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Clinical Chemistry 48: 604-612, 2002;
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(Clinical Chemistry. 2002;48:604-612.)
© 2002 American Association for Clinical Chemistry, Inc.

Erythrocyte Galactose 1-Phosphate Quantified by Isotope-Dilution Gas Chromatography-Mass Spectrometry

Jie Chen1, Claire Yager1, Robert Reynolds1, Michael Palmieri2 and Stanton Segal1,2,3a

1 Metabolic Research Laboratory and
2 Metabolic Diagnostic Laboratory, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104.

3 Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104.

aAddress correspondence to this author at: Metabolic Research Laboratory, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104. Fax 215-590-3364; e-mail segal{at}email.chop.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Measurements of {alpha}-D-galactose 1-phosphate (Gal-1-P) in erythrocytes are used to monitor the adequacy of dietary therapy in the treatment of galactosemia. We have devised a gas chromatography-mass spectrometry (GC/MS) isotope-dilution method for quantification of Gal-1-P.

Methods: We prepared trimethylsilyl (TMS) derivatives and used {alpha}-D-[2-13C]Gal-1-P as the internal standard for GC/MS. Results obtained with this method were compared with those determined by the established enzymatic method for samples from 23 healthy individuals (11 children and 12 adults), 9 suspected patients with galactosemia, 12 galactosemic patients on diet therapy, and 2 newly diagnosed toxic neonates.

Results: The method was linear up to 2.5 mmol/L with a lower limit of detection of 2.1 nmol (0.55 mg/L). Intra- and interassay imprecision (CVs) was 2.2–8.8%. In the 23 healthy individuals, values ranged from nondetectable to 9.2 µmol/L (2.4 mg/L of packed erythrocytes). Galactosemic patients on diet therapy had values of 10.9–45 mg/L of packed erythrocytes, whereas the newly identified patients had values of 166 and 373 mg/L.

Conclusions: The GC/MS method is precise and useful over the wide range of concentrations needed to assess the galactose burden in patients with galactosemia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Since Schwarz et al. (1) described the accumulation of galactose 1-phosphate (Gal-1-P) 4 in erythrocytes of patients with galactose-1-phosphate uridyltransferase (GALT; EC 2.7.7.12) deficiency galactosemia, its measurement has become standard practice for monitoring the treatment of the disorder with galactose-restricted diets (2)(3)(4)(5). In the years since the discovery by Schwarz et al. (1), numerous methods have been developed for assaying Gal-1-P in erythrocytes. Initially, Schwarz et al. (1) precipitated Gal-1-P as the barium salt, liberated the galactose by acid hydrolysis, separated the sugar by paper chromatography, and estimated the quantity by color intensity after staining. Schwarz and Simpson (6) later modified the method by determining the galactose hydrolyzed by galactose oxidase. In 1960, Kirkman and Maxwell (7) reported the first of a series of enzymatic methods. They assayed the Gal-1-P in erythrocyte hemolysates by UDP-glucose (UDPG) consumption, measuring UDPG with UDPG dehydrogenase and NADH production before and after the addition of GALT. The basic idea of using GALT to assess Gal-1-P in erythrocytes was extended to measuring the product of the reaction, {alpha}-D-glucose 1-phosphate (Glu-1-P), after enzymatic conversion to D-glucose 6-phosphate (Glu-6-P) and treatment with Glu-6-P dehydrogenase to form NADH, which was measured fluorometrically (8), or by determining the radioisotope content of the Glu-1-P when 14C-labeled UDPG was a substrate in the reaction (9). Gitzelmann (10) took another enzymatic approach, using galactose dehydrogenase to assay galactose phosphatase by spectrophotometric measurement of the difference in NADH before and after treatment of hemolysates with alkaline. Dahlqvist (11) modified this procedure and proposed assessment of NADH fluorometrically.

