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THE

EFFECTS

OF

ANOXIA

ON

THE

METABOLISM

OF

HUMAN

FETAL

TISSUES

By Claude A. Villee, Ph.D., Dwain D. Hagerman, M.D., Nils Holmberg, M.D.,

John Lind, M.D., and Dorothy B. Villee, M.D.

Departments of Biological Chemistry and Pediatrics, Harvard Medical School, Boston Lying-in Hospital

and Massachusetts General Hospital, and Wenner-Gren Research Laboratory and

S#{246}draBarnb#{246}rdshuset, Stockholm, Sweden

\Vith the assistance of Janet M. Loring, A.B., armd Frederica M. \Vellington, A.B.

(Accepted May 9) 1958; sui)mitte(l March 26.)

Aided by a grant fromu time Association for time Aid of Crippled Children amid by a grant from time

Cimarles A. Kimig and Marjorie King Fund.

ADDRESS: (CAy.) I)epartrnent of Biological Chemistry, harvard Medical Scimool, Boston 15,

Massacimusetts.

PEDIATRICS, November 1958

T

HE ABILITY of time newborn mammal

to) witimstand seyere hypoxia is well

estahlished.1 Newborn rats and mice, or

fetuses obtained by cesarean section shortly

before birtim, survive at least 45 to 50

mm-utes wimen placed in an atmosphere of pure

nitrogen. In contrast, time newborn guinea

pig, born in a more “mature” state, is killed

by 7 minutes of anoxia. The ability to

sur-vive prolonged deprivation of oxygen

de-chines rapidly after birth and disappears

after time first few weeks of life. Newborn

rats injected with cyanide and then placed

in an atnmosphere of nitrogen survived the

anoxia as well as time uninjected controls.3

In contrast, newbormm rats injected with io)doacetic acid or witim fluoride had a

pro-foummdiy decreased survival time when

placed in an atmo)spimere of nitrogen.’ Time

latter animals did not show the markedly increased concentrations of lactic acid in

tissues and blood fotmnd in the uninjected controls placed in aim atmosphere of

nitro-gen. Cyanide is known to combine with time

iron atom of the cytochromes and timus pre-vents the transfer of electrons from sub-strate molecules to oxygen. Fluoride forms

a complex with magnesium ions and will

inhibit to some extent any enzyme such as

enolase which requires magnesium as a

co-factor. lodoacetic acid inhibits a variety of

dehydrogenases, especially

3-phosphogly-ceraldehyde dehydrogenase. Himwich and

his collaborators concluded from these

ob-servations that time resistance of the

new-born to hypoxia is independent of the

cyto-chrome system and is in part dependent

upon the energy derived from accelerated

glycolysis. Enohase and

phosphoglyceralde-imyde dehydrogenase are important

mem-bers of time glycolytic cycle.

There has been wide interest in the nature

of the metabolic mechanisms which

en-able the fetus to survive periods of hypoxia

or anoxia that would be fatal to the adult.

Several theories have involved the

postula-tion that fetal tissues have some special

patimways of “anaerobic metabolism” which

enable them to survive and a number of

inFerences have been drawn from this idea.

The term anaerobic metabolism is

non-specific and simply refers to any of a host

of reactions which can proceed in the

ab-sence of molecular oxygen. R#{228}ih#{228}5has

sug-gested timat the fetus may obtain oxygen

l)y converting carbohydrate to fat:

17C,;H,2O; 2C1H9SO6 + 4H2O + 4303,

but there is no reaction known in

mam-mahian tissue in which molecular oxygen is

evolved. The over-all conversion of

glu-cose to tnipalmitin may be represented as

25C6H,206 + 502 2C51H9806 + 48C0,

+ 52H20. The conversion of glucose to fat

does not evolve oxygen which could be

utilized in other metabolic processes.

How-ever, hydrogen given off in the

dehydro-genation reactions in glycolysis or

else-where could be utilized for the synthesis of

(2)

unit, acetyl coenzyme A. In this sense the

synthesis of fat provides a “hydrogen sink,”

a depot for hydrogen atoms other than their

combination with oxygen via the

cyto-chrome system to form water. These reac-tions could make available to the fetus all the energy derivable from glycolysis

with-OUt time 1)roltmctiomm of lactic acid.

Human fetal tissues incubated

anaerobi-cahly in vitro produce carbon dioxide and

utilize more glucose and pynuvate than can l)e accounted for by time lactate produced.”

Pyruvate under anaerobic conditions may

enter into metabolic reactions other than

the formation of lactic acid and these

reac-tions may penhmaps contribute to the survival

o)f time organism. Whemm newborn rats were

injected with pyruvate labeled with

radio-active carbon and then placed in an

at-mosphene of oxygen or nitrogen,

anaeno-biosis immduced a marked rise in time

con-ceimtration of lactic acid in time blood, and

timere was rapid utilization of liver glycogemm,

but the synthesis of fatty acids from time

labeled pyruvate occurred at equal rates

aerobically ammd When liver

slices from fetal rats near term were

in-cubated in vitro with pyruvate-2-C14 or

acetate-2-C’t, labeled fatty acids were

syn-thesizeci at about equal rates in the

pres-ence or absence of oxygen.s

Analyses of the content of oxygen of fetal

i)loOd have indicated that time supply of

oxygen to time fetus may be decreased in the

last month or two of gestation.#{176} An

exten-sive study o)f tissue metabolism at different

stages of development in the rat revealed

that at 15 to 17 days of gestation the pattern of metabolism in the liver was quite

differ-(1it from time pattenim that obtains at 21

days, just before birth.’” The liver of the

fetal rat at tenmmm is cimaractenized by a imigh

content of glycogemm, a high rate of activity

of one or more enzymes in the ghycolytic cycle, which is rate-limiting in the adult and earlier in gestatioim, and a high rate of lipid symmthesis from small mnohecuhes, whicim

is deimiomistnated e(uahhy vell under

anaero-bic and aerobic conditioims imm vitro. It was

postulated that the high rates of ghycolysis

and of lipogeimesis represent an adaptation,

an adaptive change in enzyme activity or

amount, to the low oxygen tension which prevails in fetal tissue during the latter pant of gestation. Time rapid changes in the

pattern of metabolism which occur in time first 24 hours after birth may represent a

new adaptation at the enzyme level to the

higher oxygen tensions of tissue prevailing after birth.

The expenimemmts presented Imere were

undertaken to determine the metabolic

patterns of human fetal tissues incubated

aerobically and anaerobically and to look

for any qualitative or quantitative

differ-ences between the fetal and corresponding

adult tissues whicim might explain the

ob-served resistance to imypoxia.

METHODS

Samples of liver, braimi, heart, lung, kidmmey

ammd skeletal muscle were obtaimmed from fe-tuses within a few mimimmutes after delivery. The

fetuses were removed frommm the uterus eitimer by

a mimmiature cesareamm section or by vagimmal

hys-terotomy. The tissues were placed imm ice-cold

0.9% solutioi#{236} of sodium chloride and trans-ferred to the laboratory. The tissues were

sliced with a Stadie-Riggs microtome ammd an ahiqimot was takeim to be analyzed for commtemmt

of gh’cogemm. The slices of tissue were weighed omm a torsion balammce ammd placed in Warburg vessels in 3.0 ml of a medium containing, per

mmmihhihiter, 40 mole K, 80 mohe Nat, 10 mohe Mg, 100 .mohe Cl and 40 smnoie of phosphate, witim amm initial pH of 6.9. The

dium contaiimed the appropriate C ‘-labeled substrate : ummiformly labeled glucose or

fruc-tose, glucose- 1 -C1 , glucose-6-C1 ‘,

pyruvate-2-1 4, p\ruvate- 1 4, 2 , glycerol- 1-C14, or glcerol-2-C .1 4 The sohutiomm contaimming

the C”-habeled substrate was placed in the side arm of the Warburg flask amid tipped into)

the maimm reactiomm chamber after the vessels

were gassed with nitrogemm or oxvgemm for 7

mimmutes. The final concemitration of the C’4-labeled substrate in the immcubatiomm medium,

unless stated to be otherwise, was 10 .&moles

pen nml. The slices were immcubated for 1 imour at 37#{176}C.

