Stimulation of active and passive sodium absorption by sugars in the human jejunum.

The effects of glucose and fructose on water and sodium absorption in the human jejunum were compared to assess the relative contribution of active and passive sugar stimulation of sodium transport. The effect of fructose is assumed to be entirely passive, and the difference between the effects of fructose and glucose is assumed to be a measure of sugar-stimulated, active sodium absorption. Water and sodium movement with mannitol was the base line. Three sets of test solutions with differing sugar concentrations were studied. Fructose stimulated 66-100 per cent as much net sodium and water absorption as glucose. Fructose stimulated potassium absorption, whereas glucose stimulated potassium secretion. Urea absorption was stimulated by both sugars. Glucose and fructose stimulated sodium absorption when chloride was the major anion, but they had relatively little effect on net sodium movement when chloride was replaced by bicarbonate or sulfate. It is concluded that glucose stimulates passive and active sodium transport in the human jejunum. Stimulated active sodium absorption generates an electrical potential across the mucosa that causes sodium (and potassium) secretion and partly or completely nullifies the effect of active sodium transport on net sodium movement. Net sodium absorption sitmulated by glucose is mainly (66-100 per cent) the passive consequence of solvent flow. The accompanying anion determines the degree to which sugars stimulate sodium absorption (C1 greater than SO-4 greater than HCO3). The effects of bicarbonate and sugars on jejunal sodium absorption are not additive.

PIP: The carrier interaction and solvent drag components of sugar-stimulated sodium absorption were evaluated by comparing the effects of mannitol, fructose, and glucose on jejunal absorption of water, sodium, potassium, and urea. Using water and sodium movement with mannitol as the baseline, 3 sets of test solutions with differing sugar concentrations were studied. Fructose stimulated 66-100% as much net sodium and water absorption as glucose; in addition, fructose stimulated potassium absorption, whereas glucose stimulated potassium secretion. Both sugars stimulated urea absorption. When chloride was the major anion in the solution, glucose and fructose stimulated sodium absorption, however, the sugars had little effect on net sodium movement when chloride was replaced by bicarbonate or sulfate. From these observations it was concluded that glucose stimulates both passive and active sodium transport in human jejunum. Active sodium absorption generates an electrical potential causing secretion. Net sodium absorption stimulated by glucose was mainly the passive consequence of solvent flow. The effects of bicarbonate and sugars on jejunal sodium absorption were not additive.

Interrelationships of chloride, bicarbonate, sodium, and hydrogen transport in the human ileum.

Using a triple-lumen constant perfusion system, the following observations were made in normal subjects. First, chloride, bicarbonate, and sodium were found to exhibit net movement across ileal mucosa against electrochemical gradients. Second, during perfusion with a balanced electrolyte solution simulating plasma, the ileum generally absorbed, but sometimes secreted fluid. A reciprocal net movement of chloride and bicarbonate was noted when sodium movement was zero. Increasing rates of sodium absorption were associated with decreasing bicarbonate secretion rates and finally bicarbonate absorption. Even when bicarbonate was absorbed ileal contents were alkalinized (by contraction of luminal volume). Third, net chloride movement was found to be sensitive to bicarbonate concentration in ileal fluid. For instance, chloride was absorbed from solutions containing 14 or 44 mEq/liter of bicarbonate, but was secreted when ileal fluid contained 87 mEq/liter of bicarbonate. Fourth, when chloridefree (sulfate) solutions were infused, the ileum absorbed sodium bicarbonate and the ileal contents were acidified. Fifth, when plasma-like solutions were infused, the potential difference (PD) between skin and ileal lumen was near zero and did not change when chloride was replaced by sulfate in the perfusion solution. These results suggest that ileal electrolyte transport occurs via a simultaneous double exchange, Cl/HCO2 and Na/H. In this model neither the anion nor the cation exchange causes net ion movement; net movement results from the chemical reaction between hydrogen and bicarbonate. No other unitary model explains all of the following observations: (a) human ileal transport in vivo is essentially nonelectrogenic even though Na, Cl, and HCO3 are transported against electrochemical gradients, (b) the ileum can secrete as well as absorb, (c) ileal contents are alkalinized during absorption of or during secretion into a plasma-like solution, and (d) the ileum acidifies its contents when sulfate replaces chloride. Data obtained with a carbonic anhydrase inhibitor support the proposed model.

PIP: Studies using a triple-lumen perfusion system in normal subjects were conducted to elucidate mechanisms of ileal electrolyte absorption. 4 primary observations were made in this study of interrelationships of chloride, bicarbonate, sodium, and hydrogen transport in human ileum: 1) chloride, sodium, and bicarbonate all exhibited net movement across ileal mucosa against electrochemical gradients; 2) when perfusion was performed in conjunction with administration of a balanced electrolyte solution stimulating plasma, the ileum generally absorbed, but sometimes secreted fluid; 3) net chloride movement was sensitive to bicarbonate concentration in ileal fluids; and 4) infusion of chloride-free (sulfate) solutions showed that the ileum absorbed sodium bicarbonate and that the ileal contents were acidified. A model to explain these findings suggests that ileal electrolyte transport occurs via a simultaneous double exchange, Cl/HCO3 and Na/H. In this model, net movement results from the chemical reaction between hydrogen and bicarbonate, not because of anion or cationic exchange.

