The development and implementation of quality assurance programs to support nutritional measurements.

The National Institute of Standards and Technology administers quality assurance programs devoted to improving measurements of nutrients and related metabolites in foods, dietary supplements, and serum and plasma samples. These programs have been developed in collaboration with the National Institutes of Health to assist measurement communities in their efforts to achieve accurate results that are comparable among different laboratories and over time. Targeted analytes include micronutrients, botanical markers, nutritional elements, contaminants, fatty acids, and vitamin D metabolites.

Genetic variation in human erythrocyte acetylcholinesterase.

A method for solubilization of human erythrocyte membranes was developed and used to survey 70 unselected human blood samples for isozymic variation of stromal acetylcholinesterase. Three variants were observed. Pedigrees of families studied by this method indicated that this variation represented the phenotypic expression of two codominant alleles at a single locus.

Molecular basis of medium chain acyl-coenzyme A dehydrogenase deficiency. An A to G transition at position 985 that causes a lysine-304 to glutamate substitution in the mature protein is the single prevalent mutation.

We sequenced polymerase chain reaction (PCR)-amplified variant medium chain acyl-CoA dehydrogenase (MCAD) cDNAs in cultured fibroblasts from three MCAD-deficient patients. In all three patients, an A to G transition was identified at position 985 of the coding region. Since no appropriate restriction sites for detecting this point mutation were found, we devised a PCR method that amplifies an 87-bp fragment from position 955. In the 5' primer encompassing positions 955 to 984, A-981 was artificially substituted with C. With the presence of C-981 and G-985, an Nco I restriction site is introduced in the mutant copies. When cDNA or genomic DNA from fibroblasts of nine MCAD-deficient patients were tested with this method, the copies from all of them completely cleaved into two shorter fragments by Nco I, indicating their homozygosity for the A----G-985 transition. In contrast, the copies from all eight controls remained intact. Thus, this A----G-985 transition is the single prevalent mutation causing MCAD deficiency, a highly unusual feature for any genetic disorder. The PCR/Nco I digestion method is suitable for the diagnosis of MCAD deficiency.

Genetic deficiency of short-chain acyl-coenzyme A dehydrogenase in cultured fibroblasts from a patient with muscle carnitine deficiency and severe skeletal muscle weakness.

Genetic deficiency of short-chain acyl-coenzyme A (CoA) dehydrogenase activity was demonstrated in cultured fibroblasts from a 2-yr-old female whose early postnatal life was complicated by poor feeding, emesis, and failure to thrive. She demonstrated progressive skeletal muscle weakness and developmental delay. Her plasma total carnitine level (35 nmol/ml) was low-normal, but was esterified to an abnormal degree (55% vs. control of less than 10%). Her skeletal muscle total carnitine level was low (7.6 nmol/mg protein vs. control of 14 +/- 2 nmol/mg protein) and was 75% esterified. Mild lipid deposition was noted in type I muscle fibers. Fibroblasts from this patient had 50% of control levels of acyl-CoA dehydrogenase activity towards butyryl-CoA as substrate at a concentration of 50 muM in a fluorometric assay based on the reduction of electron transfer flavoprotein. All of this residual activity was inhibited by an antibody against medium-chain acyl-CoA dehydrogenase. These data demonstrated that medium-chain acyl-CoA dehydrogenase accounted for 50% of the activity towards the short-chain substrate, butyryl-CoA, under these conditions, but that antibody against that enzyme could be used to unmask the specific and virtually complete deficiency of short-chain acyl-CoA dehydrogenase in this patient. Fibroblasts from her parents had intermediate levels of activity towards butyryl-CoA, consistent with the autosomal recessive inheritance of this metabolic defect.

Relationship between unusual hepatic acyl coenzyme A profiles and the pathogenesis of Reye syndrome.

