Biochemical monitoring of pregnancy and breast feeding in five patients with classical galactosaemia--and review of the literature.

Pregnancy, delivery, and postpartal metabolic control was monitored biochemically in five patients (22-38 years of age) with clinically, enzymatically, and genotypically established classical galactosaemia and good dietary compliance. Three of the patients performed breast feeding of their newborns. Monitoring parameters were galactose-1-phosphate and galactitol concentrations in erythrocytes and urinary excretion of galactose, galactitol, galactonate, and lactose. During pregnancy, a small but steady increase of renal metabolite excretion rates was observed. After delivery, a moderate transient increase of metabolite concentrations with peak values within the first week post partum occurred, irrespective of breast feeding. Altogether, there was no evidence for clinically or subclinically significant changes of metabolic control during pregnancy, delivery, or lactation. In conclusion, a specific metabolic monitoring is apparently not required in pregnant galactosemic women, and breast feeding of the nongalactosemic offspring can be recommended.

Galactonate determination in urine by stable isotope dilution gas chromatography-mass spectrometry.

A stable isotope dilution assay was developed for the sensitive determination of D-galactonic acid. D-[U-13C(6)]galactono-1,4-lactone was prepared as internal standard. Unlabelled and U-13C-labelled D-galactonic acid species were converted to the N-(1-butyl)galactonamide pentaacetate derivatives and assessed by gas chromatography-mass spectrometry (GC-MS). Positive chemical ionisation and monitoring of the [MH-60](+)-ions in the galactonate chromatographic peak at m/z 402 and m/z 408 were used for quantification. The procedure was applied to study the variability of D-galactonate excretion in healthy subjects and galactosemic patients and to monitor the D-galactonate-D-galactitol ratio in human urine.

Endogenous galactose formation in galactose-1-phosphate uridyltransferase deficiency.

Patients with classical galactosaemia (galactose-1-phosphate uridyltransferase (GALT) deficiency) manifest clinical complications despite strict dietary galactose restriction. Therefore the significance of endogenous galactose production has been assessed. Previous in vivo studies primarily focused on patients homozygous for the most common genetic variant Q188R but little is known about other genetic variants. In the present study the endogenous galactose release in a group of non-Q188R homozygous galactosaemic patients (n = 17; 4-34 years) exhibiting comparably low residual GALT activity in red blood cells was investigated. Primed continuous infusion studies with D-[1-(13)C]galactose as substrate were conducted under post-absorptive conditions and in good metabolic control. The results demonstrate that all patients exhibiting residual GALT activity of <1.5% of control showed a comparable pathological pattern of increased endogenous galactose release irrespective of the underlying genetic variations. Possible implications of the findings towards a more differentiated dietary regimen in galactosaemia are discussed.

Age dependence of endogenous galactose formation in Q188R homozygous galactosemic patients.

The age dependence of endogenous galactose formation was investigated in Q188R homozygous galactosemic patients (n=18; 4-38 years) using the primed continuous infusion approach with D-[1-13C]galactose as a substrate. Studies were conducted under postabsorptive conditions (fasting >10h) and good metabolic control. In the patients, the release of galactose from endogenous sources into plasma (R(a)) decreased with age and ranged from 4.6 to 2.0 micromol/kg body weight per h. Galactitol and galactonate release rates paralleled the galactose R(a) but at a lower level. The mean relation of galactose, galactitol, and galactonate release was 10:5:1. Statistically, there was a highly significant (p<0.0001) inverse correlation between total galactose release (i.e., sum of R(a) plus galactitol and galactonate release) and age. The data (total galactose=y, age=t) were best fitted to the simple exponential model y=y(0)+axexp(-bt) by non-linear regression analysis. The parameter estimates were y(0)=3.0+/-0.2, a=6.5+/-0.4, and b=0.11+/-0.02. The value of y(0) provides an estimate of total galactose release in adult patients (i.e., approximately 13 mg/kg body weight per day), summation operator (y(0)+a) provides an estimate for galactosemic newborns (i.e., approximately 41 mg/kg body weight per day). The data show that significant amounts of endogenous galactose are formed in galactosemic patients with release rates being several fold higher in infants than in adults. The present findings can explain the persistently elevated galactose-1-phosphate levels in erythrocytes-and its age dependence-in galactosemic patients even when under strict dietary treatment.

Stable-isotope dilution analysis of galactose metabolites in human erythrocytes.

An established gas chromatography/mass spectrometry (GC/MS) method, devised for stable-isotope dilution analysis of plasma galactose, was developed to allow determination of erythrocyte (red blood cell, RBC) concentrations of galactose-1-phosphate and other primary metabolites relevant in galactosaemia. Galactose-1-phosphate was enzymatically converted to galactose, and the aldononitrile pentaacetate derivative was separated by gas chromatography and determined by mass spectrometry using chemical ionisation and selected ion monitoring of the [MH-60](+) ion. U-(13)C-Labelled standard was used for quantification. Comparative measurements were conducted using established fluorimetric and radiometric enzymatic methods. The GC/MS analysis for galactose-1-phosphate was linear (range examined 0-600 micromol/L(RBC), packed cells), of acceptable repeatability at low and high concentrations (within and between run CVs <15%), with a limit of quantification of 0.01 micromol/L(RBC). With samples from patients with classical galactosaemia there was a linear correlation with conventional enzymatic assays (r(2) > 0.927). In erythrocytes from post-absorptive patients under treatment, Q188R-heterozygous parents, and healthy subjects, galactose-1-phosphate concentrations (mean +/- SD) were found to be 142 +/- 38 (n = 41), 1.4 +/- 0.2 (n = 8), and 1.9 +/- 0.5 (n = 33) micromol/L(RBC), respectively. In comparison, free galactose concentrations were 3.8 +/- 1.7, 0.49 +/- 0.19, and 0.43 +/- 0.20 mol/L(RBC), respectively. The procedure allowed simultaneous galactitol analysis and proved to be useful to trace incorporation of (13)C-label into erythrocyte galactose metabolites in a D-[1-(13)C]galactose in vivo turnover study.