Ichiro Koshiishi

High-performance liquid chromatographic analysis of g;ycosaminoglycan-derived oligosaccharides

High-performance liquid chromatography of glycosaminoglycan (GAG)-derived oligosaccharides has been employed for the structural analysis and measurement of hyaluronan, chondroitin sulphate, dermatan sulphate, keratan sulphate, heparan sulphate and heparin. Recent developments in the separation and detection of unsaturated disaccharides and oligosaccharides derived from GAGs by enzymatic or chemical degradation are reviewed.

Prevention of the photodamage in the hairless mouse dorsal skin by kojic acid as an iron chelator

Kojic acid, a fungal metabolic product, has been used as a skin-depigmenting agent in skin care products marketed in Japan. Iron in the skin is known to be involved in wrinkling as a result of chronic photodamage. Kojic acid was expected to have anti-wrinkling activity, since it possesses iron-chelating activity. We now evaluated the anti-wrinkling activity of kojic acid by using hairless mice exposed to chronic solar-simulating ultraviolet (UV) irradiation as model animal. At the end of a 20-week irradiation period, topical application of kojic acid before UV irradiation was observed to dramatically prevent: (1) the wrinkling, (2) hyperplasia of the epidermis, (3) fibrosis of the lower dermis, and (4) the increase of extracellular matrix components in the upper dermis. These findings indicate that kojic acid is a typical agent preventing wrinkling of the skin due to chronic photodamage.

DEGRADATION OF DEHYDROASCORBATE TO 2,3-DIKETOGULONATE IN BLOOD CIRCULATION

In our previous paper (Biochim. Biophys. Acta 1379 (1998) 257-263), we demonstrated that bicarbonate promotes a cleavage of lactone ring of dehydroascorbate (DHA) on the basis of in vitro experiments. In the present study, we examined the degradation of DHA in blood circulation in vivo by using a high-performance liquid chromatographic method for the determination of ascorbate (AsA), DHA and 2,3-diketogulonate (2,3-DKG), which required no pretreatment of biological fluids. When DHA was intravenously administered to rats, a rapid disappearance of DHA (t1/2 < 1 min) and a concomitant appearance of 2,3-DKG in blood circulation were observed. Approximately 90% of the administered DHA were excreted into urine as resulting 2,3-DKG (55%) and AsA (31%), respectively. Furthermore, we elucidated that rat plasma lacks an enzyme having an aldonolactonase-like activity. The result of the present study suggests that this DHA disappearance is a function of both a chemical degradation to 2,3-DKG and a reduction to AsA.

Quantitative alterations of hyaluronan and dermatan sulfate in the hairless mouse dorsal skin exposed to chronic UV irradiation

The quantitative alterations of hyaluronan and dermatan sulfate in the upper dermis (fibrous tissue) and the lower dermis (adipose tissue) of the hairless mouse skin chronically exposed to the UV irradiation as solar-simulating irradiation (lambda(max) 352 nm, UV distribution: 300-310 nm, 0.9%; 310-320 nm, 2.0%; 320-420 nm, 97.1%) were evaluated. Hyaluronan and dermatan sulfate contents in each part of dermis were determined as follows: skin sections on a glass slide prepared by histological technique were processed into the upper dermis and the lower dermis with a small surgical knife, and treated with chondroitinase ABC and ACII in the presence of bacterial collagenase. The resulting unsaturated disaccharides were determined by HPLC method. By applying this method to the UV-irradiated hairless mouse skin, it was found that the chronic UV irradiation increased dermatan sulfate in the upper dermis, whereas an increase of hyaluronan content was not statistically significant. In the lower dermis, on the contrary, both hyaluronan and dermatan sulfate contents remarkably increased as compared with the control mice. Furthermore, the histological study showed the accumulation of the collagen fibers in the lower dermis of the UV-irradiated hairless mouse skin following the disappearance of adipocytes. These findings indicate that the increases of glycosaminoglycan contents in the UV-irradiated skin are related to the accumulation of the extracellular matrix components in the lower dermis.

Bicarbonate promotes a cleavage of lactone ring of dehydroascorbate

The half-life of dehydroascorbate (DHA) in human plasma is only a few minutes. This DHA disappearance is caused by a cleavage of lactone ring. Similarly, when DHA was incubated in Dulbeccos modified Eagles medium (D-MEM), which stood in atmosphere of 5% CO2-95% air, the rapid transformation of DHA into 2,3-diketogulonate (2,3-DKG) is also observed. These observations suggest that both human plasma and D-MEM contain a common component, which promotes the hydrolysis of DHA. In the present study, this component was identified to be bicarbonate which acts as a general base catalyst. Direct evidence for this mechanism was obtained as follows: (1) significant hydrolysis of DHA in the bicarbonate-free D-MEM (pH 7.40) was not observed; (2) hydrolysis of DHA in Tris-HCl buffer at constant pH (7.4) increases with increasing bicarbonate concentration; and (3) significant hydrolysis of DHA in the decarbonated ultrafiltrate of plasma was not observed. These results suggest that DHA hydrolysis may be controlled by the variation of CO2 pressure in circulating blood.

