Determination of prednisolone,prednisone, and cortisol in human plasma by high-performance liquid chromatography

A method is described for the measurement of prednisone and prednisolone in human plasma by high-performance liquid chromatography (hplc). An ether/dichloromethane extract (60:40, $\text{v}\text{v}$) of plasma is evaporated to dryness and chromatographed on a 250 × 4.5-mm Whatman Partisil column with uv detection at 239 nm. Prednisone, prednisolone cortisol, and the internal standard dexamethasone are satisfactorily resolved with the elution system of water-saturated dichloromethane/ethanol (95:4, $\text{v}\text{v}$). The hplc method can be used for plasma prednisolone concentrations as low as 25 ng/ml. The values correlate well with those obtained by a radioimmunoassay procedure for prednisolone.     

Detection of synthetic corticosteroid analogues by the competitive protein-binding radioassay

The influence of several synthetic corticosteroid analogues on the competitive protein-binding assay was examined $\text{in}$$\text{vitro}$ using 1) purified, analytic grade steroids, and 2) material extracted from pharmaceutical tablets. The two sources yielded comparable results. It was found that some of the synthetic corticosteroids were detected by the radioassay and produced competitive displacement of labelled cortisol from binding protein. Treatment with certain corticosteroid analogues may therefore interfere with the estimation of plasma cortisol concentrations by the competitive protein-binding assay.     

Plasma cortisol concentrations in two teleost fishes, Anguilla anguilla L. and Carassius auratus L.: a comparison of two assay systems

The plasma cortisol (11β, 17α, 21-trihydroxy-pregn-4-ene-3,20-dione) concentrations in the eel, $\text{Anguilla anguilla}$ and the goldfish, $\text{Carassius auratus}$ have been measured by a competitive protein binding assay (CPBA) and by a double isotope dilution derivative assay (DIDA). Results from the two techniques correlate well over the ranges encountered under most physiological conditions. The simpler CPBA is thus justified for routine use, at least in these two teleostean species.     

Comparison of ratios of covalent binding to total metabolism of the pulmonary toxin, 4-ipomeanol, In vitro in pulmonary and hepatic microsomes,and the effects of pretreatments with phenobarbital or 3-methylcholanthrene

Studies of the ratios of the amounts of 4-ipomeanol covalently bound to the total amounts metabolized support the view that the high rates of $\text{in}\text{vitro}$ pulmonary microsomal alkylation by 4-ipomeanol reflect high rates of NADPH-mediated metabolic activation of the compound rather than a relative deficiency of a microsomal detoxication pathway. Moreover, the ability of 3-methylcholanthene pretreatment, but not phenobarbital pretreatment, to shift the $\text{in}\text{vivo}$ target organ alkylation and toxicity of 4-ipomeanol from the lung to the liver in rats could not be explained by a major alteration in the balances between microsomal toxication and detoxication pathways measurable in the $\text{in}\text{vitro}$ systems examined, nor upon a major change in the nature of the reactive 4-ipomeanol metabolites produced in the lungs or livers of the pretreated animals.     

Association between plasma high density lipoprotein cholesterol and antipyrine metabolism in alcoholics

Seven patients entering an alcoholic detoxification and treatment unit exhibited elevated levels of plasma high density lipoprotein cholesterol (HDLC) and enhanced metabolic disposal of antipyrine. Followig a 2-week abstinence treatment, the HDLC levels were reduced by 28% (from 64 to 47 mg/100 ml) and the $\text{t}\text{1}\text{2}$ of antipyrine was extended from 12.4 to 13.7 hours. The extent of the HDLC reduction correlated with the antipyrine $\text{t}\text{1}\text{2}$ changes (r = ?0.753, P = 0.05).     

l-Acetylmethadol (LAM) treatment of opiate dependence: plasma and urine levels of two pharmacologically active metabolites

The metabolic conversion of α-$\text{l}$-acetylmethadol (LAM) to α-$\text{l}$-noracetylmethadol (NAM) and α-$\text{l}$-dinoracetylmethanol (NNAM), has been studied in three opiate addicts being maintained on 100 mg of LAM three times weekly. Plasma levels of NAM and NNAM were established shortly after the initial dose of LAM. The plasma level of NNAM was substantially higher following repeated dosing than following the initial dose. The combined daily urinary excretion of LAM, NAM and NNAM was 6–8 times greater after repeated dosing than after the initial dose. Since NAM and NNAM, which are formed by the sequential N-demethylation of LAM are both considerably more active morphine-like agonists than is LAM itself, it is likely that the pharmacological effects of LAM are due to NAe NAM and NNAM. Variations in the rates of formation and elimination of NAM and NNAM may partially explain the variability in response seen in LAM maintenance therapy.     

