Development of a Whole Blood Paper-Based Device for Phenylalanine Detection in the Context of PKU Therapy Monitoring

Laboratory-based testing does not allow for the sufficiently rapid return of data to enable optimal therapeutic monitoring of patients with metabolic diseases such as phenylketonuria (PKU). The typical turn-around time of several days for current laboratory-based testing is too slow to be practically useful for effective monitoring or optimizing therapy. This report describes the development of a rapid, paper-based, point-of-care device for phenylalanine detection using a small volume (40 μL) of whole blood. The quantitative resolution and reproducibility of this device with instrumented readout are described, together with the potential use of this device for point-of-care monitoring by PKU patients.Keywords: whole-blood assay, phenylketonuria, phenylalanine detection, colorimetric readout, paper-based deviceKeywords: whole-blood assay, phenylketonuria, phenylalanine detection, colorimetric readout, paper-based devic

Development of a Paper-Based Whole Blood Phenylalanine Assay for PKU Diagnosis and Monitoring in Low Resource Settings

Phenylketonuria (PKU) is a genetic inborn metabolic disorder which inhibits the functional production of the enzyme phenylalanine hydroxylase (PAH). It currently affects one in 15,000 Americans and is the most common amino acid metabolism error found in newborns. Individuals affected with PKU are incapable of metabolizing the amino acid, phenylalanine (Phe) and can face irreversible bodily damage, such as seizures or mental retardation, with elevated concentrations of Phe in the body. PKU is a lifelong disease that requires a regimented diet and total avoidance of foods containing Phe. Current methods for measuring Phe concentrations in blood require routine laboratory tests. It can take several days and even weeks for patients to get the results of these tests. This greatly limits the usefulness of measuring the patient Phe levels using current methods. The availability of these tests are limited to places with testing laboratories where patients can mail dried blood samples, which is generally only in developed nations. A point-of-care (POC) device capable of measuring blood Phe levels in low-resource settings, such as in homes or developing nations, could lower test cost and turn-around time and broaden the accessibility of Phe monitoring for affected individuals. This report describes the conversion of a laboratory-based enzymatic/colorimetric assay (ECA) into a paper-based lateral flow test (LFT) that can detect relevant Phe concentrations in a whole blood sample. The proposed assay uses a well- known, two-step chemical reaction resulting in a colorimetric response that can visually be correlated to Phe concentration. An experiment was first developed to demonstrate assay capabilities with Phe-spiked human plasma in glass fiber and cellulose substrates. A plasma separation membrane was integrated that enabled the assay to accept and process whole blood samples. The assay successfully correlated blood Phe concentrations with the intensity of a visible colorimetric response from a Phe-spiked whole blood sample

J Inherit Metab Dis

Analysis of blood phenylalanine is central to the monitoring of patients with phenylketonuria (PKU) and age-related phenylalanine target treatment-ranges (0-12\u2009years; 120-360\u2009\u3bcmol/L, and\u2009>12\u2009years; 120-600\u2009\u3bcmol/L) are recommended in order to prevent adverse neurological outcomes. These target treatment-ranges are based upon plasma phenylalanine concentrations. However, patients are routinely monitored using dried bloodspot (DBS) specimens due to the convenience of collection. Significant differences exist between phenylalanine concentrations in plasma and DBS, with phenylalanine concentrations in DBS specimens analyzed by flow-injection analysis tandem mass spectrometry reported to be 18% to 28% lower than paired plasma concentrations analyzed using ion-exchange chromatography. DBS specimens with phenylalanine concentrations of 360 and 600\u2009\u3bcmol/L, at the critical upper-target treatment-range thresholds would be plasma equivalents of 461 and 768\u2009\u3bcmol/L, respectively, when a reported difference of 28% is taken into account. Furthermore, analytical test imprecision and bias in conjunction with pre-analytical factors such as volume and quality of blood applied to filter paper collection devices to produce DBS specimens affect the final test results. Reporting of inaccurate patient results when comparing DBS results to target treatment-ranges based on plasma concentrations, together with inter-laboratory imprecision could have a significant impact on patient management resulting in inappropriate dietary change and potentially adverse patient outcomes. This review is intended to provide perspective on the issues related to the measurement of phenylalanine in blood specimens and to provide direction for the future needs of PKU patients to ensure reliable monitoring of metabolic control using the target treatment-ranges.CC999999/ImCDC/Intramural CDC HHS/United States2021-03-15T00:00:00Z31433494PMC7957320930

