A Longitudinal Analysis of Prenatal Metabolite Accumulation Through Gestation in GALT-null Rat Fetuses Open Access

Seemiller, Madelyn (Spring 2024)

Permanent URL: https://etd.library.emory.edu/concern/etds/c247dt43q?locale=en
Published

Abstract

Classic Galactosemia (CG) is an autosomal recessive disorder characterized by a deficiency in galactose-1-phosphate uridylyltransferase (GALT), an essential enzyme in the metabolism of galactose. Despite early detection via newborn screening and galactose-restricted diet intervention, individuals with CG continue to experience a range of complications, including but not limited to developmental and cognitive delays, growth restriction, cataracts, and primary ovarian insufficiency (POI). The presence of such phenotypes despite early intervention suggests that the challenges associated with CG may begin prenatally given the fetus’s inability to sufficiently metabolize endogenously-produced galactose. This study aims to investigate the prenatal accumulation of galactose metabolites in different tissues to understand the longitudinal progression of CG during the final quarter of gestation. This study also analyzes the longitudinal impact of maternal and co-gestating fetal GALT activity on prenatal metabolite accumulation based on prior studies in the Fridovich-Keil lab concerning excessive neonatal demise in GALT-null fetuses. The metabolites galactose, galactitol, and Gal-1P were measured in the fetal liver, brain, and amniotic fluid (excluding Gal-1P in AF) on the 17th, 19th, and 21st day of rat gestation (GD). The data from the varying metabolites, tissue types, cross-types, and gestational days suggests that M3/M3 fetuses from M3 x M3 crosses have elevated metabolite accumulation compared to wild-type fetuses at every GD, metabolite, and tissue type. These fetuses also differ in metabolite accumulation compared to M3/M3 fetuses from heterozygous crosses longitudinally, but only at particular metabolite and tissue types. GALT-null fetuses from M3 x M3 crosses and some from heterozygous crosses exhibit a spike in galactose accumulation from GD 17 to 19 and then a decrease from GD 19 to 21 in amniotic fluid and fetal liver, for which we hypothesize that increased endogenous galactose production, kidney function, and UGP activity may have an effect. Using fetal body mass and growth restriction as a phenotypic outcome for metabolite accumulation, growth delays for M3/M3 fetuses from M3 x M3 and M3 x Het crosses begin at GD 21, but not in fetuses from Het x M3 crosses, suggesting that maternal GALT-activity can protect gestating GALT-null fetuses from such phenotypic consequences prenatally.

Table of Contents

CHAPTER 1: BACKGROUND AND INTRODUCTION (1)

Genetics of Classic Galactosemia (1)

Primary Ovarian Insufficiency and Prenatal Analysis (2)

Galactose Metabolism (2)

The Leloir and Polyol Pathways (4)

The GALT-null Rat CG Model (4)

Rat Estrus Cycling and Husbandry (5)

Neonatal Demise and the Prenatal Gestational Environment (7)

Gestational Days (GD) (8)

CHAPTER 2: GALACTOSE METABOLITE DISTRIBUTION AND ACCUMULATION SEEN IN GALT-NULL FETUSES FROM M3XM3 CROSSES (12)

Introduction (12)

Methods (

Timed Pregnancy Estrus Cycling and Husbandry Crosses (13)

Gestational Tissue Collection (14)

Metabolite Extractions (18)

Metabolite Quantification and Dilutions (19)

High-Performance Liquid Chromatography (21)

Sample Size (23)

Statistical Methods (23)

Results (27)

Distribution of Galactose Metabolites in Different Tissues and GD, M3xM3 and WTxWT Crosses (27)

Metabolite Accumulation in M3/M3 and WT/WT Fetuses Through Gestation: Amniotic Fluid (29)

Metabolite Accumulation in M3/M3 and WT/WT Fetuses Through Gestation: Liver (30)

Metabolite Accumulation in M3/M3 and WT/WT Fetuses Through Gestation: Brain (32)

Discussion (35)

CHAPTER 3: GALACTOSE METABOLITE DISTRIBUTION AND ACCUMULATION SEEN IN M3/M3 FETUSES FROM HETEROZYGOUS CROSSES (38)

Introduction (38)

Methods (39)

Referenced Methods from Chapter 2 (39)

Results (42)

Distribution of Galactose Metabolite in Different Tissues and GD: M3/M3 Fetuses from Het Crosses (42)

Metabolite Accumulation in M3/M3 Fetuses from Het Crosses Through Gestation: Amniotic Fluid (44)

Metabolite Accumulation in M3/M3 Fetuses from Het Crosses Through Gestation: Liver (45)

Metabolite Accumulation in M3/M3 Fetuses from Het Crosses Through Gestation: Brain (47

Discussion (49)

CHAPTER 4: GALACTOSE METABOLITE DISTRIBUTION AND ACCUMULATION SEEN IN M3/WT FETUSES FROM HETEROZYGOUS CROSSES (51)

Introduction (51)

Methods (51)

Referenced Methods from Chapter 2 (51)

Results (55)

Distribution of Galactose Metabolite in Varying Tissues and GD: M3/WT Fetuses from Het Crosses (55)

Metabolite Accumulation in M3/WT Fetuses from Het Crosses Through Gestation: Amniotic Fluid (57)

Metabolite Accumulation in M3/WT Fetuses from Het Crosses Through Gestation: Liver (58)

Metabolite Accumulation in M3/WT Fetuses from Het Crosses Through Gestation: Brain (60)

Discussion (62)

CHAPTER 5: ANALYSIS OF GROWTH RESTRICTION BY FETAL BODY MASS (64)

Introduction (64)

Methods (65)

Fetal Body Mass (65)

Results (65)

Longitudinal Growth Restriction Analysis, M3/M3 Fetuses from Het and M3xM3 Crosses (65)

Longitudinal Growth Restriction Analysis, M3/WT Fetuses from Het Crosses (66)

Discussion (67)

CHAPTER 6: DISCUSSIONS AND FUTURE DIRECTIONS (69)

Discussion (69)

Limitations (71)

Future Directions (72)

REFERENCES 74

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