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Analysis of polar lipids in the urine sediment
I35
CLINICA CHIMICA ACTA
ANALYSIS
OF POLAR
LIPIDS
IN THE
URINE
SEDIMENT
J. R. WHER~ET~
De~a~t~~nt (Received
of Medicine, Universsity oj Toronto, Faculty of Medicine, Toronto (Canada~
October
roth,
1566)
SUMMARY
Analyses of the lipids in urine sediment obtained from normal adults and individuals afflicted with chronic degenerative disorders are described. Polar lipid classes were identified by their behaviour on DEAE and alumina column chromatography and on thin-layer chromatography. Quantitative estimation was by analysis of P and long-chain base in the column fractions. Complex glycolipid patterns were observed. The sediment from a patient with metachromatic leukodystrophy was found to have a 74-fold increase in the molar ratio of acidic glycolipid to total phospholipid. In three patients with the gargoylism syndrome the neutral glycolipid to total phospholipid ratio was increased 1.5 to 9 times.
INTRODUCTION
In 1921, Wittel observed metachromatic lipid in kidney tubular epithelium and casts in a forty-two year old adult dying with the metachromati~ form of diffuse cerebral sclerosis. He suggested that the finding of metachromati~ material in the urine sediment might allow the diagnosis of this condition to be made during life. In 1957, Austin” was able to demonstrate the presence of metachromatic material in the urine sediment in cases of the metachromatic form of diffuse cerebral sclerosis. The diagnostic value of his test and subsequent modifications8-7 is not universally accepted and may well be superseded by an enzymatic test 8. Histological changes have been observed in the sediment in other degenerative disorders involving the nervous system*-1’ as well as disorders primarily affecting the kidney’%. Undoubtedly, because of the complexities of such a study, there have been no reported studies of the polar lipids excreted with the urine sediment per se. This paper describes analyses of polar lipids in urine sediment obtained from normals and individuals with chronic diseases of the nervous system. MATERIALS
AND METHODS
Urine was collected in iced containers free of preservative as 24-h samples, as complete as possible. Urines were tested using the Hema-Combistix (Ames) to measure CEin. Chim. Acta, 16
(I)
135-145
WHERRETT
136
pH and detect the presence of sugar, protein or hemoglobin and aliquots were sent to the clinical routine laboratory for creatinine determination. If not used immediately the urines were stored at -zoo. Sediment was collected by centrifugation at IOOOO rev./min in the Z-34 head of a Servall SS-4 centrifuge using the KSR-3 continuous flow attachment when large volumes were encountered. The pooled, packed sediments were stored at -20’ until extraction. Complete 24-h collections could not be obtained from some individuals who could not co-operate and pooled incomplete collections For extraction,
were used. the frozen pellet was dropped
into a test
tube
homogenizer
(A. H. Thomas, Philadelphia) and homogenized with 19 vol. or more of chlorofornmethanol, 2: I, v/v. The homogenate was filtered through a funnel containing a medium-porosity sintered disc and the residue was rehomogenized in fresh solvent and filtered again. The homogenizer, filter and residue were rinsed sex-era1 times with small volumes of solvent and the extract and washings were taken to dryness. The dried lipid residue was redissolved in chloroform~methanollwater, 60: 30 : 4.5, v/v/v and transferred with suitable washings to a column containing I g of Sephadex (i-25 fine (bed dimensions 5 x go mm) which was eluted as described by Wells and Dittmer13. The rectified extract was then evaporated, the residue dried over KOH irz TjaczLoand weighed. If samples were examined by thin-layer chromatography only, the extracts were rectified
using the method
of Folch et a1.14.
c-iv,
2:
I
extract
Sephadcx COlUmn (‘-M-H&, v rectified
60:3o: total
G25 v H,O-soluble non-lipids
4.5
lipids
IO mg DEAE cellulose CCJlLUlUl
“neutral
hpids”
I
H,O-soluble non-lipids
to remove WAC
%I’
acidic phosphohpids sulphatidcs
I alumina column
I--
C-M 98:r 50 ml
---I-
~
C-M, I : I zoo ml
C-M-H@, I IO0 ml
v
f
v
neutral lipids cholesterol
CGP SM
neutral EGP
2 :5 :2
glycolipids
Fig. I. Scheme for fractionation of lipids in urine sediment by column chromatography. Abbreviations: C, chloroform; M, methanol; HAc, glacial acetic acid; NH,Ac, ammonium acetate; SGP, serine glvcerol phosphatide; CGP, choline glycerol phosphatide; EGP, ethanolamine glycerol phosphatide; SM, sphingomyelin. Clin.
Chim.
Acta,
16 (I) 135-145
POLAR
LIPIDS IN NORMAL
AND
PATHOLOGICAL
I37
URINES
Aliquots of the total lipid extract containing 10-15 mg lipid material were chromatographed on DEAE-cellulose following Rouser et &.x5, and on alumina10 to separate polar lipid classes. The DEAE-cellulose columns contained 1.5 g and the bed dimensions were 15 x 65 mm. Alumina columns contained IO g basic alumina, Brockmann activity I (Fisher Scientific, Toronto) and the bed dimensions were 13 x 80 mm, This alumina gave a pK of 9.7 when suspended I g in IO ml distilled water and no detectable solid was released on washing with the most polar solvent used. The elution scheme is shown in Fig. I. Aliquots of the fractions were taken for thin-layer and long-chain chr~)nlatogra~hy I7 determiilation of cholesterol,l~,ls pl~os~)l~~)rus~” base21. Glycolipids were detected on chromatograms using the aniline-diphenylamine spray 2z, A dichromate-reduction method was used tn determine total lipid in early studies 23. RESULTS
In order to approach the analysis of urine sediment, the amount of lipid excreted by a series of 28 adult in-patients was estimated first by the dichromatereduction method. Using a cholesterol standard, values ranged from 0.3 to 5,4 mg/g creatinine in patients without infection. Higher values were obtained if the urine contained blood cells resuIting from infection or catheterization. One mg of lipid was sufficient for analysis by thin-layer chromatography.
