Gas-liquid chromatography of nucleosides. Derivatization and chromatography.

The aims of this investigation were to establish the optimum reaction conditions for silylation of nucleotides with bis(trimethylsilyl)trifluoroacetamide (BSTFA) and to investigate the chromatographic properties of the following nucleosides: adenosine, guanosine, cytidine, thymine, inosine, xanthosine, and uridine. Closed tube silylations were performed with a 1000 molar excess of BSTFA at 25, 75, 120, 150, and 175 degrees for 15, 30, 60, 120 and 240 min. The optimal time and temperature for derivatization were found to be 150 degrees and 15 min. Using these reaction conditions, samples were then silylated with 50, 100, 200, 500, and 1000 molar excess of BSTFA; a molar excess of 225 was best. The stability of the nucleoside derivatives on standing at room temperature for 1-7 days was investigated. Quantitative gas-liquid chromatography of trimethylsilyl (TMS) nucleosides can be performed if samples are analyzed within 48 h, after which time the relative weight response for the nucleosides decreased somewhat. Chromatographic column studies were made using various liquid phases and supports. With methylsiloxanes as the liquid phase, Supelcoport as support was found to be superior. Resolution of TMS guanosine from TMS cytidine was attempted at different column lengths using 3% (w/w) SE-30 on Supelcoport, different loadings (%, w/w) of SE-30 on Supelcoport, and different polarity liquid phases, 4% (w/w) OV-11 or 3% (w/w) OV-17 or 4% (w/w) Dexsil-300 on Supelcoport. A complete separation of the six ribonucleosides including guanosine and cytidine was obtained with 1 m x 4 mm I.D. glass columns of 4% (w/w) OV-11 and 3% (w/w) OV-17 on 100-120 mesh Supelcoport. It was observed that with 2'-deoxycytidine, one obtains the TMS cytosine peak (retention temperature 150 degrees) plus another peak with a retention temperature of 120 degrees, under all derivatization conditions with BSTFA and bis(trimethylsilyl)-acetamide. Similar formation of bases from other 2'-deoxyribonucleosides occurred when the molar excess of BSTFA was greater than 500. The minimal detectable amounts obtained for all the nucleosides ranged from 5 to 10 ng injected with a signal-to-noise ratio of 3. The relative standard deviations for all nucleosides and deoxynucleosides ranged from 1.2 to 4.8% on different methylsiloxanes on Supelcoport columns.


[A new methods of gastroplasty after total gastrectomy].

The author describes a new procedure which he has studied himself in order to restore alimentary transit after total gastrectomy for carcinoma. This requires raising a jejunal loop using Roux' technique in the retrocolic position and anastomosis to the end of the duodenum below, and end-to-side anastomosis is then made with the end of the esophagus. Finally, the author discusses the advantages of this operation.


Separation of nucleic acid hydrolysis products, purines, pyrimidines, nucleosides, nucleotides, ribonucleic acid hydrolyzates, and mixtures from nucleotide syntheses by column chromatography on amberlite XAD-4.

Amberlite XAD-4 resin has been studied as a support for liquid-solid column chromatography. By coating the resin with triethylammonium bicarbonate, a new and unique separation of nucleic acid components has been achieved. Separations are accomplished with a linear gradient of this buffer from 0.1 to 0.4 M. Separation occurs in the following order: inorganic phosphate, purine or pyrimidine bases, 5'-monophosphates, nucleosides and 5'-diphosphates or 5'-triphosphates; the 2'(3')-monophosphates are eluted after either the 5'mono-, di-or triphosphates. The bases and nucleosides are separated in the order: cytosine, uracil, guanine and adenine. Inorganic phosphate and the nucleotides are eluted in the order: inorganic phosphate 5'-mono, di- and tri-phosphates. Excellent separation of the 5'-monophosphates and the 2'(3')-monophosphates is now possible. In each series of 5'-mono-, di- and tri-phosphates or 2'(3')-monophosphates, the elution order is generally cytidine, uridine, guanosine and adenosine. By use of water instead of coating the resin with triethylammonium bicarbonate, the nucleotides and inorganic phosphate are found in the void volume; adenine is eluted very slowly, whereas adenosine is not eluted. Adenosine is eluted only with ethanol-water (1:3). The method is advantageous in that the recovery is quantitative, the buffer is easily removed, the capacity of the column is large (35 mugmoles pergram of resin), flow-rates are high, the time required is short and separations of combinations of inorganic phosphate, bases, nucleosides and nucleotides are now possible that previously could not be accomplished.


Gas-liquid chromatographic determination of tienilic acid (SKF 62.698) in human plasma and urine.

A gas-liquid chromatographic method is described for the determination of tienilic acid (SKF 62.698), a diuretic with uricosuric properties, in human  plasma and urine. The method, which is based on the methylation of the compound, is rapid, specific and sensitive. The lowest level accurately determined is about 50 ng/ml in plasma and 1 mug/ml in urine. The first results from a human volunteer are given.


[Duodeno-colic fistula due to cancer of the right colon].

The author presents a case of duodeno-colic fistula revealing carcinoma of the right hepatic flexure showing the difficulty of surgery. A right hemi-colectomy enlarged to the duodenum and pancreas is the ideal operation from a curative point of view, but rarely possible owing to the age of the patient and the constitutional background. The risk of breakdown of duodenal sutures, which is sometimes technically difficult after colonic removal, is far from negligible. The results of by-pass operations, with or without exclusion of the fistula, are poor and uncertain.


Glycerolpropylsilane bonded phases in the steric exclusion chromatography of biological macromolecules.

Glycerolpropylsilane bonded phases have been found to control the adsorption and/or denaturation of proteins and nucleic acids on controlled porosity glass supports. The bonded-phase thickness is 18-19A while the amount of glycerol moiety varies from 80 to 150 mumoles/g depending on support pore diameter. It has been demonstrated that carbohydrate bonded supports may be used in the chromatography of proteins, nucleic acids, and polysaccharides.


The determination of BHT in resmethrin by high-pressure liquid chromatography.

A method was developed to determine the amount of butylated hydroxy toluene in resmethrin and resmethrin formulations by high-pressure liquid chromatography.


Pyrolysis GLC identification of food and drug ingredients. I. Saccharin.

Pyrolysis gas-liquid chromatography, used in the qualitative analysis of saccharin, has provided reproducible pyrograms which permit direct characterization of these sweetners in a variety of commerical saccharin tablets and diet beverages. This method was used also to identify the salt, after conversion to the free imide, in a commercial multivitamin product. The tablet analysis required isolation by solvent extraction. Analyses were carried out on quantities ranging from 1 to 100 mug. The sensitivity of the method could be increased by addition of a pure sample of the compound sought to the unknown.


Separation of 5'-mono-, di- and triphosphates of 1-beta-D-arabinofuranosylcytosine and of 1-beta-D-arabinofuranosyluracil by thin-layer and high pressure liquid chromatography.

The separation of all the 5'-mono-, di- and triphosphates of ara-C and ara-U by thin-layer (TLC) and high pressure liquid chromatography (HPLC) is described. Whereas for the TLC two different systems are necessary, HPLC allows a rapid separation of all the nucleotides with high resolution, reproducibility, and sensitivity within 15 minutes. An example of an HPLC separation of ara-C and ara-U nucleotides derived from a biological sample is presented.


Rapid, quantitative determination of tetrahydrocannabinol in marihuana by gas chromatography.

Using a simple injection adapter of our own design, the tetrahydrocannabinol content of marlhuana was determined by gas chromatography. The injection method obviates the need for preliminary extractions and allows analyses to be conducted on milligram quantities in less than thirty minutes.


Structure determination of polyunsaturated fatty acids by gas chromatography-mass spectrometry-- a comparison of fragmentation patterns of various derivatives.

GC-MS has been applied to the characterization of mixtures of polyunsaturated fatty acids by way of their isopropylidene, alkylboronate, methyl ether, and trimethylsilyl ether derivatives. In our studies we found a convenient and efficient method for the oxidation of mixtures of polyunsaturated fatty acid methyl esters by osmium tetroxide. The resulting polyhdroxy compounds are easily converted into apolar derivatives for gas chromatographic separation. The mass spectra of these individual compounds were recorded as to their usefulness for complete structure elucidation. Only the trimethylsilyl ethers proved to yield all information necessary for determination of chain length as well as numbers and positions of double bonds of polyunsaturated fatty acids.


Separation of benefin and trifluralin by gas chromatography using a liquid crystal column.

Benefin (N-[n-butyl]-N-ethyl-2,6-dinitro-alpha, alpha, alpha-trifluoro-p-toluidine) and trifluralin (2,6-dinitro-N,N-di-n-propyl-alpha, alpha, alpha-trifluoro-p-toluidine) (Figure 1) are isomeric preemergent herbicides (1,2). As they possess different activities, their use necessitates the development of a routine analytical method which differentiates between them. The separation of these isomers by gas-liquid chromatography (GLC) using conventional column packings has proved extremely difficult. Two partly successful separations have been previously reported to date. A partial separation was obtained using a Durapak-Carbowax 400/Poracil C column (3) and a complete separation using a column packed with a monomolecular layer of Carbowax 20M on Chromosorb W (4). Unfortunately, this latter column has a very limited life and the analysis time is 25 minutes. This paper reports a rapid and reproducible separation of benefin and trifluralin using a liquid crystal stationary phase.


Intestinal metabolism of estrogens.

Estrogen metabolism in the human intestine was studied in two ways. Firstly, by measuring the excretion of 12 estrogens in pooled human late pregnancy feces before and during the administration of ampicillin (2 g/day). Secondly, by administering 5.4 and 20 mg of 16alpha-hydroxyestrone orally to two postmenopausal women and analyzing the estrogens in simultaneously drawn portal and peripheral venous blood samples at time intervals from 0 to 150 min after steroid administration. The majority of the estrogens in normal pregnancy feces were unconjugated. The amounts of estradiol, estreon and 16-epiestriol excreted, relative to the principal estrogen estriol, were greater than in pregnancy bile or urine and 16alpha-hydroxyestrone, an important biliary estrogen, was only present in trace amounts. Considerable quantities of 15alpha-hydroxyestradiol-17beta were also found. Ampicillin administration, which decreases intestinal bacterial steroid metabolism, caused a huge increase in the fecal excretion of conjugated estrogens. In particular it caused very striking increases in the excretion of both unconjugated and conjugated, estriol, 15alpha-hydroxyestrone, 15alpha-hydroxyestradiol and 2-methoxyestrone. These findings emphasize the active role played by the intestinal microflora in estrogen metabolism under normal conditions. Administration of 16alpha-hydroxyestrone resulted in increases in portal venous unconjugated and conjugated 16alpha-hydroxyestrone, 16-oxoestradiol-17beta, 15alpha-hydroxyestrone, 16-epiestriol and conjugated estriol levels. The most significant finding in both subjects was the large increase in portal venous unconjugated 15alpha-hydroxyestrone. This would suggest that the human intestine (or intestinal contents) has the ability to carry out the transformation, 16alpha-hydroxyestrone leads to 15alpha-hydroxyestrone. Increases in the same estrogens were found in peripheral plasma, with the increase in conjugated estriol occurring in peripheral blood before it was seen in portal blood. The largest elevations in peripheral plasma values were seen in unconjugated estriol and conjugated 16alpha-hydroxyestrone in the subject who received the 20 mg dose and in unconjugated 16alpha-hydroxyestrone and 16-oxoestradiol-17beta in the subject who had the 5.4 mg dose. The intestinal and enterohepatic metabolism of estrogens is discussed in relation to these findings.


