Protein intake and blood glucose as modulators of GFR in hyperfiltering diabetic patients. Glomerular hyperfiltration has been claimed to be a risk factor for the development of diabetic nephropathy. Protein intake and hyperglycemia can both increase GFR in diabetic and normal subjects. Our study was designed to explore the relative importance of short-term changes in protein intake and glycemia on the modulation of renal hemodynamics in insulin-dependent diabetic (IDDM) patients with and without glomerular hyperfiltration. The renal hemodynamic response to a protein challenge was studied in eight hyperfiltering (HF) and eight normofiltering (NF) patients after a three week period of low or normal protein diet (LPD, NPD), each study being conducted twice, in random order, under conditions of prevailing hyperglycemia (H) and euglycemia (E). In HF patients GFR failed to increase significantly in response to protein challenge during NPD under conditions of either H or E (Baseline vs. 2 hr H: 151 +/- 4 vs. 155 +/- 6, NS; E 147 +/- 4 vs. 157 +/- 7 ml/min/1.73 m2, NS). A more normal response was restored following LPD with GFR increasing in all but one patient after challenge during H and in all patients during E (Baseline vs. 2 hr H: 130 +/- 7 vs. 145 +/- 8, P less than 0.07; E: 127 +/- 7 vs. 143 +/- 7 ml/min/1.73 m2, P less than 0.01). Changes in RPF paralleled the changes in GFR and filtration fraction remained stable under all study conditions.(ABSTRACT TRUNCATED AT 250 WORDS)


Immunochemical studies of the Alport antigen. The Alport antigen, a component of normal glomerular basement membranes (GBM) which is absent in Alport familial nephritis, is characterized as a 26 kD non-collagenous (NC1) peptide identified by a monoclonal antibody (Mab A7) and an Alport alloantibody. Both antibodies discriminate X-linkage of the Alport defect using indirect immunofluorescence of hemizygous and heterozygous Alport GBM and epidermal basement membrane (EBM). Immunoblotting of SDS-PAGE gels of collagenase-digested Alport renal BM shows absence of monomeric and dimeric components of the Alport antigen, alpha 3(IV) NC1, and alpha 4(IV) NC1. By immunoprecipitation experiments with specific antibodies, the Alport antigen is distinct from the 26 kD NC1 peptide derived from alpha 1(IV). The monoclonal antibody to the Alport antigen and rabbit antiserum to a non-consensus sequence of alpha 5(IV) NC1 react similarly by immunofluorescence with normal kidney and both fail to bind to Alport renal BM. Two dimension Western blots of collagenase-digested BM show that the anti-Alport antigen and the ant-alpha 5(IV) NC1 react similarly with monomeric and dimeric components of BM collagen. These studies are consistent with the likelihood that the Alport antigen and alpha 5(IV) NC1 are the same or are highly homologous molecules. The precise relationship will require characterization of alpha 5(IV) NC1 protein and determination of the nucleotide sequence of the Alport antigen. The associated absence of alpha 3(IV) NC1 and alpha 4(IV) (NC1) from Alport BM is consistent with other observations for a molecular association of these chains in a novel collagen network.


Expression of a monoclonal antibody (3G5) defined ganglioside antigen in the renal cortex. The monoclonal antibody (mAb) 3G5 was found, by indirect immunofluorescence, to bind to renal cortical structures in frozen sections of human, rat and calf kidneys. Double indirect immunofluorescence studies on frozen sections of rat kidneys showed that 3G5 stained only the glomerulus and the distribution of the 3G5 antigen on the glomerulus was more extensive than the staining observed with antibodies to Factor VIII antigen. 3G5 stained the proximal convoluted tubules and collecting tubules in bovine renal sections but glomeruli did not stain with 3G5. The 3G5 mAb did not stain tissue cultured bovine glomerular endothelial cells or mesangial cells, but did stain bovine glomerular epithelial cell cultures. 3G5 did not stain MDCK cell cultures. The binding of mAb 3G5 to glomeruli was investigated by immunoelectron microscopy of rat renal tissue. In contrast to the podocyte specificity on bovine glomerular cells in vitro, it was found that the specificity of 3G5 expression on rat glomerular cells in vivo was broader. No binding of mAb 3G5 was found outside the glomerulus in the rat renal cortex. Podocytes, endothelial cells and capsular epithelial cells expressed the 3G5 antigen most strongly. A lesser amount of binding was found in the glomerular basement membrane. The mesangium showed a little binding of mAb 3G5 and no binding at all was found to other cortical structures. The 3G5 antigen in rat renal tissue was found to be a glycolipid that migrated between the ganglioside markers GM2 and GM1 by immunostaining of thin layer chromatograms.(ABSTRACT TRUNCATED AT 250 WORDS)


Cervical discs are most susceptible to beta 2-microglobulin amyloid deposition in the vertebral column. Intervertebral discs of 41 chronic renal failure autopsy cases were examined histologically and immunohistochemically to assess the distribution of beta 2-microglobulin-associated (beta 2m) amyloid in the vertebral column. The results demonstrated beta 2m amyloid to appear first in the cervical discs, then in the lumbar and upper thoracic discs, and finally in the middle and lower thoracic discs as the dialysis period is prolonged. The shortest dialysis period for which beta 2m amyloid was detected was one year and seven months. Deposition of beta 2m amyloid was most remarkable in the C4-5, 5-6, and 6-7 levels, which are known to sustain severe mechanical stress in daily life. Thus it is suggested that local mechanical stress accelerates beta 2m amyloidosis. A marked macrophage reaction was observed around the amyloid in cases of severe amyloidosis, the macrophages themselves being immunohistochemically positive for IL-1 beta and TNF-alpha. Amyloid deposition and reactive inflammation mediated by cytokines appear to be closely related to the pathogenesis of destructive spondyloarthropathy.


Impaired metabolism of high density lipoprotein in uremic patients. We measured lipoproteins, apolipoproteins, lipoprotein lipase (LPL), hepatic triglyceride lipase (HTGL), lecithin: cholesterol acyltransferase (LCAT) and parameters of calcium metabolism to evaluate the roles of these enzymes and hypertriglyceridemia for impaired high-density lipoprotein (HDL) metabolism in chronic renal failure, and to examine the impact of altered calcium homeostasis on the lipoprotein-regulating enzymes. The subjects were 25 healthy volunteers and 66 uremic patients, 24 treated with hemodialysis (HD) and 42 with continuous ambulatory peritoneal dialysis (CAPD). Lipoprotein analysis revealed: (1) reduction in HDL cholesterol especially in HDL2 subfraction; (2) increase in HDL triglyceride; and (3) decreased ratio of HDL2 cholesterol to HDL3 cholesterol in both HD and CAPD patients. Simple regression analysis showed: (1) a positive correlation between VLDL triglyceride and triglyceride/cholesterol ratio of HDL; (2) positive correlations of LPL level in post-heparin plasma to cholesterol concentrations in HDL2, HDL3 and total HDL, and to apolipoproteins A-I and A-II; and (3) inverse correlations of HTGL to HDL2 cholesterol and to the ratio of HDL2 cholesterol/HDL3 cholesterol. Multiple regression analysis of HDL cholesterol indicated positive association with LPL and inverse correlation with VLDL triglyceride. Four variables including LPL, HTGL, LCAT and VLDL triglyceride explained 51.5% of the variation of HDL cholesterol. HDL2 cholesterol was associated positively with LPL and negatively with VLDL triglyceride in the model. HDL3 cholesterol was associated positively with LPL, HTGL and LCAT and inversely with VLDL triglyceride. Stepwise multiple regression analysis indicated that independent predictors of HTGL were gender, parathyroid hormone levels by a mid-portion assay, ionized calcium and age, and that those of LCAT were ionized calcium and age.(ABSTRACT TRUNCATED AT 250 WORDS)


Treatment of chronic hyponatremia in rats by intravenous saline: comparison of rate versus magnitude of correction. The role of the rate of correction in the development of demyelinating brain lesions after correction of chronic severe hyponatremia is controversial. It has been recently suggested in rats treated by intravenous (i.v.) hypertonic saline (NaCl) that both the rate and the absolute change in serum sodium represent critical risk factors. However, we previously demonstrated in rats treated by intraperitoneal (i.p.) injections of NaCl that below a threshold of serum sodium rise of 20 mEq/liter/24 hr, only 5% of the brain lesions were recorded, even in rats submitted to a rapid (1 hr) serum sodium increment following the i.p. injection. Working below this threshold (serum sodium rise less than 20 mEq/liter/24 hr) in the present work, allowed us to independently determine the role of the rate in the outcome of the correction. This was done by submitting the rats to a rapid (1 hr) intravenous infusion of NaCl. As a difference between the i.p. and i.v. route in the degree of volume expansion produced by the NaCl administration could also play a role in the pathogenesis of the brain lesions, rats treated with rapid i.v. infusion of NaCl (associated with volume expansion) were compared to a group of rats treated with water restriction (associated to volume contraction) to evaluate the role of volemia on the incidence of neurological damage. Hyponatremia was induced over three days with d-glucose in water and vasopressin. The group 1 was corrected by intravenous (i.v.) infusion of hypertonic saline over one hour.(ABSTRACT TRUNCATED AT 250 WORDS)


Anti-DNA antibodies form immune deposits at distinct glomerular and vascular sites. To investigate the capacity of lupus autoAb to produce glomerular immune deposits (ID) and nephritis, 24 murine monoclonal (m) anti-DNA antibodies (Ab), derived from either MRL-lpr/lpr, SNF1 or NZB lupus-prone mice and selected based on properties shared with nephritogenic Ig, were administered i.p. (as hybridomas) and i.v. (as purified Ig) to normal mice; at least four mice/mAb were evaluated. Three general patterns of immune deposit formation (IDF) were observed: extracellular ID within glomeruli (+/- blood vessels, N = 8); intranuclear ID (N = 5); or minimal or no ID (N = 11). The four MRL m anti-DNA Ab that produced significant extracellular ID demonstrated different disease profiles including: (a) mesangial and subendothelial ID with anti-basement membrane staining, associated with proliferative glomerulonephritis, PMN infiltration, and proteinuria; (b) diffuse fine granular mesangial and extraglomerular vascular ID, associated with proliferative glomerulonephritis and proteinuria; (c) dense intramembranous ID and intraluminal ID, associated with capillary wall thickening, mesangial interposition and expansion, aneurysmal dilatation and intraluminal occlusion of glomerular capillary loops, and heavy proteinuria; and (d) mesangial and extraglomerular vascular ID, associated with mild segmental mesangial expansion, without proteinuria. These MRL mAb were derived from four different mice, and they had variable pIs and isotypes. They all cross reacted with multiple autoantigens (autoAg), however, their autoAg binding profiles were distinguishable. Among the SNF1 derived mAb, four produced histologically and clinically indistinguishable disease characterized by diffuse mesangial and capillary wall ID, associated with cellular proliferation/infiltration and proteinuria. Three of the four mAb were derived from the same mouse and were clonally related; they were: IgG2b with SWR allotype, relatively cationic, highly cross reactive with similar Ag binding patterns, idiotypically related and encoded by identical VH and nearly identical VL sequences. We conclude that both the capacity of lupus autoAb to form ID and the location of IDF are dependent on properties unique to individual Ig. The results also indicate that the Ag binding region of the autoAb is influential in this process, and they suggest that multiple Ab-Ag interactions contribute to IDF in individuals with lupus nephritis. Furthermore, these observations raise the possibility that the pathologic and clinical abnormalities resulting from these interactions are influenced by the location of IDF, and that the dominant interaction, in a given individual, may be highly influential in the phenotypic expression of nephritis.