Fifty urine samples from 36 patients were examined by qualitative thin-layer chromatography and eight of these were the subject of more detailed analysis, Inspection of the chromatograms revealed a bewildering array of components. By observing the behaviour of these components on DEAE-cellulose columns, their relationship to components of brain lipid extracts and their staining reactions, many could be tentatively identified. Thus the cholesterol and neutral lipid bands running close to the front were about equal in size and were not further examined except for esiimation of total cholesterol. After the diphenylanline spray, cholesterol appears initially as a bright red band. The phosphatide classes could usually be identified as shades of brown when the diphenylamine spray was used. Serine-glycerol-phosphatide, which is eluted in the acetic acid-chloroform fraction, gave a pale green colour. Complex and variable patterns of blue-staining bands were encountered. In some extracts most of the slow-moving blue bands as well as slow-moving substances staining in other colours were eluted from DEAE-cellulose not with neutral solvents but with the acetic acid solvents. A pair of blue bands which ran with or just behind ethanolamine-glycerol-phosphatide were tentatively ident~~ed as ceramide dihexosides. They were alkali-stable and were eluted from DEAF,-cellulose with neutral solvents and from alumina with chloroform-methanol--water 2 :5 :2, v/v/vz4. Their position on chromatograms relative to other glycolipids was similar to that reported for ceramide dihexosides24+‘5. Further slow-running blue bands were present in the neutral chloroform-methanol eluate from DEAE-cellulose which indicated the existence of higher ceramide glycoside derivatives. When lipid containing gangliosides is run in the DEAE-cellulose column procedure, the gangliosides are eluted with the CEin.C%VZ. Ada,
16 (I) I35-Eqj
WHERRETT
138
two acetic acid-containing solvents and in the fraction containing sulphatidesa6. Gangliosides were not detected in urine by qualitative chromatographya7. The patterns from normal urine were similar one to another (Fig. 2). The most prominent neutral glycolipid bands were those running in the same region as brain cerebrosides. Next most prominent was a broad band in the same region as brain sulphatide which was eluted with both acidic and neutral glycolipids. The two bands, thought to be ceramide dihexosides, were usually detected and at times approached the intensity of the cerebrosides. Occasionally running with sphingomyelin was a more prominent band that corresponded to the major glycolipid observed in kidney extracts probably the hexosaminyl-trihexosyl ceramide described by Martenssonz8. More than the two bands characteristic of brain were almost always observed in the cerebroside region and on a number of occasions five clearly distinct bands could be seen. Several significant variations from the pattern could be distinguished. Samples from patients on medications including anti-convulsants, phenothiazines, barbiturates and others had extra blue-staining bands close to the origin, often disrupting the pattern.
A very striking
pattern
(Fig.
z) was observed
when the urine contained
ct
CMC :MH
:DH
Al
A2
A3
A4
81
82
Fig. 2. Thin-layer chromatograms of lipids in urine sediment from normal individuals (A) and urine containing polymorphonuclear leukocytes (B). Samples: AI, M 28 (see Table I) ; A2, human white matter lipid marker; Aj, F 23; A4. F 40; RI, urine containing leukocytes; B2, rat brain lipid marker. Solvent: chloroform-methanol-water, 65 : 25 : 4, v/v/v_ Detection: aniline-diphenylamine. Abbreviations: CH, cholesterol; CMH, ceramide monohexosides; CDH, ceramide dihexosides; S, sulphatides. Clin. Chirn. Acta,
16 (I)
135-145
POLAR LIPIDS IN NORMAL AND PATHOLOGICAL
=39
URINES
Fig. 3. Thin-layer chromatograms of lipids in urine sediment from patients with gargoylism syndrome and m&achromatic leukodystrophy and from normal individuals, Samples: I. Normal, F 19; 2. Gargoylism, F II (see Table I); 3. Normal, F 23; 4. Gargoylism, M 7; 5. Human white matter marker; 6. Normal, M 28; 7. Gargoylism, M 6; 8. Gargoylism, M 4; 9. Normal, F 19; IO. Metachromatic leukodystrophy, F x6. Solvent: chloroform-methanol-water, 65 : 25 : 4, v/v/v. Detection: aniline-diphenylamlne. Abbreviations: CH, cholesterol; CMH, ceramide monohexosides; CDH, ceramide dihexosides; S, sulphatides.
Amount of lipid (mg) Creatinine equivalence (mg) --... II_____.
0.63 I64 -
2.3 44
0.39 60
0.38
0.33
20
---
1.3
400
1.0 120
305 _~
-. 190 110 _______
-
white cells and it is apparent that most of the lipids observed originated in polymorphonuclear leukocytes. Here, associated with increased lipid yield, was a striking increase in the double ceramide dihexoside bands and a pair of bands of less intensity not previously detected are seen running in the region occupied also by sphingomyelin. These slower bands are eluted clearly with the neutral glycolipids and are more polar than the major kidney glycolipid. Urine sediment from four patients with the gargoylism syndrome* was exam* This term is used to designate disorders categorized clinically as “Hunter-Hurler” and related syndromes. In the classification of McKusick et aLeepatients M7 and M4 corresponded most closeiy to Type I, patient M6 to Type II and patient Frr to Type III. The urines from these cases gave a strongly positive spot test for mucopolysaccharides 30. C&z. Ci&n. Acta, 16 (I) 135-145
WHERRETT
140
ined. In all, the ceramide dihexosides were the most prominent glycolipids (Fig. 3). Other slower neutral glycolipids were present as well but the pattern differed from that of sediment taken from infected urine. Finally, in multiple specimens from a sixteen year old patient with a slowly progressive motor disturbance and dementia diagnosed as metachromatic leukodystrophy, a most unusual pattern was observed (Fig. 3). The ceramide dihexosides were slightly more intense, but there was a striking increase of glycolipid-staining spots which were eluted from DEAE-cellulose by chloroform-methanol containing ammonium acetate and ammonia with the most acidic lipid fractions. Two bands were observed which on several chromatograms were double and the slower was less intense than the faster moving. In addition, faint slower and faster bands were detected. This fraction was treated with acid as described by Stoffyn and Stoffyn3r and on chromatography, there was a decrease in all of the previously noted bands with the appearance of two cerebrosides and two ceramide dihexoside bands. As well, two further bands appeared. One gave the reaction for glycolipid and ran just in front of the two cerebroside bands and the other was a small band running in the appropriate position and giving the appropriate colour reaction for cholesterol. Quantitative
analysis
The quantitative analysis of samples from four normals, three cases of the gargoylism syndrome and a case of metachromatic leukodystrophy are shown in Table I. In the normal adults, the amount of lipid excreted in the sediment determined by weighing is again seen to be small and agrees with values obtained by the dichromate-reduction procedure. Estimations of cholesterol, phospholipid and sphingosine did not account for more than 60 “/ of the lipid, indeed the sum of these fractions varied considerably. Values for the phospholipid fractions from the DEAE column are shown in Table II. In general the distribution of phosphorus in the fractions except for normal sample F40 follows the pattern observed in many tissue;.
Total lipid mg/g cveatisine
Cholestevol mg/~oo mg lipid
Phospholipid nzgl~oo wag lipid
Glyco.sphz+zgolipid ~mzoles/~oo mg lipid _
9.2
7.8 16.7
2 Gargoylism Frr M7 MO 3
Metachromatic leukodvstrophq I216
* Sex and
5:::
0.85 0.15 0.05 0.18
9.4
6.4 15.6 II.3
2.8 5.4 9.7
0.65
16.7
IL.1
4.0
3.4
53.2 19.5 8.6
6.6
Aczdic
2.0 5.4 4.7 I.0
10.5
8.9 5.’ -
age of individual.
iVeutYa1 ~~~~~
.~______.
‘5.7 23.5
0.14
0.25
‘9.7
POLAR
LIPIDS
TABLE
AND
PATHOLOGICAL
141
URINES
II
DISTRIBUTION
Lipid
IN NORMAL
OF
PHOSPHORUS
IN
THE
COLUMN
FRACTIONS
P as o’;
fraction
of total
P in fractions Gavgoyltsm
NOrmal.
