P450 oxygenasemediated demethylation and oxidation to formaldehyde which is converted to bicarbonate and finally exhaled as 13CO2.

0

10

20

30

40

50

[13C]methacetin [13C]aminopyrine

without with red wine (10 days: 0.4 g ethanol/ kg) cirrhosis

1313COCO22--exhalation without and after red wine drinking in healthy adultsexhalation without and after red wine drinking in healthy adultsin in strikingstriking comparison to subjects suffering from liver cirrhosiscomparison to subjects suffering from liver cirrhosis

[%][%]

The impact of LThe impact of L--carnitinecarnitine supplementation on supplementation on fat oxidation and protein turnoverfat oxidation and protein turnover

LL--carnitinecarnitine

•• Point out: The primary function of LPoint out: The primary function of L--carnitinecarnitine is to is to „„shuttleshuttle““fatty acids into the mitochondria where they can be broken fatty acids into the mitochondria where they can be broken down by down by --oxidation.oxidation.

H3C – N+ – CH2 – CH – CH2 – COO -

H3C

H3C

OH

Chemical structure of LChemical structure of L--carnitinecarnitine

Aim of the studyAim of the study

Evaluation of the metabolic effect of LEvaluation of the metabolic effect of L--carnitinecarnitinesupplementation on supplementation on

•• wholewhole--body protein turnoverbody protein turnover•• [[1515N]glycine, threeN]glycine, three--compartment modelcompartment model along withalong with

•• fat oxidationfat oxidation•• [U[U--1313C]algae lipid mixture,C]algae lipid mixture,

•• in slightly obese subjects.in slightly obese subjects.

Composition of Composition of 1313CC--algae fatty acid mixturealgae fatty acid mixture““Comparatively representative for fatty acid metabolismComparatively representative for fatty acid metabolism””

50

27

913

0

10

20

30

40

50

palmitic acidC16:0

oleic acidC18:1

palmitoleicacid C16:1

linoleic acidC18:2

[%[%]]

ResultsResults1313COCO22--enrichmetenrichmet

0

2

4

6

8

10

12

14

0 2 4 6 8 10 12 14

time [hours]

DO

B

without with L-carnitine

Cumulative Cumulative 1313COCO22--exhalationexhalation

0

5

10

15

20

25

0 2 4 6 8 10 12 14

time [hours]

[%]

without with 3 g/day L-carnitine

15,8 %

19,3 %

Protein turnover ratesProtein turnover rates

3,72 3,653,41 3,41

00,5

11,5

22,5

33,5

4

synthesis breakdown

without with L-carnitine

[g/k

g/da

y[g

/kg/

day ]]

Interpretation, conclusionInterpretation, conclusion

•• Anyway, it turned out that LAnyway, it turned out that L--carnitinecarnitine seemingly increased the seemingly increased the fat oxidation in slightly obese subjects, whereas protein fat oxidation in slightly obese subjects, whereas protein turnover remains unchanged.turnover remains unchanged.

GlycosylGlycosyl--[[1313C]ureides for measuring the C]ureides for measuring the orocoecalorocoecal transit timetransit time

[[1313C]C]--Sugar Sugar ureidesureides

•• Address: NoAddress: No enzymatic degradation of the sugarenzymatic degradation of the sugar--urea bond in urea bond in the small intestine alleged.the small intestine alleged.

•• Clostridium Clostridium innocuuminnocuum is accounted for the exclusive is accounted for the exclusive degradation.degradation.

•• Ubiquitous present Ubiquitous present in the in the coecumcoecum (Mohr, BBA;1998).(Mohr, BBA;1998).•• Launched Launched in 1993 by Heine, AJG.in 1993 by Heine, AJG.•• Experienced with own laboratory synthesis:Experienced with own laboratory synthesis: LactoseLactose--

[[1313C]ureideC]ureide: marker for measuring : marker for measuring orocoecalorocoecal transit time transit time (OCTT). Credit: Wutzke, EJCN;1997, 2004, 2010.(OCTT). Credit: Wutzke, EJCN;1997, 2004, 2010.

