New investigations on hematoxylin, hematein, and hematein-aluminium complexes. I. Spectroscopic and physico-chemical properties of hematoxylin and hematein.

Analytically pure hematoxylin (Htx), pentaacetylhematoxylin (PAHtx), and hematein (Hm) were isolated and characterized by 1H-NMR spectroscopy. The VIS/UV spectra of Htx and Hm were recorded in MeOH and in H2O at various pH values. The molar extinction coefficients of the long wavelength absorption bands are reported. The pKa value for the 1st acidic dissociation step of Hm has been determined from the pH dependency of the absorption spectra of Hm in aqueous buffer solutions. Finally, the absorption spectra are qualitatively discussed.     

Spectroscopic and staining studies of the ripening and overripening of aluminum hematoxylins

Summary Spectroscopic study of alum hematoxylins during the course of ripening, optimal status and final deterioration reveals an absorption spectrum with, maxima at about 560 m and at about 430 m. Early in the course of ripening the optical density ratio D560/D430 rises above 3.0 and this ratio is maintained at 3–4 through most of the useful life of the stain batch. In the final stage of deterioration the density at 430 m, rises and that at 560 fails, so that in late deterioration stages D560/D430 may fall as low as 0.5.As ripening progresses the density at 560 m, gradually rises so that when it reaches a value of 0.350 (1 cm cell, dilution 10 mg hematoxylin/1 liter) adequate staining is achieved. This level seems the same whether ripening be done by aeration, oxygen bubbling, sodium iodate or mercuric acetate. As ripening progresses further the D560 value may rise above 0.800 with air or iodate oxidation. Maximum values are usually lower with iodate oxidation. This finding tends to agree with the ancient impression that natural ripening provides the best alum hematoxylins.In agreement with the known variable hematein content (20–100%) of comercial hematoxylins the required doses of sodium iodate to reach initial satisfactory staining performance are variable and considerably lower than the precalculated stoichiometric amount for the complete conversion of fully reduced hematoxylin to hematein. The often quoted figure of 200 mg per gram hematoxylin agrees closely with the amount of potassium iodate which would be needed to convert 1 gm pure crystalline hematoxylin (M.W. 356.32) completely to hematein. It was found that 80 mg NaIO₃ per gram of reduced hematoxylin was adequate and for commercial samples 40–60 mg usually sufficed.The final overoxidation product of hematoxylin appears to be a complex mixture, not readily reduced back to hematein. Since 185.13 mg NaIO₃ is the theoretical amount for complete conversion to hematein of fully reduced hematoxylin, the amount of iodate required to attain maximum optical density at 560 m, is always less than that. The observed amounts range from 60 to 140 mg per gram of hematoxylin, and excesses apparently operate to convert hematein to further oxidation products.

---

Zusammenfassung Alaun-Hämatoxylin zeigt bei spektroskopischen Untersuchungen während der Reifung, der besten Färbungszeit und beim endgültigen Zerfall 2 Absorptionsmaxima. Sie liegen etwa bei 560 m und bei 430 m. Das Verhältnis der optischen Dichte D560/D430 steigt im Laufe der Reifung bereits früh auf 3,0 und bleibt während der Brauchbarkeit der Farblösung beständig zwischen 3,0 und 4,0. Im letzten Stadium des Zerfalls steigt die optische Dichte der Farblösung bei dem Maximum von 430 m während die von 560 m sinkt, so daß zu dieser Zeit D560/D430 bis auf 0,5 abfallen kann.Bei der Reifung steigt die optische Dichte langsam an. Wenn ein Wert von 0,350 (1 cm Zelle, 10 mg Hämatoxylin/Liter) erreicht ist, werden die Färbungen zufriedenstellend. Es scheint gleichgültig zu sein, ob die Reifung durch Luft, durch Hindurchblasen von Sauerstoff oder chemisch mittels Natriumjodat oder Quecksilberacetat erfolgt ist. Bei fortschreitender Reifung kann der D560-Wert bei Anwendung von Luft oder Jodat auf über 0,800 steigen. Die Maximalwerte sind bei Jodatoxydation jedoch stets etwas niedriger als bei Luftoxidation. Diese Befunde scheinen mit dem alten Eindruck übereinzustimmen, daß natürliche Reifung das beste Alaun-Hämatoxylin erzeugt.Da bekanntlich der Hämateingehalt der komerziellen Hämatoxiline stark schwankt (zwischen 20 und 100%), ist auch die zur Herstellung einer zufriedenstellenden Farblösung erforderliche Menge an jodsaurem Natrium sehr unterschiedlich. Sie ist beträchtlich niedriger als die im voraus berechnete stöchiometrische Menge für die vollständige Oxydation des voll reduzierten Hämatoxylins zu Hämatein. Die oft genannte Zahl von 200 mg NaJO₃ pro Gramm Hämatoxylin stimmt genau mit der Menge von KJO₃ überein, die benötigt wird, um 1 g kristallines Hämatoxylin (M.W. 356,52) in Hämatein zu verwandeln. Es wurde festgestellt, daß pro Gramm voll reduzierten Hämatoxylins 80 mg NaJ0₃ angemessen sind und daß gewöhnlich für Handelsware 40–60 mg genügen.Ein durch endgültige Überoxydation von Hämatoxylin entstandenes Produkt scheint eine komplexe Mischung zu sein, die nicht leicht wieder zu Hämatein reduziert werden kann. Da 185,13 mg NaJO₃ die theoretische Menge für die vollständige Oxydation von voll reduziertem Hämatoxylin zu Hämatein ist, ist die benötigte Menge von Jodat, um die höchste optische Dichte (D560) zu erreichen, immer etwas geringer als 185,13 mg. Die beobachteten Mengen liegen zwischen 60 und 140 mg pro Gramm Hämatoxylin. Überschüsse scheinen Hämatein in weitere Oxydierungsprodukte zu verwandeln.

