Dyeless optical detection of ammonia in the gaseous phase using a pH-responsive polymer--characterization of the sorption process.

pH-responsive polymers enable the dyeless optical detection of acidic or basic pollutants in air. The characterization of the sorption process and the optimization of the response time of the sensitive layers were high-lighted. The swelling of a pH-responsive polysiloxane induced by sorption of gaseous ammonia was investigated by measurement techniques such as spectroscopic ellipsometry (SE) and infrared spectroscopy (IR). Furthermore, the pH-responsive polymer was applied for the detection of gaseous ammonia using the LED-based reflectometric interference spectroscopy set-up (RIfS4lambda). A limit of detection of 0.30 mg/m3 ammonia and a response time (t90%) of 35 s could be verified. The application of pH-responsive polymers can be a powerful alternative to dye-based optical sensing, since photobleaching or leaching of the sensitive functional unit cannot occur applying this approach, and since the properties of the sensitive layer proved to be very promising.

Extravascular circulation of lipoproteins: their role in reverse transport of cholesterol.

The transfer of lipoproteins out of plasma into peripheral tissues, their flow through the interstitium and their return to blood has been reviewed. In this context special emphasis has been given to reports dealing with the movement of lipoproteins into and out of the wall of arteries.

Gemfibrozil increases apolipoprotein A-I and cholesterol concentrations in human peripheral lymph.

Peripheral lymph lipoproteins were studied in four hyperlipidaemic men before and after 6 weeks of treatment with gemfibrozil, a drug which is known to increase the fractional catabolic rate of very low density lipoproteins (VLDL) by raising lipoprotein lipase activity in peripheral tissues. Decreases in plasma triglycerides of 18-60% (mean, 45%) were accompanied by increases in lymph apolipoprotein (apo) A-I concentration of 30-108% (mean, 66%; P < 0.01), and in lymph cholesterol concentration of 35-100% (mean, 59%; P < 0.05). The additional lymph cholesterol was distributed over a broad range of lipoprotein particle sizes. Effects on plasma apo A-I concentration (mean, +7%) and plasma total cholesterol concentration (-7%) were not statistically significant. No changes were observed in four untreated control subjects. These findings are compatible with the hypothesis that lipolysis of VLDL at the blood-endothelium interface increases the transfer of apo A-I from plasma to interstitial fluids, and thereby promotes cholesterol efflux from cells.

Variability of plasma apolipoprotein (apo) A-II levels associated with an apo A-II gene polymorphism in monozygotic twin pairs.

A dimorphic MspI RFLP (alleles M1 and M2) in an Alu unit 528 base pairs downstream from the apolipoprotein A-II gene on chromosome 1 was investigated for associations with dyslipoproteinaemia or coronary atherosclerosis. No significant differences were observed between the allele frequencies in healthy random controls (M2 = 0.850, n = 70) and patients with primary hypertriglyceridaemia (M2 = 0.846, n = 52) or severe coronary atherosclerosis (M2 = 0.819, n = 47). The apolipoprotein A-II gene may also contribute to the regulation of plasma levels or composition of HDL in response to environmental changes. To study the effect upon apolipoprotein A-II variability, 42 monozygotic twin pairs were genotyped for the MspI RFLP. Pairs with the genotype M2M2 (n = 28) had significantly smaller within-pair differences in plasma apolipoprotein A-II levels (2.2 vs 5.8 mg/dl, P < 0.02; Mann-Whitney) than those with other genotypes (n = 14). The M2 allele may be in linkage disequilibrium with a functional mutation that restricts the variability of plasma apolipoprotein A-II in response to environmental conditions. This provides a new example of a 'variability' gene, one of an important group of loci which may alter responses to hypolipidaemic therapy and cardiovascular risk.

Human peripheral lymph lipoproteins are enriched in sphingomyelin.

The concentrations of total choline-containing phospholipids and of sphingomyelin have been determined in peripheral lymph from six volunteers. The concentration in lymph of total phospholipids was 14.6 +/- 6.0 mg/dl and of sphingomyelin 6.1 +/- 2.7 mg/dl; the estimated concentration of phosphatidylcholine (by subtracting the concentration of sphingomyelin from that of total choline-containing lipoproteins) was 8.5 +/- 5.1 mg/dl. The concentration of free choline in lymph was less than 1 mg/dl. The ratio of the concentration in lymph to that in the subject's own plasma of sphingomyelin was 0.18 +/- 0.07 and that of (estimated) phosphatidylcholine 0.05 + 0.04; the difference between these two ratios was significant at P less than 0.002. The present data are compatible with the hypothesis that some of lipoprotein phosphatidylcholine is lost while crossing from plasma to lymph.

