Factor VII activation, apolipoprotein A-I and reverse cholesterol transport: possible relevance for postprandial lipaemia.

Postprandial lipaemia is associated with activation of factor VII (FVII) and efflux of cholesterol from tissues to nascent plasma high density lipoproteins (HDL) containing apolipoprotein A-I (apo A-I). To determine whether FVII activation and cholesterol efflux occur together in other situations, the responses to intravenous infusion of HDL-like apo A-I/phosphatidylcholine discs were measured in 10 healthy men. Disc infusion (40 mg apo A-I/kg body weight) over 4 h was followed by increases in HDL cholesteryl ester and plasma apo A-I (p <0.0001). Significant activation of FVII was apparent during infusion in fasting subjects (p = 0.03), activated FVII averaging 123% of baseline value by 12 h (p <0.0001). Plasma thrombin-antithrombin (TAT) complex increased to 156% of baseline level by 12 h (p >0.05) but individual responses differed considerably. Peak TAT post-infusion was associated inversely with peak HDL triglyceride concentration (p = 0.004). The coagulation responses to disc-infusion may be due to transfer of phosphatidylserine to cell surfaces during cholesterol efflux.

Intravenous apoA-I/lecithin discs increase pre-beta-HDL concentration in tissue fluid and stimulate reverse cholesterol transport in humans.

The extent to which plasma HDL concentration regulates reverse cholesterol transport (RCT) is not known. The principal acceptors of unesterified cholesterol (UC) from cultured cells are small pre-beta-HDL, which we have shown increase in plasma during intravenous infusion of apolipoprotein A-I/phosphatidylcholine (apoA-I/PC) discs in humans. We have now examined the effects on tissue fluid HDL and RCT. ApoA-I/PC or proapoA-I/PC discs were infused into 16 healthy males. Eleven had been given intravenous radiocholesterol to label tissue pools; in 12 prenodal leg lymph was collected throughout; and in 8 all feces were collected. The rise in small pre-beta-HDL in plasma was associated with increases in 1) pre-beta-HDL concentration in lymph (all subjects), 2) the size of other lymph HDL (four of four subjects), 3) the cholesterol content of lymph lipoproteins relative to plasma lipoproteins (P < 0.01, n = 4), 4) cholesterol-specific radioactivity in lymph (five of nine subjects), 5) plasma lathosterol (P < 0.004, n = 4), 6) plasma cholesterol esterification rate (P < 0.001, n = 4), and 7) fecal bile acid excretion (P < 0.001, n = 8). These results support the hypothesis that small pre-beta-HDL generated in plasma readily cross endothelium into tissue fluid, and thereby promote efflux of UC from peripheral cells. After delivery to the liver, peripheral cholesterol appears to be utilized more for bile acid synthesis than for biliary cholesterol secretion in humans.

Composition and ultrastructure of size subclasses of normal human peripheral lymph lipoproteins: quantification of cholesterol uptake by HDL in tissue fluids.

Peripheral lymph lipoproteins have been characterized in animals, but there is little information about their composition, and none about their ultrastructure, in normal humans. Therefore, we collected afferent leg lymph from 16 healthy males and quantified lipids and apolipoproteins in fractions separated by high performance-size exclusion chromatography. Apolipoprotein B (apoB) was found almost exclusively in low density lipoproteins. The distribution of apoA-I, particularly in lipoprotein A-I (LpA-I) without A-II particles, was shifted toward larger particles relative to plasma. The fractions containing these particles were also enriched in apoA-II, apoE, total cholesterol, and phospholipids and had greater unesterified cholesterol-to-cholesteryl ester ratios than their counterparts in plasma. Fractions containing smaller apoA-I particles were enriched in phospholipid. Most apoA-IV was lipid poor or lipid free. Most apoC-III coeluted with large apoA-I-containing particles. Electron microscopy showed that lymph contained discoidal particles not seen in plasma. These findings support other evidence that high density lipoproteins (HDL) undergo extensive remodeling in human tissue fluid. Total cholesterol concentration in lymph HDL was 30% greater (P < 0.05) than could be explained by the transendothelial transfer of HDL from plasma, providing direct confirmation that HDL acquire cholesterol in the extravascular compartment. Net transport rates of new HDL cholesterol in the cannulated vessels corresponded to a mean whole body reverse cholesterol transport rate via lymph of 0.89 mmol (344 mg)/day.

