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The nature of phospholipids in normal and rachitic costochondral plates.

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The Nature of Phospholipids in Normal and
Rachitic Costochondral Plates
By DAVID
S. HOWELL,
JUAN F. MAHQUEZ
AND
Interest in fundamental biochemical
processes ol mineralization in osteoid
led to a study of rachitic calf costal
junctions in respect to a profile of
phospholipids. Currently, chloroformmethanol extracts of rachitic and normal costochondral junctions were applied to thin-layer silica gel chromatograms, and the phospholipids thereby
separated were quantitated in terms of
phosphorus content of the eluted spots.
Striking differences between normal and
rachitic tissues were found in respect
to this profile of phospholipids.
JULIO
C. PITA
Rachitic junctiones costal de vitello esseva studiate relative a lor profilo phospholotidic. Le studio esseva interprendite conio contribution a1 investigation
del basic processos biochimic participante in le mineralisation de osteoide.
Extractos a chloroformo e methanol ex
normal e rachitic junctiones costochondral esseva applicate a systemas chromatographic a gel de d i c e in strato
tenue, e le assi-separate phospholipidos
esseva quantificate relative a1 contento
de phosphor0 del areas eluite. Frappante differentias esseva constatate inter
tissues normal e tissus rachitic.
A
CONSISTENT SELECTIVE STAINING of mineralizing sites in a variety of tissues by Sudan black'J was demonstrated by J. T. Irving and
co-workers.' Histochemical studies of extracellular sites led to a tentative
identification of the material as a lipide2
Sudan black as well as the Baker's stain for phospholipids and postulated a
distinctive role for phospholipids in formation of mineralizing matrix.3 In a
prior study of Ca4?uptake by substances in aortic walls in vitro, Fels surmised calcium binding by a phospholipid-protein c ~ m p l e x .The
~
fact that
the Sudan black staining reactions for lipids were not noted in dentinal pulp
or rapidly growing periosteal membranes directly adjacent to the sites of
mineral deposition, or in rachitic osteoid, suggested that this staining is not
a property of a nonspecific proliferating fibrous matrix but rather related
distinctively to those extracellular sites involved in the process of mineral
up take. ' , ~ 2 0
A regulatory function for lipids in cell membrane transport of electrolytes
and other substances has received considerable attention, as recently reviewed," so that a possible selective role for these compounds in mineral ion
or water transfer deserves exploration at calcifying regions.5~6~17~'s~24
The present report concerns quantitative identification of one class of
lipids in relation to histologic sites of normal mineralization in contrast to
From the Division of Arthritis, Department of A4 edicine, University of Miami S c b o l
of Medicine, Miami, Flu.
Supported by Grants AM 08662-01 and A-1155 of the National Institutes of Arthritis
und Metabolic Diseases, Bethesda, Md.
DAVID s. HOWELL,M.D.: Associate Professor of Medicine; JUAN I?. MAHQUFZ, M.D.:
Reser~rclaInstructor; JULIO C. PITA, P H . ~: .Visiting Professor of Physical Chemistry; a11
of the Depnrtmcnt of Medicine, University of Minmi School of Medicine, University of
M u i m i , Miami, Flu.
1039
1040
HOWELL, MARQUE2 AND PITA
similar poorly mineralized sites of rachitic tissues. Rachitic calf costal and adjacent costal osteoid was analyzed for phospholipid content and the results
compared to a similar assessment of normal juxtametaphyseal cartilage and
osteoid." These preliminary observations indicate that, in addition to the welldocumented electrolytic abnormalities of nutritional rickets, there are probably important differences in phospholipid profile between a rachitic and
normal cartilage and osteoid.
MATERIALSAND METHODS
I. Tissue Preparation and Histologic Description of Tissue Fractions
Rachitic calves were prepared at the Cattle Breeding Institute, Wiad, Sweden, as described elsewhere.? Normal (control) calves were raised in a similar environment and
received a similar diet with the exception that whole milk, hay, supplementary mixtures
of vitamin D, calcium, and phosphorus were added. The experimental animals developed
weakness, ataxia, lameness and excessive epiphyseal prominences of their extremities as
predicted from previous studies.8,g Blood samples from 3 of the rachitic calves, obtained
at the time of sacrifice, revealed a low serum (calcium) x (phosphate) concentration
product in each instance. Three calves were sacrificed 1-3 weeks after the first objective
clinical manifestations of rickets, which appeared at 4 to 6 months of age. A fourth animal died from an unidentified infection, and some of the rachitic tissues showed spontaneous healing. Four control animals developed satisfactorily and were sacrificed concurrently with the experimental group. Following sacrifice the costochondral tissues of
the first 9 ribs, bilaterally, were dissected free and quick-frozen in isopentane surrounded
by liquid nitrogen.
