Fractionation of serum cholesterol: a critique

A procedure for fractionation of serum cholesterol is developed by employing digitonin for the precipitation of free cholesterol in isopropanol. This method is based on the ferric acetate color reaction which is unaffected by traces of digitonin. It is influenced only negligibly by the presence of stanols in serum and is shown to be equally chromogenic with respect to both free and ester forms of cholesterol. The proposed procedure shows a three-fold improvement in precision (3.1 percent coefficient of variation [CV]) compared to that of the procedure by Leffler and McDougald (10.5 percent C.V.) which is based on the ferric chloride color reaction. The proposed free cholestrol procedure showed a mean recovery of 99.5% percent (98.1 to 102.5 percent) when cholesterol in 40 to 200 mg per dl concentrations was added to serum. The analytical performance of the proposed fractionation of serum cholesterol is critically reviewed with respect to its potential application in the diagnosis of liver diseases and in basic or experimental research.     

Nonunion using a canine model

The investigation involved a search for a model of atrophic nonunion. Fifty-two mature, adult, mongrel dogs were used to study the repair after creating a 0.5-cm bone defect in the mid-diaphysis of the radius. In addition, a 2-cm wide strip of periosteum was circumferentially resected from each osteotomy extremity. No immobilization was used thereafter. The reparative process was assessed by X-rays, histology, vascular injection, and scintigraphy. The dogs we distributed into three groups according to the time of follow-up (1, 3, and 6 months). Two kinds of repair were recognized after 3 months and were well-established after 6 months: disturbed healing with much callus (54%) and disturbed healing with absent or scanty callus (46%). In the first instance, the periosteum had regenerated and produced the external callus. The bone ends were capped with fibrocartilage; the vascularization around the defect was increased and displayed a well-defined vascular picture. In the healing pattern with absent external callus (atrophic nonunion), the bone defect was enlarged and filled with fibrous tissue, but there was no deficient vascularization in and around the osteotomy. Radioactivity counting showed an increased uptake around the osteotomy site in both types of repair, which persisted over time but was higher in the 1-month group. It was concluded that the present model yields a consistent pattern of a disturbed reparative process that mimics human cases of atrophic or hypertrophic nonunion. The differences between the two kinds of repair seemed to be related to the periosteal capacity of regeneration.     

Activity of serum sodium, potassium and calcium in vertebrates]

Of 49 patients undergoing operation for benign spontaneous pneumothorax, 28 (57%) were found to have a sharp first or second rib. In a series of 100 patients undergoing thoracotomy for other conditions, only eight (8%) were found to have a sharp rib. The association between sharp ribs, apical scars and bullae, and spontaneous pneumothorax is discussed.     

High serum calcium in human brucellosis: a case-control study

In a retrospective case-control study of 58 cases of human brucellosis, adjusted mean serum calcium levels were found to be significantly higher in patients with brucellosis compared with controls: mean (95% confidence interval) = 2.39 (2.35-2.42) mmol/L versus 2.30 (2.26-2.34) mmol/L (P = 0.0012). The possible mechanisms underlying the cause of hypercalcemia in human brucellosis are discussed.     

Effect of calcium infusions of serum calcium and gastric acid secretion

37 patients were studied with calcium infusions. Of these, 20 had previously undergone truncal vagotomy and pyloroplasty for duodenal ulcer disease, and 17 were unoperated patients with duodenal ulcer disease. Calcium was given intravenously either at a dose of 5 mg/kg/h for 3 h, or 4 mg/kg/h for 4 h. Gastric juice was collected by continuous suction. Results showed the 3-hour infusion raised calcium more than the 4-hour infusion. Top serum calcium achieved, however, did not correlate with calcium-stimulated gastric acid output, either with or without vagotomy. Stimulated gastric acid secretion was markedly less with vagotomy than without. It is suggested that the level of gastric acid stimulated by infusions might discriminate complete, from incomplete, vagotomies better than insulin, and that the 4-hour infusion is safer.     