Hüperfenüülalanineemiad ja seotud neurofüsioloogilised häired

Väitekirja elektrooniline versioon ei sisalda publikatsiooneÜks sagedasemaid ainevahetushaigusi on fenüülketonuuria (FKU), mille puhul muutuvad toksiliseks toiduvalgust saadav aminohappe fenüülalaniini (Phe) ülehulk ja selle lagundamise kõrvalsaadused. FKU tõttu tekkivad vaimsed ja füüsilised puudused on välditavad, kui sellega patsient saab varakult valguvaesele dieetravile koos Phe-vaba asendusseguga. Vastsündinuid sõeluuritakse FKU suhtes Eestis alates 1993. aastast. Selgus, et 94 Eesti PKU patsiendi seas on väga kõrge geneetiline homogeensus: tervelt 80,4% kõigist haigusega alleelidest on PAH geeni variandiga Arg408Trp; kokku leidus Eestis 17 erinevat patogeenset varianti. Nii kõrge homogeensus kajastub ka fenotüübis: 87%-l on klassikaline FKU madala Phe taluvusega. FKU sagedus Eestis on 1 : 6700 sünni kohta. Paljude Eesti FKU patsientide suguvõsa pärineb Lõuna- ja Kagu-Eestist. Eesti rahvusgruppide seas ei erine FKU sagedus ega alleelne jaotus. Eesti PKU patsientide dieetravi on jälgitav vereanalüüside kaudu, mille vastused talletatakse SA TÜK laboriinfosüsteemis. Varases lapseeas FKU patsientidel suudab enamus perekondi hoida Phe taset dieedi abil ettenähtud piirides, kuid algkoolieas on juba üle poolte patsientide analüüside mediaanväärtus soovituslikust kõrgem. Murdeeas olukord veidi paraneb ja jääb sarnaseks ka täiskasvanutel. Ka teistes riikides on täheldatud sellist tendentsi. Kõrge Phe tase võib tuleneda ka Phe lagundamise kofaktori tetrahüdrobiopteriini (BH4) puudulikkusest. BH4 häired on PKU-st palju haruldasemad ja enamasti raskema kuluga ja keerulisemad ravida. Eestis sündis 1991. aastal BH4 taastamise häirega laps, kellel diagnoositi DHPR puudulikkus, kuid seni ei õnnestunud tsütogeneetiliselt ega Sanger sekveneerimisega leida haiguse molekulaarset põhjust. Lahenduseni viis genoomi sekveneerimine, mis võimaldas leida senitundmatu 9,1 Mb suuruse inversiooni 4. kromosoomis, mis katkestas DHPR ensüümi tootva geeni. Teadaolevalt pole seni leitud nii suurt haigusseoselist struktuurset varianti genoomis.One of the most common inherited metabolic disorders is phenylketonuria (PKU), a condition where excess phenylalanine (Phe) contained in dietary protein, and its metabolites, become toxic. Mental and physical disabilities can be avoided by early low-protein dietary treatment with Phe-free protein substitution. In Estonia, newborns are screened for PKU since 1993. Very high homogeneity among 94 Estonian PKU patients was revealed: 80.4% of all the affected alleles carry the Arg408Trp variation in the PAH gene; 17 different pathogenic alleles were found. This homogeneity is reflected in phenotype: 87% of the patients have classical PKU with low Phe tolerance. The incidence of PKU in Estonia is 1 in 6700 newborns. The pedigree of many Estonian PKU patients can be traced to South or South-East Estonia. The incidence and allelic distribution of PKU does not differ among ethnic groups in Estonia. The treatment of Estonian PKU patients can be traced by the records of the blood sample analyses in the LIMS of Tartu University Hospital. In early childhood, most of the families manage to keep the Phe levels in recommended range with diet. In elementary school, the median results of more than half of the patients’ analyses exceed the recommended level. In adolescence, the situation somewhat improves and remains similar in adulthood. The tendency has been observed also in other countries. High levels of Phe may be caused also by the deficiency of tetrahydrobiopterin (BH4), a cofactor in Phe metabolism. The disorders of BH4 are rare, usually more complicated, and more difficult to treat. In 1991, a child with a disorder in BH4 regeneration was born, who was diagnosed with DHPR deficiency, but neither cytogenetic methods, nor Sanger sequencing could reveal the molecular cause. Now, by genome sequencing, an unknown inversion of 9.1 Mb in chromosome 4 was revealed, which interrupted the gene coding the DHPR enzyme. No so large pathogenic structural variant in genome has been reported.https://www.ester.ee/record=b533139

Performance of laboratory tests used to measure blood phenylalanine for the monitoring of patients with phenylketonuria

Analysis of blood phenylalanine is central to the monitoring of patients with phenylketonuria (PKU) and age‐related phenylalanine target treatment‐ranges (0‐12 years; 120‐360 μmol/L, and >12 years; 120‐600 μmol/L) are recommended in order to prevent adverse neurological outcomes. These target treatment‐ranges are based upon plasma phenylalanine concentrations. However, patients are routinely monitored using dried bloodspot (DBS) specimens due to the convenience of collection. Significant differences exist between phenylalanine concentrations in plasma and DBS, with phenylalanine concentrations in DBS specimens analyzed by flow‐injection analysis tandem mass spectrometry reported to be 18% to 28% lower than paired plasma concentrations analyzed using ion‐exchange chromatography. DBS specimens with phenylalanine concentrations of 360 and 600 μmol/L, at the critical upper‐target treatment‐range thresholds would be plasma equivalents of 461 and 768 μmol/L, respectively, when a reported difference of 28% is taken into account. Furthermore, analytical test imprecision and bias in conjunction with pre‐analytical factors such as volume and quality of blood applied to filter paper collection devices to produce DBS specimens affect the final test results. Reporting of inaccurate patient results when comparing DBS results to target treatment‐ranges based on plasma concentrations, together with inter‐laboratory imprecision could have a significant impact on patient management resulting in inappropriate dietary change and potentially adverse patient outcomes. This review is intended to provide perspective on the issues related to the measurement of phenylalanine in blood specimens and to provide direction for the future needs of PKU patients to ensure reliable monitoring of metabolic control using the target treatment‐ranges