Mechanism of bicarbonate absorption and its relationship to sodium transport in the human jejunum.

Using a constant perfusion technique, sodium and bicarbonate absorption was studied in human subjects. The following observations were made on sodium absorption from saline solution: (a) the rate of sodium absorption is markedly influenced by bulk water flow, (b) when net water flow is zero, sodium absorption is zero if there are no concentration gradients between plasma and lumen that favor net NaCl diffusion; and (c) the PD between abraded skin and jejunal lumen is near zero when saline is perfused and does not change with partial substitution of sulfate or bicarbonate for chloride. Based on these observations, we conclude that sodium absorption from saline is entirely passive in the human jejunum. On the other hand, in the presence of bicarbonate sodium is absorbed actively against electrochemical gradients. The mechanism of the link between bicarbonate and sodium absorption was studied in normal subjects and in 11 patients with pernicious anemia; the latter were chosen because they do not secrete gastric acid which can react with bicarbonate in the jejunal lumen. We observed that bicarbonate absorption (a) occurs against steep electrochemical gradients, (b) does not generate a potential difference between abraded skin and jejunal lumen, (c) is inhibited by acetazolamide, and (d) generates a high CO2 tension in jejunal fluid. These observations suggest that bicarbonate absorption is mediated by active hydrogen secretion, rather than by bicarbonate ion transport per se, and that the link between sodium and bicarbonate transport is best explained by a sodium-hydrogen exchange process.

PIP: In this study of bicarbonate and sodium absorption in the intestine, absorption in a 30-cm segment of intestine was studied by the Ingelfinger triple-lumen perfusion system, which involves perfusion of test solutions into the intestine and sampling of gut contents 10 and 40 cm beyond the infusion marker. Human subjects were used. Observations made from these experiments on the mechanism of bicarbonate absorption and its relationship to sodium transport in the jejunum from saline solutions include: 1) the rate of sodium absorption is influenced greatly by bulk water flow; 2) when net water flow is zero, sodium absorption is zero in the absence of concentration gradients betwee plasma and lumen; and 3) the potential difference between abraded skin and jejunal lumen is near zero when saline is perfused and does not change when sulfate or bicarbonate is partially substituted for the chloride. It is concluded that sodium absorption from saline is entirely passive in the human jejunum; in the presence of bicarbonate, sodium is actively absorbed against electrochemical gradients. This study also looked at the mechanism of the link between bicarbonate and sodium absorption. Normal subjects and 11 patients with pernicious anemia were studied. Bicarbonate absorption was found to 1) occur against steep electrochemical gradients; 2) not generate a potential difference between abraded skin and jejunal lumen; 3) be inhibited by acetazolamide; and 4) generate a high carbon dioxide tension in jejunal. These observations led to the conclusion that bicarbonate absorption is mediated by active hydrogen secretion rather than by bicarbonate ion transport per se, making the best explanation for the link between sodium and bicarbonate transport a sodium-hydrogen exchange process.

Influence of augmented Hageman factor (Factor XII) titers on the cryoactivation of plasma prorenin in women using oral contraceptive agents.

Prolonged cold storage of plasma may induce the conversion of plasma prorenin (inactive renin) to renin. This phenomenon is exaggerated in oral contraceptive (OC) users; the titer of Hageman factor (HF, Factor XII) in OC users is higher than in nonusers. The present study relates these observations. The increment in plasma renin activity (PRA) during cold storage, as measured by generation of angiotensin I, correlated strongly with the initial plasma titer of HF. Increasing the HF titer of nonusers to that observed in OC users by addition of purified HF increased cold-induced PRA at least twofold, while reducing the plasma HF titer of OC users correspondingly decreased cold-induced PRA. Thus, in OC users, the enhanced conversion of plasma prorenin to renin during cold storage reflects the elevated plasma titer of HF.

PIP: Prolonged cold storage of plasma may induce the conversion of plasma prorenin (inactive renin) to renin. This phenomenon is exaggerated in oral contraceptive (OC) users; the titer of Hageman factor (HF, Factor 12) in OC users is higher than in nonusers. The present study relates these observations. The increment in plasma renin activity (PRA) during cold storage, as measured by generation of angiotensin I, correlated strongly with the initial plasma titer of HF. Increasing the HF titer of nonusers to that observed in OC users by the addition of purified HF increased cold-induced PRA at least 2-fold, while reducing the plasma HF titer of OC users correspondingly decreased cold-induced PRA. Thus, in OC users, the enhanced conversion of plasma prorenin to renin during cold storage reflects the elevated plasma titer of HF.

