Blood dolichol in lysosomal diseases KALLE JOKELAINEN, KATJA S. SALMELA,KARI HUMALOJA, AND RISTO ROINE Research Unit of Alcohol Diseases, University of Helsinki; Helsinki, Finland SEPPOAUTIO Rinnekoti Institution for the Mentally Retarded, Espoo, and Department of Child Neurology, University of Helsinki, Helsinki, Finland
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MARIAARVIO Paajarvi Rehabilitation Center, Lammi, Finland IRMA JARVELA National Public Health Institute, Helsinki, Finland IRMANYKANEN Research Laboratories of the Finnish State Alcohol Company (Alko, Ltd), Helsinki, Finland
JORMA PALO Department of Neurology, University of Helsinki, Helsinki, Finland AND
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MIKKOSALASPURO Research and Treatment Unit of Alcohol Diseases, University of Helsinki, Tukholmankatu8 F, 00290 Helsinki, Finland Received June 27. 1991 JOKELAINEN, K., SALMELA, K. S., HUMALOJA, K., ROINE,R., AUTIO,S., ARVIO,M., JARVELA, I., NYKANEN,I., PALO,J., and SALASPURO, M. 1992. Blood dolichol in lysosomal diseases. Biochem. Cell Biol. 70: 481-485. Highly elevated serum total dolichol (free dolichol + dolichyl ester) concentrations have recently been found in two lysosomal storage diseases, aspartylglucosaminuria (AGU) and mannosidosis. The present study demonstrates that the increase of serum dolichol in AGU patients is caused by an increase of serum free dolichol. In 15 patients the mean serum level of free dolichol (227 k 16 ng/mL) was 1.9 times higher ( p < 0.001) than that in healthy controls (120 k 6 ng/mL), while the amounts of dolichol fatty acid esters were similar in the patients and controls (110 9 vs. 118 + 6 ng/mL). In contrast, 10 patients with neuronal ceroid-lipofuscinosis (NCL) (three with infantile, four with juvenile, and three with variant late infantile NCL) had significantly ( p < 0.01) lower mean serum levels of both free (79 k 5 ng/mL) and total (159 6 ng/mL) dolichol than age-adjusted healthy controls (free, 100 k 6 ng/mL; total, 206 k 14 ng/mL). Decreased blood dolichol has not been reported earlier for any other disease. We conclude that the increased serum free dolichol in AGU reflects disturbed lysosomal function and that the decreased free and esterified dolichols in NCLs speak against their presumed primary lysosomal nature. Key words: serum dolichol, aspartylglucosaminuria, neuronal ceroid-lipofuscinosis, lysosomal function, lysosomal storage disease.
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JOKELAINEN, K., SALMELA. K. S., HUMALOJA, K., ROINE,R., AUTIO,S., ARVIO,M., JARVELA, I., NYKANEN,I., PALO,J., et SALASPURO, M. 1992. Blood dolichol in lysosomal diseases. Biochem. Cell Biol. 70 : 481-485. Des concentrations fortement tlevtes de dolichol sbique total (dolichol libre + dolichyl ester) ont rtcemment kt6 trouvtes dans deux maladies d'emmagasinage lysosomique: l'aspartylglucosaminurie (AGU) et la mannosidose. Nous demontrons ici que l'augmentation du dolichol strique chez les malades atteints d'AGU est causte par un accroissement du dolichol strique libre. Chez 15 malades, le taux strique moyen du dolichol libre (227 k 16 ng/mL) est 1,9 fois plus tlevt ( p < 0,001) que celui des contrdles en santt (120 + 6 ng/mL) alors que les quantitts des dolichols acides gras esters sont identiques chez les malades et les contrdles (110 k 9 vs. 118 + 6 ng/mL). En revanche, 10 patients atteints de ctroide-lipofuscinose neuronale (NCL) (trois avec NCL infantile, quatre avec NCL juvenile et trois avec une variante infantile tardive) ont des t a w striques moyens significativement ( p < 0,Ol) plus bas de dolichol libre (79 + 5 ng/mL) et de dolichol total (159 k 6 ng/mL) que c e w des contrdles en santt de m&me Ige (libre, 100 k 6 ng/mL; total, 206 k 14 ng/mL). Des diminutions de dolichol sanguin n'ont t t t rapporttes pour aucune autre maladie. Nous concluons que l'augmentation du colichol strique libre dans I'AGU refltte une fonction lysosomique anormale et que la diminution des dolichols libres et esterifits dans la NCL parlent contre leur prtsumte nature lysosomique primaire. Mots elks : dolichol strique, aspartylglucosaminurie, ctro'ide-lipofuscinoseneuronale, fonction lysosomique, maladie d'emmagasinage lysosomique. [Traduit par la rtdaction]
Introduction Dolichols, synthesized in microsomes and stored in lysosomes, are a-saturated polyisoprenoid alcohols with a broad distribution among tissues, cells, a n d intracellular organelles; they are among the largest lipids i n the cell (Rip et al. 1985; Chojnacki a n d Dallner 1988). T h e main function of their phosphorylated form is to act as an essential carrier lipid in the biosynthesis of N-linked glycoproteins (Rip et al. 1985). Dolichols play also an important role in biological membranes ABBREVIATIONS: AGU, aspartylglucosaminuria; NCL, ceroid-lipofuscinosis; HPLC, high performance liquid chromatography. ' ~ u t h o rto whom all correspondence should be addressed. Printed in Canada / Im~rimCau Canada
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where both the free alcohols and their fatty acid esters may modify the structural and functional properties, such as fluidity, stability, permeability, and the fusion process (Lai and Schutzbach 1984; Valtersson et al. 1985). levels of brain in patients suffering from Alzheimer's disease have been reported by Wolfe et al. (Igg2, lgg59 lgg7) and Ng Ying Kin et 01- (l983)- Furthermore, it is well established that brain dolichols are increased in different types of NCLs (Ng Ying Kin et a/. 