Jeffrey K. Yao

Nerve Glucose, Fructose, Sorbitol, myo-Inositol, and Fiber Degeneration and Regeneration in Diabetic Neuropathy

We measured the alcohol sugars in sural nerves from 11 controls, 21 conventionally treated patients with diabetes and neuropathy, and 4 diabetics without neuropathy. The results were related to metabolic control and to clinical, neuropathological, and morphometric abnormalities in the nerves. The mean endoneurial glucose, fructose, and sorbitol values were higher in diabetic patients than in controls. Linear regression analysis revealed that nerve sorbitol content in the diabetics was inversely related to the number of myelinated fibers (P = 0.003). Mean nerve levels of myo-inositol were not decreased in the diabetic patients, with or without neuropathy, and were not associated with any of the neuropathological end points of diabetes. Our results indicate that myo-inositol deficiency is not part of the pathogenesis of human diabetic neuropathy, as had been hypothesized. Other accumulated alcohol sugars, however, were increased in diabetes and were associated with the severity of neuropathy. On repeat biopsy, six diabetics, treated for a year with the aldose reductase inhibitor sorbinil, had decreased endoneurial levels of sorbitol (P less than 0.01) and fructose (0.05 less than P less than 0.1), but unchanged levels of myo-inositol.

Microanalysis of Complex Tissue Lipids by High-Performance Thin-Layer Chromatography'

We have developed a high-performance thin-layer chromatographic method, not involving preliminary column chromatography, for separating mixtures of nonpolar lipids, glycosphingolipids, and phospholipids in one dimension. Three or four consecutive developing solvent systems are used. More than 20 different lipid subclasses can be separated using this procedure. Reproducible results have been obtained by careful control of variables. In addition, a simple and rapid fractionation procedure, using Sep-Pak silica cartridges, has been developed to concentrate neutral glycosphingolipids from biological membranes, e.g., erythrocytes, so that patients with sphingolipidosis can be easily identified.

The Sequential Alterations of Endoneurial Cholesterol and Fatty Acid in Wallerian Degeneration and Regeneration

The endoneurial nonpolar lipids were serially examined throughout Wallerian degeneration and regeneration. Following nerve crush in the rat, the endoneurial content of cholesterol falls and cholesteryl ester content rises dramatically. The maximal alteration of this ratio corresponds reasonably well with events of myelin ovoid dissolution to sudanophilic amorphous lipids. Thereafter, as regenerative events overshadow degenerative events the ratio is slowly restored toward normal. The increased cholesteryl esters are probably synthesized within endoneurium from free fatty acids which become available when myelin is degraded. The endoneurial free fatty acid content presumably represents the net effect of phospholipid degradation, cholesterol esterification, cholesteryl ester hydrolysis, and fascicular entry and exit. Free fatty acids become significantly elevated by 12 days, probably reach a peak between 16 and 60 days, and thereafter return to normal with fiber regeneration. The fatty acid composition of cholesteryl esters from crushed nerves is markedly different from those of normal sciatic nerves. The altered fatty acid composition of cholesteryl esters from myelin suggests that both synthesis and hydrolysis exhibit substrate specificity toward chain length and unsaturation, with oleate being the most favored substrate.

Adult-Onset of Tangier Disease: 1. Morphometric and Pathologic Studies Suggesting Delayed Degradation of Neutral Lipids after Fiber Degeneration

A 67-year-old woman, with the typical biochemical features of Tangier disease, had a syringomyelia-like syndrome which has now been observed in several patients with symptomatic onset in adult life. She developed progressive facial diplegia, bilateral wasting of hand muscles and loss of sensation over cranial, cervical and brachial dermatomes over 17 years. Nociception alone was first affected, then nociception and thermal discrimination and finally all modalities of sensation. Quantified tests of cutaneous sensation confirmed that sensation was normal in lower limbs but markedly abnormal in upper limbs. Biopsied fascicles of cutaneous nerves from clinically affected (forearm) and from clinically unaffected (leg) regions permitted a comparison of welladvanced and early pathologic lesions, respectively. The selective vulnerability of unmyelinated and small myelinated fibers in affected regions in this disorder has been confirmed. The earliest morphologic abnormalities of myelinated fibers, but seen infrequently, were mitochondrial enlargement and structural abnormality, aggregation of mitochondria and dense bodies and clusters of neurofilaments. Increased numbers of sudanophilic lipid droplets did not seem to form in Schwann cell cytoplasm prior to fiber degeneration. On the contrary, for myelinated fibers there appeared to be an altered process of axonal degeneration from that seen in Wallerian degeneration and in other axonal degenerations. Distinctive linear bands of closelypacked, minute, osmiophilic and clear lipid droplets formed and their further degradation appeared delayed. Although less clearly demonstrated, lipid droplets in Schwann cells of unmyelinated fibers also appeared to form following their degeneration. We would propose that in Tangier disease, the degradation of myelin ovoids to neutral lipid in Schwann cells does not appear to be delayed. However, further degradation of neutral lipid or its transport away from Schwann cells appears to be retarded.

