Blooms of the toxic dinoflagellate Karenia brevis are common on the West Florida Shelf (WFS), yet little is known of the relationships between zooplankton and K. brevis. A comprehensive analysis was undertaken to examine 1) perturbations in zooplankton community composition within K. brevis blooms 2) the contribution of zooplankton ammonium and phosphate excretion to K. brevis bloom nutrient requirements, and 3) the role of zooplankton grazing in K. brevis bloom termination. Prior to undertaking the first portion of the study, an examination of the perturbations in the normal zooplankton assemblage within K. brevis blooms, it was first necessary to define the normal zooplankton assemblage on the WFS. To this end, a seasonal analysis of abundance, biomass and community composition of zooplankton was undertaken at 6 stations on the WFS. Monthly sampling was conducted for one year at the 5, 25 and 50- m isobaths. Two major groups in community composition were observed at the near shore (5-m and 25-m) and offshore (50-m) stations. Considerable overlap was seen in community composition between the 5-m to 25-m and 25-m to 50-m isobaths, but little overlap in community composition was observed between the 5-m and 50-m isobaths. Of the 95 species identified, only 25 proved to be important (>90%) contributors to community composition. Near shore, important contributors were Parvocalanus crassirostris, Penilia avirostris, Paracalanus quasimodo, Oithona colcarva, Oikopleura dioica, Centropages velificatus and Pelecypod larvae. As distance offshore increased, important contributors to community composition were Euchonchoichiea chierchiae, Clausocalanus furcatus, Oithona plumifera, Oithona frigida, Oncaea mediteranea, Calaocalanus pavoninius, Oithona similis, and Gastropod larvae. Variations in abundance and biomass between non-bloom and bloom assemblages were evident, including the reduction in abundance of 3 key species within K. brevis blooms. One potential source of nutrients to support K. brevis blooms may be zooplankton regeneration of nutrients. To test this hypothesis, ammonium and phosphate excretion rates of several West Florida Shelf copepods (Labidocera aestiva, Acartia tonsa, Temora turbinata, and Paracalanus quasimodo) were measured and prorated to a 24-hour day. These excretion rates were then extrapolated to other West Florida Shelf zooplankton, combined with available literature excretion rates for some taxa, and applied to zooplankton abundances found for K. brevis blooms on the West Florida Shelf in 1999 and 2001. Ammonium excretion rates were found to be inadequate to support all but 104 cells l-1 of K. brevis, though phosphate excretion rates were adequate to support even 106 cells l-1 of K. brevis. Grazing assessment was conducted for three common zooplankton species that were found within two K. brevis blooms, A. tonsa, P. quasimodo, and L. aestiva, using 14C labeled K. brevis cells. Grazing rates were then applied to the zooplankton community and grazing assessed. Grazing pressure was occasionally heavy, and was capable of reducing K. brevis to background concentrations at stations in the 1999 bloom and at 1 station in the 2001 bloom. Generally, however, grazing pressure proved to be insufficient to reduce K. brevis to background concentrations during the 1999 and 2001 blooms.

The general aim of this thesis was to improve the understanding of the in vivo pharmacokinetics and, in particular, the metabolism of finasteride, a 5α-reductase inhibitor used in the treatment of enlarged prostate glands and male pattern baldness. CYP3A4 has been identified as the major enzyme involved in the sequential metabolism of finasteride to ω-OH finasteride (M1) and ω-COOH finasteride (M3). The consequences of induced and inhibited metabolism on the pharmacokinetics of finasteride and its metabolites were investigated in humans and pigs. Both studies included bile collection. The collected human and pig samples were used for the metabolite identification. As expected, induced metabolism led to reduced plasma exposure of finasteride and inhibited metabolism had the opposite effect. The interactions were investigated in detail and included examination of the biliary pharmacokinetics of finasteride and its metabolites. In pigs, the study included monitoring of the hepatic extraction over time, deconvolution and the development of a semi-physiological model for comparison of the effects on the gut wall and liver metabolism. For M3, the concentration ratios of bile to plasma and the renal clearance indicated that carrier-mediated processes are involved in the biliary and urinary excretion. This was not, however, the case for finasteride. The metabolite, M1, could not be quantified either in humans or pigs. Instead, two other OH metabolites, M1 isomers, were identified in humans. These metabolites were found to undergo glucuronide conjugation. In humans, one glucuronide was identified intact and in pigs, both glucuronides were identified intact in bile and in urine. In addition, a glucuronide of M3 was identified in human bile. In conclusion, advances have been made in the understanding of the pharmacokinetics of finasteride, in particular in relation to the metabolism. Hopefully, the findings of this comprehensive investigation can be applied to other drugs and novel chemical entities.

