Protein loading test

We have tested this hypothesis in some recent o/w thin film experiments [45]. It was not practical to reduce the protein load per unit area of interface to that found in the emulsion experiments, since the very low concentrations required would have been very slow to reach equilibrium adsorption. We circumvented this problem in a unique way. Rather than adsorb emulsifier mixtures from aqueous solution, we formed the oil droplets and the thin film in a preformed emulsion. Therefore, the adsorbed layers on the captive droplets formed by adsorption of surfactant from the continuous phase of the emulsion. The results are shown in Figure 23, where surface diffusion data of FITC-/8-lg in o/w and a/w thin films as a function of added Tween 20 are summarised. 

Increasing levels of emulsification significantly depleted protein from the fat globule in the mix. The adsorbed protein content in the mix (mg m of fat surface area) correlated with major characteristic analyses describing the fat structure in ice cream (fat agglomerate size, fat agglomeration index, solvent extractable fat Fig. 6). Thus, the measurement of protein load in the mix can be used to predict ice-cream-fat stability and related structure. Structural analyses indicated enhanced interaction between fat and air as protein adsorption decreased. It was also observed that the fat content in the dripped portion collected from a meltdown test correlated well with other indices of fat destabilization. 

Several cases of a new form of metachromatic leukodystrophy have been reported (Hahn et al., 1981, 1982 Inui et al., 1983). These cases have clinical symptoms resembling juvenile metachromatic leukodystrophy, but have only about half of the normal arylsulfohydrolase A activity in leukocytes and fibroblasts. The kinetic properties of the arylsulfohydrolase A from fibroblasts are normal. However, the cerebroside 3-sulfate loading test (Porter et al., 1971a) with growing fibroblasts shows an abnormal response, indicating a disturbance in arylsulfatase A activity. Supplementation with the activator (Stevens et al., 1981) of arylsulfatase A results in a normal cerebroside 3-sulfate loading test. This indicates that the new form of metachromatic leukodystrophy is not caused by the deficiency of arylsulfohydrolase A, but rather is caused by a deficiency of the activator protein (Shapiro et al., 1979). Fujibayashi and Wenger (1986) have studied the biosynthesis of sulfa-tide/GMj activator protein in control and mutant cultured skin fibroblasts. Their results indicate that patients with variant form of metachromatic leu-... 

TMC is supposed to possess the property of permeation enhancement, and therefore has been studied as a delivery vector for proteins and genes. For example, TMC nanoparticles were prepared by ionic crosslinking with tripolyphosphate (TPP) and used as a delivery system for ovalbumin [30]. The loading efficiency and capacity of the protein were up to 95% and 50%, respectively, with an improved integrity. Most importantly, transportation of the protein-loaded particles across nasal mucosa was confirmed by an in vivo uptake test. In another study, the influence of DQ on the property of protein-loaded TMC/TPP nanoparticles was investigated [31]. A lower DQ leads to an increased particle size and a slower release rate of protein. 

A. Rawat, D.J. Burgess, USP apparatus 4 method for in vitro release testing of protein loaded microspheres, InL J. Pharm. 409 (1-2) (2011)178-184. 

FigurelS. New bone growth in the tibial gap and internal and external callus of a rabbit tibia-fibula model on day 14 post treatment with either a pol5rmer control (PLG/NMP)((0I), PDGF-BB/IGF-I growth factor control OXATRIGEL formulation containing PDGF-BB/ IGF-I ( ), or saline (E3). ATRIGEL formulations were composed of 5050 PLG (inherent viscosity, 0.19) with NMP as the carrier solvent. Protein loads were 6.0y g of PDGF-BB and 6.0//g of IGF-I delivered in either saline or ATRIGEL formulatian per test site.

Protein load A protein load is done when a diagnosis is unclear or for heterozygote detection in OTC deficiencies. After one has determined a daily profile for pre- and postprandial ammonia and the amino acids in a self chosen diet, the protein content should be estimated per meal. The patient should not be in a catabolic but steady state for at least 4 days. For women, the test should be avoided around the period of menstruation. The protein load is, in contrast to the allopurinol test, also useful for assessing protein tolerance. False negatives have been described in conjunction with OTC heterozygote testing skewed toward a predominance of wild-type OTC. 