O

13C

H

15NH 15NH2

O

CH2OH

H

H

H OH

H OH

H

O

CH2OH

H

H

H OH

H OH

HO

O

LactoseLactose--[[1313C,C,1515NN22]ureide (DLLU)]ureide (DLLU)

galactose glucose

Gradual degradation of lactoseGradual degradation of lactose--[[1313C,C,1515NN22]ureide]ureide

galactose + glucose-[13C,15N2]ureide

glucose + [13C,15N2]urea

13CO2 + 2 15NH3

lactose-[13C,15N2]ureide

ßß--galactosidasegalactosidase

glucose ureide glucose ureide hydrolasehydrolaseClostridiumClostridium innocuuminnocuum

ureaseurease:: KlebsiellaKlebsiella,, BacteroidesBacteroides

small bowel

coecum

colon

13C,15N-measurement

kidney

IRMS

lung

0

1

2

3

4

5

6

7

8

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

time [h]

DO

B

13C-LU

Measurement of the orocoecal transit time with lactose-[13C]ureide

OCTT13C-LU

cut-off: >> 2 DOB

Utilisation of doubly stable isotope labelled Utilisation of doubly stable isotope labelled Lactobacillus Lactobacillus johnsoniijohnsonii yoghurtyoghurt in humansin humans

•• In the face of In the face of Lactobacillus Lactobacillus johnsoniijohnsonii (La1), a probiotic (La1), a probiotic lactobacillus strain of human origin, is evidently able to adherlactobacillus strain of human origin, is evidently able to adhere e to the intestinal mucosa.to the intestinal mucosa.

•• One of the important properties of probiotics is the ability to One of the important properties of probiotics is the ability to survive the dangerous journey through the intestinesurvive the dangerous journey through the intestine..

•• For evaluation of this issue (phenomenon), La1 was used for For evaluation of this issue (phenomenon), La1 was used for doubly labelling with doubly labelling with 1515N and N and 1313C to follow the metabolic fate of C to follow the metabolic fate of orally administrated doubly stable isotope orally administrated doubly stable isotope 1313CC--,,1515NN--labelled La1 labelled La1 in humans.in humans.

Aim of the studyAim of the study

•• Investigation of the metabolic fate of doubly Investigation of the metabolic fate of doubly 1313CC--,,1515NN--labelled labelled Lactobacillus Lactobacillus johnsoniijohnsonii•• the the 1313COCO22--exhalation,exhalation,•• the urinary and faecal the urinary and faecal 1313CC-- and and 1515NN--excretion, respectively,excretion, respectively,•• and the corresponding isotopic enrichment of specific blood and the corresponding isotopic enrichment of specific blood

plasma fractions.plasma fractions.

Material and methodsMaterial and methods

•• Universal Universal labellinglabelling of La1 with of La1 with 1313CC and and 1515NN•• by fermentation (by fermentation (BiostadBiostad, Braun, , Braun, MelsungenMelsungen, Germany) in a , Germany) in a

medium containingmedium containing•• [U[U--1515N]yeast extractN]yeast extract (prior (prior labelledlabelled by by [[1515N]HN]H44ClCl) and) and•• [U[U--1313CC66]glucose ]glucose ((CamproCampro Scientific, Berlin)Scientific, Berlin)

FermenterFermenter BiostadBiostad, B. Braun, , B. Braun, MelsungenMelsungen

OCTT0

2

4

6

8

0 2 4 6 8 10 12 14

time [h]

[DOB]

0

2

4

6

8

10[%]

[DOB] [%]

1313COCO22--Enrichment and cumulative percentage exhalationEnrichment and cumulative percentage exhalation

Keep an eye on it!

total-15N

OCTT0

0,05

0,1

0,15

0,2

0,25

0,3

0,35

0 6 12 18 24 30 36 42 48

time [h]

[at%exc]

15NH3

Urinary total and ammonia Urinary total and ammonia 1515NN--enrichmentenrichment

0

0,02

0,04

0,06

0,08

0,1

0,12

0,14

0,16

0,18

0,2

supernatant fibrinogen plasma proteinprecipitate

[at%exc]

15N 13C

Isotope incorporation in different fractions of the bloodIsotope incorporation in different fractions of the blood

renal faecal incorporation

12.4

50.0

37.6

Percentage total Percentage total 1515NN--excretion and excretion and 1515NN--incorporationincorporation

expiratory renal faecal incorporation

50.2

1.38.6

39.9

Percentage total Percentage total 1313CC--excretion and excretion and 1313CC--incorporationincorporation