Noninvasive sorting of stem cell aggregates based on intrinsic markers.

Noninvasive biomarkers hold important potential for the characterization and purification of stem cells because the addition of exogenous labels, probes, or reporters, as well as the disruption of cell-cell and cell-extracellular matrix interactions, can unintentionally but dramatically alter stem cell state. We recently showed that intensity of the intrinsically fluorescent metabolite, nicotinamide adenine dinucleotide (NADH), fluctuates predictably with changes in stem cell viability and differentiation state. Here, we use multiphoton flow cytometry developed in our laboratory to rapidly and noninvasively characterize and purify populations of intact stem cell aggregates based on NADH intensity and assessed the differentiation capacity of sorted populations. We found removal of aggregates with NADH intensity indicative of cell death resulted in a remaining population of aggregates significantly more likely to produce beating cardiomyocytes (26% vs. 8%, P < 0.05). Similarly, we found isolation of stem cell aggregates with NADH intensity indicative of future cardiac differentiation gave rise to more aggregates with beating cardiomyocytes at later time points (50% vs. 28%, P < 0.05). Further, coupling NADH intensity with gating based on size, enhances the enrichment for EBs capable of giving rise to cardiomyocytes (59% vs. 27%, P < 0.05). Thus, we demonstrate that endogenous properties of cell aggregates, such as NADH and size, can serve as gating parameters for large particle sorting devices to purify populations of stem cells or their progeny in a noninvasive manner, leading the way for improved therapeutic applications.

Large particle multiphoton flow cytometry to purify intact embryoid bodies exhibiting enhanced potential for cardiomyocyte differentiation.

Embryoid bodies (EBs) are large (>100 µm) 3D microtissues composed of stem cells, differentiating cells and extracellular matrix (ECM) proteins that roughly recapitulate early embryonic development. EBs are widely used as in vitro model systems to study stem cell differentiation and the complex physical and chemical interactions contributing to tissue development. Though much has been learned about differentiation from EBs, the practical and technical difficulties of effectively probing and properly analyzing these 3D microtissues has limited their utility and further application. We describe advancement of a technology platform developed in our laboratory, multiphoton flow cytometry (MPFC), to detect and sort large numbers of intact EBs based on size and fluorescent reporters. Real-time and simultaneous measurement of size and fluorescence intensity are now possible, through the implementation of image processing algorithms in the MPFC software. We applied this platform to purify populations of EBs generated from murine induced pluripotent stem (miPS) cells exhibiting enhanced potential for cardiomyocyte differentiation either as a consequence of size or expression of NKX2-5, a homeodomain protein indicative of precardiac cells. Large EBs (330-400 µm, diameter) purified soon after EB formation showed significantly higher potential to form cardiomyocytes at later time points than medium or small EBs. In addition, EBs expressing NKX2-5 soon after EB formation were more likely to form beating areas, indicative of cardiomyocyte differentiation, at later time points. Collectively, these studies highlight the ability of the MPFC to purify EBs and similar microtissues based on preferred features exhibited at the time of sorting or on features indicative of future characteristics or functional capacity.

Microfluidic sorting of microtissues.

Increasingly, invitro culture of adherent cell types utilizes three-dimensional (3D) scaffolds or aggregate culture strategies to mimic tissue-like, microenvironmental conditions. In parallel, new flow cytometry-based technologies are emerging to accurately analyze the composition and function of these microtissues (i.e., large particles) in a non-invasive and high-throughput way. Lacking, however, is an accessible platform that can be used to effectively sort or purify large particles based on analysis parameters. Here we describe a microfluidic-based, electromechanical approach to sort large particles. Specifically, sheath-less asymmetric curving channels were employed to separate and hydrodynamically focus particles to be analyzed and subsequently sorted. This design was developed and characterized based on wall shear stress, tortuosity of the flow path, vorticity of the fluid in the channel, sorting efficiency and enrichment ratio. The large particle sorting device was capable of purifying fluorescently labelled embryoid bodies (EBs) from unlabelled EBs with an efficiency of 87.3% ± 13.5%, and enrichment ratio of 12.2 ± 8.4 (n = 8), while preserving cell viability, differentiation potential, and long-term function.

