13th balkan biochemical biophysical days & meeting on metabolic disorders’ programme & abstracts
LECTURE 5 FOETAL BIOCHEMISTRY: BIOCHEMICAL DIAGNOSIS for FOETUS
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LECTURE 5FOETAL BIOCHEMISTRY: BIOCHEMICAL DIAGNOSIS for FOETUSNezih Hekim Dr.Pakize İ.Tarzi Laboratuarları Nişantaşı İstanbul Clinical chemistry laboratories are going through a challenge. With the improvement of “Point of Care” system, cinical chemistry laboratories are moving to decentralization from centralization, which means that availability of bed side testing, with the help of automatization and easiness of testing at different departments at health centers with decreasing costs clinical laboratories are moving to new areas of analysis for less requested but increasing demand tests. Some of these areas are: Pre implantation genetics, detection of metabolic diseases of foetus and screening for treatable metabolic diseases of new born. Preimplantation genetics provides the opportunity to detect 105.000 single nucleotide polymorphism for many single gene disease even before fertilization. There are two main problems: For applying this method, there is need for conception with assisted reproduction techniques like IVF and ICSI. Also, at the time, approach to disease detection is to look for mutations on the gene, but there are variations of mutations in each and every country even in the same country at different locations and sequence analysis is not a screening test. The best approach for the time seems to analyse amniotic fluid or amnion cells and/or their cultured cells during early pregnancy (CVS 15-16 week) for enzyme or metabolite with classical biochemical methods. This area is named foetal biochemistry and many biochemistry laboratories are shifting interest to this area. Without any doubt, the diagnosis of the diseases and abortion after the diagnosis will be within the frame of prenatal rules. Foetal biochemistry will be the starting point for in utero genetic treatments in near future. We have aimed to share our experiment and knowledge on foetal biochemistry with our colleagues with this presentation.
HALL C LECTURE 1AMINO ACID ANALYSIS METHODS – DETAILS AND DIFFICULTIESGürsel BİBEROĞLUGazi University, Faculty of Medicine, Department of Pediatric Metabolism and Nutrition, 06500, Ankara/TURKEY Gurselb@gazi.edu.trDisorders of amino acid metabolism constitute an important part of inborn errors of metabolism. These disorders which are mainly seen in the newborn period and early childhood are characterized by the high levels of one or more than one amino acids in the plasma or urine due to the enzyme deficiency. In amino acid metabolism disorders, like the other inborn errors of metabolism; early diagnosis is very important to prevent morbidity and permanent sequels and also for the success of the treatment. In the light of these points, accurate, sensitive and prompt amino acid analysis in biological fluids is very important. Blood, urine, cerebrospinal fluid, vitreous fluid and amnion fluid are used for the diagnosis of disorders of amino acid metabolism. The analytical techniques for the measurement of amino acids can be investigated in two parts as screening tests and quantitative methods. Screening tests including Guthrie test, thin layer chromatography, paper chromatography, photometric methods and spot tests in the urine. In the recent years a world wide and important technique, that could screen many metabolic diseases in a single analytical step named tandem mass spectrometry has been used for this purpose. Among the tests those are used for the quantitative measurement of amino acids are capillary electrophoresis, gas-liquid chromatography, high pressure liquid chromatography, ion-exchange liquid chromatography (amino acid analayzer) and tandem mass spectrometry. High resolution nuclear magnetic resonance spectroscopy and molecular analysis are also used in amino acid measurements. One of the most recent and effective technic of those is tandem mass spectrometry. Tandem mass spectrometry is a very important analytical technique that could determine many metabolic diseases from one blood sample in a very short time in a single analytical step. Phenylketonuria, hyperphenylalaninemia, maple syrup urine disease, tyrosinemia type I and II, homocystinuria, hypermethioninemia are the disorders of amino acid metabolism those could be determined by tandem mass spectrometry. In addition to amino acid disorders fatty acid oxidation disorders, organic acidemias and urea cycle defects could also be determined by tandem mass spectrometry. In the newborn period, screening tests were begun by the screening of phenylketonuria which is a kind of bacterial inhibition test progressed by Robert Guthrie. Thin layer chromatography and paper chromatography are chromatographic methods those are used for the separation and determination of amino acids. In quantitative amino acid analysis with high pressure liquid chromatography the main steps of the method are the pre-column derivatization of the amino acids with phenylisothiocyanate, o-phthalaldehyde and other similar compounds, separation in reversed-phase column and detection with either ultraviolet or fluorescence detectors. In amino acid analayzer the main part of the system is ion-exchange column, followed by gradient elution. Long period of investigation is a disadvantage for these methods. Amino acid levels in body fluids are influenced by a number of factors, such as age, physiological changes, nutritional status, diseases, medications and toxins. Also the factors such as collection time, transportation and keeping of the samples are very important. As a conclusion to gain success in the treatment and to prevent permanent sequels due to disorders of amino acid metabolism which makes up a great part of metabolic diseases, early diagnosis and treatment is highly important. Also the importance of the amino acid measurement techniques is beyond discussion for newborn screening, genetic counselling to the family and for the follow-up of the patients.
