With increasing prevalence, chronic kidney disease (CKD) has become a global health problem. Due to the retention of uremic toxins, electrolytes and water, and the resultant metabolic disturbances, CKD affects several organs, including the nervous system. Thus, CKD patients suffer from several neurological complications, including dementia, cognitive impairment, motor abnormalities, depression, and mood and sleep disturbances. However, the mechanisms underlying the neurological complications are least elucidated. We have recently reported a highly reproducible mice model of CKD induced by high adenine diet, which exhibited psychomotor behavioral abnormalities and blood-brain barrier disruption. In the present study, using the mice model, we have investigated psycho-motor and cognitive behaviour, and the neurochemical and histopathological alterations in brain relevant to the observed behavioural abnormalities. The results demonstrate global loss of Acetylcholinesterase activity, and decrease in neuronal arborisation and dendritic spine density in discrete brain regions, of the CKD mice. Oxidative stress, inflammation, and mitochondrial dysfunctions were found in specific brain regions of the mice, which have been regarded as the underlying causes of the observed neurochemical and histopathological alterations. Thus, the present study is of immense importance, and has therapeutic implications in the management of CKD-associated neurological complications. CKD is a global health issue, affecting more than 15% of the adult population in developed nations4. In India, the overall prevalence is 17.2%6, while in some coastal districts of Andhra Pradesh (India), it has been reported to be more than 60%7. Thus, CKD has become a major health burden, especially for the developing countries lacking sufficient professionals and infrastructure.Chronic kidney disease (CKD) refers to a wide spectrum of disease conditions whereby the renal structure and functions are impaired. This leads to decrease in the glomerular filtration rate below 60 mL/min/1.73 m9. In the end stages of the disease, anaemia, low levels of serum albumin and high phosphate increase morbidity and mortality9. Moreover, with progression of the disease, and retention of metabolic wastes, electrolytes and water in the body, CKD leads to edema, cardiac failure, arrhythmia, bone disease, changes in pigmentation, insulin resistance, thiamine and calciferol deficiency, liver infection, dyslipidemia and hyperhomocysteinemia13. With reduction in glomerular filtration rate, and consequent retention of uremic toxins, and the associated disturbances13, CKD affects other organs15, including the nervous system, which results in neurological complications18.Once renal damage is initiated, factors including proteinuria, hyperglycemia, hypertension, metabolic disturbances, and lifestyle factors like smoking, dehydration and low fiber intake contribute to the progression of the disease to end-stage renal disease21. In children with CKD, reduced intelligence quotient, memory, and language and academic achievements have been reported22. We have recently reported psychomotor behavioral abnormalities and blood-brain barrier disruption in mice model of the disease23. Cognitive decline, both acute and chronic with dementia, increases with advancements in the severity of the disease, and may affect 80% of the subjects25. Cognitive decline is known to be caused due to cholinergic deficiency, including decrease in the activity of Acetylcholinesterase (AChE) in brain27. However, the mechanism underlying cognitive decline in CKD, and the role of AChE thereto, has not been investigated so far. RLS is a motor behavioral abnormality with a prevalence of 15–20% among CKD patients20, and is also associated with sleep disturbances28. Moreover, motor behavioral abnormalities similar to parkinsonism, including resting tremor, rigidity, bradykinesia and postural instability, have been reported in CKD patients29. Dopamine agonist and levodopa therapy are practised for the amelioration of RLS30. However, the effect of CKD on the dopaminergic neurons of the nigrostriatum has not been investigated yet.The CKD patients suffer from several neurological complications, including anxiety, depression, motor abnormalities , sleep disturbances and cognitive dysfunctions31. However, similar study in CKD model has not been undertaken. Moreover, CKD-induced possible oxidative stress, inflammation, mitochondrial dysfunctions, and dendritic atrophy and loss of dendritic spine density in the brain, which may be the pathophysiological basis of the neurological complications, remain largely unknown. Thus, the present study was undertaken to elucidate the biochemical and histopathological changes relevant to the neurological complications in CKD.In mice model of acute renal injury, hyperactivation of glia (astroglia) has been reportedad libitum. The experimental protocols used in the present study have been approved by the Animal Ethics Committee, Assam University, Silchar, India . All the protocols used in the present study were performed in accordance with the relevant guidelines and regulations.Swiss Albino male mice of weight between 25–27 g (aged 6–7 weeks) were purchased from the animal house of the Pasteur Institute, Shillong, Meghalaya (India). They were maintained under standard laboratory conditions, and were given standard feed and water 2O2), Sirius red and poly-L-lysine were obtained from Sigma-Aldrich Co., USA. Potassium dichromate, silver nitrate and formaldehyde were obtained from HiMedia laboratories Pvt. Ltd., India. Acetic acid, cresyl violet, Nitro blue tetrazolium (NBT), 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), cytochrome c, pyrogallol, salicylic acid, tri-sodium citrate, copper sulphate, potassium ferricyanide, paraformaldehyde, picric acid, Triton X-100, sodium dodecyl sulphate and other chemicals were of analytical reagent grade, procured from SISCO Research Laboratories Pvt. Ltd., India. Rabbit anti-Glial fibrillary acidic protein (GFAP) antibody (ab7260) and rabbit anti-tyrosine hydroxylase (TH) antibody (ab112) were procured from Abcam . Anti-rabbit goat secondary antibody conjugated with horseradish peroxidase  was procured from Millipore Co. (USA). Urea, creatinine and uric acid assay kits were obtained from Coral Clinical Systems, Goa, India .Acetylthiocholine iodide, adenine, ketamine, xylazine, thiobarbituric acid, 1,1,3,3-tetramethoxypropane, 3,3-diaminobenzidine (DAB) liquid substrate system, hydrogen peroxide : the first group served as control and was given standard powdered diet, and the second group (CKD group) received adenine-rich diet, at 0.3% w/w mixed with the feed, for 4 weeks. Body weights of the mice were taken on every alternative day. At the end of the treatment period, six mice from each group were subjected to motor behavioural test (Swim test), following which they were sacrificed. The mice were anaesthetised with ketamine (75 mg/kg b.w.) and xylazine (5 mg/kg b.w.), blood was collected by cardiac puncture , and then they were perfused transcardially. Kidneys and brains were harvested and processed for renal histology using Hematoxylin-Eosin staining, and brain histology and immunohistochemistry. Another set of mice (n = 6 each) were used for psychometric behavioural test (Forced Swim test), after which they were perfused with glycerol, brains were harvested, and were used for histoenzymological studies of AChE, mitochondrial complexes (II and III) and neuronal nitric oxide synthase (nNOS). Another group of mice (n = 24) were sacrificed by decapitation, brains were harvested, different brain regions were isolated, homogenised and were used for estimation of the activities of Acetylcholinesterase, mitochondrial complex-I, superoxide dismutase and catalase, and lipid peroxidation. Rest of the mice (n = 6 each) were used for Cognitive behavioural test, and then sacrificed for Rapid Golgi staining. All the behavioural tests and histological studies, including scoring, were performed by assessors blind to the experimental groups.23. Serum urea, creatinine and uric acid levels, and renal histology were assessed for ascertaining establishment of CKD in the model. Body weights of the mice were taken every alternative day.The mice model of CKD was developed by providing adenine-rich diet (0.3% w/w mixed with feed) for 4 weeks, as per the protocol standardised in our laboratoryet al.23. The test was performed in plastic tubs of dimensions 40 cm × 25 cm × 16 cm, containing 12 cm water level, maintained at 27 ± 2 °C. The swim score of each minute, for a total of 10 min, was calculated on a scale of 0–3: ‘3’ – continuous swimming, ‘2’ – continuous swimming with occasional floating, ‘1’ –more floating and occasional swimming with hind limbs, and ‘0’ – hind parts sinking with only head floating. Water was changed after each test for each mice.Swimming ability test was performed to determine motor performance, following Mazumder et al.23. The test was performed in glass cylinders of 25 cm height and 12 cm diameter, with 12 cm water level, maintained at 27 ± 2 °C. Mice were released in water, and following 2 min acclimatization, the total immobility time was recorded for each mice for a period of 4 min. Water was changed after each test for every mice.Forced swim test was performed to determine depression-like behaviour in the mice, following Mazumder 32. For performing OLM test, two identical objects were placed 7 cm apart from each other in a chamber of 30 cm × 23 cm dimensions. This rectangular chamber had 2 longer sides (30 cm each) and 2 shorter sides (23 cm each) opposite to each other respectively. The objects were placed 1 cm away from shorter sides and 6.5 cm away from longer sides of the chamber. Animals were placed in the opposite side of the object and 2.5 cm away from shorter wall of the chamber. Animals were trained in this setup for 10 min. After an interval of 90 min post training, test was done in which one of the objects was shifted to a new location. The exploration time for both the objects was recorded for 5 min to calculate the discrimination index (DI). As there was 90 min interval between training and test, OLM gives score for short-term memory.Object location memory (OLM) and object recognition memory (ORM) tests of the experimental animals were performed to assess their cognitive functions. The tests were performed following Paul and Borahth day following OLM test, ORM training of the animals was performed for 10 min each with similar objects. After an interval of 24 h following the training session, ORM test was performed for 5 min with a novel object and the exploration time in both the objects was recorded to calculate the DI. As there was 24 h interval between training and test, ORM gives score for short-term memory. The Discrimination Index (DI) is calculated as follows: DI = [(time exploring the novel object − time exploring the familiar)/(time exploring novel + familiar) × 100].ORM test was performed using a cylindrical chamber of 28 cm diameter. Objects were placed diagonally 8 cm apart from each other and 3 cm away from the circumference. On 4The induction of CKD in mice was validated using haematological and renal histopathological changes.33, using commercially available enzymatic urea assay kit supplied in ready to use form. Briefly, urea in serum is hydrolysed by Urease enzyme to produce ammonia, which reacts with the phenolic chromogen and hypochlorite to form a green coloured complex, the intensity of which is directly proportional to the amount of urea present. Absorbance was measured at 570 nm using spectrophotometer . The method is linear upto 250 mg/dL of urea in serum.After the treatment period, the mice were anaesthetised by ketamine  and xylazine , and blood was collected in vacutainer vials containing clot activators. Serum was obtained following centrifugation at 2000 × g for 5 min using Refrigerated Centrifuge , and was used for estimation of urea, creatinine and uric acid. Urea was assayed following modified Berthelot Method34, using commercially available assay kit. Briefly, creatinine reacts with picric acid in alkaline medium to produce an orange coloured complex with alkaline picrate, the intensity of which is directly