Comparison of total tract digestibility, development of visceral organs and digestive tract of Mong cai and Yorkshire x Landrace piglets fed diets with different fibre sources.

The aim of the study was to evaluate the effects of piglet age and dietary fibre source on the development of visceral organs and the gastrointestinal tract (GIT), and on growth performance and total tract apparent digestibility (TTAD) in local [pure-breed Mong cai (MC)] and exotic [Landrace x Yorkshire (LY)] piglets. The experimental diets contained different fibre sources: C (basal diet), RB (basal diet + rice bran), SPVM (basal diet + sweet potato vine meal) and CReM (basal diet + cassava residue meal). The neutral detergent fibre (NDF) content in diet C and the fibrous diets was 8.8% and 17.1%-17.7% respectively (dry matter basis). Collection of faecal samples to determine TTAD was carried out for five consecutive days before the experiment was finished (63 days). The piglets were killed at the age of 10 days (before being given the same solid feed), 30 days (weaning, 20 days after solid feed introduced) and 63 days (33 days after being given the different fibrous diets) when the length of intestinal segments, weight of organs (liver, heart, kidneys) and empty weight of the GIT (stomach, small intestine, caecum and colon + rectum) were measured. As the age of animals increased, the relative weight of organs and the length of intestines (expressed on a mass-specific basis) decreased (p < 0.05), and the weight of GIT increased (p < 0.001). The piglets fed fibrous diets had heavier GIT than those fed diet C with the highest values in CReM (p < 0.05). The colon + rectum length was not significantly different among C, RB and SPVM, but was shorter than in CReM (p < 0.05). Coefficient of total tract apparent digestibility (CTTAD) of nutrients in the fibrous diets was lower than in C (p < 0.01). Average daily gain (ADG) and feed conversion ratio (FCR) in C, RB and CReM were not different and were better than in SPVM (p < 0.01). There were no significant differences in the weights of organs between the two breeds at day 10, 30 and 63 (p > 0.05). The weight and length of GIT were not significantly different between the two breeds at day 10 and day 30, but were greater for MC at day 63. The caecum and colon + rectum at 10 and 30 days were longer in MC than in LY (p < 0.001). The relative development of GIT post-weaning was higher than pre-weaning, the difference being most apparent in MC. As a result at 63 days, MC had heavier visceral organs and GIT, and longer intestines on fibrous diets than LY (p < 0.05). The MC at 63 days had higher CTTAD of organic matter, gross energy, crude fibre and NDF (p < 0.001) and ether extract and crude protein (p < 0.05), but lower ADG and poorer FCR than LY (p < 0.001). It can be concluded that the GIT of the MC piglets developed more rapidly than LY when they were introduced to solid feed, and that the difference was more marked on the fibrous diets and after weaning, which resulted in higher total tract digestibility of nutrients in MC compared with LY. Cassava residue meal was better digested than RB and SPVM, and supported higher live weight gains.

Digestibility and nitrogen retention of diets containing different levels of fibre in local (Mong Cai), F1 (Mong Cai x Yorkshire) and exotic (Landrace x Yorkshire) growing pigs in Vietnam.