None of these established methods uses direct chemical quantification of Gal-1-P. Using the characterization of trimethylsilyl (TMS) derivatives of sugar phosphates by gas chromatography-mass spectrometry (GC/MS) by Harvey and Horning (12), we have developed an isotope-dilution method of quantifying erythrocyte Gal-1-P by GC/MS after trimethylsylation of the sugar phosphate. The method is accurate and precise, and automated sampling permits the assay of multiple specimens over a short time period. Its description forms the basis of this report.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
materials
{alpha}-D-[2-13C]Gal-1-P ([2-13C]Gal-1-P) dipotassium salt [atomic percent excess, 99%] was purchased from Omicron Biochemicals, Inc. The atomic percent excess of [2-13C]Gal-1-P was 92.5% ± 0.14% (mean ± SD; n = 25) by GC/MS analysis. N,O-bis(Trimethylsilyl)trifluoroacetamide with trimethylchlorosilane (BSTFA + TMCS) was obtained from Pierce. Galactose dehydrogenase and alkaline phosphatase were from Roche. Gal-1-P dipotassium salt, Glu-1-P dipotassium salt, Glu-6-P dipotassium salt, galactose 6-phosphate (Gal-6-P) dipotassium salt and disodium salt, and {alpha}-D-mannose 1-phosphate (Man-1-P) dipotassium salt were purchased from Sigma. All other reagents were from Sigma and Fisher Scientific.

sample preparation and derivatization
Random nonfasting samples of venous blood collected in sodium heparin-containing tubes were centrifuged at 900g for 5 min at 4 °C. After the plasma and leukocyte-containing layers were removed by aspiration, red blood cells (RBCs) were washed three times with normal saline. Aliquots (400 µL) of packed RBCs were placed in 15-mL polystyrene tubes and stored at -80 °C until being assayed. Hemolysates were made by adding 200 µL of HPLC-grade water at 4 °C to the thawed sample, followed by the addition of 40 nmol of [2-13C]Gal-1-P in 4 µL as an internal standard. The hemolysates were then deproteinized with 2 mL of methanol, and after a 10-min centrifugation at 900g, 1 mL of the supernatant was transferred to a 13 x 100 mm screw-cap glass tube and evaporated to dryness under a stream of nitrogen. The residue was trimethylsilylated with 60 µL of BSTFA + TMCS and 60 µL of pyridine at 90 °C for 30 min. A 1-µL portion of each derivatized preparation was injected for GC/MS analysis.

gc/ms
The analysis was performed by electron ionization at 70 eV on a Hewlett-Packard 6890/5973 with an HP-5MS column (30 m x 250 µm x 0.25 µm nominal) cross-linked with 5% phenylmethylsiloxane. The helium flow was kept in a constant flow mode at 0.6 mL/min. The oven ramp was set at initial temperature 125 °C for 1 min; at ramp 1, it was increased to 175 °C at a rate of 20 °C/min, then at ramp 2 to 230 °C at a rate of 4 °C/min. Once the temperature reached 230 °C, it was kept at that temperature for 3 min. Finally, the oven temperature was increased to 300 °C at a rate of 40 °C/min. The temperature was kept at 300 °C for 5 min. The inlet temperature was set to 250 °C, and the splitless mode was applied. The purge flow was started at a rate of 50 mL/min 0.7 min after the 1-µL sample injection. The mass selective detector transfer line heater temperature was set to 280 °C. The source temperature was 230 °C. Data acquisition mode can be selected either in scan or selected-ion monitoring mode, depending on different analytical purposes. The ion intensities of m/z 204 and 205 in the Gal-1-P chromatographic peak allowed the assessment of 2-12C- and 2-13C-labeled Gal-1-P, respectively.

calibration curve
Calibrators were prepared in HPLC-grade water at concentrations of 1–200 nmol from a stock solution of 10 mmol/L [12C]Gal-1-P to which we added 40 nmol of [2-13C]Gal-1-P from a stock solution of similar concentration. The calibrators were treated by the same procedure described above (see "Sample Preparation and Derivatization" section above). The ratio Rn of m/z 204 to m/z 205 (see "Calculation" section below) was plotted vs the prepared Gal-1-P concentrations. Because the [2-13C]Gal-1-P contained a small amount of [2-12C]Gal-1-P, a correction was applied to account for this. This "blank" was determined from the ratio of m/z 204 to m/z 205 of the isotopic material.

precision and recovery
To study intra- and interassay imprecision, we added [12C]Gal-1-P to human RBC pools at concentrations of ~10 and 100 mg/L. We calculated intraassay imprecision based on a series of 12 injections of both low- and high-concentration matrix, respectively, whereas we calculated interassay imprecision based on two analyses per day of both low- and high-concentration matrix for 20 days. For this determination, we pipetted 400 µL of RBCs into tared tubes, which were weighed after the addition of the cells. The [12C]Gal-1-P concentration was calculated based on both the volume and weight of the cells to determine any imprecision attributable to variation in pipetting of packed RBCs. We evaluated recoveries by adding 10, 20, 40, or 60 nmol of 12C Gal-1-P to human RBCs and measuring the [12C]Gal-1-P before and after the additions.