THE USE OF TANDEM MASS SPECTROMETRY IN NEWBORN MASS SCREENING PROGRAMS: THE FACTS BEYOND THE MYTH. Eyskens François J.M.
Tandem mass spectrometry (TMS) is an analytical technique that can be implemented in the analysis of blood spots taken shortly after birth. Rather than testing the blood for the presence of just one compound (e.g. phenylalanine in the case of phenylketonuria), this technique can simultaneously examine a large number of metabolites (>30 metabolic disorders!) in a single blood spot by “electronically” weigh these molecules. Using this new technique the screening can be changed from the “one test-one disorder” towards the “one test-many disorders” strategy. This technology thus allows a “sea change” in newborn screening but it is important that we shouldn’t drown from it: until this moment the experience is limited and, except from some defects in fatty acid oxidation (e.g. MCAD deficiency) and some organic acidemias (e.g. glutaric aciduria type 1), the expand of the newborn screening covering a mass of metabolic disorders is not been studied thoroughly on its efficacy and utility. This technique detects well amino acids and acylcarnitines; at this moment however it is still impossible to screen for congenital hypothyroidism or biotinidase deficiency, and there is only a limited experience in screening for congenital adrenal hyperplasia using this technique. The sensitivity of the screening by TMS is high but the specificity can be rather low with a high rate of false positives resulting in a high number of retests and recalls: e.g. Belgium (Antwerp, Brussels): retests between 5 and 7% in the first two years of screening with TMS; New South Wales (Australia), Pittsburgh (USA): false positive rate of 0.15-0.26%; (for comparison: false positive rate of phenylalanine screening by an enzymatic method (Quantase, BioRad) is only 0.015%). A broad screening program also involves the detection of non-diseases (e.g. 3-methylcrotonylcarboxylase def. (Germany) and/or atypical cases whose risk of developing clinical problems is unknown (e.g. MCAD def. (Australia)). H. Levy in his editorial on “Newborn screening by Tandem Mass Spectrometry: A new era” (Clinical Chemistry (1998);44,12:2401-2) stated that until nowadays screening services are almost completely controlled by state or other governmental health departments and that these agencies generally are not distinguished by their technological innovation or by their readiness to incorporate new ideas. The great danger in my opinion of the technique of TMS is that labs that have no experience whatsoever with screening, will take over the newborn mass screening solely on the base that they possess such an instrument, without providing a screening program that serves the community, completely lacking a high technological performance (absence of quality assurance testing) and a well established structure that allows recovery of all blood cards and having good contacts with treating physicians. Last but not least one should not ignore the higher costs of TMS screening in comparison with the current used techniques (literature: $ 0.7-20; own experience: $ 6 supplementary costs/screened newborn). Conclusion: Mass screening of newborns should stay centralised in the screening labs that have the most experience and the best performance; only these labs can implement new techniques in a way that newborn screening remains on a high level. Screening is not just a job, it is a dedication. LECTURE 3DISORDERS OF CARBOHYDRATE METABOLISM: CLINICAL APPROACH, FUTURE PROSPECTS OF DIAGNOSIS AND TREATMENT Prof. Dr. Benal Büyükgebiz Pediatri Anabilim Dalı, Dokuz Eylül Üniversitesi Tıp Fakültesi Carbohydrates are the body's sugar source. Sugars used to provide energy for the body include glucose, sucrose, fructose among many others. Some sugars need to be broken down, usually by enzymes, before they can be used by the body. If the enzymes needed are not present (usually due to an inherited disorder), these sugars can build up and cause problems. The type of problem depends on the sugar involved and the localization of the enzyme defect. Most of the inherited disorders of carbohydrate metabolism fall into a few broad clinical syndromes. Hepatomegaly, convulsions, hyperbilirubinemia, cataract, mental retardation, diarrhea, episodic lactic acidosis from early infancy, failure to thrive, and hypotonia are most common signs and symptoms. The demonstration of defective enzyme activity must serve as the basis of diagnosis and treatment. LECTURE 4 DISORDERS OF CARBOHYDRATE METABOLISM: BASIC CONCEPTS, EVALUATION OF LABORATORY METHODS AND DIFFICULTIES OBSERVED Aslan AKSU
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