Lipid abnormalities in hereditary neuropathy: Part 1. Serum non-polar lipids☆

The non-polar lipids from sera of 54 patients, with various types of hereditary motor and sensory neuropathies, and from 72 healthy subjects were evaluated. A small but highly significant decrease in the percentage of linoleate to total fatty acids in both cholesteryl ester and triglyceride fractions was found in the sera of the neuropathy patients, except in those who had dominantly inherited sensory neuropathy (HSN-I) and who had spinocerebellar degeneration with retinitis pigmentosa and other features (SpC+). A significant decrease of serum lecithin-cholesterol acyltransferase activity was also found in those patients with hereditary motor and sensory neuropathies, Type I and Type II (two types of peroneal muscular atrophy). The biochemical basis of these abnormalities is not apparent. The biochemical abnormalities reported here have been found in several neurologic disorders and hence are unlikely to be disease-specific.

[14C]Acetate metabolism in the peripheral nervous system

Lipid biosynthesis was studied by incorporation of [14C]acetate into different compartments of rat sciatic nerve during development, degeneration and regeneration. Acetate incorporation was over three times higher in the sciatic endoneurium (desheathed nerve) than in epi- and perineurium. The endoneurium contained much higher contents of radioactively labeled membrane lipids (cholesterol and phospholipids) than did the epi- and perineurium (mainly triacylglycerol), indicating a benefit of utilization of endoneurium in the study of the metabolic derangements of peripheral nerve lipids. When 3H2O was used as a precursor, no incorporation was found. Endoneurial lipid biosynthesis from [14C]acetate decreased rapidly as myelination proceeded. After 4 months, the decrease continued but at a much slower rate. The total acetate incorporation found in endoneurial lipids of 6-month-old rats was predominantly in the free fatty acid fraction (40%), but was only 5% of that found in 10-day-old rats, demonstrating the importance of age-matched controls for metabolic studies of diseased nerve. During Wallerian degeneration, a decreased acetate incorporation into endoneurial lipids was observed as early as 2 days after crush injury. The profile of labeled lipids in developing and degenerating nerve revealed that the rate of lipogenesis did not change to the same extent for each lipid subclass. Cholesterol biosynthesis appeared to be the most sensitive. During regeneration, an increase in the uptake of [14C]acetate and an altered profile of labeled lipids demonstrated that the metabolic state of adult peripheral nerve, which is normally relatively inactive, can be modified by an exogenous factor such as crush injury.

Improvement of endoneurial lipid abnormalities in experimental diabetic neuropathy by oxygen modification

Endoneurial hypoxia and a high frequency of closed capillaries have been found in chronic experimental diabetes and human diabetic sural nerve, respectively. These findings have led to the hypothesis that the pathogenesis of diabetic neuropathy is due to endoneurial hypoxia. To evaluate the role of endoneurial hypoxia in experimental diabetic neuropathy, the effects of supplementation and deprivation of oxygen on peripheral nerve lipid biosynthesis were studied in normal control and streptozotocin-induced diabetic rats. Defective lipid biosynthesis in diabetic nerve was partially prevented by oxygen supplementation. When normal rats were placed in a hypoxic chamber, lipid abnormalities similar to those observed in diabetic nerves were demonstrated in the absence of changes in nerve free sugars. These findings suggest that endoneurial hypoxia may underlie some key biochemical abnormalities encountered in experimental diabetic neuropathy.

