İyon Kromatografi Kondüktivite Dedektörü İle Orotik Asidürili Hasta İdrarında Orotik Asit Tayini

Abstract

Son on yılda, biyolojik, klinik ve besin örneklerindeki organik asitlerin ölçülmesi büyüyen bir ilgi alanıdır. İnsan kan serumunda ya da idrarında bulunan bazı organik asitlerin konsantrasyonu ile bağlantılı olarak birçok hastalığın teşhisinin mümkün olduğu bilinmektedir. Orotik asit, de nova pirimidin sentezinin dördüncü ara ürünüdür. Bu sentez, glutamin, 2 mol ATP ve CO2 den meydana gelen karbomil fosfat ile başlar. Merkez bileşik üridin 5’-monofosfat (UMP)’ dır. Bu bileşik iki enzim ile katalizlenir; orotat fosforibozil transferaz (OPRT) ve oratat monofosfat dekarboksilaz (OPD). Orotik asit B13 vitamini olarak bilinir. Birçok deneysel çalışma orotik asidin karsigonesis, böbreklerde yağ depolanması ile ilgili olduğunu gösterdiği için orotik asidin biyolojik etkisi üzerine odaklanılmıştır. Orotik asidin tekrarlanan dozlarının tümör formlarının gelişmesini tetiklediği kabul edilmiştir. Orotik asidin idrar ile atılması söz konusu olduğundan kan plazmasında tayin edilmesindense idrarda tayin edilmesi daha olasıdır. Orotik asidin aşırılığının orotik asudüri denilen rahatsızlığa sebep olduğu bilinmektedir. Yetişkinlerde üre döngüsünde aksamaya sebep olur. Çocuklarda ise DNA ve RNA sentezinde kısıtlamaya yol açarak fiziksel ve sinirsel hasara sebep olabilir. Bu önemli hastalıkların teşhisinde güvenilir ve hassas bir metod geliştirilmesi gereklidir. Bu çalışmada, iyon kromatografi/suppressorlü iletkenlik dedektörü cihazı kullanarak insan idrarındaki orotik asidin miktarının tayini için seçici, güvenilir ve hassas bir yöntem geliştiriImesine çalışılmıştır. İdrar numunelerinin analizi için temizleme prosesi geliştirilmiştir. Numunelere santrifuj, kuvvetli katyon değiştirici ile muamele ve katı faz ektraksiyon işlemleri süre ve miktar optimizasyonu yapılarak uygulanmıştır. Öncelikle idrardaki süspansiyon halindeki katı parçacıkları uzaklaştırmak için santrifüj kullanılmıştır. İdrar örneklerindeki katyonları uzaklaştırmak için kuvvetli katyon değiştirici reçine kullanılmıştır. Katı faz ekstraksiyon prosedürü, idrar örneklerindeki orotik asidin ayrımının daha iyi gözlenebilmesi amacıyla uygulanmıştır. Ayrıca iyon kromatografi/suppressorlü iletkenlik dedektörü cihazı kullanarak idrardaki orotik asit miktarının hızlı, ucuz ve doğa dostu bir metodla belirlenebilmesi için kromatografik şartlar optimize edilmiştir. Optimum koşullar tespit edildikten sonra idrarın analize hazırlanması ve IC ile hızlı bir şekilde dedekte edilmesi sağlanmıştır.

