Galactitol and galactonate in red blood cells of galactosemic patients

doi:10.1016/j.ymgme.2003.08.021

Molecular Genetics and Metabolism

Volume 80, Issue 3, November 2003, Pages 283-289

https://doi.org/10.1016/j.ymgme.2003.08.021 Get rights and content

Abstract

The red blood cell (RBC) concentration of galactitol and galactonate was measured in 27 patients with galactose-1-phosphate uridyltransferase (GALT) deficiency galactosemia and 19 non-galactosemic subjects by a newly devised isotope dilution gas chromatography/mass spectrometry (GC/MS) method. The method utilizing UL[¹³C]galactitol and UL[¹³C]galactonate was reproducible with excellent precision and recovery of 99%. The RBC galactitol in galactosemic patients on galactose-restricted diets averaged 5.98 ± 1.2 μM (M ± SD) with a range of 3.54–8.81 μM. The mean in non-galactosemic patients was 0.73 ± 0.31 μM with a range of 0.29–1.29 μM. The mean of RBC galactonate in the same galactosemic patients was 4.16 ± 1.32 μM (M ± SD) with a range of 0.68–6.47, while the mean in non-galactosemic subjects was 1.94 ± 0.96 (M ± SD) with a range of 0.69–3.84. In galactosemic RBC the galactitol was higher than galactonate while this was reversed in non-galactosemic cells. RBC galactose-1-phosphate (Gal-1-P) measured at the same time as galactitol and galactonate was 30 times the level of the other two metabolites. There was no relationship between RBC Gal-1-P and galactitol or galactonate. The ability to measure all three galactose metabolites in the same procedure offers the possibility of augmented monitoring of the galactose metabolic status of patients. The measurement of RBC galactitol and galactonate presents a new means of characterizing galactosemic patients and their levels monitored over time may provide new insight in the development of long-term complications observed in afflicted patients.

Introduction

Since galactose-1-phosphate (Gal-1-P) was found to accumulate in the red blood cells of patients with galactosemia due to deficiency of galactose-1-phosphate uridyltransferase (GALT) [1], its measurement has been the currency for monitoring the dietary management and galactose metabolic status of affected individuals [2], [3], [4], [5]. Clinical experience has led to question its utility, however. With the finding of the metabolic products of alternate galactose metabolic pathways, galactitol [6] and galactonate [7] in biological fluids, the measurement of urine galactitol excretion [8], [9] has added to the patient’s dietary monitoring process. Urinary galactonate excretion measured by NMR [7] has not been routinely utilized although it may be an additional important parameter of galactose disposition.

In addition to Gal-1-P formation when galactosemic red blood cells (RBCs) are incubated with galactose in vitro, NMR analysis detected the synthesis of large amounts of galactitol and galactonate [10]. These latter metabolites were not found in the galactosemic RBC prior to incubation with galactose by the relatively insensitive NMR technique [10]. However, during the development of a new, highly sensitive gas chromatography/mass spectrometry (GC/MS) isotope dilution method for RBC Gal-1-P analysis [11], the chromatographic procedure identified the presence of galactitol and galactonate in both galactosemic and normal RBC [12]. This report describes a GC/MS isotope dilution technique for the quantitation of RBC galactitol and galactonate and their elevation in the cells from galactosemic patients on galactose-restricted diets. The technique permits their simultaneous determination with RBC Gal-1-P, which offers the possibility of augmented monitoring of the metabolic status of galactosemic patients.

Section snippets

Materials

Galactose and galactitol were obtained from Pfanstiel and Gal-1-P dipotassium salt from Sigma. Potassium galactonate was synthesized by iodine oxidation of galactose as reported by Blair and Segal [13]. UL[¹³C]galactose, 99% APE, UL[¹³C]galactitol, 99.7% APE, and 2-[¹³C]Gal-1-P, 92.5% APE, dipotassium salt, were purchased from Omicron Biochemicals. UL[¹³C]potassium galactonate was synthesized from the UL[¹³C]galactose by iodine oxidation [13] and the purity of the compound was determined by

RBC galactitol

The RBC galactitol concentration in galactosemic patients on a galactose-restricted diet is shown in Table 1. The values ranged from 3.54–8.81 μM with an average of 5.98 ± 1.2 μM (M ± SD). The lowest level in the Q188R homozygous group was 4.01 μM. Patients 2 and 15 with Gal-1-P levels under 76 μM (2 mg/dL) represent variants who probably have some ability to oxidize galactose [14]. There was no relationship of the RBC galactitol with age. For example, the two 7-month-old patients had similar levels as

