J.B. Holton

Inhibition of bilirubin conjugation in rat liver slices by free fatty acids, with relevance to the problem of breast milk jaundice

Abstract Free fatty acids inhibit bilirubin metabolism by rat liver slices and there is a correlation between the fatty acid content of breast milk and its effect on bilirubin metabolism in vitro. The action of free fatty acids and breast milk is similar and is probably due to inhibition of bilirubin conjugate formation. The relevance of these observations to the aetiology of prolonged neonatal jaundice caused by breast feeding is not certain but, since milk increases in free fatty acid content and becomes inhibitory on storage, it is important to examine only fresh milk samples in the diagnosis of this condition using in vitro tests of bilirubin metabolism.

Biochemical monitoring of treatment for galactosaemia: Biological variability in metabolite concentrations

Red cell galactose 1-phosphate (Gal-1-P) concentrations and urinary galactitol excretion have been suggested as biochemical indices of dietary compliance in classical transferase-deficient galactosaemia. We report our experience of measuring both in 32 patients over 0–10.9 years (median 3.45). A total of 438 blood specimens for Gal-1-P and 383 urine specimens for galactitol assay were received; 317 pairs of specimens were collected at the same time. Concentrations of both analytes fell rapidly over the first 2–3 months following dietary intervention, to mean (geometric SD) levels of 225 (1.60) μmol/L red cells for Gal-1-P and 388 (1.19) μmol/mmol creatinine for galactitol. Concentrations then fell exponentially over the next 7–8 years, with times to half-disappearance of 6.3 years for Gal-1-P and 6.4 years for galactitol, to levels of 104 (1.58) and 193 (1.36) respectively in patients aged over 10 years. Concentrations of both analytes were independent of the presence of the common Q188R mutation. Mean intra- and inter-individual coefficients of variation (CV) across the range of values studied were 36% and 61% for Gal-1-P, and 37% and 42% for galactitol. Analytical CVs were 3.6% for Gal-1-P and 5.5% for galactitol, indicating that the major source of variability is biological. The correlation coefficient between Gal-1-P and galactitol in paired samples overall was 0.33; the regression equation being [Galactitol] = 0.84[Gal-1-P]+176. Serial measurements of both Gal-1-P and galactitol may be valuable in monitoring galactosaemia, but high intra-individual biological variability limits their usefulness. Standardization of sample collection times may improve this. Further work is needed to assess the predictive values of both analytes for long-term outcome.

The investigation of UDPGlucose and UDPGalactose concentration in red blood cells of patients with classical galactosaemia.

UDPGlucose (UDPGlc) and UDPGalactose (UDPGal) are nucleotide sugars formed via the galactose metabolic pathway and are essential cofactors for the incorporation of galactose and glucose into complex glycoproteins and glycolipids. It has been proposed that in classical galactosaemia, where the enzyme galactose-1-phosphate uridyl transferase is deficient, the reaction product UDPGal is reduced leading to the long-term complications associated with the disease. We have measured the concentration of UDPGal and UDPGlc in red blood cells by high performance liquid chromatography (HPLC) in 16 children and 15 adult galactosaemics and compared the results with 30 and 27 control children and adults, respectively. The results indicate that UDPGal levels were found to be significantly reduced in galactosaemic patients and UDPGlc/UDPGal ratios significantly increased.

A Method of Assessing Amniotic Fluid Lecithin Concentrations and Predicting Foetal Lung Maturity, by Estimating Total Palmitic Acid Concentration Using G.L.C.

A rapid G.L.C. method for estimating total palmitic acid in amniotic fluid is described and it is shown that the principal source of this substance is lecithin. Thus, the technique is a convenient means of assessing amniotic fluid lecithin concentrations and of predicting the maturity of the foetal lung.