1983; Wolfe et al. 1983; Hall and Patrick 1987; Hall et al. 1989). Ng Ying Kin et al. (1982), as well as Wolfe et al. (1983), found raised urinary dolichols in NCLs, but this was not confirmed in two other studies (Bennett et al. 1985; Paton and Poulos 1987). The increased urinary excretion of dolichols has also been discovered in another storage disease, the HermanskyPudlak syndrome (Witkop et al. 1987). However, the clinical value of these findings remains open, since all the brain specimens were obtained postmortem and because of the controversy in the findings concerning urinary dolichols. Blood dolichol level appears to be normal in most diseases so far studied, as well as during pregnancy, and it does not undergo diurnal variation (Elmberger et al. 1988; Humaloja et al. 1991). Serum total dolichol concentration has been shown to be slightly elevated in alcoholics, exeeding the upper normal limit in 37% of the cases (Roine et al. 1989). Recently, we reported that patients suffering from two lysosomal storage diseases, AGU and mannosidosis, have almost twofold serum dolichol levels as compared with healthy controls (Salaspuro et al. 1990). Diseases or conditions with decreased blood dolichol have so far not been found. In the present study our aim was to investigate which of the two dolichol fractions, free alcohol or dolichyl fatty acid esters, is increased in serum of patients with AGU.Furthermore, we studied serum free and esterified dolichol concentrations in patients with various types of NCLs. Materials and methods Subjects The age of the six female and nine male patients with AGU ranged from 18 to 56 years (mean + SEM, 36.9 + 2.4). The diagnosis was based on the characteristic clinical picture and elevated urinary excretion of aspartylglucosamine. The group of NCL patients consisted of four female and six male patients (three with infantile, four with juvenile, and three with variant late infantile type). Their mean age was 12.6 + 2.0 years (range 6-28). The diagnosis was based in all cases on typical clinical and neuropathological findings. The mean age of the 23 healthy controls used in the NCL study was 13.0 + 1%years (range 4-18) and that of the 44 healthy con-trols used in the AGU study was 40.8 + 1.7 years (range 20-60). Methods After overnight fasting all blood samples were taken into vacuum tubes and centrifuged at 2000 x g for 10 min. The sera were kept frozen at -20°C until analyzed. Dolichols were analyzed on a Varian Vista 5500 HPLC equipped with a Varian 8085 autosampler and Varian Series 600 Data System (Varian Associates, Walnut Creek, Calif.). A reversed-phase Spherisorb S5 ODs2 column (250 x 4.6 mrn; Phase Separations Ltd. Queensferry, Clwyd, U.K.) and a gradient elution program running from 50% isopropanol plus 50% methanol to 80% isopropanol plus 20% methanol for 20 min were used. The UV detection was performed at 210 nm. As an internal standard, heneicosaprenol (Sigma Chemical Co., St. Louis, Mo.) was added to the serum. The quantification was
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based on the peak heights and the response factors of the dolichol homologues relative to heneicosaprenol. The serum levels of dolichols were expressed as the sum of the three homologues of 18, 19, and 20 isoprene units. The sample preparation for the total dolichols included liberation of dolichols from their fatty acid esters or protein complexes in a medium containing mL by alkaline hydrolysis at 980C for of serum and 5 mL of 2 M KOH in methanol. Free dolichols were then extracted with n-pentane and the extract was washed with 5% acetic acid and eva~oratedinto dryness. The residue was dissolved in 0.5 mL dichloromethane-methanol (2: 1, v/v) and purified on a bonded silica C8 extraction column (200 mg, Analytichem International, Harbor City, Calif.). Dolichols were eluted from the column with 2.5 mL ethanol-methanol-isopropanol ( 9 0 5 5 , by volume). The eluate was evaporated under nitrogen and the residue was dissolved in 40 pL of 72% isopropanol plus 28% methanol solution. This solution was then used for HPLC analyses. When free dolichols were quantitated, the drastic conditions needed for hydrolysis were avoided by carrying out the extraction with n-pentane immediately after adding the 2 M KOH solution into the serum at room temperature. After extraction the samples were prepared further as described above. Statistical calculations Statistical analysis was performed using the BMDP Statistical Software program version 1988 (VAX/VMS). Normality of variables was studied using values of skewness and kurtosis, and by W statistic. When necessary, logarithmic transformations were carried out to ensure that the distributions met the criteria for normality. Differences between patients and controls were analysed using Welch's t-test. All results are expressed as means + SEM.
Results The mean serum levels of both free (227 + 16 ng/mL; range, 117-345) and total dolichol(336 + 18 ng/mL; range, 214-457) were highly elevated in AGU patients when compared with healthy age-adjusted controls (Figs. 1 and 2). By contrast, the amounts of serum dolichyl fatty acid esters (calculated as the difference between serum total and free dolichol) were similar in the two groups (110 + 9 vs. 118 6 ng/mL). In patients with NCL, mean serum levels of both free (79 + 5 ng/rnL) pig. 1) and total dolichols (159 & 6 ng/rnL) (Fig. 2) were significantly (p < 0.01) lower than in agematched healthy controls. In this respect, there was no difference between the different forms of NCLs (data not shown). The percentage distribution of the dolichol homologues with 18, 19, or 20 isoprene units (23, 56, and 21% of the total, respectively) did not differ between the controls and the patients with either AGU or NCL.
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Discussion Dolichols serve as structural lipids of all biological membranes. Their subcellular distribution is broad. The mitochondrial inner membrane has very low concentrations, whereas in Golgi vesicles and lysosomes the dolichol content is exceptionally high. All tissues are capable of synthesizing dolichol, and endogenously produced dolichol is present in blood at low concentrations, mostly associated with the high-density lipoprotein fraction (Elmberger and Engfeldt 1985; Chojnacki and Dallner 1988). Studies on blood dolichols are complicated. Blood contains relatively high concentrations of various other lipids and, for example, the level of cholesterol is as much as
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JOKELAINEN ET AL.
FIG. 1. Serum free dolichol (mean + SEM; ng/mL) in (A) 44 healthy controls, (B) 15 patients with AGU, (C) 23 age-adjusted controls for NCL, and (D) 10 patients with NCL.
