Confirmation of Hereditary Tyrosinemia Type 1 In

Journal of Experimental Biology and Agricultural Sciences, May - 2014; Volume – 2(2S)

Journal of Experimental Biology and Agricultural Sciences

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ISSN No. 2320 – 8694

CONFIRMATION OF HEREDITARY TYROSINEMIA TYPE 1 IN NEONATAL STAGE USING SPECTROPHOTOMETRIC MICROASSAY BASED ON THE DETERMINATION OF SUCCINYLACETONE LEVEL IN DRIED-BLOOD SPOTS

Gautam Kumar*, Akanksha Sharma* and Shivam P S Y

Department of Biotechnology, Institute of Biomedical Education and Research, Mangalayatan University, Aligarh – 202145, (U.P), India.

Received – February 20, 2014; Revision – March 05, 2014, Accepted – April 26, 2014 Available Online – May 31, 2014.

KEYWORDS ABSTRACT

Hereditary Tyrosinemia Hereditary Tyrosinemia Type 1 (HT1) is a metabolic disorder below to the class of autosomal recessive Type1 inheritance caused by the dearth of enzyme fumaryl acetoacetase the last enzyme in the tyrosine catabolic pathway . Affected individuals show increased tyrosine and succinyl acetone concentration Succinyl Acetone (SA) in blood. Patients also excrete increased concentration of SA in urine. The disorder is characterized d-aminolevulinate by progressive liver disease and renal tubular defects with accompanying hypophosphatemic rickets. Symptoms of HT1 usually appear in the first few months of life and include failure to gain weight and dehydratase grow at the expected rate, diarrhea, vomiting, yellowing of the skin and whites of the eyes (jaundice). It Fumarylacetoacetase may also lead to liver and kidney failure and an increased risk of liver cancer. Liver transplantation is enzyme the only effective treatment for hereditary tyrosinemia type 1. In the present study, Succinyl Acetone is measured by using its inhibitory property on d-aminolaevulinate dehydratase enzyme for the diagnosis of HT1 in dried blood spots.

* Corresponding author (Both authors contributed equally) E-mail: [email protected] (Akanksha Sharma)

Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

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294 Akanksha et al

1 Introduction type 1 tyrosinemia affects 1 person in 1,846. The carrier rate has been estimated to be between 1 in 20 (De Braekeleer M et Type 1 Tyrosinemia, also known as hepatorenal tyrosinemia, is al.,1990). As succinyl acetone is an inhibitor of ALA the most severe form of tyrosinemia. It is caused by a (Aminolevulinate) dehydratase so its measurement is therefore deficiency of the enzyme fumarylacetoacetate hydrolase. Type reliable for early diagnosis of Hereditary Tyrosinemia (Sassa S 1 tyrosinemia an autosomal recessive disease. Worldwide, et al.,1977). prevalence of type I tyrosinemia is 0.00001%. Fumarylacetoacetate hydrolase catalyzes the final step in the 2 Materials and Methods degradation of tyrosine-fumarylacetoacetate to fumarate, acetoacetate and succinate (Lindblad B et al.,1977; Sassa S et 2.1 Chemicals Used al., 1982). Fumarylacetoacetate gets accumulated in hepatocytes and proximal renal tubal cells and causes oxidative MES Buffer (2-ethanesulfonic acid), Mercuric Chloride damage and DNA damage leading to cell death and (HgCl2), Trichloroacetic acid (TCA), Dimethyl amino dysfunctional gene expression which alters metabolic benzaldehyde, Tris, Perchloric Acid, ALA (α-linolenic acid), processes like protein synthesis and gluconeogenesis. The Dithiotreitol (DTT), Glacial Acetic Acid, Sodium hydroxide increase in fumarylacetoacetate also inhibits previous steps in and Succinyl Acetone. For the preparation of all these aqueous tyrosine degradation leading to an accumulation of tyrosine in solutions distilled water is used. the body (Holme E et al.,1992; Kvittingen EA et al.,1992). As a result of this, patients with HT shows increased blood 2.2 Samples concentrations of tyrosine and succinylacetone and excrete increased concentrations of succinylacetone and d- Dried Blood Spots (DBS) specimens from 70 neonates, aminolevulinic acid (ALA) in the urine (Grenier A et al.,1982; collected in Sir Ganga Ram Hospital by a heelstick on days 3– Jakobs C et al.,1988; Bergdahl IA et al.,1997 and Campagna D 5 (median, day 4) of life, was spotted on filter paper. et al.,1999). 2.3 Buffers and Solution Preparation to test the samples However, this assay lacks sensitivity because it requires visual judgment of the color reaction. We refined this assay as a MES Buffer (50mmol/L at pH 6.4) was prepared by dissolving semiquantitative spectrophotometric test and investigated its 900mL of distilled water into 10.66g of MES. 2mol/L NaOH is reliability as confirmatory test for HT (Laberge C et al.,1990 used to maintain the 6.4 pH of MES Buffer and its volume is and Lindstedt S et al.,1992). Tyrosine is not directly toxic to maintained upto 1000 mL with distilled water. This buffer was the liver or kidneys but causes dermatologic and kept at 40C for storing. The 30.3 g of Tris was added and neurodevelopmental problems. Type 1 tyrosinemia typically dissolved in 100 mL distilled water to make 2.5mol/L Tris. It presents in infancy as failure to thrive and hepatomegaly. The was stored at 4oC. DTT solution of 21mmol/L should be primary effects are progressive liver and kidney dysfunction. prepared fresh by dissolving 16.2 mg of Dithiotreitol in 5mL The liver disease causes cirrhosis, conjugated of already prepared 50 mmol/L MES Buffer. The 30mmol/L hyperbilirubinemia, elevated AFP, hypoglycemia and ALA (α-lenolenic acid) was also freshly prepared by coagulation abnormalities. This can lead dissolving 25.2mg of ALA in 4.5 mL of distilled water and to jaundice, ascites and hemorrhage. There is also an increased adjusting its pH to 6.0 with 2.5 mol/L Tris by maintaining a risk of hepatocellular carcinoma. The tyrosine accumulation is total volume of 5mL with distilled water. Trichloro acetic acid one of the causes for Fanconi Syndrome (TCA) and Mercuric Chloride (HgCl2) were together dissolved http://en.wikipedia.org/wiki/Fanconi_syndrome: renal tubular as 125g and 10g respectively in distilled water and brought to a acidosis, hypophosphatemia and aminoaciduria (Gentz J et total volume of 1000 mL and stored in an amber glass bottle at al.,1969; Gaull GE et al.,1970; Christensen E et al.,1981 and ambient temperature. Pronicka E et al.,1991). Cardiomyopathy, neurologic and dermatologic manifestations are also possible. The primary The modified Ehrlich reagent includes 1g of dissolved treatment for type 1 tyrosinemia is nitisinone (Orfadin). dimethylaminobenzaldehyde in 30mL of glacial acetic acid and Nitisinone inhibits the conversion of 4-OH phenylpyruvate to 8 mL of 700 g/L perchloric acid and the volume was made to homogentisic acid by 4-OH phenylpyruvate dioxygenase (as 50mL with glacial acetic acid. This reagent should be freshly shown in the figure 1), the second step in tyrosine degradation prepared and used within the 6 hours of its preparation.The (Fujita H et al.,1991). By inhibiting this enzyme, the 3mm preincubation discs of DBSs on filter paper were accumulation of the fumarylacetoacetate is prevented. punched out into microtiter wells and 50mL of 21mmol/L DTT solution was added to each of these wells. Microtiter plates Previously, liver transplantation was the primary treatment were shaken gently at 100 rpm on a plate shaker at room option and is still used in patients in whom nitisinone fails. temperature for 15 minutes. ALA-D in erythrocytes catalyzes This type of tyrosinemia is much more common in Quebec, the formation of porphobilinogen from ALA and its activity Canada. The overall incidence in Qubec is about 1 in 16,000 was estimated as an end point determination. The 10 mL of 30 individuals. In the Saguenay Lac Saint Jean region of Quebec, mmol/L ALA solution was added to each test sample and 10