Endocrine studies in a phenotypic girl with XY gonadal agenesis.

An 18-year-old phenotypic girl with XY gonadal agenesis had endocrine studies. The patient had a small amount of sexual hair, a slightly enlarged clitoris, normal labia and no posterior fusion. Her vagina was of normal length, but no cervix was palpable. At laparoscopy, no gonads, uterus or fallopian tubes were identified. The buccal smear was chromatin negative and the karyotype was 46XY. The 24 h LH plasma pattern was qualitatively similar to that found in normal late pubertal subjects, but the values were in the castrate range. The 24h mean plasma concentrations of testosterone and dihydrotestosterone were 16.4 ng/dl and 14.2 ng/dl, respectively. Urinary estrogen excretion was 15 and 19 mug/day. These clinical and endocrine findings suggest that the patient was a genetic male in whom Mullerian regression occurred normally; however, the testes probably ceased functioning early in gestation preventing normal development of the Wolffian system. This set of events resulted in agenesis of the gonaductal system and female external genitalia. The patient represents the second example of this variation of male pseudohermaphroditism associated with XY gonadal agenesis.


Serum thyroglobulin in inhabitants of an endemic goiter region of New Guinea.

Serum thyroglobulin Tg(RIA) was studied in 161 residents of the Nomane region of New Guinea. The incidence of antithyroglobulin antibodies (ATA) and antimicrosomal antibodies (anti-M) was also studied to assess the role of autoimmunity in goitrogenesis. ATA were detected in only 4 sera; these sera were excluded from the study since ATA interfere in the Tg radioimmunoassay. Anti-M antibodies were undetectable in all of 105 subjects whose sera were analyzed. Mean (+/-se) serum Tg(RIA) in the 84 nongoitrous (NG) subjects was 163.1 +/- 17.2 ng/ml, whereas that in 77 goitrous (G) subjects was 208.1 +/- 19.8 ng/ml; both values were much higher (P is less than 0.001) than that (5.1 +/- 0.49 ng/ml) in normal Californian subjects. The mean serum thyroid stimulating hormone (TSH) in the NG group (12.1 +/- 2.1 muU/ml) was not statistically different from that in the G group (10.1 +/- 1.5 muU/ml). Serum Tg(RIA) correlated positively with log TSH (r equals 0.38 P is less than 0.001). Intrigued by the finding of goiters in some residents of an endemic goiter region and its absence in other residents exposed to the same environmental factors, we evaluated the possibility that the thyroid glands of subjects who develop goiters may be inherently more responsive to any given level of TSH than those of the inhabitants without goiters. However, the slope of the correlation between serum Tg(RIA) and log TSH was only slightly (0.1 is less than P is greater than .05) higher in G than in the NG group. These studies suggested that factors other than thyroidal responsiveness to TSH must also be important in goiterogenesis of endemic goiter regions. We conclude that 1) serum thyroglobulin is a sensitive parameter of chronic as well as acute thyroidal stimulation; 2) Thyroid autoimmunity and increased thyroidal response to TSH do not explain goiterogenesis in New Guinea and attention should be focused on other possibilities.


Effects of phosphorus supplementation on serum parathyroid hormone and bone morphology in osteoporosis.

The effect of phosphorus (inorganic phosphate) supplementation was studied in seven postmenopausal women with osteoporosis. Prior to supplementation, all chemical parameters studied in serum and urine were normal. Bone density was below the fifth percentile for age in all but one patient, and the percentage of bone surface involved in resorption was higher than normal. During administration of the phosphorus supplement, fasting serum concentrations of calcium and immunoreactive parathyroid hormone showed no significant changes, while serum phosphorus, urinary calcium, and tubular reabsorption of phosphorus decreased. In four patients studied by balance techniques, calcium balance became positive or less negative. Bone-forming surface decreased and bone-resorbing surface increased in all patients. Bone-resorbing surface was highly correlated with total phosphorus intake. Density of the distal radius changed variably, while density of the midradius increased slightly in all patients.


Plasma thyrotropin, thyroxine, and triiodothyronine relationships in man.

The physiologic relationships of plasma TSH, T4 and T3 levels measured every 20 min in seven healthy young men and one healthy young woman have been investigated. A nocturnal TSH surge was observed in all subjects on both nights of the 36-48 h baseline observation period. In males the maximum plasma TSH value occurred at 2300 h. The mean peak TSH level was 2.0 +/- 0.3 (se) muU/ml compared with a mean of 1.3 +/- 0.9 muU/ml for the entire baseline records of the 8 subjects. The effect of iv infusion of 32-1000 mug of somatostatin (SRIF) for 1 1/2-3 h was investigated in four of the male subjects during 2 or 4 consecutive nights following the control period. Temporal relationships between the hormonal fluctuations observed throughout the control period and during the nights of SRIF infusion were investigated using time series analysis and Student's t test. Rapid fluctuations of plasma T4 and T3 concentration were noted, even when corrected for changes in total protein concentration, with an average coefficient of variation of 10% for T3 and 12% for T4. No increment of plasma T4 or T3 followed the nocturnal TSH surge nor were the rapid fluctuations of the thyroid hormones altered by the TSH surge. SRIF infusion commencing at 2300 h suppressed the elevated TSH levels (P is less than 0.01) while similar infusions begun at 2100 h blocked the expected nocturnal TSH rise observed during control periods in male subjects. Plasma T4 and T3 levels were not significantly affected by the administration of SRIF. The relationship of the rapid plasma T4 and T3 variations to postural changes was investigated in four euthyroid male subjects. Serum levels of TSH, T4 and T3 and total protein were determined at 15 min intervals while postural changes were carefully monitored. The ratios of T4 and T3 to total protein were relatively stable (3-4% coefficient of variation) when the subjects were kept in a supine and motionless position. A 50 mug bolus infusion of T4 raised the basal T4 level by only 1-2 mug/dl. The data suggest that short-term fluctuation of plasma T4 and T3 result from changes in protein concentration due to hemodynamic responses to alteration of posture and physical activity and not to pulsatile secretion of T4 and T3.


Hypothyroid-like alterations in testosterone metabolism in anorexia nervosa.

The metabolism of 14C-testosterone was studied in 8 severely underweight young women with anorexia nervosa. The urinary androsterone/etiocholanolone (A/E) ratio was uniformly low, in a range characteristic of hypothyroidism; the patients also showed low plasma concentrations of triiodothyronine (T3). Clinical remission as manifested by weight gain was accompanied by concomitant increases of the A/E ratio and the plasma T3 concentration to or toward normal. The administration of  T3 also resulted in a shift of the A/E ratio toward normal. These data demonstrate that the low plasma T3 concentrations in patients with anorexia nervosa may be related to the development of one of the characteristic biochemical abnormalities found in clinical hypothyroidism, namely a decreased A/E ratio. These data suggest that the "low T3 syndrome" may be associated with biochemical hypothyroidism.


Effect of the antihistaminic agents meclastine and dexchlorpheniramine on the response of human growth hormone to arginine infusion and insulin hypoglycemia.

Histamine is found in most tissues including the central nervous system. Here it reaches the highest concentrations in the hypothalamus and in the median eminence. In order to evaluate the possible role of endogenous histamine in the control of human growth hormone (hGH) secretion, we investigated the effect of two anti-histamine drugs, meclastine and dexchlorpheniramine, on the hGH response to arginine infusion and to insulin hypoglycemia in 30 normal subjects. The oral administration of meclastine for three days or the intravenous infusion of dexchlorpheniramine significantly reduced the hGH response to arginine infusion. Neither drug affected the secretion of hGH following insulin hypoglycemia. These results suggest that histamine is involved in the control of hGH release, at least in response to arginine. Our data are consistent with the finding that histamine stimulates GH release in the baboon (Meyer and Knobil, Endocrinology 80: 163, 1967) and with previous results indicating that the release of hGH in response to different stimuli is subject to different regulatory mechanisms.


Reversal of decreased human adipose tissue lipoprotein lipase and hypertriglyceridemia after treatment of hypothyroidism.

To determine whether adipose tissue lipoprotein lipase (LPL) plays a role in the regulation of triglyceride (TG) metabolism in hypothyroidism, the activity of the enzyme was measured in the subcutaneous adipose tissue of six hypothyroid patients before and during therapy with L-thyroxine. The activity of the activated form of the enzyme, measured as heparin elutable LPL, was lower in hypothyroid patients (1.54 +/- 0.93; mU/10(6) cells; mean +/- SD) than in controls (3.26 +/- 1.49; P less than .02) and increased (163 +/- 89%; P less than .01) with treatment to levels comparable to the controls. The total activity of LPL, measured in ammonium hydroxide extracts of acetone ether tissue powders, was in the low normal range in the hypothyroid patients (0.68 +/- 0.42), but not significantly different from normal (1.10 +/- 0.58) and did not increase significantly (92 +/- 105%), with treatment. Plasma post heparin lipolytic activity (PHLA) was low in hypothyroidism and increased (111 +/- 78%; P less than .05) with treatment. These increases in PHLA correlated with the increases in the activity of heparin elutable LPL (r = .88, P less than .05). In all patients fasting plasma TG levels decreased (-43 +/- 25%; P less than .02) after treatment. Serial determination of heparin elutable LPL activity, PHLA, and plasma TG during L-thyroxine treatment revealed a correlation between the per cent changes in PHLA and heparin elutable LPL activity (r = .68, P less than .05), an inverse correlation between plasma TG levels and heparin elutable LPL (r = -0.53,P less than .05) and no correlation between plasma TG and PHLA (r = -0.05). These results suggest that the low PHLA and hypertriglyceridemia of hypothyroidism are related to low adipose tissue LPL activity. All these parameters return to normal after treatment with L-thyroxine and attainment of euthyroidism.


Thyroid-pituitary feedback during iodine repletion.

The changes in serum triiodothyronine (T3), thyroxine (T4), and thyrotropin (TSH) were measured during iodine repletion in a woman who was severely iodine-deficient because of a congenital iodide-trapping defect. Serum T3 became detectable 12 h after iodine was begun and reached 68 ng/dl, a level within the normal range, by 36 h. It rose progressively during the first 9 days reaching a supra-normal level (200 ng/dl) and then fell slowly to 130 ng/dl. Serum T4 was not detected (less than 1 mug/dl) until the 9th day and then rose to 6.8 mug/dl. Serum TSH fell rapidly during the first 9 days (disappearance rate was 0.17/day); there was a significant negative correlation with serum T3 (P less than 0.01). From the 10th through the 32nd day serum TSH fell more slowly (disappearance rate was 0.05/day) and correlation was with serum T4 (P less than 0.001), not T3. It appears that either T3 or T4 may regulate TSH secretion by direct effects on the thyrotroph, although their intracellular mode of action has not been defined.


