Enzyme deficiency in cholesteryl ester storage idisease.

Cholesteryl ester storage disease has been shown to involve severe deficiency of acid cholesteryl ester hydrolase and triglyceride lipase activity in liver, spleen, and lymph node. The cholesteryl ester hydrolase was also deficient in aorta. Tissue storage of both cholesteryl esters and triglycerides is generalized. Both the lipid and enzymatic changes are very similar to those in Wolman's disease.

Fine mapping of glycerol kinase deficiency and congenital adrenal hypoplasia within Xp21 on the short arm of the human X chromosome.

We have studied patients with Duchenne muscular dystrophy (DMD), DMD together with glycerol kinase (GK) deficiency, or DMD together with both GK deficiency and congenital adrenal hypoplasia (AHC). Analysis of deletions in these patients allows the mapping of these mutations in Xp21. The following order is proposed: Xpter - L1 - AHC - GK - DMD - Xcen. One of the boys with DMD, GK, and AHC is shown by pulsed-field-gel electrophoresis to have a deletion which has a proximal endpoint at least 500 kb distal from the pERT87 (DXS164) locus.

A new micromethod for the quantification of low molecular weight oligomers of polyethylene glycol.

We describe a gas-liquid chromatographic technique for quantifying the low molecular weight (Mr 106-634) oligomers of polyethylene glycol (PEG) in clinical specimens. The deionized sample, containing tetra-ethylene glycol as an internal standard, is applied on column. This technique readily quantifies as little as 2.5 micrograms of an individual oligomer; with such a quantity, the coefficient of variation is +/- 2.5 percent (N = 25 analyses). Small volumes (250 microliter) of urine are conveniently analyzed, and a single column can be utilized for the analysis of approximately three hundred specimens. We have analyzed timed urine specimens from humans who received 0.15 g of PEG 400 per kilogram. Individuals varied markedly with regard to the total amount of PEG excreted into the urine; each subject, however, consistently excretes a uniform percentage of the ingested dose. The urinary oligomeric profile of PEG does not vary from subject to subject nor from hour to hour, during the first six hours following oral administration, so that a random urine obtained during this period provides a reliable clinical specimen. This technique should facilitate clinical studies that utilize polyethylene glycol 400 as an index of passive intestinal transport.

Thin-layer and gel permeation chromatographic separation of low molecular weight polyethylene glycol oligomers.

We describe a gel permeation and a thin-layer chromatographic technique for the complete resolution of oligomers of ethylene glycol up to a chain length of fourteen ethylene oxide [OCH2CH2] units. We employed columns of Bio-Gel P-2 with 0.02 mol/l ammonium acetate as the eluant to prepare milligram quantities of each of the individual oligomers. Thin-layer chromatography on silica gel G plates with an ethyl acetate/methanol/water solvent provides a sensitive and simple method for monitoring multiple specimens. We determined the flame ionization detector response of purified individual ethylene glycol oligomers relative to tetraethylene glycol. These data permit the accurate quantification of the oligomeric profile of commercially available mixtures of low molecular weight polyethylene glycols (PEG). When PEG 400 is administered orally to normal subjects, aged three months through adulthood, they excrete in their urine a mixture of unmetabolized oligomers with a mean molecular weight of 360 +/- 14 daltons. The ability to measure absolute, rather than relative, amounts of ethylene glycol oligomers will permit more accurate studies of passive intestinal permeability in human subjects.

Failure to conserve lactose and glucose polymers during frozen storage of fecal specimens: methods for preservation.

