Assessment’of’metabolism’inthe’ placentaby’hyperpolarized’MRI’ · vævsanalyse fandt vi en signifikant høj mRNA ekspressionen af laktat dehydrogenase (LDH) i placenta

Assessment of metabolism in the placenta by hyperpolarized MRI

Emmeli Fredsgaard Ravnkilde Mikkelsen

20104874

Emmeli Fredsgaard Ravnkilde Mikkelsen 30/11 – 2015 p. 2 of 19


Table of content

RESUMÉ ............................................................................................................................................. 3

INTRODUCTION .............................................................................................................................. 4

PLACENTAL METABOLISM ................................................................................................................. 4

HYPERPOLARIZED MAGNETIC RESONANCE IMAGING ........................................................................ 5

MATERIALS AND METHODS ...................................................................................................... 7

ANIMAL MODEL ................................................................................................................................. 7

MRI MEASUREMENTS ........................................................................................................................ 7

TISSUE ANALYSES ............................................................................................................................. 8

RNA extraction, cDNA synthesis and qPCR ................................................................................. 8

LDH activity assays and NAD+/NADH ratio ................................................................................ 9

STATISTICS ........................................................................................................................................ 9

RESULTS ......................................................................................................................................... 10

DISCUSSION ................................................................................................................................... 15

REFERENCES ................................................................................................................................. 18



Resumé Introduktion: Hyperpolariseret magnetisk resonans (MR) skanning er en metode til at måle

metaboliske processer i væv. Formålet med dette studie var at undersøge hyperpolariseret MR

skanning som en metode til at vurdere metabolismen af [1-13C]-pyruvat i placenta og foster i en

drægtig chinchilla dyremodel.

Materialer og metoder: Vi inkluderede 4 gravide chinchillaer, hvoraf to dyr fik foretaget én

skanning, og to dyr fik foretaget to skanninger mellem dag 69 og 98 i graviditeten. Efter sidste

skanning blev dyret aflivet, og organer taget ud af både mor og foster til vævsanalyse.

Resultater: Hyperpolariseret [1-13C]-pyruvat ophobede sig signifikant i placenta, og vi så tilmed et

højt signal fra [1-13C]-laktat produceret i placenta. Vi observerede ikke noget signal fra [1-13C]-

alanin og 13C-bikarbonat i placenta eller et signal fra nogle af metabolitterne i fosteret. Ved

vævsanalyse fandt vi en signifikant høj mRNA ekspressionen af laktat dehydrogenase (LDH) i

placenta.

Konklusion: Dette studie viser potentialet for brugen af hyperpolariseret MR skanning til at

vurdere metabolismen af [1-13C]-pyruvate i placenta og foster. Hyperpolariseret MR skanning viste

sig at være en lovende metode til vurdering af metabolismen i placenta men en usikker metode til

vurdering af metabolismen i fosteret.



Introduction The placenta connects the fetal and the maternal circulation and is essential for normal growth and

development of the fetus. It transfers oxygen and nutrients from the mother to the fetus and carbon

dioxide and waste products from the fetus to the maternal blood. At the same time, the placenta acts

as an endocrine organ secreting vital hormones for the pregnancy. Furthermore, the placenta has a

high metabolic function to meet its own demands. Hyperpolarized Magnetic Resonance Imaging

(MRI) is a method for assessing metabolic processes in tissues in real time. We hypothesized that it

was possible to evaluate the metabolism of pyruvate in the placenta and fetus by the use of

hyperpolarized MRI. Thus the aim of this study was to explore hyperpolarized MRI as a method to

investigate the pyruvate metabolism in the placenta and fetus in a pregnancy chinchilla model.

Placental metabolism Placental transfer of nutrients occurs via diffusion or active transport. Glucose is the main fetal

energy source and passes the placenta via facilitated diffusion through GLUT receptors, primary

GLUT1 and GLUT3 [1]. In order to meet the enhanced metabolic requirements for glucose of the

growing fetus, the placental glucose transfer must increase during pregnancy. Despite the large fetal

demand for glucose, the placenta consumes about half of the glucose supplied itself, metabolizing a

great amount to lactate [2]. Lactate is delivered to the fetus, where it is utilized as an important

substrate for fetal growth and metabolism [3]. It has been known for a long time that the anaerobic

metabolism is very active in fetal life, but what exactly stimulates the placenta to produce lactate

from glucose under aerobic conditions is unknown. Though, it is most likely regulated by the

hormones of the somatotrophic axis [4]. In the fetus the lactate is presumably used either as a fuel

for energy metabolism or converted to glucose in the gluconeogenesis [5]. The placental uptake and

metabolism of glucose depends on many different factors and determinants as maternal supply, fetal

demands, hormones, growth factors, cytokines, placental blood flow, and placental size. An altered

placental glucose metabolism can influence fetal growth, and is seen in pregnancies with e.g.