The assessment of depth of anaesthesia in animals and man. The assessment of the depth of anaesthesia in man has received much attention in recent years, following a number of reports of people being aware during surgery. A range of different measures have been suggested for determining the adequacy of anaesthesia in man, but such a critical assessment is rarely applied to laboratory animals. This article describes the methods used to assess anaesthetic depth in both man and animals, and compares the relative states of knowledge about anaesthetic depth in animals and man.


A modified anaesthetic induction chamber for rats. The anaesthetic induction chamber for rats described in this paper has been designed for use in conjunction with a controlled delivery of halothane/O2 mixture and an anaesthetic scavenger system. Using this system rapid induction of anaesthesia is achieved using low levels of anaesthetic vapour without risk to the operator.


Developmental and regressive changes in the testes of the Himalayan rabbit. The testes of 81 Himalayan rabbits, controls in toxicity studies, and 113 purpose-bred rabbits of various age groups from birth to 2 years were studied. In mature male rabbits, regressive changes in the seminiferous tubules including the multinucleated spermatid giant cells were common and showed a tendency for age-related and seasonal differences. The finding is considered as a remnant of the previous seasonal and possibly also social testicular regression in wild living animals. The giant cells develop from the syncytium of the germinative epithelium and have parallels in phylogenesis.


The effects of buprenorphine, nalbuphine and butorphanol alone or following halothane anaesthesia on food and water consumption and locomotor movement in rats. Locomotor activity and food and water consumption are potentially indices of post-operative pain in laboratory rodents, but it is important to establish whether these variables are directly affected by opioid analgesics or by halothane anaesthesia in normal rats. The effects of three opioids, buprenorphine, nalbuphine and butorphanol administered alone or following halothane anaesthesia, were studied in groups of normal non-operated adult Wistar rats. All 3 analgesics affected food intake and activity levels, but had little or no effect on water intake. Buprenorphine caused a significant elevation of activity levels and a reduction in food intake at clinical doses (0.01 and 0.05 mg/kg s/c). Nalbuphine (0.5, 1 and 2 mg/kg s/c) caused a reduction in food intake but had a smaller stimulatory effect on locomotion. Butorphanol (0.4 mg/kg s/c) caused a reduction in food intake and elevation in activity. These results suggest that water consumption is likely to be a more reliable variable to use when assessing post-operative pain and the efficacy of analgesics in rats.


Echocardiographic parameters and indices in the normal beagle dog. M-mode and two-dimensional echocardiographic measurements were made from the right sternal border of 50 healthy Beagles (25 males and 25 females) approximately 7 months old. The dogs were conscious and standing during the investigation. The following parameters, in systole and diastole, were measured on the echocardiographic images: left ventricular posterior wall thickness (LVWT); intraventricular septum thickness (IST); left ventricular internal dimension (LVID); and circumference (LVC). Fractional shortening (FS) and ejection fraction (EF) were also calculated. Mean, standard deviation, range and coefficient of variation are reported for each echocardiographic parameter and for body weight. Males and females were considered separately and together. Each parameter was analysed statistically to check for differences between the sexes and for correlations with body weight. A statistically significant difference between the sexes was only observed for LVWT in systole and diastole. A linear regression with body weight was obtained only for LVID in systole and in diastole. The results show that morphofunctional cardiac homogeneity is independent of size in dogs of this breed and age.


Anterior cervical microsurgical approach to the cranial base in the rabbit: technical note. Current trends in research on craniofacial syndromes have led to enhanced interest in the cranial base as a contributory factor in the development of normal and abnormal midfacial structure. Indeed, attention has focused upon one particular growth plate in the posterior cranial base, the spheno-occipital synchondrosis, since it has been shown that alterations in this structure are associated with profound changes in craniofacial growth. In this report we describe a surgical approach to the cranial base of the rabbit that is safe, simple and reliable. It is applicable to neonatal as well as adult rabbits.


Lymphatic drainage of Listeria monocytogenes and Indian ink inoculated in the peritoneal cavity of the mouse. The lymphatic drainage of the peritoneal cavity has been investigated by intraperitoneal inoculation of an intracellular bacterium (Listeria monocytogenes) and an inert marker (Indian ink). The results reveal that both agents are transported, either after phagocytosis by intraperitoneal macrophages or in suspension in the lymph, towards the cranial sternal lymph nodes (Lymphonodi sternales craniales) of the ventral thoracic lymphocentrum (Lymphocentrum thoracicum ventrale) and to the lymph nodes of the mediastinal lymphocentrum (Lymphocentrum mediastinale), prior to systemic dissemination. This mechanism of peritoneal lymph drainage has relevance on experimental studies involving the inoculation of pathogens, and on the investigation of metastatic diffusion of neoplasms from the peritoneum.


The effects of intracage ventilation on microenvironmental conditions in filter-top cages. Filter-top cages, while effective in reducing cross contamination by particulate material including microbes, can also cause accumulation of the waste gases carbon dioxide and ammonia as well as increased intracage relative humidity. A prototype system which provided each cage with 23 air changes per hour through a nozzle inserted in the filter lid was evaluated. The ventilated cageing system was effective in reducing intracage carbon dioxide, ammonia and relative humidity levels. Mean weekly carbon dioxide levels were 2900 ppm lower, ammonia levels 240 ppm lower and intracage relative humidity levels 8% lower in the ventilated cages than in unventilated controls.


A fast and simple technique for jugular catheterization in adult sows. Obtaining measures of an animal's physiological state with minimum disturbance to the animal is essential in animal welfare studies and it is also ethically desirable. A simple technique for implanting a temporary jugular catheter in adult sows is described. The technique involves implantation via an ear vein and can be performed in 5-7 min. Local but not general anaesthetic is required. The technique has no effect on basal levels of salivary cortisol 1-2 h later, suggesting that it has no long term stress consequences. Catheters continue to function perfectly well after 4 h.


Haematological and serum biochemical values in hairless and haired descendants of Mexican hairless dogs. Haematological and serum biochemical measurements were carried out in 1-year-old hairless and haired hybrids derived from the Mexican hairless dog (MHD). These hybrids included F1 hybrids obtained from male MHD and female Beagles, and BCF1 hybrids obtained from male hairless F1 and female Beagles. There were no significant differences between F1 and BCF1 hybrids, nor between male and female hybrids. Except for red blood cell counts, haemoglobin concentrations and packed cell volumes which were slightly higher in MHD-descendants than in Beagles, there were no differences for haematological and serum biochemical findings between hairless and haired hybrids when compared to age-matched Beagles.


Rabbit encephalitozoonosis in Kenya.

Rabbit encephalitozoonosis in Kenya. Encephalitozoon cuniculi infection was diagnosed in a laboratory rabbit breeding colony at Muguga, Kenya. This is the first report of the disease in rabbits in Kenya. Post-mortem examination showed gross renal lesions and the presence of the parasite in histological sections of the cerebrum and cerebellum. On Gram stain, spores were observed in the kidney sections.


Magnetic resonance imaging using a clinical whole body system: an introduction to a useful technique in small animal experiments. A clinical whole body magnetic resonance imaging (MRI) system with high resolution coils was used to obtain non-invasive images of the living rat. The results demonstrate the feasibility of the set-up and the advantages of this new imaging technique: detailed information, no extra costs, longitudinal studies without killing animals and simple anaesthesia. It is concluded that in small animal experimentation, this use of high resolution coils in whole body magnetic resonance systems may be particularly helpful in establishing effects of experimental procedures. Whenever non-invasive visualization is required, especially in longitudinal animal studies, e.g. biomaterial research or tumour investigation, this use of MRI will offer challenging possibilities.


Pygomelia in a C3H/Crl mouse: case report. Accessory hind limbs (pygomelia) were found in a male pup born in our colony of C3H/Crl mice. This malformation has not previously been reported in mice.


Tumor infiltrating cells in human cancer. On the possible role of CD16+ macrophages in antitumor cytotoxicity. To obtain a better understanding of the mechanism underlying different modalities of immunotherapy, we investigated the types of tumor-infiltrating cells present at the tumor site, with special attention to the presence of macrophages. Frozen sections of carcinomas of the kidney, colon, breast, lung, ovary, and thyroid gland, as well as malignant melanoma were investigated with a panel of monoclonal antibodies against macrophage, T cell and NK cell associated antigens. Both type and pattern of the tumor-infiltrating cells were analyzed. All tumor-infiltrating cells accumulated preferentially in the stromal bands between tumor cells. In all types of tumor, CD11c+, CD14+, CD68+ and alpha-naphthyl-acetate-esterase positive monocytes/macrophages accounted for most tumor-infiltrating cells. Next in frequency were T lymphocytes (CD2+, CD3+, TCR alpha beta +). Only a few B lymphocytes (CD22+), and T cells expressing the T cell receptor gamma delta (TCR gamma delta) were found. Hardly any lymphoid cells with an NK phenotype (CD3-, CD56+) were present in the tumors studied. Large numbers of CD16+ cells were found, which could be identified as macrophages on the basis of their morphology, positive staining with a panel of monocyte/macrophage markers, and the results of double staining with CD11c. We have demonstrated the presence of a large number of macrophages in the cellular infiltrates of several types of tumors. The largest numbers of CD16+ macrophages were found in renal cancer, melanoma, and colonic-carcinoma. These are the tumors that are most susceptible to immunotherapy with lymphokine activated killer cells, suggesting that these CD16+ macrophages may be involved in antitumor cytotoxicity. Furthermore, these findings suggest that new strategies of immunotherapy aimed at the use of macrophages present in many tumors could be developed.