Influence of steady-state and fluctuating salinities on the oxygen consumption and activity of some brackish water shrimps and fishes

Oxygen consumption, locomotory activity and, in some cases, osmoregulatory responses of different populations of Palaemon adspersus (Rathke) and Pomatoschistus microps (Krøyer) from the Isefjord ($\text{S 19‰}$) and Karrebaek Fjord ($\text{S 12‰}$) in Denmark and the Barther Bodden ($\text{S 6‰}$) in the G.D.R. to short-term salinity fluctuations, and after long-term adaptation, were tested. The same tests were performed on populations of Gasterosteus aculeatus (L.), Palaemonetes varions (Leach) (both from Barther Bodden, G.D.R.) and Palaemon elegans (Rathke) (Black Sea, Bulgaria, $\text{S 18‰}$). The steady-state experiments showed that the standard metabolic rates of P. adspersus and Pomatoschistus microps reach their lowest levels at mean biotope salinities at both 10 and 20°C. In contrast, the routine metabolic rates of both species are independent of salinity in the ecological salinity range.All Palaemon adspersus and Pomatoschistus microps populations responded to sudden changes in salinity with increased locomotory activity and respiration regardless of the direction of stressing. Metabolic adaptation in these euryhaline species, which is not synchronous with osmotic readjustment, takes from 5 to 12 h, depending on the salinity gradient.The polystenohaline Palaemon elegans from the Black Sea and the holeuryhaline Palaemonetes varians from the Barther Bodden exhibit similar short adaptation times (≈ 2 h) to identical salinity gradients but in different salinity zones.     

Somatocrinin,growth hormone releasing factor,stimulates secretion of growth hormone in anesthetized rats

The synthetic replicate of a 44 amino acid peptide isolated from a human pancreatic tumor which had caused acromegaly possesses high specific activity to release growth hormone (GH) in anesthetized male rats. The GH secretion induced by this peptide is dose-dependent from 50 ng to 1 μg, with plasma GH concentrations increasing more than 10-fold within 5 min of iv administration at the higher doses. Two enzymatic degradation products of the 44 residue peptide were also isolated and consist of the first 37 and 40 amino acids. All three peptides appear to possess similar potency, on a molar basis, $\text{in}\text{vivo}$, contrary to $\text{in}\text{vitro}$ results. The specificity of these peptides on GH release was shown by their failure to alter plasma concentrations of prolactin (PRL), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and corticosterone. Based on these $\text{in}\text{vivo}$ results, the three peptides with serve as powerful tools with which to investigate the mechanisms of GH secretion.     

A new antibiotic with known resistance factors,G418, inhibits plant cells

Growth rates of two lines of tobacco ($\text{Nicotiana}\text{tabacum}$) cell suspension cultures were measured in the presence or absence of G418, a new 2-deoxystreptamine antibiotic related to Gentamycin. Cell growth rates of $\text{N}$. $\text{tabacum}$ cv. Burley were inhibited at drug concentrations as low as 1.65 × 10^?7 M. At 4 × 10^?7 M, the doubling time was increased from 1.5 days (control) to 2.3 days (treatment). The drug was lethal to cells at 4 × 10^?6 M, and inhibition was irreversible. Cells of $\text{N}$. $\text{tabacum}$ cv. Wisconsin 38 also were inhibited by the drug, although at slightly higher concentrations (ca. 2–5 fold).In view of our findings, G418 and its associated resistance factors could be of great value in plant genetic engineering.     