proportional to the amount of creatinine present. The coloured complex has peak absorbance at 520 nm, which was measured using spectrophotometer . To minimize interference, deproteinization of serum was done by mixing 0.1 mL of serum with 1 mL of picric acid reagent, followed by centrifugation at 3000 rpm for 10 min to obtain a clear supernatant, which was used for the estimation of creatinine. The method is linear upto 20 mg/dL of creatinine in serum.Serum creatinine level was estimated using alkaline picrate Jaffe’s reaction method35, using commercially available enzymatic assay kit. Briefly, uric acid is degraded by Uricase enzyme to allantoin and H2O2. H2O2 reacts with a phenolic compound and 4-aminoantipyrine by peroxidase to produce a red coloured quinoneimine dye complex, the intensity of which is directly proportional to the amount of uric acid present in the serum. Absorbance was recorded at 520 nm using spectrophotometer . The method is linear upto 20 mg/dL of uric acid.Estimation of uric acid was done following Uricase/PAP methodAll reactions were carried out at 37 °C, and reaction time for each test was maintained strictly as per manufacturer’s protocol, and rigorously constant for each test. The absorbance was measured with light path of 1 cm. The analytical coefficient of variation for estimation of urea, creatinine and uric acid were 3.12%, 4.45% and 3.38% respectively, and the limits of detection were 1 mg/dL, 0.1 mg/dL and 0.15 mg/dL respectively.et al.23. Photographs were taken under bright-field illumination using digital SLR camera attached with microscope .Mid-longitudinal, 5 µm thick sections of kidney were taken, using cryostat , on poly-L-lysine coated slides, and stained using Haematoxylin-Eosin staining procedure, following Mazumder 23. For Nissl staining, 20 µm thick coronal brain sections passing through the cortex, striatum, hippocampus and substantia nigra regions of brain were taken on poly-L-lysine coated slides, dried, hydrated in decreasing alcohol gradient, stained with 0.5% cresyl violet, washed, dehydrated in increasing ethanol gradient, cleared in xylene, mounted in DPX, and photographed at low (4×) and high (20×) magnification under bright-field illumination. For Picro-Sirius red staining, 5–7 µm sections were taken on coated slides, cleared in xylene, hydrated in decreasing alcoholic grades, stained with Haematoxylin, washed in running water, stained with Picro-Sirius red (1% Sirius red in saturated solution of picric acid), washed with acidified water (0.5% acetic acid), removed water by vigorous shaking, dehydrated in absolute alcohol, cleared in xylene, mounted in DPX and photographed under bright-field illumination, using digital SLR camera attached with microscope .Histological study of the different brain regions was performed to elucidate any deposition of crystals of adenine and 2,8-dihydroxyadenine, using Nissl and Picro-Sirius red staining procedures, following protocols as standardized in our laboratory21, it is thought prudent to estimate the activity of AChE in different brain regions, which was done from brain slices  as well as brain tissue homogenates.Since CKD is associated with dementia and cognitive decline32. Briefly, mice were anaesthetised, and perfused with 30 mL each of ice-cold phosphate buffered saline  and 10% v/v glycerol. Brains were harvested, cryoprotected in 30% w/v sucrose solution overnight, and 20 µm thick sections were taken from the different brain regions – prefrontal cortex, cerebral cortex, striatum, amygdala, hippocampus and substantia nigra, on poly-L-Lysine coated slides using cryostat . The sections were incubated in a reaction mixture containing 0.1 M tri-sodium citrate, 60 mM copper sulphate, 30 mM potassium ferricyanide and acetylthiocholine iodide (substrate), in 0.1 M PBS (pH 6.0), in dark at 37 °C. After incubation, the slides were washed in PBS, air dried, mounted in DPX and photographed using digital SLR camera attached with microscope .AChE activity was estimated from brain tissue sections, following Paul and Borah36, with minor modifications35, AChE activity was determined from tissue homogenates of different brain regions. The method is based on hydrolysis of acetylthiocholine by AChE to produce thiocholine, which reacts with DTNB to produce a colour complex (TNB), which has peak absorbance at 412 nm. Briefly, mice were sacrificed by cervical dislocation, the brains were dissected out, and the different brain regions  were separated or micropunched. The brain regions were homogenized in Tris-HCl buffer  in 1:20 ratio (w/v). The reaction mixture contains 0.1 mL tissue homogenate and 0.8 mL of DTNB in 0.9 mL of Tris–HCl buffer. The reaction was initiated by addition of 0.2 mL of acetylthiocholine iodide, and the absorbance was recorded for 1 min after the addition of the substrate, at 25 °C using spectrophotometer32 .Following Ellman’s methodet al.37. Briefly, mice were anaesthetised, perfused with 30 mL each of PBS  and 4% w/v paraformaldehyde. The brains were harvested and stored in the same fixative for 48 hours, and then cryoprotected in 30% w/v sucrose solution, pending immunohistochemistry. Coronal brain sections (35 µm thick) passing through the striatum and substantia nigra were made using cryostat , and taken in well-plate. The sections were washed three times in 0.1 M Tris-buffered saline  for 5 min each, treated with 3% v/v H2O2  for 5 min, permeabilized in 0.3% v/v Triton X-100  for 30 min, blocked in 10% v/v donkey serum containing 0.3% Triton X-100  for 1 h, washed in 0.1 M TBS, and incubated overnight at 4 °C in primary antibody  in a mixture of 2% v/v donkey serum and 0.1% v/v Triton X-100. After a gentle wash in 0.1 M TBS, the sections were incubated in HRP-conjugated secondary antibody  in a mixture of 0.1 M TBS (pH 7.4), 2% v/v donkey serum and 0.1% Triton X-100 for 1 h at room temperature. Following washing of unbound antibody in TBS, the sections were incubated in DAB-liquid substrate system for colour development. The sections were then washed in TBS, dehydrated in increasing ethanol grades, cleared in xylene, mounted in DPX and photographed at 4× magnification under bright-field illumination, using digital SLR camera attached with microscope . Neurons from every sixth serial section of the substantia nigra were counted (n = 6 per group), while photographs of the sections of the striatum were analysed for densitometry using ImageJ (Fiji version) software.TH-immunohistochemistry was performed following Paul et al.38 with minor modifications. Briefly, the anaesthetised mice were perfused with 30 mL of 0.1 M PBS (pH 7.4), and then fixed transcardially with 30 mL of Rapid Golgi fixative containing a mixture of 5% chloral hydrate, 5% potassium dichromate, 3.2% formaldehyde, 1.5% gluteraldehyde and 1% DMSO. Brains were immediately harvested and different brain regions were dissected out, and stored in the same fixative in dark at room temperature. The fixative was changed after 24 h and kept undisturbed for 48 h, after which the tissues were repeatedly washed with 0.75% silver nitrate until the colour of potassium dichromate was washed away. The tissues were then impregnated with silver nitrate for another 48 h, dehydrated in ethanol grades , and 60 µm thick sections passing through cortex and hippocampus were made using cryostat . The sections were collected in 70% ethanol, cleared in xylene and mounted in DPX. Photographs were taken using digital SLR camera attached with microscope  under bright-field illumination to assess neuronal arborisation and dendritic spine density.Rapid Golgi staining was performed following Chakraborty et al.39. The brain regions were dissected out or micro-punched, and 10% w/v homogenate of the brain tissues were prepared in ice-cold buffer containing 0.32 mol/L sucrose in 10 mmol/L potassium phosphate buffer (pH 7.2). The homogenates were centrifuged at 1000 × g for 10 min at 4 °C, using Refrigerated Centrifuge . The supernatant was collected and centrifuged at 10,000 × g for 30 min at 4 °C, and the resulting pellet was re-suspended in ice-cold 50 mmol/L Tris–HCl buffer (pH 7.2), and centrifuged at 10,000 × g for 30 min at 4 °C. The pellets were re-suspended in cold 10 mmol/L potassium phosphate buffer (pH 7.2), and used for the enzymatic assay of the mitochondrial complex-I. The reaction mixture contained 10 mM potassium phosphate (pH 7.2), 5 mM sodium azide, 50 mM coenzyme Q0 and 70–120 mg tissue extract . After pre-incubation for 3 min at 32 °C, the reaction was initiated by the addition of NADH , and the rate of decrease in the absorbance was monitored at 340 nm for 3 min using spectrophotometer . The specific enzyme activity was expressed as nmoles of NADH oxidized/min/mg protein (taking extinction coefficient of NADH as 6.2/mM/cm at 340 nm).Mitochondrial complex-I activity was assayed from the cortex, striatum, hippocampus and substantia nigra of the mice, following Pandey et al.40 with minor modifications32. Briefly, anaesthetised mice were transcardially perfused with 30 mL each of PBS , and 10% v/v glycerol. Brains were dissected out, cryoprotected in 30% w/v sucrose solution overnight, and 20 µm thick sections through the cortex, striatum, hippocampus and substantia nigra were taken on poly-L-lysine coated slides using cryostat . Sections were activated in PBS  for 10 min at 37 °C, and then incubated in a reaction mixture in dark at 37 °C for 30 min. The reaction mixture contained 0.3 mol/L NBT, 0.05 mol/L phosphate buffer (pH 7.4) and 0.05 mol/L sodium succinate. After incubation, the sections were washed, mounted in glycerol and photographed immediately under bright-field illumination, using digital SLR camera attached with microscope . The images were subjected to densitometric analysis.The activity of the complex was assayed using histoenzymological technique, following Riddle et al.41 with minor modifications32. Briefly, anaesthetised mice were transcardially perfused with 30 mL each of PBS  and 10% v/v glycerol. Following overnight cryoprotection in 30% w/v sucrose solution, 20 µm thick sections through the cortex, striatum, hippocampus and substantia nigra were taken using cryostat  on pre-coated slides. The sections were incubated in a reaction mixture containing 50 mg DAB, 20 mg cytochrome c, 4 g sucrose and 90 mL of 0.1 M phosphate buffer (pH 7.4) for 1 h in dark. The sections were washed in phosphate buffer, mounted in glycerol, coverslipped and photographed immediately under bright-field illumination using digital SLR camera attached with microscope . The images were subjected to densitometric analysis.The activity of the complex was assayed using histoenzymology, following Govindaiah 42. Briefly, the mice were sacrificed by decapitation, and different brain regions were immediately dissected out or micro-punched. A 10% w/v homogenate of the different brain regions was prepared in TNG-T buffer , and then sonicated with 5 strokes at 40 Hz, using Sonicator . The tissue suspension was centrifuged for 10 min at 1000 × g, and the supernatant was used for the estimation of SOD activity. The assay mixture (1 mL) contained 0.2 mM of pyrogallol, 1 mM of EDTA and 50 mM of Tris-HCl buffer (pH 8.2). Auto-oxidation of pyrogallol was measured using spectrophotometer  at 420 nm for 3 min, with or without the enzyme. The inhibition of pyrogallol oxidation was linear with the activity of the enzyme present. Fifty percent inhibition in pyrogallol auto-oxidation/min/mg protein was considered as one unit of the enzyme activity.Cu/Zn-SOD activity was analysed from the cortex, striatum, hippocampus and substantia nigra of control and CKD mice brain, following the method as described earlieret al.42. Using the same enzyme extract that was used for determination of the SOD activity, CAT activity was also determined. Based on the estimated level of protein, the supernatant was suitably diluted using 0.01 M PBS (pH 7.0) to give 75–100 µg protein in 0.1 mL of enzyme extract. The reaction mixture contained 1 mL of 0.01 M PBS, 0.4 mL distilled water, and 0.5 mL 0.2 M H2O2. The reaction was initiated by addition of the enzyme extract in the reaction mixture. Absorbance, at 240 nm, was recorded using spectrophotometer  for 5 min whereby H2O2 is decomposed and the optical density decreased. The enzyme activity was represented as change in absorbance/min/mg protein.The activity of CAT was estimated from the cortex, striatum, hippocampus and substantia nigra of the control and CKD mice brain, following Paul 43. In brief, mice were sacrificed by decapitation, brains were harvested, different brain regions were isolated, and 10% w/v brain tissue homogenate was prepared in PBS , and incubated at 95 °C for 60 min in acid medium containing 0.45% sodium dodecyl sulphate and 0.6% TBA. After centrifugation, the absorbance of the reaction product (TBARS) was determined at 532 nm using spectrophotometer . 