Neonatal hypoglycaemia in severe succinyl-CoA: 3-oxoacid CoA-transferase deficiency.

Succinyl-CoA: 3-oxoacid CoA-transferase (SCOT) deficiency is an inborn error of ketone body utilization, characterized by intermittent ketoacidotic crises and persistent ketosis. The diagnosis was suspected in a patient who presented with hypoglycaemia, ketoacidosis and coma at 4 days of age. The hypoglycaemic tendency was only observed during the first month of life. A novel macromolecular labelling assay in cultured skin fibroblasts using D-3-hydroxy[3-14C]butyrate supported the diagnosis. Subsequently, 9% residual SCOT activity and undetectable cross-reactive protein were noted in fibroblasts and the patient was found to be homozygous for the G324E SCOT gene mutation. By 7 years of age, recurrent episodes of ketoacidosis superimposed on persistent hyperketonaemia had resulted in over 25 hospitalizations requiring intravenous fluid, glucose and sodium bicarbonate therapy. He has had normal growth but developmental delay and attention deficit-hyperactivity disorder. A continuous intravenous glucose infusion at 38 microlmol (6.8 mg)/kg per min reduced plasma total ketone levels from greater than 1.5 mmol/L to less than 0.5 mmol/L after 48 h. This indicates that patients with SCOT deficiency do not always manifest ketosis with administration of a sufficient amount of carbohydrates, but that even under such conditions hyperketonaemia is difficult to eliminate completely. The presence of hypoglycaemia does not exclude the diagnosis of SCOT deficiency in infancy.

Galactose breath testing distinguishes variant and severe galactose-1-phosphate uridyltransferase genotypes.

A galactose breath test that quantitates [1-(13)C]galactose conversion to 13CO2 provides information on the whole body galactose oxidative capacity. As there is little information on the relationship between whole body oxidation and the genotype in patients with galactosemia, we measured the 13CO2 excretion for 2 h after administration of [1-(13)C]galactose in 37 patients (3-48 y old) with galactose-1-phosphate uridyltransferase (GALT) deficiency and 20 control subjects (3-37 y old). Eleven patients with the common Q188R/Q188R genotype and no detectable erythrocyte GALT activity eliminated <2% of a bolus of [1-(13)C]galactose as 13CO2 compared with 8.47 to 28.23% in controls. This defines a severe metabolic phenotype. Seven patients with one Q188R allele and a second mutant allele such as L195P, E308K, V151A, M142K, or Q344K and one patient with a K285N/unknown genotype also released <2% as 13CO2 in 2 h. The presence of N314D or S135L as the second mutant allele does not impair total body galactose oxidation, as individuals with the GALT genotype of Q188R/N314D, K285N/N314D, and Q188R/S135L had normal 2-h galactose breath tests. Subjects with S135L/S135L, N314D/N314D, S135L/deltaT2359 as well as other rarer genotypes such as R258C/Y209C, E203K/IVSC-N314D, K285N/T138M, Q188R/D113N, S135L/F171S, R148W/N314D, and IVSC-N314D/N314D oxidized galactose comparable to controls. The dissociation of residual erythrocyte GALT activity and whole body galactose oxidative capacity is exemplified by blacks with a S135L/S135L genotype and absent erythrocyte GALT activity. An oral 2-h [1-(13)C]galactose breath test distinguishes severe and variant GALT genotypes and enables delineation of the extent of impaired galactose metabolism in an array of patients who possess diverse GALT mutations. It may prove to be useful in establishing whether a patient is capable of manifesting disease similar to patients with a Q188R/Q188R genotype.

Sugar nucleotide concentrations in red blood cells of patients on protein- and lactose-limited diets: effect of galactose supplementation.