Enteric release of vasoactive intestinal peptide after a peptone meal in the dog

Vasoactive intestinal peptide (VIP) concentrations were measured by radioimmunoassay in plasma from portal and peripheral venous blood obtained from six alert, non-anesthetized dogs before and after gastric infusion of a 10% peptone meal. Mean basal portal and cephalic vein plasma VIP concentrations were $\text{42 ± 11.7}$ and $\text{42 ± 8.0}\text{(S.E.M.) pg/ml}$, respectively. No significant changes in peripheral venous plasma VIP concentrations were noted after the peptone meal throughout the duration of the collection period. In contrast, however, the mean VIP concentration in portal plasma increased promptly after the peptone meal with a peak of $\text{79 ± 8.2}\text{pg/ml}$ ($\text{P < 0.02}$) occurring 8 min after infusion of the meal. This was followed by a gradual decline in portal plasma VIP levels, with a return to prefeeding concentrations at 60 min ($\text{44 ± 6.3}\text{pg/ml}$). Results of these studies demonstrate that following gastric infusion of a peptone meal in the dog, portal, but not peripheral, plasma VIP concentrations increase significantly. Failure to detect augmentation of peripheral vein VIP levels after the meal is probably due to hepatic clearance of VIP.     

Metabolism of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine by cell cultures

The metabolism of 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine (platelet-activating factor) was studied using various cultured cell lines. All incubations were done in the presence of bovine serum albumin and serum-free media, since albumin eliminates the adsorption of 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine to cultureware and serum enzymes interfere. Human leukemia (HL-60) cells, MDCK canine kidney cells, and transformed and nontransformed clones of mouse C3H1OT1/2 cells display varying rates of uptake, degradation, and capacities for reacylation of 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine. HL-60 cells displayed the highest uptake rate (24.6 pmol/mg cell protein/15 min). Whereas C3H10T1/2 cells in culture showed uptake rates comparable to other cells tested, they displayed a relative metabolic inertness towards 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine.     

Modification of hepatic microsomal oxidative drug metabolism in rats by the opiate maintenance drugs acetylmethadol, propoxyphene, and methadone.

The formation of cytochrome P-450 “metabolic intermediate” complexes $\text{in}$$\text{vivo}$ occurred with acetylmethadol and propoxyphene, but not methadone in both naive and phenobarbital-induced animals. The $\text{in}$$\text{vivo}$ formation correlated with the relative ability of these three compounds to form metabolic intermediate complexes and inhibit mixed-function oxidation reactions $\text{in vitro}$.     

Comparative metabolic clearance rates of beta-endorphin and beta-lipotropin in humans

In normal human subjects under basal conditions, we have reported that molar concentrations of immunoreactive β-lipotropin (IR-β-LPH) are approximately threefold greater than those of IR-β-endorphin (β-Ep). Following acute stimulation, there is a further two- to threefold disproportionate rise in plasma concentrations of IR-β-LPH as compared to those of IR-β-Ep. To begin to assess the possible factors involved in such altered IR-β-LPH/IR-β-Ep ratios in plasma, the metabolic clearance rate (MCR), volume of distribution (V_d), fractional rate of disappearance (K_d), and half-life ($\text{t}\text{1}\text{2}$) of these peptides were determined by means of bolus injection of highly purified human β-LPH and synthetic human β-Ep in normal human subjects. β-Ep was found to have an MCR and a K_d greater than that of β-LPH, and a shorter $\text{t}\text{1}\text{2}$. These differences, however, although they may in part be contributory, cannot solely account for the greater ratio of IR-β-LPH to IR-β-Ep in plasma, or for the disproportionate rise in plasma concentrations of these peptides after acute stimulation.     

Isolation of plasma membrane vesicles,derived from transverse tubules,by selective homogenization of subcellular fractions of frog skeletal muscle in isotonic media