After the iimcubatiomm period time slices of

tis-sue were remnoved, blotted dry omi filter paper,

(3)

chloroform-ARTICLES

mimethanol for extraction of lipids by the Ilmethod

of Foich.’’ The total extract of lipids was

evap-orated to dryness almd weighed ammd amm aliquot was transferred to a stainless steel plammcime’t

for C’4 analysis. Aim ahiquot of the tissue slice

after incubation was analyzed for contemmt of

glcogemm. Tissues were hydrolyzed in boilimmg

30% potassium hydroxide. The glycogelm was precipitated i)\ ethanol ammd sodiimmmm sulfate. purified, hydrolyzed to glucose and time

calm-temmt of glucose was estimated by the niethod

of Nelsomm.12

Alio1uots of the immctmbatiomm nlediumlm vere

analyzed for glucose, p\.ri.Iyate,I:l aml(l lac-tate’4 To other aliquots O)f time immclmi)ation medium, carrier glucose or pyru’ate was added

amid glucose phemmvlosazommes or pyrtmvtte

dini-trophemmlhvdrazommes were prepared and pun-fled for C’ ammalyses.’ Time respiratory carbon

dioxide was trapped jim alkali placed in the center well of the Warburg flask (0.2 ml of 5%

carbon dioxide-free solution of sodium by-droxide.) After time incubation this so)lntio)II

was ashed immto centrifuge tubes commtaining

water ammd the carbon (hioXide was precipitated as barium carbonate i)\ the additiomi of barium

chloride at pH 10.5. The barium carbommate vas wasimed witim water aIl(I ethammol ammd

trans-fenred to stainless steel piamichets for C’ ammal-ysis. Analysis for content of C’ was nmade

with a \vimmdo)\vless, propo)rtio)nal flow conmiten

amid corrected for self absorption.’ Each

saum-pie was cO)ummted lomig enough to reduce the

pro)i)ai)le error of coummting to less timami 3%.

RESU LTS

Glucose and Acetate Metabolism by Fetal

and Adult Human Liver

The statemnent is conmnmonly made that fetal tissues are characterized by a imigim rate of aerobic glycohysis. To test this iii

humamm tissues, slices of liven obtained from normal adult patients at cimolecystectomy

were inclIl)dte(l under coimditions identical

with those in which fetal liver slices were

incubated. The results of experiments in

which uniformly labeled glucose or

acetate-2-C’4 was present in the medium are sum-manized in Table I. The fetal liven came

from specimens of about 20 weeks

gesta-tional age. The ratio of vet weight to dry

weighmt for fetal and adult liver is about the

same at this age. There were 12

expeni-inemits with adtmlt liver and 12 exl)eniflmelltS with fetal liver, incubated in oxygen and in

nitrogen. The respiratory quotient for the

adult liver in these experiments was 0.95;

time respiratory quotient for the fetal liver

was well above unity, 1.31. This immdicates

that syntlmesis of fatty acids was occurring

at a rapid rate. Time nmeamm consumption of

oxygen bY fetal an(1 iohtilt liver slices proved

to be time same, 28 unoles of oxygen/gmn wet

tissue/hr. Time glycogen co)mmtent of the fetal

liver at timis age was less than that of time

adult liver. Later in fetal life the liven imas

a much higimer content of glycogemm. More

glycogen is tltiliZe(h during time incubation

period by adult liver timan by fetal liven

tmmmder botim aerobic and ammaenohic

condi-tions. This appears to be a neflectiomm of the

greater amount present initially.

The productiomm of glucose by the slices refers to the release into the incubation

mmmednmm of glucose arising fronm glycogen

wthmimm time tissime or by gluconeogemmesis

from pyruvate or from amino acids. This is

estimated by incul)ating time tissue imm

C’’-labeled glucose aimd measuring the dilution

of time radioactive glucose by time unlabeled

glucose 1)rocltIcecI by time tissue. The amount

of glucose produced is a measure of the

activity o)f the enzyme

glucose-6-phos-j)hatase vimicim converts intracellular

gIn-cose-6-pimospimate to glucose. It had heemm

shown previoush’ that

giucose-6-phos-pimatase is not present in fetal liver earlier

in development. It appears gradually and at

20 weeks is still in the process of increasing

its activity. Time fact that rate of

produc-tion of glucose is much lower immfetal liver timan in adult liver is a reflection of the lesser

activity of glucose-6-phosphatase in fetal

liven at timis age.

The PrcltIcti01m of lactic acid iii oxygen

occurs at similar rates in fetal liver and

adult liven, both in these experiments and

in comparable experiments with fetal and

adult rat liver.” Fetal liver is not

charac-tenized by a imigim rate of aerobic glycolysis. Time fetal liven is characterized, however, by

(1 higim rate of I)nt1ctin of lactic acid

(4)

pro-Fetal Liner incubated in Adult Lirer incubated in

Oxygen Nitrogen Oxygen Nitrogen

Substrate : Glucose-C’4, Uiformly Labeled

R.Q. 1.31 - (1.95

Oxygen consumption 8 .4 - 8 .4

Glycogemi utilization -54 -68 - 114 - 155

Glucose production +13 +14 + 50 + 73

Glucose utilization 0 - 1’2 - 15 - 17

Lactate production +17 +5 + 1 + 38

Conversion of carbon of glucose to

lipid 0.23 0.03 ().15 0.11

Conversion of carbon of glucose to

carI)on (lioxide 0 .41 0.08 1).2() 0.It)

Substrate : Acetate-2-C’4

R.Q. 1.19 - 0)96

-Oxygen eomssumption 8 .4 - 31).I

-Glycogen utilizatiom1 -40 -5’2 -114 -116

Lactate production +16 +40 + 18 + 34

Conversion of carbon of acetate to

lipid 1.4’2 1)08 0.042 0.0065

Conversion of carbon of acetate to

carbon dioxide 0.66 0.065 0. 45 0.074

Initial content of glycogen: Fetal liver, I 10 miioles/gm wet tissue.

Adult liver, 0O pnmoles/gnm wet tissue.

All values except R.Q. are expressed as mo1e/gm tissue/hr.

Values are eXpresse(l as /.Lmnole/grn tissue/hr.

TABLE I

METABOLISM OF HuI.N LIVEII SLICES IN \ITHO

duces three times as much lactate under

anaerobic conditions as it does under

aerobic conditions, while the increase in

adult liver is somewhat less than 100%. This

greater increase in production of lactic acid by fetal liven in response to anaerobiosis is a major difference from the adult tissue.

The results obtained with acetate as the

substrate are entirely similar to those in

which glucose was used (Table I).

