Information

Does galactose occur before or after galactose transferase?


The follow shows a protein pathway pathway.

The question is "where does galactose (GAL) first occur" i.e. which block above is galactose first contained in?

A transferase is defined as (wikipedia):

any one of a class of enzymes that enact the transfer of specific functional groups (e.g. a methyl or glycosyl group) from one molecule (called the donor) to another (called the acceptor)

This definition of transferase is confusing as:

  • The question doesn't show any functional groups does it?
  • From it, I can't figure out whether the galactose transferase is the molecule that is the donor or acceptor

I can't decide if galactose first occurs in S (as a donor) and is transfered into an acceptor upon reaching T, or if another donor forms galactose (as an acceptor) after S, causing T to contain galactose.


Classic Galactosemia

Galactosemia affects between 1:16,000[1] to 1:60,000[2] individuals.

Classic Galactosemia (ORPHA79239) is an autosomal recessive inherited metabolic disorder caused by deficient activity of galactose-1-phosphate uridyl transferase (GALT), as a result of mutations in the GALT gene. GALT is the second of three enzymes in the Leloir pathway- the main pathway of galactose metabolism.

Galactose is needed for energy metabolism and glycosylation of complex molecules. It can be derived two ways: either from exogenous (dietary) sources like lactose from dairy products, or by endogenous production. A deficiency of GALT enzyme leads to accumulation of galactose and its metabolites in the body and results in secondary glycosylation abnormalities[3].

Almost all infants born in the US with Galactosemia are diagnosed through state newborn screening programs.

The symptoms of Galactosemia present during the first few weeks of life with signs of liver and renal disease, cataracts, and an Escherichia coli sepsis. A galactose-restricted diet resolves the early signs of Galactosemia, but it cannot prevent development of later-onset complications, such as:

  • cognitive impairment
  • neurological sequelae
  • bone health abnormalities
  • primary ovarian insufficiency (POI) with subsequent infertility[3]

Almost every female with classic Galactosemia develops primary ovarian insufficiency (POI) as a diet-independent complication of the disease[4]. POI can vary from subfertility, to early development of irregular menstrual cycles and infertility, to primary amenorrhea and absence of spontaneous puberty. The mechanisms causing ovarian dysfunction are unknown. Postulated mechanisms of ovarian dysfunction include:

  • direct toxicity of metabolites (i.e. galactose-1-phosphate)
  • altered gene expression
  • aberrant function of hormone and/or receptors due to glycosylation abnormalities[3]

In general, POI is either caused by formation of a smaller primordial follicle pool or more rapid loss of primordial follicles[3]. Evidence of both has been seen in individuals with Galactosemia. Neonates have been reported with morphologically normal ovaries with abundant ooctyes and normal folliculogenesis[5,6]. Adolescents (16-17 years of age) have histological findings showing strongly reduced number of follicles, varying from far fewer follicles than expected for age[7] to almost complete absence[8,9]. Additionally, patients age 9-21 years were found by ultrasound or laparoscopy/laparotomy to have hyoplastic[9,10] or streak ovaries[8].

It is possible that a normal complement of primordial follicles forms, but undergoes atresia more rapidly and that the ovaries can be severely damaged in girls at very young prepubertal ages[3].

Fertility preservation options should be presented to females with classic Galactosemia as early as possible. Three fertility preservation procedures are currently offered to girls and women in need of fertility preservation, including:

Embryo and oocyte cryopreservation are, in theory, the best options for these patients. However, both techniques require ovarian stimulation. Gubbels et al. (2013) conducted ovarian stimulation in 15 classic Galactosemia patients, age 15-36 years, and demonstrated poor estradiol production in all patients except for one[11]. Therefore, fertility preservation in older, postpubertal classic Galactosemia patients is likely to be unsuccessful as the ovarian reserve may be poor. Considering the poor estradiol production and rapid follicle pool decline in classic Galactosemia, fertility preservation in these patients may need to take place during infancy or early childhood, seriously limiting fertility preservation options. Techniques requiring ovarian stimulation, such as embryo and oocyte cryopreservation, are not suitable for prepubertal girls because of absence of maturation of the hypothalamic-hypophyseal-ovarian axis, leaving cryopreservation of ovarian tissue the only available technique for this age group.

Females with classic Galactosemia may wish to pursue other options, including adoption or donor oocytes to achieve pregnancy. Additionally, mothers of girls with classic Galactosemia frequently propose to donate oocytes for their daughters. It may be necessary to cryopreserve the mother's oocytes for the future if the patient is not ready for children at the time of donation[3].

Spontaneous pregnancies do occur in classic Galactosemia, demonstrating that conception is possible in some patients[3]. It is important to educate patients about the occurrence of spontaneous pregnancies, allow them to try to conceive spontaneously, and avoid unplanned pregnancies. Gubbels et al. (2008) had a cohort of 22 patients with classic Galactosemia and POI. 9 patients in the cohort tried to conceive and 4 succeeded, resulting in a 44% success rate. The small sample size warrants further studies in larger cohorts. Additionally, it is important to note that most of the women who became pregnant spontaneously had gone through normal puberty and reached menarche spontaneously, indicating that these may be predictive factors for an increased chance of spontaneous conception[12].

van Erven et al. (2013) has published the following recommendations based on expert opinion regarding fertility preservation in female classic galactosemia patients:

  • Physicians should emphasize that spontaneous pregnancies occur in women with classic galactosemia, even after POI diagnosis.
  • If fertility preservation is desired, cryopreservation at an early prepubertal age as part of approved research currently seems to be the best option.
  • The ethics committee of the hospital or another independent body should review the parent's decision before the fertility preservation procedure.
  • The ethics committee of the hospital or another independent body should be involved in the decision making surrounding the use of the cryopreserved material.
  • If a patient desires pregnancy, a one-year window for attempting spontaneous pregnancy is advised to avoid unnecessary use of assisted reproductive techniques.
  • Anonymous or intrafamilial oocyte donation might be another option for classic galactosemia patients if pregnancy does not occur.

[1]Coss, K., Doran, P., Owoeye, C., Codd, M., Hamid, N., Mayne, P., . . . Treacy, E. (2012). Classical Galactosaemia in Ireland: incidence, complications and outcomes of treatment. J Inherit Metab Dis, 36, 21-27.

[2]Berry, G., & Elsas, L. (2011). Introduction to the Maastricht workshop: lessons from the past and new directions in galactosemia. J Inherit Metab Dis, 34, 249-255.

[3]van Erven, B., Gubbels, C., van Golde, R., Dunselman, G., Derhaag, J., de Wert, G., . . . Rubio-Gozalbo, M. (2013). Fertility preservation in female classic galactosemia patients. Orphanet J Rare Dis, 8, 107.

[4]Fridovich-Keil, J., Gubbels, C., Spencer, J., Sanders, R., Land, J., & Rubio-Gozalbo, E. (2011). Ovarian function in girls and women with GALT-deficiency galactosemia. J Inherit Metab Dis, 34, 357-366.