intrauterine growth restriction (IUGR) [6-8], preeclampsia [9], and diabetes [10]. However there is

inadequate knowledge about the pathological mechanisms in the placenta connected to these

conditions.

Placental glucose transfer and metabolism has been studied for several years both in vitro and in

vivo. Most in vivo measurements have so far used the Fick’s principle based on the differences in

concentrations of glucose in the maternal artery, uterine vein, fetal artery, and umbilical vein [11].

Additionally, the infusion of a tracer can determine the consumption of glucose using Fick’s



principle [11]. Radioactive and stable isotopic tracers like [14C]- and [3H]-glucose have also been

used to study placental metabolism of glucose [12]. Glucose clamp techniques have been used to

provide insight into the regulation of glucose uptake, transport, and metabolism in the placenta and

fetus according to plasma glucose and insulin concentrations in the mother and the fetus [13, 14].

However neither of these methods is potentially useful in a clinical situation, as they are highly

invasive for both the mother and the fetus.

In a clinical situation the non-invasive ultrasound examination can assess indirect measurements of

abnormal pregnancies as fetal growth and amniotic fluid volume [15]. The addition of Doppler

ultrasound can detect abnormalities in the fetoplacental blood flow, but this method has also been

shown to fail detecting placental insufficiency if present [16]. Therefore a more specific

examination is needed for detecting abnormal pregnancies in a clinical situation.

In this study we introduce a novel, non-harmful method, hyperpolarized Magnetic Resonance

Imaging, which allows monitoring of metabolic processes in tissues in real time.

Hyperpolarized Magnetic Resonance Imaging Hyperpolarized Magnetic Resonance Imaging or Spectroscopy (MRI/MRS), is a relatively new

method, which in vivo can quantify cellular uptake and metabolism of a given substrate [17]. The

method uses different carbon 13 (13C)-labeled biological substrates, which are mixed with an

electron paramagnetic agent (EPA), and hyperpolarized in a polarizer with a low temperature and a

strong magnetic field [18]. After the substrate is polarized, it is dissolved and administered

intravenous just before the initiation of the MRS sequence. The hyperpolarization enhances the

MRI signal tens of thousand fold compared to conventional MRI [18]. This significant increased

sensitivity enables evaluation of fast metabolic processes and measurements of metabolites in very

small concentrations.

There are many potential useful substrates for hyperpolarized MRS. The glycolytic pathway can be

examined by the use of 13C-pyruvate and its conversion to the derivatives 13C-lactate, 13C-alanine, 13C-bicarbonate, and the metabolites in the Krebs cycle. Depending on which carbon atom in 13C-

pyruvate is labeled and hyperpolarized, one can quantitatively determine metabolites from different

metabolic pathways (figure 1). Pyruvate contains three carbon atoms, and if the first carbon atom is

marked with 13C, denoted [1-13C]-pyruvate, it is possible to detect the metabolites [1-13C]-lactate,

[1-13C]-alanine and 13CO2, which is in equilibrium with 13C-bicarbonate (H13CO3-). These pathways

are facilitated by the enzymes lactate dehydrogenase (LDH), alanine transaminase (ALT), and

pyruvate dehydrogenase (PDH), respectively. If the second carbon atom is marked with 13C,



denoted [2-13C]-pyruvate, it is possible to detect the metabolites in the Krebs cycle after conversion

to acetyl-CoA [19].

Figure 1: The metabolism of [1-13C]-pyruvate to [1-13C]-lactate, [1-13C]-alanine and 13C-bicarbonate (H13CO3

-), and [2-13C]-pyruvate to the metabolites in the Krebs cycle.

