Kapiler elektroforez – örnek sıkıştırma yöntemi ile idrarda orotik asit tayini
Determination of orotic acid in urine by capillary elektrophoresis – sample stacking method
Yazar: MERVE TAŞER
Danışman: DOÇ. DR. NEVİN ÖZTEKİN
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Kimya Ana Bilim Dalı / Kimya Bilim Dalı
Derece: Yüksek Lisans, 2020
Konu: Kimya = Chemistry

Özet:
Son yıllarda, klinik, biyolojik ve gıda örneklerindeki organik asit içeriklerinin belirlenmesinin önemi artmakta ve giderek büyüyen bir araştırma alanı oluşturmaktadır. Biyolojik örnek olarak insan kan ve idrarında bulunan çeşitli organik asitlerin konsantrasyon seviyesinin düşük ya da yüksek olmasına bağlı olarak birçok hastalığın teşhisinin mümkün olduğu bilinmektedir. Orotik asit de bu maddelerden biridir.
Orotik asit bir pirimidindion ve bir karboksilik asittir. Tarihsel olarak B vitamini kompleksinin bir parçası olduğuna inanılıyordu ve B13 vitamini olarak adlandırıldı, ancak günümüzde bir vitamin olmadığı bilinmektedir. İnsanlarda ve diğer organizmalarda, orotik asit, dihidroorotatı orotik aside dönüştüren dihidroorotat dehidrojenaz enzimi tarafından sentezlenir.
Kalıtsal orotik asidüri enzim eksikliğinden kaynaklanan ve pirimidinleri sentezleme yeteneğinin azalmasına neden olan bir hastalıktır. De novo pirimidin sentez yolunun bilinen tek enzim eksikliğidir. Orotik asidüri, orotik asidin üridin monofosfat (UMP)'a dönüştürülememesi nedeniyle idrarda aşırı orotik asit atılımı ile karakterizedir. Çocuklarda ise megaloblastik anemiye ve DNA ile RNA sentezinde kısıtlamaya yol açarak zihinsel ve fiziksel gelişimsel gecikmelere neden olur.
Etkin bir ayırma tekniği olan kapiler elektroforez bir elektrik alan altında en küçük yapıdaki iyonlardan en büyük biyomoleküllere kadar taneciklerin yüksek voltaj uygulanarak göç ederek ayrılması olayıdır. Diğer kromatografik yöntemlerle karşılaştırıldığında kapiler elektroforez yöntemi daha yenidir ve klinik araştırmalarında giderek artan bir yere sahiptir. CE'nin avantajları; yüksek ayırma etkinliği, kısa analiz süreleri, yüksek ayırma hızı, az miktardaki örnek ve çözücü olarak su kullanılmasıdır. Silika kaplı kapiler kolonlar ise kromatografik kolonlara göre çok ucuzdur, çalışma arasında kolayca yıkanır ve dolgu kolonlarda görünen matris kirlenmesi problemi yoktur.
Bu çalışmada, bahsedilen hastalıkların teşhisinde güvenilir ve hassas bir metod geliştirilmesi amaçlanmıştır. Kapiler zon elektroforez cihazı ile iletkenlik dedektörü kullanılarak insan idrarındaki orotik asidin miktarının kantitatif tayini için seçici, güvenilir ve hassas bir yöntem geliştiriImesi üzerine çalışılmıştır. Ayırıcı tampon olarak, 20 mM morfolinoetansulfonik asit (MES), 10 mM Histidin ve 0,1 mM CTAB kullanılmıştır (pH = 6,5). İdrar ortamındaki analiz sonucunda LOD ve LOQ değerleri 0,103 mg/L ve 0,344 mg/L olarak, su ortamındaki analiz sonucunda ise LOD ve LOQ değerleri sırasıyla 0,014 mg/L ve 0,047 mg/L olarak hesaplanmıştır.

