Incorporation Of Ascorbic Acid Research Articles

Incorporation of Ascorbic Acid, Caffeine and Chloroquine Diphosphate in Dilutor Improves Structural and Functional Status of Frozen Semen

Incorporation of Ascorbic Acid, Caffeine and Chloroquine Diphosphate in Dilutor Improves Structural and Functional Status of Frozen Semen

Incorporation of Ascorbic Acid, Caffeine and Chloroquine Diphosphate in Dilutor Improves Structural and Functional Status of Frozen Semen

Effect of supplementation of ascorbic acid, caffeine and chloroquine diphosphate in the dilutor on post-thaw quality of spermatozoa was studied. Thirty-two semen ejaculates (8 each from 2 Holstein Friesian and 2 crossbred; 50% Friesian x 50% Haryana) were studied. Ascorbic acid, caffeine and chloroquine diphosphate were added in Tris-egg yolk-glycerol (control) extender at the concentration of 10 mM, 0.54 mM and 7 mM, respectively. The post-thaw semen parameters studied were structural (acrosomal integrity and livability), functional [motility, per cent sperm responding to hypoosmotic swelling test, i.e. HOS positive sperm and penetrability of sperm in bovine cervical mucus, i.e. sperm penetration distance-SPD] and measurement of activities of Glutamic oxaloacetic transaminase (GOT), Glutamic pyruvic transaminase (GPT), Alkaline transaminase (AKP) and Acid transaminase (ACP). Certain enzymes in the seminal plasma. The semen from crossbred bull had significantly lower mass activity (P ≤ 0.01), initial progressive motility (P ≤ 0.01), sperm penetration distance (SPD value) (P ≤ 0.01), HOS positive sperm and seminal plasma GOT activity, but higher abnormal sperm. Incorporation of additives in the dilutor significantly improved the post-thaw semen quality, especially the post-thaw motility, post-thaw per cent live sperm, sperm penetration distance and sperm responding to hypo-osmotic solution. The leakage of all the 4 enzymes (GOT, GPT, ACP and AKP) was significantly less in the samples frozen in presence of ascorbic acid and chloroquine as compared to control. Ascorbic acid resulted in maximum improvement to the quality of frozen-thawed semen followed by chloroquine and caffeine. The study concludes that post-thaw quality of frozen semen of HF and crossbred bulls significantly improves if the dilutor is supplemented with 10 mM concentration of ascorbic acid.

A novel voltammetric sensor for ascorbic acid based on molecularly imprinted poly(o-phenylenediamine-co-o-aminophenol)

A molecularly imprinted copolymer, poly(o-phenylenediamine-co-o-aminophenol) (PoPDoAP), was prepared as a new ascorbic acid (AA) sensor. The copolymer was synthesized by incorporation of AA as template molecules during the electrochemical copolymerization of o-phenylenediamine and o-aminophenol, and complementary sites were formed after the copolymer was electrochemically reduced in ammonium aqueous solution. The molecularly imprinted copolymer sensor exhibited a high sensitivity and selectivity toward AA. Differential pulse voltammograms (DPVs) showed a linear concentration range of AA from 0.1 to 10mM, and the detection limit was calculated to be 36.4μM. Compared to conventional polyaniline-based AA sensors, the analytical performance of the imprinted copolymer sensor was improved due to the broadened usable pH range of PoPDoAP (from pH 1.0 to pH 8.0). The sensor also exhibited a good reproducibility and stability. And it has been successfully applied in the determination of AA in real samples, including vitamin C tablet and orange juices, with satisfactory results.

Incorporation of ascorbic acid and α-tocopherol to the extender media to enhance antioxidant system of cryopreserved sea bass sperm

