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Comparison of Dietary Fructose Versus Glucose During

pregnancy on Fetal Growth and Development

Marjorie Ferqusson School of Dietetics and Human Nutrition

MacDonald College McGill University, Montreal

A Thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the

deqree of Masters of Science

Marjorie Ferqusson 1989

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l would like to thank my supervisor Dr. Kris Koski and also Louise Lanoue and Mona Cobrin. This thesis could not have been completed without their help and support.

Many thanks to D:r.:. E. Chavez, Dr. S. Kubow and Dr. L. Thibault for their constructive criticism.

The statistical advice of Dr. J.F. Hayes was grp.atly appreciated.

l would also like to thank Natalie Goyette, Chantal Garnache and Deborah Binder for their technical assistance .

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Abstract

Dietary carbohydrate during pregnancy is essential but

whether this requirement is specifie to glucose or if fructose

could substitute for glucose in the diet of pregnant rat dams

was investigated. It was concluded that the carbohydrate

requirement for the rat during pregnancy is not specifie to

glucose and the level, not the type, of carbohydrate was

critical. The potential toxicity of high fructose diets was

also investigated. Dams fed high fructose had significantly

higher liver weights than dams fed high glucose while other

toxic indicators were not affected. A third aspect was the

comparison of isocaloric, low carbohydrate diets centaining

different sources of 4% glucose equivalents: glucose, fructose

or lipid-glycerol. Fructose and lipid-glycerol were net

adequate substi tutes for glucose. The measurement of amniotic

fluid glucose, which increased as either dietary glucose or

fructose increased in the maternaI diet may be a new,

accessible nutritional indicator of carbohydrate status.

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Résumé

Les glucides alimentaires sont essentiels pour la

reproduction chez le rat. Nous avons étudié si ce besoin est

spécifique au glucose ou s~ un autre glucide, le fructose peut

rer,placer le glucose de la diète de la ratte durant la

grossesse. Nos résultats démontrent que la quantité de

glucides contenue dans la diète maternelle est plus importante

que le type de sucre durant la gestat"ion. Nous avons

également évalué les risques de toxicité d'une diète à haute

teneure de fructose. Le poids du foie des rattes qui

consomment une diète riche en fructose durant la gestation est

significativement plus élevé que celui des rattes qui . consomment une diète riche en glucose; aucun au\'.re indicateurs

de toxicité est différent entre les deux groupes. Un

troisième volet de l'étude_est la comparison de trois diètes

ont un contenu équivalent à 4% de glucose. Le fructose et le

glycérol se sont avérés de pauvres substituts du glucose

alimentaire. Finalement, la mesure du taux de glucose du

liquide amniotique, qui augmente lorsque le contenu de glucose

ou de fructose de la diète mate~nelle augmente, pourrait être

un indicateur du statut nutritionnel.

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- Table of Contents Title Page ..•••••••. . . . . . . . . . . . . . . . . . . . ................ . i

Acknowledgements ••. · .................................. . ... i i Abstract ••••••••••.• . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii Table of Contents •. · .................................. . . ... v

List of Tables ••..• · .................................. . .viii

List of Figures ........................................... X1

Chapter 1. Review of Literature

I.Essentiality of glucose for reproduction

A. Ev idence .................. . ........................ .1

B.BiochemistryjPhysiology. ........................... .3

II. Fructose Metabolism

A.Biochemistry of fructpse during pregnancy •.•....... 8

B. Effects of dietary fructose •••.•.....•...•........ 14

statement of Purpose ..••.••.•••..

Chapter 2. Materials and Methods

I.Diet Formulation

........................

A.General principles •• · ............. . B.Calculation of glucose equivalents. .............. C.Diet selection .•... .............................. D.Diet composition ..•

E.Diet preparation ••••

II.Experimental AnimaIs

A.Animal care .•••••...

B.Tissue collection •••

· ........................... . · ........................... .

· ........................... . ......................

C.Calculation of food intake. ......................

v

.22

.23

.24

.25

.27

.31

.31

.32

.33

D.Selection criteria •.•••••••••••••••••••••.•••••... 33 (

III.Analytical Procedures

A. Liver glycogen .................................... 34

B.Plasma and amniotic fluid glucose ..••••••..•.•••.. 34

C.Plasma and amniotic fluid lactate •..•••••....•.... 35

D.Plasma triglyceride .••••••...•••••..••••••••.••••• 35

E. Liver Lipid ....................................... 36

F.Plasma and amniotic fluid urie acid .•••.•.....•... 36

G.Plasma and arnniotic fluid urea nitrogen •••••••••.. 36

H. Amniotic fluid ammonia ••••..•.•••....•••.....•••.. 37

IV.statistical Analysis ••••••••....••••..••••••...•.•.• 37

Chapter 3. Results

l.Maternal Parameters

( A.Food lntake'and Body Weight Gain ••...•••••...•••.. 49

B. Reproduct ive Per f o~ance ••.••.••••..••••••..•.••.. 51

C. Li ver Weight ...................................... 52

D. Carbohydrate Metabolism ••••.•.•••••.••••••..•.••.. 54

E. Lipid Metabolism .................... , ............. 56

F.Plasma Lactate ..........••....••......•........... 56

G.Plasma Nitrogen ••••..•••••••..•••.•••••.•.....••.. 57

II.Arnniotic Fluid Parameters

A.Glucose and Lactate •.••••••••••••.••••••••..•.•••. 58

B. Ni trogen .......................................... 60

III.Fetal Pararneters

A.weight of the Fetus, Placenta and Fetal-Placental

Unit .............................................. 61

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B. Carbohydrate Metabol ism ...••....•••........•...... 63

C.Lipid Metabolism •••••••....•..••.••.•........•...• 64

D.Plasma Lactate ••••

E.Plasma Uric Acid ..

Chapter 4. Discussion •.•.•

I.Fructose Toxicity •••

.64

.65

..99

.100

II.Fructose Replacement of Glucose •..••••.•...•....... 105

III.Carbohydrate source in very low carbohydrate diets.112

Appendix 1: statistical Methods ....••.....••.•........... 118

Bibl iography ............................................. 120

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List of Tables

1-1. Effect of high fructose diets on liver lipogenic enzyme.s ••...••.••••..•.•..••••.•..•..••....•...•..• 11

11-1. Composition of triglyceride and fatty acid based carbohydrate restricted diets •••.•..••......•••.... 40

11-2. Metabolizable energy value of dietary ingredients ........................................ 41

II-J. Equicaloric substitutions of dietary supplements for oleic acid and cellulose and triglyceride and

1I-4.

1I-5.

1I-6.

1I-7.

1I-8.

11-9.

111-1.

1II-2.

cellulose .......................................... 42

Composition of vitamin mixture •...............•...• 43

Formulation of vitamin mixture •..•.•.••....•......• 44

Composition of minera1 mix ••••••...•••....•••••.••• 45

Elemental composition of rnicroelements ••.•.••.•.... 46

Elemental composition, of salt mixture •••...•••....• 47

Caiculated prote in content of ICN case in according to Kjeldahi analyses •.••.•.•••••••.•.•••...•.•..... 48

Effect of glucose and fructose during pregnancy on maternaI food intake and weight gain ..•.....•.••... 66

Effect of low carbohydrate, 4% glucose equivalent diets during gestation on maternal food intake and weight gain ........................................ 67

III-J. Effect of glucose and fructose during gestation Jn reproductive performance in dmas C-sectioned at day 21 ............................................. 68

111-4. Effect of low carbohydrate, 4% glucose equivalent diets during gestation on reproductive performance in dams C-sectioned at day 21 ••.•.••.••.•.••.•••••• 69

1II-5. Effect of glucose and fructose during pregnancy on maternaI liver weight .••..•••••......••............ 70

1II-6. Effect of 60% carbohydrate diets during pregnancy on maternal liver lipid and water content ••.••.••..... 71

1II-7. Effect of low carbohydrate, 4% glucose equivalent diets during pregnancy on maternal liver weight .•.• 72

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III-S. Effect of glucose and fructose during pregnancy on maternai parameters ••.•..••.•....••................ 73

1II-9. Effect of low carbohydrate, 4% glucose equivalent diets during pregnancy on maternaI parameters ...... 74

III-IO.Effect of glucose and fructose auring pregnancy on maternaI lactate ••••.....•.•......•................ 75

III-ll.Effect of low carbohydrate, 4% glucose equivalent diets during gestation on maternaI lactate ......... 76

III-12.Effect of glucose and fructose during pregnancy on matbrnal blood nitrogen ..•..•..................... ,77

III-13.Effect of low cdrbohydrate, 4% glucose equivalent diets during pregnancy on maternaI nitrogen ........ 78

III-14.Effect of glucose and fructose during gestation on amniotic fluid glu~ose and lactate •................ 79

III-15.Effect of low carbohydrate, 4% glucose equivalent diets during gestation on amniotic fluid glucose and lactate ...........•............................ 80

III-16.Effect of glucose and fructose during pregnùncy on amniotic fluid nitrogen ............................ 81

III-17.Effect of low carbohydrate, 4% glucose equivalent diets during pregnancy on amniotic fluid nitrogen .. 82

III-18.Effect of glucose and fructose during gestation on the weight of the fetus, the placenta and the fetal-plactental unit at day 21 .................... 83

III-19.Effect of low carbohydrate, 4% glucose equivalent diets during gestation on the weight of the fetus, the placenta, and the fetal-placental unit at dùy 21 ................................................. 84

1II-20.Effect of glucose and fructose during gestation on fetal parameters ................................... 85

1II-21.Effect of low carbohydrate, 4% glucose equivalent diets during gestation fetal parameters ............ 86

1II-22.Effect of glucose and fructose during gestation fetal plasma lactate ..•..••........................ B7

1II-23.Effect of low carbohydrate, 4% glucose equivalent diets during gestation on fetal lactate .•.•.•...... 88

ix

III-24.Effect of glucose and fructose during gestation on fetal urie acid .................................... 89

III-25.Effect o~ low carbohydrate, 4% glucose equivalent diets during gestation on fetal uric acid .......... 90

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List t)f Figures

I-l.

1-2.

Metabolism of fructose vs glucose ................... 9

Effect of fru~tose on uric acid production ......... 13

III-l.Food Intake - Graded Levels of Dietary Glucose ..... 91

1II-2.Food Intake - Graded Levels of Dietary Fructose .... 92

1II-3.Weight Gain - Graded Levels of Dietary Glucose ..... 93

1II-4.Weight Gain - Graded l..evels of Dietary Fructose .... 94

1II-5.Food Intake - 4% Glucose Equivalents ............... 95

1II-6.Weight Gain - 4% Glucose Equivalents ............... 96

1II-7.Dam liver weight ....•............•................. 97

III-S.Dam Liver weight as % Body Weight •••............... 98

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CHAPTER 1: REVIEW OF LITERATURE

ESSENTIALITY OF GLUCOSE FOR REPRODUCTION

Evidence

Dietary carbohydrate is essential during pregnancy in

the rat (Koski et al 1986). Many parameters of reproductive

performance are adversely affected when carbohydrate is absent

from the diet or only present in low concentrations. It has

been demonstrated in the dog (Romsos et al 1981) and rat

(Koski et al 1986) that there is a critical need for

carbohydrate in the diet during pregnancy for (i) fetal growth

and development (Koski et al 1986), (ii) adequate fetal liver

glycogen deposition at term (Koski et al 1986) and (iii)

postnatal survival of the neonate (Koski and Hill 1986; Romsos

et al 1981).

The requirement during pregnancy for dietary carbohydrate

is apparent after the time of implantation in the ra~ (Taylor

et al 1983); the number of implantation sites is not affected

by the lack of dietary carbohydrate early in gestation in the

dog (Romsos et al 1981) and the rat (Taylor e~ al 1983; Koski

et al 1986;1989; and Koski and Hill 1986;1989). However,

fetal survival fOllowing i.mplantation is impaired, as measured

by an increased incidence of resorption and stillbirths, when

there is a low availability of dietary carbohydrate. The

resorption rate is partic.:ularly affected by an absence of

carbohydrate in the diet during the first eight days of

pregnancy in the rat (Koski et al 1989) while the stillbirth

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rate is increased due to a lack of dietary carbohydrate near

term in dogs (Romsos et al 1981) and rats (Koski and Hill

1989: Koski et al 1989). In order to carry live fetuses to

term, dams require a level of 4% dietary glucose or i ts

equivalent (Koski et al 1986; Koski and Hill 1986).

Additionally fetal growth is retarded if dams are fed

diets lacking an adequate dietary carbohydrate. This is

demonstrated by a lower fetal weight at 19.5 days of gestation

in dams fed low carbohydrate diets (4% glucose) compared to

control levels of 62% carbohydrate (Koski and Mancini 1989).

The birth weight of rat pups (Koski and Hill 1986;1989; Koski

et al 1989) is also lower if the pups are delivered from a

mother ingesting a carbohydrate restricted diet (0 or 4 %

glucose) compared to a 62% earbohydrate diet. In the se

studies, as maternaI dietary earbohydrate inereased, so too

did birth weight. An absence of dietary car~ohydrate did not

affect the birth weight of newborn dogs (Romsos et al 1981),

but this carbohydrate deficiency was not fed throughout

pregnancy as was the case for the pregnant rats.

Finally, neonatal survival is dramatically decreased in

dogs (Romsos et al 1981) and rats (Koski and Hill 1986; 1989)

fed carbohydrate restrieted diets. In the rat, it has been

shown that increasing the concentration of dietary

earbohydrate deereases the postnatal mortality rate, thereby

establishing a specifie need for dietary carbohydrate in the

maternal diet.

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Biochemistry/Physioloqy

It has just been shown that dietary earbohydrate is

required during pregnancy and that without a eritical amount

of carbohydrate in the diet, an optimal pregnancy outeome is

not possible. However, the underlying physiological and

bioehemical mechanisms which result from a lack of dietary

earbohydrate and lead to fetal death and impaired fetal

development have not been thoroughly investigated. It is

generally thought that alterations in carbohydrate metabolism,

speeifieally perinatal hypoglyeemia, most likely underlie the

perinatal mortality which follows. This is based on the fact

that glucose is the principle metabolic fuel for the

developing embryo, the mammalian fetus, and the newborn

immediately after birth (Battaglia and Meschia ~988) and is

the preeursor for the large glyeogen reserves that aceurnulate

near terme It is presumed that these large glycogen reserves

are utilized for energy in the early postpartum period

(Shelley 1961).

MaternaI starvation during pregnancy, which also resul ts

in a lack of dietary carbohydrate, has similar effeets upon

glucose homeostasis during pregnancy as carbohydrate-free

diets. However, while maternaI starvation leads to a decrease

in the availability of aIl nutrientc;, the restriction of

maternaI dietary glucose is nutrient specifie. Two primary

indieators of glucose homeostasis ta be discussed in the

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context of dietary and fasting models during pregnancy are

blood glucose and liver glycogen.

Glucose homeostasis is al tered by pregnancy. It is known

that during the second half of pregnancy a state of relative

maternal hypoglycemia develops because of an increase in

insulin resistance (Baird 1986; Leturque et al 1987). This

normal decrease in blood glucose appears to be exacerbated by

a lack of carbohydrate in the diet during pregnancy (Romsos

et al 1981; Taylor et al 1983; Koski et al 1986). Romsos et

al (1981) first observed this in dogs where bitches receiving

0% carbohydrate displayed low blood glucose compared to

control pregnant animals. Hypoglycemia in the bi tches was

accompanied by hypoalanemia and decreased blood lactate which

further suggested a perturbation in glucose homeostasis.

Decreased blood glucose was also observed in rat dams fed 0%

carbohydrate compared te control dams (Taylor et al 1983).

Koski et al (1986) noted a trend towards a lower blood glucose

in dams fed a carbohydrate free diet. However, this was not

significant. Maternal starvation also decreases maternal

blood glucose (Metzger et al 1971; Freinkel et al 1972;

Simmons et al 1974; Philipps et al 1978; Freund et al 1980).

It appears that a lack of dietary carbohydrate, wi th or

without the presence of ether nutrients, decreases maternaI

blood glucose levels.

It is known that the blood glucose level of the mother

will affect the fetal plasma glucose concentration (Ingermann

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1987). The concentration of maternaI blood glucose alters the

maternal-fetal gradient of glucose; and the rate of transfer

of glucose acr05S the placenta to the fetus is believed to be

a function of the maternal-fetal gradient (Ingermann 1987).

Therefore a decrease in this gradient will result in a lower

rate of glucose transfer and fetal blood glucose levels are

dependent upon this transfer rate. Many studies have shown

that disturbances in the supply of dietary glucose, which is

responsible for the maintenancE:! of maternaI blood glucose

homeostasis, decrease fetal blood glucose in the rat. This

decrease occurs during maternaI fasting (Simmons et al 1974;

Philipps et al 1978; Freund et al 1980). Also with the

dietary model that specifically restricts dietary glucose,

Koski et ~l (1986) reported that as carbohydrate in the diet

decreased, fetal blood glucose tended to decrease, although

this was not statistically significant. It has been suggested

that a lack of circulating blood glucose, an important energy

source for the fetus (Battaglia and Meschia 1988), leads to

a high rate of stillbirths in dogs (Romsos et al 1981) and

rats (Koski and Mancini 1989).

While fetal blood glucose may be al tered by a lack of

carbohydrate, a more sensitive indicator of adversely affected

carbohydrate metabolism is the concentration of hepatic

glycogen in the fetus. During gestation, the maternaI system

supplies the fetus with blood glucose which, as we have seen,

can be compromised under certain conditions. One function of

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this supp1y of glucose is to a110w the fetus to lay down

carbohydrate stores as glycogen. Glycogen is synthesized in

the 1iver near the end of gestation when there is a rapid rise

in the level of fetal liver glycogen (Shelley 1960: 1961: Watts

and Gain 1976: Margo1is 1983). More specifically, the fetal

rat accumu1ates 1iver glycogen during the 1ast three days of

gestation (Margolis 1983). Mobilization of liver glycogen by

the newborn is likely the main source of blood glucose af~er

birth when the neonate is eut off from the maternaI supply of

glucose (Shelley and Neligan 1966) because neonatal hepatic

glycogen levels decrease dramatically after parturition

(Shelley 1960:1961: Watts and Gain 1976: Margolis 1983).

Shelley (1960) suggested that factors which lower the fetal

hepatie glyeogen content, may jeopardize the survival of the

neonate.

Dietary carbohydrate may be required to maintain a

cri tical level of fetal hepatie glyeogen neeessary for

neonatal survival. Severe maternal fasting ean lead to

decreased fetal liver glycogen levels due to an inhibited

synthesis of glycogen but not beeause of increased glycogen

breakdown (Gilbert and Bourbon 1980: Freund et al 1980;

Miettinen and Kliegman 1983). This inability of the fetus te

breakdown hepatic glycogen before birth suggests that glyeogen

accumulation is critica1 for the survival of the animal

(Goodner and Thompson 1967: Gilbert and Bourbon 1980).

Carbohydrate free or low carbohydrate diets aIse produce

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significantly lower levels of glycogen in the maternaI livers

of the rat at days 6-14 (Taylor et al 1983), day 19.5 (Koski

and Mancini 1989) and day 21.5 (Koski et al 1986) of

gestation. Two parallel studies examined fetal glycogen

levels and neonatal mortality in fetuses and neonates of dams

fed low carbohydrate and control diets. Interestingly, the

lower concentrations af hepatic glycagen in the day 21.5 fetus

in the gestational study (Kaski et al 1986) correlated with

100% neonatal mortality in the postnatal study (Koski and Hill

1986). This strongly suggests that fetal liver glycogen is

critical for neonatal survival. AIso, Romsas et al· (1981)

indicated that body mass glucose may be lower in bitches fed

a diet of 0% carbohydrate compared to contraIs and suggested

that this may be the cause of the high perinatal mortality.