25 000 times higher than that of the dolichol (Elmberger et al. 1988). he strong association between dolichols and blood proteins requires effective extraction procedures. However, with the sample preparation scheme and with the consequent gradient elution program in the liquid chromatograph as described in the Materials and methods, it was possible to separate dolichols from other serum lipids and protein complexes. The blood levels of free as well as esterified dolichols are very constant under normal conditions (Elmberger et al. 1988). A moderate linear increase with age has been found in some (Elmberger et al. 1988; Salaspuro et al. 1990), but not in all studies (Yamada et al. 1985; Humaloja et al. 1991). Blood dolichol level appears to be normal in most diseases studied and during pregnancy (Elmberger et al. 1988; Humaloja et al. 1991), but mean serum total dolichol concentration was found to be slightly elevated in alcoholics (Roine et al. 1989). The only diseases so far described with exceptionally high serum total dolichol levels are the two lysosomal storage diseases, AGU and mannosidosis (Salaspuro et al. 1990). No diseases or conditions with decreased blood dolichol have so far been found. AGU is an autosomal recessive lysosomal storage disorder, an example of inherited diseases accumulated in the Finnish population. There have been more than 200 patients in Finland, whereas only 20 non-Finnish patients have been reported (Aula et al. 1982). The perinatal history and early psychomotor development of AGU patients is normal. The first signs of central nervous system involvement usually appear between 1 and 4 years of age. The early symptoms consist of delayed speech development, poor ability to concentrate, and motor clumsiness. Adult patients with AGU are severely mentally retarded (Aula et al. 1982). The basic cellular change in AGU is the presence of hypertrophied storage lysosomes, which have been found in all organs so far studied. The storage material is
FIG. 2. Serum total dolichol (mean + SEM; ng/mL) in (A) 44 healthy controls, (B) 15 patients with AGU, (C) 23 age-adjusted controls for NCL, and (D) 10 patients with NCL.
aspartylglucosamine excreted in large amounts into urine. In addition, elevated urinary concentrations of other glycoproteins have also been reported (Aula et al. 1982). Aspartylglucosamine cannot be detected in the serum. The basic biochemical defect in AGU is a deficient activity of aspartylglucosaminidase, an enzyme that hydrolyzes the linkage between asparagine and N-acetylglucosamine; this linkage is essential also in the dolichol-mediated N-glycosylation of glycoproteins. In the Finnish AGU population, the defect is caused by one major mutation (AGU-Fin), which covers 98% of the mutated alleles in AGU (Syvanen et al. 1991). It has been presumed that the altered amino acid sequence of the mutant enzyme can modify the secondary structure of the protein core, leading to disturbed posttranslational modification and lysosomal transport of the enzyme (Ikonen et al. 1991; Fisher and Aronson 1991). NCLs are among the most common progressive encephalopathies of childhood in Western countries (Santavuori 1988). They are classified into four main types: infantile, (INCL), late infantile (LINCL), juvenile (JNCL), and adult NCL or Kufs' disease. All types are characterized by severe psychomotor retardation, visual failure, seizures, and accumulation of storage substance in neural and extraneural cells (Zeman and Dyken 1969; Santavuori 1988). The most common morphological feature is the intraneuronal accumulation of autofluorescent material and the destruction of cortical neurons leading to brain athrophy. The properties of the storage material are similar to lipofuscin and ceroid (Wolfe et al. 1981), but although it is also found in most organs outside the brains, these deposits are usually small and not associated with tissue destruction (Haltia 1982).
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T h e dolichol content is always elevated in brain tissue from N C L patients (Ng Ying Kin et al. 1983; Wolfe et al. 1983; Hall a n d Patrick 1987; Hall et al. 1989). I t is also increased in t h e urine of most but not all patients, making it unfeasible a s a diagnostic test (Wolfe et al. 1983). Despite new biochemical a n d genetic findings, the primary pathogenetic mechanism of NCLs still remains uncertain (Gardiner et al. 1990; Jiirvela et al. 1991). I n the present study the serum content of free a n d total dolichol was significantly lower in all forms of N C L than in healthy age-adjusted controls. These results d o not rule out accumulation of dolichols in the brain, but a t least there is n o corresponding elevation in the serum concentrations. All our patients with NCL were severely retarded and handicapped, which could be thought t o explain the decreased serum dolichol levels. We have, however, shown earlier that blood dolichol is normal in retarded a n d motorically handicapped patients (Salaspuro et al. 1990). Since dolichols are stored i n lysosomes, elevated serum free dolichol in A G U m a y be another reflection o f disturbed lysosomal function. Moreover, we suggest that t h e altered three-dimensional structure of the mutant enzyme may interfere with the normal polypeptide glycosylation process, leading t o disturbed intracellular flow a n d recirculation process of dolichols, a n d finally t o increased blood-free dolichol levels. O n the other hand, NCLs are characterized by increased tissue accumulation of dolichol and pigments like ceroid and lipofuscin, the lysosomal origin of which has been suspected but not confirmed. T h e fact that serum dolichol levels are decreased in NCLs may speak against the primary lysosomal nature o f these conditions a n d could simply be a n indicator o f a primary defect in dolichol metabolism.
Acknowledgements T h e study was supported by the Finnish Foundation f o r Alcohol Studies and by the Rinnekoti Research Foundation. T h e authors thank Ms. Tuula Heiskanen a n d Ms. Tuula Moisio f o r their skillful technical assistance. We also thank Dr. Marita Vayrynen, Kolpene Rehabilitation Centre, Rovaniemi, f o r providing patient material. Aula, P., Autio, S., Raivio, K.O., Rapola, J. 1982. Aspartylglucosaminuria. In Genetic errors of glycoprotein metabolism. Edited by P. Durand and J.S. O'Brien. Springer-Verlag, New York. pp. 123-151. Bennett, M.J., Mathers, N.J., Hemming, F.W., et al. 1985. Urinary sediment dolichol excretion in patients with Batten disease and other neurodegenerative and storage disorders. Pediatr. Res. 19(2): 213-216. Chojnacki, T., and Dallner, G. 1988. The biological role of dolichol. Biochem. J. 251: 1-9. Elmberger, P.G., and Engfeldt, P. 1985. Distribution of dolichol in human and rabbit blood. Acta Chem. Scand. 39: 323-325. Elmberger, P.G., Engfeldt, P., and Dallner, G. 1988. Presence of dolichol and its derivates in human blood. J. Lipid Res. 29: 1651-1662.