Despite the overwhelming application of sperm cryopreservation in aquaculture and broodstock management, its detrimental effects on sperm quality must be taken into account. Imbalance of reactive oxygen species is considered one of the main triggers of cell damage after cryopreservation, because the spermatozoa antioxidant system is decimated during this process, mainly because the natural antioxidants present in seminal plasma diminish when sperm is diluted in extenders. It has been demonstrated that the addition of antioxidants to the extender improves the quality of thawed sperm. Thus, the aim of the present work was to evaluate the status of the antioxidant system in cryopreserved sea bass sperm, and the possibility of enhancing this system to reduce oxidation of the membrane compounds by extender supplementation with vitamins. To do this, sperm from European sea bass (Dicentrarchus labrax) was cryopreserved using an extender control (NAM), supplemented with 0.1 mm α-tocopherol or 0.1 mm ascorbic acid. Sperm motility (computer assisted sperm analysis (CASA) parameters), viability (SYBR Green/propidium iodide (PI)), lipid peroxidation (malondialdehyde (MDA) levels) and protein oxidation (DNPH levels) were analyzed, as well as the status of the sperm antioxidant system by determining glutathione peroxidase, glutathione reductase and superoxide dismutase (GPX, GSR and SOD) activity. The results demonstrated that extenders containing vitamins significantly increased sperm motility. Total motility, velocity and linearity increased from 31.2 ± 3.0 μm/sec, 18.3 ± 1.7 μm/sec and 46.9 ± 2.0% in extender containing 0.1 mm α-tocopherol or 30.6 ± 3.9 μm/sec, 19.5 ± 1.6 μm/sec and 47.9 ± 2.2% in extender containing 1 mm ascorbic acid respect to the extender control (20.7 ± 3.3 μm/sec, 13.8 ± 1.7 μm/sec and 37.3 ± 4.1%). However, viability and levels of lipid peroxidation and protein oxidation were not affected by the presence of these antioxidants, suggesting that membrane impairment could be more associated to osmotic shock or membrane destabilization than oxidative damage. The increased activity of both GPX and GSR after cryopreservation showed that the antioxidant system of sea bass sperm must play an important role in preventing oxidation of the membrane compounds. In conclusion, the addition of α-tocopherol and ascorbic acid to the extender media, together with the antioxidant system of the spermatozoa improved sea bass sperm motility, which is one of the impairment parameters most affected by cryopreservation.

Whey Protein Film with Oxygen Scavenging Function by Incorporation of Ascorbic Acid

Residual O(2) in a package headspace can be removed by an O(2)-absorbing sachet, which can be harmful if swallowed by the consumer, or by a chemically-active plastic packaging film, which is difficult to recycle. An edible, O(2)-absorbing film would avoid these disadvantages. The objective of our research was to assess the O(2)-scavenging potential of an edible whey protein isolate (WPI) film incorporating ascorbic acid (AA). AA at 0.05, 0.1, or 0.2 M was added to 5% (w/w) heat-denatured WPI film-forming solutions with WPI : glycerol (Gly) ratio of 1: 1.00, 1: 0.80, or 1: 0.67. The pH of solutions was then adjusted to 3.5 (below pK(a1) of AA), to stabilize AA against oxidation, before film casting. The mechanical properties, O(2) permeabilities, and thermal transitions of films were measured. Activation of the O(2)-scavenging function of the AA-incorporated films was accomplished by adjustment of the films to pH ≥ 7. O(2)-scavenging ability of AA-incorporated WPI films was determined by measuring residual O(2) in the headspace of a high-barrier container. Incorporation of AA into WPI film decreased film tensile strength and further reduced O(2) permeability at each WPI : Gly ratio. AA-containing films adjusted to pH ≥ 7 demonstrated O(2) absorption proportional to AA content, consistent with theoretical O(2)-scavenging capacity. Thermal transition measurements indicated that AA was involved in WPI structural modification and decreased the degradation temperature of WPI-based film. The demonstrated O(2)-scavenging function, improved O(2) barrier and acceptable mechanical properties of AA-incorporated films indicate potential commercial usefulness. Ascorbic acid-incorporated whey protein film with oxygen scavenging function can be used to extend shelf lives of a wide variety of oxygen-sensitive products by eliminating headspace oxygen as well as oxygen permeating through the packaging wall over time. Edible oxygen-scavenger film has the advantages of avoiding both accidental consumption and nonrecyclability of conventional oxygen scavenger systems.

Active antioxidant packaging films: Development and effect on lipid stability of brined sardines

Active antioxidant food packaging films were produced by the incorporation of ascorbic acid, ferulic acid, quercetin, and green tea extract into an ethylene vinyl alcohol copolymer (EVOH) matrix. The characterisation of the thermal and barrier properties of the developed film showed that the addition of these bioactive compounds did not greatly modify their properties. However, the presence of ascorbic and ferulic acids resulted in a significant decrease in water vapour permeability, possibly due to the high affinity for water of these substances. Exposure of the films to various food simulants showed that the release from the films was dependent on the type of food simulant and the antioxidant incorporated: in the aqueous food simulant, materials containing ascorbic acid produced the largest release; in the fatty food simulant, quercetin and green tea extract presented the best performance. The efficiency of the films developed was determined by real packaging applications of brined sardines. The evolution of the peroxide index and the malondialdehyde content showed that, in general, the films improved sardine stability. Films with green tea extract offered the best protection against lipid oxidation.

