John J. Pisano

Rapid analysis of amino acid phenylthiohydantoins by high-performance liquid chromatography

Abstract All 20 amino acid phenylthiohydantoins (PTHs) can be separated in a single analysis in less than 20 min using a 25 × 0.46-cm DuPont Zorbax ODS column developed with a gradient of acetonitrile in 0.01 m sodium acetate buffer, pH 4.5. With the single solvent, 0.01 m sodium acetate (pH 4.5)-acetonitrile, 42:58 ( v v ), all PTHs are separated in less than 10 min except serine and glutamine, which coelute, and arginine which elutes at about 14 min. With this procedure it is possible to keep pace with automated Edman methods.

Determination of 3-methoxy-4-hydroxymandelic acid in urine

Abstract A specific method is described for the quantitative determination of 3-methoxy-4-hydroxymandelic (MOMA) acid in normal urine as well as in patients with pheochromocytoma. The procedure includes extraction of the acid from urine, followed by treatment of the extract with periodate to form vanillin, which is then determined spectrophotometrically.

Droplet countercurrent chromatography

A new form of countercurrent chromatography, named droplet counter-current chromatography, has been developed. This all-liquid separation technique is based on the partitioning of solute between a steady stream of droplets of moving phase and a column of surrounding stationary liquid phase. Milligram quantities of dinitrophenyl (DNP) amino acids were separated with an efficiency comparable to that of gas chromatography.

Control of sodium and potassium transport in the cortical collecting duct of the rat. Effects of bradykinin, vasopressin, and deoxycorticosterone.

Several factors interact to maintain precise control of electrolyte transport in the mammalian cortical collecting duct. We have studied the effects of deoxycorticosterone, arginine vasopressin, and bradykinin on net transepithelial sodium and potassium transport in isolated, perfused rat cortical collecting ducts. Chronic administration of deoxycorticosterone to rats increased both sodium absorption and potassium secretion above very low basal levels. Consequently, deoxycorticosterone-treated rats were used for all remaining studies. Arginine vasopressin (10(-10) M in the bath) caused a sustained fourfold increase in net sodium absorption and a sustained threefold increase in net potassium secretion. Bradykinin (10(-9) M in the bath) caused a reversible 40-50% inhibition of net sodium absorption without affecting net potassium transport or the transepithelial potential difference. In the perfusate, up to 10(-6) M bradykinin had no effect. We conclude: As in rabbits, chronic deoxycorticosterone administration to rats increases sodium absorption and potassium secretion in cortical collecting ducts perfused in vitro. Arginine vasopressin causes a reversible increase in net potassium secretion and net sodium absorption. Bradykinin in the peritubular bathing solution reversibly inhibits net sodium absorption, possibly by affecting an electroneutral sodium transport pathway.

Advances in the gas chromatographic analysis of amino acid phenyl- and methylthiohydantoins

Abstract Advances in the gas chromatography procedure for the identification of amino acid thiohydantoins include: (1) a new blend of silicone stationary phases (CFC), which provides superior resolving power than hitherto obtained with single phases or earlier blends; (2) use of a carrier gas helium, which is a superior carrier gas to the more commonly used nitrogen and argon in that it gives the best resolution and the greatest range of flow rates with no sacrifice in efficiency; and (3) high-temperature conditioning, which gives more efficient columns with significantly less bleed (baseline rise) during temperature programming. Application of the technique is demonstrated with representative analyses of phenylthiohydantoins (PTHs) obtained in the manual Edman degradation of bovine thyrocalcitonin. The methylthiodydantoins (MTHs) were also examined. All derivatives, except the seryl (unstable) and arginyl (nonvolatile) were separable on a column using the stationary phase, OV-225.

Urinary Kallikrein Excretion in Normal Man: Relationships to Sodium Intake and Sodium-Retaining Steroids

The urinary excretion of kallikrein, a renal enzyme that cleaves the potent vasodilator kinin, kallidin, from kininogen, was measured in normal volunteers by three different assays, one of which was a bioassay. When sodium intake was changed from ad libitum or 109 mEq/day to 9 mEq/day, daily kallikrein excretion increased progressively in every subject to a mean maximal value that was 271% of control by day 7; an increase in sodium intake to 259 mEq/day resulted in the return of kallikrein excretion to control values. Urinary excretion of amylase, a protein with a molecular weight similar to that of kallikrein, did not change when sodium intake was changed, but plasma renin activity and aldosterone excretion changed appropriately. In subjects on a constant-sodium (109 mEq/day), constant-potassium (100 mEq/day) diet, fludrocortisone, 0.5 mg/day for 10 days, increased kallikrein excretion to a mean maximal value that was 203% of control. In subjects on a diet containing 9 mEq sodium/day, elevated kallikrein excretion decreased during treatment with spironolactone, 400 mg/day. Rapid administration of water (1 or 2 liters in 30 minutes, orally) or rapid infusion of 2.4 liters of normal saline did not significantly alter urinary kallikrein excretion despite large changes in urine volume or sodium excretion. Thus, kallikrein excretion appears to be directly related to the effective level of circulating sodium-retaining steroid. The findings are consistent with a role for the kallikrein-kinin system in the renal response to sodium-retaining hormones.