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Confirmation of Hereditary Tyrosinemia Type 1 in neonatal stage using Spectrophotometric Microassay . . . 295 mL of 50 mmol/L MES buffer was added to each respective mixture turns brown due to solubilisation of haemoglobin. blank solutions. It should be noted that ALA should not be Blanks and samples from tyrosinemic patients remained straw- added to any of the blank sample and MES Buffer not to test coloured. The reaction was judged visually within these 5 min, samples. The enzyme was incubated for 4 hours at room but the colour development reached a maximum after 8 – 10 temperature and 100 rpm at plate shaker. After the 4 hours minutes. The absorbance of porphobilinogen was measured at incubation period, 40 mL of the TCA-Mercuric Chloride 550 nm on a microtiter plate spectrophotometer. mixture was added to each well to stop the reaction. After obtaining complete mixture by repeated aspiration, these liquid 3 Results samples were transferred into their respective properly labelled eppendorf tubes and centrifuged at 390 g for 30 seconds. The For checking out the positive cases of Hereditary Tyrosinemia 80mL of supernatant from each tube was again transferred to Type 1, 70 different dried blood specimens are processed microtiter plates and their absorbance at 550nm was observed through this procedure to find out the increased concentration after adding 100mL of freshly prepared modified Ehrlich of succinyl acetone. Out of these 70 different samples, 1 case is reagent. 70 dried blood specimens of newborns (received in found positive as it has increased concentration of succinyl month of Jan 2013) are screened by this process for the early acetone in blood as compared to the normal range i.e. >2uM/L. diagnosis of HT1. These 70 dried blood specimens are processed during two 2.4 Colour reaction different experiments in which 40 and 30 samples are processed respectively. During the first experiment with 40 For the colour reaction, 100 mL of freshly prepared modified samples, the standards of succinyl acetone (shown in Table 1) Ehrlich reagent was added. A purple colour is produced due to and their graph (Figure 1) are finally used to determine the porphobilinogen appeared within 1 min and the colour concentration of Succinylacetone in each case (Table 2). intensity increases for another 5 min before the reaction

Table 1Overall representation of 70 processed samples.

Range No. of Patients Interpretation 0-2 uM/L 69 Normal >2 uM/L 01 Affected

Table 2 Standards of Succinyl Acetone and their O.D. during first experiment.

Conc (uM/L) O.D. 0 0.615 0.25 0.484 0.5 0.433 1 0.261 2 0.151

Figure 1 Graph plotted between concentration of standards of succinyl acetone and their optical density during first experiment.

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296 Akanksha et al During first experiment in which 40 dried blood specimens of out with 30 dried blood specimens of neonates whose neonates are processed, all the cases are found to be normal standards (Table 3 and 4) and their graph (figure 2) are finally (negative) as concentration of succinyl acetone in blood is not used to determine the concentration of succinyl acetone in each >2uM/L in any of the sample. So, next experiment is carried case.

Table 3 Concentration of succinyl acetone during first experiment

S.No. O.D. Test O.D. Blank Final O.D Conc. Activity % Inhibition % 1 0.36 0.107 0.253 1.05 41.138211 58.86178862 2 0.327 0.122 0.205 1.41 33.333333 66.66666667 3 0.415 0.11 0.305 0.79 49.593496 50.40650407 4 0.371 0.1 0.271 0.98 44.065041 55.93495935 5 0.379 0.099 0.28 0.9 45.528455 54.47154472 6 0.52 0.127 0.393 0.54 63.902439 36.09756098 7 0.273 0.093 0.18 1.6 29.268293 70.73170732 8 0.185 0.117 0.068 0.04 11.056911 88.94308943 9 0.298 0.091 0.207 1.39 33.658537 66.34146341 10 0.339 0.119 0.22 1.22 35.772358 64.22764228 11 0.588 0.108 0.48 0.292 78.04878 21.95121951 12 0.468 0.127 0.341 0.68 55.447154 44.55284553 13 0.388 0.119 0.269 0.992 43.739837 56.2601626 14 0.364 0.101 0.263 0.97 42.764228 57.23577236 15 0.481 0.108 0.373 0.593 60.650407 39.3495935 16 0.537 0.124 0.413 0.48 67.154472 32.84552846 17 0.302 0.099 0.203 1.393 33.00813 66.99186992 18 0.41 0.093 0.317 0.767 51.544715 48.45528455 19 0.298 0.172 0.126 2.3 20.487805 79.51219512 20 0.318 0.092 0.226 1.25 36.747967 63.25203252 21 0.418 0.091 0.327 0.72 53.170732 46.82926829 22 0.336 0.114 0.222 1.247 36.097561 63.90243902 23 0.333 0.104 0.229 1.19 37.235772 62.76422764 24 0.531 0.097 0.434 0.441 70.569106 29.43089431 25 0.528 0.088 0.44 1.07 71.544715 28.45528455 26 0.527 0.075 0.452 1.11 73.495935 26.50406504 27 0.387 0.097 0.29 0.851 47.154472 52.84552846 28 0.418 0.119 0.299 0.838 48.617886 51.38211382 29 0.378 0.124 0.254 1.04 41.300813 58.69918699 30 0.439 0.067 0.372 0.587 60.487805 39.51219512 31 0.316 0.107 0.209 1.388 33.98374 66.01626016 32 0.322 0.11 0.212 1.378 34.471545 65.52845528 33 0.521 0.093 0.428 0.443 69.593496 30.40650407 34 0.278 0.118 0.16 1.881 26.01626 73.98373984 35 0.451 0.129 0.322 0.758 52.357724 47.64227642 36 0.287 0.108 0.179 1.61 29.105691 70.89430894 37 0.423 0.105 0.318 0.75 51.707317 48.29268293 38 0.309 0.102 0.207 1.39 33.658537 66.34146341 39 0.483 0.102 0.381 0.58 61.95122 38.04878049 40 0.323 0.113 0.21 1.34 34.146341 65.85365854

