Preservation of cocoa antioxidant activity, total polyphenols, flavan-3-ols, and procyanidin content in foods prepared with cocoa powder.

Little is known about the effects of common cooking processes on cocoa flavanols. Antioxidant activity, total polyphenols (TP), flavanol monomers, and procyanidin oligomers were determined in chocolate frosting, a hot cocoa drink, chocolate cookies, and chocolate cake made with natural cocoa powder. Recoveries of antioxidant activity, TP, flavanol monomers, and procyanidins ranged from 86% to over 100% in the chocolate frosting, hot cocoa drink, and chocolate cookies. Losses were greatest in the chocolate cake with recoveries ranging from 5% for epicatechin to 54% for antioxidant activity. The causes of losses in baked chocolate cakes were investigated by exchanging baking soda with baking powder or combinations of the 2 leavening agents. Use of baking soda as a leavening agent was associated with increased pH and darkening color of cakes. Losses of antioxidant activity, TP, flavanol monomers, and procyanidins were associated with an increased extractable pH of the baked cakes. Chocolate cakes made with baking powder for leavening resulted in an average extractable pH of 6.2 with essentially complete retention of antioxidant activity and flavanol content, but with reduced cake heights and lighter cake color. Commercially available chocolate cake mixes had final pHs above 8.3 and contained no detectable monomeric flavanols after baking. These results suggest that baking soda causes an increase in pH and subsequent destruction of flavanol compounds and antioxidant activity. Use of an appropriate leavening agent to moderate the final cake pH to approximately 7.25 or less results in both good leavening and preservation of cocoa flavanols and procyanidins.

Transplacental transfer of the opioid growth factor, [Met(5)]-enkephalin, in rats.

Placental transfer of the pentapeptide [Met5]-enkephalin, known to function as a growth regulating factor and neuromodulatory agent, was studied in pregnant Sprague-Dawley rats. Using separation by reversed phase high-performance liquid chromatography, and analysis by derivative spectroscopy, [Met5]-enkephalin was detected in 20-day-old fetal tissue including brain, heart, lung, and kidney. Fetal tissues from pregnant rats given an injection of 40 mg/kg [Met5]-enkephalin on gestation day 20 had markedly elevated levels of peptide within 1 h, indicating the transplacental transfer of this opioid. [Met5]-enkephalin levels were increased from control samples at 1, 2, 4, and 14 h post-injection of peptide, but not at 24 h. Evaluation of breakdown products of [Met5]-enkephalin, along with the related peptide [Leu5]-enkephalin, revealed that elution times differed substantially from [Met5]-enkephalin. These data indicate that [Met5]-enkephalin is present in fetal organs, crosses the placenta, does not appear to be restrictive in organ specificity, and is sustained in fetal tissues at detectable levels for at least 14 h. Given that [Met5]-enkephalin tonically inhibits DNA synthesis in the fetus, these results raise the question of whether an elevated level of this peptide (either maternally or from the fetus) may be detrimental to cellular ontogeny in the fetus, and perhaps have long-term implications for postnatal development.

Determination of conjugated linoleic acid (CLA) concentrations in milk chocolate.

The fatty acids from a series of milk-chocolate-based confectionery samples were analyzed as methyl esters by GC to determine the presence and amount of conjugated linoleic acid (CLA). A single peak corresponding to the 9-cis,11-trans isomer and ranging from less than 0.1% to nearly 0.2% of the total fatty acids, corresponding to up to 0.3 mg per g of chocolate, was observed. One of the chocolate extracts and a milk extract were subjected to silver ion HPLC and GC-MS in order to confirm the identity of the major isomer and tentatively identity minor isomers.

A rapid sample preparation method for the HPLC determination of the opioid antagonist naltrexone in serum.

HPLC with UV and electrochemical detection has routinely been employed for the determination of the opioid antagonist naltrexone in serum. Sample preparation protocols range from liquid/liquid to solid phase extraction. The sample preparation described in this communication uses ultrafiltration as the mode of sample preparation prior to HPLC analysis. The method is accurate, precise and saves considerable time compared to previously published techniques.