Therefore it appears that glycogen stores are required by the

neonate to survive after birth.

In summary, i t can be concluded that adequate dietary

carbohydrate as glucose is essential during pregnancy for the

optimal development of the fetus. It has been demonstrated

that dietary carbohydrate affects the maternaI blood glucose

level which in turn affects the fetal blood glucose and

hepatic glycogen concentrations. without these sources of

carbohydrate for the fetus and neonate, respectively, fetal

and neonatal survival appears to be compromised.

7

FRUCTOSE METABOLXSM

Biochemistry of Fructose during praqnancy

The previous studies have demonstrated the essentiality

of dietary carbohydrate during pregnancy. It is not known

whether the requirement for carbohydrate is specific to

glucose or whether another carbohydrate such as fructose can

fully replace glucose. The metabolism of fructose and

glucose differ in location,

regulation. The liver is

biochemical pathways and in

the major site of fructose

metabolism in the man and rat while glucose may be metabolized

by extrahepatic peripheral tissues (Hallfrisch 1987). After

fructose enters the liver, it is metabolized by three enzymes

which are not involved in glucose metabolism. The three

important hepatic enzymes which are required for fructose

metabolism are (see Figure I-1): fructokinase, aldolase Band

triokinase. Ballard and Oliver (1964) have suggested that

pregnancy induces a decrease in hepatic fructokinase 1 the

first enzyme to metal"lolize fructose, in the maternal system.

The physiological significance of this is unknown.

Fructokinase phosphorylates fructose forming fructose-1-

phosphate. The high activity of this enzyme results in a

rapid rate of fructose phosphorylation. Aldolase B, the

second enzyme of fructose metabolism, splits fructose-l­

phosphate, producing glyceraldehyde and dihydroxyacetone­

phosphate (DHAP). The majority of glycèraldehyde formed is

then phosphorylated by the third enzyme triokinase (Van den

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Figure I-l.Metal:x:>lism of F'IUctose versus Glucose.

GLYlOGEN

GLUCOSE --G-L-U-C-O-K-IN-A-S-E-..... GL UCi E-l-P

FRUCTOSE FRU CTOSE-1-P ! FRUCTOK'NASE l r FRUCTOSE-1-P FR U CTOSE-1,S-P P

1UDOLASE. ~~HAP l GLYCERALOEHYOE-----". .. GLYCERALOEHYDE-3-P

PEPCK

TRIOKINASE Â

9

1 ~

PEP

PYRUVATE KINASt

FRUCTOSE-'· P0 ~ PYRUfATE

LACTATE

. .,... ...

Berghe 1986). At this point the triose phosphates, DHAP and

glyceraldehyde-3-phosphate, can be directed towards

gluconeogenesis or glycolysis, depending on the physiological

state of the animal. Because fructose follows a different

biochemical pathway than glucose (Figure 1-1), it is not as

well regulated as glucose. The breakdown of glucose involves

two important regulatory enzymes, glucokinase and

phosphofructokinase (Hallfrisch 1987). Fructose bypasses

these important enzymes, allowing a faster breakdown of

fructose than glucose in the liver.

These differences in carbohydrate metabolism can

potentially alter lipid metabolism and lactate and uric acid

production.

Lipid Matabolism: The liver, where the majority of fructose

is metabolized, is also the primary site of the lipid

synthesis from carbohydrate (Reiser 1987). It appears that

fructose is more readily converted to l ipogenic substances

than glucose and this is probably due to increased activity

of lipogenic enzymes in the presence of fructose (see Table

I-1) •

Lacticacidosis: Fructose can also be rapidly converted ta

lactate because of the lack of regulation of fructose by the

liver (see Figure I-1) (Reiser 1987). Fructose is quickly

phosphorylated by fructokinase and bypasses the regulatory

enzyme phosphofructokinase. In addition, fructose-l-phosphate

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Table I-l. Eft.ct ot Hiqh Fructose Diets on Liver Lipoqenic Enzymes

High fructose vs glucose diets: effect on enyzmes

Enzyme

Pyruvate Dehydroqenase

Pyruvate Kinase

Malic Enzyme

Glucose-6-P Dehydrogenase

AcetylCoA Carboxylase

Citric Cleavage Enzyme

Fatty Acid

Synthetase

Increased Activity

Vrana et al 1983 (females) Herzberq & Rogerson 1988b

HelIer et al 1977 Naismith & Rana 1974

Fields et al 1985 Michaelis & Szepesi 1973 Chevalier et al 1972 Fukuda et al 1983 Herzberg & Rogerson 1988a,b Sugawa-Katayama & Morita

1977 (males)

Fields et al 1985 Naismith & Rana 1974 Michaelis & Szepesi 1973 Bourne et al 1975

(pregnant females) Fukuda et al 1983 Herzberg & Rogerson 1988a,b Sugawa-Katayama & Morita

1977 (males)

Fukuda et al 1983 Waterman et al 1975

Chevalier et al 1972 Herzberg & Rogerson 1988a,b

Bourne et al 1975 (pregnant females)

Bruckdorfer et al 1972 Herzberg & Rogerson 1988a,b Waterman et al 1975

*unless noted aIl rats were males

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Not Affected

Zakim et al 1967 Sugawa-Katayama &

Morita 1977 (females)

Sugawa-Katayama & Morita 1977 (females)

Zakim et al 1967

Zakim et al 1967

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stimulates pyruvate

fructose to lactate

kinase facilitating the

even further (Reiser 1987).

conversion

Uricacidosis: Fructose loading can result in an accumulation

of fructose-1-phosphate which may lead to a depletion of ATP

and inorganic phosphate in the liver (see Figure I-2) and an

eventual accumulation of uric acid. This is due to a

sequestering of phosphate as fructose-1-phosphate. ATP is

converted to ADP which in turn produces AMP. AMP breakdown

is increased because of a lack of inhibition of AMP dearninase

due ta lower concentrations of inorganic phosphate and GTP.

This is the first step in the breakdown of adenine

nucleotides. Uric acid is one breakdown product of adenine

nucleotides (Van den Berghe 1986). Thus, it appears that

dietary fructose may be a potentially toxic nutrient via its

affect upon lipid, lactate and uric acid metabolism.

Transplacental fructose movement is not significant in

the guinea pig (Karvonen and Raiha 1954), rat and rabbit

(Davies 1955). Hepatic fructokinase is low or absent (Walker

1963, Ballard and Oliver 1964, Grand et al 1974), liver

fructose-1-phosphate aldolase is not significantly active

(Burch et al 1963) and little fructose is incorporated into

glycogen in t~e fetal rat in vitro (Ballard and Oliver 1964).

This evidence suggests that fructose is a relatively foreign

nutrient ta the fetal rat that does not enter the fetal

compartment and is not metabolised by the fetus.

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Figure I-2.Effect of Fructose on Urie Aeid Production.

FRUCTOSE FRU CT<.JSE-1-P

+ .~------ ADP AMP

GPI 1 DEAMINASE

GGTP+ IMP

t INOSINE

! HYPOXAN THINE

! URIC ACID

13

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Thus, the utilization of fructose by pregnant dams may

produce lactic acid or uric acid or may alter lipid

metabolism. This in turn may harm the fetus. Although,

fructose itself is probably not directly involved in fetal

metabolism, its effects via the dam may be potentially harmful

to the developing fetus. The different metabolic pathways and

regultion of the two monosaccharides raise sorne doubts as to

the efficacy of fructose as a substitute for glucose during

pregnancy.

Effects of Dietary Fructose

There are no comprehensive studies investigating the

effect of different levels of dietary fructose, as the only

source of carbohydrate, upon pregnancy and gestation. All

investigations examining the effect of dietary fructose versus

glucose in non-pregnant rats have used only high carbohydrate,

low fat diets. Furthermore, there has been only one study

carried out where pregnant rats were fed high levels of

dietary fructose (Bourne et al 1975). Other gestational

studies, investigating the effect of fructose, have used

dietary sucrose which provides the dam with a source of bath

fructose and glucose. Otherwise fructose studies have

focussed on lactation and the neonate. Bourne's study will

be described in detail and an overview of the fructose

studies, which have been more extensively studied in the non­

pregnant rat, will be given.

14

(

Bourne et al (1975) fed pregnant rats diets of 68%

fructose or glucose throughout gestation. They examined the

impact of the different sugars upon both the maternaI and

fetal systems. They observed a tendency for the liver

weights of the fructose fed rats to be larger than the glucose

fed rats. Liver lipid concentration was not measured.

Plasma triglycerides were higher in the fructose fed dams.

No dietary differences were observed in plasma glucose. A

decrease in maternaI plasma insulin in fructose fed rats was

observed. From this study, it appeared that high levels of

fructose in the diet altered lipid metabolism in the mother

and possibly carbohydrate metabolism via insulin levels.

However, the fetal parameters measured (weight, l ipogenic

activity, plasma insulin and corticoste .• :oids) were not

affected by diet and the authors fel t the fructose in the

maternaI diet would not have a deleterious effect upon the

fetus or neonate. The authors concluded that more

investigation was required ta determine how changes in the

maternaI system affected pregnancy and whether these changes

were harmful to the dam or fetus.

The results of many investigations which will be

described below are taken from studies of the non-pregnant

rat in which a high fructose diet ranging from 50% to 80.68%

was compared to a high glucose diet. Fructose or glucose was

the only source of carbohydrate in the diet and the percent

carbohydrate was the same in both diets.

15

r -

........

Diets high in fructose have been reported to affect the

histopathology of the eye, kidney and liver of weanling and

adult rats (Poulsom 1986). weanling rats fed a 68% fructose

diet for six months displayed retinopathy (Boot-Handford and

Heath 1980). Retinal capillaries were tortuous and had

irregular diameters. The authors suggested that an increase

in retinal lactate in rats fed fructose may be involved in

the histopathology. The kidney is another organ affected by

high levels of dietary fructose (Boot-Handford and Heath

1981) • Weanling rats fed a 68% or 81% fructose diet have

increased kidney weights compared ta rats fed high glucose

(Allen and Leahy 1966; Kang et al 1979; Boot-Handford and

Heath 1981). Boot-Handford and Heath (1981) reported kidney

pathology in fructose-fed rats in the form of

glomerulosclerosis, tubular atrophy and lymphocyte

infiltrations. Of particular interest to us is the liver

morphology as this is the major site of fructose metabolism.

Hepatic steatosis develops in weanling and adult rats fed

diets of 68-69% fructose (Poulsom 1986). The liver acquires

abnormally thick lobes with blunted edges and the normal faint

mottling is replaced with heavy yellow mottling on its

surface. On closer examination, enlarged hepatocytes can be

seen with a large number of lipid containing vacuoles. This

can lead to displacement and distortion uf the hepatocyte

nuclei and cystic fusion of adjacent hepatocytes (Poulsom

1986) .

16

."' ..

(

(

In many studies, the li ver weight has been recorded

although the histopathology of the liver has not been closely

investigated. Rats (male and female) fed high fructose diets

had significantly higher liver weights th an glucose fed rats

(Allen and Leahy 1966; Bruckdorfer et al 1972; Michaelis and

szepesi 1973; Naismith and Rana 1974; Romsos and Leveille

1974; Waterman et al 1975; Aoyama et al 1980,1982,1987; Bird

and williams 1982; Vrana et al 1983; Fields et al 1985). The

increase in liver weight has been associated with and

attributed to an increase in lipid liver content (Allen and

Leahy 1966; Bruckdorfer et al 1972; Michaelis and Szepesi

1973; Naismith and Rana 1974; Romsos and Leveille 1974; Aoyama

et al 1980; 1982; Bird and Williams 1982). However, others

have not observed increased liver weight in fructose-fed

animaIs (Sugawa-Katayama and Morita 1977; Kang et al 1979;

Fukuda 1983; Herzberg and Rogerson 1988b) .

While some studies have not observed changes in lipid

metabolism in rats fed high levels of dietary fructose, the

majority of studies provide evidence of an increase in both

li ver and blood triglyceride levels in fructose fed rats.

Hepatic lipid levels have been reported to be higher in rats

fed fructose than those fed glucose (Allen and Leahy 1966;

Baron et al 1971; Bruckdorfer et al 1972; Maruhama and

MacDonald 1972; Chevalier et al 1972b: Michaelis and Szepesi

1973; Waddell and Fallon 1973: Romsos and Leveille 1974;

Naismith and Rana 1974; Vrana et al 1974ai Sugawa-Katayama

17

-------------

.... - and Morita 1977; Kang et al 1979; Aoyama et al 1980, 1982:

Bird and Williams 1982). Studies investigating fructose-fed

rats compared to glucose-fed rats have reported increased

activities of many lipogenic enzymes (see Table 1). The

greater activities of these enzymes probably lead to higher

lipid content in the liver (Reiser 1987). Others, however,

have not reported increased liver lipid concentrations in

fructose-fed rats (Zakim et al 1967; Herzberg and Rogerson

1986,1988a,b; Aoyama et al 1987).

In general, weanling and mature rats also have

significantly higher levels of serum or plasma triglycerides

when fed fructose (Zakim et al 1967; Bruckdorfer et al 1972:

Chevalier et al 1972b; Maruhama and MacDonald 1972; Waddell

and Fallon 1973; Vrana et al 1974a,b,1976, 1983,1988: Waterman

et al 1975; HelIer et al 1977; Kang et al 1979; Merkens et al

1980; Sleder et al 1980; Aoyama et al 1980,1981,1982, 1987:

Kannan et al 1981; Bird and Williams 1982; Zavaroni et al

1982; Hostmark et al 1982, 1984; Bird et al 1984; Verschoor

et al 1985; Herzberg and Rogerson 1986,1988a,b ). However,

three studies have not observed an increase in blood

triglycerides (Chevalier et al 1972b, Naismith and Rana 1974;

Kang et al 1979). It appears that weanling rats are less

likely to display an increase in blood triglycerides than more

mature rats and that meal-fed rats are more likely to show

higher blood triglycerides than ad libitum fed rats.

18

(

{ '~

The effect of dietary fructose upon carbohydrate

metabolism is not clear. While blood glucose levels in the

rat are not generally affected by dietary fructose, blood

insulin and liver glycogen are in some studies. Most

investigators have not observed differences in plasma glucose

when rats were diets high in fructose (Baron et al 1971;

Waterman et al 1975; Vrana et al 1978; Kang et al 1979: Sleder

et al 1980: Boot-Handford and Heath 1981; Zavaroni et al 1982:

Fukuda et al 1983). However, Herzberg and Rogerson

(1986;1988a,b) noted a decrease in serum glucose in fructose

fed rats which were meal-fed. In rats fed high dietary levels

of fructose, hepatic glycogen concentrations were reported to

be higher than in those fed equivalent amounts of glucose when

rats were meal-fed (Kang et al 1979; Herzberg and Rogerson

1988b) but not in rats fed ad libitum (Vrana et al 1978; Kang

et al 1979). Thus, it appears that blood glucose and liver

glycogen levels are not affected when rats are fed ad libitum

and the maj or disturb,:,,,"ces to carbohydrate metabol ism are seen

in meal-fed animaIs.

In rats fed fructose, compared to glucose, concentrations

of serum or plasma insulin have been reported to increase

(Sleder et al 1980), remain unchanged (Vrana et al 1974a;

Merkens et al 1980; Zavaroni et al 1982; Bird and Williams

1982; Fukuda et al 1983) or decrease (Bruckdorfer et al 1972;

sugawa-Katayama and Morita 1977; Herzberg and Rogerson 1986,

1988a). The reported increase in blood insulin was not

19

ft

- confirmed in a later study in the same laboratory and leads

us to question the validity of the observed rise in blood

insulin (Zavaroni et al 1982). It is not clear from these

results what effect dietary fructose has upon blood insulin

levels which in any case do not appear to affect glucose and

glycogen metabolism in ad libitum fed rats.

When fructose is administered intravenously or in oral

short-term high doses, lacticacidosis can develop because of

the potentially rapid, unregulated conversion of fructose to

lactic acid (see Figure I-l). However, long-term feeding

studies have not been done in the rat or the human to examine

this toxic effect of fructose (Reiser 1987).

Uricacidosis can also develop during fructose

administration because of the rapid accumulation of fructose-

1-phosphate which can lead to the breakdown of adenine

nucleotides (see Figure I-2) (Reiser 1987). The amount of

fructose required to produce these negative effects may not

be available through normal feeding patterns and hence the

toxicity of dietary fructose may not occur. Chronic studies

feeding fructose to the rat have not examined uric acid

levels. Human feeding studies are inconsistent because the

diets are not weIl controlled (sucrose is often fed or

fructose is not compared directly to glucose) or the subjects

are not considered healthy (obesity, carbohydrate sensitivity,

hypertriglyceridemia, gout) (Reiser 1987). Increased

concentrations of serum uric acid in three healthy subjects

20

( was demonstrated 3.n one twel ve-day study when fructose or

glucose represented about 50% of the calories (Ernmerson 1974) •

The diets used were purine-free, which do not occur naturally.

Because of the diet composition and the small number of

subjects, it is hard to conclude from this one study that

chronic ingestion of fructose is harmful ta humans.

Studies investigating carbohydrate metabolism usually do

not examine the nitrogen metabolism of these animaIs.

However, a few studies investigating dietary fructose in the

non-pregnant rat have observed that the prote in or nitrogen

concentration of the liver is similar in rats fed high glucose

and high fructose diets (Baron et al 1971; Bruckdorfer et al

1972; Sugawa-Katayama and Mor'ita 1977; Kang et al 1979; Vrana

et al 1983). The effect of low dietary carbohydrate during

pregnancy on nitrogen met~bolism has not been weIl examined.

During pregnancy in the ùog, carbohydrate free diets did not

affect the levels of plasma urea nitrogen (Romsos et al 1981) .

21

ft

-

-

STATEMENT OF PURPOSE

It has been shown that dietary carbohydrate in the form

of glucose is essential during pregnancy. It is not known

whether this requirement is specifie to glucose or whether

another carbohdyrate such as fructose can fulfill this

requirement, However, fructose is metabolized differently

than glucose, using a another biochemical pathway which is

less regulated. The differences of metabolism between the

two sugars may alter lipid and carbohydrate metabolism and

lactate and uric acid production. The effect of chronic

ingestion of graded levels of fructose, as the only source of

dietary carbohdyrate, compared to glucose during pregnancy has

not previously been investigated. A closer examination of

dietary fructose during pregnancy is required in order to know

if fructose can replace gl~cose during pregnancy and if this

replacement leads to a toxie effect because of the metabolism

of fructose. Our specifie objectives were to investigate

whether: l)at low levels fructose can fully replace glucose

and therefore be converted to glucose and glycogen in adequate

amounts to maintain glucose horneostasis in the mother and

fetusi 2)at low levels fructose can provide sufficient

precursor for glycogen deposition: and 3) at high levels

fructose will he toxic because of its effect on lipid

metabolism and lactic and uric acid production. A comparable

level of glucose was used as a control diet for each level of

dietary fructose fed to pregnant rat dams.

22

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(~

(

CHAPTER 2: MATERIALS AND METHODS

DI ET FORMULATION

General principles

The formulation of a basal carbohydrate free diet for

pregnant rat dams was adapted from work done by Koski et al

(1986). These diets are based on the concept that glucose

must be the first limiting nutrient. Prote in is minimally

adequate to supply the levels of prote in , ni trogen and

essential amine acids normally required by the carbohydrate­

sufficient rat and not in excess ta provide supplementary

glucogenic precursors that would reduce the severity of the

attainable carbohydrate deficiency. As a result, the major

dietary component of a carbohydrate-restricted diet is lipid.