Distribution of phospholipid transfer protein in human plasma: presence of two forms of phospholipid transfer protein, one catalytically active and the other inactive.

Plasma phospholipid transfer protein (PLTP) plays an important role in the maintenance of plasma high-density lipoprotein (HDL) content and remodeling of HDL in the circulation. In the present study we have used different fractionation methods to investigate the distribution of PLTP in human plasma. A novel enzyme-linked immunosorbent assay developed during the study allowed for simultaneous assessment of both PLTP mass and activity in the fractions obtained. Size-exclusion chromatography and plasma fractionation by nondenaturing polyacrylamide gel electrophoresis (PAGE) yielded similar results demonstrating that PLTP associates in native plasma with two distinct particle populations, while ultracentrifugation with high salt leads to detachment of PLTP from lipoprotein particles and loss of a majority of its phospholipid transfer activity. Interestingly, analysis of the size-exclusion chromatography fractions demonstrated that PLTP exists in the circulation as an active population that elutes in the position of HDL corresponding to an average molecular mass of 160+/-40 kDa and an inactive form with an average mass of 520+/-120 kDa. The inactive fraction containing approximately 70% of the total PLTP protein eluted between HDL and low density lipoprotein (LDL). Thus, the two PLTP pools are associated with different types of lipoprotein particles, suggesting that the PLTP activity in circulation is modulated by the plasma lipoprotein profile and lipid composition.

Expression of human apolipoprotein A-I/C-III/A-IV gene cluster in mice induces hyperlipidemia but reduces atherogenesis.

The apolipoprotein (apo)A-I/C-III/A-IV gene cluster is involved in lipid metabolism and atherosclerosis. Overexpression of apoC-III in mice causes hypertriglyceridemia and induces atherogenesis, whereas overexpression of apoA-I or apoA-IV increases cholesterol in plasma high density lipoprotein (HDL) and protects against atherosclerosis. Each gene has been studied alone in transgenic mice but not in combination as the entire cluster. To determine which phenotype is produced by the expression of the entire gene cluster, transgenic mice were generated with a 33-kb human DNA fragment. The results showed that the transgene contained the necessary elements to direct hepatic and intestinal expression of the 3 genes. In the pooled data, plasma concentrations were 257+/-9, 7.1+/-0.5, and 1.0+/-0.2 mg/dL for human apoA-I, apoC-III, and apoA-IV, respectively (mean+/-SEM). Concentrations of these apolipoproteins were higher in males than in females. Human apoA-I and apoC-III concentrations were positively correlated, suggesting that they are coregulated. Transgenic mice exhibited gross hypertriglyceridemia and accumulation of apoB(48)-containing triglyceride-rich lipoproteins. Plasma triglyceride and cholesterol concentrations were correlated positively with human apoC-III concentration, and HDL cholesterol was correlated with apoA-I concentration. In an apoE-deficient background, despite being markedly hypertriglyceridemic, cluster transgenic animals compared with nontransgenic animals showed a 61% reduction in atherosclerosis. This suggests that apoA-I and/or apoA-IV can protect against atherosclerosis even in the presence of severe hyperlipidemia. These mice provide a new model for studies of the regulation of the 3 human genes in combination.

Concentrations of electrophoretic and size subclasses of apolipoprotein A-I-containing particles in human peripheral lymph.

