Effect of CDA-II, Urinary Preparation, on Lipofuscin, Lipid Peroxidation and Antioxidant Systems in Young and Middle-Aged Rat Brain

American Journal of Chinese Medicine, Wntr, 2001 by Wen-Chuan Lin, Yueh-Wern Wu, Tung-Yuan Lai, Ming-Cheng Liau

(Accepted for publication August 7, 2000)

Abstract: The levels of lipofuscin and lipid peroxidation reflect the degree of free radical-induced oxidative damage in the brain. We examined the effects of CDA-II, a preparation of human urine, on lipofuscin and lipid peroxidation in the brain of young (3.5 months) and middle-aged rats (17 months). The rats were given CDA-II orally at dosages of 0.3 or 1.0 g/kg daily for 8 weeks. CDA-II significantly suppressed the contents of lipofuscin and lipid peroxidation in both young and middle-aged rats. CDA-II also elevated the activity of superoxide dismutase, and the amounts of glutathione and ascorbic acid in the middle-aged rats, but not in the young ones. Our results suggest that the protection against oxidative damage by CDA-II in the young rat brain may be due to its scavenging activity against free radicals. In the middle-aged rats, in addition to the scavenging activity, the levels of endogenous antioxidants were also enhanced by the CDA-II treatment.

Cell differentiation agent II (CDA-II) is an urinary preparation that is effective against cancer by inducing differentiation of neoplastic cells. Currently, CDA-II has been approved for clinical trial in China, P.R.C.. We have made an attempt to purify active anticancer components from CDA-II. The results indicated that multiple active components, such as peptides, organic acid and pigments, with different mechanisms of action act concurrently to contribute to the anticancer effect of CDA-II. Phenylacetate (one of these components), a metabolite of phenylalanine excreting in urine, acts as a differentiaring agent (Newmark and Young, 1995; Samid et al., 1992). A bioassay is used to control the quality of CDA-II, based on the ability of CDA-II to induce terminal differentiation of the human leukemia cell line (HL-60), and to inhibit colony formation of human breast carcinoma cell line-100 (HBL-100).

The use of urine and urine extracts for medical purpose has been known for centuries (Burzynski, 1988). Recently, the use of urinary remedy toward illness in Asia is gaining popularity. According to the theory of traditional Chinese medicine, urinary preparation could be used to treat a variety of ailments, including aging (Read, 1976). It is believed that the age-related deterioration of the central nervous system (CNS) is partly due to the cytotoxicity of reactive oxygen species generated in the brain (Harman, 1968). Thus, various synthetic or natural compounds having antioxidative properties have been tried to protect against the age-induced deterioration in CNS (Candice et al., 1996; Inanami et al., 1995; Santiago et al., 1992; 1993).

In our previous works, we showed that an urinary preparation (CDA-II) prevented liver injury induced by carbon tetrachloride through inhibition of increased hepatic lipid peroxidation (Lai et al., 1999). More recently, our study has shown that CDA-II was a good scavenger of hydroxyl radical, and it inhibited lipid peroxidation in brain homogenates (Lin et al., 2000). Ex vivo studies also demonstrated that oral administration of CDA-II to rat increased its plasma antioxidant capacity (Lin et al., 2000).

The aim of this experiment was to evaluate the effects of CDA-II on the age-related increase in lipofuscin and lipid peroxidation. In addition, the response of endogenous antioxidant systems to CDA-II treatment was also assayed.

Materials and Methods

Preparation of CDA-II

CDA-II was kindly supplied by EverLife Pharmaceutical Co. Ltd. (Western Hefei Anhui, China). The procedures for the preparation of CDA-II from male urine include filtration, ultrafiltration and reverse-phase chromatography. The collected urine was first acidified to a pH below 3 and finally adjusted to 2. The acidified urine was filtered, including the final step of ultrafiltration on a Millipore system with a molecular weight cut off of 10000 daltons. The filtrate was passed through a chromatographic column filled with XAD-16 (available from Rohm Hass). The column was initially washed with water and then eluted with methanol. The colored methanolic fraction was collected and evaporated under vacuum. The dried extract was reconstituted with distilled water to make 300 mg/ml.

The quality of CDA-II was based on its capability of inducing terminal differentiation of HL-60, and inhibiting colony formation of HBL-100. At 1 mg/ml of CDA-II, nitroblue tetrzolium (NBT) cells of HL-60 should be above 50% of control, and colony formation of HBL-100 cells should be less than 10% of the control.

Animals

Young (1.5 months) and middle-aged (15 months) male Sprague-Dawley rats were obtained from the National Laboratory Animal Breeding and Research Center, National Science Council, and fed with a standard laboratory diet and tap water ad libitum. The experimental animals were housed in an air-conditioned room (22 [ or -] 3 [degrees] C) with 12 hours of light. The rats were allowed free access to powdered food, and main's water that was supplied through an automatic watering system.