Tissue sites of alpha fetoprotein synthesis by the rat during pregnancy and hepatoma growth.

The tissue sites of alpha1 fetoprotein (AFT) synthesis by the rat during gestation and hepatoma growth were determined by specific incorporation of a radiolabeled amino acid precursor into AFP by tissue cultures in vitro. During gestation, AFP were produced by the yolk sac, the fetal liver, and in small amounts by the fetal gastrointestinal tract; there was no synthesis by maternal rat tissues. During growth of a transplantable hepatoma, only the hepatoma tissue synthesized AFP; the nontumor tissue of the host contained AFP but did not produce it.

PIP: Alpha fetoprotein (AFP) synthesis by adult rats during gestation and hepatoma growth was determined in vitro with specific precipitations of radiolabeled AFP antisera after incubation of Spinner cultures of various rat tissues in arginine-free culture medium containing radiolabeled arginine. In general, AFP was synthesized by fetal liver, yolk sac, small intestine, and transplantable (tumor) tissue; none of the normal adult tissues, including testis or ovary, produced AFP. AFP synthesis (measured over 22 hours) was confined to the fetal liver (367 ng), yolk sac (1,368 ng), and to a small extent, the gastrointestinal tract during 19-day gestation. None of the maternal tissues produced AFP. When measured during growth of a transplantable hepatoma, AFP was synthesized only by the hepatoma tissue, though the nontumor tissue of the host contained AFP, due to release of AFP from the cultured tissue as it degenerated in vitro, but did not produce it (noninvolved tissues of hepatoma-bearing rats did not incorporate labeled arginine into AFP in vitro). Identifying fetal organs responsible for AFP synthesis explains observed AFP concentration changes in the postpartum period in rats, since elevated AFP in the mother is caused by AFP produced by the fetus which crosses the placenta or yolk sac to maternal circulation. Elevations above normal (.06 mcg/ml) adult rat concentrations occur in 3 circumstances in the nonpregnant rat: 1) development of AFP-producing tumors; 2) proliferation by normal liver cells; and 3) exposure to chemical carcinogens.

The detection of alpha 1-fetoprotein in patients with viral hepatitis.

PIP: Alpha fetoprotein (AFP) was detected in sera (351 samples) of 128 patients with viral hepatitis by radioimmunoassay. 77 positive tests for AFP were obtained. These positive results were demonstrated on 1 or more samples taken from 40 (31%) of the 128 patients studied; the highest value obtained was 4400 ng/ml. Hepatitis B antigen (HBAg) was positive in 26/40 (65%) of patients in whom AFP was detected during the disease process. However, 58/88 (66%) who were seronegative for AFP also demonstrated HBAg in their sera. Chi-square analysis revealed no significant difference in occurrence of detectable AFP between HBAg seropositive and seronegative patients. Individuals seropositive for AFP had no statistically different concentration of the protein than patients seropositive or seronegative for HBAg. 24 patients' sera were tested serially over a 2-week period. Both the peak glutamic-pyruvic transaminase (GPT) and peak total bilirubin levels were in a higher range in those 10/24 patients seropositive (P .001) for AFP than in the 14/24 who were seronegative. Appearance of AFP was related to the severity of liver tissue destruction, as reflected by serum GPT. However, peak AFP levels were attained 5-16 days after peak serum GPT appeared in the circulation.^ieng

Early appearance of embryonic -globulin in rat serum during carcinogenesis with 4-dimethylaminoazobenzene.

PIP: Heptatic tumors were induced in male Donryo rats by feeding them a 4-dimethylaminoazobenzene diet to study the occurrence of serum alpha globulin (AG) in rats during carcinogenesis. AG was found in rat serum as early as the 3rd week after onset of feeding of the carcinogenic diet; this was designated the early-stage appearance. The embryonic protein was observed in 31 (76%) of 41 rats at the 6th week after diet introduction. Subsequently, concentrations of AG decreased, and by the 11th-12th week it disappeared from serum. After 13 weeks, the developmental protein reappeared in 27/33 rats, designated the last-stage appearance, and 26 of these animals developed hepatomas. Of the 22 rats in which AG appeared in the early stage, 20 (91%) developed hepatomas after 19 weeks. By the 6th week of the carcinogenic diet, the average serum level of AG was 2-4 mg/dl, but after 13 weeks, it reached 60-100 mg/dl, corresponding to the serum level of newborns. Since most of the rats in which AG appeared at the early stage developed hepatomas, the early appearance of the protein may be related to cancerization of liver cells; on the other hand, the early appearance of AG may simply reflect an acute liver lesion caused by the toxicity of 4-dimethylaminoazobenzene. All rats that developed hepatomas were AG positive.^ieng

Synthesis of -fetoprotein by liver, yolk sac, and gastrointestinal tract of the human conceptus.

