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The plasma pyridoxal-5'-phosphate (PLP) level of alcoholic subjects has been compared with that of non-alcoholic individuals in order to ascertain the incidence of abnormal vitamin B(6) metabolism in chronic alcohol abuse. 66 alcoholic subjects were selected on the basis that they did not exhibit abnormal liver function tests and hematologic findings. 35 of them had plasma PLP concentrations less than 5 ng/ml, the lowest value encountered in 94 control subjects, indicating a high incidence of deranged PLP metabolism in alcoholic patients even when hepatic and hematologic abnormalities are absent. The biochemical basis for the altered PLP metabolism in chronic alcohol abuse was examined. Low plasma PLP levels in alcoholics were not accompanied by decreased pyridoxal kinase and pyridoxine phosphate oxidase activities in erythrocytes. Further studies with erythrocytes demonstrated that the cellular content of PLP is determined not only by the activities of these PLP-synthesizing enzymes but also by the activity of a phosphate-sensitive, membrane-associated, neutral phosphatase, which hydrolyzes phosphorylated B(6) compounds.Acetaldehyde, but not ethanol, impaired the net formation of PLP from pyridoxal, pyridoxine, and pyridoxine phosphate by erythrocytes. However, when the B(6)-phosphate phosphatase activity was inhibited by 80 mM phosphate, this effect of acetaldehyde was abolished. By the use of broken cell preparations, it was possible to demonstrate directly that the action of acetaldehyde is mediated by the phosphatase, resulting in an acceleration of the degradation of the phosphorylated B(6) compounds in erythrocytes.

The plasma concentrations of pyridoxal phosphate (PLP) in 55 women who had been using oral contraceptive agents (OCA) for 6 months or more were compared with those of 77 nonusers. The PLP content was assayed enzymatically with tyrosine decarbosylase apoenzyme. Mean PLP of OCA users 7.8 plus or minus 3.7 ng/m1 was significantly lower than that for the controls 9.4 plus or minus 4.2 ng/m1 (p less than .05). Mean PLP of OCA users was significantly lower than controls in the 25-29 and 30-34 year age groups (p less than .001 and p less than .02 respectively). 10 subjects were followed sequentially from 2 weeks before OCA therapy for a duration of 3 months or more. There was a decrease in PLP in 9 of the subjects within 3 months followed by a return toward pretreatment levels in most of the subjects by 6 months. Tryptophan load tests were done concurrently with PLP measurements in 15 OCA users and 6 nonusers. In nonusers of comparable age (20-25) the xanthurenic acid (XA) excretion was 27.1 plus or minus 5.7 mcmoles/8hours. 11 of 15 OCA USERS HAD ELEVATED XANTHURENIC ACID EXCRETION (LESS THAN 38.5 15 OCA USERS HAD ELEVATED XANTHURENIC ACID EXCRETION (38.5 MCMOLES/8HOURS) BUT plasma PLP was less than 4.7 ng/m1 in only 4 of them. These results are consistent with the view that OCA induce an increased requirement for pyridoxine hydrochloride (vitamin B6). Although the majority of OCA users can correct the relative deficiency state naturally a substantial number remain deficient suggesting the need for vitamin B6 supplementation.

The pyridoxal 5′-phosphate content of liver in vivo and of hepatocytes in vitro remains unaltered in the presence of excess unphosphorylated vitamin B6 precursors. Studies with isolated hepatocytes and subcellular fractions show that while product inhibition of pyridoxine phosphate oxidase does not limit synthesis sufficiently to account for the phenomenon, inhibition of phosphatase activity produces striking increases in pyridoxal 5′-phosphate concentration. Protein-binding protects it against degradation by the phosphatase. The data suggest that protein-binding and the enzymatic hydrolysis of pyridoxal 5′-phosphate, synthesized in excess, act jointly to preserve the constancy of the cellular content of this coenzyme.

As many as nine isoenzymes of liver alcohol dehydrogenase have been observed, and some of them seem to be hybrid forms. Steroids and ethanol may react at different subunits in the hybrid forms.

1. Plasma renin activity, plasma adrenaline and noradrenaline concentrations, and urinary adrenaline and noradrenaline excretion rates were measured in ten subjects during 3 days of exposure to a simulated altitude of 12 000 ft. 2. In both the supine and standing positions, renin activities were suppressed during all 3 days at altitude. 3. Plasma noradrenaline and adrenaline concentrations were significantly increased by the third day at altitude. 4. Urinary adrenaline excretion tended to be increased during the entire 3 days at altitude, with no significant change between the first and third day. Noradrenaline excretion was significantly increased on the third day. 5. The finding of decreased renin levels suggests that the enhanced activity of the sympathetic nervous system at high altitude does not stimulate renin release.