Nitric oxide synthase imunolabeling in the molluscan CNS and peripheral tissues.

NOS immunoreactivity was assayed in CNS and peripheral tissues of the sea slugs Pleurobranchaea californica, Tritonia diomedea and Aplysia californica using different antisera against mammalian nitric oxide synthase in Western blots. Polyclonal anti-nNOS labeled at 250, 185, 170, 155, 100, 75, and 65 kD in extracts of Pleurobranchaea CNS, salivary gland and esophagus but not of gills or muscle. The labeling pattern for Tritonia in bands at 250, 200, 120/110, 100, 69, 65, and 60 kD differed somewhat. Anti-nNOS labeling in Aplysia was markedly different, with bands labeled only at 69 and 60 kD in CNS extracts, and at 200, 190, 69 and 60 kD in salivary and esophagus extracts. The wide variation in NOS immunoreactivity is consistent with species differences in tissue localization and biochemical properties of molluscan NOS isoforms.

Oscillation and light induction of timeless mRNA in the mammalian circadian clock.

Circadian rhythms in Drosophila melanogaster depend on a molecular feedback loop generated by oscillating products of the period (per) and timeless (tim) genes. In mammals, three per homologs are cyclically expressed in the suprachiasmatic nucleus (SCN), site of the circadian clock, and two of these, mPer1 and mPer2, are induced in response to light. Although this light response distinguishes the mammalian clock from its Drosophila counterpart, overall regulation, including homologous transcriptional activators, appears to be similar. Thus, the basic mechanisms used to generate circadian timing have been conserved. However, contrary to expectations, the recently isolated mammalian tim homolog was reported not to cycle. In this study, we examined mRNA levels of the same tim homolog using a different probe. We observed a significant (approximately threefold) diurnal variation in mTim expression within mouse SCN using two independent methods. Peak levels were evident at the day-to-night transition in light-entrained animals, and the oscillation persisted on the second day in constant conditions. Furthermore, light pulses known to induce phase delays caused significant elevation in mTim mRNA. In contrast, phase-advancing light pulses did not affect mTim levels. The mTim expression profile and the response to nocturnal light are similar to mPer2 and are delayed compared with mPer1. We conclude that temporal ordering of mTim and mPer2 parallels that of their fly homologs. We predict that mTIM may be the preferred functional partner for mPER2 and that expression of mTim and mPer2 may, in fact, be driven by mPER1.

High-performance liquid chromatographic determination of ochratoxin A in artificially contaminated cocoa beans using automated sample clean-up.

A HPLC method is described for the analysis of ochratoxin A at low-ppb levels in samples of artificially contaminated cocoa beans. The samples are extracted in a mixture of methanol-water containing ascorbic acid, adjusted to pH and evaporated to dryness. Samples in this state are then placed onto a Benchmate sample preparation workstation where C18 solid-phase extraction operations are performed. The resulting materials are evaporated to dryness and analyzed by reversed-phase HPLC with fluorescence detection. The method was evaluated for accuracy and precision with R.S.D.s for multiple injections of sample and standard calculated to 1.1% and 2.5% for sample and standard, respectively. Recoveries of ochratoxin A added to cocoa beans ranged from 87-106% over the range of the assay.

Naltrexone is not detected in preweaning rats following transplacental exposure: implications for growth modulation.

Extracts of brain and heart from rats at birth and postnatal days 2 and 10 were evaluated for naltrexone following maternal injection of 50 mg/kg opioid antagonist throughout gestation. Samples were prepared by ultrafiltration, lyophilized, reconstituted in mobile phase, and separated by reversed-phase high performance liquid chromatography with ultraviolet detection. Qualitative analysis revealed the presence of naltrexone in tissues from neonates, but not in rats of 2 and 10 days, that were transplacentally exposed to drug. These results confirm earlier reports showing that naltrexone, maternally administered, passes through the placenta and enters the fetus. Moreover, the data suggest that the somatic and neurobiological acceleration observed in offspring exposed to naltrexone during gestation is not due to opioid receptor blockade during the postnatal period.