The costochondral junctions from rachitic, as well as normal calves, were cleaned of
surrounding looye connective tissues, divided sagitally and cross-sectioned into 3 fractions under a dissecting microscope. These fractions included predominantly: ( 1) Resting
and proliferating cell cartilage (fractions A-I and B-I); ( 2 ) hypertrophic cell cartilage
and adjacent coatings of osteoid (fractions A-I1 and B-II), and ( 3 ) metaphyseal osteoid
(fraction B-111). Fraction A-I11 contained bone marrow, principally of the cellular variety,
and trabecular bone, but was not analyzed biochemically for the present study. A description of histologic methods and tissue separation has been given in detail previously.7-15 Representative samples of these tissue regions, studied biochemically, were sectioned, frozen in a cryostat, and stained with hematoxylin and eosin, as well as silver
nitrate, by the Von Kossa method to assess mineral distribution. Sudan black was
also employed, including the pyridine extraction step of Irving.1
2. Biochemical Methods
Representative samples of each fraction were diced and ground to 60 mesh, tared, wet,
and dried over calcium chloride in a dessicator to constant weight. The dried tissues
were extracted 3 times with cold 0.9 per cent buffered saline to remove water soluble
contaminants. Residues were suspended in chloroform and reground in a Virtis-45 homogenizer for 1 hour. Ten ml. samples of the suspension were placed in a separatory funnel
and washed with 5 ml. of .02 M sulfuric acid. Three such washings were followed by
2 additional extractions with chloroform-methanol, 2:l mixture. The extracts were pooled
and dried by lyophylization. Total phospholipid was cstimated from these residues by
total phosphorus content, employing a conventional phosphomolybdic acid procedure.15
For identification of individual phospholipids, ascending silica gel chromatography was
used.12 Plates were prepared according to Stah111 and were composed of a neutral thin
layer (0.25 mm. thickness) silica1 gel G# 7731, E. Merck AG Darmstadt, Germany, 20
~
*Some unmineralized juxtacartilagin~usborders justify this term in these control ribs.
1041
NATURE OF PHOSPHOLIPIDS
Gm. in 50 ml. of water. Chromatograms were developed as described by Wagner13 for
a distance of 12 cm. in chloroform-methanol-water, 65:25:4. The mean Rf for known
samples of phosphatidylethanolamine was 4.6 cm. for phosphatidylcholine, 2.4 cm. sphingomyelin, paired spots, 1.9 and 1.6 cm. respectively, and lysophosphatidylcholine 1.1 cm.
The points of migration were identified with iodine vapor. Corresponding spots were removed from the plates, digested in perchloric acid-nitric acid, 4:1, according to Bartlettl4
and total phosphorus content measured as mentioned above. Slight modification in procedure and calculation of recoveries of p on chromatogram were made according to
Slotta.10 Using purified calf brain phosphatidylethanolamine added to the starting material, 95-97 per cent recoveries were obtained in 4 analyses. This phosphatide was also
used to verify the Rf values mentioned above.*
Reproducibility of results on duplicate samples and recovery of P in phospholipid
analyses of mitochondria were compared with and without purification of starting extracts on chromatographic columns charged with silica gel; since the extra step failed
to improve reproducibility of results, the step was omitted.10 Also, partial loss of
Sudan black staining in sections, treated briefly with EDTA, interdicted steps to remove mineral from starting samplcs other than through mild hydrolysis.
RESULTS
Histologic assessments of rachitic tissue fractions B-I and B-11, in respect
to cellular composition, indicated similar cell types to those of A-I and A-I1
fractions respectively. The currently studied tissue samples were similar to
those analyzed for mucopolysaccharides previously in respect to histochemical
evidence of lipid distribution.? Thus, Sudan black staining by the method of
Irving1 including the pyridine extraction step, registered slight but consistently positive reactions in all of the cartilage cells and a strongly positive
reaction bordering bone salts in the extracellular matrices of fraction A-I1
cartilage and osteoid (fig. 1 ) . All histologic regions of fractions A-I, B-I, and
B-I1 were negative with Sudan black staining except at intracellular sites.
