Stable Isotopes in Nutrition - American Chemical Society

11 13

C-Enriched Substrates for In Situ and In Vivo Metabolic Profiling Studies by CN M R 13

J. R. BRAINARD, J. Y. HUTSON, and R. E. LONDON

Downloaded by SUFFOLK UNIV on January 20, 2018 | http://pubs.acs.org Publication Date: July 9, 1984 | doi: 10.1021/bk-1984-0258.ch011

Isotope and Nuclear Chemistry Division, University of California, Los Alamos National Laboratory, P.O. Box 1663, MS J515, Los Alamos, NM 87545 N. A. MATWIYOFF Isotope and Nuclear Chemistry Division, University of California, Los Alamos National Laboratory,P.O.Box 1663, MS J515, Los Alamos, N M 87545 and University of New Mexico/Los Alamos NMR Center for Non-Invasive Diagnosis, Albuquerque, N M 87131

The application of stable isotopes and NMR to the study of metabolism and its regulation in l i v i n g systems has unique advantages over traditional biochemical or physiological techniques. From the concentration and labeling patterns observed in labeled products and intermediates, information concerning the regulation and relative contribution of metabolic pathways can be obtained. In experiments investigating the regulation of gluconeogenesis in perfused hamster l i v e r s , the fluxes of carbon-13-labeled alanine through multiple metabolic pathways leading to glucose and glycogen have been determined and modulation of these metabolic pathways by reducing substrates demonstrated. In addition, the effects of reducing substrates on the intracellular redox potential of the perfused l i v e r have been observed by C NMR. 13

13

For over a decade, the combination of C - l a b e l e d substrates and C nuclear magnetic resonance (NMR) spectroscopy has been used to study product precursor relationships and the a c t i v i t y of metabolic pathways in microorganisms (1-3). In these studies, the C - l a b e l e d substrate is added to the nutrient medium of the microorganism and at some later time, metabolic products suspected of containing the label (often on the basis of prior studies with the radioactive isotope, C) are isolated from the medium or extracted from the microorganism, and the distribution of the C label at specific sites is quantified by high resolution C NMR or H NMR spectroscopy of an extract containing the metabolites of interest. Recently C NMR spectroscopy in conjunction with C labeling has become an important tool for studying metabolism in intact, l i v i n g tissue (4-6). 1 3

13

lk

1 3

1 3

l

1 3

1 3

0097-6156/84/0258-0157$06.00/0 © 1984 American Chemical Society

Turnlund and Johnson; Stable Isotopes in Nutrition ACS Symposium Series; American Chemical Society: Washington, DC, 1984.

158

STABLE ISOTOPES IN NUTRITION

The hallmark o f the method is t h a t m e t a b o l i t e s can be mea sured r e p e t i t i v e l y and n o n - d e s t r u c t i v e l y in i n t e r v a l s approaching r e a l t i m e , without e x t r a c t i o n o f the m e t a b o l i t e s from the t i s s u e . The development o f l a r g e bore magnets and surface c o i l s (7) is now a l l o w i n g the n o n - i n v a s i v e study o f metabolism o f C - l a b e l e d compounds in l i v e animals ( 8 ) . We a n t i c i p a t e t h a t soon C NMR spectroscopy and C - l a b e l e d s u b s t r a t e s will be used f o r non i n v a s i v e s t u d i e s o f metabolism in humans, extending the range o f p h y s i o l o g i c a l and b i o c h e m i c a l d i s o r d e r s t h a t a l r e a d y are being a c t i v e l y addressed in P NMR s t u d i e s o f humans (5t.9). The C methodology will be p a r t i c u l a r l y important in the study o f n u t r i t i o n a l d i s o r d e r s and n u t r i t i o n a l requirements under s t r e s s s i n c e many m e t a b o l i t e s o f i n t e r e s t do not c o n t a i n phosphorus atoms and cannot be s t u d i e d d i r e c t l y by P NMR. 1 3