Estimates of the Rate of Glycolysis in

Human Fetal Liver

The possible importance of glycolysis in

permitting survival during hypoxia was

shown by the experiments of Himwich and

his colleagues. 2- There are several ways of

estimating the rate of glycolysis; the

sim-plest and commonest metimod is to measure

the amount of lactic acid produced. In

these experiments the production of lactic

acid was 18.9 .moles/gm of tissue/hr in

oxygen and 51 tmoles in nitrogen, a 2.7-fold

increase (Table II). Lactic acid can come

from substrates other than glycogen and

glucose and, at least under aerobic

condi-tions, can be metabolized further. Measur-ing production of lactic acid is not the best estimate of the activity of the enzymes of the Embden-Meyerhof glycolytic cycle.

Alternatively, one can measure the

amount of pyruvic acid produced by tissues

by incubating a tissue slice in a medium

TABLE II

ESTIMATES OF RATE OF GLYCOLYSIS IN

Ht-MAN FETAL LIvER

Incubated in

Oxygen ‘sitrogen

Ratein N

Rate in 02

Lactate pro(lUCe(l 18.9 51 .‘3 .7

Pyruvate pro(Iuce(I l .I ‘39.0 1 .9

Conversion of carbon of

(5)

TABLE III

RATE OF LIPOGENESIS BY HuMAN

FETAL LIvER

Incubated in

Oxygen Nitrogen

ARTICLES

containing C’4-iabeled pyruvic acid and

measuring the dilution of the radioactive

pyruvate as unlabeled pyruvic acid is

pro-duced. In timese experiments 21 p.moles of

pyruvate were produced per hour per gram

of tissue in oxygen and 39 p.moles in

nitro-gen. Because pyruvic acid is produced from

substrates other than glycogen and glucose

and is metabolized to) many other

sub-stances, timis method of estimating the rate

of glycolysis suffers from the same

draw-backs as time measurement of lactic acid.

Perhaps the most direct measure of the

rate of ghycolysis is made by placing

C’4-labeled glucose in the incubation medium,

letting the slices metabolize this compound to pyruvic acid and isolating the pyruvic

acid at the end of the incubation period as

the pyruvate dinitrophenyihydrazone. This product is then recrystallized and its

radio-activity determined. In these experiments,

0.83 .&moles of glucose/gm of tissue/lmr and

3.1 imoles of glucose/gm of tissue/hr were

converted to pyruvate in oxygen and in

nitrogen, respectively. Thus, there is a

four-fold greater rate of ghycolysis in nitrogen

than immoxygen in human fetal liver midway through gestation. Time relatively small

ab-solute amount of glucose converted to

pyruvate compared to the total amount of

pynuvate produced reflects the fact that under time conditions of these experiments,

glycogen rather timan glucose is the chief

glycolytic substrate.

Lipogenesis by Fetal Liver

Slices of fetal liver were incubated with

a variety of C’4-iabeled substrates and the

rate at which the stmbstrate was converted

to fat was calculated from the radioactivity data (Table III). These data show timat witim

all of the substrates tested, the rate of

synthesis of fatty acid was much less under

anaerobic than aerobic conditions in this

stage of gestation, about 20 weeks. The

most active lipid precursors, as one might

expect, were pyruvic acid and acetic acid.

These substrates were converted to fatty

acids nearly 20 times faster in oxygen than in nitrogen. Thus, in comparing aerobic

Glucose-U-C’4 OH 0.01

Glucose-I-C’4 0.1’ 0.05

Glucose-6-C’4 0.1 0.03

Fruetose-IJ-C’4 0.17 0.03

Pyruvate-2-(”4 1.18 0.07

Pyruvate-1-C’4 0.03 0.002

Aeetate--(”4 I.35 0.07

(;iycerol--c’4 0.15 0.06

Citrate-1,5-C” 0.58 0.048

All values are expressed as )snmole of substrate

con-verted to hipid/gnm tissue/hr.

and anaerobic metabolism, giycohysis is

in-creased fourfold but hipogenesis is

de-creased ten- to twentyfold at this stage of

development. The carboxyl carbon of

pyruvic acid (pyruvate-1-C’4) is removed in

the conversion of pyruvate to acetyl

co-enzyme A. Thus, one would expect the

rate of incorporation of carbon-i of

pyniivate into fatty acid to be much less

than that of carbon-2. This was borne out

by time experiments. Some of the carbon

atom 1 became incorporated into fat by

an indirect metabolic route. The carbons of glucose and fructose are incorporated into

fats at about the same rate.

The fat that is synthesized in fetal tissue might be simply in a depot on it might be

utilizable by the tissues. Radioactive lipid that had been made by one sample of fetal liven was isolated, purified and used as the

substrate in a second experiment. Liver

slices from a second fetus were incubated

with this radioactive fat as the sole

sub-strate. These experiments showed that the

liven could metabolize the radioactive fat

to carbon dioxide quite rapidly. In further experiments, the radioactive fat was

sepa-rated into phosphohipid, neutral fat and

cholesterol. Some of the neutral fat was

used as substrate in a succeeding

(6)

‘rABLE V

RATE OF CONVERSION OF GLUCOSE TO CARBON DIOXIDE BY SLICES OF FETAL LmvER

Tissue

Oxygen

Liver 1 .35

Lung 0.1

Brain 0.094

Kidney 0.058

Heart 0.015

i)iaphragm 0.016

Nitrogen

0.07

0.015

0.0014

0.0O2

0.07

0.010

Au values are expressed as LII1ole of acetate uom,verted to Iipi(I/gnm of tissue/hr.

Timis experiment simows that time enzymes required for the oxidation of lipids to

car-hon dioxide are present at this stage of

de-velopment and are able to function, but it

does not prove that these reactions proceed

in vivo.

Lipogenesis in Other Fetal Tissues

In anotimer series of experiments the

lipogenetic activities of otimer fetal tissues

were compared (Table IV). The synthesis

of liI)i(IS WUS much more rapid in time liver

than in any other organ tested. The rate in

lung and brain is about one-tentim that of

the liver, and the rate of synthesis by the

heart and skeletal muscle is even lower. In all of these tissues except the heart, the rate

of lipogenesis in nitrogen is much less than

time rate in oxygen. Lipids are synthesized

1)y the heart at a very low rate in either

oxygen or nitrogen but the two rates are

comparable. This is one of the several ways in wimich the imeart differs markedly from other tissues.

Estimates of the Activity of the Hexose

Monophosphate Shunt in Fetal Tissues

A series of experiments was done in

which alternate slices of tissue were

incu-1)ated in glucose labeled at either position

1 or 6 of the molecule. By measuring the

rate at which C’4 appears in the respiratory

carbon dioxide it is possible to obtain an

estimate of what fraction of the glucose is

metabolized by way of the glycolytic

path-‘FABLE I\

RATE OF Ill’OGENESlS FROM cC’

incubated in

Jacob ated in

Oxygen Nitrogen

Glucose-U-C’4 0.31 0.032

(;lucose-6-C’ (1.19 0.0074

Glucose-1-C’ 0.45 0.057

All values are expressed as nuole of glmcose (onverted to #{237}arhoml dioxide/gui tissue/hr.

way and how mucim is Immetabolized by way

of the hexose monophosphate simunt. The

amount of glucose-i-C” that is converted

to carbon dioxide is takeim as a rough

esti-mate of the total anmount of glucose

metabo-lized to carbon dioxide by both pathways. Carbon 6 of glucose is assumed to go only

by way of the glycolytic cycle. With liver

slices incubated in oxygen, time ratio of

production of carbon dioxide from

glucose-6-C14 to that from glucose-i-C” is 0.42

(Table V). Thus about 42% of time glucose is

mnetabohized by way of time glycolytic cycle

and the rest by an alternate pathway,

pre-sumably the imexose mono)phosplmate shunt.