Fisher, K. J., and Aronson, N.N., Jr. 1991. Characterization of the mutation responsible for aspartylglucosaminuria in three Finnish patients. J. Biol. Chem 18: 12 105-12 112. Gardiner, R.M., Sandford, A., Deadman, M., et al. 1990. Batten disease (Spielmeyer-Vogt; juvenile onset neuronal ceroid lipofuscinosis) maps to human chromosome 16. Genomics, 8: 170-173.
Hall, N.A., and Patrick, A.D.
1987. Accumulation of
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phosphorylated dolichol in several tissues in ceroid-lipofuscinosis (Batten disease). Clin. Chim. Acta, 170(2-3): 323-330. Hall, N.A., Haltia, M., and Patrick, A.D. 1989. High-mannose dolichol-linked oligosaccharides in infantile ceroid-lipofuscinosis. Biochem. Soc. Trans. 17: 1032-1033. Haltia, M. 1982. Infantile neuronal ceroid-lipofuscinosis: Neuropathological aspects. In Ceroid-lipofuscinosis (Batten's disease). Edited by D. Armstrong, N. Koppang, and A. Rider. Elsevier Biomedical Press, Amsterdam. pp. 105-115. Humaloja, K., Roine, R.P., Salmela, K., et al. 1991. Serum dolichols in different clinical conditions. Scand. J. Clin. Lab. Invest. 51: 705-709. Ikonen, E., Baumann, M., Gron, K., et al. 1991. Aspartylglucosaminuria: cDNA encoding human aspartylglucosaminidase and the missense mutation causing the disease. EMBO J. 10: 51-58.
Jkvela, I., Schleutker, J., Haataja, L., et al. 1991. Infantile form of neuronal ceroid lipofuscinosis (CLN1) maps to the short arm of chromosome 1. Genomics, 9: 170-173. Lai, C.-S., and Schutzbach, J.S. 1984. Dolichol induces membrane leakage of liposomes composed of phosphatidylethanolarnineand phosphatidylcholine. FEBS Lett. 169: 279-282. Ng Ying Kin, N.M.K., and Wolfe, L.S. 1982. Presence of abnormal amounts of dolichols in the urinary sediment of Batten disease patients. Pediatr. Res. 16: 530-532. Ng Ying Kin, N.M.K., Palo, J., Haltia, M., and Wolfe, L.S. 1983. High levels of brain dolichols in neuronal ceroid-lipofuscinosis and senescence. J. Neurochem. 5: 1465-1473. Paton, B.C., and Poulos, A. 1987. Normal dolichol concentration in urine sediments from four patients with neuronal ceroid lipofuscinosis (Batten's disease). J. Inherited. Metab. Dis. 10: 28-32.
Riv, J.W., Rupar, C.A., Ravi, K., and Carroll, K.K. 1985. Distribution, - metabolism and function of dolichol and polyprenols. Prog. Lipid Res. 24: 269-309. Roine, R.P., Nykanen, I., Ylikahri, R., et al. 1989. Effect of alcohol on blood dolichol concentration. Alcoholism, 13: 519-522.
Salaspuro, M., Salmela, K., Humaloja, K., et al. 1990. Elevated levels of serum dolichol in aspartylglucosaminuria. Life Sci. 47: 627-632.
Santavuori, P. 1988. Neuronal ceroid lipofuscinoses in childhood. Brain Dev. 10: 80-83. Syvanen, A-C., Ikonen, E., Manninen, T., et al. 1992. Convenient and quantitative determination of frequency of a mutant allele using solid-phase mini-sequencing: application to aspartylglucosaminuria in Finland. Genomics, 12: 590-595. Valtersson, C.G., vanDuijn, A.J., Verkleij, T., et al. 1985. The influence of dolichol, dolichol esters, and dolichyl phosphate on phospholipid polymorphism and fluidity in model membranes. J. Biol. Chem. 260: 2742-2751. Witkop, C.J., Wolfe, L.S., Cal, S.X., et a[. 1987. Elevated urinary dolichol excretion in the Hermansky-Pudlak syndrome: indicator of lysosomal dysfunction. Am. J. Med. 82: 463. Wolfe, L.S., Ng Ying Kin, N.M.K., and Baker, R.R. 1981. Batten disease and related disorders: new findings on the chemistry of the storage material. In Lysosomes and lysosomal diseases. Edited by J.W. Callahan and J.A. Lowden. Raven Press, New York. pp 315-330. Wolfe, L.S., Ng Ying Kin, N.M.K., Palo, J., and Haltia, M. 1982. Raised levels of cerebral cortex dolichols in Alzheimer's disease. Lancet, 11: 99. Wolfe, L.S., Ng Ying Kin, N.M.K., et al. 1983. Dolichols in brain and urinary sediment in neuronal ceroid lipofuscinosis. Neurology, 33: 103-106. Wolfe, L.S., Palo, J., Bergeron, C., Kotila, M., and Varonen, S. 1985. Dolichols are elevated in brain tissue from Alzheimer's disease but not in urinary sediment from Alzheimer's disease and Down's syndrome. Neurochem. Pathol. 3: 213-221.
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Wolfe, L.S., IG,G., and Witkop, C.J. 1987. Dolichols, lysosomal membrane turnover and relationships to the accumulation of ceroid and lipofuscin in inherited diseases, Alzheimer's disease and aging. Chem. Scr. 27: 79-84. Yamada, K., Yokohama, H., Abe, S., et al. 1985. Highperformance liquid chromatographic method for the
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determination of dolichols in tissues and plasma. Anal. Biochem. 150(1): 26-31. Zeman, W., and Dyken, P. 1969. Neuronal ceroid-lipofuscinosis (Batten's disease): relationship to amaurotic familial idiocy? Pediatrics, 44: 570-583.