Background: Dietary salt intake in the South African population exceeds the physiological need. Excessive salt intake is associated with elevated blood pressure levels which may lead to hypertension and cardiovascular accidents. A lifestyle modification such as dietary salt restriction is an inexpensive, effective disease prevention option. Objective: The overall main objectives of this investigation was to: 1) compare salt intake, estimated from a short salt frequency intake questionnaire, with the 24-hour urinary salt excretion and blood pressure of young normotensive healthy white and black South Africans; and 2) compare 24-hour salt excretion and 24-hour blood pressure profiles of normotensive white and black individuals in terms of their knowledge, attitude and behaviour towards dietary salt intake. Study design: The study design was cross-sectional and nested in the baseline phase of the African Prospective Study on the Early Detection and Identification of Cardiovascular Disease and Hypertension in South Africa (African-PREDICT) study. Methods: Multiple methods of data collection were used including anthropometry, biochemical analyses, dietary intakes and cardiovascular measurements. Participants in the study completed the short salt frequency intake questionnaire, describing and quantifying habitual salt intake, and a questionnaire describing knowledge, attitude and behaviour regarding salt intake. Responses to the questionnaires were compared with actual salt intakes estimated from a single 24-hour urine sample and with the 24-hour blood pressure measurements. Results: There was no significant correlation between salt intake based on the questionnaire and 24-h urinary excretion in the white (r=0.07; p=0.40) and black (r=-0.53; p=0.56) participants before and after adjustment for covariates. Estimated salt intake from the questionnaire significantly correlated with systolic blood pressure in white participants (r=0.22, p=0.005) before adjustment for covariates and was no longer significant after adjustment. None of the correlations (unadjusted or adjusted) were significant for the black participants (all p>0.05). The Bland-Altman plots for salt intake showed that the mean difference between the methods used to determine salt intake for the white group is 0.5 g/day, and for the black group is -1.9 g/day. The urinary salt excretion may estimate salt intake to be 9.6 g/day above or 11.1 g/day below the questionnaire’s estimation in the white, and 10.8 g/day above and 18.4 g/day below in the black groups. The level of agreement (Cohen’s Kappa analyses) between the salt frequency questionnaire and the 24-hour urinary salt excretion were determined by categorising the participants in groups who meet the target of <5 grams salt per day or do not. The value of Kappa for the white participants was 0.17 (slight agreement) and for the black participant it was -0.06 (no agreement). In the white participants were a significant increase in both SBP and DBP with increasing tertiles of salt intake according to the questionnaire (p<0.006 and p<0.02 respectively). In the black participants there were no significant difference in BP levels (all p>0.05). The five foods/food groups that contributed most to dietary salt intake in both ethnic groups were discretionary salt, bread, gravy made with stock or gravy powder, soup and biltong. There were no differences in the BP levels between those who answered questions about their knowledge and attitude towards salt intake in both ethnic groups (all p>0.05). Also, there were no differences in their urinary salt excretion (all p>0.05). Only certain behaviours mentioned in the questionnaire were reflected in the salt intake levels and blood pressure. Conclusions: The short salt frequency intake questionnaire can be used to identify food items that contribute to total salt intake. However, the questionnaire considerably underestimates the dietary salt intake. The application of this questionnaire may be helpful in epidemiological studies that evaluate foods which contribute to the total salt intake in order to monitor the average salt intake of a population and to assess the proportion of the population that does not meet the target of less than 5 grams of salt intake per day. It cannot, however, be used to assess the salt intake of an individual. The knowledge, attitude and behaviour of women and men of both ethnic groups are poorly reflected in their actual salt intake and blood pressure, especially among the black participants. The majority of the participants in both ethnic groups consume dietary salt in much higher quantities than the recommended less than 5 grams per day. The current public awareness campaign to decrease salt intake to the target level of less than 5 grams per day by the South African National Department of Health and the Heart and Stroke Foundation is commendable. / MSc (Nutrition), North-West University, Potchefstroom Campus, 2015

Background: Dietary salt intake in the South African population exceeds the physiological need. Excessive salt intake is associated with elevated blood pressure levels which may lead to hypertension and cardiovascular accidents. A lifestyle modification such as dietary salt restriction is an inexpensive, effective disease prevention option. Objective: The overall main objectives of this investigation was to: 1) compare salt intake, estimated from a short salt frequency intake questionnaire, with the 24-hour urinary salt excretion and blood pressure of young normotensive healthy white and black South Africans; and 2) compare 24-hour salt excretion and 24-hour blood pressure profiles of normotensive white and black individuals in terms of their knowledge, attitude and behaviour towards dietary salt intake. Study design: The study design was cross-sectional and nested in the baseline phase of the African Prospective Study on the Early Detection and Identification of Cardiovascular Disease and Hypertension in South Africa (African-PREDICT) study. Methods: Multiple methods of data collection were used including anthropometry, biochemical analyses, dietary intakes and cardiovascular measurements. Participants in the study completed the short salt frequency intake questionnaire, describing and quantifying habitual salt intake, and a questionnaire describing knowledge, attitude and behaviour regarding salt intake. Responses to the questionnaires were compared with actual salt intakes estimated from a single 24-hour urine sample and with the 24-hour blood pressure measurements. Results: There was no