Comparable ratios of time activities of time

glycolytic pathway and imexose

monophos-phate shunt imave beelm found in liver of fetal’8 and adult rats. The different tisstmes of time body vary imm time PrP0rti0n of time

glucose metabolized by timese two pathways

(Table VI). The retiuma, for exanmple,

de-pends largely oum time imexose monopimospimate

slmunt, for 90 to 95% of time glucose utilized

is metabolized I)Y this altenimate p2

The hinmg and brain appear to) have little

#{248}rno activities of time imexose

mOlmo)phOs-phate simunt at timis stage of developmeimt.

It is evident from Table V that

glucose-i-C’4 is converted to carbon dioxide nmore

rapidly than glucose-6-C” eveim under

anaerobic conditions. The alternate

path-way requires triphosphopynidine nucleotide

(TPN) as a hydrogen acceptor and one

might infer that fetal liver has a large store of oxidized TPN or that it possesses

(7)

tn-‘l’ABLE Vt

ESTIMATES OF TIlE AcrmvITY OF THE “HEXOSE

MONO-PhOSPhATE SIleNT” IN unMAN FETAL TISSUES

Rate of Rate of Ratio of

(‘on,’ersion Conversion Rate8 of

Incuba-hon

in

liver Oxygen

Nitrogen

(IfCarbon of

Glucose-J_(’I4 to

(arIsn

J)iox-ide

0.45

(1.057

of Carbon of

Glucose-6-(” to

Carbon

Diox-ide

0.19

0.08)74

Conversion

Glucose-J_(’I4

0.42

0.13

Lumig Oxygen 0.0)3 0.04

Nitrogen 0.05 0.04

(‘erebral

cortex Oxygen

Nitrogen

Values are expresse(l 115 Inole of suh)strate convert

-ccl to carbon dioxide/gum tissue/hr.

If, under ammaenobic conditions, glycoiysis

were coupled to time synthesis of fatty acids, time metabolism of glucose to pyruvic acid

Heart Oxygen 4)059 0.0’2’2 0.37 would supply a small amount of energy. If

Nitrogen 0.01’2 0.0035 0. ‘29 time pyruvate were then converted to fatty

acids without using up all of this energy,

0:O4 0.017 0:71 both the carbons and the hydnogens of

---

-

- . . time glucose could be eliminated in the car-l)On dioxide and lipid made. This

mecha-imism would provide the fetus with a means of gaining a small amount of biologically

useful energy anaerobically without

ac-cumulating a toxic amount of lactic acid.

The experimental results, however, appear to rule out this possibility at this stage in

gestation (about 20 weeks) becatmse under

anaerobic conditions, lipogenesis is not in-creased whereas accumulation of lactate is.

Fetal Liver Incubated in

Oxygen Nitrogen

.ldult Liver

incubated in

Oxygen Nitrogen

-114

+ 35

+ 10.5

-155

+ 56 + 19

phospimopynidine mmucheotide (TPNH). Time

syntimesis of fatty acids is known to require

TPNH2’ is time synthesis of certaimm

calm-stituents of nucleic acids.2 Tissues

under-going rapid growtim, such as fetal tissues, might be expected to utilize these

meciman-isms for the reoxidization of TPNH.

Al-ternatively, fetal tissues might possess an

unusually active transimydrogenase by whicim

the hydrogen of TPNH could be transferred

to DPN. This DPNH could timen in turmm be

reoxidized by the conversion of pyruvate

to lactate immtime absemmce of oxygemm.

Carbon Balance

Since each tisstme slice inctmi)ated in vitro

is lart of a closed system, it should be

posi-ble to acco)unt for all of the cani)on atoms

that disappeared in one form (e.g.,

gly-cogen) by the carbon atoms that appear as other substances (e.g., glucose, lactic acid,

carbon dioxide, and lipid). The data for

slices of fetal liver and for slices of adult liver incubated in oxygen and nitrogen are

iresented imm Table VII. In each case, the sum of these four metabolites accounts for

ommly about omme-lmalf of the giycogen carbons timat are utilized. About 55% of the fetal

glycogen that is utilized in oxygen and 45%

of the glycogen that disappears in nitro-gen is accounted for. This difference may

indicate that there are additional reactions

which occur in nitrogen, but not in oxygen;

alternately, it may be the result of

ran-dom variations immsamples. The table points tip the fact that in these tissues, lactic acid

formation is much greater but hipogenesis

is much smaller under anaerobic conditions

than under aerobic conditions.

Time carbon atoms that are not accounted

for in the substances measured remain in

TABLE \‘Il

(‘AlIIIoN BALANCE OF HUMAN LIVEI(

(;l()gemI -54 -68

Glucose +1 + 2

Lactate/2 + 8..5 +‘6

Carhomi

cli-oxide/6 + 6. + 1.4 + 4.7 + ‘2.1

Lipid + 3.1 + 0.4 0 0

Net -‘24.’2 -38.’2 - 63.8 - 77.9

All values are expressed as .amole of (arh)on

(8)

TABLE VIII

All values are expressed as mole/gm tissue/hr. the tissue and incubation medium as other

intermediates. If measurements were made

of all of the phosphorylated intermediates

of the glycolytic cycle and of citric acid and the other members of the

tnicarboxylic-acid cycle, we would undoubtedly come

close to accounting for all of the carbon atoms.

Effect of Anaerobiosis on Subsequent

Aerobic Metabolism of Tissues

There is a widespread belief that anoxia irreparably damages tissues. It appeared

possible that when a tissue slice is

incu-bated under completely anaerobic condi-tions it metabolized for only a few minutes

and then died. Alternatively, it was possible

that the tissue slice did metabolize at a

fairly substantial rate for the entire hour

and would be able to metabolize further if replaced in oxygen at the end of the hour.

Experiments to test this point were per-formed with slices of liver from fetuses of

20 weeks gestational age. Tissue slices were

prepared and placed in eight Warburg

vessels. Four of the vessels were gassed

with oxygen and four were gassed with

nitrogen. At the end of an hour the slices

from two of the vessels originally gassed

with oxygen and two of the vessels gassed

with nitrogen were removed and placed in

fresh incubation medium in other vessels. This group of vessels was then gassed with

oxygen and incubated for a second hour.

The experiment provides a companisoim

be-tveen the metabolism during the second

hour of tissues that had spent the previous

hour in either oxygen or in nitrogen. The consumption of oxygen by the slices that had been in nitrogen for the previous

hour was about 80% as great as timat of the slices that had been in oxygen for the

previ-005 hour (Table VIII). The figures for

utilization of glycogen were calculated from

the differences in content of glycogen be-fore and after incubation. The figure given represents the total glycogen which

disap-peared over the two i-hour periods. In

order to estimate time amount of glycogen

present, one must of course digest time slice

and extract the glycogen.

During the second hour, the slices that

had been in nitrogen the previous hour

utilized less pyruvate than the ones that

had been in oxygen the previous hour. The

amount of lactic acid produced was very

similar in the two. The metabolism of

ace-tate to carbon dioxide is a measure of the activity of the enzymes of the

tricarboxylic-acid cycle. In this respect, the slices which

imad been anaerobic were not quite as good as the ones kept in oxygen time previous

hour. However, the ability to convert

labeled acetate to fatty acids was equal to or even better than the ability of the slices kept in oxygen.