When cultured cells are exposed to plasma, the initial acceptors of unesterified cholesterol are small lipid-poor apolipoprotein A-I (apoA-I)-containing high density lipoproteins (HDLs) with pre-beta electrophoretic mobility. These are converted by lecithin:cholesterol acyltransferase into larger spheroidal cholesteryl ester-rich HDLs with alpha mobility. To study the determinants of the concentration of small pre-beta HDLs in tissue fluids, we collected prenodal peripheral lymph from 34 fasted normal men. By crossed immunoelectrophoresis, the concentration of pre-beta HDLs in lymph averaged 20% of that in plasma. On multiple regression analysis, pre-beta apoA-I concentration in lymph was directly related to pre-beta apoA-I concentration in plasma and independently to alpha apoA-I concentration in lymph. Similar results were obtained when the same apoA-I-containing particles were quantified by size exclusion chromatography. Lymph pre-beta apoA-I concentration was low in a subject with familial lecithin:cholesterol acyltransferase deficiency, despite a normal plasma pre-beta apoA-I concentration, but was normal in a subject with familial lipoprotein lipase deficiency. These results suggest that the concentration of small pre-beta HDLs in human tissue fluids is determined only in part by the transfer of pre-beta HDLs across capillary endothelium from plasma. Local production, by remodeling of spheroidal alpha HDLs in tissue fluids, may be equally important. Lipolysis of triglyceride-rich lipoproteins by lipoprotein lipase appears to have little effect.

Lipid and apolipoprotein concentrations in prenodal leg lymph of fasted humans. Associations with plasma concentrations in normal subjects, lipoprotein lipase deficiency, and LCAT deficiency.

The extent to which lipid and apolipoprotein (apo) concentrations in tissue fluids are determined by those in plasma in normal humans is not known, as all studies to date have been performed on small numbers of subjects, often with dyslipidemia or lymphedema. Therefore, we quantified lipids, apolipoproteins, high density lipoprotein (HDL) lipids, and non-HDL lipids in prenodal leg lymph from 37 fasted ambulant healthy men. Lymph contained almost no triglycerides, but had higher concentrations of free glycerol than plasma. Unesterified cholesterol (UC), cholesteryl ester (CE), phosphatidylcholine (PC), and sphingomyelin (SPM) concentrations in whole lymph were not significantly correlated with those in plasma. HDL lipids, but not non-HDL lipids, were directly related to those in plasma. Lymph HDLs were enriched in UC. However, as the HDL cholesterol/non-HDL cholesterol ratio in lymph exceeded that in plasma, whole lymph nevertheless had a lower UC/CE ratio than plasma. Lymph also had a significantly higher SPM/PC ratio. The lymph/plasma (L/P) ratios of apolipoproteins were as follows: A-IV > A-I and A-II > C-III and E > B. Comparison with the L/P ratios of seven nonlipoprotein proteins suggested that apoA-IV was predominantly lipid free. Concentrations of apolipoproteins A-II, A-IV, C-III, and E in lymph, but not of apolipoproteins A-I or B, were positively correlated with those in plasma. The L/P ratios of apolipoproteins B, C-III, and E in two subjects with lipoprotein lipase (LPL) deficiency, and of apolipoproteins A-I and A-IV in a subject with lecithin:cholesterol acyltransferase (LCAT) deficiency, were low relative to those in normal subjects. Thus, the concentrations of lipids, apolipoproteins, and lipoproteins in human tissue fluid are determined only in part by their concentrations in plasma. Other factors, including the actions of LPL and LCAT, are at least as important.

Haemostatic factors in human peripheral afferent lymph.