Lipoproteins of human peripheral lymph. Apolipoprotein AI-containing lipoprotein with alpha-2 electrophoretic mobility.

Evidence from diverse sources has implicated a central role of apolipoprotein AI (apo AI), the most abundant protein of plasma high-density lipoproteins, in the transport of cholesterol from peripheral tissues to the liver (reverse cholesterol transport). Particles containing only apo AI appear to be more effective as cholesterol acceptors in tissue culture than do particles which also contain apo AII. The apo AI-containing lipoproteins of plasma have been extensively studied, but there is less information on those in tissue fluids, to which most peripheral cells are exposed. In the present study the heterogeneity of apo AI-containing particles in human peripheral lymph, collected from the dorsum of the foot, has been examined by starch block electrophoresis, exclusion chromatography and immunoelectrophoresis. The apo AI-containing particles of lymph were found to be more variable in both electrophoretic mobility and size than those of plasma from the same subjects. Of particular interest was a subpopulation which migrated on electrophoresis with the same mobility as alpha-2-macroglobulin. This fraction accounted for approximately 7% (range: 4-12%; n = 5) of lymph apo AI, contained no immunodetectable apo AII, and by exclusion chromatography was composed of particles the size of, or smaller than, albumin. Such physicochemical properties suggest that these alpha-2 migrating particles may function as the principal primary acceptors of cell cholesterol in the extracellular matrix of human peripheral tissues. By isoelectric focusing, lymph apo AI was found to contain a higher proportion of more negatively charged isoforms than the apo AI of plasma.

Lipoproteins of human peripheral lymph.

The concentration of cholesterol in peripheral lymph is roughly one tenth of that in the blood plasma of the same subject. In lymph, there is virtually no very low density lipoprotein (VLDL), probably due to low permeability of the vascular endothelium for particles of this size. More than 95% of apo B-containing lipoproteins of lymph have the density of plasma low density lipoproteins (LDL). The concentration of apo A-I and apo A-II in lymph is about 15% of that in plasma; yet about 50% of the total mass of both these main HDL apoproteins is present extravascularly. High density lipoproteins (HDL) of lymph appear square-packing, and the presence of such large HDL particles is the most conspicuous difference between lipoproteins in plasma and in the extravascular fluids. It remains to be seen whether raising plasma apo A-I concentration per se will increase the initial stages of reverse cholesterol transport and also be clinically beneficial.

The concentration of apolipoprotein A-II in human peripheral lymph.

The concentration of apolipoprotein A-I (apoA-I) and of apolipoprotein A-II (apoA-II) was determined immunoelectrophoretically in lymph and plasma of six subjects. The concentration in lymph of apoA-I was 20.3 +/- 3.1 mg/dl, that of apoA-II was 4.6 +/- 0.5 mg/dl. The ratio of the concentration in lymph over that in plasma (CL/CP) for apoA-I was 0.14 +/- 0.1, that for apoA-II 0.14 +/- 0.01. Lymph and plasma samples from two subjects were fractionated by exclusion chromatography and the concentration of both apolipoproteins in resulting fractions was determined immunoelectrophoretically. ApoA-I of lymph eluted with fractions spanning a wider range of particle sizes than plasma apoA-I, while lymph apoA-II eluted predominantly with fractions that contained particles corresponding in size to plasma apoA-II-containing particles. It appears that the largest and smallest lymph HDL represent subspecies of Lp(A-I without A-II). These findings are discussed in the context of their possible bearing on initial stages of reverse transport of cholesterol.

Urine concentrations of apolipoprotein AI in renal tubular proteinuria.

To test the hypothesis that a significant proportion of apolipoprotein AI (apoAI) metabolism occurs through glomerular filtration of free apoAI in serum and subsequent renal tubular metabolism, we have examined the urine concentration of apoAI in three situations in which proximal tubular reabsorption of another protein metabolized in this manner and retinol-binding protein (RBP) was impaired. Following infusion of a cross-linked gelatin polymer (Haemaccel) in four normal subjects, urine RBP excretion (normally about 100 micrograms/L), was between 14 and 46 mg/L, while urine apoAI excretion was less than 0.5 mg/L. On the third day following cardiac surgery involving Haemaccel infusion, urine RBP was between 27 and 159 mg/L while urine apoAI excretion was again less than 0.5 mg/L. In 16 samples from eight patients recently transplanted with allograft kidneys, urine RBP was between 9 and 70 mg/L, whereas in only two samples was apoAI detected in the urine at 0.5 mg/L. These results have been taken to indicate that significant metabolism of apoAI through glomerular filtration and tubular absorption is unlikely to occur in humans.