Hydroxyproline in serum as a homeostatic index for calcium in cattle

Hydroxyproline, calcium, magnesium, phosphorus, and alkaline phosphatase values were determined in serum over 24 h in Holstein cows. The cows represented two age groups and three percents of diet calcium. Hydroxyproline followed a cyclic pattern dipping at 0800 and 1600 h after milking at 0500 and 1530 h. Phosphorus showed a 24 h rhythm peaking at 1600 h. No other time effects were demonstrated. Hydroxyproline and alkaline phosphatase were both lower in the older cows, indicating a decreased calcium mobilization from bone with age. There was a correlation coefficient of only .20 between serum calcium and hydroxyproline. The calcium concentration in serum was maintained within a narrow range presumably as a result of homeostatic mechanisms involving bone resorption, i.e. the release of calcium and hydroxyproline may indicate the degree of homeostasis required to maintain serum calcium.     

Separation and quantification of corticosteroid-induced, bone and liver alkaline phosphatase isoenzymes in canine serum

Quantifying alkaline phosphatase (ALP) isoenzymes in canine serum would provide a useful index in a clinical laboratory. To achieve this goal, we tested a semi-automatic assay combining wheat germ lectin (WGL) precipitation and chemical inhibition of isoenzymes of the TNS gene with levamisole to quantify bone ALP (BALP) and corticosteroid-induced ALP (CALP), respectively. The liver ALP (LALP) isoenzyme was then calculated from the equation: TALP = BALP + LALP + CALP BALP, LALP and CALP standards from serum of puppies, bile-duct ligated dogs and dogs on 4.4 mg/kg/day prednisolone for 30 days, respectively, were used. The suitability of standard sera was tested by affinity electrophoresis. Levamisole (4.2 mM) inhibits 98% of BALP and LALP but only 42% CALP. Multiplying measured CALP by 1.8 gives the total CALP value in serum. WGL precipitated 92.3% BALP, 23.3% LALP and 26.8% heated CALP standards. These values were used to adjust precipitated ALP to obtain the exact levels of BALP. WGL was then tested on pooled serum standards in which the relative proportions of all the ALPs were known and controlled. BALP was adequately quantified except when LALP and CALP levels were extremely high. The assay was also applicable under conditions resulting in high ALP. Therefore, combining WGL and levamisole inhibition provides an adequate separation and quantification of canine ALP isoenzymes. The method has great potential for diagnostic use and should be tested further for routine implementation.     

Evaluation of ploidy of mature canine megakaryocytes, using Feulgen staining and microspectrophotometry

OBJECTIVE: To evaluate megakaryocyte size and ploidy, using Feulgen staining and microspectrophotometry, in adult dogs with normal platelet count. ANIMALS: Group A contained 8 and group B contained 11 adult dogs. PROCEDURE: Megakaryocytes were evaluated by light microscopy and staged according to maturation status. Stage-III megakaryocytes were measured and mapped for future relocation. Bone marrow aspirates were destained and restained, using the Feulgen method. Previously identified stage-III megakaryocytes were measured for DNA content, using microspectrophotometry. RESULTS: Megakaryocyte size correlated with ploidy values, and mean sizes within ploidy groups were significantly (P < 0.05) different from each other for both groups. The model ploidy value of stage-III megakaryocytes, which represented 18% of the total megakaryocyte population of the combined groups, was 32N. This is in contrast to results of flow cytometric studies, which indicated that the modal ploidy value for all canine megakaryocytes was 16N. CONCLUSIONS: Reasons for the disparate results between microspectrophotometric techniques and flow cytometry include maturation stage of the megakaryocyte population evaluated and percentage of megakaryocytes within that maturation stage. Flow cytometric methods, which evaluate all megakaryocytes detectable by antibody, may include cells still capable of DNA synthesis, resulting in a shift in the observed modal ploidy value. Recognition of the difference between canine and human megakaryocyte ploidy distribution is important, particularly in studies in which the dog is used as an animal model for human megakaryocytopoiesis.     