Glomerular localization of nephritogenic protein complexes on a nonimmunologic basis. In a preliminary study, we have found that a group of charge-distinct protein molecules can react with each other to form protein complexes in vitro. The present study was conducted to determine the nephritogenic effect and the mode of action of these proteins in vivo. To demonstrate the precipitating interactions, a phosphorylcholine (PC)-conjugated, cationized bovine serum albumin (BSA(+)-PC) was applied to react with dinitrophenyl (DNP)-protein conjugates by double diffusion test. The types of forces involved in binding actions were determined. The experimental groups of CDF1 mice were injected simultaneously with BSA(+)-PC and individual DNP-protein conjugates through the tail vein. Both clearance kinetics and renal tissue studies were performed. BSA(+)-PC was able to form precipitin lines with respective DNP-protein conjugates in the agarose gel without any antigen-antibody interactions. These precipitating reactions were partially prevented by the presence of either NaCl, urea, or trifluoroacetate, suggesting that electrostatic force, hydrogen bond, and/or hydrophobic force are involved in the precipitation of these modified protein molecules. The clearance kinetics of BSA(+)-PC/BSA-DNP, and BSA(+)-PC/DNP-apoferritin showed a significant acceleration of the rapid phase of elimination. All of the experimental mice had hematuria and systemic thrombotic microangiopathic changes 1 hour later. By immunofluorescence, both the BSA(+)-PC and the DNP-protein conjugate injected were present in the renal glomerular tufts of experimental groups, although these protein molecules failed to deposit in the glomerulus when injected alone. Concomitant fibrinogen deposition was noted in almost all of the glomeruli affected. Moreover, 10 to 50% of the glomeruli examined 4 hours after injection had deposition of the third component of complement (C3). These data strongly support a hypothesis that nondeposited nephritogenic protein molecules in a fashion of heterogeneous complexes can localize in the glomerulus, thereby initiating renal injury, with the lack of antibody involvement.


Impaired attachment of hepatocytes to extracellular matrix components after chronic ethanol administration. Previous studies have shown that the assembly and properties of the hepatocyte plasma membrane are altered by ethanol administration, indicating possible changes in the receptor-mediated binding of the plasma membrane to extracellular matrix substrates. In the present study, the effects of chronic ethanol consumption on the ability of hepatocytes to attach to various components of the extracellular matrix were investigated. Rats were pair-fed for 5 weeks with a liquid diet containing either ethanol (as 36% of total calories) or isocaloric carbohydrate. The effects of ethanol treatment on hepatocyte-extracellular matrix interactions was ascertained by determining the ability of isolated hepatocytes to attach to various extracellular matrix substrates. The attachment of hepatocytes, isolated from the ethanol-fed rats, to laminin-coated plates was significantly decreased compared with hepatocytes from chow-fed or pair-fed controls. Greater decreases in attachment were seen when higher numbers of hepatocytes were seeded in the plates. Similar inhibitions of attachment were also observed when fibronectin or type I collagen were used as matrices. Time-course cell attachment assays indicated that the maximum extent of attachment rather than the rate of attachment was primarily altered by chronic ethanol feeding. Hepatocytes from the ethanol-fed rats also detached more readily from the matrix-coated plates than those from the controls. A reduced number of functional surface receptors for matrix components is likely the most important factor that accounts for the ethanol-induced impairment of hepatocyte attachment. These results indicate that chronic ethanol administration impairs the interactions of hepatocytes with their extracellular matrix and that this defect could lead to alterations of hepatocyte structure and function.


Role of intercellular junctions in the passage of horseradish peroxidase across aortic endothelium. The manner in which molecules are transported across the arterial endothelial layer has been a subject open to much interpretation and controversy. Further elucidation and clarification of these mechanisms are of primary interest. To investigate the ultrastructural features of arterial endothelial junctions and to evaluate their functional roles as a transendothelial pathway for macromolecular transport, experiments were performed on the thoracic aortae of adult male Sprague-Dawley rats by using the ultrathin serial sectioning technique and horseradish peroxidase (HRP). The aorta was perfusion-fixed with or without prior intravenous injection of HRP. The intercellular clefts exhibited a great deal of variety in shape, being linear, winding, interdigitated, irregular and/or dumbbell-shaped in appearance. Besides the typical 20-nm width encountered at the uniform region of intercellular clefts, local widenings (up to several hundred nm) were quite common. The arterial endothelial junctions were highly organized. Junctional elements, including tight junctions and gap junctions, were frequently present in the same intercellular cleft, even on the same plane of sectioning. Sometimes, gap junctions were found without tight junctions, but the intercellular clefts were rarely obliterated by tight junctions alone. Some intercellular clefts were not obliterated by either gap or tight junctions, and HRP was found to reach the subendothelial space by passing through these junctionless clefts. Densitometric determination of the HRP concentration profile in such junctionless clefts showed a decreasing gradient from the luminal to the abluminal front. The serial sections provided evidence that the apparently free vesicles were actually plasmalemmal membrane invaginations open to the luminal or abluminal front in the arterial endothelium. The present study showed that the junctionless normal endothelial clefts, in addition to the transiently open junctions surrounding mitotic cells, might provide a significant pathway in the transendothelial transport of macromolecules with the size of HRP.


Endothelin. Immunohistologic localization in aorta and biosynthesis by cultured human aortic endothelial cells. Endothelin-1 (ET-1) has been shown to exist in many organs and to have various biologic functions including vasoconstriction. However, an exact location of ET gene expression of the tissues is not fully investigated. Human aortic tissue was examined to elucidate the exact location of ET gene expression. Human aortas were obtained at autopsy and fixed in either conventional 10% formalin or 3% paraformaldehyde. The aortic thin sections were subjected to examinations of an immunohistochemistry and in situ hybridization of ET-1. Human aortic endothelial cells were cultured by a previously reported method. ET-1 released in the supernatant from the cultured endothelial cells was radioimmunoassayed. Immunohistologic study of ET-1 revealed a linear staining of the endothelial monolayer and diffuse staining in the intimal and medial smooth muscle cells on human aorta except for fetal aorta. In situ hybridization signals were intense in the endothelial cells from the elderly as well as younger subjects as examined with 35S-labeled anti-sense probe RNA. Fetal aortic endothelial cells revealed the least signals that meant developing but still immature gene translation. Smooth muscle cells showed positive but weak in situ hybridization signals. Control immunohistologic and hybridization studies were negative. ET-1 biosynthesis by cultured human aortic endothelial cells was invariably low in the subjects under the age of 50, ranging from 0.23 to 0.40 pmol/1 x 10(5) cells for 3 days. On the other hand, endothelial cells from the elderly subjects generally synthesized a greater amount of endothelin in vitro. These findings indicate that ET-1 is most highly expressed in endothelial cells, although not as highly but certainly, expressed in intimal and medial smooth muscle cells. This fact gives a new insight into the biophysiologic and pathologic roles of ET. In addition, these methods are applicable to investigate the gene expression of ET-1 in all organs and tissues.


Expression of syndecan in transformed mouse keratinocytes. Malignant transformation is frequently associated with altered behavior of cells, a phenomenon that also suggests changes in cell-matrix interactions. We have studied expression of syndecan, a cell surface proteoglycan that binds extracellular matrix components and growth factors, in various chemically transformed mouse keratinocyte cell lines that differ in their morphology and tumorigenicity. A monoclonal antibody, specific for mouse syndecan, and a cDNA clone for mouse syndecan, were used to detect syndecan in seven different keratinocyte cell lines. The glycosaminoglycan composition of syndecan was studied using differential digestions of heparan sulfate and chondroitin sulfate chains. In general, the tumorigenic cells were found to express lower amounts of syndecan, both at protein and mRNA levels, than the nontumorigenic cells. The most tumorigenic cell line CarC revealed barely detectable syndecan expression. Also, molecular polymorphism of syndecan was observed, as three forms of syndecan with different molecular weights appeared on the surfaces of different keratinocytes. The highly tumorigenic cells, that expressed low amounts of syndecan, expressed syndecan with the largest molecular weight. The different molecular weights were shown to reflect an increased amount of both heparan and chondroitin sulfate chains attached to the core protein. An increased shedding of syndecan ectodomain from the membrane-associated domain was observed in cells that express high amounts of mutated Ha-ras p21. The results suggest, that transformed epithelial cells can modulate the appearance of syndecan on the cell-surface by at least two ways: (a) by altering its glycosylation or (b) by increasing its shedding from the cell surface. These modulations, together with overall suppression of syndecan expression, could be associated with malignant transformation of keratinocytes.


Ploidy independence of Ag-NOR number in neuroblastoma. Ag-NOR number and ploidy have potential use in predicting prognosis in malignancy. The issue of independence has not been conclusively studied. In contrast to other malignancies in neuroblastoma poor outcome is linked to diploidy, allowing independent analysis of the reaction of Ag-NOR status to outcome. A consecutive unselected series of 20 pediatric neuroblastoma patients was used to study the relation between ploidy and mean Ag-NOR number per nucleus. Ploidy was established by flow cytometric analysis of nuclear suspensions prepared from Formalin fixed and paraffin embedded tissue. The mean number of Ag-NORs per nucleus was determined using 3-microns paraffin sections, silver staining methods and quantification procedures detailed in the literature. The seven diploid lesions were found to have a mean Ag-NOR number per nucleus of 4.38 (range 0.86-7.5). The 13 aneuploid lesions were found to have a mean Ag-NOR number per nucleus of 2.31 (range 1.17-4.44). Probability of difference (Wilcoxon's two-tailed rank sum test) was greater than 99%. Nonsurvivors (6 of 10 diploid) had mean Ag-NOR number per nucleus of 4.34 (range 2.24-7.5). Survivors (9 of 10 aneuploid) had mean Ag-NOR number per nucleus of 1.73 (range 0.86-2.56). Probability of difference (Wilcoxon's two-tailed rank sum test) was greater than 99%. In aneuploid lesions (n = 13) a negative correlation exists between DNA index and mean number of Ag-NORs per nucleus (correlation coefficient 0.84, slope -0.74, probability of noncorrelation less than 0.010). The present observations offer the first independent support to the theory that Ag-NOR number is an independent variable to DNA ploidy based on the unique feature of neuroblastoma that diploid lesions have an established poorer prognosis than aneuploid lesion. The observations, although the number of cases is too limited for independent analysis, confirm previously published, strong negative relations between mean Ag-NOR number per cell and survival in childhood neuroblastoma. This relation is similar to findings in other solid tumor lesions.