calculation
Carbon exists in two stable, naturally occurring isotopic forms: 12C and 13C. The ratio of abundance of the 13C form to the 12C form is ~1.1%. The ratio of m/z 205 to m/z 204 in Gal-1-P, R0, was considered to represent the natural abundance of 13C in Gal-1-P, and was determined to be 0.22 ± 0.003 (mean ± SD) based on the results of 24 analyses of concentrations of 40, 80, 100, 160, 200, 400 nmol of Gal-1-P.

Subtraction of the ion intensity of m/z 205 caused by the natural abundance of 13C in all calibrators and samples was performed in the isotope dilution calculation. The equations to take into account the natural abundance are:



where

T205

P205 = Pure intensity m/z 205 of [2-13C]Gal-P

T205 = Total intensity of m/z 205

T204 = Total intensity of m/z 204

Rn, the net ratio of m/z 204 to m/z 205, is calculated by subtracting the amount of unenriched Gal-1-P in the internal standard (Rb) from each calibrator or unknown sample (Rs). The Rb found with each assay was used in the calculation, but was essentially constant at 0.08:

The Gal-1-P concentration (cGal-1-P) in a sample could be calculated by using the linear regression equation of the calibrators:

where k and b are the slope and intercept of the line relating R to the Gal-1-P concentration.

Conversion of nanomoles in the sample is:

where 260 is the molecular weight of Gal-1-P.

comparison with an enzymatic method
We used the method of Dahlqvist (11), which is used in the Children’s Hospital clinical laboratory, for comparison purposes. We analyzed 400 µL of packed cells simultaneously with the enzymatic and the GC/MS methods.

statistics
Linear regression analysis was used to determine the relationship of the m/z 204/205 ratio to the concentration of the calibrator, as well as that of the new method to that of the established enzymatic procedure. We used the Bland-Altman method (13) to compare the GC/MS with the enzymatic method. Data are presented as the mean ± SD.

participants
Blood was obtained from 12 adult volunteers (age range, 22–57 years; 6 females and 6 males). RBCs from 11 nongalactosemic children on lactose-containing diets (age range, 0.1–12.3 years) were discarded specimens after plasma removal for amino acid quantification in the Children’s Hospital clinical laboratory. Twelve specimens from patients with GALT-deficient galactosemia were obtained for regular monitoring of compliance with a galactose-restricted diet, and 2 were from newly diagnosed infants on galactose-free formulas. Nine specimens were analyzed from individuals suspected of being galactosemic either as a result of neonatal screening or were known to have reduced red cell GALT activity in the range of carriers or of compound heterozygotes for N314D/Q188R mutations. All specimens were collected in tubes containing heparin as the anticoagulant. The protocol was approved by the Institutional Review Board of The Children’s Hospital of Philadelphia.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
gc analysis of sugar phosphates
The chromatographic separation of TMS derivatives of five sugar phosphates, Gal-1-P, Gal-6-P, Glu-1-P, Glu-6-P, and Man-1-P, is shown in Fig. 1A . The peak assignment was made based on the position of each sugar phosphate in individually determined chromatograms. The 1-phosphates give a single peak, whereas 6-phosphates give two, reflecting the {alpha} and ß forms of the sugar moiety. Fig. 1B shows a chromatogram of RBCs from a GALT-deficient patient, demonstrating the presence of Gal-1-P. The peak was also identified as Gal-1-P by the corresponding position of the [2-13C]Gal-1-P peak added to the red cell extract.



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  		Figure 1. Total-ion chromatograms showing the separation of TMS-derivatized sugar phosphates.