Peripheral afferent lymph was obtained by cannulation of a collecting vessel in 17 healthy men (mean age 26 years). Lymph/plasma ratios of all vitamin K-dependent factors were lower than expected from molecular weight. Factor VII, factor IX and tissue factor pathway inhibitor (TFPI) lymph/plasma activity ratios were higher than antigen ratios. Activated factor VII (FVIIa) and TFPI-Xa complex concentrations were higher in lymph than plasma, and the raised FVIIa did not appear to be due to cannulation. The fibrinogen lymph/plasma activity (Clauss) ratio averaged about 20% of the antigen ratio. The result of an ELISA for D-dimer was higher in lymph than plasma, often more than five-fold. This high level in lymph was not explored but may indicate proteolysis of fibrinogen and fibrin with release of D-like and D-dimer-like fragments in interstitial fluid.

Very small apolipoprotein A-I-containing particles from human plasma: isolation and quantification by high-performance size-exclusion chromatography.

BACKGROUND: Very small apolipoprotein (apo) A-I-containing lipoprotein (Sm LpA-I) particles with pre-beta electrophoretic mobility may play key roles as "nascent" and/or "senescent" HDL; however, methods for their isolation are difficult and often semiquantitative. METHODS: We developed a preparative method for separating Sm LpA-I particles from human plasma by high-performance size-exclusion chromatography (HP-SEC), using two gel permeation columns (Superdex 200 and Superdex 75) in series and measuring apo A-I content in column fractions in 30 subjects with HDL-cholesterol (HDL-C) concentrations of 0.4-3.83 mmol/L. RESULTS: Three major sizes of apo A-I-containing particles were detected: an approximately 15-nm diameter ( approximately 700 kDa) species; a 7. 5-12 nm (100-450 kDa) species; and a 5.8-6.3 nm species (40-60 kDa, Sm LpA-I particles), containing 0.2-3%, 80-96%, and 2-15% of plasma total apo A-I, respectively. Two subjects with severe HDL deficiency had increased relative apo A-I content in Sm LpA-I: 25% and 37%, respectively. The percentage of apo A-I in Sm LpA-I correlated positively with fasting plasma triglyceride concentrations (r = 0. 581; P <0.0005) and inversely with total apo A-I (r = -0.551; P <0. 0013) and HDL-C concentrations (r = -0.532; P <0.0017), although the latter two relationships were largely attributable to extremely hypoalphalipoproteinemic subjects. The percentage of apo A-I in Sm LpA-I correlated with that in pre-beta-migrating species by crossed immunoelectrophoresis (r = 0.98; P <0.0001; n = 24) and with that in the d >1.21 kg/L fraction by ultracentrifugation (r = 0.86; P <0. 001; n = 20). Sm LpA-I particles, on average, appear to contain two apo A-I and four phospholipid molecules but little or no apo A-II, triglyceride, or cholesterol. CONCLUSIONS: We present a new HP-SEC method for size separation of native HDL particles from plasma, including Sm Lp A-I, which may play important roles in the metabolism of HDL and in its contribution(s) to protection against atherosclerosis. This method provides a basis for further studies of the structure and function of Sm Lp A-I.

Acute effects of intravenous infusion of ApoA1/phosphatidylcholine discs on plasma lipoproteins in humans.

To investigate the metabolism of nascent HDLs, apoA1/phosphatidylcholine (apoA1/PC) discs were infused IV over 4 hours into 7 healthy men. Plasma total apoA1 and phospholipid (PL) concentrations increased during the infusions. The rise in plasma apoA1 was greatest in small prebeta-migrating particles not present in the infusate. Total HDL unesterified cholesterol (UC) also increased simultaneously. After stopping the infusion, the concentrations of apoA1, PL, HDL UC, and small prebeta HDLs decreased, whereas those of HDL cholesteryl ester (CE) and large alpha-migrating apoA1 containing HDLs increased. ApoB-containing lipoproteins became enriched in CEs. Addition of apoA1/PC discs to whole blood at 37 degrees C in vitro also generated small prebeta HDLs, but did not augment the transfer of UC from erythrocytes to plasma. We conclude that the disc infusions increased the intravascular production of small prebeta HDLs in vivo, and that this was associated with an increase in the efflux and esterification of UC derived from fixed tissues. The extent to which the increase in tissue cholesterol efflux was dependent on that in prebeta HDL production could not be determined. Infusion of discs also reduced the plasma apoB and apoA2 concentrations, and increased plasma triglycerides and apoC3. Thus, nascent HDL secretion may have a significant impact on prebeta HDL production, reverse cholesterol transport and lipoprotein metabolism in humans.