Choline-containing Ethanolamine glycerol phosphatide Serine glycerol phosphatide Minor acidic phosphatides Other fractions 9; l
Fzo*
F23
F4o
M28
42.4 22.5
23.7 36.4 5.6 24.5 7.6 98.0
10.3
31.8
54.6 4.3 28.4
25.7
2.4 80.1
8.4 94.1
I9.0
12.9 3.4 95.0
19.0
15.1
M6
-__--___26.5 21.8 15.0 31.2 5.6 89.1
MLD
M 7
23.6 23.7 10.7 25.2 16.5 X7.1
FI6 58.4 8.6 7.6 17.9 7.6 92.0
* Sex and age of individual. TABLE MOLAR
III RATIOS
SOWCL?
OF
GLYCOLIPIDS pmoles
~__ Neutral
Normal F 20*
Gargoylism F II M7 MO Metachromatic F 16 *
PHOSPHOLIPID
__-
IN
lipid
URINE
SEDIMENT
P
Acidic
0.17 0.07
0.05 0.03 0.01
0.18
0.01
0.34
0.02
0.11
F 23 F 40 M 28
TO
Sphingosine/pmoles
0.27
0.03
0.66
0.02
leukodystrophy 0.22
1.80
Sex and age of individual.
The content of the neutral glycolipids in urine sediment is similar to that found in most tissues outside the nervous system and there is no apparent difference between the normal and abnormal specimens. If it can be assumed that the polar lipids exist primarily as components of structural lipoproteins in close association, a ratio of these components might indicate a disturbance of the metabolism of either. In Table III, the molar ratios of neutral and acidic glycolipids to phospholipid are calculated from the results in Table I. In urine sediment from patients with the gargoylism syndrome, the ratio for neutral glycolipids is elevated, in one instance over three times that of the normals whereas the ratio for acidic glycolipids is the same. The sample from the patients with metachromatic leukodystrophy shows a striking increase in acidic glycolipid content and in molar ratio of acidic glycolipid to lipid P. There is also a slight increase in the molar ratio of neutral glycolipid to lipid P. DISCUSSION
Asalytical
method
Several problems arise when attempting to analyze urine sediment lipids, not the least of which is sampling. Ideally, one would wish to obtain a timed sample of urine in which the quantities of lipid-contributing elements is known, in which the Clin. Chzm. Acta,
16
(I)
135-145
142
WHERRETT
processes of degradation are controlled and which is free of cont~ninatio~. For accurate cell counts, it would be necessary to make collections frequently throughout the 24-h period. To obtain specimens from the female uncontaminated by perineal secretions and cells, either very careful technique is required or it is necessary to resort to catheterization. The former was not practical and repeated catheterization would produce increased cell excretion. When methods of extraction and rectification suitable for many tissues were applied to urine sediment, a number of unidentified substances not encountered in other tissues were included in the extract. Some of these w-ere probably drug metabolites. An important portion of the lipid not studied is apparently neutral lipid other than cholesterol Thus the values obtained may have little significance when related to period of production or to the total material collected as lipid. It may be useful therefore, to relate lipid classes one to another. With care, phosphorus recoveries of roe% are obtained from the combination of columns used when extracts of tissue are the objects of examination. In the column runs reported, the low recovery may be attributed to tecbnica.1 errors in dealing with small samples. In one case {Sample F40), rerunning the column gave identical results suggesting that phosphorus-containing compounds may remain on the columns. By using DEAE-cellulose, the loss of acidic compounds which are known to be incompletely eluted from alumina 3%is avoided. Sphingosine recovery was not attempted but was judged satisfactory from the thin-layer chromatograms, The extent of degradation either prior to or after extraction cannot be assessed, but ethanolamine phosphatides were low in the one sample (MLD) that had been freezedried for some months prior to extraction and this effect has been noted when other tissues were analyzeda3. Source of urine se&&em! lipid Urine sediment lipid can be considered to originate from structural elements of cells including structural lipoproteins and from soluble lipoproteins derived from other sources. The cellular material will have been released from lining epithelia and glands of the urinary tract and from other cells migrating into the tract, such as blood cells. The rate of excretion of cells is known to vary significantly during the 24-h period”” and Rofea4 found that red cell excretion rate continued to increase during the day as the excretion of other types of cells diminished, Rofe’s counts, taken during a period close to the maximal rate for all types of cells other than red cells in the male, showed that about rsoooo cells were excreted per hour, of which 2% were erythrocytes, 350/ were polyrnorphonuclear leukocytes and the rest epithelial cells of various type and stage of degeneration. Epithelial cells, considered most likely to have been derived from kidney tubules comprised about 3% of total cells. If it is assumed that twice as many degenerated cells originated in the kidney tubules but could not be classified as such, then not more than 10% of the total would have originated in the kidney. Soluble lipoproteins may also be released from cells lining the tract as glandular secretions or degeneration products or be transported into the urine from a site of synthesis outside the urinary tract where they are rendered soluble. At the centrifugal force-time integral employed to collect the sediment, mucoproteins also are deposited33. Thus the pattern of urine lipids reflects, at best, a mean of the lipid make-up of the various populations of cells, cellular elements and lipoproteins. Without knowing
POLAR
LIPIDS
IN NORMAL
AND
PATHOLOGICAL
URINES
I43
the content of each lipid class in each type of cell, one cannot estimate the significance of the contribution from each type to the whole pattern. It seems unlikely, however, that lipids of red cell origin would have a significant influence on the pattern. Unless striking specificities of localization exist, the lipid pattern from cells of renal tubular origin will probably be obscured in a pattern derived from epithelial cells and to a lesser extent polymorphonuclear leukocytes. Observed &bid patterns Major attention may be directed to sphingoglycolipid classes which, in contrast to phospholipids, show specificities of tissue and cell localization3a and which are implicated in abnormalities of the urine sediment in disease. Thus the predominant pattern in the normal is a complex one in which glycolipids similar or identical to cerebrosides are most abundant components. Separation of individual cerebrosides on thin-layer chromatography using silica gel occurs on the basis of fatty acid content, the hydroxy fatty acid-containing cerebrosides having a lower RF than the nonhydroxy fatty acid cerebrosides 26~37.Other glycolipids with