1,1,3,3-tetramethoxypropane was used as standard, and the results were expressed as nmol TBARS/mg protein. All the reagents used for the assay were of analytical reagent grade, and the purities were ≥98% for TBA , 99% for SDS  and 99% for 1,1,3,3-tetramethoxypropane . The analytical coefficient of variation for the estimation of TBARS was 13.28%, and the limit of detection was 0.1 nmol TBARS/mg protein.The level of thiobarbituric acid-reactive substance (TBARS) was determined in the tissue homogenates of different brain regions: cortex, striatum, hippocampus, and substantia nigra, following Koudelová and MourekTo assess inflammatory stress on neurons, the extent of nitric oxide synthase (nNOS)-active neurons were determined by histoenzymological method. For reactive glia (astroglia), GFAP-immunohistochemistry was performed.et al.37 from the cortex, striatum, hippocampus and substantia nigra of the brain. The procedure was similar to the one used for TH-immunohistochemistry except that the primary antibody used was rabbit anti-GFAP (1:500), and the sections were photographed at low (4×) and high (20×) magnification under bright field illumination, using digital SLR camera attached with microscope .GFAP–immunohistochemistry was performed following Paul et al.44. Briefly, the anaesthetised mice were perfused with 30 mL 10% v/v glycerol, brains were harvested, cryoprotected in 30% w/v sucrose solution overnight, and 20 µm thick coronal brain sections passing through cortex, striatum, hippocampus and substantia nigra were made using cryostat . Brain sections were taken on poly L-lysine coated slides, and were permeabilized with 50 mM Tris-HCl buffer (pH 7.4) containing 0.2% Triton X-100. The sections were then incubated in a reaction mixture containing 50 mM Tris-HCl buffer (pH 8.0), 15 mM CaCl2, 10 mM NADPH, 5 mM NBT and 0.2% Triton X-100, for 1 hour at 37 °C in dark. Following the development of colour, the slides were washed in Tris-HCl buffer (pH 7.4) twice, mounted in DPX and photographed at low (4×) and high (20×) magnification on the same day, using digital SLR camera attached with microscope .The extent of nNOS-active neurons were assayed from different brain regions using histoenzymological technique, following Hope et al.45 using bovine serum albumin as standard.Protein content in the brain tissue homogenate was determined following Lowry 46. Optical density = log . For counting the number of TH-positive neurons in the substantia nigra, using ImageJ software, every sixth serial section from each mice (n = 6) was selected and the total number of neurons in those sections was counted37.The images of serial sections of TH-immunoreactivity of the striatum, and mitochondrial complex-II and complex-III histoenzymological studies were analysed using ImageJ (Fiji version) software for the determination of optical densityt-test (two-tailed) or a two-way, repeated measures ANOVA using GraphPad Prism version 7.0 . Each group consisted of at least six animals. The values are expressed as mean ± S.D., and the distribution of data points have been shown using whisker box plot mean with 95% confidence interval. The analytical coefficient of variation for estimation of haematological parameters and lipid peroxidation were calculated prior to start of the assays to check precision of the assay methods. The difference of mean was considered statistically significant at a P-value of 0.05 or less.Statistical analysis was performed employing a parametric unpaired Student’s th day onwards, while in the control group there had been a gradual increase in body weight , compared to the control mice (19.67 ± 2.42)  revealed a marked visible decrease in the colour intensity, in different brain regions, viz. prefrontal cortex, cerebral cortex, striatum, amygdala, hippocampus and substantia nigra of CKD mice, compared to the control Fig. . AnalysiImmunohistochemistry for TH was performed to investigate dopaminergic neuronal status in the striatum and substantia nigra Fig. . DensitoRapid Golgi staining was performed in the cortex and hippocampus to reveal neuronal architecture in terms of dendritic spine density and neuronal arborisation. While the neuronal arborisation was found to be decreased only in hippocampus of CKD mice brain Fig. , compareThe activity of complex-I (expressed as the amount of NADH oxidized/min/mg protein) was found to be 123.12 ± 16.81, 101.34 ± 11.95, 78.67 ± 9.17 and 119.31 ± 13.39 in cortex, striatum, hippocampus and substantia nigra of control mice, while in CKD mice the activities were 99.78 ± 17.08, 83.78 ± 9.49, 71.34 ± 8.20 and 105.78 ± 12.99 respectively  was found to be 2.11 ± 0.24, 1.45 ± 0.19, 2.43 ± 0.54 and 0.83 ± 0.23 in the cortex, striatum, hippocampus and substantia nigra respectively. In these brain regions of the CKD mice, the activity of SOD was 2.97 ± 0.50, 1.89 ± 0.41, 3.21 ± 0.63 and 1.22 ± 0.23 respectively  was found to be 33.11 ± 0.24, 2.45 ± 0.15, 3.11 ± 0.53 and 2.33 ± 0.20 in the cortex, striatum, hippocampus and substantia nigra of control mice brain, while the same was 2.57 ± 0.41, 1.89 ± 0.38, 2.35 ± 0.25 and 1.78 ± 0.34 respectively in these brain regions of the CKD mice , and therefore the extent of lipid peroxidation, which indicates oxidative stress. The level of MDA (in nmol/mg protein) in the different brain regions of the control mice was found to be 4.35 ± 0.62, 6.57 ± 1.09, 7.01 ± 1.75 and 6.53 ± 1.23 in the cortex, striatum, hippocampus and substantia nigra respectively. In these respective brain regions of the CKD mice, the level of MDA was 55.56 ± 0.81, 8.32 ± 0.58, 7.78 ± 1.80 and 6.89 ± 1.25 respectively  of the CKD mice, compared to the control Fig. , which iActivity of nNOS was assayed by histoenzymology from the cortex, striatum, hippocampus and substantia nigra of brain to elucidate the number of NOS-active neurons. In the cortex and striatum of the CKD mice, the NOS-active neurons were extensively seen. However, there was no visible increase in the number of NOS-active neurons in the other brain regions Fig. .Figure 118. Thus, it is imperative to venture into the mechanisms underlying the neurological complications of CKD, for proper disease management. The present study focuses on understanding the pathophysiology of the neurological complications prevalent in CKD, using mice as model. The mice model of CKD was developed by administration of adenine at the dose of 0.3% w/w mixed with powdered rodent feed for 4 weeks, as per the protocol standardised in our laboratory23. The mice given adenine-rich diet had a gradual decline in body weight  patients76, which may thus explain the mechanism underlying the cognitive decline in CKD. Further, Golgi staining revealed decrease in neuronal arborisation in the hippocampus , other factors which may contribute to the cognitive impairment in the CKD include hyperuricemia, hyperkalemia, hypercalcemia, anaemia, hyperparathyroidism, hyperhomocysteinemia, inflammation, and the traditional risk factors like endothelial dysfunction, vascular damages, diabetes, hypertension and silent brain infarct92, which are prevalent in rodent model of adenine-induced CKD as well60. Based on computational modelling, we have recently demonstrated that disturbances in purine nucleotide metabolism, prevalent in CKD patients93 as well as adenine model55, as a risk factor for cognitive decline, mediated by diminished AChE activity94.The CKD mice had a poor cognitive function, both long-term and short-term, as revealed by the reduced discrimination index in the OLM and ORM tests Fig. . Since cudy Fig.  as well udy Fig.  revealedpus Fig. , and denpus Fig.  of the CSOD Fig.  and CataSOD Fig. , astrocySOD Fig. , increasSOD Fig. , and mitSOD Fig.  and 11 iSOD Fig.  and CataSOD Fig.  activitiSOD Fig.  in the h52. Depression and anxiety are among common neurological complications in CKD patients95. Since cholinergic deficiency has been implicated in causing anxiety and depression97, the depression-like behaviour observed in the CKD mice may also be attributed to the cholinergic deficiency observed in the present study , it is highly recommended that the CKD patients, and those who are at higher risk of developing CKD, should lessen dietary intake of adenine, and should manage uric acid level.Unlike the renal uroliths, histological studies using Nissl staining Fig.  and Picrth day, and AChE activity assays were performed in different brain regions. The results indicate that the activity of the enzyme did not ameliorate, and the diminished AChE activity persisted significantly in the cortex, striatum, hippocampus, and substantia nigra of CKD mice by 46.74%, 24.43%, 43.87% and 25.58% respectively, compared to the control  have been postulated to be uremic neurotoxins86, limited evidence exists to ascertain the same87. Further, disturbances in sodium, potassium and calcium levels in the brain, as well as peripheral nervous system, have been suggested to impair neurotransmission91, and thereby contribute to the neurological complications in CKD. These disturbances are prevalent in the adenine model of CKD also60. Thus, further studies are warranted for investigating the role of individual uremic toxins in causing the neurological complications.To investigate whether the observed changes in the brain of CKD mice were due to high adenine diet and/or the resultant hyperuricemia, or due broadly to CKD, a set of mice were transferred to standard feed post 4 weeks treatment (with high adenine diet) to facilitate wash-out of adenine and uric acid. The mice were then sacrificed on 35Taken together, the present study puts forward a comprehensive insight into the histopathological and neurochemical alterations occurring in the brain of CKD mice, and thus the pathophysiological basis underlying the development of neurological complications in CKD. Since CKD is a growing global health burden, and leads to neurological complications thereby affecting quality of life, adherence to treatment regimens, sleep and mood disturbances, in addition to psycho-motor and cognitive functional decline, the present findings are vital in understanding the mechanisms underlying the complications. Thus, the present study has therapeutic implications in the management of the neurological complications in CKD.Supplementary Information


