Technical note: Simultaneous carotenoid and vitamin analysis of milk from total mixed ration-fed cows optimized for xanthophyll detection.

Concentrations of retinol, α-tocopherol, and major carotenoids in dairy products are often determined simultaneously by liquid chromatography. These compounds have different polarity and solubility; thus, extracting them simultaneously can be difficult and inefficient. In milks with low carotenoid concentrations, the xanthophylls lutein and zeaxanthin may not be completely resolved using common extraction techniques. A simplified method was developed to optimize extraction efficiency and the limit of detection and limit of quantification (LoQ) of lutein and zeaxanthin in bovine milk without decreasing sensitivity to other vitamins or carotenoids. The developed method evaluates lutein, zeaxanthin, ß-carotene, retinol, and α-tocopherol simultaneously by ultra-high performance liquid chromatography-photodiode array detection. Common saponification temperatures (40-60°C) and concentrations of KOH in water (10-50% KOH wt/vol) were evaluated. Multiple solvents were evaluated for optimal xanthophyll extraction (diethyl ether, dichloromethane, hexane, and tetrahydrofuran) following saponification. The limit of detection and LoQ were defined as 3:1 and 10:1 signal-to-noise ratio, respectively. All experiments were performed in triplicate. The optimal saponification procedure was a concentration of 25% KOH at either 40 or 50°C. Saponified extracts solubilized in solutions containing diethyl ether had greater concentrations of lutein- than hexane- or tetrahydrofuran-based solutions, with peak areas above LoQ values. The solution containing diethyl ether solubilized similar concentrations of retinol, α-tocopherol, and ß-carotene when compared with other solutions. The proposed optimized method allows for the simultaneous determination of carotenoids from milk with increased lutein and zeaxanthin sensitivity without sacrificing recovery of retinol, α-tocopherol, and ß-carotene.

The effect of bleaching agents on the degradation of vitamins and carotenoids in spray-dried whey protein concentrate.

Previous research has shown that bleaching affects flavor and functionality of whey proteins. The role of different bleaching agents on vitamin and carotenoid degradation is unknown. The objective of this study was to determine the effects of bleaching whey with traditional annatto (norbixin) by hydrogen peroxide (HP), benzoyl peroxide (BP), or native lactoperoxidase (LP) on vitamin and carotenoid degradation in spray-dried whey protein concentrate 80% protein (WPC80). An alternative colorant was also evaluated. Cheddar whey colored with annatto (15 mL/454 L of milk) was manufactured, pasteurized, and fat separated and then assigned to bleaching treatments of 250 mg/kg HP, 50 mg/kg BP, or 20 mg/kg HP (LP system) at 50°C for 1 h. In addition to a control (whey with norbixin, whey from cheese milk with an alternative colorant (AltC) was evaluated. The control and AltC wheys were also heated to 50°C for 1 h. Wheys were concentrated to 80% protein by ultrafiltration and spray dried. The experiment was replicated in triplicate. Samples were taken after initial milk pasteurization, initial whey formation, after fat separation, after whey pasteurization, after bleaching, and after spray drying for vitamin and carotenoid analyses. Concentrations of retinol, a-tocopherol, water-soluble vitamins, norbixin, and other carotenoids were determined by HPLC, and volatile compounds were measured by gas chromatography-mass spectrometry. Sensory attributes of the rehydrated WPC80 were documented by a trained panel. After chemical or enzymatic bleaching, WPC80 displayed 7.0 to 33.3% reductions in retinol, ß-carotene, ascorbic acid, thiamin, α-carotene, and α-tocopherol. The WPC80 bleached with BP contained significantly less of these compounds than the HP- or LP-bleached WPC80. Riboflavin, pantothenic acid, pyridoxine, nicotinic acid, and cobalamin concentrations in fluid whey were not affected by bleaching. Fat-soluble vitamins were reduced in all wheys by more than 90% following curd formation and fat separation. With the exception of cobalamin and ascorbic acid, water-soluble vitamins were reduced by less than 20% throughout processing. Norbixin destruction, volatile compound, and sensory results were consistent with previous studies on bleached WPC80. The WPC80 colored with AltC had a similar sensory profile, volatile compound profile, and vitamin concentration as the control WPC80.