The number of cells per mm.3 of fixed tissue measured in histologic sections was strikingly similar to that of normal calf plates of the same age, reported previously.15 The current counts, based on 12 fields each from 60
histologic sections, were: 75
5 (SE) for hypertrophic cell zone, 196 3
for proliferating cell zone, and 59
7 for resting cell zone in comparison
1 for respective zones in normal ~ 1 a t e s .Conl~
to 57 +- 1, 199 4,and 66
current cell counts on rachitic osteoid were 109 3.
Total nitrogen content per unit dry weight of original normal tissue (A-I),
and that of rachitic samples (B-I), lacking mineral salts as seen by histologic
examination, were comparable; total nitrogen content of fractions A-I1 and
B-I11 was lower, probably because of a large component of bone salts (table
1 ) . Total phospholipid content in the extracts of the rachitic cartilage (fractions B-I, B-11, and B-111) was markedly less than in the extracts of the corresponding regions from normal cartilage (fractions A-I and A-I1 in table 1)
In extracts of all the rachitic fractions in comparison to normal controls
the proportion of lipids with the Rf value of phosphatidylethanolamine was
lower, phosphatidylcholine the same, and sphingomyelin elevated. Conse-
*
*
*
*
*
'For detection of small amounts of other phosphatides, a two-dimensional paper chromatographic method has proved useful.21
Fig. 1.- ( A ) Zones of provisional calcification and hypertrophic cells (fraction
A-11, table 1) revealing extracellular staining at sites of mineral deposition (200x
Sudan black). ( B ) Osteoid and cartilage of distal hypertrophic cell areas of a
rachitic calf (fraction B-11, table 1) . Only cellular components stained positively
( 2 0 0 ~Sudan black). ( C ) A thin-layer silica gel chromatogram stained with ammonium molybdate-perchloric acid spray. Abbreviations: 0 = Origin; remainder
indicate phospholipid bands as described in table 1, (fraction B-11) , ( D ) Metaphyseal bone (fraction N-111) of rachitic calf, also showing wide borders of osteoid.
There was no staining in the rachitic osteoid and only weak staining of compact
bone (200 x Sudan black).
1043
NATURE OF PHOSPHOLIPIDS
quently, in all tissue fractions the ratio of phosphatidylcholine to phosphatidylethanolamine was markedly higher in the presence of rickets. In respect to
fractions A-I1 and B-11, the proportion of lipid with the Rf of lysophosphatidylcholine was less in the extracts of rachitic tissue. Only 5-10 per cent of
phospholipid phosphorus failed to migrate from the origin in the chromatograms, indicating that if phospholipid-protein complexes were an important fraction of the extract applied, this did not interfere with mobility of
the phospholipids. As in a previous report, sulfated lipids were not detected
in the rachitic tissues.7
DISCUSSION
A difference of cartilage metabolism of phospholipids between animals
with nutritional rickets and controls was demonstrated by this study. The
uniformly low concentration of phospholipids from the rachitic tissue extracts are compatible with either depressed synthesis or accelerated degradation at these tissue sites, or both. The large difference of phospholipid content between normal and rachitic tissues might have been partially a reflection of difference in cell numbers not registered by the current cell counts,
which were confined to fixed tissues-wherein heterogeneity interdicts more
than a crude assessment of this parameter. Nevertheless these counts favor a
quantitative reduction of phospholipid per unit cell in tissues lacking more
than a trace of extracellular lipids histochemically. Whether tissues other
than cartilage share in this abnormality was not examined. Although the
rachitic calves were force fed and gained weight steadily during their experimental period prior to sacrifice,7 total weights were less than that of control calves; and, by gross inspection, these calves appeared less adipose. Fat
content of diets in control and experimental calves was comparable. However,
dietary components may influence tissue phospholipid^,^^ and an effect on
the metabolism of phospholipid constituents in the experimental group receiving coconut and linseed oil supplements might have occurred.7 This explanation is unlikely, however, because similar differences in phosphatidylcholine and phosphatidylethanolamine content between rachitic and normal
rat epiphyses have been observed (unpublished observations) at age 25-30
days with employment of entirely different dietary regimens and fat constituents. It was found by Cipera et a1.I6 that during healing of rickets in chicks
there was a substantial increase in the acetone-soluble fraction of epiphyseal
cartilage per unit dry weight. Such results during healing are at least consistent with a possible relative deficiency of fatty compounds in the cartilage
of rachitic chicks.
A definite alteration in phospholipid profile seemed evident here (table 1).
