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518
lipofuscinosis (Batten's disease). J. Inherited Metab. Dis. 7: 112-1 16.
Paton, B., 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:
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28-32.
Pullarkat, R., Kim, K., Sklower, S., and Patel, V. 1988. Oligosacchary1 diphosphodolichols in the ceroid-lipofuscinoses. Am. J. Med. Genet. Suppl. 5: 243-251. Ronin, C., and Bouchillow, S. 1978. Cell-free labeling in thyroid rough microsomes of lipid-linked and protein-linked oligosaccharides. Biochim. Biophys. Acta, 539: 470-480. Sakakihara, Y., and Kamoshita, S. 1991. Partition of free dolichol into urinary sediments and supernatant. In Dolichol and other lipids related to glycoconjugate metabolism. Satellite meeting to the 11th International Symposium on Glycoconjugates, Kimberley, Ont. Abstr. 25. Salaspuro, M., and Korri, U.M. 1987. Blood acetate and urinary dolichols-new markers of heavy drinking and alcoholism. In Genetics and Alcoholism. Alan R. Liss Inc., New York. pp. 231-240. Svennerholm, L., Hagberg, B., Haltia, M., et al. 1975. Poly-
unsaturated fatty acid lipidosis. 11. Lipid biochemical studies. Acta Paediatr. Scand. 64: 489-496. Van Dessel, G., Lagrou, A., Hilderson, H.J.J., et al. 1979. Isolation and identification of polyprenols from bovine thyroid gland. Biochim. Biophys. Acta, 573: 296-300. Voets, R., Lagrou, A., Hilderson, H.J.J., et al. 1979. RNA synthesis in isolated nuclei and nucleoli from bovine thyroid. HoppeSeyler's Z. Physiol. Chem. 360: 1271-1283. Welply, J., Shenbagamurthi, P., Lennarz, W., and Naider, F. 1983. Substrate recognition by oligosaccharyltransferase. J. Biol. Chem. 258: 11 856 - 11 863. Wolfe, L., Ng, Y., Palo, J., and Haltia, M. 1983. Dolichols in brain and urinary sediment in neuronal ceroid lipofuscinosis. Neurology, 33: 103-106. Wolfe, L., Palo, J., Santavuori, P., et al. 1986. Urinary sediment dolichols in the diagnosis of neuronal ceroid-lipofuscinosis. Ann. Neurol. 19: 270-274. Wolfe, L., Gauthier, S., Haltia, M., and Palo, J. 1988. Dolichol and dolichylphosphate in the neuronal ceroid-lipofuscinosis and other diseases. Am. J. Med. Genet. Suppl. 5: 233-242. Zeman, W. 1974. Studies in the neuronal ceroid-lipofuscinoses. J. Neuropathol. Exp. Neurol. 33: 1-12.
Partition of free dolichol in human urine YOICHI SAKAKIHARA AND SHIGEHIKO KAMOSHITA Department of Pediatrics, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-Ku Tokyo, Japan Received September 3, 1991 SAKAKIHARA, Y., and KAMOSHITA,S. 1992. Partition of free dolichol in human urine. Biochem. Cell Biol. 70: 518-521.
We have demonstrated that dolichol is present in the urinary supernatant. Most of the dolichol present in the supernatant seems to be associated with cellular debris or membrane fragments. The amount of sediment in healthy subjects correlate well with the volume of urine. Although it is illogical to express urinary dolichol relative to urine volume, a good correlation between the amount of sediment and urine volume has made its use justifiable. Because of the presence of a substantial amount of dolichol in the supernatant, it seems better to use uncentrifuged whole urine as the sample for measurement of dolichol. Key words: dolichol, urine, sediment, supernatant. SAKAKIHARA, Y., et KAMOSHITA,S. 1992. Partition of free dolichol in human urine. Biochem. Cell Biol. 70 : 518-521.