Hyperpolarized [1-13C]-pyruvate has so far mostly been used to address the metabolism in the heart

under different cardiovascular diseases [20], in cancers such as lymphoma [21], prostate cancer [22]

and brain cancer [23], and in the kidneys under different diabetic conditions [24-26]. Recently, the

method has also been used to examine the placental metabolism and nutrient transport in guinea

pigs [27]. The aim of this study was to explore hyperpolarized MRI as a method of assessing the

pyruvate metabolism in the chinchilla placenta and fetus by the use of hyperpolarized [1-13C]-

pyruvate.



Materials and methods

Animal model We established a pregnancy chinchilla model. In contrast to other rodents, the chinchilla has a long

gestation period for about 111 days and usually only gives birth to 1-2 cubs. The long gestation

period is a great opportunity to measuring the metabolism in the placenta throughout a longer

period of time. The limited number of cubs makes it more human like than e.g. pigs, guinea pigs,

and rats. The chinchilla has a hemomonochorial placenta of the labyrinthine type, where a single

layer of syncytial trophoblast is directly bathed in maternal blood [28]. As the placentae of the

human species are also hemomonochorial [29], the chinchilla placenta is more comparable to

humans than e.g. the pig and sheep placentae, which are epitheliochorial [30]. We received the

pregnant chinchillas around gestation week five. All animals had free access to water and food

throughout the study. The experiments complied with the guidelines for use and care of laboratory

animals and were approved by the Danish Inspectorate of Animal Experiments (authorization

number: 2014-15-0201-00318).

Before the scans the animals were anaesthetized with 2,5% sevoflurane in 2 L atmospheric air as

breathing gas. All animals were weighed and blood glucose levels measured from tail capillary

blood with a Contour blood glucose meter. To prevent dehydration, the animals got a subcutaneous

injection of 5 mL saline water and 3 mL isotone glucose after being anaesthetized. Tail vein

catheterization was performed and an isotone glucose infusion of 6 mL/h was administered for one

hour prior to the experiment. In the scanner room the temperature, respiration and saturation of the

animal were monitored throughout the whole experiment. The aim was to examine the metabolism

in the placenta and fetus three times during the gestation period, respectively around day 40, 70 and

100. After the last experiment, the animals were sacrificed and tissue was taken out to validate the

MRI results.

MRI measurements The MRI measurements were performed on a 3T GE HDx scanner (GE Healthcare) equipped with a 1H 8-Channel MR Cardiac Array Coil for anatomical scans and a 13C Helmholtz loop coil

(PulseTeq Limited, Surrey, UK) (ø = 20cm) for hyperpolarized MRS. The placenta and fetus were

localized with a standard 3-plane localizer sequence, and T2-weighted 1H anatomic images were

acquired in a coronal, axial and oblique view. 127 mg of [1-13C]-pyruvic acid was mixed with

15mM AH111501 and polarized in a 5T SPINLab (GE Healthcare, Brøndby, DK). The sample was



polarized for at least two hours until a reproducible polarization of more than 40 %. The final

concentration of [1-13C]-pyruvic acid in the sample was 90 mM. [1-13C]-pyruvate was discharged

from the SpinLab and a volume of 2 mL was injected into the tail vein catheter over a period of 15

sec. A slice-selective 13C IDEAL spiral sequence was used for hyperpolarized [1-13C]-pyruvate

imaging, acquiring images every 5th second initiated 30 seconds after the start of [1-13C]-pyruvate

injection. A 15º flip angle, 11 IDEAL echoes and one initial spectrum per IDEAL encoding,

TR/TE/ΔTE = 100 ms/0.9 ms/0.9 ms, FOV = 120x120 mm2, 5x5 mm real resolution, and an axial

slice thickness of 20 mm covering the placenta and a part of the fetus characterized the sequence.

Regions-of-interests of placenta, fetus, maternal muscle tissue, and noise were manually drawn on

the 1H anatomic images in OsiriX [31] (figure 3) and transferred to the 13C images. The ROI signals

were translated into a signal-to-noise ratio (SNR), by dividing the sum of the pyruvate signals with

the mean of the noise signal for each metabolite. The noise signal was calculated from a region

outside the animal. Furthermore metabolite ratios were calculated by dividing the sum of the

metabolite signals with the sum of the pyruvate signals.