In the latest decade, a growing interest has been noted for the determination of organic acids in biological, clinical and food samples. Some of organic acids found in human serum or urine have known in relation to their concentrations in biological fluids, since they are intermediates formed at various stages of amino acid as indicators of a variety of disease. Orotic acid (1,2,3,6-tetrahydro-2,6-dioxo-4-pyrimidinecarboxylic acid; uracil-6-carboxylic acid) is a minor component of the diet. It is found in whey and root vegetables, such as carrots and beets. In contrast to sheep’s and goat’s milk, cow’s milk contains a relatively large amount of orotic acid, while human milk lacks this compound. The biological effects of orotic acid have been focus on considerable attention, because recent experimental studies have demonstrated that it has effects of carcinogenesis, hepatic lipid storage and renal toxicity. Several studies have shown that repeated dosing of orotic acid promotes the formation of tumours initiated by various known carcinogenic substances. Because renal excretion of orotic acid is very efficient and urinary values integrate changes over time, orotic acid measurements are more relevant in urine than in plasma. Orotic acid is an intermediate product in pyrimidine biosynthesis. It is a heterocyclic compound and an acid; it is also known as pyrimidinecarboxylic acid It was originally introduced as a vitamin, called vitamin B13, but essentiality has not been demonstrated. It is not actually a vitamin, but was originally classified as such after being added to the diets of laboratory animals in the 1960s. The addition of it to their food encouraged heart health and showed other vitamin-like benefits. Orotic acid is a very important substance but difficult to analyze in biological samples. New insights into the importance of pyrimidine biosynthesis and metabolism in humans followed the recognition of the first genetic defect, hereditary orotic aciduria. Orotic acid is an intermediary metabolite in the biosynthesis of pyrimidine nucleotides. The acid is present in healthy adults urine only at trace concentrations in urine. However, at the deficiency of enzyme uridine monophosphate synthase, which participates in the synthesis of uridylic acid from orotic acid, the excretion of orotic acid into urine increases (orotic aciduria). It may be about 1000 times higher in urine of patients having orotic acid as a diagnostic metabolite. To diagnose inborn error of organisms, it is important to use a method enabling for determination of both increased and normal levels of OA. Organic acidemias, also known as organic acidurias, are a group of disorders characterized by increased excretion of organic acids in urine. A spectrum of disorders characterized by enzymatic defects in biochemical pathways leading to toxic accumulations of molecules normally metabolized and safely excreted from the body. Numerous types of organic acidemias exist, with methylmalonic aciduria, propionic acidemia and isovaleric acidemia among the most prevalent forms. They result primarily from deficiencies of specific enzymes in the breakdown pathways of amino acids or from enzyme deficiencies in beta oxidation of fatty acids or carbohydrate metabolism. Children with an organic acidemia are susceptible to metabolic decompensation during episodes of increased catabolism, such as intercurrent illness, trauma, surgery, or prolonged episodes of fasting. Defects of pyrimidine methobolism can be lead to a variety of symptoms. The determination of the urinary orotic acid is very important to diagnose some inborn errors of metabolic pathways such as pyrimidine synthesis and urea cycle. Pyrimidines and purines are the building blocks of DNA and RNA. The pyrimidines also function as intermediates in synthesis of energy sources and essential elements of cell surface structures Pyrimidines are heterocyclic, six-membered, nitrogencontaining carbon ring structures, with uracil, cytosineand thymine being the basal structures of ribose-containing nucleosides (uridine, cytidine and thymidine respectively), or deoxyribose-containing deoxynucleosides, and their corresponding ribonucleotides or deoxyribonucleotides Pyrimidines serve essential functions in human metabolism as ribonucleotide bases in RNA (uracil and cytosine), and as deoxyribonucleotide bases in DNA (cytosine and thymine), and are linked by phosphodiester bridges to purine nucleotides in double-stranded DNA, in both the nucleus and the mitochondria. Pyrimidines, similarly to purines, are synthesized de novo from simple precursors. Synthesis occurs in six steps, with cellular compartmentalization of specific steps in the cytosol or mitochondria, enabling changes in metabolic rate with need. Pyrimidine synthesis differs from purine synthesis, in that the single pyrimidine ring is assembled first and is then linked to ribose phosphate to form UMP. This gene encodes a uridine 5 -monophosphate synthase. The encoded protein is a bifunctional enzyme