Diagnosing Metabolic Diseases Using Thin Layer Chromatography: Laboratory Experiment for both Organic Chemistry and Biochemistry Undergraduate Laboratories

Abstract

With the ability to resolve metabolites as well as proteins, Thin Layer Chromatography (TLC) is a technique that is used in both Organic Chemistry as well as Biochemistry classes. There were two goals of this project: first was to develop a virtual laboratory experience for students in Organic Chemistry lab that expanded upon a previous experiment with TLC. The second was a project in method development for Biochemistry students. Students in Organic Chemistry lab had previously completed an experiment in TLC that required identification of an analgesic by comparing Rf value to those of standards. This project produced a virtual one where students were expected to diagnose various diseases based on TLC of affected amino acids. The diseases involved were Phenylketonuria, ornithine transcarbamylase (OTC) deficiency, arginemia, tyrosinemia, maple syrup urine disease citrullinemia type I and type II which respectively resulted in increased levels of amino acids phenylalanine, ornithine, arginine, tyrosine, either leucine, isoleucine or valine, or citrulline. The procedure involved spotting the standards and “plasma from blood” samples on the TLC plate with 70/30 mixture of n‐propanol/water. Amino acids were visualized by staining with ninhydrin. Several of the relevant amino acids have Rf values that are similar. In order to correctly identify the disease, a second piece of information was needed: the relative level of urea. Four of the diseases are urea cycle disorders that produce decreased levels of urea while the other three have normal levels. Students were provided with a picture of a standard curve of urea as well as pictures of the patients; this was used to identify if the disease was a UCD or not. In order to assess learning, students were given a pre and post test assessment. Students had previously done a TLC and 73.8% were able to correctly identify the role of various components when given a copy of the procedure. There was a 5.8% increase in score in the post test. For questions that addressed the theory of how TLC works or other applications, 37.7% of students on pre test could answer correctly. This value increased to 55.0% on the post test; this result suggests were able to deepen their knowledge of TLC with a second exposure. Another approach was taken for the Biochemistry students. Method development has been shown to be a critical skill, yet it is difficult to teach. Biochemistry students were provided with “blood” of five types of patients (normal, arginemia, OTC deficiency, citrullinemia type I and II), basic procedure for separating amino acids on TLC, and the amounts of these amino acids normally found in blood as well as in the disease state. The question for these students was if it was possible to detect these diseases using TLC? Students were given a pre test assessment. The great majority (81.2%) were able to partially or completely identify the function of the components in TLC. Students were asked to list questions that must be addressed in order to answer this question when provided with blood or plasma. Student responses covered a wide range and addressed both valid questions (do the amino acids have different Rfvalues or is the amount of amino acid detectable in this method) as well as not valid questions to having no answers.