Joseph C. Touchstone

Improved separation of phospholipids in thin layer chromatography

The mobile phases described permit separation of the six major phospholipids of amniotic fluid in one dimension with either conventional or high performance thin layer chromatography. An example of this separation with an extract of amniotic fluid is given.

Thin-layer chromatographic procedures for lipid separation

This review focuses on the thin-layer chromatographic (TLC) separation aspects of lipid analysis. Since the space limitations do not permit, the quantitative aspects of the analyses are not discussed at length although some indications of appropriate methodology and detection reagents will be indicated. Many separations carried out by TLC have the prerequisite of proper sample preparation. Using proper sample clean-up prior to TLC enables one to carry out precise separation as well as sensitive quantitation. Thus, the discussions are divided into the two main topics--sample preparation and TLC. Examples of applications are limited to those which illustrate the capabilities of the technique as well as practicability. Since there are a number of reviews in the literature, the discussions herein are focused mainly on reports after 1985.

Metabolism of free and sulfoconjugated DHEA in brain tissue in vivo and in vitro.

Abstract Two rhesus monkey brains were perfused in vivo either with 14C-DHEA (dehydroepiandrosterone) and 3H-DHEA sulfate or with 3H-DHEA. Plasma from the jugular vein and the brain were analyzed for free and conjugated metabolites. Many more free than sulfoconjugated steroids were withdrawn from the blood. These were not stored to a large extent. Up to 16% of DHEA were metabolized to ring D hydroxylated Δ5-compounds. When fresh human fetal brain was incubated with 3H-DHEA, 5.9% of the free steroids were sulfurylated. 30% were converted mainly to ring D hydroxylated Δ5-metabolites.

Analysis of Saturated and Unsaturated Phospholipids in Biological Fluids

Abstract Cupric acetate (3% in 8% phosphoric acid) as a charring agent reacts only with unsaturated phospholipids while cupric sulfate (10% in 8% phosphoric acid) reacts with both saturated and unsaturated phospholipids. Thus, the amount of saturated phospholipid in a zone on a thin layer chromatogram (TLC) can be calculated by the difference in reactivity. An evaluation of methods shows that direct application of biological samples to TLC for separation and quantitation of phospholipids is reproducible. The use of these techniques for a number of different samples is described.

Urinary estriol determination in the management of prolonged pregnancy

OBSTETRICIANS and pediatricians have a common interest in prolonged pregnancy; yet confusion exists in regard to its effects and treatment. Most authors agree that even in the absence of maternal disease, such as chronic hypertension or diabetes, prolonged pregnancy increases the hazard of infant death,:?, 3, I37 I41 I’, 23 but not all do ~0.~9 15, I63 181 ” IJnless labor is prolonged, or there is superimposed toxemia, the majority in this country favor watchful waiting rather than

Separation of acidic and neutral lipids by aminopropyl-bonded silica gel column chromatography

The separation of acidic and neutral lipids by aminopropyl-bonded silica gel column chromatography is presented. Total lipid extracts from Escherichia coli and human spermatozoa were loaded onto pre-packed aminopropyl-bonded silica gel columns and the lipids separated into four fractions. Non-polar lipids including cholesterol esters, triglycerides, diglycerides, monoglycerides and cholesterol, were eluted with 4 ml of isopropanol-chloroform (1:2, v/v) (fraction 1); free fatty acids were eluted with 4 ml of 2% acetic acid in diethyl ether (fraction 2); neutral polar lipids, including phosphophatidylethanolamine, phosphatidylcholine, sphingomyelin and neutral glycolipids, were eluted with 4 ml of methanol (fraction 3); and, finally, polar acidic lipids, including phosphatidylglycerol, cardiolipin, phosphatidylinositol, phosphatidylserine, seminolipid lipid A and acidic glycosphingolipids, were eluted with 4 ml of chloroform-methanol-0.8 M sodium acetate (60:30:4.5, v/V/V) (fraction 4). The recoveries for the different lipids ranged between 89 and 98% and the intra-assay variation, expressed as the standard deviation, was less than 5%.

