The proteoglycans of the cartilaginous end-plate of the human intervertebral disc change after maturity.

Lumbar spines collected postmortem were assigned to one of two groups: group 1--three spines with healthy discs, or group 2--three spines with severely degenerated discs. The proteoglycans (PGs) of the cartilaginous end-plate (CEP) were extracted with 4 M guanidinium chloride containing protease inhibitors and were purified by caesium chloride density gradient ultracentrifugation. On Sepharose CL-2B chromatography, the most dense 20% of the gradient (the A1 fraction) showed two subfractions, one eluting near the void volume and one partitioned by the gel. Both fractions resembled those of the nucleus pulposus and the anulus fibrosus in the number of components seen on agarose-polyacrylamide gel electrophoresis. Both fractions changed with ageing/degeneration; the ratio of keratan sulphate to chondroitin sulphate, which was about 1 in group 1, increased to about 3 in group 2; the hydrodynamic volumes fell; the electrophoretically distinguishable component of lowest mobility disappeared while new, highly mobile components appeared; and the water content decreased slightly. Clearly, the PGs of the CEP of degenerated intervertebral discs differed from those of healthy discs; this supports the view that the CEP participates in the process of ageing/degeneration in the disc.

Degeneration and the chemical composition of the human lumbar intervertebral disc.

Fifteen lumbar spines were collected postmortem. The intervertebral discs were assigned morphological grades and were analyzed for water, collagen, and proteoglycan. In order of increasing degeneration, five grade 0, four grade 1, 45 grade 2, 12 grade 3A, and nine grade 3B discs were identified. The proteoglycan concentration fell progressively with increasing grade, although the concentrations of each component overlapped extensively among the grades. Grade 2 discs showed no consistent differences from adjacent grade 3A or 3B discs in the same spine. All discs in seven of the eight spines with grade 3A or 3B discs and all discs in three spines with no grade 3A or 3B discs had proteoglycan concentrations below 52 mg/g fresh weight. Four of five spines with at least one disc of proteoglycan concentration above this value contained no grade 3A or 3B discs. These observations support the hypothesis that low proteoglycan concentrations in all the discs of a spine precede degeneration.

Effect of age on the abundance and fragmentation of link protein of the human intervertebral disc.

The link proteins of the human intervertebral disc were studied in tissue extracts by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE), followed by immunoblotting, using a specific monoclonal antibody. Three link proteins were detected, corresponding in electrophoretic mobility to those present in articular cartilage. As with articular cartilage, the largest link protein predominates in the young, whereas in the adult the smallest link protein is equally abundant and internal fragmentation of the link proteins occurs. Only in the newborn is the quantity of extractable link protein comparable to that from articular cartilage. In the adult, the disc contains much less link protein than is present in autologous articular cartilage. Neither the amount nor heterogeneity of the link protein differs among different levels within the lumbar spine, although the proportions of the three proteins can differ between the anulus fibrosus and nucleus pulposus. The anulus always contained more extractable link protein relative to tissue wet weight than the nucleus, and the nuclear link protein, at least in adolescents, contained a greater proportion of the smallest link protein. Such changes in the quantity and structure of the disc link proteins may affect the properties of the proteoglycan aggregates and, thus, could influence disc function.

Distribution of glycosaminoglycans across the normal and the scoliotic disc.

This paper reports on our investigations of the glycosaminoglycans (GAGs) (hyaluronic acid, chondroitin-6-sulfate, and keratan sulfate) of 13 adjacent zones of normal and scoliotic discs obtained at necropsy. The isolation and separation of these GAGs on the microgram scale was achieved using the different solubilities of the calcium and cetyl pyridinium chloride complexes. In normal discs the distribution of these GAGs is symmetric about the nucleus pulposus where their concentration is highest, and lowest in the outer annulus regions. A shift in this distribution profile was observed for the GAGs of the scoliotic discs, particularly at the apex of the curve. On the concave side of this disc the chondroitin-6-sulfate was elevated relative to discs at a lower level. In those annulus zones on the convex side of the curve, the keratan sulfate and hyaluronic acid levels were elevated and chondroitin sulfate depressed. These results suggest the presence in these respective regions of at least two different species of proteoglycans accompanied by elevated hyaluronate levels.