Calcium transport by sarcoplasmic reticulum of vascular smooth muscle: II. Effects of calmodulin and calmodulin inhibitors.

The role of calmodulin (CaM) in modulating calcium (Ca) uptake by sarcoplasmic reticulum (SR) of vascular smooth muscle was studied in saponin skinned strips of rat caudal artery. Exogenous CaM concentrations ranging from 0.3-1.8 microM did not statistically change the steady state MgATP-dependent Ca content, the MgATP-independent Ca content, or the oxalate-stimulated Ca influx. Calmidazolium (CDZ), W-7, and trifluoperazine (TFP) were used to examine the potential effect of an endogenous CaM pool on inward Ca transport. The IC50 of these antagonists for inhibition of Ca-CaM-stimulated phosphodiesterase activity and Ca-activated superprecipitation of canine aortic actomyosin was measured and found to be in the low micromolar range with a rank order of potency for inhibition of CDZ greater than TFP greater than W-7. In skinned tissues, micromolar concentrations of antagonists that inhibited CaM-mediated reactions in isolated enzyme systems did not reduce Ca content or oxalate-stimulated Ca influx. At higher concentrations of 100-200 microM, the MgATP-dependent Ca content was significantly reduced by TFP and W-7 but not by CDZ. The order of potency for inhibition of Ca uptake was TFP greater than W-7 greater than CDZ. The MgATP-independent Ca content was significantly decreased only by 200 microM TFP. Although none of these inhibitors significantly altered Ca efflux at concentrations up to 100 microM, Ca release was significantly stimulated by all three at 200 microM. The TFP-stimulated Ca release was partially inhibited by ruthenium red. The results indicate that neither exogenous CaM nor an endogenous CaM pool directly modulates inward Ca transport by the SR of saponin skinned caudal artery. The inhibition of Ca uptake produced by hundred micromolar concentrations of CaM antagonists fails to correlate with the order of and with the potency of inhibition measured in isolated enzyme systems. This suggests that the inhibition of Ca uptake produced by high concentrations of these antagonists may be independent of a specific interaction with CaM. The activation of Ca release by high concentrations of CaM antagonists may involve a nonspecific increase in membrane permeability as well as modulation of a membrane Ca channel.

Calcium transport by sarcoplasmic reticulum of vascular smooth muscle: I. MgATP-dependent and MgATP-independent calcium uptake.

The components of 45calcium (Ca) uptake were studied in saponin skinned rat caudal artery. The steady-state Ca content increased when the free Ca concentration was varied from 10(-8) to 10(-4) M but was reduced by azide when the free Ca concentration exceeded 3.1 microM. The azide sensitivity and low affinity for Ca were consistent with functional mitochondria. The azide-insensitive component consisted of a small bound and a larger releasable Ca fraction. After skinning in Triton X-100, approximately 4 mumol Ca/kg wet tissue remained, which represented a tightly bound but slowly exchangeable Ca pool. The Ca content was independent of the free Ca concentration and MgATP, and it was not released with A-23187 or Ca. The Ca content of the larger fraction was a higher order function of the free Ca concentration and was released with A-23187, indicating it resided within a membrane-bounded structure. Ca uptake by the releasable fraction was increased by oxalate, MgATP, phosphocreatine, temperature, phosphate, and ruthenium red and represents Ca sequestered by the sarcoplasmic reticulum (SR) with little contribution from other Ca binding or storage sites. It is described by the coefficients Umax = 96.94 mumol/kg wet tissue, K1/2 = 0.75 microM, and Hill coefficient = 1.70. The SR in this preparation regulates cytosolic Ca concentrations under physiological conditions and can accumulate Ca by MgATP-dependent and MgATP-independent process. The larger, MgATP-dependent Ca uptake is described by the coefficients Umax = 72.87 mumol/kg wet tissue, K1/2 = 0.8 microM, and Hill coefficient = 2.09 and is consistent with Ca sequestered by the Ca-transport ATPase of smooth muscle SR. The smaller, MgATP-independent uptake is described by the coefficients Umax = 24.14 mumol/kg wet tissue, K1/2 = 0.56 microM, and Hill coefficient = 1.01 and represents Ca sequestered by an unidentified mechanism or by a subpopulation of SR.