Alterations of Hairless Mouse Skin Exposed to Chronic UV Irradiation and Its Prevention by Hydrocortisone

Abstract— Ultraviolet‐induced alterations of skin were investigated in a murine animal model. Groups of hairless mice were exposed to UV (black light, Λmax 352 nm; UV distribution: 300–310 nm, 0.9%; 310–320 nm, 2.0%; 320–420 nm, 97.1%) for 20 weeks at a dose of 16.3 J/cm2 five times weekly on weekdays. At the end of 20 weeks irradiation, the dorsal skins were biochemically and histologically examined. Ultraviolet caused remarkable increases in amounts of hyaluronan, chondroitin sulfates and dermatan sulfates in skin (μg/cm2). Interestingly, a significant change in a collagen content (hydroxyproline, μg/g of dry powder) caused by UV irradiation was not observed, whereas the amount of collagen (hydroxy‐proline, μg/cm2) increased remarkably. Histologically, no distinguishable thickening was observed in both upper dermis and lower dermis, but thickening of the epidermis was observed. Furthermore, the histological study indicated that UV irradiation caused a disappearance of crowds of adipocytes, alternative appearance of numerous fibroblasts and accumulation of collagen bundles and hyaluronan in lower dermis. Hydrocortisone, an anti‐inflammatory agent, prevented both the fibrosis of lower dermis and the accumulation of the extracellular matrix components. Based on these results, it seems reasonable that UV penetrates into the lower dermis and causes fibrosis there, resulting from the inflammatory responses.

Study of the measurement of chondroitin sulphates in rabbit plasma and serum

A highly sensitive high-performance liquid chromatography method, which was established by us for the determination of chondroitin sulphates in biological substances as their unsaturated disaccharides, was applied to elucidate the qualitative and quantitative differences in chondroitin sulphates in rabbit plasma and serum samples. In this work, it was found that rabbit plasma contains low-sulphated chondroitin 4-sulphate (approximately 40% sulphation at the 4-position of N-acetyl galactosamine), while serum contains the low-sulphated chondroitin 4-sulphate and fully sulphated chondroitin 4-sulphate (approximately 96% sulphation). The latter was released from platelets during coagulation of blood.

Cyanate causes depletion of ascorbate in organisms

Ascorbate-dehydroascorbate redox cycle plays a key role in protecting organisms from an excess of oxidants. Recently, we found a novel reaction of dehydroascorbate with cyanate under the conditions of neutral pH and ordinary temperature. In this report, we demonstrated that through this irreversible reaction, cyanate causes the depletion of ascorbate in the matrix, where the ascorbate-dehydroascorbate redox cycle revolves. When the leaves of weed (Erigeron canadensis) were soaked in sodium cyanate solution generally used as a herbicide, the depletion of ascorbate as well as dehydroascorbate in them was observed, followed by the change in color from green to brown. These results suggest that a possible way of cyanate toxicity is to inflict oxidative stress on organisms.

Study of the coloured substances in molybdenum blue using high-performance liquid chromatography

Abstract Methods for the separation of reduction products of phosphomolybdate and silicomolybdate were established by high-performance liquid chromatography using Syragel 60 A (Waters Assoc.) as a packed material and acetonitrile solution containing into two species, Product I (P) and Product II (P). Reduction products of silicomolybdate were also separated into tow component species, Product I (Si) and Product II (Si). From the investigation of phosphomolybdenum blue complexes produced via various different colour reactions, it was observed that the molybdenum blue reaction for phosphate proceeded in two stages as follows: H 3 PO 4 · 12MoO 3 → H 3 PO 4 · 10MoO 3 · Mo 2 O 5 → H 3 PO 4 · 8MoO 3 · 2Mo 2 O 5 . The apparent molar absorptivity of Product I (P) was 7.4 times larger that that of Product II (P). Similarly, the molybdenum blue reaction for silicate was studied and discussed.

Quantitative and qualitative alterations of chondroitin/dermatan sulfates accompanied with development of tubulointerstitial nephritis

Quantitative and qualitative alterations of renal oversulfated chondroitin/dermatan sulfates (C/DSs) accompanied by the development of tubulointerstitial nephritis were examined. The rat model with unilateral ureteral obstruction (UUO) is a suitable model for study of renal interstitial fibrosis, and was utilized in the present study. Cortical regions of serial sections of UUO kidney and sham-operated kidney on glass slides were processed using a small surgical knife under dark field microscopy. Oversulfated C/DSs in tissue sections on a glass slide were degraded to unsaturated disaccharides using chondroitinase ABC and ACII digestion in the presence of bacterial collagenase. The resulting unsaturated disaccharides were subsequently determined by HPLC. These in situ investigations yielded the following results: (1) marked increases in oversulfated C/DSs content and decreases in the oversulfation degree of C/DSs were observed in fibrous lesions, compared to non-fibrous lesions, and (2) iduronic acid content in C/DSs in fibrous lesions was significantly lower than that in non-fibrous lesions. These findings indicate that oversulfated C/DSs with low-iduronic acid content represent a potential marker for tubulointerstitial nephritis.