We studied the effects of i.v. 2 mg/kg morphine sulfate (MS) on coronary blood flow and resistance, left ventricular (LV) diameter and pressure (P), rate of change of pressure (dP/dt), and dP/dt/P in conscious dogs. An initial transient reduction in coronary vascular resistance, associated with increases in heart rate, dP/dt, dP/dt/P, and reductions in LV end-diastolic and end-systolic size were observed. This was followed by a prolonged increase in mean coronary vascular resistance, lasting from 5 to 30 min, while heart rate, arterial pressure, and LV end-diastolic diameter returned to control levels and dP/dt/P remained slightly but significantly above control. At 10 min, late diastolic coronary flow had fallen from 44 plus or minus 3 ml/min to a minimum level of 25 plus or minus 3 ml/min, while late diastolic coronary resistance had risen from 1.68 plus or minus 0.10 to 3.04 plus or minus 0.28 mm Hg/ml/min. Morphine also induced substantial coronary vasoconstriction when heart rate was held constant. Neither the MS-induced coronary vasoconstriction nor the positive inotropic response was abolished by bilateral adrenalectomy. The positive inotropic response of MS was reversed after beta blockade, but not the coronary vasoconstriction. Alpha receptor blockade abolished the late coronary vasoconstriction effects of morphine, and only dilatation occurred. In anesthetized dogs MS failed to produce late coronary vasoconstriction. Coronary after a respiratory-depressant dose of morphine, 10 mg/kg i.v. Smaller doses of MS, 0.25 mg/kg every 15 min, produced significant coronary vasoconstriction after a total dose of 0.75 mg/kg in the conscious dogs. The effects of morphine may differ in the normal dog and man and may vary depending upon the presence or absence of coronary artery disease. However, in the normal conscious dog, MS elicits a mild beta adrenergic increase in contractility and an important coronary vasoconstrictor effect, which is mediated through alpha adrenergic receptors.
S F Vatner, J D Marsh, J A Swain
To determine if propylthiouracil (PTU) inhibited extrathyroidal thyroxine (T4) to triiodothyronine (T3) conversion in man, PTU was administered to T4-treated hypothyroid patients and serial measurements of T4, T3, and thyrotropin (TSH) carried out. All patients had proven thyroidal hypothyroidism and had been receiving 0.1 or 0.2 mg T4 daily for at least 2 mo before study. Hormone measurements were made for 5 consecutive days before and daily during a 7-day treatment period with PTU, 1,000 mg/day. In eight patients receiving 0.1 mg T4 daily, administration of PTU resulted in a prompt fall in mean serum T3 concentrations from 78 plus or minus 6 ng/100 ml (SEM) to 61 plus or minus 3 ng/100 ml after 1 day. The mean serum T3 concentrations ranged from 55 to 60 ng/100 ml during the remainder of the PTU treatment period (P less than 0.01). The mean control serum TSH concentration was 29.6 muU/ml and it increased to a peak of 40 muU/ml on the 5th and 6th days. In five patients receiving 0.2 mg T4 daily, the mean control serum T3 concentration was 84 plus or minus 7 NG/100ML. It fell to 70 plus or minus 5 ng/100 ml after 1 day and 63 plus or minus 7 ng/100 ml after 2 days of PTU administration and thereafter ranged from 6) to 69 ng/100 ml (P LESS THAN 0.01). Serum TSH concentrations did not increase. No changes in serum T4 concentrations were found in either group. In five patients who received 100 mg methimazole (MMI) daily for 7 days there were no changes in serum T4, T3, or TSH concentrations. These results indicate that PTU, but not MMI, produces a prompt and sustained, albeit modest, reduction in serum T3 concentrations in patients whose sole or major source of T3 is ingested T4. These findings most likely result from inhibition of extrathyroidal formation of T3 from T4.
