Quantitative determination of peptidoglycan and (1->3)-β-D-glucan

17th April 2023   All

Definitions and significance of peptidoglycan and (1->3)-β-D-glucan 

Peptidoglycan – Peptidoglycan,1 which is also called murein, is a polymer consisting of polysaccharide chains cross-linked by short peptides. It is a component of the bacterial cell wall and supports its structural integrity. Due to its important role for the survival of bacteria, peptidoglycan is a target for certain antibiotics, such as β-lactams. Moreover, peptidoglycan is a pyrogen, which can induce fever and an inflammatory reaction when it comes in contact with the bloodstream. 

(1->3)-β-D-glucan – (1-3)-β-D-glucan is a polysaccharide that is present in the cell walls of various fungi, plant cells, and, less frequently, bacteria. It provides structural support to the cell wall of fungal cells and can stimulate the immune system to respond to fungal infections. Moreover, (1-3)-β-D-glucan has been proposed as a marker for invasive fungal infections.2

Quantification of peptidoglycan and (1-3)-β-D-glucan

The quantitative detection of peptidoglycan and (1-3)-β-D-glucan may both enable the detection of microbial contamination and provide valuable research insights. The selection of a quantification method depends on a variety of factors, including the indication for testing, required sensitivity, and sample type. 

Peptidoglycan and (1-3)-β-D-glucan determination based on the prophenoloxidase cascade

Peptidoglycan and (1-3)-β-D-glucan trigger the prophenoloxidase cascade in the hemolymph of the silkworm Bombyx mori when they come in contact with it. As prophenoloxidase is activated, toxic intermediates are produced against the invading pathogens.

This mechanism has served as a basis for the quantification of peptidoglycan and (1-3)-β-D-glucan with the silkworm larvae plasma (SLP)-reagent.3 The SLP reagent is prepared and lyophilized from silkworm hemolymph under sterile conditions and contains all factors implicated in the prophenoloxidase cascade. When peptidoglycan or (1-3)-β-D-glucan come into contact with the SLP reagent, melanization occurs via the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) into dopachrome and dopachrome’s subsequent conversion into a black melanin pigment, changing the color of the solution. The formation of the black melanin pigment can be monitored either visually or quantitatively via a colorimetric analysis. 

Kinetic colorimetric quantification of peptidoglycan and (1-3)-β-D-glucan 

Peptidoglycan and (1-3)-β-D-glucan can be quantified by monitoring the formation of melanin. The activation time of the reaction, also known as onset time, should be determined. It indicates the time point at which the absorbance at 650 nm reaches a predetermined threshold. The measured value is then aligned to a calibration curve generated using peptidoglycan standards. This method enables the determination of the absolute concentration of peptidoglycan and (1-3)-β-D-glucan with a sensitivity in the low pg/ml range. The measurements can be performed using Toxinometer® ET-7000, which enables the simultaneous assessment of up to 16 samples or, with extension modules, up to 128 samples. The Toxinometer® ET-7000 device is compact, versatile, compliant with FDA Title 21 Part 11, and requires minimal maintenance. The analysis of the acquired data is automated via the Toximaster® software.

Applications of peptidoglycan and (1-3)-β-D-glucan quantification

The quantification of peptidoglycan and (1-3)-β-D-glucan has found implications in a variety of fields. The method can be employed to determine the contamination of dialysate solutions, pharmaceuticals, biologics, medical devices, or genetically engineered products. In addition, it can be used to assess water pollution. Finally, the quantification of peptidoglycan and (1-3)-β-D-glucan can be utilized to analyze their biosynthesis, structure-activity relationships, etiological significance, and metabolism.

 

Literature sources:

  1. Garde S, Chodisetti PK, Reddy M. Peptidoglycan: Structure, Synthesis, and Regulation. EcoSal Plus. 2021;9(2).
  2. Wright WF, Overman SB, Ribes JA. (1–3)-β-D-glucan assay: a review of its laboratory and clinical application. Laboratory Medicine. 2011;42(11):679–685.
  3. FUJIFILM. SLP-HS Single Reagent Set Ⅱ. Available at: https://www.wakopyrostar.com/brands/wako/product/slp-hs-single-reagent-set