Freezing is often used to retard bacterial enzymatic activity in fecal specimens collected to quantify specific carbohydrates. The effectiveness of freezer storage on preservation of lactose and glucose polymers was assessed. The data showed that more than 50% of lactose that was added to fecal supernatants that were stored without treatment for more than 50 days at -20 degrees C was lost. Adjustment of pH with HCl (pH 4.9), with HgCl2 (pH 6.3 or 5.85), or with NaOH (pH 10) improved carbohydrate preservation (P less than 0.0004). Storage of the supernatants of fecal homogenates lessened the loss of carbohydrate compared with the total homogenates (P less than 0.001). In supernatants, degradation occurred via simple hydrolysis; in homogenates, degradation occurred by hydrolysis and fermentation to a variety of end-products. Unprocessed fecal specimens that were frozen for months, then retrieved and incubated with lactose or glucose polymers showed extensive fermentative capacity. Cumulatively, the data indicate that enzymatic activity in feces is not halted by storage in the freezer, even if bacteria have been filtered from the stool.

Lipofuscin in rabbit skin: Its occurrence after ocular herpes simplex infection.

Systemic infection, induced by intraocular inoculation of type 1 herpes simplex virus (HSV 1) in young rabbits, is accompanied by the appearance of autofluorescent pigments precisely in the midportion of their fur follicles. Histochemical and solubility reactions of the pigment led to its characterization as a lipofuscinlike pigment. Follicle fluorescence correlated with the severity of clinical symptoms; it was present in 100% of the follicles of rabbits that died but was found in only 30% to 50% of follicles of surviving animals. Similar fluorescence was also present in 10% to 20% of the follicles of uninfected rabbits. This autofluorescent material may be formed by the peroxidation of lipids from sebaceous gland secretions, but neither HSV 1 nor antigens could be found in the skin as possible initiators of this reaction.

Cultured neonatal rat oligodendrocytes are enriched in acid hydrolase activities.

We measured the activity of several acid hydrolases in oligodendrocyte and mixed glial (predominantly astrocytic) cell cultures prepared from neonatal rat cerebra. When compared with the mixed glial cultures, the cultured oligodendrocytes exhibited higher levels for all the hydrolases when activities were normalized to protein content. When enzymic activities were examined as a function of DNA content, oligodendrocytic alpha-L-fucosidase, beta-D-glucuronidase, arylsulfatase, and N-acetyl-beta-D-glucosaminidase were higher than in mixed glial cultures, whereas the activities of alpha-D-glucosidase, beta-D-galactosidase and acid phosphatase were not elevated. These differences could not be accounted for by the fetal bovine serum present in the culture medium. The enrichment in acid hydrolase specific activities in the oligodendrocytes may be associated with a rapid turnover of at least some of the extensive myelin-like membrane formed by these cultured cells. Alternatively, the enrichment of acid hydrolase activity in the oligodendrocytes may be associated with intracellular vesicles of lysosomal origin which may play a role in myelin-like membrane assembly. Exactly which of the above two processes, or possible combinations thereof, is responsible for the present finding is not known.

Lipid abnormalities in foam cell reticulosis of mice, an analogue of human sphingomyelin lipidosis.

The lipid changes in the inheritable foam cell reticulosis of mice discovered by Lyons, Hulse, and Rowe have been reexamined. The major abnormality in thymuses from homozygous-abnormal animals has been identified as an increase in the concentration (per milligram of protein) of sphingomyelin and cholesterol. This increase is associated with normal sphingomyelin-cleaving activity. The lipid compositions of the liver and spleen in the homozygous-abnormal animal and of the thymus in the heterozygous-abnormal mouse are normal. The disorder appears to be chemically analogous to those forms of human sphingomyelin lipidosis (Niemann-Pick disease) that are not accompanied by a decrease in tissue sphingomyelinase.

Glycolipids and other lipid constituents of normal human liver.

An analysis of the lipids in normal human liver is presented which is particularly designed to assist in the classification and study of lipid-storage diseases. Special emphasis has been given to a determination of the quantity and composition of the neutral glycolipid classes and predominant ganglioside (Gm(3)). The neutral glycolipid content of 0.19 (sd +/- 0.11) micro moles per g wet tissue, represented 0.4% of the total lipid in liver. Ceramide dihexoside was the most abundant neutral glycolipid. The mean contents of cholesterol, glycerides, and total phospholipids were 3.9, 19.5, and 25.1 mg/g wet weight, respectively. The relative amounts of seven different phospholipid classes were also determined; these included cardiolipin (diphosphatidylglycerol), which constituted 3.9% of the liver phospholipids.