A new technique for isolating fragmented plasma membranes from skeletal muscle has been developed that is based on gentle mechanical disruption of selected homogenate fractions. $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-stimulated}$, Mg²⁺-dependent ATPase was used as an enzymatic marker for the plasma membrane, Ca²⁺-stimulated, Mg²⁺-dependent ATPase as a marker for sarcoplasmic reticulum, and succinate dehydrogenase for mitochondria. Cell Cell segments in an amber low-speed ($\text{800 × g}$) pellet of a frog muscle homogenate were disrupted by repeated gentle shearing with a Polytron homogenizer. Sarcoplasmic reticulum was released into the low-speed supernatant, whereas most of the plasma membrane marker remained in a white, fluffy layer of the sediment, which contained sarcolemma and myofibrils. Additional gentle shearing of the white low-speed sediment extracted plasma membranes in a form that required centrifugation at $\text{100 000 × g}$ for pelleting. This pellet, the fragmented plasma membrane fraction, had a relatively high specific activity of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-stimulated}$ ATPase compared with the other fractions, but it had essentially no Ca²⁺-stimulated ATPase activity and only a small percentage of the succinate dehydrogenase activity of the homogenate.Experimental evidence suggests that the fragmented plasma membrane fraction is derived from delicate transverse tubules rather than from the thicker, basement membrane-coated sarcolemmal sheath of muscle cells. Electron microscopy showed small vesicles lined by a single thin membrane. Hydroxyproline, a characteristic constituent of collagen and basement membrane, could not be detected in this fraction.     

The rapid intermembraneous transfer of retinoids

The intermembraneous rates of retinoid (all-$\text{trans}$-retinol(al), 11-$\text{cis}$-retinol and all-$\text{trans}$-retinol palmitate) transfer from vesicle to vesicle and vesicle to erythrocyte were studied. The rates of transfer of the retinols(al) were exceedingly rapid. The rates of transfer of the retinols(al) from egg phophatidyl choline based SUV's to bovine erythrocytes had a half-time of approximately 1–2 min. The vesicle to vesicle transfer rate was too rapid to measure by conventional techniques. By contrast, all-$\text{trans}$-retinol palmitate did not undergo transfer at an appreciable rate.     

Relationship of metabolite inhibition of growth to flow-of-carbon patterns in nature

Various genetic diseases arise from biochemical imbalances that are relatively subtle in the sense that the original mutations are not lethal, that the organism is most vulnerable to damage during certain phases of rapid development, and that in well-managed cases it may be possible to avoid damaging effects through the use of appropriate nutritional manipulations. Analogous imbalances occur in lower organisms. Data obtained with $\text{Pseudomonas}$$\text{putida}$ illustrate that susceptibility to metabolic imbalance is conditionally dependent upon the nutritional regimen.Stereoisomers of leucine, isoleucine and valine, except for L-allo-isoleucine, are metabolized as sole sources of carbon and energy by $\text{P.}$$\text{putida}$. Although the cell yields calculated following utilization of $\text{D}$-leucine and $\text{L}$-leucine were similar, the rate of growth on D-leucine was seven-fold faster than on $\text{L}$-leucine. Slower growth on the $\text{L}$-isomer is not explained as 2-ketoisocaproate limitation since 2-ketoisocaproate production from $\text{L}$-leucine appears to occur more readily than from $\text{D}$-leucine. Spontaneous mutants were obtained which grew 2–10 times more rapidly than wild type on $\text{L}$-leucine, $\text{L}$-isoleucine, or $\text{L}$-valine. It is concluded that the true growth potential (rate) of wild type on any of the branched-chain amino acids is masked by a partial, sustained inhibitory effect produced by the corresponding keto acids or their derivative metabolites. Inhibition of growth rate was only found during utilization of branched-chain amino acids as the sole source of carbon and energy, indicating that the metabolite vulnerability is unique to particular flow-of-carbon patterns during growth. The partial and sustained depression of growth rate by branched-chain amino acids in the absence of other carbon sources cannot be attributed to mis-regulation events localized within the biosynthetic pathway. It is concluded that the catabolism of branched-chain amino acids produces a generalized state of metabolic imbalance owing to the existence of abnormally high levels of degradative metabolites such as keto acids of Coenzyme-A derivatives. Such compounds could (1) interfere with keto acid (e.g. pyruvate) metabolism, (ii) cause feed-forward inhibition of rate-limiting steps in the pathways of branched-chain amino acid catabolism, (iii) perturb fatty acid composition or disrupt the biochemical integrity of membrane material, or (iv) react with substrate-ambiguous enzymes, either slowing essential biochemical reactions to rates that are growth-limiting or producing erroneous products having antimetabolite properties.These effects of branched-chain amino acids in $\text{P.}$$\text{putida}$ may be quite relevant to the molecular events that characterize maple syrup urine disease in man. Metabolite inhibition is probably more common in nature than is generally appreciated, and an appreciation of the molecular basis for anomalous inhibitions of growth in prokaryotic systems should help supply insight into various molecular diseases in man, many of them yet to be described.     