(A), mixture of sugar phosphates containing Man-1-P, Gal-1-P, Glu-1-P, and Glu-6-P. (B), Gal-1-P in an RBC hemolysate from a galactosemic patient.

mass spectra of gal-1-p tms derivative
The fragmentation patterns of TMCS-derivatized [12C]Gal-1-P and [13C]Gal-1-P are shown in Fig. 2 , A and B, respectively. There was a clear 1-Da mass shift of m/z 204 to m/z 205, which was attributable to the 2-13C in Gal-1-P. The decomposition of the Gal-1-P TMS derivative in the ion source probably involves two {alpha} cleavages between C1-C2 and C3-C4, which produce a radical cation with m/z 204 (Fig. 3 ). The other major fragments, m/z 299 and m/z 315, do not show any mass shift and do not contain the isotopic carbon. As pointed out by Harvey and Horning (12) from their fragmentation pattern of Gal-1-P, these fragments possibly are TMS derivatives of phosphate.



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  		Figure 2. Mass spectra of the TMS derivatives of Gal-1-P (A) and [2-13C]Gal-1-P (B) recorded at 70 eV.

The spectra show a clear 1-Da mass shift at m/z 204 and m/z 205.



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  		Figure 3. Theoretical decomposition of the TMCS derivative of Gal-1-P.

Two cleavages occur between C1–C2 and C3–C4, which produce a radical cation fragment with m/z 204.

linearity of calibration curve
Shown in Fig. 4 is a typical calibration curve over a 200-nmol range of Gal-1-P, which encompass concentrations known to exist in RBCs of patients maintained on galactose-restricted diets as well as in RBCs of healthy individuals. The assay was linear to 1000 nmol, which would reflect cell concentrations in galactose-toxic patients. The linearity of the curve was consistently reproducible and was observed in curves constructed with each set of unknown samples. For 28 calibration curves constructed over a 3-month period in the range up to 200 nmol, the r2 was 1.00 ± 0.001 (mean ± SD). The slope of the resulting curve was 0.03 ± 0.001 (mean ± SD), and the y-intercept was -0.024 ± 0.019 (mean ± SD). The lower limit of detection was calculated by determining Gal-1-P from the equation, using the y-intercept (-0.024) and -2 SD (-0.038) divided by the slope (0.03). The resulting value was 2.1 nmol (0.55 mg/L). Because dilutions were made from a stock solution kept at -80 °C over a 3-month period, the reproducibility attests to the stability of the compound in the frozen state.



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  		Figure 4. Relationship between the ratio of the peak area of ion m/z 204 of Gal-1-P to the peak area of m/z 205 of the internal standard and the concentration of Gal-1-P.

The symbols represent single determinations. The data are representative of 28 such curves. The curve for the mean of the 28 curves is represented by the equation: y = (0.03 ± 0.0001)x - (0.024 ± 0.019), where the values in parentheses represent the mean ± SD.

precision and accuracy
The intraassay CVs (calculated from results in mg/L of RBCs) were 5.9% and 2.2% for the low and high concentrations, respectively; interassay CVs were 8.8% and 5.5%, respectively, for the low and high concentrations (Table 1 ). Also shown in Table 1 are the values calculated on the basis of weight of the RBC sample, expressed as mg/100 g of RBCs. The CV appears to be somewhat less when the weight of the sample is considered, but not enough to warrant the tedious process of weighing the samples instead of pipetting them. The relationship between the concentration expressed in mg/L of RBCs and that expressed in mg/kg of RBCs was fortuitous, indicating that 100 mL of RBCs corresponds to 100 mg of cells.


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  		Table 1. Assessment of the analytical precision of the isotope-dilution method.

The reproducibility of the Gal-1-P assay with 75–500 µL of red cells from a known galactosemic patient was similar at all of the volumes used, with a CV of 3.5% (Table 2 ). In recovery studies, 10, 20, and 40 nmol were added to each of three RBC specimens, and 60 nmol was added to five. The recoveries were similar, at 97.6% ± 4.1% (mean ± SD; n = 14).


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  		Table 2. Reproducibility of the GC/MS method.

comparison of methods
The new GC/MS method agreed well (Fig. 5 ) with the method that uses galactose dehydrogenase to determine the NADH formed in the analysis of galactose before and after treatment with alkaline phosphatase. Fig. 5A , which excludes the two high values of newly diagnosed neonates, indicates a correlation between the two methods with a r2 of 0.99. Fig. 5B is a Bland-Altman plot (13) of the averages of the two values plotted against the difference between them, which shows reasonable scatter around the mean over the range of values. The values for the two neonates by the GC/MS method (166 and 373 mg/L) compared favorably to the results obtained by the enzymatic method (154 and 351 mg/L, respectively).