This study examines the relationship between impaired fatty acid oxidation and the pathogenesis of Reye syndrome. We present a hypothesis proposing that many clinical signs of this childhood disease are caused by accumulation of unusual acyl CoA esters, precursors to deacylated metabolites found in the patients' blood and urine. A new method was developed to measure acyl CoA compounds in small human liver biopsy samples, offering several advantages over previous techniques. A major finding was an accumulation in Reye syndrome patients of short- and medium-chain acyl CoA intermediates of fatty acid and branched-chain amino acid oxidation. These metabolites included octanoyl, isovaleryl, butyryl, isobutyryl, propionyl, and methylmalonyl CoA esters. The findings were explained in a model of hepatic fatty acid oxidation involving three interrelated pathways: mitochondrial beta-oxidation, peroxisomal beta-oxidation, and omega-oxidation in the endoplasmic reticulum. The results suggest that pathogenesis in Reye syndrome stems from generalized mitochondrial damage resulting in accumulation of acyl CoA esters. High levels of these compounds lead to inhibition of mitochondrial pathways for ureogenesis, gluconeogenesis, and fatty acid oxidation. The inhibited pathways, in turn, could cause the hyperammonemia, hypoglycemia, and hypoketonemia observed in patients. The model also explains underlying biochemical differences between patients with Reye syndrome and medium-chain acyl CoA dehydrogenase deficiency, another disorder of fatty acid metabolism. Acetyl CoA levels, in the latter disease, were dramatically decreased, compared with both human controls and Reye syndrome patients.

Increase in liver acid lipase of thyroidectomized rats by thyroid hormones and its inhibition by actinomycin D.

Acid lipase activity in the livers of thyroidectomized rats is increased by administration of L-thyroxine. The response is dose-dependent and can be demonstrated within 12 h after treatment. L-Triiodothyronine also evokes a rapid increase in acid lipase activity, and this increase can be inhibited by coadministration of actinomycin D.

Acid lipase activity of human lymphocytes.

Acid lipase activity was examined in human leukocytes using 4-methylumbelliferyl esters in a fluorimetric assay. Mononuclear leukocytes had 10--15 times the acid lipase activity of polymorphonuclear leukocytes. The enzyme activity was highest using the oleate ester of 4-methylumbelliferone at pH 4.0, in the presence of L-alpha-phosphatidylcholine and taurodeoxycholic acid (sodium salt). Acid lipase activity was inhibited by diethylaminoethoxyhexestrol, sodium chloride and fluoride, potassium chloride, calcium chloride and albumin, but was unaffected by diethyl p-nitrophenyl phosphate or sulphydryl reagents. There were at least two forms of acid lipase activity: one (A form) was sensitive to heart inactivation (56 degrees C) and corresponded to the enzyme deficient in patients with Wolman's disease; the other (B form) was resistant to heat inactivation and corresponded to the residual enzyme activity found in Wolman's disease.

I-cell disease: intracellular desialylation of lysosomal enzymes using an influenza virus vector.

It has been proposed that I-cell disease results from a primary deficiency of acid neuraminidase activity. Infection by influenza virus of fibroblasts from a patient with I-cell disease resulted in the production of abundant intracellular alpha2-3 neuraminidase activity. Despite electrophoretic evidence of desialylation of intracellular and fibroblast-secreted arylsulfatase (EC 3.1.6.1) and beta-hexosaminidase (EC 3.2.1.30) from the infected cells, there was no consequent alteration of the abnormal distribution of beta-hexosaminidase activity between the intracellular spaces characteristic of I-cell disease. This suggests that deficiency of alpha2,3 neuraminidase activity is not the primary biochemical defect in I-cell disease.

Effect of thyroid hormones on human mononuclear leukocyte lysosomal acid lipase activity.

Lysosomal acid lipase (LAL), the enzyme which hydrolyzes lipoprotein cholesteryl esters, is under thyroid hormone regulation in experimental animals. To evaluate whether human LAL is also under thyroid regulation, we measured mononuclear leukocyte LAL activity in 31 hypothyroid, hyperthyroid, and euthyroid children under treatment. LAL activity was positively correlated with serum T3 and serum T4 levels, and was significantly higher in hyperthyroid children than in those who were hypothyroid. In another study, 5 adult euthyroid males were given 50 micrograms L-T3 every 8 h for 4 days. Their LAL activities increased significantly after treatment. The data suggest that LAL activity in man, as in experimental animals, is influenced by thyroid hormone status. Hormonal control of LAL activity may be important in the regulation of human cholesterol metabolism.

Effects of family history of heart disease, apolipoprotein E phenotype, and lipoprotein(a) on the response of children's plasma lipids to change in dietary lipids.