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MARIAARVIO Paajarvi Rehabilitation Center, Lammi, Finland IRMA JARVELA National Public Health Institute, Helsinki, Finland IRMANYKANEN Research Laboratories of the Finnish State Alcohol Company (Alko, Ltd), Helsinki, Finland
JORMA PALO Department of Neurology, University of Helsinki, Helsinki, Finland AND
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MIKKOSALASPURO Research and Treatment Unit of Alcohol Diseases, University of Helsinki, Tukholmankatu8 F, 00290 Helsinki, Finland Received June 27. 1991 JOKELAINEN, K., SALMELA, K. S., HUMALOJA, K., ROINE,R., AUTIO,S., ARVIO,M., JARVELA, I., NYKANEN,I., PALO,J., and SALASPURO, M. 1992. Blood dolichol in lysosomal diseases. Biochem. Cell Biol. 70: 481-485. Highly elevated serum total dolichol (free dolichol + dolichyl ester) concentrations have recently been found in two lysosomal storage diseases, aspartylglucosaminuria (AGU) and mannosidosis. The present study demonstrates that the increase of serum dolichol in AGU patients is caused by an increase of serum free dolichol. In 15 patients the mean serum level of free dolichol (227 k 16 ng/mL) was 1.9 times higher ( p < 0.001) than that in healthy controls (120 k 6 ng/mL), while the amounts of dolichol fatty acid esters were similar in the patients and controls (110 9 vs. 118 + 6 ng/mL). In contrast, 10 patients with neuronal ceroid-lipofuscinosis (NCL) (three with infantile, four with juvenile, and three with variant late infantile NCL) had significantly ( p < 0.01) lower mean serum levels of both free (79 k 5 ng/mL) and total (159 6 ng/mL) dolichol than age-adjusted healthy controls (free, 100 k 6 ng/mL; total, 206 k 14 ng/mL). Decreased blood dolichol has not been reported earlier for any other disease. We conclude that the increased serum free dolichol in AGU reflects disturbed lysosomal function and that the decreased free and esterified dolichols in NCLs speak against their presumed primary lysosomal nature. Key words: serum dolichol, aspartylglucosaminuria, neuronal ceroid-lipofuscinosis, lysosomal function, lysosomal storage disease.
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JOKELAINEN, K., SALMELA. K. S., HUMALOJA, K., ROINE,R., AUTIO,S., ARVIO,M., JARVELA, I., NYKANEN,I., PALO,J., et SALASPURO, M. 1992. Blood dolichol in lysosomal diseases. Biochem. Cell Biol. 70 : 481-485. Des concentrations fortement tlevtes de dolichol sbique total (dolichol libre + dolichyl ester) ont rtcemment kt6 trouvtes dans deux maladies d'emmagasinage lysosomique: l'aspartylglucosaminurie (AGU) et la mannosidose. Nous demontrons ici que l'augmentation du dolichol strique chez les malades atteints d'AGU est causte par un accroissement du dolichol strique libre. Chez 15 malades, le taux strique moyen du dolichol libre (227 k 16 ng/mL) est 1,9 fois plus tlevt ( p < 0,001) que celui des contrdles en santt (120 + 6 ng/mL) alors que les quantitts des dolichols acides gras esters sont identiques chez les malades et les contrdles (110 k 9 vs. 118 + 6 ng/mL). En revanche, 10 patients atteints de ctroide-lipofuscinose neuronale (NCL) (trois avec NCL infantile, quatre avec NCL juvenile et trois avec une variante infantile tardive) ont des t a w striques moyens significativement ( p < 0,Ol) plus bas de dolichol libre (79 + 5 ng/mL) et de dolichol total (159 k 6 ng/mL) que c e w des contrdles en santt de m&me Ige (libre, 100 k 6 ng/mL; total, 206 k 14 ng/mL). Des diminutions de dolichol sanguin n'ont t t t rapporttes pour aucune autre maladie. Nous concluons que l'augmentation du colichol strique libre dans I'AGU refltte une fonction lysosomique anormale et que la diminution des dolichols libres et esterifits dans la NCL parlent contre leur prtsumte nature lysosomique primaire. Mots elks : dolichol strique, aspartylglucosaminurie, ctro'ide-lipofuscinoseneuronale, fonction lysosomique, maladie d'emmagasinage lysosomique. [Traduit par la rtdaction]
Introduction Dolichols, synthesized in microsomes and stored in lysosomes, are a-saturated polyisoprenoid alcohols with a broad distribution among tissues, cells, a n d intracellular organelles; they are among the largest lipids i n the cell (Rip et al. 1985; Chojnacki a n d Dallner 1988). T h e main function of their phosphorylated form is to act as an essential carrier lipid in the biosynthesis of N-linked glycoproteins (Rip et al. 1985). Dolichols play also an important role in biological membranes ABBREVIATIONS: AGU, aspartylglucosaminuria; NCL, ceroid-lipofuscinosis; HPLC, high performance liquid chromatography. ' ~ u t h o rto whom all correspondence should be addressed. Printed in Canada / Im~rimCau Canada
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where both the free alcohols and their fatty acid esters may modify the structural and functional properties, such as fluidity, stability, permeability, and the fusion process (Lai and Schutzbach 1984; Valtersson et al. 1985). levels of brain in patients suffering from Alzheimer's disease have been reported by Wolfe et al. (Igg2, lgg59 lgg7) and Ng Ying Kin et 01- (l983)- Furthermore, it is well established that brain dolichols are increased in different types of NCLs (Ng Ying Kin et a/. 