PIP: Synthesis of serum alpha fetoprotein (AFP) was studied in 16 human embryos and fetuses from 4.2-18 weeks of gestation by incubation of selected tissues in radiolabeled amino acids followed by immunoelectrophoresis of the culture fluids and radioautography. Relatively large amounts of radioactive AFP, judged by relative intensity of AFP precipitation line on radioautography, were found in each of the liver cultures of the developing yolk sac. AFP was observed in smaller amounts in almost all gastrointestinal tract cultures studied. Labeled AFP formed in kidney cultures from 1 of 9 conceptuses and in only 1 of 14 placentas cultured. None of the cultures containing lung, thymus, pancreas, skeletal muscle, amnion, chorion, or blood produced detectable amounts of AFP.^ieng

Elevated concentrations of serum alpha-fetoprotein in rats with chemically induced liver tumors.

The study was undertaken to determine whether aflatoxin B1 (AFB1)-induced liver tumors in rats produced alpha1-fetoprotein (AFP) and whether the age of the animals would influence such as appearance, a finding suggested by data seen in man. Other liver carcinogens (N-hydroxy-N-2-fluorenylacetamide, N-2-fluorenylacetamide, and diethylnitrosamine) were tested for their ability to induce liver tumors producing AFP. The presence of AFP. The presence of AFP in the serum was determined by double diffusion in agarose and by comparison also by quantitative radioimmunoassay. Using double diffusion, AFP was detected in the majority of tumor-bearing rats that had received either N-2-fluorenylacetamide or N-hydroxy-N-2-fluorenylacetamide. Sera of diethylinitrosamine-treated rats with liver tumors were all positive, whereas sera of rats bearing AFB1-induced tumors were positive in only a few cases. However, all sera of tumor-bearing rats examined had elevated AFP levels by radioimmunoassay. Nonetheless, the average level of AFP in the sera of rats bearing AFB1-induced tumors was considerably lower, compared to the sera of rats with tumors caused by diethylnitrosamine, N-2-fluorenylacetamide, or N-hydroxy-N2-fluorenylacetamide. Rats started on AFB1 when 6 weeks old had more mixed liver tumors with neoplastic hepatocytes and bile ducts and higher AFP levels than did rats started at 26 weeks of age. However, the histological grade of differentiation of inducted tumors did not seem to influence the AFP level.

PIP: Based on findings suggested by data in humans, this study attempted to determine whether aflatoxin Bl (AFB1)-induced liver tumors in rats produced alpha fetoprotein (AFP) and whether the animal's age influenced such appearance. Other liver carcinogens such as N-hydroxy-N-2-fluorenylacetamide (h2-FAA), N-2-fluorenylacetamide (2-FAA), and diethylnitrosamine (DENA) were tested for their abilities to induce liver tumors which produced AFP. The presence of AFP in serum was determined by double diffusion in agarose and by comparison also with quantitative radioimmunoassay. Male Fischer 344/CS rats were used in all experiments. By double diffusion, the AFP was detected in a majority of tumor-bearing rats that had received either 2-FAA or h2-FAA. Sera of DENA-treated rats with hepatic tumors were all positive, whereas sera of rats with AFB1-induced tumors were positive only in a few cases. However, all sera of tumor bearing rats examined had elevated AFP levels when measured by radioimmunoassay. Still, the average level of AFP in sera of rats bearing AFB1-induced tumors was considerably lower when compared with sera of rats whose tumors were caused by DENA, 2-FAA, or h2-FAA. Younger rats suffered more severe alterations: rats started on AFB1 when 6 weeks old had more mixed liver tumors with neoplastic hepatocytes and bile ducts and higher AFP levels than did rats started at 26 weeks of age. However, histological grade of differentiation of induced tumors did not seem to influence the AFP level, although the DENA-treated rats usually had high levels of AFP and less differentiated tumors.

Radioimmunoassay for 1 -fetoprotein in the serum of rats.

PIP: A radioimmunoassay (RIA) method for alpha 1-fetoprotein (AFP) in the serum of rats is reported. The method is based on the preparation of purified and radiolabeled AFP, monospecific anti-AFP antiserum, and a modification of the coprecipitation-inhibition technique in 50% saturated ammonium sulfate. A combination of immunochemical and physiocochemical methods are used in the purification of the AFP. The purified AFP is virtually identical to the native, circulating AFP by immunological criteria and contains, at most, trace contamination with other materials. The RIA has a sensitivity that allows 25 ng AFP/ml of serum to be detected with reproducibility. This assay requires 20 mcl of serum and can be completed within a few hours. It is concluded that this RIA, dependent upon the preparation of monospecific anti-AFP, radioiodination of highly purified rat AFP and a modified Farr procedure, is a sensitive and reproducible RIA.^ieng

Radioimmunoassay of rat 1 -fetoprotein.