Human lymphedema fluid lipoproteins: particle size, cholesterol and apolipoprotein distributions, and electron microscopic structure.

The concentration of cholesterol, apolipoproteins A-I, B, and E has been determined in lymphedema fluid from nine patients with chronic primary lymphedema. The concentrations were: 38.14 +/- 21.06 mg/dl for cholesterol, 15.6 +/- 6.17 mg/dl for apolipoprotein A-I, 7.5 +/- 2.8 mg/dl for apolipoprotein B, and 1.87 +/- 0.50 mg/dl for apolipoprotein E. These values represent 23%, 12%, 6%, and 38% of plasma concentrations, respectively. The ratio of esterified to unesterified cholesterol in lymphedema fluid was 1.46 +/- 0.45. Lipoproteins of lymphedema fluid were fractionated according to particle size by gradient gel electrophoresis and by exclusion chromatography. Gradient gel electrophoresis showed that a majority of high density lipoproteins (HDL) of lymphedema fluid were larger than ferritin (mol wt 440,000) and smaller than low density lipoproteins (LDL); several discrete subpopulations could be seen with the large HDL region. Fractionation by exclusion chromatography showed that more than 25% of apolipoprotein A-I and all of apolipoprotein E in lymphedema fluid was associated with particles larger than plasma HDL2. Apolipoprotein A-I also eluted in fractions that contained particles the size of or smaller than albumin. Isolation of lipoproteins by sequential ultracentrifugation showed that less than 25% of lymphedema fluid cholesterol was associated with apolipoprotein B. The majority of apolipoprotein A-containing lipoproteins of lymphedema fluid were less dense than those in plasma. Ultracentrifugally separated fractions of lipoproteins were examined by electron microscopy. The fraction d less than 1.019 g/ml contained little material, while fraction d 1.019-1.063 g/ml contained two types of particles: round particles 17-26 nm in diameter and square-packing particles 13-17 nm on a side. Fractions d 1.063-1.085 g/ml had extensive arrays of square-packing particles 13-14 nm in size. Fractions d 1.085-1.11 g/ml and fractions d 1.11-1.21 g/ml contained round HDL, 12-13 nm diameter and 10 nm diameter, respectively. Discoidal particles were observed infrequently.

The distribution of cholesterol and apoprotein A-I between the lipoproteins in plasma and peripheral lymph from normal human subjects.

The lipoproteins of peripheral lymph and plasma from normal human subjects were separated according to their density by sequential ultracentrifugation and according to their size by gradient gel electrophoresis and gel exclusion chromatography. High density lipoproteins (HDL) carried a higher proportion of the total cholesterol in lymph than in plasma. Within the HDL fraction, the less dense and more lipid-rich component (HDL2) carried a higher proportion of the total HDL cholesterol in lymph than in plasma. Gradient gel electrophoresis showed (1) a higher proportion of large to small HDL particles in lymph than in plasma and (2) the presence of at least three populations of apo A-I-containing lipoproteins with Stokes diameters larger than the Stokes diameter of HDL2. Separation by gel exclusion chromatography showed that the proportion of large HDL particles with a high cholester: apo A-I ratio was greater in lymph than in plasma. In view of the sieving effect of the blood capillaries, which favours the passage across the capillary walls of smaller vs larger particles, we suggest that the higher ratio of large to small HDL particles in lymph than in plasma is due to the conversion of small to large HDL in the interstitial fluid by incorporation of cholesterol and other lipids from extravascular cells into the smaller particles.

Comparison of apoprotein B of low density lipoproteins of human interstitial fluid and plasma.

Virtually all apoprotein B (apoB)-containing lipoproteins of the peripheral interstitial fluid of subjects with primary lymphoedema float in the ultracentrifugal field in the density interval 1.019-1.063 g/ml; in this respect they are similar to plasma low-density lipoproteins (LDL). 2. Virtually all apo-B-containing lipoproteins of interstitial fluid migrate in the electrophoretic field with pre-beta mobility; in this respect they are similar to plasma very-low-density lipoproteins. 3. The apoB of lipoproteins of interstitial fluid does not differ in terms of Mr from apoB-100 of human plasma [Kane, Hardman & Paulus (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2465-2469] as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. Both apoB of interstitial fluid and plasma are heterogenous in terms of their charge as determined by isoelectric focusing of their complexes with the nonionic detergent Nonidet P40. ApoB of plasma LDL focuses between pH5.9 and 6.65, and that of interstitial fluid LDL between pH 5.9 and 6.1. Thus the overall charge of apoB of interstitial fluid is more negative than that of its plasma LDL counterpart.