Ascorbic Acid, Catalase and Chlorpromazine Reduce Cryopreservation-induced Damages to Crossbred Bull Spermatozoa

The present study evaluated the effectiveness of ascorbic acid, catalase, chlorpromazine and their combinations in reducing the cryodamages to crossbred bull (Bos taurus x Bos indicus) spermatozoa. A total of 32 ejaculates (eight each from four bulls) were diluted in Tris-citric acid-fructose-egg yolk-glycerol extender. Each ejaculate was split into six parts (five treatment and one control). Treatment groups included 10 mm ascorbic acid, 0.1 mm chlorpromazine, 200 IU/ml catalase, 10 mm ascorbic acid + 0.1 mm chlorpromazine or 200 IU/ml catalase + 0.1 mm chlorpromazine in the extender. Fluorescent probes (Fluorescein isothiocyanate--Pisum sativum agglutinin + Propidium iodide) were used for the assessment of spermatozoa viability and acrosomal status. The proportion of acrosome intact live (AIL), acrosome intact dead, acrosome reacted live and acrosome reacted dead sperm was assessed in fresh, equilibrated and frozen-thawed semen. The functional status of the sperm was assessed using hypo-osmotic sperm swelling test (HOSST). Activities of acrosin and hyaluronidase enzyme were also determined. Lipid peroxidation level was assayed based on the melonaldehyde (MDA) production. In cryopreserved semen, the values of AIL spermatozoa, HOSST response, hyaluronidase and acrosin activity were reduced by 53%, 47%, 34% and 54%, respectively from their initial values in fresh semen. However, MDA level was threefold higher in the frozen-thawed sperm compared with fresh sperm. Significant (p < 0.05) improvement in motility, viability, HOSST response, retention of hyaluonidase and acrosin and reduction in MDA was recorded in ascorbic acid, catalase, ascorbic acid + chlorpromazine and catalase + chlorpromazine incorporated groups. The percentage of AIL sperm was significantly (p < 0.05) higher in ascorbic acid, catalase and ascorbic acid + chlorpromazine incorporated groups compared with the control. Chlorpromazine alone did not improve the post-thaw semen quality but when combined with either ascorbic acid or catalase, improvement in semen quality was noticed. It was inferred that incorporation of ascorbic acid, catalase and ascorbic acid + chlorpromazine in semen extender improved the post-thaw semen quality in crossbred bulls.

Effect of gamma irradiation in presence of ascorbic acid on microbial composition and TBARS concentration of ground beef coated with an edible active coating

The present study was conducted to evaluate the combined effect of gamma irradiation in presence of ascorbic acid on the microbiological characteristics and thiobarbituric acid-reactive substances (TBARS) concentration of ground beef coated with an edible coating, crosslinked by gamma irradiation. The medium fat ground beef patties (23% fat ) were divided into two separate treatment groups: (i) control (ground beef without additive), (ii) ground beef with 0.5% (w/w) ascorbic acid. Meat samples were irradiated at doses of 0, 1, 2, and 3 kGy and stored at 4±2°C. The content of TBARS was evaluated. After 7 days of storage, Enterobacteriaceae, presumptive Staphylococcus aureus, presumptive Pseudomonas spp., Brochothrix thermosphacta and lactic acid bacteria were enumerated. Results showed that lactic acid bacteria and Br. thermosphacta were more resistant to irradiation than Enterobacteriaceae and Pseudomonas. The content in TBARS was stabilized during post-irradiation storage for samples containing ascorbic acid. Shelf life extension periods estimated on the basis of a limit level of 6 log CFU/g for APCs were 4, 7, and 10 days for samples irradiated at 1, 2, and 3 kGy, respectively. However, the incorporation of ascorbic acid in ground beef did not improve significantly ( p>0.05) the inhibitory effect of gamma irradiation.