Differential effects of etomidate, propofol, and midazolam on calcium and potassium channel currents in canine myocardial cells

BACKGROUND: Intravenous anesthetics etomidate, propofol, and midazolam produce negative inotropic effects of various degrees. The mechanism underlying these differences is largely unknown. METHODS: The effects of intravenous anesthetics on L-type Ca2+, transient outward and inward-rectifier K+ channel currents (ICa, IKto, and IK1) were compared in canine ventricular cells using the whole-cell voltage-clamp technique. ICa and IK were elicited by progressively depolarizing cells from -40 to +40 mV, and from -90 to +60 mV, respectively. The peak amplitude and time-dependent inactivation rate of ICa and IK were measured before, during, and after the administration of equimolar concentrations (5, 30, or 60 microM) of etomidate, propofol, or midazolam. RESULTS: Exposure to etomidate, propofol, and midazolam produced a concentration-dependent inhibition of ICa. Midazolam was the most potent intravenous anesthetic; at 60 microM, etomidate, propofol, and midazolam decreased peak ICa by 16 +/- 4% (mean +/- SEM), 33 +/- 5%, and 47 +/- 5%, respectively. Etomidate, propofol, and midazolam given in a 60-microM concentration decreased IKto by 8 +/- 3%, 9 +/- 2%, and 23 +/- 3%, respectively. IK1 was decreased by 60 microM etomidate and midazolam by 20 +/- 6% and 14% +/- 5%, respectively. Propofol had no effect on IK1. CONCLUSIONS: At equimolar concentrations, intravenous anesthetics decreased the peak ICa, IKto, and IK1 with various degrees of potency. Effects of anesthetics on ICa were significantly greater compared with their effects on K+ currents. These findings suggest that the negative inotropic actions of etomidate, propofol, and midazolam are related, at least in part, to decreased ICa. Some effects, such as IK inhibition, may partially antagonize effects of decreased ICa. Indeed, the final effect of these intravenous anesthetics on myocardium will be the sum of these and other sarcolemmal and intracellular effects.     

Supracrestal bone regeneration around dental implants using a calcium carbonate and a fibrin-fibronectin sealing system: clinical and histologic evidence

This study evaluated a new surgical technique for the treatment of an alveolar ridge deficiency in 11 patients. Twenty-two implants were placed, 15 of which presented with a combination of supracrestal and dehiscence kinds of defects, and seven presented only supracrestal bone loss. Surgical procedures were performed utilizing a combination of the resorbable space-making material calcium carbonate stabilized with a fibrin-fibronectin sealing system and the immediate placement of titanium dental implants. After implant placement, the mean height for supracrestal and dehiscence defects measured 2.57 +/- 1.41 mm and 2.47 +/- 1.54 mm, respectively. The defects were filled with calcium carbonate and a fibrin-fibronectin sealing system, and the flaps were sutured, avoiding any compression of the treated area. Healing was uneventful in all instances. At second-stage surgery at 6 months, a hard bone-like tissue was detectable at the defect sites. Histologic examination of four defects confirmed the presence of newly formed bone and revealed residual particles of calcium carbonate. There was a mean gain of 2.05 +/- 1.47 mm in the supracrestal defects and of 2.23 +/- 1.62 mm in the dehiscences. The results indicated that calcium carbonate, combined with a fibrin-fibronectin sealing system, is a viable alternative in the treatment of supracrestal and dehiscence bony defects.     

Diabetes mellitus in dogs: a study of the critical difference for canine serum fructosamine

The purpose of this study was to calculate the critical difference between two analytical measurements of serum fructosamine concentration in dogs. The critical difference can be used to judge whether the difference between two consecutive analytical results from the same animal is due to natural variation. Blood samples from 15 apparently clinically healthy beagle dogs were collected once a week for six consecutive weeks. The serum fructosamine concentration was measured by the reduction test with nitroblue tetrazolium and the critical difference was calculated from the component of variance for weeks within dogs (sigma2s) and the residual variance (sigma2e). The critical difference between two consecutive analytical results was 33.5 micromol/litre.     