Total tract digestibility and nitrogen retention of three diets containing different levels of fibre [200, 260 and 320 g/kg neutral detergent fibre (NDF) in dry matter] were determined in three breeds of growing pig at an initial age of approximately 3.5 months. The breeds were local (Mong Cai, MC), F1 crossbred (MC x Yorkshire) and exotic (Landrace x Yorkshire, LY), allocated at random within breed (block) to double 3 x 3 Latin squares. The main fibrous ingredients of the experimental diets were rice bran, cassava residue meal and non-dehulled groundnut cake meal. Digestibility of organic matter (OM), crude protein (CP), NDF, crude fibre, gross energy (GE) and ether extract (EE) decreased as the level of dietary NDF increased (p < 0.001). The r(2) values for the relationship between NDF level and digestibility of OM, CP, GE and EE were 83%, 83%, 80% and 82% respectively. On average, an increase in NDF content of 1% unit resulted in a decrease in OM, CP, GE and EE digestibility of 0.67%, 0.75%, 0.50% and 0.42% units respectively. Digestibility of energy and nutrients was the highest for MC and the lowest for LY (p < 0.01), with intermediate values for F1. There was a negative effect of NDF level on nitrogen (N) retained as a proportion of intake (p < 0.05). Nitrogen retention and utilization were significantly higher (p < 0.001) for LY than for MC and F1 pigs.

Survey on the prevalence of diarrhoea in pre-weaning piglets and on feeding systems as contributing risk factors in smallholdings in Central Vietnam.

A cross-sectional survey on the prevalence of diarrhoea in pre-weaning piglets, and on management and feeding systems under farm conditions was carried out in Thua Thien Hue Province. Faecal samples were collected from 63 piglets without, and 90 piglets with diarrhoea to determine the occurrence of Salmonella, Escherichia coli (E. coli) and different E. coli antigens (K88, K99 and 987P). The prevalence of diarrhoea was higher in the rainy season than in the dry season (33% vs 18%) and the results indicated differences in prevalence between areas. Salmonella and E. coli were found to the same extent in faeces from piglets without and with diarrhoea. All E. coli antigens were isolated from piglets without and with diarrhoea. However, the frequency was much higher in piglets suffering from diarrhoea. In piglets with diarrhoea antigen K88 was found in 26% and 20% of the samples, antigen K99 in 37% and 24% of the samples, and antigen 987P in 31% and 32% of the samples collected in the dry and rainy seasons, respectively. Nutrient supply for sows and for piglets was low in comparison with feeding standards, which may be a contributory factor to the high incidence of diarrhoea in piglets. Thus, the nutrition of sows as well as piglets could be important components in the aetiology of the disease and needs to be studied further.

Effect of supplementation on the feed intake and performance of confined and scavenging crossbred growing chickens in Burkina Faso.

An experiment was conducted to evaluate the performance of crossbred growing chickens (Rhode Island Red x indigenous Burkina Faso hens) from 6 to 17 weeks of age, under five feeding/management regimes: (1) CMx(+), confined and given a mixed feed containing cracked maize and cowpea and a vitamin-mineral premix ad libitum; (2) CS(+), confined and offered ad libitum a choice of cracked maize and cowpea with the premix; (3) ScS(+), scavenging from 09:00 to 16:00 with the diet in treatment (2) available from 16:00 to 09:00; (4) ScS(-), treatment (3) but without the premix; and (5) ScO, scavenging only, with no supplements provided. Daily dry matter (DM) intake was highest for CS(+) (43.5 g), and lowest for CMx(+) (33.6 g) (p < 0.05), with intermediate intakes for ScS(+) and Sc(-) of 36.7 g and 36.2 g, respectively. The ratios of intakes of cowpea to maize were 50:50, 21:79, 27:73 and 22:78 for CMx(+), CS(+), ScS(+) and ScS(-), respectively (p < 0.05). Dietary concentrations of crude protein (CP) were 15.7%, 11.5%, 12.3% and 11.6% of DM for CMx(+), CS(+), ScS(+) and ScS(-), respectively. Average daily gains (ADG) were 8.15 g, 5.24 g, 6.03 g, 5.36 g and 4.45 g for CMx(+), CS(+), ScS(+), ScS(-) and ScO, respectively, and were significantly higher for CMx(+) (p < 0.05). Feed conversion ratio was highest for CS(+) and lowest for CMx(+). ADG of the males (6.44 g) was significantly (p < 0.05) higher than that of the females (5.86 g). Breast and thigh muscle weights were highest for ScS(+) (p < 0.05).