significant correlation between salt intake based on the questionnaire and 24-h urinary excretion in the white (r=0.07; p=0.40) and black (r=-0.53; p=0.56) participants before and after adjustment for covariates. Estimated salt intake from the questionnaire significantly correlated with systolic blood pressure in white participants (r=0.22, p=0.005) before adjustment for covariates and was no longer significant after adjustment. None of the correlations (unadjusted or adjusted) were significant for the black participants (all p>0.05). The Bland-Altman plots for salt intake showed that the mean difference between the methods used to determine salt intake for the white group is 0.5 g/day, and for the black group is -1.9 g/day. The urinary salt excretion may estimate salt intake to be 9.6 g/day above or 11.1 g/day below the questionnaire’s estimation in the white, and 10.8 g/day above and 18.4 g/day below in the black groups. The level of agreement (Cohen’s Kappa analyses) between the salt frequency questionnaire and the 24-hour urinary salt excretion were determined by categorising the participants in groups who meet the target of <5 grams salt per day or do not. The value of Kappa for the white participants was 0.17 (slight agreement) and for the black participant it was -0.06 (no agreement). In the white participants were a significant increase in both SBP and DBP with increasing tertiles of salt intake according to the questionnaire (p<0.006 and p<0.02 respectively). In the black participants there were no significant difference in BP levels (all p>0.05). The five foods/food groups that contributed most to dietary salt intake in both ethnic groups were discretionary salt, bread, gravy made with stock or gravy powder, soup and biltong. There were no differences in the BP levels between those who answered questions about their knowledge and attitude towards salt intake in both ethnic groups (all p>0.05). Also, there were no differences in their urinary salt excretion (all p>0.05). Only certain behaviours mentioned in the questionnaire were reflected in the salt intake levels and blood pressure. Conclusions: The short salt frequency intake questionnaire can be used to identify food items that contribute to total salt intake. However, the questionnaire considerably underestimates the dietary salt intake. The application of this questionnaire may be helpful in epidemiological studies that evaluate foods which contribute to the total salt intake in order to monitor the average salt intake of a population and to assess the proportion of the population that does not meet the target of less than 5 grams of salt intake per day. It cannot, however, be used to assess the salt intake of an individual. The knowledge, attitude and behaviour of women and men of both ethnic groups are poorly reflected in their actual salt intake and blood pressure, especially among the black participants. The majority of the participants in both ethnic groups consume dietary salt in much higher quantities than the recommended less than 5 grams per day. The current public awareness campaign to decrease salt intake to the target level of less than 5 grams per day by the South African National Department of Health and the Heart and Stroke Foundation is commendable. / MSc (Nutrition), North-West University, Potchefstroom Campus, 2015

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: In this study, five trials were conducted to examine in vitro and in vivo degradation, excretion and absorption parameters of melamine (MEL) in dairy cows that have not been studied before or where limited information is available. The first two trials were in vitro studies conducted to determine the extent of MEL degradation in rumen liquor and the effects of MEL on ruminal ammonia (NH3) and volatile fatty acid (VFA) concentrations. For both trials, rumen liquor was collected from ruminally cannulated lactating Holstein cows. For the first and second trial, rumen liquor was collected from three and two cows, respectively. For both trials, Erlenmeyer flasks contained 1 g substrate and 100 mL incubation medium consisting of 20 mL rumen liquor and 80 mL reduced buffer solution. In the first trial, each flask contained 100 mg of MEL, resulting in an initial MEL concentration of 1000 mg/L. The flasks were incubated at 39° C for 0 (Control), 6, 24 or 48 hours under strictly anaerobic conditions. In all the trials, MEL concentrations were determined by LC/MSMS. MEL degradation was low after 6 and 24 h of incubation (3.2 and 5.5%, respectively) and increased to 13.6% after 48 h of incubation. In the second trial where VFA and NH3 concentrations were determined, the flasks contained either 0 (Control), 0.2 (T1) or 0.4 mg (T2) of MEL. The flasks were incubated for 6, 24 or 48 h. Treatment had no effect on individual or total VFA concentrations or NH3 concentrations at 6 and 48 h. At 24 h, T2 resulted in an inexplicable higher NH3 concentration. This study showed that the addition of melamine would not result increased rumen NH3 concentrations in vitro. Melamine would also not affect the production of different VFA’s. Therefore, it was concluded that the rumen micro-organisms present in rumen liquor would be unable to utilize MEL as a source of nitrogen and that the microbial production of VFA’s remains unaffected by the presence of MEL. In the third trial, MEL excretion in lactating cows was determined. Five cows were randomly allocated to treatments according to a 5 x 5 Latin square design. Cows received the treatment diets for 7 d followed by 8 d of MEL withdrawal during each of the five periods. The experimental treatments were formulated to provide a daily MEL intake of 0 (M0), 500 (M1), 1000 (M2), 5000 (M3) or 10000 mg (M4) via 15 kg of dairy concentrate pellets. Calculations based on the work of Newton & Utley (1978) suggested that a melamine intake of 0.16 g/kg of live weight would not result in detrimental health effects of ruminant animals. Therefore, a 600 kg lactating dairy cow should not be at risk when consuming 100 g of melamine. In this trial, the highest melamine treatment (M4 = 10 g/d) included a 10-fold safety factor from the suggested safe amount from the work of Newton & Utley (1978) and should not pose a health risk to the cows. Treatments had no effect on DMI, milk yield or milk composition. MEL was detected in the milk 8 h after initial MEL ingestion, increased rapidly and peaked on d 3 and was undetectable after 8 d. Treatments had no effect on MEL excretion efficiencies which ranged from 1.5 to 2.1%. The mean