However, two lipid choices, that differentially affect

carbohydrate metabolism, can be selected. Carbohydrate­

restricted diets with minimal but adequate prote in can be

formulated using ei ther intact triglyceride or food grade

fatty acids. The use of triglyceride-rich diets preserves the

glycerol component, which can act as a glucose precursor, and

produces a less severe carbohydrate deficiency than the food

grade oleic acid diets which lack the glucose producing

glycerol moiety. Consequently consideration of the glucose

equivalents in any carbohydrate restricted diet i5 important.

Our rationale permits us to quantify glucose equivalents

recognizing that any glucogenic amino acids provided by a

supplementary source of protein, and any glycerol provided by

23

-supplementary lipid, will have the same effect as the amount

of glucose which can be derived metabolically from them.

Calculation of glucose equivalents

The term "glucose equivalent" used in this thesis refers

to the potential glucose yield from the qlycerol moiety of the

triglyceride and potential glucose yield from glycogenic amine

acids of proteine Since glycerol and the glucogenic amine

acids are precursors of glucose particularly under the

conditions where the metabolic machinery is geared to favor

gluconeogenesis as it is in the carbohydrate-free diet, it

becomes important to calculate precisely their respective

glucogenic potential. In the calculation of the glucose

equivalent of amino acids, it is assumed that one mole of

glucogenic and variable glucogenic amine acids (including

serine, threonine and valine) could potentially yield one-half

a mole of glucose, with the exception of glycine which is

assumed to yield one quarter of a mole of glucose

(Karimzadegan et al 1979; Akrabawia and Hill 1970). The

assumption has been further substantiated by the results of

a computer based method for the estimation of energy from

amine acids and protein (Schulz 1975). Glucose equivalents

that could be derived from the glycerol moiety are calculated

based on the observation that the glycerol moiety is 10% by

weight of the intact triglyer1cle and is 90-95% digestable

24

f J

(

(Brambila and Hill 1966; Renner and Hill 1961; Karimzadegan,

Clifford and Hill 1979).

To the basal carbohydrate free diets, graded levels of

glucose or fructose can be added. The metabolizable energy

of fructose is considered to be identical to glucose. The

heat of combustion of these two monosaccharides is very

similar at 2SoC with fructose at 672.0 vs glucose at 669.94

kg cal/g molecular weight (Handbook of Chemistry and Physics

1986-7). In addition, Renner (1966) and Karimzadegan (1979)

have both determined that fructose is as effective as glucose

in stimulating growth and reducing plasma ketones in chicks

and weanling rats respectively. This suggests strongly that

metabolically, fructose is used as effectively as glucose, and

that the metabolizable energy of the two sugars is the same.

Diet Selection

Graded levels of carbohydrate were fed to the rats using

triglyceride-based diets. Triglyceride was chosen as the

lipid base because although this lipid imposes a less severe

carbohydrate restriction upon the dams than a fatty acid based

diet, it serves as a more natural source of fat in the diets.

Most dietary lipid from natural sources occur as triglyceride

and not as fatty acids. The calorie density of food grade

oleic acid, which is used in the formulation of fatty acid­

based diets, is 8.8 kcaljg. On the other hand, soybean oil,

used in the triglyceride-based diets, has a calorie density

25

of 9.24 kcaljg (Renner and Hill 1958). These numbers suggest

that the triglyceride-based diet is more easily digested and

metabolized. Therefore information gained from the

investigation of triglyeride-based diets creates a less

artificial situation as the stress imposed by the fatty acid­

based diets is not present.

The levels of dietary

triglyceride-based diets were

carbohydrate

0, 4, 12

chosen

and 60%.

for the

These

concentrations of carbohydrate have been used in prev ious

investigations (Koski et al 1986; Koski and Hill 1986), except

that the control diet used by Koski contained 62%

carbohydrate.

significant.

This difference is not considered to be

Three different diets with similar levels of glucose

equivalents were also compared. These were: (1) 4% glucose,

fatty acid-based; (2) 4% fructose, fatty acid-basedi and (3)

0% carbohydrate, triglyceride based. These diets contain,

respectively, 4% glucose, fructose and glycerol. Therefore

these diets contain similar quantities of glucose equivalents.

This allows the examination of the essentiality of glucose and

the ability of fructose and glycerol to replace glucose during

pregnancy. Only fatty acid based diets permit this type of

investigation into the essentiality of glucose because

triglyceride-based diets provide glycerol and thus introdu~e

two sources of glucose equivalents to the diet, glucose or

fructose and glycerol. Fatty acid-based diets provide only

26

r 4

glucose or fructose as a gluconeogenic precursor. At 4%

carbohydrate, these diets are approximately equivalent to the

0% carbohydrate, triglyceride-based diet which contains close

to 4% glycerol.

Diet composition

The diets are described in Table II-1.

Diets were formulated on an equicaloric basis. Each diet

was calculated to conta in 4.15 kcal of metabolizable energy

(ME) per gram dry matter. Supplements of glucose or fructose

were added to the carbohydrate-restricted diet to replace an

equal weight of (a) an equicaloric amount of oleic acid or

triglyceride plus (b) a complementary amount of cellulose to

make up the weight of the supplement (Table 1I-3). The total

metabolizable energy supplied by each diet as weIl as the

relative calorie contribution of the individual dietary

ingredients were estimated on the basis of metabolizable

energy values obtained either from the literature or estimated

from their respecti ve heats of combustion and percentage

absorbability (Table II-2).

It must be mentioned that the food grade oleic acid used

in the experiments was not pure and contained the essential

fatty acids linoleic and linolenic acids. It was estimated

that the high amount of food grade oleic acid (ranging from

34 to 69%) that was used plus the 5% soy oil would meet the

essential fatty acid requirement for linoleic acid.

27

The other component which varied amoung the diets was

cellulose. In the control diet there was 5% cellulose pr~sent

which is considered to be the satisfactory level of fiber in

the diet (NRC 1978). The quantities of protein, vitamins and

mineraIs were held constant for each diet.

The essential amine acids were supplied by 11% caseine

The percent protein in the casein was detcrmined by the

Kjeldahl procedure (AOAC, 1984). A total of 28 different

samples were do ne on four different days (see Table II-9).

The overall average was 83.86 with a range from 80.9 to 90.6.

During one of the runs a prote in standard was also analyzed

by the Kjeldahl procedure and the amount of protein was

underestimated by 3.5%. Therefore the average of 83.86%

prote in was multiplied by a factor of 1.035 in order to

compensate for the apparent loss of protein by the method

used. From this factor we estimated that the percent protein

in the casein sample was 86.45%.

In the equation shown above the percent nitrogen of the

case in prote in was presumed to be 16%. However, the ICN

Biomedical Inc. stated that in this casein the percent

nitrogen was 14.9 even though it was high-nitrogen caseine

We decided to still use the above equation in order that the

percent protein not be underestimated. It was critical that

the animaIs not be fed ù protein deficient diet. A slight

overestimation of the amount of protein present in the diet

would not yield more than half a glucose equivalent. We felt

28

(

(

confident that the percent prote in was not underestimated

because: 1) the percent nitrogen in the case in was less than

that used in the calculations and therefore protein was

overestimated: and 2) four samples were run by an automatic

Kelfast method and the percent prote in was determined to be

89.18%. Therefore we felt that 86.45 (% protein in casein)

was a reasonable number to use as i t would not resul t in a

protein deficient diet and it would not produce a diet with

substantial extra gluconeogenic precursors.

Vitamins were added liberally (approximately four times

the NRC requirements for the water soluble and 1.5-2 times

the NRC requirements for the fat soluble). These

concentrations would allow the diet to provide ample vitamins

to the dams who had a lower food intake than the control

animals. Based on additional pertinent information, the

following modifications were made. Since the dietary

requirement for choline is influenced by the lipid content of

the diet (Best et al 1954; Patek et al 1966; Salmon and

Newberne 1962: Zaki et al 1965) it has been suggested that

the choline content of high fat diets be increased (NRC 1978) .

In adequate diets, which provide 4 to 4.5 kcal/g, the

requirement is approximately 0.1%. However, in high fat diets

that contain 20% or more fat or are low in methionine,

addition of choline up to 0.4% May be required. Consequently,

the higher value for choline was added to the diets.

29

ft

...... -

-

The salt mixture (Tables 1I-6, 11-7 and 1I-8) was devised

by Koski and Hill (1986) and was based on Bernhardt and

Tomarelli (1966) and guidelines established by AIN's Ad Hoc

committee on nutrition standards (AIN 1977). The salt mix was

added ~t a dietary level of 5.5%. In general, each essential

mineraI nutrient was provided at a dietary 1evel twice that

estimated as the requirement for gestation by NRC. Exceptions

were as follows. Values for calcium and phosphorus were

chosen based on the work by Cox and Imboden (1936), such that

the calcium value was 1.2 times the phosphorus value.

Manganese and zinc values corresponded to values reported by

Keen and colleagues (1983) to have historically demonstrated

adequate reproductive performance. The copper value was

chosen in excess of 9 ppm since reports have indicated values

below this may be inadequ~te for maintaining optimal copper

levels in the weanling pup (Cerelewski 1979). Additionally,

because Waibel and colleagues (1954) have reported destruction

of thiamine by dibasic potassium phosphate and calcium

carhonate, these compounds were eliminated from the mineraI

mix formulation. The composition and formulation of the

vitamin mixture are given in Tables 11-4 and II-S,

respecti vely. In addition to NP.~ requirements and the above

modifications, molybdenum was added to prote ct the animaIs

from any possible mo1ybdenum deficiency al though there is no

requirement according to the NRC (1978) • FinaIly,

supplementary sodium bicarbonate was added at 1% dietary level

30

( to correct for any metabolic acidosis that might arise from

feedinq a carbohydrate-free diet (Lumijarvi and Hill,

unpublished) •

Diet preparation

Diets were mixed in two stages. All ingredients except

for soybean oil and oleic acid were mixed. Then the dietary

fat was added to complete the diet and this was thoroughly

mixed.

EXPERXHENTAL ANIMALS

Animal Car.

Female rats of the Sprague-Dawley strain were used in

all experiments. The rats (180-200 qrams) were time-bred at

the Charles River Inc., st. Constant, Quebec and they arrived

on day 0 or day l of the pregnancy. Upon arrival, the

pregnant rats were housed in individual suspended wire sereen

cages and were fed the experimental diet to which they were

randomly assigned (ranging from 10 to 17 per diet). Water and

experimental diets were fed ad libitum. Rats were given diets

in stainless steel cups and water was provided from individual

plastic bottles. Individual body weights and food intake were

measured every second or third day. Food spillage was

recorded. The rats were maintained in a tempe rature

controlled room at 20°C. Fluorescent lighting was provided

31

ft

...... , -

-

.......

automatically for a period of 12 hours daily from 7 am te 7

pm.

Dams were delivered of fetuses by caesarian section (C­

section) on day 21 of gestation. Day 1 represented for dams

the day after they were time-bred.

Tissue Collection

AlI dams were killed in a post-absorptive (fed) state

upon termination of the feeding periode The dams were

anaesthetized with Ketamine-HCI (Vetalar 100 mg/ml Parke­

Davis) injected at a levei of 30 mg/kg into the jugular region

to avoid anaesthetizing the fetuses. MaternaI plasma (5-7

mls) was wi thdrawn by cardiac puncture.

centrifuged and the plasma stored frozen

AlI blood was

until analyzed.

FOllowing cardiac puncture the abdomen was opened and the

liver was removed, freeze-ciamped and kept frozen at -80°C.

Uteri were removed from the abdomen but kept intact. Amniotic

fluid was collected with a tuberculin (lcc, 27G) syringe and

th en frozen at -20°C. Individual fetuses with their attached

placentas were removed from the mother. AlI uteri were

examined for implantation and resorption si tes.

Fetuses were killed by exsanguination. Fetal blood was

obtained from the axillary artery via capillary tubes. The

blood was centrifuged and the plasma stored frozen at -20°C

until analyzed. Each fetal liver was removed, frozen in

liquid air and stored at -80°C •

32

Calculation of Food Intake

The food intake for each animal was calculated on the

even days, that is days 2, 4, 6, etc. These days were chosen

because the first values for food intake can not be determined

until day 2, as rats were recei ved at the end of day 0 or day

1. AIso, food intake affects the weight gain of an animal the

day after and therefore these days are staggered against the

days on which weight gain was calculated, which were days 1,

3, 5, etc.

We received rats at the beginning of day 1 or 2,

immediately after or one day after the night of breeding.

The days were considered to begin in the morning because,

usually, the rats at this time of day were delivered, fed and

killed. Though rats were sometimes fed near the end of the

day, cumulative food intake was not different among the diets.

Selection criteria

Rats of aIl diets were excluded for the following reasons:

1) not pregnant

2) escape of dam from cage

3) rejection of diet by dam

4) d~ath of dam before C-section

5) delivery of one or more pups before C-section

6) number of pups less than 8 per dam

7) pups present or concentrated in one uterine horn

33

-

-.....

In addition, rats in the control, 60% glucose diet were

excluded if the resorption rate was greater than 5%.

The final number of dams used in each dietary treatment ranged

from 7 to 10.

ANALYTICAL PROCEDURES

Livar Glycoqan

The determination of maternaI and fetal liver glycogen

was based on the method of Lo et al (1970) with the following

modifications: 1) a total of 1 ml of KOH saturated with Na2S04

was added to the sample before boiling, 400 ~l while frozen

and, after weighing, 600 J,lol; and 2) the samples were

centrifuged at 4000 rpm.

Plasma and Amniotic Fluid Glucose

MaternaI and fetal plasma glucose and amniotic fluid

glucose were measured by hexokinase determination (Sigma, St.

Louis) using the Abbott VP Super System (Irving, Texas).

Glucose was phosphorylated to glucose-6-phosphate (G6P) by

hexokinase. G6P was then oxidized to 6-phosphogluconate by

G6P dehydrogenase with a concomitant reduction of NAD to NADH.

NADH had an absorption maximum at 340 nm. The increase in

absorbance at 340 nm was proportional to the glucose

concentration. The procedure is similar to that described by

Bondar and Mead (1974).

34

(

(~

(

Plasma and Amniotic Fluid Lactate

Maternal and fetal plasma lactate and amniotic fluid

lactate were measured by lactate dehydrogenase determination

(Sigma, st. Louis) using the Abbott VP Super System (Irving,

Texas) • The plasma was deproteinized after thawing by

perchloric acid using a 1:1 ratio of 10% perchloric acid and

plasma. Lactate dehydrogenase, in the presence of excess NAD,

converts lactate to pyruvate and pyruvate is then trapped wi th

hydrazine. At the same time NAD i5 reduced to NADH. The

increased absorbance at 340 nm due to NA OH formation becomes

a measure of the lactate originally present. This procedure

is based on the procedure of Henry (1968).

Plasma Triqlyceride

Maternal and fetal plasma triglyceride was measured by

enzymatic determination (Sigma, st. Louis) using the Abbott

VP Super System (Irving, Texas). Triglycerides were

hydrolyzed by lipoprotein lipase to glycerol and free fatty

acids. The glycerol was phosphorylated by glycerol kinase to

form glycerol-l-phasphate which was oxidized ta

dihydroxyacetone phosphate, reducing NAD to NADH. NADH was

then oxidized with the concomitant reduction of 2-[p­

iodophenyl)-3-p-nitrophenyl-S-phenyltetrazolium chloride to

formazan which has an absorbance maximum at 500 nm. The

intensity of color production was proportional ta the

35

.

,~ t' , $' -

triglyceride concentration in the sample. The procedure is

a modification of Bucolo and David (1973).

Liver Lipid

The lipid content of the maternaI livers was determined

by the Roese-Gottlieb method (AOAC, 1984).

Plasma and Amniotie Pluid Urie Acid

MaternaI and fetal plasma uric acid and amniotic fluid

uric acid were measured by enzymatic determination (Sigma,

st. Louis) using the Abbott VP Super System (Irving, Texas).

uric acid was oxidized by uricase to allantoin, producing

hydrogen peroxide. Hydrogen peroxide reacted in the presence

of peroxidase with 4-aminoantipyrine and 3, 5-dichloro-2-

hydroxybenzenesulfonate to form a quinoneimine dye wi th an

absorbance maximum at 520 nm. The intensity of color

production was proportional to the uric acid concentration in

the sample. The procedure was similar to the method of

Fossati et al (1980).

Plasma and Amniotic Pluid Urea Nitroqen

MaternaI plasma and amniotic fluid urea ni trogen were

measured by enzymatic determination (Sigma, st. Louis) using .

the Abbott VP Super System (Irving, Texas). Urea was

hydrolyzed by urease to ammonia and carbon dio>..ide. The

36

(

ammonia served to aminate a-ketogluturate to glutamate with

the concurrent oxidation of NADH to NAD. NADH had an

absorption maximum at 340 nm. The decrease in absorbance at

340 nm was proportional to the urea nitrogen concentration.

The procedure is based on the procedure of Talke and Schubert

(1965) •

Amniotic Plui4 Ammonia

Amniotic fluid ammonia was measured by glutamate

dehydrogenase determination (Sigma, st. Louis) using the

Abbott VP Super System (Irving, Texas). Ammonia aminates 2-

oxoglutarate with the concomitant reduction of NADH to NAD.

NADH had an absorption maximum at 340 nm. The decrease in

absorbance at 340 nm was proportional to the urea nitrogen

concentration. This method is based on the procedure of Oewan

(1938) •

STATISTICAL ANALYSIS

The analysis was divided into two protocols:

1) Triqlycerida ~ased diets:

The experimental design demanded a twa-way analysis of

variance, as four levels of carbohydrate (0%, 4%, 12%, 60%)

and two sources of carbohydrate (fructose or glucose) were

chosen. In the command statements summarized in Appendix 1,

CHOL was designated equivalent to carbohydrate levels in the

diet and CHOS was designated equivalent ta the source af

37

-

dietary carb~hydrate. Initially, the dams or fetuses in the

0% carbohydrate diet were randomly divided into two groups so

a 4x2 factorial design could be done. It was required that

no differences would exist between the means resulting from

this artificial division. The conunands used are given in

Appendix 1. AlI maternal data, amniotic fluid data ana fetal

data when there was one pooled fetal value (of 1-3 fetuses)

per dam, were analyzed by this 4x2 factorial design; all 0%

were regrouped and treated as one group in the presentation

of the resul ts •

If an interaction between level and source of

carbohydrate occurred, then a different set of contrasts was

used (given in Appendix 1) because the main effects could not

be analyzed separately when an interaction was pre::;ent. This

occurred with maternaI liver weight.

When the fetal data was recorded by individual fetus,

Le. fetal weight, placental weight, fetal-placental unit

weight and fetal glycogen, then the data was nested (Appendix

1). The data was examined by individual fetusi however the

variability of the data was attributed to the litter, not the

individual fetus. Contrasts were done on this nested data and

these F-values had to be estimated using hand calculations

because the SAS program declared these contrasts non­

estimatable as SAS only handles fixed observations, which the

dams were note The hand calculation for the F-values is given

in Appendix 1.

38

{

(,

,(

Liver lipid were analyzed using the t-test because only

two diets ha~ data for this parameter, 60% glucose and 60%

fructose. The commands are given in the Appendix.

2) 4% glucose equivalent dieta:

The experimental design demanded a one-way analysis of

variance, as three 4% glucose equivalent diets were chosen.

CHas was designated equivalent to the source of dietary

carbohydrate or glucosE" equivalent (glucose, fructose or

lipid-glycerol). The commands used are given in Appendix 1.

This set of commands was used with aIl maternaI data, amniotic

fluid data and the fetal data when there was one pooled fetal

value (of 1-3 fetuses) per dam. When the fetal data was

recorded by individual fetus, Le. fetal weight, placental

weight, fetal-placental unit weight and fetal glycogen, then

the data was nested (Appendix 1). The data was examined by

fetus: however the variability of the data was attributed te

the litter.

