Guthrie test

The Guthrie test is a bacterial inhibition assay for the measurement of phenylalanine. 

Figure 10.16 The Guthrie test. The similarity in structure between phenylalanine and its metabolic antagonist, /3-2-thienylalanine, provides the basis for a microbiological assay for phenylalanine.

During the past 20 years, neonatal screening programs using the Guthrie test for inborn errors of metabolism such as phenylketonuria have become more widely used. The characteristics of diseases in which mass screening is... 

Control of PKU can be achieved simply by resorting to a low PA-containing diet. However, failure to diagnose PKU will result in mental deficiency, and early diagnosis is essential. In 1968, the UK Medical Research Council Working Party on PKU recommended the adoption of the Guthrie test as a conve-... 

Fig. 25.8 (a) 3-Thienylalanine, (b) phenylalanine, (c) standard curve in Guthrie test. 

If positive, the Guthrie test provides presumptive evidence for the presence of PKU. It should be confirmed by other, chemical, means. 

The screening test for phenylketonuria (PKU) is called the Guthrie test. Based on this screening method, which of the following is the most likely explanation of a false-negative screen in a newborn ... 

Classical phenylketonuria is an hereditary defect in the synthesis of Phe hydroxylase (the enzyme may be absent or inactive), which affects about 1 infant in 10,000. These individuals are unable to convert Phe into tyrosine, and the major route of Phe metabolism is thus blocked. Phenylpyruvate and phenylacetic acid are excreted in the urine. The condition is accompanied by defective pigmentation and, if untreated, by severe mental retardation (hence the other name, phenylpyruvic oligophrenia, also known as Polling s syndrome). Tlie urine of newborn infants is now routinely tested (Guthrie test) for the presence of phenylketones the condition can be compensated by a diet low in phenylalanine, and the typic mental retardation is thereby avoided. Other types of phenylketonuria are due to defective reduction or synthesis of dihydrobiopterin (see Inborn errors of metabolism). 

The simplicity and reliability of the Guthrie test made it the basis for newborn screening programs around the world. Today, the Guthrie screen-... 

Two patients were missed in the newborn screening due to the negative Guthrie test. 

A positive Guthrie test for Phe should be repeated and confirmed by quantitative analysis. Screening for a BH4 deficiency should be done in all newborns with even slight hyperphenylalaninemia (plasma Phe >120 pmol/1) as well as in older children without hyperphenylalaninemia but with neurological symptoms suggestive of a neurotransmitter deficiency. The following protocol is suggested ... 

An aromatic amino acid found in increased levels in the blood and urine in the inborn error of metabolism, phenylketonuria. It can be estimated by microbiological assay (the Guthrie test, q.v.) or by its reaction with ninhydrin to form a fluorescent compound. This fluorescence is enhanced by the addition of the dipeptide, L-leucylalanine. 

NBS is sharing with LC-MS/MS the same sample collection (filter paper collection and handling is now referred as dried blood spot (DBS)) and sometimes this creates some confusion in the terminology. For example, Guthrie card was intended to be the filter paper where blood is collected and today, with the adoption of LC-MS/MS, this definition is still used, even if LC-MS/MS has nothing to do with the Guthrie test (bacterial growth inhibition assay). 

Staying with the comparison with the classical Guthrie test, LC-MS/MS quantifies not only phenylalanine, but also several other strategically important AA (for example, tyrosine, and the AA involved in the urea cycle) and, in the same run, the AC all with high precision and accuracy. 

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