apparent digestibility of MEL was 78%. Mean faecal and urinary MEL excretions were 22 and 54 % of ingested MEL, respectively. Higher milk, urine and faecal MEL concentrations were observed with higher levels of dietary MEL. It was concluded that MEL appeared in the milk soon after first ingestion and a withdrawal period of 8 d was required for all milk, faecal and urine samples to reach undetectable levels of MEL. Urine and faeces were the primary routes for MEL excretion. The fourth trial was conducted to determine MEL absorption by the mammary gland in lactating dairy cows through arterio-venous (A-V) difference. Five cows received 10 g of MEL/d for three consecutive days. Day 3 of the trial was selected for commencement of blood sampling as previous studies (Cruywagen et al., 2009; Shen et al., 2010; Sun et al., 2011) reported the milk melamine concentration to reach a peak on d 3 of continuous melamine consumption by dairy cows. Early on d 3, catheters were inserted into the caudal superficial epigastric vein (milk vein) and caudal auricular artery. The blood sampling period commenced after residual milk removal from the udder following oxytocin administration. Blood from both locations were collected hourly for 9 hours. Following the final blood collection, oxytocin was administered again, catheters were carefully removed and cows were milked immediately thereafter. All blood samples were centrifuged and the decanted plasma was analysed for MEL, as well as for amino acid contents to calculate mammary blood flow. The positive MEL flux (calculated from A-V difference) confirmed net absorption of MEL into the mammary gland with an efficiency of absorption of 0.29%. Melamine excretion into milk was 5.63 mg/h. The mean plasma and milk MEL concentrations were 5.2 and 3.9 mg/kg, respectively. Melamine excretion efficiency to milk, expressed as percentage of the ingested amount, was 1.47%. It was concluded that melamine ingested by cows will result in net MEL absorption by the mammary gland, but that the absorption efficiency is low. The final trial of the study aimed to determine the effects that fermentation processes during the manufacturing of cheese, yoghurt and kefir would have on their MEL content if these products were made from MEL contaminated milk. Another objective was to determine if MEL in cheese would be degraded during the curing process. Cheese, yoghurt and kefir were made from milk with a MEL content of 6.77 mg/kg. The cheese was then cured for 2 wk at 6° C. The MEL contents of the yoghurt and kefir were 6.76 and 6.78 mg/kg, respectively, indicating that the different fermentation processes used in yoghurt and kefir production had no effect on their MEL content and that MEL was not degraded during the short fermentation periods. The percentage of milk MEL partitioned to whey and cheese were 97.4 and 6.5 %, respectively. It was concluded that the different fermentation processes involved during the manufacturing of yoghurt and kefir from MEL tainted milk did not decrease the MEL concentration. The milk MEL was predominantly partitioned to whey, with little MEL transferred to cheese. It was also concluded that MEL was not degraded in cheese during a 2-wk curing period. It was finally concluded that dietary MEL is readily absorbed by dairy cows and mainly excreted via the urine. The mammary gland has a low affinity for MEL absorption and approximately 2% of ingested MEL is excreted in the milk. When cheese is made from MEL tainted milk, the majority of MEL will concentrate in the whey fraction and only 6.5% will be present in the cheese. / AFRIKAANSE OPSOMMING: Vyf proewe is gedoen om in vitro- en in vivo-degradering, uitskeiding en absorpsie parameters van melamien (MEL) na te gaan waaroor daar min of geen inligting bekend was nie. Die eerste twee proewe was in vitro-studies, uitgevoer om die mate van MEL degradeerbaarheid in rumenvloeistof na te gaan, asook die invloed van MEL op rumen-NH3 en vlugtige vetsuur (VVS)-konsentrasies. Vir beide proewe is rumenvloeistof van lakterende, rumengekannuleerde Holsteinkoeie verkry. Vir die eerste en tweede in vitro-studies, was rumenvloeistof verkry vanaf drie en twee koeie, onderskeidelik. In albei proewe is 1 g substraat in Erlen-meyerflessies afgeweeg en 100 mL inkubasiemedium bygevoeg wat uit 20 mL rumenvloeistof en 80 mL van ‘n buffermedium bestaan het. In die eerste proef is 100 mg MEL by die substraat gevoeg, sodat die aanvanklike MEL konsentrasie in die flessies 1000 mg/L was. Die flessies is by 39° C geïnkubeer vir 0 (Kontrole), 6, 24 of 48 ure, onder streng anaerobiese kondisies. Met die beïndiging van die inkubasieperiode is 100 mL van ‘n 0.2 M perchloorsuuroplossing bygevoeg om enige melamien wat nie gedegradeer was nie, op te los. In al die proewe is melamienbepalings by wyse van LC/MSMS gedoen. Melamiendegradering was laag na 6 en 24 h inkubasie (3.2 en 5.5%, respektiewelik) en teen 48 h inkubasie het dit toegeneem tot 13.6%. In die tweede proef het die flessies 0 (Kontrole), 0.2 (T1) of 0.4 mg (T2) melamien bevat. Behandeling het geen invloed op individuele of totale VVS-konsentrasies by enige van die inkubasietye gehad nie en ook nie op NH3-konsentrasies by 6 en 48 h nie. Om een of ander onverklaarbare rede het die T2-behandeling gelei tot hoër NH3-konsentrasies by 24 h. Die gevolgtrekking is gemaak dat die byvoeging van MEL geen effek op rumen NH3-konsentrasies het nie en dat die mikroorganismes in die rumen nie daartoe in staat sal wees om MEL as ‘n stikstof-bron sal kan benut nie. In die derde proef is die uitskeiding van MEL in melkkoeie ondersoek. Vyf lakterende Holsteinkoeie is ewekansig aan vyf behandelings toegeken in ‘n 5 x 5 Latynsevierkantontwerp. Gedurende elke periode het koeie die behandelings vir 7 d ontvang, gevolg deur ‘n 8 d MEL-onttrekkingsperiode. Die eksperimentele diëte is geformuleer om ‘n daaglikse MEL-inname van 0 (M0), 500 (M1), 1000 (M2), 5000 (M3) of 10000 mg (M4) per koei/dag te verseker, toegedien via 15 kg/d van ‘n suiwelkonsentraat in pilvorm. Berekeninge gebasseer op die werk van Newton & Utley (1978) stel voor dat ‘n MEL inname van 0.16 g/kg lewende massa, geen negatiewe effek op herkouers se gesondheid sal hê nie. Dus, ‘n koei wat 600 kg weeg, sal geen skade lei deur die inname van 100 g MEL nie. In hierdie proef was die hoogste MEL behandeling (M4 = 10g/d) tien keer laer as die voorgestelde veiligheidsvlak van Newton & Utley (1978). Behandeling het geen invloed op DMI, melkopbrengs of melksamestelling gehad nie. Melamien is so gou as 8 h na eerste inname in die melk