A 4x1 analysis of variance was also done to compare the

three 4% glucose equivalent diets to the 60% glucose control

diet (Appendix 1). Here, CHOS was designated equivalent te

the diet, be it one of the 4% equivalent diets or the control

diet. This allowed us to evaluate whether the three 4%

glucose equivalent diets differed significantly from the

control diet.

39

ft

• !

;.,.;

--

Table 11-1 Co.position of t~iglyceride and f.tty acid based carbohydrate restricted diets

Carbohvdrate-restricted Ingredjents Control Triglyceride Based Fatty AC1d Based

2 Carbohydrate

. l3 Soy 01

l · . d 4 o elc aCI

5 Cellulose

6 Casein

Vitamin

Mineral

. 7 mlX

. 8 mlx

Meth i on i ne

Sodium bicarbonate

Weight (g) 9 ME (kcal/g)

g

60

16

5

11. 0

1.2

5.5

0.34

1.0

100.04 4.15

1 . h b . Dry welg t aSlS (grams).

9

o

39.64

41.36

11.0

1.2

5.5

0.34

1 .0

100.04 4.15

9 Il

4 12

38.06 34.91

38.94 34.09

11 .0 11 • 0

1 .2 1 .2

5.5 5.5

0.34 0.34

1 .0 1 .0

100.04 100.04 4.15 4 . 15

9

4

5

34.69

37.31

11 .0

1.2

5.5

0.34

1 .0

100.04 4.15

2Dextrose Canhydrous), n:N Biochemicals Canada Ltd., Montréal, Québec or D-fructose, ICN Biochemicals Canada Ltd., Montréal, Québec.

3 Degummed soybean 0 il, Canada Packers 1 nc., Montréa l, Québec.

4Single distilled oleic acid: 70X C18:1, 8X C18:2, 1X C18:3, 7X C16:1 and 8X mixture of C14:0-C18:0, Canada Packers Inc., Misslssauga, Ontario.

5 Alphacel, ICN Biochemicals Canada Ltd., Montréal, Québec.

6High-nitrogen casein, ICN Biochemicals Canada Ltd., Montréal, Québec contains 86.5X protain (N x 6.25) or 89.1X (Nx6.71).

7 See Tables 1-4 and 1-5.

8 Su Tables 1-6, 1-7 and 1-8.

9 See Tables 1-2 and 1-3 for ME of various ingredients and subst1tut10n rates.

40

t:

(

Table 11-2 Metabolizable energy value of dietary ingredients'

Ingredient

Glucose

2 Fructose

Casein

Amino acid

Soyb.an oil

Qlelc aeid

Starch

HE

kcal/g dry matter

3.64

3.64

4.45

4.00

9.24

8.80

4.00

Reference/assumption

(see Anderson et al., 1958)

(Renner, 1966; Karimzegan et al 1979; Handbook of Chemistry and Physics, 67th ed)

(see Kriss and Hiller, 1934)

assuming complete absorption

(see Renner and Hill, 1958; Young, 1961)

(see Renner and Hill, 1958)

assuming 95% digestion

1Metabolizable energy values were obtained from Koski (Ph.D. Dissertation, University of California, Davis, California,

2 1985 ). See discussion.

41

ft

......

-.......

Table 11-3 Equicaloric substitutions of dietary supple.ents f~r oleic acid and cellulose and triglyceride and cellulose"

Supplement

Glucose

Fructose

Casein

Amino acid

Corn starch

Amount of supplement

9

1. 00

1. 00

1. 00

1. 00

1. 00

1 Dry weight basis.

Substituted for Substituted for

Oleic acid Cellulose TG Cellulose

9 9 9 9

0.414 0.586 .394 .606

0.414 0.586 .394 .606

0.506 0.494 .481 .518

0.454 0.546 .433 .567

0.454 0.546 .433 .567

2 . The metabolizable energy (ME) values for dietary ingredients are shown in Table 1-2.

42

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Table 11-4

Ingredient mg/kg diet

Ni ae i n 100

Calcium pantothenate 32

RibofLavln 12

Pyridoxine hvdrochloride 24

Thiamin hydrochloride '6

Folaein 4

Biotin

Cyanocobalamine (MW 1355.2) 0.2

Alpha- toeopheryl 2

60 acatate

Menaquinone 0.6

Choline chloride 4000

Chollealei ferol] 1500 lU

Vitalllin pa'mitete 4

8000 A lU

NRC requirtment mg/kg diet

20.00

8.0

3.0

6.0

4.0

1.0

0.05

30.0

0.05

tOoO

1000 lU

4000 [ U

Supplier

ICN

Anachemia

[CN

[CN

[CN

ICN

ICN

[CN

Anachemia

[CN

ICN

ICN

[CN

'Nutrition.' Requirements of Laboratorv Animals. Third Revised Edition NRC. National Academy of Sciences, washington, D.C., 1918.

2supplied as 250 lU/gram dl'alpha-tocopheryl acetate, Hoffman LaRoche, [ne., NutLev, NJ.

3supplied as 100,000 lU of vitamin 03

/ 9 , ICN Biochemicals Canada Ltd., Montréal, Québec.

4supplied as 250,000 lU/gram, ICN Biochemieals Canada Ltd., Montréal, Québec.

43

.

Teble 11-5 -Ingredient Grams g/kg diet

(A) fIt soluble

Vitelllin A plllllitate 6.1

Chollcllci ferol .292

Alphl-tocopheryl ecetete 9.1

Menequinone 0.120

Cellulose 1

784·38 Total 800.00 4

(B) Water soluble

Nilcin 20.0

CIlciu .. pentoth.nete 6.4

~

Ribofllvin 2.4 .... Pyridoxine hydrochloride 4.8

Thilllline hydrochloride 3.2

follcin 0.8

Biotin 0.2

Cyanocobelallline 40.0 mg

Butylated hydroxytoluene 20.0

Cellulose 1

l.2.L.l. Total 800.0 4

(C) !;holine !;hloride 4

1 Alphacel, ICN Nutritional Biochemicels Canada Ltd., Montréal, Québec.

44

(

(:

(

---------------------------------------------------------------------

1 nlredlent I/kll SIl t

II/kg dlet a5.5" Supplier

---------------------------------------------------------------------CaMp04 480.72 26.44 Sigma

KMC03 370.0 20.35 An.chemla

NaCI 51. 27 2.82 Il

MgS04 79.63 4.38 Il

crKCs04)2012H20 0.1163 .0064 Il

cuc03

OCUCOM)OM2

O 0.3600 .0198 Il

K!03 .0109 .0006 Il

FeS04

0 7H2

0 7.147 .3931 Il

MnC03 1.9018 .1046 \1

znco3 3.1381 .1726 Fisher

Na2

s.03 0.0091 .0005 leN

NaMo04

0 2H2

0 0.3218 .0177 Anachemia

KF o 2H2

O 0.2018 .0111 Il

Subtotal 994.46 54.71

Cci tric .cid) 5.53 .30 leN

Totll 1000.08 54.99 • 5.5"

---------------------------------------------------------------------1 2See Tlble 1-7 for elemental composition of salt mixture.

See Tlble 1-8 for elementll composition of micro elements.

45

ft

..... ~

Table 11-1

Ingredient (MW)

CaHPO (136j6)

ICHC03

(100.11)

Mg50 (120~38)

Mfcroalements

mg/100 g diet

2644

2035

282

438

(5399)

100

C. Mg Na K p Cl 5

•.••..••..•.• mg!100 g diet •..........

119 600

794

111 1 71

89 1 1 7

-----------------------------------------------------------------Tota l

1 5499 119 89 111 794 600 111 1 17 Ca) 5.49X

NRC·Reqt.1978 (b) 2

500 40 50 360 490 50 30

a:b 1.6 2.2 2.2 2.2 1.2 3.4 3.9

1Not including additional sodium as NaHC03

at a rate of 1 9 per 100 g diet ln order to correct the metabollc acidosis resulting

2fro. f •• ding lON carbohydrate diets. Nutrient Requirements of Laboratory Animals. Third Revised Edition. National Academy of Sciences, Washington, D.C., 1978.

46

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, '

Table 11-8

--------------------------------------------------------------------------------Inllredient

(MW) mil/kil diet Cr Cu Fe Mn Mo Se Zn F

--------------------------------------------------------------------------------crK(S°i)2012H20 6.4 (499.2 )

CUC03

"CU(OH)"HZ

O 19.8 (239.19)

Kro (21LOZ)

0.556

Fesof7H 2 O ]93.1 (55. 5)

MnC03

104.62 (114.94)

znco3

172.61 (125.38)

Na SeO (1J2.9~)

.483

NaMoO*o2H 2 0 11.7 (214. 0)

KF o2H 0 (194J2)

11 .06

tttric Icid 273.65

Totll (1) 100Y,00 NRC-Reqt. (1978)(b) .:b

.67

.67

.30 2.2

11. 0

77.76

11 77.76 5.0 35.0 2.2 2.2

.33

.33 0_ 15 2.2

50

50 50.0

1 .0

7.89

7.89

90.00

.22

.22 90.00

.10 12.0 2.2 7.5

1Nutrient R.quirementl of Laboratory Animill. Third Revised Edition. Nltionll ACldemy of Sciences, washington, D.C., 1978.

47

2.23

2.23 1 .00 2.2

.,..

-.....

Calculated prote,n content of ICI ca •• in accordinl to Kjeld.hl analy •••

Date 25/05/88 26/05/88 27/05/88 29/05/88

X protein 87.54- 82.90 - 89. 15- 82.08 84.76 83.20 81.39 84.62-82.68 83.86 81.87 81.57-86.11 84.02 80.91 - 83.60 84.94 84.53 83.38 82.59 83.45 81.05

90.67- 83.77 84.85 81.58 84.70 84.16

M •• n (6) 84.77*SEM (9) 84.67* (9) 83.03* (4) 82.97*

Ov.rall Mean: (4) 83.86"*

48

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(

CHAPTER XII: RESULTS

MATERNAL PARAMETERS

Maternal Pood Intaka and Body weight Gain:

Cumulative food intake and body weight gain were analyzed

and the data is presented in Tables 3.1-3.2. The daily

patterns of food intake and weight gain are shown in Figures

3.1-3.6.

Triglyceride-based di.ts:

Cumulative food intake during the entire pregnancy did

not differ among the triglyceride based diets (Table 3.1;

Figures 3.1-3.2). However, in the last two days of gestation

(days 20 and 21) there was a significant effect of the level

of carbohydrate (AOV: p < 0.0001; Table 3.1; Figures 3.1 and

3.2); as the carbohydrate content of the maternaI diet

increased, so did the food intake of the pregnant rat. The

source of carbohydrate had no effect on food intake during

this two day periode

MaternaI weight gain increased significantly with

increasing levels of either carbohydrate fed throughout

pregnancy (AOV: p < 0.0001; Table 1; Figures 3.3-3.4) and for

the last two days of gestation (AOV: p < 0.0001; Table 3.1;

Figures 3.3-3.4). No differences in weight gain could be

attributed to differences in the source of dietary

carbohydrate (glucose vs fructose).

49

., ..

-

-

4% Equivalent di.ta:

Among the four percent equivalent diets, there were

significant differences in cumulative food intake (AOV: p <

0.02; Table 3.2; Figure 3.5). When lipid-glycerol was the

source of glucose equivalent (Le. the 0% carbohydrate

triglyceride-based diet or 0% C-TG), the food intake of dams

ingesting this diet was significantly higher than those

ingesting the 4% fructose fatty acid-based diet (4% F-FA).

Food intake of rats fed the 4% glucose fatty acid-based diet

(4% G-FA) was interrnediate, and did not significantly differ

from either 0% C-TG or 4% F-FA. Food intake of the rats

during the last two days of pregnancy did not differ among

the three 4% glucose equivalent diets (Table 3.2).

MaternaI weight gain by day 21 of pregnancy differed

among the three diets (p < 0.05; Table 3.2; Figure 3.6). Rats

ingesting the 4% G-FA diet gained the greatest weight while

those ingesting the 4% F-FA diet gained the least. The

difference between these two diets was significant. The

weight gain of rats on the 0% C-TG diet fell between the other

two diets and did not differ from either of them. During the

last two days of pregnancy the dams fed the three 4% glucose

equivalent diets lost weight and this 1055 did not differ

among the three diets(Table 3.2). Far bath cumulative food

intake and total weight gain, dams fed 4% F-FA had the lowest

values. Dams fed 4% G-FA and 0% C-TG did not differ

significantly from each other.

50

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c

(

Dams fed the high carbohydrate, 60% glucose diet had

significantly greater food intake and weight gain th an the

three low carbohydrate, 4% glucose equivalent diets.

I.productive Performance:

Reproducti ve performance of the dams was measured by the

number of implantations, the resorption rate, the stillbirth

rate and the percent of live pups. The latter three values

are given as the percent of implantations. The data is

summarized on Tables 3.3-3.4.

Triqlyc.ride-based diets:

The source of carbohydrate had no significant effect upon

reproductive performance as measured by number of

implantations, resorption rate, stillbirth rate and percent

of live pups. Among the triglyceride-based diets, the number

of implantations did not differ significantly. However, the

resorption rate, stillbirth rate and percent live pups were ,

aIl affected by the level of carbohydrate in the diet (AOV:

p < 0.01, 0.0001, 0.0001, respectively; Table 3.3). The

resorption rate was greatest in the dams receiving 0%

carbohydrate, significantly less in dams on the 4%

carbohydrate diet and least in dams ingesting 12%

carbohydrate. Dams on the 60% carbohydrate diet had a

resorption rate which fell between those dams eating 4 and

12% carbohydrate and did not differ significantly from either.

However, dams fed the 12% carbohydrate diets had significantly

lower resorption rates than dams fed 4% carbohydrate. The

51

.....

.. ,

stillbirth rate was significantly greater in the dams on the

0% carbohydrate diet versus the other three levels of

carbohydrate which did not differ statistically in numbers of

stillbirths. The percent of live fetuses delivered by c-

section was least in the 0% carbohydrate fed dams. Dams fed

60% carbohydrate had a percentage of live pups which did not

differ significantly from either the 4 and 12% diets.

However, dams fed the 12% carbohydrate diets had a percent of

live pups significantly greater than dams fed the 4%

carbohydrate diets.

4% Equivalent dieta:

Among the dams fed the three 4% glucose equivalent diets,

there were no statistically significant differences in the

number of implantations or in the resorption rate (Table 3.4) •

However, statistical analysis showed significant differences

in the stillbirth rate (AOV: p < 0.05; Table 3.4) and the

percent of live fetuses born (AOV: p < 0.05; Table 3.4). The

dams ingesting the 0% C-TG diet had a statistically higher

resorption rate and lower percent of live fetuses than the two

fatty acid-based diets having 4% glucose or fructose. The

reproductive performances of aIl three 4% glucose equivalent

diets were compared to the control, 60% glucose diet which was

not significantly different.

Materna! Liver weight:

Maternal liver weight was measured as an absolute value

and calculated as a percent of total body weight. In

52

I( 1

(~

(

addition, for the two 60% carbohydrate, triglyceride-based

diets, liver lipid and water concentrations were determined.

The data is summarized in Tables 3.5-3.7 and Figures 3.7-3.8.

Triglyceride-baaed dieta:

MaternaI Iiver weight as an absolute value (Table 3.5;

Figure 3.7) was affected by the levei of carbohydrate (AOV:

p<O.OOOl), source of carbohydrate (AOV: p<0.0005) and by the

level x source of carbohydrate (AOV: p<O. 05). MaternaI liver

weight as a percent of body weight (Table 3.5; Figure 3.8) was

affected by the level of carbohydrate (AOV: p<O.OOOl), source

of carbohydrate (AOV: p<O.OOOl and by the level x source of

carbohydrate (AOV: p<O.OOOl). Therefore there was an

interaction between the level and type (i. e. glucose or

fructose) of dietary carbohydrate for both measurements of

maternaI liver weight. Because of this interaction,

interactive contrasts were done on the data to determine where

the significance of the interaction laYe The effect of the

type of dietary carbohydrate becomes significant only in the

dams fed 60% carbohydrate level, where dams fed diets

containing 60% fructose had higher liver weights than dams fed

60% glucose. However, this effect is not present in dams fed

lower concentrations of glucose and fructose. Figures 3.7 and

3.8 illustrate that the differences between dams fed glucose

and dams fed fructose are greatest at the 60% level where the

difference is significant. This is true for liver weight as

an absolute value and also as expressed as a percent of body

S3

-

-

weiqht. The livers of the dams fed 60% glucose and fructose

were further analyzed for lipid and water content, in an

attempt to discover the reason dams fed the high levels of

fructose have a greater liver weight than those fed glucose.

However, no significant differences between the two diets were

found in liver lipid, principally triglyceride, or water

content (student's t-test; Table 3.6). Glycogen content was

analyzed and is reported later (see MaternaI Carbohydrate

Metabolism) •

4% Bquivalent di.ta:

MaternaI liver weight was not affected by the source of

glucose equivalent (Table 3.7). The liver weight was not

significantly greater in the dams fed the 4% G-FA diet, versus

the dams fed the other two diets. MaternaI liver weight as

a percent of body weight was not affected by the source of

glucose equivalent.

Dams fed the high carbohydrate, 60% glucose diet had

liver weights, expressed as an absolute value, significantly

higher th an dams fed low carbohydrate, 4% glucose equivalent

diets. Expressed as a percent of the total body weight, the

liver weights of the 60% glucose fed dams were significantly

lower than the three 4% glucose equivalent diets.

MaternaI carbohydrate Metabolism:

The carbohydrate metabolism of the dam was examined by

measuring plasma glucose and liver glycogen. The data is

surnrnarized in Tables 3.8-3.9.

54

(

(

Triglyceride-based diets:

There were no significant differences in plasma glucose

among the triglyceride-based diets (Table 3.8). However,

there was a significant effect of the level of carbohydrate

upon maternaI liver glycogen (AOV: p < O.OOOli Table 3.8).

As the concentration of dietary glucose or fructose increased

so did the content Cl ~ maternaI li ver glycogen. Dams fed diets

containing 60% carbohydrate had glycogen levels significantly

higher than those fed 12% carbohydrate and these in turn were

significantly higher than dams fed 4 or 0% carbohydrate.

There were no significant differences in liver glycogen levels

between dams fed a and 4% carbohydrate. The effect of dietary

fructose or glucose upon maternaI glycogen concentrations was

not statictically significant. However, there was a trend for

the dams fed 60% fructose to have higher concentrations of

hepatic glycogen than dams fed 60% glucose.

4% Equivalent diets:

There were no significant differences in maternal plasma

glucose or liver glycogen among the 4% equivalent diets (Table

3.9) •

The high carbohydrate, 60% glucose fed dams did not have

plasma glucose concentrations different than the dams fed low

carbohydrate, 4% glucose equivalent dietsi however, the

content of liver glycogen in the high carbohdyrate fed dams

was significantly higher than dams fed very low carbohydrate

diets.

55

-

Maternal Lipid Metabolism:

The carbohydrate metabolism of the dam was examined by

measuring plasma triglycerides. The data is summarized in

Tables 3.8-3.9.

Triqlyceride-based diets:

There was a significant effect of the level of

carbohydrate upon maternal plasma triglyceride concentration

(AOV: p<O.OOOli Table 3.8), though no effect of the source

from carbohydrate. The dams fed the 4% carbohydrate diets

had significantly higher concentrations of plasma triglycet'ide

than dams fed the three other levels of carbohydrate, among

which there were no significant differences.

4% Equivalent diets:

The source of glucose equivalent affected the plasma

triglyceride concentration (AOV: p<O.Ol; Table 3.9) with 0%

C-TG dams displaying a significantly lower concentration of

plasma triglyceride than the two fatty acid-based diets having

4% glucose or fructose.

Dams fed high carbohydrate, 60% glucose diets had plasma

triglyceride levels significantly lower ~han aIl three dams

fed 4% glucose equivalent diets.