Effect of season and location on the crop contents of local and improved scavenging hens in northern Vietnam.

An experiment was carried out to determine the effects of season (dry and rainy), location (lowland and highland villages) and breed (local and improved) on the amount and physical and chemical composition of the scavenged feed. This was done by crop content analysis of a total of 192 Ri (local) and 192 Luongphuong (LP, improved) hens at 20 weeks of age. The mean fresh weights of the crop contents were 16.9 and 33.3 g, for the dry and rainy seasons, respectively (p < 0.001), 24.3 and 25.9 g, for the Ri and LP breeds, respectively (p < 0.001), and 25.0 and 25.3 g, for the lowland and highland village, respectively (p > 0.05). The proportions of contents were human food leftovers 38.4% and 24.3%, grains 20.9% and 33.7%, insects and worms 9.6% and 11.2%, and green materials 8.8% and 13.8%, for the dry and rainy seasons, respectively (p < 0.05). The proportion of green materials was higher in the LP hens and highland villages, while the proportion of grains was higher in the Ri hens and lowland village (p < 0.05). The dry matter, crude protein (CP), ether extract (EE), total ash (Ash), calcium (Ca), phosphorus, and metabolizable energy (ME) contents were significantly higher for the rainy season (p < 0.01), and CP, Ash, Ca and ME were higher for the LP hens. The crude fibre content was higher in the dry season, and in the Ri hens and highland village (p < 0.001). The total and essential amino acid concentrations were significantly higher in the rainy season, and in the LP hens (p < 0.05), but were not different between locations (p > 0.05). Nutrient intakes from scavenging were estimated at between 20% and 30% of requirements.

Utilization of ensiled sweet potato (Ipomoea batatas (L.) Lam.) leaves as a protein supplement in diets for growing pigs.

Four diets were formulated with protein from fishmeal (FM), groundnut cake (GC), ensiled sweet potato (Ipomoea batatas L. (Lam.)) leaves (SP) or ensiled sweet potato leaves with lysine (SPL). In experiment 1, 24 crossbred (Large White x Mong Cai) growing pigs were allocated randomly by sex into four groups of six pigs and given one of four diets. Experiment 2 was conducted using 16 crossbred pigs (Large White x Mong Cai) at four farms. On each farm, pigs were allocated to two experimental groups. One group was fed the FM diet and the other group the SPL diet. In experiment 1, the daily live weight gains (DLWG) were significantly higher (p < 0.05) for the FM and SPL treatments (542 and 536 g/day, respectively) than for the GC and SP treatments (464 and 482 g/day, respectively). Feed intake was highest (2.0 kg/day) for the SPL and lowest (1.7 kg/day) for the GC treatment (p < 0.05) and feed conversion ratio (FCR) was highest (3.8 kg/kg gain) for SP and lowest (3.5 kg/kg gain) for FM (p < 0.05). There were no significant differences for carcase measurements among diets (p > 0.05). The feed cost per kg live weight gain was lowest for the SP and SPL diets compared to the FM and GC diets. The results of experiment 2 show that there were no significant differences in feed intake, DLWG and FCR between the two diets (p > 0.05). In conclusion, sweet potato leaves can replace fishmeal and groundnut cake in traditional Vietnamese diets for growing pigs.

Scavenging pullets in Burkina Faso: effect of season, location and breed on feed and nutrient intake.