Blood pressure fall by angiotensin II antagonist in patients with Bartter's syndrome.

Intravenous infusion of 600 ng/kg/min of 1-sarcosine, 8-isoleucine-angiotensin II, an angiotensin II antagonist, caused a marked blood pressure fall and a decrease in plasma aldosterone in 3 patients with Bartter's syndrome. These results indicate that proximal cause of Bartter's syndrome is an arteriolar hyporesponsiveness to angiotensin II and that this angiotensin II analogue has an antagonist activity on peripheral arterioles as well as adrenal cortex.


Growth hormone, prolactin and chorionic somatomammotropin in normal and molar pregnancy.

Twenty patients with molar pregnancy, ten normal pregnant women and ten healthy non-pregnant women were given 30 g of arginine intravenously. The serum concentration of growth hormone, prolactin and chorionic somatomammotropin (CS) was determined by radioimmunoassay. In addition, serum 17beta-estradiol, estriol and progesterone were also measured. Arginine infusion induced a sharp rise of GH in patients with molar pregnancy and in nonpregnant subjects, but the response in normal pregnancy was blunted. The response of PRL was high in patients with molar pregnancy, blunted in normal pregnancy and very small in nonpregnant subjects. CS did not respond at all to arginine infusion both in normal pregnancy and molar pregnancy. The high response to argine of PRL, normal response of GH and low baseline secretion and no response of CS may be characteristic of molar pregnancy.


Cortisol-induced suppression of plasma testosterone in normal adult males.

The response of plasma testosterone, LH and FSH to oral administration of cortisol or dexamethasone was studied for 26 h in 6 groups of 12 normal adult males. Twenty-four hour treatment with cortisol (60 mg at 1000 h, followed by 30 mg at 2 h intervals) or with dexamethasone (6 mg at 1000 h, followed by 3 mg at 6 h intervals) was without effect on testosterone during the day but suppressed the nocturnal rise completely. LH and FSH did not decrease at any time during the day and showed a highly significant increase during the second part of the night. Short-term treatment (1000-1600 h, 1600-2200 h or 2400 h-0800 h) with cortisol was less effective in suppressing the nocturnal rise in testosterone. It was concluded that administration of cortisol leading to plasma levels as seen under treatment with ACTH suppresses testosterone by abolishing or flattening the nocturnal rise. This effect was not mediated by changes in LH or FSH. Our data suggest that the ACTH-induced suppression of testosterone is due to an action of cortisol.


FSH-secreting pituitary adenomas: stimulation and suppression studies in two patients.

Two patients with pituitary adenomas, partial hypopituitarism and abnormally high serum concentrations of FSH are described. In neither patient was FSH increased after the administration of LH releasing hormone and in one patient tested, there was no FSH response to clomiphene citrate. The decrease in serum FSH in response to high concentrations of testosterone (greater than 1100 ng/dl) was less than in normal men. These results indicate partial autonomy of FSH producing adenomas. Since the elevated serum FSH produced no obvious clinical symptoms, it may be undetected unless serum FSH concentrations are measured routinely in patients with pituitary tumors.


Effects of dopamine infusion on pituitary hormone secretion in humans.

Dopamine, infused at a rate of 4 mug/kg/min for 3-4 h unaccompanied by any significant changes in cardiovascular dynamics, induced a prompt and sustained suppression of circulating prolactin (PRL) levels in normal men and women as well as in patients with hyperprolactinemia. At the discontinuation of the infusion, there was a marked rebound in PRL levels in normal subjects and a rapid return to basal levels in hyperprolactinemic patients. Dopamine infusion also induced a significant fall in LH levels in the normal subjects with a marked rebound in LH levels following the infusion. No significant changes in GH, TSH, and FSH levels were observed. These data indicate that in man a dopaminergic mechanism ixists in the regulation of PRL secretion and that dopamine also exerts an inhibitory effect of LH release.


A radioimmunoassay for measurement of 3,3'-L-diiodothyronine (T2).

A sensitive, specific and reproducible radioimmunoassay (RIA) for measurement of 3,3'-L-diiodothyoride (T2) in concentrated ethanol extracts of serum and amniotic fluid is described.  The T2 binding antiserum was prepared aby immunization of rabbits with T2-human serum albumin conjugate.  Of various thyroid hormone derivatives tested, only 3-L-monoiodothyonine cross-reacted significantly with T2 binding sites on the antiserum...


17-Deoxygenation: a new pathway of cortisol metabolism. Isolation of 17-deoxycortolonic acids.

The existence of a 17-deoxylation pathway in the metabolism of cortisol has been established by isolation of two 17-deoxy metabolites of cortisol. They have been identified as the 20alpha and 20beta-hydroxy isomers of 3alpha,20-dihydroxy, 11-oxo-5beta-pregnan-21-oic acids by comparison of the nuclear magnetic resonance spectra (nmr) and high resolution mass spectra of their methyl esters with authentic samples and confirmed by reverse isotope dilution.


Effect of sulpiride-induced hyperprolactinemia on serum testosterone response to HCG in normal men.

In six normal volunteers hyperprolactinemia was induced by sulpiride (150 mg/day) for 10 days. Both before and during sulpiride hCG was injected; the higher testosterone response to hCG, when PRL levels were enhanced, suggests a possible stimulatory role of PRL on Leydig cells.


Measurement of glucose turnover in the human newborn with glucose-1-13C.

Systemic glucose production rates were measured in 6 normal newborn infants by dilution of glucose-1-13C tracer according to the prime constant-rate infusion technique. Glucose production rates were 4.4 +/- 0.39 mg/kg. min. (Mean +/- S.D.) in 4 infants at 2 hours of age, and were 3.83 and 3.86 mg/kg. min. in 2 infants at 1 day of age. Systemic glucose production accounts for 50% of the substrate utilized for oxidative metabolism in newborn infants.


Competition by 24,25-dihydroxycholecalciferol in the competitive protein binding radioassay of 25-hydroxycalciferol.

24,25-dihydroxycholecalciferol (24,25-(OH)2D3) is equipotent to 25-hydroxycholecalciferol in the displacement of 3H25-OHD3 from rat serum binding sites, and is extracted by the ethanol procedure recommended in non-chromatographic competitive protein binding radioassays for 25-OHD. Serum 24,25-(OH)2D content was measured following 24,25-(OH)2D3 isolation from lipid extracts by liquid-gel partition chromatography. Since normal serum 24,25-(OH)2D concentration is appreciable, non-chromatographic 25-OHD assays most probably overestimate serum 25-OHD levels as a result of their recognition of 24,25-(OH)2D3.


Effect of delay on culture of urine.

One hundred random urine specimens which were submitted for culture were planted using a calibrated loop within 2 h of collection and 2 and 4 h later after standing at room temperature. Colonies were counted after an 18- to 24-h incubation at 37 degrees C. Fifteen of the specimens demonstrated increases in counts exceeding 1 X log10; four increased from less than 10(5)/ml to greater than 10(5)/ml (three by the 4-h culture and a fourth by the 6-h culture), supporting the concept that delays of greater than 2 h in inoculating cultures may produce results which could cause errors in diagnosis.


Simplified method for efficient intravascular inoculation of chicken embryos.

The simple syringe-stabilizer unit described in this note provides a means for rapid intravascular inoculation of embryonated chicken eggs with minimal embryonic death from vascular trauma.


Differential recovery of Streptococcus mutans from various mitis-salivarius agar preparations.

Recoveries of Streptococcus mutans from human dental plaque were lower when plated on mitis-salivarius agar obtained from Baltimore Biological Laboratories as compared with mitis-salivarius agar obtained from Difco Laboratories. However, no difference in recoveries of established laboratory strains of S. mutans was observed between these two agar preparations.


Evaluation of a hypertonic sucrose medium for the detection of fungi in blood cultures.

A comparison was made between biphasic brain heart infusion medium without and with 15% sucrose in vented blood culture bottles. A higher recovery rate (P less than 0.01) of fungi was noted in the medium without sucrose.


Virological and serological studies of Venezuelan equine encephalomyelitis in humans.

During the 1971 epidemic of Venezuelan equine encephalomyelitis (VEE) in south Texas, 203 suspect VEE cases were evaluated by the Center for Disease Control. Sixty-seven were confirmed as cases of VEE. Laboratory confirmation was accomplished by isolation of VEE virus from a serum specimen taken during the acute illness in 50 (75%) of the confirmed cases. Serological confirmation was obtained in 17 cases (25%). Virus isolations were most often obtained from sera collected during the first 3 days of illness. Peak serum virus titers (algebraic mean, 10(5-7) suckling mouse intracranial 50% lethal doses [SMICLD50] per ml) occurred on day 2 of illness. One-half of the sera from which virus was isolated contained at least 10(5) SMICLD50/ml, which has been shown to be sufficient to infect some vector mosquitoes. Blood from 13 virus-positive VEE cases was obtained 1 and 11 months after illness. Hemagglutination-inhibiting, complement-fixing, and neutralizing antibodies were formed by all 13 patients 1 month after illness. Hemagglutination-inhibiting antibody titers were essentially unchanged 11 months after illness. Complement-fixing antibody was undetectable 11 months after illness in 23% of cases and was detectable at dilutions of 1:8 or 1:6 in 77%. Neutralizing antibody (measured by log neutralization index) was not detectable 1 year after illness in one person (8%); titers had declined from 1.0 to 2.0 in 46%, were unchanged in 39%, and were not tested in one person (8%). No evidence of intrafamilial spread of VEE virus was obtained in either of two illness and antibody surveys. A randomized household illness and antibody survey of 681 Port Isabel residents revealed an inapparent infection ratio of 1:11 and an overall antibody prevalence of 3.2%.


Comparison of three methods for grouping streptococci.

Two new methods for serological grouping of beta-hemolytic streptococci, the nitrous acid extraction procedure of El Kholy et al. and the slide agglutination method of Christensen et al., were compared with the Lancefield hot-hydrochloric acid extraction method in classifying 92 strains of groups A, B, C, and G. The nitrous acid extraction method was easily performed, specific, and sensitive when highly potent antisera were used. For the Christensen method these highly potent antisera had to be diluted to avoid cross-reactions between groups A and C and groups B and G, respectively. A few strains, most of them group B, could not be grouped by the latter method. Using these three grouping methods, two sets of commercial sera were compared with the more potent sera supplied by R. C. Lancefield. The low antibody content of these commercial sera, especially anti-group B and G sera, contributed to the inferior results obtained in some of the grouping reactions.


Evaluation of commercial systems for the identification of clinical yeast isolates.