Tissue analyses

RNA extraction, cDNA synthesis and qPCR Placental, renal and cardiac tissues from the mother as well as renal and cardiac tissues from the

fetus were used for analyzing the mRNA expression of LDHA1, PDH and ALT. First, RNA

extraction was carried out using a NucleoSpin RNA II kit (Stratagene, AH diagnostics, Aarhus,

Denmark). From the isolated RNA, complementary DNA (cDNA) was synthesized using a

RevertAid First strand cDNA synthesis kit (MBI Fermentas, Burlington, Canada). All procedures

followed the manufacturer’s instruction. 100 ng of synthesized cDNA was used as a template for

PCR amplification. qPCR was then performed using the SYBR Green qPCR Master Mix

(Stratagene, AH diagnostics, Aarhus, Denmark) according to manufacturer’s instructions. Primer

specificity of the products was verified by gel electrophoresis and melting curve analysis. The

following primer sequences were used: LDHA1: Sense 5’-GGT GGT TGA CAG TGC GTA TG-3’,

antisense 5’-TCA CAA CAT CGG AGA TTC CA-3’). ß-actin: Sense 5’-GAG ATG AAG CCC

AGA GCA AG-3’, antisense 5´-CTG GGT CAT CTT CTC ACG GT-3´. PDH: Sense 5´-TGG TCA

AAT TGA AAC AGG CA-3´, antisense 5´-TTT CAG TGA ATG TCC CCA CA-3´. ALT: Sense

5´-GCA GTC CTG ACT TCC CAG AG-3´, antisense 5´-AAG ATG TTG TTG GGG TCT GC-3´.



LDH activity assays and NAD+/NADH ratio Placental, renal and cardiac tissues from the mother as well as renal and cardiac tissues from the

fetus were dissected, instantly frozen in liquid nitrogen, and stored in -80°C. Stored tissue was used

for determination of LDH activity and NAD+/NADH ratio in each tissue type. LDH activity assay

kits were used according to the manufacturer’s instructions (Sigma-Aldrich, Brøndby, Danmark).

Briefly, tissue was homogenized and purified in LDH assay buffer prior to analysis. NAD+/NADH

quantification kits were used according to manufacturer’s instructions (Sigma-Aldrich, Brøndby,

Danmark). Stored tissue was homogenized and purified in NADH/NAD+ extraction buffer, and

filtered through a 10 kDa cutoff spin filter prior to the analysis. All analysis was performed on 96-

well costar half area plates in a PHERAstar FS micro plate reader (BMG labtech, Germany).

Statistics Data procession and statistical analyses were performed in PRISM 6. The SNRs from the different

ROIs as well as the LDH activity, NAD+/NADH ratio and mRNA expression of LDHA1, PDH and

ALT in the different tissue types were compared with an ordinary one-way ANOVA using multiple

comparisons with Fischer’s test. The lactate/pyruvate ratios throughout pregnancy were compared

with linear regression. p < 0.05 was considered as statistical significant.



Results Four pregnant chinchillas were included in the study. In total we performed six scans on the four

animals. The first MRI scan, which was planned to take place around day 40 of pregnancy, was not

carried out due to inadequate development of the fetus and the placenta. On gestational day 40 the

crown-rump length of the chinchilla fetus is less than 1 cm [32], which made it difficult to

distinguish the placenta and fetus with our setup. The planned second scan around day 70 of

pregnancy was performed on three chinchillas, and the third scan around day 100 of pregnancy was

carried out on three chinchillas as well. The missed second and third scan was due to technical

problems with the scanner and the SpinLab respectively. Animal characteristics of every scan

carried out are presented in table 1.

Gestational age Weight Blood glucose

before/after Temperature Respiration Saturation

Chinchilla 1 69 587.5 9.9/9.2 33.6 44 94 97 641.2 6.4/10.6 35.4 47 98

Chinchilla 2

80 655.3 11.6/5.1 36.1 40 100

98 687.5 11.7/3.1 34.6 46 100

Chinchilla 3

82 704.2 4.8/9.6 34.1 49 99

Chinchilla 4

96 716.3 6.3/16.1 35.7 52 96

Table 1: Information about gestational age (days), weight (g), blood glucose levels before and after the scans (mM), rectal temperature (°C), respiration frequency (/min), and blood saturation (%O2) of the mother for every scan carried out.

A characteristic MRI of a chinchilla fetus is showed in figure 2. An overlay of the metabolic maps

of [1-13C]-pyruvate and [1-13C]-lactate on axial 1H anatomic images showed a significant high

signal from [1-13C]-pyruvate and produced [1-13C]-lactate in the placenta (figure 3). During the

sequence both the lactate and pyruvate signal decreased as a result of T1 relaxation and an

irreversible destruction of the polarization from the radio frequency pulses. We did not observe any

signal from neither pyruvate nor lactate in the fetus, and no signal from alanine and bicarbonate

were observed in the placenta or the fetus.



