Development of a diagnostic kit for homocysteine, a biomarker for Cardiovascular Diseases

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1 Laurentian University Université Laurentienne Development of a diagnostic kit for homocysteine, a biomarker for Cardiovascular Diseases Ryan Mailloux, Ph.D. Laurentian University 935 Ramsey Lake Rd. (705) ext 2173 rj_mailloux@laurentian.ca 1

2 What is homocysteine? Homocysteine is a sulfur-containing amino acid Produced following the methylation of target proteins Homocysteine is metabolized in the human liver. Methylation Trans-sulfuration Elevated homocysteine levels can be quite toxic! 2

3 Why is homocysteine so toxic? NH 2 HS OH homocysteine O Highly reactive thiol (low redox potential) capable of covalently modifying various proteins and compounds involved in numerous biological processes. Blood clotting Lesion formation Inflammation Immune response HOMOCYSTEINE Increase in Cholesterol Oxidative stress CARDIOVASCULAR DISEASE 3

4 Causes of elevated homocysteine levels Age Sex Drugs/Toxins Proliferative diseases Homocysteine Smoking Alcohol Caffeine Vitamin Deficiency Genetic predisposition 4

5 Diseases associated with homocysteine Neurological disorders (Alzheimer s, Down s syndrome) Diabetes Homocysteine Cardiovascular disease Renal failure Disease distribution in Canada 5

6 CVD biomarkers Biomarker Homocysteine Lipoproteins (cholesterol) c-reactive protein Ascorbic acid Lactate dehydrogenase Drawback Cost and Time Differentiation between good cholesterol and bad cholesterol Indicator of infection and inflammation Fluctuates according to diet Circulating lactate dehydrogenase is also an indicator of liver failure, kidney disease, and heart failure High homocysteine prompts the release of c-reactive protein and the oxidation of LDL 6

7 Current assays for measuring the blood levels of homocysteine Assay Cost Drawback HPLC 65$ per assay Sophisticated equipment Highly trained staff Expensive Not high throughput Enzymatic assays Immunoassays $600 per kit Reagents are expensive Highly trained staff Sophisticated equipment Inosine dehydrogenase FDA approved 4 enzyme steps Requires a number of cofactors Lactate dehydrogenase FDA approved 4 enzyme steps Requires a number of cofactors Although homocysteine detection can be used as a biomarker for CVD and other diseases, a cost effective and accurate method of deciphering homocysteine levels in the blood is still required. 7

8 Strategy: Discovering a one-step enzymatic method for detecting homocysteine 8

9 The isolation of the homocysteine-metabolizing enzyme Isolation of microbes Isolation of the enzyme in 4 hours Microbial consortium growing in a nutrient medium Preparation of the enzyme for homocysteine assays Detection of homocysteine in one-step Amount of protein isolated from one 200mL microbial culture is 30mg/mL which can run up to 150 assays Only a crude protein fraction is required for the accurate measurement of homocysteine. Total time required to prepare the enzyme extract is 7 hours, 9

10 Enzyme identification Total protein stain Activity stain This activity assay depends on the conversion of NAD into NADH which can be detected easily by spectrophotometry Homocysteine-metabolizing enzyme MALDI-TOF for enzyme identification WG3 18 (0.599) Cn (Cen,3, 75.00, Ht); Sb (99,90.00 ); Sm (SG, 2x3.00); Cm (7:21) Biochemical profile of the enzyme TOF LD+ 5.14e %

11 Detection of homocysteine levels by spectrophotometry Homocysteine Enzyme Product NAD NADH Easy detection NADH (µm) No NADH formed using these two sulfurcontaining amino acids indicating high specificity 2 0 Homocysteine Methionine Cysteine The enzymatic detection of homocysteine in this one-step method is performed by monitoring the formation of NADH, a by-product of homocysteine metabolism, with a spectrophotometer. This instrument is very cheap and found almost every clinical setting. 11

12 Enzyme sensitivity and stability [NADH] (µm) Detect homocysteine levels between 3-20µM [Homocysteine] (µm) Enzyme activity Homocysteine consumed (µm) Time (hours) The powdered form of the enzyme is stable for 1 week 12

13 Assay optimization Reducing agents increased accuracy of the detection assay 18 25% improvement in accuracy [NADH] (µm) reducing agent + reducing agent Reactions can be performed for up to 20 minutes [NADH] (µm) Reaction time (minutes) 13

14 Detection of homocysteine in human blood Human plasma samples are required for the assay. 30 Concentration of homocysteine (µm) Human Plasma Human serum Most of the protein is removed for clinical analysis. 14

15 Use of this enzyme in the clinical detection of homocysteine No highly trained personnel required One-step Very safe Uses equipment available in any clinical setting Clinical detection of homocysteine using this novel enzyme Cost effective High throughput Production of enzyme is quick and easy 15

16 Concluding remarks: Genesis of a facile homocysteine kit Microbial Culture Enzyme extract Stabilizers Kit Clinical Applications Food and beverage industries Research Applications 16

17 Research Team Dr. Vasu Appanna Project leader Dr. Ryan Mailloux Research associate 17