1 3

1 3

3 1

1 3


3 1

Survey o f Recent

1 3

C NMR M e t a b o l i c S t u d i e s 1 3

1 3

The s t a t u s o f in s i t u and in v i v o C NMR s t u d i e s o f C - l a b e l e d s u b s t r a t e s has been reviewed w i t h i n the l a s t year (±$5,9). We survey b r i e f l y some o f the notable recent developments s i n c e those reviews appeared. S i l l e r u d and Shulman (10) were able t o q u a n t i t a t e the n a t u r a l abundance C s i g n a l s o f glycogen i r i s i t u w i t h i n the perfused l i v e r s o f r a t s and were a b l e t o compare the r e l a x a t i o n p r o p e r t i e s ( T T , and Nuclear Overhauser e f f e c t ) o f glycogen in s i t u and in v i t r o . In s p i t e o f the h i g h molecular weight o f g l y c o g e n , h i g h r e s o l u t i o n C NMR s p e c t r a were obtained and the r e l a x a t i o n parameters were found t o be i d e n t i c a l in s i t u and in v i t r o , suggesting a h i g h degree o f i n t e r n a l motion o f segments o f the polymer and a p s e u d o - i s o t r o p l c o v e r a l l motion f o r the glycogen i t s e l f . These s t u d i e s have been extended t o the in v i v o i n v e s t i g a t i o n o f h e p a t i c glycogen s y n t h e s i s in r a b b i t s (11) and in v i v o guinea p i g heart metabolism ( 1 2 ) . In the study o f guinea p i g heart metabolism, the time course o f glycogen s y n t h e s i s a f t e r intravenous i n j e c t i o n o f [ 1 - C ] - D - g l u c o s e was followed s e r i a l l y and the e f f e c t s o f anoxia on glycogen m o b i l i z a t i o n were monitored. In l i k e manner, Behar e t a l . (13) have accomplished in v i v o s e r i a l C NMR s t u d i e s o f hypoxia in r a b b i t b r a i n a f t e r intravenous i n f u s i o n o f [ 1 - C ] - D - g l u c o s e by m o n i t o r i n g the r i s e and f a l l o f the flow o f l a b e l i n t o the C-3 carbon o f l a c t a t e w i t h i n d u c t i o n and t e r m i n a t i o n o f h y p o x i a . Of s p e c i a l i n t e r e s t is the recent r e p o r t (14) o f the a c q u i s i t i o n o f l o c a l i z e d C NMR spec t r a (at natural"". abundance) o f the head and body o f humans obtained w i t h a system c o n t a i n i n g a 1.5 Τ magnet w i t h a i m b o r e . 1 3

l t

2

1 3

l3

1 3

l 3

1 3

Q u a l i t a t i v e and Q u a n t i t a t i v e Aspects o f Metabolism

1 3

C NMR Spectroscopy and

In this symposium on s t a b l e i s o t o p e s in n u t r i t i o n , it is appro p r i a t e t o i l l u s t r a t e the nature o f the metabolic i n f o r m a t i o n o b t a i n a b l e from C NMR spectroscopy w i t h an example from metab o l i s m in l i v e r , the l i v e r p l a y i n g a c e n t r a l r o l e in m a i n t a i n i n g 1 3



11.

BR AIN ARD ET AL.