Improved accuracy in diagnostic immunohistochemistry, lectin histochemistry and in situ hybridization using a gold-labeled horseradish peroxidase antibody and silver intensification. Improvements in the use of the avidin-biotin peroxidase complex technique and direct as well as indirect labeled avidin-biotin methods for application in diagnostic immunohistochemistry, lectin histochemistry and in situ hybridization are reported. The new technology combines the advantages of immunoenzyme and immunogold silver staining techniques and can be performed on routinely fixed and paraffin-embedded tissues. The basic modification of the labeling procedures was introduced at the final revealing step. The histochemical visualization of catalytic activity of horseradish peroxidase by the diaminobenzidine reaction was replaced by the detection of horseradish peroxidase immunoreactivity using anti-horseradish peroxidase-gold complexes and their intensification with silver acetate which is relatively light insensitive. The use of gold-labeled anti-horseradish peroxidase antibodies eliminates the need for quenching of endogenous peroxidase activity. Furthermore, the immunogold silver staining provides improved lateral resolution, higher contrast, and lower background staining as compared with the diaminobenzidine reaction. The new technology has been applied for the localization of different polypeptides in endocrine cells, cytoskeletal elements, cell surface receptors, basal lamina type IV collagen, endothelial cell marker, lectin binding sites, and DNA of various viruses. We concluded that the anti-horseradish peroxidase-gold complex is of general use in a variety of techniques applying horseradish peroxidase as a marker and should be a valuable alternative to existing enzyme substrate techniques.


A death attributed solely to diltiazem.

A death attributed solely to diltiazem. An elderly woman, distraught over the recent death of her husband, was found dead of an apparent suicide. Qualitative screening detected only the presence of diltiazem, an antihypertensive, antianginal agent that was prescribed for a known heart ailment. Quantitation of diltiazem was carried out on available fluids and tissues with the following results: blood 6.7 mg/L, urine 5.4 mg/L, vitreous 5.5 mg/L, and liver 79 mg/kg. Death was subsequently attributed to an overdose of this medication.


Two fatalities from ingestion of acetonitrile: limited specificity of analysis by headspace gas chromatography. A married couple was found dead in bed at their home, and the police investigation indicated that they might have ingested methanol and/or ethanol the previous evening. Postmortem examinations were performed and samples of femoral vein blood, urine, and stomach contents were submitted for toxicological analysis. Headspace gas chromatography (HSGC) using two different column packings (Carbopak B and Carbopak C) was used for qualitative and quantitative analysis of organic volatile substances. The apparent concentrations of ethanol in blood and urine were 0.20 and 0.27 g% w/v, respectively. On reanalysis of body fluids by another HSGC method and gas chromatography/mass spectrometry (GC/MS), the presence of ethanol was not confirmed. Instead, acetonitrile was identified at concentrations of 0.8 g/L in blood, 1.0 g/L in urine, and 1.3 g/L in stomach contents. Inorganic cyanide, the toxic metabolite of acetonitrile, was identified in blood from both victims at concentrations of 2.4 micrograms/g (female) and 4.5 micrograms/g (male). Despite the use of two different column packing materials for HSGC, acetonitrile was mistakenly identified as ethanol.


Evaluation of phencyclidine by EMIT d.a.u. utilizing the ETS analyzer and a 25-ng/mL cutoff. This study evaluates the usefulness of the EMIT d.a.u. phencyclidine assay using the Syva ETS instrument to reliably detect phencyclidine in urine specimens at 25 ng/mL and above. More than 1600 urine specimens were screened over a one-month period. Fifty three (53) specimens screened positive (25 of these were previously tested positive by EMIT and GC/MS at or above the 25-ng/ml level and resubmitted as unknowns). Of the 53 specimens, 52 were confirmed positive by GC/MS. Reanalysis by EMIT of the one sample that was confirmed negative by GC/MS yielded a negative result. The absorbance difference between a negative sample and a sample containing 25 ng/mL of phencyclidine averaged 53 absorbance units with a standard deviation of 7.7 units. The absorbance difference between a negative sample and a sample containing 75 ng/mL of phencyclidine averaged 105 absorbance units with a standard deviation of 6.8 units.


Evaluation of the Syva ETS-PLUS Ethyl Alcohol Assay with application to the analysis of antemortem whole blood, routine postmortem specimens, and synovial fluid. A critical evaluation of the ETS-PLUS Ethyl Alcohol Assay indicates that it is simple to perform, sensitive enough to reliably detect ethanol above a concentration of 0.010 g/dL, and dynamic enough to reliably quantitate ethanol concentrations between 0.010 and 0.400 g/dL, correlating well with headspace gas chromatography. The technique is suitable for the analysis of relatively fresh (less than 1 month old) serum, plasma, urine, bile, vitreous humor, and gastric samples. With the aid of dilution, it is adaptable to the analysis of whole blood, relatively old specimens (more than 3 months old), viscous and highly pigmented plasma, bile, and the occasional extremely viscous vitreous humor specimens. Our evaluation further indicates that samples can remain in the system's covered sample wheel for up to 2 h without significant (less than 10%) diminution in ethanol concentration. This technique was further utilized to evaluate the suitability of synovial fluid as an alternative for vitreous humor.


Cocaine and some of its products in hair by RIA and GC/MS. Noncomminuted hair samplings (100 mg) from 132 individuals were analyzed for cocaine products by both RIA and GC/MS with D3 internal standards and selected ions for cocaine, benzoylecgonine, and methylecgonine. Ethanol and pH 7 buffer washing until the washes were negative by RIA or GC/MS were followed by overnight digestion in warm 0.1M HCI. A small portion of digest buffered to pH 7 was analyzed by RIA. The remainder, internally standardized, buffered extract was TMS derivatized and analyzed on a capillary OV-1 column by GC/MS. Of the 132 specimens, 10 were positive by RIA and positive by GC/MS for cocaine only, while another 20 specimens were positive for both cocaine and benzoylecgonine by GC/MS as well as positive by RIA. One specimen that was positive by RIA was positive for cocaine and methylecgonine by GC/MS; an additional one was positive for cocaine, methylecgonine, and benzoylecgonine and positive by RIA. The RIA used (100x more reactive to cocaine than to benzoylecgonine) gave positive test results for an additional 47 of the 132 hair samples. Cutoffs used for the RIA were equivalent to 2 micrograms benzoylecgonine or 0.04 micrograms cocaine/g hair, and for GC/MS, cutoffs per gram hair were 0.1 micrograms cocaine and 0.2 micrograms benzoylecgonine or methylecgonine. Relationships between RIA and GC/MS results and distribution of values found are discussed, as are factors that appear to affect recovery from the hair.


Nonparametric assessment of toxicologic assay linearity by bootstrap analysis. An important aspect in the evaluation of toxicologic assay methodology is the assessment of calibration. This paper presents a new approach for validating calibration using bootstrap analysis. The technique is illustrated with a quantitative assay for benzoylecgonine in urine by gas chromatography/mass spectrometry (GC/MS). Because the bootstrap analysis does not rely on parametric assumptions regarding the distribution of errors about the regression line, it can be applied to situations where the parametric distribution of response errors about the regression line is unknown. This application of bootstrap methodology yields a probabilistic measure of confidence on the linearity of the calibration curve.


Alcohol-specific cocaine metabolites in serum and urine of hospitalized patients. The serum and urine from 44 consecutive patients that tested positive for the cocaine metabolite benzoylecgonine (BE) were examined for free cocaine, ecgonine methyl ester (EME), and other metabolites by gas chromatography/ion trap mass spectrometry (GC/MS). In 13 of these patients, unique ethanol-related cocaine metabolites, cocaethylene and ecognine ethyl ester (EEE), were detected in urine and serum. One was from a newborn baby whose mother's blood was positive for cocaine and negative for cocaethylene. In two other patients, isopropanol was also consumed with cocaine and ethanol. In one of these two, cocaisopropylene and ecognine isopropyl ester (EPE) were identified in urine. The urine ethanol concentration in 7 of the 13 cocaethylene-positive patients ranged from 19 to 322 mg/dL. In the other six, ethanol was not detected in the urine. However, each of these latter patients had either prior serum results that were positive for ethanol or admitted to recent alcohol abuse. In the remaining 31 of 44 cocaine-positive patients, ethanol and the alcohol-specific cocaine metabolites were absent. The detection of alcohol-related cocaine metabolites is fairly common in a cocaine-positive patient population.


Preliminary evaluation of the Abbott TDx for screening of D-methamphetamine in whole blood specimens. The Abbott TDx Urine Amphetamine/Methamphetamine II fluorescence polarization immunoassay technique was applied to the determination of D-methamphetamine in hemolyzed whole blood. The assay was found to have 100% cross-reactivity with D-methamphetamine and only an 8% cross-reactivity with L-methamphetamine. Whole blood was fortified with D-methamphetamine at concentrations ranging from 25 to 1000 ng/mL. These whole blood calibrators were used to evaluate the following sample preparation techniques: direct, diluted and buffer, and precipitated using methanol, acetone, sulfosalicylic acid, trichloroacetic acid, and zinc sulfate. Calibrators and samples were prepared by mixing 200 microL of whole blood and 200 microL precipitation reagent and centrifuging at 10,000 rpm for 5 min (9600 x g). A 50-microL aliquot of the supernatant was used for the assay. Using the zinc sulfate precipitation, blood calibration curves showed a linear range of 25-1000 ng/mL. The within-run precision for the 25-, 60-, and 200-ng/mL D-methamphetamine blood controls showed percent coefficients of variation of 17.8, 17.0, and 5.4, respectively. The TDx results were compared to RIA and GC/MS assays for the methamphetamine controls and for eight positive case specimens and found reliable for the screening of hemolyzed whole blood.


Identification of methamphetamines and over-the-counter sympathometic amines by full-scan GC-ion trap MS with electron impact and chemical ionization. An underivatized methane chemical ionization (CI) assay for measuring amphetamines in urine was evaluated against derivatized electron impact (EI) assays using a gas chromatograph-ion trap mass spectrometer. The full-scan CI mass spectra of methamphetamine, ephedrine/pseudoephedrine, and phentermine were compared with the full scan and three-ion EI mass spectra of heptafluorobutyric anhydride (HFBA) and 4-carbethoxyhexafluorobutyryl chloride (CB) derivatives. The fragmentation patterns for these compounds were nearly identical for the three major high molecular weight ions (m/z 254, 210, and 169 for EI-HFBA derivatives, and m/z 308, 262, and 280 for EI-CB derivatives). The CI mass spectra of the underivatized drugs contained more discernible differences at the higher molecular weights, including m/z 119, 148, and 150 for methamphetamine, 148, 166, and 176 for ephedrine/pseudoephedrine, and 91, 133, and 150 for phentermine. The within-run precision ranged from 7-9% for CI versus 5-6% for EI with HFBA derivatization (mean 500 ng/mL, n = 5). The limits of detection (LOD) for amphetamine and methamphetamine were 2.4 and 8.6 ng/mL, respectively, for CI versus 0.7 and 1.4 ng/mL for EI. The limits of quantitation (LOQ) were 4.5 and 19.1 ng/mL for CI versus 1.4 and 5.7 ng/mL for EI. The use of full-scan mass spectral analysis with either electron impact or chemical ionization provides additional qualitative data that may be helpful for measuring methamphetamine in the presence of other sympathomimetic amines.