M Saberi, F H Sterling, R D Utiger
Propylthiouracil (PTU) inhibits peripheral deiodination of thyroxine (T4) and triiodothyronine (T3) and decreases the metabolic effectiveness of T4 in animals. To assess the effect of PTU on extrathyroidal conversion of T4 to T3 in man, 15 studies were performed in athyreotic patients treated with 100 or 200 mug of L-T4 daily for 1 mo before the addition of PTU, 250 mg every 6 h for 8 days. serum T3, T4, and thyrotropin (TSH) were measured daily by radioimmunoassay; serum TSH response to 500-mug thyrotropin-releasing hormone (TRH) was measured before and on the last day of giving PTU. On the 100-mug LT4 dose, serum T3 fell from 120 plus or minus 5 (SE) to 83 plus or minus 6 ng/dl (P less than 0.005) with return to 113 plus or minus 5 ng/dl after stopping PTU; serum T4 (4.5 plus or minus 0.3 mug/dl) did not change. Similar results were seen in patients taking 200 mug of L-T4 daily. On the 100-mug dose of L-T4 the fall in T3 was accompanied by a reciprocal rise in serum TSH to 195 plus or minus 33% of initial concentration (P less than 0.01) with return to 104 plus or minus 8% after PTU. The serum TSH response to TRH (DELTAMUU/ml over base line) was greater during PTU therapy than during the control period. On 100-mug L-T4 DELTA TSH rose from 64 plus or minus 19 to 101 plus or minus 23 muU/ml (P less than 0.005). Expressed as percent of base-line TSH concentration, TSH rose from 140 plus or minus 52 to 280 plus or minus 44% (control vs. PTU) at 15 min, 265 plus or minus 72 to 367 plus or minus 63% at 30 min, 223 plus or minus 54 to 313 plus or minus 54% at 45 min, 187 plus or minus 45 to 287 plus or minus 51% at 60 min, and 145 plus or minus 22 to 210 plus or minus 28% at 120 min after TRH. The data suggest that PTU blocks extrathyroidal conversion of T4 to T3, thus increasing pituitary TSH secretion and augmenting the TSH response to TRH.
D L Geffner, M Azukizawa, J M Hershman
A study was made of the possible mechanism(s) underlying minoxidil-induced increase in plasma renin activity (PRA). 10 patients with essential hypertension were treated with minoxidil and subsequently with a combination of minoxidil plus propranolol. Minoxidil lowered mean arterial pressure 31.6 plus or minus 3.3 mm Hg, mean plus or minus SEM. There was an associated increase in both PRA, 6.26 plus or minus 2.43 NG/ML/H, and heart rate, 21.4 plus or minus 2.7 beats/min. The changes in PRA and heart rate were positively correlated, r, 0.79. Addition of propranolol reduced mean arterial pressure by a further 10.1 plus or minus 1.5 mm Hg and returned heart rate to control levels. Propranolol reduced PRA significantly but not to control levels. Control PRA positively correlated with PRA on minoxidil, r, 0.97, and with PRA on minoxidil plus propranolol, r, 0.98. We conclude that control PRA is a major determinant of change in PRA with minoxidil. Minoxidil increased PRA by at least two mechanisms: (a) an adrenergic mechanism closely related to change in heart rate and blocked by propranolol, and (b) a mechanism(s) not sensitive to propranolol and possibly related to decrease in renal perfusion pressure.
K O'Malley, M Velasco, J Wells, J L McNay
Vasodilating antihypertensive drugs induce hypotension with reflex tachycardia, renin release, and fluid and electrolyte retention. Propranolol can impair this renin release. The studies described here were designed to determine the hemodynamic role of vasodilatory drug-induced renin release and inhibition thereof by propranolol in two animals models, the unanesthetized, normotensive and the unanesthetized, genetically hypertensive rat. In studies with normotensive rats, propranolol impaired renin release and tachycardia resulting from hydralazine and minoxidil and potentiated their hypotensive action. Two additional interventions against the renin-angiotensin system were used in evaluating the mechanism of this potentiation. One was removal of the renin source by nephrectomy, and the second was blockade of angiotensin's vasoconstrictor action using a selective angiotensin antagonist, saralasin (1-Sar-8-Ala-angiotensin II (previously known as P113)).. Both interventions potentiated vasocilatory drug hypotension, as did propranolol, but did not prevent reflex tachycardia. When combined with saralasin propranolol did not add to protentiation by this peptide. A similar pattern of blood pressure decrement and potentiation was seen in genetically hypertensive rats when propranolol or saralasin treatment preceded hydralazine. Propranolol was demonstrated to block hydralazine-induced increases in serum renin activity in genetically hypertensive rats. We conclude that hypotensive potentiation of vasocilating drugs by propranolol in these animal models is mediated to a large extent by impairment of renin release. Persistence of hypotensive tachycardia after nephrectomy and after saralasin in normotensive rats suggests the irrelevance of angiotensin's central nervous system stimulation to this cardiac effect. Clinical studies are underway to quantify the potential importance of this beneficial drug interaction in man.
W A Pettinger, K Keeton
The hyperglucagonemia that occurs in vivo in animals made diabetic with alloxan or streptozotocin is not suppressed by high glucose but is suppressed by exogenous insulin. These observations together with other studies suggested that insulin-dependent glucose transport and metabolism by the alpha-cells serves as the primary mechanism controlling glucagon secretion. This hypothesis was tested in the present investigation. The possible interactions between glucose, insulin, and a mixture of 20 amino acids at physiological proportions were examined in the isolated-perfusin diabetic rats. Release of insulin and glucagon were used as indicators of theta-cell and alpha-cell function. According to rigid criteria the diabetic animals entering the study were severely diabetic. It was found that in vitro: (a) basal glucagon release (measured in the absence of an alpha-cell stimulus or inhibitor) was extremely low, even lower (i.e. 10%) than the basal rates seen in controls; (b) the alpha-cells of alloxanized- and streptozotocin-treated rats responded with a biphasic glucagon release to stimulation by an amino acid mixture; (c) this alpha-cell response was reduced after both streptozotocin and alloxan; (d) glucose at 5 mM was a potent inhibitor of amino acid-induced glucagon secretion in both types of experimental diabetes; (e) in alloxan diabetes alpha-cell stimulation by amino acids can be curbed by exogenous insulin, whereas glucagon secretion by the perfused pancreas of streptoxotocin diabetic rats appeared to be resistant to insulin action. The data indicate that the modulation of glucagon secretion by glucose in vitro is indipendent of insulin and that other unknown factors extrinsic to the pancreatic islets are responsible for the hyperglucagonemia observed in vivo.