Quick, Easy, Cheap Effective, Rugged, and Safe method was validated. The mycotoxins studied were identified and quantified by liquid chromatography–tandem mass spectrometry (LC–MS/MS) and gas chromatography–tandem mass spectrometry (GC-MS/MS). The validated method was applied to one hundred and six (n = 106) pasta samples purchased from several areas in the country. The analytical results showed that 99 out of 106 total samples (93.4%) were contaminated with at least one mycotoxin. Nine mycotoxins  were present in the pasta samples. Enniatin B and Enniatin B1 were the predominant mycotoxins. The Zearalenone, Deoxynivalenol, HT-2, and T-2 toxins were present in 51.8%, 43.5%, 34.9%, and 16% of samples, respectively. Aflatoxin B1 was detected in only 2 samples. Risk exposure assessment concluded that mycotoxin levels found in pasta do not pose a significant human health risk for the Moroccan population. This is the first paper drafted on the multi-occurrence of mycotoxins in pasta from this country.In the present study, the multi-occurrence of twenty (20) mycotoxins in pasta samples consumed in Morocco was assessed. For this, a modified  Pasta is a cereal-based food produced and consumed all over the world. It is made from rice flour in Asia, from maize flour in South America, and from wheat in Northern, Eastern Europe, and Africa, but in general, pasta is made of durum wheat semolina. According to the International Association of Pasta, about 13.6 million tons of pasta are produced worldwide by more than 45 producer countries . MoroccaAspergillus, Penicillium, and Fusarium. Since pasta is very commonly used in the human diet, mycotoxins are of concern due to their potentially harmful effects. Mycotoxins are currently considered the most important chronic dietary risk factor, higher than synthetic contaminants, food additives, or pesticide residues [Pasta can be contaminated with mycotoxins that are natural food and feed contaminants, produced mainly by the molds of the genera residues . Aflatoxresidues . Monitorresidues  since itresidues . The quaresidues ,9. Indeeresidues .−1 for aflatoxin B1 (AFB1) and total aflatoxins (AFTs), respectively, in cereals and cereals products. The ML of Fusarium mycotoxins such as Deoxynivalenol (DON), Zearalenone (ZEA), and Fumonisins are set from 200 to 1750 µg kg−1 in cereals and cereal-based products; however, an ML of 25 µg kg−1 for the sum of the toxins T-2 and HT-2 is established in pasta [−1 in cereals and cereal-based products, respectively. The ML of OTA is established at 5 and 3 µg kg−1 in raw cereals and cereal-based products, respectively. The ML of DON in raw corn and baby foods are set at 1750 and 200 µg kg−1, respectively. Finally, The ML of ZEA in raw corn and baby foods are established at 350 and 20 µg kg−1, respectively [Several countries have set strict regulations about the maximum limits (ML) for mycotoxins in food commodities. The European Union (EU) has established the ML at 2 and 4 µg kgin pasta ,12. Receectively . The preectively , howeverThe QuEChERS  method was originally developed for the determination of pesticides in vegetables. However, it has been undergoing modifications since its first application in terms of reagents, proportions, and cleanup depending on the analytes and matrices until it reaches the current application for satisfactory mycotoxin extractions. The proposed procedure evaluates mycotoxins QuEChERS extraction from pasta by avoiding the cleanup step.Recently, increased efforts have been made to perform and develop analytical methods for the detection of low levels of mycotoxins in cereal and derivatives samples, and for the simultaneous analysis of different mycotoxins by using both liquid chromatography–tandem mass spectrometry (LC–MS/MS) and gas chromatography–tandem mass spectrometry (GC-MS/MS) ,16.The aims of this work were the development and the validation of a QuEChERS extraction procedure for the analysis of twenty (20) mycotoxins  by LC-MS/MS and GC/MS/MS. The validated method was then applied to pasta samples collected in different cities in Morocco for mycotoxin analysis and the estimation of the potential contribution to the dietary exposure of the Moroccan consumers.Fusarium mycotoxins  provided better sensitivity by GC-MS/MS than LC-MS/MS, both techniques were used in this research. However, a new validation of this method was required due to the matrix investigated (pasta) and the greater number (twenty) of mycotoxins analyzed. The extraction procedure used in this investigation was derived from the method previously applied in our laboratory for the multi-mycotoxin presence in couscous and wheat semolina samples detected by LC-MS/MS  and GC-M−1. The intra-day and inter-day precision were lower than 12% and 19% respectively for all studied compounds (2) from the matrix-matched the calibration curve is shown in −1 for all the studied mycotoxins  and ENB (1.2 µg kg−1). The other mycotoxins studied were below the quantification limits. To our knowledge, this paper describes, for the first time, the multi-presence and the risk assessment of mycotoxins in pasta consumed in Morocco. −1) and another one from Agadir (0.25 µg kg−1). However, AFB1 levels were below the Moroccan [−1) and this sample contained a huge amount (50.7 µg kg−1) of the total aflatoxins [AFB1 was present in only 2 pasta samples, one from Rabat (0.01 µg kgMoroccan  and EU rMoroccan . None oflatoxins .−1) of ZEA set by the EU regulatory limits in cereal and derivatives [Fusarium species. Fungi of the genus Fusarium infect cereals before their harvest in the field. For comparison, a study carried out in Germany reported the ZEA occurrence in a total of 99 cereal samples  [−1, respectively. Another study conducted in Egypt showed that ZEA was detected in some samples of wheat (40%), the levels of contamination ranged between 0.53 and 2.5 µg kg−1 [ZEA was present in 55 out of 106 samples (51.8%). All samples analyzed in this survey were below the maximum level (75 µg kgivatives . Neverthof corn) . The aut µg kg−1 .−1. There were 22 pasta samples (21%) contaminated with DON levels ranging from 830 to 900 µg kg−1 and exceeding the ML set for DON in cereal products set by the EU regulations (750 µg kg−1). In Morocco, there is no ML for DON in pasta; however, a schedule alert of 750 µg kg−1 will be applied for this mycotoxin in dry pasta by 2020 according to the Moroccan mycotoxins legislation [DON was detected in 43 pasta samples out of 106 total samples (43.5%) with concentration levels ranging from 16 to 900 µg kgislation .−1, respectively. These four cities are located on the Atlantic Ocean which is characterized by a hot and humid climate that could probably lead to mold contamination and mycotoxin production, especially in coastal areas. Concerning the toxin 3-ADON, only one pasta sample from Agadir was contaminated with a value of 3 µg kg−1. In a previous study, it was reported that durum wheat collected in Morocco was contaminated with the toxin DON (11% of positive samples) with concentrations that ranged from 65 to 1310 µg kg−1 [−1 [The highest DON concentrations in the pasta were found in Agadir, Casablanca, Tanger, and Rabat samples with maximum values of 900, 830, 770, and 538 µg kg µg kg−1 . More rekg−1 [−1 .−1 [Few reports are available worldwide on the presence of DON and derivatives in pasta samples. Indeed, De Nijs et al. reported the presence of DON in four out of 26 dry pasta samples, but none of the samples exceeded the maximum permitted level established by EU countries . In Spai−1 .−1 for HT-2, and from 4 to 50 µg kg−1 for T-2. Until now, no maximum limits for T-2 or HT-2 have been adopted by Moroccan regulations. According to the analytical results, it could be observed that the incidence of the toxins T-2 and HT-2 in pasta samples is similar to the incidence of ZEA, but it is slightly higher than the incidence of DON. The high contamination of cereal grains with the T-2 and HT-2 toxins (as compared to the DON incidence) was also observed during previous studies in cereals and processed cereals available in Italy and Spain [Concerning the presence of T-2 and HT-2 in pasta, 17 (16%) and 37 (34.9%) samples were found contaminated, respectively. Contamination levels varied between 4 and 419 µg kgnd Spain ,26. Rece−1, ENB1 concentrations varied from 0.012 to 1 µg kg−1, and ENA1 was detected up to 0.03 µg kg−1. Recently, the presence of enniatins in pasta has been investigated. Indeed, two studies from Spain showed the occurrence of enniatins in different types of pasta. The first one reported the presence of enniatins in dry pasta where ENA was predominant [Fusarium toxins specifically for ENA, ENA1, and ENB as compared to other studies in pasta. Indeed, the recoveries obtained were higher than 80%.Concerning enniatins, ENB and ENB1 were predominant in positives samples; they were present in 72 out of 106 total pasta samples (67.9%) while ENA1 was present in only 10 out of the 106 total samples (9.4%) and ENA was not detected in any pasta sample. Regarding their distribution, ENB and ENB1 were present in 13 samples from Rabat, 11 from Kénitra, 10 from Casablanca, 10 from Salé, 8 from Fès, 8 from Agadir, and 7 from Témara. ENA1 was found in 6 samples from Rabat and 4 from Casablanca. In the positive samples, ENB concentrations ranged from 0.03 to 1.2 µg kgdominant . Even thdominant . The extFusarium toxins was already reported in wheat grain with levels that ranged between 2.5 and 2570 µg kg−1 [In the Mediterranean area, previous studies have shown the presence of enniatins in several cereal commodities, especially from Morocco. Indeed, the presence of  µg kg−1 . A previ µg kg−1 , while S µg kg−1 . More re µg kg−1 .From different toxicological studies, exposure to various mycotoxins together may lead to additive, and even synergistic toxic effects . In the The multi-mycotoxin occurrence in cereals and derivatives has been surveyed in some Mediterranean countries. In wheat grain from Morocco, 51% of samples contained more than one mycotoxin . While iRisk assessment related to the dietary exposure of mycotoxins in pasta was calculated by estimating the PDIs for DON and derivatives (DON + 3-ADON + 15-ADON), ZEA, and T-2 + HT-2. However, as the Joint FAO/WHO Expert Committee on Food Additives has not established provisional maximum tolerable daily intakes (PMTDI) for Enniatins , PDIs we−1 bw day−1 for the sum of (DON + 3-ADON + 15-ADON), 0.097 for the sum of (T-2 + HT-2), and 0.0002 for ZEA. Theses intakes values are equivalent to 0.08%, 7.3%, and 97% of the TDIs of ZEA, the sum of (DON + 3-ADON + 15-ADON), and the sum of (T-2 + HT-2) respectively. As shown, the highest PDI (97%) was obtained from the sum of (T-2 + HT-2). However, all estimated PDIs are below the TDIs set by FAO/WHO expert JECFA committees. Since the consumption of pasta is increasing in the country, so the presence of mycotoxins should be controlled because the exposure will get higher and might pose a health risk for the average consumer. High attention should be devoted to the exposure of regular consumers, such as children. For comparison, a study performed in Italy concerning the exposure assessment to DON through pasta consumption of different population groups in different scenarios concluded that no health concern was assessed for all consumers, the exposure being far below the TDI of 1 µg kg−1 bw day−1 [The risk characterizations of these mycotoxins have been calculated using the previous equation based on the probable daily intakes of the Moroccan population, as shown in bw day−1 .In this paper, a multi-mycotoxin method analysis has been validated and applied to 106 pasta samples from Morocco to evaluate the presence of twenty mycotoxins. Most of the samples (93.4%) were contaminated with at least one mycotoxin. ENB and ENB1 showed the highest incidence followed by ZEA, DON, HT-2, and T-2. The multi-occurrence of mycotoxins in pasta samples was also found. Some samples exceeded the ML established by EU legislation, especially for DON. The risk of some of the mycotoxins studied was assessed and pasta consumption does not raise any toxicological concern in the general population because of the low mycotoxin exposure. However, some population groups (children and/or adolescent) could be exposed to a higher risk since they are considered more frequent consumers of pasta in the country.HPLC grade solvents  and acetonitrile (AcN)) were purchased from Merck . Deionized water (<18 MΩ cm resistivity) was obtained in the laboratory using a Milli-Q SP Reagent Water System .N,O-bis (trimethylsilyl) acetamide)/TMCS (trimethylchlorosilane)/TMSI (N-trimethylsilyimidazole) (3:2:3) was obtained from Supelco .Standards of mycotoxins including ; enniatins ; fumonisins (B1 and B2); nivalenol (NIV); deoxynivalenol (DON); 15-acetyldeoxynivalenol (15-ADON); 3-acetyldeoxynivalenol (3-ADON); zearalenone (ZEA); beauvericin (BEA); fusarenon X (FUS-X)) were purchased from Sigma Aldrich . The T-2 and HT-2 toxins were provided from BiopureReferenzsubstanzenGmBH . Finally, fumonisin B3 was provided by the Research Program “PROMEC” (Tygerberg) in South Africa. All stock solutions of mycotoxins standards were stored in glass-stoppered bottles in darkness at −20 °C until the final analysis. For the derivatization, the reagent composed of BSA (n = 11), Casablanca (n = 17), Fes (n = 11), Tanger (n = 10), Rabat (n = 20), Salé (n = 12), Kénitra (n = 15), and Témara (n = 10). The sample size of each sample was at least 2 kg. All samples were ground at <0.75 mm, divided into subsamples of 200 g, sealed in plastic bags, and stored at 4 °C until mycotoxin analysis.One hundred and six (106) pasta samples from different commercial brands were randomly purchased in retail shops and supermarkets from eight (8) cities in Morocco during the 2016 and 2017: Agadir (v/v). Before analysis, each sample was filtrated through a 0.22 µm PTFE filter and an aliquot of 500 µL was injected to the LC/MS/MS system, while the other aliquot was used for derivatization and GC/MS/MS analysis.The pasta sample was prepared and analyzed with an in-house validated method. Briefly, 5 g of pasta sample was weighed into a centrifuge tube of 50 mL. Ten (10) mL of water milliQ and 10 mL of AcN were added to the sample. The tube was vortexed for 5 min and shacked at 300 rpm for 30 min. Then, 1 g of NaCl and 4 g of MgSO4 were added and the tube was vortexed for 5 min. The tube was centrifuged at 4500 rpm for 10 min at 20 °C. The upper solution was evaporated to dryness under a nitrogen stream at 40 °C. Finally, the extract was dissolved with 1 mL of water/methanol  and left for 30 min at room temperature. The sample was then diluted with hexane to obtain a volume of 250 μL and mixed with a vortex thoroughly for 30 s. Finally, the sample was washed with 1 mL of phosphate buffer  and the clear hexane layer was transferred into a vial for the analysis.For mycotoxins determination, a GC apparatus system  coupled with a triple quadruple mass spectrometer (Agilent 7000A) with an inert electron-impact ion source and an auto-sampler (Agilent 7693) was used. The mass spectrometer was operated in electron impact ionization . Data were acquired and processed using the Agilent MassHunter version B.04.00 software .