PIP: A competitive binding radioimmunoassay for rat alpha 1 fetoprotein (AFP) was developed, using Sprague-Dawley rat amniotic fluid. The assay was approximately 20,000 times more sensitive than double-diffusion in agar for AFP detection; the assay threshold was 5 ng. Further purification of apparently pure (by immunodiffusion and immunization) radiolabeled AFP was required for the specific assay. An assay for a previously undetected contaminant(s) was used to check the purity of rat AFP isolated by isoelectric focusing to obtain the purified unlabeled AFP needed to establish the standard inhibition curve. All procedures are outlined.^ieng

Early appearance of serum -fetoprotein as a function of dosage of various hepatocarcinogens.

PIP: A previously reported early appearance of alpha fetoprotein (AFP) in rats fed 3'-methyl-4-dimethylaminobenzene (3-MDAB) which was induced before definite cancers were formed and disappeared on cessation of 3-MDAB administration was further investigated using different doses of 3-MDAB as well as other hepatocellular carcinogens and hepatotoxic agents. AFP was induced after 3 weeks of ingestion of diets with 600 ppm 3-MDAB and appeared after only 2 weeks when higher doses (900 and 1200 ppm) were used. Lower levels of 300 ppm 3-MDAB gave only a transient appaerance of AFP, beginning at the 5th week and remaining detectable for 3 more weeks, but 150 ppm did not induce at all. Immunosuppression with rat lymphocyte globulin extended for 1 week the time during which positive AFP titers were maintained upon cessation of 3-MDAB (600 ppm) intake. A transient appearance of AFP was found when rats were given the carcinogens dimethyl-4-dimethylaminoazobenzene (4-DMAA; 600 ppm), aflatoxin (AFB1; 4 ppm), N-2-fluorenylacetamide (N-2-FAA; 200 and 300 ppm), and N-hydroxy-N-2-fluorenylacetamide (N-OHFAA; 213 and 320 ppm). Lower doses of AFB1 (.2 and 2 ppm), N-2-FAA (150 ppm), N-OHFAA, and diethylnitrosamine (40 ppm) did not induce detectable AFP levels in serum nor did 2'-methyl-4-DMAA (600 ppm) and CCl4 (50 mg intraperitoneally twice a week). Apparently, high levels of liver carcinogens are required to induce the early appearance, within 2-5 weeks, of detectable AFP in serum.^ieng

Early appearance of serum -fetoprotein during hepatocarcinogenesis as a function of age of rats and extent of treatment with 3'-methyl-4-dimethylaminoazobenzene.

PIP: The early appearance of serum alpha-fetoprotein (AFP) during hepatocarcinogenesis as a function of age of rats and extent of treatment with 3'-methyl-4-dimethylaminoazobenzene is reported. Administration of .06% of the benzene hepatocarcinogen in the diet of 6- to 12-week-old male rats led to the prompt appearance of AFP in the serum within 3-4 weeks. Discontinuation of treatment at Week 5 dropped the AFP in serum to undetectable levels within 2 weeks, and it remained negative over a 30-week period when, at autopsy, no liver cancer was found. Administration of azo dye to 6-week-old rats for 10 weeks also decreased AFP in serum to undetectable levels over the next 2 weeks, except in 2 of 45 rats who developed large hepatomas early and remained positive. In the remainder, AFP reappeared beginning at Week 15, and liver cancer was present at Week 20 except for 13 rats that remained negative, although 7 had hepatoma. The age of the rats played no marked role in the precocious appearance of AFP. The presence of AFP in each group was related to the histological picture of the liver at the time of autopsy. There was no detectable AFP in untreated control rats, nor was there any in rats fed .05% of the hepatotoxic but not carcinogenic alpha-naphthylisothiocyanate which led to extensive jaundice and bile duct proliferation.^ieng

Inhibition of antibody-complement-mediated killing of tumor cells by hormones.

Line 1, a chemically induced guinea pig hepatoma, is susceptible to killing by anti-Forssman immunoglobulin M antibody and guinea pig complement. When these tumor cells are pretreated at 37 degrees with 10(-4) to 10(-11) M concentrations of the polypeptide hormone insulin, with the catecholamine L-epinephrine-HCl, or with the glucocorticoid steroids hydrocortisone sodium succinate or prednisolone sodium succinate, the cells show a marked reduction in their suseptibility to killing by antibody and guinea pig complement; pretreatment at 0 degrees is ineffective. Similar results were obtained with another antigenically distinct guinea pig hepatoma (line 10) when tested with anti-Forssman immuno-globulin M or specific antitumor antibodies and human complement. The ability of the hormones to render the cells resistant is dependent on time, temperature, and hormone concentration. The effect of hormone treatment is maximal between 30 and 60 min and is reversible within 4 hr even in the continued presence of hormone. Treatment of line 1 cells with up to 10,000-fold greater concentrations of the less biologically active or inactive analogs, DL-epinephrine, beta-estradiol, testosterone, or proinsulin has no effect on the susceptibility of the cells to killing by antibody and guinea pig complement. The effect of hormone treatment is not due to a direct inactivation of bound or fluid-phase complement components by the hormones or to a decrease in the ability of the cells to bind complement-fixing antibody.