that catalyzes the final two steps of the de novo pyrimidine biosynthetic pathway. The first reaction is carried out by the N-terminal enzyme orotate phosphoribosyltransferase which converts orotic acid to orotidine-5 -monophosphate. The terminal reaction is carried out by the C-terminal enzyme OMP decarboxylase which converts orotidine-5 -monophosphate to uridine monophosphate. Defects in this gene are the cause of hereditary orotic aciduria. The enzymes that catalyse UMP synthesis, CAD [carbamoylphosphate synthetase II (CPSII), aspartate transcarbamoylase (ATCase) and dihydroorotase (DHOase)], dihydroorotate dehydrogenase (DHODH) and uridine monophosphate synthase (UMPS), are encoded by only three genes – CAD, DHODH and UMPS are encoded by only three genes. An excess of orotic acid leads to a condition known as orotic aciduria. This condition can be hereditary or brought on by excessive consumption. In adults, it causes an interruption of the urea cycle. In children, it can cause inhibition of DNA and RNA synthesis leading to mental or physical damage. Increased urinary orotic acid has been observed in patients and heterozygotes with hereditary metabolic disease. Severe trauma, pregnancy and a number of drugs have also increased the excretion of urinary orotic acid. The biological effects of orotic acid have been the focus of considerable attention, because recent experimental studies have demonstrated that it has effects of carcinogenesis, hepatic lipidstorage and renal toxicity. Orotic aciduria results from deficiency of either or both of the last two enzymes: oratate phosphoribosyl transferase and OMP decarboxylase as mentiones before, both these enzyme activities are present on a single protein as domains. Mutation in the phosphoribosyl transferase impairs its association with the other domain, resulting in loss of activity of both. This produces type I situation (orotic aciduria type I). This deficiency is treated by taking supplements of the compound uridine. In the type II, mutation in the decarboxylase occurs, but it does not effect its aggregation with phosphoribosyl transferase. In this case, only decarboxylase is inactive, whereas activity of phosphoribosyl transferase is not affected. This deficiency has been successfully treated with chronic uridine therapy. Orotic aciduria is a rare disease that is characterized by megaloblastic anemia. A devastating response to what is usually mild infection may be seen in any of the megaloblastic anemias of early life. The feature which led to the original recognition of the condition was crystalluria. Crystals of orotic acid have caused urethral and ureteral obstructions hematuria and azotemia. Disorder related to pyrimidine synthesis is hereditary orotic aciduria caused by deficiency of uridine monophosphate synthase (UMPS), a bifunctional protein having two catalytic activities: orotate phosphoribosyltransferase (OPRT) and orotidine monophosphate decarboxylase (ODC). A defect in both the activities is classified as orotic aciduri type I while ODC defect is denoted orotic aciduria type II. In most cases, orotic aciduria arises from inherited defects of enzymes involved in the urea cycle after synthesis of intramitochondrial carbamoyl phosphate. Excessice production of OA may also occur in defiency of ornithine transcarbomolayse, a urea cycle enzyme. It leads to accumulation of carbomyl phosphate, which is diverted for enhanced synthesis of OA. Increased urinary excretion of this compound follows, hence this defect is considered as a secondary orotic aciduria. Orotic acid excretion is also elevated by ammonia intoxication, during feeding of diets high in protein and in patients treated with diets high in protein and in patients treated with allopurinol or 6-azauridine. Suitable chromatographic conditions and pre-treatment method was developed for reliable analysis of orotic acid in urine in this study. Samples were analyzed by ion chromatography / conductivity detector equipped with a Dionex Ion Pac® AS20 analytical coluomn (2x250 mm) and a Dionex Ion Pac® AG20 guard column (2x50 mm) using gradient and isocritic NaOH elution. Interferences from matrix in urine were removed by strongly cation exchange resin and solid-phase extraction after centrifugation and dilution. The calibration curves showed good linearity in the ranges of 10-10000 µg/L with the r2 values of 0.999. Significant results were obtained for various spiked urine samples with % recovery in the range of 85-95 %. The method was applied to urine samples collected from both healthy volunteers and patients with orotic aciduria. The value of urinary OA excretion, expressed in µmol/mmol of creatinine are reported in both healty volunteers and patient with orotic aciduria. The advantages of the method described herein are: (i) it allows determining trace amount of orotic acid from human urine samples, (ii) the sample preparation is overly simple, inexpensive, and (iii) the test is not time demanding and could be employed in routine analysis.