A S Pagliara, S N Stillings, M W Haymond, B A Hover, F M Matschinsky
Human lysosomes were isolated from normal peripheral blood leukoyctes and characterized by electron microscopy, enzyme analysis, and assays for DNA and RNA. Stored sera from 37 unselected patients with systemic lupus erythematosus (SLE), including active and inactive, treated and untreated cases, were tested in complement fixation (CF) reactions with these lysosome preparations. 23 SLE sera exhibited positive CR reactions, as did sera from two patients with "lupoid" hepatitis. The seven SLE sera with strongest CF reactivity also demonstrated gel precipitin reactions with lysosomes. Neither CF nor precipitin reactions with lysosomes were observed with normal sera or with sera of patients with drug-induced lupus syndrome, rheumatoid arthritis (RA), polymyositis, or autoimmune hemolytic anemia. By several criteria the antilysosome CF and precipitin reactions of SLE sera cound not be attributed to antibody to DNA, RNA, or other intracellular organelles. The lysosomal component reactive with SLE sera in CF assays was sedimentable at high speed and is presumably membrane associated. The CF activity of two representative SLE sera was associated with IgG globulins by Sephadex filtration. A search for lysosomal antigen in SLE and related disorders was also made. By employing rabbit antiserum to human lysosomes in immunodiffusion, a soluble lysosomal component, apparently distinct from the sedimentable (membrane-associated) antigen described above, was identified in serum, synovial fluid, or pleural fluid from patients with SLE, RA, ankylosing spondylitis, and leukemoid reaction. An antigenically identical soluble component reactive with the rabbit antiserum could be released in vitro from intact lysosomes by repeated freeze-thaw cycles..
D A Bell, P A Thiem, J H Vaughan, J P Leddy
Studies were carried out on the effects of polyunsaturated fats on lipid metabolism in 11 patients with hypertriglyceridemia. During cholesterol balance studies performed in eight patients, the feeding of polyunsaturated fats, as compared with saturated fats, caused an increased excretion of endogenous neutral steroids, acidic steroids, or both in most patients. Increases in steroid excretions were marked in some patients and generally exceeded the decrement of cholesterol in the plasma compartment. The finding of a greater excretion of fecal steroids on polyunsaturated fats in hypertriglyceridemic patients contrasts to the lack of change in sterol balance previously reported for patients with familial hypercholesterolemia; however, other workers have found that polyunsaturated fats also enhance steroid excretion in normal subjects. In most of the patients, simultaneous studies were carried out on biliary lipid composition, hourly outputs of biliary lipids, and pool sizes of bile acids. In several but not all patients, fasting gallbladder bile became more lithogenic after institution of polyunsaturated fats. This increased lithogenicity was not due to a decrease in bile acid pools; in no case was the pool decreased by polyunsaturated fats. On the other hand, two patients showed an increased output of biliary cholesterol, and frequently there was an increase in fecal neutral steroids that were derived from cholesterol; thus, polyunsaturated fats may increase bile lithogenicity in some patients through mobilization of cholesterol into bile. Reductions in plasma cholesterol during the feeding of polyunsaturated fats was seen in most patients, and these changes were usually associated with a decrease in concentration of plasma triglycerides. In fact, the degree of cholesterol lowering was closely correlated with the extent of triglyceride reduction. Therefore, in hypertriglyceridimec patients polyunsaturated fats may contribute to cholesterol reduction by changing the metabolism of triglycerides or very low density lipoproteins. The findings of changes in the metabolism of cholesterol, bile acids, and triglycerides in the patients of this study suggests that polyunsaturated fats may cause a lowering of cholesterol through multiple mechanisms, and it seems unlikely that a single action can explain all the effects of these fats on the plasma lipids.