Determination of the 2,4-dinitrophenylhydrazones of urine ketosteroids by thin layer densitometry

Abstract A double beam scanning spectrodensitometer is used for the determination of DHEA (dehydroepiandrosterone  3β-hydroxyandrost-5-en-17-one), androsterone (3α-hydroxy-5α-androstan-17-one) and etiocholanolone (3α-hydroxy-5β-androstan-17-one) from urine. After ether solvolysis, purification and reaction with 2,4-dinitrophenylhydrazine, aliquots are developed on thin layer plates and quantitated directly.

Steroid Formation by Adrenal Tissue from Hypertensives

The adrenal cortical hormones have been thought to play a role in the pathogenesis of hypertension for the following reasons: 1) Hypertension is a characteristic finding in Cushings disease; 2) large doses of desoxycorticosterone (DCA) and salt administered to Addisonian patients produce hypertension (1); 3) hypertensive patients who developed Addisons disease had blood pressure falls to normal limits which were restored to previous levels with DCA(2) ; and 4) 80 per cent of the patients with severe hypertension have had significant improvement for three to seven years following 90 to 100 per cent adrenalectomy and limited sympathectomy (3). Moreover, animal experiments (4, 5) have shown that hypertension following renal artery occlusion cannot be produced in the absence of adrenal cortical steroids. Despite these observations, altered adrenal function in hypertension has not been clearly demonstrated. While mean levels of cortical steroids in blood and urine (6-9) are not elevated in hypertensive patients, disturbances in salt and water metabolism observed in early hypertension prior to the development of renal damage by Green, Johnson, Bridges and Lehmann (10) and BraunMenendez (11) indirectly support the idea that the pattern of adrenal corticoids is altered. Brady (12) found that slices of canine adrenal tissue produce large quantities of steroids when incubated in vitro in autologous plasma and proposed the measurement of steroid formation in vitro as a direct assessment of the functional ca-

Evidence for diplasmalogen as the major component of rabbit sperm phosphatidylethanolamine

The question of whether diplasmalogens [1,2-di(O-1′-alkenyl) phosphatidyl derivatives] make up part of the plasmalogen component of cell phospholipids was examined using rabbit epididymal spermatozoa. These cells are readily obtained as a highly homogeneous suspension and long have been known to have high plasmalogen content. Phospholipids were determined by thin layer chromatography (TLC) with CuSO4 staining. Plasmalogens were determined by hydrolysis of the phospholipids with TCA/HCl, followed by TLC and CuSO4 staining. Ethanolamine derivatives were determined by ninhydrin. The phosphatidylethanolamine (PE) content of these cells was 29±2 μg/108 cells, 90% of which was assayed as diplasmalogen and 10% as diacyl PE. No monoplasmalogen could be detected. The presence of diplasmalogen as the major component of PE was given further support from infrared and proton nuclear magnetic resonance (1H-NMR) spectroscopy, which showed the presence of O-1′-alkenyl substituents but near absence of O-acyl substituents. The phosphatidylcholine (PC) content of the cells was 104±5 μ/108 cells, of which 50% was monoplasmalogen with the 1′-alkenyl group on the 2 position of the glycerol moiety. No diplasmalogen was found in PC. The other phospholipids in rabbit sperm were phosphatidylglycerol (PG), cardiolipin (CL), sphingomyelin (SP) and lysophosphatidylcholine (LPC). Phosphatidylserine (PS) and phosphatidylinositol (PI) were present at the limits of detectability of the TLC method. None of these phospholipids contained plasmalogen. The PE component of rabbit sperm phospholipids appears to differ from that of the other cells in having the previously unreported diplasmalogen as its major constituent.

Corticosteroids in human brain

Abstract A recent report from this laboratory showed that human nerve tissues contained large quantities of cortisol (1). The present paper describes the characterization of cortisol, tetrahydrocortisol, (3α, 11β, 17α, 21-tetrahydroxy-5β-pregnan-20-one; THF) and 11β, 17α, 20β, 21-tetrahydroxy-pregn-4-ene-3-one (20β-DHF) from whole human brain.