A study on scavenging local (n = 64) and crossbred (n = 64) pullets was undertaken in the dry and rainy seasons in two villages in Burkina Faso: Korea in the arid northern Sahelian region and Bounouna in the sub-humid south. After 4 weeks of scavenging, the birds were killed and the crop contents were subjected to physical and chemical analysis. Cereal grains, brans, green and dry forages, insects/worms and household leftovers were the main physical components. The proportion of cereal grains was higher in the rainy season, whereas the proportion of bran, peanut and sand/grit was higher in the dry season (p < 0.05). The proportion of kitchen waste and millet/milo grains was higher in local than in crossbred birds, whereas the opposite was the case for millet/milo bran (p < 0.05). Rice and local beer residues were not found in the crops of birds from Korea. Dry matter, ash, crude fibre and potassium contents were higher in the dry season (p < 0.05), while calculated metabolizable energy content was higher in the rainy season (p < 0.05). There were no location or breed effects on chemical composition parameters (p > 0.05), except that potassium content was higher in Korea. Mean live weight at slaughter was 806 +/- 22.0 g (local breed) and 881 +/- 22.0 g (crossbreds) (p < 0.05). Digestive organ weights, including contents, were higher for crossbred birds (p < 0.05). Gastrointestinal tract percentage was higher in the rainy season and in Bounouna (p < 0.05). The nutrient composition of the crop contents indicates the need for supplementation, particularly of protein, for higher levels of production.

Effect of scavenging and supplementation of lysine and methionine on the feed intake, performance and carcase quality of improved dual-purpose growing chickens.

An experiment was conducted with 192 improved dual-purpose chickens from 4 to 12 weeks of age to evaluate the effects of management system (confinement and scavenging) and amino acid supplementation (with and without lysine and methionine) of a low-protein diet on the feed and nutrient intake, performance and carcase quality of improved chickens. Mean daily dry matter (DM), crude protein and metabolizable energy intakes were higher for the confined birds (65 g, 13.3 g and 966 kJ, respectively) than for the scavenging birds (58 g, 12.0 g and 868 kJ, respectively) (p < 0.001) and higher for the unsupplemented (63.3 g, 13.0 g and 945 kJ, respectively) than for the supplemented birds (59.5 g, 12.2 g and 889 kJ, respectively) (p < 0.001). Mean DM intake was 68.0 and 54.9 g/day for the males and females (p < 0.001), respectively. Average daily weight gains (ADG) were higher for scavenging (26.9g) than for confined birds (23.9g) (p < 0.001), higher for supplemented (27.6 g) than for unsupplemented birds (23.2 g) (p < 0.001) and higher for males (27.5 g) than for females (23.3 g) (p < 0.001). Carcase, breast and thigh percentages were higher for supplemented than for unsupplemented birds (p < 0.001) and higher for scavenging than for confined birds (p < 0.001). Abdominal fat percentage was significantly lower for the scavenging treatments compared to the corresponding confined treatments (p < 0.001). Feed conversion ratios (kg feed/kg gain) and feed costs/kg gain were lower for the scavenging than for the confinement treatments, and lower for the supplemented than for the unsupplemented birds (p < 0.001). Supplementation of lysine and methionine in diets that were suboptimal in terms of protein and essential amino acids improved performance and carcase quality and reduced feed costs, in particular of the confined growers.

Effects of energy and protein supplementation on the production and economic efficiency of scavenging improved (Tamhoang) and local (Ri) breed hens under smallholder conditions in Northern Vietnam.

The objectives of this study were to evaluate the effects of supplementing maize meal and soybean meal, separately or mixed, on egg production performance of two breeds of scavenging hens under on-farm conditions. Treatments were: (1) confined, with a mixed feed (75% maize meal and 25% soybean meal) ad libitum (CMF); (2) scavenging, with a supplement of 60 g/bird per day of maize meal (ScM); (3) scavenging, with a supplement of 25 g/bird per day of soybean meal (ScS); (4) scavenging, with 54 g/ bird per day of the mixed feed (ScMF). Mean hen-day production was 21.8%, 19.8%, 17.7% and 22.8% for the CMF, ScM, ScS and ScMF treatments, respectively (p <0.05), and was significantly (p <0.001) higher for the improved Tamhoang breed (24.4%) than for the local Ri hens (16.7%). Mean egg weight was significantly higher for the Tamhoang (53.2 g) compared to the Ri hens (45.0 g) (p<0.01). Feed consumption per kilogram of eggs was significantly higher for the CMF treatment compared to the other treatments and was lower for the ScS compared to the other scavenging treatments (p <0.05). Feed cost per kilogram of eggs was lowest for the ScM treatment and was 30 to 40% higher for the confined treatment compared to the scavenging treatments. Feed consumption and cost of feed per kilogram of eggs were significantly lower for the Tamhoang than for the Ri hens.