[5]Levy, H. (1996). Reproductive effects of maternal metabolic disorders: implications for pediatrics and obstetrics. Turk J Pediatr, 38(335-344).

[6]Levy, H., Driscoll, S., Porensky, R., & Wender, D. (1984). Ovarian failure in galactosemia. N Engl J Med, 310, 50.

[7]Rubio-Gozalbo, M., Gubbels, C., Bakker, J., Menheere, P., Wodzig, W., & Land, J. (2010). Gonadal function in male and female patients with classic galactosemia. Hum Reprod Update, 16, 177-188.

[8]Morrow, R., Atkinson, A., Carson, D., Carson, N., Sloan, J., & Traub, A. (1985). Ovarian failure in a young woman with galactosaemia. Ulster Med J, 54, 218-220.

[9]Robinson, A., Dockeray, C., Cullen, M., & Sweeney, E. (1984). Hypergonadotrophic hypogonadism in classical galactosemia: evidence for defective oogenesis Case report. Br J Obstet Gynaecol, 91, 199-200.

[10]Sauer, M., Kaufman, F., Paulson, R., & Lobo, R. (1991). Pregnancy after oocyte donation to a woman with ovarian failure and classical galactosemia. Fertil Steril, 55, 1197-1199.

[11]Gubbels, C., Land, J., Evers, J., Bierau, J., Menheere, P., Robben, S., & Rubio-Gozalbo, M. (2013). Primary ovarian insufficiency in classic galactosemia: role of FSH dysfunction and timing of the lesion. J Inherit Metab Dis, 36, 29-34.

[12]Gubbels, C., Land, J., & Rubio-Gozalbo, M. (2008). Fertility and impact of pregnancies on the mother and child in classic galactosemia. Obstet Gynecol Surv, 63, 334-343.

About the Author

Allison Goetsch, MS, CGC is a pediatric genetic counselor at Ann and Robert H. Lurie Children's Hospital of Chicago and a member of the Oncofertility Consortium administrative core team. She completed her graduate thesis with Dr. Teresa K. Woodruff researching oncofertility and hereditary breast and ovarian cancer (HBOC) syndrome. Allison's primary goals are to increase fertility preservation awareness and education for both health care providers and patients regarding malignant and non-malignant diseases (and/or treatments) which threaten fertility.


Newborn Screening and Follow-Up

Newborn screening for classic galactosemia is done using a small amount of blood collected from your baby’s heel. To learn more about this process, visit the Blood Spot Screening page.

During screening, a special machine measures how much of the enzyme GALT is in your baby’s blood. GALT helps break down the milk sugar known as galactose, which your body can then use for energy.

In some cases, the screening will also measure how much total galactose is in your baby’s blood. If the GALT enzyme cannot break down galactose, then there will be higher than normal levels of galactose in the body. Babies with low levels of GALT activity, either with or without high levels of total galactose, might have classic galactosemia.

If your baby’s blood spot screening result for classic galactosemia is out-of-range, your baby’s health care provider will contact you. Together, you will discuss next steps and follow-up plans.

An out-of-range screening result does not mean that your baby definitely has the condition. It does mean that your baby needs more follow-up testing. To learn more about screening results, visit the Blood Spot Screening Results page.

Your baby may need the following tests after an out-of-range screening result:

You should complete any recommended follow-up testing as soon as possible. Babies with this condition can have serious health problems very soon after birth if they are not diagnosed and treated quickly.

False-positive newborn screening results for this condition can happen.

  • Samples exposed to too much heat or took too long to get to the screening laboratory can also have false-positive results.
  • Sometimes, babies who have another condition called G6PD can screen positive for classic galactosemia, even though they do not have the condition.

Results

GAL Metabolic Genes Respond to the Ratio of Glucose and Galactose.

We grew cells in ∼500 combinations of glucose and galactose (Fig. 1 A and B) spanning a ∼1,000-fold range of glucose and galactose concentrations. We monitored the expression of a GAL1 promoter yellow fluorescent protein fusion (GAL1pr-YFP) in a derivative of the laboratory strain S288C (SI Appendix, sections I and II and Table S1). Gal1p, a galactokinase that catalyzes the first step in the Leloir pathway (10) is induced in the presence of galactose. We grew cells at low density so that the extracellular sugar concentrations are nearly constant throughout the course of the experiment, even at low sugar levels (SI Appendix, sections III and IV). Previous studies have used the average population expression level from a GAL promoter as a metric for response, which can obscure low but significant expression. To identify the decision to express GAL genes in a manner that is less dependent on absolute expression level, we used flow cytometry to quantify the percentage of cells expressing a GAL1pr-YFP reporter above basal levels (Fig. 1A). We define basal levels to be the response of cells grown in 2% (wt/vol) glucose in the absence of galactose (Fig. 1A and SI Appendix, section V).

The galactose pathway responds to the ratio of galactose and glucose. All experiments are in at least triplicate replicates in SI Appendix, Fig. S1. (A) Schematic of experiment and metric to measure steady-state GAL pathway response in S. cerevisiae in hundreds of combinations of glucose and galactose. The induced fraction (IF, hashed area) is computed (SI Appendix, section V) by estimating the fluorescence probability distribution for a given well (black curve) and taking the fraction of area outside the probability distribution of cells grown in glucose alone (green curve). (B) (Left) Flow cytometry (FCM) of response. The decision front is a linear fit to the concentrations at which 20% of all cells in the population show induction (IF >0.2). (Right) Comparison of cells monitored by live microscopy to FCM at three sugar mixtures, denoted by numbered squares (SI Appendix, Fig. S4). (C) Fraction of inducing cells as a function of the ratio of galactose and glucose concentrations. Each well in B is represented by a single dot. The line is a 1D sigmoidal curve that depends mainly on the ratio of galactose and glucose. (D) Comparison of models of signal integration (SI Appendix) by threshold sensing (Upper) and ratio sensing (Lower), displayed as in B and C. (E) Decision fronts, calculated as in B, for three strains of S. cerevisiae.

Contrary to the classic view, we found that GAL genes do not respond simply to a threshold concentration of glucose the decision to induce GAL genes instead depends on the ratio of glucose and galactose (Fig. 1 B–D replicates in SI Appendix, Fig. S1). The response was not simply a multiplicative combination of the independent behavior of cells in glucose or galactose (SI Appendix, Fig. S2). The value of the ratio was nearly constant over at least a 50-fold range of glucose and galactose concentrations (Fig. 1B). Below a glucose concentration of ∼0.006%, cells responded solely to a threshold of galactose (Fig. 1B). The result was insensitive to decreases in starting inoculum density, confirming that nutrient depletion is not significant in our experiments (SI Appendix, section IV and Fig. S3 A–C). Furthermore, modeling shows that nutrient depletion would not create the appearance of ratio sensing (SI Appendix, section IV and Fig. S3 D and E). We directly verified that the ratio-sensing behavior was a steady-state, depletion-independent, single-cell phenomenon by monitoring the kinetics of induction for 8 h at several glucose and galactose concentrations in a microfluidic device with constant nutrient replenishment (Fig. 1B, Right and SI Appendix, section VI and Fig. S4). The onset of the decision occurs within 1 h (SI Appendix, Fig. S4). Most cells are induced by 4 h, and steady-state is reached by 6 h (SI Appendix, Fig. S4). This behavior was also observed in two other strains, BC187 and YJM978, isolated from a vineyard and a clinical sample, respectively (Fig. 1E), showing that ratio sensing is not an aberrant behavior in a single laboratory strain. Furthermore, the existence of a ratio is robust to dosage perturbation of GAL genes (SI Appendix, Fig. S5).