Uridine diphosphate (UDP) galactose, a pivotal compound in the metabolism of galactose, is the obligate donor of galactose in the formation of complex glycoconjugates. The cellular UDPgalactose concentration has been thought to be maintained by the interconversion of UDPglucose and UDPgalactose by UDPgalactose-4-epimerase. However, recent findings of lower average red blood cell (RBC) UDPgalactose concentrations in galactose-1-phosphate uridyltransferase-deficient patients suggest that other factors play a role in determining its concentration. To test the hypothesis that the amount of galactose traversing the Leloir pathway contributes to the cellular UDPgalactose pool, we determined RBC UDPgalactose in patients with maple syrup urine disease (MSUD), phenylketonuria (PKU), and other metabolic diseases who were treated with a low-protein, and consequently, low-lactose diet. Six patients with MSUD were also supplemented with 19 g galactose/d and their UDPhexose concentrations were measured at intervals. We show that young patients with MSUD or PKU have decreased average RBC UDPgalactose concentrations when compared with similarly aged healthy subjects. Galactose supplementation of MSUD patients significantly increased their UDPgalactose concentrations in both RBCs and white blood cells (WBCs) from 29.5 +/- 1.5 to 42.3 +/- 5.8 nmol/g hemoglobin and from 69.0 +/- 7.5 to 193.0 +/- 49.0 nmol/g protein, respectively. Discontinuation of supplementation was associated with a return to basal values in RBCs and a reattainment of the pretreatment ratio of UDPglucose to UDPgalactose in WBCs. These observations demonstrate that dietary galactose is a factor in establishing the steady state concentrations of the uridine sugar nucleotides and imply that galactose metabolism modulates the achievement of an epimerase-mediated equilibrium.

Effect of glucose and galactose loading in normal subjects on red and white blood cell uridine diphosphate sugars.

In order to test the assumption that red blood cell (RBC) uridine diphosphate galactose content is regulated in part by dietary galactose and to help comprehend factors influencing RBC and white blood cell (WBC) uridine diphosphate hexose concentrations in vivo, oral loading studies were performed with 50 g of galactose or 75 g of glucose in normal adults. While elevations of blood glucose did not influence RBC or WBC UDPhexose levels, increased blood galactose concentrations caused transient increases in both RBC and WBC UDPgalactose. In both RBC and WBC, the UDPgalactose-4-epimerase was rate limiting. In comparison to RBC, WBC had larger changes in UDPgalactose levels, synthesized additional UDPglucose through the action of UDPglucose pyrophosphorylase and began to restore the equilibrium ratio between UDPglucose and UDPgalactose concentrations while the plasma galactose level was still increasing. Thus, galactose ingestion alters the steady-state levels of UDPhexoses in circulating cells. The modulation of UDPglucose and UDPgalactose concentrations, occurring after the presentation of galactose to cells, as a result of the combined actions of GALT and UDPgalactose-4-epimerase, may be important in determining rates of synthesis of complex glycoconjugates.

Comparison of erythrocyte uridine sugar nucleotide levels in normals, classic galactosemics, and patients with other metabolic disorders.

By limiting galactosylation mechanisms, a cellular deficiency of the uridine sugar nucleotide, UDPgalactose, has been implicated as a cause of the long-term complications seen in patients with classic galactosemia despite dietary treatment. As a result, great interest has been generated in the accurate assessment of UDPgalactose, as well as UDPglucose, from which UDPgalactose may be derived by the function of a ubiquitous, active UDPgalactose-4-epimerase. Since several series of values for the concentration of these compounds in red blood cells (RBCs) of galactosemics have been flawed by the use of methods subsequently shown to be unsuitable, we have quantified erythrocyte UDPgalactose and UDPglucose levels by an accurate high-performance liquid chromatography (HPLC) assay in 116 normals, 76 galactosemics, and 39 patients with other metabolic disorders. These large groups have permitted the evaluation of age, diet, and genotype as influential factors in the steady-state RBC levels of the sugar nucleotides. The data show that age is an important determinant of RBC levels, with children younger than 10 years having higher values than individuals older than 10 years. Mean UDPgalactose levels in galactosemic children younger than 10 years and those older than 10 years were 30% and 18% lower, respectively, than levels in comparable normals. Although the mean differences were highly significant, there was considerable overlap of individual values. There was no difference in UDPglucose levels between galactosemics and normals.(ABSTRACT TRUNCATED AT 250 WORDS)

Uridine diphosphate hexoses in leukocytes and fibroblasts of classic galactosemics and patients with other metabolic diseases.