Nous avons dtmontrt la prtsence du dolichol dans le surnageant urinaire. La plus grande partie du dolichol prtsent dans le surnageant semble associt B des dtbris cellulaires ou des fragments membranaires. Chez les sujets en santt, la quantitt de ce stdiment correspond ttroitement au volume de l'urine. I1 est peut-&treillogique d'exprimer le dolichol urinaire par rapport au volume de I'urine, mais I'ttroite relation entre la quantitk de sediment et le volume d'urine justifie son emploi. fitant donnt la prhence d'une quantitt substantiellede dolichol dans le surnageant, il semble prtftrable d'utiliser I'urine totale non centrifugke comme tchantillon pour la mesure du dolichol. Mots clks : dolichol, urine, ddiment, surnageant. [Traduit par la redaction]
Introduction Dolicol and its derivatives are lipid intermediates participating in the synthesis of N-linked glycoprotein. Its clinical implication was not recognized until Wolfe reported elevated levels of free dolichol in the brains of patients with Alzheimer's disease (Wolfe et al. 1982). Since then, dolichol accumulation has been reported in the brains of patients with several neurodegenerative disorders (Ng Yink Kin and Wolfe 1982; Sakakihara et al. 1987), in the urine of chronic alcoholics and patients with ceroid-lipofuscinosis (Ng Ying Kin and Wolfe 1982; Wolfe et al. 1983, Pullarkat et al. 1984; Wolfe et al. 1986), and in the serum from patients with aspartylglucosarninuria (Salaspuro et al. 1990). ABBREVIATIONS: PBS, phosphate buffered saline; M,, relative mass. Printed in Canada / Im~rimbau Canada
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Although the mechanism of dolichol accumulation has not been elucidated, the measurement of dolichol in these samples is clinically useful in making diagnosis and in evaluating the condition of patients, as in the case with chronic alcoholism (Roine et al. 19876). Since urinary dolichol measurement is noninvasive, some authors advocate its use in the treatment program for chronic alcoholics (Roine et al. 1987a; Salaspuro et al. 1987). However, there are several problems in urinary dolichol measurement. The first problem stems from the differences in urine preparation by various investigators. Some investigators used urinary sediment (Wolfe et al. 1983, 1986; Bennett et al. 1985; Paton and Poulos 1987), while others measured dolichol in uncentrifuged whole urine (Turpeinen 1986; Roine et al. 1987~).The second problem is related to the mode of expressing urinary dolichol. In the previous studies, dolichol in urine has been expressed as micrograms per litre of urine (Wolfe et al. 1986; Paton and Poulos 1987), micrograms per gram or millimole of creatinine (Bennett et al. 1985; Wolfe et al. 1986; Paton and Poulos 1987; Roine et al. 1987a, 1987b), and micrograms per microgram of lipid (Ng Ying Kin and Wolfe 1982; Paton and Poulos 1987). In the present study, we have examined the problems in urinary dolichol measurement as described above. First, we have explored the methods of expressing dolichol levels in both urinary sediment and supernatant. Second, we have asked whether dolichol is present in the urinary supernatant. To this end, we have analyzed the 24-h urine samples collected from 16 healthy male volunteers. Materials and methods Urine collection and preparation Twenty-four-hour urine samples were collected from 16 healthy adult male volunteers. None of the volunteers had been taking any drugs or alcohol within 24 h, nor had they suffered from illness at the time of urine collection. After vigorously stirring the urine specimens, 40 mL of urine was centrifuged at 10 000 x g for 20 min. The supernatant and sediment resuspended in 2 mL of PBS were kept frozen at - 20°C until analysis. Fractionation of urine In the experiment for dolichol partition in urine, freshly voided urine samples (100 mL) were obtained from healthy volunteers. The urine was first centrifuged at 10 000 x g for 20 min. After removing the sediment, the supernatant was filtered through a Millipore Sterifil-D filter unit (pore size, 0.22 pm; Millipore, Bedford Mass.). The filtrate was then subject to ultrafiltration through a Collodion membrane (Collodion Bags, Sartorius, Gottingen). Samples were stored at -20°C until analysis. Dolichol measurement Free dolichol in the sediment and supernatant were measured using the following methods. Urinary sediment suspended in 2 mL of PBS was homogenized with a Polytron at 4 for 20 s. An aliquot of the homogenate was taken for protein determination. After addition of 5 pL of the internal standard solution (2,2-didecaprenylethanol, 0.8 mL in dichloromethane), lipids were extracted with 3 mL of chloroform-methanol (2:1, v/v). The lower phase was collected and dried under nitrogen. Lipids were again extracted with 2 mL of boiling n-hexane. After precipitating cholesterol at 4°C for 2 h, n-hexane was removed under nitrogen, and the final residue was dissolved in 30 pL of 2-propanol-methanol-hexane (2:2:0.9, by volume) and injected into the high performance liquid chromatography system. Dolichol in the supernatant (20 mL), with 5 pL of the internal standard solution, was extracted with 20 mL of chloroform methanol (2:l, v/v) in a separatory funnel. After vigorous shak-
500
1000 1500 URINE VOLUME (muday)
FIG. 1. Correlation between the amount of urinary sediment and urine volume. A significant correlation was noted between the amount of sediment and urine volume (r = 0.733). TABLE 1. Levels of free dolichol in urinary sediment and supernatant pg dolichol in urine Per day
Per L urine
Sediment 30k11.1 30.4k13.2 Supernatant 12.4 7.8 11.4 k 6.1
*
NOTE:Levels are given as means
Per Per mg protein g creatinine 2.0k1.1
21.5k7.9 9.0 k 6.0
* SD (n = 16).
