ACTA ENDOCRINOLOGICA (BUC)

The water channels milestones include: the vague idea of “hydrophilic pores” or “water-filled channels” in the red blood cell (RBC), the proposal that water channels (WCh) are accommodated in proteins, experiments associating WCh with the major RBC membrane protein called Band 3 (the anion exchanger), and the crucial experiment performed in 1985 by Benga group in Cluj-Napoca, Romania, proving the presence and location of a minor protein of the RBC membrane involved in water transport. In the landmark papers of 1986, Benga introduced the concept of the WCh being a protein specialized in water transport, i.e. a water channel protein (WCP). The first WCP discovered by our group was re-discovered in 1992 by Agre group. In the same year two other WCPs were discovered. The name aquaporins was proposed in 1993. In subsequent years hundreds of WCPS have been discovered in organisms from all kingdoms of life. WCPs are a family of membrane proteins, belonging to the Membrane Intrinsic Proteins superfamily. WCPs family include three subfamilies: 1) aquaporins (AQPs) which are mainly water selective channels; 2) aquaglyceroporins are permeable to water and to other small uncharged molecules; 3) S-aquaporins (subcellular or superaquaporins). Benga called aquaglyceroporins and S-aquaporins the “relatives of aquaporins”. Twelve WCPs were identified in the human body, having a great importance in a lot of physiological phenomena, as well as in pathological conditions, from well defined “water channelopathies” to a wide range of diseases. Benga propose the name of aquaporinology for the domain of biomedical and natural sciences dedicated to the integrated approach of WCPs (aquaporins and relatives), which is also a chapter of Cellular and Molecular Biology.

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Phenylketonuria (PKU) is the most frequent inherited amino acid metabolic disorder, and it may also be treated by dietary means. The determination of phenylalanine (Phe) levels in the blood plasma is important not only in early diagnostic, but also in monitoring the treatment of PKU. Purpose. The aim of our work was to develop a simple, sensitive and highly accurate procedure to determine the plasma concentration of Phe. Procedure. The measurement of plasma Phe concentration involves two steps: a) separation of plasma (from the blood taken on heparin), isolation and preparation of a concentrated solution of amino acids (by ion-exchange column chromatography on Dowex-50X8 and evaporation of the eluate in vacuum at 40˚C), and b) determination of Phe concentration in the solution of amino acids by HPLC. This analysis was performed using a Dionex Ultimate 3000 instrument equipped with a Ultimate 3000 diode array detector (DAD). The values of Phe concentration in the plasma of several patients were calculated using a calibration curve made with standards of Phe (dilutions of a stock solution of 50 mg/ dL). The measurements in duplicate (plasma Phe) or a greater number of samples from the same concentration of standards of Phe showed extremely small sample to sample differences. Concentrations as low as 0.2 mg/dL could be determined. Conclusion. The whole procedure presented here is relatively simple, rather inexpensive, however very sensitive and highly accurate. Consequently, it is very adequate for confirming the diagnosis of PKU in patients with neonatal hyperphenylalaninemia, as well as for monitoring the plasma concentration of Phe in patients with PKU.

Objective. To compare two chromatographic methodologies for determination of plasma phenylalanine (Phe) and their usefulness for diagnosing hyperphenylalaninemia (HPA) and phenylketonuria (PKU). Methods. The plasma amino acids were isolated and concentrated from blood collected from infants with HPA detected by newborn screening. The plasma Phe was determined in parallel by HPLC and by image-densitometry of 2D-TLC plates. Results. Typical examples of 2D-TLC plates and HPLC chromatograms from infants with HPA and PKU are presented and evaluated. The Phe spot was visible on 2D - TLC plates at Phe concentrations higher than 300 μmol/L. The standard calibration curve traced after imagedensitometry of the Phe spot presented high dispersion of values at each concentration of Phe, high SD values, the equation of the curve having a low R-squared value (0.862). In contrast, the standard calibration curve obtained by HPLC shows linearity on the range of concentrations from 100 - 16,000 μmol/L, extremely small SD values, the equation of the curve has a very high R-squared value (0.999). Conclusions. The HPLC methodology is appropriate to confirm HPA detected by newborn or selective screening of PKU. The 2D - TLC methodology is adequate to detect patients with severe PKU.

