Mohd Hairi Bin A.Hamid Bsc.

. Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (July 2009) Whole Blood Amino Acid Screening Test: Guthrie Test

Introduction Guthrie test is one of screening test for amino acid especially to detect phenylketonuria (PKU) disease. It is also the semi-quantitative assay that used to detect the elevated level of phenylalanine (PA) in the blood. At Malaysia, the test is not mandatorily used (not common in our population). But at overseas, it is mandatorily used for newborn baby in the days (day 6 or 7) because most of their people have this disease when they become adult. Therefore, since birth; early prevention is required to reduce the phenylketonuria diseases patients. Purposes It is used to screen for phenylketonuria (PKU) disease and detect the elevated level of phenylalanine (PA) in the blood. Procedures A small amount of blood (from a heel puncture/ capillary puncture) is placed to a piece of filter paper. Then a small disc is punched out and placed on the agar gel containing -2thienylalanine and Bacillus subtilis When concentrations of PA increase, growth inhibition is overcome and zone of inhibition can be observed. The amount of growth is determined by measuring the diameter of colony present. Compare the diameter of each sample disk's colony with the colonies of reference disks. Some lab, use a fluorometric method for detection of phenylalanine (amount of fluorescent depend on the PA amount. Usually, an overnight incubation is enough to determine the abnormal level of phenylalanine in the blood.

Patient preparations The patient was recommended to fast 6-8 hours The patient should be avoided to use of drugs known to cause interference (certain antibiotic)

Mohd Hairi Bin A.Hamid Bsc. Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (July 2009)

Patient should be > 24 hours old and have adequate protein intake for 48 hours prior to test Sample preparations Before sample is collected, make sure that patient was already ingested the protein given adequately. It is because inadequate protein ingestion can cause false negative result. Venipuncture: Wipe out the first drop of blood (it may contained tissue fluid). After sample is collected, make sure the laboratory slip present of date and time, the date and time of birth, and the time of first milk feeding. If the test is delayed, store the specimens 30C and 4C for 10 days and 60 days respectively. Ages that usually tested Commonly performed for newborn baby (0 2 weeks) Children Adult Occasionally performed this test... Disorders that determined by Guthrie test Phenylketonuria (more common) o This disease refers to phenylalanine hydroxylase (PAH) deficiency. This enzyme used to convert phenylalanine (a/a) to the tyrosine (a/a). o When PAH deficient, phenylalanine is accumulates and is converted into phenylpyruvic acid, which is detected in the urine o Increase level of PA causing mental retardation, seizures and eczema. Congenital Hypothyroidism o It is due to thyroxine (growth hormone) deficiency. o Without this hormone, normal growth cannot occur. Growth hormone supplement is required. Cystic Fibrosis o Inherited condition that affects the lungs and digestive system. Medium chain acyl-CoA Dehydrogenase (MCAD) Deficiency o Is a condition in which fat metabolism is impaired. o Modification of the diet can reduce symptoms. Sickle Cell Disease o Affects the shape of RBC & reduces the ability of Hb to carry O2 around the body

Mohd Hairi Bin A.Hamid Bsc. Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (July 2009)

References range Normal blood phenylalanine level: 1 mg/dL @ < 120 mol/L (SI Units) Abnormal blood phenylalanine level: 30 - 80 mg/dL @ 120 mol/L (SI Units) When high level of phenylalanine is detected (means positive Guthrie test), therefore, further confirmatory test should be performed. Interpretation High level of PA showed that releasing of PAH is decrease, there high level of PA cant converted to Thyroxine (growth hormone); as a result, PKU will obtained. Normal level of PA showed that releasing of PAH is adequate to convert PA to PAH; therefore absent phenylketonuria disease.

Affected Factors/ Limitations Premature infants may have false-positive result due to delayed development of liver (phenylalanine hydroxylase ). Infants tested before 24 hours of age, may have false negative result due to they have not ingested enough milk containing phenylalanine. False negative result can be obtained when vomiting was occurred (feeding problem); it is because the infant may not have tolerated enough milk containing phenylalanine. Antibiotic, aspirin and salicyclate (drugs) may influence the screening result. Molecular methods to diagnose PKU 1) High Performance Liquid Chromatography (HPLC) HPLC is a form of liquid chromatography functioned to separate compounds that are dissolved in solution. Compounds are separated by injecting a plug of the sample mixture onto the column. The different components in the mixture pass through the column at different rates due to differences in their partitioning behaviour between the mobile liquid phase and the stationary phase. HPLC is the method of choice to confirm a positive screening test. It is also used to monitor therapy. It is functioned to determine the quantity of acid amino present in the sample.