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  		Figure 5. Relationship between the new GC/MS method and the established enzymatic method.

(A), linearity of values obtained by the GC/MS method vs the enzymatic procedure. (B), Bland-Altman plot (13).

reference values
Values obtained for 12 adults by the GC/MS method, based on randomly obtained blood specimens, were 1.1–2.4 mg/L (0.11–0.24 mg/dL); for the 11 children, concentrations were below the detection limit for 4 children, and values for the others ranged from 1.2 to 1.8 mg/L (0.12–0.18 mg/dL). Of eight patients tested because of mutations in the GALT gene, the results for seven were within the range of values obtained for healthy individuals.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Gal-1-P accumulates in the tissues of patients who are unable to convert it to UDP-galactose because of a deficiency of GALT. The measurement of Gal-1-P in RBCs, the most readily available tissue, has been used to assess the biochemical results of initiating galactose-restricted diets in newborns diagnosed with galactosemia as well as to monitor the adherence to such diets during later life. Indeed, Gal-1-P has been the most frequently measured galactose metabolite, and its assessment in RBCs is routine in the clinical management of galactosemic patients. Interest has also focused on the sugar phosphate as a toxicity factor and as the basis of the long-term complications associated with galactosemia (5).

Various methods have been devised to quantify RBC Gal-1-P, which can be divided into three types: one involves measuring the product Glu-1-P formed after the addition of GALT (8)(9); the second involves measuring UDPG consumed in the reaction with Gal-1-P (7); the third involves the use of galactose dehydrogenase to measure galactose liberated after alkaline phosphatase treatment of hemolysates (10)(11). We are currently using the latter method, which has widespread popularity. In this method, multiple timed fluorometric readings of NADH generated in the dehydrogenase reaction are required before and after hydrolysis of the phosphate. A direct chemical method for measuring RBC Gal-1-P based on the gas chromatographic separation of sugar phosphates has not been described until now. Ion-exchange chromatography (14) and HPLC techniques (15) have focused on phosphorylated compounds related to glucose metabolism in RBCs without consideration of galactose metabolites. Harvey and Horning (12) extensively evaluated GC/MS analysis of TMS derivatives of myriad phosphorylated sugars that are of biological interest without application to RBCs in particular. Included in their analysis was the description of the GC analysis of the TMS derivative of Gal-1-P and its mass fragmentation pattern.

Using the TMS derivatives of sugar phosphates and modern capillary columns, we devised a GC program for separating red cell sugar phosphates from Gal-1-P and the latter from Gal-6-P. The commercial availability of [2-13C]Gal-1-P along with a sophisticated bench-top mass spectrometer has enabled us to devise an accurate, sensitive method for quantifying RBC Gal-1-P by use of isotope-dilution MS. From the mass fragmentation pattern we were able to select a target ion, m/z 204, which shifted 1 mass unit to m/z 205 because of the presence of the isotopic carbon in a fragment we considered to contain C2 and C3 of the original molecule.

The isotope-dilution method is accurate and reproducible with a CV <10%. Calibration curves run with each group of specimens were remarkably reproducible and even raised the question of whether they need to be repeated with each analysis or merely periodically. Because the packed RBCs may vary in terms of viscosity, we examined the effect of weighing the RBC samples and expressing the results in terms of cell weight to reduce a difference in the results, as seen in Table 1Up . We also calculated all of the blood sample results on the basis of weight without any appreciable differences in the results. Indeed, the results of pipetting 75–500 µL of RBCs were essentially the same, indicating that pipetting was not a critical variable (Table 2Up ).

The new GC/MS method compares well with the current method using galactose dehydrogenase. The Bland-Altman plot revealed no systematic bias. There is, however, a larger percentage of difference in the reference interval, with values obtained with the GC/MS values generally being lower. Because these differences occur in the range of RBC concentrations considered to be normal, there should be no impact on the interpretation of the galactose metabolic status of the individual whose cells were analyzed.

