The role of vitamin D in ameloblasts and odontoblasts has been studied experimentally in rodents. Dental dysplasias have also been reported in clinical studies of children with rickets. Vitamin D acts via a nuclear receptor which binds the major metabolite, 1,25-dihydroxyvitamin D3, and positively or negatively controls the expression of specific genes. The most extensively studied markers of 1,25-dihydroxyvitamin D3 action are calbindin-D9k, calbindin-D28k, and osteocalcin. Therefore, to study in more detail the potential role of 1,25-dihydroxyvitamin D3 in human dental development, 1,25-dihydroxyvitamin D3 receptor (VDR) was localized by immunofluorescence in forming teeth (8-26 wk of gestation). Calbindin-D28k was also mapped by immunoperoxidase in antenatal and postnatal forming and formed teeth. VDR were detected in both dental epithelium and mesenchyme of bud, cap, and bell stages of tooth germs. Nuclei of overtly differentiated ameloblasts and odontoblasts were also immunostained. Calbindin-D28k was present in differentiated ameloblasts and odontoblasts. The presence of VDR and calbindin-D28k in ameloblasts and odontoblasts suggests that 1,25-dihydroxyvitamin D3 may contribute to the regulation of enamel and dentin formation, as classically reported for bone formation. Finally, the early appearance of VDR supports the concept that 1,25-dihydroxyvitamin D3 may also control forward stages of tooth crown development in humans.
The 2,4-dinitrophenylhydrazine ( D N P H ) method for determining ascorbic acid of Roe and co-workers f7, 8,9) has generally yielded results which agree well with those found by indophenol methods. However, studies of the homogeneity of D N P H derivatives prepared from natural products (6) as well as analyses of foods subjected to severe heat treatments (2) have indicated the procedures may lack the desired specificity.In the present study, D N P H derivatives prepared under the usual analytical conditions for determining total ascorbic acid (AA) and dehydroascorbic acid (DHAA) in vegetable extracts were purified by chrornatographic procedures. Concentrations of AA determined from the fraction most closely resembling a reference compound prepared from crystalline AA were compared with those found by the usual D N P H methods. Studies of DHAA in unoxidized vegetable extracts are presented in Part I ; those of total AA in oxidized extracts in Part 11.Determination of DHAA presents some problems different from the ones associated with the determination of total AA. Generally, DHAA constitutes a relatively small part of the total AA content of fresh vegetables ( 3 , 4 , 5 ) . Thus, if non-AA components are present the ratio of the concentration of these components to that of DHAA would be greater than that of the components to total Ah. In addition, the possibility of interference by reduced AA exists in unoxidized extracts. METHODPreparation of Vegetable Samples. Cooking methods were those described by Halliday and Noble ( 1 ) for cooking in boiling water. Cabbage samples were blended with 8 parts by weight of 6% metaphosphoric acid; asparagus with 4 parts. Blended samples were centrifuged and filtered. Cooking waters were diluted with an equal volume of 12% metaphosphoric acid. (7,8,9) were combined into a general procedure referred to here as the D N P H method. 2,4-Dinitrophenylhydrazine (DNPH) Method. Procedures of Roe and co-workersThe DNPH reagent consisted of a 1% solution of D N P H in 4.5 M. sulfuric acid containing 1% thiourea. An incubation period of 3 hours at 37.0 2 0.5" C. was used. Following incubation, 12.5 M. sulfuric acid was employed to dissolve the D N P H derivative and the percent transmission of this solution at 520 mp was determined.Chromatographic analysis of D N P H derivatives. D N P H derivatives for chromatographic analysis were prepared under the same conditions used for the D N P H method except that the amount of each component in the reaction mixture was increased 100 times. That the difference in amounts did not affect the reaction was shown by approxi-' Scientific Journal Series Paper No. 3932, Minnesota Agricultural Experiment Station.'According to Roe, Mills, Oesterling, and Damron (8) determinations of DHAA in unoxidized extracts represent a composite of DHAA and diketogulonic acid. mately equal yields of D N P H derivative being obtained (0.189 and 0.184 mg. per ml. of original solution) regardless of whether small or large amounts of reactants were used.