waargeneem, waarna die konsentrasie vinnig toegeneem het en ‘n piek na 3 d bereik het. Behandeling het geen invloed op die uitskeidingsdoeltreffendheid van melamien in melk gehad nie en waardes het gewissel van 1.5 tot 2.1%. Die gemiddelde skynbare verteerbaarheid van MEL was 78%. Die gemiddelde mis- en uriene-MEL-konsentrasies was 22 en 54%, onderskeidelik. Hoër melk-, mis- en uriene-MEL-konsentrasies is waargeneem namate die MEL-inhoud van die diëte gestyg het. Die gevolgtrekking is gemaak dat MEL spoedig na eerste inname in die melk verskyn en dat ‘n onttrekkingsperiode van 8 d benodig word voordat melk-, mis- en uriene-MEL onwaarneembare vlakke bereik. Uriene en mis is die primêre uitskeidingsroetes van ingenome MEL. Die vierde proef is onderneem om MEL-absorpsie in die melkklier met behulp van arterio-veneuse (A-V) verskille te ondersoek. Vyf koeie het elk 10 g MEL/d vir drie agtereen-volgende dae ontvang. Dag 3 van die proef is gekies vir bloedkolleksies aangesien vorige studies (Cruywagen et al., 2009; Shen et al., 2010; Sun et al., 2011) gewys het dat melk MEL op dag 3 van MEL inname, piek konsentrasies beryk. Vroeg gedurende die oggend van d 3 is kateters in die kaudale oppervlakkige epigastriese aar (melkaar) en die kaudale aurikulêre slagaar geplaas. Die bloedtrekkingsperiode het ‘n aanvang geneem direk nadat die koeie volledig uitgemelk is na toediening van oksitosien om te verseker dat soveel as moontlik residuele melk verwyder word. Monsters van veneuse-, sowel as arteriële bloed, is 9-uurliks geneem. Na die finale bloedtrekking is oksitosien weer toegedien, die kateters is versigtig verwyder en die koeie is direk daarna weer gemelk. Al die bloedmonsters is gesentrifugeer en plasmamonsters is ontleed vir MEL, asook vir aminosuursamestelling ten einde bloedtoevoer na die uier te bereken. Die positiewe fluks (bereken van A-V verskil) het bevestig dat netto MEL absorpsie in die melkklier plaasvind, met ‘n doeltreffendheid van 0.29%. Melamienuitskeiding in die melk was teen ‘n tempo van 5.63 mg/h. Die gemiddelde plasma- en melk-MEL konsentrasies was 5.2 en 3.9 mg/kg, onderskeidelik. Die uitskeidingsdoeltreffendheid van MEL na melk, uitgedruk as persentasie van ingenome MEL, was 1.47%. Die gevolgtrekking is gemaak dat MEL wat deur koeie ingeneem word, tot netto MEL-absorpsie in die melkklier sal lei, maar dat die absorpsiedoeltreffendheid baie laag is. In die finale proef is daar gepoog om die invloed van fermentasieprosesse gedurende die vervaardiging van kaas, joghurt en kefir op die produkte se melamieninhoud na te gaan indien die produkte van melamienbevattende melk gemaak sou word. ‘n Tweede doel van hierdie proef was om te bepaal of MEL in kaas gedegradeer kan word tydens rypwording. Kaas, joghurt en kefir is gemaak van melk wat ‘n MEL-inhoud van 6.77 mg/kg gehad het. Die kaas is vervolgens vir twee weke by 6° C rypgemaak. Die MEL-inhoud van die joghurt en kefir was 6.76 en 6.78 mg/kg, onderskeidelik, wat daarop dui dat die onderskeie fermentasieprosesse wat tydens die bereiding van joghurt en kefir plaasvind, geen invloed op hul MEL-inhoud gehad het nie en dat MEL nie gedurende hierdie kort fermentasieperiodes gedegradeer is nie. Die persentasie MEL na wei en kaas versprei was 97.4 en 6.5%, onderskeidelik. Die gevolgtrekking is gemaak dat die verskillende fermentasieprosesse betrokke tydens die vervaardiging van joghurt en kefir wat van melamienbesmette melk gemaak word, nie die MEL-konsentrasie verlaag het nie. Tydens die vervaardiging van kaas, word die MEL hoofsaaklik na die weikomponent versprei en baie min na kaas. Melamien word ook nie in kaas afgebreek gedurende ‘n verouderingsproses van twee weke nie. Die finale gevolgtrekkings is gemaak dat MEL maklik deur melkkoeie geabsorbeer word en dat die hoof uitskeidingsroete via urine is. Die uier het ‘n lae affiniteit vir MEL absorpsie en ongeveer 2% van ingenome MEL is in die melk uitgeskei. Wanneer kaas van MEL besmette melk gemaak word, sal die meerderheid van die MEL in die weifraksie konsentreer, met slegs 6.5% teenwoordig in die kaas. / The Hennie Steenberg Trust Fund, the Ernst and Ethel Erickson Trust and the National Research Foundation (NRF) for their financial support

Background: Studies of assessing dietary fluoride intake in children have employed different dietary methods mainly “2-day duplicate” and “3-day food diary” methods. However, none of these methods have been validated or standardised. Main aims: The main aims of the current study were to develop a better understanding of strengths and weaknesses of dietary assessment methods “2-day duplicate plate” and “3-day food diary” by comparing dietary fluoride intake estimated by each method and evaluate the validity of the two methods for estimating dietary fluoride intake in young children. Methods: Sixty one healthy 4-6 year old children living in fluoridated area of the north-east of England since birth were recruited via 10 primary schools. Dietary information was collected using “2-day duplicate plate” and “3-day food diary” methods. Two 24-h urine samples and two samples of post brushing expectorate (a mixture of saliva, toothpaste and water used to rinse after brushing) from each child. Completeness of 24-h urine samples was checked using urinary excretion of creatinine and urinary flow rate. Validity of the two dietary assessment methods was checked by measuring urinary excretion of nitrogen and potassium as independent validity checks. Total daily fluoride intake from diet and toothpaste ingestion and urinary fluoride excretion was determined for each child. Results: All participated children completed all aspects of the study. According to the validity criteria, dietary data of 58 (95%) children, when collected by the 3-day food diary, were considered valid. However, when the dietary data were collected by the 2-day duplicate plate method, the data for 46 (75%) children were viewed as valid. Mean total dietary fluoride intake was 0.533 mg/d by the 3-day food diary method and 0.583 mg/d by the 2-day duplicate plate method. No statistically significant difference in total dietary fluoride intake was observed between the two methods. The mean difference in estimated dietary fluoride intake by the two dietary assessment methods was -0.050 mg/d with 95% limits of agreement of -0.501 to + 0.401 mg/d. Conclusion: Either the 3-day food diary or the 2-day duplicate plate method can be used when investigating mean total daily dietary fluoride intake of a population. However the methods cannot be used interchangeably at the individual level.