PIP: Various aspects of hormone treatment of tumor cells are reported; it is shown that following treatment with certain hormones, the cells are less susceptible to killing by antibody and complement. The diethylnitrosamine-induced guinea pig hepatoma, designated Line 1, is susceptible to killing by anti-Forssman immunoglobulin M (IgM) antibody and guinea pig complement (GPC) but not by specific antitumor antibody and GPC. The antigenetically distinct Line 10 hepatoma, when sensitized with either antibody, is susceptible to killing by human complement (HUC) but not by GPC. Strain 2 of Servall-Wright male guinea pigs were used. 2 antigenetically distinct diethylnitrosamine-induced hepatic tumors (ascites form), Lines 1 and 10, passed in Strain 2 guinea pigs, were collected and suspended in RPMI 1640-20% FCS. Toxicity assays were performed in VBS-gel. The hormones used were hydrocortisone sodium succinate, prednisolone sodium succinate, NSC9151, bovine insulin, L-epinephrine methyl ether HC1, DL-epinephrine, beta-estradiol, testosterone, pork insulin, chicken insulin, pork proinsulin, pork DAA insulin, and the A and B chains of pork insulin. Tumor cells were cultured in 10-ml volumes of RPMI 1640-20% FCS in plastic Petri dishes. After incubation, cell cultures were washed 5 times in VBS-gel and tested for their susceptibility to killing by antibody and complement. Rabbit antiserum to sheep Forssman antigen was prepared and stored at -20 degrees until used. Tumor specific rabbit Antilines 1 and 10 antisera were prepared and similarly stored. Results of tests show that Line 1 tumor cells incubated in a medium containing the polypeptide hormone, insulin, the catecholamine, L-epinephrine HCl, or the glucocorticoid steroids, hydrocortisone sodium succinate, or prednisolone sodium succinate were rendered resistant to killing byanti-Forssman IgM antibody and GPC. This effect was dependent on hormone concentration, temperature, and time. Effects were reversible. Similar results were obtianed with Line 10 cells under attack by specific antitumor and HUC or anti-Forssman antibodies. Less physiologically active analogs of the hormones did not have this effect. Tumor cells showed maximum resistance within 30-60 minutes of exposure to the hormones and reverted to the sensitive state within 4 hours. Resistance of the cells to killing was observed at 37 degrees but not at 0 degrees. It is concluded that the effect of hormone treatment was not due to a direct inactivation of bound or fluid-phase complement components by the hormones or to a decrease in the ability of the cells to bind complement-fixing antibody.

Alpha-fetoprotein in ontogenesis and its association with malignant tumors.

PIP: The major steps in development of ontogenesis and the role of alpha fetoprotein (AFP) synthesis are outlined. AFP is defined and its physiocochemical characteristics are described including methods of detection and identification. The liver and yolk sac of fetuses are shown as the principle sites of AFP synthesis in ontogenesis, and the dynamics of AFP in ontogenesis from the early embryonic period through midpregnancy to pregnancy termination to AFP shut-down in early postnatal period are displayed. AFP synthesis during regeneration of the liver provides the model for studying the nature of AFP production. The role of AFP in hepatocellular cancer receives a great deal of attention, focusing on the site of AFP synthesis in cancer of the liver; demonstration of AFP in blood of cases of hepatic cancer (and other diseases) by agar-gel precipitation; quantitative aspects of AFP production by liver tumors; and etiologic and pathogenic influences on AFP production by hepatomas. Clinical aspects of the diagnosis of liver cancer are reviewed. The occurrence of AFP with teratocarcinomas is remarked upon. The article's central objective was to emphasize the importance of basic research on AFP, especially the development of an accessible high-sensitivity test for use in broad epidemiological surveys. Experimental approaches to some immediate problems were formulated: 1) Is there any external factor controlling AFP synthesis and determining its intensity? 2) Is synthesis performed only by certain cell types or is AFP production inherent in any hepatocyte? 3) Is control of AFP synthesis accomplished by regulating the intensity of the process in individual cells or by involvement of a varying number of cells? And 4) is AFP synthesis in a tumor due to maintained ability of the stem tumor cell to differentiate or is it the result of dedifferentiation of the mature hepatocyte??^ieng

Gossypol prevents activation of purified proenzyme of human seminal plasma acidic proteinase.

Gossypol inhibits the potential activity of the proenzyme form of human seminal plasma acidic proteinase, but has no effect on the active enzyme under the conditions tested. Inhibition of proenzyme is rapid and pH-dependent: 50% inhibition can be observed at gossypol concentrations of approx. 1.5 X 10(-5) M. SDS-polyacrylamide gel electrophoresis indicates that treatment of proenzyme with gossypol prevents the formation of active enzyme that normally occurs on acidification. Determination of free amino groups with 1-fluoro-2,4-dinitrobenzene suggests that gossypol reacts with 7.8 out of the 11.0 lysine residues in proenzyme: such a reaction could interfere with the activation process.