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Confirmation of Hereditary Tyrosinemia Type 1 in neonatal stage using Spectrophotometric Microassay . . . 297 Table 4 Standards of Succinyl Acetone and their O.D.

Conc (uM/L) O.D. 0 0.692 0.125 0.681 0.25 0.631 0.5 0.549 1 0.452 2 0.239

Table 5 Concentration of Succinylacetone in each case during second experiment of remaining 30 samples.

S.No. O.D.Blank O.D.Test Final O.D Conc Activity % Inhibition% 41 0.479 0.074 0.405 1.19 58.526012 41.47398844 42 0.442 0.055 0.387 1.22 55.924855 44.07514451 43 0.402 0.081 0.321 1.48 46.387283 53.61271676 44 0.327 0.054 0.273 1.64 39.450867 60.54913295 45 0.623 0.052 0.571 0.37 82.514451 17.48554913 46 0.414 0.071 0.343 1.41 49.566474 50.43352601 47 0.498 0.07 0.428 1.79 61.849711 38.15028902 48 0.42 0.07 0.35 1.39 50.578035 49.42196532 49 0.406 0.047 0.359 1.37 51.878613 48.12138728 50 0.419 0.063 0.356 1.38 51.445087 48.55491329 51 0.452 0.066 0.386 1.22 55.780347 44.21965318 52 0.52 0.047 0.473 0.71 68.352601 31.64739884 53 0.428 0.051 0.377 1.25 54.479769 45.52023121 54 0.388 0.057 0.331 1.48 47.83237 52.16763006 55 0.395 0.051 0.344 1.39 49.710983 50.28901734 56 0.131 0.061 0.07 >2 10.115607 89.88439306 57 0.340 0.058 0.282 1.43 17.485549 82.51445087 58 0.624 0.075 0.549 0.49 79.33526 20.66473988 59 0.542 0.042 0.5 0.63 72.254335 27.74566474 60 0.448 0.048 0.4 1.2 57.803468 42.19653179 61 0.344 0.048 0.296 1.61 42.774566 57.22543353 62 0.423 0.039 0.384 1.21 55.491329 44.50867052 63 0.633 0.058 0.575 0.38 83.092486 16.90751445 64 0.536 0.06 0.476 0.71 68.786127 31.21387283 65 0.556 0.067 0.489 0.70 70.66474 29.33526012 66 0.503 0.044 0.459 0.79 66.32948 33.67052023 67 0.432 0.065 0.367 1.28 53.034682 46.96531792 68 0.375 0.059 0.316 1.51 45.66474 54.33526012 69 0.547 0.052 0.495 0.65 71.531792 28.46820809 70 0.597 0.066 0.531 0.49 76.734104 23.26589595

During second experiment in which 30 dried blood specimens is 89.88% inhibited due to the increased concentration of of neonates are processed all the cases are found to be normal succinyl acetone during tyrosine degradation pathway. This is (negative) as concentration of succinyl acetone in blood is not the only positive case observed among the 70 dried blood > 2uM/L in any of the sample except in one case (Serial No. samples of neonates. Blanks and samples of this patient 56, table No 5). In this case concentration of succinyl acetone remained straw-coloured even after the addition of modified is >2 (which is out from normal range). Activity % of Ehrlich reagent. fumarylacetoacetic acid hydrolase enzyme is only 10.11% as it

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298 Akanksha et al

Figure 2 Graph plotted between Concentration of standards of Succinyl Acetone and their Optical Density during second experiment of remaining 30 samples.