Urinalysis of alpha-hydroxyalprazolam, alpha-hydroxytriazolam, and other benzodiazepine compounds by GC/EIMS. This procedure was developed as an overall benzodiazepine confirmation scheme and includes the detection of the most important urinary analytes encountered by clinical toxicology laboratories in North America: alpha-hydroxyalprazolam, alpha-hydroxytriazolam, 2-hydroxyethylflurazepam, oxazepam, temazepam, and lorazepam. Desmethyldiazepam (nordiazepam) was not targeted because it is metabolized to oxazepam. This procedure takes advantage of beta-glucuronidase hydrolysis for analysis of intact benzodiazepine molecules, oxazepam-2H5 as an internal standard, a newly developed extraction solvent, and a silylating moiety that may be more sensitive than trimethylsilyl (-TMS) derivatives, the tertbutyldimethylsilyl (-TBDMS) derivative. For all compounds the extraction efficiency was greater than 90% and the limit of quantitation (LOQ at a S/N of 10) was less than 10 ng/mL. Coefficients of variation for a 200-ng/mL control were less than 5% and less than or equal to 11% for within-run and between-run trials, respectively. Of 13 human specimens screened by EMIT and most with self-reported histories, alpha-hydroxyalprazolam was found in seven (range 49-1264 ng/mL), oxazepam was found in five (72-3897 ng/mL), and lorazepam (476 ng/mL), 2-hydroxyethylflurazepam (2301 ng/mL), and alpha-hydroxytriazolam (106 ng/mL) in one each.


Forensic drug testing for opiates. IV. Analytical sensitivity, specificity, and accuracy of commercial urine opiate immunoassays. Four commercial immunoassays, TDx Opiates (TDx), Coat-A-Count Morphine in Urine (CAC), Abuscreen Radioimmunoassay for Morphine (ABUS) and Emit d.a.u. Opiate Assay (EMIT), were tested for sensitivity, specificity, and accuracy with urine specimens containing known amounts of opiates and opiate metabolites. The immunoassays were evaluated in a semiquantitative mode by comparison of morphine equivalents to GC/MS assay of free and total morphine and codeine or to target concentrations. In all cases, the apparent sensitivities of the assays were higher than those required for detection of morphine at cutoffs mandated by the Health and Human Services guidelines for testing of Federal workers. The apparent specificities of the immunoassays varied considerably. The CAC assay was found to be highly selective for free morphine, whereas TDx, ABUS, and EMIT demonstrated broad cross-reactivity with other opiates. Comparison of semiquantitative results from the immunoassays with GC/MS data indicated a high degree of accuracy for determination of morphine levels. Generally, the patterns of sensitivity and cross-reactivity were unique for each assay, indicating that a detailed knowledge of assay performance characteristics is necessary for accurate interpretation of forensic urine testing data.


A possible association between fluoxetine use and suicide. Fluoxetine has been available for use as an antidepressant since early 1988. Numerous reports have indicated that it is at least as good as some of the tricyclic antidepressants and that it does not produce as serious toxicity as the tricyclics. Recently, however, case reports have suggested that it may produce suicidal tendencies in some patients. The present report provides data from a medical examiner's office regarding the number of cases in which fluoxetine has been detected and the proportion certified as suicides.


Measurement of ethylenethiourea using thermospray liquid chromatography-mass spectrometry. Reliable measurement of ethylenethiourea (ETU) is important because ETU is a potent thyroid carcinogen. A method for the separation and identification of ethylenethiourea (ETU) by applying reversed-phase high-pressure liquid chromatography (HPLC) followed by thermospray (TSP) mass spectrometry (MS) detection is described. Single ion recording detection applying HPLC-MS of ETU appeared to be highly selective and equally sensitive as an HPLC method applying UV detection reported in our earlier study (1). The detection limit for ETU was 100 pg per injection.


Determination of procaine in equine plasma and urine by high-performance liquid chromatography. The variability in plasma and urine equine procaine measurement between three independent laboratories using current methods led to the development of a sensitive, reliable, and reproducible high-performance liquid chromatographic method. Standardbred mares were administered either a penicillin G procaine preparation intramuscularly or procaine hydrochloride subcutaneously, and blood and urine were collected at defined time intervals. By HPLC the detection limits for procaine in plasma and urine were 1 and 10 ng/mL, respectively. In contrast procaine in plasma could not be detected by GC-NPD, while the urinary detection limit was 50 ng/mL. The concentration of fluoride in the collection tubes and repetitive freeze-thawing modified plasma procaine measurement. Urinary pH was a factor in estimation of urine procaine levels with greater recovery and reproducibility of results at pH 5 as compared to pH 7. This HPLC method provides a simple, sensitive, and reliable quantitation of procaine in equine plasma and urine.


Biological fate of sulfur mustard, 1,1'-thiobis(2-chloroethane). Urinary excretion profiles of hydrolysis products and beta-lyase metabolites of sulfur mustard after cutaneous application in rats. The urinary excretion profiles of some metabolites of sulfur mustard were determined by gas chromatography/mass spectrometry after cutaneous application of sulfur mustard in rats. Excretion profiles of the individual metabolites thiodiglycol and thiodiglycol sulfoxide, derived from the hydrolysis of sulfur mustard, were determined in different groups of three rats. Concentrations of thiodiglycol detected increased up to 10 fold after treatment of the urine with hydrochloric acid, presumably because of the excretion of acid-labile esters of thiodiglycol. Free thiodiglycol, free plus esterified thiodiglycol, and thiodiglycol sulfoxide excreted over 8 days accounted for less than 0.3%, 1-1.5%, and 3.4-4.3%, respectively, of the applied dose of sulfur mustard. In a further study, a modified analytical method was applied to determine these hydrolysis products and their acid-labile esters as the single analyte thiodiglycol, after treatment with acidic titanium trichloride. The excretion profile of the combined hydrolysis products was compared with the excretion profile of a different group of metabolites of sulfur mustard derived from the glutathione/beta-lyase pathway. These were also reduced to a common analyte, 1,1'-sulfonylbis-[2-(methylthio)ethane], after similar treatment with titanium trichloride. Urinary excretion of hydrolysis products determined in 4 rats over 8 days accounted for 3.7-13.6% of an applied cutaneous dose of sulfur mustard. Urinary excretion of beta-lyase metabolites accounted for 2.5-5.3% of the applied dose in the same group of rats. The excretion of beta-lyase products showed a much sharper decline than was observed for the hydrolysis products of sulfur mustard.


Continuous flow quantification of total mercury in whole blood, plasma, erythrocytes, and urine by inductively coupled plasma atomic emission spectroscopy. Mercury determination in blood and urine can be performed by inductively coupled plasma atomic emission spectroscopy (ICPAES) after dilution in an ammonia buffer and reduction by sodium borohydride. The proposed method does not need an oxidative mineralization. The sample is not nebulized into the torch, but the mercury vapor, after collection in a reactor vial, is swept into the plasma by the argon carrier gas using the described glass apparatus.


Evaluation of six commercial amphetamine and methamphetamine immunoassays for cross-reactivity to phenylpropanolamine and ephedrine in urine. We evaluated six commercially available amphetamine (A) and methamphetamine (MA) immunoassays for their relative cross-reactivities to isomers of phenylpropanolamine (PPA) and ephedrine (E) in urine: Syva EMIT, Abbott fluorescence polarization (FPIA), Roche, and Diagnostic Products Corporation (DPC) radioimmunoassays for A and MA. Two stereoisomers of PPA and four stereoisomers of E were tested using (1) drug-free urine spiked at 1,000 mg/L or 100 mg/L of each compound and (2) 60 clinical urine specimens not containing A or MA but having varying amounts of PPA and/or E. Specimens responding greater than the 1-mg/L A or MA cutoff were defined as positive. All specimens spiked at 100 mg/L were negative by all immunoassays. All specimens spiked at 1,000 mg/L were positive by EMIT and negative by FPIA, Roche A, and DPC A; 1,000 mg/L/-E and d-pseudoephedrine were also positive by Roche MA and DPC MA. Three of the 60 clinical specimens tested positive by EMIT and one specimen tested positive by DPC A and DPC MA.


A rapid, simultaneous determination of paraquat and diquat in serum and urine using second-derivative spectroscopy. A rapid, simple method based on second-derivative spectroscopy of the simultaneous analysis of paraquat and diquat in serum and urine is described. Paraquat and diquat in serum were deproteinized with sulfosalicylic acid, and those in urine were reduced with NaOH-dithionite solution. A qualitative and quantitative analysis of reduced paraquat and diquat was made at the amplitude peaks of 396-403 nm and 454-464 nm in the second-derivative spectra, respectively. The entire procedure was completed within about 10 minutes for a serum sample and within about 5 minutes for a urine sample. Application of the proposed method on a poisoned patient is also reported.


Mass spectral characterization of three anthracycline antibiotics: a comparison of thermospray mass spectrometry to particle beam mass spectrometry. Mass spectral results for three anthracyclines, doxorubicin, daunorubicin, and carminomycin are compared by using thermospray (TS) or particle beam (PB) [electron ionization (EI) and chemical ionization (CI)] instruments. Typically, positive ion TS mass spectrometry (MS) provided intense [MH]+ ions and some fragment ions, while PBMS in the EI mode provided only fragment ions. Significant [MH]+ ions were observed for carminomycin and daunorubicin when analyzed using PBMS in the positive ion CI mode. Under negative ion detection, TSMS yielded intense [M-H]- ions for these compounds while PBCIMS resulted in significant M- ions. Fragment ions observed in all three anthracyclines under positive and negative ion detection by TSMS and PBCIMS are due mainly to the cleavage of glycosidic bond, loss of H2O, and the loss of the side chain (COCH2R2) from the aglycone.


Measurement of carbamazepine and its main biotransformation products in plasma by HPLC. We present a method that permits the simultaneous analysis of carbamazepine (CBZ) and its major biotransformation products, carbamazepine-10,11-epoxide (CBZ-E) and carbamazepine-10,11-dihydroxide (CBZ-diOH), in plasma samples. The method consists of plasma extraction in alkaline medium with NaCl added using chloroform-ethyl acetate (1:1, v/v) and later purification with n-hexane. The samples were submitted to reversed-phase chromatography (RP-18) using acetonitrile-water (3:7, v/v) as the mobile phase and detection at 220 nm. Recoveries of 62.0, 99.9, and 105.4% were obtained for CBZ-diOH, CBZ-E, and CBZ, respectively, with sensitivities of 0.32 micrograms/mL for CBZ-E and CBZ-dOH and of 0.64 micrograms/mL for CBZ. The method proved to be specific, thus permitting measurements in situations of drug combinations.