Lipid abnormalities in hereditary neuropathy

The mean percentage of linoleate to total fatty acids in phosphatidylcholine and lysophosphatidylcholine fractions of serum phospholipids from neuropathic patients with HMN (hereditary motor neuropathy, also called distal type of progressive muscular atrophy), HMSN-I and HMSN-II (two types of peroneal muscular atrophy), and FA (Friedreichs ataxia) was reduced by approximately 10--20% (P less than 0.001). On the other hand, the mean percentage of nervonic acid in sphingomyelin was elevated by 9--20%. No significant difference was observed in phosphatidylethanolamine between neuropathic patients and control subjects. The mean concentration of phosphatidylcholine and sphingomyelin was also significantly reduced in neuropathic patients (except in HMN and HMSN-III). A significant correlation between endogenous 2-linoleoyl-sn-glycerol-3-phosphocholine and cholesteryl linoleate synthesis in vitro suggests that the decreased activity of phosphatidylcholine acyltransferase (EC 2.3.1.43; LCAT) in neuropathic patients is influenced by the fatty acid composition of their lipoprotein substrate. Furthermore, the reduction of phosphatidylcholine and of cholesteryl linoleate synthesis in vitro in neuropathic patients was affected by age and sex. It is unlikely that the reduced linoleate level in serum phosphatidylcholine for most, possibly all, of the inherited neuropathies studied here reflects a specific biochemical disorder. Possibly it reflects a more generalized biochemical alteration common to inherited neuropathy. One possibility is that biosynthesis of new membrane in axonal regeneration, segmental remyelination and Schwann cell hyperplasia may reduce the serum linoleate pool.

Structural alterations of peripheral nerve monogalactosylceramides during development and Wallerian degeneration.

The structural alterations of monogalactosylceramides in peripheral nerve were investigated during development, nerve fiber degeneration and regeneration. During early development, hydroxy cerebrosides and sulfatides were the main constituents of the monogalactosylceramides of immature rat sciatic endoneurium. The ratio of hydroxy to nonhydroxy cerebrosides decreased rapidly as myelination proceeded but remained fairly constant throughout adulthood. More than 50% of the adult content of endoneurial monogalactosylceramides was achieved before 21 days of age. The long-chain nonhydroxy fatty acids (above C21) had increased from under 20% to over 80% by day 20, while 24h:0 (h, hydroxy) had already reached approximately 50% of hydroxy cerebrosides by day 12. These results suggest that the biosynthesis of endoneurial monogalactosylceramides and fatty acid elongation take place preferentially at the time when peripheral nerve is undergoing active myelination. During Wallerian degeneration, the maximum decrease of monogalactosylceramides was associated temporally with axonal degeneration and demyelination and particularly with myelin conversion to sudanophilic lipids. By the time that nerve fiber regeneration was well established, both the cerebroside and sulfatide contents had returned to near control values. Cerebrosides and long-chain fatty acids (above C21) appear to be the most sensitive to fiber degeneration while fatty acid elongation is selectively increased during nerve regeneration.

Structural specificity in demyelination induced by lysophospholipids

The demyelinating activity of lysophosphatidylcholine (lysoPC) and various structural analogs in rat sciatic nerve was evaluated by following electrophysiologic changes within the first hour and 1 week after intraneural injection. The lysophospholipids tested included 1-O-hexadecanoyl-sn-glycero-3-phosphocholine (1-acyl-GPC), 3-O-hexadecanoyl-sn-glycero-1-phosphocholine (3-acyl-GPC), 1-O-hexadecanoylpropanediol-3-phosphocholine (acyl-PPC), 1-O-hexadecylpropanediol-3-phosphocholine (alkyl-PPC) and 1-acyl-sn-glycero-3-phosphoethanolamine (1-acyl-GPE). Changes in conduction velocity, width, amplitude and time integral percentage were measured. Within 1 hour, the highest demyelinating activity was observed for alkyl-PPC, followed by 3-acyl-GPC, 1-acyl-GPC and acyl-PPC. Hydrolysis products of lysoPC (glycerophosphocholine, fatty acid), lysophosphatidylethanolamine (1-acyl-GPE), biradyl choline phospholipids (1,2-di-O-alkyl-rac-glycero-3-phosphocholine, dialkyl-GPC) or sodium deoxycholate proved ineffective in these short-term experiments. One week after intraneural injection, all lysophospholipids tested caused severe electrophysiologic changes, although dialkyl-GPC and sodium deoxycholate did not. Our data suggest (i) that differences in early demyelinating activity by the choline lysophospholipids are related to their rate of turnover, as highest activity was associated with the agents that are not metabolized by lysophospholipase (e.g., alkyl-PPC) or lysolecithin acyltransferase (e.g., 3-acyl-GPC), (ii) that the lysoPC molecule as such and not its products of catabolism causes demyelination, and (iii) that demyelinating activity is not due to the general detergent action of lysoPC, but rather that specific interactions appear to trigger the processes of demyelination induced by lysophospholipids.