John H. Menkes

Adrenoleukodystrophy: Accumulation of cholesterol esters with very long chain fatty acids

An accumulation of fatty acids having chain lengths of C22 to C26 was demonstrated in the cholesterol ester fraction of white matter from two boys with adrenoleukodystrophy. A similar accumulation of very long chain fatty acids was also shown in gangliosides and cerebrosides. A disorder of the one-carbon degradation system responsible for the metabolism of some of the very long chain fatty acids may be the basic enzymatic defect in this condition.

Fatty Acid Synthesis in the Developing Brain

Extract: Fatty acid biosynthesis in the developing brain was studied. During development, total fatty acid synthesis was maximal at 15–16 days of age—a time of rapid myelination for this species. Results were expressed as mμmoles of 14C-labeled acetyl-CoA incroported into fatty acids per milligram of brain mitochondrial of microsomal protein, and as 14C-labeled malony-CoA incorporated per milligram of microsmal protein.Elongation of saturated fatty acids by microsoms followed a similar development pattern. The peak incorporation of malaony-CoA into saturated fatty acids occurred at 15 days of age, while synthesis of polyunsaturated fatty acid did not change significantly with maturation. In the mitochondrial-acetyl-CoA system, elongation of potyunsaturated fatty acids was unchanged significantly with maturation. These result support the concept that chain elongation of fatty acids is directly ralated to myelination. The marked increase in the rate of elongation of saturated fatty acid by the micosmol fraction, occurring at this time, suggests that this system is involved in synthesis of myelin fatty acids.Speculation: At the time myelination there is an increase in the activity of microsomes with resect to incroporating malony-CoA into saturated fatty asids, and into polyunsaturated fatty acids (20:2 and 20:4). This is unexpected because of the well established finding that in mature liver the microsomal fraction preferentially synthesizes polyunsaturated fatty acids, while the mitochondrial fraction forms saturated fatty acids. Polyunsaturated fatty acids constitute a relatively minor proportion of myelin fatty acids, and it is unlikely that the striking increase i microsomal activity observed at 15–16 days is necessary for their formation. It is possible that maturation changes the selectivity of the fatty acid synthesizing system, or that there are specificity differenced between the hepatic and cerebral microsomal chain elongation systems.

Polyenoic acid metabolism in cultured human skin fibroblasts

The incorporation of [1-14C]linoleic acid, and [1-14C]linoleic acid into cellular lipids of cultured human skin fibroblasts was studied. Cultured cells took up both labeled fatty acids at nearly the same rate and incorporated them into a variety of lipid classes. At the end of 1 hr incubation with [1-14C]linoleic acid, radioactivity was found in the triacylglycerol (TG) and choline phosphoglyceride (CPG) pools preferentially. Incorporation into the TG fraction decreased rapidly, while the uptake into CPG, serine phosphoglyceride (SPG), and ethanolamine phosphoglyceride (EPG) fractions increased progressively with longer incubation times. Similar results were obtained with [1-14C]linoleic acid as precursor. At the end of 24 hr, desaturation and chain elongation of 18∶3 n−3 was more extensive than conversion of 18∶2 n−6 to higher polyenoic acids. During pulse-chase experiments with either fatty acid precursor, the incorporated radioactivity was progressively lost from cellular lipids, particularly from the TG and CPG fractions, but continued to increase in the SPG and EPG pools. The similar labeling pattern of cellular phospholipids with linoleic or linolenic acids, and data from pulse-chase studies suggest that a direct transfer of fatty acids from CPG to EPG is a likely pathway in fibroblast cultures. Incorporation into the EPG pool during the pulse-chase experiments paralleled extensive desaturation and elongation of linoleic acid into 20∶4 n−6, and 22∶4 n−6; and of linolenic acid into 22∶5 n−3 and 22∶6 n−3.

