Fred J. Kieras

Analysis by high-performance liquid chromatography of hyaluronic acid and chondrotin sulfates

The use of high-performance liquid chromatography for the quantification of glycosaminoglycan disaccharides has been hampered by the inability to isocratically resolve the chondroitinase digestion products 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-glucose (delta Di-HA) and 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-galactose (delta Di-OS). To overcome this limitation, we have developed a solvent system capable of resolving delta Di-HA, delta Di-OS, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose (delta Di-6S), and 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose (delta Di-4S). Integrator responses were linear from 1 microgram down to 25 ng for delta Di-HA, delta Di-OS, and delta Di-4S and down to 100 ng for delta Di-6S. This method was used to examine changes in the content of urinary hyaluronic acid and chondroitin sulfates isolated from normal individuals and from patients with Lowe Syndrome, Werner Syndrome, and Hutchinson-Gilford Progeria Syndrome. We confirmed that the HPLC method gave results comparable to colorimetric methods.

A Comparison of Adult and Childhood Progerias: Werner Syndrome and Hutchinson-Gilford Progeria Syndrome

The Werner syndrome, also known as progeria of the adult, and the childhood Hutchinson-Gilford Progeria Syndrome (hereafter Progeria), both serve as genetic disease models of human aging (Brown, 1979). A comparison of their similarities and differences may be useful in order to gain insight into the nature of the genetic mutations underlying these conditions. Their modes of inheritance indicate the involvement of a single gene. This implies that some specific genes may lead to a phenotype of greatly accelerated senescence and that such genes may have direct effects on the rate of aging. Determining the basic mechanisms involved in producing their phenotypes may point the way to an understanding of important pathogenetic aspects underlying the aging process.

Progeria, A Model Disease for the Study of Accelerated Aging

The cause of aging is little understood at the present time, but it does appear to have a strong genetic component. This is reflected by the wide variation of approximately 50000 fold, in the maximal lifespan potential (MLP), seen in the various animal species (Brown, 1979). Among even mammals, approximately a 100-fold variation in MLP is seen. The smokey shrew has a lifespan of only about one year, while the oldest documented human died at the age of 118, and second to him the oldest human lived to 113 (McWhirter, 1984).

Urinary hyaluronic acid elevation in Hutchinson-Gilford progeria syndrome

The basic genetic defect in the Hutchinson-Gilford Progeria Syndrome (progeria), a premature aging syndrome, is unknown. To investigate possible defects in hyaluronic acid (HA) metabolism in this disease, the urinary excretion of HA was studied. Urine specimens from 11 patients with this disorder were examined for HA by a novel high performance liquid chromatography (HPLC) technique. In patients with progeria, HA excretion ranged from 169 micrograms HA/g creatinine to 1440 micrograms HA/g creatinine. In normal age-matched controls, HA excreted ranged from 0 to 77 micrograms HA/g creatinine. In all, a mean 17-fold increase in HA excretion was observed in patients with progeria when compared with age-matched normal controls. Total glycosaminoglycan (GAG) excretion was not elevated. Amongst normal controls, a modest age-related increase in HA excretion was observed. These results suggest that urinary HA levels are abnormally elevated in progeria.

Elevation of urinary hyaluronic acid in Werner's syndrome and progeria

Werners syndrome and Hutchinson-Gilford progeria syndrome (progeria) are human genetic diseases which may serve as models for the study of premature aging. The basic defects underlying these diseases are unknown. An abnormally high level of urinary hyaluronic acid (HA) excretion has been previously reported in several Werners and one progeria subject, all from Japan. To determine if a high HA level is a reliable marker for these diseases, we quantitated the urinary excretion of HA in three progeria subjects, one subject with an atypical progeroid syndrome, and a Werners syndrome subject. Compared to controls, the total urinary HA was found to be markedly increased in the three progeria samples and in the Werners syndrome sample. These findings support the previous observations indicating elevated HA may be a specific marker for these diseases.

Characterization of proteoglycans in Alzheimer's disease fibroblasts

Skin fibroblasts lines established from patients with Alzheimers disease and old normal individuals were cultured with 35S-sodium sulfate and 3H-glucosamine. Proteoglycans were isolated and characterized. Sulfate incorporation into proteoglycans increased in Alzheimers disease fibroblasts relative to normal controls. These increases changed the ratio of chondroitin sulfate to heparan sulfate proteoglycan from 1.4 to 1.7 (p = 0.0012) and decreased the ratio of cell to medium proteoglycans from 0.32 to 0.26 in normal and Alzheimer fibroblasts (p = 0.006), respectively. HPLC analysis of the disaccharides produced by chondroitinase ABC revealed no differences in composition between proteoglycans of Alzheimer and normal fibroblasts in either the cell or medium fraction. However, analysis of disaccharides produced by heparinase plus heparitinase showed differences in composition in the medium but not the cell fraction. delta UA-GlcNS was increased by 30% while delta UA-GlcNS-6S was reduced by 40% in Alzheimers disease.

Low sulfated glycosaminoglycans are excreted in patients with the Lowe syndrome

Glycosaminoglycans (GAGs) were prepared from the urine of three patients and from normal individuals by cetylpyridinium chloride precipitation and Pronase digestion. The GAGs were analyzed by electrophoresis, anion-exchange chromatography, and enzymatic and chemical degradation. Each of the three patients showed a four- to fivefold increase in urinary GAG excretion compared to normal controls and in one patient a tenfold increase was measured during a period of behavioral agitation which included joint swelling. Urinary GAGs from affected individuals were characterized by a high proportion of low sulfated molecules. The predominant low sulfated component was chondroitin-4-sulfate (C4S); however, small amounts of chondroitin-6-sulfate (C6S) were also present. Heparan sulfate (HS) was present in normal proportion (5-10%) and most of it was not low sulfated. Abnormal excretion of chondroitin (Ch), hyaluronic acid (HA), and dermatan sulfate (DS) was not detected. These findings suggest that the clinical manifestations of Lowe syndrome may be caused by a defect in GAG metabolism.

Characterization of the chondroitin sulfates in wild type Caenorhabditis elegans

The purpose of this study was to isolate and characterize the GAGs from the wild type nematode Caenorhabditis elegans in preparation for the characterization of the transgenic form constructed by Link [Proc. Natl. Acad. Sci. USA 92 (1995) 9368] which expresses various forms of beta-peptide (or A4 peptide). This peptide forms deposits very similar to the ones found in the neuritic plaques and neurofibrillary tangles in Alzheimer disease (AD). Characterization has been accomplished by degradation with specific enzymes and analysis of the products by TLC and HPLC. The results were compared with earlier works and shown to differ in disaccharide content.

Glycoprotein metabolism in neuronal ceroid lipofuscinosis fibroblasts

Human skin fibroblast lines of the infantile form of neuronal ceroid lipofuscinosis and control lines were cultured in the presence of [3H]glucosamine plus [3H]mannose and [35S]methionine. The labeled glycoconjugates were compared by quantitative polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The infantile form of the disease showed a 75% decrease of four glycoprotein components of M(r) 120-140 kDa. These components appeared to be N-linked glycoproteins as peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase F) released 86-96% of the labeled carbohydrate from the labeled protein. These results suggest that the infantile form of this disease may be characterized by abnormalities in glycoconjugate metabolism leading to reduction of specific glycoproteins.