Maternal Plasma Lactate:

The data is summarized in Tables 3.10-3.11.

Triqlyceride-based diets:

Maternal plasma lactate concentration was affected by

the level of carbohydrate (AOV: p<O.OOOl; Table 3.10), but

56

(

(

(

not the source of carbohydrate. The lactate concentrations

were significantly greater in dams fed 12 and 60% carbohydrate

compared to those fed 0 and 4% carbohydrate.

4\ Equivalent diets:

The source of glucose equivalent at the 4% level did not

affect the concentration of lactate in the maternaI plasma

(see Table 3.11).

The high carbohydrate, 60% glucose fed dams had plasma

lactate leveis which were significantly higher than the dams

fed these very low carbohydrate diets.

Kat.rnal Plasma Ni tro(Jen:

MaternaI plasma uric acid and urea nitrogen were

measc~ed. The data is summarized in Tables 3.12-3.13.

Triqlyceride-based diets:

MaternaI plasma uric acid was affected by the level of

carbohydrate in the diet (AOV: p<O.01: Table 3.12) but not

the sourc~ of carbohydrate. Dams fed the 4% carbohydrat(·

diets had the greatest concentration of uric acid and this

was significantly greater than the dams fed the 0 and 60%

carbohydrate diets, though not different from the dams fed

12% carbohydrate. The latter dJ.et did not differ

significantly from any other diet. MaternaI plasma urea

ni trogen was affected by the level of carbohydrate in the diet

(AOV: p<O.0002; Table 3.12), though not the source of

carbohydrate. As the level of carbohydrate increased, the

concentration cf urea nitrogen in the plasma decreased. Dams

S7

....

fed 0% carbohydrate had plasma urea nitrogen levels

significantly higher than the other dams. Dams fed 4%

carbohydrate had plasma urea nitrogen levels significantly

greater than dams fed 60% carbohydrate, while the dams fed

12% carbohydrate diets had intermediate values which fell

between the 4% and 60% carbohydrate diets and did not differ

significantly from elther.

4% Equivalent:

MaternaI plasma uric acid was significantly affected by

the source of glucose equivalent (AOV: p<0.02i Table 3.13).

Dams fed the 4% G-FA diet had uric acid levels significantly

higher than dams fed 4% F-FA and 0% C-TG. The latter two

diets did not differ signifieantly. Maternal plasma urea

nitrogen leveis were not affected by the source of glucose

equivalent (Table 3.13).

Dams fed 60% glucose diets had plasma urie aeid and urea

nitrogen levels significantly lower than the dams fed the

three 4% glucose equivalent diets.

AMNIOTIC PLUID PARAMETERS

AmDiotic Pluid Glucose and Lactate:

The data is summarized in Tables 3.14-3.15.

Triqlyceride-based diets:

The concentration of amniotic fluid glucose was affected

by the level of earbohydrate in the diet (AOV: p<O. 0001; Table

3.14), but not by the source of carbohydrate. As the level

58

(

(

(

of carbohydrate increased, so did the concentration of

glucose. Dams fed 60% carbohydrate had amniotic fluid glucose

concentrations significantly greater than dams fed the other

diets. Dams fed 12% carbohydrate diets had glucose levels

significantly greater than dams fed 0% carbohydrate, while the

dams fed 4% carbohydrate fell between the 0 and 12%

carbohydrate diets and did not differ significantly from

either. Amniotic fluid lactate concentrations were affected

by the level of carbohydrate in the diet (AOV: p<O.Ol: Table

3.14), but not by the source of carbohydrate. The lactate

concentration in the amniotic fluid of dams fed 60%

carbohydrate diets was significantly greater than that of dams

fed 4 and 12% carbohydrate but was not significantly greater

than that of dams fed 0% carbohydrate. There were no

significant differences among dams fed 0, 4 or 12%

carbohydrate.

4% Equivalent di.ta:

The concentration of amniotic fluid glucose was nct

affected by the source of glucose equivalent at the 4% level

(Table 3.15). The concentration of amniotic fluid lactate

was not affected by the source of glucose equivalent at the

4% level (Table 3.15) .

The dams fed high carbohydrate, 60% carbohydrate diets

had amniotic fluid glucose levels significantly higher than

dams fed the low carbohydrate, 4% glucose equivalent diets.

The high carbohydrate fed dams did not have lactate

59

ft

... •

concentrations in the amniotic fluid that differed

significantly from the dams fed very low carbohydrate diets.

Amniotic Fluid Nitrogen:

The concentrations of uric acid, urea nitrogen and

anunonia were measured in the amniotic fluid. The data is

summarized in Tables 3.16-3.17.

Triqlyceride-base4 diets:

The concentration of amniotic fluid uric aeid was

affected by the level of carbohydrate (AOV: p<O. 01; Table

3.16), but not by the source of carbohydrate. As the level

of carbohydrate in the diet increased up to 12% the urie acid

concentration in the amniotic fluid decreased. Dams fed 0%

carbohydrate had a concentration of amniotic fluid urie acid

significantly greater than dams fed 4% earbohydrate which in

turn had significantly hig~er uric acid levels than dams fed

12 and 60% carbohydrate. There were no significant

differences between the uric acid amniotic fluid

concentrations of dams fed 12 and 60% carbohydrate. The urea

nitrogen concentration in the amniotic fluid was affected by

the level of carbohydrate (AOV: p<O.005; Table 3.16), but not

by the source of carbohydrate. The concentration was

significantly greater in the amniotic fluid of dams fed the

0% carbohydrate diet compared to the other three levels of

earbohydrate, among which there were no significant

differences. The amniotic fluid ammonia concentration was not

60

(

(

(

affected by the level or source of carbohydrate in the diet

(Table 3.16).

4% Equivalent di.ts:

The source of 4% glucose equivalent did not affect the

amniotic fluid uric acid, urea nitrogen or ammonia

concentration (Table 3.17).

Amniotic fluid uric acid levels were significantly lower

in dams fed the high carbohydrate, 60% glucose diet than those

fed any of the low carbohydrate, 4% glucose equivalent diets.

Dams fed the 60% glucose diet did not have values of urea

nitrogen and ammonia which differed from dams fed 4% glucose

equivalent diets.

FE'1'AL PARAME'1'ERS

Weigbt of the Fetus, Placenta and Fetal-Placental Unit:

The data is summarized in Tables 3.18-3.19.

Triqlyceride-based diets:

Among the triglyceride-based diets, fetal wei'~ht at day

21 of gestation was affected by the level of carbohydrate in

the diet (AOV: p<0.0001; Table 3.18) but not by the source of

carbohydrate. As the concentration of carbohydrate increased

in the diet, the fetal weight increased. The weights of

fetuses of dams fed 12 and 60% carbohydrate did not differ

from each other, but were significantly higher than fetal

weights of dams fed 4% carbohydrate which in turn were

significantly higher than fetal weights of dams fed 0%

61

........

-

---."

carbohydrate. The placental weight did not differ arnong the

triglyceride-based diets (Table 3.18). The weight of fetal­

placental unit was affected by the level of dietary

carbohydrate (AOV: p < O.OOOli Table 3.18) but not by the

source of carbohydrate. The pattern of the fetal-placental

unit weights was exactly the sarne as that of the fetal weigt-.ts

described above.

4% Equivalent dieta:

Among the 4% glucose equivalent diets, aIl three weights

were affected by the source of glucose equivalent as follows

(see Table 3.19): fetal weight (AOV: p < 0.05), placental

weight (AOV: p < 0.02) and fetal-placental weight (AOV: p <

0.05). Dams fed 4% G-FA produced the greatest, while dams fed

4% F-FA produced the lowest, fetal, placental and and fetal­

placental unit weights. The differences between the two fatty

acid-based diets were always significant. Dams fed 0% C-TG

had values for these three weights which fell between the

other two diets. The 0% C-TG fetal weight value was not

significantly different from those of either fatty acid-based

diet. The 0% C-TG values for the placental and fetal­

placental unit did not differ significantly from those of dams

fed 4% F-FA and were significantly lower than those fed 4% G­

FA.

These weights were also compared to those of dams fed

high carbohydrate, 60% glucose values. The weights of fetuses

and fetal-placental units of dams fed high carbohydrate diets

62

(

(

(,

were significantly higher than those of dams fed low

carbohydrate, 4% glucose equivalent diets. The placental

weights of dams fed high carbohydrate diets did not differ

from placentas of the dams fed very low c~rbohydrate diets.

letal Carbohydrate Mataboliam:

The carbohydrate metabolism of the fetus was examined by

measuring plasma glucose and liver glycogen.

summarized in Tables 3.20-3.21.

Triglyc.rid.-baa.d di.ta:

The data is

There were no significant differences in fetal plasma

glucose among the triglyceride-based diets. The fetal liver

glycogen concentrations were signlficantly affected by the

level of carbohydrate in the diet (AOV: p < o. 001; Table

3.20), but not by the source of carbohydrate. Fetuses of dams

fed 60% carbohydrate had glycogen levels significantly higheL

than those fed 12% carbohydrate which in turn had fetal

glycogen levels significantly higher than dams fed 0%

carbohydrate. The fetal liver glycogen levels of dams fed 4%

carbohydrate had values which fell in between dams fed a and

12% carbohydratn. and which were not significantly different

from either diet.

4% Equivalent diets:

There were no significant differences in fetal plasma

glucose or liver glycogen concentrations among the 4%

equivalent diets.

63

ft

1 1 'j i ,]

1

......

-....

The plasma glucose and liver glycogen levels of fetuses

of dams fed high carbohydrate, 60% glucose diets were

significantly higher than fetuses of dams fed 4 % glucose

equivalent diets.

'etal Lipi4 Metaboliam:

The lipid metabolism of the fetus was examined by

measuring plasma triglycerides.

Tables 3.20-3.21.

Triqlyc.ri4e-based di.ta:

The data is summari~ed in

There were no significant differences in fetal plasma

triqlycerides among the triglyceride-based diets.

4% Equivalent di.ts:

There were no significant differences in fetal plasma

triglycerides among the 4% equivalent diets.

Between the high carbohydrate, 60% glucose diet and the

low carbohydrate 4% glucose equivalent diets, there was no

significant difference in fetal plasma triglycerides.

retal Plasma Lactate:

The data is summarized in Tables 3.22-3.23.

Triqlyceri4e-base4 diets:

There were no significant differences in fetal plasma

lactate among the triglyceride-based diets.

4% Bquivalent diets:

There were no significant differences in fetal plasma

lactate among the 4% equivalent diets.

64

(

(

(

Between the high carbohydrate, 60% glucose diet and the

4% glucose equivalent diets, there was no significant

difference in fetal plasma lactate.

'.tal Plasma Orio Aoi4:

The data is summarized in Tables 3.24-3.25.

Triqlyc~rid.-based diets:

There were no significant differences in fetal plasma

urie acid among the triglyeeride-based diets.

4% Bquivalent 4i.ts:

There were no signifieant differences in fetal plasma

urie acid among the 4% equivalent diets.

Between the high carbohydrate, 60% glucose diet and the

low carbohydrate, 4% glucose equivalent diets, there was no

significant differenee in fetal plasma urie acid.

65

.

0-0-

e } t ~

Dietary Ievel of cartxhvdram Cart:xil~te 0 4 12 60

awilative food intaJœ, ql Gll.JCXJSe 34B±13 (9) 373±11 (10) 362±14 (9) 403±12 (10) Fructose 374±12 (9) 3Bo±14 (7) 378±11 (la) Glu + FIu7 348±13 (9) 373±9 (19) 37l±11 (16) 390±9 (20)

food intalœ CD daya 20 + 21, qI Glucose 13±3 (9) 2B±3 (10) 3l±3 (7) 33±3 (10) Fructose 24±3 (10) 34±3 (7) 33±3 (la) Glu + FIu7 l3±3 (9)a 26±2 (20)b 32±2 (14)C 33±2 (20)C

awilative w9't gain, q5 Glucose 65±5 (9) l16±7 (10) l28±7 (9) 15o±7 (10) Fructose 106±7 (10) l4o±8 (7) l46±7 (10) Glu + FIu7 65±5 (9)a 11l±5 (20)b l33±5 (16)C l48±5 (20)C

.. j'Pt gdn CD c:Iays 20 +21, g6 + Glucose -22-4 (9) 3±4 (10) 19±4 (9) 19±4 (la)

Fructose 2±4 (10) 15±4 (7) 2o±4 (10) Glu + FIu7 -22±4 (9)a 2±2 (20,b 17±3 (16)c 19±2 (20)c

lValues are means ± SEH. Humber in paœntheses equals nJlllbpr of dams. 2Means within each parameter havi.n.J different letter superscripts followin) the nllliler differ significantly (p<O.OS). 3-6significanoe of main effects fraD analysis of variance (MN): 3MN: leve1 of cart:rltyc\rate (NS); sc:w:oe of ~te (NS); leve1 x scuroe of carbahydrate (NS). 4101: leve1 of ~te (P<O.OOOl); soo.roe of carlJà1yàrate (NS); level x sooroe of carbahydrate (NS). SNN: level of cartx:lhydrate (p<O.OOOl); soorœ of cartdlydrate (NS); level x scurœ of caz1:dlydrate (NS). 6N:N: level of carlxb~te (p<O.OOOl); souroe of cart:d1}'drate (NS); level x san-ce of carbahydrate (NS). 7~ there was no significant effect of source of ca.rbOOydrate, the statistica1 analysis treated all qluoose ard fructose at ead"l leve1 of carlx:tlydrate as a si.n.Jle groop. '1herefore, the significance is reported for these gro.lp9d means.

t !

0'>

"

~ ~

Table 3.2 Effect: of lcw au::bciIydmte, 4\ glnmep eq4valent diets œring pœgnancy CD.at:eDlal. food intaJœ ani ~ gain1,2

Food Intalœ Glucose Ecpivalent Q.mulativeT -ilays20 +21.4

4% Glucose 4% Fructose 4% Lipid-glyoerol

330±l5 (8)ab 297±13 (9)a 348±13 (9)b

16±3 (8) 15±3 (9) 13±3 (9)

Weiabt Gain Qmul.at1ve5~ ~ Iliys 20+211)

76±6 (8)a 52±5 (9)b 65±S (9)ab

-14±3 (8) -13±3 (9) -22±3 (9)

lValues are means ± SDI. NlDIiler in paœntheses ecpùs nmtJer of damS. 2Means within eadl oolUllll havin;J different letter superscripts follCJWin:;J the DFer differ significantly (p<O.05). 3-5Significance of main effects traD analysis of variance (NN): 3NN: glucose equivalent (p<O.OS). 4NN: glucose Eqrlvalent (NS). 5NN: glucose equivalent (p<O.02). 6101: glucose equivalent (NS).

"'" ,

0\ 00

~ ~ ( ~

'!able 3.3 1be effect of g)lKXlSe and f:ructoae ci.Jrln:J ~ en :t:EIRcdrtive perfODlalD! in daIIB c-sect:i.a1ed at day 211 ,

Dietary Carlxbyd"ëlte

Glucose Fructose Glu + Fru7

Glucose Fructose Glu + Fru7

Glucose Fnlct:ose Glu + Fru7

Glucose Fructose Glu + Fru7

Level of Calbilvdrate __ _____ _ o 4 12 60

average nmber of iDp1antati.c::ms per daIItl 13±0.8 (9) 14±0.7 (10) 13±0.8 (9) 14±0.7 (10)

14±0.7 (10) 14tO.9 (7) < 13±0.7 (10) 13±0.8 (9) 14±0.5 (20) 14±0.5 (16) 14±0.5 (20)

USiOI:pt.ia'l zate (' of ÎIIpl.aJltat.ÏaJ' 9.3±3.2a 5.S±1.Sb 1.4±l.SC 4.7±l.Sbc

6.3±I.Sb 1.1±2.1c 5.3±1.8bc 9.3±3.2a 6.1±1.5b 1.3±1.~ 5.0±1.sbC

stillbirth rate (\ ilIIpl.ant:atiœ) 5

26.8±7.4a 2.7±1.Ob o.O±o.ob 0.7±I.Ob 2.7±1.2b

26.8±7.4a 1.7±3.1b 1.1±3.4b

live febases (~ of iIIplantat.iœs) 6 64.0±7.7a 91.6±1.9D 98.6±2.1c

93.0±1.gb 96.l±2.3c 64.0±7.7a 92.3±3.3b 97.5±3.7c

o.o±o.Ob 0.6±I.Ob 0.2±J.ob

95.4±1.gbc 94.l±1.gbc 94.7±3.3bc

IValues are tœanS ± SEM. Nl.utiler in parentheses equals llUl'IiJer of dans. 2Means within each parameter haviBJ different letter superscripts follOlod.rg the runber differ significantly (p<0.05). 3-6Significance of main effects fram analysis of variance (AOV): 3NJV: level of cartx:hydrate (NS); soorce of cartx:lbydrate (NS); level x source of cartdlydrate (NS). 4NN: level of carbahydrate ~0.01); scmœ of cartxX1ydrate (NS); level x source of cartx:lbydrate (NS). 'ACN: level of cartx:tlydrate (p<0.0001) ; sa.u:ce of carl:x:i1.ydrate (NS) ; level x source of cartx:tlydrate (NS). 6NN: leve1 of ca.rllahyàrate (P<0.0001); sa.u:oe of carbcilydrate (NS); level x source of cartx:nydrate (NS). 7lr1len there was no significant effect of souroe of cartx:nydrate, the statistical analysis treated all glUCXlSe am f:ructose at each level of cartx:ilydrate as a sllgle group. '1herefore, the significance is reported for these grouped means.

J < 4

,.Ir .....

0-\C

~ ~

'lable 3.4 'DIe effect of loir ~te, 4. g]lDl9P eIJ.lÏ.val.ent d:iet& WriDJ ~ti.cn al rE(lI'OCb:tive perfODBl'D! in dais C sectimed at clay 211,2

Glucose Equivalent Inplant:ation3 ~ian stUlbirth Live Rate4 Rate5 Fetuses6

(% of ÏlIplantatioos)

Mean % % %

4% Glucose 12±O.8 (a) 8.l±3.4 1.D±7.8a 90.9±8.1a 4% Fructose 15±O.8 (9) 7.l±3.2 2.1±7.4a 90.8±7.7a 4% Lipid-qlyoerol i3±O.8 (9) 9.2±3.2 26.8±7.4b 64.D±7.7b

lValues are means ± S~. NuniJer in parentheses equals nmtlp..r of dams. 2Means within eaàl column havinJ different letter supeniCripts followiR) the n1lli1er differ significantly (P<O.05). 3-5Significance of main effects fJ:an analysis of variance (NN): 3NN: glucose equ.j,valent (NB). 4N:N: glucose equivalent (NS). 5NJV: glucose equivalent (P<O.05). 6NN: glUCXlSe equivalent (P<O.05).

----~~,~ --,--

""'"

~ o

~ ~<.;

'lable 3.5

Dietary carbcbydrate

Glucose Fructose

Glucose Fructose

t ~

level of carbcbvdrate o 4 12

liver weight, g2

9.8±0.4 (9j 10.3±0.4 (10) 10.8±0.4 (10)

11.l±0.4 (9) 12.2±0.6 (6)

liver weight as % lxxly weight, %3

3.6±0.1 (9) 3.2±0.1 (10) 3.4±0.1 (10)

3.3±0.1 (9) 3.5±0.1 (6)

60

11.6±0.4 (10)* l4.7±0.4 (10)*

3.3±0.1 (10)* 4.2±0.1 (10)*

IValues are means ± SEM. NurIiler in parentheses equals nuniler of px-egnant: rat dams. 2-3Significaœe of main effects fran analysis of variance (ADV): 2ADV: level of carbahydrate (p<0.0001) ~ sau:oe of carbahydrate (P<0.OO05); level x source of carbahydrate (p<0.05). MN: level of carbahydrate (p<O.OOOl); scw:ce of carbahydrate (p<0.0001); level x source of carbahydrate (p<O.OOOl).*'lhe difference in liver weights between glucose aM fructose at 60% carl:xilydrate was significant.

