Robert Kowalski

Karmienie piersią uznaje się za kluczowy czynnik wspierający prawidłową kolonizację mikrobiologiczną i dojrzewanie odporności w okresie noworodkowym, co znajduje odzwierciedlenie w zaleceniach Światowej Organizacji Zdrowia (WHO) i europejskich rekomendacjach dotyczących promowania karmienia piersią. Rozwój mikrobioty jelitowej noworodka jest procesem dynamicznym, zależnym od czynników prenatalnych, okołoporodowych i postanatalnych. Przyjmowanie pokarmu jakim jest mleko matki, drogą doustną nie zawsze jest możliwe, jedną z istotnych form leczenia stosowanych u noworodków stanowi żywienie pozajelitowe. Polega na dożylnym podawaniu mieszaniny składników odżywczych z pominięciem przewodu pokarmowego. Wdrażane jest w sytuacjach, gdy odżywianie organizmu drogą dojelitową jest niemożliwe lub niewystarczające. Przyjmuje postać płynnej mieszaniny zawierającej substancje odżywcze niezbędne do prawidłowego wzrostu i utrzymania homeostazy. Główne elementy składowe obejmują: węglowodany, lipidy, aminokwasy, witaminy, pierwiastki śladowe, elektrolity wraz z wodą. W zależności od proporcji preparaty do żywienia parenteralnego mogą charakteryzować się wysoką osmolarnością. Dobór mieszaniny odżywczej oraz szybkość podaży wymagają kontroli parametrów metabolicznych oraz bilansu płynów. Żywienie parenteralne zostaje włączone w konkretnych przypadkach klinicznych, a skład mieszaniny powinien być indywidualizowany do stanu pacjenta. Terapia żywieniowa drogą pozajelitową zapobiega niedożywieniu a także przyspiesza procesy gojenia ran i regeneracji. Odgrywa ważną rolę na oddziale neonatologii. W pierwszych dniach życia noworodka wpływa m.in. na prawidłowy rozwój neurologiczny. Długotrwałe ograniczenie żywienia drogą enteralną wiąże się jednak ze zmianami w składzie mikrobioty jelitowej. Zmniejsza się stymulacja błony śluzowej co sprzyja osłabieniu bariery jelitowej oraz zaburzeniom odporności. W konsekwencji dochodzi do spadku różnorodności bakterii jelitowych oraz rozwoju dysbiozy. Zaburzeniu ulega równowaga ilościowa poszczególnych szczepów bakterii oraz motoryka przewodu pokarmowego. Zauważalne jest większe ryzyko wystąpienia infekcji poprzez ułatwiony dostęp patogenów do nabłonka jelit. Proces prawidłowej kolonizacji przewodu pokarmowego jest odmienny a rozwój mikrobiomu opóźniony. Obecnie stosuje się odpowiednio wyselekcjonowane szczepy bakterii probiotycznych, w celu wsparcia dojrzewania przewodu pokarmowego podczas terapii żywieniem pozajelitowym. Pozwala to zmniejszyć niepożądane skutki i jednocześnie zapewnić optymalną terapie.

Neuronal N-acetylaspartate production appears in the presence of aspartate N-acetyltransferase (NAT8L) and binds acetyl groups from acetyl-CoA with aspartic acid. Further N-acetylaspartate pathways are still being elucidated, although they seem to involve neuron-glia crosstalk. Together with N-acetylaspartate, NAT8L takes part in oligoglia and astroglia cell maturation, myelin production, and dopamine-dependent brain signaling. Therefore, understanding N-acetylaspartate metabolism is an emergent task in neurobiology. This project used in in vitro and in vivo approaches in order to establish the impact of maturation factors and glial cells on N-acetylaspartate metabolism. Embryonic rat neural stem cells and primary neurons were maturated with either nerve growth factor, trans-retinoic acid or activators of cAMP-dependent protein kinase A (dibutyryl-cAMP, forskolin, theophylline). For in vivo, adult male Wistar rats were injected with theophylline (20 mg/kg b.w.) daily for two or eight weeks. Our studies showed that the N-acetylaspartate metabolism differs between primary neurons and neural stem cell cultures. The presence of glia cells protected N-acetylaspartate metabolism from dramatic changes within the maturation processes, which was impossible in the case of pure primary neuron cultures. In the case of differentiation processes, our data points to dibutyryl-cAMP as the most prominent regulator of N-acetylaspartate metabolism.