higher RF values than cerebrosides have been described 38+, but it would appear that substances other than these were observed because of their proximity on chromatograms to the more usually observed cerebrosides. They could represent new glycolipid species characteristic of cellular material released into the urinary tract or have resulted from some alteration of the usual cerebrosides on release into the urine. Alterations in the normal lipid pattern would be detected if the ratios of the contributing elements were altered or if there was a metabolic disturbance within one or more of the cell types large enough to be detected. An example of the first type occurred when infected urine was examined. Here a large increase in lipid content and a striking alteration in the glycolipid pattern occurred. The major glycolipid was ceramide dihexoside which contained both hydroxy and non-hydroxy fatty acids. An unknown glycolipid which was much more polar and which contained both hydroxy and non-hydroxy fatty acids was present also, giving a glycolipid pattern that was recognized as characteristic of human leukocytes 4c.41. In two diseases in which genetically determined errors in metabolism result in abnormalities of gly~osphingolipids, metacllromatic leukodystroph~ and the gargoylism syndrome, alterations in the urine pattern were evident. Detailed analyses of brain and kidney have been carried out in metachromatic leukodystrophy12-4J. In the urine sediment excreted by patients with this disease, sulphatides8,46 and a ceramide dihexoside sulphate47 identified by paper chromatography were found to be increased but the lipids have not been further characterized. The observations on the present case give added confirmation of the nature of the metachromatic lipids excreted in the urine sediment. The value for total lipid (in abnormally nourished patient) suggests that the increased sulphated glycolipid (twenty-two times or more) does not result from an increased excretion of a particular type of cell but rather from a disturbance within a normal number of cells shed into the urine, presumably those arising in kidney tubules. Because these cells constitute a small percentage of the total cells excreted, the chemical disturbance within the cells would be of even greater magnitude than that reflected in the sediment lipid pattern. There is some evidence that one of the extra “cerebroside” bands observed may be sulphated. The gargoylism syndrome is recognized to comprise several entities with a CEi??.ChiV%ACtU,
16
(I)
135-I45
WHERRETT
I44
disturbance in mucopolysaccharide metabolism 8s,a8. However lipid abnormalities have also been well documented, including disturbances of gangliosides in brain*+jz and complex glycolipids in liver, spieen53, and kichrey54. The finding of an altered gIycolipid pattern characterized by an increased ceramide dihexoside in urine sediment from at least some forms of gargoylism provides further evidence for a disturbance in gl~co~i~~~~~sas well as mucopolysaccharides. That this may arise in the kidney has been eon~rnle~~ in one case at ~~osi-~~l(}r~en~in which a s-fold increase in kidney ceramide dihexoside occurredZ6. Because detailed cell counts could not be done, the possibility that an increased ratio of Ieukocytes were responsible for the urine sediment pattern in these cases is not excluded. However, in three cases, the molar ratio of neutral glycolipid to phospholipid was elevated over that. observed for pure leukocytes 31 and the column fraction in which neutral glycolipids were eluted gave a pattern on thin-layer chro~natography which was unlike that of the leukocytes. Morpi~ologica~ abnormalities have been noted in the urine sediment from Krabbe’s disease and adult Gaucher’s diseases and in Fahry’s diseaselO, and it will he of interest to know if there are alterations in the urine sediment lipid pattern. The relation of the metabolism disturbances of ~nuco~~r)lysa~charid~ and of glycohpid in the gargoylism syndrome is an interesting unsolved problem.
The author is indebted to Dr. 17. Wilmington (Ol~tar~o Hospital School, Oril~~a~ and Dr. J. C. Richardson (D~partmellt of Medicine) and Dr. W. J. Whaler for generous assistance in obtaining samples for study. The technical assistance of Mrs. D. Kober is gratefuliy acknowledged. The work was supported in part by Grant MA-2235 from the Medical Research Council of Canada. The author was a Fellow of the American College of Physicians and is a John and Mary R. Markle Scholar.
Ctigz. C/&n.
Acta,
16 (I) '35-145
POLAR
21 22 23 24 “5 26 27
28 29
LIPIDS
IN NORMAL
AND
PATHOLOGICAL
URINES
I45
C. J. LAUTER A?*'DE. G. TRAMS, J. Lipid Res., 3jrg6z) 136. G. HARRIS AND 1. c. &b.C%'ILLIAMS,Cherrz. & IEd. (Londonj, (1954) 279. J. S. AMENTA, J. Lipid Xes.,5 (1964) 270. G. M. GRAY, Nafure, 207 (1965) 505. E. SVENNERHOLM AND L. SVENNERHOLM, Nature, 198 (1963) 688. J, R. WHERRETT, unpublished observation. J. R. WHERRETT ASD J. N. CUMINGS, Lliochem.j., 86 (1963) 378. E. MARTENSSOK, B&hi% Bzo#yys. Acta, I 16 (1966) 296. V. A. MCKUSICK, D. KAPLAX, D. WISE, W. B. HANLIXY, S. B. SUDD~RTH, A. E. MAIU~~IA~TEE, Xsdicinc, 44 (1965) 445.
M. B. SEVICK .%.uD
30 31 32 33
34 35 W. H. BOYCR AND M. SWANSON, J. Clin. Iwest., 34 (1955) 1581. AND B. J.\VEBIXR, A~zw. lieu. Bzochcm., 34 (1965) 109. 36 H. E. CARTER, P. JOHNSON H. JATZKKW'ITZ, Z. Ph?~srol. Chwc., 320 (1960) 134. AYD 1.S.G~~~~~n~~,Riochim.Bio~hys. Acfa, 60 (1962) 43. :;iN. Ii.KOCHETKOV, G. Z1irrxov:x 39 W. T. NOKTON AND M. BROTH, Biockem.Bzo~h~s. Rrs. Cowzmm., 12 jr903) 198. _I., 98 (1966) 782. 40 C. J. Xr~xs, J. D. X~~NTZOS AND G. M. LEVIS, Uiuchet?z. E. MARTIN, in preparation. 4I J. it.\VHISRRETT ANI) \;I'. Chenz., 311 (1955) 279. 42 H. JATZKEWITZ, Z. P!z_~~si&.
43 J. H. *JUSTIN, Newolog~, IO (1960) 470. 44 M. LEES AND H. MOSER, 111S. XI.ARONSON AND B. Cl’. VOLK (Eds.),Cerebral .Y$hingolipidoses, Academic Press,New York, 1962, p. 179. 45 I,.SVENNERHOLX, in J. FOLCH-PI .?ND H. BAUER (Eds.),Brazn Lipids md L.ipo$voteinsad the Le~rkod~~sfrophies, Elscvier,Amsterdam, 196.3,p. 104. 46 L. SVENXERHOLM, Acta Che+n. Sand., 17 (1903) 860. 47 E. MARTENSSON, Acfa Chew Scmd., 17 (1963) 1174. in J. B. STANBURY, J. B. WYNGAARDEN AND D. S. FREDGRICKSON (Ed%), The 48 i\. DORFMAN, ~4etubo~ic Basis of fnheviteed i?isense, 2nd Ed., McGraw Hill,Toronto, 1966, p. 936. ed., TV. B. Saunders, Phiis49 J. N. CUMINGS,~~ G. G..DUNC.~ (Ed.),Diseasesqiil~etabolis~,5th delphia, 1964. p. 1405-1439. 50 R. LEDEEN, K. SALSMAK, J. GON.~T~S AND A. TAGHAV~, J. NeuropathoE. Exptl. Se?woE., 24
(1965)341.
57 52 53 54
N. I<. GON~TAS AND J, GONATAS, J. Neuropa2!~ol. Ihptl. _VewoE., 24 (1965) 318. I). A. BOOTH, H. GOODWIK AND J. N. CUMINGS, J. Lipid Res., 7 (1966) 337. I,. L. UZMAN, Arch. PathoE.(Lab. fifed.), 60 (rg55) 308. E. KLENK, quoted bv F. SEITELBERGER, in J. N. CUMINGS AND A. LOWENTAXL bral Lipid&s, Black&cll, Oxford, 1957, p. 77.