Thus, the liver slice that imas spent a full hour under complete anoxia has many of its

METABOLISM OF FETAL LIVER (AGE ‘20 WEEKS) IN OXYGEN

ArrElo INCUBATION IN OXYGEN OR NITROGEN

Substrate: Acetate-2-C’4 + Pyruvate

(‘arbon of Carbon of

First flour Second hour

-

-Oxygen

Used

Glycogen

Used

Pyruvate Used

Lactate Made

Acetate Con-,‘erted to

(ar-Acetate Converted

Incubated in ban Dioxide to Lipid

Oxygen - 35 ‘26.5 ‘29 ‘2’2 0.47 1.07

Nitrogen - - 49 19 43 0.09 0.07

Oxygen Oxygen ‘29 48 ‘27 17 0.51 0.79

(9)

Clil.

TABLE IX

METABOLISM OF FETAL CEIEBRAL CORTEX (AGE ‘25 WEEKS) IN OXYGEN AFTER INcUBATIoN IN OXYGEN OR NITu0GEN

Substrate: Acetate-il-C’4 + Pyruvate

First hour Second hour

---

---Incubated in

Oxygen Used Pyruvate Used Lactate Made

Carbon of Ace-tate Converted to Carbon Dioxide

Carbon of Ace-tate Converted

to Lipid

Oxygen - 16.’2 17.6 4.0 0.19 0.005

Nitrogen - - 10.9 5.5 0.04 0.0015

Oxygen Oxygen 9.6 11.8 1.7 0.11 0.0017

Nitrogen Oxygen 7.8 7.9 ‘2.0 0.08 0.0017

All values are expressed as mole/gmn tissue/hr.

enzymatic activities intact. Its ability to

synthesize fatty acids and to oxidize

sub-strates via time tnicanboxyiic acid cycle was

essentially unimpaired.

Since the brain cells are believed to be

exceptionally sensitive to oxygen

depriva-tion, a comparable experiment was

per-formed with slices of fetal cerebral cortex

(Table IX). The consumption of oxygen by

the slice of cortex decreases with time as it

is incubated. However, the ones kept in

nitrogen the previous hour consumed about

80% as much oxygen as the ones kept in

oxy-gen the previous hour. The slices that had

been anoxic utilized less pyruvic acid and

converted somewhat less acetate to carbon

dioxide. The amount of lactic acid

pro-duced was about the same. Thus, like the

liver, the brain cells of the cerebral cortex are remarkably active after an hour of

com-plete anoxia. We have, however, no

cvi-dence that they could initiate or transmit nerve impulses.

Neither brain nor liver slices consumed

oxygen at an increased rate after anoxia. Thus, there is no evidence for an oxygen “debt” at the tissue level. Since oxygen debt represents largely an accumulation of lactic acid, these tissue slices would leave the

accumulated lactic acid and the oxygen

debt behind when they were transferred

from oime incubation flask to a fresim one.

Siimce time lactic acid was left beimind there

would be no reason for the slice to have an

increased rate of consumption of oxygen in

the subsequent hour.

Metabolism of Brain

Previous experiments had demonstrated

that early in development, the lower parts

of the brain stem of the dog are more active

metabolically than the higher parts of the

23 As the brain develops, the meta-bohic rate of the cortex increases and finally

exceeds that of the brain stem. The data

summarized in Table X show that this same

change occurs in the course of human

de-velopment. The brain stem from human

fetuses with a crown-to-rump length of

13 cm has higher rates of consumption of

oxygen and production of lactic acid, and

TABLE X

METABOLISM OF HUMAN FETAL BIAIN Substrate : Acetate-2-C’4

Brain Cerebral

Stern Cortex

Incubated in Incubated in

Oxy- Nitro- Oxy-

Nitro-gen gen gen germ

Oxygen consumption

Production of carbon

di-1 di-1.9 - 9.1

-oxide ‘24.7 - 29.1

-Lactate produced

Conversion of carbon of

4.4 9.5 4.1 6.’2

acetate to carbon

di-oxide 0.10 0.0)5 0.10 0.10

Comiversion of carl)omI of

acetate to lipi(l 0.03 0.005 0.015 0.00’2

(10)

TABLE XI

METABOLISM OF HUMAN FETAL CEREBRAL CORTEX

‘I’ABLE XII

METAW)LISM OF IIUMAN FETAL Lu NO

14.5 1.3’2

--9.’2 39.3

13.9 -1.44 -9.6 39.7

0.2() 0.15 0.14 0.04

0. 12 0.0:.? 0. 10 0.0!

Substrate:

Substrate: .lcetate-2-(”

Acetate-2-(”4 Plus Glueo.ue

Incubated in Incubated in

Oxy- Vitro- Oxy-

Vitro-gen geim geum geit

Oxygemm consumption

Production of carholl

11 .9 - 13.6

(hioxide ‘27.9 - 18.

--Laetatepro(luce(l

Conversion of carholl

4.6 7.6 14.5 ‘20.4

of acetate to carbon

dioxide 0.13 0. 10 0.09 0(15

Conversion of carholl

of acetate to lipid 0.06’2 0 0.04 0.(8)8

All values are expressed as janoie/gmn tissue/hr.

convents acetate to lipid more rapidly than the cerebral cortex. Comparable

experi-ments with tissue slices from the brains of

fetuses of 20 cm crown-to-rump length

(Table XI) reveal that the cortex has caught

up to the brain stem in the 3 on 4 weeks

of development intervening.

These data emphasize that the brain is

mucim more dependent upon substrates timan

is the lung or other tissues. Tissues from the same fetus were incubated in alternate

vessels containing radioactive acetate alone

or with acetate plus unlabeled glucose

(Table XI). Three times as much lactic acid

is produced when glucose is present in the

medium than when it is absemmt. This is true for tissues incubated in either oxygen on in

nitrogen. The brain has a very small

con-tent of giycogen and depends largely upon

exogenotms glucose for its metabolism. These

experiments simow, imowever, that the cere-bra! cortex can metabolize substances other

than glucose. It can, for example,

metabo)-lize acetate or pyruvate to carbon dioxide or to fats.

Metabolism of Lung

The fetal lung has a remarkably high

content of glycogeim early in fetal

develop-memmt, about 80 ‘.LmOles of gicogen pen gram

Oxygen comisumupt ion

R.Q.

Lactate pr(lu(e(l (‘onversion of carl)on

of acetate to carbon

(lioxide

Conversiomi of carbon of acetate to lipid

substrate: Substrate: ;lcetate-2-(’

.lcetate-2-(”4 Plus Glucose

Incubated in Incubated in

Oxj- .\itro- Ox-

.Vitr”-yen yen geii geit

All values except R.Q. are expresse(l as .tmioie/gnm us-sue/hr.

of tissue, wimereas the liver Imas 100.

Pre-sumablv because o)f this imigh conteimt of

glycogen there is only a sligimt effect oim

metabolism vhemm glucose is added to the

medium (Table XII). Slices of fetal lwmg are quite active metabolically; like the liver

they can convert some of the glycogen to

glucose wimen immcubated in vitro (Table

XI I I). U nden anaenOl)ic commditiomms there

was a net utilization of glucose.

In contrast to liver the lummg does not synthesize lipid very rapidly. Time rate of lipogenesis is less in time lumig timamm imm the liver under aerobic commditiomis and is greatly

depressed by ammoxia.

Metabolism of Cardiac and

Skeletal Muscle

The results of experiments with slices of

car(liac and skeletal muscle are sunmmmmanized

imm Table XI\T. 1mm coimtrast to liver, brain

aml(1 iiimmg. cardiac nmuscle is richly snpplie(l witim givcogemm alm(l time ainouimit of glycogen

tmtilize(1 during the incubatio)n iS higim under

both aerobic and anaero)bic conditions. The

amounts of lactic acid produced by slices

of cardiac muscle are very nearly equal

uimder aerobic am id anaerobic commditions.