Effect of pH, carbamylation and other hemoglobins on deoxyhemoglobin S aggregation inside intact erythrocytes as detected by proton relaxation rate measurements.

Measurement of the transverse water proton relaxation rate has been used to study the effect of pH, carbamylation, and other hemoglobins on the aggregation of deoxyhemoglobin S inside intact erythrocytes. Upon complete deoxygenation, cyanate-treated ($\text{S}\text{S}$) erythrocytes and erythrocytes heterozygous with respect to hemoglobin S ($\text{A}\text{S}$, $\text{C}\text{S}$, and $\text{S}\text{D}$) have high transverse water proton relaxation rates very similar to the values obtained with homozygous ($\text{S}\text{S}$) erythrocytes. These results suggest extensive intermolecular interactions between deoxyhemoglobin S molecules and a resultant increase in the correlation time for the small fraction of “irrotationally bound” water. When the transverse relaxation rate in deoxygenated ($\text{S}\text{S}$) erythrocytes was measured as a function of pH, the maximum rate was observed between pH 7.0 and 7.5. Upon increasing the pH beyond this range the observed relaxation rate decreases as does the number of sickled cells. Upon decreasing the pH, the observed transverse relaxation rate also decreases but the ratio of values from $\text{deoxy}\text{oxy}$ ($\text{S}\text{S}$) erythrocytes remains in the normal range of 4–6 and the number of sickled cells does not change. Therefore, the deoxyhemoglobin S aggregate inside sickled erythrocytes, as observed by water proton relaxation rates, is not altered by carbamylation or by the presence of nongelling hemoglobins. In addition, the enhancement of the relaxation rates as a function of pH is consistent with the number of sickled forms observed.     

The involvement of a non-'bay-region' diol-epoxide in the formation of benz(a)anthracene-DNA adducts in a rat-liver microsomal system

The metabolic activation of benz(a)anthracene was investigated by incubating [³H]-benz(a)anthracene with DNA, a NADPH-generating system and rat-liver microsomes. When hydrolysates of the DNA were chromatographed on Sephadex LH20 columns, three hydrocarbon-nucleoside adduct peaks were resolved and these were further examined using HPLC. One adduct probably results from the reaction of the non-bay-region diol-epoxide $\text{r}$-8,$\text{t}$-9-dihydroxy-$\text{t}$-10,11-oxy-8,9,10,11-tetrahydrobenz(a)anthracene $\text{(}\text{anti}$-BA-8,9-diol 10,11-oxide) with DNA. The other two adducts did not co-chromatograph with adducts formed from any of the four possible isomeric diolepoxides that can be formed in the 8,9,10,11-ring of benz(a)anthracene.     

Ultrastructural and functional differences in mitochondria isolated from Paramecium aurelia grown axenically and monoxenically.

Differences in the ultrastructure of mitochondria in $\text{P.}\text{aurelia}$ grown axenically and monoxenically have been observed. Functional mitochondria have been isolated from both cultures and various metabolic parameters have been tested; respiration rates, cytochrome content, oligomycin sensitive ATPase activity, and an attempt has been made to relate the observed structure and metabolic activity of the mitochondria to the growth conditions.     

Daily rhythm of plasma corticosterone binding activity in the white-throated sparrow,Zonotrichiaalbicollis

There is a bimodal daily rhythm of plasma corticosterone binding activity (CBA) in the white-throated sparrow, $\text{Zonotrichia}$$\text{albicollis}$, in the spring migratory condition. Low levels occurred at the beginning (0500) and at the end (2100) of a 16-hour daily photoperiod. Peak CBA occurred at 0900 and 0100, as did peak locomotor activity in this nocturnal migrant. Comparisons of CBA with total plasma corticosteroid concentrations from a previous study of the same group of birds indicate a positive correlation during most of the day but not during the early hours of darkness. The daily rhythm of locomotor activity may account for the rhythm of CBA which, in turn, may be partially responsible for the daily rhythm of plasma corticosteroid concentration.