A Porter and Silber procedure, a fluorometric procedure, a double-isotope derivative procedure, and a competitive protein-binding assay procedure were used to determine 17-hydroxycorticosteroids in plasma from normal individuals and from patients with endocrinopathies. Results from each of these procedures were intercompared. Results of the fluorometric procedure and the competitive protein-binding assay compared favorably with results obtained by the more elaborate and difficult double-isotope derivative method. The Porter and Silber method is less specific. This study should be a useful aid in comparing values with those of other laboratories and in selecting the most advantageous method for use by a particular laboratory.

Ionized calcium in serum has been determined potentiometrically by using a calcium ion-selective electrode in a flow-through configuration. For 397 normal human adults the mean value for serum ionic calcium was 1.22 ± 0.09 (2 SD) mmol/liter. Factors affecting the measurement—such as the procedure for sample collection, sample storage, the preparation of standard solutions, the type of resin employed, and the choice of the reference electrode filling solution—were examined. Magnesium ions in physiological concentrations cause a slight depression of the electrode's response to calcium ions, indicating an interference by magnesium in the method. Under the conditions used, the relative standard deviation (CV) of the method is 0.8%.

4,4′-Diaminodiphenylsulfone (DDS) can produce methemoglobinemia in vivo , apparently by the action of an unidentified metabolic product. To discern the chemical nature of such a compound, 13 p -substituted diphenylsulfones have been examined for their ability to oxidize hemoglobin (Hb) to methemoglobin (MHb) in human erythrocytes in vitro . 4-Amino-4′-hydroxyaminodiphenylsulfone (DDS-NOH) is most effective, 1·9 × 10 −5 M of drug oxidizing as much as 60 per cent of the Hb, or 100 heme equivalents/mole of DDS-NOH in 2 hr. Oxygen is required for this process, and glucose, while not required, enhances MHb formation. With dialyzed hemolysates and solutions of purified Hb, the heme equivalents oxidized/mole of drug becomes less than 10, but a ratio of 80 : 100 can be restored by adding reduced glutathione (GSH) or NADPH. Apparently DDS-NOH produces MHb by coupled oxidation with Hb and O 2 and DDS-NOH can be regenerated by reducing compounds to yield the high heme oxidation ratio. In intact cells, DDS-NOH causes a 7-fold increase in the activity of the hexose monophosphate shunt as measured by 14 CO 2 production from glucose 1- 14 C. Incubation with galactose, which is minimally oxidized by the shunt pathway, results in decreased formation of MHb, and the GSH concentration drops by 75 per cent. Depletion of intracellular GSH by a sulfhydryl oxidizing compound also reduces MHb formation by DDS-NOH. These observations implicate GSH or NADPH or both in the recycling of DDS-NOH in erythrocytes. Consistent with these interpretations, DDS-NOH produced only one-half as much MHb in erythrocytes from three individuals with glucose 6-phosphate dehydrogenase deficiency as it did in normal erythrocytes.

The correlation between surgically related mortality and preoperative condition or degree of heart failure was studied in 119 patients who had undergone ligation of the inferior vena cava for the treatment of thromboembolism. There were 18 related deaths (15%), of which 12 (10%) were associated with a poor or terminal preoperative condition, or class 4 heart failure. In patients with class 4 heart failure, related mortality was 55%, whereas in those with lesser degrees of heart failure, related mortality was 10%. When patients with class 4 heart failure were excluded from the analysis, the mortality in patients with and without heart failure was comparable (10% vs 7%). Pulmonary emboli were found in 31 of the 37 autopsies, a preoperative diagnostic accuracy of 84%. Autopsy revealed recurrence of pulmonary emboli after ligation in 3 of the 37.