Although with the mucopolysaccharides, previously studied on these tissues,7
no specific pattern of reduction of chondroitin sulfate A, C, or kerato sulfate
was distinguished, herein differences in respect to the relative content of
phosphatidylethanolamine and sphingomyelin were found (table 1) . Hyaline
cartilage, which was demonstrated to be uncalcified in terms of the Von Kossa stains, exhibited the same pattern of alterations in respect to these particu-
Resting &proliferating cell
cartilage
Hypertrophic
cell cartilage
& osteoid
Metaphyseal
osteoid
Resting & proliferating cell
cartilage
Hypertrophic
cell cartilage
& osteoid
Predominant
Histologic Region
6.16
fO.l
9.35
fO.l
10.2s
+0.111
9.51
f0.3
11.29
kO.111
%
0.137
+0.008
0.322
k0.007
0.090
t0.004
zko.02
0.715
1.290"
zk0.002
Total
Phospholipidst
mg. TO
Rachitic
16.6
16.5
19.3
4
17.8
21.6
4
20.1
33.9
f0.55
4
24.6
34.6
f0.94
32.6
i0.72
30.4
k0.94
33.4
f0.80
'ib
Phosphatidylchohe:
(PC)
i0.60
4
k 1.0
26.1
23.5
k0.84
32.8
22.1
37.5
k1.6
%
.a.
Ccmtrol
19.8
Phosphatidylethanolaminet
(PE)
11.3
4
32.8
28.5
+0.85
28.3
f1.5
18.9
21.3
17.9
k1.2
%
Sphingomyelrnl
(SPG)
Normal and Rachitic Osteoid and Cartilage
Recovery of
Phospholipid-P in
4 Fractions
Columns 5-8
of
17.5
23.0
22.0
MLg.
Total
Phospholipid-P
Applied
Phospholiuid Components
Total
Nitrogen*
.- Maior
Number samples
6
6
analyzed per fraction
*Nitrogen and phospholipids are calculated on basis of dry weight, original tissue.
+Phospholipid value = Phosphorus
25.
$Percentages in columns 5-8 are referred to total phospholipid-P applied to chromatograms.
$Mean.
I1S.D.
B-I11
B-I1
B-I
A-I1
A-I
Fraction
Number
Table 1
k0.62
4
9.43
9.80
f0.56
15.6
f1.1
16.6
f1.0
11.7
zko.90
'ib
Lysophosphatidylcholinet
(LPC)
1.38
1.32
1.38
0.89
0.89
Ratio
PC/PE
3
N
El
ro
G
z
rr
8
1045
NATURE OF PHOSPHOLIPIDS
lar phospholipids as the osteoid fractions, thereby indicating a general relationship to the rachitic state in these connective tissues. The polysaccharide
content7 was similarly low within the noncalcified as well as within mineralrich tissue fractions. It is unlikely that traces of blood or plasma lipids contained within the samples importantly influenced current data. Fraction B-I,
virtually lacking blood vessels, did not reveal, in regard to the 3 phosphatides
already discussed, phospholipid profile different from that of B-11, which
contained numerous capillaries. Inasmuch as a large component of the phospholipids of fraction A-I1 were probably extracellular ( fig. I-A), this A-I1
phospholipid profile is believed to approximate most closely that of the extracellular matrix at sites stained by Sudan black. The finding of a high proportion of lysophosphatidylcholine encourages further examination of lipid
profiles for unique features which might be amenable to study in model systems of in vitro calcification.
In view of the effect of vitamin D uptake on calcium translocation in kidney
mitochondria rich in phospholipid^,^^ the selectivity of cation transfer through
membranes coated with phospholipid,24 and the histochemical identification
of phospholipids at mineralizing sites in bone and dentine,2,3 the role of
lipids and particularly phospholipids in mineralization of these tissues demands further scrutiny.
SUMMARY
1. Phospholipid content of costal cartilage and osteoid in calves with nutritional rickets was approximately one-tenth to one-half that found in costochondral junctions dissected from normal animals of comparable age.
2. In the rachitic tissue, the proportion of phosphatidylethanolamine was
decreased and sphingomyelin increased, whereas that of phosphatidylcholine
remained constant when compared to normal.
3. One feature that distinguished normal from rachitic osteoid-cartilage
junctional tissue was a higher proportion of phospholipid with the Rf of
lysophosphatidylcholine. Possible causes of these differences in phospholipid
distribution are discussed.