S M Grundy
Changes is tubular reabsorption of uric acid in response to alterations in the extracellular fluid volume (ECFV) were examined in rats by clearance studies and by direct intratubular microinjections. Contraction of the ECFV led to a rise in the serum uric acid concentration and a 47% decrease in the clearance of uric acid. The ratio of uric acid to inulin clearance also fell, indicating an increase in the net tubular reabsorption of urate. Volume expansion resulted in an increase in the urate clearance and a 37% decrease in the net tubular reabsorption of uric acid. To localize the site in the nephron where these changes occur, microinjections of [2-14C]urate were performed. The lack of conversion of radioactive urate to allantoin after microinjections was demonstrated by thin-layer chromatography. After contraction of the ECFV, urinary recoveries of uric acid were significantly decreased after microinjections into proximal tubular sites. In contrast, recoveries were increased from these proximal sites after volume expansion. No evidence for distral reabsorption was obtained in any group of animals. These studies demonstrate that net urate reabsorption is influenced by the state of hydration of the ECFV and that these alterations are mediated by changes in the rates of reabsorption in the proximal tubule.
E J Weinman, G Eknoyan, W N Suki
Isolated renal tubules prepared from vitamin D-deficient chicks catalyze the 1 alpha-hydroxylation of 25-hydroxyvitamin D3 (250HD3) in vitro. The effect of calcium and phosphate on the rate of synthesis of the product, 1, 25-dihydroxyvitamin D3 (1,25(OH)2D3), was studied at two levels: the long-term effects of various dietary calcium and phosphate contents on the ability of the tubules to produce 1, 25 (OH)2D3, and the acute effects of different calcium and phosphate concentrations in the incubation medium on the rate of synthesis of 1,25(OH)2D3 by the tubules. Manipulation of dietary calcium and phosphate sufficient to produce marked changes in the concentration of calcium and phosphate in the serum led to altered rates of 1,25(OH)2D3 synthesis by the isolated renal tubules. The renal tubules isolated from chicks raised on a vitamin D-deficient diet containing 0.43% calcium and 0.3% P as inorganic phosphate showed the highest rate of synthesis of 1,25(OH)2D3. Diets containing more or less of either calcium or phosphate produced chicks whose renal tubules had a slower rate of 1,25(OH)2D3 production. The calcium, phosphate, and hydrogen ion content of the incubation medium were manipulated to determine the possible factors concerned with the immediate regulation of 1,25(OH)2D3 production. A calcium concentration of 0.5-1.0 mM was necessary for optimal enzymatic activity. Concentrations of calcium greater than this optimal concentration inhibited 1,25(OH)2D3 production if phosphate was also present, and this inhibition was more pronounced as the phosphate concentration was increased. The stimulation of 1,25(OH)2D3 production by calcium was less at pH 6.7 than at 7.4. Raising the phosphate concentration from 0 to 6 mM in the absence of calcium also stimulated the rate of synthesis of 1,25(OH)2D3. This stimulatory effect was blocked by 4 mM calcium. However, at 1-2 mM calciu, phosphate had a biphasic influence on 1,25(OH)2D3 production; extracellular concentrations of phosphate from 0.6 to 1.2 mM resulted in less 1,25(OH)2D3 production than higher or lower phosphate concentrations. This biphasic effect was seen both at pH 7.4 and 6.8.
D D Bikle, H Rasmussen
Isolated kidney mitochondria prepared from Vitamin D-deficient chicks catalyze the conversion of 25-hydroxyvitamin D3 to 1,25 dihydroxyvitamin D3. It wasfound that changes in the concentrations of Ca-2plus, HPO4-2minus, and Hplus altered synthesis in an interrelated fashion. Increasing the Ca-2plus concentration from 10-6 to 10-5 M caused a four- to fivefold increase in 1 alpha-hydroxylase activity when the medium pH was between 6.5 and 7.0. increasing the [Ca2+] to 10-4 M caused to furhter stimulation. At higher pH values, Ca-2plus had little effect upon 1 alpha-hydroxylase activity. In the absence of calcium [Ca2+] less than or equal to 10-7 M), a change in pH from 6.5 to 7.1 had no effect upon 1 alpha-hydroxylase activity in the presence of 10-5 M calcium, increasing the medium pH had a biphasic effect. An increase in pH from 6.5 to 6.9 caused a 1.5-fold increase in 1 alpha-hydroxylase activity, but a further increase of the pH to 7.1 caused a profound decrease in rate of hydroxylation to approximately 20% of the peak value. Neither 10-5 M LaC13 nor 10 mug/ml of oligomycin altered the effects of Ca2+ upon hydroxylate activity. However, the effect of calcium was blocked by 2.5 times 10-5 M ruthenium red, 0.83 mug/ml of antimycin A, and 500 muM dinitrophenol. The clcium ionophore, A23187, decreased but did not prevent the stimulatory effect of calcium. These data are consistent with the concept that the [Ca2+ in the mitochondrial matrix space is of importance in regulating the 1 alpha-hydroxylase. Phosphate exerted a biphasic effect on 1,25(OH)2D3 production with maximal stimulation (approximately twofold) at 1-3 mM. Calcium enhanced the stimulation by phosphate at all concentrations studied. The presence of potassium modified the interrelated effects of calcium and phosphate in two ways: 10-3 M calcium blocked the stimulation by phosphate; and in the presence of phosphate, 10-3 M calcium resulted in less 1,25(OH)2D3 production by production by isolated mitochondria are qualitatively similar to the effects of these ions on 1,25(OH)2D3 production yb isolated renal tubules.