Mohd Hairi Bin A.Hamid Bsc. Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (July 2009)

It is more popular to used due to: o More sensitive, o More accurate quantitative determination. o Suitable to separate non-volatile sample It is suitable to separate and identify the:
o

Proteins, amino acids, nucleic acids, hydrocarbons, carbohydrates, pharmaceuticals, pesticides, pigments, antibiotics, steroids, and another inorganic substance.

If identification is uncertain, GC-MS method was used.

2) Ion-exchange chromatography (IEC) The separation is based on the competition of different ionic compounds of the sample for the active sites on the ion-exchange resin (column packing) It can be used for any kind of charged molecule including large proteins, small nucleotides and amino acids. Cation exchange chromatography Anion exchange chromatography

Positively charged amino acid attracted to a negatively charged solid (stationary phase)

Negatively charged amino acid attracted to a positively charged solid (stationary phase)

Mohd Hairi Bin A.Hamid Bsc. Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (July 2009)

3) Gas liquid chromatography (GLC) In GLC, the mobile phase is a gas (helium) and the stationary phase is a high boiling point liquid absorbed onto a solid.

4) Gas chromatography mass spectroscopy (GC-MS) GC-MS is a method that combined the characteristics of gas-liquid chromatography and mass spectrometry to identify different substances within a test sample. Gas-liquid chromatography separates the components of a mixture, and mass spectroscopy characterizes each of the components individually. Function: To determine the unknown amino acid or metabolites if necessary (eg: diagnosis of equivocal data by other methods). It is also not commonly used due to quantitation methods are generally sufficient for diagnosis. Applications of GC-MS include: o Drug detection, fire investigation, environmental analysis, explosives investigation o To detect substances in luggage or on human beings (Airport security) Treatments Do screening test during newborn of the baby (can identify and treat early) Avoid eating high protein foods especially high phenylalanine (Eg: meat, fish, eggs, cheese, milk) Eat more cereals, starches, fruits, and vegetables Check the phenylalanine level in your blood frequently (consistent normal level can be maintained) Therapies: Gene therapy and injectable form of PAH.

Mohd Hairi Bin A.Hamid Bsc. Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (July 2009)

References 1. Mary Hotaling (1998), Saunders Manual of Clinical Laboratory Science (1st Edition): Amino Acids and Proteins,, W.B Saunders Company. 2. Guthrie test (2009), Wikimedia Foundation, Inc; retrieved on 26th August 2009 from http://en.wikipedia.org/wiki/Guthrie_test 3. What is the Guthrie test and why is it done? (2009), retrieved on 26th August 2009 from http://www.midwivesonline.com/parents/parents1//96 4. Phenylketonuria (2009) , retrieved on 26th August 2009 from http://www.answers.com/topic/pku 5. Chances' Choices Scene I: Michelle is saved by the starving bacteria (2007), Foundation for Blood Research, retrieved on 26th August 2009 from http://www.fbr.org/publications/cc/chances-choices.html 6. Gas Chromatography-Mass Spectroscopy Background (1998), retrieved on 26th August 2009 from http://www.gmu.edu/departments/SRIF/tutorial/gcd/gc-ms2.htm 7. Gas chromatography-mass spectrometry (2009), retrieved on 26th August 2009 from http://en.wikipedia.org/wiki/Gas_chromatography-mass_spectrometry 8. Jim Clark (2007), High performance liquid chromatography, retrieved on 26th August 2009 from http://www.chemguide.co.uk/analysis/chromatography/highperformanceliquid.htm l 9. Jim Clark (2007), Gas-Liquid Chromatography, retrieved on 26th August 2009 from http://www.chemguide.co.uk/analysis/gasliquidchromatography.html 10. Principles of Ion Exchange Chromatography, Tosoh Bioscience LLC: The Chemistry of Innovation, Montgomeryville, PA retrieved on 26th August 2009 from http://www.separations.us.tosohbioscience.com/Products/Principle