We examined the effects of family history of coronary artery disease (CAD), apolipoprotein E (apo E) phenotype, and lipoprotein(a) [Lp(a)] on the response of plasma lipids to change in dietary lipid intake after 3 mo of nutrition education in 125 children aged 4-10 y. The subjects were healthy children with elevated low-density-lipoprotein (LDL)-cholesterol concentrations who participated in the Children's Health Project, a nutrition-education program designed to lower plasma cholesterol by means of dietary modifications in accordance with recommendations of the National Cholesterol Education Program. Dietary and plasma lipids were measured by three 24-h recalls and assessments of two fasting plasma samples collected before and 3 mo after the start of intervention. Family history of CAD was determined by questionnaires administered to parents at baseline. Apo E phenotyping was done with isoelectric focusing followed by immunostaining; Lp(a) was measured with two-site immunoradiometric assays of frozen aliquots of plasma samples collected at baseline and 3 mo. After adjustment for intervention group, age, sex, and body mass index, analysis of covariance showed that baseline plasma lipid concentrations were the strongest independent predictors of change in plasma lipids after 3 mo. Plasma total and LDL-cholesterol concentrations in children with less family history of CAD were significantly more responsive to change in dietary cholesterol than concentrations in children with a stronger family history of CAD. Neither apo E phenotype nor Lp(a) significantly influenced change in plasma lipids independently or interactively with change in dietary lipids.

Genetic variation of human mononuclear leukocyte lysosomal acid lipase activity. Relationship to atherosclerosis.

Lysosomal acid lipase activity was measured in mononuclear leukocytes of patients selected on the basis of premature cardiovascular disease, with or without hyperlipidemia. Enzyme activity was significantly lower in the patient population (4.8 +/- 1.3 nmol/min/mg protein, n = 190 males) than in an age-matched control population (5.4 +/- 1.3 nmol/min/mg protein, n = 124 males). There was no effect of hypercholesterolemia or hypertriglyceridemia on the enzyme activity. In the group of patients with normal plasma lipids (n = 77), 18% had mononuclear leukocyte acid lipase activity which fell below the control population 5th percentile, and in the range of enzyme activity observed in cells from obligate heterozygotes for inherited acid lipase deficiency (Wolman disease and cholesteryl ester storage disease). Studies of acid lipase activity in families of our patients provided evidence that an autosomal mutation is associated with (or responsible for) this reduced enzymatic activity and may represent an independent risk factor for the premature development of atherosclerosis.

Prenatal diagnosis of Wolman disease.

Two pregnancies at risk for Wolman disease were monitored by assay and electrophoresis of acid lipase in cultured amniotic-fluid cells. Cells from patient 1 had 5% of control levels of acid lipase, using 14C-triolein as substrate; however, when artificial substrates (esters of 4-methylumbelliferone and p-nitrophenol) were used to measure acid lipase, these cells had 30% of control levels. Electrophoresis of cell extracts revealed the absence of the A form of acid lipase, consistent with the diagnosis of Wolman disease. Analysis of fetal tissues following prostaglandin termination of this pregnancy confirmed the diagnosis. Assay of fetal-skin fibroblasts with 14C-triolein, as well as with artificial substrates, showed marked deficiency of acid lipase activity. Electrophoresis of fetal-tissue extracts also demonstrated the absence of the A form of acid lipase. Amniotic-fluid cells from patient 2 showed normal levels of acid lipase with all substrates tested; the electrophoretic pattern of acid lipase was normal. The results suggest that the prenatal diagnosis of Wolman disease be made using the radioassay of acid lipase and/or electrophoresis.

Cholesteryl ester storage disease: pathologic changes in an affected fetus.

The prenatal diagnosis of cholesteryl ester storage disease, a rare autosomal recessive disorder, was made by demonstration of deficient lysosomal acid lipase activity in cultured amniocytes from an at-risk fetus. The histochemical and ultrastructural changes in the affected fetus (at 17 gestational weeks) are described and compared to findings in liver and duodenal biopsy specimens from a 9-year-old homozygous female. Massive lysosomal cholesterol and lipid accumulation was demonstrated in fetal hepatocytes, adrenal cells and syncytiotrophoblasts. Of particular note was the observation of extensive necrosis in the fetal adrenal glands. Necrosis of the adrenals may precede the calcification observed in some patients with cholesteryl ester storage disease and in most patients with Wolman disease, an allelic variant due to lysosomal acid lipase deficiency. Fibrosis of the liver and lipid accumulation in macrophages in liver and duodenum, which were present in the 9-year-old homozygote, were not observed in the affected fetus, and therefore, may represent later manifestations of the disease.

Effect of diabetes and insulin therapy on human mononuclear leukocyte lysosomal acid lipase activity.