How can our results be reconciled with previous work that did not report ratio sensing? All previous studies examined a relatively small range of concentrations, such that deviations from the expected threshold behavior were easily interpreted as noise our study used a concentration range that was approximately 10-fold larger than previous studies (SI Appendix, Fig. S6D). Many studies also sampled sparsely in the concentration range that they used, obscuring the differences between ratio and threshold sensing (SI Appendix, section III and Fig. S6D). The metric we use here, which deconvolves expression level from the decision (Fig. 1A and SI Appendix, section V and Fig. S6), also helps to show the behavior clearly, because it is more responsive at low concentrations where individual cells begin to induce than at high concentrations of sugar where induction is nearly saturated. With a large enough concentration range, however, the ratio-sensing behavior would have been readily observed independent of which metric was used (SI Appendix, Fig. S6).

Ratio sensing has not been previously described for carbohydrates but has been phenomenologically described for the sensing of NADH/NAD + , ATP/ADP, and X vs. autosomal chromosome levels (22 ⇓ –24). In the case of ATP/ADP, ratio sensing was proposed to result from mutually exclusive binding to the γ subunit of AMPK (25), but clarity regarding the mechanism is still lacking. In the GAL pathway, an obvious hypothesis would be that ratio sensing might be accomplished at the GAL1 or other GAL promoters (11). Glucose and galactose signals converge on these promoters through Mig1p and Gal4p, respectively (Fig. 2A). Alternatively, ratio sensing could occur upstream of either the canonical glucose or galactose signaling pathways.

The GAL pathway senses the ratio of glucose and galactose upstream of known glucose regulation. (A) The GAL regulatory network. (B) Mig1p localization as a function of glucose and galactose concentrations. Cells expressing Mig1p-GFP were grown under the same conditions as in Fig. 1B and imaged after 8 h (SI Appendix, section VII) steady-state localization was typically achieved in minutes. Images show representative cells at the indicated sugar concentration. Each concentration is the result of at least 20 cells. The number of cells with nuclear Mig1p-GFP decreases with glucose levels in a galactose-independent manner. (C) In a gal80∆ background, the ratio response is converted to a threshold response (i.e., in the absence of Gal80p the response is galactose independent). Experiment performed in duplicate. Data for no glucose conditions is not shown for clarity (Methods). (D) In a mig1∆ background, cells continue to respond to the galactose:glucose ratio. Experiment performed in duplicate. Solid line represents the decision front of the mig1∆ dashed line represents the decision front of the wild-type strain (from Fig. 1B). (E) In a mig1gal80∆ strain, the response is constitutive and does not dependent on either glucose or galactose.

Ratio Sensing Is Generated Upstream of the Canonical Gal Pathway.

To identify the mechanism for ratio sensing, we first tested whether glucose signaling is independent of galactose levels, by measuring the fraction of cells with Mig1p-GFP in the nucleus or cytoplasm in different galactose/glucose combinations (Fig. 2B and SI Appendix, Fig. S7). Mig1p localizes to the nucleus in the presence of glucose and to the cytoplasm in the absence of glucose (26). As expected, Mig1p-GFP localization is independent of galactose concentration (Fig. 2B). To further confirm the independence of the glucose branch from galactose we measured the response of a gal80∆ strain. Gal80p is a repressor of Gal4p, which in turn induces GAL1 in a gal80∆ background GAL1pr is constitutive (i.e., galactose independent) (27). Indeed, in this background the ratio sensor is broken the response is converted into a threshold sensor that depends mainly on glucose (Fig. 2C a quantitative comparison of glucose thresholds is shown in SI Appendix, Fig. S7). With respect to glucose inhibition, a gal80∆ strain therefore mimics the classic threshold expectation.

To test whether ratio sensing occurs in the canonical GAL pathway (i.e., downstream or at Gal3p) (Fig. 2A), we monitored the activity of GAL1pr-YFP in a mig1∆ mutant. Because Mig1p mediates the repression of the GAL pathway by glucose (4, 9), a mig1∆ mutant should be sensitive only to galactose levels, responding as a galactose threshold sensor regardless of whether ratio sensing through Mig1p is achieved directly or indirectly. Surprisingly, we found that even in a mig1∆ strain GAL1pr-YFP expression is still sensitive to the ratio of galactose and glucose (Fig. 2D). The ratio sensing ability of the mig1∆ strain is not due to the action of other transcription factors, because a gal80mig1∆ strain is constitutively active for GAL1pr-YFP expression that is, the activation of the GAL1 promoter is not dependent on either glucose or galactose in this strain (Fig. 2E and SI Appendix, Fig. S5C). These results are consistent with previous observations showing that glucose represses GAL1pr expression even in the absence of Mig1p (20). These results imply that either an intracellular mode by which glucose regulates the galactose pathway has been missed, or that ratio sensing is achieved neither at the GAL1 promoter nor in the canonical GAL pathway, but upstream of Gal3p.

The Galactose Transporter Gal2p Is Not Required for Ratio Sensing.

Because Gal3p directly senses internal galactose levels, ratio sensing upstream of Gal3p suggests a role for sugar transport in ratio sensing. When the GAL pathway is induced, the majority of galactose is imported through the Gal2p transporter, which transports both glucose and galactose with high affinity (Km ∼1 mM) (28, 29). Gal2p is part of the GAL pathway Gal2p levels are low in glucose media. Nevertheless, it is possible that even the low levels of Gal2p expressed in high glucose are important for ratio sensing. This would not be unprecedented: in the case of Lac induction in Escherichia coli, stochastic low-level expression of transporters is critical for the response (30). We therefore measured GAL1pr-YFP in a gal2∆ strain. Similar to the results with the mig1∆ mutant, a gal2∆ mutant does not “break” the ratio sensor (Fig. 3A) in both cases the mutation affects the ratio sensor, but neither mutant eliminates the ratio-sensing behavior. We interpret these results as strong evidence that the mechanism responsible for ratio sensing involves components outside the canonical galactose sensing pathway (Fig. 2A).