The N-acetylaspartate network begins in neurons with N-acetylaspartate production catalyzed by aspartate N-acetyltransferase from acetyl-CoA and aspartate. Clinical studies reported a significant depletion in N-acetylaspartate brain level in type 1 diabetic patients. The main goal of this study was to establish the impact of either hyperglycemia or oxidative stress on the N-acetylaspartate network. For the in vitro part of the study, embryonic rat primary neurons were treated by using a nitric oxide generator for 24 h followed by 6 days of post-treatment culture, while the neural stem cells were cultured in media with 25–75 mM glucose. For the in vivo part, male adult Wistar rats were injected with streptozotocin (65 mg/kg body weight, ip) to induce hyperglycemia (diabetes model) and euthanized 2 or 8 weeks later. Finally, the biochemical profile, NAT8L protein/Nat8l mRNA levels and enzymatic activity were analyzed. Ongoing oxidative stress processes significantly affected energy metabolism and cholinergic neurotransmission. However, the applied factors did not affect the N-acetylaspartate network. This study shows that reduced N-acetylaspartate level in type 1 diabetes is not related to oxidative stress and that does not trigger N-acetylaspartate network fragility. To reveal why N-acetylaspartate is reduced in this pathology, other processes should be considered.

N-acetylaspartate is produced by neuronal aspartate N-acetyltransferase (NAT8L) from acetyl-CoA and aspartate. In cholinergic neurons, acetyl-CoA is also utilized in the mitochondrial tricarboxylic acid cycle and in acetylcholine production pathways. While aspartate has to be shared with the malate–aspartate shuttle, another mitochondrial machinery together with the tricarboxylic acid cycle supports the electron transport chain turnover. The main goal of this study was to establish the impact of toxic conditions on N-acetylaspartate production. SN56 cholinergic cells were exposed to either Zn²⁺ overload or Ca²⁺ homeostasis dysregulation and male adult Wistar rats’ brains were studied after 2 weeks of challenge with streptozotocin-induced hyperglycemia or daily theophylline treatment. Our results allow us to hypothesize that the cholinergic neurons from brain septum prioritized the acetylcholine over N-acetylaspartate production. This report provides the first direct evidence for Zn²⁺-dependent suppression of N-acetylaspartate synthesis leading to mitochondrial acetyl-CoA and aspartate shortages. Furthermore, Zn²⁺ is a direct concentration-dependent inhibitor of NAT8L activity, while Zn²⁺-triggered oxidative stress is unlikely to be significant in such suppression.

Summary: Background: Lipoprotein X (LpX) is an abnormal lipoprotein fraction, which can be detected in patients with severe hypercholesterolaemia and cholestatic liver disease. LpX is composed largely of phospholipid and free cholesterol, with small amounts of triglyceride, cholesteryl ester and protein. There are no widely available methods for direct measurement of LpX in routine laboratory practice. We present the heterogeneity of clinical and laboratory manifestations of the presence of LpX, a phenomenon which hinders LpX detection.Methods: The study was conducted on a 26-year-old female after liver transplantation (LTx) with severely elevated total cholesterol (TC) of 38 mmol/L and increased cholestatic liver enzymes. TC, free cholesterol (FC), cholesteryl esters (CE), triglycerides, phospholipids, HDL-C, LDL-C, and apolipoproteins AI and B were measured. TC/apoB and FC:CE ratios were calculated. Lipoprotein electrophoresis was performed using a commercially available kit and laboratory-prepared agarose gel.Results: Commercially available electrophoresis failed to demonstrate the presence of LpX. Laboratory-prepared gel clearly revealed the presence of lipoproteins with γ mobility, characteristic of LpX. The TC/apoB ratio was elevated and the CE level was reduced, confirming the presence of LpX. Regular lipoprotein apheresis was applied as the method of choice in LpX disease and a bridge to reLTx due to chronic liver insufficiency.Conclusions: The detection of LpX is crucial as it may influence the method of treatment. As routinely available biochemical laboratory tests do not always indicate the presence of LpX, in severe hypercholesterolaemia with cholestasis, any discrepancy between electrophoresis and biochemical tests should raise suspicions of LpX disease.

Chronic kidney disease (CKD) associates with complex lipoprotein disturbances resulting in high cardiovascular risk. Apolipoprotein E (APOE) is a polymorphic protein with three common isoforms (E2; E3; E4) that plays a crucial role in lipoprotein metabolism, including hepatic clearance of chylomicrons and very low-density lipoprotein (VLDL) remnants, and reverse cholesterol transport. It demonstrates anti-atherogenic properties but data concerning the link between polymorphism and level of APOE in CKD patients are inconclusive. The aim of our research was to assess the relationship between APOE gene polymorphism and APOE concentration and its redistribution among lipoproteins along with CKD progression. 90 non-dialysed CKD patients were included into the study. Real time PCR was used for APOE genotyping. APOE level was measured in serum and in isolated lipoprotein fractions (VLDL; IDL + HDL; HDL). Kidney function was assessed using eGFR CKD-EPI formula. The population was divided into three APOE genotype subgroups: E2(ε2ε3), E3(ε3ε3) and E4(ε3ε4). The highest APOE level was observed for the E2 subgroup (p < 0.001). APOE concentration positively correlated with eGFR value in the E2 subgroup (r = 0.7, p < 0.001) but inversely in the E3 subgroup (r = − 0.29, p = 0.02).). A lower concentration of APOE in the E2 subgroup was associated with its diminished contents in HDL and IDL + LDL particles. In the E3 subgroup, the higher concentration of APOE was related to the increased number of non-HDL lipoproteins. In patients with CKD, APOE genotype as well as renal function are associated with the concentration of APOE and its redistribution among lipoprotein classes.