Discussion

GALT deficiency galactosemia is an enigmatic disorder [15] characterized by a neonatal toxicity syndrome and by later long-term complications despite the institution of a galactose-restricted diet [16], [17]. For many years the measurement of RBC Gal-1-P has been the prime means of monitoring the diet and assessing the galactose metabolic status of patients [1], [2], [3], [4], [5]. Although the RBC Gal-1-P determination has been a routine clinical practice, its relationship to outcome is

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Cited by (23)

Disorders of galactose metabolism
2020, Rosenberg’s Molecular and Genetic Basis of Neurological and Psychiatric Disease: Volume 1
A deficiency of each of the three enzymes important in galactose metabolism, galactose-1-phosphate uridylyltransferase (GALT), galactokinase (GALK), and UDP-galactose 4-epimerase (GALE), may result in disease with hypergalactosemia. Classic galactosemia due to severe GALT deficiency produces a multiorgan toxicity syndrome that is usually lethal with continual lactose ingestion in the first month of life. Cessation of dietary lactose will usually allow the acute complications of poor growth and feeding, jaundice, liver disease, cataracts, and overt encephalopathy to disappear. However, long-term complications involving the brain, ovaries, and bone appear later in life despite dietary treatment. The etiology is unknown and is the subject of current investigations. The severe form of GALE deficiency is extremely rare and resembles GALT deficiency following lactose exposure in the neonatal period. Most patients have an asymptomatic form. The GALK deficiency is also very rare and largely results in cataracts that may be prevented if a lactose-restricted diet is started early in life.
Disorders of Galactose Metabolism
2014, Rosenberg's Molecular and Genetic Basis of Neurological and Psychiatric Disease: Fifth Edition
A deficiency of each of the three enzymes important in galactose metabolism, galactose-1-phosphate uridyltransferase (GALT), galactokinase (GALK) and UDP-galactose 4′-epimerase (GALE), may result in disease with hypergalactosemia. Classic galactosemia due to severe GALT deficiency produces a multiorgan toxicity syndrome that is usually lethal with continual lactose ingestion in the first month of life. Cessation of dietary lactose will usually allow the acute complications of poor growth and feeding, jaundice, liver disease, cataracts, and overt encephalopathy to disappear. However, long-term complications involving the brain, ovaries and bone appear later in life despite dietary treatment. The etiology is unknown and is the subject of current investigation. The severe form of GALE deficiency is extremely rare and resembles GALT deficiency following lactose exposure in the neonatal period. Most patients have an asymptomatic form. The GALK deficiency is also very rare and largely results in cataracts which may be prevented if a lactose restricted diet is started early in life.
Metabolic fate of administered [<sup>13</sup>C]galactose in tissues of galactose-1-phosphate uridyl transferase deficient mice determined by nuclear magnetic resonance
2007, Molecular Genetics and Metabolism
The pattern of distribution of galactose and its metabolites was determined in tissues of mice deficient in galactose-1-phosphate uridyl transferase (G/G) 4 h after the administration of 1 mg/g of [¹³C]galactose. Labeled galactose was found in all the tissues examined, the highest amounts in liver and kidney. Each of the tissues had its own pattern of labeling of galactose-1-phosphate (gal-1-P), galactitol and galactonate. [¹³C]gal-1-P and galactonate concentration was highest in liver while [¹³C]galactitol was higher in kidney and heart than in other tissues. Muscle had the lowest amounts of these compounds. In contrast, no galactose was found in tissues of normal mice (N/N) except for a minute amount in muscle. No [¹³C]gal-1-P was found in liver, kidney or brain and only minute amounts in heart and muscle of N/N animals. Barely detectible, labeled galactitol was observed in these tissues except liver, where none was found. [¹³C]Galactonate was formed in liver comparable to G/G mice. Almost all of the accumulating ¹³C isotope was found in liver and kidney glucose and lactate in the normal animals. [¹³C]Glucose and lactate was also found in liver of the G/G animals, but to a lesser extent than in normals, indicating the presence of a pathway in G/G animals for circumventing the block at GALT for the normal conversion of galactose to glucose.
Of mice and men: Galactosemia
2006, Molecular Genetics and Metabolism
Urinary galactitol and galactonate quantified by isotope-dilution gas chromatography-mass spectrometry
2006, Clinica Chimica Acta
Measurements of urine galactitol have been used to monitor the adequacy of diet therapy in the treatment of galactosemia. We have devised a gas chromatographic mass spectrometry (GC/MS) isotope-dilution method for the simultaneous quantification of urine galactitol and another alternate pathway product, galactonate.