Geographic epidemiology of gonorrhea in Baltimore, Maryland, using a geographic information system

Ws/Ws rats are deficient in both mucosal- and connective tissue-type mast cells. To study the role of mast cells in active anaphylaxis, changes in vascular permeability in the trachea upon intravenous antigen challenge with Evans blue dye were examined in Ws/Ws, heterogenic Ws/+, and normal +/ + rats sensitized with the nematode Nippostrongylus brasiliensis. Antigen challenge resulted in fatal anaphylactic shock in some +/+ and Ws/+ rats, but not in Ws/Ws rats. Marked dye leakage developed within 30 min in the trachea of +/+ and Ws/+ rats, while Ws/Ws rats showed no substantial increases in the levels of vascular permeability. Ex vivo stimulation of sensitized lung fragments from +/+ animals with specific antigen induced significant releases of histamine and leukotriene (LT) C4, while sensitized Ws/Ws rat-lung fragments did not. In Ws/Ws rats, levels of nematode-specific IgE, IgG1 and IgG2a antibodies as well as levels of lung eosinophilia were not significantly different from those in +/+ rats. These results show that mast cell-deficient Ws/Ws rats fail to develop active anaphylaxis, and this is mediated probably by the lack of mast cell-derived mediators required for initiation of the reaction.     

Identification of a calcium channel modulator using a high throughput yeast two-hybrid screen

The interaction of the N-type calcium channel beta3 subunit with the alpha1B subunit alters the activation/inactivation kinetics and the maximal conductance of the channel. The defined protein-protein interaction of the human alpha1B and beta3 subunits provides a target for small-molecule modulation of N-type channel activity. We describe a high throughput screen based on a counterselection yeast two-hybrid assay, which was used to identify small molecules that disrupt alpha1B-beta3 subunit interactions and inhibit N-type calcium channel activity. These small molecules may be a new class of calcium channel antagonists with therapeutic potential.     

Direct in vivo gene transfer to canine myocardium using a replication-deficient adenovirus vector

Inter-alpha-inhibitor (I alpha I) is a serine protease inhibitor present in human plasma. It has a molecular weight of about 220 kDa which encompasses 3 chains including two heavy chains and one light chain. The light chain, known as bikunin, is responsible for the antitryptic activity of I alpha I in the inhibition of various enzymes, such as trypsin and chymotrypsin. Under physiologic or certain pathologic circumstances, several macromolecules related to I alpha I appear in plasma and urine. However, the physiologic role of I alpha I remains unclear. As far as urolithiasis is concerned, two urinary macromolecules related to I alpha I have been isolated and shown to be potent inhibitors of calcium oxalate formation. One of these inhibitors, uronic-acid-rich protein (UAP), has been identified and well characterized. The sequence of the first 18 amino acid residues of UAP is identical with that of bikunin. Furthermore, the immunoreaction between UAP and I alpha I antibody using immunoblot analysis was positive. UAP isolated from the urine of stone formers exhibited less inhibitory activity towards calcium oxalate crystallization than that derived from the urine of healthy subjects. This suggests a structural abnormality of the inhibitor obtained from stone patients. The organic matrix extracted from kidney stones contained a protein antigenically related to I alpha I. We conclude that UAP is a member of I alpha I family taking part in inhibiting calcium oxalate crystallization, and modulating the formation of stones in the urinary tract.     