(Eds.),Cere-
Clip%. C/&R. Acta, 16 (I) 135-745
CLINICA CHIMICA ACTA
ANALYSIS
OF POLAR
LIPIDS
IN THE
URINE
SEDIMENT
J. R. WHER~ET~
De~a~t~~nt (Received
of Medicine, Universsity oj Toronto, Faculty of Medicine, Toronto (Canada~
October
roth,
1566)
SUMMARY
Analyses of the lipids in urine sediment obtained from normal adults and individuals afflicted with chronic degenerative disorders are described. Polar lipid classes were identified by their behaviour on DEAE and alumina column chromatography and on thin-layer chromatography. Quantitative estimation was by analysis of P and long-chain base in the column fractions. Complex glycolipid patterns were observed. The sediment from a patient with metachromatic leukodystrophy was found to have a 74-fold increase in the molar ratio of acidic glycolipid to total phospholipid. In three patients with the gargoylism syndrome the neutral glycolipid to total phospholipid ratio was increased 1.5 to 9 times.
INTRODUCTION
In 1921, Wittel observed metachromatic lipid in kidney tubular epithelium and casts in a forty-two year old adult dying with the metachromati~ form of diffuse cerebral sclerosis. He suggested that the finding of metachromati~ material in the urine sediment might allow the diagnosis of this condition to be made during life. In 1957, Austin” was able to demonstrate the presence of metachromatic material in the urine sediment in cases of the metachromatic form of diffuse cerebral sclerosis. The diagnostic value of his test and subsequent modifications8-7 is not universally accepted and may well be superseded by an enzymatic test 8. Histological changes have been observed in the sediment in other degenerative disorders involving the nervous system*-1’ as well as disorders primarily affecting the kidney’%. Undoubtedly, because of the complexities of such a study, there have been no reported studies of the polar lipids excreted with the urine sediment per se. This paper describes analyses of polar lipids in urine sediment obtained from normals and individuals with chronic diseases of the nervous system. MATERIALS
AND METHODS
Urine was collected in iced containers free of preservative as 24-h samples, as complete as possible. Urines were tested using the Hema-Combistix (Ames) to measure CEin. Chim. Acta, 16
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pH and detect the presence of sugar, protein or hemoglobin and aliquots were sent to the clinical routine laboratory for creatinine determination. If not used immediately the urines were stored at -zoo. Sediment was collected by centrifugation at IOOOO rev./min in the Z-34 head of a Servall SS-4 centrifuge using the KSR-3 continuous flow attachment when large volumes were encountered. The pooled, packed sediments were stored at -20’ until extraction. Complete 24-h collections could not be obtained from some individuals who could not co-operate and pooled incomplete collections For extraction,
were used. the frozen pellet was dropped
into a test
tube
homogenizer
(A. H. Thomas, Philadelphia) and homogenized with 19 vol. or more of chlorofornmethanol, 2: I, v/v. The homogenate was filtered through a funnel containing a medium-porosity sintered disc and the residue was rehomogenized in fresh solvent and filtered again. The homogenizer, filter and residue were rinsed sex-era1 times with small volumes of solvent and the extract and washings were taken to dryness. The dried lipid residue was redissolved in chloroform~methanollwater, 60: 30 : 4.5, v/v/v and transferred with suitable washings to a column containing I g of Sephadex (i-25 fine (bed dimensions 5 x go mm) which was eluted as described by Wells and Dittmer13. The rectified extract was then evaporated, the residue dried over KOH irz TjaczLoand weighed. If samples were examined by thin-layer chromatography only, the extracts were rectified
using the method
of Folch et a1.14.
c-iv,
2:
I
extract
Sephadcx COlUmn (‘-M-H&, v rectified
60:3o: total
G25 v H,O-soluble non-lipids
4.5
lipids
IO mg DEAE cellulose CCJlLUlUl
“neutral
hpids”
I
H,O-soluble non-lipids
to remove WAC
%I’
acidic phosphohpids sulphatidcs
I alumina column
I--
C-M 98:r 50 ml
---I-
~
C-M, I : I zoo ml
C-M-H@, I IO0 ml
v
f
v
neutral lipids cholesterol
CGP SM
neutral EGP
2 :5 :2
glycolipids
Fig. I. Scheme for fractionation of lipids in urine sediment by column chromatography. Abbreviations: C, chloroform; M, methanol; HAc, glacial acetic acid; NH,Ac, ammonium acetate; SGP, serine glvcerol phosphatide; CGP, choline glycerol phosphatide; EGP, ethanolamine glycerol phosphatide; SM, sphingomyelin. Clin.
Chim.
Acta,
16 (I) 135-145
POLAR
LIPIDS IN NORMAL
AND
PATHOLOGICAL
I37
URINES
Aliquots of the total lipid extract containing 10-15 mg lipid material were chromatographed on DEAE-cellulose following Rouser et &.x5, and on alumina10 to separate polar lipid classes. The DEAE-cellulose columns contained 1.5 g and the bed dimensions were 15 x 65 mm. Alumina columns contained IO g basic alumina, Brockmann activity I (Fisher Scientific, Toronto) and the bed dimensions were 13 x 80 mm, This alumina gave a pK of 9.7 when suspended I g in IO ml distilled water and no detectable solid was released on washing with the most polar solvent used. The elution scheme is shown in Fig. I. Aliquots of the fractions were taken for thin-layer and long-chain chr~)nlatogra~hy I7 determiilation of cholesterol,l~,ls pl~os~)l~~)rus~” base21. Glycolipids were detected on chromatograms using the aniline-diphenylamine spray 2z, A dichromate-reduction method was used tn determine total lipid in early studies 23. RESULTS
In order to approach the analysis of urine sediment, the amount of lipid excreted by a series of 28 adult in-patients was estimated first by the dichromatereduction method. Using a cholesterol standard, values ranged from 0.3 to 5,4 mg/g creatinine in patients without infection. Higher values were obtained if the urine contained blood cells resuIting from infection or catheterization. One mg of lipid was sufficient for analysis by thin-layer chromatography.