Galactose uptake depends on the ratio of galactose and glucose even in the absence of Gal2p. (A) Gal2p and many of the Hxt1-17p family of hexose transporters import glucose and galactose but have differing relative affinities for the two sugars. Deletion of GAL2 does not eliminate ratio sensing. Black and red lines are the decision front (Fig. 1E). (B) Incorporation of 12 C-galactose and 13 C-glucose into amino acids as measured by GC-MS in a gal2Δ gal80Δ mig1Δ strain (SI Appendix). This strain constitutively expresses the GAL pathway. Error bars are the SD from three biological replicates (duplicates for the highest glucose to galactose ratio). The slope of the fitted line (black line) is 1/170 the expectation based on literature uptake measurements is 1/250 (28, 29). (Inset) Breakdown of the data into two different galactose concentrations.

In a gal2∆ strain, the family of hexose transporters [Hxt1-17p or Mal11p, Mph2p, and Mph3p (31)] are likely to be the main transporters of galactose and a likely source of ratio sensing (Fig. 3A). The HXT members transport glucose with various affinities (Km from ∼1 mM to 100 mM) (28, 29), and some also import galactose, albeit with significantly lower affinity (Km ∼250 mM) (28) Hxt14p can even support growth on galactose in a strain where all other hexose transporters have been deleted (31). Thus, ratio sensing might result from competition between the sugars during uptake. In a competitive uptake regime, the intracellular galactose concentration would depend on the ratio of the extracellular galactose and glucose concentrations (SI Appendix, section VIII and Figs. S8 and S9).

Galactose Uptake Depends on the Ratio of Extracellular Sugars Concentrations.

To directly test whether uptake of galactose through the hexose transporters depends on the extracellular ratio of galactose and glucose concentrations, we measured galactose uptake in mixtures of U- 13 C-glucose and 12 C-galactose. Because intracellular carbohydrates are rapidly metabolized, measuring the incorporation of 13 C and 12 C into amino acids using gas chromatography mass spectrometry (32) provides information on uptake rates the ratio of incorporated 12 C and 13 C is equal to the ratio of galactose and glucose uptake rates (SI Appendix, section X). To distinguish the role of hexose transporters from the effects of intracellular regulation, we constructed a gal2gal80mig1∆ strain. This strain is not responsive to glucose or galactose but constitutively expresses GAL genes (Fig. 2E). Incorporation of 13 C and 12 C thus depends solely on the relative sugar uptake rates and not on the induction of the GAL pathway. We pregrew this strain in U- 13 C-glucose medium and transferred it into media containing mixtures of U- 13 C-glucose and 12 C-galactose for two doublings (SI Appendix, Table S3). We found that the ratio between 12 C and 13 C incorporated into amino acids, and hence galactose uptake, increases as extracellular galactose:glucose ratio is increased (Fig. 3B). The 12 C: 13 C ratio increases as extracellular galactose is increased but decreases as extracellular glucose is increased (Fig. 3B).

Quantitatively, this result is consistent with a “passive” model of competitive uptake of glucose and galactose by the transporter, which predicts that relative uptake depends on the extracellular sugar ratio multiplied by the relative affinity of the transporter for each sugar (Km ratio). Our measurements yield a Km ratio of 170 (Fig. 3B), similar to the Km ratio of 250 calculated from literature reports (28, 29). The concentration of glucose at which the response changes from a ratio sensor to a galactose threshold sensor, 0.006% as measured in Fig. 1B, is close to the Km of the high affinity hexose transporter for glucose ∼1 mM, or 0.002% glucose. This is consistent with a competitive uptake model glucose concentrations below the Km of the transport have quickly diminishing effects on the uptake of galactose, thereby making galactose uptake glucose independent at low glucose concentration.

Ratio Sensing Can Provide a Fitness Advantage.

It is possible that ratio sensing in the sugar metabolism pathways in yeast evolved to compensate for an inevitable lack of perfect discrimination between different sugars in the hexose transporter. Because of substrate competition for the transporter, high galactose will partially inhibit glucose uptake, and cells that do not induce GAL gene expression cannot compensate for the decreased glucose flux by metabolizing galactose. On the other hand, it is also possible that ratio sensing is desirable for other reasons (for example, to allow the cell to sense when using multiple sugars is a better decision than using only a single sugar) and that the lack of discrimination of the hexose transporters is in itself a selected trait. Consistent with the latter possibility, there is a wide variation in the selectivity of HXT family transporters for glucose relative to other sugars, and many do not sustain growth on medium with galactose as the sole carbon source (31). Thus, a cell could evolve to express only highly selective hexose transporters if ratio sensing were undesirable.

The biological advantage of ratio sensing is most likely during a dynamic process such as depletion of glucose in mixed sugar environments. However, no mutant currently exists whose only defect is to convert the ratio response to a threshold response (e.g., a gal80∆ has a fitness disadvantage in many media). Therefore, to establish whether ratio sensing can offer a selective advantage relative to a threshold sensing response, we compared the fitness of a gal4∆ strain to that of a wild-type strain in two conditions: glucose only, and a glucose/galactose mixture (Fig. 4A). A gal4∆ strain cannot mount a transcriptional response to galactose and therefore behaves in a glucose/galactose mixture as if it were in glucose alone (33), a behavior that phenocopies a threshold sensing strain in this media regime (Fig. 4A). When cocultured in 0.016% glucose the wild-type and gal4∆ strains grew comparably (Fig. 4A) 0.016% glucose is above the concentration of glucose at which ratio sensing is observed (Fig. 1B). When 2% (wt/vol) galactose is added to the 0.016% glucose medium the wild-type strain has a significant fitness advantage of 0.1% per hour ± 0.01% SE of mean, with a P value of 0.01 (two-tailed t test Fig. 4A). At this concentration all wild-type cells induce the GAL pathway maximally. Given the steady-state advantage to the ratio response we observe here, it is likely that if a true threshold-sensing strain could be constructed we would find that it is at a disadvantage compared with the ratio-sensing strain in dynamic environments as well.

Biological implications, implementations, and regimes for ratio sensing. (A) The ability to use galactose even in the presence of glucose gives cells a fitness advantage. A wild-type strain was competed against a gal4∆ in two concentrations of carbon: (i) 0.016% glucose or (ii) 0.016% glucose and 2% (wt/vol) galactose. The 0.016% glucose concentration places cells in the ratio sensing regime (Left, green and black squares). Ten independent replicates were grown until gene expression reached steady state (8 h), then samples were taken every 2 h for 10 h to calculate cellular fitness. Error bars are the SEM. (B) Ratio output can be generally achieved by a simple module in which two input molecules, an activator (green) and a repressor (blue), bind to an integrator molecule—a promoter, transporter, scaffold protein, etc. Mutual inhibition, ε, is necessary for a robust ratio response. (C) Futile cycles, such as phosphorylation and dephosphorylation or acetylation and deacetylation, can also create ratio sensors. Ratio sensing is achieved when the enzymes are not saturated.