ACKNOWLEDGMENTS
The authors are indebted to Professors A. Hansson and A. Neimann-Sorensen as well
as to Agronomists L.-E. Liljedahl and T. Henningsson for the use of facilities in the
preparation of rachitic calves.
REFERENCES
1. Irving, J. T.: Histochemical changes in
early stages of calcification. Clin.
Orthop. 17:92, 1960.
2. Irving, J. T.: The sudanophilic material
at sites of calcification. Arch. Oral
Biol. 8:735, 1963.
3. Johnson, L. C.: Mineralization of turkey
leg tendon. 1. Histology and histochemistry of mineralization. Calcifica-
tion in biological systems. R. F.
Sognnaes, Ed. Washington, D. C.,
Am. Assoc. for the Advancement of
Science, 1960, p. 117.
4. Fels, I. G.: Binding of calcium ions by
the aorta. Nature 190:1012, 1961.
5 . Burgen, A. S. V.: Symposium on the
nature of lipoproteins. The structure
and function of cell membranes.
1046
Canad. J. Biochem. Physiol. 40: 1253,
1962.
6. Wooley, D. W., and Campbell, N. K.:
Tissue lipids as ion exchangers for
cations and the relationship to physiologic processes. Biochim. Biophys.
Acta 57:384, 1962.
7. Howell, D. S.: Histologic observations
and biochemical composition of rachitic cartilage with special reference
to niucopolysaccharides. Arthritis
Rheum. 8:337, 1965.
8. Landsburg, K. G., and Hill, 0. J.:
Rickets in calves. Penn. Agricultural
Experiment Station Tech. Bull. 291,
1963.
9. Huffman, C. F., and Duncan, C. W.:
Vitamine D studies in cattle. I. The
antirachitic value of hay in the ration
of daily cattle. J. Dairy Sci. 18:511,
1953.
10. Slottn, K. H.: Mitochondria1 phospholipids in rats of various ages. J.
Geront. 18:236, 1963.
11. Stahl, E.: Thin layer chromatography.
IV. Insertion scheme, marginal effect,
“Acid and base layers,” gradation
technic. Arch. Pharm. ( Weinheim)
292/64:411, 1959.
12. Mangold, H. K.: Thin-layer chromatography of lipids. J. Amer. Oil Chem.
Soc. 38:708, 1961.
13. Wagner, H., Horhammer, L., and
Wolff, P.: Dunnschichtchromatographie von Phosphatiden and Glykolipiden. Biochem. Z. 7 (334)175,
1961.
14. Bartlett, G. R.: Phosphorus assay in
column chromatography. J. Biol.
Chem. 234:466, 1959.
15. Howell, D. S., DelchampT, E., Reimer,
W., and Kiem, I.: A profile of electrolytes in the cartilaginous plate of
HOWELL, MARQUE2 AND PITA
growing ribs. J. Clin. Invest. 39:90,
1960.
16. Cipera, J. D., Migicovsky, B. B., and
Belanger, L. F.: Composition of
epiphyseal cartilage. I. Changes in
hexosamine and acetone extractable
contents of epiphyseal cartilage of
rachitic chicks following administration of Vitamin D3. Canad. J. Biochem. Physiol. 38:807, 1960.
17. Zambotti, V., Cescon, I., Bonferroni,
B., and Bolognani, L.: Lipids of
epiphyseal cartilage. Experentia 18:
318, 1962.
18. Leach, A. A.: The lipids of ox compact
bone. Biochem. J. 69:429, 1958.
19. Leat, W. M. F.: Serum phospholipids
of pigs given different amounts of
linoleic acid. Biochem. J. 91:437,
1964.
20. Howell, D. S., and Carlson, L.: Sulfur
metabolisin in cartilage. A study of
calcifying regions for microscopic
distribution of sulfur and relationship
to staining by Sudan black. Exp.
Cell. Res. 34:568, 1964.
21. Wuthier, R. E., and Irving, J. T.: Un
published observations.
22. Howell, D. S., Marques, J., and Rydberg, M.: The nature of phospholipids in calcifying epiphyseal plates,
Clin. Res. (abst.) 12:90, 1964.
23. De Luca, H. F., Engstrom, G. W., and
Rasmussen. H.: The action of Vitamine D and parathyroid hormone in
citro on calcium uptake and release
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Acad. Sci. (U. S. A.) 48:1604, 1962.
24. Mikulecky, D. C., Tobias, J. M.: Phospholipid-cholesterol membrane model. J. Comp. Cell Physiol. 64:151,
1964.
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