ing for 20 s, the lower phase was collected and dried under nitrogen. The remainder of the method was the same as that for sediment. Reverse-phase high performance liquid chromatography (Shimazu LC-6A, equipped with SPD-6AU spectrophotometric detector, and C-R3A recorder-integrator, Shimazu, Kyoto) was used for dolichol determination and quantitation. A reverse-phase column (C18 Microsorb, Rainin, Woburn, Mass.) was eluted with 2-propanol-methanol-n-hexane (2:2:0.9, by volume) at a flow rate of 0.4 mL/min. Dolichol was detected at 210 nm. The quantitation of dolichol was carried out using the internal standard method, with 2,2-didecaprenylethanol as an internal standard and pig liver dolichol (Sigma, St. Louis, Mo.) as authentic dolichol to construct a calibration curve. 2,2-Didecaprenylethanol was a generous gift from Eisai Company (Tokyo, Japan). Protein and creatinine measurement Protein in the sediment was measured with Bio-Rad protein assay kit. Urinary creatinine was measured by a standard alkaline picrate method adapted for an autoanalyzer.
Results The normal levels of free dolichol in the sediment are shown in Table 1. Dolichol levels were expressed as 30.4 + 13.2 pg/L urine and 21.5 + 7.9 pg/g creatinine. Although these figures were almost double those reported by Wolfe and colleagues (1986), they are comparable to the results by Paton and Poulos (1987). To ask whether dolichol in the sediment is properly expressed as micrograms per litre of urine or gram of creatinine, we studied the relationship
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ULTRAFURATE
FIG. 2. Chromatograms of free dolichol in each fraction of urine. The last peak (retention time is approximately 17 min) represents the internal standard.
between the total creatinine excretion, total urine volume, and the amount of sediment. Since we assumed that amount of protein is proportional to the number of cells which are the main component of urinary sediment, we adopted the amount of protein to represent the amount of sediment. As shown in Fig. 1, there was a significant correlation (r = 0.733) between sediment protein and urine volume. Similarly a fairly good correlation was noted between sediment protein and total creatinine excretion (r = 0.567, data not shown). These results indicate that the amount of sediment in a certain volume of urine is fairly constant in a healthy subject. We next measured the levels of free dolichol in the supernatant. As shown in Fig. 2, dolichol was detectable in the supernatant of urine. The levels of free dolichol were about one third of those in the sediments when dolichol levels were expressed as micrograms per litre. The results of free dolichol measurements in the sediment and supernatant are summarized in Table 1. To assess a possibility that dolichol in the supernatant obtained after the conventional centrifugation was derived from the unsedimented cellular debris or membrane fragments, we measured the levels of dolichol in the urinary fractions obtained with the method described in Materials and
methods. As shown in Fig. 2 and Table 2, dolichol was present in the fraction obtained after filtration of urine through a Millipore filter (0.22 pm pore size). It was also shown that the Collodion membrane, which is theoretically permeable to molecules less than 4000 Mr, was only partially permeable to dolichol (approximate Mr of 1500) present in the supernatant.
Discussion In the present study, we demonstrated that free dolichol is present both in urinary sediment and supernatant. Although the level of dolichol in the supernatant is much lower than that in the sediment, it is large enough to make it difficult to compare the result obtained from the sediment with that from the uncentrifuged whole urine. Since Roine et al. (1987a) noticed the presence of free dolichol in the supernatant, they measured the levels of free dolichol in uncentrifuged whole urine instead of the sediment. We showed that a considerable proportion of free dolichol present in the supernatant was trapped by a Millipore filter, indicating that most of the dolichol in the supernatant was probably attributable to the unsedimented cellular debris and membrane fragments after centrifugation. This could
NOTES
TABLE2. The amounts and proportions of free dolichol in urine fractions
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Dolichol Fraction
fig/L urine
"7-0
Whole urine Supernatant Filtrate Residue Ultrafiltrate Sediment
97.5 40.3 18.4 38.5 2.3 37.4
100 41 19
38
NOTE: The urine fraction which did not pass through a Collodion membrane is designated as residue. Levels are means from duplicate determinations.
