Hu Peng, Wang Fengxin, Wu Xiumei, Ouyang Jianming
(Institute of Biomineralization and Lithiasis Research, Jinan University, Guangzhou 510632)

Abstract The influence of sulfated polysaccharide (LPS) isolated from marine algae Laminarin on the morphology and phase compositions of urinary crystal calcium oxalate (CaOxa) was investigated by means of scanning electron microscopy and X-ray diffraction. LPS can inhibit the growth of CaOxa crystal, prevent the aggregation of calcium oxalate monohydrate (COM), and induce the formation of calcium oxalate dihydrate (COD). All the three changes can inhibit the formation of CaOxa stones. This result indicated that LPS may be a potential inhibitor to CaOxa urinary stones.
Keywords calcium oxalate; urinary stone; algae Laminarin

1. INTRODUCTION
Urolithiasis constitutes a serious health problem that affects a significant section of mankind. For example, about 10% of the U.S. population suffer from this illness^[1]. A survey in Shenzhen city, the most southern city in China, showed the incidence of renal calculus was 4.87%, being 6.12% in the males and 4.07% in the females^[2]. Calcium oxalate (CaOxa) is a major inorganic component of kidney stones. Though in vitro methods it should reproduce some of the stages of a real biological process, and a number of researchers are tackling the problem from different directions, the mechanism of the formation of the stones is not yet clearly understood and a number of questions about the promoting and inhibiting factors still remain unanswered^[3,4].
    Sulfated polysaccharides are widespread in nature, occurring in marine algae and in a great variety of other organisms. In marine algae, they are present as sulfate fucose (fucoidans) and as sulfate galactans (carrageenans and agars). Recently, there has been an increasing interest in systematic screening of biological activity of sulfated polysaccharides isolated from marine algae such as the antitumor, antivirus, anticoagulant and antihyperlipidemia activities^[5,6]. However, there are few report about the application of seaweed polysaccharides in the inhibition of urinary stones. Actually, in urine, there are many polysaccharide especially the glycosaminoglycans (GAGs), including chondroitin sulfate A (C₄S), chondroitin sulfate C (C₆S), heparin, hyaluronic acid, and dermatan sulfate, etc^[7]. Most of these GAGs have similar molecular structure as LPS and can inhibit the growth of urinary stones^[7-10]. With this in mind, the inhibitive action of LPS on the crystallization of urinary crystal CaOxa was investigated in this work.

2. MATERIALS AND METHODS
2.1 Reagents
All of the solutions were prepared with reagent-grade chemicals that were purchased from Shanghai Chemicals Co. Doubly distilled water was used.
2.2. Polysaccharide extraction and fraction^[5,6,11]
Marine algae Laminarin was collected on the seaside of Southern Chinese Sea, Guangdong. It was cleaned with seawater, air dried, stored in dark place at room temperature and ready for use.
    The extraction of polysaccharide from Laminarin was carried out according to the literature^[5,6,11]. The average molecular weight of LPS was 110 000. This LPS consists of linear, mannitol- or glucose-ended chains of b -(1® 3) linked glucose residues, with occasional b -(1® 6)-linked branches. The content of sulfated group in the sample was 25%, which was determined by gelatin-BaCl₂ method.
    UV-visible spectrum of LPS shows the characteristic peak of LPS at 193 nm. The fact that no peak appeared at 260-280 nm indicated that there is no protein and no nucleic acid in LPS.
2.3 Crystallization of calcium oxalate
    The crystallization experiments of CaOxa were carried out at temperature 37±1ºC in a constant temperature chamber. The concentration of both Ca²⁺ and Oxa^2- in metastable solutions equal 0.30 mmol/L. The concentration of LPS investigated in this work is 0, 0.01, 0.10, 0.40, and 1.0 mg/mL.
2.4 Measurement of calcium oxalate crystals
The morphology of CaOxa was measured by scanning electron microscopy (Phlips XL-30 ESEM) at an operating voltage of 10 KV. X-ray diffraction (XRD) results were recorded on a D/max-g A X-ray diffractometer (Rigaku, Japan) using Ni-filtered Cu-Ka radiation (l =1.54Å) and a scanning rate of 2° min^-1 at 40 KV, 30 mA.
    The phase composition of the CaOxa crystals was estimated from the intensity ratio of the major X-ray diffraction lines of COM (I_COM) and COD (I_COD), respectively, as shown in eq. (1)^[3,12] for COD, for example:
Estimated (1)
with I_COM and I_COD being the intensities of the major diffraction lines of COM and COD, respectively.

3.2 LPS inhibit aggregation of of calcium oxalate monohydrate (COM) crystals
It can be seen from Fig. 1 that the morphology of the CaOxa crystals was affected by the concentration of LPS. In the absence of LPS, most of the CaOxa crystals are aggregated (Fig. 1a). XRD showed that these aggregates crystals were calcium oxalate monohydrate (COM) crystals. In XRD pattern (Fig. 2a), COM crystals show the diffraction peaks at 0.593, 0.365, 0.298, and 0.236 nm, which can be assigned to the (), (020), (), and (130) planes of COM^[3,12].
    However, the percentage of the aggregates decreased in the presence of LPS. It indicated that LPS prevents the aggregation of COM crystals.
3.3 LPS induce the formation of calcium oxalate dihydrate (COD) crystals
It can be seen from Fig. 1 that LPS can induce the formation of bipyramidal COD crystals. Especially in the presence of 0.40 (Fig. 1d) and 1.0 mg/mL (Fig. 1e) of LPS, the bipyramidal COD crystals were the dominant phase. In the XRD patterns, the diffraction peaks at 0.618, 0.442, 0.278, and 0.224 nm were assigned to the (200), (211), (411), and (213) crystal planes of COD crystals, respectively (Fig. 2c,2d).
    Quantitative analyses indicated that in the presence of 0.01, 0.10, 0.40 and 1.0 mg/mL LPS, the percentages of COD are 5, 15, 70 and 95%, respectively. That is, in a high concentration range from about 0.4 to 1.0 mg/mL, LPS mainly induce the formation of COD crystals.
    There are many evidences that the nucleation and growth of COM in urine and subsequent trapping of the crystal within the kidney lead to stone formation, while forming COD crystals may be protective. That is, COM exhibits a greater degree of attachment to renal tubule cells in culture compared with COD^[13]. A theoretical calculation also indicated that COM has a stronger affinity for these cell membranes than COD ^[14]. So it can be concluded that COD is more easily excreted out of the body than COM. As a result, the induction of more COD crystals will be in favor of preventing the formation of urinary stone. Therefore, LPS may have the potential to inhibit CaOxa crystallization directly and may be useful in stone therapy.