Large scale preparation of selected glucose oligomers and polymers by gel filtration chromatography.

A method for isolating relatively large quantities of glucose oligomer and polymer subfractions from a corn starch hydrolysate is described. Employing large columns of Bio-Gel P-2 (40-80 microns) at room temperature, we can prepare each day 0.5 to 1.2 grams of oligomeric fractions containing three to four adjacent homologues. The columns are homemade, require no flow adapters, and are operated by gravity elution with water as the solvent. The means for avoiding and overcoming potential difficulties, such as microbial contamination and declining flow rate, are described. With the use of the described method, we can operate a single column continuously for up to twelve months.

Thiamine status in urban adolescents: effects of race.

To evaluate thiamine status in an urban adolescent population, we performed two investigations. In Study I, we compared whole blood thiamine levels in 101 healthy adolescents from varied racial backgrounds with those that had been obtained previously in 146 healthy white adults from a different geographic locale. Blood thiamine values were significantly lower in the adolescents as a group, but the differences were entirely due to the lower levels in the black adolescents. To explore further these differences (Study II), we compared thiamine status in 34 adolescents with that of their parents using measures of both whole blood thiamine content and of erythrocyte transketolase activity. White adolescents had significantly higher total whole blood thiamine values than black adolescents, and white parents had significantly higher thiamine values than black parents by both total whole blood assay and level of transketolase activity. There were no differences in thiamine status between adolescents and parents of the same race. Racial composition is an important variable to consider in population surveys of thiamine status.

Accumulation of oxygenated steryl esters in Wolman's disease.

7alpha- and 7beta-hydroxycholesteryl esters, 7-ketocholesteryl esters, and 5,6alpha- and 5,6beta-epoxycholesteryl esters have been identified in tissues of patients affected by Wolman's disease. Their structural identities were determined by mass spectroscopy and nuclear magnetic resonance spectroscopy and confirmed by chemical synthesis. It is postulated that cholesteryl ester hydrolase deficiency in Wolman's disease might lead to accumulation of oxygenated steryl esters in vivo and impairment of bile acid formation.

Comparison of the clinical and biochemical effect of increased alpha-linolenic acid in a safflower oil intravenous fat emulsion.

We report the results of a randomized comparison of two intravenous safflower oil (fat) emulsions in neonates who required total parenteral nutrition. The fat emulsions differed only in their content of alpha-linolenic acid: in one emulsion the alpha-linolenic acid content of the oil was 0.1% of fatty acids, while in the other emulsion the alpha-linolenic acid content of the oil was 3.0 +/- 1.5% (SD) of fatty acids. Group 1 and 2 patients received the "low" and "high" alpha-linolenic acid emulsions, respectively. Ten patients were studied in each group. The mean daily fat dosage was 1.70 g/kg in patients of Group 1 and 1.56 g/kg in those of Group 2. No significant difference in the clinical effects of either fat emulsion could be detected between the two study groups. Both emulsions prevented or corrected biochemical signs of essential fatty acid deficiency. The major statistically significant difference between study groups was in the level of alpha-linolenic acid and its metabolite, eicosapentaenoic acid. After 2 weeks of fat therapy, these fatty acids were increased in the high alpha-linolenic acid group; however, another metabolite of linolenic acid, docosahexaenoic acid, decreased during intravenous fat therapy in both study groups. Both study groups had significantly decreased arachidonic acid levels and increased linoleic to arachidonic acid ratios.

The jejunal absorption of glucose oligomers in the absence of pancreatic enzymes.