Calmodulin loss in vascular smooth muscle following Triton X-100 or saponin skinning.

The calmodulin (CaM) content of intact and chemically skinned strips of rat caudal artery was measured using a 125I-CaM radioimmunoassay. The total CaM measured following homogenization of arterial tissue with EGTA and EGTA/Triton X-100 was 2.58 mumol/kg wet tissue. Based on a smooth muscle volume of 40%, this value corresponds to a cellular CaM concentration of 6.5 microM. Approximately 97% of total CaM was soluble and approximately 3% was EGTA-nonextractable. Permeabilization of the plasmalemma with 0.15 mg/ml saponin or 0.5% Triton X-100 caused significant detergent-dependent loss of CaM. At the end of a 1 h skinning period, tissues exposed to saponin lost 30% of total CaM. By comparison, tissues skinned under the same conditions with Triton X-100 lost 50%. During a subsequent 4 h exposure to relaxing solution, total tissue CaM continued to decline. The exponential loss over the 5 h period was described by a first order model having diffusible and nondiffusible CaM components. The diffusible CaM component of saponin skinned tissue (59%) was significantly less than the diffusible component of those skinned with Triton X-100 (88%); however, the rate coefficients for CaM diffusion (0.78 h-1 and 0.91 h-1, respectively) did not statistically differ. The nondiffusible component of CaM was significantly larger in saponin treated strips (42%) than in Triton X-100 permeabilized tissue (12%). Arterial strips skinned with Triton X-100, which were subsequently exposed to relaxing solution for up to 22 h, lost significantly more CaM than those retained in Triton X-100 skinning solution for a comparable duration.(ABSTRACT TRUNCATED AT 250 WORDS)

A cognitive-behavioral study of self-reported stress factors in migraine headache.

Self-reported stress factors in migraine headache were examined from a cognitive-behavioral point of view. 18 migraine patients completed the Rathus Assertiveness Schedule and the Fear Survey Schedule. In addition, the migraine sufferers reported on all factors, either psychological or physical, which they felt were associated with headaches, answered a 'secondary gain' question, and completed a set of questions composed by the author. In conflict with more traditional viewpoints, migraine sufferers do not report themselves to be atypically reactive to ambiguity, uncertainty, or major life changes. Factors which do appear to be involved are quite diverse, and include tension over performed assertiveness behaviors, concern with perfectionism and evaluation, and reactions to small life changes. The impossibility of a cause and effect analysis is noted, and the quantity of reported stress factors is discussed as an argument for the author's concept of homeostatic reconditioning.

45Ca distribution and transport in saponin skinned vascular smooth muscle.