UDP-glucose and UDP-galactose concentrations in cultured skin fibroblasts of patients with classical galactosaemia

SummaryA very clear-cut reduction in UDP-galactose (UDPGal) levels in erythrocytes, skin fibroblasts and liver of patients with classical galactosaemia has been reported. As UDPGal is the galactosyl donor in glycoprotein and glycolipid synthesis, it has been suggested that an abnormality in these complex compounds may be the cause of some of the long-term complications of the disease. More recent work on erythrocytes, employing mainly HPLC rather than the enzyme methods used to measure UDPGal originally, casts doubt on the hypothesis because, although some reduction was still found, there was a large overlap between galactosaemic and normal distributions. We have reproduced the experiments on cultured skin fibroblasts at confluency, but measuring UDPGal and UDP-glucose (UDPGlc) by HPLC. There was no reduction in UDPGal levels in galactosaemic compared to control cell lines. The existence of a biologically significant depletion of UDPGal in galactosaemia remains in doubt.

Biochemical control, genetic analysis and magnetic resonance imaging in patients with phenylketonuria

Thirteen patients with phenylketonuria, detected by neonatal screening and started on diet within 16 days of age, were investigated between 10 and 18 years of age by magnetic resonance imaging (MRI) of the brain. Biochemical control was assessed from: (1) the life time blood phenylalanine (Phe) control (as determined from (a) the mean yearly exposure to Phe; (b) the accumulated time for each patient that Phe was <120 μmol/l; (c)>400 μmol/l; (d)>800 μmol/l; and (e)>1200 μmol/l); and (2) the blood Phe control over the 5 years prior to imaging (assessed for each patient by the mean yearly Phe exposure over that period). In all patients the phenylalanine hydroxylase gene locus was studied using restriction fragment length polymorphism haplotypes and mutant genes were screened for a variety of specific mutations which have been reported in other European populations or in populations of north European descent. Two patients had significant abnormalities of cerebral white matter. Although both showed poor biochemical control this did not reach statistical significance when compared to those with normal imaging. DNA haplotype patterns could be assigned to 11 patients and mutant genes were identified in 12. One patient with abnormal imaging and 4 patients without abnormalities had mutations on both chromosomes identified. In these 5 patients there was significant correlation between their genotype and biochemical control. Mutations resulting in residual in vitro enzyme activity were associated with normal imaging.

Gas-liquid chromatographic determination of galactitol in amniotic fluid for possible use in prenatal diagnosis of galactosaemia

A gas liquid chromatographic method for the estimation of galactitol in amniotic fluid from pregnancies at risk for galactosaemia is described. The method is based on the almost complete removal of glucose from the amniotic fluid by ion exchange, and the subsequent chromatography of galactitol as its hexaacetate.

Galactose-1-phosphate uridyltransferase in cultured cells

A method to measure the enzyme galactose-1-phosphate uridyltransferase in cultured cells is described. The optimun pH was 8.7 and no enzyme activity was found without preincubation with dithiothreitol. The KM values for Gal-1-P and UDPG for the wild type enzyme were found to be 0.2 mM and 0.08 mM, respectively. Values for the Duarte variant were found to be identical. No significant change in enzyme activity with time after subculture was found in either cultured skin fibroblasts or cultured amniotic fluid cells. Different transferase genotypes were clearly distinguished and reference range established. Transferase levels found in normal cultured amniotic fluid cells were the same as in normal cultured skin fibroblasts. The results of a prenatal diagnosis was obtained within 3 weeks of amniocentesis.

Prenatal Diagnosis of Disorders of Galactose Metabolism

SummaryOf three clinically significant galactose disorders, there is only a real need and experience of prenatal diagnosis in classical galactosaemia. Prenatal diagnosis for this disorder may be carried out by galactose-1-phosphate uridyl transferase assay in cultured amniotic fluid cells or in chorionic villus biopsies and by galactitol estimation in amniotic fluid supernatant. Although the long-term outcome of patients treated on a galactose-restricted diet is recognized to be unsatisfactory, prenatal diagnosis is only rarely performed with a view to terminating the affected pregnancy.

Determination of platelet serotonin by a fluorometric method

Abstract A fluorimetric method for measuring the serotonin concentration in tissues has been adapted for the determination of this amine in platelets. The characteristics of the method were assessed and found to be satisfactory. A range of platelet serotonin was established for normal children and some cases of phenylketonuria and histidinaemia were examined. In view of the results found for patients with these disorders the effect of high concentrations of phenylalanine and histidine in the method was investigated. Phenylalanine was found to add considerably to the fluorescence, but interference by histidine was not found to be significant.