As laranjas e seus derivados, principalmente os sucos, possuem compostos bioativos, tais como os flavonoides, entre eles as flavanonas hesperidina e narirutina, que podem estar relacionados à promoção e benefícios à saúde. A absorção e metabolização de flavonoides podem ser afetadas por diversos fatores como a microbiota e fatores antropométricos, o que pode afetar a sua bioatividade. Assim, o objetivo deste estudo foi comparar o metabolismo e excreção dos flavonoides entre indivíduos eutróficos e obesos após a ingestão de sucos de laranja pasteurizado obtidos das cvs. Pera e Moro. Em um estudo cross-over randomizado 20 voluntárias eutróficas e 10 voluntárias obesas, com idade entre 19 e 40 anos, consumiram em dose única 600 mL de cada suco, que contém as flavanonas narirutina e hesperidina, além das antocianinas no suco Moro. Os metabólitos de flavanonas e de antocianinas foram identificados e quantificados em urina coletada em diferentes períodos de tempo durante 24 horas. Não foi observada diferença significativa na permeabilidade intestinal entre os grupos. Foram detectados e identificados 8 metabólitos de fase II da hesperitina e naringenina, principalmente mono e diglicuronidados e sulfatos, além de três ácidos fenólicos catabólitos de flavanonas formados pela microbiota intestinal, entre elas o ácido hipúrico, ácido protocatecuico e ácido 3-(3-hidroxifenil)-3-hidroxipropiônico. Os ácidos fenólicos foram os metabólitos majoritários recuperados na urina, principalmente o ácido hipúrico. Ainda, os metabólitos de fase II apresentaram maior excreção entre o período de 4-8h e 8-12h (13 a 27% do total de metabólitos excretados). Não foi observada diferença significante (p<0,05) no total de metabólitos de naringenina e hesperitina excretados na urina durante o período de 24 h entre os dois grupos e para os sucos de laranja, nem para o total de metabólitos, provavelmente devido à grande variabilidade interindividual na excreção. Assim, não foi observada diferença entre a metabolização de flavanonas de laranja entre os eutróficos e obesos e nenhuma correlação com os parâmetros antropométricos avaliados. / Oranges and orange juices contain bioactive compounds, such as flavonoids, mainly the flavanones hesperidin and narirutin, which may be related to the promotion and health benefits. The absorption and metabolization of flavonoids can be affected by several factors such as the gut microbiota and anthropometric parameters, which may affect its bioactivity. Thus, the aim of this study was to compare the metabolism and excretion of flavonoids among eutrophic and obese people after ingestion of two pasteurized orange juice obtained from cvs. Pera and Moro. In a randomized cross-over study 20 eutrophic volunteers and 10 obese volunteers, aged 19-40 years, consumed a single dose of 600 mL of each juice. The metabolites of flavanones and anthocyanins were identified and quantified in urine collected at different time points for 24 hours. No significant difference in intestinal permeability was observed between groups. Eight Phase II metabolites of hesperitin and naringenin, mainly mono and diglycerides and sulfates, and three phenolic catabolites of flavanones formed by the gut microbiota were detected and identified, among them hippuric acid, protocatecuic acid and 3- (3-hydroxyphenyl) ) -3-hydroxypropionic acid. Phenolic acids were the major metabolites recovered in urine, mainly hippuric acid. Furthermore, phase II metabolites had greater excretion between the period of 4-8h and 8-12h (13-27% of total metabolites excreted). No significant difference (p <0.05) was observed in the total of naringenin and hesperitin metabolites excreted in the urine during the 24 h period between the two groups, probably due to interindividual variability in excretion. Thus, no difference was observed on metabolism of flavanones between the eutrophic and obese and no correlation was observed with the anthropometric parameters evaluated.

Les produits de glycation avancée (AGEs) sont trouvés en excès dans le plasma et les tissus au cours du diabète, de l'insuffisance rénale et du vieillissement. Ils se forment aussi pendant la cuisson domestique et industrielle des aliments. Actuellement, la plus forte consommation de denrées alimentaires transformées contenant des quantités importantes des AGEs augmente le risque d'exposition de la population aux AGEs et à leurs effets biologiques. C'est ainsi que l'étude de la biodisponibilité des AGEs devient un sujet d'actualité dans la compréhension de la pathogenèse de certaines maladies chroniques, particulièrement le diabète. Deux études cliniques ont été menées dans le but d'étudier l'impact de l'ingestion des AGEs alimentaires sur les fluctuations des concentrations de ces mêmes molécules retrouvées dans les fluides biologiques des individus sains étudiés. Une première étude qui compare la teneur des AGEs ingérés à travers deux régimes alimentaires administré à 62 jeunes adultes. Une deuxième étude qui évalue la teneur en AGEs d'une alimentation basée sur du lait maternel ou du lait de formules infantiles donné à 161 bébés. La quantification de la carboxyméthyllysine (CML) et des AGEs fluorescents comme indicateurs de la réaction de Maillard a été effectuée dans les aliments administrés aux sujets de l'étude ainsi que dans leurs matrices biologiques : plasma, urine et fèces. Les résultats montrent qu'un régime contenant 2,5 fois moins de CML et 1,7 fois moins des AGEs fluorescents, en comparaison avec un régime courant ou standard, peut provoquer des baisses importantes de ces indicateurs dans le plasma, l'urine et la matière fécale des individus sains. Ces diminutions sont plus accentuées au niveau de l'excrétion urinaire. D'autre part, une plus forte présence de la CML dans le lait de formules infantiles correspond à une plus forte quantité de CML dans le plasma et dans l'urine des enfants ayant ingéré cet aliment, à la différence des enfants nourris avec le lait maternel, caractérisé par de faibles quantités de la CML. Chez les adultes soumis au régime bas en AGEs ainsi que les bébés nourris avec le lait maternel, les niveaux de certains paramètres métaboliques tels que le stress oxydatif, les antioxydants importants et des facteurs de l'inflammation cellulaire et systémique ont été normaux mais inférieurs à ceux des adultes et des enfants ayant ingéré de plus fortes quantités d'AGEs. Ces effets indiquent que de plus faibles quantités de produits de Maillard alimentaires pourraient avoir un impact sur la prévention des maladies chroniques. / Advanced Glycation End Products (AGEs) are found in excess in plasma and tissus during diabetes, renal diseases and aging. AGEs are known to form in foods during industrial processes or home cooking. Nowadays, an excessive intake of technologically transformed foods riches in AGEs, increase the risk of the population to their possible pathophysiological effects. In this contexte, the study of bioavailability of AGEs formed in foods may have a significant impact in understanding the pathogenesis of some chronic diseases as diabetes.Two clinical studies were conducted to study the impact of