Fifty urine samples from 36 patients were examined by qualitative thin-layer chromatography and eight of these were the subject of more detailed analysis, Inspection of the chromatograms revealed a bewildering array of components. By observing the behaviour of these components on DEAE-cellulose columns, their relationship to components of brain lipid extracts and their staining reactions, many could be tentatively identified. Thus the cholesterol and neutral lipid bands running close to the front were about equal in size and were not further examined except for esiimation of total cholesterol. After the diphenylanline spray, cholesterol appears initially as a bright red band. The phosphatide classes could usually be identified as shades of brown when the diphenylamine spray was used. Serine-glycerol-phosphatide, which is eluted in the acetic acid-chloroform fraction, gave a pale green colour. Complex and variable patterns of blue-staining bands were encountered. In some extracts most of the slow-moving blue bands as well as slow-moving substances staining in other colours were eluted from DEAE-cellulose not with neutral solvents but with the acetic acid solvents. A pair of blue bands which ran with or just behind ethanolamine-glycerol-phosphatide were tentatively ident~~ed as ceramide dihexosides. They were alkali-stable and were eluted from DEAF,-cellulose with neutral solvents and from alumina with chloroform-methanol--water 2 :5 :2, v/v/vz4. Their position on chromatograms relative to other glycolipids was similar to that reported for ceramide dihexosides24+‘5. Further slow-running blue bands were present in the neutral chloroform-methanol eluate from DEAE-cellulose which indicated the existence of higher ceramide glycoside derivatives. When lipid containing gangliosides is run in the DEAE-cellulose column procedure, the gangliosides are eluted with the CEin.C%VZ. Ada,
16 (I) I35-Eqj
WHERRETT
138
two acetic acid-containing solvents and in the fraction containing sulphatidesa6. Gangliosides were not detected in urine by qualitative chromatographya7. The patterns from normal urine were similar one to another (Fig. 2). The most prominent neutral glycolipid bands were those running in the same region as brain cerebrosides. Next most prominent was a broad band in the same region as brain sulphatide which was eluted with both acidic and neutral glycolipids. The two bands, thought to be ceramide dihexosides, were usually detected and at times approached the intensity of the cerebrosides. Occasionally running with sphingomyelin was a more prominent band that corresponded to the major glycolipid observed in kidney extracts probably the hexosaminyl-trihexosyl ceramide described by Martenssonz8. More than the two bands characteristic of brain were almost always observed in the cerebroside region and on a number of occasions five clearly distinct bands could be seen. Several significant variations from the pattern could be distinguished. Samples from patients on medications including anti-convulsants, phenothiazines, barbiturates and others had extra blue-staining bands close to the origin, often disrupting the pattern.
A very striking
pattern
(Fig.
z) was observed
when the urine contained
ct
CMC :MH
:DH
Al
A2
A3
A4
81
82
Fig. 2. Thin-layer chromatograms of lipids in urine sediment from normal individuals (A) and urine containing polymorphonuclear leukocytes (B). Samples: AI, M 28 (see Table I) ; A2, human white matter lipid marker; Aj, F 23; A4. F 40; RI, urine containing leukocytes; B2, rat brain lipid marker. Solvent: chloroform-methanol-water, 65 : 25 : 4, v/v/v_ Detection: aniline-diphenylamine. Abbreviations: CH, cholesterol; CMH, ceramide monohexosides; CDH, ceramide dihexosides; S, sulphatides. Clin. Chirn. Acta,
16 (I)
135-145
POLAR LIPIDS IN NORMAL AND PATHOLOGICAL
=39
URINES
Fig. 3. Thin-layer chromatograms of lipids in urine sediment from patients with gargoylism syndrome and m&achromatic leukodystrophy and from normal individuals, Samples: I. Normal, F 19; 2. Gargoylism, F II (see Table I); 3. Normal, F 23; 4. Gargoylism, M 7; 5. Human white matter marker; 6. Normal, M 28; 7. Gargoylism, M 6; 8. Gargoylism, M 4; 9. Normal, F 19; IO. Metachromatic leukodystrophy, F x6. Solvent: chloroform-methanol-water, 65 : 25 : 4, v/v/v. Detection: aniline-diphenylamlne. Abbreviations: CH, cholesterol; CMH, ceramide monohexosides; CDH, ceramide dihexosides; S, sulphatides.
Amount of lipid (mg) Creatinine equivalence (mg) --... II_____.
0.63 I64 -
2.3 44
0.39 60
0.38
0.33
20
---
1.3
400
1.0 120
305 _~
-. 190 110 _______
-
white cells and it is apparent that most of the lipids observed originated in polymorphonuclear leukocytes. Here, associated with increased lipid yield, was a striking increase in the double ceramide dihexoside bands and a pair of bands of less intensity not previously detected are seen running in the region occupied also by sphingomyelin. These slower bands are eluted clearly with the neutral glycolipids and are more polar than the major kidney glycolipid. Urine sediment from four patients with the gargoylism syndrome* was exam* This term is used to designate disorders categorized clinically as “Hunter-Hurler” and related syndromes. In the classification of McKusick et aLeepatients M7 and M4 corresponded most closeiy to Type I, patient M6 to Type II and patient Frr to Type III. The urines from these cases gave a strongly positive spot test for mucopolysaccharides 30. C&z. Ci&n. Acta, 16 (I) 135-145
WHERRETT
140
ined. In all, the ceramide dihexosides were the most prominent glycolipids (Fig. 3). Other slower neutral glycolipids were present as well but the pattern differed from that of sediment taken from infected urine. Finally, in multiple specimens from a sixteen year old patient with a slowly progressive motor disturbance and dementia diagnosed as metachromatic leukodystrophy, a most unusual pattern was observed (Fig. 3). The ceramide dihexosides were slightly more intense, but there was a striking increase of glycolipid-staining spots which were eluted from DEAE-cellulose by chloroform-methanol containing ammonium acetate and ammonia with the most acidic lipid fractions. Two bands were observed which on several chromatograms were double and the slower was less intense than the faster moving. In addition, faint slower and faster bands were detected. This fraction was treated with acid as described by Stoffyn and Stoffyn3r and on chromatography, there was a decrease in all of the previously noted bands with the appearance of two cerebrosides and two ceramide dihexoside bands. As well, two further bands appeared. One gave the reaction for glycolipid and ran just in front of the two cerebroside bands and the other was a small band running in the appropriate position and giving the appropriate colour reaction for cholesterol. Quantitative
analysis
The quantitative analysis of samples from four normals, three cases of the gargoylism syndrome and a case of metachromatic leukodystrophy are shown in Table I. In the normal adults, the amount of lipid excreted in the sediment determined by weighing is again seen to be small and agrees with values obtained by the dichromate-reduction procedure. Estimations of cholesterol, phospholipid and sphingosine did not account for more than 60 “/ of the lipid, indeed the sum of these fractions varied considerably. Values for the phospholipid fractions from the DEAE column are shown in Table II. In general the distribution of phosphorus in the fractions except for normal sample F40 follows the pattern observed in many tissue;.
Total lipid mg/g cveatisine
Cholestevol mg/~oo mg lipid
Phospholipid nzgl~oo wag lipid
Glyco.sphz+zgolipid ~mzoles/~oo mg lipid _
9.2
7.8 16.7
2 Gargoylism Frr M7 MO 3
Metachromatic leukodvstrophq I216
* Sex and
5:::
0.85 0.15 0.05 0.18
9.4
6.4 15.6 II.3
2.8 5.4 9.7
0.65
16.7
IL.1
4.0
3.4
53.2 19.5 8.6
6.6
Aczdic
2.0 5.4 4.7 I.0
10.5
8.9 5.’ -
age of individual.
iVeutYa1 ~~~~~
.~______.
‘5.7 23.5
0.14
0.25
‘9.7
POLAR
LIPIDS
TABLE
AND
PATHOLOGICAL
141
URINES
II
DISTRIBUTION
Lipid
IN NORMAL
OF
PHOSPHORUS
IN
THE
COLUMN
FRACTIONS
P as o’;
fraction
of total
P in fractions Gavgoyltsm
NOrmal.