Background/Aims: Hypertriglyceridaemia (HTG) and reduction and dysfunction of high density lipoprotein (HDL) are common lipid disturbances in chronic kidney disease (CKD). HTG in CKD is caused mainly by the decreased efficiency of lipoprotein lipase (LPL)-mediated very low density lipoprotein triglyceride (VLDL-TG) lipolysis. It has not been clarified whether HDL dysfunction in CKD contributes directly to HTG development; thus, the aim of this study was to assess the impact of CKD progression on the ability of HDL to enhance LPL-mediated VLDL-TG lipolysis efficiency. Methods: VLDL was isolated from non-dialysis patients in CKD stages 3 and 4 and from non-CKD patients. The VLDL was incubated with LPL at the constant LPL:VLDL-TG ratio, in the absence or presence of HDL. After incubation, the VLDL was separated and the percentage (%) of hydrolyzed TG was calculated. Results: HDL presence increased the lipolysis efficiency of VLDL isolated from CKD and non-CKD patients, for the VLDL-TG> 50 mg/dl. Its effect was dependent on the VLDL-TG and HDL-cholesterol concentrations in the reaction mixtures: the higher the concentrations of VLDL-TG and HDL-cholesterol, the greater the effect. The positive impact of HDL on VLDL lipolysis was modified by CKD progression: the percentage of lipolyzed VLDL-TG in the presence of HDL decreased with a reduction in eGFR (r=0.43, p=0.009), and for patients with stage 4 CKD, no positive impact of HDL on lipolysis was observed. The percentage of lipolyzed TG correlated negatively with apoE and apoCs content in VLDL, and positively with HDL-apoCII, as well as with VLDL and HDL apoCII/ apoCIII ratios. The progression of CKD was associated with unfavourable changes in VLDL and HDL composition; apoE and apoCs levels increased in VLDL with a decrease in eGFR whereas the HDL-cholesterol level decreased. Conclusion: The progression of CKD affects lipoprotein composition and properties, and modulates the positive impact of HDL on VLDL lipolysis efficiency. In CKD patients, HDL deficiency and dysfunction can directly affect hypertriglyceridaemia development.

Background: The ratio of albumin to creatinine (ACR) is an important parameter used for detection of albuminuria in patients with early kidney damage. The aim of the study was to evaluate the harmonization of ACR results among Polish participants in an international external quality assessment (EQA) program, and to evaluate the impact of albumin and creatinine analytical performance on the harmonization of ACR results. Methods: We analyzed 182 results of albumin, 202 of creatinine, and 180 of ACR obtained from Polish laboratories in an EQA program organized by Labquality. The dispersion of the results in surveys and percentage differences between the results and target values were calculated. Moreover, differences between method groups were assessed. Results: The inter-laboratory coefficient of variation (CV) for ACR was 36%. Only 74% of results of Polish laboratories were within the target limits; for 11% of the results, an incorrect albuminuria category would have been reported. The inter-laboratory CV for albumin was 20%, 2.6-fold higher than for creatinine. Significant differences between method groups for albumin determination have been observed, even when the same measurement technique was used. The greatest difference between two groups was 23%, 2.5-fold greater in comparison to creatinine. Conclusions: There is an insufficient harmonization of ACR values among Polish laboratories, caused mainly by urine albumin analytical performance. Given the important role of ACR in the classification, monitoring and treatment of kidney damage, the harmonization of albumin measurements is crucial and urgently needed.

Pyruvate improves contractility of normal, hypoxic, and post-ischemic myocardium. However, sodium overload is a major problem with its therapeutic application if sodium pyruvate is used. Development of alternative forms such as N-1-methylnicotinamide (MNA) pyruvate may help to overcome this problem. The aim of the study was to investigate the effect of MNA pyruvate in a murine model of cardiac ischemia. Seven month old male ApoE-/-LDLr-/- mice that develop myocardial infarction when exposed to hypoxic stress, were used in this study. Hypoxia (8% O2 in inspired air) was maintained for 8min and was followed by reoxygenation (21% O2 in inspired air). Four groups of mice were treated 10min before the hypoxic event by intravenous injection of MNA, MNA pyruvate, sodium pyruvate, and saline as control. The myocardial ischemia and damage was recorded by ECG. Four hours following the hypoxic episode serum troponin T and creatine kinase activity were measured. Significant hypernatremia was found in the sodium pyruvate group. During hypoxia, control and MNA group developed profound STU depressions on ECG while no changes were observed in MNA pyruvate and sodium pyruvate group. Creatine kinase activity and troponin T content in the mice plasma were significantly higher in the control and MNA group as compared to the MNA pyruvate and sodium pyruvate group. This study demonstrated that administration of MNA pyruvate prior to a hypoxia-induced cardiac event was cardioprotective. This intervention did not cause hypernatremia in contrast to sodium pyruvate.