Aim. To compare the red blood cell (RBC) diffusional water permeability (Pd) in elderly human subjects and mature\r\nsubjects.\r\nPatients and methods. 58 apparently healthy subjects, aged 65-80 years, were divided into two groups: 44 mature (35-64.9 years) and 14 elderly subjects (65-80 years). The morphological characteristics of RBCs were determined from light microscopic measurements and their Pd was measured\r\nby a NMR method. The inhibition of Pd induced by p-chloromercuribenzoate (PCMB) and the activation energy (Ea,d) of water diffusion across the RBC membrane\r\nwere also determined.\r\nResults. No significant differences between the RBCs of the two groups were found in regard with morphological parameters. Pd (10-3 cm/s) was in case of mature subjects ~ 3.1 at 15?C, 3.6 at 20?C, 4.2 at 25?C, 5.0? at 30?C, 6.1 at 37?C and 7.3 at 42?C, while for elderly subjects Pd was ~ 3.4 at 15?C, 3.9 at 20?C, 4.5 at 25?C, 5.3 at 30?C, 6.6 at 37?C and 7.9 at 42?C. Although rather small these differences were statistically significant: p<0.004 to p <0.04 at various temperatures. This means that RBCs from\r\nelderly people have a higher Pd. In agreement with this suggestion, the values of inhibition of water permeability induced by PCMB were higher for the RBCs from elderly individuals; however, the differences were not statistically significant. Ea,d was the same (~23 kJ/mol) for the RBCs from both groups. After incubation with PCMB Ea,d was ~ 37 kJ/mol for the mature individuals and ~ 31 kJ/mol for\r\nelderly individuals; however, the differences were not statistically significant.\r\nConclusion. A small, but statistically significant, increase in Pd of RBCs from elderly individuals was observed. This can be correlated with peculiarities of a less physically active organism.

Newborn screening of phenylketonuria (PKU) is performed in many countries, including Romania, in addition to screening for congenital hypothyroidism. Patients affected by PKU require frequent measurements of phenylalanine (Phe) level in blood plasma. Such a determination is important not only in early diagnostic, but also in monitoring the treatment of PKU to maintain phenylalaninemia within limits that will not affect the brain. A simple, highly sensitive, accurate and rather inexpensive procedure for the simultaneous determination of Phe and Tyr plasma concentrations was previously described in this journal. The new procedure may be applied in many clinical laboratories, including those with no previous experience in diagnosis of inherited amino acid metabolic disorders. In this way the major public health problems linked to PKU not being detected in the first weeks of life (including the burden of institutionalized children with preventable mental retardation) may be avoided.

Aim. To correlate the profile of CFTR gene mutations in patients from Romania with the ethnogenesis of Rumanian people. Patients and methods. One hundred sixty-five patients with clinical diagnosis of CF and elevated values at sweat test were included in the study. Samples of EDTA-anticoagulated blood were obtained by venipuncture, sent to our laboratory and DNA was extracted from leukocytes. For the majority of blood samples we used standardized methods for analysis of at least 18 common mutations. Ten DNA samples were analyzed for 38 CFTR mutations with a kit recently introduced in our program of investigations of CFTR gene mutations.Results. The most frequent mutations in CF patients from Romania are F508del (53.6%), G542X (4.6%), W1282X (2.1%) and CFTRdele2,3(21kb) (1.2%). Other mutations were detected at frequencies less than 1.0%. The profile of the CFTR gene mutations in Rumanian patients appears to be very different from its counterpart profile in Romania’s neighbour countries and rather similar with the profile of mutations in France, Italy and Spain (which, similar to Romania, are Neo-Latin countries). A notable difference between Romania and these Neo-Latin countries is the presence of a Slavic mutation, CFTRdele2,3(21kb) in Rumanian patients; this might reflect the Slavic component in the ethnogenesis of Rumanian people.Conclusion. The profile of the CFTR gene mutations in Rumanian patients confirms the overwhelming evidence regarding the ethnogenesis of Rumanian people from the admixture of Dacians or Getae (the ancient autochtonous inhabitants of the territory of present-day Romania) with Roman (or Romanized) legionnaires and colonists, forming the Daco-Roman population (the basis of Rumanian people), who assimilated the Slavs that entered in the territory of present-day Romania in the VIth century.