~. f

~ 1-'

p.~ ~

'nIble 3.6 Effect of 60l ~t:e diets WriDJ Œ&aBIICY al a:d:oemal. liver lipid and wat:er cxmtent1

Dietary cartxnydrate N

Gluoose 8 FlUctœe 10

Lipid " DJ:y Weight

15.4tO.3 13.2tO.5

water

" 70.5tO.3 70.5±0.2

IValues are means ± SEX. T-test for each oo1U111'l was NS.

~,

........ N

} : ~

'nIble 3.7 Effect of low ~, 4' CJlucœe BJlÏ.valeDt djets clJring ~ cm lIIlteJ:nal liver ~1,2

GlUCXlSe Equivalent

4% glUCXlSe 4% fructose 4% lipid-glyoerol

Liver Weight3

(g)

lO.5±O.4 (8) lO.O±O.4 (9) 9.8±O.4 (9)

Liver Weight4

as % body wight

(%)

3.S±O.l (8) 3.9±O.l (9) 3.6±O.l (9)

lValues are means ± SEM. Nunber in parentheses ecpù.s l1UI1iJer of dams. 2MeaJls within each oolumn havin;J different letter superscripts followin;J the lUIiler differ significantly (p<O.05). 3-4Significanoe of min effects fran analysis of variance (AOV): 3NN: glucnse equivalent (NS). 4NN: glucose equivalent (NS) •

,

--.. w

~ ~

Table 3.8 Effect of Glucose and fi:uct:Œie œrirJJ pŒ9IiUICy en _temal parmEtersl,r

Diet:aJ:y lBVel of ~rtxilvdrate Carbàlydrate 0 4 12 60

plasma glUCXlSe, Dg/dl3

Glucose 100±5 (9) 107±6 (7) l12±7 (6) 100±6 (9) Fructose 98±6 (8) 120±7 (6) 11l±6 (9)

Glu + F'nl6 100±5 (9) 102±4 (15) l16±5 (12) 106±4 (18)

liver glycxgen, ngjcf' Glucose 4.94±0.95 (9) 3.45±2.66 (10) l8.53±2.8l (9) 40.l7±2.98 (8) Fructose 2.68±2.98 (8) 17.10±3.44 (6) 48.04±2.66 (10) Glu + Fru6 4.94±O.95 (9)a 3.1l±1.87 (18)a 17.96±2.05 (15)b 44.54±1.87 (18)c

plasma triglyoerides, Dg/dl.5

Glucose 484±148 (9) 985±147 (9) 487±166 (7) 269±139 (10) Fructose 889±147 (9) 511±180 (6) 235±139 (10)

Glu + Fnl6 484±148 (9)a 937±96 (18)b 498±lI3 (13)a 252±9I (20)a

IValues are œans ± SEM. N\lrli)er in paœntheses ecpùs nJJt)er of dalE. ~ within eadl parameter havirq diffeœnt letter superscripts followirg the nmber <liffer significantly (p<0.05). 3-6significance of main effects frai analysis of variance (ADV): 3N:N: level of carbohydplte (NS); sau:ce of cazbohydrate (NS); level x SCAlroe of cazbohydrate (NS). 4NN: level of cazbohydrate (p<û.OOO1); sourœ of cartxJhydrate (NS); level x scm:-oe of cartxJhydrate (NS). 5101: level of carl:xilydrate (p<O. 0001) ; SCAlroe of carl:xilydrate (NS) ; level x sruroe of carlx:t1ydrate (NS). ~ there was ~ significant effect of sruroe of carl:xilydrate, the statistica1 analysis treated aIl glUCXlSe and fructose at eadl level of carlx:t1ydrate as a sin;Jle group. 'lberefore, the significance is re;x>rted for these grouped means.

_ MYD" -Yiz ...... _~~ ........ ......:; ...... "-...... - -

lfiI'

....... ~

, ç. ,

Tcmle 3.9 Effect of 10w cadx:tJ,ydmte, 4' glua .... ~val.ent dipts œriJJ.J pœgnancy CD 1IIiltema1 ~1,2

Gluoose Equivalent

4% gluoose 4% fructose 4% lipid-glyoerol

Plasma Gluoose3

Dg/dl

92±8 (4) l06±5 (8) lOo±S (9)

Liver Plasma Glyoogen4 TriglyœridesS

Dg/g IIIJIdl

2.27±1.17 (6) 1268±157 (8)a 3.84±O.95 (9) 929±148 (9)a 4.94±O.95 (9) 484±148 (9)b

IValues are means ± SEM. Nlmiler in parenthesis ecpll.s J'VJIIiJer of dams. 2Means within eac:h ool\Dll'l havirg diffeœnt letter superscripts followirg the n1lllher differ significantly (p<O.OS). 3-6Signifiœnce of main effects fran analysis of variance (AOV): 3NN: glucose equivalent (NS). 4NN: glucose equivalent (NS). 6AOV: gluoase equivalent (p<O.Ol) •

ç

•

..... \J1

I~ ~

'DIb1e 3.10 =1~~ glucase an! tm:;tose cùr.iDj praJI1iII1CY CD Etemal

Dietary Ievei of Çêrtxi1vdrate cartxilyàrate 0 4 12 60

Dg/dll

Gluoose 26±2 (9)a 23±3 (9)a 39±3 (9)b 39±3 (9)b Fructose 25±3 (lO)a 35±4 (6)b 42±3 (9)b

Glu + Fnl4 26±2 (9)a 24±2 (19)a :i7±2 (lS)b 4o±2 (18)b

IValues are means ± SEM. N1.miJer in parentheses ecpals l'1IIliJer of dams. 2J1eans havin;J diffeœnt letter superscripts followin;J the n.miler differ significantly (P<0.05). 3SignificaJlCE of main effects frau analysis of variame (ALN): level of cart:xil~te (p<O.OOOl); sa.u:oe of caxbchydrate (NS): level. x source of c.arbà1ydrate (NS) • 4When theœ was no significant effect of source of c.arbà1yàrate, the statistical analysis treated all glucose ard fructose at eaàl level of c.aIt:làlyàrate as a sin:Jle group. 'lberefore, the significaooe is reported for these gra.JpE!d means.

",.,

..., Q\

~ ) t }

-nmle 3.11 Effect of la.r cadx:bydrate i'~ gluaJSe EfIlÏ.val.ent diets Gu:iDJ gestatim cm mtemal l.actate '

Glucose Equivalent

4% glucose 4% fructose 4% lipid-glycerol

Iactaœ (Dg/dl)

28±2 (7) 25±2 (9) 26±2 (9)

lValues are means ± SEK. Nl.DJi)er in parenthesis EqliÙs ramter of dams. 2Signifiœnce of main effects fran analysis of variaooe: glucose ecJlivalent (NS) •

1 ~

.......

.......

,-. ~

'nml.e 3.12 Effect of r'D&e an! f:nrtnae clJriDJ POq81Cy CD ..temal hlnrd nib.~'

Dietary Ievel of: cartxnvdrate carl:xtlydrate 0 4 12 60

plasma urie aeid, llIJ/dl3

Glucose 1.9±l.O (8) l.7±l.O (8) l.l±l.l (6) 1.6±O.9 (9) Fructose S.7±O.9 (10) 3.S±1.1 (6) l.S±0.9 (9)

Glu + Fnl5 1.9±1.0 (8)a 4.8±O.6 (18)b 3.3±0.7 (12)ab 1.S±0.6 (18)a

plasma urea nitrogen, ng,Idl4

Glucose 20.7±4.6 (5) Il.6±1.6 (9) 8.3±1.8 (7) 6.4±1.6 (9) FrUct:œe lO.8±2.0 (6) 8.5±2.0 (6) 6.7±1.6 (9)

Glu + Fnl5 20.7±4.6 (S)a 11.3±1.4 (lS)b 8.4±1.S (13)00 6.6±1.3 (18)e

IValues axe means ± SEM. Nl.DIber in parentheses equals rutiler of pœ;JnaI1t rat dams. 2Means within eadl parameter havirg <lil!~ letter superscripts fOllowiD) the nmi'lp..r differ significant1J! (p<O.OS). Significarx:a of main effects fraB analysis of variance (HN): HN: level of carbohydrate (p<O.Ol) i so.u:oe of cart:x.nydrate (NS); leve1 x sooroe of cart:x:nydrate (NS). 4N:N: level of cart.xilydrate (p<O.OOO2); scmœ of cazt:dlydrate (MS); leve1 x scm:œ of cartxnydrate (NS). ~ theœ was no significant effect of source of cartxnydrate, the statistical analysis t:zeated all gl\XXlSe am fructose at each level of carbohyàrate as a si.n:)le group. 'lbeœfoœ, the signifi.c:anoe is J:epOrted for these grouped meaJlS.

1'f'Ii't.\

...... 00

e _)

~e 3.13

Glu.oose Ecpivalent

4% glucose 4% fzuctose

j • \

~, '" qJnCDSe _,,01 .. -'- • Effect of l.a.I œrIJctIjdI: te pœgnancy CIl _temal. nib:uja~~ ~Yca.ua.," dJeta ci.Jr.iDJ

Plasma Urie Acid3 Plasma Urea Nitrogen4

(Dg/dl) (Dg/dl)

4% lipid-glyœrol

6.6±1.1 (6)a 3.1±1.O (7)b 1.9±l.O (8)b

17.6±3.9 (7) 15.4±3.7 (8) 20.7±4.6 (5)

IValues are means ± SEM. NuIIiJer in parentheses eqna1 s n1lllher of dams. 2Means within eaàl ool\D1ll havirq different letter superscripts followirq the nmiler differ significantly (p<O.05). 3-4Significaooe of main effects fraD analysis of variance (NJV): 3NN: glucose EqUivalent (p<O. 02). 4NN: glucose EqÙvalent (NS) •

~ !

,;s ca:z;::; -.t ....... :z:::a::;c::;w::_l~_-: ... .: 53 _ a C ... !i.@5 .. e DS4 z:;;; __ ta> ....... ":.~7'

'-A \0

~ ~

'.DIbl.e 3.14 Effect: of gl.ŒXJSe ard mrtose ct.JriJJJ gestatial al a.Jjotic fluid glucose ard J.actate1,2

Diet:aIy level O( cartrbvdrate ca.rtxnydrate 0 4 l2 60

amniotic fluid glucose, Dg/dl3

Gluoose 8±2 (9)a 13±3 (9)ab 18±2 (9)b 34±2 (8)c Fructose l2±2 (lO)ab 16±3 (7)b 33±3 (lO)e

Glu + Fru5 8±2 (9)a 12±2 (19)ab 17±2 (16)b 34±2 (18)e

amniotic fluid lactate, Dg/dl. 4

Glucose 46±4 (8)ab 38±) (8)a 42±2 (8)a 52±1 (9)b Fructose 43±3 (lO)a 45±2 (7)a 47±2 (lO)b

Glu + Fru5 46±4 (8)ab 41±2 (l8)a 44±2 (lS)a 49±2 (19)b

IValues are means ± sœ. Nul'IiJer in paœntheses eqJal.s nmber of animals. 2Means within each parameter havin;J di~~~ letter superscripts followi.n;J the npnher differ significantly (p<0.05). Significanoe of main effects fraD analysis of varian:2 CAeN): 3NN: level of cartxlhyàrate (p<O.OOOl); sooroe of cadxilydrate (NS); level X sau:œ of cartxilydrate (NS). 4NN: level of cadxilydrate {p<O.Ol); scm:ce of carlxilydrate (NS); level x sœroe of caxtxhydrate (NB). 5When there was no significant effect of source of cadxilydrate, the statistica1 analysis b:eated all glucose am fruct:œe at each leve1 of cartx:bydrate as a s~le group. '1berefoœ, the significaooe is report.ed for these grooped means.

r,

00 o

t -~

'nml.e 3.15

Gluoose Fq.t.ivalent

4% glucose 4% fructose

t $

Effect of law cadxDydrclte 4' glucose apivalent djpts œriDJ gestatim CIl aII'lÏDtiC fluid CJlmœe aIIIlai::tatel,2

Gluoose3 Iactate4

(Bq/dl) (Dg/dl)

4% lipid-çlyœrol

8±1 (8) 6±l (9) 8±2 (9)

40±4 (8) 43±2 (8) 46±4 (8)

lValues are lŒ!aJlS ± SEM. Nlmi:Jer in parentheses ecpùs nDDher of animals. 2Means within eaàl oolumn havin:] different letter superscripts followin;J the npnher differ significantly (p<O.05). 3-4Significance of main effects fran analysis of variaooe CArN): 3ArN: glucose EqUivalent (NS) 4N:N: glucose ecJlÏ.valent (NS).

( ~

~

'.lable 3.16

DietaJ:y cart:dlydrate

Glucose Fructose Glu + Fru6

Glucose F:ructose

(Xl Glu + Fru6 1-'

Glucose F:ructose Glu + Fn16

".,

Effect of gJ'JC'X'1BI! ard fnrb .. WriD.J (K8JrBIICY cm -natic fl.uid nib~,

Ievel of carlxilvdrate 0 4 12 60

uric acid, lII}/dl3

1.5±O.5 (5)a 1.0±O.4 (8)b O.3±O.1 (6)c O.4±O.1 (7)c O.5±O.1 (7)b O.4±O.1 (5)c O.3±O.1 (7)c

1.5±O.5 (5)a O.8±O.2 (15)b O.4±0.2 (1l)e O.l±O.2 (14)c

urea nitrogen, Dg/dl4

15.0±2.5 (6)a 9.2±O.9 (8)b 9.8±O.7 (8)b 8.4±O.8 (8)b lO.9±2.0 (9)b 9.l±O.9 (6)b 7.9±O.7 (lO)b

l5.0±2.5 (6)a lO.l±O.9 (l7)b 9.5±1.0 (14)b 8.l±O.9 (l8)b

. pg/Jnl.5 aDIIIOI'11.a 1

5.86±O.58 (6) 6.01±0.60 (8) 7.2o±O.89 (8) 5.48±0.73 (7) 6.58±0.94 (9) 7.47±1.26 (6) 5.40±0.45 (9)

5.86±O.58 (6) 6.31±O.49 (17) 7.32tO.54 (14) 5.44tO.50 (16)

lValues are means ± SEM. NlIlliler in paœntheses ecpùs n1lli'ler of anilM)s. 2Means within eadl parameter haviIg different letter superscripts followin) the n.œr <liffer significantly (p<O.05). 3-5significame of main effects traD analysis of variance (AOV): lN:N: level of ~te (p<O.01); srurce of cartx::b.ydrate (NS); level x source of cartxilydrate (NS). 4NN: level of cartxil~te (p<O.OO5); sooroe of carbcilydrate (NS); level x sooroe of cartx::b.ydrate (NS). 5101: level of cart:xnyç1rate (NS); sooroe of carl:xilydrate (NS); level x scmœ of cartx::b.ydrate (NS). 6when there was n;) significant effect of source of cartx::b.ydrate, the statistical analysis tJ:eated all glucose am fructOse at eaàl level of cartxilydrate as a si.n:Jle groop. 'lbe.refoœ, the significawe is t'epOrted for these graJPEd tœanS.

D' ms tt lt ..... ttébrTt!l:nt';;;.. ... *çr:lirH~~_ ............... -l .... ....a.-...-...... _~.",t...,..w\ .. _~-

1*..

00 N

;

'l'able 3.17

Glucose EqUivalent

4% ql1JCX)Se 4% fructose

~ ;

Effect of lCII cad:xiIjdmte, 4' g1.ooœe &JlÏ.valent diets clJriDJ pn!II)[1iUq al mmiotic fluid nit:mgai1

Urie Acid2 Urea Nitrogen3 ADIIDûa4

(ngJdl) (I.&g/~)

l2.4±1. 7 (7) 5.92±O.72 (8) 11. 7±2.2 (9) 4.98±O.59 (8)

4% lipid-glyoerol

(ngJdl)

O.8±O.2 (7) 1.4±O.2 (9) 1.5±O.5 (5) l5.0±2.5 (6) 5.86±O.58 (6)

lyeues are means ± 3EM. NurIiJer in pazent:hesis equa1s nmi)er of animals. 2 Significaooe of main effects fran analysis of var.iaooe (NJV): 2NN: glucose EqUivalent (NS). JNN: glucose ecJ.rlvalent (NS). 4NN: glucose ecJrlvalent (NS).

""--'-"""""'-I-':"""<i" '<~" ~ .... ~ _4_ -~ ..... .,.-.,.-"' ...... ~:; --.----... .... "iie- .... 'l'II!'3!f..

, ,

00 w

,~

Table 3.18

Oietazy

"*'

'lbe effect; of Cjluoose am fJ:ucta;e dIr:ÏJJj gestatial al tbe weiiIrt of the febJs, the placenta ard the fetal-pl.aœntal œù.t at clay 211 ,2

Level of cartxnvàrate cartxhydrate 0 4 12 60

fetal weight, gl

Gluoose 3.81±0.04 (104)a 4.65±0.03 (130)b 5.32±0.03 (116)c 5.24±0.03 (132)c Fructose 4.74±0.03 (113)b 5.33±0.04 (99)c 5.19±O.03 (124)c Glu+Fru6 3.81±O.04 (104)a 4.69±O.02 (243)b 5.32±0.03 (215)c 5.22±O.02 (256)c

placental weight, g4

Gluoase 0.39±0.01 (104) 0.4o±O.OO (130) 0.4o±O.01 (116) 0.40±O.OO (133) Fructose O.43±O.01 (113) O.4o±O.01 (99) O.40±0.01 (124) GlutFn16 0.39±0.01 (104) 0.41±0.00 (243) 0.40±0.00 (215) O.40±0.00 (257)

fetal-plaoental weight, g5

Glucose 4.2o±O.04 (109)a 4.99±O.04 (130)b 5.67±O.04 (114)c 5.65±0.04 (132)c Fructose 5.16±0.04 (113)b 5.73±0.04 (99)c 5.60±0.04 (127)c Glu+Fru6 4.2o±O.04 (109)a 5.08±0.03 (243)b 5.7o±O.03 (213)c 5.62±O.03 (259)c

1Values are means ± SEM. NlJnt)er in parentheses equals ramiler of animals. 2Means within eaàl parameter havi.nj different letter superscripts followi.nj the n1l'li1er differ significantly (p<0.05). 3-5S ignificance of Dain effects fran analysis of variance (ADV): 3ADV: leve! of cartJahydrate ~O.OOOl); sa.troe of œrt:x:ilydrate (NS); lev el x sooroe of cartxilydrate (NS). MN: leve! of caztxilydrate (NS); souroe of c:artx:tlydrate (NS); leve! x souroe of carlxilydrate (NS). 5MN: level. of cart:dl.ydrate (P<O.OOOl); SOOI.'Oe of cartxi1ydrate (lIS); leve! x swroe of cartdlydrate (NS). 6Mten there was no significant eftect of swroe of carbc.tlydrate, the statistical analysis treated all gluoase am fructose ~t eadl leve! of cat:bc:ilydrate as a sin}le gxnJp. '1herefore, the signific:arDe is œported for these grouped means.

~"

(Xl ~

.II 4 • •

iable 3.19

e ;.