Determination of nonderivatized para-hydroxylated metabolites of diazepam in biological fluids with a GC Megabore column system. A method was developed using gas chromatography (GC) and a Megabore column system capable of simultaneous detection of diazepam, N-desmethyldiazepam, temazepam, oxazepam, and their para-hydroxylated metabolites. This method does not require derivatization of para-hydroxylated metabolites. Standard curves for pure reference compounds were linear, with the minimum detectable concentration of diazepam and its metabolites as low as 0.13 ng/injection.


Simultaneous sequential determination of compounds of toxicological interest in mixtures by derivative spectroscopy. An absorption spectroscopic method was developed for the sequential determination of mixture components. The proposed method is straightforward and convenient and provides optimal results that increase the potential of spectrophotometric techniques as applied to date. The method was applied to the sequential resolution of mixtures of pure components of toxicological interest yielding close UV absorption bands by using derivative spectroscopy.


Hair analysis for drugs of abuse. III. Movement and stability of methoxyphenamine (as a model compound of methamphetamine) along hair shaft with hair growth. This paper describes the movement of methoxyphenamine (MOP, a model compound of methamphetamine) along the hair shaft at the rate of hair growth and the stability of drugs in hair for several months. Five healthy subjects (3 males and 2 females) took 50 mg of methoxyphenamine orally once a day for 7 days. Scalp hairs from the posterior vertex were collected every 2 weeks or every 8 weeks after the first dosage. The hairs were cut into 1-cm sections and extracted with methanol-5N HCl (20:1). MOP in the extract was determined by gas chromatography/mass spectrometry (GC/MS) with tetradeuterium labeled MOP as an internal standard. The drug moved along hair shaft at the rate of 2.8-3.2 mm/week, according to hair growth, without diffusion. When drug bands were extrapolated according to the sections in which drug was detected, the bands were approximately 5 mm wide, equivalent to 1.7-2.4 periods of 7-day hair growth. In the case of identical doses, the drug level was highest in the root side and lowest in the distal side. In our data, we found that the drug level in hair had decreased approximately 50% five months later. The ratio of drug levels in corresponding sections correlated well to the ratio of doses, except where the hair shafts had been damaged or the drugs had decomposed.


Divergent perceptions of lesbians: a comparison of lesbian self-perceptions and heterosexual perceptions. Social-psychological research on stereotyping was applied to lesbianism among American college students. The hypothesis that stereotypes of lesbians are often inaccurate predictors of individual lesbians was supported. Using stereotypes of lesbians identified by previous research, lesbians rated themselves on a series of bipolar stereotypical adjectives, and a comparison group of nonlesbians rated lesbians as a group on these same adjectives. Significant differences were found on 16 of the 21 adjectives. Knowing a lesbian personally did not influence heterosexuals' ratings, suggesting the resistance of stereotypes to change.


Determination of serum levels of dietary thiocyanate. A spectrophotometric method was used to determine thiocyanate (SCN-) levels in human serum, in an attempt to relate serum levels of this ion to the dietary intake of vegetables of the genus Brassica. Thiocyanate ion was measured before and after a one-week period during which the subjects ate a diet rich in these vegetables. We also determined the serum increase in SCN- 2 h after the administration of 0.5 g KSCN.


Fraisse's theory of emotion and aggression. This article shows how Fraisse's (1964) theory of emotion is a better explanation of aggression than any other theory of emotion. It discusses conceptual and methodological difficulties in existing theories of aggression and reports a study that supports a hypothesis based on Fraisse's theory. This study, which used Canadian university students equally divided by sex, supports the hypothesis that subjects would be more aggressive when faced with an unexpected annoyance than with an expected annoyance. Aggression was indexed by subjects' evaluation of the target person (confederate) as a source of annoyance.


The analysis of cocaine and benzoylecgonine in meconium. We describe an assay for measuring cocaine and benzoylecgonine in meconium of infants born to mothers suspected of using cocaine during their pregnancy. The assay involves the use of fluorescence polarization immunoassay (FPIA) to screen for benzoylecgonine in a methanolic extract of the meconium. The FPIA is sensitive to 0.6 microgram benzoylecgonine per gram meconium. Confirmation of the presence (or absence) of benzoylecgonine and cocaine in meconium samples was performed by solid phase extraction of a second methanolic extract of the meconium, derivatizing using BSTFA, followed by a gas chromatographic/mass spectrometric (GC/MS) analysis, which can detect both cocaine and benzoylecgonine. The GC/MS confirmation was sensitive to less than 0.25 microgram cocaine or 0.5 microgram benzoylecgonine per gram meconium. FPIA, which is commonly used in many toxicology laboratories, is advantageous because it precludes the need to use radioimmunoassays for the initial screen. The confirmation step provides greater certainty for the presence of cocaine and/or benzoylecgonine in meconium.


War and hunting poisons of the New World. Part 1. Notes on the early history of curare. The history to about 1850 of the muscle-relaxant poison curare is discussed, especially the developments leading to the botanical identification of the plants that yield the alkaloidal active principles: Loganiaceae (Strychnos species) and Menispermaceae (Abuta, Chondrodendron, and Curarea species). One of the earliest encounters with the poison appears to have been during the exploration of the Lake Maracaibo region in Colombia by Alonso Pérez de Tolosa in 1548. It is pointed out (yet again) that Sir Walter Ralegh did not bring back the poison to Europe in 1595 and that it was Keymis who first came across the word ourari when exploring the lower reaches of the Orinoco in 1596. Gumilla, La Condamine, Ulloa, Veigl, and others gave much additional information about the poison during the 18th century. Scientific studies began in the latter part of the century when Schreber listed the botanical identities of four of the plant components entering into the curare prepared by the Akawai Indians of Surinam. As far as is known, none of these people actually saw curare being made. Thereafter, progress was rapid. Humboldt and Bonpland were the first trained scientists to witness the preparation of the poison, at the very beginning of the 19th century. Subsequent exploration by Martius and Spix, Poeppig, Youd, the Schomburgk brothers, De Castelnau and Deville, Spruce, and others, up to the middle of the century, extended and deepened botanical and ethnological knowledge of curare. Study of its physiology started at about that time with the classical experiments of Rudolf von Koelliker and Claude Bernard.


Solid-phase extraction of drugs from biological tissues--a review. Solid-phase extraction procedures are being used increasingly in all types of drug testing laboratories, including those which carry out forensic toxicology. The advantages of such procedures include decreased operator time, reduced solvent volumes, and increased extraction efficiency. The application of existing techniques, however, has not yet been applied to tissues that are widely available postmortem. Tissue analysis can make the task of interpretation of drug levels much easier, particularly where unstable drugs are involved (e.g., cocaine). This review article explores the possibilities of applying existing extraction procedures to human tissue samples. We also consider the reasons why forensic laboratories have been slow to investigate such possibilities. We hope to stimulate interest in alternative tissue extraction procedures by reviewing reported procedures that apply to both human and animal tissue.


Recent advances on ginseng research in China. Ginseng, Panax ginseng C.A. Meyer, is a well-known Chinese traditional medicine. There have been more than 300 original papers in Chinese and in English during the last 10 years in China. This review paper summarizes some achievements from some of these published papers. Twenty-eight ginsenosides and some minor constituents were extracted and isolated from the root, root-stock, stems, leaves, flowers and flower-buds of ginseng. The chemical analysis demonstrated that the content of ginsenosides is related to the source, part and growth years of ginseng. The drug has a wide range of pharmacological and therapeutical actions, it acts on the central nervous system, cardiovascular system and endocrine secretion, promotes immune function and metabolism, possesses biomodulation action, anti-stress and anti-ageing activities, and so on. Many preparations of ginseng have been officially approved for clinical application in China. Clinical evaluation has shown that these preparations play a special role in medicinal use.


Antimicrobial activity of benzylisoquinoline alkaloids from Annona salzmanii D.C. Bark of Annona salzmanii D.C. (Annonaceae), used in Brazilian folk medicine, was found to contain four benzylisoquinoline alkaloids, namely reticuline, anonaine, laurelliptine and isoboldine. Only anonaine possesses some antibacterial property while all four alkaloids show some antifungal activity.


Uses, chemistry and pharmacology of Malouetia (Apocynaceae, subf. Apocynoideae). An outline of the uses, chemistry, and pharmacology of the South American and African genus Malouetia A.DC. (Apocynaceae-Apocynoideae) is given. The chemotaxonomy of the genus is also discussed.


Comparative study of the effects of beta-sitosterol, estradiol and progesterone on selected biochemical parameters of the uterus of ovariectomised rats. A comparative study was made of the effects of beta-sitosterol, estradiol-17 beta and progesterone, individually and in combinations, on certain biochemical parameters important to carbohydrate metabolism in the uteri of adult ovariectomised rats. beta-Sitosterol (SITO), estradiol (E2) and combined treatment (SITO + E2) induced significant increases in glycogen concentration and the activities of glucose-6-phosphate dehydrogenase (G6PDH), phosphohexose isomerase (PHI) and total lactate dehydrogenase (LDH). Progesterone (P) administration however, raised only the uterine PHI and LDH activities. Co-administration of P with beta-sitosterol (P + SITO) suppressed the SITO-induced increase in glycogen concentration and G6PDH activity. On the other hand, combined treatment (P + SITO) augmented total LDH activity.


Analysis of some Malaysian dart poisons. An investigation of nine Malaysian dart poisons has confirmed that their main active components are cardenolides from Antiaris toxicaria (Pers.) Lesch. and alkaloids probably from different forms of Strychnos ignatii P. Bergius. It is not possible to determine the ethnic origin of the poisons from the results of the analyses on their own. Two new cardiac glycosides have been isolated and their structures determined as 12 beta-hydroxycannogenin 3 beta-O-beta-D-deoxygulopyranoside and 3 beta-O-alpha-L-rhamnopyranoside, respectively.


Indigenous phytotherapy of gastrointestinal disorders in a lowland Mixe community (Oaxaca, Mexico): ethnopharmacologic evaluation. Gastrointestinal disorders are one of the major health problems in developing countries. Sixty-five plants used popularly in the treatment of such disorders in a Mixe Indian community in Oaxaca (Mexico) and collected during a fieldstudy of 15 months are described. According to indigenous criteria a plant is used in the treatment of a certain illness because of the plant's characteristic smell and taste. Plants with astringent properties are particularly valued to treat diarrhoea and dysentery. Bitter, aromatic and bitter-aromatic plants are especially employed to treat gastrointestinal cramps and pain. Additionally, the efficacy of these plants was evaluated using ethnobotanical, phytochemical and pharmacologic information on the plants. The majority of the plants contain chemicals that may produce the effects desired by the Mixe. Frequently tannin-containing drugs are used to treat diarrhoea and dysentery. A large number of the plants used by the Mixe in the treatment of gastrointestinal pain contain essential oil or bitter principles. As a result of this evaluation, plants were selected which should be studied phytochemically and pharmacologically with priority, to evaluate further their potential in the treatment of gastrointestinal disorders.


