Effect of pH on Cleavage of Glycogen by Vaginal Enzymes

Abstract

Glycogen expressed by the lower genital tract epithelium is believed to support Lactobacillus growth in vivo, although most genital isolates of Lactobacillus are not able to use glycogen as an energy source in vitro. We recently reported that α-amylase is present in the genital fluid of women and that it breaks down glycogen into small carbohydrates that support growth of lactobacilli. Since the pH of the lower genital tract can be very low, we determined how low pH affects glycogen processing by α-amylase. α-amylase in saliva degraded glycogen similarly at pH 6 and 7, but activity was reduced by 52% at pH 4. The glycogen degrading activity in nine genital samples from seven women showed a similar profile with an average reduction of more than 50% at pH 4. However, two samples collected from one woman at different times had a strikingly different pH profile with increased glycogen degradation at pH 4, 5 and 6 compared to pH 7. This second pH profile did not correlate with levels of human α-acid glucosidase or human intestinal maltase glucoamylase. High-performance anion-exchange chromatography showed that mostly maltose was produced from glycogen by samples with the second pH profile in contrast to genital α-amylase that yielded maltose, maltotriose and maltotetraose. These studies show that at low pH, α-amylase activity is reduced to low but detectable levels, which we speculate helps maintain Lactobacillus growth at a limited but sustained rate. Additionally, some women have a genital enzyme distinct from α-amylase with higher activity at low pH. Further studies are needed to determine the identity and distribution of this second enzyme, and whether its presence influences the makeup of genital microbiota.

Fig 1. Effect of pH on glycogen degradation by salivary α-amylase.

Saliva was collected from a normal donor and incubated with glycogen at pH 4–7. The percent of degradation of glycogen at pH 7 is shown on the y axis. Average of four experiments. Degradation at pH 4 was significantly different than that at pH 6 and 7 (p<0.05, Mann-Whitney test).

Fig 2. Effect of pH on glycogen degradation by lower genital tract fluids.

Genital fluids were collected by lavage from eight donors. Two donors (subject 4 and subject 9) provided samples at multiple times (approximately one week apart). Lavage samples were incubated with glycogen at pH 4–7 and the percent of degradation of glycogen at pH 7 for each sample was calculated and is shown on the y axis. Each sample was run in two separate assays and the results averaged.

Fig 3. Carbohydrate-degrading enzymes in genital fluids.

Levels of α-acid-glucosidase and intestinal maltase glucoamylase in genital fluid samples with Type I and Type II pH sensitivity were determined by ELISA. The average of triplicate ELISA wells is shown.

Fig 4. Types of commensal bacteria in genital samples.

Levels of vaginal bacteria and vaginal pH corresponding to samples with Type I and Type II pH sensitivity. Bacteria were quantified in genital samples by species-specific real time PCR. Representative results from two assays are shown. Each assay was run in triplicate.

Fig 5. Size of small carbohydrates generated by glycogen degradation.

HPAEC analysis of carbohydrates generated from glycogen by incubation with genital fluids (samples 9–1 or 11–1) or control (saline) at pH 7 (A) or pH 4 (B). The lighter trace on each graph represents the standards that were run including monomers (G1), dimers (G2), trimers (G3), tetramers (G4), and pentamers (G5). Runs were performed twice and representative results are shown.