Figure 3: Representable 1H images with overlaid metabolic maps of [1-13C]-pyruvate and [1-13C]-lactate.

Enclosed circles represent applied ROIs of the placenta, fetus, maternal muscle tissue and noise.

Figure 2: Oblique MRI scan of chinchilla fetus



By comparing [1-13C]-pyruvate SNRs from the placenta, fetus and maternal muscle tissue,

hyperpolarized [1-13C]-pyruvate was significantly supplied and accumulated in the placenta

compared to the fetus and maternal muscle tissue (figure 3+4).

We found no significant change in the lactate/pyruvate ratio between day 69 and 98 in the 111 days

gestation period (p=0.17) (figure 5). The lactate/pyruvate ratio for the two chinchillas, we managed

to scan twice in pregnancy, both decreased from 0.16 on day 69 of pregnancy to 0.113 on day 97 of

pregnancy (chinchilla 1), and from 0.22 on day 80 of pregnancy to 0.109 on day 98 of pregnancy

(chinchilla 2), respectively.

Figure 5: The placental lactate/pyruvate ratio on different days of pregnancy. Chinchilla 1 (!), chinchilla 2 (n), chinchilla 3 (u), chinchilla 4 (p).

Figure 4: Signal-to-noise ratios (SNRs) for hyperpolarized [1-13C]-pyruvate in the placenta, fetus and maternal muscle tissue. nplacenta = 6, nmuscle = 6 and nfetus = 3. *p<0.05.

60 70 80 90 100 1100.0

0.1

0.2

0.3

Days of pregnancy

Lact

ate/

pyru

vate

rat

io R2 = 0.42

Placenta Muscle Fetus0

50

100

150

200SNR

*



Animal characteristics at the day of tissue sampling are presented in table 2. The weight of the

different organs (mean ± SD) were as follows: Maternal kidney: 1.75 g ± 0.06, maternal heart: 1.53

g ± 0.17, fetal kidney: 0.15 g ± 0.06, fetal heart: 1.15 g ± 0.06, and placenta: 5.15 g ± 2.2.

Animal Gestational age

Placental weight

Fetal weight

Mother weight

Number of fetuses

Chinchilla 1 97 4.2 37.5 641.2 1

Chinchilla 2 98 4.3 33.5 687.5 1

Chinchilla 3 103 8.4 59.7 751.7 1

Chinchilla 4 96 3.7 27.7 716.3 2*

Table 2: Gestational age (days), number of fetuses and weight (g) of placenta, fetus, and mother at the day of tissue sampling. *Only one placenta and fetus was analyzed in the chinchilla carrying two fetuses.

Tissue analyses showed a significantly higher LDH activity in the maternal kidney and heart

compared to the fetal kidney and placenta (figure 6). The NAD+/NADH ratios were approximately

the same for all tissue types, and only the NAD+/NADH ratio in the maternal kidney was

significantly lower than the fetal kidney (figure 7). The transcription of LDHA1, PDH and ALT

were in general significantly lower in the fetal heart compared to all other organs, while the fetal

kidney had the same transcription of enzymes as the maternal organs and placenta (figure 8, 9 and

10). The placental mRNA expression of LDHA1 was significantly higher than the expression of

PDH and ALT (figure 11).

Placen

ta

Matern

al kid

ney

Fetal k

idney

Matern

al hea

rt

Fetal h

eart

0

10

20

30

40

50

LDH

act

ivity

U/m

l

****

*****

Figure 6: Lactate dehydrogenase (LDH) activity in U/ml, indicating a general high LDH activity in maternal tissue compared to the fetal kidney and placenta (n=4). *p<0.05 placental comparisons, **p<0.05 fetal and maternal inter-comparisons, ***p<0.05 fetal and maternal intra-comparisons.

Placen

ta

Matern

al kid

ney

Fetal k

idney

Matern

al hea

rt

Fetal h

eart

0

1

2

3

NA

D+/

NA

DH

ratio

**

Figure 7: NAD+/NADH ratios, showing approximately same ratios in all tissue types (n=4). **p<0.05 fetal and maternal inter-comparisons.