D D Bikle, E W Murphy, H Rasmussen
Pressures and flows were measured in surface glomerular capillaries, efferent arterioles, and proximal tubules of 22 Wistar rats in the early autologous phase of nephrotoxic serum nephritis (NSN). Linear deposits of rabbit and rat IgG and C3 component of complement were demonstrated in glomerular capillary walls by immunofluorescence microscopy. Light microscopy revealed diffuse proliferative glomerulonephritis, and proteinuria was present. Although whole kidney and single nephron glomerular filtration rate (GFR) in NSN (0.8 plus or minus 0.04 SE2 ml/min and 2 plus or minus 2 nl/min, respectively) remained unchanged from values in 16 weight-matched NORMAL HYDROPENIC control rats (0.8 plus or minus 0.08 and 28 plus or minus 2), important alterations in glomerular dynamics were noted. Mean transcapillary hydraulic pressure difference (deltaP) averaged 41 plus or minus 1 mm Hg in NSN versus 32 plus or minus 1 in controls (P LESS THAN 0.005). Oncotic pressures at the afferent (piA) end of the glomerular capillary were similar in both groups ( 16 mm /g) but increased much less by the efferent end (piE) in NSN (to 29 plus or minus 1 mm Hg) than in controls (33 plus or minus 1, P less than 0.025). Hence, equality between deltaP and piE, denoting filtration pressure equilibrium, obtained in control but not in NSN rats. While glomerular plasma flow rate was slightly higher in NSN (88 plus or minus 8 nl/min) than in controls (76 plus or minus 6, P greater than 0.2), the failure to achieve filtration equilibrium in NSN rats was primarily the consequence of a marked fall in the glomerular capillary ultrafiltration coefficient, Kf, to a mean value of 0.03 nl/(s times mm Hg), considerably lower than that found recently for the normal rat, 0.08 nl/(s times mm Hg). Thus, despite extensive glomerular injury, evidenced morphologically and by the low Kf, GFR remained normal. This maintenance of GFR resulted primarily from increases in deltaP, which tended to increase the net driving force for filtration, and thereby compensate for the reduction in Kf.
D A Maddox, C M Bennett, W M Deen, R J Glassock, D Knutson, T M Daugharty, B M Brenner
The role of high serum and tissue levels of unconjegated bilirubin in the pathogenesis of the impaired urinary concentrating ability was investigated in homozygous (jj) Gunn rats with the congenital absence of hepatic glucuronyl transferase. Continuous phototherapy with blue fluorescent lights at a wave length of 460 nm or oral cholestyramine feeding or both reduced serum levels of unconjugated hilirubin to levels consistently below 3.0 mg/100 ml for several weeks in both weanling and adult jj Gunn rats. The renal concentrating defect was already present in weanling jj Gunn rats by 21 days of age. In treated weanling jj animals, maximum concentrating ability and the concentration of urea and nonurea solutes in the papilla and medulla, determined after 24 h of fluid deprivation, were normal when compared to unaffected heterozygous (Jj) littermates. Solute-free water reabsorption which is reduced in jaundiced jj Gunn rats was restored to normal in treated weanling jj rats. The tissue concentration of unconjugated bilirubin was reduced throughout the papilla and inner and outer medulla in the treated jj rats in comparison with untreated jj littermates. The defect in urinary concentrating ability was only partially reversible and sometimes irreversible in adult jj rats, probably because of permanent renal parenchymal damage occurring secondary to massive crystalline deposits in the papilla and medulla. It is concluded that unconjugated bilirubin is directly involved in the pathogenesis of the concentrating defect in jaundiced jj Gunn rats.
N B Call, C C Tisher
The effect of graded doses of pentagastrin (2.7-6,000 ng/kg times h) on gastric acid secretion was measured in 20 duodenal ulcer (DU) and 20 non-DU subjects. Confirming many previous studies, the mean observed highest response and the mean calculated maximal response were significantly greater in DU than in non-DU subjects. The mean dose (plus or minus SE) in ng/kg times h for half maximal response, calculated from responses corrected for basal secretion and normalized for maximal secretion, was 92.1 plus or minus 1.7 in DU and 246.8 plus or minus 24.6 in non-DU subjects, a significant difference. By parallel line bioassay non-DU subjects required 2.8 times more pentagastrin (95% confidence limits 2.1-3.7) than DU highest response. Thus, this study shows that, compared with non-DU subjects, DU patients not only secrete more acid in response to stimulation by pentagastrin but also are more sensitive to stimulation by pentagastrin, that is, need smaller doses to achieve the same fraction of maximal response.