Preliminary evaluation of a scheme for grading the gross morphology of the human intervertebral disc.

A five-category grading scheme for assessing the gross morphology of midsagittal sections of the human lumbar intervetebral disc was developed. The ability of three observers to categorize a series of 68 discs with a wide spectrum of morphologies established the comprehensiveness of the classification. Three independent observers tested the reproducibility of the procedure by assignment of grades blindly to duplicate images of 68 discs taken from 15 spines. The intraobserver agreement ranged from 87 to 91%. The interobserver agreement was 61, 64, and 88% for the three pairs, the two low values being attributable to the bias of one observer. The agreement between the assigned and average grades was 85, 92, 68, 90, and 76% for Grades I through V, respectively. Except for Grade III, the disagreements were attributable mainly to the bias of one observer. Both the increase in the grade with age and the finding that all the discs within 14 of 15 spines had a narrow range of grades demonstrated the biologic credibility of the scheme.

Target tissue models: the proteoglycans and degeneration of the human intervertebral disc.

Seventy-five intervertebral discs from 15 spines were analyzed after morphologic grading. With degeneration, the water and proteoglycan (PG) contents decreased and the collagen content increased. The PG was isolated from 20 discs by single-stage dissociative density gradient ultracentrifugation. Neither the chondroitin 6-sulphate nor the protein contents of the PG changed with degeneration although the keratan sulphate fell slightly.

Magnetic resonance imaging reflects the chemical changes of aging degeneration in the human intervertebral disk.

T2 weighed spin echo magnetic resonance images (MRI) of the intervertebral disks of 4 lumbar spines were graded and the nuclei pulposi were analyzed for water, collagen and proteoglycan. The brightness of the nuclear image correlated directly with the proteoglycan concentration, but not with the water or collagen. The dark midnuclear cleft had a collagen concentration slightly higher and a water concentration slightly lower than the adjacent zones; no corresponding differences in proteoglycan were seen, although the relationship with MRI grade was confirmed.

Laboratory screening. A critical survey.

The diagnosis of disease is not synonymous with benefit to the patient. Early diagnosis does not necessarily facilitate cure or improve prospects for control. The discovery of what is already known or could easily have been predicted has little or no value. To evaluate screening one must evaluate the usefulness of the data collected.

Assay of keratan sulfate as anion-exchanger bound hexose.

An assay for keratan sulfate in papain digests of human intervertebral disc and other tissues has been developed. The digest is applied to the acetate form of a tertiary amine acrylic anion-exchange resin, the oligosaccharide hexose is removed by washing the resin with 0.2 M sodium acetate buffer pH 5.0, then the keratan sulfate is eluted quantitatively with 1.0 M pyridinium sulfate pH 2.5 and assayed for hexose by the anthrone reaction. The keratan sulfate content of human intervertebral disc tissues ranged from 7 to 78 mumole galactose equivalents/g fresh weight; the root mean square error was 2 mumole/g; 10-25 mg of tissue were required. The separation of oligosaccharides from keratan sulfate was confirmed by gel permeation chromatography, sugar composition, ester sulfate analysis, and nuclear magnetic resonance.

Exclusion of dextrans by meshworks of collagenous fibres.

Insoluble collagen from human dermis was equilibrated in a physiological medium with mixtures of 3H2O and fluorescein-conjugated dextrans of different molecular weights. Dextrans of mol.wts. greater than 10(5) were excluded from a volume of 3.82+/-0.87 ml(S.D.) per g of collagen; dextrans of lower molecular weight occupied a larger volume. The apparent excluded volume was proportional to the weight of the collagen. Dansylated albumin behaved similarly to dextran; the polymeric collagen from rat skin exhibited a much larger excluded volume than the insoluble collagen. These results indicated that the volume available to the plasma proteins in human dermis was limited by insoluble collagen as well as by the glycosaminoglycans of the tissue.