Placen

ta

Matern

al kid

ney

Fetal k

idney

Matern

al hea

rt

Fetal h

eart

0.6

0.8

1.0

1.2

LDH

A1/

ß-ac

tin ra

tio

******

**

Figure 8: mRNA expression of lactate dehydrogenase (LDHA1). nplacenta=4, nmaternal

kidney=4, nfetal kidney=4, nmaternal heart=2, nfetal heart=4. *p<0.05 placental comparisons, **p<0.05 fetal and maternal inter-comparisons, ***p<0.05 fetal and maternal intra-comparisons.

Placen

ta

Mater

nal kid

ney

Fetal

kidney

Mater

nal hea

rt

Fetal

heart

0.5

0.6

0.7

0.8

0.9

1.0

ALT

/ß-a

ctin

rat

io

******

**

Figure 9: mRNA expression of alanine aminotransferase (ALT). nplacenta=4, nmaternal


Placen

ta

Matern

al kid

ney

Fetal k

idney

Matern

al hea

rt

Fetal h

eart

0.55

0.60

0.65

0.70

0.75

0.80

0.85

PDH

/ß-a

ctin

ratio

*

*

***

*****

Figure 10: mRNA expression of pyruvate dehydrogenase (PDH). nplacenta=4, nmaternal


LDHA1PDH

ALT0.6

0.7

0.8

0.9

1.0

Enzy

me/

ß-ac

tin ra

tio *

Figure 11: Placental mRNA expression of lactate dehydrogenase (LDHA1), alanine aminotransferase (ALT), and pyruvate dehydrogenase (PDH), indicating a high anaerobic metabolism in the placenta (n=4). *p<0.05



Discussion Hyperpolarized [1-13C]-pyruvate significantly accumulated in the placenta compared to the fetus

and maternal muscle tissue. In the placenta, a significant amount of [1-13C]-pyruvate was converted

to [1-13C]-lactate, and the placental metabolism of pyruvate to lactate had no significant change

during the last half of pregnancy. In our experiments we did not observe any signal from [1-13C]-

alanine or 13Cbicarbonate in the placenta as well as any signal in the fetus. The LDH activity in the

fetal kidney was significantly lower compared to the maternal organs, despite their transcription of

LDHA1 was approximately the same. On the other hand the transcription of LADH1 in the fetal

heart was lower compared to the maternal organs despite any difference in their LDH activities. The

placental LDH activity was lower than in the maternal organs, and its transcription of LDHA1 was

significantly higher than the transcription of PDH and ALT.

Hyperpolarized MRI seems promising for quantitative validation of placental metabolism. The MRI

scans showed that placenta had a great supply of hyperpolarized [1-13C]-pyruvate due to its high

signal. As we did not observe any signal in the fetus, it is doubtful whether the method is able to

examine fetoplacental transport and fetal metabolism. The significant signal from [1-13C]-lactate in

the placenta is consistent with other in vivo studies, and supports the hypothesis that a lot of the

glucose delivered to the placenta is metabolized to lactate [3]. [1-13C]-pyruvate has been the most

used substrate for hyperpolarized MRS to date due to its key role in the glycolytic pathway and its

long relaxation time, in general 2 min. As the lactate/pyruvate ratio is a reflection of the LDH

activity in the placenta, the amount of produced [1-13C]-lactate depends on the amount of [1-13C]-

pyruvate delivered to the cells, the rate of [1-13C]-pyruvate transport across the cell membranes, the

cellular concentration of LDH enzyme, the cellular concentration of the co-substrate NADH, the

cellular pool size of already existing pyruvate and lactate, and the intracellular pH [33]. The high

LDH activity in the maternal organs compared to the fetal kidney suggests that the mother in

general has a higher anaerobic metabolism than the fetus. The low LDH activity in the fetal kidney

indicates that the anaerobic metabolism is not yet that active despite a high transcription of LDHA1.

The high NAD+/NADH ratio indicates that the rate-limiting step in the reaction is lack of co-

substrate (NADH). From the literature, we know that the fetus receives most of the lactate needed

for energy metabolism from the placenta [3]. This results in a large lactate pool, which can also be a

rate-limiting step for the anaerobic metabolism in the fetal kidney. The fetal heart was in the

opposite position with a high LDH activity and a low LADH1 transcription. This indicated a low

lactate pool in the cardiac cells, stimulating high enzyme activity despite low transcription of



LADH1. The placental mRNA expression of LDHA1 was high compared to PDH and ALT, which

was in accordance to our MRI results showing a significant signal from [1-13C]-lactate and no

signal from 13C-bicarbonate and [1-13C]-alanine. This also indicated a higher anaerobic than aerobic

metabolism in the placenta as shown in other in vivo studies.