partly explain the discrepancies among the previously reported normal levels of free dolichol in urinary sediment, since the speed and time of centrifugation was different among each investigator. Wolfe et al. (1986) have recommended to centrifuge the urine samples at 10 000 x g for 30 min, instead of the conventional lower speed, to minimize the unsedimented debris. However, it should be pointed out that more cells might be destroyed at the higher centrifugation speed, and there would be more cellular debris in the supernatant. Since dolichol associated with cellular debris or membrane fragments was shown to be detectable in the supernatant even with a higher speed centrifugation, we suggest that uncentrifuged whole urine might be better to represent urinary dolichol excretion. Although we acknowledge that expressing the levels of free dolichol in whole urine relative to urine volume or creatinine is illogical because these measurements are being done on different fractions of the urine (Paton and Poulos 1987), the good correlation between the amount of sediment and urine volume seems to justify this mode of expression. The inpermeability of dolichol in the filtrate through a Collodion membrane could be attributable to conjugate formation of dolichol with protein such as albumin or some specific protein as shown in the serum (Van Dessel et al. 1986). It is suggested that a portion of elevated levels of urinary dolichol is deried from lysosome of urinary epithelial cells, since dolichol was found to be accumulated in lysosome. Salaspuro et al. (1990) suggested that elevated levels of dolichol in the serum from patients with aspartylglucosaminuria is a reflection of a disturbance in lysosomal function. Whether the elevated dolichol excretion in urine is a consequence of lysosomal dysfunction in urinary epithelial cells is currently unknown. Although the elevated levels of urinary dolichol is a nonspecific phenomenon as indicated by the reports on miscel-
521
laneous conditions with raised urinary excretion of dolichol (Pullarkat et al. 1984; Wolfe et al. 1986; Wiktop et al. 1987), measurement of urinary dolichol could potentially become a useful diagnostic tool if indicated properly. Since there are still ongoing controversies about the clinical usefulness of urinary dolichol measurement, further studies are necessary to understand the origin and metabolism of urinary dolichol. Bemet, M.J., Mathers, N.J., Hemming, F.W., et al. 1985. Urinary sediment dolichol excretion in patients with Batten disease and other neurodegenerative disorders. Pediatr. Res. 19: 213-216. Ng Ying Kin, N.M.K., and Wolfe, L.S. 1982. Presence of abnormal amount of dolichol in the urinary sediment of Batten disease patients. Pediatr. Res. 16: 530-532. Paton, B.C., and Poulos, A. 1987. Normal dolichol concentration in urine sediment from four patients with neuronal-ceroidlipofuscinosis (Batten disease). J. Inherited Metab. Dis. 10: 28-32. Pullarlkat, R.K., Raguthu, S., and Pachchagiri, S. 1984. Dolichols in metastatic cancer. Trans. Am. Soc. Neurochem. 15: 171. Roine, R.P., Ylikahri, R., Koskinen, P., et al. 1987a. Effect of heavy weekend drinking on urinary dolichol levels. Alcohol (N.Y.), 4: 509-511. Roine, R.P., Turpeinen, U., Ylikahri, R., and Salaspuro, M. 19876. Urinary dolichol-a new marker of alcoholism. Alcohol. Clin. Exp. Res. 11: 525-527. Sakakihara, Y., Imabayashi, T., Suzuki, Y., and Kamoshita, S. 1987. Elevated levels of dolichol in the brains of lysosomal storage disorders. Igaku no Ayumi, 141: 1021-1022. Salaspuro, M., Korri, U.-M., Nuutinen, H., and Roine, R. 1987. Blood acetate and urinary dolichol-new markers of heavy drinking and alcoholism. In Genetics and Alcoholism. Alan R. Liss, Inc., New York. pp. 231-240. Salaspuro, M., Salmela, K., Humaloja, K., et al. 1990. Elevated levels of serum dolichol in aspartylglucosaminuria. Life Sci. 47: 627-632. Turpeinen, U. 1986. Liquid-chromatographic determination of dolichol in urine. Clin. Chem. (Winston-Salem, N.C.), 32: 2026-2029. Van Dessel, G., De Wolfe, M., Lagrou, A., et al. 1986. On the binding of dolichol by human serum. Biochim. Biopys. Acta, 878: 346-352. Wiktop, C.J., Wolfe, L.S., Cal, S.X., White, J.G., et al. 1987. Elevated urinary dolichol excretion in the Hermansky-Pudlak syndrome. Am. J. Med. 82: 463-470. 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, 2: 99. Wolfe, L.S., Ng Ying Kin, N.M.K., Palo, J., and Haltia, M. 1983. Dolichols in brain and urinary sediment in neuronal-ceroid lipofuscinosis. Neurology, 33: 103-106. Wolfe, L.S., Palo, J., Santavuori, P., et al. 1986. Urinary sediment dolichols in the diagnosis of neuronal ceroid-lipofuscinosis. Ann. Neurol. 19: 270-274.