Galactose transfer to endogenous acceptors within Golgi fractions of rat liver

The distribution of galactosyl transferase was studied using trans and cis Golgi fractions isolated by a modification of the Ehrenreich et al. procedure (1973. J. Cell Biol. 59:45-72) as well as an intact Golgi fraction isolated by a new one-step procedure. Two methods of assay were used. The first method analyzed the ability of Golgi fractions to transfer galactose (from uridine diphosphogalactose [UDP-gal] substrate) to the defined exogenous acceptor ovomucoid. The second method assessed the transfer of galactose from UDP-gal substrate to endogenous acceptors (endogenous glycosylation). The trans Golgi fraction (Golgi light) was highly active by the first method but revealed only low activity by the second method. Golgi fractions enriched in central and cis elements (the Golgi intermediate, heavy and especially the intact Golgi fraction) were highly active in both methods of assay. The endogenous glycosylation approach was validated by gel fluorography of the endogenous acceptors. For all Golgi fractions, transfer of galactose was revealed to secretory glycopeptides. It is concluded that galactosyl transferase activity in vivo occurs primarily in central and cis Golgi elements but not trans Golgi vesicles.


Follow-Up Testing

Your baby’s doctor may ask you if your baby is showing any of the signs of GALT (see Early Signs below). If your baby has certain signs, your baby’s doctor may suggest starting immediate treatment.

If your baby’s newborn screening result for classic galactosemia (GALT) was out of the normal range, your baby’s doctor or the state screening program will contact you to arrange for your child to have additional testing. It is important to remember that an out-of-range screening result does not necessarily mean that your child has the condition. An out-of-range result may occur because the initial blood sample was too small or the test was performed too early. However, a few babies do have the condition so it is very important that you go to your follow-up appointment for a confirmatory test. Because the harmful effects of untreated GALT can occur within days after birth, follow-up testing must be completed as soon as possible to determine whether or not your baby has the condition.

Follow-up testing will involve a blood test and a urine test to measure the amount of certain substances present in your baby’s body. Undigested sugars build up in the body when a child has GALT, so measuring the amounts of these sugars and other substances can help doctors determine if your baby has a condition. Individuals with GALT have low levels of GALT enzyme and high amounts of undigested sugars in their body. Genetic testing for classic galactosemia may also be necessary to confirm the diagnosis.


Case Report

A 6-month-old female patient presenting with delay of psychomotor development, bilateral ocular cataracts, hepatomegaly, and hypoattenuation of the brain white matter on CT scan was sent to our department for MR imaging and 1 H-MR spectroscopy examination to elucidate her diagnosis.

MR imaging showed diffuse and symmetrical involvement of the frontal, temporal, parietal, and occipital white matter, characterized by hypointensity on T1- and hyperintensity on T2 -weighted images, extending to “U” fibers (Fig 1A). In the temporal, frontal, and parietal regions hypointense areas associated with hyperintense white matter involvement on fluid-attenuated inversion recovery (FLAIR) images could be observed (Fig 1B). There was no gadolinium enhancement. Corpus callosum, internal and external capsules, and optic radiations were spared. The diffusion-weighted images (DWIs) showed hypointense lesions in the areas corresponding to white matter involvement with increased apparent diffusion coefficient (ADC) values (Fig 1C).

Images obtained before dietary treatment. Axial T2-weighted image (A) showing hyperintense bilateral diffuse white matter involvement reaching the “U” fibers. The internal capsules are spared. The lesions are hyperintense with hypointense areas on FLAIR images (B). ADC map (C) shows increased diffusion in the white matter lesions.

In vivo 1 H-MR spectroscopy of the parieto-occipital white matter (Fig 2A) revealed normal NAA: N-acetylaspartate (NAA)/creatine (Cr) and choline (Cho)/Cr, but reduced myo-inositol (mI)/Cr when compared with control values (Table 1). Unknown signals of very high concentration were found in the region of 3.5 to 4.0 ppm (Fig 2B). These signals were partially inverted when the spectrum was acquired with a TE of 135 milliseconds. This behavior is characteristic for carbohydrate signals. For a more detailed study of the carbohydrate composition, an in vitro 1 H-MR spectroscopy of the urine was acquired by using a high-resolution 500-MHz spectrometer, and the result was compared with the urine of a normal volunteer. We could observe a group of multiple peaks in the region of 3.6 to 3.9 ppm (Fig 3) and a triplet at 3.98 ppm, which were absent in the normal spectrum. We found also 2 clearly resolved doublets at 4.57 and 5.25 ppm in the patient’s urine, which were absent in the normal spectrum. The in vitro urine spectrum suggested the presence of high concentrations of galactose (Gal-ose) and galactitol (Gal-ol) in the patient’s urine. The diagnosis of galactosemia was later confirmed by the finding of low levels of galactose-1-phosphate uridyl transferase in the erythrocyte. Subsequently, the patient started a restricted lactose-free diet. At the age of 2 years, the follow-up MR imaging of the patient showed marked atrophy, more evident in the frontal lobes with enlarged sulci and dilation of the lateral ventricles more prominent in the anterior horns. There was marked improvement of the lesions with almost complete resolution of white matter signal intensity abnormalities. The sparse residual lesions were located in the basal temporal lobes and periventricular frontal regions, presenting hypointensity on T1-weighted and hyperintensity on T2-weighted (Fig 4) and FLAIR images, without gadolinium enhancement. On DWI, the lesions presenting increased ADC values on the previous examination had resolved. In vivo 1 H-MR spectroscopy of the parieto-occipital white matter did not show evidence of Gal-ol signal intensity (Fig 5). Metabolite ratios appeared to be normal when compared with control group (Table).

Axial localizer T2-weighted image showing the MR spectroscopy voxel location (A). STEAM (TE/TR, 30/1500 milliseconds) (B) in vivo 1 H-MR spectroscopy spectrum of the patient before treatment.

In vitro 1 H-MR spectroscopy spectrum of the urine sample, showing the region of 3.2–4.1 ppm in the patient.

Images obtained after dietary treatment. Axial T2-weighted image shows marked frontal atrophy with enlarged sulci and ventricular dilation. Note also enlargement of Sylvian fissures and improvement of white matter lesions.

STEAM (TE/TR, 30/1500 milliseconds) in vivo 1 H-MR spectroscopy spectrum of the patient after treatment.

NAA/CrCho/CrmI/CrGal-ol/Cr
Patient before treatment1.451.040.1914.30
Patient after treatment1.570.870.50
Controls * 1.53 ± 0.220.89 ± 0.140.48 ± 0.07

Note:—Control mean values are included for comparison purposes. Cho/Cr indicates choline/creatine Gal-ol/Cr, galactitol/creatine ml/Cr, myo-inositol/creatine NAA/Cr, N-acetylaspartate/creatine.

* Control group consisted of 10 healthy volunteers (3 boys and 7 girls) of mean age of 6 ± 1 years (range, 4–9 years).