1983; Wolfe et al. 1983; Hall and Patrick 1987; Hall et al. 1989). Ng Ying Kin et al. (1982), as well as Wolfe et al. (1983), found raised urinary dolichols in NCLs, but this was not confirmed in two other studies (Bennett et al. 1985; Paton and Poulos 1987). The increased urinary excretion of dolichols has also been discovered in another storage disease, the HermanskyPudlak syndrome (Witkop et al. 1987). However, the clinical value of these findings remains open, since all the brain specimens were obtained postmortem and because of the controversy in the findings concerning urinary dolichols. Blood dolichol level appears to be normal in most diseases so far studied, as well as during pregnancy, and it does not undergo diurnal variation (Elmberger et al. 1988; Humaloja et al. 1991). Serum total dolichol concentration has been shown to be slightly elevated in alcoholics, exeeding the upper normal limit in 37% of the cases (Roine et al. 1989). Recently, we reported that patients suffering from two lysosomal storage diseases, AGU and mannosidosis, have almost twofold serum dolichol levels as compared with healthy controls (Salaspuro et al. 1990). Diseases or conditions with decreased blood dolichol have so far not been found. In the present study our aim was to investigate which of the two dolichol fractions, free alcohol or dolichyl fatty acid esters, is increased in serum of patients with AGU.Furthermore, we studied serum free and esterified dolichol concentrations in patients with various types of NCLs. Materials and methods Subjects The age of the six female and nine male patients with AGU ranged from 18 to 56 years (mean + SEM, 36.9 + 2.4). The diagnosis was based on the characteristic clinical picture and elevated urinary excretion of aspartylglucosamine. The group of NCL patients consisted of four female and six male patients (three with infantile, four with juvenile, and three with variant late infantile type). Their mean age was 12.6 + 2.0 years (range 6-28). The diagnosis was based in all cases on typical clinical and neuropathological findings. The mean age of the 23 healthy controls used in the NCL study was 13.0 + 1%years (range 4-18) and that of the 44 healthy con-trols used in the AGU study was 40.8 + 1.7 years (range 20-60). Methods After overnight fasting all blood samples were taken into vacuum tubes and centrifuged at 2000 x g for 10 min. The sera were kept frozen at -20°C until analyzed. Dolichols were analyzed on a Varian Vista 5500 HPLC equipped with a Varian 8085 autosampler and Varian Series 600 Data System (Varian Associates, Walnut Creek, Calif.). A reversed-phase Spherisorb S5 ODs2 column (250 x 4.6 mrn; Phase Separations Ltd. Queensferry, Clwyd, U.K.) and a gradient elution program running from 50% isopropanol plus 50% methanol to 80% isopropanol plus 20% methanol for 20 min were used. The UV detection was performed at 210 nm. As an internal standard, heneicosaprenol (Sigma Chemical Co., St. Louis, Mo.) was added to the serum. The quantification was
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based on the peak heights and the response factors of the dolichol homologues relative to heneicosaprenol. The serum levels of dolichols were expressed as the sum of the three homologues of 18, 19, and 20 isoprene units. The sample preparation for the total dolichols included liberation of dolichols from their fatty acid esters or protein complexes in a medium containing mL by alkaline hydrolysis at 980C for of serum and 5 mL of 2 M KOH in methanol. Free dolichols were then extracted with n-pentane and the extract was washed with 5% acetic acid and eva~oratedinto dryness. The residue was dissolved in 0.5 mL dichloromethane-methanol (2: 1, v/v) and purified on a bonded silica C8 extraction column (200 mg, Analytichem International, Harbor City, Calif.). Dolichols were eluted from the column with 2.5 mL ethanol-methanol-isopropanol ( 9 0 5 5 , by volume). The eluate was evaporated under nitrogen and the residue was dissolved in 40 pL of 72% isopropanol plus 28% methanol solution. This solution was then used for HPLC analyses. When free dolichols were quantitated, the drastic conditions needed for hydrolysis were avoided by carrying out the extraction with n-pentane immediately after adding the 2 M KOH solution into the serum at room temperature. After extraction the samples were prepared further as described above. Statistical calculations Statistical analysis was performed using the BMDP Statistical Software program version 1988 (VAX/VMS). Normality of variables was studied using values of skewness and kurtosis, and by W statistic. When necessary, logarithmic transformations were carried out to ensure that the distributions met the criteria for normality. Differences between patients and controls were analysed using Welch's t-test. All results are expressed as means + SEM.
Results The mean serum levels of both free (227 + 16 ng/mL; range, 117-345) and total dolichol(336 + 18 ng/mL; range, 214-457) were highly elevated in AGU patients when compared with healthy age-adjusted controls (Figs. 1 and 2). By contrast, the amounts of serum dolichyl fatty acid esters (calculated as the difference between serum total and free dolichol) were similar in the two groups (110 + 9 vs. 118 6 ng/mL). In patients with NCL, mean serum levels of both free (79 + 5 ng/rnL) pig. 1) and total dolichols (159 & 6 ng/rnL) (Fig. 2) were significantly (p < 0.01) lower than in agematched healthy controls. In this respect, there was no difference between the different forms of NCLs (data not shown). The percentage distribution of the dolichol homologues with 18, 19, or 20 isoprene units (23, 56, and 21% of the total, respectively) did not differ between the controls and the patients with either AGU or NCL.
*
Discussion Dolichols serve as structural lipids of all biological membranes. Their subcellular distribution is broad. The mitochondrial inner membrane has very low concentrations, whereas in Golgi vesicles and lysosomes the dolichol content is exceptionally high. All tissues are capable of synthesizing dolichol, and endogenously produced dolichol is present in blood at low concentrations, mostly associated with the high-density lipoprotein fraction (Elmberger and Engfeldt 1985; Chojnacki and Dallner 1988). Studies on blood dolichols are complicated. Blood contains relatively high concentrations of various other lipids and, for example, the level of cholesterol is as much as
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JOKELAINEN ET AL.
FIG. 1. Serum free dolichol (mean + SEM; ng/mL) in (A) 44 healthy controls, (B) 15 patients with AGU, (C) 23 age-adjusted controls for NCL, and (D) 10 patients with NCL.