To examine uridine diphosphate hexose (UDPhexose) content of cells that have more complete metabolic patterns than erythrocytes, which have been commonly used in the study of galactosemia, the concentrations of uridine diphosphate galactose (UDPgalactose) and uridine diphosphate glucose (UDPglucose) were determined in white blood cells (WBC) and fibroblasts cultured from skin biopsies. Leukocyte UDPgalactose and UDPglucose values were determined in 60 normal individuals, 14 classic galactosemics, and 18 patients with other metabolic diseases on protein-restricted and low-lactose diets. There was no difference in the average concentration of these compounds between any of these groups. There was no relationship between age and WBC UDPhexose content or correlation of WBC and erythrocyte UDPhexose levels in the same blood specimens. WBC from galactosemic individuals differ from their red blood cells because the former do not show the low average UDPgalactose levels and abnormal UDPglucose to UDPgalactose ratio previously reported for erythrocytes from galactose-1-phosphate uridyltransferase-deficient individuals. Fibroblast cell lines from 10 normal and 10 galactosemic individuals, cultured and grown to confluence in glucose medium, also showed no difference in nucleotide sugar concentrations. Thus far, of the cell types easily available, red blood cells appear to be unique in showing an abnormality in nucleotide sugar metabolism. The fact that galactosemic fibroblasts demonstrate no abnormality in the concentration of these compounds suggests that the defective galactosylation that has been observed in galactosemic fibroblasts is not due to unavailability of UDPgalactose.

Renal brush border membrane lipid composition in Basenji dogs with spontaneous idiopathic Fanconi syndrome.

To comprehend the renal defect underlying idiopathic Fanconi syndrome in the Basenji dog, we have focused on delineating the lipid profiles of renal brush border membranes isolated from affected and normal Basenji dogs to establish any physical or compositional changes underlying previously observed transport and membrane-fluidity changes. The lipid composition was studied with respect to total lipid, cholesterol, and phospholipid content, cholesterol to phospholipid ratio, distribution of the major phospholipid classes, and fatty acid composition. Total phospholipid of the isolated renal brush border membranes from Fanconi syndrome dogs analyzed by 31P nuclear magnetic resonance showed no difference compared with that of normal dogs. Examination of total fatty acids in both membranes using gas-liquid chromatography analysis of fatty acid methyl esters showed no difference in the mole percents of the major fatty acids. Our data suggest that changes in bulk membrane fluidity of the Fanconi syndrome dog renal brush border as measured by 1,6-diphenyl-1,3,5-hexatriene cannot be attributed to phospholipid and fatty acid compositional change. In the membranes isolated from affected dog kidney, the cholesterol content determined by gas-liquid chromatography analysis was 66 mol% higher than in membranes isolated from normal dog kidney. This correlates with the higher cholesterol to phospholipid molar ratio of 0.82 +/- 0.08 in the affected animal as compared with 0.58 +/- 0.04 in the normal. Cholesterol content and its microdomain in the membrane bilayer may be important in modulating transport functions. Increased membrane cholesterol content may affect the conformational motility of membrane transport proteins and thus affect their function.

Uridine diphosphoglucose content of human erythrocytes: assessment by conversion to uridine diphosphoglucuronate.