PIP: Studies of the effect of gossypol on the purified proenzyme of human seminal plasma acidic proteinase suggest that, while there is no effect on the active enzyme, the potential activity of the proenzyme form is inhibited. When the purified proenzyme was incubated at pH 8.0 with 0.25 mM of gossypol, 70% of potential activity was abolished within 2.5 minutes; moreover, this inhibitory activity was progressive, to the extent that only 5% of the original activity remained 90 minutes after incubation. When the concentrations of gossypol were varied, 50% inhibition of the potential activity of the proenzyme was observed at concentrations of approximately 15 mcgM. The reaction between gossypol and proenzyme was further found to be pH-dependent; a pH of at least 6.5, optimally 8.0, was required for activity inhibition. To determine whether treatment with gossypol altered protein structure, the active enzyme and proenzyme were preincubated with gossypol and subjected to SDS-polyacrylamide gel electrophoresis. Gossypol treatment decreased the potential activity of the proenzyme by 97%, but failed to affect the active enzyme. Studies on the amino acids affected suggested that gossypol protected 7.8 of 11 lysine residues in proenzyme from reaction with FDNB, presumably by formation of Schiff's bases. The physiological significance of the gossypol inhibition of seminal plasma acidic proteinase proenzyme remains unclear. Since the acidic proteinase requires acid for activation, it is unlikely that the enzyme plays a role in fertilization. It is more probable that seminal plasma acidic proteinase disposes of the remains of the ejaculate after intercourse, at a point when the vaginal pH has become acidic again. Thus, inhibition by gossypol could interfere with this "cleaning up" process.

Oral contraceptives in women with diabetes.

We evaluated the association of oral contraceptive use with the presence and severity of diabetic retinopathy, hypertension, and glycosylated hemoglobin in women of childbearing age who have diabetes. Neither current or past use nor number of years of use of oral contraceptives was associated with severity of retinopathy, hypertension, or current glycosylated hemoglobin. In conclusion, further study of various birth control methods in young women of childbearing age should be considered.

PIP: A population-based prospective study of insulin-dependent diabetics between the ages of 14-30 southern Wisconsin examined the relationship between oral contraceptive use and presence and severity of diabetic retinopathy, hypertension and glycosylated hemoglobin (HbA1). HbA1 is a measure of overall control of hyperglycemia. Out of 10,135 diabetic patients of 452 physicians in an 11-county area of Wisconsin, 432 were women between 14-30, and were followed from 1980-1986. The exit interview and exam consisted of pupil dilatation, stereoscopic fundus photographs, blood glucose by Chemstrip, blood pressure and determination of HbA1 with a resin microcolumn. 384 of these women provided oral contraceptive use history at follow-up. 170 ever used pills, 62 for 1yr, 59 for 2-4 yr, and 49 for 5 or more years. There was a trend toward current pill use with less severe diabetic retinopathy. There was no evidence of an association between ever using pills and the severity of diabetic retinopathy, controlling for age, duration of diabetes, systolic or diastolic blood pressure, HbA1, proteinuria or body mass index. Duration of diabetes, diastolic blood pressure, proteinuria and HbA1 were significantly associated with severity of retinopathy, while age, systolic blood pressure and body mass were not. Current, prior or duration of use of pills did not show significant effects on severity of retinopathy. Number of daily doses of insulin were inversely significantly related to HbA1.

Serological survey of HIV-1, HIV-2 and human T-cell leukemia virus type 1 for suspected AIDS cases in Ghana.

OBJECTIVE: To determine seroprevalence among suspected AIDS patients in Ghana in relation to clinical manifestations. MATERIALS AND METHODS: Blood samples and medical records were collected from 290 Ghanaian patients with suspected AIDS in 1990 and 1992. Seroprevalence of HIV-1, HIV-2 and human T-cell leukemia virus (HTLV-1) were investigated by the particle agglutination method, indirect immunofluorescence assay, the monoepitope enzyme-linked immunosorbent assay and Western blot. RESULTS: The specimens were classified into five serologic categories: 78 were HIV-1-positive (26.9%), 25 were HIV-2-positive (8.6%), 17 dual-positive (5.9%), 16 indeterminate (5.5%) and 154 seronegative (53.1%). No significant difference was found between the clinical symptoms of patients with HIV-1 and HIV-2 infection. Of the patients, 14 (4.8%) were HTLV-1-seropositive, of whom 11 were also HIV-positive, indicating a significant correlation between the two groups of viral infections (P < 0.01). However, there was no evidence of an increase in severity of symptoms in cases of dual infection with HTLV-1 and HIV. CONCLUSIONS: HIV-1 infection is now dominant in Ghana in contrast to our previous survey in 1986 which showed the dominance of HIV-2. The change in seroprevalence suggests that an HIV-1 epidemic has been developing in recent years in this country, where HIV-2 was originally endemic. A relatively high prevalence of dual-reactive specimens implies the existence of highly cross-reactive strains of HIV or frequent coinfection with HIV-1 and HIV-2 in the region. The large number of seronegative patients with clinically diagnosed AIDS raises the question of the inadequacy of AIDS definitions based on clinical manifestations only.