Feeding juvenile Artemia enriched with ascorbic acid improves larval survival in the spiny lobster Jasus edwardsii

Newly hatched Jasus edwardsii phyllosoma were fed unenriched Artemia [endogenous ascorbic acid (AA) concentration of 166 μg g−1 dry weight (dw)], Artemia supplemented with algae (AA concentration 594 μg g−1 dw) or with ascorbyl-2-polyphosphate (A2P) (AA concentration 11 737 μg g−1 dw) to examine possible benefits of AA enhancement on culture. Plain or algal-enriched Artemia were fed continuously for 28 days in two treatments during the study. Four other treatments received A2P-enriched Artemia on a progressive basis starting from the commencement of the trial (D-0), the third (D-3), sixth (D-6) or ninth day (D-9) of Stage I (14 days) and similarly during Stage II (14 days). Prior to the commencement of A2P supplementation, plain Artemia were supplied to these animals. By Stage III (28 days feeding), algal, D-0 and D-3 phyllosoma had attained the largest size. The uptake and retention of AA by Stage III phyllosoma appeared to be dose-dependent with the highest concentration of AA incorporation evident in D-0 phyllosoma (1816 μg g−1 dw), while algal and plain phyllosoma contained the lowest concentrations (600 and 300 μg g−1 dw, respectively). Survival at Stage III was highest in D-0 phyllosoma (89%) and lowest in plain phyllosoma (51%). There was a positive relationship between phyllosoma AA concentration and larval survival (R2 = 0.8328, P < 0.0001). D-0 phyllosoma had the lowest stress index when subjected to an osmotic/temperature activity test, indicative of better survival in culture compared to plain, algal and D-9 phyllosoma, which had consistently higher indices. A negative relationship existed between phyllosoma AA concentration and stress indices at Stage III (R2 = 0.9263, P < 0.0001), suggesting that AA from the Artemia diet conferred stress resistance.

Biotransformation of halophenols using crude cell extracts of Pseudomonas putida F6.

Crude cell extracts of Pseudomonas putida F6 transformed 4-substituted fluoro-, chloro-, bromo- and iodo-phenol without the exogenous addition of cofactors. The rate of substrate consumption decreased with increasing substituent size (F>Cl>Br>I). Biotransformations resulted in greater than 95% utilisation of the halogenated substrate. Product accumulation was observed in incubations with 4-chloro, 4-bromo- and 4-iodo-phenol. These products were identified as the corresponding 4-substituted catechols. Transformation of 4-fluorophenol did not result in the accumulation of the corresponding catechol; however, manipulation of the reaction conditions by incorporation of ascorbic acid culminated in the formation of 4-fluorocatechol. Cell extracts of P. putida F6 also showed activity towards a 3-substituted phenol, namely 3-fluorophenol, resulting in the formation of a single product, 4-fluorocatechol.

Combined effect of ascorbic acid and gamma irradiation on microbial and sensorial characteristics of beef patties during refrigerated storage.

The present study was undertaken to evaluate the effect of ascorbic acid concentrations (0.03 to 0.5%) and irradiation doses (0.5 to 4 kGy) on microbial growth, color coordinates (L, a, and b), and sensory characteristics (taste and odor) of beef patties during storage at 4 +/- 1 degrees C. Ascorbic acid was also compared to citric acid at a similar pH value in order to differentiate the effects of ascorbic acid from those of pH reduction. Results showed significant reduction (p< or = 0.05) of aerobic plate counts (APCs) and total coliforms, and a significant interaction (p< or = 0.05) between ascorbic acid and irradiation dose was observed. The irradiation treatment had detrimental effects on redness, yellowness, and hue angle values of meat. However, incorporation of ascorbic acid into the meat before irradiation resulted in significant (p< or = 0.05) stabilization of color parameters. The color improvement obtained with ascorbic acid was not related to the pH reduction. Also, no significant detrimental effect on taste or odor was found in irradiated samples containing ascorbic acid.

Effects of L-ascorbic acid on the conversion of cassava starch

Studies have previously shown that the inclusion of ascorbic acid with starches, which are then gelatinised in excess water, causes differences in the amount of starch conversion. Also, incorporation of ascorbic acid in dilute pastes has been shown to decrease the viscosity of the paste when stored at a range of temperatures. In this study the pasting behaviour of cassava starch in the presence of ascorbic acid has been followed using a Rapid Visco Analyser. Decreases in peak and final viscosity were apparent, with the largest reductions being with the highest amounts of ascorbic acid addition (0.1%). In contrast with other published work the effects of ascorbic acid on heating pastes made with a high starch content were studied. The amount of conversion of cassava starch was estimated by assessing the volume occupied by the swollen granules and the amount of soluble material released. Viscosity measurements of the starch polysaccharides after solubilisation in alkali were also established. Pasting behaviour of the samples that had previously been heated in the presence of ascorbic acid and buffer was also assessed. Ascorbic acid inclusion seemed to affect starch conversion with the results for peak and final viscosity correlating with the alkaline viscosity estimates. The inclusion of ascorbic acid also alters the appearances of the starch sample. Ascorbic acid inclusion made the samples lighter in colour and they did not hold their shape after processing.