The primary usefulness of RBC Gal-1-P quantification has been in monitoring of the adequacy of diet therapy and evidence for breaches in the diet. Serial measurements at timely intervals are routinely performed in patients followed at metabolic centers, and experience has been gained in >40 years of determinations by various methods. In the new GC/MS method, the measurements have been made in randomized specimens from nonfasting individuals, which is the practice we use in our clinic to monitor patients’ Gal-1-P concentrations by the galactose dehydrogenase assay. The use of nonfasting specimens has been advocated as the best means of monitoring dietary compliance because unusual increases resulting from breaches in the diet and ingestion of galactose-containing foods last only 24–48 h (4). In addition, in a study in which the UDPG consumption assay was used to monitor galactosemic patients, there was no statistical difference in concentrations between fasted and nonfasted patients (3). Indeed, a fasting requirement would be very difficult in view of the variable times of patient’s clinic visits. Gal-1-P concentrations in RBCs from galactosemic patients measured by the GC/MS procedure vary little from those measured by the established enzymatic method at concentrations of 10–50 mg/L (1–5 mg/dL), which are typically observed in patients maintained on galactose-restricted diets. As such, the method is adequate to perform the needed assessment. Indeed, the GC/MS method is also accurate in determining high concentrations, as shown in two newly diagnosed patients.

We have established a range of values in random nonfasted specimens in both healthy adults and nongalactosemic children. Donnell et al. (3), using the UDPG consumption method, reported that healthy adults and children older than 18 months have undetectable RBC Gal-1-P whether fasting or not. Of 28 healthy children in whom Gal-1-P was measured by the amount of [14C]Glu-1-P formed, only 5 had measurable amounts of Gal-1-P, with the highest being 8 mg/L (0.8 mg/dL) (9). In an enzymatic assay measuring Glu-1-P formation, the range was 0.0–14.8 mg/L (0.0–1.48 mg/dL) (8).

Increased RBC Gal-1-P is not a diagnostic tool for galactosemia because the diagnosis is made by measurement of RBC GALT activity. It appears to be a measure, however, of the patient’s galactose burden and can be >=1.0 g/L (100 mg/dL) when galactose is ingested before diet therapy is initiated (5). The concentrations remain increased even on the best galactose-restricted diets, usually in the 10–50 mg/L (1–5 mg/dL) range. The consistently abnormal concentrations seen throughout the galactosemic patient’s life may be related to endogenous production of galactose (16)(17) in the face of a limited metabolic capability (18)(19). There are some galactosemic variants who can metabolize significant amounts of galactose (20)(21) and have RBC Gal-1-P concentrations above normal but not as high as in the severe forms. The detection of above-normal RBC Gal-1-P concentrations may be important, however, in assessing the need for diet therapy in those with depressed but not absent GALT activity attributable to compound heterozygosity for the Duarte and a galactosemia gene, such as the common mutations, N314D/Q188R, frequently detected in newborn screening programs (20)(21). It is interesting to note that the concentration of RBC Gal-1-P is much higher than the plasma concentrations of galactose in galactosemic patients (22).

In conclusion, we have described a GC method for separating RBC sugar phosphates by trimethylsylation with emphasis on the identification and quantification of Gal-1-P by isotope-dilution MS. The method is precise and accurate and allows for automated analysis. The method is linear over a wide range of RBC concentrations and would be useful in biochemical genetic laboratories for monitoring the galactose burden in known classic galactosemic patients as well as in determining the extent of abnormal galactose metabolism in individuals with variant genotypes of the GALT gene.


   Acknowledgments
 
We wish to thank Dr. Ian Blair, Director of the Center for Cancer Pharmacology, University of Pennsylvania, for helpful assistance.