We prepared trimethylsilyl (TMS) derivatives and used d-[UL-¹³C]galactitol and d-[UL-¹³C]galactonate as the internal standard for GC/MS. Results obtained with this method were compared with those determined by the established GC method for galactitol and the NMR method for galactonate. Thirty-three normal urine specimens were analyzed by the isotope dilution technique for galactitol and galactonate. Results of galactitol in 6 of these urine specimens along with 18 from classic galactosemics and 19 variant galactosemics were compared with the established GC method. Results for galactonate in 15 urine specimens from galactosemics were compared to the established NMR technique.
The method was linear up to 200 nmol with lower limits of detection of 1.1 nmol (1.75 mmol/mol creatinine) (Cr) and 0.8 nmol (1.28 mmol/mol Cr) for galactitol and galactonate, respectively. Intra- and Interassay imprecision ranged from 2.1–6.7% for galactitol and 3.5–8.0% for galactonate. The excretion of both metabolites was age dependent in both normal and galactosemics. In 12 normal urines from subjects under 1 year, values for galactitol ranged from 8–107 mmol/mol Cr, and in 7 over age 6, ranged from 2–5 mmol/mol Cr. Under 1 year, the range for galactonate was non-detectable to 231 and in the over 6 years group non-detectable to 25 mmol/mol Cr. In galactosemics under 1 year, the value for galactitol ranged from 397–743 and for galactonate 92–132 mmol/mol Cr while in nine patients over age 6 the range was 125–274 mmol/mol Cr for galactitol and 17–46 mmol/mol Cr for galactonate.
The GC/MS method enables the simultaneous determination of urine galactitol and galactonate and is precise and useful over the wide range of concentrations needed to assess the galactose burden in patients with galactosemia.
Pathways of galactose metabolism by galactosemics: Evidence for galactose conversion to hepatic UDPglucose
2006, Molecular Genetics and Metabolism
To determine if classic galactosemics have residual galactose-1-phosphate uridyltransferase (GALT) activity to explain their considerable ability to oxidize galactose over 24 h, we devised a method for assessing their ability to form hepatic UDPglucose (UDPglu), an intermediate in the normal Leloir pathway of galactose metabolism. The protocol involved the single oral administration of 7 mg/kg [2-¹³C]galactose concomitant with multiple small doses of acetaminophen with measurement of the extent of labeling of urinary acetaminophen glucuronide, the glucuronide moiety being formed from hepatic UDPglu. We performed the study lasting 24 h in two normal subjects and three classic galactosemics, two homozygous for the Q188R mutation and one compound for the Q188R/K258N mutation. The labeling and total excretion of acetaminophen glucuronide was measured in urine by nuclear magnetic resonance techniques. Concomitant with determination of label in the glucuronide measurement was made of galactose oxidation to ¹³CO₂ and the ¹³C enrichment of plasma glucose. All of the galactosemic patients formed ¹³C enriched acetaminophen glucuronide indicating that they had converted the labeled galactose to [¹³C]UDPglu and that residual GALT or another pathway that forms UDPglu is present in hepatic tissue. Compared to the normal whose glucuronide labeling was rapid and short-lived that of the galactosemics was delayed and extended for a long period over 10 h. The extent of isotopic enrichment of glucuronide by galactosemics was comparable to the normals, resulting in a much greater conversion of galactose to UDPglu by the galactosemics. The labeling of the UDPglu pool was reflected by the rate of ¹³CO₂ formation being rapid in the normal with peak labeling at 2–3 h with total oxidation of over 70% in 24 h. The oxidation of the galactosemics was slow with a broad peak of ¹³CO₂ at 10 h and a total excretion of 25–39% of the [¹³C]galactose administered. The normal subjects formed highly enriched plasma glucose within 30 min while no enrichment of plasma glucose was detected until after 300 min in galactosemics. The exact pathway(s) of galactose metabolism by galactosemics to UDPglu remain to be determined. Their delineation may contribute to new approaches to therapeutic strategies for this enigmatic disorder.

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Galactitol and galactonate in red blood cells of galactosemic patients

Abstract

Introduction

Section snippets

Materials

RBC galactitol

Discussion

Metabolism

Metabolism

Clin. Chim. Acta

Metabolism

Arch. Biochem. Biophys.

Some disturbance of erythrocyte metabolism in galactosemia

Biochem. J.

The value of galactose phosphate determinations in treatment of galactosemia

Arch. Dis. Child

Galactose-1-phosphate in galactosemia

Pediatrics

Observations on the management of galactosemic patients

Galactose-1-phosphate in the pathophysiology of galactosemia

Eur. J. Pediatr.

Galactitol in Galactosemia

Eur. J. Pediatr.