The effect of supplemental ascorbic acid in enriched live food for Clarias gariepinus larvae at startfeeding

The effect of three dietary ascorbic acid (AA) concentrations, each applied via two feed types, on production characteristics and physiological condition of African catfish ( Clarias gariepinus) larvae has been assessed in two 10-day culture trials. Three treatments received only Artemia nauplii enriched with an experimental emulsion containing 0, 10, or 20% ascorbyl palmitate (AP) and yielding 530, 1200 and 1600 μg AA g −1 DW Artemia, respectively; the other three treatments were fed the same Artemia diets which were partially substituted by an artificial diet containing no vitamin C (ratio 20:80). No differences in survival could be observed; however, from day 6 onwards the 20%-AP group showed significantly better growth compared to the 0%- and 10%-AP treatments. For the cofeeding series, the same positive, but not significant, influence of vitamin C on dry weight was found. Moreover, the animals receiving the highest vitamin C supplementation displayed a considerably lower stress sensitivity than those of the 0%- and the 10%-AP groups, for both the 100%- and the 20%- Artemia series. These differences had occurred by day 2, which might be indicative of the importance of AA in early development. A second trial, which was a repetition of the first one, revealed the same tendencies; however, growth differences were smaller, probably due to the higher incorporation levels of AA obtained in the live diet (530, 1700 and 2300 μg AA g −1 DW) and in the catfish larvae. Growth results of both experiments were supported with data from the ultrastructural evaluation of the hepatocytes; i.e. a more organized cell compartmentation and better-structured cell organelles in the 20%-AP group of the Artemia series compared to the control are indicative of a more active metabolism. The slow growth in the cofeeding series was documented by the poor condition of the hepatocytes. In a third experiment it was verified that the growth effect of the 20%-AP boosted Artemia diet was the result of the extra AA incorporation and not of the concomitant palmitic acid (PA), which was set free after hydrolysis of AP in the Artemia nauplii, and which could possibly be used as a supplemental energy source. The three treatments were fed Artemia nauplii enriched with 0% AP, 12% PA and 20% AP, respectively. Growth and stress resistance of the latter group were significantly higher compared to the control and the PA-supplemented fish. To our knowledge this is the first evidence for the positive role of high dietary vitamin C levels (more than 1500 μg AA g −1DW) on larval development of an aquaculture species, and more specifically of C. gariepinus.

Dietary ascorbic acid requirements during the hatchery production of turbot larvae

The effect of high ascorbic acid (AA) levels transferred through enriched live food was evaluated for turbot Scophthalmus maximus larvae in two consecutive feeding experiments. The same feeding strategy was applied to all treatments, except for the AA content of the live food which was manipulated through bioencapsulation with ascorbyl palmitate. This resulted finally in a low, medium and high‐AA treatment. The AA incorporation levels in the turbot larvae (up to 1400 μg AA g DW−1) were correlated with the AA content of the live food administered. However, feeding the high AA concentration resulted in the same values as for the medium treatment, indicating a saturation of the body AA reserves. Under standard culture conditions, no differences in growth nor overall survival could be detected among the different groups, illustrating that the dietary AA requirements of larval turbot are met by non‐enriched live food containing already 500 μg AA g DW−1. The larvae of the high‐AA treatment, however, showed a better pigmentation rate (47 and 32% for experiments 1 and 2, respectively) compared to the other groups (35 and 25%, respectively). Evaluation of the physiological condition applying a salinity stress test revealed an improvement by feeding extra AA, significantly in the medium‐AA treatment. Though not significantly different, cumulative mortalities after challenge with Vibrio anguillarum amounted to 50% for the control v. 40% for the fish fed medium and high‐AA diets, respectively. Moreover, the onset of mortalities in this study was slower (not significantly) for the fish fed the extra AA.