Plant monoterpenes do not raise plasma high-density-lipoprotein concentrations in humans.

BACKGROUND: Low plasma concentrations of HDLs are associated with an increased risk of coronary artery disease. Two uncontrolled studies suggested that plant monoterpenes may have substantial HDL-cholesterol-elevating activity in humans. Each study used a proprietary mixture of 6 monoterpenes in olive oil. OBJECTIVE: The present study was undertaken to test more rigorously the hypothesis that monoterpenes raise HDL concentrations in men with hypoalphalipoproteinemia. DESIGN: A double-blind, placebo-controlled crossover design was used. Twenty-four men aged 58-68 y (x: 62.3 y) with plasma HDL cholesterol <1.1 mmol/L, plasma triacylglycerols <3.5 mmol/L, and plasma total cholesterol <5.5 mmol/L at recruitment were randomly assigned to 6 capsules daily of a proprietary mixture of 6 monoterpenes in olive oil or 6 capsules daily of olive oil alone for 24 wk, followed by a washout period of 8 wk, and then the alternative capsules for 24 wk. RESULTS: Five men dropped out. In the others, compliance was excellent as judged by capsule counts and urinary menthol glucuronide concentrations. No significant effects were observed on plasma HDL-cholesterol or apolipoprotein A-I concentrations, nor on plasma triacylglycerol, LDL-cholesterol, or apolipoprotein B concentrations. CONCLUSIONS: Plant monoterpenes have no HDL-elevating activity of potential value for coronary artery disease prevention.

Very low activated factor VII and reduced factor VII antigen in familial abetalipoproteinaemia.

Abetalipoproteinaemia is a rare disorder of apolipoprotein B metabolism associated with extremely low plasma concentrations of triglyceride. To discover whether the general positive association between factor VII and triglyceride levels extends to this condition, 5 patients were compared with 18 controls. All patients had a triglyceride below 100 micromol/l. Plasma unesterified fatty acid concentration was normal. Although factor IX activity was only slightly reduced (mean 88% standard) and factor IX antigen was normal, mean activated factor VII in patients was strikingly reduced to 34% of that in controls, a level similar to that found in haemophilia B. The patients' mean factor VII activity and factor VII antigen were also significantly reduced to 54% and 63% of those in controls, respectively. Mean factor XI activity and tissue factor pathway inhibitor activity were reduced in patients to 70% and 75% of control values respectively, while factor XII, factor XI antigen, factor X, prothrombin and protein C were normal.

Analysis of high density lipoprotein apolipoproteins by capillary zone and capillary SDS gel electrophoresis.

We describe new methods for analyzing the apolipoproteins (apo) of the high density lipoproteins (HDL) of several species by two modes of capillary electrophoresis: size separation using a molecular sieving buffer, and capillary zone electrophoresis (CZE) using neutral coated capillaries. By either mode HDL apos were resolved within 25 min. Results for apoA-I and apoA-II mass agreed with those by electroimmunoassay; intra-assay coefficients of variation were 1.8-4.2%. The migration times of human, rat, rabbit, and bovine apoA-I during CZE were proportional to their net charge/Mr ratios. This enabled human and rabbit apoA-I to be quantified simultaneously in transgenic rabbit HDLs. CZE also resolved human apoA-I isoforms, deamidated apoA-I, and pro-apoA-I.

Do dietary phytochemicals with cytochrome P-450 enzyme-inducing activity increase high-density-lipoprotein concentrations in humans?