dietary intake of AGEs on fluctuating levels of these same molecules found in biological fluids of healthy individuals studied. A first study that compared the content of AGEs ingested through two diets administered to 62 young adults. A second study evaluates the AGE content of a diet based on breast milk or infant formula milk given to 161 babies. Quantification of CML and fluorescent AGEs as indicators of the Maillard reaction was performed in the food administered to the subjects and their biological matrices: plasma, urine and feces. The results show that a diet containing less than 2.5 times and 1.7 times of CML and fluorescent AGEs respectively, compared with a current or standard diet, can cause significant declines in these indicators in plasma, urine and fecal matter of healthy individuals. These decreases are more pronounced at the level of urinary excretion. A stronger presence of CML in the milk of infant formula is a higher amount of CML in plasma and urine of children who ingested this food, unlike the children fed with milk breast, characterized by low levels of CML. In adults subjected to diet low in AGEs and babies fed breast milk, the levels of some metabolic parameters such as oxidative stress, antioxidants and important factors of cellular and systemic inflammation, were lower than normal levels of adults and children who ingested higher amounts of AGEs. These effects indicate that lower amounts of dietary Maillard products could have an impact on chronic disease prevention

O objetivo deste estudo foi avaliar os efeitos da suplementação de Zn (na forma de ZnO nanoparticulado) em dietas para leitões recém-desmamados sobre o desempenho, a ocorrência de diarreia, a digestibilidade dos nutrientes, a excreção de Zn nas fezes, os parâmetros sanguíneos, a histologia do epitélio intestinal, a morfometria de órgãos e a contagem bacteriana no conteúdo intestinal em comparação ao uso de dose farmacológica de Zn (na forma de ZnO). Foram utilizados 192 leitões desmamados aos 21 dias, em um experimento em blocos casualizados, com 6 tratamentos, 8 repetições (blocos) e 4 animais por unidade experimental (baia). Os tratamentos foram: controle negativo - dieta basal com 100 mg de Zn/kg de ração (na forma de ZnO); controle positivo - dieta basal com 2400 mg de Zn/kg de ração (na forma de ZnO), e dieta basal com 12, 24, 48 ou 96 mg de Zn/kg de ração (na forma de ZnO-N). Os leitões foram alimentados com as dietas experimentais, durante os primeiros 21 dias de experimento. Dos 22 aos 35 dias, todos os animais foram alimentados com uma única dieta, com níveis basais de Zn. A ocorrência de diarreia foi registrada diariamente. Amostras de fezes foram coletadas para determinação da digestibilidade dos nutrientes da dieta e quantificação do Zn excretado. Amostras de sangue foram coletadas no 19° dia do experimento para análise dos parâmetros sanguíneos. No 21º dia, um animal de cada baia foi abatido para a realização da morfometria dos órgãos, histologia do epitélio intestinal e contagem bacteriana. Os dados foram submetidos à análise de variância e à regressão polinomial. Contrastes ortogonais foram utilizados para comparar o tratamento controle positivo com cada um dos níveis de Zn (na forma de ZnO-N). Não foram observados efeitos dos níveis de Zn (ZnO-N) sobre o desempenho, a excreção de Zn nas fezes, o hemograma, a morfometria de órgãos, a histologia do epitélio intestinal e a contagem bacteriana no conteúdo intestinal. O aumento dos níveis de Zn (ZnO-N) aumentou linearmente a digestibilidade aparente dos nutrientes da dieta. Foi observado efeito quadrático dos níveis de Zn (ZnO-N) sobre a frequência da ocorrência de diarreia entre os dias 1 e 7 e sobre a concentração plasmática de Zn. No período de 1 a 21 dias foi observado um menor consumo diário de ração para os níveis de Zn (ZnO-N) em comparação ao controle positivo. Não foram observados diferenças entre o controle positivo e os níveis de Zn (ZnO-N) para as variáveis de desempenho e a frequência da ocorrência de diarreia durante o período de 21 a 35 dias. Foi observada uma menor excreção de Zn nas fezes, e uma menor concentração plasmática de Zn para os níveis de Zn (ZnO-N) em comparação ao controle positivo. A suplementação de Zn, na forma de ZnO-N, não foi capaz de substituir a dose farmacológica de Zn (ZnO) no controle da diarreia após o desmame. Os níveis de Zn, na forma de ZnO-N, não ocasionaram toxicidade nos animais e propiciaram uma redução na excreção de Zn nas fezes. / The purpose of this study was to evaluate the effects of dietary Zn (as ZnO nanoparticles) on performance, diarrhea occurrence, nutrient digestibility, Zn excretion in the feces, blood parameters, histology of gut epithelium, organs morphometry and intestinal bacterial count of weanling pigs compared to the use of pharmacological doses of Zn (as ZnO). One hundred and ninety-two 21d-weaned pigs (5.90±0.83 kg BW) were used in a randomized complete block design experiment with 6 treatments, 8 replications per treatment, and 4 animals per experimental unit (pen). The treatments were: negative control (NC): basal diet (based on corn, soybean meal, dried whey and dried plasma) with 100 mg Zn (as ZnO)/kg diet; positive control (PC): basal diet with 2,400 mg Zn(as conventional ZnO, 150nm)/kg diet and basal diet with 12, 24, 48 or 96 mg Zn (as ZnO-N, 70nm)/kg diet. Pigs were fed dietary treatments from 1 to 21 d feeding period followed by a common diet (same diet for all treatments) from 22 to 35 d feeding period. Diarrhea occurrence was recorded daily. Feces samples were collected to determine the digestibility of the diet and to quantify the Zn excreted. Blood samples were collected on the 19th day of the experiment for analysis of blood parameters. On the 21th day one pig per pen was slaughtered for the analyses of organs morphometry, intestinal epithelium histology and bacterial count. ANOVA and polynomial regression analysis (for levels of Zn as ZnO-N) were performed. Orthogonal contrasts were used to compare positive control with each level of Zn (as ZnO-N). No effects of Zn levels (as ZnO-N) were observed on performance, Zn excretion on feces, blood count, organs morphometry, intestinal epithelium histology and intestinal bacterial count. Increased levels of Zn (as ZnO-N) linearly increased the apparent digestibility of nutrients. It was observed a quadratic effect of Zn levels (as ZnO-N) on the frequency of diarrhea occurrence between 1 and 7 d and on the Zn plasma concentration. From 1 to 21 d of experimental period, lower daily feed intake for Zn levels (as ZnO-N) was observed compared to positive control. No differences were observed among the positive control and levels of Zn (as ZnO-N) for performance and diarrhea occurrence during the period 21 to 35 d. Lower Zn excretion in feces and lower Zn plasma concentration were observed for Zn levels (as ZnO-N) compared to the positive control. Zn supplementation, as ZnO-N, could not to replace the pharmacological dose of Zn (ZnO) to control diarrhea after weaning. The levels of Zn, as ZnO-N, did not cause toxicity of weanling pigs and reduced Zn excretion in the feces.