l3

C-Enriched Substrates

159

the body's n u t r i e n t s w i t h i n narrow l i m i t s w h i l e being presented w i t h a h i g h l y v a r i a b l e s u p p l y . One o f the major r e g u l a t e d p r o cesses which occurs in the l i v e r is gluconeogenesls, the r e s y n t h e s i s o f glucose from the products o f glucose c a t a b o l i s m in b r a i n , muscle, and f a t c e l l s . I t is important t o understand the r e g u l a t i o n o f this process because the body n o r m a l l y s t o r e s o n l y enough glucose t o f u e l the b r a i n f o r 12 hours, and thus gluconeogenesis p l a y s a c e n t r a l r o l e in m a i n t a i n i n g energy homeostasis. The major metabolic pathways f o r conversion o f these p r o d u c t s , which c o n s i s t o f three-carbon s k e l e t o n s , i n t o glucose which has a s i x - c a r b o n s k e l e t o n , are d e p i c t e d s c h e m a t i c a l l y in F i g u r e 1. One o f the major waste products o f glucose metabolism in p e r i p h e r a l t i s s u e s is the amino a c i d a l a n i n e , and its use as a s u b s t r a t e f o r gluconeogenesls r e q u i r e s the d i s p o s a l o f ammonia in the urea c y c l e . In the f o l l o w i n g d i s c u s s i o n , we will i l l u s t r a t e how the flow o f the C l a b e l from a l a n i n e i n t o p r o d u c t s , b y p r o d u c t s , and intermediates o f gluconeogenesls can be used t o o b t a i n q u a l i t a t i v e and q u a n t i t a t i v e i n f o r m a t i o n about the r e l a t i v e a c t i v i t i e s o f these pathways. The C NMR s p e c t r a summarized in F i g u r e 2 i l l u s t r a t e the flow o f C l a b e l from [ 3 - C ] - L - a l a n i n e d u r i n g gluconeogenesls by a S y r i a n hamster l i v e r perfused w i t h a K r e b s - H e n s e l e i t buffer c o n t a i n i n g the 8 mM-labeled a l a n i n e . Although 8 mM r e p r e s e n t s an u n p h y s i o l o g i c a l l y h i g h c o n c e n t r a t i o n o f a l a n i n e (normal serum l e v e l s are approximately 300 PM), it is necessary t o c o n s i d e r the low s e n s i t i v i t y o f the NMR t e c h n i q u e . In order t o achieve g l u c o neogenic r a t e s s u f f i c i e n t t o permit d e t e c t i o n o f l a b e l e d metabo l i t e s and i n t e r m e d i a t e s in r e a l time w i t h r e l a t i v e l y s h o r t accum u l a t i o n t i m e s , a r e l a t i v e l y h i g h c o n c e n t r a t i o n o f a l a n i n e was used. The resonances observed in these s p e c t r a come from both i n t r a - and e x t r a c e l l u l a r m e t a b o l i t e s . However, s i n c e the p e r fused hamster l i v e r almost completely f i l l s the s e n s i t i v e volume o f the Rf c o i l , resonances from i n t r a c e l l u l a r m e t a b o l i t e s domi nate in these s p e c t r a . Changes in the i n t e n s i t y o f resonances from C - l a b e l e d m e t a b o l i t e s show up more c l e a r l y in s p e c t r a ( F i g u r e 3) obtained by s u b t r a c t i n g the background spectrum o f the l i v e r in the absence o f the C - l a b e l e d s u b s t r a t e . One o f the major advantages o f C NMR as a t o o l f o r metabolic s t u d i e s is t h a t i n f o r m a t i o n about the d i s t r i b u t i o n o f l a b e l at s p e c i f i c carbon s i t e s w i t h i n m e t a b o l i t e s is a v a i l a b l e d i r e c t l y from the spectrum. The d i s t r i b u t i o n o f l a b e l in products and i n t e r m e d i a t e s can be used t o p r o v i d e v a l u a b l e i n f o r m a t i o n concerning the metabolic h i s t o r y o f the product or i n t e r m e d i a t e . For example, the e a r l y appearance o f C l a b e l in C-2 and C-3 o f glutamate and g l u t a m i n e , but not in C-4 o f glutamate and g l u t a m i n e , p r o v i d e s i n f o r m a t i o n about the e a r l y a c t i v i t y o f the Krebs c y c l e and the pathway by which the C l a b e l e n t e r s it. I f l a b e l e d pyruvate (formed by transamination o f [ 3 - C ] - L - a l a n i n e ) e n t e r s the Krebs c y c l e as oxaloacetate through pyruvate c a r b o x y l a s e , then the l a b e l will appear in C-2 and C-3 o f glutamate as shown in F i g u r e 4. The appearance o f l a b e l a t two s i t e s in glutamate is a 1 3

1 3

1 3

1 3

1 3

1 3

1 3

1 3

1 3

13


S T A B L E I S O T O P E S IN N U T R I T I O N

160


ο KG

GLUT

F 16 DP

G 6Ρ

GLYCOGEN

I GLUCOSE

F i g u r e 1.

Pathways f o r gluconeogenesis from a l a n i n e .


11.

BR AIN A R D ET A L .

l3


161

CH OH


2

140 min

ço2

CH,

C3 GLUTAMATE

C 2 .