The Analytab Products Inc. (API), Micro-Drop (MD), and Uni-Yeast-Tek (UYT) systems for the presumptive identification of common clinical yeast isolates were compared with the oxidation-fermentation (OF) and a conventional procedure. With 229 coded isolates, the identification accuracies were API 94, MD 83, OF 82, and UYT 99%. The API system required the greatest technical ability. The MD materials were prone to malfunction. OF media, if incubated beyond 14 days, gave an accuracy of 87%, but this offered no advantage over the conventional procedure. The UYT system was the easiest to use.


Comparison of human fecal and serum parvo-like viruses.

Parvovirus-like particles found in the sera of two blood donors had the size and appearance on electron microscopy of a virus (B19) found in the serum of a blood donor by Cossart et al. (1975), and those of a virus found in the feces of a normal subject. Antibody to these viruses was detected by immune electron microscopy and immunoelectro-osmophoresis in the sera of 50 children aged 10 to 15 years. Of these, 36% had antibody to the fecal virus, 36% had antibody to B19, and 54% had antibody to the two other serum viruses. The results of these tests suggest that serologically the three serum viruses were similar to one another, but that the fecal virus was distinct. The two blood donors had nonspecific symptoms at the time of viremia. Both donors had developed immunoglobulin M antibody to the virus when tested 3.5 and 4.5 weeks later, but no viruses were detected in the feces or urine.


Growth of Streptococcus mutans on various selective media.

The ability of Streptococcus mutans to grow on mitis-salivarius (MS) agar, MC agar, mitis-sucrose-bacitracin (MSB), BCY agar, and MM10 sucrose agar was studied. Batch cultures of S. mutans serotype a demonstrated no growth on MSB agar. Certain serotype d and g strains did not grow on MC agar. The yield for most strains of other serotypes on these selective media was lower compared with that on MS agar. The number of total colony-forming units on BCY and MM10 sucrose agar was similar to the blood agar results. Similar data were obtained when fermenter-grown strains, harvested in the middle or the end of the logarithmic growth phase, were used for inoculation of the various media. Enumeration of S. mutans from plaque samples plated on MC and MSB agar yielded about 75% of the counts obtained on MS or the nonselective medium. When the proportions of S. mutans were expressed as a percentage of the total cultivable flora, the selective media (MC and MSB agar) showed approximately 10% lower values than the MS, BCY, and MM10 sucrose agar.


Identification of streptococcal groups A,B,C, and G by slide co-agglutination of antibody-sensitized protein A-containing staphylococci.

A total of 98.7% of streptococci, groups A,B,C, and G, isolated from various sources was correctly identified by the co-agglutination technique. The active components in this technique are protein A-containing staphylococci coated with antibodies specific for group A,B,C, and G streptococci. A suspension of streptococci belonging to one of these four groups co-agglutinates with the antibody-sensitized staphylococci specific for this group. The technique is extremely rapid and simple and requires no special equipment. It should therefore be a valuable alternative to other techniques used in the grouping of streptococci and is shown here to be as reliable as the Lancefield technique.


Enhancement of bactericidal capacity of alveolar macrophages by human alveolar lining material.

In vivo studies have shown a major role for the alveolar macrophage in the killing of inhaled bacteria. This contrasted with earlier work which showed a preservation of phagocytic properties but a loss of bactericidal capacity when alveolar macrophages were studied in vitro. Recently, alveolar lining material (ALM) from rats has been shown to enhance the in vitro bactericidal capacity of alveolar macrophages from homologous animals against Staphylococcus aureus. Utilizing a similar system, we have confirmed that rat alveolar macrophages do not kill S. aureus in vitro unless the bacteria have been incubated with rat ALM (R-ALM) before phagocytosis. In addition, human ALM (H-ALM) from 7 of 11 patients assayed showed an enhancement of bactericidal capacity by rat alveolar macrophages which was not significantly different from the results utilizing R-ALM. H-ALM from the other four patients gave results which differed significantly from results with H-ALM from the first seven patients and R-ALM (P less than 0.001). Preliminary results suggest that the factor enhancing the bactericidal capacity of rat alveolar macrophages is present in the lipid fraction of the ALM. Further characterization of the ALM is warranted in an effort to explain the enhancement of the bactericidal capacity of alveolar macrophages by most, but not all, H-ALM tested.


The effect of indomethacin and other anti-inflammatory drugs on the renin-angiotensin system.

The administration of two different doses of indomethacin, 9 and 18 mg/kg, to two different groups of rabbits was followed 6 h later by a significant decrease in plasma renin activity, and these levels were not increased by hemorrhage. The administration of 2 mg/kg of indomethacin did not alter the basal levels of plasma renin activity, but it was effective in diminishing the peripheral increase of renin produced by hemorrhage. Similar effects were obtained in other groups of rabbits treated with 9 mg/kg of meclofenamate or 18 mg or aspirin. The lowering effect of indomethacin on plasma renin activity is not specifically related to hemorrhage because it also prevented the increase in plasma renin activity elicited by 5 mg/kg of furosemide. Further studies showed that indomethacin did not exert any significant effect in vivo on the plasma level of renin substrate or on the generation of angiotensin from normal plasma by exogenous renin. And indomethacin did not interfere with the binding capacity of anti-angiotensin I for angiotensin I in the radioimmunoassay reaction or with the in vitro formation of angiotensin from hog renin-nephrectomized rabbit plasma reaction. The results thus indicate that the lowering effect of indomethacin on plasma renin activity is due to the interference with renal renin release. That this effect may be related to the blockade of prostaglandin synthesis is suggested by the similar effect exhibited by other blockers of prostaglandin synthesis.


De novo synthesis of purine nucleotides in human peripheral blood leukocytes. Excessive activity of the pathway in hypoxanthine-guanine phosphoribosyltransferase deficiency.

Human peripheral blood leukocytes were studied for the presence and the regulatory properties of the pathway of de novo synthesis of purine nucleotides. The cells were found to incorporate the labeled precursors formate and glycine into purines. The rate of [14C]-formate incorporation was decreased by several compounds known to inhibit purine synthesis by affecting the activity by glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase, the first committed enzyme in the pathway, either through decreasing the availability of PRPP, a substrate for this enzyme, or through exerting inhibition on this enzyme. PRPP availability in the leukocyte was found to be limiting for purine synthesis. Increased PRPP availability resulting from activation of PRPP synthetase by increasing inorganic phosphate (Pi) concentration caused acceleration of purine synthesis. On the other hand, no clear-cut evidence was obtained for the availability of ribose-5-phosphate in the leukocyte being rate limiting at physiological extracellular Pi concentration for PRPP generation, and thus for purine synthesis. However, the addition of methylene blue, which accelerates the oxidative pentose shunt that produces ribose-5-phosphate, resulted in acceleration of PRPP generation and of purine synthesis only when PRPP synthetase was largely activated at high Pi concentration. These results may be taken to suggest that ribose-5-phosphate availability is indeed not limiting for PRPP generation under physiological conditions. Purine synthesis de novo was accelerated more than 13-fold in the leukocytes of two gouty patients affected with partial deficiency of hypoxanthine-guanine phosphoribosyltransferase, but was normal in the leukocytes of an obligate heterozygote for this enzyme abnormality. The results domonstrate in peripheral human leukocytes the presence of the complete pathway of de novo synthesis of purine nucleotides and the manifestation in these cells of the biochemical consequences of hypoxanthine-guanine phosphoribosyltransferase deficiency, i.e., increased availability of PRPP and acceleration of purine synthesis de novo. The results indicate the usefulness of leukocytes as a model tissue for the study of purine metabolism in man.


A micropuncture study of the effect of parathyroid hormone on renal bicarbonate reabsorption.

Renal micropuncture and clearance experiments were carried out in rats to study the effect of parathyroid hormone (PTH) on renal tubular HCO-/3 reabsorption. The rats were studied during an initial period of parathyroid deficiency (acute thyroidparathyroidectomy, TPTX) and during infusion of large amounts of bovine PTH. Under normal acid-base conditions, PTH administration to TPTX rats caused a significant rise in proximal tubular fluid HCO-/3 concentration (TFHCO-/3), a decrease in fluid reabsorption, and a fall in proximal HCO-/3 reabsorption from 94.0 to 88.2% (P less than 0.01). In control experiments with mannitol infusion, a comparable reduction in proximal fluid reabsorption occurred without any significant effect on intraluminal HCO-/3 concentration. During acute intravenous HCO-/3 loading, PTH inhibited proximal HCO-/3 reabsorption. However, no change in whole kidney HCO-/3 reabsorption was observed in these experiments or in the animals studied under normal acid-base conditions. The findings are consistent with the view that PTH inhibits proximal tubular HCO-/3 reabsorption with normal or high filtered loads of HCO-/3, but distal segments of the nephron are able to reabsorb the excess delivered from the proximal tubule. Measurements of urinary ammonium and titratable acid indicate that net acid excretion (NH+/4 + TA -- HCO-/3) increases significantly after PTH administration. These results do not provide support for the view that PTH excess causes metabolic acidosis by reducing renal acid excretion.


Characteristics of stimulation of H+ transport by aldosterone in turtle urinary bladder.

Aldosterone stimulates not only Na+ absorption but also urinary acidification. In this investigation the effects of aldosterone on H+ transport are examined in vitro in turtle bladder, a urinary membrane in which several of the factors controlling H+ transport have been defined. H+ transport was increased in bladder halves  exposed to aldosterone compared to control halves. Stimulation of H+ secretion was observed as early as 1 h after addition of aldosterone and occurred before that of Na+ transport. In bladders depleted of endogenous substrate addition of glucose increased H+ transport more in aldosterone-treated halves (10.0+/-1.3 nmol/min) than in control halves (6.8+/-2.3). Addition of pyruvate failed to increase H+ transport (--0.3+/-0.7) in control halves but caused significant increments (2.4+/-0.5) in aldosterone-treated halves. In aldosterone-treated bladders glucose caused larger increments (16.5+/-2.7) in H+ transport than pyruvate (9.3+/-2.0) when halves of the same bladders were compared. Na+ transport, however, was equally increased by the two substrates. Despite the differences in time course and substrate requirements between the stimulation of H+ and Na+ transport, both increases were abolished by actinomycin-D. To examine the effect of aldosterone on the force of the H+ pump, protonmotive force, the pH gradient that would nullify the transport rate was determined with and without aldosterone. Aldosterone did not alter protonmotive force but significantly increased the slope of the H+ transport rate on the applied pH gradient. It is concluded that aldosterone stimulates H+ transport independently of Na+ transport. It increases the responsiveness of the transport rate to glucose and to a lesser extent pyruvate, an effect probably secondary to the increased transport rate. Equivalent circuit analysis indicates that aldosterone facilitates the flow of protons through the active transport pathway but does not increase the force of the pump.


The relative roles of calcium, phosphorus, and parathyroid hormone in glucose- and tolbutamide-mediated insulin release.