Heart muscles are characterized by imaviimg

(11)

METABOLISM OF SLICES OF HUMAN FETAL LUNG Subsirate: Glucose and Pyrueate

‘FABLE XIII ‘FABLE

Incubated in

Oxygen ‘s’itrogen

10.6 1 .9() 3’2 .7 +18.5

9.3

I4

11

Oxygemi consumption

R.Q.

(;lcOg(’mu mit ilizat ion

-‘glucose (;luc()s( production

Pyruvate utilization Pyruvate I)ro(hui(tioii I..actate l)r(luctioI,

Conversion of (arl)on of glucose

to carbon dioxide

Conversion of carbon of luru-vate to carbon dioxide

Conversion of carboi of glucose to lipid

Conversion of (arh)on of

yrti-vate to lipid

All values txcept EQ. are expresse(l aS j.inole/gii tissue/hr.

pyruvate to fatty acids. Time imeant imas a

metabolic pattern quite different fronm that of the liver. It is less influenced by ammaer-obic conditions than are other tissues.

‘FAh1tl XIS’

\IETAHOLISM OF A(ETATE-’2-(” ‘ icc SIirF:s OF

(AnoI.C ANI) SKELETAl MlS(LE

Cardiac M US(le Nkeletal M uscle

Incubated in Incubated in

Oxy- Nitr(,- Oxy-

Vitro-gen gert ger gen

oxygen eonsmmiptiom 2)).6 -- 9.2

--R.Q. 1 .36 - ‘2.08

(;l?()gen utilizatio,, 41 94 1 3.1 ;#{190};.‘2

I..actate production (2 .3 L5.6 6.‘2 ‘20.S

Conversion of

car-hon of acetate to

carbon (lioxide 0.51 0.057 0. 12 1)07

Conversiomi of

car-hon of acetate to

lipid 0.013 t).0’2’2 0.016 0.0096

All values except R.Q. are expressed as Inole/gIII tis-sue/hr.

METABOLISM OF Fmu-c’rosE BY SLI(ES OF

hUMAN FETAL LIVER

Fructose-C”

Fructose-C’4 +Pyruvate

Incubated in incubated in

Oxy- Nitro- Oxy-

Nitro-geit gen germ gen

41 (1

--

.

----.---.--- 9.1 oxygen consumnption ‘29.6 - ‘27.9

-8.Z R.Q. 1.16

-

1.84

--8 (;l’c)geIl utilization 38 53 31 70

‘!7 Fructose utilizatiom, 20.9 15.7 16.7 9.5

3 1 ( huicose I)rocltIct ion 1 7.9 1 7.4 ‘20.8 1 4.7

Lactate pr(iuctioIm 14.3 44.’2 24.7 46.2

0 .01.5 0.(P1 9 ( ‘onversioi of

car-l)()fl of fructose to

(I.(R)3 (1.06 1 carhn,, (lU)Xi(le (I.36 (1.01 6 (1.2 1 0.(N)))

(‘onversion of

car-I).01 9 1) 1)011 of fructose to

lipid 0.17 0.031 (1.08 0.017

1).030 0.01’2 ( omiversion of

car-.----..---.-

---

--- boll of fructose to

PruV1mt( - - 1).68 0.98

All ,lues (‘X((J)t ILQ. are expressed ais flmOle/gflI tis-su(./h,r.

Metabolism of Fructose by Fetal Liver

Fructose is known to he utilized by adult

liver and it was of interest to compare its

utilization by fetal liver with timat of

glu-cose. 1mm a series of experiments, fructose

ummiformiiiy labeled with C’ was supplied

as the substrate. Iii some of timese

expeni-ments unlabeled I)Yrtmvate was also present

in time incubation medium. Time results are

summarized in Table XV. Comparison of

these results witim those in Table I shows

that liver slices have comparable rates of

consumptioim of oxygen, utilization of

gly-cogemm, amiti prodtictiomm of lactic acid with

either glucose or fructose as substrate. Time

mlietal)Olismmm of these tW’() ileXOSes to) carbon

(1iOXi(1(. aimd to lipid also occurs at similar

rates. The effect of anaerobiosis on the

metabolism of these two substances is quite

similar. When pynimvate is added to the

in-cubatiomi mmmedium, the aerobic production

of lactic dCi(I is increased but the

(12)

TABLE XVI

Substrate: Pyrurate-2-(”4 Substrate : P!,rurate-1-(’

Acetate Absent Acetate Present Acetate Absent Acetate Pre8ent

Incubation in Incubation in Incubation in Incubation in

Oxygen Nitrogen Oxygen Nitrogen Oxygen Nitrogen Oxygen Nitrogen

Oxygen consumption

R.Q.

Glycogen utilization

Pyruvate utilization

Pyruvate production

Lactate production

Conversion of carbon of pyruvate to

carbon dioxide

Comiversioms of carbomi of pyruvate to

lipid

32.9 1.42

43.7

32.1

22.8

‘27.3

77.5

23.9

39.7

6’2.9

29.0

1.49

45.8

19.9

23.8

24.1

68.7

11.7

41.4 44.4

30.5

1.55

16

30.4

18.1

‘27.9

38.1 19.0 36.0

77.6

28.9 1.55 26.5

22.8

‘23.5 15.4

43.2

20.8

38.5

33.3

‘2.16 0.05

1.44 0.07’2

1.40 0.085

0.85 0.018

8.07 0.63

0.03’2

6.78 0.095

0.0035 0 0

All values except R.Q. are expressed as tmole/gmn tissue/hr.

carbon dioxide is greatly decreased. This finding is in keeping with the current ideas

of the metabolic path of fructose in adult tissues. Pyruvic acid is an intermediate in the steps by which fructose is metabolized

to carbon dioxide and to lipid. Thus, the addition of one of the intermediates should

decrease the rate of conversion of hexose to carbon dioxide and lipid.

Metabolism of Pyruvate by Slices

of Fetal Liver

Some of our previous experiments had

provided an indication that pyruvic acid

may be metabolized under anaerobic condi-tions to substances other than lactic acid.

This was investigated in a series of

experi-ments in which pyruvic acid labeled at

carbon-2 (the carbonyl carbon) or at

carbon-1 (the carboxyl carbon) was provided as

the sole substrate (Table XVI). The effect of the addition of acetate was tested with both of the substances. It is evident that the pres-ence of acetate had no effect on the produc-tion of pyruvate, that is, on the production

of unlabeled molecules of pyruvic acid

which diluted the labeled molecules added

as substrate. These unlabeled molecules of

pyruvate came largely from glycogen by

way of the glycolytic cycle and there is no

a priori reason to expect that acetate would affect the rate of glycolysis. However, the presence of acetate does decrease the rate

of utilization of pyruvate and the rates at which the carbon of pyruvate is converted

to lipid and to carbon dioxide. This observa-tion is in keeping with the current concepts of the metabolic paths in adult tissues, for

acetate, as acetyl coenzyme A, is an

inter-mediate in the conversion of pvnuvate to

lipid and to carbon dioxide.

It will also be noted that the presence of acetate decreased the accumulation of lactic

acid under both aerobic and anaerobic con-ditions. This finding is not readily explaina-ble, for acetate is not an intermediate in the conversion of pyruvate to lactate. The

conversion of pynuvate to lactate is a single step reduction involving diphosphopynidine

nucleotide. The decreased production of

lactate in time presence of acetate is perhaps to be explained by some reaction of acetate

which competes for the DPNH (reduced

diphosphopyridine nucleotide) wimicim is

available.