The influence of age on the canine immune system

Immune function was assessed in a group of 47 Labrador Retrievers, ranging in age from 0.8 to 11.5 years, in order to establish baseline data on canine immunosenescence. Natural killer cell activity, lymphocyte subset distributions, antibody production, and mitogen-induced lymphoproliferative responses, all of which have been demonstrated to undergo age-related changes in humans and mice, were chosen as indicators of immune function. Dogs were categorized by age as young (mean 2.4 years), middle-aged (mean 5.8 years), and old (mean 9.1 years). Natural killer cell activity was not affected significantly by age. Lymphocyte subset analysis revealed a significant age-related increase in the percentage of cells staining with a pan T-cell reagent, accompanied by a corresponding increase in the percentage of CD8 cells from youth to middle age. An age-related decrease in the percentage of B-cells was observed concomitant with the increases in T-cell percentages. A gender-related difference in pan T-cell distribution was also observed, with females having a higher percentage than males. Lymphoproliferative responses of both young and middle-aged dogs to the mitogens concanavalin A, phytohemagglutinin, pokeweed mitogen, and staphylococcal enterotoxin B were significantly higher than those of old dogs. In general, the mitogen responses of male dogs were affected more dramatically by age than those of females. A significant age-related decline in in vivo antibody responses to the protein antigen, keyhole limpet hemocyanin, was not observed, although the mean titers of the young dogs were higher than those of the old.     

Effects of iron supplementation and discontinuation on serum copper, zinc, calcium, and magnesium levels in women

The purpose of this study was: 1) to establish the prevalence of depleted iron stores, iron deficiency, and low serum levels for copper, zinc, calcium, and magnesium in a healthy female population; and 2) to examine the effects of iron supplementation and discontinuation on the serum levels of the above minerals. One hundred eleven healthy women between the ages of 18 and 40 yr reported for fasted morning blood sampling for iron, copper, zinc, calcium, and magnesium status. Forty-five subjects were either iron-deficient as defined by a hemoglobin level below 120 g.l-1 (four subjects) or iron deplete as defined by a serum ferritin value below 20 micrograms.l-1 (43 subjects). Two subjects fit both criteria. This subgroup continued with the study and were prescribed a normal therapeutic iron dose (320 mg elemental iron per day, taken as two Slow-Fe tablets.d-1 for a period of 12 wk). The subjects then discontinued the iron supplementation for a further 12 wk. The response of the various blood minerals was monitored at 6-wk intervals. Twenty-five subjects completed the full 24-wk treatment. The main conclusions to be made from this study were that: 1) For this sample population of women, iron depletion was quite common (39%), although low hemoglobin values (< 120 g.l-1) were only seen in 3.6%. No subjects fell below the criteria for low serum copper levels (< 13.3 mumol.l-1) nor low serum magnesium levels (< 0.6 mmol.l-1). Seven subjects (6.5%) fell below the criteria for low serum zinc levels (< 11.5 mumol.l-1) while two subjects (1.8%) were below the criteria for low serum calcium levels (< 2.20 mmol.l-1). 2) Therapeutic oral iron supplementation was successful in raising mean serum ferritin values from 15.9 micrograms.l-1 to 36.5 micrograms.l-1 but was not associated with decrements in serum copper or calcium levels. 3) The treatment did not significantly effect serum zinc and magnesium levels during the supplementation period, but a downward trend continued through the discontinuation phase so that at 18 and 24 wk serum zinc and magnesium levels were significantly lower than baseline. 4) Oral contraceptive use was associated with elevated serum copper and ferritin values and lowered serum magnesium levels.     

Efficient phosphate binding using a combination of gluconolactate and carbonate calcium salts

Although renal-failure-related hyperphosphataemia can be corrected by various phosphate binders, there remains a need for safer and more efficient formulations to precipitate phosphate. This work describes both a theoretical approach and a phosphate precipitation test in order to design efficient binding calcium salts formulations. The results show that the combination of a soluble calcium salt (the gluconolactate) and a proton-consuming calcium salt (the carbonate) can precipitate phosphate effectively. Furthermore, the theoretical computations correlate well with the ability of the salt to bind phosphate in vitro.     