ALA. H N a

H

C0

2

6 0 min

(CH ) 2

C=C

-N(CH ) 3

,C=0

3

f t

CH. Background

1λ

F i g u r e 2 . Proton decoupled C NMR s p e c t r a at T.Ο Τ from a p e r fused S y r i a n hamster l i v e r performing gluconeogenesis from 3 - c a l a n i n e , a , Background spectrum "before a d d i t i o n o f 8 mM a l a n i n e ; b , spectrum 60 min a f t e r a d d i t i o n o f a l a n i n e ; and c , spectrum 120 min a f t e r a l a n i n e a d d i t i o n . These s p e c t r a were accumulated under the f o l l o w i n g c o n d i t i o n s : 37 ° C , 1 . 9 s p u l s e i n t e r n a l , 65 p u l s e a n g l e , 1 6 , 0 0 0 Hz s p e c t r a w i d t h , 2 5 6 scans, 8 . 3 min total a c q u i s i t i o n t i m e , two l e v e l gated broad band de c o u p l i n g , Λ watt d u r i n g d e l a y , 7 watt d u r i n g a c q u i s i t i o n .


162


Glucose C6 \

Glutamine C2

Glutamine •


aJC

31 min v/.A-M"'Vwvv%feV

80

70

40

30

20

10

ι from TMS

Figure 3. Proton decoupled C MR spectra of perfused l i v e r during f i r s t hour of gluconeogenesis obtained by subtracting background l i v e r resonances. The dashed line indicates the chemical shift for C-U glutamate.


13


BRAINARD ET AL.

CO"

13 C) Alanine 13

CH

cor

C

13


I •CH II CH I

ç°;

—

1 1

2

m

CH,

CoA I S I

cor •c«o 11

.CHOH

2

CH,

2

l

C .

2

cor

cor l2- C)or[3- Cl

cor l2- C]or[3- C)

Fumarate

Malate

Oxaloacetate

CH

13

13

.CHOH

-

13

ο

Oxaloacetate

i-

cor 1

•CH

ι

2

•CH„

COJ

HO - C - CO~

l4- Cl Citrate 13

1

Acetyl Co A

[2 C)or[3 C] Citrate 13

1 3

•in,

.C= 0

co-

cor

(2 C l Acetyl CoA

13

m

>

I

13

I3-«CI °= Oxaloacetate

(3- Cl Malate

cor

(3- Cl Pyruvate

13

\

cor

HO - C - C O , I 2

cor

14 C1 13

c = οa-Ketoglutarate cor I

(2- C)or(3 Cl o-Ketoglutarate 13

13

•ç«

2

.C«0

ι

cor

•r.

C H2,

I

/ [2- C)orl3- Cl Glutamate 13

CH

|4 C1 Glutamate 13

\

13

•ÇH

2

.CH / \

F i g u r e h. alanine.

1

Pathways f o r glutamate b i o s y n t h e s i s from

ο

3- C



164

consequence of the equilibrium between oxaloacetate, malate and the symmetrical intermediate fumarate, which scrambles the label between C-2 and C-3. Alternately, i f pyruvate enters the TCA cycle as [2- C]-acetylCoA (through pyruvate dehydrogenase and citrate syntase), the label would appear at C-4 of glutamate also as shown in Figure 4. Since C-2 and C-3 of glutamate are labeled and C-4 is not, a l l of the [3- C]-pyruvate derived from [3- C]-alanine enters the Krebs cycle as oxaloacetate. These results indicate that acetylCoA, which is required for Krebs cycle activity, is derived from an unlabeled pool, probably from the mobilization and oxidation of endogenous lipids in the liver. Also of interest in Figure 3 Is the observation that almost 98% of the label from alanine is incorporated into the amino acids glutamate and glutamine during the first hour of perfusion. This incorporation of label into these amino acids, rather than glucose, during the early stages of gluconeogenesis reflects the requirement for a "metabolically non-toxic" sink for the amino group from alanine and suggests that urea cycle activity in the perfused liver is initially rather low. As shown in later spectra in Figures 2 and 3$ the glutamate and glutamine resonances eventually decrease slightly in intensity, and glucose resonances grow. This observation, together with the observation of urea in the perfusate at the end of the experiment, suggests that urea does serve as the major ultimate sink for nitrogen in the liver. Figures 2 and 3 also show that, in the late stages of gluconeogenesis, a l l the carbon atoms of glucose are labeled, but to different extents, indicating that more than one metabolic pathway contributes to the formation of glucose. We can use the relative enrichments for the glucose carbons to probe the contributions of various pathways to the synthesis of glucose from labeled alanine. As can be appreciated from Figure 1, there are several metabolic pathways by which alanine can be converted to glucose. In order to interpret the data quantitatively, we have used the method of mixtures. A reasonable subset of the possible metabolic pathways is selected, and the labeling pattern (distribution of isotopomers) for products and/or intermediates is associated with each pathway. The relative enrichment at each site is expressed in terms of the relative fluxes of label through the possible metabolic pathways and the expressions solved to give the relative fluxes as functions of the relative enrichments in the products and intermediates. In our analysis of gluconeogenesis, we have limited the possible pathways to the nine pathways shown together with their associated labeled products in Table I. For completeness we have included the possibility that the acetate pool may be partially labeled (Pathway M). As shown in Table I, a number of the pathways are degenerate in the sense that they lead to identical labeling patterns. Consequently, our analysis will give only the sum of relative fluxes through degenerate pathways. For convenience in 13