Globoid cell leukodystrophy (Krabbe's disease): Morphological and biochemical studies

THE FORM OF DEMYELINATING DISEASE now known as Krabbe’s disease was differentiated from other forms of diffuse sclerosis in 1916.l The synonym, globoid cell leukodystrophy, was derived from the term later applied by Collier and Greenfield2 to the characteristic cell type associated with the demyelination in this condition. The histological and histochemical findings in Krabbe’s disease, as revealed by light microscopy, have been well documented in the literature.”* Two enzymatic defects have been found in affected subjects. Bachhawat et al. documented a deficiency of cerebroside sulfotransferase activity, preventing the conversion of cerebrosides to sulfatides.7 More recently, Malone has found that leukocytes from patients with Krabbe’s disease are unable to cleave cerebrosides.8 A deficiency of a specific enzyme, galactocerebroside beta-galactosidase, has also been demonstrated in brain, liver, and spleen of patientsQ As a result, the concentration of white matter cerebrosides is increased and that of sulfatides is diminished. The concentrations of ceramide dihexosides and trihexosides are also increased; these are perhaps derived from cerebrosides. The purpose of this report is to describe clinical, routine neuropathologic, ultrastructural, chemical, and metabolic findings in three additional cases of Krabbe’s disease and to discuss briefly other recent reports dealing primarily with fine structure and chemistry.

Bilirubin interaction with ganglioside: possible mechanism in kernicterus.

Extract: Reaction of bilirubin with increasing amounts of ganglioside purified from neonatal brain significantly alters the spectral absorption of bilirubin in proportion to the quantity of ganglioside added. Increments in absorbance occur at 353 nm with a prompt but transient increase at 486 nm. A decrease in absorbance occurs which is most marked at 447 nm. When gangliosides are added to bilirubin (9.1 μg/ml or 0.016 μM/ml), the decrease in absorbance is essentially linear up to the highest concentration of purified ganglioside tested (182 μg/ml or 0.097 μ/ml), which represents a molar ratio of 6.1:1. The asymptotic nature of the bilirubin-ganglioside reaction as measured by the decrease in absorbance with time suggests a stoichiometric relationship between the two substances. An isosbestic point was demonstrated at 405 nm. Observations reported here suggest bilirubin reaction with ganglioside is at least a two-step process.Speculation: Bilirubin cytotoxicity may be related in part to plasma membrane effects which involve bilirubin interaction with ganglioside at concentrations which do not disturb mitochondrial metabolism. The difference between the ganglioside composition of infant and adult gray matter may in part explain the marked cytotoxicity of unconjugated bilirubin for the infant nervous system.

Cellular immune responses in Huntington disease Specificity of brain antigenicity detected with Huntington disease lymphocytes

This study confirms the production of migration inhibition factor by Huntington disease lymphocytes in response to antigen found in Huntington disease but not normal brain tissue. Huntington disease lymphocytes also respond to the presence of multiple sclerosis brain tissue with migration inhibition factor production.

Huntington disease: finding the gene and after.

Huntington disease is an autosomal dominant disorder that usually begins in mid-life and is characterized by progressive choreiform movements and dementia. Approximately 5% of patients develop symptoms prior to 14 years of age. In most juvenile cases, the gene is transmitted from the father. In children the clinical course is marked by mental deterioration or behavioral abnormalities, gait disturbances usually the consequence of rigidity, cerebellar signs, and seizures. The pathologic findings are highlighted by atrophy of the caudate. Atrophy also is observed on brain imaging, while positron emission tomography demonstrates marked caudate hypometabolism which antedates the appearance of the clinical disease. Cell death in the striatum primarily affects medium and small GABA-containing neurons, representing the striatal output projections. Somatostatin-containing neurons and cholinergic neurons are spared. The gene for Huntington disease has been localized in close proximity to the tip of the short arm of chromosome 4. The gene product and the manner by which it induces selective cell death is still unknown but should become evident in the near future.

Sections on Child Neurology and Neurochemistry (Joint Session)