45Ca distribution and transport were studied in chemically skinned strips of caudal artery from Kyoto Wistar rats. Sarcolemmal membranes were made hyperpermeable by exposure for 60 min to solutions containing 0.1 mg/ml of saponin. Skinned helical strips responded with graded contractions to changes in ethylene glycol bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid buffered free Ca solutions (10(-7) to 10(-5) M) and were sensitive to the Mg-ATP concentration. Tissues loaded in the presence of 10(-7) M Ca contracted in response to 10 mM caffeine. These experiments indicate the strips are skinned and possess a functional regulatory and contractile system and an intact Ca sequestering system. 45Ca distributes in three compartments in skinned caudal artery strips. The Ca contents of two components are linear functions of the Ca-ethylene glycol bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid concentration and desaturate at rapid rates. They correspond to the extracellular and cytoplasmic spaces. A significantly smaller component releases Ca at comparatively slower rates. 45Ca uptake by the slow component consists of an ATP-dependent and an ATP-independent fraction. The 45Ca content of the ATP-dependent fraction is a function of the free Ca concentration and is independent of the Ca-ethylene glycol bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid concentration. Its content was enhanced by oxalate and was abolished by Triton X-100 skinning solutions. The ATP-independent component was not affected by Triton X-100 skinning and may represent Ca binding to cytoplasmic molecules and structures. The sequestered Ca was released with caffeine or Ca but not by epinephrine. The observations indicate that the sarcoplasmic reticulum and mitochondria of vascular smooth muscle strips skinned with saponin retain their functional integrity after saponin skinning.

Effect of K ions on 45Ca fluxes in myelinated nerve.

The effects of extracellular K ions and depolarization on 45Ca fluxes were investigated in desheathed sciatic nerves of Rana pipiens and the results compared to Na-Ca countertransport models which postulate the exchange of three or more Na ions for one Ca ion. Changes in the extracellular K ion concentration ranging from 1 to 40 mM at a constant Na gradient did not affect Ca efflux significantly. In Na-Ca-free solutions maintained isotonic with sucrose, increasing K concentrations stimulated Ca efflux. Increasing K concentrations inhibit Ca influx in Na-free solutions. Although membrane potential differences was reduced by up to 40 m v during these procedures, in no instance did depolarization reduce the Ca efflux as predicted by the model and as reported for poisoned squid axon. The results suggest that K ions inhibit Ca influx and activate efflux similar to Na. Furthermore, a fraction of the "residual" Ca efflux observed in the absence of Na and Ca appears to be due to extracellular K ions. This study provides further evidence that mechanisms other than Na-Ca countertransport participate in Ca homeostasis in myelinated nerve.

The effect of monovalent cations upon 45Ca fluxes in frog sciatic nerve.

Investigation of Ca fluxes in desheathed bundles of myelinated nerve of frog indicates an intracellular Ca concentration of 5 x 10(-4) mol . kg-1 (axoplasm) and an average transmembrane flux of 6 x 10(-8) mol. kg-1 . s-1 at an extracellular Ca concentration of 1 mM. Replacement of extracellular Na by isosmotic sucrose increases Ca influx threefold and decreases efflux by 50%. Similar, but significantly smaller, effects are observed when Tris or choline are substituted for Na. Li replaces Na without significant changes in Ca fluxes. The data demonstrate that Ca transmembrane fluxes in this preparation are sensitive to changes in the Na gradient. The observed flux changes, however, are too small to establish a Na-Ca exchange as the sole homeostatic mechanism for intracellular Ca. Moreover, as Li appears to serve as a good Na substitute and even Tris and choline interact with Ca flux, the exchange does not show the specificity described for squid axon.

Effect of metabolic inhibitors on 45Ca fluxes and ATP content of myelinated nerve.

The effects of metabolic inhibitors on ATP levels and on 45Ca fluxes were studied in desheathed tibial nerves of Rana pipiens. ATP levels were significantly reduced and Ca influx and efflux increased following exposure to cyanide, mersalyl, azide, dinitrophenol, iodoacetate or ethacrynic acid. The efflux changes were relatively small compared to squid axons but are not attributable to depolarization of the cell or to changes in the transmembrane Na gradient. The magnitude of the effluxes are consistent with ideas about the amount of labile Ca sequestered in energy-dependent Ca pools. The efflux stimulated by metabolic inhibitors is reduced by removing extracellular Na or Ca, indicating that a large portion of Ca efflux in the poisoned state is mediated by Na-Ca countertransport. In the nominal absence of both these cations Ca efflux can be stimulated by metabolic inhibitors, suggesting that mechanisms in addition to countertransport participate in Ca efflux.