To settle the ongoing controversy regarding differential uridine diphosphoglucose (UDPG) and uridine diphosphogalactose (UDPGal) content of erythrocytes, which may be important in evaluating the metabolic abnormality in patients with galactosemia, we derived a combined enzymatic-high-performance liquid chromatography (HPLC) assay. Uridine diphosphoglucuronate (UDPGA), the unique product of UDPG dehydrogenase activity, was separated and quantified by HPLC in extracts of human erythrocytes. The quantity of UDPGA produced in cell filtrates incubated with the enzyme corresponds to the amount of UDPG directly determined by HPLC. The amount of UDPGA produced was independent of the enzyme purity or activity used. On the other hand, the amounts of UDPG estimated by fluorometric measurement of the production of reduced nicotinamide adenine dinucleotide varied with the enzyme purity and activity. The combined enzymatic-HPLC method confirms the direct determinations of UDPG content of normal erythrocytes. The results indicate that, under appropriate conditions, the fluorometric-based assay will give accurate estimates of UDPG, but the direct HPLC method yields consistent and correct UDPG and UDPGal determinations.

31P NMR spectra of intact red blood cells: quantitation of UDPGlucose and UDPGalactose.

The combined levels of uridine diphosphogalactose (UDPGal) and uridine diphosphoglucose (UDPGlu) were measured directly by 31P NMR spectroscopy in intact fully oxygenated erythrocytes. Quantitative analysis was obtained using a sealed capillary of methylene diphosphonate (MDP) calibrated with standard solutions of UDPGlu and UDPGal of known concentration prepared in KRB/BSA. The combined peaks of UDPGlu and UDPGal were integrated after subtraction of the underlying broad component originating from membrane phospholipids. The average concentration of 27.16 +/- 5.18 nmole/ml or 8.08 +/- 1.36 mumole/100 g hemoglobin obtained for these metabolites correlated well with their total determined by HPLC of trichloracetic acid (TCA) extracts of the same samples.

Concentration of white blood cell UDPgalactose and UDPglucose determined by high performance liquid chromatography.

We have applied a HPLC method to separate and quantitate UDPgalactose (UDPGal) and UDPglucose (UDPGlu) in human white blood cells (WBCs). Trichloroacetic acid-treated, protein-free filtrates were chromatographed on an anion-exchange column (Dionex CarboPac PA-1) using a gradient of 20-40% potassium phosphate buffer (pH 4.5). Recoveries of UDPGal and UDPGlu ranged from 93 to 106%, and the method was linear over a wide range of WBC protein concentrations. Volumes of blood as low as 2.5 ml (2.2 mg WBC protein) could be used to achieve quantitative recovery of the sugar nucleotides. The mean values and standard deviations of UDPGal and UDPGlu in 33 normal individuals ranging in age from 1 day to 65 years were 12.4 +/- 4.2 and 31.5 +/- 9.3 mumol/100 g protein, respectively. No statistical differences in UDPGal and UDPGlu values were observed between children and adults. No correlation was established between the concentrations of UDPGal and UDPGlu and either total WBC count or the number of lymphocytes obtained from Coulter counter analysis. There was no relationship between the concentrations of UDPGal and UDPGlu in WBCs and RBCs which were prepared from the same blood specimen.

Red blood cell uridine sugar nucleotide levels in patients with classic galactosemia and other metabolic disorders.

While dietary galactose restriction eliminates the life-threatening complications of classic galactosemia, central nervous system and ovarian disease are still evident in these patients, despite milk restriction. Because of the possibility that reduced tissue levels of uridine diphosphate galactose (UDPgalactose), the product of the deficient enzyme, galactose-1-phosphate uridyltransferase, are the cause of these unexplained complications, we have measured the concentration of red blood cell (RBC) uridine sugar nucleotides in these patients, comparing their values not only with those of normal subjects, but also with those of children who have other metabolic disorders. RBC UDPgalactose and uridine diphosphate glucose (UDPglucose) levels were measured by high-performance liquid chromatography (HPLC) in 35 control subjects, 24 galactosemic patients, and 19 patients with inborn errors of amino acid, organic acid, or ammonia metabolism. The last group of patients served as dietary controls, as they were all on special low-protein diets that restricted milk intake. The mean levels of UDPgalactose in galactosemic children and adults were 38% and 54% lower, respectively, than the levels in normal children and adults. While only six of 19 galactosemic children had levels below the 95% confidence limit for normals, four of five galactosemic adults had levels of UDPgalactose in the low range. The mean UDPgalactose level in children with other metabolic diseases who were on a low-milk diet was also reduced by 38%, with a mean not significantly different from galactosemics. Compared with normal adults, the level of UDPglucose in galactosemic adults was also reduced by 29%, with three of five affected adults having UDPglucose values below the 95% confidence limit.(ABSTRACT TRUNCATED AT 250 WORDS)