Biochemical Testing

Clinical Correlation and Diagnosis highlights the improvements in methodological approaches for the purposes of disease diagnosis and health research. Chapters cover such topics as serum protein electrophoresis, urinary iodine measurement, blood collection tubes, semi-solid phase assay and advancement in analytical and bioanalytical techniques, and serological diagnostic tools for Zika virus, among other subjects. All these will not be possible without a proper laboratory management where this book also includes the Tissue Bank ATMP Production as a model. The chapters are expected to provide a new perspective in health science which may trigger a further exploration into the diagnostic and research field

Molecular mechanisms of PAH function in response to phenylalanine and tetrahydrobiopterin binding

Phenylketonuria (PKU) is an autosomal recessive inborn error of metabolism (IEM) caused by mutations in the phenylalanine hydroxylase (PAH) gene. The molecular mechanism underlying deficiency of the PAH protein is, in most of the cases, loss of function due to protein misfolding. PAH mutations induce disturbed oligomerisation, decreased stability and accelerated degradation of hepatic PAH, a key enzyme in phenylalanine metabolism. Since the development of a phenylalanine-restricted diet in the 1950ies, PKU is a prototype for treatable inherited diseases. About 60 years later, the natural PAH cofactor tetrahydrobiopterin (BH4) was shown to act as a pharmacological chaperone stabilising the misfolded PAH protein. In consequence, BH4 (KUVAN®) was introduced to the pharmaceutical market as an alternative treatment for BH4-responsive PAH deficiency. Therefore, PKU is also regarded as a prototype for a pharmacologically treatable protein misfolding disease. Despite the progress in PKU therapy, knowledge on the molecular basis of PKU and the BH4 mode of action was still incomplete. Biochemical and biophysical characterisation of purified variant PAH proteins, which were derived from patient’s mutations, aimed at a better understanding of the molecular mechanisms of PAH loss of function. We showed that local side-chain replacements induce global conformational changes with negative impact on molecular motions that are essential for physiological enzyme function. The development of a continuous real-time fluorescence-based assay of PAH activity allowed for robust analysis of steady state kinetics and allosteric behaviour of recombinantly expressed PAH proteins. We identified positive cooperativity of the PAH enzyme towards BH4, where cooperativity does not rely on the presence of phenylalanine but is determined by activating conformational rearrangements. In vivo investigations on the mode-of-action of BH4 revealed differences in pharmacodynamics but not in pharmacokinetics between different strains of PAH-deficient mice (wild-type, Pahenu1/1 and Pahenu1/2). These observations pointed to a significant impact of the genotype on responsiveness to BH4. The available database information on PAH function associated with PAH mutations was based on non-standardised enzyme activity assays performed in different cellular systems and under different conditions usually focusing on single PAH mutations. These inconsistent data on PAH enzyme activity hindered robust prediction of the patient’s phenotype. Furthermore, assays on single PAH mutations do not reflect the high allelic and phenotypic heterogeneity of PKU with 89 % of patients being compound heterozygotes. In addition, the knowledge on enzyme function and regulation in the therapeutic and pathologic metabolic context was still scarce. In order to get more insight into the interplay of the PAH genotype, the phenylalanine concentration and BH4 treatment, we performed functional analyses of both, single, purified PAH variants as well as PAH full genotypes in the physiological, pathological and therapeutic context. The analysis of PAH activity as a function of phenylalanine and BH4 concentrations enabled determination of the optimal working ranges of the enzyme and visualisation of differences in the regulation of PAH activity by BH4 and phenylalanine depending on the underlying genotype. Moreover, these PAH activity landscapes allowed for setting rules for dietary regimens and pharmacological treatment based on the genotype of the patient. Taken together, precise knowledge on the mechanism of the misfolding-induced loss of function in PAH deficiency enabled a better understanding of the molecular mode of action of pharmacological rescue of enzyme function by BH4. We implemented the combination of genotype-specific functional analyses together with biochemical, clinical and therapeutic data of individual patients as a powerful tool for phenotype prediction and paved the way for personalised medicine strategies in phenylketonuria

Development of an innovative strategy based on engineered autologous erythrocytes as “enzyme replacement therapy” for the treatment of Phenylketonuria

open1openGabucci, ClaudiaGabucci, Claudi