Acid lipase activity was measured in mononuclear leukocytes obtained from 18 newly diagnosed type-I diabetics and from six newly diagnosed type-II diabetics before and five to ten days after the initiation of insulin therapy. Using either 4-methylumbelliferyl oleate or cholesteryl[1-14C]oleate as substrate for the enzyme assays, acid lipase activity in the diabetics was not found to be significantly different from that in the controls, significantly less (P less than 0.05) after insulin therapy in type-I diabetics, and unchanged following treatment in the type-II patients. These results differ from those found in previous studies of acid lipase activity in type-II diabetics, in whom insulin therapy evoked a doubling of enzyme activity. The effect of insulin treatment on the activity of lysosomal acid lipase requires further evaluation.

Kinetics of retinyl esters during postprandial lipemia in man: a compartmental model.

Orally ingested vitamin A (retinol) is incorporated into intestinal chylomicrons (CHYLO) in the form of retinyl esters (RE) along with newly absorbed dietary triglycerides (TG). As the intestinal lipoproteins undergo hydrolysis in the circulation, the majority of the RE remain with the secreted intestinal particles and have been used as a marker for intestinally derived lipoproteins during the early phase of the postprandial state. A multicompartmental model was developed for the kinetics of RE during postprandial lipemia in individuals with normal lipid levels (n = 16) and in patients with hyperlipidemia (n = 44). The assumptions used in the development of the model are presented in this report. Some of the key findings include (1) as much as 50% of the newly synthesized RE may be secreted by the intestine as very-low-density lipoprotein (VLDL)-sized particles of S(f) 20 to 400 following consumption of a test meal containing a moderate amount of fat (20 to 30 g); (2) in most individuals, approximately 50% of the RE secreted in S(f) greater than 400 are converted to smaller, less buoyant fractions, and 50% are irreversibly removed directly from the plasma; (3) as much as 5% to 20% of the ingested retinol may be secreted as small intestinal lipoproteins with the buoyance of low-density lipoprotein (LDL) in some individuals; and (4) less than 5% of RE flux through S(f) 20 to 400 is converted to S(f) less than 20, and the primary catabolic pathway for RE in this fraction is direct uptake. Comparable estimates can be obtained for the kinetic parameters when repeat studies are made in the same subjects under comparable conditions.

New developments in the diagnosis and investigation of mitochondrial fatty acid oxidation disorders.

Since the discovery of muscle carnitine palmitoyltransferase deficiency in 1973, a dozen separate defects of mitochondrial fatty acid beta-oxidation in man have been identified. With the exception of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, which occurs with a frequency approaching 1:10,000 among Caucasians of Northern European origin, the other defects are quite rare. Collectively, however, they are common causes of disease resembling Reye syndrome in early life, and some have a later and more chronic presentation with cardiomyopathy and skeletal muscle weakness. They also represent a small, but significant, proportion of cases of sudden and unexplained death within the first 2 years of life. Diagnosis of these disorders has become increasingly sophisticated, with the advent of new analytical technologies and an increased awareness of the appropriate clinical and laboratory investigations needed in order to evaluate potential defects of this pathway. The combination of provocative testing (e.g., carnitine loading, phenylpropionic acid loading, long-chain fat loading) and advanced analytical techniques for the measurement of blood and urinary metabolites (e.g., tandem fast atom bombardment-mass spectrometry, stable isotope dilution gas chromatography-mass spectrometry) permits a specific diagnosis in the case of several, although not all, of the disorders of this pathway. Methods for the measurement of all of the enzymes of beta-oxidation are now available to enhance this diagnostic capability. There remain, however, many patients in whom clinical and laboratory signs point to a defect in beta-oxidation, but in whom no specific diagnosis has yet been made.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular basis of mitochondrial fatty acid oxidation defects.

A dozen separate inherited disorders of mitochondrial fatty acid beta-oxidation have been described in humans. This represents about half of the potential sites for genetic error that can affect this important pathway of energy metabolism. As the characterization of these disorders at the clinical and biochemical levels has progressed rapidly, so has the delineation of the molecular defects that underlie them. The most commonly recognized disorder of beta-oxidation is medium-chain acyl-CoA dehydrogenase deficiency; a striking feature of this disorder is that there is a single point mutation that accounts for 90% of the variant alleles among patients with medium-chain acyl-CoA dehydrogenase deficiency. Molecular defects of other enzymes in the pathway have been identified, and it seems likely that a complete description of these defects at the molecular level is a realistic goal. In basic biological terms, such studies will lead to a better understanding of the genetic control exerted on this pathway. In clinical terms, they will lead to improved understanding of the molecular pathophysiology of these diseases and may well provide the necessary techniques to proceed with the screening of these disorders.