31P NMR analysis of red blood cell UDPGlucose and UDPGalactose: comparison with HPLC and enzymatic methods.

The levels of uridine diphosphogalactose (UDPGal) and uridine diphosphoglucose (UDPGlu) in trichloroacetic acid extracts of human red blood cells (RBC) were measured by 31P NMR spectroscopy. Individual determinations were compared to results obtained by enzymatic and high-pressure liquid chromatographic (HPLC) methods. The characteristic doublet of the P beta resonance signals of both UDPGal and UDPGlu were detected in proton-decoupled spectra of extracts. Quantitative analyses were obtained by employing a standard, methylene diphosphonate, in an external capillary tube during data acquisition for periods of 14 to 24 h using an "inverse-gated" pulse sequence. The ratio of the integrated area of each of the uridine sugar nucleotide doublets to the area of the external reference peak was linear with concentrations between 0.03 and 0.50 mM. There was no difference between the mean value obtained by 31P NMR of 6.6 +/- 1.4 mumol UDPGlu/100 g Hgb or 2.1 +/- 0.6 mumol UDPGal/100 gHgb and the corresponding levels determined enzymatically or by HPLC in identical RBC extracts. When analyzed as paired data, only UDPGlu by NMR was found to be lower than the value obtained by HPLC. As a quantitative analytical tool, NMR spectrometry validated both the enzymatic and HPLC methods used for measurement of uridine sugar nucleotides in our laboratories.

The concentration of red blood cell UDPglucose and UDPgalactose determined by high-performance liquid chromatography.

We have developed a sensitive method that employs high-performance liquid chromatography to separate and quantitate uridine diphosphogalactose (UDPGal) and uridine diphosphoglucose (UDPGlu) in human red blood cells. The trichloracetic acid extracts of red blood cells were chromatographed using a Dionex CarboPac anion-exchange resin and a 20-40% potassium phosphate buffer, pH 4.5, in a gradient-elution program. UDPGal and UDPGlu were detected spectrophotometrically at 254 nm. Recoveries of UDPGal and UDPGlu ranged from 96 to 106%. Under these conditions, there was exceptionally good reproducibility in stored specimens, and the method was sensitive in the low picamole range. The mean values and standard deviations in adults were 2.9 +/- 0.4 and 7.8 +/- 0.8 mumol/100 g Hgb for UDPGal and UDPGlu, respectively. The values in children were 4.5 +/- 1.2 and 10.2 +/- 1.7 mumol/100 g Hgb for UDPGal and UDPGlu, respectively. Values determined by the HPLC method are in excellent agreement with those obtained by enzyme analysis.

Comparison of rapid NFT and API 20E with conventional methods for identification of gram-negative nonfermentative bacilli from pharmaceuticals and cosmetics.

The accuracy of the Rapid NFT and the API 20E identification systems was evaluated by comparing them with conventional biochemical methods for the identification of gram-negative, nonfermentative bacilli. The organisms were recovered from preserved, nonsterile pharmaceutical and cosmetic products. A total of 123 test isolates that are commonly encountered in these products were used. By using the criteria of accurate and reliable identification without employing additional tests, Rapid NFT was found to be more accurate after 48 h of incubation than API 20E for characterizing isolates to the species level. Therefore, close agreement between NFT and conventional methods for identification of industrial gram-negative isolates provides evidence that the Rapid NFT system is an improved and rapid method for identifying these organisms to the species level with minimal use of supplementary tests.