Low plasma concentrations of high-density lipoprotein (HDL) are associated with increased risk of coronary heart disease. Several drugs that induce the microsomal cytochrome P-450-dependent enzyme system in liver and intestine, the sites of HDL apolipoprotein (apo) A-I and A-II synthesis, raise plasma HDL concentrations in humans. To test the hypothesis that phytochemicals with cytochrome P-450-inducing activity may also increase plasma HDL concentrations, two controlled dietary trials were undertaken in healthy nonsmoking males aged 20-28 y. One study examined the effect of replacing 300 g glucosinolate-free vegetables with 300 g Brussels sprouts/d for 3 wk. The other study examined the effects of 150 mg eugenol/d in capsule form, using a double-blind, placebo-controlled crossover design. There were no significant increases in plasma apo A-I, apo A-II, HDL cholesterol, or HDL phospholipids. These results suggest that dietary phytochemicals that induce members of the cytochrome P-450 system do not necessarily raise plasma HDL concentrations in humans, but do not exclude the possibility that some phytochemicals may have such an effect.

Effects of intravenous infusion of lipid-free apo A-I in humans.

Apolipoprotein (apo) A-I is the principal protein component of the plasma high density lipoproteins (HDLs). Tissue culture studies have suggested that lipid-free apo A-I may, by recruiting phospholipids (PLs) and unesterified cholesterol from cell membranes, initiate reverse cholesterol transport and provide a nidus for the formation, via lipid-poor, pre-beta-migrating HDLs, of spheroidal alpha-migrating HDLs. Apo A-I has also been shown to inhibit hepatic lipase (HL) and lipoprotein lipase (LPL) in vitro. To further study its functions and fate in vivo, we gave lipid-free apo A-I intravenously on a total of 32 occasions to six men with low HDL cholesterol (30 to 38 mg/dL) by bolus injection (25 mg/kg) and/or by infusion over 5 hours (1.25, 2.5, 5.0, and 10.0 mg.kg-1.h-1). The procedure was well tolerated: there were no clinical, biochemical, or hematologic changes, and there was no evidence of allergic, immunologic, or acute-phase responses. The 5-hour infusions increased plasma total apo A-I concentration in a dose-related manner by 10 to 50 mg/dL after which it decreased, with a half-life of 15 to 54 hours. Coinfusion of Intralipid reduced the clearance rate. The apparent volume of distribution exceeded the known extracellular space in humans, suggesting extensive first-pass clearance by one or more organs. No apo A-I appeared in the urine. Increases in apo A-I mass were confined to the pre-beta region on crossed immunoelectrophoresis of plasma and to HDL-size particles on size exclusion chromatography. Increases were recorded in HDL PL, but not in HDL unesterified or esterified cholesterol. Increases also occurred in LDL PL and in very low density lipoprotein cholesterol, triglycerides, and PL but not in plasma total apo B concentration. These results can all be explained by combined inhibition of HL and LPL activities. Owing to the effects that this would have had on HDL metabolism, no conclusions can be drawn from these data about the role of lipid-free apo A-I in the removal of PL and cholesterol from peripheral tissues in humans. The kinetic data suggest that the fractional catabolic rate of lipid-free apo A-I exceeds that of spheroidal HDLs and is reduced in the presence of surplus PL.

Sequential microenzymatic assay of cholesterol, triglycerides, and phospholipids in a single aliquot.

The assay of multiple analytes in a single aliquot can be advantageous from both measurement and economic standpoints. The objective of this study was to develop a simple and sensitive microenzymatic method for the determination of three biologically important lipids. Triglycerides (as glycerol), phospholipids (as choline), and total cholesterol (as unesterified cholesterol) were assayed, in that order, by sequential addition of sample, reagents, and microbial enzymes directly into a single microtiter plate well, accompanied by continuous monitoring of a common reporter reaction in which hydrogen peroxide is quantified either by colorimetry with 4-aminoantipyrine and 3-hydroxy-2,4,6-triiodobenzoic acid or by ultraviolet fluorometry with p-hydroxyphenylacetic acid. The detection limit of the method is in the subnanomole mass range for all three lipids. Results obtained with either fluorescence or colored endpoints were in excellent agreement with alternative individual chemical and enzymatic procedures.