Recently, a similar study by Friesen-Waldner et al. [27] tested the feasibility of hyperpolarized [1-13C]-pyruvate MRI for noninvasive examination of fetoplacental metabolism and nutrient transport

in guinea pigs. In contrary to our proposed single fetal-placenta model, the Friesen-Waldner study

used a multiple fetal-placenta model observing pyruvate and lactate signals in 30 placentae and fetal

livers from seven guinea pigs. Our experiments corroborate the findings of significant amounts of

[1-13C]-pyruvate and [1-13C]-lactate in the placenta, however we did not observe any signal from

the fetus during the 2 minutes of polarization. This discrepancy is believed to be due to the

difficulty to differentiate fetal tissue from surrounding tissue in this multiple fetal-placenta model

with a litter size of 4-5 cubs per guinea pig. Our animal model only contained 1-2 cubs per

chinchilla, which gave us a higher resolution in order to distinguish the signals from the placenta

and the fetus. Similar to our experiments the Friesen-Waldner study did not observe any signal from

[1-13C]-alanine or 13C-bicarbonate.

The well-saturated animals during the whole experiment were a great strength of our study. The use

of sevoflurane for sedation gave us the opportunity for quick induction and recovery from

anesthesia. Our animals were not stressed by surgical interventions, although the use of anesthesia

might have influenced the results. For example, sevoflurane has been shown to produce

vasodilatation of the feto-placental vasculature [34], but it has not yet been shown to cause any fetal

damages. A special advantage for in vivo measurements is the fact that the experiments can be

conducted many times during the gestation period, and that pupping can occur naturally if needed.

Also it is possible to measure the placental function when it is still influenced by maternal and fetal

factors as supply of hormones, metabolic substrates, growth factors, etc. [11]. However the most

important advantage for this in vivo experiment compared to other in vivo studies of the placenta is

that hyperpolarized MRS is a non-harmful imaging modality using non-ionizing endogenous

substrates for interrogating the metabolic signature of placenta. Provided that the method is

sufficiently established, it will be possible, in the future, to ethically apply it on pregnant women

most likely without teratogen concerns. However the safety of using this method on pregnant

women has not yet been confirmed.



This study also faces several limitations. One of the main limitations is our small study population,

especially when doing the statistical analyses. Furthermore, our coils for anatomical scans and MRS

are both made for human use. Therefore the sensitivity in a small animal like the chinchilla was in

risk of being too low. This could be the explanation of the lacking signal from [1-13C]-alanine and 13C-bicarbonate. Additionally, we scan the placenta as a whole structure without distinguishing

between the different cell types in the placenta. Therefore we cannot assure that the tissue analyzed

is fully trophoblastic. The placenta consists of syncytiotrophoblasts, cytotrophoblasts, mesenchyme

cells, and endothelial cells [35], which are all included in our analysis. However this seems to be a

minor quantitative problem.

Our study only considered normal pregnancies, but recently ordinary in vivo MRS has been used on

pregnant women to validate the placental metabolism during pathological conditions [36]. For

example has 31P MRS been used to study the phospholipid placental membrane metabolism in

normal and preeclamptic pregnancies [37], and 1H MRS has been used to assess the placental

choline/lipid ratio in pregnancies with placental insufficiency and IUGR [38]. Both studies reported

altered placental metabolism during preeclampsia and IUGR respectively. It is therefore supposed

to be possible also to characterize the metabolism in the placenta by hyperpolarized MRS during

different pathological conditions as IUGR, preeclampsia, and diabetes. Although it is problematic to

make interventions on a dysfunctional placenta during the pregnancy, it is of high value to have the

ability of examine the metabolism in order to suggest new regimes for treatments.

In conclusion we have introduced hyperpolarized MRI as a potential method for non-harmful

assessment of the in vivo metabolism in the placenta. The method is promising for clinical use of

diagnosing pathological conditions in the placenta. However more studies need to be done to

definitively establish the method and to examine if the method can be used to assess the metabolism

in the fetus.



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