A (Na+,K+)-ATPase presente no tecido branquial dos crustáceos osmorreguladores é um componente essencial de seu sistema de regulação iônica e osmótica. Esta enzima também apresenta um papel relevante no processo de excreção ativa de NH4+ através do tecido branquial dos crustáceos. Uma fração microsomal rica em (Na+, K+)ATPase foi preparada por centrifugação diferencial a partir de um homogeneizado do tecido branquial de Callinectes ornatus. A centrifugação em gradiente de sacarose revelou a presença de um unico pico de proteina com atividade ATPase, mas a eletroforese em gel de poliacrilamida em condições desnaturantes revelou a presença de várias bandas protéicas. O uso do anticorpo monoclonal 5 contra a subunidade da (Na+, K+) ATPase, revelou a presença de uma única banda proteica de 110 kDa com atividade (Na+, K+)?ATPase. A (Na+, K+) ATPase hidrolisou o PNPP (V= 52,0 ± 2,0 U/mg e K0,5 = 1,1 ± 0,1 mM) através de interações sítio-sítio (nH= 1,6). A modulação da enzima pelos íons magnésio (V= 52,3 ± 1,3 U/mg e K0,5 = 1,1 ± 0,05 mM), potássio (V= 51,4 ± 1,5 U/mg e K0,5 = 2,3 ± 0,1 mM) e amônio (V= 56,7 ± 2,6 U/mg e K0,5 = 9,8 ± 0,4 mM) ocorreu através de interações sítio-sítio. Os íons sódio atuaram como inibidores da atividade K+-fosfatase da enzima (Ki= 1,7 ± 0,1 mM) e a ouabaína inibiu cerca de 80% a atividade PNPPase independentemente da presença de íons amônio. A (Na+, K+) ATPase hidrolisou o ATP de acordo com cinética de Michelis-Menten, com KM= 0,16 0,01 mM e V= 116,3 5,6 U/mg, enquanto a modulação da atividade da enzima pelos íons magnésio (V= 111,0 ± 5,4 U/mg e K0,5= 0,54 ± 0,03 mM), sódio (V= 110,6 ± 5,3 U/mg e K0,5= 6,3 ± 0,3 mM), potássio (V= 116,0 ± 5,5 U/mg e K0,5= 1,5 ± 0,1 mM) e amônio (V= 173,3 ± 5,4 U/mg e K0,5= 5,4 ± 0,3 mM) ocorreram através de interações sítio-sítio. Também foi observado que na presença de concentrações crescentes de ions amonio, a estimulação da atividade (Na+,K+)-ATPase pelo íons potássio acarretou um aumento de 50% na atividade específica da enzima. A ouabaína inibiu cerca de 86% a atividade (Na+,K+)-ATPase com Ki= 74,5 ?M, sugerindo a presença de 14% de fosfatases e/ou outras ATPase contaminantes. Este é o primeiro trabalho onde se observa uma estimulação sinergística da atividade K-fosfatase da (Na+,K+)-ATPase de crustáceo pelos íons potássio e amônio. Os resultados cinéticos obtidos para a (Na+,K+)-ATPase branquial de Callinectes ornatus, analisados em conjunto com os já descritos para outras espécies de crustáceos poderão abrir novas perspectivas em relação ao papel dessa enzima na adaptação fisiológica-bioquímica, bem como para a sobrevivência desses animais em diferentes ambientes. / (Na+, K+)-ATPase present on branchial tissue osmoregulatory crustaceans is an essential component of their osmotic and ionic regulation system. Apparently, this enzyme also have a relevant role in the active excretion de NH4+ through the branchial crustacean tissue. A (Na+, K+) ATPase-rich microsomal fraction was prepared by differential centrifugation from Callinectes ornatus homogenized branchial tissue. The sucrose gradient sucrose centrifugation showed the presence of a single protein peak with ATPase activity, and SDS-PAGE revealed the presence of several proteins bands. The use of the 5 monoclonal antibody, against the ? subunit, revealed the presence of a unique protein band of 110 kDa corresponding to the (Na+, K+) ATPase. (Na+, K+) ATPase hydrolyzed the PNPP (V= 52.0 2.0 U/mg and K0.5= 1.1 0.1 mM) through the site-site interactions (nH= 1.6). The modulation of (Na+, K+) ATPase by magnesium (V= 52.3 1.3 U/mg and K0.5= 1.1 ? 0.05 mM), potassium (V= 51.4 1.5 U/mg and K0.5= 2.3 0.1 mM) and ammonia ions (V= 56.7 2.7 U/mg and K0.5= 9.8 ? 0.4 mM) followed cooperative kinetics. However, sodium ions inhibited PNPPase activity of (Na+, K+)?ATPase with Ki= 1.7 0.1 mM. Ouabain also inhibited up to 80% the total activity PNPPase independent of the presence of ammonium ions. The hydrolysis of ATP by (Na+, K+) ATPase followed Michaelis-Menten kinetics with Km= 0.16 0.01 mM and V= 116.3 5.6 U/mg, while enzyme modulation by magnesium (V= 111.0 5.4 U/mg and K0.5= 0.54 0.03 mM), sodium (V= 110.6 5.3 U/mg and K0.5= 6.3 0.3 mM), potassium (V= 116.0 5.5 U/mg and K0.5= 1.5 ? 0.1 mM) and ammonium ions (V= 173.3 . Interestingly, the stimulation of (Na+, K+)-ATPase activity by potassium ions in the presence of increasing concentration of ammonium ions to K+ resulted in a 50% higher specific activity. Ouabain inhibited approximately 86% the activity (Na+, K+) ATPase with (Ki= 74.5 M), suggesting the presence of about 14% of phosphatases and/or other ATPases. This is the first work showing synergistic stimulation of crustacean (Na+, K+) ATPase by potassium and ammonium ions when PNPP is used a substrate. The results reported herein for Callinectes ornatus branchial (Na+, K+) ATPase might open new perspectives concerning the physiological adaption and the survival of these animals in different environmental.