13

13


11.

165

l3


BRAINARD ET AL.

Table I . Subset of Pathways f o r Biosynthesis of Glucose and Intermediates During Gluconeogenesis from [ 3 - C j A l a n i n e 13


Pathway

Isotopomer(s)

13

A

[3- C]ALA .+ Pyr •. OAA

Β

(3- CjALA •> Pyr -» OAA •. ASP

.+ MAL

OAA

C

(3- C]ALA •+ Pyr -.. OAA '

D

(3- C]ALA .. Pyr .+ OAA .. Fum,. +.. χ OAA .. ASP •+-.-» (inside)

13

•+ PEP •...-.

1,6

PEP

-» Fum,. .... vOAA .+ MAL •+-•-» (inside)

13

1,6;2,5 Glucose

13

1,6;2,5 Glucose

7

13

F u n

[3- C]ALA •. Pyr .. OAA •» MAL .+

Glucose

1,6 Glucose

»(

.

o u t

i d e

)

0 A A

)

0 A A

". PEP ". ..

1,6;2,5 Glucose

. .

1,6;2,5 Glucose

13- C]ALA .. Pyr •+ OAA •. CIT -...•. aKG •. MAL -.• OAA -»• ..

3,4 Glucose; C 0

13

F u n i

[3- C]ALA .. Pyr -». OAA

ASP -»

(

£

o u t

i d e

P E P

13

OAA . CIT •+ aKG •. MAL •+ •. 1,6;2,5;3,4 [3- C]ALA - Pyr -.. OAA •. Fum (inside) Glucose; C 0 13

13- C]ALA -». Pyr -.. OAA •. CIT •+ •+ aKG •. 13

I

l3

J. [3- C]ALA -». Pyr .» OAA .+ Fum.. .. -> OAA •. CIT •+ ' (inside) Λ

Κ

2

1,6;2,5 Glucose

13

[3- C]ALA + Pyr -». Acetyl-CoA -> aKG -» MAL

2 Glutamate aKG -»

13

(3 C]ALA . Pyr -» Acetyl-CoA •+ CIT ..->.. aKG .+

2,3 Glutamate ' 4 Glutamate

Abbreviations - ALA - Alanine, Pyr - Pyruvate, OAA - Oxaloacetate, MAL - Malate, PEP - Phosphoenol Pyruvate, ASP - Aspartate CIT - C i t r a t e , aKG - aKeto-glutarate U m

(inside)

"

F u m a r a t e

n

. e q u i l i b r i u m with the OAA Pool

i n s i d e the mitochondria F

u

m

(

0

U

t

S

i

d

e

) - Fuma rate in e q u i l i b r i u m with the OAA Pool outside the mitochondria


2



166

discussing our results in terms of the cellular physiology, we have made the following substitutions: A+BsST; C+D+E+FsFum; and G+HsTCA. ST represents the relative flux of label to glucose that is not scrambled by either the intramitochondrial or the cytosolic fumarate pool. Fum is the flux of label that is scrambled by fumarase activity, either in the mitochondria or cytosol. TCA represents the flux of label around the citric acid cycle. For simplicity, we have ignored thé possibility that glucose could be derived from intermediates that have passed around the TCA cycle several times. This simplification is expected to result in negligible errors, since very l i t t l e enrichment at C-l of glutamate was observed in a l l experiments. We have also assumed that the glutamate enrichments are representative of the oxaloacetate pool from which the glucose is derived. This assumption allows the C-2/C-3 and the C-4/ÎC-2 + C-3) glutamate enrichments to serve as measures of intramitochondrial fumarase scrambling of the oxaloacetate pool and of the enrichment of the acetate pool, respectively. The expected alteration of the flux through these pathways in response to alteration in nutrient and hormone supply and the redox state of the liver provides us with a useful new probe of the regulatory mechanisms of gluconeogenesls and the health of the liver. Shown in Table II are the relative fluxes of label Table II.