New approaches to replacing failing organs.

The foremost cause of death and disability is organ failure. Failure of the heart, lungs, kidney, and liver is typically addressed by organ transplantation; however, the number of human organs available for this purpose is quite limited. For this reason, some new technologies are being advanced for the treatment of organ failure. This communication summarizes potential limitations of these technologies.

Effects of scavenging on diet selection and performance of Rhode Island red and Fayoumi breeds of chicken offered a choice of energy and protein feeds.

An experiment was conducted to evaluate the effect of scavenging on diet selection and the comparative performance of Rhode Island Red and Fayoumi breeds of chicken. The breeds were compared under three feeding/management regimes: (1) a choice of ground noug cake and cracked maize, with confinement; (2) a choice of ground noug cake and cracked maize, but being allowed to scavenge for about 6 h; and (3) a commercial layer mash, with confinement. Scavenging did not change the pattern of diet selection. About 90% of the daily intake of both the confined and scavenging birds under choice feeding comprised maize. The efficiency of feed utilization and nutrient intake were also similar in these groups. The Fayoumi were more efficient in terms of feed conversion, although the egg production performance of the two breeds did not vary significantly. Scavenging hens had a significantly lower egg production than the birds under confinement offered a choice of feeds. Scavenging significantly increased the mortality in both breeds. In conclusion, scavenging affected the performance adversely and, despite the provision of a choice of feed providing a source of energy and protein, both the scavenging and confined birds on choice feeding failed to eat sufficient to meet their protein requirements.

Evaluation of the significance of dietary folate from wild vegetables in Vietnam.

Data on the overall dietary folate intakes among high-risk groups in poor countries is very limited. Vegetables are considered good sources but the evaluation of their contribution is hampered by the lack of data on folate concentrations in many traditional foods. Data on the analysis of folate concentrations in 16 wild vegetables used in the Mekong Delta and the Central Highlands in Vietnam and an evaluation of the relative importance of different foods in folate intakes of women is presented. Vegetable samples were collected in four study villages, blanched and frozen samples were transported to Sweden for analysis. Freeze-dried samples were analysed for total folate quantification using a commercial radio protein binding assay. Daily folate intakes among women were estimated from 7-day food frequency interviews with 213 women. The folate concentration in the vegetable samples ranged from 10 to 96 microg/100 g. The mean estimated daily folate intake among the 213 women in the study areas was 251 microg. Vegetables contributed approximately one-third of the daily folate intake, of which 72% and 42%, respectively, in the two regions was from wild vegetables. A majority of the women (87%) got some dietary folate from wild vegetables and nearly one-third had mean daily folate intakes of > 50 microg from such hidden food sources. The evaluation of dietary folate is complicated by data gaps in food composition tables, the unreliability of existing food data, variations between methods used for folate analysis and limited understanding of the bioavailability of food folate.

Significance of wild vegetables in micronutrient intakes of women in Vietnam: an analysis of food variety.