Cabbage, even when cooked, is generally considered a good source of ascorbic acid, but very little work has been done to compare the various common methods of cooking as to their relative efficiency in retaining this vitamin. Brinkman, Halliday, Hinman, and Hamner (1942) reported that with coarsely shredded samples, cooking in a tightly covered pan retained in the solid portion an average of 61, in a pressure saucepan 53, and in an open kettle 40 per cent of the ascorbic acid originally present in the raw sample. Wellington and Tressler (1938), on the other hand, report retentions of only 24 and 22 per cent when finely shredded cabbage was steamed or boiled, and 58 and 38 per cent when quartered cabbage was steamed and boiled. Olliver (1941) agreed in general with the last figures, for she obtained retentions of 52 and 35 per cent when quartered cabbage was steamed and boiled, respectively.The present paper reports data from which comparisons among the following cooking methods can be made : boiling in an open and (to a limited extent) in a loosely covered pan, cooking in a tightly covered pan, in a pressure saucepan, and steaming.
Juiciness is an important factor in the palatability of meat and one o,nwhich individual judgments vary greatly. An objective method for its measurement, therefore, would be valuable. The pressometer was developed f o r this purpose, but only a few comparisons between percentage of press fluid and the quantity of juice have been reported. In one such comparison, made on 72 pork loin roasts cooked in three different ways, Smith (1933) found in each group a correlation of less than 0.1 between the percentage of press fluid and the average of the scores of three judges on quantity of juice. I n another Satorius and Child (1938a), using 39 standing rib roasts of beef and five judges, found a correlation of 0.31, which was insignificant, between the percentage of press fluid and average score for juiciness. The correlation, however, between the combined scores for flavor and aroma and the combined scores for quality and quantity of juice was 0.68, which was highly significant j on the other hand, the correlation between flavoraroma and aroma was 0.01 and nonsignificant. The authors suggest that the judges' scores on juiciness may have been affected by other palatability factors which stimulated the-flow of saliva, and that this may have ticcounted for the high correlation of flavor-aroma and the quality-quantity of juice and the lack of correlation between press fluid and jury rating on quantity of juice.I n view of the value of the objective test, if proved applicable, and of the small amount of work reported with it, another comparison was thought to be worth while. The present paper discusses results of that comparison.,
Determinations of total ascorbic acid (AA) might be expected to be less affected by interference of non-AA components than direct determinations of dehydroascorbic acid (DHAA) simply because the concentration of total AA in vegetables is generally considerably greater than that of DHAA. On the other hand, they might be influenced by the action of various oxidizing agents on non-AA components if the absorption characteristics of the latter were altered.In the present study, the specificity of the 2,4-dinitrophenylhydrazine ( D N P H ) method for determining total AA in vegetables was investigated by procedures already described (3). Thus, D N P H derivatives were purified by chromatographic techniques, concentrations of total AA calculated from the fraction most closely resembling a reference coinpound prepared froin crystalline AA, and these concentrations compared with those found by the usual D N P H methods. METHODThe D N P H method and chromatographic analysis of DN PH derivatives were carried out as described (3), except that aliquots of the same vegetable extracts used for determining D H A A were treated with indophenol ( I ) , charcoal (7.9), bromine (8,9), or hydrogen sulfide followed by bromine (8) prior to adding the D N P H reagent. In addition, the concentration of thiourea in the D N P H reagent was reduced to 0.25%, the concentration suggested by Lowry, Lopez, and Bessey ( 5 j , since preliminary studies with cabbage indicated that the same concentration of total AA was found in the presence of 1.0 and 0.25% thiourea.Standard curves were prepared from variously oxidized known solutions and were found to be essentially equivalent for the range of D H A A from 0.0015 to 0.0140 mg. per nil. Consequently, a single curve, drawn from the combined data, was used as the basis for calculating concentrations of D H A A in unknown solutions. Data for concentrations of total AA and percentage retentions were subjected to analyses ot variance, using a split plot design d i t h preliminary treatments as main effects :tnd methods of analysis as sub-effects. Duncan's multiple range test ( Z j was used to determine the significance of differences among means when significant values of F were found.
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