Relative Fluxes Through Pathways of Gluconeogenesis

Substrates 8 mM 3- C Alanine 8 mM 3- C Alanine + 20 mM Ethanol 13

13

ST 0.18 0.05

Fum 0.44 0.71

TCA 0.38 0.23

from alanine to glucose through the pathways described above, determined from the relative enrichment in the glucose and glutamate present in the perfusate at the end of tfle perfusions with and without 20 mM ethanol. In order to minimize metabolic variations from livers from different animals, each liver is used as its own control by changing the perfusion medium halfway through the experiment. In addition, spectra of the perfusates were accumulated with gated proton decoupling under fully relaxed pulsing conditions, so that the intensities observed in the spectra accurately reflect the enrichments. Two trends in this table are noteworthy. The first is the decreased flux of label through the "straight" pathways (ST) in the presence of ethanol. This trend indicates that ethanol markedly increases the scrambling of intermediates by fumarase activity as noted also by Cohen et al. (16) in isolated heptocytes. In addition, ethanol also decreases the relative flux of label through the TCA cycle. This observation suggests that the


11.

BR AIN ARD ET AL.

13

167


activity of the TCA cycle is reduced relative to gluconeogenesls in the presence of ethanol, when the redox potential of the liver is increased. The relative activity of the TCA cycle is more strongly controlled by the presence of reduced pyridine nucleotides than by the acetate units produced by ethanol oxidation. In order to more directly investigate the regulation of metabolism by the redox potential of the cell, we have recently labeled the intracellular pools of pyridine nucleotides in the liver, using the C-labeled vitamins, nicotinic acid and nicotinamide. In the design of these experiments a number of considerations are involved. In order to minimize interferences from background resonances it is desirable to label sites which give resonances in relatively clear regions of the spectrum. From this standpoint, the C-2 carbon represents an attractive labeling site, since the background spectrum of liver is relatively free of interfering resonances at 140 ppm (Table III). However, the chemical shift difference between the oxidized and reduced


13

Table III. Chemical Shifts of Selected Pyridine Nucleotide Metabolites Nicotinate Nicotinamide NAD NADP NADH NaMN NaAD +

C-2 149.87 148.29 140.72 140.83 139.15 142.03 141.52

C-5 124.83 124.96 129.69 129.70 106.22 129.34 129.32

C-6 151.17 152.48 143.35 143.34 125.12 142.26 142.39

(abbreviations shown in Figure 6 caption) resonances is only 1.6 ppm, and chemical exchange (15), or poor resolution may preclude observation of separate reduced and oxidized resonances. The chemical shift at C-5 is more sensitive to the oxidation state, but the oxidized resonance falls in the same region as the intense resonances from the fatty acyl d é finie carbons, making quantitation of the NAD resonance difficult. The relaxation behavior of the carbon site selected is also an important consideration. The selection of protonated carbons has been favored largely because the spin-lattice relaxation times are short, allowing rapid pulse repetition rates and good signal-to-noise ratios in short periods of signal averaging. Protonated carbons offer the additional advantage that their relaxation mechanisms are dominated by a single well-understood interaction, and information about the molecular dynamics of metabolites within cells can be obtained from their relaxation behavior. Narrow linewidths (long spin-spin relaxation times)