ArHtfcial Substrate CIoaving Activity of Hexosamlnidarn A and B EDWIN H. KOLODNY, Waltham, MA, and IONACY WALD. Pruszkow, Poland, and HUGO W. MOSER, Waltham, MA, and DAVID G. COOAN, Boston, and TOICHIRO KUWABARA, Waltham, MA Three forms of Gw-ganghosidosis have now been clearly defined: (I) classical Tay-Sachs disease with absence of hexosaminidase A @ex A); (2) Sandhoff-Jatzkewitz disease in which both hex A and B are lacking: and (3) juvenile G,,gangliosidosis with partial hex A deficiency. We will report here an additional variety of the disease in which the accumulation of Gm-ganglioside in brain at least equals that in these other forms, yet no deficiency occurs in hex A or B measured with artificial substrates. The initial biochemical studies of this case were reported by Sandhoff and associates (J Neurochem 18:2469,1971). The propositus was a boy of Scottish-Irish-English-American Indian descent who died at age 32 months. His clinical manifestations were intermediate between those of the classical and juvenile forms of the disease. As late as age two years, visual fixation could be shown and optic disk color was normal. Macrocephaly never developed. Certain pathologic features also distinguished this case from other forms of the disease: the neuronal cell population was not significantly altered; numerous brightly sudanophilic macrophages were widely distributed; and, in electron-microscopic studies of retinal ganglion cells, MCBs were less frequent than membrane-bound vesicles and parallel arrays of lamellae. A younger sister was afflicted with a similar clinical disorder and died at age 39 months. Serum and WBC hex A and B activity in their parents and two sibs were normal. The ability of enzyme preparations from tissues of the propositus to degrade Gm and its asialo derivative is currently being studied.

Effect of Thyroid on Fatty Acid Biosynthesis in Brain

Extract: Triodothyronine (T3) was given daily by injection to rats from 1 day of age to 2 hr prior to death. In the brain microsomal fraction of 5− to 6-day-old animals, incorporation of malonyl-CoA into total fatty acids increased from 0.9 to 2.25 mμmoles/mg protein. Although thyroid increased precursor incorporation into total fatty acids in other age groups, the difference was not statistically significant. Increased precursor incorporation was entirely due to increased formation of saturated fatty acids, and this effect was observed at all age groups.Incorporation of acetyl-CoA into saturated and unsaturated fatty acids by the mito-chondrial fraction was not increased significantly by administration of T3.In rats made hypothyroid at birth, fatty acid chain elongation was reduced in both the microsomal and the mitochondrial fractions. At 6 days of age, incorporation of malonyl-CoA by the microsomal fraction fell from 1.41 to 0.17 mμmoles/mg protein. These studies suggest that thyroid stimulates the microsomal-fatty acid-brain elongation system involved in the synthesis of saturated fatty acids characteristic of myelin lipids.Speculation: The stimulation of fatty acid biosynthesis by thyroid hormone is specifically limited to the synthesis of saturated fatty acids by the microsomal fraction. Since the effect of thyroid is probably mediated by enzyme synthesis, we conclude that the microsomal fraction contains two distinct enzyme systems involved in fatty acid chain elongation, one for the formation of polyunsaturated fatty acids, the other for the formation of saturated fatty acids. The activity of the latter system increases at time of myelination, and as a consequence of thyroid stimulation.

Effect of Temperature on Fatty Acid Metabolism in Dissociated Cell Cultures of Developing Brain

Extract: The effect of hypothermia on the incorporation and metabolism of essential fatty acids (linoleic and linolenic acids) by dissociated cell cultures of developing brain was examined. When the ambient temperature was reduced to 30°, chain elongation of linolenic acid (18:3) to 20:3, and elongation of 20:5 to 22:5 was slowed. Desaturation of 20:3 to 20:4 and 20:5 remained unaffected even at 22°. Incorporation of (1-14C)linolenic acid into the fraction containing ethanolamine phosphoglycerides and ethanolamine plasmalogens (EPG) was significantly reduced by hypothermia, whereas labeling of choline phosphoglycerides (GPG) was greater at 30° and 22° than at 37°, and fell only slightly at 15°. These observations suggest that EPG labeling depends on the availability of the higher polyunsaturated fatty acids (22:5 and 22:6), whereas CPG labeling is nonspecific. Because ethanolamine plasmalogens are major myelin constituents, the importance of hypothermia in inducing a deficiency of the higher polyunsaturated fatty acids within the developing nervous system, and thus interfering with the biosynthesis of EPG and indirectly with membrane deposition, needs to be clarified.Speculation: These studies suggest that hypothermia may induce a deficiency of the higher polyunsaturated fatty acids in the developing nervous system, which, in turn, could induce a defect in the formation of cell membranes. Increases in the concentration of essential fatty acids for formulas intended for low birth weight infants might compensate for this relative deficiency.