The proteoglycans of the human intervertebral disc.

The methods of Hascall & Sajdera (1969) were used to compare the proteoglycans of human intervertebral disc with those of bovine nasal cartilage. In contrast with cartilage, most of the hexuronate of disc could be extracted at low shear with water or dilute salt solutions. Extracts of disc with 4M-guanidinium chloride were centrifugated in 0.4M-guanidinium chloride in a CsCl gradient. Analytical ultracentrifugation of the hexuronate-containing heavy component revealed two fractions. both more polydisperse than those of cartilage. Also the more rapidly sediminting component was a much smaller fraction of the total. After prior extraction with 0.4M-guanidinium chloride, 4M-guanidinium chloride extracts of disc were found, by ultracentrifugal analysis, to be enriched in components resembling the proteoglycan monomer and aggregating factors of cartilage.

The chemical constitution of the proteoglycan of human intervertebral disc.

Proteoglycan was prepared from three pools of normal human intervertebral discs by extraction with buffered 4M-guanidinium chloride followed by CsCl-density-gradient ultracentrifugation. Chromatography on agarose (Bio-Gel A-150m) and on DEAE-cellulose suggested a single polydisperse proteoglycan species. The intrinsic viscosities of three preparations were 166, 122 and 168 ml/g. After degradation with 0.5M-KOH containing 0.02M-NaBH4, the glycosaminoglycans were recovered quantitatively and their Ca2+ salts separated into a hexuronate-rich fraction (fraction 1), which was precipitated in 0-45% (v/v) ethanol, and a hexose-rich fraction (fraction2), which was precipitated in 45-70% (v/v) ethanol. Qualitative and quantitative analyses of the glycosaminoglycans revealed fraction 1 to be chondroitin sulphate, and fraction 2 to be keratan sulphate; the latter was contaminated with protein and possibly a small amount of another glycosaminoglycan. For both glycosaminoglycans, plots of log(mol.wt.) against weight fell close to a normal distribution. The mode for chondroitin sulphate was close to 20000; that for keratan sulphate, 10000. A threefold range of molecular weight included the central 16-84% [+/- 1 S.D. of log(mol.wt.)] of the weight of both fractions.

The isolation of minimally degraded hyaluronate from rat skin.

Hyaluronate was isolated quantitatively from fresh rat skin by homogenization in an Edebo [J. Biochem. Microbiol. Technol. Eng. 2, (1960) 453-479] press, extraction with buffered saline, selective elution from DEAE-cellulose, and gel chromatography on Sephadex G-200. Traces of a protein contaminant remained.

Demonstration of link protein in proteoglycan aggregates from human intervertebral disc.

Proteoglycan aggregates free of non-aggregating proteoglycan have been prepared from the annuli fibrosi and nuclei pulposi of intervertebral discs of three human lumbar spines by extraction with 4M-guanidinium chloride, associative density gradient centrifugation, and chromatography on Sepharose CL-2B. The aggregate (A1-2B.V0) was subjected to dissociative density-gradient ultracentrifugation. Three proteins of Mr 38 900, 44 200 and 50 100 found in the fraction of low buoyant density (A1-2B.V0-D4) reacted with antibodies to link protein from newborn human articular cartilage. After reduction with mercaptoethanol, two proteins of Mr 43 000 and two of Mr 20 000 and 14 000 were seen. The A1-2B.V0-D4 fraction, labelled with 125I, coeluted with both hyaluronate and a hyaluronate oligosaccharide (HA14) on a Sepharose CL-2B column. HA10 and HA14 reduced the viscosity of A1 fractions; HA4, HA6 and HA8 did not. HA14 decreased the viscosity of disc proteoglycans less than it did that of bovine cartilage proteoglycans. Thus, although a link protein was present in human intervertebral disc, it stabilized proteoglycan aggregates less well than did the link protein from bovine nasal cartilage.

The exclusion of human serum albumin by human dermal collagenous fibres and within human dermis.