The association between food variety and nutrient intake/health status among rural women was tested in two agro-ecological settings in Vietnam. Special emphasis was placed on the significance of wild vegetables 'Rau Dai' in micronutrient supply and on the usefulness of food variety analysis in determining their current role. Data from 7-day food frequency interviews and a nutrition/health survey with 93 and 103 rural women in the Mekong Delta and the Central Highlands, respectively, were used in the analysis. Energy and nutrient intakes in the groups with the highest food variety score (FVS) (high = > or = 21) in the two regions were compared to those with the lowest food variety score (low = < or = 15). The high FVS groups in both regions also had a more diversified diet in terms of food categories. With the exception of low iron and riboflavin intakes in all groups, the high FVS groups had relatively adequate diets. A large variety of vegetables was used and only approximately half of the vegetable species were cultivated. In both regions the high FVS groups used a significantly greater variety of vegetables than the low FVS groups. Wild vegetables contributed significantly to the overall micronutrient intakes, mostly carotene, vitamin C and calcium intakes, but only the contribution to carotene intake was significantly higher in the high FVS group. Overall, we conclude that a food variety analysis is a useful tool in capturing the dietary role of wild vegetables.

Micronutrient composition and nutritional importance of gathered vegetables in Vietnam.

This article presents the nutrient composition and nutritional contribution of naturally occurring vegetables (NOV) in four villages in Vietnam. The frequency of consumption and quantities eaten were evaluated within the overall diet of 101 and 110 women in the Mekong Delta (flood period and rainy season, respectively) and of 103 women in the Central Highlands (rainy season). The concentrations of Ca, Fe and Zn were analysed in 28 species, and presence of tannins and phytic acid in 19 species. Provitamin carotenoids were determined in 11 species. Over 90% of the women consumed NOV, and they contributed 43, 72 and 75% (Central Highlands, Mekong rainy and Mekong flood period, respectively) of the total quantities of vegetables eaten. Aquatic species such as lpomoea aquatica and Limnocharis flava, and terrestrial vegetables such as Schismattoglottis calyptrata, were among the most commonly consumed vegetables. They were eaten in mean daily quantities exceeding 50 g and had high concentrations of Ca, Fe and beta-carotene. Other major aquatic vegetables included the stems of Eleocharis sp., Nelumbo nucifera and Nymphea lotus, all of which were used as salad vegetables and had relatively low nutrient density. The problems of assessing the current role of NOV due to lack of data on intakes in the context of the overall diets, as well as data on their composition, are addressed.

Aspartame effect in sickle cell anemia.

OBJECTIVE: To examine the in vitro and in vivo attributes of aspartame and to determine its efficacy for treating sickle cell anemia. RATIONALE: Aspartame (l-aspartyl-l-phenylalanine methyl ester) binds with 2 human Bence Jones proteins. The proteins (Mcg and Sea) showed phenylalanine penetrating into hydrophobic binding sites. This aspartame property suggested a potential to interfere with sickle hemoglobin fibril formation. METHODS: For the in vitro studies, blood from 20 subjects monitored for sickle cell anemia was collected in heparinized tubes. Specimens were divided in thirds and aspartame was added to 2 tubes to yield a 1 mg/mL or 2 mg/mL concentration. Sickled cells that were present after a drop from each aliquot was added to a fresh 2% metabisulfite solution were counted 3 times. For the in vivo studies, 23 subjects from the Sickle Cell Clinic (University of Oklahoma Health Sciences Center, Oklahoma City, Okla) consented to participate in a randomized single-dose administration of 1.5, 3.0, or 6 mg/kg aspartame. Heparinized blood was obtained at 0, 30, 60, 120, 240, 480, and 1440 minutes after aspartame administration. Specimens were counted in a blinded manner by means of the technique used for the in vitro method, but a photomicrograph of 1 field from each triplicate count was made. The pictures were marked and were computer counted. RESULTS: For the in vitro studies, sickled cells decreased from 28% to < 14% when 1 mg/mL aspartame was added and decreased further with 2 mg/mL. For the in vivo studies, a decreased number of sickled cells in homozygous blood (HbSS) were observed after oral administration of aspartame. Sickling was inhibited by 6 mg/kg aspartame for at least 6 hours in 15 subjects with HbSS anemia. CONCLUSIONS: Further evaluations of the efficacy of aspartame for sickle crisis and crisis prevention appears to be warranted.