Abstract:
In recent years, the importance of determining organic acid content and levels in clinical, biological and nutrient samples has been gaining attention and is a growing area of research. As a biological example, it is known that many diseases can be diagnosed depending on whether the concentration level of various organic acids in human blood and urine is low or high. Orotic acid (1,2,3,6-tetrahydro-2,6-dioxo-4-pyrimidinecarboxylic acid; uracil-6-carboxylic acid) is one of these substances.
Orotic acid is a pyrimidinedione and a carboxylic acid. It was historically believed to be part of the vitamin B complex and was called vitamin B13, but it is known to be not a vitamin today. Although not found in breast milk, cow's milk and dairy products contain orotic acid. Root vegetables such as carrots and beets are among the richest vegetable food sources of orotic acid. Studies show that orotic acid is also present in kefir, a fermented milk product, and consumption of kefir increases the level of orotic acid in the blood.
In humans and other organisms, orotic acid is synthesized by the enzyme dihydroorotate dehydrogenase, which converts dihydrorotate to orotic acid. Many experimental studies show that orotic acid is related to carcinogenesis and fat storage in the kidneys.
It is seen in the literature that there are a limited number of studies on the relationship between cholesterol and orotic acid. Limited studies have shown that orotic acid has a lowering effect on blood cholesterol levels, prevents liver problems and premature aging, and also helps in the treatment of multiple sclerosis. Scientists argue that studies on the positive effects of orotic acid on health are not sufficient and that more and more scientific studies should be conducted on this subject.
Researchers who detected significant amounts of orotic acid levels in fermented dairy products directed their studies to the relationship between orotic acid and cholesterol. But in one of the studies based on the relationship between orotic acid and cholesterol; It has been found that the synthesis of fatty acids in the livers of mice fed with orotic acid decreases and the amount of triacylglycerol, phospholipid and cholesteryl ester decreases.
Hereditary metabolic diseases can present symptoms at any time in life, affect any organ system or mimic many more common childhood diseases. Metabolic diseases are pathological pictures that develop as a result of events related to the synthesis or catabolism of protein, carbohydrate and fatty acids.
Organic acidemias are a group of diseases that develop as a result of congenital disorders in amino acid, lipid and carbohydrate metabolism. These diseases, most of which are autosomal recessive, can occur with clinical and laboratory findings such as psychosocial motor retardation, frequent infection, metabolic acidosis, ketosis, early and unexplained death.
As with other congenital metabolic diseases, in organic acidemias, with appropriate emergency treatment initiated by early diagnosis, permanent organ damage due to the disease can be prevented and the risk of death decreases.
Hereditary orotic aciduria is a disease caused by enzyme deficiency and decreasing the ability to synthesize pyrimidines. De novo is the only known enzyme deficiency of the pyrimidine synthesis pathway. Orotic aciduria is characterized by excessive excretion of orotic acid in the urine due to the inability to convert orotic acid to UMP. Since orotic acid is excreted in the urine, it is more likely to be determined in the urine than it is determined in blood plasma. It causes disruption in the urea cycle in adults. In children, it causes megaloblastic anemia and restriction in DNA and RNA synthesis, causing mental and physical developmental delays.
Orotic aciduria is divided into two types, type I and type II. It is called orotic aciduria type I, which is seen in the deficiency of both orotate phosphoribosyl transferase and orotate monophosphate decarboxylase enzymes involved in pyrimidine synthesis. Findings are seen as lack of development, megaloblastic anemia and orotic acid crystallurgy in urine. As a treatment, uridine is given by mouth.
Only in orotate monophosphate decarboxylase deficiency, orotic aciduria type II occurs. Findings of orotidinuria and megaloblastic anemia are seen. As treatment, uridine is given by mouth as in orotic aciduria type I.
Capillary electrophoresis, an effective separation technique, is the separation of particles from the smallest structure to the largest biomolecules by migration by applying high voltage under an electric field. Compared to other chromatographic methods, capillary electrophoresis method is newer and has an increasing place in clinical research. The main difference that distinguishes electrophoresis from other methods is the electric field application that causes the migration of charged particles towards one of the electrodes. The rate of migration varies according to the load / size ratio of the charged particles. In this way, it is possible to distinguish charged macromolecules commonly found in biochemical and biological research in the biotechnology industry.