Sphingomyelin lipidosis variant with cirrhosis in the pediatric age group.

Two children with a variant of sphingomyelin lipidosis had otherwise unexplained cirrhosis that was histologically inactive and appeared to run an indolent course. The primary clinical problems involved the central nervous system, with vertical supranuclear ophthalmoplegia being the most distinctive feature. Biochemical analysis of cultured skin fibroblasts obtained from one of the children revealed that sphingomyelinase activity was 42% of control values. The typical inconspicuous hepatic storage and cirrhosis, coupled with the important morphologic finding of sea-blue histiocytes in the marrow, suggested that in cases of unexplained infantile or childhood cirrhosis the marrow should be closely examined for such histiocytes. Likewise, in cases of sea-blue histiocytes without evident etiology, with or without cirrhosis, this disease should be considered.

Decreased procollagen production in cultured fibroblasts from patients with Lowe's syndrome.

The oculo-cerebro-renal syndrome described by Lowe is a catastrophic disease in children characterized by progressive eye, central nervous system and kidney degeneration. We determined procollagen production in cultured skin fibroblasts originating from patients with Lowe's syndrome as well as normal individuals after incubation of cells with [14C]proline for 1,4 and 20 h. Using [14C]hydroxyproline formation, in relation to cell protein or DNA, as an index of procollagen production, we found that cultured cells from patients synthesized collagenous protein at a substantially reduced level. This decrease in synthesis in cultured fibroblasts from patients could not be accounted for by differences in the specific radioactivity of the free intracellular proline; moreover, it could not be attributed to differences in the kinetics of growth between normal and affected cell lines. The degree of hydroxylation of proline residues in collagen was the same in both normal and affected cells.

Single radial immunodiffusion method for screening Staphylococcal isolates for enterotoxin.

A direct system for screening large numbers of staphylococcal isolates for enterotoxin production has been developed. The system employs polyvalent (serotypes A, B, C, D, and E) immunodiffusion assay slides in conjunction with a multiple-culturing system for toxin production. With the combined system, as many as 50 cultures can be screened simultaneously on a single assay slide having a sensitivity of about 0.3 microgram/ml. The system should be useful for detecting potential enterotoxin in foods containing a predominance of non-enterotoxigenic strains.

Relationship of thyroid status and serum N-acetyl-beta-glucosaminidase isoenzyme activities in humans.

Serum from 28 hyperthyroid, hypothyroid, and euthyroid pre- or early puberty females was examined for N-acetyl-beta-glucosaminidase (HEX) activity. Total, isoenzyme A (labile), and isoenzyme B (stable) levels were determined for this enzyme. A high degree of correlation (r = 0.76; p less than 0.001) exists between total hexosaminidase activity and thyroid hormone levels (as reflected by the Free Thyroxine Index). Examining each isoenzyme individually, A is selectively enhanced (r = 0.84; p less than 0.0005) whereas B displays no significant change regardless of thyroid activity. In hyperthyroid individuals, levels of total hexosaminidase (730 +/- 67 units) (mean +/- S.D.) and isoenzyme A (516 +/- 46) were significantly higher than levels found in either the hypothyroid (total: 547 +/- 30; isoenzyme A: 352 +/- 31) or euthyroid (total: 620 +/- 81; isoenzyme A: 423 +/- 45) groups. However, no change was observed in levels of isoenzyme B among hypothyroid (195 +/- 19), euthyroid (197 +/- 43) and hyperthyroid (215 +/- 32) groups. These data substantiate our earlier findings in the rat, wherein thyroxine administration evoked a similar response in the liver. They are of particular interest in light of the deficiency of HEX A in variants of GM2 gangliosidosis.