Mibefradil, a pharmacologically distinct calcium antagonist

Mibefradil is the prototype of a new class of calcium antagonists that selectively block T-type voltage-gated plasma membrane calcium channels in vascular smooth muscle. The drug is structurally and pharmacologically different from traditional calcium antagonists. It does not have negative inotropism at therapeutic concentrations, and is not associated with reflex activation of neurohormonal and sympathetic systems. In clinical studies of hypertension, mibefradil 50 and 100 mg/day reduced trough sitting diastolic and systolic blood pressures in a dose-related manner. Dosages exceeding 100 mg/day generally did not result in significantly greater efficacy, but were associated with a higher frequency of adverse events. No first-dose hypotensive phenomenon was observed. Mibefradil has antiischemic properties resulting from dilation of coronary and peripheral vascular smooth muscle, and a slight reduction in heart rate. In clinical studies of chronic stable angina pectoris, dose-related increases in exercise duration, time to onset of angina, and time to 1-mm ST-segment depression during exercise tolerance tests occurred. Mibefradil reduced the number and duration of ischemic events recorded by 48-hour ambulatory electrocardiograph (ECG) monitoring, as well as number of anginal episodes and nitroglycerin consumption. Favorable hemodynamic and clinical profiles are reported, including high trough:peak ratios (> 80%), high oral bioavailability, and long elimination half-life (17-25 hrs) permitting once/day dosing. Dizziness, headache, leg edema, and lightheadedness are frequently reported, but overall the agent is well tolerated. First-degree atrioventricular block and sinus bradycardia are the most frequent ECG changes caused by the drug. In vitro studies indicate mibefradil inhibits cytochrome P450 1A2, 2D6, and 3A4, resulting in elevated plasma concentrations of drugs metabolized by those isoenzymes. Therefore, it is contraindicated in patients receiving terfenadine, astemizole, cisapride, lovastatin, or simvastatin.     

The calcium channel blocker amlodipine raises serum dehydroepiandrosterone sulfate and androstenedione, but lowers serum cortisol, in insulin-resistant obese and hypertensive men

To determine whether the calcium channel blocker amlodipine improves glucose tolerance and alters serum adrenal androgen and glucocorticoid levels in insulin-resistant men, 24 obese and hypertensive men were enrolled into a single blind, placebo-controlled study. An amlodipine group (n = 12) and a placebo group (n = 12) were studied before and after treatment with either amlodipine (5 mg) or placebo capsule twice daily for 7 days by determining serum insulin, glucose, dehydroepiandrosterone sulfate (DHEA-S), androstenedione, and cortisol in the fasting state and during an oral glucose tolerance test. Amlodipine treatment 1) lowered fasting serum insulin (from 273 +/- 19 to 200 +/- 17 pmol/L; P < 0.0005) and glucose (from 5.4 +/- 0.1 to 5.1 +/- 0.1 mmol/L; P < 0.02), 2) reduced the area under the curve for glucose (from 1342 +/- 25 to 1198 +/- 23 mmol/L.min; P = 0.0001) and the area under the curve for insulin (from 155.5 +/- 7.8 to 103.9 +/- 4.3 nmol/L.min; P = 0.0001) during the oral glucose tolerance test, 3) increased fasting serum DHEA-S (from 5.19 +/- 0.37 to 7.95 +/- 0.58 mumol/L; P = 0.0001) and androstenedione (from 5.65 +/- 0.65 to 6.83 +/- 0.53 nmol/L; P < 0.01), and 4) decreased fasting serum cortisol (from 538 +/- 35 to 494 +/- 26 nmol/L; P < 0.05). Fasting serum androstenedione declined slightly in the placebo group (from 5.96 +/- 0.60 to 5.74 +/- 0.57 nmol/L; P < 0.005), but no change occurred in glucose tolerance, fasting serum DHEA-S, or cortisol. We conclude that amlodipine treatment improves glucose tolerance, reduces fasting and glucose-stimulated serum insulin levels, increases serum DHEA-S and androstenedione levels, and decreases circulating cortisol.