Stroma-free hemoglobin (Hb) has been modified by pyridoxylation and followed by polymerization with glutaraldehyde as a blood substitute. Nevertheless, the reaction rate of pyridoxylated Hb (PLP-Hb) with glutaraldehyde is too fast to control its molecular weight distribution. Additionally, it was reported that glutaraldehyde is cytotoxic even at low doses. To overcome these problems, another aldehyde, beta-hydroxypropionaldehyde (beta-HPA), was used in the study to polymerize hemoglobin (PLP-Hb). beta-HPA is a natural compound (reuterin) produced by Lactobacillus reuteri. It was found that the maximum degree of PLP-Hb polymerization by reuterin (RR-PLP-Hb) was approximately 40% if the formation of high molecular (>500 kDa) polymers should be prevented. In contrast, at the same reaction condition, the glutaraldehyde-polymerized PLP-Hb solution became gel-like, due to overpolymerization. This indicated that the rate of PLP-Hb polymerization by reuterin was significantly slower than that by glutaraldehyde. With increasing the reaction temperature, PLP-Hb concentration, or reuterin-to-PLP-Hb molar ratio, the time to reach the maximum degree of PLP-Hb polymerization by reuterin became significantly shorter. Removal of unpolymerized PLP-Hb from the RR-PLP-Hb solution can be effectively achieved by a gel-filtration column. The P-50 value of the unmodified Hb solution was 14 torr, while that of the RR-PLP-Hb solution was 20 torr, an indication of lower oxygen affinity. Additionally, the oxygen-Hb dissociation curves for both test solutions had a sigmodial shape and a nearly 100% saturation at 100 torr. In the in vivo study, it was found that the animals treated with the RR-PLP-Hb solution all survived and remained healthy more than 3 months. In contrast, only one out of six rats survived for the control group treated with the unmodified Hb solution. Furthermore, it was found that the RR-PLP-Hb solution resulted in a significantly longer circulation time (approximate to 12 h) than the unmodified Hb solution (approximate to 1.5 h). These results suggest that the reuterin-polymerized PLP-Hb solution may be a new option in the development of blood substitutes. (C) 2004 Wiley Periodicals, Inc.