4 Discussions transplantation was the primary treatment option and is still used in patients in whom nitisinone fails. Hereditary tyrosinemia is inherited in an autosomal recessive manner. This disorder may be manifested as an acute or Acknowledgment chronic form. During this disease succinyl acetone concentration in the blood and excretion in urine is increased. I would like to give special thanks to Dr. J Verma, Senior Concentration of tyrosine, methionine and phenylalanine is Consultant of Biochemical Genetics, Sir Ganga Ram Hospital also elevated in plasma along with the increased urinary for her constructive suggestions and help. excretion of δ-aminolevulinic acid (Christensen E et al.,1981). It is caused by either missense or deletion mutations in the References FAH gene coding for 4-fumaryl acetoacetate hydrolase. Molecular testing involves analyzing the FAH gene for Lindblad B, Lindstedt S, Steen G (1977) On the enzymic reported or rare mutations. defects in hereditary tyrosinemia. Proc Natl Acad Sci USA: 74: 4641-4645. Biochemical testing for this disease includes measurement of tyrosine, succinyl acetone and δ-aminolevulinic acid Lindstedt S, Holme E, Lock EA, Hjalmarson O, Strandvik B dehydratase activity as the activity of δ-aminolevulinic acid (1992) Treatment of hereditary tyrosinemia type 1 by (pink colouration) is inversely proportional to the inhibition of 4-hydroxyphenylpyruvate dioxygenase. Lancet: concentration of succinyl acetone (Gaull GE et al., 1970 and 340: 813- 817. Berger R et al.,1983). Straw colouration in affected patient samples results due to the elevated levels of succinyl acetone Holme E, Lindstedt S (1998) Tyrosinemia type 1 and NTBC (Gentz J et al.,1969; Grenier A et al.,1982 and Jakobs C et (2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione). J al.,1988). In unaffected individuals negligible amounts of Inherit Metab Dis: 21: 507-517. succinyl acetone results in deep pink colouration. Christensen E, Jacobsen BB, Gregersen N, Hjeds H, Pedersen As a precautionary measure the test should be carried out in JB, Brandt NJ, Baekmark UB (1981) Urinary excretion of plastic tubes as glass tubes can inhibit enzyme activity. Some succinylacetone and d-aminolevulinic acid in patients with of the severe diseases like hepatocellular carcinoma, hereditary tyrosinemia. Clin Chim Acta: 116: 331-341. aminoaciduria, rickets, jaundice, ascites, gastrointestinal bleeding and neurological disorders are the clinical features of Pronicka E, Mielniczuk Z, Rowinska E, Ksiazyk J, Hereditary Tyrosinemia Type1(Lindblad B et al.,1977; Szczygielska-Kozak M, Wieczorek E, Kulczycka H (1991) Goulden KJ et al.,1987 and Kvittingen EA et al.,1992). The Urinary succinylacetone presence and d-aminolaevulinic acid primary treatment for type1 tyrosinemia excretion in patients with type 1 tyrosinemia during treatment. is nitisinone (Orfadin). Nitisinone inhibits the conversion of 4- Mater Med Pol: 23: 136-138. OH phenylpyruvate to homogentisic acid by 4-OH phenylpyruvate dioxygenase, the second step in tyrosine Gaull GE, Rassin DK, Solomon GE, Harris RC, Sturman JA degradation. By inhibiting this enzyme, the accumulation of (1970) Biochemical observations on so called hereditary the fumarylacetoacetate is prevented. Previously, liver tyrosinemia. Pediatr Res: 4: 337-344.

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Confirmation of Hereditary Tyrosinemia Type 1 in neonatal stage using Spectrophotometric Microassay . . . 299

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Sassa S, Kappas A (1982) Succinylacetone inhibits d- Bergdahl IA, Grubb A, Schutz A, Desnick RJ, Wetmur JG, aminolevulinate dehydratase and potentiates the drug and Sassa S, Skerfving S (1997) Lead binding to d-aminolevulinic steroid induction of d-aminolevulinate synthase in liver. Trans acid dehydratase in human erythrocytes. Pharmacol Toxicol: Assoc Am Physicians: 95: 42-52. 81: 153-158.

De Braekeleer M, Larochelle J (1990) Genetic epidemiology of Campagna D, Huel G, Girard F, Sahuquillo J, Blot P (1999) hereditary tyrosinemia in Quebec and in Saguenay-Lac-St- Environmental lead exposure and activity of d-aminolevulinic Jean. Am J Hum Genet: 47: 302-307. acid dehydratase (ALA-D) in maternal and cord blood. Toxicology: 134: 143-152. Holme E, Lindstedt S (1992) Neonatal screen for hereditary tyrosinemia type 1. Lancet: 340: 850.

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