Localization and characterization of nitric oxide synthase in the rat suprachiasmatic nucleus: evidence for a nitrergic plexus in the biological clock.

Behavioral and electrophysiological evidence indicates that the biological clock in the hypothalamic suprachiasmatic nuclei (SCN) can be reset at night through release of glutamate from the retinohypothalamic tract and subsequent activation of nitric oxide synthase (NOS). However, previous studies using NADPH-diaphorase staining or immunocytochemistry to localize NOS found either no or only a few positive cells in the SCN. By monitoring conversion of L-[3H]arginine to L-[3H]-citrulline, this study demonstrates that extracts of SCN tissue exhibit NOS specific activity comparable to that of rat cerebellum. The enzymatic reaction requires the presence of NADPH and is Ca2+/calmodulin-dependent. To distinguish the neuronal isoform (nNOS; type I) from the endothelial isoform (type III), the enzyme activity was assayed over a range of pH values. The optimal pH for the reaction was 6.7, a characteristic value for nNOS. No difference in nNOS levels was seen between SCN collected in day versus night, either by western blot or by enzyme activity measurement. Confocal microscopy revealed for the first time a dense plexus of cell processes stained for nNOS. These data demonstrate that neuronal fibers within the rat SCN express abundant nNOS and that the level of the enzyme does not vary temporally. The distribution and quantity of nNOS support a prominent regulatory role for this nitrergic component in the SCN.

Identification of [Met5]-enkephalin in developing, adult, and renewing tissues by reversed-phase high performance liquid chromatography and radioimmunoassay.

Extracts of adult corneal epithelium, and developing and adult cerebellum, aorta, and heart, from rats were evaluated for [Met5]-enkephalin. Samples were prepared by ultrafiltration and solid phase extraction with a C-18 Sep-pak, separated by reversed phase high-performance liquid chromatography, and analyzed by radioimmunoassay (RIA). This qualitative analysis revealed the presence of [Met5]-enkephalin in all tissues but the adult cerebellum. These results confirm and extend earlier reports that have used RIA or immunohistochemistry with regard to the presence of this opioid peptide in developing and renewing tissues, and indicate that [Met5]-enkephalin is indeed being recognized by immunological assays.

Transplacental transfer of naltrexone in rats.

Extracts of fetal (20 days gestation) brain, heart, and liver were evaluated for naltrexone in rats 1 hour following maternal injection of 50 mg/kg opioid antagonist; adult plasma from the pregnant rats was analyzed. Samples were prepared by ultrafiltration, lyophilized, reconstituted in mobile phase, and separated by reversed phase high-performance liquid chromatography with ultraviolet detection. This qualitative analysis revealed the presence of naltrexone in all fetal tissues, as well as in adult plasma. These results indicate naltrexone, maternally administered, passes through the placenta and enters the fetus. The data would suggest that reports concerning somatic and neurobiological acceleration in offspring exposed to naltrexone during gestation may be the result of a direct opioid antagonist action in the fetus.

Resetting the biological clock: mediation of nocturnal CREB phosphorylation via light, glutamate, and nitric oxide.