Dietary ascorbic acid requirements during the hatchery production of turbot larvae

The effect of high ascorbic acid (AA) levels transferred through enriched live food was evaluated for turbot Scophthalmus maximus larvae in two consecutive feeding experiments. The same feeding strategy was applied to all treatments, except for the AA content of the live food which was manipulated through bioencapsulation with ascorbyl palmitate. This resulted finally in a low, medium and high-AA treatment. The AA incorporation levels in the turbot larvae (up to 1400 μg AA g DW−1) were correlated with the AA content of the live food administered. However, feeding the high AA concentration resulted in the same values as for the medium treatment, indicating a saturation of the body AA reserves. Under standard culture conditions, no differences in growth nor overall survival could be detected among the different groups, illustrating that the dietary AA requirements of larval turbot are met by non-enriched live food containing already 500 μg AA g DW−1. The larvae of the high-AA treatment, however, showed a better pigmentation rate (47 and 32% for experiments 1 and 2, respectively) compared to the other groups (35 and 25%, respectively). Evaluation of the physiological condition applying a salinity stress test revealed an improvement by feeding extra AA, significantly in the medium-AA treatment. Though not significantly different, cumulative mortalities after challenge with Vibrio anguillarum amounted to 50% for the control v. 40% for the fish fed medium and high-AA diets, respectively. Moreover, the onset of mortalities in this study was slower (not significantly) for the fish fed the extra AA.

The dynamics of gonad growth and ascorbate status in yellow perch, Perca flavescens (Mitchill)

Yellow perch, Perca flavescens (Mitchill), caught in the wild (Grand St Marys Lake, Ohio) and cultured in the laboratory were used to examine the relationship between gonad development and the concentration of ascorbic acid in ovaries, testes, liver and kidney. In wild fish the ovary had concentrations of ascorbate twice as high (75-102 μg g-1) as the testis (28-48 μg g-1), and when the differences in gonadosomatic indices between sexes were accounted for, females deposited 10-fold more of this essential nutrient in gonads than did males. Yellow perch cultured intensively throughout the gonadal recrudescence had shown a decrease of ascorbate concentration in the kidney, liver and ovary during winter, most likely due to the low dietary level of the vitamin. Diet enrichment with ascorbic acid resulted in a dramatic increase of ascorbate in ovary and other tissues. Incorporation of ascorbic acid may take place during the late vitellogenesis, and this vitamin deserves to be studied as a quality indicator in fish eggs.

Sialoresponsin and an antiviral action of ascorbic acid.

Several pathogens, both viral and bacterial, employ the enzyme neuraminidase (N-acetylneuraminate glycohydrolase, EC 3.2.1.1.8). The neuraminidase renders ineffective the hemagglutinin inhibitory mucins that confine the pathogens in a coating of host mucins. Sialoresponsin is a receptor "decoy" that inhibits neuraminidase. Several known antiviral agents, including ascorbic acid, inhibit neuraminidase. It is proposed that ascorbic acid may mediate an antiviral effect through the incorporation of ascorbic acid or some derivative of ascorbic acid as a part of the sialoresponsin molecule. Whether ascorbic acid works alone as a pharmacological inhibitor, or is incorporated in sialoresponsin as a physiological inhibitor; it may be useful against pathogens that employ neuraminidase.

Protection of estrogenic hormones by ascorbic acid during chromatography

Oxidative decomposition of phenolic hormones during thin-layer chromatography can be avoided by the incorporation of ascorbic acid into the plates. The application of this technique to the purification of urinary extracts is described.

Differential determination of D-isoascorbic acid and L-ascorbic acid in guinea pig organs.

To test the theory that D-isoascorbic acid (IAA) was not retained by the organs of animals, guinea pigs were fed a synthetic diet containing ascorbic acid (AA) plus IAA. The incorporation of IAA and AA was determined by making use of the different rates of osazone formation of oxidized AA and IAA with 2,4-dinitrophenylhydrazine. It was found that the organs of the guinea pigs retained a significant quantity of IAA which replaced a corresponding quantity of AA. The incorporated IAA could, in turn, be replaced by AA when only AA was subsequently given in the diet.

STABILIZATION OF THE ALCOHOLIC POTASSIUM HYDROXIDE IN COLORIMETRIC 17-KETOSTEROID DETERMINATIONS1

The 2.5 normal potassium hydroxide in absolute ethanol employed in the Callow modification of the Zimmermann reaction (Callow et ah, 1938), cannot be used for more than a few days after preparation. A marked increase in the color developed in reagent blank mixtures indicates deterioration even before the solution turns yellow. Various observations suggested that these changes are primarily oxidative. It therefore appeared probable that a suitable reducing agent would stabilize the solution. Experimental findings described below reveal that incorporation of ascorbic acid in the reagent, and maintenance of an atmosphere of nitrogen, yield preparations which remain stable for at least 3 months. Preparation of 2.5 N Alcoholic Potassium Hydroxide Protected with Ascorbic Acid and Nitrogen.—One hundred ml. of absolute alcohol are measured into a glass stoppered Pyrex bottle chilled in crushed ice. Two lots of 15 mg. each of ascorbic acid are weighed.