Parasitological and microbiological evaluation of Mixe Indian medicinal plants (Mexico). Medicinal plants are an important health resource in many regions of the Americas and are of particular importance to many Indian communities. Based on a recent ethnobotanical study in Mexico, we investigated the activity of 29 plant extracts against Entamoeba histolytica, three bacteria (Bacillus subtilis, Escherichia coli, and Micrococcus luteus) and two fungi (Cladosporium cucumerinum and Penicillium oxalicum). After separation of these extracts between CH2Cl2 and H2O the resulting phases were also evaluated.


In vivo selective monitoring of basal levels of cerebral dopamine using voltammetry with Nafion modified (NA-CRO) carbon fibre micro-electrodes. The electrochemical technique of differential pulse voltammetry (DPV) with micro-biosensors has been used for a number of years to monitor in vivo and in situ changes in the extracellular concentration of cerebral ascorbic acid, as well as that of the metabolites of dopamine (DA) and serotonin (5-HT). We have recently prepared a carbon fibre micro-electrode (mCFE) which specifically pretreated and coated with Nafion (a negatively charged polymer which repels acids such as 3,4-dihydroxyphenylacetic acid (DOPAC)) allows the direct selective detection of the oxidation of DA and 5-HT in nanomolar concentration in vitro and that of extracellular basal levels of cerebral 5-HT in vivo (peak B at +240 mV). We describe here a modified version of this micro-biosensor now called NA-CRO mCFE as its active tip (30 microns in diameter) is coated with a 50/50 (v:v) mixture of Nafion and dibenzo-18-crown-6 (Aldrich). In vitro this newly reported electrode shows insensitivity to acids (e.g., DOPAC) up to 100 microns and sensitivity to 0.5-1 nM DA. In vivo, in the striatum of anaesthetised rats, a basal oxidation peak at +80 mV (peak A, on average 0.6 nA in height), which corresponds to the oxidation potential of DA in vitro, is consistently detectable with the NA-CRO mCFE (corresponding to an estimated concentration of 1.5 nM). Experiments performed in vivo in anaesthetised rats implanted in the striatum with uncoated (normal) mCFE to measure extracellular DOPAC or with NA-CRO mCFE have been performed in order to analyse the chemical nature of peak A in vivo. It is concluded that the addition of the crown-ether compound to the Nafion coat improves the sensitivity of the micro-biosensor for DA in vitro and allows the detection of its basal extracellular levels in vivo.


A method for automatic identification of periods of muscular activity from EMG recordings. A computer-based method of identifying periods of activity in EMG recordings is described. Its application to the analysis of muscle activity patterns in freely moving human or animal subjects is illustrated and discussed.


Clinical experience with reinforced, anchored intramuscular electrodes for functional neuromuscular stimulation. Implanted intramuscular electrodes must remain functional for many years if functional neuromuscular stimulation (FNS) is to become a standard treatment in paralysed individuals. In initial trials we found that 5 of 11 coiled single-wire FNS electrodes implanted in 3 patients failed within 8 months. Consequently, we turned to a reinforced electrode comprising 2 multi-stranded, insulated wires tandem-wound on a prolene core and terminated by a prolene anchor or tine (after Mortimer et al., 1986, 1987). The electrodes were implanted with a translumbar aortogram needle, the teflon sheath of which enabled us to stimulate through the tip to guide placement. We have monitored the electrical and functional properties of 8 reinforced electrodes implanted in 2 incomplete quadriplegic patients over 22 months. Four of the electrodes were used for at least 1 h daily to exercise muscles or to provide FNS in gait. Electrical impedances, thresholds and elicited limb motion remained constant in all 8 electrodes over the test period. Disadvantages of the reinforced electrodes are (1) difficulty of eventual removal, and (2) risk of pathogenic infiltration is increased by the 3-filament structure (fortunately dense tissue encapsulation seems to mitigate infection). We conclude that tandem-wound, prolene-reinforced FNS electrodes are much more robust than previous single-coil designs and may form the basis for FNS devices of the future.


Simultaneous recording of [Ca2+]i increases in isolated olfactory receptor neurons retaining their original spatial relationship in intact tissue. A new method is described for isolating olfactory receptor neurons suitable for simultaneous recording of odorant responses in several cells. This method, called "tissue printing" by Cassab and Varner, was used to isolate cells for measurement of odorant-induced increases in cytosolic-free calcium concentration ([Ca2+]i) using the Ca2+ indicator dye fura-2. A large number of receptors could be isolated from a piece of olfactory epithelium (about 300 microns square), preserving their normal morphology and relative local topology to that in the intact olfactory tissue. The probability that there are one or more receptor cells with odorant-induced responses in [Ca2+]i per preparation was 4 times higher with cells isolated by the tissue printing than with those obtained by the pipetting method. The responses of 2 receptor cells separated by 28 microns in the recording chamber differed for 2 odorants: isoamyl acetate and citralva. The method was useful for isolating receptor neurons without losing their morphological features and for investigating the spatial distribution of odorant responsiveness of each receptor over the olfactory epithelium.


Deblurring of 3-dimensional patterns of evoked rat cerebellar cortical activity: a study using voltage-sensitive dyes and optical sectioning. One of the benefits of imaging neuronal activity is the capability of resolving spatial patterns in the x-y plane. With optical sectioning microscopy, the 3-dimensional (3-D) structure may also be studied without physical deformation by serially moving the focal plane of the microscope through the volume of interest along the focal axis. However, each image is blurred by contributions from neighboring planes. This degradation is most severe for low numerical aperture lenses and large amounts of defocus. In this study, an image restoration method using the optical properties of an aberration-free, defocused optical system has been developed for improving optical signals from voltage-sensitive dyes. Deblurring based on the optical transfer function (OTF) of the system was applied on two test sets of serially sectioned images: (1) fluorescent beads and (2) in vivo rat cerebellar cortex stained with the voltage-sensitive dye RH795. This method was shown to reduce significantly the out-of-focus contribution to the images, improving the spatial resolution not only in the x-y plane, but also the z axis. The algorithms were then applied to optical signals obtained by stimulation of the cerebellar surface. Optical signals having a distinct beam-like pattern were evoked and recorded over depths ranging from 0 to 300 microns prior to deblurring. Application of the deblurring algorithm reduced the depth of cerebellar cortex over which the optical signals were observed. In agreement, field potential recordings of the evoked parallel fiber volley and post-synaptic components were restricted to a narrow range of depths similar to the deblurred optical images. Removal of out-of-focus information is an essential step in the serial sectioning of central nervous system structures for neuronal imaging and 3-D reconstruction.


Ethidium bromide fluorescence of 28S ribosomal RNA can be used to normalize samples in northern or dot blots when analyzing small drug-induced changes in specific mRNA. Quantitative analysis of Northern blots is frequently accomplished with the aid of an internal standard. Most common is probing for an additional message the steady-state levels of which do not change in response to the experimental conditions and the signal of which is sufficiently removed from that of the target gene after gel electrophoresis. However, this strategy is not always feasible. When total RNA is immobilized on nylon, 28S ribosomal RNA on the blot can be used as an internal standard and quantitated by scanning the negative photograph of the blotted RNA stained with ethidium bromide. This procedure can also be used for RNA dot blots. The method is quick, reliable, will work with laser or white-light densitometers, and can serve as a universal internal standard, eliminating the need for additional probes.


Parametric analysis of sojourn times in conditioned place preference experiments. In conditioned place preference experiments, groups of animals are typically compared by means of the total time spent in a treatment environment. The total times are often positively skewed, which prohibits the use of parametric procedures that require normal distributions. Common response time models for such total times are also inadequate because the fit of the models to the data is often poor. The present paper assumes that the sojourn times of a single animal are independent and that they all follow the same 1-parameter exponential model. The exponential parameter allows a number of simple interpretations. We propose point estimates, confidence intervals, a goodness-of-fit test, and a test for comparing 2 samples. The procedures are also applied to single animals, and a test is given for comparing the animals of a group. The kind of parametric analysis suggested enables better interpretation than does the simple distribution-free comparison of 2 groups based on the total time spent in the treatment environment.


The search for tumor-associated proteins in pleural effusions by means of monoclonal antibodies and a dot blot assay. Liquid moiety of 61 pleural effusions was tested for tumor-associated proteins (TAP) by means of a dot immunobinding (dot blot) assay (DIA) and a panel of monoclonal antibodies (Moabs). The sensitivity of the assay was checked using a purified, serially diluted carcinoembryonic antigen (CEA) preparation and an anti-CEA monoclonal IgG system. The latter was examined using both DIA and enzyme-linked immunosorbent assay ELISA solid phase assays in simulated conditions that mimicked the protein content of effusions. Finally, the results of DIA were compared to the immunohistochemistry carried out on cell sediments from the same effusions with similar Moabs. It was found that the prevalence of several TAPs, including CEA, epithelial membrane antigen (EMA), vimentin, tenascin, and Thomsen Friedenreich antigen, was significantly higher in the malignant effusions than in the nonmalignant ones. A total, larger than 2, of detected TAPs in a given fluid, was found almost exclusively in malignant effusions (p less than 0.0001). The detection limit of the DIA for a CEA was determined at 5 ng/ml, while for the ELISA it was 1 ng/ml. Several TAPs, especially the CEA, could be detected in parallel tests, carried out on the liquid moiety and the cell sediments of malignant effusions. The evaluation of selected TAPs in pleural effusions by dot blot assay may be of clinical value.


Analysis of the structure of the muscular pulmonary arteries in patients with pulmonary hypertension and COPD: National Institutes of Health nocturnal oxygen therapy trial. We examined the cardiovascular function as well as the structure of the muscular pulmonary arteries in patients who had died while enrolled in the National Institutes of Health nocturnal oxygen therapy trial (NOTT). The cardiovascular function of the patients classified into groups based on the severity of the pulmonary hypertension was examined, and we compared the morphologic data in these groups to those obtained from age-matched controls who died with no evidence of cardiovascular disease. The groups with severe pulmonary hypertension had markedly increased pulmonary vascular resistance but similar cardiac index to the group with only mild hypertension. In the structural analyses, we found definite alterations in arterial structure from the control population: the patients who had pulmonary hypertension had markedly increased percentages of intima and media. These differences were most pronounced in the medium and larger muscular arteries. The degree of pulmonary hypertension did not appear to alter vascular structure consistently, although there was a trend towards an increase in muscle media in the smaller vessels. When the patients were classified into a group who responded to oxygen administration by a decrease in Ppa, and an age- and Ppa-matched group who did not respond, there were no differences in vascular structure between these groups, although both groups had greater percentages of intima and media compared to the control group. We conclude that, in patients with pulmonary hypertension secondary to chronic obstructive pulmonary disease (COPD), there are structural alterations of the muscular pulmonary arteries, but these do not correlate with either the severity of the pulmonary hypertension or the ability of the pulmonary vasculature to respond to oxygen administration.