J I Isenberg, M I Grossman, V Maxwell, J H Walsh
We tested the ability of human peripheral blood monocytes to kill Candida albicans and Candida parapsilosis. Evidence that multiple fungicidal mechanisms operate in normla monocytes was found. Normal monocytes ingested and killed viable C. albicans, and could iodinate heat-killed C. albicans. Both functions were defective in monocytes from subjects with myeloperoxidase deficiency or chronic granulomatous disease. Methimazole, isoniazid, and aminotriazole inhibited iodination by normal monocytes without impairing their ability to kill C. albicans, indicating that iodination was not essential to the myeloperoxidase-hydrogen peroxide-mediated fungicidal system of the monocyte. C. parapsilosis, an organism killed with supranormal efficacy by monocytes from a patient with hereditary myeloperoxidase deficiency, was selected to examine the myeloperoxidase-independent fungicidal mechanisms of monocytes. Monocytes were obtained from the blood of normal or leukemic subjects and homogenized in 0.34 M sucrose to yield fractions rich in cytoplasmic granules. These fractions were extracted with 0.01 M citric acid and the soluble components were separated by micropreparative polyacrylamide electrophoresis. Monocytes were found to contain cationic proteins, other than myeloperoxidase, that kill C. parapsilosis in vitro.
R I Lehrer
This study investigates the separate effects of age and hepatocellular liver disease on the disposition and elimination of diazepam (Valium) in man. The drug was given either by rapid intravenous injection (0.1 mg/kg) or orally (10 mg) to 33 normal volunteers rnaging in age from 15 to 82 yr as well as to 9 individuals with alcoholic cirrhosis, 8 with acute viral hepatitis, and 4 with chronic active hepatitis. In the normal individuals, the terminal plasma half-life of diazepam, (t 1/2 (B)) exhibited a striking age-dependence; at 20 yr the t 1/2 (beta) was about 20 h, but it increased linearly with age to about 90 h at 80 yr. The plasma clearance of diazepam in the majority of the normal subjects was between 20 and 32 ml/min and showed no significant age-dependence. Cigarette smoking did not affect the half-life or the clearance. Additionally, neither the plasma binding (97.4 plus or minus 1.2%, mean plus or minus SD) nor the blood/plasma concentration ratio (0.58 plus or minus 0.16) of diazepam showed any age-related changes (P greater than 0.05). By contrast, analysis of the intravenous data according to a two-compartment open model indicated that both the initial distribution space (V1) and the volume of distribution at steady state [Vd(ss)] of diazepam increased linearly with age (P less than 0.005). The increase in Vd(ss) was secondary to the change in V1. It appears then that the prolongation of t 1/2 (beta) of diazepam with age is primarily dependent on an increase in the initial distribution volume of the drug. The plasma concentration/time course of the metabolite, desmethyldiazepam, was also affected by age. In older individuals, the initial presence and the peak values of desmethyldiazepam were observed later and the metabolite was present in lower concentrations. Despite the profound prolongation of t 1/2 (theta) with age, the constancy of diazepam clearance indicates that drug plasma concentrations will not accumulate any more in the old than the young, and chronic dosage more in the old than the young, and chronic dosage modifications based on pharmacokinetic considerations are unnecessary. Data obtained in patients with liver disease were compared with those found in age-matched control groups. Patients with cirrhosis showed a more than twofold prolongation in the half-life of diazepam (105.6 plus or minus 15.2 vs. 46.6 plus or minus 14.2 h, P less than 0.001).
U Klotz, G R Avant, A Hoyumpa, S Schenker, G R Wilkinson
Human neurophysin was isolated from acetone-dried human posterior pituitaries and separated into two major neurophysin peptides by ion exchange chromatography and into four major peptides by preparative disk gel electrophoresis. Antisera raised in rabbits distinguished only two specific antigenic sites on the isolated neurophysin peptides. Individual sensitive and specific radioimmunoassays for two human neurophysins were developed. These assays were used to measure each neurophysin in unextracted human plasma. The two neurophysins are secreted independently in man. One assay measures a neurphysin that is specifically secreted in response to estrogen administration, estrogen-stimulated neurophysin (ESN). The other assay measures a neurophysin that is specifically secreted in response to cigarette smoking, nicotine-stimulated neurophysin (NSN). The mean ESN is 1.1 ng/ml plus or minus 0.7 SD in women and 1.0 ng/ml plus or minus 0.7 SD in men. The mean NSN is 0.9 ng/ml plus or minus 0.2 SD in women and 0.6 ng/ml plus or minus 0.3 SD in men. It is proposed that these may prove to be a specific human "oxytocinneurophysin," ESN, and a human "vasopressin-neurophysin," NSN.