Choline-containing Ethanolamine glycerol phosphatide Serine glycerol phosphatide Minor acidic phosphatides Other fractions 9; l
Fzo*
F23
F4o
M28
42.4 22.5
23.7 36.4 5.6 24.5 7.6 98.0
10.3
31.8
54.6 4.3 28.4
25.7
2.4 80.1
8.4 94.1
I9.0
12.9 3.4 95.0
19.0
15.1
M6
-__--___26.5 21.8 15.0 31.2 5.6 89.1
MLD
M 7
23.6 23.7 10.7 25.2 16.5 X7.1
FI6 58.4 8.6 7.6 17.9 7.6 92.0
* Sex and age of individual. TABLE MOLAR
III RATIOS
SOWCL?
OF
GLYCOLIPIDS pmoles
~__ Neutral
Normal F 20*
Gargoylism F II M7 MO Metachromatic F 16 *
PHOSPHOLIPID
__-
IN
lipid
URINE
SEDIMENT
P
Acidic
0.17 0.07
0.05 0.03 0.01
0.18
0.01
0.34
0.02
0.11
F 23 F 40 M 28
TO
Sphingosine/pmoles
0.27
0.03
0.66
0.02
leukodystrophy 0.22
1.80
Sex and age of individual.
The content of the neutral glycolipids in urine sediment is similar to that found in most tissues outside the nervous system and there is no apparent difference between the normal and abnormal specimens. If it can be assumed that the polar lipids exist primarily as components of structural lipoproteins in close association, a ratio of these components might indicate a disturbance of the metabolism of either. In Table III, the molar ratios of neutral and acidic glycolipids to phospholipid are calculated from the results in Table I. In urine sediment from patients with the gargoylism syndrome, the ratio for neutral glycolipids is elevated, in one instance over three times that of the normals whereas the ratio for acidic glycolipids is the same. The sample from the patients with metachromatic leukodystrophy shows a striking increase in acidic glycolipid content and in molar ratio of acidic glycolipid to lipid P. There is also a slight increase in the molar ratio of neutral glycolipid to lipid P. DISCUSSION
Asalytical
method
Several problems arise when attempting to analyze urine sediment lipids, not the least of which is sampling. Ideally, one would wish to obtain a timed sample of urine in which the quantities of lipid-contributing elements is known, in which the Clin. Chzm. Acta,
16
(I)
135-145
142
WHERRETT
processes of degradation are controlled and which is free of cont~ninatio~. For accurate cell counts, it would be necessary to make collections frequently throughout the 24-h period. To obtain specimens from the female uncontaminated by perineal secretions and cells, either very careful technique is required or it is necessary to resort to catheterization. The former was not practical and repeated catheterization would produce increased cell excretion. When methods of extraction and rectification suitable for many tissues were applied to urine sediment, a number of unidentified substances not encountered in other tissues were included in the extract. Some of these w-ere probably drug metabolites. An important portion of the lipid not studied is apparently neutral lipid other than cholesterol Thus the values obtained may have little significance when related to period of production or to the total material collected as lipid. It may be useful therefore, to relate lipid classes one to another. With care, phosphorus recoveries of roe% are obtained from the combination of columns used when extracts of tissue are the objects of examination. In the column runs reported, the low recovery may be attributed to tecbnica.1 errors in dealing with small samples. In one case {Sample F40), rerunning the column gave identical results suggesting that phosphorus-containing compounds may remain on the columns. By using DEAE-cellulose, the loss of acidic compounds which are known to be incompletely eluted from alumina 3%is avoided. Sphingosine recovery was not attempted but was judged satisfactory from the thin-layer chromatograms, The extent of degradation either prior to or after extraction cannot be assessed, but ethanolamine phosphatides were low in the one sample (MLD) that had been freezedried for some months prior to extraction and this effect has been noted when other tissues were analyzeda3. Source of urine se&&em! lipid Urine sediment lipid can be considered to originate from structural elements of cells including structural lipoproteins and from soluble lipoproteins derived from other sources. The cellular material will have been released from lining epithelia and glands of the urinary tract and from other cells migrating into the tract, such as blood cells. The rate of excretion of cells is known to vary significantly during the 24-h period”” and Rofea4 found that red cell excretion rate continued to increase during the day as the excretion of other types of cells diminished, Rofe’s counts, taken during a period close to the maximal rate for all types of cells other than red cells in the male, showed that about rsoooo cells were excreted per hour, of which 2% were erythrocytes, 350/ were polyrnorphonuclear leukocytes and the rest epithelial cells of various type and stage of degeneration. Epithelial cells, considered most likely to have been derived from kidney tubules comprised about 3% of total cells. If it is assumed that twice as many degenerated cells originated in the kidney tubules but could not be classified as such, then not more than 10% of the total would have originated in the kidney. Soluble lipoproteins may also be released from cells lining the tract as glandular secretions or degeneration products or be transported into the urine from a site of synthesis outside the urinary tract where they are rendered soluble. At the centrifugal force-time integral employed to collect the sediment, mucoproteins also are deposited33. Thus the pattern of urine lipids reflects, at best, a mean of the lipid make-up of the various populations of cells, cellular elements and lipoproteins. Without knowing
POLAR
LIPIDS
IN NORMAL
AND
PATHOLOGICAL
URINES
I43
the content of each lipid class in each type of cell, one cannot estimate the significance of the contribution from each type to the whole pattern. It seems unlikely, however, that lipids of red cell origin would have a significant influence on the pattern. Unless striking specificities of localization exist, the lipid pattern from cells of renal tubular origin will probably be obscured in a pattern derived from epithelial cells and to a lesser extent polymorphonuclear leukocytes. Observed &bid patterns Major attention may be directed to sphingoglycolipid classes which, in contrast to phospholipids, show specificities of tissue and cell localization3a and which are implicated in abnormalities of the urine sediment in disease. Thus the predominant pattern in the normal is a complex one in which glycolipids similar or identical to cerebrosides are most abundant components. Separation of individual cerebrosides on thin-layer chromatography using silica gel occurs on the basis of fatty acid content, the hydroxy fatty acid-containing cerebrosides having a lower RF than the nonhydroxy fatty acid cerebrosides 26~37.Other glycolipids with higher RF values than