The metabolism of carbon-i of pyruvate

(13)

(itrate Incubated in

Oxy- i’sit ro-germ geri

All values except R.Q. are expresse(l as .Lne)le/g1mm tis-sue/hr.

to carbon dioxide occurs as a single step; in timis reaction carbons 2 and 3 of pyruvate

are converted to acetyl coenzyme A. The

figures given in Table XVI, which are

ex-pressed as p.moles of carbon-i of pyruvate

converted to) carbon dioxide, provide an

estimate of time number of moles of

pyruvate converted to acetyl coenzyme A.

These figures provide evidence that the

de-carboxylation of pyruvate occurs very

rapidly ummden aerobic conditions and about

one-tenth as rapidly under anaerobic

con-ditions.

The immconporation of carbon-i of

pyru-vate into lipid proceeds at a very low rate in

either oxygen or nitrogen. This is to be ex-pected, for the metabolic path by which this

cOnversio)fl occurs is indirect and involves

the process of fixation of carbon dioxide.

Metabolism of Citrate and Glycerol by

Slices of Fetal Liver

In a series of experiments, citric acid

labeled imm carbons 1 and 5 was used as

the substrate either alone or with unlabeled

glucose. The results of these experiments

are presented in Table XVII. It will be noted timat much less citric acid is utilized

TABLE XVII

METABOLISM OF (‘ITuATE-1,5-C’4 uv SLI(ES

OF FETAL LIVEIt

Citrate and Glucose 17l(ubated in

O.ry-

Nitro-gm gert

Oxygen (onsumIption 36 .7 - 36 .‘2

-R.Q. 1.48 - 1.44

-(;lgei utihizatio, 53 83 71 96

Citrate utilization ‘29.0 15.4 ‘29.’2 9.8

Lactate pro(huittioll 16.8 46. 1 17.6 40.7

Commversion of

car-1)011 of citrate to

carbon (lioxi(Ie 3.03 0.087 3.15 0.079

Co,,versio,i of

(ar-hon of citrate to

lipid 0.58 0.0)48 0.73 0.052

under anaerobic conditions than under

aerobic conditions, as one would expect. The utilization of glycogen and the produc-tion of lactic acid are both increased by anaerobiosis. The labeled carbons of citric

acid are rapidly converted to both carbon

dioxide and to lipid by the liver. The

labeled carbons of citrate are an excellent

source of lipid carbons, exceeded in this regard only by pyruvate and acetate.

Pre-sumably, the citrate is converted by the

enzymes of the tnicanboxylic-acid cycle to

oxaloacetic acid, which is decarboxylated

first to pyruvate and then to acetyl

co-enzyme A to enter the lipogenetic cycle. The rapid metabolism of the labeled carbons

of citrate to carbon dioxide is readily under-stood; carbon-5 is given off as carbon diox-ide in the step between cx-ketoglutanic acid and succinic acid. Carbon-i of citric acid

will not appear as carbon dioxide until the second turn of the citric acid cycle. The conversion of the carbon of citric acid to

lipid occurred in nitrogen at less than 10%

of the rate of conversion in oxygen. This

is in keeping with the observations with the

other labeled substrates.

The metabolism of glycerol labeled in

carbon-2 was studied in the presence and

absence of unlabeled pynuvate (Table

XVIII). The utilization of oxygen and

gly-cogen and the production of lactate occur at rates whicim are comparable to those found when liver slices are incubated with

other substances. The metabolism of the

labeled carbon of glycerol to carbon dioxide and to) lipid occurs at rates which are simi-lar to those of glucose. Glycerol is believed to be converted to dihydroxy acetone phos-phate and to enter the glycolytic cycle at

this point. The fact that the anaerobic

con-version of glycerol to lipid is as much as

40% of the aerobic rate suggested that a

large part of it was entering the lipid as

glycerol and was not being metabolized to

acetyl coenzyme A to be synthesized into

time fatty acid moiety of the fat. The lipid

was fractionated and much of the

(14)

‘I’.llI.E XVIII

METABOLISM OF GLYCEROL BY SLICES OF HUMAN FETAL LIVER

‘l’.RIE XIX

G4icerol-?l-(’4

Incubated in

Oxy-

.Vitrn-yen geri

Glycerol -2-C”

+Pyruvate

Incubated in

Oxy-

Nitro-gers geri

35.3 1.64

37 60

‘2(1 ‘36

()xygen (olisumptioll

R.Q.

Glyeogen utilizatio,i

Lactate 1)rOdUctiOII

(‘omiversion of

car-boa of glycerol to carbon (lioxide

Conversion of

(ar-lion of glycerol to lipid

‘36.4 I .55 16 46.

15 39

METABOLISM OF SLICES OF EARLY HUMAN PLACENTA, GESTATIONAL AGE 15

TO 20 WEEKS

()xygen (0115L11111)tiOI,

R.Q.

-

---

--

-

-

---

-- Pyruvate imtilizat

- Pyruvate production

- lactate pro(Iui(tioll

(‘onversioli of carhoi of

I)ruI-vate to lipid 0. 13 (I. I 13

Conversion of carbon of glwose

to hipi(l (I.OHS ().076

Conversion of Carl)oml of

yrti-vate to carhx)I1 dioxide I.36 0 .ItS

Conversion of carbon of glucose

0.148 0).062 0.‘32 0.12 to carbon (lioxide

All valut’s (X(el)t R.Q. are expresse(l as mole/gm

tis-sue/hr.

Incubated in

Oxygen .Vitrogen

lIt

2.67

- (L(;

14.8

S.

- I I .9 ‘21 .0

13 .S

0.36 0(8)28 0.48 0.0082

All values except R.Q. are expressed as n,ole/gm I

is-sue/hr.

0. 104 0.036

Metabolism of Placenta Slices

In one series of experiments, slices of

placenta from fetuses of 15 to 20 weeks

gestation were incubated in a medium con-taming glucose and pynuvate. In alternate

vessels eitimen the glucose or time )yruvate

was labeled witim C’’. The mmmean

consump-tion of oxygen, 11.2 moles/hn/gm of tissue,

yields a figure of 2.52 when converted to

Q”2 (.&liters of gas/hr/mg dry weight of tissue). This corresponds exactly to the Q2 obtained at this gestational age in a larger

series of expenimmments.24 In four of time six

expenimemmts there was a mmet utilization of

glucose ammd production of glycogemm, which

again confirms the previous findings of time

activities of time placenta at this age. The

new observations of particular interest

(Table XIX) are timat the conversion of the

carbons of glucose on pyruvate to lipid

occur almost equally vell in oxygen and

nitrogen. Time high respiratory qimotiemmt is

remarkable and suggests that lipogenesis occurs rapidly in vitro; imoveven, time rate of conversion of labeled pyruvate or glucose

to lipid is not exceptionally imigh.

DISCUSSION

All of timese exl)eninmemmts were designed

to test time maximum activity o)f time enzyme

system involved. Amm excess of substrate was

1)rovidecl iii eacim experiment SO timat the ac-tivity of time cellular enzymes, mmot the

comm-centration of substrate, was time rate-limiting factor. The object of time experiments was to

measure the comparable activity of these

enzyme systems ummder aerobic and

anaero-bic conditions. The results of these

expeni-ments provide us witim an estimate of the limits of metabolic activity of the tissue in

oxygen and imitnogeim; timey do mmot and can-not provide an estimate of time extent of metabolism in vivo. It is possible timat the

tissues of the developimmg fetus imave a con-stant high rate of glycolvsis imm vivo. Timese

experiments demonstrate that they imave a

high rate wimen rendered anaerobic in vitro.