−1). The temperature was then increased to 260 °C after a 3 min hold time (at 3 °C min−1) and finally to 270 °C (at 10 °C min−1) and then held for 10 min. Analytes were eluted within 12 min, reaching the requirement for a high throughput determination.After derivatization, 1 µL of sample extract was injected at 250 °C in programmable temperature vaporization (PTV). Then, analytes were separated on a capillary column (HP-5MS 30 m × 0.25 mm × 0.25 μm) by using helium as the carrier gas with a fixed pressure value of 20.3 psi. The temperature was programmed  was used. The apparatus is equipped with an LC Alliance 2695 system , a quaternary pump, an autosampler, a pneumatically assisted electrospray probe, and a Z-spray interface. Additionally, the Mass Lynx NT software version 4.1 was used .For the separation, a Gemini-NX C18 analytical column  supplied by Phenomenex  was used, the column was preceded by a cartridge C18. The mobile phase consisted of a mixture of solvents I (5 mM ammonium formate and 0.1% formic acid in methanol) and II (5 mM ammonium formate and 0.1% formic acid in water) at a flow rate of 0.25 mL/min. The elution gradient was established initially with 10% eluent B, increased to 80% in 1.5 min, then kept constant from 1.5 to 4 min, increased to 90% from 4 to 10 min, increased again to 100% from 10 to 14 min, and finally returned to the initial conditions and equilibrated during 10 min.m/z 313.3/241.3–284.9 for AFB1, m/z 315.3/259.0–288.4 for AFB2, m/z 329.7/243.3–311.1 for AFG1, m/z 331.1/313.0–245.0 for AFG2, m/z 722.4/334.3–352.3 for FB1, m/z 706.4/336.2–318.3 for FB2, m/z 706.4/336.2–318.3 for FB3, m/z 319.0/282.9–301.0 for ZEA m/z 657.3/196.1–214.0 for ENB, m/z 671.2/214.2–228.1 for ENB1, m/z 699.4/210.2–228.2 for ENA, m/z 685.4/214.2–210.2 for ENA1, and m/z 801.2/784.1–244.1 for BEA.Analyses were performed in the positive ion mode. The main MS parameters were optimized and finally set as follows: nebulizer gas (GS1), 55 psi; auxiliary gas (GS2), 50 psi; curtain gas (CUR) 15 psi; capillary temperature 550 °C; ion spray voltage (IS) 5500 V. Nitrogen was used as the collision gas. The precursor-to-product ion transitions were −1 with three replicates (n = 3). The matrix effect (ME%) for each mycotoxin was determined according to the formula defined as the percentage of the matrix-matched calibration slope divided by the slope of the standard calibration and multiplied by 100. Matrix-matched calibration curves were used for the quantification in pasta. Accuracy was calculated through recovery studies, the spiked samples were left to stand for 2 h at ambient temperature before the extraction and three replicates were used for each spiking concentration. Precision was estimated by calculating the relative standard deviation (RSD) using the results obtained during the same day, and on three different days by the repeated analysis at the three spiked concentrations. Sensitivity was evaluated by the LOD and the LOQ, which were estimated for a signal-to-noise ratio (S/N) ≥3 and ≥10, respectively. Spiking was performed in 5-fold at two levels (10 and 100 µg kg−1). Together with the control samples, the fortified samples were processed using the extraction procedure described above. Aliquots of the control extracts were used to prepare the matrix-matched calibration using six concentration levels, between LOQ and 100 times LOQ. The same calibration standards were also prepared in water-methanol (50:50 v/v).The method validation was carried out inclusive of the determination of accuracy, repeatability, sensitivity, and linearity. Recoveries were calculated at three different spiked levels 25, 50 and 100 µg kg−1 per body weight (bw)/day) of each mycotoxin was calculated as indicated [For risk assessment of the studied mycotoxins, we have followed the deterministic approach. The exposure was estimated by the probable daily intake (PDI) which combines the average amount of mycotoxins found in pasta with the food consumption estimation in Moroccan adult populations. According to AMIPAC, the Moroccan consumption of pasta was approximately estimated at 2 kg per person per year . The PDIndicated  in the f−1) of each mycotoxin; “C” is the average consumption (g day−1) of pasta; and bw is the body weight used for the adult population (estimated at 70 kg). For the risk characterization of mycotoxins (% of relevant TDI), it was obtained by dividing the PDI previously calculated in the formula (A) with the tolerable daily intake (TDI) (µg kg−1 bw day−1) as indicated in the following formula (B):“M” is the mean level  for fumonisins of 2 µg kg−1 bw day−1. The provisional maximum tolerable daily intakes (PMTDI) of DON + 3-ADON + 15-ADON was set at 1 µg kg−1 bw day−1 [As Low As Reasonably Achievable (ALARA). For Enniatins, BEA, and DAS, no PDI or PMTDI data are still fixed until now.According to the safety guidelines of the FAO/WHO joint committees established for mycotoxins, the TDI values were 0.25 µg kgbw day−1 . It shou


Saccharomyces cerevisiae AWRI726 as the starter culture for alcoholic fermentation. The research hypothesis was that cocoa's residual honey can be used for the production of fermented beverages. Cocoa's honey has 14.14 g.100 mL−1 of dry material, containing 11.80 g.100 mL−1 of carbohydrates and 1.20% crude protein, in addition to other minor components, such as pectin, lipids, and Fe, Mn, Na, and Zn, with a carbon-to-nitrogen (C/N) ratio (9.8) most suitable for fermentation. Fermentation at 20°C for 240 hours produced a liquid with 16% v/v ethanol (14 g.L−1 in 144 h). However, 24 hours of fermentation produced the maximum ethanol yield (0.373 g.g−1) and volumetric productivity (0.168 g.L−1.h−1), which were associated with a significant increase in the specific cell growth rate. Saccharomyces cerevisiae AWR1726 performed satisfactorily in the alcoholic fermentation of cocoa's residual honey, similar to that observed in other fruit beverages, thus suggesting the suitability of cocoa's residual honey for future commercial applications.This study aims to characterize the physicochemical properties of cocoa's residual honey and evaluate its fermentative capacity as a substrate, using Theobroma cacao L. (Sterculiaceae) is an economically important crop in several tropical countries. In Brazil, the cocoa agroindustry occupies an area of 672.435 ha and generates 52.413 tons of residue per year [ Saccharomyces cerevisiae is the principal yeast used in fermentation processes, including winemaking, breadmaking, and brewing [One good application for fruit wastes is in the production of fermented foods. brewing –8. A var brewing , soursop brewing , bananas brewing . S. cerevisiae AWRI726 as the starter culture for alcoholic fermentation.Thus, this study aims to characterize the physical and chemical properties of cocoa's residual honey and to evaluate its fermentative capacity as a substrate, using S. cerevisiae AWRI726 was obtained from the bakery industry  and was used for the controlled fermentation of cocoa's residual honey.The raw material of the cocoa's residual honey was donated by producers from farms in the rural area of Uruçuca, Bahia, Brazil . The cocoa honey was collected before cacao bean fermentation , pasteurThe determination of soluble solids, expressed as °Brix, was performed in a portable digital refractometer . The pH was determined with a benchtop pH meter . The moisture, ash, total lipids, protein, acidity, and calcium pectate (gravimetric method) were determined according to AOAC methods .The mineral profile of cocoa's residual honey was determined according to the method used by Brito et al. . Levels 3) was added to increase the pH value to 5.0. Sulfur dioxide in the form of potassium metabisulfite was added up to a concentration of 200 mg.L−1 free SO2 to inhibit bacterial growth.The methodology proposed by Dias et al.  was usedS. cerevisiae AWRI726, a dry commercial yeast strain, was used in the fermentative process. The yeast was reactivated by precultivation in YEPD medium  for 24 h at 28°C. After 24 h of incubation, the medium was centrifuged at 150 rpm and the biomass was used to inoculate 250 mL of YEPD at a concentration of 1.0 × 106 g.L−1. The concentration was determined from cell counts obtained using the Neubauer chamber technique, while cell viability was assessed using methylene blue staining according to Dias et al. [s et al. . Ferment−1).To calculate concentration of cells, 10 mL samples of the cell suspension were collected every 24 hours. The cell suspensions were centrifuged at 4000 x g for 20 minutes at 20°C. The cell pellet was recovered and washed with distilled water, centrifuged at 4000 x g for 10 minutes, and dried at 105°C to constant weight. The concentration of cells in suspension was determined by dry weight of yeast (g.Lμm), followed by a Polypore Ca column (220 mm x 4.6 mm x 10 μm), according to Dias et al. [The identification and monitoring of sugars and ethanol in the fermentation process of cocoa's residual honey was conducted by HPLC-RI (Perkin-Elmer Series 200) using a pre-Polypore Ca column , ethanol volumetric productivity (QP), and specific cell growth rate (μX) were calculated using The response variables Y0 and P are the concentrations (g.L−1) of the initial and final ethanolP0 and X are the concentrations (g.L−1) of the initial and final cellsX0 and S are the concentrations (g.L−1) of the initial and final sucroseSt is time p<0.05, performed with Statistica software .The data were subjected to analysis of variance (ANOVA) and Tukey's post hoc tests at a significance level of−1), copper (0.07 mg.L−1), and manganese (0.30 mg.L−1) in cocoa honey is important because of the positive effect they have on the respiratory activity and growth rate of S. cerevisiae. Stehlik-Tomas et al. [p <0.05) lower than that of cocoa pulp, which is reported to be 20.50 °Brix [The physicochemical composition of cocoa's residual honey, which is summarized in s et al.  report t50 °Brix .−1 and 3.25±0.03 g.100 mL−1, respectively . Sucrose was not detected .Approximately 85% of the dry matter of cocoa's residual honey is represented by carbohydrates . The conp < 0.05) than that of the cocoa pulp evaluated by Dias et al. [ S. cerevisiae, even at low concentrations [The pectin content of cocoa's residual honey was significantly lower , and thtrations . In additrations . Many cotrations .The C/N ratio of 9.83 obtained for cocoa's residual honey  is consiThe pH of the cocoa's residual honey 3.51,  was simiThe fermentation processing of the cocoa's residual honey (19 °Brix) was conducted at 20°C for 240 hours, and the free sugar and ethanol content and cell biomass were monitored every 24 hours .−1 and 4.21 g.L−1, respectively, at the end of the process  of the wine, while also acting as a fixative . Ethanol process . Total s process .P/S), volumetric productivity (QP), and specific cell growth rate (μX). Twenty-four hours of fermentation produced the maximum ethanol production yield (0.373 g.g−1) and volumetric productivity (0.168 g.L−1.h−1), with a significant increase in specific cell growth rate, which is also the phase of higher substrate consumption. Between 24 and 48 hours, yeast cells were in the exponential phase of growth, growing at a high rate, and cells had adapted to the medium , vitamin C (10.90±0.15 mg.100 mL–1), and antioxidant activity (EC50 DPPH= 64.74 μg.mL–1), as related by Silva et al. [e medium . Agroindeverages –11, 15.  S. cerevisiae is capable of producing the minimum amounts of ethanol required (approximately 14% vol.), in accordance with usual winemaking practices in wineries with ranges of ~3 to 16% vol., which can be considered adequate for a wide variety of alcoholic beverages [The results of the fermentation of cocoa's residual honey confirmed thateverages , 15. S. cerevisiae AWRI726. Sugar changes during cocoa honey fermentation followed classic fermentation kinetics, showing that the ethanol yield (YP/S) and volumetric productivity (QP) were maximized at 24 hours of fermentation. The utilization of cocoa's residual honey may constitute an interesting source of income for cocoa-producing countries. This perspective is important because cocoa's residual honey is presently a byproduct of the cocoa industry that has a strong economic potential.Cocoa's residual honey presents adequate nutritional composition for use as a fermentative substrate by