cerebrosides have been described 38+, but it would appear that substances other than these were observed because of their proximity on chromatograms to the more usually observed cerebrosides. They could represent new glycolipid species characteristic of cellular material released into the urinary tract or have resulted from some alteration of the usual cerebrosides on release into the urine. Alterations in the normal lipid pattern would be detected if the ratios of the contributing elements were altered or if there was a metabolic disturbance within one or more of the cell types large enough to be detected. An example of the first type occurred when infected urine was examined. Here a large increase in lipid content and a striking alteration in the glycolipid pattern occurred. The major glycolipid was ceramide dihexoside which contained both hydroxy and non-hydroxy fatty acids. An unknown glycolipid which was much more polar and which contained both hydroxy and non-hydroxy fatty acids was present also, giving a glycolipid pattern that was recognized as characteristic of human leukocytes 4c.41. In two diseases in which genetically determined errors in metabolism result in abnormalities of gly~osphingolipids, metacllromatic leukodystroph~ and the gargoylism syndrome, alterations in the urine pattern were evident. Detailed analyses of brain and kidney have been carried out in metachromatic leukodystrophy12-4J. In the urine sediment excreted by patients with this disease, sulphatides8,46 and a ceramide dihexoside sulphate47 identified by paper chromatography were found to be increased but the lipids have not been further characterized. The observations on the present case give added confirmation of the nature of the metachromatic lipids excreted in the urine sediment. The value for total lipid (in abnormally nourished patient) suggests that the increased sulphated glycolipid (twenty-two times or more) does not result from an increased excretion of a particular type of cell but rather from a disturbance within a normal number of cells shed into the urine, presumably those arising in kidney tubules. Because these cells constitute a small percentage of the total cells excreted, the chemical disturbance within the cells would be of even greater magnitude than that reflected in the sediment lipid pattern. There is some evidence that one of the extra “cerebroside” bands observed may be sulphated. The gargoylism syndrome is recognized to comprise several entities with a CEi??.ChiV%ACtU,
16
(I)
135-I45
WHERRETT
I44
disturbance in mucopolysaccharide metabolism 8s,a8. However lipid abnormalities have also been well documented, including disturbances of gangliosides in brain*+jz and complex glycolipids in liver, spieen53, and kichrey54. The finding of an altered gIycolipid pattern characterized by an increased ceramide dihexoside in urine sediment from at least some forms of gargoylism provides further evidence for a disturbance in gl~co~i~~~~~sas well as mucopolysaccharides. That this may arise in the kidney has been eon~rnle~~ in one case at ~~osi-~~l(}r~en~in which a s-fold increase in kidney ceramide dihexoside occurredZ6. Because detailed cell counts could not be done, the possibility that an increased ratio of Ieukocytes were responsible for the urine sediment pattern in these cases is not excluded. However, in three cases, the molar ratio of neutral glycolipid to phospholipid was elevated over that. observed for pure leukocytes 31 and the column fraction in which neutral glycolipids were eluted gave a pattern on thin-layer chro~natography which was unlike that of the leukocytes. Morpi~ologica~ abnormalities have been noted in the urine sediment from Krabbe’s disease and adult Gaucher’s diseases and in Fahry’s diseaselO, and it will he of interest to know if there are alterations in the urine sediment lipid pattern. The relation of the metabolism disturbances of ~nuco~~r)lysa~charid~ and of glycohpid in the gargoylism syndrome is an interesting unsolved problem.
The author is indebted to Dr. 17. Wilmington (Ol~tar~o Hospital School, Oril~~a~ and Dr. J. C. Richardson (D~partmellt of Medicine) and Dr. W. J. Whaler for generous assistance in obtaining samples for study. The technical assistance of Mrs. D. Kober is gratefuliy acknowledged. The work was supported in part by Grant MA-2235 from the Medical Research Council of Canada. The author was a Fellow of the American College of Physicians and is a John and Mary R. Markle Scholar.
Ctigz. C/&n.
Acta,
16 (I) '35-145
POLAR
21 22 23 24 “5 26 27
28 29
LIPIDS
IN NORMAL
AND
PATHOLOGICAL
URINES
I45
C. J. LAUTER A?*'DE. G. TRAMS, J. Lipid Res., 3jrg6z) 136. G. HARRIS AND 1. c. &b.C%'ILLIAMS,Cherrz. & IEd. (Londonj, (1954) 279. J. S. AMENTA, J. Lipid Xes.,5 (1964) 270. G. M. GRAY, Nafure, 207 (1965) 505. E. SVENNERHOLM AND L. SVENNERHOLM, Nature, 198 (1963) 688. J, R. WHERRETT, unpublished observation. J. R. WHERRETT ASD J. N. CUMINGS, Lliochem.j., 86 (1963) 378. E. MARTENSSOK, B&hi% Bzo#yys. Acta, I 16 (1966) 296. V. A. MCKUSICK, D. KAPLAX, D. WISE, W. B. HANLIXY, S. B. SUDD~RTH, A. E. MAIU~~IA~TEE, Xsdicinc, 44 (1965) 445.
M. B. SEVICK .%.uD
30 31 32 33
34 35 W. H. BOYCR AND M. SWANSON, J. Clin. Iwest., 34 (1955) 1581. AND B. J.\VEBIXR, A~zw. lieu. Bzochcm., 34 (1965) 109. 36 H. E. CARTER, P. JOHNSON H. JATZKKW'ITZ, Z. Ph?~srol. Chwc., 320 (1960) 134. AYD 1.S.G~~~~~n~~,Riochim.Bio~hys. Acfa, 60 (1962) 43. :;iN. Ii.KOCHETKOV, G. Z1irrxov:x 39 W. T. NOKTON AND M. BROTH, Biockem.Bzo~h~s. Rrs. Cowzmm., 12 jr903) 198. _I., 98 (1966) 782. 40 C. J. Xr~xs, J. D. X~~NTZOS AND G. M. LEVIS, Uiuchet?z. E. MARTIN, in preparation. 4I J. it.\VHISRRETT ANI) \;I'. Chenz., 311 (1955) 279. 42 H. JATZKEWITZ, Z. P!z_~~si&.
43 J. H. *JUSTIN, Newolog~, IO (1960) 470. 44 M. LEES AND H. MOSER, 111S. XI.ARONSON AND B. Cl’. VOLK (Eds.),Cerebral .Y$hingolipidoses, Academic Press,New York, 1962, p. 179. 45 I,.SVENNERHOLX, in J. FOLCH-PI .?ND H. BAUER (Eds.),Brazn Lipids md L.ipo$voteinsad the Le~rkod~~sfrophies, Elscvier,Amsterdam, 196.3,p. 104. 46 L. SVENXERHOLM, Acta Che+n. Sand., 17 (1903) 860. 47 E. MARTENSSON, Acfa Chew Scmd., 17 (1963) 1174. in J. B. STANBURY, J. B. WYNGAARDEN AND D. S. FREDGRICKSON (Ed%), The 48 i\. DORFMAN, ~4etubo~ic Basis of fnheviteed i?isense, 2nd Ed., McGraw Hill,Toronto, 1966, p. 936. ed., TV. B. Saunders, Phiis49 J. N. CUMINGS,~~ G. G..DUNC.~ (Ed.),Diseasesqiil~etabolis~,5th delphia, 1964. p. 1405-1439. 50 R. LEDEEN, K. SALSMAK, J. GON.~T~S AND A. TAGHAV~, J. NeuropathoE. Exptl. Se?woE., 24
(1965)341.
57 52 53 54
N. I<. GON~TAS AND J, GONATAS, J. Neuropa2!~ol. Ihptl. _VewoE., 24 (1965) 318. I). A. BOOTH, H. GOODWIK AND J. N. CUMINGS, J. Lipid Res., 7 (1966) 337. I,. L. UZMAN, Arch. PathoE.(Lab. fifed.), 60 (rg55) 308. E. KLENK, quoted bv F. SEITELBERGER, in J. N. CUMINGS AND A. LOWENTAXL bral Lipid&s, Black&cll, Oxford, 1957, p. 77.
(Eds.),Cere-
Clip%. C/&R. Acta, 16 (I) 135-745