---

---

Supported by a Student Fellowship of the Biological Stain Commission

---

Supported in part by Research Grant No. C-4816 National Cancer Institute, National Institutes of Health.     

New investigations on hematoxylin,hematein, and hematein-aluminium complexes

Summary Analytically pure hematoxylin (Htx), penta-acetylhematoxylin (PAHtx), and hematein (Hm) were isolated and characterized by ¹H-NMR spectroscopy. The VIS/UV spectra of Htx and Hm were recorded in MeOH and in H₂O at various pH values. The molar extinction coefficients of the long wavelength absorption bands are reported. The pK _a value for the 1st acidic dissociation step of Hm has been determined from the pH dependency of the absorption spectra of Hm in aqueous buffer solutions. Finally, the absorption spectra are qualitatively discussed.     

Microbial content of commercial South African high-moisture dried fruits

AIM: The aim was to evaluate commercially available South African high-moisture dried fruits (HMDF) for the microbial, moisture and SO2 contents, as well as aw and pH. METHODS AND RESULTS: The microbial content of commercially available HMDF was evaluated using nine different growth media. The moisture content, aw) SO2 and pH of each product were determined using standard analytical methods. It was found that the highest total aerobic counts were generated from high-moisture dried (HMD) prunes and raisins. The most frequent spoilers were members of the genus Bacillus. Fungal counts were also very high in the apricot products, exceeding the limit of 1000 CFU g(-1) as set by HMDF producers. Members of the genus Staphylococcus were found in the HMD raisins and Salmonella and thermoduric organisms were isolated from the HMD prunes. CONCLUSIONS: The microbial levels of South African HMDF were within the limits set, with the exception of apricots. SIGNIFICANCE AND IMPACT OF STUDY: The study shows the presence of Salmonella, Staphylococcus and Clostridium in South African HMDF. The presence of thermoduric organisms indicated that the current pasteurization process is not adequate and that the addition of preservatives would be an additional method to ensure safety and quality.     

The content of gonadotropic hormones in commercial immunoglobulin preparations

The authors carried out a comparative quantitative and biological determination of gonadotropins in 32 batches of immunoglobulin preparations made of the abortive, placental, and donor blood sera. The maximal amounts of gonadotropins were contained in preparations obtained from the abortive blood serum. It was shown that purification by Kohn's method (variant B) led only to the partial purification of immunoglobulins from the gonadotropin admixtures.     

The reaction of unsaturated fats with the acid hematein test

Summary The reaction of saturated and unsaturated oils and fatty acids was examined in vitro with the Baker's acid hematein test. It has been found that oils whose molecules contain fatty acid components of two or more double bonds give a positive reaction with the acid hematein technique. The intensity of the reaction runs parallel with the number of double bonds.     

Relationship of melanin degradation products to actual melanin content: application to human hair

Methods not only for characterizing but also for quantitating melanin subtypes from the two types of melanin found in hair--eumelanin and pheomelanin--have been established. In relation to testing for drugs of abuse in hair, these methods will allow for correction of drug binding to specific melanin subtypes and will serve to improve drug measurement in hair. 5,6-Dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) make up the majority of the eumelanin polymer while benzothiazene units derived from 2-cysteinyl-S-Dopa (2-CysDopa) and 5-cysteinyl-S-Dopa (5-CysDopa) compose the majority of the pheomelanin polymer. Our results show that: (1) pyrrole-2,3-dicarboxylic acid (PDCA) and pyrrole-2,3,5-tricarboxylic acid (PTCA), markers for DHI and DHICA units, respectively, are produced in 0.37 and 4.8% yields, respectively, when melanins are subjected to alkaline hydrogen peroxide degradation, (2) 3-aminotyrosine (3AT) and 4-amino-3-hydroxyphenylalanine (AHP), markers for 2-CysDopa and 5-CysDopa, respectively, are produced in 16 and 23% yield, respectively, when subjected to hydriodic acid hydrolysis, and (3) that black human hair contains approximately 99% eumelanin and 1% pheomelanin, brown and blond hair contain 95% eumelanin and 5% pheomelanin; and red hair contains 67% eumelanin and 33% pheomelanin. These data will allow deeper investigation into the relationship between melanin composition and drug incorporation into hair.     