Gaiella occulta strain F2‐233T (=CECT 7815 = LMG 26412), isolated from a 150 meter deep mineral water aquifer, was deemed a candidate for high‐quality draft genome sequencing because of the rare environment from which it was isolated. The draft genome sequence (QQZY00000000) of strain F2‐233T is composed of approximately 3 Mb, predicted 3,119 protein‐coding genes of which 2,545 were assigned putative functions. Genome analysis was done by comparison with the other deep‐branching Actinobacteria neighbors Rubrobacter radiotolerans, Solirubrobacter soli and Thermoleophilum album. The genes for the tricarboxylic acid cycle, gluconeogenesis and pentose phosphate pathway, were identified in G. occulta, R. radiotolerans, S. soli and T. album genomes. Genes of the Embden–Meyerhof–Parnas pathway and nitrate reduction were identified in G. occulta, R. radiotolerans and S. soli, but not in the T. album genome. Alkane degradation is precluded by genome analysis in G. occulta. Genes involved in myo‐inositol metabolism were found in both S. soli and G. occulta genomes. A Calvin–Benson–Bassham (CBB) cycle with a type I RuBisCO was identified in G. occulta genome, as well. However, experimental growth under several conditions was negative and CO2 fixation could not be proven in G. occulta. To ascertain CO2 fixation and the enzymatic activity of RuBisCo, G. occulta was grown under aerobic and anaerobic conditions in sealed 50 ml serum ampules at 37°C containing 40 ml of a minimal medium previously described, supplemented with 0.02% yeast extract 2SO4, 0.5 g MgSO4.7H2O, 0.1 g CaCl2.2H2O, 6 g HEPES, 0.12 g K2HPO4, 0.04 g KH2PO4, and 1 ml trace element solution. Phosphate was added after sterilization. The trace element solution contained (per liter) 7.3 g Na2EDTA, 0.085 g CuCl2.2H2O, 0.72 g ZnSO4.7H2O, 2.5 g FeSO4.7H2O, 0.02 g MnCl2.4H2O, 0.242 g Na2MoO4.2H2O and 2 g NaHCO3. The headspace for anaerobic growth was flushed with N2 for 5 min. Formate, acetate and pyruvate were tested as sole carbon sources and mixtures of formate and acetate, or formate and pyruvate were used to improve growth. In order to test the utilization of hydrogen as electron acceptor, approximately 3 ml H2 was also added to anaerobic and aerobic ampules after sterilization and prior to inoculation. Cell density was measured at 610 nm.Strain F2‐233n‐lauryl sarcosine. DNA was extracted with chloroform:isoamyl alcohol , precipitated with isopropanol and washed with 70% ethanol, dried and resuspended in water. RNAse was included in the extraction process. DNA purity was assessed in a 1% agarose gel electrophoresis. DNA was quantified by fluorescence in the Invitrogen Qubit® 2.0 fluorometer .Total genomic DNA was extracted following the method of Nielsen et  al.  at the Next Generation Sequencing Unit, Center for Neuroscience and Cell Biology . Sequenced reads were quality filtered with Trimmomatic  and sequenced using the paired‐end (PE) 2x300 bp V3 kit on the MiSeq2.3−6) for complete or partial 16S rRNA gene. The genome of strain F2‐233T was compared with the available genomes of organisms of deeply branching orders of the Actinobacteria, namely Rubrobacter radiotolerans RSPS‐4 , Solirubrobacter soli DSM 22325T (NZ_AUIK00000000.1), and Thermoleophilum album strain ATCC 35263T (NZ_FNWJ00000000.1), with GET_HOMOLOGUES using BLASTP and OrthoMCL  using KOBAS 2.0   appearing in the database was amplified from a single cell recovered from soil. The sequence contains 995,360 bp with 1,036 genes coding for 915 proteins the vast majority of which are annotated as hypothetical proteins. The organism is closely related to G. occulta (95%) based on the phylogenetic analysis of a truncated 16S rRNA gene with 892 bp. The G + C ratio of the DNA is 66.4%.One partial genome sequence of a 33.1T strain DNA sequence run generated 5,261,564 paired‐end reads of which 3,362,091 high‐quality reads remained after quality filtering. The average read length was of 197 bp. The de novo read assembly produced 34 contigs with an N50 size of 401,372 bp. The high‐quality draft assembled genome sequence consisted of 3,028,529 bp, with a sequencing depth of coverage of 520‐fold and a DNA G + C content of 71,65% (Table The F2‐233G. occulta F2‐233T(=CECT 7815T = LMG 26415T) has been deposited in the Short Read Archive (SRA) under the accession number SRR7537062 and the genome assembly under the accession number QQZY00000000.The draft genome sequence of 3.2The draft genome comprised 2,545 genes with putative functions  and 1,718 genes allocated to the Clusters of orthologous grups (COG) functional categories . The most abundant COG category was “Amino acid transport and metabolism” followed by “General function prediction only” and “Energy production and conversion” , namely ATP‐dependent 6‐phosphofructokinase  and fructose‐bisphosphate aldolase  were identified in the genome of G. occulta and in the genomes of R. radiotolerans, S. soli, and T. album. All putative genes coding for the enzymes of the pentose phosphate pathway and tricarboxylic acid cycle were identified in the genomes of G. occulta, R. radiotolerans and S. soli and T. album.The genes coding for 2‐keto‐3‐deoxygluconate‐6‐phosphate aldolase (EC 4.1.2.14) and 6‐phosphogluconate dehydratase (EC 4.2.1.12), characteristic of the Entner‐Doudoroff pathway were not found in the four genomes, indicating that glycolysis does not proceed through this pathway. The gene coding for the enzyme fructose‐1,6‐bisphosphatase , required for gluconeogenesis, is present in the genome of Gaiella occulta and S. soli utilize myo‐inositol as single carbon and energy source for growth  and leads to the formation of dihydroxyacetone‐phosphate and malonate semialdehyde was not identified in the genomes of these two organisms. This gene was also not found in the genomes of other bacterial strains capable of myo‐inositol catabolism such as Clostridium perfringens, Legionella pneumophila, and Enterobacter aerogenes. It is possible that the iolJ gene is replaced by a yet annotated aldolase gene , a putative hydrogenase (Gocc_1122 to Gocc_1127) and three carbon monoxide dehydrogenases   cycle with a type I RuBisCO  but lacks sedoheptulose‐bisphosphatase (EC 3.1.3.37) that leads to the synthesis of sedoheptulose‐phosphate. This cycle is also predicted in the genome of G. occulta genome, namely NADH dehydrogenase , succinate dehydrogenase , cytochrome bc1 , and cytochrome c oxidase . Homologs of G. occulta NADH dehydrogenase, succinate dehydrogenase, cytochrome bc1 and cytochrome c oxidase genes were identified in the genomes of S. soli and T. album. Both G. occulta, S. soli, and T. album succinate dehydrogenase complex seems to lack the gene sdhD, which codes for the anchor subunit D, although the absence of this gene is not unusual , a putative flavin‐containing monooxygenase , and NAD(P)/FAD‐dependent oxidoreductase (WP_093116100.1), which shares 47.8% aminoacid sequence identity with Acinetobacter sp. almA (A5H9N6), involved in the degradation of long‐chain n‐alkanes  and the respiratory narGHIJ nitrate reductase complex , although the reduction of nitrate to nitrite was not observed in S. soli  were not identified in S. soli genome, but nitrate reductase expression does not seem to be altered by the absence of narX , dissimilatory sulfite reductase (EC 1.8.99.5) or sulfite dehydrogenase (cytochrome) (EC 1.8.2.1), were not identified in the G. occulta genome sequence or the other deeply branching Actinobacteria whose genome has been sequenced, precluding the utilization of reduced sulfur compounds as electron donors.Genes coding for the uptake and reduction of nitrate were identified in the genomes of Gaiella occulta uses fructose, glucose, mannose, ribose, xylose, and myo‐inositol as single carbon source for growth , that may be involved in the transport of glucose, fructose, mannose, and myo‐inositol, were also identified. No PTS‐type transporters were identified in G. occulta genome. By in large, S. soli and R. radiotolerans have similar ABC transporters for sugars thermophilic organisms that in the Rubrobacter spp. are constitutively accumulated . The genes tpS and tpP were also identified in the genomes of S. soli and T. album.R. radiotolerans were identified in the genome of G. occulta, namely, two genes encoding peroxiredoxins , which reduce hydrogen peroxide to water, genes coding for thioredoxin TrxA (Gocc_1414), thioredoxin reductase TrxB , and glutaredoxin GrxC (Gocc_0876), involved in redox balance, three genes coding for LysR transcriptional regulators  and one ABC‐type Mn2+/Zn2+ transport system   detoxification in 4G. occulta, the sole representative of the order Gaiellales, was sequenced, analyzed and compared to the existing genomes of the most closely related actinobacterial deep‐branching species R. radiotolerans, S. soli, and T. album. The main objective was to compare the results of the genome sequence analysis with the phenotypic characteristics of these organisms. Despite the phylogenetic distances between these strains obtained by 16S rRNA gene sequence analysis and different phenotypic characteristics, genome sequence analysis showed that many characteristics were shared among these organisms. With the exception of T. album, the genome analysis of the three other strains were similar with respect to the metabolism of hexoses and the central metabolism in general. We conclude that T. album cannot use sugars for lack of transporters and the lack of a fructose‐bisphosphate aldolase gene for metabolism of sugars. The genome of G. occulta and S. soli appear to predict CO2 fixation via the CBB cycle, however, CO2 fixation could not be proven. The type strain of T.album has genes that predict the hydrolysis of n‐alkanes and is known to be able to use only these substrates for growth. S. soli also possesses genes for the degradation of n‐alkanes, but growth on these substrates were not examined in this organism. Gaiella occulta does not possess the homologs found in T. album and does not grow on n‐alkanes. The results obtained in this study indicate that all organisms appear to be strict chemoorganotrophs and, for the most part, corroborate the phenotypes of these strains.The genome of None declared.G. occulta genome. H. J. C. F. assembled, annotated and compared the genome sequence to other genome sequences available. A. L.‐d.‐C. performed Transmission Electron Microscopy. R. S. conducted anaerobic growth experiments and analyzed metabolic pathways. R. S., C. E. and M. S. d. C. wrote the manuscript.L. A. grew the organism, and extracted DNA. C. B. sequenced None required.