The relative contributions of Ca++, phosphorus, and parathyroid hormone (PTH) on insulin secretion were evaluated in three groups of dogs. Dogs were studied with glucose infusions (group I) or standard intravenous glucose tolerance tests (IVGTT) (group II) before and after the development of diet-induced hypophosphatemia. Mean serum phosphorus levels for both groups fell from 4.1 to 1.1 mg/100 ml. Animals in group I demonstrated a fall in glucose disappearance rates (Kg) from 5.3+/-0.6% min to 3.5+/-0.5% after induction of hypophosphatemia (P less than 0.001). Mean insulin response was significantly greater in the hypophosphatemic animals than in controls in this group. In group II animals, mean insulin areas obtained during the IVGTT increased from 1,426+/-223 to 2,561+/-141 muU/ml/60 min after induction of hypophosphatemia, and were unaffected by Ca++ or PTH administration. Ca++ administration, but not hypophosphatemia or PTH infusion, increased significantly the mean insulin response to tolbutamide. Secondary hyperparathyroidism was induced by dietary manipulation in four dogs (group III). Mean PTH values increased from 71.4+/-2.1 to 3,012+/-372 pg/ml (P less than 0.001). Mean insulin response to an IVGTT was similar to group III animals, but increased from 1,352+/-128 to 1,894+/-360 muU/ml/60 min after the excessive dietary phosphorus was reduced for 3 mo, and plasma phosphorus fell from 3.2+/-0.1 to 2.8+/-0.3 mg/100 ml. PTH values decreased to 647+/-53 pg/ml. The insulin response to tolbutamide was comparable to that in group II animals, but increased significantly after calcium administration. Immunoreactive insulin disappearance rates were unaffected by hypophosphatemia or diet-induced secondary hyperparathyroidism. These data demonstrate that hypophosphatemia is associated with an augmented glucose-stimulated insulin release, without any effect on tolbutamide-stimulated insulin release. Hypercalcemia produces an augmented tolbutamide-stimulated insulin release with no apparent effect on glucose-stimulated insulin release. Finally, PTH does not appear to be an insulin antagonist and has no apparent effect on either glucose- or tolbutamide-stimulated insulin release in animals with dietary-induced secondary hyperparathyroidism.


The effects of sodium ricinoleate on small intestinal function and structure.

The mechanism of hydroxy fatty acid-induced secretion was investigated in perfused hamster small intestine in vivo. Sodium ricinoleate at an 8-mM concentration resulted in not only secretion of water and sodium, but an increase in intestinal clearance of inulin and a 16,000 mol wt dextran as well. A concentration of ricinoleate (2 mM) which did not affect water transport, however, did not alter intestinal permeability. Ricinoleate-induced intestinal secretion was also accompanied by increased mucosal cell exfoliation as measured by the appearance of DNA in the perfusate and by apparent injury to epithelial cell membranes as judged by measurement of sucrase activity and phospholipid in cell-free aliquots of luminal fluid. Light and electron microscopic studies demonstrated substantial mucosal architectural changes with 8 mM ricinoleate with villus shortening and injury to epithelial cells at the villus tips. In contrast, cholera enterotoxin caused marked secretion of sodium and water, presumably by a cyclic AMP mechanism, but did not alter inulin clearance or enhance DNA or sucrase appearance in the lumen. These studies suggest that at least a component of ricinoleate-induced intestinal secretion is related to structural alterations of the mucosa.


Effects of antihistamines on the lung vascular response to histamine in unanesthetized sheep. Diphenhydramine prevention of pulmonary edema and increased permeability.

To see whether antihistamines could prevent and reverse histamine-induced pulmonary edema and increased lung vascular permeability, we compared the effects of a 4-h intravenous infusion of 4 mug/kg per min histamine phosphate on pulmonary hemodynamics, lung lymph flow, lymph and plasma protein content, arterial blood gases, hematocrit, and lung water with the effects of an identical histamine infusion given during an infusion of diphenhydramine or metiamide on the same variables in unanesthetized sheep. Histamine caused lymph flow to increase from 6.0+/-0.5 to 27.0+/-5.5 (SEM) ml/h (P less than 0.05), lymph; plasma globulin concentration ratio to increase from 0.62+/-0.01 to 0.67+/-0.02 (P less than 0.05), left atrial pressure to fall from 1+/-1 to -3+/-1 cm H2O (P less than 0.05), and lung lymph clearance of eight protein fractions ranging from 36 to 96 A molecular radius to increase significantly. Histamine also caused increases in lung water, pulmonary vascular resistance, arterial PCO2, pH, and hematocrit, and decreases in cardiac output and arterial PO2. Diphenhydramine (3 mg/kg before histamine followed by 1.5 mg/kg per h intravenous infusion) completely prevented the histamine effect on hematocrit, lung lymph flow, lymph protein clearance, and lung water content, and reduced histamine effects on arterial blood gases and pH. 6 mg/kg diphenhydramine given at the peak histamine response caused lymph flow and lymph: plasma protein concentration ratios to fall. Metiamide (10 mg/kg per h) did not affect the histamine lymph response. We conclude that diphenhydramine can prevent histamine-induced pulmonary edema and can prevent and reverse increased lung vascular permeability caused by histamine, and that histamine effects on lung vascular permeability are H1 actions.


A role for adenosine deaminase in human monocyte maturation.

The occurrence of a deficiency of adenosine deaminase (ADA) activity in some patients with severe combined immunodeficiency suggests a possible relationship between the activity of ADA and the aberration of the immune system. To help delineate the function of ADA in the immune response we have examined its role in monocyte maturation. When incubated in vitro, peripheral blood monocytes transformed, within 3 days, to macrophagea as assessed by phase-contrast microscopy and an increase in the specific activity of the lysosomal enzyme acid phosphatase. The specific activity of ADA increased as much as ninefold, reaching a peak after the 1st day in culture, while the activities of other enzymes involved in the purine salvage pathway were not altered. Sucrose density ultracentrifugation of extracts prepared immediately after the isolation of monocytes revealed the presence of two forms of ADA with molecular weights of approximately 30,000 and 110,000. The increase in ADA specific activity during monocyte cultivation correlated with an increase in the activity of the smaller molecular species. A specific inhibitor ADA, erythro-9-(2-hydroxy-3-nonyl) adenine, prevented the increase in acid phosphatase activity, as well as the morphological changes associated with the monocyte maturation. These data suggest a role for ADA in monocyte to macrophage maturation. In view of the central role of macrophages in immune function, this observation may relate to the association of combined immunodeficiency and a deficiency of this enzyme.


Free fatty acid oxidation by forearm muscle at rest, and evidence for an intramuscular lipid pool in the human forearm.

The objects of these experiments were to determine to what extent oleic acid, removed from plasma by forearm muscles, was oxidized immediately, and to search for evidence of an intramuscular lipid pool which may be composed to triglycerides synthesized from plasma free fatty acids and which may supply substantial portions of lipid substrates for oxidation by muscle. To these ends (1-14C]oleic acid was infused at constant rate into the brachial artery of seven healthy young men at rest in the postabsorptive state. Results were: (1) muscle respiratory quotient (0.76) implied that about 80% of the oxygen consumed was for the oxidation of lipid. (2) Muscle free fatty acid uptake, had it been oxidized directly, could account for more than 100% of observed oxygen uptake. (3) There was a lag of at least 30 min before 14CO2 appeared in forearm venous blood. (4) 14CO2 release from forearm muscle tended to reach an apparent plateau after 3 h of infusion of [14C]oleic acid. (5) During the time of plateau 14CO2 release, oleic acid extracted from plasma could account for only 20% of oxygen consumption; most of the oleic acid taken up was not oxidized directly. (6) 14CO2 release persisted at a high level during the 1-3 h follow-up period after the infusion ended. (7) Neither the delay in initial appearance of 14CO2 nor its continued release after cessation of infusion was due to delay and distribution in a forearm CO2 pool, since intra-arterial infusion of NaH14CO3 in additional subjects demonstrated much more rapid distribution of 14CO2 in the forearm. Results show that most, if not all, free fatty acids taken up by resting muscle are not oxidized directly, but probably enter an intramuscular pool which has a slow turnover during resting metabolism and is the immediate source of oxidized lipid substrate.


Heterogeneity of antibody response to human platelet transfusion.

To study the antibody response to human platelet transfusions, nine thrombocytopenia patients with bone marrow failure were given 6 U (3X10(11)) of random platelet concentrates twice a week. Before transfusion, none of the patients had preexisting antibodies detectable with lymphocytotoxicity, platelet aggregation, or capillary leukoagglutination techniques. After receiving 18-78 U of platelets, they became refractory to further transfusions of random platelets and alloantibodies were detectable. Two patterns of antibody response could be identified. In three patients, the sera were not lymphocytotoxic with a panel of standard cells in which all the known HLA antigens in the first and second series were represented at least once. Yet, they caused platelet aggregation with 30, 24, and 60%, respectively, of a donor population studied. The aggregating activities were inhibited by antihuman IgG but not by antihuman IgA or antihuman IgM antiserum. The aggregating antibodies could be absorbed out with donor platelets but not lymphocytes or granulocytes. Antibodies from two of these patients aggregated platelets of their respective siblings matched for both HLA haplotypes. Transfusion of platelets from these two siblings did not increase  the platelet count while platelets obtained from aggregation-negative donors did. The sera from the remaining six patients were lymphocytotoxic with 15-100% of the panel of standard cells. They also had aggregating antibodies, which could be absorbed out by both platelets and lymphocytes, suggesting that they were HLA antibodies. These data suggest that the development of platelet-specific antibodies may play an important role in the immunological rejection of isologous platelets, and should be considered in the selection of donors for patients who are refractory to platelets from random donors.


Localization of cardiac myosin-specific antibody in myocardial infarction.

Specific localization of purified antibody against cardiac myosin has been demonstrated in areas of altered myocardial membrane permeability after experimental myocardial infarction. Intravenously administered radioiodine-labeled antimyosin was selectively localized in infarcted myocardium of seven dogs 24 h after coronary occlusion. The mean ratio (+/-SE) of antimyosin antibody in infarcted to normal myocardium in the center of the infarct was 4.2+/-0.4 for endocardial and 2.9+/-0.3 for epicardial layers. By utilizing (Fab')2 fragments of antimyosin obtained by pepsin digestion of purified antibody, the ratio of uptake was increased in eight dogs to 6.1+/-0.6 in the endocardial and 3.3+/-0.4 in the epicardial layers at the infarct center 24 h after occlusion. These ratios were further increased in the infarct center to 13.8+/-1.2 in the endocardial and 7.3+/-0.8 in the epicardial layers when eight dogs were sacrificed 72 h after coronary occlusion. The specificity of antimyosin (Fab')2 localization in infarcted myocardium was demonstrated in four dogs by simultaneous intravenous administration of 125I-labeled antimyosin (Fab')2 and 131I-labeled normal rabbit gamma globulin (Fab')2. Nonspecific trapping of normal rabbit IgG (Fab')2 was observed to be about 38% of total antimyosin (Fab')2 uptake in the central zone of infarction. Regional blood flow was related to antimyosin (Fab')2 uptake in infarcted myocardium by utilizing simultaneous administration of 85Sr-labeled microspheres. An inverse exponential relationship between antimyosin (Fab')2 uptake and regional blood flow was observed (r=0.85). The specific localization of antimyosin antibody or its (Fab')2 components in infarcted myocardium suggests a conceptually new approach to myocardial infarct localization and sizing.