Effect of lor.r cadx:iJjdrat:e, 4' CJlIKXlSe BlJÏ.valent diets mring gestatial al the ~ of the fetus, the placenta and the fetal-plaœntal. unit at day 211 ,2

WeiClht Glucose Equivalent FeblsJ ---- - Plaœnta4 Fetal-Plaoental Unit5

4% Glucose 4% Fructose 4% Lipid-glycerol

(g) (g) (g)

4.4o±O.04 (92)a 0.46±0.01 (92)a 4.86±0.04 (92)a 3.60±0.04 (llO)b 0.38±0.01 (llO)b 4.03±0.03 (126)b 3.Bl±O.04 (l04)ab 0.39±0.01 (104)b 4.20±0.04 (109)b

IValues are means ± SEH. Number in pa.rentheses equals ramiler of fetuses. 2Means within each oollnnn havirg different letter superscripts followl..rg the nJl'J'bar differ significantly (p<0.05). 3-5Significaooe of main effects fran analysis of variance ~ADV): 3ADV: gl~ EqUivalent (p<0. 05). 4NN: glucose elJlÎvalent (p<O.02). 'MN: glucose eqw.valent (p<O.05).

r \ . (

cr w- _

00 VI

~ ,

'DIble 3.20

Dietazy Cëntxilydrate

GIUCXJSe Fructose Glu + FIu6

o

58±14 (4)

58±14 (4)

~ ,

gestatia1 CD fetal

Isvel of cartx:ilvdrate 4 l2 60

blood qlUCXJSe, DIJIdl3

77±10 (9) l03±10 (9) 88±10 (9) 82±10 (9) 96±13 (5) 81±9 (10) 79±7 (18) lOl±8 (14) 84±7 (19)

liver qlyoogen, 1II3Ict' Glucose 9.9O±O.96 (26)a 38.5l±2.71 (2l)b 4l.23±2.09 (38)b 6l.56±2.21 (30)c Fructose 35.23±2.85 (22)b 35.58±1.99 (37)b 63.76±2.20 (29)c Glu + rru6 9.90±O.96 (26)a 36.87±2.54 (43)b 38.4l±1.45 (75)b 62.66±2.21 (59)c

plasma triCJlyoerides, DIJIdl5

GIUCXJSe 65±5 (6) 64±4 (10) 66±4 (9) 62±4 (10) Fruct:cse 62±4 (8) 6l±5 (6) 54±4 (9) Glu + FJ:u6 65±5 (6) 63±3 (18) 64±3 (15) 58±3 (19)

1Values are means ± sm. N\JIIi:)er in paœntl1esis nmœr of fetuses. 2Means within each parameter havÎD:) different letter superscripts follCJWÏn) the nmiler differ siqrrl.ficantly (p<O.05). 3-6giqrrl.ficaooe of main effects frcm analysis of variame (NN): 3101: level of ca.nxil~te (IfS); soucce of au:txnydrate (NS); level x swrœ of cadxbydrate (NS). 4NN: lavel of au:txnydrate (p<O.OOOl); sau:œ of carl:x:ilydrate (NS) : level x sooroe of carlxi1ydrate (NS) • 5AO\T: level of ~te (NS): sooroe of cazt:x:ilydrate (NB): level x sooroe of au:txnydrate (NB). ~ theze was no significant effect of sooroe of au:txnydrate, the statistica1 analysis tœated all qlUCXJSe and fructose at each level of au:txnydrate as a si.rgle gralp. 'lberefc,œ, the signifiœooe is reported for these grruped meaJ1S.

f ~

00 0\

~ ~ "

'nIbl.e 3.21 Effecl: of law au::IxDJIdmte 4' 1 gestat.:i.c:D cm f~-" ' 1 2 9 ''''CRe EQÙ.valent di ...... Ao~ __ -=-a. paL t:.ers, ---'"::1

Glucose Fqllvalent

Plasma Glucose3

(Bq/dl)

Liver Glyoogen4

(1IIJ/g)

4% glucose 57±10 (8) 20.70±O.85 (28) 4% fructose 52±10 (8) lO.30±O.95 (37) 4% lipid~lycerol 58±14 (4) 9.90±O.96 (26)

Plasma Triglycerides5

(Dg/dl)

61±5 (6) 57±4 (9) 65±5 (6)

lValues are means ± SEM. NI.miJer in paœnthesi.s equal.s nmtler of fetuses. 2Means within eadl oolUllll havirg different letter Slp!rSCI'ipts follawinj the nmber differ significantly (p<O.05). 3-6significaooe of main effects ftaD analysis of vari.aooe (AaJ): 3N»: glucose ecJ1lvalent (NS). 4N»: glucose BJÜvalent (NS). 5NN: glucose ecJlÏ.valent (NS).

tJI"iL,

00 .......

e )

Table 3.22

Dietazy camabydrate

Glucose Fructose

Glu + Fl:u6

ç ,

Effect of CJlWXBe ard fI:uch.. œriDJ f)EiitaUm CIl :fetal. ptAli'l1oGI3-. l.actate1,2 .

o

68±6 (6)

68±6 (6)

Isvel of Cénixilvdrate 4 12

fetal lactate, Dg/dl3

57±6 (10) 59±6 (9)

58±4 (19)

67±6 (9) 56±7 (6)

62±5 (15)

60

47±6 (9) 59±6 (10)

54±4 (19)

lValues aIe means ± Saf. NuniJer in parentheses ecpùs nmber of fetuses. 2Means within each parameter havirg diq:eœnt letter superscripts followirg the n1lllher differ significantly (p<0.05). Significance of main effects frai analysis of variance (ADV): 1evel of ~te (NB): source of caztx:ilydrate (NB): level x source of cad:xtlydrate (NS). 4tivm there was J'X) significant effect of sa.u:œ of cartxilydrate, the statistical analysis aeated all glucose am fn1ct:œe at each level of cartxilydrate as a si.rgle groop. 'lberefore, the significawe is ~orted for these grwped meaIlS.

f ~

--,

00 00

~

Table 3.23

Gluc::cse F4llvalent

4% glucose 4% fxuct:ose

-------.,.-... ". ~---.--- ~*r.:ehIIî?-; au:;:' >-, - i? waal

~

Effect. of low -~-~.-;-. -.-.-"..=---.ru-- '" g1Jxxse 8IJ.Û.valent diets --. .... '";:1 ~...-.......... al fetal. J..acta.té1,2

Plasma lactate3

(Dg/dl)

4% lipid-qlyc:erol

6l±6 (7) 58±S (9) 68±6 (6)

IValues aœ means ± SEM. Nlmi:Jer in pu:entheses Eqt;3)S l'a.IIIber of fetuses. 2Means within eac:b oolUll'll havin} different letter superscripts folloilliDJ the J'Ult)er differ significantly (p<O.OS). lSignificaJ'Dl of main effects f1:aa analysis of vari.ar'al (NJV): glucose E!CJlivalent (NS).

~

00 \0

: )

Table 3.24

DietéUy cartxilydrate

Glucose Fructose

Glu + Fnl6

~ t

Effect.,. of gllKXl68 ard fEnctDfle œriDJ 9*'tatim CD fetal urie ~,~

Level of Cartlà'lvdrate o 4 12 60

~~2

1.2±0.3 (4) 0.9±0.1 (9) 0.6±0.2 (8) 0.5±0.1 (10) 0.6±0.2 (7) 0.7±0.2 (6) 0.6±0.1 (10)

1.2±0.3 (4) 0.8±0.1 (16) 0.6±0.1 (14) 0.6±0.1 (20)

IValues are means ± SEX. Nuni:ler in parentheses equals IJ.JIItler of fetuses. 2Significan:::e of main effects fran analysis of variance (AOV) : level of cartx:b~te (NS); soorce of cartx:bydrate (NS); level x soorce of caJ:tx:bydrate (NS). ~ theœ was no significant effect of soorce of caJ:tx:bydrate, the statistical analysis treated all glucose an:i fruct:œe at eadl level of cartxJhydrate as a s.i.rçle groop. 'lberefore, the significance is replrted for these graJped means.

c

\D o

-~"~h 1 oS '., .P~........... -. --.... ~i!f .... _ =_c::c:u.::::..!. ..... ll J ... __ ..,~ ~;c:..-:; ~ ..... "'-.-..,.---- -

.-""",, ~

Table 3.25 Effect of la.r cadxiJ;ydrate 4' gl.1DB! a:pi.val.ent. diets cmrin) gest:ati.cm cm fetal urie ac:UP-,2

Glyoerol Equivalent

4% glucose 4\ fructose 4\ lipid-glycerol

Petal Urie Acid (Dg/dl)

1.0±O.2 (9) L1±O.2 (7) 1.2±O.3 (4)

IValues are means ± SEM. NuIIi:Ier in paœnthesis f"'JY'ls nJrnber of fetuses. 2Significance of main effects fran analysis of variance (AOV): 3AOV: glucose equivalent (NS).

,--

-

-

400

350

300

250

2CO

150

100

50

0

Figure 3.1 Food Intake Graded Levels of Dietary Glucose

GRAMS

--- 0 ...

-1- 4 ...

-;jE- 12 ...

-a- 60 ...

2 4 6 8 10 12 14 16

DAYS

Triglyceride baud dleta

91

18 19 20 21

1

(

(

(

Figure 3.2 Food Intake Graded Levels of Dietary Fructose

GRAMS

400 1 350 l 300 ~

~ 0'4

-+- "''4

-+- 12'4

-B- 60'4 1 250 ~ __ ...

200

150

100

50

2 4

Triglyceride ba .. d dleta

6 8 10 12 14 16 DAYS

92

18 19 20 21

.... Figure 3.3 Weight Gain Graded Levels of Dietary Glucose

GRAMS 180

60GA. c 140 - 0'

~ .. , '2%~

120 -iIP- 12 .. 4GA. b ~

100 80 ..

80

80 0% a

40

20

0 3 5 7 9 11 13 15 17 19 21 23 25

OAYS

Triglyceride b ••• d dleta

4'\>-

~

93

•

(

GRAMS

Figure 3.4 Weight Gain Graded Levels of Dietary Fructose

160~----------------------------------______ ___

3 5 7 9

Triglyceride ba .. d dleta

60% c 12% c

4% b

0% a

11 13 15 17 19 21 23 25 DAYS

94

.

Gf,AMS

FigtJre 3.5 Food Intake 4% Glucose Equivalents

400~---------------------------------------------

350 - .... Lipid-Glyc.rol

-+- .... Glueo ••

300 '""*"' .... Fructo ..

250

200

150

100

50

2 4 6 8

Fr b

10 12 14 16 18 20 22 24

DAY

95

\ Figure 3.6 Weight Gain .. 4% Glucose Equivalents

GRAMS 100

" .. Llpld-Glyc.rol

00 -+- " .. Glueo ••

""'*'" " .. Fructose GI a

60 L-G ab

Fr b

40

i

20t ~

! ! ! a 3 5 7 9 11 13 15 17 19 21 23 25

DAY

.. 1 ,

( 96

.'

........

Figure 3.7 Dam liver weight Triglyceride-based diets

GRAMS 15~------------------------------------------~

- GLUCOSE

14 -+- FRUCTose

13

9 --------------~--------------~------------~ 0% 4% 12% 60%

LEVEL OF CARBOHYDRATE

97

(

(~

(

4.4

4.2

4

3.8

Figure 3.8 Dam liver wt as %body wt Triglyceride-baaed dleta

- GLUCOSE

-+- FRUCTOSE

3~------------~--------------~--------------0'4 4'4 12~ 60%

LEVEL OF CARBOHYDRATE

98

-

.......

CKAPTER 4: DISCUSSION

The ability of dietary fructose to fully replace dietary

glucose during pregnancy in the rat has been investigated by

comparing isocaloric sU'bstitution of fructose for glucose

using a wide range of concentrations of dietary carbohydrate.

This is the first comprehensive study of the effect of dietary

fructose during pregnancy in the rat. Bourne et al in 1975

authored the only other study to investigate the effect of

dietary fructose during p.regnancy; however, they fed only very

high carbohydrate diets (68%). Bourne et al (1975) concluded

that the source of dietary carbohydrate affected maternaI

lipid metabolism during late pregnancy by significantly

increasing maternaI hepatic lipogenesis and maternaI plasma

triglyceride concentration, but they did not observe an effect

of diet upon the fetus (fetal weight, plasma insulin, and

fetal lipogenic activity). In the present study an effect of

the source of carbohydrate upon maternaI plasma triglyceride

levels was not observed, although, like Bourne' s study, no

effect of carbohydrate source upon fetal parameters was noted

in the high carbohydrate diets. This study fed fructose at

five different levels, ranging from a 4% glucose equivalent

to a 60% carbohydrate diet, and measured a number of

parameters that Bourne et al (1975) did not investigate.

These parameters were measures of carbohydrate, nitrogen and

lipid status, as weIl as body and liver weights. It appears,

from these parameters, that only at high carbohydrate

99

(

(~

(

concentrations of 60% (using triglyceride based diets) or low

concentrations of 4% glucose equivalent (using fatty acid

based diets) do dietary differences between glucose and

fructose occur. No differences due to the source of

carbohydrate, be it glucose or fructose, occurred when dams

were fed intermediate amounts of glucose or fructose. The

results from the present study en.phasize the importance of

the level of dietary carbohydrate or i ts precursors on the

essentiality of a carbohydrate source for reproduction.

Fructose Toxicity

One important aspect of our investigation into the

substitution of glucose by fructose during pregnancy, was the

potential toxicity of dietary fructose when fed at high

concentrations. Two of the blood parameters used to measure

the potential toxici ty of fructose at high dietary

concentrations have been lactic acid (Reiser 1987) and uric

acid (Bode et al 1973; Israel et al 1983; Reiser 1987).

Chronic feeding studies, measuring lactic or uric acid

concentrations in the blood, have not previously been done on

the rat, nor have lactic acid measurements been made wi th

humans in feeding studies. However, studies in humans have

shown uricacidosis but these had confounding variables.

Israel et al (1983) used carbohydrate-sensitive individuals

and Emmerson (1974) used purine free diets. It appears that

normal feeding patterns do not result in toxic concentrations

of fructose in the maternaI and fetal systems of the rat as,

100

ft

"'-'"

....

in the present study, neither of these acids were higher in

fructose fed dams compared to glucose fed dams at any level

of dietary carbohydrate.

In non-pregnant rats, it is commonly known that diets

high in fructose raise the concentration of plasma

triqlycerides (Zakim et al 1967; Bruckdorfer et al 1972;

Chevalier et al 1972b; Maruhama and MacDonald 1972; Waddell

and Fallon 1973; Vrana et al 1974a,b,1976,1983,1988; Waterman

et al 1975; Heller et al 1977; Kanq et al 1979; Merkens et al

1980; Sleder et al 1980; Aoyama et al 1980,1981,1982,1987;

Kannan et al 1981; Bird and Williams 1982; Zavaroni et al

1982; Hostmark et al 1982,1984; Bird et al 1984; Verschoor et

al 1985; Herzberg and Rogerson 1986, 1988a,b), although a tew

studies have not reported these resul ts in aIl cases

(Chevalier et al 1972b; Naismith and Rana 1974; Kang et al

1979). Bourne et al (1975) had found a significantly higher

concentration of plasma triglycerides in pregnant dams fed

high fructose diets (271 mg/dl) than dams fed high glucose

diets (149 mg/dl). The present study did not find that the

presence of fructose in the diet increased the concentration

of plasma triglyceride in th€: maternaI system. The values for

plasma triglycerides in the present study were 235 and 269

mg/dl for dams fed 60% fructose and 60% glucose respectively.

These values are higher than other values for pregnant rats

reported by Champigny and Hitier in 1987 (88-176 mg/dl) and

101

(

{

(

within the range of wapnir and stiel's in 1985 (142-280 mg/dl)

and Bourne et al in 1975 (149-271 mg/dl).

Plasma triglycerides were significantly higher in dams

fed 4% carbohydrate triglyceride based diets than dams fed

aIl the other levels of carbohydrate. It appears that the

presence of 4% carbohydrate -in very high fat diets is requirt:..l

in order to raise the plasma triglyceride levels to very high

concentrations. Dams fed a very high fat diet lacking

carbohydrate had levels of triglycerides in the maternal

plasma significantly lower (484 mg/dl) than dams fed diets

containing 4% carbohydrate. Dams fed the two fatty acid based

diets containing 4% glucose or fructose had plasma

triglyceride concentrations (929-1268 mg/dl) similar to the

dams fed the 4% glucose and fructose triglyceride based diets

(889-985 mg/dl).

From these observations, it seems that the metabolism of

glycerol in the maternaI system is different from that of

fructose and glucose. The maternaI liver's capacity to take

up glycerol is extremely high (Casado et al 1987). However,

the glycerol is bound into a triglyceride molecule and

therefore the maternaI system must be breaking down

triglycerides in order to extract the lipid-glycerol, the only

source of glucose in the diet. There 1s no source of

carbohydrate to spare the use of glycerol by the maternaI

system. Perhaps, then, the maternaI system must be very

102

..........

strongly geared to metabolize the lipid-glycerol and less

lipid as triglyceride can then circulate in the plasma.

The weight of the maternaI liver, measured as an absolute

value or as a percent of body weight, was significantly higher

in dams fed 60% fructose than dams fed 60% glucose. Bourne

et al (1975) also measured the liver weight of pregnant rats

which were fed 68% fructose or glucose and found a non­

significant trend for the increased liver weight in the

fructose fed dams. The increase in liver weight agrees with

many previous reports in the li terature where non-pregnant

rats fed high fructose diets (ranging from 66.1 to' 80.68%) had

liver weights significantly greater than rats fed a similar

concentration of dietary glucose (Allen and Leahy 1966;

Bruckdorfer et al 1972; Michaelis and Szepesi 1973; Naismith

and Rana 1974; Romsos and Leveille 1974; Waterman et al 1975;

Aoyama et al 1980,1982,1987; Bird and Williams 1982; Vrana et

al 1983; Fields et al 1985). In many studies this difference

has been accounted for by an increased liver lipid content

when rats are fed fructose (Allen and Leahy 1966; Bruckdorfer

et al 1972; Michaelis and Szepesi 1973; Naismith and Rana

1974; Romsos and Leveille 1974; Aoyama et al 1980; 1982; Bird

and Williams 1982). We could not measure any differences in

lipid content between the livers of dams fed either 60%

glucose or fructose. others have also failed to measure a

significant difference in liver lipid between rats fed high

fructose vs high glucose diets (Zakim et al 1967; Herzberg

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and Rogerson 1986,1938a,b; Aoyama et al 1987). The Roese­

Gottlieb method used to measure the liver lipid concentration

ih this study extracted mainly triglycerides, not

phospholipids. However, it would be surprising if it were the

phospholipid fraction of the liver which caused the increase

in liver weight.

We did not measur~ any differences between liver water

content so this can not account for the difference in liver

weight in this study. others have also noted that the water

content of the liver is not affected by a high fructose diet

(Bruckdorfer et al 1972: Kang et al 1979). Hepatic glycogen

concentrations have been reported to be higher in fructose

fed rats than in those fed equivalent amounts of glucose when

rats were meal-fed (Kang et al 1979; Herzberg and Rogerson

1988b) but not in rats fed ad libitum (Vrana et al 1978; Rang

et al 1979) as our dams were. In the present study there were

no significant differences in liver glycogen levels between

glucose and fructose fed dams. However, there appeared to be

a trend towards a greater liver glycogen content in dams fed

60% fructose (48 mg/g) than in dams fed 60% glucose (40 mg/g) .

This difference could possibly account for some of the

difference in liver weight between the dams fed diets high in

fructose and glucose.

The reason for the enlargement of the liver is not clear;

nor, do we know whether it is a toxic symptom. This is the

104

only parameter which indicates that at high levels fructose

May be metabolized differently than glucose.