The very-high-density lipoprotein fraction of rabbit plasma is rich in tissue-derived cholesterol.

When plasma from rabbits, which several weeks earlier had been infused with [3H]cholesterol, was subjected to equilibrium density gradient ultracentrifugation, the specific radioactivity of cholesterol in the very-high-density lipoprotein (VHDL) fraction (d 1.22-1.32 g/ml) was three to 8-fold greater (mean, 5.5-fold; P less than 0.001) than that in high-density lipoproteins (HDL; d 1.06-1.21 g/ml). On size exclusion chromatography of plasma, no increase in specific radioactivity was seen in particles smaller than HDL. These findings suggest that those apolipoprotein-lipid complexes that dissociate from HDL during ultracentrifugation to form the VHDL fraction contain proportionately more tissue-derived cholesterol than do those that are more tightly bound to HDL.

Evidence that reverse cholesterol transport is stimulated by lipolysis of triglyceride-rich lipoproteins.

The hypothesis that reverse cholesterol transport by high density lipoprotein (HDL) is augmented by lipolysis of triglyceride-rich lipoproteins received support from experiments in rabbits whose tissue cholesterol had been pre-labeled with [3H]cholesterol several weeks earlier. When lipolysis was stimulated by intravenous heparin (which releases lipoprotein lipase from vascular endothelium), reciprocal changes in plasma triglyceride and HDL cholesterol concentrations were accompanied by a rise in the specific radioactivity of HDL cholesterol, indicative of increased transfer of cholesterol into HDL from slowly exchanging cholesterol pools in extra-hepatic tissues.

Enzymatic fluorometric procedure for phospholipid quantification with an automated microtiter plate fluorometer.

We describe a new enzymatic assay for phospholipids that is rapid, sensitive, convenient, and inexpensive. The method is based on the fluorometric detection of H2O2, generated by the choline oxidase-catalyzed oxidation of choline, after liberation of choline from phospholipids by phospholipase D. Significant advantages over existing methods are that the entire reaction sequence can take place in a single vessel, a 12 x 8 well microtiter plate, and that the fluorescence intensity can be measured automatically with a Fluoroskan II (Labsystems Oy) detector. Extracting samples with organic solvents is unnecessary, although the method can be applied to extracts in isopropanol. The assay is approximately 60-fold more sensitive and has a limit of detection eightfold lower than currently available enzymatic colorimetric methods. Including solvent blanks, eight standards, and three quality-control pools, 34 samples can be pipetted and assayed in duplicate in 60 min. Results obtained by this procedure for total phospholipid choline in lipoproteins in primate plasma agreed well with those obtained for inorganic phosphorus by the Bartlett acid-digestion procedure.

Criteria to indicate testosterone administration.

A detection method for testosterone administration was developed using radioimmunoassay to measure the urinary ratios of testosterone (T) to epitestosterone (E) and to luteinizing hormone (LH). A comparative study of the effect on these ratios of a single intramuscular injection of testosterone heptanoate followed by stimulation with human chorionic gonadotrophin (HCG) in three normal men was undertaken. To allow immediate investigation, a commercially supplied epitestosterone antiserum was used. This study showed that both T/E and T/LH ratios could be used to detect testosterone administration, the latter also being an indicator of HCG use due to cross-reactivity with the LH antiserum. Subsequently, an epitestosterone antiserum of superior specificity was raised and used in a study to demonstrate the insignificant effect of exercise on these ratios. Finally, an intramuscular injection of a combined preparation of testosterone/epitestosterone heptanoates resulted in raised ratios of T/LH but not of T/E. This demonstrated the importance of the T/LH ratio in circumstances where the T/E ratio can be easily circumvented.