A G Robinson
The cytophilic activity of human myeloma proteins of different classes and subclasses for lymphocytes, monocytes, and neutrophils was investigated. Binding of both unaggregated immunoglobulins (Ig) and Ig aggregated with rabbit F(ab)2 anti-Fab fragment sera was determined. Lymphocytes bound unaggregated IgG1 and IgG3 proteins, but none of the proteins of the other classes. In contrast, after aggregation, IgG of all subclasses and IgE proteins bound to lymphocytes; aggregated proteins of the other classes did not bind. Monocytes bound unaggregated IgG1 and Ig3 better than Ig4 whereas the binding of proteins of other classes was insignificant. Neutrophils bound unaggregated IgG1 and IgG3 proteins and, in addition, IgA1, IgA2, secretory IgA, and IgG4 proteins. After aggregation, the neutrophils bound more Ig of all classes; however, the differences between the amounts bound remained similar to the amounts of unaggregated proteins. The native structure of the Ig molecule is necessary for the maintenance of complete activity, because Fc fragments bound less than intact Ig, and reduction and alkylation abolished cytophilia. The Fc receptors on all cell types tested showed no specificity for any of the respective cytophilic IgG subclasses; however, neutrophils appear to have separate receptors for IgG and IgA proteins.
D A Lawrence, W O Weigle, H L Spiegelberg
This study tests the hypothesis than obligatory cation coverage of metabolicaly generated anions is the mechanism for the sodium diuresis of fasting. Nine obese female subjects were equilibrated on a constant sodium and caloric intake and then fasted while sodium intake was maintianed. Particular activity schedule during fasting as during control. Consecutive 3-h increases in urinary sodium , ammonium, and potassium excretion during fasting were matched against simultaneously determined increases in organic acid anions (OAS) and H2PO4 minus, which would exist in combination with the cations. The changes were significantly correlated (r equals 0.891, P less than 0.001) in the relationship y equals 0.73x plus 19 where y equals increases in organic acid salts plus H2POJ minus and x equals increases in cations. As ammonium excretion rose, sodium conservation occurred with ammonium replacing sodium at the major urinary cation. Corollaries to the hypothesis were also found to be true. They were: (a) Increases in ammonium excretion lagged considerably behind increases in OAS plus H2PO4 minus during the diuretic phase making sodium coverage necessary. (b) Sodium loss was much greater than chloride although chloride balance was minimally negative. (c) After refeeding with glucose, sodium excretion promptly decreased and appeared best correlated with simultaneous decreases in OAS. Ammonium excretion also fell but much less than sodium. The data support the hypothesis that obligatory cation coverage of metabolically generated aniuns is a major mechanism responsible for the sodium diuresis of fasting.
M H Sigler
Prospective recipients of kidney transplants were tested for lymphocytotoxicity; from these we selected 102 sera that lacked cytotoxic antibodies against peripheral lymphocytes from at least 80 unrelated subjects. To detect humoral sensitization, we then reacted these with 17 cultured human lymphoid cell lines having different HL-A phenotypes. Cytotoxic antibodies reacting with these cultured cells were now detected in some of the sera. These antibodies were not directed against HL-A antigens, yet mediated lysis of target cells in the presence of rabbit but not of human or guinea pig complement. Furthermore, they activated the classical pathway of the rabbit complement system. Later, a significant association was found between occurrence of cytotoxic antibodies and rejection of the transplant. Thus, cultured human lymphoid cells, because of their great susceptibility to complement-mediated lysis, appear to be useful in detecting humoral sensitization in candidates for kidney grafts.
S Ferrone, A Ting, M A Pellegrino, P I Terasaki, R A Reisfeld
Studies were carried out during the 1st yr of life in normal infants born at term to determine the proportions of fetal hemoglobin (Hb F) and adult hemoglobin (Hb A) being synthesized, in order to describe the complete switchover from Hb F to Hb A synthesis during postnatal life. 53 blood samples from 37 infants were incubated in an amino acid mixture containing [14C]leucine and chromatographed on DEAE-Sephadex for separation of Hb F and Hb A fractions. The completeness of the CEAE-Sephadex separation of Hb A and Hb F at an age when the major portion of synthesis was of the adult type of hemoglobin was confirmed by globin chain chromatography with the use of carboxylmethyl cellulose. There was a rapid decline in Hb F synthesis postnatally until 16-20 wk of age when levels of 3.2% plus or minus SD 2.1% were reached. By combining this data with that previously published, the complete switchover from Hb F to Hb A synthesis can be described in humans in relation to postconceptional age. It follows a sigmoid curve; the steep portion, which lies between the 30th and 52nd postconceptional week, is preceded and follwoed by plateaus averaging 95% and 7% Hb F synthesis, respectively.