Synchronization between the environmental lighting cycle and the biological clock in the suprachiasmatic nucleus (SCN) is correlated with phosphorylation of the Ca2+/cAMP response element binding protein (CREB) at the transcriptional activating site Ser133. Mechanisms mediating the formation of phospho-CREB (P-CREB) and their relation to clock resetting are unknown. To address these issues, we probed the signaling pathway between light and P-CREB. Nocturnal light rapidly and transiently induced P-CREB-like immunoreactivity (P-CREB-lir) in the rat SCN. Glutamate (Glu) or nitric oxide (NO) donor administration in vitro also induced P-CREB-lir in SCN neurons only during subjective night. Clock-controlled sensitivity to phase resetting by light. Glu, and NO is similarly restricted to subjective night. The effects of NMDA and nitric oxide synthase (NOS) antagonists on Glu-mediated induction of P-CREB-lir paralleled their inhibition of phase shifting. Significantly, among neurons in which P-CREB-lir was induced by light were NADPH-diaphorase-positive neurons of the SCN's retinorecipient area. Glu treatment increased the intensity of a 43 kDa band recognized by anti-P-CREB antibodies in subjective night but not day, whereas anti-alpha CREB-lir of this band remained constant between night and day. Inhibition of NOS during Glu stimulation diminished the anti-P-CREB-lir of this 43 kDa band. Together, these data couple nocturnal light, Glu, NMDA receptor activation and NO signaling to CREB phosphorylation in the transduction of brief environmental light stimulation of the retina into molecular changes in the SCN resulting in phase resetting of the biological clock.

Ontogeny of the opioid growth factor, [Met5]-enkephalin, and its binding activity in the rat retina.

The endogenous opioid peptide [Met5]-enkephalin is a tonically active opioid growth factor (OGF) with an inhibitory action on DNA synthesis in the developing rat retina. In this study, the ontogeny of the spatial and temporal expression of OGF and its binding activity was examined. OGF-like immunoreactivity was detected in the retina at gestation day (E) 20, but not at E18, and was localized to ganglion cell and neuroblast layers; immunochemical reaction was no longer seen in the retina by postnatal day 6. Native OGF was further identified and characterized by high-performance liquid chromatography (HPLC) studies and immunodot assays, which revealed that [Met5]-enkephalin was present in the neonatal, but not adult, rat retina. OGF binding activity was detected as early as E18 using [125I]-[Met5]-enkephalin and in vitro receptor autoradiography. Little OGF binding activity was noted for prenatal retinas, but appreciable activity was observed from birth to postnatal day 4; no OGF binding could be detected after postnatal day 5 or in the adult. These results reveal the transient appearance of the OGF, [Met5]-enkephalin, and its receptor binding activity in the developing mammalian retina, and show that their ontogeny coincides with the timetable of DNA synthesis of retinal neuroblasts.

Use of capillary electrophoresis-isoelectric focusing for the determination of bovine hemoglobin variants.

A capillary electrophoretic technique was developed to monitor patterns of hemoglobin production in young calves subjected to multiple phlebotomies. The method is similar to one previously described for the determination of human hemoglobin variants in whole blood using isoelectric focusing. After a single collection of one-half the circulating blood volume, there were obvious alterations in hemoglobin chain variants. HbA levels diminished as blood loss increased with minimum values corresponding to maximum blood loss. HbF levels did not appear to be affected. Also visible during the regenerative process were atypical overlapping peaks preceding the normal hemoglobin peaks. At the conclusion of the 18-day study, most of the electropherograms had returned to initial states. These changes were found to be a sensitive indicator of accelerated erythropoiesis in contrast to the standard technique of total hemoglobin determination by colorimetric means.

The response of various hematologic parameters in the young bovine subjected to multiple phlebotomies.

Specific guidelines for the optimal collection of blood from calves maintained as donors do not exist. This investigation was conducted to document erythrocytic changes in calves subjected to controlled blood loss. Two groups of Holstein calves ranging in age from 2 to 5 months were compared. Six animals were designated controls and six were subjected to a series of three blood collections over an 8 day period. Each collection was roughly 50% of the total circulating blood volume (30 ml/kg of body weight). Hematologic values were compared for 18 days. All calves survived the study. There was no significant difference in circulating cortisol levels or growth rate between groups. Packed cell volumes (PCVs) for the phlebotomized group did not return to original levels during the monitoring period (initial 36%, final 26%), but were within normal limits at 30 days. All phlebotomized calves had a high reticulocyte count (0% control, 11% phlebotomized). Reticulocytes were not observed until the PCV fell below 20. HbF to HbA ratios were a sensitive and acute indicator of accelerated erythropoiesis. Variant ratios stabilized within 18 days. Other parameters compared were red cell indices, total erythrocyte count, total leukocyte count, total plasma protein, total hemoglobin values, and neutrophil to lymphocyte ratios.