25 000 times higher than that of the dolichol (Elmberger et al. 1988). he strong association between dolichols and blood proteins requires effective extraction procedures. However, with the sample preparation scheme and with the consequent gradient elution program in the liquid chromatograph as described in the Materials and methods, it was possible to separate dolichols from other serum lipids and protein complexes. The blood levels of free as well as esterified dolichols are very constant under normal conditions (Elmberger et al. 1988). A moderate linear increase with age has been found in some (Elmberger et al. 1988; Salaspuro et al. 1990), but not in all studies (Yamada et al. 1985; Humaloja et al. 1991). Blood dolichol level appears to be normal in most diseases studied and during pregnancy (Elmberger et al. 1988; Humaloja et al. 1991), but mean serum total dolichol concentration was found to be slightly elevated in alcoholics (Roine et al. 1989). The only diseases so far described with exceptionally high serum total dolichol levels are the two lysosomal storage diseases, AGU and mannosidosis (Salaspuro et al. 1990). No diseases or conditions with decreased blood dolichol have so far been found. AGU is an autosomal recessive lysosomal storage disorder, an example of inherited diseases accumulated in the Finnish population. There have been more than 200 patients in Finland, whereas only 20 non-Finnish patients have been reported (Aula et al. 1982). The perinatal history and early psychomotor development of AGU patients is normal. The first signs of central nervous system involvement usually appear between 1 and 4 years of age. The early symptoms consist of delayed speech development, poor ability to concentrate, and motor clumsiness. Adult patients with AGU are severely mentally retarded (Aula et al. 1982). The basic cellular change in AGU is the presence of hypertrophied storage lysosomes, which have been found in all organs so far studied. The storage material is
FIG. 2. Serum total dolichol (mean + SEM; ng/mL) in (A) 44 healthy controls, (B) 15 patients with AGU, (C) 23 age-adjusted controls for NCL, and (D) 10 patients with NCL.
aspartylglucosamine excreted in large amounts into urine. In addition, elevated urinary concentrations of other glycoproteins have also been reported (Aula et al. 1982). Aspartylglucosamine cannot be detected in the serum. The basic biochemical defect in AGU is a deficient activity of aspartylglucosaminidase, an enzyme that hydrolyzes the linkage between asparagine and N-acetylglucosamine; this linkage is essential also in the dolichol-mediated N-glycosylation of glycoproteins. In the Finnish AGU population, the defect is caused by one major mutation (AGU-Fin), which covers 98% of the mutated alleles in AGU (Syvanen et al. 1991). It has been presumed that the altered amino acid sequence of the mutant enzyme can modify the secondary structure of the protein core, leading to disturbed posttranslational modification and lysosomal transport of the enzyme (Ikonen et al. 1991; Fisher and Aronson 1991). NCLs are among the most common progressive encephalopathies of childhood in Western countries (Santavuori 1988). They are classified into four main types: infantile, (INCL), late infantile (LINCL), juvenile (JNCL), and adult NCL or Kufs' disease. All types are characterized by severe psychomotor retardation, visual failure, seizures, and accumulation of storage substance in neural and extraneural cells (Zeman and Dyken 1969; Santavuori 1988). The most common morphological feature is the intraneuronal accumulation of autofluorescent material and the destruction of cortical neurons leading to brain athrophy. The properties of the storage material are similar to lipofuscin and ceroid (Wolfe et al. 1981), but although it is also found in most organs outside the brains, these deposits are usually small and not associated with tissue destruction (Haltia 1982).
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T h e dolichol content is always elevated in brain tissue from N C L patients (Ng Ying Kin et al. 1983; Wolfe et al. 1983; Hall a n d Patrick 1987; Hall et al. 1989). I t is also increased in t h e urine of most but not all patients, making it unfeasible a s a diagnostic test (Wolfe et al. 1983). Despite new biochemical a n d genetic findings, the primary pathogenetic mechanism of NCLs still remains uncertain (Gardiner et al. 1990; Jiirvela et al. 1991). I n the present study the serum content of free a n d total dolichol was significantly lower in all forms of N C L than in healthy age-adjusted controls. These results d o not rule out accumulation of dolichols in the brain, but a t least there is n o corresponding elevation in the serum concentrations. All our patients with NCL were severely retarded and handicapped, which could be thought t o explain the decreased serum dolichol levels. We have, however, shown earlier that blood dolichol is normal in retarded a n d motorically handicapped patients (Salaspuro et al. 1990). Since dolichols are stored i n lysosomes, elevated serum free dolichol in A G U m a y be another reflection o f disturbed lysosomal function. Moreover, we suggest that t h e altered three-dimensional structure of the mutant enzyme may interfere with the normal polypeptide glycosylation process, leading t o disturbed intracellular flow a n d recirculation process of dolichols, a n d finally t o increased blood-free dolichol levels. O n the other hand, NCLs are characterized by increased tissue accumulation of dolichol and pigments like ceroid and lipofuscin, the lysosomal origin of which has been suspected but not confirmed. T h e fact that serum dolichol levels are decreased in NCLs may speak against the primary lysosomal nature o f these conditions a n d could simply be a n indicator o f a primary defect in dolichol metabolism.
Acknowledgements T h e study was supported by the Finnish Foundation f o r Alcohol Studies and by the Rinnekoti Research Foundation. T h e authors thank Ms. Tuula Heiskanen a n d Ms. Tuula Moisio f o r their skillful technical assistance. We also thank Dr. Marita Vayrynen, Kolpene Rehabilitation Centre, Rovaniemi, f o r providing patient material. Aula, P., Autio, S., Raivio, K.O., Rapola, J. 1982. Aspartylglucosaminuria. In Genetic errors of glycoprotein metabolism. Edited by P. Durand and J.S. O'Brien. Springer-Verlag, New York. pp. 123-151. Bennett, M.J., Mathers, N.J., Hemming, F.W., et al. 1985. Urinary sediment dolichol excretion in patients with Batten disease and other neurodegenerative and storage disorders. Pediatr. Res. 19(2): 213-216. Chojnacki, T., and Dallner, G. 1988. The biological role of dolichol. Biochem. J. 251: 1-9. Elmberger, P.G., and Engfeldt, P. 1985. Distribution of dolichol in human and rabbit blood. Acta Chem. Scand. 39: 323-325. Elmberger, P.G., Engfeldt, P., and Dallner, G. 1988. Presence of dolichol and its derivates in human blood. J. Lipid Res. 29: 1651-1662.