Metabolite ratios in the parieto-occipital white matter measured by the STEAM technique (TE/TR = 30/1500 ms) before and after patient’s treatment


Terminology

In 1856, the French biologist, Louis Pasteur 1822 –1895, was able to isolate galactose and called it lactose. 1 The compound was later called galactose (or “glucose lactique”) as mentioned by Pierre Eugène Marcellin Berthelot 1827–1907, a French chemist, in his book, Chimie organique fondée sur la synthèse. 2
Etymologically, galactose comes from the Ancient Greek γάλακτος (gálaktos, meaning “milk”) and‎ -ose (denoting “sugar”).

Overview

Galactose is one of the three most common monosaccharides the other two are glucose and fructose. Monosaccharides are the most fundamental type of carbohydrates. They are called simple sugars as opposed to the more complex forms such as oligosaccharides and polysaccharides. Monosaccharides can combine, though, to form complex carbohydrates via glycosidic bonds (glycosidic linkages).

Properties of galactose

Galactose is a hexose monosaccharide. It is an organic compound. Its general chemical formula is C6H12O6.The molar mass of galactose is 180.156 g/mol. The melting point is 168–170 °C. It is crystalline, water-soluble, and sweet tasting.

Galactose vs. Glucose vs. Fructose

Glucose, galactose, and fructose are the three most common natural monosaccharides. Nevertheless, glucose is the most abundant. The three have the same chemical formula: C6H12O6. Hence, they are a hexose-type of monosaccharide, owing to the six carbon atoms. Both glucose and galactose are aldoses whereas fructose is a ketose. Thus, glucose and galactose are more structurally alike. Nonetheless, glucose can be structurally identified from galactose based on the orientation of the hydroxyl group (OH) at carbon 4. Also, galactose has a higher melting point. Its melting point is 168–170 °C as opposed to glucose’s melting point of 146 °C. However, of the three, fructose has the lowest melting point (i.e. 103 °C).
Unlike glucose, galactose generally does not occur in free state. It usually is a constituent of complex biomolecules. For instance, galactose together with glucose forms lactose (milk sugar), which is a disaccharide. Thus, glucose is more often used than galactose or fructose in energy metabolism since it is more readily available. Without enough glucose, galactose enters glycolysis but galactose goes through initial steps to be converted into glucose 6-phosphate before it can proceed to glycolysis. The same principle happens to fructose fructolysis (catabolism of fructose) entails fructose phosphorylation by fructokinase to produce fructose 1-phosphate, which is then cleaved by aldolase B into two trioses, dihydroxyacetone phosphate and glyceraldehyde.

Types of galactose

Two enantiomers of galactose exist: Dextrogalactose (D-galactose) and Levogalactose (L-galalactose). This nomenclature (based on Fischer projection) designates D– when the glucose stereoisomer rotates the plane polarized light in the clockwise direction. L– is when it rotates the plane polarized light in a counterclockwise direction. The dextrotatory form of galactose is obtained from milk sugar through hydrolysis. D-galactose is also present in sugar beets, seaweeds, and nerve cell membranes. Its levorotatory form is obtained from mucilages.

Common biological reactions involving galactose

Common biological reactions involving galactose

Through dehydration synthesis, a monosaccharide, such as galactose, binds to another monosaccharide with the release of water and the subsequent formation of a glycosidic bond. The joining of two monosaccharides produces a disaccharide whereas the joining of three to ten monosaccharide units forms an oligosaccharide. Polysaccharides are produced by the joining of multiple monosaccharides. In this regard, galactose joins with another monosaccharide to form a disaccharide. For instance, lactose is formed when galactose and glucose molecules are joined together. Another is the man-made disaccharide lactulose made up of galactose and fructose. As for polymers, a galactan is a polysaccharide consisting of repeating galactose units.

Common biological reactions involving galactose

The process wherein complex carbohydrates are degraded into simpler forms, such as glucose and galactose, is called saccharification. It entails hydrolysis. In humans and other higher animals, this involves an enzymatic action. In a diet containing galactose (e.g. lactose in dairy products), digestion is aided by a β-galactosidase enzyme, lactase. Lactase catalyzes the hydrolysis of lactose and breaks β-glycosidic bond, resulting in the release of glucose and galactose in the small intestine. As for ceramide-containing diet, the lactase-glycosyleramidase complex breaks the β-glycosidic bond in glycolipids to release galactose.3 In the absence, or inadequacy, of lactase, lactose cannot be digested into simpler monosaccharides and as such, causes lactose intolerance. Lactose undigested in the small intestine moves to colon where the gut bacteria ferment it to lactic acid. As a result, methane and hydrogen gas are produced and cause discomfort, gut distention, and flatulence. Diarrhea ensues as water is drawn in to the intestine by the osmotically active lactic acid. Microorganisms, such as Escherichia coli, can metabolize lactose by producing β-galactosidase from its lac operon system.

Common biological reactions involving galactose

Galactose from dietary sources is taken up by the intestinal cells (enterocytes) through sodium-dependent glucose transporter, the same ATP-driven transport mechanism that absorbs glucose. Thus, glucose competes with galactose during intestinal absorption. Galactose leaves the intestinal cells and enters the bloodstream passively through glucose transporter- (GluT-) mediated transport.3

Common biological reactions involving galactose

Galactose is converted into glucose generally by a two-phase process. In the initial phase, β-D-galactose is converted into α-D-galactose by the enzyme mutarotase. In the last phase, α-D-galactose is converted into uridine diphosphate (UDP)-glucose. This last phase is often through the Leloir pathway. In this pathway, α-D-galactose is phosphorylated though galactokinase to produce galactose 1-phosphate. Next, galactose 1-phosphate acquires uridine monophosphate (UMP) group from UDP-glucose through the enzyme galactose-1-phosphate uridyltransferase to produce UDP-galactose. Then, UDP-galactose is interconverted into UDP-glucose by the enzyme UDP galactose-4′-epimerase. In humans and other organisms, an alternative to Leloir pathway is De Ley Duodoroff pathway. Galactose that is converted into glucose is the way by which galactose may enter the glycolytic pathway. The overall reaction would therefore be as follows:
Galactose + ATP → Glucose-1-phosphate + ADP + H +
Phosphoglucomutase catalyzes the isomerization of glucose 1-phosphate to glucose 6-phosphate. In humans, this galactose metabolism occurs in the liver.

Common biological reactions involving galactose

In humans and other mammals, some of the glucose molecules are converted into galactose so that there would more galactose to combine with glucose to produce lactose. This is especially important during milk production. The mammary gland secretes lactose as milk, especially during breastfeeding. N.B.: galactose may be obtained as well from dietary sources. The de novo synthesis of glucose and galactose in the mammary gland is called hexoneogenesis.

Common biological reactions involving galactose

Galactan is a polymer of galactose that occurs in hemicelluloses. In plants such as the axlewood ( Anogeissus latifolia) and acacia trees, galactose monomers link together and form galactans.