168


are also desirable, especially when trying to detect larger metabolites. For the pyridine nucleotides (MW « 700) we have found that methine carbons appear to represent a good compromise, giving narrow resonances with relatively short spin-lattice relaxation times. A series of C spectra showing the incorporation of nicotinate into metabolites of the pyridine salvage pathway in a perfused hamster liver is shown in Figure 5. This series of spectra was accumulated during infusion of 3 mg per hour [2- C] nicotinic acid into the perfusate. Resonances from the precursor, nicotinate, and two products, nicotinamide and NAD. are detected after approximately 1 hour of infusion. Shortly after infusion of the labeled nicotinate ceases (at approximately 255 min), the resonance from the precursor disappears. The appearance and disappearance of these resonances during this experiment are consistent with the biosynthesis of NAD. from nicotinate by the PreissHandler pathway (Figure 6) [nicotinate -> nicotinate mononucleotide (NaMN) + nicotinic acid adenine dinucleotide (NaAD) nicotinamide adenine dinucleotide (NAD.)] and the subsequent formation of nicotinamide by glycohydrocase [NAD. .. nicotinamide]. Also of note in the spectra in Figure 5 is the lack of any detectable resonance from reduced pyridine nucleotides (approximately 1.5 ppm upfield from the NAD. resonance). Figure 7 shows the response of the redox potentialina perfused hamster liver to the addition of 45 mM ethanol. Instead of the in vitro labeling strategy just described, the pyridine nucleotide pools in this hamster liver were labeled in vivo by intraperitoneal injection of 35 mg [5- C] nicotinamide 5 hours prior to sacrifice. The bottom two spectra (2.6 min and 12.8 min) were obtained prior to addition of ethanol. They show resonances from labeled NAD., natural abundance glycogen and natural abundance choline methyl groups of phospholipids but no resonance from reduced pyridine nucleotides. After addition of 45 mM 10% [1- C] ethanol (at 17.9 min), resonances from C-1 of ethanol and NADH are detectable. These data demonstrate that the pyridine nucleotide pools labeled by intraperitoneal injection are metabolically active and that addition of 45 mM ethanol results in a marked change in the redox potential of the liver as measured by NMR. Furthermore, the observation of separate resonances for the oxidized and reduced pyridine nucleotides indicates that chemical exchange between oxidized and reduced forms is slow on the NMR time scale, and demonstrate that NMR may be used to quantitate the redox potential of free pyridine nucleotides in situ. Since the chemical shifts of carbon nucleiinthe nicotinamide moiety of the di-phosphopyridine nucleotides (NAD and NADH) are almost identical to the tri-phosphopyridine nucleotides (NADP. and NADPH), the intensities of the oxidized (NAD.) and reduced (NADH) resonances determine a mean reduction charge of the cells defined by 13


13

13

13

+


11.

BRAINARD ET AL.

169

"C-Enriched Substrates

C-2 C-2 C-2 Downloaded by SUFFOLK UNIV on January 20, 2018 | http://pubs.acs.org Publication Date: July 9, 1984 | doi: 10.1021/bk-1984-0258.ch011

NICOTINAMIDE

NICOTINAMIDE / C-2

NICOTINATE OTINATE

I

N

A

D

C-2 NAD.

LC

' 170-187

306-323

136-153

M..v.irv

, 0 2

wv/ -"

9

I

J.

272-289 STOPPED NICOTINATE ADDITION 238-255 ml η

34-51 min 160

150

140 ppm

160

130

150

140 ppm

130

Figure 5. Proton decoupled 13 NMR spectra of perfused l i v e r showing synthesis and degradation of the pyridine nucleotides from nicotinate. r



170


F i g u r e 6. Proposed p y r i d i n e n u c l e o t i d e c y c l e . A b b r e v i a t i o n s : NaMN, n i c o t i n i c a c i d mononucleotide; N a A D , n i c o t i n i c a c i d adenine d i n u c l e o t i d e ; and N A D , o x i d i z e d form o f nicotinamide adenine d i n u c l e o t i d e .


11.

l3


B R A I N A R D E T AL.

Cl ETHANOL

N A D +


171

23.0

MIN

.9 MIN NAD

+

(CH ) Ν 3

\

C6

Il

Cl

GLYCOGEN

3

6LYC06EN 1

I

l y

12,8

2.6 120

100

80

MIN

MIN

60

1-3

Figure 7 . Proton decoupled C NMR spectra of perfused l i v e r showing the effect of addition of U5 mM ethanol at 1 7 . 9 min on the C-5 resonances from NAD and NADH.



172

STABLE ISOTOPESINNUTRITION

Figure 8. Proton decoupled C NMR spectra of perfused liver showing estimated upper limit for the mean reduction charge as observed by C MR.


11.