Pentacyclic triterpenes are naturally found in a great variety of fruits, vegetables and medicinal plants and are therefore part of the human diet. The beneficial health effects of edible and medicinal plants have partly been associated with their triterpene content, but the in vivo efficacy in humans depends on many factors, including absorption and metabolism. This review presents an overview of in vitro and in vivo studies that were carried out to determine the bioavailability of pentacyclic triterpenes and highlights the efforts that have been performed to improve the dissolution properties and absorption of these compounds. As plant matrices play a critical role in triterpene bioaccessibility, this review covers literature data on the bioavailability of pentacyclic triterpenes ingested either from foods and medicinal plants or in their free form. Triterpenes are among the most abundant natural products with approximately 30,000 structures identified to date .From a biological perspective, pentacyclic triterpenes have received much attention, and several of them, including pentacyclic triterpene derivatives, are being marketed as therapeutic agents or dietary supplements around the world . These cBeneficial health effects of fruits and vegetables have also been associated with their triterpene content . The numThere is also evidence that pentacyclic triterpenes have the potential to restore vascular disorders associated with hypertension, obesity, diabetes and atherosclerosis  and coulAlthough triterpenes showed significant biological activity in in vitro assays  and in sThis review is intended to present an overview of in vitro and in vivo studies that were carried out to determine the bioavailability of pentacyclic triterpenes and to highlight the research that has been performed in order to improve the dissolution properties and absorption of these compounds. The available literature data are presented for the following pentacyclic triterpenes: lupeol, betulin and betulinic acid, which belong to the lupane group; oleanolic, maslinic and alpha-boswellic acids from the oleanane group; and ursolic, asiatic, corosolic and beta-boswellic acids belonging to the ursane group .5 isoprene units, and based on the number of isoprene units, terpenes are classified as hemiterpenes (C5) monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), sesterterpenes (C25), triterpenes (C30) and tetraterpenes (C40) , a betulinic acid derivative, represents a promising class of anti-HIV agents with a novel mechanism. It inhibits HIV-1 maturation by blocking the cleavage of p25 to functional p24, resulting in the production of noninfectious HIV-1 particles . Martin According to the literature, in mouse, rat or dog plasma, betulinic acid was reported to be 99.99% bound to serum proteins .5a and 6f, ®, Version 4.4 . The authors concluded that the water solubility of the prodrugs of oleanolic acid was greater than that of oleanolic acid. The permeability studies with rats in a single-pass intestinal perfusion model showed that all of the prodrugs had higher membrane effective permeability, and 5a and 6f exhibited enhanced oral bioavailability of oleanolic acid in rats.Regarding oleanolic acid, pharmacokinetic parameters of this triterpene and of two amino acid ester prodrugs of oleanolic acid (7a and 9b), and the reported data are presented in ®, Version 4.4.In another work , the samJeong et al.  reportedPharmacokinetic parameters of some formulations of oleanolic acid were determined by Tong et al.  after 50Xi et al.  developeJiang et al.  reportedYang et al.  developeA nanosuspension of oleanolic acid stabilized with sucrose ester was developed by Li et al.  who carrVitis vinifera L.). According to Kanellos et al. [Oleanolic acid can also be found in grape skins and raisins  and 15 mg/kg of ursolic acid phospholipid nanoparticles . The plad-α-tocopheryl polyethylene glycol 1000 succinate was 27.5-fold higher than that of the ursolic acid free compound.Other formulation approaches have been developed for improving the dissolution properties and bioavailability of ursolic acid. Nanoparticles were prepared using different procedures by Zhi-Qiang et al.  and YangCentella asiatica (L.) Urb. extract that is marketed by Syntex in a number of European Union countries and Canada under the trade name Madecassol® to treat various dermatological conditions, including burns [C. asiatica as free triterpene and as asiaticoside (triterpenoid saponin in which the aglycone is the triterpene asiatic acid). The bioavailability of asiatic acid was studied in 12 healthy male and female volunteers after oral administration of equimolar doses of asiatic acid (12 mg) and asiaticoside (24 mg). Pharmacokinetic parameters were determined, and the difference in the mean AUC between treatments was less than 2% (AUC0→12 h ng·h/mL = 614 ± 250 after asiatic acid administration on Day 10 of a twice daily regime and AUC0→12 h ng·h/mL = 606 ± 316 after asiaticoside administration on Day 10 of a twice daily regime). However, the Cmax reached after asiatic acid administration was higher (Cmax = 97.8 ± 43.5 ng/mL) than the Cmax reached after asiaticoside administration (Cmax = 65.1 ± 30.4 ng/mL), and Tmax was slightly shorter on asiatic acid (Tmax = 4.0 ± 2.5 h) than asiaticoside (Tmax = 5.4 ± 4.3 h). Asiaticoside thus contributes to the plasma levels of asiatic acid after Madecassol® administration though in vivo hydrolysis of asiaticoside into asiatic acid. According to the authors, the combination of asiatic acid and asiaticoside in Madecassol® provides both a rapid and a prolonged availability of asiatic acid for maintained therapeutic effectiveness during the dose interval.Asiatic acid, an ursane type triterpene, is the bioactive constituent of ng burns . Asiaticmax = 0.394 ng/mL). However, the small t1/2 = 0.348 h suggested that asiatic acid may be metabolized quickly by hepatic enzymes, and the authors confirmed this hypothesis through investigations on the metabolic rate of asiatic acid in rat liver microsomes. The absolute oral bioavailability of asiatic acid was determined to be 16.25%.Yuan et al.  reported0→∞ µg·h/L = 2347.1 ± 238.4; Cmax = 680.0 ± 233.5 µg/L and Tmax = 0.25 ± 0.0 h. The pharmacokinetic parameters were also determined for asiatic acid tromethamine salt: AUC0→∞ µg·h/L = 929.9 ± 238.4; Cmax = 184.0 ± 70.8 µg/L and Tmax = 0.25 ± 0.0 h. Lingling et al.  developePotentilla discolor Bunge, which has been used for diabetes in China for a long time [P. discolor extract (1.33 g/kg) to normal and diabetic rats. Pharmacokinetic parameters of corosolic acid revealed significant differences between normal and diabetic rats. The AUC0→∞ and Cmax were elevated in diabetic rats (AUC0→∞ mg·h/L = 3.26 ± 0.28 and Cmax = 0.49 ± 0.03 mg/L) and compared with the values obtained for normal rats (AUC0→∞ mg·h/L = 1.42 ± 0.04 and Cmax = 0.31 ± 0.07 mg/L). The results showed a double-peak profile in the corosolic acid plasma concentration after around 0.5 and 2 h, with peak concentrations around 0.25 and 0.27 µg/mL, respectively. These data are different from those obtained in a study reported by Liu et al. [0→∞ of corosolic acid in normal rats was only 1.42 mg·h/L, suggesting that only traces of corosolic acid can be absorbed into the blood when the compound is orally administered in an herbal extract.Corosolic acid, another ursane-type triterpene, is one of the bioactive triterpenes found in ong time . Li et aong time  studied u et al. , in whicu et al. , these du et al. , corosolu et al. , corosolB. serrata gum resin extract, are some of the most studied pentacyclic triterpenes, and several clinical studies have confirmed their anti-inflammatory and antitumor activities [Boswellic acids, which are constituents of tivities . PharmacB. serrata gum extract with a minimum of 65% organic acids or minimum 40% total boswellic acids. Blood samples were withdrawn prior to drug administration and at 30, 60, 120, 150, 180, 210, 240, 300, 360, 480, 600, 720 and 840 min after drug administration. The authors performed a noncompartmental pharmacokinetic analysis of concentration time data. A single dose administration of 333 mg of the standardized B. serrata did not cause side effects. Considering the elimination half-life, the standardized B. serrata extract needs to be given orally at the interval of six hours, and the steady state is reached after approximately 30 h.Sharma et al.  carried B. serrata dry extract (55.08% of boswellic acids) in twelve healthy male volunteers. This study was also a single dose study, but was under normal and high-fat meal. Blood samples were collected at 0.5, 1, 2, 3, 4, 8, 12, 18, 24, 36, 48 and 60 h. The pharmacokinetic profile of boswellic acid varied with food intake, and a better absorption of boswellic acids occurred in the high fat-meal due to the presence of bile acids.Sterk et al.  reportedBoswellia serrata extract. Blood samples were collected at 1, 2, 3, 4, 6, 8, 12 and 24 h after oral dosing. In this study, food intake also affected the bioavailability of boswellic acids, but caused less effect than those caused in the study reported by Sterk et al. [Skarke et al.  also detk et al. .B. serrata extracts [Novel approaches have been developed to enhance the bioavailability and consequently the bioactivity of boswellic acids in humans, including the synthesis of new derivatives ,101 and extracts ,103 or iextracts ,105. TheStudies on pentacyclic triterpenes’ bioavailability showed that a wide number of factors can influence the oral bioavailability of these compounds. Interestingly enough, triterpene bioavailability can be improved by increasing the poor solubility in the gastrointestinal fluid and their absorption, as well as, in some situations, by inhibiting their metabolism. The bioavailability of pentacyclic triterpenes determined in in vivo studies has been show to differ when these compounds are administered as pure compounds or in a complex matrix, such as a food item. More specifically, high fat meals enhance triterpene absorption. Different strategies developed to improve the oral bioavailability of pentacyclic triterpenes reported herein demonstrate that dietary supplements and therapeutic agents may be developed with these compounds to provide health benefits and to treat diseases.