Effectiveness of skin absorption of tincture of I in blocking radioiodine from the human thyroid gland.

Topical application of tincture of iodine (I) was found to be effective in blocking the thyroid uptake of orally administered 131I in humans. Abdominal skin application of tincture of I resulted in an approximately 82% reduction in the uptake of 131I by the thyroid gland. The effectiveness varied among individuals and may have depended on the quantity applied and on the application site. In each study group, elevated levels of serum I were observed.

The effects of topical povidone I solution on serum iodide levels and thyroid uptake of 131I in dogs.

Topical application of povidone I solution in dogs has been found to be effective in producing significant elevations in serum iodide concentrations within 2 h after application. Among dogs treated with this preparation 2 h before oral administration of 131I, significant thyroid blocking persisted for at least 72 h.

Pharmacokinetics of cefazolin in guinea pigs.

The occurrence of antibiotic-related enterocolitis in guinea pigs restricts the use of many common antibiotics in this species. Cephaloridine, an antibiotic frequently recommended for this species, is no longer available and a substitute has yet to be explored. In this study, the potential therapeutic efficacy of cefazolin, also a first generation cephalosporin, was evaluated in guinea pigs by assessing pharmacokinetics, toxicity and the minimal inhibitory concentration for selected animal pathogens. Pharmacokinetics and toxicity were evaluated in four phases: single intramuscular injections, multiple intramuscular injections over 30 hours, multiple intramuscular injections over 5 days, and serum-protein binding studies. Antibiotic-related enterocolitis and irritation at the injection site occurred following high (100 mg/kg) repeated doses. At all dose levels, blood values exceeded the minimum inhibitory concentration for Bordetella bronchiseptica for only 1 hour postinjection. For Streptococcus and Staphylococcus sp., the drug half-life was 0.5 hours with peak concentrations occurring within 0.25 hours of injection. The volume of distribution of 0.5 l/kg indicated that there was extensive tissue distribution. Serum protein binding was approximately 85%. The short half-life and rapid plasma clearance rate (10.4 ml/min/kg) indicated that cefazolin is eliminated very rapidly from the guinea pig and may be of questionable therapeutic value.

An evaluation of ampicillin pharmacokinetics and toxicity in guinea pigs.

Sodium ampicillin was administered subcutaneously to 350-550 g male Dunkin Hartley guinea pigs at doses of 6, 8 and 10 mg/kg tid for 5 days. Over a period of 12 days, the lowest ampicillin dose appeared to be tolerated well. However, significant body weight reduction and mortality occurred with the two higher dosage regimens. Cecal cultures of dead animals confirmed the presence of Clostridium difficile, an organism associated with antibiotic-induced enterotoxemia. Assay of serum collected from ampicillin-treated animals revealed ampicillin concentrations of approximately 10 micrograms/ml at 5 minutes post-dosing which fell precipitously to less than 0.2 micrograms/ml at 60 minutes. Determination of biliary ampicillin levels during the 60 minutes after administration of a single 10 mg/kg SQ dose revealed concentrations ranging from 18 micrograms/ml to 90 micrograms/ml. Estimates of total urinary ampicillin content after a single 10 mg/kg SQ dose were less than 500 micrograms/animal at 7.5 minutes, but increased to greater than 2000 micrograms/animal at 60 minutes after dosing. Results of this study indicated that due to its short serum half-life, sodium ampicillin probably has little systemic therapeutic efficacy in guinea pigs. Because high concentrations of ampicillin accumulated in the urine and bile, the antibiotic probably would have therapeutic efficacy for urinary and intestinal infections. However, its associated toxicity at large doses probably precludes its use. In view of the rapid clearance of ampicillin in guinea pigs in comparison to other species, the pharmacokinetics of other antibiotics, especially those reported to be less toxic for guinea pigs, should be considered.