Colorimetric determination of galactose and galactose-1-phosphate from dried blood

doi:10.1016/0009-9120(92)80043-G

Clinical Biochemistry

Volume 25, Issue 1, February 1992, Pages 37-39

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A colorimetric microassay for the simultaneous quantitative determination of galactose (Gal) and galactose-1-phosphate (Gal-1-P) in dried blood spots is described. An enzymatic reaction involving alkaline phosphatase (EC 3.1.3.1) and galactose dehydrogenase (EC 1.1.1.48) produces NADH, which is coupled with diaphorase (EC 1.8.1.4) and iodonitrotetrazolium violet (INT). The colourless INT is converted to a formazan of red colour the intensity of which is quantitated either photometrically by a microplate reader or determined visually with sufficient sensitivity for screening purposes. We evaluated the assay on 200,000 blood samples in a newborn screening program, and were able to distinguish between classical and milder forms of galactosemia with ease.

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Cited by (42)

Galactokinase deficiency: lessons from the GalNet registry
2021, Genetics in Medicine
Citation Excerpt :
In both tests, Gal-1-P is converted to galactose. However, other sugar phosphates beside Gal-1-P can be a substrate for alkaline phosphatase, which could then lead to false positive results.35,36 Theoretically, patients with severe GALK1 deficiency on an unrestricted diet might have massive elevations in plasma and tissue galactose leading to the formation of other phosphorylated galactose compounds in hematopoietic cells thus leading to a false positive result with methodology that fails to exclusively measure Gal-1-P.
Galactokinase (GALK1) deficiency is a rare hereditary galactose metabolism disorder. Beyond cataract, the phenotypic spectrum is questionable. Data from affected patients included in the Galactosemias Network registry were collected to better characterize the phenotype.
Observational study collecting medical data of 53 not previously reported GALK1 deficient patients from 17 centers in 11 countries from December 2014 to April 2020.
Neonatal or childhood cataract was reported in 15 and 4 patients respectively. The occurrence of neonatal hypoglycemia and infection were comparable with the general population, whereas bleeding diathesis (8.1% versus 2.17–5.9%) and encephalopathy (3.9% versus 0.3%) were reported more often. Elevated transaminases were seen in 25.5%. Cognitive delay was reported in 5 patients. Urinary galactitol was elevated in all patients at diagnosis; five showed unexpected Gal-1-P increase. Most patients showed enzyme activities ≤1%. Eleven different genotypes were described, including six unpublished variants. The majority was homozygous for NM_000154.1:c.82C>A (p.Pro28Thr). Thirty-five patients were diagnosed following newborn screening, which was clearly beneficial.
The phenotype of GALK1 deficiency may include neonatal elevation of transaminases, bleeding diathesis, and encephalopathy in addition to cataract. Potential complications beyond the neonatal period are not systematically surveyed and a better delineation is needed.
Optimized platelet rich plasma releasate (O-rPRP) repairs galactosemia-induced ovarian follicular loss in rats by activating mTOR signaling and inhibiting apoptosis
2020, Heliyon
Citation Excerpt :
Atretic follicles were identified due to the presence of a degenerating oocyte or pyknotic granulosa cells [37]. Galactose was measured using galactose colorimetric detection kit (Invitrogen, Catalog number, EIAGALC) as a modification of a protocol described before [38], wherein measured volumes of sera and standards were used instead of dried blood spots and standards. The kit uses galactose oxidase, which reacts with galactose to produce hydrogen peroxide, and, in the presence of horseradish peroxidase, the reaction with a colorless substrate produces a colored product.
Platelet rich plasma contains a collection of growth factors, and an optimal formulation, named O-rPRP, contains the highest possible concentration of growth factors.
Challenging the healing power of O-rPRP in a high-galactose diet-induced premature ovarian insufficiency (POI) experimental rat model.
Rats were divided into four groups of ten rats each and treated for four week as follows; 1) the control group, fed with normal diet and received intraperitoneal (i.p.) injection of PBS once/week; 2) the POI group, fed with galactose diet (50%) and received PBS (i.p.) once/week; 3) the POI/O-rPRP group, fed a 50% galactose diet and received O-rPRP (i.p.) once/week; 4) the O-rPRP group (negative control), fed with a normal diet and received O-rPRP (i.p.) once/week. The levels of galactose, follicle stimulating hormone, 17 β-estradiol, anti-mullerian hormone and inhibin B were measured in serum samples. Western blotting and quantitative real-time PCR assays were employed to investigate the levels of miR-223, β1 integrin, p70S6k and MCL-1 in ovarian tissues.
After O-rPRP treatment, β1 integrin expression was enhanced, and miR-223 expression was decreased. Unlike the untreated galactose group, in the group treated with O-rPRP, p70S6k and MCL-1 expression levels were increased, indicating that the mTOR growth signaling pathway was active and that apoptosis was inactive. After the introduction of O-rPRP, the number of follicles and the follicular maturation improved, which was consistent with the improvement of inhibin B levels and subsequent inhibition of FSH.
O-rPRP inhibited galactose-induced excessive atresia and provided an overall protective effect on the ovarian follicles.
Quantification of total hexose on dry blood spot by tandem mass spectrometry
2012, Clinical Biochemistry
Citation Excerpt :