Iron deficiency in the rat. Physiological and biochemical studies of muscle dysfunction.

Work performance on a treadmill has been evaluated in normal and iron-deficient rats. Anemia was removed as a variable by adjusting the hemoglobin of all animals to the same concentration. At a hemoglobin compatible with normal work performance, iron-deficient animals showed a marked impairment of running ability as compared to control animals. Iron therapy corrected the disability within 4 days. Concentrations of the cytochrome pigments and myoglobin, and rates of oxidative phosphorylation with pyruvate-malate, succinate, and alpha-glycerophosphate as substrates were all reduced in mitochondrial preparations from skeletal muscle of iron-deficient rats, but only the rate of phosphorylation with alpha-glycerophosphate as substrate increased significantly and in parallel with the recovery in work performance of the iron-deficient rats treated with iron dextran.


The relationship between right duct lymph flow and extravascular lung water in dogs given alpha-naphthylthiourea.

The relationship between right duct lymph flow and extravascular lung water was studied in 3 normal dogs and 15 dogs with pulmonary edema induced by alpha-naphthylthiourea (ANTU). Right duct lymph was collected in a pouch created by ligating jugular, subclavian, and brachiocephalic veins. Extravascular lung water was measured in vivo by double indicator dilution and post-mortem by weighting lungs before and after drying. Cardiac output, pulmonary artery and pulmonary artery wedge pressures, and the concentration of protein and electrolytes in plasma and right duct lymph were determined. Eight lungs were examined by light and electron microscopy. There was a direct relationship between right duct lymph flow (RDLF in milliters per hour per gram dry lung) and extravascular lung water (Qwl in milliliters per gram dry lung) which was best described by the equation RDLF=0.75-0.26 Qwl+0.03 (Qwl).2 Dogs with severe ANTU-induced edema had extensive lung capillary endothelial destruction but only mild interstitial swelling and no visible damage to type I alveolar epithelial cells. Cardiac output, pulmonary artery and wedge pressures, and protein and electrolyte concentrations did not correlate with either extravascular water or right duct flow. Thus, in ANTU-induced pulmonary edema right duct lymph flow was directly related to extravascular lung water with the highest flows occurring with severe edema. The absence of a rapid increase in lymph flow with small increases in extravascular water may be due to early sequestration of fluid in the alveolar space. Hemodynamic changes did not account for changes in lung water or lymph flow. The pulmonary interstitial factors relating increased extravascular water to lymph drainage remain to be determined.


Regulation of pancreatic and gallbladder functions by intraluminal fatty acids and bile acids in man.

The effects of intraduodenal glycerol, fatty acid (FA) chain length and FA loads, and bile acid (BA) concentrations on pancreatic and gallbladder function were investigated in 31 healthy volunteers by a perfusion method. FA absorption rates in the duodenum and proximal jejunum were measured simultaneously. Pancreatic and gallbladder responses were augmented by increasing FA chain length and FA loads until the "maximal" secretory capacity of the pancreas and gallbladder emptying was attained. Glycerol had no effect. Raising BA concentrations above the critical micellar concentration accelerated FA absorption rates but decreased the magnitude of pancreatic and gallbladder responses to FA. Higher BA concentrations exerted an opposite effect, slowing FA absorption and increasing pancreatic and gallbladder responses. Indeed, a significant, inverse correlation was found between FA absorption and pancreatic and gallbladder responses to FA, suggesting a relationship between the length of intestine exposed to FA and the amount of cholecystokinin (and/or other neurohormonal factors) released, which stimulates pancreatic secretion and gallbladder contraction.


Anion transport processes in the mammalian superficial proximal straight tubule.

The experiments reported in this paper were designed to evaluate some of the characteristics of anion transport processes during fluid absorption from superficial proximal straight tubules isolated from rabbit kidney. We measured net chemical C1- flux during fluid absorption from tubules perfused and bathed with Krebs-Ringer buffers containing 113.6 mM C1-, 10 mM acetate, and 25 mM HCO-/3 at pH 7.4; assessed the effects of carbonic anhydrase inhibitors on net fluid absorption in the presence and absence of CO2; and evaluated the influx and efflux coefficients for [14C]-acetate transport at 37degreesC, at 21degreesC, and in the presence of carbonic anhydrase inhibitors. The experimental data shown that, for this nephron segment, net C1- flux accompanies approximately 27.5% of net Na+ absorption; and net C1- absorption may be accounted for by a passive transport process, primarily diffusional in nature. Fluid absorption in this nephron segment is reduced 40-60% by carbonic anhydrase inhibitors, but only when the tubules are exposed to 95% O2-5% CO2 rather than 100% O2. Thus, it seems probably that approximately half of Na+ absorption in these tubules may be rationalized in terms of a carbonic anhydrase-dependent CO2 hydration process. In addition, there may occur in these isolated proximal tubules an acetazolamide-insensitive moiety of HCO-/3 absorption comparable to that observed for proximal tubules in vivo. Finally, we provide evidence that net efflux of luminal acetate is due to metabolic energy-dependent processes other than CO2 hydration and may, under appropriate conditions, account for approximately one-fourth of net Na+ absorption.


The transport of vitamin D and its 25-hydroxy metabolite in human plasma. Isolation and partial characterization of vitamin D and 25-hydroxyvitamin D binding protein.

This study reports the isolation and partial characterization of vitamin D and 25-hydroxyvitamin D binding protein (DBP), the specific transport protein for vitamin D and its 25-hydroxy metabolite in human plasma. DBP was labeled by the addition of a tracer amount of 3H-labeled 25-OH-D3 to the original plasma used for protein fractionation. Previous experiments have shown that such 25-OH-D3 added in vitro binds to the same protein normally responsible for the transport of endogenous 25-OH-D and of vitamin D. The isolation of human DBP was achieved by an extensive sequence of procedures which resulted in a final yield of only approximately 4 mg of purified DBP from a starting volume of 34 liters of plasma. Purified DBP was homogeneous in the analytical ultracentrifuge and showed a single band of protein on analytical polyacrylamide gel electrophoresis. DBP had a sedimentation constant of 3.49s and a mol wt of approximately 52,000. The molecular weight was assessed by sedimentation equilibrium analysis and also by sodium dodecyl sulfate-disc-gel electrophoresis and by gel filtration on a standardized column of Sephadex G-150. The amino acid composition of DBP was determined and was generally consistent with the estimated extinction coefficient (E1cm1% at 280 nm) of about 9.1. The isoelectric point of DBP was estimated as 4.8 from isoelectric focusing experiments. Direct study of the binding capacity of the purified DBP for added 25-OH-D3 showed that the isolated DBP had a high affinity for 25-OH-D3, with an apparent maximum binding capacity of one molecule of 25-OH-D3 per molecule of protein.


Purification and properties of sulfite oxidase from human liver.

Sulfite oxidase has been purified to near homogeneity from human liver. Properties of the molecule have been investigated and compared to those of the rat liver enzyme which has been isolated in a pure form. Both proteins exist as dimeric molecules with one molybdenum and one cytochrome b5-type heme per sub-unit. The human enzyme has a slightly larger subunit molecular weight (61,100 vs. 57,200 daltons) and is a more negatively charged molecule. Decreased reactivity of the human enzyme with various electron acceptors suggests the presence of nonfunctional molybdenum centers in a portion of the molecules isolated. Human liver sulfite oxidase cross-reacts strongly with antibody prepared against the rat liver enzyme. Human enzyme activity is precipitated by antibody at concentrations about twofold greater than required for comparable complexation of rat sulfite oxidase.


Human sulfite oxidase deficiency. Characterization of the molecular defect in a multicomponent system.

Frozen liver tissue from an individual identified several years ago as sulfite oxidase deficient has been reexamined in light of new knowledge which has been obtained regarding the enzyme. It has been established that hepatic molybdenum levels and xanthine oxidase activity were within normal values and comparable to those observed in control samples preserved from the original study along with the deficient tissue sample. The ability of the patient's liver to synthesize the specific molybdenum cofactor required for activation of de-molybdo sulfite oxidase also appears to have been unimpaired. Using an antibody preparation directed against rat liver sulfite oxidase which also inhibits and precipitates the human enzyme, it has been determined that cross-reacting material with determinants recognized by inhibiting antibodies is absent in the liver sample from the deficient patient. Immunodiffusion experiments gave strong precipitin bands against the control liver extracts, but showed no detectable precipitin reaction between the deficient liver extract and the antibody preparation. The relationship of these findings to a second patient recently identified as sulfite oxidase deficient and to an animal model of the disease are discussed.


Stimulation of ammonia production and excretion in the rabbit by inorganic phosphate. Study of control mechanisms.

The purpose of this study was to clarify the mechanism (s) responsible for regulation of ammonia production and excretion in the rabbit. The normally low ammonia excretion rate during acute metabolic acidosis was stimulated acutely and increased approximately ninefold after infusion of sodium phosphate, but remained low if sodium sulphate or Tris was substituted for phosphate. Ammonia production was increased significantly by phosphate in rabbit renal cortex slices and in isolated renal cortex mitochondria. In isolated mitochondria, mersalyl, an inhibitor of both the phosphate/hydroxyl and phosphate/dicarboxylate mitochondrial carriers, inhibited the phosphate-induced stimulation, indicating that phosphate must enter the mitochondrion for stimulation. A malate/phosphate exchange seemed to be involved since N-ethylmaleimide, an inhibitor of the phosphate/hydroxyl exchange, did not inhibit phosphate-stimulated ammonia production, whereas there was inhibition by 2-n-butylmalonate, a competitive inhibitor of the dicarboxylate carrier. Phosphate itself was not essential since malonate stimulated ammoniagenesis in the absence of added phosphate. Similarly, citrate stimulated ammoniagenesis in isolated mitochondria in the absence of inorganic phosphate provided that it induced L-malate exit on the citrate transporter associated with inhibition of citrate oxidation by fluoroacetate. Similar results were also seen in mitochondria from rat renal cortex. A fall in mitochondrial alpha-ketoglutarate level resulted in an increase in ammonia production. This could be achieved directly with malonate or indirectly via L-malate exit. Simultaneous measurements of glutamate showed that the rate of ammonia production was reciprocally related to the glutamate content. We conclude that phosphate-induced stimulation of ammoniagenesis in the rabbit kidney is mediated by removal of glutamate, the feedback inhibitor of phosphate-dependent glutaminase. Glutamate removal is linked to phosphate-induced dicarboxylate exit across the mitochondrial membrane.


Development of glucagon sensitivity in neonatal rat liver.