Effects of starvation and plasma exchange on lecithin: cholesterol acyltransferase activity and cholesterol efflux in cholesterol-fed pigs.

The effects of starvation and of plasma exchange with a cholesterol-free substitute on efflux of tissue cholesterol and on lecithin: cholesterol acyltransferase (LCAT) activity in plasma and peripheral lymph were investigated in two pigs fed a cholesterol diet for 3-4 months. The pigs were labelled with i.v. [14C]cholesterol before plasma exchange or starvation. The cholesterol diet increased plasma total cholesterol concentration and LCAT activity in plasma and lymph, but had little effect on the rate of esterification of cholesterol in plasma or lymph. During cholesterol feeding, and when the animals were fed a normal diet, cholesterol esterification rates in plasma and lymph were much lower than the maximum rates achieved when LCAT was saturated with substrate, suggesting that LCAT in normal pig plasma and lymph is not saturated with substrate. Plasma exchange, carried out when the specific activity of tissue cholesterol exceeded that of plasma cholesterol, was followed by a brief rise in the specific activity of plasma cholesterol to a maximum value between the specific activities of muscle and adipose-tissue cholesterol, reflecting the transfer of radioactive cholesterol from tissue to plasma. During the rise in plasma total cholesterol specific activity there were no differences between the specific activities of low-density lipoprotein (LDL) cholesterol and high-density lipoprotein (HDL) cholesterol in plasma or lymph. Starvation had no effect on the plasma-cholesterol specific-activity curve. From about day 14 after labelling, cholesterol-specific activity decreased in the order: tissues greater than lymph greater than plasma. This suggests that the transfer of cholesterol from tissues to plasma was mediated by lipoproteins in the interstitial fluid.

Apoprotein B of lipoprotein(a) of human plasma.

Lipoprotein(a) was purified by agarose-gel chromatography from human plasma from which lipoproteins of Sf greater than 0 had been removed either by sequential or by density-gradient ultracentrifugation. After delipidation, the apoprotein B of this lipoprotein was analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It could not be distinguished from the apoprotein B of low-density lipoproteins (rho 1.019-1.063 g/ml). A significant increase in the concentration of apoprotein B in plasma from which the Sf greater than 0 lipoproteins had been removed was observed in six subjects 4 h after a fatty meal.

Further evidence for the role of high density lipoprotein in the removal of tissue cholesterol in vivo.

The lipoproteins of human peripheral lymph and plasma were separated according to particle size by polyacrylamide gradient gel electrophoresis. All samples of lymph contained lipoproteins that moved to the same positions on the gel as plasma LDL and plasma HDL. Some samples of lymph also contained lipoproteins with the mobility of VLDL and IDL. The lymph lipoproteins corresponding to plasma LDL reacted with anti-LDL serum and those corresponding to plasma HDL reacted with anti-HDL serum. In the lipoprotein fraction with the mobility of HDL, the proportion of particles larger than catalase was greater in lymph than in plasma. It is suggested that the shift in size distribution towards larger HDL particles in lymph compared with plasma is due to uptake of cholesterol from extravascular tissue by HDL particles after they have reached the interstitial fluid from the plasma, rather than to preferential movement of larger particles across the capillary walls.

The concentration of apolipoprotein A-I in human peripheral lymph.

The concentration of apolipoprotein A-I in peripheral lymph of eight apparently healthy subjects has been determined by quantitative immunoelectrophoresis. Under steady-state conditions the average concentration of this apolipoprotein in lymph was 15.9 +/- 3.6 mg/dl, that is 12.24 +/- 2.3% of its concentration in plasma of the corresponding subjects. Apolipoprotein A-I could not be detected immunochemically in particles smaller than haemoglobin (Mr 67 000) when lymph was subjected to gel filtration on Sephacryl S300 superfine either by thin-layer or column modification of this method. Lymph and plasma from three subjects were fractionated by column gel filtration and apolipoprotein A-I determined by quantitative immunoelectrophoresis in delipidated fractions. It was found that the distribution of apolipoprotein A-I in lymph was shifted towards larger particles when compared to its distribution in plasma.

Evidence for the presence of tissue-free cholesterol in low density and high density lipoproteins of human peripheral lymph.

The specific radioactivity of free and esterified cholesterol in the lipoproteins of human peripheral lymph was measured in 4 normal human subjects at various intervals after labelling the tissue cholesterol by a single intravenous injection of [14C]cholesterol. The free : esterified cholesterol specific-activity ratios in lymph LDL and HDL at short and long intervals after labelling in vivo suggest that both lipoproteins were capable of acting as acceptors for tissue-free cholesterol, but that in 3 of the 4 subjects the predominant acceptor was HDL.