Preparations of dermal collagenous fibres and slices of human dermis have been equilibrated with 125I-labelled monomeric human serum albumin. The space inaccessible to the albumin in the fibres and in the dermis was determined by subtraction of the accessible space, calculated from the radioactivity of the specimen, from its total fluid. For a fibre preparation examined in detail, the fluid exclusion was independent of the concentration of either albumin or collagen. Binding of albumin to the fibres was not demonstrable. Three fibre preparations excluded albumin from 3.75 +/- 0.96, 3.55 +/- 0.67, and 2.05 +/- 0.39 g of fluid/g of collagen (+/-S.D.). Slices from three specimens of dermis excluded albumin from 1.45 +/- 0.08 g of fluid/g of insoluble solids or 1.57 +/- 0.11 g of fluid/g of collagen (+/-S.D.). Thus the exclusion of albumin by dermis was much less than expected from its content of collagenous fibres. On the basis of these data and the published composition of dermis, the concentration of albumin in the accessible interstitial space was estimated to be close to that in the plasma.

Concentration of plasma albumin in its accessible space in postmortem human dermis.

This study was designed to measure the effective concentration of plasma albumin in the interstitial space of human dermis. Discs of tissue taken postmortem from four donors have been separately analyzed for their content of plasma albumin and equilibrated with 125I-labeled monomeric plasma albumin in a specially designed cell which limited tissue swelling. The equilibrated discs and their surrounding fluid were assayed for radioactivity and the tissue space accessible to albumin was calculated after correction for swelling. The albumin content of serum was also measured. The concentration of albumin in the accessible space of the tissue ranged from 0.45 to 0.93 that in serum, averaging 0.68. The fraction of the total interstitial fluid accessible to albumin averaged, for three normal dermises, 0.35 and for an overhydrated specimen, 0.51. Thus, the effect of volume exclusion should be considered in measurements of the concentrations of plasma proteins in tissue.

Admissions screening: clinical benefits.

One thousand patients admitted to a 575-bed general hospital during a 6-month period each underwent 20 chemical and hematologic tests. The potential clinical benefit was assessed. There were 2223 abnormal results found; 675 were predicted on clinical assessment, 1325 did not yield new diagnoses, and the remaining 223 led to 83 new diagnoses in 77 patients. On critical evaluation of the new diagnoses, none were unequivocally beneficial to the patient. Up to 30 patients might have benefited had these abnormal findings been followed up diligently, 39 others had findings or diagnoses of no lasting significance, and in 14 patients asymptomatic mild biochemical diabetes was discovered. Although screening may reveal many abnormal test results, the clinical benefits are not impressive.

The heterogeneity of the non-aggregating proteoglycans of the human intervertebral disc.

Non-aggregating proteoglycans of differing average hydrodynamic volumes were prepared from nuclei pulposi and anuli fibrosi of three human lumbar spines and characterized by biochemical and immunochemical analyses. The hexose-to-hexuronate and protein-to-hexuronate ratios increased with decreasing hydrodynamic volume. Analysis by composite agarose/polyacrylamide-gel electrophoresis has demonstrated two aggregating subpopulations [McDevitt, Jahnke & Green (1982) Trans. Annu. Meet. Orthop. Res. Soc. 7, 50]. In the present study, electrophoresis of the non-aggregating pools has shown three additional subpopulations, here named bands III, IV and V. The two smallest proteoglycan pools from each tissue contained two and three components respectively. These components were isolated by preparative electrophoresis and analysed. Band III was a proteoglycan richer in keratan sulphate than in chondroitin sulphate; band IV was a proteoglycan richer in chondroitin sulphate than in keratan sulphate; band V contained only chondroitin sulphate. Unsaturated disaccharides prepared from the chondroitin sulphate of all bands were predominantly 6-sulphated, with only 5-15% 4-sulphated. The molecular masses of the chondroitin sulphate and keratan sulphate did not differ between the bands. The amino acid composition of band III differed from that of band IV. Thus three distinct subpopulations of non-aggregating proteoglycan were demonstrated in the human intervertebral disc.