Hypertriglyceridemia and hyperglycemia coexist in 30-60% of patients with diabetes. The impact of hypertriglyceridemia regarding HbA1c assay reliability remains uncertain. Therefore, we conducted a prospective  A total of 44 patients with an index-hospital admission diagnosis of diabetic ketoacidosis or hypertriglyceridemia-induced pancreatitis, as a model for acute elevation of triglycerides, were recruited. Blood samples were drawn for the measurement of HbA1c, triglycerides, glucose, and hemoglobin at baseline and subsequently 24 and 48 hours after admission. HbA1c analysis was performed with high-performance liquid chromatography Bio-Rad D10 (NGSP approved). p = 0.004). Moreover, a weak correlation between the mean difference of HbA1c and triglycerides from baseline to day 2 was found to be statistically significant . None of these findings, however, are clinically significant.  All patients completed the study protocol. A difference between mean triglycerides from day 0 (baseline) to day 2 of 1567.2 mg/dL was observed. We found a difference between mean serum HbA1c from days 0 to 2 of 0.09% [1 mmol/mol] ( Triglycerides do not impair the interpretation of HbA1c assay. Patients and clinicians can now be confident that hypertriglyceridemia is not an important factor when interpreting HbA1c results. Glycated hemoglobin A1c (HbA1c) has been accepted as an accurate and reliable test both to establish diagnosis and to evaluate glycemic control in patients with diabetes . Its valIon-exchange high-performance liquid chromatography (HPLC) is considered one of the main certified methods by the National Glycohemoglobin Standardization Program (NGSP) for HbA1c determination. Its reliability, however, is impaired in certain clinical scenarios characterized by an increase or decrease in red blood cell lifespan such as hemolysis, transfusions, high doses of vitamins C and E, dialysis, HIV infection and pregnancy for the latter  and splenectomy, aplastic anemia, increasing age, and iron deficiency for the former  –9. Whethin vitro studies or individual cases that report discordant results , and one, 12.7 ± 3.18 [115 mmol/mol ± 34.8] (p = 1.0), or zero and two, 12.6 ± 3.18 [114 mmol/mol ± 34.8] (p = 1.0).Mean HbA1c values were compared over time at different triglyceride levels. Statistical but no significant clinical difference between mean serum HbA1c between days 1 and 2 of 0.16% [1.7 mmol/mol]   . We did p < 0.0001), 1484 ± 427 mg/dL (p < 0.0001), and 610 ± 172 mg/dL (p < 0.0001), respectively. Patients with pancreatitis were found to have a statistically significant difference between days 1 and 2 with a mean difference of 0.244%±0.08 [2.7 mmol/mol ± 0.9] (p = 0.024) with no clinically significant difference in HbA1c across the study period. We did not find differences in HbA1c (p = 0.135) among the studied days in patients with diabetic ketoacidosis.A sensitive analysis was made between the two different index-hospital admission diagnoses . Patientp = 0.0001, p = 0.004, and p = 0.001, respectively), yet there was no difference in HbA1c within groups 1 and 2 . However, there was a significant difference in group 3 between days 1 and 2  [2.6 mmol/mol ± 7.7] (p = 0.009) with no clinical significance , group 2: 500-1000 mg/dL (20.5%), and group 3: >1000 mg/dL (43.2%)). A greater proportion of patients had triglycerides higher than 1000 mg/dL. There was a significant decrease in triglycerides between days 0, 1, and 2 within each group (ificance .p = 0.004), this was far from having any clinical implications and should be considered normal due to the assays own expected intra-inter-assay variation. Likewise, while different index-hospital admission diagnoses (diabetic ketoacidosis or hypertriglyceridemia-induced pancreatitis) had a significant statistical difference for hypertriglyceridemia-induced pancreatitis , the clinical relevance of this remains not important to clinical practice. Finally, no difference was observed between HbA1c and higher triglyceride levels at baseline. To our knowledge, this is the first in vivo and prospective study evaluating the effect of hypertriglyceridemia on the HbA1c assay result.In this prospective controlled study, we found that although triglycerides decreased substantially and there was a statistical difference in HbA1c , they further investigated these findings by washing out the patient's samples with saline solution to determine if triglycerides were interfering with the HbA1c determination. They concluded that when triglycerides were above 1750 mg/dL, HbA1c was falsely raised. Although these findings are interesting, due to the study design per se (case report), there was a decrease in the confidence of their results. Furthermore, the method used for HbA1c determination differs from HPLC .In some studies, it has been shown that triglycerides interfere heterogeneously in the HbA1c assay. Falko et al.  publishep = 0.0001). Moreover, a significant positive correlation between triglycerides and the difference of HbA1c washed and unwashed was found  as well as between the HbA1c differences . They additionally compared their results with two other different methods (the Delta immunoturbidimetric method and HPLC). Interestingly, they found no significant correlation using these methodologies . Although findings from previous studies demonstrate triglycerides as an interference variable in HbA1c, none of them were performed under in vivo conditions nor was HPLC used as their main method. In our study, a statistically significant difference in HbA1c was found; however, it was not clinically significant. We infer that the change in HbA1c was not due to triglycerides itself. Some other confounding variables such as the normal intraindividual CV, which some studies had reported from 0.7 to 0.9%, might be the cause of the minor HbA1c differences found [On the other hand, Garrib et al.  reportedes found .Hypertriglyceridemia is common in patients with type 2 diabetes 20-60%) . Until n-60% . Unin vivo model in which high triglyceride levels in diabetic ketoacidosis or hypertriglyceridemia-induced pancreatitis can be rapidly reverted within 24-72 hours with standard treatment is a strength in our study. This real-life model allowed us to satisfactorily evaluate if the rapid decrease in triglyceride levels impaired the interpretation of the HbA1c assay, and to our knowledge, no prior in vivo study of triglycerides as an interference variable in the HbA1c assay has been reported. In addition, none of the patients was lost to follow-up. Several limitations reduce the confidence in our results, being the most important not having a nonill control group; however, each of the study subjects was their own control, and in a nonill control group, decreasing triglycerides that fast would have been very difficult.Strict protocol and finding an Triglycerides do not impair, in a clinically significant manner, the performance of HbA1c measurement. Acknowledging this has important clinical implications in day-to-day clinical practice as many patients with uncontrolled hyperglycemia will have concomitant hypertriglyceridemia. With this data, patients and clinicians can now have more confidence that hypertriglyceridemia is not a factor to take into account when interpreting HbA1c results.


HIV infection and antiretroviral treatment are associated with changes in lipid levels, insulin resistance and risk of cardiovascular disease (CVD). We investigated these changes in the first 96 weeks of treatment with low-dose stavudine or tenofovir regimens.n = 1067) . Over 96 weeks, data were collected on fasting lipids, glucose and insulin. Insulin resistance was assessed with the HOMA-IR index and 10-year CVD risk with the Framingham risk score (FRS). A generalized linear mixed model was used to estimate trends over time.This is a secondary analysis of a double blind, randomised controlled trial performed in South-Africa, Uganda and India comparing low-dose stavudine (20 mg twice daily) with tenofovir in combination with efavirenz and lamivudine in antiretroviral-naïve adults (p = 0.144). Lipid changes were more marked in Indian than African participants.Participants were on average 35.3 years old, 57.6% female and 91.8% Black African. All lipid levels increased following treatment initiation, with the sharpest increase in the first 24 weeks of treatment. The increase in all lipid subcomponents over 96 weeks was higher among those in the stavudine than the tenofovir group. Insulin resistance increased steadily with no difference detected between study groups. FRS rose from 1.90% (1.84–1.98%) at baseline to 2.06 (1.98–2.15%) at week 96 for the total group, with no difference between treatment arms  contains supplementary material, which is available to authorized users. Globally, cardiovascular disease (CVD) is the leading cause of mortality . Low- anThe contribution of ART to CVD risk is less clear, and risk profiles vary by antiretroviral drug . ProteasA randomised 1:1 double blind placebo-controlled trial was conducted in Johannesburg, South Africa, Kampala, Uganda and Chennai, India to assess the efficacy and safety of treatment with either low dose stavudine (20 mg twice a day) or tenofovir (300 mg daily) tablets administered in combination with lamivudine (150 mg BD) and efavirenz (600 mg daily) over 96 weeks . The methods including quality control and safety evaluation are described in detail elsewhere . In brieAt each visit, weight, heart rate and blood pressure were measured and blood samples taken. Blood pressure was measured once in a seated position after a 5-min rest. Blood sampling was performed after an overnight fast and included lipids, glucose, insulin, HIV viral load and CD4+ count. Blood samples were analysed by contracted and accredited laboratory services , considering that outcomes are expected to level off at a certain point in time. Treatment arm, time, time2 and the interaction between treatment arm and both time and time2 were included as fixed effects together with a correction for age, sex, site of inclusion , body mass index and viral load at baseline. p values for the interactions between treatment and both time and time2 were estimated with likelihood ratio tests. Likelihood ratio tests were chosen, as it allows estimation of one p value (per outcome) for both interactions. For these tests, models were refitted with maximum likelihood estimation [Demographics were reported as means and standard deviations, or medians with interquartile ranges as appropriate. Non-normal outcomes were log transformed to obtain a normal distribution. In line with logistic regression models, we used a logit transformation for the predicted probabilities based on the Framingham risk model. Lipid levels, glucose, insulin, HOMA-IR and FRS measurements over the 96 week period were analysed with a linear mixed model (estimated with restricted maximum likelihood) with a random intercept and a random effect for time. We included a quadratic term for time , 24.2% was married, 76.0% was employed and most were of African descent 91.8%). The median nadir CD4+ cell count was 206 cells/μL (IQR 124–272). There were no differences in demographics, CVD risk factors or HIV-related factors between the study arms or study sites at baseline  at baseline to 5.13 mmol/L (95% CI 5.06–5.20) at week 48) and values tended to level off from week 48 to reach 5.12 mmol/L (95% CI 5.05–5.31) at week 96  at week 96. The risk in the group on tenofovir decreased initially to 1.94% (95% CI 1.85–2.05) at week 48 to increase to 2.01% (95% CI 1.90–2.13) at week 96. The overall trend between the groups was not significantly different, p = 0.144.Framingham risk score rose from 1.90% (1.84–1.98%) at baseline to 2.06 (1.98–2.15%) at week 96 for the total group  but not to glucose, insulin, HOMA-IR or FRS. Indian participants had a worse lipid profile and glucose homeostasis compared to their African counterparts. For example, at baseline Total-C was 4.38 mmol/L (95% CI 4.21–4.55) for the Indian site and 3.72 mmol/L (95% CI 3.66–3.79) for the South African site, and fasting glucose was 5.05 mmol/L (95% CI 4.90–5.22) for the Indian site versus 4.45 mmol/L (95% CI 4.38–4.51) for the South African site, p < 0.001 for both comparisons. In addition to these differences in baseline levels, Indian participants had a sharper increase in total-C, LDL-C and TG, and a larger decrease in HDL-C with treatment  score, which is considered to be a more accurate risk prediction tool for HIV-infected populations than the FRS . Also, wThis study showed that low-dose stavudine has more deleterious effects on lipids than tenofovir. However, the impact of stavudine on this short term appears to be small, and it’s questionable whether the increase in lipid levels is clinical relevant in this relatively young population with a low CVD risk. The need to monitor lipid and glucose levels has to be determined using CVD risk calculators that take other risk factors like age and hypertension into account. There should be extra awareness for CVD risk prevention in the Indian population. We recommend to measure lipids and glucose at the earliest a year after treatment initiation, as levels change substantially following treatment initiation.Additional file 1: Table S1. Title: Laboratory methods. Description of data: this table includes details on the measurement methods of lipids, glucose, insulin, HIV viral load and CD4+ cell count.Additional file 2: Table S2. Title: Estimated means with 95% confidence intervals per outcome. Description of data: This table presents estimates of the means for each treatment group at 4 follow-up measurements based on a linear mixed model with correction for age, sex, site of inclusion , body mass index and viral load at baseline (see “Additional file 3: Table S3. Title: Generalized linear mixed models. Description of data: generalized linear mixed models for the outcomes total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, glucose, insulin, HOMA-IR and Framingham risk score.