The inhibition of the yeast and liver alcohol dehydrogenases' by chelating agents is accompanied by progressive changes in optical rotation.2' 3 Such alterations of the optical rotatory properties of yeast alcohol dehydrogenase are due to the binding of chelating' agents to the asymmetric zinc site of the enyme resulting in a characteristic Cotton effect. The rotatory power of this Cotton effect, when superimposed upon the background dispersion curve of the native protein, accounts for the rotational changes which appear during the course of enzymatic inhibition, and these changes can be differentiated from those due to alterations in protein conformation.4 Such observations suggested that changes in optical rotatory dispersion might be a frequent concomitant of the binding of small molecules to proteins and might, thereby, be used to identify the interactions with enzymes of optically active, chromophoric substrates, coenzymes, inhibitors, and other site specific reagents.4 To this end we have investigated the alcohol dehydrogenase of horse liver. This zinc containing enzyme5' 6 interacts stoichiometrically with two moles of coenzyme, DPN(H), to form the enzymatically active holoenzyme7 and is inhibited by a number of chelating agents.' Zinc is not removed during chelation nor does the enzyme dissociate into subunits by electrophoresis or ultracentrifugation. 1,10phenanthroline, OP, reversibly inhibits the enzyme by chelation of the zinc atom forming a mixed complex: LADH-Zn OP.9 The coenzyme binds in a specific though as yet unknown manner at or near the zinc site of the enzyme." A variety of approaches indicate that zinc is a component of the "active center" of liver alcohol dehydrogenase.'I The present report demonstrates that the binding of reduced diphosphopyridine nucleotide to liver alcohol dehydrogenase is accompanied by the appearance of a pronounced Cotton effect at 327 m,. The magnitude of this Cotton effect is a direct function of the molar ratio of the interactants, thereby permitting a quantitative assessment of coenzyme binding. Moreover, characteristic changes in the optical rotation of the enzyme-coenzyme complex identify its interaction with substrate and inhibitors. Similar effects are observed when the analogues of DPNH bind to liver alcohol dehydrogenase. The considerations pertinent to the choice of metalloenzymes for the exploration of the molecular basis of enzyme action have been discussed3 and preliminary accounts of the present studies have been reported.2' 13 Methods and Materials.-Crystalline alcohol dehydrogenase of horse liver was obtained from C. F. Boehringer and Soehne, Mannheim, W. Germany. Prior to use, the enzyme was dialyzed for 5 days against 0.1M phosphate buffer, pH 7.5 at 4? to remove low molecular weight impurities which absorb radiation at 280

The possibility that the mass spectrum and pharmacological activity of a compound may be directly related has been explored with the help of various computer-based pattern-recognition techniques. The relationship appears to hold at least for tranquilizers and sedatives, and compounds with one or the other of these two pharmacological activities can thus be classified from their mass spectra with a high degree of accuracy.

Nine of ten patients immobilized for the treatment of tractures were found to have abnormal elevations of serum ionized calcium when determined with an ion-sensitive electrode, while only three of the ten had hypercalcemic values ( 10.2, 10.4, and 11.7 mg 100 ml ) by atomic absorption spectroscopy (normal range, 8.2–10.1 mg 100 ml ). Mean serum ionized calcium values were 5.3–7.2 mg 100 ml in seven of the patients ( normal range, 4.5–5.2 mg 100 ml ), and two others had normal mean values but some individual abnormal measurements. Because of these observations, daily measurements of serum total and ionized calcium, and urinary calcium were made on four normal volunteers before, during, and after a 12-day bed rest period. Abnormal elevations of serum ionized calcium occurred during bed rest in all four subjects (peaks, 5.7–5.9 mg 100 ml ), while serum total calcium remained normal. Elevations of urinary calcium occurred in all four, but no clear temporal relation with the serum ionized calcium was seen. It is concluded that bed rest produces early and significant elevations of the serum ionized calcium, while there are minimal changes in the serum total calcium. The pathogenesis of these changes remains obscure.

The citrate in ACD blood is known to complex Ca++ and calcium gluconate is usually infused during ET to counteract this effect. We have measured Ca++ with a Ca++ selective electrode during ET with non-buffered ACD and heparinized blood and have studied the effect of administering 0.1g calcium gluconate per 100 ml ACD blood exchanged. Heparinized blood produces negligible changes in Ca++, whereas with ACD blood a profound fall in Ca++ occurs even when calcium gluconate is given. Values for Ca++ during ET and ACD blood and added calcium gluconate are much lower in premature than in full term infants (p < 0.001). Omitting calcium gluconate further lowers Ca++ levels in full term infants (p < 0.05). The injection of calcium gluconate produces a transient rise in Ca++ which is rapidly neutralized by the infused citrate. Cessation of ET produces a prompt return toward normal values. No relationship was shown between Ca++, total Ca and protein. In spite of frankly hypocalcemic levels of Ca++ (particularly in prematures) no clinical evidence of tetany was seen. Ca++ levels could not be correlated with the infant's clinical state during the ET. We conclude that in full term infants the use of calcium gluconate in ET using ACD blood exerts a small but significant effect on Ca++. The clinical significance of this observation requires further evaluation.

Serum ionic and total calcium levels were measured in 15 patients with clinical diagnosis of primary hyperparathyroidism. Ionic calcium level was measured by a calcium-selective liquid-membrane electrode and total calcium level was measured by atomic absorption spectroscopy. Concurrent determinations of ionic and total calcium in 230 blood donors served as normal values. Of the determinations of ionic calcium in the hyperparathyroid patients, 98.5% (67 of 68) fell outside the normal range of 4.48 to 5.28 mg/100 ml, while 77% (57 of 74) of the determinations of total calcium in the same patients was outside the normal range of 8.3 to 10.1 mg/100 ml. Parathyroid adenomas or hyperplasia were found in 14 of 15 patients. The calcium-selective electrode has proven to be highly discriminatory in separating hypercalcemia from normocalcemia in patients with primary hyperparathyroidism.