We compared the absorption of carbohydrate from solutions of glucose oligomers and glucose in jejunal Thiry-Vella fistulae, a preparation deprived of pancreatic secretions. The studies were performed with two concentrations (90 and 360 mg/dl) of both glucose and the glucose oligomers. Carbohydrate absorption from glucose solutions (33.1 +/- 2.8, 115.9 +/- 8.9 micrograms/cm/min) was significantly greater (P less than 0.025; P less than 0.005) than that from oligomer solutions (26.6 +/- 2.1 and 92.4 +/- 9.0 micrograms/cm/min). Thin-layer analyses of the perfusates demonstrate digestion of oligomers with a chain length up to eleven and suggest digestion of oligomers of even greater chain length. Atrophy of the jejunal mucosa occurred over the course of the study as evidenced by a decrease in the ratio of villous height to crypt depth from 3.8 to 0.3, and by a 80% decrease in the activity of maltase, sucrase, and lactase. Atrophy was accompanied by a significant decline in the absorption of both glucose oligomers (P less than 0.005) and glucose (P less than 0.01) from the more concentrated solutions but the decrement in absorption of both carbohydrates was similar: glucose oligomers, 79.3 +/- 19.4 micrograms/cm/min; and glucose, 69.8 +/- 14 micrograms/cm/min (P greater than 0.20). Water absorption was enhanced by both carbohydrates, but there was no demonstrable difference between solutions of glucose and glucose oligomers. The osmolality of the solutions clearly influenced water absorption (P less than 0.025) but failed to effect the absorption of carbohydrates.

Late clinical presentation of partial carbamyl phosphate synthetase I deficiency.

OBJECTIVE: To describe the late manifestation of partial carbamyl phosphate synthetase I deficiency in an adolescent whose previous symptoms were not distinctive enough to suggest the presence of a metabolic disease. RESEARCH DESIGN: Clinical description of one patient. SETTING: Primary care children's hospital. PARTICIPANT: An adolescent boy. SELECTION PROCEDURE: Random observation. INTERVENTIONS: Intravenous sodium benzoate and sodium phenylacetate were more successful in reversing the coma than any other intervention. MEASUREMENTS/MAIN RESULTS: The patient has had no recurrence for 2 years, but he appears to have had a partial impairment of cognitive functioning. CONCLUSIONS: General pediatricians and intensivists should be aware that partial carbamyl phosphate synthetase I deficiency, and other partial urea cycle disorders, may become manifest in adolescence, even though they usually present in neonates or infants. When patients present in hyperammonemic coma, the urea cycle disorders should be considered, especially if no obvious cause is identified.

Early changes in the permeability of the blood-brain barrier produced by toxins associated with liver failure.

Our study was designed to determine whether substances that appear in the serum during the course of liver failure have a detrimental effect on the passive permeability of the blood-brain [blood-cerebrospinal fluid (CSF)] barrier. Lactic acid, octanoic acid, and ammonia were infused into rabbits for 4 h. The permeability changes of the blood-brain barrier were quantified by infusing polyethylene glycol 400 (PEG 400) and measuring the quantity and average mol wt of the PEG 400 that entered the CSF. The lipid solubility and effective diffusional radius of the PEG molecules were also quantified to provide greater precision for measurements using this probe. None of the animals receiving toxic infusions became seriously ill during the infusions. Low dose infusions of lactic acid, octanoic acid, and ammonia increased the effective pore diameter of the blood-brain barrier from 7.3 A to an average of 8.5 A. The amount of PEG entering the CSF increased from 1.7 to 4.0 (p less than 0.025), 4.7 (p less than 0.025), and 6.7 (p less than 0.001) mmol/L, respectively. Rabbits with galactosamine-induced liver failure had 10.1 mmol/L PEG 400 in the CSF (P less than 0.001) before any evidence of cerebral edema. These changes occur soon after these toxins accumulate in the plasma and may alone or together with other toxins account for the permeability changes that allow neurotoxic substances to enter the brain during hepatic disease and encephalopathies such as Reye's syndrome.