Fisher, K. J., and Aronson, N.N., Jr. 1991. Characterization of the mutation responsible for aspartylglucosaminuria in three Finnish patients. J. Biol. Chem 18: 12 105-12 112. Gardiner, R.M., Sandford, A., Deadman, M., et al. 1990. Batten disease (Spielmeyer-Vogt; juvenile onset neuronal ceroid lipofuscinosis) maps to human chromosome 16. Genomics, 8: 170-173.
Hall, N.A., and Patrick, A.D.
1987. Accumulation of
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phosphorylated dolichol in several tissues in ceroid-lipofuscinosis (Batten disease). Clin. Chim. Acta, 170(2-3): 323-330. Hall, N.A., Haltia, M., and Patrick, A.D. 1989. High-mannose dolichol-linked oligosaccharides in infantile ceroid-lipofuscinosis. Biochem. Soc. Trans. 17: 1032-1033. Haltia, M. 1982. Infantile neuronal ceroid-lipofuscinosis: Neuropathological aspects. In Ceroid-lipofuscinosis (Batten's disease). Edited by D. Armstrong, N. Koppang, and A. Rider. Elsevier Biomedical Press, Amsterdam. pp. 105-115. Humaloja, K., Roine, R.P., Salmela, K., et al. 1991. Serum dolichols in different clinical conditions. Scand. J. Clin. Lab. Invest. 51: 705-709. Ikonen, E., Baumann, M., Gron, K., et al. 1991. Aspartylglucosaminuria: cDNA encoding human aspartylglucosaminidase and the missense mutation causing the disease. EMBO J. 10: 51-58.
Jkvela, I., Schleutker, J., Haataja, L., et al. 1991. Infantile form of neuronal ceroid lipofuscinosis (CLN1) maps to the short arm of chromosome 1. Genomics, 9: 170-173. Lai, C.-S., and Schutzbach, J.S. 1984. Dolichol induces membrane leakage of liposomes composed of phosphatidylethanolarnineand phosphatidylcholine. FEBS Lett. 169: 279-282. Ng Ying Kin, N.M.K., and Wolfe, L.S. 1982. Presence of abnormal amounts of dolichols in the urinary sediment of Batten disease patients. Pediatr. Res. 16: 530-532. Ng Ying Kin, N.M.K., Palo, J., Haltia, M., and Wolfe, L.S. 1983. High levels of brain dolichols in neuronal ceroid-lipofuscinosis and senescence. J. Neurochem. 5: 1465-1473. Paton, B.C., and Poulos, A. 1987. Normal dolichol concentration in urine sediments from four patients with neuronal ceroid lipofuscinosis (Batten's disease). J. Inherited. Metab. Dis. 10: 28-32.
Riv, J.W., Rupar, C.A., Ravi, K., and Carroll, K.K. 1985. Distribution, - metabolism and function of dolichol and polyprenols. Prog. Lipid Res. 24: 269-309. Roine, R.P., Nykanen, I., Ylikahri, R., et al. 1989. Effect of alcohol on blood dolichol concentration. Alcoholism, 13: 519-522.
Salaspuro, M., Salmela, K., Humaloja, K., et al. 1990. Elevated levels of serum dolichol in aspartylglucosaminuria. Life Sci. 47: 627-632.
Santavuori, P. 1988. Neuronal ceroid lipofuscinoses in childhood. Brain Dev. 10: 80-83. Syvanen, A-C., Ikonen, E., Manninen, T., et al. 1992. Convenient and quantitative determination of frequency of a mutant allele using solid-phase mini-sequencing: application to aspartylglucosaminuria in Finland. Genomics, 12: 590-595. Valtersson, C.G., vanDuijn, A.J., Verkleij, T., et al. 1985. The influence of dolichol, dolichol esters, and dolichyl phosphate on phospholipid polymorphism and fluidity in model membranes. J. Biol. Chem. 260: 2742-2751. Witkop, C.J., Wolfe, L.S., Cal, S.X., et a[. 1987. Elevated urinary dolichol excretion in the Hermansky-Pudlak syndrome: indicator of lysosomal dysfunction. Am. J. Med. 82: 463. Wolfe, L.S., Ng Ying Kin, N.M.K., and Baker, R.R. 1981. Batten disease and related disorders: new findings on the chemistry of the storage material. In Lysosomes and lysosomal diseases. Edited by J.W. Callahan and J.A. Lowden. Raven Press, New York. pp 315-330. Wolfe, L.S., Ng Ying Kin, N.M.K., Palo, J., and Haltia, M. 1982. Raised levels of cerebral cortex dolichols in Alzheimer's disease. Lancet, 11: 99. Wolfe, L.S., Ng Ying Kin, N.M.K., et al. 1983. Dolichols in brain and urinary sediment in neuronal ceroid lipofuscinosis. Neurology, 33: 103-106. Wolfe, L.S., Palo, J., Bergeron, C., Kotila, M., and Varonen, S. 1985. Dolichols are elevated in brain tissue from Alzheimer's disease but not in urinary sediment from Alzheimer's disease and Down's syndrome. Neurochem. Pathol. 3: 213-221.
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Wolfe, L.S., IG,G., and Witkop, C.J. 1987. Dolichols, lysosomal membrane turnover and relationships to the accumulation of ceroid and lipofuscin in inherited diseases, Alzheimer's disease and aging. Chem. Scr. 27: 79-84. Yamada, K., Yokohama, H., Abe, S., et al. 1985. Highperformance liquid chromatographic method for the
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determination of dolichols in tissues and plasma. Anal. Biochem. 150(1): 26-31. Zeman, W., and Dyken, P. 1969. Neuronal ceroid-lipofuscinosis (Batten's disease): relationship to amaurotic familial idiocy? Pediatrics, 44: 570-583.