Introduction. The determination of phenylalanine (Phe) and tyrosine (Tyr) levels in blood plasma is very important not only in early diagnostic, but also in monitoring the treatment of phenylketonuria (PKU). Purpose. We present a simple, sensitive and accurate procedure to determine simultaneously the plasma concentrations of Phe and Tyr. Procedure. The measurement involves two steps: a) separation of plasma (from blood prelevated on heparin), isolation and preparation of a concentrated solution of amino acids (by ion-exchange column chromatography on Dowex- 50X8), and b) determination of Phe and Tyr concentrations in the solution of amino acids by HPLC (using a Dionex Ultimate 3000 instrument equipped with a diode array detector). The analytical column was a Thermo Scientific Acclaim 120, C18, 5 μm Analitic (4.6 x 250 mm), coupled with an Acclaim C18 guard column. The values of Phe and Tyr concentrations in plasma of several patients were calculated using a calibration curve made with standards of Phe (1834.4 μmol/L in deionized water) and Tyr (600 μmol/L in deionized water). Concentrations as low as 24 μmol/dL of Phe and 15 μmol/dL of Tyr could be determined. Conclusion. The whole procedure presented here is relatively simple, rather inexpensive, however very sensitive and accurate. Consequently, it is very adequate for confirming the diagnosis of PKU in patients with neonatal hyperphenylalaninemia, as well as for monitoring the plasma concentrations of Phe and Tyr in patients with PKU.

Background. The modern management of phenylketonuria (PKU) consists of generalized newborn screening (NBS) for hyperphenylalaninemia (HPA), confirmation of HPA in children detected in the NBS, introduction of dietary treatment in the first weeks of life, followed by monitoring the treatment of PKU for decades to maintain phenylalaninemia within the limits that will not affect the brain. The present study aimed to evaluate the usefulness of two chromatographic methodologies for determination of plasma Phe level in the routine management of PKU: the two dimensional thin layer chromatography (2D - TLC) and the high performance liquid chromatography (HPLC) procedures, respectively. Material and Methods. Samples of blood from 23 children with HPA detected by neonatal screening or with confirmed PKU who received treatment by low-Phe diet were analyzed to estimate the plasma Phe level by the two chromatographic procedures. Results. In case of three subjects the very low concentrations of plasma Phe could not be detected by the 2D - TLC methodology, since the spot was not visible on the chromatogram. In four patients the differences between the values of plasma Phe determined by the two methodologies are not statistically significant, while in fifteen subjects the differences are highly statistically significant. This is due to the greater errors that appear in the case of 2D - TLC methodology. In the range of concentrations of plasma Phe higher than 360 μmol/L (which is the cut-off value for HPA), although in four cases there were statistically significant differences in the level of plasma Phe determined by the two methodologies, the value obtained by the 2D - TLC methodology was high enough to influence the decision of changing the diet so that HPA is kept under control. In addition, the intense spot of Phe on the 2D - TLC chromatogram may be detected even by un unexperienced laboratory specialist. Conclusion. The HPLC procedure for measurement of plasma Phe level is very suitable to be used in the routine management of PKU. The 2D - TLC procedure may be accompanied by relatively high errors; however, it detects patients with severe PKU.