The ontogenesis of the hepatic glucagon-sensitive adenylate cyclase system has been studied in the rat. With a partially purified liver membrane preparation, fetal adenylate cyclase was less responsive to glucagon than the enzyme from neonatal or adult livers. Similar results were obtained in gently prepared liver homogenates, suggesting that destruction of essential components of the fetal liver membrane did not account for the relative unresponsiveness of the adenylate cyclase enzyme to glucagon. Investigation of other factors that might account for diminished fetal hepatic responsiveness to glucagon indicate (a) minimal glucagon degradation by fetal membranes relative to 8-day or adult tissue; and (b) available adenylate cyclase enzyme, as suggested by a 13-fold increase over basal cyclic AMP formation with NaF in fetal liver membranes. These results indicate that neither enhanced glucagon degradation nor adenylate cyclase enzyme deficiency accounts for the relative insensitivity of the fetal hepatic adenylate cyclase system to glucagon. In early neonatal life, hepatic adenylate cyclase responsiveness to glucagon rapidly developed and was maximal 6 days after birth. These changes were closely paralleled by a fivefold increase in glucagon binding and the kinetically determined Vmax for cyclic AMP formation. These observations suggest that (a) fetal hepatic unresponsiveness to glucagon may be explained by a limited number of glucagon receptor sites; (b) during the neonatal period, the development of glucagon binding is expressed primarily as an increase in adenylate cyclase Vmax; (c) the ontogenesis of hepatic responsiveness to glucagon may be important in the resolution of neonatal hypoglycemia.


On the influence of the natriuretic factor from patients with chronic uremia on the bioelectric properties and sodium transport of the isolated mammalian collecting tubule.

A gel filtration fraction of urine from patients with chronic renal disease (natriuretic factor) has been shown previously to cause natriuresis in rats and to inhibit sodium transport in the isolated toad bladder. The effect of this fraction on transtubular potential difference and sodium transport was examined on the isolated perfused cortical collecting tubule of the rabbit. A rapid inhibition of potential difference from -22.5 mV to -12 mV (P less than 0.001) was observed when the fraction was applied to the peritubular surface. This effect was accompanied by a decrease in net sodium flux from 6.29 to 3.21 pmol/cm per s (P less than 0.001). Unidirectional fluxes using isotopic sodium revealed that the inhibition of net sodium transport was due to a decrease in flux from the lumen to the peritubular surface, i.e., an inhibition of active sodium transport. There was no change in sodium flux in the reverse direction. These changes were all rapidly reversed by removal of the fraction from the peritubular surface. The addition of the fraction to the lumen had no effect on potential difference or net sodium flux. Control studies using the same fraction from the urine of normal subjects had no effect on any of the parameters studies. Where both a uremic and a normal fraction were sequentially applied to the peritubular surface of the same tubule, inhibition of potential difference was obtained only with the former. In the light of evidence implicating the collecting duct fraction from normal animals, the data are consistent with the view that the natriuretic factor may be biologically important in the regulation of sodium balance via it's regulatory role in active sodium transport in the collecting tubule.


Type II hyperprolinemia. Delta1-pyrroline-5-carboxylic acid dehydrogenase deficiency in cultured skin fibroblasts and circulating lymphocytes.

Type II hyperprolinemia is an inherited abnormality in amino acid metabolism characterized by elevated plasma proline concentrations, iminoglycinuria, and the urinary excretion of delta1-pyrroline compounds. To define the enzymologic defect of this biochemical disorder, we developed a specific, sensitive radioisotopic assay for the proline degradative enzyme delta1-pyrroline-5-carboxylic acid dehydrogenase. Using this assay, we have shown an absence of delta1-pyrroline-5-carboxylic acid dehydrogenase activity in the cultured fibroblasts from three patients with type II hyperprolinemia. We confirmed this result on cultured cells by demonstrating a similar absence of delta1-pyrroline-5-carboxylic acid dehydrogenase activity in extracts prepared from the peripheral leukocytes of these patients. Additionally, we found significantly decreased levels of delta1-pyrroline-5-carboxylic acid dehydrogenase activity in the leukocyte extracts from five obligate heterozygotes for type II hyperprolinemia. We also demonstrated a reduction in leukocyte delta1-pyrroline-5-carboxylic acid dehydrogenase activity in three successive generations of a family. These results prove that an absence of delta1-pyrroline-5-carboxylic acid dehydrogenase is the enzymologic defect in type II hyperprolinemia and that this defect is inherited in an autosomal recessive fashion.


Urea secretion by the straight segment of the proximal tubule.

Studies utilizing in vitro microperfusion were designed to examine whether urea is actively or passively transported across superficial and juxtamedullary straight segments of rabbit proximal tubules. With perfusate and bath solutions containing 1 mM urea and electrolytes similar to normal plasma, the efflux (lumen-to-bath) isotopic permeability (X 10(-5) cm s-1) of superficial segments was 1.37 +/- 0.16 and of juxtamedullary segments was 2.14 +/- 0.20. In the same tubules, the influx (bath-to-lumen) isotopic permeability was 3.70 +/- 0.35 in superficial segments and 4.75 +/- 0.37 in juxtamedullary segments. Despite net water movement in the opposite direction (0.5 nl mm-1 min-1), the influx rate was significantly higher than the efflux rate of urea in both groups. With a low perfusion rate (2 nl/min) and equivalent specific activities of [14C]urea in bath and perfusate, the collected-to-perfused ratio of [14C]urea, corrected for volume marker change, was 1.07 +/- 0.01 in superficial and 1.09 +/- 0.01 in juxtamedullary nephrons, thus indicating net secretion in both segments. In separate studies urea influx was inhibited by hypothermia (decrease from 37 degrees to 28 degrees C), by phloretin (0.1 mM in bath), by cyanide (1 mM), but not by probenecid (0.2 mM). In each case the inhibition was highly significant and reversible. These data suggest that urea is actively secreted by the straight segments of both the superficial and juxtamedullary proximal tubules. These segments may, therefore, contribute significantly to the high urea concentration found at the bend of Henle's loop by micropuncture.


Cytotoxic activity of rheumatoid and normal lymphocytes against allogeneic and autologous synovial cells in vitro.

The possibility that lymphocytes from patients with rheumatoid arthritis (RA) might be sensitized to RA synovial cell antigens was investigated with a 51Cr release cytotoxicity assay. Peripheral blood lymphocytes from rheumatoid and normal donors were tested for cytotoxic activity against their own synovial cells and against allogeneic rheumatoid and nonrhemuatoid synovial cells. In the allogeneic studies, the degree of cytotoxicity was significantly influenced by the age in culture (passage number) of the synovial target cells (P less than 0.001). When the passage number of the target cells was considered in the analysis, rheumatoid lymphocytes were found to have greater cytotoxic activity than normal lymphocytes against young cultures (low passage number) of both RA and non-RA synovial cells (P = 0.0042). Differences in susceptibility to lysis between RA and non-RA synovial cells were more susceptible to both RA and normal lymphocyte-induced lysis than were non-RA synovial cells (P = 0.0048). No evidence of cytotoxicity was detected when lymphocytes from nine RA patients and two osteoarthritis patients were reacted against their own synovial cells. Although the data demonstrated an increased cytotoxic activity of peripheral blood lymphocytes from some RA patients against allogeneic synovial cells, the fact that this reactivity was seen against both non-RA and RA synovial cells and was not demonstrated against autologous synovial cells argues against the presence of an immunospecific response of RA lymphocytes to RA synovial cell antigens.


Studies on the mechanisms of food-stimulated gastric acid secretion in normal human subjects.

Liquid test meals were infused into the stomach and acid secretion was measured by intragastric titration at pH 5.0 Acid secretion after 500 or 750-ml sodium chloride meals was two to three times higher than basal secretion rates and was equivalent to 25-30% of the peak acid output in response to histamine. Since these meals did not cause a rise in serum gastrin concentration, it is assumed that they stimulate acid secretion by causing distention of the body and fundus of the stomach. Compared with this distention stimulus, glucose meals had no effect on acid secretion and fat-inhibited acid secretion; however, both glucose and fat caused an increase in serum gastrin concentration. Amino acids caused a much greater increase in serum gastrin concentration and enhanced acid secretion above that noted with distention alone. In contrast, albumin did not enhance the serum gastrin concentration or stimulate acid secretion to a statistically significant extent. There was a close correlation between the rise in serum gastrin concentration and rate of acid secretion after different test meals when average results for each test meal were plotted. However, there was a poor correlation between acid secretion and serum gastrin concentration when the responses of the individual subjects with a given test meal were compared. Our interpretations are: (a) Distention is an important stimulant of the acid-secretory response to a meal, and this is not mediated by gastrin release. (b) Gastrin is one but probably not the only mediator of the chemical phase of acid secretion, i.e., acid secretion noted with amino acids that cannot be explained by distention. (c) Glucose and fat also release gastrin; however, with glucose the rise in serum gastrin is too small and too transient to enhance acid secretion, and fat probably releases unmeasured inhibitors that overwhelm the effect of gastrin on acid secretion. (d) Albumin is not a stimulant of acid secretion.


Hematologic and clinical responses in patients with sickle cell anemia after chronic extracorporeal red cell carbamylation.

In eight patients with sickle cell anemia, weekly extracorporeal carbamylation of about 20% of the circulating red cell mass was carried out for 2 yr or longer. At each visit, a mean of 1.3+/-0.2 mol of cyanate were incorporated per mole of hemoglobin in the carbamylated erythrocytes. Within 3 mo, a stable level of about 35-50% of the circulating erythrocytes was carbamylated. This quantity and degree of hemoglobin carbamylation produced a decrease in mean whole blood P50 from 33 to 26 mm Hg. During the first 3 mo of carbamylation, the mean hemoglobin increased from 6.4 to 9.1 g/100 ml, while mean absolute reticulocytes decreased by 58% and circulating irreversibly sickled erythrocytes decreased by 65%. The mean red cell life span increased from 13 days before treatment to 21.6 days after 3 mo of carbamylation. Beyond the 3rd mo of carbamylation, blood P50, hemoglobin, and reticulocytes remained quite stable. No toxic effects of extracorporeal carbamylation of erythrocytes were noted. The capacity of blood to release oxygen at 30 mm Hg PO2 increased from 4.3 to 5.0 cm3/100 ml blood during carbamylation. The overall frequency of severe painful crises decreased by about 80% during carbamylation. Before carbamylation, 34% of the crises were induced by a concomitant illness, usually an infection. During carbamylation, the incidence of induced crises decreased 50% while spontaneous crises virtually disappeared. The marked improvements in hematologic parameters and the decreased frequency of severe painful crises observed during this study offer sufficient promise to warrant further exploration, hopefully using more efficient techniques, of the clinical efficacy of extracorporeal erythrocyte carbamylation in sickle cell anemia.


Adenosine metabolism in phytohemagglutinin-stimulated human lymphocytes.