Capillary electrophoresis (CE) is a technique that performs electrokinetic separation due to phase separation, electrophoretic mobility and molecular size differences or a few of them. This electrokinetic separation is made in cylindrical columns, the surface of which is called capillary, covered with silica, with two ends open, approximately 25-75 micrometers inner diameter and 15-100 cm long [1].
In electrophoresis, the migration rate and duration of the ion varies depending on different parameters. These parameters are; net charge, structure and size of the molecule, power of the electric field, buffer pH and concentration and ambient temperature. Electrophoretic mobility; It is directly proportional to the charge of ions and inversely proportional to the friction force. Thanks to the modifying agents of charged particles (ions) having two mobility very close to each other, the discrimination selection can be easily differentiated and the speed and direction of the electroosmatic flow can be controlled.
Capillary electrophoresis; gas chromatography is a modern analysis method developed by combining good and advantageous properties of HPLC and gel electrophoresis. Capillary electrophoresis makes it more advantageous than other electrophoresis methods due to low substance consumption, high separation efficiency (resolution) due to the flat profile of the electrosmotic flow, different separation mechanisms and selectivity of different types, and the fact that the detection takes place during separation and quantitative analysis can be made. This analytical method is used for the sensitive separation of many substances such as inorganic anions and cations, amino acids, carbohydrates, vitamins, drugs and polynucleotides. Silica coated capillary columns are much cheaper than chromatographic columns, they are easily washed between the study and there is no matrix contamination problem seen in filling columns.
Capillary electrophoresis is used in food additives, organic acids, DNA molecule, drug, protein and hydrocarbon analysis. In recent years, it has found wide use as an effective and economical method in the analysis of molecules such as medicine, protein, nucleic acid in various fields such as biochemistry, genetics, microbiology, clinical and forensic medicine.
It is important to develop an effective method to be used in the analysis of orotic acid levels of high concentrations and normal concentrations in urine in the diagnosis of congenital diseases. These measurements are made by methods such as high pressure liquid chromatography (HPLC), gas chroamtography (GC), nuclear magnetic resonance spectroscopy (NMR), capillary electrophoresis. Many studies have been conducted in the literature on the detection of orotic acid in biological samples.
Determination of orotic acid by analysis in human urine plays an important role especially in the diagnosis of some hereditary diseases. In addition, there is a connection between the progression of carcinogenic structures, especially the liver, and the amount of orotic acid in the urine. Therefore, accurate and rapid measurement of the amount of orotic acid in the urine is important in the early diagnosis of many ailments. In this study, it is aimed to develop a reliable and sensitive method in the diagnosis of said diseases. The developed method has advantages in terms of being easy, high precision and accuracy. The method is suitable for routine clinical analysis of orotic acid in human urine. It has been studied to develop a selective, reliable and sensitive method for the quantitative determination of the amount of orotic acid in human urine using a capillary zone electrophoresis device and conductivity detector. The conditions of the method were developed until the analysis with high resolution and responding as soon as possible. As a result of method development, electrokinetic injection was determined as the most appropriate method by using sample compression method. Sample compression is a method used to increase sensitivity in the capillary zone electrophoresis system. When analyzing in normal injection volume, it can be possible to decrease the determination limit obtained by using the sample compression method. With this method, quantitative analyzes in urine were performed. As separating buffer, 20 mM morpholinoethanesulfonic acid (MES), 10 mM Histidine and 0.1 mM CTAB were used (pH = 6.5). As a result of the analysis in urine environment, LOD and LOQ values were calculated as 0.103 mg / L and 0.344 mg / L, and as a result of the analysis in water environment, LOD and LOQ values were calculated as 0.014 mg / L and 0.047 mg / L, respectively.