518
lipofuscinosis (Batten's disease). J. Inherited Metab. Dis. 7: 112-1 16.
Paton, B., 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:
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by CONCORDIA UNIV on 12/11/14 For personal use only.
28-32.
Pullarkat, R., Kim, K., Sklower, S., and Patel, V. 1988. Oligosacchary1 diphosphodolichols in the ceroid-lipofuscinoses. Am. J. Med. Genet. Suppl. 5: 243-251. Ronin, C., and Bouchillow, S. 1978. Cell-free labeling in thyroid rough microsomes of lipid-linked and protein-linked oligosaccharides. Biochim. Biophys. Acta, 539: 470-480. Sakakihara, Y., and Kamoshita, S. 1991. Partition of free dolichol into urinary sediments and supernatant. In Dolichol and other lipids related to glycoconjugate metabolism. Satellite meeting to the 11th International Symposium on Glycoconjugates, Kimberley, Ont. Abstr. 25. Salaspuro, M., and Korri, U.M. 1987. Blood acetate and urinary dolichols-new markers of heavy drinking and alcoholism. In Genetics and Alcoholism. Alan R. Liss Inc., New York. pp. 231-240. Svennerholm, L., Hagberg, B., Haltia, M., et al. 1975. Poly-
unsaturated fatty acid lipidosis. 11. Lipid biochemical studies. Acta Paediatr. Scand. 64: 489-496. Van Dessel, G., Lagrou, A., Hilderson, H.J.J., et al. 1979. Isolation and identification of polyprenols from bovine thyroid gland. Biochim. Biophys. Acta, 573: 296-300. Voets, R., Lagrou, A., Hilderson, H.J.J., et al. 1979. RNA synthesis in isolated nuclei and nucleoli from bovine thyroid. HoppeSeyler's Z. Physiol. Chem. 360: 1271-1283. Welply, J., Shenbagamurthi, P., Lennarz, W., and Naider, F. 1983. Substrate recognition by oligosaccharyltransferase. J. Biol. Chem. 258: 11 856 - 11 863. Wolfe, L., Ng, Y., Palo, J., and Haltia, M. 1983. Dolichols in brain and urinary sediment in neuronal ceroid lipofuscinosis. Neurology, 33: 103-106. Wolfe, L., Palo, J., Santavuori, P., et al. 1986. Urinary sediment dolichols in the diagnosis of neuronal ceroid-lipofuscinosis. Ann. Neurol. 19: 270-274. Wolfe, L., Gauthier, S., Haltia, M., and Palo, J. 1988. Dolichol and dolichylphosphate in the neuronal ceroid-lipofuscinosis and other diseases. Am. J. Med. Genet. Suppl. 5: 233-242. Zeman, W. 1974. Studies in the neuronal ceroid-lipofuscinoses. J. Neuropathol. Exp. Neurol. 33: 1-12.
Partition of free dolichol in human urine YOICHI SAKAKIHARA AND SHIGEHIKO KAMOSHITA Department of Pediatrics, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-Ku Tokyo, Japan Received September 3, 1991 SAKAKIHARA, Y., and KAMOSHITA,S. 1992. Partition of free dolichol in human urine. Biochem. Cell Biol. 70: 518-521.
We have demonstrated that dolichol is present in the urinary supernatant. Most of the dolichol present in the supernatant seems to be associated with cellular debris or membrane fragments. The amount of sediment in healthy subjects correlate well with the volume of urine. Although it is illogical to express urinary dolichol relative to urine volume, a good correlation between the amount of sediment and urine volume has made its use justifiable. Because of the presence of a substantial amount of dolichol in the supernatant, it seems better to use uncentrifuged whole urine as the sample for measurement of dolichol. Key words: dolichol, urine, sediment, supernatant. SAKAKIHARA, Y., et KAMOSHITA,S. 1992. Partition of free dolichol in human urine. Biochem. Cell Biol. 70 : 518-521.