The association of a human genetic deficiency of adenosine deaminase activity with combined immunodeficiency prompted a study of the effects of adenosine and of inhibition of adenosine deaminase activity on human lymphocyte transformation and a detailed study of adenosine metabolism throughout phytohemagglutinin-induced blastogenesis. The adenosine deaminase inhibitor, coformycin, at a concentration that inhibited adenosine deaminase activity more than 95%, or 50 muM adenosine, did not prevent blastogenesis by criteria of morphology or thymidine incorporation into acid-precipitable material. The combination of coformycin and adenosine, however, substantially reduced both the viable cell count and the incorporation of thymidine into DNA in phytohemagglutinin-stimulated lymphocytes. Incubation of lymphocytes with phytohemagglutinin for 72 h produced a 12-fold increase in the rate of deamination and a 6-fold increase in phosphorylation of adenosine by intact lymphocytes. There was no change in the apparent affinity for adenosine with either deamination or phosphorylation. The increased rates of metabolism, apparent as early as 3 h after addition of mitogen, may be due to increased entry of the nucleoside into stimulated lymphocytes. Increased adenosine metabolism was not due to changes in total enzyme activity; after 72 h in culture, the ratios of specific activities in extracts of stimulated to unstimulated lymphocytes were essentially unchanged for adenosine kinase, 0.92, and decreased for adenosine deaminase, 0.44. As much as 38% of the initial lymphocyte adenosine deaminase activity accumulated extracellularly after a 72-h culture with phytohemagglutinin. In phytohemagglutinin-stimulated lymphocytes, the principal route of adenosine metabolism was phosphorylation at less than 5 muM adenosine, and deamination at concentrations greater than 5 muM. In unstimulated lymphocytes, deamination was the principal route of adenosine metabolism over the range of adenosine concentrations studied (0.5-250 muM). These studies demonstrate the dependence of both the unstimulated and stimulated lymphocyte on adenosine and may account for the observed sensitivity of mitogen-stimulated lymphocytes to the toxic effects of exogenously supplied adenosine in the presence of the adenosine deaminase inhibitor coformycin. A single case of immunodeficiency disease has been reported in association with purine nucleoside phosphorylase deficiency. The catabolism of guanosine was also found to be enhanced in stimulated normal lymphocytes; phosphorolysis of guanosine to guanine by intact lymphocytes increased six fold after 72-h culture with phytohemagglutinin. The specific activity of purine nucleoside phosphorylase in extracts, with guanosine as substrate, was essentially the same in stimulated and unstimulated lymphocytes after 72 h of culture.


A micropuncture study of the early phase of acute urate nephropathy.

The early pathophysiological changes in acute urate nephropathy were investigated in a rat model using micropuncture, clearance, and morphologic methods. Plasma urate was increased from 1.2 +/- 0.6 to 20.1 +/- 3.1 mg/100 ml (P less than 0.001). Urinary urate rose from 24.3 +/- 5.1 to 142.2 +/- 21.0 mg/100 ml (P less than 0.001). Renal plasma flow and glomerular filtration rate fell to 17 and 14% of control values, respectively, and urine flow rate decreased from 11.3 +/- 4.8 to 4.2 +/- 2.2 mul/min (all P less than 0.005) Superficial nephron filtration rate fell less than that of the whole kidney (70 vs. 86%). Both proximal and distal tubular pressures were increased from 10.6 to 26.1 mm Hg and from 7.2 to 24.7 mm Hg, respectively (P less than 0.005). Efferent arteriolar and peritubular capillary pressures were increased twofold. Vascular resistance beyond the peritubular capillaries increased from 4.8 X 10(9) to 21.6 X 10(9) dynes s/cm5. Extensive deposits of uric acid and urate were found in the tubular system and vasa recti from the corticomedullary junction to the tip of the papilla. It is concluded from these experiments that not only tubular obstruction in the collecting ducts, but also obstruction of the distal renal vasculature, are the primary early pathogenetic events in acute urate nephropathy.


The cytotoxicity of leukocytes and lymphocytes from patients with rheumatoid arthritis for synovial cells.

Unseparated peripheral blood leukocytes obtained from patients with rheumatoid arthritis (RA) were cytotoxic for synovial cells. The cytotoxic reactions produced by RA leukocytes were more frequent and of greater magnitude than cytotoxicity induced by leukocytes from normal persons and patients with other diseases, primarily connective tissue diseases. Furthermore, the cytotoxic activity of RA leukocytes was greater for RA synovial cells than for nonrheumatoid synovial cells, in contrast to the cytotoxicity of other leukocytes, which did not discriminate between synovial cells according to their origin. Tests with purified lymphocytes showed that the cytotoxicity of unseparated leukocytes directed against RA synovial cells was due to lymphocyte cytotoxicity. These data are consistent with the possibility that sensitized lymphocytes from patients with RA recognize a distinctive antigen present on rheumatoid synovial cells.


Cytological and enzymological characterization of adult human adipocyte precursors in culture.

Cell strains were derived from the stromal-vascular fraction of human omental adipose tissue and grown in culture. Since the purpose of this study was to isolate adipocyte precursors from adults, the cells were obtained from nonobese patients 40-60 yr of age. After treatment of adipose tissue with collagenase, mature adipocytes were separated from stromal-vascular fraction cells, and cell strains of the latter replicated in culture with a doubling time of 40-60 h. They were initially fusiform; upon reaching monolayer confluency, they accumulated lipid and became rounder. Skin fibroblasts from the same patients and grown under the same culture conditions remained fusiform and did not accumulate lipid. The stromal-vascular fraction cells of adipose tissue may be fibroblasts with the potential to become adipocyte precursors. Subcellular preparations of the cells grown from the stromal-vascular fraction revealed lipoprotein lipase activity (characterized by such properties as inhibition by 1 M NaCl) that was not detectable in skin fibroblasts. The overall specific activity of the enzymes that catalyze triglyceride synthesis was 15 times higher and that of fatty acid synthetase was 2 times higher in the cells cultured from the stromal-vascular fraction. The difference was significant in each case. Conversely, when isolated mature adipocytes were cultured, they lost considerable lipid and acquired morphological characteristics similar to those of stromal-vascular fraction cells. Thus, adipose tissue stromal-vascular fraction cells acquire in culture many of the morphological and enzymological characteristics of mature fat cells.


Neutrophil kinetics in man.

A method has been developed for measuring neutrophil cellularity in normal human bone marrow, in which the neutrophil-erythroid ratio was determined from marrow sections and marrow normoblasts were estimated by the erythron iron turnover. Neutrophil maturational categories, defined by morphologic criteria, were supported by autoradiographs of marrow flashed-labeled with 3H-thymidine. Correction for multiple counting error was empirically derived by counting serial sections through cells of each maturational category. The normal neutrophil-erythroid ratio in 13 normal human subjects was 1.5 +/- 0.07. The mean number of normoblasts in the same subjects was estimated to be 5.07 +/- 0.84 X 10(9) cells/kg. Total marrow neutrophils (X 10(9) cells/kg) were 7.70 +/- 1.20, the postmitotic pool (metamyelocytes, bands, and segmented forms) was 5.59 +/- 0.90 and the mitotic pool (promyelocytes + myelocytes) was 2.11 +/- 0.36. Marrow neutrophil ("total") production has been determined from the number of neutrophils comprising the postmitotic marrow pool divided by their transit time Transit time was derived from the appearance in circulating neutrophils of injected 3H-thymidine. The postmitotic pool comprised 5.59 +/- 0.90 X 10(9) neutrophils/kg, and the transit time was 6.60 +/- 0.03 days. From these data marrow neutrophil production was calculated to be 0.85 X 10(9) cells/kg per day. Effective production, measured as the turnover of circulating neutrophils labeled with 3H-thymidine, was 0.87 +/- 0.13 X 10(9) cells/kg per day. This value correlated well with the calculation of marrow neutrophil production. A larger turnover of 1.62 +/- 0.46 X 10(9) cells/kg per day was obtained when diisopropylfluorophosphate-32P was used to label circulating neutrophils. Studies using isologous cells doubly labeled with 3H-thymidine and diisopropylfluorophosphate-32P demonstrated a lower recovery and shorter t1/2 of the 32P label.


Composition of weight lost during short-term weight reduction. Metabolic responses of obese subjects to starvation and low-calorie ketogenic and nonketogenic diets.

The effects of starvation, an 800-kcal mixed diet and an 800-kcal ketogenic (low carbohydrate-high fat) diet on the composition of weight lost were determined in each of six obese subjects during three 10-day periods. The energy-nitrogen balance method was used to quantify the three measurable components of weight loss; protein, fat, and water. On the 800-kcal ketogenic diet, subjects lost (mean +/- SE) 466.6 +/-51.3 g/day; on the isocaloric mixed diet, which provided carbohydrate and fat in conventional proportions, they lost 277.9+/- 32.1 g/day. Composition of weight lost (percentage) during the ketogenic diet was water 61.2, fat 35.0, protein 3.8. During the mixed diet, composition of loss was water 37.1, fat 59.5, protein 3.4...


Heterogeneity of plasma glucagon. Circulating components in normal subjects and patients with chronic renal failure.

Plasma immunoreactive glucagon (IRG) concentrations were measured in 36 patients with chronic renal failure (CRF) and 32 normal subjects. In addition, the components of circulating IRG were analyzed by gel filtration in the fasting state and after physiological stimuli. Fasting IRG was elevated (P less than 0.001) in CRF patients (534 +/- 32 pg/ml) compared with the levels found in healthy subjects (113 +/- 9 pg/ml). Oral glucose suppressed plasma IRG in CRF patients from a basal level of 568 +/- 52 to a nadir of 354 +/- 57 pg/ml (120 min). This degree of suppression (38%) was comparable to that found in normal subjects (basal = 154 +/- 20 to 100 +/- 23 pg/ml) at 120 min (35%). Intravenous infusion of arginine (250 mg/kg) resulted in a 71% rise in IRG in CRF patients and a 166% increase in normal subjects. Gel filtration of fasting plasma from CRF patients showed three major peaks. The earliest (A) was found in the void volume (mol wt greater than 40,000) and constituted 16.5 +/- 4.7% of the elution profile. The middle peak (B) eluted just beyond the proinsulin marker (approximately 9,000 mol wt) and constituted the largest proportion of the elution profile (56.5 +/- 3.4%). The third peak (C) coincided with the standard glucagon and [125I]glucagon markers (3,485 mol wt) and comprised 27.0 +/- 4% of the IRG profile. In contrast, only peaks A and C were found in fasting plasma of normal subjects (53.6 +/- 10.4% in A and 46.4 +/- 10.4 in C). After oral glucose, glucagon immunoreactivity in the 3,500 mol wt peak (C) was markedly suppressed, while the B peak in patients with CRF declined to a lesser extent. The A peak in both groups was unchanged. After an arginine infusion only the C peak increased in both groups of subjects. Gel filtration of plasma in 3 M acetic acid gave similar profiles to those obtained in glycine albumin buffer. Exposure of serum to trypsin indicated that the B and C peaks were digestible, while the A peak was resistant to the action of the enzyme. In one sample, peak C increased after a 2-h exposure of serum to trypsin. We conclude that circulating IRG in normal subjects and patients with CRF is heterogenous. The hyperglucagonemia of renal failure is largely due to an increase in IRG material of approximately 9,000 mol wt, consistent with proglucagon, although the 3,500 mol wt component is also considerably elevated (threefold). The significance of circulating IRG levels should be interpreted with caution until the relative biological activity of the three components is established.