Risk of transfusion-associated HIV transmission in Kinshasa, Zaire.

Between 5 March and 12 April 1990, we assessed transfusion practices and the risk of transfusion-associated HIV transmission in all the hospitals and medical centres in Kinshasa, Zaire. Of the 733 hospitals and medical centres surveyed, 62 (8.5%) transfuse blood. Of 3741 units of blood transfused in February 1990, 1045 (27.9%) were not screened for HIV infection. Eighteen out of 62 centres (29%) received HIV test kits on a regular basis. Twenty of the centres (32.3%) recorded HIV test results. Major blood group cross-matching was done by 9.7% (six out of 62) of the centres. Bacteriological results indicated contamination in 17% (four out of 23) of stocked blood units, 6.4% (four out of 62) of solutions for disinfections, and 22% (13 out of 59) of sterilized instruments (possessed by 59 centres only). Transfusion practices in Kinshasa are associated with considerable health risks. The establishment and appropriate supervision of HIV screening facilities should be integrated into primary health-care programmes in order to increase safe transfusions in Kinshasa.

PIP: Between March 5-April 12, 1990, the authors assessed transfusion practices and the risk of transfusion-associated HIV transmission in all of the hospitals and medical centers in Kinshasa, Zaire. Of the 733 hospitals and medical centers surveyed, 62 (8.5%) transfuse blood. Of 3741 units of blood transfused in February 1990, 1045 (27.9%) were not screened for HIV infection. 18 of 62 centers (29%) received HIV test kits on a regular basis. 20 of the centers (32.3%) recorded HIV test results. Major blood group cross-matching was done by 9.7% (6 of 62) of the centers. Bacteriological results indicated contamination in 17% (4 of 23) of stocked blood units, 6.4% (4 of 62) solutions for disinfections, and 22% (13 of 59) of sterilized instruments (possessed by only 59 centers). Transfusion practices in Kinshasa are associated with considerable health risks. The establishment and appropriate supervision of HIV screening facilities should be integrated into primary healthcare programs in order to increase safe transfusions in Kinshasa.

Site-directed enzyme-linked immunosorbent assay with a synthetic simian immunodeficiency virus SIVmac peptide identifying antibodies against the HIV-2 transmembrane glycoprotein.

A 23-amino-acid-long peptide (AIEKYLEDQAQLNAWGCAFRQVC) representing the transmembranous protein gp32 in SIVmac was used in site-directed enzyme-linked immunosorbent assay (ELISA) for detection of HIV-2-specific antibodies in 567 sera from Bissau, Guinea Bissau. Ninety out of the 567 sera were identified to contain HIV-2 antibodies by whole antigen ELISA and Western blot assays. The peptide ELISA correctly identified 89 out of these 90 seropositives (sensitivity 98.9%). Three sera falsely interpreted to be positive were encountered (specificity 99.4%). The HIV-2 peptide was also used for testing of 93 HIV-1-positive Swedish sera. None of these sera reacted. Site-directed serology employing synthetic peptides should be considered for application as a screening assay.

PIP: HIV-1 is predominant in Central and East Africa, while HIV-2 is predominant in West Africa. The HIV-2 virus, originally described as human T-lymphotropic virus type IV, is identical to the simian immunodeficiency virus SIV-mac. Whole-antigen enzyme-linked immunosorbent assay (ELISA) can detect heterotypic antigens in 80% of sera, but separate tests are required for detecting HIV-1 and HIV-2. Site-directed ELISA using amino acids 586-620 of the synthetic transmembranous protein gp41 as antigen has been used for type-specific antibody determination in HIV-1. The homologous site on the 23-amino-acid-long peptide AIEKYLEDQAQLNAWGCAFRQVC representing the transmembranous protein gp32 in the simian immunodeficiency virus SIV-mac is WGCAFR, which can be used in site-directed serology of HIV-2. This approach was tested on sera from 567 Africans in Guinea-Bissau and 49 suspected AIDS patients. None of these sera had HIV-1 antibodies. 93 HIV-1-positive sera from Sweden were used as controls. The HIV-2 peptide ELISA correctly identified 89 of the 90 HIV-2 seropositives out of the sample of 567. The peptide ELISA also correctly identified all of the 93 HIV-1-positive sera as HIV-2-negative. Use of HIV-2-specific ELISA in combination with an HIV-1-specific ELISA can efficiently screen blood for both types of HIV.