Influence of exercise-induced bronchoconstriction on refractoriness. This study determined if the degree of exercise-induced refractoriness is determined by the degree of exercise-induced bronchoconstriction. In 12 patients with exercise-induced asthma (mean [SEM] age 27 [3] years) we performed 2 pairs of exercise challenges 45 min apart at different work loads on 2 days. Mean (SEM) total respiratory heat loss during low and high work loads was 3.4 (0.2) and 5.1 (0.4) kcal, respectively. After the first and second exercise challenge at low work loads, mean (SEM) SRaw increased by 107 (15) and 73 (16)% (n.s.), as compared to 361 (40) and 98 (25)% at high work loads (p less than 0.005). We found a correlation between the initial airways response and refractoriness (r = 0.58, p less than 0.005) and conclude that the degree of refractoriness after exercise-induced bronchoconstriction is in part dependent on the severity of exercise-induced bronchoconstriction.


Advances in sodium-ion coupled biogenic amine transporters. The sodium-ion coupled transporters for 5-hydroxytryptamine (5HT), noradrenaline and dopamine function to reduce extracellular levels of biogenic amines. Over the past fifteen years selective inhibitors of these transport systems have been developed including fluoxetine, citalopram, paroxetine, litoxetine (for 5HT), nisoxetine, desipramine, maprotiline (for noradrenaline) and GBR-12935 (for dopamine). Some of these inhibitors, including drugs selective for noradrenaline transport and particularly those selective for the 5HT transport system are currently widely used in the clinical management of affective disorders. Selective biogenic amine uptake inhibitors have, in addition, provided tools to undertake molecular pharmacological and biochemical studies of their respective transporters. By this means, the rat brain 5HT and dopamine transporters have been identified as polypeptides with relative molecular masses of 73,000 and 80,000, respectively, using affinity-chromatographic purification and photoaffinity-labelling techniques. Recently, the biogenic amine transporters have been cloned and a comparison of their predicted amino acid sequences reveals that these proteins share a considerable degree of similarity with notably 12-13 transmembrane spanning domains. Perspectives for future fundamental and clinical research on biogenic amine transport systems using molecular biological techniques are discussed.


Pharmacokinetic simulations of SPECT quantitation of the M2 muscarinic neuroreceptor subtype in disease states using radioiodinated (R,R)-4IQNB. Alzheimer's disease (AD) involves selective loss of muscarinic M2, but not M1, subtype neuroreceptors in the posterior parietal cortex of the human brain. Emission tomographic study of the loss of M2 receptors in AD is limited by the fact that there is currently no available M2-selective radioligand which can penetrate the blood-brain barrier. However, by taking advantage of the different pharmacokinetic properties of (R,R)-[123I]IQNB for the M1 and M2 subtypes, it may be possible to estimate losses in M2. It has previously been hypothesized that the difference between an early study and a late study should provide information on the M2 receptor population. In order to test this hypothesis, we present here the results of pharmacokinetic simulations of the in vivo localization of (R,R)-[123I]IQNB in brain regions containing various proportions of M1 and M2 subtypes. These results permit us to conclude that SPECT imaging of (R,R)-[123I]IQNB localization can potentially be used to quantitate changes in the M2 subtype in a disease state within a brain region for which the ratio M2/M1 is sufficiently high in normal individuals.


Synthesis and bioactivity studies of two isosteric acyclic analogues of melanin concentrating hormone. Salmon melanin concentrating hormone (MCH) is a cyclic heptadecapeptide. MCH stimulates perinuclear aggregation of melanosomes within integumental melanocytes of teleost fishes resulting in skin blanching. MCH contains a disulfide bridge forming a 10-residue ring [sequence: see text]. It has been proposed that the ring is necessary for maintenance of potency. In order to test this proposal, we have synthesized two pseudo-isosteric analogues of MCH that cannot cyclize. They differed only in the polarity of the side chain group of positions 5 and 14. Serine was substituted for Cys5 and Cys14 in one peptide and L alpha-aminobutyrate (Abu) was the substitution at the two positions in the other peptide. Using a fish skin bioassay we determined that these analogues exhibit less than 1/10,000th the potency of the native hormone. These results suggest that the disulfide bridge is necessary to maintain the correct conformational and topographical features of the hormone for receptor binding and transmembrane signal transduction.


Sodium-lithium countertransport and body fat distribution. The relationship between erythrocyte sodium-lithium countertransport (Na-Li CT) and body fat distribution is analyzed in a sample (n = 101) of normotensive and untreated hypertensive men participating in an epidemiological study of coronary heart disease risk factors. Na-Li CT is significantly and positively associated with both subscapular skinfold and waist to hip ratio, but not with triceps skinfold. The univariate correlation between Na-Li CT and blood pressure is diminished when adjusted for body mass index and waist to hip ratio. These findings support the existence of an association between Na-Li CT and central body fat distribution and suggest that the metabolic abnormalities associated with centrally distributed body fat could explain, at least in part, the association between Na-Li CT and blood pressure. The maximal velocity of the sodium-lithium countertransport (Na-Li CT) in erythrocytes has been reported to be directly associated with blood pressure and hypertension in numerous reports from both clinical and epidemiological studies. In most of these studies, indices of weight and/or adiposity (body mass index, in particular) have been shown to be among the most important correlates of Na-Li CT. Adiposity is an important determinant of blood pressure, and there is evidence suggesting that the patterning of the fat cells in the body is linked to a number of metabolic disturbances that could lead to hypertension and an increase in other CHD risk factors. The present report analyses the relationship between Na-Li CT and body fat distribution in a sample of normotensive and untreated hypertensive men participating in an epidemiological study.


Response to 5-hydroxytryptamine on neurons of guinea pig celiac and inferior mesenteric ganglia in primary culture. The electrophysiological effects of serotonin, a putative neurotransmitter in prevertebral sympathetic ganglia, were evaluated in cultured celiac and inferior mesenteric ganglia (IMG) neurons. Intracellular microelectrode recordings were performed in neurons that were maintained in culture an average of 26 days. Seventy-eight of 85 neurons responded when serotonin (10 microM) was applied by pressure ejection from a micropipette to the surface of the isolated cells. The majority of the neurons (n = 48) generated fast depolarizations, although slow depolarizations (n = 17), bipolar responses (n = 5), hyperpolarizations (n = 7), and a biphasic response (n = 1), were also seen. Hyperpolarizing responses were evoked in celiac neurons only. All responses were inhibited by the 5-HT3 antagonist MDL 72,222 (5 microM). Fast responses were not inhibited by tetrodotoxin (n = 3). These results demonstrate that serotonin evokes a variety of membrane potential changes in cultured prevertebral sympathetic neurons by activating 5-HT3 receptors.


Free fatty acids have nucleating effects in model biles. Nucleating factors are thought to be responsible for the more rapid nucleation of gallbladder bile from patients with gallstones as compared to controls. Biliary proteins and, in particular, mucus and non-mucus glycoproteins are the focus of current research. Non-protein nucleating factors were not extensively investigated. In this study we studied the role of free fatty acids (FFA) as possible nucleating factors. Palmitic, oleic and linoleic acid were added to model biles in increasing concentrations from 0 to 20 mu mol/ml. The nucleation time of model biles decreased to 45%-60% of the initial following the addition of 0.5 to 1 mu mol/ml of each of the three fatty acids. Only a small further decrease in the nucleation time was noted with higher concentrations of up to 20 mu mol/ml. The pronucleating effect of FFA added to whole model bile was also examined in the isolated vesicular and non-vesicular fractions. The decrease in the nucleation time at each concentration of the three fatty acids was in the following order of magnitude: whole bile greater than vesicular phase greater than non-vesicular phase. The addition of each of the three fatty acids resulted in a partial solubilization of vesicles, with transfer of their lipid contents to the non-vesicular fraction. The effect was more marked with oleic acid and least marked with linoleic acid. The vesicular cholesterol to phospholipid ratio did not change following the addition of exogenous free fatty acids. Studies with labeled FFA showed that they migrated with the non-vesicular fraction on gel chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of thermal injury on transfer of IR22 IgA myeloma protein into bile in the rat. We previously observed a 75-90% decrease in concentration of biliary IgA after thermal injury to rat skin. Decrease in biliary IgA might result from an alteration in supply of polymeric IgA delivered to the hepatocyte or from an alteration in hepatocyte transfer of polymeric IgA into bile. In the present study, we examined the transfer of intravenously administered 125I-IgA into bile. Purified IR22 rat IgA myeloma protein consisting of both monomeric and polymeric IgA was labelled with 125I. Sprague-Dawley rats (140-180 g) received a 20-30% body surface area scald-burn or sham treatment. The bile duct was cannulated 18-24 h later and 125I-IgA preparations were injected into the tail vein. Bile was collected under light ether anesthesia for 3 h. In rats injected with 125I-IR22 IgA myeloma protein there were no significant differences in total, TCA-precipitable, or immunoprecipitable radioactivity in bile from burn-injured or sham-treated animals. On Bio-Gel A-1.5 m gel permeation, the radioactivity in bile from sham-treated animals eluted in the region of polymeric IgA as expected; the radioactivity in the bile from burn-injured animals eluted equally in the same regions as polymeric IgA and monomeric IgA. In sham-treated rats injected with isolated polymeric IgA only, bile contained primarily polymeric IgA. In burn-injured rats injected with polymeric IgA only, bile contained a mixture of polymeric IgA and monomeric IgA. These findings suggest that hepatocyte processing of polymeric IgA is altered after thermal injury, resulting in the transformation of some polymeric IgA into its monomeric form.


Bile duct hemorrhage: a biopsy finding after cholangiography or biliary tree manipulation. We report on 13 patients with a distinctive bile duct lesion that presented histologically as erythrocyte engorgement of the epithelium of the small bile ducts, resulting in a red-beaded appearance. In ten of our cases, the lesion was associated with a recent history of cholangiography, and in two of our cases with vigorous manipulation of the distal biliary tree, raising the possibility that the hemorrhage might have been caused by increased pressure within the ducts during cholangiography or as a result of mechanical trauma to the biliary system.