173

l3

BRAINARD ET AL. C-Enriched Substrates

M R C

[NADH] + [NADPH]

= +

[NAD ] + [NADH] • [NADP] • [NADPH]

X =

RE

.RE . .0X

As noted previously no resonance from reduced pyridine nucleotides is detectable in well-oxygenated livers, in the absence of reducing substrates. From the signal-to-noise ratio in spectra of well oxygenated liver, an upper limit for the mean reduction charge measured by NMR can be estimated. Figure 8 shows such a spectrum, where the upper limit for the mean reduc tion charge is approximately 0.08. This estimate is five-fold lower than estimates for the MRC obtained using fluorescence spectroscopy and/or extraction techniques. However, in contrast to these techniques, NMR primarily measures metabolites in the cells which are not bound to enzymes. The differences between the reduction charge in the cell estimated from these in situ measurements may reflect the higher affinity that triphosphopyridine nucleotides have for enzymes and suggest that a large frac tion of the reduced pyridine nucleotides in the cell may be enzyme bound, and consequently do not give a high resolution NMR signal.


f

Acknowledgments This work was done under the Department of Energy.

auspices of the U n i t e d S t a t e s

Literature Cited 1. 2. 3. 4.

5. 6. 7. 8. 9. 10.

Eakin, R. T.; Morgan, L. O.; Gregg, C. T.; Matwiyoff, N. A. FEBS Lett. 1972, 28, 259-64. Den Hollander, J. Α.; Brown, T. R.; Ugurbil Κ.; Shulman, R. G. Proc. Natl. Acad. Sci. (USA) 1979, 76, 6096-6100. Walker, T. E.; Han, C. H.; Kollman, V. H.; London, R. E.; Matwiyoff, N. A. J. BIOL. Chem. 1981, 257, 1189-1195. Matwiyoff, Ν. Α.; London, R. E.; Hutson, J. Y. in "NMR Spectroscopy: New Methods and Applications"; Levy, G. C., Ed.; ACS SYMPOSIUM SERIES No. 191, American Chemical Society: Washington, DC, 1982; pp. 157-186. Iles, R. Α.; Stevens, A. N.; Griffiths, J. R. Progr. Nucl. Mag. Res. 1982, 15, 49-200. Hoekenga, D. E.; Brainard, J. R.; Hutson, J. Υ.; Matwiyoff, N. A. 2nd Annual Meeting, Soc. Mag. Res. Medicine, San Francisco CA, Aug. 1983, ABSTRACT, 159. Ackerman, J. J. H.; Grove, T. H.; Wong, L. G.; Gadiau, D. G.; Radda, G. K. Nature 1980, 283, 167-170. Alger, J. R.; Sillerud, L. O.; Behar, K. L.; Gillies, R. J.; Shulman, R. G.; Gordon. R. E.; Shaw, D.; Hanley, P. E. Science 1981, 214, 660-662. Gordon, R. E.; Hanley, P. E.; Shaw, D. Progr. Mag. Res., 1982, 15, 1-47. Sillerud, L. O.; Shulman, R. G. Biochemistry 1983, 22, 1087-1094.




174

11. Alger, J. R.; Behar, K. L.; Rothman, D. L.; Shulman, R. G. 2nd Annual Meeting, Soc. Mag. Res. Medicine, San Francisco, CA, Aug. 1983, ABSTRACTS, p. 1. 12. Neurohr, K.; Barrett, E. J.; Shulman, R. G. Proc. Natl. Acad. Sci. (USA) 1983, 80, 1603-1607. 13. Behar, K. L.; Petroff, O. A. C.; Alger, J. R.; Prichard, J. W.; Shulman, R. G. 2nd Annual Meeting Soc. Mag. Res. Medicine, San Francisco, CA, Aug. 1983. ABSTRACTS, 36-37. 14. Bottemley, P. Α.; Smith, L. S.; Edelstein, W. Α.; Hart, H. R.; Mueller, O.; Leue, W. M.; Darrow, R.; Redington, R. W. 2nd Annual Meeting Soc. Mag. Res. Medicine, San Francisco, CA, Aug. 1983. ABSTRACTS, 53-54. 15. Coleman, J. E.; Armitage, I. M.; Chlebowski, J. F.; Otvos, J. D.; Schoot Uiterkamp, A. J. M., in "Biological Applications of Magnetic Resonance"; Shulman, R.G., Ed.; Academic: New York, 1970; pp. 345-395. 16. Cohen, S. M., Glynn, P., Shulman, R. G. Proc. Natl. Acad. Sci. (USA) 1981, 78, 60-64. RECEIVED

February 9,

1984


Stable Isotopes in Nutrition - American Chemical Society

Recommend Documents