Glucose is also generated in the body by glycogen degradation and gluconeogenesis from acetone bodies and amino acids. The hexoses in human blood are predominantly glucose, which occurs in approximately ten to thousands times the concentration of galactose, and the cut-off value for diagnosing galactosemia is 1.665 mmol/L (30 mg/dL) [15,25,26]. The concentration of glucose is approximately a hundred times that of fructose [16,27] and a thousand times that of mannose [19,27].
Because hypoglycemia and hyperglycemia are harmful and not always associated with overt clinical signs, it is necessary to have methods available to screen for glucose levels to detect hypoglycemia and diabetes as early as possible. A new method for such screening and the clinical determination of blood total hexose on a dry blood spot (DBS) using tandem mass spectrometry (MS/MS) was developed.
The serum glucose controls and blood were prepared as DBS and then extracted into a methanol solution containing isotope-labeled internal standards. The methanolic extraction was subjected to HPLC, followed by MS/MS in positive ion mode. Multiple-reaction monitoring of m/z 203.1 → 23 was used to detect hexose, and m/z 209.0 → 23 was used for 13C6-D-glucose.
The recoveries of blood glucose by MS/MS were 90%–102% with an R² value of 0.999 after linear regression (p < 0.001). The controls were within an acceptable range, and the coefficients of variation were less than 10%. The blood total hexose in neonates aged 3–7 days (6.41 ± 1.46 mmol/L) was lower than that in neonates aged 8–30 days (6.66 ± 1.38 mmol/L), and it was lower in neonates than in children aged 1–72 months (7.19 ± 1.87 mmol/L).
Quantification of total hexose on a dry blood spot by MS/MS is accurate, reliable and feasible for screening and clinical tests.
Early Cataract Formation Due to Galactokinase Deficiency: Impact of Newborn Screening
2011, Archives of Medical Research
Citation Excerpt :
In addition to GALT activity, total galactose (galactose plus galactose-1-phosphate) as a screening parameter for classic galactosemia is included in the German neonatal screening program as a facultative parameter. Galactose and galactose-1-phosphate eluted from dried blood spots of 4.7 mm diameter were quantified simultaneously as described by Diepenbrock et al. (12). Enzymatic analysis of galactokinase was performed as part of the routine diagnostic work-up when GALK deficiency was suspected.
Galactokinase (GALK) deficiency is an autosomal recessive disorder causing cataract formation that can be prevented or mitigated by early diagnosis and galactose-restricted diet. The aim of this retrospective study was to explore whether GALK-deficiency meets the criteria for neonatal mass screening programs.
From 2000 until 2010, the Screening Laboratory Hannover performed newborn screening in 1,950,927 infants from Germany for galactosemia by measuring galactose-1-phosphate-uridyl-transferase and total galactose concentration (free galactose plus galactose-1-phosphate), including automatic screening for GALK deficiency.
Eleven cases were found with elevated galactose levels accompanied by normal transferase activity. Nine of 11 cases were informative; the diagnosis was established by demonstrating deficient activity of the GALK enzyme in erythrocytes. To our knowledge, screening did not produce any false negative results. All patients were treated with a galactose-restricted diet from the neonatal period or infancy. Three of nine patients suffered from congenital cataracts or eventual development of cataracts, despite normal galactose concentrations in blood.
Newborn screening for GALK deficiency prevents or at least mitigates cataract formation. As screening for GALK deficiency is technically simple, it seems to be reasonable to include this disorder in routine screening programs by simultaneous determination of transferase activity and quantification of galactose plus galactose-1-phosphate in dried blood spots.
Simultaneous diagnostic method for phenylketonuria and galactosemia from dried blood spots using high-performance liquid chromatography-pulsed amperometric detection
2010, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
We developed a simultaneous diagnostic method for phenylketonuria (PKU) and galactosemia through simultaneous determination of phenylalanine (Phe) and galactose (Gal) by high-performance liquid chromatography (HPLC) with pulsed amperometric detection (PAD). The intra- and inter-day precisions were <5.8%, with satisfactory mean recoveries (98.2–105%). For all PKU-positive samples, Phe levels were above the cut-off value (>30.0 mg/L), but Gal levels were nearly zero. For 77% of galactosemia-positive samples, Phe levels were above the cut-off value, but Gal levels were above the cut-off value (>80.0 mg/L) for all samples. Our HPLC-PAD method can reduce the false-positive rate of misdiagnosis for PKU and galactosemia.
A pulsed amperometric detection method of galactose 1-phosphate for galactosemia diagnosis
2008, Analytical Biochemistry
Galactose 1-phosphate uridyltransferase deficiency causes the accumulation of galactose and galactose 1-phosphate (Gal 1-P) in the blood. We describe a new pulsed amperometric detection method for determining Gal 1-P levels as a pathognomic marker for the diagnosis of galactosemia. The method uses high-performance anion-exchange chromatography with pulsed amperometric detection. In an anion-exchange column, the analytes were separated in 5 min by the eluent mixture of 40 mM NaOH and 40 mM Na₂CO₃. The detection limit (signal to noise ratio of 3) to Gal 1-P was 30 μg/dL. The linear dynamic range was 3.0–50 mg/dL (r²= 0.9999). The mean recoveries of Gal 1-P for intra- and interday assays were 97.55–103.78%. This method clearly separated the type I galactosemia patients from the normal group and is a practical procedure for the rapid diagnosis of galactosemia.

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AnalyticalColorimetric determination of galactose and galactose-1-phosphate from dried blood

Anal Biochem

Clin Biochem

Analytical
Colorimetric determination of galactose and galactose-1-phosphate from dried blood