Assaying the quality of cDNA libraries

Once a cDNA library has been constructed, it is very useful to be able to easily assess the quality of the library. Because actin is a ubiquitous sequence, this assessment has been accomplished by probing filter lifts and Southern blots of libraries with an actin cDNA probe. These methods provide information about the percent of actin-positive clones and the degree of completeness of cDNA clones in a library. Results of these methods are correlated with the success of finding full-length clones of interest.     

Effects of natural and commercial diets on the fatty acid content of European grayling

Eicosaenoic acid (20:1ω9) and docosaenoic acid (22:1ω11) levels were about 10 and 100 times higher in food pellets fed to cultured grayling than in the insect larvae on which wild grayling fed. Among the PUFA, docosahexaenoic acid (DHA) was very high in the pellets, resulting in an unnaturally elevated, and probably unbalanced, ω3/ω6 ratio of 7–13 in the cultured fish whereas the same ratio varied only from 4 to 6 in the wild fish. Despite very low DHA levels in the native food, wild grayling muscle tissue contained relatively high amounts of DHA. DHA is probably not essential in the diet of grayling.     

Assaying the polyadenylation state of mRNAs

The poly(A) tail present at the 3' end of most eukaryotic mRNAs can play a critical role in message translation and stability. Therefore, identifying alterations in poly(A) tail length can yield important insights into an mRNA's function and subsequent physiological impact. Here, we present three methods for assaying polyadenylation of a specific mRNA in the context of total cellular RNA. The first method described, oligo(dT)/RNase H-Northern analysis, is the classic labor-intensive assay for polyadenylation and is included for historical reference and as a potential experimental control for the poly(A) test (PAT) assays described subsequently. The PAT methods-rapid amplification of cDNA ends-PAT (RACE-PAT), and ligase-mediated PAT (LM-PAT)-are polymerase chain reaction-driven assays that allow speed, sensitivity, and length quantitation. The PAT assays can be conducted in a single day and can readily detect the poly(A) status of an mRNA present in subnanogram quantities of total cellular RNA.     

Quantitative bioreduction assays for calibrating spore content and viability of commercial Bacillus thuringiensis insecticides

The redox dyes MTT (3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyl tetrazolium bromide, thiazolyl blue) and XTT (sodium 3′- {1-[(phenylamino)-carbonyl]-3,4-tetrazolium}-bis{4-methoxy-6-nitro} benzene sulphonic acid hydrate) were used as dosimetry reporters in liquid (multi-well) and solid support (membrane) assays to estimate spore viability and content of commercial BT products derived from fermentation of Bacillus thuringiensis subsp kurstaki (Btk) and subsp israelensis (Bti). QC tests on five BT products were done using spore, protein and gene contents, and morphology (scanning electron microscopy) as indicators. Spore levels (6–40 × 10⁹ colony forming units (CFU) ml⁻¹) were approximately equivalent when based on International Units (IU) of potency. Spore viability was highly stable over a broad range of temperatures and pHs but germination and growth were restricted (optima: pH ≈ 7.5 and 37°C). Quantitative bioreduction activity (QBA) of MTT and XTT correlated with vegetative cell production. Depending on manipulation of pre-assay conditions, both dyes could discriminate doses from ∼2 to 10⁹ spores (or 10⁻³ to 10⁶ IU). Non-toxic effects of XTT and its formazan product enabled automated collection of data on growth and dose. Solid support assays also reliably estimated product dosage by in situ detection of CFU. With appropriate reference dilutions of microbe-containing products the QBA assays can provide high throughput QC monitoring of product comparisons and field release in aerial spray or water injection applications. Received 18 December 1996/ Accepted in revised form 11 March 1997     

Micromethod for Assaying Serum Levels of Erythromycin

A micromethod for assaying serum levels of erythromycin is described. The assay had the following characteristics: detection of 0.03 to 0.035 mug/ml, a long-range curve which minimizes sample dilution, 0.04 ml for a single measurement (0.3 ml required for full coverage), and utility for a variety of body fluids. The method employs radial diffusion from small paper discs which were saturated by capillary action rather than by dipping or pipetting. Although the method was designed to handle serum in which the sample volume is limited, statistical analysis demonstrated that the method has satisfactory precision for routine use. A study of 10 consecutive assays indicates a precision of +/-12% at the 95% confidence limits. The method of least squares was used to calculate the line of best fit, and the statistics were developed on this basis. The assay method is applicable to a variety of antibiotics.