Nous avons dtmontrt la prtsence du dolichol dans le surnageant urinaire. La plus grande partie du dolichol prtsent dans le surnageant semble associt B des dtbris cellulaires ou des fragments membranaires. Chez les sujets en santt, la quantitt de ce stdiment correspond ttroitement au volume de l'urine. I1 est peut-&treillogique d'exprimer le dolichol urinaire par rapport au volume de I'urine, mais I'ttroite relation entre la quantitk de sediment et le volume d'urine justifie son emploi. fitant donnt la prhence d'une quantitt substantiellede dolichol dans le surnageant, il semble prtftrable d'utiliser I'urine totale non centrifugke comme tchantillon pour la mesure du dolichol. Mots clks : dolichol, urine, ddiment, surnageant. [Traduit par la redaction]
Introduction Dolicol and its derivatives are lipid intermediates participating in the synthesis of N-linked glycoprotein. Its clinical implication was not recognized until Wolfe reported elevated levels of free dolichol in the brains of patients with Alzheimer's disease (Wolfe et al. 1982). Since then, dolichol accumulation has been reported in the brains of patients with several neurodegenerative disorders (Ng Yink Kin and Wolfe 1982; Sakakihara et al. 1987), in the urine of chronic alcoholics and patients with ceroid-lipofuscinosis (Ng Ying Kin and Wolfe 1982; Wolfe et al. 1983, Pullarkat et al. 1984; Wolfe et al. 1986), and in the serum from patients with aspartylglucosarninuria (Salaspuro et al. 1990). ABBREVIATIONS: PBS, phosphate buffered saline; M,, relative mass. Printed in Canada / Im~rimbau Canada
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Although the mechanism of dolichol accumulation has not been elucidated, the measurement of dolichol in these samples is clinically useful in making diagnosis and in evaluating the condition of patients, as in the case with chronic alcoholism (Roine et al. 19876). Since urinary dolichol measurement is noninvasive, some authors advocate its use in the treatment program for chronic alcoholics (Roine et al. 1987a; Salaspuro et al. 1987). However, there are several problems in urinary dolichol measurement. The first problem stems from the differences in urine preparation by various investigators. Some investigators used urinary sediment (Wolfe et al. 1983, 1986; Bennett et al. 1985; Paton and Poulos 1987), while others measured dolichol in uncentrifuged whole urine (Turpeinen 1986; Roine et al. 1987~).The second problem is related to the mode of expressing urinary dolichol. In the previous studies, dolichol in urine has been expressed as micrograms per litre of urine (Wolfe et al. 1986; Paton and Poulos 1987), micrograms per gram or millimole of creatinine (Bennett et al. 1985; Wolfe et al. 1986; Paton and Poulos 1987; Roine et al. 1987a, 1987b), and micrograms per microgram of lipid (Ng Ying Kin and Wolfe 1982; Paton and Poulos 1987). In the present study, we have examined the problems in urinary dolichol measurement as described above. First, we have explored the methods of expressing dolichol levels in both urinary sediment and supernatant. Second, we have asked whether dolichol is present in the urinary supernatant. To this end, we have analyzed the 24-h urine samples collected from 16 healthy male volunteers. Materials and methods Urine collection and preparation Twenty-four-hour urine samples were collected from 16 healthy adult male volunteers. None of the volunteers had been taking any drugs or alcohol within 24 h, nor had they suffered from illness at the time of urine collection. After vigorously stirring the urine specimens, 40 mL of urine was centrifuged at 10 000 x g for 20 min. The supernatant and sediment resuspended in 2 mL of PBS were kept frozen at - 20°C until analysis. Fractionation of urine In the experiment for dolichol partition in urine, freshly voided urine samples (100 mL) were obtained from healthy volunteers. The urine was first centrifuged at 10 000 x g for 20 min. After removing the sediment, the supernatant was filtered through a Millipore Sterifil-D filter unit (pore size, 0.22 pm; Millipore, Bedford Mass.). The filtrate was then subject to ultrafiltration through a Collodion membrane (Collodion Bags, Sartorius, Gottingen). Samples were stored at -20°C until analysis. Dolichol measurement Free dolichol in the sediment and supernatant were measured using the following methods. Urinary sediment suspended in 2 mL of PBS was homogenized with a Polytron at 4 for 20 s. An aliquot of the homogenate was taken for protein determination. After addition of 5 pL of the internal standard solution (2,2-didecaprenylethanol, 0.8 mL in dichloromethane), lipids were extracted with 3 mL of chloroform-methanol (2:1, v/v). The lower phase was collected and dried under nitrogen. Lipids were again extracted with 2 mL of boiling n-hexane. After precipitating cholesterol at 4°C for 2 h, n-hexane was removed under nitrogen, and the final residue was dissolved in 30 pL of 2-propanol-methanol-hexane (2:2:0.9, by volume) and injected into the high performance liquid chromatography system. Dolichol in the supernatant (20 mL), with 5 pL of the internal standard solution, was extracted with 20 mL of chloroform methanol (2:l, v/v) in a separatory funnel. After vigorous shak-
500
1000 1500 URINE VOLUME (muday)
FIG. 1. Correlation between the amount of urinary sediment and urine volume. A significant correlation was noted between the amount of sediment and urine volume (r = 0.733). TABLE 1. Levels of free dolichol in urinary sediment and supernatant pg dolichol in urine Per day
Per L urine
Sediment 30k11.1 30.4k13.2 Supernatant 12.4 7.8 11.4 k 6.1
*
NOTE:Levels are given as means
Per Per mg protein g creatinine 2.0k1.1
21.5k7.9 9.0 k 6.0
* SD (n = 16).