Elevated concentrations of orotic acid and uracil may be found in the urine of heterozygote carriers for urea cycle disorders, most commonly ornithine carbamoyltransferase (OCT) deficiency. This is due to the increased flux through the pyrimidine pathway which occurs, especially where the relevant enzymes are expressed only in liver tissue, as is the case when urea cycle enzymes are defective. A protein load was used previously to stress this route and the elevation in orotic acid excretion used as a diagnostic marker. However, the test frequently failed to detect known carriers. The al-lopurinol loading test (measurement of the increment in urinary orotic acid and orotidine in three separate 8 hour urine collections over the 24 hours following a 300 mg allopurinol tablet) is the most reliable test so far for carrier detection for such disorders [17]. 

Aminoadipic acid concentrations in the urine of the patients of Wilson et al. (1975) were 1480 /xmol (24 h) and 1500 /utmol (24 h) in his sister, compared to 520 fimol (24 h)" in the patient of Przyrembel et al. (1975) on a protein intake of 4.5 g per kg body weight (lysine 390 mg kg" ). The latter authors were able to demonstrate a greatly reduced excretion when protein intake and lysine intake were reduced, the 2-aminoadipic acid excretion falling to 155 tmol (24 h) on a protein intake of 2.3 g per kg body weight (lysine 170 mg/kg) and 49 /imol (24 h) on a protein intake of 2.3 g per kg body weight with a zero lysine intake. Controls excreted 21-29 /xmol (24 h) Przyrembel al. (1975) also showed the effects of an oral lysine load test with increased serum concentrations of both lysine and 2-aminoadipic acid. Baseline plasma lysine concentrations in their patient were low normal (0.062-0.194 mmol 1 S normal 0.227 0.091) and 2-aminoadipic acid concentrations were 0.050-0.079 mmol 1 (normal undetectable). 

S mg), dimer (peak I) and monomer (peak 2), ovalbumin (S mg) (peak 3), and cytochrome c (3 mg) (peak 4) was loaded onto a Fractogel EMD BioSEC column (600 X 16 mm) with a bed height of 600 mm. PBS (pH 7.2) was used as the eluent at a flow rate of I ml/min the sample volume was O.S ml. (B) The same protein sample as in A was injected onto a column of identical dimensions packed with unmodified Fractogel HW 6S. Without the tentacle modification the base matrix displays only a poor resolution of the test mixture. 

FIGURE 7.14 A Fractogel EMD BioSEC Superformance column (600-16) was loaded with 500 /il of BSA, ovalbumin, and cytochrome c (5/5/3 mg/ml) at I ml/min. The test covered 100 individual runs with the standard proteins as samples. The buffer system used was 20 m/VI sodium dihydrogen phosphate, 300 m/VI NaCI, pH 7.2. After each individual run the column was rinsed with I /VI NaOH (60 min with I /VI NaOH at 2 ml/min). No significant change in retention times and resolution was observed after 100 cycles. 

The Fur protein from E. coli was isolated in one step due to its high affinity for metal-chelate columns loaded with zinc. In DNase footprinting experiments, the Fur protein was shown to bind DNA in the promoter region of several iron-regulated genes. The consensus sequence, called the Fur box, is GATAATGATAATCATT ATC. In vitro binding is dependent on the divalent cations Co2+ Mn2+ /s Cd2+ Cu2+ at 150 iM, while Fe2+ seemed to be less active at this concentration, probably due to oxidation to Fe3+ (De Lorenzo et al., 1987). The unspecificity for divalent metals observed in vitro shows that the cells have to select the ions transported carefully and have to balance their active concentrations. In addition, it is a caveat for the experimenter to test a hypothesis on metal-ion specificity not only in vitro, but also in vivo. 

---