Fructose Replacement of Glucose

Another important aspect to this study was to question

the ability of fructose to fully replace glucose and maintain

the carbohydrate status of the mother and fetus. Earlier

studies had shown that dietary glucose could not be replaced

by dietary protein during pregnancy (Koski et al 1990)

indicating that the requirement was specifie to dietary

carbohydrate. The present study investigated whether the

essentiality of carbohydrate during pregnancy was specifie to

one carbohydrate, glucose, or whether another carbohydrate,

fructose, could fill the requirement. Both fructose and

protein must enter the gluconeogenic pathway in order to be

converted to glucose and glycogen, although amino acids enter

lower in the pathway (eg. alanine --> pyruvate) than fructose

(--> 3-carbon phosphates). It appears, in general, that

fructose can fill the carbohydrate requirement during

pregnancy and, like qlucose, the level, not the source, is the

most important indicator for determining optimal reproductive

outcome as measured by several parameters (maternaI and fetal

liver glycogen: maternaI, fetal and amniotic fluid glucose and

lactate) •

MaternaI and fetal liver glycogen concentration, though

not significantIy affected by the source of carbohydrate, were

profoundly affected by the levei of carbohydrate in the diet;

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as the dietary carbohydrate level increased so did the content

of maternaI liver glycogen. These results support previous

observations (Koski et al 1986). The maternaI control values

given in the former study by Koski et al in 1986 (39.4 mgjg)

were similar to those of our study (40.17 mgjg) , as were the

values of dams fed 12% carbohydrate (22.4 vs 18.53 mg/g) and

0% carbohydrate (4.2 vs 4.94 mg/g). The fetal control values

given in the study by Koski et al in 1986 (127 mgjg) were

higher than those of our study (61.56 mgjg) , as were the

values of fetuses of dams fed 12% carbohydrate and 0%

carbohydrate. This is probably due to a combination of 1) the

different methods used in glycogen determination: the present

study used the Lo et al (1971) lnethod while Koski et al (1986)

used the Seifter et al (1950) method which is less specifie

and measures total redueing equivalents: and 2)the different

fetal bleeding methods: the present study bled from the

axillary vain while Koski et al (1986) eut open the thoracic

eavity and drained the fetus of bled. The second bleeding

method would lead to more blood glucose retained in the fetal

liver whieh would be counted as glycogen in the glycogen

determination used. other authors have reporteà fetal

glycogen eoncentr3.tions similar to those observed in this

study: Ogata et al (1986) with 52.8 mgjg; Freund et al (1980)

88 mgjg; Margolis (1983) 74.67 mg/g, using a similar

methodology te the present study.

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.....

The increasing level of carbohydrate in the diet also

led to greater concentrations of glucose measured in the

amniotic fluide This pattern parallels that of the glycogen

content of both the maternaI and fetal livers. It appears

that maternaI and fetal liver glycogen and amniotic fluid

glucose are most influenced by changes of dietary carbohydrate

intake and can perhaps act as reservoirs for glucose \ihen it

is present in excess of the requi red amount needed by the

maternaI or fetal system. Amniotic fluid glucose is the Most

easily accessible parameter which could act as an nutritional

indicator of the carbohydrate status of the mother and fetus.

Another metabolite which roughly followed this pattern

was maternaI lactic acid concentrations, as dams fed 0 and 4%

carbohydrate had lactic acid levels significantly lower than

dams fed 12 and 60% carbohydrate diets (triglyceride based).

Romsos et al (1981) observed a 40% decrease in maternaI plasma

lactate levels in the pregnant dog fed a 0% carbohydrate diet

compared to bitches ingesting a 44% carbohydrate diet. This

decrease was not statistically significant. The present study

and that of Romsos et al (1981), which fed high and low

carbohydrate diets, have resul ts similar to those studies

which compare lactic acid levels in fed and fasted pregnant

rats (Freinkel et al 1972; Girard et al 1975) or guinea pigs

(Gilbert et al 1985) where fasted mothers have lower lactic

acid concentrations than the fed mothers. uteroplacental

production of lactate is dependent upon the delivery of

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glucose from the maternaI system (Battaglia and Meschia 1988)

and this would be compromised in dams fed low carbohydrate

diets.

Amniotic fluid lactate concentrations followed an unusual

pattern: in the 4% to 60% carbohydrate diets, amniotic fluid

lactate significantly increased with increasing dietary

carbohydrate. However, the amniotic fluid lactate

concentration of dams fed 0% carbohydrate did not differ from

lactate values of any ether diet and the actual lactate value

(at 0%) lay between the values at 12 and 60% carbohydrate

diets. No information could be found on amniotic fluid

lactate concentrations in the rat.

In this study the nitrogen metabolism of the maternaI

system and the amniotic fluid was also investigated. When

the availability of glucose is limited in an animal,

gluconeegenesis from amino acids can occur. The oxidation of

amine acids produces ni trogen, as ure a and ammonia (Freinkel

et al 1972). In the present study, when the dietary

carbohydrate was low and carbohydrate status impaired,

amniotic fluid urea nitrogen and urie acid and maternaI urea

nitrogen were raised.

In the fedjfasted model, where "accelerated starvation"

is observed (Freinkel et al 1972), an increase in total

urinary nitrogen on the first day of fasting was due te the

presence of both urea and ammonia. However, on the second

day of fasting the increase in urinary nitrogen was almost

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ft

..... entirely due to ammonia. Herrera et al (1969) also observed

that urinary ure a accounted for a greater portion of urinary

nitrogen than ammonia on the first day of fasting while

urinary ammonia increased by the second day of fasting.

However, in Herrera's study urea still accounted for half the

urinary ni trogen . The increase in ur inary ni trogen was

attributed to gluconeogenesis from amino acids in the liver

(urea) and the kidney (ammonia) (Freinkel et al 1972; Herrera

et al 1969). N'either study measured blood values for urea or

ammonia. In this study amniotic fluid ammonia was not

affected by diet. However, the measurements may not

accurately reflect the ammonia concentrations in vivo because

the amniotic fluid was frozen and thawed a number of times

before ammonia analysis and therefore, ammonia concentrations

could have been altered, particuiarly as arnrnonia

concentrations are not very stable (Mondzac et al 1965). The

ure a levels in both the maternaI plasma and amniotic fluid

decreased as the level of dietary carbohydrate increased which

correlated with decreased hepatic gluconeogenesis which would

be required in dams receiving increased dietary carbohydrate.

It is possible that urea was also produced from amino acids

entering the gluconeogenic pathway in the fetal liver as

Girard et al (1977) have demonstrated premature fetal glucose

synthesis in cul tured hepatic slices after four days of

maternaI fasting. However, fetal gluconeogenesis has not been

observed in vivo after 24 hours of maternaI fasting (palacin

109

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et al (1987). Therefore, it is not clear based on distinctly

different methodological approaches whether or not fetal

gluconeogenesis could occur when the pregant dams are fed very

low carbohydrate diets. Unlike the results in this study,

Romsos et al (1981) did not observe a difference in maternaI

plasma ure a ~itrogen levels between pregnant dogs fed a 0%

carbohydrate diet and bitches fed a 44% carbohydrate diet.

The concentrations of both maternaI triglyceride and urie

acid followed the same pattern in that dams with high

triglyceride levels had high uric acid levels and those with

low triglyceride levels also had low uric acid levels. Dams

fed 4% dietary glucose or fructose, regardless of the lipid

source, had the highest concentrations. This relationship is

weIl documented in humans, particularly unhealthy individuals

with a disturbance in their earbohydrate metabolism (eg.

diabetic, glucose intolerant, obese, etc.), with urie acid

levels being high when triglyceride levels are high (Fox et

al 1985; Bastow et al 1988; Modan et al 1987; Halkin et al

1988). The reasons for this relationship are not clear and

a mechanism has not been proposed. However, both have been

associated with hyperinsulinemia (Modan et al 1987; Halkin et

al 1988) and cation imbalance (Halkin et al 1988) of which

insulin is a regulator.

Early reproductive parameters were aIse affected by the

level of dietary carbohydrate in the diet. The resorption

rate was significantly higher in dams fed 0% carbohydrate

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compared ta dams fed 4, 12 or 60% carbohydrate. Therefore,

there is a requirement for dietary carbohydrate to ensure

fetal growth. Resorptions occur early in gestation, before

the pregnant animal is fully stressed by the lack of dietary

carbohydrate and the compromised carbohydrate status. The

mechanism behind the increase in resorptions is not known.

A lack of circulating glucose may be a possible signal as

glucose is considered the principle form of metabol izable

energy used by the embryo (Giroud 1970). Studies using embryo

culture have shown that exposure of the embryo to ketone

bodies can be toxic, producing abnormal morphogenesis (Horton

and Sadler 1983). Perhaps, the toxic effect of ketone bodies

could lead to increased resorptions of the embryo and early

fetus. However, it is known that in late fetal life that

ketone bodies can be an important source of energy for the

fetus when the glucose supply is compromised (Rudol f and

Sherwin 1983).

Cumulative food intake did not differ among the dams fed

the triglyceride based diets. Total weight gain jur ing

gestation increased wi th the level of carbohydrate in the

diet. Similar results were reported by Koski et al (1986)

al though the actual weight gained was less than hal f tile

values reported in this study. Weight gain was not different

between dams fed high fructose and high glucose diets in the

study of Bourne et al (1975) and no differences were found

between glucose and fructose fed dams in the present study.

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1) 1 1

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The weights of the fetus and the fetal-placental units,

like maternaI weight gain, increased as dietary carbohydrate

increased. Koski et al (1986) also reported an increasing

fetal weight with increasing concentrations of dietary

carbohydrate. As the concentration of carbohydrate in the

diet increased, the following parameters increased: maternaI

weight gain, fetal weight, fetal-placental unit weight,

resorption and in utero death rates, maternaI and fetal liver

glycogen, amniotic fluid glucose and maternaI plasma lactate;

and while others decreased: percent live pups, maternaI urea

nitrogen and amniotic fluid urea nitrogen and urie acid.

These results reinforce earlier studies (Koski et al 1986)

which demonstrated that carbohydrate is an essential component

of the diet during pregnancy in the rat.

Carbohydra~e source in very lov carbohydrate die~s

The effect of the source of carbohydrate was also

investigated when animaIs were fed low carbohydrate diets.

This experiment could then determine whether at low levels,

dietary fructose or lipid-qlycerol could fully replace dietary

glucose. Like the results from animaIs fed diets that were

triglyceride based, the source of glucose equivalent did not

affect the parameters that measured carbohydrate status.

However, there were important measurements that were impaired

by the substitution of glucose by fructose or lipid-glycerol,

Ieading to the conclusion that, though carbohydrate status per

se was not affected, the health of the dams and fetuses were

112

c::œpranised by the presence of only fructose or lipid~lycerol

in these low ca:rixlhyàrate cliets.

Fructose was a less successful carOOhydrate source than

glucose. Dams fed 4% fructose had lower focxi intake, maternaI

weight gain am fetal, placental am fetal-placental unit

weights than dams fed glucose or lipid-qlycerol. '!he low focxi

intake of dams fed fructose could have contributed ta the

lower weights of the ether parameters, aIl of which were

significantly lower than dams fed glucose. '!he diet was

of fatty acids, an unusual source of lipid, and

fnlctœe, a relatively unc::cmoon sugar, a canbination which may

have led ta the la.Ner food intake of these dams conpared ta

ethers.

Glucose fed dams also had a placental weight greater than

dams fed fl:uctose or lipid~lyc:erol. In fact, the placental

weight ~ higher than dams fed all the other diets,

incll.1Ciirç 60% carbahydrate, at 0.46 qrams conpared ta an

average of 0.40 grams, respectively. '!he reason behirrl this

enlazqed placenta is net clear. However, the enlarged

placenta! might have produc::ed a greater placental: fetal weight

ratio which pemaps was necessary for fetal sw:vi val. '!he

lower fetal weights of the fructose am lipid-glycerol fed

arûmals, pertlaps, did net imuce sudl an adaptive increase in

placental weight.

Regardi.n:;J the reproductive perfonnanca of the dams,

fructose fecl dams did net cliffer fran glucose fed dams, while

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dams fed lipid-glycerol had a high rate of in utero deaths.

It appears that qlucose at this low dietary level can not be

fully replaced by lipid-glycerol. The result of this

substitution of glucose by lipid-glycerol was a large number

of in utero deaths (27%). clearly the absence of dietary

carbohydrate, either as glucose or fructose, has a very

negative impact on fetal mortality as aIl diets containing

even 4% carbohydrate had significantly lower in utero deaths.

Dams fed very low carbohydrate diet in previous studies have

delivered one day later than dams fed high carbohydrate diets

(Koski and Hill 1986: Koski et al 1989: Koski and Hill 1989).

The delay in development may result in fetuses which can not

cope with aC-section delivery at day 21 of gestation because

maturity of the fetus is reached at day 22. Curiously, in a

previous study by Koski and Hill (1989), dams fed a 4% glucose

fatty acid based diet had a greater stillbirth rate than dams

fed a 0% carbohydrate triglyceride based diet. However, these

dams were allowed to deliver naturally and a day later than

the dams in this study.

In surveying the results of the animaIs fed the

triglyceride based diets, one notes that in three parameters

measured, the 0% carbohydrate diet behaves unusually.

MaternaI plasma triglyerides and uric acid concentrations in

dams fed 0% carbohydrate are lower than dams fed 4%

carbohydrate while maternal triglycerides and uric acid levels

decrease from 4 to 60% carbohydrate. Amniotic fluid lactate

114

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concentrations in dams fed 0% carbohydrate do not differ

significantly from dams fed any other level of carbohydrate.

However, amniotic fluid lactate levels increase in dams from

4 to 60% carbohydrate. These results may be due to the slower

development of the fetuses of 0% carbohydrate fed dams (22 day

gestation) than fetuses of dams fed diets with carbohydrate

present (21 day gestation) •

The present study did not demonstrate, in general,

differenees between pregnant rats and their fetuses fed

glucose or fructose. It appears that the mother acts as a

buffer for the fetus, efficiently converting fructose to

glucose, a fuel the fetus can use. It is known in non­

pregnant rats that there is an adaption to chronic feeding of

dietary fructose. Fructolytic enzymes, such. as fructokinase

(Chevalier et al 1972), fructose-1-phosphate aldolase (Heinz

1972) and triokinase (Heinz 1972), increase while glucose-6-

phosphata se (Freedland and Harper 1957) decreases in fructose­

fed animals. These changes allow a rapid conversion of

fructose to glucose. If this also oceurs in the pregnant

animal then dietary fructose would be essentially equivalent

to dietary glucose in its ability to supply metabolizable

glucose to the mother and fetus.

Toxic effects of fructose were not evident in the

pregnant rats and fetuses. It appears that fructose feeding

does not overload the liver as acute loads do and that the

115

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dams under chronit:: feeding conditions can metabolize fructose

without producing high amounts of lactic acid and uric acid.

Romsos and Leveille (1974) have shown that fructose feeding

increases the ATP concentrations in the liver of non-pregnant

rats. This may be a.1 adaptation to dietary fructose which

prevents the depletion of ATP which occurs with acute loads

of fructose and which also leads to increased uric acid

production.

Although the present study does not produce any results

which strongly suggest dietary fructose during pregnancy is

toxic, this does not mean that fructose as the maj or dÎE!tary

carbohydrate component can be used wi th equanimi ty . Many

parameters (eg. plasma insulin and glucagon and tissue trace

metal levels) have still not been measuredi the negative

consequences, if any, of the enlarged liver in dams fed high

fructose diets in this study; the disturbances in maternaI

lipid metabolism shown by Bourne et al (1975) have not been

elucidated: finally, it is not known whether these results

would be the same as those using diabetic animaIs or whether

the combination of diabetes and fructose could produce

markedly different effects upon carbohydrate and Iipid

metabolism in the mother and fetus. The resul ts of the

present study do suggest that moderate levels of fructose are

not harmful to the pregnant rat. Finally, it should be noted

that this study was conducted on rats and extrapolation of

these results to the human must be do ne with extreme caution,

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preferably with further evidence from human studies during

pregnancy.

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APPENDIX 1: STATISTICAL METHODS

l)Triqlycerida basad diets:

a) maternaI, amniotic fluid and fetal data wi th one pooled value per dam:

PROC GLM; CLASS CHOL CHOS; MODEL GLUCOSE = CHOL CHOS CHOL*CHOS; CONTRAST '0 VS 4'CHOL -1 1 0 0; CONTRAST '0 VS 12'CHOL -1 0 1 0; CONTRAST '0 VS 60'CHOL -1 0 0 1; CONT~ST '4 VS 12'CHOL 0 -1 1 0; CONTRAST '4 VS 60'CHOL 0 -1 0 1; CONTRAST '12 VS 60'CHOL 0 0 -1 1;

b)interaction between level and source of carbohydrate:

PROC GLM; CLASS CHOL CHOS; MODEL MLIVWT = CHOL CHOS CHOL*CHOS; CONTRAST 'INTERAVE1'CHOL*CHOS 1 -1 1 -1 1 -1 -3 3; CONTRAST 'INTERAVE2'CHOL*CHOS 1 -1 1 -1 -2 2 0 0; CONTRAST 'INTERAVE3'CHOL*CHOS 0 0 0 0 1 -1 -1 1;

c)fetal data recorded by individual fetus:

PROC GLM; CLASS CHOL CHOS; MODEL FETALWEIGHT = CHOL CHOS CHOL*CHOS DAMNO(CHOL CHOS) ; TEST H = CHOL CHOS CHOL*CHOS E = DAMNO(CHOL CHOS) ;

d) calculation of contrasts for fetal data recorded by individual fetus:

PROC GLM: CLASS CHOL CHOS; MODEL GLUCOSE = C.HOL CHOS; CONTRAST '0 VS 4'CHOL -1 1 0 0; CONTRAST '0 VS 12'CHOL -1 0 1 0: CONTRAST '0 VS 60'CHOL -1 0 0 li CONTRAST '4 VS 12'CHOL 0 -1 1 0: CONTRAST '4 VS 60'CHOL 0 -1 0 li CONTRAST '12 VS 60'CHOL 0 0 -1 1;

This program was rune The SS (sums of squares) for each contrast was then divided by the mean square of the dam. The mean square of the dam was acquired from the program run in c (above) by dividing the TYPE III S8 of the OAMNO(CHOL*CHCS) by the OF (degrees of freedom) of the

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DAMNO(CHOL*CHOS). The probability was determined by looking up a table of F values with 1 and 65 degrees of freedom.

e)liver lipids and water content

PROC TTEST~ CLASS DIET~

2)4% Glucose equivalent diets:

a)maternal, amniotic fluid and fetal data with one pooled value per dam:

PROC GLM~ CLASS CHOS~

MODEL GLUCOSE = CHOS: CONTRAST 'GLYC VS FRU'CHOS -1 0 1~

CONThAST 'GLYC VS GLU'CHOS -1 1 O~ CONTRAST 'GLU VS FRU'CHOS 0 -1 1:

b)fetal data recorded by individual fetus:

PROC GLM; CLASS CHOS~ MODEL FETALWEIGHT = CHOS DAMNO(CHOS) ; TEST H = CHOS E = DAMNO(CHOS) ~ CONTPAST '0 VS GLU'CHOS -1 1 OIE = DAMNO(CHOS); CONTRAST '0 VS FRU'CHOS -1 0 liE = DAMNO(CHOS)~ CONTRAST 'GLU VS FRU'CHOS 0 -1 liE = DAMNO(CHOS);

c)comparison of control (60% glucose) values to the three 4% glucose equivalent diets for maternaI, amniotic fluid and fetal data with one pooled value per dam:

PROC GLM; CLASS CHOS~ MODEL GLUCOSE = CHOS: CONTRAST 'CONTROL VS 4%'CHOS 1 1 1 -3~

d)comparison of control (60% glucose) values to the three 4% glucose equivalent diets for fetal data recorded by individual fetus:

PROC GLM~ CLASS CHOS; MODEL FETALWEIGHT = CHOS DAMNO(CHOS) : TEST H = CHOS E = DAMNO(CHOS) ; CONTRAST 'CONTROL VS 4%'CHOS 1 1 1 -3/E = DAMNO(CHOS) ;

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