   Footnotes
 
4 Nonstandard abbreviations: Gal-1-P, {alpha}-D-galactose 1-phosphate; GALT, galactose-1-phosphate uridyltransferase; UDPG, uridine diphosphate glucose; Glu-1-P, {alpha}-D-glucose 1-phosphate; Glu-6-P, D-glucose 6-phosphate; TMS, trimethylsilyl; GC/MS, gas chromatography-mass spectrometry; BSTFA, N,O-bis(trimethylsilyl)trifluoroacetamide; TMCS, trimethylchlorosilane; Gal-6-P, D-galactose 6-phosphate; Man-1-P, {alpha}-D-mannose 1-phosphate; and RBC, red blood cell.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Schwarz V, Goldberg L, Komrower G, Holzel A. Some disturbance of erythrocyte metabolism in galactosemia. Biochem J 1956;62:34-40.
  2. Schwarz V. The value of galactose phosphate determinations in treatment of galactosemia. Arch Dis Child 1960;35:428-432.
  3. Donnell GN, Bergren WR, Perez G, Koch R. Galactose-1-phosphate in galactosemia. Pediatrics 1963;31:802-810.[Abstract/Free Full Text]
  4. Donnell GN, Koch R, Bergren WR. Observations on the management of galactosemic patients. Hsia DY eds. Galactosemia 1969:247-268 Charles Thomas Springfield, IL. .
  5. Gitzelmann R. Galactose-1-phosphate in the pathophysiology of galactosemia. Eur J Pediatr 1995;154:S45-S49.[ISI][Medline] [Order article via Infotrieve]
  6. Schwarz V, Simpson IMN. A note on some difficulties in determination of galactose-1-phosphate in galactosemia. Clin Chim Acta 1969;24:188.[ISI][Medline] [Order article via Infotrieve]
  7. Kirkman HN, Maxwell ES. Enzymatic estimation of erythrocyte galactose-1-phosphate. J Lab Clin Med 1960;56:161-166.
  8. Perez M, Bodourian SH, Nicholson JF. A new microfluorometric method for the measurement of galactose-1-phosphate in erythrocytes. Clin Chim Acta 1982;118:177-189.[ISI][Medline] [Order article via Infotrieve]
  9. Bozkowa K, Duchnowska A, Chojnacki T, Mankowski T. The use of a radioisotopic method estimation of galactose-1-phosphate in galactosemia. Biochem Med 1977;17:24-30.[ISI][Medline] [Order article via Infotrieve]
  10. Gitzelmann R. Estimation of galactose-1-phosphate in erythrocytes: a rapid and simple enzymatic method. Clin Chim Acta 1969;26:313-316.[ISI][Medline] [Order article via Infotrieve]
  11. Dahlqvist A. A fluorometric method for assay of galactose-1-phosphate in red blood cells. J Lab Clin Invest 1971;78:931-938.
  12. Harvey DJ, Horning MG. Characterization of the trimethylsilyl derivatives of sugar phosphates and related compounds by gas chromatography and gas chromatography/mass spectrometry. J Chromatogr 1973;76:51-62.[ISI][Medline] [Order article via Infotrieve]
  13. Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986;:307-310.
  14. Bartlett GR. Phosphorus compounds in the human erythrocyte. Biochem Biophys Acta 1968;156:221-230.[Medline] [Order article via Infotrieve]
  15. Voght AM, Ackerman C, Noe T, Jansen D, Kubler W. Simultaneous detection of high energy phosphates and metabolites of glycolysis and the Krebs cycle by HPLC. Biochem Biophys Res Commun 1998;248:527-532.[ISI][Medline] [Order article via Infotrieve]
  16. Berry GT, Nissim I, Lin Z, Mazur AT, Gibson JB, Segal S. Endogenous synthesis of galactose in normal man and patients with hereditary galactosemia. Lancet 1995;346:1073-1074.[ISI][Medline] [Order article via Infotrieve]
  17. Ning C, Fenn PT, Blair IA, Berry GT, Segal S. Apparent galactose appearance rate in human galactosemia based on plasma [13C]galactose isotopic enrichment. Mol Genet Metab 2000;70:261-271.[ISI][Medline] [Order article via Infotrieve]
  18. Segal S, Blair A, Roth H. The metabolism of [14C]galactose by patients with congenital galactosemia. Am J Med 1965;38:62-70.
  19. Berry GT, Singh R, Mazur AT, Guerrero N, Kennedy MJ, Chen J, et al. Galactose breath testing distinguishes variant and severe galactose-1-phosphate uridyltransferase genotypes. Pediatr Res 2000;48:323-328.[ISI][Medline] [Order article via Infotrieve]
  20. Elsas LJ, II, Langley L, Paulk EM, Hjelm LN, Dembure PP. A molecular approach to galactosemia. Eur J Pediatr 1995;154:S21-S27.[ISI][Medline] [Order article via Infotrieve]
  21. Gitzelmann R, Bassbard NU. Partial deficiency of galactose-1-phosphate uridyltransferase. Eur J Pediatr 1995;154:S40-S44.[ISI][Medline] [Order article via Infotrieve]
  22. Ning C, Segal S. Plasma galactose and galactitol concentration in patients with galactose-1-phosphate uridyltransferase deficiency galactosemia: determination by gas chromatography-mass spectrometry. Metabolism 2000;49:1460-1467.[ISI][Medline] [Order article via Infotrieve]




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