To explore the role of urea in the urinary concentrating mechanism, the contents of vasa recta, Henle's descending limbs and collecting ducts were sampled by micropuncture of the renal papilla before and after infusion of urea in 10 protein-depleted rats. Eight protein-depleted rats not given urea were similarly studied as a control group. After urea administration, osmolality and the concentrations of urea and nonurea solute of urine from both exposed and contralateral kideny increased significantly. The osmolality and urea concentration of fluid from the end of Henle's descending limb and vasa recta plasma and the tubule fluid-to-plasma inulin ratio in the end-descending limb all increased significantly after urea infusion. We interpret these observations to indicate that urea enhances urinary concentration by increasing the abstraction of water from the juxtamedullary nephron (presumably the descending limb), in agreement with the prediction of recent passive models of the urinary concentrating mechanism. However, the concentration of urea in fluid from the descending limb after urea infusion was high (261 plus or minus 31 mM) and the difference in solium concentration between descending limb fluid and vasa recta was small and statistically insignificant.
J P Pennell, V Sanjana, N R Frey, R L Jamison
The effects of anemia upon the ventilatory responses to transient and steady-state hypoxia were studied in unanesthetized goats. Responses to transient hypoxia (inhalation of several breaths of nitrogen) were considered to reflect peripheral chemoreceptor and non-chemoreceptor influences of hypoxia upon ventilatory control. In all goats, severe anemia (hemoglobin 3.1-4.8 g/100ml) markedly heightened the responses to transient hypoxia (from a mean of 0.27 to a mean of 0.75 liter/min/percent fall in SaO2). This phenomenon was substantially reversed by alpha-adrenergic blockade (phenoxybenzamine, 5 mg/kg). In contrast, the ventilatory responses to steady-state hypoxia were unaffected by severe anemia. These data suggest that severe anemia enhances the peripheral chemoreceptor-mediated response to hypoxia through a mechanism involving the alpha-adrenergic system. It also appears that a ventilatory depressant effect of hypoxia which is not mediated by the peripheral chemoreceptors is also enhanced by severe anemia, thereby preventing an increase in the steady-state ventilatory response to hypoxia. Finally, experiments involving variation in oxygen affinity of hemoglobin suggested that O2 tension rather than O2 availability in arterial blood is the major determinant of peripheral chemoreceptor activity.
T V Santiago, N H Edelman, A P Fishman
The purpose of this study was to examine the hepatocellular extraction of taurocholate and to determine the kinetic characteristics of the uptake process. The uptake of taurocholate by the liver of the intact dog was studied by the multiple-indicator dilution method. 51Cr-labeled red blood cells (a vascular indicator), 125I-labeled albumin (an extravascular reference), and [14C]taurocholate were injected into the portal vein. Different doses of unlabeled taurocholate were included in the injection mixture. Hepatic venous dilution curves were obtained. As a consequence of the hepatic uptake, the outflow recovery of [14C]taurocholate was much reduced when compared to that of albumin, but its recovery increased with increasing doses of taurocholate, suggesting a progressive saturation of the uptake process. The analysis of the dilution curves fitted a three-compartment model system well and no return of the extracted taurocholate to the extracellular space could be detected. The initial space of distribution of taurocholate was 1.22 plus or minus 0.12 (SD) times greater than that of albumin. Analysis of the data for uptake was consistent with Michaelis-Menten kinetics. The calculated initial maximal velocity of uptake (Vmax) was 4.53 mumol times s--1 times 100 g of liver--1 and the dose yielding half-maximal velocity (DK) was 7.11 mumol times 100 g of liver--1. These results are consistent with the hypothesis that the uptake of taurocholate is carrier-mediated. The maximal vilocity of uptake was about six times the known maximal capacity of biliary secretion of taurocholate in the dog.
The content of alpha-1-antitrypsin in the serum, alveolar lavage fluid, and alveolar macrophages of smokers and nonsmokers was studied. Bronchoalveolar lavage was used to obtain alveolar fluid and macrophages from normal volunteers, and alpha-1-antitrypsin and albumin were measured using the electroimmunodiffusion technique. The serum level of inhibitor was not different between the two groups, while the total lavage fliud content of alpha-1-antitrypsin was increased in the smokers. The level of alpha-1-antitrypsin was also significantly greater (P less than 0.001) in the alveolar macrophages of the smokers suggesting the possibility of chronically increased alveolar levels in the cigarette smoker as a possible protective mechanism against proteolysis.
G N Olsen, J O Harris, J R Castle, R H Waldman, H J Karmgard
The effects of several putative mediators of inflammatory responses on the cyclic nucleotide content of mononuclear leukocytes from human peripheral blood were investigated. Incubation of mononuclear cells with 100 muM serotonin (a maximally effective concentration) for 5 min caused a five- to eightfold increase in their content of guanosine 3',5'-monophosphate (cAMP)...
J A Sandler, R I Clyman, V C Manganiello, M Vaughan