ing for 20 s, the lower phase was collected and dried under nitrogen. The remainder of the method was the same as that for sediment. Reverse-phase high performance liquid chromatography (Shimazu LC-6A, equipped with SPD-6AU spectrophotometric detector, and C-R3A recorder-integrator, Shimazu, Kyoto) was used for dolichol determination and quantitation. A reverse-phase column (C18 Microsorb, Rainin, Woburn, Mass.) was eluted with 2-propanol-methanol-n-hexane (2:2:0.9, by volume) at a flow rate of 0.4 mL/min. Dolichol was detected at 210 nm. The quantitation of dolichol was carried out using the internal standard method, with 2,2-didecaprenylethanol as an internal standard and pig liver dolichol (Sigma, St. Louis, Mo.) as authentic dolichol to construct a calibration curve. 2,2-Didecaprenylethanol was a generous gift from Eisai Company (Tokyo, Japan). Protein and creatinine measurement Protein in the sediment was measured with Bio-Rad protein assay kit. Urinary creatinine was measured by a standard alkaline picrate method adapted for an autoanalyzer.
Results The normal levels of free dolichol in the sediment are shown in Table 1. Dolichol levels were expressed as 30.4 + 13.2 pg/L urine and 21.5 + 7.9 pg/g creatinine. Although these figures were almost double those reported by Wolfe and colleagues (1986), they are comparable to the results by Paton and Poulos (1987). To ask whether dolichol in the sediment is properly expressed as micrograms per litre of urine or gram of creatinine, we studied the relationship
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ULTRAFURATE
FIG. 2. Chromatograms of free dolichol in each fraction of urine. The last peak (retention time is approximately 17 min) represents the internal standard.
between the total creatinine excretion, total urine volume, and the amount of sediment. Since we assumed that amount of protein is proportional to the number of cells which are the main component of urinary sediment, we adopted the amount of protein to represent the amount of sediment. As shown in Fig. 1, there was a significant correlation (r = 0.733) between sediment protein and urine volume. Similarly a fairly good correlation was noted between sediment protein and total creatinine excretion (r = 0.567, data not shown). These results indicate that the amount of sediment in a certain volume of urine is fairly constant in a healthy subject. We next measured the levels of free dolichol in the supernatant. As shown in Fig. 2, dolichol was detectable in the supernatant of urine. The levels of free dolichol were about one third of those in the sediments when dolichol levels were expressed as micrograms per litre. The results of free dolichol measurements in the sediment and supernatant are summarized in Table 1. To assess a possibility that dolichol in the supernatant obtained after the conventional centrifugation was derived from the unsedimented cellular debris or membrane fragments, we measured the levels of dolichol in the urinary fractions obtained with the method described in Materials and
methods. As shown in Fig. 2 and Table 2, dolichol was present in the fraction obtained after filtration of urine through a Millipore filter (0.22 pm pore size). It was also shown that the Collodion membrane, which is theoretically permeable to molecules less than 4000 Mr, was only partially permeable to dolichol (approximate Mr of 1500) present in the supernatant.
Discussion In the present study, we demonstrated that free dolichol is present both in urinary sediment and supernatant. Although the level of dolichol in the supernatant is much lower than that in the sediment, it is large enough to make it difficult to compare the result obtained from the sediment with that from the uncentrifuged whole urine. Since Roine et al. (1987a) noticed the presence of free dolichol in the supernatant, they measured the levels of free dolichol in uncentrifuged whole urine instead of the sediment. We showed that a considerable proportion of free dolichol present in the supernatant was trapped by a Millipore filter, indicating that most of the dolichol in the supernatant was probably attributable to the unsedimented cellular debris and membrane fragments after centrifugation. This could
NOTES
TABLE2. The amounts and proportions of free dolichol in urine fractions
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Dolichol Fraction
fig/L urine
"7-0
Whole urine Supernatant Filtrate Residue Ultrafiltrate Sediment
97.5 40.3 18.4 38.5 2.3 37.4
100 41 19
38
NOTE: The urine fraction which did not pass through a Collodion membrane is designated as residue. Levels are means from duplicate determinations.