Common biological reactions involving galactose

Glycosylation is the process of adding a carbohydrate component, such as galactose, to certain proteins and lipids. Galactose forms part of certain glycolipids and glycoproteins. For instance, it may serve as a constituent of cerebroside (a glycolipid comprised of a carbohydrate and a sphingolipid). Cerebrosides are of two types: glucocerebrosides and galactocerebrosides, which have glucose and galactose carbohydrate residues, respectively.

Improper metabolism

Improper metabolism of galactose results in a condition called galactosemia. It is a rare metabolic disorder. One of the typical causes is the heritable genetic mutation involving the synthesis of an enzyme in the Leloir pathway, i.e. galactose 1-phosphate uridyl transferase. Galactosemics are not advised to consume galactose- (and lactose-) containing diet. Otherwise, it could result in diarrhea, vomiting, and eventually to cirrhosis.

Biological importance/functions

Galactose is one of the most common monosaccharides and plays various biological roles. For instance, it acts as an alternative to glucose when the latter is insufficient to the metabolic demands of an organism. It can enter glycolysis to synthesize energy. However, it must go through initial steps prior to entering the glycolytic pathway.
Galactose is a constituent of lactose, the disaccharide of milk. Humans and other milk-producing animals biosynthesize lactose from galactose and glucose. Milk is a vital source of nutrients, especially of neonates.
Galactose is a component of cerebrosides, and as such are called galactocerebrosides as opposed to glucocerebrosides containing glucose instead of galactose. Galactocerebrosides are commonly found in neural tissues and they are the main glycosphingolipid in the brain — presumably, the reason galactose is referred to as the brain sugar. Galactose that is later sulfated is referred to as sulfatide. Sulfatides play a role in immune response and nervous system signaling.
In plants, galactose occurs, such as in flaxseed mucilages and sugar beet. Galactan is a polymer of galactose found in hemicellulose in plants, such as the axlewood ( Anogeissus latifolia) and acacia trees.


Systems

Genetics

Specialty Collaborations & Other Services

Pediatric Metabolics ( see NW providers [0] )

Co-management should be established with metabolic genetics.

Nutrition, Metabolic ( see NW providers [15] )

Initiates and monitors a lactose-free diet

Genetic Testing and Counseling ( see NW providers [7] )

Helps families understand the inheritance pattern and risks for subsequent children

Development (general)

Specialty Collaborations & Other Services

Early Intervention for Children with Disabilities/Delays ( see NW providers [2] )

All children who test positive for classic galactosemia should be referred to an early intervention program for evaluation and, if indicated, treatment.

Developmental - Behavioral Pediatrics ( see NW providers [1] )

An evaluation by developmental pediatrics may be helpful for children who are behind developmentally or who have attention or learning problems.

Physical Therapy ( see NW providers [0] )

May be helpful for patients with gross motor developmental delays and/or coordination problems/ataxia

Occupational Therapy ( see NW providers [0] )

May be helpful for patients with fine motor delays or problems with activities of daily living

Speech - Language Pathologists ( see NW providers [3] )

May be helpful for patients with language delay or articulation problems.

Eyes/Vision

Specialty Collaborations & Other Services

Pediatric Ophthalmology ( see NW providers [1] )

Periodic visits with pediatric ophthalmology are necessary to monitor for cataracts and treat as needed.

Endocrine/Metabolism

Specialty Collaborations & Other Services

Pediatric Endocrinology ( see NW providers [1] )

Assists in evaluation and management of girls with ovarian insufficiency


References

Ficicioglu C, Thomas N, Yager C et al. Duarte (DG) galactosemia: a pilot study of biochemical and neurodevelopmental assessment in children detected by newborn screening. Mol Genet Metab 200895:206–212.

Brenner C . Hint, Fhit, and GalT: function, structure, evolution, and mechanism of three branches of the histidine triad superfamily of nucleotide hydrolases and transferases. Biochemistry 200241:9003–9014.

Frey PA . The Leloir pathway: a mechanistic imperative for three enzymes to change the stereochemical configuration of a single carbon in galactose. FASEB J 199610:461–470.

Calderon FR, Phansalkar AR, Crockett DK, Miller M, Mao R . Mutation database for the galactose-1-phosphate uridyltransferase (GALT) gene. Hum Mutat 200728:939–943.

Tyfield L, Reichardt J, Fridovich-Keil J et al. Classical galactosemia and mutations at the galactose-1-phosphate uridyl transferase (GALT) gene. Hum Mutat 199913:417–430.

Lai K, Elsas LJ . Structure-function analyses of a common mutation in blacks with transferase-deficiency galactosemia. Mol Genet Metab 200174::264–272.

Goldstein N, Cohen Y, Pode-Shakked B et al, The GALT rush: high carrier frequency of an unusual deletion mutation of the GALT gene in the Ashkenazi population Mol Genet Metab 2011102:157–160.

Coffee B, Hjelm LN, DeLorenzo A, Courtney EM, Yu C, Muralidharan K . Characterization of an unusual deletion of the galactose-1-phosphate uridyl transferase (GALT) gene. Genet Med 20068:635–640.

Crushell E, Chukwu J, Mayne P, Blatny J, Treacy EP . Negative screening tests in classical galactosaemia caused by S135L homozygosity. J Inherit Metab Dis 200932:412–415.

Elsas LJ, Langley S, Steele E et al. Galactosemia: a strategy to identify new biochemical phenotypes and molecular genotypes. Am J Hum Genet 199556:630–639.

Carney AE, Sanders RD, Garza KR et al. Origins, distribution and expression of the Duarte-2 (D2) allele of galactose-1-phosphate uridylyltransferase. Hum Mol Genet 200918:1624–1632.

Fridovich-Keil JL, Gambello MJ, Singh RH, Sharer JD. Duarte variant galactosemia. In: Pagon RA, Adam MP, Ardinger HH et al., (eds). GeneReviews. University of Washington: Seattle, WA, 1993.

Langley SD, Lai K, Dembure PP, Hjelm LN, Elsas LJ . Molecular basis for Duarte and Los Angeles variant galactosemia. Am J Hum Genet 199760:366–372.

Thoden JB, Timson DJ, Reece RJ, Holden HM . Molecular structure of human galactokinase: implications for type II galactosemia. J Biol Chem 2005280:9662–9670.

Bergsma DJ, Ai Y, Skach WR et al. Fine structure of the human galactokinase GALK1 gene. Genome Res 19966:980–985.

Timson DJ, Reece RJ . Functional analysis of disease-causing mutations in human galactokinase. Eur J Biochem 2003270:1767–1774.

Timson DJ . The structural and molecular biology of type III galactosemia. IUBMB Life 200658:83–89.

Berry GT. Classic galactosemia and clinical variant galactosemia. In: Pagon RA, Adam MP, Ardinger HH et al, (eds). GeneReviews. University of Washington: Seattle, WA, 1993.

Webb AL, Singh RH, Kennedy MJ, Elsas LJ . Verbal dyspraxia and galactosemia. Pediatr Res 200353:396–402.