The experiments vitim human fetal

tissues, from fetuses of one-timird to

one-half of the full gestational age show that lipids are synthesized at a much lower rate in nitrogen than in oxygen. In the liver of

the rat fetus just before term the rates of

(15)

Pyruvate-2-C’4 (x’lucose-(”4 Acetate-2-(”4

Incubated in Incubated in incubated in

185

31)

Oxygen Nitrogen

35.1)

101 5

‘22

---

37.4 -.

127 158 144

I.))

.

35 14 42

0.022 0.45 0.015

0.033 0.22 0.021

All values are expressed as .tnioIe/giii tissue/hr. ‘I’ABLE XX

METABOLISM OF SLICES OF GOAT LIvEmu INCUBATED IN ‘ITRO

Liver of Fetus Liver of Fetus

48 Days 141 Days

Gestation Gestation

Incubated in Incubated in

Oxy- Vitro- Ox-

i’sitro-ger geri get. gers

Comiversion of

ace-tate-2-C” to lipid 0.675 (1.065 0.027 0.022

Conversio,, of

pyru-vate-2-C’4 to lipid 0.7.56 0.040 0.061 0.068

Lactate produced

(acetatesubstrate) 14.5 29.7 10)9 23.6

Lactate pro(lu(e(l 19.4 42.2 19.2 33.0

Thie numIll)ers are the means of six experiiiemts. cx-presSe(l as ztnole/gm wet tissue/hr.

eqtmally high.5 More recently we found timat

the tissues of rat fetuses half way throtmgh

gestation also show a marked difference in the rate of lipogenesis under aerobic and

anaerobic conditions.’#{176} In time livers of these

immature fetuses, lipogenesis under anaer-obic conditions proceeds at 10% or less of time

rate under aerobic conditions.

With the kind of assistance of Professor

Donald Barron we imave been able to do

a series of expenimmmemmts on goat fetimses early

in gestation and near term (Table XX). These

experiments demoimstrate timat the synthesis

of lipids from labeled acetate or pyruvate proceeds at equal rates in oxygen and

nitro-gen when time fetal tissues come from fetuses

mmear term. However, the tissues of fetuses

early in gestation again show a much higher

rate of lipogenesis in oxygen than in

nitno-gen. In contrast to the results of the

expeni-ments with rat tissues, the goat liver near

term has a markedly lower rate of

lipo-genesis in both oxygen and nitrogen than

has the liver early in gestation.

The only humamm tissue from a fetus near

term that has been available for

examina-tion was the liven of an anencephalic child

0)f 38 weeks gestational age. The infant did

mmot respire and time tissues were obtained

immmmediateiy after death. The liver had a

imigh content of glycogen, 325 p.moles/gm,

comparable to that expected at this age.’7

The data obtained from this experiment

are presented in Table XXI. The tissues

had a normal rate of consumption of

oxy-gemm ammd of production of lactate, glucose

and pyruvate. It is of interest, however, that

the rate of synthesis of lipid aerobically

was much less timan that observed in the

tissues of earlier fetuses. This was equally

‘FABLE XXI

METABOLISM 01 LIVER SLICES OF 3S-wEK FTus

Oxygen consumption

Glycogen utilization

Glucose productioti

Pyruvate utihizatio,, Pyruvate production

Lactate production

Conversion of carbon of sul)strate

to carbon (hioxide

Conversion of carbon of substrate to lipid

Oxygen Nitrogen Oxygen Nitrogen

u). I

1 I

31

5.3 8.5

‘24 45

1.02 0.015 (1.18

(16)

true with all of the labeled substances used.

The pattern of lipogenesis therefore is com-parable to that in the liver of a rat 24 hours or so after the completion of gestation and not to the pattern that obtains in the rat

just before birth. It will also be noted that

the rate of lipogenesis under anaerobic

con-ditions is substantially less than that under

aerobic conditions. This again is similar to the situation found in rat liver 1 day after

birth. This may be correlated with time idea

that the newborn rat is more immature than

the newborn human. It is possible that if observations were made on human tissues

of 32 to 36 weeks gestational age,

lipo-genesis might occur at high and essentially

equal rates in oxygen and nitrogen.

We have postulated’ that the difference between young and term fetal tissues

repne-sents a physiologic adaptation, by way of changes in the amount and activity of the

enzymes of the glycolytic cycle and of lipid

synthesis, to persistent hypoxia of tissues

in the latter part of gestation. This may be correlated with the reports in the literature

that the oxygen saturation of human fetal

blood is less at term than in the earlier months. It would appear that the placenta

in the latter months of pregnancy may,

under normal conditions, be unable to

supply enough oxygen to keep the fetal

blood saturated and to keep the oxygen

tension in the fetal tissues at a high level.

The fetal tissues might then become

adapted to stmch a low oxygen tension in

the tissues by altering the amounts and

ac-tivities of certain enzymes (many enzymes

might be involved) to provide more energy

without utilizing oxygen. After birth, when

the tissues are well oxygenated as an

ade-quate amount of oxygen is taken in through

the lungs, this enzymatic adaptation might be reversed and the pattern of metabolism

rapidly changed to that characteristic of

the adult. Such changes in metabolism were

observed to occur within 18 hours after

birth in the rat’#{176}and it is known that the ability of the newborn rat to resist hypoxia

disappears very quickly after birth.

An “adaptive” increase in the activity or

amount of an enzyme imas been

demon-strated in a wide variety of bacteria and in

several mammalian tissues. Timis usually

oc-curs in response to an increased

concentra-tion of its substrate2 but it may be brought

about by endocrine stimulation.27 The adap-tive response postulated imere in fetal tis-sues would occur, presumably, not directly

because of the lack of oxygen, but in

re-sponse to an increased concentraton of the

substrates of the enzymes iflVo)lVed. The

piling up of substances, e.g., timose of the

glycolytic cycle, might occur in response to

hypoxia of tissues and lead secondarily to

the adaptive increase in activity of enzymes.

As the increasing imypoxia forced the tissue

to meet more of its emmergy requirement by

glycolysis (and possibly by otimer

energy-yielding reactions whicim can proceed imm

the absence of molecular oxygen) time con-centration of these ilmternmediates, or of the

substrate of whichever enzyme is

rate-limiting, would increase. This could lead to

an increase in the amount or activity of time

rate-limiting enzyme.

The preseimt exl)enimemmts Simo)w that

lipo-genesis can be of no significammce in the

ability of a fetus imaif way tlmrougim

gesta-tion to witimstand hypoxia, for under

anaerobic conditions time rate of lipogenesis

is decreased wimereas that of glycolysis is

increased. Lipogenesis may play some role

in time resistance of time fetus to cimronic hypoxia near term, for botim rat and goat

tissues near term show rates of lipogenesis

wimich are essentially equal in oxygen and

nitrogeim. However, the rate is no greater

in nitrogen than in oxygen, as one would

expect if this were to be an important

fac-ton imm survival (luring anoxia. It would

ap-pear that the remarkable resistaimce of time

mature fetus to imypoxia is imot due to the

presence of any single, mmique metabolic

reaction but is the result of time

combina-tion of several alterations in metabolic pat-terns, each of which, taken alone, adds a

small amount to the oxygen economy of

the fetus. Together, timese metal)o)lic

adapta-tions provide a wide margin of safety to time

References

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