partly explain the discrepancies among the previously reported normal levels of free dolichol in urinary sediment, since the speed and time of centrifugation was different among each investigator. Wolfe et al. (1986) have recommended to centrifuge the urine samples at 10 000 x g for 30 min, instead of the conventional lower speed, to minimize the unsedimented debris. However, it should be pointed out that more cells might be destroyed at the higher centrifugation speed, and there would be more cellular debris in the supernatant. Since dolichol associated with cellular debris or membrane fragments was shown to be detectable in the supernatant even with a higher speed centrifugation, we suggest that uncentrifuged whole urine might be better to represent urinary dolichol excretion. Although we acknowledge that expressing the levels of free dolichol in whole urine relative to urine volume or creatinine is illogical because these measurements are being done on different fractions of the urine (Paton and Poulos 1987), the good correlation between the amount of sediment and urine volume seems to justify this mode of expression. The inpermeability of dolichol in the filtrate through a Collodion membrane could be attributable to conjugate formation of dolichol with protein such as albumin or some specific protein as shown in the serum (Van Dessel et al. 1986). It is suggested that a portion of elevated levels of urinary dolichol is deried from lysosome of urinary epithelial cells, since dolichol was found to be accumulated in lysosome. Salaspuro et al. (1990) suggested that elevated levels of dolichol in the serum from patients with aspartylglucosaminuria is a reflection of a disturbance in lysosomal function. Whether the elevated dolichol excretion in urine is a consequence of lysosomal dysfunction in urinary epithelial cells is currently unknown. Although the elevated levels of urinary dolichol is a nonspecific phenomenon as indicated by the reports on miscel-
521
laneous conditions with raised urinary excretion of dolichol (Pullarkat et al. 1984; Wolfe et al. 1986; Wiktop et al. 1987), measurement of urinary dolichol could potentially become a useful diagnostic tool if indicated properly. Since there are still ongoing controversies about the clinical usefulness of urinary dolichol measurement, further studies are necessary to understand the origin and metabolism of urinary dolichol. Bemet, M.J., Mathers, N.J., Hemming, F.W., et al. 1985. Urinary sediment dolichol excretion in patients with Batten disease and other neurodegenerative disorders. Pediatr. Res. 19: 213-216. Ng Ying Kin, N.M.K., and Wolfe, L.S. 1982. Presence of abnormal amount of dolichol in the urinary sediment of Batten disease patients. Pediatr. Res. 16: 530-532. Paton, B.C., and Poulos, A. 1987. Normal dolichol concentration in urine sediment from four patients with neuronal-ceroidlipofuscinosis (Batten disease). J. Inherited Metab. Dis. 10: 28-32. Pullarlkat, R.K., Raguthu, S., and Pachchagiri, S. 1984. Dolichols in metastatic cancer. Trans. Am. Soc. Neurochem. 15: 171. Roine, R.P., Ylikahri, R., Koskinen, P., et al. 1987a. Effect of heavy weekend drinking on urinary dolichol levels. Alcohol (N.Y.), 4: 509-511. Roine, R.P., Turpeinen, U., Ylikahri, R., and Salaspuro, M. 19876. Urinary dolichol-a new marker of alcoholism. Alcohol. Clin. Exp. Res. 11: 525-527. Sakakihara, Y., Imabayashi, T., Suzuki, Y., and Kamoshita, S. 1987. Elevated levels of dolichol in the brains of lysosomal storage disorders. Igaku no Ayumi, 141: 1021-1022. Salaspuro, M., Korri, U.-M., Nuutinen, H., and Roine, R. 1987. Blood acetate and urinary dolichol-new markers of heavy drinking and alcoholism. In Genetics and Alcoholism. Alan R. Liss, Inc., New York. pp. 231-240. Salaspuro, M., Salmela, K., Humaloja, K., et al. 1990. Elevated levels of serum dolichol in aspartylglucosaminuria. Life Sci. 47: 627-632. Turpeinen, U. 1986. Liquid-chromatographic determination of dolichol in urine. Clin. Chem. (Winston-Salem, N.C.), 32: 2026-2029. Van Dessel, G., De Wolfe, M., Lagrou, A., et al. 1986. On the binding of dolichol by human serum. Biochim. Biopys. Acta, 878: 346-352. Wiktop, C.J., Wolfe, L.S., Cal, S.X., White, J.G., et al. 1987. Elevated urinary dolichol excretion in the Hermansky-Pudlak syndrome. Am. J. Med. 82: 463-470. 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, 2: 99. Wolfe, L.S., Ng Ying Kin, N.M.K., Palo, J., and Haltia, M. 1983. Dolichols in brain and urinary sediment in neuronal-ceroid lipofuscinosis. Neurology, 33: 103-106. Wolfe, L.S., Palo, J., Santavuori, P., et al. 1986. Urinary sediment dolichols in the diagnosis of neuronal ceroid-lipofuscinosis. Ann. Neurol. 19: 270-274.