Ficicioglu C, Hussa C, Gallagher PR, Thomas N, Yager C . Monitoring of biochemical status in children with Duarte galactosemia: utility of galactose, galactitol, galactonate, and galactose 1-phosphate. Clin Chem 201056:1177–1182.

Lynch ME, Potter NL, Coles CD, Fridovich-Keil JL . Developmental outcomes of school-age children with Duarte galactosemia: a pilot study. JIMD Rep 201519:75–84.

Powell KK, Van Naarden Braun K, Singh RH, Shapira SK, Olney RS, Yeargin-Allsopp M . Long-term speech and language developmental issues among children with Duarte galactosemia. Genet Med 200911:874–879.

Welling L, Bernstein LE, Berry GT et al. International clinical guideline for the management of classical galactosemia: diagnosis, treatment, and follow-up. J Inherit Metab Dis 201640:171–176.

Hennermann JB, Schadewaldt P, Vetter B, Shin YS, Monch E, Klein J . Features and outcome of galactokinase deficiency in children diagnosed by newborn screening. J Inherit Metab Dis 201134:399–407.

Bosch AM, Bakker HD, van Gennip AH, van Kempen JV, Wanders RJ, Wijburg FA . Clinical features of galactokinase deficiency: a review of the literature. J Inherit Metab Dis 200225:629–634.

Fridovich-Keil J, Bean L, He M, Schroer R. Epimerase deficiency galactosemia. In: Pagon RA, Adam MP, Ardinger HH et al, (eds). GeneReviews. University of Washington: Seattle, WA, 1993.

Committee on Genetics Kaye CI Committee on Genetics Committee on Genetics Accurso F et al. Newborn screening fact sheets. Pediatrics 2006118:e934–963.

Kobayashi K, Saheki T, Song YZ. Citrin deficiency. In: Pagon RA, Adam MP, Ardinger HH et al, (eds). GeneReviews. University of Washington: Seattle, WA, 1993.

Peduto A, Spada M, Alluto A, La Dolcetta M, Ponzone A, Santer R . A novel mutation in the GLUT2 gene in a patient with Fanconi-Bickel syndrome detected by neonatal screening for galactosaemia. J Inherit Metab Dis 200427:279–280.

Beutler E, Baluda MC . Improved method for measuring galactose-I-phosphate uridyl transferase activity of erythrocytes. Clin Chim Acta 196613:369–379.

Adam BW, Hall EM, Sternberg M et al. The stability of markers in dried-blood spots for recommended newborn screening disorders in the United States. Clin Biochem 201144:1445–1450.

Sartippour MR, Doroudian R, Frampton G et al. Identification of galactose-1-phosphate uridyl transferase gene common mutations in dried blood spots. Clin Chim Acta 2014436:298–302.

Fujimoto A, Okano Y, Miyagi T, Isshiki G, Oura T . Quantitative Beutler test for newborn mass screening of galactosemia using a fluorometric microplate reader. Clin Chem 200046:806–810.

Sokol RJ, McCabe ER, Kotzer AM, Langendoerfer SI . Pitfalls in diagnosing galactosemia: false negative newborn screening following red blood cell transfusion. J Pediatr Gastroenterol Nutr 19898:266–268.

Therrell BL Jr., Lloyd-Puryear MA, Camp KM, Mann MY . Inborn errors of metabolism identified via newborn screening: ten-year incidence data and costs of nutritional interventions for research agenda planning. Mol Genet Metab 2014113:14–26.

Li Y, Ptolemy AS, Harmonay L, Kellogg M, Berry GT . Quantification of galactose-1-phosphate uridyltransferase enzyme activity by liquid chromatography-tandem mass spectrometry. Clin Chem 201056:772–780.

Lai K, Langley SD, Dembure PP, Hjelm LN, Elsas LJ 2nd . Duarte allele impairs biostability of galactose-1-phosphate uridyltransferase in human lymphoblasts. Hum Mutat 199811:28–38.

Centers for Disease Control and Prevention. Good laboratory practices for biochemical genetic testing and newborn screening for inherited metabolic disorders. MMWR Recomm Rep 201261:1–44.

Clinical and Laboratory Standards Institute Defining, establishing, and verifying reference intervals in the clinical laboratory Approved GuidelineThird Edition. CLSI Document EP28-A3CWayne, PA,, 2010.

De Bruyn CH, Raymakers C, Wensing A, Oei TL . Galactose-1-phosphate uridyltransferase activities in erythrocytes from a patient with galactosemia: discrepancy between two methods. Clin Chim Acta 197778:145–150.

Xu YK, Kaufman FR, Donnell GN, Ng WG . Radiochemical assay of minute quantities of galactose-1-phosphate uridyltransferase activity in erythrocytes and leukocytes of galactosemia patients. Clin Chim Acta 1995235:125–136.

Ko DH, Jun SH, Park HD et al. Multiplex enzyme assay for galactosemia using ultraperformance liquid chromatography-tandem mass spectrometry. Clin Chem 201056:764–771.

Li Y, Ptolemy AS, Harmonay L, Kellogg M, Berry GT . Ultra fast and sensitive liquid chromatography tandem mass spectrometry based assay for galactose-1-phosphate uridylyltransferase and galactokinase deficiencies. Mol Genet Metab 2011102:33–40.

Chen J, Meyers GA, Bennett MJ . An interference-free two-step enzyme assay with UPLC-tandem mass spectrometric product measurement for the clinical diagnosis of uridine diphosphate galactose-4-epimerase deficiency. J Chromatogr B Analyt Technol Biomed Life Sci 2014959:5–9.

Pesce MA, Bodourian SH, Nicholson JF . A new microfluorometric method for the measurement of galactose-1-phosphate in erythrocytes. Clin Chim Acta 1982118:177–189.

Chen J, Yager C, Reynolds R, Palmieri M, Segal S . Erythrocyte galactose 1-phosphate quantified by isotope-dilution gas chromatography-mass spectrometry. Clin Chem. 200248:604–612.

Schadewaldt P, Kamalanathan L, Hammen HW, Wendel U . Stable-isotope dilution analysis of galactose metabolites in human erythrocytes. Rapid Commun Mass Spectrom 200317:2833–2838.

Ficicioglu C, Yager C, Segal S . Galactitol and galactonate in red blood cells of children with the Duarte/galactosemia genotype. Mol Genet Metab 200584:152–159.

Berry GT, Nissim I, Lin Z, Mazur AT, Gibson JB, Segal S . Endogenous synthesis of galactose in normal men and patients with hereditary galactosaemia. Lancet 1995346:1073–1074.

Berry GT, Nissim I, Gibson JB et al. Quantitative assessment of whole body galactose metabolism in galactosemic patients. Eur J Pediatr 1997156 (suppl 1):S43–49.

Palmieri M, Mazur A, Berry GT et al. Urine and plasma galactitol in patients with galactose-1-phosphate uridyltransferase deficiency galactosemia. Metabolism 199948:1294–1302.

Richards S, Aziz N, Bale S et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 201517:405–424.