Urinary Kallikrein Excretion in Hypertensive Man: Relationships to Sodium intake and Sodium-Retaining Steroids

Urinary kallikrein excretion was measured by a radiochemical esterolytic assay in patients with essential hypertension or primary aldosteronism. Patients with essential hypertension excreted significantly less (P < 0.001) kallikrein than did normal subjects when they were allowed an ad libitum sodium intake or given 259 mEq sodium/day. When sodium intake was changed from ad libitum to 9 mEq/day, kallikrein excretion increased in the majority of patients with essential hypertension, but it remained significantly less (P < 0.001) than that in normal subjects; however, aldosterone excretion was similar in both groups. Fludrocortisone, 0.5 mg/day for 10 days, increased kallikrein excretion in three patients with essential hypertension. In patients with primary aldosteronism, mean kallikrein excretion was sevenfold higher (P < 0.001) than that in patients with essential hypertension; kallikrein excretion remained unchanged when dietary sodium was altered, but it was decreased by treatment with spironolactone. Mean kallikrein excretion in patients with primary aldosteronism was also significantly higher (P < 0.001) than that in a normotensive control population. The results show that kallikrein excretion reflects the effective level of circulating sodium-retaining steroid in patients with primary aldosteronism but suggest that it is relatively unresponsive to endogenous sodium-retaining steroid in patients with essential hypertension. The data raise the possibility that the kallikrein-kinin system is of pathogenetic significance in human hypertensive disease.

Hypotension Associated with Prekallikrein Activator (Hageman-Factor Fragments) in Plasma Protein Fraction

Thirteen lots of plasma protein fraction made by one manufacturer were implicated in 23 recent reports of hypotension in surgical patients. Four of these patients required resuscitation after rapid administration of the product in the postoperative period. All implicated lots had prekallikrein-activator activity but low levels of bradykinin and kallikrein. The prekallikrein activator was identified as Hageman-factor fragments by molecular weight (35,000 as estimated by gel chromatography), isoelectric point (4.2 to 4.4), and inhibition by antibody to Hageman factor. These data suggest that Hageman-factor fragments are potent hypotensive agents, presumably because they trigger the generation of bradykinin in recipients. Prekallikrein-activator activity, usually at levels lower than those in the initial 13 implicated lots, was frequently detected in plasma protein fraction made by other manufactures. Several of these lots were associated with additional reports of hypotension. Prekallikrein-activator activity rarely occurred in albumin.

Urinary excretion of catecholamines and their metabolites in pheochromocytoma.

Abstract Twenty-four-hourspecimens of urine from 23 patients with pheochromocytoma and a large group of hypertensive subjects were assayed for free catecholamines (norepinephrine plus epinephrine), total metanephrines (metanephrine plus normetanephrine), and 3-methoxy-4-hydroxymandelic acid. Twenty of the 23 patients with tumors had a diagnostic increase in the urinary excretion of all catecholamine metabolites. In the other 3 patients the assay of free catecholamines was the single most reliable test. Because of its over-all reliability and ease of performance, the assay of total metanephrines is favored for screening hypertensive patients for the presence of pheochromocytoma. In the occasional patient whose value is equivocal by this assay, the determination of free catecholamines is considered to be the most helpful test in confirming or excluding the diagnosis.

Hageman factor fragment inhibitor in corn seeds: Purification and characterization

Abstract A specific, potent inhibitor of Hageman factor fragment (HFf) was isolated from sweet corn kernels. Three variants with pIs of 5.1, 6.3, and 7.7, and Mr 11,000–14,000 were found. The corn HFf inhibitor (CHFI) forms a 1:1 molar complex with HFf or trypsin, and prolongs the activated partial thromboplastin time of human plasma. The Ki of CHFI 6.3 with HFf was 2.4 × 10-8 M. CHFI does not inhibit human plasma or urinary kallikreins, plasmin, α-thrombin, hog pancreatic kallikrein, bovine Factor Xa or α-chymotrypsin. CHFI appears to be identical with the corn trypsin inhibitor (CTI) previously reported (Swartz et al., J. Biol. Chem. 252 , 8105, 1977). The corn inhibitor is unique in its strong inhibition of HFf and very narrow spectrum of inhibition of other serine proteinases.