Quantification of bacterial lipopolysaccharides by the purpald assay: Measuring formaldehyde generated from 2-keto-3-deoxyoctonate and heptose at the inner core by periodate oxidation

We have adapted the purpald assay (M, S. Quesenberry and Y. C. Lee, Anal. Biochem. 234, 50-55, 1996) to quantify lipopolysaccharide (LPS) content in solution in 96-well microtiter plates at room temperature. This method employs the oxidation of unsubstituted terminal vicinal glycol groups in 2-keto-3-deoxyoctonate (Kdo) and L-(or D-)glycero-D-manno-heptose of LPS molecules by periodate to release formaldehyde. The formaldehyde is quantified at 550 nm (or 530-570 nm) by reacting with purpald reagent followed by oxidation with NaIO4. The sensitivity of the purpald assay is comparable to that of the Kdo assay for LPS determination. However, the purpald assay is superior to the Kdo assay because: (i) No acid hydrolysis of the samples and no boiling in the assay process are required; thus, it can be directly carried out with microtiter plates for a large number of samples at room temperature, (ii) The purpald assay can detect many types of LPS from various bacteria since LPS contains Kdo and heptose which possess unsubstituted terminal vicinal glycol in its structure, while the Kdo assay cannot detect LPS from certain bacteria (e.g., Haemophilus influenzae, Bordetella pertussis, and Vibrio cholerae) due to substitution at the C-4 and C-5 positions of Kdo.